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Analysis of Self-Assembling Complexes via Supramolecular Mass Spectrometry

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Permanent Link: http://ufdc.ufl.edu/UFE0021434/00001

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Title: Analysis of Self-Assembling Complexes via Supramolecular Mass Spectrometry
Physical Description: 1 online resource (156 p.)
Language: english
Creator: Barbara, Joanna E
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: ambient, assembly, cyclodextrins, desorption, electrospray, ionization, mass, molecular, noncovalent, phloroglucinol, reactive, recognition, self, spectrometry, supramolecular
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Self-assembling supramolecular complexes are stable configurations of two or more molecules stabilized by noncovalent intermolecular interactions, which aggregate independently according to molecular recognition principles. Noncovalent, e.g., cation-pi and hydrogen-bonding, interactions make important contributions to the structure and function of many biomolecules. They are vital to protein folding and stabilization and so to substrate specificity and enzyme action. Host-guest inclusion complexes stabilized by these interactions are used in areas such as enzyme mimicry, catalysis, and therapeutic drug development and delivery. Modeling and analysis of these noncovalent interactions are essential to the development of selective synthetic hosts. Thorough analytical characterization of diverse supramolecular systems is necessary to contribute to the wealth of data required to gain a better understanding of the inherent chemical behavior involved in molecular recognition and noncovalent complex formation. Mass spectrometry, as a gas-phase analytical technique, has the ability to provide vital information concerning supramolecular chemistry in the absence of interference from a solvent shell. The advancement of supramolecular mass spectrometry was the major goal of this research. A pair of designed synthetic receptors for alkali-metal cations based on the natural product 1,3,5-trihydroxybenzene (phloroglucinol) was thoroughly characterized in the gas phase using advanced supramolecular mass spectrometric techniques. Following additional solution-phase characterization using absorbance spectroscopy in the ultraviolet region, it was determined that the mass spectrometric approach and the optical spectroscopic approach combined to yield useful complementary characterization information. Following the configuration of a home-built source with a time-of-flight mass analyzer, desorption electrospray ionization (DESI) was developed and validated as a useful novel analytical tool for this area of application, through the design and implementation of a rapid screening experiment for potential guest compounds for supramolecular encapsulation by a beta-cyclodextrin host. Comparison experiments, using nuclear magnetic resonance spectroscopy as the standard solution-phase validation technique, revealed that DESI is a superior ionization technique to the commonly-employed electrospray ionization for this type of work. DESI is not as prone to the detection of false-positive nonspecific complexes resulting from the formation of artifacts during the electrospray process. Thus a useful addition to the supramolecular mass spectrometry toolkit has been contributed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Joanna E Barbara.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Eyler, John R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021434:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021434/00001

Material Information

Title: Analysis of Self-Assembling Complexes via Supramolecular Mass Spectrometry
Physical Description: 1 online resource (156 p.)
Language: english
Creator: Barbara, Joanna E
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: ambient, assembly, cyclodextrins, desorption, electrospray, ionization, mass, molecular, noncovalent, phloroglucinol, reactive, recognition, self, spectrometry, supramolecular
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Self-assembling supramolecular complexes are stable configurations of two or more molecules stabilized by noncovalent intermolecular interactions, which aggregate independently according to molecular recognition principles. Noncovalent, e.g., cation-pi and hydrogen-bonding, interactions make important contributions to the structure and function of many biomolecules. They are vital to protein folding and stabilization and so to substrate specificity and enzyme action. Host-guest inclusion complexes stabilized by these interactions are used in areas such as enzyme mimicry, catalysis, and therapeutic drug development and delivery. Modeling and analysis of these noncovalent interactions are essential to the development of selective synthetic hosts. Thorough analytical characterization of diverse supramolecular systems is necessary to contribute to the wealth of data required to gain a better understanding of the inherent chemical behavior involved in molecular recognition and noncovalent complex formation. Mass spectrometry, as a gas-phase analytical technique, has the ability to provide vital information concerning supramolecular chemistry in the absence of interference from a solvent shell. The advancement of supramolecular mass spectrometry was the major goal of this research. A pair of designed synthetic receptors for alkali-metal cations based on the natural product 1,3,5-trihydroxybenzene (phloroglucinol) was thoroughly characterized in the gas phase using advanced supramolecular mass spectrometric techniques. Following additional solution-phase characterization using absorbance spectroscopy in the ultraviolet region, it was determined that the mass spectrometric approach and the optical spectroscopic approach combined to yield useful complementary characterization information. Following the configuration of a home-built source with a time-of-flight mass analyzer, desorption electrospray ionization (DESI) was developed and validated as a useful novel analytical tool for this area of application, through the design and implementation of a rapid screening experiment for potential guest compounds for supramolecular encapsulation by a beta-cyclodextrin host. Comparison experiments, using nuclear magnetic resonance spectroscopy as the standard solution-phase validation technique, revealed that DESI is a superior ionization technique to the commonly-employed electrospray ionization for this type of work. DESI is not as prone to the detection of false-positive nonspecific complexes resulting from the formation of artifacts during the electrospray process. Thus a useful addition to the supramolecular mass spectrometry toolkit has been contributed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Joanna E Barbara.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Eyler, John R.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021434:00001


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ANALYSIS OF SELF-ASSEMBLING COMPLEXES VIA SUPRAMOLECULAR MASS
SPECTROMETRY






















By

JOANNA E. BARBARA


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007






































O 2007 Joanna E. Barbara





































To Oliver, Ella, and Jake












ACKNOWLEDGMENTS

I must acknowledge the many colleagues and friends who have been instrumental in my

graduate school journey. First, I thank my advisors, Dr. John Eyler and Dr. David Powell, for

their consistent support and direction. John Eyler has provided me with a model example of the

successful combination of family and science, and given me academic and personal freedom to

pursue the proj ects I enjoyed and balance my children with my work. Always a source of advice

and scientific insight, I truly appreciated all the sensible and thoughtful guidance I received from

him. I acknowledge David Powell both as an advisor and as a friend. He shaped the project I

undertook as the basis of my dissertation research and provided me with the necessary training

and resources to accomplish my goals. In addition, I thank him for the patience with which he

taught me instrumentation skills and introduced me to the world of tools and turbopumps; my

newly discovered ability to change a tire I attribute solely to him. As director of the service lab

where I have worked for the last two years, he has been an appreciative and approachable

supervisor, and I have thoroughly enjoyed my time in his lab.

Dr. Kathryn Williams, my mentor throughout my undergraduate and graduate career, is

acknowledged for her teaching talent and constant support. I thank her for telling me to go to

graduate school in the first place, and for supporting me as a scientist throughout my pregnancy

and the birth of my son. Dr. Williams allowed me access to all the instrumentation in her

teaching lab and consequently made several of the studies presented in this dissertation possible.

Sincere gratitude is extended to her and the other members of my committee, Dr. Richard Yost

and Dr. Peggy Borum, for the commitment of their time and energy. I also wish to acknowledge

Dr. Tim Garrett, who passed on to me his enthusiasm for mass spectrometry as my teaching

assistant several years ago.









Dr. Ben Smith is acknowledged as graduate advisor for his advice and guidance, but also

as a scientist for discussion concerning optical spectroscopy. I thank his assistant Ms. Lori Clark

for patience, support, and administrative problem-solving.

Dr. Ronald Castellano is acknowledged for designing the phloroglucinol derivatives and

furnishing his knowledge and insight as a synthetic chemist. I also thank his former student, Dr.

Andrew Lampkins, for helping introduce me to the field of supramolecular chemistry and for

sample synthesis.

I acknowledge Dr. Jodie Johnson for teaching me a great deal about fundamental mass

spectral interpretation, structural elucidation, and fragment assignment. Data analysis would

have been a much more time-consuming process without his guidance. I wish to thank Brian

Smith for engineering the DESI source and for all the consultation and machining expertise that

he contributed to its repeated modification. Sincere thanks go to Joe Shalosky for practical

advice and problem-solving associated with the instrumentation.

I thank Dr. Cynthia Cole, former director of the University of Florida Racing Lab, Patrick

Russell, and Dr. Keith Zientek who provided access to the ion trap used for the determination of

the phloroglucinol derivative dissociation pathways. Steve Miles and Larry Hartley are thanked

for electronics support, and the IT shop staff, particularly Joe Carusone, is also acknowledged.

The members of the Eyler and Powell groups, past and present, are thanked for their

support and helpful suggestions. My cheering squad and sounding boards, Jonathon, Lani, Julia,

and Soledad, are gratefully acknowledged for listening and advising on all subjects. The many

unnamed friends who have helped me make it to this point are also thanked.

Finally, I acknowledge my husband, Oliver, and my children, Ella and Jake, all of whom

have sacrificed a great deal of time with me over the past years. I want to thank Oliver for his









unfailing support and encouragement, and for taking over my responsibilities when the workload

became too much for me, despite his own responsibilities as a student and a parent. My precious

babies are acknowledged as my motivation to succeed.













TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF TABLES ................ ...............10........... ....


LIST OF FIGURES ........._.._ ........... ...............11....


AB S TRAC T ............._. .......... ..............._ 14...


CHAPTER


1 INTRODUCTION ................. ...............16.......... ......


Supramolecular Chemistry .............. ...............18....
Concepts .................... .... .. ..............1
Types of Noncovalent Bonds .............. ...............20....
lon-ion interactions .............. ...............20....
lon-dipole interactions............... ..............2
Dipole-dipole interactions ................. ...............21......_......
Hydrogen bonds .............. ...............21....
Cation-n: interactions ........._._ ...... .__ ...............21....

xn-Stacking interactions ........._._ ...... .... ...............16..
Biological and Physical Significance .............. ...............22....
Mass Spectrometric Approaches .............. ...............23....
High-Resolution Mass Spectrometry .............. ...............26....
Concepts ................. ...............26.................
In strmentati on ................. ...............27........... ....
Tandem Mass Spectrometry ................. ...............29....__. .....
Supramolecular Mass Spectrometry ................. ...............31................
Gas-Phase Supramolecular Chemistry .............. ...............31....
Survey of Recent Literature. ................. ...............33......... .....

2 CHARACTERIZING NONCOVALENT DIMERIZATION BEHAVIOR OF
DESIGNED PHLOROGLUCINOL DERIVATIVES USINTG ELECTRO SPRAY
IONIZATION HIGH RESOLUTION MASS SPECTROMETRY .............. ...................40


Introducti on ................. ...............40.................
Experim ental ................. ...............42.......... ......
Sample Preparation............... ..............4
Mass Spectrometry .............. ..... .. ...............4
Ultraviolet/Visible Absorbance Spectroscopy .............. ...............44....
Results and Discussion ................. ........ .. ...................4
Electrospray Ionization Mass Spectrometry (ESI-MS) for Dimer Detection .........._......45
Traditional Competitive Binding Approach ...._.. ................ ............... 45. ....












Dissociation Curve Approach............... ...............46
Heterodimer Studies............... ... ... .___ .. ...............48...
Ultraviolet/Visible (UV/Vis) Absorbance Spectroscopy .............. .....................5
Conclusions............... ..............5


3 ELUCIDATING THE DISSOCIATION MECHANISM OF NOVEL
PHLOROGLUCINOL DERIVATIVES .............. ...............65....


Introducti on ................. ...............65.................

Experimental ................. ...............67.................
Sample Preparation............... ..............6
M ass Spectrometry .............. ...............68....
Results and Discussion ................ ... .............. .. .. .. .......6
Electrospray Ionization Source-Skimmer Collisionally Induced Dissociation
(SCID) Justification .............. ...............68..
2-Hydroxybenzophenone Experiments .............. ...............69....
Protonated Molecule Dissociation Pathways .............. ....... .......... ........7
Alkali-Metal Cation-bound Dimer and Adducted Monomer Dissociation...................71
Conclusions............... ..............7


4 CONFIGURATION OF A HOME-BUILT DESORPTION ELECTRO SPRAY
IONIZATION SOURCE WITH A COMMERCIAL TIME-OF-FLIGHT MASS
AN ALY ZER ........._.. _..... .._ ............... 1....


Introducti on ............ ..... .._ ............... 1....
Experimental ............__... .. ..__ ... ......___ ..................94
Desorption Electrospray Ionization (DESI) Source Design ................. .....................94
M ass Spectrometry .............. ...............94....
Sample Preparation............... ..............9
Results and Discussion ................ .... .. .. .. ...... .................9
Configuration of DESI Source with Fourier Transform lon Cyclotron Resonance
Mass Spectrometer (FTICR-M S)......................... .... .. ............9
Configuration of DESI Source with Time-of-Flight Mass Spectrometer (TOF-MS).....97
Important Optimization Considerations .............. ...............100....
Conclusions............... ..............10


5 REACTIVE DESORPTION ELECTRO SPRAY IONIZATION FOR RAPID
SCREENING OF GUESTS FOR SUPRAMOLECULAR INCLUSION COMPLEXES...112


Introducti on ................. ...............112____.......

Experimental ............ ..... .._ ...............115...
Sample Preparation ............ ..... .._ ...............115...
M ass Spectrom etry ................. ........... ..... ...............116......
Nuclear Magnetic Resonance (NMR) Spectroscopy ........................... ...............117
Theoretical Calculations ................. ...............118................
Results and Discussion ................ ...............118................
Reactive DESI Receptor Screening ................. ...............118...............












Electrospray Ionization Receptor Screening ................. ........__. ........ 119.. ....
Proton NMR Spectroscopic Screening .....__.....___ ..........._ ...........12
Computational Chemistry............... ...............12
Conclusions............... ..............12


6 CONCLUSIONS AND FUTURE DIRECTIONS .............. ...............137....


LIST OF REFERENCES ............ ..... ._ ...............147...


BIOGRAPHICAL SKETCH ............ ..... .__ ...............156...











LIST OF TABLES


Table page

2-1 Phloro 1 and 2 SCID-FTICR-MS dissociation curve VCso values............... .................5

2-2 Alkali-metal cationic radii .............. ...............55....

2-3 Phloro 1 and 2 SCID-TOF-MS dissociation curve VCso values............... .................5

5-1 Intensities and ion type observed with the DESI-TOF-MS screen in methanol:water....1 27

5-2 Intensities and ion type observed with the ESI-TOF-MS screen in methanol:water. ......128

5-3 Complexes detected using DESI-MS, ESI-MS, and 1H NMR spectroscopy. .................129










LIST OF FIGURES


Figure page

1-1 Representation of a cavitate and a clathrate............... ...............3

1-2 Two possible orientations of dipole-dipole interactions .................... ............... 3

1-3 Representation of cation-n: interaction. ......___ .... .._.. ...............38..

1-4 Generalized configuration of an electrospray source ................. ............................39

1-5 Quantities used to calculate resolution .............. ...............39....

2-1 Phloroglucinol structure................ ...............5

2-2 Structures of the phloroglucinol derivatives. ......___ .... .._._. ...._.._._..........5

2-3 Electrospray ionization mass spectra of phloro 1 forming cation-bound dimers .............56

2-4 Electrospray ionization spectra of showing the presence of adventitious sodium.............57

2-5 Phloro 1 dimer di ssoci ation curves obtained using S CID-F TICR-M S............................5 8

2-6 Phloro 2 dimer dissociation curves 2 obtained using SCID-FTICR-MS .........................59

2-7 Di ssoci ati on curve s for all cati on-b ound dimers obtained using S CID-TOF -MS............60

2-8 Spectrum obtained using ESI-TOF-MS of homo and heterodimers ................. ...............61

2-9 Dissociation curves for the cation-bound heterodimers............... ..............6

2-10 Absorption spectrum for phloro 1 in methanol ................. ...............62..............

2-11 Beer' s Law plot for phloro 1................ ................................... ............62

2-12 Ultraviolet/visible absorption spectra for a range of phloro concentrations. ................... ..63

2-13 Ultaviolet/visible absorption and derivative spectra for phloro 1 and 2. ...........................64

2-14 Bar charts showing measured hLax chromic shift for phloro 1 and 2. ............. ................64

3-1 Structure of 2-hydroxybenzophenone ................. ...............77................

3-2 Dissociation of phloro 1 cation-bound dimers of to monomeric adducts ................... .......77

3-3 Dissociation of phloro 2 cation-bound dimers of to monomeric adducts ................... .......78

3-4 Proposed fragmentation pathway for 2-hydroxybenzophenone. ............. ....................79










3-5 Proposed initial dissociation mechanism for 2-hydroxybenzophenone........................8

3-6 Phloro 1 protonated molecule proposed fragmentation pathway. ............. ...................81

3-7 Phloro 2 protonated molecule proposed fragmentation pathway. ............. ...................82

3-8 Lithium cation-bound dimer of phloro 1 proposed fragmentation pathway. ........._.._........83

3-9 Lithium cation-bound dimer of phloro 2 proposed fragmentation pathway. ........._.._........84

3-10 Sodium cation-bound dimer of phloro 1 proposed fragmentation pathway. .....................85

3-11 Sodium cation-bound dimer of phloro 2 proposed fragmentation pathway. .....................86

3-12 Potassium cation-bound dimer of phloro 1 proposed fragmentation pathway ........._........87

3-13 Potassium cation-bound dimer of phloro 2 proposed fragmentation pathway. .................88

3-14 Ammonium cation-bound dimer of phloro 1 proposed fragmentation pathway. ..............89

3-15 Ammonium cation-bound dimer of phloro 2 proposed fragmentation pathway. ..............90

4-1 Schematic of the overhead view of the original home-built DESI source. ................... ...104

4-2 Desorption electrospray ionization source interfaced with the FTICR-MS. ...................105

4-3 Close-up view of DESI source configured with FTICR-MS............_._. ........._._.....105

4-4 Structure of the Rhodamine 6G preformed ion. ............_. ...._... .. ...._.._........0

4-5 First DESI-FTICR-MS spectrum showing Rhodamine 6G. .............. .....................0

4-6 Close-up view of the DESI-FTICR-MS setup. ....._._._ .... ..... .. ...._._.........0

4-7 Close-up view of the DESI source interfaced with the TOF-MS. ........._.... ..............108

4-8 Side view of the DESI-TOF-MS configuration ....._._._ .... ... .... ......._._.......10

4-9 Representation of the capillary extender for the DESI-TOF interface. .........._..............109

4-10 Mass spectrum of Rhodamine 6G obtained using DESI-TOF-MS. ............._ ..............1 10

4-11 Positive-mode DESI-TOF mass spectrum of cytochrome C. ................ ................ ...1 10

4-12 Schematic representation of the DESI spray head and inlet orifice. .........._... ..............11 1

4-13 Representative optimization data for the DESI-TOF-MS configuration. ......................111

5-1. Structures of the twelve screened guest compounds. ........._.._.. ......._ ........._......130










5-2 Desorption electrospray ionization MS receptor screen design ................. ................. 131

5-3 Reactive DESI-TOF spectrum of pcyclodextrin sprayed onto nortestosterone. ..............131

5-4 Reactive DESI-TOF mass spectra showing data for several guests. ............. ................132

5-5 Representative ESI-TOF mass spectra for the P-cyclodextrin(CD) screen. ...................133

5-6 Structure of the glucopyranose monomer of P-cyclodextrin with chemical shifts..........133

5-7 Proton NMR spectra showing the region 3.5-4 ppm chemical shift. .............. ..............134

5-8 Structure of the acetanilide guest showing chemical shift............... ..................3

5-9 Nuclear Overhauser effect spectrum of P-cyclodextrin with acetanilide. .......................135

5-10 Conformation of the P-cyclodextrin: acetanilide inclusion complex. ............. ...... ..........13 5

5-11 Representation of the toroidal P-cyclodextrin host showing cavity diameter. ................136

5-12 Optimized structure and molecular dimensions of cyclam ................. ............. .......136











Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ANALYSIS OF SELF-ASSEMBLING COMPLEXES VIA SUPRAMOLECULAR MASS
SPECTROMETRY

By

Joanna E. Barbara

December 2007

Chair: John R. Eyler
Major: Chemistry

Self-assembling supramolecular complexes are stable configurations of two or more

molecules stabilized by noncovalent intermolecular interactions, which aggregate independently

according to molecular recognition principles. Noncovalent, e.g., cation- xn and hydrogen-

bonding, interactions make important contributions to the structure and function of many

biomolecules. They are vital to protein folding and stabilization and so to substrate specifieity

and enzyme action. Host-guest inclusion complexes stabilized by these interactions are used in

areas such as enzyme mimicry, catalysis, and therapeutic drug development and delivery.

Modeling and analysis of these noncovalent interactions are essential to the development of

selective synthetic hosts. Thorough analytical characterization of diverse supramolecular systems

is necessary to contribute to the wealth of data required to gain a better understanding of the

inherent chemical behavior involved in molecular recognition and noncovalent complex

formation. Mass spectrometry, as a gas-phase analytical technique, has the ability to provide vital

information concerning supramolecular chemistry in the absence of interference from a solvent

shell. The advancement of supramolecular mass spectrometry was the maj or goal of this

research.










A pair of designed synthetic receptors for alkali-metal cations based on the natural product

1,3,5-trihydroxybenzene (phloroglucinol) was thoroughly characterized in the gas phase using

advanced supramolecular mass spectrometric techniques. Following additional solution-phase

characterization using absorbance spectroscopy in the ultraviolet region, it was determined that

the mass spectrometric approach and the optical spectroscopic approach combined to yield useful

complementary characterization information.

Following the configuration of a home-built source with a time-of-flight mass analyzer,

desorption electrospray ionization (DESI) was developed and validated as a useful novel

analytical tool for this area of application, through the design and implementation of a rapid

screening experiment for potential guest compounds for supramolecular encapsulation by a P-

cyclodextrin host. Comparison experiments, using nuclear magnetic resonance spectroscopy as

the standard solution-phase validation technique, revealed that DESI is a superior ionization

technique to the commonly-employed electrospray ionization (ESI) for this type of work. DESI

is not as prone to the detection of false-positive nonspecific complexes resulting from the

formation of artifacts during the electrospray process. Thus a useful addition to the

supramolecular mass spectrometry toolkit has been contributed.










CHAPTER 1
INTTRODUCTION

The term supramolecular chemistry was first used by Jean-Marie Lehn in 1978 to

encompass all of the previously divided areas breached by his groundbreaking work exploring

the noncovalent chemistry of macropolycycles.l He has defined it as 'the chemistry of the

noncovalent bond',2 briefly comprising intermolecular binding, catalysis, molecular recognition,

self-assembly, directed molecular design, and self-replication phenomena.3 Over the preceding

decade, various alkali and alkaline-earth cations had been observed to form stable complexes

with macrocyclic ligands, the most famous example being the crown ether family discovered by

Charles Pedersen,4 and Lehn had been working on synthesizing macropolycycles with designed

molecular recognition properties.l As more supramolecular species were synthesized and their

complexation characteristics explored, an entire discipline evolved. Lehn, Pedersen, and Donald

Cram, a synthetic chemist specializing in cyclophane chemistry, were subsequently awarded the

1987 Nobel Prize in Chemistry for work resulting in creation of the field.5 Today,

supramolecular chemistry is an accepted interdisciplinary area with far-reaching biological

significance.

Mass spectrometry, measurement of the mass-to-charge ratio of ions, is an analytical

technique applicable not only to the structural elucidation and quantitative analysis of molecules

but also to kinetic and conformational studies. Inherently a gas-phase technique, it is well-suited

to the study of supramolecular complexes because it enables the study of intrinsic properties of

ions without the interference of molecules of solvation.3,6 Recently developed soft ionization

techniques such as matrix-assisted laser desorption ionization (MALDI) and electrospray

ionization (ESI) have addressed the challenge of providing charge to the complexes without









disrupting the weak stabilizing noncovalent interactions,6 making mass spectrometry accessible

to a plethora of supramolecular applications.

The research described in this thesis contributes to the development of advanced

supramolecular mass spectrometry beyond general stoichiometric complex detection, and

demonstrates how combining useful characteristics of different types of instrumentation and

innovative experimental design can maximize acquisition of information about supramolecular

complexes via mass spectral analysis. Electrospray ionization and tandem mass spectrometry

were used to probe the noncovalent cation-bound homodimer formation behavior of a novel pair

of designed alkali-metal cation receptor synthons. Electrospray ionization mass spectral

characteristics were used to analyze the effects of structural differences between monomers on

the noncovalent interactions responsible for stabilization of the corresponding heterodimer.

Solution-phase optical spectroscopy provided insight into the supramolecular structure of the

homodimers, but highlighted the necessity of gas-phase analytical techniques for the

determination of supramolecular complexes without the complication of competition for binding

sites between the complex components and molecules of solvation. Fragmentation pathways for

alkali-metal cation adducts of some phloroglucinol derivatives were elucidated using

electrospray ionization tandem-in-time mass spectrometry.

A home-built desorption electrospray ionization (DESI) source was configured to two

high-resolution mass analyzers individually, and some varying configurations and DESI

parameters were compared. Subsequently, the recently-developed soft desorption/ionization

technique, DESI, was applied to rapid screening of supramolecular host-guest inclusion

complexes for the first time. Data obtained were compared to the results of an analogous ESI

screening experiment and demonstrated significant inconsistencies. Following solution-phase









validation using nuclear magnetic resonance (NMR) spectroscopy, reactive DESI was

determined to be the superior ionization technique for the application. Computational chemistry

confirmed that the tendency of ESI to yield nonspecific artifacts, which were detected as false-

positives in the cyclodextrin guest screening analysis, is a disadvantage of the technique which is

not shared by DESI. The reactive DESI screen resulted in positive confirmation of only those

complexes determined by NMR spectroscopy. The ESI screen did not yield detection of the

weakest binding inclusion guest, and consequently did not demonstrate comparable sensitivity to

the reactive DESI analysis. No evidence to suggest preferential detection of kinetic rather than

thermodynamic products using DESI was observed, despite the short interaction time between

host and guest molecules during the DESI event.

Supramolecular Chemistry

Concepts

Supramolecular chemistry is the structural and functional behavior of organized entities

formed through association of two or more chemical species in the absence of a traditional

covalent bond.' The species involved in the association, or complexation, can be referred to as

the host and guest' or the molecular receptor and substrate,7 and they associate uniquely via

electrostatic forces differing from full covalent bonds to form complexes.8 The host is formally

defined as the possessor of convergent binding sites such as Lewis base donor atoms while the

guest possesses divergent binding sites such as Lewis acid donor atoms or hydrogen-bond-

accepting halides.8

The complexes form through molecular recognition interactions stemming from

information pre-programmed into the associated molecules which is manifested in their

geometric and chemical characteristics and defines the nature of the governing interactions.9 This

concept of encoded molecular information is responsible for selective binding of individual









substrates by specific molecular receptors and is the principle underlying the phenomenon of

self-assembly.

The process by which molecules spontaneously form ordered aggregates with no external

interference is molecular self-assembly, but self-assembly is not limited to molecules. Self-

assembly has been reported for aggregation of varying species up to macroscopic dimensions on

the order of centimeters; the maj or pre-existing condition for the event on any scale is

complementarity in terms of geometric shape, or topology.10 Species form self-assembling

complexes if their binding sites are of complementary electronic character and are spaced

appropriately for interaction. A preorganized host will undergo minimal conformational changes

during the binding event,' which is important in terms of the thermodynamic favorability of

binding. In supramolecular complexation, alternatively referred to as noncovalent synthesis, the

products are equilibrating structures. Therefore, a complex must correspond to a thermodynamic

minimum in order to self-assemble.ll Kinetic driving forces are also relevant, and an important

consideration here is that a conformationally rigid preorganized molecule may be slow to bind a

guest because of the difficulty it experiences passing through a transition state necessary for

complexation. Thus a balance must be achieved to promote self-assembling complex formation.'

Broadly, supramolecular complexes may be divided into two categories: cavitates and

clathrates. This distinction is based on the position of the bound guest. Hosts possessing

intramolecular cavities are cavitands and guest binding, or inclusion, results in formation of a

cavitate, a complex in which the guest or guests are encapsulated by a pocket in the host

molecule. Conversely, clathrates are formed when guests are bound in extramolecular cavities

formed by aggregation of two or more molecules.12 For clarity, see Figure 1-1. The designed

synthetic alkali-metal cation receptor phloroglucinol derivative dimers characterized in chapters









two and three are clathrates binding the cation between two monomers. The cyclodextrin

complexes investigated in chapter five are cavitands with the cyclodextrin host encapsulating

guests inside a hydrophobic intramolecular cavity.

Types of Noncovalent Bonds

Non-covalent interactions are labile and confer on supramolecular complexes an important

intrinsic property, dynamic character. Practically, this attribute enables supramolecules to

reversibly associate and dissociate, yielding selective self-organization as individual molecules

are able to undergo conformational changes to promote favorable molecular recognition

interactions.9 Supramolecular complexes are stabilized by several types of noncovalent

interactions but another important consideration is the nature of their surroundings.6 The relative

influences of the presence of molecules of solvation and crystal close-packing effects can greatly

impact the thermodynamic stability of these weakly-stabilized entities.'

The maj or types of noncovalent bonds are all based on electrostatic interactions arising

from the presence of small directional and nondirectional electrical charges on the interacting

molecules. Two partial electrical charges interact to produce either attractive or repulsive forces;

charges with opposite polarities will yield an attractive force while like-charges repel.13

Ion-ion interactions

ions will interact with electrostatic dipoles resulting from the electron density on the

potential surface of a molecule. Ion-ion interactions are the strongest purely electrostatic

interactions with bond energies of ~100-3 50 kJ molf and bond strength is dependent on

interionic distance and the extent of charge delocalization.13 This is the maj or reason that crystal

close-packing forces can have such a significant effect on supramolecular complex stability and,

therefore, must be taken into consideration in the design of analytical methodologies involving

supramolecular co-crystallization.









Ion-dipole interactions

lon-dipole interaction strength is dependent on the orientation of the dipole with respect to

the ionic charge and is of the order of 50-200 kJ mol- Co-ordinate or dative bonds fit into this

category but they also incorporate some covalent contribution.'

Dipole-dipole interactions

Interactions between two appropriately-aligned dipoles can yield attractive forces with

bond energies in the region of 5-50 kJ mol l. Possible orientations include matching of a pair of

opposite poles on adj acent molecules or opposing alignment of two adj acent dipoles (Figure

1-2).5

Hydrogen bonds

A specific instance of a dipole-dipole interaction corresponding to the attractive force

between a hydrogen atom attached to an electronegative atom and a neighboring dipole results in

a hydrogen bond. Bond energies between 4-120 kJ moll are known and various bond lengths

froml1.2 to 3.2 Angstroms have been reported.' One important consideration that must be

addressed in the analysis of hydrogen-bonded supramolecular assemblies is the solvation

strategy, because protic solvents can weaken or even destroy hydrogen-bonds.3 This is another

reason that environment is a maj or factor in supramolecular chemistry.

Cation-n interactions

Aromatic rings consist of a partially positive o-scaffold and a partially negative n-cloud

above and below the plane of the ring. This confers a quadrupole moment on species such as

benzene which can result in an attractive force of the order of 5-80 kJ mol-1 between the n-cloud

and an appropriately located cation (Figure 1-3).14









7cStacking interactions

Attractive forces of 0-50 kJ mor~ can be attributed to xn-stacking of aromatic rings. The

presence of the previously described quadrupole moment enables attractive electrostatic

interactions between oppositely charged regions of the rings. Edge-to-face and face-to-face

orientations exist as well as various intermediate geometries, but face-to-face configuration never

corresponds to direct overlap which would yield a repulsive force."

Biological and Physical Significance

Noncovalent intermolecular interactions make myriad contributions to the natural world.

Structure and function of many important biomolecules are delineated by noncovalent

interactions. The immediately recognizable double-helix structure of deoxyribonucleic acid,

DNA, is partially stabilized through hydrogen bondsl6 and xn-stacking, or arene-arene,

interactions, which also govern the higher-order structure and thermal stabilization behavior of

some proteins." Active site binding between an enzyme and ligand often involves cation-n:

interactions,13 hence biological catalysis is inherently dependent on supramolecular forces.

Active transport across lipid bilayers, which form structures such as cell membranes, is also

facilitated by noncovalent interactions, cation-n: in the case of transport of ions by ionophores,

and the bilayers themselves are stabilized by hydrogen-bonds.5

Molecular devices are organized, functionally-integrated chemical systems built into

supramolecular architectures. They are capable of performing a specific function upon activation

by some external stimulus.' These molecular devices based upon supramolecular chemistry

principles are used as sensors and switches for multiple applications.l Crystal engineering is

inherently a self-assembly field, and crystal growth is based on noncovalent interactions.'









Consequently, supramolecular chemistry has considerable significance beyond the frequently-

discussed application to biologically-relevant molecules and processes.

Synthetic chemists seek to exploit supramolecular chemistry for various reasons.

Production of synthetic species capable of mimicking the catalytic and selective binding

phenomena observed in biomolecules like enzymes and proteins has significant implications to

drug design and delivery research. Host-guest inclusion complexes stabilized by noncovalent

interactions are currently used in areas such as catalysis and the encapsulation of drugs.19 Design

of hosts capable of specific molecular recognition behavior is a rapidly-expanding area of

research. Capitalization on self-assembly can aid synthetic strategy development in areas like

supramolecular catalysis and if appropriately employed can increase synthetic yields and

enhance synthetic selectivity.7,9 A current aim of supramolecular chemists is to achieve realistic

theoretical modeling of noncovalent interactions,20 and that will require an abundance of

physical data so appropriate analytical techniques are in high demand.

Mass Spectrometric Approaches

Mass spectrometry, the measurement of the mass-to-charge ratio (m/z) of ions, is an

analytical technique known for the three S characteristics: speed, sensitivity, and selectivity.21

The m/z is the ratio of the mass of a particle to the number of electrostatic charge units carried by

the particle,22 and it can be used to aid identification of the elemental composition of said

particle. Mass spectrometric analysis requires several major stages. The first stage, following

sample introduction, is ionization, for which a strategic explanation is outside the scope of this

section. The ionization stage combines, in most cases, nebulization and incorporation of a charge

onto the analyte or analytes of interest. Nebulization is necessary because mass spectrometry is

inherently a gas-phase technique as particles must be separated for detection. Ionization is

essential to charge the particles for separation according to m/z. Arguably, the second stage is









fragmentation of the ionized particles. In instances where 'hard' ionization is used, this is

combined with the ionization stage, and in some cases, for example when only the m/z ratio of

the whole particle is required, this stage is omitted. Fragmentation will be discussed in greater

detail in the tandem mass spectrometry section. Differentiation of the ions introduced on the

basis of their m/z follows and may be referred to as the mass analysis stage, and ion detection

completes the experiment.22 Data are presented as a spectrum of absolute or relative signal

intensity versus m/z, and the signal detected at each m/z is represented as a peak.

Ionization may be achieved in a variety of ways. The most appropriate method for a

specific analysis depends on the sample, the analyte, and the desired information. Techniques

used for ionization are generally classified as either hard or soft, based on the energy required

and the resulting ion types. Hard ionization methods such as electron ionization (EI) or chemical

ionization (CI) break apart the ionized particle and produce only low abundances of molecular

ions or protonated molecules but yield several characteristic fragment ions.23 Soft ionization

techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization

(MALDI) result in minimal fragmentation but yield significantly more intense signals

corresponding to the intact ion.24 Although a vast array of fragmentation strategies exist, all the

mass spectral data presented here were produced using ESI or desorption electrospray ionization

(DESI), a related technique. Consequently, the extended discussion here will be limited to a brief

description of ESI.

The ESI technique, developed by John Fenn, enables the transfer of solution-phase ions

into the gas phase,25 and so has been widely adopted as an interface for liquid chromatography

(LC)26 and capillary electrophoresis (CE).22 Electrospray ionization (see Figure 1-4) involves

electrochemical charging of a flowing liquid sample via the application of a high voltage across a









Eine-tipped spray needle and an endplate; charged droplets of solution form. The droplets exit the

spray needle in a Taylor cone, the apex of which narrows to form a liquid j et and ultimately an

aerosol.23 The mechanism by which this aerosol yields ions is the subject of debate. Two

convincing possibilities are commonly cited: droplet Hission at the Rayleigh limit and direct Hield

evaporation of ions. Both can be referred to as the desolvation mechanism or step, and

desolvation efficiency is significantly enhanced at elevated temperatures.24 For this reason,

charged droplets are introduced into a heated capillary prior to mass analysis via a potential

gradient. Generally, the opposing ends of the heated capillary are charged to aid focusing of the

ion beam into the mass spectrometer. This feature confers a useful capability on the ESI

technique, source-skimmer collisionally induced dissociation (SCID)27 which will be discussed

further in the tandem mass spectrometry section. As noted, the ESI process results in very

limited ion fragmentation and, therefore, has the benefit of promoting determination of molecular

weight. Another important advantage of ESI is its ability to produce multiply charged intact ions

which makes it amenable to the analysis of large molecules. Increasing the charge decreases the

m/z, bringing the signal into more efficient ranges for mass analysis. 24

Different instrumental approaches define the type of information obtained and the way that

mass spectrometry can be applied to a specific analytical issue. Maximizing the information

obtained in a mass spectrometric analysis is generally achieved in one, or sometimes both, of two

ways: high-resolution mass spectrometry and tandem mass spectrometry. Although a

combination of both these approaches can be used, this is not always desirable due to constraints

of expense and time, as well as control of the amount of data acquired, which is a very real and

problematic issue in terms of storage and processing capacities in mass spectrometry labs today.









High-Resolution Mass Spectrometry

Concepts

Resolution is a measurement of the degree of separation between adj acent peaks

corresponding to ions of different m/z. It is defined below, and should not be confused with

resolving power which is an indication of the theoretical resolution capability of an instrument.


R= m
Am (1.1)

In equation 1.1, m is the m/z value of the peak for which resolution is being defined and

Am is either the difference in m/z between two adj acent peaks or the width of the peak, but some

indication of the shape and separation of the spectral peaks must be included in the definition to

give it a tangible meaning. In some cases, a measure of the height of the valley between the

peaks relative to the height of the peaks themselves, commonly 10 or 50%, is used to account for

this. Alternatively, a measure of the width of the peaks at some specified fraction of their height

is substituted; often the peak width at half the peak height or full-width half maximum, FWHM,

is employed (see Figure 1-5). Resolution between 100-1,000, is usually termed low resolution,

medium resolution corresponds to R=2,000-10,000, and R>10,000 is classified as high-

resolution.2

Calculated exact mass, a term often incorrectly substituted for high-resolution, is used to

describe the sum of the exact mass of the individual isotopes that compose a single ion,29 and is

often reported to a certain point of precision, for example four decimal places. Each element has

a specific monoisotopic mass, e.g., carbon by definition has a monoisotopic mass of 12.00000

atomic mass units, the exact value of which corresponds to the mass of the lowest number of

subatomic particles one of its atoms can comprise. In mass spectrometry, the term measured

accurate mass is used to mean the measured m/z reported with four decimal places and less than









15 ppm error.29 Since each isotope of an element has a specific and precise mass, as the mass

accuracy is improved, a particle corresponding to a certain m/z can be more accurately identified

in terms of elemental composition. Fewer permutations of elemental combinations can yield a

specific m/z than a nominal m/z.30 High-resolution mass spectrometry yields accurate mass

values, so it can be one important approach to increase the useful information obtained via mass

spectrometric analysis. Combined with soft ionization techniques which produce protonated,

deprotonated, or adducted, e.g., sodiated, molecules or molecular ions, the elemental

composition of an ion can often be narrowed down to a manageable number of possibilities. Peak

distributions corresponding to the isotopic distributions and mass defect values are also valuable

contributors to the determination of elemental composition.30 The maj or disadvantage, however,

is the lack of structural information inherent in resultant spectra.

Instrumentation

There are three main types of high-resolution mass spectrometers and they are each suited

to different situations. All three are capable of high resolving power either because they rely on

detection by measurement of time, for which extremely accurate measurement capabilities are

available, or because they employ combinations of mass analyzers for superior ion beam

focusing.31 They all require frequent calibration with known mass standards to maintain

performance. Double-focusing sector instruments incorporate two mass analysis stages using

both magnetic and electric Hields to separate and focus ions on the basis of kinetic energy and

velocity, and are excellent for configuration with high-energy ionization and dissociation

techniques.22 Resolving power high enough to yield resolution of the order of 50,000 has been

reported.32 Sector instrumentation, however, was not used in this body of work and further

discussion is therefore outside the scope of this dissertation.









Time-of-flight, TOF, mass analyzers pulse packets of ions along a field-free flight tube of

known path length. The ions entering the drift tube are pulse accelerated so they all have the

same initial kinetic energy. The velocity of their flight, therefore, is dependent on their mass

according to the equation below, where E is kinetic energy, m is mass, and v is velocity.


E= -my
2 (1.2)

Since the path length is known, measurement of the flight-time of each ion enables

determination of the mass, or in this case the m/z, using the following.22


m = 2eVi! t13


In equation 1.3, m/z has been defined, e is the charge on an electron, V is the potential used

to accelerate the ions, t is the flight time, and L is the path length. Resolving powers of the order

of 10,000 have been reported for orthogonal-acceleration reflectron TOF mass analyzers33 Such

as the one used in this body of work. Manufacturer claims of 15,000 have been made.

Fourier transform ion cyclotron resonance (FTICR) mass analyzers incorporate a

superconducting magnet. Ions are introduced into the magnetic field where they are trapped

electrostatically and forced into ion cyclotron motion, a circular orbit with a frequency inversely

proportional to the m/z ratio of each ion.34 Briefly, orthogonal RF excitation of the ions yields a

coherent packet of ions traversing one circular path. The combined time domain signal of the ion

orbits is measured via the induced current on a pair of parallel plates and the signal is subj ected

to Fourier transformation for conversion to the frequency domain.35 The relationship between the

frequency and the m/z is shown below, where f is frequency and B is magnetic field strength.35

m B
: 27r f (1.4)









High mass-accuracy, typically below 5 ppm relative mass error, and resolving power of

150,000 can be achieved using an intermediate magnetic field of4.7 Tesla,31 and the Marshall

group continues to observe increased resolution as they increase the strength of the incorporated

magnetic fields.32 The trapping capabilities of this type of mass analyzer also make it suitable for

tandem mass spectrometric analysis, but the expense of acquiring and maintaining a

superconducting magnet, slow spectral acquisition, and large size of data files limit the use of

FTICR-MS.

Tandem Mass Spectrometry

Tandem mass spectrometry is the measurement of the m/z of ions prior to and following a

reaction within the mass spectrometer. The term encompasses a plethora of instrumental and

experimental approaches,36 but only a very limited discussion of the strategies relevant to this

body of work will be presented here. Maj or reasons for the popularity of tandem mass

spectrometry, sometimes referred to as MS/MS or MSn, are its usefulness in structural

elucidation of molecules and analysis of complex mixtures.36-3 As mentioned, soft-ionization

high-resolution work often only yields information about elemental compositions So structural

geometry and the configuration of functional groups remain unaddressed. In tandem work, the

term precursor, or parent, ion refers to the detected ion prior to the reaction event, and the ion or

ions generated via the reaction, for example a dissociation event, are the product, or daughter,

ions. Precursor ions are not necessarily molecular ions.36

Appropriate experimental design is an important caveat to the application of tandem mass

spectrometry. The type of problem under investigation and the information required define the

design. Traditionally, mass spectrometry may be considered tandem-in-time or tandem-in-space.

Experiments involving ion selection, reaction, and detection in different regions of the mass

spectrometer are considered tandem-in-space. Sector instruments and triple quadrupole









instruments are commonly employed for tandem-in-space experiments,40,41 although the

aforementioned SCID technique was incorporated in a tandem-in-space configuration with a

TOF mass analyzer as part of this research. Tandem-in-time approaches involve selection and

isolation of an ion, reaction, and detection in a single region of the mass spectrometer and events

occur sequentially. Ion trapping instruments such as the FTICR and various geometry ion trap

(IT) instruments are employed for this type of experiment.40,41 Data presented in Chapter 3 were

acquired using a linear quadrupole ion trap.

