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

Abstract

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.