Chapter 4 Experimental - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/446/10/10_Chapter4.pdfsilver nitrate solution. (iii) Estimation of chlorine capacity by pyridine fusion

Chapter 4 Experimental

4. 1 Preparation of Polymer Supports and Functionalization

4. 1. a. Materials and methods

The polymeric supports under study were synthesized and

characterized in the laboratory. IR spectra were recorded on a Shimadzu

IR-470 spectrophotometer using KBr pellets. I3c CP/MAS NMR was

recorded at 75.47 MHz on a Bruker 300 MSL CP/MAS instrument at the

Sophisticated Instrumentation Facility, Indian Institute of Science,

Bangalore.

4. 1. b. Source of chemicals

Styrene, TTEGDA and Polyvinyl alcohol (mol. wt. 72,000) were

purchased from Sigma Chemical Company, USA. Benzoyl peroxide was

obtained from SISCO, Bombay and was recrystallised before use.

4. I . c. Polymer synthesis

(i) Preparation of 2% TTEGDA-crosslinked polystyrene by suspension polymerization

Styrene was destabilized by washing with 1% sodium hydroxide

solution (20 ml x3) and then washed with distilled water (20 ml x 3). It

was then dried over anhydrous calcium chloride. A 1% solution of

polyvinyl alcohol (mol. wt. 72,000; 3.48 g) in water (348 ml) was prepared

and kept mechanically stirred in a cylindrical polymerization vessel under

nitrogen atmosphere on a water bath at 80°C. A mixture of styrene (22.46

ml; 98 mmol), TTEGDA (1.089 ml; 2 mmol), toluene (20 ml) as inert

diluent and benzoyl peroxide (I g) was prepared and flushed with nitrogen

gas. This mixture was then added to the PVA solution kept at 80°C with

stirring. The polymerization was allowed to proceed for 6 h. The white

shiny beads obtained were collected by filtration and washed thoroughly

with hot water, acetone (30 ml; 3 x 3 min), melhanol(30 rnl; 3 x 3 min) and

drained. The product resin was soxhleted using acetone to remove all the

low molecular weight impurities and linear polymers and dried in the oven

at 80°C. Yield 80%. Beads were sieved into different sizes using standard

sieves. Polymers of different TTEGDA crosslinks were prepared by

adjusting the relative amounts of the monomers.

4. 1. d. Functionalization of TTEGDA-crosslinked polystyrene support to incorporate chloromcthyl groups 222: Gcncral procedure.

The resin beads (200-400 mesh size; 2 g) were allowed to swell in

dry dichloromethane (20 ml) in a round bottomed flask. Chloromethyl

methylether (12 ml) and a solution of anhydrous zinc chloride in THF (1M;

0.4 ml) was added to the swollen resin under anhydrous conditions slowly

with shaking. The mixture was refluxed at 50°C with calcium chloride

guard tube for 4h. It was then cooled and filtered through a sintered glass

funnel (G-2), washed with THF (30 ml; 3 x 3 min), THFl4N HCI (30 ml; 3

x 3 min), THFIwater (30 ml; 3 x 3 min), water (till the filtrate was free of

chloride ions), finally with methanol (30 ml; 3 x 3 min) and drained. It was

soxhleted with THF and dried in oven.

(i) Preparation of chloromethyl methyl ether 223

To a mixture of formaldehyde (66 ml) and methanol (33 ml), kept

at O°C, a constant stream of dry HCI gas was passed. The formation of

ether was indicated by the appearance of oily layer, after one hour.

Administration of HCI was continued for half an hour more till the ether

was separated clearly &om the aqueous phase. The oily layer was separated

and dried over calcium chloride. Yield: 40 ml. This was used without

further purification.

(ii) Preparation of IManhydrous ZnCl2 in THF

Anhydrous zinc chloride (1.5 g) was placed in a 25 ml Erlenmeyer

flask and concentrated HC1 (3 drops) and distilled water (5 drops) were

added and the contents stirred and heated until the solid dissolved

completely. Tcmpcrature was gradually raised to cvaporatc thc watcr and

to leave a crust of solid which was then melted by stronger heating. When

the zinc chloride became a clear mobile liquid with no further evolution of

bubbles, the flask was placed in a dessicator and allowed to cool. The

resulting mass was dissolved in freshly distilled THF (10 ml). The exact

concentration of the resulting solution was determined by pipetting a sample

in to water containing several drops of nitric acid and titrating against 0.1M

silver nitrate solution.

(iii) Estimation of chlorine capacity by pyridine fusion method:224 General procedure

The chloromethyl resin (200 mg) was fused with pyridine (5 ml) in

a boiling tube at 1 10°C for 5 h. The resin was quantitatively transferred

with acetic acidlwater (1:1), diluted with water (25 ml). To this, silver

nitrate solution (O.lN, 10 ml) was added after acidifying with concentrated

nitric acid (5 ml) and titrated against ammonium thiocyanate solution

(0.1M) using ferric alum as indicator (modified Volhard's method).225 A

blank was also performed.

4. 2. Peptide Synthesis

4. 2. a. Source of chemicals

All side chain protected L-amino acids, tertiary butyl carbazate,

dicy clohexylcarbodiimide, 2(t-butyloxycarbonyloximino 2-phenyl

acetonitrile) (Boc-ON), trifluoroacetic acid, thioanisole, 1,2-ethanedithiol

and cesium carbonatc were purchased from Sigma Chcmical Company,

USA. Boc-Gly, Boc-Ala, Boc-Leu, Boc-Ile and Boc-Val were prepared in

the laboratory following Schnabel's procedure and Boc-Phe by Boc-ON bo:, .,

I 11'

method. All solvents were of reagent grade and were obtained from E.

Merck, India. They were purified following literature procedures.

4. 2. b. Purification of reageGs and solvents

All the solvents used for peptide synthesis were purified before use.

Dichloromethane was dried by adding fused calcium chloride and kept

overnight.

Diethylether was dried over fused calcium chloride overnight.

Ethanol was double distilled and stored in airtight bottles.

Diisopropyl ethylamine was refluxed over ninhydrin for lh, distilled and

stored in amber coloured bottles.

Commercial ethyl acetate was distilled and used for extraction purposes.

Tetrahydrofuran (THF) was freed from peroxide by shaking with basic

alumina, dried using sodium till the water was removed, distilled and stored

in dark coloured bottles.

4.2. c. Physical measurements

An LKB BROMA high performance liquid chromatograph with

C18 reverse phase column (preparative) was used for the purification of

peptides. For checking the fractions, a Shimadzu C-R6A liquid

chromatograph with C18 reverse phase column (analytical) was used.

NMR studies were carried out on a Bxvker AMX-400 spectrometer. CD

spectra were recorded on a JASCO-J-500 spectropolarimeter attached with

a Jasco DP-501 N data processor using 1 mm path length cells. The mass

spectra were recorded on a Kratos PC-Kompact MALDI spectrometer and

Hcwlctt Packard Elcctrospray ionization spcctromctcr. Thc absorbing

matrix used was a-cyano-4-hydroxy cinnanlic acid.

4. 2. d. Detection

TLC was used to monitor the progress of the reaction and to check

the purity of the final product. TLC was performed on glass plates pre-

coated with silica gel containing calcium sulphate binder and activated by

heating for 6 h at 1 OO°C and cooled just before use.

4.2. e. Identification of the peptides on TLC

This is an inexpensive technique and is useful for detecting small

and medium range peptides. The methanol solution of the peptide is spotted

on the plate and developed in a suitable solvent of appropriate composition.

