Histidine biosynthesis: Preparation and characterization of intermediates and analogs

Ina Lim Deras, Purdue University

Abstract

The goal of this study was to elucidate mechanistic information on the fourth step in the histidine biosynthetic pathway, the conversion of $N\sp\prime$- ((5-phosphoribosyl)-formimino) -5-aminoimidazole-4-carboxamide ribonucleotide (5$\sp\prime$-ProFAR) to $N\sp\prime$- ((5-phosphoribulosyl)-formimino) -5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR), catalyzed by 5$\sp\prime$-ProFAR isomerase. However, substrates needed for enzyme characterization and assays were not readily available, and by means of recombinant DNA technology and chemoenzymatic synthesis, 5$\sp\prime$-ProFAR and PRFAR were prepared on a millimole scale and full spectral characterization was accomplished. Fluorinated sc D-ribose-5-phosphate analogs were designed as precursors to fluorinated 5$\sp\prime$-ProFAR analogs. The fluorosugars were to serve as alternate substrates for the enzymes used in the chemoenzymatic preparation of 5$\sp\prime$-ProFAR, and the eventual role of fluorinated 5$\sp\prime$-ProFAR analogs was to serve as mechanism-based inhibitors of 5$\sp\prime$-ProFAR isomerase. Stereospecific synthesis of four fluorinated pentose phosphates was performed using two independent, divergent synthetic schemes. Full spectral characterization of the four fluorinated pentose phosphates was completed, and the presence of fluorine had significant effects on the solution forms. A significant amount (12-29%) of the fluorinated pentose phosphates existed as the hydrate of the acyclic form of the fluorinated pentoaldose phosphate. The fluorinated analogs of sc D-ribose-5-phosphate demonstrated no turnover by 5-phospho- scD-ribosyl-1-$\alpha$-pyrophosphate synthetase and served as weak inhibitors, illustrating the extreme substrate specificity of the enzyme.

Degree

Ph.D.

Advisors

Davisson, Purdue University.

Subject Area

Biochemistry|Organic chemistry

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