Valentin Köhler

Synthetic cascades are enabled by combining biocatalysts with artificial metalloenzymes

Enzymatic catalysis and homogeneous catalysis offer complementary means to address synthetic challenges, both in chemistry and in biology. Despite its attractiveness, the implementation of concurrent cascade reactions that combine an organometallic catalyst with an enzyme has proven challenging because of the mutual inactivation of both catalysts. To address this, we show that incorporation of a d6-piano stool complex within a host protein affords an artificial transfer hydrogenase (ATHase) that is fully compatible with and complementary to natural enzymes, thus enabling efficient concurrent tandem catalysis. To illustrate the generality of the approach, the ATHase was combined with various NADH-, FAD- and haem-dependent enzymes, resulting in orthogonal redox cascades. Up to three enzymes were integrated in the cascade and combined with the ATHase with a view to achieving (i) a double stereoselective amine deracemization, (ii) a horseradish peroxidase-coupled readout of the transfer hydrogenase activity towards its genetic optimization, (iii) the formation of L-pipecolic acid from L-lysine and (iv) regeneration of NADH to promote a monooxygenase-catalysed oxyfunctionalization reaction. An artificial transfer hydrogenase, based on the incorporation of a biotinylated iridium-piano-stool complex in streptavidin, is shown to be fully compatible with a range of biocatalysts. The location of the active metal centre inside the protein scaffold efficiently prevents mutual inactivation processes and enables the concurrent interplay with oxidative enzymes.

Cytochromes P450 as useful biocatalysts: addressing the limitations

Cytochrome P450 monooxygenases (P450s or CYPs) are a unique family of enzymes which are capable of catalysing the regio- and stereospecific oxidation of non-functionalised hydrocarbons. Despite the enormous synthetic potential of P450s, these enzymes have yet to be extensively employed for research purposes or in industry. Lack of stability, low activity, narrow substrate specificity, expensive cofactor requirements, limited solvent tolerance and electron supply are some of the main reasons why the academic and industrial implementation of these important biocatalysts remains a challenge. Considering the significance of P450s, many research groups have focused on improving their properties in an effort to make more robust catalysts with broad synthetic applications. This article focuses on some of the factors that have limited the exploitation of P450s and explores some of the significant steps that have been taken towards addressing these limitations.

Enantioselective Biocatalytic Oxidative Desymmetrization of Substituted Pyrrolidines

Direct activation of sp C H bonds a to a nitrogen atom represents an attractive strategy for functionalization of amines, especially those found in 5and 6-membered ring heterocycles. In particular, C H activation by oxidation, followed by nucleophilic addition, generates products of an overall oxidative Strecker or Mannich process. Recent reports have described the use of various metal-based oxidants to achieve this transformation although there are few examples of catalytic and/or enantioselective processes. The enantioselective oxidation of N-protected pyrrolidines has been reported using manganese–salen catalysts and iodosobenzene as the stoichiometric oxidant. Herein we report the enantioselective enzyme-catalyzed desymmetrization of a range of unprotected pyrrolidines to the corresponding Dpyrrolines (Scheme 1), which serve as useful building blocks for the synthesis of l-proline analogues of high enantiomeric purity.

Highly Stereoselective Synthesis of Substituted Prolyl Peptides Using a Combination of Biocatalytic Desymmetrization and Multicomponent Reactions

Time and pep-tide wait for no man: Optically pure 3,4disubstituted 1-pyrrolines, generated from the corresponding meso-pyrrolidines by biocatalytic desymmetrization (MAO-N=monoamine oxidase N), react with carboxylic acids and isocyanides in a highly diastereoselective Ugi-type multicomponent reaction to give substituted prolyl peptides of high pharmaceutical relevance. (Figure Presented)

Artificial Metalloenzymes: Reaction Scope and Optimization Strategies

The incorporation of a synthetic, catalytically competent metallocofactor into a protein scaffold to generate an artificial metalloenzyme (ArM) has been explored since the late 1970s. Progress in the ensuing years was limited by the tools available for both organometallic synthesis and protein engineering. Advances in both of these areas, combined with increased appreciation of the potential benefits of combining attractive features of both homogeneous catalysis and enzymatic catalysis, led to a resurgence of interest in ArMs starting in the early 2000s. Perhaps the most intriguing of potential ArM properties is their ability to endow homogeneous catalysts with a genetic memory. Indeed, incorporating a homogeneous catalyst into a genetically encoded scaffold offers the opportunity to improve ArM performance by directed evolution. This capability could, in turn, lead to improvements in ArM efficiency similar to those obtained for natural enzymes, providing systems suitable for practical applications and greater insight into the role of second coordination sphere interactions in organometallic catalysis. Since its renaissance in the early 2000s, different aspects of artificial metalloenzymes have been extensively reviewed and highlighted. Our intent is to provide a comprehensive overview of all work in the field up to December 2016, organized according to reaction class. Because of the wide range of non-natural reactions catalyzed by ArMs, this was done using a functional-group transformation classification. The review begins with a summary of the proteins and the anchoring strategies used to date for the creation of ArMs, followed by a historical perspective. Then follows a summary of the reactions catalyzed by ArMs and a concluding critical outlook. This analysis allows for comparison of similar reactions catalyzed by ArMs constructed using different metallocofactor anchoring strategies, cofactors, protein scaffolds, and mutagenesis strategies. These data will be used to construct a searchable Web site on ArMs that will be updated regularly by the authors.

