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Carbohydrates and Amino Acids

Permanent Link: http://ufdc.ufl.edu/UFE0022819/00001

Material Information

Title: Carbohydrates and Amino Acids Infrared Multiple Photon Dissociation Spectroscopy and Density Functional Theory Calculations
Physical Description: 1 online resource (158 p.)
Language: english
Creator: Contreras, Cesar
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ab, amino, analytical, carbohydrates, density, hydrogen, infrared, irmpd, mass, physical
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: In addition to acting as repair agents, stabilizing protein folding or early defense systems in cellular systems, saccharides are especially important in energy storage and enzymatic reactions of proteins. All these areas of research require knowledge of the saccharide structure. Spectroscopic studies of monosaccharides and amino acids were undertaken to better understand structural conformations in the gas phase. Infrared spectra were obtained by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in conjunction with infrared multiple photon dissociation (IRMPD). Sodium cation-attached phenylalanine analogs were subjected to H/D exchange before their IRMPD spectra were taken. The gas phase H/D exchange experiments of N-acetylphenylalanine indicate that of the two possible locations for exchange to occur, the O-H hydrogen is kinetically favored over the N-H hydrogen. For two larger species, O-methyl N-acetylphenylalanine and N-acetylphenylalanine O-methylglycine, exchange occurred at the N-H site since it was the only one available for exchange, but the D for H substitution only took place in solution and not in the gas phase. Theoretical calculations showed that the phenylalanine analogs, although of different size, have relatively similar structural features. The sodium cation is predicted to interact with the phenyl ring and also bind to the carbonyl oxygens. In a second project, IRMPD spectra of N-acetylglycosamines showed that the frequency of the CO stretch was indicative of the particular glycosamine conformation. A band shift of about 10 cm-1 was seen between the anomers, alpha-D-methylglucosamine and beta-D-methylglucosamine, while an 11 cm-1 shift was seen for the galactosamine anomers. Calculations indicate that the O-methyl group's position (alpha and beta, or axial and equatorial, respectively) and its close proximity to the N-acetyl group cause the orientation of the carbonyl to change in order to minimize steric hindrance, and therefore a band shift is observed for the CO stretch. A third project involved the setup and use of an optical parametric oscillator (OPO) laser to obtain IRMPD spectra of rubidium cation-bound glycosides. DFT calculations and experimental spectra showed that the anomers of D-glucoside and D-galactoside all have differing hydrogen bonding and locations of rubidium binding, consequently showing distinct spectra in the O-H stretch region.
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 Cesar Contreras.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Eyler, John R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022819:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022819/00001

Material Information

Title: Carbohydrates and Amino Acids Infrared Multiple Photon Dissociation Spectroscopy and Density Functional Theory Calculations
Physical Description: 1 online resource (158 p.)
Language: english
Creator: Contreras, Cesar
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ab, amino, analytical, carbohydrates, density, hydrogen, infrared, irmpd, mass, physical
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: In addition to acting as repair agents, stabilizing protein folding or early defense systems in cellular systems, saccharides are especially important in energy storage and enzymatic reactions of proteins. All these areas of research require knowledge of the saccharide structure. Spectroscopic studies of monosaccharides and amino acids were undertaken to better understand structural conformations in the gas phase. Infrared spectra were obtained by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in conjunction with infrared multiple photon dissociation (IRMPD). Sodium cation-attached phenylalanine analogs were subjected to H/D exchange before their IRMPD spectra were taken. The gas phase H/D exchange experiments of N-acetylphenylalanine indicate that of the two possible locations for exchange to occur, the O-H hydrogen is kinetically favored over the N-H hydrogen. For two larger species, O-methyl N-acetylphenylalanine and N-acetylphenylalanine O-methylglycine, exchange occurred at the N-H site since it was the only one available for exchange, but the D for H substitution only took place in solution and not in the gas phase. Theoretical calculations showed that the phenylalanine analogs, although of different size, have relatively similar structural features. The sodium cation is predicted to interact with the phenyl ring and also bind to the carbonyl oxygens. In a second project, IRMPD spectra of N-acetylglycosamines showed that the frequency of the CO stretch was indicative of the particular glycosamine conformation. A band shift of about 10 cm-1 was seen between the anomers, alpha-D-methylglucosamine and beta-D-methylglucosamine, while an 11 cm-1 shift was seen for the galactosamine anomers. Calculations indicate that the O-methyl group's position (alpha and beta, or axial and equatorial, respectively) and its close proximity to the N-acetyl group cause the orientation of the carbonyl to change in order to minimize steric hindrance, and therefore a band shift is observed for the CO stretch. A third project involved the setup and use of an optical parametric oscillator (OPO) laser to obtain IRMPD spectra of rubidium cation-bound glycosides. DFT calculations and experimental spectra showed that the anomers of D-glucoside and D-galactoside all have differing hydrogen bonding and locations of rubidium binding, consequently showing distinct spectra in the O-H stretch region.
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 Cesar Contreras.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Eyler, John R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022819:00001


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CARBOHYDRATES AND AMINO ACIDS: INFRARED MULTIPLE PHOTON DISSOCIATION SPECTROSCOPY AND DENSITY FUNCTIONAL THEORY CALCULATIONS By CESAR S. CONTRERAS 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 2008 1

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2008 Cesar S. Contreras 2

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To my parents, brothers and sisters, and nieces and nephews 3

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ACKNOWLEDGMENTS First and foremost, I would like to thank my research advisor, Dr. John R. Eyler. His strong work ethic has encouraged me to work harder throughout the de gree. I would like to thank Dr. Eyler for all the great opportunities he has provided, esp ecially for the great privilege to travel and conduct research in The Netherla nds. I want to acknowledge my Ph.D. committee members: Dr. Martin T. Vala, Dr. David E. Richardson, Dr. Gar Hoflund and Dr. Rodney J. Bartlett. I would like to thank them for their feedback in the disserta tion process and providing clearer explanations of the underlying fundamental knowledge related to this work. Dr. Bartlett has also provided the computationa l resources needed to complete the work in this thesis and his generosity is greatly appreciated. I would like to thank Dr. David H. Powell for allowing the use of the FTICR-MS in the Mass Spectrometry Servi ces Laboratory to conduct the experiments on the rubidium cation-attached monosaccharides. Hi s willingness to allow the research to continue into the long hours of the night is very much appreciated. Dr. Kathryn R. Williams helped me throughout the writing of the di ssertation and her advice and comments were one of the motivations that drove me forward. Discussions with Dr. Nicole A. Horenstein and her course in carbohydrate chemistry provided the basis that I needed and us ed to understand the results obtained from this work. Although the main project in collaboration with Dr. Horenstein was incomplete by the time of writing this diss ertation, her excitement toward carbohydrate chemistry was another driving force toward accomplishing my goals. I would like to thank Dr. Jos Oomens and Dr. Jeff Steill and the FOM -Institute for Plasma Physics Rijnhuizen, Nieuwegein, The Netherlands for allocating beam time and the use of the FTICR-MS in that institute. I learned a lot from interacting with everyone at FOM and that level of collaboration will be remembered whether I go I want to also acknowledge Dr. Nicolas C. Polfer for starting the work on the phenylalan ine analogs and glycosamine derivatives. His 4

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preparation for life after graduate school and post-doctoral work l eading to a facu lty position at this major university has shown me what it takes to fulfill my dreams. I greatly appreciate the friends hip of Wright L. Pearson. Many nights we stayed up trying to troubleshoot and take spectra with the OPO laser. The long discussions from anything in politics to the most obscure ja zz compositions and of course ch emistry will long be remembered, as they helped to stimulate my mind to lear n about many more different subjects outside of chemistry. I want to mention my good friend Sabri Alkis, that without his persistent of wanting to have fun would have kept me in the lab and I would not have b een able to appreciate all that Gainesville and Florida have to offer. I woul d like to thank all the Eyler and Polfer group members for the many discussions and questions th at have kept me on my toes and have pushed me to continue reading and learning about the many mass spectrometric techniques. I would like to thank my family and parents that instilled in me a hard work ethic and an ability to persist even when the future seems uncer tain. My family is always part of everything I do and this dissertation is dedicated to them. There are countless other people that helped throughout the Ph.D. degree and to all of you I am thankful. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................10 ABSTRACT...................................................................................................................................15 CHAPTER 1 INTRODUCTION................................................................................................................. .17 Saccharide Chemistry........................................................................................................... ..17 Carbohydrate Structure and Terminology.......................................................................17 Syntheses and Reactions..................................................................................................22 Biological Significance...................................................................................................22 Peptide Chemistry...................................................................................................................24 Amino Acids.................................................................................................................... 24 Phenylalanine..................................................................................................................24 Protein Structure..............................................................................................................25 Objective of Research.......................................................................................................... ...26 Overview....................................................................................................................... ..........28 2 MATERIALS AND METHODS...........................................................................................37 Mass Spectrometric Approaches............................................................................................37 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry..................................39 Tandem Mass Spectrometry............................................................................................52 Infrared Multiple Photon Dissociation...................................................................................53 Carbon Dioxide Laser......................................................................................................56 Optical Parametric Oscillator..........................................................................................58 Free Electron Laser..........................................................................................................60 Implementation of IRMPD with FTICR-MS.........................................................................62 3 THEORETICAL CALCULATIONS.....................................................................................71 Molecular Modeling............................................................................................................. ..71 Theoretical Background..................................................................................................72 Ab initio ....................................................................................................................75 Force fields...............................................................................................................77 Geometry op timization.............................................................................................79 Vibrational analysis..................................................................................................80 Molecular Dynamics.......................................................................................................81 Simulated annealing.................................................................................................81 6

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Conformational searching........................................................................................83 4 STRUCTURE DETERMINATION OF PHENYLALANINE ANALOGS..........................85 Introduction................................................................................................................... ..........85 Experimental................................................................................................................... ........87 Sample Preparation..........................................................................................................87 Instrumentation................................................................................................................ 87 Computational Details.....................................................................................................88 Results and Discussion......................................................................................................... ..89 N-acetylphenylalanine.....................................................................................................89 Hydrogen/Deuterium Exch ange Experiments.................................................................91 AcPhe.......................................................................................................................91 AcPheOMe...............................................................................................................91 AcPheGlyOMe.........................................................................................................92 Conclusions.............................................................................................................................93 5 DIFFERENTIATION OF ANOMERS OF D-GLUCOSAMINE AND DGALACTOSAMINE............................................................................................................101 Introduction................................................................................................................... ........101 Experimental................................................................................................................... ......103 Sample Preparation........................................................................................................103 Instrumentation..............................................................................................................10 3 Computational Details...................................................................................................104 Results and Discussion......................................................................................................... 105 Infrared Spectra.............................................................................................................105 Glucosamines.........................................................................................................105 Galactosamines.......................................................................................................107 Fragmentation Patterns..................................................................................................108 Conclusion............................................................................................................................109 6 DIFFERENTIATION OF O-METHYLATED GLYCOSIDE ISOMERS..........................120 Introduction................................................................................................................... ........120 Experimental Techniques.....................................................................................................121 Chemicals......................................................................................................................121 Instrumentation..............................................................................................................12 2 Laser Setup.................................................................................................................... 122 Experiment....................................................................................................................123 Computational Details...................................................................................................124 Results and Discussion......................................................................................................... 125 FELIX............................................................................................................................125 OPO...............................................................................................................................126 Glucosides..............................................................................................................126 Galactosides...........................................................................................................129 Conclusions...........................................................................................................................130 7

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7 CONCLUSIONS AND FUTURE WORK...........................................................................144 LIST OF REFERENCES.............................................................................................................149 BIOGRAPHICAL SKETCH.......................................................................................................158 8

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LIST OF TABLES Table page 4-1. Interaction distance between sodium cation and oxygen atom s on the carbonyls of the carboxyl and on the N-acetyl functional groups for calculated conformers A D......94 4-2. Summary of results from H/D exchange experiments.......................................................94 5-1. Glycosamine isomer structures obtained from the corresponding conformational searches............................................................................................................................111 9

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LIST OF FIGURES Figure page 1-1 Structure of kojibiose (2-O-D-glucopyranosyl-D-glucopyranose)................................29 1-2 Aldoses.................................................................................................................... ...........29 1-3 Fischer projection of D-glucose.........................................................................................30 1-4 D-aldoses.................................................................................................................. ..........30 1-5 Common ketoses involved in biological processes...........................................................31 1-6 Pyranose and furanose ring formation in equilibrium.......................................................31 1-7 Six-membered ring structures of the -anomers of D-hexoses.........................................32 1-8 Ring conformations, for furanoses and pyranoses.............................................................33 1-9 Disaccharide formation by glycosylation..........................................................................33 1-10 The polysaccharids amylose and cellulose........................................................................34 1-11 Amino acid................................................................................................................ .........34 1-12 The 20 amino acids used in proteins..................................................................................35 1-13 General structure of a tripeptide........................................................................................3 5 1-14 Process of inverting a -galactoside to the corresponding -galactoside by using an inverting glycosidase.........................................................................................................3 6 2-1 Electron impact ionization and chemical ionization mass spect rum of D-glucose...........63 2-2 Initial trajectory of the velocity of the ion.........................................................................63 2-3 Motion of the ion in a uniform magnetic field...................................................................64 2-4 Motion of an ion (in a uniform magnetic field) as its cyclotro n motion is excited by an alternating electric field.................................................................................................6 4 2-5 Detection of ions in an FTICR-MS....................................................................................65 2-6 Cyclotron, trapping and magnetron motions.....................................................................65 2-7 Electrospray mist.......................................................................................................... .....66 2-8. Ion transfer optics....................................................................................................... .......66 10

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2-9 Two common Penning traps..............................................................................................66 2-10 The FTICR mass spectromete r at the FOM-Institute........................................................67 2-11 Infrared spectrum of a crystalline sample of -D-glucopyranose.....................................67 2-12 Initial absorption of photon energy from the ground state to an excited vibrational level and the subsequent intramolecu lar vibrational relaxation process............................68 2-13 The CO2 laser cavity..........................................................................................................68 2-14 Vibrational energy levels of the CO2 and N2 molecules involved in the lasing mechanism...................................................................................................................... ...69 2-15 The IRMPD spectrum of Mn(CO)4CF3 -, using a tunable CO2 laser..................................69 2-16 An OPO laser.............................................................................................................. .......70 2-17 Free electron laser schematic............................................................................................. 70 4-1 Structure of the amino acid phenylalanine.........................................................................94 4-2 Phe analogs studied in this work........................................................................................95 4-3 Dihedral angles defined for the conformational search calculations of AcPhe, AcPheOMe and AcPheGlyOMe........................................................................................95 4-4 Lowest energy conformers of sodium cationized AcPhe found from conformational search calculations............................................................................................................ .96 4-5 Comparison of experimental IRMPD spectru m (red line) of AcPhe with calculated spectra of the theoretically determined lowest energy conformers A-D (blue stick spectra)....................................................................................................................... ........97 4-6 Assignment of spectral bands in the IR MPD spectrum of AcPhe using calculated bands for conformer A.......................................................................................................98 4-7 IRMPD spectrum of sodium cation-att ached AcPhe following solution phase HDX (in red) and calculated spectrum of the doubly deuterated conformer A (in blue)............98 4-8 Comparison of the singly deuterated sodium cation-attached AcPhe IRMPD spectrum with that of calculated conformer A...................................................................99 4-9 Overlap of IRMPD spectra for undeuterated and singly-deuterated sodium cationattached AcPhe...................................................................................................................99 4-10 Experimental IRMPD spectra and theoretically calculated spectra of phenylalanine analogs.............................................................................................................................100 11

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5-1 Glycosamine isomers.......................................................................................................1 12 5-2 Side and top views of lithium cationized -GlcNac........................................................112 5-3 Rotation about C-2 and N, shown with a red arrow........................................................113 5-4 IRMPD spectra of -GlcNac (black) and -GlcNac (red) bound to a lithium cation, showing the 10 cm-1 shift of the carbonyl stretch frequency...........................................113 5-5 Calculated lowest energy structures of lithium cationized -GlcNac and -GlcNac, using B3LYP/6-31+G(d).................................................................................................114 5-6 Comparison of experimental IRMPD a nd calculated infrared spectra of lithium cationized -GlcNac........................................................................................................114 5-7 Potential surface scan for rotation about (C-2)-N bond for -GlcNac............................115 5-8 Potential surface scan for rotation about the (C-2)-N bond for -GlcNac......................115 5-9 Comparison of experimental IRMPD sp ectrum (red) to the calculated infrared spectrum (blue) of lithium cationized -GlcNac.............................................................116 5-10 IRMPD spectra of lithium cationized -GalNac and -GalNac, showing the 11 cm-1 shift for the carbonyl stretch bands..................................................................................116 5-11 Calculated infrared spectra of lithium cationized -GalNac and -GalNac, indicating a 29 cm-1 shift in the C=O stretches.................................................................................117 5-12 Lowest energy structures of lithium cation-attached galactosamine anomers from B3LYP/6-31+G(d)...........................................................................................................117 5-13 Potential surface scan for -GalNac................................................................................118 5-14 Potential surface scan of -GalNac..................................................................................118 5-15 Fragmentation ion abundances as a function of laser wavelength for all four isomers...119 5-16 Possible fragmentation producing the observed MS/MS spectra of glycosamine isomers.............................................................................................................................119 6-1 The FELIX-generated IRMPD spectra (600 1700 cm-1) of all four glycoside isomers, Glc, Glc, Gal and Gal...............................................................................131 6-2 The O-methylated glycoside isomers, A) Glc, B) Glc, C) Gal and D) Gal shown in the 4C1 chair conformation...............................................................................132 6-3 The OPO optics setup......................................................................................................1 32 12

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6-4 Experimental sequence for obtaining spect ra of the O-H stretching region of the rubidium cation-attached glycosid es using OPO laser irradiation...................................133 6-5 Experiment sequence for the two laser experiment.........................................................133 6-6 Monosaccharide -D-glucoside.......................................................................................134 6-7 Lowest energy conformers of Glycosides........................................................................134 6-8 Comparison of theoretical and experimental IRMPD spectra for glycoside isomers......135 6-9 Calculated spectra for the second lowe st energy conformations of D-glucosides anomers from the conformational search.........................................................................135 6-10 IRMPD spectrum of rubidium cation-attached Glc.......................................................136 6-11 Comparison of the IRMPD spectrum of Glc in the C-H stretch region with different irradiation methods..........................................................................................................13 6 6-12 Comparison of calculated spectrum fo r conformer A (dark blue) and IRMPD spectrum (red) for rubidium cation-attached Glc..........................................................137 6-13 Experimental spectrum (red) and calculated spectrum for conformer B (green) of rubidium cation-attached Glc........................................................................................137 6-14 Spectral match between experiment (red ) and calculated conformer C of rubidium cation-attached Glc........................................................................................................138 6-15 Spectral comparison of experiment (red ) and calculated conformer D (gray) for rubidium cation-attached Glc........................................................................................138 6-16 Overlap of all four conformer spectra with IRMPD spectrum for rubidium cationbound Glc.......................................................................................................................139 6-17 Calculated conformers for Glc at the B3LYP/6-31+G(d) level of theory.....................140 6-18 Comparison of IRMPD spectra for Glc (blue) and Glc (red), clearly showing the band at 3637 cm-1 is missing for Glc.............................................................................141 6-19 Comparison of IRMPD and calculated spect ra for the lowest energy conformer of Glc.................................................................................................................................141 6-20 Overlap of calculated spectra for conf ormer A and conformer B with IRMPD spectra of Glc.............................................................................................................................142 6-21 Conformers of Glc that correlate with IRMPD spectrum.............................................142 13

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6-22 Comparison of preliminary IRMPD spectra for Gal (green) and Gal (yellow) in the O-H stretch region......................................................................................................143 6-23 Overlap of IRMPD spectra of all f our isomers in the O-H stretch region.......................143 14

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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 CARBOHYDRATES AND AMINO ACIDS: INFRARED MULTIPLE PHOTON DISSOCIATION SPECTROSCOPY AND DENSITY FUNCTIONAL THEORY CALCULATIONS By Cesar S. Contreras December 2008 Chair: John R. Eyler Major: Chemistry In addition to acting as repair agents, stabilizing protein folding or ear ly defense systems in cellular systems, saccharides are especially important in energy storage and enzymatic reactions of proteins. All these areas of research require knowledge of the saccharide structure. Spectroscopic studies of monosaccharides and ami no acids were undertaken to better understand structural conformations in th e gas phase. Infrared spectra were obtained by using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) in conjunction with infrared multiple photon dissociation (IRMPD). Sodiated phenylalanine analogs were subjec ted to H/D exchange before their IRMPD spectra were taken. The gas phase H/D exchange experiments of N-acetylphenylalanine indicate that of the two possible locations for exchange to occur, the O-H hydrogen is kinetically favored over the N-H hydrogen. For two larger specie s, O-methyl N-acetyl phenylalanine and Nacetylphenylalanine O-methylglycine, exchange o ccurred at the N-H site since it was the only one available for exchange, but the D for H substitu tion only took place in solution and not in the gas phase. Theoretical calculations showed that the phenylalanin e analogs, although of different 15

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size, have relatively similar structural features. The sodium cation is pred icted to interact with the phenyl ring and also bind to the carbonyl oxygens. In a second project, IRMPD spectra of N-acetyl glycosamines showed that the frequency of the CO stretch was indicative of the particular glycosamine conformation. A band shift of about 10 cm-1 was seen between the anomers, -D-methylglucosamine and -D-methylglucosamine, while an 11 cm-1 shift was seen for the galactosamine anomers. Calculations indicate that the Omethyl groups position ( and or axial and equatorial, respectiv ely) and its close proximity to the N-acetyl group cause the orientation of the car bonyl to change in order to minimize steric hindrance, and therefore a band shift is observed for the CO stretch. A third project involved the set up and use of an optical parametr ic oscillator (OPO) laser to obtain IRMPD spectra of rubidi um cation-bound glycosides. DFT calculations and experimental spectra showed that the anomers of D-glucos ide and D-galactoside a ll have differing hydrogen bonding and locations of rubidium binding, conseque ntly showing distinct spectra in the O-H stretch region. 16

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CHAPTER 1 INTRODUCTION Saccharide Chemistry Carbohydrates are everywhere on earth, and th ey are vital component s of our everyday lives. It has been estimate d that cellulose comprises 50% of the earths biomass.1 In the diet, sugars and starches are metabolized to pr oduce energy, which is stored as adenosine triphosphate, which also contains a sugar as part of its structure. Carbohydr ates are essential to the food industry, which uses large amount s of starch, sweet gums and monoand oligosaccharides. Carbohydrates are also important in textiles which are largely dependent on cellulose containing materials. The pharmaceu tical industry uses carbohydrates for the preparation of antibiotics and intravenous soluti ons and as components of pills and capsules. The importance of sugars was recognized by early organic chemists, who studied sugars and their derivatives as early as the 1870s, a decade before Em il Fischer began his 20-year study to classify the configurations of sugars.2 But although carbohydrate chem istry is one of the oldest fields in chemistry, interest in the field continues to increase and th is has been largely due to the sheer number and complexity of saccharide types Carbohydrate Structure and Terminology To better understand the intr icacies and complexities of carbohydrates, the naming conventions used for the isomeric structures of saccharides will be intr oduced, focusing mainly on the monosaccharide units. The naming conve ntion serves two main purposes: to allow conversion of the IUPAC nomenclature to a more readable format, and to name polymers of monosaccharide units (i.e., polysaccharides) quickl y. For example, the disaccharide shown in Figure 1-1 has the cumbersome IUPAC na me (3R,4S,5S,6R)-6-(hydroxymethyl)-3((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)t etrahydro-2H-pyran-2-yloxy)tetrahydro17

