PS-BDODMA RESIN, MERRIFIELD RESIN AND SHEPPARD RESIN IN PEPTIDE

SYNTHESIS-A COMPARATIVE STUDY

4.1. Introduction

T he PS-DVB resin though highly stable, its hydrophobic character can result in

unfavourable coupling reactions. The swelling character and hydrophobicity of

the polymer can be changed by substituting the cross-linker DVB with

BDODMA. lnfulence of BDODMA cross-linker in the polystyrene support for

polypeptide synthesis was studied by synthesizing model peptides under identical

reaction conditions. The new support was also compared with Sheppard resin in order to

evaluate its efficiency in polypeptide synthesis. The hydrophilic-hydrophobic balance of

PS-BDODMA resin influences its physicochemical properties, and the yield and purity of

the peptide reveals the advantages of the resin over conventional resins.

4.2. Results and Discussion

The rate of formation of any given peptide bond in solid phase synthesis depends

on the nature of the support, the dispersing solvent, the acylating reagent and the structure

of the protected peptide chain up to that point.' A new chemically inert PS-BDODMA

resin was synthesized and its efficiency in peptide synthesis was compared with

Merrifield and Sheppard resins. 65-74 fragment of acyl carrier protein and leucyl-alanyl-

glycyl-valine are synthesized for the comparison of PS-BDODMA with Merrifield and

Sheppard resins. HMPA handle was used as the spacer between the resin and growing

peptide. Peptides 3-7 are synthesised for the comparison of PS-BDODMA with

Merrifield resin. Boc amino acids were used for the synthesis of peptide 3 & 4. For

peptides 5-7, p-[(R,S)-a-[l-(9H-fluorene-9-yl)methoxyformamido]-2,4-dimethoxy-

benzyll-phenoxyacetamidomethyl group was used as the spacer between the peptide and

the resin. Fmoc-amino acids were used for the synthesis of peptide 1,2,5,6 & 7.

4.2.a. 4-Hydroxymethyl phenoxyacetamidomethyl2% butanediol dimethacrylate cross-linked polystyrene (PS-BDODMA-HMPA) resin

The PS-BDODMA-MA resin was prepared from aminomethyl

PS-BDODMA resin by treating with HMPA linker. HOBt/HBTU/DLEA coupling method

was used for the synthesis of PS-BDODMA-HMPA resin (Scheme. 4-1).

-NH~ + HO HOBt/HBTU DIEA

Scheme. 4-1. Preparation of PS-BDODMA-HMPA resin

Fig.4-1. IR (KBr) spectrum of PS-BDODMA-HMPA resin

The IR spectrum of the resin showed a band at 3380 cm-' for OH group, 3400 cm"

for NH group and 1643 cm-I for NHCO group (Fig.4-1). The C-terminal amino acid was

incorporated to the resin via an ester bond. The ester bond is stable enough to withstand

the repeated treatment of 20% piperidine in DMF. After the synthesis the peptide was

cleaved from the resin by using TFA at room temperature for 2 h.

.2.b. p-[(R,S)- a-[l-(9H-fluoren-9-y1) methoxyformamido]-2,4-dimethoxybenzyll- phenoxy acetamidomethyl 2% butanediol dimethacrylate cross-linked polystyrene (PS- BDODMA-Rink amide) resin

The PS-BDODMA-Rink amide resin was prepared by treating aminomethyl

butanediol dimethacrylate cross-linked polystyrene resin with p-[(R,S)-a-[I-(9H-

fluorene-9-yl)methoxyformamido]-2,4-dimethoxybey]-phenoxyacetic acid linker. The

linker was coupled to the aminomethyl resin in presence of HBTU, HOBt and DIEA

(Scheme. 4-2).

HOBt,HBTq J DIE*

Scheme. 4-2. Preparation of PS-BDODMA-Rink amide resin

The IR (KBr) spectrum of the resin showed bands at 3365 cm" 0 and 1643 cm.'

(-NHCO-) (Fig.4-2). The Fmoc group present in the resin was removed by 20% piperidine

in DMF. The amino acid corresponding to the C-terminal region was attached to the resin by

an amide bond. The resin-peptide bond is very stable even affer repeated treatment with 20%

piperidine in DMF After the synthesis the peptide was cleaved fiom the resin by using TFA

at room temperature for 2 h.

Fig.4-2. IR (KBr) spectrum of PS-BDODMA-Rink amide resin

4.2.c. Synthesis and characterization of peptides

1. Synthesis of Leu-Ala-Gly-Val

The efficiency of PS-BDODMA resin was compared with Merrifield and

Sheppard resins by synthesizing Leu-Ala-Gly-Val peptide. HMPA was used as the spacer

between the resin and the growing peptide chain. The resins with 0.16 mmol OWg was

used for the synthesis The C-terminal Val was attached to the resin by using MSNT in

presence of N-methyl irnidazole. The extent of attachment was calculated from the OD

value of the adduct formed when a pre-weighed resin was suspended in 20% piperidine in

DMF for 20 min. After removing the Fmoc-protection with piperidine-DMF (1:4), the

remaining amino acids were coupled successively till the target sequence was formed. All

acylation reactions were completed in 30 min by a single coupling step. The comparative

percentage incorporation of amino acids are given in Fig.4-3. Finally the peptide was

cleaved from the resin by using TFA. The PS-BDODMA resin yielded 12 mg, PS-DVB

yielded 9 mg and Sheppard resin yielded 12.2 mg of peptide. The HPLC profile of the

peptide from PS-BDODMA resin and Sheppard resin showed a single peak, where as that

from PS-DVB showed some small peaks in addition to a major peak (Fig.4-4). Amino

acid analysis of the peptide from PS-BDODMA resin: Leu, 1.02 (1); Ala, 1.0 (1); Gly,

0.98 (I); Val, 1.01 ( I ) .

V G Amino ac~ds A L

- -

1 PS-BDODMA-HMPA . PS-DVEHMPA Sheppard-HMPA -- .

Fig.4-3. Extent of incorporation of amino acids in PS-BDODMA-HMPA, PS-DVB- HMPA and Sheppard-HMPA resins.

Fig.4-4. HPLC timer-course analysis of peptide Feu-Ala-Gly-Val) kom (a) PS-BDODMA (b) PS-DVB and (c) Sheppard resin using the buffer (A) 0.5 mL of TFA in 100 mL of water and (B) 0.5 mL of TFA in 100 mL of acetonitri1e:water (4: 1). Flow rate: 0.5 mL/min. Gradient used: O%B in 5 min and 100% B in 50 min.

