SYNTHESIS OF BIOLOGICALLY ACTIVE PEPTIDES...

SYNTHESIS OF BIOLOGICALLY ACTIVE PEPTIDES ON PS-BDODMA SUPPORT USING

FMOC-AMINO ACIDS

6.1. Introduction

T he respective amino acids incorporation with 100% efficiency on a PS-DVB

resin is still a problem and is one of the challenging problems encountered by

polymer chemists.' Over these years this problem was overcome to an extent by

the judicious selection of polymer supports, coupling reagents and solvents. A

2% PS-BDODMA support was successfully used for the synthesis of biologically active

peptides.2 The extraordinary swelling capability of PS-BDODMA over PS-DVB resin in

various solvents is found to have a positive impact in facilitating the attachment of

respective amino aclds to the resin. The introduction of suitable linkers to the resin found

to improve the coupling reaction rate and also helps the cleavage of the target peptide in

the acid form or C-terminal modified form within a short time. Various linkers such as

4-(hydroxymethy1)benzoic acid,3 4-(hydroxymethyl)phenoxyacetic acid: 4(4-hydroxy-

methyl-3-methoxyphenoxy)butyric acid,' p-[(R,S)-a-[1-(9H-fluoren-9-y1)-methoxy-

formamidol-2,4-dimethoxybenzyll-phenoxyacetic acid @mk amide hand~e)~ were

incorporated to 2% PS-BDODMA resin for peptide synthesis. The acylation reaction

proceeds smoothly in a short time single coupling reaction using HBTU coupling method

and the side reactions such as racemisation and diketopiparazine formation were not

observed in any of these synthesis.

Amphibian skin is a rich source of biologically active compounds that are

assumed to have diverse physiological and defence functions. In addition to the range of

pharmacologically active peptides present, some of which have mammalian homologues,

skin secretions containing a broad spectrum of antimicrobial peptides. The granular

glands of amphibian skin produce many biologically active compounds." These glands

are controlled by sympathetic nerves, and discharge their content on the dorsal surface of

the animal in response to a variety of stimuli. The compounds secreted by the glands play

an important role in the regulation of physiological functions of the skin, or in defence

against predators or micro-organisms. Skin extracts of frogs are rich source of

pharmacologically active peptides such as caeruleins, tachykinins, bradykinins,

thyrotropin releasing hormone and bombesin like opoid peptides.829 Vertebrate skin has

the same embryonic-ectodermal origin as the brain, and may frog-skin peptides have

been found to have counterparts in mammalian gastro-intestinal tract and brain.'' In

addition to the peptides related to mammalian hormones andlor neurotransmitters,

amphibian skin contains numerous peptides with antimicrobial or haemolytic activities.

The production of antimicrobial peptides is part of the innate immune system and is

widespread in nature." This system was first discovered in insect haemolymph when the

synthesis of anti-microbial peptides, is induced in response to microbial infection. A

similar system was subsequently shown to operate in the lungs and gastro-intestinal tract

of mammals, the system shows striking similarities to the vertebrate acute-phase immune

response. In amphibians, the production of antimicrobial peptides appears to be

constitutive, but they are released in response to external stimulus.

A large number of antimicrobial peptides from amphibian skin can adopt an

amphipathic a-helical structure in hydrophobic environment, suggesting that oligomers

of such helices would form pores in the phospho-lipid bilayer of target membranes.

Inhibition of cell growth and cell death may then result from the disturbance of

membrane functions. The selectivity of peptides for bacterial membranes may be related

to the number and distribution of positive charges.

Recently, antimicrobial peptides have been isolated and characterized from the

skin of Xenopus laevis, Bombina variegata and Bombina orientalis, Phyllomedusa

sauvagei and Phyllomedusa bicolor, Litoria splendida and Litoria caenilea and several

species of Ranidae. A large variety of antimicrobial peptides have been isolated from

Rana species. These peptides grouped in several families on the basis of diffusing length

and distant activity. An intermolecular disulfide bridge, forming a seven membered ring,

located at the C-terminal end is common for all these peptides. The papins, from R.

pipiens, brevinin-1 and brevinin-1E from R. brevipola and R. esculenta all appear to be

members of the same family. R. esculenta secretes esculentin-1, a 46 residue peptide that

is highly potent antimicrobial agent.12 In order to study the activity of esculentin-1,

different helical regions in the sequence were selected and synthesized separately. The

helical properties of these sequences are improved by the introduction of positively

charged amino acids.

6.2. Results and Discussion

The new PS-BDODMA resin can be successfUlly employed for the synthesis of

peptides using Fmoc-amino acids. The resin is extremely stable under the conditions of

peptide synthesis. The protocol for the synthesis of peptides using different handles is

shown in Scheme-6.1. The linkers used were suitable for Fmoc-amino acids. The

C-terminal amino acid was attached to the resin through an ester bond or an amide bond.

The HOBt/ HFiTU active ester in presence DIEA was used for the C-terminal amino acid

incorporation to the resin.

The hydroxymethyl PS-BDODMA resin was also used for the synthesis of

peptides. C-terminal amino acid was attached to the resin via an ester linkage using

preformed symmetric anhydride of Fmoc-amino acid in presence of DMAP. The reaction

time was 1 h and quantitative conversion was observed by amino estimation.

Deprotection of Fmoc group was achieved by 20% piperidine in D m . M e r washing the

resin with DMF, acylation reactions were carried out in minimum quantity of DM3 by

using 2.5 equiv excess of Fmoc amino acids and HBTU, 5 equiv excess of HOBt and

2.5 equiv DIEA with respect to the amino capacity of the C-terminal amino acid. The

acylation reactions were completed in single coupling as shown by Kaiser's test.

The peptide was cleaved from the resin by treatment with TFA and suitable

scavengers at room temperature for 3-5 h. The reaction mixture was filtered and washed

with TFA and DCM. The combined filtrate and washings were evaporated under

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

thoroughly with ether to remove the scavengers. The peptide was dissolved in 1-2%

acetic acid-water mixture, passed through a sephadex column, and then lyophilized.

Purity of the peptides was analyzed using a Pharmacia LKB HPLC system having a

P-500 pump (X2) rapid spectral detector UV-M-11.

