Electrochemical and mass spectrometric investigations of biological molecules

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Electrochemical and mass spectrometric investigations of biological molecules
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viii, 209 leaves : ill. ; 29 cm.
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English
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Thompson, Maurice Vincent, 1966-
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Subjects / Keywords:
Gas chromatography   ( lcsh )
Mass spectrometry   ( lcsh )
Electrochemical analysis   ( lcsh )
Bioactive compounds   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1994.
Bibliography:
Includes bibliographical references (leaves 203-208).
Statement of Responsibility:
by Maurice Vincent Thompson.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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Full Text










ELECTROCHEMICAL AND MASS SPECTROMETRIC INVESTIGATIONS
OF BIOLOGICAL MOLECULES


















By

MAURICE VINCENT THOMPSON


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































To the memory of my mother Sylvia


Thompson


"We have only a memory dear wife and mother to cherish our whole life through"














ACKNOWLEDGMENTS






Sincere thanks go to my parents for their support and guidance. Thanks to my father

for his encouragement and understanding throughout the years. Special thanks go to my

mother for all her love, she will be always be in my heart. Thanks go to my brothers Errol,

Courtney, Peter, Karl and my sister Allison for being there and never saying no. I would like

to thank Suzette Riley for her friendship, encouragement and support.

Thanks go to my research advisor Dr. Anna Brajter-Toth for all she has taught me

over the years. I would also like to thank Dr. John Toth for all his help and direction during


the completion of this work.


Thanks to the members of the


Toth group, especially


Marino, Lisa Spurlock, ChenChan Hsueh and Quan Cheng, for their assistance over the past

four years.


Thanks go to the


Patricia Roberts Harris Fellowship


Program for funding my


graduate studies.









TABLE OF CONTENTS


ACKNOWLEDGEMENTS.


ABSTRACT .

CHAPTER 1
INTRODUCTION


Mass Spectrometric Analysis of
Background to Amino I


Amino Acids


Lcid Chemistry and Function


Mass Spectrometry of Amino Acids .
Derivatives of Amino Acids for GC/MS Analysis
M itomycinC . . . . . .C
Background and Properties . .
Drug Activity . . . . .
Electrochemical Analysis . . .
Electrochemistry On-Line with Mass Spectrometry .
Insights into Biological Redox Reactions .
Thermospray Ionization. . . .


. 1


. .15
. .15
. .24


Electrospray Ionization


CHAPTER 2
STRUCTURE


AND ELECTRON CAPTURE NEGATIVE ION MASS SPECTRA OF


FLUORINATED DERIVATIVES OF 21 AMINO ACIDS FORMED BY ONE-STEP


ACYLATION/ESTERIFICATION REACTIONS


Introduction.


Amino Acids With Hydrocarbon Side Chains
Amino Acids with Alcoholic Side Chains
Amino Acids with Acidic Side Chains


..49


Amino Acids with Amide Side Chains
Amino Acids with Basic Side Chains.
Amino Acids with Sulfur Containing S
Experimental . . . .


449


4 .
* .


ide Chains


. .60


. . .60


Reagents and Materials


. .60


Derivatization
Instrumentation


Results and Discussion


Effect of Derivatizing Reagent on Derivatives


o nnrn t- 4... n irr r x xcx\


.65


.65


`hT~na~;, ta Inn r i\ am i ~ n 1 Tnt~;lnC:n n 'h An n n








Amino


Acids with Acidic Side Chains


Amino Acids
Amino Acids
Amino Acids


with Amide Side Chains
with Basic Side Chains.


with Sulfur Containing Side Chains


Conclusions.


CHAPTER 3


ELECTROCHEMISTRY OF MITOMYCIN C.


Introduction.


Previous Studies of the Electrochemistry of Mitomycin C .
Electrochemistry in Aqueous Solutions . . .
Electrochemistry of Mitomycin C in Non-Aqueous Solutions
Graphite Electrodes . . . . .
Experimental Section


Reagents and Materials


.103
.108


Electrode Preparation and Electrochemical Methods.
Fundamentals of the Electrochemical Methods.


Results


and Discussion


Electrochemistry ofMitomycin C in Aqueous Solutions at Graphite


Electrodes .


S118


Rough Pyrolitic Graphite (RPG) Electrodes
Glassy Carbon (GC) Electrodes ..


Electrochemistry of Mitomycin C in Methanol at Graphite Electrodes


. 134
.143


Conclusions.


CHAPTER 4
THE REDUCTIVE ACTIVATION OF MITOMYCIN C BY ELECTROCHEMISTRY


ON-LINE WITH MASS SPECTROMETRY .. .
Electrochemistry On-line with Mass Spectrometry
Experimental


. . . 145


147


Samples and Reagents


Mass Spectrometry . . . .
On-Line Electrochemistry . . . .
Thermospray Mass Spectrometry of Mitomycin C. .
Electrochemistry On -Line with Mass Spectrometry (EC/MS).
Mass Spectrometric Hydrodynamic Voltammetry
Formation ofMitomycin C Deoxyguanosine Adducts


.147
.148
.151


. 162


.162
.170


* rn_ nrtrnnbnn c,,i I on Elan T rr jcnn. r Tndar^nr nn









CHAPTER


DESIGN OF THIN LAYER


CELL FOR ON-LINE


ELECTROCHEMISTRY WITH MASS SPECTROMETRY


. 182


Introduction . . .
Thin Layer Cell . . .
Design of Electrochemical Cell for EC/MS


Fabrication of Working Electrodes. .
Off-Line Electrochemistry . .
On-Line Electrochemistry/Mass Spectrometry.
Conclusions . . .


.182
.183


CHAPTER 6


SUMMARY AND FUTURE WORK .


.199


BIBLIOGRAPHY


. 203


BIOGRAPHICAL SKETCH


. 209














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

ELECTROCHEMICAL AND MASS SPECTROMETRIC INVESTIGATIONS
OF BIOLOGICAL MOLECULES

By

Maurice Vincent Thompson


December 1994


Chairperson: Dr. Anna Brajter-Toth
Major Department: Chemistry

The chemical characterization of biological molecules is very important to gain


insights


functions


of biological


systems.


Over


decades


mass


spectrometry has


become one of the most widely used


tools in analytical


chemistry.


Electrochemistry has a long history in the characterization of redox reactions of many


biological molecules.


The purpose of this work was to use mass spectrometry combined with


chromatography


(GC/MS)


detection


protein


amino


acids,


electrochemistry


and mass


spectrometry to


investigate the reductive activation


of the


anticancer drug, mitomycin C.


work study the one-step derivatization of


amino acids


was achieved


using


+*rfl..nr ratana* anb.Arrr (TPA A\ onA ;+o l bntnailaaa aA ti-fluiinrnlthlnnl (TTPP' anA ;te








capture negative ionization MS (ECNI-MS).


