SYNTHESIS OF PROTECTED PEPTIDE ACIDS, AMIDES AND ALKYL AMIDES USING

PHOTOLYTICALLY CLEAVABLE PS-BDODMA SUPPORTS

7.L Introduction

T he myriad of naturally occurring peptides and their biologically relevant

analogues, includes numerous examples where the C-terminal carboxyls are

modified as amides, aldehydes and esters. The peptide activity can be controlled

through changes at the C-terminal and therefore it is important to have efficient methods

for the solid phase synthesis of such derivatives. Chemical synthesis of C-terminal

modified peptides are important for the structure-activity relationship studies and

conformational ~tudies. ' .~ A large number of biologically active peptides contain an

amide or N-alkylamide group at the carboxy terminal. C-terminal modification of

peptides without affecting side-chain protecting groups and chiral centers has been a

great challenge in peptide chemistry. In classical Merrifield's solid phase peptide

synthesis, the C-terminal peptide amides are usually prepared by ammonolysis of the

polymer-peptide ester linkage.3 Another method for the synthesis of peptide amides is the

use of resins containing amino groups, which facilitates the coupling of the C-terminal

amino acid of the target peptide through an amide linkage and the final cleavage of the

synthesized peptide in the form of peptide amide by acid cleavage.G7 The major

limitation of these procedures is the lack of stability of side chain ester protecting groups.

Photolabile supports have been employed for 24 years in SPPS, since the

introduction of photosensitive 2-nitrobenzamido anchoring linkage between the polymer

support and the peptide.' This method permits the release of peptide amides under neutral

conditions at room temperature without affecting the side chain protecting groups and

N-protecting groups. Photolytic release of peptides offers a method of orthogonal

cleavage, which complements the traditional use of TFA or HF. The photolabile supports

have a light sensitive chromophore, which is stable to conditions of peptide ~ ~ n t h e s i s . ~ ~ ' ~

The photolabile linker can be easily introduced into the support and the C-terminal amino

acid can be easily incorporated to it. Photosensitive amino acids such as Trp and Tyr

should not remain intact at the wavelength of light used for photolysis and the by-product

formed should remain along with the polymer support. A large number of polymer

supports with various photolabile anchoring groups are used for the synthesis of peptides.

Nitration of Merrifield's resin results in very high loading of nitro group, which increases

the polarity of the resin and hence reduces the swelling property of the resin in organic

solvents with very low dielectric constants. A modified 4-bromomethyl 3-nitro and

4-aminomethyl 3-nitro tetraethyleneglycol diacrylate cross-linked polystyrene support

was used for the synthesis of protected peptide acids, amides and alkyl amides.''

PS-BDODMA resin anchored with photolabile 4-hromomethyl 3-nitro

benzamidomethyl and 4-aminomethyl 3-nitro benzamidomethyl anchoring groups were

successfully used for the solid phase peptide synthesis. This resin swells much effectively

than the Merrifield's resin in all solvents used for SPPS. This support contains only the

required number of nitro groups that are essential for the photochemical reaction. This

chapter describes the photolytic synthesis of fblly protected peptides, peptide amides and

peptide N-alkyl amides using PS-BDODMA resin.

7.2. Results and Discussion

7.2.a. Preparation of 4-chloromethyl3-nitro PS-BDODMA resin

The photolabile 4-chloromethyl 3-nitro PS-BDODMA resin was prepared by the

nitration of chloromethyl PS-BDODMA resin using fbming nitric acid at -10 "C. The

resin showed same swelling properties as that of chloromethyl PS-BDODMA resin

indicating no additional cross-linking during nitration. The resin shows characteristic IR

(Kl3r) absorption band at 690 cm-' and 1230 cm-' for chloromethyl group and 1350 cm-'

and 1545 cm-' for NO2 group (Fig.7-1).

