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A NEW STRATEGY FOR THE SYNTHESIS OF NUCLEOSIDES: ONE-POT ENZYMATIC TRANSFORMATION OF D-PENTOSES INTO NUCLEOSIDES Anatoly I. Miroshnikov,1 Roman S. Esipov,1 Tat’yana I. Muravyova,1 Irina D. Konstantinova,1 Ilja V. Fateev,1 and Igor A. Mikhailopulo,2* 1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 GSP, Moscow, B437, Miklukho-Maklaya 16/10, Russian Federation, and 2Institute of Bioorganic Chemistry, National Academy of Sciences, 220141 Minsk, Acad. Kuprevicha 5/2, Republic of Belarus. * Correspondence to: Email address
[email protected]
ABSTRACT A possibility of the one-pot synthesis of purine and pyrimidine nucleosides employing pure recombinant ribokinase, phosphopentomutase and nucleoside phosphorylases in a caskade transformation of D-ribose or 2-deoxy-D-ribose into pyrimidine and purine nucleosides is demonstrated. INTRODUCTION A chemo-enzymatic approach to the synthesis of nucleosides attracts continuously growing interest [1-3]). Recently, we have suggested a new strategy for the synthesis of nucleosides [4]. In the present communication, we report on the preparation of the recombinant E. coli phosphopentomutase (PPM) and preliminary results of one-pot enzymatic transformation of D-ribose or 2-deoxy-D-ribose into nucleosides employing recombinant E. coli ribokinase (RK) [Dpentoses → D-pentofuranose-5-phosphates (D-PFP-5)], PPM [D-PFP-5 → -D-pentofuranose-1-phosphates (-DPFP-1)], and nucleoside phosphorylases (NP’s) (-D-PFP-1 + heterobase → nucleosides) in the presence of pyrimidine or purine heterobases. The preparation of pure recombinant RK [4] as well as uridine (UP), thymidine (TP) and purine nucleoside (PNP) phosphorylases [5] was described by us earlier. RESULTS AND DISCUSSION We have found that under optimum conditions RK catalyzes the phosphorylation of the primary hydroxyl group not only of D-ribose and 2-deoxy-D-ribose, but also of Darabinose and D-xylose. These data prompted us to consider this reaction as the first stage in a cascade transformation of pentoses into nucleosides. It is noteworthy that the chemical [6] and chemo-enzymatic [2,6a] transformations of pentoses into 5-phosphates are rather laborious and low yielding. The stereospecific C5 → C1 translocation of phosphate catalyzed by PPM [7] is a reliable bridge within the strategy under investigation. With this aim in view, we have prepared recombinant PPM and studied its properties. In brief, the methodology of the plasmid preparation, containing the PPM gene DeoB from E. coli was similar to that previously described by us [4,5]. The resulting plasmid pER-PPM1 was transformed into E. coli strain ER2566. Cells were har-
vested by centrifugation and the expression level in each clone was analyzed by SDS-polyacrylamide gel electrophoresis. Two clones with the highest level of the PPM expression (up to 50% of the total cellular protein) were selected. The cells were disrupted by sonication and the resulting cell lysate was cleared by centrifugation to afford 12 mL of the PPM preparation with specific enzyme activity of 3.4 mol/min/mg protein. The PPM of clear soluble fraction was further purified by Q-Sepharose HP chromatography to afford 12.5 mL of PPM solution containing 35 mg protein with total 770 units PPM activity (22 mol/min·mg protein). The molecular weight of PPM was estimated to be ca. 43,000 (cf. [7a,b]) by SDSpolyacrylamide gel gradient electrophoresis relative to standard proteins of known molecular weight. The PPM activity was measured spectrophotometrically by following the formation of uridine resulting from the PPM catalyzed transformation of D-ribofuranose-5-phosphate into -Dribofuranose-1-phosphate followed by the condensation of the latter with uracil catalyzing by UP [5]. To assess the efficacy of PPM, we have tested the synthesis of inosine and 1-(-D-ribofuranosyl)thymine (RibThy) using D-ribose-5-phosphate and the respective heterobases, hypoxanthine or thymine, as substrates and the relevant enzymes, PPM/PNP or PPM/TP, as biocatalysts. Based on the literature data [7a,b], the following reaction conditions have been employed in these experiments after a number of preliminary tests: the molar ratio of D-ribose-5phosphate and base was 1:1; 0.05 mM MnCl2, 10 mM TrisHCl, pH 7.5; 20°C. The formation of inosine or Rib-Thy was analyzed by HPLC after 1 and 24 h. It was found that 35% of hypoxanthine is transformed into inosine after 1 h and its quantity only slightly enhanced after 24 h (39%); under similar time intervals, the yield of Rib-Thy was 8.5 and 16.9%, respectively. Analysis of the optimal reaction conditions for RK, PPM and recombinant nucleoside phosphorylases revealed rather essential differences. Bearing this in mind, we have optimized the one-pot reaction conditions aiming at the finding out a compromised composition of the components allowing satisfactory function of the enzymes under investigation. Furter experiments have been performed in one-pot fasion using D-ribose or 2-deoxy-D-ribose as starting pentoses, hypoxanthine, thymine or uracil as bases, and the RK, PPM
