WO2006030788A1

WO2006030788A1 - Intercalator and method of gene detection using the same

Info

Publication number: WO2006030788A1
Application number: PCT/JP2005/016847
Authority: WO
Inventors: Shinichiro Isobe
Original assignee: Shinichiro Isobe
Priority date: 2004-09-14
Filing date: 2005-09-13
Publication date: 2006-03-23
Also published as: JPWO2006030788A1

Abstract

An intercalator that through suppression of any fluorescence quenching of fluorochrome in the dry state, realizes operation simplification and high-sensitivity detection; and a method of gene detection using the same. There is provided an intercalator comprising a binding part capable of binding to a double-stranded DNA and at least one chromogenic part consisting of an organic EL dye and linked via a coupling part to the binding part. By virtue of the use of the organic EL dye in the chromogenic part, there can be accomplished a high-sensitivity detection even in the dry state.

Description

Specification

Inter force generator and gene detection method using the same

Technical field

[0001] The present invention relates to an intercalator used for detection of double-stranded DNA and a gene detection method using the intercalator.

Background art

[0002] In recent years, the entire genome of the human genome has been clarified, and post-genome research for gene therapy, gene diagnosis, and the like has been actively conducted. For example, DNA analysis can be used for early detection of intractable cancer. As a DNA analysis method, a hybridization method such as a Southern nose or an hybridization method is used. For example, a probe DNA immobilized on a DNA microarray substrate is hybridized with sample DNA labeled with a fluorescent dye to form a double strand, and the sample DNA is detected! /.

[0003] However, in the above hybridization method, DNA extracted from a specimen is fragmented with a restriction enzyme, fractionated by size by electrophoresis or the like, and then one sample DNA is obtained. A complicated operation of transforming into a chain is required. On the other hand, an analysis method has been proposed in which single-stranded DNA is not labeled in advance, but double-stranded DNA is directly labeled by an intercalator (for example, Patent Document 1). According to this method, there is no need to label the sample DNA in advance, and the analysis time can be shortened without a decrease in the formation speed when forming a double strand, and the occurrence of mutation is further prevented. be able to.

[0004] As the intercalator, a substance capable of forming a detectable signal itself, or a substance in which a labeling substance is bound to its side chain is used. As the labeling substance, fluorescent dyes typified by Fluorescein, Rhodamin, Cy5, Cy3, Texas Red, and ruthenium complexes are used, and fluorescence emission can be confirmed in an aqueous solution or in a wet state. However, when the sample on the microarray substrate is dried, the fluorescence of the fluorescent dye bound to the intercalator is quenched. In particular, in the case of a small amount sample such as ml to / zl, the sample is easy to dry, which is a very big problem. Also, depending on the fluorescent dye, if it takes time to measure at low temperature and light stability, problems may arise in quantitativeness. Conceivable.

Patent Document 1: JP 2002-125700 A

Disclosure of the invention

Problems to be solved by the invention

[0005] The present invention solves the above problems and suppresses the fluorescence quenching of the fluorescent dye in a dry state, thereby simplifying the operation and capable of performing highly sensitive detection, and an intercalator therefor. The object was to provide the gene detection method used.

Means for solving the problem

[0006] In order to solve the above-mentioned problems, the present inventors have intensively studied and found that an intercalator using an organic EL dye as a labeling substance emits light with high brightness without quenching even in a dry state. The present invention has been completed. That is, the intercalator of the present invention is an intercalator used for detection of double-stranded DNA, which is bonded to the binding portion through the binding portion that binds to the double-stranded DNA and the organic EL dye. It has at least one coloring part.

Conventional intercalators do not bind to double-stranded DNA, and in some cases, the binding and coloring parts cause intramolecular stacking and quench, and when bound to double-stranded DNA, intramolecular stacking is eliminated and fluorescence is emitted. . However, when the sample is dry, the fluorescence is quenched. This is thought to be due to the intermolecular stacking of the colored part as it dries. On the other hand, since the interferometer of the present invention uses an organic EL dye in the color developing portion, it does not quench even when the sample is dried, and therefore, high sensitivity detection is possible even in a dry state. Some fluorescent dyes have low temperature stability and need to be stored frozen, but organic EL dyes are stable to heat and can withstand long-term storage at room temperature, making them easy to handle.

[0007] The organic EL dye includes a compound containing a conjugated 5-membered ring compound, and the 5-membered ring compound contains one or more heteroatoms, selenium atoms, or boron atoms. Can be used. Alternatively, a condensed polycyclic compound having a force and a 6-membered ring compound having a conjugated system with the 5-membered ring compound can also be used. Furthermore, an azole derivative or an imidazole derivative can be used for the 5-membered ring compound.

In addition, a monocyclic or polycyclic aromatic group can be used for the bonding portion. For example, an anthro Sen group, phenanthrene group, pyrene group, fluorene group, biphenylene group, naphthalenediimide group, naphthalene imide group, atalidine group, phenol diimide group, benzothiazole group, benzoimidazole group, quinoline group, phenanthate One displacement force selected from the group consisting of lysine groups and indole groups can be used.

