Application of the Functionality Transfer Oligodeoxynucleotide for the Site-Selective Modification of RNA with a Divers Molecule

Abstract

Due to the importance of the RNA chemical modifications, methods for the selective chemical modification at a predetermined site of the internal position of RNA have attracted much attention. We have developed functional artificial nucleic acids that modify a specific site of RNA in a site- and base-selective manner. In addition, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been shown to introduce additional molecules on the alkynes attached to the pyridine ring. However, it was found that some azide compounds produced the cycloadduct in lower yields. Therefore, in this study, we synthesized the pyridinyl transfer group with the alkyne attached via a polyethylene glycol (PEG) linker with a different length and optimized its structure for both the transfer and CuAAC reaction. Three new transfer groups were synthesized by introducing an alkyne group at the end of the triethylene (11), tetraethylene (12) or pentaethylen glycol linker (13) at the 5-position of the pyridine ring of (E)-3-iodo-1-(pyridin-2-yl)prop-2-en-1-one. These transfer groups were introduced to the 6-thioguanine base in the oligodeoxynucleotide (ODN) in high yields. The transfer groups 11 and 12 more efficiently underwent the cytosine modification. For the CuAAC reaction, although 7 showed low adduct yields with the anionic azide compound, the new transfer groups, especially 12 and 13, significantly improved the yields. In conclusion, the transfer groups 12 and 13 were determined to be promising compounds for the modification of long RNAs.

Introduction

Nearly 150 chemically-modified nucleosides have been identified in RNA, with tRNA containing the most modification sites, ribosomal RNA (rRNA) having over 210, and the spliceosomal RNA having over 50 modification sites.¹⁾ Although the number of sites for modification in mRNA is far fewer, a variety of modified nucleosides have been found for more than decades. N6-methyladenosine (m6A) and N1-methyladenosine (m1A) are highly abundant in mRNA and have essential functions in gene regulation. 5-Methylcytosine is present in mRNA in much lower amounts, which is oxidized by the enzyme to 5-hydroxymethylcytosine (hm5C). The abundance and potential role of m5C and hm5C in mammals will be revealed in the near future.

Pseudouridine is the most common modification of intracellular RNA and is an abundant component of rRNA and tRNA, but its presence in mRNA was, until recently, somewhat ignored.²⁾ Pseudouridine is known to affect the secondary structure of RNA, and its ability to alter the reading of stop codons may also be biologically important. Ribose 2′-OMe methylation is also present as an abundant modification in tRNA and rRNA, but is also present in many mRNAs and can have a significant impact on RNA-protein interactions and RNA secondary structure.

Deamination of the amino group of cytidine to convert to uridine, called RNA editing, is catalyzed by a specific enzyme.³⁾ In an example of cytidine to uridine editing in the RNA encoding apolipoprotein B, the normal code is changed to the stop codon, resulting in the production of apoB48, which is 48% of the length of the full-length apoB100 protein. Another deamination takes place on adenosine in the double-stranded RNA to convert inosine. Inosines in the coding region function as guanosine bases that form base pairs with cytidine during translation.⁴⁾

Due to the importance of the RNA chemical modifications, incorporation of modified bases at specific positions, in particular, at an internal site of the RNA, is useful to study their effects on the structure and function. RNA specifically modified with fluorescent⁵⁾ or radioactive molecules⁶⁾ are frequently used for the study of the three-dimensional structure and subcellular localization of RNA, etc. Methods for selective chemical modification at a predetermined site of the internal position of RNA have attracted much attention.

Specifically-modified RNA can be chemically synthesized using automated synthesizers by the solid phase phosphoramidite method using a modified nucleotide precursor. However, it is difficult to apply chemical synthesis to introduce a chemical modification at a desired position in a long RNA. For this reason, the synthesized short RNA strands are ligated by an enzyme to produce long RNA molecules.^7,8) In addition, technologies for introducing chemical modifications to an already existing RNA without using a synthesizer have also been reported. One approach is the use of sequence-selective enzymes, which convert uridine to pseudouridine⁹⁾ or to replace guanosine with a 7-deazaguanine derivative.¹⁰⁾ The other has been achieved by deoxyribozyme that sequence-selectively connects the guanosine 5′-triphosphate (GTP) linkage to the 2′-OH of the adenosine ribose portion of the target site through a phosphate diester bond.¹¹⁾

In our laboratory, we are developing functional artificial nucleic acids that induce base- and site-specific functional group transfer to the target RNAs^12–15) (Fig. 1). This method is characterized by three points: sequence selectivity through double-strand formation with the target RNA, base selectivity through molecular recognition between the target base and the artificial nucleic acid, and reactivity-inducing effect triggered by the formation of metal complexes. Furthermore, our previous studies have shown that an additional molecule can be introduced onto the alkylated pyridinyl transfer group by the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC).^16,17) However, it was found that some azide compounds produced the cycloadduct in lower yields. Therefore, in this study, we synthesized the pyridinyl transfer group with the alkyne attached via an ether linker and optimized its structure for both the transfer and CuAAC reaction.

