LIPOCONJUGATES - STRUCTURE-ACTIVITY STUDIES FOR PHEROMONE ANALOGS 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 Ust ilngo maydis are reported. The pheromone analogues derived from the tw o 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(farn esyl)-OMe, -Cys(farnesyl)-OH, -Cys(prenyl)-OMe, -Cys-OMe, -Cys(n-dodec yl)-OMe and the unnatural residues -Ahds-OMe (Ahds = alpha-aminohexade canoic acid), -Ahds-OH, -Ads-OMe (Ads = alpha-aminodecanoic acid) and -N-Hdg-OMe (N-Hdg = N-hexadecylglycine), The synthesis of the unnatura l methyl ester analogues was carried out by condensation of the fully protected fragments Fmoc-G-R 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 methyle ster compounds and subsequent deprotection with TFA/scavenger and pipe ridine. Synthesis and physicochemical properties of the unnatural lipo philic amino acid methylesters are described. The preparation of the c ysteine analogues was performed by condensation of a1' or a2' with H-C ys(Trt)-OMe and subsequent deprotection with TFA/scavenger. Alkylation of the thiol function and Fmoc-deprotection was achieved in a novel o ne-pot reaction by treatment with alkyl bromide and DIPEA, quenching w ith EDT and Fmoc removal by addition of 20% piperidine (v/v). Hydrolys is of the methyl esters was carried out by treatment with NaOH in MeOH /H2O. The results of the biological assay reveal an increase in activi ty with increasing chain length of the lipophilic anchor, with alkyl b ring better than prenyl and sulfur being not essential, while the posi tion of the anchor is optimal at C-alpha and the methyl ester moiety i s important. NMR studies of two chosen analogues in DMSO and SDS/water demonstrate that the lipophilic C-terminal residue has no influence o n the structural behaviour of the peptides. Chemical-shift and NOE pat terns indicate a main alkyl ans conformation of the peptide backbone a nd a weakly populated cis conformation around the Xaa-Pro peptide bond in all eight cases without formation of a defined folded structure. N o 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 i ncrease the effective concentration of the drug in the target cell mem brane without additional structure-inducing or receptor-binding effect s. (C) Munksgaard 1996.