Novel nucleoside and oligonucleotide analogs controlled by click chemistry

Abstract

To acquire functions by releasing active molecules from silenced precursors utilizing click reactions, since control of DNA and RNA structures is essential for material development, control of biological activities, and acquisition and manipulation of functions of nucleic acids in medical applications.SOLUTION: A benzyl vinyl ether group (BVE group) or the like, which can be cleaved by a click reaction, is incorporated into a nucleoside or an oligonucleotide as a caged precursor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to novel nucleoside and nucleotide analogs that can undergo a click reaction, and more particularly to nucleotide analogs that are suitable for controlling DNA and RNA structures by click-and-release. [Background technology]

[0002] Controlling the structure of DNA and RNA is important for the development of materials, the control of biological functions, and the acquisition and manipulation of nucleic acid functions in medical applications. It is one of the most promising fields in recent years. "Click" chemistry generally involves coupling two compounds containing an alkyne group and an azide group. It is an excellent method that does not require protection or purification, reacts efficiently and specifically within cells, and is suitable for biological applications. It was awarded the Nobel Prize in Chemistry in 2022.

[0003] Recently, an approach has been reported in which the click reaction breaks chemical bonds rather than coupling them. To date, this approach has been used primarily in research into the release of active proteins, which are functionalized by releasing active molecules from silenced precursors using the click reaction. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Jimenez-Moreno, E. et al., Angew Chem Int Ed Engl. 2017, vol. 56(1), p.243-247. [Non-patent document 2] Xu et al., Chem. Commun., 2017,53, p.6271-6274. [Non-patent document 3] Carlson, et al., J Am Chem Soc. 2018,140 (10), p.3603-3612. Summary of the Invention [Problem to be solved by the invention]

[0005] The objective of the present invention is to create nucleoside and oligonucleotide molecules that can control the structure of DNA and RNA based on a click-and-release approach. [Means for solving the problem]

[0006] The present invention demonstrates that control of DNA and RNA structures is important for the development of materials, the control of biological functions, and the acquisition and manipulation of nucleic acid functions in medical applications. The present invention utilizes the click reaction to release active molecules from silenced precursors to acquire functions.

[0007] The present inventors decided to incorporate a benzyl vinyl ether group (BVE group), which can be cleaved by a click reaction, into nucleosides and oligonucleotides as a caged precursor (Figure 9).

[0008] According to the present invention, caging the nucleic acid bases disrupts the Watson-Crick or Hoogsteen base pairs of nucleic acids, preventing hydrogen bonds from forming and preventing the formation of DNA and RNA structures. According to the present invention, the BVE group is removed by the Click reaction, restoring the hydrogen bonds of the base pairs, thereby forming DNA and RNA structures, thereby activating the functions of DNA and RNA.

[0009] The present invention will be described in detail below.

[0010] The present invention encompasses caged nucleotides and caged nucleotide analogs comprising a caged purine or pyrimidine base selected from the group consisting of:

[0011] [ka]

[0012] Here, Z is represented by the following formula: Ra, Rb, Rc, Rd, Re, and Rf each independently represent H, halogen, a nitro group, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl, C1-C20 alkoxy, aryl, heteroaryl, keto, carboxy, amino, silyl, boron, and / or a target group, etc.

[0013] [ka]

[0014] The present invention provides a method for synthesizing an oligonucleotide or oligonucleotide analogue from a plurality of nucleotides or nucleotide analogues, comprising: Each of the nucleotides or nucleotide analogs contains a purine or pyrimidine base, and at least one of the nucleotides or nucleotide analogs is caged.

[0015] The present invention encompasses nucleotides or nucleotide analogs selected from:

[0016] [ka]

[0017] Here, Z is represented by the following formula: Ra, Rb, Rc, Rd, Re, and Rf each independently represent H, halogen, a nitro group, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl, C1-C20 alkoxy, aryl, heteroaryl, keto, carboxy, amino, silyl, boron, and / or a target group, etc.

[0018] [ka]

[0019] The present invention provides an oligonucleotide or oligonucleotide analogue compound comprising a sequence of nucleotides or nucleotide analogues, At least one of the nucleotides or nucleotide analogs includes a caged nucleotide or nucleotide analog selected from the group consisting of:

[0020] [ka]

[0021] Here, Z is represented by the following formula: Ra, Rb, Rc, Rd, Re, and Rf each independently represent H, halogen, a nitro group, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl, C1-C20 alkoxy, aryl, heteroaryl, keto, carboxy, amino, silyl, boron, and / or a target group, etc.

[0022] [ka]

[0023] The present invention further provides a method for (a) decaging an oligonucleotide or oligonucleotide analogue, comprising: The present invention encompasses methods of preparing a cell, tissue, or subject in vitro or in vivo comprising a plurality of nucleotides or nucleotide analogs, wherein at least one of the nucleotides or nucleotide analogs is a caged nucleotide or nucleotide analog selected from the group consisting of:

[0024] In the method (a) for uncaging an oligonucleotide or oligonucleotide analogue, (b) the oligonucleotide or oligonucleotide analogue is reacted with a tetrazine derivative to cleave the caging group Z from the base, thereby obtaining an uncaged oligonucleotide or oligonucleotide analogue.

[0025] The tetrazine derivative of the present invention (the compound on the left) is represented by the following formula: 1 and R 2 The following are available (R on the right)1 and R 2 The tetrazine derivatives are not limited to these.

[0026] [ka] [Effects of the Invention]

[0027] Oligonucleotides are useful as oligonucleotide probes, small interfering RNAs (siRNAs), antisense oligonucleotides, Z- and tetraplex-forming oligonucleotides, sensors, or aptamers.

[0028] The present invention provides spatial and temporal control of the function of oligonucleotides and oligonucleotide analogs via the Click reaction, which can be achieved in vitro and in vivo.

[0029] For example, the present invention can control the release of Z-DNA or Z-RNA and bind to related proteins. The present invention can also control quadruplex formation in vitro and in vivo and visually detect higher-order structures of nucleic acids as fluorescent probes.

[0030] The present invention also enables molecular imaging for cancer cell detection using quantum dot (QD)-functionalized caged DNA. The present invention also enables the control of blood coagulation by click-release of nucleic acid aptamers.

[0031] This click-and-release approach allows the present invention to achieve rapid response rates, excellent orthogonality, and biocompatibility. [Brief explanation of the drawings]

