Oxidation, deracemization

Assuming that the enzymatic reaction is highly enantioselective, then even after only four cycles the enantiomeric excess will have reached 93.4% whereas after seven catalytic cycles the enantiomeric excess is >99% (Figure 5.3). This type of deracemization is really a stereoinversion process in that the reactive enantiomer undergoes stereoinversion during the process. One of the challenges of developing this type of process is to find conditions under which the enzyme catalyst and chemical reactant can coexist, particularly in the case of redox chemistry in which the coexistence of an oxidant and reductant in the same reaction vessel is difficult to achieve. For this... 

Figure 5.2 Cyclic deracemization process involving sequential enzyme-catalyzed oxidation and nonenzymatic reduction.

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. 

Finally in this section on deracemization via cyclic oxidation/reduction methods, there has been some limited work carried out on the deracemization of secondary alcohols. Soda et al. [22] employed lactate oxidase in combination with sodium borohydride to deracemize D/i-lactate (18) via the intermediate pyruvate (19) (Figure 5.12). 

Carnell et al. discovered that whole cells of Cunninghamella echinulata NRRL1384 were able to deracemize racemic N-(l-hydroxy-l-phenylethyl)benzamide (24) to produce the (R) enantiomer (Figure 5.17) [30]. The deracemization involves fast, highly (S)-selective oxidation, followed by slower, partially (R)-selective reduction of the ketone (25). Optimization by removing competing extracellular amidase/prote-ase activity resulted in 82% yield and 92% ee. 

Deracemization via the biocatalytic stereoinversion is usually achieved by employing whole cells. In the case of secondary alcohols, it is believed that microbial stereoinversion occurs by an oxidation-reduction sequence... 

To obtain a better understanding of the reaction mechanism, some compounds that are considered to he intermediates were subjected to the reaction. Various reaction courses can be considered as illustrated in Fig. 21. Path A a-Methyltropic acid is oxidized to a-phenyl-a-methylmalonic acid. Then, the malonate is converted to optically active a-phenylpropionate hy arylmalonate decarboxylase. In order to confirm this assumption, incubation of the malonic acid with Rhodococcus sp. was carried out. The result obtained was the total recovery of the substrate, indicating that no decarboxylase is present in this bacterium. Path B a-Methyltropic acid is converted to racemic a-phenylpropionic acid, which is deracemized to optically active propionic acid. To examine the possibility of this route, racemic a-phenylpropionic acid was subjected to the reaction to observe... 

Voss, C.V., Gruber, C.C. and Kroutil, W. (2008) Deracemization of secondary alcohols through a concurrent tandem biocatalytic oxidation and reduction. Angewandte Chemie-International Edition, 47 (4), 741-745. 

In a different approach, Franck-Neumann et al. [24] utilized the manganese complex 14 (formed by deracemization) to obtain the enantiomerically pure target molecule 12 via Horner-Wadsworth-Emmons olefination and oxidative decomplexation of the intermediate vinylallene complex 15 (Scheme 18.6). 

Medici et al. have used a combined sequential oxidation-reduction to access a range of imsaturated secondary alcohols from their racemates [7] (Scheme 1). Here the S-alcohol 2 is oxidized by B. stereothermophilus which is displaying Prelog specificity leaving the l -enantiomer untouched. The other microorganism, Y. lipolytica contains an anti-Prelog dehydrogenase which is therefore able to reduce the ketone 1 to the l -alcohol 2. Thus the combination of the two steps effects a net deracemization of substrate 2. 

J )-Mandelic acid 3 is a useful chiral synthon for the production of pharmaceuticals such as semi-synthetic penecillins, cephalosporins and antiobesity agents and many methods have been reported for the preparation of the optically pure material. A method to deracemize the racemate which is readily available on a large scale was developed by Ohta et al. using a combination of two biotransformations. The method consists of enantioselective oxidation of (S)-... 

The results actually showed a deracemization of the racemic hydroxyester 10 as opposed to enantioselective hydrolysis with formation of optically pure (R)-hydroxyester 10 and only 20 % loss in mass balance. Small quantities of ethyl 3-oxobutanoate 9 (<5%) were also detected throughout the reaction, leading the authors to suggest a multiple oxidation-reduction system with one dehydrogenase enzyme (DH-2) catalysing the irreversible reduction to the (R)-hydroxy-ester (Scheme 5). 

Scheme 8. Resolution and deracemization of styrene oxide by fungal cells... 

For instance, styrene oxide was resolved by whole cells of Aspergillus niger and Beauveria bassiana via two different pathways showing matching enantio- and regioselectivities with excellent results (Scheme 8). Combination of the two biocatalysts employing a deracemization process in a single reactor led to R) phenylethane-l,2-diol as the sole product in 98% ee and 85% isolated yield [58]. 

Scheme 11. Deracemization of p-nitrostyrene oxide and application to the synthesis of (R)-nifenalol...

Chemoenzymatic processes involving oxidizing enzymes have been reported particularly for specific chemical syntheses. For example, industrially important amino acids can be deracemized by exploiting the enantioselectivity of amino acid oxidases a commercial process has recently been developed in which efficient... 

Kroutil et al. have recently reported [18] an elegant one-pot oxidation/reduction sequence for the deracemization of a chiral secondary alcohol using a single biocatalyst. LyophiUzed cells of a Rhodococcus sp. CBS IVJ.Ti converted racemic 2-decanol into the (S)-enantiomer in 82% yield and 92% enantiomeric excess (e.e.). via a non-specific oxidation followed sequentially by an (S)-selective reduction (Scheme 6.5). Acetone was used as the hydrogen acceptor in the first step and isopropanol as the hydrogen donor in the second step. 

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. 

Similarly to the case of amino acids, hydroxy acids can also be deracemized by combining an enantioselective oxidation with a non-enantioselective reduction with sodium borohydride. For example, the group of Soda has reported the transformation of DL-lactate into D-lactate in >99% (Scheme 5.38) [78]. 

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 application of the deracemization strategy has provided efficient entry to a novel amino acid substituent of the antifungal agents, polyoxins and nikkomycins, as shown in Scheme 8E.20. The versatile five-carbon building block was obtained from phthalimidation of the hydroxymethyl-substituted epoxide in 87% yield and 82% ee. Straightforward synthesis of polyoxamic acid was then accomplished by subsequent dihydroxylation and selective oxidation of the alkylation product. 

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