carba Nicotinamide Adenine Dinucleotide Phosphate: Robust Cofactor for Redox Biocatalysis.

Here we report a new robust nicotinamide dinucleotide phosphate cofactor analog (carba-NADP+ ) and its acceptance by many enzymes in the class of oxidoreductases. Replacing one ribose oxygen with a methylene group of the natural NADP+ was found to enhance stability dramatically. Decomposition experiments at moderate and high temperatures with the cofactors showed a drastic increase in half-life time at elevated temperatures since it significantly disfavors hydrolysis of the pyridinium-N-glycoside bond. Overall, more than 27 different oxidoreductases were successfully tested, and a thorough analytical characterization and comparison is given. The cofactor carba-NADP+ opens up the field of redox-biocatalysis under harsh conditions.

Enzymatic and Chemoenzymatic Three-Step Cascades for the Synthesis of Stereochemically Complementary Trisubstituted Tetrahydroisoquinolines.

Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4-trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step-efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3-hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet-Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)-metaraminol. Subsequent cyclization catalyzed either enzymatically by a norcoclaurine synthase or chemically by phosphate resulted in opposite stereoselectivities in the products at the C1 position, thus providing access to both orientations of the THIQ C1 substituent. This highlights the importance of selecting from both chemo- and biocatalysts for optimal results.

Biocatalytic trifluoromethylation of unprotected phenols.

Organofluorine compounds have become important building blocks for a broad range of advanced materials, polymers, agrochemicals, and increasingly for pharmaceuticals. Despite tremendous progress within the area of fluorination chemistry, methods for the direct introduction of fluoroalkyl-groups into organic molecules without prefunctionalization are still highly desired. Here we present a concept for the introduction of the trifluoromethyl group into unprotected phenols by employing a biocatalyst (laccase), tBuOOH, and either the Langlois' reagent or Baran's zinc sulfinate. The method relies on the recombination of two radical species, namely, the phenol radical cation generated directly by the laccase and the CF3-radical. Various functional groups such as ketone, ester, aldehyde, ether and nitrile are tolerated. This laccase-catalysed trifluoromethylation proceeds under mild conditions and allows accessing trifluoromethyl-substituted phenols that were not available by classical methods.

Vinylation of Unprotected Phenols Using a Biocatalytic System.

Readily available substituted phenols were coupled with pyruvate in buffer solution under atmospheric conditions to afford the corresponding para-vinylphenol derivatives while releasing only one molecule of CO2 and water as the by-products. This transformation was achieved by designing a biocatalytic system that combines three biocatalytic steps, namely the C-C coupling of phenol and pyruvate in the presence of ammonia, which leads to the corresponding tyrosine derivative, followed by deamination and decarboxylation. The biocatalytic transformation proceeded with high regioselectivity and afforded exclusively the desired para products. This method thus represents an environmentally friendly approach for the direct vinylation of readily available 2-, 3-, or 2,3-disubstituted phenols on preparative scale (0.5 mmol) that provides vinylphenols in high yields (65-83 %).

Asymmetric Biocatalytic Amination of Ketones at the Expense of NH3 and Molecular Hydrogen.

A biocatalytic system is presented for the stereoselective amination of ketones at the expense of NH3 and molecular hydrogen. By using a NAD(+)-reducing hydrogenase, an alanine dehydrogenase, and a suitable ω-transaminase, the R- as well as the S-enantiomer of various amines could be prepared with up to >99% ee and 98% conversion.

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.

Synthesis of (R)- or (S)-valinol using ω-transaminases in aqueous and organic media.

Valinol is part of numerous pharmaceuticals and has various other important applications. Optically pure valinol (ee >99%) was prepared employing different ω-transaminases from the corresponding prochiral hydroxy ketone. By the choice of the enzyme the (R)- as well as the (S)-enantiomer were accessible. Reductive amination was performed in organic solvent (MTBE) using 2-propyl amine as amine donor whereas alanine was applied in or in aqueous medium. Transformations in phosphate buffer were successfully performed even at 200 mM substrate concentration (20.4 g/L) leading to 99% (R) and 94% (S) conversion with perfect optical purity (>99% ee).

Synthesis of pharmaceutically relevant 17-α-amino steroids using an ω-transaminase.

An efficient and sustainable biocatalytic route for the synthesis of important 17-α-amino steroids has been developed using an ω-transaminase variant from Arthrobacter sp. Optimisation of the reaction conditions facilitated the synthesis of these valuable synthons on a preparative scale, affording excellent isolated yields and stereocontrol.