HBTU/HOBt -NH, + HOOC- -\OH -b DIEA

b MSNT, MeIm

Deprotection with

20% piperidine

1) Coupling with Respective Fmoc-amino acids

b 2) N-terminal Fmoc Cleavage

4 Scavengers

PS-BDODMA resin 8 Side chain protection

Scherne 6.1 General protocol for SPPS using Fmoc-amino acids Handles: H~&A, HMPB, XJMBA and Rink amide

6.2.a. Synthesis of 4-(4-hydroxymethyl-3-methoxyphenoxy)bu~amidomethyl 2% PS-BDODMA (PS-BDODMA-BMPB) resin

PS-BDODMA-HMPB resin was prepared by treating aminomethyl

PS-BDODMA resin with 4-(4-hydroxymethyl-3-methoxyphenoxy)butyic acid linker in

presence of HOBt IHBTU /DEA for 1 h (Scheme-6.2).

Scheme-6.2. Preparation of 4-(4-hydroxymethyl-3-methoxyphenoxy)butylamidomethyl 2 % PS-BDODMA resin

Fig. 6-1. IR Spectrum (KBr) of PS-BDODMA-HMPB resin

The amino acid corresponding to the C-terminal region of the peptide was

attached to the resin by an ester bond. This ester linkage was extremely stable under

repeated treatment of 20% piperidine in DMF (reagent used for the deprotection of Fmoc

group). The synthesized peptide was cleaved from the support using TFA and scavengers

at room temperature for 2 h.

6.2.b. Synthesis of Peptides

1. Synthesis of 1-15 fragment of Esculentin-l

(Gly-Ile-Phe-Ser-Lys-Leu-Gly-Arg-Lys-Lys-Ile-Lys-Asn-Leu-Leu)

The 1-15 fragment of esculentin-1 is found to be a helical region in the peptide

sequence. In order to study the activity of this fragment, it was synthesized on

2% PS-BDODMA resin using Fmoc-amino acids.

Fmoc-Leu was attached to hydroxymethyl 2% PS-BDODMA cross-linked

polystyrene support by DCC anhydride method in presence of DMAP. The quantitative

reaction was observed from the Fmoc-estimation by W absorbance method. After

(a) (b) Fig.6-2 (a) HPLC time-course analysis of the peptide Gly-Ile-Phe-Ser-Lys-Leu-Gly-Arg-

Lys-Lys-Ile-Lys-Asn-Leu-Leu 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 rnin and 100% B in 50 min (b) MALDI TOF MS of the peptide.

removing the Fmoc-protecting group by 20% piperidine in DMF, the resin was washed

thoroughly with DMF and the consecutive amino acids were incorporated by DCCIHOBt

active ester coupling method. Each coupling steps were monitored by Kaiser's ninhydrin

test. A second coupling was also performed for c o n f i i n g the quantitative reaction. Afier the

attachment of amino acids to the resin, the peptide was cleaved f?om the resin by treating with

TFA in presence of thioanisole, water, ethanedithiol and phenol. The crude peptide was

obtained in 96% yield. HPLC profile (Fig.6-2a) showed the high purity of peptide. Amino

acid analysis of the peptide also agreed with that of target peptide.

2. Synthesis of 1-15 fragment of Esculentin-1 modified at G l y ~ by Pro

(Gly-Ile-Phe-Ser-Lys-Leu-Pro-Arg-Lys-Lys-Ile-Lys-Asn-Leu-Leu)

1-15 fragment of Esculentin-1 was found to have a hghly helical secondary

structure. In order to study the changes in the helical property and the antibacterial

activity, the Gly in peptide sequence was substituted by Pro.

(a) (b) Fig.6-3. (a) HPLC time-course analysis of the peptide Gly-Ile-Phe-Ser-Lys-Leu-Pro-

Arg-Lys-Lys-Ile-Lys-Asn-Leu-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 mL1min; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS of the peptide.

Fmoc-Leu was attached to hydroxymethyl 2% PS-BDODMA resin by DCC

anhydride method in presence of DMAP. Fmoc protection was removed with piperidine

in DMF and the synthesis was continued by stepwise incorporation of respective amino

acids using DCC/HOBt active ester method. All coupling steps were monitored by

Kaiser's semi-quantitative ninhydrin test. For the confirmation of quantitative reaction a

second coupling was also performed. The peptide was cleaved from the resin by TFA in

presence of scavengers such as thioanisole, water, ethanedithiol and phenol. The crude

peptide was obtained in 95% yield. HPLC profile of the crude peptide (Fig.6-3a) showed

only one major peek corresponding to the target peptide. Amino acid analysis and

MALDI-TOF-MS of the peptide also agreed with that of target peptide.

3. Synthesis of 33-44 fragment of Esculentin 1

(Thr-Gly-Ile-Asp-lle-Ala-Gly-Cys-Lys-Ile-Lys-Gly)

Fmoc-Gly was attached to hydroxymethyl 2% PS-BDODMA support by

DCC/DMAP anhydride method. After removing the Fmoc protection by 20% piperidine

in DMF, the respective amino acids were incorporated in a stepwise manner using

DCCMOBt active ester Each coupling steps were monitored by semi-quantitative

ninhydrin test. The peptide was cleaved from the resin by TFA and water. The crude

peptide was obtained in 96% yield. The HPLC profile of the crude product showed only

one major peak corresponding to the target peptide (Fig.6-4a). The amino acid analysis

and MALDI TOF MS of the peptide also agreed with the target peptide.

The CD spectrum of the peptide showed (Fig.6-4 c) intense negative maxima at

198-202 nm (amide x+xt transition) and a small trough at 220-222 nm (amide n+n*

transition) suggesting a right-handed a-helical conformation.

(c) Fig. 6-4 (a) HPLC time-course analysis of the peptide Thr-Gly-Ile-Asp-Ile-Ala-Gly-Cys-

Lys-Ile-Lys-Gly using the buffer (A) 0.5 mL TFA in 100 mL water; (B) 0.5 mL TFA in 100 mL acetonitri1e:water (4:l); Flow rate: 0.5 d m i n ; Gradient used: 0% B in 5 min and 100% B in 50 min (b) MALDI TOF MS and (c) CD spectrum of the peptide

Mass 1 Charge

4. Synthesis of 33-44 fragment of Esculestin-1 modified at G l y ~ ~ by ALa

(Thr-Gly-Ile-Asp-Ile-Ala-Ala-Cys-Lys-Ile-Lys-Gly)

Fmoc-Gly was attached to hydroxymethyl 2% PS-BDODMA resin by

DCCDMAP anhydride method. The Fmoc protection was removed by 20% piperidine in

DMF and the successive amino acids were attached in a stepwise manner using

DCCIHOBt active ester method. The extent of coupling was monitored by Kaiser's test.

