Efficient DNA Alkylation by a Pyrrole-Imidazole CBI Conjugate with an

Gengo Kashiwazaki , Toshikazu Bando , Tomofumi Yoshidome , Seiji Masui , Toshiki Takagaki , Kaori Hashiya , Ganesh N. Pandian , Junichi Yasuoka ...
2 downloads 0 Views 510KB Size
Download PDF
Bioconjugate Chem. 2006, 17, 715−720

715

Efficient DNA Alkylation by a Pyrrole-Imidazole CBI Conjugate with an Indole Linker: Sequence-Specific Alkylation with Nine-Base-Pair Recognition Toshikazu Bando, Shunta Sasaki, Masafumi Minoshima, Chikara Dohno, Ken-ichi Shinohara, Akihiko Narita, and Hiroshi Sugiyama* Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8501, Japan. Received January 30, 2006; Revised Manuscript Received April 6, 2006

Conjugates 7, 8, and 10 of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) with a 5-amino-1H-indole-2-carbonyl linker were synthesized by Fmoc solid-phase synthesis and a subsequent liquid-phase coupling procedure. The DNA alkylating abilities of conjugates 7, 8, 6b, and 10 were examined using Texas Red-labeled PCR fragments and high-resolution denaturing gel electrophoresis. CBI conjugates 7 and 8 exhibited highly efficient sequence-specific DNA alkylation comparable with previous CBI conjugates with a vinyl linker. In particular, conjugate 10, with a 10-ringed hairpin Py-Im polyamide, alkylated at the adenine of 5′-ACAAATCCA-3′. Introduction of an indole linker greatly facilitated the synthesis of sequence-specific alkylating Py-Im polyamides.

INTRODUCTION DNA is often the target of anticancer agents, which covalently bond to or oxidatively damage bases and deoxyriboses. DNA alkylating agents, which include well-known anticancer drugs such as cyclophosphamide and mitomycin C, are used routinely in cancer treatment. However, these drugs are extremely toxic because they attack normal cells and cause severe adverse effects. One intriguing question to consider is whether the introduction of sequence selectivity to an alkylating agent can improve its efficacy as an anticancer agent (1-3). To address this question, we have developed various types of sequencespecific alkylating agents by conjugating alkylating agents and N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and have investigated their DNA alkylating activity and biological functions (4, 5). Importantly, the insertion of a vinyl linker between Segment A of DU-86 (6) and the Py-Im polyamides dramatically enhances DNA alkylating reactivity (7-10) and cytotoxicity against human cancer cell lines by more than 100fold compared with compounds with no vinyl linker (11, 12). Alkylation of the coding region of the template strand by PyIm polyamides with vinyl linkers effectively terminates transcription, producing truncated mRNA (13). We have also demonstrated that alkylating Py-Im polyamides, such as 1a, causes gene silencing of GFP in mammalian cells (14). These results encouraged us to test alkylating Py-Im polyamides in animal studies, such as in xenografts of human cancers. For this purpose, we changed the alkylating moiety to 1,2,9,9atetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI), which can be synthesized from commercially available naphthalene1,3-diol or 4-amino-1-naphthol (15-18). We demonstrated that CBI conjugate 1b tethered to Py-Im hairpin polyamides with a vinyl linker have higher sequence-specific DNA alkylating activity and cell toxicity than does CPI conjugate 1a (Figure 1) (19). More recently, we found that CBI conjugate 1b specifically alkylates at adenines, despite having a binding orientation similar to the corresponding CPI conjugate 1a (20). Additional sequence specificity relative to CPI conjugates will be useful for biological applications. a Reaction conditions: (i) Fmoc solid-phase synthesis then 1 N NaOH, DMF; (ii) HATU, iPr2NEt, DMF then 3, iPr2NEt, DMF; (iii) 5, EDCI, NaHCO3, DMF; (iv) 5% aq. NaHCO3: DMF ) 1:1.

Figure 1. (A) Structures of compounds 1a,b, 7, and 8. (B) Schematic representations of the DNA alkylation of the specific sequence by compounds 1b, 7, and 8.

Here we report the synthesis and evaluation of hairpin PyIm polyamide-CBI conjugates 7, and 8 and seco-CBI conjugates 6b and 10, which specifically alkylate DNA. In particular, compound 10, with a 10-ringed hairpin Py-Im polyamide specifically alkylated at adenine of the targeted nine-base-pair matching sequence, 5′-ACAAATCCA-3′ at nanomolar concentrations.