[0032] [Figure 1] In Examples 1 to 6, DNA-1BVE, DNA-2BVE, etc. have caged nucleotide analogs incorporated at various positions. [Figure 2]In Example 7, native DNA-1 and DNA-2 were released by the click reaction of DNA-1BVE and DNA-2BVE with a tetrazine derivative to form double-stranded DNA. This figure shows the results of HPLC analysis of the click reaction. The peaks for DNA-1BVE and released DNA-1, and DNA-2BVE and released DNA-2 are shown. [Figure 3] 1 shows CD spectra of the recovery of Z-DNA from DNABVE-dGF over the time course of the reaction in Example 8. Reaction times are indicated. [Figure 4] FIG. 10 shows the results of protein binding to Z-RNA recovered from RNABVE-rGF in Example 9. [Figure 5] This figure shows the results of efficient cancer cell detection using QD-DNA-3BVE (520), QD-DNA-4BVE (580), and QD-DNA-5BVE (620) in cells in Example 10. A red signal was observed at the quantum dot emission wavelength of 620 nm, demonstrating that efficient cancer cell detection is possible. [Figure 6] This figure shows the results of controlling quadruplex formation in cells using Cy3-DNA-3BVE and Cy5-DNA-4, and detecting quadruplex higher-order structures using fluorescent probes in Example 11. A red signal was observed at a Cy5 emission wavelength of 670 nm, and excitation at a Cy3 absorption wavelength of 550 nm reduced Cy3 emission at 570 nm. Quadruplex higher-order structures were formed in cells, demonstrating fluorescence resonance energy transfer (FRET). [Figure 7] This figure shows the control of quadruplex formation by Cy3-DNA-3BVE and Cy5-DNA-4 and the results of fluorescence imaging of mice in Example 12. Under conditions of excitation at 550 nm and emission at 670 nm, a clear fluorescent signal was observed on the left side, but almost no fluorescent signal was observed on the right side. These results indicate the formation of quadruplex higher-order structures in vivo in mice and fluorescence resonance energy transfer (FRET). [Figure 8]FIG. 13 shows the results of a prothrombin time (PT) assay of human plasma over the time course of a click reaction using TBABVE-dGF in Example 13. [Figure 9] FIG. 1 shows the incorporation of a click-cleavable benzyl vinyl ether group (BVE group) into a nucleoside or oligonucleotide as a caged precursor according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present inventors incorporated a benzyl vinyl ether group (BVE group), which can be cleaved by a click reaction, into nucleosides and oligonucleotides as caged precursors (Figure 9).

[0034] According to the present invention, caging the nucleic acid bases disrupts the Watson-Crick or Hoogsteen base pairs of nucleic acids, preventing hydrogen bonds from forming and preventing the formation of DNA and RNA structures. According to the present invention, the BVE group and other groups are removed by the Click reaction, restoring the hydrogen bonds of the base pairs, thereby forming DNA and RNA structures, thereby activating the functions of DNA and RNA.

[0035] According to the present invention, the Click reaction allows spatial and temporal control of the function of oligonucleotides and oligonucleotide analogs to be achieved in vitro and in vivo, for example, by controlling the release of Z-DNA or Z-RNA and binding to relevant proteins.

[0036] Furthermore, the present invention enables molecular imaging for cancer cell detection using quantum dot (QD)-functionalized caged DNA. The present invention also enables the control of quadruplex formation in vitro and in vivo, and the visual detection of higher-order structures of nucleic acids as fluorescent probes.

[0037] According to the present invention, blood coagulation can be further controlled by click-and-release of nucleic acid aptamers, which offers rapid response rates, excellent orthogonality, and biocompatibility.

[0038] In the present invention and specification, "click and release" refers to the spatial and temporal control of the function of oligonucleotides and oligonucleotide analogs via a click reaction in vitro or in vivo, thereby activating the function of DNA and RNA.

[0039] As used herein and in the present application, "caged" refers to a compound having a group thereon that renders the compound substantially functionally inactive in at least one intended use, and that group can be removed (e.g., by click-and-release) to restore activity to the compound.

[0040] As used herein and in this specification, "caged" refers to a compound that has a functional group that substantially disables the function of the compound in at least one intended use. This functional group can be removed (e.g., by clicking), thereby restoring activity to the compound.

[0041] As used herein and in this specification, the term "nucleotide" refers to a monomer of DNA or RNA that contains the following elements:

[0042] (a) A purine (adenine, guanine, etc.) or pyrimidine (cytosine, thymine, uracil, etc.) base. (b) Pentose (composed of deoxyribonucleotides and ribonucleotides of deoxyribose (2-deoxy D-ribose) or ribose (D-ribose)). (c) Phosphinic acid molecule.

[0043] The pentose is linked to the base of the nucleotide by a BN-glycosyl bond between carbon atom 1 of the pentose and nitrogen atom 9 of the purine base or nitrogen atom 1 of the pyrimidine base. The phosphate group of the nucleotide forms an ester bond with carbon atom 5 of the pentose.

[0044] As used herein and in this specification, "nucleotide analogs" include nucleotides in which the ribose or deoxyribose ring has been modified or replaced, as described above, and also nucleotides in which the phosphodiester bond has been modified or replaced (e.g., thiophosphate group, phosphate amidate group, etc.).

[0045] Any nucleotide (sometimes referred to as a "nucleic acid") or nucleotide analog (sometimes referred to as a "nucleic acid analog") can be used in the present invention and herein. Examples of common nucleotides include, but are not limited to:

[0046] Ribonucleotides: adenosine 5'-phosphate, guanosine 5'-phosphate, cytidine 5'-phosphate, and uridine 5'-phosphate.

[0047] Deoxyribonucleotides: deoxyadenosine 5'-phosphate, deoxyguanosine 5'-phosphate, deoxycytidine 5'-phosphate, and deoxythymidine 5'-phosphate.

[0048] Examples of "nucleotide analogs" that may be used in the present invention and specification include, but are not limited to, the following:

[0049] Peptide nucleic acid, pyranosyl-RNA, hexitol nucleic acid, mannitol nucleic acid, altritol nucleic acid, 2,5'-nucleic acid, locked nucleic acid, seco-locked nucleic acid, bicyclic nucleic acid (e.g., bicyclo[3.2.1 DNA], bicyclo[3.3.0 DNA], etc.), tricyclic nucleic acid (e.g., tricyclo-DNA), 3-hydroxy-N-acetylprolinol-substituted nucleic acid, carbocyclic nucleic acid These include nucleic acid, carbocyclic / bicyclic nucleic acid, nucleic acid with a triazole backbone, nucleic acid with an imidazole backbone, 1-phenylserinol nucleic acid, nucleic acid with an alpha anomeric backbone, and metal-linked nucleic acid.

[0050] Additional examples of "nucleotide analogs" as used herein and in the present application include, but are not limited to:

[0051] Nucleic acids include morpholino nucleic acid, cyclohexenyl nucleic acid, anhydrohexitol nucleic acid, phosphonomonoester nucleic acid, cyclobutyl nucleic acid, piperazine nucleic acid, phosphorothioate nucleic acid, 2'-O-alkyl nucleotides (e.g., 2'-O-methyl RNA, 2'-O-methoxyethyl RNA, etc.), fluoroarabinose nucleic acid, phosphorothioate nucleic acid, and N3'-P'5 phosphoroamidate nucleic acid.

[0052] As used herein and in this specification, an "oligonucleotide" refers to two or more nucleotides joined by a phosphodiester bridge, which bridge is formed between the 5'-hydroxyl group of one nucleotide and the 3'-hydroxyl group of the next adjacent nucleotide.

[0053] Oligonucleotides may be provided as single strands, or may be double stranded with complementary oligonucleotides or oligonucleotide analogs. They may also be triplex or quadruple stranded. Oligonucleotides may be polymers of the same or different nucleotides, e.g., "gapamers."

[0054] As used herein and in this specification, the term "oligonucleotide analog" refers to two or more nucleotides or nucleotide analogs (at least one of which may be a nucleotide analog) joined by a phosphodiester bond or other bond (e.g., a peptide bond in the case of a peptide nucleic acid).

[0055] Oligonucleotide analogs may be provided as single strands or may be double stranded with complementary oligonucleotides or oligonucleotide analogs. Oligonucleotide analogs may also be triplex or quadruplex. Oligonucleotide analogs may be polymers of the same nucleotide analogs or nucleotides or different nucleotide analogs. For example, "gapamers."

