Direct observation and quantitative characterization of singlet oxygen in aqueous solution upon UVA excitation of 6-thioguanines

Abstract

The incorporation of 6-thioguanine (6-TG) into DNA increases the risk of (1)O(2)-initiated skin cancer. We herein provide the first report on quantitative characterization of the photoactivity of 6-thioguanines including 6-TG and 6-thioguanosine. Time-resolved singlet oxygen luminescence was observed directly for the first time after UVA irradiation of 6-thioguanines in both CHCN(3) and aqueous solutions. Their photosensitization was characterized by the quantum yield of singlet oxygen production, showing a dramatic decrease over time from the initial 0.49-0.58 to zero. Experiments performed on both 6-TG and 6-thioguanosine did not show any significant difference in the quantum yield of singlet oxygen production, indicating that there was no potential participation of 7H- and 9H-tautomers. Our findings provide a primary basis for a better understanding of molecular events of thiopurine drugs in biological systems.

Scheme 1

Structures and metabolism of thiopurine prodrug incorporation into DNA

Scheme 2

Structures of 6-TG 7H- and 9H-amino tautomers and 6-thioguanosine

Scheme 3

Measurement and calculation of Φ_Δ

Figure 1

Extinction coefficient spectra of 6-TG in pH7.4 Tris buffer (solid black line) and in pH 10 NaOH (dash black line), 6-thioguanosine in pH 7.4 Tris buffer (solid red line) and pH 10 NaOH (dash red line), and guanine in pH 10 NaOH (blue line) solutions at ambient temperature

Figure 2

Time-resolved ¹O₂ luminescence recorded at 1270 nm upon pulsed-irradiation of 6-TG at 355 nm. 2a: ¹O₂ decay in an air-saturated pH 7.4 D₂O Tris buffer solution in the absence (solid line) and presence of 1.5 mM NaN₃ (dot line); insertion: 1^st-order kinetic fitting of ¹O₂ decay after correction with a control in the presence of NaN₃, dots: experimental data and red line: theoretical simulation. 2b: ¹O₂ decay in an air-saturated pH10 NaOH D₂O solution at time intervals of zero minute (solid line) and 3 minutes (short dash line), dot line: N₂-saturated sample; insertion: 1^st-order kinetic fitting of ¹O₂ decay after correction with a N₂-saturated control, dots: experimental data and red line: theoretical simulation.

Figure 3

³¹P NMR spectra of 9.0 μmol of phosphine and 0.15 μmol 6-TG in O₂-saturated pH 10 D₂O solutions with irradiation time of 0 (3a), 20 min (3b), 60 min (3c) and 120 min (3d) at 350 nm

Figure 4

³¹P NMR spectra from control experiments, 4a: 9.0 μmol of phosphine in O₂-saturated pH 10 D₂O solutions before irradiation, 4b: 20 min irradiation of 4a at 350 nm, 4c: 9.0 μmol of phosphine and 0.15 μmol 6-TG before irradiation, 4d: 4c kept in dark for 20 min