[0009] In addition, a sewn or embedded intercalator having a coloring portion coupled to both sides of the coupling portion via a coupling portion can be used as the intercalator.

[0010] Further, the gene detection method of the present invention binds to double-stranded DNA in the gene detection method in which an intercalator is inserted into double-stranded DNA and the double-stranded DNA labeled with the intercalator is detected. An intercalator having a coupling part, a coupling part, and a coloring part made of an organic EL dye and coupled to the coupling part via the coupling part is used.

The invention's effect

[0011] According to the present invention, the following effects can be obtained by using an organic EL dye in the coloring portion of the intercalator.

That is, the organic EL dye has a high quantum yield in the solid state (including solid and semi-solid) and high fluorescence intensity. Therefore, since the sample can emit fluorescence even in a dry state, detection of double-stranded DNA can be performed easily. In addition, no photo-retardancy, which is more thermally stable than Cy3, Cy5, and A1 exa dyes, is observed. Furthermore, since organic EL dyes are less expensive than Cy3 and Cy5, biopolymers can be detected at a lower cost. In addition, the degree of freedom in selecting the fluorescence wavelength is increased, and many fluorescence wavelengths such as orange, yellow, green, and blue can be used. This makes it possible to use two or more fluorescent dyes that have a large stochastic shift (the difference between the excitation wavelength and the fluorescence wavelength is large), so that multiple target nucleic acids contained in one sample can be detected simultaneously It is also possible. Cy3 and Cy5 must be stored frozen, whereas organic EL dyes are chemically stable and can withstand long-term storage at room temperature, making them easy to handle.

Brief Description of Drawings

FIG. 1 is an HPLC spectrum of a labeled peptide before purification (a) and after purification (b).

FIG. 2 is an example of a TOF MS spectrum of a labeled peptide.

FIG. 3 shows an example of the light emission pattern of labeled double-stranded DNA in Example 1 of the present invention. (A) is a sample with ct DNA added to an anthracene intercalator 10b, (b) and (c) are samples with ct DNA added to a fluorescein intercalator, and (d) and (e) shows the result when only 10b is spotted.

FIG. 4 is an example of the light emission pattern of labeled double-stranded DNA in Example 1 of the present invention, and shows the result of a sample in which ct DNA is added to an anthracene intercalator 10a.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

The intercalator of the present invention has a force composed of a binding part that binds to double-stranded DNA and at least one color-forming part that also forms an organic EL dye force and binds to the binding part via the linking part. Those represented by (3) are included.

(2) (D

1 -D -I-L

1 2

(3) (D—L) One I One (L One D)

1 1 2 2

Here, D and D are coloring portions, L is a connecting portion, and I is a connecting portion. (3)

1 2 1 2

This represents a so-called stitched intercalator in which the coloring part is connected to both sides of the connecting part via a connecting part. In general, the binding reaction between the intercalator and the double-stranded DNA is in an equilibrium relationship defined by the insertion and removal of the intercalator. Therefore, if the intercalator is easily detached, the fluorescence intensity decreases. However, a sewn intercalator is expensive because one colored portion passes through between the base pairs of double-stranded DNA and the other colored portion acts as a stopper that suppresses detachment from double-stranded DNA. Fluorescence intensity can be expected. In addition, since it has two coloring portions in the molecule, it has the advantage that it can be expected to have twice the fluorescence intensity as compared with the case of one coloring portion.

[0014] The binding portion (inter force rate group) binds to double-stranded DNA by intervening between the base pair of double-stranded DNA, and may contain a heteroatom. Monocyclic or polycyclic aromatic groups can be used. A polycyclic aromatic group is preferable, and a condensed aromatic group having a large planarity is more preferable. Specific examples include anthracene group, phenanthrene group, pyrene group, fluorene group, biphenylene group, naphthalene diimide group, naphthene group. Talenimide group, atalidine group, phenyldiimide group, benzothiazole group, benzimidazole group, quinoline group, phenanthridine group, and indole group group power selected Any one kind, more preferably anthracene group, atridine group or naphthalene diimide group Can be used. The naphthalenediimide group includes 1,8,4,5-naphthalenediimide and 2,3,6,7-naphthalenediimide.

[0015] In addition, a peptide compound composed of one kind of amino acid force in which the group force including lysine, arginine, histidine, and orthotin force is also selected can be used for the binding part. Furthermore, acrylidine can be introduced into the peptide compound.

