Photoresponsive deoxyribonucleoside triphosphates

Abstract

To provide photoresponsive deoxyribonucleoside triphosphate in which the initiation of the amplification reaction of a target nucleic acid can be controlled by light irradiation, and to provide its production precursor.SOLUTION: The above problem is solved by the compound represented by the general formula (1) (where, R1 and R2 each independently represent an alkyl group having 1 to 8 carbon atoms; R3 and R4 independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group; R5 represents a hydrogen atom or a group represented by the formula (7) [-P(=O)(OH)-O-P(=O)(OH)-O-P(=O)(OH)2(7)]; Y represents a hydrogen atom or a hydroxy group; and X represents a nucleobase).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a photoresponsive deoxyribonucleoside triphosphate for use in a method for light-controlled amplification of a target nucleic acid. [Background technology]

[0002] In genetic testing used in clinical diagnosis, a very small amount of target nucleic acid contained in a clinical sample is amplified to increase the signal intensity, thereby achieving highly sensitive and reproducible measurements. Examples of amplification methods when the target nucleic acid is DNA include the polymerase chain reaction (PCR) method. In addition, examples of amplification methods when the target nucleic acid is RNA include the Nucleic Acid Sequence-Based Amplification (NASBA) method (Patent Documents 1 and 2, Non-Patent Document 1), the Transcription Mediated Amplification (TMA) method (Patent Document 3), and the Transcription-Reverse Transcription Concerted (TRC) method (Patent Document 4 and Non-Patent Document 2). RNA amplification methods can be easily applied to automation because they can react at a constant temperature of relatively low temperature (e.g., 41°C to 46°C). On the other hand, the PCR method, which requires rapid temperature increase and decrease, was not easily applied to automation for the purpose of mass processing, but by adding an enzyme with strand displacement activity, DNA amplification at a constant temperature of relatively low temperature is also possible (Patent Document 5). However, in the above-mentioned reaction capable of amplifying a target nucleic acid at a constant, relatively low temperature, the reaction may also proceed at room temperature.

[0003] The digital NASBA method (Patent Document 6) is a technique for accurately quantifying minute amounts of target RNA contained in a sample, but as described above, the amplification reaction may proceed even at room temperature, that is, the amplification reaction may proceed from the preparation of the reaction solution until it is distributed to the micro-compartments, making it extremely difficult to perform highly accurate quantification. Therefore, in order to detect the extremely small amount of target RNA present in a sample more efficiently and reproducibly, it was necessary to control the amplification reaction, especially at room temperature.

[0004] As a method for controlling the amplification reaction of a target nucleic acid, there is a method of suppressing the amplification reaction by light irradiation using a substrate in which a primer or a nucleic acid component dNTP (deoxyribonucleoside triphosphate) is protected with a photolabile protecting group (Non-Patent Documents 3 and 4). However, up to now, there has been no report on a photoresponsive dNTP in which the hydroxyl group at the 3' position of a nucleic acid component dNTP is protected with a photolabile protecting group such as a 3-(dialkylamino)benzyl group (Non-Patent Document 5) or a 1-[3-(dialkylamino)phenyl]ethyl group (Non-Patent Document 6), and no report has been made on a method for photocontrolled amplification of a target nucleic acid in which this is used as a raw material for the target nucleic acid and the amplification reaction is initiated by light irradiation. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2-005864 [Patent Document 2] Special Publication No. 4-503451 [Patent Document 3] Special Publication No. 4-500759 [Patent Document 4] JP 2000-014400 A [Patent Document 5] JP 2015-116136 A [Patent Document 6] US Publication No. 2014 / 0141502 [Non-patent literature]

[0006] [Non-Patent Document 1] de Baar, MPet al., Journal of Clinical Microbiology, 39, 1895-1902 (2001) [Non-Patent Document 2] Ishiguro, T. et al., Analytical Biochemistry, 314, 77-86 (2003) [Non-Patent Document 3] Jian, W. et al., Proc. Natl. Acad. Sci. USA, 104, 42, 16462-16467 (2007) [Non-Patent Document 4] Weidong, W. et al., Nucleic Acids Research, 35, 19, 6339-6349 (2007) [Non-Patent Document 5] Ding X.et al.,J.Org.Chem.,83,7459-7466(2018) [Non-Patent Document 6] Ding X.et al.,J.Org.Chem.,83,10736-10742(2018) Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a photoresponsive deoxyribonucleoside triphosphate and a production precursor thereof for use in a light-controlled amplification method in which an amplification reaction of a trace amount of target nucleic acid present in a sample is initiated by light irradiation. [Means for solving the problem]

[0008] As a result of intensive research into solving the above-mentioned problems, the inventors have found that the above-mentioned problems can be solved by a photoresponsive deoxyribonucleoside triphosphate represented by general formula (1b), which can control the initiation of an amplification reaction of a target nucleic acid by light irradiation, and have thus completed the present invention.

[0009] That is, the present invention includes the embodiments shown in [1] to [3] below.

[0010] [1] A compound represented by general formula (1).

[0011] [ka]

[0012] (In the formula, R 1 and R 2 Each independently represents an alkyl group having 1 to 8 carbon atoms. 1 and R 2 may form a 5- to 7-membered heterocyclic ring together with the nitrogen atom to which they are attached. 3 and R 4 Each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group. R 5 is a hydrogen atom or formula (7) [-P(=O)(OH)-OP(=O)(OH)-OP(=O)(OH)2(7)] wherein Y represents a hydrogen atom or a hydroxyl group, and X represents a nucleic acid base represented by any one of the following formulas (2) to (5):

[0013] [ka]

[0014] (In the formula, R 6 represents a hydrogen atom or a methyl group, and when Y is a hydrogen atom, R 6 represents a methyl group, and when Y is a hydroxy group, R 6 represents a hydrogen atom.) [2] The compound represented by the general formula (1) according to [1], wherein Y is a hydrogen atom.

[0015] [3] R 1 and R 2 The compound represented by general formula (1) according to [2], wherein

[0016] [4] A method for producing a deoxyribonucleoside triphosphate represented by formula (1b), comprising the steps of: reacting a deoxyribonucleoside represented by formula (1a) with salicyl chlorophosphite in the presence of a base, followed by treatment with a pyrophosphate to obtain a cyclic compound represented by formula (6); and reacting the cyclic compound represented by formula (6) with an oxidizing agent in the presence of water.

[0017] [ka]

[0018] (In the formula, R 1 and R 2 Each independently represents an alkyl group having 1 to 8 carbon atoms. 1 and R 2 may form a 5- to 7-membered heterocyclic ring together with the nitrogen atom to which they are attached. 3 and R 4 Each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group, and X represents a nucleic acid base of any one of the following formulas (2), (3), (4a), or (5).

[0019] [ka]

[0020] [ka]

[0021] (In the formula, R 1 , R 2 , R 3 , R 4 and X has the same meaning as above.)

[0022] [ka]

[0023] (In the formula, R 1 , R 2 , R 3 , R 4 and X has the same meaning as above.) Effect of the Invention

[0024] By using the light-controlled amplification reagent for target nucleic acid containing the deoxyribonucleoside triphosphate (1b) of the present invention, the amplification reaction of a small amount of target nucleic acid present in a sample can be controlled by light irradiation. That is, in the amplification reaction of the target nucleic acid contained in a sample at a constant relatively low temperature, the initiation of the nucleic acid amplification reaction can be controlled by light, and the target nucleic acid can be quantified with high accuracy, which was previously difficult. In addition, the deoxyribonucleoside (1a) of the present invention is useful as a production intermediate for deoxyribonucleoside triphosphate (1b). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention will be described in detail below.

[0026] <1> Compound (1) of the present invention The compound (1) of the present invention is represented by the following general formula (1).

