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SYNTHESIS OF BIOLOGICALLY ACTIVE PEPTIDES ON PS-BDODMA RESIN
USING Boc-CHEMISTRY
5.1. Introduction
eptides composed of as few as three to 50 amino acids play an important role in l
a large number of diverse biological processes. The chemical synthesis has been
accepted as a method to generate biologically active peptides and their
analogues.'~ ' The solution phase methods have hem widely used for the synthesis of
peptides, while the curl-ent method of choice to obtain peptides is by solid phase method.
Synthesis of peptides as well as small proteins can be achieved by solid phase method.
Solid phase protocols have also been adopted for simultaneous synthesis of peptides and
peptide librarie~.'.~ The chloromethylated PS-BDODMA support with various cross-
linking densities were successfully used for the synthesis of biologically active peptides.
Even high capacity resin can accommodate the growing peptide chain because of the high
flexibility and hydrophilicity of the cross-linker. HPLC and amino acid analysis of these
peptides showed that all acylation reactions were completed in a single coupling. The
2,5-dioxopiperazine (diketopiperazine, DKP), formation from the N-terminal residue of
the peptide chain conlmonly occurs as a disturbing reaction in the synthesis and long term
storage of peptides.-7The nitrogen atom of the N-terminal deprotonated amino group can
attack the carbonyl carbon atom of the second residue causing a break down of the chain
and formation of DKPs The coupling of third amino acid was followed immediately after
the deprotection of the second amino acid can eliminate the formation of DKP.
The synthetic steps of the solid phase assembly of amino acid to peptide are
illustrated in Scheme 5-1. The C-terminal amino acid was attached to the solid support
hy an ester bond using cesium salt method. Deprotection of Boc group was achieved by
30% TFAIDCM. After the deprotection, the resin was washed thoroughly with DCM and
NMP. Acylation reactions were carried out in minimum quantity of NMP by using
2.5 equiv excess of Doc-amino acids, DCC, HOBt and DIEA with respect to the amino
capacity of the C-terminal amino acid attached resin. The coupling of each amino acids
were monitored by Kaiser test.'
The peptide was cleaved from the resin by treating with TFA and suitable
scavengers at rooin temperature After 8 h, the reaction mixture was filtered, washed with
TFA and the filtrate was evaporated to get an oily residue. The peptide was precipitated
@- CH~OOC-AA I -NH-Boc
I 1) 30% TFA in DCM 2) 5% DIEA in DCM
@CH~OOC-AA I - N H ~
1) Deprotection ' 2) Neutralisation 4 3) Coupling with respective Boc-AA
Srheme.5-1. PS-BDODMA resin based SPPS using Boc-chemistry
by adding ice-cold ether and washed with ether to remove the scavengers. The crude
peptide was passed through a sephadex column and the peptidyl fractions were collected
and lyophilized
5.2. Results and Discussion
5.2.a. Synthesis of model peptides on PS-BDODMA resin using Boc-chemistry
General procedure
The C-terminal amino acid was incorporated to chloromethylated 2%
PS-BDODMA resin by cesium salt method. After removing the Boc protection and
neutralization, the remaining Boc protected amino acids were successively added by
HOBt active ester method till the target sequence was formed. The peptide was cleaved
from the support using neat TFA in presence of scavengers. TFA was removed under
reduced pressure and the peptide precipitated by adding ice-cold ether. The peptide was
recrystallized from MeOH and the purity was checked by tlc using pyridine:acetic
acid:water (50:35: 15).
1. Synthesis of Phe-Phe-Thr-Lys-Phe-Lys-Ser-Gln
The peptide was synthesized by following the general peptide synthetic strategy
using Boc-amino acids and was obtained in 96% yield (134 mg). The peptide was
recrystallized from MeOH and the purity was checked by tlc. Rf = 0.78. The HPLC and
MALDI TOF MS are given in Fig. 5-1.
(a) (b) Fig. 5-1. (a) HPLC time-course analysis of the peptide Phe-Phe-Thr-Lys-Phe-Lys-Ser-
Gln using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 d m i n ; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide
2. Synthesis of Val-Gln-Gln-Gly-Pro-Trp-Gly-Gly-Ala-Ala-Val
The peptide was synthesized by following the general procedure and the yield of
the crude peptide was found to be 97% (141 mg). Rf=0.76. The HPLC and MALDI TOF
MS of the peptide is given in Fig. 5-2.
(a) (b) Fig. 5-2. (a) HPLC time-course analysis of the peptide Val-Gln-Gln-Gly-Pro-Trp-Gly-
Gly-Ala-Ala-Val using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 mL/min; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide
3. Synthesis of Val-Asn-Asn-Gln-Gln-Asn-Asn-DeGly-Gln-G1n-Gly-Ala-A1a-Val
The peptide was synthesized according to the general procedure and was obtained
in 96% yield (202 mg) Rt =0.69. The HPLC and MALDI TOF MS are given in Fig. 5-3.
Fig. 5-3. (a) HPLC time-course analysis of the peptide Val-Asn-Asn-Gln-Gln-Asn-Asn- Ile-Gly-Gln-Gln-Gly-Ala-Ala-Val using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0 5 rnL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 mllrnin; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide
4. Synthesis of (Val)~"
The peptide was synthesized by following the general synthetic procedure. The
yield of the peptide was 97% (133 mg). Rf=0.71. The HPLC and MALDI TOF MS are
given in Fig. 5-4.
Fig. (a) (b)
5-4. (a) HPLC time-course analysis of the peptide (Val),~ using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 d m i n ; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide
5. Synthesis of Val-Gln-Asn-Asn-Val-Val-Val-Val-Val
The peptide was synthesized by following the general synthetic strategy and the
yield of peptide was1 25 mg. Rf = 0.73.
6. Synthesis of Pro-Val-Val-Thr-Thr-Val-Val-Val-Val-Asn
The peptide was synthesized according to the general procedure (133 mg, 96%)
Rf=0.71.
7. Synthesis of Thr-Val-Val-Val-Val-Asn
The peptide was synthesized by following the general synthetic strategy (81 mg,
95%). Rr= 0.81
8. Synthesis of Pro-Met-Leu-Phe-Val-Thr
The peptide was synthesized by following the general procedure (91 mg, 95%).
Rf=0.8.
9. Synthesis of Val-Met-Leu-Phe-Leu-Pro
The peptide was synthesized by following the general synthetic strategy. The
peptide was obtained in 96% yield (94 mg) and the Rfwas 0.76.
10. Synthesis of Met-Leu-Phe-Tyr-Val-Gly
The peptide was synthesized according to the general synthetic strategy (95 mg,
96%). Rf=0.82.
5.2.b. Synthesis of protected peptides
PS-BDODMA resin was proved to be a suitable support for the synthesis of
protected peptides. The completely protected peptides can be obtained by using the trans-
esterification method.
1. Synthesis of Boc-Met-Leu-Phe-Cys(Acm)-Lys(C1-Z)-Val-OMe
The peptide was synthesized by following the general protocol using Boc-amino
acids (41 mg, 95%). The purity of the peptide was checked by tlc and HPLC (Fig. 5-5).
The Rf = 0.52.
(a) (b) Fig. 5-5. (a) HPLC time-course analysis of the peptide Boc-Met-Leu-Phe-Cys(Acm)-
Lys(C1-2)-Val-OMe using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 d / m i n ; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.
2. Synthesis of Boc-Pro-Met-Leu-Phe-Val-Thr-OMe
The peptide was synthesized according to the general procedure (1 13 mg, 92%).
Rf observed was 0.45. HPLC analysis showed a single peak for the peptide.
3. Synthesis of Boc-Val-Met-Leu-Phe-Leu-Pro-OMe
The peptide was synthesized by following the general protocol (105 mg, 93%). Rf
observed was 0.6.
4. Synthesis of Boc-Met-Leu-Phe-Tyr(Bz1)-Val-Gly-OMe
The peptide was synthesized by following the general synthetic procedure
(1 18 mg, 93%). Rf = 0.65. The HPLC and MALDI TOF MS are given in Fig. 5-6.
(a) (b) Fig. 5-6. (a) HPLC time-course analysis of the peptide Boc-Met-Leu-Phe-Tyr(Bz1)-Val-
Gly-OMe using the buffer (A) 0.5 mL. TFA in 100 mL water; (B) 0.5 mL. TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 mllmin; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.
5.2.c. Synthesis of biologically active peptides
1. Synthesis of Leucine enkephalin
(Tyr-Gly-Gly-Phe-Leu)
The peptide was synthesized by following the general procedure. The crude
peptide was obtained in 94% yield (71 mg). The purity of the peptide was checked by tlc.
Rf = 0.76. MALDI TOF MS of the peptide is given in Fig. 5-7.a.
2. Synthesis of P-casomorphin (Bovine) lo
(Tyr-Pro-Phe-Pro-Gly-Pro-lle)
The peptide was synthesized accordmg to the general synthetic strategy. The nude peptide
was obtained in 95% yield (102 mg). The purity ofthe peptide was checked by tlc. Rf = 0.73.
