As strong agonists of the purinergic receptors, endogenous dinucleoside pentaphosphates, including Ap5A, Ap5G, and Gp5G, are essential extracellular signaling molecules, and play important roles in physiological and pathological processes in cardiovascular and nervous systems [1]. Ap5A [2] and Ap5G [2b,3] exhibit both vasoregulatory effect and proliferative effect on vascular smooth muscle cells (VSMCs), and Gp5G [3,4] only induces proliferation of VSMCs. In biochemical research, the Ap5A-Zn²⁺ complex has been utilized as a probe to elucidate the configurational requirement for adenylate kinase catalysis [5]. In addition, artificial dinucleoside pentaphosphates, such as Up₅U and Ap₅T, have also been synthesized. While Up₅U showed selective agonist activity on P2Y2 receptor [6], Ap₅T exhibited strong inhibitory effects on thymidine kinase, thymidylate kinase, and ribonucleotide reductase [7].

Currently, the available synthetic methods for the preparation of dinucleoside pentaphosphates are still limited compared to those for dinucleoside di-, tri-, and tetraphosphates. The most commonly employed strategy was the direct condensation of nucleoside diphosphate with another molecule of nucleoside triphosphate by using N,N0-dicyclohexylcarbodiimide (DCC) or N,N0-carbonyldiimidazole (CDI) [5a,6-8]. But this method was typically low yielding (10%-20%) and time-consuming (1-3 days). Though Jones et al. [9] and Taylor et al. [10] reported novel methods for dinucleoside pentaphosphate synthesis, the protection of the nucleosides and preparation of specific condensing reagents limited their values in practical applications. On the basis of the P(V)-N activation strategy we developed for the synthesis for nucleoside polyphosphates [11], we report in this paper a general and facile approach for the synthesis of dinucleoside pentaphosphates (Np₅N's) via the 4,5-dicyanoimidazole (DCI)-promoted coupling of nucleoside 5'-phosphoropiperidates with nucleoside 5'-tetraphosphates (Np₄s). The P(V)-N activation method for the efficient synthesis of Np₄s [12] is also described.

All reactions were performed in anhydrous solvents under an atmosphere of dry argon. The triethylammonium salts of nucleoside 5'-phosphoropiperidates were synthesized according to the procedure described in a previous report [11b]. Tris(tetra-nbutylammonium) dihydrogen triphosphate was prepared according to a known method [12a]. Ion exchange chromatography employed DEAE Sephadex A-25 exchanger. Preparative HPLC was equipped with a RP C18 column (19 mm × 250 mm, 10 μm). NMR spectra were obtained with a 400 MHz instrument with chemical shifts reported in parts per million (ppm, δ). IR spectra were recorded on a FT-IR spectrometer. Low-resolution mass spectra were obtained with an ion trap mass spectrometer and reported as m/z.

5'-tetraphosphates (5-8) To a solution of nucleoside 5'-phosphoropiperidate (0.1 mmol) in DMF (2 mL) were added tris(tri-n-butylammonium) dihydrogen triphosphate (0.2 mmol) and 4,5-dicyanoimidazole (DCI, 0.6 mmol). The reaction was stirred at 20 ℃ for 12 h and concentrated in vacuo. The residue was dissolved in NaOAc aqueous solution (3 mol/L, 1 mL) and EtOH (50 mL) was added. The resulting white precipitate was collected by centrifuge. The crude product was dissolved in deionized H₂O (1 mL) and loaded on a DEAE Sephadex A-25 ion exchange column (1.6 cm × 25 cm). Elution with NH₄HCO₃ buffer (linear gradient 0.3-0.6 mol/L), combination of appropriate fractions, and lyophilization afforded Np₄ in ammonium salt form. For characterization, passage of the solution of the ammonium salt in deionized H₂O through a bed of Dowex 50W-X8 ion exchange resin (Na⁺ form) and lyophilization afforded nucleoside 5'-tetraphosphate as pentasodium salt. For the next step reaction, to the ammonium salt in deionized H₂O was added tetra-n-butylammonium hydroxide (3 equiv.), and the solution was repeatedly evaporated with deionized H₂O (1 mL × 3) to afford nucleoside 5'-tetraphosphate as more soluble tris(tetra-n-butylammonium) salt.

