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. / text

Enzyme dynamics has been established to play a crucial role in catalysis, and it has therefore become an important area of research to better understand enzymatic rate enhancements. The light-activated enzyme protochlorophyllide oxidoreductase (POR) is a well-studied model system where dynamics are known to be important for catalysis. The catalytic reaction involves a sequential hydride and proton transfer to reduce the C17-C18 double bond in the protochlorophyllide (Pchlide) substrate with NADPH as a cofactor to yield the chlorophyllide (Chlide) product. Both H-transfer steps are established to undergo quantum tunneling, as derived from the temperature-dependence of the kinetic isotope effects (KIEs). Furthermore, a role for ‘promoting motions/vibrations’ has been presumed from the temperature-dependence KIE data, which will be investigated further in this thesis by the study of the KIE response to pressure. A general overview of the pressure-dependence as a new experimental probe is presented and compared with temperature-dependencies of KIEs, to establish whether pressure is suitable as an alternative technique for studying the role of enzyme dynamics in catalysis. This involves a comparison of pressure data from other enzyme systems to newly collected data for POR. However, no clear trend between temperature and pressure data is observed and hence, it can be concluded that pressure effects can be difficult to interpret. A case by case analysis is required and needs to be combined with computational simulations based on structural evidence (e.g. X-ray crystallographic), which is not yet available for POR.Solvent-viscosity has been successfully used to probe enzyme dynamics in POR and provides information on the extent of any protein networks that are involved along the reaction coordinate. Here I investigate the solvent-viscosity dependence of both H-transfer reactions in POR for a range of homologous POR enzymes to obtain an evolutionary perspective of the protein dynamics required for catalysis. This has been successfully used in the past on a limited number of POR homologues and has led to the formulation of a hypothesis supporting a twin-track evolution of the two catalytic steps in POR. I observed a lack of solvent-viscosity dependence in case of the hydride transfer across all the investigated lineages, while the proton transfer was shown to be more strongly affected by viscosity in prokaryotic enzymes than in their eukaryotic counterparts. This supports the proposed theory, suggesting an early optimisation of the dynamics involved in the light-activated hydride transfer with a strong reliance on localised motion. Conversely, the proton transfer experienced selective pressure to reduce its dependence on complex solvent-slaved motion and that has led to localised dynamics in eukaryotic POR homologues. Additionally, I found that the enzymes from eukaryotic species have a higher rate of both H-transfer steps, suggesting that an optimisation of the active site architecture occurred upon endosymbiosis. Enzyme dynamics clearly have a pivotal role to play in catalysis of this unique light-activated enzyme and detection of these will only be possible by detailed structural information.

<p> AroA catalyzes a carboxyvinyl transfer reaction, forming enolpyruvyl shikimate 3-phosphate (EPSP) from shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP). Upon extended incubation, it forms EPSP ketal by intramolecular nucleophilic attack of O4H on C2' of the enolpyruvyl group. EPSP ketal was previously proposed to form by non-enzymatic breakdown of the tetrahedral intermediate (THI) which had dissociated from AroA. In this study, EPSP ketal formed in the presence of excess AroA, which demonstrated that it was formed in the active site. This eliminated non-enzymatic THI breakdown as its source, and demonstrated that AroA forms either a discrete EPSP cationic intermediate, or cl transition state with high cationic character. The pH dependence of non-enzymatic EPSP hydrolysis was examined in order to understand the intrinsic reactivity of the enolpyruvyl group. Acid catalysis accelerated EPSP hydrolysis> 10^8-fold. These results provide evidence for enolpyruvyl activation through protonation at C3', forming an unstable cationic intermediate, or a highly cation-like transition state. The incorporation of 2H into EPSP from solvent 2H20 during AreA-catalyzed hydrolysis was much slower than the hydrolysis rate, in the absence of inorganic phosphate in the reaction. This demonstrated that KIEs on AroA-catalyzed EPSP hydrolysis, when they are measured in the future, will reflect protonation of EPSP. A method was developed for KIE measurements on acid-catalyzed EPSP hydrolysis, which showed good reproducibility and no buffer dependence. Further experiments need to be completed on the acid-catalyzed KIEs and enzyme-catalyzed KIEs, followed by transition state analysis. This will precisely define the transition state structure of the enzyme-catalyzed EPSP hydrolysis reaction, and provide a good starting point for designing AroA inhibitors.</p> / Thesis / Master of Science (MSc)

