Solid phase synthesis and hplc purification of the protected 1-12 sequence of apamin for rapid synthesis of apamin analogues differing in the c-terminal region

Abstract

Abstract Apamin is a bee neurotoxin, active in the central nervous system. It is an 18-peptide whose amino acid residues 13 and 14 play an essential role for binding to its receptor and for displaying toxicity. In order to accelerate the preparation of apamin analogues differing in the C-terminal region, a new strategy was set up involving solid phase synthesis of the 1-12 segment, which, after purification, can be solid phase coupled with different 13-18 sequences. The formula of the 12-membered protected peptide is: Boc-Cys(Acm)-Asn-Cys(Acm)-Lys(Z)-Ala-Pro-Glu(Bzl)-Thr(Bzl)-Ala-Leu- Cys(Acm)-Ala-OH. It has been assembled on the photosensitive resin, α-(4-bromomethyl-3-nitro-benzamido)benzylcopoly(styrene-1%divinyl- benzene) by conventional solid phase technique with Boc-amino acids. Boc-Leu was coupled by the method of Suzuki. Photolysis of batches of 1 gram of peptide-resin In trifluoroethanol/methylene chloride (20:80) yielded 89% of cleavage. The procedure for segment purification: organic extractions (ether, chloroform and precipitation from DMF with water), gel filtration on Sephadex LH-60, and finally semi-preparative HPLC on C18 in DMF/H2O (82:18) gave excellent results and an overall purification yield of 55%. After characterization, the purified 1-12 segment was coupled with three analogous 13-18 apamin sequences assembled on benzhydrylamine resins with yields of 77, 94, and 96%. After HF cleavage, deprotection and oxidation of the cysteines, the three peptides, apamin, p-aminophenylalanine-13- apamin, and p-aminophenylalanine-14-apamin were purified on carboxy-methylcellulose CM-52 and C18 HPLC. The purified peptides (yield 14-17%), after chemical characterization, were tested for toxic activity on mice and binding on synaptic membranes. The two analogues were about 100 times less toxic to mice than apamin and about 1000 times less potent in the binding assay.