I.~Probing the mode of action of glutathione S-transferase with the use of carbon-13-NMR spectroscopy. II.~The absolute configuration of (+)- and (-)-MTPA acid: Non-empirical confirmation.

Abstract

A general probe has been developed to elucidate the chemical mode of action of glutathione $S$-transferase enzyme in glutathione conjugation of arene oxides. The combined use of $\sp{13}$C-labeled arene oxide and state-of-the-art NMR spectroscopic techniques has successfully led to the elucidation of the glutathione adducts in the enzymatic glutathione conjugation of naphthalene 1,2-oxide. Products obtained from the incubation of (2-$\sp{13}$C) naphthalene 1,2-oxide and glutathione with the mixed isozymes of glutathione $S$-transferase from rat or equine liver, in vitro, were a mixture of glutathione adducts which arose from the conjugation of the glutathionyl sulfur atom at either the benzylic or allylic carbon of the substrate. Similar results were obtained with the pure isozymes 3-3 and 4-4 of glutathione $S$-transferase, in which case we were also able to observe the regio-, stereo-, and enantioselectivity of the isozymes toward both the racemic and optically pure naphthalene 1,2-oxide. In the course of the above study, we have achieved the first synthesis of (2-$\sp{13}$C) naphthalene 1,2-oxide by employing an efficient $\sp{13}$C-labeling methodology. The salient feature of this synthesis lies in the formation of a carbon framework by use of a two-carbon-unit building block, (2-$\sp{13}$C) acetic acid, which serves as a $\sp{13}$C-labeling agent. Our synthetic effort has also been extended to a preliminary study towards the synthesis of (3-$\sp{13}$C) - and (4-$\sp{13}$C) cyclopental ($cd$) pyrene 3,4-oxide. Furthermore, the absolute configuration of the widely used chiral derivatizing agent (+)- and ($-$)-$\alpha$-methoxy-$\alpha$-(trifluoromethyl)phenylacetic (MTPA) acid has been unambiguously determined through the use of two independent, non-empirical methods as R and S, respectively. The first method of absolute configurational determination utilizes the CD exciton chirality method and the second utilizes a chemical correlation to a configurationally well-established compound. In both cases, X-ray crystallographic analysis of an MTPA ester is applied to correlate the relative stereostructure of the MTPA moiety with respect to configurationally determined stereocenters. This configurational proof taken together with previous empirical correlations leave no reasonable doubt concerning the absolute configuration of MTPA acid.