JP4645234B2 - Cross-linking agent, cross-linking method using the same, gene expression regulation method and gene function investigation method - Google Patents

Description

  The present invention relates to a crosslinking agent having a photodegradable protecting group at both ends, a method for crosslinking a double-stranded nucleic acid using the same, particularly a double-stranded RNA, a gene expression regulation method, and a gene function investigation method.

In recent years, various methods for regulating the expression of a target gene to be analyzed have been proposed as methods for analyzing gene functions.
In particular, the RNA interference (RNAi) method, which utilizes the phenomenon that double-stranded RNA (dsRNA) specifically suppresses target protein expression by promoting the specific degradation of complementary target mRNA, is simple. Moreover, it is widely used as a powerful gene function inhibition method (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5, etc.).

  On the other hand, various so-called caged compounds designed so that the activity of a target substance can be regulated by light have been developed. A caged compound is composed of a photodegradable protecting group, and the activity of the target substance can be stopped by binding to the target substance (caging). It has the property that the original activity of the target substance is recovered (uncaging) (Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Non-Patent Document 9, Patent Document 1, etc.).

In the field of biology, attention has been paid to its characteristics from an early stage, and recently, attempts have been made to use such caged compounds for regulation of gene expression (Patent Document 2, Non-Patent Document 10, etc.).
In this method, mRNA (single strand) is caged with a caged compound, introduced into the cell, and then irradiated at a desired time and site with light, etc. at a certain point to translate the target RNA (ie, protein expression). Is a method of guiding

  However, the above-described method suppresses the expression of a target gene by directly binding a caged compound to single-stranded mRNA. For example, double-stranded RNA (siRNA) used in RNAi as described above, etc. No report has yet been made on the regulation of the expression of a target gene by inhibiting the RNAi effect of siRNA by crosslinking the double-stranded nucleotide chain.

Fire, A. et. Al., (1998), Nature 391, 806-811 Svobada, P. et al., (2000) Development 127, 4147-4156 Elbashir, S. M., Lendeckel, W. and Tuschl, T., (2001) Genes and Dev. 15, 188 -200 Zamore, P. D. et al., (2000) Cell, 101, 25 33 Bernstein, E. et al., (2001) Nature, 409, 363-366 R. S. Givens, C. H. Park, Tetrahedron Lett., 37, 6259-6262 (1996) C. H. Park, R. S. Givens, J. Am. Chem. Soc., 119, 2453-2463 (1997) J. Engels, E. J. Schlaeger, J. Med. Chem. 20, 907 (1977) J. H. Kaplan, G. Forbush III, J. F. Hoffman, Biochemistry 17, 1920-1935 (1978) Biochemistry Vol.75, No.9, pp. 1251-1254, 2003 WO00 / 31588 JP 2002-315576

  The present invention provides a cross-linking agent that cross-links between double-stranded nucleic acids, between nucleic acid-protein or polypeptide, or between proteins or polypeptides, particularly between double-stranded RNAs, their cross-linking method, and expression of a target gene. Provided are a simple gene expression regulation method and a gene function investigation method that can be controlled by time and place.

The present invention has the following configuration.
1. A cross-linking agent in which 6-bromo-7-hydroxy-4-diazomethylcoumarin is linked by a linker at a distance of 8 to 50 mm .
2. A method of cross-linking double-stranded RNA with the cross-linking agent described in 1 above via a phosphate residue of the RNA strand.
3. A method for regulating gene expression, which comprises irradiating light to a double-stranded RNA to which the cross-linking agent described in 1 has been bound in advance (except for application to the human body) .
4). A method for regulating gene expression comprising the following steps (except for application to the human body) .
(a) contacting the double-stranded RNA by bringing the cross-linking agent according to 1 above into contact with the double-stranded RNA;
(b) introducing a crosslinked double-stranded RNA into a cell or organism; and
(c) A step of irradiating the introduced cell or organism with light.
5. Method for investigating gene function including the following steps (except for application to the human body) .
(a) contacting the double-stranded RNA by bringing the cross-linking agent according to 1 above into contact with the double-stranded RNA;
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(c) irradiating the introduced cell or organism with light ,
(c ′) expressing a gene in the irradiated cell or organism, and
(d) a gene expressed in step (c ′) and a gene whose expression is not substantially suppressed by the double-stranded RNA in a cell or organism having a target mRNA complementary to the double-stranded RNA. The step of comparing the expression increase / decrease or the presence / absence of expression, the increase / decrease of the expression product or the presence / absence of the expression product, or the phenotype resulting from the expression .

That is, the present inventors have found that a conventionally known caged compound is effective for suppressing expression of a target gene by binding to a single-stranded nucleotide chain, particularly RNA, When RNAi method was performed by binding to RNA (siRNA), it was found that the RNAi effect of the siRNA could hardly be suppressed and the expression of the target gene could not be controlled. .
Under such circumstances, as a result of intensive studies to achieve the above object, the present inventors cross-linked double-stranded RNA (siRNA) using a cross-linking agent having photodegradable protecting groups at both ends. As a result, it was found that the expression of the target gene can be easily and efficiently controlled at any time and place by RNAi, and the present invention has been completed.
In addition, such a crosslinking agent, the crosslinking method using this, and the gene preparation method are not performed at all, and such an idea is not known at all.

  By cross-linking double-stranded RNA using the cross-linking agent of the present invention, it is possible to easily and efficiently control the expression of the target gene at any time and place. It becomes possible to investigate or / and identify the function of a gene that is specifically expressed.

1. Compound of the Present Invention The compound of the present invention has a photodegradable protecting group at both ends, and is a cross-linking agent between nucleic acids, between nucleic acid-proteins or polypeptides, or between proteins or polypeptides.
The photodegradable protecting group is a group (hereinafter abbreviated as a leaving group) that can be bonded to a phosphate group, a carboxyl group, a hydroxyl group, an amino group, or the like to form a group that undergoes a deprotection reaction upon irradiation with light. ) Having a group capable of binding to a group selected from a phosphoric acid group, a carboxyl group, a hydroxyl group and an amino group, particularly those having a group capable of binding to phosphoric acid. preferable.
In addition, the leaving group of the photodegradable protecting group bonded to both ends is a distance sufficient for binding between nucleic acids, between nucleic acid-protein or polypeptide, or between protein or polypeptide, that is, double-stranded nucleic acid. The sense strand and the antisense strand of each of the present invention, a nucleic acid and a protein or polypeptide, respectively, two proteins or polypeptides (between proteins, between protein-polypeptide or between polypeptides) at a distance capable of cross-linking. The compound (crosslinking agent) is preferably bonded to both ends. In particular, the distance is preferably a distance sufficient to bind to the sense strand and the antisense strand of double-stranded RNA.
More specifically, since it differs depending on the type of target substance to be crosslinked, it cannot be generally stated, but the distance of the leaving group of the photodegradable protecting group bonded to both ends is, for example, the lower limit is usually 8 mm or more, preferably The upper limit is usually at most 100 mm, preferably at most 80 mm.
For example, when cross-linking between nucleic acids (double-stranded DNA, double-stranded RNA, or double-stranded hybrid of DNA and RNA), the lower limit of the distance between the leaving groups of the photodegradable protecting groups attached to both ends is It is usually at least 8 mm, preferably at least 15 mm, more preferably at least 25 mm, and the upper limit is usually at most 50 mm, preferably at most 35 mm.
When the nucleic acid and the protein or polypeptide are cross-linked, or when two proteins or polypeptides are cross-linked, the lower limit of the distance between the leaving groups of the photodegradable protecting groups bonded to both ends is usually 10 mm or more, preferably 25 mm. The upper limit is usually 100 mm or more, preferably 80 mm or more.

〔式中、Ｑ¹及びＱ²はそれぞれ独立して光分解性保護基を示し、Ａ¹及びＡ²はそれぞれ独立してアルキレン基、―O―、−NR¹−、−O−CO−、−CO−O−、−C−O−C−、−NR²−COO−、−OCO−NR²−、−NR³−CO−、−CO−NR³−又は−O−COO−（R¹〜R³はそれぞれ独立してH又はアルキル基を示す。）を、Ｔ¹はアルキレン基、アリーレン基、アラルキレン基、ヘテロ原子を含むアルキレン基、ヘテロ原子を含むアリーレン基又はヘテロ原子を含むアラルキレン基をそれぞれ示す。〕 Specifically, the compound of the present invention is represented by the general formula (1).

[Wherein, Q ¹ and Q ² each independently represent a photodegradable protecting group, and A ¹ and A ² each independently represent an alkylene group, —O—, —NR ¹ —, —O—CO—, -CO-O -, - CO- C -, - NR 2 -COO -, - OCO-NR 2 -, - NR 3 -CO -, - CO-NR 3 - or -O-COO- (R ¹ -R ³ each independently represents H or an alkyl group.), T ¹ represents an alkylene group, an arylene group, an aralkylene group, an alkylene group containing a hetero atom, an arylene group containing a hetero atom, or an aralkylene group containing a hetero atom Respectively. ]

The compound represented by the general formula (1) of the present invention is:
(a) Photodegradable protecting group: Q ¹ and Q ² ,
(b) Linker part: -A ¹ -T ¹ -A ^2-
It can be divided into two components.

1-1. Photodegradable Protecting Group In general formula (1), examples of the photodegradable protecting group represented by Q ¹ and Q ² include a protecting group that has a leaving group and undergoes a deprotection reaction upon irradiation with light. Of these, those having a group capable of binding to phosphoric acid are preferred.

〔一般式（３）中、Ｙ¹、Ｙ²、Ｘ¹、Ｘ²Ａ及びＭ¹の中の１つは、一般式（１）におけるＡ¹又はＡ²と結合する結合手を表し、残りは下記を表す。
Ｑは−O−、−NH−、−NCH₃−を、Ａは水酸基、置換アルコキシ基、非置換アルコキシ基、−OC(O)R¹¹、−NH₂、−NHCH₃、−NR¹¹R¹²を、Ｘ¹及びＸ²はそれぞれ独立して−H、水酸基、置換アルコキシ基、非置換アルコキシ基、−OC(O)R¹¹基、−NH₃基、−NR¹¹R¹²基、−R¹¹、−F、−Cl、−Br、−I、−COOH、−NO₂、−C(=O)NHR¹¹、−CN、−CHO、−C(=O)R¹¹、−SO₃Hを、Ｙ¹は−H、−Cl、−Br、−I、−C(O)OH、−NO₂、−C(O)NHR¹¹、−CN、−C(O)H、−C(O)CH₃、ベンゾキサゾール-2-イル基、−ベンゾチアゾール-2-イル、−ベンズイミダゾール-2-イルを、Ｙ²は−H、−C(O)OH、−SO₃Hを、Ｍ¹は−H、−CH₃、−NR¹²R¹³基、−C(O)NR¹²R¹³基、−COOHを、Ｚは脱離基をそれぞれ示す。また、Ｍ²は−H或いはＺと共に＝N₂、＝O又は＝NNHR¹¹であることを示す。ここで、Ｒ¹¹、Ｒ¹²及びＲ¹³はそれぞれ独立して炭素数1-20のアルキル基、炭素数2-20のアルケニル基、炭素数2-20のアルキニル基、炭素数1-20のアルコキシ基、炭素数1-20のチオアルコキシ基、炭素数1-20のアルキルスルホニル基、炭素数4-16のアリールスルホニル基、炭素及びヘテロ原子の総数2-20のヘテロアルキル基、炭素及びヘテロ原子の総数2-20のヘテロアルケニル基、炭素数3-8のシクロアルキル基、炭素数3-8のシクロアルケニル基、炭素数4-16のアリール基、炭素及びヘテロ原子の総数4-16のヘテロアリール基、炭素及びヘテロ原子の総数2-30のヘテロシクリル基から選ばれる置換官能基又は非置換官能基を示し、Ｘ¹及びＡ、Ｘ²及びＡ又はＸ¹及びＹ²は一緒になって−O−(CH₂)_n−O−基、−C−(CH₂)_n−O−基、−O−(CH₂)_n−C−基、−O−(CH₂)_n−N−基、−N−(CH₂)_n−O−基、−N−(CH₂)_n−N−基、−C−(CH₂)_n−N−基及び−N−(CH₂)_n−C−基から選ばれる基を形成してもよい。ここで、nは1又は2である。〕 More specifically, photodegradable protecting groups represented by the following general formulas (3), (3 ′), (3 ″) and (3 ′ ″) can be mentioned.

[In General Formula (3), ^one of Y ¹ , Y ² , X ¹ , X ² A and M ¹ represents a bond bonded to A ¹ or A ² in General Formula (1), and the rest Represents the following.
Q is -O -, - NH -, - NCH 3 - a, A is a hydroxyl group, a substituted alkoxy group, an unsubstituted alkoxy ^{group, -OC (O) R 11,} -NH 2, -NHCH 3, -NR 11 R 12 X ¹ and X ² are each independently —H, a hydroxyl group, a substituted alkoxy group, an unsubstituted alkoxy group, —OC (O) R ¹¹ group, —NH ₃ group, —NR ¹¹ R ¹² group, —R ¹¹ , -F, -Cl, -Br, -I , -COOH, -NO 2, -C (= O) NHR 11, -CN, -CHO, -C (= O) R 11, a -SO ₃ H, Y ¹ is -H, -Cl, -Br, -I, -C (O) OH, -NO 2, -C (O) NHR 11, -CN, -C (O) H, -C (O) CH _3, benzoxazol-2-yl group, - benzothiazol-2-yl, - benzimidazole-2-yl, Y ² is -H, -C (O) OH, a -SO ₃ H, M ¹ is ^{_{-H, -CH 3, -NR 12 R}} 13 ^{groups, -C (O) NR 12 R} 13 group, a -COOH, Z represents a leaving group, respectively. In addition, M ² together with -H or Z represents = N ₂ , = O or = NNHR ¹¹ . Here, R ¹¹ , R ¹² and R ¹³ are each independently an alkyl group having 1-20 carbon atoms, an alkenyl group having 2-20 carbon atoms, an alkynyl group having 2-20 carbon atoms, an alkoxy group having 1-20 carbon atoms. Group, thioalkoxy group having 1 to 20 carbon atoms, alkylsulfonyl group having 1 to 20 carbon atoms, arylsulfonyl group having 4 to 16 carbon atoms, heteroalkyl group having 2 to 20 carbon atoms in total, and carbon and heteroatoms A total of 2-20 heteroalkenyl groups, a C3-C8 cycloalkyl group, a C3-C8 cycloalkenyl group, a C4-C16 aryl group, a C4-hetero total number of carbons and heteroatoms A substituted functional group or an unsubstituted functional group selected from an aryl group, a carbon and a heterocyclyl group having a total number of heteroatoms of 2-30, wherein X ¹ and A, X ² and A or X ¹ and Y ² are taken together— O- (CH _{2) n} -O- group, -C- (CH _{2) n} -O- group, -O- (CH _{2) n} -C- group, -O- (CH ₂ ) _n -N- group, -N- (CH ₂ ) _n -O- group, -N- (CH ₂ ) _n -N- group, -C- (CH ₂ ) _n -N- group and -N- ( A group selected from CH ₂ ) _n —C— groups may be formed. Here, n is 1 or 2. ]

〔一般式（３'）中、Ｒ²²〜Ｒ²⁵の中の１つは、一般式（１）におけるＡ¹又はＡ²と結合する結合手を表し、残りは下記を表す。
Ｒ²¹は水素原子、−COOH、炭素数1-20のアルキル基、炭素数2-20のアルケニル基、炭素数2-20のアルキニル基、炭素数1-20のアルコキシ基、炭素数1-20のチオアルコキシ基、炭素数1-20のアルキルスルホニル基、炭素数4-16のアリールスルホニル基、炭素及びヘテロ原子の総数2-20のヘテロアルキル基、炭素及びヘテロ原子の総数2-20のヘテロアルケニル基、炭素数3-8のシクロアルキル基、炭素数3-8のシクロアルケニル基、炭素数4-16のアリール基、炭素及びヘテロ原子の総数4-16のヘテロアリール基、炭素及びヘテロ原子の総数2-30のヘテロシクリル基から選ばれる置換官能基又は非置換官能基を、Ｒ²²及びＲ²³はそれぞれ独立して炭素数1-20のアルキル基、炭素数2-20のアルケニル基、炭素数2-20のアルキニル基、炭素数1-20のアルコキシ基、炭素数1-20のチオアルコキシ基、炭素数1-20のアルキルスルホニル基、炭素数4-16のアリールスルホニル基、炭素及びヘテロ原子の総数2-20のヘテロアルキル基、炭素及びヘテロ原子の総数2-20のヘテロアルケニル基、炭素数3-8のシクロアルキル基、炭素数3-8のシクロアルケニル基、炭素数4-16のアリール基、炭素及びヘテロ原子の総数4-16のヘテロアリール基、炭素及びヘテロ原子の総数2-30のヘテロシクリル基から選ばれる置換官能基又は非置換官能基を、Ｒ²⁴及びＲ²⁵は水素原子をそれぞれ示し、Ｒ²²及びＲ²³は一緒になって−(CH₂)_n−基を形成してもよく、Ｒ²³及びＲ²⁴は一緒になって−(CH₂)_n−O−基、−(CH₂)_n−C−基及び−(CH₂)_n−N−基から選ばれる基を形成してもよく、Ｒ²²及びＲ²⁵は一緒になって−O−(CH₂)_n−基、−C−(CH₂)_n−基及び−N−(CH₂)_n−基から選ばれる基を形成してもよい。ここで、nは1又は2である。〕

[In General Formula (3 ′), one of R ^{22 to} R ²⁵ represents a bond bonded to A ¹ or A ² in General Formula (1), and the rest represents the following.
R ²¹ represents a hydrogen atom, —COOH, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, or 1-20 carbon atoms. A thioalkoxy group, an alkylsulfonyl group having 1 to 20 carbon atoms, an arylsulfonyl group having 4 to 16 carbon atoms, a heteroalkyl group having 2 to 20 carbon atoms and a total number of heteroatoms, and a heterocycle having 2 to 20 carbon atoms in total. An alkenyl group, a cycloalkyl group having 3 to 8 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, an aryl group having 4 to 16 carbon atoms, a heteroaryl group having 4 to 16 carbon atoms in total, and carbon and heteroatoms A substituted or unsubstituted functional group selected from 2-30 heterocyclyl groups, wherein R ²² and R ²³ are each independently an alkyl group having 1-20 carbon atoms, an alkenyl group having 2-20 carbon atoms, carbon C2-C20 alkynyl group, C1-C20 alkoxy group, C1-C20 thioa Coxy group, alkylsulfonyl group having 1-20 carbon atoms, arylsulfonyl group having 4-16 carbon atoms, heteroalkyl group having 2-20 total carbon and heteroatoms, and heteroalkenyl group having 2-20 total carbon and heteroatoms , A cycloalkyl group having 3 to 8 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, an aryl group having 4 to 16 carbon atoms, a heteroaryl group having 4 to 16 carbon atoms and hetero atoms, and a total number of carbon and hetero atoms A substituted or unsubstituted functional group selected from 2-30 heterocyclyl groups, R ²⁴ and R ²⁵ each represent a hydrogen atom, and R ²² and R ²³ together represent a — (CH ₂ ) _n — group. R ²³ and R ²⁴ together may be selected from the group — (CH ₂ ) _n —O—, — (CH ₂ ) _n —C— and — (CH ₂ ) _n —N—. R ²² and R ²⁵ together may form a —O— (CH ₂ ) _n — group, —C— (CH ₂ ) _n — group and —N— (CH ₂ ) _n —. A group selected from groups may be formed. Here, n is 1 or 2. ]

