Monoamine oxidases deracemization

Recently Turner and coworkers have sought to extend the deracemization method beyond a-amino acids to encompass chiral amines. Chiral amines are increasingly important building blocks for pharmaceutical compounds that are either in clinical development or currently licensed for use as drugs (Figure 5.7). At the outset of this work, it was known that type II monoamine oxidases were able to catalyze the oxidation of simple amines to imines in an analogous fashion to amino acid oxidases. However, monoamine oxidases generally possess narrow substrate specificity and moreover have been only documented to catalyze the oxidation of simple, nonchiral... 

Figure 5.8 Deracemization of a-methylbenzylamine by using a variant monoamine oxidase in combination with NH3 BH3.

Upon mutagenesis of the monoamine oxidase from Aspergillus niger (MAO-N) within several rounds of directed evolution [65], variant biocatalysts were identified with largely expanded substrate acceptance, enabling also the deracemization of tertiary amines incorporating straight-chain and cyclic structural motifs [66]. 

Monoamine Oxidase-catalysed Reactions Application Towards the Chemo-enzymatic Deracemization of the Alkaloid ( )-Crispine A... 

In order to extend the approach to include deracemization of chiral secondary amines, this group carried out directed evolution on the monoamine oxidase (MAO) enzyme MAO-N (Scheme 2.32). A new variant was identified with improved catalytic properties towards a cyclic secondary amine 64, the substrate used in the evolution experiments. This new variant had a single point mutation, lle246Met, and was found to have improved catalytic properties towards a number of other cyclic secondary amines. The new variant was used in the deracemization of rac-64 yielding (R)-64 in high yield and enantiomeric excess [34]. 

The group of Turner has reported the deracemization of amines [79]. The wild type of Type II monoamine oxidase from Aspergillus niger possesses very low but measurable activity toward the oxidation of L-a-methylbenzylamine. The oxidation of the D enantiomer is even slower. In vitro evolution led to the identification of a mutant with enhanced enantioselectivity, showing high E values (>100) for a variety of primary and secondary amines. An example is shown in Scheme 5.39. 

An important breakthrough was made very recently in this area. A chemoenzymatic method developed by Turner has allowed the cyclic deracemization of tertiary amines [80]. Enantiopure tertiary amines cannot be obtained via DKR. One of the variants obtained by directed evolution of the monoamine oxidase from Aspergillus niger showed high activity and enantioselectivity toward cyclic tertiary amines (Scheme 5.40). 

In order to be able to apply this deracemization method to a wide range of chiral amines, it was essential to identify monoamine oxidase enzymes possessing both... 

Cyclic secondary amines [102] have been successfully deracemized via directed evolution of the monoamine oxidase from A. niger. The new variant displayed high catalytic activity and enantioselectivity towards cyclic secondary amines which could be subjected to reduction using ammonia borane to give the enantiomericaUy pure amine (Scheme 4.47). 

Amine oxidases (AOs) can be used to synthesize primary, secondary and tertiary amines. Deracemization of chiral amines can be achieved by an enantioselective oxidation by an AO followed by a reduction using a nonselective reducing agent (Figure 7.12) [47d,76]. There are two types of AOs Type 1 (Cu/TOPA dependent, CAOs, EC.1.4.3.6) and Type 11 (Flavin dependent, EC 1.4.3.4). This chapter will solely focus on the flavin dependent monoamine oxidases. 

In particular, recombinant variants of the monoamine oxidase N (MAO-N) originating from Aspergillus niger have been established over the past years as excellent biocatalysts for the deracemization and stereoinversion of diverse primary, secondary, and even tertiary amines by Turner and coworkers [18]. A series of tailored variants were created by an intensive study and successive optimization of each catalyst by the combination of directed evolution, rational protein design, and novel techniques of high-throughput screenings [95,129]. 

General reaction scheme for the deracemization of 1-substituted tetrahydro-(3-carbolines (TBHCs) by the evolved monoamine oxidase N (MAO-N) in combination with a nonselective reducing agent. 

TABLE 2.11 Scope of the Deracemization of Tetrahydro-p-Carbolines (THBCs, Scheme 3.22 by Two Variants of the Monoamine Oxidase N (MAO-N) Originating from Aspergillus niger ... 

The reaction scheme for the deracemization of (substituted) 1-phenylethyl amine derivatives by the combination of a monoamine oxidase (MAO) with an co-TA ATA (amino transferase) denotes the commercially available co-TAs from Codexis. 

Deracemization of benzyliso-quinolines rac-A to berbines (S)-B, employing a monoamine oxidase (MAO), berberine bridge enzyme (BBE), and borane. 

Turner et al. first reported engineered S-stereoselective flavin-dependent monoamine oxidase variants bom Aspergillus niger for the deracemization of racemic amines producing the S-enantiomers of primary, secondary, and tertiary amines (Figure 19.8) [35]. On the other hand, Leisch et al. showed that wild-type cyclohexy-lamine oxidase from Brevibacterium oxydans IH-35A, which had been isolated and characterized by Hasegawa et al., oxidized the S-enantiomer of amines and successfully synthesized (f )-amine through a deracemization reaction [37]. 

Synthesis of (/ )-A/-methyl-2-phenylpyrrolidine via chemoe-nzymatic deracemization of its corresponding racemate with a recombinant whoie-ceii cata-iyst containing a monoamine oxidase. 

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