Org. Process Res. Dev. 2020, 24, 1763. DOI: 10.1021/acs.oprd.0c00217
Distillation of NH3/CO2
◆Experiments had shown that PAL-130 retained activity after addition of 20 vol % MeOH, and 50 vol % MeOH was required to ensure full denaturation of the enzyme
◆The suspension was particularly thick around pH 5.9, corresponding to the isoelectric point of amino acid 3, and pH 4.2, corresponding to the pKa of cinnamic acid 2
◆At lower pH values, 3 started to dissolve again, while the enzymeand substrate 2 were suspended and removed via clear filtration.
Quench with Acids
◆Very high or very low pH is known to denaturate many enzymes. In the PAL-catalyzed hydroamination reaction, the addition of NaOH led to the formation of byproducts and was thus abandoned.
◆The quench was strongly exothermic, heat generation and CO2 release occurred in a dose-controlled manner.
Extractions from Ammonia Buffer
◆As the quench with acids did not allow for the simple recycling of ammonia, we also investigated an approach relying on extraction of the basic ammonia buffer, potentially even providing a handle to further drive the equilibrium between cinnamic acid 2 and amino acid 3.
◆Using nBuOH/xylenes led to the coextraction of significant amounts of the aqueous ammonia buffer into the organic layer, and titrations indicated that the concentration of the aqueous ammonia buffer dropped to only 3.4 M NH3 when starting from 10 M NH3. Hence, recycling of ammonia proved to be difficult with this workup strategy.
Tangential Flow Filtration of the Enzyme
◆In parallel to the traditional workup strategies presented in this study, implementation of an ultrafiltration was investigated to separate the enzyme from the small molecules 2 and 3, taking advantage of the significant size difference between the enzyme (61.5 kDa per subunit of the tetramer) and the product and residual substrate.
◆While the process was technically robust, the main challenge was the short half-life of the enzyme. The positive economic impact of the enzyme recycling strategy would substantially benefit from an enzyme evolution directed toward enhanced stability
Telescope to Tetrahydroisoquinoline 4
◆The preferred workup options for the PAL step resulted in an aqueous acidic solution of amino acid 3 that was telescoped into the next step.
◆A sequence consisting of denaturation of the enzyme, NH3/CO2 removalby distillation, acidification, and telescoping to the subsequent Pictet−Spengler cyclization was developed.
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