JP2013032291A - Fluorescent probe - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent probe capable of being easily synthesized, decreasing nonspecific binding to biological molecule such as cell and being labeled and imaged with good fluorescence intensity, and a method of manufacturing such fluorescent probe.SOLUTION: This fluorescent probe capable of decreasing nonspecific binding to biological molecule such as cell and being labeled and imaged with good fluorescence intensity is obtained by condensing an intermediate obtained by bonding an alkyne derivative of cyclooctane to a fluorescent nano particle such as hydrophilic quantum dot, with a substance which can be bonded to a fluorescently-labeled object introduced with an azide group.

Description

  The present invention relates to a fluorescent probe useful for labeling or imaging cells and the like. More specifically, the present invention relates to a fluorescent probe that can be easily synthesized, reduces non-specific binding to a biomolecule such as a cell, and can be labeled and imaged with good fluorescence intensity.

  Fluorescent probes combined with proteins such as antibodies and low molecular weight compounds are useful for imaging biomolecules in living bodies and cells. Conventionally, organic fluorescent substances such as fluorescein derivatives, rhodamine derivatives, hydrazide derivatives, and cyanine dyes have been used. Widely used.

  A fluorescent probe used for labeling or imaging needs to be able to specifically detect a target molecule, and is not affected or reduced by nonspecific binding to a biomolecule other than the target molecule. However, for example, when a fluorescent probe of a fluorescein derivative is used, there is often a nonspecific binding to a cell or the like. In addition, the fluorescence intensity may not be sufficient, and the development of a fluorescent probe is not yet sufficient.

  In recent years, it has been proposed to produce a fluorescent probe using a quantum dot as a fluorescent substance (Non-Patent Document 1: S. Nie et al, Engineering Luminescent Quantum Dots for In Vivo Molecular and Cellular Imaging. Annals of Biomedical Engineering, 2006, 34, 3-14.). In producing the fluorescent probe, a fluorescently labeled object, specifically, a physiologically active substance such as an antibody or a protein or a substance such as a low molecular weight compound was directly condensed with the quantum dots. As such a condensation reaction, methods such as a thiol exchange reaction, an amidation of an amino group or a carboxylic acid are often used. However, thiol groups, amino groups, and carboxylic acids such as proteins having physiological activity are functional groups that are also important for activity. Therefore, when performing condensation reactions between quantum dots and proteins, such important functional groups are used. Needed to be protected from reacting with the quantum dots. However, if the molecular design in the condensation reaction between quantum dots and physiologically active proteins is insufficient, important functional groups will bind to the quantum dots, and the resulting fluorescent probe will be There was a case where a problem that the function / property was not reflected occurred.

  The condensation of the alkyne derivative and the azide compound is performed by a 1,3-cycloaddition reaction, also known as a Huisgen reaction. The Huisgen reaction is a reaction in which a triple bond of an alkyne compound and an azide group are condensed under heating conditions. An 8-membered ring alkyne derivative is bonded to a gel or a solid phase carrier, and an azide-modified organic fluorescent dye or enzyme (β-Gal) is reacted therewith so that the dye or enzyme becomes an 8-membered ring alkyne derivative. It has been reported that it can bind and immobilize on the gel or solid phase carrier (Non-patent document 2: K. Ebisu et al., N-terminal Specific Point-Immobilization of Active Proteins by the One-Pot NEXT. -A Method. ChemBIoChem., 2009, 10, 2460-2464). However, the fluorescent substance and the fluorescently labeled object are not bonded via an 8-membered alkyne or an azide compound.

  Development of a fluorescent probe that can be synthesized easily, has no or reduced non-specific binding to biomolecules such as cells, and can be labeled and imaged with good fluorescence intensity is desired. It was.

Annals of Biomedical Engineering, 2006, 34, 3-14 ChemBioChem., 2009, 10,2460-2464

  An object of the present invention is to provide a fluorescent probe that can be easily synthesized, has reduced non-specific binding to a biomolecule such as a cell, and can be labeled and imaged with good fluorescence intensity. And It is another object of the present invention to provide a method for manufacturing such a fluorescent probe.

  In order to solve the above-mentioned problems, the present inventors paid attention to the condensation reaction between an alkyne derivative and an azide compound, and as a result of extensive research, as a result of the quantification of cyclooctane alkyne as an 8-membered ring alkyne derivative to fluorescent nanoparticles such as quantum dots. By condensing an intermediate obtained by binding a derivative (this conjugate is hereinafter referred to as “fluorescent probe synthesis intermediate”) and a substance capable of binding to a fluorescently labeled object into which an azide group has been introduced, The present invention has been completed by finding that the problems can be solved.

