Asymmetric Routes to Chiral Secondary Alcohols

The authors describe several examples of using asymmetric hydrogenation and biocatalysis for synthesizing several secondary alcohol compounds.
Sep 01, 2010
Volume 2010 Supplement, Issue 4

Chiral secondary alcohols are important intermediates and starting materials for the pharmaceutical industry. A wide variety of methodologies have been developed for preparing these synthons in high enantiomeric excess, including resolution of racemates using enzymes such as lipases and asymmetric reduction or hydrogenation of prochiral ketones. A decision on which technology to use must be made on a case-by-case basis and should take into account several factors, including the efficiency of the technology for a particular substrate, access to the catalysts, catalyst cost, and the sensitivity of the substrates to the proposed reaction conditions. Access to a broad portfolio of technologies is beneficial to allow the best solution for a particular product. This article describes several applications of these technologies for synthesizing secondary alcohol products at laboratory scale to multikilogram manufacture. The work was performed by Chirotech Technology, Custom Pharmaceutical Services (CPS–Chirotech), a wholly owned subsidiary of Dr. Reddy's Laboratories (Hyderabad, Andhra Pradesh, India).

Alcohol dehydrogenase reduction of ketones

Figure 1: Methods of cofactor regeneration in alcohol dehydrogenase (ADH)-catalyzed ketone reduction. NADPH is the reduced form of NADP+. NADP+ is nicotinamide adenine dinucleotide phosphate. NADH is the reduced form of NAD+. NAD+ is nicotinamide adenine dinucleotide. GDH is glucose dehydrogenase. FDH is formate dehydrogenase. (FIGURE IS COURTESY OF THE AUTHOR)
The use of alcohol dehydrogenase (ADH) enzymes for synthesizing single-isomer secondary alcohols has significantly increased during the past five years. These enzymes catalyze the asymmetric reduction of a prochiral ketone and require a nicotinamide cofactor to provide the hydrogen that allows the reduction to occur. These cofactors are costly and therefore must be recycled. Several methods for recycling the cofactors are available, which include using glucose dehydrogenase (GDH), formate dehydrogenase (FDH), and isopropyl alcohol (IPA) (see Figure 1). Stable variants of GDH are available at large scale and reasonable cost although IPA is the most convenient and lowest-cost approach if IPA is a substrate for the ADH of interest.

CPS–Chirotech has developed considerable expertise in using ADH enzymes to make secondary alcohols. It has a collection of more than 250 isolated in-house and commercially available enzymes, both (S)- and (R)-selective, arrayed in 96 well plates, to allow for efficient screening. Hits with these enzymes facilitate a fast response and allow scale-up to kilogram quantities of product in weeks. For products where a more efficient enzyme is needed, CPS–Chirotech has a broad collection of thousands of wild type (WT) organisms, which originate from diverse sources and include a large number of marine organisms. There are two choices with hits from this collection—either scale-up using the WT organism as whole cells or clone and overexpress the ADH of interest. Cloning the enzyme generally leads to a lower cost catalyst, especially when expressed using an efficient expression system. CPS–Chirotech uses a platform for protein production based on Pseudomonas fluorescens (1). This expression system uses a well characterized and safe strain of P. fluorescens (i.e., MB101) that is capable of high cell-density fermentations (in excess of 100 g/L of dry cells) without oxygen enrichment. P. fluorescens can be cultivated using a medium of simple and defined mineral salts, supplemented with an inorganic nitrogen source such as ammonia and a carbon source such as glucose. Annotated published genome sequences also provide a useful source of new ADHs. Synthetic gene synthesis is routinely offered at reasonable prices (i.e., $0.50/base pair) and can be codon-optimized for a nominated expression system.

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