Recombinant Δ4,5‐Steroid 5 β‐Reductases as Biocatalysts for the Reduction of Activated CC‐Double Bonds in Monocyclic and Acyclic Molecules

It was found that Δ^4,5‐steroid 5β‐reductases are capable of reducing also small molecules bearing an activated CC double bond such as monocyclic enones and acyclic enoate esters. As preferred Δ^4,5‐steroid 5β‐reductase (5β‐StR) for this purpose, 5β‐StR from Arabidopsis thaliana was used. In part, enzyme activities are even higher than that for progesterone. Successful preliminary biotransformations with enzymatic in situ cofactor recycling were also carried out. When using the prochiral compound isophorone as a substrate, a high enantioselective reaction course (>99% ee) was observed.     

Highly Efficient Chemoenzymatic Synthesis of Methyl (R)‐o‐Chloromandelate,a Key Intermediate for Clopidogrel,via Asymmetric Reduction with Recombinant Escherichia coli

Methyl (R)‐o‐chloromandelate [(R)‐ 1 ], which is an intermediate for a platelet aggregation inhibitor named clopidogrel, was obtained in >99% ee by the asymmetric reduction of methyl o‐chlorobenzoylformate ( 2 ) with recombinant Escherichia coli overproducing a versatile carbonyl reductase. A remarkable temperature effect on productivity was observed in the whole‐cell reduction of 2 , and the optimum productivity as high as 178 g/L was attained at 20 °C on a 2‐g scale (1.0 M). The optimized reaction could be scaled up easily to transform 20 g of 2 in 100 mL of buffer. Three synthetic methods for 2 are compared.     

Asymmetric Reduction of Activated Alkenes by Pentaerythritol Tetranitrate Reductase: Specificity and Control of Stereochemical Outcome by Reaction Optimisation

We show that pentaerythritol tetranitrate reductase (PETNR), a member of the ‘ene’ reductase old yellow enzyme family, catalyses the asymmetric reduction of a variety of industrially relevant activated α,β‐unsaturated alkenes including enones, enals, maleimides and nitroalkenes. We have rationalised the broad substrate specificity and stereochemical outcome of these reductions by reference to molecular models of enzyme‐substrate complexes based on the crystal complex of the PETNR with 2‐cyclohexenone 4a . The optical purity of products is variable (49–99% ee), depending on the substrate type and nature of substituents. Generally, high enantioselectivity was observed for reaction products with stereogenic centres at Cβ (>99% ee). However, for the substrates existing in two isomeric forms (e.g., citral 11a or nitroalkenes 18 – 19a ), an enantiodivergent course of the reduction of E/Z‐forms may lead to lower enantiopurities of the products. We also demonstrate that the poor optical purity obtained for products with stereogenic centres at Cα is due to non‐enzymatic racemisation. In reactions with ketoisophorone 3a we show that product racemisation is prevented through reaction optimisation, specifically by shortening reaction time and through control of solution pH. We suggest this as a general strategy for improved recovery of optically pure products with other biocatalytic conversions where there is potential for product racemisation.     

Asymmetric Synthesis of Optically Pure Pharmacologically Relevant Amines Employing ω‐Transaminases

Various ω‐transaminases were tested for the synthesis of enantiomerically pure amines from the corresponding ketones employing D ‐ or L ‐alanine as amino donor and lactate dehydrogenase to remove the side‐product pyruvate to shift the unfavourable reaction equilibrium to the product side. Both enantiomers, (R)‐ and (S)‐amines, could be prepared with up to 99% ee and >99% conversions within 24 h at 50 mM substrate concentration. The activity and stereoselectivity of the amination reaction depended on the ω‐transaminase and substrate employed; furthermore the co‐solvent significantly influenced both the stereoselectivity and activity of the transaminases. Best results were obtained by employing ATA‐117 to obtain the (R)‐enantiomer and ATA‐113 or ATA‐103 to access the (S)‐enantiomer with 15% v v⁻¹ DMSO.     

Enantioselective Synthesis of a Positive Allosteric Modulator of the Metabotropic Glutamate Receptor 5 (mGluR5) Receptor via Dynamic Kinetic Resolution of α‐Amino Ketones

The concise synthesis of a pharmaceutical candidate is described. The chiral core of the molecule is assembled using an aza‐benzoin condensation and a dynamic kinetic resolution (DKR) as the key reactions. This enables superb control of the regio‐, diastereo‐ and enantioselectivity of the synthesis. Both biocatalysts and transition metal catalysts are remarkably effective in the key asymmetric reduction step. Similar approaches could be considered in the synthesis of other 1,2‐amino alcohols where traditional approaches based on functionalization of alkenes, epoxides or aziridines may suffer from selectivity issues.