[0027] [ka]

[0028] (In the formula, R 1 , R 2 , R 3 , R 4 , R 5 and X has the same meaning as above.) In the present invention, compound (1) includes its chemically acceptable salts and is represented by general formula (1).

[0029] R 1 and R 2The alkyl group having 1 to 8 carbon atoms represented by the formula (I) may be linear, branched or cyclic, and examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, a hexyl group, a heptyl group and an octyl group. 1 and R 2 may form a 5- to 7-membered heterocycle together with the nitrogen atom to which they are bonded, and in this case, the carbon atom of the heterocycle formed may be replaced with at least one heteroatom selected from the group consisting of nitrogen and oxygen atoms. Examples of the heterocycle include pyrrolidine, piperidine, and morpholine.

[0030] R 3 and R 4 The alkyl group having 1 to 6 carbon atoms and represented by the formula (I) may be linear, branched, or cyclic, and examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, a cyclobutyl group, a pentyl group, and a hexyl group.

[0031] Examples of the 3-(dialkylamino)benzyl group which is the protecting group for the hydroxy group at the 3'-position of compound (1) of the present invention include a 3-(dimethylamino)benzyl group, a 3-(diethylamino)benzyl group, a 3-(dipropylamino)benzyl group, a 3-(dibutylamino)benzyl group, a 3-(dioctylamino)benzyl group, a 3-(ethylmethylamino)benzyl group, a 3-(ethylpropylamino)benzyl group, a 3-pyrrolidinobenzyl group, a 3-piperidinobenzyl group, a 1-[3-(dimethylamino)phenyl]ethyl group, a 1-[3-(diethylamino)phenyl]ethyl group, a [3-(dimethylamino)phenyl](phenyl)methyl group, a [3-(diethylamino)phenyl](phenyl)methyl group, a 1-[3-(dimethylamino)phenyl]-1-methylethyl group, and a 1-[3-(diethylamino)phenyl]-1-methylethyl group.

[0032] In the compound (1) of the present invention, deprotection of the 3-(dialkylamino)benzyl group proceeds when the compound is irradiated with light having a wavelength of 200 nm or more and 400 nm or less. In terms of the high rate of photodecomposition, it is preferable to use a wavelength of 300 nm or more and 340 nm or less. The irradiance of the light is not particularly limited, but is preferably 0.1 mW / cm. 2 More than 3W / cm 2 It is preferable to use light in the following range. The light irradiation time is not particularly limited, but an irradiation time of 30 minutes or less is preferable, and an irradiation time of 5 minutes or less is more preferable. As the 3-(dialkylamino)benzyl group, a 3-(diethylamino)benzyl group is preferable in terms of high photodecomposition property.

[0033] Next, the method for producing the compound (1) of the present invention will be described.

[0034] The deoxyribonucleoside (1a) of the present invention can be produced by referring to the methods disclosed in, for example, Proceedings of the National Academy of Sciences of the United States of America, 104, 16462-16467 (2007) or Nucleic Acids Research, 35, 6339-6349 (2007).

[0035] The method for producing the deoxyribonucleoside (1b) of the present invention is a method for producing the deoxyribonucleoside triphosphate (1b) of the present invention by triphosphorylating the deoxyribonucleoside (1a) of the present invention.

[0036] [ka]

[0037] The deoxyribonucleoside (1a) of the present invention is reacted with salicyl chlorophosphite in the presence of a base, followed by treatment with pyrophosphate to obtain a cyclic compound (6) (Step-A). The deoxyribonucleoside triphosphate (1b) of the present invention can then be produced by reacting the compound with an oxidizing agent in the presence of water (Step-B). Triphosphorylation can be performed with reference to methods described in the literature (Acta Biochim. et Biophys. Acad. Sci. Hung., 16, 131-133 (1981); J. Org. Chem., 54, 631-635. (1989); Org. Lett., 13, 4156-4159 (2011); Org. Lett., 18, 580-583 (2016).).

[0038] The cyclic compound (6) obtained after step A may be isolated and purified, or may be directly subjected to the next step B without isolation and purification.

[0039] In the present invention, the cyclic compound (6) includes its chemically acceptable salts and is represented by the general formula (6).

[0040] The salicyl chlorophosphite used in the process of the present invention is commercially available.

[0041] Examples of the base that can be used in the production method of the present invention include organic bases such as triethylamine, tributylamine, pyridine, and picoline.

[0042] Examples of pyrophosphates that can be used in the production method of the present invention include bis(tetrabutylammonium) dihydrogen pyrophosphate and bis(tributylammonium) pyrophosphate, which are commercially available.

[0043] An example of the oxidizing agent that can be used in the production method of the present invention is iodine.

[0044] The production method of the present invention may be carried out in a solvent as long as the solvent does not inhibit the reaction. Examples of solvents that can be used in the production method of the present invention include ether solvents such as tetrahydrofuran, diethyl ether, 1,4-dioxane, and 1,2-dimethoxyethane, hydrocarbon solvents such as hexane, pentane, and cyclohexane, aromatic hydrocarbon solvents such as benzene, toluene, and xylene, halogenated hydrocarbon solvents such as dichloromethane, chloroform, carbon tetrachloride, and 1,2-dichloroethane, acetonitrile, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, and pyridine, and two or more of these solvents may be mixed and used. Among them, it is preferable to use dimethylformamide because of its good yield.

[0045] The molar ratio of deoxyribonucleoside (1a) to salicyl chlorophosphite used in the production method of the present invention is preferably in the range of 1:0.8 to 1:10, and more preferably 1:1 to 1:2 in terms of good yield.

[0046] The molar ratio of deoxyribonucleoside (1a) to base used in the production method of the present invention is preferably within the range of 1:0.8 to 1:10, and more preferably 1:1 to 1:2 in terms of good yield.

[0047] The molar ratio of deoxyribonucleoside (1a) to pyrophosphate used in the production method of the present invention is preferably in the range of 1:0.8 to 1:10, and more preferably 1:1 to 1:2 in terms of good yield.

[0048] The molar ratio of deoxyribonucleoside (1a) to oxidizing agent used in the production method of the present invention is preferably in the range of 1:0.8 to 1:20, and more preferably 1:1 to 1:5 in terms of good yield.

[0049] The molar ratio of deoxyribonucleoside (1a) to water used in the production method of the present invention is not particularly limited, and water may be used in an amount equal to or greater than that of deoxyribonucleoside (1a), and is preferably used as a solvent.

[0050] The reaction temperature in the production method of the present invention can be appropriately selected from the range of from −78° C. to 150° C. Among them, the range of from 0° C. to 120° C. is preferable in terms of good yield.

[0051] The deoxyribonucleoside triphosphate (1b) obtained by the production method of the present invention can be purified from the reaction solution after the reaction is completed, if necessary. The purification method is not particularly limited, but the target product can be purified by a general method such as solvent extraction, silica gel column chromatography, preparative thin layer chromatography, preparative liquid chromatography, recrystallization, or sublimation.

[0052] The deoxyribonucleoside triphosphate (1b) of the present invention (hereinafter sometimes referred to as photoresponsive dNTP (1b)) can be used as a reagent for light-controlled amplification of a target nucleic acid.

[0053] <2> Light-controlled amplification reagent The light-controlled amplification reagent for a target nucleic acid containing the light-responsive dNTP (1b) of the present invention can be used in an amplification reaction of a target nucleic acid contained in a sample at a constant relatively low temperature to control the initiation of the nucleic acid amplification reaction by light.