Fig.7-1: IR (KBr) spectrum of 4-chloromethyl 3-nitro PS-BDODhL4 resin

7.2.b. Preparation of 4-aminomethyl 3-nitro PS-BDODMA resin

4-aminomethyl 3-nitro P S - B D O D I ~ ~ resin was prepared from 4-chloromethyl

3-nitro PS-BDODMA resin by refluxing with potassium phthalimide in NMP followed

by hydrazinolysis. Amino capacity of the resin was estimated by picric acid titration method

and a quantitative reaction was observed. The resin showed IR (KBr) absorption band at

3440 c ~ ' @Hz) and 1340 cm.' and 1540 cm" corresponding to NO2 group (Fig. 7- 2).

Fig.7-2:IR (KBr) spectrum of 4-aminomethyl 3-nitro PS-BDODMA resin

7.2.c. Preparation of 4-bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin

The photolabile anchoring group 4-bromomethyl 3-nitro benzoic acid was prepared

by a two step reaction from p-toluic acid. p-Toluic acid was converted to 4-bromo-

methyl benzoic acid by the treatment with N-bromo succinimide (NBS). Nitration with

fuming nitric acid at -10 "C of 4-bromomethyl benzoic acid yielded 4-bromomethyl

3-nitro benzoic acid The pre-swollen aminomethyl resin in NMP was treated with HOBt

active ester of 4-bromomethyl 3-nitro benzoic acid yielded the photolabile

4-bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin (Scheme7-1). Estimation

of bromine capacity by Volhardt's method indicates the quantitative reaction. The resin

shows characteristic IR (KBr) bands at 1340 cm" and 1540 cm-' of the NO2 and

1650 cm'l (NHCO) (Fig.7-3) The resin shows the same extent of swelling as the

PS-BDODMA resin in all solvents used for SPPS.

I Stepwise incorporation of amino acids

Scheme. 7-1. Preparation and use of 3-nitro 4-bromomethyl benzamidomethyl PS-BDODMA resin.

Fig. 7-3.

10

n

u

I

Lad 4- am# Ism t w o IDDO YXI

, IR O<Br) spectrum of 3-nitro 4-bromomethyl benzamidomethyl PS-BDODMA resin.

7.2.d. Preparation of 4-aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin

4-Aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin can be prepared

from 4-bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin by refluxing with

potassium phthalimide in NMP followed by hydrazinolysis (Scheme. 7-2). Amino

capacity estimation by picric acid method indicates the quantitative reaction. The resin

showed characteristic IR (KBr) bands at 1342 cm-I and 1548 cm-I of the NOz, 1680 cm"

(NHCO) and 3450 cm-' (broad) of NH group (Fig. 7-4).

*0 )0

Fig. 7-4. IR (KBr) spectrum of 4-aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin

NO2 w &+&,@ O NMP, d I ~ O O C -

N2H4.H20

EtOH, 8 8 ~

0

Scheme. 7-2. Preparation of 3-nitro 4-aminomethyl benzamidomethyl PS-BDODMA resin

7.2.e Preparation of N-alkyl aminomethyl fni t ro benzamidomethyl PSBDODMA resin

4-Bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin was suspended in

DMF, dry methyl amine or ethyl amine gas was passed through the suspension at 0 'C

and the reaction mixture was shaken at room temperature for 24 h (Scheme. 7-3). The

N-alkyl resin was purified by washing and dried under vacuum. The resin shows IR

(KBr) absorption at 1342 cm-', 1540 cm-' (NOz), 3430 c ~ ' (broad) (NH) and 1640 cm-'

(NHCO). The amino capacity of the resin was estimated by picric acid method. The side

reactions like the formation of tertiary amine and quaternary ammonium salt of resin can

be eliminated by the use of large excess of amine.

