Experimental

PART A

Preparation of Polymers, F~mdionalisation and Reactivity Studies

5.1 Materials and methods

T e polymers used in these studies were synthesised based on procedures

developed earlier in this laboratory. IR spectra were recorded on a

Shimadzu IR-470 spectrophotometer using KBr pellets. UV/Vi5 spectral

measurements were made on 21 Shimadzu UV-160A spectrometer. Optical

density measurements were made on a Hitachi model 200-20 spectrophotometer

with a digital recorder-Hitachi-611 at the Centre for Cellular and Molecular

Biology, Hydembad.

5.1.1 Sources of chemicals

The monomers required for the polymer synthesis and reagents for

various physicochemical studies were obtained from the following sources.

Chemicals Sources

1. Styrene, DVB, TTEGDA, HDO'DA and Aldrich Chemical Company, USA TEGDMA

2. NNMBA BDH, England

3. Acykmide SRL, Bombay, India

4. Benzoyl peroxide S i o , Bombay, India

5. Polyvinyl alcohol (Mol. wt. 75,CQO) Fluka, AG, Switzrknd

6. Ethylenediamine and potassium BDH, India phthalirnide

5.2 Polymer synthesis ..

5.2.1 Preparation of crosslinked poly(acry1amide) resins (la-ld. 2a- 2d, SaSd and U)

(a) General procedure: DiB-mxsI:nked ply(ac~&mide)s

The crosslinking agent was washed with sodium hydroxide solution (1%)

and with diilled water (4 times) to remove the inhibitor. Aclylamide and DVB

was d i iobed in ethanoudioxane mixture (4:l; 100 ml) at 70°C. Benzoyl

peroxide (200 mg) was added to this solution as initiator and stirred

mechanicaUy for 5 h. The pre:cipitated polymer was filtered, washed with

ethanol (20 ml x 2). water (20 ml x 3) and finally with methanol (20 ml x 51 and

dried in an air oven at 50°C. Polymers containing 5, 10, 15 and 20 mole

percentage of the crosslinking agent were prepared by varying the monomer-

crosslink ratios. The amount of monomers used and the obtained yields are

given in Table 5.1.

(b) TEGDM-crmlinked poly(acrylamide)s

The crosslinking agent was destabilised by washing with 1% sodium

hydroxide solution followed by distilled water. The monomers were dissolved in

ethanol (60 ml) and potassium peroxodisulphate (100 rng) (initiator) was added

with stirring. The mixture was kept in a water bath a t 700C until the polymer got

precipitated. Water (50 ml) was added and heated for 20 min at 70°C. The

polymer was filtered and washed with water (20 ml x 3), methanol (20 ml x 5)

and dried in an air oven to concjtant weight. 5, 10, 15 and 20% TEGDMA-

crosslinked poly(acry1amide) were prepared by varying the amount of the

monomers in the polymerization mixture. The amounts of the monomers used

and yields are given in Table 5.1.

Table 5.1. Preparation of 13VB-, TEGDMA-, TIZGDA- and N'NMBA- crosslinked poly(ac ylamide)~.

Crosslinking

L I DVB I l b / 10 4.3 74.20 - 12.9

*CaJculated based on the weight of mc,nomers.

Yield* (96)

85.90

(mole%)

TTEGDA was washed with 1% sodium hydroxide solution (10 ml x 3)

and then with water to get rid of the inhibitor. The monomers were dissolved in

ethanol (65 ml) with stirring. Potassium peroxodiiulphate (100 mg) was added

Amount of monomers (9)

AA

13.49

Crosslinking agent

2.16

as initiator and the mixture heated up to 7O9C. Stirring was continued till the

polymer got precipitated. Water (50 ml) were added and heated a t 80°C for

20 rnin. The lumps of the polymer was powdered and filtered, washed with

water (20 ml x 3). methanol (20 ml x 3 x 5 min) and dried in an air oven. By

varying the amount of monomers the crosslink densities were varied to 5, 10, 15

and 20 mole percentages. The amount of monomers and yields are given in

Table 5.1.

Potassium peroxodisulphate (100 mg) was dissolved in distilled water

(100 ml) at 70°C. The monomer mixture of acylamide and N,N'-methylene-bis-

acrylamide was added to it with stirring until the whole mixture got dissolved.

Heating and stirring were continued until the polymer got precipitated. Water

(50 ml) was added and heated at 8C)"C for 20 min. The lumps of the polymers

were powdered and the polymer was filtered, washed with water (20 rnl x 3) and

methanol and dried to constant weight. Crosslink densities were varied to 5, 10,

15 and 20 mole per cent by changing the relative amounts of the monomers.

The details are given in Table 5.1.

5.2.2 Preparation of crosslinked polystyrene resins (5a-5e. 6a-6e. 7 and 8) by suspension polymerisation

(a) Generalprocedure

The monomers were washed with sodium hydroxide solution (I%, x 2)

and with distilled water (3 times) to remove the inhibitor. A 1% solution of

poly(viny1 alcohol) (WA) was prepared by d i ih r ing the required amount of

W A (molecular weight 72,000) in water (8 times the total volume of the

monomer mixture and diluent). The clear solution of W A was then transferred

to the polymerisation reactor fitted with a mechanical stirrer and the temperature

was slowly raised to 700C by iheating in a water bath. Calculated amount of

styrene, crosslinking agent, and the diluent were mixed together and the initiator,

benzoyl peroxide (200 mg) was dissolved in it. The mixture was slowly added to

the W A solution with continuous stirring at a speed of 1200 rpm. The

temperature was raised to 80°C and the stirring was continued for 8 h. The

polymer beads were couected by filtration through a sintered (G3) funnel and

washed with hot water to remove PC'A. Then washed with toluene and acetone

to remove the unreaded monomers and the soluble linear polymers. Polymer

beads were soxhleted overnight: using acetone. Dried at 50°C and sieved into

different fractions.

5.3 Functionalisation of polymers

5.3.1 Fundionalisation of poly(acrylamide)s' with ethylenediamine

Anhydrous ethylenediamine (40 ml) was taken in a round bottomed flask

and the crosslinked polymers (4 g) was added to it in small fraction with stirring.

The mixture was refluxed in an oil bath at 100-1lO"C for 9 h with stirring. The

hot mixture was poured carefuiiy into 50 ml of water containing crushed ice with

vigorous stirring. The resin was filtered, washed with 0.1 M sodium chloride

solution till free of amine (test with ninhydrin). The gel was washed with water

several times to free it from chloride ions and dried to constant weight in an air

oven at 50°C.

5.3.2 Preparation of chlor~meth~lmeth~lethe~~

A stream of dry HCI gar; was passed through a mixture of methanol

(60 ml) and formaldehyde (126 ml) which was kept cooled in an ice bath. After

about 4 h chlorornethyl methyl ether began to appear as a layer. The stream of

HCI was continued for 2 to 3 h Lonyer until the solution was saturated. The

remaining aqueous layer was separated using a separating funnel and saturated

with calcium chloride and more ether was separated. The f i h t e was d i e d at

5560°C. Yield is 75 ml.

5.3.3 Preparation of 1M ZnC12 solution in THF

Anhydrous zinc chloride (1.5 g) was heated with 3 drops of con. HCI and

5 drops of water until all solids dissolved. Temperature was gradually raised to

evaporate the water and to leave a crust of solid which was then melted by

strong heating. When zinc chloride became a clear mobile liquid with no further

evolution of bubbles, the flask was placed in a desiccator and allowed to cool.

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

concentration of the resulting solution was determined by pipetting a sample into

water, diluting with several drops of HN03 and titrating against 0.1M AgN03

solution.

