Where Chemocatalysis Meets Biocatalysis: In Water.

Chemoenzymatic catalysis, by definition, involves the merging of sequential reactions using both chemocatalysis and biocatalysis, typically in a single reaction vessel. A major challenge, the solution to which, however, is associated with numerous advantages, is to run such one-pot processes in water: the majority of enzyme-catalyzed processes take place in water as Nature's reaction medium, thus enabling a broad synthetic diversity when using water due to the option to use virtually all types of enzymes. Furthermore, water is cheap, abundantly available, and environmentally friendly, thus making it, in principle, an ideal reaction medium. On the other hand, most chemocatalysis is routinely performed today in organic solvents (which might deactivate enzymes), thus appearing to make it difficult to combine such reactions with biocatalysis toward one-pot cascades in water. Several creative approaches and solutions that enable such combinations of chemo- and biocatalysis in water to be realized and applied to synthetic problems are presented herein, reflecting the state-of-the-art in this blossoming field. Coverage has been sectioned into three parts, after introductory remarks: (1) Chapter 2 focuses on historical developments that initiated this area of research; (2) Chapter 3 describes key developments post-initial discoveries that have advanced this field; and (3) Chapter 4 highlights the latest achievements that provide attractive solutions to the main question of compatibility between biocatalysis (used predominantly in aqueous media) and chemocatalysis (that remains predominantly performed in organic solvents), both Chapters covering mainly literature from ca. 2018 to the present. Chapters 5 and 6 provide a brief overview as to where the field stands, the challenges that lie ahead, and ultimately, the prognosis looking toward the future of chemoenzymatic catalysis in organic synthesis.

Increased Readiness for Water Splitting: NiO-Induced Weakening of Bonds in Water Molecules as Possible Cause of Ultra-Low Oxygen Evolution Potential.

The development of non-precious metal-based electrodes that actively and stably support the oxygen evolution reaction (OER) in water electrolysis systems remains a challenge, especially at low pH levels. The recently published study has conclusively shown that the addition of haematite to H2 SO4 is a highly effective method of significantly reducing oxygen evolution overpotential and extending anode life. The far superior result is achieved by concentrating oxygen evolution centres on the oxide particles rather than on the electrode. However, unsatisfactory Faradaic efficiencies of the OER and hydrogen evolution reaction (HER) parts as well as the required high haematite load impede applicability and upscaling of this process. Here it is shown that the same performance is achieved with three times less metal oxide powder if NiO/H2 SO4 suspensions are used along with stainless steel anodes. The reason for the enormous improvement in OER performance by adding NiO to the electrolyte is the weakening of the intramolecular O─H bond in the water molecules, which is under the direct influence of the nickel oxide suspended in the electrolyte. The manipulation of bonds in water molecules to increase the tendency of the water to split is a ground-breaking development, as shown in this first example.

Protein engineering of lipase A from Candida antarctica to improve esterification of tertiary alcohols.

Chiral tertiary alcohols are important organic compounds in science as well as in industry. However, their preparation in enantiomerically pure form is still a challenge due to their complex structure and steric hindrances compared with primary and secondary alcohols, so kinetic resolution could be an attractive approach.  Lipase A from Candida antarctica (CAL-A) has been shown to catalyze the enantioselective esterification of various tertiary alcohols with excellent enantioselectivity but low activity. Here we report a mutagenesis study by rational design to improve CAL-A activity against tertiary alcohols. Single mutants of CAL-A were selected, expressed, immobilized and screened for esterification of the tertiary alcohol 1,2,3,4-tetrahydronaphthalene-1-ol. A double mutant V278S+S429G showed a 1.5-fold higher reaction rate than that of the wild type CAL-A, while maintaining excellent enantioselectivity.

Crystallization Assisted Dynamic Kinetic Resolution for the Synthesis of (R)-ß-Methylphenethylamine.

