A combined experimental–computational approach has been used to study the cyclopropanation reaction of N-hydroxyphthalimide diazoacetate (NHPI-DA) with various olefins, catalyzed by a ruthenium-phenyloxazoline (Ru-Pheox) complex. Kinetic studies show that the better selectivity of the employed redox-active NHPI diazoacetate is a result of a much slower dimerization reaction compared to aliphatic diazoacetates. Density functional theory calculations reveal that several reactions can take place with similar energy barriers, namely, dimerization of the NHPI diazoacetate, cyclopropanation (inner-sphere and outer-sphere), and a previously unrecognized migratory insertion of the carbene into the phenyloxazoline ligand. The calculations show that the migratory insertion reaction yields an unconsidered ruthenium complex that is catalytically competent for both the dimerization and cyclopropanation, and its relevance is assessed experimentally. The stereoselectivity of the reaction is argued to stem from an intricate balance between the various mechanistic scenarios.

The dynamic equilibria of organomagnesium reagents are known to be very complex, and the relative reactivity of their components is poorly understood. Herein, a combination of DFT calculations and kinetic experiments is employed to investigate the detailed reaction mechanism of the Pummerer coupling between sulfoxides and turbo-organomagnesium amides. Among the various aggregates studied, unprecedented heterometallic open cubane structures are demonstrated to yield favorable barriers through a concerted anion-anion coupling/ S−O cleavage step. Beyond a structural curiosity, these results introduce open cubane organometallics as key reactive intermediates in turbo-organomagnesium amide mixtures. 

Quantum chemical calculations are used to investigate the detailed reaction mechanism of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid (SEPHCHC) synthase (also known as MenD), a thiamin diphosphate-dependent decarboxylase that catalyzes the formation of SEPHCHC from 2-ketoglutarate and isochorismate. This enzyme is involved in the menaquinone biosynthesis pathway in M. tuberculosis and is thought of as a potential drug target for anti-tuberculosis therapeutics. In addition, MenD shows promise as a biocatalyst for the synthesis of 1,4-functionalized compounds. Models of the active site are constructed on the basis of available X-ray structures, and the intermediates and transition states involved in the reaction mechanism are optimized and characterized. The calculated mechanism is in good agreement with prior kinetic studies and gives new insights into the mode of action of the enzyme. In particular, the structure and role of the tetrahedral post-decarboxylation intermediate observed in X-ray structures are discussed.

In this thesis, density functional theory (DFT) is employed in the study of two enzymes and two organometallic systems.

First, the natural reaction mechanism, as well as the enantioselective formation of α-hydroxyketones catalysed by two thiamine diphosphate (ThDP)- dependent enzymes, namely benzoylformate decarboxylase (BFDC) and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid (SEPHCHC)- synthase (MenD), are investigated. To that end, different cluster models that account for the active sites of the enzymes are used. For BFDC, the calculated natural reaction mechanism clarifies the roles of various active site residues and of the cofactor. Moreover, an unprecedented tricyclic cofactor species is found to be kinetically relevant. The importance of this species is further explored in a second study, in which the relative stabilities of the different ThDP-cofactor states are assessed in different enzymatic and non-enzymatic environments. In the last study of BFDC, the enantioselective carboligation mechanism between the enamine intermediate and two different acceptors, namely benzaldehyde or acetaldehyde, is studied. Moving into MenD, the calculated natural reaction mechanism gives insight into the formation tetrahedral post-decarboxylation intermediate, which has been extensively discussed in the literature. Moreover, the proton source in the keto-enol tautomerization in the second part of the mechanism can also be elucidated. Finally, because MenD can perform 1,4- and 1,2-additions, the factors governing the regioand enantioselectivity of two non-natural reactions are covered.

Next, a Pummerer-like, C-C coupling reaction, is studied, and the calculations show that an unstable open-cubane complex yields considerably lower barriers than the more typically suggested linear complexes. In the last study, a ruthenium-catalysed cyclopropanation reaction is investigated. The calculated free-energy profiles indicate a very intricate scenario in which two cyclopropanation mechanisms and two side-reactions need to be considered. Importantly, one of these side-reactions, i.e. a migratory insertion of the carbene into the C-M bond of the ligand, results in the formation of a new catalyst, and a combined computational-experimental effort elucidates which is the active catalyst for the cyclopropanation reaction.