[0056] Oligonucleotides and oligonucleotide analogs are prepared by enzymatic synthesis or chemical synthesis (such as the phosphate phosphoramidite method). Generally, in the phosphate phosphoramidite method, oligonucleotides are prepared by solid-phase synthesis using protective chemistry.

[0057] Generally, oligonucleotides or oligonucleotide analogs (e.g., caged oligonucleotides or oligonucleotide analogs) start with one or five monomeric units (e.g., nucleotides) and can have lengths up to 20, 30, 100, 200, 1000, or more monomeric units. Longer lengths are usually achieved by enzymatic ligation of shorter segments.

[0058] The oligonucleotide or oligonucleotide analogue contains at least one or two caged purine or pyrimidine bases, as described above, and the number of caged purine or pyrimidine bases present depends on the length and purpose of the oligonucleotide or oligonucleotide analogue.

[0059] When used in vivo, this method can be carried out by administering or contacting the oligonucleotide or oligonucleotide analog with a cell (e.g., a cultured cell), a tissue (e.g., a cultured tissue such as muscle, skin, liver, nerve, bone, or pancreas).

[0060] The subject may be, for example, a fish, bird, reptile, amphibian, or human or animal subject, including, but not limited to, mammalian subjects, including, but not limited to, humans, mice, cows, horses, pigs, sheep, goats, rabbits, rats, cats, dogs, and the like.

[0061] Subjects may be at any stage of development, including prenatal, neonatal, infant, juvenile, adolescent, adult, and geriatric. Cells, tissues, and subjects may be non-animal, such as plant (including angiosperm and gymnosperm), yeast, fungal, and bacterial cells and tissues.

[0062] Oligonucleotides and oligonucleotide analogs may be formulated and administered to cells, tissues, or subjects alike, in a manner that is physiologically or pharmaceutically acceptable.

[0063] Tetrazine (Tz) is a novel bioorthogonal ligand that is expected to be applied in chemical biology. In vivo molecular imaging using Tz molecules involves the inverse electron demand Diels-Alder (IEDDA) reaction to generate reduced Tz (rTz), such as a tissue-bound product. [Example]

[0064] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0065] The oligonucleotides and oligonucleotide analogs of the present invention satisfy all of the following requirements:

[0066] (a) It allows specific probing of hydrogen bonding of oligonucleotide bases. (b) Allows the introduction of caged monomers under standard DNA synthesis conditions. (c) Click allows for the restoration of excellent DNA activity across a range of chemical and physiological conditions, providing caged oligomers that are stable over a wide range.

[0067] [Example 1] Scheme 1 The O4 atom of TIPS-protected dT was activated with a triazolyl group (Scheme 1). Subsequently, 4-vinyloxybenzyl alcohol was attached to the O4 atom via an SNAr reaction, followed by removal of the TIPS group using TABF, resulting in the formation of dT. BVE was generated.

[0068] [ka]

[0069] Synthesis of 3',5'-O-TIPS-2'-deoxythymidine (1) 2'-Deoxythymidine (1.0 g, 4.1 mmol) was dissolved in 12 mL of anhydrous pyridine. 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (1.7 mL, 2.65 mmol) was added to the mixture. The resulting mixture was stirred at room temperature for 4 hours. The reaction solution was extracted with ethyl acetate and saturated brine.

[0070] The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified by medium pressure liquid chromatography eluting with n-hexane / ethyl acetate (1:1) to give a white foam (Compound 1, 1.95 g, 97.5%).

[0071] 1HNMR(40MHz,DMSO-d6)δ11.34(1H,s),7.42(1H,d,J = 1.2Hz),4.57(1H,t,J = 7.6Hz),3.96(2H,dt,J = 3.2Hz),3.72(1H,dq,J = 2.0Hz), 2.47-2.28(2H,m), 1.78(3H,d,J = 1.2 Hz),1.08-1.01(45H,m); 22 H 40 N2O6Si2Na [M+Na] + : Calcd.507.2325; Found.507.2321.

[0072] Synthesis of O4-(1,2,4-1H-triazol-yl)-3',5'-O-TIPS-2'-deoxythymidine (2) 1,2,4-Triazole (4.24 g, 61.5 mmol) was added to a mixture of phosphorus oxychloride (1.33 mL, 14.14 mmol) and triethylamine (10.3 mL, 61.5 mmol) in 45 mL of anhydrous acetonitrile. The mixture was stirred at 0° C. for 30 minutes. 3′,5′-O-TIPS-2′-deoxythymidine (Compound 1, 1.95 g, 4.0 mmol) was added to the above solution.

[0073] The final mixture was stirred at 0°C for another 4 hours. The solid residue was filtered to obtain a filtrate, which was extracted with dichloromethane and saturated brine. The organic solution was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. A white solid was found to be the product (Compound 2, 2.1 g, 98%).

[0074] 1 HNMR(400MHz,DMSO-d6)δ9.34(1H,s),8.39(1H,s),8.18(1H,s),6.01(1H,dd,J = 2.4Hz),4.53(1H,dd,J = 8.0Hz),4.16(1H,dd,J = 9.2Hz),4.02(1H,dd,J = 6.8Hz),3.98-3.86(1H,m),2.50-2.48(2H,m),2.32(3H,s),1.08-0.97(37H,m); HRMS(ESI)for C 24 H 41 N5O5Si2Cl[M+Cl]- : Calcd.570.2346; Found.570.2322.

[0075] Synthesis of O4-(4-(vinyloxy)benzyl)-3',5'-O-TIPS-2'-deoxythymidine (3) O4-(1,2,4-1H-triazol-yl)-3',5'-O-TIPS-2'-deoxythymidine (compound 2, 1.07 g, 2.025 mmol), (4-vinyloxy)benzyl alcohol (3, 364.5 mg, 2.44 mmol), and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.61 mL, 4.05 mmol) were dissolved in 5 mL of anhydrous acetonitrile, and the mixture was stirred for 4 h.

[0076] The resulting solution was concentrated in vacuo and purified by medium pressure liquid chromatography eluting with n-hexane / ethyl acetate (1:1) to give a white solid as the product (Compound 3, 1.01 g, 80%).

[0077] 1 HNMR(400MHz,DMSO-d6)δ7.74(1H,s),7.45(2H,dd,J = 4.8 Hz),7.10-7.08(2H,m),6.88(1H,dd,J = 7.6Hz),6.00(1H,dd,J = 4.0Hz),4.75(1H,dd,J = 12.0Hz),4.55-4.50(2H,m),4.10-3.94(2H,m),3.79(1H,dt,J = 3.2Hz),2.45-2.31(2H,m),1.89(3H,d,J = 0.8Hz),1.08-0.97(33H,m); 13 CNMR(100MHz,DMSO-d6)δ163.76,150.35,148.12,137.07,135.84,127.78,116.22,11 4.65,109.37,94.33,87.25,83.83,70.42,62.58,61.28,25.58,25.30,17.65,12.16; HRMS(ESI)for C 37 H 64 N3O7Si2[M+TEA+H] + : Calcd.718.4251; Found.718.4286.

