Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage

Abstract

This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA–DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA–DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules.

Peptide nucleic acid (PNA) is a synthetic analogue of DNA in which the sugar-phosphate backbone of DNA is replaced by an artificial peptide backbone consisting of N-(2-aminoethyl)glycine units.¹⁾ PNAs hybridize to complementary sequences by the Watson–Crick base pairing rule with superior thermal stability compared to natural DNA/RNA.^2–4) This is because the neutral PNA backbone does not have the electrostatic repulsion that destabilizes DNA–DNA duplexes. The relatively rigid PNA backbone improves sequence selectivity on hybridization. PNAs are resistant to nucleases and proteases due to their unnatural backbone,⁵⁾ and are unlikely to bind to proteins.⁶⁾ These unique properties make PNAs an attractive agent for next-generation nucleic acid-based therapeutics. To improve the antisense and antigene properties of PNAs, a lot of PNA analogues have been reported, some of them possessing improved DNA/RNA binding properties.^7–10)

An approach for tailoring the hybridization behavior of PNA is to introduce substituents to the PNA backbone. Several chiral PNAs with substituents at the α-, β-, or γ-positions have been reported.⁹⁾ The effects of substituents at the α- or the γ-position have been extensively studied and revealed that γ-substitution is more effective to improve the DNA binding properties of PNAs than α-substitution.¹¹⁾ We recently reported a chiral PNA with a methyl group at the β-position (β-Me PNA) and showed that the stereochemistry of the β-carbon was critical to the hybridization ability of PNA and was strictly limited to the S-configuration.¹²⁾ Incorporation of the R-configuration monomer into PNA oligomers was detrimental to hybridization with DNA. While the introduction of a methyl group at the β-position preorganized the single-stranded PNA oligomer into a right-handed helical structure, the DNA binding ability of the β-Me PNA was slightly lower than that of unmodified PNA. To improve the DNA binding properties of β-PNA, we planned to introduce a lysine side chain at the β-position, expecting electrostatic attraction between a positively charged amino group of the PNA side chain and negatively charged phosphate of the DNA backbone.¹³⁾

Results and Discussion

Design and Synthesis of β-Lys PNA Monomers

We decided to introduce the L-lysine side chain at the β-position of the PNA backbone because our previous study revealed that β-Me PNA, in its S-configuration only, was suited for hybridizing to the complementary DNA sequence (Fig. 1). We also anticipated that the lysine side chain would be a versatile handle for attaching a variety of functional molecules without disrupting the DNA binding ability of PNAs as in the case of γ-PNA.^14,15)

Fig. 1. Structures of A) PNA, B) β-(S)-Me-PNA, and C) β-(S)-Lys PNA

Chart 1 illustrates the synthesis of chiral β-Lys PNA monomers 7 and 8. In each monomer, the amino group of the backbone was masked as an azide that can be reduced under mild conditions and should be suited for coupling with the next PNA monomer on the solid support.¹⁶⁾ The monomers were synthesized from the commercially-available Fmoc, Boc-protected L-lysinol derivative 1 derived from L-lysine. Conversion of the amino alcohol 1 to the iodide 2 using I₂, PPh₃ and imidazole (99%) followed by a displacement with NaN₃ afforded the azide 3 in 63% yield.¹⁷⁾ The Fmoc group of 3 was removed by alkaline treatment (99%) and the resulting amine 4 was alkylated with methyl bromoacetate in CH₂Cl₂ to give the protected β-Lys PNA backbone 5 in 64% yield. Coupling of the thymin-1-ylacetic acid was accomplished with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in N,N-dimethylformamide (DMF) to afford 6 in quantitative yield. Alkaline hydrolysis of the methyl ester 6 proceeded quantitatively to give β-Lys PNA monomer 7. In addition, in order to introduce functional molecules onto β-Lys PNA during solid-phase synthesis of the PNA oligomer, the Boc group of the side chain was converted to the Fmoc group that can be deprotected without affecting the azide group of the backbone. The monomer 7 was treated with 4 M HCl to remove the Boc group followed by Fmoc protection with FmocOSu to give orthogonally-protected β-Lys PNA monomer 8 in 97% yield for two steps from 7.

