Photochemistry of the Furan-Side 8-Methoxypsoralen-Thymidine Monoadduct Monoadduct? Inside the DNA Helix. Conversion to Diadduct and to Pyrone-Side

We have studied the photochemical reactions of 8-methoxypsoralen (&MOP) with calf thymus DNA. Analysis of the photoproducts formed was carried out by enzymatic digestion of the 8-MOP-modified DNA, followed by HPLC separation of photoadducts by high-pressure liquid chromatography (HPLC). The 4’,5’ (furan-side) monoadduct of &MOP bound to thymidine is converted to cross-liilked thymidine8-MOP-thymidine diadduct by 341.5 nm light with a quantum yield of 0.028 & 0.004. This is 4 times greater than the quantum yield for initial adduct formation (0.0065 f 0.0004). When low levels of %MOP are covalently bound to DNA by using 397.9 nm light, less than 10% of the adducts formed are diadducts yet nearly 70% are in 5’-TpA cross-linkable sites. The furan-side monoadducts in these sites can subsequently be converted to diadduct or to a lesser extent 3,4 (pyrone-side) monoadduct. R o r a l e n s are a class of compounds that have been used as photosensitizing agents in the treatment of psoriasis and as probes in the elucidation of nucleic acid structure and function (Song & Tapley, 1979; Thompson & Hearst, 1983a,b). This usefulness of psoralens is the result of their photoreactivity with pyrimidine bases in nucleic acids. Psoralens intercalate between base pairs in the DNA double helix and form cycloaddition products with pyrimidine bases upon irradiation with long wavelength ultraviolet light. This cycloaddition can occur between the 5,6 double bond of a pyrimidine base and the 3,4 (pyrone) or 4’3’ (furan) double bond of the psoralen (Straub et al., 1981; Kanne et al., 1982a). It is also possible to form interstrand cross-links via cycloaddition at both ends of a psoralen molecule (Kanne et al., 1982b). The structures of 8-methoxypsoralen (%MOP), of the monoaddition products of &MOP and thymidine (dT) at the furan and pyrone side, and of the dT-8-MOP-dT diadduct, or cross4inked product, are shown in Figure 1. The reaction between &MOP and calf thymus DNA occurs almost exclusively with thymidine. While a small amount of reaction with cytosine does occur, in this report we consider only those sites in the DNA containing thymidine as reactive sites. This study is primarily concerned with the quantification of the formation of the interstrand diadduct shown in structure 4 from the furan-side monoadduct shown in structure 2. 8-Methoxypsoralen was selected as the psoralen derivative to be studied for three reasons. First, &MOP is probably the most widely used psoralen derivative for both research and clinical application. Second, the photochemical binding of %MOP to DNA results in the formation of a significant proportion of pyrone-side monoadduct (ca. 20%), which is not the case with all psoralen derivatives (Kanne et al., 1984). Finally, previous work has established procedures for the isolation and quantitative analysis of the various adducts formed between 8-MOP and DNA (Kanne et al., 1982b). Using these procedures, we have analyzed the adduct popu‘This work was supported by the National Institute of General Medical Sciences, Department of Health and Human Services, Grants GM25151 and GM11180. The Nd:YAG laser used in this study was purchased with NSF Grant CHE 81 16042. 0006-2960/85/04241669$01.50/0 lation under conditions where mostly monoadducts are formed and then monitored the conversion of these monoadducts to diadducts as a function of energy absorbed. In the course of the study, an interesting observation on the source of the pyrone-side monoadduct has been made. Our results show that (1) the quantum yield for conversion of furan-side monoadduct to diadduct is approximately 4 times greater than the quantum yield for initial photoaddition of 8-MOP to DNA, (2) furan-side monoadduct is converted not only to diadduct but also to pyrone-side monoadduct, and (3) at low levels of 8-MOP covalently bound to DNA, &MOP exhibits a preference for monoaddition at 5’-TpA cross-linkable sites. While our observations are consistent with a mechanism whereby furan-side monoadduct undergoes in-helix photoisomerization to form pyroneside monoadduct, this mechanism must still be experimentally verified. We are currently addressing this problem. EXPERIMENTAL PROCEDURES Materials. 8-( [3H3] Methoxy)psoralen was obtained from HRI Associates, Inc. (Emeryville, CA). Calf thymus DNA and all hydrolytic enzymes were obtained from Sigma Chemical Co. (St. Louis, MO). Dark Binding. The dissociation constant was determined for the intercalation of &MOP into DNA by using equilibrium dialysis (Isaacs et al., 1977). Spectrapor 2 (Spectrum Medical Industries, Los Angeles, CA) dialysis tubing was boiled in a saturated sodium bicarbonate solution for ca. 20 min. A l-mL solution of 730 pg/mL DNA in 10 mM tris(hydroxymethy1)aminomethane (Tris)’ /1 mM EDTA, pH 7.5, was tied in a dialysis bag and placed in 18 mL of ca. 30 pg/mL 8-MOP solution in 10 mM Tris/l mM EDTA. This was allowed to equilibrate in the dark with stirring for 72 h. &MOP concentrations inside and outside of the dialysis bag were determined by scintillation counting, and the DNA concentration inside the bag was determined by measuring the optical density I Abbreviations: Tris, tris(hydroxymethy1)aminomethane; EDTA, ethylenediaminetetraacetic acid; HPLC, high-pressure liquid chromatography.