Laboratory Evolution of Robust and Enantioselective Baeyer−Villiger Monooxygenases for Asymmetric Catalysis

The Baeyer−Villiger Monooxygenase, Phenylacetone Monooxygenase (PAMO), recently discovered by Fraaije, Janssen, and co-workers, is unusually thermostable, which makes it a promising candidate for catalyzing enantioselective Baeyer−Villiger reactions in organic chemistry. Unfortunately, however, its substrate scope is very limited, reasonable reaction rates being observed essentially only with phenylacetone and similar linear phenyl-substituted analogs. Previous protein engineering attempts to broaden the range of substrate acceptance and to control enantioselectivity have been met with limited success, including rational design and directed evolution based on saturation mutagenesis with formation of focused mutant libraries, which may have to do with complex domain movements. In the present study, a new approach to laboratory evolution is described which has led to mutants showing unusually high activity and enantioselectivity in the oxidative kinetic resolution of a variety of 2-aryl and 2-alkylcyclohexanones which are not accepted by the wild-type (WT) PAMO and of a structurally very different bicyclic ketone. The new strategy exploits bioinformatics data derived from sequence alignment of eight different Baeyer−Villiger Monooxygenases, which in conjunction with the known X-ray structure of PAMO and induced fit docking suggests potential randomization sites, different from all previous approaches to focused library generation. Sites harboring highly conserved proline in a loop of the WT are targeted. The most active and enantioselective mutants retain the high thermostability of the parent WT PAMO. The success of the “proline” hypothesis in the present system calls for further testing in future laboratory evolution studies

Manipulating the Stereoselectivity of Limonene Epoxide Hydrolase by Directed Evolution Based on Iterative Saturation Mutagenesis

Limonene epoxide hydrolase from Rhodococcus erythropolis DCL 14 (LEH) is known to be an exceptional epoxide hydrolase (EH) because it has an unusual secondary structure and catalyzes the hydrolysis of epoxides by a rare one-step mechanism in contrast to the usual two-step sequence. From a synthetic organic viewpoint it is unfortunate that LEH shows acceptable stereoselectivity essentially only in the hydrolysis of the natural substrate limonene epoxide, which means that this EH cannot be exploited as a catalyst in asymmetric transformations of other substrates. In the present study, directed evolution using iterative saturation mutagenesis (ISM) has been tested as a means to engineer LEH mutants showing broad substrate scope with high stereoselectivity. By grouping individual residues aligning the binding pocket correctly into randomization sites and performing saturation mutagenesis iteratively using a reduced amino acid alphabet, mutants were obtained which catalyze the desymmetrization of cyclopentene−oxide with stereoselective formation of either the (R,R)- or the (S,S)-diol on an optional basis. The mutants prove to be excellent catalysts for the desymmetrization of other meso-epoxides and for the hydrolytic kinetic resolution of racemic substrates, without performing new mutagenesis experiments. Since less than 5000 tranformants had to be screened for achieving these results, this study contributes to the generalization of ISM as a fast and reliable method for protein engineering. In order to explain some of the stereoselective consequences of the observed mutations, a simple model based on molecular dynamics simulations has been proposed

An Efficient Catalyst System for the Asymmetric Transfer Hydrogenation of Ketones: Remarkably Broad Substrate Scope

A BINOL-derived diphosphonite having a xanthene backbone is an excellent bidentate ligand in Ru-catalyzed 2-propanol-based transfer hydrogenation of aryl/alkyl and alkyl/alkyl ketones (ee = 76−99%). Even notoriously difficult ketones such as isopropyl methyl ketone are reduced with extraordinarily high enantioselectivity (ee = 99%)

Solid-Phase Enzymatic Synthesis of Oligonucleotides^†

The controlled and selective synthesis of oligonucleotides on the solid phase is possible under mild aqueous conditions using the enzyme T4 RNA ligase, the resins being tentagel or kieselguhr/polydimethylacrylamide