10.1021/bc060022w CCC: $33.50 © 2006 American Chemical Society Published on Web 04/27/2006

716 Bioconjugate Chem., Vol. 17, No. 3, 2006

EXPERIMENTAL PROCEDURES Materials. Reagents and solvents were purchased from standard suppliers and used without further purification. Abbreviations of some reagents: iPr2NEt, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; HATU, O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium; EDCI, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloric salt. NMR spectra were recorded with a JEOL JNM-A 500 nuclear magnetic resonance spectrometer, and tetramethylsilane was used as the internal standard. Proton NMR spectra were recorded in parts per million (ppm) downfield relative to tetramethylsilane. Electrospray ionization mass spectrometry (ESI-MS) and electrospray ionization time-of-flight mass spectrometry (ESITOFMS) were produced on a API 150 (PE SCIEX) and BioTOF II (Bruker Daltonics) mass spectrometer. Polyacrylamide gel electrophoresis was performed on a HITACHI 5500-S DNA sequencer. Ex Taq DNA polymerase and Suprec-02 purification cartridges were purchased from Takara Co.; the Thermo Sequenase core sequencing kit and loading dye (dimethylformamide with fuschin red) from Amersham Co. Ltd; 5′-Texas Red-modified DNA oligomer from Proligo Co. Ltd; and 50% Long Ranger gel solution from FMC Bioproducts. AcImImPyPy-γ-ImPy-CO2H (2b). AcImImPyPy-γ-ImPyCO2-oxime resin was synthesized in a stepwise reaction by Fmoc solid-phase protocol (FmocPyCO2H, FmocImCO2H, FmocγCO2H, HATU, DIEA, DMF). A sample of resin was cleaved with alkali conditions (1 N NaOH in DMF, 1 h, 55 °C) and purified by HPLC using a Chemcobond 5-ODS-H column (0.1% AcOH/CH3CN 0-100% linear gradient, 0-30 min, 254 nm), to produce 2b (20 mg, 23%) as a yellow powder. 1H NMR (500 MHz, DMSO-d6) δ 10.32 (s, 1H; NH), 10.29 (s, 1H; NH), 10.23 (s, 1H; NH), 10.00 (s, 1H; NH), 9.91 (s, 1H; NH), 9.32 (s, 1H; NH), 8.02 (s, 1H; NH), 7.56 (s, 1H; Im-H), 7.50 (s, 1H; Im-H), 7.45 (s, 1H; Py-H), 7.44 (s, 1H; Im-H), 7.27 (s, 1H; Py-H), 7.16 (s, 1H; Py-H), 7.14 (s, 1H; Py-H), 6.92 (s, 1H; Py-H), 6.89 (s, 1H; Py-H), 4.00 (s, 3H; NCH3), 3.97 (s, 3H; NCH3), 3.93 (s, 3H; NCH3), 3.84 (s, 3H; NCH3), 3.81 (s, 3H; NCH3), 3.79 (s, 3H; NCH3), 3.19 (m, 2H; CH2), 2.34 (m, 2H; CH2), 2.03 (s, 3H; COCH3), 1.78 (m, 2H; CH2); ESI-TOFMS m/e calcd for C39H45N16O9 [M+ + H] 881.35, found 881.36. A synthetic protocol similar to that used for the preparation of compound 2b was followed to prepare compound 2a using Fmoc solid-phase protocol. H2N-Indole-CO2H (3). Compound 3 was synthesized by two stepwise reactions from commercially available ethyl 5-nitroindole-2-carboxylate (hydrolysis by 1 N aq. NaOH, then hydrogenation by hydrogen gas catalyzed by palladium-carbon), which was used in the next coupling step without further purification. 1H NMR (500 MHz, DMSO-d6) δ 11.22 (s, 1H; NH), 7.11 (d, 1H, J ) 8.5 Hz; CH), 6.75 (d, 1H, J ) 2.0 Hz; CH), 6.67 (s, 1H; CH), 6.65 (dd, 1H, J ) 2.0, 8.5 Hz; CH), 3.31 (brs, 2H; NH2 and H2O); ESI-TOFMS m/e calcd for C9H9N2O2 [M+ + H] 177.06, found 177.02. AcImImPyPy-γ-ImPy-Indole-CO2H (4b). To a solution of compound 2b (3.5 mg, 4.0 µmol) in DMF (0.1 mL) was added iPr NEt (1.4 µL, 8.0 µmol) and HATU (1.4 mg, 3.7 µmol), The 2 reaction mixture was stirred for 4 h at room temperature. After the conversion from 2b to activated ester was confirmed by HPLC and ESI-MS analysis, 3 (1.3 mg, 7.4 µmol) and iPr2NEt (1.3 µL, 7.5 µmol) were added to the reaction vessel. The reaction mixture was stirred for overnight at room temperature under N2 atmosphere. Evaporation of the solvent gave a yellow residue by filtration, which was washed with chloroform (2 mL × 2) and water (2 mL × 2) to produce 4b (3.0 mg, 73%) as a yellow powder. 4b was used in the next step without further purification. 1H NMR (500 MHz, DMSO-d6) δ 11.33 (brs, 1H; NH), 10.34 (s, 1H; NH), 10.33 (s, 1H; NH), 10.27 (s, 1H; NH),

Bando et al.