Fig. 1. Concept of the Functionality Transfer Reaction of the Transfer Oligodeoxynucleotide (ODN) for Site-Selective Modification of RNA

Results and Discussion

We have already reported that in the presence of NiCl₂, ODN(1) containing the pyridinyl vinyl ketone unit on the 6-thioguanine is hybridized with the RNA(2) with the complementary sequence of ODN(1) and induces the functional group transfer from the sulfur atom to the amino group of its facing cytidine (2) by Michael reaction followed by a β-elimination reaction with regenerating ODN(3)¹⁶⁾ (Chart 1). In this study, we synthesized the pyridinyl vinyl ketone transfer units furnished with the terminal alkyne, which were tested for the site-selective modification of RNA and subsequent cycloaddition reaction with a variety of azides to produce 5.

Chart 1. The Functionality Transfer Reaction of the Terminal Alkyne Containing a Transfer Group and the Subsequent Cycloaddition Reaction with the Azides

Synthesis of the Transfer Group

The transfer groups bearing the terminal alkyne and the iodoethene group were synthesized according to a previously reported method (Chart 2). (E)-3-Iodo-1-(4-ethynylpyridin-2-yl)prop-2-en-1-one (7-I) was synthesized using 4-bromo-2-cyanopyridine as the starting material. An alkyne was introduced at the 4-position of the pyridine ring by the Sonogashira coupling reaction with trimethylsilylacetylene, and subsequent deprotection of the trimethylsilyl group with tetrabutylammonium fluoride to produce 6. The cyano group of 6 was reacted with ethynylmagnesium bromide, followed by treatment with 10% hydroiodic acid to afford 7-I with the iodoethene group as a precursor of the transfer group.¹⁶⁾ To simplify the naming of nucleic acid derivatives, the structures of the pyridinyl-vinyl keto moiety are numbered and the compounds are named as their iodine compounds, as in 7-I. As our previous study determined that the E-iodoethene is essential for the transfer reaction, the predominant formation of the E-iodoethene (E : Z = 87 : 13) was confirmed by measuring its ¹H-NMR spectrum. The transfer groups with the polyethylene glycol (PEG) were synthesized by the S_NAr reaction using the mono-propargylated PEG and 5-bromo-2-cyanopyridine as starting materials to produce the corresponding cyano derivatives (8–10). Subsequently, the cyano group was converted to the E-iodoethene derivatives (11-I, 12-I, 13-I) by the reaction described for the synthesis of 7-I, and the predominant formation of the E-iodoethene was similarly confirmed by measuring its ¹H-NMR spectrum. The transfer group with 9-oxadec-11-yn-1-ol instead of the PEG was synthesized for a comparison (Supplementary Chart S1), but could not be introduced to the ODN containing 6-thioguanine. Probably due to its high hydrophobicity, it formed an aggregate in water and was not able to gain access to the ODN.

Chart 2. Synthesis of the Terminal Alkyne Containing Transfer Groups

a) 1) Trimethylsilyl (TMS)-acetylene, PdCl₂(PPh₃)₂, CuI, triethylamine (TEA), tetrahydrofuran (THF), room temperature (r.t.), 87%; 2) Tetrabuthyl ammonium fluoride, THF, CH₂Cl₂, r.t., 98%; b) Ethynylmagnesium bromide, THF, 0 °C, then 20% aqueous HI, 21%; c) HCCCH₂O(CH₂CH₂O)n-H, n = 2,3, 4, NaH, N,N-dimethylformamide (DMF), 0 °C to r.t., 74% for 8, 82% for 9, 75% for 10; d) Ethynylmagnesium bromide, THF, 0 °C, then 20% aqueous HI, 14% for 11-I, 23% for 12-I, 14% for 13-I.

Functionality Transfer Reaction

The transfer oligodeoxynucleotide probe (FT-ODN) was prepared by the reaction of ODN containing 6-thioguanine with the precursor of the transfer group (7-I, 11-I, 12-I, 13-I) at 37 °C and pH 10. Figure 2 summarizes the HPLC chart of the reaction mixture 20 min after the start. In all cases, the ODN disappeared and the corresponding FT-ODN was formed in almost quantitative yield. The number in parentheses in FT-ODN(N) represents the transfer group. Substituents, such as an ether chain on the pyridine ring, were found to have no effect on the preparation of FT-ODN, which was produced in high yield.

