Thymine-selective photoactive nucleotide analog

Abstract

To provide a novel nucleic acid photo-reaction technique based on the properties of photoactive compounds.SOLUTION: As one example, a T- and U-selective photo-crosslinking agent represented by the following compound 15.SELECTED DRAWING: Figure 5C

Description

[Technical Field]

[0001] The present invention relates to a photoresponsive nucleotide analog (photoresponsive nucleotide analog compound) having a methylpyranocarbazole skeleton and capable of photocrosslinking (photocrosslinking) with light in the visible light range. [Background technology]

[0002] Nucleic acid ligation and cross-linking are fundamental techniques in the field of molecular biology. Nucleic acid ligation and cross-linking are used, for example, in combination with hybridization, for gene introduction, detection of base sequences, or for inhibiting gene expression. For this reason, nucleic acid ligation and cross-linking techniques are extremely important not only for basic research in molecular biology, but also for use in, for example, diagnosis and treatment in the medical field, or in the development and production of therapeutic and diagnostic agents, and the like, and in the development and production of enzymes, microorganisms, and the like in the industrial and agricultural fields.

[0003] Photoreaction technologies for nucleic acids include a photolinking technology using 5-cyanovinyldeoxyuridine (Patent Document 1: Japanese Patent No. 3753938, Patent Document 2: Japanese Patent No. 3753942) and a photocrosslinking technology using modified nucleosides having a 3-vinylcarbazole structure at the base site (Patent Document 3: Japanese Patent No. 4814904, Patent Document 4: Japanese Patent No. 4940311).

[0004] Furthermore, Patent Document 5 (International Publication No. WO2020 / 158687A1) discloses a photoresponsive nucleotide analogue having a methylpyranocarbazole skeleton. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent No. 3753938 [Patent Document 2] Japanese Patent No. 3753942 [Patent Document 3] Japanese Patent No. 4814904 [Patent Document 4] Japanese Patent No. 4940311 [Patent Document 5] International Publication No. WO2020 / 158687A1 Summary of the Invention [Problem to be solved by the invention]

[0006] Given the importance of nucleic acid photoreaction technology, there is a search for new compounds that can be used in nucleic acid photoreaction technology, as well as new applications and methods of use based on the properties of these photoreactive compounds.

[0007] Therefore, an object of the present invention is to provide a new nucleic acid photoreaction technology based on the properties of photoreactive compounds. [Means for solving the problem]

[0008] The present inventors have conducted extensive research into photoreactive compounds that can be used as photoreactive crosslinkers in nucleic acid photoreaction techniques, and have found that compounds that have a methylpyranocarbazole skeleton structure in place of the base moiety of a nucleic acid base can be used as photoreactive crosslinkers in such nucleic acid photoreaction techniques.

[0009] Photoreactive crosslinkers using this compound have the advantage that they can undergo photocrosslinking by irradiation with light of a longer wavelength than conventional ones, for example, by irradiation with light in the visible light range, due to their characteristic methylpyranocarbazole structure. Therefore, when it is desired to avoid damage to DNA or cells as much as possible, photoreactive crosslinkers using this compound are particularly advantageous because they can undergo photocrosslinking by irradiation with light of a longer wavelength.

[0010] It should be noted that this photoreactive compound initiates a photoreaction when irradiated with light, but photoreactivity is sometimes referred to as photoresponsiveness to emphasize that a compound that was stable up until that point initiates a reaction in response to the signal of light irradiation.

[0011] The present inventors have conducted further research into compounds having this methylpyranocarbazole skeleton structure and have found that compounds having a methylpyranocarbazole skeleton structure in place of the base moiety of a nucleic acid base and a sugar analog skeleton structure (described below) in place of the sugar moiety of a nucleoside have high photoreactivity and also exhibit T (thymine) and U (uracil) selective photoreactivity, thereby arriving at the present invention.

[0012] Therefore, the present invention includes the following (1) and the following. (1) A T- and U-selective photocrosslinker comprising a compound represented by formula I: JPEG2025116757000002.jpg158170 (wherein in Formula I, R is a C1-C3 alkyl group, a C1-C3 halogenated alkyl group, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted cyclohexyl group; R1 is a group selected from the group consisting of a hydrogen atom, a halogen atom, an —OH group, an amino group, a nitro group, a methyl group, a methyl fluoride group, an ethyl group, an ethyl fluoride group, and a C1-C3 alkylsulfanyl group; R2 is a group selected from a hydrogen atom, a methyl group, and an ethyl group; Q1 is, A phosphate group formed together with the O attached to Q1; Nucleotides or nucleic acids linked via a phosphodiester bond formed by a phosphate group formed together with an O attached to Q1; and A protecting group selected from: Trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, trimethylsilyl group, triethylsilyl group, t-butyldimethylsilyl group, acetyl group, benzoyl group; is a group selected from the group consisting of Q2 is, A phosphate group formed together with the O attached to Q2; Nucleotides or nucleic acids linked via a phosphodiester bond formed by a phosphate group formed together with an O attached to Q2; and A protecting group selected from: 2-cyanoethyl-N,N-dialkyl(C1-C4) phosphoramidite group, methylphosphonamidite group, ethylphosphonamidite group, oxazaphospholidine group, thiophosphite group, TEA salt of -PH(=O)OH, DBU salt of -PH(=O)OH, TEA salt of -PH(=S)OH, DBU salt of -PH(=S)OH; is a group selected from the group consisting of:

[0013] (2) The T- and U-selective photocrosslinker according to (1), wherein R2 is a methyl group. (3) The T- and U-selective photocrosslinking agent according to (1), wherein R is a C1 to C3 alkyl group. (4) R1 is a hydrogen atom, The T- and U-selective photocrosslinker according to (1), wherein R is a methyl group.

