The development of cost-effective and sustainable catalytic methods for the production of enantiomerically pure chiral amines is a key challenge facing the pharmaceutical and fine chemical industries. This challenge is highlighted by the estimate that 40-45% of drug candidates contain a chiral amine, fueling a demand for broadly applicable synthetic methods that deliver target structures in high yield and enantiomeric excess. Herein we describe the development and application of a "toolbox" of monoamine oxidase variants from Aspergillus niger (MAO-N) which display remarkable substrate scope and tolerance for sterically demanding motifs, including a new variant, which exhibits high activity and enantioselectivity toward substrates containing the aminodiphenylmethane (benzhydrylamine) template. By combining rational structure-guided engineering with high-throughput screening, it has been possible to expand the substrate scope of MAO-N to accommodate amine substrates containing bulky aryl substituents. These engineered MAO-N biocatalysts have been applied in deracemization reactions for the efficient asymmetric synthesis of the generic active pharmaceutical ingredients Solifenacin and Levocetirizine as well as the natural products (R)-coniine, (R)-eleagnine, and (R)-leptaflorine. We also report a novel MAO-N mediated asymmetric oxidative Pictet-Spengler approach to the synthesis of (R)-harmicine.

A directed evolution approach has been used for the generation of variants of galactose oxidase (GOase) that can selectively oxidize glycans on glycoproteins. The aldehyde function introduced on the glycans D-mannose (Man) and D-N-acetyl glucosamine (GlcNAc) by the enzyme variants could then be used to label the glycoproteins and also whole cells that display mannosides on their surface.

Atropisomeric ligands have found numerous powerful applications in catalysis, [1] and the atropisomeric biaryl bisphosphine binap played an important role in the award of a Nobel Prize to Noyori in 2001.[2] Enantiomerically pure atropisomers commonly employed as chiral ligands are generally made by resolution: there are still relatively few effective methods for direct asymmetric coupling to form single enantiomers. [3] Kinetic resolution [4] and dynamic resolution [5] under kinetic [5a] or thermodynamic [5b] control are particularly appealing given the possibility offered by atropisomerism for thermal racemization of the less reactive enantiomer. The use of desymmetrization for the synthesis of single atropisomers is rare. [6] Following the early example of enantioselective lithiation reported by Raston and co-workers, [6b] the research groups of Hayashi [6c] and Harada [6d] also reported chemical methods for desymmetrizing biphenyl compounds. A single example of the enzymatic desymmetrization of a biaryl compound with a lipase was reported by Matsumoto et al.[6e]Herein, we report two novel and complementary biocatalytic approaches to the enantioselective synthesis of atropisomers by the desymmetrization of appropriate achiral substrates containing a pair of enantiotopic functional groups. The atropisomer in question is the diaryl ether 2, which may be formed either by enantioselective oxidation of the symmetrical diol 1 or by the corresponding reduction of the symmetrical dialdehyde 3 (Scheme 1). The enzymes we employed for these transformations were 1) a variant of galactose oxidase (GOase) which had been previously evolved to accept chiral benzylic alcohols as substrates with high enantioselectivity (1!2) [7] and 2) a family of ketoreductases that are known to possess good activity and enantioselectivity for the asymmetric reduction of benzylic ketones (3!2). [8] Atropisomeric diaryl ethers [9] form part of the structure of vancomycin [10] and are promising scaffolds for the construction for new chiral ligands.[11] Dialdehyde 3 and diol 1 were made by our published route. [9] In an initial screen, we attempted enantioselective acetylation by incubating diol 1 with Candida antarctica lipase B and vinyl acetate. Slow acylation of 1 was observed with approximately 50 % conversion after 24 h to the monoacetate 4 and modest enantioselectivity (60 % ee). In contrast, when diol 1 was incubated with the previously reported M 3-5 variant of GOase, [7] rapid oxidation to the monoaldehyde (P)-2 resulted in 80 % conversion after 24 h to material with 94 % ee.During the oxidation of 1 to 2, rapid formation of the product (P)-2 with approximately 88 % ee (see below for assignment of the absolute configuration) was observed after 1 h, followed by a slower increase in enantiomeric purity to a maximum ee value of 94 % (Figure 1). This increase in the ee value, along with the formation of the dialdehyde 3 (14 % after 24 h), suggested that the minor enantiomer (M)-2 produced in the enantioselective oxidation of 1 was ...