〔一般式（３''）中、Ｒ²¹及びＲ²⁶〜Ｒ²⁹の中の１つは、一般式（１）におけるＡ¹又はＡ²と結合する結合手を表し、残りは下記を表す。
Ｒ²¹は水素原子、−COOH、炭素数1-20のアルキル基、炭素数2-20のアルケニル基、炭素数2-20のアルキニル基、炭素数1-20のアルコキシ基、炭素数1-20のチオアルコキシ基、炭素数1-20のアルキルスルホニル基、炭素数4-16のアリールスルホニル基、炭素及びヘテロ原子の総数2-20のヘテロアルキル基、炭素及びヘテロ原子の総数2-20のヘテロアルケニル基、炭素数3-8のシクロアルキル基、炭素数3-8のシクロアルケニル基、炭素数4-16のアリール基、炭素及びヘテロ原子の総数4-16のヘテロアリール基、炭素及びヘテロ原子の総数2-30のヘテロシクリル基から選ばれる置換官能基又は非置換官能基を、Ｒ²⁶〜Ｒ²⁹は水素原子をそれぞれ示し、Ｒ²⁷及びＲ²⁸、Ｒ²⁸及びＲ²⁹又はＲ²⁷及びＲ²⁶は、一緒になって−O−(CH₂)_n−O−基、−C−(CH₂)_n−O−基、−O−(CH₂)_n−C−基、−O−(CH₂)_n−N−基、−N−(CH₂)_n−O−基、−N−(CH₂)_n−N−基、−C−(CH₂)_n−N−基及び−N−(CH₂)_n−C−基から選ばれる基を形成してもよい。ここで、nは1又は2である。。〕

[In General Formula (3 ″), one of R ²¹ and R ^{26 to} R ²⁹ represents a bond bonded to A ¹ or A ² in General Formula (1), and the rest represents the following.
R ²¹ is a hydrogen atom, —COOH, an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 2 to 20 carbon atoms, an alkynyl group having 2 to 20 carbon atoms, an alkoxy group having 1 to 20 carbon atoms, or 1-20 carbon atoms. A thioalkoxy group, an alkylsulfonyl group having 1 to 20 carbon atoms, an arylsulfonyl group having 4 to 16 carbon atoms, a heteroalkyl group having 2 to 20 carbon atoms and a total number of heteroatoms, and a heterocycle having 2 to 20 carbon atoms and a total number of heteroatoms. An alkenyl group, a cycloalkyl group having 3 to 8 carbon atoms, a cycloalkenyl group having 3 to 8 carbon atoms, an aryl group having 4 to 16 carbon atoms, a heteroaryl group having 4 to 16 carbon atoms and a total number of heteroatoms, carbon and heteroatoms A substituted functional group or an unsubstituted functional group selected from 2-30 heterocyclyl groups, wherein R ^{26 to} R ²⁹ each represent a hydrogen atom, R ²⁷ and R ²⁸ , R ²⁸ and R ²⁹ or R ²⁷ and R ²⁶ Together with —O— (CH ₂ ) _n —O— group, —C— (CH ₂ ) _n —O— group, —O— (CH ₂ ) _n -C- group, -O- (CH ₂ ) _n -N- group, -N- (CH ₂ ) _n -O- group, -N- (CH ₂ ) _n -N- group, -C- ( CH _{2) n-n-group} and -N- (CH _{2) n} -C- group may be formed selected from group. Here, n is 1 or 2. . ]

〔一般式（３'''）中、Ｒ³⁰及びＲ³²〜Ｒ³⁵の中の１つは、一般式（１）におけるＡ¹又はＡ²と結合する結合手を表し、残りは下記を表す。
Ｒ³⁰は炭素数1-20のアルキル基、炭素数2-20のアルケニル基、炭素数2-20のアルキニル基、炭素数1-20のアルコキシ基、炭素数1-20のチオアルコキシ基、炭素数1-20のアルキルスルホニル基、炭素数4-16のアリールスルホニル基、炭素及びヘテロ原子の総数2-20のヘテロアルキル基、炭素及びヘテロ原子の総数2-20のヘテロアルケニル基、炭素数3-8のシクロアルキル基、炭素数3-8のシクロアルケニル基、炭素数4-16のアリール基、炭素及びヘテロ原子の総数4-16のヘテロアリール基、炭素及びヘテロ原子の総数2-30のヘテロシクリル基から選ばれる置換官能基又は非置換官能基を、Ｒ³¹はハロゲン原子を、Ｒ³²〜Ｒ³⁵は水素原子をそれぞれ示し、Ｒ³³及びＲ³⁰は一緒になって−O−(CH₂)_n−基、−C−(CH₂)_n−基及び−N−(CH₂)_n−基から選ばれる基を形成してもよく、Ｒ³⁰及びＲ³⁴は一緒になって−(CH₂)_n−O−基、−(CH₂)_n−C−基及び−(CH₂)_n−N−基から選ばれる基を形成してもよく、Ｒ³²及びＲ³³は一緒になって−O−(CH₂)_n−O−基、−C−(CH₂)_n−O−基、−O−(CH₂)_n−C−基、−O−(CH₂)_n−N−基、−N−(CH₂)_n−O−基、−N−(CH₂)_n−N−基、−C−(CH₂)_n−N−基及び−N−(CH₂)_n−C−基から選ばれる基を形成してもよい。ここで、nは1又は2である。〕

[In General Formula (3 ′ ″), one of R ³⁰ and R ^{32 to} R ³⁵ represents a bond bonded to A ¹ or A ² in General Formula (1), and the rest represents the following: .
R ³⁰ is an alkyl group having 1-20 carbon atoms, an alkenyl group having 2-20 carbon atoms, an alkynyl group having 2-20 carbon atoms, an alkoxy group having 1-20 carbon atoms, a thioalkoxy group having 1-20 carbon atoms, carbon An alkylsulfonyl group having 1 to 20 carbon atoms, an arylsulfonyl group having 4 to 16 carbon atoms, a heteroalkyl group having 2 to 20 carbon atoms and a total number of heteroatoms, a heteroalkenyl group having 2 to 20 carbon atoms and a total number of heteroatoms, 3 carbon atoms -8 cycloalkyl group, 3-8 carbon cycloalkenyl group, 4-16 aryl group, 4-16 heteroaryl group, total number of carbon and heteroatoms, 2-30 total carbon and heteroatoms A substituted or unsubstituted functional group selected from a heterocyclyl group, R ³¹ represents a halogen atom, R ^{32 to} R ³⁵ represent a hydrogen atom, and R ³³ and R ³⁰ together represent —O— (CH ₂ ) _n -group, -C- (CH ₂ ) _n -group and -N- (CH ₂ ) _n- group to form a group R ³⁰ and R ³⁴ together represent a group selected from a — (CH ₂ ) _n —O— group, a — (CH ₂ ) _n —C— group and a — (CH ₂ ) _n —N— group. R ³² and R ³³ may be combined together to form an —O— (CH ₂ ) _n —O— group, —C— (CH ₂ ) _n —O— group, —O— (CH ₂ ) _n -C- group, -O- (CH _{2) n} -N- group, -N- (CH _{2) n} -O- group, -N- (CH _{2) n} -N- group, -C- (CH ₂ ) _n-n-group and -N- (CH _{2) n} -C- group may be formed selected from group. Here, n is 1 or 2. ]

In the general formula (3), the substituted alkoxy group and unsubstituted alkoxy group represented by A, X ¹ and X ² may be linear, branched or cyclic, but are preferably linear. And those having 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms.
Specifically, for example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, n-pentyloxy group, isopentyloxy group, sec-pentyloxy group, tert-pentyloxy group, neopentyloxy group, 2-methylbutoxy group, 1-ethylpropoxy group, n-hexyloxy group, isohexyloxy group, sec-hexyloxy group, tert-hexyloxy Group, neohexyloxy group, 2-methylpentyloxy group, 3-methylpentyloxy group, 1,2-dimethylbutoxy group, 2,2-dimethylbutoxy group, 1-ethylbutoxy group, 2-ethylbutoxy group, n -Heptyloxy group, isoheptyloxy group, sec-heptyloxy group, t rt-heptyloxy group, neoheptyloxy group, n-octyloxy group, isooctyloxy group, sec-octyloxy group, tert-octyloxy group, neooctyloxy group, n-nonyloxy group, isononyloxy group, sec -Nonyloxy group, tert-nonyloxy group, neononyloxy group, n-decyloxy group, isodecyloxy group, sec-decyloxy group, tert-decyloxy group, neodecyloxy group, n-undecyloxy group, isoundecyloxy group Group, sec-undecyloxy group, tert-undecyloxy group, neoundecyloxy group, n-dodecyloxy group, isododecyloxy group, sec-dodecyloxy group, tert-dodecyloxy group, neododecyloxy group, n-tridecyloxy group, iso Ridecyloxy group, sec-tridecyloxy group, tert-tridecyloxy group, neotridecyloxy group, n-tetradecyloxy group, isotetradecyloxy group, sec-tetradecyloxy group, tert-tetradecyloxy group, Neotetradecyloxy group, n-pentadecyloxy group, isopentadecyloxy group, sec-pentadecyloxy group, tert-pentadecyloxy group, neopentadecyloxy group, n-hexadecyloxy group, sec-hexadecyloxy group Tert-hexadecyloxy group, neohexadecyloxy group, n-heptadecyloxy group, isoheptadecyloxy group, sec-heptadecyloxy group, tert-heptadecyloxy group, neoheptadecyloxy group, n-octadecyloxy group , Isoo Kutadecyloxy group, sec-octadecyloxy group, tert-octadecyloxy group, neooctadecyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group, cycloheptyloxy group, cyclooctyloxy group, Cyclononyloxy group, cyclodecyloxy group, cyclododecyloxy group, cycloundecyloxy group, cyclotridecyloxy group, cyclotetradecyloxy group, cyclopentadecyloxy group, cyclohexadecyloxy group, cycloheptadecyloxy group, Examples include a cyclooctadecyloxy group.

  Examples of the substituent include a carboxyl group, a hydroxyl group, a sulfonic acid group, such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. Lower alkyl group having 1 to 4 carbon atoms such as fluoromethyl group, difluoromethyl group, trifluoromethyl group, chloromethyl group, dichloromethyl group, trichloromethyl group, bromomethyl group, dibromomethyl group, tribromomethyl group, Iodomethyl group, diiodomethyl group, triiodomethyl group, trifluoroethyl group, trichloroethyl group, tribromoethyl group, pentafluoroethyl group, pentachloroethyl group, pentabromoethyl group, heptafluoropropyl group, heptachloropropyl group, Nonafluorobutyl group, nonachlorobutyl group C1-C4 halo lower alkyl group such as nonabromobutyl group, nonaiodobutyl group, for example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy And a lower alkoxy group having 1 to 4 carbon atoms such as a group.

In the general formula (3), the leaving group represented by Z is a group that can be removed by photolysis by bonding to a phosphate group, carboxyl group, hydroxyl group, amino group, or the like (deprotection by light irradiation). A group capable of forming a group on which a reaction takes place).
Generally, it is a group capable of leaving from a substrate (compound to which the leaving group is bonded) together with a covalent electron pair between the leaving group and the substrate (compound to which the leaving group is bonded). Preferred leaving groups include groups in which the electron pair is stabilized by the presence of electron withdrawing, aromatic, resonant structures or combinations thereof, such as halides, or carboxylates, carbonates, amides. , A carbamate, a phosphate, a sulfonate, an amino, an aryl oxide, or a group to which a thiolate group is bonded.
More specifically, examples of the leaving group represented by Z include a halogen atom, an alkoxy group, an aryloxy group, a substituted aryloxy group, —NR ¹⁵ R ¹⁶ , —OC (O) R ¹⁴ , —OP (O). ^{R 15 R 16, -OP (O} ) (OH) R 15, -OC (O) NR 15 R 16, -NR 15 C (O) OR 16, -SR 14, -NR 15 C (O) R 16, -O ₃ SR ¹⁴ or -O-NN (O) (NR 15 R 16) can be mentioned.
Here, R ¹⁴ , R ¹⁵ and R ¹⁶ are each independently an alkyl group having 1-20 carbon atoms, an alkenyl group having 2-20 carbon atoms, an alkynyl group having 2-20 carbon atoms, or 1-20 carbon atoms. An alkoxy group having 1 to 20 carbon atoms, an alkylsulfonyl group having 1 to 20 carbon atoms, an arylsulfonyl group having 4 to 16 carbon atoms, a heteroalkyl group having 2 to 20 carbon atoms, and 2 to 20 carbon atoms Heteroalkenyl group, cycloalkyl group having 3-8 carbon atoms, cycloalkenyl group having 3-8 carbon atoms, aryl group having 4-16 carbon atoms, heteroaryl group having 4-16 carbon atoms, heterocyclyl having 2-30 carbon atoms A substituted functional group or an unsubstituted functional group selected from a group, and R ¹⁵ and R ¹⁶ may together form an alkylene group having 1 to 20 carbon atoms.

Examples of the halogen atom represented by Z include F, Cl, Br, and I.
In addition, the alkoxy group may be linear, branched or cyclic, but is preferably linear, usually having 1 to 20 carbon atoms, preferably 1 to 5 carbon atoms.
Specifically, for example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, n-pentyloxy group, isopentyloxy group, sec-pentyloxy group, tert-pentyloxy group, neopentyloxy group, 2-methylbutoxy group, 1-ethylpropoxy group, n-hexyloxy group, isohexyloxy group, sec-hexyloxy group, tert-hexyloxy Group, neohexyloxy group, 2-methylpentyloxy group, 3-methylpentyloxy group, 1,2-dimethylbutoxy group, 2,2-dimethylbutoxy group, 1-ethylbutoxy group, 2-ethylbutoxy group, n -Heptyloxy group, isoheptyloxy group, sec-heptyloxy group, t rt-heptyloxy group, neoheptyloxy group, n-octyloxy group, isooctyloxy group, sec-octyloxy group, tert-octyloxy group, neooctyloxy group, n-nonyloxy group, isononyloxy group, sec -Nonyloxy group, tert-nonyloxy group, neononyloxy group, n-decyloxy group, isodecyloxy group, sec-decyloxy group, tert-decyloxy group, neodecyloxy group, n-undecyloxy group, isoundecyloxy group Group, sec-undecyloxy group, tert-undecyloxy group, neoundecyloxy group, n-dodecyloxy group, isododecyloxy group, sec-dodecyloxy group, tert-dodecyloxy group, neododecyloxy group, n-tridecyloxy group, iso Ridecyloxy group, sec-tridecyloxy group, tert-tridecyloxy group, neotridecyloxy group, n-tetradecyloxy group, isotetradecyloxy group, sec-tetradecyloxy group, tert-tetradecyloxy group, Neotetradecyloxy group, n-pentadecyloxy group, isopentadecyloxy group, sec-pentadecyloxy group, tert-pentadecyloxy group, neopentadecyloxy group, n-hexadecyloxy group, sec-hexadecyloxy group Tert-hexadecyloxy group, neohexadecyloxy group, n-heptadecyloxy group, isoheptadecyloxy group, sec-heptadecyloxy group, tert-heptadecyloxy group, neoheptadecyloxy group, n-octadecyloxy group , Isoo Kutadecyloxy group, sec-octadecyloxy group, tert-octadecyloxy group, neooctadecyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group, cycloheptyloxy group, cyclooctyloxy group, Cyclononyloxy group, cyclodecyloxy group, cyclododecyloxy group, cycloundecyloxy group, cyclotridecyloxy group, cyclotetradecyloxy group, cyclopentadecyloxy group, cyclohexadecyloxy group, cycloheptadecyloxy group, Examples include a cyclooctadecyloxy group.
The aryloxy group represented by Z and the aryloxy group of the substituted aryloxy group include aromatic monocyclic or condensed polycyclic having 4 to 16 carbon atoms, preferably 5 to 14 carbon atoms. Examples include phenyloxy group, tolyloxy group, xylyloxy group, mesityloxy group, naphthyloxy group, anthryloxy group, phenanthryloxy group and the like.
Examples of the substituent of the substituted aryloxy group include a halogen atom (F, Cl, Br or I), a nitro group, a C 1-3 perfluoroalkyl group (trifluoromethyl group, pentafluoroethyl group, hepta). Fluoropropyl group) and the like.

In general formula (3), an alkyl group having 1 to 20 carbon atoms represented by R ^{11 to} R ¹⁶ , an alkyl group having 1 to 20 carbon atoms represented by R ^{21 to} R ²³ in general formula (3 ′), The alkyl group having 1-20 carbon atoms represented by R ²¹ in 3 ″) and the alkyl group having 1-20 carbon atoms represented by R ³⁰ in general formula (3 ′ ″) are linear or branched However, linear ones are preferable, and those having 1 to 5 carbon atoms are preferable.
Specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, sec-pentyl group Tert-pentyl group, neopentyl group, n-hexyl group, isohexyl group, sec-hexyl group, tert-hexyl group, neohexyl group, n-heptyl group, isoheptyl group, sec-heptyl group, tert-heptyl group, neoheptyl group , N-octyl group, isooctyl group, sec-octyl group, tert-octyl group, neooctyl group, n-nonyl group, isononyl group, sec-nonyl group, tert-nonyl group, neononyl group, n-decyl group, isodecyl group , Sec-decyl group, tert-decyl group, neodecyl group, n-undecyl group, isoundecyl group, sec-undecyl group, tert-undecyl group, neoundecyl group, n-dodecyl group Group, isododecyl group, sec-dodecyl group, tert-dodecyl group, neododecyl group, n-tridecyl group, isotridecyl group, sec-tridecyl group, tert-tridecyl group, neotridecyl group, n-tetradecyl group, isotetradecyl group, sec-tetradecyl group, tert-tetradecyl group, neotetradecyl group, n-pentadecyl group, isopentadecyl group, sec-pentadecyl group, tert-pentadecyl group, neopentadecyl group, n-hexadecyl group, isohexadecyl group , Sec-hexadecyl group, tert-hexadecyl group, neohexadecyl group, n-heptadecyl group, isoheptadecyl group, sec-heptadecyl group, tert-heptadecyl group, neoheptadecyl group, n-octadecyl group, isooctadecyl group, sec-octadecyl group Group, tert-octadecyl group, neooctadecyl group, n-nonadecyl group, a Examples include sononadecyl group, sec-nonadecyl group, tert-nonadecyl group, neononadecyl group, n-icosyl group, isoicosyl group, sec-icosyl group, tert-icosyl group, neoicosyl group and the like.

The alkenyl group having 2-20 carbon atoms represented by R ^{11 to} R ¹⁶ in the general formula (3), the alkenyl group having 2-20 carbon atoms represented by R ^{21 to} R ²³ in the general formula (3 ′), In 3 ″), the alkenyl group having 2 to 20 carbon atoms represented by R ²¹ and the alkenyl group having 2 to 20 carbon atoms represented by R ³⁰ in formula (3 ′ ″) have at least one double bond. It is a linear or branched, preferably linear hydrocarbon group, preferably having 2 to 5 carbon atoms.
Specifically, for example, ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 2-methyl-2-propenyl, 2- Ethyl-2-propenyl, 1-butenyl, 2-butenyl, 1-methyl-2-butenyl, 1-methyl-1-butenyl, 3-methyl-2-butenyl, 1-ethyl-2-butenyl, 3-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 1-ethyl-3-butenyl, 1-pentenyl, 2-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3- Pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 4-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 1-hexene , 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, heptenyl, octenyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, heptadecenyl, heptadecadienyl, heptadecaenyl, octadecenyl, nonadecenyl, eicosenyl, etc. It is done.