[0016] It should be noted that a conventional fluorescent reagent such as the following fluorescent dye other than an intercalator can be used in the binding portion of the present invention. Since these conventional fluorescent dyes are quenched when the specimen is in a dry state, an intercalator including the conventional fluorescent dye as a binding unit can observe only light emitted from the color developing group. Conventional fluorescent dyes include, for example, 9-chlorom ethylacridine, 9-amino-ri-chloro-2-methoxyacndine (A and ivlA), 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI), propidium iodide ^ TOTO —l—iodide (514/533), TO—PRO — 1— iodide (515/531), acridine homodimer, acridine orange ^ Hoechst 33258, and the like.

[0017] [Chemical 1]

9-ammo-o-chloro-2-racthoxy acridine

[0018] [Chemical 2]

4 ' ₅ 6-diamidiiio-2-phenylindoIe, dihydrochlonde

propidium iodide

[0020] [Chemical 4]

TOTO® »1 iodide (514/533)

[0021] [Chemical 5]

TO- PRO®- 1 iodide (515/531)

[0022] [Chemical 6]

acrtdine homodimer

[0023] [Chemical 7]

acridine orange

[0024] [Chemical 8]

Hoechst 33258

As the linking group, a compound represented by the general formula A 1 -R 2 -A (2) can be used. here,

1 1 2

A is the first linking group that binds to the inter force group, and A is the second linking group that binds to the coloring group.

1 2

The linking group, and R is a spacer group that connects the first linking group and the second linking group.

As a specific example, it is preferable to use an alkylene group or an alkylene group containing a hetero atom in the main chain as the spacer group. As the alkylene group, a methylene group, an ethylene group or a trimethylene group is preferably used. Further, as the alkylene group containing a hetero atom in the main chain, it is preferable to use an ethylene oxide group and the number of repetitions is 1 to 5. In addition, a hetero atom, preferably an oxygen atom or a nitrogen atom can be used for the first bonding group. The second linking group is a substituted or unsubstituted alkyl group, an ether group, a thioether group, a substituted or unsubstituted imino group, an amide group, and an ester group. Is preferred to use!

[0027] When a conventional fluorescent dye is used for the bonding part of the intercalator of the present invention, the intercalator can be synthesized by condensing the conventional fluorescent dye and the active ester of the chromogenic group. As shown in [9], when 9-chloromethylacridine is used as a conventional fluorescent dye, an amino alcohol is induced to alcoholate and then reacted with 9-chloromethylacridine to synthesize its amino group-containing derivative. Then, for example, by reacting with oxadiazole active ester, an intercalator having 9-chloromethylacridine as a bonding portion is obtained. As shown in [Chemical Formula 10], by reacting an ACMA derivative with an oxadiazole active ester, an intercalator having the ACMA derivative as a binding moiety can be obtained.

[0028] [Chemical 9]

Scheme 1.

[0029] [Chemical 10]

Scheme 2.

[0030] Here, the role of the linking group will be described.

The linking group ensures the connection between the coloring group and the inter force group by the first linking group and the second linking group. Furthermore, the presence of the spacer group ensures the physical distance between the chromogenic group and the inter force-rate group and ensures the freedom of selection of the molecular skeleton of the chromophore group and the inter-force group. Inhibition of stacking between the chromophore and the inter force group prevents changes in the emission wavelength of the chromophore group or a decrease in emission intensity. In addition, if a heteroatom such as a nitrogen atom is used for the first bonding group, the entire molecule can be made to have a more rigid structure, so that stacking can be further suppressed. Also, by introducing oxygen atoms, etc., a flexible molecular structure can be achieved, and the stacking strength can be controlled.

[0031] In the present invention, the connecting portion can also serve as the connecting portion, and examples thereof include peptide compounds. Here, the amino acid constituting the peptide compound is lysine, arginine, histidine, ortin, more preferably lysine, and can bind to double-stranded DNA more strongly. The peptide compound preferably further contains atalidine. This is because it can bind to double-stranded DNA more strongly. Hereinafter, an intercalator containing a peptide compound is referred to as a peptide intercalator.

[0032] The organic EL dye used in the present invention is sandwiched between a pair of an anode and a cathode in a solid state, and the energy generated when the holes injected from the anode and the electrons injected from the cathode are recombined. It is not particularly limited as long as it can emit light. For example, polycyclic aromatic compounds such as tetraphenylbutaene perylene, cyclopentagen derivatives, distyrylvirazine derivatives, attaridone derivatives, quinacdrine derivatives, stilbene derivatives, phenothiazine derivatives. A conductor, a birazinopyridine derivative, an azole derivative, an imidazole derivative, a force rubazole derivative, a tetrafluorothiophene derivative, or the like can be used.

[0033] A preferable organic EL dye used in the detection method of the present invention is a compound containing a 5-membered ring compound having a conjugated system, and the 5-membered ring compound is one or more heteroatoms, selenium atoms, or boron. The thing containing an atom can be mentioned. More specifically, a monocyclic compound composed of a 5-membered ring compound having a conjugated system and a condensed polycyclic compound composed of a 6-membered ring compound having a conjugated system with the 5-membered ring compound can be exemplified. Even in the solid state, the quantum yield is a force that exhibits strong fluorescence.