The one step formation of derivatives has an


advantage over techniques previously used. Also, the potential for the formation of cyclic

derivatives is another benefit, as these derivatives could yield abundant molecular anions

under ECNI conditions providing sensitivity and selectivity for detection by selected ion

monitoring.

Electrochemistry on-line with mass spectrometry (EC/MS) has been shown to model

the in vivo enzymatic oxidation of purines. In this work EC/MS was used to reductively

activate mitomycin C by electrochemistry and to detect by on-line mass spectrometry the

adducts formed between the electrophillic intermediate of mitomycin C and the DNA base

guanosine.

An electrochemical cell based on a thin layer cell design was also developed, this cell

allows for the resurfacing and replacement of the electrode which is a major advantage over


the electrochemical cell used previously with on-line EC/MS.


Off-line electrochemical


investigations of mitomycin C in different solvents and at different graphite electrodes were

also performed. These experiments were important to ascertain the ideal parameters for the

on-line reduction and detection of mitomycin C












CHAPTER


INTRODUCTION


The electrochemical and


mass spectrometric (MS)


investigations of biological


molecules are described in this dissertation.


The chemical characterization of biological


molecules is very important to gain insights into the functions of biological systems. Mass

spectrometry combined with gas chromatography (GC/MS), used in the detection of protein

amino acids, following one-step derivatization of the amino acid with fluorinated reagents

will be described. The electrophilic derivatives formed are highly sensitive to detection by

electron capture negative ionization MS (ECNI-MS). The use of electrochemistry combined

on-line with a mass spectrometer via a thermospray interface (EC/TSP/MS) to investigate

the reductive activation of the anticancer drug mitomycin C (MC) is also described.

The development of an electrochemical cell for on-line EC/MS is also discussed. The

electrochemical cell is based on a thin layer design and allows for the resurfacing and

replacement of the working electrode. Off-line electrochemical investigations of MC will


also be described in this disseration.


These experiments were important to determine the


ideal parameters for on-line MC reduction and MS detection.

This introductory chapter describes the background of amino acid MS analysis and


gives some discussion of


previous studies of amino acids by GC/MS. This is followed by










thermospray and electrospray ionization processes.


Mass SDectrometric Analysis of


Background to


Amino Acids


Amino Acid Chemistry and Function


Amino acids are substances from which proteins are synthesized.' All proteins are

composed of 20 'standard' amino acids known as a-amino acids, where a primary amino


group and a carboxylic acid group are on the same carbon atom.2


A side chain gives the


amino acids their chemical identity. Many amino acids are not components of proteins.

Amino acids are also metabolites of biological energetic reactions, and many are essential


nutrients.


They function as chemical messengers in communication between cells, e.g.


glycine, y-amino butyric acid (GABA) and dopamine, as intermediates of various metabolic

processes, e.g. citrulline and omithine which are intermediates of urea metabolism. Over 250

amino acids have been isolated from plants and fungi. Many of these are toxic, suggesting


they have a


protective function in nature. Some, such as azaserine, are medically useful


antibiotics.


Amino


analysis


general


received


much


attention


because


of its


importance in biological science.


Profiling of free amino acids is of great importance and


has found broad clinical applications. In the 1950s amino acid analysis was used to diagnose


heredity


disorders of amino acids metabolism.3


Amino acid analysis is used in other


processes such as low and high protein dietetic production, the determination of a degree of


I I .. ... -. ...








3

Different separation techniques, based on the charge or hydrophobicity differences

in amino acids, are used for both qualitative and quantitative analysis of amino acids. The

most common approach in the analysis of amino acid content is the amino acid analyzer.4


Manual


hydrolysis


polypeptides


or proteins


is followed


pressure


liquid


chromatography (HPLC) and on-line post-column derivatization with color or florescent-

enhancing derivatizing reagents. Although acid hydrolysis can be slow, chromatographic

analysis is rapid and works well for protein amino acids. Unusual nonprotein amino acids

are often not easily identified. Sensitivity in HPLC based amino acid analysis is often a

problem. Other techniques such as gas chromatography (GC) and mass spectrometry (MS)

has been used to analyze amino acids and can achieve greater sensitivity.

Mass Spectrometry of Amino Acids


Mass spectrometry is a powerful technique that allows for unambiguous structural

verification and identification of many substances including amino acids5. Electron impact


(EI) mass spectrometry,


molecules,


where electron beam bombardment is used to ionize gas phase


was the first ionization technique used in mass spectrometry of amino acids.


Figure 1.1 schematically illustrates electron impact ionization (EI), where ionization of the

gas phase sample molecule (M) is induced by collision with a high energy electron beam (ca.


70 eV).


Removal of an electron from the sample molecule, M, produces a radical molecular


ion (M').


Due to


the high energy involved in this process the radical molecular ion


































Figure 1.1:


Illustration of Electron Impact Ionization
A) Schematic Representation of the Ion Source.
B) Gas Phase Reaction Scheme.


















ELECTRON IMPACT(El)


M~g)


10-7torr


M*(g)


M (g,10-7torr),ow
ENERGY


HIGH
ENERGY
(70ev)


> M*(g) nG
ENERGY


2e LOW
ENERGY


(molecular)
ion


M1(g)


M3(g)


LA


~---


+ e


V
u -e










and amino acid derivatives.


The development of GC/MS allowed for the separation and


detection of amino acids in complex matrices which previously required tedious, time

consuming and difficult sample clean-up.

The high energy transfer encountered during El conditions produces an abundance


of fragmentation and ion rearrangement.


This fragmentation and rearrangement makes


molecular weight determination difficult, as molecular ion peaks are almost always absent

or at very low intensity in EI-MS. Molecular ions can be obtained using different ionization

methods. Chemical ionization (CI), field ionization (FI) and field desorption (FD) allow for


the molecular weight determination due to


the abundance of molecular ions.3 This is


important in the identification of the unknown amino acids and the analysis of amino acid

mixtures. In contrast to El these ionization techniques give little or no structural information

due to the lack of fragmentation.

Chemical ionization (CI) was developed to ionize molecules with a much reduced

energy transfer, as compared to EI, thus giving stable molecular ions.6 In this method a

reagent gas is ionized at an ion source pressure of about 0.1 kPa and the reactive ionized

plasma is used to ionize sample molecules by ion molecule reactions. Figure 1.2 illustrates


the ionization sequence involved in CI with isobutane as the reagent gas.


Isobutane is


ionized giving the principal El product. This product reacts with sample molecules (M) by


proton transfer producing protonated molecular ions (M+H')


which are called quasi-



















































CO



C)
bO



V1

C)

Ct

a)

-c
0
C,)
-4
4J

to
Cl




C)

0
4J
N


0

C)





C)

* *












C)
~oc


CO
I:
SO


Co~
1, I
o t oo


1L
Cu
zr

I -


C)
C)
C)
C)


6

aSa
IIIC
lila,
0-0-0 C-'


CO CO
1:+ I
0-o- O


- -+
O- O-- o










CI-MS


as El


can be done directly in


the mass spectrometer ion source or in


combination with gas chromatography (GC). Free protein amino acids and their methyl

esterand N-acetyl derivatives have been analyzed using both direct introduction and GC.