5.3.4 Functionalisation of crosslinked polystyrenes by chloromethylation3

The d y resin (5 g) was allowed to swell in dichloromethane for 30 min.

chloromethylmethylether (30 ml) and 1M ZnC12 in THF (1.5 ml) were added to

the resin and it was heated at 50°C for 20-24 h. The mixture was filtered

through a sintered funnel and washed with THF (30 ml x 3), THF/4N HCI (3:l;

30 ml x 3), THF/H20 (3:l; 30 rnl x .3), THF (30 ml x 2) and methanol (30 ml x

3). The resin was soxhleted overnight with THF and dried. Yield: 5.4 g.

5.3.5 Preparation of amino methyl resins4

Chloromethylated resin (1 g. 2 4 mmol) was allowed to swell in DMF

(20 ml) for 30 min. Potassium phthalimide (8.9 g, 4.8 mmol) was added and the

reaction mixture was stirred mi~gnetically at 110-120°C for 12-14 h. The resin

was filtered and washed with DIW (20 ml x 3). DMF/H20 (1:1,20 ml x 3). water-

dioxane (1:1, 20 ml x 3). ethanol (20 ml x 3) and methanol (20 ml x 3) and

dried. It was suspended in ethanol (10 mi) and hydmzine hydrate (0.6 ml,

18 mmol) was added to the solution and refluxed for 8 h. The resin was filtered

through a sintered funnel, washed with hot ethanol (20 ml x 3) and DMF

(20 ml x 3), DMF-H20 (1:1, 20 rnl x 3), water (20 ml x 3) and ethanol

(20 ml x 3). The product resin w a s dried under vacuum. IR (KBr); 3400 crn-'

(N-H).

5.4 Estimation of functional groups

5.4.1 Estimation of chlorine: capacity by pyridine fusion5 (Volhard's method)

A known amount of chlonxnet'lyl resin (200 mg) was fused with pyridine

(3 ml) in a boiling tube fitted with a lid at 100-1lO"C for 5 h. The mixture was

quantitatively transferred with acetic acid (1:1, 30 ml). Con. HN03 (5 ml) was

added to this followed by the addition of standard solution of AgN03 (0.1 M,

10 ml), water (50 ml) was added. The excess AgN03 was back titrated with

standard ammonium thiocyanate solution using ferric alum as indicator. A blank

titration was also conducted.

5.4.2 Estimation of amino capacity

The amino capacities of the resins were determined by titration method.

The aminated resin (200 mg) wa:j stirred with hydrochloric acid (0.2 M, 10 ml)

for 12 h. The resin was removed by filtration, and the excess acid was estimated

by titration against sodium hydroxide solution (0.2 M) using phenolphthalein as

indicator. A blank experiment was also performed. From the titre values, the

amount of amino groups in the polymer was calculated.

5.5 Swelling studies

The dry resin (0.5 g) was placed in a sintered glass crucible (G3) and after

placing in a beaker, was equilibrated with the solvent for 48 h. The solvent was

drained by applying mild suction and the weight of the solvent swollen resin

together with the crucible was determined. The dry weight of the crucible with

the resin was also determined. The difference between the two gave the weight

of the solvent swollen resin. Frorn these weights, swelling capacity was

calculated as the ratio of the mass of the solvent swollen resin to mass of the d y

resin. The swelling capacities of the different resins were determined

gravimetrically.

5.6 Reactivity studies

5.6.1 Preparation of pnitrophenyl ester of benzoyl glycine

(a) heparation of benzoylg&cine

Glycine (10 g, 130 mmol) was dissolved in sodium hydroxide solution

( lo%, 100 ml) in a stoppered fkisk and benzoyl chloride (22.75 ml, i90 mmolj

was added to it gradually with shaking. It was continued till all the benzoyl

chloride reacted. The precipitated benzoyl derivative was filtered, washed with

water, drained and dried. The precipitate was boiled with CCh (40 ml) carefully

to remove benzoic acid. It was cooled, filtered and washed with CCb, dried and

recrystalliied from boiling water. Yield: 14 g, m.p. 187°C.

(b) To b e m y l glycine (1.79 g. 10 mmols) in anhydrous THF (20 ml),

dicyclohexyl carbodiimide (2,OC g, 10 mmol) and p-nitrophenol (1.39 g, 10

mmol) were added. The mixture was stirred at 0°C for 60 min. The precipitated

dicyclohexyl urea (DCU) was filtczred and the solvent removed by filtration. The

precipitate was washed with ethanol to remove pnitrophenol. The solid was

d i i b e d in dichloromethane and petroleum ether was added to precipitate the

product. The clystals of p-nitrophenyl ester of benzoyl glycine was separated by

fihtion, washed with more petroleum ether and dried. Yield: 5.2 g, rn.p. 154-

155°C (Figure 5.1); IR (KBr): 1780 an-' (C=O str, ester); 1200 (C-0 str). 1650

(CO-NH); C-0 str; 3280 (N-H str); 1:350, 1520 (N=O); 760 cnil (psubstituted

aromatic ring).

Figure 5.1. IR spectrunl of pnitrophenyl ester of benzoyl glycine

5.6.2 Kinetics of aminolysis of p-nitrophenyl ester of benzoyl glycine

The active ester of benzoyl glycine (53 rng, 0.165 rnrnol) was dissolved in

1:l dioxanelwater mixture (50 rnl'~. The amino resin (0.3085 rnrnols) was added

to thii solution and the contents were stirred. 1 rnl of the reaction rnixhrre was

withdrawn at intenmk of 10 rnin !up to 80 rnin. It was diluted to 10 rnl with 1:l

dioxanelwater mixture. The concentration of the liberated p-nitrophenol in this

solution at 317.8 nrn was measured using UV spectrophotorneter. A working

curve was constructed by plotting concentration wrsus absorbance a t various

concentrations of p-nitrophenol in 1:l d i o x a n h t e r mixture. The a h r b a n c e

of the solutions was observed by UV-Vis spectxoscopy. From thii the

concentration of liberated p-nitophenol a t different intervals were obtained.

5.6.3 Estimation of functional group efficiency towards w i d e bond formation

A known amount of the amino resin (100 mg) was allowed to swell in

toluene (2 ml) for 15 min. Acetylating mixture was prepared by mixing one

volume of acetic anhydride with 8 volume of pyridine. To the amino resin five

equivalents of acetylating mixture was added and was stirred for 4 h. D i l l ed

water (10 ml) was added to the reaction mixture and refluxed for 1 h. It was

then cooled and titrated against standard sodium hydroxide solution (0.1N). A

blank was also conducted under idenhcal conditions.

Part B

Peptide Synthesis-Experimental

5.7 Source of chemicals

All L-amino acids, tertiary butyl carbazate, 2 (t-butyloxy carbonyloximino

2-phenyl acetonitrile) (Boc-ON) and dicyclohexyl carbodiimide (DCC) were

purchased from Sigma Chemical Company, USA. Boc-Gly, Boc-Leu, Boc-Phe,

Boc-lle. Boc-Met, Boc-Gln, Bcbc-Ala and Boc-Pro were prepared in the

laboratory following both Schnabel's method or Boc-ON method as detailed

below. Boc-Thr 0-benzyl, 2 chlc~robenzyloxycarbonyl Boc-Lys, forrnyl Boc-Trp.

0-benzyl BocSer and y-benzyl Em-Glu were supplied by Peninsula Inc., USA.

