Synthesis, DNA Binding, and Cleaving Properties of an Ellipticine

Bioconjugate Chem. 1997, 8, 789−792

789

Synthesis, DNA Binding, and Cleaving Properties of an Ellipticine-Salen‚Copper Conjugate Sylvain Routier,† Jean-Luc Bernier,† Jean-Pierre Catteau,† Pierre Colson,‡ Claude Houssier,‡ Christian Rivalle,§ Emile Bisagni,§ and Christian Bailly*,| Laboratoire de Chimie Organique Physique, URA 351 CNRS, USTL C3, 59655 Villeneuve d’Ascq, France, Laboratoire de Chimie Macromole´culaire et Chimie Physique, Universite´ de Lie`ge, 4000 Lie`ge, Belgium, UMR 176 CNRS, Institut Curie, Universite´ d’Orsay, Baˆt. 110-112, 91405 Orsay, France, and Laboratoire de Pharmacologie Mole´culaire Antitumorale du Centre Oscar Lambret et INSERM U-124, 59045 Lille, France. Received July 16, 1997X

The synthesis of a DNA-cutting agent that conjugates an ellipticine chromophore and a copper complex of bis(salicylidene)ethylenediamine, referred to as a salen, is reported. The presence of the salen‚Cu complex allows cleavage of DNA via oxygen-based radicals, and the ellipticine moiety serves as a DNA anchor. Spectroscopic measurements indicate that the intercalation geometry of the ellipticine chromophore is preserved with the hybrid. The cleavage is much more efficient with the conjugate than with the Schiff base copper complex alone.

Among the strategies available for the design of artificial nucleases is the use of metal complexes that create different types of DNA lesions (1). While considerable attention has been focused on the development of phenanthroline- and porphyrin-based metal complexes to tune the selectivity and reactivity of such chelates, few studies have sought to harness the potential of metal complexes of bis(salicylidene)ethylenediamine, usually referred to as salen. Complexes of tetradentate salentype Schiff bases with Cu, Co, and Mn have proved useful as synthetic catalysts to investigate nucleic acids conformation (2). In recent studies, we have investigated the capacity of different types of salen-CuII complexes to cleave DNA. Initially, we reported that a functionalized salen-CuII complex is capable of triggering singlestranded and nonspecific DNA cleavage after activation with a reducing agent (3). Subsequently, we showed that the coupling of the salen moiety to anthraquinone derivatives significantly promotes their affinity for DNA but has no effect on the cleavage reaction (4). More recently, a DNA minor groove binding element was coupled to the salen moiety in an effort to increase the specificity of cleavage, but the cleavage remained relatively weak (5). Here we report the preparation of an ellipticine-salen‚ Cu conjugate (Figure 1) and its ability to bind and to cleave DNA. The appended ellipticine chromophore serves as a DNA anchor, and as such, it potentiates significantly the capacity of the salen moiety to cleave DNA. The salen moiety functionalized with a butylamino side chain was synthesized directly as a copper complex as recently described (3). The ellipticine derivative bearing an aminopropylaminoethyl side chain (6) was first reacted with succinic anhydride to give the corresponding acid,1 which was then condensed with the salen moiety via a conventional coupling procedure using dicyclohexylcarbodiimide and N-hydroxybenzotriazole.2 * Author to whom correspondence should be addressed [fax (+33) 320 16 92 29; e-mail [email protected]]. † CNRS-USTL. ‡ Universite ´ de Lie`ge. § Institut Curie. | COL-INSERM. X Abstract published in Advance ACS Abstracts, November 1, 1997.

S1043-1802(97)00131-6 CCC: $14.00

Figure 1. Structure of the ellipticine-salen‚copper complex.

