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Expanding the uses of the NCAL and finding the spectrum of substrates best suited for such a transformation has been the main effort of my research. Previous studies had focused on aldedydes as the requisite functionality that would provide the needed electrophilicity in order to complete the aldol; however, recent advancements have introduced ketones as a viable carbonyl. With an established protocol in hand, I set out to explore various substrates that could yield Beta-lactones in good to moderate yields such as amino acid derivatives, diones, and large cyclic formations as well as simple, straight chain acids with varying groups Alpha to the ketone. In general, I was able to establish a basic framework of substrates that are highly and/or moderately susceptible towards the NCAL and current studies continue to further expand the scope. In addition to making Beta-lactones, I investigated alkyl cuprates as soft nucleophiles to afford addition at the Beta carbon yielding a variety of acids. Substrates for cuprate additions have been expanded to bulkier and multi-cyclic Beta-lactones and applied to the synthesis of a Merck IND intermediate. Additions to bi- and tri-chloro Beta-lactones due to the presence of the resulting moity in natural products are currently being studied.

The recent finding that the FDA-approved antiobesity agent orlistat (tetrahydrolipstatin, Xenical) is a potent inhibitor of the thioesterase domain of fatty acid synthase (FAS) led us to develop a concise and practical asymmetric route to pseudosymmetric 3,4-dialkyl-cis-beta-lactones. The well-documented upregulation of FAS in cancer cells makes this enzyme complex an interesting therapeutic target for cancer. The described route to 3,4-dialkyl- beta -lactones is based on a two-step process involving Calter's catalytic, asymmetric ketene dimerization of acid chlorides followed by a facialselective hydrogenation leading to cis-substituted- beta -lactones. Importantly, the ketene dimer intermediates were found to be stable to flash chromatography, enabling opportunities for subsequent transformations of these optically active, reactive intermediates. Subsequent R-epimerization and R-alkylation or acylation led to trans- beta - lactones and beta -lactones bearing alpha-quaternary carbons, respectively. Several of the ketene dimers and beta-lactones displayed antagonistic activity (apparent Ki in the low micromolar range) in competition with a fluorogenic substrate toward a recombinant form of the thioesterase domain of fatty acid synthase. The best antagonist, a simple phenyl-substituted cis- beta -lactone, displayed an apparent Ki (2.5 ( 0.5 muM) of only 10- fold lower than that of orlistat (0.28 ( 0.06 muM). In addition, mechanistic studies of the ketene dimerization process by Reaction View infrared spectroscopy support previous findings that ketene formation is rate determining. A highly diastereoselective, nucleophile-promoted bis-cyclization process, employing readily available and tractable keto-acid substrates, is described. This methodology provides concise access to bicyclic- and tricyclic-beta-lactones bearing tertiary carbinol centers and quaternary carbons, greatly extending the scope of previous routes to bicyclic-beta-lactones from aldehyde acid substrates. This and related processes may be revealing a subtle interplay between [2 plus 2] cycloaddition and nucleophilecatalyzed aldol lactonization (NCAL) reaction manifolds. An early induction period in the bis-cyclization of keto-acids is confirmed via isolation of the complex between 4- pyrrolopyridine and Modified Mukaiyama reagent N-propyl-2-bromo pyridinium triflate. Dyotropic rearrangements of tricyclic keto beta-lactones derived in high yields and >19:1 diastereoselectivity from readily available 1, 3-dione acids is described. Zn (II) salts were found to be most efficient for affecting dyotropic 1, 2-acyl migrations where as sub stoichiometric TMSOTf was found to execute a delta-lactone migration providing bis gamma-lactone in modest yields. Enantioselective desymmetrization with inexpensive (S) - tetramisole has been demonstrated to provide direct evidence of Lewis base involvement in the Nucleophile Promoted Bis-cyclization of keto-acids. Further studies using TsCl as the carboxylate activating agent instead of modified Mukaiyama reagent and catalytic tetramisole are described for achieving practical, catalytic, enantioselective synthesis of beta-lactones from keto-acids. Preliminary studies toward conjugate addition- lactonization pathway provided a hint as to the complexity involved to affect this transformation under the bis-cyclization conditions. An alternate hypotheses concerning the possibility of isomerization-dienolate formation - lactonization is experimentally proven. Additionally, applications of these and related findings in the intramolecular Morita-Baylis-Hillman reaction with cyclic ketones have been investigated which provide new avenues of synthetic methodology development.

Microbiologists have long focused on transcription as a main source of physiological regulation in bacterial adaptation. However, the time scale on which certain cellular responses must be coordinated dictates that a more rapid system be in place to deal with sudden environmental stresses. In eukaryotes, understanding the proteasome and ubiquitin-tagging has led to an appreciation for protein turnover as a mechanism for rapid adaptation. Like eukaryotes, bacteria possess several proteolytic complexes that degrade proteins into smaller polypeptides and amino acids. These enzymes were discovered as maintainers of protein quality control, through recognition of aberrant protein products, but recent studies have suggested that they play an active role in regulation of cell processes through degradation of endogenous proteins. Surprisingly, a genome wide screen for essential genes in Mycobacterium tuberculosis (Mtb) found numerous proteases to be essential for growth, providing evidence that degradative regulation may be critical for survival. One essential complex, Clp protease, was intriguing as it appeared to have a divergent structure in Mtb, and was largely dispensable for growth in most other organisms. In order to study the importance of protein turnover and degradative regulation in Mtb, I chose to study Clp as a model. I confirmed that Clp was required for normal growth in mycobacteria through targeted genetic engineering, and demonstrated that depletion of Clp was bactericidal. We hypothesized that a protease would be essential because it might prevent accumulation of toxic proteins or repressors of vital processes. To understand why Clp protease was so critical, I conducted proteomic analysis comparing wildtype and Clp-depleted cells to identify substrates of the protease. In line with our hypothesis, I identified WhiB1, a redox-sensitive transcriptional repressor. Blocking degradation of WhiB1 by Clp resulted in death, suggesting that the importance of Clp can be partially explained by its action on the repressor. Finally, taking advantage of known Clp-specific inhibitors in S. aureus, we showed that Clp could be targeted with small molecules in Mtb. The elucidation of novel drug targets and small molecules active against Mtb is crucial due to the overwhelming prevalence of the disease and rises in drug resistant forms.