Mesitylene 2-sulphonyl Bm-Arg was obtained from Applied Biosystems, CA,

USA. Trifluoroacetic acid, diisopropyl ethylamine and 1,2-ethanedithioi were

supplied by Aldrich Chemical Company, USA. Cesium carbonate and thioanisol

were purchased from Fluka, AG, Swikeriand. AII solvents were of reagent grade

and were obtained from E. Merck, India or BDH, India. They were pcrified

following literature procedure.

5.8 Purification of reagents and solvents

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

Dichloromethane was dried by adding anhydrous sodium sulphate to the solvent

and keeping over night. It was filtered through glass wool and kept in stoppered

bottles.

Ethanol was distilled ancl dried in air tight bottles. Diiipropyl ethyl

amine was diil led over ninhydrin (5-109) and stored in amber coloured bottles.

Triethyl amine was refluxed with ninhydrin for 1 h and then dii l led and stored

in amber coloured bottles.

5.9 Physical meaeurements

WWi spectral measurements were made on a Shimadzu W-160A

spectrometer and a Hitachi double beam spectrophotometer.

Crude peptides were purified by reverse phase FPLC. A Pharmacia fast

protein liquid chromatograph with FIR 35 C-18 reverse phase column (Semi

Prep) was used HPLC analysis were done using Shimadzu LC- 6A liquid

chromatograph provided with a SPD 6A UV detector and on line electronic

plotter. Biorodd C4 column wa:; used. Amino acid analysis was performed on

an LKB 4151 Alpha plus amino acid analyser.

5.10 Detection

5.10.1 Thin layer chromatography

TIC was used to monitor rhe progress of reaction and to check the purity

of the final product. TIC was perfomfed on glass plates precoated with silica gel

containing &.SO4 binder and activated by heating for 4h at 1 0 0 ' ~ and cooled

just before use.

5.10.2 Identification of the peptide on tlc

Aqueous or methanolic solution of the peptide is spotted on the plate and

developed in suitable solvent mixtures of appropriate composition. The solvent

system used include

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

2. Butanol-1 : acetic acid : water 1(4:1:1)

3. Butanol-1 : acetic acid : waterethyl acetate (1: 1: 1: 1)

4. Ethyl acetate : pyridine : acetic acid : water (30:15:3:2)

5. Methanol : chloroform (1:9)

5.11 Visualisation

The developed chromatcgram was visualid by one of the following

methods.

1. Ninhydrin spray detects th~e presence of free amino groups. A 0.2% pure

ninhydrin in acetone was sprayed on the developed chromatogmm and

heated in an air oven at 83-100°C for 15 min. Pink colour was developed

by free amino groups. (N-Tem~inal proline gives a yellow colour).

2. Iodine vapour. The tlc plates were exposed to iodine vapours in a closed

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

3. Sakaguchi reagent detects free guanidine group of arginine. The tlc

plates were thoroughly cooled and sprayed with Sakaguchi reagent A.

Sakaguchi reagent A - 0.2% solution of a-naphthol in 20% ethanol. It

was sprayed with 2.5 N NaOH solution, dried well and sprayed with

Sakaguchi B. Sakaguchi reagent B - NaOBr solution prepared by

dissolving 0.67 ml liquid bromine in ,100 ml of I N NaOH. Completely

deprotected arginine in the peptide develops a bright orange colour which

fades rapidly.

4. Rydon's reagent spray for detection of NH groups. Protected peptides

which are not visible with ninhydrin test can be well detected by thii test.

Compounds with N-H groups appear as blue-black spots. The dried

chromatogmm was exposed to chlorine gas in a tank for 3 min. Excess of

chlorine was driven from the plate by blowing cold air or nitrogen over it

and heated at 100°C for 5 min. The plate was then developed using

starch - potassium iodide spray (1:l). Descrete spots appeared on the

plate.

5.12 Preparation of Boc amino acid derivatives

5b12.1 Preparation of Bocazide from t-butyl carbazate6

t-Butyl carbazate (30 g) !was dissolved in glacial acetic acid (27 ml) and

water (37.5 ml). It was then cooled in an ice bath with constant stirring and

sodium nitrite (17.4 g) was added slowly over a period of 15 min. After 90 min,

the oily upper layer was separatszd using a separating funnel. The aqueous layer

was extracted with ether (3 x 20 ml). The ether extrads were mixed with oily

layer washed with water. sodium bicarbonate (saturated solution) and dried over

anhydrous sodium sulphate. (3n evaporating ether under reduced pressure,

Boc-azide was obtained as a golden yellow liquid and stored at 4OC. Yield -

30 ml.

5.12.2 Preparation of Boc-amino acids by Schnabel's method7

(a) Genemlpmedure

The amino acid (10 mmol) was suspended in 1:l dioxane water (10 ml)

and Boc-azide (1.6 ml, 10 mmol) was added to it. The solution was stirred at

room temperature and the pH was maintained in the alkaline range (9.0) by

adding 4N NaOH. After 24 h water (25 ml) was added to the mixture and

washed with ether. The aqueous layer was acidified to pH 2 with 2N HCI and

then extracted with ethyl acetate (3 x 20 ml). In the case of Boc-Leu and Boc-

Lys (Z) ether was used for e h c t i o n . The organic layer was dried over

anhydrous sodium sulphate and then evaporated under vacuum. The Boc-

amino acid obtained as oil, wj triturated with petroleum ether in the case of

Gly, Leu, Ala and Pro. In certain cases, seeding by a trace of pure Boc-amino

acid followed by trituration precipitated the Boc-amino acid as a white powder.

Boc-lle, Boc-Gln, Boc-Met and Hac-Phe derivatives are stored as DMF solution.

5.12.3 Preparation of Boc amino acids by Boc-ON method8

(a) General procedure

Amino acid (10 mmol) was added to the mixture of Boc-ON (11 mmol),

triethylamine (11 mmol) and 1:l dioxane-water (12 ml). The mixture was stirred

at room temperature for 12 h. The reaction mixture was diluted with water

(20 ml) and washed with ethyl acetate (2 x 25 ml). The aqueous layer was

acidified to pH 2 with 2N HCI and extracted with ethyl acetate (3 x 20 ml). The

organic layer was dried over anhydrous sodium sulphate and evaporated to

obtain the Boc-amino acid. All l k - a m i n o acid preparations were obtained with

80-90% yield.

(b) heparation of Boc-Phe by Boc-ONmethod

Phenyl alanine (0.84 g, 5 mrnol), Boc-ON (1.35 g, 5.5 mmol) and TEA

(1.39 ml, 5.5 mmol) were dissohred in I:? dioxane-water (6 ml) and the mixture

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

water (10 ml) and washed with ethyl acetate (2 x 12.5 ml). The aqueous layer

was acidified with 2N HCI to pH 2 and it was extracted with ethyl acetate (3 x

10 ml). The organic layer was dried and rotay evaporated to remove ethyl

acetate. The resulting syrupy liquid of Boc-amino acid was d i i l v e d in known

amount of DMF and its weight was found out. 85% yield was obtained.

Boc-Met, Boc-Ile, Boc-Ala and Boc-Pro were prepared by &-ON

method. List of Boc-amino acids prepared and their yields are given in

Table 5.2.

Table 5.2. Preparation of Boc-amino acids and their yields.

1 Boc-Phe 1 . . ! 85 I 0 i b 1 79-80 1 I ~oc- l le ( . , 55 ( Oily 1 66-69 1

M.P. literature ("c)

86-89 - 70-73

79-81

mr%) Amino acid derivative

Schnabel's

Boc-Leu Schnabel's 95

Boc-Ala Boc-ON 89

I Boc-Met I ,. 8 0 I oily ( 49 1

M.P. observed ("c)

85-87

70

80

5.13 Purity of Boc-amino acids4

Purity of all the Bocamino acids was tested by tlc on precoated silica gel

plates using CHC13 : CH30H : C:H3COOH (85:10:5) solvent system. Amino acids

were visualised by ninhydrin sprays after exposure to HCI vapours for 10 min.

