Codexis had earlier partnered with Merck & Co. for another biocatalytic route. In 2010, Merck and Codexis reported on the
biocatalytic asymmetric synthesis of chiral amines from ketones in the manufacture of sitagliptin, the API in Merck's antidiabetes
drug Januvia. The biocatalytic process replaced a rhodium-catalysed asymmetric enamine hydrogenation for the largescale manufacture
of sitagliptin. The researchers started from an (R)-selective transaminase that showed slight activity on a smaller truncated
methyl ketone analogue of the sitagliptin ketone. After creating this transaminase, which had marginal activity for the synthesis
of the chiral amine, they further engineered the enzyme through directed evolution to optimise its use for large-scale manufacturing
(5–8).
The initial (R)-selective transaminase was a homologue of an enzyme from Arthrobacter sp., which was previously used for (R)-specific
transamination of methyl ketones and small cyclic ketones. For the sitagliptin synthesis, the researchers generated a structural
homology model of this transaminase and found that the enzyme would not bind to the prositagliptin ketone because of steric
interference and potentially undesired interactions. The evolved transaminase was a successful biocatalyst that synthesised
the chiral amines that previously were accessible only through resolution (5–8).
Codexis and Merck were recognized in 2010 with an Environmental Protection Agency's Presidential Green Chemistry Challenge
Award, an annual recognition of advances in green chemistry. Codexis also submitted for consideration in 2010 and 2011 a biocatalytic
route for making simvastatin, the active ingredient in Merck & Co.'s anticholesterol drug Zocor, which is now off patent (5,
8, 9).
For the simvastatin route, Codexis licensed technology from Yi Tang, professor in the Department of Chemical and Biomolecular
Engineering at the University of California at Los Angeles (US). The previous synthetic routes to simvastatin involved converting
lovastatin into simvastatin by adding a methyl group that required protecting and then deprotecting other functionalities
in the lovastatin molecule in a multistep synthesis. In the first route, lovastatin was hydrolysed to the triol, monacolin
J, followed by protection with selective silylation, esterification with dimethyl butyryl chloride and deprotection. The second
route involved protecting the carboxylic acid and alcohol functionalities, methylating the C2´ carbon with methyl iodide,
and deprotecting the product. These routes were inefficient because they produced less than 70% overall yield and were mass-intensive
due to protection and deprotection (5, 8).
The route developed by Tang and his group circumvented protection and deprotection and resulted in greater atom economy, reduced
waste and overall less hazardous reaction conditions. First, they cloned LovD, a natural acyltransferase produced by Aspergillus terreus that is involved in synthesizing lovastatin and that can accept nonnatural acyl donors. Recognising that LovD might be a
type of simvastatin synthase and a starting point for creating a new biocatalytic process, they evolved the enzyme toward
commercial utility (5, 8–10). Codexis licensed Tang's technology, engineered the enzyme further and optimised the process
for pilot-scale simvastatin manufacture.
Codexis is an example of a firm specialising in biocatalysis. Another is evocatal GmbH, which was founded in 2006 as a spin-off
of the Institute for Molecular Enzyme Technology at the University of Düsseldorf (Germany) in the Research Center Jülich.
Last November, evocatal issued a carbon–carbon coupling kit using thiamine pyrophosphate-dependent (TPP) enzymes to produce
enantiopure 2-hydroxy-ketones, an important intermediate class for pharmaceutical syntheses. The kit includes seven different
TPP-enzymes with the relevant cofactor.
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