Rational Design of a De Novo 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.

Diversity-Oriented Biosynthesis Yields l-Kynurenine Derivative-Based Neurological Drug Candidate Collection

“…Enzyme Repositioning for Maximum Unlocking of New Catalytic Potential of Wild-Type Tryptophan 2,3-Dioxygenase (WT TDO): Old Path, New Frontier. As stated earlier, despite having developed an efficient and economical way to synthesize L-halokynurenine, 22 many potentially useful products, including L-Kyn analogues substituted at 3-, 5-, and 6-positions, are still challenging to obtain [Figure 1C(3)]. The limited product diversity is mainly due to the high specificity of tryptophan 2,3-dioxygenase (Tar13) used in this protocol, although Tar13 is the only tryptophan 2,3-dioxygenase reported so far that shows selectivity for chlorinated substrates.…”

“…l -Kyn derivatives are a family of nonproteinogenic amino acids that exhibit diverse therapeutic properties in neurological disorders (Figure A). − In addition, its non-natural analogue l -4-Cl-Kyn was recently identified as an amino acid building block in the lipopeptide antibiotics taromycin A and B (Figure B). , Because of their intriguing biological activities, a myriad of strategies have been employed to synthesize this pharmaceutical family. Although synthetic chemists and biologists have made significant progress in this field, there are still no productive methods known for industrialization.…”

Biocatalytic Friedel‐Crafts Reactions

“…Serine‐isotopologs can also be accepted by Pf TrpB 2B9 , which can be conveniently produced from deuterated formaldehyde and/or 13 C‐labelled glycine by L‐threonine aldolase to give tryptophan isotopologs with up to 4 isotope substitutions, on an analytical scale, augmenting previously described methods [32] . Pf TrpB 0A9 was applied in a cascade together with a ring‐opening tryptophan 2,3‐dioxygenase, Tar13, to produce chlorokynurenine and bromokynurenine, the former molecule being a current antidepressant drug candidate [33] . Co‐immobilisation of these two enzymes gave a heterogeneous catalyst capable of producing hundreds of milligrams of the products, but activity was rapidly lost upon catalyst‐recycling.…”

“…[32] Pf TrpB 0A9 was applied in a cascade together with a ring‐opening tryptophan 2,3‐dioxygenase, Tar13, to produce chlorokynurenine and bromokynurenine, the former molecule being a current antidepressant drug candidate. [33] Co‐immobilisation of these two enzymes gave a heterogeneous catalyst capable of producing hundreds of milligrams of the products, but activity was rapidly lost upon catalyst‐recycling. The same study demonstrated the antidepressant properties of both chlorokynurenine and bromokynurenine in mice.…”

Sustainable and Robust Closed-Loop Enzymatic Platform for Continuous/Semi-continuous Synthesis of Ursodeoxycholic Acid

“…The use of enzymes as catalysts for biosynthesis of chemical compounds is popular in the manufacturing of pharmaceuticals and active pharmaceutical ingredients. − Generally, biocatalysts are appealing for pharmaceutical manufacturing because their high enantio-, regio- and chemoselectivities reduce the generation of undesired side products. − Furthermore, biocatalysts are usually used under mild and environmentally friendly conditions, which means that enzymatic biotransformations are more sustainable compared with conventional chemical syntheses. , Nevertheless, the application of enzymatic systems can be limited by low reaction efficiencies, enzyme instability, and tedious optimization and scale-up processes. , …”