[0034] Specific examples of the condensed polycyclic compound will be described below.

(Diazole derivative 1)

[Chemical 11]

Here, in the formula, R, R, R, and R R each independently represent a hydrogen atom, a halogen atom,

1 2 3 4, 7

Alkyl group, alkyl group, alkyl group, alkoxy group, hydroxyl group, cyan group, An aromatic hydrocarbon group, a hydrocarbon group or a heterocyclic group which may have a substituent such as a sulfonyl group, an aromatic hydrocarbon group or a heterocyclic group, and R, R, R and RR are the same or different. One

1 2 3 4, 7

May be. The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a bur group, a allyl group, a crotyl group, a tiglyl group or a prenyl group. The above alkyl group is preferably an ethur group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenol group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group or an imidazole group. Or a thiophene group. The hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. R and R ′ are alkyl groups or alkyl groups which may contain an aromatic ring.

Five

It represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group, and the same alkyl group, alkenyl group and aromatic hydrocarbon group as those described above can be used. An- represents halide ions such as Cl-, Br ", and Γ, CFSO-, BF-, and PF-.

3 3 4 6

The same applies to the following general formulas of diazole derivatives, monoazole derivatives and triazole derivatives unless otherwise specified.

(Diazole derivative 2)

Here, in the formula, R and R are a hydrogen atom, a halogen atom, an alkyl group, and an alkyl group, respectively.

8 9

An aromatic hydrocarbon group or hydrocarbon which may have a substituent such as an alkyl group, an alkyl group, an alkoxy group, a hydroxyl group, a cyano group, a sulfol group, an aromatic hydrocarbon group or a heterocyclic group Represents a group or a heterocyclic group, and R and R may be the same or different. Above alkyl

8 9

The group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a bur group, a allyl group, a crotyl group, a tiglyl group, or a prenyl group. The alkyl group is preferably an ethur group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a full group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenol group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group, or a quinoline group, and more preferably. Or furan group, imidazole group or thiophene group. The hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. N is an integer of 1 or more, preferably 1-5.

[0038] (Diazole derivative 3)

[Chemical 13]

Here, X represents a nitrogen atom, sulfur atom, oxygen atom, selenium atom or boron atom which may have a substituent.

[0039] (Diazole derivative 4)

Here, N → 0 indicates a state in which the nitrogen atom is coordinated to the oxygen atom. (Diazole derivative 6)

[Chemical 16]

(Diazole derivative 7) [Chemical 17]

(Diazole derivative 8) [Chemical 18-1]

Here, R 1 and R 2 are each independently a hydrogen atom, a halogen atom, an alkyl group,

10 11

An aromatic hydrocarbon group or carbonization which may have a substituent such as a alkenyl group, an alkyl group, an alkoxy group, a hydroxyl group, a cyano group, a sulfol group, an aromatic hydrocarbon group or a heterocyclic group R represents a hydrogen group or a heterocyclic group, and R 1 and R 2 may be the same or different. the above

10 11

The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a vinyl group, a aryl group, a crotyl group, a tigril group. Group or prenyl group. The alkyl group is preferably an ethur group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic ring or a polycyclic ring, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenol group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group, an imidazole group or Thiophene group. The hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.

[Y 匕 18—2]

[0046]

An integer of 3, preferably 1. The same applies to the following general formulas of diazole derivatives unless otherwise specified.

[0047] (Diazole derivative 9) Wu¾004l92l

[Chemical 20]

(Monoazole derivative 2)

(Triazole derivative 1)

(Triazole derivative 2)

As the 5-membered ring compound, the following derivatives containing a thiophene group can also be used. (Thiophene derivative 1)

Here, in the formula, R, R, R, R R R each independently represents a hydrogen atom, a halogen atom,

1 2 3 4, 6, 7

Aromatic hydrocarbon group which may have a substituent such as alkyl group, alkyl group, alkyl group, alkoxy group, hydroxyl group, cyano group, sulfonyl group, aromatic hydrocarbon group and heterocyclic group Or a hydrocarbon group or a heterocyclic group, and R, R, R, and RRR may be the same or different.

1 2 3 4, 6, 7

It may be followed. The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a bur group, a allyl group, a crotyl group, a tiglyl group or a prenyl group. The alkynyl group is preferably an ethur group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentyloxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic ring or a polycyclic ring, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenyl group. In addition, the heterocyclic group is preferably a pyrrole group, a furan group, or a thiof group. It is preferably a quinone group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group, an imidazole group or a thiophene group. The hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.

R ′ represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group such as an alkyl group or an alkenyl group which may contain an aromatic ring, and the alkyl group and the alkenyl group are similar to the above. Things can be used. An- indicates halide ions such as Cl-, Br-, and Γ, CFSO-, BF-, and PF-.

3 3 4 6

The same applies to the following general formulas of thiophene derivatives unless otherwise specified (thiophene derivative 2).

