Design, synthesis, and analysis of minor groove binder pyrrolepolyamide-2'-deoxyguanosine hybrids.

Pyrrolepolyamide-2'-deoxyguanosine hybrids (Hybrid 2 and Hybrid 3) incorporating the 3-aminopropionyl or 3-aminopropyl linker were designed and synthesized on the basis of previously reported results of a pyrrolepolyamide-adenosine hybrid (Hybrid 1). Evaluation of the DNA binding sequence selectivity of pyrrolepolyamide-2'-deoxyguanosine hybrids was performed by CD spectral and T(m) analyses. It was shown that Hybrid 3 possessed greater binding specificity than distamycin A, Hybrid 1 and Hybrid 2.

1. Introduction

Organic compounds capable of controlling gene expression may potentially be used as viable gene therapy agents. In this regard, one of the most important requirements the drug must possess is the ability to selectively distinguish target sequences from all other sequences within the genome. The development of antisense or antigene drugs comprising synthetic oligonucleotides has been investigated [1]. Furthermore, it was reported that pyrrolepolyamide molecules such as distamycin A (Dst) and netropsin (Net), which bind to the minor groove of DNA, can interfere with gene expression through sequence-specific recognition of DNA [2-18] (Figure 1). Baraldi et al. reported on the design, synthesis, and biological activity of hybrid compounds comprising a combination of Dst and uramustine (uracil mustard) [14]. Uramustine interacts with GC-rich regions and can alkylate guanine-N7. The hybrid compounds exhibited enhanced antitumor activity against the K562 Human Leukemia Cell Line compared to both distamycin A and uramustine derivatives. In this case, the interaction with DNA tends to be dominated by minor groove binding of the Dst moiety. Given these findings, we expected that nucleosides bearing a minor groove binder (MGB) such as a pyrrolepolyamide, which possesses high affinity for the AAATT sequence, might be able to regulate gene expression. When the MGB polyamide-nucleoside hybrid acts on dsDNA, it is expected that complex formation would involve high affinity and sequence selectivity through minor groove-binding of the polyamide moiety and interactions between dsDNA and the nucleoside moiety of the hybrid such as those involving Hoogsteen-type hydrogen bonding. On the other hand, if assuming the hybrid is incorporated into DNA during DNA biosynthesis, it is expected that DNA replication and transcription would be obstructed through minor groove-binding of the hybrid polyamide moiety, incorporated near the target sequence of the DNA. Therefore, it is preferable that the pyrrolepolyamide be combined with the nucleoside site located on the minor groove side of a DNA duplex, for example, the 2′-position of the sugar moiety and the 2-exocyclic amino group of the guanine base. With this in mind, we previously designed and synthesized Hybrid 1, where the pyrrolepolyamide is linked at the 2′ hydroxyl group of adenosine, as a lead compound for potential use as a new gene therapy agent [19, 20]. Evaluation of the DNA-binding activity of Hybrid 1 by circular dichroism (CD) spectral and Tm value analyses showed that although the ΔT value (ca. 2°C, Table 1) was small, Hybrid 1 displayed greater binding specificity than Dst and Net.

Figure 1

Structures of Distamycin A, Netropsin, Hybrid 1, Hybrid 2, and Hybrid 3.

Table 1

ΔT values (°C) for the DNA duplexs with ligand (Hybrid 1, Hybrid 2, Hybrid 3, or Dst) at several [ligand]/[duplex] ratios in buffer consisting of 10 mM sodium phosphate, 10 mM NaCl, and 0.1 mM Na2EDTA at pH 7.0.

[ligand]/[duplex]		1 : 1	2 : 1	3 : 1
	DNA 1	2.1	2.3	—
Hybrid 1	DNA 2	− 0.1	0.2	—
	DNA 3	−0.4	0.3	—
	DNA 1	2.7	3.8	3.9
Hybrid 2	DNA 2	−0.3	−0.2	− 0.1
	DNA 3	0.2	0.3	0.3
	DNA 1	5.6	5.7	5.8
Hybrid 3	DNA 2	0.1	0.2	0.5
	DNA 3	− 0.3	−0.5	−0.2
	DNA 1	22.9	22.9	23.5
Dst	DNA 2	3.0	6.5	6.9
	DNA 3	15.2	16.3	16.3

*ΔTm value = (Tm value in the presence of ligand)-(Tm value in the absence of ligand).

**DNA duplexs: DNA 1 = d-(5′-CGCAAATTGGC-3′)-d-(3′-GCGTTTAACCG-5′) Tm = 32.2°C.

DNA 2 = d-(5′-CGCAGGC-3′)-d-(3′-GCGTCCG-5′) Tm = 39.5°C.

DNA 3 = d-(5′-GCACGACTACG-3′)-d-(3′-CGTGCTGATGC-5′) Tm = 33.9°C.

DNA 1 contains the high affinity binding site (5′-AAATT-3′) of Dst.

On the basis of these results, and for the purpose of developing a gene control agent through interaction with the DNA duplex and/or incorporation into DNA during DNA biosynthesis, we designed MGB polyamide-2′-deoxynucleoside hybrids, such as Hybrid 2 and Hybrid 3 combined with a pyrrolepolyamide using a linker at the 2-exocyclic amino group of the guanine base (Figure 1). When considering a DNA strand with incorporated hybrid during DNA biosynthesis, it is expected that a short linker might result in high stabilization of the dsDNA. Therefore, we designed Hybrid 2 containing a 3-aminopropionyl linker used to the head-to-tail hairpin polyamide reported by Mrksich et al. [21]. Moreover, for investigations concerning the stabilization of dsDNA with the complementary strand, we designed Hybrid 3 containing an alkyl linker connected directly at the 2-exocyclic amino group of the guanine base. Here we report on the synthesis and interaction of these hybrids on DNA duplexes.

2. Materials and Methods

2.1. General

Column chromatography was performed on silica gel (Silica gel N60, purchased from Kanto Chemical Co., Inc.) using methanol/chloroform as solvent systems. Melting points were determined using a Yanaco Micro-melting-point apparatus and are uncorrected. 1H-N.m.r. and 31P-n.m.r. spectra were recorded on a Bruker DRX 400 apparatus. 19F-N.m.r. spectra were recorded on a Varian MERCURY 300 apparatus. Mass spectra were recorded on a Micromass Q-Tof Ultima API apparatus. Elemental analyses were achieved using an Elemental Vavio EL apparatus. Circular dichroism (CD) spectra were recorded on a JASCO J-720 spectropolarimeter. UV melting curves were measured using a Shimazu TMSPC-8/UV-1600 apparatus. DNA oligonucleotides were purchased from Hokkaido System Science Co., Ltd. Methyl 4-nitropyrrole-2-carboxylate (5) and 4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-carboxylic acid (6) were prepared according to the procedure described by Baird and Dervan [3]. 3′,  5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-deoxyguanosine (12) and 3′,5′-di-O-acetyl-2-fluoro-O6-[2-(4-nitrophenyl)ethyl]-2′-deoxyinosine (20) were prepared from 2′-deoxyguanosine, as previously described [22-26].