Field desorption (FD) MS of free protein amino acids was first performed by Winkler and


Bleakey.


FD-MS analysis of amino acids has its limitations because it cannot be combined


with a separation technique such as GC.

Amino acids are not stable at elevated temperatures and thus require conversion to

volatile, thermally stable derivatives before they can be analyzed by GC. Amino acid


derivatization


generally


involves esterification


carboxyl


groups


with


alcohols


acylation of amino groups with halogenated anhydrides.5 The most common amino acid


derivatives


used


analysis


are tert-butyl


dimethylsilyl


(TBDMS)


trifluoroacetyl butyl ester (BTFA). All protein amino acids can be analyzed by GC after

derivatization with these compounds.8

Gas chromatography combined with mass spectrometry offers the possibility of the

unequivocal identification of amino acids even at nanomolar quantities.5 Unequivocal and

reliable identification of known and unknown amino acids even in complex mixtures can be


accomplished


use of


mass spectrometry


Selectivity


and sensitivity


mass


spectrometry allows for accurate mass measurement with the aid of selected ion monitoring

(SIM).








10

molecular ion, instead of measuring several hundred ions in a complete spectrum, the limit

of detection is increased by a factor up to 100 or possibly even more.

SIM reduces chemical noise and can be used for trace analysis. Chemical ionization

(CI) mass spectrometry is ideally suited for SIM analysis, because of the soft ionization with

little or no fragmentation which allows the molecular ions to be easily detected 4.

Derivatives of Amino Acids for GC/MS Analysis


Amino acids are non-volatile and must first be derivatized to volatile compounds for

GC/MS analysis. The carboxylic acid group is converted to an ester and the amine group is

acylated to an anhydride. N-perfluoroacyl alkyl esters (TAB) and trimethylsilyl (TMS)


derivatives are the most popular for the quantitative analysis of amino acids.


TMS derivatives have been formed from various types of amino acids.


All amino


acids except arginine have been quantitatively derivatized for GC analysis, sometimes


drastic reaction conditions."0 However, the derivatives are very easily hydrolyzed and require

well-preheated GC columns to avoid loss of the TMS groups. Therefore, they are considered

less suitable for GC/MS analysis than the TAB amino acid derivatives.

Gehre and coworkers used trifluoroacetyl (TFA) n-butyl esters (TAB amino acids)

for quantitative analysis by GC." This was extended to GC/MS analysis by Duffield and

coworkers.12 The two-step synthesis involves the esterification of the amino acid's carboxyl


groups with n-butanol.


The amino and hydroxyl groups of the resulting ester are acylated


S -








11

amino acids.8 TAB analysis was also extended to the detection of bacterial amino acids in

environmental samples, body fluids and tissues.4 Another clinical chemistry application of

TAB amino acids and GC/MS analysis was the detection of the amino acids composition of

streptococcal peptidoglycan-polysaccharide complexes.4 The amino acids were analyzed as

the butyl hepta fluorobutyl derivatives with selected ion monitoring (SIM) which enabled

the amino acids to be analyzed without interference from background noise. Amino acids in

brain and plasma samples were analyzed by GC/MS with pentafluoropropionic anhydride

and hexafluoroisopropanol derivatives.5

Trifluoroacetic anhydride (TFAA), in addition to its use as an acylating reagent for

alcohols and amines, also promotes rapid esterification of organic and inorganic oxyacids,

such as amino acids, by alcohol via a three-step pathway.13,14 This catalytic property has been


exploited


development


a one-step


acylation/esterification


procedure


polyfunctional molecules. The anhydride serves both to acylate the amino and the hydroxyl

groups and to catalyze the esterification of the oxyacid groups by an alcohol. This single-step

acylation/esterification procedure has been applied to amino acids. Para-aminobenzoyl


glutamic acid (pABG), an end product of the metabolism of folic acid in humans,


was


analyzed


GC/MS


using


one-step


acylation/eterification


procedure."5


Also


pentafluoropropanoic anhydride (PFPA)/pentafluoropropanol (PFP) mixtures were used to

derivatize ten gamma-glutamyl amino acids for GC analysis.16











































































z
U






Ca

C)

a)




C-)
0
S.-
4.
C-
C)


*
t-n





















+



+

+



O


C)
I cu
U,


+


-0ir








14

source of the mass spectrometer by resonance electron capture. Figure 1.3 illustrates how this

can be achieved in a CI ion source where the reagent gas removes the excess energy from the


high energy electron beam, producing low energy electrons (near thermal energy)


captured by the sample molecule (M).


that are


The introduction of electrophilic groups such


fluorine to amino acids enhances their electron affinity and produces derivatives that exploit


high


sensitivity


and selectivity


of the electron capture negative ion (ECNI) mass


spectrometry. For successful utilization of ECNI mass spectrometry the target molecule must

have a large cross-section for electron capture.18'19 If the analyte has an electronegative center


Cl, F, Br2, or NO2


the sensitivity of ECNI-MS is enhanced at least 100 fold versus


electron


impact


ionization.


If the


analyte


no electronegative


center,


there


enhancement in sensitivity versus electron impact ionization.


ENCI-MS


been successfully


used


for the


analysis


trace


metals


atmosphere, the analysis of polychlorobenzodioxins and related compounds in biological

samples, and the analysis of drugs and drug metabolites in biological samples.20 Low and

Duffield have applied ECNI-MS to the analysis of amino acids. Carboxy-n-butyl ester N-

(O,S)-pentafluoropropionate derivatives of 29 amino acids were analyzed and their mass

spectra determined. Positional isomers within the same class gave different NICI mass


spectra,


while the positive ion (PI) mass spectra could not distinguish these isomers.


Increased sensitivity was also observed for ECNI over PI, with derivatized leucine being








15

Mitomycin C

Background and Properties

Mitomycins, a group of antitumor antibiotics were isolated in 1956 by Hata and


coworkers.22


They


were


extracted


purified


chromatography


from


fermentation broth filtrate ofstreptomyces caespitosus. Mitomycin C (MC) was isolated in

1958 by Wakaki and co-workers.23 Mitomycin C is an antineoplastic agent and has been used

in the treatment of several types of cancers including superficial cancer of the urinary bladder

and combinations of lung, breast and stomach cancers.24

The molecular formula of mitomycin C is C15H,1N405 and the molecular weight is

334.13. The structure of MC is shown in Figure 1.4. The IUPAC name of mitomycin C is

[la, R]-6-amino-8[[(aminocarbonyl)oxy]methyl]- 1, la, 2, 8, 8a, 8b-hexahydro-8a-methoxy-


5-methyl azirino [2, 3',


4] pyrrolo [1.2-9] indole-4-7


dione. Mitomycin C is marketed


under the trade names Mutamycin, Mitomycin-C Kyowa and Ametycine.23 It is an odorless


blue-violet


crystalline powder.