Uridine 5'-tetraphosphate, pentasodium salt (5): Starting from 1 (49 mg), 5 (45 mg, 67%) was obtained as a white solid. ¹H NMR (400 MHz, D₂O): δ 7.99 (d, 1H, J = 8.0 Hz), 6.06-5.98 (m, 2H), 4.5'- 4.40 (m, 2H), 4.35-4.30 (m, 1H), 4.30-4.23 (m, 2H); ¹³C NMR (100 MHz, D₂O): δ 166.2, 152.0, 141.7, 102.8, 88.1, 83.6 (d, J_P,C = 9.0 Hz), 73.7, 69.8, 65.2 (d, J_P,C = 5.1 Hz); ³¹P NMR (D₂O, 162 MHz): δ -8.0 (d, 1P, J = 18 Hz), ^-11.1 (dd, 1P, J = 18 Hz), -22.3 (dd, 1P, J₁ = J₂ = 18 Hz), -22.5 (d, 1P, J₁ = J₂ = 18 Hz); IR (cm^-1): υ_max 3336, 2987, 2899, 1695, 1413, 1228, 1087, 924, 838; LRMS (ESI-): m/z calcd. for C₉H₁₅N₂O₁₈P₄ [M-H]^- 562.9; found: 563.0.

Cytidine 5'-tetraphosphate, pentasodium salt (6): Starting from 2 (49 mg), 6 (41 mg, 61%) was obtained as a white solid. ¹H NMR (400 MHz, D₂O): δ 7.96 (d, 1H, J = 7.2 Hz), 6.14 (d, 1H, J = 7.4 Hz), 6.01 (d, 1H, J = 4.4 Hz), 4.43-4.37 (m, 1H), 4.36-4.30 (m, 1H), 4.29- 4.20 (m, 3H); ¹³C NMR (100 MHz, D₂O): δ 166.3, 157.9, 141.7, 96.9, 89.0, 83.1 (d, J_P,C = 8.7 Hz), 74.3, 69.6, 65.0 (d, J_P,C = 5.6 Hz); ³¹P NMR (D₂O, 162 MHz): δ -9.4 (d, 1P, J = 18 Hz), ^-14.4 (d, 1P, J = 18 Hz), -24.9 (dd, 1P, J₁ = J₂ = 18 Hz), -25.7 (dd, 1P, J₁ = J₂ = 18 Hz); IR (cm^-1): υ_max 3370, 2985, 2894, 1693, 1537, 1418, 1235, 1079, 887; LRMS (ESI-): m/z calcd. for C₉H₁₆N₃O₁₇P₄ [M-H]^- 561.9; found: 562.0.

Adenosine 5'-tetraphosphate, pentasodium salt (7): Starting from 3 (52 mg), 7 (45 mg, 65%) was obtained as a white solid. 1H NMR (400 MHz, D₂O): δ 8.54 (s, 1H), 8.24 (d, 1H, J = 6.5 Hz), 6.14 (d, 1H, J = 6.1 Hz), 4.72-4.70 (m, 1H), 4.64 (dd, 1H, J₁ = J₂ = 4.8 Hz), 4.48-4.40 (m, 1H), 4.36-4.27 (m, 1H), 4.27-4.18 (m, 1H); ¹³C NMR (100 MHz, D₂O): δ 155.6, 152.8, 149.2, 139.9, 118.6, 86.5, 84.3 (d, J_P,C = 9.0 Hz), 74.2, 70.4, 65.4 (d, J_P,C = 5.3 Hz); ³¹P NMR (D₂O, 162 MHz): δ ^-11.5 (d, 1P, J = 18 Hz), ^-14.8 (d, 1P, J = 18 Hz), -25.8 (dd, 1P, J₁ = J₂ = 18 Hz), -26.2 (dd, 1P, J₁ = J₂ = 18 Hz); IR (cm^-1): υ_max 3354, 3049, 2795, 1714, 1492, 1453, 1260, 1087, 1044, 967, 924; LRMS (ESI-): m/z calcd. for C₁₀H₁₆N₅O₁₆P₄ [M-H]^- 586.0; found: 586.1. Guanosine 5'-tetraphosphate, pentasodium salt (8): Starting from 4 (54 mg), 8 (46 mg, 64%) was obtained as a white solid. 1H NMR (400 MHz, D₂O): δ 8.14 (s, 1H), 5.93 (d, 1H, J = 6.0 Hz), 4.75- 4.70 (m, 1H), 4.65-4.58 (m, 1H), 4.43-4.33 (m, 1H), 4.31-4.16 (m, 2H); ¹³C NMR (100 MHz, D₂O): δ 159.2, 154.1, 152.0, 138.0, 116.4, 86.7, 84.4, 73.7, 70.6, 65.6; ³¹PNMR(D₂O, 162 MHz): δ^-10.4 (d, 1P, J = 19 Hz), ^-14.8 (d, 1P, J = 19 Hz), -25.6 (dd, 1P, J₁ = J₂ = 18 Hz), -26.2 (d, 1P, J₁ = J₂ = 18 Hz); IR (cm^-1): υ_max 3378, 2960, 2950, 1712, 1673, 1543, 1403, 1255, 1065, 914, 812; LRMS (ESI-): m/z calcd. for C10H16N5O17P4 [M-H]^- 601.9; found: 602.0.