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2H-pyran-2,4,5-triol, but this can be shortened to 2-O-D-glucopyranosyl-D-glucopyranose. Frequently encountered disaccharides also have common names; e.g., 2-O-D-glucopyranosylD-glucopyranose is called kojibiose. Aldoses and ketoses. The term carbohydrate originated in the s ugars known in the 1870s which all had the empirical formulas Cx(H2O)y, and were thought to be hydrated carbons.3 Today, carbohydrates encompass monomers, olig omers and polymers that are derived from monosaccharides.4 Monosaccharide units have the empirical formula CH2O and are either aldoses which contain both aldehyde a nd alcohol functionalities, or ketoses, with both keto and alcohol groups. The simplest monosaccaharide al dose is glyceraldehyde, which contains three carbons (Figure 1-2). Aldoses with 4, 5, and 6 carbons are called aldote troses, aldopentoses, and aldohexoses, respectively. Monosac charides can have up to ten carbons, but the most common monosaccharides are pentoses and hexoses. Fischer projections. Linear chain monosaccharides are us ually represented by way of the Fischer projection,5 (Figure 1-3). In a Fischer proj ection, the carbon backbone is oriented vertically with the horizontal groups pointing out the front of the page. The aldehyde group CHO at C-1 is oriented at the top of the vertical chain, and the othe r carbons are numbered sequentially along the chain from this position. Monosaccharides can have many stereoisomers, since all carbons that are secondary alcohols have four different function al groups and are thus chiral centers. The chain form of D-glucos e, whose IUPAC name is (2S,3R,4S,5S)-2,3,4,5,6pentahydroxyhexanal, has four ch iral centers, (Figure 1-3). D and L sugars. Historically, the designation D or L has referred to the absolute configuration at the highest numbered chiral ca rbon (C-5 in an aldohexose). In the Fischer projection, if the hydroxyl group on C5 points to the right, the sugar is designated D. If the OH 18

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points to the left, it is an L sugar. (Not e that this should not be confused with d and l, which indicate the directi on of rotation of pl ane-polarized light: d = dextrorotatory = CW rotation; l = levorotatory = CCW rotation.) According to the newer Cahn-Ingold-Prelog convention,6 a D sugar has the R configuration at C-5, and S corresponds to L. Th e D forms of all aldoses through the aldohexoses are shown in Figure 1-4. Although the majority of monosaccharides are aldoses, a few ketoses are important in biological systems (Figure 1-5). The ketohexose D-fructose links with D-glucose to form the disaccharide sucrose. The L-fucose is a structural component of plant cell walls7 and Lrhamnose is a component of bacterial cell membra ne, but is also found in a variety of plants.8 Cyclic forms. Because aldoses and ketoses co ntain both carbonyl and hydroxyl functionalities, they readily form cyclic hemiacet als and hemiketals. shows the hemiacetals of D-glucose, which forms the six-membered ring st ructure by reaction of the C-5 oxygen with C-1, as well as the five-membered ring structure by re action of the C-4 oxygen with C-1 (Figure 1-6). Each reaction gives rise to a ne w chiral center at C-1. If the functional group at C-1, designated the anomeric carbon, is in the axial positi on, the ring structure is designated the -anomer. Likewise, if the functional group at C-1 is in the equatorial position, the ring structure is designated the -anomer. The five-membered ring is relate d to tetrahydrofuran and is called a furanose, while the six-membered ring is called pyranose, after tetrahydropyran. In an aqueous solution, the ring-closed forms ar e favored over the open-chain form, the abundance of the latter being almost negligible. An equilibrium, called mutarotation, is established between the ring structures and the open-chain form ( Figure 1-6). For D-glucose, the equilibrium mixture in an aqueous environment is 38% -pyranose, 62% -pyranose, 0.1% -furanose and less than 0.2% -furanose, with a negligible con centration of the open-chain form.4 This equilibrium ratio is 19

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unique to the D-glucose, and varies for the ot her monosaccharides, being dependent on the ring structure, functional groups, so lvent environment and the conformation of the particular Daldose.3 For example, an isomer of D-glucos e, mannose, exists predominantly as the pyranose.4 Because of the mutarotation equilibrium the aldehyde group of the open-chain form can be oxidized (positive Tollens and Benedi cts tests). Thus, all monosaccharides are classified as reducing sugars. Although Fischer projections are useful for visualization of openchain sugars, ring structures, part icularly in the chair conforma tion, are preferred for hemiacetal and hemiketal forms, (Figure 1-7) for the -anomers of the D-pyranoses Ring conformations. The ring structures of hexoses and pe ntoses are flexible and exist in many conformations, (Figure 1-8) For furanoses, th e ring can be planar and form an equilateral pentagon shape, or envelope (E), or it can be slightly bent and form a twisted pentagon (T). Sixmembered rings are more flexible and have many more conformations. For D-glucose, the chair conformation (C) is the mo st stable in an aqueous solution. It can have two forms. The one shown in Figure 1-8 is denoted 4C1, where C-4 is positioned above the plane of the ring and C-1 is positioned below the plane of the ring. The other is 1C4 and has the opposite orientation, with C-4 below and C-1 above the plane of the ring. The boat conformation (B), can have both C-1 and C-4 above the plane, in which case it is denoted 1,4B, or both C-1 and C-4 below the plane and in this case it is denoted B1,4. The chair or boat conformati ons can be perturbed, forming the half-chair (H) and skewed (S ) conformations, respectively. Polysaccharides. Carbohydrates undergo glycosylation reactions to form molecules that have multiple monosaccharide units linked togeth er. Glycosylation can be understood as the displacement of the hydroxyl group on C-1, the glycosyl donor, by the hydroxyl group, the glycosyl acceptor, of another m onosaccharide (Figure 1-9). The disa ccharide can be subjected to 20

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further glycosylation to continue the polymerization process. As part of the disaccharide, the donor is called the non-reducing monosacchar ide, since it is now bound to another monosaccharide at the anomeric carbon, and cannot undertake ring opening to expose the aldehyde group for oxidation. If the glycosidic bond does not involve the anomeric carbon of the acceptor unit, the disaccharide can still act as a re ducing sugar. The different glycosidic linkages are designated (1 1), (1 2), (1 3), (1 4) and (1 6), where the first number in the parentheses indicates the linkage position for the non-re ducing end and the second number is the linkage position of the acceptor monosaccharide. The orientation of the linkage depends on the configuration of the anomeric carbon in the donor, further increasing the number of isomers possible when forming polysaccharides through glycosylation. Oligosaccharides have 2-10 monosaccharides, with the number of monomer units often explicitly stated (i.e., disaccharides, trisaccharides, etc.). There are two classifications of oligosaccharides: true oligosaccharides, which are made up solely of simple monosaccharide units, and conjugate oligosaccharides, which have monosaccharides linked to non-saccharides, for example peptides and lipids. Oligosacchar ides can also be grouped as reducing and nonreducing sugars, depending on the linkage to the last donor. Th e degree of polymerization is small enough that oligosaccharides continue to be soluble in water, similar to monosaccharides. Polysaccharides have more than 10 monomer uni ts. Similar to oligosaccharides, they are grouped as either true or conjugate polysaccharid es. Since the degree of polymerization can be much higher for polysaccharides and there is a higher probability of having more than one type of monosaccharide unit, true polysaccharides ar e further divided into two subclasses: the homogeneous class, with only one type of m onosaccharide as the repeating unit, and the heterogeneous class with more than one type mono saccharide. There are also linear or branched 21

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polysaccharides, the latter being common in glyco lipids attached to plasma membrane, or when carbohydrates are attached to surfaces. The solubility decr eases as the size of the polymer increases, and many polysaccharides are insoluble in water. Syntheses and Reactions One of the major goals of a saccharide synthetic chemist is the production of a select type of saccharide by a one-pot synthesis, in which successive chemical reactions are conducted in one reactor, avoiding purificati on and separation of intermediates. For carbohydrates, one-pot syntheses are not common,9-11 because the multiple stereocenters provide numerous reaction sites. Efforts to develop new synthetic appro aches have also been hindered by the paucity of methods to determine the structures of products which are often formed in very low concentrations. The molecular complexities of carbohydrates al so cause problems in reactions to form derivatives and polymers. Identification of genera l patterns of reaction has been a difficult task, because product structures vary due to influen ces of the solvent, temp erature, and functional group type, as well as ring size a nd strain and intraand intermolecular forces like hydrogen bonding and interactions with metallic species.12 While solution phase and wet synthesis resear ch has provided a wealth of information on saccharide structure, it is still unknown what majo r factors influence the formation and stability of one isomeric form relative to another. It should be apparent that factors controlling the 3dimensional structure of carbohydrates must be fully studied in order to understand the many processes they mediate. Biological Significance Until recently, the role of sugars in biologica l systems was thought to be limited to energy storage and structural support. However, increased research on glycopeptides and glycoproteins 22

PAGE 23

has indicated that carbohydrates also play a role in biolog ical communication events, in processes such as egg fertilization, microbi al infection, inflammation, cancer growth and diabetes. Glucose is found in proteins synthesized in mammalian cells and is covalently bound to different amino acids in the peptide struct ure. For example, carbohydrates can be found covalently bonded to the amide nitrogen of as paragines (N-linked glycoproteins) or to the oxygen atom in serine and threon ine (O-linked glycoproteins). Carbohydrates bound to proteins affect solubility, protein fold ing and protect against protei n degradation from enzymatic processes. Carbohydrates can also bind to li pids (glycolipids), form ing components of the plasma membrane found in all vertebrate cells. More than 400 glycolipids are known, while the most common glycolipids in vertebrates are formed with only 7 distinct monosaccharides. Deoxyribonucleic acid (DNA) is a polymer that has the ribose sugar (2-deoxy-D-erythrosepentofuranose) as part of its repeating nucleotide units. Polymers. Further importance of carbohydrate stru cture in biological function is observed in polysaccharides of glucose. In amylose, -D-glucoses are connected by (1 4) glycosidic bonds (Figure 1-10). Cellulose, another pol ysaccharide of D-glucose, contains (1 4) glycosidic bonds. Each polysaccharide has a distinct function. Amylose is the soluble component of starch and is a major food source, while cellulose is the structural material in plants. Amylose, because of the (1 4) glycosidic bonds, forms a helical structure, whereas cellulose forms a ribbon-like structure. Cellulose is insoluble in water, an d it cannot be digested by humans. Adding a substituent functional group also changes the properties of the carbohydrate. Chitin is a polysaccharide of D-glucose and has the same (1-4) linkages as cellulose, but has an 23

PAGE 24

N-acetyl group at the C-2 of each glucose unit. Similar to cellulose, chitin provides molecular structural support in cells and fo rms the hard exoskeleton of in sects and shellfish. These and many other discoveries have spar ked a renewed interest in car bohydrate chemistry, and chemists having a variety of specialties are trying to solve these glycobiological prob lems. For all of these research areas, the carbohydrate 3dimensional structure is important. Peptide Chemistry Proteins, along with carbohydrat es, nucleic acids, and lipids, are one of the four main groups of molecules that are impor tant in cellular function. Prot ein chemistry has experienced a growing interest, and mass spectrometric studies have made major contributions in this area. Amino Acids The building blocks of proteins are -amino acids. The designation alpha ( ) refers to the point of attachment of the amino group (-NH2), which is always on the carbon adjacent to the carboxyl group (-COOH) (Figure 1-11). Although often shown as uncharged species (Figure 111A), amino acids in biological fluids exist as zwitterions (Figure 1-11B). Twenty amino acids are found in proteins, and each differs by the functional group R (Figure 1-12). Phenylalanine Because of its importance in this research, the amino acid phenylalanine will be described in further detail. Phenylalanine (Phe) is one of the essential amino acids, meaning that it is not synthesized by humans and must be included in the diet. Phenyl ketonuria, a genetic disorder affecting infants and young childre n, prevents the normal metabolism of phenylalanine. Instead of the normal product, tyrosine, Phe is converted to phenylpyruvate, which interferes with energy release, leading to mental retardation.13 Since phenylalanine has a nonpolar side group, it forms part of the hydrophobic region in the interior of a globular protein. Similar to other amino acids, Phe occurs as a zwitterion in 24

PAGE 25

solution. Previous results14,15 have indicated that Phe exists in the unionized form in the gas phase. However, recent mass spectrometric studies have shown that phenylalanine may also exist as a zwitterion in the gas phase, if it is bound to specific metals. Gas-phase studies on sodium cation-attached amino acids, includi ng phenylalanine, indicated that rates of deuterium/hydrogen exchange reactions were very fast.16 For phenylalanine, the exchange rates were 1.12x1011 cm3 s1molecule1 using CD3OD and 0.16x1011 cm3 s 1molecule1 using D2O. These high exchange rates were attribut ed to the presence of a protonated -amino group, indicating that the zwitter ion form was present. Protein Structure In proteins, amino acids are joined by amid e linkages (Figure 1-13), often called peptide bonds. Small polymers (up to about 30 amino acid residues) are called polypeptides, which, like polysaccharides, often carry the di-, tri-, etc. pref ixes. Proteins are polypeptides with more than 30 amino acids. The many complexities of carbohydrate structur e and conformation were described earlier in this chapter. In proteins, differences ar ise from the number and sequence of the 20 amino acids (and the presence of S-Sli nkages between cysteine residues) referred to as the primary structure. Structural patterns within segmen ts of the protein chain constitute the secondary structure, which depends strongly on the hydrogen bonding between nearby amide groups. The two common types of sec ondary structure are the -helix and the -pleated sheet. The overall shape of the protein, whether it is extended or gl obular, is called the tertiary structure. The ability to assemble proteins containing a vast number of amino acid sequences gives an organism the flexibility to tailor proteins for specific functions. Of pa rticular importance in this research is the transport of Na+ and K+ ions by specially designed proteins that bind one specific alkali metal and allow its passage throug h the cell membrane. These ions are essential 25

PAGE 26

to regulatory function within the cell. Another very specialized group of proteins, the Carbohydrate Processing Enzymes are specifically designed for the synthesis of carbohydrates in a single step without the use of protecting groups.4 There are two main types of these enzymes; one type makes glycosidic bonds (glycosyltransfe rases), and the second type breaks glycosidic bonds (glycosidases). Both enzyme types take adva ntage of binding sites that fit only a specific sugar type. The glycosyltransferase enzymes have two binding sites for the donor and acceptor sugar, respectively. When the enzyme folds toge ther, glycosylation occu rs at only one hydroxyl group of the acceptor. The other hydroxyl groups are buried in the binding site and cannot react. The glycosidase enzymes are not as specific, becau se they only have binding sites for the donor sugar, since the main purpose is to cleave th e donor. Figure 1-14 shows a mechanism where a galactoside is inverted in the glycosidas e binding site to fo rm the corresponding -galactoside. Objective of Research The biological molecules just described have been the subjects of numerous studies in solid and solution phase chemistry. However, eluc idation of the 3-dimensi onal structure in those studies has been hindered by the interactions which occur when the molecule being studied contacts other molecules and ions. Gas phase studies of carbohydrates and amino acids are increasing, driven mainly by the increased sophi stication of the gas phase instrumentation developed in recent decades. Gas phase studies ar e especially useful, because the intermolecular interactions are reduced and are almost negligible in some met hods. Therefore the molecule can be studied independent of such interactions, and the intramolecular forces that influence its shape can be studied. Gas phase studies also have the capability of investigating intermolecular interactions one at a time, for example by addition of a metal ion attached to a neutral molecule, so that the direct influence of the interaction on the ion can be studied. 26

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Fourier transform ion cyclotron mass spectro metry (FTICR-MS), in conjunction with ion fragmentation methods, has been us ed to obtain accurate structural information for gaseous ions. Infrared multiple photon dissociation (IRMPD) ha s been used by numerous groups to obtain fragmentation patterns of oligosaccharides, prot eins and peptides using a fixed-wavelength CO2 laser, and small ions have been studied using wavelength-tunable CO2 lasers. A limitation of the latter studies has been the narrow wavelength range of the tunable CO2 laser (9.1 10.9 m) with numerous gaps where lasing does not occur, resulting in IRMPD spectra obtained with incomplete ir bands. The advent of continuously tunable free electron lase rs and the subsequent implementation of them in IRMPD studies has provided the basis for obtaining complete ir spectral bands over a large wavelength range (2 250 m). Recently, another tunable laser, the optical parametric oscillator (OPO), has been used in gas phase studies to probe the conformations of ions. The OPOs in those stud ies were tunable in the ultraviolet, but newer infrared-tunable OPOs have become available, and the Eyler laboratory, in conjunction with the Mass Spectrometry Services Laboratory at the Un iversity of Florida, has implemented an OPO for use with FTICR-MS to obtain IRMPD spectra of carbohydrates. The OPO/FTICR-MS system was used for part of this thesis research, and will be presented in chapter 6. Even when studied in the gas phase, the comp lexity of carbohydrates and amino acids is evident in their spectra, and theo retical calculatio ns are crucial to decipher that information. The multiple atoms and flexible nature of amino acids and carbohydrates make them a challenge to model with theoretical methods. Algorithms have been developed to tack le such problems, but often these methods have been used without the understanding of th e underlying theoretical framework. It is imperative to understand the th eoretical basis in order to obtain accurate and meaningful results. 27

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The objective of the investigations in this thesis was to apply the techniques of both IRMPD-FTICR-MS and theoretical calculations to obtain gas phase structures of amino acid and carbohydrate ions and to differentiate carbohyd rate isomer ions. Monosaccharides and monopeptides were used to study interactions with alkali metal cations. The OPO/FTICR-MS system was implemented to obtain IRMPD spectra in the range from 2700 3800 cm-1, the ir wavelength region containi ng C-H and O-H stretching bands. Th e O-H stretches were especially sought after, since the free hydroxyl groups of sugars tend to give strong absorption features in infrared spectra. Theoretical ca lculations are key to understanding the processes that lead to the differences in the IRMPD of the respective isomers. The calculations are also used to predict how the experimental IRMPD spectral bands shift when analogs of the amino acid phenylalanine are subjected to hydrogen/deuterium exchange. Overview The next chapter will descri be the experimental methods of Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (F TICR-MS) and Infrared Multiple Photon Dissociation (IRMPD). The principles and mathematical description of FTICR-MS will be presented, as well as the instrumentation used to ionize molecules. The IRMPD mechanism will be summarized and the lasers used as irradiation sources for fragmentation will be described. A general experimental procedure, from ionization to obtaining IR MPD spectra of precursor ions will be introduced. This will be followed by chapter 3, where theoretical calculations involving ab initio and classical mechanics methods will be detailed. Hydrogen/deuterium exchange and subsequent IRMPD experiments of sodium cationattached phenylalanine analogs will be described in chapter 4. Theoretical calculations were used to predict the band shifts due to H/D exchange and the calculated structures will be discussed. Chapter 5 discusses the differentiation of lithium cation-attached N28

PAGE 29

acetylglycosamine isomers through IR MPD spectra in the 600 1800 cm-1 combined with theoretical calculations. Chapter 6 introduces the earlier work of Valle et al .,17 where IRMPD spectra in the 600 1800 cm-1 region of rubidium cation-at tached glycoside isomers were obtained. Differentiation between the isomers wa s considered inconclusive. In this work, IRMPD spectra from 2700 3800 cm-1 of the rubidium cation-attach ed glycosides are obtained. The properties of the optical parametric oscillat or used in these studies are detailed, and the modified experimental sequences to obtain IRMPD spectra are e xplained. Finally, a conclusion that summarizes the results of the work in ch apter 4, 5 and 6 and suggests future work will be presented in chapter 7. Figure 1-1. Structure of kojibiose (2-O-D-glucopyranosyl-D-glucopyranose). Figure 1-2. Aldoses, illustrating naming as chain size increases. 29

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Figure 1-3. Fischer projection of D-gl ucose with chiral carbons starred. Figure 1-4. D-aldoses 30

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Figure 1-5. Common ketoses invo lved in biological processes Figure 1-6. Pyranose and furanose ring formation in equilibrium with the linear form of Dglucose. The net abundance of the linear fo rm of D-glucose is negligible in solution. 31

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Figure 1-7. Six-membered ring structures of the -anomers of D-hexoses, showing differences of stereochemistry (highlighted in red) relative to D-glucose (bottom right). 32

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Figure 1-8. Ring conformations, for furanoses and pyranoses. Ring oxygen and carbons are numbered according to IUPAC convention.18 Figure 1-9. Disaccharide formation by glycos ylation, showing attack by a monosaccharide acceptor on a monosaccharide donor. For simp licity, the functional groups of the acceptor are symbolized with one hydroxyl group. The linkage can occur at C-1, C-2, C-3, C-4 and C-6 of the acceptor. Stereochemistry at the anomeric carbon of the donor has not been explicitly indicated, a nd both configurations are possible in the glycosylation reaction 33

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Figure 1-10. A) Representation of the polysacchar ide, amylose, showing the helical nature due to the (1 4) glycosidic bonds. B) The (1 4) linked cellulose, shown as stacked ribbon-like chains. A B Figure 1-11. Amino acid in the A) unionized and B) zwitterionic forms. The amino group is attached to the alpha carbon, which has the L configuration in a ll naturally occurring amino acids. 34

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A B Figure 1-12. The 20 amino acids found in proteins with side chain (R) groups highlighted in blue. Names and three-letter abbreviations (in parentheses) are given below each structure. A) Amino acids with nonpolar R groups. B) Amino acids with polar and/or ionizable R groups. Figure 1-13. General structure of a tripeptide, where the amide linkages are shown bracketed in red. 35

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Figure 1-14. Process of inverting a -galactoside to the corresponding -galactoside by using an inverting glycosidase. A two step SN1 process is shown but the inversion is also possible through an SN2 reaction. 36

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CHAPTER 2 MATERIALS AND METHODS Mass Spectrometric Approaches An excellent way to study gas phase ions is through the use of mass spectrometry. Modern mass spectrometry (MS) was pioneered by Dempster and Aston19,20 for separation of isotopes. They implemented ion deflecting mass spectromet ers that would become the basis of magnetic sector instruments. Other mass spectrometers quickly followed, includi ng the time of flight21 and quadrupole mass spectrometers.22 Early mass spectrometric studies of carbohydrates complemented the work of Emil Fischer and his studies of the structur e of monosaccharides.23 Development of mass spectrometry instrument ation to study amino acids, proteins and carbohydrates in the 1980s showed that MS was a robust analytical technique, useful in biochemical research. Two difficulties arise in obtaining mass spectr a of carbohydrates. The low volatility of sugars makes them difficult to vaporize and the sample may require heating to obtain gaseous molecules and ions. Heating sugar samples can cau se instabilities, leading to degradation of the sample and fragmentation of parent ions, which can pose a problem when trying to obtain the molecular weight of the sugar. One of the first ionization methods for mass spectrometry, electron impact ionization (EI),24 was used to obtain a mass spectrum of D-glucose in a quadrupole mass spectrometer.3 To obtain the molecular mass of D-glucose, the sample was heated to 130 C under reduced pressure and th en bombarded with electrons, generating positive ions of D-glucose. The EI process requires high energy electrons, most often around 70 eV. Because of the high energy electron bombardment, the molecular ion (M+) of D-glucose rearranges, leading to bond cleavag e and formation of fragment ions and neutrals. Therefore, the molecular mass cannot be determined with EI-M S. The mass spectrum of D-glucose from EI37