Stepwise incorporation of amino acids

(a & b)

(a) R=CH, (b) R=CzH5

Scheme. 7-3. Preparation 'and use of 3-nitro 4-N-alkylaminomethyl benzamidomethyl PS-BDODMA resin

7.2.f. Synthesis of protected peptide acids, amides and alkyl amides

The synthetic utility of the resins 4-chloromethyl 3-nitro PS-BDODMA,

4-aminomethyl 3-nitro PS-BDODMA, 4-bromomethyl 3-nitro benzamidomethyl

PS-BDODMA, 4-aminomethyl 3-nitro benzamidomethyl PS-BDODMA, N-methyl

aminomethyl 3-nitro benzamidomethyl PS-BDODMA and N-ethyl aminomethyl 3-nitro

benzamidomethyl PS-BDODMA are illustrated with the synthesis of some representative

peptide acids, amides and N-alkyl amides. The cesium salt method was used for the

attachment of the C-terminal amino acid to the 4-chloromethyl 3-nitro resin. The

peptides were assembled on the resin using the pre-formed HOBt active ester of Boc or

Fmoc-amino acids. For resins 4-aminomethyl 3-nitro PS-BDODMA, 4-aminomethyl

3-nitro benzamidomethyl PS-BDODMA, N-methyl aminomethyl 3-nitro

benzamidomethyl PS-BDODMA and N-ethyl aminomethyl 3-nitro benzamidomethyl

PS-BDODMA, the C-terminal amino acid was attached using its pre-formed HOBt

active ester. After the incorporation of amino acids in the target sequence, the peptides

were cleaved from the resin by photolysis carried out in TFElDCM solution. The

peptides were characterized by HPLC and amino acid analysis. The following peptide

acids, amides and N-alkyl amides were synthesized by photolysis.

1. Boc-NH-Gly-Leu-Ala-Leu-Ala-Gly

2. Boc-NH-Leu-Ala-Gly-Leu-Ala-Gly

3. Boc-NH-Gly-Ile-Cys(Acm)-Pro

4. Fmoc-NH-Ile-Leu-Ala-Gly

5. ~ m o c - ~ ~ - ~ e u - ~ s ~ ( ~ ~ u ~ - ~ e u - G l ~ - A l a - G l ~

6. Ile-Ala-Val-Gly-NH2

7. Boc-NH-Pro-Val-NHZ

8. Boc-NH-Gly-Phe-Pro-NH2

9. Boc-NH-Leu-Ala-Gly-Val-NH2

10. Boc-NH-Ala-Gly-Leu-Ile-Gly-N&

1 1. Fmoc-NH-Ala-Gly-Leu-Ile-Gly-NH2

12. Boc-NH-Leu-Ala-Val-NHMe

13. Boc-Val-Leu-Ala-Val-NHMe

14. Boc-NH-Leu-Ala-Val-NHEt

15. Boc-NH-Val-Leu-Ala-Val-NHEt

7.2.g. Mechanism of photolytic cleavage

The mechanism of photolytic cleavage of nitro benzyl and related system is well

documented The reaction involves a light induced internal oxidation-reduction reaction

of aromatic nitro compounds containing a carbon-hydrogen bond ortho to the nitro group.

The reaction follows the reduction of nitro group to the nitroso group when oxygen is

inserted to the carbon-hydrogen bond at the 2-position resulting in the oxidation of <Hz-

group to -CHO group.'2 (Scheme. 7- 4)

Scheme. 7- 4. Mechanism of photolytic cleavage of peptide from the resin

eoo eoo rooo MassICharge

Fig. 7.5. MALDI TOF MS of (a) Boc-NH-Gly-Leu-Ala-Leu-Ala-Gly (b) ~ m o c - N H - ~ e u - ~ s ~ ( ~ ~ u ' ) - ~ e u - ~ l ~ - ~ l a - ~ l ~

All the observations illustrate the applicability of the modified PS-BDODMA

resin as a photo-removable polymeric support for solid phase synthesis of fully protected

peptide acids, peptide amides and peptide alkyl amides. This method has a unique

advantage of avoiding the formation of diketopiperazine and the unwanted side reactions

in trans-esterification procedure, thus increasing the overall yield of the peptide. The

peptides obtained are in fully protected form, which can be applicable for segment

condensation. Photolytic cleavage can be conveniently employed for peptides containing

sterically hindered C-terminal amino acid like Val, Ile etc.

