Chemical synthesis of ComX pheromone and related peptides containing isoprenoidal tryptophan residues

Abstract The ComX pheromone is a post-translationally modified oligopeptide that stimulates natural genetic competence controlled by quorum sensing in Bacillus subtilis . Recently, the structure of the ComX RO-E-2 pheromone produced by strain RO-E-2 was determined. Based on the NMR analysis, a geranyl group is bound to the tryptophan residue, which results in the formation of a tricyclic ring structure. It was proposed that one of the four possible stereochemical isomers was based on a conformational search for model compounds and the assumption that amino acid residues in the natural pheromone have the l -configuration. All possible modified tryptophan residues and the corresponding ComX RO-E-2 peptides were synthesized to confirm the precise stereochemistry. Here, the synthesis of the modified tryptophan derivatives was reported in detail. It was succeeded in synthesizing four optically active modified tryptophan methyl esters from which the four diastereomeric ComX RO-E-2 peptides were prepared. Since only one of the four diastereomers was spectroscopically identical to the natural pheromone and exhibited biological activity, the absolute structure of the ComX RO-E-2 pheromone was able to be established unambiguously. Furthermore, it was noticed that two other bioactive pheromones were present in the culture broth that were co-purified with ComX RO-E-2 pheromone. These pheromones were presumed to be the N-terminal truncated peptides of ComX RO-E-2 pheromone, i.e., [2-6]ComX RO-E-2 and [3-6]ComX RO-E-2 , by LC-MS and NMR analyses. Using Fmoc solid-phase peptide synthesis, ComX RO-E-2 pheromone and the [2-6]ComX RO-E-2 and [3-6]ComX RO-E-2 peptides were prepared. The synthetic peptides were identical to the natural pheromones and also showed significant biological activity.