Benzothiazines as Major Intermediates in Enzymatic Browning Reactions of Catechin and Cysteine

The course of melanin formation is yet not thoroughly resolved on a mechanistic level. With the present study, incubations of catechin (CA)- and cysteine-derived dihydro-1,4-benzothiazine carboxylic acid derivatives were investigated for colored products during enzymatic browning. Analyses by high-performance liquid chromatography (HPLC)-mass spectrometry revealed the formation of two novel decarboxylated dihydro-1,4-benzothiazine derivatives [8-(3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl)-5-hydroxy-3,4-dihydro-2H-benzothiazine and 7-(3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl)-5-hydroxy-3,4-dihydro-2H-benzothiazine] preferentially under acidic conditions. Furthermore, in model reactions under neutral pH, a colored phenazine dimer intermediate was isolated by high-performance countercurrent chromatography and preparative HPLC when conducting the incubations in the presence of o-phenylenediamine (OPD). Mass spectrometry and nuclear magnetic resonance spectroscopy unequivocally verified the structure as (12E)-5,5'- dioxo-11a,11a'- bis(3,5,7-trihydroxy-3,4-dihydro-2H-chromen-2-yl)-3,3',4,4', 5a,5a',6,6',11,11', 11a,11a'-dodecahydro-2H,2' H,5H,5' H-12,12'-bi[1,4]thiazino[2,3-b]phenazine-3,3'-dicarboxylic acid. Enzymatically catalyzed incubations under aeration starting from the initial CA-cysteine adducts and their follow-up dihydro-1,4-benzothiazine carboxylic acids, respectively, proved that the unstable colored compound was a trichochrome-like reaction intermediate of the browning reaction cascade which can be trapped by postincubation with OPD, thus verifying their direct mechanistic relationship.

Automated summary

The mechanism of melanin formation is still not completely understood. This study investigated the formation of novel decarboxylated dihydro-1,4-benzothiazine derivatives under acidic conditions and isolated a colored phenazine dimer intermediate under neutral pH. The research confirmed the direct mechanistic relationship of these compounds in the browning reaction cascade.