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Synthesis of potential affinity labelling inhibitors of protein tyrosine kinases

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Title:
Synthesis of potential affinity labelling inhibitors of protein tyrosine kinases
Creator:
Charrier, Pierre Gilles
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English
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xv, 62 leaves : illustrations ; 29 cm

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Subjects / Keywords:
Tyrosine -- Affinity labeling ( lcsh )
Chloromethyl group -- Synthesis ( lcsh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references.
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Science, Department of Chemistry.
Statement of Responsibility:
by Pierre Gilles Charrier.

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University of Colorado Denver
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Auraria Library
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
25503020 ( OCLC )
ocm25503020
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LD1190.L46 1991m .C42 ( lcc )

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Full Text
SYNTHESIS OF POTENTIAL AFFINITY
LABELLING INHIBITORS
OF PROTEIN TYROSINE KINASES
by
Pierre Gilles Charrier
B.A., University Paris VI, 1988
Ingenieur ESCOM Paris, 1990
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado in partial fulfillment
of the requirements for the degree of
Master of Sciences
Department of Chemistry
1991
c
8
i
i
\


This thesis for the Master of Science degree by
Pierre Gilles Charrier
has been approved for the
Department of
Chemistry
by


Charrier, Pierre Gilles (M.S., Chemistry)
Synthesis of Potential Affinity Labelling Inhibitors of Protein
Tyrosine Kinases
Thesis directed by Professor Douglas Dyckes
The synthesis of chloromethylketone (CMK) peptides was
investigated in order to find new affinity labelling inhibitors of
the pp60v*src protein tyrosine kinase. The side chain of the
glutamic acid residue in an already known hexapeptide substrate
of the pp60vsrc (ELPYAG) was chosen as the site for the
incorporation of the affinity labelling group. The synthetic route
chosen was to convert the carbonyl function on side chain of the
r glutamic acid residue to a diazomethylketone (DAMK) group. This
in turn could be converted to a CMK. Procedures for the synthesis
and analysis of DAMK groups on amino acids and peptides were
optimized and the stability of the group was studied. Two
different strategies were applied: (1) the incorporation of the
DAMK glutamic acid derivative into peptides via its para-
nitrophenyl ester and (2) the derivatization of the free side
chain carboxylic function of the glutamic acid residue in ELPYAG.
Although model studies on amino acids and peptides were
successful, no hexapeptide containing the DAMK function could be
isolated. The attempted direct conversion of ELPYAG to a CMK
was also apparently unsuccessful.
in


To my parents


Acknowledgements
I wish to express my sincere thanks to Dr. Douglas F. Dyckes
for his help, constant guidance and understanding throughout this
project.
I would like also to thank Dr. John McMurry, Dr. Nihal
Obeyesekere, Dr. Joseph Kaczmarek and Dr. Joseph M. Conny for
their frienship and support during the accomplishment of this
thesis.
My special thanks go to Mike Milash for his sense of humor
and his effective cooperation.
Finally, I would like to thank all those who contributed in any
way by their moral support, assistance and patience, especially
my family, Ute and my friends.


CONTENTS
Tables................................................... x
Figures.................................................. x i
List of abbreviations.................................... x i i
CHAPTER
1. INTRODUCTION............................................. 1
A. Goals................................................. 1
B. Protein tyrosine kinases.................................. 1
C. Affinity labelling........................................ 3
D. Strategies................................................ 4
E Peptide synthesis.......................................... 6
1. Solid phase peptide synthesis....................... 6
2. Synthesis on a polyacrylamide support................ 7
3. Coupling method...................................... 8
2. EXPERIMENTAL............................................. 10
A. Material and reagents................................. 1 0
1. Distillation of solvents and reagents............ 1 0
2. Recrystallization of reagents.................... 11
3. Protected amino acids............................ 1 2


B. General method..................................... 1 2
1. Continuous flow solid phase peptide synthesis.... 12
2. Functionalization of the resin................. 1 4
3. Formation of glycine symmetric anhydride...... 1 6
4. Activation of amino acid via the BOP/HOBt
method......................................... 1 7
5. Kaiser and chlorinine tests.................... 1 7
6. Deprotection of amino acids and peptides....... 1 8
7. Diazomethane generation........................ 1 8
8. Synthesis and analysis of diazomethylketone
compounds...................................... 1 9
9. Thin layer chromatography...................... 2 0
10. Chromatographic purifications of amino acids
and peptides................................... 21
11. Amino acid analysis............................ 2 2
12. Mass spectrometry............................... 24
C. Synthetic procedures............................... 24
1. Diazomethylketone synthesis on protected
amino acids.................................... 2 4
1.1 Synthesis of Boc-L-Ala-CHN2................. 24
1.2 Analysis of the stability of Boc-L-Ala-CHN2 25
1.3 Synthesis of Fmoc-L-Ala-CHN2............... 2 6
1.4 Synthesis of Fmoc-L-Glu(CHN2)-OtBu...........2 6
1.5 Synthesis of Boc-L-Glu(CHN2)-OBzl............2 7
1.6 Synthesis of Fmoc-L-Glu(CHN2)-ONp.......... 2 7
1.6a Synthesis of Fmoc-L-Glu(OH)-ONp....... 2 7
1.6b Synthesis of Fmoc-L-Glu(CHN2)-ONp..... 2 8
vii


1.7 Synthesis of Boc-L-Glu(CHN2)-ONp..........2 9
1.7a Synthesis of Boc-L-Glu(OH)-ONp.......2 9
1.7b Synthesis of Boc-L-Glu(CHN2)-ONp..... 29
2. Diazomethylketone synthesis on protected
dipeptides.................................... 30
2.1 Synthesis of Fmoc-Glu(OBz!)-Ala-OMe........30
2.2 Debenzylation of Fmoc-Glu(OBzl)-Ala-OMe..31
2.3 Synthesis of Fmoc-Glu(CHN2)-A!a-OMe.......31
2.4 Synthesis of Fmoc-Glu(CHN2)-Ala-OMe
by coupling reaction......................3 2
2.5 Synthesis of Boc-Glu(OBzl)-Ala-OMe....... 3 2
2.6 Synthesis of Boc-G!u(OH)-A!a-OMe........ 3 2
2.7 Synthesis of Boc-Glu(CHN2)-Ala-OMe...... 3 3
D. Syntheses of hexapeptides........................ 34
1. Synthesis of
Fmoc-E(OBzl)-L-P-Y(tBu)-A-G-OH (A)............ 34
2. Synthesis of
Fmoc-E(OBzl)-L-P-Y(tBu)-A-G-OCH3.............. 3 5
3. Synthesis of
Fmoc-E(OH)-L-P-Y(tBu)-A-G-OCH3................ 3 6
4. Attempted synthesis of
E(CH2CI)-L-P-Y-A-G-OCH3....................... 3 6
5. Syntheses of
Fmoc-E(CHN2)-L-P-Y(tBu)-A-G-0-HAHL-l-
Resin(B)...................................... 3 7
6. Attempted cleavages of
E(CHN2)-L-P-Y(tBu)-A-G from the resin......... 37
vm


3. RESULTS AND DISCUSSION.................................... 40
A. Synthesis and identification of the DAMK group......... 40
B. Stability of the DAMK group............................ 4 3
C. Peptide assembly and cleavage........................... 44
1. Peptide A.......................................... 4 4
2. Peptide B........................................... 45
D. Derivatization of peptide A............................ 5 0
E Conclusion............................................. 54
F. Suggestion for further studies...........................56
LIST OF REFERENCES............................................. 5 8
ix


TABLES
Table
2.1 Sequence of acylation, washing and deprotection used
in contonuous solid phase peptide synthesis........... 1 5
2.2 Yields of protonated hexapeptides synthesis...........3 5


FIGURES
Figure
2.1 Continuous flow solid phase peptide synthesis set-up... 1 3
3.1 IR spectrum of Fmoc-Glu(CHN2)-OtBu.................. 41
3.2 NMR spectrum at 300 MHZ of Fmoc-Glu(CHN2)-OtBu...... 42
3.3 HPLC profile at 266 nm of
Fmoc-E(OBzl)-L-P-Y(tBu)-A-G-OH...................... 4 6
3.4 Mass spectrum of Fmoc-E(OBzl)-P-Y(tBu)-A-G-OH....... 47
3.5 NMR spectrum at 300 MHZ of
Fmoc-E(OBzl)-L-P-Y(tBu)-A-G-OCH3.................... 51
3.6 NMR spectrum at 300 MHZ of
Fmoc-E(OH)-L-P-Y(tBu)-A-G-OCH3...................... 5 2
3.7 Mass spectrum of Fmoc-E(OH)-L-P-Y(tBu)-A-G-OCH3.... 53
xi


List of Abbreviations
CMK
DAMK
PTK
RVS
AAA
HALH
HPLC
IR
LC/MS
MS
NMR
Rf
TLC
UV
General terms
chloromethylketone
diazomethylketone
protein tyrosine kinases
rous sarcoma virus
amino acid analysis
hyper-acid-labile-handle
high performance liquid
chromatography
infrared
liquid chromatography/mass
spectrometry
mass spectrometry
nuclear magnetic resonance
retention factor
thin layer chromatography
ultraviolet


Solvents and reagents
AcOH acetic acid
BOP benzotriazol-1 -yl-oxy-tris- (dimethyl amino)phosphonium hexafluorophosphate
DCC dicyclocarbodiimide
DCM dichloromethane
DIPCIDI diisopropylcarbodiimide
DMAP dimethylaminopyridine
DMF dimethylformamide
DMSO dimethylsulfoxide
EtOAc ethylacetate
HOBt 1-hydroxybenzotriazole
HONp para-nitrophenol
MeOH methanol
NMM N-methy I morpholine
pip piperidine
TFA trifluoroacetic acid
THF tetrahydrofuran
Xlll


Amino Acids
Name Abbreviations Structure
Alanine NH, Ala A i CH3-CH-COOH
Glutamic acid NH, Glu E l HOOC-CH2-CH2-CH-COOH
Isoleucine nh2 lie 1 1 (CH3)2-CH-CH2-CH-COOH
Leucine nh2 Leu L | CH3-CH2-CH(CH3)-CH-COOH
Proline r, CH2 NH Pro P / j ch2-ch2-ch-cooh
Tyrosine NH2 Tyr Y 1 HO-C6H4-CH2-CH-COOH
XIV


