Synthesis of d-amino acid-containing dipeptides using the adenylation domains of nonribosomal peptide synthetase

Recent papers have reported dipeptides containing d-amino acid to have novel effects, which cannot be observed with ll-dipeptides and are expected to be novel functional compounds for pharmaceuticals and food additives. Although the function of d-amino acid-containing dipeptides is gaining more attention, there are few reports on the synthetic enzymes that can accept d-amino acid as a substrate, and synthetic methods for d-amino acid-containing dipeptides are still not constructed. Previously, we developed a chemoenzymatic system for amide synthesis, which comprised enzymatic activation and a subsequent nucleophilic substitution reaction. In this study, we demonstrated the application of the system for d-amino acid-containing dipeptide synthesis. We chose six adenylation domains as targets according to our newly constructed hypothesis: an adenylation domain located upstream of the epimerization domain may activate d-amino acid, as well as l-amino acid. We successfully synthesized over 40 kinds of d-amino acid-containing dipeptides, including ld-, dl-, dd-dipeptides using only two adenylation domains, TycA-A from tyrocidine synthetase and BacB2-A from bacitracin synthetase. Furthermore, this study offered the possibility that the epimerization domain could be a clue to the activity of the adenylation domains toward d-amino acid. This paper provided additional information regarding d-amino acid-containing dipeptide synthesis through the combination of enzymatic adenylation and chemical nucleophilic reaction, and this system will be a useful tool for dipeptide synthesis. Importance Because almost all amino acids in nature are l-amino acids, the function of d-amino acids has received little attention. Thus, there is little information available on the activity of the enzymes toward d-amino acids and synthetic methods for d-amino acid-containing dipeptides. Recently, d-amino acids and d-amino acid-containing peptides have attracted attention as novel functional compounds, and d-amino acid-activating enzymes and synthetic methods are required for the development of the d-amino acid-containing peptide industry. This study provides additional knowledge regarding d-amino acid-activating enzymes and proposes a unique synthetic method for d-amino acid-containing peptides, including ld-, dl-, and dd-dipeptides.