5.14 General procedure for solid phase peptide synthesis

Manual solid phase peptide synthesis was done in a silanised glass

reaction vessel clamped horizo~~tally on a mechanical shaker. The first amino

acid at the C-terminal was esterified to the resin via a benzyl ester linkage

through cesium salt of the Boc-amino acid. The Boc-group was removed and

the second amino acid was coupled to the amino acyl resin by DCC method.

Progress of coupling was monitored at e v e y stage by semiquantitative ninhydrin

test. In all couplings a 3-fold rnolar excess of the Boc-amino acid was used to

ensure completion of reaction. DCM, DMF and N-methyl pyrrolidone (NMP)

were the solvents employed for the synthesis and the coupling times usually was

45 min. The precipitated DCU was removed by washing with CH30H followed

by DCM. Boc-group was deprotected by 30% TFA in DCM. Neuiralisation was

effected by 5% diiipropyl ethylamine in DMF or 5% triethylamine in DCM.

Final cleavage of the peptide from the support was effected by trifluoroacetic

acid.

5.14.1 First amino acid attachment by Gisin's cesium salt method9

Boc-amino acid was dii;obed in minimum quantity of ethanol. It was

diluted with 1 ml of water. An aqueous solution of cesium carbonate was added

to it dropwise with shaking till solution attained the pH of 8. It was kept for 2 h

and evaporated from dry, freshly distilled benzene in a rotary evaporator. The

process was continued till a dry white powder of cesium salt of Boc-amino acid

was obtained.

This salt was dissolved in minimum quantity of DMF or NMP. The resin

was added to it and gently warmed in an oil bath at 50°C for 24-48 h with

occasional shaking of the flask. The resin was filtered through a sintered funnel

(G3). Washed with DMF (10 ,ml x 3); DMF/H20 (1:1, 10 ml x 3), rnethanol

(10 ml x 3) and dichloromethane (10 ml x 3). The resin after dying under mild

vacuum was weighed and the weight increment noted.

5.14.2 Attachment of first amino acid by triethyl arnine method

200 mg of resin with fhe chlorine capacity z1 mmol was taken and

allowed to swell in ethyl acetate. Boc-amino acid and TEA was added to it and

refluxed in a water bath for 4 3 h. It was then filtered using sintered funnel,

washed with ethanol and water ti11 free from chlorine, rnethanol and finally with

DCM. It was dried under vacuum and the weight increment was noted.

5.14.3 Estimation of amino acid substitution level by picric acid method1'

5 mg of the Boc-aminoacyl resin was weighed out accurately into a glass

funnel. 30% TFA in DCM was added to the resin and kept for 30 min to remove

the Boc-group. It was washed with DCM (1 ml x 6) and neutralised with hiethyl

amine in DCM (5%) for 10 min and again washed thoroughly with DCM

(5 ml x 1'). In another funnel ]!he blank resin 5 mg was taken and washed with

DCM. Both the resins were treated with 0.1 M picric acid in DCM for 5 min. All

the unbound picric acid was washed off with DCM. The resin bound picrate was

eluted with 5% TEA in DCM till the eluate was clear. It was made up to a

definite volume using 95% ethanol. 0.5 ml of the solution was diluted to 5 rnl

with 95% ethanol. The optical density of the solution was measured at 358 nm.

Molar absorption coefficient (E) for picrate at 358 nm is 14,500. From the OD

values and the weight of the resin initially taken, the extent of amino acid

substitution level was estimated.

5.14.4 Deprotedion

(a) Removal of t-butyloxy ca .rb~nyl~rou~~~

Trifluoroacetic acid (30%) in dichloromethane was used for deprotection

of Bocgroup in protected amino acids or peptides. The peptide was treated

with 30% TFA in DCM (10 mi) for 15-30 min at room temperature. Excess TFA

was removed by filtration followed by washing with DCM.

5.14.5 Methods of coupling

Mainly three methods were employed for the activation of carboxyl group

by dicyclohexyl carbodiimide (DCC).

In this method the amino acid and DCC were used in 1:l ratio and the

amino acid is converted to active interemediate 0-acyl isourea which readily

reacts with 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

semiquantitative ninhydrin test. If the test is pobitive a second coupling was done

as described earlier. However any free amino groups remaining uncoupled were

capped by acetyhtion.

(b) Symmetrical anhydride method

Symmetrical anhydride was either preformed or prepared in situ. Boc-

amino acid and DCC were used in the ratio 2:l. Boc-amino acid and DCC were

d i i lved separately in minimurn quantity of DCM and cooled to 0°C. It was

mixed and allowed to stand for 1 h at 0°C. The anhydride was added to the

peptide resin through a filter to remove the DCU.

(cJ Active ester method3

In this method DCC, Boc-amino acid and active ester HOBt were used in

1:l:l ratio to get the active ester along with DCU. Active ester was prepared in

DMF and kept for 45 min. This method was very useful in the case of Asn and

Gln because the other two methods appeared to cause dehydration of amides to

nitriles. In the case of Arg thii rnethod was used because it avoids the formation

of a ladam during coupling.

5.14.6 Cleavage of the peptide from the resin

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

thioan-kol (0.1 ml), mcresol (0.1 ml) and 1.2ethanedithiol (0.1 ml) were added.

The mixture was left for 20 h at room temperature. It was filtered through a 5 ml

sintered funnel and the TFA solution was rotary evaporated to remove TFA.

The peptide was then precipitated by the addition of cold dry ether and washed

thoroughly with ether, centrifuged (10 times) and dried.

(6) FApheno&VT method

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

(0.1 ml), water (0.1 rnl), 1,2ethanedithiol (0.1 ml) and thioanisol (0.1 ml) were

added. The mixture was left for 12 h. It was filtered through a sintered funnel

and the TFA was rotary evaporated. The peptide was precipitated by the

addition of cold ether, centrifuged m r a l times and dried.

5.14.7 Purification

(a) Fast protein &quid chromatogrdphy (FPLC)

This technique was suited for the purification of biologically active

peptides and protein sequences. It has extremely high resolving power and is

very helpful in establishing the homogeneity of bioactive peptides. In isocratic

FPLC, the column is eluted with a constant buffer composition while in gradient

FPLC, the elution of the column is effected by varying the buffer composition. A

single isocmtic run alone cannot determine the homogeneity of the peptide. A

single peak may be obtained but the other components may remain adsorbed

strongly to the column. When a gradient elution is performed, peptides of

varying physicochemical properties can be separated effectively and efficiently.

A single gradient elution too is inadequate as it may give a set of compressed

peaks. But by changing the sohlent composition with time, ideal separation of

the peaks can be achieved and purity and homogeneity established. A few

nanomoles of the crude peptide was dissolved in water or methanol or acetic

acid or in a mixture of water and methanol. A suitable volume like 15-20 111 was

injected into the column. The eluted peptide was detected at 214 nm.

lb) Column chromafqpphy

Initial purification of the crude model peptides was performed using silica

gel column (70-230 mesh size). The solvent employed was CH30WCHC13

mixture of varying composition.

