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Oxidation chemistry using enzymes is approaching maturity and practical applicability in organic synthesis. Oxidoreductases (enzymes catalysing redox reactions) enable chemists to perform highly selective and efficient transformations ranging from simple alcohol oxidations to stereoselective halogenations of non‐activated C−H bonds. For many of these reactions, no “classical” chemical counterpart is known. Hence oxidoreductases open up shorter synthesis routes based on a more direct access to the target products. The generally very mild reaction conditions may also reduce the environmental impact of biocatalytic reactions compared to classical counterparts. In this Review, we critically summarise the most important recent developments in the field of biocatalytic oxidation chemistry and identify the most pressing bottlenecks as well as promising solutions.

A peroxygenase-catalysed hydroxylation of organosilanes is reported. The recombinant peroxygenase from Agrocybe aegerita (AaeUPO) enabled efficient conversion of a broad range of silane starting materials in attractive productivities (up to 300 mM h−1), catalyst performance (up to 84 s−1 and more than 120 000 catalytic turnovers). Molecular modelling of the enzyme-substrate interaction puts a basis for the mechanistic understanding of AaeUPO selectivity.

Unspecific peroxygenases (UPOs) are promising biocatalysts for oxyfunctionalisation reactions, owing to their simplicity of handling, stability and robustness. A limitation of using UPOs on a large scale is their deactivation in the presence of even rather modest concentrations of H2O2, requiring a constant and controlled supply of low amount of H2O2. Herein, we report an organometallic complex [Cp*Ir(pica)NO3] {pica=picolinamidate=κ2-pyridine-2-carboxamide ion (−1)} 1 capable of efficiently regenerating FMNH2 from FMN (TOF=350 h−1, 298 K), driven by NaHCOO; FMNH2, in turn, spontaneously reacts with O2 leading to H2O2. After having studied the compatibility of 1 with the UPO from Agrocybe aegerita (rAaeUPO PaDa-I) and individuated the best experimental conditions, we applied such a hybrid catalytic tandem in some hydroxylation, epoxidation and sulfoxidation reactions. Best performances were obtained by using a 1/rAaeUPO molar ratio of 50. TONs for the biocatalyst of up to 18933 were obtained for the transformation of ethylbenzene derivatives into (R)-1-phenylethanols (ee>99 %). 1/rAaeUPO was found to oxidise also cis-methyl styrene (TON=13488), leading exclusively (1R,2S)-cis-methyl styrene oxide (ee>99 %), cyclohexane (TON=1634) and thioanisole (TON=1369).

In this contribution, we report chemoenzymatic bromodecarboxylation (Hunsdiecker-type) of α,ß-unsaturated carboxylic acids. The extraordinarily robust chloroperoxidase from Curvularia inaequalis (CiVCPO) generated hypobromite from H2O2and bromide, which then spontaneously reacted with a broad range of unsaturated carboxylic acids and yielded the corresponding vinyl bromide products. Selectivity issues arising from the (here undesired) addition of water to the intermediate bromonium ion could be solved by reaction medium engineering. The vinyl bromides so obtained could be used as starting materials for a range of cross-coupling and pericyclic reactions.

10 páginas.- 4 figuras.- 2 tablas.- 30 referencias.- Supporting information for this article is given via a link at the end of the document https://onlinelibrary.wiley.com/doi/10.1002/anie.202217372, he hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer.The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer., This work was supported by the European Union Project grant H2020-BBI-PPP-2015-2-720297-ENZOX2, the I+D+I PID2019-106166RB-I00-OXYWAVE Spanish project funded by the Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación (AEI)/doi: 10.13039/501100011033/, the PID2020-118968RB-I00 LILI project from the Spanish MCIN/AEI/10.13039/501100011033, the ‘Comunidad de Madrid’ Synergy CAM project Y2018/BIO-4738-EVOCHIMERA-CMand the PIE-CSIC projects PIE-202040E185 and PIE-201580E042. PGS thanks the Ministry of Science, Innovation and Universities (Spain) for her FPI scholarship (BES-2017-080040) and to the Ministry of Science and Innovation for her contract as part of PTQ2020-011037 project funded by MCIN/AEI/10.13039/501100011033 within the NextGenerationEU/PRTR.DGP thanks Juan de la Cierva Incorporación contract Ref nº: IJC2020-043725-I, funded by MCIN / AEI / 10.13039/501100011033, and the EUNextGenerationEU/PRTR program. The authors thank the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance with the BL13-XALOC beamline.

