Targeting the DNA Cleavage Activity of Copper Phenanthroline and

Targeting the DNA Cleavage Activity of Copper Phenanthroline and. Clip-Phen to A‚T Tracts via Linkage to a Poly-N-methylpyrrole. Marguerite Pitié,â...
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Bioconjugate Chem. 2000, 11, 892−900

Targeting the DNA Cleavage Activity of Copper Phenanthroline and Clip-Phen to A‚T Tracts via Linkage to a Poly-N-methylpyrrole Marguerite Pitie´,† J. David Van Horn,‡ Denis Brion,‡ Cynthia J. Burrows,*,‡ and Bernard Meunier*,† Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne, 31 077 Toulouse Cedex 4, France, and Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112. Received May 19, 2000; Revised Manuscript Received September 18, 2000

To target DNA A‚T tracts, a three-ring polyamide containing an N-methylpyrrole amino acid has been linked, on solid support, to carboxylic derivatives of phenanthroline and dimers of phenanthroline: 2-Clip-Phen, 3-Clip-Phen, or 2-Clip-Phen containing a long tether. After metalation by CuCl2, the DNA cleavage activities of the different conjugates were compared on a restriction fragment. Cleavage patterns showed that the polyamide moiety of conjugates directs the cleavage activity in the vicinity of A‚T tracts but the precise cleavage selectivity of these conjugates was dependent on the type of phenanthroline residue linked to the poly-N-methylpyrrole entity.

INTRODUCTION

Molecules able to irreversibly modify nucleic acids have received considerable interest because of their potential application as biological tools or as drugs. In addition, compounds that cleave nucleic acids in a sequencespecific manner are useful as reagents for accessing structural and genetic information (1-3). 1,10-Phenanthroline is a versatile ligand able to chelate copper to give mono-Phen or bis-Phen complexes at the oxidation state + I or + II (4). The (Phen)2CuI complex in the presence of hydrogen peroxide efficiently cleaves double-stranded DNA by oxidative attack on deoxyribose units from DNA's minor groove (5, 6), while the Phen-CuI complex is less efficient (7). DNA cleavage products include 5′- and 3′-monophosphate ester termini, free bases, 5-methylenefuranone, and a small amount of 3′-phosphoglycolate (8-10). Hydrogen peroxide can be generated close to the DNA by (Phen)2CuII itself in the presence of a reductant and molecular oxygen. Since these Phen complexes have poor sequence selectivity, different conjugates between phenanthroline and targeting DNA ligands (oligonucleotides, Hoescht-33258, DNA binding protein, ...) have been prepared in order to enhance the cleavage specificity (11-17). However, these chimeric compounds contain only one phenanthroline unit, which is not the best situation for DNA cleavage since the highest cleavage activies have been observed with a Phen:Cu ratio of 2:1. We have recently prepared 2- and 3-Clip-Phen derivatives containing two Phen ligands linked at the 2′- or 3′position by a serinol bridge in order to favor the 2:1 Phen: Cu stoichiometry (18, 19). The oxidative nuclease activity of the copper complexes of these new ligands was found to be higher than with Phen itself, by a factor 2 for 2-ClipPhen but by a factor 60 for 3-Clip-Phen. Conjugates of 2-Clip-Phen with the natural polyamine spermine (a minor groove binder) (20) or intercalators such as acridine derivatives (21) have shown an enhanced nuclease activity. Analyses of the cleavage pattern of 2-Clip-Phen or its spermine conjugate have revealed, however, that † ‡

Laboratoire de Chimie de Coordination du CNRS. University of Utah.

this ligand cleaves DNA practically without sequence selectivity (20). The DNA cleavage selectivity of Clip-Phen derivatives was expected to be enhanced by covalent attachment to specific DNA recognition elements. While there are several potentially suitable DNA binding compounds, we chose to attach Clip-Phen to a well-characterized distamycin-like binder (see ref 22 and references therein). Distamycin is a tri-N-methylpyrrole derivative that tightly binds to the minor groove of DNA through a combination of electrostatic interactions, hydrogen bonds, and van der Waals contacts. While this natural product preferentially binds five successive A‚T base pairs, several modifications have recently been reported which allow the targeting of multiple sites. In addition, new methods for the preparation of such compounds on a solid support provide the basis for further flexible control of sequence specificity by hybrid nucleases (23, 24). Since several factors contribute to the specificity of chimeric reagents prepared with 1,10-phenanthroline, we describe here the preparation of four new conjugates. The distamycin analogue linked to a solid support was attached to Phen, 2- and 3-Clip-Phen or to 2-Clip-Phenhexyl derivatives (see Figure 1). Their DNA cleavage activity has been compared in the same experimental conditions on a restriction fragment. MATERIALS AND METHODS

Materials. Boc-β-alanine-4-carbonylaminomethyl)benzyl-ester-copoly(styrene-divinylbenzene)resin (Boc-βPam-resin, 0.5 mmol/g) was from Advanced Chem. Tech. Chloroform was dried on basic aluminum oxide. Other commercially available reagents and all other solvents were purchased from standard chemical suppliers and used without further purification. 1,2,3-Benzotriazol-1-yl-4-[(tert-butoxycarbonyl)amino]1-methylpyrrole-2-carboxylate (23), 2-succinoyl-amino1,3-bis(1′,10′-phenanthrolin-2′-yloxy)propane [2-ClipPhen-COOH] (20), 2-succinoyl-amino,1-(1′,10′)phenanthrolin-2′-yloxy)ethane [2-Phen-COOH] (20), 3-Clip-Phen (19), and 2-Clip-Phen-hexylamine (21) have been prepared as previously described. Boc-Py-Py-Py-β-Pam resin precursor was synthesized by manual solid-phase synthesis method (23) on Boc-β-Pam-resin (0.5 mmol/g).