7.3. Experimental

7.3.a. Preparation of 4-Chloromethyl f nitro PS-BDODMA resin

Chloromethyl PS-BDODMA resin (1 g, 0.68 mmol) was added to fuming nitric

acid (100 mL) in portion wise at -10 OC over 30 min. The suspension was kept for 2 h at

-10 "C with occasional swirling. The reaction mixture was then poured in to a beaker

containing ice-cold water. The resin was collected by filtration and washed with ice-cold

water until the washings were neutral. The resin was washed thoroughly with DCM (6 x

30 mL), methanol (6 x 30 mL) and ether (6 x 30 mL) and dried under vacuum.

7.3.b. Preparation of 4-Aminomethyl 3-nitro PS-BDODMA resin

4-Chloromethyl 3-nitro PS-BDODMA resin (500 mg, 0.66 mmol) was suspended

in NMP (20 mL), potassium phthalimide (1.2 g, 6.6 mmol) was added and the reaction

mixture was kept at 1 10- 120 "C with occasional shaking for 12 h. The resin was filtered,

washed with NMP (6 x 25 d ) , dioxane (6 x 25 mL), EtOH (6 x 25 d ) , MeOH (6 x

25 mL) and dried under vacuum. The dried resin was then suspended in EtOH (100 mL)

and refluxed with hydrazine hydrate (0.33 mL, 6.6 mmol) for 8 h, the resin was collected

by filtration, washed with EtOH (6 x 25 mL) and MeOH (6 x 25 mL). The resin was

then dried in vacuum. Amino capacity of the resin=0.65 mmol/g.

7.3.c. Preparation of 4-Bromomethyl benzoic acid

p-Toluic acid (13.6 g, 100 mmol) was suspended in dry benzene (100 mL) and a

mixture of benzoyl peroxide (200 mg) and N-bromosuccinimide (17.8 g, 100 mmol)

were added and refluxed for 24 h. The solvent was removed under vacuum and the

residue was suspended in boiling water (100 mL) for 10 min. The precipitate was filtered

and washed with boiling water (6 x 25 mL). The crude product was recrystallized from

hot MeOWDCM mixture.

mp=227.5 "C

IR W r ) : 2800-2400 cm'l, 1682 cm-' (COOH), 1556 cm-', 690 cm-'(aromatic)

7.3.d. Preparation of 4-Bromomethyl3-nitro benzoic acid

Add portion wise 4-bromomethyl benzoic acid to hming nitric acid (100 mL) at

-10 OC over 30 min. The suspension was stirred for 2 h at -10 OC. The reaction mixture

was then poured in to a beaker containing ice-cold water. The precipitate formed was

collected by filtration and washed with ice-cold water until the washings were neutral.

The precipitate was dried and recrystallised from petroleum ether. mp=125-126 "C.

IR (KBr): 2800-2300 cm-', 1688 cm-' (COOH), 1592, 700 cm-' (aromatic), 1542 cm-',

13 10 cm" (nitro).

7.3.e. Preparation of 4-Bromomethyl3-nitro benzamidomethyl PS-BDODMA resin

To the pre-swollen aminomethyl PS-BDODMA resin (1 g, 0.67 mmol) in DCM, a

mixture of 4-bromomethyl3-nitro benzoic acid ( mg, 2 mmol), HOBt (270 mg, 2 mmol),

HBTU ( 760 mg, 2 mmol) and DIEA (350 pL, 2 mmol) were added and kept at room

temperature. Afier 1 h, the resin was filtered, washed with DCM (6 x 10 mL) and a

second coupling was performed. The resin was collected by filtration, washed with DCM

(5 x 20 mL), DMF (5 x 20 rnL) and MeOH (5 x 20 mL). Bromine content of the

resin = 0.64 mmollg.