Briefly, trapping instruments for tandem work employ electrostatic trapping of ions

following ionization. A precursor ion is selected by mass or energy and the unwanted ions are

ej ected from the trapping region electrostatically. Introduction of the other components necessary

for the reaction, for example an inert gas for collision and fragmentation, into the region occurs,

and the resultant product or daughter ions are detected.42 Theoretically this process can repeat

indefinitely, yielding data following multiple events, but experimentally some limiting factor, for

example initial signal intensity or low mass cutoff, will govern the number of successive

reactions successfully monitored.36

When an ESI source comprises the front end of a mass spectrometer, tandem mass spectral

data can be acquired even in instruments not traditionally designed for it. As discussed, the exit

end of the desolvation capillary is held at a high voltage, as is the skimmer succeeding it, to force

the ions to travel into the mass analyzer region of the instrument. Increasing the voltage

difference between the exit end of the desolvation capillary and the skimmer, or skimmer-cone,

increases the corresponding energy imparted to the desolvated ions which can result in

dissociation. This strategy is called source-skimmer or nozzle-skimmer collisionally-induced









dissociation because the millibar pressure in the fragmentation region of the source promotes

collision between ambient molecules and the sprayed ions.27,43

Tandem mass spectra acquired using trapping or sequential quadrupole or sector

instruments yield spectra where parent ion selection and isolation enables determination of the

true relationships between sequentially detected ions.36 The SCID technique suffers from an

inability to select the precursor or parent ion, so ions detected following the reaction have

uncertain parentage.43 Therefore SCID is not the technique of choice for elucidating dissociation

pathways or uncovering structural information, although it is often used for these applications

because of its simplicity, low cost, and accessibility.27,44 It has found its niche, however, in

experiments designed for comparison of relative ion stabilities, and it is also useful for isomer

differentiation and improved analyte detection.27,43,45,46 IH-Cell COllisionally-induced dissociation,

CID, can be problematic for ion stability determination of noncovalent complexes because the

high-energy collisions generally employed break covalent bonds of the parent ion causing rapid

and extensive fragmentation over a very narrow energy-range, making construction of reliable

dissociation curves difficult.26

Supramolecular Mass Spectrometry

Gas-Phase Supramolecular Chemistry

Elucidation of the chemistry of gas-phase supramolecules is of vital importance to the

development of supramolecular chemistry as a field. Particles in the gas-phase are essentially

isolated entities; they experience negligible interference from surrounding gas-phase particles

and are not surrounded by molecules of solvation.6 COnsequently gas-phase analyses are capable

of characterizing the intrinsic properties of the system under investigation.3,6 In Order to further

the understanding, modeling, and ultimately synthetic utilization of the physical and chemical

bases of noncovalent bonds, analytical strategies to characterize intrinsic behavior of









supramolecular complexes are essential. In a field where stabilizing forces are relatively weak,

this is a very relevant consideration, because molecular chemistry can no longer be studied and

understood without some appreciation of environmental effects. This concept of approaching

molecules and their surroundings as complete individual chemical entities is one of the

fundamental principles underpinning supramolecular chemistry.6

Two crucial benefits are derived from the absence of a solvation shell surrounding gas-

phase molecules. One is that, using traditional analytical techniques to determine solution-phase

chemistry, direct comparisons between solution-phase and gas-phase chemistry can be made,

elucidating the direct effects of solvation on the supramolecular system.3,6,26 The second is more

specific to supramolecular chemistry. Species stabilized by noncovalent bonds such as hydrogen

bonds, which are known to compete with some solvents for binding sites, can be destabilized

upon dissolution. Hydrogen bonds may be weakened or even destroyed in the presence of protic

solvents.6,21 Hence, secondary and higher-order structures of complex molecules like proteins

can be significantly altered leading to the acquisition of entirely misleading data.

Gas-phase chemistry, therefore, has an important place in the Hield of supramolecular

chemistry as a whole, but characterization of gaseous supramolecules specifically by using the

unique analytical capabilities of mass spectrometry can be justified by several factors. As

previously discussed, mass spectral data have traditionally been used to determine elemental and

structural composition, charge states, and stoichiometric relationships pertaining to analytes of

interest. In this vein, mass spectrometry is extremely useful for topological studies to define

secondary structures and conformations of supramolecular complexes and to investigate the

stoichiometry of binding between molecular recognition elements.3,6 However, the benefits of









mass spectral analysis for supramolecular chemistry extend far beyond these traditional

approaches.

Mass spectrometry enables determination of the reactivity of noncovalent complexes, not

only in terms of fragmentation behavior, but also in comparative preferential substrate binding

and hydrogen/deuterium exchange experiments for mechanistic studies. Additionally, exciting

developments in mass spectrometry for the determination of thermodynamic data have

demonstrated the capabilities of the technique as a detection method for representation of

solution-phase equilibria and as a strategy for the calculation of both relative and absolute gas-

phase thermodynamic quantities. Supramolecular chiral analyses have been successfully

performed.3,6

Survey of Recent Literature

Some recent reviews and developments in supramolecular mass spectrometry accessible in

the literature are highlighted here for the purpose of clarifying the context of the work presented

in subsequent chapters. Two excellent reviews examining the role of gas-phase analysis within

the field of supramolecular chemistry have been presented by the Schalley group.3,6 An in-depth

overview of molecular recognition by mass spectrometry was published by De Angelis and

colleagues.26 Independent reviews concerned with mass spectral analysis of noncovalent

complexes for the determination of quantitative thermodynamic quantities were authored by

Armentrout et al 47 and the Zenobi group.21 Specific to biomolecules, Kaltashov and Eyles

reviewed mass spectrometry applied to the study of conformations and conformational

dynamics,48 Kriwacki et al. presented an overview of the mass spectral characterization of

protein complexes,49 and Liesener and Karst authored a critical review on monitoring enzymatic

conversions by mass spectrometry.5o









Supramolecular mass spectrometry for determination of the elemental composition of

synthetic products and structural elucidation continues to be prolific. Lately, novel bis-crown

ethers and rotaxanes,52 cyclic dipyridyl-glycolurils,53 large phosphorus macrocycles,54 and

mixed-metal, mixed-pyrimidine self-assembling metallacalix[n]arenes5 have been synthesized

and characterized using soft-ionization mass spectrometry. The modification of a tolylpyridine-

bridged cavitand with water-solubilizing groups for the promotion of aqueous-phase self-

assembly was determined using mass spectral analysis,56 and a detailed structure of the

amyloidogenic protein wild-type transthyretin was obtained via HPLC-nanospray MS/MS and

MALDI-MS.57 The first report of sonic spray ionization mass spectrometry for the

characterization of metal-assembled cages was published in 2006.5

Topological experiments incorporate mass spectrometry for conformational analysis and

higher-order structural elucidation. Electrospray ionization has been applied to examination of

the supramolecular architectures of complementary self-assembling host-guest complexes of the

softball type,59 Of cucurbit[n]urils,60 and the chelating bidentate catechol ligand with various

polyatomic cation guests.61 An interesting study on the dynamic topological control over

subcomponent self-assembly of synthetic helicates, macrocycles, and catenates employed ESI-

FTICR mass spectrometry for the elucidation of secondary structures.62 Additionally, ESI-MS

monitoring of the folding and assembly of hemoglobin was achieved using a novel on-line

dialysis system.63

Several groups have recently published topology and reactivity studies on biological

macromolecules. Limited proteolysis MALDI-TOF-MS was used to probe the kinetics of cyclin-

dependent kinase inhibition.64 Noncovalent complex formation between the protein Link module

from human tumor necrosis factor stimulated gene-6 and hyaluronan oligosaccharides was









studied over time using hydrogen/deuterium (H/D) amide exchange ESI-MS.65 COllisionally-

activated dissociation ESI-MS/MS experiments elucidated fragmentation and preferential

binding behaviors of differentially-substituted perylene diimide ligands completed with DNA,

revealing correlation between gas and solution-phase behavior.66 A similar proj ect focusing on

DNA completed with several drug candidates incorporated KMnO4 Oxidation with ESI-MS/MS

and compared topological and reactivity data using infrared multiple photon dissociation

(IRMPD) and CAD dissociation strategies.66 Dissociation pathways for complexes of a single-

stranded DNA with a polybasic guest were obtained using nanoelectrospray ionization and

tandem mass spectrometry.67

Mass spectral analysis of supramolecular synthon reactivities is also experiencing rapid

growth. The Dearden group, who have made myriad contributions to quantitative gas-phase

thermodynamics, have investigated the dissociation and reactivity properties of the cucurbituril

derivatives as hosts for inclusion of small-molecule guests using FTICR-MS and have exploited

the inherent trapping capabilities of the technique to study ion-molecule chemistry in the ICR

cell.68,69 Solution-phase kinetic data for the noncovalent binding between chiral resorcinarenes

and ammonium guests were obtained using FTICR-MS,70 and a series of elegant experiments

also used FTICR-MS but employed H/D exchange and ion-molecule reactions to investigate

inclusion of alkyl ammonium ions by substituted resorcarenes.20,71 Additionally, fragmentation

and rearrangement of substituted oxadiazoles and their noncovalent complexes with cobalt cation

and cyclodextrin were studied using a plethora of mass spectral approaches combined with

isotope-labeling.72 Elucidation of a ring-closure mechanism subsequently incorporated into the

design of a method for synthesis of interlocked xn-conjugated macrocycles was achieved using

FTICR-MS and tandem mass spectrometry.73 The utility of FTICR-MS in this area was also









demonstrated in the study of noncovalent complexes of the protein Mycobacterium tuberculosis

adenosine-5' -phosphosulfate reductase; reactivity and thermodynamics were investigated for the

delineation of a mechanistic model.74

Quantitative gas-phase thermodynamic data are desirable for the reasons outlined earlier.

Maj or developments in the representation of solution-phase thermodynamic behavior by ESI-MS

have been made just in the last few years. The Brodbelt group in particular has contributed to this

progress with the early design and application of a method for determination of binding constants

by ESI-MS using reference complexes with known thermodynamic data. Subsequent work

developed mathematical models based on equilibrium partitioning theory to predict the ESI-MS

response to host-guest complexation76 and to relate ESI-MS ion abundances with solution

concentrations of host-guest complexes." Complexes of crown ethers with alkali metal cations

were used for validation. A novel quinoxaline-containing crown ether ligand-metal-ligand

sandwich complex stabilized by n-stacking interactions was characterized via ESI-MS resulting

in quantitative determination of free-energy gains achieved by modification of the crown ether

with electron-rich functional groups.' Binding stoichiometry and relative binding affinities of

nucleic acid aptamer/small molecule complexes were obtained using ESI-MS and compared to

values deduced using traditional solution-phase techniques.79 Evaluation of protein-DNA binding

affinities, using the DNA-binding domain of a transcription factor, c-Myb, and several double-

stranded DNA substrates, was demonstrated using both ESIso and laser spray mass

sp ectrometry.818

Mass-spectrometric determination of gas-phase thermodynamic quantities for elucidation

of intrinsic supramolecular thermochemistry is an additional area of activity. Progress continues

toward the goal of determining absolute quantitative thermodynamic data using mass









spectrometry. Valuable contributions made by Dearden and colleagues include accurate pressure

determination strategies to enable the measurement of exchange equilibrium binding constants

for crown ethers and alkali-metal cations and entropies and enthalpies associated with

discrimination between enantiomers of chiral amines by dimethyldiketopyridino- 18-crown-6

during the molecular recognition event.83 Experimental gas-phase alkali-metal binding energies

of dibenzo-18-crown-6 obtained using FTICR-MS have been compared to theoretical data

derived computationally.84 The effects of size of noncovalent complexes on their stability during

collision-induced dissociation were studied using complexes of metallated porphyrins with

hi stidine-containing peptides and model compounds.45 More recently, energy-resolved SCID for

the evaluation of relative stabilities of noncovalent complexes of crown ethers, nucleic acid

bases, and amino acids with alkali metal cations has been investigated for determination of

solvent influences.43 COmplexes formed between alkali metals and polyether ionophore

antibiotics were analyzed in a study comparing data analysis methods for gas-phase stability

determination by CID.8

The background, significance, and recent developments in supramolecular chemistry and

mass spectrometry have been introduced. The remainder of this dissertation will describe in

detail the specific contributions to supramolecular mass spectrometry made though this research.

Chapters 2 and 3 describe the characterization of a novel alkali-metal cation synthetic receptor

platform via ESI and tandem mass spectrometry. A technical chapter presenting the

configuration of a home-built DESI source to two high-resolution mass analyzers follows.

Chapter 5 describes the development of a novel DESI technology for supramolecular

applications using a cyclodextrin model system. The work culminates in a general conclusion

and future directions section.










a b








Figure 1-1. Representation of a) a cavitand including a guest to form a cavitate and b) clathrands
forming a clathrate with a guest molecule.


16 cj

6 B


lj;- b'


~5-~


Figure 1-2. Two possible orientations of dipole-dipole interactions.


Figure 1-3. Representation of cation-n: interaction showing the quadrupole moment on an
aromatic ring and the interaction between the partially negative n: cloud and a cation.













b


Figure 1-4. Generalized configuration of an electrospray source showing a) solution flowing
through a spray needle, with co-axial nebulizing gas, across a potential difference, b)
a desolvation capillary, c) a skimmer cone, d) some ion optics (no specific type), and
e) the direction of flow into the mass analyzer.


mi mII2


Figure 1-5. Quantities used to calculate resolution where a) represents the 15% valley definition
and b) shows the full-width half maximum, FWHM, approach.










CHAPTER 2
CHARACTERIZING NONCOVALENT DIMERIZATION BEHAVIOR OF DESIGNED
PHLOROGLUCINOL DERIVATIVES USING ELECTRO SPRAY IONIZATION HIGH
RESOLUTION MASS SPECTROMETRY

Introduction

A maj or facet of supramolecular chemistry is the design of synthetic compounds capable

of supramolecular interactions with their surroundings. Synthetic supramolecular systems are

under exploration because they offer simplification of synthetic strategies and a collection of

novel, manipulable properties.86 The principle of chemical information encoded into molecules

and supramolecules is the foundation for the design of synthetic species capable of specific

molecular recognition behavior. Information can be stored at the molecular level by topological

design9,87 in a manner similar to the way that biological information is stored in DNA.

Some synthetic chemists use noncovalent interactions and self-assembly phenomena to

promote selective synthesis or for catalysis of the synthesis process.9 Alternatively, designed

synthetic receptors capable of mimicking the noncovalent interactions responsible for a plethora

of biological processes like enzyme-substrate action and active transport across cell membranes

may be sought. This type of work is of maj or importance in the improvement of drug design and

lead optimization, for example." Novel properties of supramolecular entities such as unique

photo- and electro-activity give impetus to synthesis of designed noncovalent receptor

molecules.7

Despite the wealth of macrocyclic ion receptors such as the crown ethers, few designed

xn-receptors for alkali-metal cations are known.8 Effective design of a synthetic receptor capable

of molecular recognition with a specific substrate requires that functional groups on a molecular

platform be aligned to form complementary binding interactions with the substrate. One way to

achieve this is by Eixing the conformation of the receptor by using a rigid platform such as a









benzene ring as a foundation for the functional regions of the synthon. The desired conformation

can be permanent or may only be achieved during the molecular recognition event.89 Following

this design approach, two novel synthetic compounds, derived from naturally-occurring

phloroglucinol (Figure 2-1) have been investigated for their ability to form dimers capable of

selectively binding a size-appropriate alkali-metal cation. The 1,3,5-trisubstituted

2,4,6-triethylbenzenes have demonstrated host-guest inclusion complex capabilities for both

cation and anion guests,90 So there is a good precedent for this work.

The synthetic phloroglucinol derivatives 2,4,6-tribenzoylphloroglucinol and

2,4,6-(3,5-dimethyl)-tribenzoylphloroglucnl hereafter referred to as phloro 1 and phloro 2

respectively, are shown in Figure 2-2. It was hypothesized by the Castellano group at the

University of Florida that phloro 1 could potentially form a cage-type dimer capable of housing

an alkali-metal cation in the cavity of the resultant hollow, spherical complex. Preferential

complexation of appropriately-sized cations would support this hypothesis. Conversely, the two

methyl groups on each benzoyl substituent of the central aromatic ring of phloro 2 should

prevent formation of a cage-type dimer via steric hindrance, which would be supported by

complex formation exhibiting no dependence on cation radius. These postulates were explored

using mass spectrometric strategies for determination of the order of binding preference for

several metal cations with each phloroglucinol derivative.

In the study of noncovalent preferential binding by mass spectrometry, two major

approaches are commonly employed. Traditional competitive binding experiments consist of

dissolving the host in a suitable solvent and adding equimolar amounts of the two or more

competing guests. Assuming similar ionization efficiencies and minimal signal suppression for

the resulting complex ions, subsequent comparison of mass spectral peak intensities









corresponding to the respective complexes is used to assign binding preferences. The more

intense the peak corresponding to a given complex, the higher the standing of the related guest in

the preferential binding order.19 The aforementioned assumptions can mean that this strategy is

inappropriate under some circumstances, but its speed and simplistic experimental design result

in its frequent utilization. A more rigorous approach capitalizes on the amenability of mass

spectrometry to tandem experimental design. Detection of a peak corresponding to a complex of

interest is followed by some dissociation event involving controlled introduction of energy to the

chemical system. The extent of dissociation following the addition of the energy is monitored by

comparing the absolute peak intensity after energy addition relative to the initial absolute peak

intensity. The energy applied is varied and several data points acquired. Construction of a

dissociation or stability curve which is a graphical representation of the data plotting the

previously-described normalized peak intensity against the amount of energy incorporated

follows.21,47,91 Some point on the curve, for example the point at which 50% of the original peak

intensity is lost, is used to compare the energetic stabilities of the respective complexes.21,91

Common strategies for providing the energy for dissociation include increasing the voltage gap

applied during SCID or CID, or introducing electromagnetic radiation in the form of laser light.47

Although this is the more rigorous approach, it is slower and slightly more complex in nature

than performing traditional competitive binding experiments and therefore is not always the

method of choice.

Experimental

Sample Preparation

The phloroglucinol derivatives 2,4,6-tribenzoylphloroglucinol and 2,4,6-(3,5-dimethyl)-

tribenzoylphloroglucinol were synthesized and purified by members of the Castellano group at

the University of Florida. All samples were dissolved in HPLC grade methanol (Honeywell









Burdick & Jackson, Muskegon, MI), and the cations Na Li K and NH4' were introduced as

chloride salts (Sigma-Aldrich, St. Louis, MO). The samples were prepared to concentrations of

3x10-4 M host and 3x10-5 M guest for the ESI-MS competitive binding and dissociation curve

experiments. For the heterodimer analyses, equimolar amounts of phloro 1 and 2 yielding a total

host concentration of 3x10-4 M were used. Each sample prepared for mass spectrometric analysis

contained 1% formic acid (ThermoFisher Scientific, Waltham, MA) to improve current stability

and ionization efficiency throughout the analysis.

The samples prepared for UV/Vis spectroscopic analysis did not contain additional acid.

Concentrations of 0.05-0.5 mM host in increments of 0.05 mM were used for titration-type

experiments and 3x10-5 M guest was incorporated where appropriate. Successive addition of CIL

amounts of a concentrated host stock solution to a 3 mL sample volume followed by spectral

acquisition comprised the titration approach for the chromic shift observation. The analytical

measurement of the chromic shift used solutions with the aforementioned guest concentrations

and 3x10-5 M and 3x10-4 M concentrations of the phloroglucinol derivatives.

Mass Spectrometry

All the mass spectrometry incorporated electrospray ionization. Mass spectra were

acquired using a 4.7 T Bruker Bioapex II Fourier Transform lon Cyclotron Resonance mass

spectrometer equipped with an Apollo API 100 Source (Bruker Daltonics, Billerica, MA) and an

Agilent 6210 Time-of-Flight mass spectrometer configured for ESI (Agilent Technologies, Inc.,

Santa Clara, CA).

Samples were introduced at flow rates of 2 CIL/min for the ESI-FTICR-MS and 5 CIL/min

for the ESI-TOF-MS using direct infusion via a Harvard Apparatus PHD 2000 syringe pump,

with a nebulizing N2(g) preSsure of 20 psi. The potential difference between the ESI needle and









the desolvation capillary inlet was always 3.5 kV. The capillary exit voltage was held at 90 V for

the initial FTICR-MS analyses, while the skimmer voltage was 20 V. During the dissociation

curve SCID-FTICR-MS experiments, the potential difference between the desolvation capillary

exit voltage and the succeeding skimmer was varied from 50-250 V in 10 V increments by

increasing the exit voltage while maintaining a constant skimmer voltage. Hexapole

accumulation time was 2 s and 50 scans were acquired and averaged for each data point plotted.

Each run was repeated three times using individually-prepared samples and the averaged peak

intensities were plotted versus the potential difference. For the SCID-TOF-MS dissociation curve

experiments, the potential difference was manipulated by varying the fragmentor voltage from

75-300 V in 15 V increments. Signal for each data point was acquired for 20 s and the average

integrated intensity used for data analysis. Again, the average of three runs using three different

samples was plotted for determination of VCso values.

To determine VCso values following construction of the stability curves, linear regression

was performed on the data points corresponding to the region of decline in signal intensity to

calculate the equation of the line associated with the data points. Subsequently, the equation was

solved to determine the potential difference value (the x-variable) for which the percentage

dissociation (the y-variable) was 50 % in order to obtain VCso. Standard error propagation

techniques were applied to the calculation of corresponding 95% confidence intervals.

Ultraviolet/Visible Absorbance Spectroscopy

Optical spectra were acquired using a Hewlett-Packard HP8450 Diode Array UV/visible

spectrophotometer (Agilent Technologies, Inc., Santa Clara, CA) set to absorbance or derivative

absorbance mode. Cuvettes with a path length of 1 cm were used to hold samples for absorbance

measurement. A reference solution of methanol was used for blank and background correction.

Derivative abortion spectra were used for determination of the wavelengths corresponding to









maximum absorbance. Three measurements of each wavelength of maximum absorbance were

made using three different solutions and the average was plotted for comparison.

Results and Discussion

Electrospray Ionization Mass Spectrometry for Dimer Detection

Analysis of each host compound in the presence of one of each of the cations revealed that

phloro 1 forms cation-bound dimers with Na Li K and NH4+ (Figure 2-3). Initial observation

of peak intensities corresponding to the complexes suggested a preferential binding order of

Na >Li >K > NH4 but the variation in peak intensity could also have been due to differing

ionization efficiencies for the respective complexes. Extensive carryover was noted as different

salts were incorporated and after inj section of three samples, signal suppression was significant.

Consequently, after analysis of each individual cation-bound dimer, the electrospray line and

needle were cleaned with heated HPLC water and the source region was vented for removal and

thorough cleaning of the desolvation capillary. Generally the successive skimmer and accessible

parts of the hexapole assembly also required cleaning at this point. This was an issue for both the

FTICR-MS and the TOF-MS experiments.

Traditional Competitive Binding Approach

The initial approach toward elucidating the preferential binding order of Na Li K and

NH4+ USed the traditional competitive binding experimental design. Since the ionization

technique chosen for the work was ESI, only two cations were added to each sample because

signal suppression due to high salt concentrations was a concern. Following mass spectral

analysis of samples containing all possible combinations of two of the four cations under

investigation, peak intensities corresponding to the complexes would have been compared to

determine an overall binding order. Analysis of the host compounds phloro 1 and 2, however,

revealed the presence of significant amounts of adventitious sodium (Figure 2-4). The sodium









needed to be removed to ensure controlled addition of guest cations in equimolar amounts, a

fundamental requirement for the acquisition of valid data using the traditional competitive

binding approach.

Modification of the initial experiment to incorporate a chromatographic separation step

followed by post-column addition of the competing guests was subsequently attempted, but it

proved impossible to develop a chromatographic method to remove all of the sodium. The

traditional approach was therefore abandoned in favor of the more rigorous dissociation curve

approach which would not be adversely affected by the presence of adventitious sodium.

Dissociation Curve Approach

Following the strategy of Rogniaux and colleagues,91 Solutions of one phloroglucinol in the

presence of each of the alkali metal chloride salts were electrosprayed into the FTICR mass

analyzer. The intensity of the mass spectral peak corresponding to the cation-bound dimer of

interest was recorded at a range of desolvation capillary exit voltages while the adj acent skimmer

voltage was held constant as described in the experimental section. The peak intensity relative to

its observed maximum was plotted as a percentage against the potential difference in volts, and

the equation of the linear sloping portion of the curve was used to determine the potential

difference at which 50% of the maximum signal intensity corresponding to the complex was lost.

This value, VCso,91 was calculated for each cation-bound dimer and the respective quantities

were used to rank relative stability of the complexes. The ESI-FTICR-MS dissociation curves for

phloro 1 are shown in Figure 2-5 and those corresponding to phloro 2 are presented as Figure

2-6. The calculated VCso values are presented in Table 2-1. Preferential binding order for the

cations was determined using this method to be Li >Na >K >NH4' for phloro 1 and Na >K >

Li > NH4+ for phloro 2. The Na+ and K -bound dimer VCso values for phloro 2 had to be

extrapolated from the stability curves because 50% dissociation of the phloro 2 complexes bound










by these cations was never observed even at the maximum potential difference capabilities of the

instrument. The ammonium-bound dimer of phloro 2 was not stable enough for construction of

its dissociation curve.

The alkali-metal cationic radii are given in Table 2-2, and show that cation size increases

in the order Li >Na >K >NH4 Apparent size-dependent preferential binding of the cations for

phloro 1 but not for phloro 2 supported the original hypothesis that phloro 1 would form a

sandwich-type dimer while steric hindrance should prevent phloro 2 from doing the same. A

maj or problem with the obtained stability curves, however, was poor reproducibility, evidenced

by large error bars on the plots, which show the average of three trials, and large 95% confidence

intervals in the VCso value calculations. Configured to ESI, FTICR-MS can show significant

current, and consequently peak intensity, fluctuation because only one packet of ions is detected

at a time, following hexapole accumulation. Signal averaging can be achieved using a

multiplexing strategy, but this is extremely time-consuming. The current and peak intensity

stability for an oaTOF-MS can be superior because the signal from multiple packets of ions is

detected and averaged in a very short time. For this reason, and because 50% dissociation of two

of the phloro 2 cation-bound dimers was not achieved using the FTICR-MS, the dissociation

curve experiment was repeated using the same sample preparation protocol but introducing the

electrosprayed complexes into an oaTOF mass analyzer.

The dissociation curves obtained using the TOF-MS instrument (Figure 2-7) addressed

both of the maj or problems associated with the FTICR-analysis. The reproducibility of the curve

data obtained via TOF mass analysis showed distinct improvement over that obtained using the

FTICR-MS, particularly for phloro 2. This was as expected for the reason outlined above. A

more perplexing result was that more than 80% dissociation of the Na+ and Li+ bound phloro 2









dimers was observed in the TOF analysis. This enabled the interpolation of VCso values for both

complexes. Reasons for this remain unclear.

The calculated VCso values for each observed complex determined using the TOF mass

analyzer are presented in Table 2-3. The smaller confidence intervals associated with the values

and the better reproducibility observed for the dissociation curves are the reasons that the TOF

values are assumed to be more representative of the system chemistry. The quantities calculated

using the different mass analyzers exhibit similar trends. Dimers bound with Li+ and Na+ cations

are more stable than those bound with K' and NH4' for phloro 1 supporting some size-

dependence in binding preference. The dimers observed for phloro 2 are significantly more

stable than those observed for phloro 1, with the exception of the ammonium ion bound dimers

which were not observed for phloro 2. This seems indicative of differences in binding position of

the cations by each host molecule. According to the stability curves constructed using data

acquired by the TOF mass analyzer, the binding preference order of the cations under

investigation is Li >Na >NH4' >K' for phloro 1. The order of binding preference for phloro 2 is

Li >Na >K >NH4 Therefore, size-dependent binding is again exhibited for phloro 1 but not

phloro 2.

Heterodimer Studies

The size-dependent preferential cation binding observed for phlorol but not phloro 2

supports the original conformation hypothesis, but the technique implemented does not address

the effect on cation-n: binding of the structural differences between the host molecules, so an

ESI-TOF-MS approach was used to analyze cation-bound heterodimers of phloro 1 and phloro 2.

The intention was to determine, using relative ion peak intensities, whether or not the phloro 1

and phloro 2 are a good geometric fit despite the methyl substituents on the benzoyl groups of









phloro 2. Heterodimer formation was observed with Li Na and K+ cations but not with NH4 -

Statistically, if phloro 1 and phloro 2 were a good geometric fit, the peak intensity ratio of the

cation-bound dimers should correspond to 1:2: 1 where the intensity of the signal observed for the

heterodimer should be twice that of the signal intensities corresponding to the homodimers. This

is because two distinct species can combine separately in four permutations. If we represent

phloro 1 as Pl and phloro 2 as P2, then they can combine to form PIP1, PIP2, P2P1, or P2P2,

but in this analysis PIP2 is identical to P2P I so the ratio given above would be expected unless

the phloro 1 and phloro 2 are poor complementary binders.

A representative ESI-TOF mass spectrum for a sample containing equimolar amounts of

phloro 1 and phloro 2 with the addition of LiCl salt is shown as Figure 2-8. Note the presence of

adventitious sodium. It clearly shows that the cation-bound phloro 2 dimers are the most stable

of the three dimer types. This correlates with the VCso data obtained from the dissociation curves

for the homodimers. The spectrum also shows that phloro 1 and 2 do not bind well together and

so are a poor geometric fit as the heterodimer peak intensities are smaller than those

corresponding to the phloro 2 homodimers. This supports the theory that the addition of methyl

groups to the benzoyl substituents on the phloroglucinol core has a profound effect on the cation-

x binding interactions associated with phloroglucinol derivative cation-bound dimerization, and

hence on dimer conformation. Dissociation curves constructed using SCID-TOF-MS are shown

in Figure 2-9. They show that the heterodimers exhibit the same preferential cation binding

behavior as the phloro 2 homodimer, but the large error bars suggest poor reproducibility so

specific VCso values were not determined.

The mass spectral analyses were able to elucidate the cation-binding preferences for dimers

of phloro 1 and phloro 2 as well as the related heterodimers. Size-dependent preferential binding









for phloro 1 but not phloro 2 or the heterodimers supported the postulated sandwich-type dimer

conformation for phloro 1 and end-to-end dimer conformation for phloro 2. A mass

spectrometric study into heterodimer stability revealed that the addition of methyl groups to the

benzoyl substituents of the phloroglucinol core had a significant effect on the noncovalent

cation-n: interactions responsible for stabilization of the cation-bound dimers.

Ultraviolet/Visible Absorbance Spectroscopy

The mass spectral analyses described so far yielded data elucidating the cation-binding

preferences of the phloroglucinol derivative dimers and provided some insight into the effect of

the structural differences between phloro 1 and 2 on the cation-n: interactions responsible for

dimer stabilization. So far, however, no direct information about the conformation of the dimers

under investigation had been obtained. A spectrophotometric analysis based on absorbance of

ultraviolet (UV) and short wavelength visible (vis) light was consequently employed to study

solution-phase dimer conformation. An abortion spectrum for phloro 1 in methanol is presented

as Figure 2-10, and a Beer' s Law plot constructed using a titration-type experiment is shown in

Figure 2-11. Beer' s Law is stated in equation 2. 1 where A is absorbance, E is molar absorptivity,

b is path length, and c is analyte concentration.

A = ebc (2.1)

Using a Beer' s Law plot, the molar absorptivity value at 310 nm was determined from the

slope of the line to be 2330 & 130 L moll cm-l for phloro 1 and 2200 & 27 L moll cml for

phloro 2 which yields a very similar UV/vis abortion spectrum. These molar absorptivity values

are consistent with n x*" electronic transitions.92

A titration-type experiment where UV/vis abortion spectra were obtained for phloro 1

concentrations ranging from 0.05-0.5 mM varied in 0.05 mM increments was performed to









search for observable changes in spectral characteristics which could correspond to dimerization.

The same experiment was performed for phloro 2. Representative data are presented as Figure

2-12.

Comparison of the abortion spectra acquired for low and high concentrations of phloro 1

revealed a shift of the longer wavelength peak to slightly lower wavelengths, a hypsochromic or

blue shift, and a shift of the shorter wavelength peak to slightly higher wavelengths, a

bathochromic or red shift. The spectra acquired for phloro 2, however, only evidenced

bathochromic shift of the shorter wavelength peak and no shift of the longer wavelength peak

was observed. An observed anomaly in the spacing between the spectral curves for the third

highest concentration phloro 1 solution may be due to instrumental error resulting from

accidental disturbance of the plotter, or could be the result of erroneous solution preparation.

Long-established molecular exciton theory has described two ideal homodimer

conformations: sandwich dimers and end-to-end dimers.93 When two identical molecules interact

electronically, as in dimerization, molecular orbitals which were not degenerate in the individual

molecule exist in close proximity, rendering them degenerate molecular energy levels in the

dimer form. Consequent splitting of the newly degenerate energy levels results in changes in the

magnitude of resultant electronic transitions.94 This is observed in optical spectroscopy as

chromic peak shifts corresponding to molecular aggregation. Hypsochromic shift ideally

corresponds to sandwich-type dimers while bathochromic shift corresponds to end-to-end

dimers.93

The observed chromic shift began to occur at 0.05 mM phloro 1, so an experiment was

designed to measure the wavelength of maximum absorption, hmax, of each peak before and after

the shift for both phloroglucinol derivatives in the absence and presence of each of the alkali










metal cations. Derivative spectroscopy was used to determine the hmax values for improved

precision. Specific conditions are delineated in the experimental section. The resultant data are

presented as Figure 2-14.

The bar graphs a and b show the bathochromic shift of the lower wavelength peak which

evidences end-to-end aggregation of both phloro 1 and 2, although this shift is minimal for

phloro 2 and is not observed for the samples in the presence of sodium and lithium cations. A

shift is considered to be represented by adj acent bars with non-overlapping 95% confidence

intervals which are shown as error bars. Overlap of the confidence intervals for the bars

representing the pre- and post-shift peaks is taken to mean that there is no conclusive evidence of

chromic shift.

The graphs labeled c and d show data obtained concerning the Amax of the higher

wavelength peak at concentrations preceding and succeeding that corresponding to the expected

hypsochromic shift consistent with sandwich-type dimerization. This shift is only observed in the

phloro 1 data, so sandwich-type aggregation is only evidenced for phloro 1. The optical spectra

clearly show evidence of solution-phase end-to-end aggregation of both phloroglucinol

derivatives but only show evidence of sandwich-type aggregation of phloro 1. These findings are

consistent with the original hypothesis and the gas-phase mass spectral data. The addition of

each individual alkali metal cation under investigation, in the form of a chloride salt, to the

phloroglucinol derivatives in solution appeared to have no effect on the spectroscopic data. This

is as expected because each cation should be surrounded by a solvent shell preventing cation-n:

interaction between the cation and the phloroglucinol derivative hosts.









Conclusions

Electrospray ionization mass spectrometry enabled determination of the preferential

binding order of alkali metal cations completed with dimers of two phloroglucinol derivatives

differing by the presence or absence of methyl groups on the benzoyl substituents of the

phloroglucinol core. The binding order for phloro 1 was elucidated as Li >Na >NH4+ >K+ but for

phloro 2 as Li >Na >K >NH4. Size-dependent preferential binding was therefore observed for

phloro 1 but not phloro 2. Traditional competitive binding experiments were found to yield

inconclusive results due to the presence of adventitious sodium, but the dissociation, or stability,

curve approach using SCID offered superior results. It was noted that the rapid averaging

capabilities of an oaTOF mass analyzer resulted in faster acquisition of more reproducible data

than that acquired using an FTICR instrument. Heterodimer studies via ESI-TOF showed that the

presence of additional methyl groups on phloro 2 had a profound effect on the cation-n:

interactions responsible for stabilization of the cation-bound dimers. Mass spectral analysis,

therefore, was able to characterize the intrinsic noncovalent binding chemistry between the two

phloroglucinol derivatives and the cations, but no direct information about dimeric conformation

could be obtained via this technique. Ion mobility spectrometry could potentially address this

issue in the gas-phase.

Solution-phase optical spectroscopy provided evidence of both sandwich and end-to-end

molecular aggregation for phloro 1, but only end-to-end aggregation of phloro 2. This

conformational information corroborated the gas-phase data. It also supported the original

hypothesis stating that phloro 1 should be capable of forming a sandwich or cage-type dimer

potentially housing a small cation in an intermolecular cavity while the addition of methyl

groups to the extremities of phloro 2 should sterically hinder formation of a sandwich complex.









No chemical information about cation binding was attainable via solution-phase analysis,

however, due to solvent interference.

For this analysis, it was determined that, while lacking in conformation-probing

capabilities, mass spectrometry was able to provide more useful information concerning the

chemical behavior of the supramolecular phloroglucinol derivative-cation complexes than

solution-phase spectrophotometry. It should be noted, however, that solution-phase optical

spectroscopy did yield useful complementary conformational data, despite its inability to probe

the noncovalent interactions between the organic hosts and the ionic guests.











Table 2-1. Phloro 1 and 2 dissociation curve VCso values and their associated 95% confidence
intervals for cation-bound dimers determined using SCID-FTICR-MS
Complex lon Phloro 1 VCso, V Phloro 2 VCso, V
[2M+Li] 129 & 22 151 & 26
[2M+Na] 128 & 19 177 & 213
[2M+K] 89 & 14 176 & 110
[2M+NH4]+ 71 &217-

Table 2-2. Alkali-metal cationic radii in Angstroms
Cation Radius
Li+ 0.76a
Na+ 1.02a
K 1.38a
NH4 1.43b
a Reference 95 b Reference 96

Table 2-3. Phloro 1 and 2 dissociation curve VC5o values and their associated 95% confidence
intervals for cation-bound dimers determined using SCID-TOF-MS
Complex lon Phloro 1 VC5o, V Phloro 2 VC5o, V
[2M+Li]+ 168 & 21 218 & 22
[2M+Na]' 153 & 25 190 & 21
[2M+K]' 128 & 21 162 & 23
[2M+NH4]+ 131 & 70-





























































1 ------- -- -- -- -- ---


[M+Na]*
105 461 1102





[M+K]f
477.0941


I'I ill I .'.1H 8Na+K]+ [829 [2+K]
915 2070


Figure 2-1. Phloroglucinol structure


a ~2b


OH O OH O


o'\f \/ o \


HO ~ Y OH HO' Y OH







Figure 2-2. Structures of the phloroglucinol derivatives a) 2,4,6-tribenzoylphloroglucinol (phloro
1) and b) 2,4,6-(3,5 -dimethyl)-trib enzoylphl oroglucinol (phloro 2).


b ew
3.9xl05 Inl II,,y


[M+Na]+
5.0x105S 461.1102


:. ..1+ li I2M+Na]+
; 892505


[2M+Nal
899 2505


[M+NH4] +
27x105: 456.1604


12M NTI]'



(2M+Na]+
899.2505


[M+H]+
439 1422


400 500 600 700 m/z 800 900 1000 400 500 600 700 mtz 800 900 1000


Figure 2-3. Electrospray ionization mass spectra showing phloro 1 forming cation-bound dimers
in the presence of a) Na b) Li c) K and d) NH4' cations


[M+i]l
445 1330 [M+K]+
477.0941

.I1 1


[M+HI
439 14 2


[M+Na]+
4 11102










a 8x1067 b ax1o6
[Ml+H]+ 523 2123
439 1172











524.217O
440.1214
[M+~Na]
-161.100(7 (11Na~ul'
545.1961

440 /z460 520 m/lz 540
Figure 2-4. ESI Spectra of a) phloro 1 and b) phloro 2 in the absence of added salt showing the
presence of adventitious sodium.










100




50




0 ..
50 100 150 20
100 I




50


II


50


200


0o 50


100 150
Potential difference, V


200


200


100 150
Potential difference, V


Figure 2-5. Phloro 1 dimer dissociation curves for dimers bound with a) Na', b) Li', c) K', and
d) NH4+ Obtained using SCID-FTICR-MS. Note the poor reproducibility, although
VCso values were calculated from these curves.












100




50. .




0
50 100 150 200 250 3(
100




50


50


0 150 200
Potential Difference, V


50 1--~ 00 150 200 250
Potential Difference, V

Figure 2-6. Phloro 2 dimer dissociation curves for dimers of bound with a) Na b) Li and c) K'
obtained using SCID-FTICR-MS. The NH4+ bound dimer was not stable enough for
construction of a stability curve. Note the poor reproducibility. Again VCso values
were calculated from these curves but had to be extrapolated for a) and b).



































Figure 2-7. Dissociation curves for all four cation-bound dimers of a) phloro 1 and b) phloro 2
obtained using SCID-TOF-MS. Note the improved reproducibility which translated to
smaller confidence intervals for the calculated VCso values.


--[2M +Ka]
[2M+NH4~-


0


b 1C
0ao


I


0 .


~'~b~h~


01


0O


150


200


250


-,~T 1 T


50


--[2hI+Li]+
--[2hI+Na]+
--[2hI+F;]+


100


0


150 2600
Potential Diffelrenc~e. V


2501




















[2Pl+Li]+ [2Pl+Na]+
883.2359 899.2079


- [P1+P2+Li]+

- [P1+P2+Na]+

-- [P l+P2+K]+


[2P2+Li]
1051.4247 [2P2+Na]
1067.3916


8.3x10


[Pl1+P2+Li]f
967.3337
[Pl+P2+Na]+
983.3065


800 9 400 1 000 m/ Int 11'00 12l00
Figure 2-8. Spectrum obtained using ESI-TOF-MS to analyze cation-bound homo and
heterodimers of phloro 1 (Pl) and phloro 2 (P2). Both homo and heterodimers were
observed to form with Li' and Na' although only LiCl was added to the sample.


01


Fl
o
~Tj
O
01
cn


100


150


200


250


Potential Difference, V
Figure 2-9. Dissociation curves for the cation-bound heterodimers with Li Na and K No
heterodimer formation with NH4' WAS observed.



























200 400
Wav~elength.nml
Figure 2-10. Absorption spectrum for 0.05 mM phloro 1 in methanol.


0.5


0.4


0 20 40 60 80 100 120 140 160 180
[Phloro 1], uM
Figure 2-11i. Beer' s Law plot showing absorbance intensity varying with concentration of phloro
1. Adherence to Beer' s Law is observable for several UV wavelengths.














0.8r



S0.4

0.2


0.0:
220 240 260 280 300 320 340 360 380 400
Wavelength, nm
b 10






0.0-
22 24 6 2030 2 403038 0
Waeegtn
Figure: 2-2 Ulrvoe/iil bopinsetafo ocnrtoso .505m oti
incemets f 05 m o a)phlro an b phoro2 i 5050metano~waer.Th





Figur 2highelrwavioetvsblngt paksro exiis hpschra omi hfihicesn concentrations of00-. m oti

phloro 1 but not phloro 2. The lower wavelength peak exhibits bathochromic shift for
both phloro 1 and 2.




















o.4-4 1 0l'-.01


0.2 0.02

Wdavelength. n1m Wavelengrb, nm





\~5 nm6 0. M\20 m .03m



Na K Li"--- N i 23Na K i o

CJ 6 S ~ 4 6 8 0 320 d4 32018



phloro 1 0.03 mM phlora 2 0.03 mM
26~ 3-00 mi .0.3mM ~30n 0.3 mM
3100 X-10n



2300 300


200Na+ K Lit NH1 28 Nat K Lif NH/t


th ihe aeent ek o )phloro 1 an d)0 m phloro 2.00?m










CHAPTER 3
ELUCIDATING THE DISSOCIATION MECHANISM OF NOVEL PHLOROGLUCINTOL
DERIVATIVES

Introduction

Phloroglucinol, 1,3,5-trihydroxybenzene (Figure 2-1), exists in various natural products.

Various phloroglucinol derivatives have been isolated as naturally-occurring bioactive

compounds, and synthetic phloroglucinol derivatives have been widely reported.97 Interest in

designed synthetic species based on phloroglucinol is growing.98-10 Members of this important

class of compounds have demonstrated therapeutic and pharmacological properties such as

antiviral, antibacterial, and vasodilator activities.'01 In particular, hyperforin and adhyperforin,

constituents of St. John's Wort (Hypericum perforatum), have been well-characterized for their

antidepressant action.102-10 Phloroglucinol derivatives comprise important secondary metabolites

in several dicotyledonous plants,97 and have been reported as useful environmentally-responsible

dye agents110 and feeding inhibitors.ll Additionally, acylphloroglucinols have demonstrated

potential as lead structures for degenerative disease drug development.112 ISolation, quantitation,

and characterization of diverse types of phloroglucinol derivatives are therefore relevant to many

applications, and are of increasing importance in pharmacokinetic and toxicological studies.

Pharmaceutical development relies on rapid, sensitive, and quantitative analytical

techniques for the determination of drugs and metabolites in complex biological matrices.113

High-performance liquid chromatography, HPLC, combined with mass spectrometry, MS, and

tandem mass spectrometry, MS/MS or MSn, is a widely-employed tool in this area because it

benefits from several important advantages. HPLC provides separation capabilities to improve

analysis of complex or dirty samples, and MS is useful because it enables rapid and selective

detection of compounds simultaneously.114 MS/MS is essential for structure elucidation which









can be especially important in metabolite identification.113 Another important aspect of tandem

work, however, is selected reaction monitoring, SRM, the capability of detecting a compound by

monitoring fragmentation of one ion to another following a characteristic and specific pathway

for a particular analyte.36 Multiple reactions may be monitored during a single chromatographic

separation, and SRM confers the ability to distinguish between structurally similar compounds

with different fragmentation behaviors increasing both selectivity and sensitivity of an assay

designed for a specific application.36,37 COnfident assignment of a fragment ion to its parent,

however, is an important part of method development for this type of work and consequently an

understanding of dissociation mechanisms for systems under investigation is necessary.

Elucidation of the fragmentation behavior of the phloroglucinol derivatives as a class of

compounds is therefore of growing importance due to the wealth of applications evolving for

these species. Several reports of qualitative and quantitative LC-MS for determination of

hyperforin and adhyperforin in extracts of St. John's Wort have recently been published. 102,104-108

Two synthetic phloroglucinol derivatives substituted at the 2,4 and 6 positions with

benzoyl groups (Figure 2-2) have been characterized using tandem and accurate mass

spectrometry. One compound has pure benzoyl substituents (phloro 1), but the benzoyl groups of

the second are methylated at both meta positions of the benzene ring (phloro 2). Alkali-metal-

bound dimers of both phloro 1 and 2 have been observed to form with Li Na and K and

proton and NH4 -bound dimers of phloro 1 have also been detected, as described in Chapter 2.