Drug Activity

MC anticancer activity is expressed by its ability to alkylate DNA with cross-linked

covalent bonds. To exhibit this intracellular cancer fighting ability, reduction of the quinone

moiety of MC by enzymes is essential.25 The major bioreductive catalyst for MC activation


is cytochrome P-450 reductase, a membrane bound flavoenzyme.


Chemical reducing agents

































Figure 1.4:


Structure of Mitomycin C
















H2N



H3C


O

'oCH2OCNH2

SOCH3
/1

























































0




Le



C'





lI.











C-)

S:








-d
C)
cn
0
0
I-



*























O-


I=


Z 0
I I


z 0
I I


(N
I
z
'-A -


0=


42

(9


z o
I f


Z 0
I I


I
r O


z 0
f 2


n


I \










described by Iyer


Szybalski29 then later by Moore.30


According to the mechanism


proposed by Moore, MC


is reduced in vivo to the hydroquinone 2, followed by the


elimination of methanol forming 3, and the opening of the sterically strained three-membered

aziridine ring forming 4. This gives the biologically active form of MC (4) which can react


with DNA.


DNA, acting as a nucleophile, can attack at the C-1 active site forming 5. The


C-10 position is also a possible site for the nucleophilic attack, and a crosslinked DNA

adduct 7 can be formed. The MC-DNA monoadduct 5 formation occurs on the exocyclic N-2


position of the deoxyguanosine base of the DNA and the C-l


crosslinked adduct


position of the MC.


7 has an additional deoxyguanosine from the same DNA molecule


attached at the MC C-10 position. The interaction of MC with DNA produces


more than


90% of the monofunctional adduct.24

Both the monofunctional and crosslinked adduct of MC and the DNA-base guanosine

have been isolated and their chemical structures reported. The major monofunctional adduct


was isolated from a MC-DNA complex and characterized by


Tomasz and co-workers25 as


p ,7"-diaminomitosen- 1"


a-yl)-2'-deoxyguanosine


(Figure


1.6a).


crosslinked


(bifunctional) adduct (Figure 1.6b) of MC with the DNA was also isolated and characterized


Tomasz and coworkers25 following the in vitro reductive activation of MC. MC was


activated by the chemical reducing agents hydrogen/platinum oxide (H2/PtO2) and sodium


dithionite (Na2S204) and mixed with DNA and poly(dG-dC) complexes.


The reductive


N2-(2"

































Figure 1.6:


Structure ofMitomycin C-DNA Adducts


a) Monoadduct


Bifunctional


Adduct.


















'qCOCNH2










bifunctional


adduct


was


characterized


Tomasz


coworkers


spectroscopic methods that included UV analysis, proton magnetic resonance, FTIR, and

circular dichroism.25 The bifuctional adduct was also isolated from the liver of rats that had

been injected with MC. HPLC and UV analysis was used for the identification of the isolated


adducts.


The identification of these adducts is a direct confirmation of the bifunctional


alkylation of DNA by MC and supports the Iyer-Szybalski and the Moore mechanisms.

Mass spectrometric methods have also been used to characterize the MC-DNA

adducts. Musser, Pan and Callery used HPLC/thermospray MS to analyze DNA adducts

formed from MC and calf thymus DNA.31 MC was reductively activated by hydrogen gas

with PtO2 catalyst and mixed with calf thymus DNA in tris buffer of pH 7.4. DNA was


precipitated


digested


nucleotides


mixture


was


analyzed


thermospray/HPLC-MS.


The adducts formed from the reaction of MC with DNA


were


separated from unmodified nucleosides by HPLC on a C18 column. The mobile phase used

for analysis was 0.1 M ammonium acetate/5% methanol with a 30 minute linear gradient to

0.1 M ammonium acetate/50% methanol with a flow rate of 1.2 ml/min. The thermospray


interface


temperatures


were optimized at tip


temperature of 210-220C


with a block


temperature of 2950C. The principal ions in TSP-MS of the MC-deoxyguanosine adduct,

MW= 569, were the (M+H)* molecular ion at 570 daltons and the ion at 244 daltons which

was proposed to be a protonated MC fragment.










bifunctional adduct was seen at 986 daltons. Fragments at 635, 636, 637


, 586, 435, 376, 284


and 242 were identified and the fragmentation pathway was established by MS-MS studies

of the monoadduct.

Electrochemical Analysis


The electrochemical reduction of MC at a hanging mercury drop electrode in buffered

aqueous solutions has been previously investigated.32 In the cyclic voltammetric data two

successive electrochemical reduction waves were observed at -368 and -468 mV versus SCE

respectively. They were attributed to the two-electron, two-proton quinone-hydroquinone

reduction of MC and it's ring-opened moiety 1-hydroxy-2,7-diaminomitosene (Figure 1.7).

It was proposed that the aziridine ring-opening occurred from the hydroquinone in aqueous

media, and the quinone radical intermediate was not detected as is generally the case in

aqueous media.

The electrochemical reduction of quinones in polar aprotic media such as DMF and

Me2SO is known to involve the initial formation of an anion radical prior to the formation

of the semiquinone and the hydroquinone species.33 The lipid bilayers of cellular membranes

are heterogenous, consisting of both hydrophilic and hydrophobic regions. The reduction of

MC may conceivably take place in the lipid matrix where the radical anion may have a

considerable lifetime. The aprotic environment favors mechanistic pathways different from


those in protic environments. So, the study of


reduction


in DMF and Me2SO is




































Figure 1.7.


Structure


Hydroxy


Substituted


Moiety


1-hydroxy-2,7-


diaminomitosene






26







O
o II
CH20CNH2
H2N

SOH


NH,












































en
en
rdz

0:



C)

S
0l
4-
*

0



1C.

-d




db)
4-.
3-
0
(4
Si
U,
-c
C.
4)



-e
C.


U,































I
=8




0-

'-


0= -


0=


z 0,
(' (r)
I I


z C.
(N C


z o
I I


N 0,
I


0ttz
(YOI
I


(N -4
IC


I:


z O
N CO
I I


I

O =8

I
Oi


z o
r O
N
I X


O zI
0 -


z o
N C)
XX


z 0,
(N w
I I


N


I


0


O --


O -








29

and Me2SO solutions. However, upon addition of H20 after the formation of 9, a complex

mixture of products was formed. The addition of water creates a polar environment which

favors the loss of methanol forming 10 and the subsequent opening of the three-membered


aziridine ring forming 11A.


The attack of the nucleophile OH


at C-l


leads to


15 and


subsequent reoxidation of 15 leads to 16 (pathway "A"). An alternative pathway (pathway

"B") was also proposed. The intermediate 11B generates a carbon centered free radical at C-l

instead of undergoing nucleophilic attack at C-l position. Hydrogen abstraction from H20


then takes place and 12 is formed.


