Bioconjugate Chem. zyxwvut 1991, 2, 57-66 57 Photolytic Cleavage of DNA by Nitrobenzamido Ligands Linked to 9-AminoacridinesGives DNA Polymerase Substrates in a Wavelength-DependentReaction Peter E. Nielsen,*l+Michael Egholm,t Troels Koch,t Jam B. Christensen,$ and Ole Buchardt’J Research Center for Medical Biotechnology, University of Copenhagen, Department of Biochemistry B, The Panum Institute, Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark, and Chemical Laboratory 11, The H. C. Orsted Institute, Universitetsparken 5, DK-2100 Copenhagen 0, Denmark. Received December 13, 1990 A series of reagents containing 3- or 4-nitrobenzamido ligands tethered to 9-aminoacridine via variable- length linkers have been prepared and their properties as photochemical DNA cleavers (photonu- cleases) examined. When irradiated with -300-nm light, where the nitrobenzamido ligand can absorb, they cleave DNA in an oxygen-independent reaction presumably involving oxygen transfer from the nitro group to the deoxyribose units of the DNA backbone (Nielsen et al., 1988b). This reaction is pH independent and only slightly affected by the linker length, and the DNA fragments are not substrates for DNA polymerase. When -420-nm light is used, were only the 9-aminoacridinyl ligands absorb, the DNA cleavage is also oxygen-independent but pH dependent, requires DNA saturation with the reagent (base paheagent I 2), and is most efficient with the longer linkers. The cleavage is specific for guanine residues and results in 5‘-phosphate termini and heterogeneous (more than four products) 3‘-termini. One of the products is presumably 3’-hydroxy since DNA photocleaved with nitrobenzamido acridine reagents and 420-nm radiation are substrates for DNA polymerase in a nick translation assay zyx as well as for the Klenow fragment. An electron-transfer mechanism is suggested. INTRODUCTION Synthetic DNA cleaving reagents (“syntheticnucleases”) (Sigman, 1990) are of interest in molecular biology and medicinal chemistry for designingDNA-cleavingmolecules directed at specific sequences (see, e.g., Dervan, 1986; Moser & Dervan, 1987; Chi-hong & Sigman, 1986; Chen & Sigman, 1988; Le Doan et al., 1987; Praseuth et al., 1988a,b) and as molecular probes (for a recent review on DNA probes, see Nielsen, 1990). In most cases the cleaving principle involves oxidation of the DNA (RNA) backbone deoxyriboses by transition-metal complexes (e.g., Fe(II), Cu(I), Ru(II), etc.) (Hertzberg & Dervan, 1984; Tullius & Dombroski, 1985; Sigman, 1986; Barton, 1986; Stubbe & Kozarich, 1987; Goyne & Sigman, 1987;Tullius, 1989).In some cases photochemical activation of the cleavage reaction has also been exploited (see, e.g., Freifelder et al., 1961; Friedman & Brown, 1978; Subramanian & Meares, 1985,1986;Fleisher et al., 1986;Blacker et al., 1986;Ward et al., 1986; Buchardt et al., 1987; Nielsen et al., 1988a-c; Jeppesen et al., 1988;Behr, 1989;Veal & Kill, 1988;Jeppe- sen & Nielsen, 1989a,b; Uchida et al., 1989). From a biological point of view, cleavage mechanisms which result in products that can be processed directly by conventional DNA-metabolizing enzymes would be ad- vantageous, and metal-activated hydrolytic cleavage of DNA has been reported with Zn(II)), Cd(II), and Pb(I1) complexes of trisphenanthroline derivatives (Basile et al., 1987). Thus the development of more efficient DNA- cleaving ligands is warranted and preferably with a catalytic cleavage mechanism. We have recently found that nitrobenzamido ligands linked to 9-aminoacridine photocleave DNA (Buchardt et al., 1987; Nielsen et al., 1988b)presumably via a hydrogen- + The Panum Institute. 4 The H. C. 0rsted Institute. 1043-1802/91/2902-0057$02.50/0 abstraction/oxygen-transfer mechanism. We also found that 4-nitrobenzamido derivatives are 10-30 times as efficient as the corresponding 2- or 3-substituted reagents, and that the reaction is induced by light absorbed by the nitrobenzimido chromophore (Nielsen et al., 1988b). Furthermore, it was recently shown that nitrooligopyr- roles (distamycin analogues) photocleave DNA (Nishi- waki et al., 1990). We now report that a different DNA photocleavage mechanism is in operation for the nitrobenzamido acri- dine reagents with light absorbed by the aminoacridine chromophore, and that one or more of the DNA products are substrates for zyxwv Escherichia zyxw coli DNA polymerase. A mechanism involving electron transfer between the acri- dine and the nitrobenzamido ligands is suggested. EXPERIMENTAL PROCEDURES Chemistry. General Remarks.l All solvents used for chromatography were distilled prior to use. NMR spectra were recorded on a JEOL FX 9OQ spectrometer. Mass spectrometry was performed on a MassLab VG 12-250 quadrupole instrument fitted with a VG FAB source and probe. Melting points were determined on a Buchi melting point apparatus and are uncorrected. Methyl 6-aminohexanoate hydrochloride was pre- pared according to the procedure given by Garmaise et al. (1958). 1 Abbreviations: BSA, bovine serum albumin; DMF, dime- thylformamide; DMSO, dimethyl sulfoxide; DTT, dithiothrei- tol; HMPA, hexamethylphosphorotriamide; THF, tetrahy- drofuran; Boc, tert-butoxycarbonyl; DhbtOH, 3,4-dihydro-3- hydroxy-4-0~0-1,2,3-bemtriazine; DCC, dicyclohexylcarbodiimide; SDS, sodium dodecyl sulfate; NMR, nuclear magnetic resonance; s, singlet; d, doublet;dd, doubletof doublets; t, triplet; q, quartet; m, multiplet; b, broad; 6, chemical shift. 0 1991 American Chemical Society