2.2. Methyl 4-[[4-(Tert-butoxycarbonyl)amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrole-2-carboxylate (7)

Compound 5 (3.000 g, 16.3 mmol) was dissolved in ethyl acetate (100 mL) and 10% Pd/C catalyst (1.000 g) was added to the solution. The mixture was stirred under a slight positive pressure of hydrogen for 12 hours at room temperature. The catalyst was removed by filtration through Celite and the filtrate was concentrated in vacuo to give methyl 4-amino-1-methylpyrrole-2-carboxylate, which was subsequently used without purification. Methyl 4-amino-1-methylpyrrole-2-carboxylate was dissolved in DMF (100 mL) and 4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-carboxylic acid (6) (3.920 g, 16.3 mmol), EDCI (6.250 g, 32.6 mmol) and DMAP (3.980 g, 32.6 mmol) were added to the solution. After stirring for 3 hours, the solution was diluted with ethyl acetate (200 mL) and methanol (20 mL) and washed with 3 M HCl aq. (100 mL × 3), H2O (100 mL), 5% NaHCO3 aq. (100 mL × 2) and H2O (100 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. Compound 7 was obtained in 99% yield (6.070 g) as a yellow glass; 1H-n.m.r. (DMSO-d6) δ 9.85 (s, 1H, -NHCO-), 9.09 (s, 1H, Boc-NH), 7.45 (d, 1H, J = 1.9 Hz, Py-H), 6.90 (d, 1H, J = 1.9 Hz, Py-H), 6.89 (s, 1H, Py-H), 6.84 (s, 1H, Py-H), 3.83 (s, 3H, Py-CH3), 3.81 (s, 3H, Py-CH3), 3.73 (s, 3H, -OCH3), 1.45 (s, 9H, -CH3 of the Boc group); 13C-n.m.r. (DMSO-d6) δ 160.80, 158.42, 152.85, 122.98, 122.60, 122.42, 120.72, 118.49, 117.15, 108.37, 103.82, 78.30, 50.90, 36.13, 36.01, 28.19; ESI-TOF MS calcd for C18H25N4O5 (M + H)+ 377.1825, found 377.1826; Anal. Calcd for C18H24N4O5 + 0.5 H2O: C, 56.09; H, 6.54; N, 14.54, found C, 56.18; H, 6.28; N, 14.51.

2.3. Methyl 4-[[4-[[4-(Tert-butoxycarbonyl)amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrole-2-carboxylate (8)

Compound 7 (3.000 g, 16.3 mmol) was dissolved in ethyl acetate (20 mL)/methanol (4 mL) and acetyl chloride (5.7 mL, 80 mmol) was added to the solution at 0°C. The mixture was stirred for 30 minutes. The reaction solution was concentrated in vacuo to give methyl 4-[(4-amino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrole-2-carboxylate, which was subsequently used without purification. Methyl 4-[(4-amino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrole-2-carboxylate was dissolved in DMF (50 mL) and 4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-carboxylic acid (6) (2.110 g, 8.77 mmol), EDCI (3.060 g, 15.9 mmol) and DMAP (2.920 g, 23.9 mmol) were added to the solution. After stirring for 3 hours, the solution was diluted with ethyl acetate (200 mL)/methanol (20 mL) and washed with 3 M HCl aq. (100 mL × 3), H2O (100 mL), 5% NaHCO3 aq. (100 mL × 2) and H2O (100 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. Compound 8 was obtained in 98% yield (3.900 g); 1H-n.m.r. (DMSO-d6) δ 9.92 (s, 1H, -NHCO-), 9.87 (s, 1H, -NHCO-), 9.08 (s, 1H, Boc-NH), 7.47 (d, 1H, J = 1.9 Hz, Py-H), 7.22 (d, 1H, J = 1.6 Hz, Py-H), 7.08 (d, 1H, J = 1.6 Hz, Py-H), 6.92 (d, 1H, J = 1.9 Hz, Py-H), 6.90 (s, 1H, Py-H), 6.85 (s, 1H, Py-H), 3.85 (s, 3H, Py-CH3), 3.84 (s, 3H, Py-CH3), 3.82 (s, 3H, Py-CH3), 3.74 (s, 3H, -OCH3), 1.46 (s, 9H, -CH3 of the Boc group); 13C-n.m.r. (DMSO-d6) δ 160.81, 158.52, 158.44, 152.88, 123.01, 122.83, 122.52, 122.34, 120.74, 118.52, 117.08, 108.38, 104.83, 103.85, 78.29, 50.91, 36.15, 36.04, 28.20.

2.4. 4-[[4-[[4-(Tert-butoxycarbonyl)amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrole-2-carboxylic acid (9)

Compound 8 (0.249 g, 0.5 mmol) was dissolved in methanol (2.5 mL) and 2 M NaOH aq. (2.5 mL) was added to the solution. The mixture was stirred at 60°C for 1 hour. The reaction solution was concentrated in vacuo and diluted with H2O (25 mL), washed with diethyl ether (10 mL × 3), and neutralized with 10% HCl aq. to ca. pH 2. Compound 9 was extracted with ethyl acetate (20 mL × 3) from the resulting solution. The organic layer was washed with H2O (30 mL), dried over anhydrous magnesium sulfate and evaporated to dryness. Compound 9 was obtained in 95% yield (0.230 g) as a white powder; m.p. 175–178°C; 1H-n.m.r. (DMSO-d6) δ 12.13 (s, 1H, -COOH), 9.88 (s, 1H, -NHCO-), 9.85 (s, 1H, -NHCO-), 9.08 (s, 1H, Boc-NH), 7.42 (d, 1H, J = 1.9 Hz, Py-H), 7.21 (d, 1H, J = 1.6 Hz, Py-H), 7.07 (d, 1H, J = 1.6 Hz, Py-H), 6.89 (s, 1H, Py-H), 6.85 (d, 1H, J = 1.9 Hz, Py-H), 6.84 (s, 1H, Py-H), 3.84 (s, 3H, Py-CH3), 3.83 (s, 3H, Py-CH3), 3.81 (s, 3H, Py-CH3), 1.46 (s, 9H, -CH3 of the Boc group); 13C-n.m.r. (DMSO-d6) δ 161.95, 158.45, 158.42, 152.86, 122.81, 122.70, 122.57, 122.32, 122.28, 120.25, 119.50, 118.44, 117.06, 108.39, 104.77, 103.82, 48.59, 36.11, 36.02, 28.20; ESI-TOF MS calcd for C23H29N6O6 (M + H)+ 485.2149, found 485.2152; Anal. Calcd for C23H28N6O6 + H2O: C, 54.97; H, 6.02; N, 16.72, found C, 55.18; H, 6.22; N, 16.06.

2.5. 4-[[4-[(4-Formylamino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrole-2-carboxylic acid (10)

Compound 9 (77 mg, 0.19 mmol) was dissolved in methanol (3 mL) and acetyl chloride (0.85 mL, 12 mmol) was added to the solution at 0°C. After stirring for 30 minutes, the reaction solution was concentrated in vacuo to dryness. The residue was dissolved in methanol (1 mL) and N-formylimidazole, prepared by the treatment of formic acid (12.9 μL, 0.34 mmol) with 1,1′-carbonyldiimidazole (CDI) (36 mg, 0.22 mmol) in THF (1.5 mL) at room temperature for 5 minutes [11], was added to the solution at −40°C. After stirring at −40°C for 30 minutes, the solution was diluted with 5% NaHCO3 aq. (10 mL), washed with ethyl acetate (10 mL × 2), and neutralized with 10% HCl aq. to ca. pH 2. Compound 10 was extracted with ethyl acetate (20 mL × 3) from the resulting solution. The organic layer was washed with H2O (30 mL), dried over anhydrous magnesium sulfate, and evaporated to dryness. The residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 9, v/v) solvent system. Compound 10 was obtained in 57% yield (45 mg) as a white powder; m.p. 178–181°C; 1H-n.m.r. (DMSO-d6) δ 12.17 (br, 1H, -COOH), 10.05 (d, 1H, J = 1.6 Hz, -NHCHO), 9.92 (s, 1H, -NHCO-), 9.89 (s, 1H, -NHCO-), 8.13 (s, 1H, J = 1.6 Hz, -CHO), 7.42 (d, 1H, J = 1.7 Hz, Py-H), 7.23 (d, 1H, J = 1.7 Hz, Py-H), 7.20 (d, 1H, J = 1.7 Hz, Py-H), 7.06 (d, 1H, J = 1.7 Hz, Py-H), 6.92 (d, 1H, J = 1.7 Hz, Py-H), 6.85 (d, 1H, J = 1.7 Hz, Py-H), 3.85 (s, 3H, Py-CH3), 3.84 (s, 3H, Py-CH3), 3.38 (s, 3H, Py-CH3); 13C-n.m.r. (DMSO-d6) δ 162.70, 162.13, 158.45, 158.31, 157.91, 122.98, 122.67, 122.65, 122.17, 120.76, 120.16, 119.75, 118.51, 118.44, 108.34, 104.74, 103.98, 36.13, 36.06; ESI-TOF MS calcd for C19H21N6O5 (M + H)+ 413.1573, found 413.1579.

