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

Authors: Kazuo Kamaike; Mutsumi Sano; Daisuke Sakata; Yu Nishihara; Hiroaki Amino; Akihiro Ohtsuki; Yui Okada; Takafumi Miyakawa; Makoto Kogawara; Mai Tsutsumi; Misato Takahashi; Etsuko Kawashima; Koichiro Ota; Hiroaki Miyaoka
Journal: Journal of Nucleic Acids
 (2023)

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## Abstract

MGB polyamide-oligonucleotide conjugatesON 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 Tm 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 (n=4) and ON 2 (n=4)) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA. Although Im3-oligonucleotide conjugate ON 3 (n=4) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of ON 3 (n=4) was lower compared with the pyrrole amide moiety of ON 2 (n=4). The Py3-γ-Im3-oligonucleotide conjugate ON 4 (n=2), 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 (n=2) showed high sequence recognition ability for complementary DNA.


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## Body

## 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 5′-d(AATTT)-3′/3′-(TTAAA)-5′ 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 (Tm) 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 (n=3) 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 (n=3) 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 5′- or 3′-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 (n=3), which includes a modified guanosine (G) in the 5′ direction in the oligonucleotide chain given the preferred orientation of the polyamide in the minor groove of dsDNA (C-(pyrrole polyamide)-N/3′-(TTAAA)-5′ of the complementary target DNA) [22–24], was synthesized and evaluated as a model oligonucleotide [13]. From the Tm and CD analyses, it was found that ON 1 (n=3) 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-2′-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 5′-phosphate group, which is a suitable substrate for biosynthesis, exhibited the highest inhibition.Figure 1
Structures of distamycin A, hybrids1, 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-2′-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 (n=3), 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 conjugatesON 1-4 using various aminoalkyl linkers.


## 2. Materials and Methods

Column chromatography was performed on silica gel (Kanto Chemical Silica gel N60, spherical, natural, 40-50μm). 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 (3.9mmID×150mmL) 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 2′-deoxynucleoside 16 (B = CBz) and 2′-deoxynucleoside 3′-phosphoramidites 17 (B = T, CBz, ABz, and GiBu) were purchased from Glen Research Corporation. 2′-Deoxy-2-fluoroinosine 3′-phosphoramidite 17 (B = IF,NPE) was prepared from 2′-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)

Compound2 [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. (150mL×3), 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, J=1.9 Hz, Py-H), 7.23 (d, 1H, J=1.8 Hz, Py-H), 7.05 (d, 1H, J=1.9 Hz, Py-H), 6.94-6.90 (m, 3H, Py−H̲×3), 6.06 (dd, 1H, J=2.6, 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) m/z: calcd for C19H22N5O4(M+H)+ 384.1672, found 384.1667; UV (CH3OH): λmax299, 238 nm, λmin260, and 222 nm; and ε2601.8×104.


### 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)

Compounds8, 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 v/v) solvent system to give 8 (0.19 g, 20% yield) as a white powder. 1H-NMR (CD3OD): δ 7.80 (d, 2H, J=7.5 Hz, Ar-H of the Fmoc group), 7.65 (d, 2H, J=7.5 Hz, Ar-H of the Fmoc group), 7.40 (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), 4.42 (d, 2H, J=6.6 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, J=6.0 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) m/z: calcd for C17H19N2O2(M + H)+ 283.1447, found 283.1455Compound 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 v/v) solvent system to give 10 (2.11 g, 61% yield) as a white powder. 1H-NMR (CD3OD): δ 7.79 (d, 2H, J=7.5 Hz, Ar-H of the Fmoc group), 7.64 (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=7.3 Hz, Ar-H of the Fmoc group), 4.37 (d, 2H, J=6.8 Hz, CHCH2 of the Fmoc group), 4.20 (t, 1H, J=6.8 Hz, CHCH2 of the Fmoc group), 3.15 (t, 2H, J=6.6 Hz, NCH2), 2.93 (t, 2H, J=7.4 Hz, NCH2), and 1.66-1.55 (m, 4H, CH̲2×2); 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) m/z: calcd for C19H23N2O2(M + H)+ 311.1760, found 311.1752Compound 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 v/v) solvent system to give 11 (1.64 g, 45% yield) as a white powder. 1H-NMR (CD3OD): δ 7.76 (d, 2H, J=7.5 Hz, Ar-H of the Fmoc group), 7.60 (d, 2H, J=7.4 Hz, Ar-H of the Fmoc group), 7.36 (t, 2H, J=7.4 Hz, Ar-H of the Fmoc group), 7.26 (t, 2H, J=7.4 Hz, Ar-H of the Fmoc group), 4.33 (d, 2H, J=6.8 Hz, CHCH2 of the Fmoc group), 4.17 (t, 1H, J=6.8 Hz, CHCH2 of the Fmoc group), 3.08 (t, 2H, J=6.9 Hz, NCH2), 2.87 (t, 2H, J=7.6 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) m/z: 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)