The solvent systems used include

Butanol-1 : acetic acid: water:pyridine (4: 1 : 1 : 1)

Butanol-1 : acetic acid: water (4:1:5)

Butanol-1: formic acid: water (10:3:8)

Chloroform: methanol: acetic acid (8.5: 1:0.5)

Methanol: chloroform (1 :9)

4. 2. f. Visualization

The developed chromatogram was visualized by the following

methods:

Ninhydrin spray:

Ninhydrin spray detects the presence of free amino groups. A 0.2%

pure ninhydrin in acetone was sprayed on the developed chromatogram and

heated in an air oven at 80-100°C for 10 min. Pink colour was developed

by free amino groups. N-terminal proline gives an yellow colour.

Iodine vapour.

The TLC plates were exposed to iodine vapours in a closed

chamber. Brown spots of amino acids and peptides were obtained.

4. 2. g. Preparation of reagents and amino acid derivatives

(4 Preparation of Boc-azidefrom t-butyl carbazate 226

Boc-azide was prepared from t-butyl carbazate following the

method of Carpino and coworkers. Tertiary butyl carbazate (20 g) was

dissolved in a mixture of glacial acetic acid (17.6 ml) and water (25 rnl).

Sodium nitrite (11.6 g) was added in small portions in about 15 min.

During the addition, the solution was stirred vigorously and maintained at

0°C. After 90 min, the oily layer was separated kom the aqueous layer.

The aqueous layer was extracted with ether (10 ml x 3). The ether extracts

were mixed with the oily layer, washed with water, 1 M sodium bicarbonate

(NaHC03) and dried over sodium sulphate. On evaporating ether under

reduced pressure, Boc-azide was obtained as a golden yellow liquid. It was

stored under refrigeration and used directly without purification. Yield: 20

ml.

(ii) Synthesis of Boc-amino acids: Schnabel's General procedure

The amino acid was suspended in dioxanelwater (1: 1) mixture and

Boc-azide was added to it. The mixture was stirred at room temperature,

maintained the pI~I in the alkaline range using 4N sodium hydroxide. Water

(1 5 ml) was added to the solution after 24 h and extracted with ether. The

aqueous layer was cooled in an ice-bath, acidified with 2N HC1, saturated

with sodium chloride and extracted with ethylacetate. The organic layer

was dried over anhydrous sodium sulphate and rotary evaporated to get the

Boc-amino acid. In most cases, the Boc-amino acid was precipitated by

adding dry petroleum ether followed by trituration. In certain cases,

seeding by a trace of fresh Boc-amino acid followed by trituration

precipitated the Boc-amino acid as a white powder.

In the case of leucine, the acidified aqueous layer was extracted

with ether. Purity of Boc-amino acid was tested on precoated TLC plate.

Solvent for the development of the chromatogram includes a mixture of

chloroform, methanol and glacial acetic acid in the ratio 85:10:5 (vlv). The

chromatogram was visualized by ninhydrin spray which gave a pink spot.

(iii) Preparation of Boc-glycine

Glycine (1.5 g; 20 rnmol) was suspended in 1:l dioxane-water

mixture (20 ml) and Boc-azide (3.2 ml; 20 rnmol) was added to it. The

mixture was stirred at room temperature maintaining the pH in the alkaline

range with 4 N sodium hydroxide solution. After 24 h, water (I5 ml) was

added and the solution was extracted with ether (I0 mi). The aqueous layer

was cooled in an ice-bath, acidified with 2 N HCl, saturated with NaCl and

extracted with ethyl acetate (20 ml x 3). It was dried over anhydrous

sodium sulphate and ethyl acetate was removed by rotary evaporation.

Petroleum ether was added in excess, seeded with a trace of fresh and pure

Boe-Gly to induce precipitation and triturated. The white powder of Boc-

Gly precipitate was washed with fresh petroleum ether and vacuum dried.

Yield: 1.2 g (80%)

Boc-Ala, Boc-Leu, Boc-Ile, Boc-Val, Boc-Pro were prepared following

Schnabel's method.

Table 4. 1 Preparation of Boc-amino acids

(iv) Boc-ON method: 228 General procedure

Amino acid (10 mmol), 4-tertiary butyloxycarbonyl oximino 2-phenyl

acetonitrile (Boc-ON) (2.71 g; 11 mmol) and triethylarnine (2.1 ml; 15

rnmol) in 50% aqueous dioxane (12 ml) were stirred at room temperature

for 12 h. The reaction mixture was diluted with water (20 ml) and washed

with ethylacetate (15 ml x 3). The aqueous layer was cooled to O°C,

acidified with 1N HCI and extracted with ethyl acetate (15 ml x 3). The

organic layer was dried over anhydrous sodium sulphate and rotary

evaporated to remove ethyl acetate. On adding petroleum ether, the Boc-

amino acid was obtained. All Boc-amino acid preparations were obtained

with 80-90 %yield.

(v) Preparation of Boc-Phe

Phenylalanine (0.84 g; 5 rnmol), Boc-ON (1.35 g; 5.5 mrnol) and

TEA (1.39 ml; 5.5 mrnol) were dissolved in 1.1 dioxane-water (6 ml) and

the mixture was stirred at room temperature for 12 h. The reaction mixture

was diluted with water (10 rnl) and washed with ethyl acetate (2 x 12.5 rnl).

The aqueous layer was acidified with 2N HCI to pH 2 and it was extracted

with ethyl acetate (10 ml x 3). The organic layer was dried and rotary

evaporated to remove ethyl acetate. The white powder of Boc-Phe

precipitated was washed with petroleum ether and vacuum dried.

Yield: 0.71 g (85%); M. I? (obs.): (78°C); M. I! (lit.) 80°C.

4. 2. h. Preparation of 1-Hydroxybenzotriazole (HOBt) 229

HOBt was prepared following the procedure adopted by Konig and

Geiger. 0-chloronitrobenzene (32 g) was dissolved in ethanol (100 ml). EL I ' "

Hydrazine hydrate (30 g) was added and the solution refluxed for 5 h. After

distilling off the ethanol, the residue was diluted with water (100 ml) and

extracted with ether (20 ml x 3). The aqueous layer was acidified with

concentrated HC1,when HOBt got precipitated. It was recrystallised from

hot water. Yield: 20 g (80%); M. P: 154°C.

4.2. i. Gcncral procedure for solid phase peptide synthesis

Manual SPPS using Boc-strategy was done in a glass reaction

vessel. The first amino acid of the C-terminal portion of the peptide was

esterified to the resin via a benzyl ester linkage by the cesium salt of the

Boc-amino acid. Boc group was deprotected with 33% TFA in DCM and

neutralization was effected by 5% DIEA in DCM or 5% TEA in DCM.

Second Boc-amino acid was coupled to the aminoacyl resin by DCC

coupling method or by active ester procedure. Dichloromethane or N-

methyl-2-pyrrolidone (NMP) was used as the coupling medium and the

coupling time was usually lh. The same procedure was adopted for the

coupling of all amino acids. Progress of coupling was monitored at every

stage by semiquantitative ninhydrin test. In all couplings, a 2.5 fold molar

excess of the Boc-amino acid was used and double coupling was done to

ensure completion of the reaction. Final cleavage of peptide from the

support was obtained by TFA in the presence of acid scavengers.

