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A Simple & Convenient Solid Phase Synthesis of Bacterial Origin Octapeptide Sequence, Glu-Asp-Gly-Asn-Lys-Pro-Gly-Lys-OH

The repeating octapeptide sequence, Glu-Asp-Gly-Asn-Lys-Pro-Gly-Lys-OH derived from the glycoprotein found in Staphylococcus aureus cell wall is assembled by simple solid phase peptide synthesis methodology using a base labile linker.

Protein-A, a glycoprotein obtained from Staphylococcus aureus Cowen 1. bacterial cell wall (Kronvall and Gewurz 1970) appears to be working against toxicity of certain chemical exposure (Dohadwala and Ray 1985;Singh et al. 1990) and is a potent immunomodulator (Catalona et al. 1981). It has been shown to ameliorate toxicity of cyclophosphamide, carbon tetrachloride, bacterial endotoxins (Ray et al. 1984;Kerbel and Davies 1974) and have been demonstrated to regress primary tumors in rodent and mammalian models through modulating the reactivity of cellular and humoral factors (Atherton and Sheppard 1985). The octapeptide Glu-Asp-Gly-Asn-Lys-Pro-Gly-Lys-OH was found to be the repeating sequence in protein-A structure. Toxicology and other biological studies prompted to synthesize the sequence albeit using a very simple and convenient method inspired from Merrifield's' original synthesis.
The octapeptide sequence was assembled (Scheme 1) using a base labile linker (Katti et al. 1992). The first amino acid lysine was anchored from C-terminal to amino methyl resin as its t-Boc derivative. The linker was synthesized starting from phencyl bromide, which, in turn, was prepared from acetophenone and bromine using aluminium chloride under extra cooled conditions (-78°C). The phencyl bromide (2) was coupled with chloroacetic acid and it was further extended by reacting with 2-mercaptoethanol to give the full-length liker (6). The linker (6) was coupled with t-Boc-Lys Z using dicyclohexylcarbodiimide (DCC) and 4-dimethyl aminopyridine (DMAP) to give t-Boc-Lys Z -Linker (7) and it was oxidized to sulphone (9) via reduction and subsequent oxidation of the product (8) with potassium per oxomonosulphate (oxone). The oxidized t-Boc-Lys Z -Linker (9) was coupled to aminomethyl resin using DCC and 1-hydroxybenzotriazole (HOBt) in dichloromethane-dimethyl formamide (CH 2 Cl 2 -DMF), quantified by picric acid method (Rich and Sing 1979;Stewart and Young 1984). The first loading of t-Boc-Lys Z -Linker (97.12 micromoles per gram) on solid support was sequentially extended towards n-terminus by single addition of 3-molar equivalents of suitably protected amino acids activated by DCC-HOBt. The protected amino acids were prepared during the study, purified by recrystallization and well characterised by thorough comparison with authentic reference samples. The t-Boc, Z, benzyl, methyl ester groups were employed in protecting the amino and carboxyl groups in amino acids i.e., Lys (Klieger and Gibian 1962;Schwyzer and Rittel 2004;Bezas and Zervas 1961), Glu (Bodanszky and Bodanszky 1982;Hirs and Timasheff 1979), Asp (Hirs and Timasheff 1979;Fujino andHatanaka 1967, Asn (Broadbent et al. 1967), Gly (Moroder et al. 1976) and Pro (Beyerman et al. 1968). The requisite time for each coupling was about 1 h. The assembled peptide sequence was cleaved from the solid support using 5% aq. sodium hydroxide in dioxane-methanol in 68% yield based on first lysine loading. The protected peptide obtained as yellowish syrup was thoroughly washed with organic solvents CH 2 Cl 2 , CHCl 3 , CHCl 3 -MeOH (5%), brine and dichloromethane and was deprotected in 5N HCl-dioxane with thioanisole and ethanedithiol (EDT) as scavengers and crude yield of the peptide was 87%. The octapeptide showed remarkable order of antifungal activity at micromolar dose levels in initial testing against Candida albicans.

Preparation of Boc-Lys
Boc-Lys Z (5.8 g) in dry CH 2 Cl 2 was mixed with Linker (6, 3.8 g) and DCC (3.1 g), DMAP (250 mg) under stirring at 0°C and stirring continued for 2 h at 0°C. A syrupy material was obtained and was devoid of CH 2 Cl 2 under vacuum and the solid residue was taken in EtOAc, water washed, dried over Na 2 SO 4 , EtOAc evaporated to give Boc-Lys Z -Linker (7, 7.1 g). To a solution of (7, 2.2 g) in MeOH (30 ml) was added acetic acid (2 ml) and a pinch of Zinc dust under stirring at RT After 1 hr. the mixture was filtered and syrup obtained was dissolved in EtOAc, washed with water and organic layers combined, dried over anhydrous Na 2 SO 4 and evaporated to give (8) as oil, 1.0 g.
Boc-Lys Z -O-(CH 2 ) 2 -SO 2 -CH 2 -CO 2 H (9) To the stirring solution of (8, 1 g) in MeOH was added oxone (0.60 g in 5 ml water) and stirring continued for 1 h at RT. Reaction mixture was extracted with EtOAc, H 2 O washed and organic layer concentrated in vacuum to yield (9, 1.2 g).

Sequence Loading and Quantitation
Picric acid method was used to quantify the loading of resin. The unbound amino endings were capped with Ac 2 O-DMAP. The resin was washed with CH 2 Cl 2 and DMF (20 ml 9 2) and quantified to 91% (712 mM/g) ready for coupling. The suitably protected amino acids were coupled to solid support in the following manner, (1) the cleavage of t-Boc group was carried out using 1N HCl in dioxane at 0°C, neutralized with DIEA and coupled using DCC-HOBt in DMF:CH 2 Cl 2 (1:1) at 0°C, (2) after each coupling and deprotection, the resin was washed with DMF:CH 2 Cl 2 (1:1, 15 ml 9 2, 5 min each), (3) prior to every deprotection, the coupling was monitored by picric acid method and allowed to proceed to completion generally within 2 h.
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