Enzymatic Synthesis of 2′-Deoxy-β-d-ribonucleosides of 8-Azapurines and 8-Aza-7-deazapurines

Abstract

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 chemoenzymatic synthesis of clofarabine and related 2′-deoxyfluoroarabinosyl nucleosides: the electronic and stereochemical factors determining substrate recognition by E. coli nucleoside phosphorylases

“…The cascade synthesis involves a sequential conversion of D-pentoses into their 5-monophosphates catalyzed by the recombinant E. coli ribokinase (RK; ATP co-factor) [27], stereospecific isomerization of the 5-phosphates into α-D-pentofuranose-1-phosphates catalyzed by the recombinant E. coli phosphopentomutase (PPM) [24], and the condensation of the latter with 2-chloroadenine catalyzed by PNP, which gives rise to the formation of the desired nucleoside. It should be stressed that 1,6-diphosphates of D-hexoses are not necessary for the transformation of D- 2F Ara-5P into α-D- 2F Ara-1P as we have showed in our previous works [24–26 28]. This transformation was partly optimized by using variable concentrations of ATP, D-pentoses and biocatalysts.…”

“…The very important role of the β-carboxy group of Asp204 of the E. coli PNP catalytic site was discussed earlier (see, e.g., [26,28]). It is based on the correct base positioning at the E. coli PNP catalytic site by the protonation of the sp 2 -hybridized nitrogen atom of the imidazole ring, which leads to the enhancement of the nucleophilicity of the second nitrogen atom (activation of base).…”

“…Note that 5-aza-7-deazaguanine ( 17 ) was earlier shown to be a good substrate in the enzymatic synthesis of its N 9 -2′-deoxy-β-D-ribofuranoside by using 2-deoxy-α-D-ribofuranose-1-phosphate as a co-substrate and PNP from bovine spleen as a biocatalyst [34]. Recently, an efficient transformation of 8-aza-7-deazahypoxanthine (allopurinol) into its N 9 -2′-deoxy-β-D-ribofuranoside has also been demonstrated in the transglycosylation reaction and in the cascade one-pot synthesis by using E. coli ribokinase (RK), phosphopentomutase (PPM) and nucleoside phosphorylases as biocatalysts [26]. It was found that the irreversible conversion of 5-aza-7-deazaguanine ( 17 ) into its arabinoside 4b proceeds smoothly achieving 50% yield after 24 h and remains at this point for over two weeks.…”

Enzymatic synthesis and phosphorolysis of 4(2)-thioxo- and 6(5)-azapyrimidine nucleosides by E. coli nucleoside phosphorylases

“…We have earlier shown that 2'-deoxy-β-D-ribosides of cytosine, uracil and thymine are substrates for E. coli PNP (Cd >>> Ud >> Td), whereas the corresponding ribosides devoid of substrate activity [67]. The substrate activity of 5-aza-2'-deoxycytidine ( 14 ) (but not 5-azadeoxycytidine ( 13 )) and 4-thiothymidine ( 11a ) for PNP (aCd >>> 4S Td) correlate well with that of the corresponding natural 2-deoxyribosides.…”

“…2-Thiouridine ( 9 ) (but not uridine [67] and 2-thiothymidine ( 11b )) and 4-thiothymidine ( 11a ) (like thymidine [67]) are weak substrates for E. coli PNP. The most unexpected finding consists in that the phosphorolysis of 5-aza-2'-deoxycytidine ( 14 ; anticancer drug Decitabine) catalyzed by E. coli PNP is reversible and condensation of 5-azacytosine with 2-deoxy-α-D-ribofuranose-1-phosphate resulted in the formation of nucleoside 14 .…”