2. Synthesis of (65-74) fragment of Acyl carrier protein

The efficiency of the new support was further established by comparing the purity

of 65-74 fragment of acyl carrier protein synthesized on PS-BDODMA, PS-DVB and

Sheppard resins under identical synthetic conditions. 4-Hydroxymethylphenoxyacetic

acid handle (HMPA) was attached to the amino methyl resin by using HBTU, HOBt and

DIEA. PS-DVB, Sheppard and PS-BDODMA resins attached with W A handle were

used for the synthesis. The respective resins were taken in 0.01 mmol scales. C-terminal

Fmoc-Gly was attached to the respective resins by an ester bond using MSNT in presence

of N-methyl imidazole The extent of attachment was measured from the UV absorbance

of the adduct of dibenzofulvene and piperidine formed by the treatment of accurately

weighed Fmoc-amino acid attached resin with 20% piperidine in DMF. After removing

the Frnoc-protection with 20% piperidine in D m , the remaining Fmoc-amino acids were

coupled by using 3 equiv of HBTU, HOBt and DIEA. In a particular coupling reaction

amino acid and coupling reagent required for three resins were calculated, weighed and

dissolved in a definite volume of D m , this solution was distributed equally in the

respective resins, and the coupling reaction was continued for 30 min. The percentage

incorporation of amino acids is given in Fig.4-5 a. The peptide was cleaved from the

resin using TFA in presence of scavengers. PS-BDODMA resin yielded 30 mg,

Merrifield resin yielded 25 mg and Sheppard resin yielded 3 1 mg crude peptide. HF'LC

profiles of the crude peptides obtained from different resins were as shown in Fig.4-6.

The peptide from PS-BDODMA resin and Sheppard resin showed a sharp single major

peak whereas that from Merrifield resin showed more than one major peaks. The

comparative study indicates that PS-BDODMA resin can be used as a better solid support

for peptide synthesis than the PS-DVB resin and is as efficient as Sheppard resin.

Amino acid analysis of peptide from PS-BDODMA resin: Val, 0.98 (1); Ile, 2.1 (2); Tyr,

0.89 (1) Asp, 1.92 (2); Ala, 2.1 (2); Glu, 0.93 (1); Gly, 0.98 (1). Asn and Gln are hydrolysed

to Asp and Glu.

MALDI TOF MS: nl/z 1046.3 [(M+H)+, loo%], C47H74N12Ol6, requires M+ 1045.12

PS-DVB-HMPA resrn

-- 0

O N I ) D I A A Q V Amino ands

(a) (b) Fig.4-5. (a) Extent of incorporation of amino acids in PS-BDODMA-HMPA, PS-DVB-HMPA

and Sheppard resins @) MALDI TOE MS ofthe peptide eom PS-BDODMA resin

Fig.4-6. HPLC time-course analysis of peptide (65-74 fragment of ACP) synthesized on (a) Sheppard resin; (b) PS-DVB resin and (c) PS-BDODMA resin using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4: 1); Flow rate: 0.5 mL/min; Gradient used: 0% B in 5 min and 100% B in 50 min.

3. Synthesis of Leu-Gly-Ala-Leu-Gly-Ata

The peptide was synthesized on PS-BDODMA and Merrifield resins in identical

conditions using Boc-chemistry. Cesium salt of Boc-Ala was attached to chloromethyl

PS-BDODMA and PS-DVB resins. The incorporation of amino acid to the resins were

estimated by picric acid titration method. PS-BDODMA resin (200 mg, 0.68 mmol Cl/g)

and PS-DVB resin (200 mg, 0.62 mmol ,CVg) were used for peptide synthesis. The

remaining amino acids in the target sequence were incorporated by DCCMOBt active

ester method. A single 40 min coupling was used for the synthesis. The percentage of

~ncorporation of amino acids are given in Fig.4-7 a. The peptide was cleaved from the

resins by using TFA. The peptide was obtained in 98% yield from PS-BDODMA resin as

evidenced by the amino acid analysis of residual resin after cleavage of the peptide. The

peptide obtained from PS-DVB resin was 96% yield as estimated by the amino capacity

of the resin after the removal of the peptide. The HPLC profile of the peptide obtained

from PS-BDODMA gave a single peak whereas that obtained from PS-DVB gave a small

peak along with the major peak. (Fig.4-8). Amino acid analysis of peptide from

PS-BDODMA resin: Leu, 2.0 (2); Gly, 1.98 (2); Ala, 2.04 (2). UUBS ,001 I

110 90

80 ~

100 70

i !

C 0 .- 60 5 90 %["I.

% M

S E 80

40 .- s

70

10

60 .---- - . . 7~

G 500 BOG 7W

A ci L A L Ammo acids M~u/Charge

(a) (b)

Fig.4-7. (a) Extent of incorporation of amino acids to PS-BDODMA and PS-DVB resins (b) MALDI TOF MS of the peptide from PS-BDODMA resin

Fig.4-8. HPLC time-course analysis of peptide (Leu-Gly-Ala-Leu-Gly-Ala) synthesized on (a) PS-BDODMA resin and (b) PS-DVB resin 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.

MALDI TOF MS: m/z 502.93 [(M+H)+, loo%], C22&N607, requires h/ft 501.604.

4. Synthesis of Ala-Ala-Ala-Ala

The peptide was synthesized on PS-BDODMA and PS-DVB resins in identical

conditions using Boc-chemistry. Cesium salt of Boc-Ala was incorporated to chloromethyl

PS-BDODMA and PS-DVB resins. The extent of attachment of the amino acid was

estimated by picric acid method. Boc-Ala-PS-DVB resin with 0.6 mmol Alalg and Boc-Ala-

PS-BDODMA resin with 0.67 mmol Alalg was used for the synthesis. 200 mg of each of the

resin was used for the synthesis. The remaining amino acids in the sequence were attached by

DCCiHOBt active ester method. A single 40 min coupling was used for the synthesis. The

extent of amino acid incorporation are given in Fig.4-9. The peptide was cleaved from the

PS-BDODMA and PS-DVB resins by using TFA. The crude peptide was obtained in 98%

yield from PS-BDODMA resin as revealed by the amino acid analysis of the residual resin

after cleavage of the peptide. The peptide obtained from PS-DVB was in 95% yield. The

HPLC profile of the peptide obtained from PS-BDODMA gave a single peak where as that

form PS-DVB resin gave small peaks along with a major peak (Fig.4-lo). Amino acid

analysis of peptide from PS-BDODMA resin: Ala, 4.06 (4).

-x- PS-BDODMA I - PS-DVB I A A A A

Amino acids

Fig.4-9. Extent of incorporation of amino acids to PS-BDODMA and PS-DVB resins

Fig.4-10. HPLC time-course analysis of peptide (Ala-Ala-Ala-Ala) synthesized on (a) PS-BDODMA resin and (b) PS-DVB resin using the buffer (A) 0.5 mL TFA in I00 niL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0 5 d i m i n ; Gradient used: 0% B in 5 min and 100% B in 50 min.

5. Synthesis of 25-residue fragment of NS-1 region of Hepatitis C viral polyprotein(leu-Ile-Asn-Thr-Asn-Ala-Ser-Trp-His-A1a-Asn-Arg-Thr-Ala- Leu-Ser-Asn-AspSer- Lys-Leu-Asn-Thr-Gly-Ala-NHz)

Fmoc-Ala was attached to the Fmoc-removed PS-BDODMA-Rink amide and

PS-DVB-Rink amide resins in presence of HBTU/HOBt and DIEA. In a typical coupling

reaction Fmoc-amino acid, HBTU, HOBt and DIEA required for both the resins were

calculated, weighed, dissolved in a definite amount of DMF and distributed equally to the

resins and the reaction was allowed to continue for 30 min. After each coupling, Fmoc

protection was removed by 20% piperidine in DMF. The extent of amino acid attachment

are given in Fig.4-1 I

4 5 , , , , I , i , , , , , i I I i i 8 I I I !