Protecting groups
Name
Abbreviations
Structure
t-Butyloxycarbonyl Boc
CH3
1
CH3-C-O-CO-
1
CH3
9-Fluorenylmethyl-
oxycarbonyl
Fmoc
CH2-0-C0-
H
t-Butyl
tBu
CH3
CH3-C-
CH3
Benzyl
Bzl
WCHa
para-nitrophenyl
ONp
CfeN'
/
o-
xv


CHAPTER 1
INTRODUCTION
A. Goals
The research described involves the synthesis of peptides
designed to act as inhibitors for the tyrosine kinase family of
enzymes. Tyrosine kinase activity has been implicated in some
forms of sarcoma (including those induced by the Rous sarcoma
virus). The peptides will be used to test our ideas about kinase
structure/activity relationships, and the development of kinase
inhibitors.
B. Protein Tvrosine Kinases
Protein Tyrosine Kinases (PTK) are enzymes which catalyze
the phosphorylation of proteins in tyrosine residues.1 All have a
catalytic domain capable of transfering phosphate from ATP to
proteins.2 The last ten years have seen a burgeoning of systems
in which tyrosine phosphorylation has been implicated, including
viral transformation and growth control. The first PTK activities


to be detected were associated with viral transforming proteins,
particularly those of acutely oncogenic retroviruses.3 The second
type of PTK activity was detected in association with growth
factor receptors.
In this study, we are interested in retroviral PTK and more
specifically in the pp60v-src. Rous Sarcoma Virus (RSV)
transforms chicken cells to a malignant state through the
expression of a single gene: src 4'8 This transforming gene
encodes a single 60,000 dalton phosphoprotein pp60v'src, which
appears to be the modification of the cellular polypeptide
pp60c-src.9-io Both pp60v'src and pp60c-src are PTK with the
ability to phosphorylate tyrosine in a variety of protein
substrates.11'14 All cells transformed by the RSV contain levels
of phosphotyrosine in protein which are 6-10 fold greater than
the very low levels present in uninfected cells.15 Previous work
has indicated that the pp60v'src and the pp60c'src activity is
located in the carboxy-terminal half of the protein.16'17 It
appears that the activity of pp60c'src is itself regulated by a
phosphorylation process.18'19 Like an "on/off" switch button, the
activity of the protein is increased by a phosphorylation on its
Tyr416 residue and decreased by another phosphorylation on its
Tyr527 residue.20'22 The sequence of the pp60v'src is different
from the one of the pp60c'src in various points. The main
difference is located in the carboxyl terminal of the
protein.23'25 The region of pp60c'src from residue 515 to the
2


COOH terminus, including Tyr527, has been deleted with a
different sequence in pp60v-src. Once the activity of the pp60v_
src has been stimulated, it cannot be diminished. Thus, the
increase in transforming ability, and kinase activity that occurs
in the genesis of pp60v'src most likely results from the loss of
the tyrosine involved in negative regulation.
C. Affinity labelling
A very large number of important biological processes involve
the interaction of a substrate (ligand) with a biological
macromolecule (the receptor). For instance, enzymes catalyze
reactions of their substrates and cofactors. The understanding of
how receptors and ligands interact at the molecular level is
essential and can be in part resolved using an affinity labelling
method.26 In this technique, one presents the receptor with an
analog of its natural ligand into which is incorporated a
chemically reactive group. The chemical approach to receptor
site labelling aims to simulate the natural situation:
receptor + ligand w [receptor-ligand]
with a reagent that structurally mimics the natural ligand and
preserves the specificity of the interaction. Then the receptor
binding site may thus be specifically tagged.
3


In our case, the src PTK plays the role of a receptor for
unknown substrates in order to phosphorylate the tyrosine
included in those substrates. The sequence of the src enzyme is
well known. However, the three-dimensional structure of the
protein is still unknown. Previous work has shown that small
peptides were able to be used as substrates for the src enzyme.
Following the principle of affinity labelling, a reactive chemical
group attached to the ligand peptide might form an irreversible
covalent bond with the neighboring groups of the receptor sites.
This could provide some very important information on the
conformation of the enzyme, on the structure/activity
relationships and for the development of kinase inhibitors.
D.Strategies
The peptide Glu-Leu-Pro-Tyr-Ala-Gly (ELPYAG) has been
previously synthesized27 and proved to act as a fair substrate
for the pp 60v_src, being phosphorylated on its tyrosine residue.
Based on the idea of affinity labelling, it was hoped that
incorporation of a functional group into the peptide could provide
some information concerning its interaction with the enzyme.
Other groups in the project are working on affinity labelling
associated with the tyrosine residue. For this research, the
glutamic acid was selected because its presence at the position
4


relative to the tyrosine which it occupies in ELPYAG, has been
shown to enhance phosphorylation, indicating that the glutamic
acid interacts with the enzyme. The group which was chosen as
chemically reactive group, because of the side chain carboxylic
function of the glutamic acid, was the chloromethylketone
(CMK).28'29 The protein pp60v'src might have a nucleophilic site
close to its phosphorylation site which could displace the
chlorine of the CMK and form a covalent bond with the peptide
substrate. As shown in equation (1), the CMK can be synthesized
from the diazomethylketone (DAMK) group by treatment with HCI
o o
II HCI II
Equation 1 R~CCHN2 --------- RCCH2CI + N2
Two main strategies can be applied in order to form the DAMK
on the peptide. The first one (equation 2) consists of the
Equation 2
/
II
ch2-ch2-c-o-h
R1-NH-CH
\

O
C-O-R'
/ 1. Activation of COOH
L--------------------
2. Diazomethane
R1 : N-terminus protecting group
R2 : COOH-terminus protecting group
O
ii
^,ch2-ch2- c- chn2
R1-N H-CH
^C-O-R2
v
0
I
conversion of the side chain of the otherwise fully protected
glutamic acid.
5


The incorporation of amino acid I into the target peptide
would be achieved by the deprotection of the chain carboxylic
function and its activation. The choice of protecting groups
would be dependent of the stability of the DAMK, especially to
basic and acidic condition. Thus, a pilot study was first done on
the DAMK of the simple amino acid alanine to determine its
stability.
The second strategy consists of the synthesis of the ELPYAG
peptide protected on the side chain of the glutamic acid residue
with a selectively removal group. Following deprotection, the
glutamic acid side chain could be converted into the DAMK. The
remaining protecting groups would be selected to be removed
under acidic conditions during the conversion by HCI of DAMK into
CMK (equation 1).
E. Peptide synthesis 1
1. Solid phase peptide synthesis
The principle of the solid phase peptide synthesis is the
assembly of a peptide chain by the stepwise addition of each
amino acid.30"32 In this technique, the target peptide is
elaborated from the C-terminal which has been anchored at its
carboxy-terminus to an insoluble polymeric resin support. The
6


elongation of the growing peptide chain is achieved by the
alternation of two steps. The first one consists of the coupling
reaction. The N-a-protected amino acid being added is activated
on its carboxylic function and reacts with the free amino group
of the residue previously added, to form the amide bond. The next
step consists of the deprotection on the N-a-amino function of
the amino acid just added.
2. Synthesis on a polyacrylamide support
In our studies, the peptide synthesis was carried out using a
continuous flow solid phase synthesis method3334 on a
polyacrylamide Kieselguhr resin. The addition of reactants and
the removal of excess reactants, unlike the traditional batch
method, is carried out in a continuous flow mode, using
procedures described by Atherton et al.3536 The resin consists
of a crosslinked polymer of dimethylacrylamide containing a
functionalizing agent, acryloylsarcosine methyl ester. The
polymer is held within the macropores of an inert, rigid skeleton
made of Kieselguhr, which prevents the resin from packing during
elution.
At the beginning of the synthesis, the resin is reacted with an
excess of ethylene diamine. This converts the acryloylsarcosine
methyl ester functions into amides and provides free primary
amino groups for further derivatization. The resin is then
7


modified by addition of an internal marker amino acid, followed
by a linking agent.37-38 The marker used was isoleucine but any
other amino acid not contained in the sequence of the target
peptide can be used. This reference amino acid remains on the
resin after cleavage of the peptide, allowing quantitation of the
level of resin loading and the efficacy of the cleavage reaction.
The linkage agent provides the means for attaching the first
amino acid residue. The hyper-acid-labile handle39, 3-methoxy-
4-hydroxymethyl phenoxyacetic acid was used as linker for all
the peptides syntheses described. An ester bond is created
between the carboxylic function of the amino acid and the
benzylic hydroxyl group on the linker. This allows the cleavage of
the target peptide under milder conditions than those required
for the cleavage of an amide bond.
The N-a-amino functions of the amino acids being added were
protected with the 9-fluorenylmethyloxycarbonyl (Fmoc)
group.40 They were deprotected under basic conditions by the
secondary amine, piperidine.
3. Coupling method
To couple amino acids, the carboxylic acid function has to be
preactivated.41 Castro et al 42 introduced a new combination of
reagents for the formation of the amide bond: benzotriazol-1-yl-
oxy-tris-(dimethylamino)phosphonium hexafluorophosphate (BOP)
8


and 1-hydroxybenzotriazole (HOBt). The BOP attacks the free
carboxyl function under basic conditions to give the
acyloxyphosphonium salt. This intermediate can react either
with HOBt to form the benzotriazolyl active ester or with
another free carboxyl group of amino acid or peptide to give the
symmetric anhydride. The active ester or the symmetric
anhydride then reacts with the free amino residue to form the
amide bond. The addition of the new amino acid or peptide on the
peptide chain is thus achieved.
9


