The enzymatic synthesis of theaflavin-3-gallate oxidation product and its determination

In this work, the oxidation of theaflavin-3-gallate (TF-3-G) was investigated using (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG) as substrates in a one-pot reaction. The resulting TF-3-G oxidation product was acquired by employing acetonitrile/water and ethanol/water as eluents, respectively, which was identified as theanaphthoquinone-3′-gallate (TNQ-3′-G). Surprisingly, we discovered that TNQ-3′-G could react with certain protic solvents to form new and unstable complexes through intermolecular hydrogen bond. This reactivity was also confirmed by the presence of irregular peaks in reverse-phase high-performance liquid chromatography (RP-HPLC) besides spectroscopic data. Therefore, we inferred that the number of carboxyl groups may increase through the successive oxidative polymerization of the TFs oxidation products. The high-molecular polymer could also interact with biomacromolecules in a similar manner to their interaction with protic solvents. This interaction might be one of the main factors contributing to the broad hump of thearubigins (TRs) on the RP-HPLC baseline. Additionally, these findings lay a solid foundation for interpreting the structures of TRs and understanding their generation mechanism. Theanaphthoquinone-3′-gallate (TNQ-3′-G) was synthesized via a one-pot reaction using (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG) as starting substrates. A darkening reaction occurs during the formation of TNQ-3′-G, likely due to the presence of an unstable dianion of TF-3-G. TNQ-3′-G, featuring two conjugated carbonyl groups, can form unstable complexes with specific protic solvents such as ethanol, water, acetic acid, and methanol. This discovery provides direct evidence that enhances our understanding of how high-molecular-weight black tea polyphenols interact with biomacromolecules through a similar mechanism. [Display omitted] •Theanaphthoquinone-3′-gallate (TNQ-3′-G) was successfully synthesized through a one-pot reaction using (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG) as substrates.•An unstable dianion of TF-3-G might be responsible for the darkening reaction.•TNQ-3′-G with two conjugated carbonyl group has the ability to form new unstable complexes with certain protic solvents through hydrogen bonding.•The generation of these complexes provides direct evidence that contributes to a better understanding of the connection between black tea polyphenol and biomacromolecules.