2025 Volume 73 Issue 5 Pages 457-466
New nucleoside derivatives containing the imidazole (Imd), pyridine or pyrimidine catalytic group were designed for site-specific acetylation of 2′-OH of the RNA ribose moiety. When the RNA substrate was acetylated in the presence of acetic anhydride under alkaline conditions, Probe (Imd) containing the imidazole catalytic group acetylated with a high selectivity to the 2′-OH of the uridine opposite the catalytic nucleotide. Probe (Py-4N) containing the pyridine group showed a higher catalytic activity under neutral conditions with a high selectivity for the 2′-OH group of the 5′ side of the uridine opposite the catalytic nucleotide in about 80% modification yield within 10 min. This study has shown that the oligodeoxynucleotide incorporating the new nucleotide derivative with the catalytic group can be a useful tool for site-selective acetylation of RNA 2′-OH.
RNAs in living systems, including mRNA, tRNA, and ribosomal RNA (rRNA), are chemically modified, and more than 150 different RNA modifications have been reported to date. The modified structures affect the stability and structure of the RNA and RNA–protein interactions, and play important roles in the regulation of transcription, RNA processing, nuclear export, cellular localization, and mRNA translation. Abnormalities of the RNA modifications are associated with metabolic disorders and cancer, and therefore, are regarded as therapeutic targets.1) Due to the importance of modifications of RNA, various methods have been developed to prepare modified RNA molecules. Chemical synthesis is widely used to obtain short RNA oligomers,2) and long RNA molecules are prepared by ligation of the short RNAs.3) For the enzymatic incorporation of non-natural base pairs,4–6) deoxyribozymes are utilized for modification of the internal position of RNA.7,8) Such techniques can also be applied to prepare fluorescent- or radio-labeled RNA molecules.9–11) On the contrary, acylating agents for the RNA 2′-position hydroxyl groups have been developed and used for analysis of the RNA tertiary structure, stabilization of structure, and regulation of function.12–14)
We have established a versatile method to chemically modify the internal position RNA with a high base- and sequence selectivity. This method uses oligonucleotides incorporating 6-thio-2′-deoxyguanosine (SdG) or 4-thiothymidine (SdT) with the sulfur atom attaching the transfer group. When the oligonucleotide forms a duplex with the RNA with the complementary sequence, the transfer group migrates to the 4-amino group of the cytosine base from SdG or the 6-amino group of the adenosine base from SdT. Using this method, the internal position of pre-synthesized oligonucleotides is tritium labeled site-specifically.15) The advantage of this method has been used to modify an adenosine of the premature termination codon (PTC) of the internal position of mRNA, successfully demonstrating that PTC modification induces the incorporation of a near-cognate tRNA at the stop codon to produce full-length peptides in the reconstituted Escherichia coli (E.coli) translation system.16) The sulfur atom of SdG of the oligodeoxynucleotide (ODN) was functionalized with the S-methyl ethanethioate, which selectively acetylates the 2′-hydroxy group of the target RNA substrate.17) These successful examples have established the general concept that a highly efficient and selective RNA modification can be achieved by placing reaction sites in close proximity to each other within the double strand. Thus, in this study, a new functional nucleobase was designed based on this concept to acetylate the 2′ hydroxyl group of the ribose of RNA in a site-specific manner. This paper describes in detail the site-specific acetylation of the RNA 2′ hydroxyl group using novel nucleobases with a catalytic group for acetylation.
In this study, we designed the 6-aminopurine base with a catalytic group (X) attached to the carboxamide at position 2. The catalytic group X is expected to activate and bring the acetyl group close to the 2′-hydroxyl of the opposite uridine (Fig. 1). The alkyl derivatives (C3-NH2, C3-SH and Imd) were expected to form stable acetylated intermediates, and the pyridine derivatives (Py-4N, 3N, 2N) and pyrimidine derivatives (Pym-2,4N, 3,5N) were designed to form reactive intermediates for catalysis. The synthesis of the ODN with a functional group X at the adenine base 2 position, which we labeled Probe (X), is summarized in Chart 1. The ODN was synthesized using a phosphoramidite precursor (4) with a methoxycarbonyl group at the adenine base 2 position, and the functional group X is attached to the ODN by an ester-amide exchange reaction to the ODN on a synthesis resin.
The catalytic group X is expected to activate and bring the acetyl group close to the 2′-hydroxyl of the opposite uridine.
Reagents and conditions: (a) 1) TBSCl, imidazole, N,N-dimethylformamide (DMF), room temperature (r.t.), 80%, 2) TPSCl, triethylamine (TEA), N,N-dimethyl-4-aminopyridine (DMAP), CH2Cl2, r.t., 88%; (b) CuI, I2, CH2I2, isoamyl nitrite, CH3CN, reflux, 69%; (c) NH3/H2O, tetrahydrofuran (THF), r.t., 90%; (d) nBu3SnCN, Pd(PPh3)4, DMF, 110°C, 98%; (e) 1) NaOMe, dry MeOH, r.t., 2) 10% aq. HCl, MeOH, r.t., 73%; (f) 1) benzoic anhydride, pyridine, 50°C, 45%; (g) HF/triethylamine (TEA), pyridine, r.t., 94%; (h) 1) DMTrCl, pyridine, r.t., 95%, 2) iPr2NP(Cl)OCH2CH2CN, N,N-diisopropylethylamine (DIPEA), CH2Cl2, 0°C, 68%; (i) DNA synthesizer; (j) 1) the resin, X-NH2, MeOH (for C3-NH2, C3-SH, Imd, Py-4N and Pym-2,4N), or 1) the resin, the 28% NH3 (for Py-2N, 3N, Pym-3,5N), 2) HPLC purification, 3) 5% aq. AcOH, 4) HPLC purification.