CHAPTER 2
EXPERIMENTAL
A. Material and reagents
1. Distillation of solvents and reagents
Boiling points indicated in this section are adjusted to the
pressure of 760 Torr (Temperature/Pressure nomograph).
Reagent grade N.N'-Dimethylformamide (DMF) (Aldrich) was
purified immediately prior to use in peptide synthesis by
fractional distillation under reduced pressure. It was collected
at a boiling point of c.a. 40C.
Dichloromethane (DCM) (Aldrich, reagent grade) was distilled
from potassium carbonate through a 12-inch fractionating
column
containing glass helices. Distillate was collected at a boiling
point of 41 C.
Ethylene diamine (Aldrich, 99%) was refluxed with sodium


metal and then distilled under nitrogen discarding the initial 5%
of the distillate. It was collected at a boiling point of 118C.
Trifluoroacetic acid (TFA) (Advanced ChemTech) was purified
by simple distillation. The initial 10% was rejected and the
distillate was collected at 71 C.
Piperidine (pip) (Aldrich, 99%) was distilled from KOH pellets
in the presence of a nitrogen bleed. The initial distillate (5%)
was discarded and the main fraction collected at a boiling point
of 106C.
N-Methylmorpholine (NMM) (Aldrich, 99%) was refluxed over
ninhydrin and then distilled and collected at a boiling point of
114C.
Isobutylchloroformate (Fluka, 95%) was purified by simple
distillation. The initial 5% was discarded and the distillate
collected at 128C.
Tetrahydrofuran (THF) (Burdick and Jackson Laboratories Inc.)
was filtered through alumina and fractionally distilled. It was
collected at a boiling point of 67C.
2. Recrvstallization of reagents
1-Hydroxybenzotriazole (HOBt) (Advanced ChemTech) was
recrystallized from hot/cold water and contained water of
recrystallization, m.p.: 155-157C (lit m.p.: 155-158C).43


4-Dimethylaminopyridine was recrystallized from diethyl
using activated carbon, m.p.: 109-111C (lit m.p.: 114C).44
4-Nitrophenol (HONp) (Aldrich) was recrystallized from
chloroform, m.p.: 112C (lit m.p.: 113-114C).
3. Protected amino acids
Protected amino acids were purchased from either Bachem
Bioscience or Fluka Chemika/Biochemika (95% purity).
B. General method.
1. Continuous flow solid phase peptide synthesis
Continuous flow solid phase peptide synthesis on
polyacrylamide resin was carried out using the set-up
illustrated in Figure 2.1. The resin was loaded in a 9mm x
250mm column (Rainin Instruments Co.). Solvents and reagents
were pumped through the column by a fluid pump (Fluid Metering
Inc., Model QG6) at an average rate of 50 mL/hour. The apparatus
was fitted with Teflon tubing (0.8mm ID x 1.5mm OD). DMF, 20%
piperidine in DMF, DCM, and 1% TFA in DCM were connected to the
pump through a 6-position Teflon rotary valve (Rainin) which
allowed selection of one of the solvents. Another valve (3-way
Teflon rotary valve; Rainin) allowed selection of a recirculation


solvents
Figure 2.1: Continuous flow solid
phase peptide synthesis set-up


mode for acylation reactions or a washing mode for N-a-Fmoc
cleavage59 and removal of excess reagents and by-products
between acylation steps. The sequence of acylation, washing and
Fmoc cleavage has been outlined in Table 2.1. The synthesis was
monitored by UV using a type IO Isco Detector at a wavelength of
280 nm.
2. Functionalization of the resin
Polyamide-Kieselguhr resin (1g, 0.107 mmol of active site,
Peninsula Laboratories) was suspended in DMF (20 mL), settled
and decanted to removed the fines. The procedure was repeated
several times. The initial step in the functionalization of the
resin was the agitation overnight at room temperature with
freshly distilled ethylene diamine (15 mL), which provided a free
amine group on the resin. It was then transfered to a sintered
funnel (coarse) and the ethylene diamine was filtered. The resin
was washed with DMF (15 mL) at least 10 times and transfered
to a 25 mL round bottom flask. A three-fold excess (0.3 mmol) of
N-a-Fmoc-lsoIeucine symmetric anhydride, prepared by the
BOP/HOBt method (Experimental, Section B(4)), was added to the
resin and allowed to react for 30 minutes. The Kaiser test
(Experimental, Section B(5)) confirmed the completion of the
acylation.
14


Table 2.1 Sequence of acylation, washing and deprotection used
in continuous flow solid, phase peptide synthesis.
Operation Solvent Time (min.) Mode
acylation activated amino acid in DMF 30 recirculation
removal of unreacted material DMF 10 washing
removal of unreacted material trapped in the recirculation loop activated amino acid in DMF 0.5 recirculation
removal of the unreacted material DMF 10 washing
deprotection 20% pip. in DMF 10 washing
removal of the piperidine solution DMF 20 washing
15


Unreacted reagents and by-products were removed by
washing the resin in a sintered funnel (medium) with DMF (30
mL). The protecting group was cleaved by washing with a
piperidine/DMF (20/80; v:v) solution for 10 minutes. The resin
was once again washed with DMF (4x15 mL) and transfered to a
25 mL round bottom flask. The linkage agent, 4-hydroxymethyl-
3-methoxy phenoxyacetic acid (Bachem) (0.2 mmol, 2 eq) was
next added via the BOP/HOBt method. After one hour, the Kaiser
test indicated the completion of the reaction. The resin was
poured onto a sintered funnel and washed with DMF (3 x 15 mL).
The ester bond between the linker and the first amino acid of the
target peptide (glycine) was formed via the symmetric anhydride
method (Experimental, Section B(3)). The resin was transfered to
a 25 mL round bottom flask. N-a-Fmoc-Glycine symmetric
anhydride (0.4 mmol, 4 eq) was added to the resin followed
immediately by the addition of DMAP (0.1 mmol, 1 eq) and shaken
for 30 minutes. This esterification reaction was repeated twice.
Unreacted reagents were removed by washing the resin in a
sintered funnel (medium) with DMF (30 mL). The resin was ready
for peptide synthesis and transfered to the column.
3. Formation of glvcine symmetric anhydrides
The method suggested by Dr. John McMurry was followed for
the formation of the symmetric anhydrides. An eight fold excess


of Fmoc amino acid was dissolved in 10 mL of dichloromethane
and a minimum amount of DMF was added to allow complete
dissolution (if necessary). DIPCDI (0.4 mmmol) (Aldrich) was
added and the solution was stirred for 15 minutes. The CH2CI2
was evaporated in vacuo, the anhydride was dissolved in DMF
(4 mL) and was added directly to the resin.
4. Activation of amino acid via the BQP/HQBt method
Typically for 1g of resin (0.1 mmol of binding sites), the
amino acid (0.3 mmol, 3 eq), HOBt (0.3 mmol, 3 eq) and BOP (0.3
mmol, 3 eq) were dissolved in DMF (2mL). The NMM (50 |iL, 0.45
mmol, 4.5 eq) was added to the solution. The activation of the
amino acid was allowed to proceed for 10 minutes. The activated
amino acid was then added to the free amino resin.
5. Kaiser and chlorinine tests
The Kaiser45 and chlorinine tests gave fast color reactions
with free amines and were used to monitor the end of the
acylation step in the peptide synthesis. A sample of resin beads
was removed from the column at the end of each addition step
and washed with DMF. After drying by suction, the resin beads
were tested as follows:
In the Kaiser test, a few resin beads were placed into a small
tube and 2 drops of each of the following solutions were added:
17


1) 0.5g of ninhydrin in 10 mL of ethanol; 2) 80g of phenol in 20
mL of ethanol; 3) 0.001M aqueous potassium cyanide in pyridine
(1:49, v:v). The tube was heated at 100C for 5 minutes. The
presence of free amine produced deep blue-violet beads.
The chlorinine test was used to test the acylation of the
amino acid following the proline in the target peptide. Proline
has a secondary amine and does not react very well in the Kaiser
test. The resin beads were reacted with 2 drops of acetone and 5
drops of a saturated solution of tetrachloro-1-4 benzoquinone at
room temperature for 5 minutes. As in the Kaiser test, the
presence of free amine produced dark blue beads.
6. Deprotection of amino acids and peptides
The base labile group Fmoc, was used as the N-a-protecting
group on each amino acid. After the completion of the acylation
step, it was cleaved by washing the resin with 20% piperidine in
DMF (v/v) for 10 minutes.
7. Diazomethane generation
Diazomethane was generated using the Aldrich Diazald kit. A
sample of potassium hydroxyde (2.12g) was dissolved in 3.5 mL
of water and 4.25 mL of ethanol and was poured into a 100 mL
round bottom flask. The Diazald (N-methyl-N-nitroso-p-
toluenesulfonamide) (2.12g, 9.92 mmol) in 19 mL of ether was
18


poured into the separatory funnel. Ether was placed in the 250
mL collecting flask (5 mL) and in the 125 mL Erlenmeyer (10 mL)
(Note: the tube must pass beneath the surface of the ether in the
Erlenmeyer). The Diazald set-up was placed in a hood behind a
safety shield. As the water was heated to 65C, the solution was
stirred by means of a teflon magnetic stirrer. The Diazald
solution was added dropwise at a rate of addition which
approximated the rate of distillation. When all the Diazald was
used up, 10 mL of ether were added and the distillation was
continued until the distillate became colorless. According to the
literature46'48, the yield of reaction is 65-70% which would
correspond to the generation of about 6.9 mmol of diazomethane.
8. Synthesis and analysis of diazomethvlketone compounds49'53
The amino acid with a free carboxylic function was dissolved
in the minimum amount of THF and cooled to 5C. It was
activated by addition of N-methylmorpholine and
isobutylchloroformate (3 fold excess). The mixture was allowed
to stir in a dry ice-acetone bath (-15C) and the formation of the
anhydride was followed by TLC study in system A (see following
section). If traces of starting material were still visible on TLC,
they were removed by adding another equivalent of NMM and
isobutylchloroformate. The diazomethane solution (freshly
prepared) (2 eq) was added and the reaction was allowed to stir


for 30 minutes at 0C and at room temperature for 1-2 hours
until completion of the reaction (the completion of the reaction
is easily detectable on TLC by the fact that the spots of the final
product and by-products are clearly defined under UV light,
which was not the case during the reaction). Diazomethylketone
derivatives were usually identified by TLC in solvent systems B
or C. Purification by flash chromatography was achieved with the
same systems. The yields, depending on the amino acids, were
between 30 and 40%. The diazomethylketone group was identified
by IR with its characteristic band at 2100 cm-1 and by NMR with
a peak in the 5.1-5.3 ppm region.
9. Thin layer chromatography
Thin layer chromatography (TLC) was carried out using pre-
coated silica plate with a 0.2 mm layer of silica gel 60 (F254)
(EM Science. 5735). Samples for analysis were spotted with 1 piL
pipets and the chromatograms developed in one of the following
solvent system:
- system A: chloroform/methanol (90/10)
- system B: ethylacetate/hexane (60/40)
- system C: neat ethylacetate
- system D: ethylacetate/hexane (40/60)
- system E: ethylacetate/hexane (50/50)
- system F: ethylacetate/methanol (70/30)
20


