JP7313619B2 - Compound, fluorescent label introduction agent, and fluorescent label introduction method - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law Presented on pages 392-393 of the 44th International Symposium on Nucleic Acid Chemistry published on November 14, 2017, 1st Annual Meeting of the Nucleic Acid Chemistry Society of Japan

TECHNICAL FIELD The present invention relates to a compound, a fluorescent label-introducing agent, and a fluorescent label-introducing method, and more particularly to a compound into which a fluorescent label that exhibits fluorescence when incorporated into a cell has been introduced, and a fluorescent label-introducing agent and a fluorescent label-introducing method for introducing such a fluorescent label into the compound.

Today, methods for synthesizing nucleic acids such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) have been established, and oligonucleotides, which are relatively short-chain nucleic acids, are synthesized using an automatic synthesizer. The synthetic oligonucleotides thus obtained are being routinely used for biochemical research, medical diagnosis, and the like. One useful application of synthetic oligonucleotides is nucleic acid medicine. Synthetic oligonucleotides are used, for example, in antisense methods (see Non-Patent Document 1) to inhibit the synthesis of disease-causing proteins by introducing into cells oligonucleotides having base sequences complementary to mRNA transcribed from genes encoding disease-causing proteins.

By the way, such DNA and RNA have one or more phosphate groups, and therefore have high polarity and poor cell membrane permeability. These points are one of the problems of nucleic acid drugs. In order to deal with this problem, a method of protecting the OH group contained in the phosphate group with a substituent group that can be detached by an intracellular enzyme to improve fat solubility is sometimes used. A compound modified with such a substituent becomes a prodrug that exhibits activity as a nucleic acid drug by undergoing deprotection by intracellular enzymes after passing through the cell membrane (see, for example, Non-Patent Document 2).

As described above, various attempts have been made to incorporate molecules such as synthesized DNA and RNA into cells and use them as nucleic acid medicines. As a method for evaluating the results of such attempts, a method for confirming the extent to which synthesized molecules are taken up by cells is also required. As a general technique for this purpose, a fluorescent group is attached to a synthesized molecule, labeled, added to a cell culture medium, and fluorescence is observed when cells are observed with a fluorescence microscope. With this method, molecules taken up into cells emit fluorescence, so that it is possible to directly observe whether or not molecules have been taken up into cells.

However, with this method, the labeled compound that remains unincorporated into the cells becomes a background, and there is a possibility that the accuracy of observation with a fluorescence microscope will decrease. In addition, it is not easy to distinguish between a labeling compound that is simply bound to the cell membrane and a labeling compound that has been taken up into cells.

A. M. Belikova, V.; F. Zarytova, N.; I. Grinrva, Tetrahedron Lett. , 1967, 3557-3562 P. P. Virta, H.; Lonnberg, Current Medicinal Chemistry, 2006, 13, 3441-3465 Saneyoshi et al. , Bioconjugate Chem, 2016, 2149

The present invention has been made in view of the above circumstances, and aims to provide a compound having a fluorescent label that does not show fluorescence outside the cell but emits fluorescence when taken into the cell, and a fluorescent label introduction agent for introducing such a fluorescent label.

As a result of intensive studies to solve the above problems, the present inventors have found that when a specific protective group is bound to the nitrogen atom of a fluorescent unit having a cyclic skeleton that exhibits fluorescence emission and has a nitrogen atom to cause quenching, the fluorescent unit, which is quenched outside the cell, is deprotected by the action of intracellular substances such as glutathione and enzymes and emits light when taken into the cell. By utilizing such an action, the present inventors succeeded in creating a fluorescent labeling molecule that does not emit light outside the cell but emits light when taken into the cell. The present invention has been completed based on the above findings, and provides the following.

（上記一般式（１）中、Ａは、細胞膜を通して細胞内へ取り込まれ得る生体物質又はその誘導体の構造を含む１価の基であり、ＦＬは、下記化学式（２）又は（３）で示す骨格をもつ蛍光ユニットであり、Ｌは、ＡとＦＬとを結合する２価の基であり、Ｘは、単結合又は－Ｃ（＝Ｏ）Ｏ－であり、波線で表す結合は、上記化学式（２）又は（３）に含まれる窒素原子への結合であり、Ａｒは、パラ位又はオルト位に結合を有する２価の芳香環であり、Ｒは、ニトロ基又は－Ｓ－Ｓ－Ｒ^１であり、Ｒ^１は、一価の基であり、ｎは、１～３の整数である。）

（上記化学式（２）における、アミノ基、イミノ基及びカルボキシル基の１つに上記Ｌが結合し、アミノ基及びイミノ基の少なくとも１つに上記Ｘが１つ又は２つ結合し、上記化学式（３）における、２つのアミノ基の１つに上記Ｌが結合し、残りのアミノ基に上記Ｘが１つ又は２つ結合する。）
(1) The present invention is a compound represented by the following general formula (1).

（上記一般式（１）中、Ａは、細胞膜を通して細胞内へ取り込まれ得る生体物質又はその誘導体の構造を含む１価の基であり、ＦＬは、下記化学式（２）又は（３）で示す骨格をもつ蛍光ユニットであり、Ｌは、ＡとＦＬとを結合する２価の基であり、Ｘは、単結合又は－Ｃ（＝Ｏ）Ｏ－であり、波線で表す結合は、上記化学式（２）又は（３）に含まれる窒素原子への結合であり、Ａｒは、パラ位又はオルト位に結合を有する２価の芳香環であり、Ｒは、ニトロ基又は－Ｓ－Ｓ－Ｒ ^１であり、Ｒ ^１は、一価の基であり、ｎは、１～３の整数である。）

(In the chemical formula (2), one of the amino group, the imino group and the carboxyl group is bound by the L, one or two Xs are bound to at least one of the amino group and the imino group, and the L is bound to one of the two amino groups in the chemical formula (3), and one or two Xs are bound to the remaining amino groups.)

（上記一般式（４）及び（５）中、Ａ、Ｌ、Ｘ、Ａｒ及びＲは、上記一般式（１）におけるものと同じである。）
( 2 ) Further, the present invention is a compound according to item (1) represented by the following general formula (4) or (5).

(In general formulas (4) and (5) above, A, L, X, Ar and R are the same as in general formula (1) above.)

（上記一般式（６）及び（７）中、Ａ、Ｌ、Ｘ及びＲは、上記一般式（１）におけるものと同じである。）
( 3 ) Further, the present invention is a compound according to item (1) or item (2) represented by the following general formula (6) or (7).

(In general formulas (6) and (7) above, A, L, X and R are the same as in general formula (1) above.)

（上記一般式（８）及び（９）中、Ａ、Ｌ及びＲ^１は、上記一般式（１）におけるものと同じである。上記一般式（１０）中、Ａ及びＬは、上記一般式（１）におけるものと同じである。）
( 4 ) The present invention also provides a compound according to any one of items (1) to ( 3 ) represented by any one of the following general formulas (8) to (10).

(In general formulas (8) and (9), A, L, and ^R1 are the same as in general formula (1). In general formula (10), A and L are the same as in general formula (1).)

( 5 ) The present invention also provides the compound according to any one of (1) to ( 4 ), wherein A is a nucleotide chain or an analogue thereof, or a peptide chain or an analogue thereof.

（上記一般式（１ａ）及び（１ｂ）中、ＦＬは、下記化学式（２）又は（３）で示す骨格をもつ蛍光ユニットであり、Ｌは、２価の基であり、Ｘは、単結合又は－Ｃ（＝Ｏ）Ｏ－であり、波線で表す結合は、上記化学式（２）又は（３）に含まれる窒素原子への結合であり、Ａｒは、パラ位又はオルト位に結合を有する２価の芳香環であり、Ｒは、ニトロ基又は－Ｓ－Ｓ－Ｒ^１であり、Ｒ^１は、一価の基であり、ｎは、１～３の整数である。上記一般式（１ｃ）中、ＦＬは、下記化学式（２）又は（３）で示す骨格をもつ蛍光ユニットであり、Ａ環は単環でも複数の環が縮合した構造でもよい三重結合を備えた環構造であり、Ａ環が複数の環の縮合した構造の場合、ＬからＡ環への単結合は縮合しているどの環へ結合してもよく、Ｌは、２価の基であり、Ｘは、単結合又は－Ｃ（＝Ｏ）Ｏ－であり、波線で表す結合は、前記化学式（２）又は（３）に含まれる窒素原子への結合であり、Ａｒは、パラ位又はオルト位に結合を有する２価の芳香環であり、Ｒは、ニトロ基又は－Ｓ－Ｓ－Ｒ^１であり、Ｒ^１は、一価の基であり、ｎは、１～３の整数である。）

（上記化学式（２）における、アミノ基、イミノ基及びカルボキシル基の１つに上記Ｌが結合し、アミノ基及びイミノ基の少なくとも１つに上記Ｘが１つ又は２つ結合し、上記化学式（３）における、２つのアミノ基の１つに上記Ｌが結合し、残りのアミノ基に上記Ｘが１つ又は２つ結合する。）
(6) The present invention also provides a fluorescent label introduction agent containing a compound represented by the following general formula (1a), (1b) or (1c).

（上記一般式（１ａ）及び（１ｂ）中、ＦＬは、下記化学式（２）又は（３）で示す骨格をもつ蛍光ユニットであり、Ｌは、２価の基であり、Ｘは、単結合又は－Ｃ（＝Ｏ）Ｏ－であり、波線で表す結合は、上記化学式（２）又は（３）に含まれる窒素原子への結合であり、Ａｒは、パラ位又はオルト位に結合を有する２価の芳香環であり、Ｒは、ニトロ基又は－Ｓ－Ｓ－Ｒ ^１であり、Ｒ ^１は、一価の基であり、ｎは、１～３の整数である。上記一般式（１ｃ）中、ＦＬは、下記化学式（２）又は（３）で示す骨格をもつ蛍光ユニットであり、Ａ環は単環でも複数の環が縮合した構造でもよい三重結合を備えた環構造であり、Ａ環が複数の環の縮合した構造の場合、ＬからＡ環への単結合は縮合しているどの環へ結合してもよく、Ｌは、２価の基であり、Ｘは、単結合又は－Ｃ（＝Ｏ）Ｏ－であり、波線で表す結合は、前記化学式（２）又は（３）に含まれる窒素原子への結合であり、Ａｒは、パラ位又はオルト位に結合を有する２価の芳香環であり、Ｒは、ニトロ基又は－Ｓ－Ｓ－Ｒ ^１であり、Ｒ ^１は、一価の基であり、ｎは、１～３の整数である。）

(In the chemical formula (2), one of the amino group, the imino group and the carboxyl group is bound by the L, one or two Xs are bound to at least one of the amino group and the imino group, and the L is bound to one of the two amino groups in the chemical formula (3), and one or two Xs are bound to the remaining amino groups.)

（上記一般式（４ａ）、（４ｂ）、（５ａ）及び（５ｂ）中、Ｌ、Ｘ、Ａｒ及びＲは、上記一般式（１ａ）におけるものと同じである。上記一般式（４ｃ）及び（５ｃ）中、Ａ環、Ｌ、Ｘ、Ａｒ及びＲは、前記一般式（１ｃ）におけるものと同じである。）
( 7 ) The present invention also provides a fluorescent label introduction agent according to item (6) represented by the following general formulas (4a), (4b), (4c), (5a), (5b), or (5c).

(In the general formulas (4a), (4b), (5a) and (5b), L, X, Ar and R are the same as in the general formula (1a). In the general formulas (4c) and (5c), the A ring, L, X, Ar and R are the same as in the general formula (1c).)

（上記一般式（６ａ）、（６ｂ）、（７ａ）及び（７ｂ）中、Ｌ、Ｘ及びＲは、上記一般式（１ａ）におけるものと同じである。上記一般式（６ｃ）及び（７ｃ）中、Ａ環、Ｌ、Ｘ及びＲは、前記一般式（１ｃ）におけるものと同じである。） (8) The present invention also provides a fluorescent label introduction agent according to item (6) or item (7) represented by the following general formula (6a), (6b), (6c), (7a), (7b) or (7c).