5.14.8 Amino acid analysis

It was used for characterisation and quantitation of peptides. It is an

essential requirement in determining the purity of a peptide. It does not provide

any information about the correct sequence of amino acids but it provides

quantitative information about the peptide composition. Pure peptides should

give the expected amino acid ratios for all the residues except for those which

are partially destroyed during hydrolysis like Ser, Thr or which are oxidisabk like

Cys and Met. Asn and Gln are hydrolysed to corresponding acids. Tyrosine gets

degraded if not protected before hydrolysis. Amino acid analysis was performed

on the peptidyl resin during the synthesis to ascertain the proper incorporation of

the amino acids on the suppo:?. It was also done on the purified peptide to

check its purity and homogeneity.

5.14.9 Capping of the residual amino groups by acetyl~ltion'~

The resin was suspencled in DCM (15 ml), acetic anhydride (1 g;

10 mmol) and TEA (1 g, 10 n~mol) were added. It was kept for 1 h. It was

filtered, washed with DCM (15 ml x 3 x 3'). methanol (15 rnl x 3 x 3') and dried

in vacuum. It was tested with ninhydrin which gave a negative test showing the

absence of free amino groups on the resin.

5.15 Synthesis of the peptides

5.15.1 Synthesis of Gly-Leu

fa) Attachment of Boc-Leu to the chloromethyl resin

To the 3% chloromethyl TEGDMA-PS resin (200 mg, 3.8 mmol CVg) in

ethyl acetate (10 ml), Boc-Leu (0.39 g) and TEA (0.09 ml) were added and

refluxed for 48 h. It was then filtered through a sintered funnel and washed with

ethyl acetate (20 ml x 3 x 3'), water, methanol (20 ml x 3 x 3') and DCM (20 ml

x 3 x 3') and dried overnight in vacuum desiccator. Weight increment was

120 rng. Substitution level of Ebc-arnino acid was found to be 3 rnrnoVg by

picric acid method.

(6) Synthesis of Gly-Leu

Synthesis was carried out by using 200 mg of the Boc-Leu resin. It was

taken in a solid phase reaction vessel and deproteded with 30% TFA in DCM

(30 min), washed with DCM and neutmlised with 5% TEA in DCM (10 rnin). It

was again washed with DCM. Eoc-Gly was coupled to this using K C in DCM.

Two fold molar excess of amino acid was used for coupling and the precipitated

DCU was washed with 33% methanol in DCM. Double coupling was carried

out. Resin was washed thoroughly with DCM and dried. Weight increment is

130 mg.

(c) C/ea vage and punfica~o~;~

The peptide resin (30 mg) was treated with TFA (5 ml) at room

temperature for 20 h. The resin was filtered and washed with TFA. Eltrate was

rotary evaporated to remove TFA. Dry ether was added and on cooling white

precipitate of crude peptide appeared which was washed several times with dry

ether and dried. Yield - 18 mg (60%). purity checked by tlc.

5.15.2 Synthesis of Gly-Gly-Phe-Leu

2% TEGDMA-PS resin (1.1 mrnol CVg) was used for this synthesis

(a) Repamtion of cesium salt of Boc-Leu

Boc-Leu (233 mg) was dissolved in minimum quantity of ethanol. It was

diluted with water. Then added saturated solution of cesium carbonate till the

solution became neutral. It was kept for some time and then rotary evaporated

to remove ethanol. Benzene was added and the azeotropic mixture was

evaporated. This was continued till a white powder of cesium salt of Boc-Leu

was obtained. It was dried under vacuum.

(b) Aftachrnent of cesium salt of Boc-Leu to resin

Cesium saH of Boc-Leu was d i i l w d in DMF, chloromethyl resin

(200 mg) was added and heated at 50°C for 48 h. The resin was filtered and

washed with DMF (20 ml x 3), DMF-water (1:1,20 ml x 3). water, DMF (20 ml x

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

vacuum. Weight increment was 90 mg.

The substitution level of Ihc-Leu was determined and it was found to be

0.7 mrnovg.

(c) Synthesis of Gly-Gly-Phe-Leu

Stepwise synthesis of t t i i peptide was carried out on Boc-Leu resin

(100 mg). Boc-Phe and Boc-Gly were used for synthesis. Synthetic cycle

consists of the following steps (Table 5.3).

Table 5.3. Synthesis of G-G-F-L.

Operation I Steps involved ReagentISolvent 1 Time (min)

1 Wash DCM x 6 1.5 2 I 30

3 Wash DCM x 6 1.5

Neubalise 5% TWDCM x 1 10

DCM x 6 1.5

Coupling 1 :1 DCC-Boc amino acid 45

33% methanol-DCM x 3 1.5 Wash DCM x 3 1.5

Steps 6-8 were repeated 1 I

The progress of the coupling w3s monitored by semiquantitative ninhydrin test.

For Boc-Phe and Boc-Gly double couplings were done. Dried and weight

increment was noted, i.e., 120 mg.

(d) Ckamge and purificatio~l

Cleavage of the finished peptide from the resin was done by the general

procedure. 20 mg of the peptide resin was treated with a mixture of TFA

containing thioanisol, mcresol, and 1,2-ethanedithiol in the ratio (10:l:l:l) for

24 h. The cleaved peptide was filtered and the filtrate was rotay evaporated to

remove TFA. Crude peptide was precipitated by the addition of d y ether in ice-

cold condition and washed and c:entrifuged. Yield of the crude peptide is 14 mg.

5.15.3 Synthesis of Ser-Phe-Leu-Glu

Thii peptide was synthesised on 3% TEGDMA-PS, 3% HDODA-PS, 3%

ITEGDA-PS and 3% DVB-PS resins.

(a) Attachment of cesium salt of Boc-Glu

Attachment of C-terminal amino acid to the fundionalised resin (chlorine

capacity 0.9 mmol/g, 1.01 mmol/!3, 1.14 mmol/g and 0.85 mmoVg, respectively).

The first amino acid Boc-Glu (y benzyl ester) was attached to these resins

by the cesium salt method of Gisin. Cesium carbonate was d i i l v e d in water

and the solution was added to the solution of Boc-Glu kept dissolved in

minimum quantity of ethanol till the pH reached 8. It was kept for 1 h. AU the

solvents were rotay evaporated by azeotropic distillation using d y benzene till a

white residue was obtained. It wits dried over P205 under vacuum. The powder

was dissolved in minimum quantity of NMP and resin was added to the solution.

Mixture was heated in an oil bath at 50°C for 48 h. The resin was filtered

through sintered filter, washed with NMP (10 ml x 3), NMP/H20 (1:1, 10 ml x 3),

NMP (10 ml x 3). methanol ( I D ml x 3) and finally with DCM (10 ml x 4).

Product was dried and yield was noted. The Boc-Glu substitution level of the

TEGDMA-PS, HDODA-PS, TTEGDA-PS and DVBPS were 0.81 mmol/g,

0.74 mmol/g, 0.70 mmoVg and 0.50 mrnoVg, respectively.

(6) - Stepwise synthesis of the ,wptide

100 mg of Boc-Glu resin was used for further synthesis and a 3 fold molar

excess Boc-amino acids were used for each coupling. The schedule for the

coupling of the remaining amino acids of the sequence by DCC coupling in DCM

is given below (Table 5.4).

Table 5.4. Synthesis of S-F-L-E

I Operation I Steps involved ( ReagentISolvent* ( Time (rnin) (

1 1 1 Wash I DCMx6 1 1.5 1 1 2 1 Bocdeprotection 1 30% T F W M x 1 1 30 1 1 3 1 Wash I DCMx6 1 1.5 1

I 9 1 Steps 6-8 were 1 1 1 repeated for the second coupling

*10 ml of the solvents was used.

- DCMx6

[KC-Amino acid (1:1,3 fold molar excess)

-

- MethanoWM (33%)

DCM x 3

For Ser, (OBzl) derivative was used. AU amino acids were coupled in DCM. The

peptide resin was dried. Weight increment was noted. Weight increment -

130 mg.

