Synthesis and Properties of 2'-Deoxyuridine Analogues Bearing Various Azobenzene Derivatives at the C5 Position

Abstract

Nucleic acids that change their properties upon photo-irradiation could be powerful materials for molecular sensing with high spatiotemporal resolution. Recently, we reported a photo-isomeric nucleoside bearing azobenzene at the C5 position of 2'-deoxyuridine (dU^Az), whose hybridization ability could be reversibly controlled by the appropriate wavelength of light. In this paper, we synthesized and evaluated dU^Az analogues that have various para-substitutions on the azobenzene moiety. Spectroscopic measurements and HPLC analyses revealed that the para-substitutions of the azobenzene moiety strongly affect the photo-isomerization ability and thermal stability of the cis-form. The results suggest that proper substitution of the azobenzene moiety can improve the properties of dU^Az as a light-responsive nucleic acid probe.

1. Introduction

Much attention has been focused on chemically modified nucleic acids that can alter their properties by some external stimuli [1,2,3]. Photo-responsive nucleic acids have been realized by regulation of hydrogen bonding [4] and stacking effects [5] between nucleobases. Light is considered to be a promising external stimulus due to the possibility of accurately controlling the location, dosage, and time of the irradiation. Various photochromic derivatives have been attached to nucleic acids to photo-control their properties [6]. For example, diarylethene-modified oligonucleotides (ONs) showed changes in their optical properties [7,8] and hybridization abilities [8] by photo-isomerization. However, the changes in hybridization abilities were moderate and the diarylethene modification itself destabilized the DNA duplex in both forms. To obtain a significant melting temperature difference (ΔT_m) upon photo-isomerization, ONs containing cis-trans type photochromic derivatives have been reported. Owing to their dynamic structural changes upon photo-irradiation, cis-trans type photochromic derivatives could greatly influenced the stability of nucleic acid duplexes. Although stilbene-type derivative-modified ONs have been utilized for photo-control of duplex formation, G-quadruplex formation, and gene expression, they require short wavelength photo-irradiation for cis to trans isomerization [9,10,11]. This would be disadvantageous for biomolecules due to photo-damaging reactions including the formation of pyrimidine dimers [12]. Azobenzene derivative-modified ONs are quite promising photochromic ONs because they can rapidly photo-isomerize by longer wavelengths of light [13,14]. Asanuma et al. have demonstrated that ONs modified with azobenzene moieties could be utilized as photo-responsive tweezers, RNA scission, and engines [15]. Recently, we reported that the ON containing C5-azobenzene-substituted 2'-deoxyuridine (dU^Az) could be photo-isomerized from trans to cis with an efficiency of 60% by UV light (365 nm) and from cis to trans with an efficiency of 80% by visible light (450 nm) [16]. The ON containing dU^Az showed interesting hybridization properties, namely, the T_m values of the duplexes formed between dU^Az-modified ON and complementary DNA or RNA were higher after UV irradiation than before irradiation. This may be because the hydrophobic azobenzene in the trans form extends to the outside of the groove and interferes with hydration and the formation of interstrand cation bridges to stabilize the duplexes. Meanwhile, cis-dU^Az did not affect the duplex stability due to the compact conformation of the azobenzene moiety. These results indicated that dU^Az could be a potential building block to control nucleic acid hybridization with high spatiotemporal resolution.

In this study, we describe the synthesis and properties of dU^Az analogues bearing various para-substituted azobenzene derivatives (Figure 1). The isomerization properties of photochromic compounds have been proposed to be strongly influenced by substituents [17]. Thus, the introduction of electron-donating or -withdrawing substituents into the azobenzene moiety could improve the properties of dU^Az and create unique photo-sensors for various biomolecules.

Figure 1. Para-substituted dU^Az analogues used in this study.

Figure 1. Para-substituted dU^Az analogues used in this study.

2. Experimental Section

2.1. General

Reagents and solvents were purchased from commercial suppliers and were used without purification unless otherwise specified. All experiments involving air- and/or moisture-sensitive compounds were carried out under N₂ or Ar atmosphere. All reactions were monitored with analytical TLC (Merck Kieselgel 60 F254; Merck, Darmstadt, Germany). Column chromatography was carried out using FL-100D silica gel (Fuji Silysia, Aichi, Japan). Physical data were measured as follows. NMR spectra were recorded on JNM-ECS-300, JNM-ECS-400, and JNM-ECS-500 spectrometers (JEOL, Tokyo, Japan) using CDCl₃ or DMSO-d₆ as solvents with tetramethylsilane as an internal standard. IR spectra were recorded on a FT/IR-4200 spectrophotometer (JASCO, Tokyo, Japan). Optical rotations were recorded on a JASCO P-2200 instrument. FAB mass spectra were measured on a JEOL JMS-700 mass spectrometer. Solid-phase ON synthesis was performed on an nS-8 Oligonucleotide Synthesizer (GeneDesign, Osaka, Japan). MALDI-TOF mass spectra were recorded on an ultrafleXtreme mass spectrometer (Bruker Daltonics, Billerica, MA, USA) for oligonucleotides and on a JMS-S3000 (JEOL, Tokyo, Japan) for small molecules. Photo-irradiation experiments were conducted with a Xenon lamp (MAX-303; Asahi Spectra, Tokyo, Japan) using HQBP 450-VIS ø 25 and 365-VIS ø 25 as the optical filters. UV/Vis absorption measurements and UV melting experiments were performed using a UV-1650PC UV-Vis spectrophotometer equipped with a TMSPC-8 T_m analysis accessory (Shimadzu, Kyoto, Japan). ITC experiments were performed using a Microcal iTC₂₀₀ (Malvern Instruments, Worcestershire, UK).

2.2. Preparation of 4-[4-(trimethylsilyl)ethynylphenylazo]pyridine (2)

Under an argon atmosphere, 4-(4-iodophenylazo)pyridine 1 [18] (950 mg, 3.07 mmol) was dissolved in dry THF (30 mL). Pd(PPh₃)₄ (358 mg, 0.310 mmol), CuI (59.0 mg,0.310 mmol), Et₃N (2.1 mL, 15.4 mmol), and trimethylsilylacetylene (1.1 mL, 7.68 mmol) were then added. The reaction mixture was stirred at 60 °C for 6 h. The resultant mixture was filtered over Celite. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with hexane/AcOEt (8:1) to give compound 2 (485 mg, 57%) as a red oil. IR (KBr): ν 2156 (C≡C), 1250 (N=N) cm⁻¹; ¹H-NMR (400 MHz, CDCl₃): δ 8.81 (2H, dd, J = 2.0, 4.5 Hz), 7.91 (2H, dd, J = 1.5, 6.5 Hz), 7.70 (2H, dd, J = 1.5, 4.5 Hz), 7.63 (2H, dd, J = 2.0, 6.5 Hz), 0.28 (9H, s); ¹³C-NMR (100 MHz, CDCl₃): δ 151.5, 151.4, 133.0, 132.5, 130.4, 129.3, 123.5, 123.4, 116.33, 116.31; FAB-HRMS m/z (MH⁺) calcd for C₁₆H₁₈N₃Si: 280.1265; found 280.1272.

2.3. Preparation of 4-(4-ethynylphenylazo)pyridine (5)

Under an argon atmosphere, to a solution of compound 2 [19] (319 mg, 1.14 mmol) in dry THF (5 mL) were added K₂CO₃ (79 mg, 0.57 mmol) and MeOH (5 mL) and the reaction mixture was stirred for 2 h at room temperature. The resultant mixture was diluted with AcOEt (30 mL) and washed with H₂O, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with hexane/AcOEt (3:1) to give compound 5 (205 mg, 86%) as a red powder. M.p. 160 °C (decomposed); IR (KBr): ν 2092 (C≡C), 1263 (N=N) cm⁻¹; ¹H-NMR (400 MHz, CDCl₃): δ 8.82 (2H, dd, J = 1.5, 4.5 Hz), 7.92 (2H, d, J = 8.0 Hz), 7.71 (2H, dd, J = 1.5, 4.5 Hz), 7.66 (2H, d, J = 9.0 Hz), 3.28 (1H, s); ¹³C-NMR (100 MHz, CDCl₃): δ 157.0, 151.8, 151.4, 133.1, 126.2, 123.3, 116.2, 83.0, 80.3; FAB-HRMS m/z (MH⁺) calcd for C₁₃H₁₀N₃: 208.0869; found 208.0855.