A G T N 1 , F S D N S L A T R N A H W S A N T N I L

Amim acids

Fig.4-11. Extent of incorporation of amino acids in PS-BDODMA and PS-DVB resins

The peptides were cleaved form the resins by using TFA in presence of acid

scavengers such as thioanisole, EDT, water and phenol. The crude peptide obtained from

PS-BDODMA resin was in 98% yield and that from PS-DVB resin was in 95% yield as

revealed by the amino acid analysis of the residual resin after cleavage of the peptide.

The HPLC profile of the crude peptide obtained from PS-BDODMA gave a sharp single

peak whereas that from PS-DVB resin gave some additional peaks along with the major

peak (Fig.4.12) The yield of crude peptides from PS-BDODMA and PS-DVB resins

were 94 mg and 96 mg respectively. Amino acid analysis: Ala, 4.08 (4); Gly, 0.98 (1);

Thr, 2.74 (3); Asn, 4.92 (5); Leu, 3.12 (3); Lys, 0.93 (1); Ser, 3.08 (3); Asp, 0.85 (1);

Arg, 0.93 (1); His, 0.91 (1); Trp, 0.94 (1); Ile, 1.1 (1).

MALDl TOF MS: m/z 2668 [(M+H)+, 100%], C I I ~ H I ~ ~ N ~ ~ O ~ ~ , requires M' 2666.922.

The CD spectrum, shown in the Fig.4-13 b, is characterized by a negative cotton

effect at 221 nm and a second negative band at 198-201 nrn. The long wavelength cotton

effect observed near 221 nm is ascribed to the peptide n-tx* transition, while at 198-201 nm

is the exciton-split x+n* transitions. These observations revealed the helical property for

the peptide.

Fig.4-12. HPLC time-course analysis of peptide (25-residue) synthesized on (a) PS- BDODMA resin and (b) PS-DVB resin using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 rnL acetonitri1e:water (4:l); Flow rate: 0.5 ml/min; Gradient used: 0% B in 5 min and 100% B in 50 min.

Fig.4-13. (a) MALDl TOF MS and (b) CD spectrum of the 25-residue peptide from PS-BDODMA resin

6. Synthesis of 14-residue and 10-residue fragments of NS-1 region of Hepatitis C viral polyprotein (Leu-lle-Asn-Thr-Asn-Ala-Ser- Trp-His-Ala-Asn-Arg- Thr- Ala-NH2) & (Leu-Asn-Cys(Acm)-Asn-Asp-Ser-Leu-Asn-Thr-Ala-NH~)

The peptides were synthesized on PS-BDODMA-Rink amide and PS-DVB-Rink

amide resins by the above described conditions to demonstrate the efficiency of

PS-BDODMA support in polypeptide synthesis. The extent of amino acid incorporation

of 14-residue peptide is Fig.4-14. The crude peptide was obtained in 54 mg (96%) yield

for PS-BDODMA resin and 53.5 mg (90%) yield for PS-DVB resin. The HPLC profile

of the peptide obta~ned from PS-BDODMA resin showed a single sharp peak

corresponding to target peptide whereas that from PS-DVB resin showed two small peaks

in addition to the major peak (Fig.4-15). The amino acid analysis of the purified peptide

also agreed with the target sequence.

Amino acid analysis: Ala, 3.03 (3); Thr, 1.94 (2); Arg, 0.91 (1); Asn, 2.74 (3); His, 0.89

(1); Trp, 0.90 (1); Ser, 1.08 (1); Ile, 1.1 (l);Leu, 1.12 (I).

MALDl TOF MS m/z 1568.73 [(M+H)+, loo%], C67H106N24020, requires M+ 1567.73 1.

The extent of each amino acid coupling of the 10-residue peptide (Leu-Asn-

Cys(Acm)-Asn-Asp-Ser-Leu-Asn-Thr-Ala-N&) is given in Fig.4-17. After the synthesis

the peptide was cleaved from the resin by using TFA in presence of acid scavengers such

as thioanisole, water and ethanedithiol. The crude peptide was obtained in 40 mg (98%)

yield for PS- BDODMA resin and 38.7 (90%) yield for PS-DVB resin. he HPLC profile

of the peptide obtained form PS-BDODMA resin showed only a sharp single peak

whereas that obtained from PS-DVB resin showed two small peaks along with a major

peak (Fig.4-18). The amino acid analysis of the purified peptide agreed with the target

sequence.

Amino acid analysis: Ala, 1.04 (1); Thr, 0.92 (1); Asn, 2.73 (3); Leu, 2.02 (2);Ser, 1.1

( I ) ; Asp, 0.84 (1); Cys, 0.92 (1)

MALDl TOF MS: d z 1134.4 [(M+H)+, loo%], C44H75N15018S, requires M' 1133.232.

0

A T R N A H W S A N T N I L Amino =ids

Fig.4-14. Extent of incorporation of amino acids in PS-BDODMA and PS-DVB resin

The CD spectrum of the 14-residue peptide showed (Fig. 4-16 b) a negative

cotton effect at 222 nm and a second negative band at 198-200 nm. The long wavelength

effect observed near 222 nm is ascribed to the peptide n+n* transition, while at

198-201 nm is the exciton-split x-n* transitions. These observations proposed a helical

character for the peptide.

(a) (b) Fig.4-15. HPLC time-course analysis of peptide (14-residue) synthesized on (a) PS-

BDODMA resin and (b) PS-DVB resin using the buffer (A) 0.5 mL TFA in I00 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4: 1); Flow rate: 0.5 mL/min; Gradient used: 0% B in 5 min and 100% B in 50 min.

Fig. 4-16. (a) MALDl TOF MS and (b) CD spectrum of the 14-residue peptide from PS- BDODMA resin

2 0 ! * , T , N , L , , D ,rjL 1 0 --

Amino acids

Fig.4-17. Extent of incorporation of amino acids in PS-BDODMA and PS-DVB resins

E'ig.4-18: HPLC time-course analysis of peptide (lo-residue) synthesized on (a) PS- BDODMA resin and (b) PS-DVB resin 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 mLlm111, Gradient used: 0% B in 5 rnin and 100% B in 50 min.

Fig. 4-19. MALDI TOF MS of the 10-residue peptide synthesized on PS-BDODMA resin

4.3. Experimental

4.3.a. Materials:

4-Hydroxymethylphenoxyacetic acid (HMPA), p-[(&s)dl-[l-(9H-fluorene-Pyl)

methoxyformamido]-2,4-dimethoxybenzyl] phenoxyacetic acid (Rink amide handle),

2-(1H-benzotriazole-l -y1) 1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU),

1-hydroxybenzotriazole (HOBt), Boc and Fmoc-amino acids, chloromethyl and

aminomethyl PS-DVB resins and Sheppard resin were purchased from Novabiochem

Ltd., UK. Cesium carbonate, t-butyl carbazate, dicyclohexyl carbodiimide (DCC),

diisopropylethyl amine (DIEA), piperidine, trifluoroacetic acid (TFA), thioanisole,

ethanedithiol and phenol were obtained from Sigma Chemicals Co., USA. All solvents

used were of HPLC grade purchased from E. Merck, India, BDH (India) and SISCO

chemicals (Bombay). 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 and CD was recorded

on a JASCO 71 5 spectropolarimeter.

