99790-49-5Relevant academic research and scientific papers
Flow-Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila: Integrated Systems of Reaction Control and Product Purification
Calleri, Enrica,Cattaneo, Giulia,Rabuffetti, Marco,Serra, Immacolata,Bavaro, Teodora,Massolini, Gabriella,Speranza, Giovanna,Ubiali, Daniela
, p. 2520 - 2528 (2015)
A purine nucleoside phosphorylase from Aeromonas hydrophyla (AhPNP) was covalently immobilized in a pre-packed stainless steel column containing aminopropylsilica particles via Schiff base chemistry upon glutaraldehyde activation. The resulting AhPNP-IMER (Immobilized Enzyme Reactor, immobilization yield ≈50%) was coupled on-line through a 6-way switching valve to an HPLC apparatus containing an analytical or a semi-preparative chromatographic column. The synthesis of five 6-modified purine ribonucleosides was carried out by continuously pumping the reaction mixture through the AhPNP-IMER until the highest conversion was reached, and then directing the reaction mixture to chromatographic separation. The conditions of the AhPNP-catalyzed transglycosylations (2:1 ratio sugar donor:base acceptor; 10 mM phosphate buffer; pH 7.5; temperature 37 °C, flow rate 0.5 mL min-1) were optimized by a fractional factorial experimental design. Coupling the bioconversion step with the product purification in such an integrated platform resulted in a fast and efficient synthetic process (yield=52-89%; 10 mg) where sample handling was minimized. To date, AhPNP-IMER has retained completely its activity upon 50 reactions in 10 months.
A comparison between immobilized pyrimidine nucleoside phosphorylase from Bacillus subtilis and thymidine phosphorylase from Escherichia coli in the synthesis of 5-substituted pyrimidine 2′-deoxyribonucleosides
Serra, Immacolata,Bavaro, Teodora,Cecchini, Davide A.,Daly, Simona,Albertini, Alessandra M.,Terreni, Marco,Ubiali, Daniela
, p. 16 - 22 (2013/10/22)
Pyrimidine nucleoside phosphorylase from Bacillus subtilis (BsPyNP, E.C. 2.4.2.3) and thymidine phosphorylase from Escherichia coli (EcTP, E.C. 2.4.2.4) were used, as immobilized enzymes, in the synthesis of 5-halogenated pyrimidine 2′-deoxyribonucleosides (14-18) by transglycosylation in fully aqueous medium. From the comparative study of the two biocatalysts, no remarkable differences emerged about their substrate specificity, bioconversion yield, stability in organic cosolvents (DMF and MeCN). Moreover, both biocatalysts could be recycled for at least 5 times with no loss of the productivity. Both enzymes do not accept arabinonucleosides and 2′,3′- dideoxynucleosides as substrates, whereas they catalyze bioconversions involving 5′-deoxyribonucleosides and 5-halogenated uracils. The synthesis of compounds 14-18 proceeded at a similar conversion (33-68% for BsPyNP and 25-62% for EcTP, respectively). Immobilization was found to exert, for both the biocatalysts, a dramatic enhancement of stability upon incubation in MeCN. Optimization of 5-fluoro-2′-deoxyuridine (14) synthesis (pH 7.5, 10 mM phosphate buffer, nucleoside/nucleobase 3:1 molar ratio) and subsequent scale-up afforded the target compound in 73% (EcTP) or 76% (BsPyNP) conversion (about 9 g/L).
The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy
Renck, Daiana,Ducati, Rodrigo G.,Palma, Mario S.,Santos, Diogenes S.,Basso, Luiz A.
experimental part, p. 35 - 42 (2011/10/02)
Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate (R1P). The human UP type 1 (hUP1) is a molecular target for the design of inhibitors intended to boost endogenous uridine levels to rescue normal tissues from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents, such as capecitabine and 5-fluorouracil. Here, we describe a method to obtain homogeneous recombinant hUP1, and present initial velocity, product inhibition, and equilibrium binding data. These results suggest that hUP1 catalyzes uridine phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate binds first followed by the binding of uridine, and uracil dissociates first, followed by R1P release. Fluorescence titration at equilibrium showed cooperative binding of either Pi or R1P binding to hUP1. Amino acid residues involved in either catalysis or substrate binding were proposed based on pH-rate profiles.