(In the general formulas (6a), (6b), (7a) and (7b), L, X and R are the same as in the general formula (1a). In the general formulas (6c) and (7c), the A ring, L, X and R are the same as in the general formula (1c).)

（上記一般式（８ａ）、（８ｂ）、（９ａ）及び（９ｂ）中、Ｌ及びＲ^１は、上記一般式（１ａ）におけるものと同じである。上記一般式（１０ａ）及び（１０ｂ）中、Ｌは、上記一般式（１ａ）におけるものと同じである。上記一般式（８ｃ）及び（９ｃ）中、Ａ環、Ｌ及びＲ^１は、前記一般式（１ｃ）におけるものと同じである。上記一般式（１０ｃ）中、Ａ環及びＬは、前記一般式（１ｃ）におけるものと同じである。）
( 9 ) The present invention also provides a fluorescent label introduction agent according to any one of items (6) to (8) represented by the following general formulas (8a), (8b), (8c), (9a), (9b), (9c), (10a), (10b), or (10c).

(In the general formulas (8a), (8b), (9a) and (9b), L and R ¹ are the same as in the general formula (1a). In the general formulas (10a) and (10b), L is the same as in the general formula (1a). In the general formulas (8c) and (9c), the A ring, L and R ¹ are the same as in the general formula (1c). In 0c), the A ring and L are the same as in the general formula (1c).)

( 10 ) The present invention also provides a method for introducing a fluorescent label, which comprises introducing an alkynyl group or an alkynylene group into a molecule to be labeled, and then reacting the molecule with a fluorescent label introducing agent containing a compound having an azide group among the compounds according to any one of items (6) to (9) .

( 11 ) The present invention also provides a method for introducing a fluorescent label, which comprises introducing an azide group into a molecule to be labeled, and then reacting the molecule with a fluorescent label introduction agent containing a compound having an alkynyl group or an A ring among the compounds according to any one of items (6) to (9) .

INDUSTRIAL APPLICABILITY According to the present invention, a compound having a fluorescent label that emits fluorescence when incorporated into a cell while not exhibiting fluorescence outside the cell, and a fluorescent label introduction agent for introducing such a fluorescent label are provided.

FIG. 1 is the fluorescence spectra for compound 13 without GSH, 10 mM GSH and 10 μM GSH. FIG. 2 is the fluorescence spectra for compound 20 at no GSH, 10 mM GSH and 10 μM GSH. FIG. 3 shows fluorescence spectral changes over time for compound 21 with and without nitroreductase and NADH. FIG. 4 shows fluorescence spectrum changes when fluorescence-labeled ODN1 is reacted with 10 mM GSH. FIG. 5 is a fluorescence microscopy image showing how fluorescence-labeled ODN2 is taken up into cells. FIG. 6 is a chart showing changes in the UV-visible absorption spectrum and fluorescence spectrum of fluorescence-labeled ODN2 before and after deprotection. FIG. 6(a) is a chart showing changes in the UV-visible absorption spectrum, and FIG. 6(b) is a chart showing changes in the fluorescence spectrum. FIG. 7 is a chart showing changes in fluorescence over time of fluorescence-labeled ODN2 in the presence of GSH.

One embodiment of the compound of the present invention, one embodiment of the agent for introducing fluorescent label of the present invention, and one embodiment of the method of introducing fluorescent label of the present invention are described below. It should be noted that the present invention is not limited to the following embodiments or modes, and can be implemented with appropriate modifications within the scope of the present invention.

[Compound]
The compound of the present invention is a compound represented by the following general formula (1). This compound comprises A including a biological substance or its structure that can be taken into the cell through the cell membrane, a fluorescence unit FL that emits fluorescence, and a protective group —X—CH ₂ —Ar—R that binds to FL. As will be described later, A is a biomolecule such as a nucleotide chain or peptide chain, which is labeled with a fluorescence unit FL. However, FL has quenched or attenuated fluorescence due to the presence of the protecting group —X—CH ₂ —Ar—R. When a compound having such a structure is taken into cells, the protective group —X—CH ₂ —Ar—R is removed from FL by the action of glutathione present in cells and various enzymes, and FL becomes fluorescent. Due to such a mechanism, the compound of the present invention exhibits no or only weak fluorescence when it exists outside the cell, but exhibits strong fluorescence when it is taken into the cell. Therefore, it is possible to directly observe with a fluorescence microscope how much A, which is a nucleotide chain or a peptide chain, is taken up into cells without being interfered with by background fluorescence as described in Background Art. Typically, A is a unit that becomes a so-called nucleic acid drug, and in this case, it becomes possible to evaluate how much the nucleic acid drug is taken up into cells. This makes it easier to find structures that are more likely to be taken up by cells, for example, by changing the sequence or protecting group of the oligonucleotide. In addition, it will be possible to observe the speed at which such oligonucleotides are taken up into cells and their intracellular migration over time.

In the above general formula (1), A is a monovalent group containing the structure of a biological substance or derivative thereof that can be taken up into cells through the cell membrane. Typically, A is a nucleotide chain or peptide chain. Nucleotide chains are formed by continuously binding multiple nucleotides, and are used as nucleic acid medicines and the like. A nucleotide chain is also called an oligonucleotide, and examples include those in which about 1 to 30 nucleotides are linked. This nucleotide may be deoxyribonucleic acid or ribonucleic acid, and may be not only naturally occurring nucleotides, but also nucleic acid analogs obtained by chemically modifying naturally occurring nucleotides. Peptide chains may be not only oligomers of amino acids, but also chemically modified peptide chain analogues.

L is a divalent group connecting A and FL. A, which is a biological substance or a derivative thereof, includes, for example, a functional group such as a phosphate group at the end of a nucleotide chain, an amino group or a carboxyl group at the end of a peptide chain, or various functional groups derived from various amino acid residues. Therefore, L binds to such a functional group, or binds to an alkynyl group or an azide group introduced into such a functional group by a click reaction. L connects A and FL, and its length may be short or long. L includes residues formed in a coupling reaction with A and linear groups such as alkyl chains or (poly)oxyethylene chains. For example, when A and L are combined using a click reaction, the above residue becomes a triazole ring structure. Examples of the alkyl group or (poly)oxyethylene chain include those having about 2 to 20 carbon atoms.

FL is a fluorescent unit that exhibits fluorescence emission and has a cyclic skeleton with a nitrogen atom. FL exhibits fluorescence quenching or only weak fluorescence when a protecting group —X—CH ₂ —Ar—R is attached to the nitrogen atom contained therein, while it exhibits intrinsic fluorescence when such a protecting group is removed. Preferred examples of such FL include those having a skeleton composed of rhodamine 110 or a derivative thereof represented by the following chemical formula (2) or (3). In this case, rhodamine 110 represented by the following chemical formula (2) or (3) or a derivative thereof may be modified in various ways without impairing the effects of the present invention.

When FL has a skeleton represented by the above chemical formula (2), the above L binds to at least one of an amino group, an imino group and a carboxyl group contained in the chemical formula (2), and a protecting group -X-CH ₂ -Ar-R binds to at least one of the amino group and the imino group contained in the chemical formula (2). Further, when FL has a skeleton represented by the above chemical formula (3), the above L is bound to one of the two amino groups contained in the chemical formula (3), and the protecting group -X-CH ₂ -Ar-R is bound to the remaining amino group. It is known that the rhodamine 110 skeleton represented by the chemical formula (2) takes the structure of the chemical formula (3) in which the carboxyl group is ring-closed when an electron-withdrawing substituent is bonded to the amino group or imino group.

In the above general formula (1), X is a single bond or -C(=O)O-. A wavy line connecting X and FL means a single bond to the nitrogen atom contained in FL. Ar is a bivalent aromatic ring having a bond at the para- or ortho-position. Bivalent with a bond at the para position means that two carbon atoms are included between two carbon atoms to which two bonds are respectively bonded, and bivalent with a bond at the ortho position means that the two carbon atoms to which the two bonds are respectively bonded are adjacent to each other. Ar is preferably exemplified by a p-phenylene group and an o-phenylene group. In addition, this phenylene group may further have other substituents within a range that does not impair the effects of the present invention.

R is a nitro group or —S—S—R ¹ . The protective group --X--CH ₂ --Ar--R of the present invention is released when this R is acted upon by enzymes such as glutathione and nitroreductase present in cells. The mechanism is explained by the following chemical reaction formula. The compounds shown in the following chemical reaction formulas are examples of the present invention shown for explanation.

In the above chemical reaction formula, GSH is glutathione present in cells. When the disulfide bond contained in R is cleaved by glutathione, the protective group —X—CH ₂ —Ar—R is removed from the rhodamine 110 skeleton, and the compound becomes fluorescent. Further, when R is a nitro group, the chemical reaction formula is as follows.

When R is a nitro group, the nitro group is reduced to an amino group by intracellular nitroreductase and NADH, and the protective group --X-- _CH.sub.2 --Ar--R is removed, and the compound becomes fluorescent.

In the above example, the protecting group —X—CH ₂ —Ar—R is detached by glutathione or nitroreductase present in the cell, but a method of attaching the protecting group to the fluorescent unit via an ester bond and removing it by an esterase present in the cell may also be employed.

R ¹ is a monovalent group. Preferred examples of such monovalent groups include alkyl groups having 1 to 10 carbon atoms. Among these alkyl groups, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group and tert-butyl group are preferred.

In the above general formula (1), n is an integer of 1-3. n is the number of protecting groups —X—CH ₂ —Ar—R introduced into the compound of the present invention, and may be any number of 1, 2 and 3; Among these, n is preferably 1 or 2.

More specifically, the compound of the present invention is preferably represented by the following general formula (4) or (5).

In general formulas (4) and (5) above, A, L, X, Ar and R are the same as in general formula (1) above.

More specifically, compounds represented by the following general formulas (6) and (7) are preferred as the compounds of the present invention.

The above general formulas (6) and (7) are obtained by specifying Ar in the above general formulas (4) and (5) as a p-phenylene group. In general formulas (6) and (7) above, A, L, X and R are the same as in general formula (1) above.

More specifically, compounds represented by the following general formulas (8) to (10) are preferred as the compounds of the present invention.

In general formulas (8) and (9) above, A, L, and ^R1 are the same as in general formula (1) above. In general formula (10) above, A and L are the same as in general formula (1) above.

As an example for more specifically explaining the compound of the present invention, the compound in which the 5' end of the nucleotide chain is fluorescently labeled is shown by the following chemical formulas (11) and (12). The compounds represented by chemical formulas (11) and (12) below are examples of the present invention, and the present invention is not limited thereto.

In the above chemical formulas (11) and (12), the portion corresponding to -L-FL-(X-CH ₂ -Ar) _n in general formula (1) is the portion on the left side of the alkylene chain bonded to the oxygen atom of the 5' terminal phosphate group. A wavy line attached to the phosphate group at the 5' end indicates that the following is a nucleotide chain. The portion extending from the 5' terminal phosphate group to the nucleotide chain corresponds to A in the general formula (1). Such a molecule can be obtained, for example, by synthesizing a phosphoramidite (14) having an alkynyl group as shown in the following chemical reaction formula, then introducing an alkynyl group to the end of a nucleotide chain using an automatic DNA synthesizer, and then combining it with an azide compound shown in the following chemical formula (13) by a click reaction. DIPEA shown in the following chemical reaction formula is N,N-diisopropylethylamine. A click reaction is a reaction in which an azide compound and an alkynyl compound are coupled in the presence of a Cu(I) compound. According to this reaction, a triazole ring is formed at the coupling site, as in the compound represented by the above chemical formula (12).