[0078] Synthesis of O4-(4-(vinyloxy)benzyl)-2'-deoxythymidine (4) O4-(4-(vinyloxy)benzyl)-3',5'-O-TIPS-2'-deoxythymidine (compound 3, 1.01 g, 1.62 mmol) was dissolved in 10 mL of tetrahydrofuran, followed by the addition of butylammonium fluoride (1 M / L, 3.24 mL, 3.24 mmol). The mixture was stirred at 0 °C for 10 min.

[0079] The reaction solution was evaporated in vacuo and purified by medium-pressure liquid chromatography eluting with dichloromethane / methanol (10:1). The collected fractions were concentrated in vacuo and recrystallized in n-hexane containing 3% dichloromethane. A white solid was obtained as the target compound (Compound 4, 540 mg, 87%).

[0080] 1 HNMR(400MHz,DMSO-d6)δ8.07(1H,d,J = 0.8 Hz),7.46(2H,dt,J = 2.0 Hz),7.09(2H,dt,J = 2.0Hz),6.89(1H,dd,J = 7.6Hz),6.17(1H,t,J = 6.4Hz),5.33(2H,t,J = 12.8Hz),5.25(1H,d,J = 4.4Hz),5.08(1H,t,J = 5.2Hz),4.75(1H,dd,J = 1.2 Hz),4.50(1H,dd,J = 4.8 Hz),4.25(1H,dt,J = 2.4 Hz),3.83(1H,dd,J = 3.2Hz),3.67-3.56(2H,m),2.24-2.01(2H,m),1.90(3H,d,J = 0.8Hz); 13 CNMR(100MHz,DMSO-d6)δ156.68,154.97,153.59,150.83,148.46,137.29, 135.21,127.98,116.65,116.29,94.66,87.53,82.54,70.70,62.33,61.68; HRMS(ESI)forC 19 H 22 N2O6Cl[M+Cl] -: Calcd.409.1478; Found.409.1161.

[0081] Synthesis of O4-(4-(vinyloxy)benzyl)-5'-O-dimethoxytrityl-2'-deoxythymidine (5) O4-(4-(vinyloxy)benzyl)-2'-deoxythymidine (compound 4, 500 mg, 1.33 mmol) was dissolved in 10 mL of anhydrous pyridine, followed by the addition of 4,4'-dimethoxytrityl chloride (539 mg, 1.6 mmol). The mixture was stirred at room temperature for 4 hours. The final solution was extracted with dichloromethane and saturated brine.

[0082] The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified by medium pressure liquid chromatography eluting with dichloromethane / methanol (100:1). Compound 5 was obtained as a white solid (610 mg, 67.6%).

[0083] 1 HNMR(400MHz,DMSO-d6)δ7.80(1H,d,J = 2.0Hz),7.48-7.22(12H,m),7.09(2H,dt,J = 2.0 Hz),6.92-6.87(5H,m),6.20(1H,t,J = 6.0Hz),5.36-5.33(3H,m),4.75(1H,dd,J = 12.0Hz),4.51(1H,dd,J = 4.4Hz),4.33(1H,dd,J = 1.6Hz),3.96(1H,dd,J = 3.6Hz),3.74(6H,s),3.24(2H,t,J = 2.8Hz),2.33-2.14(2H,m),1.57(3H,d,J = 6.0Hz); HRMS(ESI) for C 40 H 40 N2O8Na[M+Na] + : Calcd.699.2685; Found.699.2690.

[0084] Synthesis of 3'-O-[(2-cyanoethoxy)(diisopropylamino)phosphino]-O4-(4-(vinyloxy)benzyl)-5'-O-dimethoxytrityl-2'-deoxythymidine (6) O4-(4-(vinyloxy)benzyl)-5'-O-dimethoxytrityl-2'-deoxythymidine (compound 5, 610 mg, 0.9 mmol) was coevaporated three times with 10 mL of anhydrous acetonitrile and 10 mL of anhydrous dichloromethane. N,N-Diisopropylethylamine (0.64 mL, 3.6 mmol) and N,N-diisopropylchlorophosphoramidite (0.41 mL, 1.8 mmol) were added to the above solution. The mixture was stirred at room temperature under an argon atmosphere for 1 h. The resulting solution was extracted with dichloromethane and saturated brine.

[0085] The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified by medium-pressure liquid chromatography eluting with ethyl acetate containing 1% triethylamine. The resulting residue was recrystallized in n-hexane containing 1% dichloromethane. 600 mg (76%) of the desired product (Compound 6) was obtained as a white powder.

[0086] 1 HNMR(400MHz,DMSO-d6)δ7.89-7.87(1H,m),7.47-7.24(11H,m),7.10-7.08(2H,m),6.92-6.87(5H,m),6.20(1H,d,J = 10.4Hz),5.33(2H,s),4.76(1H,dd,J = 8.0Hz),4.52-4.50(2H,m),4.07-4.03(1H,m),3.72(6H,d,J = 8.0Hz),3.63-3.53(3H,m),3.29-3.27(2H,m),2.79-2.64(2H,m),2.35-2.33(2H,m),1.58(3H,d,J = 10.0Hz),1.21-0.99(14H,m);31PNMR(161MHz,DMSO-d6)δ147.59,147.26; HRMS(ESI)for C 49 H 57 N4O9PK[M+K] + : Calcd.915.3563; Found.915.3515.

[0087] Caged nucleosides were incorporated into a series of oligonucleotides on a 1.0 μmol scale using a DNA / RNA synthesizer via phosphoramidite chemistry. The oligonucleotides were purified and quantified by RP-HPLC and confirmed by MALDI-TOF-MS.

[0088] Using HPLC, DNA-1 BVE or DNA-2 BVE The reaction of Tz with DNA-1 was analyzed. BVE The peak corresponding to 1 completely disappeared, and another peak appeared at 7.96 min. As determined by mass spectrometry (MALDI-TOF MS), this newly observed peak was the released DNA-1.

[0089] Furthermore, HPLC and MALDI-TOF MS analyses revealed that after the reaction with Tz, DNA-2 BVE It was also revealed that the nucleoside analog was completely converted into DNA-2.

[0090] [Example 2] Scheme 2 The benzotriazol-1-yloxytris(dimethylamino)phosphonium group from the silylated dG was introduced at the O6 position of dG (Scheme 2). Subsequently, a BVE group was introduced at the O6 position of dG using (4-vinyloxy)benzyl alcohol. Removal of TBDMS with TABF afforded dG. BVE was generated.

[0091] [ka]

[0092] Synthesis of 3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (7) 2'-Deoxyguanosine (2.5 g, 9.36 mmol) was dissolved in 20 mL of anhydrous dimethylformamide and mixed with imidazole (4.15 g, 61.7 mmol) and tert-butyldimethylsilyl chloride (5.2 g, 35.5 mmol). The mixture was stirred at room temperature for 16 hours. 100 mL of ice water was added to the resulting reaction solution, which was then filtered to obtain a residue, which was then washed with 100 mL of n-hexane.

[0093] The filtration residue was extracted with ethyl acetate and saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. A white solid was obtained as the desired product (Compound 7, 4.2 g, 90.6%).

[0094] 1 HNMR (400MHz, DMSO-d6) δ10.51(1H, s), 7.77(1H, s), 6.37(2H, s), 6.00(1H, dd, J = 1.2Hz), 4.36-4.39(1H, m), 3.68-3.72(1H, m), 3.57(2H, ddd, J = C 22 H 41 NOCl[M+Cl] - HRMS(ESI): calculated 530.2397; found 530.2349.

