Design and Synthesis of the G-Quadruplex-Specific Cleaving Reagent Perylene-EDTA‚Iron(II) Wirote Tuntiwechapikul, Jeong Tae Lee, and Miguel Salazar* DiVision of Medicinal Chemistry College of Pharmacy and Institute for Cellular and Molecular Biology The UniVersity of Texas at Austin Austin, Texas 78712 ReceiVed February 7, 2001 ReVised Manuscript ReceiVed April 25, 2001 The structural uniqueness of G-quadruplex DNA makes it an ideal target for drug design. Various small molecules have been found to bind to G-quadruplex DNA. These include porphyrins, diamidoanthraquinones, carbocyanine dyes, and perylene diimides, among others. 1 The perylene diimide, N,N′-bis[2-(1-piperidino)- ethyl]3,4,9,10-perylenetetracarboxylic diimide (PIPER) has been reported to be selective for G-quadruplex structures with little affinity for either single- or double-stranded DNA. 1 Herein we report on the synthesis and characterization of a PIPER derivative, perylene-EDTA‚iron(II) that selectively cleaves G-quadruplex DNA in the presence of dithiothreitol (DTT). EDTA‚Fe(II) has been tethered to a variety of small molecules, peptides, and nucleic acids to effect cleavage of single- and double-stranded DNA in the presence of dioxygen and a reducing agent such as DTT. 2 A similar approach has been taken to effect cleavage of triple-stranded DNA. 3 Bleomycin-nickel(III) has been found to cleave loop residues in the intramolecular G-quadruplex structure formed by d(T 2 G 4 ) 4 , 4 and certain porphyrins are known to effect cleavage of G-quadruplex DNA when irradiated with UV light. 5 However, both porphyrins and bleomycin also bind to duplex DNA. Thus, although a nuclease specific for G- quadruplex DNA has been found, 6 no G-quadruplex-specific cleaving reagent has been reported to date. Among the G-quadruplex binding molecules that have been reported, PIPER appears to be one of the most selective. This molecule is known to bind by stacking on the 3′-G tetrad of parallel stranded G-quadruplex DNA and positioning the side chains in opposing grooves. 7 Indirect evidence indicates that this molecule can also bind to intramolecular G-quadruplex structures, 1 although the precise mode of binding to this type of quadruplex structure motif is not known. PIPER has also been shown to facilitate the formation of G-quadruplex structures from single- stranded DNA. 8 Furthermore, as a result of its binding to G-quadruplex structures, PIPER has been found to inhibit the extension of G-rich telomeric primers by telomerase. 1,7 Due to its strong binding to G-quadruplex DNA, it was thus natural to select this molecule as a starting point in the design of a G-quadruplex specific cleaving reagent. The synthesis of perylene-EDTA was carried out by coupling triethylester EDTA to N,N′-bis[diethylamine]-3,4,9,10-perylene- tetracarboxylic diimide, both of which were obtained following published procedures. 2,9 Cleavage of the triethylester was then carried out with NaOH (see Supporting Information). Figure 1 shows the DNA substrate used for assaying cleavage of G- quadruplex DNA by perylene-EDTA‚Fe(II). The G-quadruplex region consists of four repeats of the Tetrahymena telomeric repeat sequence T 2 G 4 , flanked by two random-sequence duplex regions consisting of 21 base pairs and 33 base pairs, respectively. Seven T residues in the bottom strand were used to bridge the G-quadruplex section in the top strand. The G-quadruplex in the top strand was preformed in 100 mM K + buffer prior to annealing to the bottom strand. The duplex-quadruplex substrate was then purified by nondenaturing PAGE. DMS methylation protection analysis (Supporting Information) of the top strand hybridized to the bottom strand in 100 mM K + confirmed that the four T 2 G 4 repeats form a stable G-quadruplex structure in which only three guanines in each of the four repeats are involved in G-tetrad formation. Figure 2 shows the cleavage pattern observed for the top strand when the duplex-quadruplex substrate in Figure 1 is incubated in the presence of perylene-EDTA‚Fe(II) and DTT. Two promi- nent cleavage sites within the G-quadruplex region centered on G4 and G16 were observed. Cleavage around G4 includes some cleavage of the two T residues of the first T 2 G 4 repeat, while cleavage around G16 includes some cleavage of the two T residues in the third T 2 G 4 repeat. Relatively minor cleavage of the second and fourth sets of guanine repeats was observed and only at high concentrations of perylene-EDTA‚Fe(II). Degradation products resulting from the radiolabeling process precluded a detailed analysis of the 3′-end of the top strand. However, except for cleavage centered at the first A immediately adjacent to the last T 2 G 4 repeat, no other significant cleavage was observed. Also, * To whom correspondence should be addressed. E-mail:m-salazar@ mail.utexas.edu. Fax: 512-232-2606. (1) (a) Kerwin, S. M. Curr. Pharm. Design 2000, 6, 441-471 and references therein. (b) Hurley, L. H.; Wheelhouse, R. T.; Sun, D.; Kerwin, S. M.; Salazar, M.; Fedoroff, O. Y.; Han, F. X.; Han, H.; Izbicka, E.; Von Hoff, D. D. Pharmacol. Ther. 2000, 85, 141-158 and references therein. (2) (a) Dervan, P. B. Science 1986, 232, 464-471. (b) Moser, H. E.; Dervan, P. B. Science 1987, 238, 645-650. (c) Sluka, J. P.; Griffin, J. H., Mack, D. P.; Dervan, P. B. J. Am. Chem. Soc. 1990, 112, 6369-6374. (d) Wade, W. S.; Mrksich, M.; Dervan, P. B. J. Am. Chem. Soc. 1992, 114, 8783- 8794. (3) Zain, R.; Marchand, C.; Sun, J-s.; Nguyen, C. H.; Bisagni, E.; Garestier, T.; He ´le `ne, C. Chem. Biol. 1999, 6, 771-777. (4) Guan, L. L.; Kuwahara, J.; Sugiura, Y. Biochemistry 1993, 32, 6161- 6145. (5) (a) Wheelhouse, R. T.; Sun, D.; Han, H.; Han, F. X.; Hurley, L. H. J. Am. Chem. Soc. 1998, 120, 3261-3262. (b) Han, F. X.; Wheelhouse, R. T.; Hurley, L. H. J. Am. Chem. Soc. 1999, 121, 3561-3570. (6) (a) Liu, Z.; Frantz, J. D.; Gilbert, W.; Tye, B.-K. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 3157-3161. (b) Liu, Z.; Gilbert, W. Cell 1994, 77, 1083- 1092. (7) Fedoroff, O. Y.; Salazar, M.; Han, H.; Chemeris, V.; Kerwin, S. M.; Hurley, L. H. Biochemistry 1998, 37, 12367-12374. (8) Han, H.; Cliff, C. L.; Hurley, L. H. Biochemistry 1999, 38, 6981- 6986. (9) Luka ´c ˇ, I.; Langhals, H. Chem. Ber. 1983, 116, 3524-3528. Figure 1. Diagram of the duplex-quadruplex substrate used to assay cleavage of G-quadruplex DNA by perylene-EDTA‚Fe(II). 5606 J. Am. Chem. Soc. 2001, 123, 5606-5607 10.1021/ja0156439 CCC: $20.00 © 2001 American Chemical Society Published on Web 05/19/2001