The 3′- and 5′-hydroxyl groups of 2′-deoxyguanosine 1 were protected with tert-butyldimethylchlorosilane (TBSCl), followed by the treatment with 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl) to form the corresponding 6-O-sulfonyl compound 1. The 2-amino group of 2 was replaced with iodine by the Zandmeyer reaction via diazotization with isoamyl nitrite to produce 2, followed by replacement of the 6-O-TPS group with ammonia to give the 2-iodo-6-amino derivative 3. Iodine was replaced by a cyano group by Stille coupling with nBu3SnCN to give 4 and the cyano group was then converted to the methoxycarbonyl derivative 5 by NaOMe addition in dry MeOH followed by hydrolysis with HCl. The 6-amino group of 5 was protected to form 6, and the TBS protecting groups were removed to form the diol derivative 7. Then 7 was converted to the phosphoramidite precursor 8 for DNA synthesis by the sequential reactions of protecting the 5′-hydroxyl group with 4,4′-dimethoxytrityl chloride (DMTr-Cl), and finally treating the 3′-hydroxyl group with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (iPr2NP(Cl)OCH2CH2CN). The precursor 8 was incorporated into the oligonucleotides on a DNA synthesizer, leaving the 5′-DMTr-protective ODN on the beads. The beads were incubated with NH2-C3-NH2, NH2-C3-SH, NH2-Imd, NH2-Py-4N, or NH2-Pym-2,4N at 55°C to simultaneously convert the ester group to a carboxamide group, deprotect the benzoyl group, and further cleave the ODN from the beads. The DMTr-protected ODN was purified by HPLC, and the DMTr group was deprotected with an aqueous solution of acetic acid and further purified by HPLC to produce Probe(X). When Py-2N, Py-3N, or NH2-Pym-3,5N was used, the ODN was not cleaved from the beads, so the beads were further treated with aqueous ammonia. The HPLC and MS data of the purified Probe (X) are summarized in “Experimental.”
Next, Probe (X) was acetylated with acetic anhydride in a pH 10 aqueous solution (Fig. 2). The HPLC peaks were isolated and assigned by measuring the molecular weight by MALDI-TOF/MS. Probes (C3-NH2) and (C3-SH) were almost completely acetylated, and the corresponding acetylated probes were stable under the stated reaction condition (Figs. 2A, 2B). Probe (Imd) was similarly acetylated, but the acetylated probe returned to the non-acetylated form after 4 h (Fig. 2C). In the case of Probe (Py-4N), the acetylated probe was formed as a minor product at 10 min, but a mixture of products formed at 60 min (Fig. 2D). Therefore, for acetylation of the RNA substrate, the acetylated forms of Probe(C3-NH2) and (C3-SH) were used, while Probes (Imd) and (Py-4N) were used without acetylation by expecting the in situ formation of their acetylated forms in a mixed solution with the RNA substrate.
Reaction conditions: Probe (X) (2.5 μM), Ac2O (12.5 mM), carbonate buffer pH 10 (23 mM), r.t. HPLC conditions: column: ODS column, 4.6 × 250 mm; solvents, A: 0.1 M TEAA buffer pH 7.0, B: CH3CN, linear gradient B 10 to 13%/20 min for Probe (C3-NH2) and Probe (Imd), B 10–17%/20 min for Probe (C3-SH), B 10–40%/20 min for Probe (Py-4N); column oven, 35°C; flow rate: 1 mL/min; UV: 254 nm.
Acetylation was performed using Probe(X) and RNA(U) in the carbonate buffer at pH 10 or in the HEPES buffer at pH 7 and 37°C, and the results are summarized in Fig. 3. Considering the stability of RNA molecules and the similarity to biological conditions, the reaction in HEPES buffer at pH 7 and 37°C is preferable. For probes with low reactivity, the reaction was performed in the carbonate buffer at pH 10. The HPLC peaks were assigned by measuring the molecular weight by MALDI-TOF/MS. Probe(C3-NHAc) did not give a new peak (Fig. 3A), while Probe(C3-SAc) gave the peak corresponding to the acetylated RNA(U) after 6 h (Fig. 3B). Since Probe(C3-SAc) is stable at pH 10, the formation of acetylated RNA(U) is due to transfer of the acetyl group from the probe to the RNA substrate. In the reaction with Probe(Imd) in the presence of acetic anhydride, the acetylated RNA(U) formed together with the acetylated Probe(Imd) (Fig. 3C). It should be emphasized that the reaction of the same concentration of Ac2O with the RNA substrate in the absence of the probe ODN did not result in any acetylation. Thus, it is clear that the RNA acetylation was catalyzed by the probe molecule. It is reasonable to explain that Probe(Imd) acetylated at pH 10 transfers its acetyl group to RNA(U), indicating that the imidazole group catalyzed the acetylation of RNA(U). Interestingly, Probe(Py-4N) produced the acetylated RNA(U) in a high yield after 10 min at pH 7 together with the diacetylated RNA(U) and the acetylated Probe (Py-4N) (Fig. 3D). Figure 4 summarizes the results of acetylation for the RNA substrate containing a different nucleobase opposite the catalytic nucleobase using a different Probe(X). Figure 4A shows the time course of acetylation for RNA(U), illustrating a high efficiency of the Probe(Py-4N); the acetylation yields reached to about 80% at 10 min. Since the acetylated Probe(Py-4N) is observed in the reaction mixture at pH 7, the acetyl group attached to the pyridine moiety is transferred to RNA(U), that is, the pyridine acts as a catalyst. Probe (Pym-2,4N) produced the acetylated RNA similar to Probe (Py-4N) (Table 1, run 5), whereas Probe (Py-3N), Probe (Py-3,5N), and Probe (Py-2N) showed a significant decrease in reactivity (Fig. 4B). The pyridine and pyrimidine moieties of these probes lack a nitrogen atom at the 4-position, indicating that this nitrogen atom contributes to the acetylation reaction. The base selectivity of Probe(Imd) and Probe(Py-4N) for the RNA substrate was examined by changing the base opposite the catalytic base to A, G, C, and U. Probe(Imd) showed a selectivity for uridine, albeit at a low ratio (Fig. 4C), and the selectivity for uridine was higher with Probe(Py-4N) (Fig. 4D). The UV melting temperatures of the duplexes formed with Probe(C3-NHAc) or Probe(Py-4N) and RNA(A, G, C, U) were measured to be the Tm’s of the duplex with Probe(C3-NHAc), A: 41.0°C, G: 41.1°C, C: 39.9°C, U: 40.4°C, and those with Probe(Py-4N), A: 46.9°C, G: 46.6°C, C: 44.0°C, U: 46.9°C. The differences in the Tm value are small, indicating that the selectivity of acetylation is not due to differences in the thermal stability of the duplexes. A local structure formed by interaction of the adenine base moiety with the uracil base may contribute to the selectivity.
