Efficient chemo-enzymatic route for enantiopure S-Pregabalin by desymmetrization of 3-isobutyl glutarimide using an evolved D-hydantoinase
Advancements in enzyme discovery and protein engineering have accelerated development of green, efficient synthetic routes to complex molecules and broadened implementation of biocatalysis for scalable manufacture of pharmaceutical intermediates. (S)-3-isobutyl-γ-aminobutyric acid (S-Pregabalin API), is a generic drug for the treatment of epilepsy and neuralgia. It has been previously synthesized via kinetic resolution and re-racemization under harsh conditions, which is inefficient and not eco-friendly. Herein, we describe the development of an enantioselective desymmetrization of 3-isobutylglutarimid utilizing an evolved D-hydantoinase to produce the Pregabalin Intermediate (IM) (R)-3-isobutyl glutaric acid monoamide en route to S-Pregabalin. Three rounds of computer-guided directed evolution were implemented towards improved activity, enantioselectivity and reduced substrate/product inhibition, providing a final variant that enables the full conversion of 240 g/L substrate into Pregabalin IM with excellent enantiomeric excess (ee) of >99.5%. The promising results demonstrated an economical and sustainable synthesis of S-Pregabalin starting from a prochiral substrate using an evolved D-hydantoinase.
Biocatalysis has become an enabling synthetic technology due to its potential to provide exquisite selectivity, evolvability, and inherent sustainability. Breakthroughs in bioinformatics, molecular biology, protein engineering and adjacent technology fields have enabled fast and reliable discovery and engineering of enzymes (1, 2) tailored towards specific process needs. As a result, an exploding number of new enzyme classes as well as chemoenzymatic processes with increasing sophistication have made their way to large-scale manufacture of chemicals and pharmaceuticals. An outstanding example is the production of (S)-3-isobutyl-γ-aminobutyric acid (S-Pregabalin, branded as Lyrica® by Pfizer), which has been a popular synthetic target for the biocatalysis community (3) since its launch in 2004. Pregabalin is a lipophilic γ-aminobutyric acid (GABA) analogue for the treatment of several nervous system disorders including epilepsy, neuralgia, fibromyalgia and anxiety disorders. The first commercial synthesis featured a classic resolution of racemic Pregabalin using (S)-mandelic acid in a three-step crystallization process (4), in which around ...