     

Dynamic Kinetic Resolution of 2‐Phenylpropanal Derivatives to Yield β‐Chiral Primary Amines via Bioamination

The amination of racemic α‐chiral aldehydes, 2‐phenylpropanal derivatives, was investigated employing ω‐transaminases. By medium and substrate engineering the optical purity of the resulting β‐chiral chiral amine could be enhanced to reach optical purities up to 99% ee. Using enantiocomplementary ω‐transaminases allowed us to access the (R)‐ as well as the (S)‐enantiomer in most cases. It is important to note that the stereopreference of the ω‐transaminases found for α‐chiral aldehydes did not correlate with the stereopreference previously observed for the amination of methyl ketones. In one case the stereopreference switched even upon exchanging a methyl substituent to a methoxy group.

     

Efficient Reduction of Ethyl 2‐Oxo‐4‐phenylbutyrate at 620 g⋅L−1 by a Bacterial Reductase with Broad Substrate Spectrum

A β‐ketoacyl‐ACP reductase (FabG) gene from Bacillus sp. ECU0013 was heterologously overexpressed in Escherichia coli and the encoded protein was purified to homogeneity. The recombinant reductase could reduce a broad spectrum of prochiral ketones including aromatic ketones and keto esters and showed the highest activity in the asymmetric reduction of ethyl 2‐oxo‐4‐phenylbutyrate (OPBE). Using E. coli cells coexpressing both FabG and glucose dehydrogenase (GDH) genes, as much as 620 g⋅L⁻¹ of OPBE was almost stoichiometrically converted to ethyl (S)‐2‐hydroxy‐4‐phenylbutyrate [(S)‐HPBE] with excellent (>99%) enantiomeric excess. More importantly, the process could be performed smoothly without external addition of an expensive cofactor as usually done and could be scaled up very easily. All these positive features demonstrate the applicability of this reductase for the large‐scale production of optically active α‐hydroxy acids/esters.     

Desymmetrisations of 1‐Alkylbicyclo[3.3.0]octane‐2,8‐diones by Enzymatic Retro‐Claisen Reaction Yield Optically Enriched 2,3‐Substituted Cyclopentanones

A series of 1‐alkylbicyclo[3.3.0]octane‐2,8‐diones was transformed by enzymatic retro‐Claisen reaction using recombinant 6‐oxocamphor hydrolase (OCH) overexpressed in Escherichia coli, to yield optically active 2,3‐substituted cyclopentanones with enantiomeric excesses of up to >95 %. Whilst the parent substrate, bicyclo[3.3.0]octane‐2,8‐dione 12 , was transformed only very slowly, derivatives 13, 14, 15, 16 and 30 with alkyl chains of varying length in the 1‐position were converted rapidly to optically active products with typically 82 % de and up to >95 % enantiomeric excess. The results confirm the apparent requirement of OCH for non‐enolisable diketone substrates, and offer a potential route to decorated cyclopentanone derivatives of multiple chiral centres. Computer modelling of 1‐methylbicyclo[3.3.0]octane‐2,8‐dione into the active site of OCH suggested that the bicyclic [3.3.0] series substrates were accommodated in the active site in similar orientation with the natural enzyme substrate, 6‐oxocamphor, and would thus yield the (2S,3S)‐product series.     

Photoenzymatic Reduction of CC Double Bonds

A simplified procedure for cell‐free biocatalytic reductions of conjugated CC double bonds using old yellow enzymes (OYEs) is reported. Instead of indirectly regenerating YqjM (an OYE homologue from B. subtilis) or NemA (N‐ethylmaleimide reductase from E. coli) via regeneration of reduced nicotinamide cofactors, we demonstrate that direct regeneration of catalytically active reduced flavins is an efficient and convenient approach. Reducing equivalents are provided from simple sacrificial electron donors such as ethylenediaminetetraacetate (EDTA), formate, or phosphite via photocatalytic oxidation. This novel photoenzymatic reaction scheme was characterized. Up to 65% rates of the NADH‐driven reaction were obtained while preserving enantioselectivity. The chemoselectivity of the novel approach was exclusive. Even when using crude cell extracts as biocatalyst preparations, only CC bond reduction was observed while ketone and aldehyde groups remained unaltered. Overall, a simple and practical approach for photobiocatalytic reductions is presented.     

