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.