Iván Lavandera

Formal Asymmetric Biocatalytic Reductive Amination

Asymmetric methods to prepare optically active a-chiral primary amines are highly demanded in asymmetric synthesis owing to the biological/pharmacological activity of many amines. Various techniques have been reported, such as asymmetric 1,2-addition to imines and asymmetric amination of a,a-disubstituted aldehydes, transformation of allylic alcohols into amines, (dynamic) kinetic resolution, and cyclic deracemization employing racemic amines as substrates. Asymmetric reductive amination of ketones has been investigated with transition-metal catalysts and organocatalysts, as well as via sulfinyl imine intermediates. Although tremendous progress in organo/metal catalysis has been achieved for the asymmetric reductive amination of ketones to access a-chiral amines, improved protocols are still required that are simple, green, and economically viable and that lead to high enantiomeric excesses. Biocatalytic reductive amination or transamination is well established for accessing a-amino acids from the corresponding a-keto carboxylic acids. However, the situation is different for primary amines that are not adjacent to a carbonic acid moiety. w-Transaminases have recently received attention for the preparation of such a-chiral unprotected amines. w-Transaminases are employed mainly in one way, namely for the kinetic resolution of racemic chiral amines; only a few reports deal with asymmetric synthesis by starting from a prochiral ketone, probably due to problems in shifting the equilibrium to the product side, as well as due to the moderate stereoselectivity of the employed w-transaminases. These asymmetric synthetic processes usually require at least stoichiometric amounts of an amine donor (for example, alanine). The latter leads to a side product (pyruvate), which has to be removed during the transformation by using, for instance, pyruvate decarboxylase or lactate dehydrogenase. Additionally, limitations due to inhibition by the product amine and by pyruvate have been reported. An ideal process would use ammonium as the amine donor, together with a cheap reducing agent (for example, formate, hydrogen, or glucose; see Scheme 1). Even

Mimicking nature: synthetic nicotinamide cofactors for C═C bioreduction using enoate reductases.

A series of synthetic nicotinamide cofactors were synthesized to replace natural nicotinamide cofactors and promote enoate reductase (ER) catalyzed reactions without compromising the activity or stereoselectivity of the bioreduction process. Conversions and enantioselectivities of >99% were obtained for C═C bioreductions, and the process was successfully upscaled. Furthermore, high chemoselectivity was observed when employing these nicotinamide cofactor mimics (mNADs) with crude extracts in ER-catalyzed reactions.

One-way biohydrogen transfer for oxidation of sec-alcohols.

Quasi-irreversible oxidation of sec-alcohols was achieved via biocatalytic hydrogen transfer reactions using alcohol dehydrogenases employing selected ketones as hydrogen acceptors, which can only be reduced but not oxidized. Thus, only 1 equiv of oxidant was required instead of a large excess. For the oxidation of both isomers of methylcarbinols a single nonstereoselective short-chain dehydrogenase/reductase from Sphingobium yanoikuyae was identified and overexpressed in E. coli.

Stereoselective Bioreduction of Bulky-Bulky Ketones by a Novel ADH from Ralstonia sp.

Ketones with two bulky substituents, named bulky-bulky ketones, as well as less sterically demanding ketones were successfully reduced to the corresponding optically highly enriched alcohols using a novel identified recombinant short-chain alcohol dehydrogenase RasADH from Ralstonia sp. DSM 6428 overexpressed in E. coli.

An Exceptionally DMSO‐Tolerant Alcohol Dehydrogenase for the Stereoselective Reduction of Ketones

A novel short-chain alcohol dehydrogenase from Paracoccus pantotrophus DSM 11072, which is applicable for hydrogen transfer, has been identified, cloned, and overexpressed in E. coli. The enzyme stereoselectively reduces several ketones in a sustainable substrate-coupled approach using 2-propanol (5% v/v) as hydrogen donor. The enzyme maintained its activity in organic co-solvents in biphasic as well as monophasic systems and was even active in micro-aqueous media (1% v/v aqueous buffer). In general, a higher conversion was observed at higher log P values of the solvent, however, DMSO, which exhibits the lowest log P value of all solvents investigated, was not only tolerated but led to a higher conversion and relative activity (110-210%). For example, the conversion after 24 h in 15% v/v DMSO was double that for the reaction performed in buffer. This tolerance to DMSO may be attributed to the ability of the wild-type strain to adapt and grow in media with high sulfur content.