This study explores a combination of the concept of enantioselective enzymatic synthesis of ß-chiral amines through transamination with in situ product crystallization (ISPC) to overcome product inhibition. Using 2-phenylpropanal as a readily available and easily racemizing substrate of choice, (R)-ß-methylphenethylamine ((R)-2-phenylpropan-1-amine) concentrations of up to 250 mM and enantiomeric excesses of up to 99 % are achieved when using a commercially available transaminase from Ruegeria pomeroyi in a fed-batch based dynamic kinetic resolution reaction on preparative scale. The source of substrate decomposition during the reaction is also investigated and the resulting unwanted byproduct formation is successfully reduced to insignificant levels.

Strategies to Design Chemocatalytic Racemization of Tertiary Alcohols: State of the Art & Utilization for Dynamic Kinetic Resolution.

The synthesis of enantiomerically pure tertiary alcohols is an important issue in organic synthesis of a range of pharmaceuticals including molecules such as the anti-HIV drug Efavirenz. A conceptually elegant approach to such enantiomers is the dynamic kinetic resolution of racemic tertiary alcohols, which, however, requires efficient racemization strategies. The racemization of tertiary alcohols is particularly challenging due to various side reactions that can occur because of their high tendency for elimination reactions. In the last few years, several complementary catalytic concepts for racemization of tertiary alcohols have been developed, characterized by efficient racemization and suppression of unwanted side-reactions. Besides resins bearing sulfonic acid moieties and a combination of boronic acid and oxalic acid as heterogeneous and homogeneous Brønsted-acids, respectively, immobilized oxovanadium and piperidine turned out to be useful catalysts. The latter two catalysts, which have already been applied to different types of substrates, also have proven good compatibility with lipase, thus leading to the first two examples of chemoenzymatic dynamic kinetic resolution of tertiary alcohols. In this review, the difficulties in racemizing tertiary alcohols are specifically described, and the recently developed complementary concepts to overcome these hurdles are summarized.

Enantioselective Synthesis of Secondary Amines by Combining Oxidative Rearrangement and Biocatalysis in a One-Pot Process.

This contribution describes the development of chemoenzymatic one-pot processes, which combine an oxidative rearrangement and a biotransformation catalyzed by an imine reductase (IRED), for the synthesis of highly enantiomerically enriched secondary amines, such as an aryl-substituted pyrrolidine and a benzazepine. The benefits of this chemoenzymatic one-pot approach include high overall conversions (up to >99%), high enantiomeric excesses (up to >99% ee), and a straightforward synthetic approach toward secondary amines without the need to isolate the formed intermediate. For the initial chemical reaction, namely, the oxidative rearrangement, PhI(OAc)2 in methanol is used as a non-natural reagent, whereas the enzymatic step requires only stoichiometric amounts of d-glucose along with catalytic amounts of IRED, glucose dehydrogenase (GDH), and the cofactor NADPH. This methodology, demonstrating the compatibility of a "classic" organic synthesis using a non-natural, highly reactive reagent and a subsequent biocatalytic step, can be applied for different amines as substrates, thus making this concept a versatile tool in synthetic organic chemistry in general and for enantioselective synthesis of heterocyclic secondary amines in particular.

Development of an efficient and scalable bioprocess for the plant hormone 12-OPDA: Overcoming the hurdles of nature's biosynthesis.

Besides its native biological function as a plant hormone, cis-(+)-12-oxo-phytodienoic acid (12-OPDA) serves as a metabolite for the cellular formation of (-)-jasmonic acid and has also been shown to have an influence on mammalian cells. In order to make this biologically active, but at the same time very expensive natural product 12-OPDA broadly accessible for further biological and medicinal research, we developed an efficient bioprocess based on the utilization of a tailor-made whole-cell catalyst by following the principles of its biosynthesis in nature. After process optimization, the three-step one-pot synthesis of 12-OPDA starting from readily accessible α-linolenic acid could be conducted at appropriate technically relevant substrate loadings in the range of 5-20 g L-1. The desired 12-OPDA was obtained with an excellent conversion efficiency, and by means of the developed, efficient downstream-processing, this emulsifying as well as stereochemically labile biosynthetic metabolite 12-OPDA was then obtained with very high chemical purity (>99%) and enantio- and diastereomeric excess (>99% ee, 96% de) as well as negligible side-product formation (<1%). With respect to future technical applications, we also demonstrated the scalability of the production of the whole cell-biocatalyst in a high cell-density fermentation process.