4.3.b. Preparation of BOC-azide?

t-Butyl carbazate was dissolved in a mixture of glacial acetic acid (27 mL) and

water (37.5 mL). NaNOz (7.4 g) was added in small amounts with vigorous stirring. The

temperature was maintained at 0 "C. After about 90 min, an oily layer was separated from

the aqueous layer. The aqueous layer was extracted with ether (3 x 30 mL) and was

mixed with oily layer. The mixture was washed with water and 0.1M NaHC03 dried over

anhydrous NazS04. Boc-azide was obtained by evaporating the ether under reduced

pressure. It was used directly with out any purification.

4.3.c. Preparation of amino acid derivatives

I. Amino acid methyllethyl ester^:^ Absolute methanoVethanol (10 mL) was cooled to -10 OC in a freezing mixture.

rhionylchloride (1 mL) was added slowly with vigorous stirring. Amino acid was added

to the reaction mixture and slowly allowed to come to room temperature. After 12 h,

alcohol was evaporated The residue was dissolved in water, saturated with Na2C03,

extracted with CHC1, (3 x 30 mL) and dried over Na2S04. An oily amino acid

methyllethyl ester was obtained on evaporation of chloroform and was used immediately.

2. Boc-amino acids (Schnabel's m e t h ~ d ) : ~

L-amino acids (10 mmol) were suspended in 1:l dioxane-water (10 mL) and

Boc-azide (1.6 mL, 10 mmol) was added to it. The mixture was stirred at room

temperature maintaining the pH in the alkaline range with 4 N NaOH. AAer 24 h, water

(25 mL) was added and the solution was extracted with ether (10 mL). The aqueous

layer was cooled in an ice bath, acidified with 2 N HCI and extracted with ethyl acetate

(3 x 20 mL) For Boc-Leu, ether was used for extraction. It was then dried over

anhydrous NazS04 and the solvent was evaporated under vacuum.

Purity of the protected amino acids was monitored by tlc on silica gel using

CHC13-MeOH-Acetic acid (85:10:5) as solvent system. The tlc plate was exposed to HCI

vapour for 10 min, and ninhydrin was sprayed to the plate forming blue spots.

TABLE.1. Preparation Boc-amino acids

tlc Rr Amino acid 1 pH I Yield (%) I Melting point

Glycine 1 - K Alanine 10 --px Phenylalan~ne -

Leucine 10

Isoleucine 1 10

4.3.d. Preparation of I-Hydroxybenzotriazole (HOBt)

o-Chloronitrobenzene (32 g) was dissolved in ethanol (100 mL). Hydrazine

Glutamine

--t- 8-9

hydrate (30 g) was added and refluxed for 5 h. Ethanol was distilled off and the residue

obtained was diluted with water (100 mL) and extracted with ether (4 x 50 mL). The

aqueous layer was acidified with conc.HC1 and HOBt precipitated was recrystallized

from hot water (Yield=20 g, 80%) mp = 157 "C.

83

89

92

90

86

4.3.e. Methods for the purification and detection of peptides

1 . Column chromatography

Sephadex G-10, G-25, G-50 were used for the gel filtration depending on the size

of the peptides. Silica gel 60 (70-200 mesh size) was used for column chromatography.

2. Thin layer chromatography

A: Chloroform (85)-Methanol (10)-Acetic acid (5) B: Chloroform (95)-Acetic acid (5)

88

84

80

75

The tlc grade silica gel coated glass plates were used for tlc analysis. The

following solvent systems were used for tlc.

1-Butanol: acetic acid: water: ethyl acetate ( I : 1:l : I)

11 . 1-Butanol: acetic acid: water (6:1:5)

~ i i . I-Butanol acetic acid: water (4: 1:l)

90

54

87

81

61

137

118

-

-

0.65

0.62

0.70

0.74

0.68

.

0.21

0.33

0.39

0.28

0.42

0.64

0.36

0.41

0.56

0.35

0.00

0.00

0.40

IV. Ethyl acetate. pyridine: acetic acid: water (60:20:6:14)

v. Chloroform. methanol (9: I)

vi. Acetonitri1e:water (3: 1)

vii. Pyridine: acetic acid: water (50:35:15)

The following detection methods were used for tlc.

a) Ninhydrin

The Boc-protection was removed by introducing the plate into a chamber

containing conc.HC1 and was heated at 80-100 "C for 10 min. The spots were developed

by spraying ninhydrin reagent (0.1% wlv solution in acetone) and the tlc plate was heated

in an oven at 80 "C for 5 min. The free amino groups gave a violet spot.

b) Iodine

The tlc plates were exposed to iodine vapour in a closed chamber. Brown spots

were observed in case of amino acids and peptides.

c) Starch-potassium iodide test

I?ydOn'~ reagent: This method was preferred for the detection of peptides larger

than tripeptide. The developed tlc plate in suitable solvent system kept at 100 OC and the

plate was exposed to chlorine gas (the chlorine chamber was prepared by adding 2 N HCI

to potassium permanganate). After 3 min, the plate was heated at 100 O C for 5 min.

Freshly prepared 1% starch and 1% potassium iodide solution in distilled water were

sprayed to the tlc plate. Discrete purple spots were appeared.

d) Sakaguchi reagent

Compounds containing free guanidine group of arginine peptides give an orange

red colour with Sakaguchi reagent. The tlc plates were cooled at 0 "C for 10 min before

spraying the reagents.

Sakaguchi-A: A 0 1 % solution of 8-hydroxy quinoline in acetone.

Sakaguchi-B: A solution of bromine (0.6 mL) in 10 mL lM NaOH.

After developing the plate in suitable solvent system, the plate and the solutions

were cooled in a deep freezer for 30 min. The plate is then sprayed with Sakaguchi-A

reagent followed by Sakaguchi-B reagent. Arginine containing peptides give bright

orange spots within 10 sec.

3. Amino acid analysis

Amino acid analysis is used as an analytical method for the characterization of the

peptide and also used for the quantitation of peptide stock solution. 10 mg of peptidyl resin was

taken in a sample tube. 200 @ of TFA and 6 N HCI (1:l) were added to the tube. The tube

was hsed under nitrogen and heated to 110-120 OC for 6 h. The tube was opened and acid

dried over NaOH and PzO5 in a desiccator under high vacuum. The amino acid mixture was

dissolved in a suitable buffer. Loading buffer and an aliquot loaded in the analyzer capsule such

that every amino acid was expected in the 3-10 mmol range.

4. Matrix Assisted Laser Desorption/Ionization Mass Spectroscopy

The matrix used was a-cyano4hydroxycinnamic acid. A saturated solution of the

matrix was made by dissolving the chemical in an aqueous solution containing 30% acetonitrile

and 0.1% trifluoroacetic acid. 5 @ of the dilute sample solution (l0-~-10" M) was mixed with

10 @ of the saturated matrix solution and then 0.5 @ of the solution applied to the sample

slide. The sample spots were air-dried and the sample holder inserted into the mass

spectrometer through a vacuum lock. The instrument was operated in the reflectron mode,

using 20 kV accelerating voltage, with detection of positive ions.