IR (KBr): 1650 cm-' (NHCO), 1340 cm-', 1540 cm-' (NOz)

7.3.f. Preparation of 4-Aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin

4-Bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin (500 mg, 0.32 mmol)

was suspended in NMP (20 mL), potassium phthalimide (550 m g, 3.2 rnmol) was added and

the reaction mixture was kept at 110-120 OC with occasional shaking for 12 h. The resin was

filtered, washed with NMP (6 x 25 mL), dioxane (6 x 25 mL), EtOH (6 x 25 mL), MeOH (6 x

25 mL) and dried under vacuum. The dried resin was then suspended in EtOH (100 rnL) and

refluxed with hydrazine hydrate (0.15 mL, 3.2 mmol) for 8 4 the resin was then colkcted by

filtration, washed with EtOH (6 x 25 mL) and MeOH (6 x 25 mL). The resin was then dried in

vacuum. Amino capacity of the resin= 0.63 mmollg.

7.3.g. Preparation of 4-Methyl aminomethyl 3-nitro benzamidomethyl PS- BDODMA resin

4-Bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin (100 mg,

0.064 mmol) was suspended in DCM (20 mL) in a stoppered bottle and was kept at

0-5 'C. Dry methyl amine gas was bubbled through the reaction mixture for 12 h. The

reaction bottle was stoppered well and shaken for 12 more hours at room temperature.

The resin was filtered, washed with DCM (3 x 2 mL x 3 min), THF (3 x 20 mL x

3 min), water (3 x 20 mL x 3 min), MeOH (3 x 20 rnL x 3 min) and dried in vacuum.

Amino capacity of the resin = 0.6 mmollg.

IR (KBr): 1650 cm-' (NHCO), 1340 cm-l, 1530 cK1 (NO& 3400 cm-I (NH)

7.3.h. Preparation 4-Ethyl aminomethyl 3-nitro benzamidomethyl PSBDODMA resin

4-Bromomethyl 3-nitro benzamidomethyl PS-BDODMA resin (100 mg,

0.064 mmol) was treated with dry ethyl mine. The above protocol was used for the

synthesis. Amino capacity of the resin= 0.6 mmol/g.

IR (KBr): 1652 cm" (NHCO), 1340 cm", 1530 cm-' (NOz), 3440 cm-' (NH).

7.3.i. General synthetic protocol for peptides using solid supports.

1. Synthesis of peptides using Boc-amino acids

Peptide synthesis was carried out manually in a silanised glass reaction vessel

with a glass filter at one end and a calcium chloride guard tube at the other end. The

C-terminal amino acid was attached to the resin by cesium salt method. The first

Boc- amino acid attached resin was taken in the reaction vessel and swelled in DCM.

Boc-protection was removed by using 30% TFA/DCM and neutralization was achieved

by 5% DIEA/DCM. The resin was washed thoroughly with DCM and D m . Boc-amino

acid (3 equiv corresponding to the halogen capacity of the resin) along with HOBt

(3 equiv), HBTU (3 equiv) and DIEA (3 equiv) were added and kept for 1 h at room

temperature. The coupling procedure was proceeds till the target peptide sequence was

completed. Each coupling was performed twice for 100% reaction. The coupling

reactions were monitored by Kaiser's test.

2. Synthesis of peptides using Fmoc-amino acids

The Fmoc-protection from the C-terminal amino acid attached resin was removed

by 20% piperidinelDMF. The resin was washed thoroughly with DMF and the

subsequent Fmoc-amino acids (3 equiv) were coupled by using HOBt (3 equiv) and

HBTU (3 equiv) in presence of DIEA (3 equiv). Each coupling steps were monitored by

ninhydrin test.

7.3.1. General procedure for photolysis

The peptidyl resin was suspended in a mixture of 30% TFE in DCM (100 mL)

and placed in an immersion type photochemical reactor. The suspension was degassed

for 1 h with dry nitrogen and irradiated with Philips HPK 125 W medium pressure

mercury lamp at 340-350 nm for 24 h. A solution of CuS04 was circulated throughthe

outer jacket of the photochemical reactor to filter off light waves below 320 nm. After

photolysis, the resin was filtered, washed with ethanol (3 x 25 mL) and DCM (3 x

25 mL). Combined filtrate and washings were evaporated on a rotary evaporator under

reduced pressure The residue was collected and purified by chromatography on a

sephadex G-10 column using suitable solvents.

