Triplex- and Duplex-Forming Abilities of Oligonucleotides Containing 2′-Deoxy-5-trifluoromethyluridine and 2′-Deoxy-5-trifluoromethylcytidine

Abstract

A facile synthesis of 2′-deoxy-5-trifluoromethyluridine and 2′-deoxy-5-trifluoromethylcytidine phosphoramidites from commercially available 2′-deoxyuridine and 2′-deoxycytidine was achieved, respectively. The obtained phosphoramidites were incorporated into oligonucleotides, and their binding affinity to double-stranded DNA (dsDNA) and single-stranded RNA (ssRNA) was evaluated by UV-melting experiments. The triplex-forming abilities of oligonucleotides including 5-trifluoromethylpyrimidine nucleobases with dsDNA were decreased. Especially, the stability of the triplex containing a trifluoromethylcytosine (^CF3C)-GC base triplet was low, likely due to the low pK_a of protonated ^CF3C by the electron-withdrawing trifluoromethyl group. A slight decrease in stability of the duplex formed with ssRNA by oligonucleotides including 5-trifluoromethylpyrimidine nucleobases was only observed, suggesting that they might be applicable to various ssRNA-targeted technologies using features of fluorine atoms.

Chemically modified oligonucleotides are expected to be the next-generation medicine owing to their specific recognition of nucleic acid sequences through formation of base pairs.^1,2) In particular, artificial oligonucleotides with high binding affinity towards double-stranded DNA (dsDNA) and single-stranded RNA (ssRNA) can be applied to antigene and antisense technologies. Therefore, chemical modifications of nucleobase, sugar and phosphate moieties have been investigated.^3,4) Among these modifications, introduction of a substituent at the 5-position of pyrimidine bases is particularly useful because the substituent does not interfere with Watson–Crick or Hoogsteen base pairings, i.e., duplex or triplex formations.⁵⁾ 5-Methylcytosine (^mC) is known to increase the stability of triplexes through stable ^mC-GC base triplet formation^5,6) (Fig. 1). The stabilization results from hydrophobic effects generated by a release of hydrating water molecules from the double helix to the bulk. Duplex stability is also enhanced by ^mC due to an increase in hydrophobic interactions with the neighboring bases^5,7,8) (Fig. 1). Analogous to ^mC, 5-bromocytosine (^BrC) stabilizes duplex formation when ^BrC-modified oligonucleotides are used.^5,8) Introduction of substituents at the C-5 position of uracil, such as a methyl group or halogen atoms, increases the stability of triplexes and duplexes formed^5,9–11) (Fig. 1). These findings indicate that the binding affinity of oligonucleotides can be regulated by hydrophobic and electronic properties of substituents at the C-5 positions of pyrimidine nucleobases.

Fig. 1. Base Triplets and Base Pairs Including 5-Substituted Pyrimidine Nucleobases, and the Order of Stabilization

Pyrimidine bases containing a trifluoromethyl (CF₃) group at the 5-position have been used for numerous nucleic acid-based technologies. For example, trifluridine, a potent inhibitor of thymidylate synthase, has been used as an antitumor and anti-herpesvirus drug in clinical practice.¹²⁾ Furthermore, Fujimoto’s group recently developed not only a DNA probe containing 5-CF₃-uracil (^CF3U) or 5-CF₃-cytosine (^CF3C) to detect DNA B–Z equilibrium using ¹⁹F-NMR,¹³⁾ but also a photo-cross-linking method using ^CF3U and 3-cyanovinylcarbazole.¹⁴⁾ Although CF₃-substituted pyrimidine nucleobases are valuable, the hybridization properties of their modified oligonucleotides have not been studied in detail. To our knowledge, there are only three reports, by the Lown,¹⁵⁾ Sugimoto¹⁶⁾ and Sigurdsson¹⁷⁾ groups, evaluating the duplex-forming abilities of oligonucleotides containing ^CF3U with ssDNA. In general, the replacement of a methyl group by a CF₃ group can modulate hydrophobic and electronic properties¹⁸⁾; thus, we were interested in the duplex- and triplex-forming abilities of oligonucleotides including 5-CF₃-pyrimidine nucleobases with ssRNA and dsDNA, respectively. Herein, we describe the syntheses of oligonucleotides containing ^CF3U and ^CF3C, and their binding affinities with dsDNA and ssRNA.

Results and Discussion

Syntheses of ^CF3U- and ^CF3C-Modified Oligonucleotides

^CF3U and ^CF3C phosphoramidites were synthesized as depicted in Charts 1 and 2, respectively. For the introduction of a CF₃ group at the 5-position of a pyrimidine base, we used direct radical trifluoromethylation developed by Baran’s group.¹⁹⁾ Treatment of 2′-deoxyuridine (1) with CF₃SO₂Na and t-BuOOH in H₂O afforded 2′-deoxy-5-CF₃-uridine (2) in 59% yield. Protection of the primary alcohol of 2 by 4,4′-dimethoxytrityl chloride (DMTrCl) followed by phosphitylation of 3 gave the ^CF3U phosphoramidite 4.