Biocatalytic oxidation reactions of toluene derivates to the corresponding aldehydes are typically challenged by regio- and chemoselectivity issues. In this contribution we address both challenges by a combined reactant- and reaction engineering approach. We demonstrate that the peroxygenase-catalysed transformation of ring-substituted toluenes proceeds highly regioselectively in benzylic position. Furthermore, neat reaction conditions not only enable attractive product concentrations (up to 185 mM) but also result in highly chemoselective oxidations to the aldehyde level.

Oxidation reactions are at the heart of organic chemistry. In industry, the epoxidation of ethylene and propylene or the hydroxylation of benzene for example is conducted at a scale of several million tonnes per year. At the same time, many of the established oxidation processes require relatively harsh reaction conditions and suffer from selectivity issues. As a consequence, the environmental impact of industrial oxidations remains high.Biocatalysis has a lot to offer here. Enzymes not only operate efficiently under very mild reaction conditions but also excel in terms of selectivity making them attractive alternatives to currently widespread transition metal catalysts for oxidation chemistry.In this special issue, we have assembled contributions from some of the leaders in the academic research community working on biocatalytic oxidation chemistry. We highlight the current state-of-the-art in the field demonstrating its enormous potential for industrial scale application but also highlighting its current limitations.Alcohol dehydrogenases have been among the first enzymes investigated for biocatalytic oxidation reactions. The manifold applications of alcohol dehydrogenases for the stereo- and regio-selective oxidation of primary and secondary alcohols are summarised in the contribution by Milagre and coworkers.While alcohol dehydrogenases transfer the reducing equivalents liberated from the starting materials to nicotinamide cofactors (NAD(P)+), oxidases utilise molecular oxygen as a sacrificial electron acceptor. Hence, oxidases appear promising, more cost-efficient alternatives to NAD(P)+-dependent dehydrogenases. The recent rapid developments in oxidase catalysis are critically summarised and discussed in the contribution by Heath and Turner.Dehydrogenases and oxidases catalyse dehydrogenation (hydride abstraction) reactions and therefore require pre-existing functional groups such as alcohols or amines for activity. In contrast, (non-) heme iron oxygenases catalyse the insertion of O-atoms (and more) even into non-activated C–H bonds. Hence, these oxygenases often accomplish chemistries, which are not achievable by existing chemical technologies.The so-called P450 monooxygenases have been forerunners for biocatalytic oxyfunctionalisation chemistry resulting in an enormous portfolio of highly regio- and stereoselective oxyfunctionalisation reactions. This wealth of possibilities is nicely summarised in the contribution by Opperman and co-workers.More recently, P450 monooxygenases have been challenged as catalysts of choice by the so-called unspecific peroxygenases (UPOs) essentially catalysing the same reactions as P450 monooxygenases but mechanistically far simpler. The rapid development of peroxygenases as catalysts for oxyfunctionalisation chemistry is summarised by Alcalde and co-workers. Xu et al. critically compare both enzyme classes and importantly highlight some limitations en route to large-scale application.Halogenases represent a truly exciting emerging class of oxygenases as for the highly selective introduction of halogens into non-activated C–H bonds where no chemical equivalents are known. The contribution by Buller and coworkers provides a concise introduction to the various halogenases known today, mechanistic insights and provides an overview over synthetic possibilities.Generally, oxidation reactions yield more reactive products such as occurs with alcohol oxidation or alkene epoxidation. Hence, the products of biocatalytic oxidation reactions themselves serve as starting materials for further transformations. The cascade catalysis concept envisions performing the oxidation step and the subsequent derivatisation step integrated together, eliminating time- and resource-consuming isolation and purification of the intermediates. This fascinating research is introduced in the contribution by Zhi and co-workers.Despite the wealth of engineered biocatalysts available, oxidative biocatalysis still is underrepresented, especially in the synthesis of commodity and bulk chemicals. Several issues still require intensive R&D efforts before oxidative enzymes become competitive also for these lower value-added compounds. In two contributions by Sieber and co-workers and Bommarius the role of molecular oxygen in enzyme- and process instability is highlighted. Molecular and physico-chemical mechanisms of enzyme inactivation are discussed together with recent strategies to overcome such limitations.Water is often considered as an ideal solvent from a sustainability point-of-view. However, for a broad range of reactants of interest, water is a sub-optimal solvent limiting traditional biocatalysis to low product titres. Sheldon and co-workers discuss this issue and present some promising green alternatives to water as a process solvent.Along these lines, the contribution of Kara and co-workers emphasises the potential of process intensification to render biocatalytic oxidation reactions both environmentally and economically more attractive.We are proud to have assembled contributions from leading researchers in the field of biocatalytic oxidation chemistry. The different aspects highlighted in this special issue give an impressive, though certainly not comprehensive, overview of this dynamic and exciting research field. We hope to enthral students and researchers alike to join us in the quest for greener industrial oxidation processes using enzymes.