Chart 1. Synthesis of β-Lys PNA Monomers

Synthesis of PNA Oligomers

To evaluate the effect of a lysine side chain at the β-position on DNA binding of PNA, one or two units of the β-Lys PNA monomer 7 were incorporated into PNA oligomers using a standard manual solid-phase peptide synthesis procedure (Table 1, P4, P5). A 10-residue mixed-base PNA, P1, was used as a reference because this sequence has been well studied.^2,18) During the course of solid-phase synthesis, the azide group was reduced by 5 min of treatment with trimethylphosphine. After deprotection and cleavage from the resin by TFA treatment, each PNA was purified by reversed-phase HPLC and characterized by matrix-assisted laser desorption/ionization (MALDI)-orbitrap or electrospray ionization time-of-flight (ESI-TOF) mass spectrometry. The synthesized PNA oligomers are listed in Table 1 (sequences) and Table 2 (mass results).

Table 1. PNA Sequences

PNA		Sequencea)
P1	aeg	H-GTAGATCACT-LLys-NH2
P4	β-(S)-Lys	H-GTAGATCACT-LLys-NH2
P5	β-(S)-Lys	H-GTAGATCACT-LLys-NH2
P6	β-(S)-Lys	H-GTAGATbpyCACT-LLys-NH2
P7		bpy-GABA-eg-LLys-TCAACGTTAGCT-LLys-NH2
P8		bpy-GABA-TCAACGTTAGCT-LLys-NH2

a) Written from N to C-terminal. PNAs terminate in a lysine amide.

Table 2. Mass Results of PNA Oligomers

	Calcd	Found
P4	1463.1365	1463.1364a)
	[M+2H]2+
P5	2996.3392	2996.3407b)
	[M+H]+
P6	3215.3442	3215.4050a)
	[M+Na]+
P7	1999.3602	1999.3611a)
	[M+2H]2+
P8	1862.7763	1862.7758a)
	[M+2H]2+

a) ESI-TOF-MS. b) MALDI orbitrap.

Hybridization Studies of PNAs with Complementary DNA

To evaluate the effect of β-Lys backbone on the DNA binding ability of PNA, the melting temperatures (T_ms) of duplexes with complementary DNA in 10 mM sodium phosphate buffer containing 1 mM ethylenediaminetetraacetic acid (EDTA) were measured. The UV melting profiles of β-Lys PNA–DNA duplexes exhibited helix-to-coil transitions (Fig. 2). The T_m values were determined from the first derivatives of the UV-melting curves and are shown in Table 3. Although the cationic amino groups of β-Lys PNAs were expected to improve the thermal stability of PNA–DNA duplexes, the modified PNA with a single β-Lys PNA unit, P4, had a lower T_m value compared to unmodified PNA P1. The PNA–DNA duplex was further destabilized when two β-Lys PNA units were incorporated (Table 3, P5). The destabilization effect of β-Lys PNA units was relatively small at lower salt concentrations. For example, P4 was destabilized by 6.4°C at 1000 mM NaCl compared to P1. However, destabilization of P4 was only 1.4°C at 0 mM NaCl. This is because the electrostatic attractions between cationic PNA side chains and negatively charged phosphates of DNA were more effective at lower salt concentrations and partially compensated for the influence of steric hindrance of the side chains. We have recently reported the PNA containing three S-form β-Me PNA units (named PNA 2 in ref. 12, H-GTAGATCACT-^LLys-NH₂, T=β-Me PNA) whose T_m value was 51°C at 0 mM NaCl. Although the methyl group of β-Me PNA induced a right-handed helical structure, the induced structure did not contribute to the total hybridization stability. This is because the benefit of the helical structure was canceled by the steric hindrance arising from the methyl group. The aminobutyl group of β-Lys PNA is significantly larger than the methyl group of β-Me PNA and a marked destabilization relative to PNA 2 was anticipated. In fact, the PNA–DNA duplexes formed by P4 or P5 were less stable than that of PNA 2. However, the destabilization was small (ΔT_m=−1°C for P4, −1.6°C for P5). Such a small difference can be explained by the electrostatic effect of positively charged lysine side chains.

