Cyclo(-D-Ala-D-Ala)

2,5-Diketopiperazines (DKPs) are widely recognized as chiral molecules with great potential in medicinal chemistry. Complete separation of DKP stereoisomers is very important for efficiently investigating the chemical characteristics of DKPs. The combination of esterification and acylation caused the enantiomers of cyclo(d-Ala-d-Ala) and cyclo(l-Ala-l-Ala) to ring-open and generate their dipeptide derivatives. These derivatives were completely separated by chiral gas chromatography (GC), and the determined isomer ratios were the same as the original isomer ratios. The derivatization also caused the ring-opening of cyclo(l-Asp-l-Phe) and cyclo(l-Met-l-Pro), and their derivatives were determined by the chiral GC method. The present study is the first report of the complete separation of cis-DKPs using chiral GC. This separation procedure can substantially contribute to the development of the chemistry of chiral DKPs.

Diketopiperazines (DKPs) are cyclic dipeptides, representing an abundant class of biologically active natural compounds. Despite their widespread occurrence in nature, little is known about their degradation. In this study, the enzymatical and microbial cleavage of DKPs was investigated. Peptidase catalyzed hydrolysis of certain DKPs was formerly reported, but could not be confirmed in this study. While testing additional peptidases and DKPs no degradation was detected, indicating peptidase stability of the peptide bond in cyclic dipeptides. Besides confirmation of the reported degradation of cyclo(l-Asp-l-Phe) by Paenibacillus chibensis (DSM 329) and Streptomyces flavovirens (DSM 40062), cleavage of cyclo(l-Asp-l-Asp) by DSM 329 was detected. Other DKPs were not hydrolyzed by both strains, demonstrating high substrate specificity. The degradation of cyclo(l-Asp-l-Phe) by DSM 40062 was shown to be inducible. Three strains, which are able to hydrolyze hydantoins and dihydropyrimidines, were identified for the degradation of DKPs: Leifsonia sp. K3 (DSM 27212) and Bacillus sp. A16 (DSM 25052) cleaved cyclo(dl-Ala-dl-Ala) and cyclo(l-Gly-l-Phe), and Rhizobium sp. NA04-01 (DSM 24917) degraded cyclo(l-Asp-l-Phe), cyclo(l-Gly-l-Phe) and cyclo(l-Asp-l-Asp). The first enantioselective cleavage of cyclo(dl-Ala-dl-Ala) was detected with the newly isolated strains Paenibacillus sp. 32A (DSM 27214) and Microbacterium sp. 40A (DSM 27211). Cyclo(l-Ala-d-Ala) and cyclo(l-Ala-l-Ala) were completely degraded, whereas the enantiomer cyclo(d-Ala-d-Ala) was not attacked. Altogether, five bacterial strains were newly identified for the cleavage of DKPs. These bacteria may be of value for industrial purposes, such as degradation of undesirable DKPs in food and drugs and production of (enantiopure) dipeptides and amino acids.