Reaction conditions: ODN (5.0 μM), RNA (2.5 μM), NaCl (100 mM), 37°C, in the carbonate buffer (25 mM) at pH 10 for (A)–(C), or in HEPES buffer (250 mM) at pH 7.0 for (D). The reaction mixture of (C) and (D) contained Ac2O (10 mM). HPLC conditions: ODS column, 4.6 × 250 mm; solvents: A: 0.1 M TEAA buffer pH = 7.0, B: CH3CN, linear gradient B 10 to 15%/20 min for probe (C3-NHAc) and probe (Imd), B 10 to 16%/20 min for probe (C3-SAc), B 9 to 15%/20 min for Probe (Py-4N); column oven: 35°C; flow rate: 1.0 mL/min; UV: 254 nm.
aAcetylation was performed using the RNA substrate and analyzed by HPLC as described in the footnote to Fig. 3.
| Probe(X) | Reactive group | Calcd ([M − H]−) | Found |
|---|---|---|---|
| Probe(C3-NH2) | NH2 | 4835.85 | 4834.37 |
| Probe(C3-NHAc) | NH2 | 4877.86 | 4879.81 |
| Probe(C3-SH) | SH | 4852.81 | 4853.9 |
| Probe(C3-SAc) | SH | 4894.82 | 4895.67 |
| Probe(Imd) | Imidazole | 4872.85 | 4871.47 |
| Probe(Imd-Ac) | Imidazole | 4914.86 | 4914.30 |
| Probe(Py-4N) | 4N-pyridine | 4855.82 | 4856.78 |
| Probe(Py-4NAc) | 4N-pyridine | 4897.83 | 4899.08 |
| Probe(Pym-2,4N) | 2,4N-pyrimidine | 4856.83 | 4859.47 |
| Probe(Py-3N) | 3N-pyridine | 4855.82 | 4858.74 |
| Probe(Pym-3,5N) | 3,5N-pyrimidine | 4856.83 | 4859.47 |
| Probe(Py-2N) | 2N-pyridine | 4855.82 | 4857.56 |
Next, the acetylation site of RNA(U) was determined by the exonuclease T inhibition assay. Exonuclease T catalyzes the removal of nucleotides from the 3′ end of a single-stranded RNA and DNA. Chemical modification of the ribose 2′-OH in RNA, such as 2′-OMe, inhibits hydrolysis of its phosphodiester linkage to the 3′-OH, thus the cleaved fragment retains a nucleotide at the 3′-side of the 2′-OMe-modified nucleotide.18) In this study, the 5′-FAM RNAs were used as the substrate, incorporating the 2′-OMe modified nucleotide at the 13, 12, and 11 positions. Hydrolysis with exonuclease T is inhibited at these positions, resulting in formation of the 14-mer, 13-mer, and 12-mer RNAs, respectively. Using these as standard substrates, the exonuclease T cleavage products of the acetylated RNA (U) obtained with Probe(X), X = Imd, Py-4N, Pym-2,4N, Pym-3,5N, Py-3N, were compared by their gel mobility (Fig. 5). The non-acetylated RNA(U) substrate was hydrolyzed to nucleotide monomers as shown in lanes 3, 9 and 16, whereas the modified RNA(U) gave bands corresponding to the inhibition at the 2′-OMe modification sites (Fig. 5, lanes 4, 5, 10, 11). Ac-RNA(U) generated by Probe(Imd) showed a band at the same position as that of the RNA-12-OMe, that is, the 13-mer marker RNA, confirming that the uridine 2′-OH of U12 was acetylated (Fig. 5, lane 6). On the contrary, Ac-RNA(U) generated by Probe(Py-4N) showed a band at the same position as that of RNA-11-OMe, that is, 12-mer marker RNA, indicating that the 2′-OH of A11 was acetylated (Fig. 5, lane 12). A minor band with the same mobility with RNA-12-OMe shows that U12 was also acetylated. The Ac-RNA(U) obtained with Probe(Pym-2,4N), Probe(Pym-3,5N), and Probe(Py3N) also showed bands corresponding to acetylation of the 2′-OH of A11 (Fig. 5, lanes 17–19). The results of the exonuclease T inhibition assay clearly indicate that Probe(Imd) with the imidazole catalytic group acetylates the 2′-OH of the uridine opposite the catalytic nucleotide and that Probe(X) with the pyridine or pyrimidine catalytic group acetylates the 2′-OH of the adenosine on the 5′ side of the uridine opposite the catalytic nucleotide.