We compared the difference in the T_m values between fully matched PNA–DNA duplexes and the duplexes containing a single G-T mismatch sequence. As shown in Table 4, the β-PNA P4 exhibited superior sequence selectivity compared to unmodified PNA P1. This is a positive effect of steric hindrance of the lysine side chain.

Fig. 2. UV-Thermal Melting Curves of the PNA–DNA Hybrid Duplexes at 5 µM Strand Concentration

All samples were prepared in 10 mM sodium phosphate buffer containing 1 mM EDTA (pH 7.4). Thermal denaturation was monitored at 260 nm at a rate of 0.2°C per min A) 0 mM NaCl and B) 100 mM NaCl.

Table 3. T_m (°C) Values of the Hybrid Duplexes of PNA–DNA^a)

[NaCl]	0 mM	100 mM	1000 mM
P1	51.4b)	49.7	45.6
P4	50.0	44.1	39.2
P5	49.4	41.5	—

a) The concentration of each strand was 5 µM, prepared in 10 mM sodium phosphate buffer containing 1 mM EDTA (pH 7.4) and the indicated concentrations of NaCl; DNA=5′-AGTGATCTAC-3′. b) Data from ref. 12 in which P1 is named PNA 1.

Table 4. T_m (°C) Values of the Mismatched Duplexes of PNA–DNA^a)

	Fullmatch DNAb)	Mismatch DNAc)	ΔTm (°C)
P1	49.7	34.6	−15.1
P4	44.1	24.2	−19.9

a) The concentration of each strand was 5 µM, prepared in 10 mM sodium phosphate buffer containing 1 mM EDTA and 100 mM NaCl (pH 7.4). b) Fullmatch DNA=5′-AGTGATCTAC-3′. c) Mismatch DNA=5′-AGTGGTCTAC-3′.

Sequence-Specific DNA Cleavage by β-PNA Bearing Bipyridine at the Side Chain

To demonstrate the lysine side chain at the β-position as a versatile handle, we chose 2,2′-bipyridine as a functional molecule to be attached because bipyridine is known to induce DNA damage in the presence of copper ion under physiological conditions. Since 1,10-phenanthroline cleaves DNA in nuclei by using endogenous copper ions,¹⁹⁾ the same mechanism can be expected for bipyridine in living cells because of the structural similarity. With this in mind, we attached bipyridine at the lysine side chain of β-PNA (Table 1, P6). Bipyridine unit 12 was prepared as depicted in Chart 2.

Chart 2. Synthesis of Boc-Protected Bipyridine Unit

P6 was synthesized on the solid support using β-Lys PNA monomer 8. After the coupling of monomer 8, the Fmoc group of the side chain was removed by piperidine treatment followed by coupling with Boc-protected bipyridine unit 12 using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine-1-ium 3-oxide·hexafluorophosphate (HATU) as a coupling agent. The subsequent procedure was the same as described in the section of PNA oligomer synthesis. To evaluate the effect of the position at which bipyridine was attached on DNA cleavage, we additionally prepared two conjugates in which bipyridine was linked to a 12-residue mixed-base PNA oligomer at the N-terminus (Table 1, P7, P8). The difference between P7 and P8 was the linker length.

DNA Cleavage by PNAs

DNA cleavage activity of P6 was tested on a 54-mer DNA substrate D1 containing a 17 bp hairpin structure for ethidium staining. The DNA substrate for P7 and P8 was a 51-mer DNA oligomer D2 containing the same hairpin structure (Fig. 3C). Cleavage reactions were carried out by incubating the DNA substrates with the PNA conjugates at 4°C for 24 h in the presence of CuSO₄ and mercaptopropionic acid (MPA). Reaction products were analyzed by denaturing polyacrylamide gel electrophoresis (Figs. 3A, B). P6 exhibited a specific DNA cleavage activity (Fig. 3A, lane 4). In contrast, neither P7 nor P8 displayed significant DNA cleavage activities (Fig. 3B, lanes 6, 7). These results demonstrated that efficient DNA cleavage was observed only when the bipyridine unit was attached to the side chain of the β-PNA unit placed at the center of the PNA oligomer. The cleavage yield with P6 on the DNA substrate D1 was 27%. No specific cleavage was observed when the pipyridine was linked to the N-terminus of the PNA irrespective of linker structure and length. In the PNA–DNA complex formed by P7 or P8 with the DNA substrate D2, the bipyridine moiety should locate close to the DNA sequence of 5′-TCT AC-3′. Since this sequence was cleaved in β-PNA–DNA duplex (Fig. 3A), the failure of DNA cleavage by bipyridine linked to the N-terminus of PNA would not be due to the DNA sequence but was probably due to the flexibility of single-stranded region of the DNA substrate.