[0014] (5) A method for selectively detecting T and U, comprising a step of forming a photocrosslink selectively with T or U among pyrimidine bases C, T, and U, without forming a photocrosslink with C, using a compound represented by formula I according to any one of (1) to (4). (6) The step of selectively forming photocrosslinks to T or U, The method according to (5), which is carried out by irradiating with light for 30 seconds or less. (7) The step of selectively forming photocrosslinks to T or U, The method according to (5), which is carried out by irradiating with light having a wavelength of 360 to 440 nm.

[0015] (8) The step of selectively forming photocrosslinks to T or U, The method according to (5), which is a step of selectively forming photocrosslinks with T or U contained in a nucleic acid that has formed a complementary strand with the compound represented by formula I. (9) A method for selectively producing a photocrosslink between a compound represented by formula I described in any one of (1) to (4) and T or U, the method comprising: a step of selectively forming a photocrosslink between a compound represented by formula I described in any one of (1) to (4) and T or U. (10) The step of selectively forming photocrosslinks to T or U, The method according to (9), wherein the method is carried out by irradiating with light for 30 seconds or less. (11) The step of selectively forming photocrosslinks to T or U, The method according to (9), which is carried out by irradiating with light having a wavelength of 360 to 440 nm. (12) The step of selectively forming photocrosslinks to T or U, The method according to (9), which is a step of selectively forming photocrosslinks with T or U contained in a nucleic acid that has formed a complementary strand with the compound represented by formula I. [Effects of the Invention]

[0016] According to the present invention, a nucleic acid photoreaction technology can be realized that achieves both high photoreactivity and selective photoreactivity to T (thymine) and U (uracil) by irradiation with light of a visible light wavelength. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is an explanatory diagram showing the flow of MEPK synthesis. [Figure 2] FIG. 2 is an explanatory diagram showing the synthesis flow up to the amidite form of MEPD. [Figure 3A] FIG. 3A is an explanatory diagram showing the base sequence of the oligo DNA used in the photocrosslinking experiment between MEPD and thymine in Example 4, and the flow of this photocrosslinking experiment. [Figure 3B]FIG. 3B is a chart showing the results of UPLC analysis of the photocrosslinking experiment of Example 4. [Figure 4A] FIG. 4A is an explanatory diagram showing the base sequence of the oligo DNA used in the photocrosslinking experiment between MEPD and uracil in Example 5, and the flow of this photocrosslinking experiment. [Figure 4B] FIG. 4B is a chart showing the results of UPLC analysis of the photocrosslinking experiment of Example 5. [Figure 5A] FIG. 5A is an explanatory diagram showing the base sequences of the oligo-DNAs used in the experiment of photocrosslinking between MEPD and thymine and the experiment of photocrosslinking between MEPK and thymine in Example 7, and the flow of this photocrosslinking experiment. [Figure 5B] FIG. 5B is a chart showing the results of UPLC analysis of the photocrosslinking experiment of Example 7. [Figure 5C] FIG. 5C is a graph showing the results of the photocrosslinking experiment of Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to the specific embodiments described below.

[0019] [T and U selective photocrosslinker] The present invention includes a T- and U-selective photocrosslinker comprising a compound represented by Formula I:

[0020] (Formula I) JPEG2025116757000003.jpg158170

[0021] [R in Formula I] In a preferred embodiment, R in formula I can be a C1-C3 alkyl group, a C1-C3 halogenated alkyl group, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted cyclohexyl group.

[0022] In a preferred embodiment, the alkyl group may be, for example, a C1-C3 alkyl group, preferably a C1-C2 alkyl group, such as a methyl group or an ethyl group. In a preferred embodiment, the halogenated alkyl group may be, for example, a C1-C3 halogenated alkyl group, preferably a C1-C2 halogenated alkyl group. Examples of halogens include Br, Cl, F, and I. Halogenation occurs when hydrogen atoms in the alkyl group are substituted with halogen atoms, and the number of substitutions can be one or more, for example, one, two, or three. In a preferred embodiment, the phenyl group may be substituted or unsubstituted, and for example, hydrogen atoms in the phenyl group can be substituted with C1-C2 alkyl groups or halogen atoms, and the number of substitutions can be one or more, for example, one, two, or three. In a preferred embodiment, the cyclohexyl group can be substituted or unsubstituted. For example, the hydrogen atoms of the cyclohexyl group can be substituted with a C1-C2 alkyl group or a halogen atom. The number of substitutions can be one or more, for example, one, two, or three.

[0023] In a preferred embodiment, R in formula I can be, for example, a C1 to C3 alkyl group, preferably a methyl group.

[0024] [R1 in Formula I] In a preferred embodiment, R1 in formula I can be a group selected from the group consisting of a hydrogen atom, a halogen atom, an -OH group (hydroxyl group), an amino group, a nitro group, a methyl group, a methyl fluoride group, an ethyl group, an ethyl fluoride group, and a C1-C3 alkylsulfanyl group.

[0025] In a preferred embodiment, examples of halogen atoms include Br, Cl, F, and I atoms. Examples of methyl fluoride groups include -CHF, -CHF, and -CF. Examples of ethyl fluoride groups include -CH-CHF, -CH-CHF, -CH-CF, -CHF-CH, -CHF-CHF, -CHF-CHF, -CHF-CF, -CF-CH, -CF-CHF, -CF-CHF, and -CF-CF. Examples of C1-C3 alkylsulfanyl groups include -CH-SH, -CH-CH-SH, -CH(SH)-CH, -CH-CH-CH-SH, -CH-CH(SH)-CH, and -CH(SH)-CH-CH. In a preferred embodiment, R1 can be a hydrogen atom, a halogen atom, an -NH2 group, an -OH group (hydroxyl group), or a -CH3 group, and preferably a hydrogen atom.

