Nitrilases desymmetrization

Scheme 1.50 Nitrilase desymmetrization approach to the atorvastatin statin side chain...

The biocatalytic differentiation of enantiotopic nitrile groups in prochiral or meso substrates has been studied by several research groups. For instance, the nitrilase-catalyzed desymmetrization of 3-hydroxyglutaronitrile [92,93] followed by an esterification provided ethyl-(Jl)-4-cyano-3-hydroxybutyrate, a useful intermediate in the synthesis of cholesterol-lowering dmg statins (Figure 6.32) [94,95]. The hydrolysis of prochiral a,a-disubstituted malononitriles by a Rhodococcus strain expressing nitrile hydratase/amidase activity resulted in the formation of (R)-a,a-disubstituted malo-namic acids (Figure 6.33) [96]. 

Bergeron, S., Chaplin, D.A., Edwards, J.H. et al. (2006) Nitrilase-catalyzed desymmetrization of 3-hydroxy-glutaronitrile preparation of a statin side-chain intermediate. Organic Process Research Development, 10, 661-665. 

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). 

Nitrilases catalyze the synthetically important hydrolysis of nitriles with formation of the corresponding carboxylic acids 7-11). Enantioselectivity is relevant in the kinetic resolution of racemic nitriles or desymmetrization of prochiral dinitriles. Both versions have been applied successfully to a number of different substrates using one of the known currently available nitrilases. Recently, scientists at Diversa expanded the collection of nitrilases by metagenome panning 150). Nevertheless, in numerous cases the usual limitations of enzyme catalysis become visible, including poor or only moderate enantioselectivity and limited activity. 

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, ... 

In another development, the statin side chain en route to Atorvastatin (Lipitor , Pfizer) is synthesized via the key intermediate alkyl 3-hydroxy-4-cyanobutyrate (Figure 13.17). Instead of the currently practiced six-step route, a much more concise three-step route starts from epichlorohydrin via Cl chain length enhancement by both nucleophilic substitution of chloride and nucleophilic ring opening of the epoxide with cyanide to yield symmetric dicyanoisopropanol. Nitrilase action desymmetrizes the dinitrile intermediate with the creation of a chiral center in C3 to yield (R)-3-hydroxy-4-cyanobutyrate, which is esterified to the key intermediate ethyl (R)-3-hydroxy-4-cyanobutyrate. 

Scheme 4.18 Desymmetrization of hydroxyglutaronitrile using a nitrilase enzyme...

A nitrilase enzyme (BD9570,0.1 unit/mg, Diversa) was used to desymmetrize prochiral substrate 14 to afford (R)-15. In this process, an aqueous solution of 100 mM NaH PO at pH 7.5 (510 mL) was added to lyophilized nitrilase enzyme powder (15.15 g). The mixture was stirred at 27°C (to rehydrate the lyophilized enzyme powder) for 40 min. 3-Hydroxyglutaronitrile (252.5 g) was then charged over 10 min. The mixture was stirred at 27°C for 16 h and then cooled to 2°C prior to acidification to give pH 2. Celite was charged (25 g) and the slurry filtered. The filtrate was extracted with methyl ethyl ketone. The combined methyl ethyl ketone extracts were evaporated in vacuo (15 mbar, 40°C) to yield the product 15 as a brown liquid (240.8 g, 81% yield ee 98.8%). 

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