Multistep Biooxidation of 5-(Hydroxymethyl)furfural to 2,5-Furandicarboxylic Acid with H2O2 by Unspecific Peroxygenases.

5-(Hydroxymethyl)furfural (HMF) is a key platform chemical derived from renewable biomass sources, holding great potential as starting material for the synthesis of valuable compounds, thereby replacing petrochemical-derived counterparts. Among these valorised compounds, 2,5-furandicarboxylic acid (FDCA) has emerged as a versatile building block. Here we demonstrate the biocatalytic synthesis of FDCA from HMF via a one-pot three-step oxidative cascade performed via two operative steps under mild reaction conditions employing two unspecific peroxygenases (UPOs) using hydrogen peroxide as the only oxidant. The challenge of HMF oxidation by UPOs is the chemoselectivity of the first step, as one of the two possible oxidation products is only a poor substrate for further oxidation. The unspecific peroxygenase from Marasmius oreades (MorUPO) was found to oxidize 100 mM of HMF to 5-formyl-2-furoic acid (FFCA) with 95 % chemoselectivity. In the sequential one-pot cascade employing MorUPO (TON up to 13535) and the UPO from Agrocybe aegerita (AaeUPO, TON up to 7079), 100 mM of HMF were oxidized to FDCA reaching up to 99 % conversion and yielding 861 mg isolated pure crystalline FDCA, presenting the first example of a gram scale biocatalytic synthesis of FDCA involving UPOs.

A multistep (semi)-continuous biocatalytic setup for the production of polycaprolactone.

Biocatalysis has gained increasing importance as an eco-friendly alternative for the production of bulk and fine chemicals. Within this paradigm, Baeyer Villiger monoxygenases (BVMOs) serve as enzymatic catalysts that provide a safe and sustainable route to the conventional synthesis of lactones, such as caprolactone, which is employed for the production of polycaprolactone (PCL), a biocompatible polymer for medicinal applications. In this work, we present a three-step, semi-continuous production of PCL using an entirely biocatalytic process, highlighting the merits of continuous manufacturing for enhancing biocatalysis. First, caprolactone is produced in batch from cyclohexanol using a coenzymatic cascade involving an alcohol dehydrogenase (ADH) and BVMO. Different process parameters and aeration modes were explored to optimize the cascade's productivity. Secondly, the continuous extraction of caprolactone into an organic solvent, needed for the polymerization step, was optimized. 3D-printed mixers were applied to enhance the mass transfer between the organic and the aqueous phases. Lastly, we investigated the ring-opening polymerization of caprolactone to PCL catalyzed by Candida antarctica lipase B (CAL-B), with a focus on eco-friendly solvents like cyclopentyl-methyl-ether (CPME). Space-time-yields up to 58.5 g L-1 h-1 were achieved with our overall setup. By optimizing the individual process steps, we present an efficient and sustainable pathway for PCL production.

Deciphering the Unconventional Reduction of C=N Bonds by Old Yellow Enzymes Using QM/MM.

The reduction of C=X (X = N, O) bonds is a cornerstone in both synthetic organic chemistry and biocatalysis. Conventional reduction mechanisms usually involve a hydride ion targeting the less electronegative carbon atom. In a departure from this paradigm, our investigation into Old Yellow Enzymes (OYEs) reveals a mechanism involving transfer of hydride to the formally more electronegative nitrogen atom within a C=N bond. Beyond their known ability to reduce electronically activated C=C double bonds, e.g., in α, ß-unsaturated ketones, these enzymes have recently been shown to reduce α-oximo-ß-ketoesters to the corresponding amines. It has been proposed that this transformation involves two successive reduction steps and proceeds via imine intermediates formed by the reductive dehydration of the oxime moieties. We employ advanced quantum mechanics/molecular mechanics (QM/MM) simulations, enriched by a two-tiered approach incorporating QM/MM (UB3LYP-6-31G*/OPLS2005) geometry optimization, QM/MM (B3LYP-6-31G*/amberff19sb) steered molecular dynamics simulations, and detailed natural-bond-orbital analyses to decipher the unconventional hydride transfer to nitrogen in both reduction steps and to delineate the role of active site residues as well as of substituents present in the substrates. Our computational results confirm the proposed mechanism and agree well with experimental mutagenesis and enzyme kinetics data. According to our model, the catalysis of OYE involves hydride transfer from the flavin cofactor to the nitrogen atom in oximoketoesters as well as iminoketoesters followed by protonation at the adjacent oxygen or carbon atoms by conserved tyrosine residues and active site water molecules. Two histidine residues play a key role in the polarization and activation of the C=N bond, and conformational changes of the substrate observed along the reaction coordinate underline the crucial importance of dynamic electron delocalization for efficient catalysis.