To a solution of nucleoside 5'-phosphoropiperidate (0.1 mmol) in N-methylpyrrolidone (2 mL) were added nucleoside 5'-tetraphosphate (tetra-n-butylammonium salt, 0.04 mmol) and DCI (0.2 mmol). The reaction was stirred at 20 ℃ for 16-18 h. The white precipitation was collected by centrifuge. The crude product was dissolved in deionized H₂O (0.5 mL) and loaded on a DEAE Sephadex A-25 ion exchange column (1.6 cm × 25 cm). Elution with NH₄HCO₃ buffer (linear gradient 0.5 to 0.9 mol/L), combination of appropriate fractions, and lyophilization afforded dinucleoside pentaphosphate in ammonium salt form. To remove the small amount of contaminated polyphosphate byproducts, the ammonium salt was further purified by a preparative RP HPLC [flow rate = 20 mL/min; linear gradient of 0-10% MeOH in TEAB buffer (10 mmol/L, pH 8.0) over 15 min; UV detection at 254 nm]. Combination of appropriate fractions and lyophilization afforded dinucleoside pentaphosphate in triethylammonium salt form. Passage of the solution of the triethylammonium salt in deionized H₂O through a bed of Dowex 50W-X8 ion exchange resin (Na⁺ form) and lyophilization afforded dinucleoside pentaphosphate as pentasodium salt.

P¹,P⁵-Diadenosine-50,5'-pentaphosphate, pentasodium salt (9): Starting from 7 (52 mg), 9 (14 mg, 35%) was obtained as a white solid. ¹H NMR (400 MHz, D₂O): δ 8.38 (s, 2H), 8.11 (d, 2H, J = 6.0 Hz), 6.06-5.98 (m, 2H), 4.75-4.68 (m, 2H), 4.60-4.51 (m, 2H), 4.39-4.32 (m, 2H), 4.31-4.15 (m, 4H); ¹³C NMR (100 MHz, D₂O): δ 154.4, 151.8, 148.1, 138.9, 117.3, 85.7, 73.7, 69.4, 67.6, 64.3; ³¹P NMR (D₂O, 162 MHz): δ ^-11.5 (m, 2P), -22.9 (m, 3P); IR (cm^-1): υ_max 3324, 3149, 2975, 1714, 1492, 1404, 1260, 1087, 1044, 967, 924; LRMS (ESI-): m/z calcd. for C20H28N10O55P⁵ [M-H]^- 915.0; found: 915.1.

P¹,P⁵-Diguanosine-50,5'-pentaphosphate, pentasodium salt (10): Starting from 8 (52 mg), 10 (14 mg, 34%) was obtained as a white solid. ¹H NMR (400 MHz, D₂O): δ 8.00 (s, 2H), 5.80 (s, 2H), 4.60-4.44 (m, 4H), 4.35-4.15 (m, 6H); ¹³C NMR (100 MHz, D₂O): δ 158.6, 153.5, 151.3, 137.4, 115.8, 86.4, 83.6, 73.1, 70.0, 65.0; ³¹P NMR (D₂O, 162 MHz): δ ^-11.0 (m, 2P), -22.4 (m, 3P); IR (cm^-1): υ_max 3388, 2964, 2922, 1673, 1543, 1413, 1250, 1065, 914, 816; LRMS (ESI-): m/z calcd. for C₂₀H₂₈N₁₀O₂₄P₅ [M-H]^- 947.0; found: 947.1.