9.92 (s, 2H; NH), 9.73 (s, 1H; NH), 9.32 (brs, 1H; NH), 8.01 (brt, 1H; NH), 7.94 (s, 1H; CH), 7.56 (s, 1H; Im-H), 7.50 (s, 1H; Im-H), 7.46 (s, 1H; Im-H), 7.40 (brd, 1H, J ) 8.5 Hz; CH), 7.31 (brd, 1H, J ) 8.5 Hz; CH), 7.30 (d, 1H, J ) 1.5 Hz; Py-H), 7.26 (d, 1H, J ) 1.5 Hz; Py-H), 7.17 (d, 1H, J ) 1.5 Hz; Py-H), 7.16 (s, 2H; Py-Hx2), 6.90 (d, 1H, J ) 1.5 Hz; Py-H), 6.85 (brs, 1H; CH), 4.00 (s, 3H; NCH3), 3.97 (s, 3H; NCH3), 3.95 (s, 3H; NCH3), 3.85 (s, 3H; NCH3), 3.84 (s, 3H; NCH3), 3.80 (s, 3H; NCH3), 3.20 (dt, 2H, J ) 6.0, 7.5 Hz; CH2), 2.36 (t, 2H, J ) 7.5 Hz; CH2), 2.04 (s, 3H; COCH3), 1.79 (qu, 2H, J ) 7.5 Hz; CH2); ESI-TOFMS m/e calcd for C48H51N18O10 [M+ + H] 1039.40, found 1039.39. AcImImPyPy-γ-ImPy-Indole-seco-CBI (6b). To a solution of compound 4b (9.5 mg, 9.2 µmol) in DMF (0.4 mL) was added seco-CBI 5 (2.1 mg, 9.2 µmol), EDCI (3.5 mg, 18.3 µmol) and NaHCO3 (1.5 mg, 18.3 µmol). The reaction mixture was stirred for 3 h at room temperature. Evaporation of the solvent gave a yellow residue, which was subjected to column chromatography (silica gel, 5-10% MeOH in CH2Cl2, gradient elution) to produce compound 6b (4.4 mg, 3.5 µmol: 38% yield) as a yellow powder. ESI-TOFMS m/e calcd for C61H61ClN19O10 [M+ + H] 1254.51; found 1254.45. AcImImPyPy-γ-ImPy-Indole-CBI (8). To a solution of compound 6b (1.1 mg, 0.9 µmol) in DMF (0.1 mL) was added 5% aqueous NaHCO3 (0.1 mL). The reaction mixture was stirred for 1 h at room temperature. Evaporation of the solvent gave a brown residue, which was subjected to column chromatography (silica gel, 5-10% MeOH in CH2Cl2, gradient elution) to produce compound 8 (1.1 mg, 0.9 µmol: quantitative yield) as a brown powder. After further purification by HPLC using a Chemcobond 5-ODS-H column (0.1% AcOH/CH3CN 0-50% linear gradient, 0-40 min, 254 nm), 8 was used in the DNA alkylation reaction.1H NMR (500 MHz, DMSO-d6) δ 11.77 (s, 1H; NH), 10.32 (s, 1H; NH), 10.29 (s, 1H; NH), 10.26 (s, 1H; NH), 9.95 (s, 1H; NH), 9.92 (s, 1H; NH), 9.82 (s, 1H; NH), 9.33 (s, 1H; NH), 8.08 (s, 1H; CH), 8.01 (brs, 1H; NH), 8.00 (d, J ) 7.5 Hz, 1H; CH), 7.60 (t, J ) 7.5 Hz, 1H; CH), 7.56 (s, 1H; CH), 7.53 (d, J ) 8.5 Hz, 1H; CH), 7.50 (s, 1H; CH), 7.46 (s, 1H; CH), 7.43 (t, J ) 7.5 Hz, 1H; CH), 7.42 (s, 1H; CH), 7.31 (s, 1H; CH), 7.27 (s, 1H; CH), 7.24 (d, J ) 8.5 Hz, 1H; CH), 7.22 (s, 1H; CH), 7.19 (s, 1H; CH), 7.16 (d, J ) 7.5 Hz, 1H; CH), 7.06 (s, 1H; CH), 6.95 (s, 1H; CH), 6.89 (s, 1H; CH), 4.62 (dd, J ) 10.0, 5.0 Hz, 1H; NCH2), 4.48 (d, J ) 10.0 Hz, 1H; NCH2), 4.00 (s, 3H; NCH3), 3.97 (s, 3H; NCH3), 3.95 (s, 3H; NCH3), 3.86 (s, 3H; NCH3), 3.84 (s, 3H; NCH3), 3.80 (s, 3H; NCH3), 3.20 (m, 2H; CH2), 2.90 (m, 1H; CH), 2.35 (m, 2H; CH2), 2.03 (s, 3H; COCH3), 1.79 (m, 2H; CH2), 1.76 (dd, J ) 7.5, 5.0 Hz, 1H; CH), 1.70 (t, J ) 5.0 Hz, 1H; CH); ESITOFMS m/e calcd for C61H60N19O10 [M++H] 1218.48, found 1218.48. AcImImPyPy-γ-PyPy-Indole-CBI (7). A synthetic procedure similar to that used for the preparation of compound 8 was followed to prepare compound 7, with a yield of 6% for two steps from 2a. After further purification by HPLC using a Chemcobond 5-ODS-H column (0.1% AcOH/CH3CN 0-50% linear gradient, 0-40 min, 254 nm), 8 was used in the DNA alkylation reaction. ESI-MS m/e calcd for C62H61N18O10 [M+ + H] 1217.5, found 1217.4. AcImImPy-β-PyPyIm-γ-PyPyP-β-Py-CO2H (9). AcImImPyβ-PyPyIm-γ-PyPyPy-β-PyCO2-oxime resin was synthesized in a stepwise reaction by Fmoc solid-phase protocol. A sample of resin was cleaved with alkali conditions (1 N NaOH in DMF, 1 h, 55 °C) and purified by HPLC using a Chemcobond 5-ODS-H column (0.1% AcOH/CH3CN 0-100% linear gradient, 0-30 min, 254 nm), to produce 9 (31 mg, 21%) as a yellow powder. 1H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 10.26 (s, 1H), 10.24 (s, 1H), 9.90 (s, 1H), 9.89 (s, 1H), 9.87 (s, 1H),