4.2. j. Attachment of first amino acid onto the resin

(9 Gisin's cesium salt method: 230 General procedure

The Boc-amino acid (2.5 mmol) was dissolved in ethanol and

neutralized with saturated solution of cesium carbonate. Ethanol was

evaporated under vacuum and the residue was dried by co-evaporating with

benzene (20 ml x 6) as an azeotrope under vacuum until the white powdery

ccsium salt of thc Boc-amino acid was obtained. It was then dissolvcd in

minimum quantity of NMP and the resin (lg; 1 mmol) was suspended in it.

The suspension was stirred at 50-60°C in an oil bath. After 24 h, the resin

was filtered out, washed successively with NMP (20 ml; 3 x 3 rnin), water

(20 ml; 3 x 3 rnin), methanol (20 ml; 3 x 3 min) and DCM (20 ml; 3 x 3

min) and dried in vacuum.

4. 2. k. Estimation of first amino acid substitution: Picric acid test:23' General procedure

The Boc-aminoacyl resin (10 mg) was deprotected using 33% TFA

in DCM for 30 min. filtered, washed with DCM (6 times) to get rid of TFA

completely. It was then neutralized with 5% TEA in DCM for 5 min.and

again washed with DCM and dried. From the deprotected resin, exactly 5

mg was taken in a Gisin tube and treated with 0.1M picric acid (2 x 5 min).

The unbound picric acid was washed off with DCM (2 ml; 3 x 2 min). The

resin bound picrate was then carefully eluted with 5% TEA in DCM till the

washings were clear. It was then made up to a definite volume (15 ml)

using 95% ethanol. The optical density (OD) of this solution was measured

at 358 nm. From the extinction coefficient of picrate (~3~~=14,500), the OD

value and the weight of the resin taken, the substitution level of the first

amino acid was estimated.

4. 2. 1. Removal of t-butyloxycarbonyl group

'The t-butyloxy (t-Boc) protection of amino acids and peptides can

be deprotected using anhydrous TFA in DCM (33%). For deprotection, the

protected amino acid or peptide was treated with the above solution at room

temperature for 30 min. Excess TFA was removed by filtration followed by

washing with DCM and the salt thus obtained was neutralized with DIEA in

DCM (5%).

4. 2. m. Methods of activation and coupling

Mainly two methods were used for the activation of carboxyl group

by dicyclohexyl carbodiimide (DCC).

(i) Dicyclohexylcarbodiimide coupling "'

In the conventional DCC coupling method, the amino acid and

DCC were used in 1:1 ratio and they form 0-acylisourea as the active

intermediate which readily reacts with other N-protected amino acids to

give the symmetrical anhydride and dicyclohexyl urea (DCU). The

precipitated DCU was removed by washing with 33% methanol in DCM.

The extent of coupling was monitored by Kaiser reagent. If the test was

positive, a second coupling was done a3 described earlier. In some cases, a

third coupling was necessary to ensure complete reaction.

(ii) Active ester method 233

Certain difficult couplings were performed by the active ester

method. Active ester can be preformed or can be prepared in situ. In this

method, the Boc-amino acid, DCC and HOBt were used in 1 : 1 : 1 ratio to get

the active ester along with DCU. The precipitated DCU was removed by

washing with 33% methanol in DCM. This method was very usefbl for

activating Asn and Gln.

4. 2. n. Cleavage of the peptide from the resin: TFA/Thioanisole method234

The peptidyl resin (100 mg) was suspended in TFA (10 ml) and to this,

thioanisole (0.1 mi), m-cresol (0.1 rnl) and 1,2-ethanedithiol (0.1 ml) were

added. The reaction mixture was left for 20 h at room temperature. It was

filtered and the TFA solution was rotary evaporated to remove TFA. The

peptide was then precipitated by addition of ice cold ether and washed

thoroughly with ether, centrifuged ( I 0-1 5 times) and dried.

4. 2. o. Purification

(i) Thin layer chromatography (TLC)

TLC was used for estimating the purity both of starting materials

for SPPS and of synthesized short peptides. Aqueous or methanolic

solution of the peptide was spotted on the silica plate and developed in a

suitable solvent mixture of appropriate composition. The following solvent

system in indicated ratios were used.

Butanol-1 : acetic acid: water (4: 1 : 5 )

Butanol-1 : acetic acid: water: pyridine (4: 1 : 1 : 1)

Chloroform: methanol: acetic acid (8.5:1:0.5)

Methanol: chloroform (1 :9)

Butanol- 1 : formic acid: water (10:3:8)

Acetonitrile: water (3: 1)

Identification sprays

The following reagents were used to visualize the spots on the TLC

plate.

Ninhydrin spray:

The Boc group was removed by exposing the plate to vapours of

conc. IICl contained in a chamber. The spots were then developed by

spraying ninhydrin reagent and heating in an oven for 5-10 minutes. Violet

spots were obscwcd in thc case of free primary amino groups.

Chlorine gas/Starch/KI reagent (Rydon 's reagent):

This spray can be used for protected peptides not visible with

ninhydrin test. This test is given by almost all compounds containing -NH

groups. The plates were exposed to chlorine gas and sprayed with a

mixture containing equal volumes of 1% (wlv) aqueous starch and

potassium iodide solutions. Blue black spots over blue background were

observed.

Iodine:

The plates were exposed to iodine vapours in a closed chamber.

Brown spots were observed in the case of amino acids and peptides.

(ii) High performance liquid chromatography

Solid phase peptide synthesis became an established procedure only

after the introduction of efficient HPLC. It has extremely high resolving

power and is very useful in establishing the homogeneity of peptides.

HPLC analyses were done using a Shirnadzu two pump system equipped

with a controller unit. A Vydac C18 column 218 TP (particle size 5 pm)

and an injection loop of 20 p1, 0.8 mllmin flow rate and detection at 226 nm

were used.

Peptide purification was carried out on LKB HPLC system. Thc

column used was C18 (4 x 250 mm, particle size, 10 pm) and an injection

loop of 50 p1 and a flow rate of 1.5 rnllmin was used. Repetitive injections

of lmg per run were carried out and the desired fractions were collected by

the use of a LKB Superrac fraction collector. Details of gradients used

were re injected on the Shimadzu HPLC to confirm their homogeneity.

Detections were done at 226 nm.

(iii) Mass spectroscopy

Mass spectrometric analysis can be used for the accurate

determination of molecular weight of proteins, peptides and

oligonucleotides. Mass spectral interpretation helps determination of

identity of a molecule by deducing information from its mass spectrum.

ES is a soft ionization technique that allows the mass spectrornctric

analysis of large biomolecules such as polypeptides as well as non-volatile

and thermally unstable compounds. It allows deposition of multiple chargcs

on a protein without causing fragmentation of the protein. This allows

accurate mass determination down to a few daltons. In the electrospray

ionization process, a flow of sample solution is pumped through a narrow

bore metal capillary held at a potential of a few kilovolts relative to a

counter electrode. Charging of the liquid occurs and as a result, it sprays

(aerosol) fi-om the capillary orifice as a mist of very fine, charged droplets.

Solvent is striped away by a heated inert gas. This spraying process takes

place at atmospheric pressure in a specially designed chamber, outside the

vacuum region of the mass spectrometer. The whole ionization process is,

in fact a form of atmospheric pressure ionization (API).

