Amine deracemization

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. 

The asymmetric amination of ketones is by far the most preferred approach for the preparahon of chiral amines using co-TAs. However, alternative methodologies may be considered if the carbonyl precursor is unstable or the synthesis of the racemic amine is easier to provide better results in economic and/or yield terms. Using racemic amines deracemization strategies allow the preparation of the desired amines in enantiopme form and a theoretical 100% yield [84- ]. This can be achieved by the combination of two stereocomplementary w-TAs. In the first step, the enantioselec-tive deamination of the racemic amine affords enantiopure untouched amine (50%) and the corresponding ketone (50%). In the second step, an enantiocomplementary -TA catalyzes the asymmetric amination of the ketone, leading to the optical pure amine in 100% theoretical yield (Scheme 2.18). 

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... 

Subsequently Turner and coworkers were able to show that the Asn336Ser variant possessed broad substrate specificity, with the ability to oxidize a wide range of chiral amines of interest [19]. They also discovered a second mutation, Ile246Met, which conferred enhanced activity toward chiral secondary amines as exemplified by the deracemization of racemic 1-methyltetrahydroisoquinoline (MTQ) (9) (Figure 5.9)[20j. 

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]. 

Figure 14.6 Enzymatic deracemization concepts for production of chiral alcohols, amines and amino acids...

Figure 11.1 Enzymatic deracemization of racemic amines via a two-step, one-pot process utilizing an enantioselective amine oxidase in combination with ammonia-borane.

Scheme 2.31 Deracemization of amines via combined use of an enantioselective amine oxidase and ammonia borane.

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]. 

Recently, Turner et al. have shown tertiary amines can also be used as substrates by using a further variant of MAO-N (MAO-N-D5) which had also been developed by directed evolution techniques (Scheme 2.33). For example, racemic N-methyl-pyrrolidine 65 was subjected to deracemization, via the intermediate iminium ion... 

An elegant four-enzyme cascade process was described by Nakajima et al. [28] for the deracemization of an a-amino acid (Scheme 6.13). It involved amine oxidase-catalyzed, (i )-selective oxidation of the amino acid to afford the ammonium salt of the a-keto acid and the unreacted (S)-enantiomer of the substrate. The keto acid then undergoes reductive amination, catalyzed by leucine dehydrogenase, to afford the (S)-amino acid. NADH cofactor regeneration is achieved with formate/FDH. The overall process affords the (S)-enantiomer in 95% yield and 99% e.e. from racemic starting material, formate and molecular oxygen, and the help of three enzymes in concert. A fourth enzyme, catalase, is added to decompose the hydrogen peroxide formed in the first step which otherwise would have a detrimental effect on the enzymes. 

Oxidoreductases are, after lipases, the second most-used kinds of biocatalysts in organic synthesis. Two main processes have been reported using this type of enzymes-bioreduction of carbonyl groups [39] and biohydroxylation of non-activated substrates [40]. However, in recent few years other processes such as deracemization of amines or alcohols [41] and enzymatic Baeyer-Villiger reactions of ketones and aldehydes [42] are being used with great utility in asymmetric synthesis. 

Recently, Turner et al. described the synthesis of the alkaloid (R)-(-t-)-crispine A, which shows cytotoxic activity against HeLa human cancer cell lines, using in the final step a deracemization procedure with the combination of an enantioselective amine oxidase obtained by directed evolution methods and a chemical non-selective reducing agent (Scheme 10.20) [48]. 

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). 

Turner has applied this deracemization process to a very interesting tandem transformation where y-amino ketones are transformed into enantiopure secondary amines via intramolecular reductive animation followed by deracemization (Scheme 5.41) [80]. 

The deracemization of a number of pharmaceutically valuable building blocks has been carried out by biocatalytic processes. This includes epoxides, alcohols, amines and acids. DKR involves the combination of an enantioselective transformation with an in situ racemisation process such that, in principle, both enantiomers of the starting material can be converted to the product in high yield and ee. The racemization step can be catalysed either enzymatically by racemases, or non-enzymatically by transition metals. 

Carr, R., Alexeeva, M., Dawson, M. J., Gotor-Fernandez, V., Humphrey, C. E., and Turner, N. J. 2005. Directed evolution of oxidase for the preparative deracemization of cyclic secondary amines. Chem. Bio. Chem.,6, 637-639. 

The method is of general applicability in the deracemization of secondary alcohols and amines and consists of a Upase-catalyzed irreversible acylation and in situ racemization of the non-reacted enantiomer catalyzed by a ruthenium catalyst. 

The coupling of these two enzymatic systems could find many more applications due to the avaUabihty of amino acid dehydrogenases of broader specificity [31]. A series of amino acid dehydrogenases with D-specificity for the preparation of D-amino acids has been applied to the reductive amination of a-keto acids [32]. However, the deracemization of rac-amino acids exploiting this type of enzyme requires an amino acid oxidase with L-specificily, which is a rare enzymatic activity. As an alternative the a-oxo acid, usually available through difficult synthetic procedures, can be used directly. 

Thus, using L-amino add oxidase from P. myxcfaciens and various amine-borane complexes or D-amino acid oxidase from porcine kidney and sodium cyanoboro-hydride, the preparation of several natural and non-natural enantiopure D- and L-amino adds was achieved, respectively [51]. In a more recent report, several P- and y-substituted a-amino adds were deracemized using D-amino add oxidase from Trigonopsis variahilis and sodium cyanoborohydride or sodium borohydride [52] (Scheme 13.20). 

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