Further, a click reaction may be performed with the azide compound represented by the above chemical formula (13) using a phosphoramidite having a triple bond in the ring and a distorted ring structure represented by the following chemical formula (14c) instead of the above compound (14). When a compound having a distorted ring structure such as the compound represented by the following chemical formula (14c) and a compound having an azide group are coupled by a click reaction, the coupling can proceed without the presence of a Cu(I) compound as a catalyst. As the compound represented by the following chemical formula (14c), for example, one commercially available from GLEN RESERCH can be used. In addition, as such a distorted ring structure, for example, one represented by the following chemical formula (A) can be mentioned.

Next, an example of a synthetic scheme for obtaining an azide compound such as compound (13) is shown below.

[Fluorescent label introduction agent]
A fluorescent label-introducing agent containing a compound represented by the following general formula (1a) or (1b) is also one aspect of the present invention. This fluorescent label-introducing agent has an azide group or an alkynyl group, and is coupled with a compound having an alkynyl group or an azide group, respectively, in the presence of a Cu(I) compound by causing a click reaction. Therefore, it can be used, for example, to introduce a fluorescent label in the compound of the present invention into a nucleotide chain or peptide chain having an alkynyl group or an azide group. That is, the fluorescent label-introducing agent of the present invention can be used to synthesize the compound of the present invention. The fluorescent label-introducing agent represented by the following general formula (1a) is used to introduce a fluorescent label into a compound having an alkynyl group, and the fluorescent label-introducing agent represented by the following general formula (1b) or (1c) is used to introduce a fluorescent label into a compound having an azide group. In particular, in the compound represented by the chemical formula (1c), the A ring is distorted due to the presence of a triple bond, and a click reaction occurs with a compound having an azide group even in the absence of a Cu(I) compound to introduce a fluorescent label. A compound into which this fluorescent label has been introduced exhibits no or only weak fluorescence outside the cell, for example, in a culture medium, but once inside the cell, the protective group —X—CH ₂ —Ar—R is eliminated and fluorescence is exhibited, as already explained in the compound of the present invention.

In the general formulas (1a), (1b) and (1c) above, FL is a fluorescent unit that emits fluorescent light and has a cyclic skeleton containing nitrogen atoms. FL exhibits fluorescence quenching or only weak fluorescence when a protecting group —X—CH ₂ —Ar—R is attached to the nitrogen atom contained therein, while it exhibits intrinsic fluorescence when such a protecting group is removed. Preferred examples of such FL include those having a skeleton composed of rhodamine 110 or a derivative thereof represented by the following chemical formula (2) or (3). In this case, rhodamine 110 represented by the following chemical formula (2) or (3) or a derivative thereof may be modified in various ways without impairing the effects of the present invention.

When FL has a skeleton represented by the above chemical formula (2), the above L binds to at least one of an amino group, an imino group and a carboxyl group contained in the chemical formula (2), and a protecting group -X-CH ₂ -Ar-R binds to at least one of the amino group and the imino group contained in the chemical formula (2). Further, when FL has a skeleton represented by the above chemical formula (3), the above L is bound to one of the two amino groups contained in the chemical formula (3), and the protecting group -X-CH ₂ -Ar-R is bound to the remaining amino group.

In the above general formulas (1a), (1b) and (1c), X is a single bond or -C(=O)O-. A wavy line connecting X and FL means a single bond to the nitrogen atom contained in FL. Ar is a bivalent aromatic ring having a bond at the para- or ortho-position. The term "bivalent having a bond at the para- or ortho-position" has already been explained for the compound of the present invention. Ar is preferably exemplified by a p-phenylene group and an o-phenylene group. In addition, this phenylene group may further have other substituents within a range that does not impair the effects of the present invention.

R is a nitro group or —S—S—R ¹ . The protective group --X--CH ₂ --Ar--R of the present invention is released when this R is acted upon by enzymes such as glutathione and nitroreductase present in cells. The reaction mechanism is as already explained for the compound of the present invention. In addition, in the fluorescent label-introducing agent of the present invention, a protective group may be bound to the fluorescent unit via an ester bond. In this case, the protective group --X-- _CH.sub.2 --Ar--R is removed from the compound into which the fluorescent label has been introduced by esterase present in the cell.

R ¹ is a monovalent group. Preferred examples of such monovalent groups include alkyl groups having 1 to 10 carbon atoms. Among these alkyl groups, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group and tert-butyl group are preferred.

In the above general formulas (1a), (1b) and (1c), n is an integer of 1-3. n is the number of protecting groups —X—CH ₂ —Ar—R introduced into the compound of the present invention, and may be any number of 1, 2 and 3; Among these, n is preferably 1 or 2.

In the above general formula (1c), the A ring is a ring structure having a triple bond, which may be a single ring or a structure in which multiple rings are condensed, and when the A ring is a structure in which a plurality of rings are condensed, the single bond from L to the A ring may be bonded to any condensed ring. Due to the presence of the triple bond, the A ring is distorted, and click reaction activity is high for compounds having an azide group. Therefore, when the compound (1c) is used, the Cu(I) compound required as a catalyst in a normal click reaction becomes unnecessary. As the structure of the A ring, an 8-membered ring structure or a structure in which it is fused with another ring, as exemplified in the following chemical formulas, can be preferably mentioned.

More specifically, the fluorescent label-introducing agent of the present invention preferably includes those represented by the following general formulas (4a), (4b), (4c), (5a), (5b) and (5c). The fluorescent label-introducing agent represented by general formula (4a) performs fluorescent labeling on a compound having an alkynyl group to give the compound represented by general formula (4). The fluorescent label-introducing agent represented by general formula (4b) or (4c) performs fluorescent labeling on a compound having an azide group to give the compound represented by general formula (4). Such a rule is the same for fluorescent label introduction agents with other general formula numbers (5a, 5b, 5c, 6a, 6b, 6c, etc.).

In general formulas (4a), (4b), (5a) and (5b), L, X, Ar and R are the same as in general formula (1a). In general formulas (4c) and (5c) above, ring A, L, X, Ar and R are the same as in general formula (1c) above.

More specifically, the fluorescent label-introducing agent of the present invention is preferably represented by the following general formulas (6a), (6b), (6c), (7a), (7b) or (7c).

General formulas (6a), (6b), (6c), (7a), (7b) and (7c) are those in which Ar in general formulas (4a), (4b), (4c), (5a), (5b) and (5c) is specified as a p-phenylene group. In general formulas (6a), (6b), (7a) and (7b) above, L, X, Ar and R are the same as in general formula (1a) above. In general formulas (6c) and (7c) above, ring A, L, X and R are the same as in general formula (1c) above.

More specifically, the fluorescent label-introducing agent of the present invention is preferably represented by the following general formulas (8a), (8b), (8c), (9a), (9b), (9c), (10a), (10b) or (10c).

In general formulas (8a), (8b), (9a) and (9b), L and R ¹ are the same as in general formula (1a). In general formulas (10a) and (10b) above, L is the same as in general formula (1a) above. In general formulas (8c) and (9c) above, ring A, L and R ¹ are the same as in general formula (1c) above. In general formula (10c) above, ring A and L are the same as in general formula (1c) above.

The synthetic scheme for obtaining the fluorescent label-introducing agent of the present invention is as already exemplified in the explanation of the compound of the present invention. Although the above examples have an azide group, those having an alkynyl group can also be synthesized by ordinary organic synthesis techniques.

[Method of introducing fluorescent label]
A method for introducing a fluorescent label, which comprises introducing an alkynyl group or an alkynylene group into a molecule to be labeled, and then reacting the molecule with the above-mentioned fluorescent label-introducing agent having an azide group, and a method for introducing a fluorescent label, comprising introducing an azide group into the molecule to be labeled, and then reacting the molecule with the above-mentioned fluorescent label-introducing agent having an alkynyl group or an A ring. Since this has already been explained, the explanation here is omitted. In the case of introducing an alkynylene group into a nucleotide chain, an alkynylene group may be introduced into the 5' end of the oligonucleotide chain using a commercially available phosphoramidite represented by the already explained chemical formula (14c) or the like when synthesizing the nucleotide chain with an automatic DNA synthesizer. The cyclic structure having an alkynylene group in chemical formula (14c) can also be replaced with one represented by chemical formula (A) below. These alkynylene group-containing cyclic structures are distorted and can cause a click reaction without a catalyst comprising a Cu(I) compound with respect to compounds having an azide group. As a cyclic structure having such an alkynylene group, an 8-membered ring can be preferably exemplified.

EXAMPLES Hereinafter, the present invention will be described in more detail by showing examples, but the present invention is not limited to the following examples. The compound numbers in the synthesis examples below correspond to the numbers attached to the chemical formulas in the above synthesis schemes.

乾燥テトラヒドロフラン（ＴＨＦ、１７０ｍＬ）にＬｉＡｌＨ_４（５．５１ｇ、１４５ｍｍｏｌ）を加えた後、氷冷下で少し撹拌し、反応容器内をアルゴンで置換した。その後、４－メルカプト安息香酸（７．５７ｇ、４９．１ｍｍｏｌ）のＴＨＦ溶液（１３０ｍＬ）を加え、４日間撹拌した。塩化アンモニウム飽和水溶液を加えて反応を停止し、メタノールを添加した後、超音波振動を加えることで鼠色の油状物質を溶解させ、セライトで溶かした。減圧下濃縮を行い、酢酸エチルを加え、水で２回、塩化ナトリウム飽和水溶液で１回洗浄を行った。有機相を回収し、無水硫酸ナトリウムで乾燥させ、濾過にて溶液を得た後これを減圧下で濃縮した。残渣をシリカゲルクロマトグラフィー（ヘキサン：酢酸エチル＝１：１で展開、クロロホルム：メタノール＝１００：０で溶出）で精製し、白色固体の（４－メルカプトフェニル）メタノール（３．０５ｇ、２１．８ｍｍｏｌ、収率４４％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＤＭＳＯ－ｄ_６） δ（ｐｐｍ）：７．２３－７．１７（４Ｈ，ｍ，Ｈ－ａｒｏｍ），５．２９（１Ｈ，ｓ，ＳＨ），５．１２－５．１１（１Ｈ，ｔ，Ｊ＝５．７Ｈｚ，ＯＨ），４．４１－４．４０（２Ｈ，ｔ，Ｊ＝５．５Ｈｚ，ＣＨ_２） ・Synthesis of (4-mercaptophenyl)methanol

After LiAlH ₄ (5.51 g, 145 mmol) was added to dry tetrahydrofuran (THF, 170 mL), the mixture was slightly stirred under ice-cooling, and the inside of the reaction vessel was replaced with argon. After that, a THF solution (130 mL) of 4-mercaptobenzoic acid (7.57 g, 49.1 mmol) was added and stirred for 4 days. A saturated aqueous solution of ammonium chloride was added to stop the reaction, methanol was added, and then ultrasonic vibration was applied to dissolve the grey-colored oily substance, which was dissolved with celite. Concentration was performed under reduced pressure, ethyl acetate was added, and the mixture was washed twice with water and once with a saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered to obtain a solution, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developed with hexane:ethyl acetate=1:1, eluted with chloroform:methanol=100:0) to give (4-mercaptophenyl)methanol (3.05 g, 21.8 mmol, yield 44%) as a white solid.
¹ H-NMR (600 MHz, DMSO-d ₆ ) δ (ppm): 7.23-7.17 (4H, m, H-arom), 5.29 (1H, s, SH), 5.12-5.11 (1H, t, J=5.7 Hz, OH), 4.41-4.40 (2H, t, J=5.5 Hz, CH ₂ )

