Nitrilases 3-hydroxyglutaronitrile

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]. 

A summary of the industrial-scale process development for the nitrilase-catalyzed [93] route to ethyl (/ )-4-cyano-3-hydroxy-butyrate, an intermediate in the synthesis of Atorvastatin (Pfizer Lipitor) from epichlorohydrin via 3-hydroxyglutaronitrile (3-HGN) was recently reported (Figure 8.15) [94], The reaction conditions were further optimized to operate at 3 m (330 gL ) substrate, pH 7.5 and 27 °C. Under these conditions, 100% conversion and product ee of 99% was obtained in 16 h reaction time with a crude enzyme loading of 6% (based on total protein, 0.1 U mg-1). It is noted that at pH < 6.0 the reaction stalled at <50% conversion and at alkaline pH a slowing in reaction rate was observed. Since the starting material is of low cost and the nitrilase can be effectively expressed in the Pfenex (Pseudomonas) expression system at low cost, introduction of the critical stereogenic center... 

Bergeron S, Chaplin D, Edwards JH, Ellis BS, Hill CL, Holt-Tiffin K, Knight JR, Mahoney T, Osborne AP, Ruecroft G (2006) Nitrilase-catalyzed desym-metrization of 3-hydroxyglutaronitrile preparation of a statin side-chain intermediate. Org Proc Res Dev 10 661-665 Burns M, Weaver J, Wong J (2005) Stereoselective enzymic bioconversion of aliphatic dinitriles into cyano carboxylic acids. WO 2005100580 DeSantis G, Zhu Z, Greenberg W, Wong K, Chaplin J, Hanson SR, Farwell B, Nicholson LW, Rand CL, Weiner DP, Robertson D, Burk MJ (2002) An enzyme library approach to biocatalysis development of nitrilases for enantioselective production of carboxylic acid derivatives. J Am Chem Soc 124 9024-9025... 

Bergeron, S., Chaplin, D.A., Edwards, J.H., Ellis, B.S.W., Hill, C.L., Holt-Tiffin, K., Knight, J.R., Mahoney, T., Osborne, A.P. and Ruecroft, G., Nitrilase-catalysed desymmetrisation of 3-hydroxyglutaronitrile preparation of a statin side-chain intermediate. Org. Proc. Res. Dev.,... 

Vink et al. (2009) used an 0-benzyl protected 3-hydroxyglutaronitrile in the presence of nitrilase from R. erythropolis NCIMB 11540 to produce the corre-... 

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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