2.6. 3-[[4-[[4-[(4-Formylamino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropionic acid (11)

Compound 9 (0.100 g, 0.206 mmol) was dissolved in DMF (2 mL) and ethyl β-alanine hydrochloride (35 mg, 0.227 mmol), EDCI (47 mg, 0.247 mmol), and DMAP (60 mg, 0.494 mmol) were added to the solution. After stirring for 3 hours, the solution was diluted with ethyl acetate (20 mL) and washed with 10% HCl aq. (10 mL × 3), H2O (10 mL), 5% NaHCO3 aq. (10 mL × 3), and H2O (10 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. Ethyl 3-[4-[[4-[[4-(tert-butoxycarbonyl)amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropionate was obtained in 85% yield (102 mg); 1H-n.m.r. (DMSO-d6) δ 9.87 (s, 1H, -NHCO-), 9.85 (s, 1H, -NHCO-), 9.07 (s, 1H, Boc-NH), 8.03 (t, 1H, J = 6 Hz, Py-NH), 7.21 (d, 1H, J = 1.3 Hz, Py-H), 7.18 (d, 1H, J = 1.5 Hz, Py-H), 7.03 (d, 1H, J = 1.3 Hz, Py-H), 6.89 (s, 1H, Py-H), 6.85 (d, 1H, J = 1.5 Hz, Py-H), 6.84 (s, 1H, Py-H), 4.07 (q, 2H, J = 7.1 Hz, CH3CH2-), 3.84 (s, 3H, Py-CH3), 3.81 (s, 3H, Py-CH3), 3.80 (s, 3H, Py-CH3), 3.40 (dt, 2H, J = 7 Hz and J = 6 Hz, -CH2NH-), 2.53 (t, 2H, J = 7 Hz, -COCH2-), 1.46 (s, 9H, -CH3 of the Boc group), 1.19 (t, 3H, J = 7.1 Hz, CH3CH2-); 13C-n.m.r. (DMSO-d6) δ 171.34, 161.27, 158.45, 158.40, 152.84, 122.82, 122.71, 122.65, 122.28, 122.16, 118.35, 117.90, 117.00, 104.27, 103.81, 59.84, 36.00, 35.89, 34.78, 34.02, 28.19, 14.06; ESI-TOF MS calcd for C28H38N7O7 (M + H)+ 584.2833, found 584.2822.

Ethyl 3-[4-[[4-[[4-(tert-butoxycarbonyl)amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropionate (71.8 mg, 0.123 mmol) was dissolved in methanol (1 mL), 2 M NaOH aq. (1 mL) was added and the solution was stirred at 60°C for 3 hours. The reaction solution was concentrated in vacuo and diluted with H2O (10 mL), washed with diethyl ether (5 mL × 3), and neutralized with 10% HCl aq. to ca. pH 2. The product was extracted with ethyl acetate (10 mL × 3) from the resulting solution. The organic layer was washed with H2O (15 mL), dried over anhydrous magnesium sulfate, and evaporated to dryness. 3-[4-[[4-[[4-(tert-Butoxycarbonyl)amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropionic acid was obtained in 74% yield (50.2 mg); 1H-n.m.r. (DMSO-d6) δ 11.90 (br, 1H, -COOH), 9.87 (s, 1H, -NHCO-), 9.85 (s, 1H, -NHCO-), 9.07 (s, 1H, Boc-NH), 8.02 (t, 1H, J = 6 Hz, Py-NH), 7.22 (d, 1H, J = 1.6 Hz, Py-H), 7.18 (d, 1H, J = 1.7 Hz, Py-H), 7.03 (d, 1H, J = 1.6 Hz, Py-H), 6.89 (s, 1H, Py-H), 6.85 (d, 1H, J = 1.7 Hz, Py-H), 6.84 (s, 1H, Py-H), 3.84 (s, 3H, Py-CH3), 3.81 (s, 3H, Py-CH3), 3.80 (s, 3H, Py-CH3), 3.37 (dt, 2H, J = 7 Hz and J = 6 Hz, -CH2NH-), 2.45 (t, 2H, J = 7 Hz, -COCH2-), 1.46 (s, 9H, -CH3 of the Boc group); 13C-n.m.r. (DMSO-d6) δ 173.05, 162.31, 158.45, 153.71, 122.84, 122.71, 122.68, 122.28, 122.15, 118.24, 117.85, 116.97, 104.67, 104.28, 103.94, 78.24, 59.74, 36.00, 35.91, 34.89, 34.17, 28.19; ESI-TOF MS calcd for C26H34N7O7 (M + H)+ 556.2520, found 556.2493.

3-[4-[[4-[[4-(tert-Butoxycarbonyl)amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropionic acid (40.6 mg, 0.073 mmol) was dissolved in methanol (3 mL) and acetyl chloride (0.85 mL, 80 mmol) was added dropwise at 0°C. After stirring for 30 minutes, the solution was concentrated in vacuo to dryness. The residue was dissolved in methanol (0.5 mL) and to this was added N-formylimidazole, prepared by the treatment of formic acid (3.3 μL, 0.088 mmol) with 1,1′-carbonyldiimidazole (14.2 mg, 0.088 mmol) in THF (1 mL) at room temperature for 30 minutes [11], at −40°C. After stirring at −40°C for 1 hour, the solution was diluted with ethyl acetate (10 mL), and washed with sat. NH4Cl aq. (5 mL × 3) and H2O (5 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 9, v/v) solvent system. Compound 11 was obtained in 85% yield (30.2 mg); 1H-n.m.r. (DMSO-d6) δ 12.00 (br, 1H, -COOH), 10.03 (d, 1H, J = 1.7 Hz, -NHCHO), 9.91 (s, 1H, -NHCO-), 9.88 (s, 1H, -NHCO-), 8.13 (d, 1H, J = 1.7 Hz, -NHCHO), 8.00 (t, 1H, J = 6 Hz, Py-NH), 7.23 (d, 1H, J = 1.7 Hz, Py-H), 7.19 (d, 1H, J = 1.7 Hz, Py-H), 7.18 (d, 1H, J = 1.7 Hz, Py-H), 7.03 (d, 1H, J = 1.8 Hz, Py-H), 6.91 (d, 1H, J = 1.8 Hz, Py-H), 6.85 (d, 1H, J = 1.7 Hz, Py-H), 3.84 (s, 3H, Py-CH3), 3.83 (s, 3H, Py-CH3), 3.80 (s, 3H, Py-CH3), 3.37 (dt, 2H, J = 7 Hz and J = 6 Hz, -CH2NH-), 2.47 (t, 2H, J = 7 Hz, -COCH2-); 13C-n.m.r. (DMSO-d6) δ 173.00, 161.24, 158.42, 158.27, 157, 85, 122.95, 122.75, 122.13, 122.04, 120.71, 118.39, 117.87, 104.63, 104.24, 103.93, 36.09, 36.05, 35.91, 34.84, 34.07; ESI-TOF MS calcd for C22H26N7O6 (M + H)+ 484.1945, found 484.1950.

2.7. N2-[3-[[4-[[4-[(4-Formylamino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropionyl]-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxyguanosine (14)

Method (1) Compound 11 was treated with 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxyguanosine (12) in the presence of EDCI and DMAP or DCC and HOBT as described for the syntheses of 7 and 18. The coupling reaction did not progress and the desired product 14 was not obtained.

