Hyphenated chromatographic system (AhPNP-IMER-LC) for the enzymatic flow synthesis of nucleoside analogues: on-line reaction monitoring and product purification

Traditionally bidimensional liquid chromatography is a powerful tool that uses a combination of several chromatography techniques, separation modes, and columns to separate multiple components. The goal of this research is to apply the flow-chemistry concept to produce an innovative bioreactor which uses a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) for the on-line monitoring, production and purification of modified nucleosides with transglycosylation reaction (Scheme 1). Therefore, one of the two dimension is composed of an Immobilized Enzyme Reactor (IMER) based on AhPNP (25 mg, 50% yield) realized immobilizing, via Schiff base chemistry, the enzyme in a 50x4.6 mm pre-packed stainless steel column containing aminopropylsilica particles (5 µm, 100 Å). AhPNP has been investigated and used by our group in several bioconversions.[1,2] The AhPNP-IMER has been be interfaced through a six way switching valve to an analytical column, for the control of the reaction progress, or to a semi-preparative column, for the separation and isolation of the pure reaction product. Coupling of transglycosylation reaction and product separation resulted in a fast and efficient process (yield=52-89%) where sample handling was minimized. To date, AhPNP-IMER has retained completely its activity upon 50 reactions and 10 months. [1] D.H. Benninger, Parkinson‘s disease, Handb. Clin. Neurol. 116 (2013) 469–483. [2] P.G. Coune, M. Craveiro, M.N. Gaugler, V. Mlynárik, B.L. Schneider, P. Aebischer, R. Gruetter, An in vivo ultrahigh field 14.1 T 1H-MRS study on 6-OHDA and α-synuclein-based rat models of Parkinson's disease: GABA as an early disease marker, NMR Biomed. 26 (2013) 43–50.