.I. 8

( 4 Fig.6-5. (a) HF'LC: time-course analysis of the peptide Thr-Gly-Ile-Asp-Ile-Ala-Ala-Cys-

Lys-lie-1,ys-Gly 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 and (c) CD spectrum of the peptide

100. 60

80.

'O . 60. - 'z - 5 40.

20

1027.6

100 K 10 20 30 40 4 10. 0 1200 , 1400 , . 1600 1800 2000 2200

The peptide was cleaved from the resin using TFA and water. The crude peptide was

obtained in 94% yield The HPLC profile of the crude product showed only one major

peak which corresponding to the target peptide (Fig.6-5a). The amino acid analysis and

MALDI TOF MS of the peptide also agreed with the target peptide sequence

The CD curve of the peptide showed (Fig. 6-5c) a relatively intense negative

maximum near 200 nm (amide R+K* transition) accompanied by a weak negative

maximum located at about 220 nm (amide n+x* transition). These observations revealed

the right-handed a-helical conformation for the peptide

5. Synthesis of 9-27 fragment of Esculestin-1

(Lys-Asn-Val-GI y-Lys-Glu-Val-Gly-Met-Asp-Val-Val-Arg-Thr-Gly-Ile-Asp-Ile-Ala)

The 9-27 fragment is a helical region in Esculestin-1. Due to the helical structure

this region plays an important role in antimicrobial activity of the peptide Esculentin-1.

4-(4-hydroxymethyl-3-methoxyphenoxy)butylamidomethy 2% PS-BDODMA resin was

used for synthesis of the peptide. Fmoc-Ala was attached to the resin using DCC

anhydride method in presence of DMAP. The quantitative reaction was observed by

measuring the optical density of adducts of dibenzohlvene and piperidine formed by the

treatment of accurately weighed Fmoc-Ala-resin with 20% piperidine in DMF. The

successive amino acids were incorporated by HBTU in presence of HOBt and DIEA. The

coupling reactions were monitored by Kaiser's test. The finished peptide was cleaved

from the resin by suspending in TFA with scavengers such as thioanisole, ethanedithiol,

phenol and water for 2 h. The crude peptide was obtained in 92% yield. HPLC profile

showed only one major peak corresponds to the target peptide (Fig.6-6a). 0.1% TFA in

water (A) and 0.1% TFA in acetonitrile (B) was used as eluent and the flow rate of the

solvent was imL/min Amino acid analysis and mass spectrum of the peptide agreed

with the peptide sequence.

The CD curve of the peptide showed (Fig.6-6c) a sharp trough near 198-200 nm

(amide rr+n* transition) accompanied by a weak negative maximum located at about

220-221 nm (amide n-m* transition). These observations indicated the right-handed

a-helical conformation for the peptide.

. .

(4 Fig.6-6. (a) HPLC time-course analysis of the peptide Lys-Asn-Val-Gly-Lys-Glu-Val-Gly-

Met-Asp-Val-Val-Arg-Thr-Gly-Ile-Asp-Ile- 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 rnin and 100% B in 50 min (b) MALDI TOF MS and (c) CD spectrum of the peptide

6. Synthesis of 9-27 fragment of Esculestin-1 modified by replacing Glu, and Asp by Lys.

The helical property of the peptide can be increased by introducing positively

charged amino acids such as Lysine and Arginine. In order to study this effect, the

rnAU -1.0

1000.

6 0 0

.so 600

LOO. ..do

100. .20

0

m 10 10 40 n(

(4 Fig.6-7. (a) HPLC time-course analysis of the peptide Lys-Asn-Val-Gly-Lys-Lys-Val-

Gly-Met-Lys-Val-Val-Arg-Thr-Gly-Ile-Lys-a using the buffer (A) 0.5 rnL 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) MALI11 TOF MS and (c) CD spectrum of the peptide

negatively charged amino acids in 9-27 sequence of Esculentin-l were replaced by

Lysine. Fmoc-Ala was attached to 4-(4-hydroxymethyl-3-methoxy-

phenoxy)butylamidomethy12% PS-BDODMA resin using DCC anhydride method in

presence of DMAP. The quantitative reaction was estimated by measuring the optical

density of adducts formed when 20% piperidine in DMF treated with accurately weighed

Fmoc-Ala attached resin. Successive amino acids were incorporated by using HBTU in

presence of HOBt and DIEA. Each coupling steps were monitored by ninhydrin test. The

peptide was cleaved from the support using TFA in presence of scavengers thioanisole,

ethanedithiol, water and phenol for 2 h. The peptide obtained was in 92% yield and

HPLC profile showed a single peak corresponding to the target peptide (Fig.6-7a). The

amino acid analysis and MALDI-TOF-MS of the peptide also agreed with the target

sequence.

The CD curve of the peptide showed (Fig.6-7c) a relatively intense negative

maximum near 200-202 nm (amide x+n* transition) accompanied by a weak negative

maximum located at about 218-201 nm (amide n+n* transition). These observations

revealed a right-handed a-helical conformation for the peptide.

7. Synthesis of 9-27 fragment of Esculestin-1 modified by replacing Gly with Ala and Asp & Glu with Lys

(Lys-Asn-Val-Ala-Lys-Lys-Val-Ala-Met-Lys-Vd-Val-~g-T~-Ala-Ile-Lys-Ile-Ala)

The presence of Gly in the peptide sequence reduces its helical property. The

helical nature can be increased by the introduction of Ala instead of Gly. The

introduction of Lys in the position of Glu and Asp improves its helical character. As the

helical nature increases, antimicrobial activity increases. Fmoc-Ala was attached to

4-(4-hydroxymethyl-3-melthoxyphenoxylbutylamidomethyl 2% PS-BDODMA resin by

DCC anhydride method in presence of DMAP. The quantitative reaction was observed by

the UV absorbance of adduct formed by the reaction of accurately weighed Fmoc-Ala-

resin and 20% piperidineIDMF. Fmoc-protection was removed by 20% piperidine in

DMF. The remaining amino acids were incorporated by HBTUHOBtDIEA method.

