Protease-Catalyzed Rational Synthesis of Uric Acid-Lowering Peptides in Non-aqueous Medium

Gout with etiological hyperuricemia deteriorates patient's quality of life, making an urgent need for effective uric acid-lowering drugs. Uric acid-lowering peptides derived from naturally-occurring proteins have great potential therapeutic applications and are found to be rich in aromatic amino acids (AAAs) and branched-chain amino acids (BCAAs). This study proposed a new method to rationally synthesize uric-acid lowering peptides from amino acids mainly composed of AAAs and BCAAs by papain in non-aqueous medium. Firstly, L-tryptophan and L-phenylalanine were screened out for the synthesis of uric acid-lowering peptides from all the 49 possible pairwise combinations of 7 amino acids dominating in reported natural uric-acid lowering peptides, which are 3 AAAs (L-tryptophan, L-phenylalanine, L-tyrosine), 2 BCAAs (L-leucine and L-alanine), 1 L-proline and 1 L-histidine. Secondly, the optimal reaction conditions of 90 mmol/L triethylamine, reaction at pH 6.0 for 1.5 h for the synthesis of uric-acid lowering peptides were determined using single factor method and response surface experiments (RSE). By means of preparative HPLC and LC–MS/MS identification, a newly synthesized tripeptide (FWF) was obtained, which inhibited xanthine oxidase activity by 18.47%. The tripeptide FWF was further confirmed by the chemical synthesis and its underlying XOD inhibition was supported by the computational molecular docking experiments. The newly identified uric acid-lowering tripeptide FWF and its protease-catalyzed rational synthesis in non-aqueous medium method provide alternative choices to developing uric acid-lowering peptides for anti-gout or anti-hyperuricemia treatments.