- Flow-Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila: Integrated Systems of Reaction Control and Product Purification
-
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.
- Calleri, Enrica,Cattaneo, Giulia,Rabuffetti, Marco,Serra, Immacolata,Bavaro, Teodora,Massolini, Gabriella,Speranza, Giovanna,Ubiali, Daniela
-
p. 2520 - 2528
(2015/08/18)
-
- Purines. XXVII. Hydrolytic Deamination versus Dimroth Rearrangement in the 9-Substituted Adenine Ring: Effect of an ω-Hydroxyalkyl Group at the 1-Position
-
In H2O at near neutrality, the 1-(ω-hydroxyalkyl)adenine derivatives 8a, b*HBr and 8c*HClO4 underwent hydrolytic deamination to give the 1-(ω-hydroxyalkyl)hypoxanthine derivatives 10a-c, in competition with the usual Dimroth rearrangement to produce the N6-(ω-hydroxyalkyl)adenine derivatives 9a-c.The rates of these competitive reactions were measured in H2O at various pH's and ionic strenght 1.0 at 40 deg C, and the relative rate of deamination with respect to Dimroth rearrangement was found to increase as the pH of the reaction medium was decreased.Under similar conditions, the corresponding 1-alkyl analogues 8d-g*HClO4 and the 1-(modified benzyl) analogues 8h, i*HBr underwent only Dimroth rearrangement to afford the N6-isomers 9d-i.In the Dimroth rearrangements of all of the substrates 8a, b, d-i*HX (X=Br or ClO4), attack of hydroxide ion on the protonated species (8a, b, d-i*H+) at the 2-position was faster than that on the neutral species by a factor of 100-640.In the reaction of the protonated species, the 1-(ω-hydroxyalkyl)analogues 8a, b*HBr rearranged faster than the corresponding 1-alkyl analogues 8e, f*HClO4 by a factor of 1.6-2.7.It has been concluded that this rate enhancement is attributable solely to the electron-withdrawing effect, and not to intramolecular participation in catalysis, of the hydroxy group in the 1-substituent chain.In the syntheses of 8a, i*HBr from 9-ethyladenine (6) according to a general 1-alkylation procedure, the 7-alkylated products 13 and 16 were also obtained as by-products in 9percent and 6percent yields, respectively.Keywords - 1,9-disubstituted adenine; 9-substituted 1-(ω-hydroxyalkyl)adenine; Dimroth rearrangement; hydrolytic deamination; 9-substituted 1-(ω-hydroxyalkyl)hypoxanthine; kinetic study; acid dissociation constant; UV; 9-ethyladenine 1-alkylation; 9-ethyladenine 7-alkylation
- Fujii, Tozo,Saito, Tohru,Terahara, Norihiko
-
p. 1094 - 1107
(2007/10/02)
-