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MS in Figure 2-1 has no corresponding peak for the M+ ion of D-glucose, which has a molecular weight of 180 Dalton (Da) or atomic mass units. The highest mass is 149 Da, corresponding to loss of CH2OH from the molecular ion. Another mass spectrometric study of D-gluc ose, using the chemical ionization (CI)25 technique, was able to yield the protonated mo lecular ion and the molecular mass of D-glucose was verified.3,26 The chemical ionization process used an ionized gas, in this case methane, for which ion/molecule reactions form CH5 + and C2H5 + which in turn react with the gaseous sugar sample, donate a proton, and form the [M+H]+ glucose ion. Figure 2-1 also shows the mass spectrum of D-glucose from the CI-MS expe riment, and the highest mass peak, 181 Da, corresponds to D-glucose w ith the added proton. Th e abundance of the [M+H]+ is low and multiple fragmentation is observed in the CI mass spectrum. The mass spectra, using fast atom bombardment ionization (FAB),27,28 of 21 amino acids were used to obtain their molecular weights.29 The solid sample containing the amino acid was dissolved in a glycerol matrix and transferred to a copper tip. Bombardment with argon atoms (4-6 keV) onto the copper tip produced ions that were analyzed using a Kratos MS 50 magnetic sector mass spectrometer. The molecular ions of the amino acids were evident in the mass spectra, but the amide terminal group fragment ion was obscured by the glycerol matrix and by background peaks from the sample. These mass spectrometers and their correspondi ng ionization techniques were able to study small carbohydrates and amino acids, but molecular ions from larger molecules were not produced due to increasing low vol atility of the larger species and congestion of peaks in the spectra for those the ions that were formed. Even with small ions, high resolution may be necessary if trying to make a distinction between OH and NH3, both having a mass of 17 Da but 38

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differing in mass if more precise measuremen ts are made. Ion cyclotron resonance mass spectrometers made high resolution experiments of biologically relevant ions possible, especially after introduction of the Fourier transf orm approach by Comisarow and Marshall.30 Fourier transform ion cyclotr on mass spectrometry (FTICR-MS),30-35 when combined with various dissociation methods, has been established as an important method for analysis of carbohydrates and proteins and was the mass spectro metric technique used in this work. The following section will deal with the theory of ion cyclotron motion, the magnetron motion that is a combination of the cyclotron and trapping moti ons, and a general descri ption of the detection of ions. The various dissociation methods, esp ecially infrared multiple photon dissociation and the corresponding infrared lasers will be subseq uently discussed. The most basic components of the FTICR mass spectrometer will also be described. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry The major difference between FTICR and ot her mass spectrometric techniques is the method by which ions are trapped and detected. Mathematical descriptio n of the trapping and detection of ions has been well documented,32 and a overview of how this occurs is beneficial to understanding the spectra obtained from FTICR. Cyclotron motion. A charged particle moves in a ci rcular fashion when subjected to a uniform magnetic field.36 The force, F, applied to the ch arged species is gi ven by Equation 2-1. BvEqF (2-1) where E is the electric field, v is velocity vector of the ion and B is the magnetic field component of the uniform electromagnetic field. The charge of the ion is q, where q and z are used interchangeably. Assuming that the ions are moving in a uniform magnetic field and no electric field is present, the first term on the ri ght side of Equation 2-1 can be neglected. The 39

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vector product of the velocity and the magnetic field can be separated into its individual Cartesian coordinates by evaluating the determinant shown in Equation 2-2: zyx zyxBBB vvv kji Bv (2-2) xyyx xzzx yzzyBvBvkBvBvjBvBviBv det (2-3) The unit vector components for x, y and z Cartesian coordinates axes are and k respectively. If the magnetic field is uniform along the z-axis with no x and y components, then the determinant can be simplified to Equation 2-6. zBB (2-4) 0 yxBBkBB (2-5) zx zyBvjBviBv det (2-6) The acceleration of the ion due to the magnetic fi eld can be described by the derivative of the velocity times the mass (m) of the ion and is equivalent to the force by the uniform magnetic field on the charged species. zx zy zyxBvjBviqBvq dt vvvd m dt vd mamF (2-7) Each acceleration component can be related to a distinct term in the determinant given in Equation 2-6. zy xBqv dt dv m (2-8) zx yBqv dt dv m (2-9) 0 dt dv mz (2-10) Rearranging Equations 2-8 and 2-9 shows that the acceleration is dependent on the mass-tocharge ratio ( m/z ) of the ion and the magnetic field strength along the z-axis. 40

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ycyz xvvB m q dt dv (2-11) xc xz yvvB m q dt dv (2-12) where c is the cyclotron frequency of the ion, equal to (q/m)B. There is no influence of the magnetic field on ion motion along the z-axis and cy clotron motion occurs only in the x-y plane. Equations 2-11 and 2-12 are a set of coupled diffe rential equations and in the general case the velocity as a function of time (t) along each ax is can be described by Equations 2-13 and 2-14. t vtvtvc xc y x cos0 sin0 (2-13) tvt vtvc xc y y sin0 cos0 (2-14) The initial velocity vector of an ion can be decom posed into initial x and y velocity vectors, with magnitudes related by 0002 2 y x pvvv (2-15) The initial angle of motion ( ) is the arctangent of the ratio of the initial y and x velocity vectors (Figure 2-2). cos00p xvv (2-16) sin00p yvv (2-17) 0 0 arctanx yv v (2-18) Substituting Equations 2-16 and 2-17 into the general equations for the velocity vectors, and expanding and simplifying Equations 2-13 and 2-14 us ing the algebraic identities in Equations 219 and 2-20 gives Equations 2-21 and 2-22. t vtvc p xcos0 (2-19) t vtvc p ysin0 (2-20) t t vtvc c p x coscos sinsin0 (2-21) t t vtvc c p y sincos cossin0 (2-22) Recognizing that the velocity is a derivative of the position of the ion wi th respect to time, equations for the x and y coordinates of th e ion, with respect to time, are obtained. 41

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tv dt dxx (2-23) tv dt dyy (2-24) 1sin 0 Ct v txc c p (2-25) 2cos 0 Ct v tyc c p (2-26) The constants of integration, C1 and C2, can be obtained in the case when time t = 0, and by rearrangement of x(t) and y(t) 1sin 0 0 C v xc p 2cos 0 C v tyc p (2-27) sin 0 01 c pv xC (2-28) cos 0 02 c pv yC (2-29) Using the identities for cos(a-b) and sin(a-b), th e equations that descri be ion cyclotron motion along the x and y Cartesian coordi nates in a uniform magnetic field along the z-axis are given by Equations 2-31 and 2-32. sin sin cos cos (2-30) t t v xtxc c c p sincos cos1sin 0 0 (2-31) t t v ytyc c c p sinsin cos1cos 0 0 (2-32) The general radial component of a vector provi des the description of the ion cyclotron motion near the origin when substituting in Equations 2-31 and 2-32 for x(t) and y(t) in Equation 2-33. tytxtr2 2 (2-33) 2 1cos1 0 2 t v trc c p (2-34) The angular component of the trajectory n ear the origin can be described with = 0 and substituting into Equations 2-31 and 2-32 yields Equati ons 2-35 and 2-36. 42

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t v txc c p sin 0 (2-35) t v tyc c p cos1 0 (2-36) The position of the ion at different time can be calculated, and plotting the position at times t = / c, / c, 3 / c, 2 / c, the ion motion is seen to be circular and periodic (Figure 2-3A). Equation 2-37 describes the periodic ity, or frequency, of the motion. 2c cv (2-37) Motion starting in the opposite direction, = gives equations for x(t) and y(t) that have opposing trajectories but have the same terms as x(t) and y(t) at = 0. t v txc c p sin 0 (2-38) t v tyc c p cos1 0 (2-39) Plotting x and y coordinates for times t = / c, / c, 3 / c, 2 / c, the ion motion is again circular and periodic (Figure 23B). Since the ICR cell is pla ced inside the bore of the magnet and parallel to the magnetic field, the ions will develop an inte rnal cyclotron motion perpendicular to the field and therefore ions wi ll move in a circular fashion, dictated by the equations just described. The mass-to-charge rati o of the ion will dictate the cyclotron frequency (Equation 2-40). For an ion with mass 221 Da a nd a charge of +1, in a magnetic field of 4.7 Tesla strength, the ion would have an angular frequency ( c) of 324.1 kHz, and a radio frequency of 51.6 kHz would be required to excite the ion into a higher orbit. B m q (2-40) 43

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Analysis of the units starting with the Lo rentz force equations, 2-1 and 2-7, shows the relationship between the frequency and ma ss-to-charge and magnetic field product. Bv m q dt vd T s m kg molC s m2, rearranges to give TC kg mol s 1 where C is the coulomb charge of the ion and T designates the magnetic field strength in Tesla units. In order for the ion cyclotron motion to be bene ficial for detecting the ions in the ICR cell, the ion cyclotron orbital radius ne eds to be excited using a uniform electric field oscillating at the cyclotron frequency of an ion with a particular m/z The presence of an alternating electric field, E can be described by the Equation 2-1. BvEq dt vd mF kBB (2-41) The alternating electric field can be described by Equation 2-42. itEEo sin1 (2-42) The magnitude of the electric field is Eo, 1 is the frequency of the al ternating electric field, and is the phase of the alternating field. Rearra nging Equation 2-41 to obt ain the acceleration and substituting in the i on cyclotron frequency, c, gives the coupled partial differentials of the velocity along the x and y axes due to the presence of the electric field. yc o xvtE m q dt dv 1sin (2-43) xc yv dt dv (2-44) Differentiating Equation 2-44, the partial second deri vative of the velocity along y is equivalent to the negative partial first derivative of th e velocity along x times the ion cyclotron frequency, c. 44

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yc oc x c yvtE m q t v t v 1 2 2sin (2-45) Equation 2-45 is a quadratic equation and can be solved using the general solutions of differential equations of the form (D2+ c 2) vy, to obtain velocity components along x and y coordinates. 0 sin2 1 2 2 yc oc yvtE m q t v (2-46) 2 1 2 1 2 1sin sin cos c occ c yt E m q tctctv (2-47) 2 1 2 1 1 2 1cos cos sin c o c c xt E m q tctctv (2-48) The components vy(t) and vx(t) are related by Equation 2-49. xc yv dt tdv (2-49) Equation 2-47 reduces to Equation 2-50 when t = 0. 2 1 2 1sin c oc yE m q ctv (2-50) Similarly, the velocity component of along the x-axis can be simplified at t = 0. 2 1 2 12cos c o xE m q ctv (2-51) Let = x(0) = y(0) = 0: 2 1 2 1 1sin sin sincos 0 cos1 sin0 0 c c c o c c p c c pt t E m q t v t v xtx (2-52) 45

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2 1 2 1 1 1 1cos cos sinsin 0 cos1 cos0 0 c c c c o c o c c p c c pt t E m q E m q t v t v yty (2-53) When =0, a situation may arise where 1 = c and the functions of x(t) and y(t) are undefined. Using LHopitals rule, the undefined terms of x(t) and y(t) can be solved. Equation 2-54 gives an example for x(t) 1 1 2 1 2 1 12 cos sin | sin sin1 1 ttt t t Limcc c ccc (2-54) Using the case where 1= c, the previously undefined terms can now be simplified. c c ccttt 2 cos sin (2-55) c c cc c o c c ptttE m q t v tx 2 cos sin sincos 0 (2-56) 2sin cos2 2 1cos 02 ttt E m q t v tyc cc c o c c p (2-57) Equations 2-56 and 2-57 give the motion of the ion as its cyclotron mo tion is excited by the alternating electric field (Figure 2-4). Analysis of the motion indicates that the important terms that change are those show n in Equations 2-58 and 2-59.32 tt E m q txc c c o cos 22 (2-58) tt E m q tyc c c o sin 22 (2-59) A schematic description of the FTICR ion de tection process follows (Figure 2-5). A broadband RF voltage is applied to two excite plates of the i on trap; if a particular radio frequency is in resonance with the ion cyclotron frequency of a trapped ion with a particular m/z the resonance condition will excite the ion into a higher cyclotron orbit. The ion cloud is now closer to the detection plates, and as the cloud sweeps by them, an alternating current is induced 46

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on the plates. The current induced by each ion with different m/z in the ion cloud can be plotted as a function of time. The image current can be very convoluted if more than a few ions with different m/z are present (Figure 2-5B). This transien t response signal of induced current as a function of time can then be deconvoluted through Fourier transformation. The Fourier analysis transforms the time domain sign als into discrete frequency do main values, which correspond to the inherent ion cyclotron resonance frequencies of each mass ion present. Using Equation 2-40, the frequencies are converted into the m/z domain spectrum, or mass spectrum. Trapping motion. So far, only ideal conditions of cyclotron motion have been discussed, and the kinetic energy of the ions as th ey enter the cell has been neglected. Ions traveling into the ion trap experience a potential barrier ( positive or nega tive voltage, depending on whether the ion is positive or negative, respectively) at the end plate of the ion trap. The potential is made large enough so that the ions are deflected toward the front of the ion trap. Another potential is raised at the front pl ate, so that the ions are deflected back and forth within the ion trap, which is called the trapping motion. With the presence of the trapping potential and the magnetic field, a 3-dimensional trap has been created and the resu lting potential is described by Equation 2-60. 22 22 2 rz a Vzrtrap (2-60) 22yxr (2-61) where r is the radial position of the ion in the xy-plane, Vtrap is the trapping voltage, a is the measure of trap size and and are constants that depend on the ion trap shape. The electric field is obtained by taking the negative derivativ e of the potential with re spect to the z direction. z a V dz d zEtrap 22 (2-62) 47

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The force on the ion applied by the electric fiel d would accelerate the ion along the z axis in a motion parallel to the magnetic field, which is uniform along th e z-axis of the laboratory reference frame. z a qV dt dv mzFtrap z 22 (2-63) The force equation indicates that the trapping motion is a simple harmonic oscillation between the trapping plates. The oscillation frequency, z, is given in Equation 2-64. 22 ma qVtrap z (2-64) Magnetron motion. The magnetic and electric fields together introduce a third fundamental motion of the trapped ion, called th e magnetron motion. The cyclotron and trapping motions are not coupled, although th e trapping potential produces an outward-directed electric force that opposes the inward-directed Lorentz force of the magnetic field. r a qV rqErFtrap 2( (2-65) Combination of both force equati ons provides the motion of an i on subjected to a 3-dimensional axial trapping quadrupolar electrostatic potential and a uniform magnetic field. r a qV rqBrmFtrap o 2 2 (2-66) 02 2 ma qV m qBtrap (2-67) Equation 2-67 is a quadratic that is independent of the ion positi on inside the trap (r). Two natural rotational frequencies are obtai ned from the solution of Equation 2-67. 2222 2 z c c (2-68) 48

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2222 2 z c c (2-69) Where + is the reduced cyclotron frequency of the ion in the presence of the d.c. trapping potential and is the magnetron frequency. The magnetron and trapping frequencies are usually less than the cyclotron frequency, so onl y the cyclotron frequenc y is detected. The cyclotron, trapping and magnetron motions of the ion (Figure 2-6). As seen from the figure, the magnetron motion is circular and superimposed on the cyclotron motion. Both motions are superimposed on the trapping motion. Instrumentation. To introduce ions into the ion trap, various sources are used for internal or external ion production. As was mentioned ear lier, electron impact ionization (EI) was one of the first internal sources, producing ions by bombard ment of a gaseous sample with electrons. In FTICR instruments the EI source is inside the va cuum chamber, near the magnetic field and the ion trap. A positively charged repeller plate in the source moves the cations, formed from the bombardment of the gaseous sample with high ener gy electrons (70 eV), into the ion trap to be detected. External sources tend to be farther from the magnet, and therefore ions produced in them need to be guided with electrostatic optics toward the ion trap. The longer path requires a vacuum pumping system that will keep the vacuum low enough so the ions do not suffer too many collisions as they travel toward the ion tr ap. This places stringent pumping requirements on FTICR-MS, since it require s very low pressures (< 10-9 torr) to keep ions trapped for long periods of time. A number of different ionization techniques can be employed to produce saccharide ions in mass spectrometers, both within the ion source and externally. Currently, the most popular ones are matrix assisted laser desorption ionization (MALDI),37 electrospray ionization (ESI),38-43 and, in the recent past, fast atom bo mbardment (FAB). The external source developed by John Fenn and co-workers, electrospray ionization (ESI), has allowed FTICR-MS 49

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to develop as a valuable tool for studying molecules of many sizes, including carbohydrates and peptides, and was the ionization method used for this work. Electrospray ionization is able to introduce liq uid samples that have a low vapor pressure, into the gas phase. ESI creates a fine mist by pushing the liquid sample through a small diameter tubing of silica. As the mist moves farther aw ay from the tip, the mist spray expands and the individual droplets become smaller and smaller due to evaporation, until the solvent and the ions are separated from each other and th e bare ions enter the capillary,44 although ions surrounded by solvent continue to be present inside the capillary (Figure 2-7). A positive or negative electrostatic potential of appr oximately 3000 V is placed on the cap illary to attr act ions of opposite charge, thus taking the i ons from an atmospheric pressu re environment to a vacuum region of 10-3 torr. As the ions travel through the capil lary, further desolva tion is facilitated by heating the capillary in the range of 100 200 C. The ions pass through a set of skimmers to improve ion abundance by removing neutral molecule s. The ions at this point have differing kinetic energies, and therefore are trapped and a ccumulated in a hexapole by an RF voltage. The ions are held for 500 1000 ms and are sent as a bunch toward the ion trap. The ion cloud needs to be kept from diverging due to the electros tatic repulsion between cations and the increasing low pressure gradient in the vacuum chamber. A set of electrostatic plates or einzel lenses, are used to focus and slightly deflect the ions, although some FTICR-MS instruments use octopole ion guides to steer the ions into the ion trap (Figure 2-8). Because of the gentle nature of the ESI proce ss, molecular ions as well as multiply-charged ions are observed in the mass spectrum. The multiply-charged ion formation is an important feature of ESI, since FTICR mass spectrometers measure m/z making it possible to observe large molecules in an instrument with a restricted mass range. 50

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An example of the use of mass spectrometry to study sugars is found in the work of Cole et al ., where the oligosaccharide ions of maltose (G2) through maltoheptaose (G7) were formed by electrospray ionization and analyzed with a quadrupole mass spectrometer.45 Using LiCl as the ionization agent, the oligosaccharide ions of malto se attached a lithium cation, but also formed complexes where multiple LiCl molecules were attached to the -glucose monosaccharide units of the oligosaccharide ions. The number of LiCl attached increased as the oligomers became larger. Theoretical calculations indicated that the lithium cation was triply coordinated to hydroxyl groups and the glucose ring oxygen atom. In another study involving proteins, the ma ss spectrum of myoglobin was observed using ESI, with myoglobin seen to form ions having multiple charge states.46 The charge states from z = 8+ to 23+ were observed in the mass spectrum. Using the m/z of one of the observed charge states, the molecular weight of horse myoglobin was determined (MW = 16,951 Da). The ion traps used with FTICR-MS are Penning traps.47 Named after F. M. Penning by Hans G. Dehmelt, the most basic Penning trap is the cubical ion trap or cell,48 which has six electrostatic plates (Figure 2-9). The front and back plates have an aperture at the center of each plate to allow the introducti on of ions or radiation into the cell. These plates serve to trap ions by placing electrostatic voltages on the plates, creati ng a potential barrier that will deflect the ions back and forth within the ion trap. Two excite plat es, placed left and right, and two detect plates, placed top and bottom, form the body of the trap. The excite plates have an RF voltage applied to them to increase the radial motion of the ions in the cell (as can be derived from Equations 258 and 2-59), while the detect plates are connected through a resi stor to capture the alternating current induced by the ion charge as the ions sweep by the plates. 51

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Similar functionality can be obtained by a closed cylindrical cell, where the trapping plates are circular instead of square or rectangular, and the detection and excite plates form a cylinder of four separate plates. A closed cylindrical cell was used in th e FTICR-MS experiments conducted at the University of Florida. Anot her common configuration is the open cylindrical cell, where the trapping plates ar e replaced by two sets of cylindrical plates, similar in design to the cylindrical detect and excite plates. Th e open cylindrical cells have the advantage of accumulating the maximum amount of ions within the restrictions of the diameter of the cell. While almost any shape of cell can be constructed to trap ions, the open cylindrical cell has lately been the preferred choice to include in comm ercial instruments due to its high S/N, ion accessibility, simplicity in design and low cost construction. An open cylindr ical cell was used in FTICR-MS experiments conducted at the FOM-Institute (Figure 2-10). Tandem Mass Spectrometry The FTICR-MS has the highest mass resolving power of any mass spectrometry technique. Separation of ions within 1 Da can quite easily be accomplished and accuracies of better than 300 ppb have been demonstrated.49 One of the drawbacks of FTICR-MS and, indeed, any mass spectrometric technique is that isomer differentia tion is not possible, since isomers have the same m/z Tandem MS methods (MSn) have been developed to overcome this limitation. As was previously discussed, trapping of the ions is essential for the detection of molecular ions. But trapping ions in the ICR cell can also serve to induce fragmentation of precursor ions. In MSn experiments, a precursor ion is usually fragme nted and may show a distinct fragmentation pattern relative to the other isomers. Further fr agmentation of a fragment ion that was produced by dissociation of the precursor ion can be done to obtain a third level of identification (MS3). The tandem MS process is limited by the S/N an d abundance of the ion being fragmented, but MS4 and higher have been conducted with FTICR-MS.46 52