Pathways A and B are competitive reactions with


pathway "A" favored at basic pH due to the increasing availability of OH- to attack C-l.

Products 16 and 12 were isolated and identified and radical formation proposed from the

formation of these products.


It was suggested that the one-electron reduction was sufficient to activate MC


reactive intermediates and was the primary mode of the bioreductive activation.


This is


because the radical anion generated products following the water hydrolysis reactions closely


resembling


the profile of products from the reduction with cytochrome P-450 reductase.


The electrochemical reduction of MC by cyclic voltammetry at mercury and platinum

electrodes was investigated by Kohn and co-workers29 to determine probable reduction

pathways in the protic non-aqueous solvent, methanol, and in buffered aqueous solutions.


Figure


1.9 shows the proposed mechanism for the reduction of MC in methanol. It was












































00




C)




C
*





IC)
S
0
*



'4-


C)





C)

C.)





C)
0)









a,


























z 0
I I


z 0
I X


z 0
I I


I: I


I








32

the addition of the nucleophile OCH3 to the C-1 position of MC forming 15. Oxidation of

15 leads to the formation of the quinone 16.

Electrochemistry On-Line with Mass Spectrometry

Insights into Biological Redox Reactions

In the past two decades research into biological systems and how they work has

increased dramatically. One type of biological reactions that is of interest to electrochemists

is redox biological reactions. Reactions at the electrode-solution interface in electrochemical

experiments can be used to model enzymatic redox reactions that take place in biological


systems.


The electrochemical reactions can as a result provide useful information about


biological reaction pathways. Enzymes such as peroxidase,34


cytochrome


P-45035 and


xanthine


oxidase36


products


are chemically


similar


to those


obtained


electrochemical reactions. The pathways of the enzymatic and the electrochemical reactions

are also similar. Insights into the enzymatic reactions are most successfully obtained when

electrochemical analysis is used in combination with other analytical methods such as HPLC,

molecular spectroscopy and mass spectrometry. Kissenger and coworkers used HPLC on-line


with


electrochemical


obtain


insights


redox


reactions.7


chromatography/mass


spectrometry


(GC/MS)


been


used


analysis


electrochemical products of biologically important reactions.38 These products are mainly

polar, non-volatile compounds that require time consuming, often tedious, derivatization








33

dried and derivatized for GC/MS analysis.

The development of liquid interfaces for mass spectrometry such as thermospray39

and more recently electrospray40 has made it possible for the use of MS for on-line detection

of non-volatile, thermally labile biological molecules. The ability to link the solution directly

to the mass spectrometer source has proven to be very useful in the analysis of thermally

labile, non-volatile compounds such as nucleotides and nucleosides.41 With the use of liquid

interfaces derivatization or extensive sample work-up is not required making LC/MS an


excellent


method


rapid


identification


of biologically


formed


adducts.


thermospray interface has allowed for the direct coupling of electrochemistry with MS.


on-line combination of MS and electrochemistry (EC/MS) offers the capability to directly

monitor reactants, short-lived intermediates and products of electrochemical reactions as a

function of electrode potential.42 The combination of electrochemistry with MS provides a

direct, sensitive and highly selective method for detecting individual components in complex

biological redox reactions.


Thermospray Ionization


Thermospray (TSP) was developed


by Vestal and Blakeley39 from efforts to make


a practical liquid chromatography-mass spectrometry (LC/MS) interface that would allow

for the analysis of non-volatile, thermally labile samples in solution. Thermospray has since

then become the most widely recognized technique for interfacing LC/MS and is routinely



































Figure 1.10:


Diagram of Thermospray Interface.






















ION SAMPLING
CONE


IONS


VAPOR
TEMPERATURE


FLANGE


ELECTRON


BEAM


HEATER


VAPORIZER
PROBE


VAPRIZER
COUPLING


MOUNTING POST


' FLANGE


BLOCK
TEMPERATURE


TO
TRAP
MECHANICAL PUMP


PROBE
HANDLE


VAPORIZER
CCNTRCU.ER








36

The principle of thermospray involves the vaporization and ionization of the analyte

solution by passing it through a heated metal capillary at a flow rate of approximately 1-2

mL/min. The mechanism of the vaporization process relies on the transfer of heat to nearly

complete the vaporization of the liquid. The vaporization process occurs in the source of the


mass spectrometer that is under vacuum.


The effluent from the capillary vaporizer is a


supersonic vapor jet consisting of a superheated mist of fine particles and solvent droplets.


Introduction of the vapor into the


MS source


is critical to


the optimum


thermospray


performance


because


ionization


sample


occurs


in the


vapor phase.


thermospray performance occurs when there is a maximum ionization and this occurs when

the vaporization is ca. 95% complete within the capillary tube. It is vital that the heat input

is properly controlled so that premature vaporization does not occur deteriorating MS

sesitivity.

In the thermospray process sample ions can be produced by gas-phase reactions

which resemble chemical ionization (CI). Ammonium acetate (NH4CH3CO2) is a typical

ionic solute used as a reagent gas in thermospray analysis of aqueous solutions. NH4CH3CO2


will ionize in the gas phase to NH44


and CH3COO-. A neutral analyte molecule M, in the


aqueous NH4CH3CO2 mobile phase, can form positive ions by the acid-base gas-phase

reaction with the ammonium ion:


NH4'


[M+H])








37

M has a lower proton affinity than the ammonium ion.

Negative ions can be formed from the TSP reactions of the analyte molecule, M, and

the acetate ion. Proton transfer (3) and adduct formation (4) reactions can occur:


+ CH3COO-


--> >


[M-H]-


+ CH3COOH


+ CHsCOO-


--> >


[M+CH3COO]-


Hambitizer and Heitbaum were the first to successfully use thermospray to interface


an electrochemical


on-line with a mass spectrometer.43


these experiments the


electroxidation ofN,N-dimethylanaline (DMA) at a Pt electrode was detected on-line by MS.

The oxidation products, dimers and trimers of DMA, were detected with a time resolution


ca. 10 seconds between the oxidation in the electrochemical cell and the detection by the


mass spectrometer.

The delay time between generation and detection and the dead volume of the transfer

lines are important parameters in EC/MS. If they are too large, the time resolution of MS

detection will be degraded and the probability of detecting short-lived intermediates will be


greatly decreased.


Volk and co-workers used EC/MS with a thermospray interface to model


the enzymatic redox reactions of purines.44 With the instrumental system used in these


experiments


time


resolution


was


improved


ca. 500


ms, therefore,


short-lived


intermediates could be detected. The oxidation of uric acid, which was one of the reactions










Volk et al. showing the power of EC/MS in giving insights into biologically complex redox

reactions.44

The fact that heat is applied to the eluent in the thermospray interface during


vaporization


ionization


leads


to several


limitations


of EC/MS.


temperature


conditions during TSP vaporization change in less than one second from ca. 25C to greater

than 200C. The elevated temperatures in the TSP interface can affect the reaction rates of

intermediates so the reaction pathways are not directly comparable to the reactions at room


temperature because the reaction kinetics can be significantly altered.


