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dissertation entitled
INVESTIGATIONS OF A ONE-STEP AQUEOUS—PHASE

CHLOROFORMATE DERIVATIZATION REACTION FOR THE
ANALYSIS OF AMINO ACIDS BY GC/GC-MS AND SMALL PEPTIDES

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Jian Wang

has been accepted towards fulﬁllment
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Ph . D . degree in Chemistry

 

 

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MSU In An Nﬂrmatlvo Action/EM Opportunly Initiation
W1

ANA

INVESTIGATIONS OF A ONE-STEP AQUEOUS-PHASE
CHLOROFORMATE DERIVATIZATION REACTION FOR THE
ANALYSIS OF AMINO ACIDS BY GC/GC-MS AND SMALL PEPTIDES
BY FAB-MS

By

Jian Wang

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Chemistry

1994

AN.

ABSTRACT

INVESTIGATIONS OF A ONE-STEP AQUEOUS-PHASE
CHLOROFORMATE DERIVATIZATION REACTION FOR THE
ANALYSIS OF AMINO ACIDS BY GC/GC-MS AND SMALL PEPTIDES
BY FAB-MS

By

J ian Wang

In gas chromatography (GCIelectron impact (EI) mass spectrometry
(MS) of organic mixtures, one of the most severe restrictions is the
requirement that sample has to be in the gas phase for separation by GC,
ionization, and subsequent analysis in the mass spectrometer.
Derivatization is generally used to reduce the polarity of analyte molecules
by chemically replacing active hydrogens, thereby increasing volatility and
promoting thermal stability. Although techniques of desorption ionization
mass spectrometry, such as fast atom bombardment (FAB), have the
capacity to analyze polar, nonvolatile, and thermally labile compounds
(such as polypeptides, oligosaccharides, oligonucleotides, and other small
biopolymers) by sampling analytes directly from a condensed phase,
derivatization still can be utilized to augment the amount of information
available from the analysis.

This dissertation focuses on: (1) derivatization-assisted amino acid

analysis by GC-MS in E1, positive CI and negative CI modes and (2)

derivatization-assisted peptide analysis by FAB-MS. For the analysis of
amino acids, the one-step aqueous medium chloroformate derivatization
method introduced by Husek for analysis by G0 has been extended to
analysis by GC-MS, and the structurally diagnostic fragmentation of the
amino acid alkyl chloroformate derivatives was also studied. Modiﬁcation
of the derivatization procedure has been conducted. An extended
examination of the derivatization method with the combination of a variety
of alkyl chloroformates and alcohols has been carried out. Fluorinated
derivatives based on a modiﬁed reaction procedure, in combination with
electron capture negative ionization (ECNI) MS analysis has been evaluated
for increasing the sensitivity of analysis. A collaborative research project to
quantitatively assess the incorporation of stable isotope-labeled amino acids
into photosynthetic proteins with the chloroformate derivatization has been
carried out. For peptide analysis, investigation of the chloroformate
derivatization for small peptides prior to analysis by FAB—MS, and a
comprehensive evaluation of the derivatization conditions have been
completed. The advantages and the limitations of the one-step aqueous

medium chloroformate derivatization method for these analytes have been

further investigated.

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ACKNOWLEDGMENTS

I would like to thank my Dad, Mom, brother for all the support they
have provided in my life. I would also like to thank my wife for her help
and love contributed during the years I was in college and in graduate
school.

I would then mention my preceptor, Dr. J .T. Watson, for his
guidance of my research and graduate study. Special thanks are due to Dr.
Zhi-Heng Huang for his critical guidance for my research.

I would like to thank my group members, present and past, and
other fellows in the chemistry department at MSU, who helped me out of all
the hard times and made my experience at MSU enjoyable.

I owe a great deal to the cooperation and patience of the Mass
Spectrometry Facility staff. These helpful people include co-director and
manger Dr. 'Douglas A. Gage; secretary Melinda Berning; electronics
specialist Mike Davenport; computer specialist Mel Micke; and technician
Bev Chamberlin.

I would also like to thank Dr. Neil R. Bowlby for the discussion on our
incorporative research project.

Finally, I would say, thank all my teachers and friends in my
kindergarten, elementary school, middle schools, high school, and college.
Thank them for all the help during different times of my twenty years in

school.

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TABLE OF CONTENTS

Ia'stofTables ...................................................................................... xi
List of Figures ................................................................................... xiv

Chapter I. Introduction and objectives
I. Introduction ................................................................................... 1
II. Derivatization of amino acids prior to analysis by GC and GC-MS

A. Introduction .......................................................................... 2

B. Review of derivatization method for amino acid analysis by GC...7

(1) Strategies ...................................................................... 7
(2) Derivatization methods by other investigators ................... 9
a) N-acyl amino acid alkyl esters ................................. 9
b) Silylation ............................................................. 10
c) Others ................................................................. 12
d) Aqueous medium derivatization ............................ 13

e) One-step aqueous medium chloroformate
derivatization .......................................................... 14

III. Derivatization of peptides prior to analysis by fast atom bombardment

(FAB)-mass spectrometry .................................................................... 1 6
A. Introduction ......................................................................... 16

B. Fast atom bombardment ......................................................... 17

(1) Principles .................................................................... 17

(2) Tandem mass spectrometry - collisionally induced

dissociation(CID) ............................................................. 21

(3) Instrumentation .......................................................... 24

C. Derivatization of peptides prior to analysis by FAB-MS ............... 28
(1) Enhancement of surface activity .................................... 29

(2) Derivatization methods by other investigators .................. 33

a) Forming more hydrophobic derivatives ................... 33

b) Enhancement of structure informative fragmentation
(modiﬁcation of fragmentation) ................................. 36

c) Functional group determination and amino acid

detection ................................................................. 37
d) Others ................................................................ 39
IV. Research objectives ....................................................................... 39
V. References ................................................................................... 41

Chapter II. Simultaneous derivatization of functional groups of amino
acids by an aqueous medium chloroformate reaction prior to analysis by GC
and GC-MS

I. Introduction .................................................................................. 48
II. Characterization of N-ethoxycarbonyl amino acid ethyl esters by EI

mass spectrometry ............................................................................. 50
A. Experimental ........................................................................ 50

B. Results and discussion ........................................................... 50

III. Modiﬁcation of the reaction procedure ......................................... 53
A. Background of chloroformate chemistry .................................. 53

(1) Reaction with amino and phenolic groups ....................... 53

(2) Reaction with carboxyl groups ....................................... 54

B. Modiﬁcation of the reaction procedure ...................................... 62

vi

(1) Experimental ............................................................... 63

(2) Results and discussion ................................................. 64

C. Effect of concentration of the alcohol in the reaction medium ...... 84

(1) Experimental ............................................................... 84

(2) Results and discussion ..................... . ............................ 85

IV. Investigation of different chloroformate derivatives of amino acids ..... 86

A. Amino acid derivatives formed by selected combinations of various

chloroformates and alcohols ...................................................... 86
(1) Experimental ............................................................... 86

(2) Results and discussion ................................................. 88

B. Evaluation of reaction conditions ............................................ 101
(1) Effect of iBuOH concentration ....................................... 101

a) Experimental ..................................................... 101

b) Results and discussion ........................................ 101

(2) Effect of H20 concentration .......................................... 102

a) Experimental ..................................................... 102

b) Results and discussion ........................................ 106

(3) Effect of iBuCF concentration ....................................... 106

a) Experimental ..................................................... 106

b). Results and discussion ........................................ 108

(4) Effect of pyridine concentration .................................... 108

a) Experimental ..................................................... 108

b) Results and discussion ........................................ 110

(5) Effect of other bases and buffer solution ......................... 111

a) Experimental ..................................................... 111

b) Results and discussion ........................................ 111

(6) Evaluation of acid I base wash during the extraction ....... 112

vii

 

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VI. C¢
VII. Re

a) Experimental ..................................................... 1 1 2

b) Results and discussion ........................................ 113
C. Reproducibility ..................................................................... 113
(1) Experimental..................... ........................................ 113
(2) Results and discussion ................................................ 114

V. Comparisons of E1, PCI, and ECNI mass spectrometry responses of
ﬂuorinated derivatives of amino acids ................................................. 11 5
A. Introduction ........................................................................ 115
B. EtCF-HFBuOH and iBuCF-HFBuOH amino acid derivatives in E1,
PCI, and ECNI modes for GC-MS analyses ................................. 118
C. EtCF-PFleOH amino acid derivatives in PCI and ECNI modes for
GC-MS analyses ...................................................................... 121
VI. Conclusions ................................................................................ 127
VII. References ................................................................................ 128

Chapter III. Application of chloroformate derivatization in the quantitative
assessment of incorporation of stable isotope-labeled amino acid into

photosynthetic proteim of Synechocystis PCC 6803

I. Introduction ............................................................................... 1 31
II. Experimental ............................................................................. 133
III. Results and discussion ............................................................... 133
A. Evidence of incorporation of labeled amino acids into cells from
cell growth curves .................................................................. 134
B. Quantitative assessment of incorporation of isotope labeled amino
acids into the photosynthetic proteins ........................................ 135
IV. Conclusions ............................................................................... 152
V. References ................................................................................. 1 54

viii

Chapter IV. Investigation of the one-step chloroformate derivatization of
small peptides prior to analysis by FAB-MS

I. Introduction ................................................................................ 155
II. Experimental .............................................................................. 156
III. Enhancement of FAB signal of model peptides by derivatization ....... 157
IV. Evaluation of derivatization efﬁciency ........................................... 162
A. Introduction ........................................................................ 162

B. Location of the underivatized carboxyl group ........................... 166

C. Effect of reaction medium composition ................................... 167

D Extraction efﬁciency ............................................................. 1 7 2

E. Effect of solvent ratio in the reaction medium .......................... 172

(1) Effect of pyridine concentration .................................... 175

(2) Effect of different bases (catalyst and buffer) ................... 183

(3) Effect of ethanol concentration ...................................... 185

(4) Effect of H20 concentration .......................................... 185

F. Effect of ethyl chloroformate concentration .............................. 188

G. Effect of reaction time ........................................................... 188

H. "Reverse" vs. “Normal"- different procedure for the reaction....190
I. Effect of FAB matrix on the responses of the derivatives ............ 194
J. Effect of multi-cycle derivatization .......................................... 196

V. Preliminary investigation of forming precharged derivatives of

peptides using the chloroformate derivatization procedure .................... 1 99
VI. Conclusions ............................................................................... 205
VII. References ................................................................................ 206

ix

Chapter V. Further investigations of the quantitative aspect of the one-step
chloroformate derivatization in an aqueous medium for amino acids and
small peptides

I. Introduction ................................................................................ 207
II. Evaluation of the reaction efficiency of ethyl and isobutyl chloroformate

with carboxyl groups of amino acids ....................... ' ............................. 2 08
A. Method I ............................................................................. 208
B. Method II ............................................................................ 214
C. Conclusions ........................................................................ 218

III. "Non-aqueous" vs. "aqueous" reaction medium for derivatization of

amino acids and peptides with an ethyl chloroformate reagent ............... 222
A. Introduction ........................................................................ 222
B. Amino acid derivatization ..................................................... 223
C. Peptide derivatization ........................................................... 226
D. Conclusions ........................................................................ 229
IV.Conclusions ................................................................................ 229
V. References .................................................................................. 230

ListofTables

Table 1.1. Structures of protein amino acids ............................................ 3

Table 1.2. Hydrophilicity / hydrophobicity index (AF) of amino acid [79] ..... 32

Table 2.1. Characteristic ion peaks in EI mass spectra of EtCF-EtOH
derivatives of amino acids .................................................................... 52

Table 2.2. Summary of results of Phe derivatives from different
chloroformate and alcohol reagents ...................................................... 80

Table 2.3. Effect of HFBuOH concentration in the reaction solution (80 ul

H20, 10 ul pyridine, and 10 ul iBuCF) on the percentage of minor
derivatization product ......................................................................... 85

Table 2.4. Chloroformate-alcohol reagents studied for amino acid
derivatization ..................................................................................... 87

Table 2.5. Ratio of peak area on GC-FID of indicated derivatives relative to
those prepared with EtCF-EtOH: (response for EtCF-EtOH derivatives were
the average of results from triplicate analyses; responses for the indicated
derivatives were the average of results from duplicate analyses) ............ 99

Table 2.6. Ratio of both peak area and peak height of reconstructed TIC
corresponding to the indicated derivatives relative to those made with EtCF-
Et0H from analyses by GC-MS (EI) ..................................................... 1 00

Table 2.7. Effect of H20 volume in the reaction medium (X ul H20, 30 ul

iBuOH, and 10 ul pyridine) on the TIC responses of iBuCF-iBuOH
derivatives of amino acids .................................................................. 107

Table 2.8. pH vs. pyridine volume in the reaction medium for amino acid
derivatization by iBuCF-iBuOH ........................................................... l 1 0

Table 2.9. Effect of reagent composition on derivatization formation and
response. Mean relative weight responses of peak area of TIC (relative to
internal standard nLeu) (RWR) and relative standard deviation (RSD) for
the indicated amino acid derivatives (Five individual samples for EtCF-
EtOH, iBuCF-iBuOH derivatization, and three individual samples for
iBuCF-HFBuOH derivatization) .......................................................... 11 6

xi

Table 2.10. Ratio of both peak area and peak height of reconstructed TIC
corresponding to the indicated derivatives relative to those made with EtCF-
EtOH from analyses by GC-MS (EI) (results from experiments of
reproducibility) ................................................................................. 1 1 7

Table 2.11. Comparison of absolute intensities of [M+1]+ in PCI and [M-1]"
in ECNI of amino acid EtCF-HFBuOH derivatives ................................. 120

Table 2.12. Summary of ECN I mass spectra of amino acid EtCF-PFleOH
derivatives ........................................................................................ 125

Table 2.13. Comparison of absolute intensities of [M-PFB]' in ECNI and
[MH-PFBCO2H]+ in PCI of amino acid EtCF-PFleOH derivatives ......... 126

Table 3.1. Quantitation of the extent of incorporation of 170-tyrosines into
the indicated proteins in Synechocystis cells grown in the presence of 170--
tyrosine (40% 170) .............................................................................. 151

Table 4.1. Estimation of FAB signal enhancement from small peptides as
their ethyl chloroformate derivatives relative to the FAB response of

underivatized peptides ....................................................................... 158
Table 4.2. FAB-MS response for indicated species as function of reaction
medium (model compound: RKDVY) .................................................. 171
Table 4.3. Extraction efficiency vs. volume of chloroform for RKDVY
(EtCF) ............................................................................................... 173
Table 4.4. pH vs. pyridine volume in the reaction mixture ..................... 184
Table 4.5. Effect of different bases for RKDVY EtCF derivatization (catalyst
and buffer) ....................................................................................... 184
Table 4.6. “Reverse” vs. “normal” - effect of different reaction procedure on
RKDVY EtCF derivatization ............................................................... 193
Table 5.1. Results from method I - esteriﬁcation efficiency with ethyl
chloroformate ................................................................................... 211
Table 5.2. Results from method I - esterification efﬁciency with isobutyl
chloroformate ................................................................................... 21 5
Table 5.3. Results from method 11 - esterification efﬁciency with ethyl
chloroformate .................................................................................. 221

Table 5.4. Relative responses of peak area from TIC and mass
chromatogram (relative to internal standard) of Leu derivatized with ethyl
chloroformate in different reaction media as indicated .......................... 225

xii

Table 5.5. Relative responses of peak area of TIC (relative to internal
standard) of amino acids derivatized with ethyl chloroformate in different
reaction medium as indicated ............................................................ 225

Table 5.6. Reaction medium compositions for RKDVY derivatization with
EtCF ................................................................................................ 227

xiii

 

 

 

 

 

 

 

 

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List of Figures

Figure 1.1. Reactions of chloroformate with functional groups of amino

acid ................................................................................................... 15
Figure 1.2. Schematic diagram for fast atom bombardment process ......... 18
Figure 1.3. View at surface of glycerol/sample solution during fast atom
bombardment ..................................................................................... 20
Figure 1.4. Peptide fragment ion designations proposed by Roepstorff and
Fohlman [66] ..................................................................................... 22
Figure 1.5. Fragment ion structures commonly observed in FAB mass
spectra of peptides [61] ......................................................................... 23
Figure 1.6. Tandem mass spectrometry (MS/MS) ................................... 25
Figure 2.1. Reactions of chloroformate with functional groups of amino
acid ................................................................................................... 49
Figure 2.2. Main fragmentation pathways for amino acid EtCF-EtOH
derivatives in EI mass Spectrometry ..................................................... 51
Figure 2.3. Thermal decomposition pathways for mixed carboxylic-carbonic
anhydride .......................................................................................... 56
Figure 2.4. Mechanism for mixed carboxylic-carbonic anhydride thermal
decomposition proposed by Tarbell [29] - ionic chain reaction ................... 58
Figure 2.5. Mechanism of esteriﬁcation through mixed carboxylic-carbonic
anhydride proposed by Kim [36] ......................................................... 61
Figure 2.6. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
with iBuCF-HFBuOH .......................................................................... 66
Figure 2.7. TIC (a) and EI mass spectrum (1)) of Phe derivatized by reaction
with iBuCF-TMSCH20H ...................................................................... 67
Figure 2.8. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
with iBuCF-TFEtOH ........................................................................... 68
Figure 2.9. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
with iBuCF-PFPrOH ........................................................................... 69

xiv

Figure 2.10. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
with iBuCF-iBuOH ........................................................................... 70

Figure 2.11. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
with MCF-TFEtOH ............................................................................. 7 7

Figure 2.12. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
with MCF-TMSCH20H ...................................................................... 78

Figure 2.13. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
with MCF-MeOH ............................................................................... 79

Figure 2.14. TIC of Phe derivatized by reaction with iBuCF in an aqueous
solution containing an equal volume mixture of seven alcohols (MeOH,
EtOH, iBuOH, TFEtOH, PFPrOH, HFBuOH, and TMSCH20H) ................ 81

Figure 2.15. TIC of Phe derivatized by reaction with iBuCF in an aqueous
solution containing an equimolar mixture of seven alcohols (MeOH, EtOH,
PrOH, TFEtOH, PFPrOH, HFBuOH, and TMSCH20H). The peak labeled
R=iBu represents a trace of the ester product formed with the alkoxyl group
corresponding to the alkyl group of the chloroformate reagent ................. 82

Figure 2.16. TIC of Phe derivatized by reaction with MCF in an aqueous
solution containing an equimolar mixture of seven alcohols (EtOH, PrOH,
iBuOH, TFEtOH, PFPrOH, HFBuOH, and TMSCH20H). The peak labeled

=Me represents a trace of the ester product formed with the alkoxyl group
corresponding to the alkyl group of the chloroformate reagent ................. 83

Figure 2.17. TICs of 20 amino acid derivatives prepared from different
chloroformatealcohol reagents (a) EtCF-EtOH. (b) PrCF-PrOH, (c) iBuCF-
iBuOH. GC-MS analysis of an aliquot of the reaction mixture containing 50
ng of each amino acid on to a 15-m, 0.25-mm i.d. column containing a 0.25-

um ﬁlm of DB-1701. GC temperature programs: (a) from 100°C to 200°C at
10°C / min, then 20°C / min to 280°C; (1)) and (c) from 120°C to 200°C at 10°C
/ min, then 20°C / min to 280°C ............................................................ 91

Figure 2.18. TICs of 20 amino acid derivatives prepared from EtCF with (a)
TFEtOH and (b) HFBuOH. Temperature programs: from 100°C to 200°C at
10°C / min, then 20°C / min to 280°C ..................................................... 92

Figure 2.19. TICs of 20 amino acid derivatives prepared from PrCF with (a)
TFEtOH, (b) PFPrOH, and (c) HFBuOH. Temperature programs: (a) from
100°C to 200°C at 10°C / min, then 20°C / min to 280°C; (1)) and (c) from
120°C to 200°C at 10°C / min, then 20°C / min to 280°C ........................... 93

Figure 2.20. TICs of 20 amino acid derivatives prepared from iBuCF with
(a) TFEtOH, (b) PFPrOH, and (c) HFBuOH. Temperature program: from

120°C to 200°C at 10°C / min, then 20°C / min to 280°C ............................ 94

XV

Figure 2.21. TIC of 21 amino acid derivatives prepared from iBuCF-
TMSCH20H. Temperature program: from 120°C to 180°C at 10°C / min,

then 20°C / min to 280°C. GC column 0V1701 10-m, 0.25-mm i.d., 0.2-um
ﬁlm. (280 ng for each amino acid. Cys was not identiﬁed.) ..................... 95

Figure 2.22. GC-FID chromatograms of 20 amino acid derivatives prepared
from different chloroformate-alcohol reagents (1) EtCF-EtOH, (2) iBuCF-
iBuOH, (3) iBuCF-HFBuOH, and (4) iBuCF-TMSCH20H. The
chromatograms result from injection of an aliquot of the reaction mixture
containing 50 ng of each amino acid on to a 15-m, 0.25-mm i.d. column

containing a 0.25-um ﬁlm of DB-1701 ..................................................... 96

Figure 2.23. TIC of 20 amino acid derivatives prepared from iBuCF-iBuOH.
GC separation is done within 6 min. GC temperature program: 120°C to
280°C at 40°C / min ............................................................................. 98

Figure 2.24. Effect of iBuOH concentration in the reaction medium for
formation of iBuCF-iBuOH derivatives of amino acids (1 ).... ................... 103

Figure 2.25. Effect of iBuOH concentration in the reaction medium for
formation of iBuCF-iBuOH derivatives of amino acids (2) ....................... 104

Figure 2.26. Effect of iBuOH concentration in the reaction medium for
formation of iBuCF-iBuOH derivatives of amino acids (3) ....................... 105

Figure 2.27. Effect of iBuCF concentration for the derivatization of amino
acids by iBuCF-iBuOH ....................................................................... 1 09

Figure 2.28. TICs of EtCF-HFBuOH derivatives of amino acids in (a) EI, (b)
PCI, and (3) NCI modes of GC-MS analysis .......................................... 119

Figure 2.29. (a) PCI and (b) ECNI mass spectra of Val EtCF-PFleOH
derivative ......................................................................................... 123

Figure 2. 30. (a) PCI and (b) ECNI mass spectra of Phe EtCF-PFleOH
derivative ......................................................................................... 124

Figure 3.1. Cell density and abundance of aromatic amino acids in the
growth medium of synechocystis cells. Panel A shows cell density as
measured by 0D730 of a culture grown in the absence of exogenously added
amino acids. In panel B is shown the cell density (0, 0 open and ﬁlled
symbols show data from two different cultures) and growth characteristic
of cells grown in the presence of phenylalanine (0.50 mM), tryptophan (0.25
mM) and 1“’O-tyrosine (0.25 mM). The aromatic amino acid absorbance (I)
was monitored at 276 nm (see Figure 3.2) in samples represented by the
filled circles on the growth curve (Reprinted from Ref. 1) ....................... 136

Figure 3.2. Absorption spectra of culture medium obtained after growth for
the number of hours indicated. Cells and other solids were removed by

xvi

centrifugation before recording the spectra. The reference cuvette
contained BG-ll medium with no added amino acids (Reprinted from
Ref.1) ............................................................................................... 137

Figure 3.3. EI mass spectra of isobutyl chloroformate derivatives of
unlabeled phenylalanine (a) and dg-phenylalanine (b) from stock solutions.
TIC/mass chromatograms of iBuCF derivatives of Phe/da-Phe mixture (1 :1)
(c). (50 nmol derivatization, 250 pmol injection for GC-MS analysis.) ...... 140

Figure 3.4. Analysis of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. EI mass spectra of the
phenylalanines in protein hydrolysates from unlabeled (a) and dg-labeled (b)
cells. TIC / mass chromatograms for Phe in protein hydrolysates from
cells that were grown in the presence of dg-Phe (c). (2 nmol protein
derivatization, 50 pmol injection for GC-MS analysis.) ........................... 141

Figure 3.5. EI mass spectra of isobutyl chloroformate derivatives of
unlabeled tyrosine (a), l70-tyrosine (40% 170) (b). and 3,5-2H-tyrosine (c)
from stock solutions. TIC / mass chromatograms of iBuCF derivatives of
Tyr and l70-Tyr mixture (d). (10 nmol derivatization, 200 pmol injection for

GC-MS analysis.) .............................................................................. 144
Figure 3.6. Analysis of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. TIC / mass

chromatograms (a) and EI mass spectrum (b) of the tyrosines in
Photosystem I proteins isolated from cells grown in the presence of 170-
tyrosine (40% 1 0). (25 pmol PS I center derivatization, 1 pmol injection for
GC-MS analysis.) .............................................................................. 145

Figure 3.7. Analysis of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. TIC / mass
chromatograms (a) and EI mass spectrum (b) of the tyrosines in
phycobiliproteins isolated from cells grown in the presence of 170—tyrosine
(40% 170). (2 nmol protein derivatization, 100 pmol injection for GC-MS

analysis.) ......................................................................................... 1 46
Figure 3.8. Analysis ‘of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. TIC / mass

chromatograms (a) and EI mass spectrum (b) of the tyrosines in
Photosystem I proteins isolated from cells grown in the presence of 1"0-
tyrosine (40% 1"0). (20 pmol PS I center derivatization, 2 pmol injection for
GC-MS analysis.) ............................................................................. 148

Figure 3.9. Analysis of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. TIC/mass
chromatograms (a) and EI mass spectrum (b) of the tyrosines in
Photosystem II proteins isolated from cells grown in the presence of 1"0-
tyrosine (40% 170). (30 pmol PS II center derivatization, 3 pmol injection for
GC-MS analysis.) ............................................................................. 149

xvii

 

 

chlo

as the

major

contai.

Flg‘un
Chlorof.
Fit'ure
Iglutath
(10“'er p;
the free
(Structu.

free H}
(Stmctur

Figure
(Figure F

the indlc
fin'l’atiz
”b one

Figure 3.10. Analysis of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. TIC / mass
chromatograms (a) and EI mass spectrum (b) of the tyrosines in
phycobiliproteins isolated from cells grown in the presence of 1"0~-tyrosine
(40% 170). (2 nmol protein derivatization, 200 pmol injection for GC-MS

analysis.) ......................................................................................... 1 50

Figure 3.11. Analysis of the growth medium separated from Synechocystis
cells at different times as indicated for Phe, Tyr, and Trp by GC-MS after
derivatization with isobutyl chloroformate ........................................... 153

Figure 4.1. Comparison of FAB mass spectra of y-ECG (glutathione)
underivatized and derivatized with ethyl chloroformate. Top panel is mass
spectrum obtained from 20 nmol of underivatized y-ECG; bottom panel is
the FAB mass spectrum obtained from 1 nmol of N,S,0-

ethoxycarbonyl/ethyl ester of “(ECG ..................................................... 159

Figure 4.2. Comparison of FAB—MS spectra of RKDVY, underivatized
(upper panel, lnmol) and derivatized (lower panel, 100 pmol) with ethyl
chloroformate ................................................................................... 160

Figure 4.3. MS/MS spectrum of fully derivatized RKDVY (MH+ 952). A
complete series of the N-terminal ions are present ................................ 161

Figure 4.4. HPLC chromatogram of derivatized RKDVY with CHaCN/H20
as the solvent. The reaction medium was H20/Et0H/Py (70/30/5). The two
major peaks represent the fully derivatized compound and the species

containing one free carboxyl group ...................................................... 163
Figure 4.5. FAB-MS spectrum of RPKPQQFFG derivatized with ethyl
chloroformate ................................................................................... 165

Figure 4.6. Product ion spectra (B/E linked scan) of fully derivatized y—ECG
(glutathione) (upper panel, MH'l' 508.5) and partially derivatized y-ECG
(lower panel, MH+ 480.5) Underlined ions correspond to the product with
the free C-terminal and the derivatized 'y-Glu side chain carboxyl group
(structure 1 in Figure 4.7); italized ions correspond to the product with the
free ‘y-Glu side chain carboxyl group and the derivatized C-terminal
(structure 2 in Figure 4.7) .................................................................. 168

Figure 4.7. Structures and fragment ions in FAB-CID-MS/MS spectrum
(Figure 6b) of partially derivatized 'y-ECG (glutathione) .......................... 169

Figure 4.8. FAB-MS spectra (partial) of 0.5 nmol of RKDVY derivatized in
the indicated ratio of solvents. The peak at m/z 952 represents the fully
derivatized derivative; peaks at m/z 924 and m/z 896 represent the products
with one or two free carboxyl groups, respectively; peaks at m/z 880 and m/z

XVIII

 

 

852 «‘
or 01

Fig"
deriV.
the Vt
rigug
of Rh

the re

Figure
of y-EC
the rear

Figure
of GHK

reaction

Figure 4
of GOP p.

reaction 1

Figure 4
derivatizat
RKDVY pr
reaction in

Figure 4.
derivatizatii

' pre;
reaction met

Figure 4.14
envatizatioz
9 VOIume of

Figure 4.17. r
(reaction) time

 

 

852 and others represent the fragmentation at the derivatization sites and/
or other incomplete derivatized products .............................................. 174

Figure 4.9. The HPLC-UV (1:214 nm) responses of the different
derivatization products of RKDVY prepared with ethyl chloroformate vs.
the volume of pyridine in the reaction medium ..................................... 1 76

Figure 4.10. The FAB-MS responses of the different derivatization products
of RKDVY prepared with ethyl chloroformate vs. the volume of pyridine in
the reaction medium ........................................................................ 179

Figure 4.11. The FAB-MS responses of the different derivatization products
of y-ECG prepared with ethyl chloroformate vs. the volume of pyridine in
the reaction medium ......................................................................... 180

Figure 4.12. The FAB-MS responses of the different derivatization products
of GHK prepared with ethyl chloroformate vs. the volume of pyridine in the
reaction medium .............................................................................. 181

Figure 4.13. The FAB-MS responses of the different derivatization products
of GGF prepared with ethyl chloroformate vs. the volume of pyridine in the
reaction medium ............................................................................. 182

Figure 4.14. The HPLC-UV (1:214 nm) responses of the different
derivatization products (a) and the yield of fully derivatized product (b) of
RKDVY prepared with ethyl chloroformate vs. the volume of ethanol in the
reaction medium .............................................................................. 186

Figure 4.15. The HPLC-UV (1:214 nm) responses of the different
derivatization products (a) and the yield of fully derivatized product (b) of
RKDVY prepared with ethyl chloroformate vs. the volume of water in the
reaction medium .............................................................................. 187

Figure 4.16. The HPLC-UV (1:214 nm) responses of the different
derivatization products of RKDVY prepared with ethyl chloroformate vs.
the volume of ethyl chloroformate added in the reaction ......................... 189

Figure 4.17. FAB-MS responses of the derivatization products of RKDVY (a)
and y—ECG (glutathione) (1)) prepared with ethyl chloroformate vs. vortexing
(reaction) time .................................................................................. 1 91

Figure 4.18. FAB-MS responses of the derivatization products of GHK (a)
and GGF (b) prepared with ethyl chloroformate vs. vortexing (reaction)
time ................................................................................................. 192

Figure 4.19. FAB-MS responses of ethyl chloroformate derivatized (a) and
underivatized (b) di-peptide DG vs. volume of matrix (glycerol/thioglycerol/
methanol; 1/1/1) ................................................................................ 195

xix

 

 

(a)

chlo

Fig‘u
of et}
Figu:
(2) CI

F i gun
CHQN;

Figure
(2) CH

Figure
efﬁcienc

Figure
{method

Hams
Chloroforr

FYI-i dill e; |
mﬁNm

Figure 4.20. HPLC chromatograms (partial) for RKDVY ethyl
chloroformate derivatives - effect of multiple exposure of analyte to
reagents ........................................................................................... 197

Figure 4.21. The HPLC-UV (1:214 nm) responses of the different
derivatization products (a) and the yield of fully derivatized product (b) of
RKDVY prepared with ethyl chloroformate vs. the number of exposure
(reaction cycle) of analyte to reagents ................................................... 198

Figure 4.22. FAB mass spectrum of Phe ethyl chloroformate-choline
derivative (M'l' 323) ............................................................................ 202

Figure 4.23. Product ion spectra (B/E linked scan) of ions from Figure 4.22:
(a) m/z 387, (1)) m/z 315, and (c) m/z 243 ................................................ 203

Figure 4.24. Possible structures of product and adduct ions from ethyl
chloroformate and choline chloride reagents ........................................ 204

Figure 5.1. Reaction scheme of method I to evaluate esterification eﬂiciency
of ethyl (isobutyl) chloroformate with amino acids ...... . .......................... 209

Figure 5.2. TIC and EI mass spectra of Phe derivatized with (1) EtCF and
(2) CH2N 2 (method I) .......................................................................... 212

Figure 5.3. TIC and EI mass spectra of Tyr derivatized with (1) EtCF and (2)
CH2N 2 (method I) .............................................................................. 21 3

Figure 5.4. TIC and EI mass spectra of Asp derivatized with (1) iBuCF and
(2) CH2N2 (method I) .......................................................................... 216

Figure 5.5. Reaction scheme of method II to evaluate esterification
efﬁciency of ethyl chloroformate with amino acids ................................ 219

Figure 5.6. TICs of Leu (a) and Len ethyl ester (b) derivatized with EtCF
(method II) ....................................................................................... 220

Figure 5.7. Partial HPLC chromatograms of RKDVY derivatized with ethyl
chloroformate in different reaction media: (a) 60 ul H20, 30 ul EtOH, 10 ul
pyridine; (b) 30 ul H20, 30 ul CH30N, 30 ul EtOH, 10 ul pyridine; (c) 60 ul
CH3CN, 30 11] EtOH, 10 ul pyridine; (d) 90 ul CH3CN, 10 pl pyridine ......... 228

XX

 

(MS)
requi
ioniza
Deriva

chemic;

 

Promoti
Spectrum
analyze ‘
Polypepn-
bjDPOIJ'me
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The ol
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Chapter I
Introduction and objectives

1. Introduction

The reasons for utilizing chemical derivatization in analyses by mass
spectrometry have been summarized by Knapp [1] as follows: (1)
enhancement of volatility; (2) degradation of the sample molecule to smaller
subunits; (3) enhancement of detectability; (4) enhancement of separability;
(5) modification of fragmentation: (a) enhancement of molecular weight-
related ions and (b) enhancement of structurally informative ions; (6)
determination of functional groups.

In gas chromatography (GC)-electron impact (EI) mass spectrometry
(MS) of organic mixtures, one of the most severe restrictions is the
requirement that sample be in the gas phase for separation by GC,
ionization, and subsequent analysis in the mass spectrometer.
Derivatization is generally used to reduce the polarity of a molecule by
chemically replacing active hydrogens, thereby increasing volatility and
promoting thermal stability. Although desorption ionization mass
spectrometry, such as fast atom bombardment (FAB), has the capacity to
analyze polar, nonvolatile, and thermally labile compounds (such as
polypeptides, oligosaccharides, oligonucleotides, and other small
biopolymers) by sampling analytes directly from a condensed phase,
derivatization still can be utilized to augment the amount of information
available from the analysis.

The objectives of this ﬁrst chapter are to 1) review and discuss the
procedures and characteristics of chemical derivatization methods for

amino acid analysis by GC and GC-MS, 2) introduce FAB ionization and

1

 

instrumei
derivatizz

FAB, and

II.Deriva
A.‘.
Th
prerequi:
hence, to
an enzyr
membra
represer
blocks 0.
the cell
vehicles
Particip.
in meta
for bios
composi

tFHnSpQ

2
instrumentation, 3) review and discuss the different strategies and

derivatization methods employed to enhance the analysis of peptides by

FAB, and 4) state the research objectives of this dissertation.

II. Derivatization of amino acids for gas phase analysis by GC and GC-MS

A. Introduction

The determination of amino acid composition is a fundamental
prerequisite to the definition of the structure of a protein or peptide and,
hence, to the understanding of functional properties, whether the protein be
an enzyme, a transport protein, or a protein crucial to the integrity of a cell
membrane. Amino acids as inherent constituents of living matter
represent more than 50% of the dry weight of a cell, mainly as building
blocks of proteins. These protein not only constitute structural elements of
the cell architecture, but also function as biocatalysts, as messengers, or as
vehicles for the selective transport of biological fuels. Amino acids also
participate in cell chemistry in their free forms, occurring as intermediates
in metabolism, or serving as nutrients, neurotransmitters, or precursors
for biosynthesis of cell constituents. A knowledge of the free amino acid
composition of biological ﬂuids is central to studies of metabolism and
transport [2,3].

Structural features common to all amino acids are an amino group,
a carboxyl group, and a hydrogen attached to a central (or) carbon atom. A
fourth substituent of variable structure confers to each amino acid its
particular chemical property. Out of twenty amino acids commonly found
in proteins (Table 1.1), fifteen amino acids are neutral with an aliphatic or
aromatic side chain determining their chemical properties. These amino

acids are soluble as zwitter ions with isoelectric points between pH 5 and 7.

 

 

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Thee
threc
meth
amin
aspar

chain

4
These side chains may also contain a hydroxyl group (as in serine,

threonine, tyrosine), a sulfur-containing group (as in cysteine and
methionine), or an amide group (as in asparagine and glutamine). Other
amino acids carry an additional carboxyl group, as in acidic amino acids
aspartic and glutamic acid, or a nitrogen-containing function on the side
chain of basic amino acids arginine, histidine and lysine.

There is no analytical panacea applicable to assaying amino acids.
Any single technique may confer advantages in speciﬁc situations, and a
separation that is readily achieved by using one method may be very
difficult if not impossible by using other means. Frequently, the technique
chosen by the individual researcher or analyst is dictated by the equipment
available.