Utilisation of fatty acids generally relies on pre-existing functional groups such as the carboxylate group or C=C-double bonds. Addition of new functionalities into the hydrocarbon part opens up new possibilities for fatty acid valorisation. In this contribution we demonstrate the synthetic potential of a peroxygenase mutant AaeUPO−Fett for selective fatty acid oxyfunctionalisation. The ω-1 hydroxy fatty acid (esters) produced are further transformed into lactones, alcohols, esters and amines via multi-enzyme cascades thereby paving the way for new fatty acid valorisation pathways.

The pulp and paper manufacturers generate approximately 50 million metric tons of lignin per annum, most of which has been abandoned or incinerated because of lignin's recalcitrant nature. Here, we report bias-free photoelectrochemical (PEC) oxidation of lignin coupled with asymmetric hydrogenation of C=C bonds. The PEC platform consists of a hematite (α-Fe2O3) photoanode and a silicon photovoltaic-wired mesoporous indium tin oxide (Si/mesoITO) photocathode. We substantiate a new function of photoelectroactivated α-Fe2O3 to extract electrons from lignin. The extracted electrons are transferred to the Si/mesoITO photocathode for regenerating synthetic nicotinamide cofactor analogues (mNADHs). We demonstrate that the reduction kinetics of mNAD+s depend on their reduction peak potentials. The regenerated mNADHs activate ene-reductases from the old yellow enzyme (OYE) family, which catalyze enantioselective reduction of α,β-unsaturated hydrocarbons. This lignin-fueled biocatalytic PEC system exhibits an excellent OYE's turnover frequency and total turnover number for photobiocatalytic trans-hydrogenation through cofactor regeneration. This work presents the first example of PEC regeneration of mNADHs and opens up a sustainable route for bias-free chemical synthesis using renewable lignin waste as an electron feedstock.

Biocatalysis has an enormous impact on chemical synthesis. The waves in which biocatalysis has developed, and in doing so changed our perception of what organic chemistry is, were reviewed 20 and 10 years ago. Here we review the consequences of these waves of development. Nowadays, hydrolases are widely used on an industrial scale for the benign synthesis of commodity and bulk chemicals and are fully developed. In addition, further enzyme classes are gaining ever increasing interest. Particularly, enzymes catalysing selective C-C-bond formation reactions and enzymes catalysing selective oxidation and reduction reactions are solving long-standing synthetic challenges in organic chemistry. Combined efforts from molecular biology, systems biology, organic chemistry and chemical engineering will establish a whole new toolbox for chemistry. Recent developments are critically reviewed.

Light-driven catalytic cycles by oxidative enzymes, such as peroxygenases, dehydrogenases or monooxygenases, perform a variety of selective oxyfunctionalization and dehydrogenation reactions with or withdrawal of reducing equivalents.