- system G: ethylacetate/methanol (80/20)
- system H: ethylacetate/methanol (90/10)
The identification of the compounds was determined by
exposure of the plate to 254 nm light or by spraying the plate
with 1% ninhydrin in acetone and heating to 100C for five
minutes.
10. Chromatographic purifications of amino acids and peptides
The three following chromatographic techniques were used
for purification of amino acids and peptides: flash
chromatography, high performance liquid chromatography (HPLC)
and gel filtration.
In flash chromatography54, a column (either 4 x 15 cm or
1x15 cm) of silica gel (Merck, grade 60, 230-400 mesh) was
filled with the appropriate solvent mixture. The amino acid or
peptide was applied on the top of the adsorbent bed. The crude
product was then eluted under pressure at a flow rate of 60
mL/min. Fractions were collected by hand in 20 x 50 mm test
tubes. Separated components were detected by spotting every
other fraction on a TLC plate. Fractions containing the purified
product were collected and the solvent was evaporated in vacuo.
The HPLC was carried out using two LDC constametric pumps
controlled by an LDC gradient master. Solvents were pumped
through a mixer, a rheodyne injector and into the column. The
21


peptides were detected by an LDC variable wavelength
spectrophotometer set at either 266 or 230 hm. The
chromatographic columns were C18 RP 318 Bio Rad or Microsorb
Rainin and C4 RP 304 Bio Rad. The solvent system used was: 1%
TFA/H2O (solvent A) and 1% TFA/CH3CN (solvent B) with a
gradient of 0-100% solvent B over one hour.
Gel filtration was performed on gel columns of Sephadex G10
(Pharmacia Fine chemicals). The peptide was dissolved in 1-2 mL
of 1% HOAc, applied to the column and eluted with 1% HOAc
pumped at a flow rate of 30 mL/hr. Fractions wei ollected on a
fraction collector ISCO Foxy and the absorbance he eluate
was monitored at a wavelength of 280 nm using,a type 10 ISCO
detector. The appropriate fractions were combined and freeze
dried.
11. Amino acid analysis
Amino acid analysis (AAA) was used to obtain qualitative and
quantitative information at the end of peptide synthesis before
and after cleavage of peptide from the resin. Samples for
analysis were removed from the resin bed and washed with DMF
and ether. They were then dried in vacuo and 2-3 mg were
weighed accurately for hydrolysis. The samples were hydrolysed
in 6N HCL (1 mL) with a small amount of phenol added to it, by
heating at 110-120C for 18 hours, in a sealed tube under
22


vacuum. At the end of the hydrolysis, the acid was removed in
vacuo. Two different techniques were used for the AAA. The first
one consists of the use of ion-exchange separation followed by
ninhydrin derivatization for detection and quantitation. Each
sample was dissolved in a known amount of 0.2 N sodium citrate
and the analysis was done on a Beckman Gold System by Dr.
James Sparrow of Baylor College of Medicine (Houston). The
second method is based on a derivatization of each amino acid
followed by a liquid chromatographic separation. The pre-column
derivatization with the 4-dimethylaminoazo-benzene-4'-
sulfonylchloride (Dabsyl chloride)55 was used. Each sample was
dissolved in 2 mL of bicarbonate solution pH 8.9. This solution
was evaporated and the dry residue was re-dissolved in 1 mL
bicarbonate solution and 1 mL of Dabsyl chloride solution. It was
allowed to react for 15 minutes at 80C. The orange colored
solution was then evaporated under a nitrogen stream and the
residue was dissolved in 2 mL 70% (v/v) aqueous ethanol.
Derivatized amino acids were analyzed by HPLC. The solvents
used were 4% DMF/NaPC>4 (pH adjusted at 6.5) (solvent A) and 4%
DMF/CH3CN (solvent B). The gradients used were 8-30% solvent B
over 40 minutes, then 30-60% solvent B over 40 minutes and
finally 60% solvent B for 5 minutes.
23


12. Mass spectrometry
Mass spectral analyses were performed by Dr. J. Kaczmarek
and Dr. J. Conny in a thermospray/electrospray LC/MS (Vestec
Corporation) according to the methods of Yamashita et al.56
C. Synthetic procedures
1. Diazomethvlketone synthesis on protected amino acids
1.1 Synthesis of Boc-L-Ala-CHNo.
Boc-L-Ala-CHN2 was synthesized according to the procedure
described by Coggins et al.28 A sample of Boc-L-Ala (0.5g, 2.65
mmol) was dissolved in THF (2.5 mL) and cooled to -5C. The
mixed anhydride was formed by the addition of triethylamine
(0.37 mL, 2.65 mmol) and isobutylchloroformate (0.306 mL, 2.65
mmol). The mixture was allowed to stir for 9 minutes at 15C.
To the mixed anhydride was added a 2-fold excess of fresh
diazomethane solution (synthezised according to the procedure
described in Experimental, Section B(7)). The reaction was
stirred for 30 minutes at 0C and for 1 hour at room
temperature. The solution was washed with 0.1 M acetic acid
(HOAc) (20 mL), saturated NaHC03 (25 mL), water (25 mL) and
then dried over MgS04. The solvent was evaporated in vacuo. The
residue which remained was triturated with benzene and hexane.
The resulting solution was evaporated yielding a yellow solid
24


weighing 0.51 g (90%). The crude product was purified by flash
chromatography on silica gel eluted in system D. Yield: 0.25g
(45%); homogeneous by TLC, Rf = 0.33 (system D); m.p.: 82C (lit
m.p.:78-81C).57 1H NMR (200 MHZ, CDCI3) 3 (ppm) 1.28 (d, 3H, B
CH3), 1.4 (s, 9H, Boc H), 4.19 (m, 1H, a CH), 5.25 (s, 1H, CHN2),
5.45 (s, 1H, NH).
1.2 Analysis of the stability of the Boc-Ala-CHNb.
The stability of the DAMK group in basic and acidic media was
determined. A sample of Boc-Ala-CHN2 was dissolved in a
solution of pip/DMF (20/80, v/v) and analyzed during a 90
minutes period by reverse phase chromatography (column C18,
gradient 20 to 30 % of acetonitrile in water over 15 minutes)
with an injection of 20 pL of the solution every 15 minutes. The
retention time of the DAMK was 5.9 minutes and stayed
unchanged in the chromatogram of each injection. Furthermore,
after this treatment the DAMK peak was still visible in NMR at
5.45 ppm. The same chromatographic analysis was performed
with a sample of Boc-Ala-CHN2 dissolved in 1% TFA CH2CI2
solution. Neither the first injection nor any of those following
showed any peak corresponding to DAMK. As a matter of fact, as
soon as the yellow solid was dissolved in the acidic medium, the
solution turned colorless (compared to yellow in the basic
medium) and bubbles of gas appeared.
25


1.3 Synthesis of Fmoc-L-Ala-CHN^.
The general procedure for diazomethylketone synthesis
described in Experimental Section B(8) was used. Fmoc-Ala (1 g,
3.2 mmol) was converted to Fmoc-Ala-CHN2. The diazomethyl-
ketone derivative was purified by flash chromatography on silica
gel eluted in system E. Yield: 0.43g (40%) of a yellow residue;
homogeneous by TLC, Rf = 0.41 (system E); m.p.: 108-110C. 1H
NMR (300MHZ, CDCI3) d (ppm) 1.35 (d, 3H, B CH3), 4.2-4.6 (m, 4H,
Fmoc CH2 and CH, a CH), 5.35 (s, 1H, CHN2), 5.6 (d, 1H, NH), 7.25-
7.45 (m, 4H, Fmoc H2(3,6,7), 7.6 (d,2H, Fmoc H4>5), 7.8 (d, 2H,
Fmoc Hi,8).
1.4 Synthesis of Fmoc-L-GlufCHN^-QtBu.
A sample of Fmoc-L-Glu(OH)OtBu (1 g, 2.35mmol) was
converted to the Fmoc-Glu(CHN2)OtBu using the procedure
described in Experimental Section B(8). The crude compound was
then purified by flash chromatography on silica gel eluted with
system E. Yield: 0.23g (21.7%) of an oily residue; homogeneous on
TLC, Rf = 0.3 (system E); 1H NMR (300 MHZ, CDCI3) d (ppm) 1.45
(s, 9H, Boc H), 1.95 (m, 1H, B CH), 2.2 (m, 1H, B CH), 2.4 (m, 2H, y
CH2), 4.25 (m, 2H, a CH and Fmoc CH), 4.38 (d, 2H, Fmoc CH2),
5.25 (s, 1H, CHN2), 5.5 (d, 1H, NH), 7.25-7.45 (m, 8H, Fmoc
H2,3,6,7), 7.6 (d, 2H, Fmoc H4,5), 7.8 (d, 2H, Fmoc H1j8).
26