3. Synthesis of P-casomorphin ( ~ u m a n ) "
(Tyr-Pro-Phe-Val-Glu-Pro-Ile)
The peptide was synthesized by following the general synthetic procedure. The Bude peptide
was obtained in 95% yield (1 12 rng). The purity of the peptide was checked by tlc. Rf= 0.74.
4. Synthesis of C-Reactive protein (77-82)'*
(Val-Gly-Gly-Ser-Glu-Ile)
The peptide was synthesized by following the general procedure. The crude
peptide was obtained in 96% yield (73 rng). The purity was checked by tlc. Rf= 0.74.
MALDI TOF MS of the ~eptide is given in Fig 5-7b.
556.6 562.1 100
1 80 : : i
60 -j %I"t. 4
40.
1 30:
i 2 0 :
3 ii 0 1 A>..* -....-- +.+.--.i.-.-
500 aao 700 0 MassICharge
iOi -&.+&i+ 600 MassJChsrge 600 c._,.._A__ 700
( 4 (b) 5-7. MALDI TOF MS of (a) Tyr-Gly-Gly-Phe-Leu (b) Val-Gly-Gly-Ser-Glu-Ile
5. Synthesis of 33-42 fragment of Alzheimer's p-amyloid peptide l3
(Gly-Leu-Met-Val-Gly-Val-lle-Ala)
The first amino acid of the target sequence was attached to chloromethylated 2%
PS-BDODMA support by using cesium salt of Boc-Ala. The nearly complete attachment
of Boc-Ala was observed by picric acid titration method. After removing Boc protection
by 30% TFA/DCM and neutralization with 5% DIEA/DCM, respective amino acids were
incorporated by DCC/HOBt active ester coupling method. Each coupling steps were
monitored by Kaiser's semi-quantitative ninhydrin test. A second coupling was carried
out in order to confirm the complete reaction. The target peptide was cleaved from the
support using TFA, in presence of thioanisole, water and ethanedithiol. The crude peptide
was obtained in 94% yield (1 18 mg). HPLC profile (Fig. 5-8. a) of the peptide showed
only a single peak. Amino acid analysis data and the MALDI TOF MS also agreed with
that of the target peptide.
Moss / Chame
(a) (b) Fig. 5-8. (a) HPLC time-course analysis of the peptide Gly-Leu-Met-Val-Gly-Val-lle-Ala
using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 mL/min; Gradient used: 0% B in 5 min and 100% B in SO min (b) MALDI TOF MS of the peptide.
6. Synthesis of Inhibitor of Ribonudeotide Reductase of Herpes Simplex V i Type L l4
(Tyr-Ala-Gly- Ala-Val-Val-Asn-Asp-Leu)
Boc-Leu was attached to chloromethylated 2% BDODMA cross-linked
polystyrene by cesium salt method. The nearly complete attachment of Boc-Leu was
observed by picric acid titration method. After removing Boc-protection with 30% TFA
in DCM and neutralization with 5% DIEA/DCM, the successive amino acids in the target
sequence were attached by DCC/HOBt active ester coupling method. Each coupling steps
were monitored by Kaiser's test. A second coupling was also performed to confirm the
complete attachment. The synthesized peptide was cleaved from the support by TFA in
presence of water, thioanisol and ethanedithiol. The crude peptide was obtained in 95%
yield (1 19 mg). The HPLC analysis showed a single peak (Fig. 5-9.a) corresponding to
the peptide Amino acid analysis data was also in agreement with that of the target
peptide. High value of Asp was due to the hydrolysis of Asn to Asp.
(a) (b) Fig.5-9. (a) HPLC time-course analysis of the peptide Tyr-Ala-Gly-Ala-Val-Val-Asn-
Asp-Leu using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 mL/min; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.
7. Synthesis of Scyliorhinin-1 peptide
(Ala-Lys-Phe-Asp-LYS-Phe-Tyr-Gly-Leu-Met)
Boc-Met was attached to chloromethylated 2% PS-BDODMA resin by cesium
salt method. The nearly complete attachment of Boc-Met was observed by picric acid
titration method AAer removing Boc-protection with 30% TFA in DCM and
neutralization with 5% DIEA/DCM, the successive amino acids in the target sequence
were attached by DCCmOBt active ester coupling method. Each coupling steps were
monitored by Kaiser's test. A second coupling was also performed to confirm the
complete attachment. The synthesized peptide was cleaved from the support by using
TFA in presence of water, thioanisole and ethanedithiol. The crude peptide was obtained
in 95% yield (119 mg). The HPLC analysis showed a single peak (Fig. 5-10)
corresponding to the peptide. Amino acid analysis data was also in agreement with the
target peptide.
(a) (b) Fig. 5-10. (a) HPLC time-course analysis of the peptide Ala-Lys-Phe-Asp-Lys-Phe-Tyr-
Gly-Leu-Met using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 mLlmin; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.
8. Synthesis of 43-residue peptide from a CD 4 binding domain of Human Immunodeficiency Virus Envelope Glycoprotein
Human immunodeficiency virus OfIV) is the etiological agent for acquired
immunodeficiency syndrome (AIDS).'~.'~ The key process in initial stage of HIV
infection and replication is the high affinity binding of its envelope glycoprotein (gp-120)
to CD 4 receptor on the extracellular surface of helper T-cells and macrophages. The gp
120 is derived from proprotein gp 160 by protwlytic cleavage and it is associated with
the transmembrane envelope glycoprotein gp 41. When binding to CD 4 receptor, gp 120
undergoes a significant wnformational change leading to the approach of fusion peptide
at the N-terminus of gp 41 to the target cell membrane, which then fuses, with viral
envelope through the mediation of fusion peptide. Confonnational alternation of gp 120
was deduced from the observation of shedding of gp120 from the transmembrane
glycoprotein gp 41 on binding to soluble CD 4.17 The nature and amino acid residues
involved in the conformational change were not explicitly reported. It is important to
~nvestigate the conformational features of CD 4 binding domain of gp 120.'~
Biologically active peptides have been used as models to study the conformation
of the region in the intact proteins homologous to the peptides as well as protein folding
studies. The peptide fragment containing 43 amino acids (381-423) play an important
role in the activity of gp 120. '~
In order to synthesize long chain peptides containing more than 20 residues,
fragment condensation is the most conveniently used technique.20 But the major draw
back of the method is the low yield of the product. Recently some new solid supports
were developed and successfully employed for the synthesis of long chain peptides with
high yield and purity. The newly developed PS-BDODMA resin can he successfully used
for the synthesis of long chain peptides and is illustrated by the synthesis of a 43-residue
peptide from the CD 4 binding domain of human immunodeficiency virus envelope
glycoprotein. The peptide synthesized was in high yield and moderate purity and can
easily be purified by HPLC.
Synthesis of 43-residue peptide from the CD 4 binding domain of HIV glycoprotein
Boc-Ile was incorporated to the chloromethylated 2% PS-BDODMA resin by
cesium salt method. The quantitative incorporation of amino acid residue was estimated
by picric acid method. The completion of the reaction was also confirmed by absence of
any detectable amount of residual chlorine by Volhardt's method. After removing Boc
protection by 30% TFA/DCM and neutralization with 5% DIEA/DCM, the remaining
amino acids in the sequence were incorporated by DCC/HOBt active ester coupling
method. A second coupling was also performed for confirming the quantitative reaction.
Each coupling steps were monitored by ninhydrin test. More coupling steps were
performed wherever necessary to bring the coupling to completion.
The synthesis was stopped at the 8'h amino acid residue from the C-terminal and 'hth
of the resin bound peptide was removed. The gp 120 416-423 peptide was cleaved from the
resin by treating with TFA in presence of thioanisole, phenol, water and ethanedithiol at
room temperature for 8 h. The cleavage yield was 98% (30 mg) as indicated by the
estimation of remaining peptide bound to the resin. The crude peptide gave a single peak on
HPLC analysis indicating high purity. The solvent system used was 0.1% TFA in water (A)
and 0.1% TFAIacetonitrile (B) at flow rate of l d m i n . The gradient used is shown in
Fig. 5-11. a. Amino acid analysis data of the peptide agreed with the target sequence.
(a) ("1 Fig. 5-11. (a) HPLC time-course analysis of the peptide (Lys-Ala-Met-Tyr-Ala-Pro-Pro-
Ile) using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitrile; Flow rate: 0.5 d l m i n ; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.
The stepwise synthesis of the gp 120 sequence was continued with the remaining
resin adopting the DCC/HOBt coupling procedure. The synthesis was stopped at the 2ofi
amino acid residue from the C-terminal to get a gp120 (404-423) peptide resin, and 'Afi of
the resin was kept aside. The gp 120 (404-423) peptide was cleaved fiom the resin by using
TFA in presence of thioanisole, water and ethanedithiol at room temperature for 8 h. The
crude peptide was obtained in 95% yield (72 mg). The peptide formed was passed
through a sephadex G-25 column and the peptidyl fractions were collected and
lyophilized. The HPLC analysis showed the moderate purity of the peptide (Fig. 5-12.a).