7.3.k Synthesis of Boc-NH-Gly-Leu-Ala-Leu-Ala-Gly, Boc-NH-Leu-Ala-Gly-Leu- Ala-Gly, Boc-NH-Gly-Ile-Cys(Acm)-Pro.

C-terminal Boc-amino acids of these peptides were attached to 4-bromomethyl

3-nitro benzamidomethyl PS-BDODMA resin (50 mg 0.64 mmol Brlg) by cesium salt

method and the extent of attachment was measured by picric acid method. After

Boc-deprotection with 30% TFA in DCM and neutralization with 5% DIEA in DCM the

remaining amino acids (2.5 mmol excess compared to the amino capacity) in the target

sequences were coupled successively as their HOBt active ester. The resin was washed

thoroughly with MeOH/DCM (6 x 10 mL), NMP (6 x 10 mL) and DCM (6 x 10 mL).

The peptidyl resin was suspended in TFEIDCM (30% vlv) and photolysed

according to the general procedure described above. The crude peptides were dissolved

in acetic acid-water mixture and eluted through a sephadex G-10 column. The peptidyl

fractions were collected and lyophilized. The purity of the peptides were checked by

HPLC and characterized by amino acid analysis.

Boc-NH-Gly-Leu-Ala-Leu-Ala-Gly (Yield= 13.8 mg, 72 %).

Amino acid analysis: Gly, 2.1 (2); Leu, 1.92 (2); Ala, 2.03 (2).

MALDI TOF MS: m/z 600.8 [(M+H)+, 100%], C27hgN609, requires M' 599.7.

Boc-MI-Leu-Ala-Gly-Leu-Ala-Gly (Yield= 14.4 mg, 75 %).

Amino acid analysis: Gly, 2.0 (2); Leu, 1.98 (2); Ala, 1.95 (2).

Boc-NH-Gly-Ile-Cys(Acm)-Pro (Yield= 11.7 mg, 68 %).

Amino acid analysis: Gly, 1.0 (I); Ile, 0.87 (1); Pro, 0.87 (1); Cys, 0.78 (1).

73.1 Synthesis of Fmoc-NH-&Leu-Ah-, F m o c - N B - L e u - ~ s ~ ( ~ ~ u ~ ~ e n - ~ ~ ~ - ~ l a ~ ~

C-terminal amino acid Boc-Gly (35 mg, 0.2 mrnol) was attached to the resin (100 mg

0.64 mmol Brlg) by cesium salt method and the extent of incorporation was estimated by

picric acid method. The resin was washed thoroughly with DCM and Boc-protection was

removed by 30% TFNDCM (10 d ) , the resin was washed thoroughly with DCM (6 x

10 d ) , NMP (6 x 10 d ) and DMF (6 x 10 d ) . Fmoc-Ala was attached to the resin

by HOBVHBTU method. After removing Fmoc protection with 20% piperidine /Dm, it

was washed with DMF and the remaining amino acids (Fmoc-protected) were assembled

till the target sequences were formed.

The resins were suspended in TFE/DCM (30% vtv) mixture and photolysed

according to the general procedure. The crude peptides were dissolved in 5% acetic

acidtwater and purified by passing through a sephadex G-10 column. The purity of the

peptides was monitored by HPLC.

Fmoc-NH-Ile-Leu-Ala-Gly (Yield= 13 mg, 68%)

Amino acid analysis: Ile, 0.93 (1); Leu, 1.03 (1); Ala, 1.1 (1); Gly, 1.04 (1).

~ m o c - ~ e u - ~ s ~ ( ~ ~ u ' ) - ~ e u - ~ l ~ - A l a - G l ~ (Yield= 17 mg, 65%)

Amino acid analysis: Leu, 2.1 (2); Asp, 0.91 (1); Gly, 2.12 (2); Ala, 0.98 (1).