エタノール（２０ｍＬ）に２，２’－ジピリジルジスルフィド（１１．８ｇ、５３．６ｍｍｏｌ）を加え、さらに酢酸（１．５３ｍＬ、２６．８ｍｍｏｌ）及び２－プロパンチオール（５．０ｍＬ、５３．６ｍｍｏｌ）を加えて室温で撹拌した。２日後、薄層クロマトグラフィー（ＴＬＣ）で反応の進行を確認し、減圧下で濃縮した。残渣をシリカゲルクロマトグラフィー（ヘキサン：酢酸エチル＝１：１）で精製し、淡黄色液体の２－（イソプロピルジスルファニル）ピリジン（８．０４ｇ、４３．３ｍｍｏｌ、収率８１％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＤＭＳＯ－ｄ_６） δ（ｐｐｍ）：８．４５－８．４４（１Ｈ，ｍ，Ｈ－ａ），７．７７－７．７５（１Ｈ，ｍ，Ｈ－ｃ），７．６５－７．６１（１Ｈ，ｍ，Ｈ－ｄ），７．０８－７．０５（１Ｈ，ｍ，Ｈ－ｂ），３．１８－３．１０（１Ｈ，ｍ，ｉＰｒ－ＣＨ），１．３４－１．３２（６Ｈ，ｄ，Ｊ＝６．９Ｈｚ，ｉＰｒ－ＣＨ_３） ・Synthesis of 2-(isopropyldisulfanyl)pyridine

2,2′-Dipyridyl disulfide (11.8 g, 53.6 mmol) was added to ethanol (20 mL), and acetic acid (1.53 mL, 26.8 mmol) and 2-propanethiol (5.0 mL, 53.6 mmol) were added and stirred at room temperature. After 2 days, the progress of the reaction was confirmed by thin layer chromatography (TLC) and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane:ethyl acetate=1:1) to give pale yellow liquid 2-(isopropyldisulfanyl)pyridine (8.04 g, 43.3 mmol, yield 81%).
¹ H-NMR (600 MHz, DMSO-d ₆ ) δ (ppm): 8.45-8.44 (1H, m, H-a), 7.77-7.75 (1H, m, H-c), 7.65-7.61 (1H, m, H-d), 7.08-7.05 (1H, m, H-b), 3.18-3.10 (1H, m, iPr-CH), 1.34-1.32 (6H, d, J = 6.9 Hz, iPr-CH ₃ )

エタノール（２ｍＬ）に（４－メルカプトフェニル）メタノール（９４５ｍｇ、６．７４ｍｍｏｌ）を加え、撹拌しながら２－（イソプロピルジスルファニル）ピリジン（２．５６ｇ、１３．８ｍｍｏｌ）及び酢酸（１００μＬ、６．１２ｍｍｏｌ）を加えた後、室温で３０分間撹拌した。ＴＬＣで原料の消失を確認し、減圧下で濃縮した。残渣をシリカゲルクロマトグラフィー（ヘキサン：酢酸エチル＝３：１で展開、ヘキサン：酢酸エチル＝１００：０で溶出）で精製し、透明液体の（４－（イソプロピルジスルファニル）フェニル）メタノール（１．１９ｇ、５．５４ｍｍｏｌ、収率８３％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：７．５４－７．５３（２Ｈ，ｄ，Ｊ＝８．２Ｈｚ，Ｈ－ｃ），７．３２－７．３０（２Ｈ，ｄ，Ｊ＝８．６Ｈｚ，Ｈ－ｂ），４．６７（２Ｈ，ｓ，Ｈ－ａ），３．０８－３．０４（１Ｈ，ｍ，ｉＰｒ－ＣＨ），１．３０（６Ｈ，ｄ，Ｊ＝６．５，ｉＰｒ－ＣＨ_３） Synthesis of (4-(isopropyldisulfanyl)phenyl)methanol

(4-Mercaptophenyl)methanol (945mg, 6.74mmol) was added to ethanol (2mL), and 2-(isopropyldisulfanyl)pyridine (2.56g, 13.8mmol) and acetic acid (100μL, 6.12mmol) were added with stirring, followed by stirring at room temperature for 30 minutes. Disappearance of the starting material was confirmed by TLC, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel chromatography (developed with hexane:ethyl acetate=3:1, eluted with hexane:ethyl acetate=100:0) to give (4-(isopropyldisulfanyl)phenyl)methanol (1.19 g, 5.54 mmol, yield 83%) as a clear liquid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 7.54-7.53 (2H, d, J=8.2 Hz, Hc), 7.32-7.30 (2H, d, J=8.6 Hz, Hb), 4.67 (2H, s, Ha), 3.08-3.04 (1H, m, iPr-CH), 1.30 (6H, d, J = 6.5, iPr- _CH3 )

塩化メチレン（２ｍＬ）にトリホスゲン（２１９ｍｇ、７３８μｍｏｌ）を加え、０℃で撹拌しながら、（４－（イソプロピルジスルファニル）フェニル）メタノール（５０３ｍｇ、２．３５ｍｍｏｌ）及びピリジン（１８７μＬ、２．３２ｍｍｏｌ）を加え、室温で９時間撹拌した。ＴＬＣで原料の消失を確認し、酢酸エチルを加え、水で３回洗浄した。有機相を回収し、無水硫酸ナトリウムで乾燥させ、濾過にて溶液を得た後これを減圧下で濃縮した。残渣をシリカゲルカラムクロマトグラフィー（ヘキサン：酢酸エチル＝３：１で展開、ヘキサン：酢酸エチル＝１００：０で溶出）で精製し、透明液体の１－（４－（クロロメチル）フェニル）－２－イソプロピルジスルファン（３２０ｍｇ、１．３７ｍｍｏｌ、収率５９％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：７．５２－７．５１（２Ｈ，ｄ，Ｊ＝８．６Ｈｚ，Ｈ－ｃ），７．３２－７．３１（２Ｈ，ｄ，Ｊ＝８．２Ｈｚ，Ｈ－ｂ），４．５５（２Ｈ，ｓ，Ｈ－ａ），３．０９－３．０２（１Ｈ，ｍ，ｉＰｒ－ＣＨ），１．２９（６Ｈ，ｄ，Ｊ－６．５Ｈｚ，ｉＰｒ－ＣＨ_３） ・Synthesis of 1-(4-(chloromethyl)phenyl)-2-isopropyldisulfane

Add triphosgene (219 mg, 738 μmol) to methylene chloride (2 mL), add (4-(isopropyldisulfanyl)phenyl)methanol (503 mg, 2.35 mmol) and pyridine (187 μL, 2.32 mmol) while stirring at 0° C., and stir at room temperature for 9 hours. Disappearance of the starting material was confirmed by TLC, ethyl acetate was added, and the mixture was washed with water three times. The organic phase was collected, dried over anhydrous sodium sulfate, filtered to obtain a solution, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developed with hexane:ethyl acetate=3:1, eluted with hexane:ethyl acetate=100:0) to give 1-(4-(chloromethyl)phenyl)-2-isopropyldisulfane (320 mg, 1.37 mmol, yield 59%) as a clear liquid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 7.52-7.51 (2H, d, J=8.6 Hz, Hc), 7.32-7.31 (2H, d, J=8.2 Hz, Hb), 4.55 (2H, s, Ha), 3.09-3.02 (1H, m, iPr-CH), 1.29 (6H, d, J-6.5Hz, iPr- _CH3 )

ローダミン１１０塩酸塩（３９４ｍｇ、１．０７ｍｍｏｌ）を反応容器に加え、内部をアルゴンで置換した。これをＤＭＦに溶解させ、ＮａＨ（３２ｍｇ、１．３ｍｍｏｌ）を加えて室温で１時間撹拌した。その後、ジ－ｔｅｒｔ－ブチルジカーボネート（２２１μＬ、９６２μｍｏｌ）を加え、１日間撹拌した。ＴＬＣにより原料が薄くなっていることを確認し、ヘキサン：酢酸エチル＝１０：１の混合溶媒（６６ｍＬ）中へ反応溶液を少しずつ滴下し、得られた溶液をヘキサン：酢酸エチル＝２０：１の混合溶媒（６３ｍＬ）へ少しずつ滴下し、さらに得られた溶液をヘキサン：酢酸エチル＝２０：１の混合溶媒（６３ｍＬ）へ少しずつ滴下した。溶媒を留去して残渣を回収し、ヘキサン：酢酸エチル：メタノール＝１：１：１の混合溶媒（９０ｍＬ）に溶解させた。析出した赤い固体を濾別し、溶液の方を回収した。溶媒を留去し、残渣をシリカゲルカラムクロマトグラフィー（クロロホルム：メタノール＝９０：１０で展開、ヘキサン：酢酸エチル＝６０：４０～４０：６０で溶出）で精製し、燈色固体の化合物１５（１８８ｍｇ、４３４μｍｏｌ、収率４５％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：８．２３－８．２１（２Ｈ，ｍ，Ｈ－ａｒｏｍ），８．０２－８．００（１Ｈ，ｍ，Ｈ－ｂ），７．６７－６．６６（１１Ｈ，ｍ，Ｈ－ａｒｏｍ，Ｈ－ａ），１．５２（９Ｈ，ｓ，Ｂｏｃ－ＣＨ_３） ・Synthesis of compound 15

Rhodamine 110 hydrochloride (394 mg, 1.07 mmol) was added to the reaction vessel and the interior was purged with argon. This was dissolved in DMF, NaH (32 mg, 1.3 mmol) was added and stirred at room temperature for 1 hour. Di-tert-butyl dicarbonate (221 μL, 962 μmol) was then added and stirred for 1 day. It was confirmed by TLC that the starting material had become thin, and the reaction solution was added dropwise little by little into a mixed solvent (66 mL) of hexane:ethyl acetate = 10:1, the obtained solution was added dropwise little by little to a mixed solvent (63 mL) of hexane:ethyl acetate = 20:1, and the obtained solution was further dropped little by little into a mixed solvent (63 mL) of hexane:ethyl acetate = 20:1. The solvent was distilled off to collect the residue, which was dissolved in a mixed solvent (90 mL) of hexane:ethyl acetate:methanol=1:1:1. The precipitated red solid was separated by filtration, and the solution was recovered. The solvent was distilled off, and the residue was purified by silica gel column chromatography (developed with chloroform:methanol=90:10, eluted with hexane:ethyl acetate=60:40-40:60) to give compound 15 (188 mg, 434 μmol, yield 45%) as an orange solid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 8.23-8.21 (2H, m, H-arom), 8.02-8.00 (1H, m, H-b), 7.67-6.66 (11H, m, H-arom, Ha), 1.52 (9H, s, Boc-CH ₃ )

ＴＨＦ（１．５ｍＬ）に化合物１５（５８ｍｇ、１３４μｍｏｌ）を溶解させ、トリホスゲン（３２ｍｇ、１０８μｍｏｌ）及びＮ，Ｎ－ジイソプロピルエチルアミン（ＤＩＰＥＡ、６８．４μＬ、４０２μｍｏｌ）を加え、室温で５時間撹拌した。その後、反応溶液に塩化アンモニウム飽和水溶液（１５ｍＬ）を加えて反応を停止させた。これに塩化メチレンを加え、塩化アンモニウム飽和水溶液で３回洗浄した。有機相を集めて減圧下で濃縮し、クロマトグラフィーで精製することで、淡黄色液体の化合物１６（１６ｍｇ、２３．９μｍｏｌ、収率１８％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：８．００（１Ｈ，ｄ，Ｊ＝７．９Ｈｚ，Ｈ－ｃ），７．６６－６．６６（１５Ｈ，ｍ，Ｒｈ－Ｂｎ，Ｈ－ｂ），５．１７（２Ｈ，ｓ，Ｈ－ａ），３．０９－３．０２（１Ｈ，ｍ，ｉＰｒ－ＣＨ），１．５２（９Ｈ，ｓ，Ｂｏｃ－ＣＨ_３），１．２９（６Ｈ，ｄ，Ｊ＝６．９Ｈｚ，ｉＰｒ－ＣＨ_３） ・Synthesis of compound 16

Compound 15 (58 mg, 134 μmol) was dissolved in THF (1.5 mL), triphosgene (32 mg, 108 μmol) and N,N-diisopropylethylamine (DIPEA, 68.4 μL, 402 μmol) were added and stirred at room temperature for 5 hours. After that, saturated aqueous ammonium chloride solution (15 mL) was added to the reaction solution to stop the reaction. Methylene chloride was added to this, and it wash|cleaned 3 times with ammonium chloride saturated aqueous solution. The organic phase was collected, concentrated under reduced pressure, and purified by chromatography to give compound 16 (16 mg, 23.9 μmol, 18% yield) as a pale yellow liquid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 8.00 (1H, d, J=7.9 Hz, Hc), 7.66-6.66 (15H, m, Rh-Bn, H-b), 5.17 (2H, s, Ha), 3.09-3.02 (1H, m, iPr-CH), 1.52 (9H, s , Boc-CH ₃ ), 1.29 (6H, d, J=6.9 Hz, iPr-CH ₃ )

化合物１６（２．５ｍｇ、３．７３μｍｏｌ）に４Ｍ塩酸水溶液（５００μＬ）を加え、室温で３時間撹拌した。ＴＬＣで原料の消失を確認し、減圧下で濃縮し、酢酸エチルで３回、クロロホルムで３回共沸除去を行って、未精製のままの化合物１７を赤色固体（２．０ｍｇ、３．５μｍｏｌ、収率９４％）として得た。これを未精製のまま次の反応に用いた。 ・Synthesis of compound 17

A 4M hydrochloric acid aqueous solution (500 μL) was added to compound 16 (2.5 mg, 3.73 μmol), and the mixture was stirred at room temperature for 3 hours. After confirming the disappearance of the starting material by TLC, it was concentrated under reduced pressure and azeotropically removed three times with ethyl acetate and three times with chloroform to give crude compound 17 as a red solid (2.0 mg, 3.5 μmol, yield 94%). This was used in the next reaction without being purified.