2.4. Preparation of 4-ethynyl-4'-methoxyazobenzene (6)

Under an argon atmosphere, to a solution of compound 3 [19] (750 mg, 2.44 mmol) in dry THF (10 mL) were added K₂CO₃ (169 mg, 1.22 mmol) and MeOH (10 mL) and the reaction mixture was stirred for 2 h at room temperature. The resultant mixture was diluted with AcOEt (40 mL) and washed with H₂O, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with hexane/CH₂Cl₂ (4:1) to give compound 6 (576 mg, 99%) as an orange powder. M.p. 76–78 °C; IR (KBr): ν 2102 (C≡C), 1252 (N=N) cm⁻¹; ¹H-NMR (400 MHz, CDCl₃): δ 7.81 (2H, d, J = 8.5 Hz), 7.73 (2H, d, J = 8.0 Hz), 7.51 (2H, d, J = 8.0 Hz), 6.89 (2H, d, J = 9.0 Hz), 3.76 (3H, s), 3.11 (1H, s); ¹³C-NMR (100 MHz, CDCl₃): δ 162.3, 152.3, 146.9, 132.9, 124.9, 123.9, 122.4, 114.2, 83.4, 79.1, 55.5; FAB-HRMS m/z (MH⁺) calcd for C₁₅H₁₃N₂O: 237.1022; found 237.1021.

2.5. Preparation of 4-ethynyl-4'-methylazobenzene (7)

Under an argon atmosphere, to a solution of compound 4 [19] (403 mg, 1.38 mmol) in dry THF (6 mL) were added K₂CO₃ (95 mg, 0.69 mmol) and MeOH (6 mL) and the reaction mixture was stirred for 2 h at room temperature. The resultant mixture was diluted with AcOEt (20 mL) and washed with H₂O, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with hexane to give compound 7 (203 mg, 92%) as an orange powder. M.p. 138–140 °C; IR (KBr): ν 2156 (C≡C), 1250 (N=N) cm⁻¹; ¹H-NMR (300 MHz, CDCl₃): δ 7.87–781 (4H, m), 7.61 (2H, d, J = 8.5 Hz), 7.30 (2H, d, J = 8.5 Hz), 3.21 (1H, s), 2.42 (3H, s); ¹³C-NMR (125 MHz, CDCl₃): δ 152.2, 150.7, 142.0, 132.9, 129.8, 124.3, 123.0, 122.7, 83.3, 79.3, 21.5; MALDI-TOF-HRMS m/z (MH⁺) calcd for C₁₅H₁₃N₂: 221.1073; found 221.1075.

2.6. Preparation of 5-[4-(4-pyridyl)diazenylphenyl]ethynyl-2'-deoxyuridine (11)

Under an argon atmosphere, compound 5 (281 mg, 1.35 mmol) was dissolved in dry DMF (15 mL). Pd(PPh₃)₄ (156 mg, 0.135 mmol), CuI (26 mg, 0.135 mmol), Et₃N (940 µL), and 2'-deoxy-5-iodouridine 10 (466 mg, 1.35 mmol) were then added. The reaction mixture was stirred at 60 °C for 12 h. The resultant mixture was filtered over Celite. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with DMF to give compound 11 (295 mg, 50%) as a red powder. M.p. 235 °C (decomposed); IR (KBr): ν 3250 (NH, OH), 1636 (C=O); [α]_D²⁴ −19.6 (c 1.00, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): δ 11.7 (1H, br s, NH), 8.82 (2H, d, J = 4.5 Hz), 8.48 (1H, s, H-6), 7.96 (2H, d, J = 8.0 Hz), 7.75–7.68 (4H, m), 6.13 (1H, t, J = 6.0 Hz, H-1'), 5.27 (1H, d, J = 4.0 Hz, OH), 5.21 (1H, t, J = 4.5 Hz, OH), 4.29–4.25 (1H, m, H-3'), 3.84–3.82 (1H, m. H-4'), 3.67–3.58 (2H, m, H-5'), 2.21–2.17 (2H, m, H-2'); ¹³C-NMR (125 MHz, DMSO-d₆): δ 161.3, 156.5, 151.5, 150.8, 149.4, 144.68, 132.3, 126.8, 123.5, 115.9, 97.7, 91.4, 87.6, 85.4, 85.0, 69.8, 60.8, 40.3; MALDI-TOF-HRMS m/z (MH⁺) calcd for C₂₂H₂₀N₅O₅: 434.1459; found 434.1451.

2.7. Preparation of 5-[4-(4-methoxyphenyl)diazenylphenyl]ethynyl-2'-deoxyuridine (12)

Under an argon atmosphere, compound 6 (303 mg, 1.28 mmol) was dissolved in dry DMF (15 mL). Pd(PPh₃)₄ (148 mg, 0.128 mmol), CuI (24 mg, 0.128 mmol), Et₃N (1 mL), and 2'-deoxy-5-iodouridine 10 (441 mg, 1.28 mmol) were then added. The reaction mixture was stirred at 60 °C for 12 h. The resultant mixture was filtered over Celite. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (10:1) to give compound 12 (360 mg, 61%) as a light-orange powder. M.p. 253 °C (decomposed); IR (KBr): ν 3345 (NH, OH), 2102 (C≡C), 1638 (C=O) cm⁻¹; [α]_D²⁴ −21.6 (c 1.00, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): δ 11.7 (1H, br s, NH), 8.47–8.44 (1H, m, H-6), 7.90–7.85 (4H, m), 7.64 (2H, d, J = 6.0 Hz), 7.13 (2H, d, J = 5.0 Hz), 6.18–6.11 (1H, m, H-1'), 5.31–5.25 (1H, m,OH), 5.24–5.18 (1H, m, OH), 4.32–4.24 (1H, m, H-3'), 3.89–3.81 (4H, m, H-4', Ph-OMe), 3.70–3.59 (2H, m, H-5'), 2.23–2.14 (2H, m, H-2'); ¹³C-NMR (125 MHz, DMSO-d₆): δ 162.3, 161.3, 151.2, 149.4, 146.2, 144.3, 132.2, 124.8, 122.6, 114.7, 97.9, 91.6, 87.6, 85.1, 84.9, 69.9, 60.8, 55.6, 40.3; FAB-HRMS m/z (MH⁺) calcd for C₂₄H₂₃N₄O₆: 463.1612; found 463.1613.

2.8. Preparation of 5-[4-(4-methylphenyl)diazenylphenyl]ethynyl-2'-deoxyuridine (13)

Under an argon atmosphere, compound 7 (280 mg, 1.27 mmol) was dissolved in dry DMF (13 mL). Pd(PPh₃)₄ (173 mg, 0.127 mmol), CuI (29 mg, 0.127 mmol), Et₃N (1.33 mL), and 2’-deoxy-5-iodouridine 10 (292 mg, 0.846 mmol) were then added. The reaction mixture was stirred at 60 °C for 12 h. The resultant mixture was filtered over Celite. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (10:1) to give compound 13 (284 mg, 75%) as a light-orange powder. M.p. 255 °C (decomposed); IR (KBr): ν 3345 (NH, OH), 2091 (C≡C), 1641 (C=O) cm⁻¹; [α]_D²⁴ −20.6 (c 1.00, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): δ 11.7 (1H, br s, NH), 8.47–8.44 (1H, m, H-6), 7.92–7.79 (4H, m), 7.69–7.63 (2H, m), 7.43–7.38 (2H, m), 6.17–6.11 (1H, m, H-1'), 5.30–5.24 (1H, m, OH), 5.22–5.16 (1H, m, OH), 4.30–4.23 (1H, m, H-3'), 3.85–3.79 (1H, m, H-4'), 3.71–3.57 (2H, m, H-5'), 2.40 (3H, s), 2.22-2.13 (2H, m, H-2'); ¹³C-NMR (125 MHz, DMSO-d₆): δ 161.3, 151.1, 150.1, 149.4, 144.4, 142.2, 132.2, 130.0, 125.1, 122.8, 97.7, 91.4, 87.6, 85.4, 85.0, 69.8, 60.8, 40.3, 21.1; MALDI-TOF-HRMS m/z (MH⁺) calcd for C₂₄H₂₃N₄O₅: 447.1663; found 447.1663.

2.9. Preparation of 5-[4-(4-trifluoromethylphenyl)diazenylphenyl]ethynyl-2'-deoxyuridine (14)

Under an argon atmosphere, compound 8 [20] (369 mg, 1.35 mmol) was dissolved in dry DMF (15 mL). Pd(PPh₃)₄ (156 mg, 0.135 mmol), CuI (26 mg, 0.135 mmol), Et₃N (1 mL), and 2'-deoxy-5-iodouridine 10 (465 mg, 1.35 mmol) were then added. The reaction mixture was stirred at 60 °C for 12 h. The resultant mixture was filtered over Celite. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (10:1) to give compound 14 (415 mg, 62%) as a light-orange powder. M.p. 255 °C (decomposed); IR (KBr): ν 3365 (NH, OH), 1659 (C=O) cm⁻¹; [α]_D²⁴ −16.7 (c 1.00, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): δ 11.8 (1H, br s, NH), 8.50 (1H, s, H-6), 8.08 (2H, d, J = 8.0 Hz), 7.99 (4H, d, J = 8.5 Hz), 7.71 (2H, d, J = 8.0 Hz), 6.15 (1H, t, J = 6.5 Hz, H-1'), 5.29 (1H, d, J = 3.5 Hz, OH), 5.24–5.21 (1H, m, OH), 4.29 (1H, s, H-3'), 3.84 (1H, d, J = 3.5 Hz, H-4'), 3.71–3.61 (2H, m, H-5'), 2.22–2.18 (2H, m, H-2'); ¹³C-NMR (125 MHz, DMSO-d₆): δ 161.3, 154.0, 150.8, 149.4, 144.6, 132.3, 130.9, 126.8, 126.3, 123.4, 123.2, 97.7, 91.4, 87.6, 86.2, 85.0, 79.2, 69.8, 60.8, 40.3; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₂₄H₁₉N₄O₅F₃Na: 523.1200; found 523.1200.