Each coupling reactions were monitored by Kaiser's test. The finished peptide can be

cleaved from the resin by TFA in presence of scavengers such as thioanisole, water,

ethanedithiol and phenol at room temperature for 2 h. The peptide obtained was in 93%

yield and HPLC profile showed a single peak corresponding to the target sequence

(Fig.6-8a). The amino acid analysis and MALDI-TOF-MS of the peptide also agreed

with the target peptide sequence.

(4 Fig.6-8. (a) HPLC: time-course analysis of the peptide Lys-Asn-Val-Ala-Lys-Lys-Val-

Ala-Met-Lys-Val-Val-Arg-Thr-Ala-Ile-Lys-IeAa 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 M S and (c) CD spectrum of the peptide

The CD curve of the peptide showed (Fig.6-8c)a relatively intense negative

maximum in 198-200 nm (amide x+x* transition) accompanied by a weak negative

maximum located at about 219-222 nm (amide n-tx* transition). These observations

revealed a right-handed a-helical conformation for the peptide.

6.3. Experimental

Materials

Fmoc-amino acids, HMPB, HOB< HBTU and DIEA were purchased from

Novabiochem Ltd., UK. Trifluoroacetic acid, thioanisole, ethanedithiol, phenol,

dicyclohexyl carbodiimide, fluorenyl methyl chloroformate, fluorenyl methyl

succinimidyl carbonate and DMAP were purchased from Aldrich Chemical Co., USA.

All solvents used were of HPLC grade purchased from E. Merck, India and SISCO

Chemicals, Bombay

6.3.a. Preparation of reagents and amino acid derivatives

6.3.a.l. Synthesis of Fmoc-amino acids using fluorenyl methyl chloroformate

Ammo acid (10 mmol) was dissolved in a mixture of dioxane (10 mL) and 10%

sodium carbonate (30 mL), stirred vigorously at 0 OC. Fluorenylmethylchloroformate

(12.5 mmol) in dioxane was added dropwise over a 15 min period with stirring. The

reaction mixture was allowed to stir at room temperature for 1 h. The extent of reaction

was monitored by tlc using the solvent system chloroform-methanol-acetic acid

(85: 10:s vlv), followed by development with ninhydrin. Water (100 mL) was added to

the reaction mixture and the clear solution extracted with ether (3 x 50 mL). The aqueous

solution was then acidified with hydrochloric acid to pH = 3 and the white precipitate

formed were extracted with ethyl acetate (3 x 50 mL), which was dried over anhydrous

sodium sulphate. The precipitate was filtered and the filtrate was evaporated.

Fmoc-amino acid was crystallized from ethyl acetatelpetroleum ether mixture.

6.3.a.2. Synthesis of Fmoc-amino acids using fluorenyl methyl succinimidyl carbonate

The amino acid (10 mmol) and sodium carbonate (10 mmol) were dissolved in a

mixture of water (15 mL) and acetone (15 mL). Fluorenylmethylsuccinimidyl carbonate

(9.9 mmol) was added over a period of 60 min to the vigorously stirred solution of amino

acid and Na~C03. The pH was kept between 9 and 10 by the addition of 1M NazC03.

The stirring was continued for 24 h, ethyl acetate (60 mL) was added and the mixture was

acidified with 6 M HCI. The ethyl acetate layer was separated and washed with water

(4 x 50 mL), dried over anhydrous magnesium sulphate and then evaporated

approximately to 15 mL. Petroleum ether was added to the solution till a precipitate

obtained. Cooling the solution to 0 OC, crystalline Fmoc-amino acid was obtained.

6.3.a.3. Preparation of 4-(4-hydroxymethyl-fmethoxypbenoxy)butylamidomethyl 2% PS-BDODMA (PS-BDODMA-HMPB) resin

4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid (0.052 g, 0.22 mmol),

HBTU (0.082 g, 0.22 mmol), HOBt (0.029 g, 0.22 mmol) and DIEA (0.028 g,

10.22 mmol) were added to pre-swollen aminomethyl resin (0.500 g, 0.12 mmoYg) in

Dh@ and the reaction mixture was kept at room temperature for 1 h with occasional

swirling. The resin was filtered, washed with DMF (3 x 30 mL), dioxane:H~O (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.

IR (KBr): 3420 cm-' (NH), 3400 c~-'(oH), 1680 cm-' (ester), 1640 cm-' W C O ) .

Estimation of hydroxyl group in PS-BDODMA-HMPB resin

200 mg of the resin was acetylated with measured amount of acetic anhydride-

piperidine mixture (1.4, 3 mL) for 6 h. 10 mL distilled water was added and the reaction

mixture was refluxed for 3 h. The mixture was cooled, filtered and acetic acid formed

was back titrated with standard (0.1N) NaOH. A blank titration was also carried out.

From the titre values, hydroxyl capacity of the resin can be calculated.

Capacity = 0.1 1 mmol, OWg

63.a.4. Preparation of F m o c - A l a O C a r 6 r q ( ~ ~ ~ C a 2 ~ N H C O - C H r C & r e s i n

Fmoc-Ala (0.187 g, 0.6 mmol), dissolved in minimum volume of DCM was

mixed with DCC (0.062 g, 0.3 mmol) and the mixture was stirred well for 1 h. DCU

formed was filtered off, evaporated the DCM and the Fmoc-Ala anhydride formed was

dried in vacuum.

PS-BDODMA-HMPB resin (500 mg, 0.06 mmol) swollen in DMF (10 MI) for 1 h

and the excess DMF was decanted. F m o ~ A l a anhydride was dissolved in minimum volume

of DMF and added to the swollen resin. DMAP (7.3 mg 0.06 mmol) was added to the

mixture and shaken for 1 h. The resin was filtered, washed with DMF (3 x 40 mL), isoamyl

alcohol (3 x 30 ml-), diethyl ether (3 x 30 mL) and dried under vacuum

Estimation of amino group in the resin

Fmoc-Ala-O-CH~-C6H3(OCH~)-O-(CH~)~-NHCO-CH2-C6fi-resin (10 mg) was

suspended in 20% piperidine in DMF (3 mL) for 30 min and then the OD of the solution was

measured at 290 nm. From the OD values, the extent of Ala attached to the resin can be

calculated [I0 mg Fmoc-Ala resin suspended in a solution of 20% piperidine in DMF (3 mL)

for 30 min has an optical density 1.65 at 290 nm, if the amino capacity of the resin is

0.1 mmol/g]. Amino capacity of Ala-PS-BDODMA-HMPB resin = 0.11 mmoYg resin.