The computational study of enantioselective reactions is a challenging task that requires high accuracy, as very small energy differences have to be reproduced. Quantum chemical methods, most commonly density functional theory, are today an important tool in this pursuit. This Perspective describes recent efforts in modeling asymmetric reactions in enzymes by means of the quantum chemical cluster approach. The methodology is described briefly and a number of illustrative case studies performed recently at our laboratory are presented. The reviewed enzymes are limonene epoxide hydrolase, soluble epoxide hydrolase, arylmalonate decarboxylase, phenolic acid decarboxylase, benzoylformate decarboxylase, secondary alcohol dehydrogenase, acyl transferase, and norcoclaurine synthase. The challenges encountered in each example are discussed, and the modeling lessons learned are highlighted.

Thiamin diphosphate (ThDP)-dependent enzymes constitute a large class of enzymes that catalyze a diverse range of reactions. Many are involved in stereospecific carbon-carbon bond formation and, consequently, have found increasing interest and utility as chiral catalysts in various biocatalytic applications. All ThDP-catalyzed reactions require the reaction of the ThDP ylide (the activated state of the cofactor) with the substrate. Given that the cofactor can adopt up to seven states on an enzyme, identifying the factors affecting the stability of the pre-reactant states is important for the overall understanding of the kinetics and mechanism of the individual reactions. In this paper we use density functional theory calculations to systematically study the different cofactor states in terms of energies and geometries. Benzoylformate decarboxylase (BFDC), which is a well characterized chiral catalyst, serves as the prototypical ThDP-dependent enzyme. A model of the active site was constructed on the basis of available crystal structures, and the cofactor states were characterized in the presence of three different ligands (crystallographic water, benzoylformate as substrate, and (R)-mandelate as inhibitor). Overall, the calculations reveal that the relative stabilities of the cofactor states are greatly affected by the presence and identity of the bound ligands. A surprising finding is that benzoylformate binding, while favoring ylide formation, provided even greater stabilization to a catalytically inactive tricyclic state. Conversely, the inhibitor binding greatly destabilized the ylide formation. Together, these observations have significant implications for the reaction kinetics of the ThDP-dependent enzymes, and, potentially, for the use of unnatural substrates in such reactions.

Benzoylformate decarboxylase (BFDC) is a thiamin-diphosphate enzyme that catalyzes the decarboxylation of benzoylformate to yield benzaldehyde and carbon dioxide. In addition to its natural reaction, BFDC is able to catalyze carboligation reactions in a highly enantioselective fashion, making the enzyme a potentially important biocatalyst. Here we use density functional theory calculations to investigate the detailed mechanism of BFDC-catalyzed carboligation and to elucidate the sources of the enantioselectivity. Benzaldehyde and acetaldehyde are studied as acceptors, for, when reacting with a benzaldehyde donor, they yield products with opposite enantiospecificity. For each of the acceptors, several possible binding modes to the active site are initially examined before the individual reaction paths leading to the two enantiomeric products are followed. The calculated energies are in good agreement with the experimental results, and the analysis of the transition states gives insight into the origins of the enantioselectivity.

Density functional theory calculations are used to investigate the detailed reaction mechanism of benzoylformate decarboxylase, a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the nonoxidative decarboxylation of benzoylformate yielding benzaldehyde and carbon dioxide. A large model of the active site is constructed on the basis of the X-ray structure, and it is used to characterize the involved intermediates and transition states and evaluate their energies. There is generally good agreement between the calculations and available experimental data. The roles of the various active site residues are discussed and the results are compared to mutagenesis experiments. Importantly, the calculations identify off-cycle intermediate species of the ThDP cofactor that can have implications on the kinetics of the reaction.

Quantum chemical calculations are used to investigate the detailed reaction mechanism of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid (SEPHCHC) synthase (called MenD), a thiamin diphosphate (ThDP)-dependent transketolase that catalyzes the formation of SEPHCHC from  a-ketoglutarate and isochorismate. Models of the active site are constructed on the basis of available X-ray structures, and the intermediates and transition states involved in the reaction mechanism are optimized and characterized. The calculated mechanism is in good agreement with available kinetics studies, and gives new insights into the mode of action of the enzyme. The source of the proton necessary for the second half of the reaction is discussed, and the structure of the post-decarboxylation tetrahedral intermediate is clarified. Furthermore, the reactions of the  a-ketoglutarate donor with two non-natural acceptors are also studied, and the factors governing the regio- and enantioselectivity are analyzed.