The hydroboration of simple alkenes with BH3 preferentially occurs in an anti- Markovnikov fashion. The standard explanation for this preference, reproduced in all general organic chemistry textbooks, is that the selectivity arises from a greater stability for the anti-Markovnikov transition state. This explanation presupposes the applicability of the transition-state theory model for reactivity and selectivity. This dissertation explores the applicability of transition state theory to selectivity in hydroborations and finds that in some cases transition state theory fails to accurately account for observations. Experimental results for the hydroboration of propene-d6 and styrene-d8 with excess BH3 was analyzed by 2H-NMR to determine the percentage of the Markovnikov product for the BH3-mediated reaction. The experimental selectivities were then compared with predictions based on very high-level calculations using transition state theory. It was observed that the regioselectivity of the hydroboration of these alkenes is lower than can be accounted for by transition state theory. The regioselectivity discrepancy was explored through dynamic trajectory analysis. It is proposed here that the observed regioselectivity is that of a “hot” reaction, resulting from an exothermic association of alkene with borane to form an intermediate complex. This complex then overcomes low-energy barriers to form anti-Markovnikov and Markovnikov products faster than excess energy is lost to solvent. This hypothesis was explored for the hydroboration of internal disubstituted and trisubstituted alkenes. The applicability of transition state theory and the role of dynamics in determining the selectivity was gauged by determining product ratios in the presence of large excesses of borane and by considering the energetics of the calculated hydroboration reaction path. In all cases the enthalpic barriers for the rate-limiting association step and the formation of products from the intermediate π -complex were small. Isotope effects were determined experimentally and were found to be too small for the conventional mechanism to be the predominate pathway. When the hydroboration reaction of propene with BH2Cl or BHCl2 was explored through a series of experimental and theoretical studies, we observed that the regioselectivity was lower than that predicted from transition state theory. However, the calculated pathways indicated that energy barriers for product formation were too large for this reaction to be considered a “hot” reaction. The regioselectivity discrepancy was attributed to the chloroboranes undergoing equilibration with selective reaction of the most highly reactive forms of the borane.

In the design of novel materials, tunable properties are parameters such as composition or structure that may be adjusted in order to enhance a desired chemical or material property. Trends in tunable properties can be accurately predicted using computational and combinatorial chemistry tools in order to optimize a desired property. I present a study of tunable properties in materials and employ a variety of algorithms that ranges from simple screening to machine learning. In the case of tuning a nanocomposite membrane for olefin/paraffin separations, I demonstrate a rational design approach based on statistical modeling followed by ab initio modeling of the interaction of olefins with various nanoparticles. My simplified model of gases diffusing on a heterogeneous lattice identifies the conditions necessary for optimal selectivity of olefins over paraffins. The ab initio modeling is then applied to identify realistic nanomaterials that will produce such conditions. The second case, [alpha]-Fe₂O₃, commonly known as hematite, is potential solar cell material. I demonstrate the use of a screened search through chemical compound space in order to identify doped hematite-based materials with an ideal band gap for maximum solar absorption. The electronic structure of hematite is poorly treated by standard density functional theory and requires the application of Hartree-Fock exchange in order to reproduce the experimental band gap. Using this approach, several potential solar cell materials are identified based on the behavior of the dopants within the overall hematite structure. The final aspect of this work is a new method for identifying low-energy chemical processes in condensed phase materials. The gap between timescales that are attainable with standard molecular dynamics and the processes that evolve on a human timescale presents a challenge for modeling the behavior of materials. This problem is particularly severe in the case of condensed phase systems where the reaction mechanisms may be highly complicated or completely unknown. I demonstrate the use of support vector machines, a machine-learning technique, to create transition state theory dividing surfaces without a priori information about the reaction coordinate. This method can be applied to modeling the stability of novel materials. / text

With advancements in computer technology and processing power, the ability to examine chemical systems using theory continues to be more practicable. Using ab initio methods, such as density functional theory, we are now able to routinely simulate hundreds of atoms. This system size allows us to directly simulate surfaces and nano-materials that are industrially relevant. With the expansion of accessible systems comes the opportunity to develop new computational methods to extract their chemical properties. Of particular interest is bridging the time scale gap between simulation and experiment. The evolution of a system chemical in time can be directly simulated using classical dynamics, however, molecules vibrate on the order of femtoseconds and interesting transitions tend to happen on much longer time scales: milliseconds to seconds. In condensed phase chemical systems these interesting transitions are hindered by energy barriers so state to state dynamics are dominated by rare evens. Luckily, rare event transitions tend to happen through mountain passes in the potential energy landscape. Within harmonic transition state theory, the transition states between minima can be characterized by saddle points. Finding saddle points is a challenging problem which has not been satisfactorily solved; nevertheless, there are algorithms currently being used despite their deficiency. In particular, my work strives to improve the efficiency and stability of the nudged elastic band method and compare its performance to similar algorithms on a variety of test systems. In addition, I present a method to predict how energy-based chemical properties change with respect to the chemical composition of the system. This is achieved by taking a derivative of the property with respect to the atomic numbers of the atoms present in the system. The accuracy and predictive quality of these derivatives are assessed for both model and industrially relevant systems. With this information, we can follow these derivatives to optimize a desired property in the space of chemical composition. This method is a step toward using theory to rationally design compounds with desirable properties. / text