ＤＭＦ（１０ｍＬ）に化合物１７（１８９ｍｇ、３３１μｍｏｌ）を溶解させ、１－エチル－３－（３－ジメチルアミノプロピル）カルボジイミド塩酸塩（ＥＤＣ、６３４ｍｇ、３．３２ｍｍｏｌ）及び１１－アジド－３，６，９－トリオキサウンデカン酸（３９４μＬ、１．９９ｍｍｏｌ）を順次加え、室温で２日間撹拌した。ＴＬＣにより原料消失を確認した後、酢酸エチルを加え、塩化ナトリウム飽和水溶液で２回洗浄した。有機相を集めて共沸脱水（ピリジン５回、トルエン３回）を行った。クロマトグラフィーで精製し、白色固体の化合物１３（１５．８ｍｇ、１２．４μｍｏｌ、収率６．１％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：８．９３（１Ｈ，ｓ，Ｈ－ｉ），８．０３－６．７２（１５Ｈ，ｓ，Ｈ－ｈ，Ｒｈ－Ｂｎ，Ｂｎ’），５．１８（２Ｈ，ｓ，Ｈ－ｊ），４．１３（２Ｈ，ｓ，Ｈ－ｇ），３．７８－３．６３（１０Ｈ，ｍ，Ｈ－ｂ，ｃ，ｄ，ｅ，ｆ），３．３３（２Ｈ，ｍ，Ｈ－ａ），３．０８－３．０４（１Ｈ，ｍ，ｉＰｒ－ＣＨ），１．３０（６Ｈ，ｄ，ｉＰｒ－ＣＨ_３） ・Synthesis of compound 13

Compound 17 (189 mg, 331 μmol) was dissolved in DMF (10 mL), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 634 mg, 3.32 mmol) and 11-azido-3,6,9-trioxaundecanoic acid (394 μL, 1.99 mmol) were sequentially added and stirred at room temperature for 2 days. After confirming the disappearance of the raw materials by TLC, ethyl acetate was added, and the mixture was washed twice with a saturated aqueous sodium chloride solution. The organic phase was collected and subjected to azeotropic dehydration (pyridine 5 times, toluene 3 times). Purification by chromatography gave compound 13 (15.8 mg, 12.4 μmol, 6.1% yield) as a white solid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 8.93 (1H, s, Hi), 8.03-6.72 (15H, s, H-h, Rh-Bn, Bn'), 5.18 (2H, s, H-j), 4.13 (2H, s, H-g), 3.78-3.63 (10H, m, H-b , c, d, e, f), 3.33 (2H, m, Ha), 3.08-3.04 (1H, m, iPr-CH), 1.30 (6H, d, iPr-CH ₃ )

ＤＭＦ（１ｍＬ）に化合物１５（５３ｍｇ、１２３μｍｏｌ）を溶解させ、ＥＤＣ（６１ｍｇ、３１８μｍｏｌ）及び１１－アジド－３，６，９－トリオキサウンデカン酸（７８．２μＬ、３６９μｍｏｌ）を順次加え、室温で８時間撹拌した。ＴＬＣにより原料が薄くなったことを確認し、酢酸エチルを加え、塩化ナトリウム飽和水溶液で２回洗浄した。有機相を集めて、濾過し、減圧下で濃縮した。残渣をシリカゲルクロマトグラフィー（ヘキサン：酢酸エチル＝１：１で展開、ヘキサン：酢酸エチル６５：３５～５５：４５で溶出）で精製し、燈色固体の化合物１８（４５ｍｇ、６９．７μｍｏｌ、収率５７％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：８．９３（１Ｈ，ｓ，Ｈ－ｉ），８．０２（１Ｈ，ｄ，Ｊ＝７．６Ｈｚ，Ｈ－ｊ），７．６９－６．６５（１０Ｈ，ｍ，Ｈ－ａｒｏｍ），４．１３（２Ｈ，ｍ，Ｈ－ｇ），３．６９（８Ｈ，ｍ，Ｈ－ｃ～ｆ）、３．３４（２Ｈ，ｔ、Ｊ＝５．０Ｈｚ，Ｈ－ｂ）、１．５２（９Ｈ，ｓ，Ｂｏｃ－ＣＨ_３）、１．２６（２Ｈ，ｔ，Ｊ＝７．０Ｈｚ，Ｈ－ａ） ・Synthesis of compound 18

Compound 15 (53 mg, 123 μmol) was dissolved in DMF (1 mL), EDC (61 mg, 318 μmol) and 11-azido-3,6,9-trioxaundecanoic acid (78.2 μL, 369 μmol) were sequentially added and stirred at room temperature for 8 hours. It was confirmed by TLC that the raw material had become thin, ethyl acetate was added, and the mixture was washed twice with a saturated aqueous sodium chloride solution. The organic phases were collected, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developed with hexane:ethyl acetate=1:1, eluted with hexane:ethyl acetate 65:35-55:45) to give compound 18 (45 mg, 69.7 μmol, yield 57%) as an orange solid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 8.93 (1H, s, Hi), 8.02 (1H, d, J=7.6 Hz, Hj), 7.69-6.65 (10H, m, H-arom), 4.13 (2H, m, H-g), 3.69 (8H, m, Hc-f), 3.3 4 (2H, t, J = 5.0 Hz, Hb), 1.52 (9H, s, Boc-CH ₃ ), 1.26 (2H, t, J = 7.0 Hz, Ha)

塩化メチレン（７５０μＬ）にトリフルオロ酢酸２５０μＬを添加した溶液に化合物１８（３０ｍｇ、４６．５ｍｍｏｌ）を溶解させ、室温で９０分間撹拌した。ＴＬＣにより原料が薄くなったことを確認した後、反応溶液にクロロホルムを加え、炭酸水素ナトリウム飽和水溶液で１回、塩化ナトリウム飽和水溶液で１回洗浄を行った。有機相を集めて、減圧下で濃縮した。その後、共沸脱水（ピリジン３回、トルエン２回）を行い、酢酸エチルで共沸除去を行った。減圧下で濃縮を行い、真空乾燥を行うことで、ピンク色固体の化合物１９（４５ｍｇ、６９．７μｍｏｌ、収率５７％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：８．４０－６．５６（１０Ｈ，ｍ，Ｒｈ－Ｂｎ），４．１１（２Ｈ，ｄ，Ｈ－ｇ），３．７５－３．６２（１０Ｈ，ｍ，Ｈ－ａ，ｃ，ｄ，ｅ，ｆ），３．３１（２Ｈ，ｔ，Ｊ＝５．０Ｈｚ，Ｈ－ｂ） ・Synthesis of compound 19

Compound 18 (30 mg, 46.5 mmol) was dissolved in a solution of 250 μL of trifluoroacetic acid in methylene chloride (750 μL) and stirred at room temperature for 90 minutes. After confirming that the raw material became thin by TLC, chloroform was added to the reaction solution, and the reaction solution was washed once with a saturated sodium bicarbonate aqueous solution and once with a saturated sodium chloride aqueous solution. The organic phases were collected and concentrated under reduced pressure. After that, azeotropic dehydration (pyridine 3 times, toluene 2 times) was performed, and azeotropic removal was performed with ethyl acetate. Concentration was performed under reduced pressure and vacuum drying was performed to obtain compound 19 (45 mg, 69.7 μmol, yield 57%) as a pink solid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 8.40-6.56 (10H, m, Rh-Bn), 4.11 (2H, d, H-g), 3.75-3.62 (10H, m, Ha, c, d, e, f), 3.31 (2H, t, J = 5.0 Hz, H-b)

アセトニトリル（１ｍＬ）に化合物１９（３９．４ｍｇ、７２．４μｍｏｌ）を溶解させ、トリエチルアミン（ＴＥＡ、１００μＬ、７２．１μｍｏｌ）及び１－（４－（クロロメチル）フェニル）－２－イソプロピルジスルファン（１５５ｍｇ、５６０μｍｏｌ）を加えて７０℃で１日間撹拌した。ＴＬＣにより反応が進行していることを確認した後、クロマトグラフィーで精製し、燈色固体の化合物２０（１０．８ｍｇ、１１．５μｍｏｌ、収率１６％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：８．８７（１Ｈ，ｓ，Ｈ－ｈ），７．９８－６．４０（６Ｈ，ｍ，Ｈ－ａｒｏｍ），４．６１（４Ｈ，ｓ，Ｈ－ｉ），４．１１（２Ｈ，ｓ，Ｈ－ｇ），３．６５（１２Ｈ，ｍ，Ｈ－ａ，ｃ～ｆ），３．２６（２Ｈ，ｓ，Ｈ－ｂ），３．０５（２Ｈ，ｄ，Ｊ＝６．９Ｈｚ，ｉＰｒ－ＣＨ），１．２９（１２Ｈ，ｓ，ｉＰｒ－ＣＨ_３） ・Synthesis of compound 20

Compound 19 (39.4 mg, 72.4 μmol) was dissolved in acetonitrile (1 mL), triethylamine (TEA, 100 μL, 72.1 μmol) and 1-(4-(chloromethyl)phenyl)-2-isopropyldisulfane (155 mg, 560 μmol) were added and stirred at 70° C. for 1 day. After confirming the progress of the reaction by TLC, it was purified by chromatography to obtain compound 20 (10.8 mg, 11.5 μmol, yield 16%) as an orange solid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 8.87 (1H, s, Hh), 7.98-6.40 (6H, m, H-arom), 4.61 (4H, s, Hi), 4.11 (2H, s, H-g), 3.65 (12H, m, Ha, cf), 3.26 (2H, s, Hb), 3.05 (2H, d, J = 6.9 Hz, iPr-CH), 1.29 (12H, s, iPr-CH ₃ )