The acetylated RNA(U) samples were prepared and isolated by HPLC as described in the footnote to Fig. 3 and hydrolyzed in the buffer containing exonuclease T at 25°C. The hydrolysates were analyzed by electrophoresis on a 20% denatured polyacrylamide gel, and bands were visualized by measuring the fluorescence at 518 nm with excitation at 494 nm.
The differences in the acetylation sites due to differences in the catalytic groups are discussed based on the molecular model of the ODN/RNA duplex by assuming a tetrahedral intermediate between the 2′-OH group and the acetyl group attached to the catalytic group. Figure 6A shows the structure of a tetrahedral intermediate of the acetyl group formed between the imidazole group of Probe(Imd) and the 2′-OH of 12U. As a part of the molecular modeling simulated structure is shown in Fig. 6A, the intermediate can be formed without a significant conformational change in the duplex structure, probably due to the flexibility and length of the ethylene linker of the imidazole group. In a similar molecular modeling, the pyridine catalytic group of Probe (Py-4N) can form a preferred intermediate with the 2′-OH of the 5′-side 11A (Fig. 6B). Although it is difficult to discuss the difference in the acetylation site in a quantitative manner, conformational restriction of the catalytic group may be responsible for the selectivity.
(A) Intermediate of Probe(Imd) with the 2′-OH of the uridine opposite to the catalytic nucleoside; (B) Intermediate of Probe(Py-4N) with the 2′-OH of the adenosine on the 5′ side of the uridine opposite to the catalytic nucleoside. The duplex consisting of RNA(U) and Probe(X) was generated and optimized by the Amber Force Field installed in HyperChem7.5.
In this study, new nucleoside derivatives containing the imidazole (Imd), pyridine, or pyrimidine catalytic group were designed for site-specific acetylation of the 2′-OH of the RNA ribose. When the RNA substrate was acetylated in the presence of acetic anhydride under alkaline conditions, the probe (Imd) containing the imidazole catalytic group acetylated with a high selectivity to the 2′-OH of the uridine opposite the catalytic nucleotide. Probe (Py-4N) containing the pyridine group showed a higher catalytic activity under neutral conditions with a high selectivity for the 2′-OH group of the 5′ side of the uridine opposite the catalytic nucleotide: about an 80% modification yield within 10 min. This study has shown that the oligodeoxynucleotide incorporating the new nucleotide derivative with the catalytic group can be a useful tool for site-selective acetylation of RNA 2′-OH.
IR spectra were measured on a Perkin-Elmer SpectrumOne FT Infrared Spectrometer. 1H-NMR spectra were measured on a Varian Unity 400 (400 MHz) and a Bruker advanced III (500 MHz) NMR spectrometers using solvent peaks as the internal standards. 13C-NMR spectra were measured on a Bruker advanced III (125 MHz) NMR spectrometer using the solvent peaks as the internal standards. 31P-NMR spectra were measured using 10% phosphate D2O solution (0.00 ppm) as an external standard on a Varian Unity 400 (161 MHz) NMR spectrometer. Low-resolution electrospray ionization (ESI)-MS and high-resolution ESI-MS were primarily measured on an Applied Biosystems Mariner Biospectrometry Workstation mass spectrometer. MALDI-TOFMS was measured on a BRUKER DALTONICS microflex-KS linear mass spectrometer. The matrix was a 1/1 solution of 3-hydroxypicolinic acid in water/CH3CN with a 10:1 ratio of 10% aqueous diammonium hydrogen citrate or an ethanol solution of 2′-4′-6′-trihydroxyacetophenone monohydrate in ethanol. Kanto Chemical Silicagel 60N, FUJI SILYSIA CHROMATOREX Silicagel NH or Fuji Gel FL-60D were used for the column chromatography. JASCO LC-2000PLUS series was used for HPLC, using SHISEIDO CAPCELL PAK C18 MG (4.6 × 250 nm) for analysis and Nacalai Tesque COSMOSIL 5C18-AR-II for isolation. The oligodeoxynucleotides incorporating the amidite precursor 4 were synthesized using an NTS H-Series DNA/RNA Synthesizer. The RNA substrates used in the experiments were purchased from Gene Design and Japan Bioservice.
6-O-TPS-3′,5′-O-diTBS Derivative of 2′-Deoxyguanosine (1)To a suspension of 2′-deoxyguanosine (20 g, 70.1 mmol) in dry DMF (400 mL), TBSCl (42.2 g, 280 mmol) and imidazole (30 g, 441 mmol) were added at room temperature under an argon atmosphere, then the mixture was stirred for 20 h. 200 mL of ice water was added to the reaction mixture and then stirred for 1 h. The precipitates were collected by filtration and dried under reduced pressure. The product was purified by recrystallization from MeOH to give 3′,5′-O-diTBS derivative of 2′-deoxyguanosine 1 as white crystals (28 g, 80%). The above compound (1.0 g, 2.02 mmol) was dried by azeotrope using dry CH3CN, and suspended in dry CH2Cl2 (40 mL). To this suspension, TPSCl (1.2 g, 3.96 mmol), DMAP (25 mg, 0.21 mmol), and TEA (560 μL, 4.02 mmol) were added at 0°C under an argon atmosphere, then the mixture was stirred for 27 h. The reaction solution was washed with water and saturated brine, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (hexane:ethyl acetate = 10 : 1 to 5 < : 1) to afford 1 as a white foamy substance (1.36 g, 89%). 1H-NMR (500 MHz, CDCl3) δ: 7.97 (1H, s), 7.20 (2H, s), 6.29 (1H, dd, J = 6.6, 6.6 Hz), 4.86 (2H, s), 4.57 (1H, ddd, J = 3.1, 3.1, 5.9 Hz), 4.31 (2H, sept, J = 6.7 Hz), 3.97 (1H, ddd, J = 3.4, 3.6, 3.6 Hz), 3.80 (1H, dd, J = 4.2, 11.2 Hz), 3.74 (1H, dd, J = 3.2, 11.2 Hz), 2.91 (1H, sept, J = 6.9 Hz), 2.56 (1H, m), 2.34 (1H, ddd, J = 3.6, 6.1, 13.1 Hz), 1.28–1.25 (18H, m), 0.91 (9H, s), 0.89 (9H, s), 0.10 (6H, s), 0.07 (3H, s), 0.06 (3H, s); 13C-NMR (125 MHz, CDCl3) δ: 158.4, 155.6, 155.2, 154.2, 150.9, 140.0, 131.5, 123.8, 116.7, 87.8, 83.9, 72.0, 62.8, 40.9, 34.3, 29.8, 26.0, 25.8, 24.7, 24.6, 23.6, 18.4, 18.0, −4.7, −4.8, −5.4, −5.5; IR (ATR) cm−1: 2955, 1634, 1574; ESI-HRMS m/z (M + H+): 762.4147 (Calcd for C37H64N5O6SSi2+: 762.4110).
