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

Abstract

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 NH 3 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, NH 3 /CO 2 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.

Robust, site-specifically immobilized phenylalanine ammonia-lyases for the enantioselective ammonia addition of cinnamic acids

“… 26 Despite the increased attention for the industrial applicability of PAL-based procedures, no other studies reported immobilized PAL preparations with high operational stability in ammonia addition, while an attempt described within the PAL process development for EMA401 (ref. 7 ) has supported the existence of such interests. Accordingly, using SWCNT-based supports, the best performing in our previous studies, a site-oriented immobilization procedure was established using a maleimide moiety containing linker that was attached directly to amino-functionalized carbon nanotubes (SWCNT NH 2 ) or to carboxy-functionalized carbon nanotubes (SWCNT COOH ), priorly activated with carbonyldiimidazole and functionalized with 1,3-diaminopropane ( Fig.…”

“… 4 The PAL-based ammonia addition reaction of cinnamic acid derivatives has multiple advantages over other synthetic methods, such as a 100% atom economy, high enantioselectivity, low-cost, easily accessible substrates, and no requirement for a cofactor or its regeneration. 4,5 PALs are already used in established industrial processes, such as the multi-ton scale production of ( S )-2,3-dihydro-1 H -indole-2-carboxylic acid by DSM 6 or of (3 S )-5-(benzyloxy)-2-(diphenylacetyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (EMA401) by Novartis 7 ( Fig. 1 ).…”

Perspectives and Opportunities in Medicinal Chemistry: A View from the Novartis Institute for BioMedical Research Institute in Basel, Switzerland

“…The use of such reagents was however historically limited by the biotransformations achieved by natural enzymes, and by their substrate specificity. Recently, the engineering of novel enzymes optimized for specific reactions [10] enabled the extension of their application range, including unconventional biotransformations such as stereo-controlled halogenations [11] (Fig. 2).…”

Toward a Scalable Synthesis and Process for EMA401, Part I: Late Stage Process Development, Route Scouting, and ICH M7 Assessment

“…Out of the routes described in this publication, the asymmetric hydrogenation as published by Burk et al was the preferred option for early phase deliveries, while the biocatalytic approach using phenylalanine ammonia lyase (PAL) described in part III of this series of manuscripts would have been the preferred option for late phase clinical trials. 21 For the theoretical impurity BCME, potentially formed in the penultimate bond forming step, we analyzed its formation based on literature and compared the results from spiking experiments with the purge factor obtained by applying ICH M7 control option 4. Our results for both strategies showed that BCME was purged below levels of toxicological concern in the drug substance.…”

“…These findings are disclosed in parts II and III of this publication series. 20,21 Investigations on the Theoretical Impurity Bis-(chloromethyl) ether. Bis(chloromethyl) ether (BCME; CAS #542-888-1) is a highly toxic compound and known mutagen, classified as an ICH M7 class 1 impurity.…”