Chart 1. Synthesis of ^CF3U Phosphoramidite 4

Chart 2. Synthesis of ^CF3C Phosphoramidite 11

The protected ^CF3C phosphoramidite 11 was synthesized from commercially available 2′-deoxycytidine (5). Radical trifluoromethylation of 5 afforded 2′-deoxy-5-CF₃-cytidine (6) in 55% yield. Subsequent silylation of 3′- and 5′-hydroxyl groups gave compound 7, which was treated under N-acetylation conditions to furnish mono-acetylated 8 and di-acetylated 9 in 46 and 51% yields, respectively. Treatment of a mixture of 8 and 9 with tetrabutylammonium fluoride (TBAF) followed by dimethoxytritylation led to compound 10 in 53% yield over 2 steps. Finally, the synthesis of phosphoramidite 11 was carried out by phosphitylation of 10. The phosphoramidites 4 and 11 were incorporated into oligonucleotides using an automated DNA synthesizer with common phosphoramidite chemistry, and their oligonucleotides were purified by reversed-phase HPLC (RP-HPLC) and characterized by electrospray ionization-time-of-flight (ESI-TOF) mass spectrometry.

UV-Melting Experiments

Initially, the triplex-forming ability of oligonucleotides containing ^CF3U and ^CF3C with dsDNA targets (YZ=AT, GC) was evaluated by UV-melting experiments and compared to that of 5-methyl and 5-unsubstituted congeners (Table 1). Unfortunately, the melting temperature (T_m) of the triplex-forming oligonucleotide (TFO) 12c (X=^CF3U) for the AT base pair decreased by 4–5°C compared to that of TFOs 12a (X=U) and 12b (X=T). For the GC base pair, the T_m of the ^CF3C-modified TFO 12f was 18°C, which was a drastic decrease of 11–13°C compared to TFOs 12d (X=C) and 12e (X=^mC). To investigate the cause of this decrease, we measured the pK_a of 6 (Fig. 2). The absorbance at 277 nm (A₂₇₇), which is the maximum absorption wavelength of 6 at pH 1.92, was plotted at each pH unit. The curve-fitting analysis based on the Henderson–Hasselbalch equation demonstrated that the pK_a of protonated 6 was approximately 2.6 (see Experimental for details). Therefore, the protonated form of 6 was found to be much more acidic than those of 2′-deoxycytidine 5 (pK_a=4.4) and 2′-deoxy-5-methylcytidine (pK_a=4.5).²⁰⁾ This result implies that protonation of the N-3 position of ^CF3C would not occur at neutral pH; therefore, recognition of the ^CF3C base for a GC base pair would be limited to one hydrogen bond. On the other hand, the pK_a (7.4) of ^CF3U is also lower than those of uracil (9.5) and thymine (9.8).¹²⁾ A partial deprotonation of ^CF3U under the measurement conditions might significantly affect a decrease in the T_m of triplex including a ^CF3U-AT base triplet. Since it is known that T or C can interact with a CG base pair through a single hydrogen bond,²¹⁾ the triplex-forming ability towards dsDNA including a CG base pair was investigated. Interestingly, whereas the TFO 12c containing ^CF3U led to a decrease (3–4°C) in the T_m compared with those containing U (12a) and T (12b), the T_m of the TFO 12f containing ^CF3C was almost same as those containing C (12d) and ^mC (12e). The T_m of the triplex including a ^CF3C-GC base triplet was comparable to that including a ^CF3C-CG base triplet, which also strongly suggests that ^CF3C forms only a single hydrogen bond with a GC base pair at neutral pH. In addition, the obtained results suggest that ^CF3C can be a newly promising scaffold for CG base pair recognition in triplex DNA formation though C or ^mC analogs have been explored towards recognition of a CG base pair.^4,22,23)

Table 1. T_m (°C) of Triplexes between Oligonucleotides 12a–f and Hairpin dsDNA^a)

TFO (X)	YZ		TFO (X)	YZ
	AT	CG		GC	CG
12a (U)	34	20	12d (C)	29	18
12b (T)	35	19	12e (mC)	31	18
12c (CF3U)	30	16	12f (CF3C)	18	17

a) Conditions: 10 mM sodium cacodylate buffer (pH 7.4), 100 mM KCl, 50 mM MgCl₂ and 1.89 µM of each oligonucleotide. C=2′-Deoxy-5-methylcytidine.