The absorption spectrum of the conjugate is significantly modified in the presence of DNA (Figure 2). The absorption band centered at 308 nm corresponding to the ellipticine moiety is shifted by 11 nm when the ligand is fully bound to DNA, and a marked hypochromism is observed. The interaction of the salen‚Cu moiety with DNA causes a weak hypochromism in the 360 nm band. Circular dichroism (CD) and electric linear dichroism (ELD) experiments were performed to define the orientation of the ligand chromophore with respect to the DNA helix. In contrast to what we previously reported with the functionalized salen‚Cu complex (3), the CD spectra obtained with the ellipticine-salen‚Cu hybrid in the presence of increasing DNA concentrations show no isodichroic point, indicating that the binding is geometrically heterogeneous (spectra not shown). The CD 1 The succinyl side chain was introduced by reacting 1-[[(ethylamino)propyl]amino]-9-methoxyellipticine (376 mg, 1 mmol) with succinic anhydride (100 mg, 1 mmol) in 200 mL of toluene for 5 h under reflux. After evaporation of the solvent, the functionalized ellipticine derivative was recrystallized in acetone. Yield: 310 mg, 63%, mp 150 °C; 1H NMR (DMSO-d6) δ 1.19 (t, 3H, J ) 6.9 Hz, CH3-CH2), 1.88 (q, 2H, J ) 6.8 Hz, CH2-β), 2.67 (s, 3H, CH3-5), 3.38 (s, 3H, CH3-11), 3.4-3.5 (m, 10H, CH2-R, CH2-8, CH2-CH3, CO-CH2, CH2-CO2H), 3.92 (s, 3H, OCH3), 6.57 (s large, 1H, NH-1), 7.02 (d, 1H, J ) 6.2 Hz), 7.16 (dd, 1H, J ) 2.4 and 8.7 Hz), 7.47 (d, 1H, J ) 8.7 Hz, H-7), 7.77 (d, 1H, J ) 6.2 Hz, H-3), 7.79 (d, 1H, J ) 2.4 Hz, H-10), 11.04 (s, 1H, NH-6). Anal. Calcd for C27H32N4O4‚H2O: C, 65.57; H, 6.93; N, 11.33. Found: C, 65.28; H, 6.88; N, 11.05.

© 1997 American Chemical Society

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Routier et al.

Figure 2. Absorption spectra of the ellipticine-salen‚CuII hybrid (35 µM) in the absence (full line) and presence (dashed line) of calf thymus DNA (175 µM) in 1 mM sodium cacodylate buffer, pH 6.5. The spectrum corresponding to the drug bound to DNA was referenced against a DNA solution of exactly the same concentration and was adjusted to a common baseline. Figure 4. Dependence of the reduced dichroism ∆A/A on (A) the wavelength and (B) the DNA-phosphate-to-drug ratio. Conditions: (A) (9) calf thymus DNA, (2) poly(dA-dT)‚poly(dAdT), (b) poly(dG-dC)‚poly(dG-dC), P/D ) 10, 12.5 kV/cm; (B) (O) 310 nm, (b) 350 nm, 12.5 kV/cm, in 1 mM sodium cacodylate buffer, pH 6.5.

Figure 3. Dependence of the CD ∆A on the DNA-phosphateto-drug ratio. The CD was monitored at 320 nm in the presence of 50 µM drug and decreasing concentrations of calf thymus DNA in 1 mM sodium cacodylate buffer, pH 6.5.

signal monitored at 320 nm first increases with increasing P/D ratios, until a P/D value of 0.5-1 is reached, and then rapidly decreases to become negative at a P/D value >5 and remains constant at P/D > 10 (Figure 3). The same biphasic evolution of the CD at 320 nm was previously reported with a structurally related ellipticine derivative (7). The decrease of the CD peak at P/D ratio G 1 is attributable to decrease of excitonic coupling arising from a distance increase between adjacent intercalated molecules. Similar variation of molar dichroism with the P/D ratio has been reported with other typical intercalating agents such as ethidium bromide and acridine orange (8). 2 N,N′-Dicyclohexylcarbodiimide (7.6 mg, 36.8 mmol) and N-hydroxybenzotriazole (5.1 mg, 37.7 mmol) dissolved in 1 mL of DMF were added to a cold solution of the ellipticine acid (16 mg, 32.6 µmol) in CH2Cl2. The mixture was stirred for 2 h at 0 °C before a solution containing the aminosalen‚Cu complex (20.3 mg, 39.4 mmol) and triethyamine (6 µL, 49.3 mmol) was added. The reaction was continued for 1 h at 0 °C and then for 4 days at room temperature. After evaporation of the solvent in vacuo, the crude residue is dissolved in pure CH2Cl2 and washed twice in turn with 10% citric acid (25 mL), 1 N sodium bicarbonate (10 mL), and water (20 mL). The organic layer is dried over Na2SO4 and evaporated under reduced pressure to yield the final product as a brown solid: 22 mg, 65%, mp 131-133 °C; IR ν 3400 (CONH), 2930 (CH2, CH3), 1730 (CONH), 1690 (CONH), 1620 (NH) cm-1; MS (laser desorption) 859.3 (M + 1)+, 921.2 (M + Cu)+, 881.4 (M + Na)+. Anal. Calcd for C47H53N7O5‚Cu: C, 65.71; H, 6.22; N, 11.42. Found: C, 65.80; H, 6.25; N, 11.35.

