Synthesis and Evaluation of MGB Polyamide-Oligonucleotide Conjugates as Gene Expression Control Compounds

Abstract

MGB polyamide-oligonucleotide conjugates ON 1-4 with linked MGB polyamides at the 2-exocyclic amino group of a guanine base using aminoalkyl linkers were synthesized and evaluated in terms of binding affinity for complementary DNA containing the MGB polyamide binding sequence using T_m and CD analyses. The MGB polyamides comprised pyrrole polyamides (Py₄- and Py₃-), which possess binding affinity for A-T base pairs, and imidazole (Im₃-) and pyrrole-γ-imidazole (Py₃-γ-Im₃-) polyamide hairpin motifs, which possess binding affinity for C-G base pairs. It was found that the stability of modified dsDNA was greatly influenced by the linker length. Py₄- and Py₃-oligonucleotide conjugates (ON 1 () and ON 2 ()) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA. Although Im₃-oligonucleotide conjugate ON 3 () containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of ON 3 () was lower compared with the pyrrole amide moiety of ON 2 (). The Py₃-γ-Im₃-oligonucleotide conjugate ON 4 (), which possesses binding affinity for C-G base pairs via a pyrrole/imidazole combination and contains a 2-aminoethyl linker, showed high binding ability for complementary DNA. Furthermore, the DNA sequence recognition of MGB polyamide-oligonucleotide conjugates was investigated using single-base mismatch DNAs, which possess a mismatch base in the MGB polyamide binding sequence. The Py₃-γ-Im₃-oligonucleotide conjugate ON 4 () showed high sequence recognition ability for complementary DNA.

1. Introduction

Numerous nucleic acid analogues have been synthesized and characterized as potential gene therapy agents [1, 2]. We previously designed and synthesized nucleoside (Hybrid 1) linked to pyrrole polyamide minor groove binder (MGB) comprising modified distamycin A, which possesses a high affinity for the -d(AATTT)-/-(TTAAA)- sequence of double-stranded DNA (dsDNA) [3–8], as a lead compound for the development of potential gene therapy agents [9–12]. When the MGB polyamide-nucleoside hybrid interacts with dsDNA, it is expected that complex formation would involve high affinity and sequence selectivity. If 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. The dsDNA binding ability of Hybrid 1 was investigated via melting temperature (T_m) and circular dichroism (CD) analyses (Figure 1) [5, 6]. It was shown that Hybrid 1 possessed greater binding specificity compared with distamycin A [12]. Then, in an effort to examine the development of potential antisense drugs, we synthesized oligonucleotide ON 1 () conjugated to Hybrid 2in lieu ofHybrid 1 containing the formyl group which is unstable under the basic conditions of deprotection during oligonucleotide solid-phase synthesis, and subsequently examined the binding ability of ON 1 () to complementary DNA (Figure 2) [13]. Dervan et al. [14], Zamecnik et al. [15], Novopashina et al. and Boutorine et al. [16–21] have reported the synthesis and evaluation of oligonucleotides conjugated with one or two MGB polyamides to either the - or -ends. Sequence-specific stabilization of DNA duplexes and DNA triplexes by MGB polyamides conjugated to one DNA strand was shown. It was expected that oligonucleotides conjugated with the MGB polyamide to the 2-exocyclic amino group of a guanine base, which is positioned above the floor of the minor groove of the DNA duplex, would possess high DNA binding ability. ON 1 (), which includes a modified guanosine (G) in the direction in the oligonucleotide chain given the preferred orientation of the polyamide in the minor groove of dsDNA (C-(pyrrole polyamide)-N/-(TTAAA)- of the complementary target DNA) [22–24], was synthesized and evaluated as a model oligonucleotide [13]. From the and CD analyses, it was found that ON 1 () formed stable dsDNA with complementary DNA via action of the pyrrole amide moiety. From this result, it is expected that MGB polyamide-oligonucleotide conjugates could be effective gene expression control compounds and that MGB polyamide--deoxyguanosine hybrid might be of potential use as a sequence-specific gene therapy agent based on potential obstruction of DNA replication and transcription. The inhibition of mouse mammary carcinoma FM3A cell growth by pyrrole polyamide compounds (Hybrids and distamycin A) has been evaluated (Figure 1) [25]. It was found that hybrids induce dose-dependent inhibition of cell growth. In particular, Hybrid 3 bearing a -phosphate group, which is a suitable substrate for biosynthesis, exhibited the highest inhibition.

Figure 1 

Structures of distamycin A, hybrids 1, 2, and 3.

Figure 2 

MGB polyamide-oligonucleotide conjugates.

The binding ability of pyrrole polyamide-oligonucleotide conjugates to target DNA, and the inhibition of cell growth by pyrrole polyamide--deoxyguanosine hybrids should be greatly influenced by the chain length of the pyrrole polyamide moiety and the length of the linker between the pyrrole polyamide moiety and the guanine base. Although we previously reported the synthesis of MGB polyamide-oligonucleotide conjugate ON 1 (), with linked pyrrole amide tetramer (Py₄-) at the 2-exocyclic amino group of a guanine base using 3-aminopropyl linker and evaluated the stability of modified dsDNA as described above [13], an examination of the length of the chain or linker connecting the pyrrole polyamide moiety to the guanine base and the DNA sequence recognition ability had not been investigated. In an effort to improve the activity of pyrrole polyamide-oligonucleotide conjugates, we performed the synthesis and evaluation of ON 1 and ON 2 with linked pyrrole polyamides (Py₄- and Py₃-) using various aminoalkyl linkers in terms of binding affinity for complementary DNA (Figure 2).

In addition to pyrrole polyamides, which possess high affinity for A-T base pairs of dsDNA, pyrrole-imidazole polyamides, which possess high affinity for C-G base pairs of dsDNA, were reported by Dervan et al. [14, 22–24, 26–31]. Furthermore, the synthesis and evaluation of MGB polyamide-oligonucleotide conjugates which possess binding affinity for C-G base pairs are an important aspect of the study. We performed the synthesis and evaluation of ON 3 and ON 4 with conjugated MGB polyamides (imidazole polyamide (Im₃-) and pyrrole-γ-imidazole polyamide hairpin motif (Py₃-γ-Im₃-)) at the 2-exocyclic amino group of a guanine base using various aminoalkyl linkers (Figure 2).

Herein, we report on the synthesis and evaluation of MGB polyamide-oligonucleotide conjugates ON 1-4 using various aminoalkyl linkers.

2. Materials and Methods

Column chromatography was performed on silica gel (Kanto Chemical Silica gel N60, spherical, natural, 40-50 μm). Precoated silica gel plates with a fluorescent indicator (Merck 60 F254) were used for analytical TLC. HPLC was performed on a Waters liquid chromatograph (600E system) equipped with a UV-VIS detector (2487 Dual), data module (741 type), and fraction collector. A μBondasphare C18 5 μm 100A () column with gradients of 5-50% CH₃CN in water (0.01 M TEAA, pH 7) was used. ¹H-NMR and ¹³C-NMR spectra were recorded using Bruker DRX 400 and a Bruker Biospin AVANCE III HD 400 instruments. Mass spectra were recorded on a Micromass Q-Tof Ultima API and a Micromass LCT spectrometer with a time-of-flight analyser. Elemental analyses were performed 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 Shimadzu TMSPC-8/UV-1600 apparatus. UV spectra were recorded using a Shimadzu UV-1200 apparatus. DNA oligonucleotides were purchased from Hokkaido System Science Co., Ltd. Compounds 2, 3, 4, 5, 7, 9, and 12 were prepared as previously described [9–13]. The CPG support-bound -deoxynucleoside 16 (B = C^Bz) and -deoxynucleoside -phosphoramidites 17 (B = T, C^Bz, A^Bz, and G^iBu) were purchased from Glen Research Corporation. -Deoxy-2-fluoroinosine -phosphoramidite 17 (B = I^F,NPE) was prepared from -deoxyguanosine as previously described [32–35]. Ethyl 1-methylimidazole-2-carboxylate (20) was prepared according to the procedure described by Baird and Dervan. [30].

2.1. Methyl 1-Methyl-4-[1-Methyl-4-(1-Methyl-1H-Pyrrole-2-Carbonyl)Amino-1H-Pyrrole-2-Carbonyl]Amino-1H-Pyrrole-2-Carboxylate (6)

Compound 2 [10–13] (2.83 g, 9.06 mmol) and 1-methyl-1H-pyrrole-2-carboxylic acid (5) [13] (1.26 g, 9.97 mmol) were dissolved in dichloromethane (45 mL), and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (2.61 g, 13.6 mmol) and 4-(N,N-dimethylamino)pyridine (DMAP) (1.66 g, 13.6 mmol) were added to the solution. After stirring at room temperature for 14 h, the solution was diluted with chloroform (300 mL) and washed with 2 M HCl aq. (150 mL x 3), H₂O (150 mL x3), 5% NaHCO₃ aq. (), and H₂O (150 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 6 (2.79 g, 80% yield) as a slightly brown glass. ¹H-NMR (DMSO-d₆): δ 9.92 (s, 1H, CONH), 9.82 (s, 1H, CONH), 7.46 (d, 1H,  Hz, Py-H), 7.23 (d, 1H,  Hz, Py-H), 7.05 (d, 1H,  Hz, Py-H), 6.94-6.90 (m, 3H, ), 6.06 (dd, 1H, , 3.9 Hz, Py-H), 3.88 (s, 3H, NCH₃), 3.85 (s, 3H, NCH₃), 3.84 (s, 3H, NCH₃), 3.74 (s, 3H, OCH₃); ¹³C-NMR (DMSO-d₆): δ 160.8, 158.6, 158.5, 128.1, 125.5, 123.0, 122.5, 122.2, 120.7, 118.5, 112.6, 108.4, 106.6, 104.8, 50.9, 36.2, and 36.1, 36.0; HRMS (ESI-TOF) : calcd for C₁₉H₂₂N₅O₄()⁺ 384.1672, found 384.1667; UV (CH₃OH): , 238 nm, , and 222 nm; and .

2.2. 2-[(9H-Fluoren-9-yl)Methoxycarbonylamino]Ethanaminium Chloride (8), 4-[(9H-Fluoren-9-yl)Methoxycarbonylamino]Butanaminium Chloride (10), and 5-[(9H-Fluoren-9-yl)Methoxycarbonylamino]Pentanaminium Chloride (11)

Compounds 8, 10, and 11 were prepared according to the synthetic procedure of 3-[(9H-fluoren-9-yl)methoxycarbonylamino]propanaminium chloride (9) [10–13].

Compound 8: (9H-Fluoren-9-yl)methyl phenyl carbonate (7) (0.95 g, 3.0 mmol) was suspended in methanol (12.5 mL), and then ethylenediamine (0.20 mL, 3.0 mmol) was added to the solution. After stirring for 4 h at room temperature, pyridinium hydrochloride (0.75 g, 6.5 mmol) was added, and the solution stirred for 10 min. The solution was concentrated in vacuo, and the residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 4 ) solvent system to give 8 (0.19 g, 20% yield) as a white powder. ¹H-NMR (CD₃OD): δ 7.80 (d, 2H,  Hz, Ar-H of the Fmoc group), 7.65 (d, 2H,  Hz, Ar-H of the Fmoc group), 7.40 (t, 2H,  Hz, Ar-H of the Fmoc group), 7.31 (t, 2H,  Hz, Ar-H of the Fmoc group), 4.42 (d, 2H,  Hz, CHCH₂ of the Fmoc group), 4.22 (t, 1H, J = 6.6 Hz, CHCH₂ of the Fmoc group), 3.32-3.30 (m, 2H, NCH₂), and 2.92-2.90 (t, 2H,  Hz, NCH₂); ¹³C-NMR (CD₃OD): δ 157.9, 143.8, 141.2, 127.4, 126.8, 124.8, 119.6, 66.7, 47.1, 39.8, and 38.3; HRMS (ESI-TOF) : calcd for C₁₇H₁₉N₂O₂(M + H)⁺ 283.1447, found 283.1455