Amino acid analysis data of the peptide agreed with the target sequence
Fig. (a) (b)
5-12. (a) HPLC time-course analysis of the peptide (Ile-Lys-Gln-Ile-Ile-Asn-Met- Trp-Gln-Lys-Val-Gly-Lys-Ala-Met-Tyr-Ala-Pro-Pro-Ile) using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitrile; Flow rate: 0.9 mL/min; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.
The stepwise synthesis was continued with the remaining resin using the DCCmOBt
coupling method. The synthesis was stopped at the 3 5 ~ amino acid residue ffom the
C-terminal to get a gp 120 (389423) peptide resin. 'Afi of the resin was removed and the peptide
was cleaved fiom the resin by using TFA in presence of thioanisole, water, phenol and
ethanedithiol at room temperature for 8 h (Yield=130 mg, 93%). The crude peptide was
passed through a sephadex G-25 column and KPLC analysis was carried out (Fig. 5-13.a).
Amino acid analysis data ofthe peptide agreed with the target sequence.
(a) (b) Fig. 5-13. (a) HPLC time-course analysis of the peptide (35 residue) using the buffer (A)
0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitrile; Flow rate: 0 5 d m i n ; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.
The remaining amino acids in the gp 120 (381-3) sequence were attached in a
stepwise manner using DCC/HOBt active ester method. The finished peptide was cleaved
from the resin by using TFA in presence of thioanisole, water, phenol and ethanedithiol
(a) (b) Fig. 5-14. (a) HPLC time-course analysis of the peptide (43-residue) using the buffer (A)
0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitrile; Flow rate: 0.5 mL/min; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.
for 8 h at room temperature. The uude peptide was obtained in 90% yield (520 mg). The
peptide formed was purified on HPLC using C-18 reverse phase column. The solvent used was
0.1% W w a t e r (A) and 0.1% TFNacetonitrile (B) at a flow rate of 1 mUmin (Fig. 5-14 a)
5.3. Experimental
Materials
Cesium carbonate, t-butyl carbazate, dicyclohexyl carbodiimide (DCC),
diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), thioanisole, ethanedithiol and
phenol were purchased from Aldrich Chemical Co., USA and Boc-amino acids were
purchased from Novabiochem Ltd., UK. All solvents used were of HPLC grade
purchased from E. Merck (India) and SRL (India). HPLC was done on a Pharmacia Akta
purifier instrument using C-18 reverse phase semi. preparative HPLC column. The amino
acid analysis was carried out on an LKB 4151 Alpha plus amino acid analyzer. Mass
spectra of peptides were performed in a Kratos PC Kompact MALDI TOF MS
instrument.
5.3.a. General procedure for solid phase peptide synthesis
The following steps are involved in the synthesis of peptides using Boc-
chemistry.
The C-terminal Boc-amino acid (3 mmol excess than the capacity of the resin)
was dissolved in minimum amount of ethanol and a saturated solution of CszC03 was
added till the pH become 7. Ethanol was evaporated under pressure and water was
removed by azeotropic distillation with dry benzene. The white powdery cesium salt of
Boc-amino acid was dissolved in minimum amount of NMP and added to the pre-swollen
chloromethyl resin (200 mg, 0.136 mmol C1) in NMP. The reaction mixture was kept at
50 "C for 24 h. The resin was washed with NMP (6 x 30 mL), NMP: water (I:], 6 x
30 mL), methanol (5 x 25 mL), DCM (5 x 25 mL), ether (5 x 25 mL) and dried under
vacuum. The Boc protection was removed by 30% TFA in DCM. The resin was washed
with DCM (6 x 30 mL) and neutralized with 5% DIEA in DCM. The resin was washed
DCM (5 x 25 mL) and NMP (5 x 25 mL). The extent of incorporation of amino acid was
estimated by picric acid titration method.
The stepwise incorporation of amino acids to the resin was carried out manually
in a specially designed silanised glass vessel clamped to a mechanical shaker. The
successive amino acids in the target sequence were coupled to the resin as their HOBt
active ester. After 30 min add DMSO (2.4 pL) and shaken for 5 min and then add one
drop of DIEA and kept for 5 min. Each steps of coupling reactions were monitored by
Kaiser's test.' After the synthesis, the peptidyl resin was washed with NMP (5 x 25 mL),
methano1:DCM (33:67, 10 x 25 mL), DCM (5 x 25 mL), ether (5 x 25 mL) and dried in
vacuum. The peptide was cleaved by suspending in TFAIscavenger mixture for 8 h. The
reaction mixture was filtered and the filtrate was evaporated till an oily residue obtained.
The peptide was precipitated by adding ice-cold ether and the precipitate was washed
with ether until the scavengers are removed and dried in vacuum. A small amount of
peptide was dissolved in water, injected to C-18 RPC column, and eluted with 0.1% TFA
in water (A) and 0.1% TFA in acetonitrile: water (80:20) (B) in a linear gradient.
The following peptides were synthesized using the above protocol,
1. Phe-Phe-Thr-Lys-Phe-Lys-Ser-Gln
Amino acid analysis: Phe, 3.08 (3); Thr, 0.89 (1); Lys, 1.92 (2); Ser, 0.86 (1); Glu,
0.97 (1). Gln hydrolyzed to Glu.
MALDI TOF MS: m/z 1033.3 [(M+H)+, 100%], C ~ ~ H ~ ~ N ~ I O L Z , requires M+ 1032.15.
2. Val-Gln-Gln-Pro-Trp-Gly-Gly-Ala-AIa-Val
Amino acid analysis: Val, 2.13 (2); Glu, 1.93 (2); Pro, 0.97 (1); Gly, 2.02 (2); Ala,
1.98 (2). Gln is hydrolyzed to Glu and Trp destroyed during acid hydrolysis.
MALDI TOF MS: m/z 1070.8 [(M+H)+, loo%], C47H74NI~016, requires M' 1069.35.
3. Val-Asn-Asn-Gln-Gln-Asn-Asn-Ile-Gly-Gln-Gln-Gly-Ala-Ala-Va1
Amino acid analysis: Val, 2.03 (2); Asp, 3.98 (4); Glu, 4.1 (4); Ile, 1.0 (1); Gly,
2.14 (2); Ala, 2.01 (2). Asn and Gln hydrolyzed to Asp and Glu.
MALDI TOF MS: mlz 1556 [(M+H)+, loo%], CSXH,~~NZ~OZX, requires M' 1554.58.
4. Val-Val-Val-Val-Val-Val-Val-Val-Val-Val
Amino acid analysis: Val, 9.84 (10).
MALDI TOF MS: m/z 1010.4 [(M+H)+, 100°?], CSOH~~NIOOII , requires M' 1009.3
5. Val-Gln-Asn-Asn-Val-Val-Val-Val-Val
Amino acid analysis: Val, 5.93 (6); Glu, 1.04 (1); Asp, 2.1 (2). Asn and Gln are
hydrolyzed to Asp and Glu.
6. Pro-Val-Val-Thr-Thr-Val-Val-Val-Val-Gln
Amino acid analysis: Pro, 0.89 (1); Val, 5.84 (6); Thr, 1.52 (2); Glu, 1.01 (1). Gln
hydrolyzed to Glu and Thr was lost during hydrolysis.
7. Thr-Val-Val-Val-Val-Asn
Amino acid analysis: Thr, 0.85 (1); Val, 4.1 (4); Asp, 0.93 (1). Asn was
hydrolysed to Asp.
8. Pro-Met-Leu-Phe-Val-Thr
Amino acid analysis: Pro, 0.95 (l), Met, 0.91 (1); Leu, 1.03 (1); Phe, 0.98 (1);
Val, 0.97 (1); Thr, 0.89 (1).
9. Val-Met-Leu-Phe-Leu-Pro
Amino acid analysis: Val, 1.01 (1); Met, 0.92 (1); Leu, 2.1 (2); Phe, 0.91 (1); Pro,
0.93 (1).
10. Met-Leu-Phe-Tyr-Val-Gly
Amino acid analysis: Met, 0.93 (1); Leu, 0.98 (1); Phe, 0.93 (1); Tyr, 0.91 (1);
Val, 1.01 (1); Gly, 1.0 (1).