The separation of the protein amino acids by ion exchange
chromatography led to the ﬁrst automated instruments some three decades
ago [4]. A separation of the twenty protein amino acids took about 2 hours.
Modern amino acid analyzers cut the analysis time in half. However, their
purchase price and the cost of operation are high, and they are dedicated
instruments with little ﬂexibility for other tasks.

The advances in high-performance liquid chromatography (HPLC)
have led to rapid and sensitive procedures for assaying amino acids.
Determinations of amino acids can now be achieved at levels reaching 10'18
mol using ﬂuorimetric [5-8] or voltameric detection [9-11] combined with
HPLC or capillary electrophoresis [7,8]. Although one-purpose amino acid
analyzers are no longer essential, routine automated measurements of
amino acids with modern, ﬂexible HPLC instruments are not possible with
relatively inexpensive systems [11]. Furthermore, although in LC analysis
not all the active hydrogen-containing groups need to be treated so that the

 

averal

anal ys

analys

l

amino

analysi
instrun
advante
The resc
theoretic
long is u
the total

brought
thousand
mPfibilit)
amino aci
short Capi
using any
in Standar
a few mm]
about 20 c
a function

5
average time for sample pretreatment is shorter than in GC, one hour

analysis time is considered as an average for LC performed amino acid
analysis.

Soon after the advent of gas liquid chromatography, its application to
amino acid analysis was proposed. High sensitivity, great speed of
analysis, high resolving power, low cost, and great versatility of the
instrument are its expedient features. Perhaps the most important
advantage offered by GC over other methods is unsurpassed resolution.
The resolving power of HPLC columns is of the order of tens of thousands of
theoretical plates per meter, yet it is rare indeed that a column even 1 m
long is used. Unlike GC analysis, use of capillary columns in LC lengthens
the total time of the chromatographic run [6,10]. Thus, the resolving power
brought to bear on the analytical problem is only of the order of a few
thousand theoretical plates. In this regard, no other technique offers the
capability of long capillary GC column. Complete baseline resolution of the
amino acids in a protein hydrolysate can be achieved in about 10 min using
short capillary columns, but resolution of this order has not been achieved
using any other method in an acceptable analysis time. Furthermore, even
in standard amino acid assays, the analyst is faced with not only resolving
a few components but also simultaneously resolving and precisely assaying
about 20 components. Since the precision and accuracy of the analysis are
a function of the separation of the sample components, the importance of
achieving Optimal resolution is axiomatic. The analysis is much more
complex when the sample is a physiological ﬂuid in which any one or more
of hundreds of nonproteic amino acids may be present in addition to the
standard protein amino acids. In this context, resolving power is a crucial

factor in determining the success of the assay. In biological samples, the

 

nature 01
speciﬁc I
from nor
variety 1
chromatr
ﬂexibilit;
analysis.
so many
analysis
mdmwl
acids in 1
ﬁne cou;
rEipid ide
Adt
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SPECIFIC 5
dEIvECtor (
Gus) pr,
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applied to
(3n.
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be quanti
The goal

6
nature of the sample matrix can often require the development of a tissue-

speciﬁc procedure for sample cleanup prior to GC to eliminate interference
from non-amino acid components. The analyst face with assaying a wide
variety of sample types must be prepared to select derivatives and
chromatographic columns to suit the purpose of the analysis. This
ﬂexibility is not always available when using other methods of amino acid
analysis. Furthermore, no other technique offers the potential for resolving
so many components in one analysis in less than 1 hour. An ion-exchange
analysis of a physiological ﬂuid typically requires 5 hours. The ﬂexibility
and resolving power make GC the method of choice for assaying free amino
acids in physiological ﬂuids [2]. Another advantage is the possibility of on-
line coupling of a gas chromatograph to a mass spectrometer, enabling
rapid identiﬁcation of unknowns.

Although most gas chromatographic analyses are conducted using a
nonspeciﬁc ﬂame ionization detector, other detectors can be used to gain
speciﬁc structural information. Thus a nitrogen- or phosphorus-speciﬁc
detector could conﬁrm the presence of speciﬁc element. Mass spectrometry
(MS) provides structural information and a mass spectrometer is '
unquestionably the most powerful detector that can be coupled to a gas
chromatograph. Advances in HPLC/MS in recent years have made this
technique valuable for other classes of compounds, but it is not as well
established or as generally available as GC/MS, nor has it been as widely
applied to the identiﬁcation of amino acids.

One perceived disadvantage of CC for amino acid analysis is a
consequence of the inherent nonvolatility of amino acids. Derivatives must
be quantitatively formed to block or remove the polar functional groups.
The goal of these derivatization reactions is to reduce the polarity and

 

an5113‘s:

mrbox).
blocked
into Sui
conﬁde:
gas Chrc
Since the
fully blo
suited f0:

to mas},-

7
increase the vapor pressure by chemically replacing active hydrogens,

thereby increasing volatility and promoting thermal stability of amino
acids. Vapor pressure of a compound is inﬂuenced by intermolecular
attractions due to dispersion (van der Waals) forces, ionic interactions, and
hydrogen bonds. The total dispersion forces increase with molecular size
and little can be done with respect to these forces to increase volatility other
than reduce the size of the molecule. Chemical derivatization for volatility
enhancement is aimed at reducing ionic and hydrogen bond interactions by

conversion of ionizable groups to nonionizable derivatives (e.g., carboxyl '
groups to esters); replacing hydrogens bound to heteroatoms (N-H, 0-H, S-
H) with alkyl, acyl, silyl or other groups; and reducing the polarity of
hydrogen bond accepting groups

B. Review of derivatization methods of amino acids for gas phase

mlysisbygaschmmatographw
(1) Strategies of amino acids derivatization

Amino acids, as the name indicates, contain an amino and a
carboxyl group. These and other polar groups on the side chains must be
blocked to reduce intermolecular attractions and to convert amino acids
into sufﬁciently volatile derivatives. This area of research has received
considerable attention, as derivatization is decisive for success or failure of
gas chromatographic amino acid analysis. It has been intensively studied
since the early 19608. Since the ﬁrst report 36 years ago of the formation of
fully blocked N-triﬂuoroacetyl amino acid methyl esters which are well
suited for gas chromatography [12], a wide range of reagents has been used
to mask the functional groups of amino acids. However, most

derivatization schemes have either failed to be applicable to all the standard

 

amino
chrom
be sufl
that tl
Thus,
column
V
hypothr
derivati
1.
2.

50.00am

10.
The
dem'atizat

takins'l hc

8
amino acids to be expected in a protein hydrolysate or, because of their

chromatographic properties or the properties of the column used, failed to
be sufﬁciently resolved during chromatography. It should be borne in mind
that the protein amino acids do not represent a single homologous series.
Thus, determining the combination of derivatives and chromatographic
column to effect the resolution of these derivatives was not an easy task.
When summarizing criteria aimed at the establishment of a
hypothetical, ideal procedure for amino acid determination, an "ideal"

derivatization should fulﬁll the following requirements:

1. Formation of only one derivative per compound

2. Formation of derivatives of high chemical and thermal
stability

3. Complete and reproducible derivatization over a wide range of
concentrations

4. N o interference with other derivatization

5. Derivatization of polyfunctional compounds in minimum

number of steps
A rapid reaction proceeding at room temperature
Ability to derivatize in an aqueous medium

Reagents more volatile than derivatives

99°99)

Short GC analysis with a good resolution for all the amino acid
derivatives

10. Low reagent and instrumental cost

The main problem in CC analysis for amino acids is the need for
derivatization, commonly involving laborious and multi-step procedures

taking 1 hour or more [13], thereby losing the advantage of the speed of GC

 

itself.
method

T
was lai
formatic
These a
most co
based ti
include
isobutyl
lSOpropy
Dentyl e:
anhydu‘d
ﬂuorinat

9
itself. Looking for more convenient and less time-consuming derivatization

method has been the goal for many researchers in this area.

(2) Derivatization methods by other investigators
a) N-acyl amino acid alkyl ester

The foundation for the most commonly used derivatization procedure
was laid by Gehrke, who developed the procedures for quantitative
formation of N(0,S)—triﬂuoroacetyl (TFA) n-butyl amino acid esters [13-15].
These are but one set of N-acyl amino acid alkyl esters which represent the
most Commonly used class of derivatives for amino acid analysis by GC
based the two-step reactions. Other variants by different research groups
include the N -heptaﬂuorobutyryl (HFB) isopropyl esters [16], the N-HFB
isobutyl esters [17,18], the N-TFA n-propyl esters [19], and the N-TFA
isopropyl esters [20]. The alkyl group has ranged from the methyl to the
pentyl ester while the acyl group, usually derived from the corresponding
anhydride, has been almost exclusively conﬁned to the acetyl derivatives, its
ﬂuorinated analog, or a perﬂuorinated homolog. N-acyl amino acid alkyl
esters are usually formed in two separate reactions, acid-catalyzed
esteriﬁcation followed by acylation. The typical procedure to form n-HFB
amino acid isobutyl esters involves transferring the standard amino acid
solution and internal (standard solution to a sample tube and evaporating to
dryness under nitrogen stream at 50°C. To the dried sample, 200 pl
isobutanol-3 N HCl is added, then sample tube is capped and heated for 45
min at 120°C. After cooling, the sample is evaporated to dryness under a
nitrogen stream at 40°C, 80 ul ethyl acetate and 20 ul heptaﬂuorobutyric
anhydride (HFBA) are added, and the mixture is heated 20 min at 110°C.

After cooling, evaporate excess HFBA at room temperature under nitrogen

 

is Inc
reacti
the ri
Purifi.
the pc

the 0V:

methoc

[2324}.c

10
stream [2,17,21]. Gehrke demonstrated that capillary GC analysis of amino

acids using fused silica bonded-phase columns provides data with good
precision and in general excellent agreement with ion-exchange analysis
[21]. One problem of these procedures based on N -acyl amino acid alkyl
esters is that they cannot be used to assay asparagine and glutamine
because of conversion to aspartic and glutamic acids, respectively, during
acid-catalyzed esteriﬁcation [2,3]. A third reaction is usually necessary to
block the imidazole ring for quantitation of histidine [3,22]. The solubility of
the amino acids decrease in higher alcohols.

Naturally, complete derivatization of all functional groups in one step
is most attractive. If volatile derivatives could be formed in a single
reaction, signiﬁcant beneﬁts would accrue. These include simpliﬁcation of
the reaction mixtures. Consequent reduction of the number of highly
puriﬁed reagents and solvents to be obtained or prepared would minimize
the potential for introduction of impurities into the reaction. Presumably,

the overall derivatization time would be as short as possible.

b) Silylation
One of the one-step approaches is the silylation method, a widely used
method to make volatile derivatives. Under proper conditions, amino,
carboxyl, hydroxyl, carbonyl, and thiol groups are converted to the
corresponding trimethylsilyl (TMS) ether or ester by trimethylsilylation
[23 ,24].(Eq.1 .1 ):

Iﬂ-Si(CH3)3
R R

(Equation 1.1)

 

enuﬂ
TBDI
ETOu;
GMT}
have
Orgar
Ykﬂd
THIN
anah

Shita

11
Silylation of seventeen of the amino acids was achieved in a closed

tube reaction in 15 min at 150°C using bis(TMS)-triﬂuoroacetamide
(BSTFA) as the silyl donor. However, 2.5 hours at 150°C was necessary for
the reproducible derivatization of glycine, arginine, and glutamic acid, and
similar reaction conditions were recommended for all twenty protein
amino acids [25]. Stable derivatives are also formed with glutamine and
asparagine, enabling their distinction from glutamic acid and asparatic
acid, respectively. The drawbacks are the inherent instability of the
resulting TMS derivatives toward hydrolysis. Furthermore, the excessive
silylation of nitrogen atoms lead to the formation of multiple derivatives for
amino acids [25-27]: especially glycine, glutamic acid, lysine, arginine,
histidine and tryptophan present difficulties [3]. Coinjection of excess TMS
reagent is required in order to block active sites of the columns. This is
often necessary for protection of N-TMS derivatives because
trimethylsilylamines and imidazoles are potent reagents themselves and
transfer the TMS group easily to hydroxyl groups [3].

The tert.-butyldimethylsilyl (TBDMS) function group was ﬁrst
employed as a more moisture stable alternative to the TMS group. The
TBDMS is now widely used for the silylation of hydroxyl and carboxyl
groups using N-methyl-N-(tert-butyldimethylsilyl)triﬂuoroacetamide
(MTBSTFA) as silylating reagent. The TBDMS derivatives were found to
have superior GC and mass spectral properties [28,29]. Multifunctional
organic acids were quantitatively converted to their TBDMS derivatives,
yielding a single peak for each organic acid [30]. In recent years, the
TBDMS derivatization has been successfully extended to amino acid
analysis, which have been shown to be suitable for assaying asparagine,

glutamine, and most of the other protein amino acids [23,31-37].

 

 

Quanti‘
dimcth)
spectral
Mass 5p
each am
predomir
moiety w
peaks are
the derive
step for m
is prerequ
amino aci.
Furthermor
Spectra of 1
potential {0
acid N-acyl

Sm'table for

 

 

12
Quantitative silylation has been achieved using TBDMS in

dimethylformamide (DMF) by heating at 75°C for 30 min [22]. Mass
spectral (EI, CI) analysis of TBDMS amino acids have been conducted.
Mass spectra display a characteristic and unique [M-57] fragment ion for
each amino acid which often dominates the EI mass spectrum [33], and
predominant ions in the spectra reﬂect losses of part or all of the TBDMS
moiety with or without the associated functional group [31]. However, two
peaks are obtained for arginine and either derivatization is incomplete or
the derivatives degrade in the chromatographic system [22]. A painstaking
step for moisture removal from the hydrochloride salts of amino acids still
is prerequisite for TMS derivatives. Meanwhile, relatively few nonproteic
amino acids have been chromatographed as the TBDMS derivatives.
Furthermore, it was pointed out that the de novo interpretation of the mass
spectra of TBDMS amino acid derivatives is fraught with somewhat more
potential for ambiguity than is the interpretation of the spectra of amino
acid N-acyl alkyl esters. Therefore, these derivatives are perhaps less

suitable for the identiﬁcation of unknown compounds [2].

c) 0thas

The cyclic oxazolidinone derivatives of amino acids are formed by
condensation with 1,3-dichlorotetraﬂuoroacetone at room temperature for
15 min in acetonitrile-pyridine solvent. Further treatment with either
pentaﬂuoropropionic anhydride (PFPA) or heptaﬂuorobutyric anhydride
(HFBA) in a benzene-methanol solvent for at least 30 seconds at room
temperature results in derivatives of protein amino acids that can be
separated in less than 10 min. Arginine, glutamine, and asparagine

cannot be analyzed without additional acylation in heptane at 80°C for 2-3

 

 

min [38,3S
reactions.
A
hexaﬂuorc
acid HFIP
one hour :
amino acic

purpose bu

Prior
isolated In
exchange te
There appe;

Maki
quantitativ
selective b1.
and Phenol
(iBuCF). ’
diazometha.
F” the chi
25% SOdiurI
shaken for

that it Was

 

Kim e

 

13
min [38,39]. Although the total reaction is not long, it actually involve three

reactions.

A modiﬁed procedure using a mixture of PFPA and
hexaﬂuoroisopropanol (HFIP) was introduced to provide N-PFPA amino
acid HFIP ester in one reaction [40,41]. The derivatization reaction needs
one hour at 100°C. These derivatives do not form stable anions for all
amino acids. They have been used for certain amino acids for special

purpose but are not recommended for general analysis.

d) Derivatization in an aqueous medium

Prior to conversion to suitable volatile derivatives, amino acids are
isolated from complex aqueous samples mainly by the multi-step ion-
exchange technique although its inherent drawbacks are well known [42].
There appears to be a need for improvement of sample puriﬁcation.

Makita et a1. [43-46] demonstrated that amino acids were
quantitatively extracted with diethyl ether from aqueous media after
selective blocking of the active hydrogens on the amino, thiol, imidazole,
and phenolic hydroxyl groups by reaction with isobutyl chloroformate
(iBuCF). The remaining carboxyl groups were then methylated with
diazomethane to form N(0,S)-isobutoxycarbonyl amino acid methyl esters.
For the chloroformate reaction, the amino acid solution was buffered by
2.5% sodium carbonate, iBuCF was added, and the reaction mixture was
shaken for 10 min at room temperature. Arginine was an exception, in
that it was converted into N-isoBOC ornithine methyl ester by treatment
with arginase, followed by the above derivatization procedure.

Kim et al. [47,48] combined Makita‘s N(0,S)-isoBOC procedure for the

selective puriﬁcation of protein (and non protein) amino acids from aqueous

 

samples,

amide or :
silylation

60°C for
reaction i
protein a
derivatizat
amino acic
and amide
derivatives

was exclud.

in aqueons

Huse
amino acids
be Perform
mhmn
and the an

ethyl (met

 

medium (6
SUIﬂIydryL
demaﬁz e d.
groups were

hm, 1.1

 

14
samples, and the TBDMS derivatization of the carboxyl and remaining

amide or aliphatic hydroxyl groups in the N(0,S)-isoBOC amino acids. The
silylation reaction producing optimal overall derivatization by heating at
60°C for 15 min in acetonitrile and MTBSTFA. The aqueous isoBCF
reaction is an alternative to the conventional ion-exchange clean-up for
protein amino acids from aqueous samples prior to the TBDMS
derivatization. The TBDMS derivatization of the resulting N(0,S)-isoBOC
amino acids offers advantages over the methylation, since polar hydroxyl
and amide functions as well as carboxyl groups are converted to TBDMS
derivatives which give better results for the GC analysis. Again, arginine
was excluded from the study.

e) One-step chloroformate derivatization of amino acids
in aqueous medium.

Husek [49-51] developed a one-step aqueous medium procedure for
amino acids derivatization which is uniquely rapid. The derivatization can
be performed in a few seconds, and the subsequent capillary GC analysis
can be carried out in a few minutes. The total time of sample preparation
and the analysis can be as short as 5 min. Amino acids were treated by
ethyl (methyl) chloroformate in a water-ethanol(methanol)-pyridine
medium (60:32:8) for 3-5 sec. In a single step, the amino, carboxyl,
sulfhydryl, phenolic, and imino group on the side chain of histidine are
derivatized. Carboxyls were converted to esters, while the other functional
groups were converted to N ,0- and S-eth(meth)oxycarbonyl derivatives (see

Figure 1.1 for the reaction schemes). Arginine analyses requires an

 

Anuno

RNH2 +

Carbox

RCOOH

 

Phenoh

Ph0H+t

Thiol gr

Riﬂii-CL

 

Figure

 

Amino group

R-NH2 + ClCOOR'

Carboxyl group

R-COOH + ClCOOR'

Phenolic group

Ph-OH + ClCOOR'

Thiol group

R-SH + ClCOOR'

15

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Carbamate (N—alkoxycarbonyl-)

R-NHCOOR'

Ester

R-CO0R'

Carbonate (0-alkoxycarbonyl-)

Ph-OCOOR'

Thiocarbonate (S-alkoxycarbonyl-)

R-SCOOR'

Figure 1.1. Reactions of chloroformate with functional groups of amino acid.

 

additional
procedure
has the be
derivatizatiu
comment in

was qouted

   
  
  
  
     
  
 
  
 
  

and are be

beneﬁt of

mass Spec‘
ionization t
(SIMS), {1
desorption“;

ionization

A

to create
Without thr
thermolysis
Successful,
need for c},

less Critica

__4

researCher:
important 1
I57].

16
additional step such as that in Makita‘s method [43-46]. A similar

procedure was also used to derivatize fatty acids [52-56]. This procedure
has the beneﬁts of simple sample handling, the ability to conduct
derivatization in aqueous solutions, and the use of inexpensive reagents. A
comment in an annual review, <Amino Acids and Peptides> V.24 (1991),
was qouted as: "N-acyl derivatives are continuing in use after many years,
and are being shadowed by N-alkoxycarbonyl derivatives that have the
beneﬁt of being formed from an alkyl chloroformate very rapidly".

III. Derivatization of peptides prior to analysis by fast atom bombardment
(FAB)-mass spectrometry

A. Introduction

Over the past ﬁfteen years, many new “soft ionization” techniques for
mass spectrometry have emerged, among them desorption chemical
ionization (DCI), ﬁeld desorption (FD), secondary ion mass spectrometry
(SIMS), fast atom bombardment (FAB), matrix assisted laser
desorption/ionization (MALDI), 252Cf plasma desorption (PD), thermospray
ionization (TSP), and electrospray (ES). The goal of all of these methods is
to create gas-phase ions of polar or thermally fragile molecules, often
without the introduction of excessive internal energy that would cause
thermolysis or extensive fragmentation. The methods have been extremely
successful. One might suppose that such methods would eliminate the
need for chemical derivatization; certainly the volatility enhancement is
less critical when using a desorption ionization method. However, many
researchers are ﬁnding that chemical derivatization can still play an

important role in increasing the sensitivity of ion production or analysis

[57].

 

I

E
electror
to incre
develop
LlAll‘l4
These
fragmer
derivatl
trimeth
formati
introduc
“'35 p05:

little to l

for ioni
TEprodm
han'ng S
hmas
Pmbe, er
and mat
prOcess.
ﬁOnvolati

FAB-MS (

17

B. Fast atom bombardment mass spectrometry [58-61]

Before the introduction of FAB-MS [62,63], in order to be analyzed by
electron impact mass spectrometry (El-MS), peptides had to be derivatized
to increase volatility and thermal stability. In the late 1950s, Biemann
developed a derivatization procedure using acetylation and reduction with
LiAlH4 to form polyamino alcohols which could be analyzed by GC-MS [64].
These derivatized peptides displayed improved volatility, and also
fragmentation of speciﬁc bonds in the peptide backbone. Later, this
derivatization procedure was modiﬁed to yield N-trifluoroethyl-O-
trimethylsilyl polyamino alcohols. Another procedure employed the
formation of N -acetyl-N,O-permethylated derivatives which were
introduced by direct probe insertion for analysis by EI-MS [65]. Although it
was possible to analyze peptides by EI-MS, mass spectrometry cOntributed
little to peptide analysis until the 1980s.

(1) Principles

Fast atom bombardment (FAB) provided for the ﬁrst time a technique
for ionizing nonvolatile samples that is both simple to use and gives
reproducible results. The FAB employs a neutral Ar or Xe atom beam,
having several keV of transitional energy, to sputter ions (secondary ions )
from a sample dissolved in a liquid matrix. When the fast atoms strike the
probe, energy is transferred to the matrix causing molecules of both sample
and matrix to undergo desorption and ionization. Figure 1.2 shows the
process. FAB-MS allows the desorption, ionization, and analysis of
nonvolatile and thermally labile compounds with derivatization. As result,

FAB-MS can be used to analyze peptides without derivatization.

18

 

Fast Atom

 

 

9 Atom Beam

“J

O
6

9 Secondary Ion Beam
6

ll K

f) f) '9 Mass Analyzer
Sample .9 '9 |

Mount

 

 

Figure 1.2. Schematic diagram for fast atom bombardment process.

 

compc
the 32
chaml
stable
sputte.
manm
aunple
by the
ﬁscous
addition
anahte
A
mepms
l0 sevex
detectit
from t
Certail

compo

not c0.
these
Plat—On;
”10160111

[‘lse/l'edg

19
Liquid matrices, such as glycerol, or other suitable viscous organic

compounds are mixed with the sample in order to maintain the ﬂuidity of
the sample during the introduction process into the high vacuum source
chamber and throughout the analysis period. The matrix promotes a
stable, reproducible ion current lasting for long periods of time. The
sputtering process leading to the production of ions is dependent on the
maintenance of a liquid surface. Figure 1.3 represents a closer view of the
sample surface. This ﬁgure illustrates the surface destruction of a sample
by the high-ﬂux particle beam and the desorbed species produced. The
viscous liquid matrix replenishes the destroyed area and provides
additional analyte to the sample surface. Without this renovating eﬂ‘ect, the
analyte signal would not last nearly as long nor be as stable.

Although the FAB matrix provides beneﬁts for desorption ionization,
the presence of a viscous matrix material in a high concentration gives rise
to several signiﬁcant problems. These include poor sensitivity and limit of
detection, high background ion counts at every mass, intense cluster ions
from the matrix, and ion suppression eﬁ‘ects whereby the formation of
certain ions from the sample are inhibited by the presence of other
compounds in the sample.

Although the mechanism of desorption and ion formation in FAB is
not completely understood, it is based on a combination of factors. Among
these mechanisms are desorption of preformed ions, including both
protonated and metal ion (e.g. Na“, K") adducts, and desorption of neutral
molecules followed by gas-phase ionization in the high-pressure region

[“selvedge”] directly above the vacuum-solution interface.

 

 

 

Glyce
of r‘

Fig
fas

(Ml

Fast Atom Beam

Glycerol Solutio
of Analyte

 

Probe Surface

 

Figure L3. View at surface of glycerol/sample solution during
fast atom bombardment.

(MH+: positive ions, (M-H)' : negative ions, N:neutrals, G: glycerol)

 

 

 

 

 

 

 

 

 

 

 

Fas
weight in‘
in the po
hachhone
chains a
retain ti
the C-u

is haset
upperi
fragme
shown

residu

fragn
ion in
adsor
most

indUCl

3111011]
Conce;
by the
inert g

the col

21
Fast atom bombardment of peptides provides abundant molecular

weight information, usually in the form of a protonated molecule, [M+H]+,
in the positive mode. Limited fragmentation of peptides occurs along the
backbone and at side chains, giving information about the amino acid
chains and the amino acid sequence of the peptide. Peptide fragments can
retain the charge at the N-terminus (producing an, b... cn, and dn ions) or at
the C-terminus (producing xn, yn, zn, vmand wn ions). This nomenclature
is based on a system proposed by Roepstorff [66] (labeling fragment ions by
upper-case letters) (Figure 1.4) and modiﬁed by Biemann [67,68] (labeling
fragment ions by lower-case letters). The structures of these ions are
shown in Figure 1.5, the subscript n represents the number of amino acid

residues present in the fragment.

(2) Tandem mass spectrometry - collisionally induced
dissociation (CID)

FAB is considered a soft ionization technique because very little
fragmentation occurs in the desorption process. Methods of supplying an
ion with enough energy to dissociate include collision with a surface,
adsorption of energy from a laser beam, use of an electron beam, and the
most commonly used method of collisions with an inert gas, collisionall
induced dissociation (CID).

The use of tandem mass spectrometry (MS/MS) can increase the
amount of structural information available from FAB ionization. The
concept is illustrated in Figure 1.6. In MS/MS, a precursor ion is selected
by the ﬁrst mass analyzer and directed into a collision cell to collide with an
inert gas, such as He or Ar, then the fragments (product ions) emerge from

the collision cell will be analyzed by the second mass-selective device to

 

 

 

 

 

H2N '

X3 Y3 Z3 x2 Y2 X1 Y1 Z]

‘51 el 152 el es el e
HzN-C-C-N—C-C-N-C-C -N-C—COOH
Hl lHlHl lHlHl lHlH
A1 3101 Aszcz A3 B1303

Figure 1.4. Peptide fragment ion designations proposed by
Roepstorﬁ‘ and Fohlman [66].

 

Fig“!
Spectr

o o
n u .
m-aa-c-macu-c-m—?e-coou (+u )
I

 

 

a n a
[ W m m]
o o
u u
woes-uniﬁed *oac-m-cu-c-m-cuwoou
a n n n
o o
u + . ll
mu-ou-c-m-oe—cao mu-ou-c-m-cn-coou
a a n a
o o o
u u , . ll
tau-cu-c.m-cu.c-m ca-c-m—c'm-coou
a a n a
,0 o
I , , u
workmen-$4 (+ H ) e5»:a4-c-m-?e.coou
a cm' a

0

ll
ﬁne-c-m—cu-coou (_+ H‘)
can- a

E]

Figure 1.5. Fragment ion structures commonly observed in FAB mass
spectra of peptides [61].

 

 

 

 

 

 

preside
require
hlS.'“.\lE
sith t
second
the ti
mass
lbetw
scam
magi

H1855

sou)
sax:
thei

& Sig

Vole.

Whei

Stiles

mOm

24
provide additional characteristic data for the precursor ion. It is this

requirement for two mass-selective devices that led to the terminology
MS/MS. With a triple quadrupole instrument, a precursor ion selected
with the ﬁrst quadrupole undergoes collisions and fragmentation in the
second quadrupole (collision cell), and the product ions are analyzed with
the third quadrupole. When a two-sector instrument is used for tandem
mass spectrometry analysis, CID occurs in the ﬁrst ﬁeld free region
(between the ion source and the ﬁrst sector) and analysis is performed by
seaming the electric and magnetic sectors with the ratio between the
magnetic and electric ﬁelds (B/E) held at a constant value determined by the

mass of the precursor ion [58,60].

(3) Instrumentation

All mass spectrometers consist of three basic components: the ion
source, the mass analyzer, and the detector. Ions are produced from the
sample in the ion source, the mass analyzer separates ions according to
their mass to charge ratio, m/ 2. Each ion strikes the detector and produces
a signal proportional to its relative abundance.

Singly charged ions with mass of m, subjected to an accelerating
voltage V, acquire a translation energy:

eV=(1/2) mv2

where e is the electronic charge and v is the velocity of the ion m+.

Double focusing mass spectrometers have an electric sector (E) for
selecting monoenergetic ions as well as a magnetic sector (B) to analyze the

momentum (and thereby the mass) of the ions.

25
\ | /

Ionization & Fragmentation

/ | \

Mass Analyzer I

Precursor Ion

J

:------

 

r
’ ---------

 

 

m/z E
x ,‘v
\ l /
Collisionally Induced Dissociation

/ / \
Mass Analyzer II H
l . l. l. .

m/z

Product Ion Spectrum

Figure 1.6. Tandem Mass Spectrometry (MS/MS).

 

the
ﬁel

pro

dis;

to-ch

stren

The 1

meme

lfBis

detect

“Mast:
these p
legethe]
strengtk

ln

Sect”), .

%

W

Ions leave the ion source with a small spread of kinetic energy due to
the ionization process and other factors. The electric sector with a ﬁxed
ﬁeld E deﬂects the ions in a circular path. The deﬂection radius, Re, is
proportional to the energy. The electric sector acts as a device for
dispersing ions according to their kinetic energies.

eE=mv2/R.,=2eV/R.3

Magneticsecm:

A magnetic analyzer separate ions according to their relative mass-
to-charge ratios. When accelerated, m ions enter a magnetic ﬁeld of
strength B, the ions follow a circular path of radius Rap

R1n=mvleB
The radius taken by an ion in a magnetic ﬁeld is proportional to its
momentum. Combing these equations gives:

m/e=Rm2B2/2V
If B is scanned at ﬁxed value of Rm, ions of different m/z will pass through a

detector slit to give a mass spectrum.

Linkmugms

Double focusing instruments can be scanned in special ways so that
metastable ion decomposition of a precursor ion can be observed. To obtain
these product ions, a B/E linked scan in which both B and E are scanned
together, such that the ratio of the magnetic ﬁeld strength and electric ﬁeld
strength is held constant.

In the lst ﬁeld free region (between the ion source and the electric

sector), fragmentation is induced by collisional activation to produce

 

Du

ens
anc'
whe

ast

sect<

thus,
Simil

are:

31)ng
Comb

magne

ofBE
of the

dElEClec

27
product ions (m2+, m3+, m4+,...) from a precursor ion ml" (with Mn, Mn',

Mn" as the neutral loss):
m2+ + Mn
m1+ ------- > m3+ + Mn’
mi + M.”
During fragmentation, m1+ discharges internal energy and adds kinetic
energy. However; this energy is known to be approximately 1 eV or less,
and is very small compared with the initial kinetic energy of m1+ (10 KeV
when accelerated by 10 kV). The velocity of m2+ is approximately the same
as that Of m1+ (Mn: neutral loss).
When the ion m1+ fragments to m2+, the conditions of the electric

sector to pass m1+ and m2+ are:

for m1+ eEl = mlvlz’Re

for m2+ eE2 = m2v12/Re
thus, it can be easily shown that m2/m1 = E2/E1
Similarly, the conditions to pass m1+ and m2+ through the magnetic sector
are:

for m1+ eB1 = m1v1/Rm

for my eB2 = m2v1/Rm
giving rise to the conclusion: m2/m1 = B2/Bl
Combining the two equations m2/m1 =E2/E1 =B2/Bl thus

B1/E1 = B2/E2 = constant

With this method, by simultaneously scanning (linked-scanning) the

magnetic ﬁeld strength B and electrostatic ﬁeld strength E so that the ratio
of B/E is maintained constant while the m1+ precursor ions are detected, all
of the product ions that are generated from the precursor ion can be

detected.

 

in
at

$11

50!

int:

3118

adv:
dete.
stru<
more
are l:
deriv
large
shoul

deﬁvz

Z

C. Derivatization of peptides prior to analysis by FAB-MS

Fast atom bombardment mass spectrometry has made a signiﬁcant
impact on the approach to the structure analysis of proteins. But, in fast
atom bombardment mass spectrometry of peptides, some factors prevent
successful detection and sequence analysis of peptides. One of them is the
poor ion production and detection (ion desorption/ionization efﬁciency) of
some hydrophilic peptides, and the second factor is the ambiguity in
interpretation of the spectra when key sequence ions are weak or absent.

Investigators [69-72] soon realized that chemical modiﬁcation of an
analyte to enhance its hydrophobic and/or ionic character improved its
signal-to-background ratio during FAB analysis.

In the analysis of peptides by FAB-MS, there are two independent
advantages that result from derivatization: (i) enhancement of
detectability, and (ii) modiﬁcation of fragmentation (enhancement of
structurally informative ions). These advantages result from forming
more hydrophobic and/or precharged derivatives. Certainly, researchers
are looking for derivatization methods to fulﬁll both of these aspects. Useful
derivatization should be speciﬁc, modifying only the intended portion of the
target molecule, and yield a single product. The modiﬁcation procedure
should be fast, simple, and applicable to small sample sizes. Ideally, the
derivatization reagent should be safe and stable.

 

ill
rna

lay

desi
cont
anal
hydi

mole

inter
throc
comp
surfa
Thus.

molec

alkyl .
siren:
COrres
ObServ

Wen

29
(1) Enhancement of surface activity

The penetration depth of the incident particle into the liquid is
believed to lie within 50 to 100 angstroms of the gas/liquid interface (73).
When the sample volume is approximated as a portion of a sphere, the
maximum depth of the sampled volume is equivalent to ﬁve molecular
layers (74). All the processes associated with the energy deposition and
collision cascade are centralized near the surface of the matrix. Since
desorption depends on sputtering from the matrix surface, the surface
concentration of the peptide is an important factor. The capability of an
analyte to occupy the surface area is dependent upon interactions (e.g.,
hydrophobic, hydrophilic, hydrogen bonding.) between the matrix
molecules and the analyte. Most of the matrices tend to be hydrophilic in
nature. The more hydrophilic compounds, which are surface inactive, can
interact favorably with the matrix molecules and distribute themselves
throughout the entire matrix volume. Meanwhile, the more hydrophobic
compounds, which are surface active, move themselves towards the
surface and away from the interaction between the matrix molecules.
Thus, the surface-active compounds will have a higher concentration of
molecules near the surface than surface-inactive compounds.

When working on a series of related surfactants differing only in
alkyl chain length by LSIMS, Ligon et. al. [75] observed differences in signal
strength attributable to differences in surface activity; the spectra
correspond to real surface concentrations. Ligon concluded that the
observed ratio between two analyte molecules will depend on their bulk

concentration and on their relative surface activity.

 

93C
C0l’
hid
Sun
has a

 

 

so
Physical and chemical parameters such as pH, surface tension, and

the hydrophilicity/hydrophobicity of the matrix, charge, size, stability, and
solubility of the peptide aﬂ'ect its surface activity [76].

Clench et. al. [77] found that if two dipeptides with grossly diﬁ‘ering
surface activity were present in a mixture, the one with lesser surface
activity would not be observed. In the positive FAB spectrum of a 0.01 M
Ala-Gly and 0.001 M Phe-Leu, the MH+ of Phe-Leu dominated the mixture
spectrum despite the fact that Ala-Gly was present at 10 times, the
concentration of Phe-Leu.

Naylor et. al. [78] pointed out the limitation in the use of FAB-MS for
analysis is that the hydrophilic peptides in a mixture are suppressed. (i)
The hydrophilic peptides alone give a relative poor signal response. (ii)
Hydrophilic peptides are further suppressed in the presence of hydrophobic
peptides that initially occupy the surface of the matrix. These attributes
result from the intrinsic tendency of even a pure hydrophilic peptide to
avoid the matrix/"vacuum" interface; in a mixture of hydrophilic and
hydrophobic peptides, the hydrophobic components occupying the interface
tend to suppress any signal that might otherwise arise from the low
concentrations of the hydrophilic component at or near the surface. (iii)
Hydrophilicity/hydrophobicity index (AF values) can be used to indicate
which peptide may be suppressed. These indices for peptides are calculated
by adding AF values of Bull and Breese indices for amino acids [79], and
each sum is divided by the number of amino acids in the peptide. This
correlation of the FAB mass spectrometric sensitivity to a
hydrophobicity/hydrophilicity scale seems useful to predict the peptide
suppression. The peptide that is suppressed in the analysis of the mixture

has a more positive AF value.