Compounds13, 14, and 15 were prepared according to the synthetic procedure of 12 [10–13].Pyrrole amide tetramer4 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, J=1.8 Hz, and Py-H), 7.25-7.24 (m, 2H, and Py-H × 2), 7.08 (d, 1H, J=1.8 Hz, and Py-H), 7.06 (d, 1H, J=1.8 Hz, and Py-H), 6.95-6.91, (m, 3H, and Py-H × 3), 6.06 (dd, 1H, J=2.6 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), 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) m/z: calcd for C25H28N7O5(M + H)+ 506.2152, found 506.2151; UV (CH3OH): λmax 306, 238 nm; λmin 262, 223 nm; and ε2602.6×104.Pyrrole amide tetramer4 (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, J=1.9 Hz, and Py-H), 7.25-7.24 (m, 2H, and Py-H × 2), 7.07 (d, 1H, J=1.8 Hz, and Py-H), 7.05 (d, 1H, J=1.8 Hz, and Py-H), 6.95 (d, 1H, J=2.2 Hz, and Py-H), 6.93-6.91 (m, 1H, and Py-H), 6.85 (d, 1H, J=1.9 Hz, and Py-H), 6.06 (dd, 1H, J=2.6 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) m/z: 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.69 (d, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.40 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.30-7.23 (m, 3H, CONHCH2, and Py-H × 2), 7.18 (d, 1H, J=1.8 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, J=2.5 Hz, J =3.9 Hz, and Py-H), 4.29 (d, 2H, J=6.9 Hz, and CHCH2 of the Fmoc group), 4.22-4.19 (t, 1H, J=6.9 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) m/z: 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, J=7.5 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.5 Hz, and Ar-H of the Fmoc group), 7.41 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.4 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, J=2.6 Hz, J=3.9 Hz, and Py-H), 4.29 (d, 2H, J=6.9 Hz, and CHCH2 of the Fmoc group), 4.21 (t, 1H, J=6.9 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) m/z: 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 trimer6 (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, J=1.9 Hz, and Py-H), 7.26 (d, 1H, J=1.8 Hz, and Py-H), 7.08 (d, 1H, J=1.8 Hz, and Py-H), 6.95-6.94 (m, 1H, and Py-H), 6.92 (dd, 1H, J=1.7, and 3.9 Hz Py-H), 6.85 (d, 1H, J=1.9 Hz, and Py-H), 6.06 (dd, 1H, J=2.5, 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) m/z: 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.40 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.30-7.28 (m, 1H, and CONH), 7.23 (d, 1H, J=1.8 Hz, and Py-H), 7.17 (d, 1H, J=1.8 Hz, and Py-H), 7.03 (d, 1H, J=1.8 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, J=1.6 Hz, and Py-H), 6.06 (dd, 1H, J=2.5, 3.8 Hz, and Py-H), 4.29 (d, 2H, J=6.8 Hz, and CHCH2 of the Fmoc group), 4.20 (t, 1H, J=6.8 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) m/z: 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 v/v) solvent system to give 21 (2.28 g, 59% yield) and ethyl 1-methyl-5-nitroimidazole-2-carboxylate (22) (920 mg, 24% yield).Compound20: Rf 0.20 (ethyl acetate/hexane (2 : 1 v/v) solvent system)Compound21 (white powder): Rf 0.42 (ethyl acetate/hexane (2 : 1 v/v) solvent system), 1H-NMR (DMSO-d6): δ 8.63 (s, 1H, and Im-H), 4.35 (q, 2H, J=7.1 Hz, and CH2CH3), 3.99 (s, 3H, and NCH3), and 1.33 (t, 3H, J=7.1 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) m/z: 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, 21.09Compound22 (white powder): Rf 0.49 (ethyl acetate/hexane (2 : 1 v/v) solvent system), 1H-NMR (DMSO-d6): δ 8.15 (s, 1H, and Im-H), 4.38 (q, 2H, J=7.1 Hz, and OCH2CH3), 4.19 (s, 3H, and NCH3), and 1.33 (t, 3H, J=7.1 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) m/z: 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) m/z: 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, J=7.1 Hz, and OCH2CH3), 3.92 (3H, s, and NCH3), 2.96 (brs, NH2, and 2H), and 1.40 (3H, t, J=7.1 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) m/z: calcd for C7H12N3O2(M + H)+ 170.0930, found 170.0938.


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

Compound24 (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 (100mL×3). 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 v/v) 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, J=7.2 Hz, and OCH2CH3), 3.88 (s, 3H, and NCH3), 1.44 (s, 9H, and OC(CH3)3), and 1.28 (t, 3H, J=7.2 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) m/z; 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)

Compound25 (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) m/z: 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)

Compounds24 (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. (100mL×2) 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 v/v) 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, J=7.2 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, J=7.2 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) m/z: 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)

Compound27 (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, J=7.1 Hz, and OCH2CH3), 3.99 (s, 3H, and NCH3), 3.94 (s, 3H, and NCH3), and 1.29 (t, 3H, J=7.1 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) m/z: 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. (100mL×2) 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 v/v) 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, J=7.1 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 J=7.1 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) m/z: calcd for C17H21N8O4(M + H)+ 401.1686, found 401.1682; UV (CH3OH): λmax311nm, λmin236nm, ε2601.2×104.


### 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)

Compound28 (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) m/z: 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 (50mL×3). 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, J=7.4 Hz, and Ar − H × 2 of the Fmoc group), 7.69 (d, 2H, J=7.4 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, J=7.4 Hz, and Ar-H × 2 of the Fmoc group), 7.34 (t, 2H, J=7.4 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, J=6.9 Hz, and CHCH2 of the Fmoc group), 4.22 (t, 1H, J=6.9 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) m/z: 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 (100mL×3). 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, J=7.5 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.5 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, J=7.5 Hz, and Ar-H of the Fmoc group), 7.33 (t, 2H, J=7.5 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, J=6.8 Hz, and CHCH2 of the Fmoc group), 4.20 (t, 1H, J=6.8 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) m/z: 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 (50mL×3). 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.67 (d, 2H, J=7.4 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, J=7.5 Hz, and Ar-H of the Fmoc group), 7.33 (t, 2H, J=7.5 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, J=6.9 Hz, and CHCH2 of the Fmoc group), 4.19 (t, 1H, J=6.9 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 CH2×2), 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) m/z: 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 trimer6 (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. (80mL×2), 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 v/v) 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, J=1.8 Hz, and Py-H), 7.12 (d, 1H, J=1.8 Hz, and Py-H), 6.78-6.77 (m, 1H, and Py-H), 6.71 (d, 1H, J=1.8 Hz, Py-H), 6.68-6.66 (m, 1H, and Py-H), 6.52 (d, 1H, J=1.9 Hz, and Py-H), 6.24-6,22 (m, 1H, and CONH), 6.13 (dd, 1H, J=2.6 Hz, J =4.0 Hz, and Py-H), 4.15 (q, 2H,J=7.2 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, J=7.1 Hz, and COCH2CH2-), 1.95-1.88 (m, 2H, and -CH2CH2CH2-), and 1.25 (t, 3H, J=7.2 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) m/z: 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)

