Synthesis of S-alkyl and C-terminal analogs of the Saccharomyces cerevisiae a-factor. Influence of temperature on the stability of Fmoc and OFm groups toward HF

The a-mating factor of Saccharomyces cerevisiae Tyr-Ile-Ile-Lys-Gly-Val-Phe-Trp-Asp-Pro-Ala-Cys(farnesyl)OCH3, and 10 analogs modified at the cysteine side chain and/or the terminal carboxyl were synthesized using a combination of solid phase and solution phase methodologies. The strategy of synthesis involved the condensation of an amine terminal protected decapeptide with a carboxyl terminal S-alkylated dipeptide ester or amide using benzotriazol-l-yloxy-tris(methylamino)-phosphonium hexafluorophosphate as the coupling agent. The protected decapeptide was assembled on a PAM-resin using 9-fluorenylmethoxycarbonyl (Fmoc) for the protection of the Tyr alpha-amine and Lys epsilon-amine and 9-fluorenylmethyl ester (OFm) for the protection of the Asp beta-carboxyl. Premature loss of the OFm group from the HF cleavage was observed at 0-2 degrees, whereas no loss occurred when the cleavage reaction was conducted at -5 degrees. In contrast to these results, the OFm group in Asp(OFm) was partially removed by HF at -5 degrees and was completely stable to HF only at -20 degrees. The S-alkylated dipeptide esters were prepared, in yields from 64% to 88%, via thioalkylation of amine protected or unprotected dipeptide esters using potassium fluoride dihydrate as the base. The use of a tertiary amine as the base of thiohexadecanylation resulted in low reactivity.