Organic reactions are systematically examined experimentally and theoretically to determine the role dynamics plays in the outcome of the reaction. It is shown that trajectory studies are of vital importance in understanding reactions influenced by dynamical motion. This dissertation discusses how a combination of kinetic isotope effects, theoretical calculations, and quasiclassical dynamics trajectories aid in the understanding of the solvolysis of p-tolyldiazonium cation in water, the cycloadditions of cyclopentadiene with diphenylketene and dichloroketene, and the cycloaddition of 2- methyl-2-butene with dichloroketene. In the solvolysis of p-tolyldiazonium cation, significant 13C kinetic isotope effects are qualitatively consistent with a transition state leading to formation of an aryl cation, but on a quantitative basis, the isotope effects are not adequately accounted for by simple SN1 heterolysis to the aryl cation. The best predictions of the 13C isotope effects for the heterolytic process arise from transition structures solvated by clusters of water molecules. Dynamic trajectories starting from these transition structures afford products very slowly. The nucleophilic displacement process for aryldiazonium ions in water is determined to be at the boundary of the SN2Ar and SN1 mechanisms. The reaction of cyclopentadiene with diphenylketene affords both [4 + 2] and [2 + 2] cycloadducts directly. This is surprising. There is only one low-energy transition structure for adduct formation. Investigation of this reaction indicates that quasiclassical trajectories started from a single transition structure afford both [4 + 2] and [2 + 2] products. Overall, an understanding of the products, rates, selectivities, isotope effects, and mechanism in these reactions requires the explicit consideration of dynamic trajectories.

This thesis is an investigation of the relative differences of acid ionization constants and ion mobility in D2O versus H2O under hydrothermal conditions, for acetic acid and phosphoric acid. Values of specific conductivity were measured for each acid in H2O and D2O, as well as for a series of strong electrolytes using a high-temperature high-pressure AC conductivity flow cell that was originally designed at the University of Delaware. The Fuoss-Hsai-Fernández-Prini (FHFP) equation was fitted to the experimental values of molar conductivity, Λ, to obtain molar conductivities at infinite dilution, Λo. The molar conductivities at infinite dilution for each acid were used to calculate degrees of dissociation and ionization constants in D2O versus H2O from 298 K to 571 K. Measured values of differences in pKaq in D2O vs H2O, ΔpKaq = [pKaq,D2O – pKaq,H2O], become relatively independent of temperature above 423 K, with values of: ΔpKaq 0.45 for acetic acid and ΔpKaq 0.35 for phosphoric acid. The Density Model was then fitted to the values of pKaq in H2O and D2O to represent their temperature dependence to a precision of ± 0.01 in ΔpKaq. Comparisons of the molar conductivities and ionic molar conductivities at infinite dilution for the strong electrolytes in H2O and D2O as a function of temperature have also been made, based on Walden’s rule correlations, (λ°•η)D2O / (λ°•η)H2O. Changes in values of (λ°•η)D2O / (λ°•η)H2O as a function of temperature are consistent with a change in the relative hydration behavior of ions, where the effective Stokes’ radii of the ions in D2O versus H2O changes at temperatures above ~ 450 K. Changes in values of (λ°•η)D2O / (λ°•η)H2O for D+/H+ and OD-/OH- suggest that proton hopping “Grotthuss” mechanisms become more efficient in D2O versus H2O with increasing temperature. / University Network of Excellence in Nuclear Engineering, Ontario Power Generation Ltd, Natural Science and Engineering Research Council of Canada

