Biotechnology Letters 26: 18471850, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.

1847

An improved method for the enzymatic transformation of nucleosides into 50 -monophosphates

Vladimir N. Barai 1, Sergei V. Kvach 1, Anatoli I. Zinchenko 1 & Igor A. Mikhailopulo2,*
Institute of Microbiology, National Academy of Sciences, 220141 Kuprevicha 2, Minsk, Belarus Institute of Bioorganic Chemistry, National Academy of Sciences, 220141 Kuprevicha 5, Minsk, Belarus *Author for correspondence and present address: Department of Pharmaceutical Chemistry, University of Kuopio, P.O.Box 1627, FIN-70211 Kuopio, Finland (Fax: +358-17-162456; E-mail: Igor.Mikhailopulo@ uku.)
2 1

Received 26 August 2004; Revisions requested 22 September 2004; Revisions received 11 October 2004; Accepted 12 October 2004

Key words: enzymatic phosphorylation, Erwinia herbicola, nucleoside, nucleoside-50 -monophosphate, nucleoside phosphotransferase Abstract An improved method to transform nucleosides into 50 -monophosphates using nucleoside phosphotransferase from Erwinia herbicola is reported. The method is based on the shift in the equilibrium state of the reaction to the formation of desired product due to its precipitation by Zn2+. Under optimal conditions, the extent of nucleoside transformations into nucleoside-50 -monophosphates were 4191% (mol). Introduction Naturally occurring and modied nucleoside-50 monophosphates (NMP) are valuable compounds for diverse biochemical studies and as active pharmacological ingredients (see, for example, Kuninaka & Shoyu 1976, Becheur et al. 1998, Boogaerts et al. 2001). NMP are obtained by microbial synthesis or by isolation from hydrolysates of nucleic acids, which are the main sources of these compounds (Kuninaka & Shoyu 1976). Methods for the production of modied NMP are based either on chemical (Scheit 1980, Burgess & Cook 2000) or on an enzymatic phosphorylation of corresponding nucleosides (Humble et al. 1984, Zinchenko et al. 1990, Rutkowski & Draminski 1991, Zaitseva et al. 1994). The yields of NMPs by chemical phosphorylation of modied nucleosides vary widely depending on the method employed, the structure of initial compounds, and from assay to assay. On the other hand, the enzymatic 50 monophosporylation is advantageous over the chemical methods rst of all owing to a stringent regioselectivity. The reactions of the enzymatic 50 -monophosphorylation are, however, either reversible or they are inhibited by the reaction products, NMP, which necessitates one to use a phosphate donor in manifold molar excess versus a nucleoside (Giziewicz & Shugar 1975, Humble et al. 1984, Zinchenko et al. 1990, Zaitseva et al. 1994). Microbial and plant nucleoside phosphotransferases (EC 2.7.1.77; NPase) are most widely applied as biocatalysts for the 50 -monophosphorylation of nucleosides. As distinct from nucleoside kinases, these enzymes have a broader specicity with respect to both the phosphate donors and acceptors, and they can accept the low-energy monoesters of phosphoric acid such as, for example, natural NMP and p-nitrophenylphosphate (p-PNP), as substrates. In the present paper, a previously described method for the enzymatic transformation of nucleosides into NMP using the whole cells Erw. herbicola 47/3 (Zinchenko et al. 1990, Zaitseva et al. 1994) as a biocatalyst, was improved by an application of Zn2+ ions for the precipitation of NMP from the phosphorylating mixture during the course of reaction.

1848 Materials and methods Reagents Adenosine (Ado) and p-NPP were purchased from Sigma (USA). Activated charcoal Norit A, as well as Silufol UV254 aluminum sheets for TLC were obtained from Serva (Germany). 1-(b-D Arabinofuranosyl)cytosine (ara-C) and 1-(b-D arabinofuranosyl)uracil (ara-U) were purchased from Fluka (Germany). Preparation of 9-(b-D arabinofuranosyl)guanine (ara-G) was described previously (Zinchenko et al. 1990). Syntheses of 9-(b-D -arabinofuranosyl)adenine (ara-A), 9-(b-D arabinofuranosyl)-2-aminoadenine (ara-A2NH2) and 9-(b-D -arabinofuranosyl)-2-uoroadenine (ara-A2F) were synthesized by an enzymatic transglycosylation as it was described for the preparation of the base- and sugar-modied nucleosides (Barai et al. 2002). The purity of all compounds was checked by TLC and high-performance liquid chromatography (HPLC), and the structure was proved by 1H and 13C NMR spectroscopy. The UV spectra were recorded in ethanol. HPLC was carried out using a Nova-Pac C-18 (3.9 300 mm) column (Waters) and an isocratic elution with the following buer: 7% acetonitrile in 0.1 M KH2PO4 (v/v) at 0.7 ml min)1 (time of analysis was 15 min). Microorganisms Erwinia herbicola 47/3 cells were grown in 250 ml Erlenmeyer asks containing 50 ml of the following medium (g l)1): 6 Na2HPO4; 3 KH2PO4; 0.5 NaCl; 1 NH4CL; 0.12 MgSO4; 3 meat peptone (Sigma, USA); 5 yeast extract (Serva, Germany). The initial pH of the culture medium was adjusted to 7. Cultivation was carried out on a circular shaker (180200 rpm) at 28 C for 24 h. Biomass was harvested by centrifugation (4500 g, 10 min), washed twice with two volumes of 0.85% (w/v) NaCl, estimated by weighing an aliquot after drying at 105 C for 24 h, and suspended in a small volume of the same solution. Standard reaction conditions for the enzymatic synthesis of NMPs The reaction mixture (3 ml) consisted of 30 mM nucleoside, 90 mM p-NPP, 0.2 M sodium acetate buer (pH 4.5), 0.1 M salt of cation studied and wet paste of bacterial cells (1%, w/v; calculated as abs. dry weight). The mixture was incubated at 37 C with gentle stirring. Aliquots of the reaction medium were diluted twice with 0.25 M EDTA (pH 7.5) and analyzed by TLC as described by Zinchenko et al. (1990). Preparative synthesis of NMPs In the case of preparative (10 ml scale) NMPs synthesis, after the reaction was completed, the pH of reaction medium was adjusted to 7 with 1 M NaOH. The mixture was heated to 80 C and centrifuged (5000 g, 10 min). The sediment obtained was washed with hot (5060 C) water (5 ml) and dissolved in 0.1 M EDTA (pH 7; 8 ml), then the cells were removed by centrifugation and washed with 0.1 M EDTA (pH 7; 2 ml). Two supernatants were combined and NMP was isolated using a procedure described by Rideout et al. (1979). The NMP contained solution was stirred with Norit A (500 mg) for 10 min, charcoal was centrifuged and then washed by centrifugation two times with distilled water (2 5 ml). The NMP was eluted from charcoal with 0.1 M NH4OH in 30% (v/v) ethanol. The eluate was loaded on to a column (2.5 cm3) with Dowex 1 8 ion-exchange resin in the Cl)-form. After washing with 10 mM HCl, the NMP was eluted from a column with 0.2 M NaCl in 40 mM HCl. The eluate was evaporated to 2.5 ml under reduced pressure and the NMP was precipitated with 68 vol. ethanol at 4 C for 24 h. The produced precipitate was ltered o, washed with ethanol (5 ml), and dried under reduced pressure to constant mass.