[0054] The light-controlled amplification reagent is a reagent containing the following components (A) to (I). Note that either one of the following (A) and (B) has the following promoter sequence (G) added to its 5'-end. In addition, other components that are more preferable for reasons other than those required for commonly used amplification reactions may be added. (A) a first single-stranded oligonucleotide (first primer) having a sequence complementary to a portion of a target nucleic acid; (B) a second single-stranded oligonucleotide (second primer) having a sequence homologous to a portion of the target nucleic acid; (C) an enzyme having RNA-dependent DNA polymerase activity; (D) Photoresponsive dNTP (1b), (E) an enzyme having ribonuclease H (RNase H) activity; (F) an enzyme having DNA-dependent DNA polymerase activity; (G) an enzyme having DNA-dependent RNA polymerase activity, (H) Deoxyribonucleoside triphosphates (dNTPs: dATP, dTTP, dGTP, dCTP), provided that dNTPs having the same nucleic acid base as the photoresponsive dNTP (1b) described in (D) are not included, or may be included to the extent that the amplification reaction does not start before light irradiation. (I) Ribonucleoside triphosphates (NTPs: ATP, UTP, GTP, CTP).

[0055] As described above, either one of (A) and (B) has the promoter sequence (G) added to its 5'-terminus. An enhancer sequence consisting of several to several tens of nucleotides may be inserted between the added primer and the promoter sequence.

[0056] The enzymes (C), (E), and (F) may be different from each other, or a part or all of them may be the same. For example, avian myeloblastoma virus (AMV) reverse transcriptase is an enzyme that encompasses all of the enzymatic activities of (C), (E), and (F), and is a particularly preferred embodiment as a light-controlled amplification reagent.

[0057] Examples of the enzyme (G) include T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. The promoter sequence added to either (A) or (B) may be a sequence corresponding to the component (polymerase) used in (G).

[0058] The photoresponsive dNTP (1b) of (D) provides an unmodified deoxynucleoside triphosphate (dNTP) that can be used as a substrate for DNA elongation upon irradiation with light. The photoresponsive dNTP (1b) used may be any one, two, three, or all of the photoresponsive dNTPs (1b) having four types of nucleic acid bases (adenine base (2), guanine base (3), thymine base (4), or cytosine base (5)) represented by formulas (2) to (5).

[0059] The photoresponsive dNTP (1b) of (D) may be contained in the light-controlled amplification reagent of the present invention in the form of a free deoxyribonucleoside triphosphate (i.e., the form of (H) above), or in the form of a deoxyribonucleotide constituting the primers (A) and / or (B). Specifically, the primers (A) and / or (B) may be primers represented by the following general formula (0) in which the 3'-end side of the primer is modified with a 3-(dialkylamino)benzyl group.

[0060] [ka]

[0061] In general formula (0), Z represents an oligodeoxyribonucleotide constituting the first or second primer, X represents a nucleic acid base selected from the group consisting of adenine base (2), guanine base (3), thymine base (4), and cytosine base (5), and Q represents a 3-(dialkylamino)benzyl group represented by general formula (8).

[0062] [ka]

[0063] In the general formula (8), R 1 , R 2 , R 3 and R 4 has the same meaning as above.

[0064] <3> Method for amplifying a target nucleic acid The above-mentioned light-controlled amplification reagent can be used in a light-controlled amplification reaction of a target nucleic acid.

[0065] The target nucleic acid may be DNA or RNA having any base sequence, and may be any as long as it contains a sequence portion in (A) and (B) that is specific enough to be distinguished from other nucleic acids. The origin of the target nucleic acid is not particularly limited, and may be, for example, a polynucleotide synthesized by a nucleic acid amplification reaction such as the PCR method, a chemically synthesized oligonucleotide, a polynucleotide extracted from blood, tissue, cells, etc., or a polynucleotide extracted from food, soil, wastewater, etc.

[0066] When the target nucleic acid is DNA, the PCR method or the like can be used to amplify the target nucleic acid.

[0067] When the target nucleic acid is RNA, the amplification method of the target nucleic acid can be NASBA (Nucleic Acid Sequence-Based Amplification), TMA (Transcription Mediated Amplification), TRC (Transcription-Reverse Transcription Concerted), etc. These methods use a primer containing a promoter sequence for the target RNA, reverse transcriptase and ribonuclease H (RNase H) to generate double-stranded DNA containing a promoter sequence, generate RNA containing a specific base sequence with RNA polymerase, and then perform a chain reaction using the generated RNA as a template for synthesizing double-stranded DNA containing the promoter sequence.

[0068] Although there is no particular limitation on the solvent used in the amplification reaction of the target nucleic acid, it is preferable to use a buffer solution. Examples of buffer solutions that can be used include Tris-HCl buffer solution, sodium acetate buffer solution, HEPES (2-[4-(2-HydroxyEthyl)-1-Piperazinyl]EthaneSulfonic acid)-KOH buffer solution, sodium phosphate buffer solution, and potassium phosphate buffer solution. An inorganic salt or a carboxylate salt may be added to the above-mentioned buffer solution at any concentration. Examples of the inorganic salt include sodium chloride, magnesium chloride, potassium chloride, sodium bromide, magnesium bromide, potassium bromide, sodium iodide, and potassium iodide, and examples of the carboxylate salt include magnesium acetate and manganese acetate. Two or more of these inorganic salts and carboxylate salts may be mixed and used. The concentration of the inorganic salt or the carboxylate salt added is preferably in the range of 0M to 5M. Furthermore, an organic solvent miscible with the buffer solution may be added at a ratio of 0% to 80%. Examples of the organic solvent include dimethyl sulfoxide (DMSO), methanol, ethanol, hexamethylphosphoric triamide, N,N-dimethylformamide, N,N-dimethylacetamide, 1,4-dioxane, tetrahydrofuran, ethylene glycol, and glycerin. Two or more of these organic solvents may be mixed and used.

[0069] The amplification reaction of the target nucleic acid is initiated by mixing the target nucleic acid with a light-controlled amplification reagent and irradiating the target nucleic acid with light. That is, when the light-responsive dNTP (1b) is contained as a free deoxyribonucleoside triphosphate, dNTPs having the same nucleic acid base as the light-responsive dNTP (1b) are not present in the amplification reaction system without light irradiation. Therefore, even if the target nucleic acid is added to the light-controlled amplification reagent in this state, the amplification reaction does not proceed. On the other hand, when the light-responsive dNTP (1b) is irradiated with light, the light-responsive protecting group is released, and unmodified dNTPs, which are one of the substrates for DNA elongation, are present in the light-controlled amplification reagent. Therefore, the amplification reaction of the target nucleic acid proceeds by adding the target nucleic acid to the light-controlled amplification reagent in this state and setting the reagent under appropriate amplification reaction conditions (such as appropriate temperature).

[0070] Furthermore, when photoresponsive dNTP (1b) is contained as a deoxyribonucleotide constituting the first and / or second primer (i.e., an embodiment of general formula (0)), the photolabile protecting group modified to photoresponsive dNTP (1b) inhibits hybridization between the primer and the target nucleic acid in the absence of light irradiation, and the amplification reaction does not proceed even when the target nucleic acid is added. On the other hand, when photoresponsive dNTP (1b) (general formula (0)) is irradiated with light, the photoresponsive protecting group is released, and the primer becomes capable of hybridizing with the target nucleic acid. Therefore, the amplification reaction of the target nucleic acid proceeds by setting the light-controlled amplification reagent to which the target nucleic acid has been added under appropriate amplification reaction conditions (such as appropriate temperature).

[0071] The photoresponsive dNTP (1b) or the 3-(dialkylamino)benzyl group of general formula (0) undergoes deprotection by irradiation with light having a wavelength of 200 nm or more and 400 nm or less, and the amplification reaction begins. It is preferable to use a wavelength of 300 nm or more and 340 nm or less in terms of the fast photolysis rate. The irradiance of the light is not particularly limited, but is preferably 0.1 mW / cm. 2 More than 3W / cm 2 It is preferable to use light in the following ranges: The light irradiation time is not particularly limited, but an irradiation time of 30 minutes or less is preferable, and an irradiation time of 5 minutes or less is more preferable.

