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Financial support from the Spanish Ministry of Science and Innovation (MCI, PID2019-109253RB-I00) and the Asturian Regional Government (AYUD/2021/51542) are gratefully acknowledged. Technical support from the Scientific-Technical Services (UNIOVI) is acknowledged. We thank Prof. Wolfgang Kroutil (University of Graz, Austria) for providing us with alcohol dehydrogenases overexpressed in E. coli cells. S.G.-G. thanks the University of Oviedo for a predoctoral fellowship (PAPI-21-PF-14) inside the “Programa de Apoyo y Promoción de la Investigación”.

A bienzymatic cascade has been designed and optimized to obtain enantiopure chlorohydrins starting from the corresponding 1-aryl-2-chlorobut-2-en-1-ones. For the synthesis of these α-chloroenones, a two-step sequence was developed consisting of the allylation of the corresponding aldehyde with 3-dichloroprop-1-ene, followed by oxidation and further isomerization. The selective cooperative catalytic system involving ene-reductases (EREDs) and alcohol dehydrogenases (ADHs) afforded the desired optically active chlorohydrins under mild reaction conditions in excellent conversions (up togt;99%) and selectivities (up togt;99:1 diastereomeric ratio (dr),gt;99% enantiomeric excess (iee/i)).

Financial support from the Spanish Ministry of Science and Innovation (MCI,PID2019-109253RB-I00) and the Asturian Regional Government (AYUD/2021/51542) are gratefully acknowledged. L.R.-F. thanks the Asturian regional government for a predoctoral fellowship inside the Severo Ochoa programme (BP21-057). The technical support of the Scientific-Technical Services of the University of Oviedo is acknowldged.

This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 860414

The synthesis of enantioenriched β-chlorohydrins is highly appealing due to their relevance as building-blocks in organic synthesis. However, the approximation to aliphatic derivatives is particularly challenging due to the difficulties to get access to the α-chloroketone precursors. Herein, we propose a straightforward and scalable approach combining in a concurrent manner gold(I) and redox enzyme catalysis through a hydration-bioreduction cascade. A total of nine aliphatic β-chlorohydrins bearing different functional groups were obtained with very high yields (63-88%) and stereoselectivities (99% ee).

Financial supports from the Spanish Ministry of Science and Innovation (MCI, PID2019-109253RB-I00) and the Asturian Government (AYUD/2021/51542) are acknowledged.S. G.-G. thanks University of Oviedo for a predoctoral fellowship (PAPI-21-PF-14).

Dynamic kinetic resolutions of racemic compounds provide elegant synthetic possibilities for the preparation of valuable enantiopure organic molecules with a theoretical maximum 100% yield. This chapter describes the combination of stereoselective enzymatic methods with suitable conditions for the racemization of the slow-reacting enantiomer from racemates of various types of compounds, such as alcohols, amines, and amino acids, for the synthesis of biologically active compounds and natural products. This contribution has been divided into three main topics based on the enzyme that catalyzes the asymmetric transformation and the racemization conditions of choice. These are: (i) the use of hydrolases and metal species; (ii) the use of hydrolases without requiring a metal catalyst for the racemization step; (iii) the use of other enzyme classes. A selection of scalable experimental procedures is provided in each case to demonstrate the robustness of the methodology described.

The development of methods to engineer and immobilize amine transaminases (ATAs) to improve their functionality and operational stability is gaining momentum. The quest for robust, fast, and easy-to-use methods to screen the activity of large collections of transaminases, is essential. This work presents a novel and multiplex fluorescence-based kinetic assay to assess ATA activity using 4-dimethylamino-1-naphthaldehyde as an amine acceptor. The developed assay allowed us to screen a battery of amine donors using free and immobilized ATAs from different microbial sources as biocatalysts. As a result, using chromatographic methods, 4-hydroxybenzylamine was identified as the best amine donor for the amination of 5-(hydroxymethyl)furfural. Finally, we adapted this method to determine the apparent Michaelis-Menten parameters of a model immobilized ATA at the microscopic (single-particle) level. Our studies promote the use of this multiplex, multidimensional assay to screen ATAs for further improvement.