[Chemical 25]

[0056] Here, in the formula, R and R each independently represent a hydrogen atom, a halogen atom, an alkyl group,

8 9

Aromatic hydrocarbon group or carbonization which may have a substituent such as alkenyl group, alkyl group, alkoxy group, hydroxyl group, cyano group, sulfol group, aromatic hydrocarbon group or heterocyclic group R represents a hydrogen group or a heterocyclic group, and R and R may be the same or different. the above

8 9

The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a vinyl group, an aryl group, a crotyl group, a tiglyl group, or a prenyl group. The alkyl group is preferably an ethur group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic ring or a polycyclic ring, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenol group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group, an imidazole group or Thiophene group. The hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.

The same applies to the following general formulas of thiophene derivatives unless otherwise specified.

[0057] (thiophene derivative 3)

In the case of a thiophene derivative, it is a non-condensed compound, and a 2,3,4,5-tetraphenylthiophene derivative represented by the following general formula can also be used.

[0058] [Chemical 26]

[0059] Here, R 1, R 2 and R 3 are each independently a hydrogen atom, a linear, branched or cyclic

13 14 15

Represents an alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted aralkyl group, Ar and Ar represent a substituted or unsubstituted aryl group, and Ar and

1 2 1 2 may form a nitrogen-containing heterocycle together with the nitrogen atom to which it is bonded. Y and Y are

1 2 Hydrogen atom, halogen atom, linear, branched or cyclic alkyl group, linear, branched or cyclic alkoxy group, substituted or unsubstituted aryl group, substituted or unsubstituted aralkyl group, substituted or unsubstituted Represents a substituted amino group.

[0060] (thiophene derivative 4)

It is also possible to use a 2,3,4,5-tetraphenylthiophene derivative represented by the following general formula.

[0061] [Chemical 27]

[0062] Here, in the formula, Ar to Ar each independently represents a substituted or unsubstituted aryl group,

1 6

In addition, Ar and Ar, and Ar and Ar and Ar together with the nitrogen atom bonded to the nitrogen-containing complex

1 2 3 4 5 6

A ring may be formed.

[0063] Alternatively, imidazole may be used as the five-membered ring compound and an imidazole derivative represented by the following general formula may be used. Here, the imidazole group constituting the imidazole derivative preferably has a quaternary amino group. It is because water solubility can be improved. Further, when it contains a pyridino group, the pyridino group may also have a quaternary amino group in order to improve water solubility.

[Imidazole derivative 1] [Chemical 28]

[0065] (Imidazole derivative 1)

[Chemical 29]

[0066] Here, in the formula, R, R, R, and R R each independently represent a hydrogen atom, a halogen atom,

1 2 3 4, 5

Aromatic hydrocarbons that may have substituents such as alkyl, alkyl, alkyl, alkoxy, hydroxyl, cyano, sulfonyl, aromatic hydrocarbon and heterocyclic groups Group, hydrocarbon group or heterocyclic group, R, R, R, RR are the same or different. May be. The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a bur group, a allyl group, a crotyl group, a tiglyl group or a prenyl group. The above alkyl group is preferably an ethur group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a phenol group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group or an imidazole group. Or a thiophene group. The hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. R ′ and R ′ each represents an aliphatic hydrocarbon group or an aromatic hydrocarbon group such as an alkyl group or a alkenyl group which may contain an aromatic ring, and the alkyl group, the alkenyl group and the aromatic hydrocarbon group include A thing similar to the above can be used. An- represents halide ions such as Cl-, Br ", and Γ, CFSO-, BF-, and PF-.

3 3 4 6

The same applies to the following general formulas of imidazole derivatives unless otherwise specified.

(Imidazole derivative 2)

(Imidazole derivative 3) [Chemical 31— 1]

Here, in the formula, R, R, R, and R R R each independently represent a hydrogen atom or a halogen atom.

6 7 8 9, 10, 11

, An aromatic hydrocarbon group which may have a substituent such as an alkyl group, an alkyl group, an alkyl group, an alkoxy group, a hydroxyl group, a cyano group, a sulfonyl group, an aromatic hydrocarbon group or a heterocyclic group Or a hydrocarbon group or a heterocyclic group, and R, R, R and RRR are the same

6 7 8 9, 10, 11 May be different. The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. In addition, the above alkenyl group is preferably a bur group or an aryl group. Group, crotyl group, tiglyl group or prenyl group. The alkyl group is preferably an ethur group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, more preferably a phenyl group. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group or an imidazole group. Or a thiophene group. The hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.

In addition, the imidazole skeleton can be placed at any position on the central benzene ring R, R, R, R

8 9 10 11

The knit may be bonded. In addition, R is an olefin group or a substituent which may have a substituent.

12

A raffin group, and n is an integer of 1 to 3, preferably 1.

[0071] (Power rubazole derivative)

Moreover, the force rubazole derivative shown with the following general formula can also be used.