Molecular simulation is an essential tool in understanding complex chemical and biochemical processes as real-life experiments prove increasingly costly or infeasible in practice . This thesis is devoted to methodological aspects of molecular simulation, with a particular focus on computing transition paths and their associated free energy profiles. The first part is dedicated to computational methods for reaction path and transition state searches on a potential energy surface. In Chapter 3 we propose an improvement to a widely-used transition state search method, the Activation Relaxation Technique (ART). We also present a local convergence study of a prototypical algorithm. The second part is dedicated to free energy computations. We focus in particular on an adaptive importance sampling technique, the Adaptive Biasing Force (ABF) method. The first contribution to this field, presented in Chapter 5, consists in showing the applicability to a large molecular system of a new parallel implementation, named multiple-walker ABF (MW-ABF). Numerical experiments demonstrated the robustness of MW-ABF against artefacts arising due to poorly chosen or oversimplified reaction coordinates. These numerical findings inspired a new study of the longtime convergence of the ABF method, as presented in Chapter 6. By studying a slightly modified model, we back our numerical results by showing a faster theoretical rate of convergence of ABF than was previously shown

Abstract Histidine acid phosphatases are characterized by the presence of a conserved RHGXRXP motif. One medically important acid phosphatase is the Prostatic Acid Phosphatase (PAP). PAP has been associated with prostate cancer for a long time and has been used as a marker to stage prostate carcinoma. Yet, there is no clear understanding on the functioning of the enzyme in vivo. This thesis work focuses on the characterization of putative ligands and elucidation of the reaction mechanism of PAP using computational methods. The ligand-enzyme complexes were generated by docking and molecular dynamics simulations. The complexes showed that the conserved arginines of RHGXRXP motif are important for binding the highly negatively charged phosphate group. The complexes also highlighted that the active site aspartate (Asp258) should be neutral in the complex and is involved as a general acid-base in the reaction. The studies support that PAP could dephosphorylate the growth factor receptors EGFR and ErbB-2. The studies also found that the majority of tyrosine phosphorylated peptides from these growth factor receptors could bind to PAP. The affinities were assessed based on theoretical calculations and were further confirmed by experimental measurements in the feasible cases. To clearly understand the mechanism of PAP, quantum mechanical methods were employed. The enzymatic reaction involves two steps. In the first step, the phosphate moiety is transferred from the ligand to the conserved histidine. The calculations on the first step of the reaction involved generating the transition state (TS) structures and estimating the respective barriers. The calculations clearly support that Asp258 becomes neutral by picking up the proton from the monoanionic ligand entering the binding site. The proton from neutral Asp258 is later transferred to the leaving group via a water bridge, restoring the negative state of Asp258. The second step involves the hydrolysis of phosphohistidine enzyme intermediate. Using hybrid quantum mechanics/molecular mechanics calculations, it was found that the Asp258 accepts a proton from the nucleophilic water only after the TS is crossed. This proton is possibly then transferred to the free phosphate while it leaves the binding site, restoring the enzyme to its free state. The study highlights the importance of active site arginines in the binding as well as the stabilization of TS. Further, the analysis of TS structures in both the steps showed an associative mechanism, based on the distance of the nucleophilic and the leaving atoms to the phosphate atom. These distances are much smaller than what has been found in other well studied nonmetallo-phopshatases. Thus, the study finds a novel mechanism of enzymic phosphotransfer in PAP mediated catalysis.

Transglutaminases (TGases) are a group of enzymes that catalyze the formation of an amide bond between the γ-carboxamide group of a glutamine residue and an amine donor, usually an ε-amino group of the lysine residue, leading to the formation of ε-(γ-glutamyl)lysine crosslinks. Owing to the roles that transglutaminases such as tissue transglutaminase (TG2) and Factor XIIIa (FXIIIa) have been found to play in a wide range of disease states, efforts have been directed towards the study of these proteins. The study of enzymes to better understand their function and mode of action is facilitated through the use of tools such as protein labelling, enzyme inhibition, and substrate analogue kinetic studies among others. Transition state analogues have been effective inhibitors in the study of enzyme activity. Sulfoxide inhibitors can efficiently mimic transition states leading to the tetrahedral intermediate of an acyl transfer reaction and we discuss the synthesis towards sulfoxide transition state analogue inhibitors of TG2 in chapter 2. Novel sulfoxide compounds were synthesized, though the desired target compounds proved difficult to isolate due to their instability. Fluorescent probes are effective in protein labelling as a means of discerning activity. This technique was applied in order to elucidate intracellular TG2 activity, which is a topic of controversy. To that end, the synthesis of a fluorescent, TG2-specific, cell permeable probe is discussed in chapter 3. However, preliminary in vivo results show that while the probe is cell permeable and fluorescent, it was not TG2-specific. Molecular modelling suggests that the hexa-arginine tag, designed to improve cell permeability, decreases the affinity of the probe for its intended target. Finally, FXIIIa has become a new addition to the study of transglutaminases in the Keillor group. Given our interest in this enzyme, we had three goals for this work as explained in chapter 4. Firstly, owing to the anticipated high demand for FXIIIa required for later experiments, our primary aim was the development of an optimized method for the expression and purification of recombinant FXIIIA. After evaluating different conditions for FXIIIA expression, the Studier auto-induction ZYP media1 at 20 °C for 24 h was found to provide the optimal conditions for the expression of recombinant GST-tagged FXIIIA, typically giving a total of 1.5 mg of protein/L of culture. Secondly, a variety of different peptides were synthesized and tested using a glutamate dehydrogenase (GDH)-based assay to identify a high affinity sequence for a substrate of FXIIIa. The two peptides with the highest affinity for FXIIIa were Ac-DQMMMAF-OH and Ac-DQMML-OH. Testing with TG2 displayed negligible reactivity, confirming their use as orthogonal peptides, results reinforced by modelling studies of the peptides with both FXIIIa and TG2. This discovery represents the first time peptides orthogonal to TG2 with affinity for FXIIIa have been kinetically characterized with both transglutaminase enzymes. Lastly, our work towards a fluorogenic activity assay by incorporating a coumarin ester through attachment to a glutamic acid residue into a peptide sequence recognized by FXIIIa, will be discussed.