The charged droplets upon solvent evaporation, decreases in size

until they become unstable and explode (coulomb explosion) to form a

number of small droplets. Finally, at a still smaller size, the field due to the

excess charge is large enough to cause the desorption of ionized sample

molecules from the droplet. These ions, which are field desorbed from the

droplets at atmospheric pressure, are then sampled through a series of

skimmers and lenses focus the ions into a beam. The nebulising and drying

gases are pumped away transferring the ions into the vacuum system for

mass analysis. A particular feature of the ES ionization spectra is that the

molecular ions recorded are multiple charged, (M+nH),"'in the positive-ion

mode, or (M-nH)"- in the negative-ion mode and also cover a range of

charge states. On average, one charge is added per 1000 Da in mass. ES

method offers methods for molecular weight determination of proteins and

other biopolymers with acceptable accuracy, convenient access, particularly

by insource dissociation, to some fragment ion information.

ESMS characteristics are:

Determination of mol. weights from 50 to morc than 100,000 Da with

0.02% accuracy.

Femtogram to picogram sensitivity.

Compatible with a wide range of compounds.

Compatible with a wide range of inlets such as HPLC.

MALDI is a soft ionization technique that produces (quasi)

molecular ions from large non-volatile molecules such as proteins,

oligonucleotides, polysaccharides and synthetic polymers with minimum

fragmentation. The first attempt to use organic matrices to facilitate laser

desorption and ionisation of non-volatile aminoacids and peptides was

reported by Karas, Bachmann and Hillenkamp in 1985 using nicotinic acid

as the organic matrix. Hillenkamps group further developed this approach

and produced molecular ion mass spectra of proteins with masses in excess

of 10,000 Da .

The use of organic matrices has become routine for MALDI, which

is one of the most powerhl tools for mass analysis of high molecular

weight compounds. MALDI generates high mass ions by irradiating a solid

mixture of an analyte dissolved in a suitable matrix compound with a pulsed

laser beam. The laser pulse desorbs and indirectly ionises the analyte

molecules. A short-pulse UV laser is typically used for the desorption.

Different wavelengths such as IR have been investigated recently as

alternatives. MALDI consists of two steps, sample preparation and mass

spectral analysis. The key to a successful MALDI analysis depends

primarily on uniformly mixing the matrix and the analyte. The samples are

typically prepared in a suitable solvent such as water, acetone or THF. A

few microlitres of this mixture is deposited onto a substrate and dried, and

the solid matrix is then placed into the mass spectrometer.

4. 2. p. Synthesis of model peptides

0) Synthesis of Ala-Phe-Gly

Attachment of Boc-Gly to the chloromethyl resin

Boc-Gly (83.lmg; 0.475 mmol) was dissolved in ethanol and

converted to cesium salt by adding a saturated solution of cesium carbonate

till the solution is neutral. EtOH was evaporated under pressure and water

removed by azeotropic distillation with benzene. The cesium salt was

dissolved in minimum volume of NMP and chloromethyl resin (100 mg;

0.19 mmol) was added and kept at 50°C for 48 h. The resin was washed

with NMP (20 ml x 3), 1:l NMP-water (20 ml x 3), water, methanol (20 ml

x 3), DCM (20 ml x 3) and dried in vacuum.

Synthesis of Ala-Phe-GIy

The Boc-protection on the resin was removed with 33% TFA in

DCM. The resin was washed with DCM (20 ml x 3) and then neutralized

with 5% DIEA in DCM (20 ml x 6) . The resin was washed with DCM (20

ml x 4) and NMI' (20 ml x 2). Then, Boc-Phe (100.8 mg; 0.38 mmol) atid

Boc-Ala (71.82 mg; 0.38 mmol) were coupled successively to the resin as

their HOBt active ester. Active ester was prepared by shaking the

respective Boc-amino acid with HOBt (51.3 mg; 0.38 mmol) and DCC

(78.28 mg; 0.38 mrnol) in NMP. DCU formed was filtered off and the

active ester was added to the resin. After the synthesis, the peptide resin

was washed with NMP (20 ml x 3), MeOH (20 ml x 3), DCM (20 ml x 3)

and dried in vacuum. The procedure for one synthetic cycle is given below.

1. Wash the resin with DCM (1 x 5 min)

2. Deblocking using 33% TFA in DCM (1 x 30)

3. Wash with DCM (6 x 1.5 min)

4. Prewash using 5% DIEA in DCM

5. Neutralization with 5% DIEA in DCM (I x 5 min)

6. DCM wash (4 x 1.5 min)

7. NMP wash (2 x 1.5 min)

8. Coupling of Boc-amino acid in presence of DCC and HOBt (60 min) (1:l:l)

9. DCU removal by washing with 33% methanol in DCM (3 x 2 min)


11. Repetition of steps 7-10 to ensure maximum coupling, tested by Kaiser reagent.

Cleavage of the IJe~tide from the resin

50 mg of the peptidyl resin was treated with TFA (5 ml),

thioanisole (0.05 ml) and 1,2-ethanedithiol (0.05 ml) at room temperature

for 18 h. The resin was removed by filtration and TFA removed by rotary

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

The precipitated peptide was washed several times with ether to get rid of

TFA and scavengers , dried and weight noted (1 8 mg).

(ii) Synthesis of Gly-Gly-Gly

Attachment of Boc-Gly to the chloromethvl resin

Boc-Gly (83.1 mg; 0.475 mmol) was dissolved in minimum

quantity of ethanol. Then, saturated solution of cesium carbonate is added

till the solution becomes neutral. It was kept for sometime and rotary

evaporated to remove ethanol. Water was removed by azeotropic

distillation with benzene. This was continued till a white powder of cesium

salt of Boc-Gly was obtained. It was dried under vacuum. Cesium salt of

Boc-Gly was dissolved in NMP, chloromethyl resin (100 mg; 0.19 mmol)

was added and heated at 50°C for 48 h. The resin was filtered, washed with

NMP (20 ml x 3), NMP-water (I: I; 20 ml x 3), water, methanol (20 ml x

3), DCM (20 ml x 3), drained and dried under vacuum.

Svnthesis of Gly-Gly-Glv

The deprotection of Boc-Gly on the resin was done with 33% TFA

in DCM for 30 minutes and the resin was washed with DCM (20 ml x 6)

and neutralized with 5% DIEA in DCM. The resin was washed with DCM

(20 ml x 4) and NMP (20 ml x 2). The Boc-glycines were then

successively coupled by HOBt active ester method. Boc-Gly (66.5 mg;

0.38 rnmol) was dissolved in NMP, HOBt (5 1.3 mg; 0.38 mmol) was added,

and the solution was shaken with DCC (78.28 mg; 0.38 mmol) for lh, DCU

formed was filtered off and the filtrate added to the resin. The mixture was

shaken for 60 minutes. After synthesis, peptidyl resin was washed with

NMP (20 ml x 3), MeOH (20 ml x 3), DCM (20 ml x 3) and dried in

vacuum. The procedure for one synthetic cycle is given below.

1. Wash with DCM (1 x 5 min)



4. Prewash with 5% DIEA in DCM

5. Neutralization with 5% DIEA in DCM (1 x 5 min)


7. Wash with NMP (2 x 1.5 min)

8. Coupling of Boc-amino acid in presence of DCC and 11OBt (60 min)

9. Washing off DCU with 33% methanol in DCM (3 x 2 min)


I I . Repetition of steps 7-10 for maximum coupling, tested by Kaiser reagent.