Compound 10: (9H-Fluoren-9-yl)methyl phenyl carbonate (7) (3.16 g, 9.99 mmol) was suspended in methanol (42 mL), and then butane-1,4-diamine (1.0 mL, 9.95 mmol) was added to the solution. After stirring for 4 h at room temperature, pyridinium hydrochloride (2.51 g, 21.7 mmol) was added, and the solution stirred for 10 min. The solution was concentrated in vacuo, and the residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 4 ) solvent system to give 10 (2.11 g, 61% yield) as a white powder. ¹H-NMR (CD₃OD): δ 7.79 (d, 2H,  Hz, Ar-H of the Fmoc group), 7.64 (d, 2H,  Hz, Ar-H of the Fmoc group), 7.39 (t, 2H,  Hz, Ar-H of the Fmoc group), 7.31 (t, 2H,  Hz, Ar-H of the Fmoc group), 4.37 (d, 2H,  Hz, CHCH₂ of the Fmoc group), 4.20 (t, 1H,  Hz, CHCH₂ of the Fmoc group), 3.15 (t, 2H,  Hz, NCH₂), 2.93 (t, 2H,  Hz, NCH₂), and 1.66-1.55 (m, 4H, ); ¹³C-NMR (DMSO-d₆): δ 156.4, 144.1, 140.9, 127.8, 127.3, 125.4, 120.3, 65.5, 46.9, 39.8, 38.6, 26.46, and 24.39; HRMS (ESI-TOF) : calcd for C₁₉H₂₃N₂O₂(M + H)⁺ 311.1760, found 311.1752

Compound 11: (9H-Fluoren-9-yl)methyl phenyl carbonate (7) (3.16 g, 9.99 mmol) was suspended in methanol (42 mL), and then pentane-1,5-diamine (1.17 mL, 10.0 mmol) was added to the solution. After stirring for 4 h at room temperature, pyridinium hydrochloride (2.51 g, 21.7 mmol) was added and the solution stirred for 10 min. The solution was concentrated in vacuo, and the residue was subjected to silica gel column chromatography using a methanol/chloroform (1 : 4 ) solvent system to give 11 (1.64 g, 45% yield) as a white powder. ¹H-NMR (CD₃OD): δ 7.76 (d, 2H,  Hz, Ar-H of the Fmoc group), 7.60 (d, 2H,  Hz, Ar-H of the Fmoc group), 7.36 (t, 2H,  Hz, Ar-H of the Fmoc group), 7.26 (t, 2H,  Hz, Ar-H of the Fmoc group), 4.33 (d, 2H,  Hz, CHCH₂ of the Fmoc group), 4.17 (t, 1H,  Hz, CHCH₂ of the Fmoc group), 3.08 (t, 2H,  Hz, NCH₂), 2.87 (t, 2H,  Hz, NCH₂), 1.64-1.60 (m, 2H, CH₂), 1.51-1.47 (m, 2H, CH₂), and 1.38-1.34 (m, 2H, CH₂); ¹³C-NMR (CD₃OD): δ 158.9, 145.3, 142.6, 128.7, 128.1, 126.1, 120.1, 67.5, 48.4, 41.2, 40.6, 30.3, 28.2, and 24.5; HRMS (ESI-TOF) : calcd for C₂₀H₂₅N₂O₂(M + H)⁺ 325.1916, found 325.1925

2.3. (9H-Fluoren-9-yl)Methyl 4-{1-Methyl-4-[1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Pyrrole-2-Carbonyl)Amino-1H-Pyrrole-2-Carbonyl)Amino-1H-Pyrrole-2-Carbonyl]Amino-1H-Pyrrole-2-Carbonyl}Aminobutylcarbamate (13)

Compounds 13, 14, and 15 were prepared according to the synthetic procedure of 12 [10–13].

Pyrrole amide tetramer 4 was prepared as previously described [13]. ¹H-NMR (DMSO-d₆): δ 9.96 (s, 1H, and CONH), 9.95 (s, 1H, and CONH), 9.85 (s, 1H, and CONH), 7.48 (d, 1H,  Hz, and Py-H), 7.25-7.24 (m, 2H, and Py-H × 2), 7.08 (d, 1H,  Hz, and Py-H), 7.06 (d, ¹H,  Hz, and Py-H), 6.95-6.91, (m, 3H, and Py-H × 3), 6.06 (dd, ¹H,  Hz,  Hz, and Py-H), 3.88 (s, 3H, and NCH₃), 3.86 (s, 3H,and NCH₃), 3.85 (s, 3H, and NCH₃), 3.84 (s, 3H, and NCH₃), and 3.74 (s, 3H, and OCH₃); ¹³C-NMR (DMSO-d₆): δ 161.0, 158.8, 158.7, 128.3, 125.6, 123.2, 122.9, 122.7, 122.5, 122.3, 121.0, 118.8, 118.7, 112.9, 108.6, 106.9, 105.0, 104.9, 51.2, 36.44, 36.38, 36.32, and 36.28; HRMS (ESI-TOF) : calcd for C₂₅H₂₈N₇O₅(M + H)⁺ 506.2152, found 506.2151; UV (CH₃OH): λ_max 306, 238 nm; λ_min 262, 223 nm; and .

Pyrrole amide tetramer 4 (881 mg, 1.74 mmol) was dissolved in methanol (8.7 mL), and then 2 M NaOH aq. (8.7 mL) was added to the solution. After stirring at 60°C for 3 h, Dowex 50WX8 (H⁺-form) was added. Dowex 50WX8 was removed by filtration and the solution evaporated to give Py₄-carboxylic acid (856 mg, quantitative yield), which was subsequently used without purification. ¹H-NMR (DMSO-d₆): δ12.12(s, 1H, COOH), 9.95 (s, 1H, and CONH), 9.91 (s, 1H, and CONH), 9.84 (s, 1H, and CONH), 7.43 (d, 1H,  Hz, and Py-H), 7.25-7.24 (m, 2H, and Py-H × 2), 7.07 (d, 1H,  Hz, and Py-H), 7.05 (d, 1H,  Hz, and Py-H), 6.95 (d, 1H,  Hz, and Py-H), 6.93-6.91 (m, 1H, and Py-H), 6.85 (d, 1H,  Hz, and Py-H), 6.06 (dd, 1H,  Hz, J =3.8 Hz, and Py-H), 3.88 (s, 3H, and NCH₃), 3.86 (s, 3H, and NCH₃), 3.85 (s, 3H, and NCH₃), and 3.82 (s, 3H, and NCH₃); ¹³C-NMR (DMSO-d₆): δ 162.0, 158.6, 158.51, 158.47, 128.5, 125.5, 122.77, 122.71, 122.6, 122.3, 122.1, 120.3, 119.5, 118.5, 112.7, 108.4, 106.7, 104.77, 104.72, 36.24, 36.14, 36.12, and 36.08; HRMS (ESI-TOF) : calculated for C₂₄H₂₆N₇O₅(M + H)⁺ 492.1995, found 492.1993.

Py₄-carboxylic acid (492 mg, 1.00 mmol), 10 (520 mg, 1.50 mmol), and 1-hydroxybenzotriazole (HOBt) (270 mg, 2.00 mmol) were dissolved in DMF (5 mL), and then N,N-dicyclohexylcarbodiimide (DCC) (310 mg, 1.50 mmol) and N-ethyldiisopropylamine (N,N-diisopropylethylamine: DIEA) (240 μL, 1.38 mmol) were added to the solution. After stirring for 16 h, the precipitate was removed by filtration. The filtrate was diluted with chloroform (200 mL) and washed with 2 M HCl aq. (100 mL × 3), H₂O (100 mL), 5% NaHCO₃ aq. (100 mL × 3), and H₂O (75 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 13 (555 mg, 71% yield) as a slightly brown glass. ¹H-NMR (DMSO-d₆): δ 9.93 (s, 1H, CONH), 9.88 (s, 1H, and CONH), 9.82 (s, 1H, and CONH), 8.00-7.97 (m, 1H, and CONH), 7.88 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.69 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.40 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.30-7.23 (m, 3H, CONHCH₂, and Py-H × 2), 7.18 (d, 1H,  Hz, and Py-H), 7.06-7.04 (m, 2H, and Py-H x 2), 6.95-6.95 (m, 1H, and Py-H), 6.93-6.92 (m, 1H, and Py-H), 6.88-6.87 (m, 1H, and Py-H), 6.06 (dd, 1H,  Hz, J =3.9 Hz, and Py-H), 4.29 (d, 2H,  Hz, and CHCH₂ of the Fmoc group), 4.22-4.19 (t, 1H,  Hz, and CHCH₂ of the Fmoc group), 3.89 (s, 3H, and NCH₃), 3.86 (s, 3H, and NCH₃), 3.85 (s, 3H, and NCH₃), 3.80 (s, 3H, and NCH₃), 3.19-3.14 (m, 2H, and NHCH₂), 3.03-2.98 (m, 2H, and NHCH₂), and 1.52-1.39 (m, 4H, and CH₂ × 2); ¹³C-NMR (DMSO-d₆): δ 161.2, 158.6, 158.50, 158.46, 156.1, 143.9, 140.7, 128.1, 127.5, 127.0, 125.5, 125.1, 123.0, 122.8, 122.2, 122.11, 122.09, 120.1, 118.4, 117.7, 112.6, 106.6, 104.72, 104,69, 104.2, 65.2, 46.8, 38.1, 36.2, 36.1, 35.9, 27.0, and 26.7; HRMS (ESI-TOF) : calculated for C₄₃H₄₆N₉O₆(M + H)⁺ 784.3571, found 784.3578.

2.4. (9H-Fluoren-9-yl)Methyl 5-{1-Methyl-4-[1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Pyrrole-2-Carbonyl)Amino-1H-Pyrrole-2-Carbonyl)Amino-1H-Pyrrole-2-Carbonyl-Amino-1H-Pyrrole-2-Carbonyl]Aminopentylcarbamate (14)

Py₄-carboxylic acid (390 mg, 0.79 mmol), 11 (430 mg, 1.19 mmol), and 1-hydroxybenzotriazole (161 g, 1.19 mmol) were dissolved in DMF (4 mL), and then DCC (246 mg, 1.19 mmol) and N-ethyldiisopropylamine (190 μL, 1.09 mmol) were added. After stirring for 18 h, the precipitate was removed by filtration. The filtrate was diluted with chloroform (200 mL) and washed with 2 M HCl aq. (100 mL × 3), H₂O (100 mL), 5% NaHCO₃ aq. (100 mL × 3), and H₂O (75 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 14 (530 mg, 84% yield) as a slightly brown glass. ¹H-NMR (DMSO-d₆): δ 9.93 (s, 1H, and CONH), 9.88 (s, 1H, and CONH), 9.82 (s, 1H, and CONH), 7.98-7.95 (m, 1H, and CONH), 7.88 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.41 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.30-7.24 (m, 3H, CONH, and Py-H ×2), 7.18 (s, 1H, and Py-H), 7.05-7.04 (m, 2H, and Py-H × 2), 6.95-6.94 (m, 1H, and Py-H), 6.93-6.91 (m, 1H, and Py-H), 6.87 (s, 1H, and Py-H), 6.06 (dd, 1H,  Hz,  Hz, and Py-H), 4.29 (d, 2H,  Hz, and CHCH₂ of the Fmoc group), 4.21 (t, 1H,  Hz, and CHCH₂ of the Fmoc group), 3.89 (s, 3H, and NCH₃), 3.86 (s, 3H, and NCH₃), 3.85 (s, 3H, and NCH₃), 3.80 (s, 3H, and NCH₃), 3.18-3.13 (m, 2H, and NHCH₂), 3.01-2.96 (m, 2H, and NHCH₂), 1.52-1.39 (m, 4H, and CH₂ × 2), and 1.31-1.24 (m, 2H, and CH₂); ¹³C-NMR (DMSO-d₆): δ 161.2, 158.6, 158.50, 158.46, 156.1, 143.9, 140.7, 128.9, 128.1, 127.6, 127.3, 127.0, 125.5, 125.1, 123.1, 122.8, 122.2, 122.1, 121.4, 120.1, 118.4, 117.7, 112.6, 106.6, 104.72, 104.68, 104.1, 65.1, 46.8, 38.3, 36.2, 36.1, 35.9, 29.11, 29.05, and 23.7; HRMS (ESI-TOF) : calculated for C₄₄H₄₈N₉O₆(M + H)⁺ 798.3728, found 798.3733.