2.10. Preparation of 5-[4-(4-nitrophenyl)diazenylphenyl]ethynyl-2'-deoxyuridine (15)

Under an argon atmosphere, compound 9 [21] (302 mg, 1.20 mmol) was dissolved in dry DMF (15 mL). Pd(PPh₃)₄ (139 mg, 0.120 mmol), CuI (23 mg, 0.120 mmol), Et₃N (1 mL), and 2'-deoxy-5-iodouridine 10 (414 mg, 1.20 mmol) were then added. The reaction mixture was stirred at 60 °C for 12 h. The resultant mixture was filtered over Celite. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (5:1) to give compound 15 (545 mg, 95%) as a red powder. M.p. 270 °C (decomposed); IR (KBr): ν 3403 (NH, OH), 1631 (C=O) cm⁻¹; [α]_D²⁴ −22.5 (c 1.00, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): δ 11.8 (1H, br s, NH), 8.50–8.44 (3H, m), 8.10–8.08 (2H, m), 8.02–7.97 (2H, m), 7.74–7.68 (2H, m), 6.17–6.11 (1H, m, H-1'), 5.30–5.28 (1H, m, OH), 5.22 (1H, d, J = 4.5 Hz, OH), 4.28 (1H, m, H-3'), 3.84 (1H, m, H-4'), 3.68–3.62 (2H, m, H-5'), 2.22–2.18 (2H, m, H-2'); ¹³C-NMR (125 MHz, DMSO-d₆): δ 161.3, 155.0, 150.9, 149.4, 148.6, 144.7, 132.3, 126.7, 125.1, 123.6, 123.5, 97.7, 91.5, 87.6, 86.5, 85.0, 69.8, 60.8, 40.3; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₂₃H₁₉N₅O₇Na: 500.1177; found 500.1169.

2.11. Preparation of 5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-pyridyl)diazenylphenyl]ethynyl-2'-deoxyuridine (16)

To a solution of compound 11 (147 mg, 0.339 mmol) in dry pyridine (4 mL) was added DMTrCl (138 mg, 0.406 mmol) at room temperature, and the reaction mixture was stirred for 16 h. The reaction was quenched by the addition of MeOH with 10 min stirring. The solvent was removed in vacuo, and the residue was partitioned between CHCl₃ and H₂O. The separated organic layer was washed with H₂O, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (20:1 with 0.5% Et₃N) to give compound 16 (223 mg, 89%) as a red foam. IR (KBr): ν 3323 (NH, OH), 1705 (C=O), 1252 (N=N) cm⁻¹; [α]_D²⁴ −4.3 (c 1.00, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): δ 11.7 (1H, br s, NH), 8.67 (2H, d, J = 4.5 Hz), 8.05 (1H, s, H-6), 7.68 (2H, d, J = 8.5 Hz), 7.59 (2H, d, J = 6.0 Hz), 7.28 (2H, d, J = 7.0 Hz), 7.17–7.09 (8H, m), 7.02–7.00 (1H, m), 6.70–6.68 (4H, m), 6.01 (1H, t, J = 6.5 Hz, H-1'), 5.23–5.21 (1H, m,OH), 4.19–4.17 (1H, m, H-3'), 3.82 (1H, s, H-4'), 3.49 (6H, s, OMe), 3.06–3.04 (2H, m, H-5'), 2.16–2.13 (2H, m, H-2'); ¹³C-NMR (125 MHz, DMSO-d₆): δ 161.3, 158.1, 156.5, 151.6, 150.7, 149.3, 144.7, 143.7, 135.5, 135.4, 132.2, 129.6, 127.9, 127.6, 126.7, 126.6, 123.1, 115.9, 113.2, 98.1, 86.1, 86.0, 85.8, 85.3, 70.35, 63.5, 54.96, 40.4; MALDI-TOF-HRMS m/z (MH⁺) calcd for C₄₃H₃₈N₅O₇: 736.2766; found 736.2759.

2.12. Preparation of 5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-methoxyphenyl)diazenylphenyl] ethynyl-2'-deoxyuridine (17)

To a solution of compound 12 (101 mg, 0.218 mmol) in dry pyridine (3 mL) was added DMTrCl (89 mg, 0.262 mmol) at room temperature, and the reaction mixture was stirred for 16 h. The reaction was quenched by the addition of MeOH with 10 min stirring. The solvent was removed in vacuo, and the residue was partitioned between CHCl₃ and H₂O. The separated organic layer was washed with H₂O, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (20:1 with 0.5% Et₃N) to give compound 17 (152 mg, 90%) as an orange foam. IR (KBr): ν 3437, 3410 (NH, OH), 1701 (C=O), 1272 (N=N) cm⁻¹; [α]_D²⁴ 28.4 (c 1.00, CHCl₃); ¹H-NMR (500 MHz, CDCl₃): δ 10.2 (1H, br s, NH), 8.28 (1H, s, H-6), 7.88 (2H, d, J = 7.0 Hz), 7.64 (2H, d, J = 7.0 Hz), 7.46 (2H, d, J = 7.0 Hz), 7.36 (4H, d, J = 7.5 Hz), 7.26–7.24 (3H, m), 7.11 (1H, d, J = 8.0 Hz), 6.81–6.74 (4H, m), 6.45–6.37 (1H, m, H-1'), 4.60–4.58 (1H, m, H-3'), 4.22–4.19 (1H, m, H-4'), 3.84 (3H, s), 3.49 (6H, s, OMe), 3.45 (1H, d, J = 10.0 Hz, H-5'), 3.31 (1H, d, J = 9.0 Hz, H-5'), 2.68–2.57 (1H, m, H-2'), 2.40–2.28 (1H, m, H-2'), 1.29–1.19 (1H, m, OH); ¹³C-NMR (125 MHz, CDCl₃): δ 162.1, 161.8, 158.4, 151.6, 149.5, 146.9, 144.4, 135.5, 135.4, 132.3, 129.9, 129.8, 128.0, 127.8, 124.3, 122.1, 114.1, 113.2, 100.3, 93.5, 86.9, 86.0, 86.0, 82.2, 72.2, 60.3, 55.4, 55.0, 41.6; MALDI-TOF-HRMS m/z (MH⁺) calcd for C₄₅H₄₁N₄O₈: 765.2919; found 765.2912.

2.13. Preparation of 5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-methylphenyl)diazenylphenyl] ethynyl-2'-deoxyuridine (18)

To a solution of compound 13 (200 mg, 0.446 mmol) in dry pyridine (5 mL) was added DMTrCl (182 mg, 0.536 mmol) at room temperature, and the reaction mixture was stirred for 16 h. The reaction was quenched by the addition of MeOH with 10 min stirring. The solvent was removed in vacuo, and the residue was partitioned between CHCl₃ and H₂O. The separated organic layer was washed with H₂O, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with hexane/AcOEt (1:2) to give compound 18 (208 mg, 62%) as an orange foam. IR (KBr): ν 3425 (NH, OH), 1707 (C=O), 1253 (N=N) cm⁻¹; [α]_D²⁴ −27.9 (c 1.00, CHCl₃); ¹H-NMR (500 MHz, CDCl₃): δ 9.09 (1H, br s, NH), 8.27 (1H, s, H-6), 7.80 (2H, d, J = 8.0 Hz), 7.67 (2H, d, J = 7.5 Hz), 7.45 (2H, d, J = 8.0 Hz), 7.35 (4H, d, J = 8.5 Hz), 7.31–7.24 (4H, m), 7.15 (1H, t, J = 7.0 Hz), 7.11 (2H, d, J = 8.5 Hz), 6.81–6.78 (4H, m), 6.38 (1H, t, J = 4.0 Hz, H-1'), 4.60–4.57 (1H, m, H-3'), 4.17–4.09 (1H, m, H-4'), 3.77–3.65 (6H, m, OMe), 3.50–3.46 (1H, m, H-5'), 3.40–3.28 (1H, m, H-5'), 2.67–2.52 (2H, m, H-2'), 1.86–1.82 (1H, m, OH); ¹³C-NMR (125 MHz, CDCl₃): δ 161.2, 158.6, 151.7, 150.7, 149.2, 144.4, 141.9, 135.4, 132.4, 130.0, 129.9, 129.8, 128.1, 127.9, 127.1, 124.7, 122.9, 122.3, 113.4, 100.4, 93.6, 87.1, 86.8, 85.9, 72.4, 63.5, 55.2, 41.2, 21.5; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₄₅H₄₀N₄O₇Na: 771.2789; found 771.2780.

