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Intramolecular Isotope Effects for the Study of Reactions with MassTransfer LimitationsWagner, Joshua G. 16 January 2010 (has links)
The research presented provides a method to use the comparison of intermolecular isotope effects vs. the intramolecular isotope effects for the study of reactions in which study of the rate limiting step is ambiguous due to interfering mass transfer effects. The oxidation of unfunctionalized hydrocarbons at mild conditions developed by Sir Derek Barton, the Gif reaction is the model used. The history is provided to demonstrate the relevance of using this model as one which could show the usefulness of this method. Evidence has been provided and used to theorize that the rate limiting step of the reaction may be diffusion of the reactants, not a chemical change. Starting materials were made which would allow for the measurement for both the intermolecular and intramolecular KIE and those values were compared. The results show that there is little difference between the intermolecular and intramolecular KIE, therefore the reaction is not diffusion controlled.
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Intramolecular Isotope Effects for the Study of Reactions with MassTransfer LimitationsWagner, Joshua G. 16 January 2010 (has links)
The research presented provides a method to use the comparison of intermolecular isotope effects vs. the intramolecular isotope effects for the study of reactions in which study of the rate limiting step is ambiguous due to interfering mass transfer effects. The oxidation of unfunctionalized hydrocarbons at mild conditions developed by Sir Derek Barton, the Gif reaction is the model used. The history is provided to demonstrate the relevance of using this model as one which could show the usefulness of this method. Evidence has been provided and used to theorize that the rate limiting step of the reaction may be diffusion of the reactants, not a chemical change. Starting materials were made which would allow for the measurement for both the intermolecular and intramolecular KIE and those values were compared. The results show that there is little difference between the intermolecular and intramolecular KIE, therefore the reaction is not diffusion controlled.
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Application of kinetic isotope effects and theoretical calculations to interesting reaction mechanismsHirschi, Jennifer Sue 15 May 2009 (has links)
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.
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The experimental and theoretical determination of combinatorial kinetic isotope effects for mechanistic analysisChristian, Chad F. 15 May 2009 (has links)
Unfortunately, chemists can never experimentally unravel a full reaction pathway.
Even our ability to define key aspects of mechanisms, such as short-lived intermediates
and the even more ephemeral transition states, is quite limited, requiring subtle
experiments and subtle interpretations. Arguably the most important knowledge to be
gained about the mechanism of a reaction is the structure and geometry of the transition
state at the rate-limiting step, as this is where a reaction’s rate and selectivity are
generally decided. The Singleton group has developed a methodology for predicting the
combinatorial kinetic isotope effects (KIEs) at every atomic position, typically carbon or
hydrogen, at natural abundance. A combination of experimental isotope effects and
density functional theory (DFT) calculations has greatly aided our ability to predict and
understand a reaction’s pathway and transition state geometries. Precise application of
this method has allowed for the mechanistic investigation of a myriad of bioorganic,
organic, and organometallic reactions. The technique has been applied in the analysis of
the catalytic borylation of arenes via C-H bond activation, dynamic effects in the enyne
allene cyclization, palladium catalyzed allylic alkylation, the nature of proton transfer in
orotate decarboxylase, and the epoxidation of enones with t-butyl hydroperoxide.
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Evaluation of transition state models using chlorine kinetic isotope effects and high resolution vibrational measurementsJulian, Robert Lynn, January 1976 (has links)
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).
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Mechanistic Investigations into the Origin of Selectivity in Organic ReactionsThomas, Jacqueline Besinaiz 15 May 2009 (has links)
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.
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Studies of the chemical mechanisms of flavoenzymesSobrado, Pablo 30 September 2004 (has links)
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.
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Mechanisms of Decarboxylation: Internal Return, Water Addition, and Their Isotope EffectsMundle, Scott Owen Chelmsford 31 August 2010 (has links)
2-(2-mandelyl)thiamin (MTh), the adduct of benzoylformate and thiamin, is an accurate model of 2-(2-mandelyl)thiamin diphosphate, the initial covalent intermediate in the decarboxylation of benzoylformate by benzoylformate decarboxylase (BFDC). The first order rate constant for spontaneous decarboxylation of MTh is about 106 times smaller than the enzymic rate (kcat) for the BFDC reaction. Based on the similarities of MTh and the corresponding enzymic intermediate, as well as the inherent nature of the intermediate, it is not obvious why the enzyme-catalyzed reaction is so much faster. However, earlier studies showed that the decarboxylation of MTh is catalyzed by protonated pyridines and this was proposed to occur through a preassociation mechanism. If this explanation is correct, then the observed 12C/13C kinetic isotope effect (CKIE) will increase in the presence of the catalyst as a more favorable forward commitment is made possible. This provides a specific model for the enzyme-catalyzed process.
We developed a technique using headspace analysis and compound specific isotope analysis (CSIA) to determine the CKIE for the decarboxylation of MTh in the presence and absence of pyridinium. We found that the CKIE increases in the presence of the catalyst, as predicted for the preassociation mechanism.
In a related study, we investigated the kinetics of decarboxylation of pyrrole-2-carboxylic acid, which was known to be subject to acid catalysis in highly acidic solutions. In the expected mechanism, protonation of the pyrrole ring at C2 destroys the aromaticity of the ring. C-C bond cleavage in the process of decarboxylation will re-establish the aromatic pyrrole. However, the overall reaction rate would not increase as it is counteracted by a larger concentration of the undissociated carboxyl group compared to carboxylate ion necessary for decarboxylation.
