Browsing by Subject "Mechanism"
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Item Biosynthetic studies of thiosugar-containing natural products, BE-7585A and Lincomycin A(2011-05) Sasaki, Eita; Liu, Hung-wen, 1952-; Anslyn, Eric V; Bielawski, Christopher; Fast, Walter L; Whitman, Christian PSulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been well understood. On the other hand, a wide variety of sugar derivatives commonly found in natural products are often vital components for the efficacy and specificity of their parent molecules. Elucidation of such unusual sugar biosyntheses is important both for understanding their intriguing chemical mechanisms and creating unnatural compounds by altering their biosynthetic machineries, which could potentially exhibit enhanced or novel biological activities. This dissertation describes biosynthetic studies of two thiosugar-containing natural products, BE-7585A and lincomycin A, produced by Amycolatopsis orientalis and Streptomyces lincolnensis, respectively. While the former possess a C-2-thiosugar-containing disaccharide moiety, the latter contains a C-1-thio substituent on a characteristic eight-carbon backbone sugar. The focus of this research is to characterize the biological pathways and mechanisms responsible for the sulfur incorporation and the unique sugar scaffolds. BE-7585A, an angucycline-type natural product, contains the rare C-2-thiosugar moiety. PCR-based screening of a cosmid library constructed from the genomic DNA of A. orientalis led to the identification of the BE-7585A biosynthetic gene cluster. A gene, bexX, was found to be a candidate for a thiosugar synthase with moderate sequence similarity to a thiazole synthase. The gene, bexX, and a glycosyltransferase homologue, bexG2, were heterologously expressed in Escherichia coli. A variety of biochemical experiments provided a wealth of evidence supporting the proposed biosynthetic pathway for the C-2-thiodisaccharide moiety. Finally, whole genome sequencing and a genome mining approach led to the identification of a sulfur carrier protein to accomplish the in vitro enzymatic synthesis of the C-2-thiosugar for the first time. Lincomycin A is a lincosamide antimicrobial natural product with a C-1 methylthio substituent. Although the lincomycin A biosynthetic gene cluster has been reported, biochemical verification of the biosynthetic pathway has remained elusive. In this dissertation, the complete methlthiolincosamide biosynthetic pathway including the potential C-1 sulfur incorporation mechanism was proposed. Furthermore, two early intermediates of the pathway were characterized for the first time by demonstrating the LmbR (transaldolase) and LmbN (isomerase) reactions in vitro.Item Cartel Mechanism Design: Nonratifiable Conditions of Collusive Behavior(2012-02-14) Hsueh, Shao-ChiehThis dissertation is about an open question of cartel ratifiable conditions. My research goal is to establish a mechanism which is able to detect and explain cartels' activities. My research question in the second chapter is whether or not an efficient cartel mechanism is ratifiable in the first-price sealed-bid auction format with participation costs. R. McAfee and J. McMillan study the coordinated bidding strategies in a strong cartel, which is incentive-compatible and efficient. This chapter investigates this efficient collusive mechanism with two important conditions (1) bidders can update their information through a cartel's prior auction and (2) they have to pay participation costs to participate in seller's auction. The concept of ratifiability, introduced by P. Cramton and T. Palfrey, is applied to explore the first-price sealedbid auction with participation costs. I discovered that the efficient cartel mechanism, such as pre-auction knockout, would be ratified when either of the two conditions exists. However, this mechanism is no longer ratifiable when both conditions exist. The bidder with the highest value in the cartel would have incentive to betray, since doing so sends a credible signal of high value. Hence, the other bidders will be discouraged from participating in the seller's auction and the highest-value bidder maximizes his revenue. In the third chapter, I studied the seller's strategy when she faces a cartel in an auction mechanism. An active seller's optimal strategy is to raise the reserve price to a level that is higher than her own valuation. The collusive mechanism is sustainable even though its revenue is extracted by the higher reserve price. If the seller is authorized to change the auction mechanism, she can receive the expected payoff, prevent the formation of a ring and keep the auction efficient. Further, I presented two methods that could deter a cartel under specific conditions. One is the residual claimants method as proposed by Y. Che and J. Kim and the other is to set a positive participation cost as outlined in the first chapter. The residual claimants method can inhibit a ring in many cases, but it may have some trouble in preventing an efficient cartel mechanism when there is only one participant in the seller's auction. In the fourth chapter, I investigated