Fig. 3. PAGE Analysis of DNA Cleavage by (A) P6 or (B) P7 or P8. (C) DNA Substrates Used

The major products of the sequence selective cleavage by P6 were analyzed by MALDI-TOF mass spectrometry. To simplify the product analysis, a 10-mer DNA oligonucleotide D3 (5′-AGT GAT CTA C-3′) was used as a substrate for cleavage by P6. We assign the cleavage product at m/z=1212.4 as a 4-mer DNA with a 5′-phosphate terminus and the product at m/z=1605.5 as a 5-mer DNA with a 3′-phosphate terminus, respectively (Fig. 4). DNA fragments with a terminal phosphate are the most common products observed with oxidative cleavage.^20,21) Thus, these two major products indicate that P6 cleaved the 10-mer DNA through oxidative destruction of the deoxythymidine that is the fifth residue from the 3′-terminus. By linking bipyridine to the side chain of β-Lys PNA, the metal-binding ligand was forced to project along the minor groove passing close to hydrogen atoms that can be abstracted. The bipyridine can reach the deoxyribose of the deoxythymidine directly below the deoxyadenine paired to the thymine of β-Lys PNA in the 3′-direction. Molecular modeling also supported hydrogen abstraction from the deoxythymidine in the minor groove (Fig. 5).

Fig. 4. MALDI-TOF Mass Analysis of Cleavage Products

Fig. 5. Molecular Modeling of β-Lys PNA/DNA Duplex

The bipyridine unit attached to the PNA side chain projects along the minor groove.

In summary, we synthesized two new β-Lys PNA monomers, 7 and 8, and incorporated them into a 10-residue PNA sequence. PNA containing the β-Lys PNA units formed a stable hybrid duplex with complementary DNA. However, incorporation of β-Lys PNA units destabilized PNA–DNA duplexes more than unmodified PNA did. Electrostatic attractions between cationic lysine side chains of β-PNA and negatively charged phosphates of DNA can reduce the destabilization effect caused by steric hindrance of the side chains. The bipyridine-conjugated β-Lys PNA sequence selectively cleaved DNA. When bipyridine was appended at the N-terminus of an unmodified PNA, no specific cleavage was observed. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety was located within the minor groove of the PNA–DNA duplexes and cleaved DNA efficiently. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules. We hope that the β-PNAs reported here will expand the scope of application of PNAs in the areas of therapeutics and nanotechnologies.

Experimental

¹H- and ¹³C-NMR spectra were recorded on JEOL JNM-ECS 400 NMR (400 MHz) spectrometer. ¹H-NMR spectra were referenced with (CH₃)₄Si (δ 0.00 ppm) as an internal standard. ¹³C-NMR spectra were referenced with deuterated solvent (δ 77.0 ppm for CDCl₃). High resolution (HR)-MS were obtained on an LCMS-IT-TOF mass spectrometer (Shimadzu) in ESI method or MALDI DUO orbitrapXL mass spectrometer (Thermofisher Scientific). Optical rotations were measured on a JASCO-P-2200 digital polarimeter. Column chromatography was performed on silica gel 60 N (Kanto Chemical Co., Inc., 100–210 µm). HPLC was carried out on a Hitachi HPLC system consisting of the following equipment: pump, LC-2130; detector, L-2400; column, Mightysil RP-8 GP (250 mm×4.6 mm, 5 µm) for analysis and Wakopak Navi C18-5 (250 mm×10 mm) for purification. All experiments were performed under anhydrous conditions in an atmosphere of argon, unless otherwise mentioned.

