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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

Hardegger, Leo and Beney, Pascal and Bixel, Dominique and Fleury, Christian and Gao, Feng and Grand-Guillaume-Perrenoud, Alexandre and Gu, Wayne and Haber, Julien and Hong, Tao and Humair, Roger and Kaegi, Andreas and Kibiger, Michael and Kleinbeck-Riniker, Florian and Luu, Van Tong and Padeste, Lukas and Rampf, Florian and Ruch, Thomas and Schlama, Thierry and Sidler, Eric and Udvarhelyi, Aniko and Wietfeld, Bernhard and Yang, Yao (2020) 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, 24 (9). pp. 1763-1771. ISSN 1083-61601520-586X

Abstract

A process using engineered phenylalanine ammonia lyase (PAL) enzymes was developed as part of an alternative route to a key intermediate of olodanrigan (EMA401). In the first part of the manuscript, the detailed results from a screening for the optimal reaction conditions are presented, followed by the discussion of several work-up 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. Based on our screenings, carbonate/carbamate buffered ammonia at 9–10 M NH3 concentration and pH 9.5–10.5 were found as the optimal conditions. Enzyme loadings down to 2.5wt% (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 and achieving maximum conversion. This temperature gradient also allowed balancing enzyme stability (low at 45 °C, high at 20 °C) with 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 work-up operations investigated, a sequence consisting of denaturation of the enzyme, followed by 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.

Item Type: Article
Keywords: olodanrigan, phenylalanine ammonia lyase, scale-up, process development, biocatalysis, phenylalanine derivative
Date Deposited: 10 Oct 2020 00:45
Last Modified: 10 Oct 2020 00:45
URI: https://oak.novartis.com/id/eprint/42582

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