11. Boc-Met-Leu-Phe-Cys(Acm)-Lys(C1-Z)-Val-OMe
Boc-Val (88 mg, 0.4 mmol) was attached to the chloromethylated resin by cesium
salt method. Afier removing the Boc group by 30% TFNDCM and neutralization with
5% DIEAIDCM, Boc-Lys(C1-Z) (142 mg, 0.34 mmol), HOBt (46 mg, 0.34 mmol) and
DCC (70 mg, 0.34 mmol) in NMP were added and shaken for 30 min. DMSO (2.4 pL)
was added, shaken for 5 min followed by the addition of a drop of DIEA and kept for
5 min. After washing with 33% MeOH/DCM (5 x 25 mL), DCM (5 x 25 mL), NMP (5 x
25 mL), a second coupling was performed using the same procedure. The remaining
amino acids Boc-Cys(Acm) (100 mg, 0.34 mmol), Boc-Phe (90 mg, 0.34 mmol),
Boc-Leu (79 mg, 0.34 mmol) and Boc-Met (85 mg, 0.34 mmol) were successively added
till the target sequence was formed. After the synthesis, the peptidyl resin was suspended
in anhydrous methanol (1 5 mL) in presence of DIEA (1.75 mL) for 8 h with occasional
stirring at 50 'C, The polymeric material was filtered off and washed with dry methanol
and DCM. The resin was subjected to three cycles of trans-esterification for complete
recovery of peptide. The filtrate was evaporated to get an oily residue and was
precipitated as a white powder by the addition of cold ether (41mg). The peptide was
washed thoroughly with ether to remove DIEA and passed through a sephadex G-10
column, the peptidyl fractions were collected and lyophilized. tlc analysis showed a
single spot corresponding to the peptide. HFLC analysis gave a single peak
corresponding to the peptide. The solvent system used HFLC were 1% TFNwater (A)
and 1% TFAtacetonitrile (B).
Amino acid analysis: Met, 0.94 (1); Leu, 1.1 (1); Phe, 0.91 (1); Cys, 0.84 (1); Lys, 0.91
(1); Val, 0.94 (1).
MALDI TOF MS: m/z 1095 [(M+H)+, loo%], C47H74N120~6, requires M+ 1094.762.
12. Boc-Pro-Met-Leu-Phe-Val-Thr-OMe
Cesium salt of Boc-Thr (126 mg, 0.4 mmol) was prepared by treating with a
saturated solution of CszCO3 till the pH become 7. The solvent was removed by
azeotropic distillation with dry benzene and the cesium salt of Boc-Thr formed was dried
over PzOs under vacuum. The cesium salt was dissolved in minimum amount of NMP
and chloromethyl resin (200 mg, 0.14 mmol C1) was added and kept at 50 'C for 24 h
with occasional shaking The resin was washed with NMP (5 x 25 mL), NMP:water ( l : l ,
5 x 25 mL), DCM (5 x 25 mL), methanol (5 x 25 mL), ether (5 x 25 mL) and dried in
vacuum. The amount of Thr attached to the resin was estimated by picric acid titration
method and was observed as 0.56 mmol/g.
The Boc protection was removed by 30% TFA/DCM and after neutralization with
5% DJEA/DCM, Boc-Val(74 mg, 0.34 mmol), HOBt (46 mg, 0.34 mmol) and DCC (70 mg,
0.34 mmol) were added and shaken for 30 min. DMSO (2.4 $)was added, shaken for 5 min
followed by the addition of a drop of DJEA and kept for 5 min. After washing with 33%
methanol/DCM (5 x 25 mL), DCM (5 x 25 mL), NMP (5 x 25 mL) a second coupling was
performed using the same procedure. The remaining amino acids Boc-Phe (90 mg,
0.34 mmol), Boc-Leu (79 mg, 0.34 mmol), BooMet (84.7 mg, 0.34 mmol), Boc-Pro
(73 mg, 0.34 mmol) were successively coupled using HOBt active ester method. After
the synthesis the peptide was cleaved from the resin by suspending in anhydrous
methanol (15 mL) and DIEA (1.75 mL). The suspension was kept at 50 'C with
occasional stirring for 8 h. The polymeric material was filtered and washed with dry
methanol and DCM. The peptidyl resin was subjected to three cycles of trans-
esterification for quantitative removal of the peptide. The filtrate and washings were
evaporated under vacuum till an oily residue obtained. It was then precipitated by adding
ice cold-ether (1 13 mg). The peptide formed was washed thoroughly with ether to
remove DEA. The crude peptide was passed through a sephadex G-10 column, the
peptidyl fractions were collected and lyophilized. tlc analysis gave a single spot
corresponding to the peptide. HPLC analysis using 1% TFNwater (A) and 1%
TFNacetonitrile (B) as eluent, a single peak was observed corresponding to the peptide.
Amino acid analysis: Pro, 0.92 (1); Met, 0.93 (1); Leu, 1.1 (1); Phe, 0.98 (1); Val, 1.2 (1);
Thr, 0.68 (1). Thr had a low value because it undergoes degradation under hydrolytic
conditions.
13. Boc-VaCMet-Leu-Phe-Leu-Pro-OMe
Boc-Pro (88 mng, 0.4 mmol) was dissolved in minimum amount of ethanol, a
saturated solution of Cs~C03 was added with stirring till pH become 7. The ethanol was
evaporated and the water was removed by azeotropic distillation with dry benzene. The
white powder obtained was kept over P205 under vacuum. Cesium salt was dissolved in
minimum amount of NMP and chloromethyl resin (200 mg, 0.14 mmol Cl) was added to
it and kept at 50 "C for 24 h with occasional shaking. The resin was washed with NMP
(5 x 25 mL), NMP.water (1.1, 5 x 25 mL), DCM (5 x 25 mL), methanol (5 x 25 mL),
ether (5 x 25 mL) and dried under vacuum. The extent of incorporation of Boc-Pro was
estimated by picric acid method and was observed to be 0.59 mmollg.
After the deprotection of Boc group with 30% TFA/DCM, followed by
neutralization with 5% DIEA/DCM, Boc-Leu (79 mg, 0.34 mmol), HOBt (46 mg,
0.34 mmol) and DCC (70 mg, 0.34 mmol) in NMP were added and shaken for 30 min.
DMSO (2.4 pL) was added, shaken for 5 min followed by addition of a drop of DlEA
and kept for 5 min. After washing with 33% methanol/DCM (5 x 25 mL), DCM (5 x
25 mL) and NMP (5 x 25 mL), a second coupling was conducted using the same
procedure. The remaining amino acids Boc-Phe (90 mg, 0.34 mmol), Boc-Leu (79 mg,
0.34 mmol), Boc-Met (84.7 mg, 0.34 mmol), and Boc-Val (74 mg, 0.34 mmol) were
successively attached by HOBt active ester coupling method. After the synthesis, the
peptide was cleaved from the resin by suspending in dry methanol (I5 mL) and DIEA
(1 75 mL). The reaction mixture was kept at 50 OC with occasional shaking for 8 h. The
polymeric material was filtered off and washed with methanol and DCM. The filtrate along
with the washings was evaporated under vacuum to obtain an oily residue. The peptide was
precipitated by the addition of cold ether and was washed thoroughly with ether to remove
DIEA (105 mg). The crude peptide was passed through a sephadex G-10 column and the tlc
analysis showed a single spot corresponding to the peptide. The HPLC analysis of the
peptide using 1% TFAIwater (A) and 1% TFAIacetonitrile (B) showed a single peak
supporting the extra purity of the peptide.
Amino acid analysis: Val, 0.9 (1); Met, 0.89 (1); Leu, 2.12 (2); Phe, 1.1 (1); Pro, 0.93 (1).
14. Boc-Met-Leu-Phe-Tyr(Bz1)-Val-Gly-OMe
Boc-Gly (72 mg, 0.4 mmol) was dissolved in minimum amount of ethanol and
saturated solution of Cs2C03 was added to it with stirring till the pH become 7. Ethanol
was evaporated and water was removed by azeotropic distillation with dry benzene till a
white reside was obtained and it was kept over P205 in vacuum. The residue was
dissolved in minimum amount of NMP and chloromethylated resin (200 mg, 0.14 mmol CI)
was added and kept at 50 OC for 24 h with occasional shaking. The resin was washed with
NMP (5 x 25 mL), NMP:water (1:1, 5 x 25 mL), DCM (5 x 25 mL), methanol (5 x
25 mL), ether (5 x 25 mL) and dried under vacuum (0.6 mmol Glylg)
Boc-protection was removed by 30% TFADCM and after neutralization with 5%
DIEAIDCM, Boc-Val(74 mg, 0.34 mmol), HOBt (46 mg, 0.34 mmol) and DCC (70 mg,
0.34 mmol) in NMP were added and shaken for 30 min. DMSO (2.4 pL) was added,
shaken for 5 min fbllowed by addition of a drop of DIEA and kept for 5 min. The resin
was washed with 33% methanol in DCM (5 x 25 mL) and NMP (5 x 25 mL), a second
coupling was also conducted following the same procedure. The remaining amino acids
Boc-Tyr(Bz1) (126 mg, 0.34 mmol), Boc-Phe (90 mg, 0.34 mmol), Boc-Leu (79 mg,
0.34 mmol) and Boc-Met (84.7 mg, 0.34 mmol) were successively coupled by HOBt
active ester method. The peptide was cleaved from the support by suspending in dry
methanol (15 mL) and DIEA (1.75 mL). The reaction mixture was kept at 50 OC for 8 h.