化合物１５（８８ｍｇ、２０３μｍｏｌ）を無水ピリジンで共沸脱水し、ピリジン（３ｍＬ）及び４－ニトロクロロギ酸ベンジル（９４ｍｇ、４３６μｍｏｌ）を加え、１５０分間撹拌した。ＴＬＣにより原料が薄くなったことを確認し、酢酸エチルに溶解し、水で１回、炭酸水素ナトリウム水溶液で１回洗浄を行った。有機相を回収し、硫酸ナトリウムで乾燥させ、濾過にて溶液を得た後これを減圧下で濃縮した。残渣をシリカゲルクロマトグラフィー（ヘキサン：酢酸エチル＝１：１で展開、ヘキサン：酢酸エチル＝７５：２５～６０：４０で溶出）で精製し、白色固体の化合物２１（５６ｍｇ、９１．６μｍｏｌ、収率４５％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：８．２２（２Ｈ，ｄ，Ｊ＝１０．０Ｈｚ，Ｈ－ａ，ｂ），８．０２－６．６６（１４Ｈ，ｍ，Ｒｈ－Ｂｎ，Ｂｎ’），５．２９（２Ｈ，ｓ，ＣＨ_２），１．５２（９Ｈ，ｓ，Ｂｏｃ－ＣＨ_３） ・Synthesis of compound 21

Compound 15 (88 mg, 203 μmol) was azeotropically dehydrated with anhydrous pyridine, pyridine (3 mL) and benzyl 4-nitrochloroformate (94 mg, 436 μmol) were added and stirred for 150 minutes. After confirming that the raw material became thin by TLC, it was dissolved in ethyl acetate and washed once with water and once with an aqueous sodium hydrogen carbonate solution. The organic phase was collected, dried over sodium sulfate, filtered to obtain a solution, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developed with hexane:ethyl acetate=1:1, eluted with hexane:ethyl acetate=75:25-60:40) to give compound 21 (56 mg, 91.6 μmol, yield 45%) as a white solid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 8.22 (2H, d, J=10.0 Hz, Ha, b), 8.02-6.66 (14H, m, Rh-Bn, Bn'), 5.29 (2H, s, CH ₂ ), 1.52 (9H, s, Boc-CH ₃ )

５－ヘキシン－１－オール（３２．３μＬ、３００μｍｏｌ）を塩化メチレンに溶解させた後、反応溶液内をアルゴン置換し、Ｎ，Ｎ－ジイソプロピルエチルアミン（ＤＩＰＥＡ、１０２μＬ、６００μｍｏｌ）及びシアノエチルジイソプロピルクロロホスホロアミダイト（１００μＬ、４５０μｍｏｌ）を順次加え、室温で１５分間撹拌した。原料の消失をＴＬＣにより確認した後、反応溶液に塩化メチレンを追加して、飽和炭酸水素ナトリウム水溶液で１回、飽和塩化ナトリウム水溶液で１回洗浄した。有機相を集めて、無水硫酸ナトリウムで乾燥させ、濾過にて溶液を得た後これを減圧下で濃縮した。残渣をシリカゲルクロマトグラフィー（ＮＨシリカゲル、ヘキサン：酢酸エチル＝１００：０～５０：５０で展開、ヘキサン：酢酸エチル＝１００：０～９０：１０で溶出）で精製し、透明液体の化合物１４（７７ｍｇ、２５８μｍｏｌ、収率８６％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：４．０５（１Ｈ，ｍ，－Ｃ≡ＣＨ），３．８２－３．７０（２Ｈ，ｍ，Ｈ－ｆ），３．６６－３．４９（４Ｈ，ｍ，Ｈ－ｄ，ｅ），２．５８（２Ｈ，ｔ，Ｊ＝６．６Ｈｚ，ｉＰｒ－ＣＨ），２．１６（２Ｈ，ｍ，Ｈ－ａ），１．７７－１．５２（４Ｈ，ｍ，Ｈ－ｂ，ｃ），１．１８（２Ｈ，ｍ，ｉＰｒ－ＣＨ），１．１２（１２Ｈ，ｍ，ｉＰｒ－ＣＨ_３）
^３１Ｐ－ＮＭＲ（２４３ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：１４８．０１ ・Synthesis of compound 14

After dissolving 5-hexyn-1-ol (32.3 μL, 300 μmol) in methylene chloride, the reaction solution was replaced with argon, N,N-diisopropylethylamine (DIPEA, 102 μL, 600 μmol) and cyanoethyldiisopropylchlorophosphoramidite (100 μL, 450 μmol) were sequentially added, and the mixture was stirred at room temperature for 15 minutes. After confirming the disappearance of the raw materials by TLC, methylene chloride was added to the reaction solution, and the mixture was washed once with a saturated aqueous sodium hydrogencarbonate solution and once with a saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered to obtain a solution, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (NH silica gel, developed with hexane:ethyl acetate=100:0-50:50, eluted with hexane:ethyl acetate=100:0-90:10) to give compound 14 (77 mg, 258 μmol, yield 86%) as a clear liquid.
¹ H-NMR (600 MHz, CDCl ₃ ) δ (ppm): 4.05 (1H, m, -C≡CH), 3.82-3.70 (2H, m, H-f), 3.66-3.49 (4H, m, H-d, e), 2.58 (2H, t, J = 6.6 Hz, iPr-CH), 2.16 (2H, m, H-a ), 1.77-1.52 (4H, m, Hb, c), 1.18 (2H, m, iPr-CH), 1.12 (12H, m, iPr-CH ₃ )
³¹ P-NMR (243 MHz, CDCl ₃ ) δ (ppm): 148.01

チミジン（３．３５ｇ、１３．８ｍｍｏｌ）を無水ピリジンで３回共沸脱水し、反応容器をアルゴン置換してピリジン（２０ｍＬ）に溶解させた。４，４－ジメトキシトリチル（ＤＭＴｒ）クロリド（６．０９ｇ、１８．０ｍｍｏｌ）を加え、室温で９時間撹拌した。ＴＬＣにより原料消失を確認し、エタノールを少量加え、溶液を減圧下で濃縮した。残渣をクロロホルムに溶解させ、飽和炭酸水素ナトリウム水溶液で２回、飽和塩化ナトリウム水溶液で１回洗浄した。有機相を集めて、無水硫酸ナトリウムで乾燥させ、濾過にて溶液を得た後これを減圧下で濃縮した。残渣をシリカゲルクロマトグラフィー（クロロホルム：メタノール＝９：１で展開、クロロホルム：メタノール＝９８：２で溶出）で精製し、淡黄色固体の化合物２２（４２７ｍｇ、７８４μｍｏｌ、収率６％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＤＭＳＯ－ｄ_６） δ（ｐｐｍ）：１１．３２（１Ｈ，ｓ，ＮＨ），７．４９１－７．４９０（１Ｈ，ｄ，Ｊ＝０．６Ｈｚ，Ｈ－６），７．３８－６．８６（１３Ｈ，ｍ，ＤＭＴｒ－ａｒｏｍ），６．２０－６．１８（１Ｈ，ｔ，Ｊ＝６．０Ｈｚ，Ｈ－１’），５．３１－５．３０（１Ｈ，ｄ，Ｊ＝６．０Ｈｚ，ＯＨ－３’），４．３２－４．２９（１Ｈ，ｍ，Ｈ－４’），３．８７－３．８６（１Ｈ，ｑ，Ｊ＝２．０Ｈｚ，Ｈ－３’），３．７２（６Ｈ，ｓ，ＯＣＨ_３－ＤＭＴｒ），３．２１－３．１４（２Ｈ，ｍ，Ｈ－５’），２．２５－２．１１（２Ｈ，ｍ，Ｈ－２’），１．４２９－１．４２８（３Ｈ，ｄ，Ｊ＝０．６Ｈｚ，ＣＨ_３－５） ・Synthesis of compound 22

Thymidine (3.35 g, 13.8 mmol) was azeotropically dehydrated with anhydrous pyridine three times, the reaction vessel was purged with argon and dissolved in pyridine (20 mL). 4,4-Dimethoxytrityl (DMTr) chloride (6.09 g, 18.0 mmol) was added and stirred at room temperature for 9 hours. After confirming the disappearance of the raw materials by TLC, a small amount of ethanol was added and the solution was concentrated under reduced pressure. The residue was dissolved in chloroform and washed twice with a saturated aqueous sodium bicarbonate solution and once with a saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered to obtain a solution, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developed with chloroform:methanol=9:1, eluted with chloroform:methanol=98:2) to give compound 22 (427 mg, 784 μmol, yield 6%) as a pale yellow solid.
^１ Ｈ－ＮＭＲ（６００ＭＨｚ、ＤＭＳＯ－ｄ _６ ） δ（ｐｐｍ）：１１．３２（１Ｈ，ｓ，ＮＨ），７．４９１－７．４９０（１Ｈ，ｄ，Ｊ＝０．６Ｈｚ，Ｈ－６），７．３８－６．８６（１３Ｈ，ｍ，ＤＭＴｒ－ａｒｏｍ），６．２０－６．１８（１Ｈ，ｔ，Ｊ＝６．０Ｈｚ，Ｈ－１'），５．３１－５．３０（１Ｈ，ｄ，Ｊ＝６．０Ｈｚ，ＯＨ－３'），４．３２－４．２９（１Ｈ，ｍ，Ｈ－４'），３．８７－３．８６（１Ｈ，ｑ，Ｊ＝２．０Ｈｚ，Ｈ－３'），３．７２（６Ｈ，ｓ，ＯＣＨ _３ －ＤＭＴｒ），３．２１－３．１４（２Ｈ，ｍ，Ｈ－５'），２．２５－２．１１（２Ｈ，ｍ，Ｈ－２'），１．４２９－１．４２８（３Ｈ，ｄ，Ｊ＝０．６Ｈｚ，ＣＨ _３ －５）

化合物２２（４．１９ｇ、７．６９ｍｍｏｌ）を無水ピリジンで３回共沸脱水し、無水トルエンで３回共沸除去した後、反応容器をアルゴン置換して塩化メチレン（２６ｍＬ）に溶解させた。Ｎ，Ｎ－ジイソプロピルエチルアミン（ＤＩＰＥＡ、２．６ｍＬ、１４，９ｍｍｏｌ）及びビス（ジイソプロピルアミノ）クロロホスフィン（３．１３ｇ、１１．７ｍｍｏｌ）を順次加え、室温で２時間撹拌した。原料の消失をＴＬＣにより確認した後、反応溶液に塩化メチレンを追加して、飽和炭酸水素ナトリウム水溶液で１回、飽和塩化ナトリウム水溶液で１回洗浄した。有機相を集めて、硫酸ナトリウムで乾燥させ、濾過にて溶液を得た後これを減圧下で濃縮した。残渣をシリカゲルクロマトグラフィー（ＮＨシリカゲル、ヘキサン：酢酸エチル＝１００：０～２０：８０で展開、ヘキサン：酢酸エチル＝６０：４０～５０：５０で溶出）で精製し、白色泡状固体の化合物２３（３．９７ｇ、５．１２ｍｍｏｌ、収率６７％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：８．２３（１Ｈ、ｓ、ＮＨ），７．６６（１Ｈ，ｄ、Ｊ＝１．１Ｈｚ，Ｈ－６），７．４１－６．８１（１３Ｈ，ｍ、ＤＭＴｒ－ａｒｏｍ）、６．４４（１Ｈ，ｍ，Ｈ－１’），４．５３（１Ｈ，ｍ，ＯＨ－３’），４．２４（１Ｈ，ｍ，Ｈ－４’），３．７９（６Ｈ，ｓ，Ｊ＝２．０Ｈｚ，Ｈ－５’，５”，Ｎ－ＣＨ（ｉ－Ｐｒ）_２），３．５９－３．４２（６Ｈ，ｍ，ＯＣＨ_３－ＤＭＴｒ），２．５３（１Ｈ，ｍ，Ｈ－５’），２．３２－２．２６（１Ｈ，ｍ，Ｈ－２’），１．２９（３Ｈ，ｄ，Ｊ＝０．６Ｈｚ，５－ＣＨ_３），１．１１（２４Ｈ，ｍ，ｉＰｒ－ＣＨ_３）
^３１Ｐ－ＮＭＲ（２４３ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：１１６．６０ ・Synthesis of compound 23