Since its discovery in 2017, the fatty acid decarboxylase (FAP) photoenzyme has been the focus of extensive research, given its ability to convert fatty acids into alka(e)nes using merely visible blue light. Unfortunately, there are still some drawbacks that limit the applicability of this biocatalyst, such as poor solubility of the substrates in aqueous media, poor photostability, and the impossibility of reusing the catalyst for several cycles. In this work, we demonstrate the use of FAP in non-conventional media as a free enzyme and an immobilized preparation. Namely, its applicability in deep eutectic solvents (DESs) and a proof-of-concept immobilization using a commercial His-tag selective carrier, a thorough study of reaction and immobilization conditions in each case, as well as reusability studies are shown. We observed an almost complete selectivity of the enzyme towards C18 decarboxylation over C16 when used in a DES, with a product analytical yield up to 81 % when using whole cells. Furthermore, when applying the immobilized enzyme in DES, we obtained yields >10-fold higher than the ones obtained in aqueous media.

Abstract Oleate hydratase catalyses the addition of water to the CC double bond of oleic acid to produce (R)-10-hydroxystearic acid. The enzyme requires an FAD cofactor that functions to optimise the active site structure. A wide range of unsaturated fatty acids can be hydrated at the C10 and in some cases the C13 position. The substrate scope can be expanded using ‘decoy’ small carboxylic acids to convert small chain alkenes to secondary alcohols, albeit at low conversion rates. Systematic protein engineering and directed evolution to widen the substrate scope and increase the conversion rate is possible, supported by new high throughput screening assays that have been developed. Multi-enzyme cascades allow the formation of a wide range of products including keto-fatty acids, secondary alcohols, secondary amines and α,ω-dicarboxylic acids. Key points • Phylogenetically distinct oleate hydratases may exhibit mechanistic differences. • Protein engineering to improve productivity and substrate scope is possible. • Multi-enzymatic cascades greatly widen the product portfolio.

[EN] In this study, we developed a new bienzymatic reaction to produce enantioenriched phenylethanols. In a first step, the recombinant, unspecific peroxygenase from Agrocybe aegerita (rAaeUPO) was used to oxidise ethylbenzene and its derivatives to the corresponding ketones (prochiral intermediates) followed by enantioselective reduction into the desired (R)- or (S)-phenylethanols using the (R)-selective alcohol dehydrogenase (ADH) from Lactobacillus kefir (LkADH) or the (S)-selective ADH from Rhodococcus ruber (ADH-A). In a one-pot two-step cascade, 11 ethylbenzene derivatives were converted into the corresponding chiral alcohols at acceptable yields and often excellent enantioselectivity., European Research Commission (ERC consolidator grant,No. 648026),the NetherlandsOrganizationfor ScientificResearch(VICIgrant,No. 724.014.003)and the GuangzhouElite Project.

Enantioenriched azido alcohols are precursors for valuable chiral aziridines and 1,2-amino alcohols, however their chiral substituted analogues are difficult to access. We established a cascade for the asymmetric azidohydroxylation of styrene derivatives leading to chiral substituted 1,2-azido alcohols via enzymatic asymmetric epoxidation, followed by regioselective azidolysis, affording the azido alcohols with up to two contiguous stereogenic centers. A newly isolated two-component flavoprotein styrene monooxygenase StyA proved to be highly selective for epoxidation with a nicotinamide coenzyme biomimetic as a practical reductant. Coupled with azide as a nucleophile for regioselective ring opening, this chemo-enzymatic cascade produced highly enantioenriched aromatic α-azido alcohols with up to >99% conversion. A bi-enzymatic counterpart with halohydrin dehalogenase-catalyzed azidolysis afforded the alternative β-azido alcohol isomers with up to 94% diastereomeric excess. We anticipate our biocatalytic cascade to be a starting point for more practical production of these chiral compounds with two-component flavoprotein monooxygenases., A one-pot enzymatic cascade for the asymmetric azidohydroxylation of styrenes leads to chiral 1,2-azido alcohols with up to two stereocenters.

