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.

Abiotic transformation products of tetracycline and chlortetracycline in salt solutions and manure.

Tetracyclines belong to the group of the most applied antibiotics in veterinary medicine worldwide. Due to their incomplete absorption and/or metabolism in the animal gut, tetracyclines are frequently detected in manure samples. Within the matrix, an elimination of these compounds has been reported in several studies. However, only little information about potential transformation products of tetracyclines in manure and the environment is available. Therefore, the fate of tetracycline (TC) and chlortetracycline (CTC) was investigated in aqueous solutions and manure. Abiotic incubation of TC in phosphate buffer led to a remarkable red-brown coloring of the solution. Subsequent compound isolation and structure elucidation by MS/MS and NMR techniques revealed the formation of seco-cycline A, a compound formerly described as a fungal biotransformation product of TC. For CTC, two comparable products were identified which were derived from its isomeric form isoCTC. All transformation products showed no antimicrobial activity for concentrations up to 500 mg L-1. When TC and CTC were incubated in cow manure for 7 d, the above mentioned three transformation products were also formed in this complex matrix (up to 5.1 mg kg-1). Manure, soil and leachate samples from Lower Saxony revealed the presence of seco-cycline A in manure and soil, but not in water. To obtain a better insight in the fate of tetracyclines in environmental matrices, future analytical and ecotoxicological studies dealing with this subject should include the analysis especially of seco-cycline A.