Both phloro 1 and phloro 2 were observed as protonated molecules and as molecular adducts of

each alkali-metal cation. Following fragmentation pathway elucidation for a simple compound,

2-hydroxybenzophenone (Figure 3-1), which contains similar functional groups to phloro 1 but

has no C3 character, a dissociation mechanism for the initial fragmentation of the cationized









molecules of phloro 1 and 2 was deduced. Dissociation pathways for the alkali-metal cation and

proton-bound dimers, as well as the protonated and ammonium adducted molecules of phloro 2,

were determined in this research. A recent publication elucidated the fragmentation behavior of

protonated and deprotonated hyperforin and adhyperforin,109 but to date no dissociation

mechanism information has been made available for alkali metal adducts of phloroglucinol

derivatives. This information is very valuable because electrospray ionization is an extremely

important interface for HPLC with MS. Alkali metal adducts, particularly sodiated and

potassiated species, are known to be prevalent in mass spectra obtained using ESI. Additionally,

in analytes lacking a significantly acidic or basic site, formation of an alkali metal cation adduct

may be the only available route to ionization and consequently detection where ESI is the sole

accessible ionization technique.

Experimental

Sample Preparation

The phloroglucinol derivatives 2,4,6-tribenzoylphloroglucinol and 2,4,6-(3,5-dimethyl)-

tribenzoylphloroglucinol were synthesized and purified by members of the Castellano group at

the University of Florida. Structures were confirmed by ESI-FTICR-MS and 1H and 13C HUClarT

magnetic resonance spectroscopy. All samples were dissolved in HPLC grade methanol

(Honeywell Burdick & Jackson, Muskegon, MI), and the cations Na Li K and NH4+ wer

introduced as chloride salts (Sigma-Aldrich, St. Louis, MO). The samples were prepared at

concentrations of 3x10-4 M host and 3x10-5 M guest, and contained 1% formic acid

(ThermoFisher Scientific, Waltham, MA) to improve current stability and ionization efficiency

throughout the analysis. The test compound 2-hydroxybenzophenone (Sigma-Aldrich, St. Louis,

MO) was prepared in the same solvent at a concentration of 3x10-4 M.









Mass Spectrometry

All mass spectra were acquired using direct infusion electrospray ionization with an

appropriate mass analyzer. The tandem mass spectral data for phloroglucinol derivative

fragmentation pathway elucidation were obtained using an LTQ linear ion trap mass

spectrometer, monitoring the fragmentation through MS5 (ThermoFisher Scientific, Waltham,

MA). The 2-hydroxybenzophenone fragmentation was observed via an LCQ Deca quadrupole

ion trap (ThermoFisher Scientific, Waltham, MA). The precursor mass selection window was set

to 1 amu and optimal CID cone voltages between 25 and 30 V were applied. High-resolution

exact mass measurements of precursor and product ions were made using SCID-ESI-MS with

either a 4.7 T Bruker Bioapex II Fourier Transform lon Cyclotron Resonance mass spectrometer

equipped with an Apollo API 100 Source (Bruker Daltonics, Billerica, MA) or an Agilent 6210

Time-of-Flight mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA). Samples were

introduced at flow rates of 2 CIL/min for the FTICR-MS and 10 CIL/min for all other mass

spectrometers using direct infusion. The potential difference between the ESI needle and the

desolvation capillary inlet was 3.5 kV. Each run was repeated three times using individually-

prepared samples for mapping of dissociation pathways. The ESI-SCID-TOF data presented are

data obtained via the experiments described in Chapter 2; consequently, experimental

configurations and conditions may be found in the Chapter 2 experimental section.

Results and Discussion

Electrospray Ionization Source-Skimmer Collisionally Induced Dissociation Justification

Chapter 2 describes ESI-SCID-TOF experiments yielding dissociation curves for the

alkali-metal cation-bound dimers of the phloroglucinol derivatives phloro 1 and 2. The data were

reanalyzed by plotting the ratio of cation-bound dimeric peak intensity to adducted monomeric

peak intensity against the SCID fragmentation voltage difference. The curves for phloro 1 are










presented as Figure 3-2. Comparison of the shapes of these curves highlighted a difference

between the sigmoidal decline of the curves corresponding to the Na' and Li' adducts and the

plateaus corresponding to the K+ and NH4+ adducted species. Analogous curves for phloro 2 are

shown in Figure 3-3; all three curves corresponding to the Na Li and K' adducts are of a

similar shape and differ predominantly in magnitude. This type of plot represents the dissociation

of an alkali-metal cation-bound phloroglucinol derivative dimer to a monomeric adduct of the

same cation, so differences in shape could be indicative of different cation-dependent

dissociation pathways for the phloro 1 host. Initial attempts at dissociation pathway elucidation

based on the ESI-SCID-TOF data were hindered by ambiguity concerning the origin of apparent

fragment ions, which is a limitation of SCID. These observations illustrated the need for a

tandem mass spectrometric investigation incorporating specific precursor ion selection

capabilities, as opposed to SCID which does not enable direct determination of precursor-

product ion relationships. Consequently, fragmentation of the mass selected cation-bound dimers

of phloro 1 and phloro 2 was investigated using CID in an ion trap mass analyzer, but to aid in

dissociation pathway elucidation, a similar experiment with 2-hydroxybenzophenone was first

performed. The SCID-TOF data were used to obtain exact mass measurements for the fragment

ions which were observed via both tandem mass spectrometric approaches.

2-Hydroxybenzophenone Experiments

The compound 2-hydroxybenzophenone (Figure 3-1) was selected to aid in the elucidation

of the dissociation behavior of the phloroglucinol derivatives. It has similar structural

characteristics to the phloro 1 molecule as it is comprised of a benzene ring modified by a

benzoyl substituent, and so has the important functional groups involved in the phloro compound

fragmentation. The two phloroglucinol derivatives, however, have a repeating pattern of central

benzene ring substituents which confers C3 Symmetry on their molecular structures, and which









complicates determination of dissociation mechanisms and pathways because structural

rearrangement during the high-energy CID event becomes a significant issue. The smaller and

less complex 2-hydroxybenzophenone has minimal structural reconfiguration capabilities and so

proved extremely useful as a tool for the derivation of the initial fragmentation mechanism of

both phloro 1 and 2.

Following MS3 Of electrosprayed 2-hydroxybenzophenone, a fragmentation pathway for

the protonated molecule was determined and is presented as Figure 3-4. Primary cleavages were

observed between the carbonyl carbon and each of its neighboring carbon atoms, as was

expected. Interestingly, the maj or fragment ion observed following the initial CID event

corresponded to the loss of a benzene ring and indicated the involvement of keto-enol

tautomerization in the dissociation process. This was evidenced by the loss of a complete

benzene ring which would only be possible if cleavage of the bond between the carbonyl carbon

and carbon 1 on the benzene ring were accompanied by the abstraction of a hydrogen atom from

a different region of the precursor ion. As stated previously, the rearrangement capabilities of

2-hydroxybenzophenone are limited so the most accessible hydrogen atom available for

abstraction would be that originating in the alcohol group, following keto-enol tautomerization.

This deduction coupled with the similar fragmentation behavior of hyperforin and adhyperforin

reported by Sleno et all09 enabled elucidation of the primary mechanism of covalent dissociation

which is shown in Figure 3-5 for 2-hydroxybenzophenone.

Protonated Molecule Dissociation Pathways

Dissociation pathways for the protonated molecules of phloro 1 and phloro 2 are presented

as Figures 3-6 and 3-7, respectively. Both phloroglucinol derivatives exhibit identical

fragmentation behavior and adhere to the mechanism deduced for the smaller

2-hydroxybenzophenone. Following keto-enol tautomerization, the complete neutral benzene










ring is lost from one of the phloro 1 benzoyl groups and the complete neutral dimethylated

benzene ring is lost from the corresponding substituent of phloro 2. This neutral loss occurs for

all three benzoyl groups until only the protonated core of the molecule C906 TemainS, suggesting

that the proton is noncovalently bound to one or more of the core oxygen atoms and is not solely

stabilized by arene interaction, as the central carbon cycle is no longer conjugated at this point.

No evidence of structural rearrangement beyond the initial keto-enol tautomerization was

observed.

Alkali-Metal Cation-bound Dimer and Adducted Monomer Dissociation

The dissociation pathways for the lithium cation-bound dimers of phloro 1 and phloro 2

were determined using the same strategy and are shown in Figures 3-8 and 3-9. The lithium

cation-bound dimer of phloro 1 dissociates to a monomeric lithium cation adduct via the neutral

loss of a phloro 1 molecule. The monomeric lithium adduct is subsequently fragmented in one of

three different ways. The neutral loss of one complete benzene ring, previously observed for the

protonated molecules, is the primary pathway observed, but neutral loss of either a water

molecule or carbon dioxide also occurs. Analogous to the fragmentation of the protonated

molecules, the benzene ring loss is followed by successive benzene losses until only the oxygen

and carbon monoxide-substituted cyclohexane core remains. The lithium cation, however,

remains a component of the detected ion through the entire process, suggesting that it is

interacting with the lone electron pair(s) of one, or more probably, several of the core oxygen

atoms. Evidence that the lithium cation remains is based not only on the fragment nominal and

measured accurate m/z data, but also on the isotopic distribution observed in the detected ions.

Lithium cation is the only one of the cations employed which has a nonstandard isotopic

distribution. This characteristic combined with the fact that the less abundant monoisotope has a

high enough abundance for detection above the noise threshold aid in tracking of the cation









throughout fragmentation. The loss of carbon dioxide from the monomeric lithium cation adduct

is of particular interest because it provides evidence for structural rearrangement of the

phloroglucinol derivative during the high-energy CID process. Carbon dioxide can only be

cleaved from phloro 1 following relocation of the benzene ring portion of one of the benzoyl

groups, but the position of the benzene ring after the rearrangement is currently indeterminate.

The phloro 2 lithium cation adducts exhibit analogous behavior. The lithium cation-bound

dimer dissociates to the lithium cation adducted monomer, but the monomeric adduct only

fragments via neutral loss of either a dimethylated benzene substituent or a carbon dioxide

molecule; no water loss occurs. Successive cleavages of the bonds between the carbonyl carbons

and the dimethylated benzene ring portion of the benzoyl groups follow until only the core

remains, similar to the dissociation behavior of the other phloroglucinol derivative compound.

The sodium cation-bound dimers of both phloro 1 and phloro 2 adhere to the same

dissociation pathway as those bound by lithium cation; the specific pathways are presented as

Figures 3-10 and 3-11, respectively. Smaller initial signal intensity, however, meant that the

dissociation process could not be observed through MS5 so only two of the benzoyl substituents

were observed to fragment. Both monomeric sodium cation adducts exhibit water and carbon

dioxide loss, further evidencing the structural rearrangement during CID observed for the lithium

adducts .

Fragmentation behavior for the less stable phloroglucinol derivative dimers bound by the

potassium or ammonium cations proved somewhat different to the previously characterized

dissociation process. For both phloro 1 and phloro 2 dimers bound by potassium cation, the

initial step yields monomeric potassium adducts and the successive step, for which the

mechanism has been proposed, involves the neutral loss of either the conventional or









dimethylated benzene ring. The respective pathways are delineated in Figures 3-12 and 3-13. No

alternative dissociation pathways are evidenced; neither water nor carbon dioxide losses from the

monomeric adducts are observed, hence there is no evidence to suggest that the potassium cation

phloroglucinol derivative adducts undergo structural rearrangement during CID. Low initial

signal intensity only enabled detection through MS3, So only one fragmentation step for the

monomeric adducts could be followed.

Adducts of the phloroglucinol derivatives with ammonium cation showed the most

deviation from the dissociation pattern observed for the cationized host compounds. The

corresponding pathways are presented as Figures 3-14 and 3-15. Although the initial

fragmentation of the phloro 1 ammonium cation-bound dimer involves the neutral loss of a

phloro 1 monomer yielding the monomeric ammonium cation adduct, consistent with the

dissociation behavior already described, no stable ammonium cation-bound phloro 2 dimer could

be observed. Consequently, the dissociation pathway for phloro 2 in the presence of ammonium

chloride is shown for the monomeric precursor ion. The successive step for phloro 1, however,

results in detection of the protonated molecule following the neutral loss of ammonia. No loss of

a neutral benzene ring occurs during the dissociation process, but subsequent to the ammonia

loss, cleavage of the bond between the carbonyl carbon of one benzoyl substituents and a carbon

atom on the core benzene ring is observed, yielding a carbonium cation. This can be further

fragmented to yield a carbonium cation of one of the benzoyl substituents carrying the charge on

the carbonyl carbon.

The fragmentation behavior of the ammonium cation adducted phloro 2 monomer shows

two alternative dissociation pathways. The traditional phloro 2 neutral loss of a dimethylated

benzene ring occurs, consistent with data obtained for each of the other phloro 2 monomeric









adducts. An alternative competing dissociation pathway involves the loss of neutral ammonia

yielding the protonated molecule, consistent with data obtained for the phloro 1 ammonium

cation adducted molecule.

Conclusions

Dissociation behavior for two phloroglucinol derivative compounds has been elucidated

using ESI-MS and a combination of tandem mass spectrometric techniques. Coupling of SCID

followed by high resolution mass analysis and mass selective ion trapping followed by CID led

to the acquisition of complementary data for thorough delineation of the dissociation pathways

associated with the C3 Symmetric molecules.

Initial justification for the dissociation pathway study was based on the increasing

pharmaceutical significance of phloroglucinol derivative compounds, but data obtained during

the relative stability experiments conducted on the alkali metal cation-bound dimers of phloro 1

and phloro 2 also highlighted a need for this information. Graphical representations of the

dissociation of the dimers to monomeric adducts suggested the possibility that different cations

could result in different fragmentation behavior.

Since no information concerning dissociation characteristics of alkali metal adducts of

phloroglucinol derivatives is currently available, the goal was initially pursued through

fragmentation of the smaller and less complex, but structurally similar, 2-hydroxybenzophenone.

Elucidation of the dissociation pathway observed via CID of the protonated molecule was

achieved and yielded useful data which led to the determination of the mechanism for the initial

dissociation step. The proposed mechanism involves keto-enol tautomerization followed by the

neutral loss of an alkyl group.

Adherence to the proposed mechanism was ultimately observed for CID of the protonated

molecules of both phloroglucinol derivatives. Successive occurrences of the fragmentation step










corresponding to the determined mechanism were observed until no benzoyl substituents

remained. The carbon and oxygen core of the molecule retained the charge-supplying proton

throughout all the cleavages associated with this behavior suggesting that the proton

noncovalently interacts with the core region of the host molecule.

Dissociation pathways for both phloroglucinol derivative dimers bound by Na Li and

K' were delineated, and showed that the dimers fragment to the corresponding cation-adducted

monomers. The primary route to fragmentation of the monomeric adducts adhered to the

dissociation mechanism observed for the 2-hydroxybenzophenone. Again, retention of the cation

throughout dissociation showed that the major stabilizing noncovalent interactions probably

involve the lone electron pairs on the oxygen atoms and not only the arene system, which is

predominantly destroyed by the loss of the final benzoyl substituent. Competing pathways

involving water loss and carbon dioxide loss were also evidenced, but only for the lithium and

sodium cation adducts. The loss of neutral carbon dioxide from the complete cation-adducted

monomers provided evidence for structural rearrangement of the host molecules during the CID

process.

The ammonium adducts behaved differently to the other adducts under investigation.

Although the dimeric adduct of phloro 1 initially dissociated to the analogous monomer, no

stable phloro 2 ammonium cation-bound dimer could be isolated for fragmentation. The

monomeric adduct of phloro 1 was only observed to fragment via the loss of neutral ammonia,

and successive steps showed no evidence of adherence to the dissociation mechanism determined

for the other adducts. The monomeric ammonium cation adduct did evidence fragmentation by

the proposed mechanism, but a competing pathway involving the loss of neutral ammonia was

also observed.










Electrospray ionization mass spectrometry was successfully employed to characterize the

dissociation behavior of the noncovalent adducts of the two phloroglucinol derivatives for future

pharmaceutically-relevant LC-MS and LC-MSn analyses. Differences in fragmentation behavior

of protonated and differentially cation-adducted species have been highlighted for this class of

compounds. Evidence for stabilizing noncovalent interactions between the oxygen-rich core of

the phloroglucinol derivatives and the cations rather than between the arene system and the

cations has been obtained.


































Ck
B
o
o

9
B

2
-
E


NH


~-~~+**


100 200 150
Potential Difference, V


Potential Di~ffeence, V


Figure 3-2. Dissociation of dimers of phloro 1 bound by a) Na b) Li c) K and d) NH4' to
their respective monomeric alkali-metal cation adducts as potential difference voltage
is varied in an ESI-SCID-TOF experiment. These curves are based on data presented
in Chapter 2.


Na







50 100 200 150 25

,


3
** Li1



1


Figure 3-1. Structure of the test compound 2-hydroxybenzophenone.


50


bE


C


2.


2.


50










50


251


-
3


5


d -


5











Lif







100 150 200 25
Potential Difference, V


I Na


100 150 200 25
5
Kt
4


a


s
4


as





Ob
O


100 150 200 ~ 2 50~;
Potential Difference, V
Figure 3-3. Dissociation of dimers of phloro 2 bound by a) Na b) Li' and c) K' to their
respective monomeric alkali-metal cation adducts as potential difference voltage is
varied in an ESI-SCID-TOF experiment. The NH4 -bound dimer was not stable
enough for curve construction. These curves are based on data presented in Chapter 2.











H


-C6H6


-CO


:C7H 02


[C6H,O]
m/z = 93


Formula = C13H 02
m/z = 199


-C6H60








Formula = C7H,O+
m/z = 105


Formula =
m/7 = 121


-C3H40


m/z = 65


-C2HO
~ [C3H40]
m/z= 56


-CH,
2 6H30]+
m/z = 91


-C4H4
[C3HO]t
m/z = 53


-CO








Formula = C6H,+
m/z = 77

Figure 3-4. Proposed fragmentation pathway for protonated 2-hydroxybenzophenone .


4CHO]+




































Figure 3-5. Proposed initial dissociation mechanism for protonated 2-hydroxybenzophenone .
The cationized molecules of the phloroglucinol derivatives phloro 1 and 2 also adhere
to this mechanism.



















































IIFonnula = C9HO6+
OTheoretical m/z = 204.9768
Experimental m/z = 204.9787
Mass error = 9.3ppm



S-C604


[C3HO2] Theoretical m/z = 68.9971
Experimental m/z = 68.9999
Mass error = 40.6 ppm

Figure 3-6. Proposed fragmentation pathway for the protonated molecule of phloro 1 showing
observed fragments with exact masses and mass error values where appropriate.
MS/MS stages were obtained using the LTQ instrument and exact mass data were
determined via SCID-TOF.


H"


OH



Formula = C21H1306+
Theoretical m/z = 361.0707
Experimental m/z = 361.0701
Mass error = -1.7 ppm


H

/ -C6H



Formula = C27H1906
Theoretical m/z = 439.1176
Experimental m/z = 439.1170
M~n =rr 14 n


or ppm


S-2(C6H6)






O OH

OFormula
Theoretica
Experimer
Mass error


'C zHO6
l1 m/z = 283.0237
ntal m/z = 283.0250
r = 4.6 ppm


-C302


[C6HO4] Theoretical m/z = 136.9869
Experimental m/z = 136.9882
Mass error = 9.5 ppm













OH O
CH3



OH


Molecular Formula = Czz,06 ,O
Theoretical ni z =523.2115
Experimental ni z =523.2110
Mass error = 1.0 ppni


S-2(CH ,)


OH


Molecular Formula = CHO,
Theoretical ni z =417.1333
Experimental ni z =417.1324
Mass error = 2.2 ppni


HI


Molecular Formula = CH 06,
Theoretical ni z =311.0550
Experimental ni z =311.0562
Mass error = 3.9 ppni




S-CHz,,


-CO,
~ [C6HO,]


Theoretical n z = 136.9869
Experimental n z = 136.9882
Mass error = 9.5 ppni


IIMolecular Formula = C 9HO6+
O Theoretical ni z =204.9768
Experimental ni z =204.9771
Mass error = 1.5 ppni


S-C604

[C3HO,]+ Theoretical m/z = 68.9971
Experimental m/z =68.9999
Mass error = 40.6 ppm

Figure 3-7. Proposed fragmentation pathway for the protonated molecule of phloro 2 showing
observed fragments with exact masses and mass error values where appropriate.
MS/MS stages were obtained using the LTQ instrument and exact mass data were
determined via SCID-TOF.

















Formula=2(C,7H gO6)Li+
Theoretical ni z =883.2363
Experimental ni z =883.2350
Mass error = -1.5 ppni


I-:;~,2


[C,6H, ,O, ]Li'
m/z = 401 Da


-COf -,


L~i


[C zH160O,]Li+
m/z= 427 Da


Formula = C,,H gO6Li
Theoretical ni z= 445.1259
Experimental ni z =445.1239
Mass error = -4.5 ppni


6 6H


-2(C6H6)


Li


-C6H6


~O Formula = C,,H606Li'
Theoretical n z = 289.0319
Experimental n z = 289.0302

1 CHM ass error = -5.9 ppni







Formula = C906Li
I Theoretical n z = 210.9850
O Experimental n z = 210.9829
Mass error = -10.0 ppni


Formula = CH zO6L1
Theoretical n z = 367.0789
Experilnental n z = 367.0751
Mass error = -10.4 ppin


Figure 3-8. Proposed fragmentation pathway for the lithium cation-bound dimer of phloro 1
showing observed fragments with exact masses and mass error values where
appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass
data were determined via SCID-TOF.


SLi




















Fonnula=2(C,,HoO,)Li
O ~Theoretical m z = 1051.4243
Experimental m z = 1051.4208
Mass error = -3.3 ppm




S-C,,H3006






-CO,
S[C31HoO,]Li
OH n 1/z= 485

OFormula = C,,H3006Li+
Theoretical m z= 529.2200
Experimental m z = 529.2159
Mass error = -7.7 ppm

-C,Ho


Li


-C,Ho





Formula = C,H2006Li
Theoretical m z =423.1415
Experimental m z =423.1397
Mass error = -4.3 ppm


Formula = C,,HoO6Li
OTheoretical m z =317.0632
Experimental m z =317.0610
Mass error = -6.9 ppm

-C Ho


O




Formula = C906Li+
m z = 2 11


Figure 3-9. Proposed fragmentation pathway for the lithium cation-bound dimer of phloro 2
showing observed fragments with exact masses and mass error values where
appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass
data were determined via SCID-TOF.



















Formula =2(C27HgO6 Na'
Theoretical m/z =899.2105
2 Experimental m/z = 899.2124
Mass error = 2.1 ppm


-CO2


[C27H160,]Na

m/z= 443


Formula = C27H gO6Na'
Theoretical m/z= 461.0996
Experimental m/z =461.0984
Mass error = -2.6 ppm


26,H1404]Na
m/z= 413


Formula= C21H120 Na'
S Theoretical m/z =383.0526
Experimental m/z =383.0552
Mass error = 6.8 ppm

-C6H6







HOO

/ 0Formula = C ,H606N
m/z = 305

Figure 3-10. Proposed fragmentation pathway for the sodium cation-bound dimer of phloro 1
showing observed fragments with exact masses and mass error values where
appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass
data were determined via SCID-TOF.


Na


































I~f -H,O


IFormula=2(C 11,,0* 5.
2 heoretical m z =1067.3977
Experimental m z =1067.4071
Mass error = 8.8 ppm


-CzzH,,06


a [C33H gO,]Na'
Theoretical m z= 527.1829
Experimental m z =527.1776
MaSS eTTOr = 10.0 ppm


S-COz

[C32H3C)O,]Na
m/z= 501


Formula = CHz,,06Na
Theoretical m z= 545.1935
Experimental m z =545.1978
Mass error = 7.9 ppm


-H,O
[C3,H,,O3]N
m/z= 483


HO OH Formula= C,,H,,006
Theoretical m z =439.1152
O Experimental m z =439.1172
Mass error = 4.6 ppm





O N



OOH

/ O Formula = CP 1006
m z = 333


Figure 3-11i. Proposed fragmentation pathway for the sodium cation-bound dimer of phloro 2
showing observed fragments with exact masses and mass error values where
appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass
data were determined via SCID-TOF.
























Theoretical m/z = 915.1838
2Experimental m/z = 915.1861
Mass error = 2.5 ppm

S-C27H 06,




O OH

o-C9H602
~a [C,,H1204 K
HO- Y OH m/z = 331



/Fonnula= C27H gO6K
Theoretical m/z =477.0735
Experimental m/z =477.0741

S-6H6 Mass error = 1.3 ppm













Formula = C21H1206K
m/z = 399

Figure 3-12. Proposed fragmentation pathway for the potassium cation-bound dimer of phloro 1
showing observed fragments with exact masses and mass error values where
appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass
data were determined via SCID-TOF.

















Formula = 2(C33 006)K
Theoretical m/z = 1083.3716
2Experimental m/z = 1083.3826
Mass error = 9.7 ppm


I-C33300


K







Formula= C33H3006Kt
Theoretical m/z =561.1674
Experimental m/z = 561.1658
Mass error = -2.9 ppm


S-C H,0


0 Kal


Formula = C25006,Kt
m/z = 455
Figure 3-13. Proposed fragmentation pathway for the potassium cation-bound dimer of phloro 2
showing observed fragments with exact masses and mass error values where
appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass
data were determined via SCID-TOF.






















Formula = 2(C i i,l Ia Ii,
Theoretical m z =894.2545
Experimental m z =894.2504
Mass error = -4.6 ppm


' 2 i


NH,+








SFormula =C II, II ITp
-Theoretical m z = 456.1442
Experimental m z = 456.1462
Mass error = 4.4 ppm








:1-C9H60,
0 a [C,,H130 ]
OH In z = 293


u, I -C ,HO3


-NH3


-CH60


Formula = CH ,O
m z =317


S-C13HO3


Formula = CH,06
Theoretical m z = 439 1176


[CH O]


Exeietlmz=4917 m z = 105
c* Mass error = -1.4 ppm

Formula = CH,O+ xeietlmz4917
m/z= 105

Figure 3-14. Proposed fragmentation pathway for the ammonium cation-bound dimer of phloro 1
showing observed fragments with exact masses and mass error values where
appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass
data were determined via SCID-TOF.













NH4


Formula=2(C33H3006)NH4
mz = 1062


12


INH4



7 -NH3


Formula = C33H3006 4 +
Theoretical m/z= 540.2381
Experimental m/z = 540.2376
Mass error = -0.9 ppm


H"


Formula = C33Hz zO6
Theoretical m/z= 523.2115
Experimental m/z = 523.2143
Mass error = 5.4 ppm


m/z = 434




Figure 3-15. Proposed fragmentation pathway for the ammonium cation adducted monomer of
phloro 2 showing observed fragments with exact masses and mass error values where
appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass
data were determined via SCID-TOF. The ammonium cation-bound dimer was too
unstable to be mass selected for dissociation.










CHAPTER 4
CONFIGURATION OF A HOME-BUILT DESORPTION ELECTRO SPRAY IONIZATION
SOURCE WITH A COMMERCIAL TIME-OF-FLIGHT MASS ANALYZER

Introduction

In 2004, the Cooks group reported the development of desorption electrospray ionization,

DESI,11 which, combined with direct analysis in real time (DART) formed the basis of an

innovative platform for ambient ionization.116 The term ambient is used here to imply that it is an

atmospheric pressure technique maintaining sample accessibility throughout analysis. Consisting

of a pneumatically-assi sted electrospray solution used to desorb and ionize analytes from an

ambient surface, the DESI technique has demonstrated ESI-like characteristics in the ionization

of analytes amenable to electrospray ionization such as peptides. DESI also benefits from an

ability to ionize species traditionally ill-suited to ESI, however, via an apparent additional

ionization mechanism."' Inherent sample accessibility throughout analysis confers on the

technique the advantages of real-time manipulation of ionization conditions such as temperature

and solvent composition and also the configuration referred to as reactive DESI, a variant of the

technique which is described in greater detail in Chapter 5. The DESI technique is related to both

ESI and desorption ionization techniques such as MALDI and desorption/ionization on porous

silicon, DIOS."'S Currently, DESI involves a nebulized solvent, usually water or a mixture of

water and methanol, under high voltage conditions sprayed in an optimal geometry at an analyte

adsorbed onto a surface or an in situ analyte. Desorbed ions are focused into a mass spectrometer

via an atmospheric pressure interface.115,118-121

Two mechanisms of ionization have been postulated, but they are each relevant to different

applications.115,118-121 One involves the formation of charged solvent droplets which ionize

analyte molecules on the desorption surface by charge transfer; the resultant buildup of static









charge forces desorption of the analyte ions from the surface, a phenomenon known as chemical

sputtering.121 The second mechanism explains the strong resemblance in spectral characteristics

of DESI data to that obtained via ESI. Electrosprayed droplets impact the surface resulting in the

dissolution of analyte within the droplet; subsequent evaporation of the solvent yields analyte

ions. Both ESI and DESI generally yield significant amounts of the protonated molecular ion,

multiply charged ions, and alkali metal adducts in positive mode.ll An interesting study

investigating droplet dynamics and ionization mechanisms of DESI using phase Doppler particle

analysis was recently published by Venter et al. 122

Primarily, DESI is advantageous because it is an ambient method, which requires little or

no sample pretreatment.ll It is sensitive, fast, and versatile,115,118s-121 being suitable for solid,

liquid, and adsorbed gaseous samples.ll Traditional DESI has been applied to an enormous

number of analytes, including pharmaceuticals, chemical warfare agents, plant alkaloids, and

lipids.116 Novel sampling surfaces like porous silicon, thin-layer chromatography plates,123 and

solid-phase microextraction fibersl24 have been analyzed.125,126 A maj or application area of the

DESI technique is the trace level detection of explosives on ambient surfaces. Native explosives

and their plastic compositions have been analyzed on metal, plastic, paper, polymer, and living

human tissue surfaces; promising results were obtained using complex matrices such as

lubricants and detergents.120 Pharmaceutical applications include high throughput analysis of

various commercial dosage forms, drug and metabolite identification in blood and urine,119 and

coupling of DESI to thin layer chromatography for over-the-counter preparations analysis.125

Food chemistry applications and determination of alkaloids in plant tissues have been

demonstrated.ll Mass spectrometric profiling of intact, untreated bacteria employing DESI has

been described.12 A study describing the complementary application of nuclear magnetic









resonance spectroscopy and DESI-MS, and incorporating principal component analysis for data

interpretation, for urine metabolite determination in patients with inborn errors of metabolism

was recently contributed by the Cooks group.128 Imaging mass spectrometry under ambient

conditions has been achieved by the application of DESI,129 and tissue imaging

demonstrated.129'130

The preceding applications employed standard bench top ion trap or triple quadrupole

mass analyzers. An interesting addition to the types of trapping instruments with which the DESI

technology has been interfaced is a custom-built portable mass spectrometer fitted with a

miniature cylindrical ion trap mass analyzer.13 The Cooks group has also demonstrated the

capabilities of DESI coupled to an Orbitrap mass spectrometer for exact mass measurements on

therapeutic drugs and peptides,132 and several other mass analyzers have been configured for

high resolution and exact mass DESI applications. DESI has been interfaced with TOF-MS for

the direct determination of counterfeit, commercial, and illicit drug tabletsl33,134 and for the

determination of CID fragmentation pathways of over-the-counter drugs.135 An atmospheric

pressure interface for the coupling of ESI, electro-sonic spray ionization, ESSI, an extremely soft

form of ESl utilizing a supersonic gas j et, 136 and DESI to FTICR-MS was developed by the

Bruce group,137 and successful analysis of proteins and peptides was recently achieved using

DESI-FTICR-MS.138

A home-built DESI source originally designed for configuration with a Bruker Bioapex

FTICR-MS has been successfully interfaced with first the FTICR-MS instrument and

subsequently an Agilent TOF-MS. The DESI technique has compatibility issues with FTICR-

MS, hence the DESI-TOF-MS configuration was undertaken. The unique geometry of the source

housing on the Agilent TOF mass spectrometers poses particular challenges for DESI source










integration, as do some of the instrument-control-specific features of the accompanying Analyst

software. Strategies for addressing those challenges are described herein.

Experimental

Desorption Electrospray Ionization Source Design

The DESI source was constructed by the University of Florida Chemistry Department

Machine Shop. It was fabricated using anodized aluminum (6061-T6), and consists of one xyz-

stage for the manipulation of an electrospray head and a second xyz-stage for the manipulation

of a sample holder platform. A schematic showing an overhead view is presented as Figure 4-1,

and photographs of the source configured with the FTICR-MS are included as Figures 4-2 and

4-3. The dimensions shown in the schematic are those used for configuration with the FTICR-

MS; alterations made for configuration with the TOF-MS are described in the results and

discussion section. The electrospray head is mounted on a cylindrical block which can be rotated

for manipulation of the angle made between the spray needle and the deposition surface. A

commercial ESI needle is housed inside the coaxial gas needle which is visible in the schematic.

A Teflon@ spacer between the block and the solution/gas introduction port insulates the

remainder of the source from the high-voltage applied to the solution. Solution introduction is

via direct infusion using a Harvard Apparatus PHD 2000 syringe pump (Harvard Apparatus,

Holliston, MA), and high-voltage is applied to the spray solution via an external 210-10R high

voltage power supply (Bertan Associates, Hicksville, NY). The sample surface is chassis ground.

The sample holder is a Teflon@ platform mounted on an aluminum steel block with an

indentation for housing of a deposition surface the size and shape of a standard microscope slide.

Mass Spectrometry

Mass spectra were acquired using a Bruker Bioapex II 4.7 T Fourier Transform lon

Cyclotron Resonance mass spectrometer (Bruker Daltonics, Billerica, MA), or an Agilent 6210









MSD Time-of-Flight mass spectrometer configured for ESI (Agilent Technologies, Inc., Santa

Clara, CA).

Sample Preparation

Rhodamine 6G (Figure 4-4) samples were prepared by drawing a layer of red Sharpie

(Sandford Corporation, Oak Brook, IL) ink onto the sampling surface. Cytochrome C (horse

heart, Sigma-Aldrich, St. Louis, MO) samples were prepared by dissolving 1 mg/mL of the solid

in 50:50 methanol:water. Ten microliter spots were deposited onto the sampling surface using a

micropipettor and were dried under ambient conditions for thirty minutes. HPLC grade solvents

were employed (Honeywell Burdick & Jackson, Muskegon, MI).

Results and Discussion

Configuration of DESI Source with FTICR-MS

Initially the DESI source was interfaced with the FTICR using the original dimensions and

attachment mechanism specifically designed for this purpose. The FTICR-MS instrument used is

ordinarily equipped with an ESI source, and so has an atmospheric pressure inlet orifice

accessible for DESI. The hollow center of the desolvation capillary is the inlet and must be

immediately adj acent to the deposition surface for DESI compatibility. The ESI spray head

housing attaches to the desolvation capillary housing via a hinged door and latch, so the whole

spray head can be removed easily. Removal of the ESI spray head housing makes the inlet

orifice accessible. The original design of the DESI source included a hinged door, of the same

dimensions as that of the ESI spray head housing, placed adj acent to the sample holder platform

(see Figure 4-2). A circular hole cut in the door enabled access to the desolvation capillary for

ion entrance.

The applied high-voltage necessary to charge the spray solution is ordinarily achieved in

the standard ESI source for this instrument by maintaining the spray needle at ground potential









and applying a voltage of 3-4 kV to the desolvation capillary entrance. The applied voltage is

negative for detection of positive ions and positive for detection of negative ions. A safety

feature of this instrument prevents the high-voltage from being applied unless a certain switch in

the capillary housing is closed; the switch is depressed when the hinged door is closed and

tightly latched. If the high-voltage does not turn on, the software will not allow the instrument to

acquire data. Therefore, the switch must also be depressed during DESI spectral acquisition. To

overcome this issue, a small screw was placed through the DESI source door and screwed into

position to depress the switch upon latching the replica door. This can be seen in Figure 4-3. The

screw is above and to the right of the desolvation capillary. Interestingly, to obtain DESI signal,

the high-voltage had to be applied to the electrospray solution while the desolvation capillary

was held at 100 V. Reducing the desolvation capillary entrance to ground potential resulted in

unstable current and loss of signal.

When the DESI source was first interfaced with the FTICR-MS, it was observed that the

dimensions of the source did not allow the full range of motion that should have been available

to the spray head on its mounting block, and the spray head was too far away from the inlet for

DESI signal to be attained. This issue was unforeseen during the design process because the

additional length of the spray needle and the length of the desolvation capillary were difficult to

measure accurately in position in the enclosed ESI spray head housing. A useful feature of the

DESI source design was exploited to address the problem. Between the spray head mounting

block and the x-y-z stage is an entirely removable piece, labeled "a" in Figure 4-1. An analogous

piece is used to connect the sample holder platform and its x-y-z stage. Piece "a", which was

originally 9 cm long, was replaced with a 12.75 cm piece. This enabled the movement of the

spray head towards the inlet orifice, and subsequently DESI signal of Rhodamine 6G was









observed. The first DESI-FTICR-MS data acquired using the DESI source are spectrally

represented in Figure 4-5, and a close-up view of the DESI needle coaxial gas tubing adj acent to

the orifice inlet is shown as Figure 4-6, to provide some idea of the small distance between the

needle tip and the inlet of the mass spectrometer necessary for spectral acquisition. Also visible

is a portion of the sample holder stage and the deposition surface from which the detected ions

were desorbed.

Optimization of DESI parameters is an involved process which requires signal stability.

Unfortunately, sample ablation during DESI can be quite fast, of the order of tens of seconds.

The small colorless spots on the slide shown in Figure 4-6 are areas where sample ablation via

DESI has occurred. An important step in the acquisition of mass spectral data via FTICR-MS is

the hexapole accumulation of ions prior to their transfer to the ICR cell for detection. This

usually takes between 1 and 2 seconds. Additionally, several complete scan events involving

accumulation time, ion transmission time, and time for cyclotron resonance detection and

multiplexing are necessary to obtain usable signal intensity and signal-to-noise ratio, S/N, values.

This makes optimization of DESI parameters for FTICR-MS extremely challenging, as, during

the time it takes to determine the effect of slight variation of one parameter using the FTICR-

MS, sample ablation has resulted in loss of signal. For this reason, it was determined that it

would be useful to configure the home-built DESI source with an Agilent TOF-MS, which has

the advantage of a very high scan rate, conferring the ability to maintain high-resolution while

acquiring around 1 spectrum per second, depending on the selected mass range.139

Configuration of DESI Source with TOF-MS

To capitalize on the fast spectral acquisition capabilities of the TOF mass analyzer, the

DESI source was moved onto the TOF-MS described in the experimental section. The original









design was not compatible with the unusual geometry of the Agilent source (see Figure 4-7), so

several modifications had to be made.

The first maj or obstacle to the DESI-TOF configuration was the height of the desolvation

capillary housing on the TOF. To address this, a specially built platform for the DESI source had

to be made out of stainless steel. The 13" stand is visible in Figure 4-8. However, although the

stand brought the source to an appropriate height, the slanting front of the desolvation capillary

housing made positioning of the sample holder platform adj acent to the inlet orifice difficult. The

front of the base of the DESI source was held at a certain distance from the capillary by the

protruding lower edge of the desolvation capillary housing, and the original dimensions of the

arm connecting the sample holder platform and its x-y-z stage limited movement of the platform

toward the mass spectrometer, so that 7 cm between the sample surface and the inlet orifice was

the minimum distance achievable. This issue was overcome by exchanging the removable 7.5 cm

long piece labeled b in Figure 4-1 with a new piece of length 14 cm. Since the spray head was

now incapable of reaching either the sample surface or the inlet orifice of the mass spectrometer,

piece "a" was replaced again, this time with a 19 cm long piece.

At this point, the spray head was able to reach the inlet orifice, but the slanting geometry of

the desolvation capillary housing was still preventing movement of the sample holder platform to

within the necessary distance of a few mm from the inlet orifice. At its nearest point, the edge of

the sample platform or the arm of its mounting block j arred against the edge of the capillary

housing, 1.25 cm away from the orifice. As stated briefly in the introduction section of this

chapter, in 2006 the Bruce group at Washington State University reported the use of a flared

capillary adapter to improve collection efficiency of ions resulting from ESI, ESSI, and DESI

ionization techniques.137 This strategy was modified to address the issue of the distance









remaining between the needle of the home-built DESI source and the inlet orifice of the TOF

mass analyzer. A capillary extender designed to fit snugly onto the end of the desolvation

capillary and extend to the sample surface was used to bring the orifice inlet to the sample (see

Figure 4-9). A gold canted coil spring was placed into a groove milled on the inside of the piece

which fits over the capillary to hold the extender firmly in place while promoting conduction for

the electrospray. This was essential, because in the absence of the spring, the capillary extender

will be pulled off the end of the desolvation capillary and stick to the edge of the sample holder

stage due to charge build-up upon application of the potential difference necessary to achieve an

electrospray. An additional useful feature of the capillary extender is that it enlarges the area of

the sampling orifice, which has been shown to improve ion transmission and spectral

characteristics.137 The diameter of the desolvation capillary inlet orifice is 0.6 mm, but the

diameter of the capillary extender inlet orifice is 1.5 mm.

The Agilent TOF mass spectrometer can only acquire spectra when it is configured to a

source. Since several commercial atmospheric ionization sources have been designed for it, a

source recognition mechanism incorporating strategically positioned magnets is employed to

allow control of the sources via one software suite. Magnets on the inside of the source

attachments, which interface by latching onto the desolvation capillary housing, are in locations

specific to each source type. Consequently, when a nonstandard source is interfaced with the

desolvation capillary, a configuration of appropriately-sized magnets is necessary to enable

source recognition by the software. The standard ESI source has the simplest magnetic

configuration, so the mass spectrometer was configured for ESI whenever the DESI source was

attached. One small bar magnet on the inside of the ESI spray head housing is used for ESI

source recognition. The addition of a small bar magnet correctly positioned external to the









desolvation capillary source housing and held in place with Scotch tape enabled the instrument to

"recognize" the ESI source and allowed spectral acquisition in the absence of a commercial

source. A second issue in terms of nebulizer gas settings also had to be addressed prior to data

acquisition. The ESI nebulizing gas is not used for the DESI source, and the outlet has to be

covered to minimize unnecessary loss of N2(g). However, for the instrument to come out of

standby mode, the internal flow meter has to register the flow rate set by the user. This is

achieved by setting the nebulizing gas flow rate to the number that is registered prior to spectral

acquisition. A setting of zero may or may not be appropriate, despite the fact that nebulizing gas

is not flowing.

Following the described modifications and conditions, DESI-TOF-MS data were obtained

for the Rhodamine 6G sample. A representative mass spectrum is shown as Figure 4-10.

Following the Rhodamine 6G tests, another standard, the protein cytochrome c, was used for

confirmation of the DESI-TOF ionization capabilities. Cytochrome C was successfully analyzed

using the DESI-TOF-MS configuration and provided evidence of the success of the interface for

compounds other than Rhodamine 6G which is known to exist as a preformed ion. The first

spectral data obtained for cytochrome C are presented as Figure 4-11.

Important Optimization Considerations

Optimization strategies, and corresponding effects on spectral characteristics, for the

geometric and physical parameter conditions important in DESI mass spectral analyses have

been extensively documented elsewhere.118,119,122,137,1014011 COnsequently, the discussion here is

limited to important considerations in the optimization process which are frequently omitted

from the published literature. DESI conditions must be optimized specifically for each type of

analyte and surface, although similar analytes often have similar optimal conditions. Following

the determination of the specific surface used for sample deposition, parameters requiring










optimization may be broadly grouped into two categories: electrospray conditions and geometric

parameters.

Examples of the electrospray conditions requiring optimization include solution-phase

composition, solution flow rate, applied high voltage difference, and nebulizing gas flow rate.

These can be optimized to some extent using real-time visual cues, such as the extent of sample

ablation or surface wetting. Monitoring the current measured on the desolvation capillary for

stability is a significant aid in optimization of solution composition and applied voltage values.

Solution and nebulizing gas flow rate optimization can be performed speedily by monitoring

wetting of the sample surface. An additional aid in significantly increasing the efficiency of

optimization of the electrospray conditions is monitoring the total ion chromatogram (TIC)

instead of the signal corresponding to a particular analyte or analytes. This is useful because it

renders the ESI optimization process largely independent of the geometric DESI parameters,

upon which analyte-specific signal intensity and S/N are dependent.

Important geometric DESI parameters have been defined by the Cooks group,""s and are

delineated in Figure 4-12. Optimization of these parameters is a very interactive and iterative

process. Signal intensity and S/N are particularly sensitive to variation of the angle a, which is

additionally the most difficult parameter to manipulate using the home-built DESI source, so this

is the most challenging parameter to optimize. A good starting point for analytes traditionally

amenable to ESI is a = 60o. Again, monitoring the TIC instead of signal intensity or S/N is an

efficient way of making an initial assessment of the appropriate range of geometric parameter

values for subsequent, more rigorous optimization.

For each type of DESI analysis, it is important to optimize parameters for maximum signal

intensity and maximum S/N specific to the analysis. This is because the variable values yielding









maximal signal intensity sometimes do not correspond directly to those yielding maximal S/N,

although overlap in terms of a value ranges for some parameters may be observed. Figure 4-13

presents some representative parameter optimization data and shows the effects on signal

intensity and S/N resulting from variation of the electrospray flow rate. The optimal flow rate

range for maximizing both signal intensity and S/N is 4-6 CIL/min, but the peak maxima do not

necessarily correspond. Consequently, in some situations 5 CIL/min could be most desirable flow

rate, but for analysis of a limited amount of sample, where SCID would be employed for

structural elucidation or determination of relative stabilizing forces, it is conceivable that the

lower flow rate would be selected for the advantage of increased spectral acquisition time it

would confer.