 

fl

lllC

ina;

hyd
laye
hydi
anal
wate
sens
facto
layer
Pepti
activi
homo
lﬁﬂer

mixtu

31
The scale of Bull and Breese is derived from the preference or

reluctance of an amino acid to transfer from aqueous solution to an air-
water interface. Bull and Breese [79] measured the surface tensions of
amino acids in 0.10 M NaCl as a function of the concentration of the amino
acids at 30°C. From the experimental results, the free energies of transfer
of the amino acid residues from the solution to the surface have been
calculated to yield a hydrophobicity index for each residue. (Table 1 .2 ) The
more positive the index, the more hydrophilic is the amino acid.
Furthermore, the hydrophobicity/hydrophilicity index of a peptide
may not be sufﬁcient in itself to predict ion suppression effects. A number
of physical/chemical properties of peptides, including their hydrophobic or
hydrophilic nature, play important roles in their tendency to occupy surface
layers of sample and therefore their capacity to form ions by FAB-MS. For
hydrophilic peptides, other factors such as charge state of the peptide in the
analysis matrix and its tendency to form secondary structure in the
water/glycerol solution in addition to the hydr0philic index affect the
sensitivity of the FAB measurements. Whatever the balance of these
factors, it is clear that competition of the various species for the surface
layers of the liquid sample remains the dominate factor [59]. One goal of
peptide derivatization for analysis by FAB-MS is to enhance the surface
activity of hydrophilic peptides. Derivatization inherently increases the
homogeneity of molecular mixture. This technique can equalize
differences in surface activity for individual components of a peptide

mixture.

 

Leu

Met
Phe

Table 1.2. HydrOphilicity / hydrophobicity index (AF) of amino acid [79].

 

 

Amino Acid AF (call mole)
Ala +610
Arg +690
Asn +890
Asp +610
Cys +360
Gln +970
Glu +510
Gly +810
His +690
Ile ~1450
Leu -1650
Lys +460
Met -660
Phe -1520
Pro -170
Ser +420
Thr +290
Trp -1zoo
Tyr 4430
Val ~750

 

 

 

by
all

3m

COD

 

33
(2) Derivatimtion methods by other investigators

a) Forming hydrophobic derivatives

Various methods have been employed to increase the FAB signal
response of hydrophilic peptides. One successful approach used to increase
the FAB signal response has involved formation of hydrophobic derivatives
of hydrophilic peptides. These derivatization methods include a variety of
peptide modiﬁcations, ranging from esteriﬁcation to the attachment of a
single hydrophobic moiety to a speciﬁc site on the peptide

Although not widely used, esteriﬁcation can greatly reduce the
detection limits of hydrophilic peptides. Falick, et. al. [80] derivatized the
hydrophilic peptides for LSIMS by preparing alkyl and benzyl esters of
peptides. He reported a factor of 25 increase in sensitivity of hydrOphilic
peptides at 20 pmol level (Thr-Lys-Pro-Arg). The derivatives were prepared
as follows: Peptides collected from the HPLC were lyophilized. A volume of
5 ul of dry alcohol 0.2 M in acetyl chloride was added. The reaction was
allowed to proceed for 1 hour at 45°C. Relative yields of MH" ions from
peptides esteriﬁed with various alcohols (methanol, 2-propanol, 1-butanol,
1-hexanol, l-octanol, and benzyl alcohol) were compared. Although the
signal-to-background ratio increased for peptide esteriﬁed with alcohols of
increasing alkyl chain length, the best combination of ion yield and ease of
reagent removal was obtained with l-hexanol. The alkyl chain increased
the surface activity of peptides and eliminated the discrimination against
hydrophilic peptides. A mixture of peptides of different hydrophilicity was
analyzed after derivatization. The procedure did not affect side-chain
amides. Partial derivatization was sometimes observed with peptides

containing more than one carboxyl group.

34
Naylor et. al. [78] converted the peptides to their isopropyl esters by

esteriﬁcation with 1 M HCl in 2-propanol (at 37°C for 24 h). After the
esteriﬁcation, the more hydrophilic derivatives were detected in the
complex enzymatic digests of proteins. This was possible because the
derivatization converts -COz' to -COOCH(CH3)2’ which will produce a
maximum index change of about -1000 (the difference in the Bull and
Breese value for aspartic acid and leucine). '

Ligon et. al. [81] derivatized several dipeptides by treating them with
dodecanal to attach a long hydrocarbon tail on the peptides to increase their
hydrophobicity. It was also pointed out that the term "surface activity"
included such phenomena as hydrophobicity, solubility, and solute-induced
variations in surface tension. The more polar peptides were improved by
derivatization. The samples were prepared by treating an aqueous solution
(30 [11, 0.1 M) of each dipeptide with an equal volume of a 0.1 M solution of
dodecanal dissolved in methanol. The resulting solution was warmed to
the boiling point of methanol. Under the conditions, dodecanal forms a
Schiff base or imine with peptides having a primary amine function.

The analysis is greatly complicated if the aldehyde contains
homologues.‘ The analysis may fail entirely if the aldehyde has been
partially oxidized to the corresponding acid, which can by itself entirely
dominate the SIMS spectrum. Furthermore, it was found that relative
hydrophobic peptides (and probably most large peptides) may not beneﬁt
from this derivatization.

Chai and Zhao [82,83] reported the analysis of positive FAB of seven
amino acids, dipeptides, and tripeptides as di-isopropylphosphorylated
derivatives. Results showed an improvement insensitivity by factors of 4-

29, mostly above 10, for the derivatized amino acids compared to that for the

35
underivatized ones. Also improved sensitivity and decreased background

noise from the glycerol matrix were observed after derivatization of
peptides. It is probably because the combination of the enhanced surface
activity and increased proton afﬁnity by the derivatization. N-terminal
fragment ions dominated the fragmentation of N-diisopropyloxy phosphoryl
derivatized peptides, giving evidence of directed fragmentation. Derivatized
peptides also displayed suppressed fragmentation of amino acid side
chains. The dialkylphosphite reagent used for derivatization also can be
used for peptide synthesis [84] and more studies are underway for this
derivatization [85].

Baillie and Nelson [86-88], in their work on glutathione conjugates by
FAB/MS, derivatized the amino terminus with ethyl or benzyl
chloroformates and methylated the carboxyl group with HCl/MeOH to form
the alkyloxycarbonyl (ethyl or benzyl) methyl ester. The derivatization
procedure using chloroformate reagents was similar to that for amino
acids [43,46]. (pH 9 by 0.5 M Na2CO3 and 0.1 M NaHCO3, vortex for 1 min.
The reaction was allowed to proceed at room temperature for 30 min.) The
methylation was completed after 30 min at room temperature by
methanolic HCl. The chloroformate derivative facilitated the puriﬁcation of
the conjugate which had better chromatographic properties in HPLC, and
showed informative mass spectral fragmentation under CID conditions.
N-benzyloxycarbonyl derivatized glutathione had higher sensitivity in FAB-
MS detection compared to that of an underivatized species.

The two examples above [82,88] also demonstrate the effect of

derivatization for modiﬁcation of fragmentation of peptides.

 

(modiﬁ
P
chlorid
(Bdbs-C
NaHCC
acetone
at 40°C
acidifyin
contrast
derivatis
Which rel
of the pe;
Roi
addition
glass tip
acetylatic
(SO-70%.
0f the sir
Peptides l

 

Was facii;
Thh

imerprec

the C~te

leaving ll

 

36
b) Enhancement of structure informative fragmentation

(modiﬁcation of fragmentation)

Renner et. al. [89,90] made derivatives of peptides with dansyl
chloride (DnsCl) or 2-bromo-5-(dimethylamino)benzensulfonyl chloride
(Bdbs-CI) at the amino terminus. The peptide (1 nmol) is dissolved in 0.2 M
NaHCO;; (60 pl). After addition of a 50 mM solution of Dns-Cl or Bdbs—Cl in
acetone (mole ratio peptide: reagent=1 :3), the mixture is incubated for 1.5 h
at 40°C. The procedure is followed by distilling off the acetone and
acidifying with 10% H3PO4. The derivatized peptide is isolated by HPLC. In
contrast to those of underivatized peptides, the spectrum of the dansyl
derivatives exhibit intense and structure-speciﬁc cleavage patterns, all of
which relate to the derivatized end of the molecule and result from cleavage
of the peptide bond.

Roepstorff [91] did an on-probe acetylation of the peptides by the
addition of acetic anhydride with careful mixing into the matrix with a
glass tip at ambient temperature for 10 min. The estimated degree of
acetylation for some tri- to hexa-peptides were 40-100%, most of them were
60-70%. The acetylation conditions were insufficient for effective acetylation
of the side-chain amino group of lysine. The spectrum of the acetylated
peptides gave more sequence information than the spectra of the free
peptides; especially the assignment of the N-terminal amino acid residue
was facilitated by the acetylation.

The C-terminus derivatization of a peptide is also useful in the
interpretation of MS/MS spectra because of the selective mass shift of all of
the C-terminal fragments relative to the underivatized peptides, while

leaving the N-terminal fragments unchanged [78,80].

 

 

spel
PUKI
proc
grou
pepti
befbr

incre:

surﬂac
molecr
gas pl
cation:
"prefor-
of large
C
been 81
enhance
charge
anlino a
appear-a]
elucidatj
When a
more ab .
ions are

fragment

 

37
To increase the amount of sequence information from FAB-MS

spectra, peptides converted to polyamino alcohols were analyzed by tandem
FAB-MS [92]. The peptide YAGFL was reduced using diborane. In this
procedure, the amide groups were converted to amines and the carboxyl
group was reduced to an alcohol. The MS/MS spectra of the reduced
peptide had complete b... y... and zn ion series, which were incomplete
before reduction. Therefore, the simplicity of spectral interpretation was
increased markedly with reduction of peptides to polyamino alcohols.

The "preformed" ions produced with peptide modiﬁcation are more
surface active in the FAB matrix than the unmodiﬁed analytes. If
molecules carry a charge initially, they are thought to be sputtered into the
gas phase directly from the matrix by the primary beam. Protonation,
cationation with metal ions, and chemical modiﬁcation to produce a
"preformed" charge are derivatization techniques to enhance the analysis
of large, highly polar, and nonvolatile compounds.

Chemical derivatization to introduce a charge into a molecule has
been suggested as means to improve the sensitivity in FAB-MS and to
enhance the detectability of molecular-weight related ions. The localized
charge has a stronger effect in directing fragmentation than any basic
amino acids that may be present in the peptide, drastically altering the
appearance of the MS/MS spectra of peptides [93] and facilitating structure
elucidation by enhancing the formation of structurally informative ions.
When a preformed charge is introduced into peptides, an and (In ions are
more abundant for N ~terminally derivatized peptides, while vn, yn, and Wu
ions are formed when peptides are derivatized at the C-terminus. The

fragmentation pattern produced by derivatization with a ﬁxed charge

38
simpliﬁes mass spectral interpretation. There are a number of

derivatization schemes which attach a ﬁxed charge to peptides [58,93-100].

c) Functional group determination and amino acid
detection

The presence of functional groups in a peptide can be identiﬁed by
measuring the mass shift after reacting the peptide with a site-speciﬁc
reagent.

In order to distinguish between the isobaric Gln and Lys residues, as
well as to conﬁrm a proposed structure, Kausler [101] methylated free
carboxylic groups by treatment of methanol/H01, resulting in a mass shift
of 14 u. Peptides were mixed with 100 pl methanol/2 N HCl and after
heating at 100°C for 1 hour, the sample was lyophilized. The amide groups
of Asn as well as Gln were also quantitatively converted to esters, indicated
by a mass shift of 15 u per amide function.

Fragmentations of N-benzyloxycarbonyl-protected tripeptide ethyl
esters have been investigated by negative-ion FAB-MS [102]. A signiﬁcant
difference was found among the intensities of the fragment ions formed by
cleavage of the benzyloxycarbonyl group, depending on the numbers and
positions of proyl residues in the derivatives. So the fragmentation pattern
of N-benzyloxycarbonyl-protected tripeptide ethyl esters can be used to
predict the numbers and positions of proline residues in the peptides.

FAB was employed to identify and quantitate dansyl amino acids
obtained in the N-terminal analysis of proteins [103]. After a time-
consuming dansylation process, the protein was hydrolyzed for 12 hours at
105°C with 6 N HCl. The spectra of N-terminal dansyl amino acids are
characterized by protonated molecules and fragment ions produced by

IQ
cleavage of the bonds on either side of the sulfanyl group. The dansyl

amino acids can be determined quantitatively at a level of 0.1 nmol with the
response being a linear function of concentration up to approximately 10-20

nmol/ill.

dJOthers

The t-butyloxycarbonyl (t-BOC) protected peptides [104,105] and amino
acids [106] were investigated. Prominent peaks due to subsequent loss of
C4H3 [M+H-56]+ and C02 [M+H-100]+ are always accompanied with
[M+H]+ ions. The [M+H]+ ion abundances of t-BOC peptides are smaller
than those of the corresponding underivatized species. Due to the facile
fragmentation of the t-BOC group, especially during CID-MS/MS, the
spectra are more complex and yield less sequence information than those of
the underivatized peptides. Fragmentations of benzyloxycarbonyl protected
amino acids and peptides with FAB ionization have also been investigated

[107].

N.Reseamhobjectives

This dissertation focuses on: (1) derivatization-assisted amino acid
analysis by GC-MS in El, positive CI and ECNI modes and (2)
derivatization-assisted peptide analysis by FAB-MS.

For the analysis of amino acids, the one-step aqueous medium
chloroformate derivatization method introduced by Husek [49-51] for
analysis by G0 has been extended to analysis by GC-MS, and the
structurally diagnostic fragmentation of the amino acid alkyl
chloroformate derivatives in EI-MS was also studied. Modiﬁcation of the

derivatization procedure has been conducted. An extended examination of

40
the derivatization method with the combination of a variety of alkyl

chloroformates and alcohols has been carried out. Fluorinated derivatives
prepared with a modiﬁed reaction procedure, in combination with ECNI
analysis has been evaluated for increasing the sensitivity of analysis. A
collaborative research project to quantitatively assess the incorporation of
stable isotope-labeled amino acids into photosynthetic proteins with the
chloroformate derivatization has been carried out.

For peptide analysis, investigation of the chloroformate derivatization
for small peptides prior to analysis by FAB-MS to enhance the FAB signal of
peptides, and a comprehensive evaluation of the derivatization conditions
have been completed.

The advantages and the limitations of the one-step aqueous medium

chloroformate derivatization method have been further investigated.

V. References

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Chapter II
Simultaneous derivatization of functional groups of amino acids by an
aqueous medium chloroformate reaction prior to analysis by GC and
GC-MS

I. Introduction

The one-step aqueous medium ethyl chloroformate derivatization
procedure for analysis of amino acids by GC [1 -3] is a very attractive method
based on the criteria for a hypothetical, ideal procedure for amino acid
determination by GC. In this procedure, using an aqueous medium
containing water / ethanol / pyridine, in a single step, the amino, carboxyl,
sulfhydryl, phenolic and imino groups on the side chain of histidine are
derivatized by ethyl chloroformate. Carboxyls are converted to esters, while
the other functional groups are converted to N,O- and S-ethoxycarbonyl
derivatives (Figure 2.1 ). I

This chapter includes: 1) characterization of N-ethoxycarbonyl ethyl
esters of amino acids by EI mass spectrometry; 2) a modiﬁcation of the
reaction procedure; 3) an extended examination of the derivatization
method with a combination of a variety of alkyl chloroformates and
alcohols; and 4) comparisons of El, PCI, ECNI ionization for the
ﬂuorinated derivatives of amino acids formed through a modiﬁed reaction

procedure.

Amino group

R-NH2 + ClCOOR'

Carboxyl group

R-COOH + ClCOOR'

Phenolic group

Ph-OH + ClCOOR'

Thiol group

R-SH + ClCOOR'

‘Jv

 

 

Carbamate (N-alkoxycarbonyl-)

R-NHCOOR'

Ester

R-COOR'

Carbonate (O-alkoxycarbonyl-)

Ph-OCOOR'

Thiocarbonate (S-alkoxycarbonyl-)

R-SCOOR'

Figure 2.1. Reactions of chloroformate with functional groups of amino acid.

50
II. Characterization of N-ethoxycarbonyl amino acid ethyl esters by El

massspectmmetry [4]

A. Experimental

A solution of 20 amino acids in 0.1M HCl at a concentration of 0.5
rig/pl each was prepared. The N(O,S) ethoxycarbonyl ethyl ester (ECEE)
derivatives of amino acids were prepared by adding 10 pl of the amino acid
mixture to a H20 / ethanol (EtOH) / pyridine (Py) (50 pl / 30 ul / 10 ul)
solution. Ethyl chloroformate (EtCF) (5-10 ul) was then added and the
reaction mixture was vortexed for 5-10 8 and extracted with 100-200 ul
CHCl3. A 1-ul aliquot of the CHCl3 layer was injected for analysis.

Analyses by GC-MS were carried out on a JEOL AX-505H double
focusing mass spectrometer coupled to a Hewlett-Packard 5890J gas
chromatograph. GC separation was achieved on a DB-1701 (15-m length x
0.25-mm i.d.) fused silica capillary column with a 0.25 am ﬁlm coating
from J. & W. Scientiﬁc (Rancho Cordova, CA). Direct (Splitless) injection
was used. Helium gas ﬂow was approximately 1 ml/min. MS conditions
were as follows: interface temperature 275°C, ion source temperature ca.
150-20000, electron energy was 70 eV, scan rate of the mass spectrometer
was 1 s/scan over the range of m/z 50-500.

B. Results and discussion

Interpretation of the spectra of this family of new derivatives can
facilitate recognition of individual amino acids.

Table 2.1 lists the mass value of the characteristic peaks in the
spectra of individual amino acid derivatives. The main fragmentation
pathway for EtCF-EtOH amino acid derivatives are shown in Figure 2.2,
two fragmentation routes (a and b) are possible through the rupture of a

 

 

+
NHCOZEF"
12’; :~ 002m
‘5 '6
-'R1 8 -’COzEt b
‘l'NHCOZEt +NHCOZEt
KC02Et R/l
m/z 174 M'73
-72 l -72
+NH2 4‘er
l\CO2Et R)
m/z 102 M145

Figure 2.2. Main fragmentation pathways for amino acid EtCF-EtOH
derivatives in EI mass spectrometry

 

 

 

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as 5.. can an an 95.
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3 sum .ﬁm o3 as m: 93
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8
.82 .m2 .m: 8 a: a: 8n n: 8m
8 8 m3 as mu m: .am
8 2 m3 «3 as m: 8a
«S 8 8H s: as 5 s:
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muses 0:38 we $333.86 moﬁéoum mo 38on 398 E 5 $33 :3 onerous—“~30 Ad 03a“.

53
carbon-carbon bond or to the amine group. Generally, path b is preferred

over a because 'COgEt, the fragment having higher ionization energy
compared to that of the alkyl chain, is favored energetically to retain the
unpaired electron and to become the neutral product. Subsequent
fragmentation of the even-electron ions (EE+) thus formed is dominated by
the loss of 72 u ('COgEt - H'), giving rise to ions m/z 102 and [M-145],
respectively. For detailed fragmentation pathways for each type of amino
acid (aliphatic, cyclic, hydroxyl, sulfur-containing, acidic, basic, and

aromatic amino acids) see Ref. 4.

III. Modiﬁcation of the reaction procedure
A. Background of chloroformate chemistry
(1) Reaction with amino and phenolic groups

Chloroformates have been used as the protecting group for N-
terminus during peptides synthesis. The alkoxycarbonyl derivatives
formed can be cleaved under mild conditions (by careful alkaline
hydrolysis) [5,6]. Benzyl- and tert.-butyl chloroformate are generally used
as reagents [5].

In analytical chemistry, chloroformates have been used extensively
to treat amino, phenolic hydroxyl groups. Derivatization of amino groups
in basic aqueous solution by chloroformates has been a commonly used
approach (as reviewed for derivatization of amino acids in chapter I [7-11]).
Other than those discussed in chapter I, applications for the derivatization
of catecholamines in aqueous solution by methyl chloroformate have also
been reported [12,13].

Chloroformate derivatization has also been applied in LC analysis.

One example is that amino acids were derivatized at amino groups by 9-

54
ﬂuorenylmethyl chloroformate (FMOC-Cl) separated by reverse-phase

HPLC and quantitated based on the ﬂuorescent properties of the derivatives
[14-17]. The protein hydrolysates were dissolved in 0.1 M sodium
bicarbonate, pH 8.0. The derivatization was achieved by the addition of 4.0
mM solution of FMOC-Cl in dry acetone. The mixture was quickly shaken
and the reaction was allowed to proceed for 10 min at room temperature
(22°C). The excess reagent was removed by extraction with pentane/ethyl
acetate (90:10) [14].

A slightly different derivatization procedure was used by Betner [15].
Borate buffer (0.5 M boric acid solution adjusted to pH 7.7 with 30% sodium
hydroxide solution) was used. The reaction mixture was incubated for 45 s
after vortexing. 1-aminoadamantane (ADAM) of 40 mM in water-acetone
(1:3, v/v) was added to react with the excess reagent by incubation for at
least 45 8. An advantage of using 1-aminoadamantane (ADAM) was that
the risk of partial extraction of the hydrophobic amino acids into the
organic phase was eliminated [15].

Chiral chloroformates as reagents for the resolution of metoprolol
enantiomers [18], separation of amino acid enantiomers and chiral amines
using pre-column derivatization with (+)-1-(9-ﬂuorenyl)ethyl chloroformate

[19] by reversed-phase liquid chromatography are also in the literature.

(2) Reaction withcarboxylgroups
Previously, the only application of chloroformate to react with a
carboxylic group was the formation of the mixed anhydride as an activated
intermediate to separate carboxylic acid enantiomers by GC [20], and to
determine the enantiomers of indoprofen [21] and ketoprofen [22] by HPLC.

Both cases used ethyl chloroformate.

55
In 1990, Husek demonstrated a method to derivatize carboxylic

groups in fatty acid analysis [23-27], and to derivatize both amino and
carboxyl groups in one reaction in an aqueous medium for analysis of
amino acids by GC [1 -3]. In this method, Husek derivatized short-chain
fatty acids by methyl and ethyl chloroformate (MCF and EtCF) to form
esters or alkoxycarbonyl esters which were analyzed by GC. The esters can
be formed instantaneously and almost quantitatively, at least from the
standpoint that no other volatile derivatives were produced. The reaction
can proceed in both non-aqueous solvent and in solvent containing water
[23]. In the case of non-aqueous solvent: 5 ill of the appropriate
chloroformate was added and the reaction medium was shaken for 2-3 s.
The medium consists of acetonitrile/pyridine/alcohol (methanol, ethanol) in
volume ratio of 22:2:1. In the case of solvent containing water: to 50 ul of a
water-pyridine (5:1) solution was added 50 ul of an acetonitrile-methanol
(5:1) solution for MCF treatment, or 50 pl of an acetonitrile-ethanol (2:3)
solution for EtCF treatment. The derivatizations were believed to proceed
through the mechanism of decarboxylation of the intermediates: the mixed
anhydrides.

Chloroformates have been known in organic chemistry as a possible
source of mixed carboxylic-carbonic anhydride formation since the
beginning of this century [28]. These reagents and the thermal
decomposition of mixed carboxylic-carbonic anhydride were intensively
investigated in the 19508 and 19608. Esteriﬁcation of carboxylic acids using
chloroformates through mixed carboxylic-carbonic anhydrides with new

catalysts was investigated in the 1980s.

 

The reaction of a chloroformate with a carboxylic acid leads the
formation of an ester as the main product when heating and / or acidic or
basic catalysts are employed [6]. The mechanism and kinetics of the
thermal decomposition of the intermediate mixed carboxylic-carbonic
anhydride have been investigated [6,29-35]. The mixed anhydrides are
stable at room temperature, but decompose around 150-170 °C [32] in the
absence of catalysts, and at lower temperature and faster in the presence of
catalysts [29]. The decomposition proceeds by paths A and B (Figure 2.3).
For certain mixed anhydrides, when the temperature is 250°C, however,

the ester is the only decomposition product (path C) [34].

A
—> R OR' + (:02

liaise—

B
—> (R 0),,0 + R'OCOR' + (:02

l (3

R301? + 002

 

Figure 2.3. Thermal decomposition pathways for mixed anhydride.

Tarbell [6,29,30] suggested that the decomposition of mixed benzoic n-
butyl carbonic anhydride is through a series of ionic chain reactions which
are initiated by catalysts acting as nucleophiles. (Figure 2.4) OR‘ generated
according to (1) and (2) is the chain carrier; it can attack at either carbonyl,
as in (3) and (4) to generate products. C5H5COO' generated in (2) and (4)
attacks unchanged anhydride at the carboxylic carbonyl to form the acid

57
anhydride in (5). The decomposition according to both path A and B

proceeds by a single rate-determining step [29]. This rate-determining
stage of thermal decomposition is the attack of nucleophile 3:, according to
(1) and (2). Reactions (3) and (4) are very fast. For this compound, the
proportions of products (path A and B) are not altered by changes in solvent,
temperature, or presence of catalysts.

It was also pointed out that for CsHsCOOCOOR, path A is favored
when the point of attachment of the alkyl group (from chloroformates) is a
secondary or a primary carbon with extensive substitution on the B-carbon
[32].

Another researcher, Windholz [34], reported that for a mixed
anhydride RlCOOCOORz, structures of both carboxylic (R1) and carbonic
(R2) components have directing inﬂuences on the possible path of
decomposition. A number of mixed anhydrides derived from aliphatic
acids decompose exclusively by path A while aromatic derivatives tend to
decompose equally along path A and path B. The differences were
explained by steric factors. The directing inﬂuences of alkyl radicals R2
have also been examined. Experiments indicated that the more electron-
releasing isopropyl group favors easier decomposition by path A, while
phenyl as R2 favors path B. For benzoic ethyl carbonic anhydride, it was
reported that heating the mixed anhydride in the presence of triethylamine
hydrochloride lowered the decomposition temperature and favored ester
formation. Boron trifluoride etherate lowered the decomposition
temperature considerably and caused exclusively ethyl benzoate formation
[34]. Various catalysts have been investigated; the results have some
controversy. Overall, the outcome of the mixed anhydride decomposition

reaction was disappointing as a means for the preparation" of esters.

58

CsHs'g-O- 'OR + B:- = C6H5-Ji'0' 'OR (1)
B

l

(.3ng83 + '0' '0R

1

co2 + OR‘

Cngg-O- '0R + B2. = 06H580°£0R (2)
/ B \

CsHs-g-O- -B + OR’ CsHs-g-O' + B-C-OR

06115-80- -OR + OR" —» 06115- -OR +002 + OR“ (3)

CsHs‘g‘O‘g'OR + 0R’—--- CGH5- -0' + R - -OR (4)
C61'I5-g-0- -OR + C6H5-g.0’ ——>C6H5-g-O- -CgH5 + 002 +OR- (5)

Figure 2.4. Mechanism for mixed carboxylic-carbonic anhydride thermal
decomposition proposed by Tarbell [29] - ionic chain reaction. "

In 1980s, several papers were concerned with forming esters of
carboxylic acids through carboxylic-carbonic mixed anhydride
intermediate by accelerating the reaction via path A [36-39].

Kim [36,37] demonstrated a simple and mild esteriﬁcation method for
carboxylic acids using chloroformate through the mixed carboxylic-
carbonic anhydrides. Typically, a solution of equal molar amount of acid,
alkyl chloroformate, and triethylamine in methylene chloride at 0°C was
added 4-(dimethylamino)pyridine (DMAP) in a catalytic amount. The '
resulting solution was stirred at 0°C for 30 min. Simple aliphatic
carboxylic esters were prepared in high yields by the reaction of acids with
equal molar amounts of various chloroformates and triethylamine in the
presence of a catalytic amount of 4-(dimethylamino)pyridine without
contamination of the symmetrical acid anhydrides and the carbonates.
Although aromatic acids gave a mixture of the ester, the acid anhydride,
and the carbonate under normal conditions used, it was found that
increasing the amount of 4-(dimethylamino)pyridine drastically decreased
the formation of the acid anhydride and the carbonate. The method
reached a limit with sterically hindered acids such as pivalic acid and
mesitoic acid. Pavalic acid yielded approximately a 1:1:1 mixture of ester,
anhydride and carbonate upon treatment with benzyl chloroformate. In the
case of mesitoic acid, exclusive formation of mesitoic anhydride was
obtained without the formation of a trace of the ester. The same method

was applied to esteriﬁcation of N-protected oc-amino acids which gained

high yields (88-98%) for ten amino acids.

so
The reaction mechanism was suggested as in Figure 2.5 which is

similar, but different, from that suggested by Tarbell [29,30] for thermal
decomposition of mixed anhydrides. The esteriﬁcation proceeds via a
mixed carboxylic-carbonic anhydride as the intermediate. The mixed
anhydride is converted into an acylpyridinium species (2a) by nucleophilic
attack of DMAP on the carboxyl carbonyl center of the mixed anhydride as a
major pathway along with an alkoxycarbonylpyridinium species (3b) by
nucleophilic attack of DMAP on the carbonate center as a minor pathway in
most carboxylic acids. The carbon dioxide evolution would provide a
driving force for the major pathway. Nucleophilic attack by the alcohol on
the acyl group of 2a gives the ester and DMAP, which is reused in the
formation of 2a. Also, the acid anhydride, which can be formed via
nucleophilic attack by the carboxylate anion of 3b on the carboxyl carbonyl
center of the mixed anhydride, can be converted into 2b by DMAP to raise
the yield of the ester, while the reaction of 3a with the alcohol affords the
carbonate.

In the case of aromatic acids, it is assumed that increasing the
amount of DMAP increases the reaction rate of conversion of the acid
anhydride into 2b and would, therefore, prevent the formation of the
carbonate by consuming the alcohol due to fast reaction of 2b with the
alcohol. However, in the case of sterically hindered acids: the nucleophilic
attack by DMAP on the two reactive carbonyl centers of the mixed
anhydride occurs with roughly a 1:1 ratio and the acid anhydride is inert
toward DMAP for pivalic acid. The reaction proceeds exclusively via the
intermediate 3b for mesitoic acid due to the steric hinderance on the

carboxyl carbonyl center of the mixed anhydride.

EB SE .3 38.3.5 outages waxes canons... :ozmoetmumm do Emamﬁez .md Pun—urn

OOOmnxdu :Ouxaﬂ OOOmuNdN :OuNﬁN

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a

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an a
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Arena + 23m + 30005 + mOOOm

62

Domagala [38] also described a convenient esteriﬁcation of a-keto
acids by chloroformate. Triethylamine was added to a dichloromethane
solution of or-keto acid. then methyl chloroformate was added at 20°C. The
loss of 002 was reported to be completed in 10 min with a 95% yield.

Boltanski [39] esteriﬁed the half ester of malonic acid using ethyl
chloroformate. Triethylamine was added to the monomethyl malonate in
dry THF. Ethyl chloroformate was added at 4°C and the reaction mixture

was stirred for 30 min, during which time the loss of 002 was complete

with a yield of 99%.

B. Modiﬁcation of the reaction procedure

In Husek's approach, it was concluded that esters are formed though
the decarboxylation of the mixed anhydride with pyridine as a catalyst. The
role of the alcohol in the reaction medium was not clearly explained. Both
ethyl and methyl esters were present in the derivatization products when
fatty acids were derivatized with methyl chloroformate in a reaction
medium containing 2% pyridine in chloroform which was “stablized” with
1% ethanol, and the GC signals of ethyl esters were more than double of
those from methyl esters [23]. It was explained that ethyl esters were
formed by activation of the trace amount of ethanol present by the HCl

released from methyl chloroformate, as shown in reaction 2-1.

HCl
R- ,-OH + HO-CZH5 ——-—>R- .()(32H5

(Reaction 2-1)

63
In Kim's esteriﬁcation mechanism [36,37], the ester is believed to

form through the nucleophilic attack by the alcohol on the acyl group of the
intermediate 2a (Figure 2.5). Experiments were designed to clarify the role

of the alcohol in the reaction medium for Husek's derivatization method.

(1) Experimental
D . |° I' .
a)Phe iBuCF-ROE
To ﬁve H2O-alcohol-pyridine (60-10-10 ul) solutions, 5 pl of Phe (10 ug/
pl) were added. The alcohols were isobutanol (iBuOH), triﬂuoroethanol
(TFEtOH), n-pentaﬂuoropropanol (PFPrOH), n-heptafluorobutanol
(HFBuOH), and trimethylsilylmethanol (TMSCH20H). To each solution, 5
ul of isobutyl chloroformate (iBuCF) were added and reaction mixtures
were vortexed for 5-10 8. Each derivative was extracted by 200 p0 CHCls; the
solvent was removed (evaporated) under N2 stream. Each derivative was

redissolved in 100 ul CHCla.

b)Phe MCF-ROH
Similar to a), except that the iBuCF was replaced by methyl
chloroformate (MCF). Three alcohols: MeOH, TFEtOH, and TMSCH2OH,

were used.

c) Phe EtCF-EtOH
Similar to a), but only ethyl chloroformate was applied, and EtOH

was in the reaction medium.

64
d)Phe iBuCF-mixedalcoholwithequal volume

. Similar to a), but instead of using one alcohol in the reaction medium
in each derivatization, a mixture of seven alcohols (MeOH, EtOH, iBuOH,
TFEtOH, PFPrOH, HFBuOH, and TMSCH20H), each 10 pl, was added to

the reaction medium.

e) Phe iBuCF-mixed alcohol with equimolar ratio

i) 10 ug of Phe were added to a solution of H20-
mixed alcohol-pyridine (80-30-10 ul). The mixed alcohol contained seven
alcohols (MeOH, EtOH, PrOH, TFEtOH, PFPrOH, HFBuOH, and
TMSCH20H) with equal molar amount, iBuOH was not included. The
reaction proceeded by vortexing for 10 s after adding 10 ul of iBuCF. 200 pl
CH013 were used to extract the derivatives and lul of the chloroform layer
was injected to GC-MS for analyses.

ii) Similar to i), except that MCF was used instead
of iBuCF. In the mixed alcohol, MeOH was replaced by iBuOH.

GIL-MS:
Column: DB1701 (15 m x 0.25 mm ID.) fused-silica capillary column
with a 0.25 pm ﬁlm. Source temperature was ~200 °C, interface
temperature was 275°C, electron energy 70 eV, scan rate of the mass

spectrometer was 1 s/scan over the range of m/z 50-750.

(2) Results and discusdon
a) In these series of experiments, Phe were derivatized
with iBuCF in a solution containing different alcohols (TFEtOH, PFPrOH,
HFBuOH, TMSCH2OH, iBuOH) in each case.

65
The results indicated that the type of ester formed during the

derivatization process with chloroformate reagents is directly dependent
upon the type of alcohol present in the reaction medium. When using an
alcohol (TFEtOH, PFPrOH, HFBuOH, and TMSCH20H) with an alkyl group
different from that in the alkyl chloroformate (iBuCF in these experiments),
the alkoxy group found in the ester part of the major derivative corresponds
to the alcohol in the reaction medium and not to the alkyl group of the
chloroformate reagent. For example, when phenylalanine is reacted with
isobutyl chloroformate (iBuCF) in an aqueous medium also containing
HFBuOH, the major derivative produced is that in which the carboxylic
group is esteriﬁed with the heptaﬂuorobutyl group, not the isobutyl group.
The TIC and the mass spectrum of this derivative and its structure are
shown in Figure 2.6. Only a small amount of derivative is formed in which
the alkyl group in the ester moiety is the same as that in the isobutyl
chloroformate reagent. It is 4% by both the peak area and the peak height of
the total intensity. Similarly, when Phe is derivatized with isobutyl
chloroformate in the presence of trimethylsilylmethanol
((CH3)3SiCH20H.-=TMSCH2OH) in an aqueous reaction medium, the ester
derivative formed includes a trimethylsilylmethyl group. The TIC and the
mass spectrum of this derivative and its structure are shown in Figure 2.7
The same results were found for Phe treated with iBuCF in the presence of
TFEtOH (Figure 2.8) and PFPrOH (Figure 2.9). The TIC and mass
spectrum of Phe derivatized with iBuCF in the presence of iBuOH is in
Figure 2.10 for comparison.

The results presented in Figures 2.6 to 2.9 cannot be explained very
well by the simple decarboxylation mechanism proposed by Husek, et. al. [1-
3]. According to the Husek mechanism, the mixed anhydride formed by the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.7(?7.0 4 6 8 10 12 15 RT.
moo-s- ~ hr. * ‘ 797.c-
:2 i Phe iBuCF-HFBuOH
ta 80+
, ; NHCO2iBu
V t
e 60+ CH2—CHC02HFB
I
n 4
t 40+
e t
n
{5 20-
t .
Y - - JL l '6
260 490 600 800 Scan
(a)
100‘ mm 000.3 330 .
R + 2 ' “ Phe rBuCF-HFBuOH
e . 91
| 8 04 MW447 _
a ‘ P
.t ‘ ‘
I q .
v 604 "
e l p
+ .
Q . 432 M _
U 40‘ l 447 ,
n ‘57 . - -l ,
d , ‘20.0 »
2 2°? T
c l
° 6? ‘9? l l"? zi" 2s at“
0.1+ , . - . .A; . f - re s w - - .
1oo zoo 400 33%
(b)

Figure 2.6. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction

with iBuCF-HFBuOH.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.10149.9 g 1 - q - - 3. ‘ 1p - ‘ - RI
R100‘ L A A A A 1149.9
{9 « Phe iBuCF-TMSCH20H
a L
.t 8°i
l (
v . NHCOgiBu
e °°lj OCH2-CHC02CH2TMS
I i ‘
n 1
t 404
e 4
n 4
is 20-
t 4 JJ
Y .
AL A A A t
he 4.- s.f---.-lc
100 200 300 400 500 600 Scan
(a)
Scan#(529)
100 73
R « Phe iBuCF-TMSCH20H
e . MW 351
r 804
a q
t .
I < .
v 60, 91 ,
e l 57 t
C 401 131 M-NH2COOiBu 351 L
d 1 160 '10.0 .
a 20. I; .
2 : 103 1 6 4 0 336
e . " .
0'l YAI'v'rI‘ﬁ'Y'I
so 100 150 200 250 800 350 $
(b)

Figure 2.7. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
wrth iBuCF-TMSCH2OH.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

”if”? 1 1 . I I ‘2 q .19 .1? . R-T-
2 . 562.
| I Phe iBuCF-TFEtOH
a 80-
1 .
I .
V -1 .
a 60~ EIHCOzIBu
' ‘ CH2-CHC02TFE
n d
t 40~
e 1
n 1
.5 20~
‘ I
Y I
_LW__ 4-_h‘__Lw_ﬁl A
T v r I ' I v TIC
100 200 300 400 500 600 700 Scan
(a)
100
R ; M-NHZCOOiBu 23° Phe iBuCF-TFEtOH ’
9 ‘ 91 MW 347
I 80«
a <
E .
I .
V 60‘
e <
A i 347
b 404 J
u . j
n . 5 ,# f;
d . '20.0
a I
n 20‘ 16
I 83 103 l 274
e y 11.4.1,1f.119,;ziol
so 100 150 200 250 300 350 :33
(b)

F igure 2.8. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
With iBuCF-TFEtOH.

