Lozhnikova N.V.², Beresnev A.I.¹, Kucharskaya T.A.¹,

Kvach S.V.¹, Eroshevskaya L.A.¹, ZinchenkoÀ.I.^1,2

¹Institute of Microbiology, National Academy of Sciences of Belarus, Minsk,

²International Sakharov Environmental University, Minsk

biocatalitic synthesis of 5-fluoro-2′-deoxyuridine by thymidine phosphorylase

Non-natural nucleosides are of interest as antiviral and antitumor agents ([for a review, see [1]). In most cases these molecules have been synthesized by a stereoselective coupling reaction of the base analogue and the protected pentose. Bioconversion reactions catalyzed by the action of microbial enzymes (including nucleoside phosphorylases), as reported in literature, make an alternative approach for preparing nucleosides, nucleotides and their modified analogues instead of conventional chemical syntheses, plagued by formation of regio- and stereochemical isomers and by low overall yields [2].

Thymidine phosphorylase of Escherichia coli (EC 2.4.2.4; TP) catalyzes conversion of thymidine to thymine and 2-deoxy-D-ribose-1α-phosphate [3–5]. TP can also perform the transglycosylation of thymidine and its analogues, including 2′-deoxyuridine (2'-dUrd), because the phosphorolysis reaction is reversible. The synthesis of modified nucleosides using bacterial TP is well documented, including our papers [4, 6, 7] but in most cases it was conducted with non-recombinant enzymes.

5-Fluoro-2'-deoxyuridine (5-FdUrd), a derivative of 5-fluorouracil (5-FUra), is an anti-metabolite showing a significant cytotoxic activity [8]. This drug is widely used for the treatment of some solid tumors [9, 10]. Chemical synthesis is the primary method for 5-FdUrd production. It usually involves environmentally unfriendly procedures and has low enantioselectivity, thus reducing process efficiency and increasing downstream costs.

The present study was undertaken in order to develop a practical synthesis of 5-FdUrd using reaction of enzymatic transglycosylation.

Materials and methods. 2'-dUrd was chosen as a donor of the ß-D-deoxyribofuranose moiety and 5-FUra as its acceptor. Recombinant TP isolated from cells of the newly engineered strain E. coli TDP served as biocatalyst [11]. Optimal procedure for the reaction: 2'-dUrd (0.1 mmol, 22.8 mg), 5-FUra (0.1 mmol, 13 mg), and TP (15 units) from E. coli were added to 10 mL of 2 mM phosphate buffer (pH 7.0). The reaction mixture was stirred at 40ºC for 1 h. The reaction was monitored by thin-layer chromatography on Merck F254 silica gel 60 aluminium sheets as described in [11].

Results and discussion. First, we seeked for the most suitable reaction conditions for each concentration of the substrates (2'-dUrd, 5-FUra, and phosphate ion). The studied reaction catalyzed by TP proceeds in two steps (Fig. 1, step 1 and step 2) and both are reversible. Therefore, the produced uracil and the concentration of phosphate buffer inhibit step 2 under «one-pot» reaction conditions.

Fig. 1. Synthesis of 5-FdUrd from 2'-dUrd and 5-FUra by recombinant TP

Fig. 2 shows the effects of substrate concentrations (2'-dUrd and 5-FUra). As can be seen from Fig. 2a, with increase in 5-FUra concentration, the initial velocity decreased, and the conversion level increased. But 5-FUra at 100 mM concentration shifted the equilibrium and inhibited the production of 5-FdUrd.

Fig. 2b shows the effect of the 2'-dUrd concentration. The conversion and initial velocity of 5-FdUrd production increased with increasing concentration of 2'-dUrd. However, starting from 5:1 ratio of 2'-dUrd to 5-FUra the reaction yield reached the quantitative level.

Fig. 2. Effects of variation of substrate concentration on the conversion rate.

(A) Effect of changes in 5-FUra concentration (2'-dUrd concentration was kept constant at 10 mM). (B) Effect of changes in 2'-dUrd concentration (5-FUra concentration was kept constant at 10 mM).

The increasing concentration of phosphate ions inhibited the catalytic conversion of 2'-dUrd to 5-FdUrd (not shown). We suggest to use the 5:1 ratio of 5-FUra (50 mM) to 2'-dUrd (10 mM) and 2 mM phosphate buffer (pH 7.0).

In conclusion, we report in this communication the first application of recombinant E. coli TP to produce 5-FdUrd from 2'-dUrd and 5-FUra. The obtained results open up possibilities for engineering of industrial technology for production of the title pharmaceutically valuable modified nucleoside.

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