Results and discussion At the rst stage, we studied the bivalent metal ions, which can form water-insoluble salts with AMP and p-NPP in the standard reaction medium without biocatalyst. Among the bivalent metal ions studied (Ba2+, Ca2+, Cu2+, Co2+, Zn2+, and Ni2+), only Zn2+ and Cu2+ have shown the ability to precipitate AMP and, to a much lesser extent, p-NPP (see Table 1). The higher solubility of the corresponding salts of p-NPP is rather important because its precipitation would

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Table 1. Eect of bivalent metal ions on solubility of AMP and p-NPP, and eciency of the AMP synthesis by the Erwinia herbicola cells as wella. Added salt in the medium Solubility (mM ) AMP CuCl2 ZnCl2 None
a b

Max. degree of Ado conversion to AMP (mol%) p-NPP 25 12

b

Time of attaining max. conversion (h)

0.42 0.17
b

65 83 60

6 6 13

The AMP/p-NPP ratio was 1:3 (mol) throughout. The reactions were conducted at 37 C. Precipitate was not formed.

Table 2. Eect of Zn2+ ions on solubility of NMP and eciency of the NMP syntheses by the Erwinia herbicola cellsa. Initial nucleoside NMP solubility (mM ) +Zn2+ Ara-A Ara-A2F Ara-A2NH2 Ara-G Ara-C Ara-Ub Ado2NH2
a b

Max. degree of nucleoside conversion to NMP (mol%) )Zn2+ 36 33 35 32 26 29 65 +Zn2+ 59 55 58 41 42 27 91

Time of attaining max. conversion (h) )Zn2+ 11 11 11 7 7 6 8 +Zn2+ 12 12 12 8 8 6 7

0.15 0.14 0.17 2.8 3.6 30 0.19

The AMP/p-NPP ratio was 1:3 (mol) throughout. The reactions were conducted at 37 C. Precipitate was not formed.

essentially slow down the desired enzymatic phosphorylation. The results of AMP synthesis are also presented in Table 1. As can be seen from the data shown in Table 1, both cations, Zn2+ and Cu2+, increase the AMP yield along with the considerable decrease of the reaction time. Taking into account that Zn2+ displayed higher eciency versus Cu2+, further experiments have been carried out using ZnCl2. At the next stage, we have studied the eciency of the synthesis of the base- and sugar-modied NMP employing the optimal conditions elaborated for the AMP synthesis (Table 2). In all (except for one) reactions studied, we have observed an enhancement of the yield of NMP in the presence of Zn2+ ion. This fact, to our opinion, denotes a shift in the equilibrium state of the reaction to the formation of NMP (principal products) due to their precipitation in the form of water-insoluble salts. This assumption is corroborated by the observation that Zn2+ ions did not aect the yield of ara-UMP because it lacks the ability to form insoluble precipitate with Zn2+ ions under the reaction conditions.

Conclusion The method proposed may be used for the synthesis of dierent NMP capable to form water insoluble salts with zinc cations. Under such conditions, the transformation of nucleosides into their 50 -monophosphates in the presence of zinc ions results in remarkable increasing the yields of desired NMP. References
Barai VN, Zinchenko AI, Eroshevskaya LA, Kalinichenko EN, Kulak TI, Mikhailopulo IA (2002) An universal biocatalyst for the preparation of base- and sugar-modied nucleosides via an enzymatic transglycosylation. Helv. Chim. Acta 85: 19011908. Becheur H, Valla D, Loriot MA, Attar A, Bloch F, Petite JP (1998) Concurrent emergence of hepatitis B e antigennegative hepatitis B virus variant and autoimmune hepatitis cured by adenine arabinoside monophosphate. Dig. Dis. Sci. 43: 24792482. Boogaerts MA, Van HA, Catovsky D, Kovacs M, Montillo M, Zinzani PL, Binet JL, Feremans W, Marcus R, Bosh F (2001) Activity of oral udarabine phosphate in previously treated chronic lymphocytic leukemia. J. Clin. Oncol. 19: 42524258.

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