[0026] In a preferred embodiment, R1 in formula I can be a hydrogen atom.

[0027] [R2 in Formula I] In a preferred embodiment, R2 in formula I can be a group selected from a hydrogen atom, a methyl group, and an ethyl group.

[0028] In a preferred embodiment, R2 in formula I can be a methyl group.

[0029] [Q1 and Q2 in Formula I] In a preferred embodiment, Q1 in Formula I is A phosphate group formed together with the O attached to Q1; Nucleotides or nucleic acids linked via a phosphodiester bond formed by a phosphate group formed together with an O attached to Q1; and A protecting group selected from: Trityl group, monomethoxytrityl group, dimethoxytrityl group, trimethoxytrityl group, trimethylsilyl group, triethylsilyl group, t-butyldimethylsilyl group, acetyl group, benzoyl group; It can be a group selected from the group consisting of:

[0030] In a preferred embodiment, Q2 in Formula I is A phosphate group formed together with the O attached to Q2; Nucleotides or nucleic acids linked via a phosphodiester bond formed by a phosphate group formed together with an O attached to Q2; and A protecting group selected from: 2-cyanoethyl-N,N-dialkyl(C1-C4) phosphoramidite group, methylphosphonamidite group, ethylphosphonamidite group, oxazaphospholidine group, thiophosphite group, TEA salt of -PH(=O)OH, DBU salt of -PH(=O)OH, TEA salt of -PH(=S)OH, DBU salt of -PH(=S)OH; It can be a group selected from the group consisting of:

[0031] The 2-cyanoethyl-N,N-dialkyl(C1-C4) phosphoramidite group has the following structure:

[0032] JPEG2025116757000004.jpg18170

[0033] The R and R' groups that form the dialkyl group can each be a C1 to C4 alkyl group. Examples of such 2-cyanoethyl-N,N-dialkyl(C1 to C4)phosphoramidite groups include a 2-cyanoethyl-N,N-dimethylphosphoramidite group, a 2-cyanoethyl-N,N-diethylphosphoramidite group, and a 2-cyanoethyl-N,N-diisopropylphosphoramidite group.

[0034] The methylphosphonamidite group has the following structure:

[0035] JPEG2025116757000005.jpg19170

[0036] The R and R' groups can each be a hydrogen atom or a C1 to C4 alkyl group.

[0037] The ethylphosphonamidite group has the following structure:

[0038] JPEG2025116757000006.jpg19170

[0039] The R and R' groups can each be a hydrogen atom or a C1 to C4 alkyl group.

[0040] An oxazaphospholidine group has the following structure:

[0041] JPEG2025116757000007.jpg40170

[0042] In the above structure, substitution products in which hydrogen atoms are substituted with C1 to C4 alkyl groups are also included.

[0043] The thiophosphite group has the following structure:

[0044] JPEG2025116757000008.jpg38170

[0045] In the above structure, substitution products in which hydrogen atoms are substituted with C1 to C4 alkyl groups are also included.

[0046] The TEA salt of -PH(=O)OH and the TEA salt of -PH(=S)OH are salts of triethylamine (TEA).

[0047] The DBU salt of -PH(=O)OH and the DBU salt of -PH(=S)OH are the respective salts of diazabicycloundecene (DBU).

[0048] In a preferred embodiment, Q1 can be a nucleotide or nucleic acid linked via a phosphodiester bond formed by the phosphate group formed together with the O attached to Q1.

[0049] In a preferred embodiment, Q1 can be the above-mentioned protecting group, preferably a dimethoxytrityl group, a trityl group, a monomethoxytrityl group, or a trimethoxytrityl group, and particularly preferably a dimethoxytrityl group.

[0050] In a preferred embodiment, Q2 can be a nucleotide or nucleic acid linked via a phosphodiester bond formed by the phosphate group formed together with the O attached to Q2.

[0051] In a preferred embodiment, Q2 can be the above-mentioned protecting group, preferably a 2-cyanoethyl-N,N-dialkyl(C1-C4) phosphoramidite group, an oxazaphosphoridine group, or a thiophosphite group, and particularly preferably a 2-cyanoethyl-N,N-diisopropylphosphoramidite group.

[0052] [Sugar analog backbone structure in formula I] In a preferred embodiment, the compound of formula I is represented by the following formula X:

[0053] JPEG2025116757000009.jpg54170

[0054] The backbone structure represented by the formula:

[0055] JPEG2025116757000010.jpg63170

[0056] D-threoninol structure represented by: The following expression:

[0057] JPEG2025116757000011.jpg67170

[0058] or an L-threoninol structure represented by The following expression:

[0059] JPEG2025116757000012.jpg62170

[0060] The serinol structure can be represented by the following formula:

[0061] [Photoreactive nucleoside analogs] The compound represented by the above formula I is a modified nucleic acid in which a photoresponsive nucleoside analogue, in which the sugar backbone of a ribose (or deoxyribose) structure in a natural nucleoside is replaced with the backbone structure shown in the above formula I, is linked to the base sequence of a nucleic acid by a phosphodiester bond via the Q1 and Q2 groups.