DNA Alkylation by a Pyrrole-Imidazole CBI Conjugate

9.86 (s, 1H), 9.85 (s, 1H), 9.81 (s, 1H), 9.34 (s, 1H), 8.07 (s, 1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.55 (s, 1H), 7.49 (s, 1H), 7.48 (s, 1H), 7.31 (s, 1H), 7.28 (s, 1H), 7.23 (s, 1H), 7.23 (s, 1H), 7.22 (s, 1H), 7.18 (s, 1H), 7.15 (s, 1H), 7.06 (s, 1H), 7.02 (s, 1H), 6.96 (s, 1H), 6.88 (s, 1H), 6.86 (s, 1H), 6.82 (s, 1H), 6.65 (s, 1H), 3.99 (s, 3H), 3.97 (s, 3H), 3.93 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.78 (s, 3H), 3.43 (m, 6H), 2.50 (m, 4H), 2.27 (m, 2H), 2.03 (s, 3H), 1.81 (m, 2H); ESI-TOFMS m/e calcd for C69H79N26O15 [M+ + H] 1511.61; found 1511.63. AcImImPy-β-PyPyIm-γ-PyPyPy-β-Py-Indole-seco-CBI (10). A synthetic procedure similar to that used for the preparation of compound 6b was followed to prepare compound 10, with a yield of 14% for two steps from 9. After further purification by HPLC using a Chemcobond 5-ODS-H column (0.1% AcOH/ CH3CN 0-50% linear gradient, 0-40 min, 254 nm), 10 was used in the DNA alkylation reaction. ESI-TOFMS m/e calcd for C91H95ClN29O16 [M+ + H] 1884.71; found 1884.70. Preparation of 5′-Texas Red-Modified DNA Fragments. The 5′-Texas Red-modified 537-bp DNA fragment, λ-DNA F10960*-11496, was prepared by polymerase chain reaction (PCR) with 5′-TexasRed-modified-ATCAGGGCAACTCAACCCTGTCC-3′ (λ-DNA forward, 10960-10982) and 5′-CAGGACGACCAATATCCAGC-3′ (λ-DNA reverse, 37007-37026). The 5′-Texas Red-modified 488-bp DNA fragment, λ-DNA F30990*-31477, was prepared by polymerase chain reaction (PCR) with 5′-Texas Red-modified-TGCATTCCGTGGTTGTCATACC-3′ (λ-DNA forward, 30990-31011) and 5′-GCATTATGTCGTGATTGAGC-3′ (λ-DNA reverse, 17045-17064). Fragments were purified by filtration using Suprec-02, and their concentrations were determined by UV absorption. The asterisk indicates Texas Red modification and the nucleotide numbering starts with the replication site. High-Resolution Gel Electrophoresis. The 5′-Texas Redlabeled DNA fragments (10 nM) were alkylated by various concentration of 7, 8, and 6b in 10 µL of 5 mM sodium phosphate buffer (pH 7.0) containing 10% DMF at 23 °C for 15 h. The reaction was quenched by the addition of calf thymus DNA (1 mM, 1 µL) and heating for 5 min at 90 °C. The DNA was recovered by vacuum centrifugation. The pellet was dissolved in 8 µL of loading dye (formamide with fuschin red), heated at 94 °C for 20 min, and then immediately cooled to 0 °C. A 2-µL aliquot was subjected to electrophoresis on a 6% denaturing polyacrylamide gel using a Hitachi 5500-S DNA Sequencer. Molecular Modeling Studies. Density functional calculations were performed at the B3LYP/6-31G* level utilizing Spartan 04 (Wave function, Irvine, CA) program. Energy minimizations of d(ATACAAATCCAAT)/d(ATTGGATTTGTAT)-11 were performed with the Discover (Accelrys, San Diego, CA) program using CFF force-field parameters. The starting structure was built on the basis of the NMR structure of the ImPyPy-γPyPyPy-d(CGCTAACAGGC)/d(GCCTGTTAGCG) complex (21) and the Duo-Dist-octamer complex (22). The connecting parts between them were built using standard bond lengths and angles. The CBI unit of the assembled initial structure was energy minimized using a distance-dependent dielectric constant of  ) 4r (r stands for the distance between atoms i and j) and with convergence criteria having an RMS gradient of less than 0.001 kcal/mol Å. Eighteen Na cations were placed at the bifurcating position of the O-P-O angle at a distance of 2.51 Å from the phosphorus atom. The resulting complex was soaked in a 10-Å layer of water. The whole system was minimized without any constraint, to the stage where the RMS was less than 0.001 kcal/mol Å.