The combination of gold(I) and enzyme catalysis is used in a two-step approach, including Meyer-Schuster rearrangement of a series of readily available propargylic alcohols followed by stereoselective bioreduction of the corresponding allylic ketone intermediates, to provide optically pure β,β-disubstituted allylic alcohols. This cascade involves a gold N-heterocyclic carbene and an enzyme, demonstrating the compatibility of both catalyst types in aqueous medium under mild reaction conditions. The combination of [1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene][bis(trifluoromethanesulfonyl)-imide]gold(I) (IPrAuNTf2 ) and a selective alcohol dehydrogenase (ADH-A from Rhodococcus ruber, KRED-P1-A12 or KRED-P3-G09) led to the synthesis of a series of optically active (E)-4-arylpent-3-en-2-ols in good yields (65-86 %). The approach was also extended to various 2-hetarylpent-3-yn-2-ol, hexynol, and butynol derivatives. The use of alcohol dehydrogenases of opposite selectivity led to the production of both allyl alcohol enantiomers (93->99 % ee) for a broad panel of substrates.

Polymeric materials containing ionic liquid fragments, like those found in bulk ILs, are excellent solid media for the immobilisation of biocatalysts. Herein, the entrapment of the enzymatic system formed by alcohol dehydrogenase from Rhodococcus ruber (ADH-A) overexpressed in E. coli and its coenzyme has been studied. The activity, stability and reusability of these preparations have been investigated in the bioreduction of prochiral ketones finding excellent levels of conversion and selectivity. Interestingly, the immobilised enzyme remained active and exhibited excellent stability in aqueous solutions after several recycling uses. More importantly, these biopolymer materials retained most of their activity after consecutive reaction cycles, prolonged storage and under flow conditions.

Financial support of this work by the Spanish Ministerio de Economía y Competitividad (MINECO, CTQ-2013-44153-P project) is gratefully acknowledged. D.G.-M. thanks also Asturian Regional Government for a predoctoral fellowship. we also thank Prof. Wolfgang Kroutil for the donation of overexpressed alcohol dehydrogenases.

The authors thank the national Council for Scientific and Technological Development (CNPq, Brazil, Proc. 456834/2014), the Coordination for the Improvement of Higher Level Personnel (CAPES, Brazil), the Spanish Ministry of Economy and Competitiveness (MINECO, CTQ-2013-44153-P project) and the Government of the Principado de Asturias (FC-GRUPIN-IDI/2018/000181) for financial supports. P. T. B thanks CAPES for her PDSE fellowship (Edital No 47/2017). Prof. Wolfgang Kroutil is also acknowledged for the donation of overexpressed alcohol dehydrogenases.

The compatibility between gold(I) catalysts and amine transaminases has been explored to transform racemic propargylic alcohols into enantioenriched allylic amines in a straightforward and selective manner. The synthetic approach consists of a gold(I)-catalysed Meyer-Schuster rearrangement of a series of 2-arylpent-3-yn-2-ols and a subsequent stereoselective enzyme-catalysed transamination of the resulting alpha,beta-unsaturated prochiral ketones. The design of cascade processes involving sequential or concurrent approaches has been studied in our search for ideal reaction conditions to produce the desired amines. Thus, the N-heterocyclic carbene complex [1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene]-[bis(trifluoromethanesulfonyl)-imide]gold(I) ([Au(IPr)(NTf2)] (A) in aqueous medium was found to be an ideal catalyst, while selective, made-in-house and commercial amine transaminases permitted the asymmetric synthesis of both (E)-4-arylpent-3-en-2-amine enantiomers in good isolated yields (53-84%) and excellent stereoselectivities (97 to >99% enantiomeric excess).

A series of optically active β-hydroxy sulfones has been obtained through an oxosulfonylation-stereoselective reduction sequence in aqueous medium. Firstly, β-keto sulfones were synthesized from arylacetylenes and sodium sulfinates to subsequently develop the carbonyl reduction in a highly selective fashion using alcohol dehydrogenases as biocatalysts. Optimization of the chemical oxosulfonylation reaction was investigated, finding inexpensive iron(III) chloride hexahydrate (FeCl3 ⋅ 6H2 O) as the catalyst of choice. The selection of isopropanol in the alcohol-water media resulted in high compatibility with the enzymatic process for enzyme cofactor recycling purposes, providing a straightforward access to both (R)- and (S)-β-hydroxy sulfones. The practical usefulness of this transformation was illustrated by describing the synthesis of a chiral intermediate of Apremilast. Interestingly, the development of a chemoenzymatic cascade approach avoided the isolation of β-keto sulfone intermediates, which allowed the preparation of chiral β-hydroxy sulfones in high conversion values (83-94 %) and excellent optical purities (94 to >99 % ee).