The ELD spectra of the hybrid bound to calf thymus DNA and to the alternating copolymers poly(dA-dT)‚poly(dA-dT) and poly(dG-dC)‚poly(dG-dC) are shown in Figure 4A. The mode of binding of the drug was analyzed only on the basis of the highest ELD values, obtained when the drug molecules are fully bound to DNA, i.e., for P/D ratios >10. At lower P/D ratio, the measured ELD values fall significantly due to the appearance of unbound molecules in the solution (Figure 4B). The reduced dichroism is always negative in sign, even in the 350-400 nm region where the salen‚Cu unit aborbs the light. The reduced dichroism values do not vary markedly when the drug is bound to calf thymus DNA that contains about 42% A‚T base pairs or to the AT and GC polynucleotides; the ellipticine moiety retains much the same orientation whatever the DNA composition. The negative LD measured with the drug-DNA complex at 310 nm (i.e. in the ellipticine absorption band) is equally intense as that measured with the DNA alone at 260 nm (∆A/A ) -0.38), but the LD measured in the salen absoprtion band (360-400 nm) is only half of that obtained with DNA. This indicates that the ellipticine ring is tilted close to the plane of the DNA bases, consistent with an intercalative mode of binding, whereas the salen substituent does not insert between the base pairs. The orientation of the copper complex can be determined by measuring the angles R and β between the transition moments (of the bases and the dye chromophore, respectively) and the orientation axis of the DNA molecules. With calf thymus DNA, the ELD value in the 350-400 nm band was found to be -0.2, which corresponds to an orientation of the salen‚Cu moiety chromophore inclined at about 65° (β) to the helix axis. The angle is estimated by comparing the reduced dichroism at a given electric field strength for the DNA bases and for the hybrid in their respective absorption bands, assuming a theoretical angle of 90° (R) for the bases with respect to the orientation axis of the particles (9). The angle is 62° if an experimental angle of 72° is taken for

Communications

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Figure 5. (A) Cleavage of closed circular DNA. Supercoiled DNA (0.7 µg) was incubated at 37 °C for 2 h with the drug at the indicated concentration in the presence of 100 µM MPA. Control lanes refer to the plasmid DNA incubated without drug in the absence (DNA) and presence (MPA) of 2-mercaptopropionic acid. Forms I, II, and III refer to the supercoiled, nicked, and linear DNA forms, respectively. (B) Comparison of the cleavage efficiency of the conjugate (b) and the salen (O). The plots show the formation of nicked DNA (form II) as a function of the drug concentration. Three gels as shown in (A) were scanned and the results averaged.

