SYNTHESIS OF BIOLOGICALLY ACTIVE PEPTIDES ON PS-BDODMA RESIN USING Boc-CHEMISTRY

Transcription

1 SYNTHESIS OF BIOLOGICALLY ACTIVE PEPTIDES ON PS-BDODMA RESIN USING Boc-CHEMISTRY

2 5.1. Introduction l eptides composed of as few as three to 50 amino acids play an important role in 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

3 @- CH~OOC-AA I I -NH-Boc 1) 30% TFA in DCM 2) 5% DIEA in I -NH~ ' 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

4 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 = The HPLC and MALDI TOF MS are given in Fig (a) (b) Fig (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 dmin; 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.

5 (a) (b) Fig (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 Fig (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

6 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 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 dmin; 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 = 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= Synthesis of Thr-Val-Val-Val-Val-Asn The peptide was synthesized by following the general synthetic strategy (81 mg, 95%). Rr= Synthesis of Pro-Met-Leu-Phe-Val-Thr The peptide was synthesized by following the general procedure (91 mg, 95%). Rf=0.8.

7 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 Synthesis of Met-Leu-Phe-Tyr-Val-Gly The peptide was synthesized according to the general synthetic strategy (95 mg, 96%). Rf= 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 transesterification 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 = (a) (b) Fig (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/min; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.

8 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 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 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 = The HPLC and MALDI TOF MS are given in Fig (a) (b) Fig (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.

9 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 = 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 = Synthesis of P-casomorphin (~uman)" (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= 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= MALDI TOF MS of the ~eptide is given in Fig 5-7b : : i 60 -j %I"t ii 1 30: i i : 01 A>..* 500 aao 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

10 5. Synthesis of 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 (118 mg). HPLC profile (Fig 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 (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.

11 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 (119 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.

12 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 (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.

13 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 ( ) 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

14 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 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 ldmin. The gradient used is shown in Fig a. Amino acid analysis data of the peptide agreed with the target sequence. (a) ("1 Fig (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 dlmin; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.

15 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 ( ) peptide resin, and 'Afi of the resin was kept aside. The gp 120 ( ) 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 a). Amino acid analysis data of the peptide agreed with the target sequence Fig. (a) (b) (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 a). Amino acid analysis data ofthe peptide agreed with the target sequence.

16 (a) (b) Fig (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 dmin; 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 (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.

17 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% Wwater (A) and 0.1% TFNacetonitrile (B) at a flow rate of 1 mumin (Fig 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 Bocchemistry. 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, 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.

18 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 [(M+H)+, 100%], C ~~H~~N~IOLZ, requires M 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 [(M+H)+, loo%], C47H74NI~016, requires M' 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'

19 4. Val-Val-Val-Val-Val-Val-Val-Val-Val-Val Amino acid analysis: Val, 9.84 (10). MALDI TOF MS: m/z [(M+H)+, 100?], CSOH~~NIOOII, requires M' 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). hydrolysed to Asp. Asn was 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

20 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 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.

21 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 transesterification 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 (113 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),

22 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)

23 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 [(M+H)+, loo%], CS~H~~N~O~~SZCI, requires Id 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, 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,

24 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 preswollen 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 [(M+H)+, loo%], C28H37Nj07, requires Mt 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, 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

25 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 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

26 (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 (150 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.

27 18. Synthesis of C-reactive Protein (Val-Gly-Gly-Ser-Glu-Ile) Boc-Ile (95 mg, 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, 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 [(M+H)+, loo%], C23&N6ol0, requires M

28 19. Synthesis of fragment of Alzheimer's P-amyloid peptide. (Gly-Leu-Met-~al-~l~-~l~-~al-~al-~le-~la) Boc-Ala (64 mg, 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, 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. Mer 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

29 collected and lyophilized to yield 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 [(M+H)+, 67%], [(M+Na)', 100%], [(M+K)+, 55%], C41H74N10011S, requires M' 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, 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 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.

30 The peptide was cleaved from the resin using TFA (2.625 ml) in presence of ethanedithiol (75 &), water (150 &) and thioanisole 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 [(M+H)+, loo%], C ~~H~~NIZOI~, requires M' 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, 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)