10.1021/bc000050t CCC: $19.00 © 2000 American Chemical Society Published on Web 10/28/2000

DNA Cleavage Activity of Cu Phenanthroline and Clip-Phen

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Figure 1. Structures of poly-N-methylpyrrole-Clip-Phen (or Phen) conjugates.

HPLC analyses were performed on a reversed-phase Alltima C18 100A 10 µm column (250 × 4.6 mm) from Alltech in 0.1% (v/v) TFA with acetonitrile eluant at a flow rate of 1 mL/min, gradient elution 1.5% acetonitrile/ min; the chromatograms were monitored at 255 nm. Preparative HPLC was carried out on a reversed-phase Alphabond C18 10 µm preparative column (150 × 22.5 mm) from Alltech, 0.1% TFA, 1.5% acetonitrile/min, 5 mL/min, or on a reversed-phase Nucleosyl C18 10 µm semipreparative column (250 × 6.2 mm) from Interchim, 0.1% TFA, with 21-28% acetonitrile in 30 min linear gradient, 3 mL/min. 2-Succinoyl-amino-1,3-bis(1′,10′-phenanthrolin-3′yloxy)propane (3-Clip-Phen-COOH). To a solution of 3-Clip-Phen (59 mg, 0.13 mmol) in 6 mL of dry CHCl3 was added succinic anhydride (30 mg, 0.36 mmol), and the mixture was heated at reflux for 1 h. The product was precipitated after cooling by addition of diethyl ether

and centrifuged at 3000 rpm. The pellet was then redissolved in 2 mL of a solution of CHCl3/CH3OH (9/1) then precipitated again by diethyl ether to give 3-ClipPhen-COOH as a pale brown powder (48 mg, 66%). 1H NMR (CDCl3, 250 MHz): δ 9.10 (dd, 2 H, J ) 4.5 and 2.0 Hz), 8.85 (d, 2 H, J ) 3.0 Hz), 8.47 (d, 1 H, J ) 8.0 Hz), 8.17 (dd, 2 H, J ) 8.0 and 2.0 Hz), 7.68 and 7.53 (AB, 2 × 2 H, J ) 9.0 Hz), 7.55 (dd, 2 H, J ) 8.0 and 4.5 Hz), 7.29 (d, 2 H, J ) 3.0 Hz), 4.95 (m, 1 H), 4.21 (m, 4 H), 2.84 (m, 4 H). MS (FAB, positive mode, MNBA) m/z 548 (M + H). UV-vis (CH3OH): 238 nm ( ) 75 200 M-1 cm-1), 272 nm ( ) 54 400 M-1 cm-1), 294 nm (sh,  ) 29 300 M-1 cm-1), 314 nm (sh,  ) 10 000 M-1 cm-1), 328 nm ( ) 7000 M-1 cm-1), 348 nm ( ) 4700 M-1 cm-1). 2-Clip-Phen-hexyl-COOH. To a solution of 2-ClipPhen-hexyamine (200 mg, 0.35 mmol) in 40 mL of CHCl3, succinic anhydride (100 mg, 0.96 mmol) was added and the mixture was heated at reflux for 1 h. After evapora-