In the general formula (3), an alkynyl group having 2 to 20 carbon atoms represented by R ^{11 to} R ¹⁶ , an alkynyl group having 2 to 20 carbon atoms represented by R ^{21 to} R ²³ in the general formula (3 ′), 3 ″) the alkynyl group having 2-20 carbon atoms represented by R ²¹ and the alkynyl group having 2-20 carbon atoms represented by R ³⁰ in formula (3 ′ ″) have at least one triple bond. , A linear, branched or cyclic, preferably a linear aliphatic hydrocarbon group, preferably having 2 to 5 carbon atoms.
Specifically, for example, ethynyl group, 1-propynyl, 2-propynyl group, propargyl, 1-butynyl group, 2-butynyl group, pentynyl group, hexynyl group, octynyl group, 2-ethylhexynyl group, decynyl group, dodecynyl group, An octadecynyl group etc. are mentioned.

Formula alkoxy group having 1 to 20 carbon atoms represented by R ¹¹ to R ¹⁶ in (3), the general formula (3 ') in the alkoxy group having 1 to 20 carbon atoms represented by R ²¹ to R ²³ of general formula ( The C 1-20 alkoxy group represented by R ²¹ in 3 ″) and the C 1-20 alkoxy group represented by R ³⁰ in the general formula (3 ′ ″) are linear or branched. Alternatively, it may be cyclic, but is preferably linear, and preferably has 2 to 5 carbon atoms.
Specifically, for example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, isobutoxy group, sec-butoxy group, tert-butoxy group, n-pentyloxy group, isopentyloxy group, sec-pentyloxy group, tert-pentyloxy group, neopentyloxy group, 2-methylbutoxy group, 1-ethylpropoxy group, n-hexyloxy group, isohexyloxy group, sec-hexyloxy group, tert-hexyloxy Group, neohexyloxy group, 2-methylpentyloxy group, 3-methylpentyloxy group, 1,2-dimethylbutoxy group, 2,2-dimethylbutoxy group, 1-ethylbutoxy group, 2-ethylbutoxy group, n -Heptyloxy group, isoheptyloxy group, sec-heptyloxy group, t rt-heptyloxy group, neoheptyloxy group, n-octyloxy group, isooctyloxy group, sec-octyloxy group, tert-octyloxy group, neooctyloxy group, n-nonyloxy group, isononyloxy group, sec -Nonyloxy group, tert-nonyloxy group, neononyloxy group, n-decyloxy group, isodecyloxy group, sec-decyloxy group, tert-decyloxy group, neodecyloxy group, n-undecyloxy group, isoundecyloxy group Group, sec-undecyloxy group, tert-undecyloxy group, neoundecyloxy group, n-dodecyloxy group, isododecyloxy group, sec-dodecyloxy group, tert-dodecyloxy group, neododecyloxy group, n-tridecyloxy group, iso Ridecyloxy group, sec-tridecyloxy group, tert-tridecyloxy group, neotridecyloxy group, n-tetradecyloxy group, isotetradecyloxy group, sec-tetradecyloxy group, tert-tetradecyloxy group, Neotetradecyloxy group, n-pentadecyloxy group, isopentadecyloxy group, sec-pentadecyloxy group, tert-pentadecyloxy group, neopentadecyloxy group, n-hexadecyloxy group, sec-hexadecyloxy group Tert-hexadecyloxy group, neohexadecyloxy group, n-heptadecyloxy group, isoheptadecyloxy group, sec-heptadecyloxy group, tert-heptadecyloxy group, neoheptadecyloxy group, n-octadecyloxy group , Isoo Kutadecyloxy group, sec-octadecyloxy group, tert-octadecyloxy group, neooctadecyloxy group, cyclopropyloxy group, cyclobutyloxy group, cyclopentyloxy group, cyclohexyloxy group, cycloheptyloxy group, cyclooctyloxy group, Cyclononyloxy group, cyclodecyloxy group, cyclododecyloxy group, cycloundecyloxy group, cyclotridecyloxy group, cyclotetradecyloxy group, cyclopentadecyloxy group, cyclohexadecyloxy group, cycloheptadecyloxy group, Examples include a cyclooctadecyloxy group.

A thioalkoxy group having 1 to 20 carbon atoms represented by R ^{11 to} R ¹⁶ in the general formula (3), a thioalkoxy group having 1 to 20 carbon atoms represented by R ^{21 to} R ²³ in the general formula (3 ′), The thioalkoxy group having 1-20 carbon atoms represented by R ²¹ in the formula (3 ″) and the thioalkoxy group having 1-20 carbon atoms represented by R ³⁰ in the general formula (3 ′ ″) are linear. May be branched or cyclic, but is preferably linear, and preferably has 2 to 5 carbon atoms.
Specifically, for example, specifically, for example, thiomethoxy group, thioethoxy group, thiopropoxy group, thiobutoxy group, thiopentyloxy group, thiomethylbutoxy group, thioethylpropoxy group, thiohexyloxy group, thiomethylpentyloxy group Thiodimethylbutoxy group, thioethylbutoxy group, thioheptyloxy group, thiooctyloxy group, thiononyloxy group, thiodecyloxy group, thioundecyloxy group, thiododecyloxy group, thiotridecyloxy group, thiotetra Decyloxy, thiopentadecyloxy, thiohexadecyloxy, thioheptadecyloxy, thiooctadecyloxy, thiocyclopropyloxy, thiocyclobutyloxy, thiocyclopentyloxy, thiocyclohexyloxy, Loheptyloxy group, thiocyclooctyloxy group, thiocyclononyloxy group, thiocyclodecyloxy group, thiocyclododecyloxy group, thiocycloundecyloxy group, thiocyclotridecyloxy group, thiocyclotetradecyloxy group, Examples thereof include a thiocyclopentadecyloxy group, a thiocyclohexadecyloxy group, a thiocycloheptadecyloxy group, and a thiocyclooctadecyloxy group.

1 to 20 alkylsulfonyl groups represented by R ^{11 to} R ¹⁶ in the general formula (3), 1 to 20 alkylsulfonyl groups represented by R ^{21 to} R ²³ in the general formula (3 ′), The alkylsulfonyl group having 1-20 carbon atoms represented by R ²¹ in the formula (3 ″) and the alkylsulfonyl group having 1-20 carbon atoms represented by R ³⁰ in the general formula (3 ′ ″) are linear. May be branched or cyclic, but is preferably linear, and preferably has 2 to 5 carbon atoms.
Specifically, for example, methylsulfonyl group, ethylsulfonyl group, propylsulfonyl group, butylsulfonyl group, pentylsulfonyl group, hexylsulfonyl group, heptylsulfonyl group, octylsulfonyl group, nonylsulfonyl group, decylsulfonyl group, undecylsulfonyl group , Dodecylsulfonyl group, tridecylsulfonyl group, tetradecylsulfonyl group, pentadecylsulfonyl group, hexadecylsulfonyl group, heptadecylsulfonyl group, octadecylsulfonyl group, nonadecylsulfonyl group, icosylsulfonyl group and the like.

4-11 arylsulfonyl group represented by R ^{11 to} R ¹⁶ in the general formula (3), 4-16 arylsulfonyl group represented by R ^{21 to} R ²³ in the general formula (3 ′), The arylsulfonyl group having 4 to 16 carbon atoms represented by R ²¹ in the formula (3 ″) and the arylsulfonyl group having 4 to 16 carbon atoms represented by R ³⁰ in the general formula (3 ′ ″) are monocyclic or A condensed polycycle may be used, and preferably those having 5 to 14 carbon atoms.
Specific examples include a phenylsulfonyl group, a tolylsulfonyl group, a xylylsulfonyl group, a mesitylsulfonyl group, a naphthylsulfonyl group, an anthrylsulfonyl group, a phenanthrylsulfonyl group, and the like.

Formula (3) in R ¹¹ to R ¹⁶ heteroalkyl group of carbon and the total number of heteroatoms 2-20 represented by the general formula (3 ') in the total number of carbon and hetero atoms represented by R ²¹ to R ²³ 2 -20 heteroalkyl groups, carbons represented by R ²¹ in general formula (3 '') and the total number of heteroatoms 2-20 heteroalkyl groups and carbons represented by R ³⁰ in general formula (3 ''') and The heteroalkyl group having 2 to 20 heteroatoms in total includes at least one heteroatom selected from O, S, N and P in the main chain residue as described above or a cycloalkyl group described below It may be linear, branched or cyclic, and preferably has 2 to 10 carbon and hetero atoms in total. Moreover, the number of the hetero atoms contained is 1-5.

2 to 20 heteroalkenyl groups in total number of carbons and heteroatoms represented by R ^{11 to} R ¹⁶ in the general formula (3), 2 in total number of carbons and heteroatoms represented by R ^{21 to} R ²³ in the general formula (3 ′) -20 heteroalkenyl group, the general formula (3 '') carbon and heteroalkenyl groups and the general formula of the total number of carbon and hetero atoms represented by R ²¹ 2-20 (3 in 'the'') represented by R ³⁰ The heteroalkenyl group having 2 to 20 heteroatoms in total includes at least one heteroatom selected from O, S, N and P in the main chain residue as described above or a cycloalkenyl group described later It may be linear, branched or cyclic, and preferably has 2 to 10 carbon and hetero atoms in total. Moreover, the number of the hetero atoms contained is 1-5.

A cycloalkyl group having 3 to 8 carbon atoms represented by R ^{11 to} R ¹⁶ in the general formula (3), a cycloalkyl group having 3 to 8 carbon atoms represented by R ^{21 to} R ²³ in the general formula (3 ′), The cycloalkyl group having 3-8 carbon atoms represented by R ²¹ in the formula (3 ″) and the cycloalkyl group having 3-8 carbon atoms represented by R ³⁰ in the general formula (3 ′ ″) are preferably The thing of carbon numbers 5-14 is mentioned.
Specific examples include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, and the like.

Formula (3) in R ¹¹ cycloalkenyl group 3-8 carbon atoms represented by to R ^16, Formula (3 ') in the carbon number of 3-8 represented by R ²¹ to R ²³ cycloalkenyl group, generally In formula (3 ″), a C 3-8 cycloalkenyl group represented by R ²¹ and a C 3-8 cycloalkenyl group represented by R ³⁰ in formula (3 ′ ″) are a double bond A cycloalkyl group having a carbon number of 5 to 14 is preferable.
Specific examples include cyclopentenyl, methylcyclopentenyl, cyclohexenyl, methylcyclohexenyl, dimethylcyclohexenyl, ethylcyclohexenyl, butylcyclohexenyl, cycloheptenyl, cyclooctenyl, cyclodecenyl, and cyclododecenyl groups.

In the general formula (3), an aryl group having 4 to ¹⁶ carbon atoms represented by R ^{11 to} R ¹⁶ , an aryl group having 4 to ¹⁶ carbon atoms represented by R ^{21 to} R ²³ in the general formula (3 ′), The aryl group having 4-16 carbon atoms represented by R ²¹ in 3 ″) and the aryl group having 4-16 carbon atoms represented by R ³⁰ in general formula (3 ′ ″) are aromatic monocyclic or condensed It is polycyclic and preferably has 5 to 14 carbon atoms.
Specifically, for example, phenyl group, o-tolyl group, m-tolyl group, p-tolyl group, 2,3-xylyl group, 2,4-xylyl group, 2,5-xylyl group, 2,6-xylyl group Group, 3,5-xylyl group, mesityl group, naphthyl group, anthryl group, phenanthryl group and the like.

In formula (3), the total number of carbon atoms and heteroatoms represented by R ^{11 to} R ¹⁶ in 4-16 heteroaryl groups, and in formula (3 ′), the total number of carbon atoms and heteroatoms represented by R ^{21 to} R ²³ 4 -16 heteroaryl groups, carbons represented by R ²¹ in general formula (3 '') and the total number of heteroatoms 4-16 heteroaryl groups and carbons represented by R ³⁰ in general formula (3 ''') and The heteroaryl group having a total number of heteroatoms of 4-16 is an aryl group as described above containing at least one heteroatom selected from O, S, N and P in the ring, and is monocyclic or condensed polycyclic The total number of carbon and heteroatoms is preferably 5 to 14. Moreover, the number of the hetero atoms contained is 1-5.
Specifically, for example, furyl group, thienyl group, pyrrolyl group, azepinyl group, pyrazolyl group, imidazolyl group, oxazolyl group, isoxazolyl group, thiazolyl group, isothiazolyl group, 1,2,3-oxadiazolyl group, triazolyl group, tetrazolyl group Group, thiadiazolyl group, pyranyl group, pyridyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, morpholinyl group, thiomorpholinyl group, pyrrolidinyl group, pyrrolinyl group, imidazolidinyl group, imidazolinyl group, pyrazolidinyl group, pyrazolinyl group, piperidinyl group, etc. Monocyclic, for example, cinnolinyl, benzofuranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, indolizinyl, isoindolyl, indolyl, indazolyl Group, prynyl group, quinolidinyl group, isoquinolyl group, quinolyl group, phthalazinyl group, naphthyridinyl group, quinoxalinyl group, quinazolinyl group, carbazolyl group, carbolinyl group, acridinyl group, isoindolinyl group, benzoxazolyl group (benzoxazol-2-yl group) Benzothiazolyl group (benzothiazol-2-yl group), benzoimidazolyl group (benzoimidazol-2-yl group), a group in which the above-mentioned monocycle such as pteridinyl group and other cyclic groups are condensed.

The total number of carbon and hetero atoms represented by R ^{11 to} R ¹⁶ in the general formula (3) is 2-30 heterocyclyl groups, the total number of carbon and hetero atoms represented by R ^{21 to} R ²³ in the general formula (3 ′) 2 − 30 heterocyclyl groups, carbons represented by R ²¹ in general formula (3 ″) and the total number of heteroatoms of 2-30 heterocarbon groups and carbons and heteroatoms represented by R ³⁰ in general formula (3 ′ ″) A total of 2-30 heterocyclyl groups are saturated, partially unsaturated or aromatic 5-10 membered heterocycles containing at least one heteroatom selected from O, S and N, and are monocyclic Or a condensed polycycle may be sufficient, Preferably the thing whose total number of carbon and a hetero atom is 5-14 is mentioned.
Specifically, for example, furyl group, thienyl group, pyrrolyl group, azepinyl group, pyrazolyl group, imidazolyl group, oxazolyl group, isoxazolyl group, thiazolyl group, isothiazolyl group, 1,2,3-oxadiazolyl group, triazolyl group, tetrazolyl group Group, thiadiazolyl group, pyranyl group, pyridyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, morpholinyl group, thiomorpholinyl group, pyrrolidinyl group, pyrrolinyl group, imidazolidinyl group, imidazolinyl group, pyrazolidinyl group, pyrazolinyl group, piperidinyl group, etc. Monocyclic, for example, cinnolinyl, benzofuranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, indolizinyl, isoindolyl, indolyl, indazolyl Group, prynyl group, quinolidinyl group, isoquinolyl group, quinolyl group, phthalazinyl group, naphthyridinyl group, quinoxalinyl group, quinazolinyl group, carbazolyl group, carbolinyl group, acridinyl group, isoindolinyl group, benzoxazolyl group (benzoxazol-2-yl group) Benzothiazolyl group (benzothiazol-2-yl group), benzoimidazolyl group (benzoimidazol-2-yl group), a group in which the above-mentioned monocycle such as pteridinyl group and other cyclic groups are condensed.

The alkylene group having 1 to 20 carbon atoms formed by combining R ¹⁵ and R ¹⁶ in the general formula (3) may be linear, branched or cyclic, and preferably has 1 to 3 carbon atoms. Is mentioned.
Specifically, methylene group, ethylene group, methylmethylene group, ethylmethylene group, trimethylene group, propylene group, 2-propylene group, propylmethylene group, isopropylmethylene group, dimethylmethylene group, tetramethylene group, butylene group, 2 -Methylpropylene group, pentamethylene group, pentylene group, 1-methyltrimethylene group, 2-methyltrimethylene group, 3-methyltrimethylene group, 1-ethylethylene group, 2-ethylethylene group, ethylmethylethylene group, 1-methyltetramethylene group, 2-methyltetramethylene group, 3-methyltetramethylene group, 4-methyltetramethylene group, 1,1-dimethyltrimethylene group, 1,2-dimethyltrimethylene group, 1,3- Dimethyltrimethylene group, 2,2-dimethyltrimethylene group, 2-ethyl Limethylene group, hexamethylene group, hexylene group, 1-ethyltrimethylene group, undecamethylene group, 1-methyldecamethylene group, 1-methylpentamethylene group, 2-methylpentamethylene group, 3-methylpentamethylene group, 1,2-dimethyltetramethylene group, 1,3-dimethyltetramethylene group, 2,3-dimethyltetramethylene group, 1,1-dimethyltetramethylene group, 1-ethyltetramethylene group, 2-ethyltetramethylene group, 1-ethyl-2-methyltrimethylene group, 1-methylhexamethylene group, 1-methylheptamethylene group, 1-methyloctamethylene group, 1-methylnonamethylene group, heptamethylene group, heptylene group, octamethylene group, Octylene group, 2-ethylhexylene group, nonamethylene group, nonylene group, decame Len group, Decylene group, Hendamemethylene group, Dodecamethylene group, Tridecamethylene group, Tetradecamethylene group, Pentadecamethylene group, Hexadecamethylene group, Optadecamethylene group, Octadecamethylene group, Nonadecamethylene group, Examples include an eicosamethylene group, a cyclopropylene group, a cyclopentylene group, a cyclohexylene group, a cycloheptylene group, a cyclooctylene group, a cyclononylene group, and a cyclodecylene group, and an ethylene group is particularly preferable.

In the general formula (3 ′ ″), examples of the halogen atom represented by R ³¹ include F, Cl, Br, and I.

In the present invention, among the photodegradable protecting groups as described above, those represented by the general formula (3) are preferable, and those represented by the following general formulas (4) and (5) are particularly preferable.
The photodegradable protecting groups Q ¹ and Q ² may be the same or different.

[Wherein, Z ¹¹ represents a halogen atom, an imidazolyl group or a 4-nitrophenoxy group, and Q, A, Y ¹ , Y ² , M ¹ , M ² , X ¹ and X ² are the same as described above. ]

Examples of the halogen atom represented by Z ¹¹ in the general formula (5) include F, Cl, Br, and I.