MALDI TOF MS: d z 822.9 [(M+H)+, 100°h], C ~ ~ H S ~ N ~ O I ~ , requires ~ + 8 2 1 . 2 .

7.3.m. Synthesis of Ile-Ala-Val-Gly-NHt

Fmoc-Gly (45 mg, 0.15 mmol) was attached to the 4-aminomethyl 3-nitro

benzamidomethyl PS-BDODMA resin (75 mg, 0.05 mmol) by HOBt active ester

method Afier Fmoc-deprotection, the remaining amino acids in the target sequences

were incorporated by Fmoc-solid phase synthetic strategy. Fmoc-protection from the

peptidyl resin was removed by 20% piperidine1DMF (10 d ) , washed with DMF (6 x

10 d ) , DCM (6 x 10 mL) and dried. The peptidyl resin was suspended in TFE/DCM

mixture and photolysed. The crude peptide was passed through a sephadex G-10 column

and the purity was checked by HPLC.(Yield= 11 mg, 69%)

Amino acid analysis: Ile, 0.93 (1); Ala, 1.05 (1); Val, 0.92 (1); Gly, 1.1 (1).

7.3.11. Synthesis of Boc-NH-Pro-Val-NHz, Boc-NH-Gly-Phe-Pro-NHz, Boc-Leu-Ala- Gly-Val-NHz, Boc-Ala-Gly-Leu-Ile-Gly-NH2

The C-terminal amino acid (0.15 mmol) was attached to the 4-aminomethyl

3-nitro benzamidomethyl PS-BDODMA resin (75 mg, 0.05 mmol) by using HOBt

(20 mg, 0.15 mmol), HBTU (57 mg, 0.15 mmol) in presence of DIEA (26 pL). After the

Boc-deprotection with 30% TFAfDCM (10 mL), the remaining amino acids in the target

sequences were coupled by Boc-solid phase synthetic protocol. The peptidyl resin was

suspended in TFElDCM mixture and photolysed. The crude peptide obtained was

dissolved in 5% acetic acidlwater and passed through a sephadex G-10 column. The

peptidyl fractions were collected and lyophilized. The purity of the peptides was

monitored by HPLC.

Boc-NH-Pro-Val-NH2 (Yield= 10.4 mg, 74%)

Amino acid analysis: Pro, 0.91 (1); Val, 1.0 (1).

Boc-NH-Gly-Phe-Pro-NHz (Yield= 15 mg, 80%)

Amino acid analysis: Gly, 0.96 (1); Phe, 1.0 (1); Pro, 0.98 (1).

Boc-Leu-Ala-Gly-Val-Nfi (Yield= 14.8 mg, 71%)

Amino acid analysis. Leu, 1.0 (1); Ala, 0.95 (1); Gly, 1.02 (1); Val, 0.97 (1).

Boc-Ala-Gly-Leu-Ile-Gly-N& (Yield= 17.5 mg, 73%)

Amino acid analysis: Ala, 1.12 (1); Gly, 2.02 (2); Leu, 0.91 (1); Ile, 1.04 (1).

7.3.0. Synthesis of Fmoc-NH-Ala-Gly-Leu-Ile-Gly-NH2

The C-terminal Fmoc-Gly (45 mg, 0.15 mmol) was attached to the 4-aminomethyl

3-nitro benzamidomethyl PS-BDODMA resin (75 mg, 0.05 mmol) by using HOBt (20 mg,

0.15 mmol), HBTU (57 mg, 0.15 mmol) in presence of DIEA (26 pL). The extent of

attachment was determined by measuring the OD of the adduct formed by treating a definite

amount of amino acid attached resin with 20% piperidine in DMF (10 mL). The remaining

amino acids were coupled by Fmoc-synthetic strategy. Atter the synthesis the peptidyl resin

was suspended in TFEiDCM and photolysed. The crude product was eluted through a

sephadex G-10 column. The peptidyl fractions were collected and lyophilized. The purity of

the peptide was checked by HPLC.(Yield= 22.5 mg, 75%)

Amino acid analysis: Ala, 1.06 (1); Gly, 1.92 (2); Leu, 1 .O1 (1); Ile, 1.1 (1).

