Synthesis and applications of stereospecifically labeled compounds: Stereochemical course of the imidazole glycerol phosphate dehydratase reaction
Abstract
Stereospecifically labeled compounds play an important role in the study of biochemical transformations. Chirally-labeled oxirane is a synthon for the synthesis of such intermediates. Several approaches to this compound have been investigated with the common feature of these syntheses being the generation of oxirane by a double desilylation of a 2,3-bis(silyl)oxirane. Paracoccus NCIB 11086 is a microorganism which was reported to be capable of living anaerobically on benzoic acid as its sole source of cell carbon, with nitrate as the ultimate oxidant. An unusual transformation reported in this biodegradative pathway is the oxidation of cyclohexanone to adipic acid. To facilitate studies on the mechanism of this unusual conversion, (1-$\sp{13}$C) -, (2,2,6,6-$\sp2{\rm H}\sb4,$1-$\sp{13}$C) -, and (3,3,5,5-$\sp2{\rm H}\sb4,$1-$\sp{13}$C) cyclohexanone samples were synthesized. Approaches to several ($\sp2$H,$\sp{13}$C) -labeled adipic acid standards were also developed. Imidazole glycerol phosphate (IGP) dehydratase is responsible for the conversion of IGP to imidazole acetol phosphate in the histidine biosynthetic pathway. Studies of the S. typhimurium and E. coli proteins have provided only the most basic physical and chemical properties. Very little is known about the mechanism of action of IGP dehydratase. To further characterize this enzyme's catalytic mechanism we have investigated the stereochemical course of the E. coli IGP dehydratase reaction. To facilitate these studies, several routes to I (3-$\sp2$H) GP have been investigated. A total synthetic route, although promising, was long, and incorporation of label was problematic. A more efficient, coupled enzymatic route was thus developed, beginning with chemically-synthesized (3-$\sp2$H) ribose-5-phosphate. The stereochemical course of the enzymatic reaction was analyzed by using a coupled, enzymatic system generating (2S)- (3-$\sp2{\rm H}\sb1$) histidinol as the final product. To facilitate assignment of the C-3 resonances of this product, (2S*,3S*)- (3-$\sp2{\rm H}\sb1$) histidinol was synthesized. Proton NMR analysis showed that the 3-proR proton resonates downfield relative to the 3-proS proton. Analysis of enzymatically-derived (2S)- (3-$\sp2{\rm H}\sb1$) histidinol showed that deuterium from (2R,3S)-I (3-$\sp2$H) GP occupies the 3-proR position of the product. Complementary studies with unlabeled IGP in a D$\sb2$O-based medium showed that a single deuterium had been incorporated into the 3-proS position of the product. Thus, in the conversion of IGP to histidinol, a "new" solvent-derived hydrogen is added at C-3, with net inversion of configuration.
Degree
Ph.D.
Advisors
Schwab, Purdue University.
Subject Area
Biochemistry
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