[0062] According to the present invention, a compound that is a modified nucleic acid into which such a photoreactive nucleoside analogue has been introduced can be used as a T- and U-selective photocrosslinker. The present invention includes precursors that can be used to synthesize such T- and U-selective photocrosslinkers, for example, precursors protected by protecting groups. That is, the present invention includes an embodiment of a reagent for producing modified nucleic acids. To be used as a reagent for producing modified nucleic acids, it is sufficient to make it in a form that can be used by known nucleic acid synthesis means, and it can be made into a reagent for synthesizing modified nucleic acids (monomer for synthesizing modified nucleic acids) that can be used by, for example, the phosphoramidite method and the H-phosphonate method.

[0063] [Photocrosslinking] In a preferred embodiment, the compound of Formula I has a pyranocarbazole moiety capable of forming a crosslink by photoreaction. When the compound of Formula I is formed as a single-stranded modified nucleic acid, it can form a double helix with a complementary single-stranded nucleic acid, and the pyranocarbazole moiety can form a crosslink by photoreaction, resulting in the formation of a photocrosslink (photocrosslink) between the strands of the double helix.

[0064] In a preferred embodiment, when the compound of Formula I is used as a single-stranded nucleic acid, it can hybridize with a complementary single-stranded nucleic acid to form a double helix. In forming a double helix, the nucleobase at the position where it should form a base pair with the pyranocarbazole structure moiety in the complementary strand is not particularly restricted and can be freely selected. When the formed double helix is irradiated with light, a crosslink can be formed between the nucleic acid strands forming the double helix by a photoreaction. This photocrosslink is formed between the pyranocarbazole structure and a nucleobase located one base away from the position of the pyranocarbazole structure moiety as the nucleobase in the complementary strand, at a position where it should form a base pair with the pyranocarbazole structure moiety in the complementary strand. In other words, this photocrosslink is formed between the pyranocarbazole structure and a nucleobase located one base away from the nucleobase located one base away from the position of the pyranocarbazole structure moiety in the complementary strand, at a position where it should form a base pair with the pyranocarbazole structure moiety in the complementary strand, at a position where it should form a base pair with the pyranocarbazole structure moiety in the complementary strand.

[0065] [Base specificity of photocrosslinking] In a preferred embodiment, the pyranocarbazole structure of the compound of formula I can form a photocrosslink with thymine (T) and uracil (U). In a preferred embodiment, the compound of formula I is a compound ( MEP D).

[0066] The present inventors have previously discovered compounds similar to the compound of formula I, which have a methylpyranocarbazole structure as the base moiety and a deoxyribose structure as the sugar skeleton moiety ( MEP We have investigated the photocrosslinkability of modified nucleic acids containing K. MEP It was found that modified nucleic acids containing K have specificity in that they form photocrosslinks with natural nucleic acid bases, cytosine, uracil, and thymine, but do not form photocrosslinks with guanine and adenine. MEPThe specificity of modified nucleic acids containing K can be said to be the property of forming photocrosslinks with bases having a pyrimidine ring but not with bases having a purine ring. It has been thought that such specificity is achieved by having a pyranocarbazole structure as the base moiety.

[0067] However, according to the discovery of the present invention, the compound of formula I, although having a pyranocarbazole structure as the base moiety, not only does not form photocrosslinks with guanine and adenine, which are bases having a purine ring, but also does not form photocrosslinks with cytosine (C), which is a base having a pyrimidine ring. On the other hand, according to the discovery of the present invention, the compound of formula I forms photocrosslinks with thymine (T) and uracil (U). That is, according to the present invention, the compound of formula I has the specificity of forming photocrosslinks with thymine (T) and uracil (U), which are natural nucleobases, but not with cytosine (C), guanine (G), and adenine (A).

[0068] Thus, contrary to the conventional common technical knowledge that it was believed that the base moiety (pyranocarbazole structure) itself determines the base specificity of photocrosslinking, the present inventors modified the sugar structure moiety to modify the base properties of photocrosslinking, thereby realizing a T- and U-selective photocrosslinking agent, thereby arriving at the present invention.

[0069] That is, the present invention also provides a T- and U-selective photocrosslinking reagent comprising a compound represented by formula I.

[0070] Therefore, in a preferred embodiment, the present invention also relates to a method for selectively detecting T and U, which comprises a step of forming a photocrosslink selectively with T or U among pyrimidine bases C, T, and U using a compound represented by formula I, without forming a photocrosslink with C.

[0071] That is, the present invention also relates to an agent for selectively detecting T and U, and a reagent for selectively detecting T and U, which comprise a compound represented by formula I.

[0072] In a preferred embodiment, the present invention also relates to a method for selectively forming a photocrosslink between a compound of formula I and T or U, the method comprising the step of selectively forming a photocrosslink between a compound of formula I and T or U.

[0073] That is, the present invention also provides a reagent for selectively producing photocrosslinks with T and U, which comprises a compound represented by formula I.

[0074] [Sequence selectivity of T- and U-selective photocrosslinkers] In a preferred embodiment, a T- and U-selective photocrosslinker comprising a compound represented by formula I can be converted into a photocrosslinkable single-stranded modified nucleic acid having the structure of formula I. In a preferred embodiment, a photocrosslinkable single-stranded modified nucleic acid having the structure of formula I can be hybridized with a nucleic acid having a base sequence complementary to the modified nucleic acid to form a double helix, and then photocrosslinked. This allows the photocrosslinking reaction to occur only with a specific target base sequence. In other words, the T- and U-selective photocrosslinker of the present invention can be imparted with extremely high base sequence selectivity by sequence design as desired.

[0075] That is, the present invention also relates to a photocrosslinking agent specific to a base sequence containing T or U, which comprises a compound represented by formula I, a photocrosslinkable probe specific to a base sequence containing T or U, and a reagent for producing a photocrosslink specific to a base sequence containing T or U.