Thermally labile


intermediates and products may also be affected by the elevated temperature of the interface.

These compounds can be degraded complicating the interpretation of mass spectral data.

Electrospray Ionization

The evolution of electrospray (ESP) as a viable liquid interface for MS provides a


new


technique for interfacing EC/MS. The ESP interface heat requirement of ca. 700C is


much lower than that of TSP making ESP more useful for detection of thermally unstable


compounds.


Vaporization and ionization are accomplished through application of a large


potential, typically


kV. Therefore, the reaction kinetics of the solution analytes should


become comparable to the room temperature kinetics, and thermally labile compounds

degradation should be slowed down. Another advantage of ESP is its extended mass range


of greater than


100,000 daltons. Electrospray-MS has been used to determine accurate










Figure 1.11 shows a diagram of the electrospray interface. The electrospray process


requires two steps:


dispersal


of highly


charged droplets at near atmospheric pressure


followed by droplet evaporation. Electrospray is produced by the application of a high

electric field to a small flow of liquid (typically 1-1 OL/min) in a capillary tube. The electric

field results in charge accumulation on the liquid surface resulting in the formation of highly

charged liquid droplets. After desolvation of these droplets, the ions formed are sampled by

the mass spectrometer through a small orifice.46

The lower heat requirement and the extended mass range of electrospray makes it a

viable interface for EC/MS, as thermally unstable reactants, intermediate and products of


electrochemical reactions should be more easily monitored.


With the capability of the


extended mass range, complex redox reactions of large biological molecules such as proteins

and DNA fragments may be investigated by EC/MS.

































Figure 1.11:


Diagram of Electrospray Interface.40


















ELECTROSTATIC
LENSES


CYLINDRICAL
ELECTRODE


r=i=i


UQUID
SAMPLE


NEEDLE


DR 'rN


CAPILLARY


SKIMMER


1st PUMPING


STAGE


QUADRUPLE
MASS SPECTROMETER


2nd PUMPING
STAGE














CHAPTER


STRUCTURES AND ELECTRON CAPTURE NEGATIVE ION MASS SPECTRA OF
FLUORINATED DERIVATIVES OF 21 AMINO ACIDS FORMED BY ONE-STEP
ACYLATION/ESTERIFICATION REACTIONS

Introduction


Amino acid analysis is important in biological science with the profiling of free


amino


acids


finding


broad


clinical applications especially in


disease


diagnosis.


Chromatography/Mass Spectrometry (GC/MS) analysis of amino acids has generated a lot

of interest because of the analytical power of MS.6 Mass spectrometry has the potential to

provide definitive qualitative and quantitative information about many organic molecules.3


combination


provides


the sensitivity


selectivity


needed for the


separation and detection of amino acids in complex matrices, such as bodily fluids, with

limited sample clean-up.4 HPLC is the most popular technique for analyzing amino acids.

Unusual non- protein amino acids are often difficult to identify by HPLC as sensitivity is

often a problem.


As has been described in Chapter


trimethylsilyl (TMS) derivatives and the N-


perfluoroacyl alkyl esters (TAB amino acids) are the most important derivatives that allow


qualitative and quantitative analysis of amino acids.


5 However the ease of hydrolysis of TMS


groups makes them much less suitable for GC/MS analysis than the


TAB amino acids.


TAB arnino acids.








43

carboxyl groups are esterified with n-butanol and the amino and hydroxyl groups of the

resulting ester are acylated with trifluoroacetic anhydride (TFAA) producing N-TFA n-butyl

esters.5

The high sensitivity and selectivity of electron capture negative ion (ECNI) mass

spectrometry can be exploited for amino acid analysis with the introduction of electrophilic

groups as in N-TFA n-butyl esters. For ECNI MS analysis the analyte must have a positive

electron affinity and a large cross section for electron capture. These conditions are met in


N-TFA n-butyl


esters because of the presence of the


fluorine atoms


which are very


electronegative.19


It is known that trifluoroacetic anhydride


TFAA and its homologs promote rapid


esterification of organic and inorganic oxyacids by alcohols via a three-step pathway 13,14

(Figure 2.1). The component responsible for this rapid esterification is the unsymmetrical

anhydride (reaction 1), which dissociates to form the acylium cation and the trifluroacetate

ion (reaction 2). Even though the concentration of the acylium cation is small its reactivity

is sufficiently fast so that rapid esterification from the nucleophilic attack by the alcohol can

still occur (reaction 3).


This


catalytic


property


TFAA,


maybe


used


simultaneous


acylation/esterification of polyfunctional compounds.


acylate the compounds,


TFAA or its higher homologs can


while esterification is by the reaction with an alcohol such

























































*<

h
-D
ct

-d
*
C.





C-



'C
c.)
*
I-
C.)
Ct





tCu

$1-





Cu






















0


u

+

OU



O U

A


en


0


0


OO0
0-O
,,,,,6


O ro
(^










derivatives that are highly sensitive to detection by ECNI-MS.


These reactions have been


exploited to produce analytically useful derivatives of several polyfunctional molecules.47'4815

In this study, the applicability of simultaneous derivatization with TFAA and TFE


for the GC/MS analysis of amino acids using ECNI detection is explored.


The one step


formation of TFAA/TFE derivatives is an advantage over other techniques previously used,


where


acylation and esterification


was carried


out in two separate


steps.


Single-step


acylation/esterification was used to obtain the amino acid derivative, para-aminobenzoyl

glutamic acid (pABG), an end product of the metabolism of folic acids in humans.'5 In this


study


TFAA promotes the esterification reaction of the alpha carboxyl group by


TFE and


an internal


cyclization


reaction.


This


stabilizes


the derivative's


molecular anion


producing a single ion for ECNI detection. The cyclic derivative provides a sensitive and

selective ion that may be detected by selected ion monitoring (SIM). This, along with the


time


material


conserved,


major


advantages


single-step


acylation/esterification derivatization.

In this chapter the results of simultaneous acylation and esterification of the 20

'standard' amino acids, and cystine, using three combinations of fluorinated reagents will be


reported and discussed. The reagent combinations used are: 1) TFAA/TFE,


2) TFAA/PFP,


3) PFPA/ TFE. Structures of the reagents are shown in Figure 2.2.

In the following description the 20 standard amino acids and cystine are classified

































Figure 2.2


Flourinated Reagents





















CF3-C-O--C--CF3


TRIFLUOROACETIC ANHYDRIDE (TFE)


CF3CH2--OH


TRIFLUROETHANOL (TFE)


0
II
CF3CF2- C


0
II
-O-C-CCF2CF3


CF3CF2CH2-OH


PENTAFLUOROPROPIONIC ANHYDRIDE (PFP)


PENTAFLUOROPROPANOL (PFP)










Amino Acids With Hydrocarbon Side Chains


The structures of the six amino acids with hydrocarbon side chains are shown in

Figures 2.3a and 2.3b. Glycine, alanine, valine, leucine and isoleucine have aliphatic side

chains ranging from an H atom in glycine to an iso-butyl group for leucine and isoleucine.