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tion of the solvent, the crude product was redissolved in hot methanol, precipitated at -20 ° C and filtered to give 2-Clip-Phen-hexy-COOH as a white powder (133 mg, 56%). 1H NMR (CD2Cl2, 500 MHz): δ 9.15 (dd, 2 H, J ) 4.0 and 1.5 Hz), 8.42 (d, 1 H, J ) 6.5 Hz), 8.30 (dd, 2 H, J ) 8.0 and 1.5 Hz), 8.09 (d, 2 H, J ) 9.0 Hz), 7.73 and 7.67 (AB, 2 × 2 H, J ) 8.5 Hz), 7.65 (dd, 2 H, J ) 8.0 and 4.0 Hz), 7.13 (m, 1 H), 7.12 (d, 2 H, J ) 9.0 Hz), 5.06 (m, 2 H), 4.86 (m, 3 H), 3.12 (m, 2 H), 2.55 (t, 2 H, J ) 7.0 Hz), 2.43 (t, 2 H, J ) 7.0 Hz), 2.22 (t, 2 H, J ) 7.0 Hz), 1.49 (m, 2 H), 1.35 (m, 2 H), 1.19 (m, 2 H). MS (FAB, positive mode, MNBA) m/z 661 (M + H). UV-vis (CH3OH): 226 nm ( ) 96 500 M-1 cm-1), 276 nm ( ) 63 800 M-1 cm-1), 296 nm (sh,  ) 20 300 M-1 cm-1), 322 nm ( ) 4000 M-1 cm-1), 346 nm ( ) 2200 M-1 cm-1). General Protocol of Conjugate Synthesis. Boc-PyPy-Py-β-Pam resin (ca 0.05 or 0.15 mmol, estimated from initial resin substitution) was placed in a glass peptide synthesis vessel and the Boc group was removed just before use with a 80% TFA/CH2Cl2/0.5 M thiophenol solution as previously described (23). The resin was then washed with DMF (1×) and CH2Cl2 (2×). The carboxylic acid derivative of phenanthroline (40 mM) was activated with 3 equiv of HOBT (1-hydroxybenzotriazole) and 1.7 equiv of DCC in DMF/DIEA (diisopropylethylamine) (2/ 1) for 5 min; then this mixture was added to the resin and agitated (20 h, room temperature). The synthesis was monitored by analytical HPLC with an aliquot of resin heated in DMAPA [3-(dimethylamino)-1-propylamine] 5 min at 90 ° C (disappearance of the Py-Py-Py-β-Dp peak at 22 min associated to the formation of the conjugateproduct peak at 25, 26, or 27 min for conjugates 1, 2 and 3, and 4, respectively). Resin was then isolated by filtration and washed sequencially with an excess of DMF (1×) and CH2Cl2 (2×) before being heated in DMAPA for 15 h at 55 ° C. The reaction was then filtered to remove the resin. Phen-Py-Py-Py-β-Dp (1). Phen-COOH (123 mg, 0.36 mmol) was activated by DCC/HOBT and added to the Py-Py-Py-β-Pam-resin (ca 0.15 mmol). The crude product was obtained in 3 mL of DMAPA then diluted with H2O and purified by preparative HPLC. Since the isolated product was contaminated by DMAPA, 50% of the material was repurified by preparative HPLC to give, after lyophilization, Phen-Py-Py-Py-β-Dp (1) as a yellow powder (22 mg, 25% recovery).1H NMR (DMSO-d6, 500 MHz): δ 9.90 (s, 2 H), 9.88 (s, 1 H), 9.37 (br s, 1 H), 9.24 (br s, 1 H), 8.90 (br s, 1 H), 8.51 (d, 1 H, J ) 8.5 Hz), 8. 47 (br s, 1 H), 8.13 (br s, 1 H), 8.06 (m, 3 H), 7.36 (d, 1 H, J ) 8.5 Hz), 7.22 (s, 1H), 7.17 (s, 1 H), 7.15 (s, 1 H), 7.05 (s, 1 H), 6.88 (m, 2 H), 4.67 (m, 2 H), 3.84 (s, 3 H), 3.82 (s, 3 H), 3.81 (s, 3 H), 3.61 (m, 2 H), 3.39 (m, 2 H), 3.12 (m, 2 H), 3.03 (m, 2 H), 2.75 (d, 6 H, J ) 4.5 Hz), 2.36 (t, 2 H, J ) 7.0 Hz), 1.75 (m, 2H). MS (ES, positive mode, in CH3CN/H2O [1/1] with 0.1% TFA) m/z 861.6 (862 calcd for M + H), 431.6 (z ) 2, M + 2 H). UV-vis (H2O): 222 nm ( ) 53 400 M-1 cm-1), 280 nm ( ) 42 600 M-1 cm-1), 310 nm (sh,  ) 29 200 M-1 cm1). tR(analytical HPLC): 25 min. 2-Clip-Phen-Py-Py-Py-β-Dp (2). 2-Clip-Phen-COOH (172 mg, 0.32 mmol) was activated by DCC/HOBT and added to the Py-Py-Py-β-Pam-resin (ca 0.15 mmol). The crude product was obtained in 3 mL of DMAPA then precipitated by addition of diethyl ether. The pellet was redissolved in water acidified with formic acid and purified by preparative or semipreparative HPLC. The conjugate was obtained after lyophilization as a yellow powder (24 mg, 11% recovery). 1H NMR (DMSO-d6, 500 MHz): δ 9.93 (s, 1 H), 9.89 (s, 2 H), 9.31 (br s, 1 H), 9.16

Pitie´ et al.