the bases (10). Such an orientation is not consistent with a minor groove binding (which usually gives an angle of about 45°). It is more likely that the salen‚Cu moiety extends out from the surface of the double helix.3 DNA cleavage was analyzed by monitoring the conversion of supercoiled plasmid DNA (form I) to the nicked circular molecules (form II) and linear DNA (form III). The tests were performed under aerobic conditions in the presence of 2-mercaptopropionic acid (MPA) as a reducing agent. As shown in Figure 5A, the ellipticine derivative equipped with a salen‚CuII functionality is able to catalyze reductive cleavage of DNA. Incubation of the plasmid for 2 h with 20 µM conjugate suffices to completely convert the form I to the nicked form II, whereas in the same conditions only ∼50% of the DNA is cleaved with the salen moiety lacking the ellipticine. The reduced electrophoretic mobility of the nicked DNA reflects the intercalation of ellipticine molecules. The quantitative analysis of the cleavage data reveals that the hybrid compound is considerably more efficient than the salen‚Cu complex in producing nicked DNA species (Figure 5B). In other words, the ellipticine moiety 3 The CuII atom is tetracoordinated. The ESR spectrum of the conjugate-Cu complex was obtained from a frozen, aqueous solution (0.1 mM) at 77 K and 9.32 GHz. The magnetic parameters are A| ) 191 G, g| ) 2.23, and g⊥ ) 2.06. 4 Spin-trapping techniques have shown that the drug can serve as a source of oxygen-based free radicals, with hydroxy radicals OH• probably the ultimate reactive species. In the presence of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), solutions of the conjugate-Cu complex containing hydrogen peroxide generate an ESR spectrum characteristic of the DMPO-OH radical adduct (aN ) aH ) 15.2 G).

Figure 6. DNase I footprinting of the ellipticine-salen‚CuII conjugate bound to the 265- and 117-mer EcoRI-PvuII restriction fragments cut out from plasmid pBS. In each case the DNA was 3′-end labeled with [R-32P]dATP in the presence of AMV reverse transcriptase. The drug concentration (micromolar) is shown at the top of the appropriate gel lanes. The tracks labeled “Ct” contained no drug. The tracks labeled “GA” represent formic acid-piperidine markers specific for purines. Numbers on the left side of the gels refer to the numbering scheme of the fragments. The sequences protected from cleavage are indicated.

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contributes significantly to reinforce the single-strand cleavage efficiency of the salen complex.4 The capacity of the conjugate to recognize specific sequences in DNA was studied by DNase I footprinting. The gel in Figure 6 shows that the attachment of the salen moiety does not hinder the capacity of the ellipticine chromophore to recognize specific sequences. Two well-resolved footprints encompassing the sequences 5′CCCTCACT and 5′-AGTCACGA can be detected using micromolar drug concentrations. Complementary DNase I footprinting experiments were carried out with the 160 base pair tyrT fragment and, again, binding was shown to occur at various sequences with a preference for GCrich regions of DNA (not shown). The drug strongly discriminates against runs of adenines or thymines. The sequence selectivity of the conjugate is reminiscent to that of the ellipticine alone (11). The intercalating chromophore imposes its preference for GC-rich sequences. In conclusion, the experimental results show that the attachment of an intercalating ellipticine chromophore was the correct way to proceed to confer highly efficient cleavage of DNA, the first time this has been achieved with a hybrid intercalator-salen‚copper complex conjugate. On the basis of this success, we intend to develop a general strategy for constructing other salen-based molecules as synthetic conjugates that will afford an independent approach to the design of artificial nucleases. ACKNOWLEDGMENT

This work was supported by grants (to C.B.) from the Association pour la Recherche sur le Cancer and (to C.H. and P.C.) from the Actions de Recherches Concerte´es contract 95/00-93 and the FNRS, Te´le´vie 7/4526/96. Support by the “convention INSERM-CFB” is acknowledged. LITERATURE CITED (1) (a) Sigman, D. S., Mazumder, A., and Perrin, D. M. (1993) Chemical nucleases. Chem. Rev. 93, 2295-2316. (b) Pratviel, G., Bernadou, J., and Meunier, B. (1995) Carbon-hydrogen bonds of DNA sugar units as targets for chemical nucleases and drugs. Angew. Chem., Int. Ed. Engl. 34, 746-769. (2) (a) Muller, J. G., Chen, X., Dadiz, A. C., Rokita, S. E., and Burrows, C. J. (1992) Ligands effects associated with the intrinsic selectivity of DNA oxidation promoted by nickel(II) macrocyclic complexes. J. Am. Chem. Soc. 114, 6407-6411. (b) Woodson, S. A., Muller, J. G., Burrows, C. J., and Rokita, S. E. (1993) A primer extension assay for modification of guanine by Ni(II) complexes. Nucleic Acids Res. 21, 55245525. (c) Cheng, C.-C., Rokita, S. E., and Burrows, C. J. (1993) Nickel(III)-promoted DNA cleavage with ambient dioxygen. Angew. Chem., Int. Ed. Engl. 32, 277-278. (d) Muller, J. G., Paikoff, S. J., Rokita, S. E., and Burrows, C. J. (1994) DNA modification promoted by water-soluble nickel(II) salen