Fig. 2. HPLC Charts of the Reaction for the Synthesis of FT-ODN

(A) ODN only, (B) ODN with 7-I, (C) ODN with 11-I, (D) ODN with 12-I, (E) ODN with 13-I. The reaction was performed using ODN (50 µM) and the precursor of the transfer group (7-I, 11-I, 12-I, 13-I) (1 mM) in 25 mM carbonate buffer (pH 10) at 37 °C for 20 min. HPLC analysis: column: OSAKA SODA C18, 4.6 × 250 mm; solvents: A: 0.1 M TEAA buffer, B: CH₃CN, B: 8 to 28%/20 min, 28 to 100%/25 min, linear gradient; column oven: 35 °C; flow rate: 1.0 ml/min; UV: 254 nm.

Next, we investigated the effect of substituents of the pyridine ring on the transfer reaction to the amino group at the cytosine 4-position. The synthesized FT-ODN was mixed with 30 mer RNA (5′ agagagaaagaagaa-c-aaagacggcugcga 3′), in which the bolded sequence is complementary with the 16 mer ODN shown in Chart 3 and the underlined cytidine is the target site for the modification (Chart 4). A solution of RNA (5 µM) in HEPES buffer solution (50 mM, pH 7) containing a NaCl solution (100 mM) was heated at 65 °C for 3 min, then rapidly cooled at 0 °C. Aqueous solutions of FT-ODN (7.5 µM) and NiCl₂ (75 µM) were added to this solution at 0 °C, and the mixture was incubated at 37 °C for 1 h. The concentration shown in parentheses is that of the final solution. The reaction progress was followed by HPLC. Figure 3 summarizes the HPLC chart of the reaction mixture 60 min after the start. The HPLC charts indicate the presence of un-reacted RNA, regenerated ODN, modified RNA(N) and remaining FT-ODN(N). Our previous studies have shown that prolonged reaction time does not increase the yield of the modified RNA and that FT-ODN containing the transfer group with its non-reactive Z-stereochemistry remains.¹⁷⁾ The modification yields calculated from the ratio of unmodified to modified RNA are as follows, 74% yield for RNA(7) with FT-ODN(7) (A), 82% yield for RNA(11) with FT-ODN(11) (B), 80% yield for RNA(12) with FT-ODN(12) (C) and 75% yield for RNA(13) with FT-ODN(13) (D). The modification efficiencies with FT-ODN(11) and (12) were slightly higher than the other two. These results indicated that the ether chain structure has no significant effect on the functionality transfer reaction.

Chart 3. Synthesis of the Functionality-Transfer ODN (FT-ODN) by the Introduction of the Transfer Group to the Thioguanine Base of the ODN

The number in parentheses in FT-ODN(N) represents the transfer Group.

Chart 4. The Functionality Transfer Reaction to Produce the Modified RNA(N)

The number in parentheses in RNA(N) represents the transfer group.

Fig. 3. HPLC Charts of the RNA Modification Reaction with FT-ODN

The number in parentheses in RNA(N) represents the transfer group. (A) with FT-ODN(7), (B) with FT-ODN(11), (C) with FT-ODN(12), (D) with FT-ODN(13). The reaction was performed using RNA (5 µM), FT-ODN (7.5 µM) and 75 µM NiCl₂ in 50 mM N-(2-hydroxyethyl) piperazine-N′-2-ethanesulfonic acid (HEPES) containing 100 mM NaCl at pH 7.0 and 37 °C for 60 min. HPLC analysis: column: OSAKA SODA C18, 4.6 × 250 mm; solvents: A: 0.1 M TEAA buffer, B: CH₃CN, B: 8 to 28%/20 min, 28 to 100%/25 min, linear gradient; column oven: 35 °C; flow rate: 1.0 ml/min; UV: 254 nm.

The modified RNA(N) was next subjected to a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC reaction). The azide compounds (14–17) were used in this study. A reaction mixture of the functionality transfer reaction was used without purification, and was mixed with a dimethyl sulfoxide (DMSO) solution of the azide compound (400 µM), a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (300 µM), an aqueous solution of sodium ascorbate (300 µM), and an aqueous solution of copper sulfate (150 µM) at 25 °C. The mixture was allowed to stand for 40 min. The HPLC charts of the reaction of RNA(13) modified with the transfer group 13 are shown as examples in Fig. 4. The azide compound bonded in the click reaction is indicated by a hyphen followed by a number.