Computational Study of the Fries Rearrangement Catalyzed by Acyltransferase from Pseudomonas protegens.

The acyltransferase from Pseudomonas protegens (PpATase) catalyzes in nature the reversible transformation of monoacetylphloroglucinol to diacetylphloroglucinol and phloroglucinol. Interestingly, this enzyme has been shown to catalyze the promiscuous transformation of 3-hydroxyphenyl acetate to 2',4'-dihydroxyacetophenone, representing a biological version of the Fries rearrangement. In the present study, we report a mechanistic investigation of this activity of PpATase using quantum chemical calculations. A detailed mechanism is proposed, and the energy profile for the reaction is presented. The calculations show that the acylation of the enzyme is highly exothermic, while the acetyl transfer back to the substrate is only slightly exothermic. The deprotonation of the C6-H of the substrate is rate-limiting, and a remote aspartate residue (Asp137) is proposed to be the general base group in this step. Analysis of the binding energies of various acetyl acceptors shows that PpATase can promote both intramolecular and intermolecular Fries rearrangement towards diverse compounds.

Peptide and Enzyme Catalysts Work in Concert in Stereoselective Cascade Reactions-Oxidation followed by Conjugate Addition.

Enzymes and peptide catalysts consist of the same building blocks but require vastly different environments to operate best. Herein, we show that an enzyme and a peptide catalyst can work together in a single reaction vessel to catalyze a two-step cascade reaction with high chemo- and stereoselectivity. Abundant linear alcohols, nitroolefins, an alcohol oxidase, and a tripeptide catalyst provided chiral γ-nitroaldehydes in aqueous buffer. High yields (up to 92 %) and stereoselectivities (up to 98 % ee) were achieved for the cascade through the rational design of the peptide catalyst and the identification of common reaction conditions.

Correction to "Strategies for Transferring Photobiocatalysis to Continuous Flow Exemplified by the Photodecarboxylation of Fatty Acids".

[This corrects the article DOI: 10.1021/acscatal.2c04444.].

Biocatalytic characterization of an alcohol dehydrogenase variant deduced from Lactobacillus kefir in asymmetric hydrogen transfer.

Hydrogen transfer biocatalysts to prepare optically pure alcohols are in need, especially when it comes to sterically demanding ketones, whereof the bioreduced products are either essential precursors of pharmaceutically relevant compounds or constitute APIs themselves. In this study, we report on the biocatalytic potential of an anti-Prelog (R)-specific Lactobacillus kefir ADH variant (Lk-ADH-E145F-F147L-Y190C, named Lk-ADH Prince) employed as E. coli/ADH whole-cell biocatalyst and its characterization for stereoselective reduction of prochiral carbonyl substrates. Key enzymatic reaction parameters, including the reaction medium, evaluation of cofactor-dependency, organic co-solvent tolerance, and substrate loading, were determined employing the drug pentoxifylline as a model prochiral ketone. Furthermore, to tap the substrate scope of Lk-ADH Prince in hydrogen transfer reactions, a broad range of 34 carbonylic derivatives was screened. Our data demonstrate that E. coli/Lk-ADH Prince exhibits activity toward a variety of structurally different ketones, furnishing optically active alcohol products at the high conversion of 65-99.9% and in moderate-to-high isolated yields (38-91%) with excellent anti-Prelog (R)-stereoselectivity (up to >99% ee) at substrate concentrations up to 100 mM.

Concise synthesis of (R)-reticuline and (+)-salutaridine by combining early-stage organic synthesis and late-stage biocatalysis.

Efficient access to the morphinan scaffold remains a major challenge in both synthetic chemistry and biotechnology. Here, a biomimetic chemo-enzymatic strategy to synthesize the natural promorphinan intermediate (+)-salutaridine is demonstrated. By combining early-stage organic synthesis with enzymatic asymmetric key step transformations, the prochiral natural intermediate 1,2-dehydroreticuline was prepared and subsequently stereoselectively reduced by the enzyme 1,2-dehydroreticuline reductase obtaining (R)-reticuline in high ee and yield (>99% ee, up to quant. conversion, 92% isol. yield). In the final step, membrane-bound salutaridine synthase was used to perform the selective ortho-para phenol coupling to give (+)-salutaridine. The synthetic route shows the potential of combining early-stage advanced organic chemistry to minimize protecting group techniques with late-stage multi-step biocatalysis to provide an unprecedented access to the medicinally important compound class of promorphinans.