Bioconjugate Chem., Vol. 17, No. 3, 2006 717

Figure 2. Geometry optimized structures of (E)-3-(4-formamido-1methyl-1H-pyrrol-2-yl)acrylamide (A) and 5-formamido-1H-indole-2carboxamide (B) and their superimposition (C) obtained by density functional calculation at the B3LYP/6-31G* level.

RESULTS AND DISCUSSION Molecular Design of Linker Unit. A vinyl linker between the Py-Im polyamides and CBI moiety is highly advantageous for DNA alkylation, as it facilitates highly specific DNA alkylation by adjusting the position of the CBI moiety in the minor groove. However, the vinyl linker moiety has certain disadvantages in the molecular design of alkylating Py-Im polyamides, as it is relatively unstable under acidic and basic conditions, and it suffers from low chemical yields. Because ethyl 3-(4-amino-N-methyl-pyrrol-2-yl)acrylate is extremely unstable, Py-Im polyamides prepared by a solid phase method cannot easily be employed in the synthesis of alkylating agents which possess vinyl linkers. Therefore, we synthesized various alkylating Py-Im polyamides by a combination of timeconsuming liquid-phase coupling reactions.4 To overcome these problems, we searched for a new linker that could provide almost the same geometry as Py with a vinyl linker and is stable. We chose a 5-amino-1H-indole-2-carbonyl linker (indole linker) as the new linker, because amide linkages of this unit are approximately superimposable with Py with a vinyl linker. Similarity of structures between 5-formamido-1H-indole-2carboxamide and (E)-3-(4-formamido-1-methyl-1H-pyrrol-2-yl)acrylamide was estimated by density functional calculation at the B3LYP/6-31G* level (Figure 2). We expected that Py-Im polyamide-indole-CBI conjugates 7 and 8 would alkylate DNA in a sequence-specific fashion (Figure 1b). Indole linker unit has already been used in the synthesis of derivatives of CC1065 (23). Synthesis of Hairpin Py-Im Polyamide-CBI Conjugates with Indole Linker, 7 and 8. Conjugates 7 and 8 were synthesized as shown in Scheme 1. The six-ring Py-Im polyamides 2a and 2b, both of which have terminal carboxylic acid groups, were prepared by Fmoc solid-phase synthesis using an oxime resin (24-26), and purified by reversed-phase HPLC. Indole linker unit 3 was synthesized by hydrolysis of commercially available ethyl 5-nitro-1H-indole-2-carboxylate, followed by reduction using palladium-carbon and H2 gas. The carboxylic acids 2a and 2b were converted to activating esters with HATU and iPr2NEt, followed by coupling with 3 to give 4a and 4b. The DNA alkylating moiety, seco-CBI 5, was prepared according to procedures from the literature (16). Conjugates 6a and 6b were synthesized by the coupling of 4a and 4b with seco-CBI 5 using EDCI and NaHCO3. Treatment of 6a and 6b with aqueous 5% NaHCO3 provided 7 and 8, respectively. Use of Fmoc solid phase synthesis greatly facilitates the synthesis of alkylating Py-Im polyamides. For example, conjugate 8 was synthesized with an overall yield of 6.4%. In contrast, conjugate 1b with a vinyl linker, prepared by the 22

