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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. (Figure presented.).

Homochiral 2-hydroxy-4-butyrolactone derivatives are important structural motifs frequently found in naturally occurring biologically active products, synthetic drugs, biodegradable polymers as well as building blocks and chiral auxiliaries in asymmetric organic synthesis (1). Among the methods described in the literature, the stereoselective reduction of 4-hydroxy-2-oxoacid derivatives to produce 2-hydroxy-4-butyrolactone derivatives remains unexplored. In this communication we report a tandem biocatalytic stereodivergent route for the preparation of these compounds using two stereocomplementary aldolases and oxidoreductases, using simple and achiral starting materials. The strategy comprises (Scheme 1) (i) aldol addition reaction of 2-oxoacids to formaldehyde using both 3-methyl-2-oxobutanoate hydroxymethyltransferase and 2-keto-3-deoxy-L-rhamnonate aldolase and variants thereof, and (ii) subsequent 2-oxogroup reduction of the aldol adduct by both ketopantoate reductase and a delta-1-piperidine-2-carboxylate/delta-1-pyrroline-2-carboxylate reductase with uncovered promiscuous ketoreductase activity. Finally, the formation of the corresponding lactone takes place during the work up and purification steps. Yields, enantiomeric excesses and diasteromeric ratios for a total of 23 structurally diverse 2-hydroxy-3-substituted-4-butyrolactones will be discussed. Moreover, one-pot one-step cascade reactions with the aldolases and reductases operating in tandem will be presented.

NAD(P)H-dependent ¿1-piperideine-2-carboxylate/¿1-pyrroline-2-carboxylate reductase from Pseudomonas syringae pv. tomato (DpkAPsyrin, EC 1.5.1.21) catalyzes the stereoselective reduction of ¿1-piperideine-2-carboxylate and ¿1-pyrroline-2-carboxylate to L-pipecolic acid and L-proline respectively. Furthermore, DpkAPsyrin is also able to catalyze the enantioselective synthesis of N-methyl-L-amino acids from methylamine and various 2-oxo acids [1]. In our research group, we observed that in addition to the imine reductase activity, DpkAPsyrin have a promiscuous ketoreductase activity and, consequently, we consider interesting to exploit its underdeveloped synthetic capabilities for the synthesis of 2-hydroxy-4-arylbut-3-enoic acid (4). These 2-hydroxyacids are intermediates in the synthesis of 2-hydroxy-4-arylbutanoic acid, important building blocks for the production of a variety of angiotensin converting enzyme (ACE) inhibitors (e.g. enalapril, lisinopril, cilapril, and benazepril) [2]. In this communication we reported the preparation of structurally diverse (S)-2-hydroxy-4-arylbut-3-enoic acid (4) (isolated yield: 57%-78% and ee: 87%->99%) from aromatic aldehydes (1) and pyruvate (2) by enzymatic cascade reaction using trans-o-hydroxybenzylidene pyruvate hydratase-aldolase from Pseudomonas putida HBPAPputida) [3] and DpkAPsyrin (Scheme 1). The cascade process was possible because it was found that the ketoreductase preferentially reduced the aldol condensation adducts (3) rather than the pyruvate (2). This allows for high levels of 2-hydroxy-4-arylbut-3-enoic acids (4) conversions ( >95% after 24 h).

ATAs engineered for having an enlarged small binding pocket were applied for the synthesis of enantiomerically pure (R)-benzo[1,3]dioxol-5-yl-butylamine, a chiral component of human leukocyte elastase inhibitor DMP 777 (L-694,458). Kinetic resolution of the racemic amine was performed by using the L59A variant of the (S)-selective ATA from Chromobacterium violaceum (Cv-ATA), providing the residual (R)-enantiomer in excellent yield and >99% ee. At moderate enzyme loading and absence of co-solvent, high volumetric productivity of 0.22 mol L-1 h(-1) (42.5 g L-1 h(-1)) was achieved. Complementarily, the (S)-enantiomer was generated via kinetic resolution using the (R)-selective ATA-117-Rd11 from Arthrobacter sp. with acetone as the amino acceptor. In an alternative approach, we employed ATA-117-Rd11 for the asymmetric amination of the prochiral ketone precursor, which at 86% conversion gave the (R)-benzo[1,3]dioxol-5-yl-butylamine with excellent >99% ee. We further evaluated the utility of Cv-ATA L59A for the asymmetric synthesis of pharmaceutically relevant (S)-1-phenylbutan-1-amine, a chiral component of the deubiquitinase inhibitor degrasyn (WP1130). The enzyme showed good tolerance to high concentrations of isopropylamine, producing (S)-1-phenylbutan-1-amine in enantiomerically pure form (>99% ee).

ATAs engineered for having an enlarged small binding pocket were applied for the synthesis of enantiomerically pure (R)‐benzo[1,3]dioxol‐5‐yl‐butylamine, a chiral component of human leukocyte elastase inhibitor DMP 777 (L‐694,458). Kinetic resolution of the racemic amine was performed by using the L59A variant of the (S)‐selective ATA from Chromobacterium violaceum (Cv‐ATA), providing the residual (R)‐enantiomer in excellent yield and >99% ee. At moderate enzyme loading and absence of co‐solvent, high volumetric productivity of 0.22 mol L⁻¹ h⁻¹ (42.5 g L⁻¹ h⁻¹) was achieved. Complementarily, the (S)‐enantiomer was generated via kinetic resolution using the (R)‐selective ATA‐117‐Rd11 from Arthrobacter sp. with acetone as the amino acceptor. In an alternative approach, we employed ATA‐117‐Rd11 for the asymmetric amination of the prochiral ketone precursor, which at 86% conversion gave the (R)‐benzo[1,3]dioxol‐5‐yl‐butylamine with excellent >99% ee. We further evaluated the utility of Cv‐ATA L59A for the asymmetric synthesis of pharmaceutically relevant (S)‐1‐phenylbutan‐1‐amine, a chiral component of the deubiquitinase inhibitor degrasyn (WP1130). The enzyme showed good tolerance to high concentrations of isopropylamine, producing (S)‐1‐phenylbutan‐1‐amine in enantiomerically pure form (>99% ee).