Enantioselective High-Throughput Assay Showcased for the Identification of (R)- as well as (S)-Selective Unspecific Peroxygenases for C-H Oxidation.

Identifying (bio)catalysts displaying high enantio-/stereoselectivity is a fundamental prerequisite for the advancement of asymmetric catalysis. Herein, a high-throughput, stereoselective screening assay is reported that gives information on enantioselectivity, stereopreference and activity as showcased for peroxygenase-catalyzed hydroxylation. The assay is based on spectrophotometric analysis of the simultaneous formation of NAD(P)H from the alcohol dehydrogenase catalyzed enantioselective oxidation of the sec-alcohol product formed in the peroxygenase reaction. The assay was applied to investigate a library comprising 44 unspecific peroxygenases (UPOs) containing 25 UPOs not reported yet. Thereby, previously non-described wild-type UPOs displaying (S)- as well as (R)-stereoselectivity for the hydroxylation of representative model substrates were identified, reaching up to 98 % ee for the (R)- and 94 % ee for the (S)-enantiomer. Homology models with concomitant docking studies indicated the structural reason for the observed complementary stereopreference.

Chemoenzymatic Synthesis of Tenofovir.

We report on novel chemoenzymatic routes toward tenofovir using low-cost starting materials and commercial or homemade enzyme preparations as biocatalysts. The biocatalytic key step was accomplished either via stereoselective reduction using an alcohol dehydrogenase or via kinetic resolution using a lipase. By employing a suspension of immobilized lipase from Burkholderia cepacia (Amano PS-IM) in a mixture of vinyl acetate and toluene, the desired (R)-ester (99% ee) was obtained on a 500 mg scale (60 mM) in 47% yield. Alternatively, stereoselective reduction of 1-(6-chloro-9H-purin-9-yl) propan-2-one (84 mg, 100 mM) catalyzed by lyophilized E. coli cells harboring recombinant alcohol dehydrogenase (ADH) from Lactobacillus kefir (E. coli/Lk-ADH Prince) allowed one to reach quantitative conversion, 86% yield and excellent optical purity (>99% ee) of the corresponding (R)-alcohol. The key (R)-intermediate was transformed into tenofovir through "one-pot" aminolysis-hydrolysis of (R)-acetate in NH3-saturated methanol, alkylation of the resulting (R)-alcohol with tosylated diethyl(hydroxymethyl) phosphonate, and bromotrimethylsilane (TMSBr)-mediated cleavage of the formed phosphonate ester into the free phosphonic acid. The elaborated enzymatic strategy could be applicable in the asymmetric synthesis of tenofovir prodrug derivatives, including 5'-disoproxil fumarate (TDF, Viread) and 5'-alafenamide (TAF, Vemlidy). The molecular basis of the stereoselectivity of the employed ADHs was revealed by molecular docking studies.

Robust Light Driven Enzymatic Oxyfunctionalization via Immobilization of Unspecific Peroxygenase.

Unspecific peroxygenases have attracted interest in synthetic chemistry, especially for the oxidative activation of C-H bonds, as they only require hydrogen peroxide (H2 O2 ) instead of a cofactor. Due to their instability in even small amounts of H2 O2 , different strategies like enzyme immobilization or in situ H2 O2 production have been developed to improve the stability of these enzymes. While most strategies have been studied separately, a combination of photocatalysis with immobilized enzymes was only recently reported. To show the advantages and limiting factors of immobilized enzyme in a photobiocatalytic reaction, a comparison is made between free and immobilized enzymes. Adjustment of critical parameters such as (i) enzyme and substrate concentration, (ii) illumination wavelength and (iii) light intensity results in significantly increased enzyme stabilities of the immobilized variant. Moreover, under optimized conditions a turnover number of 334,500 was reached.

Rapid, Label-Free Screening of Diverse Biotransformations by Flow-Injection Mass Spectrometry.

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1-50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.

Mechanistic Insights into the Ene-Reductase-Catalyzed Promiscuous Reduction of Oximes to Amines.

The biocatalytic reduction of the oxime moiety to the corresponding amine group has only recently been found to be a promiscuous activity of ene-reductases transforming α-oximo ß-keto esters. However, the reaction pathway of this two-step reduction remained elusive. By studying the crystal structures of enzyme oxime complexes, analyzing molecular dynamics simulations, and investigating biocatalytic cascades and possible intermediates, we obtained evidence that the reaction proceeds via an imine intermediate and not via the hydroxylamine intermediate. The imine is reduced further by the ene-reductase to the amine product. Remarkably, a non-canonical tyrosine residue was found to contribute to the catalytic activity of the ene-reductase OPR3, protonating the hydroxyl group of the oxime in the first reduction step.