Conclusions

A home-built DESI source engineered by machinists at the University of Florida

department of chemistry machine shop specifically for configuration with a Bruker Bioapex

FTICR-MS instrument has been successfully interfaced with an Agilent 6210 API Time-of-

Flight mass spectrometer. Initial configuration of the DESI source with the FTICR was

successful in that DESI mass spectral acquisition was achieved; mass spectra of the dye

Rhodamine 6G were acquired. A significant challenge, the fact that sample ablation during DESI

often occurred prior to adequate data acquisition to determine the effects of parameter variation

during optimization, highlighted the poor compatibility of DESI with FTICR-MS in terms of

slow scan rate of the FTICR instrument.

Several modifications to the original design were necessary to accommodate the significant

height difference between the inlet orifice of the FTICR instrument and that of the TOF mass

spectrometer, and the unorthodox geometry of the desolvation capillary housing. The fabrication










of a 13" custom built stand overcame the first issue. Extension of the range of motion in the

direction of ion transmission of both the DESI spray head and the sample holder platform

coupled with the fabrication and incorporation of a specifically-designed capillary extender

addressed the latter. Following some additional steps to compensate for the source recognition

and condition-monitoring processes integral to the function of the commercial instrument,

successful acquisition of DESI-TOF mass spectra of deposited Rhodamine 6G and cytochrome C

was attained.

DESI optimization is an iterative and interactive process which has been documented

elsewhere, but some important considerations and strategies for increasing the efficiency of the

process have been outlined to aid the novice user.





























~~prampl holderhld
~Kdoo/ MS I inle

Fiue -.Sceaico heoehedvewo heoiinlhmebit EIsoreshwn

mao pat n imninin cetmees for coniguatin it te TIR-S

Thepice mrked aI a.nd b wr lee o ofgrto ihteTFM n h
door au~lr. n d ig eermvd










Tf"
lllrlll[llrrrrlll
Ir
,I~ sp'"~' Ilrnd


Figure 4-2. Desorption electrospray ionization source interfaced with the FTICR-MS showing
door and hinge for attachment to the desolvation capillary housing.


Figure 4-3. Close-up view of DESI source configured with FTICR-MS showing the spray head,
the sample holder platform, and the front end of the desolvation capillary.


sanlple platfoH111
s-!~-z ~taes











































444.23 66


Figure 4-4. Structure of the Rhodamine 6G preformed ion.


2.5x106


MS
443.2327


440 m/z460
Figure 4-5. First spectrum obtained using the home-built DESI source configured to the FTICR-
MS, showing the molecular ion of Rhodamine 6G.











106



































Figure 4-6. Close-up view of the DESI-FTICR-MS setup used to acquire the spectrum presented
in Figure 4-5. The relative positions of the DESI spray needle and the desolvation
capillary orifice are shown. The deposition surface, a sandblasted glass slide, and its
layer of Rhodamine 6G are visible and small colorless spots on the sample surface
show areas of sample ablation.







































Figure 4-7. Close-up view of the DESI source interfaced with the TOF-MS showing the unusual
slanting geometry of the desolvation capillary housing. Note that the capillary itself is
not visible, as only the capillary extender protrudes from the housing. The DESI
spray head solution/gas introduction port and fluid connections can be seen. The bar
magnet taped to the upper right side of the desolvation capillary housing is
responsible for ESI source recognition by the software.






































Figure 4-8. Side view of the DESI-TOF-MS configuration. The 13" stand constructed to bring
the DESI source to the height of the desolvation capillary is shown and the
connection for the application of the high voltage to the electrospray solution is
visible.


8 14.2 b



1.5 1
1.6S canted coil
spnng
Figure 4-9. Representation of the capillary extender, with dimensions given in mm, for the
DESI-TOF interface showing a) the side view and b) the view from the back. A
canted coil spring is fitted into a circular groove on the inside of the extender to hold
the extender firmly in place on the desolvation capillary.


































12'
1030.9
11+
1124.5 10 9tg
123.X 3740


1200

77:

17$
728.0
18+
687.6


I I I I I


M+ '
443.2351


5000


400 420 440 n/z 460 480 500
Figure 4-10. Mass spectrum of Rhodamine 6G obtained using the DESI-TOF-MS configuration.


15+
824.91

I.5 83~ 951.713


400


800 mz 1000


1200


600


1400


Figure 4-11i. Positive-mode DESI-TOF mass spectrum of cytochrome C desorbed from a glass
slide. A multiprotonated envelope containing charge states ranging from +9 to +19
was observed.























Figure 4-12. Schematic representation of the DESI spray head and inlet orifice showing the
maj or geometric DESI parameters which require optimization.


O 2 4 6 8 1 0 2 4 6 8 10
Flow rate, AL/min Flow rate, LAL/min
Figure 4-13. Representative optimization data for the DESI-TOF-MS configuration using
Rhodamine 6G. The effects of variation of the solution flow rate on a) signal intensity
and b) S/N for the molecular ion are shown.










CHAPTER 5
REACTIVE DESORPTION ELECTRO SPRAY IONIZATION FOR RAPID SCREENING OF
GUESTS FOR SUPRAMOLECULAR INCLUSION COMPLEXES

Introduction

The need for a better understanding of noncovalent molecular recognition interactions is

fueling the development of analytical techniques appropriate for the characterization of both

natural and synthetic supramolecular systems.26 Methods like optical and nuclear magnetic

resonance spectroscopies and circular dichroism have proven extremely useful for solution-phase

analyses, and X-ray crystallography is indispensable for solid phase experimentation.17,21 Gas-

phase techniques, on the other hand, enable characterization of supramolecular species without

the added complications of interference from solvating molecules and crystal close packing

effects.3,6,26 Mass spectrometry is therefore an appropriate tool for this application and has the

benefits of sensitivity, selectivity, and the ability to provide complex stoichiometric

information. 19,26

ESI is inherently applicable to complexation based on solution-phase self-assembly, and

ESI mass spectrometry has been reported for the analysis of a plethora of noncovalent systems

from enzyme-substrate complexes26,79 to macrocycles, such as calixareneS140 and

cucurbiturils,60,68,69 which are capable of forming inclusion complexes with both neutral and

charged guest molecules. ESI is known for a tendency to form nonspecific artifacts which can be

confused with true supramolecular complexes in this type of work;143,144 this can be particularly

problematic in the study of alkali-metal complexation. Additionally, while ionization occurs at

atmospheric pressure, the technique does not satisfy a recent definition of the term ambient,

which includes the requirement of sample accessibility for manipulation during analysis." In

this application area, sample accessibility is useful because it enables real-time manipulation of









parameters like solvent character and availability of different guest molecules for faster analysis

in terms of guest screening.

A maj or impetus for the development of high-throughput noncovalent receptor screening

techniques is the wide applicability of molecular recognition interactions to fields like drug

discovery and lead optimization.17,26 High-throughput enzyme-substrate assays are well-

documented, but predominantly reliant on solution-phase analytical techniques such as

immunoassays using spectrophotometric or radiochemical detection, and consequently often

subject to interference from solvent and assay reagent moleculeS.5014 Structural modifications

of ligands are sometimes required to yield required chromophoric properties for detection of the

binding event which can alter natural kinetic behavior, and the use of radiochemicals for

scintillation-based assays involves the generation of radioactive waste.'o Mass spectrometry has

the selectivity to minimize some of these effects and consequently reduce the instance of false

positive results, particularly when high-resolution or tandem mass analyzers are employed. Mass

spectrometry has been applied to receptor screening as a technique for the analysis of

combinatorial libraries,146-148 but in this context complexation is often driven by solution-phase

equilibria in complex matrices, complicating spectra and increasing the likelihood of signal

suppression from competing analytes. All of the above suffer from an inability to refine

experimental design on a real-time basis for faster method development. A fast mass

spectrometric screening method for supramolecular receptors with an appropriately soft

ionization technique, manipulable chemical conditions, and minimizing interference from

competing species would address some of these issues.

The DESI technique appeared promising for supramolecular applications initially because

of its reported similarity to ESI characteristics in the ionization of some analytes well-known to









be amenable to electrospray ionization. Sample accessibility throughout analysis confers on the

technique a maj or advantage in the form of reactive DESI, a variant of the technique in which

reagents are incorporated into the spray solution for reaction with the deposited analyte at the

sampling surface. Reactive DESI using hydrochloric acid and trifluoroacetic acid has been

shown to improve selectivity and detection limits.120,121 Traditional DESI is not well suited to the

study of supramolecular complexation because deposition and drying of pre-mixed host and

guest can add the complication of close-crystal packing forces to the analysis, potentially

resulting in the detection of complexes which would not form under useful and realistic

conditions. Reactive DESI, however, does not incorporate this disadvantage and so is the more

promising configuration of the technique for this application.

Conventional DESI has been applied to an enormous number of analytes, including

pharmaceuticals, chemical warfare agents, plant alkaloids, and lipids.116 Large biomolecules such

as cytochrome C have been shown to maintain nativelike folded conformations during DESI

with appropriate conditions,149 prOViding evidence for the applicability of DESI to

supramolecular systems. Reactive DESI, predominantly incorporating alkali metal and

ammonium cations as spray reagents but also using chloride anions, has been reported for the

analysis of pharmaceuticals and metabolites,140 and for cation adduction ionization of explosives

and chemical warfare agents.iso Trace detection of the explosive triacetone triperoxide further

demonstrated the utility of reactive DESI for analysis of alkali metal complexes," and

heterogeneous reactions between ions formed by the DESI spray head and solids deposited on a

surface have been shown to yield covalently-bound products identified by mass analysis.152,153 A

natural progression, therefore, is the application of reactive DESI for receptor screening in small-

molecule host:guest inclusion complex studies. Very recently, a reactive DESI supramolecular









screen for the determination of counterfeit antimalarial tablets was described by Nyadong et

al.,154 but to date DESI has not been applied to the analysis of cyclodextrin host:guest inclusion

complexes. Additionally, no direct comparison between DESI and ESI for supramolecular

applications or for characterization of solution-phase representative data has been made.

Cyclodextrins, cyclic polymers of monosaccharides, are extremely well-characterized

toroidal molecules with a hydrophilic exterior and a hydrophobic intramolecular cavity in which

a small neutral guest molecule can be encircled to form a true inclusion complex which self-

assembles under suitable solution-phase conditions.26,15 The cyclodextrins have been shown to

complex a multitude of guest species and several ESI-MS studies of this behavior have been

reported, 19,143,144,156-158 making them a useful model system for reactive DESI method

development. P-cyclodextrin is composed of seven ot-1,4-linked glucopyranose units and

consequently has a versatile cavity size for small molecule encapsulation. It has been shown to

house various pharmaceuticals and nitro-compounds among other candidates. Its versatility led

to its selection as the test host compound. The initial test guest nortestosterone was selected for

the documented ability of similar steroids to complex with P-cyclodextrinl44159 and the guests

used in the screening analysis were chosen on the basis of size and appropriate functional groups.

Experimental

Sample Preparation

All solutions were prepared using HPLC grade solvents (Honeywell Burdick & Jackson,

Muskegon, MI). P-cyclodextrin (Trappsol, CTD, Inc., High Springs, FL) was diluted to

0.05 mM with 50:50 methanol:water. Nortestosterone was used for proof-of-concept and the

twelve screened potential guest compounds were acenaphthalene (Gl), acetanilide (G2), caffeine

(G3), 4-chloro-3 -nitrobenzonitrile (G4), cyclam (G5), 1,3 -dinitronaphthalene (G6),










diphenylglyoxime (G7), formanilide (G8), guanosine (G9), L-arginine (G10),

2-methylnaphthalene (G11), and 4'-nitroacetanilide (Gl2) shown in Figure 5-1 (G5,G10: Acros

Organics, ThermoFisher Scientifie, Waltham, MA; all others: Sigma-Aldrich, St. Louis, MO).

They were selected as potential guests on the basis of their sizes and appropriate functional

groups.

Mass Spectrometry

In preparation for the DESI experiments, the guest compounds were diluted to 1 mg/mL,

approximately 5 mM, in 50:50 methanol:water, spotted in 10 C1L aliquots onto a sandblasted

glass slide and allowed to dry at ambient temperature and pressure for 30 minutes. The DESI

spray solvent was a 0.05 mM P-cyclodextrin solution in 50:50 or 80:20 methanol:water. ESI

samples comprised 0.05 mM P-cyclodextrin in either methanol or 50:50 methanol:water with

one guest compound at 1 mg/mL. Maltohexaose (Supelco, Bellefonte, PA), 1 mg/mL in 50:50

methanol:water, was used to test for ESI artifacts. For all ESI experiments, 0.5% formic acid was

incorporated into the solvents, and the P-cyclodextrin solution used as the DESI spray solvent

contained 0.5 mg/mL of NaCl; both additives (ThermoFisher Scientifie, Waltham, MA) were

used to improve current stability and ionization efficiency.

Mass spectra were acquired using an Agilent 6210 MSD Time-of-Flight mass spectrometer

configured for ESI (Agilent Technologies, Inc., Santa Clara, CA). Acquisition of DESI spectra

required an external syringe-pump connected to a home-built DESI source, for which the

desolvation capillary of the TOF was adapted using a specifically designed capillary extender to

enable sampling close to the desorption surface. The capillary extender was not flared as in the

Bruce design,137 but its orifice was three times the diameter of that of the desolvation capillary.

DESI experiments were performed using a spray flow rate of 3 CIL/min and 100 psi head









pressure of nebulizing N2(g). A voltage of 4 kV was applied to the spray solution via an external

210-10R high voltage power supply (Bertan Associates, Hicksville, NY), while the sample

surface was held at ground and the desolvation capillary at 100 V. The spray needle was

positioned 1.5 mm above the deposition surface at a 55o angle from the horizontal. A schematic

of the experimental configuration is included as Figure 5-2.

ESI mass spectra were acquired using a 5 C1L sample loop inj section for sample

introduction, with a flow rate of 0.5 mL/min and a desolvation capillary voltage of 3.5 kV. The

drying gas temperature was 325oC and the fragmentor voltage was held at 175 V for all

experiments. Reference mass correction was enabled throughout the ESI data acquisition to

maximize mass accuracy.

Nuclear Magnetic Resonance Spectroscopy

An Inova NMR spectrometer operating at 500 MHz was used for acquisition of all 1H

spectra (Varian, Inc., Palo Alto, CA). Deuterium oxide (ThermoFisher Scientific, Waltham, MA)

was selected as the solvent and proton chemical shifts were referenced to the HDO signal at

4.780 ppm. Samples consisting of I and 2 mg/mL of a single guest were analyzed alone and in

the presence of P-cyclodextrin (ThermoFisher Scientific, Waltham, MA). A titration approach

covering the concentration range of 0.5-20 mM P-cyclodextrin was employed for each guest to

maximize chances of complex detection. The 2D correlation spectroscopy (COSY) NMR

technique was used for unambiguous proton assignment, and 2D nuclear Overhauser effect

spectroscopy (NOESY) was employed to elucidate the conformation of the

P-cyclodextrin: acetanilide complex for validation of the 1D approach to inclusion complex

confirmation. For a basic overview of basic two dimensional NMR techniques the reader is

directed to a review article by Reynolds and colleagues.160









Theoretical Calculations

Optimized structures for the guest compounds were obtained using Gaussian 03161 with the

HF/6-31G* method and basis set. Corresponding dimensions were measured using Molekel 5.0,

the latest version of a free molecular visualization software package developed by the Swiss

National Supercomputing Center (Manno, Switzerland).

Results and Discussion

Reactive Desorption Electrospray Ionization Receptor Screening

To confirm the applicability of reactive DESI for the analysis of P-cyclodextrin inclusion

compounds, a steroid guest, nortestosterone, was used as a model. Steroid and cyclodextrin

host:guest inclusion complexes have been well-characterized and the nortestosterone was readily

available. The host compound, P-cyclodextrin, was sprayed in methanol:water at the deposited

nortestosterone guest under the conditions previously described. The methanol:water solvent

system was used here because a maj or application of cyclodextrin inclusion is the aqueous

solvation of small molecules. Therefore, it seemed appropriate to demonstrate the utility of this

screening technique using likely real-world conditions. The P-cyclodextrin: nortestosterone 1:1

complex was observed and confirmed using exact mass, showing that complexation on a

timescale suitable for the reactive DESI host:guest interaction can occur. A representative

spectrum is shown in Figure 5-3.

Following this proof-of-principle experiment, the receptor screening analysis was

performed for all twelve potential guest compounds illustrated in Figure 5-1 using the reactive

DESI setup described in the experimental section. The sample surface which was mounted on an

x-y-z-stage was manually moved in the x-direction (see Figure 5-2) for spatial guest selection,

and blank areas between deposited guests were used for blanks. No carryover problems were










experienced. The guests G2, G3, G8, and Gl2 were detected at m/z ratios corresponding to

theoretical 1:1 complexes with the sodiated P-cyclodextrin host. None of the other eight guests

exhibited any evidence of complexation via reactive DESI-TOF-MS. Representative spectra are

shown in Figure 5-4 and summarized results are presented in Table 5-1. Sample preparation took

about ten minutes, but an additional thirty minutes of drying time were required. Instrumental

acquisition time was only twelve minutes including sampling of the blanks.

A concern with this experimental design was that the solvent system could be responsible

for false negative results as several of the guests are only sparingly soluble in water, the primary

component of the electrosprayed droplets of the P-cyclodextrin solution by the time they reach

the deposition surface. One of the maj or benefits of DESI, its ambient nature, was exploited to

rapidly explore this issue. The only alteration necessary to explore the effect of the solvent

system was replacing the original spray solution with one containing a higher percentage of

methanol. This was done at the end of the screen adding fourteen minutes to the acquisition time,

but again only G2, G3, G8, and Gl2 were detected as complexes.

Electrospray Ionization Receptor Screening

For validation of the DESI screen, the twelve guests were screened using ESI, which is an

accepted ionization technique for this type of work, in methanol:water solvent. Under these

conditions, compounds G2, G5, G7, G8, G9 and G10 were observed to form detectable 1:1

complexes, confirmed via accurate mass calculations, while the other five guests showed no

evidence of complexation. Figure 5-5 shows representative mass spectra for analysis of G2

which completed and G4 which did not for comparison, and results of the ESI screen are

summarized in Table 5-2. Similar relative intensities between the sodiated cyclodextrin and the

sodiated complex using both DESI and ESI were noted. Again, solvent system effects were a









concern, so the screen was repeated using only methanol for dissolution. Similar results

evidencing only marginal changes in signal intensity were obtained.

The entire twelve compound screen took fifty-one minutes of acquisition time, including

blank injections, and approximately ten minutes of sample preparation time resulting in an

analysis time of sixty-one minutes excluding data analysis. Changing the solvent system required

preparation of new samples and repetition of the entire screen, doubling the total analysis time.

Although the total analysis time for the DESI screen was significantly longer than for the ESI

analysis, this is solely due to the drying time for the deposited guests which does not contribute

to either labor or instrument time. Appropriate automation and efficient planning in terms of

running screening analyses during drying time for subsequent screens would yield a considerable

improvement in analysis time over the analogous ESI experiment.

Comparison of the results obtained using DESI and ESI highlighted significant disparities,

challenging the validity of the reactive DESI screen. Both techniques resulted in complex

detection for two of the guests, G2 and G8, and four guests, Gl, G4, G6, and G1 1, were not

detected in complex form using either technique. Conflicting results were obtained for the six

remaining guests. Compounds G3 and Gl2 were detected as complexes by DESI only, and the

other four guests were only observed to complex using ESI. Electrospray ionization is known to

be prone to artifacts,143,144 HOnspecific aggregates formed during the electrospray process and

consequently detected as adducts. This is a significant issue in supramolecular receptor screening

where artifact formation can potentially lead to identification of false positive receptor

candidates, and it may provide a potential explanation for the conflicting results. Initially,

maltohexaose, a linear sugar composed of six glucopyranose units, was substituted for the

P-cyclodextrin and another ESI screen performed to identify nonspecific complex formation









following the strategy ofBakhtiar and Hop.144 This strategy, however, yielded inconclusive

information and highlighted the need for a more rigorous validation approach.

Proton Nuclear Magnetic Resonance Spectroscopic Screening

ESI has been shown to be representative of solution-phase equilibria in some

circumstances,76,77 but the relationship between ESI spectra and solution-phase chemistry

remains under scrutiny. A recent study investigating the influence of shrinking electrospray

droplets on chemical equilibria reported observation of a significant effect on the equilibrium for

one fluorescent dye pair, yet no apparent effect on the monomer-dimer equilibrium for a

different fluorescent species.162 COnsequently, the host and guests previously described were

analyzed using solution-phase 1H NMR spectroscopy to determine which of the ionization

techniques yielded the results most consistent with solution-phase behavior. Conditions used for

the NMR screen are delineated in the experimental section. The experiments were conducted in

deuterium oxide, consistent with the composition of the DESI droplets upon interaction with the

surface, as deuterated methanol gives rise to an interferent peak within the spectral range of

interest. Acquisition of each spectrum took about Hyve minutes, so one hour was required for the

entire screen, omitting sample preparation and data analysis which took approximately one more

hour.

Previous studies have shown that the 1H NMR signals corresponding to the P-cyclodextrin

cavity protons exhibit significant upfield shift upon inclusion of a guest molecule into the

cavity.163,164 Proton NMR spectra for P-cyclodextrin and each of the twelve guests were obtained

at the concentration ranges previously described. Proton assignments were made with the aid of

COSY data, and the labeling convention is detailed in Figure 5-6. Representative spectra are

presented as Figure 5-7. NMR spectroscopy revealed evidence, in the form of a spectral shift of










the signals corresponding to the H13', HS5', and to some extent H16' protons of P-cyclodextrin, of

true inclusion complex formation for the guests G2, G3, G8, and Gl2. The H13', HS5', and H16'

protons are the protons which line the surface of the hydrophobic cavity of the toroidal host, and

therefore would be the protons experiencing environmental change in the event of true inclusion

of a guest. No evidence of inclusion of the other eight potential guests was observed. As a

secondary validation technique for the evidence of inclusion complexation between

P-cyclodextrin and the guests under investigation, 2D NOESY was used to determine complex

conformation. Proton assignments for the acetanilide guest are shown in Figure 5-8. An

expanded view of the NOESY data obtained for the P-cyclodextrin: acetanilide complex is

presented as Figure 5-9. NOESY data indicated interaction between the host cavity protons and

the ortho and meta protons on the acetanilide benzene ring, which revealed the complex

conformation illustrated in Figure 5-10. It was expected that the aromatic portion of the

acetanilide would be encapsulated upon complexation as it is the most hydrophobic region of the

guest, so the 2D NOESY derived complex conformation supported the predicted complex, and

validated the simpler 1D approach to inclusion complex determination which was used to screen

the twelve potential guests.

Solution-phase NMR spectroscopic screening yielded identical results to those obtained

via DESI. The four guests detected as 1:1 complexes in the DESI screen were validated as

included guests in solution and the eight remaining guests which were not detected as complexes

via DESI showed no evidence of inclusion in the NMR experiments. Conversely, the ESI screen

proved erroneous for 50% of the guests, resulting in the detection of two false negatives and four

false positives. A summary of results obtained for the three screening methods is presented as

Table 5-3.









The false positives were thought to be attributable to the aforementioned nonspecific

adduct formation commonly associated with ESI. Computational chemistry was used to

investigate this hypothesis and is discussed in detail in the following section. Determination of

the two false negatives was more difficult to explain. Interestingly, the two guests detected using

NMR and DESI but not ESI exhibited the smallest upfield shifts in the NMR experiments.

Assuming the magnitude of the signal shift is related to the extent of complexation, an

assumption for which there is some precedent,165 this would mean that these two guests are the

least thermodynamically favorable. The organic methanol solvent is in competition with the

guests for inclusion in the hydrophobic host cavity, and in ESI is present in higher concentration

at the time of host:guest interaction than in DESI. Consequently, for weakly binding guests, the

methanol could preferentially move into the cavity, preventing inclusion of the weaker guests

during the DESI screen. As previously discussed, this problem would be minimized using

reactive DESI. The droplets interacting with the guest compounds would be predominantly

aqueous, a substantial fraction of the more volatile organic phase having evaporated during the

electrospray process.

The NMR screen showed that reactive DESI is less prone to adduct formation resulting in

the detection of false positives in the screening of supramolecular complexes than ESI.

Additionally, since all four complexes determined via NMR were identified in the DESI screen,

there is no evidence to suggest that complexation during the DESI event is purely kinetically-

driven. In fact, if the extent of NMR signal shift is taken as representative of the extent of

binding, it appears that the DESI screen spectral intensities may even be consistent with relative

binding constants.










Computational Chemistry

To investigate the theory that the four false positive inclusion complexes detected solely

using the ESI screen were nonspecific adducts of guest exterior to the host cavity, structures of

the twelve guests were optimized using the theoretical calculation method previously outlined.

The host compound, P-cyclodextrin, has known dimensions, and the cavity diameter at its widest

point is only 6.5 Angstroms (see Fig. 5-11). Since the potential guests were selected only on the

basis of potentially appropriate molecular weight and functional groups for P-cyclodextrin

inclusion, optimized structures and specific molecular dimensions were desirable to determine

whether the screened compounds were actually viable inclusion guests. The calculations revealed

that eleven of the guests could theoretically adopt conformations suitable for at least partial

encapsulation by the host. Cyclam (G5), however, which has a relatively rigid cyclic structure,

cannot fit into the P-cyclodextrin cavity; its optimized structure, with the corresponding

dimensions, is presented as Figure 5-12. Repulsion between the hydrogen atoms on the inside of

the ring hinders the molecule from adopting a narrower conformation for inclusion. The cyclam

guest exceeds the internal cavity diameter of P-cyclodextrin in two dimensions; therefore, it is

only capable of forming a nonspecific complex exterior to the cavity and complex detection must

be due to nonspecific artifact formation. The P-cyclodextrin:cyclam complex was only detected

in the ESI screen experiment and not during either the DESI or the NMR screens, providing

evidence for the formation of nonspecific adducts resulting in false positives using ESI, but no

evidence to suggest that DESI suffers from a similar drawback in this type of application.

Conclusions

Initially, evidence of complexation between sprayed P-cyclodextrin and deposited

nortestosterone steroid, a member of a known family of steroid guests for the cyclodextrin host,









confirmed that inclusion complexation on a suitable timescale for the DESI heterogeneous ion-

molecule reaction is possible. A subsequent screening experiment for inclusion of a group of

twelve deposited potential guest compounds by the same sprayed host yielded four positive and

eight negative results. The appearance of an ion peak at the exact m/z corresponding to the

protonated or sodiated complex following the interaction was necessary for a positive result,

while absence of a peak at the expected m/z yielded a negative result. The guests observed to

complex during the reactive DESI event were three anilide compounds and caffeine.

An analogous screen using ESI, which has been widely reported for supramolecular

applications, was conducted. Solutions of pre-mixed host and an individual guest were

electrosprayed and again a previously absent peak at the exact m/z of a complex was taken as a

positive result. The ESI screen identified six complexes, only two of which had previously been

observed using DESI-MS. Disparities between the ESI and DESI screens highlighted the need

for further validation, as ESI is known for a tendency to form nonspecific adducts which can

result in false positives in supramolecular applications.

Solution-phase validation via NMR was used to assess which of the two ionization

techniques yielded more representative data. Upfield shift of the chemical shift values of NMR

signals corresponding to the P-cyclodextrin protons known to comprise the surface of the

hydrophobic cavity upon guest addition was used to confirm true inclusion complexation of four

of the potential guest molecules. A lack of shift of the host signals in the presence of guest

indicated that inclusion of the guest into the hydrophobic host cavity was not detected and was

recorded as a negative result. Two dimensional NMR techniques were used to confirm guest

inclusion and conformation of detected complexes. The NMR screen yielded identical positive










and negative results to the DESI screen, confirming the technique as superior to ESI for

supramolecular applications.

The two weaker binders of the four guests observed as inclusion complexes by DESI and

NMR were not detected in complex form using ESI. This suggests that ESI may not be as

sensitive as reactive DESI for supramolecular complex screening. Further investigation is

necessary to corroborate this claim. Computational chemistry helped confirm that ESI is more

prone to the formation of nonspecific artifacts detected as false positives in this type of

application than reactive DESI.

The utility of reactive DESI-MS for rapid supramolecular complex receptor screening has

been demonstrated and the technique has been compared to ESI-MS, an accepted tool for

supramolecular mass spectrometry. Comparison between results obtained via the DESI and ESI

screens and solution-phase NMR data revealed the superiority of DESI for supramolecular

complex guest screening. Reactive DESI has been confirmed as a promising useful tool for

supramolecular mass spectrometry.













Table 5-1. Absolute intensities (x104 COunts) and ion type observed with the DESI-TOF-MS
screen in methanol:water solvent
Guest [M+H]+ [M+Na]+ [CD+M+H]+ [CD+M+Na]+ [CD+G+2H]2+
G1
G2 0.35 6.6 -0.77-
G3 71 6.0 -0.12-
G 4-----
G5 5.0 2.0---
G 6-----
G 7-----
G8 1.7 50 -0.20-
G 9-----
G10 1.4----
G 11 -----
Gl2 8.0 4.0 0.28-













Table 5-2. Absolute intensities (x104 COunts) and ion type observed with the ESI-TOF screen in
methanol:water solvent.
Guest [G+H]+ [G+Na]+ [CD+G+H]+ [CD+G+Na]+ [CD+G+2H]2+
Gl -
G2 120 16 0.22 0.06-
G3 140 50---
G 4-----
G5 180 12 --1.4
G6 -0.09---
G7 8.0 2.0 0.20 0.02-
G8 80 2.3 -0.10-
G9 13 53 -1.8-
G10 140 20 1.5 0.27-
G 11 -----
Gl2 9.7 0.79---











Table 5-3. Potential guests detected as complexes using DESI-MS, ESI-MS, and 1H NMR
spectroscopy.
Guest DESI-MS ESI-MS 1H NMR
G1
G2 Complex detected Complex detected Complex detected
G3 Complex detected -Complex detected
G4 ---
G5 -Complex detected -
G6 ---
G7 -Complex detected -
G8 Complex detected Complex detected Complex detected
G9 -Complex detected -
G10 Complex detected-
G 11 ---
Gl2 Complex detected Complex detected










OI


G3






G7


G2



G6 :


G1


--N--NHH NHHN


G5


HO


"NH NH"
NH


H2N' 'N


HO
G9


G10


G11


Figure 5-1. Structures of the twelve screened guest compounds: G1 is acenaphthalene
(monoisotopic mass 152.0626 Da), G2 is acetanilide (135.0684 Da), G3 is caffeine
(194.0804 Da), G4 is 4-chloro-3 -nitrobenzonitrile (181.9883 Da), G5 is cyclam
(200.2001 Da), G6 is 1,3 -dinitronaphthalene (218.0328 Da), G7 is diphenylglyoxime
(240.0899 Da), G8 is formanilide (121.0528 Da), G9 is guanosine (283.0917 Da),
G10 is L-arginine (174. 1117 Da), G11 is 2-methylnaphthalene (142.0783 Da), and
Gl2 is 4'-nitroacetanilide (180.0535 Da).


G4





G8


G12










Host
solution in N (g) in
from pump

Capillary adapter
\ HV



Deposited potentialToms
guest compounds :':: i ~ aaye



Canted
Movement in x- coil spring
direction for
guest selection
Heated
Deposition
capillary
surface
Figure 5-2. Receptor screen DESI-MS design showing spray head, sample surface, desolvation
capillary, and capillary adapter. The host solution was 0.05 mM P-cyclodextrin
solution in 50:50 methanol:water containing 0.5 mg/mL of NaCl and the twelve
guests Gl-Gl2 were deposited on a sandblasted glass slide for spectral acquisition.


[CDL+H]+ [CD+Nor+H]+
6.5xl04 1135 3680 6500 1409 5770


8 [CD+Nor+Na]+
2' i I 11431.5523




1157.3648 1400 1420 m/z 14402~---~:4





[CD+Nor+H] +
1409.5770
1100 1200 mz 1300 1400
Figure 5-3. Reactive DESI-TOF spectrum of P-cyclodextrin sprayed onto a deposited sample of
nortestosterone. The inset shows a close-up view of the mass range of interest.





[CD 1157.3671


1 610 0


~~J\,


1295 iz, 1300


[CD+C;2 iN3]+
1292.4274


b
1.h


[C'I+Nul*`
1157.3671


175
No evidence of [CD+G7? I N1]"
i Ii 1Theoretical mh: 1397.4489






1390 1395 miz 1400


4100








1?7.


[C D G ;5 INa i









II


m:I I121?


1300


1400


1100 1200


Figure 5-4. Reactive DESI-TOF mass spectra showing data for the guests a) G2, b) G7, and c)
G8. Insets show close-up views of the complex ion mass ranges of interest.


785 1'"1 7



S1291.42~78


[CD)+NaCl+Nal*
1215.3203


ICD+NaCl+Na]+
1215.3203





























[CD+Na]+
1157.3588


[CD+Na]+
1157.3588


2.8x104









1


[CD+NaOOCH+Na] '[CD G2 H]'
1225.3463 1270.4389


[CD+H]+
134.3767


No evidence of [CD+G4+Na]+
Theoretical m/z: 1339.3473


[CD+NaOOCH+Na]
1225.3463


1300


1100


Figure 5-5. Electrospray ionization TOF mass spectra for the P-cyclodextrin(CD) guest screen in
methanol shown for potential guest compounds a) G2 and b) G4 which yielded
positive and negative complexation data, respectively.


HI'


Figure 5-6. Structure of the glucopyranose monomer of P-cyclodextrin showing the labels used
to represent the protons observed via NMR spectroscopy.


[CD+H]f
1134.3767
















































4.00 3.90 3.80 6;, ppm 3.70 3.60 3.50

Figure 5-7. Proton NMR spectra showing the region 3.5-4 ppm chemical shift for a) lone
P-cyclodextrin and b) P-cyclodextrin in the presence of acetanilide (G2). The signals
corresponding to H3', H5', and to some extent H6', exhibit an upfield shift consistent
with inclusion of a guest upon the addition of acetanilide.







134












7.18 N
2.15
7,41 7.45Cz

Figure 5-8. Structure of the acetanilide guest, G2, showing the observed chemical shift values for
the NMR spectral peaks corresponding to the stable protons.


In Oln ~I ~t~
O 0\00 ~ ~ln
In rclrcl rcl ~~I


5.0 4.X 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0
P-Cyclodextrin, ppm
Figure 5-9. Nuclear Overhauser effect spectrum of P-cyclodextrin in the presence of acetanilide.
Signal at the intersection of a line corresponding to the acetanilide with a line
corresponding to the host indicates correlation. The signal diameter is indicative of
the strength of the through-space interactions.


Figure 5-10. Conformation of the P-cyclodextrin: acetanilide inclusion complex derived using 2D
NOESY.











1 6.5a~
i


Figure 5-11i. Representation of the toroidal P-cyclodextrin host showing cavity diameter.

6. 870 1 3


Figure 5-12. Optimized structure and corresponding molecular dimensions of cyclam, the G5
guest which was observed to complex using ESI supramolecular complex screening
only.











CHAPTER 6
CONCLUSIONS AND FUTURE DIRECTIONS

Mass spectrometry, currently experiencing an explosion of applications, has become an

essential tool in the development of the field of supramolecular chemistry. Its sensitivity,

specificity, and ability to obtain information about the inherent supramolecular chemistry of

systems in the absence of solvent interference make it a powerful analytical technique with the

potential to fuel rapid advances in this area. The research presented here was conducted to

contribute to the evolution of supramolecular mass spectrometry as a technique which can be

diversified to address very different needs arising from the growth of the multidisciplinary field

of supramolecular chemistry. The maj or unifying theme is that specific instrumental

characteristics and capabilities can be exploited to yield useful information beyond elemental

composition or structural elucidation, with a focus on expanding the scope of supramolecular

mass spectrometry.

Despite advances in mass spectrometric instrumentation and software for instrumental

control and data analysis, the efficient incorporation of mass spectrometry into many labs is

limited by the tendency to use mass spectrometers as workhorses where generation of large

amounts of data is perceived as more desirable than the acquisition of useful and meaningful

information. For some applications, this may be an appropriate use of the techniques comprising

mass spectrometry as a whole, but in an economically-driven society, this poses several

problems. Primarily, valuable resources of instrument and scientist time can be wasted through

poor experimental design. Additional analysis time for superfluous tandem mass spectrometry

stages, and time spent wading through unnecessary data acquired due to inefficient planning are

a few common examples of laboratory waste. Additional concerns are data handling and storage.

Generation of large amounts of data increases the time and resources needed to extract useful









information, but also creates pressure in terms of hardware and software laboratory requirements.

Many labs, particularly those in biomedical areas, would benefit significantly in terms of

efficiency and productivity by employing a trained and experienced instrumentalist for

experimental design and efficient utilization of complex analytical instrumentation.

This research was initially directed toward the characterization of novel designed synthetic

receptors for alkali-metal cations based on naturally-occurring phloroglucinol using accepted

supramolecular mass spectrometric techniques. Relative binding constants were determined for

the homodimers of two phloroglucinol derivatives bound by Li Na K and NH4' USing an

SCID-ESI dissociation curve approach. The preferential binding order for

2,4,6-tribenzoylphloroglucinol (phloro 1) was determined to be Li' Na > K >NH4' and for

2-4-6-(3,5 -dimethyl)trib enzoylphl oroglucinol (phloro 2) was revealed as Na > K > Li >NH4 -

This size-dependent binding for phloro 1 but not phloro 2 supported the hypothesis that phloro 1

should be capable of forming a cage-type dimer encapsulating an alkali metal cation in the

intermolecular cavity, while the additional methyl groups on the benzoyl substituents of phloro 2

should sterically hinder that dimeric conformation. No direct conformational information was

obtained via mass spectrometry, so UV/vis absorbance spectroscopy was used to probe dimeric

conformation in solution. Chromic shifts corresponding to dimerization provided further

evidence supporting the hypothesis. In this proj ect, mass spectrometry and solution-phase optical

spectroscopy yielded complementary information, but the characterization might have been

achievable solely via mass spectrometric techniques, had ion mobility spectrometry (IMS) been

available for determination of the dimeric cross-sectional areas.

Characterization of the phloroglucinol derivatives as designed xn-receptors benefits

supramolecular chemistry in several ways. A significant step towards providing a new synthetic










receptor for encapsulation of alkali metal cations, which has implications in applications like

cation scavenging in waste cleanup and active transport mimicry, has been made. Additionally,

the effect of the addition of small substituents, like methyl groups, upon the nature of

noncovalent xn-system interactions has been shown to be dramatic. This fact highlights the need

for more in-depth investigations into the effects on chemical behavior of substituent modification

of arene systems, and could ultimately impact synthetic receptor design strategies based on

molecular recognition principles. On a more general level, the complementary nature of solution-

phase optical spectroscopic techniques and mass spectrometry was demonstrated for

supramolecular applications.

A useful addition to the work presented here would be the implementation of an IMS study

to explore the dimeric conformations. Following measurement of the cross-sectional area of the

homodimers of phloro 1 and phloro 2 in the presence of each of the four cations, comparison

between data obtained for each host in the presence of the same guest should enable

determination of the dimeric conformations. However, a vital future step for this area of research

is expansion of the study to a larger group of phloroglucinol derivative hosts substituted with

different heteratoms and functional groups. Comparison of dimeric stability using the SCID

dissociation curve approach, for a much greater number of host compounds could significantly

benefit the Hield of synthetic design based on supramolecular recognition principles. The group

would have to be large enough to allow the determination of statistically relevant patterns of

behavior related to specific types of substitution. If patterns and trends in relative complex

stability were observed to correlate with substituent characteristics, this could significantly

impact molecular-recognition based designed chemical synthesis, resulting in simplification of

the therapeutic drug design process.









Dissociation pathways were proposed for ESI-CID-MS analysis of the cation-bound

phloroglucinol derivative dimers. With the aid of the simple 2-hydroxybenzophenone compound,

a mechanism for the initial dissociation step was derived, and the more complicated C3

phloroglucinol derivatives were observed to adhere to this mechanism. The ammonium adducted

phloroglucinol derivatives demonstrated slight differences in fragmentation behavior from that

observed with the other cations employed. Ion trap mass spectrometry was used for unambiguous

determination of the precursor-product ion relationships, and SCID tandem mass spectrometry

using one of two high-resolution mass analyzers provided accurate mass measurements for

unambiguous determination of elemental composition. The two useful instrumental capabilities

of tandem mass spectrometry and high-resolution mass analysis were incorporated into the

analysis to maximize the usefulness of the mass spectral data acquired. The growing

pharmaceutical relevance of the phloroglucinol derivative family provided a need for this

information, as LC-MS and LC-MS/MS analytical methods for determination of this type of

compound in various matrices are under rapid development. The most common interface

between LC and MS is ESI, and alkali-metal adducts are frequently observed via ESI, so

fragmentation information for alkali-metal adducts is vital. The wealth of applications for

phloroglucinol derivatives outlined in Chapter 3 renders dissociation pathway information

specific to alkali-metal adducts of phloroglucinol derivatives of substantial significance. The

contributions of a proposed mechanism for the initial fragmentation step and dissociation

pathways for the alkali-metal cation-bound dimers and monomeric adducts have been made, but

future application-specific method development could benefit from further insight.

Now that the first mechanism for the initial dissociation of alkali-metal cation-adducted

phloroglucinol derivatives has been proposed, the next logical step would involve a detailed









mechanistic study. Exchange of one or all of the 1H atoms of the hydroxyl groups with 2H

followed by an analogous fragmentation study would help to clarify the location of the hydrogen

which is abstracted as part of the loss of the benzene or dimethylated benzene ring from the

monomeric phloroglucinol derivative adducts. Additionally, in a separate study, strategic

replacement of successive 12C atoms with 13C atoms could be used to investigate the structural

reconfiguration observed via carbon dioxide neutral loss from the monomeric sodium and

lithium cation adducts of the two host molecules. Both these experiments would provide detailed

information about the dissociation of phloroglucinol derivatives which could be valuable for

LC-MS/MS method development for the quantitative and qualitative analysis of this compound

class.

An unrelated experiment could help to determine one missing and vital piece of

information which could potentially yield insight into the cation-n: interactions which are

involved in ESI adduction by metal cations. The location of the cation, although clearly in the

core phloroglucinol region, was not determined by the studies comprising this body of work, but

a possible strategy to resolve this issue has been devised. A combination of computational

chemistry and gas-phase infrared ion spectroscopy could yield the desired information.

Theoretical geometry optimization of the phloroglucinol derivative hosts with an alkali-metal

cation in one of a select few positions would enable determination of the adduct conformations

corresponding to the lowest energy structures. Theoretical vibrational spectra for each structure

could then be obtained. Gas-phase ion spectroscopy using an infrared laser for multiple photon

dissociation of the electrosprayed alkali metal cation-adducted phloroglucinol derivatives would

provide experimental vibrational spectra. Comparison between the theoretical and experimental









vibrational spectra could confirm the location of the cation. This is a proj ect for which our group

has both the resources and the expertise.

Prior to applying the recently-developed ambient ionization technique DESI to the analysis

of supramolecular inclusion complexes, a home-built DESI source was interfaced with an

Agilent TOF-MS instrument. The source was originally designed for a Bruker FTICR-MS

instrument, so several modifications were necessary to accommodate the unusual geometry of

the Agilent source housing. Commercial DESI sources have yet to be successfully interfaced

with the Agilent TOF instruments, so this work provides a basic approach for possessors of

Agilent TOF mass spectrometers aiming to incorporate the DESI technique into their work. The

superior scan rate of TOF-MS over FTICR-MS was observed to be useful for DESI parameter

optimization, demonstrating a significant advantage of TOF mass analyzers over FTICR mass

analyzers for DESI analyses. Successful configuration of the DESI-TOF-MS system was

demonstrated using Rhodamine 6G and the protein cytochrome c.

The next step in characterization of the DESI-TOF-MS configuration described in this

dissertation is the demonstration of its performance for a diverse range of compound classes. An

optimization study resulting in the determination of optimal conditions and surface chemistry for

structurally-related compounds such as differentially substituted benzenes or carbonyls could

provide useful insight into the effect of different functional groups on the DESI process, and

would aid the wider implementation of the DESI technique. Additionally, the high mass range

capabilities of the TOF-MS make it very appropriate for the study of large organometallics,

which have not been extensively analyzed by DESI, so a future collaborative proj ect with a

prolific inorganic synthetic chemist could prove extremely successful. Many large metal









complexes degrade in the presence of acid and DESI is an ionization technique that does not

necessarily require acid addition.

The potential of the DESI source interfaced with the FTICR-MS instrument was not

thoroughly explored, and recent successful configuration of DESI-FTICR-MS by several groups

suggests that the challenges encountered are not insurmountable. The system should be revisited

and the optimization procedure exhaustively performed using a standard that is not as rapidly

ablated as Rhodamine 6G. During the cyclodextrin work, it was observed that both caffeine and

acetanilide yield a relatively long-lived and stable DESI signal; therefore, they could prove to be

appropriate analytes for this purpose. Successful integration of the DESI source with the

University of Florida FTICR-MS could result in some very exciting work involving infrared

dissociation of trapped ions produced using the DESI technique. To date, neither DESI followed

by infrared multiple photon dissociation nor DESI followed by gas-phase ion spectroscopy have

been reported. Since DESI-FTICR-MS is very desirable for proteomics applications, protein

conformation studies using this setup seem a logical and attainable aim.

The reactive DESI technique was subsequently used for the development of a rapid

screening technology for supramolecular receptors for inclusion by P-cyclodextrin hosts.