[0095] Synthesis of O6-(benzotriazol-1-yl)-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (8) 3',5'-Di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (compound 7, 2.8 g, 5.65 mmol) was dissolved in 45 mL of anhydrous acetonitrile, followed by the addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (1.73 mL, 11.3 mmol) and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (5.0 g, 11.3 mmol). The mixture was stirred at room temperature for 1 h.

[0096] The resulting solution was extracted with ethyl acetate and saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The residue was purified by medium-pressure liquid chromatography eluting with dichloromethane / methanol (20:1) to give a white solid (Compound 8, 2.7 g, 80%).

[0097] 1 HNMR (400MHz, DMSO-d6) δ8.17(1H,s), 8.04-8.07(1H,m), 7.39-7.65(3H,m), 6.62( 2H,s), 6.13(1H,t,J=6.8Hz), 4.41~4.44(1H,m), 3.71~3.74(1H,m), 3.59(2H,ddd,J C 28 H 44 N8O4Si2Cl[M+Cl] - HRMS(ESI): calculated 647.2724; found 647.2728.

[0098] Synthesis of O6-(4-(vinyloxy)benzyl)oxy)-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (9) O6-(Benzotriazol-1-yl)-3',5'-di-O-(tert-butyldimethylsilyl)-2'-deoxyguanosine (compound 8, 2.7 g, 4.4 mmol) was dissolved in 50 mL of anhydrous dimethoxyethane. Cesium carbonate (2.82 g, 8.66 mmol) and (4-vinyloxy)benzyl alcohol (3, 1.3 g, 8.66 mmol) were added to the above solution. The mixture was stirred at 60 °C for 24 h.

[0099] The reaction solution was extracted with dichloromethane and saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude sample was purified by medium-pressure liquid chromatography eluting with n-hexane / AcOEt (3:1) to give a white solid (Compound 9, 2.0 g, 73%).

[0100] 1HNMR (400MHz, DMSO-d6) δ8.06(1H, s), 7.52(2H, dd, J = 4.8Hz), 7.08-7.11(2H, m), 6.88(1H, dd, J = 7.8Hz), 6.52(2H, s), 6.21(1H, dd, J = 1.2Hz), 5.46(2H, s), 4.75(1H, dd, J = 12Hz), 4.49-4.53(2H, m), 3.82-3.85(1H, m), 3.63~3.75(2H, m), 2.68~2.77(1H, m), 2.23~2.29(1H, m), 0.87(18H, d, J=12Hz), 0.05(12H, min)t, J = 2Hz); 13 CNMR (100MHz, DMSO-d6) δ154.79, 154.09, 153.91, 152.05, 151.88, 148.54, 147.75, 147.23, 136.19, 13 3.64, 126.88, 115.32, 113.75, 106.43, 93.42, 87.59, 84.51, 68.51, 61.66, 59.59, 24.68, 24.40, 16.74. C 31 H 49 N5O5Si2Cl[M+Cl] - HRMS(ESI): calculated 662.2272; found 662.2953.

[0101] Synthesis of O6-(4-(vinyloxy)benzyl)oxy)-2'-deoxyguanosine (10) The compound (Compound 9, 2.0 g, 3.2 mmol) was dissolved in 40 mL of tetrahydrofuran, followed by the addition of tetrabutylammonium fluoride (1 M / L, 0.24 mL, 0.24 mmol). The mixture was stirred at room temperature for 1.5 hours.

[0102] The resulting solution was concentrated in vacuo and the relatively crude product was purified by medium pressure liquid chromatography eluting with dichloromethane / methanol (10:1) to give the product as a white solid (Compound 10, 1.15 g, 90%).

[0103] 1HNMR (400MHz, DMSO-d6) δ8.11(1H, s), 7.50-7.54(2H, m), 7.07-7.11(2H, m), 6.88(1H, dd, J = 6Hz), 6.50(2H, s), 6.22(1H, dd, J = 1.6Hz), 5.47(2H, s), 5.28(1H, d, J = 3.2 Hz), 5.00(1H, t, J = 5.2Hz), 4.75(1H, dd, J = 1.2Hz), 4.50(1H, dd, J = 4.4Hz), 4.36(1H, dd, J = 3.2Hz), 3.83(1H, dt, J = 2.8Hz), 3.48~3.61(2H, m), 2.55-2.69(1H, m), 2.19-2.24(1H, m); 13 CNMR (100MHz, DMSO-d6) δ163.69, 154.97, 150.42, 148.46, 137.29, 136.06, 127.98, 116.28, 109.31, 94.66, 87.21, 83.70, 70.39, 62.32, 61.30, 12.21; C 19 H 21 N5O5Br[M+Br] - HRMS(ESI): calculated 478.0743; found 478.0719.

[0104] Synthesis of O6-(4-(vinyloxy)benzyl)oxy)-N2-dimethylformamidyl-2'-deoxyguanosine (11) O6-(4-(vinyloxy)benzyl)oxy)-2'-deoxyguanosine (compound 10, 1.15 g, 2.87 mmol) and N,N-dimethylformamide dimethyl acetal (2.31 mL, 17.17 mmol) were dissolved in 15 mL of DMF.

[0105] The mixture was stirred at 70° C. for 8 hours and concentrated in vacuo. The crude sample was purified by medium pressure liquid chromatography eluting with dichloromethane / methanol (15:1) to give the product as a white solid (Compound 11, 1.1 g, 90%).

[0106] 1HNMR (400MHz, DMSO-d6) δ8.65(1H, s), 8.29(1H, s), 7.49-7.55(2H, m), 7.08-7.12(2H, m), 6.89(1H, dd, J) = 7.6 Hz), 6.35(1H, dd, J = 1.6Hz), 5.56(2H, s), 5.32 (1H, t, J = 4.0Hz), 5.12 (1H, dd, J = 0.8Hz), 4.75(1H, dd, J = 1.6Hz), 4.49-4.51(1H, m), 4.39-4.43(1H, m), 4.11(1H, dd, J = 5.2Hz), 3.86-3.89(1H, m), 3.50-3.64(2H, m), 3.16-3.19(3H, m), 3.04(3H, m), 2.64-2.71(1H, m), 2.23-2.29(1H, m). C 22 H 27 N6O5[M+H] + HRMS(ESI): calculated 455.2965; found 455.2193.

[0107] Synthesis of O6-(4-(vinyloxy)benzyl)oxy)-5'-O-dimethoxytrityl-N2-dimethylformamidyl-2'-deoxyguanosine (12) O-(4-(vinyloxy)benzyl)oxy)-N-dimethylformamidyl-2'-deoxyguanosine (compound 11, 1.1 g, 2.39 mmol) and 4,4'-dimethoxytrityl chloride (965 mg, 1.12 mmol) were dissolved in 7 mL of anhydrous pyridine. The mixture was stirred at room temperature for 17 hours.

[0108] The resulting solution was concentrated in vacuo and purified by medium pressure liquid chromatography eluting with dichloromethane / methanol (20:1) to give the product as a white solid (Compound 12, 628 mg, 35%).