Anaerobic demethylation of guaiacyl-derived monolignols enabled by a designed artificial cobalamin methyltransferase fusion enzyme.

Lignin-derived aryl methyl ethers (e.g. coniferyl alcohol, ferulic acid) are expected to be a future carbon source for chemistry. The well-known P450 dependent biocatalytic O-demethylation of these aryl methyl ethers is prone to side product formation especially for the oxidation sensitive catechol products which get easily oxidized in the presence of O2. Alternatively, biocatalytic demethylation using cobalamin dependent enzymes may be used under anaerobic conditions, whereby two proteins, namely a methyltransferase and a carrier protein are required. To make this approach applicable for preparative transformations, fusion proteins were designed connecting the cobalamin-dependent methyltransferase (MT) with the corrinoid-binding protein (CP) from Desulfitobacterium hafniense by variable glycine linkers. From the proteins created, the fusion enzyme MT-L5-CP with the shortest linker performed best of all fusion enzymes investigated showing comparable and, in some aspects, even better performance than the separated proteins. The fusion enzymes provided several advantages like that the cobalamin cofactor loading step required originally for the CP could be skipped enabling a significantly simpler protocol. Consequently, the biocatalytic demethylation was performed using Schlenk conditions allowing the O-demethylation e.g. of the monolignol coniferyl alcohol on a 25 mL scale leading to 75% conversion. The fusion enzyme represents a promising starting point to be evolved for alternative demethylation reactions to diversify natural products and to valorize lignin.

Biocatalytic Transamination of Aldolase-Derived 3-Hydroxy Ketones.

Although optical pure amino alcohols are in high demand due to their widespread applicability, they still remain challenging to synthesize, since commonly elaborated protection strategies are required. Here, a multi-enzymatic methodology is presented that circumvents this obstacle furnishing enantioenriched 1,3-amino alcohols out of commodity chemicals. A Type I aldolase forged the carbon backbone with an enantioenriched aldol motif, which was subsequently subjected to enzymatic transamination. A panel of 194 TAs was tested on diverse nine aldol products prepared through different nucleophiles and electrophiles. Due to the availability of (R)- and (S)-selective TAs, both diastereomers of the 1,3-amino alcohol motif were accessible. A two-step process enabled the synthesis of the desired amino alcohols with up to three chiral centers with de up to >97 in the final products.

Strategies for Transferring Photobiocatalysis to Continuous Flow Exemplified by Photodecarboxylation of Fatty Acids.

The challenges of light-dependent biocatalytic transformations of lipophilic substrates in aqueous media are manifold. For instance, photolability of the catalyst as well as insufficient light penetration into the reaction vessel may be further exacerbated by a heterogeneously dispersed substrate. Light penetration may be addressed by performing the reaction in continuous flow, which allows two modes of applying the catalyst: (i) heterogeneously, immobilized on a carrier, which requires light-permeable supports, or (ii) homogeneously, dissolved in the reaction mixture. Taking the light-dependent photodecarboxylation of palmitic acid catalyzed by fatty-acid photodecarboxylase from Chlorella variabilis (CvFAP) as a showcase, strategies for the transfer of a photoenzyme-catalyzed reaction into continuous flow were identified. A range of different supports were evaluated for the immobilization of CvFAP, whereby Eupergit C250 L was the carrier of choice. As the photostability of the catalyst was a limiting factor, a homogeneous system was preferred instead of employing the heterogenized enzyme. This implied that photolabile enzymes may preferably be applied in solution if repair mechanisms cannot be provided. Furthermore, when comparing different wavelengths and light intensities, extinction coefficients may be considered to ensure comparable absorption at each wavelength. Employing homogeneous conditions in the CvFAP-catalyzed photodecarboxylation of palmitic acid afforded a space-time yield unsurpassed by any reported batch process (5.7 g·L-1·h-1, 26.9 mmol·L-1·h-1) for this reaction, demonstrating the advantage of continuous flow in attaining higher productivity of photobiocatalytic processes.

Transmembrane Shuttling of Photosynthetically Produced Electrons to Propel Extracellular Biocatalytic Redox Reactions in a Modular Fashion.

Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time-consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme- or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.

Development of a novel chemoenzymatic route to enantiomerically enriched ß-adrenolytic agents. A case study toward propranolol, alprenolol, pindolol, carazolol, moprolol, and metoprolol.

