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.

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.

Power of Biocatalysis for Organic Synthesis.

Biocatalysis, using defined enzymes for organic transformations, has become a common tool in organic synthesis, which is also frequently applied in industry. The generally high activity and outstanding stereo-, regio-, and chemoselectivity observed in many biotransformations are the result of a precise control of the reaction in the active site of the biocatalyst. This control is achieved by exact positioning of the reagents relative to each other in a fine-tuned 3D environment, by specific activating interactions between reagents and the protein, and by subtle movements of the catalyst. Enzyme engineering enables one to adapt the catalyst to the desired reaction and process. A well-filled biocatalytic toolbox is ready to be used for various reactions. Providing nonnatural reagents and conditions and evolving biocatalysts enables one to play with the myriad of options for creating novel transformations and thereby opening new, short pathways to desired target molecules. Combining several biocatalysts in one pot to perform several reactions concurrently increases the efficiency of biocatalysis even further.

The role of biocatalysis in the asymmetric synthesis of alkaloids - an update.

Alkaloids are a group of natural products with interesting pharmacological properties and a long history of medicinal application. Their complex molecular structures have fascinated chemists for decades, and their total synthesis still poses a considerable challenge. In a previous review, we have illustrated how biocatalysis can make valuable contributions to the asymmetric synthesis of alkaloids. The chemo-enzymatic strategies discussed therein have been further explored and improved in recent years, and advances in amine biocatalysis have vastly expanded the opportunities for incorporating enzymes into synthetic routes towards these important natural products. The present review summarises modern developments in chemo-enzymatic alkaloid synthesis since 2013, in which the biocatalytic transformations continue to take an increasingly 'central' role.

Asymmetric Biocatalytic Synthesis of 1-Aryltetrahydro-ß-carbolines Enabled by "Substrate Walking".

Stereoselective catalysts for the Pictet-Spengler reaction of tryptamines and aldehydes may allow a simple and fast approach to chiral 1-substituted tetrahydro-ß-carbolines. Although biocatalysts have previously been employed for the Pictet-Spengler reaction, not a single one accepts benzaldehyde and its substituted derivatives. To address this challenge, a combination of substrate walking and transfer of beneficial mutations between different wild-type backbones was used to develop a strictosidine synthase from Rauvolfia serpentina (RsSTR) into a suitable enzyme for the asymmetric Pictet-Spengler condensation of tryptamine and benzaldehyde derivatives. The double variant RsSTR V176L/V208A accepted various ortho-, meta- and para-substituted benzaldehydes and produced the corresponding chiral 1-aryl-tetrahydro-ß-carbolines with up to 99 % enantiomeric excess.

Sequence-Based In-silico Discovery, Characterisation, and Biocatalytic Application of a Set of Imine Reductases.

Imine reductases (IREDs) have recently become a primary focus of research in biocatalysis, complementing other classes of amine-forming enzymes such as transaminases and amine dehydrogenases. Following in the footsteps of other research groups, we have established a set of IRED biocatalysts by sequence-based in silico enzyme discovery. In this study, we present basic characterisation data for these novel IREDs and explore their activity and stereoselectivity using a panel of structurally diverse cyclic imines as substrates. Specific activities of >1 U/mg and excellent stereoselectivities (ee>99 %) were observed in many cases, and the enzymes proved surprisingly tolerant towards elevated substrate loadings. Co-expression of the IREDs with an alcohol dehydrogenase for cofactor regeneration led to whole-cell biocatalysts capable of efficiently reducing imines at 100 mM initial concentration with no need for the addition of extracellular nicotinamide cofactor. Preparative biotransformations on gram scale using these 'designer cells' afforded chiral amines in good yield and excellent optical purity.

Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules.

The review compiles artificial cascades involving enzymes with a focus on the last 10 years. A cascade is defined as the combination of at least two reaction steps in a single reaction vessel without isolation of the intermediates, whereby at least one step is catalyzed by an enzyme. Additionally, cascades performed in vivo and in vitro are discussed separately, whereby in vivo cascades are defined here as cascades relying on cofactor recycling by the metabolism or on a metabolite from the living organism. The review introduces a systematic classification of the cascades according to the number of enzymes in the linear sequence and differentiates between cascades involving exclusively enzymes and combinations of enzymes with non-natural catalysts or chemical steps. Since the number of examples involving two enzymes is predominant, the two enzyme cascades are further subdivided according to the number, order, and type of redox steps. Furthermore, this classification differentiates between cascades where all reaction steps are performed simultaneously, sequentially, or in flow.