Fig. 4. HPLC Charts of the Azide-Alkyne Cycloaddition Using RNA (13) and Azides (14–17)

The azide compound bonded in the click reaction is indicated by a hyphen followed by a number. Peaks marked with asterisk(*) correspond to the cycloadduct with FT-ODN. (A) The reaction mixture to form RNA(13). (B) The CuAAC reaction with 14. (C) The CuAAC reaction with 15. (D) The CuAAC reaction with 16. (E) The CuAAC reaction with 17. The reactions were performed using R-N₃ (400 µM, DMSO), sodium ascorbate (300 µM), TBTA (300 µM, DMSO) and CuSO₄ (150 µM) at r.t., for 40 min. HPLC Conditions: column, OSAKA SODA C18, 4.6 mm × 250 mm; solvent, A: 0.1 M TEAA buffer, B: CH₃CN, B: 8% to 28%/20 min, 28% to 100%/25 min, linear gradient; column oven at 35 °C; flow rate, 1.0 ml/min; UV, 254 nm.

The newly appeared peak was isolated, the solvents were removed by freeze-drying, and the adduct formation was confirmed by UPLC/QTof-MS measurements (Supplementary Materials). In the UPLC charts, the regenerated RNAs were observed, which may be due to the removal of the modified structure in the UPLC column heated at 60 °C or lyophilization after the HPLC isolation. The CuAAC reaction yield for RNA(N)-M was calculated as the conversion yield from the modified RNA(N) with respect to the unmodified RNA, and are summarized in Table 1. The transfer group 7 showed lower yields of four azides compared to the other transfer groups, probably because the RNA strand in close proximity prevents the azide compound from approaching the alkyne. In addition to that, instability of the 7-modified structure may also be the cause of the low yield, since a part of the RNA(7) modified with 7 was found to revert to unmodified RNA. The particularly low yield with azide 15 may be due to prevention of its access by ionic repulsion between the carboxylate of 15 and the phosphodiester group of RNA. The reaction yields were improved with the transfer groups 11, 12 and 13, which have the alkyne at the end of the PEG linker. Since the alkyne reaction site is far from the RNA strand, the access of the azide compound is no longer affected by the steric or ionic repulsion.

Table 1. The Conversion Yield (%) from RNA(N) to RNA(N)-M by the Azide-alkyne Cycloaddition^a)

Transfer group	7	11	12	13
Azide
14	69	87	87	93
15	65	85	>95	>95
16	82	>95	>95	>95
17	86	>95	>95	>95

a) The reaction and HPLC conditions are described in the footnote of Fig. 4. The conversion yields were calculated from the peak area ratio of the RNA(N) and the clicked product RNA(N)-M.

Conclusion

Due to the importance of the RNA chemical modifications, methods for selective chemical modification at a predetermined site of the internal position of RNA have attracted much attention. In addition, the CuAAC has been shown to introduce additional molecules on the alkynes attached to the pyridine ring. However, it was found that some azide compounds produced the cycloadduct in lower yields. Therefore, in this study, we synthesized the pyridinyl transfer group with the alkyne attached via a PEG linker with a different length and optimized its structure for both the transfer and CuAAC reaction. Three new transfer groups were synthesized by introducing an alkyne group at the end of the triethylene (11), tetraethylene (12) or pentaethylen glycol linker (13) at the 5-position of the pyridine ring of (E)-3-Iodo-1-(pyridin-2-yl)prop-2-en-1-one. These transfer groups were introduced into the 6-thioguanine base in ODN in high yields. The transfer groups 11 and 12 more efficiently underwent cytosine modification. For the CuAAC reaction, although 7 showed low adduct yields with the anionic azide compound, the new transfer groups, especially 12 and 13, significantly improved the yields. In conclusion, the transfer group 12 and 13 have been determined as promising compounds for the modification of long RNAs. These applications for the modification of a long RNA will be reported in the near future.

Experimental

General

The ¹H-NMR spectra (500 MHz) and ¹³C-NMR (125 MHz) spectra were measured by a Bruker Ascend 500 using solvent peaks as internal standards. The high-resolution electrospray ionization (ESI)-MS measurement was performed using an Applied Biosystems Mariner Biospectrometry Workstation mass spectrometer with physalaemin, neurotensin, angiotensin I, bradykinin as internal standards. UPLC/MS measurements were performed using a Waters ACQUITY UPLC equipped with a Xevo G2-XS Qtof. Column chromatography was performed using Kanto Chemical Silica gel 60N. HPLC was performed by a JASCO LC-2000 PLUS series equipped with an OSAKA SODA CAPCELL PAK C18 MG (4.6 × 250 nm). The RNAs and ODN were purchased from Gene Design (Japan), GeneNet (Japan) or Nihon Bioservice (Japan). The concentrations of the ODN and RNA were determined by measuring the absorbance at 260 nm and 25 °C based on the sum of the molar absorbance coefficients of the nucleobases multiplied by 0.9: adenine, 15300; guanine, 11800; thymine, 9300; cytosine, 7400; uracil, 10000; 6-thioguanine, 11800.