1.5 Synthesis of Boc-L-Glu(CHN£)-OBzl.
The general procedure for diazomethylketone synthesis
described in Experimental Section B(8) was used. Boc-L-
Glu(CHN2)-OBzl was synthesized from Boc-L-Glu(OH)-OBzl (1 g,
3 mmol). The diazomethylketone derivative was then purified by
flash chromatography on silica gel eluted with solvent system D.
Yield: 0.39g (35%) of a yellow residue; homogeneous by TLC, Rf =
0.55 (system D) m.p.: 62C 1H NMR (300 MHZ, CDCI3) 3 (ppm) 1.4
(s, 9H, Boc H), 1.95 (m, 1H, B CH), 2.18 (m,1H, B CH), 2.3 (m, 2H, y
CH2), 4.3 (m, 1H, a CH), 5.1-5.3 (m, 4H, Bzl CH2, CHN2 and NH),
7.35 (s, 5H, Bzl Ph).
1.6 Synthesis of Fmoc-L-GlufCHNgl-QNp.
1.6a Synthesis of Fmoc-L-Glu(OH)-ONp
The procedure described by Bodanszky et al.58 was followed in
the esterification of the y -t-Butyl protected glutamic acid by
the p-nitrophenol. Fmoc-L-Glu(Ot.Bu)-OH (3g, 7.05 mmol) was
dissolved in ethylacetate (22.5 ml_), a 20% excess of p-
nitrophenol was added (1.18g, 8.46 mmol) and the mixture was
stirred and cooled in an ice-water bath while
dicyclohexylcarbodiimide (DCC) (1.46g, 7.05 mmol) was added.
Stirring was continued at 0C for 0.5 hour and for 3 hours at
room temperature. The disappearence of DCC was monitored by
its IR band at 2100 cm-1. The reaction was also followed by a
TLC study in system A. The Rf of the p-nitrophenyl ester = 0.7. A
27


small amount of DCC was added after 2.5 hours of stirring. At
that point some of the starting material was still visible on TLC,
but not the DCC. The completion of the reaction was achieved
after the addition of a small spatula-full of DCC. The precipitate
N-N'-dicyclohexylurea was removed by filtration. The solution
was extracted 10 times with a solution of 5% sodium carbonate
and once with brine (100mL), and dried over MgS04 overnight.
MgS04 was removed by filtration and the solvent was evaporated
in vacuo. The remaining oil was immediately dissolved in cold
trifluoroacetic acid (TFA). The TFA was evaporated under high
vacuum. A white solid precipitated with the addition of
anhydrous ether (50 mL). It was triturated 3 times with
anhydrous ether and dried over calcium chloride. Yield: 1.7g,
(51%) m.p.: 112C; Rf = 0.48 (system A).
1.6b Synthesis of Fmoc-L-Glu(CHN2)-ONp
The synthesis was carried out according to the procedure
described in Experimental Section B(8). Fmoc-L-Glu(OH)-ONp (1 g,
2 mmol) was derivatized into Fmoc-L-Glu(CHN2)-ONp. The crude
product of the reaction was purified by flash chromatography
(system B) yielding to 0.33g (32%) of a yellow residue;
homogeneous on TLC, Rf = 0.42 (system B). IR: characteristic
band of the DAMK at 2070 cm1.
28


1.7 Synthesis of Boc-L-GIu(CHNq1-QNd.
1.7a Synthesis of Boc-L-Glu(OH)-ONp
A sample of Boc-Glu(OBzl)-ONp (2g, 4.36 mmol) was dissolved
in a methanol/ethylacetate (70/30; v/v) solution (200 mL). A
catalyst of 10% Pd/C (200 mg) was added and H2 was bubbled for
2 hours. The completion of the reaction was checked by TLC in
system F. The Rf of Boc-L-Glu(OH)-ONp = 0.7. The catalyst was
removed by filtration through celite and the solvent evaporated
in vacuo. The brown residue was triturated and washed with
ether (100 mL) and used without any further purification. Yield:
1.2 g (75%); m.p.: 105C. 1H NMR (300 MHZ, DMSO) 3 (ppm) 1.13 (s,
9H, Boc CH), 1.6 (m, 1H, 6 CH), 1.78 (m, 1H, I3 CH), 2.1 (m, 2H, y
CH), 3.9 (m, 1H, a CH), 6.3 (d, 2H, ONp Hi,5), 6.45 (d, 2H, ONP,
H2,4 ), 7.2 (d, 1H, NH).
1.7b Synthesis of Boc-L-Glu(CHN2)-ONp
Boc-L-Glu(OH)-ONp (1 g, 2.7 mmol) was converted to Boc-L-
Glu(CHN2)-ONp following the procedure described in
Experimental Section B(8). The crude product was purified by
flash chromatography in system D yielding to 0.43 g (40%) of a
yellow residue. Rf = 0.7 in system D. IR: characteristic band of
the DAMK at 2070 cm-1.
29


2. Diazomethvlketone synthesis on protected dioeptides
2.1 Synthesis of Fmoc-GlufQBzn-Ala-QMe.
To Fmoc-Glu(OBzl)-OH (4 g, 8.54 mmol) and HOBt (1.15 g, 8.54
mmol) dissolved in DMF (50 mL), diisopropylcarbodiimide
(DPICDI) (1.34 mL, 8.54 mmol) was added and the preactivation
allowed to proceed for 1 hour.
Ala-OMe.HCI (1.19 g, 8.54 mmol) was poured into CH2CI2 (17
mL) and NMM (0.94 mL, 8.54 mmol) was added. The solution was
stirred for 2-3 minutes to dissolve the amino acid. To this was
added the previous preactivated Fmoc-Glu(OBzl)-OH solution and
the reaction was allowed to proceed overnight under agitation.
The solvent was evaporated in vacuo. The oily residue was
redissolved in ethylacetate (80 mL). Diisopropylurea, which had
precipitated, was removed by filtration and the organic phase
washed with 0.1 HCI solution (3x10 mL), 5% NaHCC>3 (3 x 20 mL)
and brine (20 mL). The ethylacetate layer was dried over MgS04
and evaporated in vacuo. The crude product was recrystallized in
petroleum ether yielding 3.1 g (67%) of a white solid;
homogeneous by TLC, Rf = 0.84 (system F). 1H NMR (300 MHZ,
CHCI3) d (ppm) 1.4 (d, 3H, Ala 8 CH3), 2 (m, 1H, Glu 6 CH), 2.18
(m, 1H, Glu 8 CH), 2.58 (m, 2H, y CH), 3.75 (s, 3H, OCH3), 4.2 (t,
1H, Fmoc CH), 4.31 (m, 1H, Glu a CH), 4.39 (d, 2H, Fmoc CH2), 4.55
(m, 1H, Ala a CH), 5.15 (s, 2H, Bzl CH2), 5.65 (d, 1H, Glu NH), 6.75
(d, 1H, Ala NH), 7.3-7.45 (m, 9H, Bzl Ph and Fmoc H2,3,6,7). 7.6 (d,
2H, Fmoc H4,5), 7.8 (d, 2H, Fmoc Hi>8).
30


2.2 Debenzvlation of Fmoc-Glu(OBzn-Ala-OMe.60-62
Fmoc-Glu(OBzl)-Ala-OMe (2 g, 3.68 mmol) was dissolved in
methanol/ethylacetate (70/30, v/v) (100mL). A 10% Pd/C (200
mg, 10% w/w) was added and H2 was bubbled through the
reaction mixture for 90 minutes. The catalyst was removed by
filtration through celite and the solvent evaporated in vacuo. The
crude product was purified by flash chromatography on silica gel
eluted with 1% acetic acid in ethylacetate/methanol (80/20).
Yield: 1.2 g (72%) m.p.:114-116C; homogeneous by TLC, Rf = 0.58
(system G). 1H NMR (300 MHZ, CHCI3) 3 (ppm) 1.4 (d, 3H, Ala
8 CH3), 2 (m, 1H, Glu 6 CH), 2.18 (m, 1H, Glu B CH), 2.58 (m, 2H, 7
CH), 3.7 (s, 3H, OCH3), 4.2 (t, 1H, Fmoc CH), 4.38 (m, 3H, Glu a CH
and Fmoc CH2), 4.58 (m, 1H, Ala a CH), 5.9 (d, 1H, Glu NH), 7.05 (d,
1H, Ala NH), 7.25-7.4 (m, 4H, Fmoc H2,3I6,7), 7.58 (d, 2H, Fmoc
H4,5), 7.65 (d, 2H, Fmoc Hi,8).
2.3 Synthesis of Fmoc-GlufCHN^-Ala-OMe.
A sample of Fmoc-Glu(OH)-Ala-OMe (1 g, 2.2 mmol) was
converted to Fmoc-Glu(CHN2)-Ala-OMe using the procedure
described in Experimental Section B(8). The crude compound was
then purified by flash chromatography on silica gel eluted with
system C. Yield = 0.32 g (30%) of a yellow residue; Rf = 0.61
(system C), m.p.: 77-80 C. IR: characteristic band of the DAMK at
2070 cm-1.
31


2.4 Synthesis of Fmoc-GlufCHN^-Ala-OMe bv coupling
reaction.
A sample of Fmoc-Glu(CHN2)-ONp (0.51 g, 1 mmol) was added
to a solution of Ala-OMe (0.1 g, 1 mmol) (Experimental Section
C(2.1)) in 2 ml_ of CH2CI2 and reacted for 30 minutes. A TLC in
system C gave two spots with respective Rf: 0.7 (corresponding
to the free para-nitrophenol) and 0.61 (corresponding to the
DAMK of the dipeptide).
2.5 Synthesis of Boc-GlufOBzn-Ala-OMe.
Boc-Glu(OBzl)-OH (4 g, 11.85 mmol) was dissolved in CH2CI2
(30 mL), HOBt (1.6 g, 11.85 mmol) and DIPCDI (1.85 mL, 11.85
mmol) were added to the solution and the activation was allowed
to proceed for 30 minutes under agitation. A solution of Ala-OMe
(1.66 g, 11.85 mmol) was prepared using the procedure described
in Experimental Section C (2.1). The preactivated Boc-Glu(OBzl)-
OH solution was added to the free alanine methyl ester and the
reaction was stirred for 10 hours. The solvent was evaporated in
vacuo and the oily residue was redissolved in ethylacetate (80
mL). The precipitate of diisopropylurea was removed by
filtration and the organic phase washed with 0.1 HCI solution (3
x 10 mL), 5% NaCOa (3 x 20 mL) and saturated brine (20 mL). The
EtOAc layer was dried over MgSC>4 and evaporated in vacuo. A
white solid precipitated with the addition of petroleum ether.
The dipeptide was filtered and used without any further
32