Enantioselective Oxidative Aerobic Dealkylation of N-Ethyl Benzylisoquinolines by Employing the Berberine Bridge Enzyme.

N-Dealkylation methods are well described for organic chemistry and the reaction is known in nature and drug metabolism; however, to our knowledge, enantioselective N-dealkylation has not been yet reported. In this study, exclusively the (S)-enantiomers of racemic N-ethyl tertiary amines (1-benzyl-N-ethyl-1,2,3,4-tetrahydroisoquinolines) were dealkylated to give the corresponding secondary (S)-amines in an enantioselective fashion at the expense of molecular oxygen. The reaction is catalyzed by the berberine bridge enzyme, which is known for CC bond formation. The dealkylation was demonstrated on a 100 mg scale and gave optically pure dealkylated products (ee>99 %).

Stereoselective synthesis of γ-hydroxynorvaline through combination of organo- and biocatalysis.

An efficient route for the synthesis of all four diastereomers of PMP-protected α-amino-γ-butyrolacton to access γ-hydroxynorvaline was established. The asymmetric key steps comprise an organocatalytic Mannich reaction and an enzymatic ketone reduction. Three reaction steps could be integrated in a one-pot process, using 2-PrOH both as solvent and as reducing agent. The sequential construction of stereogenic centres gave access to each of the four stereoisomers in high yield and with excellent stereocontrol.

Deracemization by simultaneous bio-oxidative kinetic resolution and stereoinversion.

Deracemization, that is, the transformation of a racemate into a single product enantiomer with theoretically 100% conversion and 100% ee, is an appealing but also challenging option for asymmetric synthesis. Herein a novel chemo-enzymatic deracemization concept by a cascade is described: the pathway involves two enantioselective oxidation steps and one non-stereoselective reduction step, enabling stereoinversion and a simultaneous kinetic resolution. The concept was exemplified for the transformation of rac-benzylisoquinolines to optically pure (S)-berbines. The racemic substrates were transformed to optically pure products (ee>97%) with up to 98% conversion and up to 88% yield of isolated product.

Recent trends and novel concepts in cofactor-dependent biotransformations.

Cofactor-dependent enzymes catalyze a broad range of synthetically useful transformations. However, the cofactor requirement also poses economic and practical challenges for the application of these biocatalysts. For three decades, considerable research effort has been devoted to the development of reliable in situ regeneration methods for the most commonly employed cofactors, particularly NADH and NADPH. Today, researchers can choose from a plethora of options, and oxidoreductases are routinely employed even on industrial scale. Nevertheless, more efficient cofactor regeneration methods are still being developed, with the aim of achieving better atom economy, simpler reaction setups, and higher productivities. Besides, cofactor dependence has been recognized as an opportunity to confer novel reactivity upon enzymes by engineering their cofactors, and to couple (redox) biotransformations in multi-enzyme cascade systems. These novel concepts will help to further establish cofactor-dependent biotransformations as an attractive option for the synthesis of biologically active compounds, chiral building blocks, and bio-based platform molecules.

The role of biocatalysis in the asymmetric synthesis of alkaloids.

Alkaloids are not only one of the most intensively studied classes of natural products, their wide spectrum of pharmacological activities also makes them indispensable drug ingredients in both traditional and modern medicine. Among the methods for their production, biotechnological approaches are gaining importance, and biocatalysis has emerged as an essential tool in this context. A number of chemo-enzymatic strategies for alkaloid synthesis have been developed over the years, in which the biotransformations nowadays take an increasingly 'central' role. This review summarises different applications of biocatalysis in the asymmetric synthesis of alkaloids and discusses how recent developments and novel enzymes render innovative and efficient chemo-enzymatic production routes possible.