[0072] The temperature for the amplification reaction of the target nucleic acid may be appropriately set in the range of 0° C. to 80° C., taking into consideration the composition of the solvent used. When the amplification reaction is carried out by the TRC method, it is preferably carried out in the range of 40° C. to 50° C.

[0073] <4> Detection Method In the above-mentioned method for amplifying a target nucleic acid, the amplified target nucleic acid can be detected by the following method. The target nucleic acid amplified with the light-controlled amplification reagent may be detected by adding a detection component before or after the amplification reaction and measuring the fluorescence intensity or chemiluminescence intensity derived from the component. A preferred embodiment of the detection component is an oligonucleotide probe whose fluorescence properties change when it forms a complementary double strand with a part of the target nucleic acid compared to before the formation.

[0074] An example of the probe is a DNA bound to an intercalative fluorescent dye having a sequence complementary or homologous to a portion of the target nucleic acid. The sequence of the DNA portion must be a sequence present in the target nucleic acid, and complementary or homologous to a portion that can be sufficiently distinguished from nucleic acids other than the target nucleic acid. The length of the DNA portion is preferably 6 nucleotides to 100 nucleotides, more preferably 10 nucleotides to 30 nucleotides, for specific analysis of the target nucleic acid. It is preferable that the 3' end of the DNA portion has a sequence non-complementary to the target nucleic acid added thereto, or the 3' end is chemically modified (e.g., aminated) so that extension from the 3' end by an enzyme having RNA-dependent DNA polymerase activity does not occur when the DNA portion forms a complementary bond with the amplified target nucleic acid.

[0075] The fluorescent intercalating dye changes its fluorescent properties by intercalating into the double-stranded portion when the DNA portion forms a complementary bond with another nucleic acid. For this purpose, for example, the fluorescent intercalating dye may be bound to DNA via a linker of an appropriate molecular length that does not prevent the intercalation into the double-stranded portion. There are no particular limitations on such a linker, so long as it does not prevent the fluorescent intercalating dye from intercalating into the double-stranded portion. In particular, a linker molecule selected from bifunctional hydrocarbons having functional groups at both ends is convenient and preferable for modifying oligonucleotides. A commercially available reagent set (e.g., C6-Thiolmodifier manufactured by Clontech) may also be used.

[0076] There are no particular limitations on the intercalative fluorescent dye as long as its fluorescent properties change, for example, the emitted fluorescent wavelength changes, by intercalating into a double strand, but from the viewpoint of ease of measurement, etc., it is particularly preferable for the dye to have a property of increasing its fluorescent intensity by intercalation. Specifically, thiazole orange and oxazole yellow, which show particularly significant changes in fluorescent intensity, and their derivatives can be exemplified as preferred intercalative fluorescent dyes.

[0077] The position at which the intercalative fluorescent dye is bound to the DNA portion via a linker is not particularly limited, and may be the 5' end, 3' end, or center of the DNA portion, as long as it does not prevent the intercalation of the intercalative fluorescent dye into the double strand and does not inhibit the complementary binding between the DNA portion and RNA.

[0078] Another example of an oligonucleotide probe whose fluorescence properties change when it forms a complementary double strand with a part of the target nucleic acid compared to before formation is the molecular beacon. A molecular beacon is a DNA having a sequence complementary or homologous to a part of the target nucleic acid, and has a stem-loop structure with a fluorescent dye and a quencher at both ends. In the stem-loop structure, the fluorescence of the fluorescent dye is suppressed by the quencher, but when a region complementary to the target RNA present in the loop sequence hybridizes with the amplification product generated during the reaction, the stem part is cleaved and emits fluorescence. The stem can be formed by forming a double strand of DNA within the molecule and introducing a pair of a fluorescent dye (such as Cy3) and a quencher (such as an azo compound) with high association ability into the DNA.

[0079] Another example of an oligonucleotide probe whose fluorescence properties change when it forms a complementary double strand with a part of a target nucleic acid, compared to before it is formed, is a FRET (Fluorescence Resonance Energy Transfer) probe. In this case, two probes designed inside the first and second primers used for amplification in the sequence of the target nucleic acid are used. Of the two probes, one probe is modified with a donor fluorescent dye at the 3' end and the other probe is modified with an acceptor fluorescent dye at the 5' end. By designing the donor fluorescent dye and the acceptor fluorescent dye to be close to each other when hybridized, the target nucleic acid can be detected by the FRET phenomenon that occurs when the two probes hybridize with the amplification product generated during the reaction.

[0080] Yet another example of an oligonucleotide probe whose fluorescence properties change when it forms a complementary double strand with a part of a target nucleic acid, compared to before it is formed, is the TaqMan probe. In this case, a single probe designed inside the first and second primers used for amplification in the sequence of the target nucleic acid is used. The 5' end of the TaqMan probe is labeled with a fluorescent dye (reporter dye), and the 3' end is labeled with a quencher. The fluorescence of the reporter dye is suppressed by the quencher, but if the probe is hybridized to the target RNA during the primer extension reaction by DNA polymerase, the probe is decomposed by the 5'→3' exonuclease activity, and the reporter dye is released, allowing detection by the fluorescence generated. EXAMPLES

[0081] The present invention will now be described in more detail with reference to examples and reference examples. However, the present invention is not limited thereto. Reference Examples do not constitute the present invention. The reagents and anhydrous solvents used in the reaction were commercially available products (Tokyo Chemical Industry Co., Ltd., Kanto Chemical Co., Ltd., Fujifilm Wako Pure Chemical Industries, Ltd., Sigma-Aldrich Co., Ltd.) and were used without purification. Nuclear magnetic resonance (NMR) spectra were measured with 400MHz Bruker AVANCE III 400 and 400MHz Bruker AVANCE III HD. Chemical shift values ​​were expressed in ppm using tetramethylsilane (TMS) as an internal standard. s represents singlet, d represents doublet, t represents triplet, q represents quartet, quint represents quintet, brs represents broad singlet, and m represents multiplet.

[0082] Reference example 1 N 6 ,N 6 -Bis(tert-butoxycarbonyl)-5'-O-tert-butyldimethylsilyl-2'-deoxyadenosine (2.88g, 5.1mmol) was dissolved in methylene chloride (20mL), sodium iodide (100mg, 0.67mmol), 1.5M aqueous tetrabutylammonium hydroxide solution (6.78mL, 10mmol), 1.0M aqueous sodium hydroxide solution (7.63mL, 7.6mmol) and distilled water (10mL) were added and stirred at room temperature for 5 minutes. To the resulting mixture was added a solution of m-(diethylamino)benzyl chloride (3.02g, 15mmol) in methylene chloride (9.0mL), and the mixture was reacted at room temperature for 15 hours under light shielding. The aqueous and organic phases were separated, and the aqueous phase was extracted twice with methylene chloride (50mL). The organic phases were combined and dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane:ethyl acetate=70:30) and N 6 ,N 6 -Bis(tert-butoxycarbonyl)-5'-O-tert-butyldimethylsilyl-3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (625 mg, 17%) was obtained as a yellow solid.