7.3.p. Preparation of Boc-NH-Val-N(CH3)-Resin

4-Methyl aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin (100 mg,

0.06 mmol) was suspended in NMP (10 mL). The HOBt active ester of Boc-Val (61 mg,

0.18 mmol) was added to the reaction mixture and kept for 1 h with occasional shaking. The

resin was collected by filtration and washed with NMP (6 x 10 mL), DCM (6 x 10 mL),

MeOH (6 x 10 mL) and ether (6 x 10 mL). Amino capacity= 0.58 mmol/g.

7.3.q. preparation of Boc-NH-Val-N(CzH5)-resin

4-Ethyl aminomethyl 3-nitro benzamidomethyl PS-BDODMA resin (100 mg,

0.06 mrnol) was suspended in NMP (10 mL). The HOBt active ester of Boc-Val(61 rng,

0.18 mmol) was added to the reaction mixture and kept for 1 h with occasional shaking.

The resin was collected by filtration and washed with NMP (6 x 10 mL), DCM (6 x 10 mL),

MeOH (6 x 10 mL) and ether (6 x 10 mL). Amino capacity= 0.57 mmoYg

7.3.r. Synthesis of Boc-NH-Leu-Ala-Val-NHMe, Boc-NH-Val-Leu-Ala-Val-NHMe, Boc-NH-Val-Leu-Ala-Val-NHEt, Boc-NH-Leu-Ala-Val-NHEt

Boc-Val was attached to the resins N-methyl aminomethyl 3-nitro

benzamidomethyl PS-BDODMA (50 mg, 0.03 rnmol) and N-ethyl aminomethyl 3-nitro

benzamidomethyl PS-BDODMA (50 mg, 0.03 mmol) by using HOBt (13.5 mg,

0.09 mmol), HBTU (37.9 mg, 0.09 mmol) and DIEA (14 pL). The successive amino

acids (3 equiv) were coupled and finally the peptides were detached kom the resins by

photolysis. The purity of the peptides was checked by HPLC.

Boc-NH-Leu- Ala-Val-NHMe

Amino acid analysis: Ala, 1.0 (1); Leu, 0.9 (1); Val, 1.12 (1).

Boc-NH-Val-Leu-Ala-Val-NHEt

Amino acid analysis. Ali, 1.0 (1); Leu, 1.21 (1); Val, 1.98 (2).

References

1. Stimson, E. R.; Meinwald, Y. G.; Montelione, G. T.; Scheraga, H. A. Inf. J Peptide Protein Res. 1986, 27, 569.

2. Mammi, S.; Goodman, M. Int. J. Peptide Protein Res. 1986,28, 29.

3. Manning, M. J. Am. Chem. Soc. 1968,90, 1348.

4. Matsueda, G. R.; Stewart, J. M. Peptides 1970, 2, 45.

5. Pietta, P. A,; cavello, P. F.; Takahashi, K.; Marshall, G. R. J. Org. Chem. 1974,39,44.

6 . Orlowski, R. C.; Walter, R.; Winkler, D. J. Org. Chem. 1976,41,3701.

7. Penke, B.; Rivier, J. J. Org. Chem. 1987, 52, 1197.

8. Rich, D. H.; Gunvara, S. K. J. Am. Chem. Soc. 1975,97,1575.

9. Pillai, V. N. R. Synthesis 1980, 1

10. Pillai, V. N. R. "Organic Photochemishy" Padwa, A Eds., Vo1.9 ,Marcel Dekker, New York, 1987, pp.225-312.

11. Kumar, K. S.; Pillai, V. N. R. Tetrahedron, 1999, 55,10437.

12. Patchornik, A,; Amit, B.; Woodward, R. B. J Am. Chem. Soc. 1970,92, 6333.