Abstract
Two of the key enzymes in the biosynthetic pathway of sialic acids were studied. A highly expressed CMP-sialic acid synthetase with a decapeptide tag attached at the C-terminal of the enzyme and the wild type enzyme from E. coli were purified and studied for their properties including their kinetic parameters, stability, pH-rate profiles, metal ions effect, and substrate specificities. Sialic acid derivatives with modifications at the C-5 and C-9 positions were synthesized by combined enzymatic and chemical methods. Sialic acid aldolase which catalyzes the aldol condensation of ManNAc and pyruvate has a broad substrate specificity, and is able to accept a variety of derivatives of ManNAc. Several ManNAc derivatives were prepared from subtilisin catalyzed transesterification reactions. The C-6 position of ManNAc was regioselectively acylated with several activated acyl donors. Similar type of reactions were also applied to the other monosaccharides and disaccharides. Regioselective acylation of nucleotides at the C-5' position of the ribonucleosides catalyzed by subtilisin followed by radical deoxygenation at the C-3' position provides a method for the preparation of the 2',3- dideoxy nucleotides which are potential anti-viral reagents for HIV. Several subtilisin mutants were engineered to increase their stabilities. Their stabilities in organic solvent were studied and these enzymes were applied to the synthetic purposes. Polymerization of methionine methyl ester and resolution of unnatural amino acids were carried out with the mutant enzymes and the wild type BPN'. The modified CMP-sialic acid synthetase has been used in the synthesis of several CMP-NeuAc derivatives from modified ManNAc derivatives and CIP. Sialyl N-acetyllactosamine (NeuAca2,6Galb1,4GlcNAc) was synthesized by a multi-enzyme system with in situ generation of NeuAc and regeneration of CTP and CMP-NeuAc.
Liu, Jennifer Lin-Chun (1992). Enzymes in organic synthesis : uses of CMP-sialic acid synthetase, sialic acid aldolase and subtilisin. Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -1365906.