Chiral methyl groups, stereochemical analysis using

A method for the determination of carbon-linked methyl groups (and, by extension, used in the analysis of chiral methyl groups in stereochemical studies of enzyme-catalyzed reactions acting on methyl groups). In this procedure, the methyl groups are converted to acetic acid by oxidation of the metabolite with chromic and sulfuric acids. Milder versions are also available. 

A. Stereochemical Analysis Using Chiral [ H, H, H)Methyl Groups. 356... 

Since these early studies, numerous additional procedures have been developed for the synthesis of chiral labeled acetic acid and other chiral labeled molecules by chemical (I41-15I) and enzymic methods 152-155). The malate synthase/fumarase system described above continues to be the most widely used method for the stereochemical analysis of chiral labeled acetic acid resulting from the degradation of product molecules containing chiral methyl groups. 

Considerable ingenuity was required in both the synthesis of these chiral compounds695 697 and the stereochemical analysis of the products formed from them by enzymes.698 700 In one experiment the phospho group was transferred from chiral phenyl phosphate to a diol acceptor using E. coli alkaline phosphatase as a catalyst (Eq. 12-36). In this reaction transfer of the phospho group occurred without inversion. The chirality of the product was determined as follows. It was cyclized by a nonenzymatic in-line displacement to give equimolar ratios of three isomeric cyclic diesters. These were methylated with diazomethane to a mixture of three pairs of diastereoisomers triesters. These dia-stereoisomers were separated and the chirality was determined by a sophisticated mass spectrometric analysis.692 A simpler analysis employs 31P NMR spectroscopy and is illustrated in Fig. 12-22. Since alkaline phosphatase is relatively nonspecific, most phosphate esters produced by the action of phosphotransferases can have their phospho groups transferred without inversion to 1,2-propanediol and the chirality can be determined by this method. 

The analysis of stereochemical problems in both chemistry and biochemistry has benefited greatly from the use of compounds that contain a methyl group with one atom each of H, and H. Such compounds exist as a pair of enantiomers, identified by R and S, and early work in this area will always be associated with the names of Arigoni and Cornforth. Recently a very efficient five-stage synthesis of chiral acetate has been reported (in which the penultimate reaction uses supertritide) with an enantiomeric purity of 100%. 

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