(d, 2 H, J ) 3.5 Hz), 8.82 (dd, 2 H, J ) 4.5 and 1.2 Hz), 8.82 (1 H), 8. 39 (d, 2 H, J ) 9.0 Hz), 8.05 (m, 2 H), 8.00 and 7.93 (AB, 2 × 2 H, J ) 8.5 Hz), 7.93 (1 H), 7.33 (d, 2 H, J ) 9.0 Hz), 7.21 (d, 1 H, J ) 1.5 Hz), 7.16 (s, 2 H), 7. 05 (d, 1 H, J ) 2.0 Hz), 6.88 (d, 1 H, J ) 1.5 Hz), 6.87 (d, 1 H, J ) 2.0 Hz), 4.92 (m, 3 H), 4. 83 (m, 2 H), 3.84 (s, 3 H), 3.81 (s, 3 H), 3.80 (s, 3 H), 3.38 (m, 2 H), 3.11 (m, 2 H), 3.01 (m, 2 H), 2.75 (d, 6 H, J ) 4.5 Hz), 2.63 (m, 4 H), 2.35 (t, 2 H, J ) 7.0 Hz), 1.74 (m, 2 H). MS (ES, positive mode, in CH3CN/H2O [1/1] with 0.1% TFA) m/z 1069.9 (1070 calcd for M + H), 535.7 (z ) 2, M + 2 H). UV-vis (H2O): 218 nm ( ) 75 300 M-1 cm-1), 276 nm ( ) 51 700 M-1 cm-1), 316 nm (sh,  ) 28 400 M-1 cm-1). tR (analytical HPLC): 26 min. After DMAPA/diethyl ether precipitation, the conjugate could also be redissolved in DMSO and precipitated by addition of water to give the unprotonated conjugate (17% of recovery). 1H NMR (DMSO-d6, 250 MHz): δ 9.93 (br s, 2 H), 9.91 (s, 1 H), 9.21 (dd, 2 H, J ) 4.2 and 1.5 Hz), 8.85 (d, 1 H, J ) 6.5 Hz), 8.56 (dd, 2 H, J ) 8.1 and 1.5 Hz), 8.47 (d, 2 H, J ) 8.7 Hz), 8.03 and 7.95 (AB, 2 × 2 H, J ) 8.6 Hz), 8.02 (m, 1 H), 7. 95 (m, 1 H), 7.83 (dd, 2 H, J ) 8.1 and 4.2 Hz), 7.36 (d, 2 H, J ) 8.7 Hz), 7.32 (d, 1 H, J ) 1.5 Hz), 7.29 (d, 1 H, J ) 1.5 Hz), 7.24 (d, 1 H, J ) 1.5 Hz), 7.14 (d, 1 H, J ) 1.5 Hz), 6.99 (d, 1 H, J ) 1.5 Hz), 6.95 (d, 1 H, J ) 1.5 Hz), 4.92 (m, 5 H), 3.96 (s, 3 H), 3.93 (br s, 6 H), 3.52 (m, 2 H), 3.19 (m, 2 H), 2.70 (m, 4 H), 2.45 (t, 2 H, J ) 7.0 Hz), 2.42 (t, 2 H, J ) 7.0 Hz), 2.25 (s, 6 H), 1.65 (m, 2 H). UV-vis (CH3OH): 230 nm ( ) 114 600 M-1 cm-1), 276 nm ( ) 85 000 M-1 cm-1), 292 nm (sh,  ) 23 500 M-1 cm-1). 3-Clip-Phen-Py-Py-Py-β-Dp (3). 3-Clip-Phen-COOH (48 mg, 0.09 mmol) was activated by DCC/HOBT and added to the Py-Py-Py-β-Pam-resin (ca 0.05 mmol). The crude product was obtained in 1 mL of DMAPA then precipitated by addition of diethyl ether. The pellet was redissolved in water acidified with formic acid and purified by preparative or semipreparative HPLC. The conjugate was obtained after lyophilization as an orange powder (7 mg, 10% recovery). 1H NMR (DMSO-d6, 500 MHz): δ 9.92 (s, 1 H), 9.89 (s, 1 H), 9.88 (s, 1 H), 9.45 (br s, 1H), 9.13 (d, 2 H, J ) 4.0 Hz), 8.96 (d, 2 H, J ) 2.0 Hz), 8.85 (m, 2 H), 8.57 (d, 1 H, J ) 7.0 Hz), 8.23 (d, 2 H, J ) 2.0 Hz), 8.07 (m, 5 H), 7.98 (m, 2 H), 7.19 (s, 1H), 7.17 (s, 1 H), 7.15 (s, 1 H), 7.03 (s, 1 H), 6.88 (s, 1 H), 6.84 (s, 1 H), 4.73 (m, 1 H), 4.52 (m, 4 H), 3.84 (s, 3 H), 3.81 (s, 3 H), 3.79 (s, 3 H), 3.39 (m, 2 H), 3.12 (m, 2 H), 3.02 (m, 2 H), 2.75 (d, 6 H, J ) 4.5 Hz), 2.56 (m, 4 H), 2.35 (t, 2 H, J ) 7.0 Hz), 1.75 (m, 2H). MS (ES, positive mode, in CH3CN/H2O [1/1] with 0.1% TFA) m/z 535.6 (z ) 2, 535.5 calcd for M + 2 H), 357.5 (z ) 3, M + 3 H). UV-vis (H2O): 234 nm ( ) 69 400 M-1 cm-1), 278 nm ( ) 48 600 M-1 cm-1), 298 nm (sh,  ) 41 500 M-1 cm-1). tR (analytical HPLC): 26 min. 2-Clip-Phen-(hexyl)-Py-Py-Py-β-Dp (4). 2-Clip-Phenhexyl-COOH (59 mg, 0.09 mmol) was activated by DCC/ HOBT and added to the Py-Py-Py-β-Pam-resin (ca 0.05 mmol). The crude product was obtained in 1 mL of DMAPA then precipitated by addition of diethyl ether. The pellet was redissolved in water acidified with formic acid and purified by preparative HPLC. The conjugate was obtained after lyophilization as a yellow powder (5 mg, 7% recovery). 1H NMR (DMSO-d6, 500 MHz): δ 9.89 (s, 2 H), 9.85 (s, 1 H), 9.30 (br s, 1 H), 9.15 (d, 2 H, J ) 4.5 Hz), 8.80 (br s, 2 H), 8.63 (d, 1 H, J ) 6.5 Hz), 8. 40 (d, 2 H, J ) 9.0 Hz), 8.05 (m, 2 H), 8.00 and 7.93 (AB, 2 × 2 H, J ) 8.5 Hz), 7.96 (m, 1 H), 7.83 (t, 1 H, J ) 7.0 Hz), 7.33 (d, 2 H, J ) 9.0 Hz), 7.20 (s, 1 H), 7.16 (s, 1 H), 7. 14 (s, 1 H), 7. 05 (s, 1 H), 6.88 (s, 1 H), 6.85 (d, 1 H),

