Efficient enzymatic synthesis of theaflavin and its production mechanism

Abstract In this study, a novel preparation method of theaflavin (TF) has been established. Our findings indicated that the formation of TF was significantly enhanced by using an ice bath (2–3°C). Additionally, increasing the ratio of (−)‐epigallocatechin (EGC) under the ice bath could further improve its yield. This approach prevented the appearance of a dark solution within 3 h, effectively protecting TF from oxidation. Our study on the generation mechanism of TF suggested that EGC‐quinone I (EGC‐Q‐I) with two carbanions could potentially serve as one of synthons based on the retrosynthetic analysis of the bicyclo[3.2.1]octane‐type intermediate. Subsequently, quantum mechanical calculations further supported this hypothesis. Practical Application : In this study, we have developed a novel method for the synthesis of theaflavin (TF), demonstrating that the use of ice bath significantly enhanced its yield. Increasing the ratio of (−)‐epigallocatechin (EGC) under the ice bath further improved TF yields and prevented darkening of the solution for at least 3 h, thereby protecting TF from oxidation. Our study suggested that EGC‐quinone I is a potential synthon based on the retrosynthetic analysis of the bicyclo[3.2.1]octane‐type intermediate (BOI). This hypothesis is supported by QM calculations.