Fig. 2. The Plot of the Absorbance at 277 nm (A₂₇₇) vs. pH and the Fitting Curve (R²=0.98)

Next, the duplex-forming ability of ^CF3U and ^CF3C modified oligonucleotides with ssRNA was examined (Table 2). T_ms of the oligonucleotides containing 5-methyl analogs, 12b (X=T) and 12e (X=^mC), increased by 1°C compared to those of the oligonucleotides containing 5-unsubstituted analogs, 12a (X=U) and 12d (X=C). In contrast, the T_ms of ^CF3U- and ^CF3C-modified oligonucleotides, 12c and f, were slightly lower than those of 12a and d.

Table 2. T_m (°C) of Duplexes between Oligonucleotides 12a–f and ssRNA^a)

ssDNA (X)	Tm (Y=A)	ssDNA (X)	Tm (Y=G)
12a (U)	50	12d (C)	56
12b (T)	51	12e (mC)	57
12c (CF3U)	48	12f (CF3C)	55

a) Conditions: 10 mM sodium cacodylate buffer (pH 7.4), 100 mM NaCl and 3 µM of each oligonucleotide. C=2′-Deoxy-5-methylcytidine.

The duplex-forming ability of oligonucleotides containing three ^CF3U and ^CF3C bases with ssRNA was also investigated (Table 3, Fig. S1). The T_m of 12g bearing three U bases was 56°C, which was comparable to that of the thymine congener 12e. On the other hand, the triply ^CF3U-modified oligonucleotide 12h, the T_m of which was 51°C, destabilized the duplex by 1.7°C per modification. Since it was previously reported by Sigurdsson’s group that replacement of thymine by ^CF3U led to a decrease of 3.1°C in the T_m of the duplex with ssDNA,¹⁷⁾ these results demonstrate that the duplex-forming ability of ^CF3U-modified oligonucleotide with ssRNA is similar to its behavior with ssDNA. Replacement of three C bases (12i) with ^mC ones (12e) resulted in an increase in the T_m by 2°C. The triply ^CF3C-modified oligonucleotide 12j exhibited slightly decreased binding affinity with a T_m of 53°C. The ΔT_m per modification of ^CF3C-modified oligonucleotide was –0.7°C, which was slightly improved compared to that of ^CF3U (−1.7°C). These results suggest that an oligonucleotide containing ^CF3C as well as that containing ^CF3U might become an important material for nucleic acid technologies, especially targeting ssRNA, using features of fluorine atoms.

Table 3. T_m (°C) of Duplexes between Oligonucleotides 12e and g–j, and ssRNA^a)

Sequence of ssDNA	Tm	ΔTm per mod.
5′-TTTTUCUCUCTCTCT-3′ (12g)	56	—
5′-TTTTTCTCTCTCTCT-3′ (12e)	57	+0.3
5′-TTTTCF3UCCF3UCCF3UCTCTCT-3′ (12h)	51	−1.7
5′-TTTTTCTCTCTCTCT-3′ (12i)	55	—
5′-TTTTTCTCTCTCTCT-3′ (12e)	57	+0.7
5′-TTTTTCF3CTCF3CTCF3CTCTCT-3′ (12j)	53	−0.7

a) Conditions: 10 mM sodium cacodylate buffer (pH 7.4), 100 mM NaCl and 3 µM of each oligonucleotide. C=2′-Deoxy-5-methylcytidine. The sequence of target ssRNA is 5′-r(AGAGAGAGAGAAAAA)-3′.

Conclusion

^CF3U and ^CF3C phosphoramidites were easily synthesized and incorporated into 15-mer oligonucleotides. The UV-melting experiments revealed that introduction of a CF₃ group at the C-5 position of pyrimidine bases resulted in a decrease in the triplex-forming affinity with dsDNA in comparison with that observed in methyl and unsubstituted analogs. Although the duplex-forming ability of oligonucleotides including ^CF3U and ^CF3C with ssRNA also slightly decreased, the stability of the duplex was considered to be sufficiently practicable. Therefore, we expect that these oligonucleotides would be applicable to ssRNA-targeted technologies using features of fluorine atoms such as ¹⁹F-NMR spectroscopy and ¹⁸F-labeled positron emission tomography (PET) imaging. Further studies on the application of ^CF3U- and ^CF3C-modified oligonucleotides are currently underway in our laboratory.