The tetrahydro-β-carboline (THBC) ring system is an important structural motif found in a large number of bioactive alkaloid natural products. Herein we report a broadly applicable method for the synthesis of enantiomerically pure β-carbolines via a deracemization procedure employing the D9 and D11 variants of monoamine oxidase from Aspergillus niger (MAO-N) in combination with a nonselective chemical reducing agent. Biotransformations were performed on a preparative scale, leading to the synthesis of optically enriched products in excellent enantiomeric excess (e.e.; up to 99%) and isolated yield (up to 93%). Interestingly, a switch in enantioselectivity associated with the MAO-N variants is observed as the nature of the C-1 substituent of the THBC is varied. Molecular modeling provided an explanation for this observation and highlighted key active site residues which were modified, resulting in an increase in (R)-selectivity associated with the enzyme. These results provide insight into the factors which influence the selectivity of the MAO-N variants, and may offer a platform for future directed evolution projects aimed toward the challenge of engineering (R)-selective amine oxidase biocatalysts.

We have previously reported a general method for the deracemisation of racemic chiral amines (primary, [1] secondary [2] and tertiary [3] ) by using variants of the enzyme monoamine oxidase N (MAO-N) from Aspergillus niger. This deracemisation process employs a combination of an enantioselective enzymatic oxidation of the amine to afford the corresponding imine or iminium ion, together with a non-selective chemical reduction of the imine or iminium ion back to the racemic starting material (Scheme 1). The use of an (S)-selective MAO-N enzyme leads to accumulation of the (R)-amine in high enantiomeric excess and yield through several rounds of oxidation and reduction.Previously, we have applied this chemo-enzymatic approach to the deracemisation of the alkaloid (AE)-crispine A. [4] Crispine A (1) was first isolated from extracts of the plant Carduus crispus (welted thistle), along with the cytotoxic crispine B (2, Figure 1) and three other bicyclic isoquinoline alkaloids. [5] Although the deracemisation of (AE)-crispine A resulted in the generation of the R enantiomer in > 97 % ee, the reaction required 40 h to proceed to completion with a MAO-N-5 variant in an overall yield of 48 %. This previous study also highlighted that less-functionalised analogue 3 was more reactive with the same variant, taking only 6 h to reach completion (> 97 % ee), thus indicating that the two methoxy groups of racemate (AE)-1 resulted in lower activity. We reasoned that this drop in activity was due to steric interference between these two groups and residues within the active site of the enzyme. As a result, we employed a combination of molecular modelling and a rational re-design of the MAO-N-5 variant to identify and develop potential new MAO-N variants that could have enhanced activity towards enantiomer (S)-1. In addition, we sought to improve the efficiency of the overall synthesis of enantiomer (R)-1 by utilising the microwave synthesis of the racemic amine coupled with the enhanced deracemisation that is brought about by changes to the MAO-N enzyme.To identify MAO-N variants that have improved activity towards compound (AE)-1, we modelled (S)-crispine A into the active site of the MAO-N-5 enzyme (PDB code 2VVM). [6] Four residues (Phe210, Leu213, Met242 and Met246), which are located at the entrance to the active site channel, were identified as providing possible steric interactions with the methoxy groups of crispine A (1). To optimise these residues, two randomised libraries were created: The first library targeted amino acids Phe210 and Leu213 (library A) and the second library targeted Met242 and Met246 (library B). Both libraries were screened against compound (AE)-1 by using our previously reported solid-phase assay [7] (Figure 2); sixteen active "hits" were collected from each of the two libraries (A and B), which were then subjected to a second round of screening to eliminate any false positives. After the second round of screening, the two MAO-N variants from each library that had the highest activity, as judged by t...