[0109] 1HNMR (400MHz, DMSO-d6) δ9.35 (1H, d, J = 8.0 Hz), 8.58 (1H, s), 7.55 (2H, d, J = 8.0 Hz), 7.28 (2H, d, J = 7.6Hz), 7.09~7.18 (9H, m), 6.84~6.92 (1H, m), 6.73~6.80 (4H, m), 6.36 (1H, t, J=6.0Hz), 5.57 (2H, s), 5.36(1H, d, J=4.8Hz), 4.75(1H, dd, J=1.2Hz), 4.46~4.51(3H, m), 3.96(1H, d, J=2.8Hz), 3.71(6H, d), J = C 43 H 44 N6O7Na [M+Na] + HRMS(ESI): calculated 779.3371; found 779.3309.

[0110] Synthesis of 3'-O-[(2-cyanoethoxy)(diisopropylamino)phosphino]-O6-(4-(vinyloxy)benzyl)oxy)-5'-O-dimethoxytrityl-N2-dimethylformamidyl-2'-deoxyguanosine (13) The compound (compound 12, 628 mg, 0.83 mmol) was coevaporated with 10 mL of anhydrous acetonitrile three times and dissolved in 10 mL of anhydrous dichloromethane. N,N-Diisopropylethylamine (0.58 mL, 3.33 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.36 mL, 1.66 mmol) were added to the mixture. The mixture was stirred at room temperature under an argon atmosphere for 1 hour. The reaction mixture was extracted with dichloromethane and saturated brine.

[0111] The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The remaining residue was purified by medium-pressure liquid chromatography eluting with n-hexane / ethyl acetate (2:1) containing 1% triethylamine to give the product as a white solid, which was subsequently recrystallized in n-hexane containing 1% dichloromethane. 471 mg (60%) of the desired product (Compound 13) was obtained as a white powder.

[0112] 1HNMR (400MHz, DMSO-d6) δ9.38(1H, d, J = 7.2Hz), 8.33(1H, d, J = 6.4Hz), 7.56(2H, d, J =8.8Hz), 7.29(2H), dd, J = 8.0Hz), 7.09~7.19(10H, m), 6.88(1H, d, J = 7.6Hz), 6.72~6.80(4H, m), 6.37(1H, dd, J = 5.6Hz), 5.57(2H, d, J = 2.4Hz), 4.75(2H, dd, J = 1.2Hz), 4.50(1H, dd, J = 1.6Hz), 4.02~4.11(2H, m), 3.70~3.72(8H, m), 3.52-3.64(5H, m), 3.16- 3.28(5H, m), 2.98-3.09(2H, m), 2.65-2.77(5H, m), 1.17-1.25(14H, m). 31 PNMR (161MHz, DMSO-d6) δ147.91, 147.38; C 52 H 62 N8O8P[M+H] + HRMS(ESI): calculated 957.4550; found 957.4503.

[0113] [Example 3] Scheme 3 Synthesis of 3',5'-O-TIPS-2'-deoxyadenosine (14) 2'-Oxyadenosine (500 mg, 2.0 mmol) was dissolved in 6 mL of anhydrous pyridine (Scheme 3). 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (0.85 mL, 2.58 mmol) was added to the above solution. The mixture was stirred at room temperature for 6 hours. The resulting solution was extracted with ethyl acetate and saturated brine.

[0114] The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude residue was purified by medium pressure liquid chromatography eluting with n-hexane / ethyl acetate (1:2) to give compound 14 as a white solid in the amount of 667 mg (67%).

[0115] 1H NMR(400 MHz, DMSO-d6)δ8.22(1H,s),8.08(1H,s),7.30(2H,s),6.28(1H,dd,J = 5.6 Hz),5.20(1H,dd,J = 7.2 Hz),3.92 (2H,t,J = HRMS(ESI)for C 22 H 40 N5O4Si2[M+H] + : Calcd. 494.2641; Found.494.2624.

[0116] [ka]

[0117] Synthesis of N6-(4-(vinyloxy)benzyl)oxycarbonyl-3',5'-O-TIPS-2'-deoxyadenosine (15) 3',5'-O-TIPS-2'-deoxyadenosine (compound 14, 158 mg, 0.32 mmol) and 3-ethyl-1-(((4-(vinyloxy)benzyl)oxy)carbonyl)-1H-imidazole-tetrafluoroborate 3-ium (6, 140 mg, 0.35 mmol) were dissolved in 5 mL of anhydrous dichloromethane. The mixture was stirred at room temperature and reacted for 24 hours.

[0118] The resulting solution was extracted with dichloromethane and saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified by medium-pressure liquid chromatography eluting with n-hexane / ethyl acetate (1:1) to give 82 mg (38%) of compound 15 as a white solid.

[0119] 1H NMR(400 MHz,DMSO-d6)δ8.68(1H,s),8.45(1H,s),8.11(1H,s),7.25(2H,d,J = 4.8Hz),6.92(2H,dd,J = 6.0 Hz),6.68(1H,dt,J = 4.0Hz),6.42(1H,t,J = 5.Hz),4.65(1H,dt,J = 7.2Hz),4.46(2H,s),4.38(1H,dt,J = 6.4 Hz),3.95(1H,dd,J = 3.6Hz),3.71-3.45(2H,m),0.82(16H,s),0.01-0.00(12H,m); 13 C NMR(100MHz,DMSO-d6)δ154.11,147.22,143.71,143.17,142.21,136.17, 126.88,115.33,93.42,69.53,64.15,61.70,60.35,24.68,24.40,16.75; HRMS(ESI)for C 32 H 48 N5O7Si2[M+H] + : Calcd. 670.2434; Found.670.2446.

[0120] Synthesis of N6-(4-(vinyloxy)benzyl)oxycarbonyl-2'-deoxyadenosine (16) N6-(4-(vinyloxy)benzyl)oxycarbonyl-3',5'-O-TIPS-2'-deoxyadenosine (compound 15, 82 mg, 0.122 mmol) was dissolved in 5 mL of a mixture of anhydrous pyridine and hydrogen fluoride. Pyridine (approximately 70% hydrogen fluoride, 27.3 μL, 0.488 mmol) was added. The mixture was stirred at 60 °C for 12 h.

[0121] The resulting solution was extracted with ethyl acetate and saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified by medium-pressure liquid chromatography eluting with dichloromethane / methanol (10:1) to give 31 mg (60%) of compound 16 as a white solid.

[0122] 1H NMR(400MHz,DMSO-d6) 8.35(1H,s),8.14(1H,s),7.31(4H,d,J = 4.8Hz),7.01(2H,dt,J = 4.8Hz),6.85(1H,dd,J = 3.6Hz),5.33(1H,d,J = 1.6 Hz),5.27(1H,dd,J = 5.2Hz),5.17(1H,t,J = 2.8Hz),4.70(1H,dd,J = 4.8Hz),4.47-4.40(5H,m),3.90(1H,dd,J = 6.4Hz),3.66-3.51(2H,m),2.74(1H,dt,J = 6.8Hz),2.28(1H,dtJ = 6.8Hz) 13 C NMR(100MHz,DMSO-d6)δ156.08,154.97,152.33,148.87,148.47,139.50,1 37.30,127.98,119.25,116.29,94.66,87.97,83.91,70.95,62.33,61.88; HRMS(ESI)for C 20 H 22 N5O6[M+H] + : Calcd.450.1792; Found.450.1798.