Compound27 (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. (100mL×2) 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 v/v) 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, J=7.1 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, J=7.1 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) m/z: 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)

Compound32 (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, J=1.8 Hz, and Py-H), 7.18 (d, 1H, J=1.8 Hz, and Py-H), 7.02 (d, 1H, J=1.9 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, J=1.9 Hz, and Py-H), 6.06 (dd, 1H, J=2.6 Hz, J=3.9 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, J=7.4 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) m/z: calcd for C22H27N6O5(M + H)+ 455.2043, found 455.2039.Compound33 (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, J=7.1 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, J=7.1 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) m/z: 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. (20mL×2) 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, J=1.5 Hz, and Py-H), 7.18 (d, 1H, J=1.5 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, J=7.1Hz, 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, J=6.7 Hz, and COCH2CH2-), 2.07-2.04 (m, 2H, -CH2CH2CH2-), and 1.40 (t, 3H, J=7.1 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) m/z: calcd for C39H46N15O8(M + H)+ 852.3654, found 852.3651; UV (CH3OH): λmax300nm, λmin257nm, ε2603.0×104.


### 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)

Compound34 (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, J=1.5 Hz, and Py-H), 7.19 (d, 1H, J=1.5 Hz, and Py-H), 7.06 (d, 1H, J=1.7 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, J=1.7 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, J=7.2 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) m/z: 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. (20mL×2), 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.66 (d, 2H, J=7.4 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.29 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.22 (d, 1H, J=1.7 Hz, and Py-H), 7.18 (d, 1H, J=1.6 Hz, and Py-H), 7.04 (d, 1H, J=1.6 Hz, and Py-H), 6.94 (s, 1H, and Py-H), 6.92-6.91 (m, 1H, and Py-H), 6.89 (d, 1H, J=1.9 Hz, and Py-H), 6.05 (dd, J=2.6 Hz, J=3.8 Hz, 1H, and Py-H), 4.28 (d, 2H, J=7.0 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, J=6.5 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) m/z: 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. (20mL×2), 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.4 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.30-7.27 (1H, m, and CONH), 7.22 (d, 1H, J=1.7 Hz, and Py-H), 7.17 (d, 1H, J=1.7 Hz, and Py-H), 7.04 (d, 1H, J=1.7 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, J=1.7 Hz, and Py-H), 6.05 (dd, J=2.5 Hz, J=3.9 Hz, 1H, and Py-H), 4.31 (d, 2H, J=6.8 Hz, and CHCH2 of the Fmoc group), 4.21(t, 1H, J=6.8 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 NHCH2CH2-), 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) m/z: 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. (20mL×2) 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.4 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, J=7.1 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.1 Hz, and Ar-H of the Fmoc group), 7.29-7.27 (1H, m, and CONH), 7.23 (d, 1H, J=1.8 Hz, and Py-H), 7.18 (d, 1H, J=1.8 Hz, and Py-H), 7.04 (d, 1H, J=1.8 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, J=1.8 Hz, and Py-H), 6.05 (dd, 1H, J=2.6 Hz, J=3.9 Hz, and Py-H), 4.29 (d, 2H, J=6.8 Hz, CHCH2 of the Fmoc group), 4.19 (t, 1H, J=6.8 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) m/z: calcd for C56H62N17O9 (M + H)+ 1116.1926, found 1116.4918.


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

ConjugatesON 1-4 were synthesized by the postsynthetic modification method as previously described for the synthesis of ON 1 (n=3) [13].CPG support-bound oligonucleotide18 (11-mer: 5′-d(CGIF,NPEAATTTGGC)-3′ or 5′-d(CGIF,NPEACCCTGGC)-3′) was synthesized using a syringe-based system. CPG support-bound 2′-deoxynucleoside 16 (B=CBz, 1000 Å, purchased from Applied Biosystems Pty Ltd.) (2 μmol) was treated with 3% w/v Cl3CCO2H in dichloromethane (1.0mL×2) for 1 min, followed by washing with acetonitrile (2.0mL×2). A 0.1 M solution of 2′-deoxynucleoside 3′-phosphoramidite 17 (B=GiBu) 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 (2.0mL×2) 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 (2.0mL×2). 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 (2.0mL×2) to give the CPG support-bound phosphorotriester dimer. Following chain elongation using 2′-deoxynucleoside 3′-phosphoramidites 17 (B=T, CBz, ABz, GiBu, and IF,NPE), as described above, the terminal DMTr protecting group of the oligonucleotide was removed by treatment with 3% w/v Cl3CCO2H in dichloromethane (1.0mL×2) for 1 min, and the CPG support was washed with acetonitrile (2.0mL×2). 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 (2.0mL×2). 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 (2.0mL×2). The generated oligomer was then cleaved from the CPG support by treatment with conc. NH4OH (1.5mL×2) 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 (5.0mL×3), and the aqueous layer was evaporated. The MGB polyamide-oligonucleotide conjugates ON 1-4 were then purified by reversed-phase HPLC.ON 1 (5′-d(CGGAATTTGGC)-3′, G = Py4-NH(CH2)n-G (n=3−5)).ON 1 (n=3) yields 48.8 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C135H168N50O69P10(M +2H)2+ 1951.4276, found 1951.4158. ON 1 (n=4): yields 34.0 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C136H170N50O69P10(M +2H)2+ 1958.4353, found 1958.4075. ON 1 (n=5): yields 31.4 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C137H173N50O69P10(M +3H)3+ 1310.6314, found 1310.5460.ON 2 (5′-d(CGGAATTTGGC)-3′, G = Py3-NH(CH2)n-G (n=4).ON 2 (n=4) yields 22.5 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C130H165N48O68P10 (M +3H)3+ 1265.2768, found 1265.2252.ON 3 (5′-d(CGGACCCTGGC)-3′, G = Im3-NH(CH2)n-G (n=3−5)).ON 3 (n =3) yields 21.8 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C123H156N51O67P10 (M + H)+ 3728.7744, found 3728.7866. ON 3 (n=4) yields 20.7 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C124H158N51O67P10 (M + H)+ 3742.7900, found 3742.7827. ON 3 (n=5) yields 28.7 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C125H160N51O67P10 (M + H)+ 3756.8057, found 3756.8152.ON 4 (5′-d(CGGACCCTGGC)-3′: G = Py3-γ-Im3-NH(CH2)n-G (n=2−4)).ON 4 (n=2) yields 8.4 A260 units from 16 (B = CBz) (1 μmol). HRMS (ESI-TOF) m/z calcd for C144H179N58O71P10 (M + H)+ 4165.9555, found 4165.9580. ON 4 (n=3) yields 11.0 A260 units from 16 (B = CBz) (1 μmol). HRMS (ESI-TOF) m/z calcd for C145H181N58O71P10 (M + H)+ 4179.9712, found 4179.9858. ON 4 (n=4) yields 11.3 A260 units from 16 (B = CBz) (1 μmol). HRMS (ESI-TOF) m/z 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, Tm values were obtained using a TMSPC-8 system with Tm 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].