Routier et al. complexes: a switch to DNA alkylation. J. Inorg. Biochem. 54, 199-206. (e) Burrows, C. J., and Rokita, S. E. (1994) Recognition of guanine structure in nucleic acids by nickel complexes. Acc. Chem. Res. 27, 295-301. (f) Gravert, D. J., and Griffin, J. H. (1993) Specific DNA cleavage mediated by [salenMn(III)]. J. Org. Chem. 58, 820-822. (g) Gravert, D. J., and Griffin, J. H. (1996) Steric and electronic effects, enantiospecificity, and reactive orientation in DNA binding/ cleaving by substituted derivatives of [salenMnIII]+. Inorg. Chem. 35, 4837-4847. (h) Bhattacharya, S., and Mandal, S. S. (1995) Ambient oxygen activating water soluble cobaltsalen complex for DNA cleavage. J. Chem. Soc., Chem. Commun., 2489-2490. (i) Sato, K., Chikira, M., Fujii, Y., and Komatsu, A. (1994) Stereospecific binding of chemically modified salen-type Schiff base complexes of copper(II) with DNA. J. Chem. Soc., Chem. Commun., 625-626. (3) Routier, S., Bernier, J.-L., Waring, M. J., Colson, P., Houssier, C., and Bailly, C. (1996) Synthesis of a functionalized salen-copper complex and its interaction with DNA. J. Org. Chem. 61, 2326-2331. (4) Routier, S., Cotelle, N., Catteau, J.-P., Bernier, J.-L., Waring, M. J., Riou, J.-F., and Bailly, C. (1996) Salen-anthraquinone conjugates. Synthesis, DNA-binding and cleaving properties, effects on topoisomerases and cytotoxicity. BioOrg. Med. Chem. 4, 1185-1196. (5) Routier, S., Bernier, J.-L., Catteau, J.-P., and Bailly, C. (1997) Recognition and cleavage of DNA by a distamycinsalen‚copper conjugate. BioOrg. Med. Chem. Lett. 7, 17291732. (6) Rivalle, C., Wendling, F., Tambourin, P., Lhoste, J.-M., Bisagni, E., and Chermann, J.-C. (1983) Antitumor aminosubstituted pyrido[3′,4′:4,5]pyrrolo[2,3-g]isoquinolines and pyrido[4,3-b]carbazole derivatives: Synthesis and evaluation of compounds resulting from new side chain and heterocycle modifications. J. Med. Chem. 26, 181-185. (7) Bourdouxhe, C., Colson, P., Houssier, C., Sun, J.-S., Montenay-Garestier, T., He´le`ne, C., Rivalle, C., Bisagni, E., Waring, M. J., He´nichart, J.-P., and Bailly, C. (1992) Binding of a distamycin-ellipticine hybrid molecule to DNA and chromatin: spectroscopic, biochemical and molecular modeling investigations. Biochemistry 31, 12385-12396. (8) (a) Houssier, C., Hardy, B., and Fredericq, E. (1974) Interaction of ethidium bromide with DNA. Optical and electrooptical study. Biopolymers 13, 1141-1160. (b) Fredericq, E., and Houssier, C. (1972) Study of the interaction of DNA and acridine orange by various optical methods. Biopolymers 11, 2281-2308. (9) Houssier, C. (1981) Investigating nucleic acids, nucleoproteins, polynucleotides, and their interactions with small ligands by electrooptical systems. In Molecular Electro-Optics (S. Krause, Ed.) pp 363-398, Plenum Publishing, New York. (10) Chou, P.-J., and Johnson, W. C., Jr (1993) Base inclinations in natural and synthetic DNAs. J. Am. Chem. Soc. 115, 12051214. (11) Bailly, C., OhUigin, C., Rivalle, C., Bisagni, E., He´nichart, J. P., and Waring, M. J. (1990) Sequence-selective binding of an ellipticine derivative to DNA. Nucleic Acids Res. 18, 6283-6291.

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