As the compounds of the above general formulas (3), (4) and (5), ^one of Y ¹ , Y ² , X ¹ , X ² A and M ¹ is A ¹ or / and in general formula (1). A bond to A ² , wherein Q is —O—, A is a hydroxyl group, —OCH ₃ or —OC ₂ H ₅ , X ² is —H, Y ¹ is —H, Y ² is —H, X ¹ is -Br, Z is -OC (O)-(4-nitrophenyl), -OC (O) O- (4-nitrophenyl), -NR ¹⁵ C (O) R ¹⁶ , -OC (O) NR ¹⁵ R ¹⁶ , —OP (O) R ¹⁵ R ¹⁶ , —OC (O) Z ¹¹ or M ² is preferably ═N ₂ , and in particular, Q is —O— and A is represented by formula (1). A bond that binds to A ¹ and / or A ² , X ² is —H, Y ¹ is —H, Y ² is —H, X ¹ is —Br, and Z is —OC (O)-(4-nitrophenyl) ), -OC (O) O- (4-nitrophenyl), -NR ¹⁵ C (O) R ¹⁶ , -OC (O) NR ¹⁵ R ¹⁶ , -OP (O) R ¹⁵ R ¹⁶ , -OC (O ) what is Z ¹¹ or = N ₂ with M ² being particularly preferred.

  The photodegradable protecting group of the present invention as described above is, for example, WO00 / 31588 International Publication Pamphlet, JP 2002-315576 A, T. Furuta, et al., Proc. Natl. Acad. Sci., USA., Vol. 96, pp. 1193-1200, February 1999, Chemistry, Neurobiology, RS Givens, CH Park, Tetrahedron Lett. 37, 6259-6262 (1996), CH Park, RS Givens, J. Am. Chem. Soc. 119, 2453-2463 (1997), J. Engels, EJ Schlaeger. J. Med. Chem. 20, 907 (1977), JH Kaplan, G. Forbush III, JF Hoffman, Biochemistry 17, 1920-1935 (1978), etc. According to the above, it can be appropriately synthesized, or a commercially available product may be used. In particular, the compounds of the above general formulas (3), (4) and (5) are, for example, WO00 / 31588 international publication pamphlet, JP 2002-315576 A, T. Furuta, et al., Proc. Natl. Acad. Sci. , USA., Vol. 96, pp. 1193-1200, February 1999, Chemistry, Neurobiology.

1-2. Linker part In the general formula (1), the structural part represented by -A ¹ -T ¹ -A ^2- is a linker part for binding the photodegradable protecting groups represented by Q ¹ and Q ² .
That is, A ^{1 of the} linker moiety is bonded to the photodegradable protective group Q ¹ as described above, and A ² is bonded to the photodegradable protective group Q ^{2 as} described above, whereby ^two photodegradable protective groups Q ¹ are bonded. A compound is formed in which ¹ and Q ² are linked via T ¹ .
More specifically, for example, when the photodegradable protecting group is the general formula (3), any ^one of Y ¹ , Y ² , X ¹ , X ² A and M ^{1 in} the general formula (3) One of them becomes a bond, and A ¹ or / and A ^{2 of the} linker binds to the site. It is preferable that A in the general formula (3) is a bond and A ¹ or / and A ^{2 of the} linker moiety is bonded to the site A.
When the photodegradable protecting group is the general formula (3 ′), any one of R ²¹ , R ²² , R ²³ , R ²⁴ and R ^{25 in} the general formula (3 ′) is a bond. Thus, A ¹ or / and A ^{2 of the} linker moiety are bound to the site. In the general formula (3 ′), R ²³ is preferably a bond and A ¹ or / and A ^{2 of the} linker moiety is bonded to the R ²³ site.
When the photodegradable protecting group is the general formula (3 ″), any one of R ²¹ , R ²⁶ , R ²⁷ , R ²⁸ and R ^{29 in} the general formula (3 ″) is a bond. Thus, A ¹ or / and A ^{2 of the} linker moiety are bound to the site. In the general formula (3 ″), R ²⁸ is preferably a bond and A ¹ or / and A ^{2 of the} linker moiety is preferably bonded to the site of R ²⁸ .
When the photodegradable protecting group is the general formula (3 ′ ″), any one of R ³⁰ , R ³² , R ³³ , R ³⁴ and R ^{35 in} the general formula (3 ′ ″) is A ¹ or / and A ^{2 of the} linker moiety binds to the site as a bond. In the general formula (3 ′ ″), R ³⁰ is preferably a bond and A ¹ or / and A ^{2 of the} linker is bonded to the R ³⁰ site.

The length of the linker is long enough for Q ¹ and Q ² bound to both ends to be bound between nucleic acids, between nucleic acid-protein or polypeptide, or between proteins or polypeptides, ie double-stranded. Each of the sense strand and the antisense strand of the nucleic acid is preferably of a length capable of cross-linking each of the nucleic acid and the protein or polypeptide, each of the two proteins or polypeptides (between protein, protein-polypeptide or between polypeptides). . In particular, it is preferable that the length is sufficient to bind to the sense strand and the antisense strand of double-stranded RNA.
More specifically, since it varies depending on the type of the target substance to be crosslinked, it cannot be generally stated, but the length of the linker part is, for example, the lower limit is usually 2 mm or more, preferably 9 mm or more, and the upper limit is usually 94 mm or less, Preferably it is 74 mm or less.
For example, in the case of cross-linking between nucleic acids (double-stranded DNA, double-stranded RNA or DNA-RNA double-stranded hybrid), the lower limit of the length of the linker part is usually 2 mm or more, preferably 9 mm or more, more The upper limit is usually at most 44 mm, preferably at most 29 mm.
When the nucleic acid and the protein or polypeptide are cross-linked, or when two proteins or polypeptides are cross-linked, the length of the linker part is usually at least 4 mm, preferably at least 19 mm, and the upper limit is usually at least 94 mm, Preferably it is 74 mm or more.

The alkylene group represented by A ¹ , A ² and T ¹ may be linear, branched or cyclic, and usually has 1 to 10 carbon atoms, preferably 1 to 8 carbon atoms.
Specifically, for example, methylene group, ethylene group, methylmethylene group, ethylethylene group, trimethylene group, propylene group, tetramethylene group, butylene group, 1-methyltrimethylene group, 2-methyltrimethylene group, pentamethylene group , Pentylene group, 2,2-dimethylpropylene group, 2-methylpropylene group, 2-ethylpropylene group, 1-methyltetramethylene group, 2-methyltetramethylene group, 1,1-dimethylethylene group, 1,2- Dimethylethylene group, 1,3-dimethyltrimethylene group, 2,2-dimethyltrimethylene group, 2-ethyltrimethylene group, hexamethylene group, hexylene group, heptylene group, octylene group, 2-ethylhexylene group, nonamethylene Group, nonylene group, decamethylene group, decylene group, 1,4-dimethyltetramethylene group, , 3-dimethyltetramethylene group, 1,2,3-trimethyltrimethylene group, 1,2-diethylethylene group, heptamethylene group, 1,5-dimethylpentamethylene group, 3-ethylpentamethylene group, octamethylene group 1,6-dimethylhexamethylene group, cyclopropylene group, cyclopentylene group, cyclohexylene group, cycloheptylene group, cyclooctylene group, cyclononylene group, cyclodecylene group, cyclopropane-1,2-dimethylene group, cyclopentane- 1,3-dimethylene group, cyclohexane-1,4-dimethylene group, cyclohexane-1,4-diethylene group, cyclooctane-1,5-dimethylene group, adamantanediyl group, tricyclo [5.2.1.02.6 ] Decandiyl group, norbornanediyl group, methylnorborna A didiyl group, an isobornanediyl group, a decalindiyl group, and the like.

The arylene group represented by T ¹ in the general formula (1) may be monocyclic, condensed polycyclic or non-condensed polycyclic, and usually includes 5 to 14 carbon atoms.
Specific examples include a phenylene group, a tolyl group, a xylyl group, a mesityl group, a naphthylene group, an anthracene diyl group, a phenanthracene diyl group, and a biphenyldiyl group.

Examples of the aralkylene group represented by T ¹ in the general formula (1) include a group formed by combining an alkylene group and an arylene group, which may be linear or branched, and usually has 7 to 10 carbon atoms.
Specifically, for example, _{-CH 2 -C 6 H 4 -,} - CH 2 CH 2 -C 6 H 4 -, - CH 2 -C 6 H 4 -CH 2 -, - CH 2 CH 2 -C 6 H _{4 -CH 2 -, - CH 2} CH 2 CH 2 -C 6 H 4 -, - CH (CH 3) -CH 2 -C 6 H 4 -, - CH 2 CH 2 CH 2 CH 2 -C 6 H 4 _{-, - CH 2 CH 2 CH} (CH 3) -C 6 H 4 - , and the like.

In the general formula (1), an alkylene group containing a hetero atom represented by T ¹ , an arylene group containing a hetero atom or an aralkylene group containing a hetero atom is a carbon at any position of the alkylene group, arylene group and aralkylene group as described above. Those in which the atom is substituted with a divalent group having a hetero atom, in other words, those containing a divalent group having a hetero atom at any position in the chain of the alkylene group, arylene group and aralkylene group as described above And has no or very low reaction activity for a leaving group, a phosphoric acid, a carboxyl group, a hydroxyl group, an amino group or the like represented by Z. The divalent group having a hetero atom includes, for example, a reaction with a leaving group represented by Z, a phosphoric acid, a carboxyl group, a hydroxyl group or an amino group having a hetero atom such as a nitrogen atom, a sulfur atom or an oxygen atom. Examples include groups having no activity or very low activity. Specifically, for example, carbonyl group, thiocarbonyl group, imino group, malonyl group, -S-, -O-, -N-,

等が挙げられる。

Etc.

In the general formula (1), the alkyl group represented by R ¹ , R ² and R ³ may be linear, branched or cyclic, and usually has 1 to 12 carbon atoms, preferably 1 to 1 carbon atoms. 6, more preferably 1-4.
Specifically, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, sec-pentyl group. , Tert-pentyl group, neopentyl group, n-hexyl group, isohexyl group, sec-hexyl group, tert-hexyl group, neohexyl group, n-heptyl group, isoheptyl group, sec-heptyl group, tert-heptyl group, neoheptyl group N-octyl group, isooctyl group, sec-octyl group, tert-octyl group, neooctyl group, n-nonyl group, isononyl group, sec-nonyl group, tert-nonyl group, neononyl group, n-decyl group, isodecyl group , Sec-decyl group, tert-decyl group, neodecyl group, n-unde Group, isoundecyl group, sec-undecyl group, tert-undecyl group, neoundecyl group, n-dodecyl group, isododecyl group, sec-dodecyl group, tert-dodecyl group, neododecyl group, cyclopropyl group, cyclobutyl group, cyclopentyl group, Examples thereof include a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, a cyclononyl group, a cyclodecyl group, a cycloundecyl group, and a cyclododecyl group.

In the linker part of the present invention, the lower limit of the number of atoms constituting the main chain in -A ¹ -T ¹ -A ^2- is usually 5 or more, preferably 7 or more, and the upper limit is usually 80 or less, preferably Is preferably 70 or less.

Linker moiety -A ¹ -T ¹ -A ² of the present invention described above - ^{is, -A 3 - (T 2 -E} ) p -T 3 -A 4 - is what is preferably represented by.
Here, A ³ and A ⁴ are each independently an alkylene group, —O—, —NR ¹ —, —O—CO—, —CO—O—, —C—O—C—, —NR ² —COO. —, —OCO—NR ² —, —NR ³ —CO—, —CO—NR ³ — or —O—COO— (R ^{1 to} R ³ are the same as described above), T ² and T ³ are independent of each other. An alkylene group, and E represents a bond, a nitrogen atom, a sulfur atom, an oxygen atom, —O—CO— or —CO—O—. In addition, p represents an integer of 1 or more, and p (T ² -E) — may be the same or different.

The alkylene group represented by A ³ , A ⁴ , T ² and T ³ may be linear, branched or cyclic, and usually has 1 to 10 carbon atoms, preferably 1 to 8 carbon atoms. Specific examples thereof are the same as the alkylene groups represented by A ¹ , A ² and T ¹ .

The lower limit of p is 1 or more, and the upper limit is usually 6 or less, preferably 4 or less, more preferably 2 or less.
In addition, although p pieces of — (T ² -E) — may be the same or different, T ² bonded (adjacent) to A ³ is preferably the same as T ³ .

The above linker moiety has a lower limit of the number of atoms constituting the main chain in -A ^3- (T ² -E) _p -T ³ -A ⁴ -of usually 5 or more, preferably 7 or more, Is usually 80 or less, preferably 70 or less.

  More specifically, examples of the linker of the present invention include those represented by the following general formula.

[Wherein, R ^{1 to} R ³ are as described above, and m represents an integer of 3 or more. Here, the two R ^{1 s} may be the same or different, and the two R ^{2 s} may be the same or different. Two R ^{3 s} may be the same or different. ]
The lower limit of m is usually 3 or more, preferably 5 or more, and the upper limit is usually 78 or less, preferably 68 or less.

1-3. Specific compound (crosslinking agent)
As described above, the compound of the present invention (crosslinking agent) is a compound in which a photodegradable protecting group is bonded to both ends of a linker, and is represented by the general formula (1), preferably the general formula (2). It is.

[Wherein, Q ¹ , Q ² , A ¹ , A ² , A ³ , A ⁴ , T ¹ , T ² , T ³ , E and p are the same as described above. ]

〔式中、Ｑ、Ｙ¹、Ｙ²、Ｍ¹、Ｚ、Ｍ²、Ｘ¹、Ｘ²、Ａ³、Ａ⁴、Ｔ²、Ｔ³、Ｅ及びｐは前記と同じである。〕 Specific examples of the compound (crosslinking agent) of the present invention include those represented by the following general formula (6).

[Wherein, Q, Y ¹ , Y ² , M ¹ , Z, M ² , X ¹ , X ² , A ³ , A ⁴ , T ² , T ³ , E and p are the same as above]. ]

〔式中、Ｑ、Ｙ¹、Ｙ²、Ｍ¹、Ｚ¹¹、Ｍ²、Ｘ¹、Ｘ²、Ａ³、Ａ⁴、Ｔ²、Ｔ³、Ｅ及びｐは前記と同じである。〕 Among the compounds (crosslinking agents) of the present invention, those that bind to the phosphate group of the target object (nucleic acid, protein, or polypeptide) and crosslink the target object include, for example, the following general formula (7) What is shown.

[Wherein, Q, Y ¹ , Y ² , M ¹ , Z ¹¹ , M ² , X ¹ , X ² , A ³ , A ⁴ , T ² , T ³ , E and p are the same as above]. ]

〔式中、Ｑ、Ｙ¹、Ｙ²、Ｍ¹、Ｚ¹¹、Ｍ²、Ｘ¹、Ｘ²、Ａ³、Ａ⁴、Ｔ²、Ｔ³、Ｅ及びｐは前記と同じである。〕 Among the compounds (crosslinking agents) of the present invention, those which bind to the carboxyl group, hydroxyl group or amino group of the target object (nucleic acid, protein or polypeptide) and crosslink the target object include, for example, the following general formula ( 8).

[Wherein, Q, Y ¹ , Y ² , M ¹ , Z ¹¹ , M ² , X ¹ , X ² , A ³ , A ⁴ , T ² , T ³ , E and p are the same as above]. ]

〔式中、Ｑ、Ｙ¹、Ｙ²、Ｍ¹、Ｚ¹¹、Ｍ²、Ｘ¹、Ｘ²、Ａ³、Ａ⁴、Ｔ²、Ｔ³、Ｅ及びｐは前記と同じである。〕 Among the compounds (crosslinking agents) of the present invention, those that bind to the phosphate group and the carboxyl group, hydroxyl group or amino group of the target object (nucleic acid, protein or polypeptide) to crosslink the target object, One of the two photodegradable protecting groups is bonded to the phosphate group of the target object, and the other is bonded to the carboxyl group, hydroxyl group or amino group of the target object to crosslink the target object. Examples thereof include those represented by the following general formula (9).

[Wherein, Q, Y ¹ , Y ² , M ¹ , Z ¹¹ , M ² , X ¹ , X ² , A ³ , A ⁴ , T ² , T ³ , E and p are the same as above]. ]

1-4. Synthesis of Compound (Crosslinking Agent) of the Present Invention The compound (crosslinking agent) of the present invention represented by the general formula (1) can be easily synthesized, for example, according to the following synthesis scheme.
In the following synthesis scheme, Q, Y ¹ , Y ² , M ¹ , Z, Z ¹¹ , M ² , X ¹ , X ² , R ¹ , R ² , R ³ and m are the same as described above. Moreover, the formal name of the abbreviation used in the following synthetic scheme is as follows.
Et ₃ N: Triethylamine DMF: N, N-dimethylformamide

（Ｒ²がHの場合、例えば上記の合成スキームに従えば合成し得る。）

(When R ² is H, it can be synthesized, for example, according to the above synthesis scheme.)

2. Crosslinking method of the present invention When the compound (crosslinking agent) of the present invention is used, it is between double stranded nucleic acids, between nucleic acid-protein or polypeptide, or between proteins or polypeptides [ie, sense strand and antisense of double stranded nucleic acid. Each of the chain, each of the nucleic acid and the protein or polypeptide, each of the two proteins or polypeptides (between protein, protein-polypeptide or between polypeptides) (hereinafter abbreviated as “complex according to the present invention”) Can be easily cross-linked.
That is, the compound of the present invention (crosslinking agent) has a group capable of binding to a group selected from a phosphate group, a carboxyl group, a hydroxyl group, an amino group, and the like in the photodegradable protective group. The target object (nucleic acid, protein or polypeptide) constituting the complex according to the invention is bonded to a group selected from a phosphate group, a carboxyl group, a hydroxyl group and an amino group to crosslink the complex according to the present invention. be able to.
In other words, phosphorous of one target object (nucleic acid, protein or polypeptide) in which one of the two photodegradable protecting groups of the compound (crosslinking agent) of the present invention forms a complex according to the present invention. A group selected from an acid group, a carboxyl group, a hydroxyl group, and an amino group, and the other photodegradable protective group is a group selected from a phosphate group, a carboxyl group, a hydroxyl group, and an amino group of the other target object; By bonding, the complex according to the present invention (two target objects constituting the complex) can be crosslinked.
As a result, the complex according to the present invention (two target objects constituting the complex) is converted to the compound of the present invention (crosslinking agent) via its phosphate residue, carboxyl group, hydroxyl group or amino group. Thus, it will be cross-linked.
Therefore, the cross-linking method of the present invention is selected from a phosphate group, a carboxyl group, a hydroxyl group, and an amino group between the double-stranded nucleic acid, between the nucleic acid-protein or polypeptide, or between the protein or polypeptide. The compound of the present invention (crosslinking agent) as described above via a group, preferably a compound represented by the general formula (1), more preferably a compound represented by the general formula (2), more preferably a general formula (6) It is characterized by crosslinking with the compounds shown.
More specifically, the cross-linking method of the present invention comprises 1) a complex according to the present invention (especially a double-stranded RNA) and a phosphate via the phosphate residue of the complex (especially a double-stranded RNA). Cross-linking with a cross-linking agent having a photodegradable protecting group having a group capable of binding to both ends or a compound represented by the general formula (7), 2) converting the complex according to the present invention to phosphoric acid of the complex Cross-linking with a compound represented by the general formula (8) via a residue and a carboxyl group, a hydroxyl group or an amino group, and 3) the complex according to the present invention is a carboxyl group, a hydroxyl group of the complex. Or it bridge | crosslinks with the compound shown by General formula (9) through an amino group, It is characterized by the above-mentioned.