2.5. (9H-Fluoren-9-yl)Methyl 4-{1-Methyl-4-[1-Methyl-4-(1-Methyl-1H-Pyrrole-2-Carbonyl)Amino-1H-Pyrrole-2-Carbonyl]Amino-1H-Pyrrole-2-Carbonyl}Aminobutylcarbamate (15)

Pyrrole amide trimer 6 (515 mg, 1.34 mmol) was dissolved in methanol (6.7 mL), and then 2 M NaOH aq. (6.7 mL) was added to the solution. After stirring for 3 h at 60°C, Dowex 50WX8 (H⁺-form) was added. Dowex 50WX8 was removed by filtration and the solution evaporated to give the Py₃-carboxylic acid (495 mg, quantitative yield), which was subsequently used without purification. ¹H-NMR (DMSO-d₆): δ12.16 (s, 1H, COOH), 9.93 (s, 1H, and CONH), 9.86 (s, 1H, and CONH), 7.46 (d, 1H,  Hz, and Py-H), 7.26 (d, 1H,  Hz, and Py-H), 7.08 (d, 1H,  Hz, and Py-H), 6.95-6.94 (m, 1H, and Py-H), 6.92 (dd, 1H, , and 3.9 Hz Py-H), 6.85 (d, 1H,  Hz, and Py-H), 6.06 (dd, 1H, , and 3.9 Hz Py-H), 3.92 (s, 3H, and NCH₃), 3.89 (s, 3H, and NCH₃), and 3.86 (s, 3H, and NCH₃); ¹³C-NMR (DMSO-d₆): δ 162.4, 159.1, 158.9, 128.6, 125.9, 123.2, 123.1, 122.6, 120.7, 120.0, 119.0, 113.1, 108.9, 107.1, 105.2, 36.7, 36.6, and 36.5; HRMS (ESI-TOF) : calculated for C₁₈H₂₀N₅O₄(M + H)⁺ 370.1515, found 370.1509.

Py₃-carboxylic acid (495 mg, 1.34 mmol), 10 (697 mg, 2.01 mmol), and 1-hydroxybenzotriazole (362 mg, 2.68 mmol) were dissolved in DMF (13.4 mL), and then DCC (553 mg, 2.68 mmol) and N-ethyldiisopropylamine (300 μL, 1.74 mmol) were added. After stirring for 16 h, the precipitate was removed by filtration. The filtrate was diluted with chloroform (200 mL) and washed with 2 M HCl aq. (100 mL × 3), H₂O (100 mL), 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 subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 15 (538 mg, 61% yield) as a slightly brown glass. ¹H-NMR (DMSO-d₆): δ 9.87 (s, 1H, and CONH), 9.81 (s, 1H, and CONH), 7.99-7.97 (m, 1H, and CONH), 7.88 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.40 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.30-7.28 (m, 1H, and CONH), 7.23 (d, 1H,  Hz, and Py-H), 7.17 (d, 1H,  Hz, and Py-H), 7.03 (d, 1H,  Hz, and Py-H), 6.95-6.94 (m, 1H, and Py-H), 6.92-6.91 (m, 1H, and Py-H), 6.87 (d, 1H,  Hz, and Py-H), 6.06 (dd, 1H, , 3.8 Hz, and Py-H), 4.29 (d, 2H,  Hz, and CHCH₂ of the Fmoc group), 4.20 (t, 1H,  Hz, and CHCH₂ of the Fmoc group), 3.88 (s, 3H, and NCH₃), 3.85 (s, 3H, and NCH₃), 3.80 (s, 3H, and NCH₃), 3.19-3.14 (m, 2H, and NHCH₂), 3.02-2.98 (m, 2H, and NHCH₂), and 1.53-1.37 (m, 4H, and CH₂ × 2); ¹³C-NMR (DMSO-d₆): δ 161.2, 158.6, 158.4, 156.1, 143.9, 140.7, 128.1, 127.6, 127.0, 125.5, 125.1, 123.0, 122.8, 122.09, 122.06, 120.1, 118.4, 117.7, 112.6, 106.6, 104.6, 104.1, 65.2, 46.8, 38.1, 36.2, 36.1, 35.9, 27.0, and 26.7; HRMS (ESI-TOF) : calcd for C₃₇H₄₀N₇O₅(M + H)⁺ 662.3091, found 662.3054.

2.6. Ethyl 1-Methyl-4-Nitroimidazole-2-Carboxylate (21)

Ethyl 1-methylimidazole-2-carboxylate (20) [30] (2.97 g, 19.3 mmol) was dissolved in chloroform (19 mL), and then tetramethylammonium nitrate (5.26 g, 38.6 mmol) and trifluoroacetic anhydride (10.7 mL, 77.2 mmol) were added to the solution at 0°C. After stirring for 2.5 h at room temperature, 5% NaHCO₃ aq. (300 mL) was added. Products were extracted with chloroform (900 mL) from the resulting solution. The organic layer was washed with 5% NaHCO₃ aq. (200 mL × 2) and H₂O (100 mL), dried over anhydrous magnesium sulfate, and evaporated to dryness. The residue was subjected to silica gel column chromatography using an ethyl acetate/hexane (3 : 5~1 : 1 ) solvent system to give 21 (2.28 g, 59% yield) and ethyl 1-methyl-5-nitroimidazole-2-carboxylate (22) (920 mg, 24% yield).

Compound 20: Rf 0.20 (ethyl acetate/hexane (2 : 1 ) solvent system)

Compound 21 (white powder): Rf 0.42 (ethyl acetate/hexane (2 : 1 ) solvent system), ¹H-NMR (DMSO-d₆): δ 8.63 (s, 1H, and Im-H), 4.35 (q, 2H,  Hz, and CH₂CH₃), 3.99 (s, 3H, and NCH₃), and 1.33 (t, 3H,  Hz, and CH₂CH₃); ¹³C-NMR (DMSO-d₆): δ 157.7, 144.9, 134.8, 126.8, 61.7, 36.7, and 13.9; HRMS (ESI-TOF) : calculated for C₇H₁₀N₃O₄(M + H)⁺ 200.0671, found 200.0664. Anal. Calcd for C₇H₉N₃O₄: C, 42.21; H, 4.55; N, 21.10, found. C, 42.23; H, 4.59; and N, 21.09

Compound 22 (white powder): Rf 0.49 (ethyl acetate/hexane (2 : 1 ) solvent system), ¹H-NMR (DMSO-d₆): δ 8.15 (s, 1H, and Im-H), 4.38 (q, 2H,  Hz, and OCH₂CH₃), 4.19 (s, 3H, and NCH₃), and 1.33 (t, 3H,  Hz, and OCH₂CH₃); ¹³C-NMR (DMSO-d₆): δ 158.0, 140.7, 138.9, 131.4, 62.1, 35.0, and 13.9; HRMS (ESI-TOF) : calcd for C₇H₁₀N₃O₄(M + H)⁺ 200.0671, found 200.0669. Anal. Calcd for C₇H₉N₃O₄: C, 42.21; H, 4.55; N, 21.10, found. C, 42.13; H, 4.50; and N, 21.01

2.7. 1-Methylimidazole-2-Carboxylic Acid (23)

Ethyl 1-methyl-1H-imidazole-2-carboxylate (20) (3.08 g, 20.0 mmol) was dissolved in ethanol (50 mL)/pyridine (50 mL), and then 2 M NaOH aq. (100 mL) was added to the solution. After stirring at room temperature for 1 h, Dowex 50WX8 (H⁺-form) was added. Dowex 50WX8 was removed by filtration, and the solution evaporated to give 23 (2.52 g, quantitative yield) as a white powder, which was subsequently used without purification. ¹H-NMR (CD₃OD): δ 7.49 (s, 1H, and Im-H), 7.39 (s, 1H, and Im-H), and 4.15 (s, 3H, and NCH₃); ¹³C-NMR (CD₃OD): δ 158.0, 141.9, 126.6, 119.6, and 37.1; HRMS (ESI-TOF) : calcd for C₅H₇N₂O₂(M + H)⁺ 127.0508, found 127.0512.

2.8. Ethyl 4-Amino-1-Methylimidazole-2-Carboxylate (24)

Ethyl 4-nitro-1H-imidazole-2-carboxylate (21) (2.54 g, 12.7 mmol) was dissolved in ethanol (64 mL)/ethyl acetate (64 mL), and then 10% Pd/C (0.49 g) was added. The mixture was stirred under a slight positive pressure of hydrogen at room temperature for 3 h. Pd/C was removed by filtration through celite and washed with ethyl acetate (50 mL). The filtrate was evaporated to dryness to give 24 (2.14 g, quantitative yield) as a white powder, which was subsequently used without purification. ¹H-NMR (CDCl₃): δ 6.37 (s, 1H, and Im-H), 4.38 (2H, q,  Hz, and OCH₂CH₃), 3.92 (3H, s, and NCH₃), 2.96 (brs, NH₂, and 2H), and 1.40 (3H, t,  Hz, and OCH₂CH₃); ¹³C-NMR (CDCl₃): δ 159.0, 145.6, 131.6, 109.5, 61.3, 35.7, and 14.5; HRMS (ESI-TOF) : calcd for C₇H₁₂N₃O₂(M + H)⁺ 170.0930, found 170.0938.

2.9. Ethyl 4-(tert-Butoxycarbonylamino)-1-Methyl-1H-Imidazole-2-Carboxylate (25)

Compound 24 (2.09 g, 12.4 mmol) was dissolved in DMF (15 mL), and then a solution of di-tert-butyldicarbonate (5.42 g, 24.8 mmol) in DMF (10 mL) was added. After stirring for 19 h at room temperature, H₂O (50 mL) was added to the solution. The solution was diluted with chloroform (300 mL) and washed with H₂O (). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to silica gel column chromatography using an ethyl acetate/hexane (2 : 3 ) solvent system to give 25 (3.34 g, quantitative yield) as a white powder. ¹H-NMR (DMSO-d₆): δ 9.68 (s, 1H, and CONH), 7.30 (s, 1H, and Im-H), 4.25 (q, 2H,  Hz, and OCH₂CH₃), 3.88 (s, 3H, and NCH₃), 1.44 (s, 9H, and OC(CH₃)₃), and 1.28 (t, 3H,  Hz, and OCH₂CH₃); ¹³C-NMR (DMSO-d₆): δ 158.4, 152.7, 138.1, 130.9, 113.8, 78.9, 60.42, 35.4, 28.0, and 14.1; HRMS (ESI-TOF) ; calcd for C₁₂H₂₀N₃O₄(M + H)⁺ 270.1454, found 270.1439; Anal. Calcd for C₁₂H₁₉N₃O₄: C, 53.52; H, and 7.11; N, 15.60, found. C, 53.22; H, 7.05; and N, 15.55.

2.10. 4-(tert-Butoxycarbonylamino)-1-Methyl-1H-Imidazole-2-Carboxylic Acid (26)

Compound 25 (2.63 g, 9.80 mmol) was dissolved in ethanol (24.5 mL)/pyridine (24.5 mL), and then 2 M NaOH aq. (49 mL) was added to the solution. After stirring at room temperature for 1 h, Dowex 50WX8 (H⁺-form) was added. Dowex 50WX8 was removed by filtration, and the solution evaporated to give 26 (2.36 g, quantitative yield) as a white powder, which was subsequently used without purification. ¹H-NMR (DMSO-d₆): δ 9.52 (s, 1H, and CONH), 7.16 (s, 1H, and Im-H), 3.88 (s, 3H, and NCH₃), and 1.44 (s, 9H, and OC(CH₃)₃); ¹³C-NMR (DMSO-d₆): δ 160.4, 152.5, 137.1, 133.7, 112.2, 79.0, 35.3, and 28.1; HRMS (ESI-TOF) : calcd for C₁₀H₁₅N₃O₄Na (M + Na)⁺ 264.0960, found 264.0950.

2.11. Ethyl 4-(4-(tert-Butoxycarbonylamino)-1-Methyl-1H-Imidazole-2-Carboxamido)-1-Methyl-1H-Imidazole-2-Carboxylate (27)

Compounds 24 (660 mg, 3.90 mmol) and 26 (1.13 g, 4.70 mmol) were dissolved in DMF (39 mL), and then N-ethyldiisopropylamine (1.36 mL, 7.80 mmol), 1-hydroxybenzotriazole (1.05 g, 7.80 mmol), and N,N-diisopropylcarbodiimide (DCI) (3.60 mL, 23.3 mmol) were added to the solution. After stirring for 18 h, the reaction solution was diluted with chloroform (200 mL) and washed with 5% NaHCO₃ aq. () and H₂O (100 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using an ethyl acetate/hexane (2 : 1 ) solvent system to give 27 (1.34 g, 88% yield) as a slightly brown glass. ¹H-NMR (CDCl₃): δ 9.45 (s, 1H, and CONH), 7.54 (s, 1H, and Im-H), 7.25 (s, 1H, and Im-H), 6.71 (s, 1H, and CONH), 4.43 (q, 2H,  Hz, and OCH₂CH₃), 4.025 (s, 3H, and NCH₃), 4.016 (s, 3H, and NCH₃), 1.52 (s, 9H, and OC(CH₃)₃), 1.44 (t, 3H,  Hz, and OCH₂CH₃); ¹³C-NMR (CDCl₃): δ 159.0, 156.2, 152.6, 137.2, 136.8, 133.2, 132.0, 114.9, 112.7, 81.1, 61.7, 36.2, 35.7, 28.5, and 14.6; HRMS (ESI-TOF) : calcd for C₁₇H₂₅N₆O₅(M + H)⁺ 393.1886, found 393.1902.