718 Bioconjugate Chem., Vol. 17, No. 3, 2006 Scheme 1

Bando et al.

a

steps of a convergent liquid-phase synthesis, gave an overall yield of 0.1% (19). The purity and identity of conjugates 7 and 8 were verified by reversed-phase analytical HPLC and ESITOF mass spectrometry. Evaluation of DNA Alkylating Activity. Sequence-selective alkylation by compounds 7, 8, and 6b was investigated using the 5′-Texas Red-labeled 537 bp DNA fragments λ-DNA F10960*-11496 using an automated DNA sequencer. Alkylation was carried out at 23 °C for 15 h, followed by quenching by the addition of calf thymus DNA. The samples were heated at 94 °C under neutral conditions for 20 min. The alkylation sites were visualized by thermal cleavage of the DNA strand at the alkylated sites. Under these heating conditions, all the purine N3 alkylated sites in the DNA almost quantitatively produced cleavage bands on the gel. Sequencing analysis of the alkylated DNA fragments after heat treatment is shown in Figure 3. The conjugates with indole linkers, 7 and 8, produced discrete cleavage bands at nanomolar concentrations. Efficient alkylation by 7 occurred at site 1, 5′-AATCCA-3′, and site 2, 5′-TAACCA3′, only at a concentration of 10 nM (lane 3). Similarly, alkylation by 8 was observed at the four match sites, 3-6, 5′WGWCCA-3′ (W ) A or T; lanes 7-10) with no alkylation at the mismatch site. These results clearly indicate that 7 and 8 efficiently alkylate DNA in a sequence-specific fashion at nanomolar concentrations, as does 1b (19), and the indole moiety acts as an appropriate substitute for the vinyl linker. We confirmed that seco-CBI conjugate 6b, which is quantitatively converted to 8 under basic conditions, efficiently alkylates DNA at target sequences, 5′-WGWCCA-3′ (lanes 1214), as does CBI conjugate 8. It is assumed that 6b readily converts to 8 in aqueous buffered solution. Analogous in situ cyclization to reactive aziridines for DNA alkylation was found

Figure 3. (A) Thermally induced strand cleavage of the 5′-Texas Redlabeled 537 bp DNA fragment λ-DNA F10960*-11496 by conjugates 7, 8, and 6b at 23 °C for 15 h: Lane 1 ) DNA control; Lanes 2-6 ) 20, 10, 5, 2, and 1 nM of 7; Lanes 7-11 ) 20, 10, 5, 2, and 1 nM of 8; Lanes 12-16 ) 20, 10, 5, 2, and 1 nM of 6b. (B) Schematic representation of the recognition of matching sequences by 7 and 8. The arrows indicate the site of alkylation by 7 and 8.

for both mitomycin C and nitrogen mustard (23). In addition, this result is consistent with the previous results for Py-Im derivatives with seco-CBI (27). Synthesis and Evaluation of DNA Alkylating Activity using seco-CBI Conjugate 10. Toward the goal of sequencespecific alkylating agents that target specific genes, alkylating polyamides that recognize longer sequences are desirable. Thus, we designed and synthesized seco-CBI conjugate 10 by coupling of carboxylic acid 9, prepared by Fmoc solid-phase synthesis

DNA Alkylation by a Pyrrole-Imidazole CBI Conjugate

Figure 4. (A) Chemical structures of compound 9, 10, and 11. (B) Thermally induced strand cleavage of the 5′-Texas Red-labeled 488 bp DNA fragment, λ-DNA F30990*-31477, by conjugate 10 at 37 °C for 15 h: Lane 1 ) DNA control; Lanes 2-8 ) 1000, 800, 600, 400, 200, 100, and 50 nM of 10. Sites 7 and 8 ) match sequences, sites 9-12 ) mismatch sequences. (C) Schematic representation of the recognition in matching sequences by 10. The arrows indicate the site of alkylation. The alkylating base is shown in red bold, and the mismatch-binding base is shown in italics.

in 21% yield, with 3 and successively with seco-CBI 5 (Figure 4a). It is important to note that the synthesis of the larger polyamide proceeded as smoothly as for 7 and 8. Sequencing gel analysis clearly indicated that the seco-CBI conjugate 10 alkylated predominantly at the adenine of the nine-base-pair matching sequence 5′-ACAAATCCA-3′ (site 7) and 5′TCATTTCCA-3′ (site 8, lanes 2-6), together with faint