The polymeric material was filtered, washed with TFA and DCM. The filtrate was
evaporated under vacuum till an oily residue was obtained. The peptide was precipitated
by the addition of ice-cold ether and was washed thoroughly with ether to remove DIEA
(yield=118 mg). The crude peptide was passed through a sephadex G-10 column and tlc
showed a single spot for the peptide. HPLC analysis gave a single peak for the peptide.
Amino acid analysis: Met, 0.91 (1); Leu, 0.93 (1); Phe, 0.95 (1); Tyr, 0.66 (1); Val, 1.1
(1); Gly, 1.13 (1). Tyr had a low value because it undergoes degradation in hydrolytic
conditions.
MALDI TOF MS: d z 934.4 [(M+H)+, loo%], C S ~ H ~ ~ N ~ O ~ ~ S Z C I , requires Id 933.13.
15. Synthesis of Leucine Enkephalin
(Tyr-Gly-Gly-Phe-Leu)
Boc-Leu (95 mg, 0.4 mmol) was dissolved in minimum amount of ethanol and
saturated solution of cesium carbonate was added dropwise till the pH become 7.
Ethanol was evaporated under reduced pressure, water was removed by azeotropic
distillation with benzene and the cesium salt was dried over PzOs. The cesium salt was
added to the pre-swollen chloromethyl resin (200 mg, 0.136 mmol C1) in NMP and
heated to 50 "C for 24 h in an oil bath. The resin was filtered and washed with NMP (6 x
25 mL), 1: 1 NMP:water (6 x 25 mL), MeOH (6 x 25 mL), DCM (6 x 25 mL), ether (6 x
25 mL) and dried under vacuum,
Boc protection was removed by 30% TFA in DCM for 30 min. After washing
with DCM (6 x 25 mL), the resin was neutralized with 5% DIEA in DCM for 10 min.
The resin was washed with DCM (6 x 25 mL) and NMP (6 x 25 mL). The remaining
amino acids in the target sequence were attached by HOBt active ester method. The
HOBt active ester was prepared by dissolving HOBt (46 mg, 0.34 mmol) and DCC
(70 mg, 0.34 mmol) in NMP and shaking the solution with respective Boc-amino acids
for 1 h. The precipitated DCU was filtered off and the active ester was added to pre-
swollen Boc-deprotected Leu-resin. After 30 min, DMSO (2.4 pL) was added followed
by DIEA (6 pL) and shaken for 10 min. Boc-Phe (90 mg, 0.34 mmol), Boc-Gly (60 mg,
0.34 mmol) and Boc-Tyr(Bz1) (126 mg, 0.34 mmol) were used for the preparation of
HOBt active esters. Each coupling steps were monitored by semi-quantitative ninhydrin
test. A second coupling was also performed for the confirmation of quantitative coupling.
The peptidyl resin was washed with NMP (6 x 25 mL), 1.1 NMP:water (6 x 25 mL),
MeOH (6 x 25 mL), DCM (6 x 25 mL), ether (6 x 25 mL) and dried under vacuum.
The peptide was cleaved from the resin by using TFA (2.85 mL) and water
(150 pL) After 8 h, the solution was filtered and the filtrate was concentrated
under vacuum The peptide was precipitated by adding ice-cold ether and the peptide
was thoroughly washed with cold ether (6 x 25 mL) yielded 71 mg peptide.
Amino acid analysis. Leu, 1.04 (1); Phe, 1.0 (1); Gly, 2.14 (2) Tyr, 0.72 (1). Tyr had a
low value because it undergoes degradation in hydrolytic conditions.
MALDI TOF MS: m/z 556.8 [(M+H)+, loo%], C28H37Nj07, requires Mt 555.641.
16. Synthesis of P-casomorphin (Bovine)
(Tyr-Pro-Phe-Pro-Gly-Pro-Ile)
To a solution of Boc-Ile (95 mg, 0.4 mmol) in minimum amount of ethanol, a
saturated solution of cesium carbonate was added drop wise till the pH become 7.
Ethanol was evaporated under pressure, water was removed by azeotropic distillation
with dry benzene and the white powdery cesium salt was dried over PzOs. The cesium
salt was added to the pre-swollen chloromethyl resin (200 mg, 0.136 mmol CI) in NMP
and kept at 50 'C for 24 h. The resin was filtered and washed with NMP (6 x 30 mL), 1: 1
NMP:water (6 x 30 mL), methanol (6 x 30 mL), DCM (6 x 30 mL), ether (6 x 30 mL)
and dried under vacuum.
The Bo~protection was removed by keeping the resin in 30% TFA in DCM for
30 min. The resin was washed with DCM (6 x 30 mL) and NMP (6 x 30 mL), the remaining
amino acids in the target sequence were coupled by HOBt active ester method. The HOBt
active ester was prepared by dissolving HOBt (46 mg, 0.34 mmol), DCC (70 mg, 0.34 mmol)
in minimum amount of NMP and stirred with the respective Bo~amino acid for 1 h. DCU
formed was filtered off and the active ester was added to the pre-swollen Boc-deprotected
Ile-resin. Atter 30 min treatment of active ester with the resin, DMSO (2.4 pL) was
added and kept for 5 min. A drop of DIEA was also added to the reaction mixture and
kept for 5 min. Boc-Pro (73 mg, 0.34 mmol), Boc-Gly (60 rng, 0.34 mmol), Boc-Phe
(90 mg, 0.34 mmol) Boc-Tyr(Bz1) (126 mg, 0.34 mmol) were used for the preparation of
HOBt active esters. Each coupling steps were monitored by Kaiser's test. A second
coupling was also performed for confirming the quantitative coupling. The peptidyl resin
was washed with NMP (6 x 25 mu, MeOH (6 x 25 mL), DCM (6 x 25 mL), ether (6 x
25 mL) and dried under vacuum.
The detachment of peptide from the resin was achieved by treating with TFA
(2.65 mL), EDT (75 pL), thioanisole (150 @) and water (150 bL). After 6 h, the solution
was filtered and the filtrate was concentrated under vacuum. The peptide was precipitated
by adding ice-cold ether and thoroughly washed with ether to remove the scavengers. The
peptide was dissolved in 1% acetic acid in water and passed through a sepha& G-10
column. The peptidyl fractions were collected and lyophilized to yield 102 mg of peptide.
Amino acid analysis: Ile, 0.97 (1); Pro, 2.94 (3); Phe, 0.95 (1); Tyr, 0.71 (1). Tyr had a
low value because it undergoes degradation in hydrolytic conditions.
17. Synthesis of P-casomorphin (Human)
(Tyr-Pro-Phe- Val-Glu-Pro-Ile)
Boc Ile (95 mg, 0.4 mmol) was dissolved in minimum amount of ethanol and a
saturated solution of cesium carbonate was added drop wise till the pH become 7.
Ethanol was removed under pressure and water was removed by azeotropic distillation
with dry benzene. The cesium salt was added to the pre-swollen chloromethyl resin
(200 mg, 0.34 mmol) in NMP and heated to 50 OC for 24 h. The resin was filtered washed
with Nh4P (6 x 25 mL), 1:l NMP:H20 (6 x 25 mL), MeOH (6 x 25 mL), DCM
(6 x 25 mL), ether (6 x 25 mL) and dried under vacuum.
The Boc-protection was removed by 30% TFA in DCM for 30 min. After
washing with DCM (6 x 25 mL), the resin was neutralized with 5% DIEA/DCM for
10 min. The resin was washed with DCM (6 x 25 mL) and NMP (6 x 25 mL). The
remaining amino acids in the target sequence were successively attached by HOBt active
ester method. HOBt (46 mg, 0.34 mmol) and DCC (70 mg, 0.34 mmol) in NMP along
with Boc-amino acids were used for the preparation of active ester. The precipitated DCU
was filtered off and active ester solution was added to pre-swollen Boc-deprotected
Ile-resin. After 30 min treatment of the resin with active ester DMSO (2.4 &) and DIEA
(6 &) were added and kept for 10 min. Boc-Pro (73 mg, 0.34 mmol), Boc-Glu(Bzl)
(115 mg, 0.34 mmol),Boc-Val (74 mg, 0.34 mmol), Boc-Phe (90 mg, 0.34 mmol),
Boc-Tyr(Bz1) (126 mg, 0.34 mmol) were used for the preparation of HOBt active esters.
Each coupling steps were monitored by ninhydrin test. A second coupling was also
performed for confirming the quantitative reaction. The peptidyl resin was washed with
NMP (6 x 25 mL), MeOH (6 x 25 d ) , DCM (6 x 25 d ) , ether (6 x 25 mL) and dried
under vacuum.
The peptide was cleaved from the resin using TFA (2.7 mL), ethanedithiol
(75 pL), thioanisole (1 50 FL) and water (75 pL). After 8 h, the solution was filtered
and the filtrate was evaporated to get an oily residue. The peptide was precipitated
by the addition of' ice-cold ether and washed thoroughly with ether to remove the
scavengers. The crude yield is 112 mg. The peptide was dissolved in 1% acetic acid in
water and passed through a sephadex G-10 column. The peptidyl fractions were collected
and lyophilized.