3′,5′-O-diTBS-2-iodo Derivative (2)To a suspension of 1 (1.1 g, 1.44 mmol) in dry CH3CN (85 mL), CuI (322 mg, 1.73 mmol), I2 (732 mg, 5.77 mmol), CH2I2 (700 μL, 7.22 mmol), and isoamyl nitrite (970 μL, 7.22 mmol) were added, then the mixture was heated at 75°C for 45 min. The reaction solution was brought to room temperature and the solvent was evaporated under reduced pressure. The residue was dissolved in CHCl3, washed with saturated aqueous Na2S2SO3 and saturated brine, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (hexane : ethyl acetate = 10 : 1) to afford 2 as a white foamy substance (0.87 mg, 69%). 1H-NMR (400 MHz, CDCl3) δ: 8.25 (1H, s), 7.20 (2H, s), 6.38 (1H, dd, J = 6.4, 6.4 Hz), 4.59 (1H, m), 4.24 (2H, sept, J = 6.3 Hz), 3.98 (1H, ddd, J = 3.4, 3.4, 3.4 Hz), 3.84 (1H, dd, J = 4.0, 11.0 Hz), 3.74 (1H, dd, J = 3.4, 11.3 Hz), 2.91 (1H, sept, J = 6.9 Hz), 2.58 (1H, m), 2.41 (1H, m), 1.19–1.27 (18H, m), 0.89 (9H, s), 0.88 (9H, s), 0.09 (6H, s), 0.06 (3H, s), 0.06 (3H, s); 13C-NMR (125 MHz, CDCl3) δ: 154.5, 154.2, 153.3, 150.9, 143.4, 131.4, 123.8, 123.2, 115.5, 88.3, 85.0, 71.8, 62.7, 41.3, 34.4, 29.9, 26.0, 25.8, 24.8, 24.8, 23.6, 23.6, 18.4, 18.0, −4.6, −4.8, −5.4, −5.5; IR (ATR) cm−1: 2926; ESI-HRMS m/z (M + Na+): 895.2792 (Calcd for C37H61IN4O6SSi2Na+: 895.2787).
3′,5′-O-diTBS-2-iodo-2′-deoxyadenosine (3)A 28% aque-ous solution of NH3 (7.0 mL, 8673 mmol) was added to a solution of 2 (1.0 g, 1.145 mmol) in THF (13 mL), and the mixture was stirred for 69 h at room temperature. The reaction solution was diluted with saturated aqueous NH4Cl and extracted with ethyl acetate. The extract was washed with saturated aqueous NaHCO3, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (hexane:ethyl acetate = 3 : 1 to 5 : 2) to afford 3 as a white foamy substance (619 mg, 89%). 1H-NMR (400 MHz, CDCl3) δ: 7.99 (1H, s), 6.33 (1H, dd, J = 6.4, 6.4 Hz), 5.61 (2H, s), 4.61 (1H, m), 3.96 (1H, ddd, J = 3.8, 3.8, 3.8 Hz), 3.86 (1H, dd, J = 4.5, 11.1 Hz), 3.75 (1H, dd, J = 3.4, 11.3 Hz), 2.63 (1H, m), 2.38 (1H, ddd, J = 4.1, 6.3, 13.3 Hz), 0.90 (9H, s), 0.90 (9H, s), 0.09 (6H, s), 0.08 (6H, s); 13C-NMR (125 MHz, CDCl3) δ: 155.2, 150.0, 139.5, 120.1, 119.8, 88.1, 84.7, 72.0, 62.9, 41.3, 26.1, 25.9, 18.6, 18.2, −4.5, −4.6, −5.2, −5.3; IR (ATR) cm−1: 2956, 1649, 1589; ESI-HRMS m/z (M + Na+): 628.1614 (Calcd for C22H40IN5O3Si2Na+: 628.1607).
