A variety of biological and organic reaction mechanisms are studied using powerful tools from experimental and theoretical chemistry. These tools include the precise measurement of kinetic isotope effects (KIEs) and the use of theoretical calculations to predict KIEs as well as determine factors that contribute to reaction acceleration and selectivity. Theoretical analysis of the Swain-Schaad relationship involves the prediction of a large number of isotope effects and establishes the semiclassical boundaries of the relationship. Studies on the mechanism of oxidosqualene cyclase involve the determination of a large number of precise KIEs simultaneously. Transition state models for the Sharpless asymmetric epoxidation have been developed that explain the versatility, high selectivities, and ligand accelerated catalysis of the reaction. Theoretical predictions on the proposed enzymatic mechanism of flavin dependent amine oxidation suggest a hydride transfer mechanism and rules out mechanisms involving covalent intermediates. Finally, a theoretical analysis of Diels-Alder reactions successfully describes the unexpected exo selectivity in some of these reactions.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1976. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 211-217).

Detailed mechanistic studies were conducted on several organic reactions that exhibit product selectivity (regio-, peri-, or enantioselectivity). The organic reactions studied were electrophilic aromatic substitutions, Diels-Alder cycloadditions of 1,3- dienes with cyclopentadieneone, Lewis acid catalyzed ene reactions with olefins, chlorinations of alkynes, and the enantioselective intramolecular Stetter reaction. Analyses of these systems were conducted by measurement of kinetic isotope effects, standard theoretical calculations, and in some cases dynamic trajectories. Mechanistic studies of electrophilic aromatic substitution, Lewis acid catalyzed ene reaction with olefins, the chlorination of alkynes, and the Diels-Alder cycloadditions of 1,3-dienes with cyclopentadienones, suggest that the origin of selectivity is not always a result of selectivity result from a kinetic competition between two closely related pathways to form distinct products. All of these systems involve one transition state on a potential energy surface that bifurcates and leads to two distinct products. In these systems, experimental kinetic isotope effects measured using natural abundance methodology, theoretical modeling of the potential energy surfaces, and trajectory analyses suggests that selectivites (regio- and periselectivities) are a result of influences by momenta and steepest-descent paths on the energy surface. The work here has shown that in order to understand selectivity on bifurcating surfaces, transition state theory is not applicable. In place of transition state energetics, the guiding principles must be those of Newtonian dynamics. In the mechanistic studies for the enantioselective intramolecular Stetter reaction, the origin of selectivity is a result of multiple transition states and their relative energies. Experimental H/D kinetic isotopes effects had lead to the conclusion that two different mechanisms were operating for reactions where carbenes were generated in situ versus reactions using free carbenes. However, 13C kinetic isotope effects and theoretical modeling of the reaction profile provide evidence for one mechanism operating in both cases.

Both bromodeprotonation and bromodesulphonation occur during aqueous bromination of sodium p-methoxybenzenesulphonate, A, and potassium l-methylnaphthalene-4-sulphonate, B. Extensive kinetic studies reported here suggest that bromodesulphonation of A proceeds by a two-step process with Br2 as the brominating species, but do not completely exclude Br+ (or H2OBr+) acting in either a one- or two-step process. For B, the kinetic data can be interpreted by either a one- or two-step process with Br2 as the brominating species. Kinetic sulphur isotope effects have been measured for the bromodesulphonation of A and B and found to vary with bromide-ion concentration, thus strongly supporting the two-step process involving molecular bromine. The kinetic results for the bromodeprotonation of A cannot distinguish between a one- and two-step process involving Br2l the two-step mechanism has been confirmed by the observation of a variation in kinetic hydrogen isotope effect with bromide-ion concentration. / Thesis / Doctor of Philosophy (PhD)

