Biocatalysis in API synthesis
In addition to chemocatalysis, biocatalysis is used to synthesize APIs or intermediates. Historically, pharmaceutical companies
have been reluctant to use biocatalysts because naturally occurring biocatalysts have not been viable economically for commercial
production. This situation, however, is changing as biocatalysts may be engineered to address some of the drawbacks of conventional
chemistry. Enzyme-catalyzed reactions can exhibit higher enantioselectivity, regioselectivity, substrate specificity, and
stability, and therefore require mild conditions (i.e., ambient pressures and temperatures) to react while achieving higher
reaction efficiency and product yields.
Biocatalysis is part of the toolbox of major pharmaceutical companies and fine-chemical producers, such as DSM (Heerlen, the Netherlands), Evonik (Düsseldorf, Germany), Dowpharma (Midland, MI), Kaneka (Osaka, Japan), Lonza (Basel, Switzerland), and PCAS (Longjumeau, France). It has also generated companies and researchers to specialize in biocatalysis.
A case in point is Codexis (Redwood City, CA). In April 2008, Codexis filed a Form S-1 with the US Securities and Exchange Commission for an initial
public offering for $100 million. Codexis has carved out a niche in biocatalysis, including biocatalysis for pharmaceutical
applications. The company was founded in 2002 as a wholly owned subsidiary of Maxygen (Redwood City, CA). Codexis added to its toolbox in pharmaceutical manufacture with the acquisitions of Jülich Fine Chemicals
in 2005 and BioCatalytics in 2007. Codexis's pharmaceutical customers have included Arch Pharmalabs (Hyderabad, India), Bristol-Myers Squibb (New York), Dr. Reddy's Laboratories (Hyderabad), Merck & Co., Pfizer (New York), Ranbaxy Laboratories (Gurgaon, India), Schering-Plough (Kenilworth, NJ), and Teva Pharmaceutical (Jerusalem).
As an example of its work in pharmaceuticals, Codexis developed four enzymes and reduced the cost of two intermediates for
producing atorvastatin, the API in Pfizer's "Lipitor." It supplies Pfizer with one of these intermediates and generic atorvastatin
manufacturers with the other intermediate, according to the company's S-1 filing.
The key advanced chiral intermediate in atorvastatin is tert-butyl (4R, 6R)-6-cyanomethyl-2, 2-dimethyl-1,3-dioxane-4-acetate (ATS-8 or TBIN). Pfizer's traditional ATS-8 process uses a sodium borohydride
(NaBH4) reduction of the corresponding (5R)-hydroxy-3-ketoester (ATS-6 or HK) under cryogenic conditions to give, after quenching, the (3R, 5R)-dihydroxyester (ATS-7 or diol). The ATS-6 is first converted in situ to a diastereodirecting boron chelate by treating diethylmethoxyborane that is reacted with NaBH4 below 85 °C to promote diastereoselectivity.
After the reaction, the borane reagent is regenerated and recovered by repeated methanol quenches and vacuum distillations.
Several percent of the undesired (3S)-diastereomer is formed. Subsequently, the ATS-7 diol, an oil, is protected as its acetonide, ATS-8. ATS-8's diastereopurity
is upgraded by crystallization with concomitant product loss (2).
To improve the synthesis, Codexis developed a biocatalytic route. The process involves reducing ATS-6 to stereopure ATS-7.
It uses a ketoreductase biocatalyst specifically evolved to reduce ATS-6 with perfect diastereoselectivity under green reaction
conditions (300 g/L in water at ambient temperature and pressure) with a previously evolved glucose dehydrogenase biocatalyst
that returns the oxidized cofactor nicotinamide adenine dinucleotide phosphate (NADP+) to its reduced state (NADPH). This process eliminates the use of boron reagents, reduces solvent use by 85%, decreases waste
by 60%, and increases the yield of ATS-7 with greater stereopurity. Codexis's biocatalytic process is used commercially to
supply ATS-8 to generic atorvastatin manufacturers (2).