Reduction of α,β-Unsaturated Ketones by Old Yellow Enzymes: Mechanistic Insights from Quantum Mechanics/Molecular Mechanics Calculations

Enoate reductases catalyze the reduction of activated CC bonds with high enantioselectivity. The oxidative half-reaction, which involves the addition of a hydride and a proton to opposite faces of the CC bond, has been studied for the first time by hybrid quantum mechanics/molecular mechanics (QM/MM). The reduction of 2-cyclohexen-1-one by YqjM from Bacillus subtilis was selected as the model system. Two-dimensional QM/MM (B3LYP-D/OPLS2005) reaction pathways suggest that the hydride and proton are added as distinct steps, with the former step preceding the latter. Furthermore, we present interesting insights into the reactivity of this enzyme, including the weak binding of the substrate in the active site, the role of the two active site histidine residues for polarization of the substrate CO bond, structural details of the transition states to hydride and proton transfer, and the role of Tyr196 as proton donor. The information presented here will be useful for the future design of enantioselective YqjM mutants for other substrates

New Uses of Amino Acids as Chiral Building Blocks in Organic Synthesis

N,N-Dibenzylamino aldehydes have emerged as a highly useful class of chiral building blocks in synthetic organic chemistry. We envisioned the transformation of the N,N -dibenzylamino aldehydes to the corresponding aldimines followed by diastereoselective methylene transfer with a sulfonium ylide to obtain α-amino aziridines in high yields

Enantiotopic Group Recognition: Direct Evidence for Selective Complexation of Enantiotopic Groups by a Chiral Host

Enantiotopic Group Recognition:  Direct Evidence for Selective Complexation of Enantiotopic Groups by a Chiral Hos

Laboratory Evolution of Enantiocomplementary Candida antarctica Lipase B Mutants with Broad Substrate Scope

Candida antarctica lipase B (CALB) is a robust and easily expressed enzyme used widely in academic and industrial laboratories with many different kinds of applications. In fine chemicals production, examples include acylating kinetic resolution of racemic secondary alcohols and amines as well as desymmetrization of prochiral diols (or the reverse hydrolytic reactions). However, in the case of hydrolytic kinetic resolution of esters or esterifying kinetic resolution of acids in which chirality resides in the carboxylic acid part of the substrate, rate and stereoselectivity are generally poor. In the present study, directed evolution based on iterative saturation mutagenesis was applied to solve the latter problem. Mutants with highly improved activity and enantioselectivity relative to wild-type CALB were evolved for the hydrolytic kinetic resolution of p-nitrophenyl 2-phenylpropanoate, with the selectivity factor increasing from E = 1.2 (S) to E = 72 (S) or reverting to E = 42 (R) on an optional basis. Surprisingly, point mutations both in the acyl and alcohol pockets of CALB proved to be necessary. Some of the evolved CALB mutants are also efficient biocatalysts in the kinetic resolution of other chiral esters without performing new mutagenesis experiments. Another noteworthy result concerns the finding that enantiocomplementary CALB mutants for α-substituted carboxylic acid esters also show stereocomplementarity in the hydrolytic kinetic resolution of esters derived from chiral secondary alcohols. Insight into the source of stereoselectivity was gained by molecular dynamics simulations and docking experiments

Enantiotopic Group Recognition: Direct Evidence for Selective Complexation of Enantiotopic Groups by a Chiral Host

Enantiotopic Group Recognition:  Direct Evidence for Selective Complexation of Enantiotopic Groups by a Chiral Hos

BINOL-Based Diphosphonites as Ligands in the Asymmetric Rh-Catalyzed Conjugate Addition of Arylboronic Acids

BINOL-based diphosphonites having achiral backbones are useful ligands in the Rh-catalyzed conjugate addition of arylboronic acids to α,β-unsaturated carbonyl compounds. The nature of the achiral backbone determines the direction and degree of enantioselectivity, with er values of up to 99.5:0.5 possible