Experimental

General

All moisture-sensitive reactions were conducted in well-dried glassware under Ar atmosphere. Anhydrous CH₂Cl₂, tetrahydrofuran (THF), pyridine and N,N-dimethylformamide (DMF) were used as purchased. ¹H-NMR, ¹³C-NMR, ¹⁹F-NMR and ³¹P-NMR spectra were recorded on a Bruker AVANCE III HD 500 equipped with a BBO cryoprobe or Agilent 400-MR. Chemical shift values were reported in ppm downfield from internal tetramethylsilane (δ=0.00 ppm) for ¹H-NMR, residual CDCl₃ (δ=77.00 ppm) for ¹³C-NMR, internal hexafluorobenzene (δ=0.00 ppm) for ¹⁹F-NMR, and external 5% H₃PO₄ (δ=0.00 ppm) for ³¹P-NMR. IR spectra were recorded on a JASCO FT/IR-4200 spectrometer. High-resolution mass spectrometry (HR-MS) was performed on a Waters SYNAPT G2-Si (Quadrupole/TOF). For column chromatography, silica gel PSQ 60B (Fuji Silysia, Japan) was used. The progress of the reaction was monitored by analytical TLC on pre-coated aluminum sheets (Silica gel 60 F₂₅₄ by Merck, Germany). For HPLC, a JASCO EXTREMA (PU-4180, CO-4060 and UV-4075) with a fraction collector CHF122SC (ADVANTEC), was used. Syntheses of oligonucleotides were performed on an automated DNA synthesizer (Gene Design nS-8II). UV-melting experiments were carried out using JASCO V-730 UV/Vis spectrophotometer equipped with a T_m analysis accessory. Solution pH values were measured with a pH meter LAQUAact D-41 (HORIBA, Japan).

Synthesis of Phosphoramidites 4 and 11

2′-Deoxy-5-trifluoromethyluridine (2)

To a solution of 2′-deoxyuridine (1, 381 mg, 1.67 mmol) and CF₃SO₂Na (782 mg, 5.01 mmol) in H₂O (5 mL), t-BuOOH (70% solution in H₂O, 0.81 mL, 8.35 mmol) was slowly added at 0°C. The reaction mixture was then warmed up to room temperature. After being stirred for 3 h, the reaction mixture was concentrated in vacuo. The crude residue was purified by column chromatography (CHCl₃–MeOH=10 : 1 to 5 : 1) to give compound 2 as a white solid (264 mg, 59%). The NMR spectral data were identical to those reported in the literature.¹⁹⁾

2′-Deoxy-5′-O-(4,4′-dimethoxytrityl)-5-trifluoromethyluridine (3)

To a solution of compound 2 (70 mg, 0.24 mmol) in pyridine, DMTrCl (96 mg, 0.28 mmol) was added at room temperature under Ar atmosphere. After being stirred for 41.5 h, the reaction was quenched with MeOH and then concentrated in vacuo. The crude residue was purified by column chromatography (CHCl₃–MeOH=100 : 1) to give compound 3 as a white solid (101 mg, 72%). The NMR spectral data were identical to those reported in the literature.²⁴⁾

3′-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-trifluoromethyluridine (4)

To a solution of compound 3 (232 mg, 0.39 mmol) and i-Pr₂NEt (0.41 mL, 2.34 mmol) in CH₂Cl₂ (5.0 mL), i-Pr₂NP(Cl)OCH₂CH₂CN (0.13 mL, 0.59 mmol) was added at 0°C under Ar atmosphere. The reaction mixture was stirred at room temperature for 3 h. After addition of sat. NaHCO₃ aq., the reaction mixture was extracted with AcOEt. The combined organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated in vacuo. The crude residue was purified by column chromatography (hexane–AcOEt=2 : 1) to give compound 4 as a white form (202 mg, 65%). ¹H-NMR (400 MHz, CDCl₃) δ: 1.07 (3H, d, J=7.0 Hz), 1.15–1.18 (9H, m), 2.19–2.28 (1H, m), 2.44 (1H, t, J=6.5 Hz), 2.62 (1H, t, J=6.0 Hz), 2.62 and 2.70 (1H, ddd, J=14.0, 5.5, 2.5 Hz), 3.31 (1H, td, J=10.5, 4.0 Hz), 3.40 and 3.45 (1H, dd, J=10.5, 3.5 Hz), 3.51–3.87 (10H, m), 4.20–4.28 (1H, m), 4.49–4.55 (1H, m), 6.20 and 6.22 (1H, dd, J=5.5, 5.5 Hz), 6.80–6.85 (4H, m), 7.20–7.30 (7H, m), 7.37–7.39 (2H, m), 8.12 and 8.17 (1H, s). ¹⁹F-NMR (376 MHz, CDCl₃) δ: 98.42, 98.45. ³¹P-NMR (162 MHz, CDCl₃) δ: 148.7, 149.1. HR-MS (ESI-TOF): Calcd for C₄₀H₄₇F₃N₄O₈P [M+H]⁺ 799.3084. Found 799.3088.