Following deposition of several potential guest compounds onto a desorption surface, successful

pickup of some of the guests by the host, observed via detection of an ion associated with the

specific host:guest complexes, during the DESI event was achieved. An analogous screen using

electrospray ionization resulted in the detection of complexes of different guests to those

observed to complex via DESI, and illustrated the need for solution-phase validation of the DESI

technique for this application. Proton NMR spectroscopic techniques yielded data corresponding

to the DESI results and invalidating the ESI results, and computational chemistry showed that









some of the complexes detected via ESI could only be nonspecific adducts. Reactive DESI was

confirmed as a valuable analytical technique for supramolecular screening, providing solution-

phase-consistent data, and was demonstrated as superior to ESI for the screening of guests for

P-cyclodextrin inclusion complexes.

Some additional information would greatly enhance the diversity of potential analytical

applications for this technique. Consequently, some suggestions for future experiments are

delineated here. The molecular recognition interaction between sprayed P-cyclodextrin and

nortestosterone was initially employed to demonstrate that inclusion complexation can occur on

the DESI timescale. An attempt at inclusion complexation of several other hormones was made

concurrently, but proved inconclusive. The hormones estriol, estrone, dehydrotestosterone,

testosterone, and epitestosterone were deposited, but no evidence of desorption was observed. It

remains unclear, however, whether the age of the compounds, which had been stored for several

years under unknown conditions, was responsible for the lack of signal obtained with these

hormones. The fact that no uncomplexed signal was observed for any of these analytes suggests

the possibility that unfavorable storage conditions could have led to degradation. It would be

extremely useful, however, to determine if this were the case using another ionization technique.

A comprehensive analysis of the inclusion complexation of new samples of these

hormones, particularly the testosterone, using a variety of surface chemistries and geometric

configurations would be a valuable addition to the data presented here. To increase the chances

of successful complexation, a derivatized P-cyclodextrin such as hydroxypropyl-P-cyclodextrin

could be used as the spray reagent. Comparison of the complexation behavior of the related guest

molecules would be extremely useful both to determination of the scope of this specific

technique and to characterization of the capabilities and limitations of reactive DESI in general.









Real-world matrix effects on reactive DESI for supramolecular complexes have yet to be

explored. Development of this type of analytical technique in the area of metabolite screening

and toxicological applications would require molecular recognition interactions during the DESI

process to be robust in biological matrices. A very preliminary experiment to gauge the

adaptability of the technology to this type of work would be a simple screen using P-cyclodextrin

sprayed at deposited spots of various compositions. Since nortestosterone is known to complex

under reactive DESI conditions, solutions of urine, blood plasma, and serum spiked with

2 mg/mL nortestosterone would be appropriate for initial tests. The previ ously-determined

water:methanol testosterone solution would be a necessary control to ensure favorable

conditions. Signal corresponding to therapeutic drugs has been obtained using traditional DESI

for all three of the suggested matrices, as noted in Chapter 4.

The supramolecular complex screening work presented here has evident potential in the

development of high-throughput screening experiments for lead optimization in drug discovery

applications. The demonstrated benefits, and the simplicity, rapidity, and potential for

automation, confer upon the described technology inherent analytical utility in this area. Prior to

expansion into a marketable technique for this type of application, however, several important

steps are necessary.

In order to demonstrate the applicability of the technique to real-world receptor substrate

chemistry, molecular recognition interactions between a known receptor and substrate must be

determined to be specific. An experiment incorporating the receptor region of some well-

characterized protein into the spray for interaction with two deposited substrates, one specific

substrate for the receptor, and one similar substrate would address this issue. It would also









demonstrate the utility of the technique for systems beyond the small molecule inclusion

complex model system, which is essential to its development in biomedical applications.

Following this, the logical approach would be a systematic study of one receptor with a

family of known substrates. Subsequently, potential for different types of receptor molecules

would have to be investigated.

An additional step involves the determination of the effect of surface chemistry upon the

molecular recognition interactions exploited in reactive DESI. To design and implement a screen

that would be competitive with a 96-well plate optical spectroscopic assay, for example, the

design of a specialized deposition surface would have to be carefully considered. An experiment

observing complexation between sprayed P-cyclodextrin and acetanilide on a variety of different

surfaces including Teflon, polymethylmethacrylate, and porous silicon is essential to enable the

future fabrication of a miniature sample holder along the lines of a MALDI target or desorption

ionization on porous silicon (DIOS) chip. These surfaces have the advantage of amenability to

the development of a miniature tray with shallow wells drilled or etched onto the surface. The

P-cyclodextrin and acetanilide system is suggested as the complex system which resulted in the

most intense and stable complex ion signal.

Through careful experimental design and appropriate utilization of different types of mass

spectrometric instrumentation, contributions to the development of advanced supramolecular

mass spectrometry have been made. Several specific contributions have been detailed and an

attempt to define their real-world relevance has been undertaken. The overall goal of this

dissertation, however, was promotion of the idea of maximizing the usefulness and efficiency of

mass spectrometry as an analytical technique for diverse supramolecular chemistry applications,

through the rational implementation of appropriate and complementary instrumental capabilities.










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BIOGRAPHICAL SKETCH

Joanna Barbara was born and grew up in England, but moved to the United States with her

husband and then 1-year old daughter to undertake an undergraduate degree in chemistry at the

University of Florida in the spring of 2001. Graduating with her B.S. in 2003, and following the

birth of her son, she remained at UF to continue her studies in graduate school, pursuing

concurrently a non-thesis M.S and a Ph.D. in analytical chemistry. Following coursework

completion, she began research in the area of supramolecular mass spectrometry under the joint

direction of Dr. John Eyler and Dr. David Powell, and worked as a research assistant in the Mass

Spectrometry Service Lab throughout that period. Upon graduation, Joanna will enter the

industrial workforce in a metabolism research position with XenoTech in Lenexa, Kansas.





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ANALYSIS OF SELF-ASSEMBLING COMPLEXES VIA SUPRAMOLECULAR MASS SPECTROMETRY By JOANNA E. BARBARA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

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2007 Joanna E. Barbara 2

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To Oliver, Ella, and Jake 3

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ACKNOWLEDGMENTS I must acknowledge the many colleagues and friends who have been instrumental in my graduate school journey. First, I thank my advisors, Dr. John Eyler and Dr. David Powell, for their consistent support and dire ction. John Eyler has provided me with a model example of the successful combination of family and science, and given me academic and personal freedom to pursue the projects I enjoyed and balance my child ren with my work. Always a source of advice and scientific insight, I truly appreciated all th e sensible and thoughtful gui dance I received from him. I acknowledge David Powell both as an adviso r and as a friend. He shaped the project I undertook as the basis of my disse rtation research and provided me with the necessary training and resources to accomplish my goals. In addition, I thank him for the patience with which he taught me instrumentation skills and introduced me to the world of tools and turbopumps; my newly discovered ability to change a tire I attribute solely to him. As director of the service lab where I have worked for the last two years, he has been an appreci ative and approachable supervisor, and I have thoroughly enjoyed my time in his lab. Dr. Kathryn Williams, my mentor throughout my undergraduate and graduate career, is acknowledged for her teaching talent and constant support. I thank her for telling me to go to graduate school in the first place, and for s upporting me as a scientist throughout my pregnancy and the birth of my son. Dr. Williams allowed me access to all the instrumentation in her teaching lab and consequently made several of the studies presented in this dissertation possible. Sincere gratitude is extended to her and the ot her members of my committee, Dr. Richard Yost and Dr. Peggy Borum, for the commitment of thei r time and energy. I also wish to acknowledge Dr. Tim Garrett, who passed on to me his enthus iasm for mass spectrometry as my teaching assistant several years ago. 4

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Dr. Ben Smith is acknowledged as graduate ad visor for his advice and guidance, but also as a scientist for discussion concerning optical spectroscopy. I thank his a ssistant Ms. Lori Clark for patience, support, and admi nistrative problem-solving. Dr. Ronald Castellano is acknowledged for de signing the phloroglucinol derivatives and furnishing his knowledge and insight as a synthetic chemist. I also thank his former student, Dr. Andrew Lampkins, for helping introduce me to the field of supramolecular chemistry and for sample synthesis. I acknowledge Dr. Jodie Johnson for teaching me a great deal about fundamental mass spectral interpretation, structural elucidation, and fragment assi gnment. Data analysis would have been a much more time-consuming process without his guidance. I wish to thank Brian Smith for engineering the DESI source and for al l the consultation and m achining expertise that he contributed to its repeated modification. Si ncere thanks go to Joe Shalosky for practical advice and problem-solving associat ed with the instrumentation. I thank Dr. Cynthia Cole, former director of the University of Florida Racing Lab, Patrick Russell, and Dr. Keith Zientek who provided access to the ion trap used fo r the determination of the phloroglucinol derivative dissociation pathwa ys. Steve Miles and Larry Hartley are thanked for electronics support, and the IT shop staff, pa rticularly Joe Carusone, is also acknowledged. The members of the Eyler and Powell groups, past and present, are thanked for their support and helpful suggestions. My cheering squa d and sounding boards, Jonathon, Lani, Julia, and Soledad, are gratefully acknowledged for listening and advising on all subjects. The many unnamed friends who have helped me make it to this point are also thanked. Finally, I acknowledge my husband, Oliver, and my children, Ella and Jake, all of whom have sacrificed a great deal of time with me over the past years. I want to thank Oliver for his 5

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unfailing support and encouragement, and for taking over my responsibilities when the workload became too much for me, despite his own responsib ilities as a student and a parent. My precious babies are acknowledged as my motivation to succeed. 6

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ........10 LIST OF FIGURES.......................................................................................................................11 ABSTRACT...................................................................................................................................14 CHAPTER 1 INTRODUCTION................................................................................................................. .16 Supramolecular Chemistry.....................................................................................................18 Concepts..........................................................................................................................18 Types of Noncovalent Bonds..........................................................................................20 Ion-ion interactions..................................................................................................20 Ion-dipole interactions..............................................................................................21 Dipole-dipole interactions........................................................................................21 Hydrogen bonds.......................................................................................................21 Cationinteractions................................................................................................21 -Stacking interactions.............................................................................................16 Biological and Phys ical Significance..............................................................................22 Mass Spectrometric Approaches............................................................................................23 High-Resolution Mass Spectrometry..............................................................................26 Concepts...................................................................................................................26 Instrumentation.........................................................................................................27 Tandem Mass Spectrometry............................................................................................29 Supramolecular Mass Spectrometry.......................................................................................31 Gas-Phase Supramolecular Chemistry............................................................................31 Survey of Recent Literature.............................................................................................33 2 CHARACTERIZING NONCOVALENT DIMERIZATION BEHAVIOR OF DESIGNED PHLOROGLUCINOL DERIVATIVES USING ELECTROSPRAY IONIZATION HIGH RESOLU TION MASS SPECTROMETRY.......................................40 Introduction................................................................................................................... ..........40 Experimental................................................................................................................... ........42 Sample Preparation..........................................................................................................42 Mass Spectrometry..........................................................................................................43 Ultraviolet/Visible Absorbance Spectroscopy................................................................44 Results and Discussion......................................................................................................... ..45 Electrospray Ionization Mass Spectrometr y (ESI-MS) for Dimer Detection.................45 Traditional Competitive Binding Approach....................................................................45 7

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Dissociation Curve Approach..........................................................................................46 Heterodimer Studies........................................................................................................48 Ultraviolet/Visible (UV/Vi s) Absorbance Spectroscopy................................................50 Conclusions.............................................................................................................................53 3 ELUCIDATING THE DISSOCIATION MECHANISM OF NOVEL PHLOROGLUCINOL DERIVATIVES................................................................................65 Introduction................................................................................................................... ..........65 Experimental................................................................................................................... ........67 Sample Preparation..........................................................................................................67 Mass Spectrometry..........................................................................................................68 Results and Discussion......................................................................................................... ..68 Electrospray Ionization Source-Skimme r Collisionally Induced Dissociation (SCID) Justification.....................................................................................................68 2-Hydroxybenzophenone Experiments...........................................................................69 Protonated Molecule Dissociation Pathways..................................................................70 Alkali-Metal Cation-bound Dimer and Adducted Monomer Dissociation.....................71 Conclusions.............................................................................................................................74 4 CONFIGURATION OF A HOME-BUILT DESORPTION ELECTROSPRAY IONIZATION SOURCE WITH A CO MMERCIAL TIME-OF-FLIGHT MASS ANALYZER....................................................................................................................... ....91 Introduction................................................................................................................... ..........91 Experimental................................................................................................................... ........94 Desorption Electrospray Ioni zation (DESI) Source Design............................................94 Mass Spectrometry..........................................................................................................94 Sample Preparation..........................................................................................................95 Results and Discussion......................................................................................................... ..95 Configuration of DESI Source with Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (FTICR-MS).................................................................................95 Configuration of DESI Source with Time -of-Flight Mass Spectrometer (TOF-MS).....97 Important Optimization Considerations........................................................................100 Conclusions...........................................................................................................................102 5 REACTIVE DESORPTION ELECTRO SPRAY IONIZATION FOR RAPID SCREENING OF GUESTS FOR SUPRAMO LECULAR INCLUSION COMPLEXES...112 Introduction................................................................................................................... ........112 Experimental................................................................................................................... ......115 Sample Preparation........................................................................................................115 Mass Spectrometry........................................................................................................116 Nuclear Magnetic Resonance (NMR) Spectroscopy.....................................................117 Theoretical Calculations................................................................................................118 Results and Discussion......................................................................................................... 118 Reactive DESI Receptor Screening...............................................................................118 8

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Electrospray Ionization Receptor Screening.................................................................119 Proton NMR Spectroscopic Screening..........................................................................121 Computational Chemistry..............................................................................................124 Conclusions...........................................................................................................................124 6 CONCLUSIONS AND FUTURE DIRECTIONS...............................................................137 LIST OF REFERENCES.............................................................................................................147 BIOGRAPHICAL SKETCH.......................................................................................................156 9

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LIST OF TABLES Table page 2-1 Phloro 1 and 2 SCID-FTICR-MS dissociation curve VC50 values....................................55 2-2 Alkali-metal cationic radii................................................................................................ .55 2-3 Phloro 1 and 2 SCID-TOF-MS dissociation curve VC50 values........................................55 5-1 Intensities and ion type observed with the DESI-TOF-MS screen in methanol:water....127 5-2 Intensities and ion type observed with the ESI-TOF-MS screen in methanol:water.......128 5-3 Complexes detected using DESI-MS, ESI-MS, and 1H NMR spectroscopy..................129 10

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LIST OF FIGURES Figure page 1-1 Representation of a cav itate and a clathrate.......................................................................38 1-2 Two possible orientations of dipole-dipole interactions....................................................38 1-3 Representation of cationinteraction...............................................................................38 1-4 Generalized configuration of an electrospray source.........................................................39 1-5 Quantities used to calculate resolution..............................................................................39 2-1 Phloroglucinol structure................................................................................................... ..56 2-2 Structures of the phloroglucinol derivatives......................................................................56 2-3 Electrospray ionization mass spectra of phloro 1 forming cation-bound dimers..............56 2-4 Electrospray ionization spectra of showi ng the presence of adventitious sodium.............57 2-5 Phloro 1 dimer dissociation curv es obtained using SCID-FTICR-MS..............................58 2-6 Phloro 2 dimer dissociation curv es 2 obtained using SCID-FTICR-MS...........................59 2-7 Dissociation curves for all cation-bound dimers obtained using SCID-TOF-MS.............60 2-8 Spectrum obtained using ESI-TOF -MS of homo and heterodimers..................................61 2-9 Dissociation curves for the cation-bound heterodimers.....................................................61 2-10 Absorption spectrum for phloro 1 in methanol..................................................................62 2-11 Beers Law plot for phloro 1.............................................................................................. 62 2-12 Ultraviolet/visible abso rption spectra for a range of phloro concentrations......................63 2-13 Ultaviolet/visible absorption and derivative spectra for phloro 1 and 2............................64 2-14 Bar charts showing measured max chromic shift for phloro 1 and 2................................64 3-1 Structure of 2-hydroxybenzophenone................................................................................77 3-2 Dissociation of phloro 1 cation-boun d dimers of to monomeric adducts..........................77 3-3 Dissociation of phloro 2 cation-boun d dimers of to monomeric adducts..........................78 3-4 Proposed fragmentation pathway for 2-hydroxybenzophenone........................................79 11

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3-5 Proposed initial dissociation mechanism for 2-hydroxybenzophenone.............................80 3-6 Phloro 1 protonated molecule proposed fragmentation pathway......................................81 3-7 Phloro 2 protonated molecule proposed fragmentation pathway......................................82 3-8 Lithium cation-bound dimer of phloro 1 proposed fragmentation pathway......................83 3-9 Lithium cation-bound dimer of phloro 2 proposed fragmentation pathway......................84 3-10 Sodium cation-bound dimer of phloro 1 proposed fragmentation pathway......................85 3-11 Sodium cation-bound dimer of phloro 2 proposed fragmentation pathway......................86 3-12 Potassium cation-bound dimer of phloro 1 proposed fragmentation pathway..................87 3-13 Potassium cation-bound dimer of phloro 2 proposed fragmentation pathway..................88 3-14 Ammonium cation-bound dimer of phloro 1 proposed fragmentation pathway...............89 3-15 Ammonium cation-bound dimer of phloro 2 proposed fragmentation pathway...............90 4-1 Schematic of the overhead view of the original home-built DESI source.......................104 4-2 Desorption electrospray ionization source interfa ced with the FTICR-MS....................105 4-3 Close-up view of DESI sour ce configured with FTICR-MS...........................................105 4-4 Structure of the Rhoda mine 6G preformed ion................................................................106 4-5 First DESI-FTICR-MS spectrum showing Rhodamine 6G.............................................106 4-6 Close-up view of the DESI-FTICR-MS setup.................................................................107 4-7 Close-up view of the DESI s ource interfaced with the TOF-MS....................................108 4-8 Side view of the DESI-TOF-MS configuration...............................................................109 4-9 Representation of the capillary ex tender for the DESI-TOF interface............................109 4-10 Mass spectrum of Rhodamine 6G obtained using DESI-TOF-MS.................................110 4-11 Positive-mode DESI-TOF mass spectrum of cytochrome C...........................................110 4-12 Schematic representation of the DESI spray head and inlet orifice.................................111 4-13 Representative optimization data for the DESI-TOF-MS configuration.........................111 5-1. Structures of the twelve screened guest compounds........................................................130 12

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5-2 Desorption electrospray ioniza tion MS receptor screen design.......................................131 5-3 Reactive DESI-TOF spectrum of cyclodextrin sprayed onto nortestosterone...............131 5-4 Reactive DESI-TOF mass spectra showing data for several guests................................132 5-5 Representative ESI-TOF mass spectra for the cyclodextrin(CD) screen....................133 5-6 Structure of the glucopyranose monomer of -cyclodextrin with chemical shifts..........133 5-7 Proton NMR spectra showing the region 3.5-4 ppm chemical shift................................134 5-8 Structure of the acetanilide guest showing chemical shift...............................................135 5-9 Nuclear Overhauser effect spectrum of -cyclodextrin with acetanilide........................135 5-10 Conformation of the -cyclodextrin:acetanilide inclusion complex...............................135 5-11 Representation of the toroidal -cyclodextrin host showi ng cavity diameter.................136 5-12 Optimized structure and mol ecular dimensions of cyclam..............................................136 13

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Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ANALYSIS OF SELF-ASSEMBLING COMPLEXES VIA SUPRAMOLECULAR MASS SPECTROMETRY By Joanna E. Barbara December 2007 Chair: John R. Eyler Major: Chemistry Self-assembling supramolecular complexes are stable configurations of two or more molecules stabilized by noncovalent intermolecula r interactions, which ag gregate independently according to molecular recognition pr inciples. Noncovalent, e.g., cationand hydrogenbonding, interactions make important contributio ns to the structure and function of many biomolecules. They are vital to protein folding an d stabilization and so to substrate specificity and enzyme action. Host-guest inclusion complexes stabilized by these inte ractions are used in areas such as enzyme mimicry, catalysis, and therapeutic drug development and delivery. Modeling and analysis of these noncovalent interactions are esse ntial to the development of selective synthetic hosts. Thorough analytical characteri zation of diverse supramolecular systems is necessary to contribute to the wealth of data required to gain a better understanding of the inherent chemical behavior involved in molecular rec ognition and noncovalent complex formation. Mass spectrometry, as a gas-phase analyt ical technique, has the ability to provide vital information concerning supramolecular chemistry in the absence of interference from a solvent shell. The advancement of supramolecular ma ss spectrometry was the major goal of this research. 14

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A pair of designed synthetic receptors for al kali-metal cations based on the natural product 1,3,5-trihydroxybenzene (phloroglucinol) was thoroughl y characterized in the gas phase using advanced supramolecular mass spectrometric tec hniques. Following additional solution-phase characterization using absorbance spectroscopy in th e ultraviolet region, it was determined that the mass spectrometric approach and the optical sp ectroscopic approach combined to yield useful complementary characterization information. Following the configuration of a home-built so urce with a time-of-flight mass analyzer, desorption electrospray ionization (DESI) was developed and validated as a useful novel analytical tool for this area of application, through the desi gn and implementation of a rapid screening experiment for potential guest compounds for supramolecular encapsulation by a cyclodextrin host. Comparison experiments, using nuclear magnetic resonance spectroscopy as the standard solution-phase valida tion technique, revealed that DESI is a superior ionization technique to the commonly-employed electrospray ionization (ESI) for this type of work. DESI is not as prone to the detection of false-pos itive nonspecific complexes resulting from the formation of artifacts during the electrospray process. Thus a useful addition to the supramolecular mass spectrometry toolkit has been contributed. 15

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CHAPTER 1 INTRODUCTION The term supramolecular chemistry was fi rst used by Jean-Marie Lehn in 1978 to encompass all of the previously divided ar eas breached by his groundbreaking work exploring the noncovalent chemistry of macropolycycles.1 He has defined it as the chemistry of the noncovalent bond,2 briefly comprising intermolecular binding, catalysis, molecular recognition, self-assembly, directed molecular design, and self-replication phenomena.3 Over the preceding decade, various alkali and alkaline-earth cations had been observed to form stable complexes with macrocyclic ligands, the most famous exam ple being the crown ether family discovered by Charles Pedersen,4 and Lehn had been working on synthe sizing macropolycycles with designed molecular recognition properties.1 As more supramolecular species were synthesized and their complexation characteristics explored, an entire disc ipline evolved. Lehn, Pe dersen, and Donald Cram, a synthetic chemist specializing in cycl ophane chemistry, were s ubsequently awarded the 1987 Nobel Prize in Chemistry for work resulting in creation of the field.5 Today, supramolecular chemistry is an accepted interd isciplinary area with far-reaching biological significance. Mass spectrometry, measurement of the mass-to-charge ratio of ions, is an analytical technique applicable not only to the structural elucidation and quantitative analysis of molecules but also to kinetic and conforma tional studies. Inherently a gas-pha se technique, it is well-suited to the study of supramolecular complexes because it enables the study of in trinsic properties of ions without the interference of molecules of solvation.3,6 Recently developed soft ionization techniques such as matrix-assisted laser de sorption ionization (MAL DI) and electrospray ionization (ESI) have addressed the challenge of providing char ge to the complexes without 16

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disrupting the weak stabiliz ing noncovalent interactions,6 making mass spectrometry accessible to a plethora of supramolecular applications. The research described in this thesis c ontributes to the development of advanced supramolecular mass spectrometry beyond genera l stoichiometric complex detection, and demonstrates how combining usef ul characteristics of different types of instrumentation and innovative experimental design can maximize acquisition of info rmation about supramolecular complexes via mass spectral analysis. Electro spray ionization and tandem mass spectrometry were used to probe the noncovalent cation-bound homodimer formation behavior of a novel pair of designed alkali-metal cation receptor synt hons. Electrospray ionization mass spectral characteristics were used to analyze the effect s of structural differences between monomers on the noncovalent interactions responsible for st abilization of the corr esponding heterodimer. Solution-phase optical spectrosc opy provided insight into the supramolecular structure of the homodimers, but highlighted the necessity of gas-phase analytical techniques for the determination of supramolecular complexes without the complication of competition for binding sites between the complex components and molecu les of solvation. Fragmentation pathways for alkali-metal cation adducts of some phlorogl ucinol derivatives we re elucidated using electrospray ionization tande m-in-time mass spectrometry. A home-built desorption electrospray ionization (DESI) sour ce was configured to two high-resolution mass analyzers individually, and some varyin g configurations and DESI parameters were compared. Subsequently, the recently-developed soft desorption/ionization technique, DESI, was applied to rapid screening of supramolecula r host-guest inclusion complexes for the first time. Data obtained were compared to the results of an analogous ESI screening experiment and demons trated significant inconsistencies. Following solution-phase 17

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validation using nuclear magnetic resona nce (NMR) spectroscopy, reactive DESI was determined to be the superior ionization techni que for the application. Computational chemistry confirmed that the tendency of ES I to yield nonspecific artifacts, which were detected as falsepositives in the cyclodextrin guest screening analys is, is a disadvantage of the technique which is not shared by DESI. The reactive DESI screen resulted in positive confirmation of only those complexes determined by NMR spectroscopy. The ES I screen did not yiel d detection of the weakest binding inclusion guest, and consequently did not demonstrate comparable sensitivity to the reactive DESI analysis. No ev idence to suggest preferential de tection of kinetic rather than thermodynamic products using DESI was observed, despite the short interaction time between host and guest molecules during the DESI event. Supramolecular Chemistry Concepts Supramolecular chemistry is the structural a nd functional behavior of organized entities formed through association of two or more chem ical species in the absence of a traditional covalent bond.7 The species involved in the associati on, or complexation, can be referred to as the host and guest5 or the molecular receptor and substrate,7 and they associate uniquely via electrostatic forces diffe ring from full covalent bonds to form complexes.8 The host is formally defined as the possessor of conve rgent binding sites such as Le wis base donor atoms while the guest possesses divergent binding sites such as Lewis acid donor atoms or hydrogen-bondaccepting halides.8 The complexes form through molecular recognition interactions stemming from information pre-programmed into the associated molecules which is manifested in their geometric and chemical characteristics and defi nes the nature of the governing interactions.9 This concept of encoded molecular information is re sponsible for selective binding of individual 18

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substrates by specific molecular receptors a nd is the principle underl ying the phenomenon of self-assembly. The process by which molecules spontaneously form ordered aggregates with no external interference is molecular self-assembly, but self-assembly is not limited to molecules. Selfassembly has been reported for aggregation of varying species up to macroscopic dimensions on the order of centimeters; the major pre-exis ting condition for the ev ent on any scale is complementarity in terms of geometric shape, or topology.10 Species form self-assembling complexes if their binding sites are of comp lementary electronic ch aracter and are spaced appropriately for interaction. A preorganized host will undergo minimal conformational changes during the binding event,5 which is important in terms of the thermodynamic favorability of binding. In supramolecular complexation, alternativ ely referred to as noncovalent synthesis, the products are equilibrating struct ures. Therefore, a complex must correspond to a thermodynamic minimum in order to self-assemble.11 Kinetic driving forces are al so relevant, and an important consideration here is that a c onformationally rigid preorganized molecule may be slow to bind a guest because of the difficulty it experiences passing through a transition state necessary for complexation. Thus a balance must be achieved to promote self-assembling complex formation.5 Broadly, supramolecular complexes may be di vided into two categories: cavitates and clathrates. This distinction is based on the position of the bound guest. Hosts possessing intramolecular cavities are cavitands and guest binding, or inclusion results in formation of a cavitate, a complex in which the guest or guest s are encapsulated by a pocket in the host molecule. Conversely, clathrates are form ed when guests are bound in extramolecular cavities formed by aggregation of two or more molecules.12 For clarity, see Figure 1-1. The designed synthetic alkali-metal cation rece ptor phloroglucinol deri vative dimers characterized in chapters 19

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two and three are clathrates binding the cat ion between two monomers. The cyclodextrin complexes investigated in chapter five are ca vitands with the cyclodextrin host encapsulating guests inside a hydrophobic intramolecular cavity. Types of Noncovalent Bonds Non-covalent interactions are labile and confer on supramol ecular complexes an important intrinsic property, dynamic character. Practically, this attribute enables supramolecules to reversibly associate and dissociate, yielding sele ctive self-organization as individual molecules are able to undergo conformational changes to promote favorable molecular recognition interactions.9 Supramolecular complexes are stabili zed by several types of noncovalent interactions but another impor tant consideration is the nature of their surroundings.6 The relative influences of the presence of molecules of solv ation and crystal close-packing effects can greatly impact the thermodynamic stability of these weakly-sta bilized entities.5 The major types of noncovalent bonds are all based on electrostatic interactions arising from the presence of small directional and nondir ectional electrical char ges on the interacting molecules. Two partial electrical charges interact to pr oduce either attractive or repulsive forces; charges with opposite polarities will yield an at tractive force while like-charges repel.13 Ion-ion interactions Ions will interact with electrostatic dipoles resulting from the electron density on the potential surface of a molecule. Ion-ion interactions are the strongest purely electrostatic interactions with bond energies of ~100-350 kJ mol-1, and bond strength is dependent on interionic distance and the exte nt of charge delocalization.13 This is the major reason that crystal close-packing forces can have su ch a significant effect on supram olecular complex stability and, therefore, must be taken into consideration in the design of analyti cal methodologies involving supramolecular co -crystallization. 20

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Ion-dipole interactions Ion-dipole interaction strength is dependent on the orientation of the dipole with respect to the ionic charge and is of the order of 50-200 kJ mol-1. Co-ordinate or dative bonds fit into this category but they also incorpor ate some covalent contribution.5 Dipole-dipole interactions Interactions between two appr opriately-aligned dipoles can yi eld attractive forces with bond energies in the region of 5-50 kJ mol-1. Possible orientations incl ude matching of a pair of opposite poles on adjacent molecules or opposing a lignment of two adjacent dipoles (Figure 1-2).5 Hydrogen bonds A specific instance of a dipole-dipole interaction correspond ing to the attractive force between a hydrogen atom attached to an electron egative atom and a neighboring dipole results in a hydrogen bond. Bond energies between 4-120 kJ mol-1 are known and various bond lengths from1.2 to 3.2 Angstroms have been reported.5 One important consideration that must be addressed in the analysis of hydrogen-bonded supramolecular assemblies is the solvation strategy, because protic solvents can weaken or even destroy hydrogen-bonds.3 This is another reason that environment is a major f actor in supramolecular chemistry. Cationinteractions Aromatic rings consist of a partially positive -scaffold and a partially negative -cloud above and below the plane of the ring. This c onfers a quadrupole moment on species such as benzene which can result in an attractive fo rce of the order of 5-80 kJ mol-1 between the -cloud and an appropriately loca ted cation (Figure 1-3).14 21

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-Stacking interactions Attractive forces of 0-50 kJ mol-1 can be attributed to -stacking of aromatic rings. The presence of the previously described quadr upole moment enables a ttractive electrostatic interactions between oppositely charged regions of the rings Edge-to-face and face-to-face orientations exist as well as various intermedia te geometries, but face-to -face configuration never corresponds to direct overlap whic h would yield a repulsive force.15 Biological and Physical Significance Noncovalent intermolecular inte ractions make myriad contri butions to the natural world. Structure and function of many important biomolecules are delineated by noncovalent interactions. The immediately recognizable double-helix structure of deoxyribonucleic acid, DNA, is partially stabilized through hydrogen bonds16 and -stacking, or arene-arene, interactions, which also govern th e higher-order structure and ther mal stabilization behavior of some proteins.17 Active site binding between an en zyme and ligand often involves cationinteractions,13 hence biological catalysis is inherently dependen t on supramolecular forces. Active transport across lipid bilaye rs, which form structures such as cell membranes, is also facilitated by noncovalent interactions, cationin the case of transport of ions by ionophores, and the bilayers themselves are stabilized by hydrogen-bonds.5 Molecular devices are organized, functionally-integrated chemical systems built into supramolecular architectures. They are capable of performing a specific function upon activation by some external stimulus.7 These molecular devices base d upon supramolecular chemistry principles are used as sensors and switches for multiple applications.18 Crystal engineering is inherently a self-assembly fiel d, and crystal growth is base d on noncovalent interactions.5 22

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Consequently, supramolecular chemistry has considerable significance beyond the frequentlydiscussed application to biologically-r elevant molecules and processes. Synthetic chemists seek to exploit supr amolecular chemistry for various reasons. Production of synthetic species capable of mimicking the ca talytic and se lective binding phenomena observed in biomolecules like enzymes a nd proteins has signific ant implications to drug design and delivery researc h. Host-guest inclusion comple xes stabilized by noncovalent interactions are currently used in areas such as catalysis and the encapsulation of drugs.19 Design of hosts capable of specific molecular recogn ition behavior is a ra pidly-expanding area of research. Capitalization on self-a ssembly can aid synthetic strategy development in areas like supramolecular catalysis and if appropriatel y employed can increase synthetic yields and enhance synthetic selectivity.7,9 A current aim of supramolecular chemists is to achieve realistic theoretical modeling of noncovalent interactions,20 and that will require an abundance of physical data so appropriate analy tical techniques are in high demand. Mass Spectrometric Approaches Mass spectrometry, the measurem ent of the mass-to-charge ratio (m/z) of ions, is an analytical technique known for the three S char acteristics: speed, sensitivity, and selectivity.21 The m/z is the ratio of the mass of a particle to the number of electrostatic charge units carried by the particle,22 and it can be used to aid identificati on of the elemental composition of said particle. Mass spectrometric anal ysis requires several major stag es. The first stage, following sample introduction, is ionization, for which a strategic explanation is outside the scope of this section. The ionization stage combines, in most cas es, nebulization and inco rporation of a charge onto the analyte or analytes of interest. Nebuliz ation is necessary because mass spectrometry is inherently a gas-phase technique as particles must be separated for detection. Ionization is essential to charge the particle s for separation according to m/z. Arguably, the second stage is 23

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fragmentation of the ionized par ticles. In instances where hard ionization is used, this is combined with the ionization stage, and in some cases, for example when only the m/z ratio of the whole particle is required, this stage is omitted. Fragmentation will be discussed in greater detail in the tandem mass spectrometry secti on. Differentiation of the ions introduced on the basis of their m/z follows and may be referred to as the mass analysis st age, and ion detection completes the experiment.22 Data are presented as a spectrum of absolute or relative signal intensity versus m/z, and the signal detected at each m/z is represented as a peak. Ionization may be achieved in a variety of ways. The most appropriate method for a specific analysis depends on the sample, the an alyte, and the desired information. Techniques used for ionization are generally classified as either hard or soft, ba sed on the energy required and the resulting ion types. Hard ionization met hods such as electron ionization (EI) or chemical ionization (CI) break apart the ionized particle and produce only low abundances of molecular ions or protonated molecules but yield several characteristic fragment ions.23 Soft ionization techniques, such as electrospra y ionization (ESI) and matrix-assi sted laser desorption ionization (MALDI) result in minimal fragmentation but yield significantly more intense signals corresponding to the intact ion.24 Although a vast array of fragment ation strategies exist, all the mass spectral data presented here were produced using ESI or desorption electrospray ionization (DESI), a related technique. Consequently, the ex tended discussion here will be limited to a brief description of ESI. The ESI technique, developed by John Fenn, enab les the transfer of solution-phase ions into the gas phase,25 and so has been widely adopted as an interface for liquid chromatography (LC)26 and capillary el ectrophoresis (CE).22 Electrospray ionization (see Figure 1-4) involves electrochemical charging of a fl owing liquid sample via the applic ation of a high voltage across a 24

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fine-tipped spray needle and an endplate; charged droplets of solution form. The droplets exit the spray needle in a Taylor cone, the apex of which narrows to form a liquid jet and ultimately an aerosol.23 The mechanism by which this aerosol yiel ds ions is the subject of debate. Two convincing possibilities are commonly cited: droplet fission at the Rayleigh limit and direct field evaporation of ions. Both can be referred to as the desolvation mechanism or step, and desolvation efficiency is significantly enhanced at elevated temperatures.24 For this reason, charged droplets are introduced into a heated capillary prior to mass an alysis via a potential gradient. Generally, the opposing ends of the heat ed capillary are charged to aid focusing of the ion beam into the mass spectrometer. This feature confers a useful capability on the ESI technique, source-skimmer collisionally induced dissociation (SCID)27 which will be discussed further in the tandem mass spectro metry section. As noted, the ESI process results in very limited ion fragmentation and, theref ore, has the benefit of promoting determination of molecular weight. Another important advantage of ESI is its ability to produce multiply charged intact ions which makes it amenable to the analysis of larg e molecules. Increasing the charge decreases the m/z, bringing the signal into more efficient ranges for mass analysis. 24 Different instrumental approaches define the type of information obt ained and the way that mass spectrometry can be applied to a specific analytical issue. Maximizing the information obtained in a mass spectrometric analysis is gene rally achieved in one, or sometimes both, of two ways: high-resolution mass spectrometry and tandem mass spectrometry. Although a combination of both these approaches can be used, this is not always desirable due to constraints of expense and time, as well as control of the am ount of data acquired, whic h is a very real and problematic issue in terms of storage and processing capacities in mass spectrometry labs today. 25

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High-Resolution Mass Spectrometry Concepts Resolution is a measurement of the degr ee of separation between adjacent peaks corresponding to ions of different m/z. It is defined below, and should not be confused with resolving power which is an indication of the theoretical resolution capability of an instrument. m R m (1.1) In equation 1.1, m is the m/z value of the peak for which resolution is being defined and m is either the difference in m/z between two ad jacent peaks or the width of the peak, but some indication of the shape and separatio n of the spectral peaks must be included in the definition to give it a tangible meaning. In some cases, a m easure of the height of the valley between the peaks relative to the height of the peaks themse lves, commonly 10 or 50%, is used to account for this. Alternatively, a measure of the width of the peaks at some specified fraction of their height is substituted; often the peak wi dth at half the peak height or full-width half maximum, FWHM, is employed (see Figure 1-5). Resolution between 100-1,000, is usually termed low resolution, medium resolution corresponds to R=2,00010,000, and R>10,000 is classified as highresolution.28 Calculated exact mass, a term often incorrec tly substituted for high-resolution, is used to describe the sum of the exact mass of the in dividual isotopes that compose a single ion,29 and is often reported to a certain point of precision, for example four decimal places. Each element has a specific monoisotopic mass, e.g., carbon by definition has a monoi sotopic mass of 12.00000 atomic mass units, the ex act value of which corresponds to th e mass of the lowest number of subatomic particles one of its atoms can compri se. In mass spectrometry, the term measured accurate mass is used to mean the measured m/z reported with four decimal places and less than 26

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15 ppm error.29 Since each isotope of an element has a specific and precise mass, as the mass accuracy is improved, a particle corresponding to a certain m/z can be more accurately identified in terms of elemental composition. Fewer permuta tions of elemental combinations can yield a specific m/z than a nominal m/z.30 High-resolution mass spectrometry yields accurate mass values, so it can be one important approach to increase the useful information obtained via mass spectrometric analysis. Combined with soft ionization techniques which produce protonated, deprotonated, or adducted, e.g., sodiated, mol ecules or molecular ions, the elemental composition of an ion can often be narrowed down to a manageable number of possibilities. Peak distributions corresponding to the isotopic distributions and mass def ect values are also valuable contributors to the determina tion of elemental composition.30 The major disadvantage, however, is the lack of structural informa tion inherent in resultant spectra. Instrumentation There are three main types of high-resolution mass spectrometers and they are each suited to different situations. All thr ee are capable of high resolving pow er either because they rely on detection by measurement of time, for which extremely accurate measurement capabilities are available, or because they employ combinati ons of mass analyzers for superior ion beam focusing.31 They all require frequent calibration with known mass standards to maintain performance. Double-focusing sector instrument s incorporate two mass analysis stages using both magnetic and electric fields to separate a nd focus ions on the basis of kinetic energy and velocity, and are excellent for configurati on with high-energy ioni zation and dissociation techniques.22 Resolving power high enough to yield reso lution of the order of 50,000 has been reported.32 Sector instrumentation, however, was not used in this body of work and further discussion is therefore outside th e scope of this dissertation. 27

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Time-of-flight, TOF, mass analyz ers pulse packets of ions alo ng a field-free flight tube of known path length. The ions entering the drift tube are pulse accelerated so they all have the same initial kinetic energy. The velocity of thei r flight, therefore, is dependent on their mass according to the equation below, where E is ki netic energy, m is mass, and v is velocity. 21 = 2 E mv (1.2) Since the path length is known, measuremen t of the flight-time of each ion enables determination of the mass, or in this case the m/z, using the following.22 22 mt eV z L (1.3) In equation 1.3, m/z has been defined, e is the charge on an electron, V is the potential used to accelerate the ions, t is the f light time, and L is the path length. Resolving powers of the order of 10,000 have been reported for orthogonal-acceleration reflectron TOF mass analyzers33 such as the one used in this body of work. Ma nufacturer claims of 15,000 have been made. Fourier transform ion cycl otron resonance (FTICR) ma ss analyzers incorporate a superconducting magnet. Ions are introduced into the magnetic field where they are trapped electrostatically and forced into ion cyclotron mo tion, a circular orbit with a frequency inversely proportional to the m/z ratio of each ion.34 Briefly, orthogonal RF excita tion of the ions yields a coherent packet of ions traversing one circular path. The combined time domain signal of the ion orbits is measured via the induced current on a pair of parallel plates a nd the signal is subjected to Fourier transformation for c onversion to the frequency domain.35 The relationship between the frequency and the m/z is shown below, where f is frequency and B is magnetic field strength.35 2 mB z f (1.4) 28

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High mass-accuracy, typically below 5 ppm relative mass error, and resolving power of 150,000 can be achieved using an intermediate magnetic field of 4.7 Tesla,31 and the Marshall group continues to observe increased resolution as they increase th e strength of the incorporated magnetic fields.32 The trapping capabilities of this type of mass analyzer also make it suitable for tandem mass spectrometric analysis, but th e expense of acquiring and maintaining a superconducting magnet, slow spectral acquisition, a nd large size of data f iles limit the use of FTICR-MS. Tandem Mass Spectrometry Tandem mass spectrometry is the measurement of the m/z of ions prior to and following a reaction within the mass spectrometer. The term encompasses a plethora of instrumental and experimental approaches,36 but only a very limited discussion of the strategies relevant to this body of work will be presented here. Major reasons for the popularity of tandem mass spectrometry, sometimes referred to as MS/MS or MSn, are its usefulness in structural elucidation of molecules and an alysis of complex mixtures.36-38 As mentioned, soft-ionization high-resolution work often only yields information about elemental composition39 so structural geometry and the configuration of functional groups remain unaddressed. In tandem work, the term precursor, or parent, ion refers to the detected ion prior to th e reaction event, and the ion or ions generated via the reaction, for example a dissociation event, are th e product, or daughter, ions. Precursor ions are not necessarily molecular ions.36 Appropriate experimental desi gn is an important caveat to the application of tandem mass spectrometry. The type of problem under investig ation and the information required define the design. Traditionally, mass spectrometry may be considered tandem-in-time or tandem-in-space. Experiments involving ion selec tion, reaction, and detection in different regions of the mass spectrometer are considered tandem-in-space. Sector instruments and triple quadrupole 29

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instruments are commonly employed for tandem-in-space experiments,40,41 although the aforementioned SCID technique was incorporated in a tandem-in-space configuration with a TOF mass analyzer as part of this research. Tandem-in-time approaches involve selection and isolation of an ion, reaction, and detection in a single region of the mass spectrometer and events occur sequentially. Ion trapping in struments such as the FTICR and various geometry ion trap (IT) instruments are employed for this type of experiment.40,41 Data presented in Chapter 3 were acquired using a linear quadrupole ion trap. Briefly, trapping instruments for tandem work employ elec trostatic trapping of ions following ionization. A precursor ion is selected by mass or energy and the unwanted ions are ejected from the trapping region electrostatica lly. Introduction of the ot her components necessary for the reaction, for example an in ert gas for collision and fragme ntation, into the region occurs, and the resultant product or daughter ions are detected.42 Theoretically this process can repeat indefinitely, yielding data following multiple even ts, but experimentally some limiting factor, for example initial signal intensity or low mass cutoff, will govern the number of successive reactions successfully monitored.36 When an ESI source comprises the front end of a mass spectrometer, tandem mass spectral data can be acquired even in instruments not tr aditionally designed for it. As discussed, the exit end of the desolvation capillary is held at a high voltage, as is the skimmer succeeding it, to force the ions to travel into the mass analyzer re gion of the instrument. Increasing the voltage difference between the exit end of the desolvatio n capillary and the skimmer, or skimmer-cone, increases the corresponding energy imparted to the desolvated ions which can result in dissociation. This strategy is called sourceskimmer or nozzle-skimmer collisionally-induced 30

