Pfizer, Inc.'s (New York) "Lipitor" (atorvastatin) is the pharmaceutical industry's top-selling drug, with 2006 sales of $13.6 billion, according to IMS Health (Fairfield, CT). A key intermediate in Lipitor is ethyl (R)-4-cyano-3-hydroxybutyrate, which can be produced through biocatalytic routes.Dowpharma (Midland, MI) uses a nitrilase developed by Diversa Corporation (San Diego) for the asymmetric hydrolysis of 3-hydroxyglutaronitrile to make ethyl (R)-4-cyano-3-hydroxybutyric acid, which is then converted to ethyl (R)-4-cyano-3-hydroxybutyrate. The route to making ethyl (R)-4-cyano-3-hydroxybutyrate starts with epichlorohydrin, which is subjected to a cyanide reaction. This step is followed by an enzyme desymmetrization (100% theoretical yield) using a nitrilase engineered by Diversa to work at 3-M substrate concentration to give 99% conversion and 99% enantiomeric excess (1, 2).
A key part of the process is the scale-up of the production of the nitralase, which Dowpharma does via its "Pfenex" expression technology to give soluble, active enzyme in titers in excess of 25 g/L fermentation broth. The final step is a simple esterification (1, 2).
Codexis, Inc. (Redwood City, CA) also developed a biocatalytic process for making ethyl (R)-4-cyano-3-hydroxybutyrate. The process involves two enzymes that catalyze the enantioselective reduction of ethyl 4-chloroacetoacetate by glucose to form an enantiopure chlorohydrin. In the second step, a third evolved enzyme catalyzes the biocatalytic cyanation of the chlorohydrin to cyanohydrin under neutral conditions (2).
Production for colesevelam