Cleavage . of the ue~tide from the resin



for 20 h. The resin was removed by filtration and TFA evaporated by

rotary evaporation. The peptide was precipitated by ice-cold ether. The

precipitated peptide was washed with ether several times to remove TFA

and scavengers, dried and weight noted (18 mg). A small amount of the

peptide was dissolved in methanol and spotted onto a TLC plate to check

the purity.

(iii) Synthesis of Gly-Ala-Ala

Attachment of Boc-Ala to the chloromethvl resin

A 2% TTEGDA-PS resin was used for this synthesis. Boc-Ala

(52.5 mg; 0.475 mmol) was dissolved in minimum volume of ethanol.

Then, saturated solution of cesium carbonate is added to make the solution

neutral. Ethanol was rotary evaporate$ water was removed by azeotropic

distillation with benzene. This was repeated to get a white powder of

cesium salt and was dried under vacuum. Cesium salt of Boc-Ala was

dissolved in NMP, chloromethyl resin (100 mg; 0.19 mmol) was added and

heated at 50°C for 48h. The resin was filtered, washed with NMP (20 ml x

3); NMP-water (20 mi x 3); water; methanol (20 ml x 3), DCM (20 ml x 3),

drained and dried under vacuum. The substitution level of Boc-Ala was

determined.

Svnthesis of Glv-Ala-Ala

Deprotection of Boc-Ala resin was done using 33% TFA in DCM

for 30 minutes. After washing with DCM (20 ml x 6), the resin was

neutralized with 5% DIEA in DCM. The rest of the amino acids were

coupled in NMP by HOBt active ester method. Active ester was prepared

by shaking the respective amino acids with HOBt (5 1.3 mg; 0.38 mmol) and

DCC (78.28; 0.38 mmol) in NMP. DCU formed was filtered off and the

active ester was added to the resin. After the synthesis, the peptide resin

was washed with NMP (20 ml x 3), MeOH (20 mi x 3), DCM (20 rnl x 3)

and dried under vacuum. The protocol for the synthesis is given below.

Table 4. 2. Protocol for the synthesis of Gly-Ala-Ala

Cleavage of the DeDtide from the resin

50 mg of the peptidyl-resin was treated with TFA (5 ml),

thioanisole (0.05ml) and 1,2-ethanedithiol (0.05 ml) at room temperature

for 18 h. The resin was removed by filtration and TFA by rotary

evaporation. ?'he peptide was precipitated by the addition of ice-cold ether.

'The precipitated peptide was washed several times with ether to remove

TFA and scavengers, dried and yield noted (18 mg).

DCC: HOBt in NMP

(iv) Synthesis of Ac.Ala-Ala-Ala

9

10

Attachment of Boc-Ala to the chloromethyl resin

4% TTEGDA-PS (1.5 mmollgm) was used for this synthesis. Boc-

Ala (70.87 mg; 0.375 mmol) was dissolved in minimum volume of ethanol.

Wash

Steps 6-9 repeated for second coupling

DCM 3 x 2

Then, saturated solution of cesium carbonate was added till solution became

neutral. It was stirred for sometime and rotary evaporated to remove

ethanol. Benzene was added and the azeotropic mixture was evaporated till

a white powder of cesium salt of Boc-Ala was obtained. It was dried under

vacuum. The cesium salt was dissolved in NMP, chloromethyl resin (100

mg; 0.15 mmol) was added and heated at 50°C for 48h. The resin was

filtered, washed with NMP (20 ml x 3), NMP-water (1:l; 20 ml x 3), water,

methanol (20 ml x 3) and DCM (20 ml x 3), drained and dried under

vacuum.

Synthesis of Ala-Ala-Ala

The deprotection of Boc-Ala resin was done with 33% TFA in

DCM for 30 minutes. The resin washed with DCM (20 ml x 6) and

neutralized with 5% DIEA in DCM. The resin was washed with DCM (20

ml x 4) and NMP (20 ml x 2). The remaining residues were attached by the

HOBt active ester method. Boc-Ala (56.7 mg; 0.30 mmol) was dissolved in

NMP, HOBt (40.5 mg; 0.30 mmol) was added and the solution was shaken

with DCC (61.8 mg; 0.30 mmol) for 45 minutes. DCU formed was filtered

off and the filtrate added to the resin. The mixture was shaken for 60

minutes. After synthesis, the peptidyl-resin was washed with NMP (20 ml

x 2), MeOH-DCM mixture (20 ml x 6), DCM (20 ml x 6) and dried in

vacuum. The steps involved in the synthesis are given below.

1. Wash with DCM (1 x 5 min)

2. Boc-deprotection using 33% TFA in DCM (1 x 30)

3. DCM wash (6 x 1.5 rnin)



- - 6. Wash with DCM (4 x 1.5 min)

7. NMP wash (2 x 1.5 rnin)

8. Coupling with 1 : 1 : 1 Boc-amino acid, DCC and HOBt (60 ~~ .. ~~ ~ ...

9. Washing off DCU with 33% methanol in DCM (3 x 2 min) r . .


I I . Rcpcat steps 7-10 to cnsurc maximum coupling, tcstcd by Kaiscr reagent.

Acetylation of Ala-Ala-Ala resin (Acetic anhydride method)235

Suspend the peptidyl resin in DMF. Prepare an acetylating mixture

by adding 5 mmol (0.47 ml) acetic anhydride and 5 mmol (0.70 ml) TEA to

15 ml of DMF. Add the acetylating mixture to the resin suspension and

shake for 30 minutes until the Kaiser test is negative.236

Cleavarzc of the veutide from the resin

50 mg of the peptidyl-resin is treated with TFA (5 ml), thioanisole

(0.05 ml), and 1,2-ethanedithiol (0.05 ml) at room temperature for 20 h.

The resin was removed by filtration, TFA evaporated by rotary evaporation.

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

precipitated peptide was washed several times with ether to remove TFA

and scavengers, dried and weight noted (18 mg).The purity of the

synthesized peptide was checked by TLC.

(v) Synthesis of Asn-Ala-Gly-Ala

Attachment of Boc-Ala to the chloromethvl resin

Boc-Ala (89.75 mg; 0.475 mmol) was dissolved in ethanol and

converted to cesium salt by adding a saturated solution of cesium carbonate

till the solution becomes neutral. Ethanol was evaporated under pressure

and water removed by azeotropic distillation with benzene. The cesium salt

was dissolved in minimum volume of NMP and chloromethyl resin (100

mg; 0.19 mmol) was added and kept at 50°C for 48 h. The resin was

washed with NMP (20 ml x 3), 1:l NMP-water (20 ml x 3), water,

methanol (20 ml x 3), DCM (20 ml x 3) and dried under vacuum.

Synthesis of Asn-Ala-Gly-Ala

The Boc-Ala protection was removed by 33% TFA in DCM. The

resin was washed with DCM (20 ml x 6) and then neutralized with 5%

DIEA in DCM (20 ml x 6). The resin was washed with DCM (20 ml x 4)

and NMP (20 ml x 2). Then, Boc-Gly (66.5 mg; 0.38 rnmol), Boc-Ala

(71.82 mg; 0.38 mmol) and Boc-Asn (88.25 mg; 0.38 mmol) were

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

was prepared by shaking the respective Boc-amino acid with HOBt (51.3

mg; 0.38 mmol) and DCC (78.28 mg; 0.38 mmol) in NMP. DCU formed

was filtered off and the active ester was added to the resin. After the

synthesis, the peptide resin was washed with NMP (20 ml x 3), MeOH (20

ml x 3), DCM (20 ml x 3) and dried in vacuum. The steps involved in the

synthesis of Asn-Ala-Gly-Ala are shown in Table 4.3.