Method (2) N-(9-Fluorenylmethoxycarbonyl)-β-alanine (4.580 g, 14.7 mmol) was dissolved in DMF (0.25 mL)/THF (25 mL) and oxalyl chloride (1.8 mL, 20 mmol) was added to the solution at 0°C. After stirring for 30 minutes at room temperature, the solution was concentrated in vacuo to give 3-[(9-fluorenylmethoxycarbonyl)amino]propionyl chloride. The residue was dissolved in THF (10 mL) and added to a solution of 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxyguanosine (12) in pyridine (80 mL). After stirring for 72 hours at room temperature, H2O (10 mL) was added to the solution. The solution was diluted with ethyl acetate (300 mL) and washed with H2O (100 mL × 3), 5% NaHCO3 aq. (100 mL × 3), and H2O (100 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was recrystallized from ethyl acetate. The resulting white crystals were collected by vacuum filtration to give N2-[(9-fluorenylmethoxycarbonyl)amino]propionyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-2′-deoxyguanosine (13) in 43% yield (3.420 g); m.p. 199-200°C; 1H-n.m.r. (DMSO-d6) δ 12.02 (br, 1H, N2-H), 11.76 (br, 1H, N1-H), 8.13 (s, 1H, H-8), 7.85 (d, 2H, J = 7.5 Hz, Ar-H of the Fmoc group), 7.66 (dd, 2H, J = 2.6 Hz, J = 7.3 Hz, Ar-H of the Fmoc group), 7.42 (t, 1H, J = 6 Hz, -CH2NH-Fmoc), 7.37 (t, 1H, J = 7.5 Hz, Ar-H of the Fmoc group), 7.30 (m, 2H, Ar-H of the Fmoc group), 6.12 (m, 1H, H-1′), 4.66 (m, 1H, H-3′), 4.29 (d, 2H, J = 6.7 Hz, =CH-CH2- of the Fmoc group), 4.21 (t, 1H, J = 6.7 Hz, =CH-CH2- of the Fmoc group), 3.82–3.92 (m, 3H, H-4′, 5′, and 5′′), 3.32 (dt, 2H, J = 6 Hz, J = 6,4 Hz, -CH2CH2NH-Fmoc), 2.64 (m, 1H, H-2′), 2.54 (t, 2H, J = 6,4 Hz, -COCH2CH2-), 2.50 (m, 1H, H-2′′), 1.01 (m, 28H, -CH(CH3)2 of the TIPDS group); 13C-n.m.r. (DMSO-d6) δ 174.39, 156.02, 154.78, 148.06, 147.87, 143.82, 140.68, 136.68, 136.73, 127.50, 126.96, 125.07, 120.29, 120.01, 84.64, 81.37, 70.57, 65.36, 61.99, 46.67, 36.50, 36.26, 17.29, 17.23, 17.02, 16.84, 16.76, 12.66, 12.51, 12.17, 11.94; ESI-TOF MS calcd for C40H55N6O8Si2 (M + H)+ 803.3620, found 803.3674; Anal. Calcd for C40H54N6O8Si2 + H2O: C, 58.51; H, 6.87; N, 10.24, found C, 58.67; H, 6.91; N,10.43.

Compounds 10 (214 mg, 0.27 mmol) and 13 (100 mg, 0.24 mmol) were dissolved in DMF (5 mL) and EDCI (93 mg, 0.48 mmol) and DMAP (150 mg, 1.23 mmol) were added to the solution. After stirring for 24 hours, the solution was diluted with ethyl acetate (50 mL)/methanol (3 mL) and washed with H2O (30 mL × 3). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 9, v/v) solvent system to give 14 (195 mg, 83% yield); 1H-n.m.r. (DMSO-d6) δ 12.05 (br, 1H, N2-H), 11.79 (br, 1H, N1-H), 10.04 (s, 1H, -NHCHO), 9.90 (s, 1H, -NHCO-), 9.88 (s, 1H, -NHCO-), 8.13 (s, 1H, H-8), 8.13 (s, 1H, -CHO), 8.11 (t, 1H, J = 5.7 Hz, -CH2NHCO-), 7.22 (d, 1H, J = 1.6 Hz, Py-H), 7.20 (m, 2H, Py-H × 2), 7.03 (d, 1H, J = 1.7 Hz, Py-H), 6.91 (d, 1H, J = 1.8 Hz, Py-H), 6.86 (d, 1H, J = 1.6 Hz, Py-H), 6.15 (dd, 1H, J = 3.5 Hz, J = 7.1 Hz, H-1′), 4.66 (dt, 1H, J = 7 Hz, J = 7.4 Hz, H-3′), 3.83–3.93 (m, 3H, H-4′, 5′, and 5′′), 3.84 (s, 6H, Py-CH3× 2), 3.79 (s, 3H, Py-CH3), 3.50 (dt, 2H, J = 6 Hz, J = 6.4 Hz, -CH2CH2NH-), 2.75 (m, 2H, H-2′ and 2′′), 2.70 (t, 2H, J = 6,4 Hz, -COCH2CH2-), 1.01 (m, 28H, -CH(CH3)2 of the TIPDS group); 13C-n.m.r. (DMSO-d6) δ 174.77, 161.36, 158.43, 158.28, 157.85, 154.83, 148.14, 148.01, 136.76, 122.96, 122.74, 122.65, 122.19, 122.07, 120.73, 120.27, 118.41, 117.97, 104.65, 104.34, 103.94, 84.63, 81.37, 70.55, 61.96, 36.59, 36.10, 36.06, 35.91, 34.63, 17.32, 17.16, 17.05, 16.87, 16.80, 12.67, 12.52, 12.18, 11.96; ESI-TOF MS calcd for C44H63N12O10Si2 (M + H)+ 975.4329, found 975.4271.

2.8. N2-[3-[[4-[[4-[(4-Formylamino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropionyl]-2′-deoxyguanosine (Hybrid 2)

Compound 14 (50 mg, 0.051 mmol) was treated with 0.2 M tetraethylammonium fluoride in THF (3 mL) and stirred at room temperature for 30 minutes. The solution was concentrated in vacuo to dryness and the residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 4, v/v) solvent system to give Hybrid 2 (37.3 mg, quantitative yield); 1H-n.m.r. (DMSO-d6) δ 12.04 (br, 1H, N2-H), 11.74 (br, 1H, N1-H), 10.03 (s, 1H, -NHCHO), 9.90 (s, 1H, -NHCO-), 9.88 (s, 1H, -NHCO-), 8.23 (s, 1H, H-8), 8.13 (s, 1H, -CHO), 8.11 (t, 1H, J = 5.5 Hz, -CH2NHCO-), 7.22 (d, 1H, J = 1.6 Hz, Py-H), 7.20 (m, 2H, Py-H × 2), 7.02 (d, 1H, J = 1.7 Hz, Py-H), 6.91 (d, 1H, J = 1.7 Hz, Py-H), 6.85 (d, 1H, J = 1.6 Hz, Py-H), 6.20 (t, 1H, J = 6.7 Hz, H-1′), 5.28 (br, 1H, 5′-OH), 4.66 (t, 1H, J = 5.4 Hz, H-3′), 4.36 (br, 1H, 3′-OH), 3.84 (s, 6H, Py-CH3 × 2), 3.82 (m, 1H, H-4′), 3.79 (s, 3H, Py-CH3), 3.54 (m, 2H, H-5′, and 5′′), 3.50 (dt, 2H, J = 5.5 Hz, J = 6.5 Hz, -CH2CH2NH-), 2.71 (t, 2H, J = 6,5 Hz, -COCH2CH2-) 2.28 (m, 2H, H-2′ and 2′′); 13C-n.m.r. (DMSO-d6) δ 174.74, 161.35, 158.41, 158.27, 157.85, 154.83, 148.35, 147.87, 137.45, 122.95, 122.73, 122.65, 122.16, 122.04, 120.71, 120.18, 118.37, 117.96, 104.64, 104.33, 103.94, 87.72, 82.95, 70.45, 61.46, 36.58, 36.08, 36.05, 35.89, 34.63; ESI-TOF MS calcd for C32H37N12O10 (M + H)+ 733.2806, found 733.2841; Anal. Calcd for C32H36N12O9 + 4 H2O: C, 47.76; H, 5.51; N, 20.89, found C, 47.79; H, 5.84; N, 21.26.