Biocatalytic artificial photosynthesis integrates photocatalysis and redox biocatalysis through inspiration from plants to synthesize value-added chemicals using solar energy. The nature-inspired approach, however, suffers from sluggish rates due to the challenging water oxidation kinetics. Here, we report photoelectrochemical (PEC) biosynthetic reactions that use nonrecyclable real-world PET microplastics as an electron feedstock. A Zr-doped hematite photoanode (i) extracts electrons from hydrolyzed PET solutions obtained from post-consumer PET wastes (e.g., drinking bottles) and (ii) transfers the electrons to the bioelectrocatalytic site. Carbon-based cathodes receive the electrons to (i) activate redox enzymes [e.g., unspecific peroxygenase (UPO), L-glutamate dehydrogenase (GDH), ene-reductase from the old yellow enzyme family (OYE)] and (ii) drive various organic synthetic reactions, such as oxyfunctionalization of C-H bonds, amination of C=O bonds, and asymmetric hydrogenation of C=C bonds. These photoelectrocatalyst/biocatalyst hybrids achieve benchmark total turnover numbers of 362,000 (UPO), 144,000 (GDH), and 1,300 (OYE). This work presents a photoelectrocatalytic approach for integrating environmental remediation and biocatalytic photosynthesis towards sustainable solar-to-chemical synthesis.

Propargylic alcohols and amines are versatile building blocks in organic synthesis. We demonstrate a straightforward enzymatic cascade to synthesize enantiomerically pure propargylic alcohols and amines from readily available racemic starting materials. In the first step, the peroxygenase from Agrocybe aegerita converted the racemic propargylic alcohols into the corresponding ketones, which then were converted into the enantiomerically pure alcohols using the (R)-selective alcohol dehydrogenase from Lactobacillus kefir or the (S)-selective alcohol dehydrogenase from Thermoanaerobacter brokii. Moreover, an enzymatic Mitsunobu-type conversion of the racemic alcohols into enantiomerically enriched propargylic amines using (R)-selective amine transaminase from Aspergillus terreus or (S)-selective amine transaminase from Chromobacterium violaceum was established. The one-pot two-step cascade reaction yielded a broad range of enantioenriched alcohol and amine products in 70-99% yield.

Chemical communications: ChemComm 58, 10540-10543 (2022). doi:10.1039/D2CC02411J, Published by The Royal Society of Chemistry, Cambridge

The fatty acid photodecarboxylase from Chlorella variabilis NC64A (CvFAP) promotes the elimination of CO2 from fatty acids (Cn) producing the corresponding hydrocarbon (Cn-1). Therefore, this enzyme is of great biotechnological interest since it can be used in alternative biofuel production routes matching the concept of green chemistry. However, due to its recent discovery, this reaction still requires optimizations, which was the focus of the present work together with the application of continuous flow system. The results in batch reactors showed the importance of using high power LED lamps (300 W) to reduce the reaction time for full conversion (30 min, >99%). In another approach, a continuous flow system demonstrated high potential, as it enabled full conversion with a half concentration of enzyme extract in a very short residence time of 15 min. Furthermore, the use of less expensive and sustainable light sources, not previously reported for reactions with CvFAP, were evaluated with full conversion (>99%) after 1 h for continuous flow reactions using 300 W common white LED lamp and based preliminary batch reactions investigations using direct sunlight. Thus, important advances and new perspectives for CvFAP photodecarboxylation reactions could be achieved with the present report.

Phthalic acid esters (PAEs) are widely used as plastic additives to increase the flexibility and durability of plastics. Constantly leaching out from plastics, PAEs are ubiquitously found in the environment. As PAEs exhibit biological activities such as being endocrine disruptive, the quest for efficient degradation strategies continues. Here, we report a bienzymatic degradation system for PAEs to phthalic acid (PA) using a cascade comprising two hydrolases, EstJ6 and P8219. The reaction conditions were optimized with respect to concentrations of both enzymes, temperature, and initial pH. Finally, the substrate scope of the new cascade was investigated, revealing that particularly PAEs with relatively small alcohols were degraded to more than 90 %. This present study provides a potential doable biocatalytic strategy for the complete hydrolysis of PAEs.

A photochemoenzymatic halodecarboxylation of ferulic acid was achieved using vanadate-dependent chloroperoxidase as (bio)catalyst and oxygen and organic solvent as sole stoichiometric reagents in a biphasic system. Performance and selectivity were improved through a phase transfer catalyst, reaching a turnover number of 660.000 for the enzyme.

Process intensification aims at enabling bridging the gap between fundamental research such as identification of new catalysts and reactions and their implementation in industrial environments. Especially the field of biocatalysis has seen some tremendous improvements and the development of new tools and approaches to bridge this gap. In this contribution we highlight some recent developments as selected case studies.