 

 

 

 

 

 

 

 

 

 

 

 

 

M . 1.

3088 g A . g A q 6 3 10 RT.
R. “ ““J"““‘é01.'8
l9 Phe iBuCF-PFPrOH
a 80-:

I .
i, . 17110021311
° 5°: OCHz-CHCOﬂ’FP

I d
n 1
1 40-

e 1
n .
is 20«
‘ .
y J
v '160' ' '260' V ' '30'0' - 400 - V '560' 660 ‘rlgcan
(a)

100
R j M-NHZCOOiBu 23° .

a ‘ 91 Phe iBuCF-PFPrOH
l 80- MW397 _
a 1 I
I < »
I 1 .
V 60‘ .
9 4 I
A I i
3 4o- 3 7 »
n :57 t
g - '2o.o .
n 20: 120 206 ’b

C « 220
e 55 1 3 J 324 ’

0] illLLVIYA'lIVT Mir
100 200 300 400 500
M/Z
(b)

Figure 2.9. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
with iBuCF-PFPrOH.

70

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

R ‘ 1207.9
:9 I Phe iBuCF-iBuOI-I
f 801
L 1 Ifncoziau
«3 504 OCHg-CHCOgiBu
| .
n d
1 404
a 1
" I
1" em
1 I
y 1 I
7 ‘ ? IAtﬂ‘AA'Arkv f ' ' Ti V ' ' I f ﬁt ‘ I ' f mo
100 200 300 400 500 600 Scan
(a)
100 1
a j I Phe iBuCF-iBuOH ,
e ‘ MW321 »
I 80" 91 I-
a 1 >
t .
i I 57 .
v 604 I-
e 1 240 _
A ‘ . I
b 40.1 M°NH2COOIBU 321 ,_
g 120 204 r 1 - .I- I *
d I 74 220 '20.0
a
n 2°? 13° 1
c 1
° 1 65 1°3 r 192 211°
0‘ Tvkvjv‘lv "'11' 'r'* 71" 'r
so 100 150 200 250 300 350 400
M12
(b)

Figure 2.10. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction

with iBuCF-iBuOH.

71
reaction between the alkyl chloroformate and carboxyl group should

decarboxylate (-COz) to yield the ester containing the alkyl group derived
from the alkyl chloroformate. Also, it does not seem to be possible to form
the major ester product so exclusively in such a short time by the acid
(released from the chloroformate) catalyzed esteriﬁcation suggested by
Husek [23].

It turned out that with a slight modification, the mechanism
suggested by Kim [36 ] for the esteriﬁcation of fatty acids with chloroformate
can be used to explain our experimental results.

In Kim's mechanism (Figure 2.5), DMAP catalyst attacks the mixed
anhydride on the carboxyl carbonyl center to form a acylpyridinium species
(2a); then the attack by the alcohol released from the mixed anhydride on
the acyl group of 2a gives the ester product. In that reaction medium, no
additional alcohol is added. The reaction proceeds in CHzClz with 1:1 ratio
of fatty acid and ethyl chloroformate.

Following the idea of this mechanism, it is not hard to explain what
we observed in our experiments. Since we added an alcohol in which the
alkyl group was different from that in the chloroformate used; and also, the
amount of this alcohol was in great excess relative to the amount of the
analyte present, which would be converted to the mixed carboxylic-carbonic
anhydride. The amount of alcohol released from the process of forming 2a
was no more than the amount of the mixed anhydride, which was the same
amount as the analyte if the yield of forming the mixed anhydride was one
hundred percent. In this case, there were two alcohols in the reaction
medium to compete the attack on the acyl group of 2a. The huge difference
in the quantity of these two alcohols explain why the ester with the alkyl
group from the added alcohol formed exclusively. The relative nucleophilic

72
reactivity of the alcohols should also be a factor to affect the formation of the

ester product.

We believe that this mechanism explains our experimental results
better than the acid catalyzed esteriﬁcation does, and the simple
decarboxylation of the mixed anhydride [1 -3] is not a complete and detailed
explanation for ester formation during reaction of carboxyl groups with
chloroformates.

Other information from the literature also supports our explanation.
Mixed carboxylic-carbonic anhydrides are considered as active acylating
agents. Their reaction with nucle0philes proceeds readily under mild
conditions [6]. The use of chloroformates as esteriﬁcation catalysts in the
presence of alcohol has been reported [6,40-42]. Although no detailed
mechanism was proposed, it was believed that the reaction was through the
formation of the mixed anhydride which then reacted rapidly with the
alcohol with tertiary amine base as the catalyst [40-42]. An attempt was
made to avoid the competition of the alcohol released during ester formation
by employing an excess of the alcohol to be esteriﬁed [40]. Kim's
mechanism should also ﬁt those experiments.

Combining all the information, we can conclude that for the
esteriﬁcation of the carboxyl groups with chloroformates in Husek's
method, the overall (result is an exchange reaction between the mixed
anhydride and the alcohols in the reaction medium (Reaction 2-2). The
mixed anhydride reacts with the alcohol in the reaction medium to undergo
an exchange reaction as illustrated via path A leading to the principal
product. A small amount of derivative in which the alkyl group in the ester
moiety is the same as that in the alkyl chloroformate reagent is formed

through path B as a minor product (since R"OH is in great excess relative

73
to R'OH). R'OH is released from path A‘and is also produced from the

hydrolysis of the chloroformate reagent in the reaction medium.

 

 

Rn'OH’ Ron- -O-C-OR"
A NHC-OR' Major
R(‘r‘H- -O-C-OR" —
NHC'OR' RI-OH
B = R H- -O-C-OR' Minor
NHC-OR'
(Reaction 2-2)

It also can be seen from the series of experiments of Phe derivatized
with iBuCF and different alcohols (ROH) (Experimental a)), that
ﬂuorinated moieties increased the volatility of the derivatives. The Phe
derivative from iBuCF-TFEtOH eluted at scan 423 (Figure 2.8); the Phe
derivatives from iBuCF-PFPrOH (Figure 2.9) and iBuCF-HFBuOH (Figure
2.6) eluted at scan 411 and scan 420, respectively, while the iBuCF-iBuOH
derivative of Phe eluted at scan 520 (Figure 2.10).

In the EI mass spectra of Phe derivatives from ﬂuorinated alcohols,
(M+1- NHzcoOiBu) ions (m /z 230 in Figure 2.8, m/z 280 in Figure 2.9, and
m/z 330 in Figure 2.6) are more predominant than that in the spectrum of
Phe derivative with iBuOH (m/z 204 in Figure 2.10). These spectra are also
relatively simpler than that of Phe iBuCF-iBuOH derivative while the Phe
iBuCF-TMSCH20H derivative produces more fragmentation in its

spectrum (Figure 2.7).

74
For these series experiments (Experimental 8)), the peak area and

peak height of the derivatives, intensity percentages of the minor products

are in section I of Table 2.2.

b) In the series of experiments of Phe derivatized with iBuCF and
different alcohols (ROH) (Experimental a)), the minor product is less than
4% of the total intensity by area for each derivative with iBuCF. In the
second series of experiments (Experimental b)), MCF derivatives of Phe
were made in the presence of TFEtOH (Figure 2.11), and in the presence of
TMSCH20H (Figure 2.12) to compare the intensity percentages of the minor
products in these two reactions with what was observed for the
corresponding reactions from the iBuCF derivatization (Experimental a)).
The MCF derivative of Phe in the presence of MeOH was also made as a
control (Figure 2.13).

The results of MCF derivatization (Experimental b)) have the same
trend as what was observed from iBuCF derivatization (Experimental a))
for Phe with different alcohols: the ester moiety of the major derivatization
product is from the alcohol added in the reaction medium. But from the
results summarized in section II of Table 2.2, we can determine that the
relative intensity of the minor product by area is 9.7% for the Phe derivative
with MCF-TFEtOH and 8.7% for the Phe derivative with MCF-TMSCHon.
These percentages are higher than those obtained for the corresponding
derivatives from iBuCF. (3.6% for Phe iBuCF-TFEtOH derivative, and <1%
for Phe iBuCF-TMSCHgoH derivative.)

These differences may indicate the different nucleophilic reactivities

of MeOH and iBuOH for the attack on the acyl group of the acylpyridinium

75
intermediate and / or that MCF is easier to hydrolyze to produce more

MeOH in the reaction medium.

0) EtCF-EtOH derivative of Phe was made for the comparison with
other derivatives made from iBuCF (Experimental a)) and MCF
(Experimental b)) in the presence of different alcohols. The result is in
section III of Table 2.2.

From Table 2.2, except for the different retention times for the
different derivatives, different percentages of the minor products for the
different derivatives, we also can see that the derivatives from iBuCF-
HFBuOH, -PFPrOH, and -iBuOH have higher responses by areas which are
twice as much as that from the EtCF-EtOH derivative. This may indicate
that iBuCF derivatives of amino acids provide higher sensitivity for
detection than EtCF derivatives do. This aspect will be evaluated later in
this chapter.

(1) and 6) Additional evidences for the direct involvement of alcohol
constituents in the aqueous reaction medium containing the chloroformate
reagent are from experiments in (Experimental d)) (Figure 2.14), and
(Experimental e)) (Figure 2.15 and 2.16).

These data were obtained during the analysis of Phe after treatment
with isobutyl chloroformate in an aqueous solution: (1) containing seven
alcohols with equal volume (Experimental d)): pentafluoropropanol
(PFPrOH), heptaﬂuorobutanol (HFBuOH), trifluoroethanol (TFEtOH),
methanol (MeOH), ethanol (EtOH), isobutanol (iBuOH), and
trimethylsilylmethanol (TMSCH20H) (Figure 2.14); and (2) containing

equimolar amounts of seven alcohols (the ﬁrst experiment in Experimental

76
e)): pentaﬂuoropropanol (PFPrOH), heptaﬂuorobutanol (HFBuOH),

trifluoroethanol (TFEtOH), methanol, ethanol, propanol, and
trimethylsilylmethanol (no iBuOH) (Figure 2.15).

In Figure 2.14, derivatives from all seven alcohols were produced.
Figure 2.15 is more meaningful since each alcohol was present with an
equimolar amount. Figure 2.15 is a reconstructed total ion current
chromatogram resulting from analysis of the reaction mixture by GC-MS. '
Seven major peaks are obtained corresponding to the esters formed by
reactions with the alcohols in the reaction medium. The different
intensities of the peaks result from the differential nucleophilic reactivity of
these alcohols with the acylpyridinium species and/or responses of the
corresponding derivatives under EI-MS conditions. A minor peak is also
present which derives from the esteriﬁcation with the alcohol released from
iBuCF.

The second experiment in (Experimental e)) resulted in Figure 2.16.
In this experiment, Phe was derivatized by MCF with equimolar amounts
of seven alcohols (no MeOH). The same explanation for Figure 2.15 is
applicable to Figure 2.16

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.4é8.6l ? 4‘ A q A q 4 L R.T.
R100, 458.6
f1 Phe MCF-TFEtOH
1‘ 803
i ' : 1"IHC02M8
V 4
e 60, ~CH2-CHC02TFE
I I
n 4
t 401
e 1
n 1
is 2m
‘ I
y .
O I i":v 7“ ‘ﬁme—u' Y “C
100 200 300 400 500 Scan
(a)
100
R 1 9‘ Phe MCF-TFEtOH
e . MW305
l 30. M-NchOOMe 230
a 4
I
I 4
v 60~
e 1
A 1
U ..
n
d . '3o.o .
a 20
n I 59 131 17s I
C . 65 83 10319L1‘15 l 214 .
9 , I
045‘I T‘Lrlvlvv‘yj;¢vttvlv VT.‘ 11' .1
50 100 150 200 250 300 3‘51;
/
(b)

Figure 2.11. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction

with MCF-TFEtOH.

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

Manages 2 4 6 l FLT.
a1ooLL-4+-"‘*"m-
f I Phe MCF-TMSCH20H
a 80:
E . PfHCOzMe
; 60; OCHz-CHCOzCHgTMS
. I
n 1
1 40-
g 1
" I
f 20-
1 .
y u ___._._A_IIJL~,_ _. A-.I L_. C
* '100' r'20'0 900 460 sJJ'Sm
(a)
100 73
a . Phe MCF-TMSCH20H ;
e . MW309 .
I 801 I
a 1 I
I j 91
v 60-
° ‘ 17s
A . 309
b 40‘ 59 1 3 1 M-NH;COOMe
u .
n .
d . 219 / 2 * 0.0
g 20.. 294
c . I
. . 21’ I .
0- .3‘....,v f.,r .j
50 100 150 200 so 300 350 400
M
(b)

Figure 2.12. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction

with MCF-TMSCH20H.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max. 48 7.3 1‘ L 21 1 A 4‘ J 61 81 ‘ R.T.
R100. 417.3
I9 1 Phe MCF-MeOH
:1 804
1, $110on1:
9 60-1 Qan-CH002MO
| .
n 4
t 40-4
a It
n ‘1
is 20-1
I I
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100 200 300 400 Scan
(a)
100
1 ‘152 M-NHZCOOMe
R 1 Phe MCF-MeOH
e ‘ I
I 80. MW237 ,
a 3 I
I « 206 I
1 1 91
V 60q
a q
A . 146 237
b 401
U J
n . , ﬂ
d . 173 '20.0
I11 2°: 59 131
C
1 103 119
50 100 '1s'o'f 700' Y ' '2éo' ‘300' '1 '350
MIZ
(b)

Figure 2.13. TIC (a) and EI mass spectrum (b) of Phe derivatized by reaction
With MCF-MeOH.

 

 

 

 

 

. m Hv . - N Hv . . mvm H Scum-
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Max.0278.2 7 s 9 ,9 . + R.T.

1°“"“““L‘ ‘2782

R * R=CH TMS

I Phe iBuCF-ROH 2

a l

1 8°. 1"IHC02iBu

I .

v . Gong-cucozR

e 60" IR=M8

I

n 1

t 40‘ R=iBu

3 ‘ R=PFP R‘HFB R=Et

s ‘ R=TFE

i 20-

t I

y I w
0 IC
3C0 350 400 450 500 550 600 Scan

Figure 2.14. TIC of Phe derivatized by reaction with iBuCF in an aqueous
solution containing an equal volume mixture of seven alcohols (MeOH,
EtOH, iBuOH, TFEtOH, PFPrOH, HFBuOH, and TMSCH20H).

 

100+ -7” -- - $.... A, -9--- - ‘ -19- RT.
I Phe iBuCF-ROH R=CH2TMS

801 .
: IfHCOzIBu
1 CHZ-CH002R I

 

~<--m:m~:- o<-'~m-m:n
ca
‘2

 

 

 

 

 

 

 

5%ogvv,-,-.i .- . --.H”TIC
550 600 650 700 75° Scan

Figure 2.15. TIC of Phe derivatized by reaction with iBuCF in an aqueous
solution containing an equimolar mixture of seven alcohols (MeOH, EtOH,
PrOH, TFEtOH, PFPrOH, HFBuOH, and TMSCH20H). The peak labeled
R=iBu represents a trace of the ester product formed with the alkoxyl group
corresponding to the alkyl group of the chloroformate reagent.

 

 

 

 

 

 

 

Max. Q6.0 10 1 1 RHT
R1m‘4LL4114L4#4L1114.11 LLJL4IAAP$CAAP141MS 226.0
{9 ~ PheMCF-ROH 2
a 80:
g 1 Ichoznae
v . CH CHCOR
e 604 2- 2
I 1
n q
1
e
n
s
I
1
y
0 r
V ' ‘ ' j ' 1 a ' T f f ‘ ‘ 1% a ‘ ' I ' ' ' ' 1 F F ' ' C
0 650 700 750 300 850 903'Scan

Figure 2.16. TIC of Phe derivatized by reaction with MCF in an aqueous
solution containing an equimolar mixture of seven alcohols (EtOH, PrOH,
iBuOH, TFEtOH, PFPrOH, HFBuOH, and TMSCH20H). The peak labeled
R=Me represents a trace of the ester product formed with the alkoxyl group
corresponding to the alkyl group of the chloroformate reagent.

84
C. Eﬂ'ect of concentration of the alcohol in the reaction medium on

the percentage of the minor derivatization product
(1) Experimental

12 . |° |°
a) Phe iBuCF-HFBuOH (0, 10, 30, 60, 70 pl)

To each of the ﬁve solutions containing 70 pl H20, 10 pl pyridine, and
various amounts of HFBuOH: 0, 10, 30, 50, 70 pl, were added 10 pl of Phe (10
nmol/pl). To each solution, 10 pl of iBuCF were added and the reaction
mixtures were vortexed for 30 s. 200 pl chloroform were used to extract the
derivatives, and 2 pl of chloroform layer was injected to GC-MS for

analyses.

b) Leu iBuCF-HFBuOH (0, 10, 30, 50, 70 pl)

To each of the ﬁve solutions containing 60 pl H20, 10 pl pyridine, and
various amounts of HFBuOH: 0, 10, 30, 50, 70 pl, were added 20 pl of solution
containing Leu, Phe, Tyr, and Lys (each 2.5 nmol/pl). 10 pl of methyl
stearate (0.25 nmol/pl) were added as an internal standard. To each
solution, 10 pl of iBuCF were added and the reaction mixtures were
vortexed for 30 s. 200 pl chloroform were used to extract the derivatives, and
50 pl of 1 M HCl were added as the counter phase. The chloroform solvent
was evaporated under N2 stream. The derivatives were redissolved in 100

pl chloroform, and 2 pl of solution were injected to GC-MS for analysis.

39.-MS:
Instrument: HPSB92 MSD Column: DB-5MS 30 m x 0.25mm I.D.
Injection Temperature: 250-2800C Interface Temperature: 280°C

85
(2)Results and discussion

When Phe and Len were treated with iBuCF with a different
concentration of HFBuOH in the reaction medium, the percentages of the
minor products (in which the ester moieties are from the iBuCF) were
different. Increasing the amount of HFBuOH in the reaction medium, the
amount of the minor products decreased. Under the experimental
conditions speciﬁed, when HFBuOH was more than 30 pl in the reaction
medium, the percentages of the minor products were below 1% for both
derivatives. This indicated that the major products were formed

exclusively with higher concentration of alcohols. See Table 2.3.

Table 2.3. Effect of HFBuOH concentration in the reaction solution (H20 80

pl, pyridine 10 pl, iBuCF 10 pl) on the percentage of minor derivatization
product.

 

Percentage of minor derivatizatiorgroduct

 

HFBuOH (pl) Phe Leu

0 - -

10 10% 8.9%
30 3.2% I 3.0%
50 <1% <1%

70 <1% <1%

86
IV. Investigation of diﬂ‘erent chloroformate derivatives of amino acids

A. Amino acid derivatives formed by selected combinations of various

chloroformates andaleohols
(1) Experimental

A solution of 20 amino acids in 0.1M HCl at a concentration of 0.5
pg/pl each was prepared. The ethyl chloroformate (in the presence of
different alcohols) derivatives of amino acids were prepared by adding 10 pl
to the amino acid mixture to a H20-alcohol-pyridine (Py) (50 pl-30 pl-lO pl)
solution. 10 pl of ethyl chloroformate (EtCF) were then added to each
solution and the reaction mixtures were vortexed for 5-10 8 and extracted
with 200 pl CHCl3. A Z-pl aliquot of the CH013 layer was injected for
analysis. Other chloroformate (in the presence of different alcohols)
derivatives were prepared by adding 10 pl of the amino acids and 10 pl of the
chloroformate reagent to a solution of HzO-alcohol-Py (70 pl-3O pl-10 pl)
following the same procedure. See group 1 to group 5 in Table 2.4 for the
different combinations of chloroformates and alcohols from which the
derivatives of the amino acids were made.

Analyses by GC-MS were carried out on a JEOL AX-505H double
focusing mass spectrometer coupled to a Hewlett-Packed 5890J gas
chromatograph. GC separation was achieved on a DB-1701 (15-m length x
0.25-mm i.d.) fused silica capillary column with a 0.25 pm ﬁlm coating
from J. & W. Scientiﬁc (Rancho Cordova, CA). Direct (splitless) injection
was used. Helium gas ﬂow was approximately 1 ml/min. MS conditions
were as follows: interface temperature 275°C, ion source temperature ca.
150-2000C, electron energy was 70 eV, scan rate of the mass spectrometer
was 1 s/scan over the range of m/z 50-750. GC-FID (flame ionization

detection) was carried out on the same gas chromatographic column with

Table 2.4. Chloroformate-alcohol reagents studied for amino acid

 

 

 

 

 

 

derivatization.
Reagents Derivatives
I
EtCF-EtOH N(0,S)«ethoxycarbonyl ethyl ester
PrCF-PrOH N (O,S)—propoxycarbonyl propyl ester
iBuCF-iBuOH N (O,S)-isobutoxycarbonyl isobutyl ester
I I
EtCF -TFE N (O,S)-ethoxycarbonyl triﬂuoroethyl ester
-PFP N(0,S)-ethoxycarbonyl pentaﬂuoropropyl ester
-HFB N(0,S)-ethoxycarbonyl heptaﬂuorobutyl ester
I I I ,
PrCF -TFE N(0,S)-propoxycarbonyl triﬂuoroethyl ester
-PFP N(0,S)-propoxycarbonyl pentaﬂuoropropyl ester
-HFB N(0,S)-propoxycarbonyl heptaﬂuorobutyl ester
I V
iBuCF-TFE N(0,S)-isobutoxycarbonyl trifluoroethyl ester
-PFP N(0,S)-isobutoxycarbonyl pentaﬂuoropropyl ester
-HFB N(0,S)-isobutoxycarbonyl heptaﬂuorobutyl ester
V
iBuCF-TMSCHzOH N (O,S)-isobutoxycarbonyl trimethylsilymethyl ester
-TMS(CH2)20H N(0,S)-isobutoxycarbonyl trimethylsilyethyl ester
-TMS(CH2)3OH N(0,S)-isobutoxycarbonyl trimethylsilypropyl ester

 

88
injector and detector temperatures 260°C and 280°C; N2 was the carrier

gas.

(2) Results and discusm‘on

The one-step chloroformate derivatization of amino acids in an
aqueous medium has been extended with the use of a variety of alkyl
chloroformate and alcohol reagents. In Husek's method, only methyl and
ethyl chloroformate were applied and the application of other chloroformate
derivatives were not investigated. In the previous section, we discovered
that the ester moiety of the amino acid derivatives is directly dependent
upon the type of alcohol used in the aqueous reaction medium. These
results provide new insight into the one-step derivatization reaction and
provide the basis for preparing a variety of chloroformate derivatives that
can be assessed for optimizing the analysis of amino acids by GC with FID
or by GC-MS. We also noticed that isobutyl chloroformate derivatives of Phe
showed higher responses than that of ethyl chloroformate derivative of Phe
(Table 2.2). Based on these results, various combinations of chloroformate
reagents and alcohols were used to generate a wide variety of N(0,S)-
alkoxycarbonyl amino acid alkyl esters for analyses by GC or GC-MS with
the objectives of obtaining higher sensitivity for detection, optimizing the
chromatographic separation of the amino acid derivatives, and evaluating
the inﬂuence of the alkyl group of the chloroformate (alkyl carbamate in the
derivative) and also the structure of the alcohol (alkoxyl group in the ester of
the derivative) in the reaction medium on the response of the derivatives
detected by FID or by EI-MS. Chloroformate with larger alkyl groups
(propyl and isobutyl), fluorinated alcohol (TFEtOH, PFPrOH, and

$
HFBuOH), and trimethylsilyl alcohols (TMSCH20H, TMS(CH2)20H, and

TMS(CH2)3OH) have been used for the investigation.

Figure 2.17 to 2.21 show the reconstructed TIC of each group of
chloroformate-alcohol derivatives of 20 amino acids. In group 1, the
derivatives formed by the reagents generate a response in MS analysis and
by FID that increases slightly with the size of the alkyl groups for the
groups studied: (isobutyl > propyl > ethyl) in the chloroformate reagent as
well as in the derivatizing alcohol. Although it is not true for each amino
acid, the responses of the iBuCF-iBuOH amino acid derivatives are higher
than those of EtCF-EtOH derivatives. Meanwhile, in other series, the trend
is not very clear, and not the same for every amino acid, or the difference is
not signiﬁcant. Generally, for each alcohol (HFBuOH, PFPrOH, and
TFEtOH), the responses of the PrCF and iBuCF derivatives are higher than
those of EtCF derivatives. For each chloroformate (EtCF, PrCF, and
iBuCF), the responses from HFBuOH derivatives are higher, although the
factors of difference are generally within 2-3. The overall highest
detectability is produced by iBuCF-iBuOH, iBuCF-HFBuOH, and iBuCF-
TMSCH20H derivatization reagents. Figure 2.22 shows that the GC-FID
responses of the 20 amino acids derivatized with iBuCF-iBuOH, iBuCF-
HFBuOH, and iBuCF-TMSCH20H are higher than those prepared with
EtCF-EtOH. Tyr and Hyp also can be derivatized and separated from the
other 20 amino-acids (even though these two were not included in the
mixture represented in Figure 2.17-2.22. The guanidino group on the side
chain of Arg is not derivatized by the reaction mixture described here as
veriﬁed by detection with FAB; Arg in this form cannot be eluted from the
GC column. A quantitative comparison of the detector response to various

derivatives is given in Table 2.5, which lists the ratio of GC-FID'peak area

90
produced by the derivatives made from PrCF-PrOH, iBuCF-iBuOH or

iBuCF-HFBuOH to those made from EtCF-EtOH. Table 2.6 compares the
reconstructed TIC responses of different groups of derivatives relative to
those prepared from EtCF-EtOH. In nearly all cases, derivatives prepared
from EtCF-EtOH gave a lower response. The mass spectra of the amino
acid derivatives reported herein are produced through fragmentation
pathways similar to those described earlier for mass spectra of EtCF-EtOH
derivatives of amino acids in section II of this chapter and Ref. 4. The
amino acid derivatives described here are expected to have recoveries
similar to those of amino acids derivatized with EtCF-EtOH [1-3]. The
inclusion of a suitable internal standard, such as norleucine (Figure2.17-
2.22) or stable isotope labeled amino acids, would make the approach
described here suitable for quantitative analyses.

In group 5, with TMS alcohols, it is found that an increase of the size
of the alcohol (TMS(CH2)3OH>TMS(CH2)20H>TMSCH20H) causes greater
production of the derivatives esteriﬁed with the alkyl group of the
chloroformate reagent. The increased competition from this side reaction
may result from the weaker nucleophilic attack on the intermediate
acylpyridinium species for the bulkier TMS alcohols. In general, the
formation of minor products through path B of Reaction 2-2 are typically
much less than 10% as indicated by the total ion current chromatogram of
the derivatives; however, the yields of side products were not systematically
investigated for each amino acid with each derivatization reagent
combination.

Fast GC temperature programs were applied to EtCF-EtOH and

iBuCF-iBuOH derivatives of amino acids. Complete separation of the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

352 o(a) EtCF-EtOH
° § 10 15 RT.
1% l
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t w
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Figure 2.17. TICs of 20 amino acid derivatives prepared from different
chloroformate-alcohol reagents (a) EtCF-EtOH, (b) PrCF-PrOH, (c) iBuCF-
iBuOH. GC-MS analysis of an aliquot of the reaction mixture containing 50
ng of each amino acid on to a 15-m, 0.25-mm i.d. column containing a 0.25-

pm ﬁlm of DB-1701. GC temperature programs: (a) from 100°C to 200°C at
10°C / min, then 20°C / min to 280°C; (b) and (c) from 120°C to 200°C at 10°C
/ min, then 20°C / min to 280°C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(a) EtCF-TFEtOH
Max.275 4‘ 61 g 1,0 - A 1? - - 1,4 R.T.
R1001 - ~ - | - 275721
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v 0
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n 1 H
I 401
e ‘ G T N
2 . . o
i 20" ﬁ
1 j E o
y . U I»
. L_ (.11
200 ' I 460 600 30° 1000Scan
(b) EtCF-HFBuOH
Max.425. 4 Q 8 10 12 14 FLT.
100 1 1 - ‘ A * ‘ ‘ ‘ ‘ i ‘ ‘ ‘ 4 ‘ ‘ ‘ g
n I U 425.6
+ F
e I
I . c
ta 80‘ V M
i I PML 0 w
V . H+K
e 60" G
I 1 T
n ‘ A N O
t 40* s
e t
n I
S J .7
; 2°: J1 E a
V 200 V ' ' 400 I 600 800 1000Scan

Figure 2.18. TICs of 20 amino acid derivatives prepared from EtCF with (a)
TFEtOH and (b) HFBuOH. Temperature programs: from 100°C to 200°C at
10°C / min, then 20°C / min to 280°C.

93

(a) PrCF-TFEtOH
mm.

 

3‘
$883

8

 

 

““'.3."3" 0“".-.

 

 

 

 

 

 

 

 

«19 200

(b) PrCF-PFPrOH
“I. ”I 1 A § 9 ‘9 ‘3 P RT.

 

t

4

.<-o:o~:- O<""I".3

 

 

 

 

 

 

 

 

 

L
F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

kl. IO
“V 200 350 400 500 000 700 000 9138“.
(c) PrCF-HFBuOH
RT
5 Q 7 ‘° ‘3 m.
5 Mel
" c: III
N 0
TI
5 0
LL k “I LJJLLJ

Figure 2.19. TICs of 20 amino acid derivatives prepared from PrCF with (a)
TFEtOH, (b) PFPrOH, and (c) HFBuOH. Temperature programs: (a) from

100°C to 200°C at 10°C / min, then 20°C / min to 280°C; (b) and (c) from
120°C to 200°C at 10°C I min, then 20°C / min to 280°C.

(aﬁBuCF-TFEtOH

M401 RT.
am i 1 § q 19 '3

my C

§

K0"

5

  
 
 

8

 

'("".:I."'3" ."""."O
0
:

 

 

 

 

 

 

 

 

 

r
PO
L——.———e

 

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(b) iBuCF-PFPrOI-I
$3771 1 9 g ,9 A LL L RT.
3 c 377.1
u' i
I M F
t
i W
V
o 60 Lu 9 c» u
' u.
" 404‘
‘0 a V "x
'2 7 o
t 20‘ A E . ‘ o
Y J L _ L4
, i , v TIC
‘05 zoo 300 400 soo 600 no soo 900 Sc."
(0) iBuCF-HFBuOH
mace. RI.
5 § Q ‘9 ‘3
in
If M P 9 F a WF
. W
3 v “
I G u W
1 so« “
I n o
2 T r
3 s
.- L °
* n Ls
v hm Lu“:
460 500 630 760 060 was“.

 

Figure 2.20. TICs of 20 amino acid derivatives prepared from iBuCF with
(a) TFEtOH, (b) PFPrOH, and (c) HFBuOH. Temperature program: from

120°C to 200°C at 10°C / min, then 20°C / min to 280°C.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

iBuCF-TMSCH20H
Max:156! .6 a A a m n A A? FLT.
1o ,. W U 0 IV 1‘
R 4561.6
.° ‘ s
. H w
a 80 )l a
t
i | NL E K
v 4 G V P
e 60l L
l ‘ A T
n < C-C
1 4m 3 o
B
n
is zol N
. U N w
y bde LA) -
' w ' ' ' ' ' ' ' ' ' ' """" ‘ TIC
100 zoo 300 400 500 600 700 800 Scan

Figure 2.21. TIC of 21 amino acid derivatives prepared from iBuCF-
TMSCH20H. Temperature program: from 120°C to 180°C at 10°C / min,

then 20°C / min to 280°C. GC column 0V1701 10-m, 0.25-mm i.d., 0.2-um
ﬁlm. (280 ng for each amino acid. Cys was not identiﬁed.)

(1) EtCF-EtOH

If

 

 

 

 

 

 

 

 

5 (min)

O'- "i"
:1.

. 0
110°C 101so°c @1o°C/min.1nen 30°C/m1nto 280 C

 

 

 

 

 

 

 

 

 

 

 

 

(2) iBuCF-iBUOH
: 1- F
m. c
c 1 D
A V '- M
r N o x
s w
E 0
w H
.-.- UULLU J'“
E“
1 1 1 I
0 5 10 14
(min)

 

. 0
140°C to 200°C @ 10°C/min. then 30°C/m1nto 280 C

Figure 2.22. GC-FID chromatograms of 20 amino acid derivatives prepared
from different chloroformate-alcohol reagents (1) EtCF-EtOH, (2) iBuCF-
iBuOH, (3) iBuCF-HFBuOH, and (4) iBuCF-TMSCH20H. The
chromatograms result from injection of an aliquot of the reaction mixture
containing 50 ng of each amino acid on to a 15-m, 0.25-mm i.d. column

containing a 0.25-um ﬁlm of DB-1701.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(3) iBuCF-HFB
:r. F c
c ' u w
l
v NI.
L D “
- o ‘
. ‘ ‘r
u
S
I Q

l 1 l 115
0 S ‘0 (min)

100°C to 200°C @ 10°C/min.1nen 15°C/m1n to 280°C

(4) iBuCF-TMSCH20H

c "L n '

2.:
A

 

 

 

 

 

 

 

 

 

 

 

 

 

“—
d
0

d

.

(min)

150°C to 200°C @1o°C/min. then 40°C/min to 280°C.

Figure 2.22. GC-FID chromatograms of 20 amino acid derivatives prepared
from different chloroformate-alcohol reagents (1) EtCF-EtOH, (2) iBuCF-
iBuOH, (3) iBuCF-HFBuOH, and (4) iBuCF-TMSCH20H. The
chromatograms result from injection of an aliquot of the reaction mixture
containing 50 ng of each amino acid on to a 15-m, 0.25-mm i.d. column

containing a 0.25-um ﬁlm of DB-1701 .

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

iBuCF-iBuOH

Max.169%9 3 4 5 RT
R‘OO‘ ' 4 ﬁg; M F 4 L ‘ We
9 .
I q 0
ta 80‘ L
i .
v .
e 60- A PT 0

* v
I 1 S
n 1 K
t 40. V C 0
9 ‘ Q
n J

1
f’ 204 E w
t .

1 H
y Add JN M

1 ~ v . 1 , - r - 1 ~ v a - ,m _ C
150 200 250 300 350 400 4531868"

Figure 2.23. TIC of 20 amino acid derivatives prepared from iBuCF-iBuOH.
GC separation is done within 6 min. GC temperature program: 120°C to

280°C at 40°C / min.

Q

Table 2.5. Ratio of peak area on GC-FID of indicated derivatives relative to
those prepared with EtCF-EtOH: (response for EtCF-EtOH derivatives were
the average of results from triplicate analyses; responses for the indicated
derivatives were the average of results from duplicate analyses).

 

 

PrCF-PrOH iBuCF-iBuOH iBuCF-HFBuOH
lEtCF-EtOH lEtCF-EtOH lEtCF-EtOH
Ala 1.5 2.0 1.4
Gly 1.8 2.6 2.4
Val 1.2 1.6 1.5
Leu 1 .3 1 .7 1 .4
Ile 1 .1 1.6 1 .4
n-Leu 1 .5 1.7 1 .9
Pro 1.2 1.7 1 .2
Thr 1.3 1.7 1.5
Ser 1 .8 1 .3 2.0
A s n 1 .5 1 .9 1 .6
Asp 0.5 3.1 2.9
Met 1 .7 1.5 1 .4
Glu 1 .3 2.2 4.3
Phe 1.2 1.5 1.4
Cys 1.3 1.4 2.0
Gln 4.1 3.2 4.5
Orn 1 .1 1 .2 1 .4
Lys 1 .0 1 .1 1 .2
His 0.7 0.4 5.7

Trp 0.9 1.2 2.7

 

100

Table 2.6. Ratio of both peak area and peak height of reconstructed TIC
corresponding to the indicated derivatives relative to those made with EtCF-
EtOH from analyses by GC-MS (EI).

 

 

 

PrCF-PrOH iBuCF-iBuOH iBuCF-HFBuOH

lEtCF-EtOH IEtCF-EtOH IEtCF-EtOH

A H A H A H
Ala 0.99 0.95 1.79 2.09 2.05 1.93
Gly 1.13 1.13 1.98 224 3.33 3.02
Val 0.90 0.81 1.09 1.21 1.70 1.57
Leu 0.97 1.01 1.49 1.58 2.24 2.96
Ile 0.78 0.81 0.87 0.91 1.37 1.75
Leu 0.98 0.92 1.42 1.45 2.01 1.83
Pro 1.06 1.16 1.60 1.92 1.88 1.88
Thr 1.14 1.39 1.58 1.70 1.87 2.02
Ser 0.85 0.67 1.10 1.08 1.05 1.43
Asn 1.23 1.18 1.69 1.72 2.49 2.42
Asp 0.37 0.37 1.88 2.66 3.51 2.81
Met 1.81 1.55 1.52 1.53 1.84 1.52
Glu 1.20 1.30 0.94 0.83 2.33 2.16
Phe 1.22 1.79 1.41 1.90 1.83 1.82
Cys 1.48 1.85 0.62 0.83 2.02 2.25
Gln 2.03 1.96 1.70 1.68 1.40 1.22
Orn 1.22 1.87 1.01 1.58 1.02 127
Lys 1.07 1.42 0.95 1.14 0.71 0.67
His 1.68 1.65 1.17 0.83 2.93 3.17
Trp 1.10 0.84 1.20 0.86 1.52 1.05

 

A=Area; H=Height

101
derivatives of 20 amino acids can be accomplished within 6 minutes in both

cases (Figure 2.23).

