Directed evolution of enantioselective enzymes as catalysts in the production of chiral pharmaceuticals

Abstract

Publisher Summary This chapter discusses the directed evolution of enantioselective enzymes as catalysts in the production of chiral pharmaceuticals. A number of industrial processes for the enantioselective synthesis of intermediates needed in the production of chiral pharmaceuticals are based on the application of enzymes. A prominent example is the lipase-catalyzed kinetic resolution of an epoxy-ester used in the production of the anti-hypertensive therapeutic Dilthiazem. The chapter proposes a radically different approach to the development of enantioselective catalysts, which does not rely on any knowledge regarding the structure of the enzyme or on any speculations concerning enzyme mechanism. The combination of proper molecular biological methods for random mutagenesis and gene expression coupled with high-throughput screening systems for the rapid identification of enantioselective variants of the natural (wild-type) enzyme forms the basis of the concept. The idea is to start with a wild-type enzyme showing an unacceptably low enantiomeric excess ( ee ) or selectivity factor ( E ) value for a given transformation of interest, to create a library of mutant genes, to identify the most enantioselective enzyme-variant following expression, and to repeat the process as often as necessary using in each case, an improved mutant gene for the next round of mutagenesis. Because the inferior mutant genes and enzyme variants are discarded, an evolutionary “pressure” in the overall process builds up.