## 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)

Compound2 [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. (150mL×3), 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, J=1.9 Hz, Py-H), 7.23 (d, 1H, J=1.8 Hz, Py-H), 7.05 (d, 1H, J=1.9 Hz, Py-H), 6.94-6.90 (m, 3H, Py−H̲×3), 6.06 (dd, 1H, J=2.6, 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) m/z: calcd for C19H22N5O4(M+H)+ 384.1672, found 384.1667; UV (CH3OH): λmax299, 238 nm, λmin260, and 222 nm; and ε2601.8×104.


## 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)

Compounds8, 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 v/v) solvent system to give 8 (0.19 g, 20% yield) as a white powder. 1H-NMR (CD3OD): δ 7.80 (d, 2H, J=7.5 Hz, Ar-H of the Fmoc group), 7.65 (d, 2H, J=7.5 Hz, Ar-H of the Fmoc group), 7.40 (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), 4.42 (d, 2H, J=6.6 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, J=6.0 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) m/z: calcd for C17H19N2O2(M + H)+ 283.1447, found 283.1455Compound 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 v/v) solvent system to give 10 (2.11 g, 61% yield) as a white powder. 1H-NMR (CD3OD): δ 7.79 (d, 2H, J=7.5 Hz, Ar-H of the Fmoc group), 7.64 (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=7.3 Hz, Ar-H of the Fmoc group), 4.37 (d, 2H, J=6.8 Hz, CHCH2 of the Fmoc group), 4.20 (t, 1H, J=6.8 Hz, CHCH2 of the Fmoc group), 3.15 (t, 2H, J=6.6 Hz, NCH2), 2.93 (t, 2H, J=7.4 Hz, NCH2), and 1.66-1.55 (m, 4H, CH̲2×2); 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) m/z: calcd for C19H23N2O2(M + H)+ 311.1760, found 311.1752Compound 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 v/v) solvent system to give 11 (1.64 g, 45% yield) as a white powder. 1H-NMR (CD3OD): δ 7.76 (d, 2H, J=7.5 Hz, Ar-H of the Fmoc group), 7.60 (d, 2H, J=7.4 Hz, Ar-H of the Fmoc group), 7.36 (t, 2H, J=7.4 Hz, Ar-H of the Fmoc group), 7.26 (t, 2H, J=7.4 Hz, Ar-H of the Fmoc group), 4.33 (d, 2H, J=6.8 Hz, CHCH2 of the Fmoc group), 4.17 (t, 1H, J=6.8 Hz, CHCH2 of the Fmoc group), 3.08 (t, 2H, J=6.9 Hz, NCH2), 2.87 (t, 2H, J=7.6 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) m/z: 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)