[0083] 1H NMR (400MHz, CDCl3): δ8.84(s,1H),8.42(s,1H),7.19(t,J=7.9Hz,1H),6.64-6.61 (m,3H),6.55(m,1H),4.53(d,J=1.4Hz,2H),4.37(m,1H),4.27(m,1H),3.89(dd,J= 11.1,4.0Hz,1H),3.78(dd,J=11.1,3.2Hz,1H),3.36(q,J=7.1Hz,4H),2.70-2.59( m,2H),1.45(s,18H),1.16(t,J=7.1Hz,6H),0.88(s,9H),0.07(s,3H),0.06(s,3H). Example 1 Obtained in Reference Example 1, N 6 ,N 6 -Bis(tert-butoxycarbonyl)-5'-O-tert-butyldimethylsilyl-3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (625 mg, 0.87 mmol) was weighed out into a light-shielding container, dissolved in methylene chloride (30 mL), activated (vacuum dried at 80°C for 3 hours) silica gel (6.0 g) was added, and the solvent was distilled off under reduced pressure. The resulting residue was heated under reduced pressure at 80°C for 13 hours. The resulting solid was washed with methanol and chloroform, and the filtrate was concentrated to obtain 5'-O-tert-butyldimethylsilyl-3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (369 mg, 81%) as a yellow solid.

[0084] 1 H NMR (400MHz, CDCl3): δ8.32(s,1H),8.14(s,1H),7.17(t,J=7.8Hz,1H),6.63-6.59(m ,3H),6.47(t,J=6.5Hz,1H),6.05(brs,2H),4.51(d,J=3.7Hz,2H),4.34(m,1H),4.24( m,1H),3.88(dd,J=11.0.4.2Hz,1H),3.76(dd,J=11.0,3.2Hz,1H),3.33(q,J=7.0Hz, 4H),2.64-2.61(m,2H),1.13(t,J=7.0Hz,6H),0.89(s,9H),0.06(s,3H),0.05(s,3H). Next, 5'-O-tert-butyldimethylsilyl-3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (369 mg, 0.70 mmol) was dissolved in tetrahydrofuran (3.0 mL) and cooled to 0°C. 1M tetrabutylammonium fluoride-tetrahydrofuran solution (0.70 mL, 0.70 mmol) was slowly added dropwise to the resulting mixture, and the temperature was raised to room temperature. The reaction was carried out at room temperature for 2 hours, and methanol (3.0 mL) was added to stop the reaction. The reaction solution was concentrated, and the resulting residue was purified by flash silica gel column chromatography (ethyl acetate:methanol=90:10) to obtain 3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (compound A, 202 mg, 70%) as a yellow solid.

[0085] 1 H NMR (400MHz, CDCl3): δ8.32(s,1H),7.81(s,1H),7.20(t,J=8.1Hz,1H),6.65-6.62(m,3H),6.57(m,1H),6.28(m,1H),5.74(brs,2H),4. 54(s,2H),4.49(m,1H),4.39(s,1H),3.99(m,1H),3.70(m,1H),3.36(q,J=7.0Hz,4H),3.01(m,1H),2.44(m,1H),1.17(t,J=7.0Hz,6H). Example 2 Compound A (70.0 mg, 0.17 mmol) obtained in Example 1 was azeotropically distilled twice with dehydrated toluene (3.0 mL) and three times with dehydrated pyridine (3.0 mL) to remove water. Chlorophosphite salicyl (40.0 mg, 0.20 mmol) and 4-dioxane (1.0 mL) were added to a solution of the dried solid in dehydrated pyridine (2.0 mL) and reacted at room temperature for 1.5 hours. Then, a solution of bis(tributylammonium) pyrophosphate (82.0 mg, 0.15 mmol) in DMF (N,N-dimethylformamide) (1.0 mL) and tributylamine (0.12 mL, 0.50 mmol) were added and reacted at room temperature for 2 hours. After the reaction, a solution of iodine (32 mg, 0.13 mmol) / pyridine (9.0 mL) / distilled water (1.0 mL) was added until the reaction solution turned red. After confirming that the reaction solution did not fade even after stirring at room temperature for about 15 minutes, distilled water (2.0 mL) was added and reacted at room temperature for 12 hours. The reaction solution was washed with ethyl acetate (60 mL) and the aqueous phase was concentrated. The resulting residue was purified by octadecyl-modified silica gel column chromatography (water:methanol = 9:1), and the solvent was distilled off under reduced pressure. The resulting residue was suspended in distilled water (1.3 mL) and methanol (0.5 mL), and a solution of sodium perchlorate (850 mg) in acetone (10 mL) was added and centrifuged, and the supernatant was removed, and the solvent was distilled off under reduced pressure. The same operation was performed twice to obtain 3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine triphosphate (compound B, 13 mg, 12%) as a white solid.

[0086] 1 H NMR(D2O,400MHz):δ8.14(m,1H),7.56(m,3H),7.53(m,1H),7.41(m,1H),6.40(m,1H),4.60(m,2H,obscured by the residual proton of D2O),4.58(m,1H,obscured by the residual proton of D2O),4.40(m,1H),4.11(m,2H),3.57(m,4H),2.66(m,2H),1.01(m,6H); 31P NMR(D2O,162MHz):δ-8.0(m,1P),-11.4(d,1P),-21.5(m,1P);HR-MS(ESI-TOF):molecular formula C 21 H 30 NO 12 P3;[M-H+]-:651.1179(calculated:651.118).

[0087] [ka]

[0088] Reference example 2 2-Nitro-p-xylene glycol (5.00 g, 27.3 mmol) and imidazole (3.72 g, 54.6 mmol) were dissolved in DMF (15.0 mL) under an argon atmosphere and cooled to 0°C. To the resulting solution, a solution of t-butyldimethylsilyl chloride (4.25 g, 27.3 mmol) in DMF (10.0 mL) was added. The resulting mixture was warmed to room temperature and reacted for 1.5 hours, after which methanol (20 mL) was added and the mixture was stirred at room temperature for 30 minutes to stop the reaction. The solvent in the reaction solution was distilled off under reduced pressure, and distilled water (200 mL) was added to the resulting residue, which was then extracted five times with ethyl acetate (200 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane:ethyl acetate=4:1) to obtain 4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitrobenzenemethanol (1.9 g, 23%) as a whitish yellow solid.

[0089] [ka]

[0090] 1H NMR(400MHz,CDCl3):δ8.06(s,1H),7.66(d,J=8.0Hz,1H),7.60(d,J=8.0Hz,1H),4.93(d,J=6.7Hz, 2H,CH2-OH),4.79(s,2H,CH2-OTBS),2.51(t,J=6.7Hz,1H,OH),0.95(s,9H,TBS),0.11(s,6H,TBS). Reference example 3 4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitrobenzenemethanol (648 mg, 2.17 mmol) obtained in Reference Example 2, triphenylphosphine (1.71 g, 6.51 mmol), carbon tetrachloride (2.16 g, 6.51 mmol) and diisopropylethylamine (929 mg, 7.16 mmol) were dissolved in THF (tetrahydrofuran) (7.3 mL) under an argon atmosphere and stirred at room temperature for 1 hour. Distilled water (50 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was purified by flash silica gel column chromatography (hexane:ethyl acetate=15:1) to obtain 4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitrobenzyl bromide (747 mg, 83%) as a white solid.

[0091] [ka]

[0092] 1 H NMR (400MHz, CDCl3): δ8.00(s,1H),7.55-7.51(m,2H),4.82(s,CH2-Br),4.79(s,2H,CH2-OTBS),0.95(s,9H,TBS),0.11(s,6H,TBS). Reference example 4 5'-O-(4,4'-dimethoxytrityl)thymidine (2.72g, 5.0mmol) was dissolved in a mixed solvent (25mL) of dichloromethane / dimethylacetamide (10:1), triethylamine (607mg, 6.0mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Benzoyl chloride (773mg, 5.5mmol) was added and the mixture was reacted at room temperature for 15 hours. After the reaction, the mixture was diluted with ethyl acetate (50mL) and washed with saturated saline (30mL). The solvent was removed under reduced pressure, and the resulting residue was purified by flash silica gel column chromatography (toluene:ethyl acetate=3:1→1:1) to give a mixture of N-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine and 3'-O-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine (1.78 g, 55%, N-Bz form:O-Bz form=6.5:1) as a colorless solid.