OsO4⋅Streptavidin: A Tunable Hybrid Catalyst for the Enantioselective cis‐Dihydroxylation of Olefins

Enzymatic and homogeneous catalysis have evolved independently to address the challenges in the synthesis of enantiopure products. With the aim of complementing these fields, artificial metalloenzymes, which combine the structural diversity of biocatalysts with the wealth of metal-catalyzed reactions, have attracted increasing attention. [1] In homogeneous catalysis the cis-selective, OsO4-dependent asymmetric dihydroxylation (AD) of olefins ranks among the most powerful methods for the synthesis of vicinal diols. Ligands for homogeneous catalysis have been largely developed by Sharpless and co-workers, and are, with few exceptions, almost exclusively based on quinidine or quinine derivatives. [2] Although most classes of prochiral olefins are dihydroxylated with good activity and selectivity, the cissubstituted olefins are problematic. Nature relies on nonheme iron dioxygenases such as naphthalene dioxygenase (NDO) to perform a related reaction. These enzymes display broad substrate scope. [3] It is believed that both the OsO4- and NDO-catalyzed dihydroxylations proceed by an outer sphere [3+2] mechanism in which the substrate is not bound to the

Improving the Catalytic Performance of an Artificial Metalloenzyme by Computational Design.

Artifical metalloenzymes combine the reactivity of small molecule catalysts with the selectivity of enzymes, and new methods are required to tune the catalytic properties of these systems for an application of interest. Structure-based computational design could help to identify amino acid mutations leading to improved catalytic activity and enantioselectivity. Here we describe the application of Rosetta Design for the genetic optimization of an artificial transfer hydrogenase (ATHase hereafter), [(η(5)-Cp*)Ir(pico)Cl] ⊂ WT hCA II (Cp* = Me5C5(-)), for the asymmetric reduction of a cyclic imine, the precursor of salsolsidine. Based on a crystal structure of the ATHase, computational design afforded four hCAII variants with protein backbone-stabilizing and hydrophobic cofactor-embedding mutations. In dansylamide-competition assays, these designs showed 46-64-fold improved affinity for the iridium pianostool complex [(η(5)-Cp*)Ir(pico)Cl]. Gratifyingly, the new designs yielded a significant improvement in both activity and enantioselectivity (from 70% ee (WT hCA II) to up to 92% ee and a 4-fold increase in total turnover number) for the production of (S)-salsolidine. Introducing additional hydrophobicity in the Cp*-moiety of the Ir-catalyst provided by adding a propyl substituent on the Cp* moiety yields the most (S)-selective (96% ee) ATHase reported to date. X-ray structural data indicate that the high enantioselectivity results from embedding the piano stool moiety within the protein, consistent with the computational model.

Protein-based hybrid catalysts--design and evolution.

Artificial metalloenzymes result from the introduction of a catalytically competent non-native metal cofactor within a protein environment. In the present contribution, we summarize the recent achievements in the design and the optimization of such protein-based hybrid catalysts, with an emphasis on enantioselective transformations. The second part outlines the milestones required to achieve en masse production, screening and directed evolution of artificial metalloenzymes. In the spirit of Darwinian evolution, this will allow the full potential of such protein-based hybrid catalysts to be fully unraveled, thus complementing both homogeneous and enzymatic catalysis.

Recent Trends in Biomimetic NADH Regeneration

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate constitute a major cost factor in preparative biotransformations. The development of efficient methods for their regeneration with cheap reducing equivalents has been an area of intense research in the last decades. Methods explored include chemical, electrochemical, and photochemical approaches. None of the methods to regenerate NADH has reached an efficiency comparable with enzymatic regeneration (e.g. formate dehydrogenase) which remains the method of choice for most applications. In this review, we summarize primarily organometallic-based approaches for NADH regeneration methods which include non-enzymatic steps, before moving on to the most recent developments in synthetic NADH related transformations. We highlight the frequent problem of mutual inactivation between the organometallic catalyst for NADH regeneration and the corresponding NADH dependent downstream enzyme. Potential remedies are discussed, such as the compartmentalization of the organometallic complex.

Chimeric Self-sufficient P450cam-RhFRed Biocatalysts with Broad Substrate Scope

Summary A high-throughput screening protocol for evaluating chimeric, self-sufficient P450 biocatalysts and their mutants against a panel of substrates was developed, leading to the identification of a number of novel biooxidation activities.