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Magnetic multi-enzymatic system for Cladribine manufacturing Cruz, Guillermo Saiz, Laura Pilar Bilal, Muhammad Eltoukhy, Lobna Loderer, Christoph Fernández Lucas, Jesús 577.15 577.2 60 661.12 Cascade synthesis Magnetic catalysts Enzyme immobilization Transglycosylation reaction Nucleoside analogues Bioquímica (Biología) Biología molecular (Biología) Biotecnología Medicamentos 2403 Bioquímica 2415 Biología Molecular 2302.09 Enzimología 3302 Tecnología Bioquímica 3209.01 Análisis de Medicamentos 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 Leishmania mexicana (LmPDT) and Escherichia coli hypoxanthine phosphoribosyltransferase (EcHPRT) were immobilized onto Ni2+-prechelated magnetic microspheres (MagReSyn®NTA). Among the resulting derivatives, MLmPDT3 (activity: 11,935 IU/gsupport, 63% retained activity, operational conditions: 40 °C and pH 5–7) and MEcHPRT3 (12,840 IU/gsupport, 45% retained activity, operational conditions: pH 5–8 and 40–60 °C) emerge as optimal catalysts for further synthetic application. Moreover, the MLmPDT3/MEcHPRT3 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 MLmPDT3), 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, MLmPDT3/MEcHPRT3 was reused for 16 cycles, which displayed a 75% retained activity. Ministerio de Ciencia e Innovación (España) National Science Centre Deutsche Forschungsgemeinschaft Depto. de Bioquímica y Biología Molecular Fac. de Ciencias Biológicas TRUE pub 2024-10-10T15:27:13Z 2024-10-10T15:27:13Z 2022-11 journal article VoR https://hdl.handle.net/20.500.14352/108867 1422-0067 10.3390/ijms232113634 eng PID2020-117025RB-I00 info:eu-repo/grantAgreement/NCN//2020%2F37%2FK%2FST8%2F03805 info:eu-repo/grantAgreement/DFG//LO 2678%2F2-1 Cruz, Guillermo, et al. «Magnetic Multi-Enzymatic System for Cladribine Manufacturing». International Journal of Molecular Sciences, vol. 23, n.o 21, noviembre de 2022, p. 13634. DOI.org (Crossref), https://doi.org/10.3390/ijms232113634. Attribution 4.0 International http://creativecommons.org/licenses/by/4.0/ open access application/pdf MDPI