2.9. 9-Fluorenylmethyl 3-aminopropyl-1-carbamate hydrochloride (17)

9-Fluorenylmethyl chloroformate (15) (5.174 g, 20 mmol) was dissolved in 1,4-dioxane (50 mL), and sodium phenoxide 3 H2O (3.573 g, 21 mmol) was added to the solution. After stirring for 1.5 hours at room temperature, the solution was concentrated in vacuo and ethyl acetate (100 mL) was added. The solution was washed with H2O (50 mL × 2) and sat. NaOH aq. (50 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to crystallization from ethyl acetate, collected by vacuum filtration, and dried in vacuo to yield 9-fluorenylmethyl phenyl carbonate (16) (5.825 g, 92% yield) as a white solid, m.p. 132-133°C; 1H-n.m.r. (DMSO-d6) δ 7.92 (d, 2H, J = 7.5 Hz, Ar-H of the Fmoc group × 2), 7.68 (d, 2H, J = 7.5 Hz, Ar-H of the Fmoc group × 2), 7.15–7.46 (m, 9H, Ph-H × 5 and Ar-H of the Fmoc group × 4), 4.68 (d, 2H, J = 6.1 Hz, =CH-CH2- of the Fmoc group), 4.39 (t,1H, J = 6.1 Hz, =CH-CH2- of the Fmoc group); 13C-n.m.r. (DMSO-d6) δ 152.87, 150.61, 143.15, 140.84, 129.57, 127.78, 127.18, 126.13, 124.94, 121.11, 120.22, 69.38, 46.28; ESI-TOF MS calcd for C21H16O3 (M + Na)+ 339.0997, found 339.0986; Anal. Calcd for C21H16O3: C, 79.73; H, 5.10, found C, 79.79; H, 5.11.

9-Fluorenylmethyl phenyl carbonate (16) (3.763 g, 12 mmol) was suspended in methanol (50 mL) and propane-1,3-diamine (1.0 mL, 12 mmol) was added to the solution. After stirring for 4 hours at room temperature, pyridinium hydrochloride (3.005 g, 26 mmol) was added and the solution stirred for 10 minutes. The solution was concentrated in vacuo and the residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 4, v/v) solvent system to give 17 (3.250 g, 86% yield) as a white powder; m.p. 129-130°C; 1H-n.m.r. (DMSO-d6) δ 7.79 (d, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.63 (d, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.39 (t, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.31 (t, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 4.40 (d, 2H, J = 6.4 Hz, =CH-CH2- of the Fmoc group), 4.20 (t, 1H, J = 6.4 Hz, =CH-CH2- of the Fmoc group), 3.20 (t, 2H, J = 6.4 Hz, -CH2-CH2-CO-), 2.91 (t, 2H, J = 7.3 Hz, -CH2-CH2-N), 1.77–1.83 (m, 2H, -CH2-CH2-CH2-); 13C-n.m.r. (DMSO-d6) δ159.49, 145.41, 142.80, 128.94, 128.27, 126.21, 121.10, 67.83, 48.64, 38.42, 29.32; ESI-TOF MS calcd for C18H20N2O2 (M + H)+ 297.1603, found 297.1615; Anal. Calcd for C18H20N2O2 Cl + 0.75 H2O: C, 62.42; H, 6.55; N, 8.09, found C, 62.65; H, 6.76: N, 8.61.

2.10. 9-Fluorenylmethyl 3-[4-[[4-[[4-(Tert-butoxycarbonyl)amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropyl-1-carbamate (18)

Compounds 10 (50 mg, 0.1 mmol) and 17 (38 mg, 0.11 mmol) were dissolved in DMF (3 mL) and dicyclohexylcarbodiimide (26 mg, 0.12 mmol) and 1-hydroxybenzotriazole (17 mg, 0.12 mmol) were added to the solution. After stirring for 24 hours at room temperature, the solution was diluted with ethyl acetate (50 mL) and washed with 3 M HCl aq. (25 mL × 3), H2O (25 mL), 5% NaHCO3 aq. (25 mL × 2), and H2O (25 mL). The organic layer was dried over anhydrous magnesium sulfate, evaporated to dryness, and the residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 49, v/v) solvent system to give 18 (75.6 mg, 96% yield); 1H-n.m.r. (DMSO-d6) δ 9.87 (s, 1H, -NHCO-), 9.85 (s, 1H, -NHCO-), 9.08 (s, 1H, Boc-NH), 7.95 (t, 1H, J = 5.5 Hz, -CH2NHCO-), 7.88 (d, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.69 (d, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.41 (t, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.32 (t, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.26 (t, 1H, J = 5.6 Hz, -CH2NH-Fmoc), 7.21 (d, 1H, J = 1.7 Hz, Py-H), 7.18 (d, 1H, J = 1.5 Hz, Py-H), 7.04 (d, 1H, J = 1.7 Hz, Py-H), 6.89 (s, 1H, Py-H), 6.86 (d, 1H, J = 1.5 Hz, Py-H), 6.84 (s, 1H, Py-H), 4.32 (d, 2H, J = 6.6 Hz, =CH-CH2- of the Fmoc group), 4.22 (t, 1H, J = 6.6 Hz, =CH-CH2- of the Fmoc group), 3.84 (s, 3H, Py-CH3), 3.81 (s, 3H, Py-CH3), 3.80 (s, 3H, Py-CH3), 3.17 (m, 2H, -NHCH2CH2-), 3.03 (m, 2H, -CH2CH2NH-), 1.62 (m, 2H, -CH2CH2CH2-), 1.46 (s, 9H, -CH3 of the Boc group); 13C-n.m.r. (DMSO-d6) δ 161.25, 158.45, 158.41, 156.10, 152.80, 143.91, 140.72, 127.56, 127.02, 125.09, 122.89, 122.82, 122.73, 122.29, 122.15, 120.08, 118.34, 117.77, 117.02, 104.68, 104.49, 103.81, 78.35, 65.19, 46.76, 38.03, 36.00, 35.89, 29.64; ESI-TOF MS calcd for C41H47N8O7 (M + H)+ 763.3568, found 763.3591; Anal. Calcd for C41H46N8O7 + 2 H2O: C, 61.64; H, 6.31; N, 14.03, found C, 62.12; H, 6.10; N, 14.09.