DNA Cleavage Activity of Cu Phenanthroline and Clip-Phen

4.87 (m, 5 H), 3.84 (s, 3 H), 3.81 (s, 3 H), 3.80 (s, 3 H), 3.38 (m, 2 H), 3.11 (m, 2 H), 2.99 (m, 4 H), 2.75 (d, 6 H, J ) 4.5 Hz), 2.46 (m, 2 H), 2.35 (m, 4 H), 2.25 (t, 2 H, J ) 7.5 Hz), 1. 74 (m, 2 H), 1.54 (m, 2 H), 1.35 (m, 2 H), 1. 23 (m, 2 H). MS (ES, positive mode, in CH3CN/H2O [1/1] with 0.1% TFA) m/z 592.0 (z ) 2, 592.0 calcd for M + 2 H), 497.1 (z ) 2, M + 2 H - 197), 395.2 (z ) 3, M + 3 H). UV-vis (H2O): 222 nm ( ) 73 800 M-1 cm-1), 276 nm ( ) 51 500 M-1 cm-1), 308 nm (sh,  ) 33 200 M-1 cm-1). tR (analytical HPLC): 27 min. Boc-Py-Py-Py-β-Dp (5). Boc-Py-Py-Py-β-Pam-resin (ca 0.03 mmol) was heated in 0.5 mL of DMAPA 15 h at 55 ° C. The resin was removed by filtration and the crude product was precipitated by addition of diethyl ether. The pellet was washed with diethyl ether and dried under vacuum before being redissolved in CH2Cl2 and precipitated by addition of diethyl ether. The pellet was dissolved in water acidified with trifluoroacetic acid and purified on semipreparative HPLC (tR ) 20 min) and lyophilized to give Boc-Py-Py-Py-β-Dp as a white powder (9 mg, 40% recovery). 1H NMR (D2O, 250 MHz): δ 6.75 (m, 2 H), 6.56 (br s, 1 H), 6.46 (d, 1 H, J ) 1.5 Hz), 6.40 (d, 1 H, J ) 1.5 Hz), 6.29 (br s, 1 H), 3.54 (s, 6 H), 3.53 (s, 3 H), 3.42 (t, 2 H, J ) 6.6 Hz), 3.20 (t, 2 H, J ) 6.6 Hz), 3.00 (m, 2 H), 2.72 (s, 6H), 2.41 (t, 2 H, J ) 6.5 Hz), 1.83 (m, 2 H), 1.38 (s, 9 H). MS (ES, positive mode, in CH3CN/H2O [1/1] with 0.1% TFA) m/z 640.4 (640.77 calcd for M + H). UV-vis (H2O): 226 nm ( ) 21 500 M-1 cm-1), 306 nm ( ) 25 800 M-1 cm-1). tR (analytical HPLC): 35 min. Cleavage of 5′-32P-End-Labeled DNA. End-labeled restriction fragment was prepared by sequential digestion of supercoiled pBR322 plasmid DNA with EcoRI restriction endonuclease, alkaline phosphatase, [γ-32P]ATP and T4 polynucleotide kinase and then RsaI restriction endonuclease (25). The 32P-end-labeled 167 base pair fragment was purified by 8% preparative nondenaturing gel electrophoresis and isolated. Metalations of the different ligands used in DNA cleavage experiments were carried out using a 1.25 mM CuCl2 concentration with 1 equiv of ligand in water for 1 h at room temperature before dilution of the complex with water to 125 or 25 µM. Cleavage reactions were carried out in 50 µL total volume containing calf thymus DNA (86 µM nucleotide concentration) and 104 cpm of the 32P-end-labeled restriction fragment in 10 mM sodium phosphate buffer (pH 7.2), 100 mM NaCl. After preincubation with complex (1 or 5 µM) for 30 min at room temperature, DNA cleavage was initiated by addition of an aqueous solution of ascorbate (2 µL, 2.5 mM) and samples were incubated for 1 h at 37 °C. 2 µL of a 0.1 M EDTA solution and 8 µL of sodium acetate buffer (3 M, pH 5.2) were then added and samples were precipitated with 120 µL of ethanol. Pellets were rinsed with ethanol and lyophilized. One part of the samples was redissolved in 50 µL of piperidine 0.2 M and heated for 30 min at 90 °C before being lyophilized. Fragments of DNA were analyzed by denaturing 8% polyacrylamide gel electrophoresis. The gels were dried under vacuum and then analyzed using phosphorimagery (Molecular Dynamics). Fragments were identified with respect to Maxam-Gilbert G-sequencing (26). RESULTS AND DISCUSSION

To increase the sequence selectivity of DNA cleavage by Clip-Phen derivatives on a succession of A‚T base pairs, four conjugates with the distamycin analogue Py-