6.3.a.5. Preparation of Fmoc-Leu-0-CHt-C&-resin

Fmoc-Leu (272 mg, 0.77 mmol) dissolved in minimum volume DCM and shaken

for 1 h with DCC (79 mg, 0385 mmol) dissolved in minimum volume of DCM. DCM

was filtered off, evaporate DCM from the filtrate forming Fmoc-Leu anhydride, which

was dried in vacuum.

Hydroxymethyl 2% PS-BDODMA resin (350 mg, 0.22 mmol OWg) was

suspended in DMF. Excess DMF was removed from the resin after 1 h. Fmoc-Leu

anhydride was dissolved in minimum amount of DMF and added to the swollen resin.

DMAP (9.4 mg, 0 077 mmol) was dissolved in DMF, added to the reaction mixture and

kept for 2 h with occasional shaking. The resin was filtered and washed with DMF (3 x

20 mL), isoamyl alcohol (3 x 20 mL), acetic acid (3 x 20 mL), isoamyl alcohol (3 x

20 mL) and diethyl ether (5 x 20 mL) and dried in vacuum.

Capacity = 0.2 mmol Leulg resin.

6.3.a.6. Preparation of Fmoc-Gly-0-CH2-CsH4-resin

Hydroxymethyl 2% PS-BDODMA resin (400 mg, 0.09 mmol) was suspended in

DMF (10 mL). Excess DMF was removed from the resin after 1 h. Fmoc-Gly-OPfp ester

was dissolved in minimum amount of DMF and added to the swollen resin. DMAP (10.9

mg, 0.09 mmol) was added to the reaction mixture and kept for 1 h at room temperature

with occasional swirling The resin was filtered and washed with DMF (3 x 20 mL),

diethyl ether (5 x 20 rnL) and dried in vacuum

Capacity of the resin= 0.2 mmol Glytg. resin.

6.3.a.7. Synthesis of Peptides

1. Synthesis of 1-15 fragment of Esculentin-1

(Gly-Ile-Phe-Ser-Lys-Leu-Gly-Arg-Lys-Lys-Ile-Lys-Asn-Leu-Leu)

Fmoc-Leu-0-CHZ-C&-resin (150 mg, 0.2 mmol OWg) was swelled in DMF for

1 h. Fmoc protect~on was removed by using 20% piperidine in DMF (1 x 10 mL,

20 min), wash the resin with DMF (6 x 10 mL). Fmoc-Leu (35.3 mg, 0.1 mmol), DCC

(21 mg, 0.1 mmol) and HOBt (14 mg, 0.1 mmol) dissolved in DMF was added to the

reaction mixture and kept at room temperature. The resin was filtered after 40 min and

washed thoroughly with DMF (6 x 10 mL). The remaining amino acids, Fmoc-Asn(Trt)

(60 mg, 0.1 mmol), Fmoc-Lys(Boc) (46.8 mg, 0.1 mmol), Fmoc-Ile (35.3 mg, 0.1 mmol),

Fmoc-Arg(Mtr) (60.8 mg, 0.1 mmol), Fmoc-Gly (29.7 mg, 0.1 mmol), ~ m o c - ~ e r ( ~ u ' )

(38.3 mg, 0.1 mmol), and Fmoc-Phe (38.7 mg, 0.1 mmol) were successively incorporated

by treating the Fmoc-removed resin with DCC (21.6 mg, 0.1 mmol) and HOBt (14 mg,

0.1 mmol). The resin was washed with DMF (6 x 10 mL). All acylation reactions were

performed twice for confirming the quantitative conversion. The Fmoc-deprotection and

extent of coupling in each cycle were monitored by Kaiser test. Atter the attachment of

all amino acids, Frnoc-protection was removed and the resin was washed with DMF (6 x

10 mL), ether (6 x 10 mL) and dried in vacuum.

The peptide was cleaved from the resin by suspending in TFA (2.7 pL), water

(150 pL), thioanisole (150 pL) and ethanedithiol (75 pL) for 6 h at room temperature.

The resin was filtered, washed with TEA and DCM and the combined filtrate was

evaporated. The peptide was precipitated by adding ice-cold ether. The peptide was

washed thoroughly with ether to remove the scavengers added. The yield of crude peptide

is 49 mg (96%). The peptide was dissolved in 1% acetic acid in water and passed through

a sephadex G-15 column. The peptidyl fractions were collected and lyophilized.

Amino acid analysis: Gly, 2.1 (2); Ile, 1.98 (2); Phe, 0.97 (1); Ser, 0.78 (1); Lys, 3.92 (4);

Leu, 3.1 (3); Arg, 0.94 (I); Asp, 0.97 (1). Low value of Ser is due its degradation during

acid hydrolysis and Asn is hydrolyzed to Asp.

MALDI TOF MS: m/z 1720. [(M+H)+, 100°?], CaoH143N2301g, requires h4+ 1719.18.

2. Synthesis of 1-15 fragment of Esculentin-1 modified at Gly by Proline

(Gly-Ile-Phe-Ser-Lys-Leu-Pro-Arg-Lys-Lys-Ile-Lys-Asn-Leu-Leu)

Fmoc-Leu-O-CH2-C&&-resin (150 mg, 0.2 mmol/g) was swelled in DMF for 1 h.

Fmoc-protection was removed by using 20% piperidine in DMF (1 x10 mL, 20 min). Wash

the resin thoroughly with DMF (6 x 10 mL) and coupling reactions were carried out in a

minimum volume of DMF as solvent. Fmoc-Leu (35.3 mg, 0.1 mmol) was attached to the

resin in presence of DCC (21 mg, 0.1 mmol) and HOBt (14 mg, 0.1 mmol) dissolved in

DMF and the reaction mixture was kept at room temperature. The resin was filtered after 40

min and washed thoroughly with DMF (6 x 10 mL). The remaining amino acids in the target

sequence, Fmoc-Asn(Trt) (60 mg, 0.1 mmol), FmooLys(Boc) (46.8 mg, 0.1 mmol), Fmoc-

Ile (35.3 mg, 0.1 mmol), Fmoc-Pro (33.7 mg, 0.1 mmol), Fmoc-Arg(Mtr) (60.8 mg,

0.1 mmol), F m o ~ G l y (29.7 mg, 0.1 mmol), FmocSer(~ut) (38.3 mg, 0.1 mmol), and Fmoc-

Phe (38.7 mg, 0.1 mmol) were successively incorporated by treating the Fmoc removed resin

with DCC (21.6 mg, 0.1 mmol) and HOBt (14 mg, 0.1 mmol) for 40 min. Atter 40 min the

resin was washed with DMF (6 x 10 mL). All acylation reactions were performed twice for

confirming the quantitative conversion. Each coupling and deprotection step was monitored

by Kaiser test. M e r the synthesis, Fmoc-protection was removed and the resin was washed

with DMF (6 x 10 mL), ether (6 x 10 mL) and dried in vacuum.