Flavocytochrome b2 catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate OH and CH bonds by the wild type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The DVmax and D(V/Klactate) values are both 3.0, 3.6 and 4.5 for the wild type enzyme, flavin domain and D282N enzymes, respectively. The D20Vmax values are 1.38, 1.18, and 0.98 for the wild type enzyme, the flavin domain, and the D282N enzyme; the respective D20(V/Klactate) values are 0.9, 0.44, and 1.0. The Dkred value is 5.4 for the wild type enzyme and 3.5 for the flavin domain, whereas the D2Okred is 1.0 for both enzymes. The V/Klactate value for the flavin domain increases 2-fold at moderate concentrations of glycerol. The data are consistent with the lactate hydroxyl proton not being in flight in the transition state for CH bond cleavage and there being an internal equilibrium prior to CH bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium. Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide, carbon dioxide and water. Sequence alignments identified this enzyme as a member of the L-amino acid oxidase family. The tyrosine and arginine residues in L-amino acid oxidase that bind the carboxylate of o-aminobenzoate are conserved and correspond to Tyr413 and Arg98 in tryptophan 2-monooxygenase. Mutation and characterization of the Y413A, Y413F, R98K and R98A enzymes indicate that these residues are in the active site and interact with the substrate. Deletion of the OH group of Tyr413 increases the Kd for the substrate and makes CH bond cleavage totally rate limiting. The pH V/Ktrp rate profile for the Tyr413 mutant enzymes shows that this residue must be protonated for activity. For both the R98A and R98K enzymes flavin reduction is rate limiting. The Vmax and V/Ktrp pH profiles indicate that the unprotonated form of the substrate is the active form for activity.

Flavocytochrome b2 catalyzes the oxidation of lactate to pyruvate. Primary deuterium and solvent kinetic isotope effects have been used to determine the relative timing of cleavage of the lactate OH and CH bonds by the wild type enzyme, a mutant protein lacking the heme domain, and the D282N enzyme. The DVmax and D(V/Klactate) values are both 3.0, 3.6 and 4.5 for the wild type enzyme, flavin domain and D282N enzymes, respectively. The D20Vmax values are 1.38, 1.18, and 0.98 for the wild type enzyme, the flavin domain, and the D282N enzyme; the respective D20(V/Klactate) values are 0.9, 0.44, and 1.0. The Dkred value is 5.4 for the wild type enzyme and 3.5 for the flavin domain, whereas the D2Okred is 1.0 for both enzymes. The V/Klactate value for the flavin domain increases 2-fold at moderate concentrations of glycerol. The data are consistent with the lactate hydroxyl proton not being in flight in the transition state for CH bond cleavage and there being an internal equilibrium prior to CH bond cleavage which is sensitive to solution conditions. Removal of the hydroxyl proton may occur in this pre-equilibrium. Tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide, carbon dioxide and water. Sequence alignments identified this enzyme as a member of the L-amino acid oxidase family. The tyrosine and arginine residues in L-amino acid oxidase that bind the carboxylate of o-aminobenzoate are conserved and correspond to Tyr413 and Arg98 in tryptophan 2-monooxygenase. Mutation and characterization of the Y413A, Y413F, R98K and R98A enzymes indicate that these residues are in the active site and interact with the substrate. Deletion of the OH group of Tyr413 increases the Kd for the substrate and makes CH bond cleavage totally rate limiting. The pH V/Ktrp rate profile for the Tyr413 mutant enzymes shows that this residue must be protonated for activity. For both the R98A and R98K enzymes flavin reduction is rate limiting. The Vmax and V/Ktrp pH profiles indicate that the unprotonated form of the substrate is the active form for activity.

Transition 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.

Tyrosine hydroxylase (TyrH) catalyzes the pterin-dependent hydroxylation of tyrosine to form dihydroxyphenylalanine. The enzyme requires one atom of ferrous iron for activity. Using deuterated 4-methylphenylalanine substrates, intrinsic primary and secondary isotope effects of 9.6 ± 0.9 and 1.21 ± 0.08 have been determined for benzylic hydroxylation catalyzed by TyrH. The large, normal secondary isotope effect is consistent with a mechanism involving hydrogen atom abstraction to generate a radical intermediate. The similarity of the isotope effects to those measured for benzylic hydroxylation catalyzed by cytochrome P-450 suggests that a high-valent, ferryl-oxo species is the hydroxylating species in TyrH. Uncoupled mutant forms of TyrH have been utilized to unmask isotope effects on steps in the aromatic hydroxylation pathway which also implicate a ferryl-oxo intermediate. Inverse secondary isotope effects were seen when 3,5-2H2-tyrosine was used as a substrate for several mutant enzyme forms. This result is consistent with a direct attack by a ferryl-oxo species on the aromatic ring of tyrosine forming a cationic intermediate. Rapid-freeze quench Mössbauer studies have provided preliminary spectroscopic evidence for an Fe(IV) intermediate in the reaction catalyzed by TyrH. The role of the iron atom in the regulatory mechanism has also been investigated. The iron atom in TyrH, as isolated, is in the ferric form and must be reduced for activity. The iron can be reduced by a number of one-electron reductants including tetrahydrobiopterin, ascorbate, and glutathione; however, it appears that BH4 (kred = 2.8 ± 0.1 mM-1 s-1) is the most likely candidate for reducing the enzyme in vivo. A one-electron transfer would require a pterin radical. Rapid-freeze quench EPR experiments aimed at detecting the intermediate were unsuccessful, suggesting that it decays very rapidly by reducing another equivalent of enzyme. The active Fe(II) form can also become oxidized by oxygen (210 ± 30 M-1 s-1); this increases the affinity of catecholamine inhibitors. Serine 40 can be phosphorylated to relieve the inhibition; however, results with S40E TyrH show phosphorylation does not have an effect on the rate constant for reduction of the enzyme but causes a 40% decrease in the rate constant of oxidation.