[0072] [32]

[0073] In addition, a 5-membered ring compound having a conjugated system and a monocyclic compound containing one or more hetero atoms, selenium atoms, or boron atoms can also be used. Although not particularly limited, for example, an imidazole derivative represented by the following general formula can be used.

[0074] [Y 匕 33]

[0075] Here, in the formula, R, R, and R each independently represent a hydrogen atom, a halogen atom, or an alkyl.

1 4 5

Group, alkyl group, alkyl group, alkoxy group, hydroxyl group, cyano group, sulfol group, aromatic hydrocarbon group, heterocyclic group which may have a substituent such as a heterocyclic group Or a hydrocarbon group or a heterocyclic group, and R, R and R may be the same or different.

1 4 5

The alkyl group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms. The alkenyl group is preferably a vinyl group, an aryl group, a crotyl group, a tiglyl group, or a prenyl group. The above alkyl group is preferably an ethur group or a propargyl group. The alkoxy group is preferably a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a pentoxy group or a phenoxy group. The aromatic hydrocarbon group includes a monocyclic or polycyclic ring, preferably a biphenyl group, a phenyl group, a tolyl group, a xylyl group, or a naphthyl group, and more preferably a biphenyl group or a phenyl group. It is. The heterocyclic group is preferably a pyrrole group, a furan group, a thiophene group, an imidazole group, an oxazole group, a thiazole group, a pyrazole group, a pyridine group or a quinoline group, more preferably a furan group or an imidazole group. Or a thiophene group. The hydrocarbon group is preferably a linear or branched alkyl group having 1 to 6 carbon atoms.

[0076] The organic EL dye used in the intercalator of the present invention is not particularly limited as long as it is a condensed polycyclic compound and a monocyclic compound as described above, but a diazole derivative represented by the following general formula: Alternatively, an imidazole derivative can be preferably used.

[0077] [Yi 34]

(8)

[0079] More preferably, it is a diazoguchi pyridine derivative or an imidazolopyridine derivative. Here, among diazoguchi pyridine derivatives, oxaziazo pyridine derivatives represented by the following general formula can be preferably used.

[0080] [Chemical 36]

[0081] After synthesizing the carboxylic acid derivative, the oxadiazo-mouth pyridine derivative is prepared by using, for example, Ν, に '-dicyclohexylcarbodiimide (DCC) as a condensing agent by the reaction shown in the following scheme 3. Those derived into an active ester containing a succinimide ester can be used.

[0082] [Chemical 37]

Scheme 3.

[0083] Here, preferred examples of the intercalator include an anthracene group, a naphthalene diimide group, and an atalidine group for the bonding part, and an azole derivative or imidazole derivative, more preferably an azole derivative, for the coloring part. More preferred is an intercalator using an oxaziazo oral pyridine derivative or a benzothiadiazole derivative represented by [Chemical Formula 15].

[0084] Further, in order to improve the water solubility of the intercalator, a charged group such as a sulfol group or a quaternary amino group can be introduced into the color developing portion. The charged group can be introduced, for example, by the following method.

An intercalator having no charge can be synthesized by treating a compound having a primary, secondary or tertiary amino group at the connecting part of an anthracene group or naphthalene diimide group under basic conditions. On the other hand, in order to introduce a charged group, a charged intercalator can be synthesized by forming a quaternary amino group by treatment under acidic conditions. Alternatively, a primary or secondary amino group is introduced at the end of the connecting part of the anthracene group or naphthalenediimide group, and two amino groups are present in the molecule. An intercalator with one amino group at one end is synthesized by reacting an amino group at one of the most active sites with an active ester. Intercalators without charge can be synthesized by treatment under basic conditions. Alternatively, a quaternary amino group can be formed by treatment under acidic conditions, and a charged intercalator can be synthesized.

[0085] The detection method of the present invention can be applied to any detection method as long as it is a detection method for measuring fluorescence of a labeled solid or semi-solid state biomolecule. For example, when used for gene analysis using a DNA microarray, the following procedure can be used. For probe nucleic acid to be immobilized on the substrate, cDNA, etc. should be used when examining gene expression. A library prepared by amplification by PCR using a whole library, a genomic library or a whole genome as a template can be used. In addition, when examining gene mutations and the like, those obtained by synthesizing various oligonucleotides corresponding to mutations and the like based on known standard sequences can be used.

[0086] For immobilization of the probe nucleic acid on the substrate, an appropriate method can be selected according to the type of nucleic acid and the type of substrate. For example, it is possible to use a method in which DNA is charged and electrostatically bonded to a substrate surface-treated with a cation such as polylysine.

[0087] First, target nucleic acids are spotted on a substrate, and hybridization is performed on the substrate. The hybridization is preferably performed at room temperature to 70 ° C. for 2 to 48 hours. The target nucleic acid having a base sequence complementary to the probe nucleic acid selectively binds to the probe nucleic acid to form a double strand by hybridization. Next, an intercalator prepared to an arbitrary concentration is poured onto the substrate. After that, the substrate is cleaned and the excess interferometer is removed and dried at room temperature.