[0076] [Light wavelength] The wavelength of the light irradiated for photocrosslinking may be, for example, in the range of 360 to 440 nm, preferably 380 to 420 nm, more preferably 390 to 410 nm, or 380 to 440 nm, or 360 to 420 nm, and light having a wavelength of 400 nm or more is particularly preferred. In a preferred embodiment, a single-wavelength laser beam within these wavelength ranges can be used.

[0077] Thus, in the present invention, photocrosslinking can be formed by irradiation with light having a wavelength in the visible light range. Conventional photoreactive crosslinking agents require irradiation with light having a wavelength shorter than this range. According to the present invention, photocrosslinking can be formed by irradiation with light having a wavelength longer than that of conventional photoreactive crosslinking agents, which is advantageous in that adverse effects of light irradiation on nucleic acids and cells can be minimized.

[0078] [Photoreaction time] Photocrosslinking according to the present invention proceeds extremely rapidly. For example, while psoralen, a known photoreactive compound, requires several hours (irradiation with 350 nm light), photocrosslinking can be achieved by irradiation with light of a much longer wavelength, for example, in as little as 30 seconds or less (irradiation with 400 nm light) or as little as 20 seconds or less (irradiation with 400 nm light). That is, when the photocrosslinking agent according to the present invention is used, the photoreaction can proceed and photocrosslinking can be formed by irradiation with light for, for example, 0.1 to 30 seconds, 0.1 to 20 seconds, 0.1 to 15 seconds, 0.1 to 10 seconds, or 0.1 to 5 seconds, for example, 0.5 to 30 seconds, 0.5 to 20 seconds, 0.5 to 15 seconds, 0.5 to 10 seconds, or 0.5 to 5 seconds, for example, 1 to 30 seconds, 1 to 20 seconds, 1 to 15 seconds, 1 to 10 seconds, or 1 to 5 seconds.

[0079] [Photoreaction temperature] In a preferred embodiment, in order to allow the photocrosslinking reaction to proceed, light irradiation can be carried out at a temperature in the range of, for example, 0 to 50°C, alternatively 0 to 40°C, alternatively 0 to 30°C, alternatively 0 to 20°C, alternatively 0 to 10°C, or alternatively 0 to 5°C.

[0080] As described above, the present invention is advantageous in that photocrosslinking can be formed by light irradiation under temperature conditions within a living body or temperature conditions suitable for culturing or storing cells or nucleic acids, thereby minimizing adverse effects on cells or nucleic acids due to temperature changes caused by photocrosslinking.

[0081] [Photoreaction conditions] In a preferred embodiment, photocrosslinking utilizes a photoreaction, and therefore is not subject to any particular restrictions on pH, salt concentration, etc., and can be carried out by light irradiation in a solution with a pH and salt concentration that allows biopolymers such as nucleic acids to exist stably. [Example]

[0082] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the examples illustrated below.

[0083] [ MEP D and MEP K] The photoresponsive artificial nucleoside of the present invention MEP For the synthesis and comparison experiment of D MEP K was synthesized as described below, and then oligonucleotides containing these were synthesized and experiments were carried out.

[0084] [Example 1] [ MEP Synthesis of K] Figure 1 shows: MEP The synthesis flow of K is shown below. The conditions for (a) to (e) in Figure 1 are as follows: (a) Ethyl acetoacetate,H2SO4,EtOH,90℃,2h, (b) KOH,TDA-1,Chlorosugar,CH3CN,rt,8h (c) NaOCH3,CH3OH,CHCl3,rt,10h. (d) DMTrCl,DMAP,Pyridine,rt,24h. (e) (iPr2N)2PO(CH2)2CN,tetrazole,CH3CN,rt,4h.

[0085] JPEG2025116757000013.jpg103170

[0086] [Synthesis of Compound 2] Compound 1 (5.00 g, 27.3 mmol), ethyl acetoacetate (3.79 mL, 30.0 mmol), and EtOH (30 mL) were placed in a recovery flask and stirred on ice. Concentrated H2SO4 (7 mL) was added dropwise. EtOH (10 mL) was added and stirred at 90°C for 2 hours. After confirming the disappearance of the raw materials by TLC (CHCl3:MeOH = 9:1), stirring was stopped. Acetone was added to the solution and recrystallization was carried out. The compound obtained by recrystallization was filtered, washed with chloroform, and then dried to obtain compound 2 (4.90 g, 19.6 mmol, 72%). 1 H-NMR(400MHz,DMSO-d6)δ11.64(s,1H),8.53(s,1H),8.25(d,1H,J=7.68Hz),7.53(d,1H,8.00 Hz),7.44(t,1H,J=7.56Hz),7.40(s,1H),7.24(t,1H,J=7.36Hz),6.26(s,1H),2.59(s,3H)SALDI-MS:Calc'd for C 16 H 11 NNaO2[M+Na] + =272.0681,Found 272.0682.

[0087] JPEG2025116757000014.jpg52170

[0088] [Synthesis of Compound 3] Compound 2 (300 mg, 1.20 mmol) and KOH (260 mg, 10.1 mmol) were added and the atmosphere was replaced with N2. CH3CN (50 mL) and TDA-1 (250 μL) were added and stirred. After 30 minutes, chlorosugar (1.17 g, 3.00 mmol) was added and stirred at room temperature for 6 hours. The reaction was stopped by checking with TLC (CHCl3). The precipitate was removed by suction filtration, and the filtrate was evaporated to remove the solvent. 1H-NMR(400MHz,DMSO-d6)δ11.67(s,1H),8,47(s,1H),8,17(dt,2H,J=11.4Hz),7.5 0-7.43(m,2H),7.25(dt,1H,J=8.54Hz),6.33(d,1H,J=4.75Hz),SALDI-MS:Calc'd for C 16 H 11 NNaO2[M+Na] + =272.0681,Found 272.0682.