2.11. 9-Fluorenylmethyl 3-[4-[[4-[(4-Formylamino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropyl-1-carbamate (19)

Compound 18 (0.696 g, 0.91 mmol) was dissolved in methanol (12 mL) and acetyl chloride (3 mL, 42 mmol) was added to the solution at 0°C. After stirring for 30 minutes, the reaction solution was concentrated in vacuo to dryness. The residue was dissolved in methanol (5 mL)/THF (10 mL) and N-formylimidazole, prepared by the treatment of formic acid (41 μL, 1.09 mmol) with 1,1′-carbonyldiimidazole (177 mg, 1.09 mmol) in THF (4 mL) at room temperature for 5 minutes [11], was added to the solution at −40°C. After stirring at −40°C for 1 hour, the solution was diluted with 5% NaHCO3 aq. (10 mL), washed with ethyl acetate (10 mL × 2), and neutralized with 10% HCl aq. to ca. pH 2. Compound 10 was extracted with ethyl acetate (20 mL × 3) from the resulting solution. The organic layer was washed with H2O (30 mL), dried over anhydrous magnesium sulfate, and evaporated to dryness. The residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 19, v/v) solvent system. Compound 19 was obtained in 88% yield (0.555 g); 1H-n.m.r. (DMSO-d6) δ 10.04 (d, 1H, J = 1.7 Hz, -NHCHO), 9.91 (s, 1H, -NHCO-), 9.88 (s, 1H, -NHCO-), 8.13 (d, 1H, J = 1.7 Hz, -NHCHO), 7.96 (t, 1H, J = 5.6 Hz, -CH2NHCO-), 7.88 (d, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.65 (d, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.41 (t, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.33 (t, 2H, J = 7.4 Hz, Ar-H of the Fmoc group), 7.25 (t, 1H, J = 5.6 Hz, -CH2NH-Fmoc), 7.23 (d, 1H, J = 1.8 Hz, Py-H), 7.20 (d, 1H, J = 1.8 Hz, Py-H), 7.19 (d, 1H, J = 1.5 Hz, Py-H), 7.04 (d, 1H, J = 1.8 Hz, Py-H), 6.92 (d, 1H, J = 1.8 Hz, Py-H), 6.87 (d, 1H, J = 1.5 Hz, Py-H), 4.32 (d, 2H, J = 6.7 Hz, =CH-CH2- of the Fmoc group), 4.22 (t, 1H, J = 6.7 Hz, =CH-CH2- of the Fmoc group), 3.85 (s, 6H, Py-CH3 × 2), 3.80 (s, 3H, Py-CH3), 3.16 (m, 2H, -NHCH2CH2-), 3.03 (m, 2H, -CH2CH2NH-), 1.61 (m, 2H, -CH2CH2CH2-); 13C-n.m.r. (DMSO-d6) δ 161.24, 158.42, 158.27, 157.85, 143.91, 140.72, 128.89, 127.56, 127.03, 125.09, 122.95, 122.76, 122.05, 120.71, 120.08, 118.34, 117.77, 104.63, 104.08, 103.93, 65.19, 46.76, 38.88, 38.03, 36.09, 35.89; ESI-TOF MS calcd for C37H39N8O6 (M + H)+ 691.2993, found 691.2974; Anal. Calcd for C37H38N8O6 + H2O: C, 62.70; H, 5.69; N, 15.81, found C, 63.05; H, 5.69; N, 16.00.

2.12. N2-[3-[[4-[[4-[(4-Formylamino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropyl]-3′,5′-di-O-acetyl-O6-[2-(4-nitrophenyl)ethyl]-2′-deoxyguanosine (21)

Compound 19 (150 mg. 0.22 mmol) was dissolved in 10% piperidine/DMF (1 mL). After stirring at room temperature for 20 minutes, the solution was concentrated in vacuo to dryness. The residue and 3′,5′-di-O-acetyl-2-fluoro-O6-[2-(4-nitrophenyl)ethyl]-2′-deoxyinosine (20) (0.219 g, 0.43 mmol) were dissolved in DMF (2 mL) and triethylamine (60 μL, 0.43 mmol) was added to the solution. After stirring at 60°C for 12 hours, the reaction solution was concentrated in vacuo to dryness. The residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 19, v/v) solvent system to give 21 (0.168 g, 81% yield); 1H-n.m.r. (DMSO-d6) δ 10.19 (d, 1H, J = 1.7 Hz, -NHCHO), 9.91 (s, 1H, -NHCO-), 9.87 (s, 1H, -NHCO-), 8.16 (d, 2H, J = 8 Hz, Ar-H of the NPE group), 8.13 (d, 1H, J = 1.7 Hz, -NHCHO), 8.03 (s, 1H, H-8), 8.01 (t, 1H, J = 5.4 Hz, -CH2NHCO-), 7.60 (d, 2H, J = 8 Hz, Ar-H of the NPE group), 7.22 (d, 1H, J = 1.7 Hz, Py-H), 7.19 (d, 1H, J = 1.8 Hz, Py-H), 7.16 (d, 1H, J = 1.7 Hz, Py-H), 7.04 (d, 1H, J = 1.7 Hz, Py-H), 7.01 (t, 1H, J = 5.7 Hz, N2-H), 6.91 (d, 1H, J = 1.9 Hz, Py-H), 6.90 (d, 1H, J = 1.3 Hz, Py-H), 6.24 (t, 1H, J = 6.7 Hz, H-1′), 5.41 (m, 1H, H-3′), 4.68 (t, J = 6.6 Hz, -CH2CH2- of the NPE group), 4.31 (m, 1H, H-4′), 4.19 (m, 2H, H-5′ and 5′′), 3.84 (s, 6H, Py-CH3 × 2), 3.80 (s, 3H, Py-CH3), 3.36 (m, 2H, -NHCH2CH2-), 3.25 (m, 4H, -CH2CH2- of the NPE group and -CH2CH2NH- ), 3.12 (m, 1H, H-2′), 2.45 (m, 1H, H-2′′), 2.08 (s, 3H, the Ac group), 1.99 (s, 3H, the Ac group), 1.76 (m, 2H, -CH2CH2CH2-); 13C-n.m.r. (DMSO-d6) δ 170.10, 169.99, 161.31, 159.99, 158.70, 158.42, 158.28, 157.86, 146.71 146.22, 130.20, 123.38, 122.95, 122.77, 122.16, 120.72, 118.37, 117.72, 104.64, 104.13, 103.94, 81.35, 65.41, 63.64, 36.22, 36.04, 35.89, 34.31, 20.77, 20.47; ESI-TOF MS calcd for C44H50N13O12 (M + H)+ 952.3702, found 952.3738.

2.13. N2-[3-[[4-[[4-[(4-Formylamino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropyl]-2′-deoxyguanosine (Hybrid 3)

Compound 21 (0.116 g, 0.12 mmol) was treated with 0.5 M DBU/pyridine (2 mL). After stirring at room temperature for 12 hours, ammonium chloride (80 mg, 1.5 mmol) was added to the solution. The solution was diluted with chloroform (60 mL) and washed with H2O (25 mL). The organic layer was dried over anhydrous magnesium sulfate, evaporated to dryness, and the residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 4, v/v) solvent system to give N2-[3-[[4-[[4-[(4-formylamino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropyl]-3′,5′-di-O-acetyl-2′-deoxyguanosine (71 mg, 73% yield); 1H-n.m.r. (DMSO-d6) δ 10.70 (br, 1H, N1-H), 10.07 (d, 1H, J = 1.5 Hz, -NHCHO), 9.91 (s, 1H, -NHCO-), 9.87 (s, 1H, -NHCO-), 8.12 (d, 1H, J = 1.5 Hz, -NHCHO), 8.04 (t, 1H, J = 5.8 Hz, -CH2NHCO-), 7.87 (s, 1H, H-8), 7.22 (d, 1H, J = 1.6 Hz, Py-H), 7.19 (d, 1H, J = 1.6 Hz, Py-H), 7.17 (d, 1H, J = 1.6 Hz, Py-H), 7.06 (d, 1H, J = 1.7 Hz, Py-H), 6.91 (d, 1H, J = 1.7 Hz, Py-H), 6.90 (d, 1H, J = 1.6 Hz, Py-H), 6.64 (t, 1H, J = 5 Hz, N2-H), 6.18 (t, 1H, J = 7 Hz, H-1′), 5.46 (m, 1H, H-3′), 4.30 (m, 1H, H-4′), 4.17 (m, 2H, H-5′ and 5′′), 3.84 (s, 6H, Py-CH3 × 2), 3.80 (s, 3H, Py-CH3), 3.35 (m, 2H, -NHCH2CH2-), 3.25 (m, 2H, -CH2CH2NH- ), 3.05 (m, 1H, H-2′), 2.47 (m, 1H, H-2′′), 2.08 (s, 3H, the Ac group), 1.99 (s, 3H, the Ac group), 1.74 (m, 2H, -CH2CH2CH2-); ESI-TOF MS calcd for C36H43N12O10 (M + H)+ 803.3225, found 803.3201.

