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PLP-enzyme inactivators: Design, enabling methodology development and evaluation
Hydrogen sulfide (H2S), one of three known gaseous neurotransmitters, has been associated with neuronal cell death pursuant to cerebral ischemia. As the PLP-dependent enzyme, cystathionine β-synthase (CBS), is the primary mediator of H2S biogenesis in the brain, there is currently great interest in CBS inhibition. Chapter 1 of this dissertation will present a new class of CBS-target mechanism-based inhibitors designed to engage the PLP cofactor in a non-traditional linkage (hydrazone, oxime or possibly nitrone) and relies on an exceptionally slow off-rate to produce effective inhibition. Additional highlights of this work include the exploitation of C2-symmetry both for the design (cystathionine mimicry) and as a simplifying element of the synthesis, as well as the use of modified Mitsunobu chemistry to bring in the reactive inhibitor X-Y functionalities. Preliminary studies have identified the fully saturated (L,L)- bis-hydrazino acid 6S to be the most potent inhibitor (Ki ∼50 µM) of the set. Presented, are results from recent collaborative studies with the group of Peter T.-H. Wong at National University of Singapore (NUS) on the β-eliminase enzyme, hCBS. It is demonstrated that 6S reduced H2S production in SH-SY5Y cells overexpressing CBS in the presence of high substrate concentrations and thus attenuated the consequential increase in cell death. In vivo studies reveal the ability of 6S to dramatically reduce infarct volume under conditions of pre-treatment (∼80% reduction) and post-treatment (∼66% reduction) using a rat transient middle cerebral artery occlusion (tMCAO) model for ischemia.  Pyridoxal 5′-phosphate (PLP)-dependent enzyme reactions (145 activities) comprise ∼4% of all enzymatic transformations. Chapter 2 will highlight a new strategy to inhibit PLP-dependent enzymes, with a focus on PLP-dependent decarboxylases i.e. for which the Cα-CO2- bond is cleaved. For this reason, this class of enzyme serves as a useful platform for the study of so-called “quaternary” AA suicide substrates, in which the a-proton is replaced by an “actuatable trigger.” The work herein will describe the design of an α-(1′-fluoro)vinyl trigger and the development of the first synthetic methodology that allows for its installation at the α-carbon of a wide range of amino acids. This methodology was predicated on the development of a previously unknown “1-fluorovinyl cation” equivalent. Finally, Chapter 3 will feature efforts toward development of a complementary “(2′Z-methxoxy)vinyl cation” equivalent,  motivated by the appearance of methoxyvinylglycine, in nature. Highlights include capture of the methoxyvinyl cation equivalent with a wide range of anionic nucleophiles of the organomagnesium or lithiated acetylide variety. Treatment of the resulting largely syn-β-hydroxysilyl alcohols with base leads to the corresponding Z-configured methoxyvinyl products, presumably via a Peterson olefination-type mechanism. In the future, “(2 ′Z-methxoxy)vinyl cation” equivalent, may be applicable to the synthesis of new PLP-dependent enzyme inhibitors outfitted with the methoxyvinyl trigger.  McCune, C. D., Chan, S. J., Beio, M. L., Shen, W., Chung, W. J., Szczesniak, L. M., Chai, C., Koh, S. Q., Wong, P. T. H., Berkowitz, D. B., "Zipped Synthesis" by Cross-Metathesis Provides a Cystathionine β-Synthase Inhibitor that Attenuates Cellular H2S Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model. ACS Central Science 2016, Ahead of Print.  McCune, C. D., Beio, M. L., Friest, J. A., Ginotra, S., Berkowitz, D. B., A useful methoxyvinyl cation equivalent: α-t-butyldimethylsilyl-α-methoxyacetaldehyde. Tetrahedron Letters 2015.
McCune, Christopher D, "PLP-enzyme inactivators: Design, enabling methodology development and evaluation" (2016). ETD collection for University of Nebraska - Lincoln. AAI10103364.