Horst Lechner

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.

Amination of benzylic and cinnamic alcohols via a biocatalytic, aerobic, oxidation–transamination cascade

The amination of benzylic and cinnamic alcohols was achieved via a biocatalytic, one-pot oxidation–transamination cascade in aqueous medium at physiological conditions. Alcohol oxidation by galactose oxidase at the expense of O2 furnished the corresponding aldehydes, which were aminated using ω-transaminases in situ. The applicability of this method was demonstrated by a short synthesis of the antifungal agent naftifine.

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.

Nitrile as Activating Group in the Asymmetric Bioreduction of β-Cyanoacrylic Acids Catalyzed by Ene-Reductases.

Asymmetric bioreduction of an (E)-β-cyano-2,4-dienoic acid derivative by ene-reductases allowed a shortened access to a precursor of pregabalin [(S)-3-(aminomethyl)-5-methylhexanoic acid] possessing the desired configuration in up to 94% conversion and >99% ee. Deuterium labelling studies showed that the nitrile moiety was the preferred activating/anchor group in the active site of the enzyme over the carboxylic acid or the corresponding methyl ester.

A system for ω-transaminase mediated (R)-amination using L-alanine as an amine donor

Chiral amines are important building blocks for fine chemicals and pharmaceuticals. Consequently, various biocatalytic routes in particular using ω-transaminases (ω-TAs) have been developed recently. Although catalysts for the synthesis of both enantiomers are available, the application of alanine dependent (R)-selective ω-TAs is less favourable due to the requirement of the more expensive D-alanine as an amine donor. Here we describe an efficient method for (R)-amination using ω-TAs in combination with an alanine racemase (AlaR). In this case, the readily available L-alanine can be used as an amine donor leading to improved atom efficiency and significantly reduced costs.

Asymmetric Biocatalytic Cannizzaro‐Type Reaction

Typically, disproportionation reactions furnish 1:1 mixtures of products and are often plagued by unfavorable equilibria; thus, they are commonly considered to be inefficient and are rarely used in organic synthesis. Prominent disproportionation reactions are the Kornblum–DeLaMare rearrangement, the Meerwein–Ponndorf–Verley/Oppenauer reduction/oxidation, the Boudouard reaction, and the catalytic disproportionation of toluene. Among the various disproportionation procedures, the Cannizzaro reaction (CR), which is catalyzed by a strong base (e.g. , KOH), is of special interest, because it allows the transformation of an easily accessible—but somewhat unstable—starting material (an aldehyde) into equimolar amounts of an alcohol and a carboxylic acid, both of which are considerably more stable. In view of its preparative applicability, it is notable that the overall DG value of the CR is negative, which provides a strong driving force in favor of the product formation (see the Supporting Information), as shown in Equation (1).

Biocatalysts for the formation of three- to six-membered carbo- and heterocycles.

During the last decade, the number of different types of enzymes applicable for organic synthesis as biocatalysts has significantly increased. Consequently, the spectrum of reactions has significantly expanded also for cyclisations. This review highlights heterologously expressable biocatalysts transforming non-natural substrates for the formation of three- to six-membered carbo- and heterocycles, excluding terpene cyclases as well as SAM-dependent enzymes. The review focuses on the non-natural substrate scope and the mechanism of the selected enzymes.

Library of Norcoclaurine Synthases and Their Immobilization for Biocatalytic Transformations

Norcoclaurine synthases (NCS), catalyzing a Pictet-Spengler reaction in plants as one of the first enzymes in the biosynthetic benzylisoquinoline pathway, are investigated for biocatalytic transformations. The library of NCS available is extended by two novel NCSs from Argemone mexicana (AmNCS1, AmNCS2) and one new NCS from Corydalis saxicola (CsNCS); furthermore, it is shown that the NCS from Papaver bracteatum (PbNCS) is a highly productive catalyst leading to the isoquinoline product with up to >99% e.e. Under certain conditions lyophilized whole Escherichia coli cells containing the various overexpressed NCS turned out to be suitable catalysts. The reaction using dopamine as substrate bears several challenges such as the spontaneous non-stereoselective background reaction and side reactions. The PbNCS enzyme is successfully immobilized on various carriers whereby EziG3 proved to be the best suited for biotransformations. Dopamine showed limited stability in solution resulting in the coating of the catalyst over time, which could be solved by the addition of ascorbic acid (e.g., 1 mg ml-1 ) as antioxidant.

Asymmetric Synthesis of (R)‐1‐Alkyl‐Substituted Tetrahydro‐ß‐carbolines Catalyzed by Strictosidine Synthases

Abstract Stereoselective methods for the synthesis of tetrahydro‐ß‐carbolines are of significant interest due to the broad spectrum of biological activity of the target molecules. In the plant kingdom, strictosidine synthases catalyze the C−C coupling through a Pictet–Spengler reaction of tryptamine and secologanin to exclusively form the (S)‐configured tetrahydro‐ß‐carboline (S)‐strictosidine. Investigating the biocatalytic Pictet–Spengler reaction of tryptamine with small‐molecular‐weight aliphatic aldehydes revealed that the strictosidine synthases give unexpectedly access to the (R)‐configured product. Developing an efficient expression method for the enzyme allowed the preparative transformation of various aldehydes, giving the products with up to >98 % ee. With this tool in hand, a chemoenzymatic two‐step synthesis of (R)‐harmicine was achieved, giving (R)‐harmicine in 67 % overall yield in optically pure form.