Phenylalanine is aromatic and has a phenyl moiety. Proline (Figure 2.3b) which has a

secondary amine and is actually a cyclic a-imino acid is commonly referred to as an amino

acid and can also be classified in this group. The side chains in this group of amino acids are

expected to be unreactive during the derivatization procedure, therefore, only acylation of

the amine group and esterification of the acid is expected.

Amino Acids with Alcoholic Side Chains


Three amino acids serine, threonine and tyrosine have side chains with alcoholic

groups. Their structures are shown in Figure 2.4. Serine and threonine have hydroxyl side


chains of different sizes while tyrosine has a phenolic side chain.


The hydroxy groups are


expected to undergo acylation by the anhydride during derivatization.

Amino Acids with Acidic Side Chains


The structures of the two acidic amino acids, aspartic acid and glutamic acid are


shown in Figure


They are both dicarboxylic acids which makes ring closure possible


during derivatization.

Amino Acids with Amide Side Chains




















































Ui

C




Vt
Co
C

-o



-d
z

-c
-J

U,





C


0


4-h



Co

Cu


rn
a




















rCI-
U-U U--=
tdI
z


O
O
u

0
I
a? -
S-u-fI
z
C.


O
0
0
O





O
z I
U~ -
u




O





X --o -
a

0
0
o

I
z
a~-I-
z


w-0-\^
-UC-


m
mU c-
U --U--U -
1 1
u-o-u-


I
I


a
o
o
0
0
C-)
S


































Figure 2.3 b


Structure of


a Cyclic Imino Acid, Proline
























UN


COOH


Proline
(pro)




























Figure 2.4


Structures of Amino Acids with Alcoholic Side Chains.












CH3
I
HC-OH


-OH


H2N C- COOH

H


H2N C- COOH

H


Threonine


Serine


(ser)


(thr)


OH


H2N C- COOH

H


Tyrosine

































Figure 2.5


Structures of Amino Acids with Acid


Side Chains.
















O
II
C- NH2


CH2

H2N- C- COOH

H


- NI'2


CH2
CH2

H2N-C-COOH

H


Aspartic Acid


Glutamic Acid


(asp)


(glu)

































Figure


Structures of Amino Acids with Amide Side Chains














O
II
C-NH2
CH2

H2N-C-COOH

H


Asparagine
(asn)


-NH2


CH2
CH2

H2N- C COOH

H


Glutamine
(gin)










Amino Acids with Basic Side Chains


Figure 2.7 shows the structure of the four amino acids which are classified as having


basic


side chains:


lysine,


which has


butylammonium


group;


argmnine,


which


bears a


guanidino group; histidine, which carries an imidazolium side chain and tryptophan, which


has an indole group as its side chain.


The basic side chains may undergo acylation by the


anhydride at the amine group during derivatization.

Amino Acids with Sulfur Containing Side Chains

Cysteine and methionine are amino acids with sulfur containing side chains (Figure

2.8). Cysteine has a thiol group and is unique among the standard' amino acids because it

can form a disulfide bind with another cysteine to make the dimer, cystine. Methionine has

a thiol ether side chain. The sulfur containing side chain should be acylated at the thiol group

during derivatization.


ExDerimental


Reagents and Materials


Amino


standards


were


obtained


from


Sigma


Chemicals.


Trifluoroacetic


anhydride (TFE),


pentafluoropropionic


anhydride (PFPA),


trifluoroethanol


(TFE) and


pentafluoropropanol (PFP) were obtained from PCR Chemicals. Other reagents and solvents

were obtained from Fisher Scientific. Ethyl acetate and the fluorinated alcohols were stored

over molecular sieves. All other materials were used as received.

































Figure 2.7


Structures of Amino Acids with Basic Side Chains.












NH2
CH2
I
CH2
CH2
CH2

H2N- -COOH

H


NH2
C= NH
I
NH
CH2
I
CH2
CH2

H2N- -COOH


Lysine
(lys)


Arginine
(arg)


N


CH2

H2N -COOH

H


CH2

H2N-C -COOH

H


Tryptophan


Histidine


(trp)


fl\c~n\



































Figure 2.8


Structure of Amino Acids with Sulfur Containing Side Chains.

















SH
CH2

H2N- C COOH

H


CH3
S

CH2
CH2

H2N- C -COOH


Cysteine
(cys)


Methionine


(met)










Derivatization


Derivatives were prepared by evaporating 50 jiL aliquots of amino acid solutions to

dryness under a stream of dry nitrogen in glass culture tubes, then adding 200 ,/L TFAA and


00 uL


TFE, tightly capping the tubes, and heating in a metal block for one hour at 80-


100C.


After


1 hour, excess reagents were evaporated under dry nitrogen, and the dry


residue was dissolved in 100 pL dry ethyl acetate.

Instrumentation


GC/MS data were obtained using a Finnigan Model 4500 GC/MS system (Finnigan


MAT, San Jose, CA, USA). Typically, two pL injections representing 2-8 nanomole of the

derivative, were made into a 30 m x 0.25 mm DB-17 capillary GC column (J & W Scientific,


Folsom,


USA)


interfaced


directly


to the


mass


spectrometer


temperature-


programmed from 50"C to 300C at


12C/min.


Chemical ionization (CI) and electron


capture negative ion (ECNI) spectra were obtained using methane as the reagent gas at a

pressure of ca. 0.4 Torr.

Derivative structures were inferred from molecular weights obtained from CI data

and from observed shifts in molecular weight when PFPA was substituted for TFAA and

PFP substituted for TFE. Structures of negative ions in ECNI spectra were elucidated in the

same manner.


Results and Discussion










combination of reagents. However, there were differences in


the ENCI


fragmentation


mechanisms depending on which of the three anhydrides were used. For about half of the

TFAA/TFE and TFAA/PFP derivatives formed, the base peak in the ECNI spectrum is the

[CF3CONH]J ion, which is unrelated to the structure of the parent amino acid and therefore

provides no analytically useful information. The analogous PFPA/TFE derivatives produce

an analytically more useful [M-H] ion as the base peak. Only the more analytically useful

PFPA/TFE derivatives are reported and discussed here. The choice of alcohol had no effect

on the ECNI mass spectra.

Negative Ion Chemical Ionization Mass Spectrometry (NICI-MS)

Structures of Derivatives.


Derivatives with Hydrocarbon


Side Chains


amino


acids


hydrocarbon


chains:


alanine,


valine,


leucine,


isoleucine, glycine and phenylalanine have no reactive functional groups other than the alpha

amino and carboxyl groups which undergo the acylation/esterification reaction. A single

derivative with the general structure I (Figure 2.9) is formed in each case.