A series of amino alcohols have been prepared in a chemo-, diastereo- and enantioselective fashion starting from the corresponding (het)aryl diketones, avoiding tedious chemical protection and deprotection steps. Different alcohol dehydrogenases have been able to selectively reduce the more reactive trifluoroacetyl groups under optimized conditions, while amine transaminases catalyzed the biotransamination of the less hindered acetyl groups. Based on the different reactivity of the acetyl and trifluoroacetyl groups, the design of sequential and concurrent cascades was investigated. The proper selection of the enzymes permits the synthesis of amino alcohol stereoisomers in high to excellent yields (86->99% conversion) and remarkable stereocontrol (up to >99% de and >99% ee) using an aqueous medium and mild reaction conditions., Financial support of this work by the Spanish Ministry of Economy and Competitiveness (MINECO, CTQ2016-75752-R project) and Asturian Regional Government (FC-GRUPINIDI/2018/000181) are gratefully acknowledged. D.G.-M. thanks also Asturian Regional Government for a predoctoral fellowship.

A sequential two-step chemoenzymatic methodology for the stereoselective synthesis of (3E)-4-(het)arylbut-3-en-2-amines in a highly selective manner and under mild reaction conditions is described. The approach consists of oxidation of the corresponding racemic alcohol precursors by the use of a catalytic system made up of the laccase from Trametes versicolor and the oxy-radical TEMPO, followed by the asymmetric reductive bio-transamination of the corresponding ketone intermediates. Optimisation of the oxidation reaction, exhaustive amine transaminase screening for the bio-transaminations and the compatibility of the two enzymatic reactions were studied in depth in search of a design of a compatible sequential cascade. This synthetic strategy was successful and the combinations of enzymes displayed a broad substrate scope, with 16 chiral amines being obtained in moderate to good isolated yields (29-75 %) and with excellent enantiomeric excess values (94 to >99 %). Interestingly, both amine enantiomers can be achieved, depending on the selectivity of the amine transaminase employed in the system.

In this work, the first example of a radical stereodivergent reaction directed towards the stereoselective synthesis of both (R*,R*)- and (R*,S*)-2,2'-biflavanones promoted by samarium diiodide is reported. Control experiments showed that the selectivity of this reaction was exclusively controlled by the temperature. It was possible to generate a variety of 2,2'-biflavanones bearing different substitution patterns at the aromatic ring in good-to-quantitative yields, being both stereoisomers of the desired compounds obtained with total or high control of selectivity. A mechanism that explains both the generation of the corresponding 2,2'-biflavanones and the selectivity is also discussed. The structure and stereochemistry determination of each isomer was unequivocally elucidated by single-crystal X-ray diffraction experiments.

A collection of fusion biocatalysts has been generated that can be used for self-sufficient oxygenations or ketone reductions. These biocatalysts were created by fusing a Baeyer-Villiger monooxygenase (cyclohexanone monooxygenase from Thermocrispum municipale: TmCHMO) or an alcohol dehydrogenase (alcohol dehydrogenase from Lactobacillus brevis: LbADH) with three different cofactor regeneration enzymes (formate dehydrogenase from Burkholderia stabilis: BsFDH; glucose dehydrogenase from Sulfolobus tokodaii: StGDH, and phosphite dehydrogenase from Pseudomonas stutzeri: PsPTDH). Their tolerance against various organic solvents, including a deep eutectic solvent, and their activity and selectivity with a variety of substrates have been studied. Excellent conversions and enantioselectivities were obtained, demonstrating that these engineered fusion enzymes can be used as biocatalysts for the synthesis of (chiral) valuable compounds. (C) 2019 The Authors. Published by Elsevier Ltd.

Deep eutectic solvents (DESs) nowadays represent a sustainable alternative to traditional organic solvents in (bio)transformations. Herein, the use of a solvent composed of an aqueous buffer and choline chloride : glucose (1.5 : 1 mol/mol) is proposed, a natural DES (NADES) serving as both a cosolvent and efficient system to recycle the nicotinamide cofactor. Thus, glucose from the NADES served as a co-substrate required for several alcohol dehydrogenases to reduce different prochiral ketones, and also helped to solubilise the organic compounds to develop effective biotransformations at higher substrate concentrations.