[0089] JPEG2025116757000015.jpg70170

[0090] [Synthesis of Compound 4] Compound 3 (1.58 g, 3.82 mmol) was placed in a recovery flask, and MeOH (40 mL), CHCl3 (30 mL), and NaOMe (1.00 g) were added. The mixture was stirred at room temperature for 6 hours. The solvent was then removed using an evaporator, and the residue was purified by column chromatography (CHCl3:MeOH = 9:1). After purification, the residue was dried to obtain compound 4 (360 mg, 0.985 mmol, 82%). 1 H-NMR(400MHz,DMSO-d6)δ8.62(d,1H,3.04Hz),8.30(d,1H,7.68Hz),7.89-7.80 (m,2H),7.48(t,1H,7.78Hz),7.31(t,1H,J=5.96Hz),6.71(t,1H,J=7.8Hz),6.3 0(s,1H),5.42(s,1H),5.16(s,1H),4.50(d,1H,3.44Hz),3.89(d,1H,3.72Hz),3 .78(s,2H),2.59(s,3H),2.17-2.12(m,1H),1.14-1.06(m,1H)SALDI-MS:Calc'd for C 21 H 19 NNaO5[M+Na] + =388.1155,Found 388.1152.

[0091] JPEG2025116757000016.jpg69170

[0092] [Synthesis of Compound 5] Compound 4 (375 mg, 1.03 mmol) and DMAP (12.3 mg, 0.101 mmol) were added to a recovery flask, and the atmosphere was replaced with N2. Dry pyridine (10 mL) was then added in an ice bath. DMTrCl (525 mg, 1.55 mmol) was then added. The mixture was then stirred at room temperature for 24 hours. After confirming the disappearance of the raw materials by TLC (CHCl3:MeOH = 9:1), the reaction solution was concentrated using an evaporator. Azeotropic distillation was performed several times with Toluene. The product was then purified by column chromatography (CHCl3:MeOH = 19:1) to yield a white powder (128 mg, 0.192 mmol, 18.6%). 1 H-NMR(400MHz,DMSO-d6)δSALDI-MS:Calc'd for C 21 H 19 NNaO5[M+Na] + =,Found.

[0093] JPEG2025116757000017.jpg77170

[0094] [Synthesis of Compound 6] To compound 5 (129 mg, 0.192 mmol) in a recovery flask, CHCl (4.17 mL) was added under N. Then, 0.25 M tetrazole (800 μL, 0.211 mmol) and (iPrN)PO(CH)CN (121 μL, 0.384 mmol) were added dropwise and stirred at room temperature for 1 hour. The reaction was monitored by TLC (CHCl:MeOH = 9:1) and stirring was stopped. The reaction solution was transferred to an analytical funnel and washed several times with NaCl. The organic layer was then dried with NaSO, and the solvent was removed using an evaporator to give compound 6 (95.1 mg, 0.112 mmol, 58.3%). 1 H-NMR(400MHz,DMSO-d6)δSALDI-MS:Calc'd for C 51 H 54 N3NaO8P [M+Na] + =890.3541,Found 890.3544.

[0095] JPEG2025116757000018.jpg77170

[0096] [Example 2] [ MEP Synthesis of D] Figure 2 shows: MEP The synthesis flow up to the amidite form of D is shown below.

[0097] JPEG2025116757000019.jpg106170

[0098] [Synthesis of Compound 13] The synthesis of methylpyranocarbazole (compound 2) is as follows: MEP The synthesis was carried out in the same manner as in K.

[0099] Methylpyranocarbazole was added to a recovery flask and the atmosphere was purged with nitrogen. DMF anhydrate (20 mL) was added and dissolved. NaH (187.2 mg, 4.681 mmol, 1.1 eq.) was added in an ice bath and stirred for 1 hour. Ethyl bromoacetate (943.8 μL, 8.511 mmol, 2 eq.) was added dropwise in an ice bath. The reaction was stirred at room temperature for 6 hours, and completion was confirmed by TLC (CHCl3:MeOH = 95:5). The reaction was quenched with water in an ice bath. Extraction with AcOEt was performed, and the solvent was removed using an evaporator. Purification by column chromatography (CHCl3:MeOH = 99:1) yielded the desired product as a white solid.

[0100] JPEG2025116757000020.jpg52170

[0101] [Synthesis of compound 14] Compound 13 (454 mg, 1.356 mmol) was placed in a recovery flask and dissolved in a THF / MeOH / HO mixed solvent (3:2:1, 12 mL). NaOH (271.2 mg, 6.780 mmol, 5 eq.) was added and the mixture was stirred for 6 hours. Completion of the reaction was confirmed by TLC (CHCl3:MeOH = 95:5), and the mixture was quenched by adding 5 M aqueous HCl. The pH of the reaction solution was adjusted to approximately 2, saturated brine was added, and the mixture was extracted with THF. The target product was obtained as a pale pink solid (490 mg, 118%).