Since the reaction occurs readily, there must be an alternative pathway for the acid-catalyzed reaction. This can be achieved in an associative mechanism that is initiated by addition of water to the carboxyl group of the carboxyl-protonated reactant. C-C bond cleavage results in formation of pyrrole and protonated carbonic acid, a species that has been recognized as a viable intermediate in related processes. Protonated carbonic acid is spontaneously converted to H3O+ and carbon dioxide. The associative mechanism is consistent with solvent-deuterium kinetic isotope effects and 12C/13C kinetic isotope effects.
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Mechanisms of Decarboxylation: Internal Return, Water Addition, and Their Isotope EffectsMundle, Scott Owen Chelmsford 31 August 2010 (has links)
2-(2-mandelyl)thiamin (MTh), the adduct of benzoylformate and thiamin, is an accurate model of 2-(2-mandelyl)thiamin diphosphate, the initial covalent intermediate in the decarboxylation of benzoylformate by benzoylformate decarboxylase (BFDC). The first order rate constant for spontaneous decarboxylation of MTh is about 106 times smaller than the enzymic rate (kcat) for the BFDC reaction. Based on the similarities of MTh and the corresponding enzymic intermediate, as well as the inherent nature of the intermediate, it is not obvious why the enzyme-catalyzed reaction is so much faster. However, earlier studies showed that the decarboxylation of MTh is catalyzed by protonated pyridines and this was proposed to occur through a preassociation mechanism. If this explanation is correct, then the observed 12C/13C kinetic isotope effect (CKIE) will increase in the presence of the catalyst as a more favorable forward commitment is made possible. This provides a specific model for the enzyme-catalyzed process.
We developed a technique using headspace analysis and compound specific isotope analysis (CSIA) to determine the CKIE for the decarboxylation of MTh in the presence and absence of pyridinium. We found that the CKIE increases in the presence of the catalyst, as predicted for the preassociation mechanism.
In a related study, we investigated the kinetics of decarboxylation of pyrrole-2-carboxylic acid, which was known to be subject to acid catalysis in highly acidic solutions. In the expected mechanism, protonation of the pyrrole ring at C2 destroys the aromaticity of the ring. C-C bond cleavage in the process of decarboxylation will re-establish the aromatic pyrrole. However, the overall reaction rate would not increase as it is counteracted by a larger concentration of the undissociated carboxyl group compared to carboxylate ion necessary for decarboxylation.
Since the reaction occurs readily, there must be an alternative pathway for the acid-catalyzed reaction. This can be achieved in an associative mechanism that is initiated by addition of water to the carboxyl group of the carboxyl-protonated reactant. C-C bond cleavage results in formation of pyrrole and protonated carbonic acid, a species that has been recognized as a viable intermediate in related processes. Protonated carbonic acid is spontaneously converted to H3O+ and carbon dioxide. The associative mechanism is consistent with solvent-deuterium kinetic isotope effects and 12C/13C kinetic isotope effects.
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The chemical mechanisms of flavin-dependent amine oxidases and the plasticity of the two-his one-carboxylate facial triad in tyrosine hydroxylaseRalph, Erik C. 15 May 2009 (has links)
Despite a number of kinetic and spectroscopic studies, the chemical mechanisms
of amine oxidation by flavoenzymes remain widely debated. The mechanisms of by Nmethyltryptophan
oxidase (MTOX) and tryptophan 2-monooxygenase (TMO) were
probed using a combination of pH and primary deuterium, solvent, and 15N kinetic
isotope effects. Slow substrates were chosen for these studies; MTOX was characterized
with N-methylglycine and TMO was characterized with L-alanine. Primary deuterium
kinetic isotope effects of 7.2 and 5.3 were observed for sarcosine oxidation by MTOX
and for alanine oxidation by TMO, respectively, independent of the substrate
concentration and pH. Monitoring the reduction of flavin spectroscopically revealed no
intermediate flavin species with both enzyme-substrate systems. Furthermore, the
magnitudes of the 15N kinetic isotope effects observed with both systems suggest that
nitrogen rehybridization and C-H bond cleavage are concerted. These results are
consistent with both enzymes utilizing a hydride transfer mechanism for amine
oxidation.
The role of the iron ligands of tyrosine hydroxylase (TyrH) was also investigated. TyrH contains one iron per monomer, which is held by three conserved amino acid
residues, two histidines and a glutamate. As a probe of the plasticity of the metal binding
site, each of the metal ligands in TyrH was substituted with glutamine, glutamate, or
histidine. The resulting proteins were characterized for metal content, catalytic activity,
and dopamine binding. The H336E and H336Q enzymes retain substantial catalytic
activity. In contrast, the E376Q enzyme retains about 0.4% of the wild-type catalytic
activity, and the E376H enzyme has no significant activity. The H331E enzyme oxidizes
tetrahydropterin in a tyrosine-independent manner. The position of the charge-transfer
absorbance band for the H336E and H336Q enzyme-inhibitor complexes is shifted
relative to that of the wild-type enzyme, consistent with the change in the metal ligand.
In contrast, the E376H and E376Q enzymes catalyze dopamine oxidation. These results
provide a reference point for further structural studies of TyrH and the other aromatic
amino acid hydroxylases, and for similar studies of other enzymes containing this ironbinding
motif.
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