Flavan-4-ols are a subclass of flavonoids that are present in complex molecules with application in the industrial sector as pigments, antioxidants, or antimitotics, among many others. The most traditional way to achieve their synthesis is from naturally abundant flavanones, asymmetric transfer hydrogenation reactions or bioreduction being well known strategies, while their preparation from racemic flavan-4-ols has been less explored. In this article, we have focused on the synthesis of a series of trans-flavan-4-ols bearing different substitution patterns in the aromatic ring to explore later the potential of lipases as biocatalysts for stereoselective acylation reactions. Therefore, a series of flavanones have been chemically prepared, starting from the corresponding benzaldehydes by aldol condensation with 2′-hydroxyacetophenone in a strongly basic medium, and later transformed into the corresponding racemic trans-flavan-4-ols following a carbonyl reduction, Mitsunobu reaction, and ester deprotection sequence. A screening of lipases and optimization of the reaction conditions for the stereoselective acylation of racemic 2-phenylchroman-4-ol were performed before expanding the best reaction conditions to the kinetic resolution of other 2-arylchroman-4-ols. Interestingly, the combination of AK lipase from Pseudomonas fluorescens as enzyme and vinyl acetate as both acyl donor and solvent allowed the performance of highly asymmetric transformations (E &gt, 200, 50–99% eeS and &gt, 99% eeP) under mild reaction conditions (30 °C and 250 rpm).

The transfer hydrogenation of flavanones and ortho-hydroxychalcones catalyzed by ruthenium pincer complexes RuCl(CNNPh)(disphosphine) has allowed the synthesis of ortho-hydroxy 1,3-diarypropanols in 80–88 % yield, under mild reaction conditions and short reaction times (1 h) in 2-propanol. The amount of the co-catalyst NaOiPr has been found crucial for the selective reduction of flavanones to ortho-hydroxy 1,3-diarypropanols vs. flavan-4-ols. Preliminary results show that with pincer catalysts bearing (S,R)-Josiphos, flavanone is reduced to the corresponding (S)-alcohol in moderate conversion (36 %) and up to 92 % ee., This work was supported by the Spanish Ministry of Science and Innovation (PID2019-109253RB-I00) and from the Ministero dell’Università e della Ricerca (MIUR).

Financial support from the Spanish Ministry of Economy and Competitiveness (MEC, Project CTQ2016-75752-R), the Spanish Ministry of Science and Innovation (MCI, PID2019- 109253RB-I00) and the Asturian regional government (FC-GRUPIN-IDI/2018/000181) are gratefully acknowledged. J. A.-V. also thanks the Asturian regional government for a predoctoral fellowship inside the Severo Ochoa programme (BP17-32). Prof. Wolfgang Kroutil (University of Graz, Austria) is acknowledged for the donation of overexpressed alcohol dehydrogenases and amine transaminases.

A one-pot/two-step bienzymatic asymmetric amination of secondary alcohols is disclosed. The approach is based on a sequential strategy involving the use of a laccase/TEMPO catalytic system for the oxidation of alcohols into ketone intermediates, and their following transformation into optically enriched amines by using transaminases. Individual optimizations of the oxidation and biotransamination reactions have been carried out, studying later their applicability in a concurrent process. Therefore, 17 racemic (hetero)aromatic sec-alcohols with different substitutions in the aromatic ring have been converted into enantioenriched amines with good to excellent selectivities (90–99% ee) and conversion values (67–99%). The scalability of the process was also demonstrated when two different amine donors were used in the transamination step, such as isopropylamine and cis-2-buten-1,4-diamine. Satisfyingly, both sacrificial amine donors can shift the equilibrium toward the amine formation, leading to the corresponding isolated enantioenriched amines with good to excellent results.

Financial support from the Spanish Ministry of Economy and Competitiveness (MEC, Project CTQ2016-75752-R) is gratefully acknowledged. D.G.-M. thanks the Asturian regional government for a predoctoral fellowship inside the Severo Ochoa program

Financial supports from the Spanish Ministry of Economy and Competitiveness (MEC,Project CTQ2016-75752-R) and the Asturian Regional Government(FC-GRUPIN-IDI/2018/000181)are gratefully acknowledged.

The synthesis of chiral amines is of central importance to pharmaceutical chemistry, and the inclusion of fluorine atoms in drug molecules can both increase potency and slow metabolism. Optically enriched β-fluoroamines can be obtained by the kinetic resolution of racemic amines using amine transaminases (ATAs), but yields are limited to 50%, and also secondary amines are not accessible. In order to overcome these limitations, we have applied NADPH-dependent reductive aminase enzymes (RedAms) from fungal species to the reductive amination of β-fluoroacetophenones with ammonia, methylamine and allylamine as donors, to yield β-fluoro primary or secondary amines with >90% conversion and between 85 and 99% ee. In addition, the effect of the progressive introduction of fluorine atoms to the β-position of the acetophenone substrate reveals the effect of mono-, di- and tri-fluorination on the proportion of amine and alcohol in product mixtures, shedding light on the promiscuous ability of imine reductase (IRED)-type dehydrogenases to reduce fluorinated acetophenones to alcohols.