B. Evaluation of reaction conditions

The reaction conditions of iBuCF-iBuOH derivatives of amino acids
have been evaluated.

(1) Effect of iBuOH concentration in the reaction medium
a) Experimental

A solution of 50 ul of 24 amino acids (2 jig/each) was added to each of
the six solutions containing 30 ul H20, 10 111 pyridine, and various amount
of iBuOH (O, 10, 20, 30, 40, 50 111). To each solution, 10 pl of iBuCF were
added and reaction mixtures were vortexed for 30 s. The derivatives were
extracted by chloroform twice, (200 Ill and 100 111 for each time); the solvent
was removed (evaporated) under N2 stream. Derivatives were redissolved
in 100 111 chloroform. A 2111 aliquot of chloroform solution were injected for
analysis. The GC-MS conditions were the same as those in the previous

experiments.

b) Results and discussion

The TIC responses of the derivatives of Phe, 4-Cl-Phe, Tyr, nLeu,
Asn, Trp were approximately constant over the entire range of iBuOH (O to
50 1.11) added in the reaction solution (Figure 2.24). The TIC responses of the
derivatives of Ala, Gly, Val, Leu, Ile, Pro, Thr, and Asp increased when the
amount of iBuOH added increased from 0 to 20 111, and their responses did
not change when the amount of iBuOH added increased from 20 to 50 111
(Figure 2.25). The TIC responses of Orn, Lys, and Met decreased as the
amount of iBuOH added increased (Figure 2.26). These results indicated

102
that the addition of 20-40 pl of iBuOH in the reaction solution containing 80

1.11 of water was appropriate to achieve good responses from all the amino
acids. In these experiments, His, Gln and Cys-Cys derivatives were not
observed. Cys was strange in these experiments. The response of Cys
derivative was good when no iBuOH was added in the reaction solution, but
Cys peak disappeared in the case of 10 and 20 pl of iBuOH were added. Cys
peak was detected again when 30, 40, 50 ul of iBuOH were added. In the
other parallel experiments, when the amino acid mixture was newly made
from each amino acid solution, the responses of Cys derivative was
approximately constant over the entire range of 0 to 50 ul of iBuOH added.
Also from the newly prepared amino acid mixture, response of Hyp
derivative was approximately constant when the amount of iBuOH added

was from 10 to 40 1.11 while its response was relatively lower in the case that

no iBuOH was added.

(2) Eﬁ'ect of concentration 011120 in the reaction medium
a) Experimental

A solution of 10 ul of 20 amino acids (0.5 ug/ul) were added to each of
six solutions containing 30 111 iBuOH, 10 111 pyridine, and various amounts
of H20 (0, 10, 20, 30, 40, 50 111). To each solution, 10 1.11 of iBuCF were added
and reaction mixtures were vortexed for 30 s. The derivatives were
extracted by chloroform twice, 200 1.11 and 100 111 for each time; the solvent
was removed (evaporated) under N2 stream. Derivatives were redissolved
in 200 111 chloroform. A 2-111 aliquot of chloroform solution was injected for
analysis. The GC-MS conditions were the same as those in the previous

experiments.

103

Effect of iBuOH

Phe

 

 

 

 

 

'13::-
0.0 “.1,.1“,....,....,....,....l
0 1O 20 30 40 50 60
iBuOH(ul)
150.0 nLeu
$100.0 Asn
1:
O
3‘ 1
£
50.0-5
g},—D——.D”’D~“D“0Trp
0.04Hm].n1,....,....,..4.,r...l
0 1O 20 3O 40 50 60
iBuOH(ul)

Figure 2.24. Effect of iBuOH concentration in the reaction medium for
formation of iBuCF-iBuOH derivatives of amino acids (1).

104

 

Effect of iBuOH

 

 

 

 

 

   

 

 

D=Val
E=Leu
Ala
0.0 ,.,..,....,....,.-..,....,...q
0 10 20 30 4O 50 60
iBuOH(ul)
180.0—
. Pro
135.0; Asp
g 1 ----- Ile
S
g, 900—
3
Ci
45.0
\UThr
0.0'TYIYIIIYIIIITYIUIIYIIY rlYYII]
0 10 20 30 40 50 60

iBuOH (111)

Figure 2.25. Effect of iBuOH concentration in the reaction medium for
formation of iBuCF-iBuOH derivatives of amino acids (2).

105

Effect of iBuOH

Response

 

 

0.0 I I I I l I I I I l I I I I l I I I I I I I I I I
O 10 20 3O 4O 50 60
iBuOH (ul)

 

Figure 2.26. Effect of iBuOH concentration in the reaction medium for
formation of iBuCF-iBuOH derivatives of amino acids (3).

106
b) Results and discussion

The results of TIC responses from these experiments did not follow
very smooth curves, but the trend of the TIC responses for each amino acid
derivative as a function of the volume of H20 added in the reaction solution
still can be described. The overall trend of TIC responses for most of the
derivatives was that the TIC responses increased slightly when the amount
of H20 added increased, and then the responses were about constant over a
range of volume of water added. The ranges were diﬁ‘erent for different
derivatives, but each amino acid derivative was among one of the several
groups. Approximately, the TIC responses of Asn, Cys, Met, Thr, Ser, Glu,
Om, and Trp were about constant when 20 to 100 111 of water were present in
the reaction medium. The corresponding range for constant responses was
from 40 to 100 111 for Ala, Gly, Val, and Len, and was from 60 to 100 111 for
Phe, nLeu, and Pro, while the range was 20 to 60 111 for Lys. The TIC
responses of Ile and Asp decreased slightly over the range of 10 to 100 111 of
water. The results are summarized in Table 2.7. His and Gln derivatives
were not detected in these experiments. These experiments indicated that
40 to 80 11.1 of H20 were appropriate for the reaction solution containing 30 111
of iBuOH, and the amount of H20 in the reaction solution did not seem to be

critical for the chloroformate derivatization of amino acids.

(3) Eﬁ'ect of iBuCF concentration in the derivatization
a) Experimental
A solution of 50 111 of 24 amino acids (2 ug/each) were added to each of
ﬁve solutions containing 30 111 H20, 30 1.11 iBuOH, and 10 111 pyridine. To
each solution, various amounts of iBuCF (5, 10, 15, 20, 30 111) were added

and reaction mixtures were vortexed for 308. The derivatives were

107

Table 2.7. Effect of H20 volume in the reaction medium (X 111 H20, 30 1,11

iBuOH, and 10 111 pyridine) on the TIC responses of iBuCF-iBuOH
derivatives of amino acids.

 

Amino acids Range of volume of H20 added to
produce constant TIC responses of
amino acid derivatives

Asn, Thr, Ser, Met, Glu, Cys, Orn, 20-100111

Trp

Ala, Gly, Val, Leu 40 - 100 111
nLeu, Pro, Phe 60 - 100 1.11
Lys 20 - 60 1.1.1

Ile, Asp slightly decrease from 10 - 100 111

 

108
extracted by chloroform twice, 200 1.11 and 100 1.11 for each time; the solvent

was removed (evaporated) under N2 stream. Derivatives were redissolved
in 100 111 chloroform. A 2-111 aliquot of chloroform solution was injected for
analysis. The GC-MS conditions employed were the same as those in the

previous experiments.

b) Resultsanddiscusdon

The TIC responses of most of the derivatives of amino acids were
constant when the iBuCF added was 5 to 10 111. Their responses dropped
signiﬁcantly when the amount of iBuCF added was more than 10 to 15 111.
Part of the results are shown in Figure 2.27. The results from these
experiments can be explained by the hydrolysis of the large excess of
iBuCF. The HCl released from the hydrolysis lowered the pH of the
reaction solution. (See discussions in the next section: Effect of

concentration of pyridine.)

(4) Eﬂ‘ect of pyridine concentration in the reaction medium
a) Experimental

A 10—111 solution of 24 amino acids (each 42 rig/111) was added to each of
the six solutions containing 70 111 H20, 30 1.11 iBuOH, and various amounts of
pyridine: 0, 2, 5, 10, 15, and 20 1.11. To each solution, 10 111 of iBuCF were
added and the reaction mixtures were vortexed for 30 s. The derivatives
were extracted by chloroform twice, 200 1.11 and 100 111 for each time; the
solvent was removed (evaporated) under N2 stream. Derivatives were
redissolved in 25 1.11 chloroform. A 2-1.11 aliquot of chloroform solution was
injected for analysis. The GC-MS conditions were the same as those used

in the previous experiments.

Effect of iBuCF concentration

 

 

 

 

250.0:
200.0;
.1 .
2150.0:
0 ..
3 :
33100.07
50.0-
1
0.0 . .
015 30 35
iBuCF(ul)
300.0:
250.0:
200.0—
8 1
8
5150-03
g .
100.05
50.05
0.0 .. .
° 30 35
iBuCF(uol)

Figure 2.27. Effect of iBuCF concentration for the derivatization of amino
acids by iBuCF-iBuOH.

110
b) Results and discussion

Without pyridine, there were no derivatives detected. When the
amount of pyridine was 2 111, weak signals for some amino acid derivatives
(such as Gly, Val, and Phe) were detected. When the amount of pyridine
increased up to 5 111, the responses of all the amino acid derivatives
increased. When the amount of pyridine was from 10 to 20 1.11, there were no
signiﬁcant changes for the TIC responses for all the amino acid
derivatives. (His, Trp, and Cys-Cys derivatives were not detected in these
experiments.)

The pHs of the reaction solutions before and after the addition of
iBuCF in each experiment with pyridine from 0 to 20 111 are listed in

Table 2.8.

Table 2.8. pH vs. pyridine volume in the reaction medium for amino acid
derivatization by iBuCF-iBuOH

 

 

Pyridine 0 2 5 10 15 Z)
(111)

pH 1-2 5—6 6 67 7 7
(before)

pH 1-2 .1-2 2 4-5 5 5
(after)

 

Pyridine did not only act as a catalyst, but also as a buffer to control

the pH of the reaction solution for the chloroformate derivatization reaction.

When the amount of pyridine added was between 10 to 20 1.11, the pHs before
the addition of pyridine were the same (~7) for each solution, and the pHs
after the addition of iBuCF were also the same (~5) for each solution. The

111
constant pH changes of the reaction solutions paralleled the constant TIC

responses of the amino acid derivatives in the range of addition of 10 to 20 111
of pyridine . With a lesser amount of pyridine added in the reaction
solution, the pHs of the reaction solutions after the addition of iBuCF were
low (~1-2). The carboxyl and amino groups were exclusively protonated at
these low pH which might be the reason why there were less or no
derivatives formed. The amount of pyridine should be no less than 8 to 10 1.11

in the reaction solution containing 80 111 H20 for the amino acid

derivatization with iBuCF.

(5) Effect of other bases and buﬂt‘er solutiom
a) Experimental

A solution of 50 1.11 of 24 amino acids (2 11g/each) was added to each of
the two solutions containing 30 1.11 H20, 30 1.11 iBuOH. Instead of pyridine, 10
1.11 of 2,4,6-trimethylpyridine or 100 111 of DMAP (10 1.1g/1.1l in chloroform) were
added. To each solution, 10 1.11 of iBuCF were added and reaction mixtures
were vortexed for 30 s. The derivatives were extracted by chloroform twice,
200 1.11 and 1 00 1.11 for each time; the solvent was removed (evaporated) under
N2 stream. Derivatives were redissolved in 100 111 chloroform. A 2-111
aliquot of chloroform solution was injected for analysis. The GC-MS
conditions were the same as those in the previous experiments.

Instead of any organic base, 50 1.11 of buffer: (i) 0.2 M sodium
phosphate (pH=7 .68), (ii) 0.1 M sodium borate (pH=9.5), (iii) 0.1%
HAc/NH4Cl (pH 4.0) were used in the same experiments as above.

112
b) Results and discussion

Although DMAP was a stronger base (pKa~10), no derivatives of
amino acids were detected in GC-MS analysis. The pH of the reaction
solution before and after the addition of iBuCF was «8 and ~5 respectively.
Derivatives of amino acids were formed when using 2,4,6-
trimethylpyridine. The results were about the same as what was observed
when pyridine was added. The pHs of the reaction solution before and after
the addition of iBuCF were both ~7-8. These bases did not provide any
advantages over pyridine.

No derivatives of amino acids were detected in each of the cases of
using buffer solutions. This further conﬁrmed the importance of pyridine

as a catalyst in the chloroformate derivatization of amino acids.

(6) Evaluation of acid I base wash of the chloroform phase
during the extraction of derivatives
a) Experimental
A mixture of amino acids were derivatized by 10 1.11 iBuCF in a
reaction solution containing 80 1.11 H20, 30 1.11 of iBuOH, and 10 1.11 pyridine.
Afterwards, different procedures were followed:
i) "Normal": The derivatives were extracted by 200 1.11 chloroform.
ii) "Acid wash": A 100 pl of 1 M HCl was added in the reaction
solution at the time of chloroform extraction.
iii) "Acid wash + base wash": The chloroform solution from ii) was
washed by 100 1.11 of 0.5 M NaHCOa.
The "acid wash" experiment was repeated for the derivatization of
the amino acid mixture with 10 111 of EtCF. The solution contained 60 111
H20, 30 111 EtOH, and 10 111 pyridine. "

113

b) Results and discussion
During the chloroform extraction, HCl solution can transfer the
remaining pyridine into the upper aqueous phase. For iBuCF derivatives of
amino acids, the TIC of the amino acid derivatives prepared with i), ii), and
iii) were compared. N 0 signiﬁcant difference in responses and separations
were found. The same result was found for the EtCF derivatives of amino

acids.

C. Reproducibility of the derivatization and analyses of amino acids
with chloroformate-alcohol reagents
(1) Experimental
a) iBuCF-iBuOH
Five samples were made following the procedure below. A mixture
of 24 amino acids (2 1.1g/each) were derivatized by 10 111 iBuCF in a solution
containing 60 111 H20, 30 111 iBuOH, and 20 111 of pyridine. The derivatives
were extracted by chloroform three times, 200 111, 100 111, and 100 1.1.1 for each
time; the solvent was removed (evaporated) under N 2 stream. Derivatives
were redissolved in 100 111 chloroform. A 2-111 aliquot of chloroform solution
was injected for analysis. The GC-MS conditions were the same as those in
the previous experiments.
b) EtCF-MB
Five samples were made following the procedure in a), but iBuCF
was replaced by EtCF.
c) iBuCF-HFBuOH
Three samples were made following the procedure in a), but iBuOH

was replaced by HFBuOH.

114

(2) Results and discussion

The reproducibility of the whole process of chloroformate
derivatization and GC-MS analysis of amino acids was evaluated for three
chloroformate-alcohol reagents. The results are listed in Table 2.9 which
includes the mean relative weight responses (RWR) of peak area and height
in TIC (relative to the internal standard, nLeu), and relative standard-
deviations (RSD%). The RSDs for EtCF derivatives were around 10-20%.
Ser did not show a good peak resulting in a high RSD. Most of the RSD’s
were around 10% for iBuCF-iBuOH derivatives and were below 10% for
iBuCF-HFBuOH derivatives. The reproducibilities were not very good
because each step of the whole derivatization procedure could contribute
some variations. The reaction proceeded in a small narrow glass tube
which was about 4 cm long and 0.3 to 0.4 mm in the diameter. The starting
materials and derivatives may attach on to the wall of the glass tube
although vortexing has been performed to mix the solution during the
reaction. During the extraction-separation step, technically, it was very
hard to exactly separate the entire chloroform layer from the aqueous layer.
It also may result in losing part of the most volatile derivatives to remove
the chloroform solvent under N2 stream.

Table 2.10 compares the TIC responses of the other two derivatives
relative to those prepared from EtCF-EtOH. For each amino acid, the TIC
response of iBuCF-HFBuOH derivative seems to be higher than those from
EtCF-EtOH. The factors of the difference can be up to 3 to 4 for Ala, Gly,
Val, Leu, and Ile. Most derivatives of iBuCF-iBuOH show higher TIC
responses than those from EtCF-EtOH except for Met, Om, Lys, Tyr, and

Trp.

115
In these experiments, Hyp, Gln derivatives were not detected from

these three derivatization reagents. (Hyp was detected in the earlier
experiments in section B when the amino acid solution was newly made.)
His was only detected from iBuCF-HFBuOH derivatization reagents. Cys
was not detected from iBuCF-iBuOH reagents.

The analyses of ten times diluted solution showed that the iBuCF-
HFBuOH derivatives had much better signal to background ratios than
those of EtCF-EtOH derivatives.

V. Comparisons of E1, PCI, and ECNI mass spectrometry responses of
ﬂuorinated derivatives of amino acids

A. Introduction

Introducing fluorinated moieties into amino acid derivatives from
ﬂuorinated alcohols through our modiﬁed chloroformate derivatization
procedure is one of the most important applications of our investigation of
the mechanism of the one-step chloroformate derivatization for amino
acids. The ﬂuorinated derivatives prepared by this simple method have the
potential to facilitate analyses by electron capture negative ionization mass
spectrometry. Characterization of EtCF-TFEtOH amino acid derivatives
with positive chemical ionization (PCI) and electron capture negative
ionization (ECNI) GC-MS based on our modiﬁed reaction mechanism was

reported earlier this year by Moini [43 ] paralleling our research.

116

Table 2.9. Effect of reagent composition on derivatization formation and
response. Mean relative weight responses of peak area of TIC (relative to
internal standard nLeu) (RWR) and relative standard deviation (RSD) for
the indicated amino acid derivatives (Five individual samples for EtCF-
EtOH, iBuCF-iBuCF derivatization, and three individual samples for
iBuCF-IFBuOH derivatization).(* Leu and Ile coelute.; “ Lys peak contains His.)

 

Amino l EtCF- EtOH

 

 

iBuCF- iBuOH iBuCF- HFBuOH
Acid I RWR RSD% RWR RSD% RWR RSD%
Ala 0.393 20 0.505 20 0.676 6.8
Gly 0.882 17 0.983 7.6 1.17 2.0
Val 0.641 15 0.996 10 1.17 4.4
Leu 0.725 6.1 0951 4.3 0830* 0.48
Ile 0.646 11 0.830 3.6 0.830 0.48
Pro 1.21 16 1.08 3.6 1.00 2.3
Thr 0.959 20 0.461 10 0.545 0.65
Ser 0.395 62 0.195 a) 0.246 2.7
Asn 1.19 16 0.707 4.8 0.749 4.6
Asp 1.40 16 1.03 7.1 .873 4.1
Met 1.83 15 0268 28 1.07 ' 4.3
Glu 0.384 15 0254 12 0.316 12
Phe 2.35 17 1.20 7.2 1.55 2.8
Cys 0.734 18 - - 0.390 5.2
4-Cl-Phe 2.15 20 0.962 9.0 1.16 6.9
Orn 0.984 21 0275 15 0.538 17
Lys 1.46 23 0.359 17 0.686** 16*
Tyr 1.93 21 0.505 18 0.950 11
Trp 0.656 38 0.165 30 0.646 11
Cys-Cys 0.421 %

 

117

Table 2.10. Ratio of both peak area and peak height of reconstructed TIC
corresponding to the indicated derivatives relative to those made with EtCF-
EtOH from analyses by GC-MS (EI) (results from experiments for
reproducibility).

 

iBuCF-iBuOH/EtCF-EtOH iBuCF-HFB/EtCF—EtOH

 

Area Height Area Heiﬂt
Ala 3.0 3.2 4.1 4.4
Gly 2.6 2.9 3.1 3.6
Val 3.5 4.5 4.2 5.2
Leu 2.9 3.2 2.6 3.1
Ile 2.9 3.3 3.0 3.8
nLeu 2.3 2.2 2.3 2.2
Pro 2.0 2.8 2.0 2.6
Thr 1.1 1.0 1.4 2.1
Asn 1.4 1.7 1.5 1.9
Met 0.30 0.49 1.4 1.7
Asp 1.7 2.4 1.5 1.5
Phe 1.2 1.5 1.6 1.4
Glu 1.5 2.0 1.9 2.4
Cl-Phe 1.0 1.3 1.3 1.8
Orn 0.64 0.84 1.4 2.0
Lys 1.8 0.68 1.1 1.5
Tyr 0.60 0.55 1.2 1.4
Trp 0.59 0.58 2.4 3.6

Cys - - 1.3 2.1

118
B. EtCF-HFBuOH and iBuCF-HFBuOI-I derivatives of amino acids in

E1, PCI, and ECNI modes of GC-MS analyses

EtCF-HFBuOH amino acid derivatives have been compared in EI,
PCI, and ECNI modes. EI analyses were performed under conditions of 70
eV and 100 11A ionization current. The PCI and ECNI conditions were ~100
eV and 300 11A.

Comparing the El and PCI modes for the EtCF-HFBuOH derivatives
of amino acids showed similar TIC and mass spectra except for the higher
abundance of [M+H]+ in PCI spectra (Figure 2.28a and 2.28b). In the ECNI
mode the maximum intensity (base peak) of TIC was higher than that in
the PCI mode. Ala, Gly, Val, Leu, Ile, Pro, and nLeu derivatives that
eluted at a lower temperature, showed relatively low intensities (Figure
2.28c). The reason for this was not clear, however; the same situation was
found for iBuCF-HFBuOH derivatives in ECNI mode as well. The ions, [M-
HFB]‘, ([M-l 831‘), and [M-28]' ions, were observed in relatively high
abundance for some amino acid derivatives in the ECNI spectra. But the
spectra of the EtCF-HFBuOH amino acid derivatives in the ECNI mode
were not simpliﬁed as expected and the base peak in each spectrum was not
from one speciﬁc fragmentation pathway common to all the spectra of these
derivatives. The spectra did not have simple, clear patterns.

No dominating characteristic ions, formed from a common speciﬁc
fragmentation pathway for each of the EtCF-HFBuOH amino acid
derivatives, can be used for quantitation in ECNI mode to increase the
sensitivity of detection for amino acids. The intensities of each [M+1]+ ion
in PCI mode and each [M-1]' ion in ECNI mode from their mass
chromatograms for the amino acid EtCF-HFBuOH derivatives are listed in
Table 2.11. It can be noted that regarding the [M+1]+ ion in PCI and the

119

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(a)EI
100mg 4 6 or.--‘.°-..‘?-““ .RT.
R AAAAAAAAAAA w
1' 1 11.11 c-c
1.0044 CI-F
l . F
V
C ‘1:
1
ﬂ
1
0
ﬂ
1'
‘ LL...
560 660 760 060 s 1
1o - 12 - - 14 8.1.
"'°° * ‘ .1... 1
I 3 m4
' em
1 i L“ s 1“
V 1 ‘ P P
' “l G 1 .. were
1 1
N M
94d A +0
. 4
" I
l. 204 S d L!
1 ‘ .
Y 1 UL] MU
o
260 360 460 500 can 760 s
(c)ECNI
1005A.°A-e184-§---§---‘.°-A,L2AA14 RT.
5 1 N ““511
l 1-5
1 , F
' 30* I:1+T
’ ‘ c
' ‘ or
V 1
° 60‘ 0 K414
1 1
n 1
r 401
. 1
n 1
4 s
1‘ 20~ V 1.44an w l
‘ 4
’ .1
‘55 203 350 460 560 660 760 960 s

Figure

 

2.28. TICs of EtCF-HFBuOH derivatives of amino acids in (a) El, (b)

PCI, and (3) ECNI modes of GC-MS analysis.

120

Table 2.11. Comparison of absolute intensities of [M+1]+ in PCI and [M-1]‘

in ECNI of amino acid EtCF-HFBuOH derivatives.

 

 

EtCF-HFBuOH PCI ECNI
Amino acid MW I [M+1]+ I [M-ll‘
Ala 343 22.2 13.1}
Gly 329 54.1 13.4
Val 371 17.4 17.9
Leu + Ile 385 27.9 24.2
nLeu 385 ~% ~17
Pro 370 33.4 12.5
Thr 373 7.9 4.3
Asp 5% 14.7 12.0
p-Glu 311 46.2 4.4
Ser 359 5.4 2.5
Asn 368 (-H20) 29.2 119.5
Glu 583 2.3 0.8
Met 403 17.9 (M+) 21.6
Phe 419 20.7 25.9
Cys 447 25.6 8.2
Cl-Phe 453 20.9 50 / 469.4 (by M)
Gm 458 ~8 (M"') ~8
His 481 53.3 18.4
Lys 472 10.4 21.4
Trp 458 31.2 (M"') 34.0
Gln 4(1) - 4.8
Cys-Cys 748 2.3 28.7

 

121
[M-1]‘ ion in ECNI modes, the negative ion mode does not show any

advantage for most of the N-ethoxycarbonyl amino acid heptaﬂuorobutyl
esters. A similar result was reported for EtCF-TFEtOH amino acid
derivatives [43].

The same chromatographic and spectral results were found for
iBuCF-HFBuOH derivatives of amino acids when they were compared in

EI, PCI, and ECNI modes of analyses by GC-MS.

C. EtCF-pentaﬂuorobenzyl alcohol (PFleOH) derivatives of amino
acids in PCI and ECNI modes of GC-MS analyses

EtCF-PFleOH amino acid derivatives have been compared in PCI
and ECNI modes of GC-MS analyses. The PCI spectra of these derivatives
followed general fragmentation pathways as described in section I of this
chapter and in Ref. 4. The major fragmentation pathway resulted from
[MH-PFBCOzHT" ([MH-226]+) plus an additional PFB+ (m/z 181) ion. (Thr,
Ser, Gln, Glu, His, Trp, Cys-Cys peaks were not detected or not identiﬁed in
this experiment). In the ECNI mode, the [M-PFB]' ([M-181]') ion dominated
each spectrum of the derivative, and the spectra were simple with only a
few ions. (Ser, Gln, Trp, and Cys-Cys peaks were not detected or not
identiﬁed). Representative mass spectra of each ionization mode are shown
in Figure 2.29 for the Val derivative and in Figure 2.30 for the Phe
derivative. It can be seen that both Val and Phe EtCF-PFleOH derivatives
show only two ions in their ECNI spectra while their PCI mass spectral
peaks represent more fragmentation. The ECN I spectrum of Val is
dominated by ion of m/z 188, the [M-181]' ion, and the ECNI spectrum of
Phe is also dominated by the [M-181]' ion (m/z 236). The ECNI spectra of

122
EtCF-PFleOH derivatives of amino acids are summarized in Table 2.12.

The

intensities of the base peaks, [M-PFBJ‘, in ECN I and the intensities of [MH-
PFBCOzHJ+ ions (the base peaks in most spectra) in PCI of amino acid
EtCF-PFleOH derivatives are listed in Table 2.13. These intensities are
from the mass chromatogram for each ion. It is probable that most of the
base peaks in the ECNI spectra are oﬂ'scale since they all reach a count of
1600 which might be the maximum for the instrument utilized. The
diﬁ‘erent intensities of these two ions indicate that sensitivity is increased by
at least one to two order of magnitude in the ECNI mode over PCI ion
detection for most of the amino acid derivatives detected in both modes. The
[M-181]‘ ions can be used as the trace ions for proﬁling and quantitation of
amino acids in the ECNI mode of GC-MS analysis after derivatization with
EtCF-PFleOH reagents.

Although an increase in sensitivity of detection of amino acid
derivatives in the ECNI mode compared to PCI mode by EtCF-PFleOH
derivatization was observed, there are still some practical problems for this
derivatization scheme: 1) In ECNI, the resolution of TIC was not as good as
that in PCI, and there were peaks which were not identiﬁed. 2) It was
veriﬁed from the TIC and spectra in PCI mode, that for some amino acids,
the TIC response of the derivatization product in which the ester part was
an ethyl group from ethyl chloroformate was half of that of the major
product of pentafluorobenzyl ester. These may come from the weak
nucleophilic reactivity of pentaﬂuorobenzyl alcohol. 3) PFleOH was not

volatile and cannot be removed under N2 stream.

123

 

 

 

 

 

 

 

 

 

 

(a)PCI
100‘ 1‘
R
,e 80; Val EtCF-PFleOH
f . MW369
I 4
v 60-
° 1
A I
b 40. .
U 1 1
n , 181 ,
d .
a 201
n .
c 296
116 '
98 324 1
a YTJiLl‘ '1 “11103918 . . .
100 200 300 400 500 600
M12
(b)ECNI
100 1
R j T“ M481
9 , Val EtCF-PFleOH
L em MW369
t «
t .
V 601
a 4
A .
b 40<
U < .
n 142 L
d . .
a 204
n .
c L
6 1
1oo'n'ado'w'ao'ovh'4do'"'so'o'ﬂ'edo'v'v'ioo'ﬁ
M/Z

Figure 2.29. (a) PCI and (b) ECNI mass spectra of Val EtCF-PFleOH
derivative.

124

 

 

 

 

 

 

 

 

 

 

 

 

 

(a) PCI
100+ 3:33 .
R I Phe EtCF-PFleOH ;
? em MW 417 .
a ‘ ’
1 « '
t . ,
v 60‘ *
e « ’
. 181192 ’
A . ,
b 40- *
U 4 .
3 ‘ D
a 20: 91 372 418 1
n . 233 344 .
c , »
e . 65 1 147 ft 354' ‘33 l l 4416 ’
o 100 200 300 1 ' '460 r ' ' r560 3‘0];
(b) ECNI
1oo
« 29‘ M-181
R 4
f so: Phe EtCF-PFleOH
a ~ MW417
t .1
i 4
v 60‘
a 1
c 1
40"
u , 190
n 1
d .
a 20.
n .
c
9 l
(300-...260....360....‘60-...560 ado 1760113

Figure 2.30. (a) PCI and (b) ECNI mass spectra of Phe EtCF-PFleOH

derivative.

125

Table 2.12. Summary of ECNI mass spectra of amino acid EtCF-PFleOH
derivatives.

 

 

Amino acid [M-181]‘ Base peak Other ions

Ala m/z 160 m/z 160 m/z 114

Gly m/z 146 m/z 146 m/z 100

Val m/z 188 m/z 188 m/z 142

Leu m/z 202 m/z 202 m/z 156

Ile m/z 202 m/z 202 m/z 156

nLeu m/z 202 m/z 202 m/z 156

Pro m/z 186 m/z 186

Thr m/z 190 m/z 190 m/z 163

p-Glu m/z 128 m/z 128

Asn m/z 185 m/z 185 m/z 139, 227
Met m/z 220 m/z 220 m/z 174

Phe m/z 236 m/z 236 m/z 190

Cys m/z 264 m/z 264 m/z 218, 158, 174
Cl-Phe m/z 270 m/z 270 m/z 224

Asp m/z 384 m/z 384 m/z 186, 338
Orn m/z 275 m/z 275 m/z 229

Glu . m/z 398 m/z 398 m/z 200, 218, 352
Lys m/z 289 m/z 289 m/z 180, 243

His

m/z 298

m/z 298

m/z 180, 252

126

Table 2.13. Comparison of intensities of [M-PFBJ' in ECNI and [MH-
PFBC02H]+ in PCI of amino acid EtCF-PFleOH derivatives.

 

 

Amino EtCF- ECNI Intensity PCI Intensity
acid PFleOH [MH-
[M-PFB]’ PFBCOzHT"
(MW) ([M-1811') J[M-181]‘ [MK-2261+ [MB-2261+

Ala 341 m/z 160 1600* m/z 116 64
Gly 327 m/z 146 400 m/z 102 Q
Val 3$ m/z 188 1600* m/z 144 1(1)
Leu 383 m/z 202 1600* m/z 158 ~70-80
Ile 383 m/z 202 1600* m/z 158 1%
nLeu 383 m/z 202 1600* m/z 158 ~90-100
Pro 367 m/z 186 1600* m/z 142 100
Thr 371 m/z 190 350 - -
p-Glu 309 m/z 128 400 - -
Asn 366 (-H20) m/z 185 1600* m/z 141 200
Met 401 m/z 220 1600* m/z 176* 12
Phe 417 m/z 236 1600* m/z 192* %

m/z 328 (bp) 73
Cys 445 m/z 264 1600* m/z 220* 42
Cl-Phe 451 m/z 270 1600* m/z 226* 16

m/z 362 (bp) 48
Asp 565 m/z 384 1600* m/z 340 18
Gm 456 m/z 275 768 m/z 142 7.0
Glu 579 m/z 398 1% - -
Lys 470 m/z 289 4(1) m/z 156 3.0
His 479 m/z 298 152 - -

 

*. maximum intensity

#. not base peak

127
VI. Conclusions

Characterization of chloroformate-alcohol derivatives of amino acids
has been conducted to facilitate the identiﬁcation of amino acids in analysis
by GC-MS using the chloroformate derivatization method. The one-step
chloroformate derivatization of amino acids in an aqueous medium has
been extended with the use of a variety of alkyl chloroformate and alcohol
reagents. It was discovered that the ester moiety of the amino acid
derivatives is directly dependent upon the type of alcohol used in the
aqueous reaction medium. Based on these ﬁndings, a new mechanism for
ester formation is proposed to involve an alcohol exchange reaction with an
intermediate mixed anhydride of the carboxyl group. These results have
provided new insight into the one-step derivatization reaction and have
provided the basis for preparing a variety of derivatives that can be assessed
for optimizing the analysis of amino acids by CO with FID or by GC-MS.
Discovering the inﬂuence of the alcohol on the chloroformate reaction in an
aqueous medium opens the possibility for preparing a wide variety of ester
derivatives that can be tailored to the analytical needs of a speciﬁc problem.
The derivatization conditions of amino acids with isobutyl chloroformate-
isobutanol have been completely investigated. Fluorinated moiety was
introduced into the amino acid derivatives through a modiﬁed reaction
procedure. Comparisons of responses from El, PCI, and ECNI ionization
for these ﬂuorinated derivatives of amino acids in analysis by GC-MS have
been conducted. Although the reaction conditions need improvement ethyl
chloroformate-pentaﬂuorobenzyl alcohol derivatives of amino acids showed
increased sensitivity of detection in ECNI mode than in PCI mode. The
one-step aqueous medium chloroformate derivatization method can apply to

the gas phase analysis of a variety of compounds having amino, carboxyl

128

polar functional groups. Its characters of simple and fast show advantages

over other derivatization methods for analysis of amino acids by GO or GC-

MS. In next two chapters, we will discuss the applications of the method

and its further extension to peptide analysis in desorption mass

spectrometry.

VII. References

1 . P. Husek, FEBS Lett., 280 (1991) 354.

2. P. Husek and C.C. Sweeley, J. High Resolut. Chromatogr., 14 (1991)
751.

3. P. Husek, J. Chromatogr., 552 (1991) 289.

4. Z.-H. Huang, J. Wang, D.A. Gage, J .T. Watson, C.C. Sweeley and P.
Husek, J. Chromatogr., 635 (1993) 271.

5. PD. Bailey, An introduction to Peptide ChemistryJohn Wiley &
Sons, 1990.

6. M. Matzner, R.P. Kurkjy, and R.J. Cotter, Chem. Rev., 64 (1964) 645.

7. M. Makita, S. Yamamoto, and M. Kono, J. Chromatogr., 120 (1976)
129.

8. M. Makita, S. Yamamoto, and S. Kiyama, J. Chromatogr., 237 (1982)
279. I

9. P.G. Pearson, M.D. Threadgill, W.N. Howald, T.A. Baillie, Biomed.
Environ. Mass Spectrom., 16 (1988) 51.

10. K.J. Hoﬁ'mann, T.A. Baillie, Biomed. Environ. Mass Spectrom., 15
(1988) 637.

11.

P.G. Pearson, W.N. Howald, S.D. Nelson, Anal. Chem., 62 (1990)
1827. "

12.
13.

14.

15.
16.

17.
18.
19.

25.

26.

29.
30.
31 .
32.

129
A.P.J.M. De Jong and C.A. Cremers, J. Chromatogr., 276 (1983) 267.

O. Gyllenhaal, L. Johansson, and J. Vessman, J. Chromatogr., 190
(1980) 347. ‘

E.J. Miller, A.J. narkates, M.A. nieman, Anal. Biochem. 190 (1990)
92.

B. Gustavsson, I. Bentner, J. Chromatogr., 507 (1990) 647.

S. Einarsoon, B. Josefsson, and S. Lagerkvist, J. Chromatogr., 282
(1983) 609.

I. Betner and P. Foldi, Chromatographia, 22 (1986) 381.

M. Ahnoﬁ‘, S. Chen, I. Grundevik, J. Chromatogr., 506 (1990) 593.
S. Einarsson, B. Josefsson, P. Moller, and D. Sanchez, Anal. Chem.,
59 (1987) 1191 .

A. Carlson and O. Gyllenhaal, J. Chromatogr., 508 (1990) 333.

S. Bjorkman, J. Chromatogr., 339 (1985) 339.

S. Bjorkman, J. Chromatogr., 414 (1987) 465.

P. Husek, J .A. Rijks, P.A. Leclerg and C.A. Cramers,

J. High Resolut. Chromatogr., 13 (1990) 633.

P. Husek, J. Chromatogr., 547 (1991) 307.

H.M. Liebich, E. Gesele, H.G. Wahl, C. Wirth, J. W011 and P. Husek,
J. Chromatogr., 626 (1992) 289.