how to achieve external efficiency in a repeated game. In particular, I looked into the allocation of the budgeted that allows an authority, such as the government, to differentiate collusive behavior and to expose agents to external threats. A threshold level of the budget payment is found in an incentive compatible collusive mechanism for which the government can prevent an agent from participating. In a two-stage model, I showed that if the government can boost exemption or have more budget to subsidize agents, it is less likely that a ring will be formed.Item Mechanisms of transition-metal catalyzed additions to olefins(Texas A&M University, 2005-08-29) Nowlan, Daniel ThomasTransition metal catalyzed reactions have an important place in synthetic chemistry, but the mechanistic details for many of these reactions remain undetermined. Through a combination of experimentally determined 13C kinetic isotope effects (KIEs) and density functional theory (DFT) calculations, some of these reactions have been investigated. The cyclopropanation of an olefin catalyzed by rhodium (II) tetrabridged complexes has been shown to proceed through an asynchronous, but concerted mechanism. DFT does not provide an accurate transition structure for the reaction of an unstabilized carbenoid with an olefin, but it does predict an early, enthalpically barrierless transition state which is consistent with the reactivity of unstabilized carbenoids. For the case of stabilized carbenoids, the theoretical structures predict the KIEs accurately and a new model is proposed to explain the selectivity observed in Rh2(S-DOSP)4-catalyzed cyclopropanations. The chain-elongation step of atom transfer radical polymerization (ATRP) has been shown to be indistinguishable from that of free radical polymerization (FRP) for the CuBr/2,2??-bipyridine system. While DFT calculations predict an earlier transition state than observed, the calculations suggest that with increasing levels of theory the predicted KIEs come closer to the observed KIEs. A recently proposed [2 + 2] mechanism for the cyclopropenation of alkynes catalyzed by Rh2(OAc)(DPTI)3 has been shown not to be a viable pathway. Rather, the experimental KIEs are predicted well by canonical variational transition state theory employing the conventional mechanism for cyclopropenation via a tetrabridged rhodium carbenoid. DFT calculations also suggest an alternative explanation for the observed enantioselectivity. The 13C KIEs for metal-catalyzed aziridination have been measured for three separate catalytic systems. While the KIEs do not completely define the mechanism, all of the reactions exhibit similar KIEs, implying similar mechanisms. A surprising feature of this system is the presumed nitrene intermediate??s triplet spin state. This complicates the DFT analysis of this system.Item Mechanistic characterization of members of the amidohydrolase superfamily(2009-05-15) Marti Arbona, RicardoThe amidohydrolase superfamily is a functionally diverse group of enzymes found in every organism sequenced to date. The landmark for this superfamily is the conservation of a (beta/alpha)8-barrel structural fold. Isoaspartyl dipeptidase (IAD) from Escherichia coli catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. Structural studies of the wild-type enzyme demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for all four metal-substituted forms of the wild-type enzyme and the site-directed mutants, E77Q and Y137F. Structural analysis of IAD with the bound substrate and site-directed mutagenesis shows the importance of the side chains of residues Glu-77, Tyr-137, Arg-169, Arg-233, Asp- 285, and Ser-289 in the substrate binding and hydrolysis. The reaction mechanism for the hydrolysis of dipeptides by IAD is initiated by the polarization of the amide bond via complexation to the beta-metal and the hydrogen bond to Tyr-137. Asp-385 participates in the activation of the bridging hydroxide for nucleophilic attack at the peptide carbon center. The lately protonated Asp-285 donates the proton to the alpha-amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond. N-formimino-L-glutamate iminohydrolase (HutF) from Pseudomonas aeruginosa acts in the deimination of the fourth intermediate of the histidine degradation pathway, N-formimino-L-glutamate. An amino acid sequence alignment between HutF and other members of the amidohydrolase superfamily containing mononuclear metal centers suggests that the residues Glu-235, His-269, and Asp-320 are involved in substrate binding and deimination. Site-directed mutagenesis of Glu-235, His-269, and Asp-320, in conjunction with the analysis of the four metalsubstituted enzyme forms and pH-rate profiles provides valuable information toward the proposal of a mechanism for deimination of N-formimino-L-glutamate by HutF. This information suggests that the reaction is initiated by the activation of the hydrolytic water through base catalysis via His-269. The enhanced nucleophile attacks the formimino carbon center. In a concerted reaction, Asp-320 deprotonates the hydroxide nucleophile, and His-269 donates a proton to the terminal amino of the iminium group resulting