That is, this invention consists of the following.
1. A fluorescent probe represented by the following general formula (I).
[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
Ring A represents a derivative of cyclooctane, and the cyclooctane moiety may be further condensed with an aromatic ring;
R represents an alkyl group, an alkoxy group or a halogen atom, and n is an integer of 0 to 2;
Z represents a substance that can bind to the fluorescently labeled object;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]

2. 2. The fluorescent probe according to item 1, represented by the following general formula (III):
[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
Z represents a substance that can bind to the fluorescently labeled object;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]
3. 3. The fluorescent probe according to item 2, wherein Y is —NH—CO—O— in the fluorescent probe represented by the general formula (III).
4). A fluorescent probe synthesis intermediate represented by the following general formula (IV).
[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
Ring A ′ represents an alkyne derivative of cyclooctane, and the cyclooctane moiety may be further condensed with an aromatic ring;
R represents an alkyl group, an alkoxy group or a halogen atom, and n is an integer of 0 to 2;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]
5). 5. The fluorescent probe synthesis intermediate according to item 4, represented by the following general formula (V):
[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]
6). 6. The fluorescent probe synthesis intermediate according to item 5, wherein Y is —NH—CO—O— in the fluorescent probe synthesis intermediate represented by the general formula (V).
7). The method for producing a fluorescent probe synthesis intermediate according to any one of the preceding items 4 to 6, comprising the following steps:
1) A step of preparing fluorescent nanoparticles into which a carboxylic acid group, amino group, thiol group, maleimide group, carbonate group, or active ester group is introduced;
2) A functional group capable of reacting with various functional groups introduced into the fluorescent nanoparticles in the step 1) is introduced into the cyclooctane alkyne derivative, and the cyclooctane alkyne derivative into which the functional group is introduced is obtained. Preparing step;
3) A step of binding the fluorescent nanoparticles into which various functional groups prepared in the steps 1) and 2) are introduced and an alkyne derivative of cyclooctane.
8). The method for producing a fluorescent probe according to any one of items 1 to 3, including the following steps:
A) a step of introducing an azide group into a substance (Z) that can bind to a fluorescently labeled object;
B) A step of binding the alkyne derivative of cyclooctane in the fluorescent probe synthesis intermediate according to any one of 4 to 6 above and the azide group introduced in the step A).

  The fluorescent probe of the present invention uses fluorescent nanoparticles such as hydrophilic quantum dots and is very bright and excellent in light stability as compared with organic fluorescent dyes. In addition, since there are a plurality of reactive sites such as amino groups on the surface of the fluorescent nanoparticle, a large number of substances capable of binding to the fluorescent labeling target can be introduced on the surface of the fluorescent nanoparticle of the present invention. Therefore, the fluorescent probe of the present invention can be said to be a highly specific fluorescent probe that suppresses non-specific binding. The fluorescent probe of the present invention can be used for analysis by flow cytometry and microscopic analysis.

  Furthermore, according to the method for producing a fluorescent probe of the present invention, since the selectivity of the reaction is high and the reaction efficiency is good, a fluorescent probe library and a target fluorescent probe can be easily produced.

FIG. 3 is a diagram showing an example of a cyclooctane derivative condensed with a triazole ring in the formula (I). It is a figure which shows the example of the alkyne derivative of cyclooctane in Formula (III). FIG. 3 is a diagram showing examples of raw material compounds of an alkyne derivative of cyclooctane that can be used for the synthesis of a fluorescent probe synthesis intermediate of formula (III) and further a fluorescent probe of formula (I). It is a figure which shows the reaction aspect of the fluorescent nanoparticle of this invention, and the alkyne derivative of cyclooctane. It is a figure which shows the synthetic scheme of the fluorescent probe synthetic intermediate of this invention. Example 1 It is a figure which shows the synthetic scheme of the fluorescent probe (QD-Biotin) of this invention. (Example 2) It is a figure which shows the synthetic scheme of the fluorescent probe (QD-NP) of this invention. (Example 3) It is a figure which shows the binding effect with the streptavidin of the fluorescent probe (QD-Biotin) of this invention. (Experimental example 1) It is a figure which shows the analysis result by the flow cytometry with the cell surface antibody of the fluorescent probe (QD-NP) of this invention. (Experimental example 2) It is a figure which shows the cell surface antibody imaging effect of the fluorescent probe (QD-NP) of this invention. (Experimental example 3) The figure which shows the analysis result by the flow cytometry when the antibody is expressing on the cell surface (A20 / BCR) and when not expressing (A20 (-)) about the fluorescent probe (QD-NP) of the present invention It is. (Experimental example 4) It is a figure which shows the analysis result by a flow cytometry when the fluorescent probe (QD-NP) of this invention and its unlabeled antagonist or non-antagonist are added. (Experimental example 5) It is a figure which shows the analysis result by the flow cytometry when not using the antagonist with respect to the fluorescent probe (QD-NP) of this invention, or the probe (fluorescent NP) by an organic type fluorescent substance, respectively. (Experimental example 6 and comparative example)

  The present invention relates to a fluorescent probe useful for labeling or imaging cells and the like. More specifically, the present invention relates to a fluorescent probe that can be easily synthesized, reduces non-specific binding to a biomolecule such as a cell, and can be labeled and imaged with good fluorescence intensity. The fluorescent probe of the present invention has a structure represented by the following general formula (I), and can bind to fluorescent nanoparticles (X), a derivative of cyclooctane condensed with a triazole ring (ring A), and a fluorescently labeled object. Composed of substance (Z), X is linked to ring A via Y.