Compounds13, 14, and 15 were prepared according to the synthetic procedure of 12 [10–13].Pyrrole amide tetramer4 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, J=1.8 Hz, and Py-H), 7.25-7.24 (m, 2H, and Py-H × 2), 7.08 (d, 1H, J=1.8 Hz, and Py-H), 7.06 (d, 1H, J=1.8 Hz, and Py-H), 6.95-6.91, (m, 3H, and Py-H × 3), 6.06 (dd, 1H, J=2.6 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), 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) m/z: calcd for C25H28N7O5(M + H)+ 506.2152, found 506.2151; UV (CH3OH): λmax 306, 238 nm; λmin 262, 223 nm; and ε2602.6×104.Pyrrole amide tetramer4 (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, J=1.9 Hz, and Py-H), 7.25-7.24 (m, 2H, and Py-H × 2), 7.07 (d, 1H, J=1.8 Hz, and Py-H), 7.05 (d, 1H, J=1.8 Hz, and Py-H), 6.95 (d, 1H, J=2.2 Hz, and Py-H), 6.93-6.91 (m, 1H, and Py-H), 6.85 (d, 1H, J=1.9 Hz, and Py-H), 6.06 (dd, 1H, J=2.6 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) m/z: 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.69 (d, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.40 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.30-7.23 (m, 3H, CONHCH2, and Py-H × 2), 7.18 (d, 1H, J=1.8 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, J=2.5 Hz, J =3.9 Hz, and Py-H), 4.29 (d, 2H, J=6.9 Hz, and CHCH2 of the Fmoc group), 4.22-4.19 (t, 1H, J=6.9 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) m/z: 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, J=7.5 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.5 Hz, and Ar-H of the Fmoc group), 7.41 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.4 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, J=2.6 Hz, J=3.9 Hz, and Py-H), 4.29 (d, 2H, J=6.9 Hz, and CHCH2 of the Fmoc group), 4.21 (t, 1H, J=6.9 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) m/z: 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 trimer6 (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, J=1.9 Hz, and Py-H), 7.26 (d, 1H, J=1.8 Hz, and Py-H), 7.08 (d, 1H, J=1.8 Hz, and Py-H), 6.95-6.94 (m, 1H, and Py-H), 6.92 (dd, 1H, J=1.7, and 3.9 Hz Py-H), 6.85 (d, 1H, J=1.9 Hz, and Py-H), 6.06 (dd, 1H, J=2.5, 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) m/z: 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.40 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.30-7.28 (m, 1H, and CONH), 7.23 (d, 1H, J=1.8 Hz, and Py-H), 7.17 (d, 1H, J=1.8 Hz, and Py-H), 7.03 (d, 1H, J=1.8 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, J=1.6 Hz, and Py-H), 6.06 (dd, 1H, J=2.5, 3.8 Hz, and Py-H), 4.29 (d, 2H, J=6.8 Hz, and CHCH2 of the Fmoc group), 4.20 (t, 1H, J=6.8 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) m/z: 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 v/v) solvent system to give 21 (2.28 g, 59% yield) and ethyl 1-methyl-5-nitroimidazole-2-carboxylate (22) (920 mg, 24% yield).Compound20: Rf 0.20 (ethyl acetate/hexane (2 : 1 v/v) solvent system)Compound21 (white powder): Rf 0.42 (ethyl acetate/hexane (2 : 1 v/v) solvent system), 1H-NMR (DMSO-d6): δ 8.63 (s, 1H, and Im-H), 4.35 (q, 2H, J=7.1 Hz, and CH2CH3), 3.99 (s, 3H, and NCH3), and 1.33 (t, 3H, J=7.1 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) m/z: 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, 21.09Compound22 (white powder): Rf 0.49 (ethyl acetate/hexane (2 : 1 v/v) solvent system), 1H-NMR (DMSO-d6): δ 8.15 (s, 1H, and Im-H), 4.38 (q, 2H, J=7.1 Hz, and OCH2CH3), 4.19 (s, 3H, and NCH3), and 1.33 (t, 3H, J=7.1 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) m/z: 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) m/z: 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, J=7.1 Hz, and OCH2CH3), 3.92 (3H, s, and NCH3), 2.96 (brs, NH2, and 2H), and 1.40 (3H, t, J=7.1 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) m/z: calcd for C7H12N3O2(M + H)+ 170.0930, found 170.0938.


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

Compound24 (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 (100mL×3). 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 v/v) 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, J=7.2 Hz, and OCH2CH3), 3.88 (s, 3H, and NCH3), 1.44 (s, 9H, and OC(CH3)3), and 1.28 (t, 3H, J=7.2 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) m/z; 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)

Compound25 (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) m/z: 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)

Compounds24 (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. (100mL×2) 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 v/v) 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, J=7.2 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, J=7.2 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) m/z: 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)

Compound27 (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, J=7.1 Hz, and OCH2CH3), 3.99 (s, 3H, and NCH3), 3.94 (s, 3H, and NCH3), and 1.29 (t, 3H, J=7.1 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) m/z: 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. (100mL×2) 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 v/v) 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, J=7.1 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 J=7.1 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) m/z: calcd for C17H21N8O4(M + H)+ 401.1686, found 401.1682; UV (CH3OH): λmax311nm, λmin236nm, ε2601.2×104.


## 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)

Compound28 (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) m/z: 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 (50mL×3). 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, J=7.4 Hz, and Ar − H × 2 of the Fmoc group), 7.69 (d, 2H, J=7.4 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, J=7.4 Hz, and Ar-H × 2 of the Fmoc group), 7.34 (t, 2H, J=7.4 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, J=6.9 Hz, and CHCH2 of the Fmoc group), 4.22 (t, 1H, J=6.9 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) m/z: 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 (100mL×3). 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, J=7.5 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.5 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, J=7.5 Hz, and Ar-H of the Fmoc group), 7.33 (t, 2H, J=7.5 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, J=6.8 Hz, and CHCH2 of the Fmoc group), 4.20 (t, 1H, J=6.8 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) m/z: 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 (50mL×3). 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.67 (d, 2H, J=7.4 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, J=7.5 Hz, and Ar-H of the Fmoc group), 7.33 (t, 2H, J=7.5 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, J=6.9 Hz, and CHCH2 of the Fmoc group), 4.19 (t, 1H, J=6.9 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 CH2×2), 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) m/z: 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 trimer6 (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. (80mL×2), 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 v/v) 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, J=1.8 Hz, and Py-H), 7.12 (d, 1H, J=1.8 Hz, and Py-H), 6.78-6.77 (m, 1H, and Py-H), 6.71 (d, 1H, J=1.8 Hz, Py-H), 6.68-6.66 (m, 1H, and Py-H), 6.52 (d, 1H, J=1.9 Hz, and Py-H), 6.24-6,22 (m, 1H, and CONH), 6.13 (dd, 1H, J=2.6 Hz, J =4.0 Hz, and Py-H), 4.15 (q, 2H,J=7.2 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, J=7.1 Hz, and COCH2CH2-), 1.95-1.88 (m, 2H, and -CH2CH2CH2-), and 1.25 (t, 3H, J=7.2 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) m/z: 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)

Compound27 (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. (100mL×2) 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 v/v) 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, J=7.1 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, J=7.1 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) m/z: 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)

Compound32 (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, J=1.8 Hz, and Py-H), 7.18 (d, 1H, J=1.8 Hz, and Py-H), 7.02 (d, 1H, J=1.9 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, J=1.9 Hz, and Py-H), 6.06 (dd, 1H, J=2.6 Hz, J=3.9 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, J=7.4 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) m/z: calcd for C22H27N6O5(M + H)+ 455.2043, found 455.2039.Compound33 (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, J=7.1 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, J=7.1 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) m/z: 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. (20mL×2) 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, J=1.5 Hz, and Py-H), 7.18 (d, 1H, J=1.5 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, J=7.1Hz, 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, J=6.7 Hz, and COCH2CH2-), 2.07-2.04 (m, 2H, -CH2CH2CH2-), and 1.40 (t, 3H, J=7.1 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) m/z: calcd for C39H46N15O8(M + H)+ 852.3654, found 852.3651; UV (CH3OH): λmax300nm, λmin257nm, ε2603.0×104.


