Use of Nucleoside Phosphorylases for the Preparation of Purine and Pyrimidine 2′‐Deoxynucleosides

Abstract

Enzymatic transglycosylation – the transfer of the carbohydrate moiety from one heterocyclic base to another – is being actively developed and applied for the synthesis of practically important nucleosides. This reaction is catalyzed by nucleoside phosphorylases (NPs), which are responsible for reversible phosphorolysis of nucleosides to yield the corresponding heterocyclic bases and monosaccharide 1‐phosphates. We found that 7‐methyl‐2′‐deoxyguanosine (7‐Me‐dGuo) is an efficient and novel donor of the 2‐deoxyribose moiety in the enzymatic transglycosylation for the synthesis of purine and pyrimidine 2′‐deoxyribonucleosides in excellent yields. Unlike 7‐methylguanosine, its 2′‐deoxy derivative is dramatically less stable. Fortunately, we have found that 7‐methyl‐2′‐deoxyguanosine hydroiodide may be stored for 24 h in Tris‐HCl buffer (pH 7.5) at room temperature without significant decomposition. In order to optimize the reagent ratio, a series of analytical transglycosylation reactions were conducted at ambient temperature. According to HPLC analysis of the transglycosylation reactions, the product 5‐ethyl‐2′‐deoxyuridine (5‐Et‐dUrd) was obtained in high yield (84–93%) by using a small excess (1.5 and 2.0 equiv.) of 7‐Me‐dGuo over 5‐ethyluracil (5‐Et‐Ura) and 0.5 equiv. of inorganic phosphate. Thymidine is a less effective precursor of α‐d‐2‐deoxyribofuranose 1‐phosphate (dRib‐1p) compared to 7‐Me‐dGuo. We synthesized 2′‐deoxyuridine, 5‐Et‐dUrd, 2′‐deoxyadenosine and 2′‐deoxyinosine on a semi‐preparative scale using the optimized reagent ratio (1.5:1:0.5) in high yields. Unlike other transglycosylation reactions, the synthesis of 2‐chloro‐2′‐deoxyadenosine was performed in a heterogeneous medium because of the poor solubility of the initial 2‐chloro‐6‐aminopurine. Nevertheless, this nucleoside was prepared in good yield. The developed enzymatic procedure for the preparation of 2′‐deoxynucleosides may compete with the known chemical approaches.magnified image

Synthesis of Ribavirin, Tecadenoson, and Cladribine by Enzymatic Transglycosylation

“…Therefore, the availability of 7-methyl-2 -deoxyguanosine iodide would represent a valuable tool for an alternative synthetic approach, as proven by the bioconversion of Cladribine (see Table 1). However, this sugar donor was found to be less stable than 7-methylguanosine iodide in aqueous medium, in agreement with a very recent report by Mikhailov et al [49]. We have found that this molecule is unstable also in DMSO at room temperature, as indicated from the appearance of extra signals in the 13 C NMR spectrum recorded at two-hour intervals in DMSO, clearly showing that the decomposition of the nucleoside had occurred (see Supplementary Materials, Figure S1).…”

“…While this research was ongoing, an E. coli PNP-catalyzed transglycosylation for the preparation of 2 -deoxynucleosides, including Cladribine, was developed by Mikhailov and co-workers who exploited a 7-methyl purine nucleoside iodide as the sugar donor [49].…”

MutT homologue 1 (MTH1) catalyzes the hydrolysis of mutagenic O6-methyl-dGTP

“…Based on this information and the fact that N7-methyl-dGTP has been described to be the major product formed after MMS or iodomethane treatment of dGTP ( 37 , 38 ), we successfully generated N7-methyl-dGTP using this route. The formation of N7-methylated dGTP from iodomethane methylation of dGTP was verified by NMR disclosing the presence of a large H8-proton shift arising when the methylation occurs at the N7 position ( 39 , 40 ) ( Supplementary Figure S3A and B ). By combining HPLC, NMR and LC–MS analysis we could conclude that N7-methyl-dGTP indeed is the major product formed upon MMS treatment of dGTP.…”

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

“…Hydroiodide was obtained according to the literature procedure. [18] (pH 7.5) and 9.9 mL of 50 mM buffer solution of potassium dihydrophosphate at ambient temperature in one portion. The reaction mixture was stirred (orbital shaker) for 5 h at ambient temperature.…”