Biocatalytic synthesis of enantiopure building blocks for pharmaceuticals.

Biocatalytic transformations have emerged as a viable alternative to other asymmetric chemical methods due to the intrinsic high stereoselectivity of the enzymes and the mild reaction conditions. Just a decade ago, the reaction scope of applicable biotransformations for organic synthesis was limited to a handful of reaction types. Tremendous progress has been made in the meantime so that this review presents only a small selection of the broad range of possible biotransfromations for organic synthesis available today. Lyases (hydroxynitrile lyase, aldolases) and redox enzymes like alcohol dehydrogenases, Baeyer­Villiger monooxygenase, dioxygenases, ene reductases, berberine bridge enzyme and v-transaminases are discussed besides hydrolases.

Asymmetric Preparation of prim-, sec-, and tert-Amines Employing Selected Biocatalysts.

This account focuses on the application of ω-transaminases, lyases, and oxidases for the preparation of amines considering mainly work from our own lab. Examples are given to access α-chiral primary amines from the corresponding ketones as well as terminal amines from primary alcohols via a two-step biocascade. 2,6-Disubstituted piperidines, as examples for secondary amines, are prepared by biocatalytical regioselective asymmetric monoamination of designated diketones followed by spontaneous ring closure and a subsequent diastereoselective reduction step. Optically pure tert-amines such as berbines and N-methyl benzylisoquinolines are obtained by kinetic resolution via an enantioselective aerobic oxidative C-C bond formation.

Chemoenzymatic synthesis of all four diastereomers of 2,6-disubstituted piperidines through stereoselective monoamination of 1,5-diketones.

The regioselectivity of various enantiocomplementary ω-transaminases was evaluated for the stereoselective monoamination of designated 1,5-diketones; excellent conversions, enantio- and regioselectivities were observed. The resulting amino-ketones underwent spontaneous intramolecular ring closure to afford Δ1-piperideines, which served as precursors for the cis- and anti-piperidine scaffold as demonstrated for the synthesis of the alkaloids dihydropinidine and epi-dihydropinidine. Key to the success of accessing the trans-piperidines was a Lewis acid mediated conformational change of the Δ1-piperideines in the reduction step. Thus, all four diastereomers of 2,6-disubstituted piperidines could successfully be prepared.

Concise Chemoenzymatic Three Step Total Synthesis of Isosolenopsin Through Medium Engineering.

A short and efficient total synthesis of the alkaloid isosolenopsin and its enantiomer has been achieved. In the key step, a ω-transaminase catalyzed the regioselective mono-amination of the diketone pentadecane-2,6-dione which was obtained in a single step via Grignard reaction. Initial low conversions in the biotransformation could be overcome by optimisation of the reaction conditions employing suitable cosolvents. In the presence of 20 vol% DMF or n-heptane best results were obtained employing two enantio-complementary ω-transaminases originating from Arthrobacter between 30-40 °C; under these conditions conversions of >99% and perfect stereocontrol (ee > 99%) were achieved. Diastereostelective chemical reduction (H2/Pd/C) of the biocatalytic product gave the target compound. The linear three step synthesis provided the natural product isosolenopsin in diastereomerically pure form (ee > 99%, d.r. = 99:1) with an overall yield of 64%.

TTC-based screening assay for ω-transaminases: a rapid method to detect reduction of 2-hydroxy ketones.

A rapid TTC-based screening assay for ω-transaminases was developed to determine the conversion of substrates with a 2-hydroxy ketone motif. Oxidation of the compounds in the presence of 2,3,5-triphenyltetrazolium chloride (TTC) results in a reduction of the colourless TTC to a red-coloured 1,3,5-triphenylformazan. The enzymatic reductive amination of a wide range of various aliphatic, aliphatic-aromatic and aromatic-aromatic 2-hydroxy ketones can be determined by the decrease of the red colouration due to substrate consumption. The conversion can be quantified spectrophotometrically at 510 nm based on reactions, e.g. with crude cell extracts in 96-well plates. Since the assay is independent of the choice of diverse amine donors a panel of ω-transaminases was screened to detect conversion of 2-hydroxy ketones with three different amine donors: l-alanine, (S)-α-methylbenzylamine and benzylamine. The results could be validated using HPLC and GC analyses, showing a deviation of only 5-10%. Using this approach enzymes were identified demonstrating high conversions of acetoin and phenylacetylcarbinol to the corresponding amines. Among these enzymes three novel wild-type ω-transaminases have been identified.