Compound 22 (4.19 g, 7.69 mmol) was azeotropically dehydrated three times with anhydrous pyridine and azeotropically removed three times with anhydrous toluene, then the reaction vessel was purged with argon and dissolved in methylene chloride (26 mL). N,N-diisopropylethylamine (DIPEA, 2.6 mL, 14.9 mmol) and bis(diisopropylamino)chlorophosphine (3.13 g, 11.7 mmol) were added sequentially and stirred at room temperature for 2 hours. After confirming the disappearance of the raw materials by TLC, methylene chloride was added to the reaction solution, and the mixture was washed once with a saturated aqueous sodium hydrogencarbonate solution and once with a saturated aqueous sodium chloride solution. The organic phases were collected, dried over sodium sulfate, filtered to obtain a solution, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (NH silica gel, developed with hexane:ethyl acetate=100:0-20:80, eluted with hexane:ethyl acetate=60:40-50:50) to give compound 23 (3.97 g, 5.12 mmol, yield 67%) as a white foamy solid.
^１ Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ _３ ） δ（ｐｐｍ）：８．２３（１Ｈ、ｓ、ＮＨ），７．６６（１Ｈ，ｄ、Ｊ＝１．１Ｈｚ，Ｈ－６），７．４１－６．８１（１３Ｈ，ｍ、ＤＭＴｒ－ａｒｏｍ）、６．４４（１Ｈ，ｍ，Ｈ－１'），４．５３（１Ｈ，ｍ，ＯＨ－３'），４．２４（１Ｈ，ｍ，Ｈ－４'），３．７９（６Ｈ，ｓ，Ｊ＝２．０Ｈｚ，Ｈ－５'，５”，Ｎ－ＣＨ（ｉ－Ｐｒ） _２ ），３．５９－３．４２（６Ｈ，ｍ，ＯＣＨ _３ －ＤＭＴｒ），２．５３（１Ｈ，ｍ，Ｈ－５'），２．３２－２．２６（１Ｈ，ｍ，Ｈ－２'），１．２９（３Ｈ，ｄ，Ｊ＝０．６Ｈｚ，５－ＣＨ _３ ），１．１１（２４Ｈ，ｍ，ｉＰｒ－ＣＨ _３ ）
³¹ P-NMR (243 MHz, CDCl ₃ ) δ (ppm): 116.60

化合物２３を反応容器に加えてアルゴン置換し、１Ｈ－テトラゾール（１４４ｍｇ、２．０６ｍｍｏｌ）、３－フェニル－１－プロパノール（３３１μＬ、２．４３ｍｍｏｌ）及びジイソプロピルアミン（２７９μＬ、１．９９ｍｍｏｌ）のアセトニトリル溶液（１０ｍＬ）を加えた。２時間撹拌し、原料消失をＴＬＣにより確認した後、反応溶液に塩化メチレンを追加して、飽和炭酸水素ナトリウム水溶液で１回、飽和塩化ナトリウム水溶液で１回洗浄した。有機相を集めて、無水硫酸ナトリウムで乾燥させ、濾過にて溶液を得た後これを減圧下で濃縮した。残渣を少量の酢酸エチルに溶解させ、多量のヘキサンを加えて懸濁液にした。濾過を行って得た白色固体を酢酸エチルに溶解させ、シリカゲルクロマトグラフィー（ヘキサン：酢酸エチル＝１００：０～６０：４０で展開、ヘキサン：酢酸エチル＝７０：３０～６０：４０溶出）で精製し、白色泡状固体の化合物２４（２６０ｍｇ、３２１μｍｏｌ、収率１５％）を得た。
^１Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：７．６４（１Ｈ，ｓ，ＮＨ），７．４０（１Ｈ，ｓ，Ｈ－６），７．２９－７．１１（１８Ｈ，ｍ，ＤＭＴｒ－ａｒｏｍ，Ｐｈ－Ｈ），６．４３（１Ｈ．ｍ，Ｈ－１’），４．６６（１Ｈ，ｄ，Ｈ－４’），３．７８－３．７５（６Ｈ，ｄ，ＤＭＴｒ－ＯＭｅ），３．６０－３．４７（６Ｈ，ｍ，Ｈ－５’，５”，Ｎ－ＣＨ（ｉ－Ｐｒ）_２），２．７１－２．５８（２Ｈ，ｍ，Ｐｈ－［ＣＨ_２］－ＣＨ_２），２．３０（２Ｈ，ｍ，Ｐｈ－ＣＨ_２－［ＣＨ_２］），１．９２－１．７９（２Ｈ，ｍ，Ｈ－２’，２”），１．４０（３Ｈ，ｓ，５－ＣＨ_３），１．１７－１．１４（１２Ｈ，ｍ，ｉＰｒ－ＣＨ_３）
^３１Ｐ－ＮＭＲ（２４３ＭＨｚ、ＣＤＣｌ_３） δ（ｐｐｍ）：１４７．６７ ・Synthesis of compound 24

Compound 23 was added to the reaction vessel and purged with argon, and 1H-tetrazole (144 mg, 2.06 mmol), 3-phenyl-1-propanol (331 μL, 2.43 mmol) and diisopropylamine (279 μL, 1.99 mmol) in acetonitrile solution (10 mL) were added. After stirring for 2 hours and confirming disappearance of the raw materials by TLC, methylene chloride was added to the reaction solution, and the mixture was washed once with a saturated aqueous sodium hydrogencarbonate solution and once with a saturated aqueous sodium chloride solution. The organic phase was collected, dried over anhydrous sodium sulfate, filtered to obtain a solution, and concentrated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate, and a large amount of hexane was added to form a suspension. The white solid obtained by filtration was dissolved in ethyl acetate and purified by silica gel chromatography (developed with hexane:ethyl acetate = 100:0 to 60:40, eluted with hexane:ethyl acetate = 70:30 to 60:40) to obtain compound 24 (260 mg, 321 µmol, yield 15%) as a white foamy solid.
^１ Ｈ－ＮＭＲ（６００ＭＨｚ、ＣＤＣｌ _３ ） δ（ｐｐｍ）：７．６４（１Ｈ，ｓ，ＮＨ），７．４０（１Ｈ，ｓ，Ｈ－６），７．２９－７．１１（１８Ｈ，ｍ，ＤＭＴｒ－ａｒｏｍ，Ｐｈ－Ｈ），６．４３（１Ｈ．ｍ，Ｈ－１'），４．６６（１Ｈ，ｄ，Ｈ－４'），３．７８－３．７５（６Ｈ，ｄ，ＤＭＴｒ－ＯＭｅ），３．６０－３．４７（６Ｈ，ｍ，Ｈ－５'，５”，Ｎ－ＣＨ（ｉ－Ｐｒ） _２ ），２．７１－２．５８（２Ｈ，ｍ，Ｐｈ－［ＣＨ _２ ］－ＣＨ _２ ），２．３０（２Ｈ，ｍ，Ｐｈ－ＣＨ _２ －［ＣＨ _２ ］），１．９２－１．７９（２Ｈ，ｍ，Ｈ－２'，２”），１．４０（３Ｈ，ｓ，５－ＣＨ _３ ），１．１７－１．１４（１２Ｈ，ｍ，ｉＰｒ－ＣＨ _３ ）
³¹ P-NMR (243 MHz, CDCl ₃ ) δ (ppm): 147.67

[Evaluation of fluorescence change accompanying deprotection]
The change in fluorescence due to elimination of the protective group from the fluorescently labeled portion of the compound of the present invention was evaluated. Compounds 13, 20 and 21 were used for evaluation. Although any compound does not have A in general formula (1) and has only a fluorescently labeled moiety, it is possible to evaluate the change in fluorescence due to elimination of the protective group. Compound 13 has a disulfide bond and a protecting group having a carbamate structure that is eliminated by glutathione. Compound 20 has a disulfide bond and a protective group with a non-carbamate structure that is eliminated by glutathione. Compound 21 has a nitrophenyl group and a protective group that is released by nitroreductase.

To 30 μL of a 200 μM aqueous solution of compound 13, 400 μL of 10×PBS (phosphate buffered saline) and 1000 μL of pure water were added, and 710 μL of each was divided into two containers. 400 μL of 50 mM glutathione solution (GSH) and 890 μL of pure water were added to one container to adjust the GSH concentration to 10 mM. 1290 μL of pure water was added to the other container to obtain a GSH-free solution. A solution of compound 13 having a GSH concentration of 10 μm was prepared in the same manner. These three solutions of GSH-free, GSH 10 mM and GSH 10 μM were allowed to stand in a 37° C. constant temperature bath and reacted at pH=7.4 (PBS buffer). Fluorescence measurement was performed at a reaction time of 480 minutes to examine each fluorescence spectrum. The results are shown in FIG. FIG. 1 shows fluorescence spectra of compound 13 with no GSH added, GSH 10 mM and GSH 10 μM.

To 30 μL of a 200 μM aqueous solution of compound 20, 600 μL of 10×PBS (phosphate buffered saline) and 1200 μL of pure water were added, and 610 μL of each was divided into two containers. 400 μL of 50 mM glutathione solution (GSH) and 890 μL of pure water were added to one container to adjust the GSH concentration to 10 mM. 1390 μL of pure water was added to the other container to obtain a GSH-free solution. A solution of compound 20 having a GSH concentration of 10 μm was prepared in the same manner. These three solutions of GSH-free, GSH 10 mM and GSH 10 μM were allowed to stand in a 37° C. constant temperature bath and reacted at pH=7.4 (PBS buffer). Fluorescence measurement was performed at a reaction time of 480 minutes to examine each fluorescence spectrum. The results are shown in FIG. FIG. 2 shows the fluorescence spectra of compound 20 with no GSH added, GSH 10 mM and GSH 10 μM.

As shown in FIGS. 1 and 2, for both compound 13 and compound 20, an increase in fluorescence derived from rhodamine 110 was confirmed at 10 mM, which corresponds to the intracellular GSH concentration. This indicates that the protecting group of the fluorescent unit, which had been quenched by the protecting group, was removed by GSH, and thereby showed fluorescence again. On the other hand, in the case of GSH concentration (10 μM), which exists in a very small amount outside the cells, or when GSH was not added, the increase in fluorescence was very slight or remained quenched.

Sample A containing 50 μg/mL nitroreductase and 1 mM NADH and sample B containing no nitroreductase and NADH at a concentration of 10 μM compound 21 in phosphate buffer (pH=7.0) were prepared. These samples were allowed to react at 37° C. for 2 hours, and changes in fluorescence spectra were observed. For sample A, changes in the fluorescence spectrum were also observed immediately after the addition of nitroreductase and NADH (that is, the reaction time was 0 hours). The results are shown in FIG. FIG. 3 shows fluorescence spectral changes over time for compound 21 with and without nitroreductase and NADH.

As shown in FIG. 3, compound 21 was observed to increase fluorescence derived from rhodamine 110 in the presence of the intracellular enzyme nitroreductase. This indicates that the protecting group of the fluorescent unit, which had been quenched by the protecting group, was removed by the reducing action of nitroreductase, and thus fluorescence was again exhibited.

[Fluorescent Labeling of Oligonucleotide (T ₂₀ )]
Next, fluorescent labeling was performed on the oligonucleotides. This fluorescently labeled oligonucleotide corresponds to the compound of the present invention. Oligonucleotides were synthesized by solid-phase synthesis according to the phosphoramidite method on an automatic DNA synthesizer. When synthesizing oligonucleotides, compound 23 (phosphoramidite of thymine base) was used to synthesize an oligonucleotide having 20 consecutive thymidines (referred to as _T20 ), and finally compound 14 (phosphoramidite having an ethynyl group) was bound. This resulted in the introduction of an ethynyl group at the 5' end of _T20 . The resulting oligonucleotide is called ODN1.