[0102] JPEG2025116757000021.jpg54170

[0103] [Synthesis of Compound 15] Compound 14 (490 mg, 1.596 mmol) was added to a recovery flask and the atmosphere was purged with nitrogen. Ultra-dehydrated DMF (6 mL) was added to dissolve the starting material. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (305.9 mg, 1.596 mmol, 1 eq.) was added and stirred for 10 minutes. After confirming the precipitation of a solid, 1-hydroxybenzotriazole (215.7 mg, 1.596 mmol, 1 eq.) was added and stirred for 10 minutes. After confirming the disappearance of the precipitated solid, D-threoninol (204.4 mg, 1.915 mmol, 1.2 eq.) was added and stirred overnight. Completion of the reaction was confirmed by TLC (CHCl3:MeOH = 95:5), and saturated brine cooled in an ice bath was added to precipitate a solid. The product was collected by suction filtration to give the desired product as a light brown solid (532.9 mg, 85%).

[0104] JPEG2025116757000022.jpg72170

[0105] [Synthesis of Compound 16] Compound 15 (532.9 mg, 1.352 mmol) was added to a recovery flask, dried under vacuum, and then purged with nitrogen. Dry pyridine (15 mL) was added, followed by DMAP (26.2 mg, 0.3245 mmol, 0.24 eq.) in an ice bath. DMTrCl (502.8 mg, 1.487 mmol, 1.1 eq.) was added in an ice bath and stirred overnight. Completion of the reaction was confirmed by TLC (CHCl3:MeOH = 9:1), and the mixture was quenched by adding methanol in an ice bath. The solvent was removed by evaporation, and the mixture was extracted with CHCl3. Purification by column chromatography (AcOEt:Hexane = 3:2, TEA 1%) afforded the desired product as a pale yellow solid (684.1 mg, 73%).

[0106] JPEG2025116757000023.jpg71170

[0107] [Synthesis of compound 17] Compound 16 (531.4 mg, 0.7632 mmol) was added to a recovery flask, purged with nitrogen, and then dissolved in dry MeCN (8 mL). 0.25 M Tetrazole (6.1 mL, 1.526 mmol, 2 eq.) and (iPrN)PO(CH)CN (499.8 μL, 1.526 mmol, 2 eq.) were added dropwise and the mixture was stirred for 2 hours. Completion of the reaction was confirmed by TLC (AcOEt:Hexane = 3:2, TEA 1%), and the reaction solution was extracted with deacetylated AcOEt. The desired product was obtained as a pale yellow solid (694.5 mg, quant.).

[0108] JPEG2025116757000024.jpg68170

[0109] [Example 3] [ MEP Synthesis of oligonucleotides containing D] Oligonucleotides with various base sequences were synthesized using an oligo synthesizer.

[0110] After the synthesis reaction using the oligo synthesizer was completed, cleavage was performed twice using 28% aqueous ammonia (1 mL) for 30 minutes, followed by deprotection at 65 °C for 4 hours. The solvent was then removed using a speed vac, and the residue was dissolved in 100 μL of purified water and purified by HPLC. Analysis by MALDI-TOF-MS was then performed to identify the target product.

[0111] [Photocrosslinking test] Oligo DNA MEP After introducing D, a photocrosslinking test was carried out as described below.

[0112] [Example 4] [ MEP Photocrosslinking of D with thymine] MEP To verify the photocrosslinking between D and thymine, the following experiment was carried out.

[0113] ODN(A MEP D) The final concentrations were adjusted to 10 μM, ODN (GT) 10 μM, and dU (50 μM) with 50 mM cacodylic acid buffer (pH 7.4) containing 100 mM NaCl, followed by annealing and leaving at 4°C.

[0114] Thereafter, the film was irradiated with light at 400 nm and 4°C using a UV-LED (OmniCure, LX405-S).

[0115] FIG. 3A shows the base sequences of the oligo DNAs used and an explanatory diagram showing the flow of the photocrosslinking experiment. MEP D forms a photocrosslink with the base (T) adjacent to its 3' end, rather than with the base (G) at the complementary position in the complementary strand.

[0116] FIG. 3B is a chart showing the results of UPLC analysis of this photocrosslinking experiment.

[0117] As shown in Figure 3B, the peak of the raw material decreased depending on the light irradiation time, and the peak of the photo-crosslinked product was confirmed. The crosslinking reaction had progressed to over 80% in 10 seconds. MEPIt was confirmed that D photocrosslinks with thymine in an extremely short time.

[0118] [Example 5] [ MEP Photocrosslinking of D with uracil] next MEP The photocrosslinking of D with uracil was verified. MEP The experiment was carried out using the same procedure as in the experiment verifying the photocrosslinking between D and thymine.

[0119] FIG. 4A shows the base sequences of the oligo DNAs used and an explanatory diagram showing the flow of the photocrosslinking experiment. MEP D forms a photocrosslink not with the base (G) at the complementary position in the complementary strand, but with the base (U) adjacent to it on the 3'-terminal side.

[0120] FIG. 4B is a chart showing the results of UPLC analysis of this photocrosslinking experiment.

[0121] As shown in Figure 4B, the peak of the raw material decreased depending on the light irradiation time, and the peak of the photo-crosslinked product was confirmed. The crosslinking reaction had progressed to over 80% in 10 seconds. MEP It was confirmed that D photocrosslinks with uracil in an extremely short time.

[0122] [Example 6] [ MEP Photocrosslinking of D with cytosine] next MEP The photocrosslinking reaction between ODN(G) and uracil was also verified. MEP K),ODN(GC) MEP The experiment was carried out using the same procedure as in the experiment verifying the photocrosslinking between D and thymine.

[0123] As a result, MEP Photocrosslinking of D with cytosine did not proceed at all.

[0124] [ MEP Regarding the results of photocrosslinking experiments between D and each base] From the above results, MEPIt was revealed that D photocrosslinks with thymine and uracil, but not with cytosine.