Statistical models like Transition State Theory (TST) and Rice-Ramsperger-Kassel-Marcus (RRKM) Theory have generally been successful in predicting the rates and selectivities of chemical reactions. However, these statistical models can fail to explain experimental results of ordinary organic reactions. For these reactions, consideration of nonstatistical dynamic effects or the detailed motion and momenta of the atoms is necessary to account for the experimental observations. Dynamic effects have been found to be important in a growing number of reactions and the nature of these effects can be varied. One of the most interesting reactions investigated is the ozonolysis of vinyl ethers. Ozonolysis of a homologous series of vinyl ethers in solution exhibit experimental product ratios wherein the selectivity among cleavage pathways increases with the size of the alkyl group to an extent that is far less than RRKM theory would predict. Trajectory studies account for the observed selectivities and support a mechanism involving a competition between cleavage of the primary ozonide and intramolecular vibrational energy redistribution. A recent theoretical study from our group predicted that a highly asynchronous organocatalytic Diels-Alder (DA) reaction, which is concerted in the potential energy surface, is stepwise in the free energy surface. Kinetic isotope effects (KIEs) were measured for three DA reactions. We envision that the entropic barrier may have several experimental consequences such as unusual isotope effects due to extensive recrossing. Preliminary results for the organocatalytic reaction show an intramolecular KIE close to unity that cannot be reconciled with statistical theories. This is in contrast with Lewis-acid catalyzed and thermal DA reactions, which exhibit substantial "normal" intramolecular KIEs that are in accord with TST predictions. Finally, the Baeyer-Villiger oxidation of cylohexanone in water was investigated. KIEs were measured for the oxidation of cyclohexanone with peracetic acid and trifluoroperacetic acid. When using peracetic acid as the oxidant, the alkyl migration was determined to be the rate-determining step based on significant intermolecular KIEs on the carbonyl and alpha-methylene carbons. A change in the rate-determining step is seen when trifluoroperacetic acid is used. Only the carbonyl carbon exhibits a significant isotope effect. Theoretical predictions provide an experimental picture of the transition states and qualitatively support these conclusions.

Non-statistical recrossing in ketene cycloadditions with alkenes, heavy-atom tunneling and the mechanism of the decarboxylation of Mandelylthiamin is investigated in this dissertation. A combination of experimental kinetic isotope effects and theoretical models and kinetic isotope effects is utilized for this endeavor. This dissertation also describes how the use of quasiclassical dynamic trajectories, microcanonical RRKM calculations, and canonical variational transition state theory in combination with small-curvature tunneling approximations is utilized to help advance our research methodology to better understand mechanism. In the cycloaddition of dichloroketene with cis-2-butene, significant amounts of recrossing is observed using quasiclassical dynamic trajectories. An unusual inverse 13C intramolecular KIE lead us to investigate the role that heavy atoms play in non-statistical recrossing. More importantly, this discovery has uncovered a new phenomena of entropic intermediates that not only applies to ketene cycloadditions, but can also be applicable to other "concerted" reactions such as Diels-Alder reactions. The ring-opening of cyclopropylcarbinyl radical has revealed that heavy-atom tunneling plays a major role. The intramolecular 13C kinetic isotope effects for the ring-opening of cyclopropylcarbinyl radical were unprecedentedly large and in combination with theoretical predictions and multidimensional tunneling corrections, the role of tunneling in this reaction can be better understood. The mechanism decarboxylation of mandelylthiamin has been extensively studied in the literature. However, until the use of theoretically predicted KIEs and theoretical binding motifs the rate-limiting step of this reaction has been hotly debated. In this dissertation, a discussion of how the theoretical KIEs indicate the initial C-C bond as the rate-limiting step and chelating binding motifs of pyridinium and mandelylthiamin to explain the observed catalysis is given.

<p> The c^13 isotope effect in acid and alkaline hydrolysis of both ethyl and tertiary butyl benzoate-carboxyl-c^14 have been measured. Significant isotope effects were found in all the hydrolyses except acid hydrolysis of the tertiary butyl oster. These results are additional evidence for the suggestion of Choen and Schneider that alkyl oxygen rather than normal acyl oxygen bond cleavage occurs when tertiary butyl benzoate is hydrolyzed in acid solution. No appreciable c^13 isotope effect was found to occur in thermal, aqueous, or acid-catalysed decarboxylation of anthranilic acid. From this observation and other experimental evidence, it is concluded that anthranilic acid decarboxylates by a bimolecular process in which the attack of the hydrogen ion is rate controlling. It is further suggested that the hydrogen ion attack is on the zwitterion at the c carbon position. Both the c^13 and the c^14 isotope effect in the decarboxylation of mesitoic acid were measured using a mass spectrometer. The c^14 isotope effect was found to be two and one half times the magnitude of the c^13 isotope effect instead of twice as present theory predicts. </p> / Thesis / Doctor of Philosophy (PhD)