5. Circular Dichroism

Solut~ons containing the peptide concentration between and 4 x 10" M were

prepared from accurately weighed peptide samples dissolved in 0.1 M phosphate buffer

(pH=7). CD spectra were measured at room temperature on a JASCO 715 spectropolarimeter

using 2 mm quartz cells. Dry purified nitrogen was employed to keep the instrument oxygen

free during the experiments. A complete base line was recorded for each measurement using

the same cell in which the sample solution had been replaced with pure solvent. The results

were plotted as the mean residue ellipticity (@),degree cm2 dmol-'.

4.3.f. Preparation of PS-BDODMA-HMPA Resin

4-Hydroxymethylphenoxyacetic acid (218.6 mg, 1.2 mmol), HBTU (455 mg,

1.2 mmol), HOBt (162 mg, 1.2 mmol), DIEA (200 FL, 1.2 mmol) were added to the pre-

swollen aminomethyl resin (2 g, 0.4 mmol) in DMF and the reaction mixture was kept at

room temperature for I h with occasional shaking. The quantitative conversion was

estimated by ninhydrin test. The resin was filtered and washed with DMF (3 x 30 mL),

dioxane/H20 (1 :I , 3 x 30 mL), MeOH (3 x 30 mL) and ether (3 x 30 mL). The resin was

collected and dried in vacuum.

1R (KBr): 3400 cm-' (NH), 3380 cm-' (OH), 1643 cm-' (NHCO).

4.3.g. Preparation of PS-DVB-HMPA resin

4-Hydroxymethylphenoxyacetic acid (240 mg, 1.32 mmol), HBTU (500 mg,

1.32 mmol), HOBt (178.3 mg, 1.32 mmol) and DIEA (230 pL, 1.32 mmol), were added

to the pre-swollen aminomethyl resin (2 g, 0.44 mmol) in DMF. The reaction mixture

was kept at room temperature for 1 h with occasional shaking. A second coupling

reaction was conducted for quantitative conversion. The resin was filtered and washed

with DMF (3 x 30 mL), dioxanelwater (1:1, 3 x 30 mL), MeOH (3 x 30 mL) and ether

(3 x 30 mL). The resin was collected and dried in vacuum.

4.3.h. Preparation of Sheppard-HMPA resin

Sheppard resin (2 g, 0.42 mmol) was swelled in DMF for 1 h. 4-Hydroxymethyl

phenoxy acetic acid (230 mg, 1.26 mmol), HBTU (478 mg, 1.26 mmol), HOBt (170 mg,

1.26 mmol) and DIEA (240 pL, 1.26 mmol) were added to the swollen resin. The

reaction mixture was kept at room temperature for 1 h with occasional shaking. The

quantitative conversion was estimated by ninhydrin test. The resin was filtered and

washed with DMF (3 x 30 mL), dioxanelwater ( l : l , 3 x 30 mL), MeOH (3 x 30 mL) and

ether (3 x 30 mL) The resin was collected and dried in vacuum.

4.3.i. Preparation of Fmoc-Val-0-CHZ-G&-O-CH~-CO-NH-C~-PSBDOD resin

PS-BDODMA-HMF'A resin (0.5 g, 0.19 mmollg) was swelled in dry CHzC12.

Dry Fmoc-Val (129 mg, 0.38 mmol) in a septum-stoppered flask was dissolved in dry

CHzC12 with the help of appropriate volume of MeIm (24 pL, 0.29 mmol) and a few

drops of dry THF. This solution was transferred to a stoppered flask containing MSNT

(121 mg, 0.38 mmol). The mixture was immediately added to the swollen polymer

support. After 30 min, the reactants were washed out first with CH2C12 (6 x 30 mL)

followed by DMF (6 x 30 mL). The Fmoc-protection was cleaved off with

20% piperidine in DMF and coupling yield was determined by the W absorption of the

dibenzofblvene-piperidine adduct collected. The estimation indicated the quantitative

reaction (0.18 mmollg)

4.34. Preparation of Fmoc-Val- O-CHZ-C~J&-O-CH~-CO-NH-C~H~ -PS-DVB resin

PS-DVB-HMPA resin (0.5 g, 0.2 mmoWg) was swelled in dry CH2Clz. Dry

Fmoc-Val (136 mg, 0.4 mmol) in a septum-stoppered flask was dissolved in dry CHzC12

along with MeIm (25 pL, 0.3 mmol). The Fmoc-Gly was dissolved completely by the

addition of few drops of dry THF. This solution was transferred to a stoppered flask

containing MSNT (123 mg, 0.4 mmol). The mixture was immediately added to the

swollen polymer support. M e r 30 min, the reactants were washed out with DCM (6 x

30 mL) and DMF (6 x 30 mL). The extent of the reaction was determined by measuring

the UV absorbance of dibenzofblvene-piperidine adducts formed by treating a measured

amount of resin with of 20% piperidine in DMF (3 mL). A second coupling reaction was

required for complete conversion (0.18 mmollg).

4.3.k. Preparation of Fmoc-Val- 0-CH2-CsH4-0-CHz-CO-NH-C& -Sheppard resin

Sheppard-HMPA resin (0.5 g, 0.23 mmol) was swelled in dry CHzClz. Dry Fmoc-

Val (156 mg, 0.46 mmol) in a septum-stoppered flask was dissolved in dry CHzC12 with

the help of appropriate volume of MeIm (29 pL, 0.35 mmol) and a few drops of dry THF.

This solution was transferred to a stoppered flask containing MSNT (136 mg,

0.46 mmol). The mixture was immediately added to the swollen polymer support. Atter

30 min the reactants were washed out first with CH2C12 (6 x 30 mL) followed by DMF

(6 x 30 mL) The Fmoc protection was cleaved off with 20% piperidine in DMF and

coupling yield was determined by the UV absorption of the dibenzofulvene-piperidine

adduct collected. The estimation indicated the quantitative reaction (0.22 mmoltg)

4.3.1. Preparation of F~~~~-G~~-O-CHZ-C~~&-O-CH~-CO-NH-C~~-PS-BDODMA resin

PS-BDODMA-HMPA resin (0.5 g, 0.19 mmollg) was swelled in dry CH2Clz.

Dry Fmoc-Gly (120 mg, 0.38 mmol) in a septum-stoppered flask was dissolved in dry

CH~CIZ with the help of appropriate volume of MeIm (24 BL, 0.29 mmol) and a few

drops of dry THF This solution was transferred to a stoppered flask containing MSNT

(121 mg, 0.38 mmol). The mixture was immediately added to the swollen polymer

support. AAer 30 min, the reactants were washed out first with CH2Clz (6 x 30 mL)

followed by DMF (6 x 30 mL). The Fmoc protection was cleaved off with 20%

piperidine in DMF and coupling yield was determined by the UV absorption of the

dibenzohlvene piperidine adduct collected. The estimation indicated the quantitative

reaction (0.18 mmollg).

4.3.m. Preparation of Fmoc-Gly- 0-CH2-CsH4-0-CH2-CO-NH-C6H4 -PS-DVB resin

PS-DVB-HMPA resin (0.5 g, 0.2 mmol) was swelled in dry CH2C12. Dry Fmoc-

Gly (122 mg, 0.4 mmol) in a septum-stoppered flask was dissolved in dry CHzClz along

with MeIm (25 pL, 0.3 mmol). The Fmoc-Gly was dissolved completely by the addition

of few drops of dry THF. This solution was transferred to a stoppered flask containing

MSNT (123 mg, 0.4 mmol). The mixture was immediately added to the swollen polymer

support. AAer 30 min, the reactants were washed out with DCM (6 x 30 mL) and DMF

(6 x 30 mL) The extent of the reaction was determined by measuring the UV absorbance

of dibenzohlvene-piperidine adducts formed by treating a measured amount of resin with

20% piperidine in DMF (3 mL). A second coupling reaction was required for complete

conversion (0.18 mmollg).