5-((3,6-Dioxanona-8-yn-1-yl)oxy)picolinonitrile (8)

Sodium hydride (60%, dispersion in paraffin liquid, 161 mg, 4.03 mmol) was washed twice with HPLC grade hexane and dried in vacuum. Under an argon atmosphere, a solution of diethylene glycol (713 mg, 6.72 mmol) in dry tetrahydrofuran (3.2 mL) was added to this solid at 0 °C, and the mixture was stirred for 45 min. Into this mixture, a solution of 9.2 M propargylic bromide in toluene (400 mg, 3.36 mmol) was then added and the mixture was stirred for 2 h. The reaction mixture was allowed to warm to room temperature and stirred for 15 h. The reaction was stopped by adding 50 mL of pure water and extracted five times with dichloromethane. The organic layers were washed with brine and dried over anhydrous sodium sulfate. The solvents were evaporated, and the residue was purified by silica gel column chromatography (Kanto 60N, hexane: ethyl acetate = 2 : 3 to 0 : 1) to afford 3,6-dioxanona-8-yn-1-ol (235 mg, 48%) as a yellow oil. ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 4.20 (2H, d, J = 2.44 Hz), 3.72 (2H, t, J = 8.30 Hz), 3.70 (2H, d, J = 2.44 Hz), 3.61 (4H, J = 4.88 Hz), 2.44 (1H, t, J = 2.44 Hz); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 79.33, 74.61, 72.40, 69.97, 68.94, 61.43, 58.18; ESI-HRMS (m/z) Calcd for C₇H₁₂O₃Na [M + Na]⁺ : 167.0679. Found : 167.0695.

Sodium hydride (60%, dispersion in paraffin liquid, 161 mg, 4.03 mmol) was washed twice with HPLC grade hexane and dried in vacuum. Under an argon atmosphere, a solution of the above oil (238 mg, 1.65 mmol) in dry DMF (2.0 mL) was added to this solid at 0 °C, and the mixture was stirred for 40 min, followed by the addition of 5-bromo-2-cyanopyridine. The reaction mixture was allowed to warm to room temperature and stirred for 17 h. The reaction was stopped by adding 50 mL of a saturated aqueous ammonium chloride solution (50 mL) and extracted five times with hexane : ethyl acetate = 4 : 1. The organic layers were washed with brine and dried over anhydrous sodium sulfate. The solvents were evaporated, and the residue was purified by silica gel column chromatography (Kanto 60N, hexane : ethyl acetate = 9 : 1, 8 : 2 to 0 : 1) to afford 8 (201 mg, 74%) as a yellow solid. ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 8.40 (1H, d, J = 2.93 Hz), 7.63 (1H, d, J = 8.30 Hz), 7.28 (1H, dd, J = 8.78 Hz, 2.93 Hz), 4.25 (2H, t, J = 4.39 Hz), 4.20 (2H, d, J = 2.44 Hz), 3.90 (2H, t, J = 4.39 Hz), 3.76–3.70 (4H, m), 2.43 (1H, J = 2.44 Hz); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 157.64, 140.87, 129.87, 125.81, 120.98, 117.84, 79.83, 75.07, 71.15, 69.74–69.47, 68.71, 58.85; ESI-HRMS (m/z) Calcd for C₁₃H₁₅N₂O₃ [M + H]⁺ : 247.1077. Found : 247.1090.

5-((3,6,9-Trioxadodec-11-yn-1-yl)oxy)picolinonitrile (9)

3,6,9-Trioxadodec-11-yn-1-ol was obtained as a yellow oil in a 76% yield using triethylene glycol and propargylic bromide in a similar manner as already described for the synthesis of 3,6,9-trioxadodec-11-yn-1-ol. ¹H-NMR (500 MHz, CHCl₃) δ (ppm): 4.18 (2H, d, J = 2.44 Hz), 3.71–3.62 (10H, m), 3.56–3.59 (2H, m) 2.42 (1H, t, J = 2.44 Hz); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 79.53, 74.70, 72.54, 70.55, 70.28, 70.23, 69.01, 61.60, 58.34; ESI-HRMS (m/z) Calcd for C₉H₁₆O₄ [M + H]⁺ : 189.1121. Found : 189.1121.

3,6,9-Trioxadodec-11-yn-1-ol was reacted with 5-bromo-2-cyanopyridine in a similar manner as already described for the synthesis of 8 to give 9 as a yellow solid in 82% yield. ¹H-NMR (500 MHz, CHCl₃) δ (ppm): 8.38 (1H, d, J = 2.44 Hz), 7.62 (1H, d, J = 9.28 Hz), 7.28 (1H, dd, J = 8.50, 2.93 Hz), 4.23 (2H, t, J = 2.44 Hz), 4.18 (2H, d, J = 2.44 Hz) 3.89 (2H, t, J = 4.41 Hz), 3.72–3.64 (8H, m), 2.42 (1H, d, J = 2.5 Hz); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 174.04, 140.85, 130.04, 129.70, 120.89, 117.82, 71.33, 71.03, 70.87, 69.91, 69.74, 69.49, 68.78, 58.81, 58.79; ESI-HRMS (m/z) Calcd for C₁₅H₁₈N₂O₄ [M + H]⁺ : 290.1339. Found : 291.1330.