purification. Yield: 4 g (80%); homogeneous by TLC, Rf = 0.67
(system E). 1H NMR (300 MHZ, CHCI3), 3 (ppm) 1.38 (d, 3H, Ala 8
CH3), 1.4 (s, 9H, Boc H), 1.95 (m, 1H, Glu 8 CH), 2.15 (m, 1H, Glu 8
CH), 2.5 (m, 2H, y CH), 3.7 (s, 3H, OCH3), 4.25 (m, 1h, Glu a CH),
4.5 (m, 1H, Ala a CH), 5.1 (s, 2H, Bzl CH2), 5.5 (d, 1H, Glu NH),
7.05 (d, 1H, Ala NH), 7.35 (s, 5H, Ph H).
2.6 Synthesis of Boc-GlufOHI-Ala-QMe.
Boc-Glu(OBzl)-Ala-OMe (3 g, 7.11 mmol) was dissolved in
methanol (50 mL). A 10 % Pd/C (300 mg, 10% w/w) was added
and the mixture was bubbled with H2 for 2 hours. The catalyst
was removed by filtration and the solvent evaporated in vacuo.
The white residue was homogeneous by TLC, Rf = 0.45 (system H).
It was used without any further purification. Yield: 1.73 g (73%)
m.p.: 104C. 1H NMR (300 MHZ, CHCI3), 3 (ppm) 1.4 (m(d+s), 12H,
Ala 8 CH3 and Boc H), 1.9 (m, 1H, Glu 8 CH), 2.1 (m, 1H, Glu 8 CH),
2.5 (m, 2H, y CH), 3.7 (s, 3H, OCH3), 4.38 (m, 1H, Glu a CH), 4.55
(m, 1H, Ala a CH), 5.55 (d, 1H, Glu NH), 7.4 (d, 1H, Ala NH).
2.7 Synthesis of Boc-Glu(CHN^-Ala-OMe.
A sample of Boc-Glu(OH)-Ala-OMe (1 g, 3 mmol) was converted
to the Boc-Glu(CHN2)-Ala-OMe using the procedure described in
Experimental Section B(8). The crude product was then purified
by flash chromatography on silica gel eluted with system C.
Yield: 0.41 g (38%) of a yellow residue; Rf = 0.76 (system C) m.p.:
64-66C. IR: characteristic band at 2070 cm'1.
33


IL_Svntheses of hexapeptides
1^ Synthesis of Fmoc-E(OBzh-L-P-Y(tBu)-A^G-OH (A)
All syntheses of A were carried out using the continuous flow
solid phase peptide synthesis technique. After batchwise
functionalization of the resin following the procedure described
in Experimental Section B(2), the derivatized resin was poured
onto a column using the solid phase peptide synthesis set-up
described in Experimental Section B(1). The Fmoc protecting
group of glycine was removed under basic conditions and the
stepwise addition proceeded for the following amino acids:
Fmoc-Ala, Fmoc-Tyr(tBu), Fmoc-Pro, Fmoc-Leu, Fmoc-Glu(OBzl).
The yields of the 6 syntheses which were performed are
indicated in Table 2.2. An AAA using the ninhydrin method
(Experimental Section B(11) was performed on the resin bound
product of experiment 6. The AA/lle ratios with lie as an
internal marker were for Glu, Leu, Pro, Tyr, Ala, Gly respectively
equal to: 0.97, 0.87, 0.85, 0.85, 0.88 and 0.88.
34


Table 2.2: Yields of protonated hexapeptides synthesis
Experiment number Yield(%)
1 29
2 45
3 46.5
4 44
5 49
6 46
HPLC (Experimental Section B(10)) confirmed the high purity
of the synthesized peptides giving a sharp peak at a retention
time of 38 minutes with a peak area of 91% of the total area.
Mass spectrometry (MS) analysis (Experimental Section
B(12)) showed peaks at m/z = 1017 (M+H) and m/z = 1039 (M+Na).
Two other peaks corresponding to impurities were also visible at
m/z = 1054 and 1070. The peptides were purified in the next
step.
2. Synthesis of Fmoc-EfOBzl)-L-P-Y(tBu1-A-G-OCHo 63-66
A sample of Fmoc-E(OBzl)-L-P-Y(tBu)-A-G-OH (100 mg, 9.85
10-5 mol) was dissolved in 5 mL of CH2CI2. To this was added a
3-fold excess of freshly prepared diazomethane solution
(Experimental Section B(7)) (0.3 mmol). The reaction was stirred
for 5 minutes. TLC indicated the disappearence of the starting
material and thus the completion of the reaction (Rf of the
methylated peptide = 0.58 (system A)). The solvent was
35


evaporated in vacuo. The crude peptide was purified by flash
chromatography using 0.1% MeOH/CHCl3 (200 mL) followed by
0.3% MeOH/CHCl3 (200 mL) and 0.5% MeOH/CHCI3 (200 mL) as the
eluant system. Yield = 86 mg (85%). The high purity of the
peptide was confirmed by HPLC, showing a single peak at a
retention time of 40 minutes.
3. Synthesis of Fmoc-EfOm-L-P-YttBul-A-G-OCHa
A sample of Fmoc-E(OBzl)-L-P-Y(tBu)-A-G-OCH3 (85 mg, 8.25
10-5 mol) was dissolved in methanol (5 mL), 10% Pd/C (0.85 mg,
10% w/w) was added and H2 was bubbled through the mixture for
2 hours. The catalyst was removed by filtration and MeOH
evaporated in vacuo yielding 65 mg (69%) of a white residue. MS
analysis showed peaks at m/z = 941 and 963 corresponding to
(M+H) and (M+Na). HPLC confirmed the high purity of the peptide
(single peak at a retention time of 38 minutes).
4. Attempted synthesis of E(CH£Cn-L-P-Y-A-G-0CH3 67
A sample of Fmoc-E(OH)-L-P-Y(tBu)-A-G-OCH3 was dissolved
in 3 mL of THF and the synthesis of the DAMK was carried out
using the procedure described in Experimental Section B(8).
After evaporation of the solvent, the crude yellow residue was
purified by flash chromatography using 0.1% MeOH/CHCl3 (200
mL) followed by 0.3% MeOH/CHCI3 (200 mL) and 0.5% MeOH/CHCI3
36


(200 mL) as the eluant system. Three major components were
isolated with the following Rf (system A): 0.86 (sample 1), 0.23
(sample 2), 0.14 (sample 3). All three samples were treated
separately with a 20% pip/DMF solution to remove the Fmoc
group. The basic solutions were evaporated under high vacuum.
The three residues were then each dissolved in THF and HCI was
bubbled slowly for 15 minutes. The solvents were evaporated and
each sample analyzed by MS.
5. Syntheses of Fmoc-EfCHN^-L-P-YftBul-A-G-Q-HALH-l-
ResinfBI
All syntheses of B were carried in the same way as those of
A. The leucine was deprotected and Fmoc-Glu(CHN2)-ONp in DMF
(2 mL) was added directly to the growing peptide. The resin
became bright yellow with the liberation of p-nitrophenol and,
after 1 hour of recirculation mode, the Kaiser test indicated the
completion of the coupling reaction. The peptide-resin was
washed to prepare it for the cleavage of the peptide from the
resin.
6. Attempted cleavages of E(CHNo1-L-P-Y(tBu1-A-G from the
resin
The different procedures described below were carried out to
cleave the peptide from the resin. In experiments 7 and 8 the
Fmoc group of the glutamic acid was removed prior to attempted
37


cleavage from the resin. In the other experiments (9-12), the
attempts to cleave from the resin were performed on Fmoc
protected peptides. In all of these experiments except the last
one (experiment 12), the resin was taken out of the column and
poured in a 125 mL Erlenmeyer for cleavage treatment. In the
experiment 12, the resin-peptide was directly treated in the
column which allowed the cleavage reaction to be followed by UV
(wavelength at 280 nm).
Experiment 7: A 1 M HCI solution in ethanol (100 mL) was added
to the resin and shaken for 15 minutes. The acidic solution was
neutralized with 1 M NMM solution (90 mL). The solvent was
evaporated and the orange residue was purified by gel
chromatography (Experimental Section(10)). Fractions showing
absorption at 280 nm were collected and lyophilized to be
analysed by electrospray mass spectrometry.
Experiment 8: A 1 M HCI solution in CH2CI2 (100 mL) was added
to the resin and stirred for 3 hours. The suspension was filtered
and the filtrate was evaporated. The green residue obtained was
ready for MS analysis. The dry resin settled in 2 different layers
in the frit. A sample of the top layer dissolved in water, had
some absorption in UV at 280 nm. It was dissolved in water and
analyzed by MS.
Experiment 9: CH2CI2 (100 mL) was added to the resin and HCI
was bubbled in the suspension for 15 minutes. After filtration,
the solvent was evaporated. No residue was obtained from this
38


evaporation suggesting that the peptide was still in the resin.
The resin was swollen in DMF(50 mL) and filtered. The DMF
solution was evaporated in vacuo and the residue submitted to
MS analysis.
Experiment 10: The same experiment as experiment 8 was
performed with a different solvent: THF. The evaporation of the
solvent led to an oily residue. A white precipitate was formed
with the addition of anhydrous ether. It was filtered and
analyzed by electrospray mass spectrometry.
Experiment 11: The same experiment as experiment 9 was
performed with THF as the solvent. The workup procedure was
identical to the one from the experiment 10.
Experiment 12: The cleavage was carried out under continuous
flow in the column and followed by UV. The resin was washed
with CH2CI2, HCI bubbled in CH2CI2, THF. At this point, the flow
was stopped and the column stored in the fridge for a week. The
resin was then washed with HCI bubbled in THF, DMF and finally
20 % TFA in CH2Cl2. For all the different solvents, fractions
were collected, evaporated and analyzed by electrospray mass
spectrometry analysis.
39


CHAPTER 3
RESULTS AND DISCUSSION
A. Synthesis and identification of the DAMK group
The procedure for the DAMK synthesis was studied and tests
were evaluated using 3 different variables: time, concentration
of the reagents and temperature of the reaction. The improved
synthesis of DAMK on derivatized amino acids is described in
Experimental Section B(8). The diazomethylketone group of all
synthezised compounds was identified by two analytical
techniques: IR and NMR. All DAMK products were analyzed by IR
and showed a characteristic band of N2 stretching at about 2070
cm-1. NMR spectra were also taken for DAMK products. With the
exception of DAMK p-nitrophenyl esters of glutamic acid, all of
them showed a single peak in the region of 5.2-5.45 ppm. The IR
and NMR spectra of the Fmoc-Glu(CHN2)-OtBu indicated in
Figures 3.1 and 3.2 provide good examples of those analytical
methods.
Concerning the DAMK p-nitrophenyl esters, their NMR spectra
were not clear enough in the 5.0-5.4 ppm region to allow