ODN1 was fluorescently labeled by a click reaction. To a solution (38 μL) of ODN1 (50 nmol) was added a methanol solution (25 μL) containing 50 mM CuSO ₄ and 100 mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), and a 150 mM sodium L-ascorbate aqueous solution (25 μL). L), methanol (50 μL), THF (25 μL) and pure water (15 μL) were added, stirred, and allowed to stand at room temperature for 24 hours. A saturated aqueous solution of ethylenediaminetetraacetic acid (EDTA) (300 μL) and acetonitrile (150 μL) were added to the reaction solution, Cu(I) was precipitated by centrifugation (14000 rpm, 4° C., 30 minutes), and the supernatant was collected. A HPLC preparative column was used to isolate and purify the target peak to obtain fluorescence-labeled ODN1.

The resulting fluorescence-labeled ODN1 was reacted with GSH. The reaction conditions were 1.0 μM fluorescence-labeled ODN1 and 10 mM GSH in a sodium phosphate buffer (pH 7.4) at 37° C. for 480 minutes. The results are shown in FIG. FIG. 4 shows fluorescence spectrum changes when fluorescence-labeled ODN1 is reacted with 10 mM GSH.

As shown in FIG. 4, it was confirmed that the quenched fluorescence of the fluorescence-labeled ODN1 was recovered by reacting with 10 mM GSH. From this, it is understood that even in the fluorescent label bound to the oligonucleotide chain, the protective group is removed by the action of GSH and the fluorescence is recovered.

[Fluorescent Labeling of Oligonucleotide Analog (T′ ₂₀ ) and Intracellular Introduction Experiment]
In addition to compound 23 (phosphoramidite of thymine base), compound 24 (phosphoramidite of thymine base, in which a phenylpropyl group is bound to the OH of phosphoric acid, represented by X) was used to synthesize a nucleotide chain of (5' end) XXXTTXXXXTXXXTTTXXXT (3' end) (referred to as T'20) in the same manner as ODN1 above. , and finally compound ₁₄ (a phosphoramidite having an ethynyl group) was attached. As a result, an ethynyl group was introduced at the 5' end of _T'20 . The resulting oligonucleotide is called ODN2. ODN2 is more lipid-soluble than ODN1, and is thought to improve the efficiency of its introduction into cells.

ODN2 was fluorescently labeled by a click reaction. A methanol solution (25 μL) containing 50 mM CuSO ₄ and 100 mM TBTA, and 150 mM sodium L-ascorbate aqueous solution (25 μL) were added to a solution (50 μL) of ODN2 (100 nmol), and the volume of the solution was reduced once by distilling off under reduced pressure. A THF solution (20 μL) of compound 13 (1000 nmol), methanol (160 μL), THF (60 μL) and pure water (80 μL) were added thereto, stirred, and allowed to stand at 37° C. for 48 hours. A saturated aqueous solution (400 μL) of ethylenediaminetetraacetic acid (EDTA) and acetonitrile (1000 μL) were added to the reaction solution, Cu(I) was precipitated by centrifugation (14000 rpm, 4° C., 30 minutes), and the supernatant was collected. A HPLC preparative column was used to isolate and purify the target peak to obtain fluorescently labeled ODN2.

Hela cells cultured in a CO ₂ incubator were transferred to 2 wells of a 24-malware plate and passaged on coverslips. HeLa cells were adhered to the coverslip and incubated, and two days later, an intracellular transport experiment using fluorescence-labeled ODN2 was performed. The medium was aspirated and washed with Opti-MEM (registered trademark, the same applies hereinafter). One of the two wells was added with 200 μL of Opti-MEM, and the other was added with 10% PBS/Opti-MEM (200 μL) with N-ethylmaleimide (NEM) dissolved therein. NEM has the effect of blocking intracellular GSH and weakening the reactivity to the S—S bond contained in the protecting group of the present invention. That is, one of the two holes is in a state in which intracellular GSH acts, and the other is in a state in which GSH does not act. After standing for 30 minutes in a 37° C. incubator, the solution was aspirated, and 200 μL of fluorescence-labeled ODN2 (10% PBS/Opti-MEM solution) was added to each well. After the malware plate was left in a CO ₂ incubator for 1 hour, it was observed under a fluorescence microscope and an image was obtained. The resulting image is shown in FIG. FIG. 5 is a fluorescence microscopy image showing how fluorescence-labeled ODN2 is taken up into cells.

The upper left image in FIG. 5 is a bright field image showing the position of the cells. The lower left image is the fluorescence image. It can be seen that fluorescence is observed at the position of the cells. The lower right image is a fluorescence image in cells treated with NEM. No clear fluorescence was observed in the NEM-treated cells, indicating that protective groups were removed by intracellular thiols (GSH) to generate fluorescence.

[Changes in UV-visible absorption spectrum and fluorescence spectrum before and after deprotection of fluorescence-labeled ODN2]
For the fluorescence-labeled ODN2 obtained by the above procedure, the ultraviolet-visible absorption spectrum and fluorescence spectrum before deprotection and the ultraviolet-visible absorption spectrum and fluorescence spectrum after deprotection were observed. The ultraviolet-visible absorption spectrum measurement was performed at a concentration of 10 μM of the fluorescently labeled ODN2 in a 20 mM sodium phosphate aqueous solution (pH 7.4) at a liquid temperature of 23° C., and the fluorescence spectral measurement was performed at a liquid temperature of 37° C. in a 100 mM sodium phosphate aqueous solution (pH 7.4) at a concentration of 500 nM of the fluorescently labeled ODN2 and an excitation light wavelength of 495 nm. The results are shown in FIG. FIG. 6 is a chart showing changes in the UV-visible absorption spectrum and fluorescence spectrum of fluorescence-labeled ODN2 before and after deprotection. FIG. 6(a) is a chart showing changes in the UV-visible absorption spectrum, and FIG. 6(b) is a chart showing changes in the fluorescence spectrum. The structures of the fluorescence-labeled ODN2 before and after deprotection are shown in the chemical formulas below.

As shown in FIGS. 6(a) and 6(b), along with the deprotection, strong absorption appeared around 500 nm and strong fluorescence was observed around 530 nm.

[Fluorescence change over time in the presence of GSH of fluorescence-labeled ODN2]
Fluorescence-labeled ODN2 obtained by the above procedure was observed for changes in fluorescence over time in the presence of GSH. Fluorescence spectrum changes were measured in a 100 mM sodium phosphate aqueous solution (pH 7.4) at a liquid temperature of 37° C. at a concentration of 500 nM for fluorescence-labeled ODN2, an excitation wavelength of 495 nm, a fluorescence detection wavelength of 520 nm, and GSH concentrations of 0 μM, 10 μM, 1.0 mM, and 10 mM in the aqueous solution. The results are shown in FIG. FIG. 7 is a chart showing changes in fluorescence over time of fluorescence-labeled ODN2 in the presence of GSH.

As shown in FIG. 7, in the GSH-added solution, an increase in fluorescence intensity was observed over time, indicating that the fluorescence-labeled ODN2 was deprotected in the presence of GSH, an intracellular substance, and began to emit fluorescence.

Claims (11)

A compound represented by the following general formula (1).

（上記一般式（１）中、Ａは、細胞膜を通して細胞内へ取り込まれ得る生体物質又はその誘導体の構造を含む１価の基であり、ＦＬは、下記化学式（２）又は（３）で示す骨格をもつ蛍光ユニットであり、Ｌは、ＡとＦＬとを結合する２価の基であり、Ｘは、単結合又は－Ｃ（＝Ｏ）Ｏ－であり、波線で表す結合は、前記化学式（２）又は（３）に含まれる窒素原子への結合であり、Ａｒは、パラ位又はオルト位に結合を有する２価の芳香環であり、Ｒは、ニトロ基又は－Ｓ－Ｓ－Ｒ ^１であり、Ｒ ^１は、一価の基であり、ｎは、１～３の整数である。）

(In the above chemical formula (2), one of the amino group, the imino group and the carboxyl group is bound to the L, one or two of the Xs are bound to at least one of the amino group and the imino group, and the L is bound to one of the two amino groups in the chemical formula (3), and one or two of the Xs are bound to the remaining amino groups.) The compound according to claim 1, represented by the following general formula (4) or (5).

(In general formulas (4) and (5) above, A, L, X, Ar and R are the same as in general formula (1) above.) The compound according to claim 1 or 2, represented by the following general formula (6) or (7).

(In general formulas (6) and (7) above, A, L, X and R are the same as in general formula (1) above.) The compound according to any one of claims 1 to 3, represented by any one of the following general formulas (8) to (10).

(In general formulas (8) and (9), A, L, and ^R1 are the same as in general formula (1). In general formula (10), A and L are the same as in general formula (1).) A compound according to any one of claims 1 to 4, wherein A is a nucleotide chain or analogue thereof or a peptide chain or analogue thereof. A fluorescent label introduction agent containing a compound represented by the following general formula (1a), (1b) or (1c).

（上記一般式（１ａ）及び（１ｂ）中、ＦＬは、下記化学式（２）又は（３）で示す骨格をもつ蛍光ユニットであり、Ｌは、２価の基であり、Ｘは、単結合又は－Ｃ（＝Ｏ）Ｏ－であり、波線で表す結合は、前記化学式（２）又は（３）に含まれる窒素原子への結合であり、Ａｒは、パラ位又はオルト位に結合を有する２価の芳香環であり、Ｒは、ニトロ基又は－Ｓ－Ｓ－Ｒ ^１であり、Ｒ ^１は、一価の基であり、ｎは、１～３の整数である。上記一般式（１ｃ）中、ＦＬは、下記化学式（２）又は（３）で示す骨格をもつ蛍光ユニットであり、Ａ環は単環でも複数の環が縮合した構造でもよい三重結合を備えた環構造であり、Ａ環が複数の環の縮合した構造の場合、ＬからＡ環への単結合は縮合しているどの環へ結合してもよく、Ｌは、２価の基であり、Ｘは、単結合又は－Ｃ（＝Ｏ）Ｏ－であり、波線で表す結合は、前記化学式（２）又は（３）に含まれる窒素原子への結合であり、Ａｒは、パラ位又はオルト位に結合を有する２価の芳香環であり、Ｒは、ニトロ基又は－Ｓ－Ｓ－Ｒ ^１であり、Ｒ ^１は、一価の基であり、ｎは、１～３の整数である。）

(In the above chemical formula (2), one of the amino group, the imino group and the carboxyl group is bound to the L, one or two of the Xs are bound to at least one of the amino group and the imino group, and the L is bound to one of the two amino groups in the chemical formula (3), and one or two of the Xs are bound to the remaining amino groups.) 7. The fluorescent label introduction agent according to claim 6, represented by the following general formula (4a), (4b), (4c), (5a), (5b) or (5c).

(In the general formulas (4a), (4b), (5a) and (5b), L, X, Ar and R are the same as in the general formula (1a). In the general formulas (4c) and (5c), the A ring, L, X, Ar and R are the same as in the general formula (1c).) 8. The fluorescent label introduction agent according to claim 6 or 7, represented by the following general formula (6a), (6b), (6c), (7a), (7b) or (7c).

(In the general formulas (6a), (6b), (7a) and (7b), L, X and R are the same as in the general formula (1a). In the general formulas (6c) and (7c), the A ring, L, X and R are the same as in the general formula (1c).) The fluorescent label introduction agent according to any one of claims 6 to 8, represented by the following general formulas (8a), (8b), (8c), (9a), (9b), (9c), (10a), (10b) or (10c).

(In the general formulas (8a), (8b), (9a) and (9b), L and R ¹ are the same as in the general formula (1a). In the general formulas (10a) and (10b), L is the same as in the general formula (1a). In the general formulas (8c) and (9c), the A ring, L and R ¹ are the same as in the general formula (1c). In 0c), the A ring and L are the same as in the general formula (1c).) After introducing an alkynyl group or an alkynylene group into a molecule to be labeled, the molecule is reacted with a fluorescent label introduction agent containing a compound having an azide group among the compounds according to any one of claims 6 to 9. After introducing an azide group into a molecule to be labeled, the molecule is reacted with a fluorescent label introduction agent containing a compound having an alkynyl group or an A ring among the compounds according to any one of claims 6 to 9.

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