4.3.11. Preparation of Fmoc-Gly- 0-CH2-C6H4-0-CH2-CO-NH-C6H4 -Sheppard resin

Sheppard-HMPA resin (0.5 g, 0.23 mmollg) was swelled in dry CHzC12. Dry

Fmoc-Gly (I36 mg, 0.46 mmol) in a septum-stoppered flask was dissolved in dry CH2Ch

with the help of appropriate volume of MeIm (29 pL, 0.35 mmol) and a few drops of dry

' T This solution was transferred to a stoppered flask containing MSNT (136 mg,

0.46 mmol). The mixture was immediately added to the swollen polymer support. Atter

130 min, the reactants were washed out first with CH2C12 (6 x 30 mL) followed by DMF

(6 x 30 mL) The Fmoc-protection was cleaved off with 20% piperidine in DMF and

coupling yield was determined by the W absorption of the dibenzohlvene-piperidine

adduct collected The estimation indicated the quantitative reaction (0.22 mmollg).

4.3.0. Preparation of PS-BDODRZA-Rink amide resin

p-[(Qs)-a-[I -(9H-fluorene-9-yl)methoxyformamido]-2,4-dimethoxybe~yl]

phenoxyacetic acid (323 mg, 0.6 mmol) HBTU (227 mg 0.6 mmol) HOBt (81 mg,

0.42 mmol) and DIEA (I00 pL, 0.6 mmol) were added to the pre-swollen aminomethyl

resin (1 g, 0.2 mmolig) in DMF and the reaction mixture was kept at room temperature

for 1 h with occasional shaking. The quantitative conversion was estimated by ninhydrin

test. The resin was filtered and washed with DMF (3 x 30 mL), dioxanem0 (I:], 3 x 30

mL), MeOH (3 x 30mL) and ether (3 x 30 mL). The resin was collected and dried in

vacuum. The resin (10 mg) was suspended in 20 % piperidine/DMF (3 mL) for 30 min

and the OD of the solution was measured at 290 nm. From the OD value, the amino

capacity can be calculated and was observed as 0.18 mmollg.

IR (KBr). 3410 cm-I (NH), 1720 cm-' (ester), 1602 cm-' (NHCO).

4.3.p. Preparation of PS-DVB-Rink amide resin

p-[(~,~)-~~-[l-(9H-flu0rene-9-yl)methoxyforrnamido]-2,4-dimethoxybem~l]

phenoxyacetic acid (356 mg, 0.66 mmol), HBTU (250 mg, 0.66 mmol), HOBt (89 mg,

0.66 mmol) and DIEA (1 15 pL, 0.66 mmol) were added to the pre-swollen aminomethyl

resin (1 g, 0.22 mmol/g) in DMF and the reaction mixture was kept at room temperature

for 1 h with occasional shaking. A second coupling reaction was conducted for

quantitative conversion. The ninhydrin test was conducted for the estimation of the extent

of reaction. The resin was filtered and washed with DMF (3 x 30 mL), dioxanelwater

(1: 1, 3 x 30 mL), MeOH (3 x 30 mL) and ether (3 x 30 mL). The resin was collected and

dried in vacuum.

The resin (I0 mg) was suspended in 20% piperidine in DMF (3 mL) for 30 min.

The OD of the solution was measured at 290 nm and the amino capacity of the resin was

calculated to be 0 19 mmollg

4.3.q. Preparation of F ~ O C - A ~ ~ - N H - ( H ~ C O ) Z C ~ H ~ CH-C6Hq-0-CH2-CO-NH-CH2- CsH4-PS-BDODMA-resin

Fmoc-Ala (140 mg, 0.45 mmol), HBTU (170 mg, 0.45 mmol), HOBt (60 mg,

0.45 mnlol) and DIEA (83 hL, 0.45 mmol) were added to the pre-swollen resin

(1 g, 0.18 mmol) in DMF and the reaction mixture was kept for 1 h at room temperature. A

quantitative reaction was observed by ninhydrin test. The resin was filtered and washed with

DMF (3 x 30 mL), MeOH (3 x 30 mL), ether (3 x 30 mL) and dried in vacuum. The amino

capacity of the resin was estimated as 0.17mmoYg.

4.3.r. Perparation of F~OC-A~~-NH-(H~CO)~C~H~'CH-C~H~-O-CH~-CO-NH-CH~- CsH4-PS-DVB-resin

Fmoc-Ala (148 mg, 0.48 mmol), HBTU (180 mg, 0.48 mmol) HOBt (64 mg,

0.48 mmol) and DIEA (0.042 g, 0.32 mmol), were added to pre-swollen resin (1 g,

0.19 mmol) and the reaction mixture was kept for that room temperature. The second

coupling reaction was also conducted for quantitative conversion. The resin was filtered,

washed with DMF (3 x 30 mL), MeOH (3 x 30 mL) ether (3 x 30 mL) and dired in

vacuum. The amino capacity of the resin was estimated as 0.18 mmollg.

4.3s. Peptide Synthesis

1. Synthesis of Leu-Ala-Gly-Val

F~O~-V~~-O-CH~-C~H~-O-CHZ-CO-NH-C~&-PS-BDODMA resin (200 mg,

0.18 mmolig) was swelled in DMF for 1 h. Fmoc protection was removed by 20%

piperidine in DMF (10 mL, 20 min) and washed the resin with DMF (6 x 20 mL).

Coupling reactions were carried out in a minimum volume of DMF as solvent. Fmoc-

amino acid (3.5 mmol excess) was added to the swollen resin. HBTU (47.8 mg,

0.13 mmol), HOBt (17 mg, 0.13 mmol) and DlEA (23 pL, 0.13 mmol) were added to it

and kept for 40 min at room temperature. The resin was filtered and washed with DMF

(6 x 20 mL). The coupling and deprotection steps were monitored by ninhydrin test. The

synthetic operations are as follows;

1. Wash with DMF (6 x lmin)

2. Fmoc-deprotection with 20% piperidine in DMF (10 rnL x 20 min)

3 . Wash with DMF (6 x 1 min)

4. Acylation was carried out with 3.5 mmol excess of Fmoc-amino acid, HBTU, HOBt

and DIEA relative to the amino capacity of the C-terminal amino acid.

5. Wash with DMI; (6 x 1 min)

After the synthesis, Fmoc protection was removed and the resin was washed with

DMF, ether and dried in vacuum.

The peptide was cleaved from the resin by suspending in TFA (2.85 mL) and

water (150 WL) for 2 h at room temperature. The reaction mixture was filtered and the

resin was washed with TFA and DCM. The combined filtrate was evaporated under

reduced pressure. The peptide was precipitated by the addition of ice-cold ether and

washed thoroughly with ether to remove the scavengers added. The peptide was hrther

purified by dissolving in 1% acetic acid in water and passed through a sephadex G-10

column. The eluting fractions containing the peptide were collected and lyophilized.

