Nitrilases
Nitrilases (E.C. 3.5.5.1) promote the mild hydrolytic conversion of organonitriles directly to the corresponding carboxylic acids.19 However, less than 20 microbially derived nitrilases had been characterized at the start of this work, despite their potential synthetic value. The paucity of enzymes and the limited substrate scope of the handful of enzymes available have limited practical commercial development of nitrilase-catalyzed conversions, except in a few cases. Accordingly, we engaged in a discovery effort centered around exploiting the natural diversity available within our environmental DNA libraries and have discovered and characterized more than 200 new sequence unique nitrilases.20 All of the newly discovered nitrilases possess the conserved catalytic triad Glu-Lys-Cys that is characteristic for this enzyme class.19
The nitrilase collection was evaluated for the production of a-hydroxy acids formed through hydrolysis of cyanohydrins. Since cyanohydrins racemize readily under basic conditions,21 a dynamic kinetic resolution (DKR) process is possible, permitting access to a-hydroxy acids in 100% theoretical yield. One important application of this type involves commercial production of (R)-mandelic acid from mandelonitrile.22,23 Mandelic acid and derivatives find broad use as intermediates and resolving agents for production of many pharmaceutical and agricultural products.24 The nitrilase library was screened for activity and enantioselectivity in the hydrolysis of mande-lonitrile to mandelic acid (Scheme 20.1) and several enzymes afforded mandelic acid with >90% ee. One enzyme (nitrilase I) generated (R)-mandelic acid quantitatively with 98% ee. The broad substrate scope of this enzyme was demonstrated on a range of mandelic acid derivatives as well as aromatic and heteroaromatic analogues (Table 20.1). Nitrilase I was effectively applied to a series of ortho-, meta-, and para-substituted mandelonitrile derivatives, and products generally were produced with very high enantioselectivities. Other larger aromatic groups, such as 1-naphthyl and 2-naphthyl, as well as hetero aromatic 3-pyridyl and 3-thienyl analogues also were accommodated within the active site, and hydrolysis with nitrilase I yielded glycolic acid products with high ee. This was the first reported demonstration of a nitrilase that affords such a broad range of mandelic acid derivatives and heteroaromatic analogues with high levels of enantioselectivity. It is noteworthy that for several of these processes the reactions were run in such a manner that product concentrations of up to 2M were achieved.
Average user rating: 5 stars out of 1 votes
Post a comment