P¹-Adenosine-5'-P⁵-guanosine-5'-pentaphosphate, pentasodium salt (11): Starting from 7 (52mg), 11 (13 mg, 31%) was obtained as a white solid. ¹H NMR (400 MHz, D₂O): δ 8.33 (s, 1H), 8.04 (s, 1H), 7.91 (s, 1H), 5.95 (d, 1H, J = 5.8 Hz), 5.70 (d, 1H, J = 6.3 Hz), 4.63 (m, 2H), 4.45 (m, 2H), 4.25-4.20 (m, 2H), 4.13-4.10 (m, 4H); ¹³C NMR (100 MHz, D₂O): δ 159.0, 155.6, 154.0, 153.0, 151.8, 149.2, 140.1, 137.9, 118.6, 116.3, 86.8 (×2), 84.2 (×2), 74.4, 73.5, 70.5 (×2), 65.5 (×2); ³¹PNMR (162 MHz,D₂O): δ^-11.0(m, 2P), -22.8 (m, 3P); IR (cm^-1): υ_max 3783, 3455, 2950, 2673, 2480, 1722, 1706, 1618, 1442, 1380, 1243, 1122, 1060, 948, 800, 732; LRMS (ESI-): m/z calcd. for C₂₀H₂₈N₁₀O₂₃P₅ [M-H]^- 931.0; found: 931.1.

As shown in Scheme 1, nucleoside 5'-tetraphosphates (5-8) were efficiently synthesized by treating nucleoside 5'-phosphoropiperidates (1-4) with 2.0 equiv. of tris(tetra-n-butylammonium) triphosphate and 6.0 equiv. of DCI in anhydrous DMF at 20 ℃ for 12 h. The precipitated crude products were separated by filtration. Ethanol precipitation of the sodium salts followed by ion exchange chromatography afforded 5-8 in good isolated yields ranging from 61% to 67%. The ³¹P NMR spectrum of crude reaction mixture of 5 (Fig. 1) showed that the conversion of 1-5 was smooth and clean. The calculated ³¹P NMR yield of 5 was close to 90%. Compared to nucleoside 5'-triphosphate synthesis [11a], longer reactiontimewas required, indicating that the nucleophilicity of triphosphate reagent was lower than that of pyrophosphate. However, due to the highly negatively charged nature, the isolated yields of 5-8 were slightly lower than those of nucleoside di- and tri-phosphates. It was also noticed that Np₄s were less stable than their NTP counterparts. When the aqueous solution of 7 was stored at -20 ℃, about 10% of 7 decomposed after 6 months.

Fig. 1

	Download: JPG larger image
Fig. 1. The 31P NMR spectrum of crude reaction mixture of Up4 (5).

In the following research, adenosine 5'-phosphoropiperidate (3) was coupled with Ap₄ (7) in 1:0.5 molar ratio [11d] in Nmethylpyrrolidone at 20 ℃ with or without DCI. ³¹P NMR tracing experiments showed that there was almost no reaction between the reactants after 24 h without DCI. This result was in accordance with the observed low reactivity of phosphoropiperidate toward phosphate nucleophiles in our previous research. In contrast, addition of acidic DCI (2.0 equiv.) promoted the reaction to completion in 16 h. But when 3 disappeared, there was still significant amount of 7 (~20%) unreacted due to its low nucleophilicity and the self-condensation of 3. Decrement of the equivalent of 7 to 0.4 effectively promoted its consumption, and the ³¹P NMR yield of Ap5A (9) was improved to around 50%. But we found that ion exchange chromatography alone was difficult to completely separate the desired Np₅N0 products from other closely related long chain polyphosphate impurities. Therefore, RP-HPLC was employed afterward to ensure the purity of these highly negatively charged compounds. As shown in Table 1, three naturally occurring Np₅N's (9-11) were synthesized and isolated in 31%-35% yields following this P(V)-N activation method.

Table 1

Table 1 The P(V)–N activation method for the synthesis of dinucleoside pentaphosphates (9–11).

Table 1 The P(V)–N activation method for the synthesis of dinucleoside pentaphosphates (9–11).

In summary, we developed efficient P(V)-N activation methods for the synthesis of both nucleoside 5'-tetraphosphates and dinucleoside pentaphosphates. Compared to the known methods, these new approaches feature easily accessible starting materials, mild reaction conditions, and moderate to good isolated yields, and provide facile and reliable access to Np₄s and Np₅N's.

We thank the National Natural Science Foundation of China (Nos. 21002041 and 21262014), Key Project of Chinese Ministry of Education (No. 212092), Scientific Research Foundation of Chinese Ministry of Human Resources and Social Security for Returned Chinese Scholars (2011), and Research Funds (Nos. ky2012zy08 and 2013QNBJRC001) and Startup Funds for PhDs (2010) from JXSTNU for financial support.