In the above method, the double-stranded nucleic acid to be crosslinked is not particularly limited as long as it forms a double strand.
Specifically, for example, selected from plasmid DNA, genomic DNA, synthetic DNA synthesized by a known amplification method such as PCR, DNA such as cDNA, RNA such as mRNA and antisense RNA, such as cyclic nucleotide, etc. Examples include those composed of two nucleic acids (for example, double-stranded DNA, double-stranded RNA, DNA-RNA hybrid, etc.). Of these, double-stranded RNA is preferable.

The protein to be crosslinked is not particularly limited as long as it can form a complex with a nucleic acid as described above, or can form a complex with another protein or polypeptide.
Specifically, examples include transcription factors such as NFκB, NFAT, and STAT, RNA polymerase, and the like.

The polypeptide to be cross-linked is not particularly limited as long as it can form a complex with a nucleic acid as described above, or can form a complex with another polypeptide or protein.
Specific examples include PKCε V1-2 inhibitor peptide, RGD peptide, Zn finger peptide, leucine zipper peptide, bHLH peptide and the like.

  The present invention is particularly useful for double-stranded RNA among the above-mentioned cross-linking targets.

In order to crosslink the complex according to the present invention as described above using the compound (crosslinking agent) of the present invention, the compound of the present invention (crosslinking agent) and the complex according to the present invention may be brought into contact with each other. .
Examples of the method for bringing the compound of the present invention (crosslinking agent) into contact with the complex according to the present invention include, for example, a solution containing the compound of the present invention (crosslinking agent) and a solution containing the complex according to the present invention. The lower limit is usually 1 hour or more, preferably 4 hours or more, more preferably 8 hours or more, the upper limit is 36 hours or less, preferably 24 hours or less, and the lower limit is usually 10 ° C. or more, preferably 15 ° C. or more. More preferably, it is 20 ° C. or higher, and the upper limit is usually 40 ° C. or lower, preferably 35 ° C. or lower, more preferably 25 ° C. or lower.
The amount of the compound of the present invention (crosslinking agent) used in the above cannot be generally specified depending on the type of the compound of the present invention (crosslinking agent) used or the type of the complex according to the present invention to be crosslinked. However, the lower limit is usually 1 mol or more with respect to 1 mol of the cross-linking target (complex according to the present invention), preferably more than 1 mol with respect to 1 mol of the normal cross-linking target (complex according to the present invention). And more than 1/4 of the number of combinations of crosslinkable reactive groups (phosphate group, carboxyl group, hydroxyl group, amino group, etc.) in the crosslink target, more preferably a combination of crosslinkable reactive groups in the crosslink target The molar amount corresponds to the number. The upper limit is usually a molar amount 10 times the number of crosslinkable reactive group combinations in the crosslinkable object, and preferably a molar amount 5 times the number of crosslinkable reactive group combinations in the crosslinkable object.
Here, the combination of reactive groups capable of cross-linking in the cross-linking target is the compound (cross-linking agent) of the present invention possessed by one target object (nucleic acid, protein or polypeptide) forming the complex according to the present invention. One of the two photodegradable protective groups possessed by A is a reactive group (for example, a group selected from a phosphate group, a carboxyl group, a hydroxyl group, and an amino group) and the other that forms the complex according to the present invention. Reactive groups (for example, phosphate groups, carboxyl groups) to which the other of the two photodegradable protecting groups of the compound (crosslinking agent) of the present invention (nucleic acid, protein or polypeptide) can bind , A group selected from a hydroxyl group and an amino group).
The number of combinations of reactive groups capable of crosslinking in the crosslinking target is the total number of reactive groups as described above (the number of all combinations) that the compound of the present invention (crosslinking agent) can bind to. Yes, in other words, a reactive group to which one of the two photodegradable protecting groups of the compound of the present invention (crosslinking agent) possessed by one target object forming the complex according to the present invention can bind. The other of the two photodegradable protecting groups possessed by the compound of the present invention (crosslinking agent) possessed by the other target object forming the complex according to the present invention can be bound. The smaller number of reactive groups.
In the above, the solution containing the compound of the present invention (crosslinking agent) or / and the complex according to the present invention is not particularly limited as long as it is usually used in this field. For example, water, DMSO, Examples of the buffer solution include Tris buffer solution, Good buffer solution, TE buffer solution, TAE buffer solution, TBE buffer solution, and TBS buffer solution.
In addition, the complex according to the present invention obtained by binding the compound of the present invention (crosslinking agent) obtained as described above is generally subjected to a purification method known per se such as column chromatography and unreacted. It is desirable to remove the compound (crosslinking agent) of the present invention and purify the complex according to the present invention to which the compound (crosslinking agent) of the present invention is bound.

In the present invention, the complex according to the present invention to which the compound of the present invention (crosslinking agent) is bonded (crosslinked), that is, the compound of the present invention (crosslinking agent) is bonded (crosslinked by). Also included are double stranded nucleic acids, nucleic acid-protein complexes, nucleic acid-polypeptide complexes, two proteins, protein-polypeptide complexes, and two polypeptides.
Among them, the complex according to the present invention to which the compound of the present invention (crosslinking agent) is bound (crosslinked by) is a double-stranded nucleic acid to which the compound of the present invention (crosslinking agent) is bound (crosslinked by). In particular, double-stranded RNA to which the compound of the present invention (crosslinking agent) is bound (crosslinked) is preferable. Preferred examples of the compound of the present invention (crosslinking agent) are as described above.

The compound according to the present invention (crosslinking agent) crosslinks the complex according to the present invention, whereby the complex according to the present invention, that is, double-stranded DNA such as double-stranded DNA, double-stranded RNA, and DNA-RNA hybrid. The functions originally possessed by the nucleic acid, nucleic acid-protein complex, nucleic acid-polypeptide complex, protein complex, polypeptide complex, or protein-polypeptide complex are suppressed.
For example, when the complex according to the present invention is a double-stranded DNA, transcription of the nucleic acid molecule is suppressed in a host having a transcription system, and as a result, gene expression is suppressed.
In addition, when the complex according to the present invention is a double-stranded RNA, the specific degradation action of complementary target mRNA possessed by siRNA is suppressed in RNAi.
When the complex according to the present invention is a nucleic acid-protein complex, transcription of a nucleic acid molecule downstream of the nucleic acid molecule is suppressed or activated in a host having a transcription system, and as a result, gene expression is suppressed. Or activated.
When the complex according to the present invention is a nucleic acid-polypeptide complex, transcription of a nucleic acid molecule downstream of the nucleic acid molecule is suppressed or activated in a host having a transcription system. Suppressed or activated.
When the complex according to the present invention is a protein complex, when the complex is a protein complex, the function of the protein is suppressed or activated in the cell.

By irradiating the complex according to the present invention crosslinked with the compound according to the present invention (crosslinking agent) with light such as ultraviolet rays or visible light, the compound according to the present invention (crosslinking agent) is irradiated from the complex according to the present invention. ) Can be eliminated. Thereby, the function suppression of the complex according to the present invention can be released.
That is, the complex according to the present invention crosslinked by the compound of the present invention (crosslinking agent) has a function that the complex originally has been suppressed, but the complex according to the present invention and the present complex are irradiated with light. Since the bond with the compound (crosslinking agent) of the invention is lost [because the crosslinking with the compound (crosslinking agent) of the invention is lost), the function of the complex is restored.

The light irradiated in the present invention means an electromagnetic wave having a wavelength longer than that of X-rays and having a wavelength range of about 1 to 900 nm. Among them, light on the long wavelength side, for example, in the range of 350 to 400 nm is preferable, and light having a wavelength of about 365 ± 6 nm is more preferable.
Further, the irradiation time of the light cannot be generally described because it varies depending on the type of the compound of the present invention (crosslinking agent) used, the type of the complex according to the present invention to be crosslinked, etc., but the irradiation time is long. And the nucleic acid molecule may be mutagenized, or cells or organisms introduced with the complex of the present invention crosslinked by the compound of the present invention (crosslinking agent) may be damaged. There is a possibility that the compound of the invention (crosslinking agent) cannot be sufficiently removed. Specifically, for example, light of 4 mJ / cm ² s has a lower limit of 1 minute or more, and the upper limit is usually 10 minutes or less, preferably 3 minutes or less, or light of 376 mJ / cm ² s is irradiated. The lower limit is usually 0.1 seconds or longer, preferably 1 second or longer, and the upper limit is usually 30 seconds or shorter, preferably 10 seconds or shorter.

As described above, according to the compound of the present invention (crosslinking agent) and the crosslinking method using the same, the complex according to the present invention having various functions (double-stranded DNA, double-stranded RNA, DNA-RNA hybrid, etc.) Of double-stranded nucleic acid, nucleic acid-protein complex, nucleic acid-polypeptide complex, protein complex, polypeptide complex, or protein-polypeptide complex).
Furthermore, by irradiating the complex according to the present invention crosslinked with the compound of the present invention (crosslinking agent) with light, the function of the complex according to the present invention that has been suppressed can be controlled at any time and place. Recovery, in other words, the function of the complex according to the present invention can be controlled at any time and place.
For example, double-stranded nucleic acid such as double-stranded DNA, double-stranded RNA, DNA-RNA hybrid, nucleic acid-protein complex, nucleic acid-polypeptide complex, protein complex, polypeptide complex, or protein-polypeptide The unknown function of the complex can be specifically expressed at any time, specifically expressed at any location, or specifically expressed at any time and location.
The compound of the present invention (crosslinking agent) and the crosslinking method using the same can be used for purposes other than those described above.
For example, a complex according to the present invention formed in vivo, in vitro, etc. (double-stranded nucleic acid such as double-stranded DNA, double-stranded RNA, DNA-RNA hybrid, nucleic acid-protein complex, nucleic acid-polypeptide A complex, a protein complex, a polypeptide complex, or a protein-polypeptide complex) with the compound of the present invention (crosslinking agent), and after isolating the crosslinked complex, the complex is irradiated with light. Then, each component (nucleic acid chain, protein, polypeptide) constituting the complex can be isolated without changing its function. Therefore, according to this method, not only the components constituting the complex are clarified, but also the functions of the respective components are retained, so that the complex forming action (cells, tissues, in vivo, etc.) It is extremely useful for analyzing the interaction between components) and its function.
Further, for example, when the complex according to the present invention has a medicinal effect (for example, a so-called RNAi drug using RNAi), the complex is crosslinked with the compound of the present invention (crosslinking agent) to suppress the medicinal effect. In addition, after the cross-linked complex is administered to cells, tissues, in vivo, and the like, it is possible to treat the disease by recovering the suppressed medicinal effect by irradiating light. According to this method, by controlling the range and time of light irradiation, a specific medicinal effect is produced at a specific target site (such as a lesion) or a medicinal effect is produced at an arbitrary time (time). Is possible.

3. The gene expression regulation method of the present invention The compound (crosslinking agent) of the present invention and the crosslinking method using the same can be used for various applications as described above. In particular, the RNAi method using double-stranded RNA Useful for.

The gene expression regulation method of the present invention is characterized by irradiating light to a double-stranded RNA to which a compound of the present invention (crosslinking agent) as described above is bound in advance.
That is, first, by binding the compound of the present invention (crosslinking agent) to double stranded RNA, in other words, by linking the double stranded RNA with the compound of the present invention (crosslinking agent), double stranded RNA Gene expression inhibitory action (degradation action of target mRNA complementary to the double-stranded RNA) can be suppressed. That is, the gene of the present invention (cross-linking agent) is bound to a double-stranded RNA (siRNA) against a predetermined gene (target mRNA) to cross-link the double-stranded RNA (siRNA), whereby the gene in the cell or organism (Target mRNA) can be expressed. Subsequently, the compound (crosslinking agent) of the present invention was desorbed from the complex by irradiating the double-stranded RNA crosslinked with the compound (crosslinking agent) of the present invention with light. The gene expression inhibitory action (degradation action of the target mRNA complementary to the double-stranded RNA) of the double-stranded RNA can be recovered, and as a result, the expression of the gene (target mRNA) can be suppressed. Thereby, the expression of a specific gene (target mRNA) can be regulated (controlled) at an arbitrary time and place.

  The gene expression regulation method of the present invention is a compound expression according to the present invention in which a double-stranded RNA used in a gene expression regulation method known per se using a double-stranded RNA such as RNAi method using a double-stranded RNA is used in advance. (When cross-linking with (cross-linking agent) to regulate gene expression, that is, when suppressing gene expression, other than irradiating light to double-stranded RNA cross-linked with the compound of the present invention (cross-linking agent) May be carried out according to a method known per se, and materials, reagents and the like used may be those used in a method known per se.

The gene expression regulation method of the present invention specifically includes the following steps (a) to (c).
(a) contacting the double-stranded RNA by bringing the compound (cross-linking agent) of the present invention into contact with the double-stranded RNA,
(b) introducing a crosslinked double-stranded RNA into a cell or organism; and
(c) A step of irradiating the introduced cell or organism with light.

3-1. Double-stranded RNA cross-linking step (step (a))
(1) Double-stranded RNA
The double-stranded RNA used in the method of the present invention is capable of mediating RNAi (having the ability to generate RNAi), that is, the double-stranded RNA promotes specific degradation of target mRNA complementary to the double-stranded RNA. As long as it causes a phenomenon that specifically suppresses the expression of the target protein (having the ability to degrade the target mRNA), RNA extracted from natural products such as various cells, organisms, tissues, synthetic RNA, Any recombinant RNA or the like can be used.
The ribonucleotide constituting the double-stranded RNA is, for example, a ribonucleotide containing a non-naturally occurring nucleobase (for example, uridine modified at 5-position such as 5- (2-amino) propyluridine, 5-bromouridine, etc. Or cytidine, eg adenosine and guanosine modified in position 8 such as 8-bromoguanosine, deazanucleotides such as 7-deaza-adenosine, eg O- and N-alkylated nucleotides such as N6-methyladenosine), sugars Modified ribonucleotides (eg, those in which the 2′OH group of the sugar is substituted with a group selected from the group consisting of H, OR, halogen atom, SH, SR ⁴¹ , NH ₂ , NHR ⁴¹ , NR ⁴¹ ₂ or CN) Te, R ⁴¹ _is C 1 -C ₆ alkyl, alkenyl or alkynyl, halogen atom, F, Cl, r or I), backbone-modified ribonucleotides (for example, phosphoester groups that bind adjacent ribonucleotides replaced with, for example, phosphothioate group modifying groups), and ribonucleotides that combine these You may go out.
In addition, the double-stranded RNA sequence used in the method of the present invention must have sufficient identity to the target mRNA. Preferably, this sequence has at least 50%, preferably at least 70%, more preferably at least 85%, particularly preferably 100% identity to the target mRNA in the double-stranded part of the double-stranded RNA Is.
The length of the double-stranded RNA is not particularly limited as long as it is capable of mediating RNAi (having the ability to generate RNAi). Specifically, each RNA strand has a lower limit of usually 19 bases or more, preferably 20 bases or more, more preferably 25 bases or more, and an upper limit of usually 1500 bases or less, preferably 1000 bases or less, more preferably Is 500 bases or less in length. When the cell or organism into which the double-stranded RNA is introduced is an animal, the upper limit is preferably 30 bases or less, more preferably 27 bases or less.
Examples of such double-stranded RNA include those described in JP-T 2003-529374, JP-T 2004-526422, JP-T 2002-516112, JP-A 2004-261002, and the like.

  The double-stranded RNA as described above is a method known per se usually used in this field (for example, JP 2003-529374, JP 2004-526422, JP 2002-516112, JP 2004-261002 A. Etc.] and can also be obtained using a commercially available kit.

  Double-stranded RNA generally has a lower limit of usually 1 μM or more, preferably 10 μM or more, more preferably 20 μM or more in an appropriate solvent, and an upper limit is usually 100 μM or less, preferably 50 μM or less, more preferably 30 μM or less. Prepared as a solution containing double-stranded RNA at a concentration of

(2) Contact between the compound of the present invention (crosslinking agent) and double-stranded RNA In order to crosslink double-stranded RNA by bringing the compound of the present invention (crosslinking agent) into contact with double-stranded RNA, for example, A solution containing the compound of the present invention (crosslinking agent) and a solution containing double-stranded RNA are mixed, and the lower limit is usually 1 hour or longer, preferably 4 hours or longer, more preferably 8 hours or longer, and the upper limit is 36 hours or less, preferably 24 hours or less, the lower limit is usually 10 ° C or higher, preferably 15 ° C or higher, more preferably 20 ° C or higher, and the upper limit is usually 40 ° C or lower, preferably 35 ° C or lower, more preferably 25 What is necessary is just to make it react at degrees C or less.
The compound of the present invention (crosslinking agent) used in the above method is preferably a crosslinking agent having a photodegradable protective group having a group capable of binding to phosphoric acid at both ends, represented by the general formula (7). The compound (crosslinking agent) is particularly preferred.
The amount of the compound of the present invention (crosslinking agent) used in the above cannot be generally stated depending on the type of the compound of the present invention (crosslinking agent) used, etc., but the lower limit is usually 1 mol of double-stranded RNA to be crosslinked. The reactive group (phosphate group, carboxyl group, hydroxyl group, amino group) that can crosslink in double-stranded RNA in an amount of 1 mole or more, preferably more than 1 mole per mole of double-stranded RNA And the like, and more preferably a molar amount corresponding to the number of reactive group combinations capable of crosslinking in double-stranded RNA. The upper limit is usually 10 times the molar amount of the combination of reactive groups capable of crosslinking in double-stranded RNA, preferably 5 times the molar amount of the combination of reactive groups capable of crosslinking in double-stranded RNA. .
Here, the combination of reactive groups capable of cross-linking in double-stranded RNA refers to two photodegradable protecting groups possessed by the compound of the present invention (cross-linking agent) possessed by one RNA strand forming double-stranded RNA. The compound of the present invention (crosslinking agent) possessed by a reactive group (for example, a group selected from a phosphate group, a hydroxyl group and an amino group) to which one of them can be bound and the other RNA strand forming the double-stranded RNA ) Means a combination with a reactive group (for example, a group selected from a phosphoric acid group, a hydroxyl group and an amino group) to which the other of the two photodegradable protecting groups can be bonded.
The number of reactive group combinations capable of crosslinking in double-stranded RNA refers to the total number of reactive groups as described above that can be bound to the compound of the present invention (crosslinking agent) possessed by double-stranded RNA (all combinations). In other words, one of the two photodegradable protecting groups of the compound of the present invention (crosslinking agent) possessed by one RNA strand forming a double-stranded RNA can be bound. A reactive group to which a group can bind and a reactive group to which the other of the two photodegradable protective groups of the compound (crosslinking agent) of the present invention possessed by the other RNA strand forming a double strand can bind The lesser of the numbers.
In the above, the solution (solvent) containing the double-stranded RNA or / and the compound of the present invention (crosslinking agent) is not particularly limited as long as it is usually used in this field. For example, water, DMSO And buffer solutions (for example, Tris buffer solution, Good buffer solution, TE buffer solution, TAE buffer solution, TBE buffer solution, TBS buffer solution, etc.).
In the above method, the reaction solution is subjected to a known purification method such as column chromatography, the unreacted compound (crosslinking agent) of the present invention is removed, and the compound (crosslinking agent) of the present invention is bound. It is desirable to purify single-stranded RNA.

3-2. Double-stranded RNA introduction step (step (b))
Double-stranded RNA to which the compound of the present invention (crosslinking agent) obtained as described above is bound is introduced (transfected) into cells or organisms.