2.12. Ethyl 1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxylate (28)

Compound 27 (510 mg, 1.30 mmol) was dissolved in ethanol (19.5 mL)/chloroform (6.5 mL), and then acetyl chloride (2.80 mL, 39.0 mmol) was added to the solution at room temperature. After stirring for 2 h at 40°C, the reaction solution was concentrated in vacuo to give Im₂-amine compound, which was subsequently used without purification. ¹H-NMR (DMSO-d₆): δ 10.45 (1H, s, and CONH), 7.71 (s, 1H, and Im-H), 7.47 (s, and 1H, Im-H), 4.28 (q, 2H,  Hz, and OCH₂CH₃), 3.99 (s, 3H, and NCH₃), 3.94 (s, 3H, and NCH₃), and 1.29 (t, 3H,  Hz, and OCH₂CH₃); ¹³C-NMR (DMSO-d₆): δ 158.1, 154.9, 135.7, 135.0, 131.4, 129.9, 117.6, 115.9, 60.8, 48.6, 35.75, 35.66, and 14.1; HRMS (ESI-TOF) : calcd for C₁₂H₁₇N₆O₃(M + H)⁺ 293.1362, found 293.1365.

Im₂-amine compound (1.30 mmol) and 23 (214 mg, 1.70 mmol) were dissolved in DMF (13 mL), and then EDCI (1.02 g, 5.30 mmol) and DMAP (490 mg, 4.00 mmol) were added to the solution. After stirring at room temperature for 18 h, the solution was diluted with chloroform (200 mL) and washed with H₂O (10 mL), 5% NaHCO₃ aq. () and H₂O (100 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using an ethyl acetate/hexane (2 : 1 ) solvent system to give 28 (440 mg, 84% yield) as a slightly brown glass. ¹H-NMR (CDCl₃): δ 9.48 (s, 1H, and CONH), 9.40 (s, 1H, and CONH), 7.56 (s, 1H, and Im-H), 7.50 (s, 1H, and Im-H), 7.11 (s, 1H, and Im-H), 7.02 (s, 1H, and Im-H), 4.44 (q, 2H,  Hz, and OCH₂CH₃), 4.10 (s, 3H, and NCH₃), 4.08 (s, 3H, and NCH₃), 4.03 (s, 3H, and NCH₃), and 1.45 (t, 3H, and  Hz, and OCH₂CH₃); ¹³C-NMR (CDCl₃): δ 158.1, 156.5, 156.2, 138.5, 136.8, 136.0, 133.6, 132.1, 128.4, 126.4, 115.1, 114.6, 61.8, 36.2, 35.90, 35.85, and 14.6; HRMS (ESI-TOF) : calcd for C₁₇H₂₁N₈O₄(M + H)⁺ 401.1686, found 401.1682; UV (CH₃OH): , , .

2.13. (9H-Fluoren-9-yl)Methyl 3-(1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)Propylcarbamate (29)

Compound 28 (500 mg, 1.20 mmol) was dissolved in ethanol (6 mL)/pyridine (6 mL) and then 2 M NaOH aq. (12 mL) was added to the solution. After stirring at room temperature for 3 h, Dowex 50WX8 (H⁺-form) was added. Dowex 50WX8 was removed by filtration, and the solution evaporated to give Im₃-carboxylic acid (450 mg, quantitative yield) as a white powder, which was subsequently used without purification. ¹H-NMR (DMSO-d₆): δ 10.09 (s, 1H, and CONH), 9.90 (s, 1H, and CONH), 7.61 (s, 1H, and Im-H), 7.55 (s, 1H, and Im-H), 7.44 (s, 1H, and Im-H), 7.07 (s, 1H, and Im-H), 4.01 (s, 3H, and NCH₃), 4.00 (s, 3H, and NCH₃), and 3.94 (s, 3H, and NCH₃); ¹³C-NMR (DMSO-d₆): δ 160.2, 155.9, 155.4, 137.8, 135.3, 135.0, 134.3, 133.4, 127.7, 127.0, 114.6, 113.8, 35.4, 35.2, and 35.1; HRMS (ESI-TOF) : calcd for C₁₅H₁₇N₈O₄(M + H)⁺ 373.1373, found 373.1366.

Im₃-carboxylic acid (330 mg, 0.90 mmol) and 9 (440 mg, 1.30 mmol) were dissolved in DMF (9 mL), and then N-ethyldiisopropylamine (210 μL, 1.20 mmol), 1-hydroxybenzotriazole (360 mg, 2.70 mmol), and N,N-diisopropylcarbodiimide (800 μL, 5.40 mmol) were added to the solution. After stirring for 19 h, the solution was diluted with chloroform (100 mL) and washed with H₂O (). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 29 (290 mg, 50% yield) as a slightly brown glass. ¹H-NMR (DMSO-d₆): δ 10.05 (s, 1H, and CONH), 9.56 (s, 1H, and CONH), 8.31-8.29 (m, and 1H, CONH), 7.88 (d, 2H,  Hz, and Ar − H × 2 of the Fmoc group), 7.69 (d, 2H,  Hz, Ar − H × 2 of the Fmoc group), 7.64 (s, 1H, and Im-H), 7.51 (s, 1H, and Im-H), 7.45 (s, 1H, and Im-H), 7.42 (t, 2H,  Hz, and Ar-H × 2 of the Fmoc group), 7.34 (t, 2H,  Hz, and Ar − H × 2 of the Fmoc group), 7.30-7.27 (m, 1H, and CONH), 7.08 (s, 1H, and Im-H), 4.32 (d, 2H,  Hz, and CHCH₂ of the Fmoc group), 4.22 (t, 1H,  Hz, and CHCH₂ of the Fmoc group), 4.02 (s, 3H, and NCH₃), 4.00 (s, 3H, and NCH₃), 3.96 (s, 3H, and NCH₃), 3.24-3.20 (m, 2H, and NHCH₂), 3.05-3.01 (m, 2H, and NHCH₂), and 1.66-1.59 (m, 2H, and CH₂); ¹³C-NMR (CDCl₃): δ 159.7, 156.9, 156.5, 156.0, 144.2, 141.4, 138.5, 135.9, 135.5, 134.4, 133.8, 128.3, 127.7, 127.1, 126.3, 125.2, 120.1, 114.6, 113.9, 66.7, 47.5, 38.0, 36.0, 35.81, 35.78, 35.76, and 30.2; HRMS (ESI-TOF) : calcd for C₃₃H₃₅N₁₀O₅(M + H)⁺ 651.2792, found 651.2820.

2.14. (9H-Fluoren-9-yl)Methyl 4-(1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)Butylcarbamate (30)

Im₃-carboxylic acid (740 mg, 2.00 mmol) and 10 (1.04 g, 3.00 mmol) were dissolved in DMF (20 mL), and then N-ethyldiisopropylamine (400 μL, 2.60 mmol), 1-hydroxybenzotriazole (810 mg, 6.00 mmol), and N,N-diisopropylcarbodiimide (1.80 mL, 12.0 mmol) were added to the solution. After stirring for 19 h, the solution was diluted with chloroform (200 mL) and washed with H₂O (). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 30 (430 mg, 33% yield) as a slightly brown glass. ¹H-NMR (DMSO-d₆): δ 9.68 (s, 1H, and CONH), 9.56 (s, 1H, and CONH), 8.28-8.26 (m, 1H, and CONH), 7.88 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.64 (s, 1H, and Im-H), 7.51 (s, 1H, and Im-H), 7.45 (s, 1H, and Im-H), 7.39 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.33 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.29-7.297 (m, 1H, and CONH), 7.08 (s, 1H, and Im-H), 4.29 (d, 2H,  Hz, and CHCH₂ of the Fmoc group), 4.20 (t, 1H,  Hz, and CHCH₂ of the Fmoc group), 4.02 (s, 3H, and NCH₃), 4.00 (s, 3H, and NCH₃), 3.95 (s, 3H, and NCH₃), 3.24-3.19 (m, 2H, and NHCH₂), 3.02-2.98 (m, 2H, and NHCH₂), and 1.49-1.41 (m, 4H, and CH₂ × 2); ¹³C-NMR (CDCl₃): δ 159.3, 156.7, 156.5, 156.0, 144.2, 141.5, 138.5, 135.9, 135.4, 134.6, 133.7, 128.4, 127.8, 127.2, 126.3, 125.2, 120.1, 114.5, 113.8, 66.7, 47.5, 40.9, 38.8, 35.86, 35.83, 35.80, 27.5, and 27.2; HRMS (ESI-TOF) : calcd for C₃₄H₃₇N₁₀O₅(M + H)⁺ 665.2948, found 665.2973.

2.15. (9H-Fluoren-9-yl)Methyl 5-(1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)Pentylcarbamate (31)

Im₃-carboxylic acid (450 mg, 1.20 mmol) and 11 (650 mg, 1.80 mmol) were dissolved in DMF (12 mL), and then N-ethyldiisopropylamine (300 μL, 1.60 mmol), 1-hydroxybenzotriazole (490 mg, 3.60 mmol) and N,N-diisopropylcarbodiimide (1.10 mL, 7.20 mmol) were added to the solution. After stirring for 19 h, the solution was diluted with chloroform (100 mL) and washed with H₂O (). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 31 (580 mg, 71% yield) as a slightly brown glass. ¹H-NMR (DMSO-d₆): δ 10.03 (s, 1H, and CONH), 9.56 (s, 1H, and CONH), 8.26-8.24 (m, 1H, and CONH), 7.87 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.67 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.64 (s, 1H, and Im-H), 7.50 (s, 1H, and Im-H), 7.45 (s, 1H, and Im-H), 7.39 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.33 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.26-7.24 (m, 1H, and CONH), 7.08 (s, 1H, and Im-H), 4.29 (d, 2H,  Hz, and CHCH₂ of the Fmoc group), 4.19 (t, 1H,  Hz, and CHCH₂ of the Fmoc group), 4.01 (s, 3H, and NCH₃), 4.00 (s, 3H, and NCH₃), 3.95 (s, 3H, and NCH₃), 3.23-3.18 (m, 2H, and NHCH₂), 3.00-2.95 (m, 2H, and NHCH₂), 1.54-1.39 (m, 4H, and ), and 1.30-1.24 (m, 2H, and CH₂); ¹³C-NMR(CDCl₃): δ 159.2, 156.6, 156.3, 155.9, 144.1, 141.3, 138.4, 135.8, 135.3, 134.6, 133.7, 128.2, 127.6, 127.0, 126.1, 125.1, 120.0, 114.4, 113.7, 66.5, 47.4, 40.9, 38.8, 35.69, 35.66, 35.64, 29.6, 29.4, and 24.1; HRMS (ESI-TOF) : calcd for C₃₅H₃₉N₁₀O₅(M + H)⁺ 679.3105, found 679.3085.

2.16. Ethyl 4-(1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)Butanoate (32)

Pyrrole amide trimer 6 (1.51 g, 3.93 mmol) was dissolved in ethanol (17 mL)/pyridine (17 mL), and then 2 M NaOH aq. (34 mL) was added to the solution. After stirring at room temperature for 3 h, Dowex 50WX8 (H⁺-form) was added. Dowex 50WX8 was removed by filtration, and the solution evaporated to give Py₃-carboxylic acid (1.45 g, quantitative yield), which was subsequently used without purification.