Amino acid analysis: Ile, 1.01 (1); Pro, 1.98 (2); Glu, 0.94 (1); Val, 0.97 (1); Phe, 1.02
(1); Tyr, 0.74 (1). Tyr had a low value because it undergoes degradation in hydrolytic
conditions.
18. Synthesis of C-reactive Protein
(Val-Gly-Gly-Ser-Glu-Ile)
Boc-Ile (95 mg, 0.408 mmol) was dissolved in minimum amount of ethanol and a
saturated solution of cesium carbonate was added dropwise till the pH become 7.
Ethanol was evaporated under vacuum and water was removed by azeotropic distillation with
dry benzene. The cesium salt was added to the preswollen chloromethyl resin (200 mg,
0.136 mmol CI) in NMP and heated to 50 OC for 24 h. The resin was filtered and washed with
NMP (6 x 25 mL), 1 :I NMF':water (6 x 25 mL), MeOH (6 x 25 mL), DCM (6 x 25 mL), ether
(6 x 25 mL) and dned under vacuum.
The Boc-protection was removed by 30% TFA in DCM for 30 min. AAer
washing with DCM (6 x 25 mL), the resin was neutralized with 5% DIEA/DCM for
10 min. The HOBt active esters of the remaining amino acids in the target sequence was
prepared and successively attached. HOBt (46 mg, 0.34 mmol), DCC (70 mg,
0.34 mmol) in NMP along with Boc-amino acids were used for the preparation of active
ester. The DCU was filtered off and active ester solution was added to the pre-swollen
Boc-deprotected Ile-resin. Boc Glu(Bz1) (115 mg, 0.34 mmol), Boc-Ser(Bzl) (100 mg,
0.34 mmol), Boc-Gly (60 mg, 0.34 mmol), Boc-Val (74 mg, 0.34 mmol) were used for
the preparation of HOBt active esters. The coupling time was 40 min and each coupling
steps were monitored by semi-quantitative ninhydrin test. A second coupling was also
performed. The resin was washed with NMP (6 x 25 mL), MeOH (6 x 25 mL), DCM
(6 x 25 mL), ether (6 x 25 mL) and dried under vacuum.
The peptide was cleaved from the resin by treating with TFA (2.7 mL),
ethanedithiol (75 wL), water (75 pL) and thioanislole (150 pL). After 8 h, the solution
was filtered and the filtrate was evaporated to get an oily residue. The peptide was
precipitated as a white powder by adding ice-cold ether and washed thoroughly with
ether to remove the scavengers. The peptide was dissolved in 1% acetic acidlwater and
passed through a sephadex G-10 column. The peptidyl fractions were collected and
lyophilized to yield 73 mg of peptide.
Amino acid analysis: Ile, 1.03 (I); Glu, 0.93 (1); Ser, 0.79 (I); Gly, 2.1 (2); Val, 0.96 (1).
Ser had a low value because of loss during acid hydrolysis.
MALDl TOF MS: ml'z 562.1 [(M+H)+, loo%], C23&N6ol0, requires M+ 560.614.
19. Synthesis of 33-42 fragment of Alzheimer's P-amyloid peptide.
(Gly-Leu-Met-~al-~l~-~l~-~al-~al-~le-~la)
Boc-Ala (64 mg, 0.408 mmol) was dissolved in minimum amount of ethanol and
a saturated solution of cesium carbonate was added drop-by-drop till the pH become 7.
Ethanol was evaporated under reduced pressure, water was removed by azeotropic
distillation with benzene and the cesium salt was dried under vacuum. The white
powdery cesium salt was added to the pre-swollen chloromethyl resin (200 mg,
0.136 mmol CI) in NMP and heated to 50 OC for 24 h in an oil bath. The resin was
filtered and washed with NMP (6 x 25 mL), 1:l NMP:water (6 x 25 A ) , MeOH (6 x
25 mL), DCM (6 x 25 mL), ether (6 x 25 mL) and dried under vacuum.
M e r removing the Boc groups with 30% TFAlDCM for (30 min), the resin was
washed thoroughly with DCM (6 x 25 mL) and neutralized with 5% DIEAIDCM for
10 min. The remaining amino acids in the target peptide sequence were attached
successively by HOBt active ester method. The active ester was prepared by treating
HOBt (46 mg, 0.34 mmol) and DCC (70 mg, 0.34 mmol) in NMP with Boc-amino acid
for 1 h. The precipitated DCU was removed by filtration and the active ester was added
to the pre-swollen Boc removed Ala-resin in NMP. After 30 min DMSO (2.4 pL) and
DIEA (6 pL) were added to the reaction mixture and kept for 10 min. Boc-Ile (79 mg,
0.34 mmol), Boc-Val (74 mg, 0.34 mmol), Boc-Gly (60 mg, 0.34 mmol), Boc-Met
(85 mg, 0.34 mmol) and Boc-Leu (79 mg, 0.34 mmol) were used for the preparation of
active esters. Each coupling steps were monitored by ninhydrin test. A second coupling
was also performed for the quantitative reaction. The peptidyl resin was washed with
NMP (6 x 25 mL), MeOH (6 x 25 mL), DCM (6 x 25 mL), ether (6 x 25 mL) and dried
under vacuum.
The peptide was detached from the resin using TFA (2.45 mL) in presence of
scavengers such as ethanedithiol (75 pL), water (150 pL), thioanisole (150 pL) and
phenol (150 pL). After 8 h, the solution was filtered and the filtrate was evaporated to get
an oily residue. The peptide was precipitated by adding ice-cold ether and washed
thoroughly with ether to remove the scavengers. The peptide was dissolved in 2% acetic
acidlwater and passed through a sephadex G-10 column and the peptidyl fractions were
collected and lyophilized to yield 118.3 mg of peptide. The purity of the peptide was
checked by passing through a C-18 RPC HPLC column using 0.1% TFA in water (A) and
0.1% TFA in 80% acetonitrile/20% water (B) in a gradient used as shown in Fig. 5-8a.
Amino acid analysis: Ala, 1.0 (1); Ile, 0.98 (1); Val, 2.89 (3); Gly, 3.16 (3); Met, 0.79 (1);
Leu, 1.09 (1). MALDI TOF MS: mlz 916.2 [(M+H)+, 67%], 937.6 [(M+Na)', 100%],
955.9 [(M+K)+, 55%], C41H74N10011S, requires M' 915.158.
20. Synthesis of Inhibitor of Ribonucleotide reductase of Herpes Simplex Virus-Type I
(Tyr-Ala-Gly -Ala-Val-Val-Asn-Asp-Leu)
Boc-Leu (95 mg, 0.4 mmol) was dissolved in minimum quantity of ethanol and a
saturated solution of cesium carbonate was added drop wise till the pH become 7. The
ethanol was evaporated under reduced pressure and water was removed by azeotropic
distillation with berrzene. The white powdery cesium salt was dried over P20s. The
cesium salt was added to the pre-swollen chloromethyl resin (200 mg, 0.136 mmol C1) in
M and kept at 50 OC for 24 h in an oil bath. The resin was filtered and washed with
NMP (6 x 25 mL), 1 : l NMP:water (6 x 25 mL), MeOH (6 x 25 mL), DCM (6 x 25 mL),
ether (6 x 25 mL) and dried under vacuum.
The Boc group was removed by 30% TFA/DCM for 30 min. The resin was washed
thoroughly with DCM (6 x 25 mL) and neutralized with 5% DIEA/DCM for 10 min. The
successive amino acids in the target sequence were attached by HOBt active ester method.
The active ester was prepared by treating HOBt (46 mg 0.34 mmol), DCC (70 mg,
0.34 mmol) in NMP with Boc-amino acids for 1 h. The DCU formed was removed by
filtration and the active ester was added to the pre-swollen Boc- removed Leu-resin. After
30 min, DMSO (2.4 &) and DIEA (6 @) were added and kept for 10 min. Boc-Asp(Bz1)
(1 10 mg, 0.34 mmol), Boc-Asn (79 mg, 0.34 mmol), Boc-Val(74 mg, 0.34 mmol), Boc-Ala
(64 mg, 0.34 mmol), Hoc-Gly (60 m& 0.34 mmol), Boc-Tyr(Bzl) (126 mg, 0.34 mmol) were
used for the preparation of HOBt active ester. The extent of coupling was monitored by
ninhydrin test and a second coupling was performed to confirm the quantitative reaction. The
resin was filtered and washed with NMP (6 x 25 mL), MeOH (6 x 25 mL), DCM (6 x
25 mL), ether (6 x 25 mL) and dried under vacuum.
The peptide was cleaved from the resin using TFA (2.625 mL) in presence of
ethanedithiol (75 &), water (150 &) and thioanisole (150 @). After 8 h, the polymeric
material was filtered off and the filtrate was concentrated under pressure. The peptide was
precipitated by adding ice-cold ether and was washed thoroughly with ether to remove the
scavengers. The peptide was dissolved in 2% acetic acidlwater and passed through a
sephadex G- 10 column. The peptidyl fractions were collected and lyophilized to yield 56 mg
of peptide. The purity of the peptide was checked by HPLC using a C-18 RPC column. 0.1%
TFA in water (A) and 0.1% TFA in acetonitrilelwater (4:l) (Ei) was used as the eluent. The
gradient used is as shown in Fig. 5-9a.