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It has been shown for carbohydr ate isomers that depending on the conditions of tandem MS experiments, the fragmentation patterns obtai ned for different isomers may or may not be distinct. Leary et al obtained low-energy collision indu ced dissociation (CID) tandem mass spectra of monolithiated disaccharides that showed many cleavages of the reducing end monosaccharide, but there was little diffe rentiation between the different linkages.50 In the same study, each dilithiated disaccharide did produce di stinct fragmentation ion patterns and it was possible to assign the (1 6) glycosidic bond linkages instead of the (1 4) linkages to four glucose oligosaccharide isomers. The binding strength of alkali metal ions to carbohydrates was obtained using infrared multiple photon dissociation (IRMPD) and co llision induced dissociation tandem mass spectrometric techniques. Cancilla and co-workers determined that the binding energy of alkali metal ions to oligosaccharides decrease d going down group IA of the periodic table.51,52 Lithium is bound strongly enough to the carbohydrates that during irradiation and subsequent fragmentation its direct loss is much less relative to that of the other alkali metal cations. For ions containing K+, Rb+ and Cs+ only loss of the alkali meta l cation was seen when the complexes were subjected to IRMPD. Using CID, other fragmentation ch annels were observed, even for precursor ions containing the larger alkali metal cations. Infrared multiple photon dissociation can also be used to obtain infrared spectra of ions in the gas phase, and therefore will be discussed next. Infrared Multiple Photon Dissociation Detailed knowledge of the primary structure of sugars and amino acids is the major goal of this work and tandem MS techniques that ar e complementary with gas phase infrared spectroscopy were utilized. But typical infrared spectroscopy ba nds of solid and solution phase samples are too broad to distinguish characteristics for sugar or amino acid isomers. The FTIR 53

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spectrum of -D-glucopyranose obtained from a crystallin e sample in a KBr pellet illustrates the broad peaks in the infrared spectrum (Figure 2-11). For gas phase photon absorbance spectroscopy th e abundance of the ions is too low to detect a change in the incident radiation intensity. Action or consequence spectroscopy must be used and is distinct from absorbance spectrosco py in that the absorption of radiation is not explicitly detected, but instead, the action or consequence of absorption of radiation is monitored. One particular type of action sp ectroscopy, infrared multiple photon dissociation (IRMPD), is used to obtain infrared spectra of ga s phase molecules or ions. By monitoring the fragmentation of the precursor ion and its signal loss in the ma ss spectrum at a particular irradiation wavelength, a spectrum of the loss as a function of different wavelengths can be constructed. Mechanism Infrared multiple photon dissociation (IRMPD) occurs because the fluence, or density over time, of the laser intensity is large enough that multiple photons of the same ir wavelength are absorbed by an ion, causing the ion to fragment. This phenomenon was first observed in the 1960s as the CO2 laser became more powerful and had higher photon intensities. The collisionless environment that the FTICRMS technique provides was used by Beauchamp and co-workers, who initially demonstrated the use of low intensity CO2 lasers to induce fragmentation of ions in low pressure ICR traps.53,54 Absorption of photons whose frequency is in resonance with the freque ncy of a vibrational mode (vi), excites the ion into a higher vibrational level, (vi+1) (Figure 2-12). Intramolecular vibr ational redistribution (IVR) distributes the energy throughout the other normal modes, and the original vibrational mode returns to its ground state. Another photon with the same wa velength can again be ab sorbed into the ground state of vi, and intramolecular redistribution of the photon energy occurs again. The sequential 54

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absorption process is slow, and the FTICR-MS provides a collisionless environment so that collisional relaxation does not occur. Photon energy absorption and IVR continue so that in essence the process reaches a qua si-continuum of vibrational energy levels. Some vibrational modes may have shallow potentia l curvature, and as IVR occurs one of the modes may be shallow enough that the energy being redistributed into this mode will be large enough to cause dissociation. The quasicontinuum condition a llows absorption of photon energy at any ir wavelength into the vibrational mode that has the lowest potential energy barrier toward dissociation, facilitati ng bond cleavage. Fragmentation can be observed from this weakest bond or other modes with relatively low potentia l energy barriers, and in FTICR-MS these fragmentations are observed in the tandem mass spectru m. It is important to note that if initially the laser frequency is not in res onance with at least one vibrational mode of the molecule, then the IVR process will be inefficient, and ther efore subsequent dissociation cannot occur and fragmentation will be minimal. The IRMPD experiment uses a high fluence, frequency tunable infrared laser and the overall fragmentation as a function of laser fr equency, or IRMPD spectrum, can be obtained. The most common tunable infrared lasers for obtaining IRMPD spectra will be discussed in the next section. These include the CO2, optical parametric oscillator and free electron lasers. For the most part all lasers have the following features: A lasing medium An optical resonant cavity A pumping mechanism that provides excitation to the medium to achieve population inversion There are numerous configurations for each laser, so only the basic properties will be described and the features used in this work will be highlighted. 55

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Carbon Dioxide Laser One of the first lasers to be used for spectroscopy, the CO2 laser55,56 is used throughout science, the manufacturing indus try and medical fields in multip le applications. The lasing medium is a gas mixture that is mainly composed of the following gases: CO2, N2, He, Xe and H2O. The gas mixture fills a chamber enclosur e where a discharge is applied. The optical resonator includes one totally reflective surf ace and one semi-reflective mirror that form the chamber enclosure ends. For wavelength tunable CO2 lasers, the totally reflective surface is replaced by a Brewster window and a diffraction grating.57 The grating is rotated so that a different lasing wavelength will be in resonance in the optical cavity at a particular grating setting. The basic mechanism of lasing follows. The gas discharge excites the CO2 molecules from the ground vibrational level to higher vibrationa l levels. The discharge also excites the N2 gas, and when N2 collides with the CO2 gas, energy is tran sferred causing the CO2 molecule to go into a higher vibrational state (Figure 2-14). The N2 vibrations are in close resonance with the v3 vibrational mode of CO2 (asymmetric stretch), and therefore energy is transferred into this state. Relaxation occurs into lower lying excited vibrat ional levels, and photons are released within the resonant cavity. The photons with wavelengths that are resonant with the cavity length will dominate (constructive interference), and will induce the CO2 molecules to absorb more radiation and stimulate emission of radiation with the same wavelength. This process of amplification continues to the point where th e population of molecu les occupying the higher energy v3 state is greater than the molecules occupying the other v1 and v2 states, or what is termed population inversion. The ga in saturation is exceeded if th e coherent beam of photons is intense enough to overcome the threshold of th e resonant cavity, and the laser beam exits through the semi-reflective mirror. For a tunable CO2, the length of the cavity can be changed so 56

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that any of the rotational-vibrational modes of the CO2 molecule can be in resonance within the cavity, and lasing occurs at the wa velength equal to the energy leve l of the rotational-vibrational mode. The gas discharge increases the temperature of the lasing medium and degrades the lasing efficiency over time if the laser is not cooled. Most CO2 lasers have a cooling system that runs water or a coolant through an isolated outer chamber of the resonance cavity, but some low power CO2 lasers are air-cooled. Cooli ng is also obtained with the introduction of He in the gas mixture. He gas cools the gas discharge by increasing the thermal conductivity of the gas mixture, so that heat is quickly transferred to the walls of the resonance cavity, which is surrounded by the coolant chamber. The ionizati on potential of He (20 eV) is too high to influence the discharge (1 3 eV), but the di scharge current can now be increased since the temperature of the gas mixture is lower due to the addition of He The resulting effect is higher lasing efficiency and power. The addition of Xe to the gas mixture (0.5 torr) also increases laser efficiency and power by affecting the discharge conditions. The low ioni zation potential of Xe (12 eV) facilitates new electron production and maintains a consta nt discharge with a constant current. Because the current is constant, the elec tric field in the medium decreases and the mean energy of the electrons in the discharge is reduced. This is favorable for the CO2 and N2 gases, since their collision cross section increases when the electron energy is less than 4 eV. The resonance cavity can be sealed or the gas mixtur e can be allowed to flow through the chamber. In a sealed chamber, the electric discharge induces the breakdown of CO2 to form CO, which degrades the lasing capabilities over time. Addition of 0.2 torr of H2O is crucial, since it virtually eliminates the formation of CO and increases the lifetime of the laser56 through the chemical reaction of CO + H2O CO2 + H2. The constant concentration of CO2 obtained by 57

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the addition of H2O maintains the pumping rate and therefore the lasing efficiency is not reduced. When the gas mixture flows through the chamber, a gas pumping system is used to carry away the degraded mixture. This requires a constant supply of the gas mixture, which can increase the cost of daily opera tion of the laser. The fast fl ow setup has the potential of obtaining higher powers, but advances in both ty pes have been made and either setup can produce laser beams with high intensities adequate for IRMPD experiments. Earlier work of Eyler and co-workers to obtain IRMPD spectra was done with CO2 lasers.58,59 In a study by Shin and Beauchamp, the IRMPD spectrum of Mn(CO)4CF3 was obtained by monitoring the loss of two carbon monoxide molecules as a function of CO2 laser irradiation at different wavelengths.60 One spectrum from that st udy (Figure 2-15) shows that CO2 lasers do not provide continuous coverage of infrared bands. Nevertheless, the CO2 laser has many uses, one of which is to obtain IR MPD fragmentation patte rns of carbohydrates and amino acids. In chapter 5, the CO2 laser will also be used as an off resonance photon source for IRMPD experiments using an optical parametric os cillator (OPO) laser, which will be discussed next. Optical Parametric Oscillator An optical parametric oscillator (OPO)61 consists of an optical resonator cavity and a nonlinear crystal. The resonance cavity can be a two-mirror cavity or one end of the crystal can serve as the second reflective surf ace of the resonator (Figure 2-16) The OPO uses a pump laser and takes advantage of the prope rties of a non-linear crystal to obtain cohe rent radiation at wavelengths longer than the pump lasers wavele ngth. The pump photon is converted into two photons within the non-linear crys tal, and their resultant laser beams are called the signal and idler beams. Due to conservation of energy, the idler and signal beam frequencies are equal to the pump lasers frequency. 58

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p = i + s (2-70) Only the frequencies that satisfy the conserva tion of momentum will be generated efficiently. p = i + s (2-71) j = 2 njhj (2-72) npp = nii + nss (2-73) The term is the wave vector of the frequency n is the index of refraction at frequency j, and h is Plancks constant. This phase matching condition allows for lasing at almost any wavelength, limited by the transparen cy region of the non-linear crys tal. Because the OPO itself is not involved in a stimulated emission pro cess to obtain a populati on inversion, it requires a pump laser with good beam quality and high powe r density. For a continuous-wave (cw) OPO, the threshold is met when the gain exceeds loss es in the cavity resonator. The OPO can be constructed so that a single id ler beam is output, or both idle r and signal beams are present, termed a double resonant OPO. In the double resonant OPO, both beams depend on the configuration of the reso nator so each beam radius is decreased to maximize the gain. The size of the beam radii is limited by the laser induced damage threshold and birefringence of the crystal. The interacting wave lengths in the resonance cavity and the crystal deposit heat throughout the crystal, establishing a gradient of hot and cold regions with the surface. Phase matching is dependent on a constant temperatur e and therefore cannot be maintained throughout the crystal, limiting the power output. The temper ature gradient can be minimized if the crystal is placed within an oven and kept at a constant temper ature. Fabricated n on-linear crystals, such as a periodically poled lithium niobate (ppLiNbO3) crystal, have high damage thresholds, so that Nd/YAG lasers of a few watts can be used as pump lasers. The high damage threshold of ppLiNbO3 allows the crystal to be heated to high temperatur es (150 C), increasing the efficiency and power of the output beams. Tuning of an OPO can be accomplished by changing 59

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the temperature of the crystal, changing the phase matching cond itions of the idler and signal beams. The ppLiNbO3 crystal has multiple poling periods corresponding to different wavelength ranges that allow for lasing from 1.38-2.0 and 2. 28-4.67 microns at a set temperature between 50 150 C. An etalon, placed within the resonance cav ity, is used to tune a particular set of idler and signal wavelengths within a particular poling period and temp erature range. To enhance one resonance mode within the cavity, a piezo-elect ric mirror can be used as one end of the resonance cavity and combined with lock-in electronics to keep the laser wavelength in resonance. Optical parametric oscillators and amplifie rs are relatively ne w technology and IRMPD spectra of carbohydrates are ra re. Ultraviolet pulsed-OPOs have been used in ion dip spectroscopy studies,62 and Simons and co-workers have studied the glycosidic linkages of lactosides,63 and hydrogen bonding of the monosaccharides mannose, galactose, glucose and lactose.64 In the chapter 5, IRMPD sp ectra of the glycoside anomers of glucose and galactose, obtained with an infrared cw-OPO, will be presented. Free Electron Laser Ever since an FTICR-MS was coupl ed to a free electron laser (FEL)65 at the FOM-Institute for Plasma Physics in The Netherlands (FOM-Institute) by Valle et al.,66 other national laboratories have followed. There are a total of 13 locations that house FELs for scientific research67 and 3 of these are able to conduct IRMP D studies using the FEL with an FTICRMS68,69 or other ion trap mass spectrometers.70,71 In the following section, the general lasing and tuning properties of the free el ectron laser will be summarized. Detailed properties of the FOMInstitutes FEL, the F ree E lectron L aser for e X periments (FELIX), will be discussed in chapters 3 and 4 and therefore only relevant information re garding specific properties will be included in this summary. 60

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An accelerating electron gun produces a beam of el ectrons that are guided into a resonant cavity and passed through an undulating magnetic fi eld (Figure 2-17). In the resonance cavity, the electrons are accelerated with the magnetic field to relativis tic speeds, and release photons. The electrons tend to bunch and thus radiate in phase. The bunching characteristic of the electrons makes all FELs pulsed-wave lasers. Gain amplification is required to overcome cavity losses and slight detuning of th e resonant wavelength is done to increase the net gain of the optical wave. Because of the nature of the fr ee electron beam, FELs are continuously tunable and have large output wavelength ra nges (FELIX is tunable between 2.0 250 m). A high energy electron beam (1 800 MeV, 2 500 Amp) and optimizing for maximum gain can obtain femtosecond pulses with 4 MW 36 GW, producing laser power of 0.1 10 MW at 1% efficiency. An FEL produces s hort laser pulses of 500 fs 10 ps that are separated by a few hundred ps ns. Tuning can be accomplished in a number of different ways: The resonant wavelength can be changed by changing the electron beam energy, and this is necessary when the undulator magnets are fixed. Changing the magnetic field strength will alter the resonant wavelength, and the power output will be independent of the laser wave length for a large ra nge. Tuning by changing the magnetic field requires a high quality B field for fine wavelength selection. The laser can be operated at an odd harmonic of the fundamental wavelength, where the gain can be slightly higher. The FEL can be continuously tuned by m oving the undulator magnets or wigglers a certain distance apart so that the free elec trons and their released photons undulate at a specific wavelength. A study by vonHelden et al. looked at the mid-ir spectra of si x gas-phase conformers of phenylalanine.72 A neutral molecular beam was formed by laser desorption (1064 nm irradiation) of a graphite-phenylalanine solid mixture. A UV resonant 2-photon ionization (R2PI) experiment was conducted that scanned th e wavelength of a Nd/YAG pumped dye laser (37500 37700 cm-1) and indicated that six conformers of phenylalanine were present. The IR61

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UV ion dip spectrum of each conformer was obtained by monitoring the ion signal loss as FELIX was scanned from 300 1900 cm-1 followed by irradiation with the dye laser set to a specific conformers ionization wavelength. The ir spectrum of each conformer was distinctly different and density functional th eory calculations indicated that the phenylalanine conformers have a puckered structure. Implementation of IRMPD with FTICR-MS General experimental procedure. An external ESI source coupled to an FTICR with a superconducting 4.7 tesla magnet was employed. To reduce collisions, pressures of approximately 10-9 torr were obtained near the ICR cell in the vacuum chamber, allowing for long trapping times without loss of i on signal due to dephasing collisions.32 Depending on the nature of the experiment, the solvent (mainly a methanol and water mixt ure) included either a dilute protic acid or an alkali me tal salt to ionize the sample. The solution was electrosprayed into the FTICR and through the ai d of electrostatic optics and differentia l pumping stages the ions were transferred to th e ICR cell in the high homogene ity field of the superconducting magnet. Once the precursor ion was isolated, by ejection of all othe r ions using stored waveform inverse Fourier transform excitation (SWIFT),73 the laser beam was introduced into the ICR cell. After laser fragmentation occurred, the fragment ions were excited by an RF broadband pulse and an image current was induced at the detection plates of the ICR cell so that the transient response could be obtained, Fourier transformed, and the mass spectrum processed and stored digitally. The laser was then tuned to a different lasing mode so that the fragmentation process could be repeated at this new lasing frequency. The lasers used provided infrared radiation between 600 2000 cm-1 and 2600 3900 cm-1 depending on the laser type used. The IRMPD spectrum, while not directly indicating the amount of photon energy absorbed, does indicate what vibrational modes are infrared active. In other words, if a 62

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theoretical vibrational spectrum of the molecu lar ion were obtained, those modes with the highest infrared intensities shoul d be the most active in the ab sorption process of IRMPD, and would be predicted to produce th e largest abundance of the fragment ions. Before obtaining a theoretical infrared spectrum of the ion of inte rest, the most stable conformations of the ions need to be predicted by theoretical calculations. B A Figure 2-1. Mass spectrum of D-glucose by A) Electron impact ionization and B) chemical ionization, clearly showing the [M+H]+ ion at 181 m/z Images from Carbohydrate Chemistry: Monosaccharides and their Oligomers by El Khadem et al ., Copyright 1997.3 Reprinted with permission of Academic Press, Inc. y vp(0) vx(0) vy(0) x Figure 2-2. The initial trajectory of the ion, showing the initial velocity vector and angle of motion in the x-y plane. 63

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-1 -0.5 0.5 1 x -2 -1.5 -1 -0.5 y 1 0.5 0 .5 1 x 0.5 1 1.5 2 y B A Figure 2-3. Motion of the ion in a uniform magnetic field, for the conditions when A) = 0 and B) = For simplicity the values for vp and c were taken as unity. x y Figure 2-4. The motion of an ion (in a uniform magnetic field) as its cyclotron motion is excited by an alternating electric field. 64

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Figure 2-5. Schematic for detection of ions in an FTICR-MS. Image courtesy of Environmental Molecular Sciences Laboratory website.74 B A Figure 2-6. A) The superimposed cyclotron and magnetron motions, and B) the superimposed cyclotron, trapping and magnetron motions from Fourier transform ion cyclotron resonance mass spectrometry: A primer, by Marshall et al., Mass Spectrometry Reviews, Copyright 1998.32 Reprinted with permission of John Wiley & Sons, Inc. 65

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Figure 2-7. Electrospray mist. Reprodu ced by permission from Creative Commons.75 to cell Figure 2-8. Drawing of ion transfer optics and names of each optic component. Figure 2-9. Two common Penning traps, a cubi c cell and an open-ended cylindrical cell are shown orientated relative to the unifor m magnetic field. Reproduced by permission of Valle et al .17 66

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FOM-Institute Figure 2-10. FTICR mass spectrome ter at the FOM-Institute. The open-ended cylindrical cell is shown in the inset. Drawing courtesy of Dr. Jos Oomens. Figure 2-11. Infrared spectrum of a crystalline sample of -D-glucopyranose in a KBr pellet. Images from Carbohydrate Chemistry: Monosaccharides and their Oligomers, by El Khadem et al ., Copyright 1997.3 Reprinted with permission of Academic Press, Inc. 67

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Figure 2-12. The initial absorption of photon energy from the ground state to an excited vibrational level and the subsequent intramolecular vibrational relaxation process. Figure 2-13. The CO2 laser cavity. Shown are electrodes for gas discharge, the water jacket part of the cooling system, diffraction grating a nd a diaphragm to select one harmonic of a lasing mode. Brewster window is not shown. Adapted from The CO2 Laser by Witteman et al ., Copyright 1987.56 Reprinted with permi ssion of Springer-Verlag, Inc. 68

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Figure 2-14. Vibrational energy levels of the CO2 and N2 molecules involved in the lasing mechanism. Excitation of CO2, by collision with N2, activates the asymmetric stretch (001). Photons are released when the mol ecule relaxes down to (I) symmetric stretch or (II) one of the angle-bending modes. Only the fundamental vibrations are shown, but each mode is coupled with rotations, and relaxation can occur to a rotationalvibrational mode with a slightly different wavele ngth than the fundamental. Figure 2-15. IRMPD spectrum of Mn(CO)4CF3 -, using a tunable CO2 laser. Image reproduced from Infrared multiphoton dissociation spectrum of CF3Mn(CO)3(NO)-, by S. K. Shin and J. L. Beauchamp, Journal of American Chemical Society, Copyright 1990. Reprinted with permission of American Chemical Society. 69

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1 1 1 Signal Pump Nd/YAG Idler etalon Figure 2-16. Diagram of an OPO laser, showi ng the pump beam of an Nd/YAG laser (yellow) being converted into the idle r (blue) and signal beams (red) by a periodically poled LiNbO3 crystal. The resonan ce cavity is defined by one semi-reflective mirror (right end) and a reflective coating on the surface of the crystal (left end). An intracavity etalon is shown, that helps tune a particul ar set of idler and signal wavelengths. Image courtesy of Wright L. P earson and LINOS Photonics, Inc. crystal FOM-Institute Figure 2-17. Free electron laser schema tic, courtesy of the FOM-Institute. 70

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CHAPTER 3 THEORETICAL CALCULATIONS Molecular Modeling Theoretical chemistry is crucial in obtain ing structural information from an IRMPD experiment and can also serve to probe the dynamics of the molecular species In order to obtain the theoretical vibrational spectrum of a species, a number of points need to be considered. First, the correct structure needs to be found, or at l east one that will reprodu ce the spectrum within reasonable error.76 The existence of multiple minima on the potential energy surface of a molecule makes finding the most stable structur es the first priority when trying to obtain theoretical vibrational frequenc ies of carbohydrates and amino acids This requires exploring the potential energy surface of the dissociating i on to find a global minimum structure. Assignment of vibrational modes in the IRMPD spectrum of a molecular ion requires that either 1) the bands are well reso lved and/or can be deconvoluted to obtain all 3N-6 vibrational modes, or 2) theoretical calculations reproduce the experimental infrared spectrum. Option 1 is difficult to obtain with action spectroscopy for a number of reasons. Typical IRMPD experiments are conducted at room temperature, where a range of excited rotational energy levels couple with vi brations of the molecule so each band is slightly broadened. The conditions of FTICR trapping lead to magnetron and trapping motions of the ions which produce a spatial distri bution of the tightly-packed ion cloud, so that non-uniform irradiation of the ions results. Fluctuation in ion signals due to inherent electronic noise will introduce an uncertainty that will be reflected in the IRMPD spectrum. Some modes may be degenerate, and cannot be resolved with the current resolution of the FTICR-MS/IRMPD experiment. An example would be C-H stretches that have similar molecular environments such that they have the similar stretching fr equencies. This is evident in sugars, wher e the carbons 2 4 of 71

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a six-membered D-glucoside can have similar el ectron density throughout the region, and the CH stretches form one band around 3000 cm-1 with a full width at half maximum of 250 cm-1. If correction of these conditions were possible, assignment of bands w ould continue to be more time consuming without the use of theore tical calculations. The following section will introduce methods to obtain theoretical structures and their vi brational spectra. The general Hartree-Fock (HF) and density functional theory (DFT) methods in term s of the Hamiltonian will be described. Gaussian or Pople type basis sets used to describe the wavefunctions of the molecule will be introduced, focusing on those that are used to describe carbohydrates and amino acids. The empirical and semi-empirical force fields parameters that were used for the random torsion angle search algorithm will be discus sed as an efficient way to obtain multiple conformations of the molecule. The conformational algorithm itself will be subsequently summarized. Finally, obtaining the theoretical infrared spectra will be discussed. Theoretical Background Both quantum and molecular mechanics met hods examine potential energy surfaces with single point calculations, geometry optimizati ons and molecular dynamics simulations. In quantum mechanics, the Schrdinger equation gi ves the wavefunctions and energies of a molecule. E H (3-1) The term H is the Hamiltonian, is the wavefunction and E is the energy. The Schrdinger equation is an eigenvalue equation of the form in Equation 3-2. cfOf (3-2) The operator, O, acts on the function (f) producing an eigenvalue and the function itself. The molecular Hamiltonian is composed of the following operators: 72