## 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)

Compound34 (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, J=1.5 Hz, and Py-H), 7.19 (d, 1H, J=1.5 Hz, and Py-H), 7.06 (d, 1H, J=1.7 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, J=1.7 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, J=7.2 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) m/z: 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. (20mL×2), 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.66 (d, 2H, J=7.4 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.29 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.22 (d, 1H, J=1.7 Hz, and Py-H), 7.18 (d, 1H, J=1.6 Hz, and Py-H), 7.04 (d, 1H, J=1.6 Hz, and Py-H), 6.94 (s, 1H, and Py-H), 6.92-6.91 (m, 1H, and Py-H), 6.89 (d, 1H, J=1.9 Hz, and Py-H), 6.05 (dd, J=2.6 Hz, J=3.8 Hz, 1H, and Py-H), 4.28 (d, 2H, J=7.0 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, J=6.5 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) m/z: 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. (20mL×2), 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.4 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.4 Hz, and Ar-H of the Fmoc group), 7.30-7.27 (1H, m, and CONH), 7.22 (d, 1H, J=1.7 Hz, and Py-H), 7.17 (d, 1H, J=1.7 Hz, and Py-H), 7.04 (d, 1H, J=1.7 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, J=1.7 Hz, and Py-H), 6.05 (dd, J=2.5 Hz, J=3.9 Hz, 1H, and Py-H), 4.31 (d, 2H, J=6.8 Hz, and CHCH2 of the Fmoc group), 4.21(t, 1H, J=6.8 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 NHCH2CH2-), 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) m/z: 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. (20mL×2) 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, J=7.4 Hz, and Ar-H of the Fmoc group), 7.68 (d, 2H, J=7.4 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, J=7.1 Hz, and Ar-H of the Fmoc group), 7.32 (t, 2H, J=7.1 Hz, and Ar-H of the Fmoc group), 7.29-7.27 (1H, m, and CONH), 7.23 (d, 1H, J=1.8 Hz, and Py-H), 7.18 (d, 1H, J=1.8 Hz, and Py-H), 7.04 (d, 1H, J=1.8 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, J=1.8 Hz, and Py-H), 6.05 (dd, 1H, J=2.6 Hz, J=3.9 Hz, and Py-H), 4.29 (d, 2H, J=6.8 Hz, CHCH2 of the Fmoc group), 4.19 (t, 1H, J=6.8 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) m/z: calcd for C56H62N17O9 (M + H)+ 1116.1926, found 1116.4918.