[0088] Next, the fluorescence intensity of the surface of the dried substrate is measured by a fluorescence laser scanner method.

The level of gene expression can be monitored by fluorescence intensity.

Example

Hereinafter, the present invention will be described in more detail using examples.

Synthesis example 1.

A synthesis example of a naphthalene diimide intercalator is shown. Ethylene glycol diamine 1 was converted to Boc of one amino group in dioxane to obtain 2. Next, a condensation reaction was carried out between Boc-terminated 2 at one end and naphthalene dianhydride 3 to obtain the target 4 in a yield of 64%. Thereafter, the Boc group was removed from TFA to obtain naphthalenediimide derivative 5 in a yield of 82% (Scheme 4).

[0090] [Chemical 38] Di xiine

H

Boc Reagent

it lOh Y ₊ 60%

Y, 82 ^ft o

Scheme 4.

[0091] Next, a condensation reaction between naphthalenediimide derivative 5 and oxadiazole active ester 6 was performed to obtain monosubstituted intercalator 7a and disubstituted intercalator 7b (Scheme 5).

[0092] [Chemical 39]

7a Y. 40% 7b Y. 36 ^ft o Scheme 5.

Synthesis example 2

A synthesis example of an anthracene intercalator is shown. Anthracene derivative 9 induces the corresponding amino alcohol to alcoholate using NaH at low temperature, and 9,10-biscuc there. It was synthesized by introducing Loloanthracene (8). After NaCl produced by the reaction was filtered off, DMF was distilled off under reduced pressure, and the residue was dissolved in chloroform. Water was added thereto, and the target compound was extracted under TFA acidic conditions. Thereafter, water was concentrated, and the target compound was obtained by reprecipitation using an organic solvent. Next, intercalators 10a and 10b in which one end and both ends of the linkage were substituted by a condensation reaction of anthracene derivative 9 and oxadiazole active ester 6 were synthesized (Scheme 6).

[0094] [Chemical 40]

Scheme 6.

[0095] Synthesis Example 3.

For comparison, an intercalator having a conventional fluorescent dye fluorescein was also synthesized. Specifically, the anthracene derivative 9 synthesized in Synthesis Example 2 was synthesized by substituting one end of the anthracene derivative 9 with (fluorescein) with (fluorescein).

[0096] Synthesis Example 4.

The synthesis example of a peptide intercalator is shown.

1. Synthesis of Ac-Lvs (EL) -Lys-Lys-Lvs (Acr) -Lys-Lvs-Lys (Acr) -Lys-Lys-NH

2

(1) Ac-Lys (Mtt)-(Lys (Boc)) Lys (Acr) — (Lvs (Boc)) — Lys (Acr) — (Lys (Boc)) — Resin

2 2 2

(Experimental operation)

Add 0.15 g (0.61 mmol / g) of Fmoc-NH-SAL Resin to the reaction vessel, add 0.26 g of Fmoc-Lys (Acr) -OH to cartridges 3, 6, and cartridges 1,2,4,5, 7, Fmoc-Lys (Boc) —OH was added to 0.18 g in 8 and 0.23 g of Fmoc-Lys (Mtt) -OH was added to cartridge 9. After that, Applied Synthesis was performed using Biosystems (i: (7) 431 A peptide synthesizer. Methodi was also performed using the standar d Fmoc method, and the N-terminal was acetylated. A yellow solid peptide resin was obtained and the yield was 0.30. g.

[0097] (2) Ac-Lys (Mtt)-(Lys (Boc)) — Lys (Acr) — (Lvs (Boc)) — Lys (Acr) — (Lys (Boc)) — Resin M

2 2 2 tt group deprotection, EL modification and excision of resin, and side chain deprotection

(Experimental operation)

i) Deprotection of Mtt group

0.30 g of the peptide resin synthesized in 1 was placed in a screw tube, and excess dichloromethane (DCM) was added thereto and allowed to swell for 30 minutes, and then excess DCM was removed with nitrogen gas. Thereafter, 4 ml of a mixed solution of DCM: TFA: TIPS (triisopropylpropylsilane) = 94: 1: 5 was added and stirred for 2 minutes, and the solvent was removed with nitrogen gas. This operation was repeated 5 times, then suction filtered, washed with DCM, triethylamine, DCM, and dried under reduced pressure.

[0098] ii) Modification of methoxy type organic EL dye

6 ml of NMP was added to the peptide resin dried under reduced pressure and stirred for 30 minutes to swell, and 0.15 ml of triethylamine was added and stirred. Further, 0.2 g of oxadiazole active ester 6 was added and stirred at room temperature for 24 hours. Thereafter, the solution was filtered with suction, washed with NMP and DCM, and dried under reduced pressure.

