Toward a Scalable Synthesis and Process for EMA401, Part III: Using an Engineered Phenylalanine Ammonia Lyase Enzyme to Synthesize a Non-natural Phenylalanine Derivative,Organic Process Research & Development

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Toward a Scalable Synthesis and Process for EMA401, Part III: Using an Engineered Phenylalanine Ammonia Lyase Enzyme to Synthesize a Non-natural Phenylalanine Derivative
Organic Process Research & Development ( IF 3.1 ) Pub Date : 2020-08-20 , DOI: 10.1021/acs.oprd.0c00217
Leo A. Hardegger ₁ , Pascal Beney ₁ , Dominique Bixel ₁ , Christian Fleury ₁ , Feng Gao ₂ , Alexandre Grand-Guillaume Perrenoud ₁ , Xingxian Gu ₂ , Julien Haber ₁ , Tao Hong ₂ , Roger Humair ₁ , Andreas Kaegi ₁ , Michael Kibiger ₁ , Florian Kleinbeck ₁ , Van Tong Luu ₁ , Lukas Padeste ₁ , Florian A. Rampf ₁ , Thomas Ruch ₁ , Thierry Schlama ₁ , Eric Sidler ₁ , Anikó Udvarhelyi ₃ , Bernhard Wietfeld ₁ , Yao Yang ₂

Affiliation

A process using an engineered phenylalanine ammonia lyase (PAL) enzyme was developed as part of an alternative route to a key intermediate of olodanrigan (EMA401). In the first part of this report, the detailed results from a screening for the optimal reaction conditions are presented, followed by a discussion of several workup strategies investigated. In the PAL-catalyzed reaction, 70–80% conversion of a cinnamic acid derivative to the corresponding phenylalanine derivative could be achieved. The phenylalanine derivative was subsequently telescoped to a Pictet–Spengler reaction with formaldehyde, and the corresponding tetrahydroisoquinoline derivative was isolated in 60–70% yield with >99.9:0.1 er. On the basis of our screenings, carbonate/carbamate-buffered ammonia at an NH₃ concentration of 9–10 M and pH 9.5–10.5 was found to be optimal. Enzyme loadings down to 2.5 wt % (E:S = 1:40 w/w) could be achieved, and substrate concentrations between 3–9 v/w (1.17–0.39 M) were found to be compatible with the reaction conditions. A temperature gradient was applied in the final process: a pre-equilibrium was established at 45 °C, before making use of the temperature dependence of the entropy term with subsequent cooling to 20 °C to achieve maximum conversion. This temperature gradient also allowed balancing of the enzyme stability (low at 45 °C, high at 20 °C) with the activity (high at 45 °C, low at 20 °C) in order to achieve optimal conversion (low at 45 °C, high at 20 °C). From the various workup operations investigated, a sequence consisting of denaturation of the enzyme, NH₃/CO₂ removal by distillation, acidification, and telescoping to the subsequent Pictet–Spengler cyclization was our preferred approach. The process presented in this study is a more sustainable, shorter, and more cost-effective alternative to the previous process.

中文翻译：

迈向EMA401的可扩展合成和方法，第三部分：使用工程化的苯丙氨酸氨分解酶合成非天然的苯丙氨酸衍生物

开发了一种使用工程化的苯丙氨酸氨裂合酶（PAL）酶的方法，作为通往olodanrigan（EMA401）关键中间体的替代途径的一部分。在本报告的第一部分中，介绍了筛选最佳反应条件的详细结果，然后讨论了所研究的几种后处理策略。在PAL催化的反应中，肉桂酸衍生物向相应的苯丙氨酸衍生物的转化率可达到70-80％。随后将苯丙氨酸衍生物与甲醛进行伸缩反应，以进行Pictet-Spengler反应，并分离出相应的四氢异喹啉衍生物，收率60-70％，> 99.9：0.1 er。根据我们的筛选，碳酸盐/氨基甲酸酯缓冲的氨为NH ₃发现最佳浓度为9-10 M，pH值为9.5-10.5。可以实现低至2.5 wt％的酶负载（E：S = 1:40 w / w），并且发现3–9 v / w（1.17–0.39 M）之间的底物浓度与反应条件兼容。在最后的过程中应用了温度梯度：在利用熵项的温度依赖性并随后冷却至20°C以实现最大转化率之前，在45°C时建立了预平衡。该温度梯度还可以平衡酶的稳定性（在45°C时较低，在20°C时较高）与活性（在45°C时较高，在20°C时较低）之间保持平衡，以实现最佳转化率（在45°C时较低） C，最高20°C）。从所研究的各种后处理操作中，发现了一个由变性酶NH ₃ / CO组成的序列₂通过蒸馏，酸化和伸缩进行随后的Pictet-Spengler环化反应的去除是我们的首选方法。本研究中提出的过程是以前过程的更可持续，更短且更具成本效益的替代方法。

更新日期：2020-09-20

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