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

ConjugatesON 1-4 were synthesized by the postsynthetic modification method as previously described for the synthesis of ON 1 (n=3) [13].CPG support-bound oligonucleotide18 (11-mer: 5′-d(CGIF,NPEAATTTGGC)-3′ or 5′-d(CGIF,NPEACCCTGGC)-3′) was synthesized using a syringe-based system. CPG support-bound 2′-deoxynucleoside 16 (B=CBz, 1000 Å, purchased from Applied Biosystems Pty Ltd.) (2 μmol) was treated with 3% w/v Cl3CCO2H in dichloromethane (1.0mL×2) for 1 min, followed by washing with acetonitrile (2.0mL×2). A 0.1 M solution of 2′-deoxynucleoside 3′-phosphoramidite 17 (B=GiBu) 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 (2.0mL×2) 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 (2.0mL×2). 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 (2.0mL×2) to give the CPG support-bound phosphorotriester dimer. Following chain elongation using 2′-deoxynucleoside 3′-phosphoramidites 17 (B=T, CBz, ABz, GiBu, and IF,NPE), as described above, the terminal DMTr protecting group of the oligonucleotide was removed by treatment with 3% w/v Cl3CCO2H in dichloromethane (1.0mL×2) for 1 min, and the CPG support was washed with acetonitrile (2.0mL×2). 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 (2.0mL×2). 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 (2.0mL×2). The generated oligomer was then cleaved from the CPG support by treatment with conc. NH4OH (1.5mL×2) 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 (5.0mL×3), and the aqueous layer was evaporated. The MGB polyamide-oligonucleotide conjugates ON 1-4 were then purified by reversed-phase HPLC.ON 1 (5′-d(CGGAATTTGGC)-3′, G = Py4-NH(CH2)n-G (n=3−5)).ON 1 (n=3) yields 48.8 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C135H168N50O69P10(M +2H)2+ 1951.4276, found 1951.4158. ON 1 (n=4): yields 34.0 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C136H170N50O69P10(M +2H)2+ 1958.4353, found 1958.4075. ON 1 (n=5): yields 31.4 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C137H173N50O69P10(M +3H)3+ 1310.6314, found 1310.5460.ON 2 (5′-d(CGGAATTTGGC)-3′, G = Py3-NH(CH2)n-G (n=4).ON 2 (n=4) yields 22.5 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C130H165N48O68P10 (M +3H)3+ 1265.2768, found 1265.2252.ON 3 (5′-d(CGGACCCTGGC)-3′, G = Im3-NH(CH2)n-G (n=3−5)).ON 3 (n =3) yields 21.8 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C123H156N51O67P10 (M + H)+ 3728.7744, found 3728.7866. ON 3 (n=4) yields 20.7 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C124H158N51O67P10 (M + H)+ 3742.7900, found 3742.7827. ON 3 (n=5) yields 28.7 A260 units from 16 (B = CBz) (2 μmol). HRMS (ESI-TOF) m/z calcd for C125H160N51O67P10 (M + H)+ 3756.8057, found 3756.8152.ON 4 (5′-d(CGGACCCTGGC)-3′: G = Py3-γ-Im3-NH(CH2)n-G (n=2−4)).ON 4 (n=2) yields 8.4 A260 units from 16 (B = CBz) (1 μmol). HRMS (ESI-TOF) m/z calcd for C144H179N58O71P10 (M + H)+ 4165.9555, found 4165.9580. ON 4 (n=3) yields 11.0 A260 units from 16 (B = CBz) (1 μmol). HRMS (ESI-TOF) m/z calcd for C145H181N58O71P10 (M + H)+ 4179.9712, found 4179.9858. ON 4 (n=4) yields 11.3 A260 units from 16 (B = CBz) (1 μmol). HRMS (ESI-TOF) m/z 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, Tm values were obtained using a TMSPC-8 system with Tm 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 (n=3, 4, and 5) (5′-d(CGGAATTTGGC)-3′: G = Py4-NH(CH2)n-G) using 3-aminopropyl [13], 4-aminobutyl, and 5-aminopentyl linkers.Pyrrole amide tetramer4 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 (n=3, 4, and 5) were synthesized by a postsynthetic modification method using 2′-deoxy-2-fluoroinosine 3′-phosphoramidite 17 (B=IF,NPE) [32–35] and pyrrole amide tetramers 12, 13, and 14 as previously described (Scheme 4) [13]. 2′-Deoxy-2-fluoroinosine (IF,NPE) was incorporated into CPG support-bound oligonucleotide 18 (5′-d(CGIF,NPEAATTTGGC)-3′) 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 (n=3, 4, and 5) were purified by reversed-phase HPLC to yield 48.8, 34.0, and 31.4 A260 units, respectively, from 16 (B=CBz) (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.ConjugatesON 1 (n=3, 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 Tm and CD analyses. From the Tm values, it was found that the stability of modified dsDNA was greatly influenced by the linker length (ON 1 (n=3, Tm=59.5°C, ΔTm=+25.4°C), (n=4, Tm=60.2°C, ΔTm=+26.1°C), and (n=5, Tm=52.9°C, ΔTm=+18.8°C)) (Table 1). ON 1 (n=4) showed high binding ability for complementary DNA, similar to ON 1 (n=3) previously reported [13]. In the CD spectrum for dsDNA [ON 1 (n=4)/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
Tm values of modified dsDNAs and respective ΔTm values.
dsDNAsComplementary DNAMismatch DNAd)3′-d(GCCTTAAACCG)-5′3′-d(GCCTTcAACCG)-5′3′-d(GCaTTcAACCG)-5′Tm(°C)b)ΔTm(°C)c)Tm(°C)b)ΔTm(°C)c)Tm(°C)b)ΔTm(°C)c)Unmodified DNA34.1—22.1—n.d.e)—Modified DNAa)ON 1 (n=3)59.5+25.4f)45.6+23.5n.d.e)—ON 1 (n=4)60.2+26.1ON 1 (n=5)52.9+18.8ON 2 (n=4)50.8+16.739.1+17.0n.d.e)—a)modified DNA: 5′-d(CGGAATTTGGC)-3′, ON 1 (G = Py4-NH(CH2)n-G); ON 2 (G = Py3-NH(CH2)n-G). b)dsDNA (4.3 μM) in 10 mM sodium phosphate buffer (pH 7.0) containing 10 mM NaCl and 0.1 mM Na2EDTA. c)ΔTm (°C) = Tm [modified dsDNA]-Tm (unmodified dsDNA). d)mismatch base: a: adenine; c: cytosine. e) n.d.: not detected. f)it was confirmed that ON 1 (n=3) formed stable dsDNA with complementary DNA [13].Figure 3