ABSORBANCE
2.5
3.0
3.5
4.0 MICRONS 50
6.0
0
0
0.0
.10
.20
.30
.40
.50
.60
.70
1.0


3.2 NMR spectrum at 300 MHZ of Fmoc-Glu(CHN2)-OtBu


identification. However, the strong IR absorbance of the N2 band
provided sufficient evidence for the presence of the DAMK
function.
B. Stability of the DAMK group
The predicted stability of DAMK derivatives in basic media
was confirmed. However, the experiments in acidic solutions
showed the complete unstability of the DAMK group. This
characteristic changed the strategy for the introduction of a
side chain DAMK Fmoc glutamic acid derivative. Because of the
destruction of the DAMK in acidic media, the derivatized
glutamic acid has to be synthesized with a non-acid-labile
carboxy protecting group. However, to achieve the coupling
reaction during the peptide synthesis, the a-carboxylic function
of the amino acid introduced has to be activated. The carboxy
protecting group has thus to be removed. The use of a good
leaving group which could protect the chain COOH during the
derivatization of the side chain carboxylic function was chosen
and achieved by forming the a-para-nitrophenyl ester (-ONp) of
the DAMK glutamic acid. Comparisons were done between the
product resulting from the coupling of Fmoc-Glu(CHN2)-ONp and
Ala-OMe and the dipeptide Fmoc-Glu(CHN2)-Ala-OMe. TLC
analysis of the coupling reaction, showed two major spots, with
43


the respective Rf of 0.7 and 0.61. The first one was identified as
HONp and the second one corresponded exactly to the Rf (System
C) found for the DAMK of the dipeptide. This result indicated that
the para-nitrophenyl ester group could be used as both protecting
group and leaving group during the activation and coupling
reactions.
Mass spectrometry could have been useful for the analysis of
the DAMK group. Unfortunately, each sample used in the
electrospray positive ionization mode mass spectrometry had to
be dissolved in an acidic solution (3% AcOH in MeOH/water,
50/50, v/v) in order to be protonated. The instability of the
DAMK toward acidic solution did not allow the use of this
analytical technique.
C. Peptide assembly and cleavage
1. Peptide A
The use of base labile N-fluorenylmethyloxycarbonyl amino
acids combined with t-butyl and benzyl side chain protecting
groups provided a simple and rapid strategy in solid phase
peptide synthesis of the peptide A. However, due to the size of
the column, only small amounts of peptides could be synthesized
at a time. The yields (Table 2.2) were moderate, but their high
44


purity, demonstrated by HPLC (Figure 3.3) enables the subsequent
derivatization steps to proceed without any further purification.
The peptide obtained from the experiment 1 gave good
quantitative and qualitative AAA results (ninhydrin method,
Experimental Section B(10)). Furthermore, the AAA showed that
92% of the hexapeptide has been cleaved from the hyper-acid-
labile handle under low acidic conditions. AAA on experiments 7-
11 were performed using the Dabsylation method (Experimental
Section B(10)). Qualitative results were satisfactory showing
the presence of the six amino acids in approximately the same
proportion. However, quantitative cleavage results were not
possible due to the fact that the lie internal marker was not
giving sufficient response on HPLC.
HPLC and mass spectrometry were used to confirm the AAA
results. All peptides synthesized (experiments 1-6) were
analyzed by HPLC and gave reproducible results (see previous
Figure 3.3). MS on experiments 2 and 4 gave expected results for
the mass of the peptides (Figure 3.4). The impurities showed on
the spectrum were purified in the following derivatization steps
of the peptide.
2. Peptide B
The assembly of peptide B proceeded as with peptide A. The
coupling reaction between the DAMK p-nitrophenyl glutamic
45


LC B 266,10 550,100 of SEQ R00R.D
3.3 HPLC profile at 266 nm of Fmoc-E(OBzl)-L-P-Y(tBu)-A-G-OH


intensity m/z = 1039 (M+Na)
3.4 Mass spectrum of Fmoc-E(OBzl)-L-P-Y(tBu)-A-G-OH
4 7
m/z


ester was followed with a special attention. The use of the p-
nitrophenyl ester as a leaving group for the coupling reaction had
already been studied in solution. The yellow color of the resin
and the negative Kaiser test indicated the addition of the
derivatized DAMK glutamic acid on the peptide. Treatment with
HCI was then required in order to cleave the peptide from the
resin and transform the DAMK to CMK.
Different cleavages have been tried in experiments 7 through
12. Experiment 7 was carried out with an HCI/ethanol solution.
This required the neutralization of the peptide solution after
cleavage, creating a salt. A gel chromatography was then
performed in order to isolate the peptide. Only the first
fractions collected indicated, by MS, traces of the expected
peptide (m/z = 682). Attempted purification by HPLC resulted in
extremely low yield and MS still showed noninterpretable peaks.
Experiments 8 and 9 were done using HCI in CH2CI2 solution. In
experiment 8, the formation of a 1M solution was tried by
bubbling HCI in the solvent and weighing the solution before and
after. The accuracy of the experiment was questionable due to
the rapid evaporation of the dichloromethane. Experiment 9 was
not controlled with respect to the concentration of HCI in
CH2CI2.
Electrospray MS did not give any positive results for the
products of experiment 8 and 9. The resin from experiment 9 was
washed with DMF and a MS indicated a peak at 900 instead of 904
48


(m/z of the Fmoc peptide). Because of the preceding fact, CH2CI2
was thought not to be a good solvent. The peptide could have been
cleaved but not dissolved and still imprisoned in the resin.
Experiments 10 and 11 were tried with a different solvent: THF.
Both experiments gave three major peaks in MS at m/z = 722,
611 and 557 which were not the expected mass (904). Both
residues were analyzed by AAA and showed Ala, Gly, Leu, Pro and
Tyr residues which confirmed the presence of a peptide. The
glutamic acid could not be seen under these AAA conditions
because of its derivatized side chain. In experiment 12, cleavage
was performed directly in the column by replacing the usual
solvents with the ones treated with HCI. Progress of the reaction
was followed by the UV detector of the peptide synthesizer.
CH2CI2 and THF treated with HCI, then DMF and finally 20% TFA
in CH2CI2 were used to wash the resin but none of them indicated
a good cleavage reaction by an high UV absorbance. The peptide
seemed to be liberated from the resin in very low concentration
which could explain the low response recorded on the UV for each
different solvent. MS of all the collected solutions showed peaks
in the m/z = 650-750 region but none corresponding to the
expected mass. AAA of each sample indicated the presence of
peptide and also complete cleavage from the resin.
49


D. Derivatization of peptide A
Another use of the diazomethane was the O-methylation of A.
It provided an easy synthetic way in order to esterify the
carboxy-terminus of the hexapeptide. The substitution of the -OH
by the -OCH3 group increased the hydrophobicity of A. The
peptide was more mobile on TLC which allowed its purification
by flash chromatography. HPLC analysis revealed the high purity
of A-methyl ester. The subsequent debenzylation was carried out
with the same conditions used for amino acids and dipeptides.
The stability of the Fmoc group toward hydrogenolysis was once
again studied. A supposedly partially poisoned catalyst could be
active enough to reduce a benzyl group but sufficiently inactive
to leave an Fmoc derivative intact, at least for a certain period
of time which was determined by TLC for each reaction. The
debenzylation was confirmed by NMR spectra taken before and
after reaction (Figures 3.5 and 3.6). The comparison between the
two spectra showed the disappearance of the CH2 benzyl peak at
5.12 ppm and of the phenyl benzyl peak at 7.35 ppm. The purity of
the methylated debenzylated peptide was confirmed by
electrospray mass spectrometry as shown in Figure 3.7 and by
HPLC.
The DAMK synthesis of derivatized A showed uncertain
results. The activation of the side chain carboxylic function of
the glutamic acid was not so clear as with the same activation
50


3.5 NMR spectrum at 300 MHZ of Fmoc-E(OBzl)-L-P-Y(tBu)-A-G-OCH3




Intensity m/z = 963 (M+Na)
3.7 Mass spectrum of Fmoc-E(OH)-L-P-Y(tBu)-A-G-OCH3


on amino acids or dipeptides. TLC taken during the reaction
showed formation of at least 2 different components instead of
the one seen in the synthesis of smaller derivatives. The three
products separated by flash chromatography gave inconsistent
results. The product in fraction 1 showed no IR band in the region
of 2100 cm1 on the first spectrum taken but indicated such a
band on a repeated analysis. This fa'ct was initially attributed to
a difference of concentration of the samples between the two
experiments. The other two products showed no -N2 absorbance
by IR. After the Fmoc deprotection step and the HCI treatment,
the three fractions were analyzed by MS but none of them showed
components with the theoretical mass of m/z = 682 expected for
the protonated CMK hexapeptide. Peaks were seen for fraction 1
in the 530-550 region, for fraction 2 at 679 and 754 and for
fraction 3 at 646 and 678. All three fractions showed the 5
following amino acids: Ala, Gly, Leu, Pro, Tyr in AAA which
confirmed the presence of peptides.
E. Conclusion
The research continued an investigation into the structure-
activity relationships that exist between PTK and new affinity
labelling peptide substrates. Incorporation of diazomethylketone
group into the side chain of amino acids and peptides as a new
54


site for affinity labelling has been developed. Synthetic
procedures for the DAMK group have been optimized and analyses
on the stability of this group were conducted. Because of its
instability in acidic media, the use of para-nitrophenyl esters
as both a-carboxy protecting group and activating group for the
peptide synthesis was explored. The coupling reaction using the
DAMK para-nitrophenyl Fmoc glutamic acid has been tested in
solution and solid phase synthesis. A number of means were used
in attempts to convert the DAMK to CMK and cleave the peptides
from the resin. Cleavages and recovery of peptides were achieved
but no evidence confirming the presence of the desired peptide
possessing a chloromethylketone group was found.
In another experiment, the incorporation of the CMK was tried
by derivatization of protected peptide. Model studies on amino
acids and dipeptides exhibited evidence of the DAMK group. The
synthesis of the benzylated ELPYAG and its further derivatiza-
tions have been achieved with success up to the point preceding
the conversion of the y-carboxyl group to a DAMK function.
Standard procedures for the synthesis of the DAMK group have
been performed on the hexapeptide but no evidence has shown its
presence. Detection through the conversion of DAMK to CMK also
failed to provide positive evidence.
55