2′-Deoxy-5-trifluoromethylcytidine (6)

To a solution of 2′-deoxycytidine (5, 909 mg, 4.00 mmol) and CF₃SO₂Na (1.87 g, 12.0 mmol) in H₂O (8 mL), t-BuOOH (70% solution in H₂O, 2.72 mL, 20.0 mmol) was slowly added at 0°C. After being stirred for 4 h at room temperature, the reaction mixture was quenched with sat. NaHCO₃ aq. and concentrated in vacuo. The residue was filtered through a short pad of silica gel and the filtrate was concentrated in vacuo. The crude residue was purified by column chromatography (CHCl₃–MeOH=10 : 1 to 5 : 1) to give compound 6 as a white solid (650 mg, 55%). The NMR spectral data were identical to those reported in the literature.²⁵⁾

3′,5′-O-Bis(tert-butyldimethylsilyl)-2′-deoxy-5-trifluoromethylcytidine (7)

To a solution of compound 6 (495 mg, 1.68 mmol) in DMF (15 mL), imidazole (686 mg, 10.1 mmol) and TBSCl (758 mg, 5.03 mmol) were added at room temperature under Ar atmosphere. After being stirred for 12 h, the reaction mixture was quenched with MeOH and extracted with AcOEt. The combined organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated in vacuo. The crude residue was purified by column chromatography (hexane–AcOEt=3 : 2) to give compound 7 as a white solid (659 mg, 75%). IR (attenuated total reflectance (ATR)) cm⁻¹: 3075, 2953, 2930, 2858, 1660, 1514, 1494, 1472, 1417, 1353, 1324, 1279, 1255. ¹H-NMR (500 MHz, CDCl₃) δ: 0.07 (6H, s), 0.08 (6H, s), 0.88 (9H, s), 0.89 (9H, s), 1.94–1.99 (1H, m), 2.53–2.58 (1H, m), 3.76 (1H, dd, J=11.5, 2.0 Hz), 3.88 (1H, dd, J=11.5, 2.0 Hz), 4.05 (1H, d, J=2.0 Hz), 4.35 (1H, dd, J=3.0, 3.0 Hz), 5.53 (1H, br s), 6.18 (1H, dd, J=6.5, 6.5 Hz), 8.24 (1H, s), 8.57 (1H, br s). ¹³C-NMR (125 MHz, CDCl₃) δ: −5.71, −5.67, −4.9, −4.7, 18.0, 18.3, 25.7, 25.8, 42.8, 62.9, 72.5, 87.4, 88.8, 96.3 (q, J=35.0 Hz), 123.2 (q, J=270.0 Hz), 142.1, 154.3, 161.3. ¹⁹F-NMR (376 MHz, CDCl₃) δ: 100.2. HR-MS (ESI-TOF): Calcd for C₂₂H₄₁F₃N₃O₄Si₂ [M+H]⁺ 524.2588. Found 524.2590.

N-Acetyl-3′,5′-O-bis(tert-butyldimethylsilyl)-2′-deoxy-5-trifluoromethylcytidine (8) and 3′,5′-O-Bis(tert-butyldimethylsilyl)-2′-deoxy-N,N-diacetyl-5-trifluoromethylcytidine (9)

To a solution of compound 7 (914 mg, 1.74 mmol) in pyridine (15 mL), Ac₂O (0.33 mL, 3.49 mmol) and N,N-dimethyl-4-aminopyridine (DMAP) (21.3 mg, 0.17 mmol) were added at room temperature under Ar atmosphere. After being stirred for 7 h, another Ac₂O (0.33 mL, 3.49 mmol) and DMAP (21.3 mg, 0.17 mmol) were added, and then the reaction mixture was further stirred for 13 h. After addition of sat. NaHCO₃ aq., the reaction mixture was extracted with AcOEt. The combined organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated in vacuo. The crude residue was purified by column chromatography (hexane–AcOEt=5 : 1) to give compounds 8 (446 mg, 46%) and 9 (536 mg, 51%).