N 2-[3-[[4-[[4-[(4-Formylamino-1-methylpyrrol-2-yl)carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carboxyl]aminopropyl]-3′,5′-di-O-acetyl-2′-deoxyguanosine (71.4 mg, 0.089 mmol) was dissolved in pyridine (0.2 mL) and 0.05 M sodium methoxide/methanol (0.8 mL) was added to the solution. After stirring at room temperature for 2 hours, the solution was neutralized with Dowex-50WX2 ion-exchange resin (H+ form) after which time the resin was immediately removed by filtration. The filtrate was evaporated to dryness and the residue was crystallized from methanol. The resulting white crystals were collected by vacuum filtration to give Hybrid 3 (51.1 mg, 80% yield); 1H-n.m.r. (DMSO-d6) δ 10.80 (br, 1H, N1-H), 10.05 (d, 1H, J = 1.6 Hz, -NHCHO), 9.91 (s, 1H, -NHCO-), 9.87 (s, 1H, -NHCO-), 8.12 (m, 2H, -NHCHO and H-8), 8.07 (t, 1H, J = 5.6 Hz, -CH2NHCO-), 7.22 (d, 1H, J = 1.8 Hz, Py-H), 7.19 (d, 1H, J = 1.8 Hz, Py-H), 7.18 (d, 1H, J = 1.8 Hz, Py-H), 7.04 (d, 1H, J = 1.8 Hz, Py-H), 6.91 (d, 1H, J = 1.8 Hz, Py-H), 6.89 (d, 1H, J = 1.8 Hz, Py-H), 6.62 (br, 1H, N2-H), 6.17 (t, 1H, J = 6.8 Hz, H-1′), 4.36 (m, 1H, H-3′), 3.84 (s, 3H, Py-CH3), 3.83 (s, 3H, Py-CH3), 3.82 (m, 1H, H-4′), 3.80 (s, 3H, Py-CH3), 3.56 (m, 1H, H-5′), 3.50 (m, 1H, H-5′′), 3.33 (m, 2H, -NHCH2CH2-), 3.25 (m, 2H, -CH2CH2NH- ), 2.61 (m, 1H, H-2′), 2.24 (m, 1H, H-2′′), 1.75 (m, 2H, -CH2CH2CH2-); 13C-n.m.r. (DMSO-d6) δ 161.43, 158.43, 158.23, 157.85, 156.17, 152.78, 150.19, 135.69, 122.94, 122.88, 122.75, 122.13, 122.05, 120.71, 118.37, 117.82, 104.65, 104.65, 104.23, 103.95, 87.71, 83.16, 70.65, 61.65, 38.21, 36.09, 36.05, 35.90. 29.15; ESI-TOF MS calcd for C32H39N12O8 (M + H)+ 719.3014, found 719.3005; Anal. Calcd for C37H38N8O6 + 1.5 H2O: C, 51.54; H, 5.54; N, 22.54, found C, 51.36; H, 5.50; N, 22.26.

2.14. Circular Dichroism (CD) Spectroscopy

CD spectra were measured using a JASCO J-720 spectropolarimeter equipped with a thermoelectrically controlled cell holder and a cuvette with a path length of 10 mm. CD titrations were conducted at 20°C by incrementally injecting 15 μL aliquots of a 270 μM ligand (Hybrid 2, Hybrid 3, or Dst) solution into 3 mL of a 5.8 μM duplex [DNA 1: d-(5′-CGCAAATTGGC-3′)-d-(3′-GCGTTTAACCG-5′)], DNA 2: d-(5′-CGCAGACTGGC-3′)-d-(3′-GCGTCTGACCG-5′)], or DNA 3: d-(5′-GCACGACTACG-3′)-d-(3′-CGTGCTGATGC-5′)] solution in the same buffer [19, 20]. The resulting ligand concentration in the DNA duplex solution increased from 0 to 18.1 μM. After each injection, the CD spectrum was recorded between 220 and 380 nm.

2.15. UV Absorption Spectroscopy

In an effort to assess the effect of the bound ligand (Hybrid 1, Hybrid 2, Hybrid 3, or Dst) on the thermal stability of the three duplexes (DNA 1–3), UV melting experiments were conducted in the absence and presence of each ligand. Absorbance versus temperature profiles were measured using a Shimazu TMSPC-8/UV-1600 spectrophotometer equipped with a thermoelectrically controlled cell holder at 260 nm and a heating rate of 1.0°C/min. The concentration of each 11mer duplex was 4.3 μM, while the ligand concentrations varied from 0 to 12.9 μM.

3. Results and Discussion

Hybrid 2 incorporating the 3-aminopropionyl linker was synthesized by condensation of N2-(Fmoc-β-alanyl)-3′,5′-O-TIPDS-guanosine (12) with pyrrole amide trimer 10 as shown in Scheme 2.

Scheme 2

Reaction conditions (i) 3-(Fmoc-amino)propionyl chloride (prepared from N-Fmoc-β-alanine and SOCL2), THF/Pyridine, rt, 72 hours. (ii) 10, EDCI, DMAP, DMF, rt, 24 hours. (iii) 0.2 M TEAF, THF, rt, 30 minutes.

Pyrrole amide trimer 10 was prepared using modifications of the method of Baird and Dervan [3]. The monomer building blocks methyl 4-nitropyrrole-2-carboxylate (5) and 4-(tert-butoxycarbonyl)amino-1-methylpyrrole-2-carboxylic acid (6) were synthesized from N-methylpyrrole via 2-trichloroacetylation, 4-nitration and esterification to give 5, and subsequent alkaline hydrolysis of ester 5 to give 6. Using pyrrole monomers 5 and 6, pyrroleamide trimer 10 was synthesized as shown in Scheme 1. Monomer 5 was reduced to give methyl 4-aminopyrrole-2-carboxylate, which was then coupled with 6 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-(dimethylamino)pyridine (DMAP) to give dimer 7. Next, following deprotection of the Boc group, dimer 7 was coupled with 6 to give trimer 8. Trimer 8 was then converted to trimer 10 through alkaline hydrolysis of the ester moiety, deprotection of the Boc group, and finally N-formylation [11].

Scheme 1

Reaction conditions (i) CL3CCOCL, Et2O, rt, 1 hour. (ii) HNO3, Ac2O, −10°C ∼ rt, 1 hour. (iii) NaOMe, MeOH, rt, 15 minutes. (iv) 1. H2, 10% Pd/C, AcOEt, rt, 12 hours; 2. (Boc)2O, Et2O, rt, 2 hours. (v) 1. 1 M NaOH, H2O/MeOH, 60°C, 1 ~ 2 hours; 2. 10% HCL aq. (vi) 1. H2, 10% Pd/C, AcOEt, rt, 12 hours; 2. 6, EDCI, DMAP, DMF, rt, 3 hours. (vii) 1. AcCL, MeOH, 0°C, 30 minutes. 2. 6, EDCI, DMAP, DMF, rt, 3 hours. (viii) 1. AcCL, MeOH, 0°C, 30 minutes; 2. HCOOH, CDI, THF, −40°C, 30 minutes. (ix) ethyl β-alanine, EDCI, DMAP, DMF, rt, 3 hours.