<p>This thesis describes the use of kinetic isotope effects, linear free energy relationships and stereochamical studies to distinguish between different mechanistic alternatives and to obtain information about transition state structure.</p><p>In the first part fluorine and deuterium kinetic isotope effects were determined for the base promoted HF elimination from 4-fluoro-4-(4’-nitrophenyl)butane-2-on. During this work a new method for the determination of fluorine kinetic isotope effects was developed. The results from the study demonstrates that the reaction proceeds via an E1cB_ip mechanism.</p><p>In the second part the transition state structure for the S_N2 reaction between ethyl chloride and cyanide ion in DMSO was studied. Kinetic isotope effects for six different positions in the reacting system, both in cyanide and ethyl chloride, were determined. The experimental isotope effects were then compared with the theoretically predicted isotope effects. </p><p>The third part describes the use of Hammett type free-energy relationships and stereochemical evidence to study the mechanism of the copper catalysed alkene aziridination. The results from the study support a model that involves the simultaneous presence of two different copper nitrene intermediates. One which reacts non-stereospecifically via a radical intermediate and one which reacts stereospecifically via a concerted mechanism.</p><p>In the fourth part a mechanistic study of the Ru(aminoalcohol) catalysed transfer hydrogenation of acetophenone in isopropanol is described. Kinetic isotope effects were determined for both proton and hydride transfer. The observation of significant primary deuterium kinetic isotope effects for both proton and hydride transfer support a mechanism where the proton and hydride are transferred simultaneously in a concerted mechanism.</p>

The use of gas chromatography isotope ratio mass spectrometry (GC-IRMS) for compound specific stable isotope analysis is an underutilized technique because of the complexity of the instrumentation and high analytical costs. However stable isotopic data, when coupled with concentration measurements, can provide additional information on a compounds production, transformation, loss, and cycling within the biosphere and atmosphere. A GC-IRMS system was developed to accurately and precisely measure δ13C values for numerous oxygenated volatile organic compounds (OVOCs) having natural and anthropogenic sources. The OVOCs include methanol, ethanol, acetone, methyl ethyl ketone, 2-pentanone, and 3-pentanone. Guided by the requirements for analysis of trace components in air, the GC-IRMS system was developed with the goals of increasing sensitivity, reducing dead-volume and peak band broadening, optimizing combustion and water removal, and decreasing the split ratio to the IRMS. The technique relied on a two-stage preconcentration system, a low-volume capillary reactor and water trap, and a balanced reference gas delivery system. Measurements were performed on samples collected from two distinct sources (i.e. biogenic and vehicle emissions) and ambient air collected from downtown Miami and Everglades National Park. However, the instrumentation and the method have the capability to analyze a variety of source and ambient samples. The measured isotopic signatures that were obtained from source and ambient samples provide a new isotopic constraint for atmospheric chemists and can serve as a new way to evaluate their models and budgets for many OVOCs. In almost all cases, OVOCs emitted from fuel combustion were enriched in 13C when compared to the natural emissions of plants. This was particularly true for ethanol gas emitted in vehicle exhaust, which was observed to have a uniquely enriched isotopic signature that was attributed to ethanol’s corn origin and use as an alternative fuel or fuel additive. Results from this effort show that ethanol’s unique isotopic signature can be incorporated into air chemistry models for fingerprinting and source apportionment purposes and can be used as a stable isotopic tracer for biofuel inputs to the atmosphere on local to regional scales.