The peptidyl resin was suspended in TFA (2.7 mL) and a mixture of scavengers

thioanisole (150 PI,), water (150 pL) and ethanedithiol (75 pL) for 8 h at room

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

was evaporated under pressure to obtain an oily residue. The peptide was precipitated by

adding ice-cold ether to the oily residue. The peptide formed was washed thoroughly with

ether to remove the scavengers and was dissolved in 1% acetic acid in water. The peptide

solution was passed through a sephadex G-15 column and the peptidyl fractions were

collected and lyophilized. The yield of crude peptide is 50 mg (95%).

Amino acid analysis: Gly, 1.02 (1); Ile, 2.1 (2); Phe, 0.95 (1); Ser, 0.76 (1); Lys, 3.9 (4);

Leu, 3.1 1 (3); Pro, 0.94 (1); Arg, 0.96 (1); Asp, 0.98 (1). Low value of Ser is due its

degradation during acid hydrolysis and Asn is hydrolyzed to Asp.

MALDI TOF MS: m/z 1756.246 [(M+H)+, loo%], C83H147N230~8. requires M+ 1755.246.


(Thr-Gly-Ile-Asp-Ile-Ala-Gly-Cys(Acm)-Lys-Ile-Lys-Gly)

Fmoc-Gly-0-CH2-C&-resin (150 mg, 0.2 mmolfg) was suspended in DMF (10 mL)

for 1 h. Fmoc-protection was removed by using 20% piperidine in DMF (10 mL) for 20 min

and wash the resin thoroughly with DMF (6 x 10 mL). Coupling reactions were carried out in

minimum volume of DMF as solvent. Fmo~Lys(Boc) (46.8 mg, 0.1 rnmol) was attached to

the resin in presence of DCC (21 mg, 0.1 mmol) and HOBt (14 mg, 0. I mmol) dissolved in

DMF and the reaction mixture was kept at room temperature. The resin was filtered after

40 min and washed thoroughly with DMF (6 x 10 mL). The remaining amino acids, Fmoc-

Ile (35.3 mg, 0.1 mmol), Fmoc-Lys(Boc) (46.8 mg, 0.1 mmol), Fmo~Cys(Acm) (41.4 mg,

0 1 mmol), Fmoc-Ala (3 1.1 mg, 0.1 mmol), ~mooAs~(Bu'), Fmoc-Gly (29.7 mg, 0.1 mmol)

and ~moc-~hr (Bu~) (39.7 mg, 0.1 mmol) were successively incorporated by treating the

Fmoc removed resin with DCC (21.6 mg, 0.1 mmol) and HOBt (14 mg, 0.1 mmol). After

40 min, the resin was washed with DMF (6 x 10 mL). All acylation reactions were

performed twice for confirming the quantitative conversion. Each coupling and Fmoc-

deprotection steps were monitored by Kaiser test.

The Fmoc-deprotected peptidyl resin was suspended in TFA (2.8 mL) and water

(200 pL) for 8 h at room temperature, the resin was filtered and washed with TFA and

DCM. The filtrate was evaporated and ice-cold ether was added to it. The precipitated

peptide was washed thoroughly with ether. The peptide was dissolved in acetic acid/

water mixture and passed through a sephadex G-10 column. The peptidyl fractions were

collected and lyophilized. Yield of crude peptide is 36 mg (96%).

Amino acid analysis: Thr, 0.95 (I); Gly, 3.13 (3); Ile, 2.96 (3); Asp, 0.93 (1); Ala, 1.08

(1); Cys, 0.91 (I); Lys, 1.94 (2).

MALDI TOF MS: d z 1247.512 [(h4+H)+, loo%], C S ~ H ~ S N ~ ~ O I ~ S , requires M+

1246.505.

4. Synthesis of 33-44 fragment of Esculentin-1 modified at Gly 39 by Ala

(Thr-Gly-Ile-Asp-Ile-Ala-Ala-Cys-Lys-Ile-Lys-Gly)

Fmoc-Gly-0-CH2-CsH4-resin (150 mg, 0.2 mmollg) was suspended in DMF

(10 mL) for 1 h Fmoc-protection was removed by 20% piperidine in DMF (10 mL,

20 min) and washed the resin thoroughly with DMF (6 x 10 mL). Coupling reactions

were carried out in a minimum volume of DMF as solvent. Fmoc-Lys(Boc) (46.8 mg,

0.1 mmol) was attached to the resin in presence of DCC (21 mg, 0.1 mmol) and HOBt

(14 mg, 0.1 mmol) dissolved in DMF and the reaction mixture was kept at room

temperature. The resin was filtered after 40 min and washed thoroughly with DMF (6 x

10 mL). The remaining amino acids, Fmoc-Ile (35.3 mg, 0.1 mmol), Fmoc-Lys(Boc)

(46.8 mg, 0.1 mmol), Fmoc-Cys(Acm) (41.4 mg, 0.1 mmol), Fmoc-Ala (31.1 mg,

0.1 mmol), ~ m o c - ~ s ~ ( ~ u ' ) , Fmoc-Gly (29.7 mg, 0.1 mmol) and ~moc-~hr(Bu')

(39.7 mg, 0.1 mmol) were successively incorporated by treating the Fmoc removed resin

with DCC (21.6 mg, 0.1 mmol) and HOBt (14 mg, 0.1 mmol). Atter 40 min, the resin

was washed with DMF (6 x 10 mL). All acylation reactions were performed twice for

confirming the quantitative conversion. The coupling and deprotection steps were

monitored by Kaiser test. After the incorporation of all amino acids, Fmoc-protection was

removed and the resin was washed with DMF (6 x 10 mL), ether (6 x 10 mL) and dried

in vacuum.

