A wide range of natural purine analogues was used as probe to assess the mechanism of recognition by the wild-type (WT) E. coli purine nucleoside phosphorylase (PNP) versus its Ser90Ala mutant. The results were analyzed from viewpoint of the role of the Ser90 residue and the structural features of the bases. It was found that the Ser90 residue of the PNP 1) plays an important role in the binding and activation of 8-aza-7-deazapurines in the synthesis of their nucleosides, 2) participates in the binding of α-D-pentofuranose-1-phosphates at the catalytic site of the PNP, and 3) catalyzes the dephosphorylation of intermediary formed 2-deoxy-α-D-ribofuranose-1-phosphate in the trans-2-deoxyribosylation reaction. 5-Aza-7-deazaguanine manifested excellent substrate activity for both enzymes, 8-amino-7-thiaguanine and 2-aminobenzothiazole showed no substrate activity for both enzymes. On the contrary, the 2-amino derivatives of benzimidazole and benzoxazole are substrates and are converted into the N1- and unusual N2-glycosides, respectively. 9-Deaza-5-iodoxanthine showed moderate inhibitory activity of the WT E. coli PNP, whereas 9-deazaxanthine and its 2'-deoxyriboside are weak inhibitors.
4.02 br. m 3.63 dd / 4.12 dd J 5,4 = 2.12 J 5',4 = 1.10 gem J 5,5' = 12.82 3.04 J P,H1 = 6.90 J P,H2 = 1.74 5.50 dd J 1,2 = 4.70 4.18 m J 2,3 = 3.40 3.93 m J 3,4 1.0 4.19 m 3.83 dd / 3.71 dd J 5,4 = 3.36 J 5',4 = 5.96 gem J 5,5' = 12.28 2.49 J P,H1 = 5.18 J P,H2 < 1.0 -D-Ribofuranose 5.63 dd J 1,2 = 5.55 4.04 dd J 2,3 = 5.96 4.09 q J 3,4 = 4.15 4.21 dt 3.78 br.dd / 3.66 br.dd 3.03 J P,H1 = 4.30 J P,H2 < 1.0
The enzymatic synthesis of 8-azapurine and 8-aza-7-deazapurine 2′-deoxyribonucleosides has been studied. Two methods have been used: (i) transglycosylation employing 2′-deoxyguanosine, 2′-deoxycytidine, 2′-deoxyuridine, and 2′-deoxythymidine as 2-deoxy-D-ribofuranose donors and recombinant E. coli purine nucleoside phosphorylase (PNP) as biocatalyst, and (ii) one-pot synthesis from 2-deoxy-D-ribose and nucleobases employing recombinant E. coli ribokinase (RK), phosphopentomutase (PPM) and PNP as biocatalysts. Good substrate activity was observed for all bases studied except 2-amino-8-aza-6-chloro-7-deazapurine, which afforded the desired N 9 -nucleoside in moderate yield due to very low solubility of the base and partial replacement of C6-chloro atom of the base and formed nucleoside with a hydroxy group. The participation of Ser90 O γ of E. coli PNP in the binding of 8-aza-7-deazapurines in the catalytic center of PNP followed by the formation of a productive complex and glycosidic bond is suggested.Nucleosides with 8-azapurines and 8-aza-7-deazapurines (purine numbering throughout) are applicable as drugs 1 and tools in chemistry, chemical biology, and molecular diagnostics. 2-5 Chemical syntheses of these nucleoside shape mimics suffer from the formation of mixtures of isomers, which makes it necessary to conduct time-consuming separation procedures. As a result, the desired nucleosides are obtained in moderate or low yields (for example see refs. 4,6 and those in the Supporting Information). Nucleoside phosphorylases (NP), in particular E. coli NP, are very efficient biocatalysts in glycosylation reactions. 7 Doskocil and Holy 8 have shown that 8-azaguanine is a good ribosyl acceptor in the enzymatic reaction catalyzed by E. coli purine nucleoside phosphorylase (PNP; product of deoD gene; EC 2.4.2.1; 7,9 ). Later, Votruba et al. studied alginate gel entrapped cells of an auxotrophic thyminedependent strain of E. coli as a biocatalyst for the transfer of the 2′-deoxy-D-ribofuranosyl moiety of 2′-deoxyuridine to purine and pyrimidine bases including aza and deaza analogues. 10 The reaction proceeds regio-and stereospecifically, affording 8-aza-2′-deoxyadenosine and 8-aza-2′-deoxyguanosine. Furthermore, it was found that the presence of nitrogen-7 of purines and their isosteric analogues is a prerequisite for the reaction. 10 However, there are several exceptions; 5-aza-7-deazaguanine 11 (1), 5-aza-7-deazaisoguanine 12 (2) and N-(1,3,4-thiadiazol-2-yl)-cyanamide 13 (3) are substrates for bacterial purine nucleoside phosphorylases (Figure 1). Figure 1 Some unusual substrates of E. coli purine nucleoside phosphorylase in synthetic reactionsWe applied the enzyme-catalyzed glycosylation to diverse range of heterocyclic bases belonging to the classes of 8-azapurines and 8-aza-7-deazapurines. The work presented here reports on the use of various donors of the 2′-deoxy-D-ribofuranosyl residue and recombinant E. coli PNP 14 as a biocatalyst (transglycosylation reaction; Scheme 1, path A) and enzymatic cascade transforma...
The trans-2-deoxyribosylation of 4-thiouracil (4SUra) and 2-thiouracil (2SUra), as well as 6-azauracil, 6-azathymine and 6-aza-2-thiothymine was studied using dG and E. coli purine nucleoside phosphorylase (PNP) for the in situ generation of 2-deoxy-α-D-ribofuranose-1-phosphate (dRib-1P) followed by its coupling with the bases catalyzed by either E. coli thymidine (TP) or uridine (UP) phosphorylases. 4SUra revealed satisfactory substrate activity for UP and, unexpectedly, complete inertness for TP; no formation of 2’-deoxy-2-thiouridine (2SUd) was observed under analogous reaction conditions in the presence of UP and TP. On the contrary, 2SU, 2SUd, 4STd and 2STd are good substrates for both UP and TP; moreover, 2SU, 4STd and 2’-deoxy-5-azacytidine (Decitabine) are substrates for PNP and the phosphorolysis of the latter is reversible. Condensation of 2SUra and 5-azacytosine with dRib-1P (Ba salt) catalyzed by the accordant UP and PNP in Tris∙HCl buffer gave 2SUd and 2’-deoxy-5-azacytidine in 27% and 15% yields, respectively. 6-Azauracil and 6-azathymine showed good substrate properties for both TP and UP, whereas only TP recognizes 2-thio-6-azathymine as a substrate. 5-Phenyl and 5-tert-butyl derivatives of 6-azauracil and its 2-thioxo derivative were tested as substrates for UP and TP, and only 5-phenyl- and 5-tert-butyl-6-azauracils displayed very low substrate activity. The role of structural peculiarities and electronic properties in the substrate recognition by E. coli nucleoside phosphorylases is discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.