Bioconjugate Chem., Vol. 17, No. 3, 2006 719

Figure 5. The energy-minimized structure of d(ATACAAATCCAAT)/ d(ATTGGATTTGTAT)-11 (A). The alkylating base is shown in orange and the minor-groove-binding Py-Im polyamide is shown in blue. The indole linker is shown in purple, and the CBI moiety is shown in yellow. Schematic representation of distance and angle of N3 of adenine and CBI (B).

mismatch alkylations at the A of 5′-CGGATATTA-3′ (site 9), 5′-AAATACCCA-3′ (site 10), 5′-TATTATTTA-3′ (site 11) and 5′-CGTTTATTA-3′ (site 12). It is assumed that 10 readily converts to 11 under the reaction conditions. Differences in reaction rates for each sequence can be a consequence of many associated factors, which require further investigation for detailed understanding. To the best of our knowledge, the present result is the first example of the sequence-specific Py-Im alkylating polyamide recognizing a nine-base-pair sequence.

720 Bioconjugate Chem., Vol. 17, No. 3, 2006

Model of Binding of 11 to Target Oligonucleotides. To gain an insight into the location of the seco-CBI conjugate 10 in the DNA minor groove, we constructed the d(ATACAAATCCA11AT)/d(ATTGGATTTGTAT)-11 complex, which is based on the predictable structure of the CBI conjugate in the DNA minor groove. The energy-minimized structures suggest that binding of 11 to the minor groove of d(ATACAAATCCA11AT)/d(ATTGGATTTGTAT) is located in close proximity to the nucleophilic N3 of the A11 residues: 2.95 Å, with the angle of the N3-C9-C8b bond ) 151° (Figure 5). The similar binding orientations of the vinyl-linked CPI and CBI conjugates would indicate induction of alkylation at a specific nine-basepair sequence.

CONCLUSIONS We have successfully synthesized Py-Im hairpin polyamide CBI conjugates 7 and 8, and the corresponding seco-CBI conjugates with indole linker, 6a and b, together with the longer alkylating conjugate 10 using Fmoc solid phase synthesis and a subsequent liquid phase coupling procedure. We have evaluated these DNA alkylating polyamides in detail using highresolution denaturing gel electrophoresis employing DNA fragments. The results clearly indicate that Py-Im CBI conjugates 7, 8, 6b, and 10 efficiently alkylate at specific DNA sequences. Importantly, the selectivity and the efficiency of DNA alkylation by 8 were as good as those of the corresponding CBI conjugate 1b. In particular, we have clearly demonstrated successful sequence-specific DNA alkylation by the seco-CBI conjugate 10, which alkylates a specific nine-base-pair sequence, 5′-ACAAATCCA-3′. Furthermore, introduction of an indole linker greatly facilitated the synthesis of sequence-specific alkylating Py-Im polyamides. To open the next door toward the goal of developing antitumor agents, the design of new indole-CBI conjugates will allow further biological studies.

ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Priority Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

LITERATURE CITED (1) Denison, C., and Kodadek, T. (1998) Small-molecule-based strategies for controlling gene expression. Chem. Biol. 5, R129-145. (2) Hurley, L. H. (2002) DNA and its associated processes as targets for cancer therapy. Nat. ReV. Cancer. 2, 188-200. (3) Baraldi, P. G., Bovero, A., Fruttarolo, F., Preti, D., Tabrizi, M. A., Pavani, M. G., and Romagnoli, R. (2004) DNA minor groove binders as potential antitumor and antimicrobial agents. Med. Res. ReV. 24, 475-528. (4) Dervan, P. B. (2001) Molecular recognition of DNA by small molecules. Bioorg. Med. Chem. 9, 2215-2235. (5) Dervan, P. B., and Edelson, B. S. (2003) Recognition of the DNA minor groove by pyrrole-imidazole polyamides. Curr. Opin. Struct. Biol. 13, 284-299. (6) Nagamura, S., Asai, A., Kanda, Y., Kobayashi, E., Gomi, K., and Saito, H. (1996) Synthesis and antitumor activity of duocarmycin derivatives: modification of segment A of duocarmycin B2. Chem. Pharm. Bull. 44, 1723-1730. (7) Tao, Z.-F., Saito, I., and Sugiyama, H. (2000) Highly cooperative DNA dialkylation by the homodimer of imidazole-pyrrole diamideCPI conjugate with vinyl linker. J. Am. Chem. Soc. 122, 16021608. (8) Bando, T., Iida, H., Saito, I., and Sugiyama, H. (2001) Sequencespecific DNA interstrand cross-linking by imidazole-pyrrole CPI conjugate. J. Am. Chem. Soc. 123, 5158-5159.

