Recognition of Artificial Nucleobases by E. coli Purine Nucleoside Phosphorylase versus its Ser90Ala Mutant in the Synthesis of Base‐Modified Nucleosides

Abstract

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.

Enzymatic Synthesis of 2‐Deoxyribose 1‐Phosphate and Ribose 1 Phosphate and Subsequent Preparation of Nucleosides

“…Practically irreversible phosphorolysis of 7-Me-Guo and 7-Me-dGuo in the presence of PNP ensures the quantitative transformation of starting nucleosides to anomerically pure Rib-p and dRib-p. Rib-p and dRib-p are obtained with 74-96 % yields after isolation and purification (48-81 % overall, taking into account the stage of preparation of the initial 7-Me-Guo/7-Me-dGuo). The structure of the obtained compounds was confirmed by 1 H, 13 C, 31 P NMR, and mass-spectroscopy.…”

“…[20] Signals of protons at 5.68 (dd, 1H, J 1,2 = 4.9 Hz, J H-P = 4.4 Hz, H1) and 5.84 (ddd, 1H, J 1,2a = 6.6 Hz, J 1,2b = 1.4 Hz, J H-P = 6.1 Hz, H1) demonstrated characteristic J H,P couplings typical for Rib-p and dRib-p 1 H-NMR spectra. Moreover, J C,P coupling constants were observed in 13 C-NMR spectra. The purity of Rib-p and dRib-p was also confirmed via enzymatic reaction of the obtained substances with allopurinol or adenine in the presence of PNP (see supplementary materials).…”

“…NPs are frequently used for the stereoselective synthesis of Rib-p and dRib-p: enzymatic phosphorolysis of thymidine in the presence of TP (yield of 12-37 % [12,13] ); phosphorolysis of uridine in the presence of UP (yield of 22-31 %. [13,14] ). Phosphorolysis of inosine in the presence of PNP, xanthine oxidase and catalase (yield of 12 % [15] ) and phosphorolysis of guanosine in the presence of PNP (yield of 11 % [16] ) have also been used.…”

An Expedient Synthesis of Flexible Nucleosides through Enzymatic Glycosylation of Proximal and Distal Fleximer Bases

“…[42,43] . In the case of flex-bases 3 and 4,H PLC monitoring showed that the glycosylation reactions were more effective than in the case of 1 ( Figure S2 and Ta ble 1, entries [15][16][17][18][19][20][21]. Only one isomer,t he N1-glycosylated product 3a or 4a was formed, even if the enzyme concentration was reduced tenfold (entries [19][20][21].…”

“…[11,12] NPs have been reportedt oa ccept modified bases as well as unnatural glycosyldonors. [6][7][8][9][13][14][15] NDTs( EC 2.4.2.6) catalyzet he transfer of 2-deoxyriboseb etween purine and pyrimidine bases. [16] TwoN DT types have been defined based on their substrate specificity:N DT-I (also named PTD), which is specific for purines, [17] and NDT-II, which accepts either purine or pyrimidine but has as trong preference for 2'-deoxyribosyl-pyrimidinea st he donor substrate.…”