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Ne ee NN eNUUUKKH (3-3) KN is the kinetic energy of the nuclei (N), Ke is the kinetic energy of the electrons (e), UNN is the nuclear-nuclear repulsion term, Uee is the electron-electron repulsion term and UNe is the attractive potential between nuclei and electrons Nuclei are many times more massive than electrons, and the motion of the nuclei can be co nsidered fixed relative to the motion of the electrons. This approximation, termed the Born-Oppenheimer approximation, indicates that the electron distribution is only dependent on the fixed positions of the nuclei and not their velocities. The molecular Hamiltonian then is s implified to only electronic terms, and is called Helec. Ne eee elecUUKHH (3-4) This allows the electronic portion and the nucle ar portion of the Schrdinger equation to be solved independently. Solving for the electronic portion produces an eff ective nuclear potential, Eeff that depends on nuclear coordinates and describes the potential energy surface for the system. elec eff elecelecE H (3-5) The effective nuclear potential is also used as part of the nuclear Hamiltonian (Hnucl) in the Schrdinger equation of nuclear moti on to describe vibrations, tran slations and rotations of the nuclei. Many different wavefunctions ar e solutions to the Schrding er equation, corresponding to the different stationary states of the molecule. The wavefunc tion must meet the following two requirements for the eigenvalue equa tion to be solvable: 1) The should be normalizable, so that integration over all space gives the probability density of all electrons. 1)(2drri (3-6) 73

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The probability density for all electrons within a system is given by 2 and r is an arbitrary coordinate. 2) The wavefunction needs to be antisymmetric since all electrons are indistinguishable but cannot occupy the same space. ijji ,, (3-7) The wavefunction of the electronic Schrdinger e quation is termed a molecular orbital of the molecule and each molecular orbital is approxim ated with a linear combination of atomic orbitals. v vvc ic (3-8) This wavefunction is not antisymmetric and would not constitute a solution to the Schrdinger equation. The electron spin has yet to be included in the description of the electron. For simplicity, electrons can have spin up (+1/ 2) or spin down (-1/2) and are designated and respectively. For each molecular orbital, two elec trons of opposite spin can occupy one orbital. A determinant of the spin-orbit functions will form an antisymmetric wavefunction. For a closed shell molecule, the determinant is represented in Equation 3-9 where each row represents all possible assignments of an electron i to all orbital-spin combinations. )()()()()()()()( )2()()2()( )1()()1()(...)1()()1()( 12 2 1 1 21 21 1 2 1 2 11 11nrnr nrnr r r r r r r nnn nn n n n n (3-9) Basis sets. The linear combination of atomic orbitals that make up the molecular orbital can themselves be approximated with a finite set of one electron basis functions. N i ic1 (3-10) 74

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N is the number of finite basis functions and the ci are molecular orbital expansion coefficients. Electronic structure programs most often use Gaussian type basis functions, where the s type function is given in Equation 3-11. 24 32r se g (3-11) The atomic orbital exponent is different for each atom in the molecule and r is the spherical coordinate that defines the radial extension of the function. Linear combinations of primitive Gaussians (Equation 3-11) are used to form the entire basis function, and are called contracted Gaussians. p p pgd (3-12) The molecular orbital with contracted Gaussians is given in Equation 3-13. p ppi igdc (3-13) Ab initio Hartree-Fock (HF) is the most common qu antum mechanical method used to obtain properties of molecules. Although not an ab initio method, density functional theory (DFT) is included in this section due to its similarity to the Hartree-Fock method. Hartree-Fock. The Hamiltonian for the one electron wavefunctions just described is designated the Fock operator (F). The Fock operator is a sum of the kinetic energy of a single electron, the potential on the electron by the fixed nuclei and the average potential from the other N-1 electrons. To solve the equation, electronic structure al gorithms proceed as follow: Evaluate the integrals and account for the atomic positions Form an initial guess for the molecular expansion coefficients 75

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Form the Fock matrix Solve the Fock equation Repeat the procedure until the wavefunction of on e electron is consistent with that of the other electrons in an average field produced by the N-1 electrons The last step indicates that the wavefunction has converged. The above discussion has dealt with closed shell systems or restricted HF. For open shell molecules, the unrestricted method is used to treat unpaired electrons. The electron can have different spin states and the molecular orbital includes the extra factors, give n by Equations 3-14 and 3-15. i ic (3-14) i ic (3-15) The different spin states produce tw o sets of orbitals that permit proper dissociation to separate atoms and correct delocalized orbitals. One drawback is that the eigenfunctions are not pure spin states and suffer from spin contamination from higher spin states. Density functional theory. The wavefunction probability density 2 describes the probability of all electrons in a system. Density functional theory uses the probability density to model electron correlation by using functionals to describe the molecule. From the work of Kohn and Sham, the functionals currently used partition the electronic ener gy into several terms in Equation 3-16. XCJVT E E E E E (3-16) JVTEEE (3-17) T is the kinetic energy, V is th e nucleus-electron attraction and nuc leus-nucleus repulsion, J is the electron-electron repulsion and XC repres ents the exchange-correlation. The terms JVT E E E correspond to the ch arge distribution of and are similar to the potentials that were described for Hartree-Fock. The term XC E is the exchange-correlation term and includes a 76

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portion of the electron-electron interactions. The exchange-correlation te rm is usually divided into two parts, referred to as excha nge and correlation, respectively. )()()(C X XCEEE (3-18) All three terms are functionals of the electr on density, and many exchange and many correlation functionals have been parametrized to be used for DFT calculations. One of the more popular functionals to use for molecular systems and for carbohydrates and amino acids is the hybrid functional, B3LYP. It is called a hybrid functio nal since it includes the Hartree-Fock exchange term. XC DFT DFT X HFHF XCEcEcE (3-19) In general DFT calculations proceed similar to HF calculations, except with the added XC E term. Force fields In molecular mechanics methods the electron energy is represented explicitly through force field parameters. Molecular mechanics force fiel ds use the equations of classical mechanics to describe the potential energy surface. Simple an alytical functions are used to describe the molecule. For example, the compression and stre tching of a bond is described as a simple harmonic oscillator, and is analogous to a mass on a spring. 22 1orrkV (3-20) The potential energy is V, k is the force constant and ro and r are the equilibrium and displacement distances of the atoms in the bond, respectively. Both k and ro are known quantities for a pair of atoms a nd are called force field paramete rs. The known quantities have been obtained by fitting the force field to experimental data for specific types of atoms. Some recent modifications to force fields have included the use of ab initio methods to obtain the fitting parameters. The potential energy of the molecular system is the sum of all the individual 77

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components of the potential. Using the AM BER (Assisted Model Building and Energy Refinement) force field, the components of the potential describe bonds, angles, torsions, improper torsions, van derWaals interactions, electrostatics and hydrogen bonding (lone pair interactions are included for sulfur). lonepair Hbond estatic vdW dihedral angle bond AMBEREEEEEEEE (3-21) bonds r bondrrkE2 0 (3-22) esan anglekElg 2 0 (3-23) dihedrals o n dihedraln V Ecos1 2 (3-24) vdWij ij ij ij ij vdWR B R A E612 (3-25) estaticij ij ji estaticR qq E (3-26) 10 12ij ij ij ij bHbondR D R C E (3-27) Other force fields have similar energy com ponents and some may add an extra term in a component. All force fields have a different set of parameter values for all the constants. The force fields that have been modified to study carbohydrates are molecular mechanics (MM),77 AMBER,78 OPLS79,80 and CHARMM.81 MM and AMBER force fields have been used extensively to model large proteins, peptides and carbohydrates, since these molecular systems are too large to be studied with ab initio methods. The classical mechanic force fields cannot describe interactions that are pur ely quantum mechanical. Nevertheless, these force fields can be used to obtain an initial set of struct ural isomers that can be analyzed by ab initio methods. 78

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Geometry optimization The geometry optimization is a minimization of the energy in the Schrdinger equation used to describe the molecule. It is impl emented in robust comput er molecular modeling software, and at the core it has the following steps: Define an adequate basis set to desc ribe the wavefunction of the molecule Solve the eigenvalue equation by minimizing the eigenvalue (energy) that is described by the wavefunction through th e variational principle Use an algorithm that steps the atoms positi ons in the direction that lowers the total potential energy. At each step the eige nvalue is minimized and a new optimized wavefunction is obtained Vary the atoms position toward the minimum of the potential energy surface, until the energy difference from one step compared to that of the next step is zero, or more practically, a set threshold limit The algorithm implemented in software uses an y number of trajectory finding methods to numerically minimize the energy.82-84 This is distinct from the energy minimization to obtain the normalized wavefunction. The algorithm can be thought of as, although not strictly, following the potential energy surface of the molecule to find the minimum of the potential well. The algorithm is designed so that if the Emin(n+1) > Emin(n), where n is the optimization step, then the step will be discarded and the calculation will return to the previous atomic positions of Emin(n) and a new step will be taken until Emin(n+1) ~ Emin(n), and the energy is said to have converged to a stable point. The derivative, dE/dq ~ 0 is a stationary point on the potential energy surface. A stable point can be defined to be a minimum, a maximum or an nth order saddle point. The second derivative of the equation is obtained to indicate that the stationary point is a minimum on the potential energy surface. The s econd derivative also desc ribes the motion of the nuclei along the potential energy surface and is us ed to calculate molecular vibrations. 79

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Vibrational analysis The equilibrium positions of the atoms ar e determined by solving the electronic Schrdinger equation. The kinetic energy (T) of nuclei about their equilibrium positions is given in Equation 3-28. 2 3 12 1 N i it q T (3-28) eq iiiixxmq 2 1 (3-29) Here qi are the mass-weighted Cartesian displ acement coordinates (shown only for the x coordinates in Equation 3-29) and N is the number of nuclei in the molecule. The potential energy (U) can be expanded as a Taylor series about the equilibrium positions, and to a first approximation only the quadratic terms are used Although the energies can be evaluated quantum mechanically, the vibrational motions can be solved using Newtons second law (the first derivative term vanishes at the equilibrium position). ... 2 13 1 2 3 1 3 1 ji eq N j ji N i i N i eq i eqqq qq U q q U UU (3-30) ji eq N j ji N i eqqq qq U UU 3 1 2 3 12 1 (3-31) By approximating to the second order, one can obtain harmonic approximations of the vibrations, and until recently 2nd order approximations were the major method implemented in quantum chemistry codes to solve for the normal modes of molecular systems. The eigenvalue equation uses the wavefunction and potential energy surface obtained from the geometry optimization, starting at the converged point a nd perturbs the system by moving the atoms along 80

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the potential energy surface. The normal modes will be used to obtain calculated spectra to compare to experimental IRMPD data. Molecular Dynamics Carbohydrates are complex molecules that can have more than one local minimum. For example, a hexose monosaccharide can exist in a lin ear form, but can also be in a five or sixmembered ring structure. A monosaccharide like glucose can have up to five chiral centers, whose functional groups have rotatable bonds. The six-membered ring structure can have different configurations, with chair, boat, envelope, twist, and skewed the most stable configurations, but others are possible. A study of L-ideronate had to include the 4C1, 1C4 and 2S0 ring structures to obtain the aver age structure from the experiment.85 Although not as complicated to model, amino acids are also linear chain polyatomics and have multiple conformations. There is a set of robust methods to deal with global minimum optimization or the multiple minima problem, most of which i nvolve molecular dynamics theory implementation in chemistry computer algorithms and codes. The most common are simulated annealing,86 replica exchange molecular dynamics(REMD)87 and dihedral angle searching.88 Although not reported in this work, simulated annealing will be summarized to contrast to the dihedral angle search method. Simulated annealing Simulated annealing requires that a molecule start at a local minimum so that its energy can be increased to extract the molecule from its potential well, in a st ate well above the local minimum. Exploring the conformation space to find the global minimum is done by taking the geometry optimized starting structure and incr easing the system temperature above 300 K and even higher than 1500 K, depending on the molecule size and flexibility. Heating occurs for only a few ps (in the time scale of the calculation and not real time). By virtual heating of the 81

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glycoside, local minima barriers will be overc ome, and thus the molecule will sample many states on the potential energy surface. Controll ed cooling allows the molecule to find lower energy configurations than the initial configuration, and give n sufficient simulation time the global minimum can potentially be reached.89 To find the global minimum, the system is slowly cooled to 0 K for the rest of the simulated ann ealing calculation. Adequate cooling time is 100 1000 ps depending on molecular size. The time step of the simulation is crucial and should be on the order of a few femtoseconds since the inte gration step needs to be smaller than the vibrational frequencies or the integration will be inadequate. The major drawback to using this method is the short time step in the simulati on. For a 30 atom monosaccharide, using an empirical force field, a simulated annealing run for 1 ns of simulated time will take approximately 15 days on a Pentium IV 1.3 GHz processor with 500 MB of RAM. While codes exist that will allow for multiple cpu processing, this usually requires access to a supercomputer or a network cluster of computers. If using ab initio molecular dynamics, large amount of computing time will be required even while using high performance multiple processor systems. Similar to simulated annealing, REMD requires a small time step for proper integration. REMD also requires a large amount of computational power since multiple simulations need to be processed simultaneously, and it is mainly implemented in algorithms for use on high performance multiple processor systems. Since this is the first step in obtaining the theoretical vibrational frequencies of the sugars and amino acids in this work, the bulk of the time should not be spent finding different conformations. Therefore the computational method needs to proceed quickly in obtaining different conformations, so that th ey can be further analyzed with ab initio methods to describe 82

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the amino acids and carbohydrates adequately. A method that will search the conformational space adequately while keeping computa tional time manageable is needed. Conformational searching Torsion angle searching is a global minimum search method that provides multiple conformations so that they can be compared with each other to find the lowest energy structure. A strict conformational analysis (CA) searches all the dihedral angles of the molecule, incrementing each by a few degrees at a time and at each point a geometry optimization is performed. For an ion with a few torsion a ngles, because of high symmetry or because it contains only a few atoms, the conformational anal ysis (CA) will be relatively simple. But for molecules with many degrees of freedom (e .g., glycosides), a quantum mechanical conformational analysis of all torsion angles ca n be extremely computationally expensive, even at low levels of ab initio theory. Random conformational searching is a method wh ere the dihedral angl es can be altered by a software algorithm that emphasizes finding low energy structures. For faster results while using a single processor, the empirical or cl assical mechanics force fields are employed. Ab initio or density functional geometry optimization is done only in the last step of the overall process, to overcome the limitations of the em pirical and semi-empiric al force fields. The algorithm as implemented in the Hyperchem suite of programs is based on the usage directed approach90 and has the following general scheme: Definition of dihedral angles by the user Randomization of the dihedral angl es to create a new structure Geometry optimization for the new structure Comparison of the energy converged structure to the set of other structures that have been made by the randomization step, to check for duplicate structures and high energy species 83

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The algorithm relies on comparing the structures crea ted in the search to verify that all of the structures are distinct from one another. The spatial orientation of torsions angles and bonds are compared to a specified precision to check for th e duplicates. The total energy obtained from the geometry optimization is compared to that of othe r structures and if they are within a specified limit, the structure may be considered the same if the other criteria are also met. The usagedirected method proceeds depending on the following scenarios: If the structure is found to be the same as one of the others in the search, then it is tabulated as such and the search continue s with this structure being used for the randomization of the dihedral angles If the structure is distinct from any of the ot hers, and is within the total energy limit, then it is tabulated as a new structure and the sear ch continues with this structure being used for the randomization of the dihedral angles If the structure is distinct but its total en ergy is higher than a user specified limit, the structure is discarded and the search conti nues by applying randomiza tion of the dihedral angles to the previous structur e that was accepted by the search The number of created structures can be modifi ed, to keep the computation time reasonable (default value is 1000 struct ures). A table is created with al l the distinct conformers arranged from lowest to highest energy with the final valu es for the dihedral angl es of each structure are listed. Individual conformers are extracted for further analysis with hi gher levels of theory. 84

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CHAPTER 4 STRUCTURE DETERMINATION OF PHENYLALANINE ANALOGS Introduction Hydrogen deuterium exchange (H/D) is th e method in which particularly labile hydrogens atoms are substituted with deuterium at oms in the molecule or ion of interest.91 H/D exchange has been used in solution phase stud ies, mainly NMR work, to obtain structural information.92 H/D exchange studies have also been conducted in the gas phase and early work from Beauchamp, et al. probed the proton affinity and excha nge rates of labile hydrogen atoms.93 Ion-molecule reactions involving H/D exchange have also been followed in mass spectrometry experiments, for example to investigate the dynamics of unsaturated platinum complexes.94 Utilizing the technique of infrared multiple photon dissociation (IRMPD),58 infrared spectra of gas phase deuterated ions can be obtained, and th ese have recently been used to obtain structural information for small ions59,69,95-98 and biologically relevant ions66,99-103 by observing the spectral shifts that occur in the infrared spectra due to deuteration. H/D exchange experiments can provide a great d eal of structural information, especially when coupled with mass spectrometric and/or infrared spectral techniques. Substitution of hydrogen by deuterium has primarily been used to obtain informati on on the tertiary structure of proteins,104 but can also provide primar y structural information for smaller species such as the phenylalanine analogs studied in this work. In the case of amino acids, exchange is seen for hydrogen atoms bound to the Lewis base oxygen and nitrogen atoms.105 Phenylalanine (Phe), one of the essential am ino acids, has been studied extensively both in solution and in the gas phase.14,101,106-111 Recent work has shown that Phe behaves differently in each phase, in part due to structural differences of the molecule in the two phases.15 The binding of alkali metals to Phe in the gas pha se has been especially well characterized.14,106,110 85

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However, much less is known of the effect that protecting groups or modifications to the basic amino acid composition have on the phenylalanine structure. Recent work by Dunbar and coworkers101 showed that by extending the amino acid chai n, by forming a dipeptide, an attached alkali cation has a higher possibili ty of forming a chelating complex. The sodium cation tends to interact with electronegative sites on the extended chain, subject to steric hindrance constraints. For sodiated PheAla and AlaPhe, the most energe tically favorable structures involved a sodium cation interaction with the phenyl ring. The phe nylalanine moiety tends to pucker to allow the alkali metal to interact with both the phenyl ring and the lone pair electrons on the oxygen or nitrogen atoms. To further investigate the eff ect that modification of phenylalanine has on the chelating complexes, in the work reported here phenylalanine analogs bound to sodium cations were studied in the gas phase after either solutionor gasphase hydrogen/deuterium exchange. Mass spectrometric techniques us e low quantities of material and are ideally suited for the low abundance yield of produc ts obtained from organic synthe sis of the amino acid analogs. Fourier transform ion cyclotron re sonance mass spectrometry (FTICR-MS)31-35,112-114 has the advantage of trapping ions for extended periods of time so that laser irradiation of the sample can be performed without deactivation of excited ions by collisions with background gas. In this work sodium cations were attached to the pheny lalanine analogs in solution, and the resulting charged complexes were desolvated during the electrospray ionization process39,42,43 to produce isolated ions. Infrared multiple photon dissoci ation spectra of the Phe analogs cationized by sodium then provided structural information, when combined w ith theoretical calculations. Density functional theory (DFT)76,115,116 calculations were used to predict the lowest energy structures of each analog. The hybrid functional B3LYP, which has been shown to be adequate for predicting infrared spectra of Phe, 72,101 was used for all calculations. 86

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Neutral Phe has three potential hydrogen atoms which can undergo H/D exchange (Figure 4-1). Limiting the deuter ation sites to one or two locati ons, by the addition of protecting groups, allows observed spectral shifts to be cl early defined and simplif ies band assignment and subsequent structure elucidation. The analogs of Phe studied in this wo rk had protecting groups on all but one or two of the possible excha nge sites. The analogs, N-acetylphenylalanine (AcPhe), O-methyl N-acetylphenylalanine (AcPheOMe) and N-acetylphenylalanine Omethylglycine (AcPheGlyOMe) (Figure 4-2). Ac Phe has two possible exchangeable hydrogens (in the O-H and N-H groups), AcPheOMe has one, and AcPheGlyOMe has two. Experimental Sample Preparation Phe analogs were synthesized from a commercial sample of L-phenylalanine (SigmaAldrich Co.) by Mr. Alfred Chung at the Proteomics Division of the Interdisciplinary Center for Biotechnology Research at the Univ ersity of Florida. Stock solutions were made by dissolving 0.01 mg/mL of the analogs in an 80:20 CH3OD:D2O solution. For solution phase H/D exchange, a solution of 80:20 CH3OD:D2O was used to dilute the stock sample to 1 mmol, and an equimolar amount of NaCl was added. Samples were allowed to undergo exchange for a minimum of 20 minutes followed by electr ospray introduction into the FTICR-MS. For gas phase exchange, ND3 was introduced into the ICR vacuum chamber at a background pressure of 5E-7 torr, and allowed to react with the cationized Phe analogs for 1-5 seconds before laser irradiation occurred. For species where no gas phase exchange was detected, reaction time was increased up to 35 sec onds to verify that no reaction had taken place. Instrumentation To deduce sites of sodium interaction and to assist in ionizing the peptide molecules, the phenylalanine analogs were electrosprayed wi th sodium, producing the amino acid sodium 87

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cation complexes. Electrospray ioniza tion was conducted with a Z-spray source (Micromass/Waters Corporation, Milford, MA) at a solution flow rate of 20 L/min, with the flow rates of the nebulizing gas and the desolvation gas (N2) set to 32 and 150 l/hr, respectively, and the electrospray needle-skimmer voltage di fference set at 3 kV. A 4.7T superconducting magnet (Cryomagnetics Inc., Oak Ridge, TN) was used with a laborator y-constructed pumping system, ion trap and electronics console. The FTICR-MS system has been described previously66. Precursor ions were isolated using the stored waveform inverse Fourier transform (SWIFT)73,117-120 technique to eject all unwanted ions. Ions were detected using the broadband detection mode covering a mass range from 20 to 2000 Da. For the IRMPD activation, the F ree E lectron L aser for I nfrared eX periments (FELIX)65,121,122 was used with the wavelength scanned over the range of 5.5 12.5 m (800 1800 cm-1). Mass-selected precursor ions were irradiated in the open cylindrical FTICR ion trap for a peri od of 4 seconds at each wavelength with an average power of 50 60 mJ per macropulse and a laser repetition rate of 5 macropulses per second. Typically, four individual transients were accumulated at each wavelength to improve the signal-to-noise ratio. The depletion signal of the prec ursor ion was monitored throughout the experiment. Computational Details The Hyperchem suite of programs123 was used to assign trial structures to the phenylalanine analogs. Depending on the analog, me thyl or acetyl groups were added to a core Phe structure to produce trial AcPhe, AcPheOMe and AcPheGlyOMe structures. Sodium was initially placed near the carboxyl or carbonyl group of the Ph e analogs, and the complex was given an overall charge of +1. It should be noted that although the s odium was placed near the carboxyl or carbonyl group, no bond was defined between sodium and any other atom, and 88

