ABSTRACT
2,5-diketopiperazines (DKPs) are cyclic dipeptides ubiquitously found in nature. In particular, cyclo(Phe-Pro), cyclo(Leu-Pro), and cyclo(Val-Pro) are frequently detected in many microbial cultures. Each of these DKPs has four possible stereoisomers due to the presence of two chirality centers. However, absolute configurations of natural DKPs are often ambiguous due to the lack of a simple, sensitive, and reproducible method for stereochemical assignment. This is an important problem because stereochemistry is a key determinant of biological activity. Here, we report a synthetic DKP library containing all stereoisomers of cyclo(Phe-Pro), cyclo(Leu-Pro), and cyclo(Val-Pro). The library was subjected to spectroscopic characterization using mass spectrometry, NMR, and electronic circular dichroism (ECD). It turned out that ECD can clearly differentiate DKP stereoisomers. Thus, our ECD dataset can serve as a reference for unambiguous stereochemical assignment of cyclo(Phe-Pro), cyclo(Leu-Pro), and cyclo(Val-Pro) samples from natural sources. The DKP library was also subjected to a biological screening using assays for E. coli growth and biofilm formation, which revealed distinct biological effects of cyclo(D-Phe-L-Pro).
Subject(s)
Dipeptides/chemistry , Peptides, Cyclic/chemistry , Circular Dichroism , Diketopiperazines/chemistry , Dipeptides/chemical synthesis , Dipeptides/pharmacology , Escherichia coli/drug effects , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/pharmacology , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Microbes use signaling factors for intraspecies and interspecies communications. While many intraspecies signaling factors have been found and characterized, discovery of factors for interspecies communication is lagging behind. To facilitate the discovery of such factors, we explored the potential of a mixed microbial culture (MMC) derived from wheatgrass, in which heterogeneity of this microbial community might elicit signaling factors for interspecies communication. The stability of Wheatgrass MMC in terms of community structure and metabolic output was first characterized by 16S ribosomal RNA amplicon sequencing and liquid chromatography/mass spectrometry (LC/MS), respectively. In addition, detailed MS analyses led to the identification of 12-hydroxystearic acid (12-HSA) as one of the major metabolites produced by Wheatgrass MMC. Stereochemical analysis revealed that Wheatgrass MMC produces mostly the (R)-isomer, although a small amount of the (S)-isomer was also observed. Furthermore, 12-HSA was found to modulate planktonic growth and biofilm formation of various marine bacterial strains. The current study suggests that naturally derived MMCs could serve as a simple and reproducible platform to discover potential signaling factors for interspecies communication. In addition, the study indicates that hydroxylated long-chain fatty acids, such as 12-HSA, may constitute a new class of interspecies signaling factors.