5-((3,6,9,12-Tetraoxapentadec-14-yn-1-yl)oxy)picolinonitrile (10)

3,6,9,12-Tetraoxapentadec-14-yn-1-ol was obtained as a yellow oil in a 65% yield using tetraethylene glycol and propargylic bromide in a similar manner as already described for the synthesis of 3,6-dioxanona-8-yn-1-ol. ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 4.20 (2H, d, J = 2.44 Hz), 3.73–3.64 (14H, m), 3.61 (2H, t, J = 4.64 Hz), 2.42 (1H, t, J = 2.44 Hz); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 79.52, 74.47, 72.47, 70.49–70.18, 68.98, 61.56–61.45, 58.24; ESI-HRMS (m/z) Calcd for C₁₁H₂₀O₅Na [M + Na]⁺ : 255.1203. Found : 255.1201.

3,6,9,12-Tetraoxapentadec-14-yn-1-ol was reacted with 5-bromo-2-cyanopyridine in a similar manner as already described for the synthesis of 8 to give 10 as a yellow solid in 82% yield. ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 8.39 (1H, d, J = 2.93 Hz), 7.63 (1H, d, J = 8.30 Hz), 7.28 (1H, dd, J = 8.30 Hz, 2.93 Hz), 4.24 (2H, t, J = 4.64), 4.19 (2H, d, J = 2.44 Hz), 3.89 (2H, t, J = 4.64 Hz), 3.72-3.64 (12H, m), 2.42 (1H, t, J = 2.44 Hz); ¹³C-NMR (125 MHz, CDCl3) δ (ppm): 163.93, 147.12, 136.11, 131.88, 127.23, 124.08, 82.25, 84.01–83.49, 75.87–75.00, 64.95; ESI-HRMS (m/z) Calcd for C₁₇H₂₂N₂O₅Na [M + Na]⁺ : 357.1421. Found : 357.1430.

(E)-3-Iodo-1-(5-((3,6-dioxanona-8-yn-1-yl)oxy) Pyridin-2-yl)prop-2-en-1-one (11-I)

A solution of ethylmagnesium bromide in tetrahydrofuran (0.5 M, 3.36 mL, 1.68 mmol) was added to 8 (103 mg, 0.42 mmol) at 0 °C under an argon atmosphere. The mixture was allowed to warm to room temperature and stirred for 2 h, followed by the addition of a 20% aqueous hydrogen iodide solution. After 1 h, the reaction mixture was quenched by the addition of a saturated aqueous sodium hydrogen carbonate solution, and the mixture was stirred for an additional 30 min, then filtered through a celite pad. The mixture was extracted with ethyl acetate, the organic layers were successively washed with an aqueous ammonium chloride solution and brine, then dried over anhydrous sodium sulfate. The solvents were evaporated, and the residue was purified by silica gel column chromatography (Kanto 60N, hexane : ethyl acetate = 7 : 3, 1 : 1 to 0 : 1) to afford 11-I (26.3 mg, 14%) as a dark brown solid. ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 8.61 (1H, d, J = 15.13 Hz), 8.37 (1H, d, J = 2.93 Hz), 8.13 (1H, d, J = 14.64 Hz), 8.11 (1H, d, 8.78 Hz) 7.32 (1H, dd, J = 8.78 Hz, 2.93 Hz), 4.27 (2H, t, J = 4.64 Hz) 4.21 (2H, d, J = 2.44 Hz), 3.92 (2H, t, J = 4.88 Hz), 3.75–3.72 (4H, m), 2.43 (1H, t, J = 4.39 Hz); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 185.62, 158.06, 145.75, 140.22, 137.77, 128.05, 124.97, 121.15, 100.72, 79.63, 74.81, 70.93, 69.43, 68.29, 58.63, 29.84; ESI-HRMS (m/z) Calcd for C₁₅H₁₆INO₄Na [M + Na]⁺ : 424.0016. Found : 424.0046.