Table 4. 3. Synthesis of Asn-Ala-Gly-Ala

:DCC:HOBt in NMP

Cleavage of the ueutide from the resin




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


TFA and scavengers, dried and weight noted (16 mg).

(vi) Synthesis of Val-Gly-Val-Ala-Pro-Gly

Attachment of Boc-Gly to the chloromethvl resin

A 2% TTEGDA-PS resin was used for this synthesis. Boc-Gly

(83.125 mg; 0.475 mmol) was dissolved in ethanol and converted to cesium

salt. Ethanol was evaporated under pressure and water removed by

azeotropic distillation with benzene. The cesium salt was dissolved in

minimum volume of NMP and chloromethyl resin (100 mg; 0.19 mmol)

was added and kept at 50°C for 48 h. The resin was washed with NMP (20

ml x 3), 1 : 1 NMP-water (20 ml x 3), water, MeOH (20 ml x 3), DCM (20

ml x 3) and dried under vacuum.

Synthesis of Val-Glv-Val-Ala-Pro-Gly

Boc-protection was removed with 33% TFA in DCM. The resin

was washed with DCM (20 ml x 6) and then neutralized with 5% DIEA in

DCM. The resin was washed with DCM (20 ml x 4) and NMP (20 ml x 2).

Then, Boc-Pro (81.7 mg; 0.38 mmol), Boc-Ala (71.82 mg; 0.38 mmol) ,

Boc-Val (82.56 mg; 0.38 mmol), Boc-Gly (66.5 mg; 0.38 mmol) and Boc-

Val (82.56 mg; 0.38 mmol) were coupled successively to the resin as their

HOBt active esters. Active ester was prepared by shaking the respective

Boc-amino acid with HOBt (51.3 mg; 0.38 mmol) and DCC (78.24 mg;

0.38 mmol) in NMP. DCU formed was filtered off and the active ester was

added to the resin. Atter the synthesis, the peptide resin was washed with

NMP (20 ml x 3), MeOH (20 ml x 3), DCM (20 ml x 3) and dried in

vacuum. The protocol followed for the synthesis is shown below.

105

Table 4.4. Protocol for the synthesis of Val-Gly-Val-Ala-Pro-Gly

Cleavage of the ueutide from the resin


thioanisole (0.05 ml), and 1,2-ethanedithiol (0.05 ml) at room temperature

for 20 h. The resin was removed by filtration and TFA by rotary

evaporation. Then, the peptide was precipitated by the addition of ice-cold

ether . The precipitated peptide was washed with ether several times to get

rid of TFA and scavengers, dried and weight noted (19 mg).

DCC: HOBt in NMP

4. 2. q. Synthesis of hydrophobic peptides

9

10

(i, Synthesis of Ala-Cys-Leu-Phe- Val-Dpro-Gly-Leu- Val- Val-Cys-Ala

Attachment of Boc-Ala to the chloromethyl resin

Wash

Steps 6-9 repeated for second coupling

DCM 3x2

Boc-Ala (1 13.4 mg; 0.6 mmol) was dissolved in ethanol and the

cesium salt was prepared by adding a saturated solution of cesium

carbonate. Ethanol was evaporated under vacuum and water removed by

azeotropic distillation with benzene. The cesium salt was dissolved in

minimum volume of NMP and chloromethyl resin (400 mg; 0.24 mmol)

was added and kept at 50°C for 48 h. The resin was washed with NMP (40

ml x 2), 1:l NMP-water (40 ml x 3), water, methanol (40 ml x 3), DCM

(40 ml x 3) and dried under vacuum.

Synthesis of Ala-Cvs-Leu-Phe-Val-Dvro-Glv-Leu-Val-Val-Cys-Ala

The Boc-protection on the resin was removed by 33% TFA in

DCM. After neutralization with 5% DIEA in DCM, the resin was washed

with DCM (40 ml x 4) and NMP (40 ml x 2). Then, Boc-Cys.Acm (140

mg; 0.48 mmol), Boc-Val (104 mg; 0.48 mmol), Boc-Val (104 mg; 0.48

mmol), Boc-Leu (11 1.0 mg; 0.48 mmol), Boc-Gly (84 mg; 0.48 mmol),

Boc-Dpro (103.2 mg; 0.48 mmol), Boc-Val (104 mg; 0.48 mmol), Boc-Phe

(127.3 mg; 0.48 mmol), Boc-Leu (1 11.0 mg; 0.48 mmol), Boc-Cys.Acm

(140 mg; 0.48 mmol) and Boc-Ala (90.7 mg; 0.48 mmol) were successively

coupled to the resin as their HOBt active esters. Active ester was prepared

by shaking the respective Boc-amino acid with HOBt (64.8 mg; 0.48 mmol)

and DCC (98.8 mg; 0.48 mmol) in NMP. DCU formed was filtered off and

the active ester was added to the resin. After the synthesis, the peptide resin

was washed with NMP (40 ml x 3), MeOH-DCM mixture (40 ml x 3),

DCM (40 ml x 3) and dried in vacuum. The steps involved in the synthesis

are shown below.

Table 4. 5. Protocol for the synthesis of 12 residue peptide

Cleavage and purification


thioanisole (0.2 ml) and 1,2-ethanedithiol (0.2 ml) at room temperature for

22 h. The resin was removed by filtration and TFA removed by rotary

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


TFA and scavengers, dried and yield noted (80 mg). A small aliquot of the

peptide dissolved in methanol was injected to a Shimadzu C-R6A liquid

chromatograph with C18 rpc and eluted using 0.1% TFA in 100% water (A)

and 0.1% TFA in 80% acetonitrile: 20% water (B) to check the purity of the

peptide. The peptide was then purified to homogeneity by C18 reversed

phase preparative HPLC. The purified peptide was analyzed by mass

spectrometry, CD and NMR. [Section 3. 2 (ii) (a)]

(ii) Synthesis of Val-Leu-Gly-Phe-Leu-Gly-Phe-Leu-Ala-Thr-Ala-Gly- Ser-Ala-Met-Gly-Ala-Ala-Ser-Leu

Attachment of Boc-Leu to the chloromethvl resin

Boc-Leu (103.95 mg; 0.45 mmol) was dissolved in ethanol and

converted to the ccsium salt by adding a saturated solution of cesium

carbonate till the solution is neutral. Ethanol was evaporated under vacuum



mg; 0.45 mmol) was added and kept at 50°C for 48 h. The resin was


methanol (20 ml x 3), DCM (20 ml x 3) and dried. The substitution level

of Boc-Leu was determined.

Synthesis of Val-Leu-Gly-Phe-I~eu-Gl~he-Leu-Ala-Thr-Ala-Glv-Ser-Ala- Met-Glv-Ala-Ala-Ser-Leu

The deblocking of Boc-Leu resin was carried with 33% TFA in

DCM. The resin was washed with DCM (20 ml x 6) and neutralized with

5% DIEA in DCM. The resin was washed with DCM (20 ml x 4) and with

NMP (20 ml x 2). Then, Boc-Ser.OBzl (106.3 mg; 0.36 mmol), Boc-Ala

(68.04 mg; 0.36 mmol), Boc-Ala (68.04 mg; 0.36 mmol), Boc-Gly (63.0

mg; 0.36 mmol), Boc-Met (89.74 mg; 0.36 mmol), Boc-Ala (68.04 mg; 0.36

mmol), Boc-Ser.OBzl (106.3 mg; 0.36 mmol), Boc-Gly (63.0 mg;0.36

mrnol), Boc-Ala (68.04 mg; 0.36 mmol), Boc-Thr.OBzl (1 11.38 mg; 0.36

mmol), Boc-Ala (68.04 mg; 0.36 mmol), Boc-Leu (83.16 mg; 0.36 mmol),

Boc-Phe (95.5 mg; 0.36 mmol), Boc-Gly (63.0 mg; 0.36 mrnol), Boc-Leu

(83.16 mg; 0.36 mmol), Boc-Phe (95.5 mg; 0.36 mmol), Boc-Gly'(63.0 mg;

0.36 mmol), Boc-Leu (83.16 mg; 0.36 mmol), and Boc-Val (78.2 mg; 0.36

mmol) were successively coupled to the resin as their HOBt active esters.