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Py-Py-β-Dp have been prepared (Figure 1). Conjugate 1, having a less voluminous and sterically constrained nuclease part with only one phenanthroline, has been synthesized for comparison with the efficacy of the two phenanthrolines of Clip-phen derivatives in the cleavage pattern of the conjugates. Conjugates 2 and 3, with, respectively, 2- and 3-Clip-Phen, allowed analysis of the effect of the bridge position between the two phenanthroline entities of Clip-Phen on the cleavage pattern. The aliphatic linker between Clip-Phen and poly-Nmethyl-pyrroles is relatively short (only four single bonds) in conjugates 2 and 3. Previous reports have been shown that the length of the junction influences both cleavage efficiency and cleavage pattern. Since significant increases in specificity have been obtained in different laboratories for conjugates of poly-N-methyl-pyrroles including longer aliphatic linkers (see ref 21 and references therein), conjugate 4 (with 10 single bonds in the spacer) has been prepared for comparison with conjugate 2. We chose a long 5-methylene spacer in order to increase the flexibility of the linker. Synthesis Strategy (Figure 2). Phenanthroline amine derivatives were transformed in carboxylic acids by reaction with succinic anhydride. The poly-N-methylpyrrole portion was synthesized manually on solid support in seven sequential steps from Boc-β-Pam-resin and the Boc-N-methyl-pyrrole-OBt synthon (obtained in eight steps from pyrrole) according to Dervan methodology (23). It is known that poly-N-methyl-pyrroles are unstable when their N-terminal amine function is unprotected, so we chose to attach the phenanthroline part on this fragile amine in order to protect it without any other modification of conjugates (23). This synthetic strategy allowed us to realize the coupling reaction with phenanthroline derivatives on the solid support. This method also provided facile purification (excess of reagents being easily removed and washed by filtration) and avoided isolation of the unstable peptide with the unprotected N-terminal primary amine. Optimization of the coupling conditions was conducted with the 2-Clip-Phen-COOH derivative. Various attempts to obtain a corresponding activated ester in pure form failed. They included activation of small quantities of 2-Clip-Phen-COOH (less than 0.2 mmol) with DCC/ HOBT (1-hydroxybenzotriazole), BOP [(benzotriazol-1yloxy)tris(dimethylamino)phosphonium]/HOBT or DCC/ NHS (N-hydroxysuccinimide). In the end, 2 equiv of the carboxylic acid were activated by DCC/HOBT in a DMF/ DIEA (diisopropylethylamine) solution, and the crude activation mixture was directly injected into the reaction vessel containing the resin supporting the poly-N-methylpyrrole which had been deprotected just before use. After a washing step, the conjugate was cleaved from the support by aminolysis of the resin ester linkage in hot (55 °C) DMAPA [3-(dimethylamino)-1-propylamine] (23). We tried to purify the different conjugates by diluting the crude mixture dissolved in DMAPA with 4 vol of water followed by separation of the product by HPLC on a C18 reversed-phase column. This strategy was tested for purification of conjugate 1, but it appeared that, at the end of the purification, the conjugate was contaminated with DMAPA and usual small quantity of TFA used in the elution solvant (0.1%; v/v) was unable to neutralize all the amine. Thus, another step of HPLC purification was necessary to remove residual DMAPA from the conjugate. Since alkalinization can damage the C18 column, and conjugates with 2- or 3-Clip-Phen are insoluble in a DMAPA/water mixture, the conjugate dissolved in DMAPA was instead precipitated by addition

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Figure 2. Solid-phase synthetic scheme for poly-N-methylpyrrole-Clip-Phen (or Phen) conjugates: (i) 80% TFA/CH2Cl2, 0.4 M PhSH; (ii) Boc-Py-OBt, DIEA, DMF; (iii) 80% TFA/CH2Cl2, 0.4 M PhSH; (iv) Boc-Py-OBt, DIEA, DMF; (v) 80% TFA/CH2Cl2, 0.4 M PhSH; (vi) Boc-Py-OBt, DIEA, DMF; (vii) 80% TFA/CH2Cl2, 0.4 M PhSH; (viii) Phen-COOH, DCC, HOBT, DIEA, DMF; (ix) DMAPA, 55 °C. (Box) Boc-N-methylpyrrole-OBt ester (Boc-Py-OBt) used for peptide synthesis. Phen-COOH: 2-Phen-COOH, 2-Clip-Phen-COOH, 3-Clip-Phen-COOH, or 2-Clip-Phen-hexy-COOH.

of diethyl ether in order to remove the major part of the excess amine. The crude product was then dissolved in acidified water and purified by HPLC to give a watersoluble protonated conjugate in a global yield ranging from 7 to 25%. Modifications of the reaction times between the phenanthroline derivative and the peptide, the use of increased equivalents of phenanthroline derivative and activators, or activation of the carboxylic acid group with BOP/HOBT or HBTU [O-(benzotriazol-1-yl)N,N,N′,N′-tetramethyluronium hexafluorophosphate] led to similar yields. Only the global recovery yield of the nine successive steps of the conjugate synthesis can be estimated from the manufacturer’s information about the resin substitution. The yield of the coupling reaction between the phenanthroline derivative and the peptide was difficult to determine: (i) The coupling reaction between the phenanthroline derivative and the peptide was monitored by HPLC on aliquots of resin heated in DMAPA by observing the appearance of the conjugate and the disappearance of the unreacted peptide during the reaction. However, various attempts to isolate the unprotected peptide from the resin failed. Only degradated product has been isolated after addition of TFA on the resin in order to remove Boc residues followed by cleavage with DMAPA. Thus, the quantity of unreacted peptide might be underestimated during HPLC monitoring. (ii) Peptide 5 (protected on the N-terminal amine for stability reason), corresponding to the last step before reaction with the phenanthroline derivative, was purified under the same conditions of the conjugates 1-4. During the HPLC purification step, the synthesis seemed efficient since only traces of aborted peptide sequence were

Figure 3. Sequence of the EcoRI-RsaI restriction fragment. Preferential binding sites for the distamycin analogue (5 succesive A‚T base pairs) are underlined. A secondary binding site observed during cleavage is shown (dashed underline).