Enantioselective Phase‐Transfer Catalytic α‐Benzylation and α‐Allylation of α‐tert‐Butoxycarbonyllactones

A new enantioselective α‐benzylation and α‐allylation of α‐tert‐butoxycarbonyllactones was devloped. α‐Benzylation and α‐allylation of α‐tert‐butoxycarbonylbutyrolactone and α‐tert‐butoxycarbonylvalerolactone under phase‐transfer catalytic conditions (50% cesium hydroxide, toluene, −60 °C) in the presence of (S,S)‐3,4,5‐trifluorophenyl‐NAS bromide (1 mol%) afforded the corresponding α‐substituted α‐tert‐butoxycarbonyllactones in very high chemical yields (up to 99%) and optical yields (up to 99% ee). The synthetic potential of this method has been successfully demonstrated by the asymmetric synthesis of unnatural α‐quaternary homoserines, 3‐alkyl‐3‐carboxypyrrolidine and 3‐alkyl‐3‐carboxypiperidine.     

Enantioselective Synthesis of α‐Amino‐γ‐sulfonyl Phosphonates with a Tetrasubstituted Chiral α‐Carbon via Quinine‐Squaramide‐Catalyzed Michael Addition of Nitrophosphonates to Vinyl Sulfones

α‐Nitro‐γ‐sulfonyl phosphonates with a key tetrasubstituted chiral α‐carbon center have been synthesized for the first time in high yield and enantioselectivity through a quinine‐squaramide‐catalyzed conjugate addition of α‐nitro phosphonates to aryl vinyl sulfones. Representative examples presented here for the transformation of nitrosulfonyl phosphonates to aminosulfonyl phosphonates, alkylation at the α‐position of the sulfonyl group followed by desulfonation and scale‐up of the conjugate addition highlight the practical applications of the methodology.     

Chemoenzymatic One‐Pot Synthesis of γ‐Butyrolactones

The synthesis of enantio‐ and diastereomerically pure γ‐butyrolactones is described using a one‐pot, two‐enzyme cascade. Ethyl 2‐methyl‐4‐oxopent‐2‐enoate ( 2 ) was reduced selectively first in a 1,4‐reduction using the old yellow enzyme (OYE1) [EC 1.6.99.1] and consecutively in a 1,2‐reduction by an alcohol dehydrogenase [EC 1.1.1.2].     

Cutting Short the Asymmetric Synthesis of the Ramatroban Precursor by Employing ω‐Transaminases

Starting from an adequate ketone precursor previous reports required three steps for the preparation of (R)‐2,3,4,9‐tetrahydro‐1H‐carbazol‐3‐amine, a key intermediate for the synthesis of the antiallergic drug ramatroban. A single biocatalytic step was sufficient to prepare the target amine with >97% ee (HPLC) via reductive amination of the corresponding ketone using an ω‐transaminase as biocatalyst. Since the ketone was barely soluble under the reaction conditions employed, it was provided as a solid and still the reaction went to completion within 4 h at 50 mM substrate concentration. Although 2‐propylamine is regarded as an ideal amine donor, it turned out to be detrimental for the specific ketone precursor leading to the formation of various side products. These could be avoided by using (R)‐1‐phenylethylamine as the best suited amine donor. An alternative work‐up was developed via freeze‐drying of the reaction mixture, enabling the isolation of the desired (R)‐amine in excellent yield (96%) and enantiopure form on a preparative scale (500 mg). No purification steps (e.g., column chromatography, crystallisation) were required.

     

Loop‐Grafted Old Yellow Enzymes in the Bienzymatic Cascade Reduction of Allylic Alcohols

The enzymatic reduction of C=C bonds in allylic alcohols with Old Yellow Enzymes represents a challenging task, due to insufficient activation through the hydroxy group. In our work, we coupled an alcohol dehydrogenase with three wild‐type ene reductases—namely nicotinamide‐dependent cyclohex‐2‐en‐1‐one reductase (NCR) from Zymomonas mobilis, OYE1 from Saccharomyces pastorianus and morphinone reductase (MR) from Pseudomonas putida M10—and four rationally designed β/α loop variants of NCR in the bienzymatic cascade hydrogenation of allylic alcohols. Remarkably, the wild type of NCR was not able to catalyse the cascade reaction whereas MR and OYE1 demonstrated high to excellent activities. Through the rational loop grafting of two intrinsic β/α surface loop regions near the entrance of the active site of NCR with the corresponding loops from OYE1 or MR we successfully transferred the cascade reduction activity from one family member to another. Further we observed that loop grafting revealed certain influences on the interaction with the nicotinamide cofactor.     

