Magnetic Multi-Enzymatic System for Cladribine Manufacturing

Abstract

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/g_support, 63% retained activity, operational conditions: 40 °C and pH 5-7) and MEcHPRT3 (12,840 IU/g_support, 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.

Keywords: cascade synthesis; enzyme immobilization; magnetic catalysts; nucleoside analogues; transglycosylation reaction.

Figure 1

Enzymatic synthesis of Cladribine catalyzed by: (A) LmPDT. (B) LmPDT/EcHPRT. dIno: 2′-deoxyinosine; 2-ClAde: 2-chloroadenine; Hyp: hypoxanthine; PRPP: 5-phospho-α-D-ribosyl-1-pyrophosphate; LmPDT: Leishmania mexicana purine 2′-deoxyribosyltransferase; EcHPRT; Escherichia coli hypoxanthine phosphoribosyltransferase.

Figure 2

Biochemical characterization of MLmPDT3 and MEcHPRT3. (A) Effect of temperature on MLmPDT3 activity (●). (B) Effect of pH on MLmPDT3 activity, (●) 50 mM sodium citrate (pH 4–6), (△) 50 mM sodium phosphate (pH 6–8), (○) 50 mM MES (pH 6–7), (■) 50 mM Tris-HCl (pH 7–9), (□) 50 mM sodium borate (pH 8–10). (C) Effect of temperature MEcHPRT3 activity (●). (D) Effect of pH on MEcHPRT3 activity, (●) 12 mM sodium citrate (pH 4–6), (△) 12 mM sodium phosphate (pH 6–8), (○) 12 mM MES (pH 6–7), (■) 12 mM Tris-HCl (pH 7–9), (□) 12 mM sodium borate (pH 8–10).

Figure 3

Biochemical characterization of the MLmPDT3/MEcHPRT3 system. (A) Effect of temperature on cladribine synthesis (●). (B) Effect of pH on cladribine synthesis, (●) 50 mM sodium citrate (pH 4–6), (△) 50 mM sodium phosphate (pH 6–8), (○) 50 mM MES (pH 6–7), (■) 50 mM Tris-HCl (pH 7–9), (□) 50 mM sodium borate (pH 8–10).

Figure 4

Thermal inactivation profile of MLmPDT3/MEcHPRT3 on cladribine synthesis at 40 °C (●) and 50 °C (○).

Figure 5

Effect of MLmPDT3/MEcHPRT3 ratio (μg of immobilized LmPDT/μg of immobilized EcHPRT) in the product formation. (A) cladribine (reaction 1). (B) Hyp converted into IMP (reaction 2).

Figure 6

Reusability of the MLmPDT3/MEcHPRT3 system. 2′-deoxyribosyltransferase activity, reaction 1 (black bar); phosphoribosyltransferase activity, reaction 2 (white bar).