Recent Advances in Cofactor Regeneration Systems Applied to Biocatalyzed Oxidative Processes

Nowadays, the design of sustainable processes applicable at industrial scale is highly desirable due to environmental reasons. The use of biocatalytic reactions to carry out oxidative transformations is one of the possible strategies framed in here due to the mildness and usually high selectivities achieved with these methods. Anyway, while implementing this type of system at industrial scale several drawbacks must be overcome in order to obtain efficient setups. Historically, one of the main issues has been the cofactor-dependency of these biocatalysts to be active. Herein, we will show the state-of-the-art concerning the recent efforts developed in the design of (potentially) efficient methods to regenerate the cofactor in oxidative transformations. Thus, the more studied enzymatic methods will be discussed to highlight some recent examples dealing with the co-expression of both oxidative and recycling enzymes in one host or the development of self-sufficient biocatalysts. Furthermore, novel applications of these systems to couple two productive synthetic reactions will also be reviewed. Subsequently, we will focus on some recent examples related with the employment of electrochemical, photochemical, and chemical strategies to carry out the nicotinamide coenzyme or the flavin/heme prosthetic group regeneration. In the case of biocatalysts that use NAD(P) as electron shuttle, these systems have also been employed to replace it, allowing the design of nicotinamide-free recycling setups. In all cases, the (dis)advantages that these methodologies present will be briefly discussed.

Ketone–Alcohol Hydrogen‐Transfer Equilibria: Is the Biooxidation of Halohydrins Blocked?

To ensure the quasi-irreversibility of the oxidation of alcohols coupled with the reduction of ketones in a hydrogen-transfer (HT) fashion, stoichiometric amounts of α-halo carbonyl compounds have been employed as hydrogen acceptors. The reason that these substrates lead to quasi-quantitative conversions has been tacitly attributed to both thermodynamic and kinetic effects. To provide a clear rationale for this behavior, we investigate herein the redox equilibrium of a selected series of ketones and 2-propanol by undertaking a study that combines experimental and theoretical approaches. First, the activity of the (R)-specific alcohol dehydrogenase from Lactobacillus brevis (LBADH) with these substrates was studied. The docking of acetophenone/(R)-1-phenyethanol and α-chloroacetophenone/(S)-2-chloro-1-phenylethanol in the active site of the enzyme confirms that there seems to be no structural reason for the lack of reactivity of halohydrins. This assumption is confirmed by the fact that the corresponding aluminum-catalyzed Meerwein-Ponndorf-Verley-Oppenauer (MPVO) reactions afford similar conversions to those obtained with LBADH, showing that the observed reactivity is independent of the catalyst employed. While the initial rates of the enzymatic reductions and the IR ν(C=O) values contradict the general belief that electron-withdrawing groups increase the electrophilicity of the carbonyl group, the calculated ΔG values of the isodesmic redox transformations of these series of ketones/alcohols with 2-propanol/acetone support the thermodynamic control of the reaction. As a result, a general method to predict the degree of conversion obtained in the HT-reduction process of a given ketone based on the IR absorption band of the carbonyl group is proposed, and a strategy to achieve the HT oxidation of halohydrins is also shown.

Simple and quick preparation of α-thiocyanate ketones in hydroalcoholic media. Access to 5-aryl-2-imino-1,3-oxathiolanes

A simple preparation on gram-scale of thiocyanate derivatives via nucleophilic substitution of halogenated compounds with SCN salts at high substrate concentrations in a few minutes and excellent yields was successfully accomplished in hydroalcoholic media. The obtained compounds were employed for the efficient synthesis of valuable 5-aryl-2-imino-1,3-oxathiolane derivatives (a one-pot approach is also presented).