Enantioselective Construction of Cyclic Indolyl α‐Amino Esters via a Friedel–Crafts Alkylation Reaction

An enantioselective Friedel–Crafts alkylation reaction of indoles with cyclic N‐sulfonyl ketimino esters was developed. Under the optimized conditions using a chiral copper(II) triflate‐bisoxazoline complex as the catalyst, a range of N‐sulfonyl ketimino ester derivatives and indoles reacted smoothly to afford indole‐containing chiral cyclic α‐amino esters bearing tetrasubstituted α‐stereogenic centers [3‐ethoxycarbonyl‐3‐(3‐indolyl)‐2,3‐dihydrobenzo[d]isothiazole 1,1‐dioxides] in excellent yields and with high enantioselectivities (up to 99% ee). Pyrrole and N,N‐dimethylaniline were also investigated as aromatic substrates to afford the corresponding products with good results. An asymmetric induction model was then proposed on the basis of the observed absolute configuration of the product 3‐ethoxycarbonyl‐3‐(5‐bromo‐3‐indolyl)‐2,3‐dihydrobenzo[d]isothiazole 1,1‐dioxide. Synthetic transformations to convert the products into cyclic chiral N‐sulfonamido alcohols and the deprotection of the sulfonamides were performed. This study provides an efficient approach to chiral α‐tetrasubstituted indolic α‐amino acids as potential building blocks for peptides and biologically active molecules.

     

Insights into Sequence–Activity Relationships amongst Baeyer–Villiger Monooxygenases as Revealed by the Intragenomic Complement of Enzymes from Rhodococcus jostii RHA1

The Rhodococcus jostii RHA1 genome encodes a number of enzymes that can be exploited as biocatalysts. Study of the substrate spectrum and enantioselectivity of Baeyer–Villiger monooxygenases from R. jostii allowed the identification of short amino acid sequences specific to groups displaying certain catalytic characteristics. The gel illustrates the substrate acceptance spectra and selectivities of the different proteins.

     

Phylogenetic Studies,Gene Cluster Analysis,and Enzymatic Reaction Support Anthrahydroquinone Reduction as the Physiological Function of Fungal 17β‐Hydroxysteroid Dehydrogenase

17β‐Hydroxysteroid dehydrogenase (17β‐HSDcl) from the filamentous fungus Curvularia lunata (teleomorph Cochliobolus lunatus) catalyzes NADP(H)‐dependent oxidoreductions of androgens and estrogens. Despite detailed biochemical and structural characterization of 17β‐HSDcl, its physiological function remains unknown. On the basis of amino acid sequence alignment, phylogenetic studies, and the recent identification of the physiological substrates of the homologous MdpC from Aspergillus nidulans and AflM from Aspergillus parasiticus, we propose an anthrahydroquinone as the physiological substrate of 17β‐HSDcl. This is also supported by our analysis of a secondary metabolite biosynthetic gene cluster in C. lunata m118, containing 17β‐HSDcl and ten other genes, including a polyketide synthase probably involved in emodin formation. Chemoenzymatic reduction of emodin by 17β‐HSDcl in the presence of sodium dithionite verified this hypothesis. On the basis of these results, the involvement of a 17β‐HSDcl in the biosynthesis of other anthrahydroquinone‐derived natural products is proposed; hence, 17β‐HSDcl should be more appropriately referred to as a polyhydroxyanthracene reductase (PHAR).     

Base–Base Bifunctional Catalysis: A Practical Strategy for Asymmetric Michael Addition of Malonates to α,β‐Unsaturated Aldehydes

Lewis base–Brønsted base bifunctional catalysis is a novel and practical strategy for the asymmetric Michael addition. The addition of malonates to a series of α,β‐unsaturated aldehydes can take place under base–base bifunctional catalytic conditions using 0.5–5 mol% of (S)‐2‐[diphenyl(trimethylsilyloxy)methyl]pyrrolidine as catalyst and 5–30 mol% of lithium 4‐fluorobenzoate as additive base with up to 99% ee.     

Conjugate Reduction of α,β‐Unsaturated Carbonyl and Carboxyl Compounds with Poly(methylhydrosiloxane) Catalyzed by a Silica‐Supported Compact Phosphane–Copper Complex

A silica‐supported, cage‐type, compact phosphane (Silica‐SMAP) was used for the copper‐catalyzed conjugate reduction of α,β‐unsaturated carbonyl and carboxyl compounds with poly(methylhydrosiloxane) (PMHS). The heterogeneous catalyst system showed high activity and chemoselectivity, and was easily separable from the reaction mixture after the reaction. Furthermore, the catalyst was reusable without loss of its high catalytic activity or selectivity.