observed but only 40% of peptide was recovered. This means that the theoretical peptide quantity can be overestimated in the coupling with the phenanthroline derivative. (iii) During HPLC purification, conjugates were eluted as very broad peaks difficult to collect without loss of material. The peak resolution was better for compound 5 compared to that of conjugate 1, the worse being for conjugate 4. Yields were in good correlation with the difficulty of purification. To reduce the loss of product during the HPLC step, another purification method was optimized in the case of conjugate 2. After diethyl ether precipitation, the crude product was redissolved in DMSO and precipitated with water. In this case, the conjugate was obtained in the unprotonated form with a better yield (17% versus 11% for HPLC purification). DNA Cleavage Activity. Conjugates 1-4 were metalated with 1 equiv of CuCl2, and their DNA cleavage activities were studied. Comparison of the DNA cleavage

DNA Cleavage Activity of Cu Phenanthroline and Clip-Phen

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Figure 4. Cleavage of the EcoRI-RsaI restriction fragment (104 cpm in the presence of 86 µM of random DNA) by conjugates 1, 2, 3, and 4 (1 or 5 µM). The cleavage reaction proceeded for 1 h at 37 °C in the presence of 100 µM ascorbate in 10 mM sodium phosphate buffer (pH 7.2) with 100 mM NaCl. The final concentration of conjugate and piperidine treatment (0.2 M, 30 min at 90 °C) are indicated on the top of the gel. Control experiments were performed with 5 µM of conjugate without ascorbate. Lanes 1 and 30: Maxam-Gilbert G. Lane 2: control DNA. Lane 3: control DNA with piperidine treatment. Lane 28: control ascorbate. Lane 29: control ascorbate with piperidine treatment.

selectivity of these complexes was carried out with the EcoRI/RsaI restriction fragment of 167 base pairs from the plasmid pBR322 that was 5′-end labeled with 32P (Figure 3). This restriction fragment contains three sites of high affinity for the distamycin analogue with five, six, and seven successive A‚T base pairs, respectively. This sequence has been previously tested for DNA cleavage with (2-Clip-Phen)CuCl2 (results not shown) and with the copper complex of a 2-Clip-Phen-spermine conjugate (20), and in both cases, no cleavage specificity was observed. The DNA target (104 cpm) was incubated in the presence of random double-stranded calf thymus DNA (86 µM in nucleotide) with different concentrations of conjugates, and results obtained for 1 and 5 µM of complexes are shown Figure 4. Initiation of the redox activity of these copper complexes was realized by addition of 100 µM ascorbate in the presence of air. The resulting samples were analyzed by PAGE before and after piperidine treatment. Such treatment was per-

formed to reveal all damage mediated by conjugates on their DNA target [direct cleavages (1, 27) and alkalilabile oxidations including base damage (28)]. Cleavage sites were determined by comparison with a MaxamGilbert G-sequencing lane (26). The four conjugates induced a nonrandom cleavage pattern as confirmed by phosphorimager scanning (Figure 5). Cleavages were confined to localized sites around the three regions containing the successive A‚T base pairs highlighted. A secondary binding site was also observed at another A‚T rich sequence in which 10 out of 12 base pairs are A‚T. This region is also known to be a binding site for distamycin since cleavages at this sequence have been previously observed with the iron complex of EDTA-distamycin conjugate (29). No modification of the cleavage pattern was observed after piperidine treatment, indicating that these conjugates performed direct DNA breaks, probably due to deoxyribose oxidation, as is precedented for copper complexes of phenanthroline (1).

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Figure 5. Phosphorimager scanning between G28 and G127 of the cleavage patterns of the EcoRI-RsaI restriction fragment by copper complexes of conjugates 1, 2, 3, and 4 then piperidine treatment. Lane A: Maxam-Gilbert G. Lane B: 1 µM of 2. Lane C: 5 µM of 1. Lane D: 1 µM of 3. Lane E: 5 µM of 4.

Differences have been observed in the cleavage pattern of each conjugate. As expected, conjugate 3, with 3-ClipPhen, was the more active; with 5 µM of the hybrid molecule, nearly all the target was cleaved (compare the residual full-length target in lanes 19 and 21 with other cleavage patterns, Figure 4). At this concentration of 3, the cleavage pattern was less selective and quantity of small fragments increased dramatically in comparison with cleavage pattern observed for 1 µM of 3 (lanes 18 and 20, Figure 4). This was probably due to multiple cleavages of fragments generated from the target by initial oxidation damage because, for 1 µM of complex, breaks were selectively located around the poly-N-me-

thylpyrrole binding sites (see also lane D, Figure 5). Conjugates 1 and 4 were less active DNA cleavers since breaks were only observed with 5 µM complex (lanes 13 and 15 for 1 and lanes 25 and 27 for 4, Figure 4). The poor DNA cleavage activity of 1 can be explained by the presence of only one phenanthroline unit in the conjugate, complexes of (Clip-Phen)CuCl2 or with two 1,10phenanthrolines for one copper being known to be more active. The lower cleavage efficiency observed with conjugate 4 indicates that the increase of the tether length between the two parts of conjugate has a negative effect on the cleavage activity. The cleavage pattern obtained with conjugate 4 also showed more diffuse

DNA Cleavage Activity of Cu Phenanthroline and Clip-Phen

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Figure 6. Phosphorimager scanning between G28 and G127 of the cleavage patterns of the EcoRI-RsaI restriction fragment by 1 and 5 µM of copper complex of conjugate 2 then piperidine treatment.