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dissociation because the millibar pressure in the fragmentation region of the source promotes collision between ambient molecules and the sprayed ions.27,43 Tandem mass spectra acquired using trappi ng or sequential quadrupole or sector instruments yield spectra where pa rent ion selection and isolati on enables determination of the true relationships between sequentially detected ions.36 The SCID technique suffers from an inability to select the precursor or parent i on, so ions detected following the reaction have uncertain parentage.43 Therefore SCID is not the technique of choice for elucidating dissociation pathways or uncovering structural information, although it is ofte n used for these applications because of its simplicity, low cost, and accessibility.27,44 It has found its niche, however, in experiments designed for comparison of relative ion stabilities, and it is also useful for isomer differentiation and improved analyte detection.27,43,45,46 In-cell collisionally -induced dissociation, CID, can be problematic for ion stability dete rmination of noncovalent complexes because the high-energy collisions generally em ployed break covalent bonds of the parent ion causing rapid and extensive fragmentation over a very narrow energy-range, making cons truction of reliable dissociation curves difficult.26 Supramolecular Mass Spectrometry Gas-Phase Supramolecular Chemistry Elucidation of the chemistry of gas-phase s upramolecules is of vital importance to the development of supramolecular chemistry as a fi eld. Particles in the gas-phase are essentially isolated entities; they experi ence negligible interference from surrounding gas-phase particles and are not surrounded by molecules of solvation.6 Consequently gas-phase analyses are capable of characterizing the intrinsic proper ties of the system under investigation.3,6 In order to further the understanding, modeling, and ultimately synthetic utilization of the physical and chemical bases of noncovalent bonds, analytical strategies to charact erize intrinsic behavior of 31

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supramolecular complexes are essential. In a fiel d where stabilizing forces are relatively weak, this is a very relevant consideration, because molecular chemistry can no longer be studied and understood without some appreciation of environm ental effects. This concept of approaching molecules and their surroundings as complete individual chemical en tities is one of the fundamental principles underp inning supramolecular chemistry.6 Two crucial benefits are derived from the absence of a solvati on shell surrounding gasphase molecules. One is that, using traditional analytical techniques to determine solution-phase chemistry, direct comparisons between solutionphase and gas-phase chemistry can be made, elucidating the direct effects of so lvation on the supramolecular system.3,6,26 The second is more specific to supramolecular chemistry. Species stabilized by noncovalent bonds such as hydrogen bonds, which are known to compete with some solvents for binding sites, can be destabilized upon dissolution. Hydrogen bonds may be weakened or even destroyed in the presence of protic solvents.6,21 Hence, secondary and higher-order structur es of complex molecu les like proteins can be significantly altered leading to the acquisition of entirely misleading data. Gas-phase chemistry, therefore, has an important place in the field of supramolecular chemistry as a whole, but characterization of gaseous supramolecules specifically by using the unique analytical capabilities of mass spectrometry can be jus tified by several factors. As previously discussed, mass spectral data have traditionally been used to determine elemental and structural composition, charge stat es, and stoichiometric relationshi ps pertaining to analytes of interest. In this vein, mass spectrometry is extr emely useful for topologi cal studies to define secondary structures and conformations of s upramolecular complexes an d to investigate the stoichiometry of binding between molecular recognition elements.3,6 However, the benefits of 32

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mass spectral analysis for supramolecular ch emistry extend far beyond these traditional approaches. Mass spectrometry enab les determination of the reactivity of noncovalent complexes, not only in terms of fragmentation be havior, but also in comparativ e preferential substrate binding and hydrogen/deuterium exchange experiments fo r mechanistic studies. Additionally, exciting developments in mass spectrometry for the determination of thermodynamic data have demonstrated the capabilities of the technique as a detection method fo r representation of solution-phase equilibria and as a strategy for th e calculation of both rela tive and absolute gasphase thermodynamic quantities. Supramolecular chiral analyses have been successfully performed.3,6 Survey of Recent Literature Some recent reviews and developments in su pramolecular mass spectrometry accessible in the literature are highlighted here for the purpose of clarifying th e context of the work presented in subsequent chapters. Two excellent reviews ex amining the role of gas-phase analysis within the field of supramolecular chemistry ha ve been presented by the Schalley group.3,6 An in-depth overview of molecular recognition by mass spectrometry was published by De Angelis and colleagues.26 Independent reviews concerned with mass spectral analysis of noncovalent complexes for the determination of quantitat ive thermodynamic quantities were authored by Armentrout et al.47 and the Zenobi group.21 Specific to biomolecules, Kaltashov and Eyles reviewed mass spectrometry applied to the study of conformations and conformational dynamics,48 Kriwacki et al. presented an overview of the mass spectral char acterization of protein complexes,49 and Liesener and Karst authored a cri tical review on monitoring enzymatic conversions by mass spectrometry.50 33

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Supramolecular mass spectrometry for determ ination of the elemental composition of synthetic products and structural elucidation co ntinues to be prolific Lately, novel bis-crown ethers51 and rotaxanes,52 cyclic dipyridyl-glycolurils,53 large phosphorus macrocycles,54 and mixed-metal, mixed-pyrimidine self-assembling metallacalix[ n]arenes55 have been synthesized and characterized using soft-ionization mass spect rometry. The modification of a tolylpyridinebridged cavitand with water-solubilizing groups for the promotion of aqueous-phase selfassembly was determined using mass spectral analysis,56 and a detailed structure of the amyloidogenic protein wild-type transthyretin was obtained vi a HPLC-nanospray MS/MS and MALDI-MS.57 The first report of sonic spray ionization mass spectrometry for the characterization of metal-assemb led cages was published in 2006.58 Topological experiments incorporate mass spect rometry for conformational analysis and higher-order structural elucidat ion. Electrospray ionizat ion has been applied to examination of the supramolecular architectures of complementar y self-assembling host-guest complexes of the softball type,59 of cucurbit[ n]urils,60 and the chelating bidentate catechol ligand with various polyatomic cation guests.61 An interesting study on the dyna mic topological control over subcomponent self-assembly of synthetic helicat es, macrocycles, and catenates employed ESIFTICR mass spectrometry for the eluc idation of secondary structures.62 Additionally, ESI-MS monitoring of the folding and assembly of hemoglobin was achieved using a novel on-line dialysis system.63 Several groups have recently published topology and reactivity studies on biological macromolecules. Limited proteoly sis MALDI-TOF-MS was used to probe the kinetics of cyclindependent kinase inhibition.64 Noncovalent complex formation between the protein Link module from human tumor necrosis factor stimulate d gene-6 and hyaluronan oligosaccharides was 34

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studied over time using hydrogen/deuterium (H/D) amide exchange ESI-MS.65 Collisionallyactivated dissociation ESI-MS/MS experiments elucidated fragmenta tion and preferential binding behaviors of differentially-substituted perylene diimide ligands complexed with DNA, revealing correlation between gas and solution-phase behavior.66 A similar project focusing on DNA complexed with several drug candidates incorporated KMnO4 oxidation with ESI-MS/MS and compared topological and reactivity data using infrared multiple photon dissociation (IRMPD) and CAD dissociation strategies.66 Dissociation pathways for complexes of a singlestranded DNA with a polybasic guest were obtained using nanoelectrospray ionization and tandem mass spectrometry.67 Mass spectral analysis of supramolecular sy nthon reactivities is al so experiencing rapid growth. The Dearden group, who have made myriad contributions to qu antitative gas-phase thermodynamics, have investigated the dissociation and reactivity properties of the cucurbituril derivatives as hosts for inclusion of small-molecule guests using FTICR-MS and have exploited the inherent trapping capabilities of the technique to study ion-molecule chemistry in the ICR cell.68,69 Solution-phase kinetic data for the noncovalent binding betw een chiral resorcinarenes and ammonium guests were obtained using FTICR-MS,70 and a series of elegant experiments also used FTICR-MS but employed H/D exchange and ion-molecule reactions to investigate inclusion of alkyl ammonium ions by substituted resorcarenes.20,71 Additionally, fragmentation and rearrangement of substituted oxadiazoles and their noncovalent complexes with cobalt cation and cyclodextrin were studied using a plethora of mass spectral approaches combined with isotope-labeling.72 Elucidation of a ring-closure mechanis m subsequently incorporated into the design of a method for synthesis of interlocked -conjugated macrocycles was achieved using FTICR-MS and tandem mass spectrometry.73 The utility of FTICR-MS in this area was also 35

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demonstrated in the study of nonc ovalent complexes of the protein Mycobacterium tuberculosis adenosine-5-phosphosulfate reductase; reactivity and thermodynamics were investigated for the delineation of a mechanistic model.74 Quantitative gas-phase thermodynamic data are desirable for the reasons outlined earlier. Major developments in the representation of solution-phase thermodynamic behavior by ESI-MS have been made just in the last few years. The Brodbelt group in particular has contributed to this progress with the early design and application of a method for determination of binding constants by ESI-MS using reference complexes with known thermodynamic data.75 Subsequent work developed mathematical models based on equilibrium partitioning theory to predict the ESI-MS response to host-guest complexation76 and to relate ESI-MS i on abundances with solution concentrations of host-guest complexes.77 Complexes of crown ethers with alkali metal cations were used for validation. A novel quinoxalinecontaining crown ether ligand-metal-ligand sandwich complex stabilized by -stacking interactions was char acterized via ESI-MS resulting in quantitative determination of free-energy gains achieved by modification of the crown ether with electron-rich functional groups.78 Binding stoichiometry and re lative binding affinities of nucleic acid aptamer/small molecule complexes were obtained using ESI-MS and compared to values deduced using traditional solution-phase techniques.79 Evaluation of pr otein-DNA binding affinities, using the DNA-binding domain of a transcription factor, c-Myb, and several doublestranded DNA substrates, was demonstrated using both ESI80 and laser spray mass spectrometry.81,82 Mass-spectrometric determination of gas-pha se thermodynamic quantities for elucidation of intrinsic supramolecular thermochemistry is an additional area of act ivity. Progress continues toward the goal of determining absolute quantitative thermodynamic data using mass 36

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spectrometry. Valuable contributions made by Dear den and colleagues include accurate pressure determination strategies to enable the measur ement of exchange equilibrium binding constants for crown ethers and alkali-metal cations a nd entropies and enthalpies associated with discrimination between enantiomers of chiral amines by dimethyldike topyridino-18-crown-6 during the molecular recognition event.83 Experimental gas-phase alka li-metal binding energies of dibenzo-18-crown-6 obtained using FTICR-MS have been compared to theoretical data derived computationally.84 The effects of size of noncovalent complexes on thei r stability during collision-induced dissociation were studied using complexes of metallated porphyrins with histidine-containing peptid es and model compounds.45 More recently, energy-resolved SCID for the evaluation of relative stab ilities of noncovalent complexes of crown ethers, nucleic acid bases, and amino acids with alkali metal cations has been investigated for determination of solvent influences.43 Complexes formed between alkali metals and polyether ionophore antibiotics were analyzed in a study comparing data analysis methods for gas-phase stability determination by CID.85 The background, significance, and recent deve lopments in supramolecular chemistry and mass spectrometry have been introduced. The rema inder of this dissertation will describe in detail the specific contributions to supramolecular mass spectrome try made though this research. Chapters 2 and 3 describe the characterization of a novel alkali-metal cation synthetic receptor platform via ESI and tandem mass spectro metry. A technical chapter presenting the configuration of a home-built DESI source to two high-resolution mass analyzers follows. Chapter 5 describes the development of a novel DESI technology for supramolecular applications using a cyclodextrin model system. The work culminates in a general conclusion and future directions section. 37

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Figure 1-1. Representation of a) a cavitand including a guest to form a cavitate and b) clathrands forming a clathrate with a guest molecule. Figure 1-2. Two possible orientations of dipole-dipole interactions. Figure 1-3. Representation of cationinteraction showing th e quadrupole moment on an aromatic ring and the interacti on between the partially negative cloud and a cation. 38

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Figure 1-4. Generalized configur ation of an electrospray sour ce showing a) solution flowing through a spray needle, with co -axial nebulizing gas, across a potential difference, b) a desolvation capillary, c) a skimmer cone, d) some ion op tics (no specific type), and e) the direction of flow into the mass analyzer. Figure 1-5. Quantities used to calculate resolution where a) represents the 15% valley definition and b) shows the full-width ha lf maximum, FWHM, approach. 39

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CHAPTER 2 CHARACTERIZING NONCOVALENT DIME RIZATION BEHAVIOR OF DESIGNED PHLOROGLUCINOL DERIVATIVES US ING ELECTROSPRAY IONIZATION HIGH RESOLUTION MASS SPECTROMETRY Introduction A major facet of supramolecular chemistry is the design of synthetic compounds capable of supramolecular interactions with their su rroundings. Synthetic supramolecular systems are under exploration because they offer simplificatio n of synthetic strategi es and a collection of novel, manipulable properties.86 The principle of chemical info rmation encoded into molecules and supramolecules is the foundation for the desi gn of synthetic species capable of specific molecular recognition behavior. Information can be stored at the molecular level by topological design9,87 in a manner similar to the way that bi ological information is stored in DNA. Some synthetic chemists use noncovalent in teractions and self-assembly phenomena to promote selective synthesis or for catalysis of the synthesis process.9 Alternatively, designed synthetic receptors capable of mi micking the noncovalent interactio ns responsible for a plethora of biological processes like enzyme-substrate action and active transpor t across cell membranes may be sought. This type of work is of major importance in the improvement of drug design and lead optimization, for example.17 Novel properties of supramol ecular entities such as unique photoand electro-activity give impetus to synthesis of designed noncovalent receptor molecules.7 Despite the wealth of macrocyc lic ion receptors such as the crown ethers, few designed -receptors for alkali-metal cations are known.88 Effective design of a synthetic receptor capable of molecular recognition with a specific substrate requires that functional groups on a molecular platform be aligned to form complementary bindi ng interactions with the substrate. One way to achieve this is by fixing the conformation of th e receptor by using a rigid platform such as a 40

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benzene ring as a foundation for the functional regions of the synthon. The desired conformation can be permanent or may only be achiev ed during the molecular recognition event.89 Following this design approach, two novel synthetic co mpounds, derived from naturally-occurring phloroglucinol (Figure 2-1) have been investigated for their ability to form dimers capable of selectively binding a size-appropriate al kali-metal cation. The 1,3,5-trisubstituted 2,4,6-triethylbenzenes have demonstrated hostguest inclusion comple x capabilities for both cation and anion guests,90 so there is a good precedent for this work. The synthetic phloroglucinol derivatives 2,4,6-tribenzoylphloroglucinol and 2,4,6-(3,5-dimethyl)-tribenzoylphloroglucinol, hereafter referred to as phloro 1 and phloro 2 respectively, are shown in Figure 2-2. It wa s hypothesized by the Castellano group at the University of Florida that phloro 1 could poten tially form a cage-type dimer capable of housing an alkali-metal cation in the cavity of the re sultant hollow, spherical complex. Preferential complexation of appropriately-sized cations w ould support this hypothesis. Conversely, the two methyl groups on each benzoyl substituent of th e central aromatic ri ng of phloro 2 should prevent formation of a cage-type dimer via steric hindrance, which would be supported by complex formation exhibiting no dependence on ca tion radius. These postulates were explored using mass spectrometric strategies for determin ation of the order of binding preference for several metal cations with each phloroglucinol derivative. In the study of noncovalent preferential binding by mass spectrometry, two major approaches are commonly employed. Traditional competitive binding experiments consist of dissolving the host in a suitabl e solvent and adding equimolar amounts of the two or more competing guests. Assuming similar ionization e fficiencies and minimal signal suppression for the resulting complex ions, subsequent comparison of mass spectral peak intensities 41

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corresponding to the respective complexes is used to assign binding preferences. The more intense the peak corresponding to a given complex, the higher the standing of the related guest in the preferential binding order.19 The aforementioned assumptions can mean that this strategy is inappropriate under some circumstances, but its speed and simplistic experimental design result in its frequent utilization. A more rigorous a pproach capitalizes on th e amenability of mass spectrometry to tandem experimental design. Detec tion of a peak corresponding to a complex of interest is followed by some dissociation event in volving controlled introd uction of energy to the chemical system. The extent of dissociation foll owing the addition of the energy is monitored by comparing the absolute peak intensity after energy addition relative to the initial absolute peak intensity. The energy applied is varied and se veral data points acqui red. Construction of a dissociation or stability curve which is a graphical representa tion of the data plotting the previously-described normalized peak intensity against the amount of energy incorporated follows.21,47,91 Some point on the curve, for example the point at which 50% of the original peak intensity is lost, is used to compare the en ergetic stabilities of the respective complexes.21,91 Common strategies for providing the energy for dissociation incl ude increasing the voltage gap applied during SCID or CID, or introducing electromagnetic radiati on in the form of laser light.47 Although this is the more rigorous approach, it is slower and slightly more complex in nature than performing traditional competitive binding experiments and therefore is not always the method of choice. Experimental Sample Preparation The phloroglucinol derivatives 2,4,6-tribenz oylphloroglucinol and 2,4,6-(3,5-dimethyl)tribenzoylphloroglucinol were synthesized and purified by members of the Castellano group at the University of Florida. All samples were dissolved in HPLC grade methanol (Honeywell 42

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Burdick & Jackson, Muskegon, MI), and the cations Na+, Li+, K+, and NH4 + were introduced as chloride salts (Sigma-Aldrich, St. Louis, MO). The samples were prepared to concentrations of 3x10-4 M host and 3x10-5 M guest for the ESI-MS competitive binding and dissociation curve experiments. For the heterodimer analyses, equimolar amounts of phloro 1 and 2 yielding a total host concentration of 3x10-4 M were used. Each sample prepared for mass spectrometric analysis contained 1% formic acid (ThermoFisher Scientif ic, Waltham, MA) to impr ove current stability and ionization efficiency throughout the analysis. The samples prepared for UV/Vis spectroscopic analysis did not cont ain additional acid. Concentrations of 0.05-0.5 mM host in incremen ts of 0.05 mM were used for titration-type experiments and 3x10-5 M guest was incorporated where ap propriate. Successive addition of L amounts of a concentrated host stock solution to a 3 mL sample volume followed by spectral acquisition comprised the titration approach for the chromic shift observation. The analytical measurement of the chromic shift used solutions with the aforementioned guest concentrations and 3x10-5 M and 3x10-4 M concentrations of the phloroglucinol derivatives. Mass Spectrometry All the mass spectrometry incorporated el ectrospray ionization. Mass spectra were acquired using a 4.7 T Bruker Bioapex II Fourie r Transform Ion Cyclotron Resonance mass spectrometer equipped with an Apollo API 100 Source (Bruker Da ltonics, Billerica, MA) and an Agilent 6210 Time-of-Flight mass spectrometer c onfigured for ESI (Agilent Technologies, Inc., Santa Clara, CA). Samples were introduced at flow rates of 2 L/min for the ESI-FTICR-MS and 5 L/min for the ESI-TOF-MS using direct infusion via a Harvard Apparatus PHD 2000 syringe pump, with a nebulizing N2(g) pressure of 20 psi. The potential difference between the ESI needle and 43

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the desolvation capillary inlet wa s always 3.5 kV. The capillary exit voltage was held at 90 V for the initial FTICR-MS analyses, while the skim mer voltage was 20 V. During the dissociation curve SCID-FTICR-MS experiments, the potential difference between the desolvation capillary exit voltage and the succeedi ng skimmer was varied from 50-250 V in 10 V increments by increasing the exit voltage while maintain ing a constant skimmer voltage. Hexapole accumulation time was 2 s and 50 scans were acquired and averaged for each data point plotted. Each run was repeated three times using indivi dually-prepared samples and the averaged peak intensities were plotted versus the potential difference. For the SCID-TOF-MS dissociation curve experiments, the potential diffe rence was manipulated by varying the fragmentor voltage from 75-300 V in 15 V increments. Signal for each data point was acquired for 20 s and the average integrated intensity used for data analysis. Agai n, the average of three r uns using three different samples was plotted for determination of VC50 values. To determine VC50 values following construction of the stability curves, linear regression was performed on the data points corresponding to the region of decline in signal intensity to calculate the equation of the line associated with the data points. Subse quently, the equation was solved to determine the potential difference value (the x-variable) for which the percentage dissociation (the y-variable) was 50 % in order to obtain VC50. Standard error propagation techniques were applied to the calculation of correspondi ng 95% confidence intervals. Ultraviolet/Visible Absorbance Spectroscopy Optical spectra were acquired using a Hewlett-Packard HP8450 Diode Array UV/visible spectrophotometer (Agilent Technol ogies, Inc., Santa Clara, CA) se t to absorbance or derivative absorbance mode. Cuvettes with a path length of 1 cm were used to hold samples for absorbance measurement. A reference solution of methanol was used for blank and background correction. Derivative absortion spectra were used for de termination of the wavelengths corresponding to 44

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maximum absorbance. Three measurements of each wavelength of maximum absorbance were made using three different solutions a nd the average was plotted for comparison. Results and Discussion Electrospray Ionization Mass Spectrometry for Dimer Detection Analysis of each host compound in the presence of one of each of the cations revealed that phloro 1 forms cation-bound dimers with Na+, Li+, K+, and NH4 + (Figure 2-3). Initial observation of peak intensities corresponding to the complexes suggested a pr eferential bind ing order of Na+>Li+>K+> NH4 +, but the variation in peak intensity could also have been due to differing ionization efficiencies for the respective complexes. Extensive carryover was noted as different salts were incorporated and afte r injection of three samples, si gnal suppression was significant. Consequently, after analysis of each individu al cation-bound dimer, the electrospray line and needle were cleaned with heated HPLC water a nd the source region was vented for removal and thorough cleaning of the desolvati on capillary. Generally the suc cessive skimmer and accessible parts of the hexapole assembly also required cleani ng at this point. This wa s an issue for both the FTICR-MS and the TOF-MS experiments. Traditional Competitive Binding Approach The initial approach toward elucidati ng the preferential bi nding order of Na+, Li+, K+, and NH4 + used the traditional competitive binding experimental design. Since the ionization technique chosen for the work was ESI, only two cations were added to each sample because signal suppression due to high salt concentr ations was a concern. Following mass spectral analysis of samples containing all possible combinations of tw o of the four cations under investigation, peak intensities corresponding to the complexes w ould have been compared to determine an overall binding order. Analysis of the host compounds phloro 1 and 2, however, revealed the presence of significant amounts of adventitious sodium (F igure 2-4). The sodium 45

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needed to be removed to ensure controlled addition of guest cations in equimolar amounts, a fundamental requirement for the acquisition of valid data using the traditional competitive binding approach. Modification of the initial experiment to in corporate a chromatographic separation step followed by post-column addition of the competing guests was subsequently attempted, but it proved impossible to develop a chromatographi c method to remove all of the sodium. The traditional approach was therefore abandoned in favor of the more rigorous dissociation curve approach which would not be adversely affect ed by the presence of adventitious sodium. Dissociation Curve Approach Following the strategy of Rogniaux and colleagues,91 solutions of one phloroglucinol in the presence of each of the alkali metal chloride salts were electrosprayed into the FTICR mass analyzer. The intensity of the mass spectral peak corresponding to the cation-bound dimer of interest was recorded at a range of desolvation capillary exit voltages while the adjacent skimmer voltage was held constant as described in the ex perimental section. The peak intensity relative to its observed maximum was plotted as a percentage against the potential difference in volts, and the equation of the linear sloping portion of th e curve was used to determine the potential difference at which 50% of the maximum signal intensity corresponding to the complex was lost. This value, VC50,91 was calculated for each cation-bound di mer and the respective quantities were used to rank relative stab ility of the complexes. The ESI-F TICR-MS dissociation curves for phloro 1 are shown in Figure 2-5 and those corr esponding to phloro 2 are presented as Figure 2-6. The calculated VC50 values are presented in Table 2-1. Preferential binding order for the cations was determined using this method to be Li+ Na+>K+>NH4 + for phloro 1 and Na+>K+> Li+> NH4 + for phloro 2. The Na+ and K+-bound dimer VC50 values for phloro 2 had to be extrapolated from the stability curves because 50% dissociation of the phloro 2 complexes bound 46

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by these cations was never observed even at the maximum potential difference capabilities of the instrument. The ammonium-bound dimer of phloro 2 was not stable enough for construction of its dissociation curve. The alkali-metal cationic radii are given in Table 2-2, and show that cation size increases in the order Li+>Na+>K+>NH4 +. Apparent size-dependent preferen tial binding of the cations for phloro 1 but not for phloro 2 supported the original hypothesis that ph loro 1 would form a sandwich-type dimer while steric hindrance s hould prevent phloro 2 from doing the same. A major problem with the obtaine d stability curves, however, wa s poor reproducibility, evidenced by large error bars on the plots, which show the av erage of three trials, an d large 95% confidence intervals in the VC50 value calculations. Configured to ESI, FTICR-MS can show significant current, and consequently peak intensity, fluctuati on because only one packet of ions is detected at a time, following hexapole accumulation. Signal averaging can be achieved using a multiplexing strategy, but this is extremely tim e-consuming. The current and peak intensity stability for an oaTOF-MS can be superior becaus e the signal from multiple packets of ions is detected and averaged in a very short time. Fo r this reason, and because 50% dissociation of two of the phloro 2 cation-bound dimers was not achieved using the FTICR-MS, the dissociation curve experiment was repeated using the same sa mple preparation protocol but introducing the electrosprayed complexes into an oaTOF mass analyzer. The dissociation curves obtained using the TO F-MS instrument (Figure 2-7) addressed both of the major problems associated with the FTICR-analysis. The reproducibility of the curve data obtained via TOF mass analysis showed dis tinct improvement over that obtained using the FTICR-MS, particularly for phloro 2. This was as expected for the reason outlined above. A more perplexing result was that more than 80% dissoci ation of the Na+ and Li+ bound phloro 2 47

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dimers was observed in the TOF analysis This enabled the interpolation of VC50 values for both complexes. Reasons for this remain unclear. The calculated VC50 values for each observed complex determined using the TOF mass analyzer are presented in Table 2-3. The smaller confidence intervals associated with the values and the better reproducib ility observed for the dissociation cu rves are the reasons that the TOF values are assumed to be more representative of the system chemistry. The quantities calculated using the different mass analyzers exhi bit similar trends. Dimers bound with Li+ and Na+ cations are more stable than those bound with K+ and NH4 + for phloro 1 supporting some sizedependence in binding preference. The dimers observed for phloro 2 ar e significantly more stable than those observed for phloro 1, with the exception of the ammonium ion bound dimers which were not observed for phloro 2. This seems indicative of differences in binding position of the cations by each host molecule. According to the stability curves constructed using data acquired by the TOF mass analyz er, the binding preference order of the cations under investigation is Li+>Na+>NH4 + >K+ for phloro 1. The order of bi nding preference for phloro 2 is Li+>Na+>K+>NH4 +. Therefore, size-dependent binding is again exhibited for phloro 1 but not phloro 2. Heterodimer Studies The size-dependent preferential cation bi nding observed for phl oro1 but not phloro 2 supports the original conforma tion hypothesis, but the technique implemented does not address the effect on cationbinding of the structural differences between the host molecules, so an ESI-TOF-MS approach was used to analyze ca tion-bound heterodimers of phloro 1 and phloro 2. The intention was to determine, using relative io n peak intensities, whether or not the phloro 1 and phloro 2 are a good geometric fit despite the methyl substituents on the benzoyl groups of 48

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phloro 2. Heterodimer formation was observed with Li+, Na+, and K+ cations but not with NH4 +. Statistically, if phloro 1 and phlor o 2 were a good geometric fit, the peak intensity ratio of the cation-bound dimers should correspond to 1:2:1 wher e the intensity of the signal observed for the heterodimer should be twice that of the signal intensities corresponding to the homodimers. This is because two distinct species can combine sepa rately in four permutations. If we represent phloro 1 as P1 and phloro 2 as P2, then they can combine to form P1P1, P1P2, P2P1, or P2P2, but in this analysis P1P2 is identical to P2P1 so the ratio given above would be expected unless the phloro 1 and phloro 2 are poor complementary binders. A representative ESI-TOF mass spectrum for a sample containing equimolar amounts of phloro 1 and phloro 2 with the addition of LiCl salt is shown as Figure 2-8. Note the presence of adventitious sodium. It clearly shows that th e cation-bound phloro 2 dimers are the most stable of the three dimer types. Th is correlates with the VC50 data obtained from the dissociation curves for the homodimers. The spectrum also shows that phloro 1 and 2 do not bind well together and so are a poor geometric fit as the heterodime r peak intensities are smaller than those corresponding to the phloro 2 homodimers. This s upports the theory that the addition of methyl groups to the benzoyl substituents on the phlorogl ucinol core has a profound effect on the cationbinding interactions associated with phlorogl ucinol derivative cati on-bound dimerization, and hence on dimer conformation. Dissociation curves constructed using SCID-TOF-MS are shown in Figure 2-9. They show that the heterodime rs exhibit the same preferential cation binding behavior as the phloro 2 homodi mer, but the large error bars suggest poor reproducibility so specific VC50 values were not determined. The mass spectral analyses were able to eluc idate the cation-binding preferences for dimers of phloro 1 and phloro 2 as well as the related heterodimers. Size-depend ent preferential binding 49

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for phloro 1 but not phloro 2 or the heterodimers supported the postulated sandwich-type dimer conformation for phloro 1 and end-to-end dimer conformation for phloro 2. A mass spectrometric study into heterodimer stability reveal ed that the addition of methyl groups to the benzoyl substituents of the phloroglucinol core had a significant effect on the noncovalent cationinteractions responsible for stab ilization of the cation-bound dimers. Ultraviolet/Visible Absorbance Spectroscopy The mass spectral analyses described so far yielded data elucida ting the cation-binding preferences of the phloroglucinol derivative dimers and provided so me insight into the effect of the structural differences betw een phloro 1 and 2 on the cationinteractions responsible for dimer stabilization. So far, however, no direct in formation about the conformation of the dimers under investigation had been obtained. A spectro photometric analysis based on absorbance of ultraviolet (UV) and short wavelength visible (vis) light was consequently employed to study solution-phase dimer conformation. An absortion sp ectrum for phloro 1 in methanol is presented as Figure 2-10, and a Beers Law plot constructed using a titration-type experiment is shown in Figure 2-11. Beers Law is stated in equation 2.1 where A is absorbance, is molar absorptivity, b is path length, and c is analyte concentration. Ab c (2.1) Using a Beers Law plot, the molar absorptivity value at 310 nm was determined from the slope of the line to be 2330 130 L mol-1 cm-1 for phloro 1 and 2200 27 L mol-1 cm-1 for phloro 2 which yields a very similar UV/vis abso rtion spectrum. These molar absorptivity values are consistent with n electronic transitions.92 A titration-type experiment where UV/vis absortion spectra were obtained for phloro 1 concentrations ranging from 0.05-0.5 mM varied in 0.05 mM increments was performed to 50

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search for observable changes in spectral characteristics which could correspond to dimerization. The same experiment was performed for phloro 2. Representative data are presented as Figure 2-12. Comparison of the absortion spectra acquired fo r low and high concentrations of phloro 1 revealed a shift of the longer wavelength peak to slightly lower wavelengths, a hypsochromic or blue shift, and a shift of th e shorter wavelength peak to s lightly higher wavelengths, a bathochromic or red shift. The spectra acquired for phloro 2, however, only evidenced bathochromic shift of the shorter wavelength p eak and no shift of the longer wavelength peak was observed. An observed anomaly in the spaci ng between the spectral curves for the third highest concentration phloro 1 solution may be due to instrumental error resulting from accidental disturbance of the plotter, or could be the result of erroneous solution preparation. Long-established molecular exciton theo ry has described two ideal homodimer conformations: sandwich dimers and end-to-end dimers.93 When two identical molecules interact electronically, as in dimerization, molecular orbitals which were not degenerate in the individual molecule exist in close proxim ity, rendering them degenerate mo lecular energy levels in the dimer form. Consequent splitting of the newly dege nerate energy levels results in changes in the magnitude of resultant electronic transitions.94 This is observed in optical spectroscopy as chromic peak shifts corresponding to molecula r aggregation. Hypsoc hromic shift ideally corresponds to sandwich-type dimers while bathochromic shift corresponds to end-to-end dimers.93 The observed chromic shift began to occur at 0.05 mM phloro 1, so an experiment was designed to measure the wavelength of maximum absorption, max, of each peak before and after the shift for both phloroglucinol derivatives in the absence and presence of each of the alkali 51

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metal cations. Derivative spectroscopy was used to determine the max values for improved precision. Specific conditions are delineated in the experimental section. The resultant data are presented as Figure 2-14. The bar graphs a and b show the bathochromic shift of the lower wavelength peak which evidences end-to-end aggregati on of both phloro 1 and 2, although this shift is minimal for phloro 2 and is not observed for the samples in the presence of sodium and lithium cations. A shift is considered to be represented by adjacent bars with non-overlapping 95% confidence intervals which are shown as e rror bars. Overlap of the conf idence intervals for the bars representing the preand post-shif t peaks is taken to mean that there is no conclusive evidence of chromic shift. The graphs labeled c and d show data obtained concerning the max of the higher wavelength peak at concentrations preceding a nd succeeding that corresponding to the expected hypsochromic shift consistent with sandwich-type dimerization. This shift is only observed in the phloro 1 data, so sandwich-type a ggregation is only ev idenced for phloro 1. The optical spectra clearly show evidence of solution-phase end-to-end aggregation of both phloroglucinol derivatives but only show evidence of sandwich-t ype aggregation of phloro 1. These findings are consistent with the original hypothesis and th e gas-phase mass spectral data. The addition of each individual alkali metal cation under investiga tion, in the form of a chloride salt, to the phloroglucinol derivatives in solu tion appeared to have no effect on the spectroscopic data. This is as expected because each cation should be surrounded by a solvent shell preventing cationinteraction between the cation and th e phloroglucinol derivative hosts. 52

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Conclusions Electrospray ionization mass spectrometry enab led determination of the preferential binding order of alkali metal cat ions complexed with dimers of two phloroglucinol derivatives differing by the presence or absence of met hyl groups on the benzoyl substituents of the phloroglucinol core. The binding order for phloro 1 was elucidated as Li+>Na+>NH4 + >K+ but for phloro 2 as Li+>Na+>K+>NH4. Size-dependent preferential bi nding was therefore observed for phloro 1 but not phloro 2. Traditional competiti ve binding experiments were found to yield inconclusive results due to the presence of adventitious sodium, but the disso ciation, or stability, curve approach using SCID offered superior results. It was noted that the rapid averaging capabilities of an oaTOF mass anal yzer resulted in faster acquisi tion of more reproducible data than that acquired using an FTICR instrument. He terodimer studies via ESI-TOF showed that the presence of additional methyl groups on phloro 2 had a profound effect on the cationinteractions responsible for stabilization of the cation-bound dimers. Mass spectral analysis, therefore, was able to characterize the intrin sic noncovalent binding ch emistry between the two phloroglucinol derivatives and th e cations, but no direct information about dimeric conformation could be obtained via this technique. Ion mobility spectrometry could potentially address this issue in the gas-phase. Solution-phase optical spectroscopy provided evidence of both sandw ich and end-to-end molecular aggregation for phloro 1, but only end-to-end aggregation of phloro 2. This conformational information corrobo rated the gas-phase data. It also supported the original hypothesis stating that phloro 1 should be capab le of forming a sandwich or cage-type dimer potentially housing a small cati on in an intermolecular cavity while the addition of methyl groups to the extremities of phloro 2 should ster ically hinder formation of a sandwich complex. 53

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No chemical information about cation binding was attainable via so lution-phase analysis, however, due to solvent interference. For this analysis, it was determined th at, while lacking in conformation-probing capabilities, mass spectrometry was able to pr ovide more useful information concerning the chemical behavior of the supramolecular phlo roglucinol derivative-cation complexes than solution-phase spectrophotometry. It should be noted, however, that solution-phase optical spectroscopy did yield useful complementary conf ormational data, despite its inability to probe the noncovalent interactions between the organic hosts and the ionic guests. 54

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Table 2-1. Phloro 1 and 2 dissociation curve VC50 values and their associated 95% confidence intervals for cation-bound dimers determined using SCID-FTICR-MS Complex Ion Phloro 1 VC50, V Phloro 2 VC50, V [2M+Li]+ 129 22 151 26 [2M+Na]+ 128 19 177 213 [2M+K]+ 89 14 176 110 [2M+NH4]+ 71 217 Table 2-2. Alkali-metal ca tionic radii in Angstroms Cation Radius Li+ 0.76a Na+ 1.02a K+ 1.38a NH4 + 1.43b a Reference 95 b Reference 96 Table 2-3. Phloro 1 and 2 dissociation curve VC50 values and their associated 95% confidence intervals for cation-bound dimers determined using SCID-TOF-MS Complex Ion Phloro 1 VC50, V Phloro 2 VC50, V [2M+Li]+ 168 21 218 22 [2M+Na]+ 153 25 190 21 [2M+K]+ 128 21 162 23 [2M+NH4]+ 131 70 55

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O HO OH H Figure 2-1. Phlorogl ucinol structure O O OH OH OH O O O OH OH OH O a b Figure 2-2. Structures of the phl oroglucinol derivatives a) 2,4,6tribenzoylphloroglucinol (phloro 1) and b) 2,4,6-(3,5-dimethyl)-trib enzoylphloroglucinol (phloro 2). Figure 2-3. Electrospray ionizati on mass spectra showing phloro 1 forming cation-bound dimers in the presence of a) Na+, b) Li+, c) K+, and d) NH4 + cations 56

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Figure 2-4. ESI Spectra of a) phlor o 1 and b) phloro 2 in the absence of added salt showing the presence of adventitious sodium. 57

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Figure 2-5. Phloro 1 dimer dissociati on curves for dimers bound with a) Na+, b) Li+, c) K+, and d) NH4 + obtained using SCID-FTICR-MS. No te the poor reproducibility, although VC50 values were calculated from these curves. 58

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Figure 2-6. Phloro 2 dimer dissociation curves for dimers of bound with a) Na+, b) Li+, and c) K+ obtained using SCID-FTICR-MS. The NH4 + bound dimer was not stable enough for construction of a stability curve. Note the poor reproducibility. Again VC50 values were calculated from these curves but had to be extrapolated for a) and b). 59

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Figure 2-7. Dissociation curves fo r all four cation-bound di mers of a) phloro 1 and b) phloro 2 obtained using SCID-TOF-MS. Note the improved reproducibility which translated to smaller confidence intervals for the calculated VC50 values. 60

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Figure 2-8. Spectrum obtained using ESI-TOF -MS to analyze cation-bound homo and heterodimers of phloro 1 (P1) and phloro 2 (P2). Both homo and heterodimers were observed to form with Li+ and Na+ although only LiCl was added to the sample. Figure 2-9. Dissociation curves for th e cation-bound heterodimers with Li+, Na+, and K+. No heterodimer formation with NH4 + was observed. 61

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Figure 2-10. Absorption spectrum for 0.05 mM phloro 1 in methanol. Figure 2-11. Beers Law plot showing absorbance intensity varying with concentration of phloro 1. Adherence to Beers Law is obs ervable for several UV wavelengths. 62

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Figure 2-12. Ultraviolet/ visible absorption spectra for con centrations of 0. 05-0.5 mM host in increments of 0.5 mM of a) phloro 1 and b) phloro 2 in 50:50 methanol:water. The higher wavelength peak exhibits hypsochromi c shift with increasing concentration of phloro 1 but not phloro 2. The lower wavelengt h peak exhibits bathochromic shift for both phloro 1 and 2. 63

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Figure 2-13. Ultraviole t/visible absorption spectra for 0.03 mM and 0.3 mM solutions of a) phloro 1 and c) phloro 2. The correspondi ng derivative spectra are shown as b) and d), respectively. Figure 2-14. Bar charts showing measured max chromic shift with corresponding 95% confidence intervals of the lower wavelength peak for a) phloro 1 and b) phloro 2 and the higher wavelength peak fo r c) phloro 1 and d) phloro 2. 64

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CHAPTER 3 ELUCIDATING THE DISSOCIATION MECH ANISM OF NOVEL PHLOROGLUCINOL DERIVATIVES Introduction Phloroglucinol, 1,3,5-trihydroxybenzene (Figure 21), exists in various natural products. Various phloroglucinol derivativ es have been isolated as naturally-occurring bioactive compounds, and synthetic phloroglucinol deri vatives have been widely reported.97 Interest in designed synthetic species base d on phloroglucinol is growing.98-100 Members of this important class of compounds have demonstrated therap eutic and pharmacological properties such as antiviral, antibacterial, and vasodilator activities.101 In particular, hyperforin and adhyperforin, constituents of St. Johns Wort ( Hypericum perforatum ), have been well-cha racterized for their antidepressant action.102-109 Phloroglucinol derivatives compri se important secondary metabolites in several dicotyledonous plants,97 and have been reported as us eful environmen tally-responsible dye agents110 and feeding inhibitors.111 Additionally, acylphlorogluc inols have demonstrated potential as lead structures for de generative disease drug development.112 Isolation, quantitation, and characterization of diverse t ypes of phloroglucinol derivatives are therefore relevant to many applications, and are of increasing importance in pharmacokinetic and toxicological studies. Pharmaceutical development relies on rapid, sensitive, and quantitative analytical techniques for the determination of drugs a nd metabolites in complex biological matrices.113 High-performance liquid chromatography, HPLC, combined with mass spectrometry, MS, and tandem mass spectrometry, MS/MS or MSn, is a widely-employed tool in this area because it benefits from several important advantages. HPLC pr ovides separation capabilities to improve analysis of complex or dirty samples, and MS is useful because it enables rapid and selective detection of compounds simultaneously.114 MS/MS is essential for structure elucidation which 65

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can be especially important in metabolite identification.113 Another important aspect of tandem work, however, is selected reac tion monitoring, SRM, the capabi lity of detecting a compound by monitoring fragmentation of one ion to another following a characteristic and specific pathway for a particular analyte.36 Multiple reactions may be monitored during a single chromatographic separation, and SRM confers the ab ility to distinguish between structurally similar compounds with different fragmentation behaviors increasing both selectivity and sensitivity of an assay designed for a specific application.36,37 Confident assignment of a fr agment ion to its parent, however, is an important part of method developmen t for this type of work and consequently an understanding of dissociation mechanisms fo r systems under investigation is necessary. Elucidation of the fragmentati on behavior of the phloroglucinol derivatives as a class of compounds is therefore of growing importance due to the wealth of applications evolving for these species. Several reports of qualitative and quantitative LC-MS for determination of hyperforin and adhyperforin in extracts of St Johns Wort have recently been published.102,104-108 Two synthetic phloroglucinol de rivatives substituted at th e 2,4 and 6 positions with benzoyl groups (Figure 2-2) have been ch aracterized using tandem and accurate mass spectrometry. One compound has pure benzoyl substituents (phloro 1), but the benzoyl groups of the second are methylated at both meta positions of the benzene ring (phloro 2). Alkali-metalbound dimers of both phloro 1 and 2 have been observed to form with Li+, Na+, and K+, and proton and NH4 +-bound dimers of phloro 1 have also been detected, as described in Chapter 2. Both phloro 1 and phloro 2 were observed as protonated molecules and as molecular adducts of each alkali-metal cation. Following fragmentation pathway elucidation for a simple compound, 2-hydroxybenzophenone (Figure 3-1), which contains similar functional groups to phloro 1 but has no C3 character, a dissociation mechanism for the initial fragmentation of the cationized 66

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molecules of phloro 1 and 2 was deduced. Dissoc iation pathways for the alkali-metal cation and proton-bound dimers, as well as the protonated and ammonium adducted molecules of phloro 2, were determined in this resear ch. A recent publication elucidated the fragmentation behavior of protonated and deprotonated hyperforin and adhyperforin,109 but to date no dissociation mechanism information has been made available for alkali metal addu cts of phloroglucinol derivatives. This information is very valuable because electrosp ray ionization is an extremely important interface for HPLC with MS. Alkali metal adducts, particularly sodiated and potassiated species, are known to be prevalent in mass spectra obtained using ESI. Additionally, in analytes lacking a significantl y acidic or basic site, formation of an alkali metal cation adduct may be the only available route to ionization and consequently detection where ESI is the sole accessible ionization technique. Experimental Sample Preparation The phloroglucinol derivatives 2,4,6-tribenz oylphloroglucinol and 2,4,6-(3,5-dimethyl)tribenzoylphloroglucinol were synthesized and purified by members of the Castellano group at the University of Florida. Structur es were confirmed by ESI-FTICR-MS and 1H and 13C nuclear magnetic resonance spectroscopy. All samples were dissolved in HPLC grade methanol (Honeywell Burdick & Jackson, Mu skegon, MI), and the cations Na+, Li+, K+, and NH4 + were introduced as chloride salts (Sigma-Aldrich, St Louis, MO). The samples were prepared at concentrations of 3x10-4 M host and 3x10-5 M guest, and contained 1% formic acid (ThermoFisher Scientific, Waltham MA) to improve current stabil ity and ionization efficiency throughout the analysis. The test compound 2hydroxybenzophenone (Sigma-Aldrich, St. Louis, MO) was prepared in the same so lvent at a concentration of 3x10-4 M. 67

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Mass Spectrometry All mass spectra were acquired using direct infusion electrospray ionization with an appropriate mass analyzer. The tandem mass sp ectral data for phloroglucinol derivative fragmentation pathway elucidation were obt ained using an LTQ linear ion trap mass spectrometer, monitoring the fragmentation through MS5 (ThermoFisher Scientific, Waltham, MA). The 2-hydroxybenzophenone fragmentation was observed via an LCQ Deca quadrupole ion trap (ThermoFisher Scientific, Waltham, MA). The precursor mass selection window was set to 1 amu and optimal CID cone voltages betw een 25 and 30 V were applied. High-resolution exact mass measurements of precursor and produ ct ions were made using SCID-ESI-MS with either a 4.7 T Bruker Bioapex II Fourier Transfor m Ion Cyclotron Resonance mass spectrometer equipped with an Apollo API 100 Source (Bruke r Daltonics, Billerica, MA) or an Agilent 6210 Time-of-Flight mass spectrometer (Agilent Technol ogies, Inc., Santa Clara, CA). Samples were introduced at flow rates of 2 L/min for the FTICR-MS and 10 L/min for all other mass spectrometers using direct infusion. The potential difference between the ESI needle and the desolvation capillary inlet was 3.5 kV. Each run was repeated three times using individuallyprepared samples for mapping of dissociation pa thways. The ESI-SCID-TOF data presented are data obtained via the experiments described in Chapter 2; consequently, experimental configurations and conditions may be found in the Chapter 2 experimental section. Results and Discussion Electrospray Ionization Source-Skimmer Collis ionally Induced Dissociation Justification Chapter 2 describes ESI-SCID-TOF experime nts yielding dissociation curves for the alkali-metal cation-bound dimers of the phloroglu cinol derivatives phloro 1 and 2. The data were reanalyzed by plotting the ratio of cation-bound dimeric peak intensity to adducted monomeric peak intensity against the SCID fragmentation voltage difference. The curves for phloro 1 are 68

