Sustainable Flow‐Synthesis of (Bulky) Nucleoside Drugs by a Novel and Highly Stable Nucleoside Phosphorylase Immobilized on Reusable Supports

The continuous synthesis of valuable nucleoside drugs was achieved in up to 99 % conversion by using a novel halotolerant purine nucleoside phosphorylase from Halomonas elongata (HePNP). HePNP showed an unprecedented tolerance to DMSO, usually required for substrate solubility, and could be immobilized on agarose microbeads through disulfide bonds, via a genetically fused Cystag. This covalent yet reversible binding chemistry showcased the reusability of agarose microbeads in a second round of enzyme immobilization with high reproducibility, reducing waste and increasing the sustainability of the process. Finally, the flow synthesis of a Nelarabine analogue (6‐O‐methyl guanosine) was optimized to full conversion on a 10 mm scale within 2 min residence time, obtaining the highest space‐time yield (89 g L−1 h−1) reported to date. The cost‐efficiency of the system was further enhanced by a catch‐and‐release strategy that allowed to recover and recirculate the excess of sugar donor from the downstream water waste. Nucleoside synthesis: A novel purine nucleoside phosphorylase (HePNP) from Halomonas elongata is characterized, showing a high stability towards co‐solvents. HePNP is immobilized by reversible covalent bonds, allowing the reuse of the support for another immobilization cycle once HePNP becomes inactive. The immobilized HePNP is integrated into a flow reactor for the synthesis of (bulky) nucleoside drugs, also enabling the separation and recycling of the sugar donor excess.