Py₃-carboxylic acid (1.45 g, 3.93 mmol) and ethyl 4-aminobutanoate (0.774 g, 5.90 mmol) were dissolved in dichloromethane (20 mL), and then EDCI (1.50 g, 7.86 mmol) and DMAP (960 mg, 7.86 mmol) were added to the solution. After stirring at room temperature for 10 h, the solution was diluted with chloroform (300 mL) and washed with H₂O (80 mL), 5% NaHCO₃ aq. (), and H₂O (80 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using an ethyl acetate/hexane (2 : 1~8 : 1 ) solvent system to give 32 (1.56 g, 82% yield) as a slightly brown glass. ¹H-NMR(CDCl₃): δ 7.60 (s, 1H, and CONH), 7.50 (s, 1H, and CONH), 7.15 (d, 1H,  Hz, and Py-H), 7.12 (d, 1H,  Hz, and Py-H), 6.78-6.77 (m, 1H, and Py-H), 6.71 (d, 1H,  Hz, Py-H), 6.68-6.66 (m, 1H, and Py-H), 6.52 (d, 1H,  Hz, and Py-H), 6.24-6,22 (m, 1H, and CONH), 6.13 (dd, 1H,  Hz, J =4.0 Hz, and Py-H), 4.15 (q, 2H Hz, and OCH₂CH₃), 3.98 (s, 3H, and NCH₃), 3.93 (s, 3H, and NCH₃), 3.90 (s, 3H, and NCH₃), 3.44-3.39 (m, 2H, and NHCH₂CH₂-), 2.41 (t, 2H,  Hz, and COCH₂CH₂-), 1.95-1.88 (m, 2H, and -CH₂CH₂CH₂-), and 1.25 (t, 3H,  Hz, and OCH₂CH₃); ¹³C-NMR(CDCl₃): δ 173.8, 161.8, 159.5, 1589.0, 128.5, 125.4, 123.4, 123.2, 121.5, 121.2, 119.3, 118.9, 112.0, 107.4, 103.8, 103.2, 60.7, 38.9, 36.8, 36.65, 36.57, 31.9, 24.7, and 14.2; HRMS (ESI-TOF) : calcd for C₂₄H₃₁N₆O₅ (M + H)⁺ 483.2356, found 483.2354.

2.17. Ethyl 1-Methyl-4-(1-Methyl-4-(1-Methyl-4-(tert-Butoxycarbonylamino)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxylate (33)

Compound 27 (2.22 g, 5.65 mmol) was dissolved in ethanol (19.5 mL)/chloroform (6.5 mL), and then acetyl chloride (2.80 mL, 39.0 mmol) was added to the solution at room temperature. After stirring for 2 h at 40°C, the reaction solution was concentrated in vacuo to give Im₂-amine compound, which was subsequently used without purification.

Im₂-amine compound (5.65 mmol) and 26 (2.27 g, 8.48 mmol) were dissolved in dichloromethane (56.5 mL), and then EDCI (3.25 g, 17.0 mmol) and DMAP (2.07 g, 17.0 mmol) were added to the solution. After stirring at room temperature for 19 h, the solution was diluted with chloroform (500 mL) and washed with H₂O (100 mL), 5% NaHCO₃ aq. () and H₂O (100 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using an ethyl acetate/hexane (2 : 1 ) solvent system to give 33 (2.05 g, 70% yield) as a slightly brown glass. ¹H-NMR (CDCl₃): δ 9.53 (s, 1H, and CONH), 9.17 (s, 1H, and CONH), 7.57 (s, 1H, and Im-H), 7.49 (s, 1H, and Im-H), 7.19 (s, 1H, and Im-H), 6.92 (s, 1H, and CONH), 4.44 (q, 2H,  Hz, and OCH₂CH₃), 4.07 (s, 3H, and NCH₃), 4.05 (s, 3H, and NCH₃), 4.03 (s, 3H, and NCH₃), 1.53 (s, 9H, and OC(CH₃)₃), 1.45 (t, 3H,  Hz, and OCH₂CH₃); ¹³C-NMR (CDCl₃): δ 158.8, 155.9, 155.8, 152.5, 137.0, 136.6, 135.7, 133.3, 133.0, 131.8, 114.8, 114.3, 112.7, 80.8, 61.5, 36.0, 35.7, 35.6, 28.3, and 14.3; HRMS (ESI-TOF) : calcd for C₂₂H₃₀N₉O₆(M + H)⁺ 516.2319, found 516.2318.

2.18. Ethyl 1-Methyl-4-(1-Methyl-4-(1-Methyl-4-(4-(1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)Butanamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxylate (34)

Compound 32 (1.44 g, 2.99 mmol) was dissolved in ethanol (7.5 mL)/pyridine (7.5 mL), and then 2 M NaOH aq. (15 mL) was added to the solution. After stirring at room temperature for 3 h, Dowex 50WX8 (H⁺-form) was added. Dowex 50WX8 was removed by filtration and the solution evaporated to give Py₃-NH(CH₂)₃CO₂H (1.36 g, quantitative yield), which was subsequently used without purification. ¹H-NMR (DMSO-d₆): δ 9.89 (s, 1H, and CONH), 9.83 (s, 1H, and CONH), 8.06-8.03 (m, 1H, and CONH), 7.23 (d, 1H,  Hz, and Py-H), 7.18 (d, 1H,  Hz, and Py-H), 7.02 (d, 1H,  Hz, and Py-H), 6.95-6.93 (m, 1H, and Py-H), 6.92-6.91 (m, 2H, Py − H × 2), 6.87 (d, 1H,  Hz, and Py-H), 6.06 (dd, 1H,  Hz,  Hz, and Py-H), 3.88 (s, 3H, and NCH₃), 3.85 (s, 3H, and NCH₃), 3.79 (s, 3H, and NCH₃), 3.19-3.16 (m, 2H, and NHCH₂CH₂-), 2.24 (t, 2H,  Hz, and COCH₂CH₂-), and 1.72-1.69 (m, 2H, and -CH₂CH₂CH₂-); ¹³C-NMR(DMSO-d₆): δ 174.5, 161.4, 158.6, 158.5, 128.2, 125.5, 123.0, 122.8, 122.15, 122.12, 118.5, 117.8, 112.7, 106.7, 104.7, 104.2, 37.9, 36.3, 36.1, 36.0, 31.3, and 24.8; HRMS (ESI-TOF) : calcd for C₂₂H₂₇N₆O₅(M + H)⁺ 455.2043, found 455.2039.

Compound 33 (54 mg, 0.104 mmol) was dissolved in ethanol (1.56 mL)/chloroform (0.52 mL), and then acetyl chloride (220 μL, 3.14 mmol) was added to the solution at room temperature. After stirring for 3 h at 40°C, the reaction solution was concentrated in vacuo to give Im₃-amine compound, which was subsequently used without purification. ¹H-NMR (CD₃OD): δ 7.81 (s, 1H, and Im-H), 7.65 (s, 1H, and Im-H), 7.35 (s, 1H, and Im-H), 4.49 (q, 2H,  Hz, and OCH₂CH₃), 4.14 (s, 3H, and NCH₃), 4.11 (s, 3H, and NCH₃), 4.10 (s, 3H, and NCH₃), and 1.45 (t, 3H,  Hz, and OCH₂CH₃); ¹³C-NMR (DMSO-d₆): δ 158.2, 155.6, 154.7, 135.9, 135.2, 134.8, 133.5, 131.5, 131.4, 115.9, 115.5, 115.1, 60.8, 35.7, 35.7, 35.2, and 14.1; HRMS (ESI-TOF) : calcd for C₁₇H₂₁N₉O₄(M + H)⁺ 416.1795, found 416.1788.

Im₃-amine compound (0.104 mmol) and Py₃-NH(CH₂)₃CO₂H (71 mg, 0.156 mmol) were dissolved in dichloromethane (2.1 mL), and then EDCI (60 mg, 0.312 mmol) and DMAP (38 mg, 0.312 mmol) were added to the solution. After stirring at room temperature for 14 h, the solution was diluted with chloroform (50 mL) and washed with H₂O (20 mL), 5% NaHCO₃ aq. () and H₂O (20 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 2% methanol/chloroform solvent system to give 34 (50 mg, 57% yield) as a slightly brown glass. ¹H-NMR(CDCl₃): δ 9.51 (s, 1H, and CONH), 9.24 (s, 1H, and CONH), 8.87 (s, 1H, and CONH), 7.93 (s, 1H, and CONH), 7.58-7.54 (m, 2H, Im-H,and CONH), 7.46 (s, 1H, and Im-H), 7.44 (s, 1H, and Im-H), 7.20 (d, 1H,  Hz, and Py-H), 7.18 (d, 1H,  Hz, and Py-H), 6.75-6.74 (m, 2H, and Py − H × 2), 6.60-6.59 (m, 1H, and Py-H), 6.52-6.44 (m, 2H, Py-H, and CONH), 6.10-6.09 (m, 1H, and Py-H), 4.39 (q, 2H, Hz, and OCH₂CH₃), 4.02 (s, 3H, and NCH₃), 4.01 (s, 3H, and NCH₃), 4.00 (s, 3H, and NCH₃), 3.97 (s, 3H, and NCH₃), 3.93 (s, 3H, and NCH₃), 3.92 (s, 3H, and NCH₃), 3.52-3.48 (m, 2H, and NHCH₂CH₂-), 2.52 (t, 2H,  Hz, and COCH₂CH₂-), 2.07-2.04 (m, 2H, -CH₂CH₂CH₂-), and 1.40 (t, 3H,  Hz, and OCH₂CH₃); ¹³C-NMR(CDCl₃): δ 171.2, 162.6, 159.8, 159.3, 158.7, 155.9, 155.8, 136.5, 136.4, 135.8, 135.7, 133.3, 133.1, 131.9, 128.4, 125.5, 123.09, 123.05, 121.9, 121.7, 119.4, 119.0, 115.0, 114.6, 112.5, 107.3, 104.2, 103.9, 61.6, 38.9, 36.8, 36.6, 36.5, 36.1, 35.63, 35.56, 33.8, 25.5, and 14.2; HRMS (ESI-TOF) : calcd for C₃₉H₄₆N₁₅O₈(M + H)⁺ 852.3654, found 852.3651; UV (CH₃OH): , , .

2.19. (9H-Fluoren-9-yl)Methyl-(3-(1-Methyl-4-(1-Methyl-4-(1-Methyl-4-(4-(1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)Butanamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)Ethyl)Carbamate (35)

Compound 34 (350 mg, 0.41 mmol) was dissolved in ethanol (2 mL)/pyridine (2 mL), and then 2 M NaOH aq. (2 mL) was added to the solution. After stirring at room temperature for 6 h, Dowex 50WX8 (H⁺-form) was added. Dowex 50WX8 was removed by filtration, and the solution evaporated to give Py₃-γ-Im₃-carboxylic acid (338 mg, quantitative yield) as a white powder, which was subsequently used without purification. ¹H-NMR(DMSO-d₆): δ 10.48 (s, 1H, and CONH), 10.22 (s, 1H, and CONH), 9.93 (s, 1H, and CONH), 9.89 (s, 1H, and CONH), 9.47 (s, 1H, and CONH), 8.09-8.06 (m, 1H, and CONH), 7.62 (s, 1H, and Im-H), 7.60 (s, 1H, and Im-H), 7.54 (s, 1H, and Im-H), 7.24 (d, 1H,  Hz, and Py-H), 7.19 (d, 1H,  Hz, and Py-H), 7.06 (d, 1H,  Hz, and Py-H), 6.97-6.95 (m, 1H, and Py-H), 6.94-6.93 (m, 1H, and Py-H), 6.91 (d, 1H,  Hz, and Py-H), 6.05-6.04 (m, 1H, and Py-H), 4.01 (s, 3H, and NCH₃), 3.98 (s, 3H, and NCH₃), 3.94 (s, 3H, and NCH₃), 3.88 (s, 3H, and NCH₃), 3.84 (s, 3H, and NCH₃), 3.80 (s, 3H, and NCH₃), 3.22-3.16 (m, 2H, and NHCH₂CH₂-), 2.39 (t, 2H,  Hz, and COCH₂CH₂-), and 1.82-1.79 (m, 2H, and -CH₂CH₂CH₂-); ¹³C-NMR(DMSO-d₆): δ 169.6, 160.8, 158.1, 158.0, 155.0, 154.9, 149.1, 136.0, 135.1, 134.5, 133.0, 132.9, 132.1, 127.6, 125.0, 123.4, 122.5, 122.3, 121.7, 121.6, 117.9, 117.3, 114.3, 114.0, 112.3, 106.1, 104.3, 103.8, 37.6, 35.8, 35.6, 35.5, 35.1, 34.8, 34.6, 32.4, and 25.0; HRMS (ESI-TOF) : calcd for C₃₇H₄₂N₁₅O₈(M + H)⁺ 824.3341, found 824.3337.