CD spectra of unmodified and modified dsDNAs. modified DNA:5′-d(CGGAATTTGGC)-3′, ON 1 (G = Py4-NH(CH2)n-G) and ON 2 (G = Py3-NH(CH2)n-G).Using single-mismatch DNA (3′-d(GCCTTcAACCG)-5′), which contains a mismatch base in the recognition sequence (5′-d(AATTT)-3′/3′-(TTAAA)-5′) of the pyrrole amide moiety, and 2-base mismatch DNA (3′-d(GCaTTcAACCG)-5′) which does not form dsDNA with the unmodified DNA, the DNA sequence recognition ability of ON 1 (n=4) was investigated by Tm analysis (Table 1). ON 1 (n=4) formed dsDNA with single-base mismatch DNA and displayed stabilization of the dsDNA (Tm=45.6°C, ΔTm=+23.5°C,) by the pyrrole amide moiety. On the other hand, ON 1 (n=4) 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 (n=4) (5′-d(CGGAATTTGGC)-5′: 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 (n=4) was purified by reversed-phase HPLC and yielded 22.5 A260 units from 16 (B=CBz) (2 μmol) (Scheme 4).The stability of the modified dsDNA ofON 2 (n=4) and complementary DNA was investigated (Table 1 and Figure 3(II)). The stability of the modified dsDNA (ON 2 (n=4)/complementary DNA: Tm=50.8°C, ΔTm=+16.7°C) was lower compared with modified dsDNA (ON 1 (n=4)/complementary DNA: Tm=60.2°C, ΔTm=+26.1°C). The DNA sequence recognition ability of ON 2 (n=4) was investigated using single- and 2-base mismatch DNAs (Table 1). ON 2 (n=4) formed dsDNA with single-base mismatch DNA and displayed stabilization of the dsDNA (Tm=39.1°C, ΔTm=+17.0°C). On the other hand, ON 2 (n=4) 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 (n=4) and ON 2 (n=4)) 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 > Tm (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 (n=3, 4, and 5) (5′-d(CGGACCCTGGC)-3′: G = Im3-NH(CH2)n-G) as model modified oligonucleotides, which form dsDNA with complementary DNA (3′-d(GCCTGGGACCG)-5′) that includes the imidazole polyamide binding sequence (Figure 2).ConjugatesON 3 (n=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.Compounds24 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 (n=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 = CBz) (2 μmol).ConjugatesON 3 (n=3, 4, and 5) were converted into modified dsDNAs by annealing with complementary DNA.The stability of modified dsDNAs was investigated by Tm and CD analyses as described above. ON 3 (n=4) formed more stable dsDNA with complementary DNA (Tm=46.5°C, ΔTm=+5.1°C) compared with ON 3 ((n=3, Tm=43.7°C, ΔTm=+2.3°C) and (n=5, Tm=45.6°C, ΔTm=+2.2°C)) (Table 2). Moreover, it was determined that the imidazole amide moiety of ON 3 (n=4) 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 (n=4) formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of ON 3 (n=4) was lower compared with the pyrrole amide moiety of ON 2 (n=4) (ΔTm=+16.7°C, Table 1)]. The DNA sequence recognition ability of ON 3 (n=4) was investigated using two single-base mismatch DNAs (3′-d(GCCTGaGACCG)-5′ (mismatch base, a: adenine), which have a mismatch base in the sequence recognized by the imidazole amide moiety, and 3′-d(GCCTGGGACtG)-5′ (mismatch base, t: thymine)) (Table 2). ON 3 (n=4) formed modified dsDNA with two single-base mismatch DNAs and showed the same stabilization (Tm=35.4°C, ΔTm=+1.5°C, and Tm=37.1°C, ΔTm=+1.6°C) given the low DNA sequence recognition ability of the imidazole amide moiety.Table 2
Tm values of modified dsDNAs and respective ΔTm values.
dsDNAsComplementary DNAMismatch DNAd)3′-d(GCCTGGGACCG)-5′3′-d(GCCTGaGACCG)-5′3′-d(GCCTGGGACtG)-5′Tm(°C) b)ΔTm(°C)c)Tm(°C)b)ΔTm(°C)c)Tm(°C)b)ΔTm(°C)c)Unmodified DNA41.4—33.9—35.5—Modified DNAa)ON 3 (n=3)43.7+2.335.4+1.537.1+1.6ON 3 (n=4)46.5+5.1ON 3 (n=5)43.6+2.2ON 4 (n=2)63.3+21.941.1+7.255.8+20.3ON 4 (n=3)53.6+12.2ON 4 (n=4)49.8+8.4a) modified DNA:5′-d(CGGACCCTGGC)-3′, ON 3 (G = Im3-NH(CH2)n-G); ON 4 (G = Py3-γ-Im3-NH(CH2)n-G). b) dsDNA (4.3 μM) in 10 mM sodium phosphate buffer (pH 7.0) containing 10 mM NaCl and 0.1 mM Na2EDTA. c) ΔTm (°C) = Tm (modified dsDNA)-Tm (unmodified dsDNA). d) mismatch base, a: adenine, t: thymine.Figure 4
CD spectra of unmodified and modified dsDNAs. modified DNA:5′-d(CGGACCCTGGC)-3′, 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 theγ-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 5′- or 3′-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 DNAsON 4 (n=2, 3, and 4) (5′-d(CGGACCCTGGC)-3′: 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 (n=2, 3, and 4) were synthesized by a postsynthetic modification method to yield 8.4, 11.0, and 11.3 A260 units, respectively, from 16 (B=CBz) (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 conjugatesON 4 (n=2, 3, and 4) were investigated by Tm analysis (Table 2). It was found that modified dsDNAs comprising ON 4 (n=2, 3, and 4)/complementary DNA possessed higher stability compared with modified dsDNA comprising ON 3 (n=4)/complementary DNA (Tm=46.5°C), and that ON 4 (n=2) formed the most stable dsDNA with complementary DNA (Tm=63.3 ̊C, ΔTm=+21.9°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 (n=2) was bound in the minor groove of dsDNA (Figure 4(II)) [5, 6, 13].The DNA sequence recognition ability ofON 4 (n =2) was investigated using two single-base mismatch DNAs [3′-d(GCCTGaGACCG)-5′ and 3′-d(GCCTGGGACtG)-5′] (Table 2). It was found that ON 4 (n=2) possessed higher DNA sequence recognition ability, since the mismatch dsDNA [ON 4 (n=2)/3′-d(GCCTGGGACtG)-5′, Tm=55.8°C, ΔTm=+20.3°C] possessed higher stability compared with the mismatch dsDNA (ON 4 (n=2)/3′-d(GCCTGaGACCG)-5′, Tm=41.1°C. ΔTm=+7.2°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 byTm 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 (n=4) and ON 2 (n=4)] containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA via binding of the MGB polyamide moiety. Although Im3-oligonucleotide conjugate ON 3 (n=4) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of ON 3 (n=4) was lower compared with the pyrrole amide moiety of ON 2 (n=4). The Py3-γ-Im3-oligonucleotide conjugates ON 4 (n=2), 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 conjugatesON 1 (n=4) and ON 2 (n=4) was investigated by Tm analysis. ON 1 (n=4) formed dsDNA with single-base mismatch DNA and resulted in stabilization of the dsDNA. In the case of 2-base mismatch DNA, ON 1 (n=4) did not form dsDNA and the pyrrole amide moiety displayed no activity. Examination of ON 2 (n=4), containing a pyrrole amide moiety with short chain length, showed the same results as ON 1 (n=4). 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 (n=4) and ON 4 (n=2) was investigated by Tm analysis using two single-base mismatch DNAs in lieu of complementary DNA. Stabilization of the duplex was observed in dsDNAs comprising ON 3 (n=4) and single-base mismatch DNA, which possess a mismatch base in the imidazole polyamide binding sequence. ON 4 (n=2) 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.



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*Source: 2447998-2023-03-14.xml*