Magnetic Multi-Enzymatic System for Cladribine Manufacturing.

Enzyme-mediated processes have proven to be a valuable and sustainable alternative to traditional chemical methods. In this regard, the use of multi-enzymatic systems enables the realization of complex synthetic schemes, while also introducing a number of additional advantages, including the conversion of reversible reactions into irreversible processes, the partial or complete elimination of product inhibition problems, and the minimization of undesirable by-products. In addition, the immobilization of biocatalysts on magnetic supports allows for easy reusability and streamlines the downstream process. Herein we have developed a cascade system for cladribine synthesis based on the sequential action of two magnetic biocatalysts. For that purpose, purine 2'-deoxyribosyltransferase from (PDT) and hypoxanthine phosphoribosyltransferase (HPRT) were immobilized onto Ni-prechelated magnetic microspheres (MagReSynNTA). Among the resulting derivatives, MPDT3 (activity: 11,935 IU/g, 63% retained activity, operational conditions: 40 °C and pH 5-7) and MHPRT3 (12,840 IU/g, 45% retained activity, operational conditions: pH 5-8 and 40-60 °C) emerge as optimal catalysts for further synthetic application. Moreover, the MPDT3/MHPRT3 system was biochemically characterized and successfully applied to the one-pot synthesis of cladribine under various conditions. This methodology not only displayed a 1.67-fold improvement in cladribine synthesis (compared to MPDT3), but it also implied a practically complete transformation of the undesired by-product into a high-added-value product (90% conversion of Hyp into IMP). Finally, MPDT3/MHPRT3 was reused for 16 cycles, which displayed a 75% retained activity.