Py₃-γ-Im₃-carboxylic acid (330 mg, 0.40 mmol) and 8 (190 mg, 0.60 mmol) were dissolved in dichloromethane (6 mL), and then EDCI (230 mg, 1.20 mmol) and DMAP (70 mg, 0.60 mmol) were added to the solution. After stirring at room temperature for 17 h, the solution was diluted with chloroform (50 mL) and washed with H₂O (20 mL), 5% NaHCO₃ aq. (), and H₂O (20 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 35 (240 mg, 55% yield) as a slightly brown glass. ¹H-NMR (DMSO-d₆): δ 10.39 (s, 1H, and CONH), 9.89 (s, 1H, and CONH), 9.81 (s, 1H, and CONH), 9.64 (s, 1H, and CONH), 9.60 (s, 1H, and CONH), 8.26-8.24 (m, 1H, and CONH), 8.03-8.01 (m, 1H, and CONH), 7.87 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.66 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.64 (s, 1H, and Im-H), 7.53 (s, 1H, and Im-H), 7.50 (s, 1H, and Im-H), 7.41-3.9 (m, 1H, and CONH), 7.39 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.29 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.22 (d, 1H,  Hz, and Py-H), 7.18 (d, 1H,  Hz, and Py-H), 7.04 (d, 1H,  Hz, and Py-H), 6.94 (s, 1H, and Py-H), 6.92-6.91 (m, 1H, and Py-H), 6.89 (d, 1H,  Hz, and Py-H), 6.05 (dd,  Hz,  Hz, 1H, and Py-H), 4.28 (d, 2H,  Hz, and CHCH₂ of the Fmoc group), 4.20 (t, 1H, J =7.0 Hz, and CHCH₂ of the Fmoc group), 4.01 (s, 3H, and NCH₃), 3.97 (s, 3H, and NCH₃), 3.93 (s, 3H, and NCH₃), 3.88 (s, 3H, and NCH₃), 3.85 (s, 3H, and NCH₃), 3.80 (s, 3H, and NCH₃), 3.30-3.15 (m, 6H, and NHCH₂CH₂- × 3), 2.36 (t, 2H,  Hz, and COCH₂CH₂-), and 1.82-1.78 (m, 2H, and -CH₂CH₂CH₂-); ¹³C-NMR (CDCl₃): δ 171.1, 162.3, 159.7, 159.6, 159.1, 157.2, 155.7, 155.6, 143.7, 141.1, 136.4, 135.5, 135.3, 134.1, 133.5, 133.1, 128.4, 127.6, 126.9, 125.4, 124.9, 123.1, 122.8, 121.8, 121.5, 119.9, 119.5, 119.1, 115.0, 114.4, 113.9, 112.5, 107.3, 104.0, 103.9, 66.8, 47.0, 39.2, 38.92, 38.85, 38.79, 36.8, 36.6, 36.4, 35.5, 33.7, 29.7, and 25.3; HRMS (ESI-TOF) : calcd for C₅₄H₅₈N₁₇O₉(M + H)⁺ 1088.4603, found 1088.4604.

2.20. (9H-Fluoren-9-yl)Methyl (3-(1-Methyl-4-(1-Methyl-4-(1-Methyl-4-(4-(1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)Butanamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)Propyl)Carbamate (36)

Py₃-γ-Im₃-carboxylic acid (338 mg, 0.41 mmol) and 9 (205 mg, 0.62 mmol) were dissolved in dichloromethane (10 mL), and then EDCI (236 mg, 1.23 mmol) and DMAP (90 mg, 0.74 mmol) were added to the solution. After stirring at room temperature for 17 h, the solution was diluted with chloroform (50 mL) and washed with H₂O (20 mL), 5% NaHCO₃ aq. (), and H₂O (20 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 36 (240 mg, 53% yield) as a slightly brown glass. ¹H-NMR((DMSO-d₆): δ 10.40 (s, 1H, and CONH), 9.88 (s, 1H, and CONH), 9.81 (s, 1H, and CONH), 9.63-9.58 (m, 2H, and CONH × 2), 8.26-8.24 (m, 1H, and CONH), 8.03-8.01 (m, 1H, and CONH), 7.87 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.64 (s, 1H, and Im-H), 7.54 (s, 1H, and Im-H), 7.51 (s, 1H, and Im-H), 7.40 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.30-7.27 (1H, m, and CONH), 7.22 (d, 1H,  Hz, and Py-H), 7.17 (d, 1H,  Hz, and Py-H), 7.04 (d, 1H,  Hz, and Py-H), 6.95-6.94 (m, 1H, and Py-H), 6.92-6.90 (m, 1H, and Py-H), 6.89 (d, 1H,  Hz, and Py-H), 6.05 (dd,  Hz,  Hz, 1H, and Py-H), 4.31 (d, 2H,  Hz, and CHCH₂ of the Fmoc group), 4.21(t, 1H,  Hz, and CHCH₂ of the Fmoc group), 4.01 (s, 3H, and NCH₃), 3.97 (s, 3H, and NCH₃), 3.95 (s, 3H, and NCH₃), 3.88 (s, 3H, and NCH₃), 3.84 (s, 3H, and NCH₃), 3.80 (s, 3H, and NCH₃), 3.22-3.20 (m, 4H, and NHCH₂CH₂- × 2), 3.03-3.01 (m, 2H, and NHCH₂CH_2-), 2.39-2.35 (m, 2H, and COCH₂CH₂-), 1.82-1.78 (m, 2H, and -CH₂CH₂CH₂-), and 1.64-1.62 (m, 2H, and -CH₂CH₂CH₂-); ¹³C-NMR (DMSO-d₆): δ 170.0, 161.4, 158.7, 158.5, 158.5, 156.2, 155.6, 155.4, 143.9, 140.8, 136.5, 135.2, 134.6, 134.4, 133.3, 132.8, 128.1, 127.6, 127.1, 125.5, 125.1, 123.0, 122.8, 122.2, 122.1, 120.1, 118.4, 117.8, 114.8, 114.6, 114.0, 112.7, 106.7, 104.7, 104.3, 65.3, 46.8, 38.2, 37.9, 36.2, 36.1, 36.01, 35.97, 35.3, 35.1, 35.0, 32.9, 29.6, and 25.5; HRMS (ESI-TOF) : calcd for C₅₅H₆₀N₁₇O₉ (M + H)⁺ 1102.4760, found 1102.4775.

2.21. (9H-Fluoren-9-yl)Methyl (4-(1-Methyl-4-(1-Methyl-4-(1-Methyl-4-(4-(1-Methyl-4-(1-Methyl-4-(1-Methyl-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)-1H-Pyrrole-2-Carboxamido)Butanamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)-1H-Imidazole-2-Carboxamido)Butyl)Carbamate (37)

Py₃-γ-Im₃-carboxylic acid (41 mg, 0.048 mmol) and 10 (28 mg, 0.080 mmol) were dissolved in dichloromethane (3 mL), and then EDCI (28 mg, 0.14 mmol) and DMAP (12 mg, 0.096 mmol) were added to the solution. After stirring at room temperature for 14 h, the solution was diluted with chloroform (50 mL) and washed with H₂O (20 mL), 5% NaHCO₃ aq. () and H₂O (20 mL). The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The residue was subjected to chromatographic separation on a column of silica gel using a 0-5% methanol/chloroform solvent system to give 37 (14 mg, 27% yield) as a slightly brown glass. ¹H-NMR (DMSO-d₆): δ 10.40 (s, 1H, and CONH), 9.88 (s, 1H, and CONH), 9.81 (s, 1H, and CONH), 9.63-9.58 (m, 2H, and CONH × 2), 8.25-8.23 (m, 1H, and CONH), 8.04-8.02 (m, 1H, and CONH), 7.87 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H,  Hz, and Ar-H of the Fmoc group), 7.65 (s, 1H, and Im-H), 7.54 (s, 1H, and Im-H), 7.52 (s, 1H, and Im-H), 7.40 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H,  Hz, and Ar-H of the Fmoc group), 7.29-7.27 (1H, m, and CONH), 7.23 (d, 1H,  Hz, and Py-H), 7.18 (d, 1H,  Hz, and Py-H), 7.04 (d, 1H,  Hz, and Py-H), 6.95-6.94 (m, 1H, and Py-H), 6.93-6.91 (m, 1H, and Py-H), 6.90 (d, 1H,  Hz, and Py-H), 6.05 (dd, 1H,  Hz,  Hz, and Py-H), 4.29 (d, 2H,  Hz, CHCH₂ of the Fmoc group), 4.19 (t, 1H,  Hz, CHCH₂ of the Fmoc group), 4.01 (s, 3H, and NCH₃), 3.97 (s, 3H, and NCH₃), 3.95 (s, 3H, and NCH₃), 3.88 (s, 3H, and NCH₃), 3.85 (s, 3H, and NCH₃), 3.80 (s, 3H, and NCH₃), 3.22-3.20 (m, 4H, NHCH₂CH₂- × 2), 3.00-2.99 (m, 2H, and NHCH₂CH₂-), 2.38-2.35 (m, 2H, and COCH₂CH₂-), 1.81-1.78 (m, 2H, and -CH₂CH₂CH₂-), 1.46-1.30 (m, 4H, and -CH₂CH₂CH₂- × 2); ¹³C-NMR (DMSO-d₆): δ 170.0, 161.3, 158.6, 158.5, 158.4, 156.1, 155.6, 155.4, 143.9, 140.7, 136.5, 135.2, 134.6, 134.5, 133.3, 132.8, 128.1, 127.6, 127.0, 125.5, 125.1, 123.0, 122.8, 122.2, 122.1, 120.1, 118.4, 117.8, 114.8, 114.6, 114.0, 112.7, 106.7, 104.7, 104.2, 65.2, 46.8, 38.2, 38.1, 36.2, 36.1, 36.0, 35.3, 35.1, 35.0, 32.9, 26.9, 26.6, 25.5, and 24.0; HRMS (ESI-TOF) : calcd for C₅₆H₆₂N₁₇O₉ (M + H)⁺ 1116.1926, found 1116.4918.

2.22. Synthesis of MGB Polyamide-Oligonucleotide Conjugates ON 1-4

Conjugates ON 1-4 were synthesized by the postsynthetic modification method as previously described for the synthesis of ON 1 () [13].

CPG support-bound oligonucleotide 18 (11-mer: -d(CGI^F,NPEAATTTGGC)- or -d(CGI^F,NPEACCCTGGC)-) was synthesized using a syringe-based system. CPG support-bound -deoxynucleoside 16 (, 1000 Å, purchased from Applied Biosystems Pty Ltd.) (2 μmol) was treated with 3% Cl₃CCO₂H in dichloromethane () for 1 min, followed by washing with acetonitrile (). A 0.1 M solution of -deoxynucleoside -phosphoramidite 17 () in acetonitrile (0.5 mL) and 0.5 M 1H-tetrazole in acetonitrile (0.5 mL) were then delivered to the column. Following 10 min, coupling agents were ejected from the column, and the CPG support was washed with acetonitrile () to give the phosphite dimer. Following this, 1 : 1 : 8 acetic anhydride/2,6-lutidine/THF (1.0 mL) and 16% 1-methylimidazole/THF (1.0 mL) were delivered to the column, coupling agents were ejected from the column, and the CPG support was washed with acetonitrile (). The resultant CPG support-bound phosphite dimer was treated with 0.02 M I₂ in 1 : 2 : 7 H₂O/pyridine/THF (1.0 mL) for 1 min and washed with acetonitrile () to give the CPG support-bound phosphorotriester dimer. Following chain elongation using -deoxynucleoside -phosphoramidites 17 (, C^Bz, A^Bz, G^iBu, and I^F,NPE), as described above, the terminal DMTr protecting group of the oligonucleotide was removed by treatment with 3% Cl₃CCO₂H in dichloromethane () for 1 min, and the CPG support was washed with acetonitrile (). Resultant CPG support-bound oligonucleotide 18 was treated with 0.1 M Fmoc-NH(CH₂)_nNH-MGB polyamide (12, 13, 14, 15, 29, 30, 31, 35, 36, or 37) in 1 : 5 Et₃N/1,4-dioxane (1.0 mL) at 60°C for 24 h and then washed with acetonitrile (). Following this, the CPG support-bound oligomer was treated with 0.5 M DBU in pyridine (2.0 mL) at room temperature for 12 h and washed with acetonitrile (). The generated oligomer was then cleaved from the CPG support by treatment with conc. NH₄OH () for 2 h at room temperature. The resulting solution was then heated in a sealed vial at 55°C for 6 h. Following evaporation, the residue was dissolved in H₂O (5.0 mL) and washed with ethyl acetate (), and the aqueous layer was evaporated. The MGB polyamide-oligonucleotide conjugates ON 1-4 were then purified by reversed-phase HPLC.