3′,5′-O-diTBS-2-cyano-2′-deoxyadenosine (4)A solution of 3 (700 mg, 1.156 mmol) and nBu3SnCN (548 mg, 1.73 mmol) in dry DMF (20 mL) was bubbled with argon gas for 20 min at room temperature. To this solution, Pd(PPh3)4 (160 mg, 0.138 mmol) was added and the mixture was stirred at 110°C for 1 h under an argon atmosphere. The reaction solution was brought to room temperature, diluted with ethyl acetate, washed with aqueous saturated NH4Cl, dried over Na2SO4, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (hexane to hexane:ethyl acetate = 2 : 1) and further purified by silica gel chromatography (hexane:ethyl acetate = 5 : 2) to afford 4 as a white foamy substance (572 mg, 98%). 1H-NMR (400 MHz, CDCl3) δ (ppm): 8.26 (1H, s), 6.37 (1H, dd, J = 6.3, 6.3 Hz), 5.69 (2H, s), 4.62 (1H, m), 4.00 (1H, ddd, J = 3.7, 3.7, 3.7 Hz), 3.88 (1H, dd, J = 4.3, 11.3 Hz), 3.76 (1H, dd, J = 3.1, 11.3 Hz), 2.65 (1H, m), 2.44 (1H, m), 0.91 (9H, s), 0.89 (9H, s), 0.10 (3H, s), 0.10 (3H, s), 0.07 (3H, s), 0.07 (3H, s); 13C-NMR (125 MHz, CDCl3) δ: 155.7, 148.8, 141.8, 137.7, 121.5, 116.6, 88.3, 85.2, 71.8, 62.8, 41.4, 26.1, 25.9, 18.6, 18.2, 13.7, −4.5, −4.7, −5.3, −5.4; IR (ATR) cm−1: 2929, 2856, 1656, 1594; ESI-HRMS m/z (M + Na+): 527.2596 (Calcd for C23H40N6O3Si2Na+: 527.2593).
3′,5′-O-diTBS-2-methoxycarbonyl-2′-deoxyadenosine (5)To a solution of 4 (540 mg, 1.07 mmol) in dry MeOH (26 mL), a 0.5 M NaOMe/MeOH solution (1.0 mL, 0.50 mmol) was added at room temperature under an argon atmosphere, then the mixture was stirred for 28 h at room temperature. The reaction solution was neutralized with Dowex 50 (H+), filtered through a celite pad, and the filtrate was evaporated under reduced pressure. The residue was dissolved in MeOH, then water (6.5 mL) was slowly added to this solution at room temperature, followed by the addition of 10% aqueous hydrochloric acid (590 μL). After stirring for 20 min, the reaction solution was neutralized with saturated aqueous NaHCO3, and extracted with ethyl acetate, dried over NaSO4, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 1 : 1 to 1 : 2) to afford 5 (420 mg, 73%, two step yield) as a white foamy substance. 1H-NMR (400 MHz, CDCl3) δ: 8.28 (1H, s), 6.55 (1H, dd, J = 6.4, 6.4 Hz), 5.73 (2H, s), 4.62 (1H, m), 4.00 (3H, s), 3.98 (1H, ddd, J = 3.4, 3.4, 3.4 Hz), 3.88 (1H, dd, J = 3.7, 11.3 Hz), 3.77 (1H, dd, J = 3.1, 11.3 Hz), 2.58 (1H, ddd, J = 6.5, 6.5, 6.5 Hz), 2.48 (1H, m), 0.90 (9H, s), 0.90 (9H, s), 0.09 (6H, s), 0.08 (6H, s); 13C-NMR (125 MHz, CDCl3) δ: 164.7, 155.6, 150.6, 149.7, 141.5, 121.1, 88.2, 84.4, 71.9, 62.9, 53.5, 42.0, 26.1, 25.9, 18.6, 18.2, −4.5, −4.7, −5.2, −5.4; IR (cm−1): 2953, 2928, 2856, 1739, 1646, 1591; ESI-HRMS m/z (M + Na+): 560.2724 (Calcd for C24H43N5O5Si2Na+: 560.2695).
3′,5′-O-diTBS-6-N-benzoyl-2-methoxycarbonyl-2′-deoxyadenosine (6)Benzoic acid anhydride (105 mg, 0.46 mmol) was added to a solution of 5 (50 mg, 0.093 mmol) in dry pyridine (470 μL) under an argon atmosphere and the mixture was stirred at 50°C for 86 h. The reaction solution was diluted with CHCl3, washed with saturated aqueous NH4Cl and dried over Na2SO4. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 3 : 1) to afford 6 as a white foamy substance (26.7 mg, 45%). 1H-NMR (400 MHz, CDCl3) δ: 9.01 (1H, s), 8.51 (1H, s), 8.01 (2H, d, J = 7.6 Hz), 7.59 (1H, t, J = 7.3 Hz), 7.50 (2H, t, J = 7.5 Hz), 6.64 (1H, t, J = 6.4 Hz), 4.63 (1H, m), 4.10 (1H, m), 4.04 (3H, s), 3.88 (1H, dd, J = 3.8, 11.1 Hz), 3.78 (1H, dd, J = 3.1, 11.3 Hz), 2.62 (1H, m), 2.51 (1H, m), 0.91 (9H, s), 0.89 (9H, s), 0.10 (6H, s), 0.07 (6H, s); 13C-NMR (125 MHz, CDCl3) δ: 164.7, 164.0, 152.4, 149.7, 149.4, 144.0, 133.1, 133.0, 128.9, 128.0, 125.1, 88.3, 84.6, 71.9, 62.9, 53.6, 41.8, 26.0, 25.8, 18.5, 18.1, −4.6, −4.8, −5.3, −5.5; IR (ATR) cm−1: 2928, 2857, 1740, 1697, 1605, 1575, 1252; ESI-HRMS m/z (M + Na+): 664.2939 (Calcd for C31H47N5O6Si2Na+: 664.2957).