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presented as Figure 3-2. Comparison of the shap es of these curves highlighted a difference between the sigmoidal decline of the curves corresponding to the Na+ and Li+ adducts and the plateaus corresponding to the K+ and NH4 + adducted species. Analogous curves for phloro 2 are shown in Figure 3-3; all three curves corresponding to the Na+, Li+, and K+ adducts are of a similar shape and differ predominantly in magnitude. This type of plot repr esents the dissociation of an alkali-metal cation-bound phl oroglucinol derivative dimer to a monomeric adduct of the same cation, so differences in shape could be indicative of different cation-dependent dissociation pathways for the phloro 1 host. Init ial attempts at dissociation pathway elucidation based on the ESI-SCID-TOF data were hindered by ambiguity concerning the origin of apparent fragment ions, which is a limita tion of SCID. These observations illustrated the need for a tandem mass spectrometric investigation inco rporating specific pr ecursor ion selection capabilities, as opposed to SCID which does not enable direct determination of precursorproduct ion relationships. Conseque ntly, fragmentation of the mass selected cation-bound dimers of phloro 1 and phloro 2 was inves tigated using CID in an ion trap mass analyzer, but to aid in dissociation pathway elucidation, a similar experiment with 2-hydroxybenzophenone was first performed. The SCID-TOF data were used to ob tain exact mass measurements for the fragment ions which were observed via both ta ndem mass spectrometric approaches. 2-Hydroxybenzophenone Experiments The compound 2-hydroxybenzophenone (Figure 3-1) was selected to aid in the elucidation of the dissociation behavior of the phloroglucinol derivatives. It has similar structural characteristics to the phloro 1 molecule as it is comprised of a benzene ring modified by a benzoyl substituent, and so has the important f unctional groups involved in the phloro compound fragmentation. The two phloroglucin ol derivatives, however, have a repeating pattern of central benzene ring substituents which confers C3 symmetry on their molecula r structures, and which 69

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complicates determination of dissociation m echanisms and pathways because structural rearrangement during the high-ener gy CID event becomes a significant issue. The smaller and less complex 2-hydroxybenzophenone has minimal st ructural reconfigurat ion capabilities and so proved extremely useful as a tool for the deriva tion of the initial fragmentation mechanism of both phloro 1 and 2. Following MS3 of electrosprayed 2-hydroxybenzophenone a fragmentation pathway for the protonated molecule was determined and is presented as Figure 3-4. Primary cleavages were observed between the carbonyl carbon and each of its neighboring carbon atoms, as was expected. Interestingly, the major fragment i on observed following the initial CID event corresponded to the loss of a benzene ring a nd indicated the involvement of keto-enol tautomerization in the dissociation process. Th is was evidenced by the loss of a complete benzene ring which would only be possible if cleavage of the bond between the carbonyl carbon and carbon 1 on the benzene ring were accompanied by the abstraction of a hydrogen atom from a different region of the precursor ion. As stated previously, the rearrangement capabilities of 2-hydroxybenzophenone are limited so the most accessible hydrogen atom available for abstraction would be th at originating in the alcohol group, following keto-enol tautomerization. This deduction coupled with the similar fragment ation behavior of hyperforin and adhyperforin reported by Sleno et al109 enabled elucidation of the primary mechanism of covalent dissociation which is shown in Figure 3-5 for 2-hydroxybenzophenone. Protonated Molecule Dissociation Pathways Dissociation pathways for the protonated mol ecules of phloro 1 and phloro 2 are presented as Figures 3-6 and 3-7, respectively. Both phloroglucinol derivatives exhibit identical fragmentation behavior and adhere to the mechanism deduced for the smaller 2-hydroxybenzophenone. Following keto-enol tautomer ization, the complete neutral benzene 70

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ring is lost from one of the phloro 1 benzoyl groups and the complete neutral dimethylated benzene ring is lost from the corresponding substituent of phloro 2. This neutral loss occurs for all three benzoyl groups until only the protonated core of the molecule C9O6 remains, suggesting that the proton is noncovalently boun d to one or more of the core oxygen atoms and is not solely stabilized by arene inte raction, as the central ca rbon cycle is no longer co njugated at this point. No evidence of structural rearrangement beyond the initial keto-enol tautomerization was observed. Alkali-Metal Cation-bound Dimer a nd Adducted Monomer Dissociation The dissociation pathways for the lithium cation-bound dimers of phloro 1 and phloro 2 were determined using the same strategy and are shown in Figures 3-8 and 3-9. The lithium cation-bound dimer of phloro 1 dissociates to a monomeric lithium cation adduct via the neutral loss of a phloro 1 molecule. The monomeric lithium adduct is subsequently fragmented in one of three different ways. The neutral loss of one co mplete benzene ring, previously observed for the protonated molecules, is the primary pathway observed, but neutral loss of either a water molecule or carbon dioxide also occurs. Analogo us to the fragmentation of the protonated molecules, the benzene ring loss is followed by successive benzene losses until only the oxygen and carbon monoxide-substituted cyclohexane core remains. The lithium cation, however, remains a component of the detected ion through the entire process, s uggesting that it is interacting with the lone elec tron pair(s) of one, or more probably, several of the core oxygen atoms. Evidence that the lithium cation remains is based not only on the fragment nominal and measured accurate m/z data, but also on the isotop ic distribution observed in the detected ions. Lithium cation is the only one of the catio ns employed which has a nonstandard isotopic distribution. This characteristic combined with the fact that th e less abundant monoisotope has a high enough abundance for detection above the no ise threshold aid in tracking of the cation 71

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throughout fragmentation. The loss of carbon dioxid e from the monomeric lithium cation adduct is of particular interest b ecause it provides evidence for st ructural rearrangement of the phloroglucinol derivative duri ng the high-energy CID process. Carbon dioxide can only be cleaved from phloro 1 following relocation of th e benzene ring portion of one of the benzoyl groups, but the position of the benzene ring after th e rearrangement is currently indeterminate. The phloro 2 lithium cation adducts exhibit analogous behavior. The lithium cation-bound dimer dissociates to the lithium cation adduc ted monomer, but the monomeric adduct only fragments via neutral loss of either a dime thylated benzene substituent or a carbon dioxide molecule; no water loss occurs. Successive cleav ages of the bonds betw een the carbonyl carbons and the dimethylated benzene ring portion of the benzoyl groups follow until only the core remains, similar to the dissoci ation behavior of the other phloroglucinol derivative compound. The sodium cation-bound dimers of both phl oro 1 and phloro 2 a dhere to the same dissociation pathway as those bound by lithium cation; the specific pathways are presented as Figures 3-10 and 3-11, respectively. Smaller init ial signal intensity, how ever, meant that the dissociation process could not be observed through MS5 so only two of the benzoyl substituents were observed to fragment. Both monomeric sodium cation adducts exhibit water and carbon dioxide loss, further evidencing the structural rearrangement dur ing CID observed for the lithium adducts. Fragmentation behavior for the less stable phloroglucinol derivative dimers bound by the potassium or ammonium cations proved somewhat different to the previously characterized dissociation process. For both phloro 1 and phloro 2 dimers bound by potassium cation, the initial step yields monomeric potassium adducts and the successive step, for which the mechanism has been proposed, involves the ne utral loss of either the conventional or 72

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dimethylated benzene ring. The respective pathwa ys are delineated in Figures 3-12 and 3-13. No alternative dissociation pathways are evidenced; neither water nor carbon dioxide losses from the monomeric adducts are observed, he nce there is no evidence to sugge st that the potassium cation phloroglucinol derivative adducts undergo structural rearrange ment during CID. Low initial signal intensity only enabled detection through MS3, so only one fragmentation step for the monomeric adducts could be followed. Adducts of the phloroglucinol derivatives with ammonium cation showed the most deviation from the dissociati on pattern observed for the cationized host compounds. The corresponding pathways are presented as Figures 3-14 and 3-1 5. Although the initial fragmentation of the phloro 1 ammonium cation-bound dimer involves th e neutral loss of a phloro 1 monomer yielding the monomeric ammo nium cation adduct, consistent with the dissociation behavior already described, no stable ammonium cation-bound phloro 2 dimer could be observed. Consequently, the dissociation pathway for phloro 2 in the presence of ammonium chloride is shown for the monomeric precursor ion. The successive step for phloro 1, however, results in detection of the protona ted molecule following the neutral loss of ammonia. No loss of a neutral benzene ring occurs during the dissocia tion process, but subseq uent to the ammonia loss, cleavage of the bond between the carbonyl ca rbon of one benzoyl substituents and a carbon atom on the core benzene ring is observed, yi elding a carbonium cation. This can be further fragmented to yield a carbonium ca tion of one of the benzoyl subs tituents carrying the charge on the carbonyl carbon. The fragmentation behavior of the ammoni um cation adducted phloro 2 monomer shows two alternative dissociation pathways. The traditi onal phloro 2 neutral loss of a dimethylated benzene ring occurs, consistent with data obt ained for each of the other phloro 2 monomeric 73

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adducts. An alternative competi ng dissociation pathway involves the loss of neutral ammonia yielding the protonated molecule consistent with data obtained for the phloro 1 ammonium cation adducted molecule. Conclusions Dissociation behavior for two phloroglucino l derivative compounds ha s been elucidated using ESI-MS and a combination of tandem mass spectrometric techniqu es. Coupling of SCID followed by high resolution mass analysis and ma ss selective ion trapping followed by CID led to the acquisition of complementary data for thorough delineation of the dissociation pathways associated with the C3 symmetric molecules. Initial justification for th e dissociation pathway study was based on the increasing pharmaceutical significance of phloroglucinol derivative compounds, but data obtained during the relative stability experiments conducted on the alkali metal cation-bound dimers of phloro 1 and phloro 2 also highlighted a need for this information. Graphical representations of the dissociation of the dimers to m onomeric adducts suggested the possi bility that different cations could result in different fragmentation behavior. Since no information concerning dissociation ch aracteristics of alka li metal adducts of phloroglucinol derivatives is currently available, the goal was initially pursued through fragmentation of the smaller and less complex, but structural ly similar, 2-hydroxybenzophenone. Elucidation of the dissociation pathway obser ved via CID of the pr otonated molecule was achieved and yielded useful data which led to the determination of the mechanism for the initial dissociation step. The proposed mechanism involve s keto-enol tautomerization followed by the neutral loss of an alkyl group. Adherence to the proposed mechanism was ultimately observed for CID of the protonated molecules of both phloroglucinol derivatives. Successive occurrences of the fragmentation step 74

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corresponding to the determined mechanism were observed until no benzoyl substituents remained. The carbon and oxygen core of the mo lecule retained the charge-supplying proton throughout all the cleavages asso ciated with this behavior suggesting that the proton noncovalently interacts with the co re region of the host molecule. Dissociation pathways for both phlorogl ucinol derivative dimers bound by Na+, Li+, and K+ were delineated, and showed that the dimers fragment to the corresponding cation-adducted monomers. The primary route to fragmentati on of the monomeric adducts adhered to the dissociation mechanism observed for the 2-hydr oxybenzophenone. Again, retention of the cation throughout dissociation showed that the major st abilizing noncovalent interactions probably involve the lone electron pa irs on the oxygen atoms and not only the arene system, which is predominantly destroyed by the loss of the final benzoyl substituent. Competing pathways involving water loss and carbon dioxide loss were al so evidenced, but only for the lithium and sodium cation adducts. The loss of neutral carb on dioxide from the complete cation-adducted monomers provided evidence for st ructural rearrangement of the host molecules during the CID process. The ammonium adducts behaved differently to the other adduct s under investigation. Although the dimeric adduct of phl oro 1 initially dissociated to the analogous monomer, no stable phloro 2 ammonium cation-bound dimer could be isolated for fragmentation. The monomeric adduct of phloro 1 was only observed to fragment via the loss of neutral ammonia, and successive steps showed no evidence of adhere nce to the dissociation mechanism determined for the other adducts. The monomeric ammonium cation adduct did evidence fragmentation by the proposed mechanism, but a competing path way involving the loss of neutral ammonia was also observed. 75

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Electrospray ionization mass spectrometry was successfully employed to characterize the dissociation behavior of the noncova lent adducts of the two phlorogl ucinol derivatives for future pharmaceutically-relevant LC-MS and LC-MSn analyses. Differences in fragmentation behavior of protonated and differentially ca tion-adducted species have been highlighted for this class of compounds. Evidence for stabilizing noncovalent interactions between th e oxygen-rich core of the phloroglucinol derivatives and the cations ra ther than between the arene system and the cations has been obtained. 76

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Figure 3-1. Structure of the test compound 2-hydroxybenzophenone. Figure 3-2. Dissociation of di mers of phloro 1 bound by a) Na+, b) Li+, c) K+, and d) NH4 + to their respective monomeric alkali-metal cati on adducts as potentia l difference voltage is varied in an ESI-SCID-TOF experiment. These curves are based on data presented in Chapter 2. 77

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Figure 3-3. Dissociation of di mers of phloro 2 bound by a) Na+, b) Li+ and c) K+ to their respective monomeric alkali-metal cation a dducts as potential di fference voltage is varied in an ESI-SCIDTOF experiment. The NH4 +-bound dimer was not stable enough for curve construction. These curves ar e based on data presented in Chapter 2. 78

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OHO O O -C6H6C+O C+ -CO-CO-C2HO-C3H4O[C4HO]+[C3H4O]+Formula = C7H5O+m/z = 105 Formula = C6H5 +m/z = 77 H+ H+ [C6H5O]+ -C6H6O-C4H4-CH2[C6H3O]+[C3HO]+ Formula = C13H11O2 +m/z = 199 Formula = C7H5O2 +m/z = 121 m/z = 65 m/z= 56 m/z = 91 m/z = 53 m/z = 93 Figure 3-4. Proposed fragmentation pathway for protonated 2-hydroxybenzophenone. 79

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OO H O O H O O H+ H+ H++ Figure 3-5. Proposed initial dissociation mechanism for protonated 2-hydroxybenzophenone. The cationized molecules of the phloroglucinol derivatives phloro 1 and 2 also adhere to this mechanism. 80

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O O O O O O -C6H6-2(C6H6) H+ -C6H6-C6H6 -C3O2-C6O4[C6HO4]+[C3HO2]+Formula = C27H19O6 +Theoretical m/z = 439.1176 Experimental m/z = 439.1170 Mass error = 1.4 ppmFormula = C21H13O6 +Theoretical m/z = 361.0707 Experimental m/z = 361.0701 Mass error = -1.7 ppmFormula = C15H7O6 +Theoretical m/z = 283.0237 Experimental m/z = 283.0250 Mass error = 4.6 ppmFormula = C9HO6 +Theoretical m/z = 204.9768 Experimental m/z = 204.9787 Mass error = 9.3ppm Theoretical m/z = 68.9971 Experimental m/z = 68.9999 Mass error = 40.6 ppm Theoretical m/z = 136.9869 Experimental m/z = 136.9882 Mass error = 9.5 ppm H+ H+O O O O OH O H+O O O OH OOH O O OH OH OH O Figure 3-6. Proposed fragmentati on pathway for the protonated molecule of phloro 1 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. 81

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O O O O O O -C8H10-C8H10-C8H10 H+ -2(C8H10)Molecular Formula = C9HO6 +Theoretical m/z = 204.9768 Experimental m/z = 204.9771 Mass error = 1.5 ppm Molecular Formula = C33H31O6 +Theoretical m/z = 523.2115 Experimental m/z = 523.2110 Mass error = 1.0 ppm Molecular Formula = C25H21O6 +Theoretical m/z = 417.1333 Experimental m/z = 417.1324 Mass error = 2.2 ppm-C6O4[C3HO2]+Theoretical m/z = 68.9971 Experimental m/z = 68.9999 Mass error = 40.6 ppm -C3O2[C6HO4]+Theoretical m/z = 136.9869 Experimental m/z = 136.9882 Mass error = 9.5 ppmMolecular Formula = C17H11O6 +Theoretical m/z = 311.0550 Experimental m/z = 311.0562 Mass error = 3.9 ppm H+ H+ H+ O O OH OH OH CH3CH3CH3CH3O CH3CH3 O O O OH CH3CH3O CH3CH3OH OO O CH3CH3O OH O Figure 3-7. Proposed fragmentati on pathway for the protonated molecule of phloro 2 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. 82

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O OOH O O O -C6H6-2(C6H6) Li+ -C6H6-C6H6O O OH OH OH O Li+ 2-C27H18O6 Li+ Li+ Li+Formula=2(C27H18O6)Li+Theoretical m/z = 883.2363 Experimental m/z = 883.2350 Mass error = -1.5 ppmFormula = C27H18O6Li+Theoretical m/z= 445.1259 Experimental m/z = 445.1239 Mass error = -4.5 ppmFormula = C21H12O6Li+Theoretical m/z = 367.0789 Experimental m/z = 367.0751 Mass error = -10.4 ppmFormula = C15H6O6Li+Theoretical m/z = 289.0319 Experimental m/z = 289.0302 Mass error = -5.9 ppmFormula = C9O6Li+Theoretical m/z = 210.9850 Experimental m/z = 210.9829 Mass error = -10.0 ppm -H2O -CO2[C26H18O4]Li+[C27H16O5]Li+ m/z= 427 Dam/z = 401 Da O O OH OH OH O O O OH OOH O O O O O O O Figure 3-8. Proposed fragmenta tion pathway for the lithium cation-bound dimer of phloro 1 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. 83

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O OOH O O O Li+-C8H10-C8H10O O OH OH OH O Li+ 2-C33H30O6 Li+ Li+ Li+Formula = C33H30O6Li+Theoretical m/z= 529.2200 Experimental m/z = 529.2159 Mass error = -7.7 ppmFormula = C17H10O6Li+Theoretical m/z = 317.0632 Experimental m/z = 317.0610 Mass error = -6.9 ppm Formula = C25H20O6Li+Theoretical m/z = 423.1415 Experimental m/z = 423.1397 Mass error = -4.3 ppm Formula = C9O6Li+m/z = 211 -CO2[C31H30O4]Li+m/z= 485 O O OH OH OH O O O OH OOH O O O O O O O -C8H10 Formula=2(C33H30O6)Li+Theoretical m/z = 1051.4243 Experimental m/z = 1051.4208 Mass error = -3.3 ppm Figure 3-9. Proposed fragmenta tion pathway for the lithium cation-bound dimer of phloro 2 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. 84

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O O O O O OH -C6H6-C6H6 O O O OH OH OH Na+ 2-C27H18O6 Formula =2(C27H18O6)Na+Theoretical m/z = 899.2105 Experimental m/z = 899.2124 Mass error = 2.1 ppmFormula = C27H18O6Na+Theoretical m/z= 461.0996 Experimental m/z = 461.0984 Mass error = -2.6 ppmFormula = C21H12O6Na+Theoretical m/z = 383.0526 Experimental m/z = 383.0552 Mass error = 6.8 ppmFormula = C15H6O6Na+m/z = 305Na+ -H2O -CO2[C26H14O4]Na+[C27H16O5]Na+Na+m/z= 413 m/z= 443 Na+ O O O OH OH OH O O O OH O OH Figure 3-10. Proposed fragmentation pathway fo r the sodium cation-bound dimer of phloro 1 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. 85

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Na+-C8H10O O OH OH OH O Na+ 2-C33H30O6 Formula=2(C33H30O6)Na+Theoretical m/z = 1067.3977 Experimental m/z = 1067.4071 Mass error = 8.8 ppmFormula = C33H30O6Na+Theoretical m/z= 545.1935 Experimental m/z = 545.1978 Mass error = 7.9 ppmFormula = C17H10O6Na+m/z = 333Formula = C25H20O6Na+Theoretical m/z = 439.1152 Experimental m/z = 439.1172 Mass error = 4.6 ppm -CO2[C32H30O4]Na+m/z= 501 O O OH OH OH O O O OH OOH O -C8H10 -H2O[C33H28O5]Na+ [C32H28O3]Na+m/z= 483-H2O Na+O OOH O O O Na+Theoretical m/z= 527.1829 Experimental m/z = 527.1776 Mass error = 10.0 ppm Figure 3-11. Proposed fragmentation pathway fo r the sodium cation-bound dimer of phloro 2 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. 86

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K+O OOH OH OH O 2 Formula = 2(C27H18O6)K+Theoretical m/z = 915.1838 Experimental m/z = 915.1861 Mass error = 2.5 ppmFormula = C27H18O6K+Theoretical m/z = 477.0735 Experimental m/z = 477.0741 Mass error = 1.3 ppmm/z = 331-C27H18O6-C9H6O2K+ K+Formula = C21H12O6K+m/z = 399 -C6H6[C18H12O4]K+ O OOH OH OH O O OOH OHO O Figure 3-12. Proposed fragmentation pathway fo r the potassium cation-bound dimer of phloro 1 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. 87

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K+O OOH OH OH O 2 Formula = C33H30O6K+Theoretical m/z = 561.1674 Experimental m/z = 561.1658 Mass error = -2.9 ppm-C33H30O6K+ K+Formula = C25H20O6K+m/z = 455 -C8H10 O OOH OH OH O O OOH OHO O Formula = 2(C33H30O6)K+Theoretical m/z = 1083.3716 Experimental m/z = 1083.3826 Mass error = 9.7 ppm Figure 3-13. Proposed fragmentation pathway fo r the potassium cation-bound dimer of phloro 2 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. 88

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-NH3 H+O OOH OH OH O 2 C+O C+O O OH OH Formula = C27H18O6NH4 +Theoretical m/z = 456.1442 Experimental m/z = 456.1462 Mass error = 4.4 ppmFormula = C27H19O6 +Theoretical m/z = 439.1176 Experimental m/z = 439.1170 Mass error = -1.4 ppmFormula = C20H13O4 +m/z = 317Formula = C7H5O+m/z = 105-C27H18O6 m/z = 105-C9H6O2-C7H6O2-C13H8O3-C11H8O3Formula = 2(C27H18O6)NH4 +Theoretical m/z = 894.2545 Experimental m/z = 894.2504 Mass error = -4.6 ppm m/z = 293[C18H13O4]+[C7H5O]+NH4 + O OOH OH OH O NH4 + O OOH OH OH O Figure 3-14. Proposed fragmentation pathway fo r the ammonium cation-bound dimer of phloro 1 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. 89

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-C8H10O O OH OH OH O NH4 + 2 Formula=2(C33H30O6)NH4 +m/z = 1062Formula = C33H30O6NH4 +Theoretical m/z= 540.2381 Experimental m/z = 540.2376 Mass error = -0.9 ppmFormula = C25H20O6NH4 +m/z = 434 O O OH OH OH O O O OH OOH O -NH3 NH4 +NH4 + Formula = C33H31O6 +Theoretical m/z= 523.2115 Experimental m/z = 523.2143 Mass error = 5.4 ppmO O OH OH OH O H+ Figure 3-15. Proposed fragmentation pathway fo r the ammonium cation adducted monomer of phloro 2 showing observed fragments with exact masses and mass error values where appropriate. MS/MS stages were obtained using the LTQ instrument and exact mass data were determined via SCID-TOF. The ammonium cation-bound dimer was too unstable to be mass selected for dissociation. 90

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CHAPTER 4 CONFIGURATION OF A HOME-BUILT DE SORPTION ELECTROSPRAY IONIZATION SOURCE WITH A COMMERCIAL TIME-OF-FLIGHT MASS ANALYZER Introduction In 2004, the Cooks group reported the developmen t of desorption electrospray ionization, DESI,115 which, combined with direct analysis in real time (DART) formed the basis of an innovative platform for ambient ionization.116 The term ambient is used here to imply that it is an atmospheric pressure technique maintaining samp le accessibility throughout analysis. Consisting of a pneumatically-assisted electrospray solution used to desorb and ionize analytes from an ambient surface, the DESI technique has demonstrat ed ESI-like characteri stics in the ionization of analytes amenable to electrospray ionization such as peptides. DESI also benefits from an ability to ionize species traditi onally ill-suited to ESI, however, via an apparent additional ionization mechanism.117 Inherent sample accessibility th roughout analysis confers on the technique the advantages of real -time manipulation of ionization conditions such as temperature and solvent composition and also the configuration referred to as reactive DESI, a variant of the technique which is described in gr eater detail in Chapter 5. The DESI technique is related to both ESI and desorption ionization techniques such as MALDI and desorption/ionization on porous silicon, DIOS.115 Currently, DESI involves a nebulized so lvent, usually water or a mixture of water and methanol, under high voltage conditions sp rayed in an optimal geometry at an analyte adsorbed onto a surface or an in situ analyte. Desorbed ions are focused into a mass spectrometer via an atmospheric pressure interface.115,118-121 Two mechanisms of ionization ha ve been postulated, but they ar e each relevant to different applications.115,118-121 One involves the forma tion of charged solvent droplets which ionize analyte molecules on the desorption surface by char ge transfer; the result ant buildup of static 91

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charge forces desorption of the analyte ions from the surface, a phenomenon known as chemical sputtering.121 The second mechanism explains the str ong resemblance in spectral characteristics of DESI data to that obtained via ESI. Electrospr ayed droplets impact the surface resulting in the dissolution of analyte within the droplet; subseq uent evaporation of the solvent yields analyte ions. Both ESI and DESI generally yield signif icant amounts of the protonated molecular ion, multiply charged ions, and alkali metal adducts in positive mode.118 An interesting study investigating droplet dynamics a nd ionization mechanisms of DESI using phase Doppler particle analysis was recently published by Venter et al .122 Primarily, DESI is advantageous because it is an ambient method, which requires little or no sample pretreatment.117 It is sensitive, fast, and versatile,115,118-121 being suitable for solid, liquid, and adsorbed gaseous samples.118 Traditional DESI has been applied to an enormous number of analytes, including pharmaceuticals, ch emical warfare agents, plant alkaloids, and lipids.116 Novel sampling surfaces like porous silicon, thin-layer chromatography plates,123 and solid-phase microextraction fibers124 have been analyzed.125,126 A major application area of the DESI technique is the trace level detection of ex plosives on ambient surfaces. Native explosives and their plastic compositions have been analyzed on metal, plastic, paper, polymer, and living human tissue surfaces; promising results were obtained using complex matrices such as lubricants and detergents.120 Pharmaceutical applications in clude high throughput analysis of various commercial dosage forms, drug and metabolite identificati on in blood and urine,119 and coupling of DESI to thin layer chromatogra phy for over-the-counter preparations analysis.125 Food chemistry applications and determination of alkaloids in plan t tissues have been demonstrated.118 Mass spectrometric profiling of intact, untreated bacteria employing DESI has been described.127 A study describing the complementar y application of nuclear magnetic 92

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resonance spectroscopy and DESI-MS, and incorporating principal component analysis for data interpretation, for urine metabolite determination in patients with inborn errors of metabolism was recently contributed by the Cooks group.128 Imaging mass spectrometry under ambient conditions has been achieved by the application of DESI,129 and tissue imaging demonstrated.129,130 The preceding applications employed standard bench top ion trap or triple quadrupole mass analyzers. An interesting addition to the type s of trapping instruments with which the DESI technology has been interfaced is a custom-bu ilt portable mass spectrometer fitted with a miniature cylindrical ion trap mass analyzer.131 The Cooks group has also demonstrated the capabilities of DESI coupled to an Orbitrap mass spectrometer for exact mass measurements on therapeutic drugs and peptides,132 and several other mass analyzer s have been configured for high resolution and exact mass DESI applications. DESI has been interfaced with TOF-MS for the direct determination of counterfeit, commercial, and illicit drug tablets133,134 and for the determination of CID fragmentation pathways of over-the-counter drugs.135 An atmospheric pressure interface for the coupling of ESI, electro-sonic spray ionization, ESSI, an extremely soft form of ESI utilizi ng a supersonic gas jet,136 and DESI to FTICR-MS was developed by the Bruce group,137 and successful analysis of proteins and peptides was recently achieved using DESI-FTICR-MS.138 A home-built DESI source originally designe d for configuration with a Bruker Bioapex FTICR-MS has been successfully interfaced with first the FTICR-MS instrument and subsequently an Agilent TOF-MS. The DESI te chnique has compatibility issues with FTICRMS, hence the DESI-TOF-MS configuration was unde rtaken. The unique geometry of the source housing on the Agilent TOF mass spectrometers po ses particular challenges for DESI source 93

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integration, as do some of the instrument-contro l-specific features of the accompanying Analyst software. Strategies for addressing th ose challenges are described herein. Experimental Desorption Electrospray Ionization Source Design The DESI source was constructed by the Univ ersity of Florida Chemistry Department Machine Shop. It was fabricated using anodized aluminum (6061-T6), and consists of one xyzstage for the manipulation of an electrospray head and a second xyz-stage for the manipulation of a sample holder platform. A schematic showi ng an overhead view is presented as Figure 4-1, and photographs of the source conf igured with the FTICR-MS are included as Figures 4-2 and 4-3. The dimensions shown in the schematic are those used for configuration with the FTICRMS; alterations made for confi guration with the TOF-MS are described in the results and discussion section. The electrospray head is mount ed on a cylindrical block which can be rotated for manipulation of the angle made between th e spray needle and the deposition surface. A commercial ESI needle is housed inside the coaxial gas needle which is visible in the schematic. A Teflon spacer between the block and the solution/gas introduction port insulates the remainder of the source from the high-voltage ap plied to the solution. So lution introduction is via direct infusion using a Harvard Apparatus PHD 2000 syringe pump (Harvard Apparatus, Holliston, MA), and high-voltage is applied to the spray solution via an external 210-10R high voltage power supply (Bertan Associates, Hicksv ille, NY). The sample surface is chassis ground. The sample holder is a Teflon platform m ounted on an aluminum steel block with an indentation for housing of a deposition surface the size and shape of a standard microscope slide. Mass Spectrometry Mass spectra were acquired using a Bruke r Bioapex II 4.7 T Four ier Transform Ion Cyclotron Resonance mass spectrometer (Bruker Daltonics, Billerica, MA), or an Agilent 6210 94

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MSD Time-of-Flight mass spectrometer configured for ESI (Agilent Technologies, Inc., Santa Clara, CA). Sample Preparation Rhodamine 6G (Figure 4-4) samples were pr epared by drawing a layer of red Sharpie (Sandford Corporation, Oak Brook, IL) ink onto the sampling surface. Cytochrome C (horse heart, Sigma-Aldrich, St. Louis, MO) samples we re prepared by dissolvin g 1 mg/mL of the solid in 50:50 methanol:water. Ten microliter spots were deposited onto the sampling surface using a micropipettor and were dried under ambient conditi ons for thirty minutes. HPLC grade solvents were employed (Honeywell Burdick & Jackson, Muskegon, MI). Results and Discussion Configuration of DESI Source with FTICR-MS Initially the DESI source was interfaced with the FTICR using the original dimensions and attachment mechanism specifically designed for th is purpose. The FTICR-MS instrument used is ordinarily equipped with an ESI source, and so has an atmospheric pressure inlet orifice accessible for DESI. The hollow center of the deso lvation capillary is the inlet and must be immediately adjacent to the deposition surface fo r DESI compatibility. The ESI spray head housing attaches to the desolvation capillary housing via a hinged door a nd latch, so the whole spray head can be removed easily. Removal of the ESI spray head housing makes the inlet orifice accessible. The original design of the DESI source included a hinged door, of the same dimensions as that of the ESI spray head housin g, placed adjacent to the sample holder platform (see Figure 4-2). A circular hole cut in the doo r enabled access to the desolvation capillary for ion entrance. The applied high-voltage necessary to charge the spray solution is or dinarily achieved in the standard ESI source for this instrument by maintaining the spray n eedle at ground potential 95

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and applying a voltage of 3-4 kV to the desolv ation capillary entrance. The applied voltage is negative for detection of positive ions and positive for detection of negative ions. A safety feature of this instrument prev ents the high-voltage fr om being applied unless a certain switch in the capillary housing is closed; the switch is de pressed when the hinged door is closed and tightly latched. If the high-voltage does not turn on, the software will not allow the instrument to acquire data. Therefore, the switch must also be depressed during DESI spectral acquisition. To overcome this issue, a small screw was placed through the DESI source door and screwed into position to depress the switch upon latching the replic a door. This can be seen in Figure 4-3. The screw is above and to the right of the desolvatio n capillary. Interestingly, to obtain DESI signal, the high-voltage had to be applied to the elect rospray solution while the desolvation capillary was held at 100 V. Reducing the desolvation cap illary entrance to ground potential resulted in unstable current and loss of signal. When the DESI source was first interfaced w ith the FTICR-MS, it was observed that the dimensions of the source did not allow the full range of motion that should have been available to the spray head on its mounting block, and the spray head was t oo far away from the inlet for DESI signal to be attained. This issue was unforeseen during the design process because the additional length of the spray need le and the length of the desolvation capillary were difficult to measure accurately in position in the enclosed ESI spray head housing. A useful feature of the DESI source design was exploi ted to address the problem. Be tween the spray head mounting block and the x-y-z stage is an entirely removable piece, labeled a in Figure 4-1. An analogous piece is used to connect the sample holder plat form and its x-y-z stage. Piece a, which was originally 9 cm long, was replaced with a 12.75 cm piece. This enabled the movement of the spray head towards the inlet orifice, and s ubsequently DESI signal of Rhodamine 6G was 96

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observed. The first DESI-FTICR-MS data acqui red using the DESI source are spectrally represented in Figure 4-5, and a cl ose-up view of the DESI needle coaxial gas tubing adjacent to the orifice inlet is shown as Figure 4-6, to provide some idea of the small distance between the needle tip and the inlet of the mass spectrometer necessary for spectral acquisition. Also visible is a portion of the sample holder stage and the deposition surface from which the detected ions were desorbed. Optimization of DESI parameters is an i nvolved process which requires signal stability. Unfortunately, sample ablation during DESI can be quite fast, of the order of tens of seconds. The small colorless spots on the slide shown in Figure 4-6 are areas where sample ablation via DESI has occurred. An important step in the acqu isition of mass spectral data via FTICR-MS is the hexapole accumulation of ions prior to thei r transfer to the ICR cell for detection. This usually takes between 1 and 2 seconds. Additionally, several complete scan events involving accumulation time, ion transmission time, and ti me for cyclotron resonance detection and multiplexing are necessary to obtain usable signal intensity and signal-to-noise ratio, S/N, values. This makes optimization of DESI parameters for FTICR-MS extremely challenging, as, during the time it takes to determine the effect of s light variation of one pa rameter using the FTICRMS, sample ablation has resulted in loss of signal. For this reason, it was determined that it would be useful to configure the home-built DESI source with an Agilent TOF-MS, which has the advantage of a very high scan rate, conferri ng the ability to maintain high-resolution while acquiring around 1 spectrum per second, depending on the selected mass range.139 Configuration of DESI Source with TOF-MS To capitalize on the fast spectral acquisiti on capabilities of the TO F mass analyzer, the DESI source was moved onto the TOF-MS describe d in the experimental section. The original 97

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design was not compatible with the unusual geomet ry of the Agilent source (see Figure 4-7), so several modifications had to be made. The first major obstacle to the DESI-TOF conf iguration was the height of the desolvation capillary housing on the TOF. To address this, a specially built pl atform for the DESI source had to be made out of stainless steel. The 13 st and is visible in Figure 4-8. However, although the stand brought the source to an appropriate height the slanting front of the desolvation capillary housing made positioning of the sample holder platfo rm adjacent to the inle t orifice difficult. The front of the base of the DESI source was held at a certain distance from the capillary by the protruding lower edge of the desolvation capillary housing, and the original dimensions of the arm connecting the sample holder platform and its x-y-z stage limited movement of the platform toward the mass spectrometer, so that 7 cm betw een the sample surface a nd the inlet orifice was the minimum distance achievable. This issue was overcome by exchanging the removable 7.5 cm long piece labeled b in Figure 4-1 with a new pi ece of length 14 cm. Since the spray head was now incapable of reaching either the sample surf ace or the inlet orifice of the mass spectrometer, piece a was replaced again, this time with a 19 cm long piece. At this point, the spray head was able to reac h the inlet orifice, but the slanting geometry of the desolvation capillary housing wa s still preventing movement of the sample holder platform to within the necessary distance of a few mm from the inlet orifice. At its nearest point, the edge of the sample platform or the arm of its mounting block jarred against the edge of the capillary housing, 1.25 cm away from the orifice. As stated briefly in the introduction section of this chapter, in 2006 the Bruce group at Washington State University reported the use of a flared capillary adapter to improve collection efficiency of ions resulting from ESI, ESSI, and DESI ionization techniques.137 This strategy was modified to address the issue of the distance 98

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remaining between the needle of the home-built DESI source and the inlet orifice of the TOF mass analyzer. A capillary extend er designed to fit snugly onto the end of the desolvation capillary and extend to the sample surface was used to bring the orifice in let to the sample (see Figure 4-9). A gold canted coil spring was placed into a groove milled on the inside of the piece which fits over the capillary to hold the extende r firmly in place while promoting conduction for the electrospray. This was essentia l, because in the absence of the spring, the capillary extender will be pulled off the end of the desolvation capillary and stick to the edge of the sample holder stage due to charge build-up upon application of th e potential difference necessary to achieve an electrospray. An additional useful feature of the cap illary extender is that it enlarges the area of the sampling orifice, which has been show n to improve ion transmission and spectral characteristics.137 The diameter of the desolvation capil lary inlet orifice is 0.6 mm, but the diameter of the capillary ex tender inlet orifice is 1.5 mm. The Agilent TOF mass spectrometer can only acqui re spectra when it is configured to a source. Since several commercial atmospheric ioni zation sources have been designed for it, a source recognition mechanism incorporating stra tegically positioned magnets is employed to allow control of the sources via one software suite. Magnets on the inside of the source attachments, which interface by la tching onto the desolvation capilla ry housing, are in locations specific to each source type. Consequently, wh en a nonstandard source is interfaced with the desolvation capillary, a configur ation of appropriately -sized magnets is necessary to enable source recognition by the software. The standa rd ESI source has the simplest magnetic configuration, so the mass spectro meter was configured for ESI whenever the DESI source was attached. One small bar magnet on the inside of the ESI spray head housing is used for ESI source recognition. The addition of a small bar magnet correctly positioned external to the 99

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desolvation capillary source housing and held in place with Scotch tape enabled the instrument to recognize the ESI source and allowed spectral acquisition in the absence of a commercial source. A second issue in terms of nebulizer gas settings also had to be addressed prior to data acquisition. The ESI nebulizing gas is not used fo r the DESI source, and the outlet has to be covered to minimize unnecessary loss of N2(g). However, for the instrument to come out of standby mode, the internal flow meter has to regi ster the flow rate set by the user. This is achieved by setting the nebulizing gas flow rate to the number that is registered prior to spectral acquisition. A setting of zero may or may not be a ppropriate, despite the fact that nebulizing gas is not flowing. Following the described modifications and conditions, DESI-TOF-MS data were obtained for the Rhodamine 6G sample. A representa tive mass spectrum is shown as Figure 4-10. Following the Rhodamine 6G tests, another standa rd, the protein cytochrome c, was used for confirmation of the DESI-TOF i onization capabilities. Cytochrome C was successfully analyzed using the DESI-TOF-MS configuration and provided evidence of the success of the interface for compounds other than Rhodamine 6G which is known to exist as a preformed ion. The first spectral data obtained for cytochrome C are presented as Figure 4-11. Important Optimization Considerations Optimization strategies, and corresponding effects on spectr al characteristics, for the geometric and physical parameter conditions impor tant in DESI mass spectral analyses have been extensively documented elsewhere.118,119,122,137,140,141 Consequently, the discussion here is limited to important considerations in the optimization process which are frequently omitted from the published literature. DESI conditions must be optimized specifically for each type of analyte and surface, although similar analytes of ten have similar optimal conditions. Following the determination of the specific surface used for sample deposition, parameters requiring 100

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optimization may be broadly grouped into two ca tegories: electrospray conditions and geometric parameters. Examples of the electrospray conditions re quiring optimization include solution-phase composition, solution flow rate, applied high volta ge difference, and nebulizing gas flow rate. These can be optimized to some extent using realtime visual cues, such as the extent of sample ablation or surface wetting. Monitoring the curren t measured on the desolvation capillary for stability is a significant aid in optimization of solution compos ition and applied voltage values. Solution and nebulizing gas flow rate optimizat ion can be performed speedily by monitoring wetting of the sample surface. An additional aid in significantly increasing the efficiency of optimization of the electrospray conditions is monitoring the total ion chromatogram (TIC) instead of the signal corresponding to a particular analyte or analytes. This is useful because it renders the ESI optimization process largely in dependent of the geometric DESI parameters, upon which analyte-specific signal in tensity and S/N are dependent. Important geometric DESI parameters have been defined by the Cooks group,118 and are delineated in Figure 4-12. Optimization of these pa rameters is a very interactive and iterative process. Signal intensity and S/N are partic ularly sensitive to variation of the angle which is additionally the most difficult parameter to mani pulate using the home-built DESI source, so this is the most challenging parameter to optimize. A good starting point for analytes traditionally amenable to ESI is = 60 Again, monitoring the TIC instead of signal intensity or S/N is an efficient way of making an ini tial assessment of the appropriate range of geometric parameter values for subsequent, more rigorous optimization. For each type of DESI analysis, it is importa nt to optimize parameters for maximum signal intensity and maximum S/N specific to the analysis This is because the variable values yielding 101

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maximal signal intensity sometimes do not correspond directly to those yielding maximal S/N, although overlap in terms of a value ranges for some parameters may be observed. Figure 4-13 presents some representative parameter optimization data and show s the effects on signal intensity and S/N resulting from variation of the electrospray flow rate. The optimal flow rate range for maximizing both signal intensity and S/N is 4-6 L/min, but the peak maxima do not necessarily correspond. Consequent ly, in some situations 5 L/min could be most desirable flow rate, but for analysis of a limited amount of sample, where SCID would be employed for structural elucidation or determin ation of relative stabilizing for ces, it is conceivable that the lower flow rate would be selected for the a dvantage of increased sp ectral acquisition time it would confer. Conclusions A home-built DESI source e ngineered by machinists at the University of Florida department of chemistry machine shop specifica lly for configuration with a Bruker Bioapex FTICR-MS instrument has been successfully interfaced with an Agilent 6210 API Time-ofFlight mass spectrometer. Initial configura tion of the DESI source with the FTICR was successful in that DESI mass spectral acquisi tion was achieved; mass spectra of the dye Rhodamine 6G were acquired. A significant challenge, the fact that sample ablation during DESI often occurred prior to adequate data acquisition to de termine the effects of parameter variation during optimization, highlighted the poor compatib ility of DESI with FTICR-MS in terms of slow scan rate of the FTICR instrument. Several modifications to the original design were necessary to accommodate the significant height difference between the inlet orifice of the FTICR instrument and that of the TOF mass spectrometer, and the unorthodox ge ometry of the desolvation capil lary housing. The fabrication 102

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of a 13 custom built stand overcame the first issue. Extension of the range of motion in the direction of ion transmission of both the DESI spray head and the sample holder platform coupled with the fabrication a nd incorporation of a specifical ly-designed capillary extender addressed the latter. Following some additional steps to compen sate for the source recognition and condition-monitoring processes integral to the function of the co mmercial instrument, successful acquisition of DESI-T OF mass spectra of deposited R hodamine 6G and cytochrome C was attained. DESI optimization is an iterative and inte ractive process which has been documented elsewhere, but some important considerations and strategies for increasing the efficiency of the process have been outlined to aid the novice user. 103

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Figure 4-1. Schematic of the overhead view of the original home-built DESI source showing major parts and dimensions, in centimeters for configuration with the FTICR-MS. The pieces marked a and b were altered for configuration with the TOF-MS and the door and hinge were removed. 104

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Figure 4-2. Desorption electrospr ay ionization source interfaced with the FTICR-MS showing door and hinge for attachment to th e desolvation capillary housing. Figure 4-3. Close-up view of DESI source config ured with FTICR-MS showing the spray head, the sample holder platform, and the fr ont end of the desolvation capillary. 105

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O NH O NH+O Figure 4-4. Structure of the R hodamine 6G preformed ion. Figure 4-5. First spectrum obtained using the home-built DESI source configured to the FTICRMS, showing the molecular ion of Rhodamine 6G. 106

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Figure 4-6. Close-up view of the DESI-FTICR-MS setup used to acquire the spectrum presented in Figure 4-5. The relative pos itions of the DESI spray needle and the desolvation capillary orifice are shown. The deposition surf ace, a sandblasted glass slide, and its layer of Rhodamine 6G are visible and sm all colorless spots on the sample surface show areas of sample ablation. 107