Proline forms two products (Figure 2.10).


In addition to the expected product of


acylation of the amino group and esterification of the imino acid II, there is also a second

product III which is the unsaturated analog of II.

Amino Acids with Alcoholic Side Chains



































Figure 2.9


Structure of PFPA/TFE Derivatives with Unreactive Side Chains

















CF3CF2C


OC


2CF3
































Figure 2.10


Sructure of Proline PFPA/TFE Derivatives













o0 C


-C


/ COC


2CF3


CF2CF3


COCH2CF3


CF2CF3


\N/

































Figure 2.11


Structure of Serine PFPA/TFE Derivative.
















C





CF3CF2CN C
II


)-CCF2CF3


1H2


--C


OCH2CF3
j.f -










tyrosine form analogous derivatives.


Tyrosine forms only a single derivative immediately


after preparation. After a few days, a second compound which is consistent with a product

with an underivatized phenolic hydroxyl group V (Figure 2.12) was identified. It has been

reported that trifluoroacyl derivatives of phenolic compounds establish an equilibrium for

the hydrolysis reaction after a few days.47


Amino


Acids with Acidic Side Chains


The dicarboxylic acids, glutamic acid and aspartic acid, form derivatives with the

structure VI (Figure 2.13). The derivative is formed from the acylation of the amine group

and the esterification of both carboxylic groups by the anhydride and alcohol respectively.

Aspartic acid forms a single derivative while glutamic acid forms two major derivatives: one

analogous to that formed by aspartic acid and a cyclic derivative VII (Figure 2.14).


The cyclic glutamic derivative,


VII, is consistent with the ring closure reactions


observed with pAGB 5 in the formation of a lactam structure. Ring closure is possible during

derivatization if the amino acid contains an additional reactive site on its side chain. The

proposed pathway for the formation of the glutamic acid lactam derivative is shown in Figure

2.15. This pathway is consistent with that of Bourne et al'1314 where the anhydride, PFPA,

catalyzes the esterification of the carboxyl acids by alcohols. First, there is formation of an

asymmetric anhydride (B), which ionizes to a pentafluoroacetate ion (C), and an acylium ion

(D) which undergoes nucleophilic attack by the alcohol, TFE (E). In the case of glutamic

































Figure 2.12


Sructure of Tyrosine PFPA/TFE Derivative.

















O

CF3CF2'


O-CCF2F3





;H2

,O


SCH2CF3
i~ "^OCH2CF3


OH


CF3CF2


-I


/o
Nt C ri rT ,-t n

































Figure 2.13


General Structure of PFPA/TFE Derivatives with Acidic and Amide Side


Chains













-C- OCH2CF3


(CH2)n
I2


CF3CF2CN-- C


-C


OCH2CF3


VI


Asp: n = 1


Glu: n


=2
































Figure 2.14


Structure of Proposed Cyclic PFPA/TFE Derivative of Glutamic Acid.














O

C--- OCH2CF3

C2F5C-N


VII

































Figure 2.15


Proposed Pathway for the Formation of PFPA/TFE Lactam Derivative of


Glumatic Acid.








C

CH--C


CH2

CH2

C- OH


-OH


PFPA


O O

H2N- H-C-OCCF2CF3

CH2

CH2

C-OCCF2CF3

0 0


2CF2CF302


H2N- CH-

CH2

CH2

C+

O


S-OCH2CF3


CF3CH20H


H-N


PFPA


O
nr l r C TII


O

C-OCH2CF3


H2N-










Amino Acids with Amide Side Chains


Asparagine and glutamine produced derivatives are identical to those formed by


aspartic acid and glutamic acids respectively (Figures 2.13 and 2.14).


This suggests that


under the reaction conditions used the amide groups of asparagine and glutamine are

converted to their acids and subsequently derivatized to the products mentioned above. The

difficulty in obtaining derivatives of amides has been discussed previously.47

Amino Acids with Basic Side Chains.


Lysine, tryptophan, arginine and histidine are amino acids with basic side chains. The

imidazole group of the histidine is unreactive and, therefore, forms the simple derivative I

as previously discussed for amino acids with hydrocarbon side chains. Lysine forms a single

derivative in which the amino group of the side chain is acylated by the anhydride VIII

(Figure 2.16).


Arginine forms one major product with a lactam structure IX (Figure 2.17).


proposed pathway for the formation of the this derivative is similar to that described above


for the lactam derivative of glutamic acid (Figure 2.15).


The proposed mechanism for the


formation of this derivative is similar to that described above for glutamic acid. In ECNI

there is evidence of derivatives of omithine X, XI (Figure 2.18) being formed, which elutes

slightly earlier than the lactam derivative with about 50% of the intensity of the lactam

derivative. Omithine XII (Figure 2.19) is an unessential amino acid which is an intermediate

































Figure 2.16


Structure of Lysine PFPA/TFE Derivative.














IN-- CCF2CF3

(H12)4


CF3CF2CN-- C
II I
3T^ 2^_r
3^rL1i 1


--C


OCH2CF3


VIII

































Figure 2.17


Structure of Arginine PFPA/TFE Derivative.
















F3


F2


IX

































Figure 2.18


Structure of Omithine PFPA/TFE Derivative.











(CH2)3N- CCF2CF3


CF3CF2C


--C

0


- OCH2CF3


C2F5 ,C


C

































Figure 2.19


Structure of the Amino Acid Omithine













(CH2)3NH2


-CHC OH


0

XII








91

Tryptophan forms two major products. In one form the nitrogen of the indole group

is acylated by the anhydride XIV (Figure 2.20) as reported by Duffield et al.2' and in the

other form, the indole group is underivatized XIII. This is analogous to the equilibrium set-

up in the case of tyrosine. The sensitivity of detection of the fully derivatized species XIV


compared


to the


partially


derivatized


species


is much


greater,


even


though


concentrations may be equal. This due to the presence of an additional C2F


site in


XIV


which results in a larger electron capture cross section.

Amino Acids with Sulfur Containing Side Chains


Methionine, cysteine and


side chains.


cystine are the three amino acids with sulfur containing


The methylmercapto side chain of methionine is unreactive and a simple


derivative, analogous to those of the aliphatic amino acids I, is formed. Cysteine forms a


major product with the structure XIV


(Figure 2.21) where in addition to the expected


acylation/esterification there is also acylation of the thiol group by the anhydride.


Cystine


forms a derivative identical to the cysteine derivative suggesting that under the reaction

conditions cystine is cleaved to cysteine prior to derivatization. Cysteine also forms several

low concentration minor products with high sensitivity to ECNI detection. The structures of

these compounds cannot be inferred from their ECNI spectra.




Conclusions

































Figure 2.20


Structures of Tryptophan PFPA/TFE Derivatives.