6-N-Benzoyl-2-methoxycarbonyl-2′-deoxyadenosine (7)To a solution of 6 (179 mg, 0.28 mmol) in dry pyridine (1 mL), TEA (170 μL, 1.22 mmol) and TEA·3HF (200 μL, 1.23 mmol) were added at 0°C under an argon atmosphere, then the mixture was stirred for 9 h at room temperature. The reaction solution was applied to a silica gel column and purified (CHCl3 : MeOH = 50 : 1 to 45 : 1) to afford 7 as a white powder (108.4 mg, 94%). 1H-NMR (400 MHz, CD3OD) δ: 8.79 (1H, s), 8.08 (2H, d, J = 7.0 Hz), 7.65 (1H, t, J = 7.3 Hz), 7.55 (2H, dd, J = 7.5, 7.5 Hz), 6.63 (1H, t, J = 6.6 Hz), 4.64 (1H, m), 4.07 (1H, m), 4.01 (3H, s), 3.87 (1H, dd, J = 3.4, 12.2 Hz), 3.79 (1H, dd, J = 4.3, 12.2 Hz), 2.86 (1H, m), 2.53 (1H, m); 13C-NMR (125 MHz, DMSO) δ: 165.7, 163.5, 152.2, 150.6, 149.1, 145.1, 133.0, 132.6, 128.6, 128.5, 127.1, 88.2, 83.7, 70.7, 61.5, 52.8; IR (cm−1): 3350, 2932, 1730, 1608, 1521, 1261; ESI-HRMS m/z (M + Na+): 436.1248 (Calcd for C19H19N5O6Na+: 436.1228).
5′-O-DMTr-3′-O-(2-cyanoethyl)diisopropylphoshoramidite Derivative of 6-N-Benzoyl-2-methoxycarbonyl-2′-deoxyadenosine (8)The diol derivative 7 (30 mg, 0.07257 mmol) was dried by azeotrope using dry pyridine and dissolved in dry pyridine (600 μL). To this solution, DMTr chloride (49 mg, 0.1446 mmol) was added under an argon atmosphere and the mixture was stirred at room temperature for 75 min, then MeOH (1 mL) was added and stirred for an additional 10 min, The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (CHCl3 : MeOH = 100 : 1) to afford the 5′-O-DMTr protected derivative of 7 as a white powder (49.5 mg, 95%). 1H-NMR (500 MHz, CDCl3) δ: 8.96 (1H, s), 8.37 (1H, s), 8.04 (2H, dd, J = 8.6, 8.6 Hz), 7.62 (1H, t, J = 7.4 Hz), 7.53 (2H, dd, J = 8.8, 16.3 Hz), 7.39 (2H, t, J = 7.4 Hz), 7.3–7.2 (3H, m), 6.81 (4H, m), 6.67 (1H, m), 4.73 (1H, m), 4.15 (1H, m), 4.04 (3H, s), 3.78 (6H, s), 3.48 (1H, dd, J = 4.5, 10.3 Hz), 3.41 (1H, dd, J = 4.65, 10.4 Hz), 2.78 (1H, m), 2.68 (1H, m)), 4.62 (1H, m); ESI-MS m/z (M + H+): 716.9859 (Calcd for C40H37N5O8 + H+: 716.27).
The above compound (138 mg, 0.19 mmol) was dried by azeotrope using dry toluene and dry CH3CN then suspended in dry CH2Cl2 (2.8 mL). To this suspension, iPr2NEt (200 μL, 1.15 mmol) and iPr2NP(Cl)OCH2CH2CN (130 μL, 0.58 mmol) were added under an argon atmosphere, and the mixture was stirred at 0°C for 75 min. iPr2NEt (133 μL, 0.77 mmol) and iPr2NP(Cl)OCH2CH2CN (87 μL, 0.39 mmol) were added again and the mixture was stirred for a further 25 min at 0°C. The reaction solution was diluted with CHCl3, and the entire mixture was washed with aqueous NaHCO3, brine, and dried over Na2SO4. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane : ethyl acetate = 3 : 2 to 1 : 1 to 2 : 3). The residue was dissolved in dry CH2Cl2, added to hexane cooled at −78°C and precipitated, then the supernatant was removed and the residue was vacuum dried to afford 8 as a white foamy substance (120 mg, 68%). 1H-NMR (500 MHz, CDCl3) δ: 8.95 (1H, s), 8.02 (1H, s), 7.59 (2H, m), 7.53 (2H, m), 7.37 (2H, m), 7.30–7.15 (8H, m), 6.78 (4H, m), 6.66 (1H, m), 4.79 (1H, m), 4.28 (1H, m), 4.23–4.07 (2H, m), 4.04 (3H, s), 3.77 (6H, s), 3.55–3.42 (4H, m), 2.63 (1H, m), 2.44 (1H, m); 31P-NMR (162 MHz, CDCl3) δ: 149.1; ESI-MS m/z (M+): 915.9497 (Calcd for C49H54N7O9P+: 915.3721).
Synthesis of Probe(C3-NH2)The oligonucleotide probes were synthesized using the automated DNA synthesizer according to the conventional amidite chemistry and the standard synthesis protocol. A bottle containing a solution of 4 in dry CH3CN was installed in the synthesizer together with the amidite precursors of the natural nucleotides, and 4 was incorporated at the X position of the sequence (5′ DMTr-d(CTT TXTTC TCC TTT CT)-3′). After the synthesis, the resins were heated at 55°C for 12 h in anhydrous MeOH (1 mL) containing TEA (50 μL). The solvents were evaporated and the residue was dissolved in 0.1 M triethylammonium acetate (TEAA) buffer, the insoluble materials were filtered off through the membrane filter, then the filtrate was purified by HPLC (column: Cosmosil 5C18-AR-II, 10 × 250 mm, solvent: A: 0.1 M TEAA buffer, B: CH3CN, B: 10 to 40%/20 min, linear gradient; flow rate: 1.0 mL/min; column oven: 35°C; monitored at UV 254 nm) to give the DMTr-Probe(C3-NH2). The DMTr group was deprotected in 5% aqueous AcOH for 30 min at room temperature, and Probe(C3-NH2) was separated by HPLC. HPLC (Column: Cosmosil 5C18-AR-II, 10 × 250 mm, solvent: A: 0.1 M TEAA buffer, B: CH3CN, B: 12 to 40%/20 min, linear gradient; flow rate: 1.0 mL/min; column oven: 35°C; monitored at UV 254 nm).