[0125] [Example 7] [ MEP D and MEP Photocrosslinking rate of K MEP Photocrosslinking of D with thymine, MEP The following experiment was carried out to verify the photocrosslink formation between K and thymine.

[0126] MEP Photocrosslinking experiments between D and thymine, and MEP The photocrosslinking experiment between K and thymine was carried out as described in Example 4. MEP The experiment was carried out using the same procedure as in the experiment verifying the photocrosslinking between D and thymine.

[0127] Figure 5A shows the base sequence of the oligo DNA used and an explanatory diagram showing the flow of this photocrosslinking experiment. X in the sequence represents: MEP D or MEP Indicates K. MEP D and MEP K forms a photocrosslink not with the base (G) at the complementary position in the complementary strand, but with the base (T) adjacent to it on the 3'-terminal side.

[0128] FIG. 5B is a chart showing the results of UPLC analysis of this photocrosslinking experiment.

[0129] Figure 5C is a graph showing the results of this photocrosslinking experiment. As shown in the graph of Figure 5C, when photocrosslinking was performed by irradiation with light at 400 nm, MEP In D MEP Compared to K, a high crosslinking rate was achieved with extremely short light irradiation times. MEP In D, a crosslinking rate of over 90% was achieved with 30 seconds of light irradiation. MEP In the case of K, even after 30 seconds of light irradiation, the crosslinking rate was less than 50%. MEP In D, a photocrosslinking rate of 20% or more was achieved with 1 second of light irradiation, a photocrosslinking rate of 50% or more was achieved with 2 seconds of light irradiation, and a photocrosslinking rate of 70% or more was achieved with 10 seconds of light irradiation.MEP In the case of K, the photocrosslinking rate was 5% or less after 1 second of light irradiation, and 20% or less after 5 seconds of light irradiation. [Industrial Applicability]

[0130] The present invention provides a nucleic acid photoreaction technology that achieves both high photoreactivity and selective photoreactivity to thymine (T) and uracil (U) upon irradiation with visible light, making it an industrially useful invention.

Claims

1. A T- and U-selective photocrosslinker comprising a compound represented by the following formula I: (However, in Formula I, R is a C1-C3 alkyl group, a C1-C3 halogenated alkyl group, a substituted or unsubstituted phenyl group, or a substituted or unsubstituted cyclohexyl group; R1 is a group selected from the group consisting of a hydrogen atom, a halogen atom, an —OH group, an amino group, a nitro group, a methyl group, a fluorinated methyl group, an ethyl group, a fluorinated ethyl group, and a C1-C3 alkylsulfanyl group; R2 is a group selected from a hydrogen atom, a methyl group, and an ethyl group; Q 1 teeth, Q 1 a phosphate group formed together with the O attached to Q 1 Nucleotides or nucleic acids linked via a phosphodiester bond formed by a phosphate group formed together with an O bound to A protecting group selected from: a trityl group, a monomethoxytrityl group, a dimethoxytrityl group, a trimethoxytrityl group, a trimethylsilyl group, a triethylsilyl group, a t-butyldimethylsilyl group, an acetyl group, a benzoyl group; is a group selected from the group consisting of Q 2 teeth, Q 2 a phosphate group formed together with the O attached to Q 2 Nucleotides or nucleic acids linked via a phosphodiester bond formed by a phosphate group formed together with an O bound to A protecting group selected from: 2-cyanoethyl-N,N-dialkyl(C1-C4) phosphoramidite group, methylphosphonamidite group, ethylphosphonamidite group, oxazaphospholidine group, thiophosphite group, TEA salt of —PH(═O)OH, DBU salt of —PH(═O)OH, TEA salt of —PH(═S)OH, DBU salt of —PH(═S)OH; is a group selected from the group consisting of:

2. The T- and U-selective photocrosslinker according to claim 1, wherein R2 is a methyl group.

3. 2. The T- and U-selective photocrosslinker according to claim 1, wherein R is a C1-C3 alkyl group.

4. R1 is a hydrogen atom, 2. The T- and U-selective photocrosslinker according to claim 1, wherein R is a methyl group.

5. A method for selectively detecting T and U, comprising a step of forming a photocrosslink selectively with T or U among pyrimidine bases C, T, and U with a compound represented by formula I according to any one of claims 1 to 4, without forming a photocrosslink with C.

6. The step of selectively forming photocrosslinks to T or U comprises: The method of claim 5, wherein the method is carried out by irradiating with light for 30 seconds or less.

7. The step of selectively forming photocrosslinks to T or U comprises: The method according to claim 5, wherein the method is carried out by irradiating with light having a wavelength of 360 to 440 nm.

8. The step of selectively forming photocrosslinks to T or U comprises: The method according to claim 5, which is a step of selectively forming photocrosslinks with T or U contained in a nucleic acid that has formed a complementary strand with the compound represented by formula I.

9. A method for selectively producing a photocrosslink between a compound represented by formula I according to any one of claims 1 to 4 and T or U, the method comprising the step of selectively forming a photocrosslink between a compound represented by formula I according to any one of claims 1 to 4 and T or U.

10. The step of selectively forming photocrosslinks to T or U comprises:

10. The method of claim 9, wherein the method is carried out by irradiating with light for 30 seconds or less.

11. The step of selectively forming photocrosslinks to T or U comprises: The method according to claim 9, wherein the method is carried out by irradiating with light having a wavelength of 360 to 440 nm.

12. The step of selectively forming photocrosslinks to T or U comprises: The method according to claim 9, which is a step of selectively forming photocrosslinks with T or U contained in a nucleic acid that has formed a complementary strand with the compound represented by formula I.

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