[0093] [ka]

[0094] N-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine; 1 H NMR (400MHz, CDCl3): δ7.88(dd,J=7.2,1.2Hz,2H),7.73(d,J=0.84Hz,1H),7.55(t,J=7. 4Hz,1H),7.37-7.42(m,4H),7.25-7.30(m,5H),7.19-7.23(m,2H),6.82(d,J=8.0Hz,4H), 6.34(t,J=7.2Hz,1H),4.49(quint,J=2.5Hz,1H),3.97(q,J=2.5Hz,1H),3.74(s,6H),3. 42(dd,J=10.6,2.7Hz,1H),3.31(dd,J=10.6,2.7Hz,1H),2.23-2.34(m,2H),1.41(s,3H). 3'-O-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine; 1H NMR (400MHz, CDCl3): δ8.02(t,J=6.9Hz,2H),7.66(d,J=0.96Hz,1H),7.55(t,J =7.4Hz,1H),7.37-7.42(m,4H),7.25-7.30(m,5H),7.09-7.14(m,2H),6.82(d, J=8.0Hz,4H),6.50(dd,J=5.6Hz,3.0Hz,1H),5.68(d,J=5.6Hz,1H),4.26(d,J= 1.8Hz,1H),3.73(s,6H),3.53(t,J=2.4Hz,1H),2.47-2.64(m,1H),1.37(s,3H). Reference example 5 4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitrobenzyl bromide (377 mg, 1.05 mmol) obtained in Reference Example 3 was dissolved in methylene chloride (2.0 mL) and stirred at room temperature for 10 minutes, and then a mixture of N-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine and 3'-benzoyl-5'-O-(4,4'-dimethoxytrityl)thymidine (521 mg, 0.81 mmol, N-Bz form: O-Bz form = 4:1) obtained in Reference Example 4, tetrabutylammonium iodide (236 mg, 0.64 mmol), 1M aqueous sodium hydroxide solution (1.62 mL, 1.62 mmol) and methylene chloride (6 mL) was added dropwise and reacted at room temperature for 3 hours. The reaction solution was diluted with dichloromethane (20 mL), washed with saturated saline (20 mL), and dried over anhydrous sodium sulfate. The residue obtained by distilling off the solvent under reduced pressure was purified by flash silica gel column chromatography (toluene: ethyl acetate = 30: 1) to obtain the product (146 mg). The product obtained was then dissolved in a 2M ethanol / methylene chloride solution of ammonia (16 mL, ethanol: methylene chloride = 2.5: 1) and stirred at room temperature for 6.5 hours, after which the solvent was distilled off under reduced pressure and the residue obtained was purified by flash silica gel column chromatography (hexane: ethyl acetate = 1: 1) to obtain 3'-O-[4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitro]benzyl-5'-O-(4,4'-dimethoxytritylyl)thymidine (46.7 mg, 2 steps 7%) as a colorless solid.

[0095] [ka]

[0096] 1 H NMR (400MHz, CDCl3): δ8.05(brs,1H),7.98(brs,1H),7.68(d,J=8.0Hz,1H),7.59(d,J=4.6Hz,2H),7.39(d,J= 8.6Hz,2H),7.21-7.31(m,7H),6.83(dd,J=8.9,1.2Hz,4H),6.39(dd,J=8.4,5.6Hz,1H),4.85(q,J=14.6Hz,2H) ,4.76(brs,2H),4.33(d,J=5.6Hz,1H),4.22(d,J=2.4Hz,1H),3.78(s,6H),3.52(dd,J=10.6,3.2Hz,1H),3.37( dd,J=10.6,3.2Hz,1H),2.53-2.58(m,1H),2.21-2.29(m,1H),1.47(d,J=0.8Hz,3H),0.95(s,9H),0.12(s,6H). Reference example 6 A 1M THF solution (0.23mL, 0.23mmol) of tetrabutylammonium fluoride was added to a THF (3.8mL) solution of 3'-O-[4-[[(t-butyldimethylsilyl)oxy]methyl]-2-nitro]benzyl-5'-O-(4,4'-dimethoxytritylyl)thymidine (157mg, 0.19mmol) obtained in Reference Example 5, and the mixture was reacted at 0°C for 2 hours. The mixture was diluted with ethyl acetate (20mL) and washed with saturated saline (20mL). The residue obtained by distilling off the solvent under reduced pressure was purified by flash silica gel column chromatography (hexane:ethyl acetate=1:1→1:4), to obtain 3'-O-[4-(hydroxymethyl)-2-nitro]benzyl-5'-O-(4,4'-dimethoxytritylyl)thymidine (124mg, 92%) as a colorless oil.

[0097] [ka]

[0098] 1 1H NMR (400 MHz, CDCl3): δ 8.10 (d, J = 1.4 Hz, 1H), 7.91 (brs, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.61 (dd, J = 8.6, 1.1 Hz, 2H), 7.38 (dd, J = 7.0, 1.5 Hz, 2H), 7.31 - 7.20 (m, 7H), 6.82 (dd, J = 8.9, 3.4 Hz, 4H), 6.39 (dd, J = 8.4, 5.6 Hz, 1H), 4.86 (q, J = 14.9 Hz, 2H), 4.79 (s, 2H), 4.31 (d, J = 5.9 Hz, 1H), 4.23 (d, J = 2.5 Hz, 1H), 3.79 (s, 6H), 3.53 (dd, J = 10.6, 2.7 Hz, 1H), 3.35 (dd, J = 10.6, 2.7 Hz, 1H), 2.54 (dq, J = 13.6, 5.4 Hz, 1H), 2.17 - 2.28 (m, 1H), 1.94 (t, J = 6.0 Hz, 1H), 1.49 (d, J = 0.96 Hz, 3H). Reference Example 7 To a methylene chloride (3.6 mL) solution of 3'-O-[4-(hydroxymethyl)-2-nitro]benzyl-5'-O-(4,4'-dimethoxytrityl)thymidine (130 mg, 0.18 mmol) obtained in Reference Example 6, succinic anhydride (22 mg, 0.21 mmol), triethylamine (36 mg, 0.36 mmol) and 4-dimethylaminopyridine (22 mg, 0.18 mmol) were added and reacted at room temperature for 18 hours. The mixture was diluted with ethyl acetate (20 mL) and washed with purified water (20 mL). The solvent was distilled off under reduced pressure to obtain the product (78.2 mg) as a colorless oil. A solution of the obtained product (21.6 mg) in dimethylformamide (3.6 mL) was added with 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (90 mg, 0.55 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (70 mg, 0.37 mmol) and reacted at room temperature for 18 hours. The mixture was diluted with ethyl acetate (20 mL) and washed with purified water (20 mL). The solvent was distilled off under reduced pressure and the resulting residue was purified by flash silica gel column chromatography (chloroform:ethyl acetate = 3:1) to give 3'-O-[[[[[4-(3,4-dihydro-4-oxo-1,2,3-benzotriazin)-3-yloxy]carbonylethyl]carbonyl]oxymethyl]-2-nitro]benzyl-5'-O-(4,4'-dimethoxytritylyl)thymidine (65 mg, 38% for two steps) as a colorless oil.