and relevant nucleoside phosphorylaes (NP) as biocatalysts (Scheme; Table 1). OH
O
HO OH
O
OH
OH
X
HO
RK ATP/KCl/MgCl2
O HO P O O
X
O HO
OH
O R HO
O O HO
X
O P OH O
-D-PF-1P
PPM
X
D-PF-5P
D-Ribose: X = OH 2-Deoxy- D-ribose: X = H
HO
HO -phosphate Hypoxanthine/PNP
NH N
O
Thymine/TP or Uracil/UP
Universal Glycosylating Agents for the Enzymatic Synthesis of Pyrimidine and Purine Nucleosides
O
X
R = CH3 Thymidine: X = H Ribothymidine: X = OH R=H Uridine: X = OH 2'-Deoxyuridine: X = H O N HO
N
O
Recombinant E. coli Enzymes: RK - Ribokinase PPM - Phosphopentomutase TP - Thymidine Phosphorylase UP - Uridine Phosphorylase PNP - Purine Nucleoside Phosphorylase
action of RK, PPM and PNP in one-pot transformation of 2deoxy-D-ribose in the presence of 2-chloroadenie into 2chloro-2′-deoxyadenosine (Cladribine) depending on the ratio of substrates are illustrated in Figure 1.
HO
NH N
Figure. 1. Progress of the synthesis of 2-chloro-2′-deoxyadenosine in the cascade one-pot enzymatic reactions; the mM concentrations of ATP/2-chloroadenine/2-deoxy-D -ribose: A – 0.6/0.5/0.5; B – 0.9/0.5/0.75.
X
Inosine: X = OH 2'-Deoxyinosine: X = H
It is remarcable that the formation of inosine proceeds faster vs that of 2′-deoxyinosine and reached maximum yield after 30 min. We have earlier shown that the trans-2′deoxyribosylation of purines and deazapurines procceds with higher efficiency vs the trans-ribosylation (e.g., [1]). Table 1. Nucleoside Syntheses in the Cascade One-Pot Enzymatic Reactions at the Equimolar Ratio of Pentose & Base, and 20°C (a,b) Nucleoside Pentose Yield of Time of Reaction NP Nucleoside (%) (h) Ino Rib/PNP 45.9 0.5 dIno dRib/PNP 38.3 24 Thd dRib/TP 34.7 44 Rib-Thy Rib/TP 19.9 44 Urd Rib/UP 27.6 0.5 dUrd dRib/UP 33.2 44 (a) Standard reaction conditions: total volume of the reaction mixture 2 mL; 2 mM ATP, 50 mM KCl, 3 mM MnCl2, 20 mM Tris·HCl (pH 7.5), 2 mM pentose, 2 mM heterobase; run at 20°C; enzymes (respective units): RK 7.65; PPM 3.9; TP 4.5; UP 5.4; PNP 4.68. (b) HPLC Analyses: Breeze chromatograph (Waters); column: Nova-Pak C18, 4 um, 4.6 × 150 mm; isocratic elution with 1.4 % MeCN and 0.1 % TFA at a flow rate of 1 mL/min, run time 15 min; UV-detector, eluates were monitored at 254 nm
The formation of thymidine proceeds with higher efficacy and gives rise to the higher final yield of thymidine vs that of Rib-Thy (Table 1). The observed differences in the formation of thymidine and Rib-Thy may be explained by the lower substrate activity of -D-ribofuranose-1phosphate vs its 2-deoxy-counterpart for TP. In harmony with this suggestion is the formation of corresponding uracil nucleosides catalyzed by UP. Indeed, as might be expected, -D-ribofuranose-1-phosphate manifests much higher substrate activity towards UP than 2-deoxy--D-ribofuranose 1-phosphate does. However, the final concentrations of uridine and 2′-deoxyuridine are rather similar. Taking into accout the fuctional peculiarities of the enzymes under consideration, a careful optimization of the reaction conditions is necessary to achieve high yield of the desired nucleosides. Thus, e.g., the results of the concerted
CONCLUSION The efficient strain-producers of the recombinant E. coli RK, PPM and nucleoside phosphorylases were created. A possibility of the one-pot synthesis of purine and pyrimidine nucleosides employing the aforementioned enzymes in a cascade transformation of D-pentoses into nucleosides was demonstrated. The studies directed towards assessment of the scope and limitations of this strategy are now in progress. Acknowledgements: Financial support by the International Science and Technology Centre (project #B-1640) is gratefully acknowledged. IAM is thankful to the Alexander von Humboldt-Stiftung (Bonn – Bad-Godesberg, Germany) for partial financial support of this study. REFERENCES 1. 2. 3.
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