Amino acid analysis: Leu, 0.96 (1); Asp, 2.1 (2); Val, 1.94 (2); Ala, 2.0 (2); Gly,1.02 (1);
Tyr, 0.73 (1). Tyr had a low value because it undergoes degradation in hydrolytic
conditions.
MALDI TOF MS: m/z 922.2 [(M+H)+, loo%], C ~ ~ H ~ ~ N I Z O I ~ , requires M' 921.01.
21. Synthesis of Scyliorhinin-1 peptide
(Ala-Lys-Phe-Asp-Lys-Phe-Tyr-Gly-Leu-Met)
Boc-Met (100.3 mg, 0.4 mmol) was dissolved in minimum quantity of ethanol
and a saturated solution of cesium carbonate was added drop wise till the pH become 7.
The ethanol was evaporated under reduced pressure and water was removed by
azeotropic distillation with benzene. The white powdery cesium salt was dried over PzOs.
The cesium salt was added to the pre-swollen chloromethyl resin (200 mg,
0.136 mmol C1) in W and kept at 50 OC for 24 h in an oil bath. The resin was filtered
and washed with NMP (6 x 25 mL), 1:l NMP:water (6 x 25 mL), MeOH (6 x 25 mL),
DCM (6 x 25 mL), ether (6 x 25 mL) and dried under vacuum.
The Boc group was removed by 30% TFADCM for 30 min. The resin was washed
thoroughly with DCM (6 x 25 mL) and neutralized with 5% DIEA/DCM for 10 min. The
successive amino acids in the target sequence were attached by HOBt active ester method.
The active ester war prepared by treating HOBt (46 mg, 0.34 mmol), DCC (70 mg,
0.34 mmol) in NMP with Boc-amino acids for 1 h. The DCU formed was removed by
filtration and active ester was added to the pre-swollen Boc- removed Met-resin. ARer
30 min DMSO (2.4 pL) and DIEA (6 pL) were added and kept for 10 min. Boc-Asp(Bz1)
(110 mg, 0.34 mmol), Boc-Ala (64 mg, 0.34 mmol), Boc-Gly (60 mg, 0.34 mmol),
Boc-Tyr(Bz1) (126 mg, 0.34 mmol) Boc-Leu (79 mg, 0.34 mmol), Boc-Phe (90 mg,
0.34 mmol), Boc-Lys(C1-Z) (142 mg, 0.34 mmol) were used for the preparation of HOBt
active ester. The extent of coupling was monitored by ninhydrin test and a second
coupling was performed to confirm the quantitative reaction. The resin was filtered and
washed with NMP (6 x 25 mL), MeOH (6 x 25 mL), DCM (6 x 25 mL), ether (6 x
25 mL) and dried under vacuum.
The peptide was cleaved from the resin using TFA (2.625 mL) in presence of
ethanedithiol (75 pL), water (150 pL) and thioanisole (150 pL). After 8 h, the polymeric
material was filtered off and the filtrate was concentrated under reduced pressure. The
peptide was precipitated by adding ice-cold ether and was washed thoroughly with ether
to remove the scavengers. The peptide was dissolved in 2% acetic acidlwater and passed
through a sephadex G-10 column. The peptidyl fractions were collected and lyophilized
to yield 63 mg of peptide. The purity of the peptide was checked by HPLC using a
C-18 RPC column. 0.1% TFA in water (A) and 0.1% TFA in acetonitrilelwater (4: 1) (B)
was used as the elue,nt. The gradient used is as shown in Fig. 5-10a.
Amino acid analysis: Leu, 0.96 (1); Asp, 1.1 (1); Ala, 1.0 (1); Gly,1.02 (1); Tyr, 0.73 (1);
Phe, 1.98 (2); Lys, 1.94 (2); Met, 0.89 (1). Tyr had a low value because it undergoes
degradation in hydrolytic conditions.
MALDI TOF MS: m / ~ 1220.6 [(M+H)+, loo%], C59H86N~2014S, requires M+ 1219.483.
22. Synthesis of 43-residue peptide from the CD 4 binding domain of Human Immunodeficiency Virus Envelope Glycoprotein
(Asn-Ser-Thr-Trp-Ser-Thr-Lys-Gly-Ser-Asn-Asn-Tb-Glu-Gly-Ser-Asp-Thr-Ile-Th-
Leu-Pro-Cys(Acm)-Arg-Ile-Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln-Lys-Val-Gly-Lys-Ala-
Met-Tyr-Ala-Pro-Pro-Ile)
The peptides were synthesized on a 2% chloromethylated PS-BDODMA resin
(200 mg) having a chlorine capacity 0.68 mmol/g. Boc-Ile was first attached to the resin
by cesium salt method Boc-Ile (95 mg, 0.4 mmol) was dissolved in minimum amount of
ethanol and a saturated solution of cesium carbonate was added dropwise till the pH
become 7. Ethanol was evaporated under vacuum and water was removed by azeotropic
distillation with dry benzene. The cesium salt was added to the pre-swollen chloromethyl
resin (200 mg, 0.136 mmol CI) in NMP and heated to 50 "C for 24 h. The resin was
filtered and washed with NMP (6 x 25 mL), 1.1 Nh4P:water (6 x 25 mL), MeOH (6 x
25 mL), DCM (6 x 25 mL), ether (6 x 25 mL) and dried under vacuum.
The Boc-protection was removed by 30% TFA in DCM for 30 min. After
washing with DCM (6 x 25 mL), the resin was neutralized with 5% DIEA/DCM for
10 min. The HOBt active esters of the remaining amino acids in the target sequence were
prepared and successively incorporated. HOBt (46 mg, 0.34 mmol), DCC (70 mg,
0.34 mmol) in NMP along with the Boc-amino acid were used for the preparation of
active ester. The DCU was filtered off and the active ester solution was added to the pre-
swollen Boc-deprotected Ile-resin. Boc-Pro (73 mg), BooAla (64 mg), Boc-Tyr(Bzl)
(126 mg), Boc-Met (85 mg), Boc-Lys(C1-Z) (141 mg), Boc-Gly (60 mg, 0.34 mmol),
Boc-Val (74 mg, 0.34 mmol), Boc-Gln (84 mg), Boc-Trp (103 mg), Boc-Asn (79 mg),
Boc-Ile (79 mg), Boc-Arg(Mts) (155 mg), Boc-Cys(Acm) (100 mg), Boc-Leu (79 mg),
Boc-Thr(Bzl) (105 mg), Boc-Asp(0Bzl) (1 10 mg), Boc-Ser(Bzl) (100 mg, 0.34 mmol),
Boc Glu(OBz1) (115 mg, 0.34 mmol) were used for the preparation of HOBt active
esters The coupling time was 40 min and each coupling steps were monitored by semi-
quantitative ninhydrin test. The coupling steps were repeated wherever necessary
(Table.5-I). The resin was washed with NMP (6 x 25 mL), MeOH (6 x 25 mL), DCM
(6 x 25 mL), ether (6 x 25 mL) and dried under vacuum.
The synthesis was stopped at the 8& amino acid residue from the C-terminal and
Yi~ of the peptide hound resin was removed. The gp120 (416-423) peptide (Lys-Ala-Met-
Tyr-Ala-Pro-Pro-Ile) was cleaved from the resin by treating with TFA (2.7 mL),
ethanedithiol(75 pL), water (75 pL) and thioanisole (150 pL). After 8 h, the solution was
filtered and the filtrate was evaporated to get an oily residue. The peptide was
precipitated as white powder by adding ice-cold ether and washed thoroughly with ether
to remove the scavengers The peptide was dissolved in 1% acetic acidwater and passed
through a sephadex G-10 column. The peptidyl fractions were collected and lyophilized
to yield 30 mg of peptide. HPLC analysis was carried out using 0.1% TFA in water (A)
and 0.1% TFA in 80% acetonitrile/20% water (B) as eluent. The gradient used as shown
in Fig.5-lla.
Amino acid analysis: Lys, 0.94 (1); Ala, 2.1 (2); Met, 0.91 (1); Tyr, 0.74 (1); Pro,
01.96 (2); Ile, 1.03 (1). Tyr had a low value because of degradation in hydrolytic
conditions.
MALDI TOF MS: mlz 891.5 [(M+H)+, 100%], C4~&7N9010S, requires Mf 890.126.