We initially attempted the synthesis of the pyrrolepolyamide-2′-deoxyguanosine hybrid (Hybrid 2) by condensation of 3′,5′-O-TIPDS-guanosine (12) and pyrrole amide trimer 11, prepared by coupling pyrrole amide trimer 9 with ethyl β-alanine, alkaline hydrolysis of the ester moiety, deprotection of the Boc group, and finally N-formylation. The desired compound was not obtained when using EDCI/DMAP or dicyclohexylcarbodiimide (DCC)/hydroxybenztriazole (HOBT) as condensation reagents due to the low reactivity of the exocyclic amino group of the guanine base. We then attempted the synthesis of Hybrid 2 using pyrrole amide trimer 10 and N2-[3-(9-fluorenylmethoxycarbonylamino)propioyl]-3′,5′-O-TIPDS-guanosine (13). Compound 13 was prepared by reaction of 3′,5′-O-TIPDS-guanosine (12) with 3-(Fmoc-amino)propionyl chloride, prepared from N-Fmoc-β-alanine and SOCl2, in 43% yield. Treatment of 13 with pyrroleamide trimer 10 in the presence of EDCI and a large excess of DMAP generated pyrrolepolyamide-2′-deoxyguanosine derivative 14 in 83% yield through deprotection of the Fmoc group of 13 and condensation of the amine product with 10. Hybrid 2 was obtained in quantitative yield from 14 by treatment with 0.2 M tetraethylammonium fluoride (TEAF) in THF.

Hybrid 3 incorporating the 3-aminopropyl linker was synthesized using 3′,5′-di-O-acetyl-2-fluoro-O6-[2-(4-nitrophenyl)ethyl]-2′-deoxyinosine (20) and pyrrole amide trimer 19 as shown in Scheme 4. Monoprotected propane-1,3-diamine containing the Fmoc group, which can be deprotected under mild basic conditions and is stable during deprotection of the Boc group, was designed as the linker reagent. Monoprotection of propane-1,3-diamine by treatment with 9-fluorenylmethyl chloroformate (Fmoc-Cl) under basic conditions in the presence of a large excess of propane-1,3-diamine and Na2CO3 was initially examined. The desired compound was not obtained due to the low selectivity of monoprotection and basic lability of the Fmoc group. Pittelkov et al. reported on the monoprotection of alkanediamines [H2N-(CH2)n-NH2: n = 0–4] with Boc, Cbz or allyloxycarbonyl groups using the corresponding phenyl carbonates. Monoprotected alkanediamines were obtained in 46%–86% yield [27]. Using a modification of the method of Pittelkov et al., we then treated propane-1,3-diamine with 9-fluorenylmethyl phenyl carbonate (16), prepared from Fmoc-Cl and sodium phenoxide in 87% yield, and then 1 M HCl aq. to give the HCl salt of 3-(Fmoc-amino)propylamine as shown in Scheme 3. Linker reagent 17 was obtained in 69% yield. Pyrrole amide trimer 10 was condensed with linker reagent 17 in the presence of DCC, HOBT, and diisopropylethylamine (DIEA) in DMF to generate pyrrole amide trimer 18, obtained in 96% yield. Pyrrole amide trimer 18 was then subjected to deprotection of the Boc group and subsequent N-formylation to give pyrrole amide trimer 19. Treatment of pyrrole amide trimer 19 with 2′-deoxy-2-fluoroinosine derivative 20 in the presence of triethylamine in DMF at 60°C for 12 hours generated the desired pyrrolepolyamide-2′-deoxyguanosine derivative 21 in 81% yield through deprotection of the Fmoc group of 19 and condensation of the amine product with 20. Cleavage of the NPE protecting group using 0.5 M DBU in pyridine [28] and subsequent deacetylation using sodium methoxide in methanol generated Hybrid 3 in good yield.

Scheme 4

Reaction conditions (i) 1. piperidine, DMF, rt, 20 minutes. 2. 19, Et3N, DMF, 60°C 12 hours. (ii) 0.5 M DBU, pyridine, rt, 12 hours. (iii) 0.05 M NaOMe, MeOH/pyridine, rt, 2 hours.

Scheme 3

Reaction conditions (i) sodium phenoxide, 1,4-dioxane, rt, 1.5 hours. (ii) 1. Propane-1,3-diamine (1.1 eq.), rt, 3.5 hours; 2. pyridinium hydrochloride. (iii) 17, DCC, HOBT, DIEA, DMF, rt, 24 hours. (v) 1. AcCL, MeOH, 0°C, 30 minutes; 2. HCOOH, CDI, THF, −40°C, 1 hour.

For our investigations concerning the interaction of DNA duplexes with Hybrid 2 and Hybrid 3 using CD and UV melting data (Tm value) [29], DNA 1 [d-(5′-CGCAAATTGGC-3′)-d-(3′-GCGTTTAACCG-5′)], which contains the high affinity binding site of Dst, DNA 2 [d-(5′-CGCAGGC-3′)-d-(3′-GCGTCCG-5′)], which included changes at A5 to G5 and T7 to C7, and DNA 3 [d-(5′-GCACGACTACG-3′)-d-(3′-CGTGCTGATGC-5′)], which represents a random sequence, were used (Figure 2 and Table 1). Moreover, experiments employing Dst and compound 11 were conducted for comparison. Typical CD spectra of the uncomplexed DNA duplexes (DNA 1–3), indicative of DNA in the B-form conformation, are shown in Figure 2 (ligand = 0 μM). The addition of Hybrid 2, Hybrid 3, or Dst resulted in marked changes in the ellipticity θ, unlike the case with compound 11. In particular, an induced Cotton effect of bound ligand is indicated by the presence of an extra CD band centered at ∼325 nm for Hybrid 2 and Hybrid 3, and ∼333 nm for Dst. Hybrid 3 showed selective binding affinity only with DNA 1, which contains the AAATT sequence (Figure 2), and stabilization of the DNA duplex (Table 1). On the other hand, Hybrid 2, combined with a pyrrolepolyamide at the 2-exocyclic amino group of the guanine base through an amide bond using a 3-aminopropionyl linker, and Dst containing the cationic amidine edge which contributes to high DNA binding affinity for ligand binding [30-33], did not show any marked differences in sequence-specific binding affinity with the tested DNA.

Figure 2

CD spectra for the DNA duplexs with ligand (Hybrid 2, Hybrid 3, Dst, or compound 11) at several [Iigand]/[duplex] ratios in buffer consisting of 10 mM sodium phosphate, 10 mM NaCL, and 0.1 mM Na2EDTA at pH 7.0 and 20°C: 3.0 mL of duplex solution (5.8 μM) titrated ligand (Hybrid 2, Hybrid 3, and Dst: 0–14.5 μM) and 3.0 mL of duplex solution (4 μM) titrated ligand (compound 11: 0–12 μM). *DNA duplexs: DNA 1  = d-(5′-CGCAAATTGGC-3′)-d-(3′-GCGTTTAACCG-5′), DNA 2  = d-(5′-CGCAGGC-3′)-d-(3′-GCGTCCG-5′), DNA 3  = d-(5′-GCACGACTACG-3′)-d-(3′-CGTGCTGATGC-5′), DNA 1 contains the high affinity binding site (5′-AAATT-3′) of Dst.

4. Conclusions

We synthesized and evaluated pyrrolepolyamide-2′-deoxyguanosine hybrids (Hybrid 2 and Hybrid 3) incorporating 3-aminopropionyl or 3-aminopropyl linkers. Hybrid 3, combined with a pyrrolepolyamide at the 2-exocyclic amino group of the guanine base through an N-alkyl bond using a 3-aminopropyl linker, showed high recognition of DNA containing the pyrrolepolyamide binding sequence. These results suggest that improvements in stability might be effected by varying the length of the linker. On the other hand, in a DNA strand with incorporated hybrid during DNA biosynthesis, the presence of a short linker, such as the 3-aminopropionyl moiety, might result in high stabilization of the dsDNA. These studies are currently underway and will be reported elsewhere. The minor groove binder pyrrolepolyamide-2′-deoxyguanosine hybrid presented in this report might potentially be of use as a sequence-specific gene therapy agent and could provide the basis for the development of a new series of antisense or antigene drugs.