ON 1 (-d(CGGAATTTGGC)-, G = Py₄-NH(CH₂)_n-G ()).

ON 1 () yields 48.8 A₂₆₀ units from 16 (B = C^Bz) (2 μmol). HRMS (ESI-TOF) calcd for C₁₃₅H₁₆₈N₅₀O₆₉P₁₀(M +2H)²⁺ 1951.4276, found 1951.4158. ON 1 (): yields 34.0 A₂₆₀ units from 16 (B = C^Bz) (2 μmol). HRMS (ESI-TOF) calcd for C₁₃₆H₁₇₀N₅₀O₆₉P₁₀(M +2H)²⁺ 1958.4353, found 1958.4075. ON 1 (): yields 31.4 A₂₆₀ units from 16 (B = C^Bz) (2 μmol). HRMS (ESI-TOF) calcd for C₁₃₇H₁₇₃N₅₀O₆₉P₁₀(M +3H)³⁺ 1310.6314, found 1310.5460.

ON 2 (-d(CGGAATTTGGC)-, G = Py₃-NH(CH₂)_n-G ().

ON 2 () yields 22.5 A₂₆₀ units from 16 (B = C^Bz) (2 μmol). HRMS (ESI-TOF) calcd for C₁₃₀H₁₆₅N₄₈O₆₈P₁₀ (M +3H)³⁺ 1265.2768, found 1265.2252.

ON 3 (-d(CGGACCCTGGC)-, G = Im₃-NH(CH₂)_n-G ()).

ON 3 (n =3) yields 21.8 A₂₆₀ units from 16 (B = C^Bz) (2 μmol). HRMS (ESI-TOF) calcd for C₁₂₃H₁₅₆N₅₁O₆₇P₁₀ (M + H)⁺ 3728.7744, found 3728.7866. ON 3 () yields 20.7 A₂₆₀ units from 16 (B = C^Bz) (2 μmol). HRMS (ESI-TOF) calcd for C₁₂₄H₁₅₈N₅₁O₆₇P₁₀ (M + H)⁺ 3742.7900, found 3742.7827. ON 3 () yields 28.7 A₂₆₀ units from 16 (B = C^Bz) (2 μmol). HRMS (ESI-TOF) calcd for C₁₂₅H₁₆₀N₅₁O₆₇P₁₀ (M + H)⁺ 3756.8057, found 3756.8152.

ON 4 (-d(CGGACCCTGGC)-: G = Py₃-γ-Im₃-NH(CH₂)_n-G ()).

ON 4 () yields 8.4 A₂₆₀ units from 16 (B = C^Bz) (1 μmol). HRMS (ESI-TOF) calcd for C₁₄₄H₁₇₉N₅₈O₇₁P₁₀ (M + H)⁺ 4165.9555, found 4165.9580. ON 4 () yields 11.0 A₂₆₀ units from 16 (B = C^Bz) (1 μmol). HRMS (ESI-TOF) calcd for C₁₄₅H₁₈₁N₅₈O₇₁P₁₀ (M + H)⁺ 4179.9712, found 4179.9858. ON 4 () yields 11.3 A₂₆₀ units from 16 (B = C^Bz) (1 μmol). HRMS (ESI-TOF) calcd for C₁₄₆H₁₈₃N₅₈O₇₁P₁₀ (M + H)⁺ 4193.9868, found 4194.0024.

2.23. Melting Temperature Experiments

Absorbance versus temperature profiles of duplexes in 10 mM sodium phosphate buffer (pH 7.0) containing 10 mM NaCl and 0.1 mM Na₂EDTA were measured using a TMSPC-8/UV1600 (Shimadzu Co., Ltd.) instrument equipped with a thermoelectrically controlled cell holder at 260 nm and a heating rate of 1.0°C/min. The concentration of each duplex was 4.3 μM [5, 36]. From these melting curves, values were obtained using a TMSPC-8 system with T_m analysis software.

2.24. Circular Dichroism (CD) Spectropolarimetry

CD spectra of duplexes in 10 mM sodium phosphate buffer (pH 7.0) containing 10 mM NaCl and 0.1 mM Na₂EDTA were measured using a JASCO J-720 spectropolarimeter equipped with a thermoelectrically controlled cell holder (at 20°C) and a cuvette with a path length of 10 mm. The concentration of each duplex was 5.8 μM [5].

3. Results and Discussion

In an effort to examine the effect of linker length or the distance between the guanine base and pyrrole polyamide on the stability of the modified dsDNA (ON 1/complementary DNA), we synthesized ON 1 (, 4, and 5) (-d(CGGAATTTGGC)-: G = Py₄-NH(CH₂)_n-G) using 3-aminopropyl [13], 4-aminobutyl, and 5-aminopentyl linkers.

Pyrrole amide tetramer 4 and the linker reagent 9 were prepared as previously described (Schemes 1 and 2) [10–13]. Linker reagents 8, 10, and 11 were prepared according to the synthetic procedure of 9. Pyrrole amide tetramers 12, 13, and 14 bearing 3-aminopropyl, 4-aminobutyl, and 5-aminopentyl linkers were synthesized via hydrolysis of the ester moiety of 4 and coupling with linker reagents 9, 10, and 11, respectively, as shown in Scheme 3. ON 1 (, 4, and 5) were synthesized by a postsynthetic modification method using -deoxy-2-fluoroinosine -phosphoramidite 17 () [32–35] and pyrrole amide tetramers 12, 13, and 14 as previously described (Scheme 4) [13]. -Deoxy-2-fluoroinosine (I^F,NPE) was incorporated into CPG support-bound oligonucleotide 18 (-d(CGI^F,NPEAATTTGGC)-) using a standard procedure [37]. Resultant CPG support-bound oligonucleotide 18 was treated with 0.1 M 12, 13, or 14 in 1 : 5 Et₃N/1,4-dioxane at 60°C for 24 h. The generated CPG support-bound oligonucleotide was then treated with 0.5 M DBU in pyridine to remove the NPE and CE protecting groups, and then treated with concentrated NH₄OH to cleave the oligomer from the CPG support and remove the Bz and iBu protecting groups. Conjugates ON 1 (, 4, and 5) were purified by reversed-phase HPLC to yield 48.8, 34.0, and 31.4 A₂₆₀ units, respectively, from 16 () (2 μmol).

Scheme 1 

Synthesis of pyrrole polyamides. Reagents and conditions: i EDCI, DMAP, CH₂Cl₂, rt, 6 (80%). Compounds 2, 3, 4, and 5 were prepared as previously described [10–13].

Scheme 2 

Synthesis of aminoalkyl linker reagents. Reagents and conditions: i H₂N(CH₂)_nNH₂, MeOH, rt; (ii) pyridinium hydrochloride, 8 (20%); 10 (61%); 11 (45%). Compound 9 was prepared as previously described [10–13].

Scheme 3 

Synthesis of pyrrole polyamides bearing aminoalkyl linker. Reagents and conditions: i 1 M NaOH aq./MeOH, 60°C; Dowex 50WX8 (H⁺-form); ii 9, 10, or 11, DCC, HOBt, DIEA, DMF, and rt; 13 (71%); 14 (84%); 15 (61%). Compound 12 was prepared as previously described [10–13].

Scheme 4 

Synthesis of MGB polyamide-oligonucleotide conjugates. Reagents and conditions: i Oligonucleotide assembly on CPG support by the phosphoramidite method, 3% Cl₃CCO₂H, CH₂Cl₂; 0.05 M phosphoramidite 17, 0.25 M 1H-tetrazole, CH₃CN, or CH₂Cl₂; Ac₂O, 2,6-lutidine, 1-methylimidazole, THF; and 0.02 M I₂, H₂O/pyridine/THF. (ii) 3% Cl₃CCO₂H, CH₂Cl₂; 0.1 M FmocNH-(CH₂)_nNH-MGB polyamide 12, 13, 14, 15, 29, 30, 31, 35, 36, or 37, 1 : 5 Et₃N/1,4-dioxane, 60°C, 24 h; 0.5 M DBU, pyridine, rt, 12 h. Conc. NH₄OH, rt., 2 h–55°C, and 6 h.

Conjugates ON 1 (, 4, and 5) were converted to modified dsDNAs (ON 1/complementary DNA) by annealing with complementary DNA. The stability of modified dsDNAs was investigated by T_m and CD analyses. From the values, it was found that the stability of modified dsDNA was greatly influenced by the linker length (ON 1 (, °C, °C), (, °C, °C), and (, °C, °C)) (Table 1). ON 1 () showed high binding ability for complementary DNA, similar to ON 1 () previously reported [13]. In the CD spectrum for dsDNA [ON 1 ()/complementary DNA], a strong additional CD band centered at 331 nm resulting from an induced Cotton effect of the bound pyrrole amide moiety was observed (Figure 3(I)) [5, 6, 13].

Table 1 

values of modified dsDNAs and respective values.

Figure 3 

CD spectra of unmodified and modified dsDNAs. modified DNA: -d(CGGAATTTGGC)-, ON 1 (G = Py₄-NH(CH₂)_n-G) and ON 2 (G = Py₃-NH(CH₂)_n-G).

Using single-mismatch DNA (-d(GCCTTcAACCG)-), which contains a mismatch base in the recognition sequence (-d(AATTT)-/3-(TTAAA)-) of the pyrrole amide moiety, and 2-base mismatch DNA (-d(GCaTTcAACCG)-) which does not form dsDNA with the unmodified DNA, the DNA sequence recognition ability of ON 1 () was investigated by analysis (Table 1). ON 1 () formed dsDNA with single-base mismatch DNA and displayed stabilization of the dsDNA (, °C,) by the pyrrole amide moiety. On the other hand, ON 1 () did not form dsDNA with 2-base mismatch DNA and the pyrrole amide moiety did not show any activity.

We surmised that shortening the pyrrole amide chain of the modified DNA would be effective in reducing activity and increasing recognition of the target DNA sequence. ON 2 () (-d(CGGAATTTGGC)-: G = Py₃-NH(CH₂)_n-G) was synthesized by a postsynthetic modification method using pyrrole amide trimer 15, which was prepared via coupling of pyrrole amide dimer 2 and pyrrole-2-carboxylic acid 5, hydrolysis of ester product 6, and condensation with linker reagent 10 (Schemes 1 and 3). ON 2 () was purified by reversed-phase HPLC and yielded 22.5 A₂₆₀ units from 16 () (2 μmol) (Scheme 4).

The stability of the modified dsDNA of ON 2 () and complementary DNA was investigated (Table 1 and Figure 3(II)). The stability of the modified dsDNA (ON 2 ()/complementary DNA: °C, °C) was lower compared with modified dsDNA (ON 1 ()/complementary DNA: °C, °C). The DNA sequence recognition ability of ON 2 () was investigated using single- and 2-base mismatch DNAs (Table 1). ON 2 () formed dsDNA with single-base mismatch DNA and displayed stabilization of the dsDNA (°C, °C). On the other hand, ON 2 () did not form dsDNA with 2-base mismatch DNA. The DNA sequence recognition ability of pyrrole polyamide-oligonucleotide conjugates was not improved. However, from the result of 2-base mismatch DNA, it was thought that conjugates (modified DNAs ON 1 () and ON 2 ()) did not act on single-base mismatch DNA under conditions where dsDNA (unmodified DNA/single-base mismatch DNA) did not form (e.g., processing temperature > T_m (unmodified DNA/single-base mismatch DNA)).

Polyamides containing 1-methylpyrrole (Py) and 1-methylimidazole (Im) can be combined in antiparallel side-by-side dimeric complexes with the minor groove of dsDNA [22–24]. An imidazole ring on one ligand complemented by a pyrrole ring on a second ligand (Im/Py combination) recognizes G-C base pairs, while a Py/Im combination targets C-G base pairs. A Py/Py combination is partially degenerate and binds either A-T or T-A base pairs. Based on the results of ON 1 and ON 2 described above, it was expected that imidazole polyamide-oligonucleotide conjugates should possess high binding ability for DNA that includes a guanine (G) base. Next, we synthesized and evaluated imidazole polyamide-oligonucleotide conjugates ON 3 (, 4, and 5) (-d(CGGACCCTGGC)-: G = Im₃-NH(CH₂)_n-G) as model modified oligonucleotides, which form dsDNA with complementary DNA (-d(GCCTGGGACCG)-) that includes the imidazole polyamide binding sequence (Figure 2).