2.14. Preparation of 5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-trifluoromethylphenyl)diazenylphenyl] ethyn-yl-2'-deoxyuridine (19)

To a solution of compound 14 (114 mg, 0.228 mmol) in dry pyridine (3 mL) was added DMTrCl (93 mg, 0.273 mmol) at room temperature, and the reaction mixture was stirred for 16 h. The reaction was quenched by the addition of MeOH with 10 min stirring. The solvent was removed in vacuo, and the residue was partitioned between CHCl₃ and H₂O. The separated organic layer was washed with H₂O, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃ with 0.5% Et₃N) to give compound 19 (118 mg, 65%) as an orange foam. IR (KBr): ν 3372 (NH, OH), 1669 (C=O), 1253 (N=N) cm⁻¹; [α]_D²⁴ 26.9 (c 1.00, CHCl₃); ¹H-NMR (300 MHz, CDCl₃): δ 10.04 (1H, br s, NH), 8.25 (1H, s, H-6), 7.85 (2H, d, J = 8.5 Hz), 7.69–7.55 (4H, m), 7.41–7.12 (8H, m), 7.07–6.95 (3H, m), 6.71–6.69 (4H, m), 6.36–6.29 (1H, m, H-1'), 4.55–4.47 (1H, m, H-3'), 4.15–4.07 (1H, m, H-4'), 3.57 (6H, s, OMe), 3.42–3.34 (1H, m, H-5'), 3.25–3.18 (1H, m, H-5'), 2.60–2.42 (2H, m, H-2’), 2.32–2.21 (1H, m), 0.99–0.90 (1H, m, OH); ¹³C-NMR (75 MHz, CDCl₃): δ 161.7, 158.5, 154.2, 151.2, 149.5, 135.5, 132.4, 129.88, 129.86, 128.0, 127.9, 127.0, 126.22, 126.18, 126.0, 125.9, 123.0, 122.7, 113.3, 100.2, 93.3, 87.0, 86.9, 86.1, 83.7, 83.1, 75.2, 72.3, 55.1, 24.7; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₄₅H₃₇N₄O₇F₃Na: 825.2507; found 825.2497.

2.15. Preparation of 5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-nitrophenyl)diazenylphenyl] ethynyl-2'-deoxyuridine (20)

To a solution of compound 15 (150 mg, 0.314 mmol) in dry pyridine (3 mL) was added DMTrCl (128 mg, 0.377 mmol) at room temperature, and the reaction mixture was stirred for 16 h. The reaction was quenched by the addition of MeOH with 10 min stirring. The solvent was removed in vacuo, and the residue was partitioned between CHCl₃ and H₂O. The separated organic layer was washed with H₂O, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (20:1 with 0.5% Et₃N) to give compound 20 (115 mg, 47%) as a red foam. IR (KBr): ν 3452 (NH, OH), 1703 (C=O), 1251 (N=N) cm⁻¹; [α]_D²⁴ −16.9 (c 1.00, CHCl₃); ¹H-NMR (500 MHz, CDCl₃): δ 9.98 (1H, br s, NH), 8.38-8.22 (3H, m), 7.95 (2H, d, J = 7.0 Hz), 7.69 (2H, d, J = 7.0 Hz), 7.49-7.14 (8H, m), 7.06 (2H, d, J = 7.0 Hz), 6.83-6.72 (5H, m), 6.43-6.38 (1H, m, H-1'), 4.61-4.57 (1H, m, H-3'), 4.21-4.16 (1H, m, H-4'), 3.65 (6H, s, OMe), 3.50-3.45 (1H, m, H-5'), 3.33-3.26 (1H, m, H-5'), 2.66-2.59 (1H, m, H-2'), 2.34-2.27 (1H, m, H-2'), 1.28-1.17 (1H, m, OH); ¹³C-NMR (125 MHz, CDCl₃): δ 161.6, 158.5, 155.5, 151.2, 148.6, 144.4, 135.5, 135.4, 132.4, 132.3, 129.9, 128.0, 127.9, 127.8, 127.0, 124.6, 124.5, 123.4, 123.3, 123.0, 122.9, 113.3, 113.2, 100.1, 87.0, 86.9, 72.2, 55.1; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₄₄H₃₇N₅O₉Na: 802.2483; found 802.2501.

2.16. Preparation of 3'-O-[2-cyanoethyl(diisopropylamino)phosphino]-5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-pyridyl)diazenylphenyl]ethynyl-2'-deoxyuridine (21)

Under an argon atmosphere, to a solution of compound 16 (223 mg, 0.312 mmol) in dry CH₂Cl₂ (3 mL) was added N,N-diisopropylamine (160 µL, 0.935 mmol) and 2-cyanoethyl-N,N'-diisopropylchlorophosphoramidite (105 µL, 0.468 mmol) at room temperature, and the reaction mixture was stirred for 3 h. The resultant mixture was partitioned between AcOEt and H₂O. The separated organic layer was washed with saturated aqueous NaHCO₃, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (20:1 with 0.5% Et₃N) to give a 1:1 diastereomeric mixture of 21 (219 mg, 75%) as a red foam. IR (KBr): ν 3603 (NH), 1712 (C=O), 1251 (N=N) cm⁻¹; [α]_D²⁴ 32.3 (c 1.00, CHCl₃); ¹H-NMR (300 MHz, CDCl₃): δ 8.84 (2H, m), 8.42 (0.3H, s, H-6), 8.37 (0.7H, s, H-6), 7.74–7.70 (4H, m), 7.51–7.50 (2H, m), 7.41–7.35 (4H, m), 7.31–7.16 (3H, m), 7.04 (2H, dd, J = 9.0, 8.5 Hz), 6.83–6.77 (4H, m), 6.42–6.36 (1H, m, H-1'), 4.72–4.61 (1H, m, H-3'), 4.27–4.19 (1H, m, H-5'), 3.93–3.74 (1H, m, H-5'), 3.71 (6H, s, OMe), 3.67–3.49 (4H, m, CH₂CH₂CN), 3.36–3.24 (1H, m, H-4'), 2.72–2.52 (2H, m, H-2'), 2.49–2.35 (2H, m), 1.28 (8.4 H, t, J = 7.0 Hz, ((CH₃)₂CH)₂N), 1.17 (3.6H, t, J = 7.0 Hz, ((CH₃)₂CH)₂N); ¹³C-NMR (125 MHz, CDCl₃): δ 158.5, 157.1, 151.1, 144.33, 144.29, 132.8, 135.5, 135.4, 135.3, 132.4, 132.3, 129.9, 128.0, 127.9, 127.8, 127.0, 126.8, 122.91, 122.89, 117.5, 116.2, 113.2, 100.1, 93.0, 87.0, 85.8, 63.1, 58.4, 58.2, 58.1, 57.9, 55.1, 43.23, 43.17, 43.07, 43.01, 40.9, 24.6, 24.54, 24.48, 24.44, 24.39, 21.5, 20.4, 20.3, 20.2; ³¹P-NMR (120 MHz, CDCl₃): δ 148.8, 148.4; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₅₂H₅₄N₇O₈NaP: 958.3664; found 958.3647.