(1) Cell or organism The cell or organism used in the present invention is capable of transmitting the light to be irradiated and supplying the energy of the light to the introduced (transfected) double-stranded RNA. There is no particular limitation as long as it is sufficient.
Examples of such cells include eukaryotic cells or cell systems such as plant cells or animal cells (mammalian cells such as humans and rats, nematode cells, insect cells, etc.), eg embryo cells (of early Xenopus laevis). Embryo cells, zebrafish embryo cells, Drosophila cells, etc.), pluripotent stem cells, tumor cells such as teratocarcinoma cells or virus-infected cells, and monolayer-like cultured cells derived from various organisms.
Examples of organisms include eukaryotes, plants, or animals (for example, mammals such as humans and rats, nematodes, insects, etc.).

(2) Introduction of double-stranded RNA into cells or organisms As a method for introducing (transfecting) double-stranded RNA having the compound of the present invention (crosslinking agent) bound into cells or organisms as described above, it is known per se. Can be used.
Examples of such a method include a method using a calcium phosphate method, DEAE-dextran method, electroporation and microinjection, virus method, cationic liposome [eg, Tfx50 (Promega), Lipofectamine 2000 (Life Technologies), etc.]. (Graham, FL and van der Eb, AJ (1973) Virol. 52, 456, McCutchan, JH and Pagano, JS (1968) J. Natl. Cancer Inst. 41, 351, Chu, G. et al. (1987). Nucl. Acids Res. 15, 1311, Fraley, R et al. (1980) J. Biol. Chem. 255, 10431, Capechi, MR (1980) Cell 22, 479, Felgner, PL et al. (1987), Proc. Natl. Acad Sci. USA 84, 7413).
According to a method known per se as described above, the double-stranded RNA to which the compound of the present invention (crosslinking agent) is bound can be easily introduced (transfected) into a cell or an organism, and a commercially available transfection kit It can also be easily performed using

  In cells or organisms into which double-stranded RNA to which the compound of the present invention (crosslinking agent) is bound is introduced, the compound of the present invention (crosslinking agent) mediates RNAi inherent in the double-stranded RNA. The ability to obtain (i.e. the ability to generate RNAi), i.e. the ability of the double-stranded RNA to specifically inhibit target protein expression by promoting the specific degradation of complementary target mRNA (target mRNA (The ability to degrade) is suppressed, and the same gene (the target mRNA complementary to the double-stranded RNA) is expressed in a state where no double-stranded RNA is introduced (normal state) .

3-3. Light irradiation process (process (c))
Subsequently, the present invention is made from the double-stranded RNA to which the compound (cross-linking agent) of the present invention is bound by irradiating light to the cell or organism into which the double-stranded RNA to which the compound of the present invention (cross-linking agent) is bound is introduced The compound (crosslinking agent) is removed.
That is, the gene expression inhibitory action (decomposition action of the target mRNA complementary to the double-stranded RNA) of the double-stranded RNA that has been suppressed by the binding of the compound of the present invention (cross-linking agent) is reduced by light irradiation. The binding between the compound of the present invention (crosslinking agent) and double-stranded RNA is removed and recovered, thereby suppressing the expression of the gene (target mRNA).

(1) Light irradiation The light used is the same as described above, and is an electromagnetic wave in the wavelength range of about 1 to 900 nm, preferably on the long wavelength side, for example, in the range of 350 to 400 nm, more preferably in the wavelength range. Is about 365 ± 6nm.
Further, the irradiation time of the light is also the same as described above, specifically, for example, the lower limit is 1 minute or more, for example, 4 mJ / cm2s, the upper limit is usually 10 minutes or less, preferably 3 minutes or less, Alternatively, 376 mJ / cm 2 s of light may be irradiated at a lower limit of usually 0.1 seconds or longer, preferably 1 second or longer, and an upper limit of usually 30 seconds or shorter, preferably 10 seconds or shorter. If the irradiation time is long, mutations may be induced in the double-stranded RNA, or cells or organisms introduced with the double-stranded RNA crosslinked by the compound of the present invention (crosslinking agent) may be damaged. If the irradiation time is short, the compound (crosslinking agent) of the present invention may not be sufficiently removed, so care must be taken.

The irradiation with light may be performed on the whole or the whole of the cell or organism into which the double-stranded RNA to which the compound of the present invention (crosslinking agent) is bound is introduced, or a part thereof.
By irradiating the whole (all) or part of the cell or organism into which the double-stranded RNA to which the compound of the present invention (crosslinking agent) is bound is introduced, the whole (all) or part of the cell or organism Can suppress gene expression.
In addition, when irradiating light to all (the whole) of the cell or organism, the cell or organism may be opposed to a light source or light irradiation means (for example, ultraviolet irradiation means such as a mercury lamp). Thereby, the expression of the target gene (target gene) can be suppressed in the whole cell or organism into which the double-stranded RNA to which the compound of the present invention (crosslinking agent) is bound has been introduced.
In addition, when irradiating light to a part of the cell or organism, the light emitted from the light source and light irradiation means (for example, ultraviolet irradiation means such as a mercury lamp) is converted into spot light by an optical system including an objective lens. The light spot may be irradiated only to a predetermined region of the cell or organism. Thereby, the expression of (target gene) can be suppressed only in a predetermined region in a cell or organism into which a double-stranded RNA to which a compound of the present invention (crosslinking agent) is bound is introduced.

As described above, by performing steps (a) to (c) of the present invention, the expression of a specific gene (target mRNA) can be regulated (controlled) at an arbitrary time and place.
That is, even if double-stranded RNA to which the compound of the present invention (crosslinking agent) is bound is present in the cell or organism, the expression of the target mRNA (target gene) complementary to the double-stranded RNA is suppressed. The expression of the gene can be specifically suppressed by irradiating light at a desired time and / or place thereafter.
In addition, the expression of a specific target gene (target mRNA) is carried out by subjecting a cell or organism into which double-stranded RNA to which a compound of the present invention (crosslinking agent) has been bound after light irradiation has been introduced into the cell or organism according to a conventional method. When the gene in the cell or organism is expressed by culturing or growing under suitable conditions where the compound can grow, the cell is caused by the action of double-stranded RNA from which the compound of the present invention (crosslinking agent) has been removed by light irradiation. Alternatively, it is suppressed by the RNAi effect that occurs in the organism.
Therefore, the gene expression regulation method including the steps (a) to (c) of the present invention further comprises, after the step (c), a step (c ′) a step of expressing a gene in a light-irradiated cell or organism. The inclusion is preferred.

4). Method for investigating gene function of the present invention In the method as described above, by comparing the expression of a gene in a cell or organism into which a double-stranded RNA to which a compound of the present invention (crosslinking agent) is bound before and after light irradiation is introduced. Analysis, identification, identification, etc. of gene functions can be performed.
Therefore, the gene function investigation method of the present invention comprises (a) a step of bringing a compound (crosslinking agent) of the present invention into contact with a double-stranded RNA to cross-link the double-stranded RNA, and (b) a cross-linked double strand. A step of introducing a strand RNA into a cell or an organism, (c) a step of irradiating the introduced cell or organism with light, (c ′) a step of expressing a gene in the irradiated cell or organism, and (d) Comparing the gene expressed in step (c ′) with a control.

In the present invention, the control refers to RNAi produced by the action of a crosslinked double-stranded RNA by the irradiation of the double-stranded RNA (the double-stranded RNA from which the compound of the present invention (crosslinking agent) has been removed by the light irradiation). Expression is substantially suppressed by the double-stranded RNA in a cell or organism having the same gene as the gene (target gene) whose expression is suppressed by the effect (ie, the target mRNA complementary to the double-stranded RNA) Not mean the gene.
Such controls include, for example, (i) a cell or organism into which cross-linked double-stranded RNA has been introduced, and that is expressed in a cell or organism that has not been irradiated with light (cross-linked double-stranded RNA Is the same type of cells or organisms into which is introduced, and has been introduced with cross-linked double-stranded RNA but not irradiated with light. Or a gene expressed in the cell or organism before light irradiation in the organism), (ii) the same type as the cell or organism into which the crosslinked double-stranded RNA has been introduced, and the crosslinked double strand A gene expressed in a cell or organism into which RNA has not been introduced, or (iii) a cell or organism into which cross-linked double-stranded RNA has been introduced, which is irradiated with light in a cell or organism that is irradiated with light Expression in no part (region) Gene, and the like.
These controls may be used alone or in appropriate combination.

In the present invention, the comparison between the gene expressed in step (c ′) and the control is, for example, between the cell or organism introduced with the double-stranded RNA to which the compound of the present invention (crosslinking agent) bound after light irradiation has been introduced and the control. Increase / decrease (expression level) or presence / absence of expression of target gene (target mRNA complementary to double-stranded RNA) or increase / decrease in expression product (protein, etc.) of target gene (target mRNA) in cells or organisms (protein amount) ) Or the presence or absence of an expression product (protein, etc.), the phenotype produced by the expression of the target gene (target mRNA), etc. are measured and observed according to a method known per se, and the results are compared.
In the present invention, in order to express a gene, a cell or organism into which a double-stranded RNA to which a compound of the present invention (crosslinking agent) is bound has been introduced, and / or a control cell or organism, are prepared according to a conventional method. Alternatively, the gene in the cell or organism may be expressed by culturing or growing under suitable conditions that allow the organism to grow.

Specific examples of the gene function investigation method of the present invention include the following steps (a) to (d).
(a) contacting the double-stranded RNA by bringing the compound (cross-linking agent) of the present invention into contact with the double-stranded RNA,
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(c) irradiating the introduced cell or organism with light,
(c ′) expressing a gene in a light-irradiated cell or organism,
(c '') expressing the gene in the cell or organism without irradiating the introduced cell or organism, and
(d) A step of comparing the gene expressed in step (c ′) with the gene expressed in step (c ″).
Note that the cells or organisms that are irradiated with light in the step (c) and the cells or organisms that are not irradiated with light in the step (c ″) may be the same [one (group) cells / individuals]. These may be of the same kind (different cells / individuals), but generally they are of the same kind and different cells / individuals.

Other specific examples include those including the following steps (a) to (d).
(a) contacting the double-stranded RNA by bringing the compound (cross-linking agent) of the present invention into contact with the double-stranded RNA,
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(b ′) expressing the gene in the introduced cell or organism before irradiating with light,
(c) irradiating the introduced cell or organism with light,
(c ′) expressing a gene in the irradiated cell or organism, and
(d) A step of comparing the gene expressed in step (b ′) with the gene expressed in step (c ′).
The cell or organism in step (b ′) and the cell or organism in step (c ′) are the same [one (group) cell / individual].

Other specific examples include those including the following steps (a) to (d).
(a) contacting the double-stranded RNA by bringing the compound (cross-linking agent) of the present invention into contact with the double-stranded RNA,
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(c) irradiating the introduced cell or organism with light,
(c ′) expressing a gene in a light-irradiated cell or organism,
(c '') expressing a gene in a cell or organism into which cross-linked double-stranded RNA has not been introduced, and
(d) A step of comparing the gene expressed in step (b ′) with the gene expressed in step (c ′).
The cell or organism in step (c '') is the same type (different cell / individual) as the cell or organism into which the double-stranded RNA crosslinked in step (b) has been introduced, that is, the same type. There are different cells / individuals.

  In the above method, the compound of the present invention (crosslinking agent), double-stranded RNA, the method of contacting the compound of the present invention (crosslinking agent) with double-stranded RNA, and the crosslinked double-stranded RNA introduced. Cells or organisms, methods of introducing cross-linked double-stranded RNA into the cells or organisms, light, light irradiation methods, methods of expressing genes in cells or organisms, preferred embodiments of these structural requirements, specific examples, etc. Is the same as described above.

In addition, as described above, also in the gene function investigation method of the present invention, the light irradiation may be performed on all cells or organisms into which the double-stranded RNA to which the compound of the present invention (crosslinking agent) is bound is introduced (whole). Moreover, the one part may be sufficient. By irradiating a part of the cell or organism with light, the function of the gene only in a predetermined region in the cell or organism into which the double-stranded RNA to which the compound of the present invention (crosslinking agent) is bound is introduced is investigated. be able to.
As a specific example in such a case, for example, one including the following steps (a) to (d) can be mentioned.
(a) contacting the double-stranded RNA by bringing the compound (cross-linking agent) of the present invention into contact with the double-stranded RNA,
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(c) irradiating a predetermined region of the introduced cell or organism with light,
(c ′) expressing a gene in the irradiated cell or organism, and
(d) A step of comparing the gene in the region irradiated with the light expressed in step (b ′) with the gene in the region not irradiated with light.
The cell or organism in the above is one (group) cell / individual.

5. Kit of the Present Invention The kit of the present invention is used for carrying out the crosslinking method, gene expression regulation method or gene function investigation method of the present invention as described above.
As such a kit, at least the compound of the present invention (crosslinking agent) as described above, preferably the compound represented by the general formula (1), more preferably the compound represented by the general formula (2), more preferably the general formula A compound represented by (6), particularly preferably one compound selected from a compound represented by formula (7), a compound represented by formula (8) and a compound represented by formula (9), most preferably It comprises a compound represented by the general formula (7).
Preferred embodiments and specific examples of these constituent elements are as described above.

  Furthermore, reagents other than those described above can be added to form the kit of the present invention. Examples of such reagents include, but are not limited to, transfection reagents for introducing double-stranded RNA into cells or organisms.

  In addition, instructions for use in the crosslinking method, gene expression regulation method or gene function investigation method of the present invention as described above may be included. The “instructions” refers to instruction manuals, package inserts, pamphlets (leaflets), etc. of the kits in which the features, principles, operation procedures, etc. in the method of the present invention are substantially described by sentences or diagrams. means.

The method of the present invention has the following effects, for example.
(1) Since the function of the target gene can be searched for by destroying the mRNA (target mRNA) of the target gene whose sequence is known, it is effective for functional analysis of the target gene.
(2) It is effective for searching unknown genes (for example, disease-related genes) and narrowing down candidate target genes.
(3) Rapid analysis of gene function and phenotype in a state where the gene is not expressed is possible.
(4) It can also be used for the development of so-called RNAi pharmaceuticals that use RNAi to treat diseases. In particular, according to the present invention, it is possible to develop an RNAi drug capable of producing a drug effect in a tissue or site-specific manner.

  Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

EXAMPLE 1 Glutaric acid bis (6-bromo-4-diazomethyl-2-oxo-2H-chromen-7-yl) ester (Pentanedioic acid bis (6-bromo-4-diazomethyl-2-oxo-2H-chromen-7) -yl) ester) (compound 1 of the invention)

In a 10 ml eggplant flask, add 24.9 mg (0.0898 mmol) of 6-bromo-7-hydroxy-4-diazomethylcoumarin, 1 ml of dichloromethane, 17.4 μl (0.1078 mmol) of triethylamine, 5.726 μl (0.0449 mmol) of glutaryl chloride. And stirred at room temperature for 1 day. Thereafter, the reaction mixture was diluted with chloroform, and the organic layer was washed with distilled water to stop the reaction. After drying with magnesium sulfate, the solvent was removed to obtain a crude product of Compound 1 of the present invention (0.2753 mg, 0.0418 mmol, yield 93%, yellow solid).
6-Bromo-7-hydroxy-4-diazomethylcoumarin was synthesized according to the method described in the WO00 / 31588 international publication pamphlet.
¹ H NMR (270 MHz, DMSO-d ₆ )
δ2.21 (2H, q, H3 ′), 2.89 (4H, t, 2H2 ′, 2H4 ′), 6.00 (H, S, −CH = N), 6.73 (H, S, H3), 7.54 (H, S, H8), 8.23 (H, S, H5)

Example 2 Glutaric acid (6-bromo-4-diazomethyl-2-oxo-2H-chromene) -7-yl 2- [4- (6-bromo-4-diazomethyl-2-oxo-2H-chromene-7- Ileoxycarbonyl) butyryloxy] ethyl ester (Pentanedioic acid (6-bromo-4-diazomethyl-2-oxo-2H-chromen) -7-yl 2- [4- (6-bromo-4-diazomethyl-2-oxo-) 2H-chromen-7-yloxycarbonyl) butyryloxy] ethyl ester) (compound 2 of the present invention)

(1) Synthesis of Pentanedioic acid mono [2- (4-carboxybutyryloxy) ethyl] ester (Compound 1)

To a 50 ml eggplant flask, add 2.5668 g (22.496 mmol) of glutaric anhydride, 20 ml of dichloromethane, 564.5 μl (10.12 mmol) of ethylene glycol, and 3.245 ml (23.28 mmol) of triethylamine, and stir at room temperature for 2 hours. Stir for 18.5 hours. Then, 4.1907 g, 9.7724 g, and 1.4920 g of ion exchange resin [Dow Chemical Co., Ltd., Dowex 1 × 2 (50-100 mesh)] were added in three portions, filtered with dichloromethane after the first addition, and after the second addition. After the third addition with dichloromethane, each was filtered with methanol. Subsequently, the solvent was removed to obtain a crude product of Compound 1 (3.036 g, 10.46 mmol, 93% yield from ¹ H NMR).
¹ H NMR (270 MHz, CDCl ₃ )
δ1.97 (4H, tt, 2H3 ', 2H10'), 2.43 (4H, t, 2H2 ', 2H11', J = 7.3Hz), 2.47 (4H, t, 2H4 ', 2H9', J = 7.3Hz) 4.31 (4H, S, 2H6 ', 2H7')

(2) Synthesis of 4-chlorocarbonylbutyric acid [2- (4-chlorocarbonylbutyryloxy) ethyl] ester (compound 2)

To a 100 ml eggplant flask, 268.8 mg (0.9260 mmol) of the compound 1 obtained in (1) above, 168.8 μl (2.3151 mmol) of thionyl chloride and 1 drop of DMF were added and stirred at 70-80 ° C. for 2 hours. Thereafter, thionyl chloride, DMF and HCl were removed, and a crude product of compound 2 was obtained from ¹ H NMR (296.9 g, 0.9075 mmol, 98%, white solid).
¹ H NMR (270 MHz, CDCl ₃ )
δ2.05 (4H, tt, 2H3 ', 2H10'), 2.45 (4H, t, 2H2 ', 2H11', J = 7.3Hz), 2.75 (4H, t, 2H4 ', 2H9', J = 7.3Hz) 4.31 (4H, S, 2H6 ', 2H7')

(3) Glutaric acid (6-bromo-4-diazomethyl-2-oxo-2H-chromene) -7-yl 2- [4- (6-bromo-4-diazomethyl-2-oxo-2H-chromene-7- Yloxycarbonyl) butyryloxy] ethyl ester (Pentanedioic acid (6-bromo-4-diazomethyl-2-oxo-2H-chromen) -7-yl 2- [4- (6-bromo-4-diazomethyl-2-oxo-) 2H-chromen-7-yloxycarbonyl) butyryloxy] ethyl ester) (compound 2 of the present invention)