(E)-3-Iodo-1-(5-((3,6,9-trioxadodec-11-yn-1-yl)oxy)pyridin-2-yl)prop-2-en-1-one (12-I)

12 was obtained as a yellow oil in 23% yield using 9, ethylmagnesium bromide and hydrogen iodide in a similar manner as already described for the synthesis of 11-I. ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 8.62 (1H, d, J = 15.13 Hz), 8.36 (1H, d, J = 2.93 Hz), 8.11 (1H, d, J = 14.68 Hz), 8.11 (1H, d, J = 8.78 Hz), 7.31 (1H, dd, J = 8.78, 2.93 Hz), 4.26 (2H, t, J = 4.88 Hz), 4.19 (2H, d, J = 2.44 Hz), 3.91 (2H, t, J = 4.88 Hz), 3.74–3.71 (2H, m), 3.70–3.66 (6H, m), 2.42 (1H, t, J = 2.44 Hz); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 185.57–185.54, 180.05, 145.69, 140.20–140.63, 137.55–137.49, 125.12, 121.11, 100.66, 79.73, 74.69, 71.06–70.59, 69.54–69.21, 68.29, 58.53; ESI-HRMS (m/z) Calcd for C₁₇H₂₁INO₅ [M + H]⁺ : 446.0459. Found : 446.0488.

(E)-3-Iodo-1-(5-((3,6,9,12-tetraoxapentadec-14-yn-1-yl)oxy)pyridin-2-yl)prop-2-en-1-one (13-I)

13 was obtained as a dark brown solid in a 14% yield using 9, ethylmagnesium bromide and hydrogen iodide in a similar manner as already described for the synthesis of 11-I. ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 8.62 (1H, d, J = 14.64 Hz), 8.37 (1H, d, J = 2.93 Hz), 8.12 (1H, d, J = 14.64 Hz), 8.12 (1H, d, J = 9.27 Hz), 7.32 (1H, dd, J = 8.78 Hz, 2.93 Hz) 4.27 (2H, t, J = 4.39 Hz), 3.91 (2H, t, J = 4.88 Hz), 3.74-3.72 (2H, m), 3.71–3.66 (12H, m), 2.42 (1H, t, J = 2.20 Hz); ¹³C-NMR (125 MHz, CDCl₃) δ (ppm): 185.28, 157.86, 145.36, 139.94, 137.28, 124.94, 121.02, 100.52, 79.53, 74.42, 70.51, 69.30, 68.10, 58.30; ESI-HRMS (m/z) Calcd for C₁₉H₂₄INO₆Na [M + Na]⁺ : 512.0541. Found : 512.0534.

Preparation of the Functionality-Transfer ODN, FT-ODN(N) (Chart 3 and Fig. 2)

The sequence of ODN is 5′ dCTTT-X-TTCTCCTTTCT, in which X represents 2′-deoxy-6-thioguanosine. A general procedure of the preparation of the functionality-transfer ODN is described for the reaction between ODN and 7-I. At room temperature, an aqueous stock solution of ODN (105 µM, 1.90 µL, 200 pmol) was added to carbonate buffer (125 mM, 0.8 µL, pH 10.0) and diluted with ultrapure water (1.1 µL). To this solution, 7-I ((E)-3-iodo-1-(4-ethynylpyridin-2-yl)prop-2-en-1-one) (20 mM, 0.2 µL, 4 nmol) was added. After 20 min at room temperature, and the reaction progress was followed by HPLC. Final concentrations: ODN (50 µM), 7-I (1.0 mM), carbonate buffer (25 mM). The reaction solution was used for the functionality-transfer reaction without purification. The HPLC analysis conditions were as follows: column: OSAKA SODA C18, 4.6 mm × 250 mm and eluent solvents: A: 0.1 M TEAA buffer, B: CH₃CN, B: 8% to 28%/20 min, 28% to 100%/25 min, linear gradient; column oven: 35 °C; flow rate: 1.0 ml/min; UV: 254 nm. The HPLC charts obtained using the transfer group (7-I, 11-I, 12-I, 13-I) are summarized in Fig. 2.

Functionality Transfer Reaction to Modify RNA (Chart 4 and Fig. 3)

The sequence of RNA is 5′ agagagaaagaagaa-c-aaagacggcugcga, in which the underlined c indicates the target site for the modification. A general procedure of the functional group transfer reaction to RNA is described for the reaction between FT-ODN(7) and RNA. A stock solution of RNA (106 µM, 0.94 µL, 100 pmol) was added to a HEPES buffer solution (0.5 M HEPES, 2 µL, pH 7), followed by the addition of an aqueous NaCl solution (1 M, 2 µL, 2.0 µmol) and ultrapure water (9.06 µL). The solution was heated at 65 °C for 3 min, then rapidly cooled at 0 °C. Aqueous solutions of FT-ODN (50 µM, 3 µL, 150 pmol) and NiCl₂ (500 µM, 3 µL, 1.50 nmol) were added to this solution at 0 °C, and the mixture was incubated at 37 °C for 1 h. Final concentrations: RNA (5 µM), FT-ODN (7.5 µM), NiCl₂ (75 µM), HEPES buffer (50 mM), NaCl (100 mM). The reaction progress was followed by HPLC using the following conditions: column: OSAKA SODA C18, 4.6 × 250 mm and eluent solvents: A: 0.1 M TEAA buffer, B: CH₃CN, B: 8% to 28%/20 min, 28% to 100%/25 min, linear gradient; column oven: 35 °C; flow rate: 1.0 ml/min; UV: 254 nm. The HPLC charts obtained using FT-ODN(7, 11, 12, 13) are summarized in Fig. 3. The newly appeared peak was isolated, the solvents were removed by freeze-drying, and subject to UPLC/QTof-MS measurements. The modification yield was calculated from the peak area ratio of the unreacted RNA to the modified RNA.