This thesis describes the use of kinetic isotope effects, linear free energy relationships and stereochamical studies to distinguish between different mechanistic alternatives and to obtain information about transition state structure. In the first part fluorine and deuterium kinetic isotope effects were determined for the base promoted HF elimination from 4-fluoro-4-(4’-nitrophenyl)butane-2-on. During this work a new method for the determination of fluorine kinetic isotope effects was developed. The results from the study demonstrates that the reaction proceeds via an E1cBip mechanism. In the second part the transition state structure for the SN2 reaction between ethyl chloride and cyanide ion in DMSO was studied. Kinetic isotope effects for six different positions in the reacting system, both in cyanide and ethyl chloride, were determined. The experimental isotope effects were then compared with the theoretically predicted isotope effects. The third part describes the use of Hammett type free-energy relationships and stereochemical evidence to study the mechanism of the copper catalysed alkene aziridination. The results from the study support a model that involves the simultaneous presence of two different copper nitrene intermediates. One which reacts non-stereospecifically via a radical intermediate and one which reacts stereospecifically via a concerted mechanism. In the fourth part a mechanistic study of the Ru(aminoalcohol) catalysed transfer hydrogenation of acetophenone in isopropanol is described. Kinetic isotope effects were determined for both proton and hydride transfer. The observation of significant primary deuterium kinetic isotope effects for both proton and hydride transfer support a mechanism where the proton and hydride are transferred simultaneously in a concerted mechanism.

No description available.

L’effet isotopique cinétique (KIE) est un outil puissant pour obtenir un aperçu sur le mécanisme d'une grande variété réactions. Nous avons observé différentes mesures de l’effet isotopique cinétique primaire du 13C pour la formation des α-, et β-mannopyranosides et des α- et β-glucopyranosides, en partant du sulfoxyde de glycosyle protégé par le groupement 4,6-O-benzylidène, par la spectroscopie RMN à ultrahaut champ (13C à 200 MHz et 1H à 800 MHz). Nous avons aussi calculé les KIE pour ces réactions en collaboration avec le Prof. Pratt à l'Université d'Ottawa. Les valeurs expérimentale et calculée (B3LYP / 6-31G (d, p) avec un modèle de continuum polarisable) sont en bon accord sauf pour l’α-mannopyranoside. Trois cas (-mannopyanoside,  et -glucopyranosides) parmi les quatre ont montré un caractère “SN2-like“, mais la formation de l'-mannopyranoside suggère fortement un mécanisme dissociatif (SN1). Une telle différence de mécanisme nécessite une authentification par des mesures cinétiques. Nous avons ensuite porté notre attention sur le développement d'une réaction intramoléculaire, comme horloge intramoléculaire, afin d’évaluer la cinétique relative des réactions de glycosylation. La formation des produits tricycliques fournit une grande évidence de l'existence d'un ion mannosyloxocarbénium comme un intermédiaire transitoire. Les réactions de compétition avec de l'isopropanol et du méthallyltriméthylsilane sont interprétées comme indiquant que la β-O-mannosylation passe par un mécanisme associatif (SN2-like), tandis que l’α-O-mannosylation et le β-C-mannosylation sont dissociative (SN1-like). Ceci est en plein accord avec nos résultats expérimentaux sur l’effet isotopique cinétique. Cette approche de la détermination de la cinétique relative des réactions de glycosylation est une méthode directe et est potentiellement applicable à une large variété de donneurs de glycosyle. / Kinetic isotopic effects (KIEs) are powerful tools to obtain insight into the mechanism of a great range of reactions. We demonstrated differing primary 13C kinetic isotope effect (KIE) measurements for the formation of α-, β-mannopyranosides and α-, β-glucopyranosides from the 4,6-O-benzylidene protected mannosyl and glucosyl sulfoxides by NMR (13C at 200 MHz and 1H at 800 MHz). We have also calculated the KIEs in collaboration with the Pratt group at the University of Ottawa for these reactions. Experimental and calculated (B3LYP/ 6-31G (d,p) values with a polarizable continuum model) were in good agreement, except for the -mannopyranoside. Three of (-mannpyanoside and the -, -glupyranosides) four cases showed a SN2-like character. The formation of the -mannopyranoside on the other hand suggests a strongly dissociative mechanism (SN1). Such a difference in mechanism necessarily demands authentication by kinetic measurements. We turned then our attention to the development of an intramolecular clock reaction with which to probe the relative kinetics of glycosylation reactions and to the formation of the tricyclic products that provides strong evidence for the existence of a mannosyl oxocarbenium ion as a transient intermediate. Competition reactions with isopropanol and trimethylmethallylsilane are interpreted as indicating β-O-mannosylation to proceed via an associative SN2-like mechanism, whereas α-O-mannosylation and β-C-mannosylation are dissociative and SN1-like. This is in full agreement with our experimental KIE results. This approach to the determination of relative kinetics of glycosylation reactions, is straightforward and is potentially applicable to a broad range of glycosyl donors.

