Systematic analysis of the Saccharomyces cerevisiae alpha-factor containing lactam constraints of different ring size

Eight cyclic analogs and corresponding linear homologs of the alpha-factor mating pheromone (WHWLQLKPGQPMY) of Saccharomyces cerevisiae were synthesized using solid-phase procedures on a phenylacetamidomethyl support. On-resin lactamization of the side chains of residues 7 and 10 to form rings containing from 14 to 18 atoms was effected by the BOP reagent. All peptides were highly homogeneous and gave expected molecular ions by FAB mass spectrometry. The constrained analogs had biological activities varying from 10% to less than 0.1% of that of [Nle12]-alpha-factor. In all cases, cyclic analogs with Glu in position 10 were more active than the homolog with Asp at this position. This trend was also found with the corresponding linear pheromones, suggesting that a gamma-carbonyl in position 10 is an important determinant of pheromone potency. The cyclic peptides had from 50- to 20000-fold lower affinities for the alpha-factor receptor than for [Nle12]-alpha-factor, as judged using a competition binding assay. Circular dichroism studies indicate that the cyclic lactam-containing region of cyclo7.10[Orn7, Glu10,Nle12]-alpha-factor retains a beta-turn-like structure similar to that found in the corresponding model tetrapeptide. The results show that covalently constrained analogs of the linear pheromone can maintain biological activity, despite binding poorly to the receptor, and indicate that a beta-turn-like structure in the center of the pheromone allows signal transduction.