P. Husek, J. Chromatogr., 615 (1993) 334.

P. Husek, J. Chromatogr., 630 (1993) 429.

A. Einforn, Ben, 42 (1909) 2772.

E.J. Longosz and D.S. Tarbell, J. Org. Chem., 26 (1961) 2161.

D.S. Tarbell, Acc. Chem. Res., 2 (1969) 296.

TB. Windholz, J. Org. Chem., 23 (1958) 2044.

D.S. Tarbell and E.J. Longosz, Ibid., 24 (1959) 774

33.

35.
36.
37.

39.

42.

130 .
C.J. Michejda, D.S. Tarbell, and W.H. Saunders, Jr., J. Am. Chem.

Soc., 84 (1962) 4113.

T.B. Windholz, J. Org. Chem., 25 (1960) 1703.

D.S. Tarbell, N.A. Leister, J. Org. Chem., 23 (1958) 1149.

S. Kim, J .1. Lee, Y.C. Kim, J. Org. Chem., 50 (1985) 560.

S. Kim, Y.C. Kim, J .1. Lee, Tetrahedron. Lett., 24 (1983) 3365.

J .M. Domagala, Tetrahedron. Lett., 21 (1980) 4997.

L. Gutman, A. Boltanski, Tetrahedron. Lett., 26 (1 985) 1537.
R.L.Barnden, R.M. Evans, J .C. Hamlet, B.A. Heins, A.B.A. Jansen,
M.E. Trevett, and GB. Webb, J. Chem. Soc., 3733 (1953).

D.A. Johnson, J.Am. Chem. Soc., 75 (1953) 3636.

K. Freudenberg and W. Jacob, Ber. , B74 (1941) 1001.

M. Vatanhah and M. Moini, Biol. Mass. Spectrom., 23 (1994) 277.

Chapter III
Application of chloroformate derivatization in the quantitative assessment
of incorporation of stable isotope-labeled amino acids into photosynthetic
proteins of WW PCC 6803

I. Introduction

This chapter will describe the work of the author in a collaborative
research project with Dr. N. R. Bowlby who worked with Dr. L. McIntosh in
the MSU-DOE Plant Research Laboratory and Dr. C. Hoganson from the
laboratory of Dr. G.T. Babcock in the Chemistry Department at MSU [l].
The goal of the research project was to develop analytical procedures for
quantitative assessment of the incorporation of stable-isotope labeled amino
acids into photosynthetic proteins of the cyanobacterium Synechocystis
6803. The one-step aqueous medium amino acid derivatization / GC-MS
analysis method was applied as part of a quantitative assessments for some
aspects the research project.

The conversion of the energy of light into chemical energy is one of
the fundamental process of life. In plant and algae photosynthesize,
Photosystem II (PS II) centers were found to independently store oxidizing
power and catalyze the formation of dioxygen [2,3]. The mechanism of
photosynthesis has not been well understood. The introduction of
biochemical techniques for the puriﬁcation of photosynthetic complexes at
the end of 708 made it possible to resolve the structure/function relationship
in these protein complexes by using spectroscopic techniques.

Isotopic substitution of particular amino acids thought to be
important in the catalytic process of the photosynthetic proteins can provide

information about events at the atomic level. Aromatic amino acids have

131

132
distinctive electronic and chemical properties, they may perform important

functions within enzymes. Many non-photosynthetic organisms are unable
to synthesize aromatic amino acids and require that they be present in their
diet or growth medium. For such an organism, providing the
appropriately labeled amino acid will ensure that the proteins of interest
become labeled, a phenomenon that has been widely exploited in
characterizing molecular processes in these systems. Photosynthetic
organisms, including cyanobacteria, however, are usually capable of
synthesizing de novo all of the amino acids, and show little tendency to take
up exogenous amino acids from the growth medium. This property has
prevented researchers in photosynthesis from achieving quantitative
isot0pic labeling of speciﬁc amino acids.

In Synechocystis, however, an excess of phenylalanine in the
medium inhibits the biosynthesis of tyrosine and tryptophan, as well as that
of phenylalanine [4]. In this collaborative research project, incorporation of
isotope-labeled aromatic amino acids into the photosynthetic proteins of
Synechocystis by the addition of these amino acids to the growth medium
has been investigated. The analytical procedures that were used to quantify
the extent of incorporation of labeled amino acids into proteins of the
photosynthetic apparatus will be described.

The experiments in this project were undertaken in different
laboratories. The incorporation of labeled amino acids into the proteins and
the electron paramagnetic resonance spectroscopy (EPR) experiments were
carried out by other collaborators. The quantitative assessment of the
incorporation of labeled amino acids was accomplished by the author.

Experiments and results from the other collaborators will also be described

133
and discussed briefly in order to give an overall picture of the research

accomplished.

11. Experimental

Blue-green cyanobacterium Synechocystis 6803 cells were grown by
the standard method. Phenylalanine was added in the growth medium to
inhibit the endogenous aromatic amino acid biosynthesis [4]. Cell density
was measured at various times following inoculation. Cell harvest was
followed by protein isolation, puriﬁcation and hydrolysis. The amount of
amino acids in the culture was monitored by UV spectroscopy. These
experiments were carrried out by Dr. N. R. Bowlby. The relative change in
the amount of amino acids in the culture was also monitored by the direct
derivatziation of the amino acids in the clariﬁed growth medium and GC-
MS analysis with the method described in chapter II. Amino acid analysis
of the protein hydrolysates was performed by the method of derivatization
with isobutyl chloroformate and analysis by GC-MS as discussed in chapter
II. GC-MS analysis was performed with a Hewlett-Parkard 5890J gas
chromatograph-JOEL AX-505H double-focusing mass spectrometer. The
amino acids thus generated were derivatized with isobutyl chloroformate
and 0.05 to 0.25 nmol of sample was subjected to GC-MS analysis. Controls
were performed to ensure that the isotopic labeled amino acids were not
degraded during the acid hydrolysis. For the experimental details see
Reﬁl .

III. Results and discussion
Techniques have been developed to monitor the ﬂux of exogenous

amino acid labels in Synechocystis through the use of induced obligate

134
auxotrophy, protein puriﬁcation, and GC-MS quantiﬁcation of individual

amino acids. Kinetics of growth and amino acid uptake in otherwise
photoautotrophic cultures of Synechocystis 6803 have been conducted.
Isotope labeled amino acid (”0 Tyr) was used in the detailed study of
the properties and environment of the redox active tyrosines Y1) and Yz by
EPR [5]. To use the isotope labeling approach effectively, however, it is
necessary to have the capability of quantifying the extent of incorporation of
the labeled amino acids into the photosynthetic apparatus. Amino acid
analysis with the simple derivatization method played a signiﬁcant role in

this part of the project.

A. Evidence of incorporation of labeled amino acids into cells from
cell growth curves

The standard procedure for monitoring the growth of Synechocystis
cells is to measure the apparent optical density at 730 nm. Typical growth
curves for Synechocystis cells grown in the absence and presence of the
amino acids are shown in Figure 3.1. The growth curve in the absence of
amino acids (panel a) has a normal shape characterized by a lag phase
followed by logarithmic growth. In the presence of the amino acids (panel
b), the growth rate is slower than fully autotrophic cells, as observed by
Barry and Babcock [4]. A stationary phase is reached at about 0.6 0D730
when the cells grow in the presence of the isotope label. If the cells are
allowed to grow beyond the plateau at 160 hours, the growth again becomes
logarithmic, and a second plateau is reached corresponding to the normal
cell density at stationary phase (i.e. 1 .2 to 1 .5 OD730).

Thus, when cells are grown in the presence of exogenous amino

acids it is useful to quantify amino acids present in the growth medium to

135
ensure that functional auxotrophy is maintained. Figure 3.1b also shows

the quantity of the aromatic amino acids present in the growth medium at
the times when cell density was determined. UV absorption spectra, at
selected times, of the growth medium after separation from the cells are
presented in Figure 3.2. The spectra show a strong absorbance at 276 nm
arising from aromatic amino acids. (While the absorption at 276 nm is a
combination of absorbances from all three amino acids, it is dominated by
tryptophan). As the cells enter the log phase of growth, the quantity of
exogenous amino acids in the growth medium declines rapidly, with nearly
complete depletion of these amino acids as the cells reach the ﬁrst
stationary phase. After remaining in stationary phase for several days, the
cells will again enter a logarithmic growth phase and reach the true
stationary phase, as noted above. During the second growth ‘spurt’ the
cells synthesize the needed amino acids since the growth medium has been
depleted of the exogenous phenylalanine to a level that does not maintain
auxotrophy. Continuation of cell growth beyond the ﬁrst stationary phase
thus results in the accumulation of non-labeled amino acid in proteins. It
was found that harvesting the cells within 24 hours of amino acid depletion
is optimal. This generally corresponds to an OD730 of between 0.45 and 0.65.
Samples collected at the second stationary phase and subjected to GC-MS

analysis show a nearly complete loss of the labeled amino acid (see below).

B. Quantitative assessment of incorporation of isotope labeled amino
As mentioned in the introduction, derivatization / GC-MS analysis of
amino acids played an important role in this research project. In order to

quantify the extent of incorporation of isotopic label, and to follow the time

136

 

.4.
0"

F3
01

 

ABSOEBANCE
t)

 

 

 

O 100 200 300 400
TIME (hrs)

 

21)

UJ
CJLS

1

1L}

SORBAN

EMIS-

A

 

 

 

 

o 160 260 300 400
TIME (hrs)

Figure 3.1. Cell density and abundance of aromatic amino acids in the
growth medium of Synechocystis cells. Panel A shows cell density as
measured by 0D73o of a culture grown in the absence of exogenously added
amino acids. In panel B is shown the cell density (0, 0 open and ﬁlled
symbols show data from two different cultures) and growth characteristic
of cells grown in the presence of phenylalanine (0.50 mM), tryptophan (0.25
mM) and l"O-tyrosine (0.25 mM). The aromatic amino acid absorbance (I)
was monitored at 276 nm (see Figure 3.2) in samples represented by the
ﬁlled circles on the growth curve (Reprinted from Ref. 1).

137

 

87 hours

   
 
    
 

111 hours

135 hours

 

Absorbance

160 hours

wavelength (nm)

 

 

 

Figure 3.2. Absorption spectra of culture medium obtained after growth for
the number of hours indicated. Cells and other solids were removed by
centrifugation before recording the spectra. The reference cuvette
contained BG-ll medium with no added amino acids (Reprinted from

Ref.1).

138
course of incorporation, the one-step aqueous medium amino acid

derivatization and GC-MS analysis method has been applied. The amino
acid analysis by GC-MS functioned to: 1) quantify the incorporation (uptake)
of isotope labeled amino acids into the photosynthetic-proteins by analyzing
the photosynthetic protein hydrolysates, monitor the time course of the
amino acid incorporation by analyzing the hydrolysates of proteins from
cells harvested at the different time of growth; 2) monitor the quantities of
amino acids present in the growth medium to verify those results obtained
from the UV absorption experiments; 3) verify if there is a discrimination of
uptake between the different isotopes of the same amino acid (i.e., 160 Tyr
and 170 Tyr); 4) ensure no exchange of hydrogens and deuteriums between
different amino acids during the acid hydrolysis. The EPR experiments
from the project partner failed to answer these questions because of the lack
of sensitivity of the technique to low levels of unlabeled amino acid.

Derivatization of amino acids in protein hydrolysates with iBuCF
leads to the formation of N(0,S)-isobutoxycarbonyl amino acid isobutyl
ester, which is amenable to GC-MS analysis.

We will discuss the GC-MS amino acid analysis results for the
representative photosynthetic protein fractions obtained from cells grown

on different labeled amino acids.

.1.

 

 

_l .
NHCOleu +
. . ‘I . '1‘?
0021811 / COleu / COZH
~NH2C02iBu (117)~ '04H8 (56) 6
m/z 321 (Mi') m/z 204 m/z 148

(Scheme 3.1)

139

Figure 3.3 shows the mass spectra of isobutyl chloroformate
erivatives of unlabeled phenylalanine (a), and dg-phenylalanine (b) from
tock solutions. For phenylalanine iBuCF derivative, one series of
ragmentation reactions is shown in Scheme 3.1. Elimination of
NH2C02iBu from Mt. gives the conjugated ion m/z 204. Subsequent loss of
C4113 leads to the formation of the high abundance ion m/z 148, which can

be used for the quantiﬁcation. Per-deuterated phenylalanine (d3-
phenylalanine) shows a shift of seven mass units for m/z 148 and m/z 204
(one 2H is lost during cleavage of the NH2COziBu moiety). Figure 3c shows
the TIC and mass chromatograms of the derivatives of the mixture of
phenylalanine and dg-phenylalanine (1 :1) from the stock solutions. These
two derivatives eluted as one peak in the TIC, but m/z 148 and m/z 155
reached their maximum at a different scan number. Figure 3.4 shows the
mass spectra of the derivative of phenylalanine in the hydrolysates of
phycobiliproteins recovered from control cells (a) and from cells that were
grown in the presence of dg-phenylalanine (b). Figure 3.4c shows the TIC /

mass chromatograms for the case with dg-phenylalanine in the growth
medium. In the dg-phenylalanine experiment, the phycobiliproteins used

for analysis were isolated from cells that had been allowed to grow well
beyond the initial plateau at ~0.6 OD730 and had reached the second
stationary phase at about 1 .2 OD730. The results clearly show the absence of
118-phenylalanine in these proteins. This observation cannot be explained by

simple dilution of label, as the cell number increased by only about 3-4-fold

(from the ﬁrst stationary phase to beyond the second stationary phase).

Rather, these results suggest that, when endogenous aromatic amino acid

 

 

*1!

140

100-

Phe

A El‘ A A E 14‘ A

74

m

 

ooanancc> "m"’-°3

 

 

 

 

5‘:
3%
53*

 

 

 

 

 

-0" -0
aoamaﬂcv) °‘ . I

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.51.3 1
Lo R.T
R GSLS l
1; Phe / d,-Phe
1
2 1
‘11 TIC ‘
e 5L3 ‘
I m/z 155 (d,-Phe) »
2
g 155
I m/z 148 (Phe) 36' l
t
Y
630 ——_ I """"""" *IgJ'l'q
640 8:: 660 670Scan

Figure 3.3. EI mass spectra of isobutyl chloroformate derivatives of
unlabeled phenylalanine (a) and dg-phenylalanine (b) from stock solutions.
TIC/mass chromatograms of iBuCF derivatives of Phe/dg-Phe mixture (1 :1)
(c). (50 nmol derivatization, 250 pmol injection for GC-MS analysis.)

141

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
   

 

 

 

 

 

 

1001?
R ‘ 148
i, so1 1311?. T
f 1 (phycobilisomes) ,
i 1 .
v 60- .
° ‘ :
A ‘0' 1
b T
2‘, 1‘ 220
d .
I 20-
n 1
° ‘ 1 1°
0 c “Emlzvrr VI. - A;
(a) rwz
10c 57 1P8 .
E . dg-Phe ;
' 80‘ (phycobilisomes) ~
8
1
i 1
v so-
9 .
C 40‘ 1120
q 204
u ‘ 7‘ 91
n
d ‘ 1
a 204 1
n
° 65 1 1 2 210
e o 1.: 1.3:“ L kﬁgﬂ Mi . L.
5° ‘°° ‘50 200 250
(b) rwz
Max.200 O 10 R.T.
R AAAAA A A A A 1403.
6; da-Phe
a phycobilisomes
t
i ,TIC
Z 200.0
m/z 148
I
n
f; 148
o.
2 m/z 155
1
l'.
y
- -- ,Hvlssr'lt
6‘0 ' ' 640 as; 660 1570Scan

Figure 3.4. Analysis of protein hydrolysates from Synechocystis by GC—MS
after derivatization with isobutyl chloroformate. EI mass spectra of the
phenylalanines in protein hydrolysates from unlabeled (a) and dg-labeled (b)
cells. TIC/mass chromatograms for Phe in protein hydrolysates from cells
that were grown in the presence of ds-Phe (c). (2 nmol protein
derivatization, 50 pmol injection for GC-MS analysis.)

 

 

142
biosynthesis resumes in the cells, the labeled phenylalanine is broken down

and recycled for use in other biosynthetic processes.

4.

 

 

‘l .
NHCOziBu _I +
C02iBu / COziBn . CO $331.:-
-NH2C02iBu (117) -(C02iBu-H) (100)
= >
OCOziBu OCOziBu OH
m/z 437 (M"') m/z 320 m/z 220
-C4H3 (56)

11 +

2 I C02 —l .

th107 HV2164

(Scheme 3.2)

Incorporation of labeled tyrosines into photosynthetic proteins has
also been carried out. Similar fragmentation as described for the
phenylalanine derivative produces the high abundant ions of m/z 220 and
m/z 164; m/z 107 provides a third speciﬁc fragmentation ion (see Scheme
3.2). These fragmentation ions are easily observed in the mass spectrum of
the iBuCF derivative of 16O-tyrosine (unlabeled) (Figure 3.5a). Figure 3.5b
and 3.5c show the mass spectra of l"Oe‘tyrosine (40% 17O) and 3,5, d2—
tyrosine (99% 2H) derivatives, respectively. 16O-Tyr and 17O-'I'yr coeluted
and reached their maximum at the same scan number (Figure 3.5d).

Figure 3.6 show the TIC / mass chromatograms (a) and mass spectrum (b)

143

of the iBuCF derivative of tyrosine in a Photosystem I preparation that was
isolated from cells grown on 1'7O-Tyr to about 0.6 OD730. After correction for
the contribution from natural isotope abundance, it was estimated
approximately 14% incorporation of 17O-Tyr into the Photosystem I
proteins. Interestingly, phycobilisomes isolated from these cells showed a
somewhat lower abundance of the 1"’O-label (approximately 10% label

incorporation), supporting the idea that these nitrogen-rich proteins

additionally serve as a transient repository for amino acids that are rapidly
taken up from the medium (Figure 3.7). In these experiments, the
incorporation of 17O-Tyr is signiﬁcantly less than the extent of labeling of
the 1'7O-Tyr stock solution (40% 17O) that was supplied to the cells. It may
reﬂect isotope discrimination against the 17O-hydroxyl isotope by one or
more of the enzymes involved in the import and incorporation of the
exogenous amino acids. Degradation of the label during growth can be
excluded by the observation that 1'70-'I‘yr incubated in an alkaline (pH 11)
aqueous solution remains stable for at least seven days. Since the cells
were harvested at about 0.6 OD730, they were expected to have a high extent
of incorporation. Other contamination in the growth medium may be part
of the reason for low incorporation of label. The quantitative analysis by
GC-MS for the incorporation of isotope labeled amino acids helped to
evaluate the successfulness of the individual cell growth experiments.

Cell growth was repeated for l7O--Tyr incorporation into
photosynthetic proteins under well controlled conditions. Figure 3.8-3.10
show the T103 / mass chromatograms and mass spectra of tyrosine iBuCF
derivatives from hydrolysates of Photosystem I, Photosystem II, and
phycobiliprotein, respectively, that were isolated from cells grown on 170-

Tyr to about 0.8 OD730. The ratios of 16O-Tyr/l'7O-Tyr/180-Tyr, after

 

 

144

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

“7‘ Tn To 1 o
It .
0 Tyr ,
1 so ,
C >
c 57
1
v 601
C
A
b 401
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“u
n
c 1 ° . J 6
2 4
. -JIAA’JI. LLJ J-‘4 11 i 215 v _1 ‘60
151: zoo zoo
m:
(I)
13‘
I T
. ol :1
‘1. ' O-‘l’yr
I
i ‘0. _ 21'0 b
. l 7 104
A
b so
\I
2
‘ 20' 7‘ 3 1
n L 11: I
C
91 1 9 1 0 3 6
. -J J.“ L14 ‘lA'AA. 4A4. l Av... _ 1L v ‘ A;
100 zoo 300 100
(b) ﬁll
133
n 1 9 1156 L
o 'r 35-‘H-
1 .01 57 2.1 . TY?
I
t
1
v 60-
O
A
b 40-
U
3
: "7 N 3f
c I l 0
1 2 at J o
' thinning-1 ia,x
100 zoo 300 coo
(C) III!
“8.14.. 11 p.13
11 ' ‘ 616..
I Tyr/‘OJI‘yr
I.
t
‘ __ me
I 12.1
x ..
2 “mob-m)
° Iii-1.2
n 1!.l
3
1
; IJIIO‘N'DI')
. Y no?
no .55 do us «o us when

 

((1)

Figure 3.5. EI mass spectra of isobutyl chloroformate derivatives of
unlabeled tyrosine (a), 17O--tyrosine (40% 170) (b), and 3,5-2H-tyrosine (c)
from stock solutions. TIC/mass chromatograms of iBuCF derivatives of
Tyr and 17O-'I‘yr mixture ((1). (10 nmol derivatization, 200 pmol injection for
GC-MS analysis.)

145

Max.29.9

 

    
 

A11 A R . T .
a 388
7

i ‘O-Tyr
a Photosystem I
t
i ,rIc
e 7 . 1

m/z 108 ("O-hit)

 

 

 

KUPUDOHSH
N
on
43L
Q
N

 

 

 

 

 

 

 

 

 

 

 

 

 

m/2107(Tyr)

Y Y V f V , f 1 V ﬁ 107

810 815 820 325 330 835 3405can

(a)
100‘ 107 L
R r
e so 1r4 220 llO-Tyr ’
,1. . 57 ’ PhotosystemI ,
t ,
1 1 .
v 60+ ~
8 ‘ >
A l .
b 401 ~
U 1 .
n .
d .
a 20 ' ~
n l 714 3 0 ,
C I 1 o .
1 o
. . i 97 “ah 1...... .1 E16212 2,. ii“ »
0W w . “:‘r r ‘4‘ A a 1 ‘f‘r‘ ‘“ :‘- ; ‘4 . v ‘r‘;

50 100 150 200 250 300 350
(b) M/Z

Figure 3.6. Analysis of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. TIC/mass
chromatograms (a) and EI mass spectrum (b) of the tyrosines in
Photosystem I proteins isolated from cells grown in the presence of 17O-
tyrosine (40% 170). (25 pmol PS I center derivatization, 1 pmol injection for
GC-MS analysis.)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.282.1 11 arr.
R A 1557 .9
e 17
l . O O
f: phycobilisomes
i 1 TIC
v 51 .o
e
I m/z 108 ("O-Tyr)
n
t
e 1U 5.5
n 282.
3
i m/z 107 (Tyr)
t
y
- . - 107
310 820 830 840 850 Scan
(a)
100 107 .
R I
e 4 VIC-Tyr r
1 80A phycobilisomes .
a ‘ 1Y4 2o ’
t. 57 ,
1 ‘ >-
v 60‘ ’
e 1 .
A 1 :
b 401 ’
U
n r
d 1 .
a .
n 20 3 o t
c 74 130 l 1 o 336 1
23s 2 2
e 0:91“ . .Jl ”315111.11 n 5L1.|.Jl‘_..l.'__u I”. .143. a. ‘ _..._4. LL LLn'lsn 1.. 4.3. 4_ ‘ A _ in ‘
so 100 150 200 250 300 350
M/Z
(b)

Figure 3.7. Analysis of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. TIC/mass
chromatograms (a) and EI mass spectrum (b) of the tyrosines in
phycobiliproteins isolated from cells grown in the presence of 1'7O-tyrosine
(40% 17O). (2 nmol protein derivatization, 100 pmol injection for GC-MS
analysis.)

147
correction of natural isotope abundance, in the growth medium and in each

proteins are listed in Table 3.1. These data were calculated from the peak
areas and peak heights of the mass chromatograms for each characteristic
abundant ion of 16O-Tyr, 17O-Ty'r, and 18O-Tyr (m/z 107/108/109, m/z
164/165/166, m/z 220/221/222, and m/z 320/321/322). The results from these
experiments showed that although the isotope discrimination against 170-
Tyr in the incorporation still exits, the extent of incorporation of labeled
tyrosines into photosynthetic proteins (PS I, PS II, and phycobilisomes) are
much higher than those obtained from the previous experiment. In PS I
and PS II, the ratio of 1"O-Tyr to 16O-'I3'r is about 65% which is close to that
in the starting growth medium (~80%). For these two photoproteins, if 160-
Tyr is fully incorporated, the percentage for 17O-Tyr incorporation is ~80%.
Again, phycobiliprotein showed a lower abundance of 17O-label. PS I and
PS II took 2.5-fold more 1"’O-Tyr than phycobiliprotein did.

In the cell growth experiment, UV absorption spectra used to
monitor the concentration of the amino acids in the growth medium (as in
Figure 3.2) did not show consistent results. In the second experiment just
described above, the UV spectra did not show a signiﬁcant decrease of the
response from the aromatic amino acids in the growth medium as the
growth time increased. Derivatization / GC-MS analyses of the growth
medium for aromatic amino acids, phenylalanine, tyrosine, and
tryptophan were carried out to clarify the UV absorption result. Growth
medium of 40 111 (20 nmol Phe, 10 nmol Tyr, and 10 nmol Trp for the
starting medium) separated from the cells at different time: "0 day", "8
days", "16 days" were directly treated with 10 ul of iBuCF with the addition
of 40 111 water, 30 111 isobutanol, and 10 111 pyridine. 10 nmol of 4-Cl-
phenylalanine were added as an internal standard. A 2-111 aliQuot of the

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.73.2 11 R.T.
R 1771.1
‘1’ PhotosystemI
a
r. ,TIC
1 - 42.é
Z m/2109("0-'lyr)
1 Man
n 17 52.
t m/2108( 02m)
e
n
s r‘xesnA
i 73.?
c m/2107(Tyr)
y
4““,—
- anew“ ,,,,,,,,,,, ---t107
710 720 730 740 750Scan
(a)
10 164 2 o
R .
e 57 +
1 8 0‘ 1 O 7 Photosystem I ~
a
t
1 .
V 60" L
e t
A
b 40
U
n
d
a 2m
n 1
C 1
e 422
W L A
400
M/Z

 

Figure 3.8. Analysis of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. TIC/mass
chromatograms (a) and EI mass spectrum (b) of the tyrosines in
Photosystem I proteins isolated from cells grown in the presence of 17O-
tyrosine (40% 17O). (20 pmol PS I center derivatization, 2 pmol injection for
GC-MS analysis.)

149

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.94.3 11 R.T.
a i ‘ ‘ ‘ A A A A 2007.3
‘1‘ Photosystem II
a
t . .~_________wr1c
1 57.0
V
e m]: 109(“o-'1‘yr)
I :6 =1.7
n 67.3
2 m/z 108 ("O-Tyr)
2 561m . 4
1 94.3
t m/z 107 (Tyr)
y
a , a. . 107
710 720 730 740 750Scam
(a)
10° 57 220 .
R 1 07 164 *
e 1 Photosystem II ’
1 807 .
a ‘ ’
t .
i 1 t
v 60% "
e i s
A I .
b 401 F
U 1 »
n
d 1 74 3 o
a 20* 130
fl
C 1 1 9 189 252 335
e 91 276
.1 Lullﬁ! L.
100 200 300 400
M/Z
(b)

Figure 3.9. Analysis of protein hydrolysates from Synechocystis by GCoMS
after derivatization with isobutyl chloroformate. TIC/mass
chromatograms (a) and EI mass spectrum (b) of the tyrosines in
Photosystem II proteins isolated from cells grown in the presence of 17O-
tyrosine (40% 1"0). (30 pmol PS 11 center derivatization, 3 pmol injection for
GC-MS analysis.)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.210.8
AAAAAAAAA 11 R T
R A A A AAAAAAAAA 2A77As A2
:3 phycobilisomes
a
ti . rrc
v 47.6
e 111/2109(180-1311')
I
n 17
t m/2108( Oo’l‘yr)
e
n
S
; m/2107('13'r)
y
- . - - , - - Y ,,,,,,,,,,,,,,,,, - . 107
710 720 730 740 750
Scan
(a)
10
, 1‘54 2 0
R o o
e , 107 phycobilisomes ‘
1 so a
a * l
t ‘ 1
1 .
v 60-57
e A I
A 1 ‘
b 401 l A
U l b
n .
d P
a D
n .
C L
e b
100 200 300 A 400 A
(b) M/Z

Figure 3.10. Analysis of protein hydrolysates from Synechocystis by GC-MS
after derivatization with isobutyl chloroformate. TIC/mass
chromatograms (a) and EI mass spectrum (b) of the tyrosines in
phycobiliproteins isolated from cells grown in the presence of 17O-tyrosine
(40% 17O). (2 nmol protein derivatization, 200 pmol injection for GC-MS

analysis.)

151

Table 3.1. Quantitation of the extend of incorporation of 1“’O-tyrosines into
the indicated proteins in Synechocystis cells grown in the presence of 170-
tyrosine (40% 170) (after correction of natural isotope abundance)

 

 

1‘5O-/17O-/130- Tyr 15041704130 Tyr

(bxpeak area) (by peak height)
Growth medium (start) 100 / 8O / 78 100 / 79 I77
Photosystem I 100 /64 I 62 100 /62 / 60
Photosystem II 100/66/66 100/65/65

Phycobilisomes 100 / 25/ 24 100 / 25 / 22

152
ﬁnal 20-111 of chloroform solution was analyzed by GC-MS. The relative

responses (relative to internal standard 4-Cl-phenylalanine) of peak areas
and peak heights of T108 and relative responses of peak areas and peak
heights of the characteristic ions from the mass chromatograms, for each
amino acid in the growth medium at different growth times were
calculated. No signiﬁcant changes of the relative responses were found
(Figure 3.11), which supported the results from the UV absorption for the
second experiment. The inconsistency of the change of the concentration of
the aromatic amino acids in the growth medium may be explained by the
presence of another organism besides the cyanobacteria in the cell growth
medium which consumed the amino acids to form other proteins. .

Amino acid analysis also conﬁrmed that no exchange of label
between different amino acids, i.e., 3,5-d2-tyrosine in the cell growth

medium did not result in 3,5-d2-phenylalanine in the proteins.

IV. Conclusions

With the rise of popularity of Synechocystis as an experimental
organism for the study of PS I and PS II, and its ease of manipulation, both
in terms of site-directed mutagenesis and inducible auxotrophy, it has
become more important to study the kinetics and extent of amino acid
labeling. The procedure described in this chapter, and the results show
that protein isolation, hydrolysis, and GC-MS analysis is an analytical
methodology that is capable of providing quantitative insight into these
issues. The convenient one-step aqueous medium chloroformate
derivatization / GC-MS analysis of amino acids contribute signiﬁcantly to
the procedure to quantitate the extent of incorporation of isotope labels.

Amino acid derivatization has been conducted directly in the growth

153

 

 

 

A120.O

(,2: Phe

23100.0 4

2

8 80 0 Tyr

E -

§‘ 60.02

“ i

O .

a 40.0: C}_ Trp

:9 [3} ‘‘‘‘‘‘‘‘‘‘‘‘‘‘‘ ~13

kg 20.0:

2 1
0.0...1...[...,.,,I,ﬁ,l

O 8 16
Time (day)

Figure 3.11. Analysis of the growth medium separated from Synechocystis
cells at different times as indicated for Phe, Tyr, and Trp by GC-MS after
derivatization with isobutyl chloroformate.

 

154
medium separated from the cells. The conditions necessary for successful

incorporation of isotopically labeled aromatic amino acids in vivo, and the
limit in cell density necessary to achieve the labeling have been veriﬁed by
GC-MS amino acid analysis. While the induced auxotrophy described in
this chapter is limited to the aromatic amino acids phenylalanine, tyrosine
and tryptophan, these amino acids have been implicated as participants in
important reactions in PS II [6,7]. This labeling approach should prove
valuable in the elucidation of the structure / function relationships of these
amino acids. In addition, although the focus of this study is on the roles of
aromatic amino acids in PS II and PS I, the techniques developed are
applicable to the study of any enzyme or protein complex that contains these

amino acids as important components.

V. References

1. NE. Bowlby, M. Espe, R. Bhatnagar, J. Wang, C. Hoganson, L.
McIntosh, and G.T. Babcock, Photosynthesis Research, 38 (1993) 379.

2. B. Kok, B. Forbush, and M. McGloin, Photochem Photobiol., 11 (19700

57.

3. P. J oliot, G. barberi, and R. Chabaud, Photochem Photobiol., 10 (1969)
309.

4. BA. Barry andiG.T. Babcock, Pro. Natl. Acad. Sci. USA, 84 (1987)
7099.

5. C.W. Hoganson and G.T. Babcock, Biochemitry, 31 (1992) 11874.

6. G.H. Noren, R.J. Boemer, and B.A. Barry, Biochemistry, 30 (1991)
3943.

7. B. Svensson, I. Vass, E. Cedergren, and S. Styring, EMBO J., 9 (1990)
2051.

Chapter IV
Investigation of the one-step chloroformate derivatization of small

peptides prior to analysis by FAB-MS

I. Introduction

Fast atom bombardment mass spectrometry of peptides is often used
for molecular weight determinations and sometimes for amino acid
sequencing. However, experimental factors, such as the poor ionization
efﬁciency of some hydrophilic peptides, can prevent successful analysis. The
ionization efﬁciency can be improved by preparing derivatives of the peptides
prior to analysis by FAB-MS. Derivatization methods involve a variety of
peptide modiﬁcations to increase peptide hydrophobicity, ranging from
esteriﬁcation to the attachment of a hydrophobic moiety, or a moiety
containing a ﬁxed charge, to a speciﬁc site on the peptide as were reviewed in
chapter I of this dissertation. However, none of the previously reported
methods simultaneously derivatizes both amino and carboxyl groups in a
single reaction, and most of the methods are time-consuming.

Prior to this investigation, the one-step chloroformate derivatization
reaction in an aqueous medium developed [6] and reﬁned [7-9] for analysis of
amino acids has not been applied to peptide derivatization. In this chapter,
the results of an investigation of the one-step chloroformate derivatization
procedure for preparing N(0,S)«ethoxycarbonyl ethyl ester derivatives of
peptides to enhance the FAB response and to generate sequencing ions by
FAB-MS/MS during analysis of low-level samples will be reported. A detailed
investigation of the reaction conditions will be described. A preliminary
investigation of forming precharged derivatives of peptides using the
chloroformate derivatization procedure will be discussed.

155

156

II. Experimental

Materials

The peptides were purchased from SIGMA Chemical Company (St.
Louis, MO), and the alkyl chloroformates and alcohols were purchased from
Aldrich Chemical Company (Milwaukee, WI).

D . |° |°
Typically, 1-10 nmol of peptide were added to a 100411 volume of the

reaction medium (H20/EtOH/pyridine (Py)=60/30/5-10). Ethyl chloroformate
(5-10 111) were added and the reaction mixture was vortexed for 5-40 seconds.
Chloroform (100-300 pl) was added to extract the derivative; the chloroform
was removed by vacuum (Speed-Vac, Savant Instruments Inc., Farmingdale,
NY).

HELL:

The HPLC-UV experiments were carried out on a Beckman HPLC (a
112 Solvent Delivery Module, a 420 Controller, a 340 Organizer) and a
Spectroﬂow 757 absorbance (1:214 nm) detector (Kratos). The HPLC column
(length = 25 cm, ID. = 4.6 pm) was packed with 5-um particles (Econosphere)
with a chemically bonded Cm stationary phase. The mobile phase was a
gradient of CH3CN and H20 (0.1% TFA); CHsCN concentration increased

from 0 to 100% in 30 minutes.

Wm

Secondary ions were produced with a 15-keV primary beam of Xe atom
in a JEOL HX-llO double-focusing mass spectrometer operated in the
positive ion mode. The accelerating voltage was 10 kV and the resolution was
set to 1000 (10% valley). For CID MS/MS, helium was used as the collision

gas in a cell located in the ﬁrst ﬁeld-free region. The helium pressure was

157
adjusted to reduce the abundance of the precursor ions by 50%. A JEOL DA-

5000 data system generated linked scans at constant B/E. The derivative
was dissolved in 1/1 CH3CN/I120, and 1 ul of this solution (100 pmol to

lnmol) was added to 1 pl of glycerol lthioglycerol /methanol (1/1/1) matrix.

III. Enhancement of FAB signal of model peptides by derivatization
The FAB response of most of the ethyl chloroformate derivatives, as
N(0,S)-ethoxycarbonyl and ethyl esters, of the model compounds (di-peptides
to penta-peptides) increased five- to ﬁfty-fold relative to that of the
underivatized analytes (see Table 4.1). In Figure 4.1, comparison of the FAB
mass spectra of derivatized and underivatized glutathione (y-ECG) shows an
approximately 40-fold signal enhancement after derivatization by ethyl
chloroformate. Furthermore, the mass shift of the MI-I+ ion shows that the
terminal amino and carboxyl groups as well as the thiol and carboxylic side
chain groups are derivatized. A penta-peptide, RKDVY, also shows
enhancement of FAB response by a factor of about 50 after derivatization by
ethyl chloroformate (see Figure 4.2). From the mass of the protonated
molecule (MI-1+=952) and the CID-MS/MS spectrum (Figure 4.3) of the ethyl
chloroformate-ethanol derivative of RKDVY, it is apparent that the N-
terminal, the C-terminal, the amino group on the side chain of Lys (K), the
carboxyl group on the side chain of Asp (D), and the phenolic group on the
side chain of Tyr (Y) are derivatized during the one-step reaction with ethyl
chloroformate. Blockage of the hydrophilic functional groups (at terminals
and on side chains) by the derivatization reagent is likely the reason for the
enhancement of the FAB signal. The derivatization increases the

hydrophobicity of the small peptide and hence its surface concentration in

158

Table 4.1. Estimation of FAB signal enhancement from small peptides as
their ethyl chloroformate derivatives relative to the FAB response of
underivatized peptides.