[0099] [ka]

[0100] 11H NMR (400 MHz, CDCl3): δ 8.36 (dd, J = 8.0, 1.0 Hz, 1H), 8.22 (dd, J = 8.2, 0.48 Hz, 1H), 8.08 (dd, J = 13.0, 1.6 Hz, 1H), 8.00 (dt, J = 7.4, 1.4 Hz, 2H), 7.84 (dt, J = 7.4, 1.1 Hz, 1H), 7.74 (d, J = 8.0, 2.5 Hz, 1H), 7.63 (dd, J = 8.0, 1.7 Hz, 1H), 7.59 (d, J = 1.1 Hz, 1H), 7.38 (dd, J = 8.5, 1.5 Hz, 2H), 7.30 -7.21 (m, 7H), 6.82 (dd, J = 8.9, 2.0 Hz, 4H), 6.39 (dd, J = 8.4, 5.6 Hz, 1H), 5.29 (s, 2H), 5.24 (s, 1H), 4.86 (q, J = 15.0 Hz, 2H), 4.32 (d, J = 5.9 Hz, 1H), 4.22 (d, J = 2.4 Hz, 1H), 3.78 (s, 6H), 3.52 (dd, J = 10.6, 2.7 Hz, 1H), 3.36 (dd, J = 10.6, 2.7 Hz, 1H), 3.13 (t, J = 6.6 Hz, 2H), 2.91 (t, J = 6.6 Hz, 2H), 2.50 - 2.58 (m, 1H), 2.16 - 2.28 (m, 1H), 1.48 (d, J = 1.0 Hz, 3H). Reference Example 8

[0101]

Chem.

[0102] Amino-SynBase CPG 1000 / 110 (LCAA) (LGC LINK Technologies) (400 mg, loading amount 105 μmol / g) was placed in a plastic centrifuge tube and set in a solid-phase synthesizer, and a DMF solution of 3'-O-[[[[[4-(3,4-dihydro-4-oxo-1,2,3-benzotriazine)-3-yloxy]carbonylethyl]carbonyl]oxymethyl]-2-nitro]benzyl-5'-O-(4,4'-dimethoxytritylyl)thymidine (61.6 mg, 64.5 μmol) and diisopropylethylamine (8.4 mg, 64.5 μmol) obtained in Reference Example 7 was added and reacted at room temperature for 3 days. After the reaction was completed, the solid phase was removed by suction filtration, washed with ethyl acetate, and dried to obtain a solid-phase-supported thymidine derivative (349.5 mg). The loading amount of the thymidine derivative in the solid phase was 100.5 μmol / g.

[0103] In the following, the nucleic acid amplification method is TRC (Transcription-Reverse transcription The present invention will be described using an example in which the "announcement concered" method was used. The present invention will be described in more detail below, but is not limited to these examples.

[0104] Example 3 Effect of dATP concentration on TRC reaction The effect of the dATP concentration on the detection time in the TRC method was examined.

[0105] (1) The standard RNA (SEQ ID NO: 1) of hepatitis C virus was prepared by in vitro transcription from a plasmid into which the gene of the standard RNA of hepatitis C virus was inserted. The standard RNA was diluted with water for injection for 10 3 The solution was diluted to copies / 2 μL and used as an RNA sample.

[0106] (2) 12 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and then 2 μL of the RNA sample was added. The molecular beacon probe (SEQ ID NO: 2), which is a probe for detecting the standard RNA, contains a portion of the homologous strand of the standard RNA [from base 108 to base 123 of SEQ ID NO: 1]. The first primer (SEQ ID NO: 3) is an oligonucleotide in which a T7 promoter sequence (SEQ ID NO: 6) has been added to the 5'-end of a portion of the complementary strand of the standard RNA (specifically, from base 125 to base 145 of SEQ ID NO: 1: SEQ ID NO: 5).

[0107] Composition of reaction solution: The concentration is the final concentration after adding the initiator (in 20 μL) described below. 66mM Tris-HCl buffer (pH8.36) 0.33 mM each dCTP, dGTP, dTTP 2.0mM each ATP, CTP, GTP, UTP 3.3mM ITP 150mM Trehalose 50 nM molecular beacon probe (containing a portion of the homologous strand of standard RNA [from 108 to 123 of SEQ ID NO: 1]: SEQ ID NO: 2) 1.0 μM first primer (SEQ ID NO:3) 1.0 μM second primer (a part of the homologous strand of standard RNA [the first base of SEQ ID NO: 1] to 16th position]: SEQ ID NO: 4) 12.8U AMV reverse transcriptase 166U T7 RNA polymerase Various concentrations of dATP (from 0 to 3300 μM) (3) After incubating the above reaction solution at 46° C. for 3 minutes, 6 μL of an initiator having the following composition was added.

[0108] Composition of initiator: The concentration is the final concentration after adding the initiator (in 20 μL) 18.4mM Magnesium chloride 90.0mM Potassium Chloride 0.1% (w / v) Tween 20 (Fujifilm Wako Pure Chemical Industries, Ltd.) 9.0% (v / v) DMSO 2.5% (w / v) glycerol (4) Using a thermostatically controlled fluorescence spectrophotometer capable of direct measurement of the PCR tube, the reaction was allowed to proceed at 46°C, while the fluorescence intensity of the reaction solution (excitation wavelength 470 nm, fluorescence wavelength 520 nm) was measured over time for 30 minutes. The time when the initiator was added was set as 0 minutes, and the time when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value at a specified time by the background fluorescence intensity ratio) exceeded 2.3 was defined as the detection time.

[0109] The results are shown in Table 1. It can be seen that if the dATP concentration in the reaction solution is 165 μM or higher, there is no effect on the amplification reaction.

[0110] [Table 1]

[0111] Example 4 Effect of photolabile protecting groups on the TRC reaction The effect of compound A synthesized in Example 1 on the TRC reaction was examined.

[0112] (1) According to Example 1, 3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (Compound A) was synthesized by modifying the 3'-terminal hydroxyl group of dATP with a 3-(diethylamino)benzyl group.

[0113] (2) A solution containing 3'-O-[3-(diethylamino)benzyl]-2'-deoxyadenosine (Compound A) synthesized in (1) was dissolved in DMSO to a constant concentration.

[0114] (3) 8 μL of the reaction solution having the composition shown below was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and 2 μL of the RNA sample prepared in Example 3(1) was added thereto.

[0115] Composition of reaction solution: The concentration is the final concentration after adding the initiator (in 20 μL) described below. 66mM Tris-HCl buffer (pH8.36) 0.33mM each dATP, dCTP, dGTP, dTTP 2.0mM each ATP, CTP, GTP, UTP 3.3mM ITP 150mM Trehalose 50 nM Molecular Beacon Probe (SEQ ID NO: 2) 1.0 μM first primer (SEQ ID NO:3) 1.0 μM second primer (SEQ ID NO: 4) 12.8U AMV reverse transcriptase 166U T7 RNA polymerase (4) After incubating the reaction solution at 46°C for 3 minutes, add 10 μL of the initiator consisting of the following composition: Added.

[0116] Composition of initiator: The concentration is the final concentration after adding the initiator (in 20 μL) 18.4mM Magnesium chloride 90.0mM Potassium Chloride 0.1% (w / v) Tween 20 (Fujifilm Wako Pure Chemical Industries, Ltd.) 2.5% (w / v) glycerol 10% (v / v) DMSO (containing 0 to 200 μM of photolabile protecting group, (2) (Prepared with (5) Measurement was performed in the same manner as in Example 3(4) to determine the detection time.

[0117] The results are shown in Table 2. When the diethylaminobenzyl group was used as the photodegradable protecting group, It can be seen that there is no effect on the TRC reaction at least up to a concentration of 200 μM.