The Copper-Catalyzed Azide-Alkyne Cycloaddition Reaction (Chart 5, Fig. 4)

Chart 5. The Azide-Alkyne Cycloaddition to Produce the Clicked RNA

A general procedure for the copper-catalyzed azide-alkyne cycloaddition reaction to RNA is described for the reaction between the modified RNA(13) and benzyl azide. To a reaction mixture containing the modified RNA(13), FT-ODN(13) and ODN (18 µL), a DMSO solution of benzyl azide (10 mM, 1 µL), a DMSO solution of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (25 mM, 0.3 µL), an aqueous solution of sodium ascorbate (25 mM, 0.3 µL), and an aqueous solution of copper sulfate (10 mM, 0.38 µL) were added at 25 °C, the reaction mixture was allowed to stand for 40 min. Final concentrations: benzyl azide (400 µM), TBTA (300 µM), sodium ascorbate (300 µM), copper sulfate (150 µM). The reaction progress was followed by HPLC using the following conditions; column: OSAKA SODA C18, 4.6 × 250 mm and eluent solvents: A: 0.1 M TEAA buffer, B: CH₃CN, B: 8 to 28%/20 min, 28 to 100%/25 min, linear gradient; column oven: 35 °C; flow rate: 1.0 mL/min; UV: 254 nm. The HPLC charts obtained using the modified RNA(13) and the azide compounds (14–17) are summarized in Fig. 4. The newly appeared peak was isolated, the solvents were removed by freeze-drying, and subject to UPLC/QTof-MS measurements. The modification yields were calculated from the peak area ratio of the modified RNA to the clicked RNA, and are summarized in Table 1. The UPLC/QTof-MS of the modified RNA is reported in the Supporting Information and the data are summarized in Table 2.

Table 2. UPLC/QTof-MS Data of the Modified RNA

Modified RNA	FT-ODN(N)	Azide	Calcd ([M − H]−)	found
RNA(7)	7	—	10026.13	10028
RNA(7)-14	7	14	10159.28	10158
RNA(7)-15	7	15	10261.23	10260
RNA(7)-16	7	16	10283.43	10282
RNA(7)-17	7	17	10227.31	10226
RNA(11)	11	—	10144.09	10145
RNA(11)-14	11	14	10277.24	10277
RNA(11)-15	11	15	10379.19	10379
RNA(11)-16	11	16	10401.39	10401
RNA(11)-17	11	17	10345.27	10345
RNA(12)	12	—	10188.14	10189
RNA(12)-14	12	14	10321.29	10321
RNA(12)-15	12	15	10423.24	10422
RNA(12)-16	12	16	10445.44	10445
RNA(12)-17	12	17	10389.32	10389
RNA(13)	13	—	10232.19	10233
RNA(13)-14	13	14	10365.34	10364
RNA(13)-15	13	15	10467.29	10466
RNA(13)-16	13	16	10489.49	10491
RNA(13)-17	13	17	10433.37	10433

UPLC/MS Measurement

UPLC/MS measurements were performed by a Waters ACQUITY UPLC equipped with a Xevo G2-XS Qtof. UPLC conditions: Column: Oligonucleotide BEH C18 Column, 130 Å, 1.7 µm, 2.1 × 50 mm, column temperature: 60 °C; eluents: A: 15 mM TEA, 400 mM HFIP pH 7.9, B: A/MeOH = 1/1, B: 40 to 100%/10 min, 0.2 mL/min. MS Conditions: mass range: 450–3000 Da; mode: ESI negative; resolution: continuum; cone voltage: 40 V; capillary voltage: 4 kV; desolvation: 600 °C; desolvation gas flow: 700 L/hr.

Acknowledgments

This study was partially supported by a Grant-in-Aid for Scientific Research (B) (Grant Number 15H04633 for S. S.) and a Grant-in-Aid for Specially Promoted Research (Grant Number 19H05458 for S. S.) from the Japan Society for Promotion of Science (JSPS).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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