Conjugates ON 3 (, 4, and 5) were synthesized by a postsynthetic modification method using imidazole amide trimers (29, 30 and 31) as described above (Scheme 4). Imidazole amide trimers (29, 30, and 31) bearing 3-aminopropyl, 4-aminobutyl, and 5-aminopentyl linkers, respectively, were synthesized as shown in Scheme 5.

Scheme 5 

Synthesis of imidazole polyamide derivatives. Reagents and conditions: i (CH₃)₄N⁺ NO₃⁻, TFAA, CHCl₃, 0°C-rt, 21 (59%), and 22 (24%). ii 1 M NaOH aq./EtOH/pyridine, rt; Dowex 50WX8 (H⁺-form), 23 (quant.); 26 (quant.). iii H₂, 10% Pd/C, 1 : 1 AcOEt/EtOH, rt, 24 (quant.). iv (Boc)₂O, DMF, rt, 25 (quant.); (v) DCI, HOBt, DIEA, DMF, rt, 27 (88%). vi AcCl, EtOH/CHCl₃, rt-40°C. vii 23, EDCI, DMAP, DMF, rt, 28 (84%). viii 9, 10, or 11, DCI, HOBt, DIEA, DMF, rt, 29 (50%); 30 (33%); 31 (71%). Compound 20 was prepared according to the procedure described by Baird and Dervan [30].

Baird and Dervan have reported the nitration of ethyl 1-methylimidazole-2-carboxylate (20) by treatment with concentrated sulfuric acid/90% nitric acid [30]. The reaction mixture was refluxed for 50 min and then quenched by pouring on ice. Ethyl 1-methyl-4-nitroimidazole-2-carboxylate (21) was extracted with dichloromethane and recrystallized from 21 : 1 CCl₄/ethanol in 22% yield. We attempted an improvement of the nitration method of 20 using tetramethylammonium nitrate/trifluoroacetic anhydride as a nitrating agent [38]. The reaction was performed at room temperature for 2.5 h. Following the extraction process, the reaction mixture was subjected to silica gel column chromatography using an ethyl acetate/hexane solvent system. Compound 21 and ethyl 1-methyl-5-nitroimidazole-2-carboxylate (22) were readily isolated in 59% and 24% yields, respectively.

Compounds 24 and 26 were prepared from 21 according to the procedure described by Baird and Dervan [30]. Imidazole amide trimers (29, 30, and 31) bearing 3-aminopropyl, 4-aminobutyl, and 5-aminopentyl linkers, respectively, were synthesized via coupling of 24 and 26 to give imidazole amide dimer 27, deprotection of the Boc group of 27, coupling with 23 to give imidazole amide trimer 28, hydrolysis of the ester moiety of 28, and condensation with 3-aminopropyl, 4-aminobutyl, and 5-aminopentyl linker reagents (9, 10 and 11), respectively. Conjugates ON 3 (, 4, and 5) were synthesized using imidazole amide trimers (29, 30, and 31) to yield 21.8, 20.7, and 28.7 A₂₆₀ units, respectively, from 16 (B = C^Bz) (2 μmol).

Conjugates ON 3 (, 4, and 5) were converted into modified dsDNAs by annealing with complementary DNA.The stability of modified dsDNAs was investigated by and CD analyses as described above. ON 3 () formed more stable dsDNA with complementary DNA (°C, °C) compared with ON 3 ((, °C, °C) and (, °C, °C)) (Table 2). Moreover, it was determined that the imidazole amide moiety of ON 3 () was bound in the minor groove of dsDNA, since an induced CD band of the imidazole amide moiety centered at 314 nm was observed (Figure 4(I)) [5, 6, 13]. Although ON 3 () formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of ON 3 () was lower compared with the pyrrole amide moiety of ON 2 () (°C, Table 1)]. The DNA sequence recognition ability of ON 3 () was investigated using two single-base mismatch DNAs (-d(GCCTGaGACCG)- (mismatch base, a: adenine), which have a mismatch base in the sequence recognized by the imidazole amide moiety, and -d(GCCTGGGACtG)- (mismatch base, t: thymine)) (Table 2). ON 3 () formed modified dsDNA with two single-base mismatch DNAs and showed the same stabilization (°C, °C, and °C, °C) given the low DNA sequence recognition ability of the imidazole amide moiety.

Table 2 

values of modified dsDNAs and respective values.

Figure 4 

CD spectra of unmodified and modified dsDNAs. modified DNA: -d(CGGACCCTGGC)-, ON 3 (G = Im₃-NH(CH₂)_n-G) and ON 4 (G = Py₃-γ-Im₃-NH(CH₂)_n-G).

The MGB polyamide hairpin motifs that link the side-by-side MGB polyamides using the γ-aminobutyric acid (GABA) linker to favor the heterodimeric binding site have been reported by Dervan et al. [29–31]. A code for the binding of MGB polyamide hairpin motifs has been proposed wherein Py/Im, Im/Py, Hp (3-hydroxy-1-methylpyrrole)/Py and Py/Hp combinations recognize C-G, G-C, T-A, and A-T base pairs, respectively [39–43]. MGB polyamide hairpin motifs can recognize many different sequences of dsDNA and bind in the minor groove of dsDNA according to a set of pairing rules. Novopashina et al. and Boutorine et al. have reported that oligonucleotides conjugated with MGB polyamide hairpin motifs to either the - or -end formed stable dsDNA with target DNA by sequence-specific dsDNA stabilization of MGB polyamide hairpin motifs [16–21].

As a further study examining stabilization and recognition abilities, modified DNAs ON 4 (, 3, and 4) (-d(CGGACCCTGGC)-: G = Py₃-γ-Im₃-NH(CH₂)_n-G) with conjugated pyrrole-imidazole polyamide hairpin motifs, which recognize C-G base pairs via a pyrrole/imidazole combination, at the 2-exocyclic amino group of a guanine base were synthesized and evaluated (Figure 2). Pyrrole-γ-imidazole polyamide derivatives (35, 36, and 37) were synthesized as shown in Scheme 6. Pyrrole trimer 32 was synthesized via hydrolysis of the ester moiety of pyrrole trimer 6 and coupling with ethyl 4-aminobutanoate. Pyrrole trimer 32 was converted into the carboxylic acid compound. Imidazole trimer 33 was synthesized via removal of the Boc group of imidazole dimer 27 and coupling of imidazole monomer 26. The Boc group of imidazole trimer 33 was removed and then coupled with the carboxylic acid compound to give pyrrole-imidazole amide 34. Following hydrolysis of the ester moiety of 34, 2-aminoethyl, 3-aminopropyl, and 4-aminobutyl linker reagents (8, 9, and 10) were coupled to give pyrrole-γ-imidazole amide derivatives (35, 36, and 37), respectively. Using pyrrole-γ-imidazole amide derivatives (35, 36, and 37), conjugates ON 4 (, 3, and 4) were synthesized by a postsynthetic modification method to yield 8.4, 11.0, and 11.3 A₂₆₀ units, respectively, from 16 (^z) (1 μmol) (Scheme 4).

Scheme 6 

Synthesis of pyrrole-γ-imidazole polyamide derivatives. Reagents and conditions: i 1 M NaOH aq./EtOH/pyridine, rt; Dowex 50 WX8 (H⁺-form). ii H₂N(CH₂)₃CO₂Et, EDCI, DMAP, CH₂Cl₂, rt, 32 (82%). iii AcCl, CH₃OH, rt–40°C. iv 26, EDCI, DMAP, CH₂Cl₂, rt, 33 (70%). v EDCI, DMAP, CH₂Cl₂, rt, 34 (57%). vi 8, 9 or 10, EDCI, DMAP, CH₂Cl₂, rt, 35 (55%); 36 (53%); 37 (27%).

The DNA binding ability of conjugates ON 4 (, 3, and 4) were investigated by analysis (Table 2). It was found that modified dsDNAs comprising ON 4 (, 3, and 4)/complementary DNA possessed higher stability compared with modified dsDNA comprising ON 3 ()/complementary DNA (°C), and that ON 4 () formed the most stable dsDNA with complementary DNA ( ̊C, °C). Furthermore, we attempted an examination of a modified oligonucleotide using the aminomethyl linker, although the modified oligonucleotide was not synthesized by the same synthetic procedure. From the CD spectra, it was determined that the pyrrole-γ-imadazole amide moiety of ON 4 () was bound in the minor groove of dsDNA (Figure 4(II)) [5, 6, 13].

The DNA sequence recognition ability of ON 4 (n =2) was investigated using two single-base mismatch DNAs [-d(GCCTGaGACCG)- and -d(GCCTGGGACtG)-] (Table 2). It was found that ON 4 () possessed higher DNA sequence recognition ability, since the mismatch dsDNA [ON 4 ()/-d(GCCTGGGACtG)-, °C, °C] possessed higher stability compared with the mismatch dsDNA (ON 4 ()/-d(GCCTGaGACCG)-, °C. °C].

4. Conclusions

We synthesized MGB polyamide-oligonucleotide conjugates with linked MGB polyamides at the 2-exocyclic amino group of a guanine base using various aminoalkyl linkers by a postsynthetic modification method and evaluated the binding affinity for complementary DNA that included the MGB polyamide binding sequence by and CD analyses. The MGB polyamides comprised pyrrole polyamides (Py₄- and Py₃-), which possess binding affinity for A-T base pairs, and imidazole (Im₃-) and pyrrole-γ-imidazole (Py₃-γ-Im₃-) polyamide hairpin motifs, which possess binding affinity for C-G base pairs. It was found that the stability of the modified dsDNA was greatly influenced by the linker length. Py₄- and Py₃-oligonucleotide conjugates (ON 1 () and ON 2 ()] containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA via binding of the MGB polyamide moiety. Although Im₃-oligonucleotide conjugate ON 3 () containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of ON 3 () was lower compared with the pyrrole amide moiety of ON 2 (). The Py₃-γ-Im₃-oligonucleotide conjugates ON 4 (), which possesses binding affinity for C-G base pairs via a pyrrole/imidazole combination, and contains a 2-aminoethyl linker, showed high binding ability for complementary DNA.

Furthermore, using single-base mismatch DNA, which possess a mismatch base in the pyrrole polyamide binding sequence, and 2-base mismatch DNA, which does not form dsDNA with unmodified DNA, the DNA sequence recognition of conjugates ON 1 () and ON 2 () was investigated by analysis. ON 1 () formed dsDNA with single-base mismatch DNA and resulted in stabilization of the dsDNA. In the case of 2-base mismatch DNA, ON 1 () did not form dsDNA and the pyrrole amide moiety displayed no activity. Examination of ON 2 (), containing a pyrrole amide moiety with short chain length, showed the same results as ON 1 (). However, from the result of 2-base mismatch DNA, it was thought that modified DNA conjugates did not act on single-base mismatch DNA under conditions where dsDNA (unmodified DNA/single-base mismatch DNA) does not form. On the other hand, the DNA sequence recognition of conjugates ON 3 () and ON 4 () was investigated by analysis using two single-base mismatch DNAs in lieu of complementary DNA. Stabilization of the duplex was observed in dsDNAs comprising ON 3 () and single-base mismatch DNA, which possess a mismatch base in the imidazole polyamide binding sequence. ON 4 () showed high sequence recognition ability for DNA that included the binding sequence of the pyrrole-γ-imidazole polyamide hairpin motif. A binding code has been proposed for MGB polyamide hairpin motifs whereby Py/Im, Im/Py, Hp/Py, and Py/Hp combinations recognize C-G, G-C, T-A, and A-T base pairs, respectively [39–43]. MGB polyamide hairpin motif-oligonucleotide conjugates may be utilized to act on dsDNA of various sequences.

It is expected that these results could lead to the development of effective gene expression control compounds and novel anticancer and/or antiviral nucleoside drugs.

Data Availability

Supporting data for the results of this report are available in the provided supplementary materials.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgments

The authors would like to thank Dr. Yasuo Shida and Mr. Haruhiko Fukaya for the elemental analyses and mass measurements (Analytical Center, Tokyo University of Pharmacy and Life Sciences).

Supplementary Materials

Figure S1: mass, ¹H-NMR, and ¹³C-NMR spectra of the synthesized compounds. Figure S2: UV spectra of MGB amide compounds 4, 6, 28, and 34. Figure S3: HPLC charts of MGB polyamide-oligonucleotide conjugates. Figure S4: mass spectra of MGB polyamide-oligonucleotide conjugates. Figure S5: UV melting curves of modified dsDNAs. Figure S6: CD spectra of modified dsDNAs. (Supplementary Materials)