To a 10 ml eggplant flask, 61.6 mg (0.219 mmol) of 6-bromo-7-hydroxy-4-diazomethylcoumarin and 3 ml of dichloromethane were added. To this solution, 33.6 μl (0.241 mmol) of triethylamine and 33.5 μg (0.0991 mmol) of the compound 2 obtained in the above (2) were added and stirred at room temperature for 1 day. Thereafter, the reaction mixture was diluted with chloroform, and the organic layer was washed with distilled water to stop the reaction. After drying with magnesium sulfate, the solvent was removed to obtain a crude product of Compound 2 of the present invention (82.4 mg, 0.101 mmol, yield 92%, yellow solid).
6-Bromo-7-hydroxy-4-diazomethylcoumarin was synthesized according to the method described in the WO00 / 31588 international publication pamphlet.
¹ H NMR (270 MHz, DMSO-d ₆ )
δ2.05 (4H, tt, 2H3 ', 2H10'), 2.45 (4H, t, 2H2 ', 2H11', J = 7.3Hz), 2.75 (4H, t, 2H4 ', 2H9', J = 7.3Hz) 4.31 (4H, S, 2H6 ′, 2H7 ′), 5.95 (H, S, −CH = N), 6.69 (H, S, H3), 7.45 (H, S, H8), 8.17 (H, S, H5)

Example 3 3,3-Dimethylglutaric acid (6-bromo-4-diazomethyl-2-oxo-2H-chromene) -7-yl 2- [4- (6-bromo-4-diazomethyl-2-oxo-2H) -Chromen-7-yloxycarbonyl) -3,3-dimethylbutyryloxy] ethyl ester (3,3-dimethylpentanedioic acid (6-bromo-4-diazomethyl-2-oxo-2H-chromen) -7-yl 2 -[4- (6-bromo-4-diazomethyl-2-oxo-2H-chromen-7-yloxycarbonyl) -3,3-dimethylbutyryloxy] ethyl ester) (compound 3 of the present invention)

(1) 3,3-Dimethylglutaric acid mono [2- (4-carboxy-3,3-dimethylbutyryloxy) ethyl] ester (3,3-dimethylpentanedioic acid mono [2- (4-carboxy-3,3 -dimethylbutyryloxy) ethyl] ester) (compound 3)

Into a 50 ml eggplant flask, 4.3785 g (30.799 mmol) of dimethylglutaric anhydride was added, 20 ml of dichloromethane, 837.0 μl (15.00 mmol) of ethylene glycol and 4.939 ml (35.42 mmol) of triethylamine were sequentially added, and the mixture was stirred at room temperature for 2 hours. The mixture was stirred at 40 ° C. for 24 hours. Thereafter, the temperature was returned to room temperature, 6 g of an ion exchange resin (Dow Chemical Co., Dowex 1 × 2 (50-100 mesh)) was added, and the mixture was filtered with dichloromethane. Subsequently, the solvent was removed to obtain a crude product of compound 3 (4.5085 g, 13.009 mmol, 88% yield from ¹ H NMR).
¹ H NMR (270 MHz, CDCl ₃ )
δ = 1.15 (12H, s, 3'-2CH3,10'-2CH3), 2.48 (4H, s, 2H 2 ', 2H 11), 2.51 (4H, s, 2H 4', 2H 9 '), 4.28 ( 4H, s, 2H ₆ ', 2H ₇ ')

(2) 4-chlorocarbonyl-3,3-dimethylbutyric acid [2- (4-chlorocarbonyl-3,3-dimethylbutyryloxy) ethyl] ester (4-chlorocarbonyl-3,3-dimethylbutyric acid [2- ( 4-chlorocarbonyl-3,3-dimethylbutyryloxy) ethyl] ester) (compound 4)

To a 100 ml eggplant flask, 299.7 mg (0.9260 mmol) of the compound 3 obtained in (1) above, 158 μl (2.3151 mmol) of thionyl chloride, and 1 drop of DMF were added and stirred at 70-80 ° C. for 2 hours. Thereafter, thionyl chloride, DMF, and HCl were removed to obtain a crude product of compound 4 (309.1 mg, 13.009 mmol, 88% yield from ¹ H NMR).
¹ H NMR (270 MHz, CDCl ₃ )
δ = 1.15 (12H, s, 3'-2CH3,10'-2CH3), 2.47 (4H, s, 2H ₂ ', 2H ₁₁ '), 3.15 (4H, s, 2H ₄ ', 2H ₉ '), 4.29 (4H, s, 2H ₆ ', 2H ₇ ')

(3) 3,3-dimethylglutaric acid (6-bromo-4-diazomethyl-2-oxo-2H-chromene) -7-yl 2- [4- (6-bromo-4-diazomethyl-2-oxo-2H) -Chromen-7-yloxycarbonyl) -3,3-dimethylbutyryloxy] ethyl ester (3,3-dimethylpentanedioic acid (6-bromo-4-diazomethyl-2-oxo-2H-chromen) -7-yl 2 -[4- (6-bromo-4-diazomethyl-2-oxo-2H-chromen-7-yloxycarbonyl) -3,3-dimethylbutyryloxy] ethyl ester) (compound 3 of the present invention)

In a 10 ml eggplant flask, 28.27 mg (0.0737 mmol) of compound 4 obtained in (2) above, dichloromethane 1 ml, triethylamine 33.9 μl (0.243 mmol), 6-bromo-7-hydroxy-4-diazomethylcoumarin 62.2 mg (0.2213 mmol) was added, and the mixture was stirred at 40 ° C. for 24 hours. Thereafter, the temperature was returned to room temperature, 1 ml of water was added, extraction was performed alternately with chloroform and dichloromethane, and the organic layer was dried over magnesium sulfate, and then the solvent was removed to obtain a crude product of compound 3 of the present invention ( 55.5 mg, 0.0636 mmol, 87% yield from ¹ H NMR).
6-Bromo-7-hydroxy-4-diazomethylcoumarin was synthesized according to the method described in the WO00 / 31588 international publication pamphlet.
¹ H NMR (270 MHz, DMSO)
δ = 1.08 (12H, s, 3'-2CH ₃ and 10'-2CH ₃ ), 2.68 (4H, s, H _{2 '} and H _11' ), 3.22 (4H, s, H _{4 '} and H _9' ) 4.19 (4H, s, H _{6 '} and H _7' ), 5.85 (H, s, −CH = N), 6.60 (H, s, H ₃ ), 7.30 (H, s, H ₈ ), 8.06 ( H, s, H ₅ )

Example 4 Cross-linking of double-stranded RNA by the compound of the present invention It was confirmed whether the compound of the present invention cross-links double-stranded RNA.
(1) Crosslinking agent solution The compound 3 of the present invention obtained in Example 3 was dissolved in a DMSO solution so as to have a concentration of 21.1 μM to obtain a crosslinking agent solution.
(2) Sample A crosslinker solution or DMSO and a siRNA (double-stranded RNA) solution (20 μM siRNA-containing TE interference solution) were mixed and allowed to stand for 8 hours under light-shielding conditions so as to have the composition shown in Table 1 below. . Among these samples, sample Nos. 5 and 6 were irradiated with UV (350 nm) for 3 minutes using a Rayonet Photochemical Reactor apparatus manufactured by Southern NE Ultraviolet.
Thereafter, 2 μl of 5% formamide was added to each sample, and sample Nos. 2, 4 and 6 were heated at 65 ° C. for 15 minutes and then allowed to stand in ice for 10 minutes.
The siRNA was 22 bp (QIAGEN) consisting of the following base sequence targeting GFP.
5'- GCAAGCUGACCCUGAAGUUCAU-3 '
3'-GCCGUUCGACUGGGACUUCAAG -5 '

(3) Electrophoresis Each sample obtained in (2) above was prepared by mixing 2 μl of sample buffer (3 ml of glycerin, 0.1 ml of 0.5 M EDTA-HCl aqueous solution (pH 8.0) and 6.9 ml of purified water. The mixture was mixed and then electrophoresed at 7 mA (constant current) for 1 hour using a MODEL BE-211 electrophoresis apparatus manufactured by BIO CRAFT, 20% polyacrylamide gel, and x1 TBE buffer.
Thereafter, the gel was stained with SYBR GOLD (Molecular Probes) and observed with a transilluminator (Funakoshi).

(4) Results The results are shown in FIG. In the figure, lane No. 1 uses the results when sample No. 6 is used, lane No. 2 uses the results when sample No. 4 is used, and lane No. 3 uses the sample No. 5. Lane No. 4 shows the result when sample No. 3 is used, lane No. 5 shows the result when sample No. 2 is used, and lane No. 6 shows the result when sample No. 1 is used. The results when used are shown, “dsRNA →” indicates the migration position of double-stranded RNA, and “ssRNA →” indicates the migration position of single-stranded RNA.

As is clear from FIG. 1, from the results of lanes No. 5 and 6, double-stranded RNA (siRNA) not reacted with the compound of the present invention (crosslinking agent) (lane No. 5: sample No. 2) It can be seen that it cannot withstand heat denaturation and dissociates into a single strand. In contrast, the double-stranded RNA (siRNA) (lane No. 2: sample No. 4) reacted with the compound of the present invention (crosslinking agent) was heat denatured unless irradiated with UV. The two-strands are kept in the single-stranded form after being reacted with the compound of the present invention (crosslinking agent), irradiated with UV, and further heated (lane No. 1: sample No. 6). You can see that they are dissociated.
From the above, it is clear that the compound (cross-linking agent) of the present invention cross-links double-stranded RNA (siRNA), and the cross-linking is released by UV irradiation. It can be seen that the thermal stability of RNA is increased.

Example 5 Confirmation of RNAi Effect Inhibitory Ability by Compounds of the Present Invention (1) Reagents / HeLa Cells (American Type Culture Collection (ATCC, Rockville, MD))
・ DMEM medium (Nissui Pharmaceutical Co., Ltd., Dulbecco's modified Eagle medium Nissui 2)
・ Opti-MEM medium (GIBCO)
・ Trypsin solution (0.38mg / ml EDTA aqueous solution containing 2.5mg trypsin: GIBCO)
・ Lipofectamine 2000 (manufactured by Invitrogen)
・ PEGFP-N1 vector solution
A pEGFP-N1 vector (manufactured by CLONTEC, BD Living colors) dissolved in TE buffer so as to be 1714 μg / ml was used.
・ PDsRed2-N1 vector solution
A pDsRed2-N1 vector (manufactured by CLONTEC, BD Living colors) dissolved in TE buffer so as to be 1612 μg / ml was used.
・ SiRNA solution:
A double-stranded RNA having the following sequence (targeted with the EGFP gene) dissolved in water to a concentration of 200 nM was used.
5'- GCAAGCUGACCCUGAAGUUCAU-3 '
3'-GCCGUUCGACUGGGACUUCAAG -5 '
・ Crosslinking agent solution:
A compound obtained by dissolving the compounds 1 to 3 of the present invention obtained in Examples 1 to 3 in a DMSO solution so as to have a concentration of 4 μM was used as a crosslinking agent solution.

(2) Preparation of Hela cells for transfection
HeLa cells (Hela cells and 500 μl of 10% FBS-containing DMEM medium) were spread at a density of 3 × 10 ⁴ cells / well in a 24-well plate. After 24 hours, the old medium was removed, and the Hela cells were washed with a PBS (−) solution. Then, the PBS (−) solution was removed, and 500 μl of 10% FBS-containing DMEM medium was added per well. This was used as the Hela cell plate for transfection.

(3) Preparation of Transfection Sample i) Crosslinking of siRNA As shown in Table 2 below, 0.2 μl of the crosslinker solution and 0.20 μl of the siRNA solution were mixed and allowed to stand for 8 hours under light-shielding conditions. Among these, Nos. 2, 4 and 6 were irradiated with UV (350 nm) for 3 minutes using a Rayonet Photochemical Reactor apparatus manufactured by Southern NE Ultraviolet.

ii) Preparation of sample As shown in Table 3 below, 90 µl of Opti-MEM medium, 0.36 µl of pRGFP vector solution, 0.60 µl of pDsRed vector solution and 0.40 µl of the reaction solution prepared in i) above were mixed and allowed to stand for 15 minutes. This was used as a sample for transfection.
In addition, instead of the reaction solution prepared in i), 0.20 μl of siRNA solution (double-stranded RNA not crosslinked with the compound of the present invention) was used as a control sample.
Here, pEGFP 0.36 μl contains 617 ng pEGFP, pDsRed 0.60 μl contains 985 ng pDsRed, and 200 nM siRNA 0.20 μl contains 62.6 pg siRNA.

(4) Transfection
0.8 μl of Lipofectamine 2000 was added to 9.2 μl of Opti-MEM medium and allowed to stand for 10 minutes.
Subsequently, this and the transfection sample obtained in the above ii) were mixed and allowed to stand for 20 minutes.
The obtained mixture was added to the transfection Hela cell plate prepared in (2) above, and cultured at 37 ° C. for 48 hours using a CO ₂ incubator (NAPCO, Automatic CO ₂ Incubators 5400).

(5) Results After the culture, observation of a transmission image of cells, fluorescence images of EGFP and DsRed was performed.
Using Meta Morph analysis software (manufactured by Meta Imaging Software), the total area of each fluorescent area of EGFP and DsRed was determined, and the EGFP / DsRed ratio was determined.
The result is shown in FIG.
In addition, in FIG. 2, the area of EGFP-expressing cells per area and the area of DsRed-expressing cells were counted using the analysis software, and the expression ratio when co-transfecting EGFP and DsRed was taken as 100%. The expression level of EGFP is expressed as a relative value.

  As is apparent from the results of FIG. 2, it can be seen that any of the compounds of the present invention can suppress the RNAi effect of siRNA by binding to siRNA, and can recover the RNAi suppression by UV irradiation. . Among them, the compounds 2 and 3 of the present invention have a high inhibitory action on the RNAi effect, and the action is found to be the highest for the compound 3 of the present invention.

Example 6 Examination of the reaction time of the compound of the present invention and siRNA (1) Reagent As a cross-linking agent solution, the compound 3 of the present invention obtained in Example 3 was dissolved in a DMSO solution to a concentration of 4 μM. The same thing as Example 5 was used except having used what.

(2) Preparation of Hela cells for transfection Prepared by the same method as in Example 5.

(3) Preparation of transfection sample i) Cross-linking of siRNA Mix 0.2 μl of cross-linking agent solution and 0.20 μl of siRNA solution, and leave it under light-shielded conditions for 0 hours, 1 hour, 2 hours, 4 hours, 8 hours or 24 hours. did.

ii) Preparation of sample As shown in Table 4 below, 90 µl of Opti-MEM medium, 0.36 µl of pRGFP vector solution, 0.60 µl of pDsRed vector solution and 0.40 µl of the reaction solution prepared in i) above were mixed and allowed to stand for 15 minutes. This was used as a sample for transfection.
In addition, instead of the reaction solution prepared in i), 0.20 μl of siRNA solution (double-stranded RNA not crosslinked with the compound of the present invention) was used as a control sample.
Here, pEGFP 0.36 μl contains 617 ng pEGFP, pDsRed 0.60 μl contains 985 ng pDsRed, and 200 nM siRNA 0.20 μl contains 62.6 pg siRNA.

(4) Transfection It was carried out in the same manner as in Example 5.

(5) Results After the culture, observation of a transmission image of cells, fluorescence images of EGFP and DsRed was performed.
Using Meta Morph analysis software (manufactured by Meta Imaging Software), the total area of each fluorescent area of EGFP and DsRed was determined, and the EGFP / DsRed ratio was determined.
The result is shown in FIG.
In FIG. 3, the area of EGFP-expressing cells per area and the area of DsRed-expressing cells are counted using the analysis software, and the expression ratio when co-transfecting EGFP and DsRed is 100%. The expression level of EGFP in is shown as a relative value.

  As is clear from the results of FIG. 3, it was found that the expression of EGFP was suppressed to 9.0% by siRNA. In addition, when the reaction time between the siRNA and the compound 3 of the present invention is short, the reaction does not proceed, and the siRNA cannot be sufficiently crosslinked, and further, no significant difference is observed between the reaction time of 8 hours and 24 hours. It was found that the reaction time of siRNA and the compound 3 of the present invention reached a plateau in about 8 hours.

Example 7 Confirmation of effectiveness of compound of the present invention (1) Photodegradable protecting group
(a): Compound 3 of the present invention
(b): 3,3-dimethylglutaric acid bis (6-bromo-4-methyl-2-oxo-2H-chromene) -7-yl 2- [4- (6-bromo-4-methyl-2-oxo -2H-chromen-7-yloxycarbonyl) -3,3-dimethylbutyryloxy] ethyl ester (3,3-Dimethylpentanedioic acid (6-bromo-4-methyl-2-oxo-2H-chromen) -7- yl 2- [4- (6-bromo-4-methyl-2-oxo-2H-chromen-7-yloxycarbonyl) -3,3-dimethylbutyryloxy] ethyl ester) (hereinafter abbreviated as bis-Bhc-CH ₃ ). )

The compound was synthesized according to the method of Example 3 except that 6-bromo-7-hydroxy-4-methylcoumarin was used.
Incidentally, 6-bromo-7-hydroxy-4-methylcoumarin is obtained from T. Furuta, H. Takeuchi, M. Isozaki, Y. Takahashi, M. Sugimoto, M. Kanehara, T. Watanabe, K. Noguchi, TM Dore. , M. Iwamura, RY Tsien, Bhc-cNMPs as either water-soluble or membrane-permeant photo-releasable cyclic nucleotides for both one and two-photon excitation, ChemBioChem, 5, 1119-1128 (2004) And synthesized.
¹ H NMR (CD3OD) δ 2.13 (4h, quintet, J = 7.3 Hz), 2.41 (6H, s), 2.55 (4H, t, J = 7.3 Hz), 2.76 (4H, t, J = 7.3 Hz), 4.34 (4H, s), 6.29 (2H, s), 7.15 (2H, s), 7.81 (2H, s); ¹³ C NMR (DMSO-d6) δ 18.65 (q), 19.75 (t), 32.81 (t ), 32.95 (t), 62.24 (t), 111.56 (s), 112.57 (d), 115.46 (d), 119.45 (d), 150.17 (s), 150.81 (s), 153.02 (s), 159.68 (s) ), 169.92 (s), 172.43 (s); IR (ATR) 2953, 1770, 1727, 1396, 1384, 1360, 1147, 1114

(c): 6-bromo-7-hydroxy-4-diazomethylcoumarin (hereinafter abbreviated as Bhc-diazo)
It was synthesized according to the method described in the WO00 / 31588 international pamphlet.

(2) Reagent As the cross-linking agent solution, the same one as in Example 5 was used except that the following was used.
-Compound 3-containing crosslinking agent solution of the present invention The compound 3 of the present invention obtained in Example 3 was dissolved in DMSO solution to a concentration of 4 µM.
・ Bisc-CH ₃ containing crosslinker solution
A solution in which bis-Bhc-CH ₃ was dissolved in a DMSO solution to a concentration of 4 μM was used.
・ Bhc-diazo-containing crosslinking agent solution
Bhc-diazo dissolved in DMSO solution to a concentration of 8 μM was used.

(3) Preparation of Hela cells for transfection Prepared by the same method as in Example 5.
(4) Preparation of transfection sample i) Cross-linking of siRNA As shown in Table 5 below, 0.2 μl of a cross-linking agent solution and 0.20 μl of siRNA solution were mixed and allowed to stand for 8 hours under light-shielding conditions. Among these, No. 2, 4 and 6 were irradiated with UV (350 nm) for 3 minutes using a Rayonet Photochemical Reactor apparatus manufactured by Southern NE Ultraviolet.

ii) Preparation of sample As shown in Table 6 below, 90 μl of Opti-MEM medium, 0.36 μl of pRGFP vector solution, 0.60 μl of pDsRed vector solution and 0.40 μl of the reaction solution prepared in i) above were mixed and allowed to stand for 15 minutes. This was used as a sample for transfection.
In addition, instead of the reaction solution prepared in i), 0.20 μl of siRNA solution (double-stranded RNA not crosslinked with the compound of the present invention) was used as a control sample.
Here, pEGFP 0.36 μl contains 617 ng pEGFP, pDsRed 0.60 μl contains 985 ng pDsRed, and 200 nM siRNA 0.20 μl contains 62.6 pg siRNA.

