Synthesis of sterol biosynthesis inhibitors and metabolism of isotopically labeled sterols by Saccharomyces cerevisiae

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Title: Synthesis of sterol biosynthesis inhibitors and metabolism of isotopically labeled sterols by Saccharomyces cerevisiae
Author: Zhou, Wen
Abstract: The dissertation focuses on the chemistry and mechanism of sterol biomethylation catalyzed by (S ) -adenosyl -L -methionine :sterol methyl transferase (SMT ) enzyme . The research is composed of two parts : synthesis of sterol methylation inhibitors and isotopically labeled substrates and mechanism studies of the SMT enzyme . Part 1 describes preparation of ^H and ^^C -isotopically labeled sterols , and the design , synthesis and enzymatic evaluation of three types of sterol methylation inhibitors : (1 ) substrate analogs which act as product inhibitors of the reaction ; (2 ) substrate analogs which act as mechanism -based inactivators ; and (3 ) transition state analogs . The synthetic work focused on modification of the side chains of cholesterol , zymosterol , lanosterol , and cycloartenol which are natural substrates in C -methylation reactions . The sterol side chain was modified at C -20 , C -21 , and C -22 positions to study the effect of configuration and conformation of the sterol side chain on biomethylation . A series of novel sterol methylation inhibitors containing aza , aziridine , and ammonium groups at positions C -22 to C -25 were synthesized to serve as transition state analogs . Substrate analogs with the 26 , 27 -cyclopropylidene functional group were also synthesised and discovered to be potent irreversible mechanism -based inhibitors of the SMT enzyme . Several new 24 , 25 -ethano and 24 , 28 -methano sterols , 24 - inyl and 29' , 29" -cyclopropylidene lanosterols , and 24 (25 ) , 26 (26' ) -diene and 24 (25 ) -en -25 -thylnyl sterols have been synthesized . All the inhibitors were characterized by gas chromatography -mass spectrometry , high pressure liquid chromatography , and 'H and ^^C nuclear magnetic resonance spectroscopy . Part 2 describes research on the coupled methylenation -deprotonation of C -24 of the sterol side chain in plant and fungal sterol C -methylation reactions . These studies involved determination of the stereochemistry of hydrogen migration from C -24 to C -25 during biomethylation and of C -28 deprotonation . To accomplish our aims , [27 -'^C] , [24 -^H] and [28 -^H2] labeled sterols were prepared and assayed with SMT enzymes from a fungus {Saccharomyces cerevisiae ) and a plant (Arabidopsis thaliana ) . As a result , migration of the hydrogen from C -24 to C -25 was found to be introduced from Re -facQ of the 24 , 25 -double bond of the sterol side chain to generate the similar 25R stereochemistry in S . cerevisiae and A . thaliana , suggesting a similar topography of the SMT active site .
URI: http : / /hdl .handle .net /2346 /11825
Date: 1998-05

Citation

Zhou, Wen Synthesis of sterol biosynthesis inhibitors and metabolism of isotopically labeled sterols by Saccharomyces cerevisiae. Doctoral dissertation, Texas Tech University. Available electronically from http : / /hdl .handle .net /2346 /11825 .

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