The synthesis and evaluation of a molecularly imprinted polymer has been undertaken using an oxazine-based tryptophanamide transition state analogue (TSA) as template. An efficient route to the synthesis of oxazine-based TSAs for the reaction of pyridoxamine and indole-3-pyruvic acid has been established, with yields of up to 80%. NMR titration studies were performed to examine the interactions between the functional monomer, methacrylic acid and the template. Complexation of the template by functional monomer in the presence of crosslinker showed an apparent KD of 0.63-0.79 ± 0.04 M (293 K, acetonitrile-d3) based upon the chemical shift of the template amide protons. TSA-imprinted and non-imprinted reference polymers were synthesized by free radical polymerization in acetonitrile. Polymer monoliths were ground and fractionated into a 25-63 μm size range. Polymer-ligand recognition studies were conducted using the polymers as HPLC stationary phases. An imprinting factor (IF) of 2.93 was observed for the TSA, indicating the selectivity of the imprinted sites for the template. Studies using the D- and L-enantiomers of the phenylalaninamide analogue of the template showed enantioselectivity in the case of the imprinted polymer, α = 1.10, though not in the case of the non-imprinted reference polymer (1.00). Using UV-spectroscopy based polymer-ligand binding studies, a maximum theoretical capacity (Bmax) of 0.059 ± 0.004 mmol·g-1 was observed for the imprinted polymer. Conclusively, an imprinted polymer with binding sites selective for the TSA was successfully prepared and shall subsequently be studied with respect to its capacity to catalyse the transamination reaction between pyridoxamine and indole-3-pyruvic acid to yield pyridoxal and tryptophan.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18448号 / 理博第4008号 / 新制||理||1578(附属図書館) / 31326 / 京都大学大学院理学研究科化学専攻 / (主査)教授 林 重彦, 教授 寺嶋 正秀, 教授 松本 吉泰 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Type 2 diabetes is a major health concern worldwide. One of its complications is postprandial hyperglycemia, i.e., high blood glucose concentrations, caused by glucose fast release from dietary polysaccharides into the bloodstream after meals. α-Glucosidase inhibitor drugs reduce postprandial hyperglycemia by inhibiting maltase/glucoamylase (MGAM) and sucrase/isomaltase (SI). MGAM and SI transform polysaccharides into absorbable monosaccharides, and inhibiting them delays monosaccharide release into the blood. The three commercially available α-glucosidase inhibitors are limited by their absorption abilities, inhibition efficacies, and side effects, which highlights the need for more specific α-glucosidase inhibitors. Because enzymes catalyze their reactions by tightly binding to their cognate transition states (TS), TS analogs can be powerful inhibitors and potential drugs. The measurement and interpretation of kinetic isotope effects (KIEs) is the only method that can directly determine TS structures on large molecules. In this work, methods to prepare radioisotopically labelled maltose were developed, as well as methods to measure KIEs on acid- and enzyme-catalyzed maltose hydrolysis. However, the methods developed did not achieve the required precision for TS analysis. Also, KIEs were calculated computationally for a model reaction of maltose hydrolysis. / Thesis / Master of Science (MSc)

Brief explanation and history of cinchona based Phase Transfer Catalysis (PTC). Studied aryl acetates in PTC, encompassing napthoyl, 6-methoxy napthoyl, phenyl and protected 4-hydroxy phenyl acetates. Investigated means of controlling the selectivity of the PTC reaction by changing the electrophile size, the ether side group size or by addition of inorganic salts. Found that either small or aromatic electophiles increased enantioselectivity more than aliphatic electrophiles, and that increasing the size of ether protecting group also increased selectivity. Positive effects of salt addition included either decreasing reaction time or increasing enantiomeric excess. Applied findings towards the synthesis of S-equol. Computational experiments working towards deducing the transition state between PTC and aryl acetate substrates.