(5) Transfection It was carried out in the same manner as in Example 5.

(6) Results After the culture, the permeation image of the cells and the fluorescence images of EGFP and DsRed were observed.
Using Meta Morph analysis software (manufactured by Meta Imaging Software), the total area of each fluorescent area of EGFP and DsRed was determined, and the EGFP / DsRed ratio was determined.
The result is shown in FIG.
In FIG. 4, the area of EGFP-expressing cells per area and the area of DsRed-expressing cells are counted using the analysis software, and the expression ratio when co-transfecting EGFP and DsRed is 100%. The expression level of EGFP in is shown as a relative value.

As is apparent from FIG. 4, it can be seen that the compound 3 of the present invention can suppress the siRNA action without UV irradiation, and the suppression is restored by UV irradiation. In contrast, bis-Bhc-CH ₃ has a methyl group instead of an azo group that can be covalently bonded to siRNA, and therefore cannot bind to siRNA. It can be seen that the action of siRNA can hardly be suppressed. In addition, Bhc-diazo has an azo group that can be covalently bound to siRNA, but it does not have a linker moiety, so it cannot crosslink the duplex of siRNA and almost suppresses the action of siRNA. It turns out that it is not possible.
From the above, in order to suppress the action of siRNA, the photodegradable protective group has a group capable of binding to double-stranded RNA, and the two photodegradable protective groups are double-stranded. It can be seen that it is important to have structures located at both ends of the linker moiety so that RNA can be cross-linked.

Example 8 Investigation in cell (1) Reagent The same reagent as in Example 6 was used.

(2) Preparation of Hela cells for transfection
HeLa cells (Hela cells and 10% FBS-containing DMEM 500 μl) were spread on a 24-well plate at 5 × 10 ³ cells / well. After 24 hours, the old medium was removed, and the Hela cells were washed with a PBS (−) solution. Then, the PBS (−) solution was removed, and 500 μl of DMEM containing 10% FBS was added per well. This was used as the Hela cell plate for transfection.

(3) Preparation of transfection sample i) 0.2 μl of siRNA crosslinker solution and 0.20 μl of siRNA solution were mixed and allowed to stand for 8 hours under light-shielding conditions.
ii) Preparation of sample As shown in Table 7 below, 90 µl of Opti-MEM medium, 0.36 µl of pRGFP vector solution, 0.60 µl of pDsRed vector solution and 0.40 µl of the reaction solution prepared in i) above were mixed and allowed to stand for 15 minutes. This was used as a sample for transfection.
In addition, instead of the reaction solution prepared in i), 0.20 μl of siRNA solution (double-stranded RNA not crosslinked with the compound of the present invention) was used as a control sample.
Here, pEGFP 0.36 μl contains 617 ng pEGFP, pDsRed 0.60 μl contains 985 ng pDsRed, and 200 nM siRNA 0.20 μl contains 62.6 pg siRNA.

(4) Transfection
0.8 μl of Lipofectamine 2000 was added to 9.2 μl of Opti-MEM medium and allowed to stand for 10 minutes.
Subsequently, this and the transfection sample obtained in the above ii) were mixed and allowed to stand for 20 minutes.
The obtained mixed solution was added to the transfection Hela cell plate prepared in (2) above, and cultured at 37 ° C. for 6 hours using a CO ₂ incubator (NAPCO, Automatic CO ₂ Incubators 5400).

(5) UV irradiation After the culture, the medium was removed, and the Hela cells were washed with a PBS (−) solution. Then, the PBS (−) solution was removed, and 500 μl of Opti-MEM medium was added per well. Next, UV (335-385 nm) was irradiated to the center of the well for 1 second using an inverted fluorescent microscope IX71 (objective lens: UPLAPO × 10) manufactured by Olympus Corporation.
After UV irradiation, the Opti-MEM medium was removed, 500 μl of 10% FBS-containing DMEM per well was added, and the cells were cultured at 37 ° C for 42 hours using a CO ₂ incubator (NAPCO, Automatic CO ₂ Incubators 5400). .

(6) Results After the culture, the permeation image of the cells and the fluorescence images of EGFP and DsRed were observed.
Using Meta Morph analysis software (manufactured by Meta Imaging Software), the total area of each fluorescent area of EGFP and DsRed was determined, and the EGFP / DsRed ratio was determined.
The result is shown in FIG.
In FIG. 5, the area of EGFP-expressing cells and the area of DsRed-expressing cells per a certain area are counted using the analysis software, and the expression ratio when co-transfecting EGFP and DsRed is 100%. The expression level of EGFP in is shown as a relative value.
Moreover, the result of having observed the fluorescence image of EGFP and DsRed at the time of using the compound 3 of this invention is shown in FIG.

As is clear from FIGS. 5 and 6, siRNA cross-linking by the compound 3 of the present invention is not released in the UV-irradiated part, and the RNAi effect of siRNA is suppressed, whereas in the UV-irradiated part, the compound of the present invention is suppressed. It can be seen that siRNA cross-linking by 3 is released and the RNAi effect of siRNA is restored.
From the above, it is possible to release the siRNA cross-linking by the compound of the present invention specifically at any place by narrowing the UV irradiation site and locally irradiating, that is, specific at any place. It can be seen that gene expression can be suppressed.

Example 9 Confirmation of Effect of Compound of the Present Invention (Crosslinking Agent) and Light Irradiation on RNAi Introducing siRNA reacted with the compound of the present invention has a lower expression level than that transfected with EGFP alone. In order to investigate this cause, it was confirmed whether the transfection efficiency was lowered due to the compound of the present invention, or whether transfection efficiency was already lowered by transfection with siRNA.

(1) Reagent A cross-linker solution obtained by dissolving the compound 3 of the present invention obtained in Example 3 in a DMSO solution to a concentration of 4 μM is used. In addition to siRNA, the following sequence is used as a control siRNA. The same thing as Example 5 was used except having used the control siRNA solution which melt | dissolved in RNA so that it might become a density | concentration of 200 nM.
5'- UUCUCCGAACGUGUCACGUdTdT-3 '
3'-dTdTAAGAGGCUUGCACAGUGCA -5 '

(2) Preparation of Hela cells for transfection Prepared by the same method as in Example 5.

(3) Preparation of transfection sample i) Cross-linking of siRNA As shown in Table 8 below, 0.2 μl of cross-linking agent solution and 0.20 μl of siRNA solution or 0.20 μl of control siRNA solution were mixed and allowed to stand for 8 hours under light-shielding conditions. .

ii) Preparation of sample As shown in Table 9 below, 90 μl of Opti-MEM medium, 0.36 μl of pRGFP vector solution, 0.60 μl of pDsRed vector solution and 0.40 μl of the reaction solution prepared in i) above were mixed and allowed to stand for 15 minutes. This was used as a sample for transfection.
Further, instead of the reaction solution prepared in i), siRNA solution (double-stranded RNA not crosslinked with the compound of the present invention) 0.20 μl or control siRNA solution (double-stranded RNA not crosslinked with the compound of the present invention) A control sample was prepared by performing the same operation as described above using 0.20 μl.
Here, pEGFP 0.36 μl contains 617 ng pEGFP, pDsRed 0.60 μl contains 985 ng pDsRed, and 200 nM siRNA 0.20 μl contains 62.6 pg siRNA.

(4) Transfection
0.8 μl of Lipofectamine 2000 was added to 9.2 μl of Opti-MEM medium and allowed to stand for 10 minutes.
Next, this was mixed with the transfection sample or control sample obtained in the above ii) and allowed to stand for 20 minutes.
The obtained mixed solution was added to the transfection Hela cell plate prepared in (2) above, and cultured at 37 ° C. for 6 hours using a CO ₂ incubator (NAPCO, Automatic CO ₂ Incubators 5400).

(5) UV irradiation After the culture, the medium was removed, and the Hela cells were washed with a PBS (−) solution. Then, the PBS (−) solution was removed, and 500 μl of Opti-MEM medium was added per well. Next, an inverted fluorescence microscope IX71 (objective lens: UPLAPO × 10) manufactured by Olympus Co., Ltd. was used in the wells in which the Hela cells into which the transfection sample d was introduced and the Hela cells into which the transfection sample f had been introduced. ) Was irradiated with UV (335-385 nm) for 1 second.
After UV irradiation, the Opti-MEM medium was removed, 500 μl of 10% FBS-containing DMEM per well was added, and the cells were cultured at 37 ° C for 42 hours using a CO ₂ incubator (NAPCO, Automatic CO ₂ Incubators 5400). .

(6) Results After the culture, the permeation image of the cells and the fluorescence images of EGFP and DsRed were observed.
Using Meta Morph analysis software (manufactured by Meta Imaging Software), the total area of each fluorescent area of EGFP and DsRed was determined, and the EGFP / DsRed ratio was determined.
The result is shown in FIG.
In FIG. 7, the area of EGFP-expressing cells per area and the area of DsRed-expressing cells are counted using the analysis software, and the expression ratio when co-transfecting EGFP and DsRed is 100%. The expression level of EGFP in is shown as a relative value.

  As is apparent from FIG. 7, the expression rate when only the control siRNA is transfected and the expression rate when the control siRNA is cross-linked with the compound 3 of the present invention are comparable. Therefore, the decrease in the expression level is not caused by the decrease in transfection efficiency due to the transfection of the compound of the present invention or siRNA itself, that is, that the siRNA exhibits a specific effect, the transfection efficiency. Was confirmed not to be decreased by siRNA and the compound of the present invention.

Example 10 Examination of Photoregulation of Endogenous Gene by Compound 3 of the Present Invention (1) Reagent / HeLa Cell (American Type Culture Collection (ATCC, Rockville, MD))
・ DMEM medium (Nissui Pharmaceutical Co., Ltd., Dulbecco's modified Eagle medium Nissui 2)
・ Opti-MEM medium (GIBCO)
・ Trypsin solution (0.38mg / ml EDTA aqueous solution containing 2.5mg trypsin: GIBCO)
・ Lipofectamine 2000 (manufactured by Invitrogen)
・ SiRNA solution:
A double-stranded RNA having the following sequence (targeted with the Lamin B1 gene) dissolved in water to a concentration of 20 nM was used.
5'- CGCGCUUGGUAGAGGUGGAdTdT-3 '
3'-dTdTGCGCGAACCAUCUCCACCU -5 '
・ Crosslinking agent solution:
A solution obtained by dissolving the compound 3 of the present invention obtained in Example 3 in a DMSO solution to a concentration of 40 μM was used as a crosslinking agent solution.

(2) Preparation of Hela cells for transfection
HeLa cells (Hela cells and 10% FBS-containing DMEM 500 μl) were spread on a 24-well plate at 1 × 10 ³ cells / well. After 24 hours, the old medium was removed, and the Hela cells were washed with a PBS (−) solution. Then, the PBS (−) solution was removed, and 500 μl of DMEM containing 10% FBS was added per well. This was used as the Hela cell plate for transfection.

(3) Preparation of transfection sample i) Cross-linking of siRNA 4.6 μl of cross-linking agent solution and 0.46 μl of siRNA solution were mixed and allowed to stand for 8 hours under light-shielding conditions.
ii) Preparation of Sample As shown in Table 10 below, 90 μl of Opti-MEM medium and 5.06 μl of the reaction solution prepared in i) above were mixed and allowed to stand for 15 minutes, which was used as a sample for transfection.
Moreover, what performed operation similar to the above using 0.46 microliters of siRNA solutions (double stranded RNA which is not bridge | crosslinked with the compound of this invention) instead of the reaction liquid prepared by i) was made into the control sample.
Here, 62.6 pg of siRNA is contained in 0.46 μl of 20 nM siRNA.

(4) Transfection It carried out like Example 8.

(5) UV irradiation It carried out similarly to Example 8.

(6) Results After culture, the amount of Laminin B1 protein in each sample was quantified by Western blotting.
The results of Western blotting of Lamin B1 protein in each sample and the results of Western blotting of β-actin protein in each sample as a control are shown in FIG. In the figure, lane No. 1 uses the results when sample No. 1 is used, lane No. 2 uses the results when sample No. 2 is used, and lane No. 3 uses the sample No. 3. Lane No. 4 shows the results when Hela cells into which siRNA and compound 3 of the present invention were not introduced and UV was not irradiated were used.
Further, the amount of Laminin B1 protein in each sample was quantified from the results of Western blotting.
The result is shown in FIG. FIG. 9 shows the amount of Laminin B1 protein in each sample transfected with siRNA alone and the relative amount of Laminin B1 protein in each sample.

As is apparent from the results of FIGS. 8 and 9, β-actin, which is not a siRNA control gene, is the gene in any case regardless of the presence or absence of siRNA, the presence or absence of compound 3 of the present invention, and the presence or absence of UV irradiation. It can be seen that is expressed. On the other hand, Lamin B1, which is a control of siRNA, can suppress the RNAi effect of siRNA when its expression is suppressed to 12% by siRNA (lane No. 3) and compound 3 of the present invention binds to siRNA. (Lane No. 2), and further, it can be seen that RNAi suppression by the compound 3 of the present invention can be recovered by UV irradiation (lane No. 1).
From the above, it can be seen that by using the compound (crosslinking agent) of the present invention, not only the gene that was introduced from the outside and overexpressed, but also the expression of the endogenous gene can be controlled by the presence or absence of light irradiation.

The present invention relates to a cross-linking agent that cross-links between double-stranded nucleic acids, between nucleic acids-proteins or polypeptides, or between proteins or polypeptides, particularly between double-stranded RNAs, cross-linking methods thereof, gene expression regulation methods, and gene functions. It provides a survey method.
According to the present invention, it is possible to easily perform cross-linking and release of cross-linking between double-stranded nucleic acids, between nucleic acid-proteins or polypeptides, or between proteins or polypeptides, particularly between double-stranded RNAs. Expression can be controlled at any time and place. In addition, the RNAi effect of double-stranded RNA (siRNA), which was difficult to suppress with conventional caged compounds, can be suppressed, and the expression of the target gene can be easily controlled at any time and place.

The result of electrophoresis of each sample obtained in Example 4 is shown. The expression level of EGFP in each sample is shown as a relative value when the expression ratio obtained when co-transfecting EGFP and DsRed obtained in Example 5 is 100%. The expression level of EGFP in each sample when the expression ratio when co-transfecting EGFP and DsRed obtained in Example 6 is 100% is shown as a relative value. The expression level of EGFP in each sample is shown as a relative value when the expression ratio obtained when co-transfecting EGFP and DsRed obtained in Example 7 is 100%. The expression level of EGFP in each sample is shown as a relative value when the expression ratio obtained when co-transfecting EGFP and DsRed obtained in Example 8 is 100%. The result of having observed the fluorescence image of EGFP and DsRed in the cell obtained in Example 8 is shown. The expression level of EGFP in each sample when the expression ratio when co-transfecting EGFP and DsRed obtained in Example 9 is 100% is shown as a relative value. The result of the Western blot obtained in Example 10 is shown. The expression level of EGFP in each sample is shown as a relative value when the expression ratio obtained when co-transfection of EGFP and DsRed obtained in Example 10 is 100%.

Claims (10)

A cross-linking agent in which 6-bromo-7-hydroxy-4-diazomethylcoumarin is linked by a linker at a distance of 8 to 50 mm . The double-stranded RNA, a method of crosslinking with a crosslinking agent according to claim 1 via a phosphate residue of the RNA strand. A method for regulating gene expression, which comprises irradiating light to a double-stranded RNA to which the cross-linking agent according to claim 1 is bound in advance (except for application to the human body) . A method for regulating gene expression comprising the following steps (except for application to the human body) .
(a) contacting the double-stranded RNA by bringing the cross-linking agent according to claim 1 into contact with the double-stranded RNA;
(b) introducing a crosslinked double-stranded RNA into a cell or organism; and
(c) A step of irradiating the introduced cell or organism with light. The method for regulating gene expression according to claim 4 , further comprising the step (c ') of expressing a gene in a light-irradiated cell or organism following the step (c). The method according to claim 4 , wherein the light is applied to a predetermined area of the introduced cell or organism. Method for investigating gene function including the following steps (except for application to the human body) .
(a) contacting the double-stranded RNA by bringing the cross-linking agent according to claim 1 into contact with the double-stranded RNA;
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(c) irradiating the introduced cell or organism with light,
(c ′) expressing a gene in the irradiated cell or organism, and
(d) a gene expressed in step (c ′) and a gene whose expression is not substantially suppressed by the double-stranded RNA in a cell or organism having a target mRNA complementary to the double-stranded RNA. The step of comparing the expression increase / decrease or the presence / absence of expression, the increase / decrease of the expression product or the presence / absence of the expression product, or the phenotype resulting from the expression . The method according to claim 7 , wherein the light is irradiated to a predetermined area of the introduced cell or organism. Method for investigating gene function including the following steps (except for application to the human body) .
(a) contacting the double-stranded RNA by bringing the cross-linking agent according to claim 1 into contact with the double-stranded RNA;
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(c) irradiating the introduced cell or organism with light,
(c ′) expressing a gene in a cell or organism irradiated with light,
(c '') expressing the gene in the cell or organism without irradiating the introduced cell or organism, and
(d) Expressions resulting from expression of the gene expressed in step (c ') and the gene expressed in step (c''), with or without expression, with or without expression, with or without expression product or with or without expression product Comparing the molds;
Or
(a) contacting the double-stranded RNA by bringing the cross-linking agent according to claim 1 into contact with the double-stranded RNA;
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(b ′) expressing the gene in the introduced cell or organism before irradiating with light,
(c) irradiating the introduced cell or organism with light,
(c ′) expressing a gene in the irradiated cell or organism, and
(d) The gene expressed in step (b ′) and the gene expressed in step (c ′), the increase / decrease or presence / absence of expression, increase / decrease of expression product / presence / absence of expression product, or phenotype generated by expression Comparing for;
Or
(a) contacting the double-stranded RNA by bringing the cross-linking agent according to claim 1 into contact with the double-stranded RNA;
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(c) irradiating the introduced cell or organism with light,
(c ′) expressing a gene in a cell or organism irradiated with light,
(c '') expressing a gene in a cell or organism into which cross-linked double-stranded RNA has not been introduced, and
(d) Expressions resulting from expression of the gene expressed in step (c ') and the gene expressed in step (c''), with or without expression, with or without expression, with or without expression product or with or without expression product The process of comparing molds. Method for investigating gene function including the following steps (except for application to the human body) .
(a) contacting the double-stranded RNA by bringing the cross-linking agent according to claim 1 into contact with the double-stranded RNA;
(b) introducing a crosslinked double-stranded RNA into a cell or organism,
(c) irradiating a predetermined region of the introduced cell or organism with light,
(c ′) expressing a gene in the irradiated cell or organism, and
(d) Increase / decrease of expression, presence / absence of expression product, increase / decrease of expression product, presence / absence of expression product of gene in region irradiated with light expressed in step (c ′) and gene in region not irradiated with light Or comparing phenotypes that occur upon expression.

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