Compound 8

Colorless oil. IR (ATR) cm⁻¹: 3425, 2953, 2931, 2858, 1722, 1691, 1657, 1579, 1541, 1473, 1363, 1254. ¹H-NMR (500 MHz, CDCl₃) δ: 0.07–0.09 (12H, m), 0.87–0.90 (18H, m), 1.96–2.05 (1H, m), 2.25–2.74 (4H, m), 3.75–3.79 (1H, m), 3.87–3.93 (1H, m), 4.10 (1H, br s), 4.37 (1H, br s), 6.18 (1H, br s), 7.58 (0.7H, br s), 8.28 (0.3H, br s), 8.47 (0.7H, br s), 13.1 (0.3H, br s). ¹³C-NMR (125 MHz, CDCl₃) δ: −5.83, −5.78, −5.0, −4.8, 17.9, 18.2, 25.6, 25.7, 26.6, 28.8, 42.3, 42.7, 62.8, 72.5, 86.9, 88.3, 89.2, 97.2 (q, J=34.0 Hz), 104.4 (q, J=31.5 Hz), 121.6 (q, J=270.0 Hz), 122.6 (q, J=270.0 Hz), 141.0, 143.8, 146.7, 153.0, 153.7, 156.5, 171.7, 188.1. ¹⁹F-NMR (376 MHz, CDCl₃) δ: 99.4, 102.0. HR-MS (ESI-TOF): Calcd for C₂₄H₄₃F₃N₃O₅Si₂ [M+H]⁺ 566.2693. Found 566.2696.

Compound 9

White foam. IR (ATR) cm⁻¹: 2954, 2930, 2859, 1732, 1699, 1643, 1515, 1471, 1464, 1448, 1406, 1369, 1349, 1315, 1274, 1255, 1236. ¹H-NMR (500 MHz, CDCl₃) δ: 0.06 (6H, s), 0.10 (3H, s), 0.11 (3H, s), 0.87 (9H, s), 0.91 (9H, s), 2.11 (1H, ddd, J=13.5, 7.0, 6.0 Hz), 2.33 and 2.36 (6H, br s), 2.76 (1H, ddd, J=13.5, 6.0, 2.5 Hz), 3.79 (1H, dd, J=11.5, 2.5 Hz), 3.95 (1H, dd, J=11.5, 2.5 Hz), 4.16–4.18 (1H, m), 4.40–4.42 (1H, m), 6.19 (1H, dd, J=6.5, 6.5 Hz), 8.74 (1H, s). ¹³C-NMR (125 MHz, CDCl₃) δ: −5.7, −5.6, −4.9, −4.6, 18.0, 18.4, 25.7, 25.8, 26.0, 42.9, 62.9, 72.7, 89.5, 89.9, 106.2 (q, J=35.0 Hz), 121.9 (q, J=270.0 Hz), 146.2 (q, J=6.5 Hz), 153.7, 163.1, 170.4, 171.1. ¹⁹F-NMR (376 MHz, CDCl₃) δ: 100.6. HR-MS (ESI-TOF): Calcd for C₂₆H₄₄F₃N₃NaO₆Si₂ [M+Na]⁺ 630.2618. Found 630.2617.

N-Acetyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-trifluoromethylcytidine (10)

To a solution of a mixture of 8 and 9 (982 mg, 1.67 mmol, ca. 8 : 9=1 : 1.2) in THF (20 mL), TBAF (1 M in THF, 5.0 mL 5.0 mmol) was added at room temperature. After being stirred for 0.5 h, the reaction mixture was concentrated in vacuo. The crude residue was purified by column chromatography (CHCl₃–MeOH=10 : 1) to give the appropriate compound, which was dissolved in pyridine (15 mL); Et₃N (2.3 mL, 16.7 mmol) and DMTrCl (2.83 g, 8.35 mmol) were added at room temperature under Ar atmosphere. After being stirred for 6 h, the reaction mixture was quenched with sat. NaHCO₃ aq. and extracted with AcOEt. The combined organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated in vacuo. The crude residue was purified by column chromatography (CHCl₃–MeOH=30 : 1) to give compound 10 as a pale yellow foam (566 mg, 53% in 2 steps). IR (ATR) cm⁻¹: 3418, 2936, 1717, 1656, 1607, 1577, 1542, 1508, 1486, 1446, 1370, 1250. ¹H-NMR (500 MHz, CDCl₃) δ: 2.18–2.32 (2H, m), 2.43 (0.8H, br s), 2.55–2.62 (0.2H, m), 2.70 (2.2H, s), 2.80–2.86 (0.8H, m), 3.34–3.44 (2H, m), 3.79 (6H, s), 4.14 and 4.22 (1H, br s), 4.43 (1H, br s), 6.17 (1H, dd, J=6.0, 6.0 Hz), 6.82 (1H, d, J=8.0 Hz), 7.20–7.40 (9H, m), 7.56 (0.8H, br s), 8.16 (0.2H, br s), 8.41 (0.8H, br s), 13.1 (0.2H, br s). ¹³C-NMR (125 MHz, CDCl₃) δ: 26.7, 28.9, 41.5, 42.0, 55.1, 63.3, 72.2, 86.8, 87.3, 88.3, 97.7 (q, J=35.0 Hz), 104.7 (q, J=35.0 Hz), 113.14, 113.15, 121.4 (q, J=270.0 Hz), 122.3 (q, J=270.0 Hz), 126.9, 127.76, 127.84, 129.81, 129.83, 135.21, 135.23, 140.7, 143.6, 144.2, 146.8, 153.5, 153.7, 156.7, 158.6, 171.6, 188.4. ¹⁹F-NMR (376 MHz, CDCl₃) δ: 99.1, 101.7. HR-MS (ESI-TOF): Calcd for C₃₃H₃₂F₃N₃NaO₇ [M+Na]⁺ 662.2090. Found 662.2089.