Merrifield's model tetra peptide was also synthesized on Fmoc-Val-0-CHz-C&-

O-CH2-CO-~-C6& PS-DVB resin (200 mg, 0.18 mmollg) and Fmoc-Val-0-CHz-

C&-0-CH2-CO-NH-C&-Sheppard resin (200 mg, 0.22 mmollg) under the identical

conditions that were used in PS-BDODMA resin.

2. Synthesis of Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly

Fmoc-Gly-0-CHz-C6&-0-CH2-CO-NH-C6& PS-BDODMA resin (200 mg,

0.18 mmollg) was swelled in DMF for 1 h. Fmoc protection was removed by 20%

piperidine in DMF (10 mL, 20 min) and washed the resin with DMF (6 x 20 mL).

Coupling reactions were carried out in a minimum volume of DMF as solvent. Fmoc-

amino acid (3.5 mmol excess) was added to Gly-resin. HBTU (47.8 mg, 0.13 mmol),

HOBt (17 mg, 0.13 mmol) and DIEA (23 pL, 0.13 mmol) were added to it and kept for

30 rnin at room temperature. The resin was filtered and washed with DMF (6 x 20 mL).

'The coupling and deprotection steps were monitored by ninhydrin test. The synthetic

operations are as follows,

I Wash with DMF (6 x 1 min)

2 Fmoc-deprotection with 20% piperidine in Dh@

3 Wash with DMF (6 x 1 min)

4 . Acylation was carried out with 3.5 mmol excess of Fmoc-amino acid, HBTU, HOBt

and DIEA relative to the amino capacity of the C-terminal amino acid.

5 Wash with DMF (6 r 1 min)

After the synthesis, Fmoc protection was removed and the resin was washed with

DMF, ether and dried in vacuum.

The peptide was cleaved from the resin by suspending in TFA (2.85 mL), EDT

(75 pL) and water (75 pL) for 2 h at room temperature. The resin was filtered and

washed with TFA and DCM. The combined filtrate was evaporated under reduced

pressure. The peptide was precipitated by the addition of ice-cold ether. The peptide was

washed thoroughly with ether to remove the scavengers added. The peptide was further

purified by dissolving in 2% acetic acid in water and passed through a sephadex G-10

column. The eluting fractions containing the peptide were collected and lyophilized.

The 65-74 fragment of acyl carrier protein was also synthesized on Fmoc-Gly-0-

CH~-C~H~-O-CHZ-CO-NH-C~& PS-DVB resin (200 mg, 0.18 mmol) and Fmoc-Gly-0-

CH2-C6H4-0-CH2-CO-NH-C6& PS-Sheppard resin (200 mg, 0.22 mmol) under the

identical conditions that were used in PS-BDODMA resin.

3. Synthesis of Leu-Gly-Ala-Leu-Gly-Ala

Cesium salt of Boc-Ala was prepared by adding a saturated solution of cesium

carbonate to Boc-Ala dissolved in minimum amount of ethanol till the pH become 7.0.

Ethanol was evaporated under reduced pressure and water was removed by azeotropic

distillation with benzene. The cesium salt was dissolved in minimum volume of NMP

and added to the pre-swollen chloromethyl resin (200 mg, 0.14 mmol) in NMP, the

mixture was kept at 50 O C for 24 h. The resin was washed with NMP (3 x 30 mL), 1:l

NMPJwater (3 x 30 mL), MeOH (3 x 30 mL), DCM (3 x 30 mL), ether (3 x 30 mL) and

dried under vacuum. Boc-protection was removed by treating with 30% TFA in DCM.

The resin was washed with DCM (5 x 50 mL) and then neutralized with 5% DIEA in

DCM. The resin was washed thoroughly with DCM and Nh4P. Boc-Gly (60 mg,

0.34 mmol), Boc-Leu (78.7 mg, 0.34 mmol) and Boc-Ma (63 mg, 0.34 mmol) were

coupled successively to the resin as their HOBt active esters. The active ester was

prepared by mixing Boc-amino acid with HOBt (46mg, 0.34 mmol) and DCC (70 mg,

0.34 mmol) in NMP. DCU formed was filtered off and the active ester was added to the

resin. After the synthesis, the peptide resin was washed with NMP (3 x 40 mL), MeOH

(3 x 40 mL), DCM (3 x 40 mL), ether (3 x 40 mL) and dried in vacuum.

The peptide was cleaved from the resin by adding TFA (2.85 mL) and water

(150 pL) for 8 h. The polymeric material was filtered off and the filtrate was

concentrated to get an oily residue. By adding ice-cold diethyl ether, the peptide was

precipitated as white powder and was washed thoroughly with ether. The purity of the

peptide was checked by injecting an aqueous solution of peptide to C-18 RPC column

and eluted using 0.1% TFA in water (A) and 0.1% TFA in acctonitrilelwater (80:20) (B)

The HPLC profile gave a single peak corresponding to the target peptide.

The same peptide sequence was also synthesized on PS-DVB resin. Boc-Ala-PS-

DVB resin (200 mg, 0.7 mmol/g) was used for the synthesis. The amino acids Boc-Gly

(63 mg, 0.38 mmol), Boc-Leu (81 mg, 0.38 mmol), Boc-Ala (65 mg, 0.38 mmol) were

successively coupled to the Boc deprotected Ala-PS-DVB resin by HOBt active ester

mehtod. HOBt (50 mg, 0.38 mmol) and DCC (74 mg, 0.38 mmol) were used for the

preparation of the active ester. DCU formed was filtered off, the filtrate was added to the

resin, and the reaction mixture was kept for 40 min. The peptide was cleaved from the

resin by using TFA for 8 h. The purity of the peptide was checked by injecting the

aqueous solution of peptide to C-18 RPC column and eluted using 0.1% TFA in water

(A) and 0.1% TFA in acetonitrilelwater (80:20) (B) The HPLC profile gave a major peak

along with a small peak.

4. Synthesis of Ala-Ala-Ala-Ala

Boc-protection was removed from Boc-Ala-PS-BDODMA resin (200 mg,

0.14 mmol) by treating with 30% TFA in DCM. The resin was washed with DCM (5 x

50 mL) and then neutralized with 5% DIEA in DCM. The resin was washed thoroughly

with DCM and NMP. Boc-Ala (63 mg, 0.34 mmol) was coupled successively to the resin

as its HOBt active ester. Active ester was prepared by mixing Boc-amino acid with HOBt

(46mg, 0.34 mmol) and DCC (70 mg, 0.34 mmol) in M. DCU formed was filtered off

and the active ester was added to the resin. Atter the synthesis, the peptide bound resin

was washed with NMP (3 x 40 mL), MeOH (3 x 40 mL), DCM (3 x 40 mL), ether (3 x

40 mL) and dried in vacuum.

The peptide was cleaved from the resin by using TFA (2.85 mL) and water

(150 pL) for 8 h The polymeric material was filtered off and the filtrate was

concentrated to get an oily residue. By adding ice-cold diethyl ether, the peptide was

precipitated as a white powder and was washed thoroughly with ether. The purity of the

peptide was checked by injecting an aqueous solution of peptide to C-18 RPC column

and eluted using 0.1% TFA in water (A) and 0.1% TFA in acetonitrilelwater (80:20) (B).

The HPLC profile gave a single peak corresponding to the target peptide.