Design of 3D-Printed Heterogeneous Reactor Systems To Overcome Incompatibility Hurdles when Combining Metal and Enzyme Catalysis in a One-Pot Process.

Combining chemo- and biocatalysis enables the design of novel economic and sustainable one-pot processes for the preparation of industrial chemicals, preferably proceeding in water. While a range of proofs-of-concept for the compatibility of such catalysts from these two different "worlds of catalysis" have recently been demonstrated, merging noncompatible chemo- and biocatalysts for joint applications within one reactor remained a challenge. A conceptual solution is compartmentalization of the catalytic moieties by heterogenization of critical catalyst components, thus "shielding" them from the complementary noncompatible catalyst, substrate or reagent. Exemplified for a one-pot process consisting of a metal-catalyzed Wacker oxidation and enzymatic reduction as noncompatible individual reactions steps, we demonstrate that making use of 3D printing of heterogeneous materials containing Cu as a critical metal component can overcome such incompatibility hurdles. The application of a 3D-printed Cu-ceramic device as metal catalyst component allows an efficient combination with the enzyme and the desired two-step transformation of styrene into the chiral alcohol product with high overall conversion and excellent enantioselectivity. This compartmentalization concept based on 3D printing of heterogenized metal catalysts represents a scalable methodology and opens up numerous perspectives to be used as a general tool also for other related chemoenzymatic research challenges.

Merging High-Pressure Syngas Metal Catalysis and Biocatalysis in Tandem One-Pot Processes for the Synthesis of Nonchiral and Chiral Alcohols from Alkenes in Water.

While simultaneously proceeding reactions are among the most fascinating features of biosynthesis, this concept of tandem processes also offers high potential in the chemical industry in terms of less waste production and improved process efficiency and sustainability. Although examples of one-pot chemoenzymatic syntheses exist, the combination of completely different reaction types is rare. Herein, we demonstrate that extreme "antipodes" of the "worlds of catalysis", such as syngas-based high-pressure hydroformylation and biocatalyzed reduction, can be combined within a tandem-type one-pot process in water. No significant deactivation was found for either the biocatalyst or the chemocatalyst. A proof-of-concept for the one-pot process starting from 1-octene was established with >99 % conversion and 80 % isolated yield of the desired alcohol isomers. All necessary components for hydroformylation and biocatalysis were added to the reactor from the beginning. This concept has been extended to the enantioselective synthesis of chiral products by conducting the hydroformylation of styrene and an enzymatic dynamic kinetic resolution in a tandem mode, leading to an excellent conversion of >99 % and an enantiomeric ratio of 91 : 9 for (S)-2-phenylpropanol. The overall process runs in water under mild and energy-saving conditions, without any need for intermediate isolation.

Synthetic Study of Dragmacidin E: Enantioselective Construction of the Seven-Membered Ring-Fused Indole Skeleton with Contiguous Stereocenters.

We developed an enantioselective synthetic method of constructing a seven-membered ring-fused indole skeleton with contiguous stereocenters for the synthesis of dragmacidin E. Introduction of chirality at the benzylic position was achieved by Ir-catalyzed asymmetric hydrogenation. After construction of the tricyclic molecular framework using Pd-catalyzed cascade cyclization, the tetrasubstituted carbon center was created using the Ag nitrene-mediated C-H amination reaction. The developed method provided access to the functionalized seven-membered ring-fused indole skeleton with a hydroxymethyl branch in the tetrasubstituted carbon.

Reticular Nanoscience: Bottom-Up Assembly Nanotechnology.

The chemistry of metal-organic and covalent organic frameworks (MOFs and COFs) is perhaps the most diverse and inclusive among the chemical sciences, and yet it can be radically expanded by blending it with nanotechnology. The result is reticular nanoscience, an area of reticular chemistry that has an immense potential in virtually any technological field. In this perspective, we explore the extension of such an interdisciplinary reach by surveying the explored and unexplored possibilities that framework nanoparticles can offer. We localize these unique nanosized reticular materials at the juncture between the molecular and the macroscopic worlds, and describe the resulting synthetic and analytical chemistry, which is fundamentally different from conventional frameworks. Such differences are mirrored in the properties that reticular nanoparticles exhibit, which we described while referring to the present state-of-the-art and future promising applications in medicine, catalysis, energy-related applications, and sensors. Finally, the bottom-up approach of reticular nanoscience, inspired by nature, is brought to its full extension by introducing the concept of augmented reticular chemistry. Its approach departs from a single-particle scale to reach higher mesoscopic and even macroscopic dimensions, where framework nanoparticles become building units themselves and the resulting supermaterials approach new levels of sophistication of structures and properties.