(9H-Fluoren-9-yl)methyl tert-Butyl(6-azidohexane-1,5-diyl)-(S)-dicarba-mate (3)

Compound 3 was prepared according to the literature¹⁷⁾; ¹H-NMR (400 MHz, CDCl₃) δ: 1.43 (s, 9H), 1.33–1.53 (m, 6H), 3.07–3.14 (m, 2H), 3.34–3.45 (m, 2H), 3.72–3.79 (m, 1H), 4.20–4.23 (m, 1H), 4.37–4.47 (m, 2H), 4.57 (s, 1H), 4.91 (s, 1H), 7.31 (t, J=7.4 Hz, 2H), 7.40 (t, J=7.4 Hz, 2H), 7.59 (d, J=7.3 Hz, 2H), 7.76 (d, J=7.6 Hz, 2H); HR-MS (ESI) Calcd for C₂₆H₃₃N₅O₄Na [M+Na]⁺ 502.2425. Found 502.2429.

Methyl (S)-{1-Azido-6-[(tert-butoxycarbonyl)amino]hexan-2-yl}glycinate (5)

To a solution of 3 (802.7 mg, 1.67 mmol) in MeOH (30 mL) was added 2 M NaOH (6 mL, 12 mmol) at 0°C with stirring at room temperature (r.t.) for 1.5 h. H₂O (15 mL) was added and the pH was adjusted to 4 by addition of 2 M HCl followed by evaporation of MeOH. After addition of H₂O (50 mL), the aqueous phase was washed with CH₂Cl₂ (2×30 mL). The aqueous phase was then made strongly alkaline by addition of 2 M NaOH and extracted with CH₂Cl₂ (4×30 mL). The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo to afford 4 (429.6 mg, 1.67 mmol, quant.) as a colorless oil. To a solution of 4 (429.6 mg, 1.67 mmol) in CH₂Cl₂ (30 mL) was added NaHCO₃ (561 mg, 6.68 mmol) and methyl bromoacetate (0.63 mL, 6.68 mmol) at 0°C under argon. The solution was stirred at r.t. for 8 d. After addition of H₂O (60 mL), the aqueous phase was extracted with CH₂Cl₂ (3×30 mL). The organic phase was dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (hexane : EtOAc=1 : 1) to give 5 (435.2 mg, 1.32 mmol, 79%) as a colorless oil.; [α]_D²⁶+4.5 (c 1.03, CHCl₃); ¹H-NMR (400 MHz, CDCl₃) δ: 1.44 (s, 9H), 1.32–1.52 (m, 6H), 2.65–2.70 (m, 1H), 3.10–3.15 (m, 2H), 3.38 (dd, J=12.5, 4.6 Hz, 1H), 3.23 (dd, J=12.5, 6.0 Hz, 1H), 3.46 (s, 2H), 3.73 (s, 3H), 4.57 (s, 1H); ¹³C-NMR (100 MHz, CDCl₃) δ: 22.8, 28.4, 30.1, 32.0, 40.2, 48.3, 51.8, 54.4, 56.7, 79.0, 155.8, 172.6; HR-MS (ESI) Calcd for C₁₄H₂₈N₅O₄ [M+H]⁺ 330.2136. Found 330.2136.

Methyl (S)-N-{1-Azido-6-[(tert-butoxycarbonyl)amino]hexan-2-yl}-N-[2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl]glycinate (6)