in the collapse of the tetrahedral intermediate, the cleavage of the carbon-nitrogen bond and the release of the products.Item Mechanistic investigations of SpnF- and SpnL-catalyzed cyclizations in the biosynthesis of spinosyn A(2013-12) Kim, Nam Ho, 1975-; Liu, Hung-wen, 1952-Spinosyn A is a particularly interesting natural product due to its structural complexity and potent insecticidal activity. The biosynthetic pathway of spinosyn A is interesting as it has two unusual features, the SpnF-catalyzed (4+2) cycloaddition and the SpnL-catalyzed cyclization to produce the perhydro-as-indacene core. The work described in this dissertation focuses on elucidating the mechanisms of the SpnF- and SpnL-catalyzed reactions. SpnF has attracted significant interest as a possible Diels-Alderase. To explain how SpnF catalyzes the formation of cyclohexene ring, three plausible mechanisms have been proposed, the Diels-Alder reaction mechanism, the ionic rearrangement mechanism, and the biradical rearrangement mechanism. Kinetic isotope effect studies were performed using four deuterium-labeled mechanistic probes, specially the C4-D, C7-D, C11-D, and C12-D analogs. Currently, the ionic rearrangement mechanism can be excluded, based on the results using the C4-D and C7-D analogs. In addition, how SpnF accelerates the reaction was studied to assess the contribution of an entropic x preorganization compared to enthalpic transition state stabilization. To measure the relative rate enhancements due to structural perturbations, three mechanistic probes were synthesized, the linear analog, the C13-14 Unc analog, and the C2-3 Unc analog. Unfortunately, the linear analog and C13-14 Unc analog didn’t show any turnover activity under either non-enzymatic or enzymatic conditions. Thus, no conclusion could be drawn from incubation with these substrate analogs. Mechanistic studies of SpnL-catalyzed cyclization were devoted to differentiating between the Rauhut-Currier type mechanism and the Michael addition mechanism. Biochemical studies using the C13-F analog as a mechanism-based inhibitor showed the formation of a covalent adduct with SpnL, which is consistent with the Rauhut-Currier type mechanism. Additional experimental data obtained from isotope trace experiments and kinetic isotope effect studies using C12-D analog supports the Rauhut-Currier type mechanism. Biochemical studies concerning the role of SAM in SpnF and SpnL showed that SAM is required for the activity of SpnL, and were inconclusive for SpnF. SpnL mutant studies showed that Cys60 and Glu96 may be important for the catalysis of SpnL. Chemoenzymatic total synthesis of spinosyn A was completed by chemical etherification of 17-pseudoaglycone and D-forosamine.Item The development of metal-organic frameworks using palladium metal complexes for catalysis and the utilization of a dihaloimidazolidinedione for easy acid chloride generation(2016-08) Nguyen, An Ngoc-Michael; Humphrey, Simon M.; Jones, Richard APart 1. Metal-Organic Frameworks (MOFs) have been extensively studied due to their functional versatility and well-defined, porous structures. It is, however, exceptionally rare to find MOFs that use late transition metal complexes as a building block. Incorporation of these complexes would have immediate applications in catalysis and enhanced gas adsorption/storage. Furthermore, because of the MOF’s well-defined structures, heterogeneous catalysis properties can be more readily studied and improved. Herein, 1,2-bis(bi(para-carboxyl)phenylphosphino)benzene palladium dichlo-ride’s (PdCl2(BBCB)) catalytic ability is studied using the Mizoroki-Heck reaction, and 1,2-bis(bi(4-carboxy-biphenyl-4’-phosphino))ethane palladium dichloride (PdCl2(BBCE-1L)) is used to make a MOF with an enlarged pore size and greater catalytic ability. BBCB, a tetra-para-carboxylic acid derivative of dppb, is used to form a palladium dichloride bis(phosphine) complex. This complex was found to perform a Mizoroki-Heck reaction in decent yields despite formation of palladium black. BBCE-1L, a tetra-4-carboxylic acid biphenyl derivative of dppe, was then developed to deter palladium black formation and enlarge the pore size of the MOF. A MOF was made using a Zn(II) paddlewheel as the metal node and PdCl2(BBCE-1L) as the organic linker. This MOF was found to have moderate thermal stability and potentially high pore volume. Part 2. Acid chloride generation is a widely researched field for the synthesis of amides and esters, motifs found throughout nature and pharmaceuticals. Recently, we found that dihaloimidazolidinediones could substitute a variety of alcohols with halogens through an amide-stabilized carbocation. As an extension of this chemistry, we were able to activate most carboxylic acids into the corresponding acid chloride with near quantitative yields. Mechanistic studies of electronic and steric effects suggest that the rate-determining step is the nucleophilic attack of a free chloride ion on the carbonyl center to produce the corresponding acid chloride. Electronic effects of ring size on electrophilicity was also observed and explained through analysis of a simplified Walsh diagram.