Extracts of suspension-cultured Zea mays (cv. Black Mexican Sweet) cells treated with [14C]benoxacor for 0.25−24 h were analyzed by HPLC and TLC to investigate the metabolic fate of benoxacor. Thin layer chromatography determined that benoxacor was rapidly metabolized to six detectable metabolites within 0.5 h. Twelve metabolites were detected in extracts from cells treated for 24 h. Analysis of cell extracts by reversed phase HPLC determined that the glutathione conjugate [mono(GSH)] of benoxacor was present in all samples analyzed, based on cochromatography with a mono(GSH) conjugate standard. The abundance of the mono(GSH) conjugate increased as treatment time increased. The presence of a di(GSH) conjugate was detected in extracts of cells treated for 0.5 h and reached a maximum level 2 h after treatment. Three predominant metabolites present in samples treated with benoxacor for 1 h were subjected to structural analysis by 1H-NMR or mass spectrometry following purification by conventional HPLC methodologies. These structural analyses determined that two of the metabolites were the catabolic formylcarboxamide and carboxycarboxamide derivatives of benoxacor. A third metabolite was determined to be the mono(GSH) conjugate of benoxacor. This metabolite consisted of a single glutathione molecule linked via the cysteinyl sulfhydryl group to the N-dichloroacetyl α-carbon of benoxacor. Structures of the metabolites and postulated pathways of their biosynthesis in vivo are presented. Keywords: Benoxacor; herbicide safener; CGA-154281; glutathione S-transferase; BMS cells; xenobiotic metabolism; xenobiotic detoxification; maize; dehalogenase/dechlorinase enzymes; glutathione conjugation; dichloroacetamide; substituted benzoxazine; herbicide safener metabolism

The metabolic fate of the herbicide safener benoxacor was studied in suspension cultures of Zea mays (cv. Black Mexican Sweet) 3 and 24 h after treatment (HAT) with [14C]benoxacor. Following purification by reversed phase HPLC, metabolites were subjected to 1H-NMR or mass spectrometry for structural analysis. A catabolic α-hydroxyacetamide derivative was detected as well as several amino acid conjugates either containing GSH or presumably derived from GSH. A relatively abundant di(GSH) conjugate, which consisted of two GSH molecules linked to benoxacor, was biosynthesized following the apparent addition of a suitable activating group to the benzoxazine-ring C2 atom of the previously described mono(GSH) conjugate. The remaining metabolites identified were all presumably derived from either the mono(GSH) or di(GSH) conjugates of benoxacor. A disaccharide conjugate was identified as S-O-(diglycoside)GSH and was apparently formed by one or more glycosyl transferases which used the mono(GSH) conjugate as a substrate. Two additional metabolites identified were the mono- and di(γ-GluCys) conjugates of benoxacor, the formation of which was most likely the result of peptidase activity on the mono- and di(GSH) conjugates of benoxacor, respectively. An S-(S‘-Cys)GSH conjugate was also identified and was likely formed by both peptidase and dipeptidase activities on the di(GSH) conjugate. Metabolite structures and postulated pathways of their biosynthesis in vivo are presented. Keywords: Benoxacor; herbicide safener; CGA-154281; glutathione S-transferase; BMS cells; xenobiotic metabolism; xenobiotic detoxification; maize; glutathione conjugation; glutathione conjugate metabolism; dichloroacetamide, substituted benzoxazine; herbicide safener metabolism

The stability constants were determined by potentiometric titrations in aqueous solutions (7 = 0.1; T = 25°) for the binary 1:1 complexes between Mn(II), Co(II), Zn(II), or Cu(II) and ¿/-biocytin (e-TV-biotinyl-L-lysine), its sulfoxide and sulfone, A'-acetyl-L-lysine, or L-alanine. Logs of the stability constants, log with biocytin are

The syntheses of flavinyl peptides, in which L‐tryptophan, L‐tyrosine, or L‐phenylalanine are attached via peptide linkage to the isoalloxazine system with ω‐carboxyalkyl groups in position 3 or 10, are described. Lumiflavin was carboxymethylated by known methods to yield N‐3‐carboxymethyllumiflavin. Oxidation of 10‐ω‐hydroxyhexyl‐, 10‐ω‐hydroxypentyl‐, 10‐ω‐hydroxybutyl‐, and 10‐formylmethylflavins gave the corresponding 10‐ω‐carboxyalkylflavins. The 10‐ω‐carboxyethylflavin was obtained by condensation of 2‐amino‐4,5‐dimethyl‐N‐ω‐carboxyethylaniline with alloxan. Activations of the carboxyl group of the flavins were achieved with N,N′‐carbonyldiimidazole and p‐nitrophenyltrifluoroacetate to form the corresponding acyl imidazoles and p‐nitrophenyl esters. 10‐Carboxymethylflavin was hydrogenated to form 10‐carboxymethyldihydroflavin and activated by carbodiimide. Reaction of the carboxy‐activated flavins with the appropriate amino acid methyl esters, followed by air oxidation in the case of dihydroflavin, gave the corresponding flavinyl peptides. Interaction of the flavin with aromatic amino acids results in a broadening of the visible flavin absorption towards the green, without the appearance of discrete new maxima, and in quenching of the flavin fluorescence. The fluorescence efficiency increases with increasing numbers of methylene groups in the flavin side chain. The nonlinear dependency of fluorescence quenching versus number of methylene groups indicates that different types of intramolecular interactions are involved.

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