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transcript
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.
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Giin, B. F. (1972) Anal. C15em. .4cta. 58,348.
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Rebek, J. and Feihr, D. (1974) cL An: Chem. Soc.. 96, 1606.
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