Enantiodivergent Chemoenzymatic Dynamic Kinetic Resolution: Conversion of Racemic Propargyl Alcohols into Both Enantiomers.

Natural lipases typically recognize enantiomers of alcohols based on the size differences of substituents near the carbinol moiety and selectively react with the R enantiomers of secondary alcohols. Therefore, lipase-catalyzed dynamic kinetic resolution (DKR) of racemic secondary alcohols produces only R enantiomers. We report herein a method for obtaining S enantiomers by DKR of secondary 3-(trialkylsilyl)propargyl alcohols by using a well-known R-selective Pseudomonas fluorescens lipase in combination with a racemization catalyst VMPS4, in which the silyl group reverses the size relationship of substituents near the carbinol moiety. We have already reported R-selective DKR of the corresponding propargyl alcohols without substituents on the ethynyl terminal carbon, and the presence of an easily removable silyl group has enabled us to produce both enantiomers of propargyl alcohols in high chemical yields and with high enantiomeric excess. In addition, immobilization of the lipase on Celite was found to be important for achieving a high efficiency of the DKR.

Enantioselective Synthesis of Thiomorpholines through Biocatalytic Reduction of 3,6-Dihydro-2H-1,4-thiazines Using Imine Reductases.

In this work, an enantioselective biocatalytic synthesis of chiral thiomorpholines using imine reductases (IREDs) is described. As substrates, four prochiral and one chiral 3,6-dihydro-2H-1,4-thiazines were synthesized in a modified Asinger reaction and subsequently reduced using imine reductases as a biocatalyst, NADPH as a cofactor, and a glucose dehydrogenase (GDH)-glucose cofactor regeneration system. As a result, chiral thiomorpholines with a stereogenic center created in 3-position were obtained under mild process conditions with high conversions and excellent enantioselectivities of up to 99%. Furthermore, as a proof of concept, a sequential one-pot process combining both individual reaction steps was achieved.

Lipase-Catalyzed Kinetic Resolution of C1-Symmetric Heterocyclic Biaryls.

The highly enantioselective lipase-catalyzed kinetic resolution (KR) of racemic C1-symmetric biaryl compounds including heterocyclic moieties, such as carbazole and dibenzofuran, has been achieved for the first time. This enzymatic esterification was accelerated by the addition of disodium carbonate while maintaining its high enantioselectivities, and was particularly effective for biaryls having N-substituted carbazole moieties. Furthermore, mesoporous silica-supported oxovanadium-catalyzed cross-dehydrogenative coupling of 3-hydroxycarbazole and 2-naphthol was followed by the lipase-catalyzed KR in one-pot to synthesize the optically active heterocyclic biaryl compounds with high optical purity.

Expanding the Toolbox of R-Selective Amine Transaminases by Identification and Characterization of New Members.

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d-ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.

Synthetic Processes toward Nitriles without the Use of Cyanide: A Biocatalytic Concept Based on Dehydration of Aldoximes in Water.

While belonging to the most fundamental functional groups, nitriles represent a class of compound that still raises challenges in terms of an efficient, cost-effective, general and, at the same time, sustainable way for their synthesis. Complementing existing chemical routes, recently a cyanide-free enzymatic process technology based on the use of an aldoxime dehydratase (Oxd) as a biocatalyst component has been developed and successfully applied for the synthesis of a range of nitrile products. In these biotransformations, the Oxd enzymes catalyze the dehydration of aldoximes as readily available substrates to the nitrile products. Herein, these developments with such enzymes are summarized, with a strong focus on synthetic applications. It is demonstrated that this biocatalytic technology has the potential to "cross the bridge" between the production of fine chemicals and pharmaceuticals, on one hand, and bulk and commodity chemicals, on the other.