Several organic reactions that could potentially involve coarctate transition states were investigated by a combination of experimental and theoretical studies. In the thermal fragmentation of ∆-1,3,4-oxadiazolines, the mechanism supported by kinetic isotope effects and theoretical calculations is a three-step process that does not demonstrate any special stabilization in coarctate transition states. Rather than undergoing a direct coarctate conversion to product, the mechanism avoids coarctate steps. The last step is a concerted coarctate reaction, but being concerted may be viewed as being enforced by the necessity to avoid high-energy intermediates. In the deoxygenation of epoxides with dichlorocarbene, the stabilization from the transition state aromaticity is not great enough to compete with the preference for asynchronous bonding changes. KIEs and calculations suggested that the reaction occurs in a concerted manner but with a highly asynchronous early transition state with much more Cα-O bond breaking than Cβ-O bond breaking. In the Shi epoxidation, a large β-olefinic 13C isotope effect and small α-carbon isotope effect indicated an asynchronous transition state with more advanced formation of the C-O bond to the β-olefinic carbon. The calculated lowest-energy transition structures are generally those in which the differential formation of the incipient C-O bonds, the "asynchronicity," resembles that of an unhindered model, and the imposition of greater or less asynchronicity leads to higher barriers. In reactions of cis-disubstituted and terminal alkenes using Shi's oxazolidinone catalyst, the asynchronicity of the epoxidation transition state leads to increased steric interaction with the oxazolidinone when a π-conjugating substituent is distal to the oxazolidinone but decreased steric interaction when the π-conjugating substituent is proximal to the oxazolidinone. Dynamic effects were studied in Diels-Alder reaction between acrolein and methyl vinyl ketone. This reaction yields two products in a ratio of 3.0 ± 0.5. Theoretical studies shows that only one transition structure is involved in the formation of both. Quasiclassical trajectory calculations on an MP2 surface give a prediction of a product ratio of 45:14 (3.2:1), which is in good agreement with the experimental observation.

In order to rationally optimize a reaction, it is necessary to have a thorough understanding of its mechanism. Consequently, great effort has been made to elucidate a variety of reaction mechanisms. However, the fundamental ideas needed to understand reaction mechanisms are not yet fully developed. Throughout the literature, one encounters numerous examples of experimental observations that are not explainable by conventional mechanistic ideas and methods. The research described in this dissertation employs a unique approach towards the identification and analysis of systems whose observations cannot be explained by conventional transition state theory (TST). The nucleophilic substitution of 4,4-dichloro-but-3-en-2-one by sodium-para-tolyl-thiolate was explored. It was deduced that the reaction was concerted and consequently, the product selectivity observed in the reaction cannot be explained by TST. Dynamic effects play a major role in the observed selectivity and this is further supported by the results of dynamic trajectory simulations. Using computational studies, the ethanolysis of symmetric aryl carbonates was also shown to be concerted, provided that the substrate possesses good leaving groups. Furthermore, extensive precedence has been set by Gutthrie, Santos, Schelgel, and others, detailing concerted substitutions at acyl carbon. The Fujiwara hydroarylation is thought to occur by either a C-H activation mechanism or an electrophilic aromatic substitution (EAS). The KIEs associated with this reaction have been determined and provide strong support for the latter. Computational studies also displayed fair agreement with experimentally determined KIEs, further supporting the EAS mechanism. Isotopic perturbation of equilibria is invaluable in helping to determine whether a structure exists as a single structure or whether it is a time average of two equilibrating structures. The bromonium cation of tetramethylethylene and hydrogen pthalate have been wrongly reported as existing as equilibrating structures. The time averaged geometries have been determined in each case, via a variety of methods and the myth of equilibrating structures in the above cases has been debunked.