EPR spectroscopy and isotopic substitution were used to gain increased knowledge about the proton-coupled electron transfer (PCET) mechanism for the reduction of the tyrosine D radical (YD*) in photosystem II. pL dependence (where pL is either pH or pD) of both the rate constant and kinetic isotope effect (KIE) was examined for YD* reduction. Second, the manner in which protons are transferred during the rate-limiting step for YD* reduction at alkaline pL was determined. Finally, high field electron paramagnetic resonance (EPR) spectroscopy was used to study the effect of pH on the environment surrounding both the tyrosine D radical and the tyrosine Z radical (YZ*). At alkaline pL, it was determined that the proton and electron are both transferred in the rate-limiting step of YD* reduction. At acidic pL, the proton transfer occurs first followed by electron transfer. Proton inventory experiments indicate that there is more than one proton donation pathway available to YD* during PCET reduction at alkaline pL. Additionally, the proton inventory experiments indicate that at least one of those pathways is multiproton. High field EPR experiments indicate that both YD* and YZ* are hydrogen bonded to neutral species. The EPR gx component for YD* is invariant with respect to pH. Analysis of the EPR gx component for Yz* indicates that its environment becomes more electropositive as the pH is increased. This is most likely due to changes in the hydrogen bond strength

<p>This thesis concerns the study of reaction mechanisms by means of kinetic isotope effects (KIEs). Studies of the nucleophilic bimolecular substitution (S_N2) reaction had the dual purpose of improving our fundamental understanding of molecular reactivity and assessing the ability of kinetic isotope effects to serve as mechanistic tools. The transition state of the S_N2 reaction between a cyanide ion and ethyl chloride in tetrahydrofuran was found to be reactant like and only slightly tighter than has been found previously for the same reaction in dimethyl sulphoxide. One conclusion was that the transition-state structure in this reaction was predicted fairly well by the theoretical calculations, even without solvent modelling. The S_N2 reactions between cyanide ions and <i>para</i>-substituted benzyl chlorides were found to have reactant-like transition states, of which the C_α-Cl bond was most influenced by the <i>para</i>-substitution. Theoretical calculations indicated that the chlorine KIEs could be used as probes of the substituent effect on the C_α-Cl bond if bond fission was not too advanced in the transition state. Furthermore, the nucleophile carbon ¹¹C/¹⁴C KIEs were determined for the reactions between cyanide ions and various ethyl substrates in dimethyl sulphoxide.</p><p>Precision conductometry was employed to estimate the aggregation status of tetrabutylammonium cyanide in tetrahydrofuran and in dimethyl sulphoxide, which is of interest as tetrabutylammonium cyanide is frequently used as the nucleophilic reagent in mechanistic investigations and synthetic reactions. The tendency for ion-pair formation was found to be very slight, significant, and very strong in dimethyl sulphoxide, water, and tetrahydrofuran, respectively. </p><p>The nitrogen kinetic isotope effect on monoamine oxidase B catalysed deamination of benzylamine was determined in an attempt to obtain conclusive evidence regarding the mechanism of the oxidation. Monoamine oxidase is an important drug target in connection with the treatment of, for example, depression and Parkinson’s disease, and knowledge on how the enzyme effects catalysis would facilitate the design of highly selective and efficient inhibitors.</p>