[0118] [Table 2]

[0119] Reference Example 9 Photoregulation of TRC reaction using a primer modified with a photodegradable protecting group (1) Using the solid-phase-supported thymidine derivative obtained in Reference Example 8, a first primer consisting of the nucleotide sequence set forth in SEQ ID NO: 7, in which the hydroxyl group at the 3' position of the thymidine at the 3' end (the 49th position) was modified with a photolabile protecting group (hereinafter, also referred to simply as "photolabile protecting group modified primer") was synthesized (outsourced to Gene Design). Of the first primer consisting of the sequence set forth in SEQ ID NO: 7, the 28 bases on the 5' end side are the T7 promoter sequence (SEQ ID NO: 6). The synthesized product was purified using HPLC, and the molecular weight was confirmed using an electrospray ionization mass spectrometer (ESI-MS), and then it was diluted to 100 μM with TE buffer and stored at -20°C.

[0120] (2) 8 μL of the reaction solution having the following composition was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).

[0121] Composition of reaction solution: The concentration is the final concentration after adding the initiator (in 20 μL) described below. 0.1% (w / v) Tween 20 (Fujifilm Wako Pure Chemical Industries, Ltd.) 66mM Tris-HCl buffer (pH8.36) 2.5% (w / v) glycerol 0.33mM each dATP, dCTP, dGTP, dTTP 2.0mM each ATP, CTP, GTP, UTP 3.3mM ITP 94.5mM Trehalose 50 nM Molecular Beacon Probe (SEQ ID NO: 2) 1.0 μM second primer (SEQ ID NO: 4) 45.4U AMV reverse transcriptase 100U T7 RNA polymerase (3) The photolabile protecting group-modified primer solution synthesized in (1) or the first primer solution consisting of the nucleotide sequence set forth in SEQ ID NO: 7 (without modification with a photolabile protecting group), prepared to 10 μM using water for injection, was added at 2 μL to the above reaction solution without exposure to ultraviolet light (ultraviolet light exposure time 0 minutes) (final concentration after addition of the initiator described below: 1 μM).

[0122] (4) The 10 μM photodegradable protective group-modified primer solution prepared in (3) was irradiated with ultraviolet light (wavelength 365 nm, intensity 1 mW / cm) using an ultraviolet light irradiation device (Omnicure LX405S UV LED spot curing device, Sanei Tech Co., Ltd.). 2 ) was irradiated for 1 or 10 minutes. 2 μL of the irradiated primer solution was added to the above reaction solution.

[0123] (5) Standard RNA (SEQ ID NO: 1) was diluted with TE buffer (containing 0.01% (w / v) cholic acid) for 10 min. 5 After diluting to copies / 2 μL, 2 μL each was added to the reaction solutions prepared in (3) and (4) above to which the primer solutions had been added.

[0124] (6) After the addition of the standard RNA, the reaction solution was incubated at 46° C. for 3 minutes, and then 8 μL of the initiator having the composition described in Example 3(3) that had been similarly incubated at 46° C. for 3 minutes was added.

[0125] (7) Measurement was performed in the same manner as in Example 3(4) to determine the detection time.

[0126] The results are shown in Table 3. From the results with 0 minutes of UV irradiation, it can be seen that when the photolabile protective group-modified primer synthesized in (1) is used as the first primer, the detection time is delayed by about 1 minute compared to when the first primer not modified with a photolabile protective group is used. This shows that the TRC reaction can be inhibited by modifying the deoxyribonucleotide at the 3' end of the primer with a photolabile protective group. On the other hand, even when the photolabile protective group-modified primer synthesized in (1) is used as the first primer, the detection time is equivalent to that when the first primer not modified is used when the primer is irradiated with UV light for 1 minute or 10 minutes to decompose the protective group. This shows that the TRC reaction can be controlled by light by modifying the deoxyribonucleotide at the 3' end of the primer with a photolabile protective group.

[0127] [Table 3]

[0128] Reference Example 10: Effect of light control of TRC reaction on TRC reactivity In Reference Example 9, only the first primer was irradiated with ultraviolet light, but in this experiment, the reaction solution containing the first primer was irradiated with ultraviolet light to examine the effect of the irradiation on the TRC reaction.

[0129] (1) 8 μL of the reaction solution having the composition described in Reference Example 9(2) was dispensed into a 0.5 mL PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI).

[0130] (2) 2 μL each of a 10 μM solution of a photolabile protecting group-modified primer (SEQ ID NO: 7) or a solution of a first primer (not modified with a photolabile protecting group) (SEQ ID NO: 7) prepared in Reference Example 9(3) was added to the above reaction solution (to give a final concentration of 1 μM after addition of the initiator described below).

[0131] (3) Standard RNA (SEQ ID NO: 1) was diluted with TE buffer (containing 0.01% (w / v) cholic acid) for 10 min. 5After diluting to copies / 2 μL, 2 μL each was added to the reaction solution prepared in (2) above to which the primer solution had been added.

[0132] (4) After the addition of the standard RNA, the reaction solution was incubated at 46° C. for 3 minutes, and then 8 μL of an initiator having the composition described in Example 3(3) that had been similarly incubated at 46° C. for 3 minutes was added, followed by irradiation with ultraviolet light (wavelength 365 nm, intensity 1 mW / cm 2 ) for 1 minute.

[0133] (5) Measurement was performed in the same manner as in Example 3(4) to determine the detection time.

[0134] The results are shown in Table 4. Even when the entire reaction solution was irradiated with UV light, the detection time was the same as in Reference Example 9. This shows that irradiating the entire reaction solution with UV light does not affect the TRC reaction.

[0135] [Table 4]

[0136] In Reference Examples 9 and 10, a 2-nitrobenzyl group (o-nitrobenzyl group) is used as the photodegradable protecting group. However, it is easily conceivable that the TRC reaction can be controlled by light even if a 3-(dialkylamino)benzyl group (an embodiment of compound (1) of the present invention) is used as the photodegradable protecting group instead of the 2-nitrobenzyl group.

Claims

1. A compound represented by general formula (1): 【Chemistry 1】 (In the formula, R １ and R ２ each independently represents an alkyl group having 1 to 8 carbon atoms; or R １ and R ２ may form a 5- to 7-membered heterocyclic ring together with the nitrogen atom to which they are attached. ３ and R ４ Each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group. ５ is a hydrogen atom or a group represented by the formula (7) [-P(=O)(OH)-OP(=O)(OH)-OP(=O)(OH) ２ (7)], Y represents a hydrogen atom or a hydroxyl group, and X represents any one of the nucleic acid bases represented by the following formulas (2) to (5): 【Chemistry 2】 (In the formula, R ６ represents a hydrogen atom or a methyl group, when Y is a hydrogen atom, R represents a methyl group, and when Y is a hydroxy group, R ６ represents a hydrogen atom.)

2. The compound represented by the general formula (1) according to claim 1, wherein Y is a hydrogen atom.

3. R １ and R ２ The compound represented by the general formula (1) according to claim 2, wherein is an ethyl group.

4. A deoxyribonucleoside represented by the general formula (1a) and chlorophosphite salicyl are used as a base. and then treating with pyrophosphate to obtain a compound represented by the general formula (6). obtaining a cyclic compound; and A method for producing a deoxyribonucleoside triphosphate represented by formula (1b), comprising a step of reacting a cyclic compound represented by general formula (6) with an oxidizing agent in the presence of water. 【Chemistry 3】 (In the formula, R １ and R ２ each independently represents an alkyl group having 1 to 8 carbon atoms; or R １ and R ２ may form a 5- to 7-membered heterocyclic ring together with the nitrogen atom to which they are attached. ３ and R ４ Each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a phenyl group, and X represents a nucleic acid base of any one of the following formulas (2), (3), (4a), or (5). 【Chemistry 4】 【Chemistry 5】 (In the formula, R １ , R ２ , R ３ , R ４ and X has the same meaning as above. 【Chemistry 6】 (In the formula, R １ , R ２ , R ３ , R ４ and X has the same meaning as above.

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