Rational Design of a <i>De Novo</i> Enzyme Cascade for Scalable Continuous Production of Antidepressant Prodrugs

Abstract

Natural products and their derivatives provide a promising source for drug discoveries. However, several natural products and derivatives are still difficult to advance to in vivo drug testing due to the requirement of an ample supply of drug candidates. Herein, we describe an immobilized enzyme cascade for the scalable continuous production of neuropharmaceutical prodrugs, L-4-chlorokynurenine (L-4-Cl-Kyn) and its non-natural analogue L-4-Br-Kyn. This synthetic route features a highly efficient one-pot C–H activation/oxidation/hydrolyzation cascade for the assembly of the Kyn core structure, inexpensive substituted aryl sources for the abundance of Kyn scaffold types, and easily available gram-scale MOF–hydrogel hybrid material for the immobilization of enzymes to recycle biocatalysts and maximize their catalytic efficiency. As a result, our method accumulates 370 mg of L-4-Cl-Kyn and 365 mg of L-4-Br-Kyn, 250 mL product volume per cycle for 5 cycles, which is nearly twice the yield of free enzymatic catalysis in the same enzyme amount frame. Therefore, this study establishes the applicability of synthetic biology strategies and immobilization biotechnology in synthesizing high value-added chemicals and narrows the gap in moving cell-free immobilized enzyme cascade systems from academic studies to industrial applications. Moreover, animal experiments demonstrate that L-4-Br-Kyn displays a slightly better antidepressant effect than the phase II drug L-4-Cl-Kyn, which further emphasizes the importance of our work for the facile scale-up synthesis to provide large amounts of pharmaceutical products in facilitating the development or discovery of drug candidates. These practical features of our study will undoubtedly be welcomed by academic and industrial researchers.

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