Development of an efficient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates

Abstract

A process is reported for efficient, enantioselective production of key intermediates for the common chiral side chain of statin-type cholesterol-lowering drugs such as Lipitor (atorvastatin) and Crestor (rosuvastatin). The process features a one-pot tandem aldol reaction catalyzed by a deoxyribose-5-phosphate aldolase (DERA) to form a 6-carbon intermediate with installation of two stereogenic centers from 2-carbon starting materials. An improvement of almost 400-fold in volumetric productivity relative to the published enzymatic reaction conditions has been achieved, resulting in a commercially attractive process that has been run on up to a 100-g scale in a single batch at a rate of 30.6 g͞liter per h. Catalyst load has been improved by 10-fold as well, from 20 to 2.0 wt % DERA. These improvements were achieved by a combination of discovery from environmental DNA of DERAs with improved activity and reaction optimization to overcome substrate inhibition. The two stereogenic centers are set by DERA with enantiomeric excess at >99.9% and diastereomeric excess at 96.6%. In addition, downstream chemical steps have been developed to convert the enzymatic product efficiently to versatile intermediates applicable to preparation of atorvastatin and rosuvastatin. Biocatalysis has received attention recently as a tool of enormous potential for the synthesis of pharmaceutical, industrial, and agricultural chemicals and intermediates (1-5). To capitalize further on this potential, enzymes must demonstrate not only superior performance characteristics such as high enantioselectivity and low catalyst loading but also costeffectiveness and scalability. A successful industrial biocatalytic process requires enzymes that can be produced cheaply in kilogram quantities and are stable and active at very high substrate and product concentrations, frequently in the presence of organic solvents. Although many remarkable biocatalytic transformations have been reported on the milligram-to-gram scale, especially in light of recent advances in directed evolution and high-throughput screening for novel enzymes (6-8), the number of processes that have been applied on an industrial scale remains limited (5, 9). This is especially true for biocatalytic processes involving formation of carbon-carbon bonds.The aldol reaction is an extremely useful transformation that allows the formation of new carbon-carbon bonds and the introduction of up to two new stereocenters into the product. Controlling the stereochemistry of aldol reactions can be challenging and has been an area of intense research. Although several elegant asymmetric methods have been developed (10-12), there remains a need for additional methods, and in recent years enzyme-catalyzed approaches using aldolases have received attention as alternatives to chemical methodologies (13).Several years ago, Wong and coworkers (14, 15) reported unprecedented one-pot tandem aldol reactions catalyzed by a deoxyribose-5-phosphate aldolase (DERA), in which 2 eq of acetaldehyde were added in sequen...

Development of an organo- and enzyme-catalysed one-pot, sequential three-component reaction

“…2g In addition, an aldolase variant has been used to catalyse two sequential aldol reactions to yield a precursor of the statin side-chain. 3 The combination of organocatalysts and enzymes is, however, rare. 4 Here, we report the first example of a three-component reaction in which two carboncarbon bond-forming steps are catalysed using the specific combination of an organocatalyst and an enzyme.…”

Biocatalytic Synthesis of Chiral Pharmaceutical Intermediates

“…The two stereogenic centers are set by DERA with enantiomeric excess at greater than 99.9 % and diastereomeric excess at 96.6 %. In addition, downstream chemical processes have been developed to convert the enzyme product efficiently to versatile intermediates applicable to preparation of atorvastatin and rosuvastatin (84).…”

Aldolase-catalysed stereoselective synthesis of fluorinated small molecules

“…[5,6] For example, an efficient and scalable aldolasecatalysed process has been developed for the enantioselective synthesis of precursors of the side chain found in the statin drugs. [7] Furthermore, protein engineering has been shown to be valuable for increasing further the synthetic value of aldolase enzymes, for example by broadening the range of substrates accepted or by modifying the stereochemistry of carboncarbon bond formation. [8][9][10][11][12][13][14] Recently, aldolases have emerged as a useful class of catalysts for controlling the formation of fluorine-bearing stereocentres.…”