The peptidyl resin was suspended in a mixture of TFA (2.8 mL) and water

(200 1L) at room temperature for 8 h. The resin was filtered and washed with

TFA and DCM The filtrate was evaporated and the peptide was precipitated by

adding ice-cold ether The yield of crude peptide is 35.5 mg (94%). The peptide

was dissolved in 1 % acetic acid water and passed through a sephadex G-10

column. The peptidyl fractions were collected and lyophilized.

Amino acid analysis: Thr, 0.83 (1); Gly, 2.11 (2); Ile, 3.13 (3); Asp, 0.91 (1); Ma, 2.00

(2); Cys, 0.79 (1); Lys, 1.89 (2). Low value of Thr is due to its degradation during acid

hydrolysis.

MALDI TOF MS: mlz 1261.512 [(M+H)+, lo%], C53H98N,5017S, requires M+

1260.512.


(Lys-Asn-Val-Gly-Lys-Glu-Val-Gly-Met-Asp-Val-Val-~g-Th-Gly-Ile-Asp-Ile-Ala)

F~OC-A~~-O-CH~-C~H~(OCH~)-O-(CHZ)~-CONH-CH~-C~~-~~S~~ (175 mg,

0.12 mmollg) was swollen in DMF for 1 h. 20% piperidine in DMF (1 x 10 mL, 20 min)

was used for the removal of Fmoc-protecting group. The resin was washed thoroughly

with DMF (6 x 10 mL) and the coupling reactions were carried out in minimum volume

of DMF as solvent. Fmoc-Ile (28.3 mg, 0.08 mmol) was attached to the resin in presence

of HBTU (30 3 mg, 0.08 mmol), HOBt (11.2 mg, 0.08 mmol) and DIEA (14 pL,

0.08 mmol) dissolved in DMF and the reaction mixture was kept at room temperature.

The resin was filtered after 40 min and washed thoroughly with DMF (6 x 10 mL). The

remaining amino acids in the sequence, ~ m o c - A s ~ ( 0 ~ u ~ ) (32.8 mg. 0.08 mmol),

Fmoc-Ile (28 3 mg, 0.08 mmol), Fmoc-Gly (23.7 mg, 0.08 mmol), ~rnoc-~hr@u')

(31.7 mg, 0.08 mmol), Fmoc-Arg(Mtr) (48.6 mg, 0.08 mmol), Fmoc-Val (27.3 mg,

0.08 mmol), Fmoc-Met (29.7 mg, 0.08 mmol), F~OC-GIU(OBU~) (34.5 mg, 0.08 mmol),

Fmoc-Lys(Boc) (37 5 mg, 0.08 mmol) and Fmoc-Asn(Trt) (47.8 mg, 0.08 mmol) were

successively incorporated to the resin using the coupling agent HBTU (30.3 mg,

0.08 mmol) in presence of HOBt (1 1.2 mg, 0.08 mmol) and DIEA (14 pL, 0.08 mmol)

for 40 min at room temperature. After each coupling the resin was washed with DMF

(16 x 20 mL). The coupling and Fmoc deprotection steps were monitored by ninhydrin

test. After the attachment of all amino acids, Fmoc-protection was removed and the resin

was washed with DMF (6 x 10 mL), ether (6 x 10 mL) and dried in vacuum.

The peptidyl resin was suspended in TFA (2.35 mL), thioanisole (150 pL), water

( 1 50 pL), phenol (200 pL) and ethanedithiol (150 pL) for 2 h at room temperature. The

resin was filtered, washed with TFA and DCM. The filtrate was evaporated and the

peptide was precipitated by adding ice-cold ether. The peptide was washed thoroughly

with ether to remove the scavengers added. The yield of crude peptide is 38.6 mg (92%).

The peptide was purified by passing through a sephadex G-25 column by dissolving it in

acetic acid/water mixture. The peptidyl fractions were collected and lyophilized.

Amino acid analysis: Ala, 1.00 (1); Ile, 2.01 (2); Thr, 0.83 (1); Arg, 0.96 (1); Met, 0.94

(1); 1 , 3 (3); Val, 4.12 (4); Lys, 1.92 (2); Asp, 2.93 (3); Glu, 0.93 (1). Asn is

hydrolyzed to Asp.

MALDI TOF MS: m/z 2002.512 [(M+H)+, IOP.], CajH149Nz50z&, requires M'

2001.355.

6. Synthesis of 9-27 fragment of Esculentin-1 modified by replacing Glu 14, Aspir and Asp 2s by Lys

(Lys-Asn-Val-Gly -Ly s-Lys-Val-Gly-Met-Lys-Val-Val-Arg-T~-Gly-Ile-Lys-Ile-~a)

F~oGA~~-O-C~H~-~~(OCH~>OCH~~-CONHCH&-~ (200 mg,O. 12 mmollg)

was suspended in DMF for 1 h. 2O0% pipendine in DMF (1 x 10 mL, 20 min) was used for

the removal of Fmoc-protecting group. The resin was washed well with DMF (6 x

10 mL) and the coupling was conducted in a minimum volume of DMF as solvent.

Fmoc-Ile (32 mg, 0.09 mmol) was attached to the resin in presence of HBTU (34.1 mg,

0.09 mmol), HOBt (12.6 mg, 0.09 mmol) and DIEA (16 WL, 0.09 mmol) dissolved in

DMF and the reaction mixture was kept at room temperature. The resin was filtered after

40 min and washed thoroughly with DMF (6 x 10 mL). The remaining amino acids in the

sequence, Fmoc-Ile (32 mg, 0.09 mmol), Fmoc-Gly (26.7 mg, 0.09 mmol), Fmoc-

~ h r ( ~ u ' ) (35.7 mg, 0.09 mmol), Fmoc-Arg(Mtr) (54.7 mg, 0.09 mmol), Fmoc-Val

(30.5 mg, 0.09 mmol), Fmoc-Met (33.4 mg, 0.09 mmol), Fmoc-Lys(Boc) (42.1 mg,

0.09 mmol) and Fmoc-Asn(Trt) (53.8 mg, 0.09 mmol) were successively incorporated to

the resin using the coupling agent HBTU (34.1 mg, 0.09 mmol) in presence of HOBt

(12.6 mg, 0.09 mmol) and DIEA (16 pL, 0.09 mmol) for 40 min at room temperature.