Active ester was prepared by shaking the respective Boc-amino acid with

HOBt (48.6 mg; 0.36 mmol) and DCC (74.16 mg; 0.36 mmol) in NMP.

DCU formed was filtered off and the active ester was added to the resin.

After the synthesis, the peptide resin was washed with NMP (30 mi x 3),

MeOH-DCM mixture (30 ml x 3), DCM (30 ml x 3) and dried in vacuum.

The protocol for the synthesis is given below.

Table 4. 6. Protocol for the synthesis of 20 residue peptide

mino acid in NMP

Cleavage and purification of the pe~tide







A small aliquot of the peptide was dissolved in acetonitrile-water

mixture and injected to a Shimadzu C-R6A liquid chromatograph with C 18

rpc and eluted using 0.1% TFA in 100% water (A) and 0.1% TFA in 80%

acetonitrile : 20% water (B) to check the purity of'the peptide. The peptide

was then purified to homogeneity by C18 reversed phase preparative HPLC.

The purified peptide was analyzed by MALDI mass spectroscopy [Section

3. 2 (ii) (b)] .

(iii) Synthesis of Glu-Thr-Thr-Ala-Leu- Val-Ala-Asp-Asn-Gly

Attachment of Boc-Gly to the chlorometh~l resin

Boc-Gly (78.75 mg; 0.45 rnmol) was dissolvcd in ethanol and

converted to the cesium salt by adding a saturated solution of cesium




mg; 0.18 rnmol) was added and kept at 50°C for 48 h. The resin was



Synthesis of Glu-Thr-Thr-Ala-Leu-Val-Ala-Asv-Asn-Gly



with 5% DIEA in DCM. The resin was washed with DCM (20 ml x 4) and

NMP (20 ml x 2). Then, Boc-Asn (104.49 mg; 0.45 mmol), Boc-Asp.OBzl

(145.48 mg; 0.45 mmol), Boc-Ala (85.05 mg; 0.45 mmol), Boc-Val (97.78

mg; 0.45 mmol), Boc-Leu (93.6 mg; 0.45 mmol), Boc-Ala (85.05 mg; 0.45

mmol), Boc-Thr.OBz1 (139.23 mg; 0.45 mmol), Boc-Thr.OBzl(139.23 mg;

0.45 mmol) and Boc-Glu.OBz1 (151.83 mg; 0.45 mmol) were successively

coupled to the resin as their HOBt active esters. Active ester was prepared

by shaking the respective Boc-amino acid with HOBt (60.75 mg; 0.45

mmol) and DCC (92.70 mg; 0.45 mmol) in NMP. DCU formed was filtered

off and the active ester was added to the resin. After the synthesis, the

peptide resin was washed with NMP (30 ml x 3), MeOH-DCM mixture (30

ml x 3), DCM (30 ml x 3) and dried in vacuum. The steps involved in the

synthesis are shown below.

Table 4. 7 . Protocol for the synthesis of 10 residue peptide

Cleavage and ~urification






TFA and scavengers, dried and yield noted (130 mg). A small aliquot of the

peptide was dissolved in acetonitrile-water mixture and injected tn a

Shimadzu C-R6A liquid chromatograph with C18 rpc and eluted using 0.1%

TFA in 100% water (A) and 0.1% TFA in 80% acetonitrile: 20% water (B)

to check the purity of the peptide. The peptide was then purified to

homogeneity by C18 reversed phase preparative WLC. The purified

peptide was analyzed by MALDI mass spectra [Section 3. 2 (ii) (c)].

(iv) Synthesis of Tyr-Ala-Gly-AIa-Val-Val-Asn-Asp-Leu-Tyr-Gly-Ala- Val- Val-Asn-Asp-Leu

Attachment of Boc-Leu to the chloromethvl resin

Boc-Leu (219.45 mg; 0.95 rnmol) was dissolved in ethanol and

converted to the cesium salt by adding a saturated solution of cesium




mg; 0.38 rnmol) was added and kept at 50°C for 48 h. The resin was



Synthesis of Tyr-Ala-Glv-Ala-Val-Val-Asn-~~-Leu-Tvr-Gly-Ala-Val-Val- Asn-Asv-Leu

The Boc-protection was removed by 33% TFA in DCM.

The resin was washed with DCM (30 ml x 6) and then neutralized with 5%

DIEA in DCM. The resin was washed with DCM (30 ml x 4) and NMP (30

ml x 2). Then, Boc-Asp.OBzl (310 mg; 0.95 mmol), Boc-Asn (220 mg;

0.95 mmol), Boc-Val (206 mg; 0.95 mmol), Boc-Val (206 mg; 0.95 mmol),

Boc-Ala (180 mg; 0.95 mmol), Boc-Gly (170 mg; 0.95 mmol), Boc-

Tyr.OBzl (355 mg; 0.95 mmol) Boc-Leu (219.45 mg; 0.95 mmol), Boc-

Asp.OBz1 (310 mg; 0.95 mmol), Boc-Asn (220 mg; 0.95 mmol), Boc-Val

(206 mg; 0.95 mmol), Boc-Val (206 mg; 0.95 mmol), Boc-Ala (180 mg;

0.95 rnmol), Boc-Gly (170 mg; 0.95 mmol), Boc-Ala (180 mg; 0.95 mmol)

and Boc-Tyr.OBz1 (355 mg; 0.95 mmol) were successively coupled to the

resin as their HOBt active esters. Active ester was prepared by shaking the

respective Boc-amino acid with HOBt (130 mg; 0.95mmol) and DCC (195

mg; 0.95 mmol) in NMP. DCU formed was filtered off and the active ester

was added to UIG resin. Aner the synthesis, the peplide resin was washed

with NMP (30 ml x 3), MeOH-DCM mixture (30 ml x 3), DCM (30 ml x

3) and dried in vacuum. The protocol for the synthesis is given below.

1. DCM wash (1 x 5 min)

2. Deprotection of Boc groups using 33 % TFA in DCM (1 x 30)



5. Neutralization with 5% DIEA in DCM ( I x 5 min)


7. NMP wash (2 x 1.5 min)

8. Coupling of Boc-amino acid with DCC and HOBt in 1 : 1 : 1 ratio (60 min)


1 0. Wash with DCM (2 x 1.5 min)

I I. Repeating steps 7-1 0 to ensure maximum coupling.