Three simultaneous isotope effects have been determined for the Tschugaeff reaction, i.e., the thermal decomposition of an xanthate ester. The ratio of the rate constants, k32/k34, was found to be 1.009 for the thion sulphur atom and 1.002 for the thio-ether sulphur atom. The rate ratio k12/k13, for the xanthate carbon atom was found to have a value of 1.000. The findings of previous investigators of the Tschugaeff reaction had been consistent with either of two mechanisms. However, with the determination of the isotope effects referred to above, it has been possible to distinguish between the two possible mechanisms and to gain considerable information about the transition state of the Tschugaeff reaction. The measured isotope effects have been discussed in terms of Bigeleisen's isotopic rate equation. In addition, it has been possible to carry out theoretical calculations for each reaction mechanism by assuming an appropriate potential function and in this manner make the interpretation of the measured isotope effects more quantitative. / Thesis / Master of Science (MS)

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The high-resolution 195Pt NMR signals (128.8 MHz) of most of the octahedral mixed-ligand Pt(IV) complexes in the series [PtCl6-n(OH)n]2-, n = 0-6, have been recorded in aqueous solutions at 293 K. These signals show characteristic 35/37Cl isotope-induced fine structure that results from the presence of several isotopologues in samples with a natural chlorine isotope distribution; each 37Cl isotope incorporated into the Pt coordination sphere of one of these complexes affords a fixed upfield (low frequency) isotope shift of between 0.17 and 0.22 ppm, depending on the particular complex. This assignment is confirmed by the excellent agreement between the natural abundances of the various isotopologues and the relative contributions of the corresponding signals to the overall area of the experimental spectrum of the particular isotoplogue set, obtained by a non-linear least-squares line fitting procedure. These results confirm that the 195Pt magnetic shielding in isotopomers differing only in the combination of the two chlorine isotopes coordinated in sites trans to hydroxido-ligands are indistinguishable under these experimental conditions, unlike those of similar isotopomers in the related series of aqua-complexes [PtCln(H2O)6-n]4-n, n = 3-5, as reported by Koch and co-workers. Moreover, the order of 195Pt shielding for the members of all stereoisomer pairs in the series of hydroxido-complexes is the reverse of that reported for the corresponding pairs in the aqua-series. These and other observations are interpreted qualitatively in terms of the relative strengths of the trans-influences of aqua-, hydroxido- and chlorido-ligands and the effect of these on bond displacements in these complexes. The 195Pt NMR spectra of especially the complexes cis-[PtCl2(OH)4]2- and [PtCl(OH)5]2- show remarkable fine structure in a ca. 45 % 18O-enriched aqueous solution; apart from additional signals resulting from 18O-containing isotopologues, the resonance signals of isotopomers differing in the combination of 16/18O isotopes in sites trans to chlorido-ligands are partially resolved. The effect of temperature on the 35/37Cl isotope-induced fine structure in the 195Pt signals of [PtCl6]2- and [PtCl(OH)5]2- was investigated in the range 283-308 K; some interesting differences are observed. 195Pt relaxation time measurements for [PtCl6]2- in this temperature range reveal that line-broadening is at least partially responsible for the loss of resolution between the signals of isotopologues of this complex as the temperature is increased, possibly due to the spin-rotation relaxation mechanism. The temperature coefficient of 195Pt shielding and the magnitude of isotope shifts in the spectra of the complexes in this series show interesting correlations with the 195Pt shielding itself; an interpretation of these observations is presented. / AFRIKAANSE OPSOMMING: Die hoëresolusie 195Pt NMR seine (128.8 MHz) van die oktaëdriese gemengde-ligand Pt(IV) komplekse in die reeks [PtCl6-n(OH)n]2- is waargeneem in waterige oplossing by ʼn temperatuur van 293 K. Hierdie seine toon ʼn karakteristieke 35/37Cl isotoop-geïnduseerde fynstruktuur as gevolg van die teenwoordigheid van verskeie isotopoloë in monsters met ʼn natuurlike chloor isotoopverspreiding. Die verplasing van ʼn 35Cl isotoop deur ʼn 37Cl isotoop in die Pt koördinasiesfeer van hierdie komplekse lei tot ʼn laefrekwensie isotoopverskuiwing van die 195Pt resonansiesein van tussen 0.17 en 0.22 ppm, afhangend van die spesifieke kompleks. Die toekenning van resonansieseine in hierdie spektra word ondersteun deur die goeie ooreenstemming tussen die berekende natuurlike verspreiding van isotopoloë en die persentasie area bydrae van die ooreenstemmende pieke tot die area van volledige stel seine van die chemiese spesie, soos bepaal deur ʼn nie-linieêre kleinste-kwadrate passingsmetode. Hierdie resultate bevestig dat vir isotopomere waarvan slegs die kombinasie van chloorisotope wat in posisies trans tot hidroksido-ligande gekoördineer is ʼn ononderskeibare 195Pt magnetiese skerming waargeneem word, m.a.w. ʼn enkele resonansiesein word vir hierdie isotopomere gemeet, anders as gerapporteer deur Koch en medewerkers vir die verwante aqua-komplekse [PtCln(H2O)6-n]4-n waar n = 3-5. Verder is die order van 195Pt magnetiese skerming vir stereoisomere in hierdie hidroksido-komplekse in elke stereoisomer paar die teenoorgestelde van dit waargeneem vir die ooreenstemmende aqua-komplekse. Hierdie waarnemings word kwalitatief geïnterpreteer in terme van die verskillende trans-invloede van die chlorido-, aqua- en hidroksido-ligande en die effekte daarvan op bindingslengtes in die komplekse. In ʼn ongeveer 45 % 18O-verrykte monster toon die 195Pt seine van veral die komplekse cis-[PtCl2(OH)4]2- en [PtCl(OH)5]2- uitsonderlike fynstruktuur vanweë die addisionele seine van 18O-bevattende isotopoloë en die parsiële resolusie van die seine van isotopomere wat verskil in die kombinasie van 16/18O isotope wat trans tot chlorido-ligande gekoördineer is. ʼn Studie is gemaak van die uitwerking van temperatuur op die 35/37Cl isotoop-geïnduseerde fynstruktuur in die 195Pt seine van die komplekse [PtCl6]2- en [PtCl(OH)5]2- in die gebied 283-308 K; interessante verskille is waargeneem. 195Pt magnetiese relaksasietyd metings vir die kompleks [PtCl6]2- in waterige oplossing in hierdie temperatuurgebied toon dat verbreeding van resonansieseine ten minste gedeeltelik verantwoordelik is vir die waargenome verlies aan resolusie tussen die seine van isotopoloë namate die temperatuur styg; die verbreeding van seine kan waarskynlik aan die spin-rotasie relaksasiemeganisme toegeskryf word. Die temperatuurkoëffisiënt van 195Pt magnetiese skerming en die grootheid van isotoopverskuiwings in die spektra van die hidroksido-komplekse in hierdie reeks toon interessante korrelasies tot die 195Pt magnetiese skerming; ʼn interpretasie van hierdie waarnemings word voorgestel.