N-Acetyl-3′-O-[2-cyanoethoxy(diisopropylamino)-phosphino]-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-trifluoromethylcytidine (11)

To a solution of compound 10 (438 mg, 0.68 mmol) and i-Pr₂NEt (0.71 mL, 4.08 mmol) in CH₂Cl₂ (5.0 mL), i-Pr₂NP(Cl)OCH₂CH₂CN (0.23 mL, 1.02 mmol) was added at 0°C under Ar atmosphere. The reaction mixture was stirred at room temperature for 3 h. After addition of sat. NaHCO₃ aq., the reaction mixture was extracted with AcOEt. The combined organic layer was washed with water and brine, dried over Na₂SO₄, and concentrated in vacuo. The crude residue was purified by column chromatography (hexane–AcOEt=3 : 2 to 1 : 2) to give compound 11 as a white form (291 mg, 51%). ¹H-NMR (400 MHz, CDCl₃) δ: 1.07 (3H, d, J=7.0 Hz), 1.15–1.18 (9H, m), 1.65 (0.7H, br s), 2.22–2.34 (1.3H, m), 2.45 (1H, t, J=6.5 Hz), 2.62 (1H, t, J=6.0 Hz), 2.64–2.96 (3H, m), 3.27–3.34 (1H, m), 3.44 and 3.47 (1H, dd, J=10.5, 3.0 Hz), 3.52–3.88 (10H, m), 4.26 and 4.31 (1H, br s), 4.48–4.56 (1H, m), 6.15 and 6.17 (1H, dd, J=6.5, 6.5 Hz), 6.80–6.83 (4H, m), 7.19–7.30 (7H, m), 7.35–7.36 (2H, m), 7.55 (0.7H, br s), 8.14–8.50 (1H, m), 13.1 (0.3H, br s). ¹⁹F-NMR (376 MHz, CDCl₃) δ: 99.1, 101.8. ³¹P-NMR (162 MHz, CDCl₃) δ: 148.6, 149.3. HR-MS (ESI-TOF): Calcd for C₄₂H₅₀F₃N₅O₈P [M+H]⁺ 840.3349. Found 840.3351.

Synthesis of Oligonucleotides 12c, f, h and j

Phosphoramidites 4 and 11, dT-phosphoramidite (Sigma) and d^mC(Ac)-phosphoramidite (Sigma) were used. Syntheses of oligonucleotides were performed on a 0.2 µmol scale using a standard phosphoramidite protocol (DMTr-ON mode), except for a prolonged coupling time of 10 min for 4 and 11. Cleavage from the CPG support and removal of the protecting groups were carried out by 0.05 M K₂CO₃ in MeOH at room temperature for 4 h in order to prevent undesired transformations of CF₃ group under the standard deprotection conditions using conc. NH₃ aq.,¹⁷⁾ and the mixture was diluted with 2 M triethylammonium acetate (TEAA) buffer. After removal of volatile components in vacuo, the crude oligonucleotides were purified with Sep-Pak^® Plus C18 cartridges (Waters), followed by reversed-phase HPLC (Waters XBridge™ Prep Shield RP18 5 µm, 10×50 mm). The compositions of the oligonucleotides were confirmed by ESI-TOF-MS analysis. The deconvoluted ESI-TOF-MS data [M] for TFOs 12c, f, h and j: 12c, Found 4551.40 (Calcd 4550. 99); 12f, Found 4550.50 (Calcd 4550.01); 12h, Found 4658.50 (Calcd 4657.95); 12j, Found 4658.20 (Calcd 4657.95).

UV-Melting Experiments

In the triplex-forming experiment, oligonucleotides and hairpin dsDNA were dissolved in 10 mM sodium cacodylate buffer (pH 7.4) containing 100 mM KCl and 50 mM MgCl₂ to give a final concentration of 1.89 µM, respectively. In the duplex-forming experiment, oligonucleotides and ssRNA were dissolved in 10 mM sodium cacodylate buffer (pH 7.4) containing 100 mM NaCl to give a final concentration of 3 µM, respectively. The samples were annealed in boiling water followed by slow cooling to 5°C. The melting profiles at 260 nm were recorded from 5 to 90°C (for triplex) and from 10 to 80°C (for duplex) at a scan rate of 0.5°C/min. The two-point average method was employed to obtain the T_m, and the final values were determined by averaging three independent measurements, which were accurate within a 1°C range.

pK_a Calculation

The absorbance of the solution of 6 in citrate buffer (prepared at each pH) was measured in the wavelength range of 220 to 340 nm using JASCO V-730. The pK_a of 6 was calculated by fitting the plot to a rearrangement of the Henderson–Hasselbalch Eq. 1.   

