Engineering a Transaminase for the Efficient Synthesis of a Key Intermediate for Rimegepant,Organic Process Research & Development

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Engineering a Transaminase for the Efficient Synthesis of a Key Intermediate for Rimegepant
Organic Process Research & Development ( IF 3.1 ) Pub Date : 2022-01-20 , DOI: 10.1021/acs.oprd.1c00376
Yulei Ma ₁ , Xuecheng Jiao ₁ , Zujian Wang ₁ , Huiyan Mu ₁ , Kaihua Sun ₁ , Xiang Li ₁ , Tong Zhao ₁ , Xufeng Liu ₁ , Na Zhang ₁

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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.

中文翻译：

设计用于高效合成 Rimegepant 关键中间体的转氨酶

转氨酶越来越多地被用作合成药物中间体的高效生物催化剂，但其主要缺点是其底物接受性差，尤其是限制了空间位阻手性胺的合成。在这里，我们报告了一种转氨酶的工程，它可以转化酮 (6 S ,9 R )-6-(2,3-difluorophenyl)-9-hydroxy-6,7,8,9-tetrahydro-5 H -cyclohepta[ b ]pyridin-5-one to (5 S ,6 S ,9 R )-5-amino-6-(2,3-difluorophenyl)-6,7,8,9-tetrahydro-5 H -cyclohepta[ b]pyridin-9-ol，一种合成 rimegepant 的关键中间体，一种 CGRP 拮抗剂，用于治疗偏头痛。从对所需酮没有可检测活性的酶骨架开始，合理的设计方法使我们能够生产具有可检测微量活性的酶变体。然后，通过遵循各种进化策略，包括以关键循环为重点的迭代饱和诱变和全序列的随机诱变，进一步提高了活性。所得到的变体显示出 99.0% 的转化率和 >99.5% 的转化率，用于克级和千克级的所需反应，得到产物 (5 S ,6 S ,9 R )-5-amino-6-(2 ,3-二氟苯基)-6,7,8,9-四氢-5 H-cyclohepta[ b ]pyridin-9-ol，产率80.2%，HPLC纯度99.9%，具有广阔的工业应用潜力。

更新日期：2022-01-20

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