[0123] [Example 4] Scheme 4 Synthesis of 3',5'-O-TIPS-2'-deoxycytidine (17) 2'-Deoxyadenosine (500 mg, 2.2 mmol) was dissolved in 5 mL of anhydrous pyridine (Scheme 4). 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (0.94 mL, 2.86 mmol) was added to the above mixture. The mixture was stirred at room temperature for 6 hours and extracted with ethyl acetate and saturated brine.

[0124] The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified by medium pressure liquid chromatography eluting with dichloromethane / methanol (10:1) to give 1.07 g (99%) of compound 17 as a white solid.

[0125] 1H NMR(400MHz,DMSO-d6)δ7.63(1H,d,J = 7.6Hz),7.13(2H,d,J = 20.0Hz),5.99(1H,dd,J = 4.0Hz),5.70(1H,d,J = 7.6Hz),4.48(1H,dd,J = HRMS(ESI)for C 20 H 22 N5O6[M+H] + : Calcd.493.2328; Found.492.232.

[0126] [ka]

[0127] Synthesis of N6-(4-(vinyloxy)benzyl)oxycarbonyl-3',5'-O-TIPS-2'-deoxycytidine (18) 3',5'-O-TIPS-2'-deoxycytidine (compound 17, 150 mg, 0.32 mmol) was dissolved in 5 mL of anhydrous dichloromethane, followed by the addition of 3-ethyl-1-(((4-(vinyloxy)benzyl)oxy)carbonyl)-1H-imidazol-3-ium tetrafluoroborate (6, 140 mg, 0.35 mmol). The mixture was stirred at room temperature and reacted for 24 h.

[0128] The reaction mixture was extracted with ethyl acetate and saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified by medium-pressure liquid chromatography eluting with dichloromethane / methanol (10:1) to give 122 mg (59%) of compound 18 as a white solid.

[0129] 1HNMR(400MHz,DMSO-d6)δ8.64(1H,s),8.35(1H,d,J = 4.8Hz),7.26(2H,d,J = 7.2Hz),6.92(2H,d,J = 6.6Hz),6.68(1H,dd,J = 7.6Hz),6.19(1H,d,J = 3.2Hz),6.11(1H,t,J = 8.0Hz),4.65(1H,dd,J = 5.6Hz),4.46(2H,s),4.38(1H,dd,J = 7.6Hz),4.29(1H,dd,J = 5.2Hz),3.89(1H,dd,J = 3.6Hz),3.63(2H,ddd,J = 9.6Hz),2.74(1H,dt,J = 8.8Hz),2.10(1H,dt,J = 8.8Hz),0.83(15H,s),0.01-0.00(12H, m); 13 C NMR(100MHz,DMSO-d6)δ159.22,155.01,148.12,146.45,144.37,137.08,127.78 ,116.23,94.33,93.59,88.16,85.88,69.79,62.59,60.65,25.59,25.30,17.66; HRMS(ESI)for C 31 H 46 N3O8Si2[MH] - : Calcd.644.2675; Found.644.2609.

[0130] Synthesis of N6-(4-(vinyloxy)benzyl)oxycarbonyl-2'-deoxycytidine (19) N6-(4-(vinyloxy)benzyl)oxycarbonyl-3',5'-O-TIPS-2'-deoxycytidine (compound 18, 122 mg, 0.182 mmol) was dissolved in 6 mL of anhydrous pyridine and hydrogen fluoride (approximately 70% hydrogen fluoride, 40.8 μL, 0.728 mmol). The mixture was stirred at 60 °C for 12 h.

[0131] The resulting solution was extracted with ethyl acetate and saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude residue was purified by medium-pressure liquid chromatography eluting with dichloromethane / methanol (10:1) to afford 40.3 mg (55%) of the target compound 19 as a white foam.

[0132] 1 H NMR(400 MHz, DMSO-d6)δ7.83(1H,d,J = 4.2 Hz),7.73(1H,d,J = 2.4Hz),7.16(2H,d,J = 10.6Hz),7.04(2H,d,J = 6.4Hz),6.87(1H,dd,J = 7.2 Hz),6.19(1H,t,J = 6.4Hz),5.75(1H,d,J = 7.2Hz),5.20(2H,s),5.01(1H,s),4.73(1H,dd,J = 5.8 Hz),4.47(3H,t,J = 4.8 Hz),4.23(1H,dt,J = 6.8 Hz),3.80(1H,dd,J = 2.8 Hz),3.58 (2H,ddd,J = 8.8 Hz),2.13(1H,ddd,J = 6.4 Hz),1.96(2H,dt,J = 7.6 Hz); 13 C NMR(100 MHz,DMSO-d6)δ165.51,155.05,154.97,148.47,140.92,137.30,127.98,116.29,94.65,93.85,87.15,84.85,70.38,62.33,61.36; HRMS(ESI)for C 19 H 21 N3O7K[M+K] + : Calcd.442.1467; Found.442.1441.

[0133] [Example 5] Scheme 5 Synthesis of 3-(6-methyl-1,2,4,5-tetrazin-3-yl)benzyl alcohol (20) 3-Cyanobenzyl alcohol (460 mg, 3.76 mmol), nickel(II) trifluoromethanesulfonate (268 mg, 0.75 mmol), and hydrazine monohydrate (65% aqueous solution, 11.4 mL, 188 mmol) were added to 1.96 mL of acetonitrile and stirred overnight at 60° C. The reaction solution was cooled to room temperature (Scheme 5).

[0134] 5 mL of an aqueous solution of sodium nitrite (1.4 g, 18.8 mmol) was added dropwise to the above mixture. 1 M / L HCl solution was slowly added until pH = 3. The mixture was extracted with dichloromethane and saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated in vacuo. A pink solid was obtained as the target compound (Compound 20, 273 mg, 36%).

[0135] 1 H NMR(400 MHz,CDCl3)δ8.52(1H, dt,J = 1.2Hz),8.50 (1H,t,J = 1.6 Hz), 7.66-7.57 (2H,m),4.84(2H,d,J = 5.6 Hz),3.10 (3H, s),1.95(1H,t,J = 6.0Hz); HRMS(ESI)for C 10 H 11 NO[M+H] + : Calcd.203.0927; Found.203.0924.

[0136] [ka]

[0137] [Example 6] Caged nucleosides were incorporated into a series of oligonucleotides on a 1.0 μmol scale using a DNA / RNA synthesizer via phosphoramidite chemistry. The oligonucleotides were purified and quantified by RP-HPLC and confirmed by MALDI-TOF-MS (Figure 1).

[0138] 1. DNA-1 BVE 5'-d(T BVE T BVE G BVE CTA TGT CAA)-3' (12 bases, SEQ ID NO: 1) 2. DNA-2 BVE 5'-d(T BVE T BVE G BVE ACA TAG CAA)-3' (12 bases, SEQ ID NO: 2) 3. DNABVE -dG F 5'-d(CG BVE CG F CG)-3' (6 bases) 4. RNA BVE -rG F 5'-d(CG BVE CG F CG F CG)-3' (8 bases) 5. QD-DNA-3 BVE (520) 5'-d(T BVE G BVE G BVE GGT CGT CAT CTT CAT CTG TC)-3' (23 bases, SEQ ID NO: 3) 6. QD-DNA-4 BVE (580) 5'-d(T BVE G BVE G BVE CTC CCA GCC ACT TAC TCG T)-3' (22 bases, SEQ ID NO: 4) 7. QD-DNA-5 BVE (620) 5'-d(T BVE T BVE C CAG CTC CTT TCA TCA CTC G)-3' (22 bases, SEQ ID NO: 5) 8. Cy3-DNA-7 BVE Cy3-5'-d(TAG BVE GGT)-3' (6 bases, SEQ ID NO: 6) 9. Cy5-DNA-8 Cy5-5'-d(GGG TTA GGG TTA GGG T)-3' (16 bases, SEQ ID NO: 6) 10. TBA BVE -dG F 5'-d(G BVE GT TGG TGT GGT TG F G)-3' (15 bases, SEQ ID NO: 7)

[0139] Benzyl vinyl ether groups (BVE groups) are incorporated into nucleosides and oligonucleotides.