F. Suggestion for further studies
The strategy employing the orthogonal combination of base-
labile 9-fluorenylmethyloxycarbonyl for N-a-amino protection
and acid-labile t-butyl derivatives for side chain protection has
been employed during the peptide syntheses. A hyper-acid-labile
handle (HALH) was used as the linkage agent between the peptide
and the resin. Barany et al.68 recently introduced a new
hypersensitive acid-labile anchoring agent: tris(alkoxy)benzyl
ester. This linker can be used under milder conditions (0.05-0.1%
(v/v) TFA) than those used with the HALH (1% (v/v) TFA) in the
protected peptide synthesis. The stability of the DAMK has to be
tested under those conditions. Furthermore, the deprotection of
the t-butyl protecting group on the tyrosine residue would not
occur. This might improve the DAMK synthesis on the glutamic
acid residue of the hexapeptide. The deprotection of the tyrosine
could be carried out after the CMK conversion.
Due to the stability of DAMK towards basic medium, a change
in the peptide synthesis technique could be tried. Instead of
using an acid-labile handle as linkage agent, a base labile agent
could be employed, allowing cleavage of the peptide under
stronger basic conditions (ammonolysis) than those required
during deprotection steps. Thus, the synthesis of peptide, still
using the Fmoc strategy, will be the same through the
incorporation of the DAMK glutamic acid. At this point the
56


peptide could be cleaved from the resin by amminolysis and
recovered with the DAMK side chain group on it for further
derivatizations. This will suppress the problem of a two
reactions (cleavage and conversion) step allowing a better
control on the peptide synthesis. The recovery of the DAMK
peptide will also allow the testing of the degree of inhibition of
the enzyme with both DAMK and CMK Substrates.
57


LIST OF REFERENCES
1. Eckhart, W.; Hutchinson, M. A.; Hunter, T. Cell 1979, 18, 925.
2. Hunter, T.; Cooper, J. A. Ann. Rev. Biochem. 1985, 54, 897.
3. Bishop, J. M. Science 1987, 235, 305.
4. Hunter, T; Sefton, B. M. Proc. Natl. Acad. Sci. USA 1980, 77,
1311.
5. Collet, M. S.; Purchio, A. F.; Erikson, R. L. Nature 1980, 285,
167.
6. Levinson, A. D.; Oppermann, H.; Varmus, H. E.; Bishop, J. M.
J. Biol. Chen). 1980, 255, 11973.
7. Schwartz, D. E. ; Tizard, R. ; Gilbert, W. Cell 1983, 32, 853.
8. Takeya, T.; Hanafusa, H. Ce//1983, 32, 881.
9. Rubsamen, H.; Friis, R. R.; Bauer, H. Proc. Natl. Acad. Sci. USA
1979, 76, 967.
10. Sefton, B. M.; Beeman, K.; Hunter, T. J. Virol. 1978, 28, 957.
11. Brugge, J. S.; Erikson, R. L. Nature 1977, 269, 346.
12. Levinson, A. D.; Oppermann, H.; Levintow, L.; Varmus, H. E.;
Bishop, J. M. Cell 1978, 15, 561.
13. Purchio, A. F.; Erickson, E.; Brugge, J. S.; Erickson, R. L. Proc.
Natl. Acad. Sci. USA 1978, 75, 1567.
14. Collet, M. S.; Erickson, R. L. Proc. Natl. Acad. Sci. USA 1978,
75, 2021.
15. Sefton, B. M.; Hunter, T.; Beeman, K.; Eckhart, W. Cell 1980,
20, 807.


16. Levinson, A. D.; Courtneidge, S. A.; Bishop, J. M. Proc. Natl.
Acad. Sci. USA 1981, 78, 3, 1624.
17. Brugge, J. S.; Darrow, D. J. Biol. Chem. 1984, 259, 7, 4550.
18. Smart, J. E.; Oppermann, H.; Cernilofsky, A. P.; Purchio, A. F.;
Erickson, R. L.; Bishop, J. M. Proc. Natl. Acad. Sci. USA 1981,
78, 10, 6013.
19. Patschinsky, T.; Hunter, T.; Esch, F. S.; Copper, J. A.; Sefton, B.
M. Proc. Natl. Acad. Sci. USA 1982, 79, 973.
20. Copper, J. A.; Gould, K. L.; Cartwright, C. A.; Hunter, T. Science
1986, 231, 1431.
21. Piwnica-Worms, H.; Saunders, K. B.; Roberts, T. M.; Smith, A.
E.; Cheng, S. H. Cell 1987, 49, 75.
22. Sato, M.; Kato, J.; Takeya, T. J. Virol. 1989, 63, 2, 683.
23. Kmiecik, T. E.; Shalloway, D. Cell 1987, 49, 65.
24. Cartwright, C. A.; Eckhart, W.; Simon, S.; Kaplan, P. L. Cell
1987,49,83.
25. Jove, R.; Kornbluth, S.; Hanafusa, H. Cell 1987, 50, 937.
26. Knowles, J. R. Accounts of Chemical Research 1972, 5, 155.
27. Levin, V. A. Unpublished results.
28. Coggings, J. R.; Kray, W.; Shaw, E. Biochemistry 1974, 138,
579.
29. Sekura, R.; Meister, A. J. Biol. Chem. 1977, 252, 8, 2606.
30. Merrifield, R. B. Fed. Proc. Fed. Amer. Soc. Exp. Biol. 1962, 21,
412.
31. Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
59


32. Merrifield, R. B. Biochemistry 1964, 3, 1385.
33. a. Atherton, E.; Brown, E.; Sheppard, R. C.; Rosevear, A. J.
Chem. Soc., Chem. Commun. 1981, 1151.
b. Sheppard, R. C. Chem. Brit. 1983, 402.
34. Lukas, T. J.; Pryskowsky, M. B.; Erikson, B. W. Proc. Natl. Acad.
Sci. USA. 1981, 78, 2791.
35. Atherton, E.; Fox, H.; Harkiss, D.; Logan, C. J.; Sheppard, R. C.;
Williams, B. J. J. Chem. Soc., Chem. Commun. 1978, 537.
36. Atherton, E.; Logan, C. J.; Sheppard, R. C. J. Chem. Soc., Perkin
Trans. 11981, 538.
37. Atherton, E.; Gait, M. J.; Sheppard, R. C.; Williams, B. J. Bioorg.
Chem. 1979, 8, 351.
38. Atherton, E.; Clive, D. L. J.; Sheppard, R. C. J. Amer. Chem. Soc.
1975, 97, 6585.
39. Sheppard, R. C.; Williams, B. J. J. Chem. Soc., Chem. Commun.
1982, 587.
40. a. Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92, 5478.
b. ibkL J. Org. Chem. 1972, 37, 3404.
c. Ibid. J. Org. Chem. 1973, 38, 4318.
41. Khorana, H. G. Chem. Rev. 1953, 53, 145.
42. Castro, B.; Dormoy, J. R.; Evin, G.; Ogura, H. Tetrahedron Lett.
1975, 14, 1219.
43. Nietzki, R.; Braunschweig, E. Chem. Ber. 1894, 27, 3381.
60


44. Koenigs, E.; Friedrich, H.; Jurany, H. Chem. Ber. 1925, 58,
2571.
45. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.
46. Pizey, J. S. In Synthetic Reactions; Halsted; New York 1970,
Vol II, Chapter 4.
47. Hopps, H. B. Aldrichim. Acta 1970, 3, 4.
48. Black, T. H. Ibid 1983, 16, 1 ,3.
49. Shaw, E.; Mares-Guia, M.; Cohen, W. Biochemistry 1965, 4, 10.
50. Shoellmann, G.; Shaw, E. Biochemistry 1963, 2, 2.
51. Kettner, C.: Shaw, E. Methods in Enzymology 1981, 80, 826.
52. Penke, B.; Czombos, J.; Balaspiri, L.; Petres, J.; Kovacs, K.
Helv. Chim. Acfa 1970, 53, 5, 122.
53 Kettner, C.; Shaw, E. Biochemistry 1978, 17, 4778.
54. Still, C. W.; Khan, M.; Mitra, A J. Org. Chem. 1978, 43, 14.
55. Hill, D. W.; Waters, F. H.; Wilson, T. D.; Stuart, J. D. Anal. Chem.
1979, 51, 1338.
56. Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 4451.
57. Birch, P. L.; El-Obeid, H. A.; Akhtar, M. Arch. Biochem. Biophys.
1972, 148(2), 447.
58. Bodanszky, A.; Bodanszky, M.; Chandramouli, N.; Kwei, J. Z.;
Martinez, J.; Tolle, J. C. J. Org. Chem. 1980, 45, 72.
59. Fields,G. B.; Noble, R. L. Int. J. Peptide Protein Res. 1990, 35,
161.
61


60. Martinez, J.; Tolle, J. C.;Bodanszky, M. J. Org. Chem. 1979, 44,
20, 3596.
61. Atherton, E.; Bury, C.; Sheppard, R. C.; Williams, B. J.
Tetrahedron Lett. 1979, 32, 3041.
62. Carpino, L. A.; Tunga, A. J. Org. Chem. 1986, 51, 1932.
63. Wade, P. A.; Pillay, M. K. J. Org. Chem. 1981, 46, 5425.
64. Guzman, A.; Mendoza, S. Synthesis Comm. 1981, 12, 32, 989.
65. Fusco, R.; Sannicolo, F. J. Org. Chem. 1982, 47, 1691.
66. Chavis, C.; Dumont, F.; Wightman, R. H.; Ziegler, J. C.; Imbach,
J. L. J. Org. Chem. 1982, 47, 202.
67. Abo-Ghalia, M.; Flegel, M.; Blanot, D.;Van Heijenoort, J. Int. J.
Peptide Protein Res. 1988, 32, 208.
68. Albericio, F.; Barany, G. Tetrahedron Lett. 1991, 32, 8, 1015.
62


Full Text

PAGE 2

91

PAGE 15

O!
PAGE 37

1j'

PAGE 48

a Pd/C a a

PAGE 51

Pd/C 10%

PAGE 69

mlz