(1)

where A₂₇₇ is the absorbance at 277 nm, C₀ is the concentration of 6 (6.77×10⁻⁵ M), and ε_H and ε are the molar absorptivity of protonated and neutral forms of 6 at 277 nm, respectively. SigmaPlot (Systat Software, Inc.) was used for the fitting. The data obtained were 2.56±0.11 for pK_a, 6807±161 for ε_H and 12718±514 for ε (R²=0.98).

Acknowledgment

This work was partially supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grants (JP17K15431 and JP17K05943).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) Morihiro K., Kasahara Y., Obika S., Mol. Biosyst., 13, 235–245 (2017).
• 2) Wan W. B., Seth P. P., J. Med. Chem., 59, 9645–9667 (2016).
• 3) Sharma V. K., Sharma R. K., Singh S. K., MedChemComm, 5, 1454–1471 (2014).
• 4) Hari Y., Obika S., Imanishi T., Eur. J. Org. Chem., 2012, 2875–2887 (2012).
• 5) Luyten I., Herdewijn P., Eur. J. Med. Chem., 33, 515–576 (1998).
• 6) Xodo L. E., Manzini G., Quadrifoglio F., van der Marel G. A., van Boom J. H., Nucleic Acids Res., 19, 5625–5631 (1991).
• 7) Butkus V., Klimašauskas S., Petrauslriené L., Maneliene Z., Janulaitis A., Minchenkova L. E., Schyolkina A. K., Nucleic Acids Res., 15, 8467–8478 (1987).
• 8) Sanghvi Y. S., Hoke G. D., Freier S. M., Zounes M. C., Gonzalez C., Cummins L., Sasmor H., Cook P. D., Nucleic Acids Res., 21, 3197–3203 (1993).
• 9) Povsic T. J., Dervan P. B., J. Am. Chem. Soc., 111, 3059–3061 (1989).
• 10) Amosova O. A., Fresco J. R., Nucleic Acids Res., 27, 4632–4635 (1999).
• 11) Theruvathu J. A., Kim C. H., Rogstad D. K., Neidigh J. W., Sowers L. C., Biochemistry, 48, 7539–7546 (2009).
• 12) Heidelberger C., Cancer Res., 30, 1549–1569 (1970).
• 13) Nakamura S., Yang H., Hirata C., Kersaudy F., Fujimoto K., Org. Biomol. Chem., 15, 5109–5111 (2017).
• 14) Nakamura S., Fujimoto K., Chem. Commun., 51, 11765–11768 (2015).
• 15) Gmeiner W. H., Pon R. T., Lown J. W., J. Org. Chem., 56, 3602–3608 (1991).
• 16) Sugimoto N., Matsumura A., Sasaki M., Satake H., Takeda S., Wataya Y., Chem. Express, 7, 405–408 (1992).
• 17) Markley J. C., Chirakul P., Sologub D., Sigurdsson S. T., Bioorg. Med. Chem. Lett., 11, 2453–2455 (2001).
• 18) Purser S., Moore P. R., Swallow S., Gouverneur V., Chem. Soc. Rev., 37, 320–330 (2008).
• 19) Ji Y., Brueckl T., Baxter R. D., Fujiwara Y., Seiple I. B., Su S., Blackmond D. G., Baran P. S., Proc. Natl. Acad. Sci. U.S.A., 108, 14411–14415 (2011).
• 20) Karino N., Ueno Y., Matsuda A., Nucleic Acids Res., 29, 2456–2463 (2001).
• 21) Rusling D. A., Brown T., Fox K. R., “Sequence-specific DNA Binding Agents,” Chap. 1, ed. by Neidle S., Waring M. J., RSC Publishing, Cambridge, 2006, pp. 1–28.
• 22) Hari Y., Akabane M., Obika S., Chem. Commun., 49, 7421–7423 (2013).
• 23) Akabane-Nakata M., Obika S., Hari Y., Org. Biomol. Chem., 12, 9011–9015 (2014).
• 24) Hansen A. S., Thalhammer A., El-Sagheer A. H., Brown T., Schofield C. J., Bioorg. Med. Chem. Lett., 21, 1181–1184 (2011).
• 25) Musumeci D., Irace C., Santamaria R., Montesarchio D., MedChemCommun., 4, 1405–1410 (2013).