1.5

45

1.5

1.5

(c) Cleavage and purification

Final cleavage was done by treating the peptidyl resin with TFA

containing scavengers like mcresol, thioanisol and 1,2ethanedithiol in the ratio

(10:l:l:l) for 24 h. The cleaved peptide was filtered. Filtrate was rotary

evaporated to remove TFA. The crude peptide was precipitated by the addition

of dry cold ether. It was centrifuged. Yield was found to be 78, 63, 58 and 50%

for TEGDMA-PS, HDODA-PS, TTEGDA-PS and DVB-PS, respectively .

5.15.4 Synthesis of Val-Thr-Leu-Val-Val-Gly segment (1 12-1 17) from catch01 0-methyl transferase

(a) Attachment of first amino acid to the chloromethyl resin

The first amino acid k:-Gly was esterified to the chloromethyl resin

(TEGDMA-PS, chlorine capacity 1.06 mmoVg) following Gisin method. Cesium

carbonate solution was added carefully to Boc-GLy d i i l v e d in ethanoV water

mixture till the pH reached 7-8. It was kept for 1 h and the solvents removed

completely by rotarg evaporation using dry benzene. The white powder

obtained was dried under vacuum. It was dissolved in minimum quantity of

DMF. To this chloromethyl resin (200 mg; 0.212 mmols) was added and heated

in oil bath at 50°C for 48 h. Re:jin was filtered, washed with DMF (10 ml x 3).

DMF/H20 (10 mi x 3). DMF (10 ml x 3) and CH30H (10 ml x 3). The product

was dried and weight increment rvas noted (30 mg).

(b) Stepwise addition of Boci~rnino acids

The target peptide was assembled on the Boc-Gly resin using the

remaining Boc-amino acids of the sequence in a stepwise manner. (150 mg,

0.16 mmol) of Boc-Gly resin used for the synthesis. For each coupling 3

fold molar excess of Boc-amino acids was used to ensure completion of coupling.

In most cases double couplings were required. Val-Val coupling was found to be

very diicuk For that 3 couplings were done. In the case of Boc-Thr (OM) derivative was used for coupling. One complete synthetic cyck consisted of the

fobwing steps.

1. Wash with dichloromethane (10 ml x 6 x 1.5 min)

2. Bocdeprotection using 30% TFA in DCM (10 ml x 30 min)

3. Wash with dichloromethane (10 ml x 6 x 1.5 min)

4. Neutralisation with 5% TEA in DCM (10 ml x 1 x 10 min)

5. Wash with DCM (10 ml ,: 6 x 1.5 min)

6. Equilibration with Boc-amino acids for 10 min followed by coupling of

Boc-amino acids in presence of K C in DCM for 45 min.

7. Wash with 33% methanol in W M (10 ml x 6 x 1.5 min)

8. Repetition of steps 5-7 to ensure completion of coupling tested by

semiquantitative ninhydrin test.

fc) CIeavage and purifictio~r

The finished peptide was cleaved from the resin by following the method

suggested by Bodanszky and Btdanszky. Peptidyl resin (50 mg) was treated

with a mixture of TFA, thioani:;ol, ~ncresol and 1,Zethanedithiol in the ratio

10:1:1:1 for 22 h. The peptide was cleaved from the resin, which was d i i l v e d

in TFA. The filtrate was evaporated. The crude peptide was precipitated by the

addition of ice cold dry ether. It ,was centrifuged and dried.

The crude peptide was dissolved in CH,OH and it was purified by fast

protein liquid chromatography using reverse phase C18 column. Solvent A

consisted of water containing 0.1% TFA and solvent B consisted of acetonitrile

containing 0.1% TFA. The flow rate was regulated at 0.5 mumin. The FPLC

profile consist of one major peak

(dl Amino acid anaiysis

The fraction from FPLC was collected and dried. This purified peptide

was hydrolysed in glass tubes using 6N HCI and TFA (2:l) at 110°C for 22 h and

analysed. The amino acid analysis gave values which agreed with the expected

values.

5.15.5 Synthesis of segment (204-210) from catechol 0-methyl transferase ValhspGly-Leu-Glu-Lys-Ala

(a) Attachment of fhe C-terminal amino acid to the resin

The first amino acid was attached to the chloromethyl resin by the cesium

salt method. Cesium carbonate was dissolved in water and added to the solution

of Boc-Ala (0.3 g, 1.62 mmol) in a minimum quantity of ethanol till the pH

becomes neutral. The mixture u ~ a s kept for 1 h and the solvents rotary

evaporated by azeotropic diilli~tion using benzene. The white powder obtained

was dried under vacuum over P2O5. It was d i i l v e d in minimum quantity of

DMF and the resin (500 rng, 0.81 mmol! was added to the above solution and

the mixture was then heated at 50°C in an oil bath for 48 h. The resin was

filtered through a sintered funnel, washed with DMF (10 ml x 3 x 3 rnin).

DMF/H20 (1:1, 10 ml x 3 x 3 min), DMF (10 ml x 3 x 3 min), methanol (10 ml x

3 x 3 rnin) and DCM (10 ml x 5 x 5 min). The product was dried and weight

increase was noted 600 mg.

Boc-Ala substitution bzvel was determined by picric acid method:

0.5 mmoUg.

(bj Stepwise synthesis of the pepMe

Boc-Ah (500 mg, 0.25 mmovg) resin was used for the synthesis of

remaining amino acids. A 3-fold molar excess of amino acid was used for the

fist coupling. The schedule for the coupling of Boc-arnino acids by DCC

method is given below (Table 5.5).

Table 5.5. Synthesis of V-D-G-L-E-K-A.

/ operation I Steps involved I ReagentISolwnt* I Erne (min) No.

1 1 Wash I DCM x 6 1 1.5 1 2

3

4

5

6

7

8

'10 ml of the solvents were used.

9

Boc-amino acids were used for coupling except in the case of side chain

protected ones. Boc-Glu (y-benzyl), Boc-Lys (CLZ) and Boc-Asp (benzyl) were

the side chain protected amino acids used. Boc-Ah to Boc-Lys coupling was

found to be v e q difficult. After 3 couplings the residual amino groups are

capped by acetylation. Asp to Val coupling was also found to be difficult.

Bocdeprotection 30% TFA/DCM x 1

DCMx6

5% TEA/DCM x 1

DCMx6

Coupling DCC-Amino acid (1 : 1 3 fold molar excess)

Wash MethanoVDCM (33%)

Wash DCM x 3

Steps 6-8 were repeated for the second coupling

(c) Ckavage and purification

30

1.5

10

1.5

14

1.5

1.5

The peptide was cleaved from the peptide resin (200 mg) by

trifluoroacetic acid containing scavengers like mcresol, thioanisol and

1.2-ethanedithiol in the ratio 10::l:l:l for 24 h. The mixture was filtered and the

TFA was removed from the filtrate by rotary evaporation. The peptide was

precipitated by the addition of dry ether. it was centrifuged and dried. Yield:

50 mg.

The crude peptide was purified by FPLC using pep RPC Clg column.

Solvent A consists of water containing 0.1% TFA and B consisted of acetonitrile

containing 0.1% TFA. The flow rate was 0.5 mumin and sensitivity was 0.1.

The major peak was separate15 collected. The collected fractions were dried in

speed vac and hydrolysed using 6N HCI and TFA (2:l). This was heated at

110°C for 24 h and subjected to amino acid analysis.