Peptide Enhancement

RKDVY 50 / 80 *(one free -COOH)
y—ECG 35**

GHK 50*1

YGG 20*

DC 30*

VGDE 10 (one free -COOH)
KYK 25 I40 (one free -COOH)
LGG 5*

GGF 5**

SY 20***

* Average from analyses of 3 samples, each done in duplicate. ** Average from analyses of 4
samples, each done in duplicate. *** Average from analyses of 2 samples.

1. side chain of His is not derivatized

159

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100 * 1&5

F: ‘ é? underivatized 'y-ECG

I . * i: 20 nmol

ta 80. 75 277

i - HgN-(1-§)-?-G-COOH

:50: . HOOC SH

A l * 119

b ‘57

u 40. *

g . , 241 MH+=308

‘ 1 1 5 t
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c . ' '
U 40J * 149 201 277
n 57 t 230 480
d 115
g 20 e 405
, 5 t 435
g 165 257 81 333 $9 I 464 530
1 0 0 200 m0 0 o o o MIZ

Figure 4.1. Comparison of FAB mass spectra of y-ECG (glutathione)
underivatized and derivatized with ethyl chloroformate. Top panel is mass

spectrum obtained from 20 nmol of underivatized y—ECG; bottom panel is the
FAB mass spectrum obtained from 1 nmol of N,S,O-ethoxycarbonyl/ethyl

ester of y-ECG.

160

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

n *
1m I A
R 5?” underivatized RKDVY
f W 1 nmol
:3 80‘ coon
‘ HZN-R-x-D-v-r-coon
I; so: 329 NH2 PhOI-I
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° . 429
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. 0. 1.0 .0 0“. ' ‘ 'm' ' ‘ 'm' ' ' v“ mid
. .
100 '
a” 00:“ derivatized RKDVY
n . EtOOCNH-a-xnv-r-coost 100 pmol
$801 EtOOCNH PhOCOOEt
V ‘ 924
8 604
A :- MH+=952J\
u 40.
n
d
a
n 20.
C
e 539 see
”0 0 0 O

 

 

Figure 4.2. Comparison of FAB-MS spectra of RKDVY, underivatized (upper

panel, lnmol) and derivatized (lower panel, 100 pmol) with ethyl
chloroformate.

 

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162
the matrix on the probe tip [2]. RKDVY derivatives prepared by other

chloroformate-alcohol reagents, such as EtCF-HFBuOH, iBuCF-iBuCF,
iBuCF-HFBuOH, iBuCF-(3-pyridinemethanol) (PyCHzOH), and iBuCF-
PyCH20H have been synthesized. However, the FAB response of derivatives
formed by chloroformate reagents that introduce a larger alkyl group (e.g.,
isobutyl) did not show greater enhancement of the FAB signal over that from
an analogous derivative containing an ethyl group.

IV. Evaluation derivatization efficiency

A. Introduction

In Figure 4.2b, the FAB spectrum of ethyl chloroformate-ethanol
derivatized RKDVY, a peak at m/z 924 has a relatively high abundance and it
appears 28 u lower than the peak at m/z 952 for MH+. Similarly, in the mass
spectrum of derivatized y-ECG (Figure 4.1b), there is a signiﬁcant peak at
m/z 480, which is 28 u lower than that at m/z 508 for MK". These ions could
be fragments of the MH+ or could be individual components resulting from
the derivatization reactions. The product ion spectrum (B/E linked scan) of
MH+ 508, the ethyl chloroformate derivatized 'y-ECG, showed no fragment ion
peak at m/z 480. The m/z 480 ion must represent an individual product from
derivatization. Although the ion of m/z 924 was found in the product ion
spectrum of the derivatized RKDVY (MI-1+ = 952), one component in the
RKDVY ethyl chloroformate derivatization product mixture was separated by
HPLC and conﬁrmed by FAB-MS to have a peak at m/z 924 for the NIH".
Figure 4.4 is the HPLC chromatogram of the RKDVY ethyl chloroformate
reaction mixture (products). Analysis by FAB-MS indicated that the two
larger, late-eluting components have peaks at m/z 924 and m/z 952,
respectively, representing two different MH+ species. These results indicated

163

ﬁilly derivatized

solvent '—

1 free carboxyl
\

 

 

 

 

2 free carboxyls b
\

 

 

j I

10 20 (min)

Figure 4.4. HPLC chromatogram of derivatized RKDVY with CH3CN/I120 as
the solvent. The reaction medium was H20/EtOH/Py (70/30/5). The two

major peaks represent the fully derivatized compound and the species
containing one free carboxyl group.

164
that the derivatization of RKDVY by ethyl chloroformate does not form a

single product, and peptides are not quantitatively derivatized under the
conditions that were assumed to achieve full derivatization of amino acids in
a simple one-step reaction [6]. The two components with peaks at m/z 924
and m/z 952 as their protonated molecules, represent the derivative with one
free carboxyl group and the derivative with both carboxyl groups fully
derivatized, respectively. Another small peak in the chromatogram
represents the derivative with two free carboxyl groups. None of the peaks
correspond to a species with a free amino or a free phenolic group. A similar
result was found for y-ECG derivatization by ethyl chloroformate; the peak at
m/z 480 in the FAB-MS spectrum corresponds to the derivative with one free
carboxyl group. Using the same procedure, HPLC separation and FAB-MS
analysis, other small peptides, such as GGF, KYK, and GHK, were also
conﬁrmed to be incompletely derivatized under conditions for amino acid
derivatization in a one-step reaction. In the case of GHK, the reaction on the
side chain of histidine was also incomplete. All these results indicated that
derivatization of amino (N-terminal and side chain), thiol, and phenolic
groups is complete, but that conversion of carboxyl groups (C-terminal and
side chain) is not complete.

Results of other experiments demonstrated that the size of the peptide
is not a major factor in causing incomplete reaction of the carboxyl group.
When three peptides of different sizes (SY, RKDVY, RPKPQQFFG) were
derivatized by ethyl chloroformate, subsequent analysis by FAB-MS indicated
that even the di-peptide, SY, had two derivatization products: one with the
carboxyl group derivatized and one with a free carboxyl group. Similar
results were obtained for the penta-peptide, RKDVY, and the ninemer,
RPKPQQFFG. Figure 4.5 shows the FAB-MS spectrum of theutwo ethyl

165

 

 

 

 

 

 

 

BP: m/z 228.4 Int. 18.0

R50 “7}.4
'9 derivatized RPKPQQFFG
a 40 200 pmol
t
i
V
8 30
A
3 2° MH+=1276
n
d
:1 m/z=1248
c 1
e

l 85L -1 ”EQAA: 11§§5hﬂLLlAL‘

860 who 1200 M

 

Figure 4.5. FAB-MS spectrum
chloroformate.

of RPKPQQFFG derivatized with ethyl

166
chloroformate derivatization products of RPKPQQFFG (derivatized at the

lnmol level, with 200 pmol transferred to the probe tip). The peak at m/z
1276 corresponds to MH+ for the fully derivatized species; the peak at m/z
1248 (28 u lower) corresponds to a species in which one of the carboxyl groups
has not be converted to an ethyl ester. Thus, the incomplete reaction of
carboxyl groups is a problem, regardless of the size of the peptide (from di-
peptide to ninemers in our model compounds).

A thorough investigation to evaluate the derivatization efﬁciency and
to ﬁnd the reaction conditions for complete reaction for both amino and
carboxyl groups of small peptides in the one-step chloroformate derivatization

has been carried out.

B. Location of the underivatized carboxyl group

To determine whether the free carboxyl group in an incomplete
derivatized peptide is on a residue side chain or at the C-terminal, MS/MS
spectra of the MRI of y-ECG(glutathione)de1ivatives were obtained. Figure
4.6a shows the MS/MS spectrum of the fully derivatized product (MH+=508.5
u). For the derivative with carboxyl groups partially derivatized there are
two possibilities: (a) free C-terminal; derivatized y-glutamate side chain. (b)
derivatized C-terminal; free y-glutamate side chain, or a mixture of both
cases. The structures are shown in Figure 4.7 together with m/z values for
possible fragment ions. If the C-terminal is not derivatized (structure 1 in
Figure 4.7), N-terminal ions (a, b, c, (1) will be the same as those in the
spectrum of the fully derivatized case, but C-terminal ions (1:, y, 2) will be
shifted 28 u lower. If ‘y-Glu side chain -COOH is not derivatized (structure 2
in Figure 4.7), C-terminal ions will be the same as those for the fully

derivatized species, but N-terminal ions will be shifted 28 11 lower in mass.

167
Peaks for both cases were found in the FAB-MS/MS spectrum (Figure 4.6b),

so the reaction mixture contained two products, each having one free carboxyl
group, either at the C-terminal or on the side chain (or vice versa). For
example, part of the ya ion current (m/z 279) in Figure 4.6a (fully derivatized)
is shifted 28 u to m/z 251 (13) in Figure 4.6b (incomplete reaction), but the
same b1 ion (m/z 230) and the b2 ion (m/z 405) were found in both spectra (In
and ha in Figure 4.6b). These peaks provide evidence for a free C-terminal in
the incomplete reaction product. Meanwhile, peaks at m/z 279 (3",) and m/z
305 (x',) found in Figure 4.6b were the same as those for the y, and the x2
ions in Figure 4.6a, while the a2 ion (m/z 377) in Figure 4.6a is shifted 28 u to
m/z 349 (a',) in Figure 4.6b, which is consistent with the free 'y-Glu side chain
-COOH. It should be noted in Figure 4.6b that the an ions (corresponding to
the free C-terminal) cannot be distinguished from the U, ions (corresponding
to the free y—Glu side chain -COOH), because the mass difference of 28 u
between corresponding b and a ions is the same as that between an ethyl
ester and a free carboxyl group. Fortunately, by combining information from
other sequence ion series, we can draw the above conclusion that for the

reaction product with one free carboxyl group, the underivatized position was

both at the C-terminal and on the y-Glu side chain.

C. Effect of reaction medium composition

The effect of solvent composition on the emciency of derivatization was
examined. Three reaction media were studied: H20/EtOH/Py (60/30/10),

H20/EtOH/Py/CH30N (60/30/10/10), and H20/EtOH/Py/THF (60/30/10/10).
The suitability of the derivatization was assessed with the model compound,

RKDVY, by comparing the FAB-MS response of the derivatives produced in

the different media with 5 ul of ethyl chloroformate. Two" series of

168

 

 

 

 

 

 

 

 

 

 

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e , r, ,3 15:279‘317 35
‘ 104 1 . . .
9'0 100 150 200 250 300 350 400 450Wz

 

Figure 4.6. Product ion spectra (B/E linked scan) of fully derivatized y-ECG
(glutathione) (upper panel, MH+ 508.5) and partially derivatized y—ECG

(lower panel, MH+ 480.5) Underlined ions correspond to the product with the

free C-terminal and the derivatized y-Glu side chain carboxyl group
(structure 1 in Figure 4.7); italized ions correspond to the product with the

free y-Glu side chain carboxyl group and the derivatized C-terminal
(structure 2 in Figure 4.7).

169

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170
experiments for each reaction medium composition were carried out. In one

series, the FAB spectrum was taken after the derivatives were extracted by
100 pl chloroform, then dried and redissolved in CH3CN. In the other series,

the derivatives were not extracted with chloroform. Instead, the reaction
mixture was dried under vacuum and then redissolved in CH30N. The
results are listed in Table 4.2. Within each series for the three reaction
media, no signiﬁcant difference in the yield of derivatives or the ratio of FAB
response for the different derivatives was found. Adding acetonitrile or
tetrahydrofuran did not affect the reaction much. In the ﬁrst series, the ratio
of the response of the fully derivatized product to that with one free -COOH is
similar for each reaction medium composition (about 1:3). This ratio for the
second series is about 1:6. The difference between these two ratios might be
because of the chloroform extraction of the derivatives. In this case,
extraction by 100 pl of chloroform might not be sufficient to efficiently
partition the derivatives into the chloroform layer, especially the more polar
derivatives, e.g., those with one or two free carboxyl groups. That is, the
derivative with one free -COOH may be less soluble in the chloroform layer '
than the fully derivatized one. The ratio of the two species in the sample
with no chloroform extraction should reﬂect more accurately the ratio after
the derivatization reaction. (No experimental results were obtained to
explain the absolute response of the fully derivatized product with no
chloroform extraction being lower than that from the series with extraction
by chloroform. Both series were done on the same day with same instrument

conditions.)

171

Table 4.2. FAB-MS response for indicated species as function of reaction
medium (model compound: RKDVY).

 

W/E/P W/E/P/A W/E/P/T
AI NI AI NI AI NI

 

I (with chloroform extraction)

fully derivatized 27 34 25 28 34 34
1 free -COOH 80 100 87 100 100 100
2 free -COOH 15 18 25 29 24 24

II (no chloroform extraction)

fully derivatized 1 3 1 8 1 1 15 9.3 1 7
1 free -COOH 70 100 74 100 56 100
2 free -COOH 23 33 28 38 24 43

 

*W=water; E=ethanol; P=pyridine; A=acetonitrile;T=tetrahydroﬁ1ran

Al=absolute intensity, NI=normalized intensity.

1 7 2
D. Extraction efficiency

During the experiment described above, it was noticed that variations
in the mode of chloroform extraction affected the relative and also the
absolute FAB response of the samples. Experiments were designed to
evaluate the extraction efﬁciency as a function of the volume of chloroform
used. Table 4.3 shows the results of using different volumes of chloroform for
the model compound RKDVY ethyl chloroformate derivatives. The ratios of
the FAB responses (of MH+ ion) in the water layer (after extraction) to that
in the chloroform layer were listed. The eﬁiciencies of single extractions by
100 pl, 200 pl, 300 pl, and 500 pl were compared with those of multiple
extractions by 200 p1/100 pl, 300 pl/lOO pl, and 100 pl/100 pl/100 pl. The
multiple extraction protocol made the extraction quite complete.

For the ethyl chloroformate derivative of the penta-peptide RKDVY, no
detectable underivatized species were found by FAB-MS and HPLC-UV, in
either the chloroform extract layer or the aqueous layer after the extraction.
The same result was found for the case with no chloroform extraction when

the reaction mixture was dried directly in vacuum.

E. Effect of solvent ratio in the reaction medium

The solvent ratio in the reaction medium was adapted from the
procedure for amino acid analysis [69] which is about 60/30/10 (v/v/v) for
H20/EtOH/Py. To ﬁnd more suitable conditions for small peptides,
experiments were carried out with different solvent ratios, and the results
are listed in Figure 4.8 for the ethyl chloroformate derivatives of the model
compound RKDVY . From the point of view of absolute response or relative
response of different derivatives or the reaction yield, a solvent ratio of

60/30/10 was optimal for small peptide derivatization. A more detailed and

173

Table 4.3. Extraction efﬁciency vs volume of CHC13 for RKDVY (EtCF) .
(MI-1+ intensity in H20 layer / MH+ intensity in CHCl3 layer)

 

 

 

 

(Single Extraction)
Volume (pl) 100 200 300 500
fully derivatized 25% 5.4% 2.4% 2.0%
1 free -COOH 34% 11% 5.9% 8.6%
2 free -COOH 22% 14% 18% 13%
(Multiple Extraction)
Volume (pl) 200/100 300/100 100/100/100
fully derivatized 0% 0% 0%
1 free -COOH 2.6% 2.2% 1.2%

2 free -COOH 21% 14% 11%

 

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175
systematic evaluation of the effect of each component in the reaction medium

on the efﬁciency of the derivatization reaction has been carried out.

(1) Effect of pyridine concentration
Based on the results of the derivatization of amino acids by
chloroformates in chapter 11, pyridine functions as a catalyst and as a buffer
for the chloroformate derivatization reaction. The concentration or volume of
pyridine has an important effect on the reaction. The effect of pyridine
concentration on the efﬁciency of ethyl chloroformate derivatization of

peptides was examined. The experiment was carried out for different
volumes of pyridine in a solution of H20/EtOH (70/30; v/v). The HPLC—UV

(1:214 nm) responses of the different derivatization products of RKDVY
derivatized by ethyl chloroformate are shown in Figure 4.9, and the
corresponding pH values of the reaction mixture before and after the addition
of ethyl chloroformate as a function of the volume of the pyridine added are
listed in Table 4.4. First of all, these data demonstrated that varying the
volume of pyridine does not make the reaction go to completion to form a
single product. When the volume of pyridine in the mixture was less than
about 3.5 v.1, the recovery of the fully derivatized product decreased
signiﬁcantly. The fully derivatized product reached the highest recovery
when 3 to 5 pl of pyridine were added to the reaction mixture. When the
amount of pyridine added to the reaction mixture increased from 5 to 10 pl,
the recovery of the fully derivatized product decreased. The recovery of the
product with one free -COOH increased as the amount of pyridine added
increased from 0 to 10 1.11. When 10 to 20 pl of pyridine were added to the
reaction mixture, recoveries of the two products described above were almost

constant although the recovery of the fully derivatized productl'decreased

176

RKDVY (EtCF) vs. Pyridine (HPLC)

  
 

1 free -COOH

fully derivatized

 

 

 

HPLC-UV Response Area (Relative)

V ‘3“ ~61
______ OO
................. 0“” 2free-COOH
o.oo “W.H...ﬁwﬂ,
O 4 8 12 16 20

Pyridine (111)

Figure 4.9. The HPLC-UV (1:214 nm) responses of the different
derivatization products of RKDVY prepared with ethyl chloroformate vs. the
volume of pyridine in the reaction medium.

177

Table 4.4. pH vs. pyridine volume in the reaction mixture

 

Before: before the addition of ethyl chloroformate.

After: after the addition of ethyl chloroformate.

H20/EtOH/Py (H20/EtOH=7O/30)

Pyml) 0.0 1.0 2.5 3.5 5.0 10.0 15.0 20.0
pH(before) 6 6-7 7 7 7 7 7 -8 7-8

pH(aﬁ:er) 3 0-1 1 3-4 4-5 5-6 5-6 5-6

178
slightly. In Table 4.4, as prridine increased from 1 to 5 pl, the pH (after)

increased from 1-2 to about 4-5, and when prﬁdine increased from 10 to 20
pl, the pH (after) was stable at about 5-6 which paralleled the constant
recoveries for both derivatization products in this range of pyridine volume.
Recovery of the product with two free -COOH groups was less sensitive to the
quantity of pyridine used than was that described above for the other two
reaction products.

The same trend in recovery as above was found when a similar
experiment was repeated on FAB-MS for RKDVY ethyl chloroformate
derivatization. The experiment was carried out with different volumes of
pyridine (5, 10, 15, 20 pl) added in a solution of H20/EtOH (60/30; v/v). The
FAB-MS responses of different derivatization products of RKDVY derivatized
by ethyl chloroformate are shown in Figure 4.10. Each data point was the
average of duplicate or triplicate analyses. The only difference between the
HPLC and the FAB results was that the decrease of the recovery of the fully

derivatized product was more signiﬁcant when prﬁdine increased from 10
to 20 pl for the FAB result in Figure 4.10. The results from both HPLC and
FAB-MS indicated that the addition of about 5-10 pl of pyridine is more
appropriate for the derivatization of RKDVY by 5 pl of ethyl chloroformate
although varying the volume of pyridine did not result in a complete reaction
that forms only one product. The amount of pyridine required to achieve a
higher absolute and relative yield for the derivatization of RKDVY is similar
to what was found'in the derivatization of amino acids.

A result similar to that of RKDVY derivatization from FAB-MS was
found for the derivatization of y-ECG by ethyl chloroformate (Figure 4.11).
The results from the experiments on FAB-MS for GHK (Figure 4.12) and
GGF (Figure 4.13) ethyl chloroformate derivatives showed more constant

179

RKDVY (EtCF) vs. Pyridine (FAB-MS)

 

 

 

601
3 lfree-COOH
501
3 c
g 40—j
m _
$2 1
m 3 0‘; \ \0 fully derivatized
2. 2 \
10— ’,,,4
1 -—-—""
$.__..__..————- 2free-COOH
07 IIIIIIIII I 111111111 l IIIIIIIII 1
5 10 15 20

Figure 4.10. The FAB-MS responses of the different derivatization products
of RKDVY prepared with ethyl chloroformate vs. the volume of pyridine in
the reaction medium.

180

Glutathione (EtCF) vs. Pyridine

   

 

 

50
3 40?
5 1
53‘ 30a
g j fully derivatized
m 2
5. 2°:
3 a ”\\w
/
10“ /
f?‘ ~ x o/ 1 free -COOH
o-‘Tf rrrrr III i ITIITTIIIIIﬁ]
5 1O 15 20
Pyridine(ul)

Figure 4.11. The FAB-MS responses of the different derivatization products
of y—ECG prepared with ethyl chloroformate vs. the volume of pyridine in the
reaction medium.

181

GHK (EtCF) vs. Pyridine

  
 

 

 

a 8 O —
g C terminus: -COOEt
Q.
E 6 o —
m I
5. 4 o —
Q j C terminus: -COOH
in .
2 0 — . .A """""""" ‘ ........... ‘
1 fully derivatized
_. _. _ — — 4 ___________
o l YYYYYYYYY I TTTTTTTTT $- IIIIIIIII 1
5 1 O 1 5 2 0

Pyridine (111)

Figure 4.12. The FAB-MS responses of the different derivatization products
of GHK prepared with ethyl chloroformate vs. the volume of pyridine in the
reaction medium.

182

GGF (EtCF) vs. Pyridine

 

 

 

Q)
U)
5 ‘ C terminus: -COOEt
g; 1
a) 11 ‘45 - - _ -
2. ‘ ""“n --------- A ----------- A
g C terminus: -COOH

6 _

o YYYYYYY I T j Tﬁ I IIIIIIIII l

5 1 0 1 5 20

Pyridine (ul)

Figure 4.13. The FAB-MS responses of the different derivatization products
of GGF prepared with ethyl chloroformate vs. the volume of pyridine in the
reaction medium.

 

183
recoveries over the entire range of 5 to 20 pl of pyridine added to the reaction

mixtures. Each data point in these figures are the average of duplicate or

triplicate analyses.

(2) Effect of different bases (catalyst and buffer)

The effect of 4-dimethylaminopyridine (4-DMAP) on the completion of
the derivatization of RKDVY by ethyl chloroformate has been evaluated. The
results are summarized in Table 4.5. In A of Table 4.5, the experiment was
carried out for different volumes of 4-DMAP and pyridine in a solution of
H20/EtOH (60/30; v/v); 10 nmol RKDVY were derivatized by 10 pl ethyl
chloroformate. The derivatization products were separated by HPLC, and
conﬁrmed by FAB-MS. The m/z values of MH+ for each product are listed.
In B, 4-DMAP solution 10, 30, and 50 pl replaced the 4-DMAP / pyridine
mixture in the derivatization reactions in A. The m/z values of the ions
related to the derivatives found in each FAB-MS spectrum are listed. These
results indicated that 4-DMAP in the reaction medium reduces the
derivatization efﬁciency. No fully derivatized product was detected with only
4-DMAP (not combined with pyridine) in the reaction medium.

The derivatization of RKDVY by 10 pl of ethyl chloroformate was also
carried out for 5, 10, and 20 ml of 2,4,6-trimethylpyridine in a solution of
H20/EtOH (60/30; v/v). The separation of the derivatization products by
HPLC and molecular weight confirmation by FAB-MS indicated that the
reactions were incomplete although the fully derivatized product was formed.
In the presence of 2,4,6-trimethylpyridine more peaks were detected in the
HPLC chromatograms and in the FAB spectra of the derivatization products.
Additional peaks at m/z 906 and m/z 878 were present and had not been seen

in the spectra of derivatives formed with pyridine in the reaction medium.

184

Table 4.5. Effect of different bases for RKDVY EtCF derivatization (catalyst
and buffer)

 

A. (4oDMAP) (10 pg / pl CHC13)/ Pyridine

4-DMAP/Py (pl) m/z value of MB" of peaks in HPLC
chromatogram

0/10 952 924 896

2/8 952 924 896 824

5/5 952 924 896 824

10/0 ----- ----- ----- 824 752
B. 4-DMAP
4-DMAP (pl) m/z of ions in FAB spectra

10 ----- 924 896 824

30 ----- ---- 896 824

50 ---- 924 896 824

185
(3) Effect of ethanol concentration

The derivatization of 10 nmol of RKDVY by 10 pl of ethyl
chloroformate was carried out for different volumes of ethanol (0, 10, 30, 50,
70 pl) in a solution containing 60 pl H20 and 10 pl pyridine. The HPLC-UV
responses (peak height) of the different derivatization products of RKDVY
and the yield of the fully derivatized product as a function of the volume of
ethanol added are shown in Figure 4.14. Figure 4.14 shows that no complete
derivatization has been achieved for the amount of ethanol added. The
response for each derivatization product is quite constant when 10 to 50 pl of
ethanol are added. The yield of the fully derivatized product is about 25%
when 10 to 70 pl of ethanol are added. The yield is evaluated as the ratio of
the response of the fully derivatized product over the sum of the responses of
the different products. Each data point in the ﬁgures is an average of
duplicate analyses.

(4) Effect. of H20 concentration

Following the same strategy of the experiment above, the effect of the
amount of water in the reaction medium on the derivatization efﬁciency was
evaluated. Similarly, the derivatization of 10 nmol of RKDVY by 10 pl of
ethyl chloroformate was carried out for different volumes of water (20, 40, 60,
80, 100 pl) in a solution containing 30 pl ethanol and 10 pl pyridine. The
HPLC-UV responses (peak height) of the different derivatization products of
RKDVY and the yield of the fully derivatized product as a function of the
volume of ethanol added are shown in Figure 4.15. Again, it shows that
complete derivatization has not been achieved for the amount of water added.

The response for each derivatization product is quite constant when 60 to 80

186

RKDVY (EtCF) vs. EtOH

200.0 j (a) H20/EtOH/Pyridine
60/0-70/10 (ul)

  
 
  
 
 
   

.5
01
O
O
l

1 free ~COOH

.I.
O
O
0

fully derivatized

01

O

O
l

 

n 1

  

 

HPLC-UV Response (Peak Height)

\U‘_r__C]——-
000] IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
o1I0 2I0 3I0 4I0 5I0 6I0 7Io sIo
EtOH(ul)
50.01 (b)
40.05

30.01

Yield (%)

 

 

 

0.0‘ ........ , ........ , ...............
0 1I0 20 30 40 sIo 6Io 7I0 sIo
EtOH(ul)

Figure 4.14. The HPLC-UV (1:214 nm) responses of the different
derivatization products (a) and the yield of fully derivatized product (b) of
RKDVY prepared with ethyl chloroformate vs. the volume of ethanol in the
reaction medium.

187

RKDVY (EtCF) vs. H 20

H20/EtOH/Pyridine

(3) 204008000 (ul)

N
01
O
O
miJ

  
 
 
 
 
  
 

N
O
O
O
in

1 free -COOH

.5
O
O
O
1

fully derivatized

 

HPLC-UV Response (Peak Height)

 

50.0
Zﬁ‘ee-COOH
-—-‘—D‘-‘-—_D""~—C}-—_-‘O
0.0 IrTYII YYYYYYYY ITTVI1II'III'IYI IIIIIIII I
2I0 30 4I0 50 60 70 80 9I0100
H20(ul)
SOoOﬁ (b)

Yield (%)
00
P
o

 

‘
d
0.0 IIIIIIIIIIIIIIIIIIIIIIIIIIIIII

20 3I0 4I0 5I0 6I0 7I0 8I0 9I0100
H20(ul)

 

Figure 4.15. The HPLC-UV (1:214 nm) responses of the different
derivatization products (a) and the yield of fully derivatized product (b) of
RKDVY prepared with ethyl chloroformate vs. the volume of water in the
reaction medium.

188
pl of water are added. The yield of the fully derivatized product is about 20%

when 40 to 100 pl of water are added.

F. Effect of ethyl chloroformate concentration on the
derivatization

Does the addition of more ethyl chloroformate reagent drive the
reaction to completion? In the derivatization of amino acids, it was noticed
that adding more chloroformate reagents would lower the TIC responses of
the amino acid derivatives which probably resulted from the pH conditions of
the reaction medium. The effect of the amount of ethyl chloroformate added
on the derivatization efﬁciency was assessed for RKDVY. Figure 4.16 shows
the HPLC-UV responses (peak height) of the different derivatization products
of RKDVY as a function of the volume of ethyl chloroformate added. These
results were from the derivatization of 10 nmol of RKDVY by different
amounts of ethyl chloroformate (5, 10, 20, 30 pl) in a solution containing 60 pl
H20, 30 pl ethanol and 10 pl pyridine. The conclusion of the effect of the
concentration of ethyl chloroformate is the same as those for the water and

ethanol: complete derivatization has not been achieved.

G. Effect of reaction time

The kinetics of the derivatization reaction were investigated. For
RKDVY and y-ECG (glutathione), the FAB responses of the derivatives as a
function of the vortexing time for the reaction are shown in Figure 4.17. (The
reaction is fast and therefore the vortexing time was considered as the
reaction time). Vortexing times ranging from 2 seconds to 3 minutes gave a
constant yield of each product indicating that the reaction was essentially
instantaneous although incomplete. In the case of GHK IIand GGF

189

RKDVY (EtCF) vs. EtCF

200.0

  
   
   

H20/EtOH/Pyridine
60/30/10 (111)

J

.6
0|
0
O
l

1 free -COOH

 

 

  

1 00.0 i
: fully derivatized
50.0 '\\.
n g ___________ «a
D ‘I - {I- 2 free -COOH

 

HPLC-UV Response (Peak Height)

0.0[TTIUIIYIFITIIVTIITIITTTIITTIYI

5 1 0 1 5 2 0 2 5 3 0 3 5
Ethyl chloroformate (111)

Figure 4.16. The HPLC-UV (1:214 nm) responses of the different
derivatization products of RKDVY prepared with ethyl chloroformate vs. the
volume of ethyl chloroformate added in the reaction.

'190
derivatization by ethyl chloroformate, the FAB responses of the derivatives

decreased slightly when the vortexing time increased (Figure 4.18), but the
yield of the fully derivatized products were approximately constant when the
vortexing time varied. Each experiment above was carried out by
derivatizing 10 to 20 nmol peptide with 5 pl of ethyl chloroformate in a
reaction medium containing 60 pl H20, 30 pl ethanol and 10 pl pyridine. All
the data points in the ﬁgures are averages of duplicate or triplicate analyses.

H. "Reverse" vs. "normal" - different procedure for the
derivatization

It was suggested that reversing the sequence of the addition of
pyridine and chloroformate reagents might achieve better yields for the
derivatization of hydroxy fatty acids by chloroformates [10]. The same
strategy was tested for the derivatization of the peptide RKDVY by ethyl
chloroformate. The derivatization of 10 nmol RKDVY by 10 p1 ethyl

chloroformate in a solution containing 60 pl H20, 30 pl ethanol and 10 pl
pyridine was carried out in both "normal" and "reverse" modes. "Normal" is
deﬁned as the addition of ethyl chloroformate after the addition of pyridine as
all of the experiments described earlier; "reverse" refers to changing the
sequence of the addition of these two reagents. The HPLC-UV responses of
the derivatization products from the experiments of both modes are listed in
Table 4.6. Two samples for each mode were prepared and each was analyzed
in duplicate . The results indicated that regarding both the yield of fully
derivatized product and the absolute response for each derivatization

product, both "normal" and "reverse" modes provided the same results.

191

(a) RKDVY (EtCF) vs. Reaction Time
6 0 7 ‘

 

 

 

 

 

 

 

 

1— "l
5 0 ﬂ 1 free -COOH
3
c:
:33" 4 0 1
Q)
m 3 0 a
a)
2 fully derivatized
ab, 2 o
<:
r.
1 O -— {P , __ .. - 43 ————— a
313‘ “D” '” 2 free -COOH
o l T l ﬂ
0 5 O 1 O O 1 5 O 2 0 0
Vortexing time (second)
(b) Glutathione (EtCF) vs. Reaction Time
8 0 —
7 0 a
8 6 0 ~ fully derivatized
g A
g 5 O —
Q)
E: 4 0 ‘c//.
2' 3 O _ 1 free -COOH
m
E 2 0 1
1 0 —
o l l l l
0 5 0 1 0 0 1 5 O 2 0 O

Vortexing time (second)

Figure 4.17. FAB-MS responses of the derivatization products of RKDVY (a)
and y—ECG (glutathione) (b) prepared with ethyl chloroformate vs. vortexing
(reaction) time.

192

(a) GHK (EtCF) vs. Reaction Time

  

 

 

 

 

 

 

6 0 1
5 0 -
8
g 4 0 _ c terminus: -COOEt
.3
m 3 O —
g 20 I- C terminus: -COOH
1 0 ‘ fully derivatized
D-D-“G---~—D—-———~D
0 I l 1 ﬂ
0 5 0 1 0 0 1 50 200
Vortexing time (second)
(b) GGF (EtCF) vs. Reaction Time
6 0 a
5 0 -
8
g 4 o —
'0 Cterminus: -COOEt
‘2 3 0 ~
to
2.
g 2 0 -
m 1 O _ C terminus: -COOH
0 r I 1 ﬂ
0 5 0 1 00 1 50 200

Vortexing time (second)

Figure 4.18. FAB-MS responses of the derivatization products o.fIGH.K (a)
and GGF (b) prepared with ethyl chloroformate vs. vortexing (reaction) time.

193

Table 4.6. “Reverse” vs. “normal” - effect of different reaction procedure on
RKDVY EtCF derivatization

 

 

 

 

2 free 1 free fully yield
-COOH -COOH derivatized H3 /
H1 H2 H3 H1+H2+H3

N1 42 172 40

N1 42 220 50

Ave. N1 42 196 45

N2 36 160 37

N2 40 210 48

Ave. N2 38 185 43

Ave. N 40 190 44 16%

R1 30 134 38

R1 32 142 33

Ave. R1 31 138 35

R2 34 184 37

R2 34 184 37

Ave. R2 34 184 37

 

Ave. R 33 161 36 16%

194
L Effect of FAB matrix on the responses of the derivatives

It was found from experiments that glycerol and thioglycerol were a
better matrix than nitrobenzyl alcohol (NBA) for the ethyl chloroformate
derivatives of the small model peptides. To quantitatively transfer the
matrix to the probe tip, a mixture of glycerol, thioglycerol, and methanol with
1 :1 :1 ratio of volume was prepared.

The effect of the volume of the matrix mixture on the FAB responses of
both derivatized and underivatized peptides were evaluated with a dipeptide
Asp-Gly (DG) as the model compound. Twenty nmol of DG were derivatized
by 5 ul ethyl chloroformate in a solution of 60 Ill H20, 30 ul ethanol, and 10 pl
pyridine. After extraction by chloroform and evaporation of the chloroform
under vacuum, the derivatives were dissolved in 20 pl acetonitrile / water
(1 :1). A l-ul aliquot of this solution was transferred to the probe tip on which
different amount of the matrix, 0.5, 1.0, 1.5, 2.0, 2.5 ul, were added. The FAB
responses of DG ethyl chloroformate derivatives as a function of the different
volume of the matrix are shown in Figure 4.19a. For each volume of matrix,
the sample was analyzed in duplicate. The results indicated that when the
volume of the matrix was less than 1.5 ul, the FAB responses of the analytes
(DG ethyl chloroformate derivatives) were relative higher, but the
reproducibility of FAB responses of the analytes were more dependent on the
accuracy of the matrix volume on the probe tip. When the volume of the
matrix was between 1.5 and 2.5 ul, the FAB responses of the analytes were
relatively lower, but were less sensitive to the volume of the matrix; that is to
say the responses were about constant for the different volumes of matrix
added in this range. There was a compromise between sensitivity and

reproducibility from the results of these experiments. The experimental

195

(a) DG (EtCF) vs. Matrix

 

 

 

 

90.0—
1 u
3 67.5;
s: .
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O) 45.0:
g I
Q . I
33., 22.52 I
0.0 WIUITUTTIUITTIFITTIFVIIIIIIW]
O 0.5 1 1.5 2 2.5 3
Matrix (ul) '
(b) DG vs. Matrix
45.0—
‘ I
1
8 33.7—
s: .
O
a.
£3 .
m 22.5:- I
2.
Q . I
a, 11.2- I
0.0 I fl T r U T V l I I T I T I F l l l I I I l I l
0 0.5 1 1.5 2 2.5
Matrix (ul)

Figure 4.19. FAB-MS responses of ethyl chloroformate derivatized (a) and
underivatized (b) di-peptide DG vs. volume of matrix (glycerol/thioglycerol/
methanol; 1/1/1).

196
results for the underivatized DG is shown in Figure 4.19b which shows the

same trend as in Figure 4.19a.

