Fluorescent 2′-Deoxyuridine (dU) Analogue: Tropolonyl triazolyl-dU (tt-dU) Exhibits Solvatochromism/HeLa Cell Internalization and Its Triphosphate (tt-dUTP) Is Incorporated into DNA Enzymatically

Abstract

This era has witnessed the development and extensive application of modified nucleosides, including fluorescent nucleosides that clinically served humankind. Most fluorescent nucleoside analogues are derived from benzenoid aromatic scaffolds. However, the non-benzenoid aromatic moiety, tropolone, which exhibits unique hydrogen bonding and metal chelating properties, also occurs in nature. Recently, we introduced the tropolone unit at deoxyuridine through an ethyne linker and prepared its DNA analogues, which are fluorescent. This report describes the synthesis of a new troponyl triazolyl-dU (tt-dU) analogue, possessing a triazolyl linker, through click chemistry. tt-dU exhibits fluorescence with solvatochromism and enters into Hela cells without any cytotoxicity. Its triphosphate (tt-dUTP) was also synthesized and incorporated enzymatically into DNA, as shown in primer extension experiments. The unique photophysical properties and metal-chelating ability of the tropolone group make tt-dU a promising modified nucleoside.

Key words

DNA - fluorescent nucleosides - tropolone - click chemistry - enzymatic incorporation - solvatochromism - cell imaging

Publication History

Received: 27 July 2023

Accepted after revision: 30 October 2023

Accepted Manuscript online:
30 October 2023

Article published online:
27 November 2023

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• 38 Synthesis of Nucleoside tt-dU (5): Compound 4 (50 mg, 0.05 mmol) was dissolved in MeOH (3 mL) and two drops of benzene. To the stirring solution, ammonia solution (1 mL) was added slowly at 0 °C. After addition, the mixture was removed from the ice bath and stirred at room temperature for ca. 1.5 hours. Upon completion of the reaction, solvents were evaporated under reduced pressure and the residue was co-evaporated with DCM and hexane. The product was precipitated from methanol/diethyl ether and dried to give 5 (37 mg, 93% yield) as a yellow solid. ¹H NMR (400 MHz, DMSO-d ₆): δ = 8.63 (s, 1 H), 7.96 (s, 1 H), 7.87 (d, J = 7.6 Hz, 2 H), 7.51 (d, J = 7.0 Hz, 1 H), 7.45 (t, J = 7.3 Hz, 1 H), 7.35 (s, 1 H), 6.25 (t, J = 6.6 Hz, 1 H), 5.31 (s, 1 H), 5.06 (s, 1 H), 4.30 (s, 1 H), 3.87 (s, 1 H), 3.62 (s, 2 H), 2.21 (d, J = 5.3 Hz, 2 H). ¹³C NMR (101 MHz, DMSO-d ₆): δ = 161.58, 150.15, 140.21, 137.03, 121.32, 105.33, 88.14, 85.30, 71.10, 61.85. HRMS (ESI-TOF): m/z calcd for C₁₈H₁₇N₅O₇+Na: 439.1104; found: 439.2038.
• 39 Synthesis of tt-dUTP (6): To a solution of 4 (70 mg, 0.134 mmol, 1.0 equiv) in trimethyl phosphate (3 mL), freshly distilled POCl₃ (31 μL, 0.337 mmol, 2.5 equiv) was added under an argon atmosphere while cooling with ice. The solution was stirred for 24 h at ca. 4 °C. After 24 h, the starting material was not completely consumed. Bis(tributylammonium) pyrophosphate (370 mg, 0.674 mmol, 5.0 equiv) in DMF and tributylamine (0.351 mL, 1.48 mmol, 11.0 equiv) were simultaneously added to the reaction mixture in ice-cold condition. The reaction was continued for 30 min at 4 °C, quenched with 1 M triethyl ammonium bicarbonate buffer (TEAB, 15 mL), and washed with ethyl acetate. The aqueous layer was evaporated and purified using a DEAE Sephadex-A25 anion exchange column (0.1–1 M TEAB buffer, pH 7.5) followed by HPLC (TEAB buffer and acetonitrile solvent system). Evaporation of the appropriate fraction gave the desired triphosphate (10mg, 11% yield) as the triethyl ammonium salt. ¹H NMR (400 MHz, D₂O): δ = 8.38 (s, 2 H), 7.55 (s, 1 H), 7.51 (d, J = 13.1 Hz, 1 H), 6.68 (d, J = 31.2 Hz, 1 H), 6.37–6.21 (m, 1 H), 5.92 (d, J = 20.6 Hz, 1 H), 4.05 (s, 1 H), 2.90 (d, J = 8.5 Hz, 2 H), 2.54 (s, 3 H). ³¹P NMR (162 MHz, D₂O): δ = 6.36 (d, J = 10.0 Hz), –10.43 (d, J = 25.7 Hz), –22.9 (t). HRMS (ESI-TOF): m/z calcd for C₁₈H₂₀N₅O₁₆P₃–H: 652.9956; found: 652.9969.