2.17. Preparation of 3'-O-[2-cyanoethyl(diisopropylamino)phosphino]-5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-methoxyphenyl)diazenylphenyl]ethynyl-2'-deoxyuridine (22)

Under an argon atmosphere, to a solution of compound 17 (152 mg, 0.199 mmol) in dry CH₂Cl₂ (2 mL) was added N,N-diisopropylamine (103 µL, 0.600 mmol) and 2-cyanoethyl-N,N'-diisopropylchlorophosphoramidite (67 µL, 0.299 mmol) at room temperature, and the reaction mixture was stirred for 3 h. The resultant mixture was partitioned between AcOEt and H₂O. The separated organic layer was washed with saturated aqueous NaHCO₃, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (20:1 with 0.5% Et₃N) to give a 3:1 diastereomeric mixture of 22 (72 mg, 38%) as an orange foam. IR (KBr): ν 3624 (NH), 1700 (C=O), 1253 (N=N) cm⁻¹; [α]_D²⁴ 19.4 (c 1.00, CHCl₃); ¹H-NMR (500 MHz, CDCl₃): δ 9.76 (1H, br s, NH), 8.37–8.28 (1H, m, H-6), 7.89 (2H, d, J = 7.5 Hz), 7.66 (2H, t, J = 8.0 Hz), 7.51–7.47 (2H, m), 7.41–7.31 (4H, m), 7.31–7.25 (2H, m), 7.19–7.12 (1H, m), 7.08 (2H, dd, J = 6.0, 10.0 Hz), 7.01–6.96 (2H, m), 6.84–6.78 (4H, m), 6.41–6.31 (1H, m, H-1'), 4.70–4.62 (1H, m, H-3'), 4.31–4.29 (0.25H, m, H-4'), 4-18-416 (0.75H, m, H-4'), 3.87–3.82 (4H, m, CH₂CH₂CN), 3.82 (3H, s), 3.68 (6H, s, OMe), 3.64–3.48 (2H, m, H-5'), 2.76–2.58 (2H, m, H-2'), 2.47–2.35 (2H, m, ((CH₃)₂CH)₂N), 1.28 (9H, t, J = 7.0 Hz, ((CH₃)₂CH)₂N), 1.17 (3H, t, J = 7.0 Hz, ((CH₃)₂CH)₂N); ¹³C-NMR (125 MHz, CDCl₃): δ 162.0, 158.4, 151.6, 149.2, 146.8, 144.2, 144.2, 135.28, 135.25, 132.18, 132.17, 129.8, 129.78, 129.75, 127.9, 127.85, 127.82, 127.7, 126.9, 124.6, 124.3, 121.97, 121.95, 117.3, 114.1, 113.2, 100.2, 100.1, 93.3, 86.8 (d, J (C, P) = 3.5 Hz), 77.2, 77.0, 76.7, 63.0, 62.9, 58.2, 58.1, 55.4, 54.9, 44.9 (d, J (C, P) = 5.0 Hz), 24.4, 24.3, 22.8, 22.7, 20.2 (d, J (C, P) = 7.0 Hz); ³¹P-NMR (200 MHz, CDCl₃): δ 149.5, 149.1; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₅₄H₅₇N₆O₉NaP: 987.3817; found 987.3826.

2.18. Preparation of 3'-O-[2-cyanoethyl(diisopropylamino)phosphino]-5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-methylphenyl)diazenylphenyl]ethynyl-2'-deoxyuridine (23)

Under an argon atmosphere, to a solution of compound 18 (170 mg, 0.227 mmol) in dry CH₂Cl₂ (3 mL) was added N,N-diisopropylamine (119 µL, 0.681 mmol) and 2-cyanoethyl-N,N'-diisopropylchlorophosphoramidite (78 µL, 0.341 mmol) at room temperature, and the reaction mixture was stirred for 3 h. The resultant mixture was partitioned between AcOEt and H₂O. The separated organic layer was washed with saturated aqueous NaHCO₃, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with hexane/AcOEt (1:1) to give a 2:1 diastereomeric mixture of 23 (120 mg, 55%) as an orange foam. IR (KBr): ν 3610 (NH), 1699 (C=O), 1272 (N=N) cm⁻¹; [α]_D²⁴ 29.6 (c 1.00, CHCl₃); ¹H-NMR (500 MHz, CDCl₃): δ 9.74 (1H, br s, NH), 8.35–8.26 (1H, m, H-6), 7.84–7.77 (2H, m), 7.71–7.63 (2H, m), 7.51–7.44 (2H, m), 7.41–7.33 (4H, m), 7.32–7.25 (5H, m), 7.12–7.03 (2H, m), 6.84–6.75 (4H, m), 6.41–6.31 (1H, m, H-1'), 4.72–4.60 (1H, m, H-3'), 4.29–4.19 (1H, m, H-4'), 3.93–3.74 (4H, m, CH₂CH₂CN), 3.70 (4H, s, OMe), 3.63 (2H, s, OMe), 3.37–3.12 (2H, m, H-5'), 2.72–2.50 (2H, m, H-2'), 2.50 (3H, s), 2.41–2.26 (2H, m, ((CH₃)₂CH)₂N), 1.22–1.14 (8H, m, ((CH₃)₂CH)₂N), 1.12–1.06 (4H, m, ((CH₃)₂CH)₂N); ¹³C-NMR (125 MHz, CDCl₃): δ 153.6, 146.7, 145.7, 139.3, 137.4, 136.8, 130.4, 127.4, 124.93, 124.91, 124.74, 124.72, 123.0, 122.9, 122.0, 121.9, 119.8, 117.9, 119.3, 112.5, 108.3, 88.5, 82.1, 80.9 (d, J (C, P) = 3.5 Hz), 68.8, 68.7, 58.2, 53.4, 53.3, 50.2, 48.4, 38.2, 35.9, 35.8, 19.5 (d, J (C, P) = 7.0 Hz), 16.5, 15.4, 15.3; ³¹P-NMR (200 MHz, CDCl₃): δ 149.6, 149.2; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₅₄H₅₇N₆O₈NaP: 971.3868; found 971.3875.

2.19. Preparation of 3'-O-[2-cyanoethyl(diisopropylamino)phosphino]-5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-trifluoromethylphenyl)diazenylphenyl]ethynyl-2'-deoxyuridine (24)

Under an argon atmosphere, to a solution of compound 19 (98 mg, 0.122 mmol) in dry CH₂Cl₂ (2 mL) was added N,N-diisopropylamine (63 µL, 0.366 mmol) and 2-cyanoethyl-N,N'-diisopropylchlorophosphoramidite (41 µL, 0.183 mmol) at room temperature, and the reaction mixture was stirred for 3 h. The resultant mixture was partitioned between AcOEt and H₂O. The separated organic layer was washed with saturated aqueous NaHCO₃, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (20:1 with 0.5% Et₃N) to give a 1:1 diastereomeric mixture of 24 (106 mg, 87%) as an orange foam. IR (KBr): ν 3640 (NH), 1674 (C=O), 1251 (N=N) cm⁻¹; [α]_D²⁴ 32.3 (c 1.00, CHCl₃); ¹H-NMR (300 MHz, CDCl₃): δ 8.39 (0.4H, s, H-6), 8.34 (0.6H, s, H-6), 8.01–7.95 (2H, m), 7.81–7.68 (4H, m), 7.51–7.45 (2H, m), 7.41–7.32 (4H, m), 7.21–7.13 (1H, m), 7.09–6.99 (2H, m), 6.85–6.75 (6H, m), 6.42–6.31 (1H, m, H-1'), 4.70–4.59 (1H, m, H-3'), 4.29–4.15 (1H, m, H-4'), 3.75–3.64 (4H, m, CH₂CH₂CN), 3.48 (6H, s, OMe), 3.43–3.18 (2H, m, H-5'), 2.70–2.60 (2H, m, H-2'), 2.50–2.33 (2H, m, ((CH₃)₂CH)₂N), 1.21–1.14 (7.2H, m, ((CH₃)₂CH)₂N), 1.10-1.04 (4.8H, m, ((CH₃)₂CH)₂N); ¹³C-NMR (125 MHz, CDCl₃): δ 161.2, 158.6, 154.3, 151.3, 149.1, 146.9, 144.4, 144.3, 142.7, 135.5, 135.4, 132.4, 132.3, 32.0, 129.97, 129.92, 129.7, 128.0, 127.9, 127.8, 127.0, 126.3, 126.2, 126.0, 125.9, 125.6, 123.0, 122.7, 120.3, 117.6, 117.4, 116.7, 113.3, 100.4, 100.2, 93.2, 87.2, 87.0, 86.3, 85.9, 85.8, 83.0, 77.4, 77.2, 77.0, 76.6, 75.0, 74.9, 73.7, 63.1, 58.4, 58.2, 58.1, 55.2, 55.4, 53.4, 50.7, 43.3, 43.2, 43.1, 43.0, 40.8, 24.8, 24.6, 24.56, 24.51, 24.47, 24.41, 22.8, 20.4, 20.3, 20.1; ³¹P-NMR (120 MHz, CDCl₃): δ 148.9, 148.4; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₅₄H₅₄N₆O₈F₃NaP: 1025.3585; found 1025.3596.