J. Effect of multi-cycle derivatization

To improve the efficiency of the ethyl chloroformate derivatization
reaction with peptides, multi-cycle derivatization was assessed with RIGDVY
as a model compound. After removal of the chloroform in the ﬁnal step of the
derivatization described in the experimental section, the residues of the
reaction mixture were exposed to the derivatization procedure one to three
additional times. The relative responses of the different products following
one to four cycles of derivatization are compared in Figure 4.20 and 4.21 as
obtained from analysis by HPLC-UV. Two samples were prepared for each
cycle and each was analyzed in duplicate. As shown in these ﬁgures, multi-
cycle derivatization does not drive the derivatization reaction to completion,
but each additional cycle of derivatization does increase the yield of the fully
derivatized product. Furthermore, the product with two free carboxyls is
absent or in relatively low abundance after the second exposure to EtCF
reagents. The ratio of A3/A2 (peak area of the fully derivatized product/peak
area of the product with one free -COOH) increases from about 0.3 (single
cycle) to about 4 after four cycles of derivatization (an increase of more than
10-fold). The ratio of A3/A3+A2+A1 (peak area of the fully derivatized
product/sum of the peak areas of all the products) increases from about 0.2
(single cycle) to about 0.8, which means that the total yield of the reaction
reaches about 80% after four cycles of derivatization (an increase of about 4-
fold) assuming the molar absorbance for the different derivatization products
of RKDVY are the same. Because of the very short time for the reaction in

each cycle, the total time for all multi-cycles is still considerably short. It also

197

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fully derivatized E]

)

d
a
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o

   
  

E 120.0
100.0
80.0
60.0
40.0
20.0

 

 

HPLC-UV Response (Peak

 

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0.0

80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0

0.0

Yield (%)

lllllllnAllllllllllnllllnlnlllllillllllnl

 

1 2 3 4
Number of exposures to reagent

Figure 4.21. The HPLC-UV (1:214 nm) responses of the different
derivatization products (a) and the yield of fully derivatized product (b) of

199
can be noticed from Figure 4.21a that with the completion of the second cycle,

the absolute response of the fully derivatized product doubles compared to
that from the ﬁrst cycle. Comparing the result after the fourth cycle with
that after three cycles, the response of the fully derivatized product does not
increase, but the response of the derivative with one free -COOH still
decreases. Thus, the yield of the fully derivatized product which is the ratio
of A3 over the sum of A1+A2+A3 is increasing. If this increase of the yield is
real after the fourth cycle of derivatization, the low absolute responses for
both products (after the 4th cycle) may be explained by the losses during the
extraction and / or the variations of the individual samples. It is also possible
that the increase of the yield (A3 / A1+A2+A3 ) after the fourth cycle comes
from the relatively more severe loss of the derivative with one free -COOH
during the extraction, but not the real increase of the fully derivatized
product. Regarding the efﬁciency of the extraction, more than four

derivatization cycles are not recommended.

V. Preliminary investigation of forming precharged derivatives of
peptides using the chloroformate derivatization procedure

As reviewed in chapter I, precharged peptide derivatives can increase
the ionization efﬁciency in analyses of peptides by FAB. Furthermore,
precharged peptide derivatives may inﬂuence the fragmentation in CID-
MS/MS experiments to form one or several complete series of structurally
informative fragment ions to assist in peptide sequencing [3-5] . With our
modiﬁed one-step chloroformate derivatization method, a charged moiety can
be introduced into the peptide derivatives by applying a charged alcohol, such

as choline, in the reaction medium. See Reaction 4-1.

200
HzN-(CO-(IZH-NH)n-C'JH-COOH
R R1

H20/ Pyridine (CH3)3N*(CH2)20H or

EtCF l

Et02CHN-(CO-(I'JH-NH),,-('3H-COO(CH2)2N+(CH3)3 or
R R1

(Reaction 4-1)

First, Phe was chosen as the model compound to react with ethyl
chloroformate-choline chloride reagents to test the proposed idea. A 10-u1
volume of Phe solution (100 ug) was added to a solution containing 80 ul of
choline chloride / water solution (1:1; w/w) and 10 ul pyridine. Ethyl
chloroformate of 10111 volume was added to the solution. The solution was
vortexed for 10 s, and 100 pl of chloroform were added. Both the chloroform
and the aqueous layers were analyzed by FAB. The expected derivatization
product, N-ethoxycarbonyl-Phe choline ester (M+ at m/z 323 in Figure 4.22)
was detected in the aqueous layer.

An ion of m/z 238 was also detected in the chloroform layer which
might be N-ethoxycarbonyl-Phe (the species with a free carboxyl group). N 0
or very low signal was detected for N-ethoxycarbonyl-Phe ethyl ester in the
chloroform layer. It can be concluded that almost all the ester derivatization
product came from choline. It also can be noticed in Figure 4.22 there were
some overscaled ions, m/z 387, m/z 315, m/z 243. m/z 176, and m/z 104, which
did not come from the FAB matrix glycerol. Product spectra (B/E linked

201
scan) (Figure 4.23) of these ions revealed that they may come from the

reaction between the derivatization reagents, ethyl chloroformate and choline
chloride, or may come from the adducts of the reagents and their reaction
products. The possible structures of these ions are in Figure 4.24. If not
separated from the amino acid or the peptide derivatization products, these
ions which have high concentration may suppress the signal from the
analytes. 100 nmol of tri-peptide GGF were derivatized following the
procedure described above, and 1111 of the aqueous layer was analyzed by
FAB. No corresponding N -ethoxycarbonyl-GGF choline ester was detected.
No further experiment has been attempted.

The preliminary conclusions are, precharged choline moiety can be
attached on the carboxyl group of Phe by the simple ethyl chloroformate
derivatization reaction based on the mechanism described in chapter II. N-
ethoxycarbonyl Phe was also detected. The efﬁciency of the derivatization
with peptides needs further investigation. The enhancement of FAB signal of
peptides by this derivatization procedure needs to be assessed. The
conditions to separate the peptide derivatization products from the reaction
products derived from the reagents need to be investigated. Furthermore, the
choline may attach to the carboxyl group on side chains of aspartic acid and
glutamic acid which results in doubly or even more highly charged products
when more carboxyl groups exist on side chains of peptides. This aspect of

the derivatization needs investigation.

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202

M; 323 Phe EtCF-Choline

311/ 38619

 

g.
s

 

287.1
359.0

. A

 

A
O

 

 

 

O

 

 

 

 

 

 

 

 

 

 

 

.‘ ‘ ‘ ' 1
'0 150 200 250 300 350 400 450 “/2500

Figure 4.22. FAB mass spectrum of Phe ethyl chloroformate-choline
derivative (M"' 323).

203

(a) Product ion spectrum of Mt 387

 

8

18

176

8

.3

3

245

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381

 

 

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160 150 260 250

(b) Product ion spectrum of M+ 31 5

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315

 

 

 

(c) Product ion spectrum of Mt 243

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104

533%

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243

 

 

Figure 4.23. Product ion spectra (B/E linked scan) of ions from Figure 4.22:

(a) m/z 387, (b) m/z 315, (c) m/z 243.

 

 

 

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VI. Conclusions

The results of this study indicate that preparation of the ethyl ester,
N,O,S-ethoxycarbonyl derivative of small peptides prior to analysis by FAB-
MS increases the detectability of the derivatized peptide by 5- to 50-fold
relative to that of the underivatized peptide. The chloroformate reagent can
be used in an aqueous medium to react with both the amino and carboxyl
groups as well as phenolic and sulfhydryl groups, but not aliphatic hydroxyl
groups. Amino, phenolic and sulfhydryl groups are completely derivatized,
but carboxyl groups are only partially esteriﬁed regardless of location on side
chains or at the C-terminus. From a thorough investigation of the
derivatization conditions, a multi-cycle derivatization improves the eﬁiciency
of esteriﬁcation reaction; four cycles of derivatization drives the reaction to
80% completion while other variations do not make the reaction complete.
The one-step chloroformate derivatization method has the possibility of
bringing charged moieties into the peptide structure based on our modiﬁed
derivatization procedure. Preliminary results from reacting the amino acid
Phe with ethyl chloroformate-choline reagents proved the idea. More
investigations need to be performed.

The results from small peptides indicate that carboxyl group
derivatization with ethyl chloroformate does not go to completion in the one-
step aqueous medium chloroformate derivatization. This suggests that the
reaction efficiency of ethyl chloroformate with carboxyl groups of amino acids

should be examined. This work will be described in chapter V.

VII. References

1 A.M. Falick, and D.A. Maltby, Anal. Biochem., 182(1989)165.

2 S. N aylor, A.F. Findeis, B.W. Gibson and DH. William, J. Am. Chem.
Soc. , 108 (1986) 6359.

3 D.S. Wagner, Ph.D. Dissertation, Dept. of Chemistry, Michigan State
University, 1992.

4. J .E. Vath, K. Biemann, Int. J. Mass Spectrom. Ion Proc., 100 (1990)
287.

5. J .T. Stults, J. Lai, S. McCune, and R. Wetzel, Anal. Chem., 65 (1983)
1703.

6 P. Husek, J. Chromatogr. , 552 (1991) 289.

7 Z.-H. Huang, J. Wang, P. Husek, D.A. Gage, J .T. Watson and C.C.
Sweeley, J. Chromatogr., 635 (1993) 271.

8 ‘ J. Wang, Z.-H. Huang, D.A. Gage, and J .T. Watson, The 40th ASMS
Con. Mass Spectrom. Allied Topics, p1579.

9 J. Wang, Z.-H. Huang, D.A. Gage, and J .T. Watson, J. Chromatogr. ,
663 (1994) 71.

10. P. Husek, J. Chromatogr. , 630 (1993) 429.

206

Chapter V
Further investigations on the quantitative aspect of the one-step
chloroformate derivatization in an aqueous medium for amino acids and
small peptides

I. Introduction

In chapter IV, we saw that even with all the variations of reaction
conditions, the derivatization of RKDVY, more specifically, the
esteriﬁcation of its carboxyl groups with ethyl chloroformate, was
incomplete. These results initiated a further investigation to evaluate the
esteriﬁcation efﬁciency of ethyl chloroformate with amino acids. No
reaction yields for the ethyl chloroformate derivatization with amino acids
were reported in the original method development by Husek [1-3]. Also in
our earlier investigations for derivatization of amino acids by
chloroformate-alcohol reagents, the underivatized amino acids were not
detected. The incompletely derivatized amino acids may remain in the
aqueous phase during the chloroform extraction, or they may not be volatile
enough to elute from the GC column during the GC-MS analyses.

In this chapter, two reaction schemes will be used to evaluate the
esteriﬁcation efﬁciency of ethyl chloroformate with amino acids in order to
have a better knowledge of the advantages and the limitations of the one-
step aqueous medium chloroformate derivatization method. Further
investigations to improve the reaction yield for amino acid and peptide

derivatization with ethyl chloroformate were pursued.

207

$8
11. Evaluation of the reaction efficiency of ethyl and isobutyl

chloroformate with carboxyl groups of amino acids

A.Methodl

In this method, a two-step reaction procedure was applied to evaluate
the reaction efﬁciency of carboxyl groups of amino acids in the one-step
ethyl chloroformate and isobutyl chloroformate derivatization method. The
ﬁrst reaction followed the general chloroformate derivatization procedure
outlined in section II of chapter II of this dissertation. One variation was
that no chloroform extraction of the derivatization products was performed.
Instead, the solvent and the reagent in the reaction mixture were removed
under vacuum. By doing this, all the amino acid derivatization products ﬂ
(carboxyl groups derivatized and underivatized) by the chloroformate
reagents remained in the reaction vial; thus, partial or total loss of the
underivatized species during the chloroform extraction was avoided. In the
following step, conventional methylation of carboxyl groups by
diazomethane (CH2N2) was performed. The reaction and the condition of
methylation of carboxyl groups by diazomethane were elucidated in
Reaction 5-1. Any remaining free acid from the ﬁrst reaction was
converted to its methyl ester. The overall reaction scheme is outlined in
Figure 5.1. The derivatization products after the two-step reaction were
analyzed by GC-MS.

CH2N2
R-COOH > R-COOCH3

ether / MeOH
room temperature

(5-1)

 

.mEom 058m ﬁrs 32:8825 232:8: 136 we b.5650
cosmomtoemo 36:35 3 H v2.88 .6 25:3 couowom 46 055E

 

mmmbmcm 92-00 mo EmpmoumEoEo
meE .8 DE. 5 no.8 xmoa u<

 

 

 

 

 

 

3+3
s2: x u 22»
J.
m m
3V goooémgmznvooum a a EOOO-EQ-EZOOOBH Anoaomom hon—ma
+ 538% 2 m0 :osoaom moan.” M
Ar + .l - - a
. . i ﬂooom z m
3 ﬁoooiﬁmznvoosm «new ﬂoooioizneooﬁ 28m
_
m

m

210
The efficiencies of esteriﬁcation of Phe, Tyr, Asp, Glu, and Lys by

reaction with ethyl chloroformate have been evaluated by the method
described above. The results indicated that reaction of ethyl chloroformate
with the carboxyl groups of these amino acids by the one-step aqueous
medium reaction is incomplete to different extents dependent on the types of
amino acid. The results are summarized in Table 5.1. The completion of
the -COOH reaction (yield) was evaluated as the ratio of the peak area for
the ethoxycarbonyl amino acid ethyl ester over the sum of the peak areas for
the ethoxycarbonyl amino acid ethyl ester and methyl ester (equation
below).
Aa

Yield = x 100%
Aa'l-Ab

 

A: peak area in TIC or mass
chromatogram of GC-MS analysis

a: ethyl ester; b: methyl ester

The peak areas were from either the TICs or the mass
chromatograms of the characteristic ions of these derivatives.
Representative TIC and mass spectra are shown in Figure 5.2 for Phe and
in Figure 5.3 for Tyr. The esteriﬁcation efﬁciencies for Phe, Tyr, and Lys
were fairly good (70-90%) while the esteriﬁcation of the acidic amino acids,
Asp and Glu, were far from complete based on our experimental results
under optimal reaction conditions.

The esteriﬁcation efficiency of isobutyl chloroformate with carboxyl
groups of Phe, Asp, Glu, Gln, and Ser was also examined by method I
described above. The results are summarized in Table 5.2. The results

again indicated that the esteriﬁcation efﬁciency for Asp and" Glu were

211

Table 5.1. Results from method I - esteriﬁcation efﬁciency with ethyl

 

 

chloroformate.
Amino Sample Injection Yield (%)
Acid (lug) (us)
Phe 2.0 0.200 ~70 (by TIC)
~85 (by m/z 192)
~70 (by m/z 176
and m/z 162)
Tyr 20.0 0.900 ~90-95* (by TIC and
mass chrom)
Lys 2.0 0.200 ~90*
Asp 2.0 0.200 ~10 (by TIC)
~30 side chain:
free COOH
~20 C-terminus:
free COOH
~40 2 free -COOH
Glu 2.0 0.200 <1 (by TIC)
~20 side chain:
free COOH
~20 C-terminus:
free COOH
~60 2 free -COOH

 

"‘ by both TIC and mass chromatogram

 

 

 

 

 

 

 

 

 

 

 

 

Max.152.7 6 RT.
R A x A # l 1 A A A A A A P
'9 TIC
a methyl ester
’ __/£
I
,, . v ,Tlc
° qul76
I
n
l
9 A— 7 ~ >176
n 43.6
5
l
t
y
"r - . -1 ‘7. @‘35
410 430 «to
Scariuasz) (a) TIC 3““
100‘ 162 ’
R ‘ r
6 4 .
f 3 .COOMe 1162 '
v 60$ —’ F
e ‘ 192 z
A L
b
U
n
d
a
n
C
8

 

 

 

 

 

 

 

 

 

 

 

 

100‘ 16 b
l 80% - ---------- b
a i ICOOEt 1176 l
l i —l .
u .
v 601 192
e 1
A 1 102 192 M+‘=265
b 40‘ 91 .
u - ,
3 ‘ 120 ’
< 148 ‘20.0
a 20‘ 74 1 1
n 4
c , 55
9 . 1 20 >
o . . . . J. . . , . -
50 100 150 200 250 300
M

(c) Phe ethoxycarbonyl ethyl ester

Figure 5.2. TIC and EI mass spectra of Phe derivatized with (1) EtCF and

(2) CH2N2 (method I).

213

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.6724 9 RT.
R ............... l x P. A ”417.4
? TIC Tyr ethyl ester
3 methyl ester\
1 A A A TIC
2 672.4
I ‘ m/z 264
n
3 ~ “~26.
n 61.6
,5 m/z 250
l
y
— 256 0.9
550 640
Sam: (590)
100
J
R J
a < """""" 250
t . :COzMel
i 4 _‘
v 60-1 280
a 1 17s
A .
b 40" r
U 4
a 20: 135
n 4
c 147 206
‘ 280
e G‘sﬁgq _ .. fl 1... wjlvivlv .f‘.l:.1,ﬁ-
53 100 150 200 250 300 350 (13%
Scan; (603) (b) 1371' ethoxycarbonyl methyl ester
1oo+ 1 7
Fl J EtozCn/Nj—ICOZEt
9
I 804 192 .-'. ......... 264
f ‘ :COZEt l
' -—l
I -1
v 604 280
e 4
A l 254 M+=353
b 40:
U <
3 1 135 '20.0
a .1
n 20. J 74 220 mo
6 . 74 117 1 J l
. 9‘ 308
° Gish. .L. J.W.LM . .1. . . . , . . ..
so 100 150 200 250 300 350 m

(c) Tyr ethoxycarbonyl ethyl ester

Figure 5.3. TIC and EI mass spectra of Tyr derivatized with (1) EtCF and (2)
CH2N2 (method I).

214
poor relative to those for the other amino acids tested. Figure 5.4 shows the

TIC, mass chromatograms, and mass spectra of different Asp
derivatization products with isobutyl chloroformate and diazomethane.

It should be pointed out that method I may under-estimate the
esteriﬁcation efﬁciency of amino acids with chloroformate reagents.
Partial loss of the ester products from the ﬁrst step may occur during the
relatively long process of removing the solvents and reagents under
vacuum. Experiments indicated that these losses may have occurred,

resulting in inconsistent recovery of the derivatives.

B. Method II

A different experimental scheme, outlined in Figure 5.5, was
designed to avoid the potential problem in method I created by the vacuum
during the drying process. The procedures are as follows, commercially
available amino acid ethyl and methyl esters were used as standards to
evaluate the esteriﬁcation efﬁciency of ethyl and methyl chloroformate with
Phe, Leu, Asp, and Glu. 100 nmol of Phe, Leu, Glu, and their ethyl esters
were derivatized individually with ethyl chloroformate following the
standard procedure. Methyl stearate (25 or 50 nmol) was added to each
reaction medium as an internal standard for quantitation. A 2411 aliquot of
the ﬁnal chloroform solution of the derivatives was injected for analysis by
GC-MS (0.2 nmol of amino acid derivative, 0.1 or 0.05 nmol of methyl

stearate). Asp was examined by methyl chloroformate reaction.

215

Table 5.2. Results from method I - esteriﬁcation efficiency with isobutyl
chloroformate.

 

 

Amino Acid Yield (%)

Phe ~90

Gln ~95

Ser ~95

Asp ~10
~45 side chain: free COOH
~20-25 C-terminus: free COOH
~20-25 2 free COOH

Glu ~5
~35 side chain: free COOH
~20 C-terminus: free COOH
~40 2 free COOH

 

"‘ 10 ug for each derivatization and 500 ngfor each injection

“ yield is calculated from peak areas of TIC

 

216

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Max.400.0
A '1 A g A q R . T
R 1795.6
6 Asp ,
1 Me-iBu esters i’Bu-Me esters
2 Me-Me esters iBu-iQu estersi
1 TIC
V
e 100.0
m/z 244
I
n
t
e " *4.0 f
n 400.0
8 ml: 202
1
t:
Y L
V 1 v v v— ‘
400 450 500 550$can }
(a) TIC and mass chromatograms
1001 202
R . l
e * IVIICKJzHBU l
1 80* _
a ‘ M8020 :. (3021“e .
t_ 102 .2 ’
1 ‘ m/z 202 ’
v 601 r
e 1 .
57 .
A « MH+-=261 »
b 4 01 ~
1.1 1 l .
n 1.
d 1 *10.0 »
a 204 1 6 .-
l'l 1 ,
c 86 128 160 >
e J 1 8 r
O A A T AL f V Y I f Y
50 100 150 200 250 300 350 400
M/Z

(b) Asp isobutoxycarbonyl dimethyl esters

Figure 5.4. TIC and EI mass spectra of Asp derivatized with (1) iBuCF and
(2) CH2N2 (method I).

(continued to next page)

217

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100‘ 2 2 ’
2 1 1 2 NHCOgiBu :
1 so- MeO
a . 57 T 2C :COziBu
t -J
1 P
v 60-1 111/2202 .
e Rim-sacs C
A 1
b QO-( 1-
U r
n D
d '10.0 1
a 20.1 :16 ,
n ' 174 ’
C 4 .
160
e JJJL 8.: Y J A :l l 42 Y .1 1. ‘1' VA V 1
so 100 150 200 250 300 350 400
(c) Asp isobutoxycarbonyl B—methyl isobutyl esters W z
10
s 818 2 4 ,
R NHCOziBu ,
e iBuO C ’
1 eoi 11.4 2 :C02Me -
a —l .
l 111/2244 ’
v 60 .
e MH“:-303 1
A .
b
U
n >
d 110.0 .
a 1-
n b
C
a
so 100 150 200 250 $00 350 400
(d) Asp isobutoxycarbonyl Bcisobutyl methyl esters W z
100 2W
2 NHCOziBu I
1 so- 57 ' - _
a lBUOzC : C0218“
t .4 .
1 111/2244 .
v so< -
6 1
A 88 MH+w346
b 401 i
U
n 144
d -1o.o
1; 20<
1 o
C
7° 11* 1 11° ..
e d L 4:1{4 v . - fa : r1 1 212 - . - - r. 1
so 100 150 200 250 300 350 400
an

Figure 5.4. TIC and EI mass spectra of Asp derivatized with (1) iBuCF and

(e) Asp isobutoxycarbonyl diisobutyl esters

(2) CH2N2 (method I).

218
A (analyte) / A (1.8.)
Yield = x 100%

A (standard) / A (1.8.)

 

A: peak area in TIC or mass chromatogram of GC-MS analysis
I.S.: internal standard (methyl stearate)

As outlined in Figure 5.5 and the equation above, the responses of the
TIC and the characteristic ion of the ethyl chloroformate derivative from
each amino acid were compared with the responses of those from the
corresponding derivative derived from its ethyl ester. The TICs of EtCF
derivatives of Phe and Phe ethyl ester are shown in Figure 5.6 as a
representative example. The results are summarized in Table 5.3. Each
data was from the average of analysis of two individually prepared
samples, each analyzed in duplicate. The esteriﬁcation efficiency of Phe
derived from method II was the same as that calculated from method I.
The efﬁciency of methylation of Asp by methyl chloroformate (~40%) derived
from method II was higher than the efficiency of ethylation of Asp by ethyl
chloroformate (~10%) derived from method I. This may result from the
different efﬁciencies of methylation and ethylation of amino acids by
chloroformate reagents, or from loss of the derivative during the vacuum

drying process as we discussed earlier.

C. Conclusions
The results from both method I and method II proved that although

the one-step aqueous medium chloroformate derivatization for the analysis
of amino acids is a simple derivatization procedure, the reaction is not

complete for each amino acid. The esteriﬁcation efficiency for acidic amino

219

 

 

 

Methyl Stearate (1.8.)
(1) HzN-(llH-COOIEI >- EtOOCNH-CH—COOEE
R EtCF Reaction R
(Analyte)
Methyl Stearate (IS)
(2) HzN-CH-COOEQ > EtOOCNH-(IIH-COOﬂ
fl; EtCF Reaction R
(Standard)
A (analyte) / A (I.S.)
Yield = x 100%

 

A (standard) / A (IS)

A: peak area in TIC or mass chromatogram of GC-MS analysis

 

 

 

Figure 5.5. Reaction scheme of method II to evaluate esteriﬁcation
efficiency of ethyl chloroformate with amino acids

 

 

Leu (EtCF)
.l
H

‘q ' ll‘lv l1 li‘ll .0 .Q... .. I‘ll‘l I‘ll- '7‘ .u l ‘
I

w M

1 CI

V'f‘ﬁ1r17_‘

 

358
5.83

(a)

.Iv‘k

 

 

rlllclfllltrlllr. lllll
8.. ll!
I

)

at

C

t

Du

(

r

m

S

e 1 it 1-1!

‘yl llntlll Ill '0 1.11.11.11.11

.m

e

m

1803

TOT~

 

,
----'nv‘--..- —— - P...- -¢.--~d .—

.- ’-— _

1
S“.
..4.b
ﬁl

v

 

(b)

Figure 5.6. TICs of Leu (a) and Leu ethyl ester (b) derivatized with EtCF

(method H).

Table 5.3. Results from method I - esteriﬁcation efﬁciency with ethyl
chloroformate.

 

 

Amino Acid Yield (%)
Phe 76 (by TIC)
73 (by m/z 192)
72 (by m/z 176)
Leu 43 (by TIC)
47 (by m/z 158)
Asp* 37 (by TIC)
40-45 (by m/z 188)
Glu <10 (by TIC)

 

* Asp was examined by methyl chloroformate reaction.
Each datum is the average of two individually prepared samples, each analyzed in
duplicate.

10 nmol for each derivatization and 0.2 nmol for each injection for GC-MS analyses.

 

222
acids are low by the chloroformate derivatization. These experimental

results also answeredrthe questions raised in the derivatization of small
peptides by ethyl chloroformate. Both amino acid and peptide esteriﬁcation
by the chloroformate reagents under the current derivatization procedure
are incomplete.

Although not totally identical, results from other researchers agree
with our ﬁndings. Esteriﬁcation yield of some dicarboxylic acids, namely
those containing 4 and 5 carbon atoms (succinic, glutamic) was very low.
Correspondingly, the yield of their substituents, Asp and Glu, were low [4].
The yields of esteriﬁcation of amino acids by methanol or ethanol in chiral

menthyl chloroformate derivatization were reported as 95% [5].

III. "Non-aqueous" vs. "aqueo ' reaction medium for derivatization of
amino acids and peptides with ethyl chloroformate reagent

A. Introduction

Since incomplete esteriﬁcation is a common result for the one-step
aqueous medium chloroformate derivatization of amino acids and peptides,
experiments to investigate and improve the yield of esteriﬁcation have been
attempted.

The one-step aqueous medium chloroformate derivatization method
for amino acids in analytical scale developed by Husek was partially based
on the method of esteriﬁcation of fatty acids by the chloroformate reaction
on organic macro scale [6,7]. In the original method, the reaction
proceeded in a non-aqueous medium. It was also noticed that for
esteriﬁcation of short chain fatty acids, higher yields were easier to achieve
in a non-aqueous reaction medium (such as, CH3CN, CH2C12, and hexane

in the presence of alcohol). Even with a higher concentration of 'EtOH, the

 

223
esteriﬁcation of fatty acids by ethyl chloroformate in the reaction medium

containing water reached around 90%. Lowering the EtOH concentration
drastically decreased the reaction yield [8].

These results might be explained by the esteriﬁcation mechanism
discussed in the chapter II (Figure 2.5). In that mechanism, the ester is
formed by nucleophilic attack of the alcohol in the reaction medium on the
acylpyridinium species 21. If water exists in the reaction medium,
nucleophilic attack of water on 23 (hydrolysis of 23) would compete with the
attack of alcohol. This process drives the intermediate 23 back to the free
acid. The esteriﬁcation may reach a certain level of equilibrium which
depends on the concentration and the nucle0philic reactivity of the alcohol
in the reaction medium. If this explanation is true, the yield of
esteriﬁcation of amino acids by chloroformate reagents may be improved in
the non-aqueous reaction medium although this would complicate the
method somewhat.

Experiments were designed to verify this idea by comparing the
results from aqueous and non-aqueous reaction media for derivatization of

amino acids and the penta-peptide RKDVY.

B.Aminoacid derivatization

Leu (50 nmol) with 25 nmol of methyl stearate as an internal
standard was derivatized by 10 pl of ethyl chloroformate in each of three
reaction media: (1) a solution of 60 pl H20, 30 pl EtOH, and 10 pl pyridine
(the conventional aqueous medium); (2) a solution of 60 pl CH3CN, 30 pl
EtOH, and 10 pl pyridine; (3) a solution of 90 pl CH30N and 10 pl pyridine.
The derivatives from (1) were extracted with 200 pl chloroform, and the

chloroform was removed under N2 stream. The derivatives were

224

redissolved in 110 pl of chloroform. No extraction was made for derivatives
from reaction media (2) and (3). A 2-pl aliquot of each solution was injected
for analysis by GC-MS.

Ratios of the peak areas (Leu relative to methyl stearate) from both
the TICs and mass chromatograms (m/z 158 for Leu, m/z 74 for methyl
stearate) are listed in Table 5.4 for the results from the analysis of three
reaction media. Comparing the results from (1) and (3), it can be seen that
the relative response of Leu ethyl chloroformate derivative increased (0.25 to
0.50) when water was absent from the reaction medium. Among the three
reaction media, the relative response of Leu ethyl chloroformate derivative
was the highest in the non-aqueous reaction medium (2) which also
contained EtOH. If the amino group reaction yield did not change in the
three reaction media, we can say that the yield of esteriﬁcation of amino
acid is higher in a non-aqueous medium. The Leu derivative from reaction
medium (2) was rerun one hour after the ﬁrst run. Its relative response
from TIC and the characteristic ion did not change. This indicated that the
reaction did not proceed further, and the reaction yield did not increase
with time.

Similar experiments with amino acids, Leu, Phe, Tyr, Lys, each 50
nmol, and 5 nmol methyl stearate, in the three reaction media were carried
out. The derivatives were extracted by 200 pl chloroform with 100 pl water
as a counter phase in each case. A 2-pl aliquot of the chloroform layer was
injected for analysis by GC-MS.

The relative responses of peak areas of TIC for these amino acid ethyl
chloroformate derivatives are listed in Table 5.5. These results are similar
to that in Table 5.4. For Leu and Phe derivatives, the relative responses

increased in a non-aqueous media compared with those from an aqueous

 

Table 5.4. Relative responses of peak area from TIC and mass
chromatogram (relative to internal standard) of Leu derivatized with ethyl
chloroformate in different reaction medium as indicated.

 

 

Leu (EtCF) H20/EtOH/Py CH3CN/EtOH/Py CH3CN/Py
from TIC 0.25 0.75 0.50

from mass

chromatogram 0.30 0.94 0.60

 

Table 5.5. Relative responses of peak area of TIC (relative to internal
standard) of amino acids derivatized with ethyl chloroformate in different
reaction medium as indicated.

 

Amino Acid H20/EtOH/Py CH3CN/EtOH/Py CH3CN/Py

Leu ~2.2 ~3.6 ~3.9
Phe ~2.7 ~4.2 ~4.7
Lys* ~0.15 ~0.07 ~0.09

 

* ratio of peak height

226
medium. But, for Tyr and Lys, with a side chain amino or phenolic group,

the relative responses were higher when the reaction was conducted in an
aqueous medium. This may indicate that amino and phenolic group
derivatization by ethyl chloroformate achieves a better yield in an aqueous
medium while the carboxyl group reaction gives a better yield in a non-
aqueous medium. Similar results were observed when RKDVY
derivatization by ethyl chloroformate was compared in aqueous and non-

aqueous reaction media.

0. Peptide derivatization

The problem of incomplete esteriﬁcation in the one-step
chloroformate derivatization was raised when the derivatization method
was applied to the analyses of small peptides by FAB-MS. Reaction
conditions have been systematically reported in chapter IV; no reaction
condition for complete esteriﬁcation has been found. In a series of
experiments to evaluate the effect of water concentration in the reaction
medium, 20 pl water in a solution containing 30 pl EtOH and 10 pl pyridine
has been tested. In these experiments, the response of the fully derivatized
RKDVY product (Figure 4.15) was the same as, while the response of the
product with one free -COOH was lower than those obtained from the
reaction media containing more water. Other experiments also indicated
as seen in Figure 4.8 (FAB-MS spectra) that when the water volume was 10
pl in the reaction medium, the absolute responses of the derivatization
products were lower than those from other reaction medium compositions
with more water. All these results suggest that less water did not favor the
RKDVY derivatization with ethyl chloroformate.

 

226
medium. But, for Tyr and Lys, with a side chain amino or phenolic group,

the relative responses were higher when the reaction was conducted in an
aqueous medium. This may indicate that amino and phenolic group
derivatization by ethyl chloroformate achieves a better yield in an aqueous
medium while the carboxyl group reaction gives a better yield in a non-
aqueous medium. Similar results were observed when RKDVY
derivatization by ethyl chloroformate was compared in aqueous and non-

aqueous reaction media.

0. Peptide derivatization

The problem of incomplete esteriﬁcation in the one-step
chloroformate derivatization was raised when the derivatization method
was applied to the analyses of small peptides by FAB-MS. Reaction
conditions have been systematically reported in chapter IV; no reaction
condition for complete esteriﬁcation has been found. In a series of
experiments to evaluate the effect of water concentration in the reaction
medium, 20 pl water in a solution containing 30 pl EtOH and 10 pl pyridine
has been tested. In these experiments, the response of the fully derivatized
RKDVY product (Figure 4.15) was the same as, while the response of the
product with one free -COOH was lower than those obtained from the
reaction media containing more water. Other experiments also indicated
as seen in Figure 4.8 (FAB-MS spectra) that when the water volume was 10
p1 in the reaction medium, the absolute responses of the derivatization
products were lower than those from other reaction medium compositions
with more water. All these results suggest that less water did not favor the
RKDVY derivatization with ethyl chloroformate.

 

227
Further investigation of the reaction conditions were undertaken by

partially and totally replacing the water, and even the ethanol, in the

reaction medium by CHaCN to compare the effect of a non—aqueous medium

with other reaction media. The experiments were carried out as: 20 nmol

of RKDVY were derivatized by 10 pl (210 pmol) EtCF in four different
reaction media which are listed in Table 5.6. Two individual samples were

prepared for each reaction medium composition.

Table 5.6. Reaction medium compositions for RKDVY derivatization with

 

 

EtCF.
H20 (:11ch EtOH Pyridine
(pl) (pl) (111) (pl)

1 a) 0 3) 10

2 3O 3) 3) 10

3 0 a) 30 10

4 O 90 0 10

 

The reaction mixtures were separated by HPLC (with the similar
conditions as in the chapter 1V), fractions corresponding to the derivatives
were collected and analyzed by FAB-MS. Partial HPLC chromatograms of
the reaction products from the four reaction media are shown in Figure 5.7.

These results showed that the total reaction yield of ethyl
chloroformate derivatization of RKDVY decreased when water in the
reaction medium was partially replaced by CH3CN (a to b in Figure 5.7,
V1120: 60 to 30 pl). The yield kept decreasing as water was totally replaced
by CH3CN, and the chromatographic pattern changed indicating the

change of the structures of the major derivatization products (b to c in

229
Figure 5.7). In chromatogram c (V1120 =0), m/z values of MH+ of the major

components were identiﬁed to be 880 and 852 (peak 2+3 and 1 in c). Ion of
m/z 880' was conﬁrmed by MS/MS spectra to be the derivative with phenolic
group not derivatized, and the ion of m/z 852 corresponded to the derivative
with phenolic and C-terminus carboxyl groups not derivatized. Also in
chromatogram c, component with MH+ at m/z 896 was not identiﬁed and
component with MH+ at m/z 924 was insigniﬁcant which was part of peak
3. These may indicate the same conclusion as that from the amino acid
experiments in last section: in a non-aqueous reaction medium,
esteriﬁcation efﬁciency with ethyl chloroformate increased, but phenolic
group derivatization efficiency decreased. The overall effect was that the
total derivatization efﬁciency decreased. From chromatogram c to d in
Figure 5.7 (VEtOHi 30 to 0 pl), the peak with MR" at m/z 852 increased (peak
1 in c and d) which may indicate that without EtOH in the reaction medium
((1) the esteriﬁcation efﬁciency will be lower than that with EtOH (c).

D. Conclusions

The overall conclusion is that the water in the reaction medium
lowers the esterification efficiency in the one-step chloroformate
derivatization. However, elimination of water in the reaction medium
results in overall lower yield of derivatization for amino acids and small

peptides with multi—functional groups.

IV. Conclusions
The reaction efﬁciency of ethyl and isobutyl chloroformate with
carboxyl groups of amino acids has been examined. It was found that the

esteriﬁcation of amino acids by the one-step aqueous medium ethyl

geese a S .7636 a 8 as 652.3 3 S .moem .1 8 .76emo a 8 E ”enema
3 S .moem a om. .7636 3 cm .oem 3 8 EC 85.93 a S 466 a 8.03:1 8 3 "sees 2:88
39:56 5 Suﬁaououozo 350 ﬁrs 633.36% :3 mo mﬁaumSmEoEo 04mm Eaten S6 charm

 

 

 

on 55 S 8 55 2
1l 4 T _
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unmemz

 

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230
(isobutyl) chloroformate reaction is also incomplete as found for peptide

derivatization in the chapter IV with the current reaction conditions. The
esteriﬁcation efﬁciency varies with different types of amino acid, typically
40-95% for the amino acids examined. Acidic amino acids have lower
esteriﬁcation efﬁciency. Derivatization of amino acids and peptide RKDVY
in a non-aqueous reaction medium has been investigated. The results
indicated that esteriﬁcation efﬁciency by ethyl chloroformate may increase,
but amino and phenolic group derivatization may decrease in a non-
aqueous medium. For overall reaction yield, RKDVY derivatization with
ethyl chloroformate needs water in the reaction medium although the

esteriﬁcation reaction is incomplete.

V. References

1 . P. Husek, FEBS Lett., 280 (1991) 354.

2. P. Husek and C.C. Sweeley, J. High Resolut. Chromatogr., 14 (1991)
751.

3. P. Husek, J. Chromatogr., 552 (1991) 289.

4. P. Husek, personal communication.

5. N. Domergue, M. Pugniere, A. Previero, Anal. Biochem., 214 (2)
(1993) 420.

6. s. Kim, J.I. Lee,'iY.C. Kim, J. Org. Chem., 50 (1985) 560

7. S. Kim, Y.C. Kim, J .1. Lee, Tetrahedron Lett., 24 (1983) 3365.

8. P. Husek, J .A. Rijks, P.A. Leclerg and C.A. Cramers,
J. High Resolut. Chromatogr., 13 (1990) 633.

IGAN STRTE UN IV.

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