How the fast (femtosecond-picosecond, fs-ps) protein dynamics contribute to enzymatic function has gained popularity in modern enzymology. With multiple experimental and theoretical studies developed, the most challenging part is to assess both the chemical step kinetics and the relevant motions at the transition state (TS) on the fast time scale. Formate dehydrogenase (FDH), which catalyzes a single hydride transfer reaction, is a model system to address this specific issue. I have crystallized and solved the structure of FDH from Candida boidinii (CbFDH) in complex with NAD+ and azide. With the guidance of the structure information, two active site residues were identified, V123 and I175, which could be responsible for the narrow donor-acceptor-distance (DAD) distribution observed in the wild type CbFDH. This thesis describes studies using kinetic isotope effects (KIEs) and their temperature dependence together with two-dimensional infrared spectroscopy on the recombinant CbFDH and its V123 and I175 mutants. Those mutants were designed to systematically reduce the size of their side chain (I175V, I175A, V123A, V123G and double mutant I175V/V123A), leading to broader distribution of DADs. The kinetic experiments identified a correlation between the DAD distribution and the intrinsic KIEs. The contribution of the fs-ps dynamics was examined via two-dimensional infrared spectroscopy (2D IR) by measuring the vibrational relaxation of TS analog inhibitor, aizde, reflecting the TS environmental motions. Our results provide a test of models for the kinetics of the enzyme-catalyzed reaction that invokes motions of the enzyme at the fs-ps time scale to explain the temperature dependence of intrinsic KIEs.

