Deracemization useful enzymes

Deracemization of Alcohols Using Two Enzyme/Microorganism Systems... 

For the deracemization of phenylethanol derivatives using G. candidum under aerobic conditions (Figure 8.41b), the (S)-specific enzyme was reversible and (R) enzyme was irreversible, so (R)-alcohol accumulated when the cell and racemic alcohols were mixed [31b,c]. Para-substituted phenylethanol derivatives gave better results than meta-substituted derivatives. Sphingomonas was used for... 

The use of D-AAO from the yeast Rhodotorula gracilis to deracemize naphthyl amino acids has been studied in some detail by the groups of Servi and Pollegioni, who compared the kinetic properties of the enzyme with racemic 1- and 2-naphthylalanine (1 and 2) and 1- and 2-naphthylglycine (3 and 4). 

The same group have used the enzyme combination employed in the aspartate deracemization cited above to deracemize 2-naphthylalanine, hut have made use of an interesting innovation introduced by Helaine et al to pull over the poised equilibrium of the transamination reaction. Cysteine sulphinic acid was used as the amino donor in the transamination. The oxoacid product spontaneously decomposes in to pyruvic acid and SO2 (Scheme 3). 

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]. 

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. 

It should be mentioned that the great majority of dynamic kinetic resolutions reported so far are carried out in organic solvents, whereas all cyclic deracemizations are conducted in aqueous media. Therefore, formally, this latter methodology would not fit the scope of this book, which is focused on the synthetic uses of enzymes in non-aqueous media. However, to fully present and discuss the applications and potentials of chemoenzymatic deracemization processes for the synthesis of enantiopure compounds, chemoenzymatic cyclic de-racemizations will also be briefly treated in this chapter, as well as a small number of other examples of enzymatic DKR performed in water. 

The first example of chemoenzymatic DKR of allylic alcohol derivatives was reported by Williams et al. [37]. Cyclic allylic acetates were deracemized by combining a lipase-catalyzed hydrolysis with a racemization via transposition of the acetate group, catalyzed by a Pd(II) complex. Despite a limitation of the process, i.e. long reaction times (19 days), this work was a significant step forward in the combination of enzymes and metals in one pot Some years later, Kim et al. considerably improved the DKR of allylic acetates using a Pd(0) complex for the racemization, which occurs through Tt-allyl(palladium) intermediates. The transesterification is catalyzed by a lipase (Candida antarctica lipase B, CALB) using isopropanol as acyl acceptor (Scheme 5.19) [38]. 

The method of combined enzyme- and transition metal-catalyzed reactions widely applied to the DKR of secondary alcohols has also been applied to the DKR of a-hydroxy acid esters rac-1. The principle is based on the enantioselective acylation catalyzed by Pseudomonas species lipase (PS-C from Amano Ltd) using p-Cl-phenyl acetate as an acyl donor in cyclohexane combined with in situ racemization of the non-acylated enantiomer catalyzed by ruthenium compounds [7]. Under these conditions, various a-hydroxy esters of type 1 were deracemized in moderate to good yields and high enantioselectivity (Scheme 13.2). 

Microbial stereoinversion consists of a deracemization process in which a single whole cell system is applied for the two-step inversion of the configuration of one enantiomer, usually a compound containing a secondary alcohol group. Examples of a two-enzyme system is known for deracemization of mandelates where a single microorganism is used [18]. 

Schema 13.9 Deracemization by stereoinversion using three different enzymes in an equilibrium-shifted transamination.

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. 

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