Controlling stereoselectivity by enzymatic and chemical means to access enantiomerically pure (1S,3R)-1-benzyl-2,3-dimethyl-1,2,3,4-tetrahydroisoquinoline derivatives.

A chemoenzymatic strategy for the synthesis of enantiomerically pure novel alkaloids (1S,3R)-1-benzyl-2,3-dimethyl-1,2,3,4-tetrahydroisoquinolines is presented. The key steps are the biocatalytic stereoselective reductive amination of substituted 1-phenylpropan-2-one derivatives to yield chiral amines employing microbial ω-transaminases, and the diastereoselective reduction of a Bischler-Napieralski imine intermediate by catalytic hydrogenation in the presence of palladium on charcoal, leading exclusively to the desired cis-isomer.

Inverting the regioselectivity of the berberine bridge enzyme by employing customized fluorine-containing substrates.

Fluorine is commonly applied in pharmaceuticals to block the degradation of bioactive compounds at a specific site of the molecule. Blocking of the reaction center of the enzyme-catalyzed ring closure of 1,2,3,4-tetrahydrobenzylisoquinolines by a fluoro moiety allowed redirecting the berberine bridge enzyme (BBE)-catalyzed transformation of these compounds to give the formation of an alternative regioisomeric product namely 11-hydroxy-functionalized tetrahydroprotoberberines instead of the commonly formed 9-hydroxy-functionalized products. Alternative strategies to change the regioselectivity of the enzyme, such as protein engineering, were not applicable in this special case due to missing substrate-enzyme interactions. Medium engineering, as another possible strategy, had clear influence on the regioselectivity of the reaction pathway, but did not lead to perfect selectivity. Thus, only substrate tuning by introducing a fluoro moiety at one potential reactive carbon center switched the reaction to the formation of exclusively one regioisomer with perfect enantioselectivity.

Biocatalytic organic synthesis of optically pure (S)-scoulerine and berbine and benzylisoquinoline alkaloids.

A chemoenzymatic approach for the asymmetric total synthesis of the title compounds is described that employs an enantioselective oxidative C-C bond formation catalyzed by berberine bridge enzyme (BBE) in the asymmetric key step. This unique reaction yielded enantiomerically pure (R)-benzylisoquinoline derivatives and (S)-berbines such as the natural product (S)-scoulerine, a sedative and muscle relaxing agent. The racemic substrates rac-1 required for the biotransformation were prepared in 4-8 linear steps using either a Bischler-Napieralski cyclization or a C1-Cα alkylation approach. The chemoenzymatic synthesis was applied to the preparation of fourteen enantiomerically pure alkaloids, including the natural products (S)-scoulerine and (R)-reticuline, and gave overall yields of up to 20% over 5-9 linear steps.

Novel carbon-carbon bond formations for biocatalysis.

Carbon-carbon bond formation is the key transformation in organic synthesis to set up the carbon backbone of organic molecules. However, only a limited number of enzymatic C-C bond forming reactions have been applied in biocatalytic organic synthesis. Recently, further name reactions have been accomplished for the first time employing enzymes on a preparative scale, for instance the Stetter and Pictet-Spengler reaction or oxidative C-C bond formation. Furthermore, novel enzymatic C-C bond forming reactions have been identified like benzylation of aromatics, intermolecular Diels-Alder or reductive coupling of carbon monoxide.

Recent biocatalytic oxidation-reduction cascades.

The combination of an oxidation and a reduction in a cascade allows performing transformations in a very economic and efficient fashion. The challenge is how to combine an oxidation with a reduction in one pot, either by running the two reactions simultaneously or in a stepwise fashion without isolation of intermediates. The broader availability of various redox enzymes nowadays has triggered the recent investigation of various oxidation-reduction cascades.

Simultaneous iridium catalysed oxidation and enzymatic reduction employing orthogonal reagents.

An iridium catalysed oxidation was coupled concurrently to an asymmetric biocatalytic reduction in one-pot; thus it was shown for the first time that iridium- and alcohol dehydrogenase-catalysed redox reactions are compatible. As a model system racemic chlorohydrins were transformed to enantioenriched chlorohydrins via an oxidation-asymmetric reduction sequence.