The stepwise synthesis of the gp 120 sequence was continued with the remaining
resin adopting the DCCtHOBt coupling procedure. The synthesis was stopped at the 2 0 ~
amino acid residue from the C-terminal to get a gp 120 (404-423) peptide resin, and 'Ath of
the resin was kept aside. The gp 120 (404-423) peptide was (Ile-Lys-Gln-Ile-Ile-Asn-Met-
Trp-Gln-Lys-Val-Gly-Lys-Ala-Met-Tyr-Ala-Pro-Pro-Ile) cleaved from the resin by TFA
(2.7 mL), ethanedithiol (75 pL), water (75 pL) and thioanisole (150 pL). Atter 8 h, the
solution was filtered and the filtrate was evaporated to get an oily residue. The peptide
was precipitated as a white powder by adding ice-cold ether and washed thoroughly with
ether to remove the scavengers. The peptide was dissolved in 1% acetic acidlwater and
passed through a sephadex G-10 column. The peptidyl fractions were collected and
lyophilized to yield 75.6 mg of peptide. HPLC analysis was carried out using 0.1% TFA
in water (A) and 0.1% TFA in 80% acetonitrile/20% water (B) as eluent. The gradient
used as shown in Fig. 5-12a.
Amino acid analysis: Ile, 3.93 (4); Lys, 2.98 (3); Glu, 1.93 (2); Asp, 0.97 (1); Met, 1.45
(2); Val, 1.1 (1); Gly, 1.08 (1); Ala, 2.1 (2); Tyr, 0.71 (1); Pro, 1.94 (2). Asn and Gln
hydrolyzed to Asp and Glu. Trp is destroyed during acid hydrolysis. Low value of Tyr is
due to its degradation in acid hydrolysis.
MALDI TOF MS: mlz 2330.9 [(M+H)+, loo%], C , ~ ~ H , ~ T N ~ O ~ ~ S ~ , requires M+
2329.927.
The stepwise synthesis was continued with the remaining resin using the
DCCiHOBt coupling method. The synthesis was stopped at the 35* amino acid residue
from the C-terminal to get the gp 389.423 peptide resin. Vith of the resin was removed and
the peptide (Ser-Asn-Asn-Thr-Glu-Gly-Ser-Asp-Thr-Ile-Th-Leu-Pro-Cys(Acm)-~g-Ile-
Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln-Lys-Val-Gly-Lys-Aa-Met-Tyr-Ala-Pro-Pro-Ile) was
cleaved from the resin using TFA (2.7 mL), ethanedithiol (75 pL), water (75 pL) and
thioanisole (150 FL). ARer 8 h, the solution was filtered and the filtrate was evaporated
to get an oily residue. The peptide was precipitated as a white powder by adding ice-cold
ether and washed thoroughly with ether to remove the scavengers. The peptide was
dissolved in 1% acetic acidlwater and passed through a sephadex G-10 column. The
peptidyl fractions were collected and lyophilized to yield 135 mg of peptide. HPLC
analysis was carried out using 0.1% TFA in water (A) and 0.1% TFA in 80%
acetonitrile/20% water (B) as eluent. The gradient used as shown in Fig. 5-13a.
Amino acid analysis: Ser, 1.64 (2); Asp, 3.96 (4); Thr, 2.56 (3); Glu, 2.9 (3); Gly, 2.2 (2);
Ue, 4.92 (5); Leu,, 0.97 (1); Pro, 2.89 (3); Cys, 0.91(1); Arg, 0.95 (1); Lys, 2.87 (3);
Met,1.76 (2); Val, 1.12 (1); Ala, 2.2 (2); Tyr, 0.71 (1). Asn and Gln hydrolyzed to Asp
and Glu. Trp is destroyed during acid hydrolysis. Low value of Tyr is due its degradation
during acid hydrolysis.
MALDI TOF MS: m/z 3992.3 [w+H)+, loo%], C ~ ~ ~ H X + ~ N ~ S O ~ Z S ~ , requires M+
3990.721.
TABLES-1. Amino acid attachment requiring more than one coupling steps in the synthesis of 43-residue peptide
Amino acids
Tyr419-Met418 -
L . Y S ~ I ~ - G ~ ~ I Z
C;IIL~IZ-TT~I I
Trp411-Met410
Asnios-Ile40s
Ile408-Ile407
Gln406-Ly~405
Ile404-Arg~
ATg403-cys402
A~n39~-Asn3~0
Ly~387-Thr386
St:r3~~-Trp384
Trp3~4-Thr383
Number of couplings
2
2
2
2
2
2
2
4
3
3
2
2
3
The remaining amino acids in the gp381-423 sequence were attached in a stepwise
manner using DCC/HOBt active ester method. The finished peptide was cleaved from the
resin using TFA (2.7 mL), ethanedithiol(75 pL), water (75 pL) and thioanisole (I50 bL).
ARer 8 h, the solution was filtered and the filtrate was evaporated to get an oily residue.
The peptide was precipitated as a white powder by adding ice-cold ether and washed
thoroughly with ether to remove the scavengers. The peptide was dissolved in 1% acetic
acidlwater and passed through a sephadex G-10 column. The peptidyl fractions were
collected and lyophilized to yield 582 mg of peptide. HPLC analysis was carried out
using 0.1% TFA in water (A) and 0.1% TFA in 80% acetonitrile/20% water (B) as eluent.
The gradient used is as shown in Fig. 5-14a.
Amino acid analysis: Asp, 4.88 (5); Ser, 3.56 (4); Thr, 4.48 (5); Lys, 3.96 (4); Gly, 3.3
(3); Glu, 2.89 (3); Ile, 4.78 (5); Leu, 0.91 (1); Pro, 2.83 (3); Cys, 0.9 (1); Arg, 0.94 (1);
Met, 1.64 (2); Val, 1 . 1 (1); Ala, 2.21 (2); Tyr, 0.73 (1). Asn and Gln hydrolyzed to Asp
and Glu. Trp is destroyed during acid hydrolysis. Low value of Tyr is due its degradation
in acid hydrolysis.
MALDI TOF MS: m/z 4854.6 [(M+H)+, 10O0h], C~llH336N~8067S3, requires M+
4853.579.
References
1. Barany, G.; Merrifield, R. B. in "Peptides: Analysis, Synthesis, Biology" Gross, E; Meienhofer, J: Eds., New York. Academic Press, 1980, Vo1.2 pp 3-285.
2. Merrifield, R. B. Science 1986, 232, 341.
3. Fodor, S. P. A,; Leighton Reed, J.; Pirmng, M. C.; Stryer, L.; Tsai Lu, A.; Solas, D. Science 1991, 251, 767.
4. Eichler, J.; Houghten, R. A. Mol. Med To-day 1995,1, 174.
5. Bodanzky, M. "Principles of peptide synthesis"; Springer Verlag: Berlin. 1984, pp 158-201
6. Bettersby, J . E.; Hascock, W. S.; Canova Davis, E.; Osewein, J.; 0' Conner, B. Int. J. Peptide Protein Res. 1994, 44,2 15.
7. Marsden, B. J.; Nguyer, T. M. D.; Schiller, P. W. Int. J Peptiide Protein Res. 1993,43,3 13.
8. Kaiser, E.; Colescott, R C.; Bossinger, C. D.; Cook, P. I. Anal. Biochem. 1970,34,594.
9. Hughes, J . Nature 1975, 258, 577.
10. Henschen, A, Z. Physiol. Chem. 1979, 360, 121 1
1 1 . Greenberg, R. -1. Biol. Chem. 1984,259, 5132.
12. Shephard, E. G. Immunology 1992, 76,79.
13. Gregori, L. J. Biol. Chem. 1995,270, 19702.
14. Vasilakos, J. P.; Michael, J. G. J. Immunol. 1993, 150, 2346.
15. Barre-Sinoussi, F.; Chermann, J. C.; Rey, F.; Nugeyre, M. T.; Chamaret, S.; Gruest, J.; Dauguet, C.; Axler-Blinc, C.; Vezinet-Bmn, F.; Rouzioux, C.; Rosenbaum, W.; Montagnier, L. Science 1983,220, 868.
16. Gallo, R. C.; Salahuddin, S. 2.; Popovic, M.; Shearer, G. M.; Kaplan, M.; Hayer, B. F.; Palker, T. J. :, Redfield, R.; Oleske, J.; Safari, G.; White, G.; Foster, P.; Markham, P. D. Science 1984, 224, 500.
17. Hart, T. K.; Kirsh, R.; Ellens, H.; Sweet, R.W.; Lambert, D. M.; Pettaway, S. R.; Leaq, J.; Buzelski, P. Proc. Null. Acad. Sci. 1991, 88,2189.
18. Sun,N. C.;Ho,D.;Sun,C.R.Y.;Liou,R.S.,Gordon,W.,Fung,M. S.C.,Li,X.L., Tiny, R. C., Lee, T. H., Chang, N. T.; Chang, T. W. J. Virol. 1989, 3579.
19. George, D. G.; Barker, W. C.; Mewes, H. W.; Pfeiffer, F.; Tsugita, A. Nucl. Acidr Res. 1994, 22, 3569.
20. Kaiser, E. T.; Mihara, H.; Laforet, G. A.; Kelly, J. W.; Walters, L.; Findies, M. A.; Sasaki, T. Science 1989, 243, 187.