The same peptide sequence was also synthesized on Boc-Ala-PS-DVB resin

(200 mg, 0.7 mmol/g). After the synthesis, the peptidyl resin was washed with NMP (3 x

40 mL) , MeOH (3 x 40 mL), DCM (3 x 40 mL), ether (3 x 40 mL) and dried under

vacuum. The peptide was cleaved from the resin by using TFA for 8 h. The purity of the

peptide was checked by injecting an aqueous solution of the peptide to C-18 RPC column

and eluted using 0.1% TFA in water (A) and 0.1% TFA in acetonitrilelwater (80:20) (B)

The HPLC profile gave a major peak along with a small peak.

5. Synthesis of Leu-Ile-Asn-Thr-Asn-Ala-Ser-Trp-His-Ala-Asn-Arg-Thr-Ala-Leu- Ser-Asn-Asp-Ser-Lys-Leu-Asn-Thr-Gly-Ala-NHz

Fmoc-Ala-NH-(H~CO)~C~&)CH-~-O-CHz-CO-~-CH~-C~-PS-BDOD~-

resin (200 mg, 0.17 mmollg) was swelled in Dh4F for 1 h. Fmoc protection was removed

by 20% piperidine in DMF (10 mL, 20 min) and the resin was washed thoroughly with

DMF (6 x 20 mL) Couping reactions were carried out in a minimum volume of DMF as

solvent. Fmoc-amino acid (3.5 mmol excess) was added to Ala-resin. HBTU (45.1 mg,

0.12 mmol), HOBt (16 mg, 0.12 mmol) and DIEA (20 pL, 0.12 mmol) were added to it

and kept for 30 mln at room temperature. The resin was filtered and washed with DMF

(6 x 20 mL) The coupling and deprotection steps were monitored by Kaiser's test. The

synthetic operations are as follows,

1 . Wash with DMF (6 x 1 min)

2. Fmoc-deprotection with 20% piperidine in DMF (1 x 20 min)

3. Wash with DMF (6 x 1 min)

4. Acylation was carried out with 3.5 mmol excess of Fmoc-amino acid, HBTU, HOBt

and DlEA relative to the amino capacity of the C-terminal amino acid.

5. Wash with DMF (6 x lmin).

After the attachment of all the amino acids, Fmoc protection was removed and

the resin was washed with D m , ether and dried in vacuum.

The peptide was cleaved from the resin by suspending in TFA (2.35 mL), phenol

(200 pL), EDT (1 50 pL) for 2 h at room temperature. The resin was filtered and washed

with TFA and DCM. The combined filtrate was evaporated under reduced pressure till an

oily residue was obtained. The peptide was precipitated by the addition of ice-cold ether.

The peptide was washed thoroughly with ether to remove the scavengers added. The

peptide was further purified by dissolving in 1% acetic acid in water and passed through

a sephadex G-25 column. The eluted fractions containing the peptide were collected and

lyophilized.

The same peptide was synthesized on F~oc-A~~-NH-~~CO)~C~H~)CH-C~&-O-

CH2-CO-NH-CHz-C6H4-PS-DVB-resin (200 mg, 0.18 mmollg) under identical

conditions that were used in PS-BDODMA resin.

6. Synthesis of Leu- Ue-Asn-Thr-Asn-Ala-Ser-Trp-His-Ala-Asn-Ang

F~~C-A~~-NH-(H~CO)~C~H~)CH-C~H~-O-CH~-CO-NH-CH~-C~&-PS-BDODMA-

resin (200 mg, 0 17 mmol/g) was swelled in DMF for1 h. Fmoc-protection was removed

by 20% piperidine in DMF (10 mL, 20 min) and the resin was washed thoroughly with

DMF (6 x 20 mL) All coupling reactions were carried out in minimum volume of DMF.

3 5 mmol excess of Fmoc-amino acid, HBTU (45.1 mg, 0.12 mmol), HOBt (16 mg,

0.12 mmol) and DlEA (20 pL, 0.12 mmol) were added to the swollen resin and kept for

30 min at room temperature. The resin was filtered and washed with Dh4F (6 x 20 mL).

Kaiser's test was used for monitoring the coupling and deprotection steps. The synthetic

steps are same as that described in the synthesis of peptide 5.

After removing the Fmoc protection with 20% piperidine in DMF (10 mL,

20 min) and DMF wash (6 x 30 mL), the peptidyl resin was suspended in TFA (2.35

mL), water (150 pL), phenol (200 pL), EDT (150 pL) and thioanisole (150 pL) for 2 h at

room temperature. The resin was filtered and washed with TFA and DCM. The combined

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 peptide was further purified by dissolving in 1% acetic acid in water and passed

through a sephadex G-15 column. The eluted fractions containing the petitide were

collected and lyophilized

The same peptide was synthesized on Fmoc-Ala-NH-(H3C0)2C6H3)CH-C6H4-0-

CHZ-CO-W-CH~-C~H~-PS-DVB-~~S~~ (200 mg, 0.18 mmolfg) under identical

conditions that were used in PS-BDODMA resin.

7. Synthesis of Leu-Asn-Cys(Acm)-Asn-Asp-Ser-Leu-Asn-Thr-Ala-NH2

Fmoc-A~a-NW-(~~C0)~C6H~)CH-C6tI4-O-cH2-CO-NH-CHZ-C~Ij4-PS-BDOD~-

resin (200 mg, 0.17 mmoWg) was swelled in DMF for 1 h. Fmoc-protection was removed

by 20% piperidine in DMF (10 mL x 20 min) and the resin was washed with DMF (6 x

20 mL). The coupling reactions were carried out in a minimum volume of DMF as

solvent. Fmoc amoino acid (3.5 mmol excess) was added to Ala-resin along with HBTU

(45.1 mg, 0.12 mmol), HOBt (16 mg, 0.12 mmol) and DIEA (20 pL, 0.12 mmol) and

kept for 30 min at room temperature. The resin was filtered and washed with DMF (6 r

20 mL) The coupling and deprotection steps were monitored by Kaiser test. The

synthetic steps are same as that described in the synthesis of peptide 5.

The peptide was cleaved from the resin by suspending in TFA (2.7 mL), EDT

(150 pL) and water (150 pL) for 2 h at room temperature. The resin was filtered and

washed with TFA and DCM. The combined filtrate was evaporated under reduced

pressure till an oily residue was obtained. The peptide was precipitated by adding ice-

cold ether and washed thoroughly with ether to remove the scavengers. The peptide was

further purified by dissolving in 1% acetic acid in water and passed through a sephadex

(3-10 column. The eluted fractions containing the peptide were collected and lyophilized.

The same peptide was synthesized on F~O~-~~-NH-(H,CO)~C~H~)CH-C~~I~-O-

<:HI-CO-NH-CH2-C6H4-PS-DVB-resin (200 mg, 0.18 mmollg) under identical

conditions that were used in PS-BDODMA resin.

References

1. Cameron, L. R.; Holder, J. L.; Meldal, M.; Sheppard, R. C. J. Chem. Soc., Perkin

Trans. I , 1988, 2895.

2. Kent, S. B. H. Annu. Rev. Biochem.1988,57,959.

3 . Brenner, M . ; Huber, W. Helv. Chem. Acta. 1953, 36, 1109.

4 . Schnabel, E. A?m. Chem. 1967,702, 188.