breaks (lanes 25 and 27 Figure 4 and lane E Figure 5). This fact can be attributed to the tether being too long, which allowed the nuclease entity of the conjugate to oxidize a large number of nucleotides around the binding site. A loss of specificity was also observed with 4. Major cleavages were observed in the G33 to G71 region which includes the higher affinity binding site with seven successive A‚T base pairs (three overlapping primary binding sites for the poly-N-methylpyrrole entity of the conjugates) in the vicinity of other primary binding sites of five and six successive A‚T base pairs (Figures 4 and 5). Cleavages were essentially observed on nucleosides on the 5′- and 3′-sides of the higher affinity binding site with a nucleoside preference specific to each conjugate. In the case of conjugate 2, cleavage was also observed inside the A‚T box (lanes 6-9, Figure 4, and lane B, Figure 5). A comparison of the phosphorimager scanning of the cleavage patterns obtained for 1 and 5 µM of 2

(Figure 6) showed that the cleavage inside the seven A‚ T base pairs box was preferential for 1 µM of 2, the quantity of cleavage on the 5′- and 3′-sides of this higher affinity site, the five and six A‚T boxes or in the secondary binding site (where 10 out of 12 base pairs are A‚T) increasing dramatically for higher concentration of 2 (5 µM). A comparison of cleavage patterns obtained with copper complexes of 2 and 3 has been done for different experimental conditions including the variation of the concentration of copper complexes, the nature of the buffer (phosphate or Tris), salts (NaCl and MgCl2), and reductant (ascorbate or mercaptopropionic acid), in the presence or not of random double-stranded calf thymus DNA. As it can be observed in Figures 4 and 5, copper complex of 3 (the conjugate with 3-Clip-Phen) cleaved always on the 5′- and 3′-sides of the seven A‚T base pairs box when the preferential cleavage site for the copper

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complex of 2 (the conjugate with 2-Clip-Phen) was inside this seven A‚T box (results not shown). Another difference in the cleavage pattern was also observed in the case of conjugate 1 (with only one phenanthroline) which was unable to cleave efficiently the sequence between the seven and the five A‚T boxes (lanes 13 and 15, Figure 4, and lane C, Figure 5). These results show that when the peptide part of the conjugates can orient the cleavage around a binding site, a fine specificity of cleavage can be modulated by the position of the bridge joining C2 or C3 of the phenanthroline units of Clip-Phen or by the choice of a system with only one phenanthroline. CONCLUSION

This work shows that Clip-Phen copper complexes are versatile DNA cleavers which can be easily targeted on a particular DNA sequence by linkage to a specific DNA binder like a poly-N-methylpyrrole. The fine-tuning of the cleavage is due to the type of Clip-Phen used in these conjugates and to the tether length. It must be noted that the conjugate based on the 3-Clip-Phen was the most efficient one, indicating that the position of the serinol bridge between the two Phen units has a key role in the redox activity of the ligated copper. Our next goal will be to study if the conjugation of Clip-Phen derivatives also has an influence on the DNA cleavage chemistry of these artificial nucleases. ACKNOWLEDGMENT

A collaborative CNRS-NSF grant is gratefully acknowledged. We thank Rachel Jameton for her technical assistance during PAGE experiments. LITERATURE CITED (1) Sigman, D. S., Mazumder, A., and Perrin, D. M. (1993) Chemical nucleases. Chem. Rev. 93, 2295-2316. (2) Dervan, P. B. (1986) Design of sequence-specific DNAbinding molecules. Science 232, 464-471. (3) Meunier, B., Ed. (1996) DNA and RNA Cleavers and Chemotherapy of Cancer and Viral Diseases, Kluwer, Dordrecht. (4) James, B. R., and Williams, R. J. P. (1961) The oxidationreduction potentials of some copper complexes. J. Chem. Soc. 2007-2019. (5) Marshall, L. E., Graham, D. R., Reich, K. A., and Sigman, D. S. (1981) Cleavage of deoxyribonucleic acid by the 1,10phenanthroline-cuprous complex. Hydrogen peroxide requirement and primary and secondary structure specificity. Biochemistry 20, 244-250. (6) Veal, J. M., Merchant, K., and Rill, R. L. (1991) Noncovalent DNA binding of bis(1,10-phenanthroline)copper(I) and related compounds. Biochemistry 30, 1132-1140. (7) Veal, J. M., Merchant, K., and Rill, R. L. (1991) The influence of reducing agent and 1,10-phenanthroline concentration on DNA cleavage by phenanthroline + copper. Nucleic Acids Res. 19, 3383-3388. (8) Kuwabara, M., Yoon, C., Goyne, T. E., Thederahn, T., and Sigman, D. S. (1986) Nuclease activity of 1,10-phenanthroline-copper ion: reaction with CGCGAATTCGCG and its complexes with netropsin and EcoRI. Biochemistry 25, 74017408. (9) Goyne, T. E., and Sigman, D. S. (1987) Nuclease activity of 1,10-phenanthroline-copper ion. Chemistry of deoxyribose oxidation. J. Am. Chem. Soc. 109, 2846-2848. (10) Zelenko, O., Gallagher, J., and Sigman, D. S. (1997) Scission of DNA with bis(1,10-phenanthroline)copper without intramolecular hydrogen migration. Angew. Chem., Int. Ed. Engl. 36, 2776-2778.

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