5.15.6 Synthesis of Leu-Arg-Lys-Gly-Thr-Val-Leu-Leu-Ala-Asp-Asn-Val (160-171) segment from catechol O-methyl transferase

(a) Aftachmenf of firsf amino acid and synthesis of COMT (160-1 71)

The first amino acid was attached to the chloromethyl resin by cesium salt

method. The rest of the amino acids were attached in a stepwise manner

following the standard solid ph.ase rnethodoiogy in DCM. The protocol for the

entire synthesis was given below.

DCM wash (10 ml x 6 x 1.5 min)

30% TFAIDCM-Bocdeprotedion (10 ml x 1 x 30 min)

DCM wash (10 mix 6 x '1.5 min)

5% TEAIDCM-Neutraliition (10 ml x 1 x 10 min)

DCM wash (10 ml x 6 x 1.5 rnin)

Coupling of amino acid in DC:M followed by DCC (45 min)

33% methanol1DCM (10 ml x 6 x 1.5 min)

DCM wash (10 ml x 6 x 1.5 min)

Repetition of 6, 7 and 8 for the second coupling.

Boc-amino acids were mupled in ! X M using DCC. But for Arg and Asn

active ester method using HOBt in DMF was used for coupling. Boc-Asp

(benzyl), Boc-Thr (obenzyl), Boc-Lys (CE) and Boc-Arg (Mts) were the side chain

protected amino acid used. Lys to Arg coupling was difficult, for that 3 couplings

were done. AU the couplings were monitored by ninhydrin test.

(b) Cleavage and purificatiori

The peptidyl resin (100 mg) was treated with a mixture of TFA (2 ml), m-

cresol (0.2 mi), thioan'ml (0.2 ml) and 1,2ethanedithiol (0.2 ml) and kept for 24

h. The resin was removed by filtration. The filtrate on rotary evaporation

removes TFA. Peptide was precip~tated by the addition of cold dry ether.

Centrifuged, Dried, Yield - 40 m'g.

The peptide was dissolved in CH30H and purified by high performance

liquid chromatography. Buffer A consisted of millipore water containing 0.1%

TFA and B consisted of acetonitrile containing 0.1% TFA. Flow rate:

0.5 mumin.

(c) Amino acid analysis

The purified samples were subjected to amino acid analysis by

hydrolysing the samples with 6N HCI and TFA (2:l) at 110°C for 22 h.

5.15.7 Synthesis of Met-Leu-Cys-Cys-Met-kg-kg-%-Lys-Gln-Trp- Gly (1-12) residues of GAP-43 protein

(a) Attachment of C-terminal amino acid to the hnctionalised resin

The first amino acid Boc-Gly was attached to the chlorornethyl resin by

cesium salt method. Saturated solution of cesium carbonate was added carefully

to a solution of Boc-Gly d i i h r e d in minimum quantity of alcohol till the pH of

the mixture reached to 8. The mixture was kept for 1 h and the solvents rotary

evaporated by azeotropic distillation using dry benzene. A white residue was

obtained. It was dried over P2C15 and dissolved in minimum quantity of DMF.

Chloromethyl resin was added to the above solution and the mixture heated in

an oil bath a t 500C for 24 h with occasional shaking. The resin was filtered

through a sintered funnel washed with DMF (10 ml x 3), DMF/H20 (1:1, 10 ml

x 3) DMF (10 ml x 3), methanol (10 ml x 3) and DCM (10 ml x 4). The product

was dried in vacuum. Yield -5 noted.

The Boc-Gly substitution level of the 3 resins were determined by the

picric acid method.

(6) Stepwise synthesis of peptides

Boc-Gly resin was used for the further synthesis and a three fold excess of

Boc-amino acids was used for each couphng. The schedule for the coupling of

the remaining amino acids of the sequence by HOBt active ester method is given

below (Table 5.6).

Table 5.6. Synthesis of M-1-(1-C-M-R-R-T-K-Q-W-G

Operation No.

3 DCM x 4 1.5

4

Wash t 5% DlPElVDMFx2 1 Neuhlisation 5

1

Steps involved I ReagenWSolvent*

1 1 wash 1 DCMx4 1 1 . 5 1 Steps 5,6, 7 and 8 were repeated for

Time (min) I

2 I Bocdeprotection Wash t 30%TFIWCMx 1 30

DCM x 4

DMF x 5

lAA+ lHOBt+ 1DCC+ DMF

DMSO DlPEA

Wash Methanol x 3

1 the second coupling 1 1 I *10 ml of the solvents were used.

1.5

1.5 -

20

8' 5'

15

Double couplings were carried out. For the first coupling a three-fold molar

excess of amino acid and for cecond coupling a two fold molar excess of amino

acids were used. Only Boc-amino acids were used for coupling except in the

case of side chain protected ones. Boc-Cys (Acm), Boc-Arg (Mts), Boc-Thr

(Om), Boc-Lys (CE) and h - T r p (CHO) were the side chain protected amino

acids used. AU amino acids were coupled in DMF solutions through the

formation of their active esters using H O B W C for 40 min. Couplings were

done in 3 stages.

(i) First stage using preformed active ester in DMF for 20 min

(ii) Second stage add DMS,O 15% of the total volume of the solvent used for

synthesis

(iii) DlPEA was added and allowed to couple for 5 min.

(c) Cleavage of the finished pptide from the resin

The peptide was cleaved from the resin following TFA cleavage procedure

using the cleavas mixture phenoVEDT/thimnisol%eionised water and TFA. To

the peptide resin (50 mg) a mixture of phenol (50 PI), EDT (25 HI), thioanisol

(25 PI), deionised water (50 pi) and TFA (825 HI) were added and kept for 6 h.

The cleaved peptide resin was filtered. The filtrate was removed by rotary

evaporation and the crude peptide was precipitated by the addition of ice cold

ether and kept for 1 h. Centrifuged and yield was noted.

(d) finfiation of the peptide

The crude peptide purified by FPLC using a pep RPC C18 column.

Sohrent A consisted of 80% water and 20% acetonitrile containing 0.1% TFA

and solvent B consisted of a&onitrile containing 0.1% TFA. The flow rate was

0.5 mVmin and the sensitivity was 0.1 chart speed was maintained at 0.5 cm/min.

One major and a short peak was obtained in the FPLC profile of the crude

peptide. The peaks were collected and evaporated to get the purified peptide.

Thii was hydrolysed by 6N HCl and TFA (1:2) a t 110°C for 22 h and subjected

to amino acid analysis. The results are summarked in chapter 4.

5.15.8 Synthesis of Src family member GQ

Gly-Cys-Thr-LeuSer-Ala-GIu-Glu-ArgAla-Ala-Leu-Glu-hgSer-T~

(a) Altachment of C-terminal amino acid BOG T p (CHO) to the chloromethyl resin

The Boc-Trp (CHO) was attached to the chloromethyl resin (chlorine

capacity: 0.6 mmoug) by Gisin method. Cesium carbonate solution was added

to a solution of Boc-Trp (CHO) in ethanoywater mixture till the pH becomes 8.

It was kept for 1 h and the solvents removed by rotary evaporation using

benzene. A white powder of cesium salt was obtained and it was dried. The salt

was dissolved in minimum quantity of DMF and chloromethyl resin (130 rng;

0.078 rnmol) was added to it. The mixture was heated at 50°C in an oil bath for

30 h. The resin was filtered through a sintered funnel and washed with DMF (10

ml x 3). DMFM20 (1:1, 10 rnl x 3). DMF (10 ml x 3), methanol (10 ml x 3) and

DCM (10 ml x 3). It was dried and weight increment is 150 mg. Boc-Trp

substitution level of the above resin was determined and it was found to be

0.29 mmovg.