2.20. Preparation of 3'-O-[2-cyanoethyl(diisopropylamino)phosphino]-5'-O-(4,4'-dimethoxytrityl)-5-[4-(4-nitrophenyl)diazenylphenyl]ethynyl-2'-deoxyuridine (25)

Under an argon atmosphere, to a solution of compound 20 (111 mg, 0.142 mmol) in dry CH₂Cl₂ (2 mL) was added N,N-diisopropylamine (74 µL, 0.426 mmol) and 2-cyanoethyl-N,N’-diisopropylchlorophosphoramidite (48 µL, 0.213 mmol) at room temperature, and the reaction mixture was stirred for 3 h. The resultant mixture was partitioned between AcOEt and H₂O. The separated organic layer was washed with saturated aqueous NaHCO₃, followed by brine. The organic layer was dried (Na₂SO₄) and concentrated in vacuo. The residue was purified by silica gel column chromatography and eluted with CHCl₃/MeOH (20:1 with 0.5% Et₃N) to give a 2:1 diastereomeric mixture of 25 (137 mg, 99%) as a red foam. IR (KBr): ν 3576 (NH), 1712 (C=O), 1252 (N=N) cm⁻¹; [α]_D²⁴ −15.0 (c 1.00, CHCl₃); ¹H-NMR (500 MHz, CDCl₃): δ 9.45 (1H, br s, NH), 8.45–8.29 (3H, m), 8.04–7.99 (2H, m), 7.76–7.70 (2H, m), 7.53–7.47 (2H, m), 7.42–7.36 (4H, m), 7.31–7.26 (2H, m), 7.20–7.14 (1H, m), 7.11–7.03 (2H, m), 6.85–6.79 (4H, m), 6.42–6.35 (1H, m, H-1'), 4.71–4.63 (1H, m, H-4'), 4.30–4.25 (0.33H, m, H-3'), 4.24–4.20 (0.67H, m, H-3'), 3.89–3.75 (4H, m, CH₂CH₂CN), 3.72–3.69 (6H, m, OMe), 3.65–3.50 (2H, m, H-5'), 2.75–2.61 (2H, m, H-2'), 2.47–2.35 (2H, m, ((CH₃)₂CH)₂N), 1.22–1.16 (8H, m, ((CH₃)₂CH)₂N), 1.16–1.06 (4H, m, ((CH₃)₂CH)₂N); ¹³C-NMR (125 MHz, CDCl₃): δ 156.5, 153.3, 150.6, 146.2, 144.3, 143.7, 139.4, 137.8, 130.5, 127.5, 124.9, 123.0, 122.9, 122.0, 121.8, 119.7, 119.53, 119.52, 118.0, 112.6, 103.3, 93.2, 81.9 (d, J (C, P) = 3.5 Hz), 68.7, 68.4, 62.9, 62.8, 58.1, 58.0, 55.4, 54.9, 38.9 (d, J (C, P) = 5.0 Hz), 24.4, 24.3, 22.8, 22.7, 19.5 (d, J (C, P) = 7.0 Hz); ³¹P-NMR (200 MHz, CDCl₃): δ 149.6, 149.3; MALDI-TOF-HRMS m/z (MNa⁺) calcd for C₅₃H₅₄N₇O₁₀NaP: 1002.3567; found 1002.3570.

2.21. Synthesis of dU^Az-Modified Oligodeoxynucleotides

Solid-phase oligonucleotide synthesis was performed using commercially available reagents and phosphoramidites with activator 42 (Sigma-Aldrich) as the activator. para-Substituted-dU^Az phosphoramidites were chemically synthesized as described above. All of the reagents were assembled, and the ONs were synthesized according to the standard synthesis cycle (trityl on mode). Cleavage from the solid support and deprotection were accomplished with concentrated ammonium hydroxide solution at 55 °C for 12 h. The crude oligonucleotides were purified with Sep-Pak Plus C18 cartridges (Waters, MA, USA) followed by RP-HPLC on a XBridge^TM OST C18 column, 2.5 μm, 10 × 50 mm (Waters) using MeCN in 0.1 M triethylammonium acetate buffer (pH 7.0). The purified oligonucleotides were quantified by UV absorbance at 260 nm and confirmed by MALDI-TOF mass spectrometry (Table 1).

Table 1. Yields and MALDI-TOF MS data of dU^Az-modified ONs.

ON a	X	Yield/%	MALDI-TOF MS
			Calcd. [M-H]−	Found [M-H]−
28	dUAz-OMe	21	3852.6	3853.2
29	dUAz-Me	27	3836.6	3836.2
30	dUAz-CF3	15	3890.6	3890.2
31	dUAz-NO2	22	3866.6	3866.9
32	dUAz-pyridyl	21	3823.6	3824.2

^a ON sequence is 5'-d(GCGTTXTTTGCT)-3'.

Table 1. Yields and MALDI-TOF MS data of dU^Az-modified ONs.

ON a	X	Yield/%	MALDI-TOF MS
			Calcd. [M-H]−	Found [M-H]−
28	dUAz-OMe	21	3852.6	3853.2
29	dUAz-Me	27	3836.6	3836.2
30	dUAz-CF3	15	3890.6	3890.2
31	dUAz-NO2	22	3866.6	3866.9
32	dUAz-pyridyl	21	3823.6	3824.2

^a ON sequence is 5'-d(GCGTTXTTTGCT)-3'.

2.22. UV Melting Experiments

Equimolecular amounts of the target DNA/RNA and ONs were dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl to give a final strand concentration of 4.0 µM. The melting samples were denatured at 100 °C and annealed slowly to room temperature. Absorbance was recorded in the forward and reverse directions at temperatures of 5–90 °C at a rate of 0.5 °C/min.

2.23. Photo-Isomerization of dU^Az Analogues

ONs were dissolved in a 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl to give a final strand concentration of 4.0 µM. The ON solution was exposed to the appropriate wavelength of monochromic light for 10 seconds and subsequently analyzed by RP-HPLC. The ratio of cis/trans isomers was obtained from the HPLC peak areas at 260 nm.

2.24. Thermal Isomerization of cis-dU^Az Analogues

ONs were dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl to give a final strand concentration of 4.0 µM. The oligonucleotide solution was exposed to the appropriate wavelength of monochromic light for 10 seconds and subsequently heated to 60 °C. The change of absorbance at 365 nm or 400 nm was monitored by a UV-Vis spectrophotometer and plotted to calculate the half-life time of cis-dU^Az analogues.

2.25. Isothermal Titration Calorimetry

Prior to experiments, all solutions (titrant and titrand) were denatured at 100 °C and annealed slowly to room temperature. Titrations were carried out by injecting 2 µL portions of the complementary RNA strand into 250 µL of the dU^Az-modified ON solution in the calorimetric cell. The dU^Az-modified ON concentrations in the cell were 0.1 µM, and the solutions in the syringe were 1 µM. The equilibration period after each injection was 360 s. The measurements were performed with and without the photo-isomerization of dU^Az-modified ONs.

3. Results

3.1. Synthesis of Para-Substituted dU^Az Phosphoramidites and Modified Oligonucleotides

The synthetic route for para-substituted ethynyl-azobenzene derivatives is outlined in Scheme 1. 4-4-[(Trimethylsilyl)ethynylphenylazo]pyridine 2 was synthesized from the prepared 4-(4-iodophenylazo)pyridine 1 [18] through a palladium-catalyzed cross-coupling reaction [22] with trimethylsilylacetylene. TMS-protected ethynyl-azobenzene derivatives 2–4 [19] were converted to compounds 5–7 by removal of the silyl group. Compounds 8 and 9 were also synthesized according to the reported procedures [20,21].

Scheme 1. Route for the synthesis of para-substituted ethynyl-azobenzene derivatives.

Scheme 1. Route for the synthesis of para-substituted ethynyl-azobenzene derivatives.

The preparation of para-substituted dU^Az phosphoramidites commenced with the coupling of 4-ethynyl-azobenzene derivatives 5–9 with 2'-deoxy-5-iodouridine 10 (Scheme 2). Tritylation at the primary hydroxyl group of 11–15 with DMTrCl and phosphitylation at the secondary hydroxyl group yielded phosphoramidites 21–25. The amidites 21–25 were incorporated into ONs using conventional solid-phase phosphoramidite synthesis and purified by reverse-phase HPLC. The ON sequences used in this study are shown in Table 2.

Scheme 2. Route for the synthesis of para-substituted dU^Az phosphoramidites.

Scheme 2. Route for the synthesis of para-substituted dU^Az phosphoramidites.

Table 2. The oligonucleotides used in this study.

ON	Sequence	X
26	5'-d(GCGTTTTTTGCT)-3'	-
27	5'-d(GCGTTXTTTGCT)-3'	dUAz
28	5'-d(GCGTTXTTTGCT)-3'	dUAz-OMe
29	5'-d(GCGTTXTTTGCT)-3'	dUAz-Me
30	5'-d(GCGTTXTTTGCT)-3'	dUAz-CF3
31	5'-d(GCGTTXTTTGCT)-3'	dUAz-NO2
32	5'-d(GCGTTXTTTGCT)-3'	dUAz-pyridyl
33	5'-d(AGCAAAAAACGC)-3'	-
34	5'-r(AGCAAAAAACGC)-3'	-

Table 2. The oligonucleotides used in this study.