Cleavage of the vevtide from the resin

250 mg of the peptidyl resin was treated with TFA (25 mi),






Transfer hydrogenation with formic acid

The protected peptide was dissolved in 98% formic acid in a 50 ml

round bottomed flask containing 10% palladium charcoal. The mixture was

continuously stirred while hydrogen gas was bubbled slowly through the

solution for 6 h. The reaction can be followed by TLC. After completion,

the catalyst is filtered off and washed with formic acid. The washings were

collected and evaporated in vacuo at room temperature. The residue

obtained was purified by IIPLC. 'l'he purified peptide was analyzed by

mass spectroscopy [Section 3. 2 (ii) (d)].

4.2.r. Synthesis of cystine peptides

(i) Synthesis of Cys-Pro-Leu-Cys-Gly-Ala

Attachment of Boc-Ala to the chloromethvlated resin

Boc-Ala (340 mg; 1.8 mmol) was attached to the chloromethylated

resin (400 mg; 0.72 mmol) by cesium salt method. The resin was washed

with NMP (40 rnl x 2); 1 : 1 NMP-water (40 rnl x 3); water, methanol (40 ml

x 3), DCM (40 rnl x 3) and dried under vacuum.

Svnthesis of Cys-Pro-Leu-Cvs-Glv-Ala



with 5% DIEA in DCM. The resin was washed with DCM (40 ml x 4) and

NMP (40 ml x 2). Then, Boc-Gly (315 mg; 1.8 mmol), Boc-Cys.Acm (526

mg; 1.8 mmol), Boc-Leu (416 mg; 1.8 mmol), Boc-Pro (387 mg; 1.8 mmol),

Boc-Cys.Acm (526 mg; 1.8 mmol) were successively coupled to the resin as

their HOBt active esters. Active ester was prepared by shaking the

respective Boc-amino acid with HOBt (243 mg; 1.8 mmol) and DCC (370

mg; 1.8 rnrnol) in NMP. DCU formed was filtered off and the active ester

was added to the resin. After the synthesis, the peptide resin was washed

with NMP (40 ml x 3), MeOH-DCM mixture (40 ml x 3); DCM (40 ml x

3) and dried in vacuum. The protocol used for the synthesis is given below

in Table 4. 8.

Table 4. 8. Protocol for the synthesis of Cys-Pro-Leu-Cys-Gly-Ala

Cleavage and ~urification



22 h. TFA was removed by rotary evaporation. The peptide was

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

washed several times with ether to get rid of TFA and scavengers, dried and

yield noted (90 mg). The crude peptide was purified by HPLC. A

preparative C18 rpc column was used. The solvent system used was

acctonitrilc-water containing 0.1% TFA and water containing 0.1% TFA.

The fraction corresponding to the major peak was collected and solvent

evaporated and lyophilized to get the pure peptide. Purity was further

confirmed by TLC. The purified peptide was subjected to NMR, CD and

mass spectral analysis [Section 3. 2 (iii) (a)].

Oxidation and disulfide bond formation

A mixturc of acctic acid and water in a 4: 1 ratio was prcparcd. 10

pmol (7.04 mg) of the purified bis (Acm) protected peptide was dissolved in

15 ml of the aforementioned mixture. Solid iodine (mol. wt. 253.8; 5 mg)

corresponding to 2 equiv./S-Acm function was dissolved in minimum

amount of 80% acetic acid and added to the peptide solution with vigorous

mixing at 25'C. M e r 2 h, the reaction was quenched by dilution with

water. The iodine was then removed with a 10% solution of sodium

thiosulphate and the mixture was concentrated by evaporation. It was then

purified by HPLC. The fraction corresponding to the oxidized peptide was

collected and the purity of the peptide was checked in three different

solvent systems.

(ii) Synthesis of Cys-Tyr-Zle-Gln-Asn-Cys-Pro-Leu-Gly

Attachment of Boc-Glv to the chloromethvlated resin

Boc-Gly (207 mg; 1.18 mmol) was converted to cesium salt. The

cesium salt was dissolved in minimum amount of NMP and chloromethyl

resin (250 mg; 0.475 mmol) was added and kept at 50°C for 48 h. The resin

was washed with NMP (30 ml x 3), 1:l NMP-water (30 ml x 3), water,


Synthesis of Cvs-Tyr-Ile-Gin-Asn-Cys-Pro-Leu-Gly

Thc Boc-group on thc rcsin was rcmovcd with 33% 'I'l:A in DCM.

The resin was washed with DCM (30 ml x 6) and then neutralized with 5%

DIEA in DCM. The resin was washed with DCM (30 ml x 4) and NMP (30

ml x 2). Then, Boc-Leu (220 mg; 0.95 rnmol), Boc-Pro (220 mg; 0.95

mmol), Boc-Cys.Acm (280 mg; 0.95 mmol), Boc-Asn (220 mg; 0.95

mmol), Boc-Gln (235 mg; 0.95 mmol), Boc-Ile (220 mg; 0.95 mmol), Boc-

'l'yr.0-Hz1 (307 mg; 0.95 mmol), Boc-Cys.Acm (280 mg; 0.95 mmol) were

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

ester was prepared by shaking the respective Boc-amino acid with HOBt

(128 mg; 0.95 rnmol) and DCC (196 mg; 0.95 mmol) in NMP. DCU

formed was filtered off and the active ester was added to the resin. After

the synthesis, the peptide-resin was washed with NMP (30 ml x 2), MeOM

(30 ml x 6), DCM (30 ml x 3) and dried under vacuum. The steps involved

in the synthesis are given below.

1. Wash with DCM (I x 5 min)






7. Wash with NMP (2 x 1.5 min)

8. Coupling of Boc-amino acid in presence of DCC and HOBt (60 min)



11. Repeat steps 7-10 to for maximum coupling, tested by Kaiser reagent.

Cleavage ofthe ue~tide from the resin

300 mg of the peptidyl-resin was treated with 1'FA (30 ml),

thioanisole (0.3 ml) and l,2-ethanedithiol (0.3 ml) at room temperature for

20 h. The resin was removed by filtration and TFA by rotary evaporation.

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

washed several times with ether to remove TFA and scavengers, dried, yield

noted (145 mg).

Transfer hydrogenation with formic acid

The cleaved peptide was subjected to hydrogenation to remove the

benzyl group. A solution of the peptide in 98% formic acid was treated

with 10% palladium charcoal (5 times). The mixture was stirred

continuously while hydrogen gas was slowly bubbled through the solution

for 6 h. The mixture was filtered and the filtrate evaporated in vacuo. The

residue obtained was purified by HPLC. The purified peptide was analysed

by NMR, CD and mass spectroscopy [Section 3. 2 (iii) (b)].

Oxidation and simultaneous devrotection of Cvs-Tvr-Ile-Gln-Asn-Cvs-Pro- Leu-Gly

A mixture of acetic acid and water in a 4: 1 ratio was prepared. 10

~pmol (5.5 mg) of the purified S-Acm protected peptide was dissolved in 15

ml of the aforementioned mixture. Solid iodine (mol.wt.253.8; 2.5 mg)

corresponding to 2 equiv./S-Acm function was dissolved in minimum

volume of 80% acetic acid and added to the peptide solution with vigorous

mixing at 25OC. After 2 h, the reaction was quenched by dilution with

water. The iodine was removed by treatment with 10% solution of sodium

thiosulphate. The mixture was concentrated by evaporation. It was then

purified by HPLC and the fraction corresponding to oxidised pept~de was

collected, solvent evaporated and lyophilized. The purity was checked by

'I'LC in three different solvent systems.

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Chapter 4 Experimental - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/446/10/10_Chapter4.pdfsilver nitrate solution. (iii) Estimation of chlorine capacity by pyridine fusion