(6) Stepwise addition of Boc-amino acids

The desired peptide sequence was assembled on the Boc-Trp (CHO) resin

using the remaining Boc-amino .acids of the sequence in a stepwise manner. The

resin (130 mg, 0.03 mrnol) was used for further synthesis. A three-fold molar

excess of amino acid was used for first coupling and a two fold molar excess for

second coupling. One complete synthetic cyck consists of the foUowing steps.

1. Activation of AA+ 1 eq HOBt -+ 1 eq. DCC in DMF (5 ml), 40 min

2. Bocdeprotection-30% TFA/DCM (10 ml x 15 min)

3. Wash with DCM (4 x 10 rnl)

4. Neutralisation--5% DIPEA in DMF (2 x 10 ml x 5 min)

5. Wash with DMF (5 x 10 ml)

6. (a) Coupling-addition of activated AA from (1) (40 min)

(b) Addition of DMSO to :make up to 15% by volume (10 min)

(c) Addition of DIPEA (4 equivalents x 5 min)

7. Wash with CH30H (2 x 10 ml x 1 min)

8. Wash with DCM (4 x 10 rnl x I min)

9. Repetition of steps 5 ,6 , 7 and 8 for second coupling.

Boc-amino acids were used for coupling except in the case of side chain

protected ones. BocSer (OBzl), Bx-Arg (Mts), Boc-Glu (y-benzyl), Boc-Thr

(OBzl) and Boc-Cys (Acm) were the side chain protected amino acids used. All

amino acids were coupled in DMF.

(c) Cleavage of the pepfide horn the peptidyl resin

(i) The peptidyl resin (90 mg) was suspended in TFA containing phenol,

thioanisol, deionised water and 1,2ethanedithiol in the ratio

1650:100:100:100:50 pl itt room temperature for 9 h. The solution was

filtered and the filtrate on rotary evaporation to remove TFA. The crude

peptide was precipitated by the addition of precooled dry ether. The

mixture was allowed to stand for 1 h. Centrifuged (810 times) and dried.

The peptide was dissolved in 1 :1 mixture of acetic acid-methanol and the

optical density of Trp was observed at 280 nm. Yield - 3 mg.

(ii) The resin was subjected to second cleaving using the same cleaving

mixture for overnight. The mixture was filtered. Filtrate was rotary

evaporated and the peptide was precipitated by addition of dry ether. It

was isolated by centrifugation, washed and dried.

The peptide was d i i l ved in 1:l mixture of acetic acid and methanol

optical density of Trp at 280 nm was obsewed. Yield - 8 mg. i.e., Total yield - 11 mg.

(d) firification of the peptide

The crude peptide solution was purified by fast protein liquid

chromatography using a reverse phase C18 column. Solvent A consisted of

millipore water containing O.I% TFA and solvent B conskts of acetonitrile

containing 0.1% TFA. The flow rate was 0.5 mumin. The FPLC profile shows

only one major peak which was collected separately.

(e) Amino acid anabjs

The fraction collected from FPLC was evaporated in a speed Vac and

hydrolysed with 6N HC1 and TFA (2:l) at 1lO"C for 22 h and analysed. The

amino acid analysis gave values which agreed with the expected values.

5.15.9 Synthesis of Ro-1.ys-Leu-Leu-Thr-Lys-Phe-LeuCpSer-Tr- Lys-Ile-Gly (segment from seminal plasmin)

(a) Attachment of C-terminalamino acid Bcx-Giy to the resin

The first amino acid BCK-Gly was attached to the 3% TEGDMA-PS resin

(C1 capacity 0.7 rnmoUg) by (;isin's cesium salt method. The remaining Boc-

amino acids were attached in a stepwise manner following the standard solid

phase methodology in DMF solution. Preformed active esters of Boc-amino

acids were used for coupling. The protocol was given below.

1. Activation of AA+ 1 eq HOBt + 1 eq. DCC in DMF (5 ml), 40 rnin

2. Bocdeprotection30Y~ TFNDCM (15 ml x 15 min)

3. Wash with DCM (4 x 10 ml)

4. Neutralisation4W DIF'EA in DMF (2 x 10 rnl x 5 min)

5. Wash with DMF (5 x 10 ml)

6. (a) Addition of preformed active ester (40 min)

(b) Add DMSO (10 min)

(c) Add DIPEA (4 equkalenk x 5 min)

7. MeOH wash (1 x 10 ml x 1 min)

8. DCM wash (4 x 10 ml x 1 min)

9. Repetition of steps 5,6, 7 and 8 for second coupling.

The synthesis was carried out in a solid phase reaction vessel with a

shaker. Almost all couplings proceed smoothly in a single stage itself, as revealed

by the ninhydrin test. A three fold molar excess of amino acid was used for first

coupling and a two fold molar excess for second coupling Boc-Lys (CIZ), Boc-

Trp (CHO), BocSer (OBzl), and Boc-Thr (OM) were the side chain protected

amino acids used for the synthesis.

(b] Ckavage of the peptide

The peptidyl resin (200 mg) was suspended in a mixture of TFA (3.3 ml),

thioanisol (0.5 ml), mcresol (0.5 ml) and 12ethanedithiol (0.2 ml) for 24 h at

room temperature. The resin was removed by fithation. F A was evaporated

and the crude peptide was precipitated by adding precooled dry ether. It was

centrifuged 8 1 0 times and dried. Crude yield - 80%.

(c] FZIrification of the cmdz peptide

The crude peptide was purified by FPLC using Pep RPC C1, column.

Solvent A consisted of pure water containing 0.1% TFA and solvent B consisted

of acetonitrile containing O.1Sb TFA. The flow rate was 0.5 mllmin. Sensitivity

was 0.1. The peptide was soluble in water. One major peak and 4 small peaks

were observed in the FPLC profile of the crude peptide. These peaks were

collected. The fraction afte:r rotary evaporation was hydrolysed using HCI

containing F A at 1 10°C for 22 h and subjected to amino acid analpiis.

References

1. Inman. J. K and Dink, H. M. (1969, &dern&by, 8,4074.

2. Marvel, C. S. and Porter, R. K (1x1) Yhg. Synth. Coil.," Vol. 1 (Edn. 2) John W i i y and Sons, Nev~ York, p. 37.

3. Feinberg, R. S. and Merrifield, R. B. (1374) Tebahedmn, 30,3209.

4. Mitchell, A R. and Menifwld, R. B. (1476) J. Chg. &m., 41,2015.

5. Stewart, J. M. and Young, J. D. (I=) "Solid Phase Peptide Synthesii," (2nd Edn.) W. H. Freeman and Co., .%n F r c m i i , p. 114.

6. Calpino, L A, Giza, C. A. and C a m , B. A (1959) J. Am. Chem. h., 81, 955.

7. Schnabel E. (1x7) Ann. C k m , 702 188.

Itoh, M., Hagiwam. 0. and Kaniya, T 1975) Tebahedmn, 16,4393.

Gisin, B. F. (1973) Hetv. Cfwm. Acta. 56, 1476.

Giin, B. F. (1972) Anal. C15em. .4cta. 58,348.

Gutte, B. and Memifield, R. B. (1971) ..' Bid Chem.., 246. 1922.

Rebek, J. and Feihr, D. (1974) cL An: Chem. Soc.. 96, 1606.

Mojisov, S., Mitchell, A. R. and Merrif~id, R. B. (1980) J. Gg. Chem., 45,555.

Bodanszky. M. and Bodamrky, A (193) Int. J. Pept. ht. Res., 23,287.

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