Regulus et al. 10.1073/pnas.0706044104. |
Fig. 5. 1H NMR spectrum of the slowest eluting isomer 4d obtained by chemical synthesis and purified by HPLC. The isomer 4d is contaminated (≈ 10%) with isomer 4c.
Fig. 6. 2D COSY NMR spectrum of 4d. Interaction between protons 7 and 8 together with interactions among protons 2, 3, and 10 are indicated.
Fig. 7. UV absorption spectra of the four isomers of 4 in water.
Fig. 8. Circular dichroism spectra of the four isomers of 4. An almost opposite Cotton effect is observed at 190 nm for isomers 4a and 4b on one hand and for isomers 4c and 4d on the other hand, strongly suggesting that the cytosine adducts 4 exist as four diastereomers.
Fig. 9. Typical HPLC-MS/MS chromatograms showing the formation of dCyd adducts 4 in cells incubated with increasing concentrations of bleomycin for 1 h at 37°C. DNA was extracted by using a recently optimized protocol (29) and then digesting to nucleosides by using a mixture of endo- and exonuclease and alkaline phosphatase. Then samples were analyzed by HPLC-MS/MS to determine the amount of 4. Before each series of analyses, three different quantities of purified 4 were injected onto the HPLC-MS/MS system to allow us to perform an external calibration and thus to determine the levels of 4 in the hydrolyzed DNA samples. The reported chromatograms correspond to the 342 ® 226 transition used to quantified 4 in control cells (dotted line) or cells treated with either 50 mM (dashed line) or 100 mM (solid line) bleomycin for 1 h.
Fig. 10. Inhibition of the formation of 4 by methoxyamine (MX). Levels of 4 were determined by HPLC-MS/MS upon g-irradiation of aerated aqueous solution of isolated DNA irradiated either in the absence (0 mM, dot-dash line) or in the presence of increasing concentrations 1.5 mM (dotted line), 3 mM (dashed line) and 5 mM (solid line) of MX. As expected, the radiation-induced formation of 8-oxodGuo is not decreased by the presence of MX during the irradiation (data not shown). In addition, no decomposition of dCyd adducts 4 was observed upon incubation with MX, indicating that the observed decrease is not due to direct reaction of MX with 4 but, rather, as expected, by its reaction with reactive aldehyde 2.
SI Text
Chemical synthesis and purification of 4. Synthesis.
Furfuryl ester and 3-chloroperbenzoic acid (mCPBA) were obtained from Sigma (St. Quentin Fallavier, France). A solution of furfuryl ester 5 (4 g) in methylene chloride (60 ml) was added over 15 min to a stirred solution of mCPBA (8 g) in methylene chloride (60 ml) at 0°C. The solution was then stirred for an additional 1-h period at 0°C before extraction with saturated aqueous sodium hydrogen carbonate solution (2 ´ 80 ml) and water (80 ml), dried by using Na2SO4, and lyophilized to give crude 2,5-dioxo-penten-3-yl acetate 6. The resulting products were suspended in water (5 ml) containing dCyd (2 mM), and the solution was maintained at 37°C under continuous stirring for 24 h. The resulting solution containing 7 was dried under reduced vacuum and treated with 1 ml of a 50:50 ammonia/methanol solution at room temperature for 30 min to generate 4. Then the solution was dried under reduced pressure, and the resulting products were purified by HPLC.
Purification of 4.
HPLC separations were performed by using a Spectra Physic P2000 HPLC pump and a UV2000 variable wavelengths detector purchased from Thermo Electro Corporation (Paris, France). Purification of 4 was achieved at a total flow rate of 1 ml/min in the gradient mode on a Uptishere ODB (5 mm, 250 ´ 4.6 mm) octadecylsilyl silica gel column (Interchim, Montluçon, France), the UV detector being set-up at 270 nm. The proportion of acetonitrile in 5 mM ammonium formate, starting from 0%, reached 15% within 30 min. Under these conditions, the four isomers of 4 elute at 14.9 min for both the two first nucleosides 4a and 4b and 16.6 min and 17.5 min for the two other isomers 4c and 4d, respectively. Collected fractions of either 4c or 4d; that were found to be contaminated by the other isomers, because of the difficulty of achieving a complete purification, were pooled and freeze-dried twice to remove ammonium formate (≈3% yield relative to dCyd for 4c and 4d diastereomers). Small amounts of purified 4a and 4b were obtained through successive enrichments by collecting the beginning and the end of the peak, respectively. This procedure was repeated three times to obtain the two diastereomers with a purity estimated to be ≈90%, as inferred from HPLC-MS/MS analysis. The purified amounts were sufficient to obtain UV absorption spectra of the different isomers (SI Fig. 7) and to perform circular dichroism analysis (SI Fig. 8), which strongly suggest that 4a and 4b on one hand and 4c and 4d on the other hand are likely two pairs of diastereomers.