Efficient chemoenzymatic routes toward the synthesis of both enantiomers of adrenergic ß-blockers were accomplished by identifying a central chiral building block, which was first prepared using lipase-catalyzed kinetic resolution (KR, Amano PS-IM) as the asymmetric step at a five gram-scale (209 mM conc.). The enantiopure (R)-chlorohydrin (>99% ee) subsequently obtained was used for the synthesis of a series of model (R)-(+)-ß-blockers (i.e., propranolol, alprenolol, pindolol, carazolol, moprolol, and metoprolol), which were produced with enantiomeric excess in the range of 96-99.9%. The pharmaceutically relevant (S)-counterpart, taking propranolol as a model, was synthesized in excellent enantiomeric purity (99% ee) via acetolysis of the respective enantiomerically pure (R)-mesylate by using cesium acetate and a catalytic amount of 18-Crown-6, followed by acidic hydrolysis of the formed (S)-acetate. Alternatively, asymmetric reduction of a prochiral ketone, namely 2-(3-chloro-2-oxopropyl)-1H-isoindole-1,3(2H)-dione, was performed using lyophilized E. coli cells harboring overexpressed recombinant alcohol dehydrogenase from Lactobacillus kefir (E. coli/Lk-ADH-Lica) giving the corresponding chlorohydrin with >99% ee. Setting the stereocenter early in the synthesis and performing a 4-step reaction sequence in a 'one-pot two-step' procedure allowed the design of a 'step-economic' route with a potential dramatic improvement in process efficiency. The synthetic method can serve for the preparation of a broad scope of enantiomerically enriched ß-blockers, the chemical structures of which rely on the common α-hydroxy-N-isopropylamine moiety, and in this sense, might be industrially attractive.

Biocatalytic hydrogen-transfer to access enantiomerically pure proxyphylline, xanthinol, and diprophylline.

Alcohol dehydrogenases (ADHs; EC 1.1.1.1) have been widely used for the reversible redox reactions of carbonyl compounds (i.e., aldehydes and ketones) and primary or secondary alcohols, often resulting in optically pure hydroxyl products with high added value. In this work, we report a concise chemoenzymatic route toward xanthine-based enantiomerically pure active pharmaceutical ingredients (API) - proxyphylline, xanthinol, and diprophylline employing various recombinant short-chain ADHs with (R)- or (S)-selectivity as key biocatalysts. By choosing the appropriate ADH, the (R)- as well as the (S)-enantiomer of proxyphylline was prepared in excellent enantiomeric excess (99-99.9% ee), >99% conversion, and the isolated yield ranging from 65% to 74%, depending on the used biocatalyst (ADH-A from Rhodococcus ruber or a variant derived from Lactobacillus kefir, Lk-ADH-Lica). In turn, E. coli/ADH-catalyzed bioreduction of the carbonylic precursor of xanthinol and diprophylline furnished the corresponding (S)-chlorohydrin in >99% ee, >99% conversion, and 80% yield (in the case of Lk-ADH-Lica); while the (R)-counterpart was afforded in 94% ee, 64% conversion, and 41% yield (in the case of SyADH from Sphingobium yanoikuyae). After further chemical functionalization of the key (S)-chlorohydrin intermediate, the desired homochiral (R)-xanthinol (>99% ee) was obtained in 97% yield and (S)-diprophylline (>99% ee) in 90% yield. The devised biocatalytic method is straightforward and thus might be considered practical in the manufacturing of title pharmaceuticals.

Regioselective Biocatalytic C4-Prenylation of Unprotected Tryptophan Derivatives.

Regioselective carbon-carbon bond formation belongs to the challenging tasks in organic synthesis. In this context, C-C bond formation catalyzed by 4-dimethylallyltryptophan synthases (4-DMATSs) represents a possible tool to regioselectively synthesize C4-prenylated indole derivatives without site-specific preactivation and circumventing the need of protection groups as used in chemical synthetic approaches. In this study, a toolbox of 4-DMATSs to produce a set of 4-dimethylallyl tryptophan and indole derivatives was identified. Using three wild-type enzymes as well as variants, various C5-substituted tryptophan derivatives as well as N-methyl tryptophan were successfully prenylated with conversions up to 90 %. Even truncated tryptophan derivatives like tryptamine and 3-indole propanoic acid were regioselectively prenylated in position C4. The acceptance of C5-substituted tryptophan derivatives was improved up to 5-fold by generating variants (e. g. T108S). The feasibility of semi-preparative prenylation of selected tryptophan derivatives was successfully demonstrated on 100 mg scale at 15 mM substrate concentration, allowing to reduce the previously published multistep chemical synthetic sequence to just a single step.