One-pot synthesis of 2,5-diketopiperazine with high titer and versatility using adenylation enzyme

2,5-Diketopiperazine (DKP) is a cyclic peptide composed of two amino acids and has been recently reported to exhibit various biological activities. DKPs have been synthesized using various methods. In chemical synthesis, a multi-step reaction requiring purification and racemization is problematic. Although enzymatic synthesis can overcome these problems, there has been no example of a general-purpose synthesis of DKPs with high titers. Therefore, we propose a chemoenzymatic method that can synthesize DKPs in a general-purpose manner with high efficiency under mild conditions. The adenylation domain of tyrocidine synthetase A (TycA-A) catalyzes the adenylation reaction of amino acids, and various amides can be synthesized by a nucleophilic substitution reaction with any amine. On the other hand, DKPs can be produced via intramolecular cyclization reactions from dipeptide esters. Based on these observations, we expected a one-pot synthesis of DKPs via dipeptide ester synthesis by TycA-A and cyclization reactions. This method enabled the synthesis of more than 128 types of DKPs without racemization. Importantly, the intramolecular cyclization reaction proceeded largely depending on the pH. In particular, the cyclization reaction proceeded well in the pH range of 6.5-9.5. Based on these results, we constructed a bioreactor with pH-stat for purified enzyme reaction; cyclo(L-Trp-L-Pro) was produced at 4.07 mM by controlling the reaction pH over time using this reactor. The DKPs obtained using this method will provide deeper insights into their structures and functions in future studies. KEY POINTS: • Adenylation enzyme enabled one-pot synthesis of arbitrary 2,5-diketopiperazine. • Little or no racemization occurred during 2,5-diketopiperazine synthesis. • Bioreactor with pH-stat for purified enzymes improved the reaction rate.