Hardegger, Leo, Beney, Pascal, Bixel, Dominique, Fleury, Christian, Gao, Feng, Grand-Guillaume-Perrenoud, Alexandre, Gu, Wayne, Haber, Julien, Hong, Tao, Humair, Roger, Kaegi, Andreas, Kibiger, Michael, Kleinbeck-Riniker, Florian, Luu, Van Tong, Padeste, Lukas, Rampf, Florian, Ruch, Thomas, Schlama, Thierry, Sidler, Eric, Udvarhelyi, Aniko, 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