Bando et al. (9) Bando, T., Narita, A., Saito, I., and Sugiyama, H. (2002) Molecular design of pyrrole-imidazole hairpin polyamide for effective DNA alkylation. Chem.-Eur. J. 8, 4781-4790. (10) Bando, T., Narita, A., Saito, I., and Sugiyama, H. (2003) Highly efficient Sequence-specific DNA interstrand cross-linking by pyrrole/ imidazole CPI conjugates. J. Am. Chem. Soc. 125, 3471-3485. (11) Bando, T., Iida, T., Tao, Z.-F., Narita, A., Fukuda, N., Yamori, T., and Sugiyama, H. (2003) Sequence-specificity, reactivity and antitumor activity of DNA alkylating pyrrole-imidazole diamides. Chem. Biol. 10, 751-758. (12) Bando, T., Narita, A., Iwai, A., Kihara, K., and Sugiyama, H. (2004) C-H to N substitution dramatically alters the sequencespecific DNA alkylation, cytotoxicity, and expression of human cancer cell lines. J. Am. Chem. Soc. 126, 3406-3407. (13) Oyoshi, T., Kawakami, W., Narita, A., Bando, T., and Sugiyama, H. (2003) Inhibition of transcription at a coding sequence by alkylation polyamide. J. Am. Chem. Soc. 125, 4752-4754. (14) Shinohara, K., Narita, A., Oyoshi, T., Bando, T., Teraoka, H., and Sugiyama, H. (2004) Sequence-specific gene silencing in mammalian cells by alkylating pyrrole-imidazole polyamides. J. Am. Chem. Soc. 126, 5113-5118. (15) Boger, D. L., Ishizaki, T., Kitos, P. A., and Suntornwat, O. (1990) Synthesis of N-(tert-butyloxycarbonyl)-CBI, CBI, CBI-CDPI1, and CBI-CDPI2: enhanced functional analogues of CC-1065 incorporating the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) left-hand subunit. J. Org. Chem. 55, 5823-5832. (16) Boger, D. L., Yun, W. Y., and Teegarden, B. R. (1992) An improved synthesis of 1, 2, 9, 9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI): a simplified analog of the CC-1065 alkylation subunit. J. Org. Chem. 57, 2873-2876. (17) Boger, D. L., and McKie, J. A. (1995) An efficient synthesis of 1, 2, 9, 9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI): an enhanced and simplified analog of the CC-1065 and duocarmycin alkylation subunits. J. Org. Chem. 60, 1271-1275. (18) Kastrinsky D. B., and Boger, D. L. (2004) Effective asymmetric synthesis of 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI). J. Org. Chem. 69, 2284-2289. (19) Bando, T., Narita, A., Asada, K., Ayame, H., and Sugiyama, H. (2004) Enantioselective DNA alkylation by a pyrrole-imidazole S-CBI conjugate. J. Am. Chem. Soc. 126, 8948-8955. (20) Bando, T., Narita, A., Sasaki, S., and Sugiyama, H. (2005) Specific adenine alkylation by pyrrole-imidazole CBI conjugates. J. Am. Chem. Soc. 127, 13890-13895. (21) de Clairac, R. P. L., Geierstanger, B. H., Mrksich, M., Dervan, P. B., and Wemmer, D. E. (1997) NMR characterization of hairpin polyamide complexes with the minor groove of DNA. J. Am. Chem. Soc. 119, 7909-7916. (22) Sugiyama, H., Lian, C. Y., Isomura, M., Saito, I., and Wang, A. H.-J. (1996) Distamycin A modulates the sequence specificity of DNA alkylation by duocarmycin A. Proc. Natl. Acad. Sci. U.S.A. 93, 14405-14410. (23) Wolkenberg S. E., and Boger, D. L. (2002) Mechanisms of in situ activation for DNA-targeting antitumor agents. Chem. ReV. 102, 2477-2495. (24) Baird, E. E., and Dervan, P. B. (1996) Solid-phase synthesis of polyamides containing imidazole and pyrrole amino acids. J. Am. Chem. Soc. 118, 6141-6146. (25) Wurtz, N. R., Turner, J. M., Baird, E. E., and Dervan, P. B. (2001) Fmoc solid-phase synthesis of polyamides containing pyrrole and imidazole amino acids. Org. Lett. 3, 1201-1203. (26) Belitsky, J. M., Nguyen, D. H., Wurtz, N. R., and Dervan, P. B. (2002) Solid-phase synthesis of DNA binding polyamides on oxime resin. Bioorg. Med. Chem. 10, 2767-2774. (27) Chang A. Y., and Dervan, P. B. (2000) Strand selective cleavage of DNA by diastereomers of hairpin polyamide-seco-CBI conjugates. J. Am. Chem. Soc. 122, 4856-4864. BC060022W