Elizabeth L. Noey

Catalytic Asymmetric Intermolecular Stetter Reactions of Enolizable Aldehydes with Nitrostyrenes: Computational Study Provides Insight into the Success of the Catalyst

Over the past decade, N-heterocyclic carbenes (NHCs) have been used as catalysts in a variety of C-C bond forming reactions.[1] Our group has been interested in the development of chiral NHCs as catalysts for the asymmetric intra-molecular Stetter reaction[2,3] and more recently the inter-molecular variant.[4,5] We recently reported that hetaryl aldehydes and enals react efficiently with nitroalkenes in the Stetter reaction, leading to β-nitro ketones with high enantioselectivity.[4d] Crucial to the success of this method was the development of a fluorinated triazolium salt pre-catalyst that provides significantly enhanced enantioselectivity over des-fluoro analogues.[6] Although this new catalyst system greatly expands the scope of this method, these conditions are not amenable to the use of unactivated aliphatic aldehydes. Due to their lower electrophilicity than aryl aldehydes, aliphatic aldehydes have rarely been used successfully in the asymmetric inter-molecular Stetter reaction.[7,8]

Mechanism of Gold(I)-Catalyzed Rearrangements of Acetylenic Amine-N-Oxides: Computational Investigations Lead to a New Mechanism Confirmed by Experiment

Quantum mechanical studies of the mechanism of gold-catalyzed rearrangements of acetylenic amine-N-oxides to piperidinones or azepanones have revealed a new mechanism involving a concerted heteroretroene reaction, formally a 1,5 hydrogen shift from the N-alkyl groups to the vinyl position of a gold-coordinated methyleneisoxazolidinium or methyleneoxazinanium. Density functional calculations (B3LYP, B3LYP-D3) on the heteroretroene mechanism reproduce experimental regioselectivities and provide an explanation as to why the hydrogen is transferred from the smaller amine substituent. In support of the proposed mechanism, new experimental investigations show that the hydrogen shift is concerted and that gold carbenes are not involved as reaction intermediates.

Quantum Mechanical Investigation of the Effect of Catalyst Fluorination in the Intermolecular Asymmetric Stetter Reaction

The asymmetric intermolecular Stetter reaction was investigated using the B3LYP and M06-2X functionals. Fluorination of a triazolium bicyclic catalyst had been found to significantly influence reaction yields and enantiomeric ratios. Computations indicate that the improved reactivity of the fluorinated catalyst is due to better electrostatic interactions between the nitroalkene and catalyst. Computational investigations of preferred conformations of the ground state catalyst and acyl anion equivalent, and the transition structures leading to both enantiomers of the products, are reported.

Engineering synthetic recursive pathways to generate non-natural small molecules.

Recursive pathways are broadly defined as those that catalyze a series of reactions such that the key, bond-forming functional group of the substrate is always regenerated in each cycle, allowing for a new cycle of reactions to begin. Recursive carbon-chain elongation pathways in nature produce fatty acids, polyketides, isoprenoids and α-keto acids (αKAs), which all use modular or iterative approaches for chain elongation. Recently, an artificial pathway for αKA elongation has been built that uses an engineered isopropylmalate synthase to recursively condense acetyl-CoA with αKAs. This synthetic approach expands the possibilities for recursive pathways beyond the modular or iterative synthesis of natural products and serves as a case study for understanding the challenges of building recursive pathways from nonrecursive enzymes. There exists the potential to design synthetic recursive pathways far beyond what nature has evolved.

Selective Gold(I)-Catalyzed Formation of Tetracyclic Indolines: A Single Transition Structure and Bifurcations Lead to Multiple Products

Several alkynylindoles undergo gold(I)-catalyzed cyclization reactions to form a single isomer in each case. Density functional theory shows why this reaction is favored over the many possible regio- and stereoisomeric reaction pathways. This transformation involves a two-step no-intermediate mechanism with surface bifurcations leading to two or three products. Such bifurcations could explain reactivity in many gold(I)-catalyzed enyne cyclization reactions.

Molecular dynamics explorations of active site structure in designed and evolved enzymes.

This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP, a noncatalytic arrangement of the catalytic triad is dominant. Unnatural truncated substrates are inactive because of the lack of protein-protein interactions provided by the ACP. Directed evolution is able to gradually restore the catalytic organization of the active site by motion of the protein backbone that alters the active site geometry. In the third case, we demonstrate the key role of MD in combination with crystallography to identify the origins of substrate-dependent stereoselectivities in a number of Codexis-engineered ketoreductases, one of which is used commercially for the production of the antibiotic sulopenem. Here, mutations alter the shape of the active site as well as the accessibility of water to different regions of it. Each of these examples reveals something different about how mutations can influence enzyme activity and shows that directed evolution, like natural evolution, can increase catalytic activity in a variety of remarkable and often subtle ways.

Origins of stereoselectivity in evolved ketoreductases.

Significance Ketoreductases are the most commonly used enzymes in industrial pharmaceutical synthesis. We investigated the nature of enantioselectivity in closely related mutant ketoreductases that reduce almost-symmetrical 3-oxacyclopentanone and 3-thiacyclopentanone, which are difficult to reduce enantioselectively by other means. We present the efficiencies of select variants and their crystallographic structures. Our experimental and theoretical studies reveal how mutations modulate the stereoselectivity of the reduction. Molecular dynamics simulations of the Michaelis–Menten and transition state-bound complexes were used to rationalize the observed stereochemical outcomes. We discovered that the closed conformation of the flexible substrate binding loop is likely the catalytically active one imparting the stereochemical preferences. Our molecular dynamics approach reveals how each enzyme stabilizes the diastereomeric transition structures by altering the active site size. Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP+-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.

Origins of Regioselectivity in the Fischer Indole Synthesis of a Selective Androgen Receptor Modulator

The selective androgen receptor modulator, (S)-(7-cyano-4-(pyridin-2-ylmethyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-2-yl)carbamic acid isopropyl ester, LY2452473, is a promising treatment of side effects of prostate cancer therapies. An acid-catalyzed Fischer indolization is a central step in its synthesis. The reaction leads to only one of the two possible indole regioisomers, along with minor decomposition products. Computations show that the formation of the observed indole is most favored energetically, while the potential pathway to the minor isomer leads instead to decomposition products. The disfavored [3,3]-sigmatropic rearrangement, which would produce the unobserved indole product, is destabilized by the electron-withdrawing phthalimide substituent. The most favored [3,3]-sigmatropic rearrangement transition state is bimodal, leading to two reaction intermediates from one transition state, which is confirmed by molecular dynamics simulations. Both intermediates can lead to the observed indole product, albeit through different mechanisms.