[0099] iii) Resin cutting and side chain deprotection

Peptide resin dried under reduced pressure was charged with 0.08 ml of m-taresol, 0.48 ml of thio-anol, and 3.44 ml of TFA and stirred at room temperature for 1.5 hours. Thereafter, the mixture was suction filtered and washed with TFA. After TFA was distilled off under reduced pressure, 15 ml of ether was added in an ice bath. After sonication, the solution was left for a while and the supernatant was removed. Next, 15 ml of ethyl acetate was collected in an ice bath and left for a while after sonication. Thereafter, the solution was suction filtered, washed with ether, and dried under reduced pressure.

[0100] A yellow-orange solid was obtained, and the yield was 0.29 g. Figure 1 shows the HPLC vector before and after product purification. When a TOF-Mass measurement was performed on a sample with a peak near RT = 12.5 min, a peak corresponding to the molecular weight of the EL dye-peptide complex (EL-Peptide): 2055.30 was observed at 2057.33. The production of the target product was confirmed. (Matrix: a-CHCA; Fig. 2)

[0101] Example 1. (Detection method)

The synthesized anthracene intercalator 10b and fluorescein intercalator were dissolved in DMSO to prepare ImM solutions. Next, 995 μL of ultrapure water was weighed into a 5 mL sample tube, and 1 μL of anthracene intercalator 10b was added thereto. The same concentration of fluorescein intercalator was prepared in the same procedure. Next, 4 L of 32 mM ct DNA was added as double-stranded DNA and stirred thoroughly. The prepared sample solution was spotted by 1 nL (relative dye concentration of 1 pmol) on a glass substrate with a microarrayer. After drying, observation was performed with a fluorescent scanner. Here, BIO-RAD molecular imager FX Pro was used as the detection device. The laser wavelength is 488 nm and the scan interval is 50 nm.

[0102] (Result)

Figure 3 shows the observation results of labeled double-stranded DNA. Here, in Fig. 3, (a) is a sample with ct DNA added to an anthracene intercalator 10b, and (b) and (c) are ct DNA added to a fluorescein intercalator. Samples, and (d) and (e) are the observation results when only 10b is spotted. 10b bound to the double-stranded DNA emitted light even in the dry state and could be observed stably. In contrast, the fluorescein intercalator was quenched in the dry state and could not be observed.

[0103] Example 2.

The fluorescence of the synthesized anthracene intercalator 10a was measured in the same manner as in Example 1. Figure 4 shows the result of spotting the same sample multiple times. It was possible to observe stably even in a dry state.

[0104] Example 3.

Fluorescence was measured for the synthesized peptide intercalator by the same method as in Example 1. As a result, as in Examples 1 and 2, stable observation was possible even in a dry state.

[0105] Comparative example.

Fluorescence was measured by the same method as in Example 1 using a conventional fluorescent dye (4 ′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI)). However, when dry, it causes quenching, Observation was impossible.

Claims

The scope of the claims

[1] Intercalator used for detection of double-stranded DNA, comprising a binding portion that binds to double-stranded DNA, and at least one color that is bound to the binding portion via an organic EL dye component. And an intercalator having a portion.

[2] The organic EL dye according to claim 1, wherein the organic EL dye includes a 5-membered ring compound having a conjugated system, and the 5-membered ring compound includes one or more heteroatoms, selenium atoms, or boron atoms. Intercalator.

[3] The intercalator according to claim 1, wherein the organic EL dye is a condensed polycyclic compound comprising the 5-membered ring compound and a 6-membered ring compound having a conjugated system.

4. The intercalator according to claim 2 or 3, wherein the 5-membered ring compound is an azole derivative or an imidazole derivative.

[5] The intercalator according to any one of [1] to [4], wherein the bonding portion is a monocyclic or polycyclic aromatic group.

[6] The above-mentioned bonding part includes an anthracene group, a phenanthrene group, a pyrene group, a fluorene group, a biphenylene group, a naphthalene diimide group, a naphthalene imide group, an atalidine group, a phenol diimide group, a benzothiazole group, a benzimidazole group, a quinoline. The inter force modulator according to claim 1, wherein the group force is selected from a group force consisting of a group, a phenanthridine group and an indole group force.

[7] The interforcer according to [1], wherein the binding portion is a peptide compound composed of one kind of amino acid force in which a group force including lysine, arginine, histidine, and an orthotin force is also selected.

[8] The intercalator according to any one of claims 1 to 7, wherein the intercalator is a stitched intercalator having a coloring portion coupled to both sides of the coupling portion via a coupling portion.

[9] A double strand in which an intercalator is inserted into double-stranded DNA and labeled with the intercalator

In a gene detection method for detecting DNA, an intercalator having a binding portion that binds to double-stranded DNA, a linking portion, and a coloring portion that is composed of an organic EL dye and is bonded to the linking portion via the linking portion. The gene detection method used.