[0140] [Example 7] Using HPLC, DNA-1BVE or DNA-2 BVE The reaction of DNA-1 with Tz was analyzed (Figure 2). BVE When DNA-1 was reacted with Tz, the corresponding peak at 27.58 min completely disappeared, and another peak appeared at 7.96 min. As determined by MALDI-TOF MS, this newly observed peak was determined to be DNA-1, which had undergone the click reaction to remove the BVE group. BVE was also completely converted to DNA-2.

[0141] [Example 8] The conformation of the oligonucleotide was investigated in parallel with the reaction by circular dichroism (CD) spectroscopy (Figure 3). After the reaction, a significant increase in the CD peak at around 290 nm, which is the characteristic CD peak of Z-DNA, was observed. This indicates the conversion of the caged DNA to Z-DNA via click-and-release.

[0142] [Example 9] Proteins such as ADAR1, DLM1, PKZ, and E3L specifically bind to Z-DNA and regulate transcription and gene inhibition. Recent studies have demonstrated that ADAR1 has a Zα domain and can bind to Z-RNA and Z-DNA, suggesting the possibility of immunotherapy for cancer. We investigated the interaction between Z-RNA and ADAR1 protein.

[0143] As a result of the click reaction, RNA BVE -rG F The bands decreased and new bands appeared (Fig. 4). BVE -rG F This indicates that the protein was converted into Z-RNA and bound to ADAR1.

[0144] [Example 10] Functionalized quantum dots (QDs) were obtained by conjugating biotinylated caged oligonucleotides with Tz. QDs and Tz were mixed with HeLa cells, incubated for 24 hours, and then imaged using a confocal microscope (Figure 5).

[0145] It was found that the FRET signal was excited at 405 nm and emitted at 620 nm. In contrast, control cells in the absence of Tz showed no fluorescence at 620 nm. The observation of the FRET signal demonstrated that it enables efficient detection of cancer cells.

[0146] [Example 11] Cy3-DNA-3 BVE , Cy5-DNA-4 and Tz were mixed with cells and incubated for 24 hours before being imaged using a confocal microscope (Figure 6).

[0147] A red signal is observed at the Cy5 emission wavelength of 670 nm, and when excited at the Cy3 absorption wavelength of 550 nm, the Cy3 emission at 570 nm decreases, indicating the FRET phenomenon between the two DNA molecules within the cell. A quadruplex higher-order structure is formed within the cell, demonstrating fluorescence resonance energy transfer (FRET).

[0148] [Example 12] In vivo fluorescence imaging of mice was performed. Cy3-DNA-3 BVE and Cy5-DNA-4 were subcutaneously injected into the left and right sides of the backs of mice, followed by injection of Tz and PBS buffer (pH 7.4) into the same left and right sides, respectively. Mice were imaged using an in vivo imaging system.

[0149] Under excitation at 550 nm and emission at 670 nm, a clear fluorescent signal was observed on the left side, but almost no fluorescent signal was observed on the right side (Figure 7). These results suggest that quadruplex formation can be controlled in vivo by the click reaction.

[0150] [Example 13] Aptamers are nucleic acid ligands with high affinity for target molecules. Thrombin-binding aptamers (TBAs) are DNA quadruplexes that can strongly bind to thrombin and inhibit fibrin clot formation. It is essential to prevent TBAs from inducing their anticoagulant activity.

[0151] The click reaction destroys the non-activated TBA and restores the activated TBA structure. Prothrombin time assays showed that increasing the reaction time improved the anticoagulant activity, with the observed clotting time being 15.5 seconds (TBA) compared to the 0-hour click reaction result (11.76 seconds). BVE -dG) and 26.06 seconds (TBA BVE -dG F ) was confirmed to increase (Figure 8).

Claims

1. Caged nucleotides and caged nucleotide analogs containing the following caged purine or caged pyrimidine bases: 【Chemistry 1】 (where Z is expressed by the following formula: Ra, Rb, Rc, Rd, Re and Rf each independently represent H, halogen, a nitro group, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl, C1-C20 alkoxy, aryl, heteroaryl, keto, carboxy, amino, silyl, boron, and / or a target group. 【Chemistry 2】

2. 2. The caged nucleotide and caged nucleotide analog of claim 1, wherein at least one of Ra, Rb, Rc, Rd, Re, and Rf is a target group.

3. 2. The caged nucleotide and caged nucleotide analog of claim 1, wherein at least one of Ra, Rb, Rc, Rd, Re, and Rf is a phosphoramidite.

4. 1. An oligonucleotide or oligonucleotide analog compound comprising a sequence of nucleotides or nucleotide analogs, an oligonucleotide or oligonucleotide analog compound, wherein at least one of the nucleotides or nucleotide analogs is a caged nucleotide or nucleotide analog and is selected from the group consisting of: 【Transformation 3】 (where Z is expressed by the following formula: Ra, Rb, Rc, Rd, Re, and Rf each independently represent H, halogen, a nitro group, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl, C1-C20 alkoxy, aryl, heteroaryl, keto, carboxy, amino, silyl, boron, and / or a target group. 【Chemistry 4】

5. 1. An oligonucleotide or oligonucleotide analog compound comprising a sequence of nucleotides or nucleotide analogs, 5. An oligonucleotide or oligonucleotide analog compound, wherein at least one of the nucleotides or nucleotide analogs is a caged nucleotide or nucleotide analog according to claim 4, and at least one of Ra, Rb, Rc, Rd, Re and Rf is a target group.

6. 1. An oligonucleotide or oligonucleotide analog compound comprising a sequence of nucleotides or nucleotide analogs, 5. An oligonucleotide or oligonucleotide analog compound, wherein at least one of the nucleotides or nucleotide analogs is a caged nucleotide or nucleotide analog according to claim 4, and at least one of Ra, Rb, Rc, Rd, Re and Rf is a phosphoramidite.

7. 1. A method for uncaging an oligonucleotide or oligonucleotide analogue, comprising: (a) providing in a cell, tissue, or subject, in vitro or in vivo, an oligonucleotide or oligonucleotide analog comprising a plurality of nucleotides or nucleotide analogs; At least one of the nucleotides or nucleotide analogs is a caged nucleotide or nucleotide analog and is selected from the group consisting of: (b) reacting the oligonucleotide or oligonucleotide analogue with a tetrazine derivative to cleave the caging group Z from the base to provide a deprotected or uncaged oligonucleotide or oligonucleotide analogue. 【Transformation 5】 (where Z is expressed by the following formula: Ra, Rb, Rc, Rd, Re, and Rf each independently represent H, halogen, a nitro group, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl, C1-C20 alkoxy, aryl, heteroaryl, keto, carboxy, amino, silyl, boron, and / or a target group. 【Transformation 6】

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