ON	Sequence	X
26	5'-d(GCGTTTTTTGCT)-3'	-
27	5'-d(GCGTTXTTTGCT)-3'	dUAz
28	5'-d(GCGTTXTTTGCT)-3'	dUAz-OMe
29	5'-d(GCGTTXTTTGCT)-3'	dUAz-Me
30	5'-d(GCGTTXTTTGCT)-3'	dUAz-CF3
31	5'-d(GCGTTXTTTGCT)-3'	dUAz-NO2
32	5'-d(GCGTTXTTTGCT)-3'	dUAz-pyridyl
33	5'-d(AGCAAAAAACGC)-3'	-
34	5'-r(AGCAAAAAACGC)-3'	-

3.2. Isomerization Properties of Para-Substituted dU^Az Analogues

The influence of para-substitution on the efficiency of the dU^Az cis-trans photo-isomerization in ONs was investigated by UV spectroscopy and HPLC analysis. The photo-isomer ratio was measured at 260 nm because ONs 27–32 have the same extinction coefficient before and after irradiation at this wavelength (Figure S1). The wavelengths of light used for photo-isomerization and their efficiencies are shown in Table 3. Ten seconds of irradiation was confirmed to be enough for reaching photostationary state (PSS); the cis/trans ratios of ONs 27–32 after longer irradiation times (30 and 120 min) were the same as that after 10 s of irradiation. Compared with ON 27 containing dU^Az, ONs 28 and 29 containing electron-donating substituted dU^Az were photo-isomerized from trans to cis more effectively. Irradiation of ON 28 (X = dU^Az−OMe) using 365 nm afforded a mixture with 79% of the cis-form. In contrast, ON 30 bearing the electron-withdrawing CF₃ group showed a lower trans to cis isomerization efficiency. However, the cis to trans isomerization efficiency was higher than that of ON 27 containing dU^Az. ONs 31 and 32 containing dU^Az-NO₂ and dU^Az-pyridyl were barely photo-isomerized. The low efficiency of photo-isomerization was due to the low electron density at the azo-structure [23]. In case of dU^Az-pyridyl, electrons tend to be delocalized at the nitrogen of the pyridine ring [24]. ONs 28–31 were photo-isomerized trans to cis and vice versa for at least three cycles without any attenuation of efficiency.

Table 3. Photo-isomerization properties of para-substituted dU^{Az a}.

ON b	X	Before Photo-Irradiation	Trans to Cis		Cis to Trans
		Trans-Isomer/% b	Wavelength c/nm a	cis-isomer/% d	Wavelengthc /nm a	Trans-Isomer/% d
27	dUAz	82	365	58	450	82
28	dUAz-OMe	59	365	79	450	59
29	dUAz-Me	77	365	61	450	77
30	dUAz-CF3	91	365	28	450	91
31	dUAz-NO2	96	400	14	450	96
32	dUAz-pyridyl	100	-	0	-	-

^a Conditions: 10 mM Na₂HPO₄ (pH 7.0), 100 mM NaCl, 4 μM ON; ^b ON sequence is 5'-d(GCGTTXTTTGCT)-3'; ^c The wavelength of monochromic light corresponds to π-π * transition (trans to cis) or n-π * (cis to trans). Photo-irradiation was performed for 10 s at room temperature; ^d The ratio of isomers is calculated from the peak area of HPLC traces (260 nm).

Table 3. Photo-isomerization properties of para-substituted dU^{Az a}.

ON b	X	Before Photo-Irradiation	Trans to Cis		Cis to Trans
		Trans-Isomer/% b	Wavelength c/nm a	cis-isomer/% d	Wavelengthc /nm a	Trans-Isomer/% d
27	dUAz	82	365	58	450	82
28	dUAz-OMe	59	365	79	450	59
29	dUAz-Me	77	365	61	450	77
30	dUAz-CF3	91	365	28	450	91
31	dUAz-NO2	96	400	14	450	96
32	dUAz-pyridyl	100	-	0	-	-

^a Conditions: 10 mM Na₂HPO₄ (pH 7.0), 100 mM NaCl, 4 μM ON; ^b ON sequence is 5'-d(GCGTTXTTTGCT)-3'; ^c The wavelength of monochromic light corresponds to π-π * transition (trans to cis) or n-π * (cis to trans). Photo-irradiation was performed for 10 s at room temperature; ^d The ratio of isomers is calculated from the peak area of HPLC traces (260 nm).

Next, the thermo-stability of ONs with various para-substituted dU^Az in the cis-form was investigated [25]. The ONs modified with dU^Az analogues were cis-isomerized by the appropriate wavelength of light and subsequently heated to 60 °C. The change in absorbance at 365 nm was monitored by a UV-Vis spectrophotometer and plotted to calculate the half-life of the cis-isomers (Table 4). With regard to ONs 28 and 29 containing electron-donating substituted dU^Az, thermo-stabilities of the cis-isomer were significantly decreased. This phenomenon could be attributed to the para-electron-donating substituent and ethynyl linker creating a push-pull configuration of the azobenzene moiety, which is well known in easily thermo-isomerized azobenzene derivatives [18]. On the other hand, ONs 30 and 31 containing electron-withdrawing substituted dU^Az showed long half-life times. The cis-isomer of ON 31 (X = dU^Az−NO₂) scarcely isomerized from cis to trans even when heated to 60 °C for more than 10 h. In the case of ONs 30 and 31, both the ethynyl linker and para-substitution work as 4,4'-di-electron-withdrawing moieties. The 4,4'-di-electron-withdrawing substitution of azobenzene strengthens the thermal stability of the cis-form [26].

Table 4. Half-life time of the cis-form of para-substituted-dU^Az at 60 °C ^a.

ON b	X	t1/2 of cis-isomer/min. c,d
27	dUAz	44
28	dUAz-OMe	9
29	dUAz-Me	24
30	dUAz-CF3	65
31	dUAz-NO2	>300
32	dUAz-pyridyl	- e

^a Conditions: 10 mM Na₂HPO₄ (pH 7.0), 100 mM NaCl, 4 μM ON; ^b ON sequence is 5'-d(GCGTTXTTTGCT)-3'; ^c trans- ONs were photo-isomerized to the cis-form by a 10 second irradiation of 365 nm (ONs 27-30) or 400 nm (ON 31) monochromic light; ^d Half-life times of cis-isomers were measured at 60 °C; ^e t_1/2 could not be determined because dU^Az-pyridyl did not isomerize to the cis-form.

Table 4. Half-life time of the cis-form of para-substituted-dU^Az at 60 °C ^a.

ON b	X	t1/2 of cis-isomer/min. c,d
27	dUAz	44
28	dUAz-OMe	9
29	dUAz-Me	24
30	dUAz-CF3	65
31	dUAz-NO2	>300
32	dUAz-pyridyl	- e

^a Conditions: 10 mM Na₂HPO₄ (pH 7.0), 100 mM NaCl, 4 μM ON; ^b ON sequence is 5'-d(GCGTTXTTTGCT)-3'; ^c trans- ONs were photo-isomerized to the cis-form by a 10 second irradiation of 365 nm (ONs 27-30) or 400 nm (ON 31) monochromic light; ^d Half-life times of cis-isomers were measured at 60 °C; ^e t_1/2 could not be determined because dU^Az-pyridyl did not isomerize to the cis-form.

The para-substitution would sterically and electronically affect the stability of the duplexes formed by dU^Az analogue-modified ONs with complementary strands. Therefore, we investigated the T_m values of the duplexes formed between ON 28–32 and full match DNA or RNA (Table 5 and Table 6). It was found that para-substitution of dU^Az slightly destabilized the DNA/DNA duplexes but had little influence on the DNA/RNA duplexes.

Table 5. T_m values of duplexes formed by dU^Az analogue-modified ONs with complementary DNA ^a.

Duplex	X	Tm [°C]	ΔTm [°C] b
27/33	dUAz	47	-
28/33	dUAz-OMe	44	−3
29/33	dUAz-Me	45	−2
30/33	dUAz-CF3	45	−2
31/33	dUAz-NO2	45	−2
32/33	dUAz-pyridyl	44	−3

^a Conditions: 10 mM Na₂HPO₄ (pH 7.0), 100 mM NaCl, 4 μM ON. The T_m values given are the average of at least three data points. The T_m value of natural DNA 26/DNA 33 duplex is 52 °C; ^b The ΔT_m indicates the difference from ON 27/DNA 33 duplex.

Table 5. T_m values of duplexes formed by dU^Az analogue-modified ONs with complementary DNA ^a.

Duplex	X	Tm [°C]	ΔTm [°C] b
27/33	dUAz	47	-
28/33	dUAz-OMe	44	−3
29/33	dUAz-Me	45	−2
30/33	dUAz-CF3	45	−2
31/33	dUAz-NO2	45	−2
32/33	dUAz-pyridyl	44	−3

^a Conditions: 10 mM Na₂HPO₄ (pH 7.0), 100 mM NaCl, 4 μM ON. The T_m values given are the average of at least three data points. The T_m value of natural DNA 26/DNA 33 duplex is 52 °C; ^b The ΔT_m indicates the difference from ON 27/DNA 33 duplex.

