Quantification of Thiopurine/UVA-Induced Singlet Oxygen Production

Abstract

Thiopurines were examined for their ability to produce singlet oxygen ((1)O(2)) with UVA light. The target compounds were three thiopurine prodrugs, azathioprine (Aza), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), and their S-methylated derivatives of 6-methylmercaptopurine (me6-MP) and 6-methylthioguanine (me6-TG). Our results showed that these thiopurines were efficient (1)O(2) sensitizers under UVA irradiation but rapidly lost their photoactivities for (1)O(2) production over time by a self-sensitized photooxidation of sulfur atoms in the presence of oxygen and UVA light. The initial quantum yields of (1)O(2) production were determined to be in the range of 0.30-0.6 in aqueous solutions. Substitution of a hydrogen atom with a nitroimidazole or methyl group at S decreased the efficacy of photosensitized (1)O(2) production as found for Aza, me6-MP and me6-TG. (1)O(2)-induced formation of 8-oxo-7,8-dihydro-2'-dexyguanosine (8-oxodGuo) was assessed by incubation of 6-methylthiopurine/UVA-treated calf thymus DNA with human repair enzyme 8-oxodGuo DNA glycosylase (hOGG1), followed by apurinic (AP) site determination. Because more 8-oxodGuo was formed in Tris D(2)O than in Tris H(2)O, (1)O(2) is implicated as a key species in the reaction. These findings provided quantitative information on the photosensitization efficacy of thiopurines and to some extent revealed the correlations between photoactivity and phototoxicity.

Figure 1

Extinction coefficient spectra measured at ambient temperature in 50 mM TE buffer (pH 7.4) solutions for adenine (green solid line), Aza (blue solid line), 6-MP (red solid line), me6-MP (red dot line), 6-TG (black solid line) and me6-TG (black dot line)

Figure 2

Time-resolved ¹O₂ phosphorescence recorded at 1270 nm upon pulsed-irradiation of thiopurines at 355 nm. 2a–2e: ¹O₂ decay in air-saturated CD₃CN solutions 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 the same sample but in the presence of NaN₃ as a control, dots: experimental data and red line: theoretical simulation. Each decay curve is an average of data points obtained from 2–5 laser pulses. 2a: Aza at OD_{355 nm} = 0.21, 2b: 6-MP at OD_{355 nm} = 0.19, 2c: 6-TG at OD_{355 nm} = 0.19, 2d: me6-MP at OD_{355 nm} = 0.15, 2e: me6-TG at OD_{355 nm} = 0.07

Figure 2

Time-resolved ¹O₂ phosphorescence recorded at 1270 nm upon pulsed-irradiation of thiopurines at 355 nm. 2a–2e: ¹O₂ decay in air-saturated CD₃CN solutions 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 the same sample but in the presence of NaN₃ as a control, dots: experimental data and red line: theoretical simulation. Each decay curve is an average of data points obtained from 2–5 laser pulses. 2a: Aza at OD_{355 nm} = 0.21, 2b: 6-MP at OD_{355 nm} = 0.19, 2c: 6-TG at OD_{355 nm} = 0.19, 2d: me6-MP at OD_{355 nm} = 0.15, 2e: me6-TG at OD_{355 nm} = 0.07

Figure 3

The effect of D₂O and H₂O on AP site formation upon UVA (~10 W lamp) irradiation of 0.10 mg/mL DNA in the presence of 6-methylthiopurines. The data represent the mean of three to six repeating experiments in D₂O or H₂O of 50 mM TE buffer (pH 7.4) solutions. 1. DNA in D₂O, 2. DNA in H₂O, 3. DNA and me6-TG (0.05 mM) in D₂O, 4. DNA and me6-TG (0.05 mM) in H₂O, 5. DNA and me6-TG in D₂O prior incubation with hOGG1, 6. DNA and me6-TG (0.05 mM) in H₂O prior incubation with hOGG1, 7. DNA and me6-MP (0.15 mM) in D₂O, 8. DNA and me6-MP (0.15 mM) in H₂O, 9. DNA and me6-MP (0.15 mM) in D₂O prior incubation with hOGG1, 10. DNA and me6-MP (0.15 mM) in H₂O prior incubation with hOGG1.

Scheme 1

Structures and metabolism of thiopurine prodrugs. Azathioprine (Aza) can convert to 6-mercaptopurine (6-MP) by cleavage of nitroimidazole group. Thiopurine prodrugs undergo extensive metabolism to 6-thioguanine (6-TG) nucleotides (6-TGN). 6-TG is also directly converted to 6-TGN by hypoxanthine phosphoribosyltransferase (HPRT). 6-TGN becomes incorporated into DNA. Thiopurine methyltransferase (TPMT) can convert 6-MP to 6-methylmercaptopurine (me6-MP) and 6-TG to 6-methylthioguanine (me6-TG).