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I. Development of a new assay and inhibitors for human cystathionine beta-synthase. II. Asymmetric catalyst development guided by in situ enzymatic screening (ISES)
Abstract
hCBS is one of the two key enzymes of the transsulfuration pathway, which connects dietary methionine to the biosynthesis of cysteine and eventually to glutathione, the major intracellular redox buffer. hCBS catalyzes a PLP dependent beta-replacement reaction of L-serine with L-homocysteine to form (L,L)-cystathionine. High levels of plasma homocysteine are commonly referred to as hyperhomocysteinemia, which is now generally accepted as an independent risk factor for atherosclerotic disease in the coronary, cerebral and peripheral vessels. Mutations in hCBS have been shown to be the single most common cause of hereditary hyperhomocystinemia. A continuous UV assay for hCBS has been developed, employing an unnatural substrate for the enzyme, and tapping into a three enzyme coupled assay for the product thereby obtained. This ultimately results in the reduction of oxaloacetate with NADH by malate dehydrogenase for each CBS turnover, resulting in a continuous UV spectroscopic signal. This assay is then applied to the purification of hCBS, as well as for kinetic characterization of the purified enzyme. Three series of functionalized (L,L)-cystathionine analogues have been designed to target the PLP cofactor in the CBS active site. By exploiting the pseudo-C2-symmetry of (L,L)-cystathionine, a streamlined synthetic approach has been developed, utilizing Grubbs cross metathesis chemistry. Modified Mitsunobu conditions were successfully applied to the introduction of all three classes of heteroatomic functionality at the α-position. Interestingly, a classic slow-binding inhibition profile was observed, particularly in the α-N-hydroxyamino acid series, also the most potent series. Model spectroscopic studies of representative members of each inhibitor class are consistent with the formation of the expected cofactor-centered nitrone, hydrazone and oxime adducts. In Situ Enzymatic Screening (ISES) was employed for catalyst discovery. For the Ni(0)-mediated intramolecular allylic amination, an array of chiral phosphinooxazoline ligands was synthesized with a new methodology and evaluated using ISES technique. Together with S. Dey and K. Karukurichi, we have developed the second generation ISES for predicting the relative rate and enantioselectivity of asymmetric reactions. From the second generation ISES, a novel chiral scaffold derived from β-fructopyranose was discovered that can induce high enantioselectivity for epoxide HKR.
Subject Area
Biochemistry|Organic chemistry
Recommended Citation
Shen, Weijun, "I. Development of a new assay and inhibitors for human cystathionine beta-synthase. II. Asymmetric catalyst development guided by in situ enzymatic screening (ISES)" (2007). ETD collection for University of Nebraska-Lincoln. AAI3256642.
https://digitalcommons.unl.edu/dissertations/AAI3256642