Protein motions are complex, including occurring at different time scales, and their roles in enzyme-catalyzed reactions have always been of great interest among enzymologists. In order to characterize the potential factors that play a role on the chemical step of enzymatic reactions, variants of dihydrofolate reductase have been used as a benchmark system to study the motions of proteins correlated with the chemical step. A “global dynamic network” of coupled residues in Escherichia coli dihydrofolate reductase (ecDHFR), which assists in catalyzing the chemical step, has been demonstrated through quantum mechanical/molecular mechanical and molecular dynamic (QM/MM/MD) simulations, as well as bioinformatic analyses. A few specific residues — M42, G121, and I14 — were shown to function synergistically with measurements of single turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. Although similar networks have been found in other enzymes, the general features of these networks are still unclear. This project focuses on exploring homologous residues of the proposed global network in human DHFR through computer simulations and measurements of the temperature dependence of KIEsint. The mutants M53W and S145V, both remote residues, showed significant decreases in catalytic efficiency. Non-additive isotope effects on activation energy were observed between M53 and S145, indicating their synergistic effect on hydride transfer in human DHFR. Apart from the effects of the conserved residues, we also extend our studies to exploring three potential phylogenetic events that account for the discrepancies between E. coli and human DHFR. They are L28, PP insertion and PEKN insertions by phylogenetic sequence analysis. Two of them (N23PP and G51PEKN E. coli DHFR) have been proved to be important both by MD simulation and experimental probe of KIEs measurement. The experiments have found that PP insertion itself rigidified the M20 loop and motions coupled to hydride transfer were impaired, however, loop rigidification was improved after incorporating PEKN. Furthermore, deletion of PP and PEKN of the engineered human enzyme also show a similar outcome. However, the effect of the key residue of L28 is not clear. In this project, we have step-wise engineered the human DHFR to be like hagfish (F31M) and E. coli (F32L). And it is found out that there is an increase in the temperature dependence of KIEs when the enzyme was bacterilized into a more primitive variant. This indicates that not only is residue F32 important and correlated with the chemical step as indicated by bioinformatic studies, but it is possible to trace the evolutionary trajectory. A triple mutation F32L-PP26N-PEKN62G on the human DHFR was also conducted, and it is not surprising to find out that the temperature dependence of KIEs has retained its behavior like wild-type human DHFR. These results suggest that the three predicted phylogenetically coherent events coevolved together to maintain the evolutionary preservation of the protein dynamics to enable H-tunneling from well-reorganized active sites. As has been indicated by the previous project, as the enzyme evolves, the active site of the enzyme will “reorganize” to form the optimal transition state for chemical step (from F32L-F32M-wild type DHFR). Here in this project, we aimed to systematically address this point of view through a series of cyclic permutation DHFR from directed evolutions. As this primitive enzyme is 7 orders of magnitude less efficient than the well-evolved human DHFR, together with four generations of evolved variants (cp, cp’ and cp”), this provides a good model system for explorations of the molecular basis of enzyme evolution. It is found that the organizations of transition state are improved before the catalytic efficiency is enhanced as the enzyme evolves.

The present dissertation seeks to deepen our understanding of hydrogen transfers and especially C-H bond activations in enzymes. Hydrogen transfers are ubiquitous in chemistry and biology and a thorough understanding of how they occur and what factors influence them will facilitate developments in biomimetic catalysis, rational drug design, and other fields. A particular difficulty with H-transfers is the importance of nuclear quantum effects to the reaction, particularly tunneling. The overall scope of the work here aims to examine how experimental kinetic isotope effects (KIEs) can be interpreted with a particular type of tunneling model, referred to as Marcus-like models, to yield a semi-quantitative picture of the physical mechanisms of H-transfers. Previous work had used this kind of model to qualitatively interpret experimental data using a combination of intuition and generalized theories. The work here examines these theories in quantitative detail, testing and calibrating our intuition in the context of several experimental systems. The first chapter of research (ch. II) focusses on the temperature dependence of primary KIEs and how these experiments can be quantitatively interpreted as a probe for certain kinds of enzyme or solvent dynamics. The subsequent chapters (ch. III-VI) focus on the use of secondary KIEs to determine the detailed structures of tunneling ready states (TRSs) and how the dynamics of H-tunneling affect those structures. These chapters focus primarily on the TRS of the enzyme alcohol dehydrogenase, but by examining an uncatalyzed analogue to that reaction (ch. VI), the work gains some insight about similarities and differences between catalyzed and uncatalyzed reactions. In summary, the work uncovers some principles of catalysis, not just the mechanism of a catalyzed reaction. The mechanism of C-H activation presented here provides an elegant solution to problems that have been vexing to accommodate within traditional models. This work constitutes some initial steps in making Marcus-like models quantitatively useful as a supplement or even replacement for traditional models of reactivity.