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Figure 4-7. Close-up view of th e DESI source interfaced with the TOF-MS showing the unusual slanting geometry of the desolvation capillary housing. Note that the capillary it self is not visible, as only the ca pillary extender protrudes from the housing. The DESI spray head solution/gas introduction port and fluid connections can be seen. The bar magnet taped to the upper right side of the desolvation capillary housing is responsible for ESI source r ecognition by the software. 108

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Figure 4-8. Side view of the DESI-TOF-MS conf iguration. The 13 stand constructed to bring the DESI source to the height of the desolvation capillary is shown and the connection for the applicati on of the high voltage to th e electrospray solution is visible. Figure 4-9. Representation of the capillary extender, with dime nsions given in mm, for the DESI-TOF interface showing a) the side vi ew and b) the view from the back. A canted coil spring is fitted into a circular gr oove on the inside of th e extender to hold the extender firmly in place on the desolvation capillary. 109

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Figure 4-10. Mass spectrum of Rhodamine 6G obtained usin g the DESI-TOF-MS configuration. Figure 4-11. Positive-mode DESI-TOF mass spectru m of cytochrome C desorbed from a glass slide. A multiprotonated envelope containi ng charge states ranging from +9 to +19 was observed. 110

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Figure 4-12. Schematic representation of the DESI spray head and inlet orifice showing the major geometric DESI parameters which require optimization. Figure 4-13. Representative optimization data for the DESI-TOF-MS configuration using Rhodamine 6G. The effects of variation of the solution flow rate on a) signal intensity and b) S/N for the molecular ion are shown. 111

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CHAPTER 5 REACTIVE DESORPTION ELECTROSPRAY I ONIZATION FOR RAPID SCREENING OF GUESTS FOR SUPRAMOLECULAR INCLUSION COMPLEXES Introduction The need for a better understanding of noncova lent molecular recognition interactions is fueling the development of analytical techniqu es appropriate for the characterization of both natural and synthetic supramolecular systems.26 Methods like optical and nuclear magnetic resonance spectroscopies and circular dichroism have proven extremely useful for solution-phase analyses, and X-ray crystallography is indispensable for solid pha se experimentation.17,21 Gasphase techniques, on the other ha nd, enable characterization of s upramolecular species without the added complications of interference from solvating molecules and crystal close packing effects.3,6,26 Mass spectrometry is theref ore an appropriate tool for this application and has the benefits of sensitivity, sel ectivity, and the ability to pr ovide complex stoichiometric information.19,26 ESI is inherently applicable to complexa tion based on solution-phase self-assembly, and ESI mass spectrometry has been reported for the analysis of a plethora of noncovalent systems from enzyme-substrate complexes26,79 to macrocycles, such as calixarenes140 and cucurbiturils,60,68,69 which are capable of forming inclus ion complexes with both neutral and charged guest molecules. ESI is known for a te ndency to form nonspecific artifacts which can be confused with true supramolecular complexes in this type of work;143,144 this can be particularly problematic in the study of alkali-metal comple xation. Additionally, while ionization occurs at atmospheric pressure, the technique does not sati sfy a recent definition of the term ambient, which includes the requirement of sample accessibility for manipulation during analysis.115 In this application area, sample accessibility is usef ul because it enables re al-time manipulation of 112

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parameters like solvent character and availability of different guest molecules for faster analysis in terms of gu est screening. A major impetus for the development of hi gh-throughput noncovalent receptor screening techniques is the wide applicab ility of molecular recognition interactions to fields like drug discovery and lead optimization.17,26 High-throughput enzyme-substrate assays are welldocumented, but predominantly reliant on solu tion-phase analytical techniques such as immunoassays using spectrophotometric or radi ochemical detection, and consequently often subject to interferences from solvent and assay reagent molecules.50,145 Structural modifications of ligands are sometimes required to yield require d chromophoric properties for detection of the binding event which can alter natural kinetic behavior, and the use of radiochemicals for scintillation-based assays involves the generation of radioactive waste.50 Mass spectrometry has the selectivity to minimize some of these effect s and consequently reduce the instance of false positive results, particularly when high-resolution or tandem mass analyzers are employed. Mass spectrometry has been applied to receptor sc reening as a technique for the analysis of combinatorial libraries,146-148 but in this context complexation is often driven by solution-phase equilibria in complex matrices, complicating spectra and increasing the likelihood of signal suppression from competing analytes. All of the above suffer from an inability to refine experimental design on a real-time basis fo r faster method development. A fast mass spectrometric screening method for supramolecu lar receptors with an appropriately soft ionization technique, manipulab le chemical conditions, and minimizing interference from competing species would address some of these issues. The DESI technique appeared promising for s upramolecular applications initially because of its reported similarity to ESI characteristics in the ionization of some analytes well-known to 113

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be amenable to electrospray ionization. Sample accessibility throughout analysis confers on the technique a major advantage in the form of react ive DESI, a variant of the technique in which reagents are incorporated into the spray soluti on for reaction with the de posited analyte at the sampling surface. Reactive DESI using hydrochloric acid and trifluoroacetic acid has been shown to improve selectivity and detection limits.120,121 Traditional DESI is not well suited to the study of supramolecular complexation because deposition and drying of pre-mixed host and guest can add the complication of close-crystal packing forces to the analysis, potentially resulting in the detection of complexes which would not fo rm under useful and realistic conditions. Reactive DESI, however, does not incor porate this disadvantage and so is the more promising configuration of the t echnique for this application. Conventional DESI has been applied to an enormous number of analytes, including pharmaceuticals, chemical warfare ag ents, plant alkaloids, and lipids.116 Large biomolecules such as cytochrome C have been s hown to maintain nativelike folded conformations during DESI with appropriate conditions,149 providing evidence for the applicability of DESI to supramolecular systems. Reactive DESI, pre dominantly incorporating alkali metal and ammonium cations as spray reagen ts but also using chloride ani ons, has been reported for the analysis of pharmaceuticals and metabolites,140 and for cation adduction io nization of explosives and chemical warfare agents.150 Trace detection of the explosiv e triacetone triperoxide further demonstrated the utility of reactive DESI for analysis of alkali metal complexes,151 and heterogeneous reactions between ions formed by the DESI spray head and solids deposited on a surface have been shown to yield covalent ly-bound products identified by mass analysis.152,153 A natural progression, therefore, is the application of reactive DESI for receptor screening in smallmolecule host:guest inclusion complex studies. Very recently, a reactive DESI supramolecular 114

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screen for the determination of counterfeit antimalarial ta blets was described by Nyadong et al. ,154 but to date DESI has not been applied to th e analysis of cyclodext rin host:guest inclusion complexes. Additionally, no direct comparis on between DESI and ESI for supramolecular applications or for characterization of solu tion-phase representative data has been made. Cyclodextrins, cyclic polymers of monosacch arides, are extremely well-characterized toroidal molecules with a hydrophilic exterior and a hydrophobic intramolecular cavity in which a small neutral guest molecule can be encircled to form a true inclusion complex which selfassembles under suitable solution-phase conditions.26,155 The cyclodextrins have been shown to complex a multitude of guest species and several ESI-MS studies of this behavior have been reported,19,143,144,156-158 making them a useful model sy stem for reactive DESI method development. cyclodextrin is composed of seven 1,4-linked glucopyranose units and consequently has a versatile cavity size for sma ll molecule encapsulation. It has been shown to house various pharmaceuticals and nitro-compounds among other candidates. Its versatility led to its selection as the test hos t compound. The initial test guest nor testosterone was selected for the documented ability of simila r steroids to complex with cyclodextrin144,159 and the guests used in the screening analysis were chosen on th e basis of size and appropriate functional groups. Experimental Sample Preparation All solutions were prepared using HPLC grade solvents (Honeywell Burdick & Jackson, Muskegon, MI). cyclodextrin (Trappsol, CTD, Inc., High Springs, FL) was diluted to 0.05 mM with 50:50 methanol:water. Nortestoster one was used for proof-of-concept and the twelve screened potential guest compounds were acenaphthalene (G1), acetanilide (G2), caffeine (G3), 4-chloro-3-nitrobenzonitrile (G4), cyclam (G5), 1,3-dinitronaphthalene (G6), 115

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diphenylglyoxime (G7), formanilide (G8), guanosine (G9), L-arginine (G10), 2-methylnaphthalene (G11), and 4-nitroacetanilide (G12) shown in Figure 5-1 (G5,G10: Acros Organics, ThermoFisher Scientific, Waltham, MA; all others: Sigma-Aldrich, St. Louis, MO). They were selected as potential guests on the basis of their sizes and appropriate functional groups. Mass Spectrometry In preparation for the DESI experiments, the guest compounds were diluted to 1 mg/mL, approximately 5 mM, in 50:50 methanol:water, spotted in 10 L aliquots onto a sandblasted glass slide and allowed to dry at ambient temp erature and pressure for 30 minutes. The DESI spray solvent was a 0.05 mM cyclodextrin solution in 50:50 or 80:20 methanol:water. ESI samples comprised 0.05 mM cyclodextrin in either methanol or 50:50 methanol:water with one guest compound at 1 mg/mL. Maltohexaose (Supelco, Bellefonte, PA), 1 mg/mL in 50:50 methanol:water, was used to test for ESI artifact s. For all ESI experiments, 0.5% formic acid was incorporated into the solvents, and the cyclodextrin solution used as the DESI spray solvent contained 0.5 mg/mL of NaCl; both additives (ThermoFisher Scientific, Waltham, MA) were used to improve current stabil ity and ionization efficiency. Mass spectra were acquired using an Agilent 6210 MSD Time-of-Flight mass spectrometer configured for ESI (Agilent Technologies, Inc., Santa Clara, CA). Acquisition of DESI spectra required an external syringe-pump connected to a home-built DESI source, for which the desolvation capillary of the TOF was adapted us ing a specifically designe d capillary extender to enable sampling close to the desorption surface. Th e capillary extender was not flared as in the Bruce design,137 but its orifice was three times the diamet er of that of the desolvation capillary. DESI experiments were performed using a spray flow rate of 3 L/min and 100 psi head 116

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pressure of nebulizing N2(g). A voltage of 4 kV was applied to the spray solution via an external 210-10R high voltage power supply (Bertan Asso ciates, Hicksville, NY), while the sample surface was held at ground and the desolvation capillary at 100 V. The spray needle was positioned 1.5 mm above the deposition surface at a 55 angle from the horizontal. A schematic of the experimental configurat ion is included as Figure 5-2. ESI mass spectra were acquired using a 5 L sample loop injection for sample introduction, with a flow rate of 0.5 mL/min a nd a desolvation capillary voltage of 3.5 kV. The drying gas temperature was 325 C and the fragmentor voltage was held at 175 V for all experiments. Reference mass correction was en abled throughout the ESI data acquisition to maximize mass accuracy. Nuclear Magnetic Resonance Spectroscopy An Inova NMR spectrometer operating at 500 MHz was used for acquisition of all 1H spectra (Varian, Inc., Palo Alto, CA). Deuteriu m oxide (ThermoFisher Scientific, Waltham, MA) was selected as the solvent and proton chemical shifts were referenced to the HDO signal at 4.780 ppm. Samples consisting of 1 and 2 mg/mL of a single guest were an alyzed alone and in the presence of cyclodextrin (ThermoFisher Scientific, Waltham, MA). A titration approach covering the concentration range of 0.5-20 mM cyclodextrin was employed for each guest to maximize chances of complex detection. Th e 2D correlation spectroscopy (COSY) NMR technique was used for unambiguous proton assi gnment, and 2D nuclear Overhauser effect spectroscopy (NOESY) was employed to elucidate the conformation of the -cyclodextrin:acetanilide comple x for validation of the 1D a pproach to inclusion complex confirmation. For a basic overview of basic two dimensional NMR techniques the reader is directed to a review article by Reynolds and colleagues.160 117

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Theoretical Calculations Optimized structures for the guest co mpounds were obtained using Gaussian 03161 with the HF/6-31G* method and basis set. Corresponding dimensions were measured using Molekel 5.0, the latest version of a free molecular visualiz ation software package developed by the Swiss National Supercomputing Cent er (Manno, Switzerland). Results and Discussion Reactive Desorption Electrospr ay Ionization Receptor Screening To confirm the applicability of reactive DESI for the analysis of cyclodextrin inclusion compounds, a steroid guest, nortest osterone, was used as a mode l. Steroid and cyclodextrin host:guest inclusion complexes have been well-characterized and the nortestosterone was readily available. The host compound, cyclodextrin, was sprayed in meth anol:water at the deposited nortestosterone guest under the conditions prev iously described. The methanol:water solvent system was used here because a major applicat ion of cyclodextrin incl usion is the aqueous solvation of small molecules. Ther efore, it seemed appropriate to demonstrate the utility of this screening technique using likel y real-world conditions. The cyclodextrin:nortestosterone 1:1 complex was observed and confirmed using ex act mass, showing that complexation on a timescale suitable for the reactive DESI host:guest interaction can occu r. A representative spectrum is shown in Figure 5-3. Following this proof-of-principle experime nt, the receptor screening analysis was performed for all twelve potential guest com pounds illustrated in Figure 5-1 using the reactive DESI setup described in the experimental sect ion. The sample surface which was mounted on an x-y-z-stage was manually moved in the x-direction (see Figure 52) for spatial guest selection, and blank areas between deposited guests were used for blanks. No carryover problems were 118

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experienced. The guests G2, G3, G8, and G12 we re detected at m/z ratios corresponding to theoretical 1:1 complexes with the sodiated cyclodextrin host. None of the other eight guests exhibited any evidence of comp lexation via reactive DESI-TOF-M S. Representative spectra are shown in Figure 5-4 and summarized results are presented in Table 5-1. Sample preparation took about ten minutes, but an additional thirty minutes of drying ti me were required. Instrumental acquisition time was only twelve minutes including sampling of the blanks. A concern with this experimental design was that the solvent system could be responsible for false negative results as several of the guests are only sparingly soluble in water, the primary component of the electros prayed droplets of the cyclodextrin solution by the time they reach the deposition surface. One of the major benefits of DESI, its ambient nature, was exploited to rapidly explore this issue. The only alteration necessary to expl ore the effect of the solvent system was replacing the origin al spray solution with one containing a higher percentage of methanol. This was done at the end of the screen adding fourteen minutes to the acquisition time, but again only G2, G3, G8, and G12 were detected as complexes. Electrospray Ionization Receptor Screening For validation of the DESI scree n, the twelve guests were scr eened using ESI, which is an accepted ionization technique for this type of work, in methanol:water solvent. Under these conditions, compounds G2, G5, G7, G8, G9 and G 10 were observed to form detectable 1:1 complexes, confirmed via accurate mass calcula tions, while the other five guests showed no evidence of complexation. Figure 5-5 shows repr esentative mass spectra for analysis of G2 which complexed and G4 which did not for comparison, and results of the ESI screen are summarized in Table 5-2. Similar relative intens ities between the sodiated cyclodextrin and the sodiated complex using both DESI and ESI were noted. Again, solvent system effects were a 119

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concern, so the screen was repeated using onl y methanol for dissolution. Similar results evidencing only marginal changes in signal intensity were obtained. The entire twelve compound screen took fift y-one minutes of acquisition time, including blank injections, and approximately ten minutes of sample preparation time resulting in an analysis time of sixty-one minutes excluding data analysis. Changing the solvent system required preparation of new samples and repetition of th e entire screen, doubling the total analysis time. Although the total analysis time for the DESI scr een was significantly longer than for the ESI analysis, this is solely due to the drying tim e for the deposited guests which does not contribute to either labor or instrument time. Appropriate automation and efficien t planning in terms of running screening analyses during drying time for s ubsequent screens would yield a considerable improvement in analysis time over the analogous ESI experiment. Comparison of the results obtained using DESI and ESI highlighted significant disparities, challenging the validity of the reactive DESI screen. Both techniques resulted in complex detection for two of the guests, G2 and G8, and four guests, G1, G4, G6, and G11, were not detected in complex form using either techni que. Conflicting results were obtained for the six remaining guests. Compounds G3 and G12 were de tected as complexes by DESI only, and the other four guests were only observed to comple x using ESI. Electrospra y ionization is known to be prone to artifacts,143,144 nonspecific aggregates formed during the electrospray process and consequently detected as adducts. This is a sign ificant issue in supramolecular receptor screening where artifact formation can potentially lead to identification of false positive receptor candidates, and it may provide a potential explanation for the conflicting results. Initially, maltohexaose, a linear sugar composed of six glucopyranose units, was substituted for the cyclodextrin and another ESI screen perfor med to identify nonspecific complex formation 120

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following the strategy of Bakhtiar and Hop.144 This strategy, however, yielded inconclusive information and highlighted the need fo r a more rigorous validation approach. Proton Nuclear Magnetic Resonance Spectroscopic Screening ESI has been shown to be representative of solution-phase equilibria in some circumstances,76,77 but the relationship between ESI sp ectra and solution-phase chemistry remains under scrutiny. A recent study investiga ting the influence of shrinking electrospray droplets on chemical equilibria reported observation of a significant effect on the equilibrium for one fluorescent dye pair, yet no apparent effect on the monomer-dimer equilibrium for a different fluorescent species.162 Consequently, the host and gues ts previously described were analyzed using solution-phase 1H NMR spectroscopy to determine which of the ionization techniques yielded the results most consistent with solution-phase behavior. Conditions used for the NMR screen are delineated in the experiment al section. The experiments were conducted in deuterium oxide, consistent with the composition of the DESI dr oplets upon interaction with the surface, as deuterated methanol gives rise to an interferent peak within the spectral range of interest. Acquisition of each spectrum took about five minutes, so one hour was required for the entire screen, omitting sample preparation and da ta analysis which took approximately one more hour. Previous studies have shown that the 1H NMR signals corresponding to the -cyclodextrin cavity protons exhibit significant upfield shift upon inclusion of a guest molecule into the cavity.163,164 Proton NMR spectra for -cyclodextrin and each of the twelve guests were obtained at the concentration ranges prev iously described. Proton assignments were made with the aid of COSY data, and the labeling convention is detail ed in Figure 5-6. Representative spectra are presented as Figure 5-7. NMR spectroscopy revealed evidence, in the form of a spectral shift of 121

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the signals corresponding to the H3, H5, and to some extent H6 protons of -cyclodextrin, of true inclusion complex formation for the guest s G2, G3, G8, and G12. The H3, H5, and H6 protons are the protons which line the surface of the hydrophobic cavity of the toroidal host, and therefore would be the protons experiencing environmental change in the event of true inclusion of a guest. No evidence of inclusion of the other eight potential guests was observed. As a secondary validation technique for the evid ence of inclusion complexation between -cyclodextrin and the guests under investigatio n, 2D NOESY was used to determine complex conformation. Proton assignments for the acetanilide guest are shown in Figure 5-8. An expanded view of the NOESY data obtained for the -cyclodextrin:acetanilide complex is presented as Figure 5-9. NOESY da ta indicated interaction between the host cavity protons and the ortho and meta protons on the acetanilide benzen e ring, which revealed the complex conformation illustrated in Figure 5-10. It was expected that the aromatic portion of the acetanilide would be encapsulated upon complexa tion as it is the most hydrophobic region of the guest, so the 2D NOESY derive d complex conformation supporte d the predicted complex, and validated the simpler 1D approach to inclusion complex determination which was used to screen the twelve potential guests. Solution-phase NMR spectroscopic screening yi elded identical results to those obtained via DESI. The four guests detected as 1:1 complexes in the DESI screen were validated as included guests in solution and the eight remaini ng guests which were not detected as complexes via DESI showed no evidence of inclusion in th e NMR experiments. Conversely, the ESI screen proved erroneous for 50% of the gue sts, resulting in the detection of two false negatives and four false positives. A summary of results obtained for the three screening methods is presented as Table 5-3. 122

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The false positives were thought to be attr ibutable to the aforementioned nonspecific adduct formation commonly associated with ES I. Computational chemistry was used to investigate this hypothesis and is discussed in de tail in the following section. Determination of the two false negatives was more difficult to expl ain. Interestingly, the tw o guests detected using NMR and DESI but not ESI exhibited the smalle st upfield shifts in the NMR experiments. Assuming the magnitude of the signal shift is related to the extent of complexation, an assumption for which there is some precedent,165 this would mean that these two guests are the least thermodynamically favorable. The organic me thanol solvent is in competition with the guests for inclusion in the hydrophob ic host cavity, and in ESI is pr esent in higher concentration at the time of host:guest interact ion than in DESI. Consequently, for weakly binding guests, the methanol could preferentially move into the cavi ty, preventing inclusion of the weaker guests during the DESI screen. As pr eviously discussed, this prob lem would be minimized using reactive DESI. The droplets in teracting with the guest compounds would be predominantly aqueous, a substantial fraction of the more volat ile organic phase having evaporated during the electrospray process. The NMR screen showed that reactive DESI is less prone to adduct fo rmation resulting in the detection of false positives in the screen ing of supramolecular complexes than ESI. Additionally, since all fo ur complexes determined via NMR were identified in the DESI screen, there is no evidence to suggest that complexation during the DESI event is purely kineticallydriven. In fact, if the extent of NMR signal shift is taken as re presentative of the extent of binding, it appears that the DESI sc reen spectral intensities may ev en be consistent with relative binding constants. 123

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Computational Chemistry To investigate the theory that the four fals e positive inclusion complexes detected solely using the ESI screen were nonspecific adducts of gue st exterior to the host cavity, structures of the twelve guests were optimized using the th eoretical calculation method previously outlined. The host compound, -cyclodextrin, has known dimensions, and the cavity diameter at its widest point is only 6.5 Angstroms (see Fig. 5-11). Since the potential guests were selected only on the basis of potentially appropriate molecu lar weight and functional groups for -cyclodextrin inclusion, optimized structures and specific molecular dimensions were desirable to determine whether the screened com pounds were actually viable inclusion guests. The calculations revealed that eleven of the guests could theoretically a dopt conformations suitable for at least partial encapsulation by the host. Cyclam (G5), however, wh ich has a relatively ri gid cyclic structure, cannot fit into the -cyclodextrin cavity; its optimized structure, with the corresponding dimensions, is presented as Figure 5-12. Repulsi on between the hydrogen atoms on the inside of the ring hinders the molecule from adopting a na rrower conformation for inclusion. The cyclam guest exceeds the internal cavity diameter of -cyclodextrin in two dimens ions; therefore, it is only capable of forming a nonspeci fic complex exterior to the cav ity and complex detection must be due to nonspecific artifact formation. The -cyclodextrin:cyclam complex was only detected in the ESI screen experiment and not during e ither the DESI or the NMR screens, providing evidence for the formation of nonspecific adducts resulting in false positives using ESI, but no evidence to suggest that DESI suffers from a si milar drawback in this type of application. Conclusions Initially, evidence of complexation between sprayed -cyclodextrin and deposited nortestosterone steroid, a member of a known family of steroid guests for the cyclodextrin host, 124

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confirmed that inclusion complexation on a suita ble timescale for the DESI heterogeneous ionmolecule reaction is possible. A subsequent sc reening experiment for inclusion of a group of twelve deposited potential guest compounds by the same sprayed host yielded four positive and eight negative results. The appearance of an ion peak at the exact m/z corresponding to the protonated or sodiated complex following the in teraction was necessary for a positive result, while absence of a peak at the expected m/z yi elded a negative result. The guests observed to complex during the reactive DESI event we re three anilide compounds and caffeine. An analogous screen using ESI, which has b een widely reported for supramolecular applications, was conducted. Solutions of pr e-mixed host and an individual guest were electrosprayed and again a previously absent peak at the exact m/z of a complex was taken as a positive result. The ESI screen identified six comp lexes, only two of which had previously been observed using DESI-MS. Disparities between th e ESI and DESI screens highlighted the need for further validation, as ESI is known for a te ndency to form nonspecific adducts which can result in false positives in supramolecular applications. Solution-phase validation via NMR was used to assess which of the two ionization techniques yielded more representative data. Upfield shift of the chemical shift values of NMR signals corresponding to the -cyclodextrin protons known to comprise the surface of the hydrophobic cavity upon guest addition was used to c onfirm true inclusion complexation of four of the potential guest molecules. A lack of shif t of the host signals in the presence of guest indicated that inclusion of th e guest into the hydrophobic host cav ity was not detected and was recorded as a negative result. Two dimensiona l NMR techniques were used to confirm guest inclusion and conformation of detected comple xes. The NMR screen yielded identical positive 125

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and negative results to the DESI screen, conf irming the technique as superior to ESI for supramolecular applications. The two weaker binders of the four guests observed as inclusion complexes by DESI and NMR were not detected in complex form using ESI. This suggests that ESI may not be as sensitive as reactive DESI for supramolecular complex screening. Further investigation is necessary to corroborate this clai m. Computational chemistry helped confirm that ESI is more prone to the formation of nonspeci fic artifacts detected as fals e positives in this type of application than reactive DESI. The utility of reactive DESI-MS for rapid su pramolecular complex r eceptor screening has been demonstrated and the technique has been compared to ESI-MS, an accepted tool for supramolecular mass spectrometry. Comparison betw een results obtained via the DESI and ESI screens and solution-phase NMR data revealed the superiority of DESI for supramolecular complex guest screening. Reactive DESI has been confirmed as a promising useful tool for supramolecular mass spectrometry. 126

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Table 5-1. Absolute intensities (x104 counts) and ion type observed with the DESI-TOF-MS screen in methanol:water solvent Guest [M+H]+ [M+Na]+ [CD+M+H]+ [CD+M+Na]+ [CD+G+2H]2+ G1 G2 0.35 6.6 0.77 G3 71 6.0 0.12 G4 G5 5.0 2.0 G6 G7 G8 1.7 50 0.20 G9 G10 1.4 G11 G12 8.0 4.0 0.28 127

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Table 5-2. Absolute intensities (x104 counts) and ion type observed with the ESI-TOF screen in methanol:water solvent. Guest [G+H]+ [G+Na]+ [CD+G+H]+ [CD+G+Na]+ [CD+G+2H]2+ G1 G2 120 16 0.22 0.06 G3 140 50 G4 G5 180 12 1.4 G6 0.09 G7 8.0 2.0 0.20 0.02 G8 80 2.3 0.10 G9 13 53 1.8 G10 140 20 1.5 0.27 G11 G12 9.7 0.79 128

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Table 5-3. Potential guests detected as comp lexes using DESI-MS, ESI-MS, and 1H NMR spectroscopy. Guest DESI-MS ESI-MS 1H NMR G1 G2 Complex detected Complex detected Complex detected G3 Complex detected Complex detected G4 G5 Complex detected G6 G7 Complex detected G8 Complex detected Complex detected Complex detected G9 Complex detected G10 Complex detected G11 G12 Complex detected Complex detected 129

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Figure 5-1. Structures of the twelve screened guest com pounds: G1 is acenaphthalene (monoisotopic mass 152.0626 Da), G2 is acetanilide (135.0684 Da), G3 is caffeine (194.0804 Da), G4 is 4-chloro-3-nitrobe nzonitrile (181.9883 Da), G5 is cyclam (200.2001 Da), G6 is 1,3-dinitronaphthalene (218.0328 Da), G7 is diphenylglyoxime (240.0899 Da), G8 is formanilide (121.0528 Da), G9 is guanosine (283.0917 Da), G10 is L-arginine (174.1117 Da), G11 is 2-methylnaphthalene (142.0783 Da), and G12 is 4-nitroacetanilide (180.0535 Da). 130

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Figure 5-2. Receptor screen DESI-MS design show ing spray head, sample surface, desolvation capillary, and capillary adapte r. The host solution was 0.05 mM cyclodextrin solution in 50:50 methanol:water contai ning 0.5 mg/mL of NaCl and the twelve guests G1-G12 were deposited on a sandblaste d glass slide for spectral acquisition. Figure 5-3. Reactive DESI-TOF spectrum of cyclodextrin sprayed onto a deposited sample of nortestosterone. The inset shows a closeup view of the mass range of interest. 131

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Figure 5-4. Reactive DESI-TOF mass spectra showi ng data for the guests a) G2, b) G7, and c) G8. Insets show close-up views of th e complex ion mass ranges of interest. 132

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Figure 5-5. Electrospray ioniza tion TOF mass spectra for the cyclodextrin(CD) guest screen in methanol shown for potential guest com pounds a) G2 and b) G4 which yielded positive and negative complexation data, respectively. Figure 5-6. Structure of th e glucopyranose monomer of -cyclodextrin showing the labels used to represent the protons observed via NMR spectroscopy. 133

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Figure 5-7. Proton NMR spectra showing the regi on 3.5-4 ppm chemical shift for a) lone -cyclodextrin and b) -cyclodextrin in the presence of acetanilide (G2). The signals corresponding to H3, H5, and to some extent H6, exhibit an upfield shift consistent with inclusion of a guest upon the addition of acetanilide. 134

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Figure 5-8. Structure of the acetani lide guest, G2, showing the observed chemical shift values for the NMR spectral peaks corresponding to the stable protons. Figure 5-9. Nuclear Overhaus er effect spectrum of -cyclodextrin in the pres ence of acetanilide. Signal at the intersection of a line corresponding to the acetanilide with a line corresponding to the host i ndicates correlation. The signal diameter is indicative of the strength of the thro ugh-space interactions. Figure 5-10. Conformation of the -cyclodextrin:acetanilide inclus ion complex derived using 2D NOESY. 135

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Figure 5-11. Representation of the toroidal -cyclodextrin host showing cavity diameter. Figure 5-12. Optimized structure and correspondi ng molecular dimensions of cyclam, the G5 guest which was observed to complex usi ng ESI supramolecular complex screening only. 136

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CHAPTER 6 CONCLUSIONS AND FUTURE DIRECTIONS Mass spectrometry, currently experiencing an explosion of applica tions, has become an essential tool in the developm ent of the field of supramolecular chemistry. Its sensitivity, specificity, and ability to obtai n information about the inherent supramolecular chemistry of systems in the absence of solvent interferences make it a powerful analytical technique with the potential to fuel rapid advances in this area. The research presented here was conducted to contribute to the evolu tion of supramolecular mass spectrometry as a technique which can be diversified to address very different needs arising from the growth of the multidisciplinary field of supramolecular chemistry. Th e major unifying theme is that specific instrumental characteristics and capabilities can be exploited to yield usef ul information beyond elemental composition or structural elucid ation, with a focus on expandi ng the scope of supramolecular mass spectrometry. Despite advances in mass spectrometric inst rumentation and software for instrumental control and data analysis, the efficient incorpor ation of mass spectrometry into many labs is limited by the tendency to use mass spectrometers as workhorses where generation of large amounts of data is perceived as more desirable than the acquisition of useful and meaningful information. For some applications, this may be an appropriate use of th e techniques comprising mass spectrometry as a whole, but in an ec onomically-driven society, this poses several problems. Primarily, valuable resources of instru ment and scientist time can be wasted through poor experimental design. Additional analysis time for superfluous tandem mass spectrometry stages, and time spent wading th rough unnecessary data acquired due to inefficient planning are a few common examples of laboratory waste. Additional concerns are data handling and storage. Generation of large amounts of da ta increases the time and resources needed to extract useful 137

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information, but also creates pressure in terms of hardware and software laboratory requirements. Many labs, particularly those in biomedical areas, would bene fit significantly in terms of efficiency and productivity by employing a trained and experienced instrumentalist for experimental design and effici ent utilization of complex analytical instrumentation. This research was initially directed toward the characterization of novel designed synthetic receptors for alkali-metal cations based on natu rally-occurring phlorogluc inol using accepted supramolecular mass spectrometric techniques. Rela tive binding constants were determined for the homodimers of two phlorogl ucinol derivatives bound by Li+, Na+, K+, and NH4 + using an SCID-ESI dissociation curve approach. The preferential binding order for 2,4,6-tribenzoylphloroglucinol (phlor o 1) was determined to be Li+ Na+> K+>NH4 + and for 2-4-6-(3,5-dimethyl)tribenz oylphloroglucinol (phloro 2) was revealed as Na+> K+> Li+>NH4 +. This size-dependent binding for phloro 1 but not phloro 2 supported the hypothesis that phloro 1 should be capable of forming a cage-type dime r encapsulating an alkali metal cation in the intermolecular cavity, while the additional methyl groups on the benzoyl substituents of phloro 2 should sterically hinder that di meric conformation. No direct conformational information was obtained via mass spectrometry, so UV/vis absorbance spectroscopy was used to probe dimeric conformation in solution. Chromic shifts co rresponding to dimerization provided further evidence supporting the hypothesis. In this projec t, mass spectrometry and solution-phase optical spectroscopy yielded complementary information, but the charac terization might have been achievable solely via mass spectrometric techniqu es, had ion mobility spectrometry (IMS) been available for determination of the dimeric cross-sectional areas. Characterization of the phlorogl ucinol derivatives as designed -receptors benefits supramolecular chemistry in several ways. A significant step towards providing a new synthetic 138

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receptor for encapsulation of alkali metal cations which has implications in applications like cation scavenging in waste cleanup and active tr ansport mimicry, has been made. Additionally, the effect of the addition of small substituents, like methyl groups, upon the nature of noncovalent -system interactions has been shown to be dramatic. This fact highlights the need for more in-depth investigations into the effect s on chemical behavior of substituent modification of arene systems, and could ultimately impact synthetic receptor design strategies based on molecular recognition principles. On a more general level, the complementary nature of solutionphase optical spectroscopic techniques and mass spectrometry was demonstrated for supramolecular applications. A useful addition to the work presented here would be the implementation of an IMS study to explore the dimeric conformations. Following m easurement of the cross-sectional area of the homodimers of phloro 1 and phloro 2 in the pr esence of each of the four cations, comparison between data obtained for each host in the presence of the same guest should enable determination of the dimeric conformations. However, a vital future step for this area of research is expansion of the study to a larger group of phloroglucinol derivative hosts substituted with different heteratoms and functional groups. Comp arison of dimeric stability using the SCID dissociation curve approach, for a much greater number of host compounds could significantly benefit the field of synthetic design based on supramolecular recognition principles. The group would have to be large enough to allow the determ ination of statistically relevant patterns of behavior related to specific type s of substitution. If patterns and trends in relative complex stability were observed to correla te with substituent characteris tics, this could significantly impact molecular-recognition base d designed chemical synthesis, resulting in simplification of the therapeutic drug design process. 139

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Dissociation pathways were proposed fo r ESI-CID-MS analysis of the cation-bound phloroglucinol derivative di mers. With the aid of the simple 2-hydroxybenzophenone compound, a mechanism for the initial dissociation step was derived, and the more complicated C3 phloroglucinol derivatives were observed to adhere to this mechanism. The ammonium adducted phloroglucinol derivatives demons trated slight differences in fragmentation behavior from that observed with the other cations employed. Ion tr ap mass spectrometry was used for unambiguous determination of the precurso r-product ion relatio nships, and SCID tandem mass spectrometry using one of two high-resolution mass analyz ers provided accurate mass measurements for unambiguous determination of elemental compositi on. The two useful instru mental capabilities of tandem mass spectrometry and high-resolution mass analysis were incorporated into the analysis to maximize the usefulness of the mass spectral data acquired. The growing pharmaceutical relevance of the phloroglucinol derivative family provided a need for this information, as LC-MS and LC-MS/MS analytical methods for determination of this type of compound in various matrices are under rapid development. The most common interface between LC and MS is ESI, and alkali-metal adducts are frequently observed via ESI, so fragmentation information for alkali-metal adducts is vital. The wealth of applications for phloroglucinol derivatives outlined in Chapter 3 renders dissociation pathway information specific to alkali-metal adducts of phlorogluci nol derivatives of subs tantial significance. The contributions of a proposed mechanism for the initial fragmentation step and dissociation pathways for the alkali-metal cation-bound dimers and monomeric adducts have been made, but future application-specific method devel opment could benefit from further insight. Now that the first mechanism for the initial dissociation of alkali-metal cation-adducted phloroglucinol derivatives has b een proposed, the next logical st ep would involve a detailed 140

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mechanistic study. Exchange of one or all of the 1H atoms of the hydroxyl groups with 2H followed by an analogous fragmentation study would help to clarify the location of the hydrogen which is abstracted as part of the loss of th e benzene or dimethylated benzene ring from the monomeric phloroglucinol derivative adducts. Additionally, in a separate study, strategic replacement of successive 12C atoms with 13C atoms could be used to investigate the structural reconfiguration observed via ca rbon dioxide neutral loss from the monomeric sodium and lithium cation adducts of the two host molecules. Both these experiments would provide detailed information about the dissociation of phloroglucin ol derivatives which could be valuable for LC-MS/MS method development for the quantitativ e and qualitative analysis of this compound class. An unrelated experiment could help to determine one missing and vital piece of information which could potentially yield insight into the cationinteractions which are involved in ESI adduction by metal cations. The location of the cation, although clearly in the core phloroglucinol region, was not determined by the studies comprising this body of work, but a possible strategy to resolve this issue has been devised. A combina tion of computational chemistry and gas-phase infrared ion spectro scopy could yield the desired information. Theoretical geometry optimization of the phlorog lucinol derivative hosts with an alkali-metal cation in one of a select few positions would enable determination of the adduct conformations corresponding to the lowest energy structures. Theoretical vibrational spectra for each structure could then be obtained. Gas-phase ion spectroscopy using an infrared laser for multiple photon dissociation of the elec trosprayed alkali metal cation-adducte d phloroglucinol derivatives would provide experimental vibrational spectra. Compar ison between the theoretic al and experimental 141

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vibrational spectra could confirm the location of the cation. This is a project for which our group has both the resources and the expertise. Prior to applying the recently-developed ambien t ionization technique DESI to the analysis of supramolecular inclusion complexes, a home-built DESI source was interfaced with an Agilent TOF-MS instrument. The source was originally designed for a Bruker FTICR-MS instrument, so several modifications were neces sary to accommodate the unusual geometry of the Agilent source housing. Commercial DESI sources have yet to be su ccessfully interfaced with the Agilent TOF instruments, so this work provides a basic appr oach for possessors of Agilent TOF mass spectrometers aiming to incorpor ate the DESI technique into their work. The superior scan rate of TOF-MS over FTICR-MS was observed to be useful for DESI parameter optimization, demonstrating a significant a dvantage of TOF mass analyzers over FTICR mass analyzers for DESI analyses. Successful conf iguration of the DESI-TOF-MS system was demonstrated using Rhodamine 6G and the protein cytochrome c. The next step in characterization of the DE SI-TOF-MS configuration described in this dissertation is the demonstration of its performance for a divers e range of compound classes. An optimization study resulting in the determination of optimal conditions and surface chemistry for structurally-related compounds such as different ially substituted benzenes or carbonyls could provide useful insight into the effect of different functional groups on the DESI process, and would aid the wider implementation of the DESI technique. Additionally, the high mass range capabilities of the TOFMS make it very appropriate for the study of large organometallics, which have not been extensively analyzed by DE SI, so a future collaborative project with a prolific inorganic synthetic chemist could prove extremely successful. Many large metal 142

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complexes degrade in the presence of acid and DE SI is an ionization technique that does not necessarily require acid addition. The potential of the DESI source interfaced with the FTICR-MS instrument was not thoroughly explored, and recent successful conf iguration of DESI-FTICR-MS by several groups suggests that the challenges encountered are not insurmountable. The system should be revisited and the optimization procedure exhaustively perfor med using a standard that is not as rapidly ablated as Rhodamine 6G. During the cyclodextri n work, it was observed that both caffeine and acetanilide yield a relatively long-lived and stable DESI signal; therefore, they could prove to be appropriate analytes for this purpose. Successful integration of the DESI source with the University of Florida FTICR-MS could result in some very exciting wo rk involving infrared dissociation of trapped ions produ ced using the DESI technique. To date, neither DESI followed by infrared multiple photon dissociation nor DESI followed by gas-phase ion spectroscopy have been reported. Since DESI-FTICR-MS is very de sirable for proteomics applications, protein conformation studies using this setup seem a logical and attainable aim. The reactive DESI technique was subsequent ly used for the development of a rapid screening technology for supramolecular receptors for inclusion by -cyclodextrin hosts. Following deposition of several potential guest co mpounds onto a desorption surface, successful pickup of some of the guests by the host, observed via detection of an ion associated with the specific host:guest complexes, during the DESI event was achieved. An analogous screen using electrospray ionization resulted in the detection of complexe s of different guests to those observed to complex via DESI, and illustrated the need for solution-phase validation of the DESI technique for this application. Proton NMR spectroscopic tec hniques yielded data corresponding to the DESI results and invalida ting the ESI results, and comput ational chemistry showed that 143

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some of the complexes detected via ESI could only be nonspecific adducts. Reactive DESI was confirmed as a valuable analytical technique for supramolecular screen ing, providing solutionphase-consistent data, and was demo nstrated as superior to ESI for the screening of guests for -cyclodextrin inclusion complexes. Some additional information would greatly e nhance the diversity of potential analytical applications for this technique. Consequently, some suggestions for future experiments are delineated here. The molecular reco gnition interaction between sprayed -cyclodextrin and nortestosterone was initially employed to demons trate that inclusion co mplexation can occur on the DESI timescale. An attempt at inclusion complexation of several other hormones was made concurrently, but proved inconclusive. The hormones estriol, estrone, dehydrotestosterone, testosterone, and epitestosterone were deposited, but no evidence of desorption was observed. It remains unclear, however, whether the age of th e compounds, which had been stored for several years under unknown conditions, was responsible for the lack of signal obtained with these hormones. The fact that no uncomplexed signal wa s observed for any of th ese analytes suggests the possibility that unfavorable storage condition s could have led to degradation. It would be extremely useful, however, to determine if this were the case using anothe r ionization technique. A comprehensive analysis of the inclusi on complexation of new samples of these hormones, particularly the testosterones, usi ng a variety of surface chemistries and geometric configurations would be a valuab le addition to the data presented here. To increase the chances of successful complexation, a derivatized -cyclodextrin such as hydroxypropyl-cyclodextrin could be used as the spray reagent. Comparison of the complexation behavior of the related guest molecules would be extremely useful both to de termination of the scope of this specific technique and to characterization of the capabilitie s and limitations of reactive DESI in general. 144

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Real-world matrix effects on reactive DESI for supramolecular complexes have yet to be explored. Development of this type of analytic al technique in the area of metabolite screening and toxicological applications would require molecular recogniti on interactions during the DESI process to be robust in biological matrices. A very preliminary experiment to gauge the adaptability of the technology to this type of work would be a simple screen using -cyclodextrin sprayed at deposited spots of various compositions. Since nortestosterone is known to complex under reactive DESI conditions, solutions of ur ine, blood plasma, and serum spiked with 2 mg/mL nortestosterone would be appropriate for initial test s. The previously-determined water:methanol testosterone solution would be a necessary control to ensure favorable conditions. Signal corresponding to therapeutic drugs has been obt ained using traditional DESI for all three of the suggested ma trices, as noted in Chapter 4. The supramolecular complex screening work pr esented here has evident potential in the development of high-throughput screening experime nts for lead optimization in drug discovery applications. The demonstrated benefits, a nd the simplicity, rapidi ty, and potential for automation, confer upon the described technology inhere nt analytical utility in this area. Prior to expansion into a marketable technique for this type of application, however, several important steps are necessary. In order to demonstrate the applicability of the technique to real-world receptor substrate chemistry, molecular recognition interactions be tween a known receptor and substrate must be determined to be specific. An experiment incorporating the recepto r region of some wellcharacterized protein into the spray for interac tion with two deposited substrates, one specific substrate for the receptor, and one similar substrate would address this issue. It would also 145

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demonstrate the utility of the technique fo r systems beyond the small molecule inclusion complex model system, which is essential to its development in biomedical applications. Following this, the logical approach would be a systematic study of one receptor with a family of known substrates. Subsequently, potential for different types of receptor molecules would have to be investigated. An additional step involves the determinati on of the effect of surface chemistry upon the molecular recognition interactions exploited in reactive DESI. To design and implement a screen that would be competitive with a 96-well plate optical spectroscopic assay, for example, the design of a specialized deposition surface would have to be carefully considered. An experiment observing complexation between sprayed -cyclodextrin and acetanilide on a variety of different surfaces including Teflon, polymethylmethacrylate, and porous silicon is essential to enable the future fabrication of a miniature sample holder along the lines of a MALDI target or desorption ionization on porous silicon (DIOS) chip. These surfaces have the advantage of amenability to the development of a miniature tr ay with shallow wells drilled or etched onto the surface. The -cyclodextrin and acetanilide syst em is suggested as the comple x system which resulted in the most intense and stable complex ion signal. Through careful experimental de sign and appropriate utilizatio n of different types of mass spectrometric instrumentation, contributions to the development of advanced supramolecular mass spectrometry have been made Several specific c ontributions have b een detailed and an attempt to define their real-world relevance has been undertaken. The overall goal of this dissertation, however, was promotion of the idea of maximizing the usefulness and efficiency of mass spectrometry as an analytical technique fo r diverse supramolecular chemistry applications, through the rational implementation of appropriate and complementary instrumental capabilities. 146

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BIOGRAPHICAL SKETCH Joanna Barbara was born and grew up in England, but moved to the United States with her husband and then 1-year old daught er to undertake an undergraduate degree in chemistry at the University of Florida in the spring of 2001. Gr aduating with her B.S. in 2003, and following the birth of her son, she remained at UF to cont inue her studies in gr aduate school, pursuing concurrently a non-thesis M.S and a Ph.D. in analytical chemistry. Following coursework completion, she began research in the area of supramolecular mass spectrometry under the joint direction of Dr. John Eyler and Dr David Powell, and worked as a research assistant in the Mass Spectrometry Service Lab throughout that peri od. Upon graduation, Joanna will enter the industrial workforce in a metabolism research position with XenoTech in Lenexa, Kansas. 156


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