To a solution of 5 (592 mg, 1.80 mmol) and thymin-1-ylacetic acid (661.9 mg, 3.59 mmol) in DMF (15 mL) was added EDCI (689 mg, 3.59 mmol) at 0°C under argon. The solution was stirred at r.t. for 18 h. After addition of H₂O (60 mL), the mixture was extracted with EtOAc (30 mL×3), washed with 1 M HCl, H₂O, saturated NaHCO₃, H₂O, and brine, then dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (hexane : EtOAc=1 : 6–0 : 1) to give 6 (930.1 mg, 1.80 mmol, quant.) as a viscous oil; ¹H-NMR (600 MHz, CDCl₃) δ: 1.43 and 1.45 (each s, total 9H), 1.27–1.59 (m, 6H), 1.91 and 1.92 (each d, J=1.1 Hz, total 3H), 3.08–3.13 (m, 2H), 3.45 (dd, J=12.9, 4.1 Hz, 0.5H), 3.51–3.52 (m, 1.5H), 3.74 and 3.83 (each s, total 3H), 3.83–3.87 and 4.01–4.04 (m, total 1H), 4.15–4.28 (m, 2H), 4.50–4.58 (m, 1.5H), 4.84 and 4.87 (each s, total 0.5H), 4.95 and 5.05 (br, total 1H), 7.07 (d, J=1.1 Hz, 1H), 9.60 (s, 1H); HR-MS (ESI) Calcd for C₂₁H₃₃N₇O₇Na [M+Na]⁺ 518.2334. Found 518.2330.

(S)-N-{1-Azido-6-[(tert-butoxycarbonyl)amino]hexan-2-yl}-N-[2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl]glycine (7)

To a solution of 6 (118 mg, 0.24 mmol) in MeOH (7.2 mL) was added 2 M NaOH (2.4 mL, 4.8 mmol) at 0°C with stirring at r.t. for 40 min. H₂O (5 mL) was added and MeOH was evaporated. The pH was adjusted to 4 by addition of 1 M HCl and the aqueous phase was extracted with EtOAc (3×30 mL), washed with brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (CH₂Cl₂: MeOH=9 : 1) to give 7 (quant.) as a powder; ¹H-NMR (400 MHz, CDCl₃) δ: 1.44 (s, 9H), 1.27–1.57 (m, 6H), 1.88 and 1.89 (each s, total 3H), 3.00–3.14 (m, 2H), 3.42–3.58 (m, 2H), 3.82–4.02 (m, 1H), 4.12–4.77 (m, 4H), 5.00 and 5.15 (each s), 7.16 (s, 1H), 10.15 and 10.26 (each s); HR-MS (ESI) Calcd for C₂₀H₃₁N₇O₇Na [M+Na]⁺ 504.2177. Found 504.2174.

(S)-N-[6-({[(9H-Fluoren-9-yl)methoxy]carbonyl}amino)-1-azidohexan-2-yl]-N-[2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetyl]glycine (8)

To a solution of 7 (221.6 mg, 0.46 mmol) in dioxane (8 mL) was added conc. HCl (4 mL) at 0°C with stirring at r.t. for 1 h. To the solution were added H₂O (8 mL), NaHCO₃ (6 g), and FmocOSu (162.9 mg, 0.48 mmol) in MeCN (4 mL) at 0°C with stirring at r.t.. After completion of the reaction, the reaction mixture was poured into 1 M HCl (50 mL) and extracted with EtOAc (3×30 mL), washed with H₂O and brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (CH₂Cl₂ : MeOH=1 : 0–99 : 1–9 : 1) to give 8 (quant.) as a powder; ¹H-NMR (400 MHz, CDCl₃) δ: 1.26–1.51 (m, 6H), 1.80 and 1.86 (each s, total 3H), 3.03–3.17 (m, 2H), 3.37–3.55 (m, 2H), 3.86–3.93 (m, 1H), 4.09–4.69 (m, 7H), 5.20 and 5.41 (each t, J=5.5 Hz), 7.29 (t, J=6.7 Hz, 2H), 7.37 (t, J=7.1 Hz, 2H), 7.55–7.59 (m, 2H), 7.73 (d, J=6.9 Hz, 2H), 9.75 and 9.93 (each s); HR-MS (ESI) Calcd for C₃₀H₃₃N₇O₇Na [M+Na]⁺ 626.2334. Found 626.2319.

