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.

Natural product libraries with a remarkable range of biological activities play pivotal roles in drug discoveries due to their extraordinary structural complexity and immense diversity. l-Kynurenine (l-Kyn)-based derivatives are privileged pharmacophores that exhibit diverse therapeutic implications in neurological disorders. However, the difficulty in obtaining l-Kyn analogues with different skeletal structures has recently led to a decline in its medicinal research. Herein, we report a two-step, one-pot protocol for diversity-oriented biosynthesis of a collection of previously intractable l-Kyn-like compounds. The success of these challenging transformations mainly depends on unlocking the new catalytic scope of tryptophan 2,3-dioxygenases, followed by rational site-directed mutagenesis to modify the substrate domains further. As a result, 18 kynurenine analogues with diverse molecular scaffolds can be rapidly assembled in a predictable manner with 20–83% isolated yields, which not only fill the voids of the catalytic profile of tryptophan 2,3-dioxygenases with an array of substituent groups (e.g., F, Cl, Br, I, CH3, OCH3, and NO2) but also update the current understanding of its substrate spectrum. Our work highlights the great potential of existing enzymes in addressing long-standing synthetic challenges for facilitating the development or discovery of new drug candidates. Furthermore, our approach enables translating the reaction parameters from Eppendorf tubes to 1 L scale, affording l-4-Cl-Kyn and l-5-Cl-Kyn both on a gram scale with more than 80% isolated yields, and provides a promising alternative to further industrial applications.