Lipoconjugates: structure-activity studies for pheromone analogues of Ustilago maydis with varied lipophilicity

The synthesis, biological activities and conformational behaviour of a variety of analogues of the mating pheromones of the basidomycete Ustilago maydis are reported. The pheromone analogues derived from the two allelic forms H-G-R-D-N-G-S-P-I-G-Y-S-S-Xaa-Z (a1) and H-N-R-G-Q-P-G-Y-Y-Xaa-Z (a2), with Xaa-Z being an unidentified lipophilic cysteine derivative, all differ in the C-terminal residue and include -Cys(farnesyl)-OMe, -Cys(farnesyl)-OH, -Cys(prenyl)-OMe, -Cys-OMe, -Cys(n-dodecyl)-OMe and the unnatural residues -Ahds-OMe (Ahds=alpha-aminohexadecanoic acid), -Ahds-OH, -Ads-OMe (Ads=alpha-aminodecanoic acid) and -N-Hdg-OMe (N-Hdg=N-hexadecylglycine). The synthesis of the unnatural methyl ester analogues was carried out by condensation of the fully protected fragments Fmoc-G-R(Pmc)-D(tBu)-N(Trt)-G-S(tBu)-P-I-G-Y(tBu)-S(tBu)-S(tBu)-OH (a1') and Fmoc-N(Trt)-R(Pmc)-G-Q(Trt)-P-G-Y(tBu)-Y(tBu)-OH (a2') respectively, prepared by Fmoc-SPPS, with the appropriate methylester compounds and subsequent deprotection with TFA/scavenger and piperidine. Synthesis and physicochemical properties of the unnatural lipophilic amino acid methylesters are described. The preparation of the cysteine analogues was performed by condensation of a1' or a2' with H-Cys(Trt)-OMe and subsequent deprotection with TFA/scavenger. Alkylation of the thiol function and Fmoc-deprotection was achieved in a novel one-pot reaction by treatment with alkyl bromide and DIPEA, quenching with EDT and Fmoc removal by addition of 20% piperidine (v/v). Hydrolysis of the methyl esters was carried out by treatment with NaOH in MeOH/H2O. The results of the biological assay reveal an increase in activity with increasing chain length of the lipophilic anchor, with alkyl being better than prenyl and sulfur being not essential, while the position of the anchor is optimal at C7 and the methyl ester moiety is important. NMR studies of two chosen analogues in DMSO and SDS/water demonstrate that the lipophilic C-terminal residue has no influence on the structural behaviour of the peptides. Chemical-shift and NOE patterns indicate a main all-trans conformation of the peptide backbone and a weakly populated cis conformation around the Xaa Pro peptide bond in all eight cases without formation of a defined folded structure. No evidence is seen that the membrane-simulating system SDS/water has a structure-inducing effect on the bound peptide. We therefore conclude that the lipomodification in mating pheromones of U. maydis acts to increase the effective concentration of the drug in the target cell membrane without additional structure-inducing or receptor-binding effects.