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

Abstract

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 =α‐aminohexadecanoic acid), ‐Ahds‐OH, ‐Ads‐OMe (Ads =α‐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 Cα 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. © Munksgaard 1996.

Thea2Mating-Type Locus Geneslga2andrga2Direct Uniparental Mitochondrial DNA (mtDNA) Inheritance and Constrain mtDNA Recombination During Sexual Development ofUstilago maydis

“…Early Golden Bantam; Olds Seed, Madison, WI) was done as described (Holliday 1974;Banuett and Herskowitz 1989;Basse 2005). For pheromone stimulation, synthetic Mfa2 pheromone (Koppitz et al 1996; kindly provided by M. Feldbrügge, Max-Planck-Institute, Marburg, Germany) was added at a final concentration of 2.5 mg/ml to a logarithmically growing culture transferred to fresh CM and then incubation proceeded on a vertical rotary platform (10 rpm) at 28°for 6 hr. The antibiotics hygromycin B (hyg), carboxin (cbx), and nourseothricin (nat) were purchased from Roche (Mannheim, Germany), Riedel-de Haën (Hannover, Germany), and Werner BioAgents ( Jena-Cospeda, Germany), respectively.…”

Biogenesis of the Saccharomyces cerevisiae Pheromone a -Factor, from Yeast Mating to Human Disease

“…Notably, S. commune pheromone-receptor gene pairs have been expressed in S. cerevisiae and can substitute for the endogenous S. cerevisiae receptors and pheromones (92). Genetic and biochemical evidence supports the importance of prenylation for several Basidiomycete pheromones (145), and a few of these pheromones have been purified and/or synthesized to demonstrate their activity (6,144,197).…”

“…For R. toruloides, the precursor gene encodes multiple copies of the pheromone, and an initial processing step is required to reveal C-terminal CAAX motifs for each (3,63). Other Basidiomycetes for which pheromones are known are Usitlago maydis (32,144,244,252), Usitlago hordei (6,145), Cryptococcus neoformans (72,177,189,243), Schizophyllum commune (92,267), Coprinus cinereus (38,52,197), and Sporisorium reilianium (235). For S. reilianium, at least 8 pheromone precursor genes have been identified, ranging in size from 10 to 74 residues, all encoding CAAX motifs.…”

Thea2mating-type-locus genelga2ofUstilago maydisinterferes with mitochondrial dynamics and fusion, partially in dependence on a Dnm1-like fission component

“…CM contains 1% (w/v) glucose and CM/Ara contains 1% (w/v) arabinose instead of glucose. For pheromone stimulation, synthetic Mfa1 pheromone (kindly provided by Michael Feldbrügge, MPI Marburg, Germany) was added to a logarithmically growing culture at a final concentration of 2.5 μg/ml (Koppitz et al, 1996) for 4 hours of incubation in CM.…”