Engineering a Transaminase for the Efficient Synthesis of a Key Intermediate for Rimegepant
Yulei Ma, Xuecheng Jiao, Zujian Wang, Huiyan Mu, Kaihua Sun, Xiang Li, Tong Zhao, Xufeng Liu, Na Zhang
Abstract
Transaminases have been increasingly utilized as efficient biocatalysts in the synthesis of pharmaceutical intermediates, but a major drawback is their poor substrate acceptance, especially the limitation for the synthesis of sterically hindered chiral amines. Herein we report the engineering of a transaminase that can convert the ketone (6S,9R)-6-(2,3-difluorophenyl)-9-hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one to (5S,6S,9R)-5-amino-6-(2,3-difluorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-ol, a key intermediate for the synthesis of rimegepant, a CGRP antagonist for the treatment of migraine. Starting from an enzyme backbone with no detectable activity toward the desired ketone, a rational design approach enabled us to produce an enzyme variant with detectable trace activity. Then, by following various evolution strategies, including iterative saturation mutagenesis focused on a key loop and random mutagenesis of the whole sequence, further improvement of the activity was achieved. The resultant variant showed 99.0% conversion and >99.5% de for the desired reaction at the gram scale as well as at the kilogram scale to afford the product (5S,6S,9R)-5-amino-6-(2,3-difluorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9-ol in 80.2% yield with 99.9% HPLC purity, thus showcasing promising potential for industrial application.