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therefore sodium was free to move during the geometry optimization steps of the calculation. Explicit definition of a bond was considered unnecessary since the qua ntum mechanical AM1 force field was used initially to model the intera ctions of the analog complexes. The dihedral angles which were varied in the conformational search were explicitly defined (Figure 4-3). Using the usage directed appro ach as described in chapter 3,90 1000 structures were created within the Hyperchem software to ade quately probe the conformational space of each analog. During the conformational search, th e algorithm eliminated duplicate structures by comparing energies, torsion angles, and RMS fit residual errors between corresponding atoms. The comparison threshold values were set to 0.05 kcal/mol, 10 and 0.25 respectively. All conformers within 15 kcal/mol of the lowest en ergy conformer were further refined by geometry optimization using Gaussian03124 at the B3LYP/6-31G(d) level of theory.125,126 The conformations were compared and duplicate conformations having the same energy (within 0.0001 hartree), and the same vibrational spectrum as the comparison conformer, were discarded. Using the B3LYP/6-311++G(d,p) level of theory,127,128 an average of 45 conformers were further geometry optimized, and a vibrational frequency calculation, at the same level of theory, was performed for each of these conformations. Th e calculated frequencies were scaled by 0.965, followed by comparison to experimental spectra. Results and Discussion N-acetylphenylalanine Calculated infrared spectra for the four lowe st energy conformers (Figure 4-4) of sodiumcationized AcPhe are compared to the experimental IRMPD spectrum obtained for this complex in Figure 4-5. Conformer A has a cationinteraction (Figure 4-4) while conformer B does not, although the conformers have comparable energies (0.04 kcal/mol). The calculated spectrum of conformer B gives a poorer match to the experiment al data relative to th at of conformer A. 89

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When compared to the experimental C=O stretching bands (1660 and 1720 cm-1), the calculated C=O stretches for conformer B are red shifted. The interaction between the sodium cation and the carbonyl groups is stronger for this conformer since the cationinteraction does not occur in conformer B. The C=O stretch frequencies of conformers C and D also do not fit the experimental data as well as those for conformer A. For both conformers C and D, one calculated C=O stretch band is red shifted and one is blue shifted when compared to the experimental spectrum. The calculated struct ures for conformers C and D have only one carbonyl interacting with the sodium cation an d the carboxyl carbonyl group does not interact with either the sodium cation or the phenyl group. The free carboxyl carbonyl stretch shows a large blue shift, while the carbonyl interacting with the sodium cation is red shifted due to a stronger interaction between the oxyg en and the nitrogen atoms. Table 4-1 gives the calculated distances between the sodium cation and th e oxygens of the n-acetyl and carboxyl groups, indicative of the strength of electrostatic interaction. With the scaling factor correction, the rms differences between the e xperimental data and the calculated conformer C=O stretch frequencies are 5 cm-1 for A, 20 cm-1 for B, 29 cm-1 for C and 40 cm-1 for D, with frequency values for the e xperimental spectra taken from the peak maxima of the experimental bands. All othe r calculated bands for conformers C and D do not agree well with experimental data (except that conformer D shows reasonable agreement with the bands at approximately 1150 and 1525 cm-1). The observed IRMPD spectral bands can be assigned using the calculated results for confor mer A (Figure 4-6) although small contributions to the IRMPD spectrum from the other conformers is possible, due to the similar relative energies of the structures. Bands I, II and V w ould be expected to shift following substitution of 90

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a deuterium atom for a hydrogen atom and are used extensivel y in the discussion of IRMPD spectra of H/D-exchanged AcPhe analogs below. Hydrogen/Deuterium Exchange Experiments AcPhe For the solution phase H/D exchange of AcPhe, the mass spectrum indicated that, as would be expected, two exchanges had taken pl ace, one involving th e amine hydrogen and one the carboxylate hydrogen. The IRMPD spectrum of sodium cation-attached AcPhe following solution phase HDX (Figure 4-7). In experiments involving gasphase H/D exchange of sodium cation-attached AcPhe, the resultant mass spectra showed only a 1 Da mass increase, indicating that only one D for H substitution had occurred. An IRMPD spectru m of the singly deuterated complex ion was obtained and the calculated spectra for sodium cation-attached AcPhe with one D substituted for either the amide or carboxylate hydrogen were co mpared (Figure 4-8). Agreement with the experimental spectrum is much better for the spectrum calculated for D exchange with the carboxylate hydrogen. This conclu sion is further supported by the presentation in Figure 4-9, where the spectra for undeuterated and singly-de uterated sodium cation-attached AcPhe are overlaid, with C=O stretching a nd O-H, O-D, N-H, and N-D be nding mode regions indicated. AcPheOMe For sodium cation-attached AcPheOMe, no gas-phase H/D exchange was observed. AcPheOMe does not have a carboxylate hydrogen ( it has been replaced by the methyl group), and exchange of the amide hydrogen is apparently quite slow in the gas phase (as was seen for sodium cation-attached AcPhe). One H/D excha nge took place in solution-phase experiments, presumably at the amide hydrogen, as confirmed by the agreement between the IRMPD spectrum of mono-deuterated sodium cation-at tached AcPheOMe and the calculated spectrum 91

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for the amide-deuterated species (Figure 4-10A). The lowest energy sodium cation-attached AcPheOMe structure used for DFT calculations of the mono-deuterated spectrum (Figure 410C). AcPheGlyOMe Two exchanges of D for H were observed for AcPheGlyOMe in solution, and none in gas-phase experiments. This protected dipeptide has two amide hydrogens, and DFT calculations predict the most stab le structure of the sodium cation-attached complex (Figure 410D). Agreement between the IRMPD spectru m obtained for the sodium cation-attached doubly-deuterated ion from solution-phase HDX experiments and the calculated spectrum (Figure 4-10B) for the structure in Fig. 410D with both amide hydrogens replaced with deuterium is quite good. The most stable structures calculated fo r both sodium cation-attached AcPheOMe and AcPheGlyOMe involve interaction of the sodium cation with the phenyl ring of the phenylalanine side chain and lone pairs on a ll carbonyl oxygens. Figures 4-10C and 4-10D predict that these complexes will have a puckered conformation. The structures in Figure 4-10 correspond to the undeuterated species of AcPheOMe and AcPheGlyOMe, and the corresponding spectra are for the deuterated ions. The optimized structures for the undeuterated ions were used to calculate the sp ectral shift of the deuterated ions. Results for H/D exchange experiments involving the Phe anal ogs are summarized in Table 4-2. The results found in this study and others of phenylalanine indicate th at the species behaves quite differently when interacting with a chelatin g metal, which seems to fix the structure into one conformation. Gas phase H/D exchange has been observed for the amide hydrogen on other amino acids.105,129 Direct substitution of the amide hydr ogen with deuterium is slow, and earlier studies indicate that if a hydr oxyl group is available, the exch ange will occur at the hydroxyl 92

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hydrogen followed by deuterium migration to the amide position. Formation of a chelated sodium cation complex apparently impedes migr ation of deuterium to the amide hydrogen, as seen for sodium cation-attached AcPhe, where exchange occurred at the carboxylate hydrogen but not at the amide hydrogen. In preliminar y gas-phase H/D exchange experiments involving protonated (as opposed to sodium cation-attached ) AcPhe, three hydrogen atoms were exchanged with deuterium. Deut erium migration from the carboxylate group to the amide nitrogen was not impeded for this species. Conclusions Shifts of vibrational spectr al bands upon H/D exchange with sodium cation-attached phenylalanine analogs have been predicted theore tically and compared to those observed in gasphase infrared multiple photon dissociation spectr a. The H/D exchange experiments not only provided band assignment confirmation, but simultaneou sly helped identify st ructural features of the Phe analogs being studied. Structural characte ristics common to all phenylalanine analogs in this study include the following. Chelating complexes form when the sodium cation binds to Phe analogs The lowest energy structures for all three analogs include cation interactions Torsion of carbonyls toward the phenyl ring, in order to interact with the sodium cation, causes the conformation to stay relati vely rigid and in a puckered state Complexation with the sodium cation hinders D for H exchange at the amide group hydrogen(s). These results differ from those found for other am ino acids in the gas phase, where gas phase exchange can be observed at the amine groups. 93

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Table 4-1. Interaction distance between sodium cation and oxygen atoms on the carbonyls of the carboxyl and on the N-acetyl functional groups for calculated conformers A D. AcPhe ConformerCarboxyl Carbonyl ON-acetyl Carbonyl O A 2.22 2.35 B 2.26 2.17 C n/a 2.14 D n/a 2.14 Calculated Distances between Sodium and Oxygen Atoms Distance is reported in Angstroms. Table 4-2. Summary of result s from H/D exchange experiments, where no D for H substitution was observed for sodium cation-attached AcPheOMe and AcPheGlyOMe in the gas phase N-acetylphenylalanine species Deuteration method Deuteration sites Gas phase 1 OH Sodiated (AcPhe) Solution 2 OH, NH Gas phase None Sodiated (AcPheOMe) Solution 1 NH Gas phase None Sodiated (AcPheGlyOMe) Solution 2 NH Figure 4-1. Phenylalanine with excha ngeable hydrogens highlighted in red 94

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Figure 4-2. Phe analogs studied in this work. The portion highlighted in blue represents the Nacetyl adduct, highlighted in red is the methyl group, and the portion highlighted in purple indicates the O-methylated glycine. Possible exchangeable hydrogen atoms are indicated by asterisks. Figure 4-3. Dihedral angles defined for the conformationa l search calculations of AcPhe, AcPheOMe and AcPheGlyOMe (shown in red arrows). The labellling scheme for the dihedral angles is similar to that of References 133 and 134.130,131 95

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A. (0) B. (0.04 kcal/mol) D. (2.7 kcal/mol) C. (0.5 kcal/mol) Figure 4-4. Lowest energy conformers of sodi um cationized AcPhe found from conformational search calculations. Relative energies (giv en in parentheses) were obtained at the B3LYP/6-31++G(d,p) level of theory and are zero-point corrected. An alternate view is shown for conformer A to show the puckering of the structure. 96

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Calculate d Ex p eriment Na+ [AcPhe] Conformer A 90011001300150017001900 Wavenumber (cm-1) conformer A experiment Na+ [AcPhe] Conformer B 90011001300150017001900 Wavenumber (cm-1) conformer B experiment Wavenumber/c m -1 Wavenumber/c m -1 Conformer A Conformer B Na+ [AcPhe] Conformer C 90011001300150017001900 Wavenumber (cm-1) conformer C experiment Wavenumber/c m -1 Conformer C Na+ [AcPhe] Conformer D 900110013001500170019 Wavenumber (cm-1) 00 conformer D experiment Wavenumber/c m -1 Conformer D Figure 4-5. Comparison of expe rimental IRMPD spectrum (red line) of AcPhe with calculated spectra of the theoretically determined lowest energy conformers A-D (blue stick spectra). Calculated vibrational freque ncies are scaled by a factor of 0.965. 97

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Figure 4-6. Assignment of spectral bands in the IRMPD spectrum of AcPhe using calculated bands for conformer A. The calculated spectrum has been convoluted with a 20 cm-1 Gaussian profile. 90011001300150017001900Wavenumber (cm-1) conformer A experiment Figure 4-7. IRMPD spectrum of sodium cati on-attached AcPhe follo wing solution phase HDX (in red) and calculated spectrum of the doubl y deuterated conformer A (in blue). Deuterium substitution occurred at both the N-H and COOH hydrogens. 90011001300150017001900Vibration assignments I OH bend II NH bend, CH2 wag III CO bend IV CH2 bends, CH phenyl ring wags V NH bend VI C=O stretch VII C=O stretch I II III IV V VI VIIWavenumber (cm-1) Wavenumber/c m -1 conformer A experiment Wavenumber/cm1 98

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A Experiment Calculated ND B Figure 4-8. Comparison of the singly deuter ated sodium cation-attached AcPhe IRMPD spectrum with that of calculated conformer A if exchange had occurred at A) the N-H hydrogen or at B) the O-H hydrogen. Figure 4-9. Experimental spectr a for undeuterated and singly-deut erated sodium cation-attached AcPhe, indicating that deuterium was ex changed with the carboxylate hydrogen. Experiment Calculated OD Wavenumber/cm-1 Undeuterated Ex p erimen t Deuterated Ex p erimen t Wavenumber/cm-1 99

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9001100130015001700 Wavenumber (cm-1) calculated experiment 1000110012001300140015001600 yg y A BWavenumber/cm-1 Figure 4-10. Experimental IR MPD spectra and theoretically calculated spectra for monodeuterated sodium cation-attached AcPheOMe (A) and doubly-deuterated sodium cation-attached AcPheGlyOMe (B). The lowest energy structures found for sodium cation-attached AcPheOMe and AcPheGlyOMe are shown as (C) and (D), respectively. --calculated --experiment Wa-1venumber/cm D C 100

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CHAPTER 5 DIFFERENTIATION OF ANOMERS OF DGLUCOSAMINE AND D-GALACTOSAMINE Introduction Gas-phase experiments continue to gain pr ominence in chemistry and biology since mass spectrometric techniques have evolved to allow routine study of prot eins and other large molecules.132,133 Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) experiments,30,31,33,113,114 when combined with sophisticat ed ionization techniques such as electrospray ionization (ESI),38-41 permit the study of sugar complexes without requiring sample heating for volatilization, which can often fragme nt or otherwise destabilize precursor ions of interest. As stated in chapter 2, sugars can be studied at low pressures (below 10-9 torr) in FTICR-MS, providing a collisionl ess environment where fragment ation processes due to laser irradiation can be studied. Collision induced di ssociation and single-frequency infrared multiple photon dissociation (IRMPD) methods have been used to study carbohydrate isomers in tandem MS experiments (MS/MS).51,52,134 These two methods frequently produce similar fragmentation patterns for isomeric ions, making differentia tion difficult. Tandem mass spectrometry at varying laser irradiation wavelengt hs has been shown to provide an extra degree of analysis that can help in differentiation of sugars, and recen t studies using FTICR-MS in conjunction with IRMPD have shed new light on the structures of carbohydrates.17,66,135 Carbohydrates, even simple monosaccharides, are flexible and can assume many configurations, sometimes favoring one partic ular conformation over another depending on environmental influences such as interaction with different solvents, metals, and intramolecular hydrogen bonding. Glycan conformation can strongly influence th e functions of proteins to which they are bound, as glycans are important in cell-cell signaling and have been found to aid in protein folding thus stabilizing the protein structure. 101

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To obtain a complete picture of the sugars role in protein-based systems, the interactions between alkali metals and sugars must be understood. Sodium and potassium play a crucial role in regulating cell functions and their blood concentration levels are indicators of well-functioning biological systems. Lithium ions are used as strong prescription drugs to regulate mood. Practically, alkali metal-attached sugars are easily ionizable with the electrospray process and yield high abundances of the precursor ions whic h allow MS/MS experiments to be carried out reliably. If the alkali metal-suga r interaction is strong, fragmentat ion pathways other than simple alkali ion loss may become more prevalent when ions are submitted to irradiation with an infrared laser. Intramolecular fragmentati on is usually observed for lithium-bound saccharides.52 136 Identification and differen tiation of conformational isomer s, specifically methylated and D-glucosamines (-GlcNac and -GlcNac) and methylated and D-galactosamines (GalNac and -GalNac) bound to the lithiu m cation (Figure 5-1) has b een made possible in this work through observing their infrar ed spectra and fragmentation pattern differences as a function of irradiation wavelength. Plotting the fragmentation of the glycosamines as a f unction of laser irradiation wavelength yields IRMPD action sp ectra. These gas phase infrared spectra can be compared with theoretical calculations of the vibrational frequencies to obtain band assignments and structural information for each glycosamine isomer. Molecular mechanics conformational searches using the AMBER force field137 were conducted and dens ity functional theory (DFT)115,116,138 geometry optimizations and frequency calcu lations were used to identify spectral features of each glycosamine isomer. The results obtained are expected to provide the basis for future experimental work involving diand trisaccharides. 102

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Experimental Sample Preparation Glycosamines were obtained from Prof. Brad Bendiak, Department of Cellular and Structural Biology, University of Colorado Health Sciences Center. Stoc k solutions were made by dissolving 0.01 mg/mL of each glycosamine in 80:20 CH3OH:H2O solution. Stock samples were diluted by a factor of 10 and were mixe d with an equimolar amount of LiCl before introduction in the electrospray apparatus. Instrumentation All ESI-FTICR-MS experiments were carried out using a laboratory built FTICR spectrometer equipped with a 4.7 T superconduc ting magnet (Cryomagnetics Inc., Oak Ridge, TN), which has been described previously.66 An external Z-spray source (Micromass/Waters Corporation, Milford, MA) was used for sample injection at a flow rate of 10 L/ min. Electrospray ionization efficiency was incr eased through the use of both nebulizer and desolvation gas (N2 ), with a continuous flow of 35 and 155 l/hr, respectively. Source temperature was set to 52 C and the desolvation gas temperature was 125 C. The electrospray needle-skimmer voltage difference was set to 3 kV. Precursor ions were isolated using stored waveform inverse Fourier transform (SWIFT) waveforms 118-120 to eject all other ions. Ions were detected using a broadband dete ction mode covering a mass range from 20 to 2000 Da. For the IRMPD activation, a free electron laser was used to scan the infr ared fingerprint region of 900 1800 cm-1 of each glucosamine anomer. Four indivi dual transients were accumulated at each wavelength to improve the signal-to-noise ratio. Three IRMPD spectra were averaged for each anomer and the resultant spectrum used to determ ine the peak position of the C=O stretch band. The appearance of fragment ions with m/z 210, 132, 127, and 122 and were monitored as a function of irradiation wavelength. 103

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Computational Details Using the structure database provi ded in the Hyperchem software,123 each anomer of DGlcNac and D-GalNac was created with a cloc kwise hydrogen bonding network, with has been shown in literature to be the most stable solution phase structure for D-glucopyranosides.139-141 Lithium was initially centered above the ring system of the glycan approximately 2.5 from the ring oxygen (O1) (Figure 5-2). Each glycosamine was given an overall charge of +1. A conformational search of torsion angles wa s done to probe the rearrangement of each glycosamine isomer as it interacted with the lith ium cation. All calculat ions were performed in an isolated environment. All H-C-O-H torsion angles were defined so that only the O-H bonds rotated about the CO bonds, thus keeping the struct ural definitions for each glycosamine intact. The ring torsion angles were defined and were varied in the conformational search, while ensuring that the chiral center orientation did not change. Usi ng the usage-directed approach,90 1000 different structures were created with the algorithm. For each step, the dihedral angles were randomly changed to create a new struct ure, and a geometry optimization using the AMBER force field was performed to verify existe nce of a stable structure. This optimized structure was then compared to the other stab le structures previously obtained from the conformational search, and duplicate structures were discarded. On average, the 100 most stable structures within 15 kcal/mol of the lowest ener gy structure found were then geometry optimized using Gaussian 03124 with the B3LYP/6-31+G(d) level of theory.126 Similar to the procedure described in Chapter 3, duplicate structures were discarded and a frequency calculation, at this same level of theory, was carried out for each stru cture to verify minima and to obtain theoretical infrared spectra for comparison with experimentally obtained spectra. This level of theory has been used previously to study sugars in the gas phase A scaling factor of 0.98 was used for the calculated frequencies, and is within the reported average scaling factor using this basis set.142,143 104

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For further verification that the lowest energy conformer had been found for each glycosamine isomer, a rigid potential surface scan of the H-(C-2)-N-H di hedral angle was done (Figure 5-3) using Gaussian 03 with B3LYP/6-31+G(d). Sing le point calculations were performed as the amide group was rotated in 10 degree increments about the (C-2)-N bond, while keeping the rest of the molecule fixed. Results and Discussion Infrared Spectra Glucosamines The IRMPD spectra of both lithium cationized and -GlcNac indicate that the spectral bands in the range from 800 to 1500 cm-1 are broad and there is no clear distinction between the two anomers (Figure 5-4). For the band near 1652 cm-1, however, a shift in position in this band between the two anomers can be clearly seen. Us ing the average of 3 i ndependently determined IRMPD spectra an average shift of 10 cm-1 was calculated. Calculations indicate that the band near 1652 cm-1 is the C=O stretch of the carbonyl on the acetamido (-NHCO(CH3)) group. There is a corresponding shift of 20 cm-1 between the theoretically calculated frequencies of the two anomers of D-GlcNac, but the experimental and theoretical shifts agree gi ven the rms error of 34 cm-1 reported for B3LYP/ 6-31G(d) vibrational frequencies.144 The lowest energy structures for lithium cationized -GlcNac and -GlcNac were calculated and both anomers have the chair 4C1 conformation, where carbon 4 is above the plane of the sugar ring and carbon 1 is below th e plane of the ring (Figure 5-5). Hydrogen bonding is evident. For -GlcNac, two hydrogen bonds are form ed between the hydroxyl groups from C-3 through C-6, while for -GlcNac only one hydrogen bond form s between C-3 and C-4. 105

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Analysis of hydrogen bonding and the most probabl e interaction locations for the lithium for each anomer follows. -GlcNac. The calculated spectrum of the lo west energy structure found for lithium cationized -GlcNac is similar to the experimental spectrum, having the same broad features from 800 1500 cm-1 (Figure 5-6). The calculated C=O st retch frequency after scaling is 1648 cm-1. With the O-methyl group at C-1 in the axial position, the amide hydrogen in-GlcNac is located nearest to the oxygen of the O-methyl. To minimize steric hindrance, the carbonyl is oriented away from the O-methyl group. A rigi d potential surface scan of the dihedral angle rotation about the (C-2)-N bond led to one stable minimum, with three other possible stationary points along the potential energy surface (Figure 5-7). The point of highest energy corresponds to the structure in which the carbonyl oxygen is in close contact with the o-methyl group. Structures with the cation at other locations of the sugar were higher in energy by more than 10 kcal/mol. -GlcNac. For -GlcNac, the theoretical calculations show that the O-me thyl group is in an equatorial position, that the acetamido group has reoriented so that the amide hydrogen is located nearest to the oxygen of the O-methyl gr oup, and steric hindrance is minimized between the carbonyl and O-methyl groups. Results of th e potential surface scan (Figure 5-8) indicate that a torsion angle of 6 fo r the H-(C-2)-N-H dihedral angl e is found for the lowest energy structure in the complete range of angles from 0 to 360, corresponding to the lowest energy structure from the conformational search. Another minimum is found in the dihedral scan (4 kcal/mol higher in energy), with large potential energy barriers between the two minima (54 and 242 kcal/mol, respectively). The highest energy structure occurs for a dihedral angle which places the N-acetyl methyl group closest to th e hydrogen of the C-3 hydroxyl group. In the 106