Afier each coupling the resin was washed with DMF (16 x 20 mL). The coupling and

Fmoc deprotection steps were monitored by ninhydrin test. M e r the attachment of all

amino acids, Fmoc-protection was removed and the resin was washed with DMF (6 x

10 mL), ether (6 x 10 mL) and dried in vacuum.

The peptidyl resin was suspended in TFA (2.45 mL), water (150 a ) , phenol (200 a ) ,

thioanisole (150 &) and ethanedithiol (150 pL) for 2 h at room temperature. The resin was

filtered, washed with TFA and rinsed with DCM. The filtrate was evaporated and the peptide

was precipitated by adding ice-cold ether. The peptide was washed thoroughly with ether to

remove the scavengers added. The yield of crude peptide is 44.6 mg (92%). The peptide was

again purified by dissolving in 1% acetic acid water mixture and the solution was passed

through a sephadex (3-25 column. The elution !?actions containing the peptide were collected

and lyophilized.


(1); Gly, 3.01 (3); Val, 4.24 (4); Lys, 4.88 (5); Asp, 0.91 (1). Asn is hydrolyzed to Asp.

Low value of Thr is due to its degradation during hydrolysis.

MALDI TOF MS: m/z 2027.6 [(M+H)+, 100°/o], C90H168N28022S, requires Mf 2026.582.

7. Synthesis of 9-27 fragment of Esculentin-1 modified by replacing Glyl~, GIy16, Glyz3 with Ala and ASPI~ASPZS, GIu14 with Lys

(Lys-Asn-Val-ALa-Lys-Lys-Val-Ala-Met-Lys-Val-Val-Arg-Thr-Ala-Ile-Lys-Ile-Ala)

FmocAla-O-CH~-Car,(OCH~~O-CH~~CONH-CHT~-reSin (230 mg, 0.12 mmollg)

was suspended in DMF for 1 h. 20% piperidine in DMF (1 x 10 mL, 20 min) was used

for the removal of Fmoc-protecting group. The resin was washed well with DMF (6 x

10 mL) and the coupling reactions were conducted in a minimum volume of DMF as

solvent. Fmoc-lle (35.5 mg, 0.1 mmol) was attached to the resin in presence of HBTU

(37.9 mg, 0.1 mmol), HOBt (14 mg, 0.1 mmol) and DIEA (17 pL, 0.1 mmol) dissolved

in DMF and the reaction mixture was kept at room temperature. The resin was filtered

after 40 min and washed thoroughly with DMF (6 x 10 mL). The remaining amino acids

in the sequence, Fmoc-Ile (35.5 mg, 0.1 mmol), Fmoc-Ala (3 1.1 mg, 0.1 mmol), Fmoc-

~ h r ( ~ u ' ) (39.7 mg, 0.1 mmol), Fmoc-Arg(Mtr) (60.8 mg, 0.1 mmol), Fmoc-Val

(33.9 mg, 0.1 mmol), Fmoc-Met (37.1 mg, 0.1 mmol), Fmoc-Lys(Boc) (46.8 mg,

0.1 mmol) and Fmoc-Asn(Trt) (59.7 mg, 0.1 mmol) were successively incorporated to the

resin using the coupling agent HBTU (37.9 mg, 0.1 mmol) in presence of HOBt (14 mg,

0.1 mmol) and DIEA (17 pL, 0.1 mmol) for 40 min at room temperature. Atter each

coupling the resin was washed with DMF (16 x 10 mL). The coupling and Fmoc

deprotection steps were monitored by ninhydrin test. After the attachment of all amino

acids, Fmoc-protection was removed and the resin was washed with DMF (6 x 10 mL),

ether (6 x 10 mL) and dried in vacuum.

The peptide bearing resin was suspended in TFA (2.45 mL), ethanedithiol

(150 pL), water (150 wL), phenol (250 pL) and thioanisole (150 pL) for 2 h at room

temperature. The polymeric material was filtered and washed with TFA and DCM. The

filtrate was evaporated and the peptide was precipitated by adding ice-cold ether. The

peptide was washed thoroughly with ether, dissolved in 1% acetic acid in water, and

passed through a sephadex G-25 column. The peptidyl fractions were collected and

lyophilized. Yield of crude peptide = 47 mg (92%).


(1); Val, 4.04 (4); Lys, 4.92 (5); Asp, 0.93 (1). Asn is hydrolyzed to Asp. Low value of

Thr is due to its degradation during hydrolysis.

MALDI TOF MS: m/z 2069.6 [(M+H)+, 100%], C ~ ~ H ? ~ ~ N ~ ~ O Z Z S , requires Mf 2068.667.

References

1 . Barany, G.; Merrifield, R. B. "The Peptides", Gross, E.; Meinhofer, J. Eds., Vo1.2.

Academic Press, New York, 1979, pp 1-289

2. Roice, M.; Kumar, K. S.; Pillai, V. N. R. Macromolecules 1999, 32, 8807.

3. Wang, S. S. .I Am. Chem. Soc. 1973,95, 1328.

4. Sheppard, R. C. ; Williams, B. J. Int. J. Peptide Protein Res. 1982, 20,451.

5. Florsheimer, A.; Riniker, B. in "Peptides 1990, Proc. 21" European Peptide

Symposium" Giralt, E.; Andrew, D. Eds., ESCOM, Leiden, 1991, p.131.

6. Rink, H. Tetrahedron Lett. 1987,28, 8787.

7. Daly, J . W. Proc. Natl. Acad Sci., USA 1995, 92, 9.

8. Erspamer, V. in "Amphibian Biology" Vol.1, Heatwole, H. ed. Surrey Beatty & Sons,

1994, pp. 1 7 8 3 5 0

9. Bevins, C. L.; ZaslofT, M. Annu. Rev. Biochem. 1990,59,395.

10. Erspamer, V., Melchiorri, P. Pure Appl.Chem. 1973, 35, 464.

1 1 . Boman, H. G, Cell 1991, 65, 205.

12. Simmaco, M.; Mignogna, G.; Barra, D.; Bossa, F. J. Biol.Chem. 1994, 11956.

SYNTHESIS OF BIOLOGICALLY ACTIVE PEPTIDES...

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