Table 6. T_m values of duplexes formed by dU^Az analogue-modified ONs with complementary RNA ^a.

Duplex	X	Tm [°C]	ΔTm [°C] c
27/34	dUAz	42	-
28/34	dUAz-OMe	41	−1
29/34	dUAz-Me	43	+1
30/34	dUAz-CF3	41	−1
31/34	dUAz-NO2	41	−1
32/34	dUAz-pyridyl	42	0

^a Conditions: 10 mM Na₂HPO₄ (pH 7.0), 100 mM NaCl, 4 μM ON. The T_m values given are the average of at least three data points. The T_m value of natural DNA 26/RNA 34 duplex is 47 °C; ^b The ΔT_m indicates the difference from ON 27/RNA 34 duplex.

Table 6. T_m values of duplexes formed by dU^Az analogue-modified ONs with complementary RNA ^a.

Duplex	X	Tm [°C]	ΔTm [°C] c
27/34	dUAz	42	-
28/34	dUAz-OMe	41	−1
29/34	dUAz-Me	43	+1
30/34	dUAz-CF3	41	−1
31/34	dUAz-NO2	41	−1
32/34	dUAz-pyridyl	42	0

^a Conditions: 10 mM Na₂HPO₄ (pH 7.0), 100 mM NaCl, 4 μM ON. The T_m values given are the average of at least three data points. The T_m value of natural DNA 26/RNA 34 duplex is 47 °C; ^b The ΔT_m indicates the difference from ON 27/RNA 34 duplex.

The changes in T_m values of dU^Az-modified duplexes upon photo-irradiation could not be determined due to the low thermal stability of the cis-form itself (ONs 28 and 29, Table 4) or low trans to cis photo-isomerization efficiency (ONs 30–32, Table 3). Therefore, we investigated the changes in the thermodynamic stabilities of 12-bp DNA/RNA duplexes containing dU^Az-OMe and dU^Az-Me upon photo-irradiation by isothermal titration calorimetry (ITC) at 25 °C (Table 7). 1 µM of complementary RNA strand in the syringe was titrated to 0.1 µM dU^Az analogue-modified ON in the cell and the heat transfer during binding was measured to determine the thermodynamic parameters of DNA/RNA duplex formation. The experiments were conducted for dU^Az analogue-modified ONs both before and after irradiation. Duplex 28/34 showed a lower Gibbs free energy change (ΔG) after photo-irradiation than that before photo-irradiation. This trend was also observed in duplex 29/34. These results indicate that the hybridization abilities of ONs modified with dU^Az-OMe and dU^Az-Me can be regulated by the appropriate wavelength of light, as with the case of dU^Az.

Table 7. Thermodynamic parameters of duplexes formed by dU^Az analogue-modified ONs with complementary RNA ^a.

Duplex	X	Ka × 107 [M]	∆H [kcal/mol]	∆S [kcal/mol/deg]	ΔG [kcal/mol]
28/34	dUAz-OMe	2.65 ± 0.317	−54.1 ± 0.839	−0.148	−9.97
	dUAz-OMe b	3.03 ± 0.600	−54.8 ± 1.50	−0.150	−10.1
29/34	dUAz-Me	2.17 ± 0.414	−55.3 ± 1.60	−0.152	−9.98
	dUAz-Me b	4.15 ± 0.798	−52.1 ± 1.20	−0.140	−10.4

^a Conditions: 5 mM Na₂HPO₄ (pH 7.0), 50 mM NaCl, 0.1 µM dU^Az modified ON (titrand); 5 mM Na₂HPO₄ (pH 7.0), 50 mM NaCl, 1 µM complementary RNA strand (titrant). ^b After photo-irradiation at 365 nm for 10 s.

Table 7. Thermodynamic parameters of duplexes formed by dU^Az analogue-modified ONs with complementary RNA ^a.

Duplex	X	Ka × 107 [M]	∆H [kcal/mol]	∆S [kcal/mol/deg]	ΔG [kcal/mol]
28/34	dUAz-OMe	2.65 ± 0.317	−54.1 ± 0.839	−0.148	−9.97
	dUAz-OMe b	3.03 ± 0.600	−54.8 ± 1.50	−0.150	−10.1
29/34	dUAz-Me	2.17 ± 0.414	−55.3 ± 1.60	−0.152	−9.98
	dUAz-Me b	4.15 ± 0.798	−52.1 ± 1.20	−0.140	−10.4

^a Conditions: 5 mM Na₂HPO₄ (pH 7.0), 50 mM NaCl, 0.1 µM dU^Az modified ON (titrand); 5 mM Na₂HPO₄ (pH 7.0), 50 mM NaCl, 1 µM complementary RNA strand (titrant). ^b After photo-irradiation at 365 nm for 10 s.

4. Discussion

As shown above, para-substitution strongly affected the photo-isomerization efficiency of dU^Az. It is important to modulate the electron nature of the azo structure for the optimization of dU^Az photo-isomerization properties. Notably, the electron-donating OMe group could strongly bias the equilibrium to the cis-isomer after irradiation at 365 nm. This would contribute to the enhancement of the properties of dU^Az in the “on” state. On the other hand, electron-withdrawing CF₃ and NO₂ groups strongly biased the equilibrium to the trans form before irradiation. This could suppress the undesired expression of dU^Az properties such as the hybridization ability before irradiation.

Modulating the rate of the thermal relaxation of the switched state is also very important because it realizes the spatiotemporal control of nucleic acid properties [27]. The thermal stability of the cis-isomer influences the method of utilization of photo-switches. cis-ON 28 (X = dU^Az-OMe) showed the shortest thermal half-life among the dU^Az analogue-modified ONs that we evaluated. This property is crucial for a photo-switch that thermally relaxes the hybridization ability. This would enable repeated activation with spatiotemporal resolution. On the other hand, ONs 30 and 31 (X = dU^Az–CF₃ and dU^Az–NO₂) have a long half-life of the cis-form. In this case, it is possible to turn the hybridization property of dU^Az on and off using the appropriate wavelength of light.

The para substitutions decreased the affinity of ONs modified with dU^Az analogues against DNA but had little influence on that against RNA. These results suggest that para substitutions influence the duplex stability not electronically but sterically. The difference in the stability between DNA/DNA duplexes and DNA/RNA duplexes is attributed to the difference in the duplex structures. DNA/DNA duplexes tend to have B-form structures whereas DNA/RNA duplexes tend to have more A-form structures. Thus, the DNA/DNA duplex has a comparatively shallow major groove and para-substitution of the azobenzene moiety may sterically destabilize the duplex. The destabilization of duplexes in the trans-form may lead to an increase in the difference of hybridization ability between trans- and cis-dU^Az and improve the property of dU^Az for photo-switching hybridization.

We introduced various para substitutions into dU^Az and modulated the photo- and thermo-sensitivities for trans-cis isomerization. Our results represent an important strategy for optimization of the properties of photo-switches. For example, dU^Az-OMe showed better trans to cis isomerization efficiency and lower thermal stability of the cis-isomer than dU^Az. This analogue could be utilized as a useful photo-switch for situations in which a fast drop in activity is desired. dU^Az-modified ON shows an interesting hybridization property, namely the affinity of the cis-form against complementary strands is higher than that of the trans-form, different from most other photo-switches [16]. Para-substituted dU^Az analogues have the potential for unique nucleic acid probes and drugs that reversibly capture the target DNA and RNA with spatiotemporal control.

5. Conclusions

We synthesized photo-isomeric dU^Az analogues bearing various para-substituted azobenzene derivatives and investigated the influence of the para substitution on the photo-isomerization properties, thermal stabilities of the cis-isomer, and hybridization abilities. Electron-withdrawing substituents on dU^Az lowered the efficiency of photo-isomerization while improving the thermal stability of the cis-isomer. On the other hand, electron-donating substituents improved the efficiency of photo-isomerization while decreasing the thermal stability of the cis-isomer. According to UV-melting experiments, para-substitution of dU^Az tended to destabilize DNA/DNA duplexes but had little effect on the stabilities of the DNA/RNA duplexes regardless of the nature of the substituents. ITC experiments revealed that the duplex formed between dU^Az-OMe-modified ON and complementary RNA are more stable after photo-irradiation than that before photo-irradiation. This trend was observed in the duplex formed between dU^Az-Me-modified ON and complementary RNA. These results indicated that dU^Az-OMe and dU^Az-Me could regulate the stability of the DNA/RNA duplex by photo-irradiation similar to dU^Az and appropriate substitution could optimize the properties of dU^Az as light-responsive nucleic acid probes.