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lowest energy structure, the lithium cation inte racts with the O1 oxygen as well as the oxygens of the O-methyl and C-6 hydroxyl groups. The Mulliken calculated charge145 on the lithium cation (0.62) is close to the calculated charge of lithium on -GlcNac (0.66), although the interaction in this case is with three oxygens instead of two oxygens. The sma ll difference in lithium charge between the two anomers can arise from the stro ng interaction of the lithium cation with the carbonyl oxygen lone pa ir electrons of -GlcNac, reducing the total charge by a similar magnitude to that of the three -GlcNac oxygens intera cting with the lithium cation. Other structures where the lithium cation is located at different positions relative to -GlcNac were calculated to be higher in energy by more than 9 kcal/mol a nd the calculated and experimental spectra were compared (Figure 5-9). Galactosamines Based on analysis of the glucosamines discussed above, where the major distinguishing feature in the IRMPD spectra was the carbonyl stretching band, spectra for the anomers of galactosamine were only obtained in the range 1400 1700 cm-1 (Figure 5-10). An 11 cm-1 shift in band position was seen between the carbonyl stretching peaks of lithium cationized -GalNac and -GalNac. The calculated spect ra indicate a shift of 29 cm-1 for the C=O stretch frequencies (Figure 5-11). The lowest energy structures from the DFT calculations indicate a 4C1 ring conformation (Figure 4-12). B3LYP/6-31+G(d) calculations of lithium cation-attached -GalNac indicate that hydrogen bonding occurs between the C-3 and C-4 hydroxyl groups, and with the carbonyl oxygen and the hydroxyl group at C-3. The other potential hydrogen bonds are interrupted due to the lithium cation located between the C-4 and C-5 functional groups. There is also a hydrogen bond between the amide and the O-methyl oxygen. -GalNac has one hydrogen bond 107

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between the C-3 and C-4 O-H groups. The position of the acetamido group relative to the Omethyl group is similar to that found for the gl ucosamines. Interaction of the amide hydrogen with the O-methyl oxygen and the lessening of steric hindrance cause the carbonyl to change position relative to the O-methyl gr oup; these are the two major feat ures that influence the shift of the C=O stretch frequencies. Potential surface scans of -GalNac and -GalNac (torsion about the (C-2)-N bond), similar to those carried out for the glucosamin es, indicated that the conformers found in the conformational search and used throughout this analysis were the lowest points on the 1dimensional surface (Figures 5-13 and 5-14, respectively). The highest energy points on the surface correspond to structures in which the carbonyl oxygen is in close proximity to the Omethyl oxygen at C-1 for both anomers of D-GalNac. These are analogous to the highest energy points on the potential surfaces of the D-GlcNac anomers. The lithium cation in the complex with -GalNac is bound to the O-1, C-6 and C-4 hydroxyl oxygens, and has a charge of 0.57. The interactions of the lithium cation in -GalNac are the same as in -GalNac (O-1, C-6 and C4 hydroxyl oxygens) corresponding to a similar char ge of 0.58 on the lithium cation. Other structures with a different lithium position in the -GalNac complex have a calculated energy of 10 kcal/mol or higher, while for -GalNac the more energetic locati ons are 5 kcal/mol or higher in energy. The charge on the lithium cation is less for the galactosamines than for the glucosamines, as is evident from the interactions just reported. Table of the relative energies using DFT for the structures from the conforma tional search (Table 5-1) are given below. Fragmentation Patterns Four fragments were seen for each of the sugars in the tandem mass spectra produced by IRMPD fragmentation (Figure 5-15). The frag mentation channels i nvolved the loss of CH4O 108

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(210 m/z), fragments resulting from cross ring cleavage at C-1 O1 and C-2 C-3(127 m/z) and (132 m/z) and fragments resulting from cross ring cleavage at C-1 O1 and C-2 C-3 (122 m/z) (Figure 5-16). The proposed fragment ions include the lithium cation, except m/z 132. From Figure 5-15, the abundance of the 122 m/z fragment ion for -GlcNac is higher than for the other three isomers, and the DFT calculated structures suggest that the incr ease in abundance may be due to the lithium cation being located between the acetamido and C-3 functional groups. As cross ring cleavage occurs to produce the 122 m/z fragment (which includes the acetamido group), attachment of lithium to the leaving group would be facile. For -GlcNac and /GalNac, fragmentation ions and their relative abundances are similar, showing no discernable differences in the MS/MS spectra. DFT calcula tions support these results, since calculated structures show the lithium cation in a similar position for each isomer, thus predicting that the lithium cation-bound fragments would have similar abundance ratios. Conclusion Glycosamine isomer differentiation in the ga s phase is possible with IRMPD spectra if each isomer has distinct infrared spectral featur es or fragmentation patterns in the tandem mass spectrum. In this study, for the lithium cationi zed anomers of glucosamine and galactosamine, C=O stretch band shifts we re observed (10 and 11 cm-1, respectively) and can be used to differentiate the anomers. The shifts between the alpha and beta anomers were explained using theoretically calculated spectra as being due to the difference in location of the acetamido carbonyl, which is influenced by the repulsion of the O-methyl oxygen on carbon atom 1 and the carbonyl. The favorable inter action of the amide hydrogen w ith the O-methyl oxygen also influences the location of the car bonyl, since both are part of the acetamido group. Calculated structures indicate that the lithium cation is located between C-3 and the acetamido group for 109

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GlcNac. This is corroborated by the tandem mass spectrum fragmen tation pattern that indicates a larger abundance of the acetamido-lithium fragment ion for -GlcNac than for the other isomers. As discussed in the results section, the other 3 isomers have a similar location of the lithium cation. In the calculated structures of -GlcNac and -GalNac, the lithium cation is found to interact with 3 oxygens, near the ring oxygen. Fragmentation patterns in the MS/MS spectrum of the three isomers are similar, possibly due to the similar position of the lithium cation for all three. Other defining spectral f eatures, which might be seen in different ir wavelength ranges, could be used to further differentiate the isom ers. A study of glycosides and their isomer differentiation using bands in the midir and O-H stretch regions will be presented in chapter 6. 110

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Table 5-1. Glycosamine isomer structures obtained from the corr esponding conformational searches. Relative energies were obtained at the B3LYP/6-31+G(d) level of theory. Conformer 1 of each isomer appears in Figures 5-5 and 5-12. Conformer # -GlcNac -GlcNac -GalNac -GalNac 1 0.00 0.00 0.00 0.00 2 4.67 0.01 2.70 3.42 3 5.60 1.79 15.66 5.00 4 15.56 1.80 17.21 5.01 5 17.45 2.60 17.30 6.65 6 18.66 2.61 17.41 11.03 7 24.55 2.94 17.42 12.41 8 30.06 5.19 19.09 12.56 9 30.37 5.27 19.10 13.21 10 30.62 5.31 25.56 13.37 11 34.25 5.53 25.72 13.92 12 35.65 6.78 27.71 15.41 13 36.04 7.72 32.75 16.38 14 36.34 8.86 34.95 18.30 15 38.24 15.16 18.33 16 38.48 21.34 17 39.37 22.58 18 40.82 19 47.42 20 50.99 indicates that the 15 kcal/mol cutoff was me t with the specified number of structures 111

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C B D A Figure 5-1. Isomers A) -GlcNac, B) -GlcNac, C) -GalNac and D) -GalNac. The differences between and structures at carbon 1 and those of glucose and galactose on carbon 4 are shown in red and purple, respectively. 2.5 Figure 5-2. Side and top views of lithium cationized -GlcNac. The lithium cation was initially placed above the ring, approximately 2. 5 from O1. The clockwise hydrogen bonding network is visible in the top view a nd is encircled in red. The carbons and ring oxygen are numbered. 112

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Figure 5-3. Rotation about C-2 a nd N, shown with a red arrow. 100011001200130014001500160017001800 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 beta-O-Methyl-N-acetylglucosamine 10 cm-1 shiftRelative yieldWavenumber / cm-1 10 cm-1 shift Wavenumber/cm-1 1 Figure 5-4. IRMPD spectra of lithium cation-bound -GlcNac (black) and -GlcNac (red), showing the 10 cm-1 shift of the carbonyl stretch frequency. 113

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+ + -GlcNac -GlcNac Figure 5-5. Calculated lowest energy structures of lithium cationized -GlcNac and -GlcNac, using B3LYP/6-31+G(d). Lithium is shown in blue and hydrogen bonding is indicated with dashed dots. Figure 5-6. Comparison of e xperimental IRMPD and calculated infrared spectra of lithium cationized -GlcNac. The calculated spectrum has been scaled by 0.98, and a 20 cm1 Gaussian band profile has been used. Wavenumber/c m -11100 1200 1300 1400 1500 1600 1700 1800 1000 calculated experiment 114

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0 50 100 150 200 250 -180-140-100-60-202060100140180 Torsion angle (degrees)Relative energy (kcal/mol) Figure 5-7. Potential surface scan for rotation about (C-2)-N bond for -GlcNac. 0 50 100 150 200 250 -180-140-100-60-202060100140180 Tosion angle (degrees)Relative energy (kcal/mol) Figure 5-8. Potential surface scan for rotation about the (C-2)-N bond for -GlcNac. 115

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Wavenumber/cm1 1000 1100 1200 1300 1400 1500 1600 1700 1800 calculated experiment Figure 5-9. Comparison of e xperimental IRMPD spectrum (red ) to the calculated infrared spectrum (blue) of lithium cationized -GlcNac. 1350 1450 1550 1650 1750 Wavenumber/c m -111 cm-1 shift Figure 5-10. IRMPD spectra of lithium cationized -GalNac and -GalNac, showing the 11 cm1 shift for the carbonyl stretch bands. 116

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140014501500155016001650170017501800 29 cm-1 calculated shift avenumber (cm-1) W alpha-GalNac beta-GalNac Wavenumber/c m -1 Figure 5-11. Calculated infrared spectra of lithium cationized -GalNac and -GalNac, indicating a 29 cm-1 shift in the C=O stretches. + + -GalNac -GalNac Figure 5-12. Lowest energy structures of lithium cation-attached galactosamine anomers from B3LYP/6-31+G(d), showing hydrogen bonding. Lithium is shown in blue. 117

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0 50 100 150 200 250 -180-140-100-60-202060100140180 Torsion angle (degrees)Relative energy (kcal/mol) Figure 5-13. Potential surface scan for -GalNac. 0 5 10 15 20 25 30 35 40 -180-140-100-60-202060100140180 Torsion angle (degrees)Relative energy (kcal/mol) Figure 5-14. Potential surface scan of -GalNac. 118

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6 78 91 0 210 132 127 1226 78 91 0 210 132 127 122 6.06.57.07.5 8.08.59.09.510.010.5 210 132 127 1226.06.57.07.5 8.08.59.09.510.010.5 210 132 127 122 5.866.26.4 6.6 6.8 7 210 132 127 122 210 132 127 1227.2 7 6.8 6.6 5.8 66.2 6.4 Figure 5-15. Fragment ion abundances as a function of laser wavelength for all four isomers. The abscissa has units of wavelength (m). Figure 5-16. Possible fragmentation producing the observed MS/MS spectra of glycosamine isomers. Black dashed dots indicate lithiu m cation-attached fragment ions and red dashed dots indicate fragment i ons without the lithium cation. 119

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CHAPTER 6 DIFFERENTIATION OF O-METHYLATED GLYCOSIDE ISOMERS Introduction Mass spectrometry has been used succe ssfully to obtain sequence and linkage information about oligosaccharides.135 However, as many oligosaccharides are structural isomers with identical mass, differ entiation of their isomeric struct ures can be challenging, if not impossible, with this approach. Chapters 1 a nd 2 mention that one of the recently developed methods for obtaining structural information about sugars combin es infrared spectroscopy with theoretical calculations.17,146-150 With solid and solution phase sa mples, infrared bands of sugars and many other large molecules are broad, and therefore it is difficult to observe multiple identifying features in their spectra.151 A gas phase experiment may have less spectral broadening since there is no solvent present a nd therefore provide more identifying spectral features that allow for differentiation of isomers. In the gas phase, a typical infrared absorption experiment is difficult to conduct, since concentra tion of the species of in terest is too low to detect absorbance of radiation of the incident laser beam by the gaseous sample. Nevertheless, as has been discussed throughout this work, a gas phase spectrum is obtainable using action spectroscopy methods. Infrared multiple photon dissociation (IRMPD)152 spectra were obtained by Valle et al. for the rubidium cation-attached glycoside isomers O-methyl--Dgluco side (Glc), O-methyl--Dgluco side (Glc), O-methyl--Dgalacto side (Gal) and O-methyl-Dgalacto side (Gal) at the FOM-Institute for Plasma Physic s Rijnhuizen, The Netherlands using the F ree E lectron L aser for I nfrared e Xperiments (FELIX).17,66,122 One major band for each isomer is observed in the 600 1700 cm-1 range, with few distinguis hing features (Figure 6-1). 120

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However, the O-methylated glycosides have four hydroxyl groups (Figure 6-2), which are predicted to give rise to intense stretching bands in the 3500 cm-1 wavenumber region of the infrared spectrum. Therefore, IRMPD spectra of the O-H stretching region were obtained to positively differentiate between all four isomers. Extensive calculations were carried out with density functional theo ry using the B3LYP hybrid func tional and 6-31+G(d) basis set.126,128 This chapter presents IRMPD spectra of the same four O-methyl D-glycoside isomers studied by Valle et al., but the spectra were take n using an FTICR-MS coupled with a continuous wave optical parametric oscillator (cw-OPO) laser.57,61,153 The OPO optics setup and the experiment sequence are introduced in the experimental techniques section. Results and spectral characteristics of each isomer in the mid-ir and the O-H stretching region are discussed, as well as hydrogen bonding and the rubidium cation intera ctions that influen ce the O-H stretch band positions in the IRMPD spectra. Setup of th e OPO optics and acquisition of IRMPD spectra using the OPO laser was done in conjunction wi th Wright L. Pearson. All figures of experimental spectra were plotted by Mr. Pearson. In this chapte r, the appropriate abbreviation, for example Glc for Rb+-O-methyl--D-glucopyranoside, will be used to indicate the rubidium cation-attached monosaccharide as opposed to the neutral molecule. Experimental Techniques Chemicals O-methyl D-glycoside samples were provide d by Prof. Brad Bendiak, Department of Cellular and Structural Biology, University of Co lorado Health Sciences Center. Solvents and inorganic salts were obtained from Sigma-Aldrich, and were used without further purification. Solid 10 mg samples of D-glycosides were dissolved in a 10 mL solv ent mixture of 80:20 MeOH:H2O. Stock solutions were diluted to 1x10-4M with the same solvent mixture, and an equimolar amount of RbCl was a dded as the ionization agent. 121

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Instrumentation An FTICR 4.7T Apex II mass spectrometer (Bru ker, Billerica, MA) coupled with an ESI source (Analytica of Branford, Inc., Branford, CT ), which is located in the Mass Spectrometry Services laboratory at the University of Florid a was used for experiments with the OPO laser. The ESI source had a flow rate of 2 L/ min, and ionization effi ciency was aided with a nebulizer and a desolvation gas, both N2 gas, with continuous flows of 35 and 155 l/hr, respectively. The electrospray needle-capillary voltage differen ce was set to 3.6 kV. Precursor ions were isolated using swept frequency ejection pulses to remove all other ions. Ions were detected using the broadband detection mode covering a mass range from 70 to 500 Da. Data was collected with the Bruker X-MASS data acquisition system. For IRMPD spectra obtained with FELIX, a 4.7T supeconducting magnet (Cryomagnetics Inc., Oak Ridge, TN) was used with a laboratory-constructed pumping system, ion trap and electronics console and with a Z-spray electrospray ionization source. The FELIX setup was mentioned in chapters 3 and 4, and is described in the article by Valle et al.66 Laser Setup The tunable, continuous-wave OS 4000 optical parametric oscillator (OPO) laser (LINOS Photonics, Mnchen, Germany) uses a Nd/YAG pump laser (2W, 1064 nm) to produce two output beams from the nonlinear OPO crystal, th e idler and signal beams. The two beams are dependent on the pump radiation and are correlated by Equation 6-1. isp111 (6-1) The p, s and i are the wavelengths of the pump la ser and the signal and idler beams, respectively. Tuning the OPO is accomplished by changing the poling period of the OPO crystal. A total of 18 poling periods, with differe nt wavelength ranges, allow for lasing in the 122

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wavelength ranges from 1.38-2.0 and 2.28-4.67 micr ons. Within a poling period, finer tuning is accomplished by changing the temperature of the OPO crystal from 50 150 C. An etalon is used to minimize and discriminate between the co mpeting lasing wavelengths, allowing for fine tuning of a specified wavelength. A piezo-ele ctric mirror makes up one end of the resonance cavity and uses lock-in electronics to keep the laser wavelength in resonance. The current filtering lenses allow the use of the two idler beam s created at both ends of the resonance cavity. Figure 6-3 shows idler beam 1 exiting at the front of the OPO enclosure box and idler beam 2 exiting at the back of the OPO enclosure box. Both idler beams are oriented by a set of mirrors to enter the ICR cell collinearly. Two Uniblitz CS25 shutters (Vincent Associates, Rochester, NY) block the idler beams from entering the cell wh en closed and divert the beams to a detector to record the power output. The signal beam exits the OPO enclosure box adjacent to idler beam 1 and is directed into a wave meter (WA-1500, EXFO Electro-Optical Engineering Inc., Plano, TX). A 10.6 micron continuous wave CO2 laser (Synrad, Inc., Mukilteo, WA) is aligned to enter the ICR cell and is collinear with the OPO idler beams. The CO2 laser can be used independently or as an off-resonanc e laser in a two-laser experiment.154,155 The entire optics setup, with the exception of a short path of th e signal beam before it enters the wavemeter, is enclosed in a nitrogen gas purge box designed by Mr. Pearson to eliminate absorbance of laser radiation from water vapor in the air. Experiment OH Stretches. After electrospray ionization, trapping and isolation in the ICR cell, rubidium cationattached glycoside ions were irradiated for 10 s by the OPO id ler beams, followed by excitation and detection of fragment ions. A typical experimental sequence at each wavelength, the appearance of Rb cation signal as well as the lo ss of precursor ion signal were monitored via the 123

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mass spectrum (Figure 6-4). The peak s due to each isotope of rubidium, 85Rb and 87Rb, and their corresponding complex glycoside io ns, were monitored and used to form the composite IRMPD spectrum. CH Stretches. Weak absorption by the C-H stretc hing modes of the rubidium cationattached glycosides produced little or no lo ss of the rubidium cations even after 10 s of irradiation with the OPO lasers. Using a fixed wavelength (10.6 m) CO2 laser, an onresonance, off-resonance two laser ex periment was conducted, where the CO2 laser was the offresonance laser used to provide more photon flue nce for dissociation. The mid-ir spectra in Figure 6-1 show little or no absorption at or near the 10.6 m (~940 cm-1) wavelength, and fragmentation is not expected to occur at this wavelength. The CO2 irradiation time and power were adjusted such that none of the glycos ide-rubidium cation complexes were observed to fragment due to the off-resonance radiation alone. The experimental sequence included irradiation for 10 s with OPO idler laser beams, and irradiation with the CO2 laser during the last 3 s of the irradiation period (Figure 6-5). In regions where it was suspected that this two laser sequence did not provide enough photons for abso rbance by the C-H stretch bands to induce dissociation of the rubidium cation, total irradiation time was increased to 15 s, with the last 7 s including the CO2 laser. At each wavelength, ten indivi dual transients were accumulated to improve the signal-to-noise ratio. For D-glucosides, three IRMPD spectra (conducted on different days) were used to calculate the average spectrum for each isomer. Preliminary IRMPD spectra of D-galactosides will also be discussed. Computational Details Using the Hyperchem123 drawing database, O-methylated -D-glucosides were drawn with a clockwise hydrogen bonding networ k, with has been shown in literature to stabilize 124

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monosaccharides.139,140,156 For each isomer, the rubidium cation was placed centered above the glycoside ring system, approximately 3 from O1 (Figure 6-6). The dihedral angles within the ring atoms were defined and were varied in the conformational se arch by using the built-in ring flex and torsion algo rithm of Hyperchem.90 The H-C-O-H torsion angl es were defined so that the O-H functional group would rotate about the CO bonds, keep ing the torsion angle changes separate from the ring conformation changes. All torsion angles were randomly varied for 1000 searches. For each of the 1000 structures created, a geometry optimization using the AMBER77,137 force field was performed. Comparison of conformers was done to find duplicate structures. The parameters used to test for duplicate structures were the atomic positions, dihedral angles and total energy, whose thres hold values were set to 0.25 10, and 0.05 kcal/mol, respectively. All conformers within 15 kcal/mol of the lowest energy conformer were further geometry optimized using B3 LYP/6-31+G(d), plus LANDL2DZ basis157,158 for Rb, in Gaussian03.124 For the glucoside anomers, further ri ng conformations were explored whenever the conformational search did not provide adeq uate structures to account for IRMPD spectra bands of each anomer. Chair (4C1) conformations with differing rubidium cation position were only explored for D-galactosides. Vibrational an alysis was then done for all the conformations at the B3LYP/6-31+G(d) level of theory. Freque ncies were scaled by 0.97 for O-H stretches and mid-ir region, and 0.96 for C-H stretches. These scaling factor values are well within the error for factors used for similar sugars.85,140,141,143,146,156,159-162 Results and Discussion FELIX The lowest energy conformations for each isom er from the conformational search (Figure 6-7) and the calculated infrared spectra of the conformers were used to compare to FELIX IRMPD spectra (Figure 6-8). Using a 20 cm-1 Gaussian band profile to mimic the width of the 125

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experimental spectral bands, the ca lculated spectra of the lowest energy structures seem to agree with the experimental spectra (Figure 6-8). Using the next set of higher energy structures calculated for each isomer and comparing them to the corresponding IRMPD spectrum, it can be seen that these also seem to agree with the experimental spectra (Fig ure 6-9). Although the experimental band that appears for each isomer in the 900 1400 cm-1 range has a few small features that can potentially be used for diffe rentiation between isomers, all the calculated spectra include those same features and therefor e a distinction between isomers is unambiguous. The experimental spectra band is too broad to be used for identification and differentiation of each isomer. OPO Glucosides -D-glucoside. The IRMPD spectrum of rubidium cation-attached O-methyl-Glc from 2750 3750 cm-1 (Figure 6-10) with the ge neral region for C-H and O-H stretches are indicated. The most common region for free O-H stretches,163 namely for those hydroxyl groups that are not interacting with another atom, molecule, or i on, is expected to be in the range of 3500 3700 cm-1. Hydroxyl groups that are involved in hydr ogen bonding are expected to have O-H stretches in the lower energy range of the O-H stretch region (3200 3600 cm-1). For Glc in Figure 6-10, the O-H stretch region show s four major bands, 3450, 3560, 3637 and 3677 cm-1, labeled I, II, III and IV, respectively. The me thylated sugar has four O-H groups, accounting for the number of bands observed in the spectrum. The bandwidths are 32, 20, 9, and 21 cm-1 full width at half maximum, respec tively. The side band near band II has a bandwidth of 17 cm-1. The C-H stretch region has two bands from 2800 3050 cm-1. Figure 6-11 shows a comparison of the C-H stretch region spectrum when using only the OPO laser and the spectrum from the 126