Oxidoreductases Working Together: Concurrent Obtaining of Valuable Derivatives by Employing the PIKAT Method

Oxidoreductases are an important class of enzymes that catalyze redox processes, transferring electrons from a reductant to an oxidant. These biocatalysts are widely applied due to their usually exquisite chemo-, regio-, and stereoselectivities through mild and environmentally friendly procedures. Probably the oxidoreductases most often employed are the alcohol dehydrogenases (ADHs, EC 1.1.1.x.), which are able to perform stereoselective carbonyl reductions or enantioselective alcohol oxidations. Another type of redox biocatalysts are Baeyer–Villiger monooxygenases (BVMOs; EC 1.14.13.x.) that catalyze the oxidation of ketones, sulfides, and other heteroatoms by atmospheric oxygen. Besides all the advantages that biocatalyzed oxidations present over chemical methods, the requirement of the expensive nicotinamide NADPH cofactor necessitates effective cofactor regeneration by, for example, chemical, electrochemical, photochemical, or enzymatic methods. The methodology most often exploited is the ‘enzyme-coupled’ approach, in which a second, preferably irreversible, enzymatic reaction is used to shift the equilibrium towards the desired product. Recently, “designer bugs,” whole cells containing the overexpressed genes of the desired enzymes (ADH/BVMO plus enzyme for the recycling system), or “self-sufficient” BVMOs, in which the recycling enzyme has been covalently linked to the monooxygenase, have been developed with very promising results. Nevertheless, such enzyme-coupled transformations depend on a sacrificial coupled reaction which lowers the atom-efficiency environmental factor, E, of the overall process. We have recently developed a system in which two productive redox reactions are connected through internal cofactor recycling. In this manner , it was possible to obtain simultaneously up to three enantioenriched derivatives starting either from two racemic mixtures or a racemate plus a prochiral compound, maximizing the redox efficiency of the whole process and allowing parallel interconnected kinetic asymmetric transformations (PIKAT; Scheme 1). Herein we have broadened the scope of the system combining the stereoselective oxidation of several sulfides with the enantioselective oxidation of different sec-alcohols. The cofactor concentration employed in these processes was optimized, which resulted in good performance, even when using micromolar concentrations of the NADP connector. Firstly, the enzymatic resolution of ( )-2-octanol (1a, 2 equivalents) catalyzed by two commercially available ADHs (LBADH from Lactobacillus brevis and ADH-T from Thermoanaerobacter sp.) was coupled with the sulfoxidation of different sulfides (4a–e, 1 equivalent) in the presence of the Baeyer–Villiger monooxygenases PAMO from Thermobifida fusca, its M446G mutant or HAPMO from Pseudomonas fluorescens ACB (Scheme 2). The results are summarized in Table 1. For these reactions, PAMO and M446G were used at 30 8C and HAPMO at 20 8C. Scheme 1. Concurrent obtaining of enantioenriched derivatives through parallel interconnected kinetic asymmetric transformation (PIKAT) method.

Remote interactions explain the unusual regioselectivity of lipase from Pseudomonas cepacia toward the secondary hydroxyl of 2'-deoxynucleosides.

Lipase from Pseudomonas cepacia (PCL) surprisingly favors acylation of the secondary hydroxyl at the 3′‐position over the primary hydroxyl at the 5′‐position in 2′‐deoxynucleosides by up to >98:1. Catalytically productive tetrahedral intermediate analogues for both orientations were found by molecular modeling. However, acylation of the 3′‐hydroxyl places the thymine base in the alternate hydrophobic pocket of PCLs substrate‐binding site where it can hydrogen bond to the side‐chain hydroxyls of Tyr23 and Tyr29 and the main chain carbonyl of Leu17. Conversely, acylation of the 5′‐hydroxyl leaves the thymine base in the solvent where there is no favorable binding to the enzyme. We propose that these remote stabilizing interactions between the thymine base and PCLs substrate‐binding site stabilize the 3′‐acylation transition state and thus account for the unusual regioselectivity.