Abstract: To elucidate the mechanisms by which catechin structure regulates theaflavin (TF) synthesis, this study utilized eight catechin monomers to construct high-trans and high-cis substrate systems according to the differences in catechin profiles between Camellia ptilophylla from Nankun Mountain and Camellia sinensis (large-leaf tea) from Yunnan. Polyphenol oxidase (PPO) was used to catalyze the enzymatic synthesis of TFs. The reaction kinetics, substrate consumption, and product formation patterns were analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The impact of substrate structure on TF synthesis was investigated using equimolar pairwise mixtures of catechin monomers. The results indicated that high-cis systems oxidized significantly faster than high-trans systems, with cis-catechins exhibiting higher consumption rates than their trans-counterparts. Furthermore, the consumption rates of catechol-type and pyrogallol-type catechins were heavily modulated by their specific stereo-conformations. A total of six known TFs and six novel isomers were generated from the two substrate systems, and their yields were determined by the relative proportions and conformational features of the substrates. Specifically, the yield of TFs was significantly higher when both substrates or the catechol-type catechin participating in TF synthesis were in the cis-conformation. These findings systematically reveal the impact of catechin structure, particularly its stereo-conformations, on TF synthesis, providing a scientific basis for understanding TF formation and exploring novel TF components in C. ptilophylla.

Key words: black tea; Camellia ptilophylla; theaflavins; catechins; conformation