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two laser experiment. All that is observed in the latter is enha ncement of the bands with no new bands or shifting of the bands seen in the one laser experiment. The simulated spectra were compared to the OPO-IRMPD spectrum. For the lowest energy conformer (A) (Figure 6-7) there is mini mal agreement with the experimental spectrum (Figure 6-12). Comparing the simulated spectrum of the next lowest energy conformer (B) (0.8 kcal/mol higher in energy) with that of the e xperiment, the agreement is similar to that of conformer (A) (Figure 6-13). Two other conformati ons that are within 7 kcal/mol of the lowest energy structure are compared to the experimental data (Figures 6-14 and 6-15). An overlap of spectra for all four calculated conf ormers with the IRMPD spectrum of Glc shows that these four conformers account for all the spectral featur es in the IRMPD spectru m, and all are within the margin of error for the DFT calculations at this level of theory (Figure 6-16). The the first three conformers of Glc (Figure 6-17) have a 4C1 ring conformation, and a fourth conformer D has a 1C4 ring conformation. The carbohydrate nomenclature was discussed in chapter 1. To account for bands I and II in the IRMPD spectrum, the la rge red shift of the OH stretches must be explained. Conformer D has hydrogen bonds between C-3 and C-6 hydroxyl groups and C-6 and the oxygen of the O-methyl group of carbon atom 1. This results in the shifting of the O-H stretching frequencies for the respective hydroxyl gr oups to band I (3450 cm1) and II (3560 cm-1) for conformer D. The location of the rubidium cation is similar for conformers A and B, but changes for conformers C and D. For conformers A and B, the rubidium cation is above the plan e of the ring. For conformer C, the rubidium cation is between C-4 and C-5 functional groups, and for conformer D, the rubidium cation is below the plane of ring. In all cases, the rubidium cation is in teracting with the maximum possible number of oxygen atoms. For conformer C, the positi on of the rubidium cation and hydrogen bonding 127

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network cause the O-H stretch of the C-4 hydroxyl to red shift toward ba nd II. For all four conformations of Glc, the free O-H stretches were of highest energy, corresponding to the band at 3677 cm-1. Band IV, although indicating that the O-H stretch has not been perturbed, cannot be generally assigned to a pa rticular hydroxyl group since each conformation has unperturbed hydroxyl groups at different car bon atoms. It should be noted that conformers A D have relative energies up to 6.49 kcal/mol, which is with in the error for DFT calculations at this level of theory,144,164-166 and potentially all conformations can be present in the gas phase experiment. The region from 2800 3050 cm-1 has multiple C-H stretching modes, and similar to the mid-ir spectra, the peaks are too broad to contribu te in making a distinction between the isomers, and will not be discussed. An extensive conformational search for all rubidium cation-attached Glc, where all torsion angles a nd rings conformations, continues to be explored by the author and Wright L. Pearson. The spectrum for Glc will be used as a basis of comparison for spectra of the other three isomers. -D-glucoside. The O-H stretch region of rubidium cation-attached Glc shows three bands, with the band at 3637 cm-1 that appears for Glc is clearly missing. The three bands have similar positions to those of Glc (Figure 6-18). The bandwidths of the Glc O-H stretch bands are 45, 54 and 21 cm-1, respectively. The C-H stretch regi on shows two bands in the range of 2900 3050 cm-1 and the intensities of the two bands ha ve changed with re spect to those of Glc. Two O-H stretch bands of Glc overlap bands I and II of Glc and are indicative that the hydroxyl groups contributing to these bands ar e involved in strong hydrogen bonding and are perturbed by the rubidium cation. From the results of Glc, the third band of Glc at 3677 cm-1 should correspond to an O-H stretch of a hydr oxyl group not involved in hydrogen bonding. Similar to Glc, the calculated sp ectrum of the lowest energy structure found for Glc did not 128

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account for all bands present in the IRMPD spect rum (Figure 6-19). The theoretical spectrum for two other conformations of Glc, one having a 1C4 ring conformation a nd the other with a O,3S ring conformation, have better correla tion to the experimental spectrum of Glc (Figure 620), and the conformations are shown in Figure 6-21. The calculated bands of these conformers are significantly red shifted, but do not comple tely overlap all O-H st retch bands, and further conformational calculations ar e required for better correlation between calculated and experimental spectra. Galactosides Spectra for the rubidium cation-attached O-me thyl-D-galactosides was also taken, although only a single pass in the range of 3500 3800 cm-1 has been completed, and therefore will not be compared to calculated structures. For Gal, the range from 3400 to 3580 cm-1 has been scanned twice and the average of the two spect ra was calculated and the IRMPD spectra of Gal and Gal was compared (Figure 6-22). A quick summary of the IRMPD spectral features for the galactosides is discussed, relative to the findings of the glucoside isomers. -D-Galactoside. Two bands appear for Gal, centered at 3580 and 3650 cm-1, with bandwidths of 17 and 30 cm-1, respectively. The two bands fa ll within the region for bands II and III of Glc, and it is expected that the gas phase conformations for Gal have hydroxyl groups involved in hydrogen bonding and rubidium interactions. -D-galactoside. The O-H region has 3 distinguishable bands that are broad, appearing at 3550, 3600 and 3660 cm-1, with respective bandwidths of 38, 27 and 30 cm-1. In Figures 22 and 23, Gal is shown to have distinct spectrum relativ e to the other three isomers. The band near 3600 cm-1 differentiates Gal and Gal, since Gal has a nearby band that is shifted to 3580 cm1. Similar to Gal, the O-H stretches also fall within bands II and III of Glc, with a slight 129

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overlap of band IV. The gas phase conformations for Gal are expected to have hydroxyl groups whose stretches are perturbed by the rubidium cation and hydrogen bonding, but the perturbations are not expected to be as strong as those for the glucoside isomers. A spectrum showing IRMPD spectra for all four isomers indicates that each isomer has peaks that overlap band II and band III of Glc, with a slight overlap at band IV (Figure 6-23). Comparison of all four isomer spectra, there is a clear indication that di fferentiation is possible using the infrared spectra of the O-H stretch region. Differentiation between the glucoside anomers, Glc and Glc, can be easily accomplished if th e region of band III is scanned, since it is in this wavelength range that no O-H stretch is expected for Glc. Differentiation between the galactoside anomers, Gal and Gal, is possible due to the band shift of Gal near 3580 cm-1 to 3600 cm-1 of Gal. Finally, differentiation of the glucos ides and galactosides is possible using their respective IRMPD spectra, since both galactos ides do not have band p eaks in the region of band I of Glc,. A peak appearing in the region of band I of Glc can be used as an analytical marker to indicate the presence of rubidium catio n-attached glucosides, a nd not galactosides, in an unidentified sample. Conclusions To differentiate the rubidium cati on-attached isomers of O-methyl--D-glucoside, Omethyl--D-glucoside, O-methyl--D-galactoside and O-methyl--D-galactoside, Fourier transform ion cyclotron resonance mass spectrometry in conjunction with infrared multiple photon dissociation was used to obtain infrared sp ectra of the rubidium-bound isomers in the gas phase. Previous work of Valle et al. at the FOM-Institute for Pl asma Physics Rijnhuizen, The Netherlands, showed few discernabl e differences in the spectra of all four isomers in the spectral range of 600 1700 cm-1, so that differentiation of c onformational isomers using DFT 130

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calculations was dubious. Work was done to couple an optical parametric os cillator laser system with an FTICR-MS instrument to obtain IRMPD spectra at 2600 3900 cm-1. Theoretical calculations and experimental spect ra indicate that the spatial orientation which distinguishes each isomer (or, glucose or galactose) influences the strength of the hydrogen bonding network. The rubidium cation location also perturbs the O-H stretches and the hydrogen bonding network, making differentiation of all four glycoside isomers possible. 600 800 1000 1200 1400 1600 6008001000120014001600 600 800 1000 1200 1400 1600Glc Glc Wavenumber/cm-1 Wavenumber/cm-1 Figure 6-1. FELIX-generate d IRMPD spectra (600 1700 cm-1) of all four glycoside isomers, Glc, Glc, Gal and Gal. Spectra were obtained by Valle et al. and included in Jose J. Valles dissertation.17 Gal Gal 500700 1500 1700 90011001300Wavenumber/c m -1Wavenumber/c m -1 131

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B A C D Figure 6-2. O-methylated glycoside isomers, A) Glc, B) Glc, C) Gal and D) Gal shown in the 4C1 chair conformation. A difference in structure for beta isomers is shown in red, and differences between glucose a nd galactose are highlighted in blue. N2 purge b ox front back CO2 laser Power detector Figure 6-3. OPO optics setup, showing idler beams (as well as CO2 laser) directed into the ICR cell and signal beam directed to the waveme ter. The setup include s two iris shutters that divert the idler beams into the power detector when they are closed (for clarity, only one iris shutter is shown). 132

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Figure 6-4. Experimental seque nce for obtaining spectra of th e O-H stretching region of the rubidium cation-attached glycosid es using OPO laser irradiation. Time Figure 6-5. Experiment sequence fo r the two laser experiment. The CO2 laser output was tuned to be off-resonance with any vibrational modes, and did not promote dissociation when used alone. Time 133

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3 Figure 6-6. The monosaccharide -D-glucoside (side and top view s) with the rubidium cation centered above ring and ~ 3 from O1. Ring oxygen (O1) and the carbons of the monosaccharide are numbered. A B D C Figure 6-7. Lowest energy conformers of A) Glc B) Glc C) Gal and D) Gal obtained from each isomers conformational search. Rubidium is shown in purple. 134

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500 700 900 1100 1300 1500 1700 Wavenumber/cm-1 500 700 900 1100 1300 1500 1700 Wavenumber/cm-1 B A 500 700 900 1100 1300 1500 1700 Wavenumber/cm-1 500 700 900 1100 1300 1500 1700 Wavenumber/cm-1 C D Figure 6-8. Comparison of theo retical (gray) and experimental IRMPD spectra for the isomers A) Glc, B) Glc, C) Gal and D) Gal. Theoretical spectral frequencies have been scaled by 0.97 and a 20 cm-1 Gaussian band profile has been applied. 500 700 900 1100 1300 1500 1700 Wavenumber/cm-1 500 700 900 1100 1300 1500 1700 Wavenumber/cm-1 B A Figure 6-9. Calculated spectra for the second lowest energy c onformations of D-glucosides anomers from the conformational search. Labe ling is the same as in Figure 6-8. Theoretical spectra of each isomer are shown in purple. 135

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Wavenumber/c m -1II II IV I Figure 6-10. IRMPD spectrum of rubidium cation-attached Glc, with the typical ranges for CH and O-H stretches indicated. The four major bands in the O-H stretch region are labeled. Image courtesy of Wright L. Pearson. CO2 Laser A B Wavenumber/c m -1 Figure 6-11. Comparison of the IRMPD spectrum of Glc in the C-H stretch region with A) irradiation with the on-resonance OPO laser and B) irradiation with both the OPO and CO2 laser (as described in the experimental section) showing the enhancement of the peaks. Image courtesy of Wright L. Pearson. 136

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Wavenumber/cm-1 Figure 6-12. Comparison of calculated spect rum for conformer A (dark blue) and IRMPD spectrum (red) for rubidium cation-attached Glc. Image courtesy of Wright L. Pearson. Wavenumber/cm-1 Figure 6-13. Experimental spect rum (red) and calculated spectrum for conformer B (green) of rubidium cation-attached Glc. Image courtesy of Wright L. Pearson. 137

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Wavenumber/cm-1 Figure 6-14. Spectral match betw een experiment (red) and calculated conformer C of rubidium cation-attached Glc. The red shift toward the band near 3560 cm-1 is clearly evident. Image courtesy of Wright L. Pearson. Wavenumber/cm-1 Figure 6-15. Spectral comparison of experiment (red) and calculated conformer D (gray) for rubidium cation-attached Glc, showing a red shift for O-H stretches of C-3 (3450 cm-1) and C-6 (3560 cm-1). Image courtesy of Wright L. Pearson. 138

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Wavenumber/cm-1 Figure 6-16. Overlap of all four conformer sp ectra with IRMPD spectrum for rubidium cationbound Glc, accounting for all bands present in experimental spectrum. Spectrum for conformer A is in dark blue, conformer B is the green line, conformer C is the blue line, and the gray line is the spectrum of conformer D. Image courtesy of Wright L. Pearson. 139

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A B C D Figure 6-17. Calculated conformers for Glc at the B3LYP/6-31+G(d) level of theory that account for experimental spectral bands, s hown in top and side views. Hydrogen bonding is shown with red dashed dots (top view only) and the rubidium cation is colored tan. 140

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Wavenumber/cm-1 Figure 6-18. Comparison of IRMPD spectra for Glc (blue) and Glc (red), clearly showing the band at 3637 cm-1 is missing for Glc. Image courtesy of Wright L. Pearson. 33003350340034503500355036003650370037503800 Wavenumber/cm-1 Figure 6-19. Comparison of IRMPD and calculate d spectra for the lowest energy conformer of Glc. 141

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33003350340034503500355036003650370037503800 Wavenumber/cm-1 Figure 6-20. Overlap of calculated spectra for conformer A and conformer B with IRMPD spectra of Glc. A B Figure 6-21. Conformers of Glc that correlate with IRMPD sp ectrum, shown in top and side views. Hydrogen bonding is marked with red dashed dots (top view only) and the rubidium cation is colored tan. Relative energies are in parenthesis and were calculated at the B3LYP/6-31+G(d) level of theory. 142

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335034003450350035503600365037003750Wavenumber/cm-1 Figure 6-22. Comparison of preliminary IRMPD spectra for Gal (dark red) and Gal (green) in the O-H stretch region. A shift occurs for the band near 3580 cm-1, and more spectral features are evident for the Gal anomer. 335034003450350035503600365037003750Wavenumber/cm-1 Alpha Glc Beta Glc Alpha Gal Beta Gal I II III IV Figure 6-23. Overlap of IRMPD sp ectra of all four isomers in th e O-H stretch region. The major spectral bands are labeled. 143

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CHAPTER 7 CONCLUSIONS AND FUTURE WORK Fourier transform ion cyclotron resonance ma ss spectrometry and infrared multiple photon dissociation have been used to obtain gas phase infrared spectra of alkali cation-attached glycosides, N-acetylglycosamines and phenylal anine derivatives. The experiments were combined with theoretical calculations to obtain the best predictions of the structures of the respective ions. The IRMPD-FTICR-MS experiments proved in valuable in differentiating carbohydrate isomers and in providing well-resolved spectra of phenylalanine analogs. The IRMPD spectra obtained in the 800 1800 cm-1 were used in conjunction with theoretical calculations to predict the band shifts due to deuterium/hydrogen exchange of sodium cation-attached phenylalanine analogs. It was also possi ble to differentiate the and -anomers of N-acetylglycosamines by observing the shift in the C=O stretc h. IRMPD spectra in the 8001800 cm-1 were also obtained for rubidium cation-attached glycosides, but the band features were quite broad and differentiation between the respective isomers wa s inconclusive. Instead, using an optical parametric oscillator coupled to an FT ICR-MS, IRMPD spectra in the 2750 3800 cm-1 range were obtained for the rubidium cation-attached glycosides. Differentiation was possible because the bands were narrow (12 45 cm-1), and each isomer displayed distinct peaks in the O-H stretch region (3400 3800 cm-1). Fragmentation of the precursor ion as a functi on of irradiation wavele ngth was also useful in obtaining structural informa tion and alkali metal cation binding locations. Binding of the lithium cation to carbohydrates induces fragmentati on of the sugar when s ubjected to irradiation with an infrared laser. For the lithium cation-at tached N-acetylglycosamines, the fragment ions with m/z 210, 132, 127 and 122 were observed after irradiation and subsequent fragmentation of 144

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each precursor ion. Fragmentation ion abundances for -GalNac, -GalNac and -GlcNac were similar, where the order of abundance of the fragment ions was 127 > 210 > 132 ~ 122. However, -GlyNac displayed a distinct abundance order: 122 ~ 127 > 132 > 210. The 122 m/z fragment ion arises from the lithium cation attached to a cross ring cleavage fragment containing the N-acetyl group. The calculated structures su pport this result, indica ting that the lithium cation is located between the C-3 hydr oxyl group and the N-acetyl group for -GlcNac. For the other three isomers, the Li+ is located near the ring oxygen. Theoretical calculations were an integral part of this work. The Conformational searches, in conjunction with the DFT cal culations, provided good spectral matching with experimental spectra for the phenylalanine analogs. The lowest energy structures obtained in the conformational searches gave the best matches to the IRMPD spectra for all three phenyalanine analogs, and band assignments were based on the results of theDFT calculations. For deuterium/hydrogen exchange experiments, the ba nds that involved R-NH and R-OH vibrations in the undeuterated analog were expected to shift after each analog was subjected to H/D exchange and subsequent IRMPD spectra were take n. The bands shifts were correctly predicted by theory. After confirmation with experiment, the calculated structures of the phenylalanine analogs were found to have a puckered struct ure, in which all the carbonyl oxygens interact with Na+ and the phenyl ring. A cationinteraction was found for all of the lowest energy structures. The conformational search and DFT calculations approach was also adequate to obtain structures for N-acetylglycosamines. The calculati ons indicated that the spectral band shifts are due to the location of the carbonyl of the N-acetyl group. The or ientation of the N-acetyl group and the relative position of th e carbonyl are influenced by the repulsion between the O-methyl 145

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oxygen and the carbonyl oxygen. The C=O band shift seems to be a localized effect so that differentiation of anomers is possible. Howe ver, IRMPD could not differentiate between DGalNac and D-GlcNac. For exampl e, in the IRMPD spectra of -GlcNac and -GalNac, the C=O stretch bands overlap almost completely. A similar situation was observed with -GlcNac and -GalNac, whose C=O stretch bands overlap, thus preventing differentiation of the two isomers using only their IRMPD spectra. However, theoretical calculations were able to predict the band shifts by finding the orientation of the carbonyl in the N-acetyl group of each glycosamine isomer. Conformational searches and DFT calculations did not provide an adequate match for the IRMPD spectra of the rubidium cation-attached glycosides Although similar to the Nacetylglycosamines, the free glycoside sugars can form extensive hydrogen bonded networks that may not be adequately modeled with the levels of theory used. The rubidium cation requires that the empirical AMBER force field be used in th e conformational search, instead of the semiempirical AM1 force field used fo r other two projects. Probably mo re important is the need to use an effective core potential basis set in the DFT calculations involving rubidium. This most likely increases the discrepancies between calcula ted and experimental spectra of the rubidium cation-attached glycosides. In contrast to the N-acetylglycosamines, all the hydroxyl groups were probed in the IRMPD spectra of the Rb+glycosides and therefore theoretical calculations were required to find the corre ct orientations. The hydroxyl groups are distinct from the carbonyl group, in that they may be involved in extensive hydrogen bonded networks and are easily influenced by external perturbations, such as the rubidium cation. The ring conformation also affects the hydroxyls orientation and the ring flexing is more cumbersome to model. Aside from these drawbacks, the calculations for the rubidium cation-attached glycosides did seem to 146

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indicate that the major influences in O-H band shif ts are due to the spatial orientation particular to each isomer (or, glucose or galactose) and the resu lting effects on the strength of the hydrogen bonded network. Theoretical calculations also indicate that the rubidium cation location perturbs the O-H stretches a nd the hydrogen bonded network, thus making differentiation of all four glycoside isomers possible As with any project, future st udies need to be undertaken to gain deeper insight into the chemistry of the molecules being studied. For phenyalanine analogs, IRMPD spectra of deuterated and undeuterated proton-bound Phe anal ogs would provide structural information about the ions. The experime nts and calculations would help explain why gas phase H/D exchange at the acetamido group is observed for proton-bound AcPhe but not for the corresponding sodium cation-attached AcPhe. Ca lculated and IRMPD spectra of sodium cationattached diand tripeptides containing phenylalanine are needed to observe if lack of exchange at the amide group occurs for the larger species. For the N-acetylglycosamines, it would be usef ul to obtain IRMPD spectra in the range of 2600 3900 cm-1 using the OPO/FTICR-MS setup, in order to confirm the lowest energy calculated structures used as a compar ison to IRMPD spectra in the 800 1800 cm-1. IRMPD spectra of rubidium cation-bound GlcNac and GalNac s isomers would be useful to determine if the C=O stretch undergoes a similar shifting pattern observed for the Li+ attached glycosamines. This would be especially helpful for -GlcNac, whose lowest ener gy structure has lithium bound to the carbonyl group itself, and is probably a ffecting the shift. A gas phase H/D exchange experiment and IRMPD spectra are also needed for the deuterated GlcNac and GalNac ions. Although all four isomers of the D-glycosides can be differentiated by IRMPD, theoretical structures are needed for -D-glucoside and -D-glucoside encompassing all the IRMPD 147

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spectral features. Theoretical calculations of Dgalactosides are also needed and are underway to explore more of the conformational space than th e previous conformational searches. Molecular dynamic simulations that mimic the experimental conditions may prove useful, so that a more representative set of conformations can be obtained. Experiments similar to those of lithium cation-attached N-acetylglycosamines, but using lithium cation-attached glycosides, are needed to obtain fragmentation patterns as a function of irradi ation wavelengths to provide structural information. As a more extensive and long te rm project, the OPO/FT ICR-MS setup could be used to obtain IRMPD spectra for the other D-hexose s to test if differentiation can be established for the entire set of isomeric monosaccharid es. Theoretical calculations, H/D exchange experiments and IRMPD spectra of lithium-bound sp ecies have proven useful for all the work herein presented, and they would also be used in the analysis of the D-hexoses to obtain structural information. 148

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BIOGRAPHICAL SKETCH Born in Tijuana, Baja California, Mexico to Roberto G. Contreras and Maria Elia Contreras Sanchez, Cesar S. Contreras spent the first five years of ch ildhood living in Tijuana with his siblings: Roberto, Ma rtha, Diana, Maria, Elizabeth, Sandra and Daniel. Cesar and his family migrated to United States and settled in San Diego, California, which he called home for the next 23 years. Cesar graduated from Clai remont High with honors in Spanish language and literature and political science. After attending San Diego Mesa Community College, and earning an associate degree in liber al arts and sciences, Cesar br iefly attended CSU San Marcos, before enrolling at San Diego St ate University (SDSU). At SD SU he conducted research with Dr. Andrew L. Cooksy, and was chosen as a MA RC scholar, two events that would help fulfill his dreams of attending graduate school in Chemistr y. He obtained his Bachelor of Science from SDSU in 2002, and after a year off, decided to con tinue his studies at the University of Florida. Throughout his educational career, Ce sar held several jobs that c ontributed to his growth as a person and a scientist. Cesar was employed as a chemical research technician for Schumacher Industries, a division of Air Products and Chemic al, Inc. Cesar was also employed at the San Diego Research Laboratory, before heading to gra duate school at UF. 158