A series of enantioenriched (hetero)aromatic secondary allylic alcohols has been synthesized through deracemization of the corresponding racemic mixtures combining a non-selective chemoenzymatic oxidation (laccase from Trametes versicolor and oxy-radical TEMPO) and a stereoselective biocatalyzed reduction (lyophilized cells of E. coli overexpressing an alcohol dehydrogenase, ADH). Both steps were performed in aqueous medium under very mild reaction conditions. After optimization, a sequential one-pot two-step protocol was set up, obtaining the corresponding chiral alcohols in moderate to high conversions (48–95%) and enantiomeric excess (65->99% ee). Depending on the ADH stereopreference, both antipodes from these valuable chiral synthons could be prepared, even at preparative scale (119−178 mg), in a straightforward manner.

This is the presentation of the Special Issue “Biocatalysis in Spain”, covering the work of several Spanish groups in the field of Biocatalysis and Biotransformations. Thus, both research articles and reviews allow one to draw an accurate view of the state-of-the-art development in Spain.

The design of catalytic oxidative methodologies in aqueous medium under mild reaction conditions and using molecular oxygen as final electron acceptor represents a suitable alternative to the traditional oxidative transformations. These methods are especially relevant if other functionalities that can be oxidized are present within the same molecule, as in the case of allylic alcohols. Herein we apply a simple chemoenzymatic system composed of the laccase from Trametes versicolor and 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) to oxidize a series of racemic allylic sec-alcohols into the corresponding α,β-unsaturated ketones. Afterward, these compounds react with different commercially available ene-reductases to afford the corresponding saturated ketones. Remarkably, in the case of trisubstituted alkenes, the bioreduction reaction occurred with high stereoselectivity. Overall, a bienzymatic one-pot two-step sequential strategy has been described with respect to the synthesis of saturated ketones sta...

Biocatalysis is gaining increasing attention in the academic and industrial sector due to the possibility of developing highly stereoselective transformations in a sustainable manner. The creation of stereogenic centres in organic synthesis is not trivial and multiple approaches have been disclosed based on organometallic and organocatalytic methods with the use of day by day more complex catalysts to induce asymmetry in selected transformations. The intrinsic chirality of enzymes makes them powerful tools for the development of stereoselective transformations, catalysing a wide range of chemical reactions due to the high abundance and diversity of enzymes in nature. In addition, the enormous advances in rational design and molecular biology methods have opened up the possibility to create more robust and versatile biocatalysts, which have improved the initial activities displayed by wild-type enzymes. Therefore, their applicability has been widely increased in terms of reaction conditions, substr...

A chromatographic method for the separation of volatile compounds in Asturian cider apple juices has been developed. For this separation purpose, a monocationic imidazolium-based ionic liquid bearing a reactive terminal iodine atom was synthesized by a quaternization-anion exchange chemical sequence. Next, the gas chromatography (GC) stationary phase was prepared by covalently linking the imidazolium monolith to the reactive silanol groups of the inner capillary wall at 70 °C. This coated GC column exhibited good thermal stability (290 °C), as well as good efficiency (2000 plates/m) in the separation of volatile compounds from Asturian apple cider juices, and was characterized using the Abraham solvation parameter model. The intra-day and inter-day precision of the chromatographic method was evaluated, obtaining relative standard deviations from 3.7 to 12.9% and from 7.4 to 18.0%, respectively. Furthermore, recoveries from 82.5 to 122% were achieved. Graphical Abstract Covalent bonding of an ionic liquid to inner column wall led to a great improvement of the separation efficiencies of stationary phases in gas chromatography.

The lipase catalyzed resolution of several cis and trans 3-aminopyridinylmethyl-4-hydroxypyrrolidines by acylation or hydrolysis has been studied. Some of these pryrrolidines are precursors in the synthesis of selective nNOS inhibitors; for this purpose, the best result is obtained in the resolution of (±)-trans-5via a CAL-A catalysed acylation. The high enantioselectivity of this process allows one to obtain in high yield the optically pure (3S,4R)-configured product that has the correct configuration to complete the synthesis of nNOS inhibitors. The study described here highlights the effect of structural variations remote from the stereogenic centers of the molecule on the reactivity and enantioselectivity of the enzymatic processes.