Ethyl 5′-({2-[(tert-Butoxycarbonyl)amino]ethyl}carbamoyl)-[2,2′-bipyridine]-5-carboxylate (11)

To a mixture of 9²²⁾ (207 mg, 0.76 mmol) and 10 (181 µL, 1.14 mmol) in DMF (10 mL) were added EDCI (218.5 mg, 1.14 mmol), N,N-dimethyl-4-aminopyridine (DMAP) (28 mg, 0.23 mmol), and Et₃N (159 µL, 1.14 mmol) at 0°C with stirring at r.t. for 41 h. The reaction mixture was poured into 1 M HCl (50 mL) and extracted with EtOAc (2×40 mL), washed with H₂O, sat. NaHCO₃, H₂O, and brine, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (CH₂Cl₂ : MeOH=19 : 1) to give 11 (93.4 mg, 0.23 mmol, 30%) as a white wax; ¹H-NMR (400 MHz, CDCl₃) δ: 1.45 (s, 12H), 3.43–3.47 (m, 2H), 3.59–3.63 (m, 2H), 4.45 (q, J=7.1 Hz, 2H), 5.03 (s, 1H), 7.65 (s, 1H), 8.27 (dd, J=8.2, 2.1 Hz, 1H), 8.43 (dd, J=8.3, 2.0 Hz, 1H), 8.56 (dd, J=8.3, 4.2 Hz, 2H), 9.16 (s, 1H), 9.29 (d, J=1.5 Hz, 1H); ¹³C-NMR (100 MHz, CDCl₃) δ: 14.3, 28.4, 39.8, 42.7, 61.5, 80.2, 121.0, 121.2, 126.3, 129.8, 135.6, 137.9, 148.2, 150.4, 157.1, 157.8, 165.1, 165.5; HR-MS (ESI) Calcd for C₂₁H₂₇N₄O₅ [M+H]⁺ 415.1976. Found 415.1971.

5′-({2-[(tert-Butoxycarbonyl)amino]ethyl}carbamoyl)-[2,2′-bipyridine]-5-carboxylic Acid (12)

To a solution of 11 (161.5 mg, 0.39 mmol) in CH₂Cl₂ (3 mL) and MeOH (4 mL) was added 2 M NaOH (1 mL, 2 mmol) with stirring at r.t. for 1 h. H₂O (5 mL) was added and the pH was adjusted to 3 by addition of 1 M HCl. The mixture was extracted with CH₂Cl₂–MeOH (9 : 1) and concentrated in vacuo to give 12 (147.1 mg, 0.38 mmol, 98%) as a white powder; ¹H-NMR (400 MHz, d₆-DMSO) δ: 1.31 (s, 9H), 3.09–3.13 (m, 2H), 3.27–3.32 (m, 2H), 6.91–6.94 (m, 1H), 8.33–8.39 (m, 2H), 8.45–8.50 (m, 2H), 8.82–8.85 (m, 1H), 9.10 (s, 1H), 9.13 (d, J=1.3 Hz, 1H); ¹³C-NMR (100 MHz, d₆-DMSO) δ: 28.7, 78.2, 121.2, 121.4, 127.5, 131.0, 137.0, 138.9, 149.1, 150.8, 156.3, 156.5, 158.0, 165.0, 166.6; HR-MS (ESI) Calcd for C₁₉H₂₃N₄O₅ [M+H]⁺ 387.1663. Found 387.1650.

Oligomer Synthesis

PNA and β-PNA oligomers were manually synthesized from β-Lys PNA monomers, unmodified PNA monomers, and Nα-Fmoc-Nε-Boc-lysines using the standard Fmoc-protected solid-phase synthesis protocol. Fmoc-PAL-PEG-PS resins were used as a solid support. HATU, N,N-diisopropylethylamine and 2,6-lutidine were used for coupling each monomer. Completion of the coupling reaction was monitored by the Kaiser test. Twenty percent piperidine in DMF was used for deprotection of the Fmoc-protecting group from the terminal amino group. The unreacted amines were capped with acetic anhydride. After coupling the last monomer, the resins were treated with TFA containing 13.9% m-cresol and 2.8% H₂O to cleave the oligomers from the resins as well as to remove the protecting groups from the nucleobases and amino groups (Boc). The solvent was removed, and the residue was washed with Et₂O three times, dissolved in H₂O, and purified by reversed-phase HPLC.