Flow Biocatalysis: A Challenging Alternative for the Synthesis of APIs and Natural Compounds.

Biocatalysts represent an efficient, highly selective and greener alternative to metal catalysts in both industry and academia. In the last two decades, the interest in biocatalytic transformations has increased due to an urgent need for more sustainable industrial processes that comply with the principles of green chemistry. Thanks to the recent advances in biotechnologies, protein engineering and the Nobel prize awarded concept of direct enzymatic evolution, the synthetic enzymatic toolbox has expanded significantly. In particular, the implementation of biocatalysts in continuous flow systems has attracted much attention, especially from industry. The advantages of flow chemistry enable biosynthesis to overcome well-known limitations of "classic" enzymatic catalysis, such as time-consuming work-ups and enzyme inhibition, as well as difficult scale-up and process intensifications. Moreover, continuous flow biocatalysis provides access to practical, economical and more sustainable synthetic pathways, an important aspect for the future of pharmaceutical companies if they want to compete in the market while complying with European Medicines Agency (EMA), Food and Drug Administration (FDA) and green chemistry requirements. This review focuses on the most recent advances in the use of flow biocatalysis for the synthesis of active pharmaceutical ingredients (APIs), pharmaceuticals and natural products, and the advantages and limitations are discussed.

Rationalizing the Unprecedented Stereochemistry of an Enzymatic Nitrile Synthesis through a Combined Computational and Experimental Approach.

In this contribution, the unique and unprecedented stereochemical phenomenon of an aldoxime dehydratase-catalyzed enantioselective dehydration of racemic E- and Z-aldoximes with selective formation of both enantiomeric forms of a chiral nitrile is rationalized by means of molecular modelling, comprising in silico mutations and docking studies. This theoretical investigation gave detailed insight into why with the same enzyme the use of racemic E- and Z-aldoximes leads to opposite forms of the chiral nitrile. The calculated mutants with a larger or smaller cavity in the active site were then prepared and used in biotransformations, showing the theoretically predicted decrease and increase of the enantioselectivities in these nitrile syntheses. This validated model also enabled the rational design of mutants with a smaller cavity, which gave superior enantioselectivities compared to the known wild-type enzyme, with excellent E-values of up to E>200 when the mutant OxdRE-Leu145Phe was utilized.

Segmented Flow Processes to Overcome Hurdles of Whole-Cell Biocatalysis in the Presence of Organic Solvents.

In modern process development, it is imperative to consider biocatalysis, and whole-cell catalysts often represent a favored form of such catalysts. However, the application of whole-cell catalysis in typical organic batch two-phase synthesis often struggles due to mass transfer limitations, emulsion formation, tedious work-up and, thus, low yields. Herein, we demonstrate that utilizing segmented flow tools enables the conduction of whole-cell biocatalysis efficiently in biphasic media. Exemplified for three different biotransformations, the power of such segmented flow processes is shown. For example, a 3-fold increase of conversion from 34 % to >99 % and a dramatic simplified work-up leading to a 1.5-fold higher yield from 44 % to 65 % compared to the analogous batch process was achieved in such a flow process.

Effect of Particle Wettability and Particle Concentration on the Enzymatic Dehydration of n-Octanaloxime in Pickering Emulsions.

Pickering emulsion systems have emerged as platforms for the synthesis of organic molecules in biphasic biocatalysis. Herein, the catalytic performance was evaluated for biotransformation using whole cells exemplified for the dehydration of n-octanaloxime to n-octanenitrile catalysed by an aldoxime dehydratase (OxdB) overexpressed in E. coli. This study was carried out in Pickering emulsions stabilised solely with silica particles of different hydrophobicity. We correlate, for the first time, the properties of the emulsions with the conversion of the reaction, thus gaining an insight into the impact of the particle wettability and particle concentration. When comparing two emulsions of different type with similar stability and droplet diameter, the oil-in-water (o/w) system displayed a higher conversion than the water-in-oil (w/o) system, despite the conversion in both cases being higher than that in a "classic" two-phase system. Furthermore, an increase in particle concentration prior to emulsification resulted in an increase of the interfacial area and hence a higher conversion.