Synthesis of Probe(C3-SH)Probe(C3-SH) was synthesized in a manner similar to Probe(C3-NH2) except for the following procedures. The resins were at heated at 55°C for 15 h in anhydrous MeOH (1 mL) containing 3-amino-1-propanethiol hydrochloride and N,N-diisopropyethylamine. The DMTr-deprotected product was treated with 100 mM solution of dithiothreitol in HEPES buffer, then purified by HPLC.
Synthesis of Probe(Imd), Probe(Py-4N), and Probe(Pym-2,4N)These probes were synthesized in a similar manner to Probe(C3-NH2), except that the following amines were used instead of TEA: histamine for Probe(Imd), 4-aminopyridine for Probe(Py-4N), or 4-aminopyrimidine for Probe(Pym-2,4N).
Synthesis of Probe(Py-2N), Probe(Py-3N), and Probe-(Pym-3,5N)These probes were synthesized in a similar manner to Probe(C3-NH2), except that the following amines were used instead of TEA: 2-aminopyridine for Probe(Py-2N), 3-aminopyridine for Probe(Py-3N), or 5-aminopyrimidine for Probe(Pym-3,5N), and that the DMTr-protected probes were cleaved from the resin by treatment in 28% aqueous ammonia for 30 min at room temperature. The MALDI-TOF MS data of Probe(X) are summarized in Table 1.
The Acetylation Reaction of Probes (Fig. 2)To a carbonate buffer solution (25 mM Na2CO3, 25 mM NaHCO3, pH 10.0, 188 μL) of Probe (X) (2.5 μM) was added to a DMF solution of acetic anhydride (25 mM, 200 μL, 5 μmol), and the mixture was kept at room temperature for 1 h. The mixture was then diluted with 3.6 mL 0.1 M TEAA buffer and analyzed by HPLC (column: Nacalai COSMOSIL 5C18-AR-II waters, 4.6 × 250 mm; solvent: A: 0.1 M TEAA buffer pH = 7.0, B: CH3CN, linear gradient B 10 to 13%/20 min for Probe (C3-NH2) and (Imd), B 10–17%/20 min for Probe (C3-SH), B 10–40%/20 min for Probe (Py-4N); column oven: 35°C; flow rate: 1.0 mL/min; monitored at UV: 254 nm). The MALDI-TOF MS data of the acetylated Probe(C3-NHAc), Probe(C3-SAc), Probe(Imd-Ac), and Probe(Py-4NAc) are summarized in Table 1.
The Acetylation Reaction of the RNA Substrate Reaction Using Probe(C3-NHAc) or Probe(C3-SAc) (Figs. 3A, 3B)A solution of Probe (X) (5 μM) and RNA(U) (2.5 μM) in carbonate buffer (12. 5 mM, pH 10) containing NaCl (100 mM) was incubated at 37°C. The reaction mixture was analyzed by HPLC (column: SHISEIDO CAPCELL PAK C18 MG, 4.6 × 250 mm; solvent: A: 0.1 M TEAA buffer pH = 7.0, B: CH3CN, linear gradient B 10 to 15%/20 min for Probe (C3-NHAc), B 10 to 16%/20 min for Probe (C3-Ac); column oven: 35°C; flow rate: 1.0 mL/min; monitored at UV: 254 nm).
Reaction Using Probe(Imd) (Figs. 3C, 4)A solution of Probe(Imd) (5 μM), the RNA substrate (2.5 μM), and Ac2O (10 mM) in carbonate buffer (12. 5 mM, pH 10) containing NaCl (100 mM) was incubated at 37°C. The reaction mixture was analyzed by HPLC (column: SHISEIDO CAPCELL PAK C18 MG, 4.6 × 250 mm; solvent: A: 0.1 M TEAA buffer pH = 7.0, B: CH3CN, linear gradient B 10 to 15%/20 min; column oven: 35°C; flow rate: 1.0 mL/min; UV: 254 nm).
Reaction Using Probe(Py-4N), Probe(Py-2N), Probe(Py-3N), Probe(Pym-2,4N), or Probe(Pym-3,5N)A solution of Probe (X) (5 μM), the RNA substrate (2.5 μM), and Ac2O (10 mM) in HEPES buffer (25 mM, pH 7) containing NaCl (100 mM) was incubated at 37°C. The reaction mixture was analyzed by HPLC (column: SHISEIDO CAPCELL PAK C18 MG, 4.6 × 250 mm; solvent: A: 0.1 M TEAA buffer pH = 7.0, B: CH3CN, linear gradient B 9 to 15%/20 min for Probe(Py-4N); column oven: 35°C; flow rate: 1.0 mL/min; UV: 254 nm).
The MALDI-TOF MS data of RNA(U) are summarized in Table 2.
| RNA | Nucleoside opposite catalytic nucleoside |
Calcd ([M − H]−) | Found |
|---|---|---|---|
| RNA(U) | U | 5258.83 | 5259.61 |
| Ac-RNA(U) | U | 5300.83 | 5301.40 |
| FAM-RNA(U) | U | 5827.78 | 5828.74 |
| FAM-Ac-RNA(U) | U | 5869.79 | 5870.52 |
Part of the duplex used in the experiment, 5′-d(CTTTXTTCTCCTT)/3′-r(GAAAUAAGAGGAA), was generated in the 3′-endo conformation with an adenosine at position X by HyperChem7.5. The adenine base of X was modified to the imidazole or the pyridine catalytic group to form the tetrahedral intermediate with the acetyl group 2′-OH of 12U or 11A, respectively, and optimized by the Amber force field installed in HyperChem7.5.
This study was partially supported by a Grant-in-Aid for Specially Promoted Research (Grant Nos: 18H02558 and 24K21282 for S.S.) from the Japan Society for Promotion of Science (JSPS).
The authors declare no conflict of interest.
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