Azide Reduction-Coupling

The resin was treated with a 0.9 M PMe₃ in THF–H₂O (9 : 1; 1 mL) for 5 min then washed with NMP (3×1 mL). Subsequent coupling was performed with double coupling.

Attachment of Bipyridine Unit 12 onto β-PNA

Piperidine (20%) in DMF was used for deprotection of the Fmoc-protecting group from the side chain amino group. Subsequent coupling of Boc-protected bipyridine unit 12 was performed with double coupling using HATU as a coupling agent.

Thermal Denaturation Analysis

All PNA stock solutions were prepared in Milli-Q water. The concentrations of the PNAs were determined at 85°C using the molar extinction coefficients of the corresponding nucleobases (T=8.6 cm²/µmol; C=6.6 cm²/µmol; G=11.7 cm²/µmol and A=13.7 cm²/µmol). Prior to the experiment, all buffers were degassed by heating to 90°C for 10 min. The samples were prepared in 10 mM sodium phosphate buffer containing 1 mM EDTA (pH 7.4) and the indicated concentrations of NaCl. The concentration of each strand was 5 µM. All samples were annealed by heating at 90°C for 10 min followed by a gradual cooling to r.t.. Thermal denaturation was monitored at 260 nm at a rate of 0.2°C/min. The T_m values were determined from the first derivatives of the UV-melting curves.

Electrophoretic Mobility Shift Assays of DNA Cleavage

The DNA cleavage reactions by PNA were studied by use of electrophoretic mobility shift assays. Cleavage reactions were prepared by mixing 1 µL of 100 mM phosphate buffer pH 7.4, 1 µL of 20 µM DNA and 2 µL of 10 µM PNA with water to a total volume of 8 µL.

D1:

DNA sequence for P6 5′-TTT TTT TTT TAG TGA TCT ACC GGC TGA TCG AAT TCG TAC GAA TTC GAT CAG CCG-3′.

D2:

DNA for P7 and P8 5′-AGC TAA CGT TGA TCT ACC GGC TGA TCG AAT TCG TAC GAA TTC GAT CAG CCG-3′.

Solutions were heated at 95°C for 5 min and then slowly cooled to 4°C. Cleavage reaction was initiated by addition of 1 µL of 100 µM CuSO₄ and 1 µL of 30 mM MPA (3-mercaptopropionic acid). Reactions were incubated at 4°C for 24 h and quenched by addition of 1 µL of 20 mM EDTA. Reaction products were separated and quantified by denaturing polyacrylamide gel electrophoresis on a 17% polyacrylamide, 5.8 M urea, 29 : 1 cross-linked gel. Gels were stained with ethidium bromide and visualized by UV illumination and densitometry (Printgraph AE-6932GXCF, ATTO).

DNA Cleavage Detection by MALDI-TOF Mass Spectrometry

DNA cleavage experiments were performed on a 100 µL scale with 2 µM PNA, 2 µM DNA oligonucleotide D3 (5′-AGT GAT CTA C-3′), 4 µM CuSO₄, 3 mM MPA (3-mercaptopropionic acid) in 10 mM phosphate buffer at pH 7.4. Reactions were incubated at 4°C for 48 h and quenched by addition of 10 µL of 20 mM EDTA. The DNA cleavage reaction mixtures were desalted using the ZipTip procedure. The oligonucleotides were bound to ZipTip, washed with 10 mM trimethylammonium acetate buffer and eluted with 10 mM trimethylammonium acetate–acetonitrile (1 : 1). The MALDI-TOF mass spectra were measured on an AB Sciex TOF/TOF™5800 system (AB Sciex) using 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as the matrix.

Molecular Modeling

Molecular modeling was performed by MacroModel (Schrödinger). X-Ray structure (PDB code: 1NR8) in ref. 23 was used as an initial structure. Models were built by replacing beta-hydrogen with the side chain possessing bipyridine unit, and conformation of the side chain was optimized with conformational search method of MacroModel.

Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research (C) (Grant No. 15K07869 for A.K. and No. 24590135 for T.S.) from the Japan Society for the Promotion of Science (JSPS).

Conflict of Interest

The authors declare no conflict of interest.

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