Desymmetrization Directed evolution

The initial results of an early directed evolution study are all the more significant, because no X-ray data or homology models were available then to serve as a possible guide [89]. In a model study using whole E. coU cells containing the CHMO from Adnetohacter sp. NCIM B9871,4-hydroxy-cydohexanone (3 5) was used as the substrate. The WT leads to the preferential formation of the primary product (i )-36, which spontaneously rearranges to the thermodynamically more stable lactone (R)-37. The enantiomeric excess of this desymmetrization is only 9%, and the sense of enantioselectivity (R) is opposite to the usually observed (S)-preference displayed by simple 4-alkyl-substituted cydohexanone derivatives (see Scheme 2.10) [84—87]. 

This screening system has also been applied successfully in the directed evolution of enantioselective EHs acting as catalysts in the kinetic resolution of chiral epoxides 95,96) (Section IV.A.4). Moreover, the firm Diversa has applied the MS-based method in the desymmetrization of a prochiral dinitrile (l,3-dicyano-2-hydroxypropane) catalyzed by mutant nitrilases 46). In this industrial application, one of the nitrile moieties was labeled selectively with as in N-17, which means that the two pseiido-eaaniiovaenc products (S)- N-18 and (J )-18 differ by one mass unit. This is sufficient for the MS system to distinguish between the two products quantitatively 46). 

The Bacillus subtilis lipase A (BSLA) is an unusual lipase because it lacks the so-called lid structural unit 13(3). Moreover, it is a small enzyme composed of only 181 amino acids. The initial results of an ongoing study are remarkable and illustrate the power of directed evolution 45,137). The desymmetrization of meso-, 4-diacetoxy-2-cyclopentene (15) was chosen as the model reaction, and the MS-based ee assay using an appropriately Ds-labeled substrate (Section III.C) provided a means to screen thousands of mutants. 

In the first reported case of the directed evolution of an enantioselective nitrilase, an additional limitation had to be overcome that is sometimes ignored when enzymes are used as catalysts in synthetic organic chemistry product inhibition and/ or decreased enantioselectivity at high substrate concentrations 46). A case in point concerns the desymmetrization of the prochiral dinitrile 35 with preferential formation of the ( /-configurated acid 18, which is known to be a chiral intermediate in the synthesis of the cholesterol-lowering therapeutic drug 36 (Lipitor, ... 

Directed Evolution of a Lipase for Desymmetrization of meso-l,4-Diacetoxycyclopentene [33]... 

The second MS-based approach does not require any derivatization reaction and has in fact been applied several times in the area of directed evolution [20,33-36]. It makes use of deuterium-labeled pseudo enantiomers or pseudo meso compounds. This practical method is restricted to studies involving kinetic resolution of race-mates and desymmetrization of prochiral compounds bearing reactive enantiotopic groups (Figure 9.2) [20]. 

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