[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
Ring A represents a cyclooctane derivative, and the cyclooctane moiety may be further condensed with an aromatic ring such as a benzene ring;
R represents an alkyl group, an alkoxy group or a halogen atom, and n is an integer of 0 to 2;
Z represents a substance that can bind to the fluorescently labeled object;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]

  The present invention also extends to a fluorescent probe synthesis intermediate for producing the fluorescent probe. The fluorescent probe synthesis intermediate of the present invention has a structure represented by the following general formula (IV), and is composed of fluorescent nanoparticles (X) and an alkyne derivative of cyclooctane (ring A ′).

[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
Ring A ′ represents an alkyne derivative of cyclooctane, and the cyclooctane moiety may be further condensed with an aromatic ring such as a benzene ring;
R represents an alkyl group, an alkoxy group or a halogen atom, and n is an integer of 0 to 2;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]

  In Y represented by the above general formula (I) or (IV), when NH and CO are bonded via a linker, the linker is not particularly limited as long as it can bond NH and CO. Examples thereof include compounds having an amino group or a carboxylic acid group at both ends of an alkylene group or alkyleneoxy group having 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms. Specific examples include compounds represented by the following formula (VI) or formula (VII).

  Among Ys represented by the above general formula (I) or (IV), when W is an alkylene group, an alkyleneoxy group or an alkylenecarbonyl group, the alkylene group preferably has 1 to 3 carbon atoms, particularly preferably 1. When W is -Ph-B-, the alkylene group or alkyleneoxy group of B preferably has 1 to 3 carbon atoms, particularly preferably 1. In the present invention, it is particularly preferred that W is an oxygen atom.

  When R represented by the general formula (I) or (IV) is an alkyl group or an alkoxy group, the number of carbon atoms is preferably 1 to 3, particularly preferably 1. Moreover, when R is a halogen atom, it is preferably a fluorine atom.

  A preferred fluorescent probe is represented by the following formula (III), and a fluorescent probe synthesis intermediate is represented by formula (V). Further, in these formulas, Y is preferably —NH—CO—O—.

  The present invention also includes the use of the fluorescent probe synthesis intermediate (IV) for producing the fluorescent probe or fluorescent probe library represented by the formula (I). Here, the fluorescent probe library means a probe library in which Z in the fluorescent probe of the present invention represented by the formula (I), that is, a probe library in which a plurality of types of probes having different substances that can bind to the fluorescently labeled object are combined. .

  Hereinafter, the fluorescent nanoparticle (X), the cyclooctane derivative (ring A) and the fluorescent labeling object constituting the configuration of the fluorescent probe of the present invention will be described in detail, and the preparation of the fluorescent probe of the present invention will be further described. The method is also described in detail.

1. Fluorescent nanoparticles (X) The fluorescent nanoparticles in the fluorescent probe of the present invention usually have a particle size of 40 nm or less, preferably about 10 to 30 nm, and have a short wavelength such as ultraviolet rays or X-rays. Examples thereof include particles that emit fluorescence when irradiated with light. The fluorescent nanoparticles of the present invention are preferably water-dispersible. For example, the surface of the particles is preferably subjected to a hydrophilic treatment. For example, hydrophilic quantum that is a so-called fluorescent nanoparticle having a core-shell structured particle whose surface is covered with a semiconductor material as a core (core) and whose electrons are confined in all three dimensions. Dots are preferred.

  In the present invention, the hydrophilic quantum dot means, for example, the surface of a nanoparticle having a size of several nanometers of a core-shell structure having a core of a semiconductor such as cadmium selenide (CdSe) and coated with zinc sulfide (ZnS). The particle size obtained by hydrophilization is usually 40 nm or less, preferably about 10 to 30 nm. Hydrophilic quantum dots have extremely strong fluorescence compared to organic fluorescent dyes and fluorescent proteins, and emit different fluorescence depending on the particle size of the core-shell structure. For example, the particle size of the core-shell structure is blue at 2 nm, green at 3 nm, yellow at 4 nm, and red at 5 nm. In addition, such quantum dots have characteristics such as excellent light stability and difficulty in fading compared to organic fluorescent dyes, a very narrow fluorescence spectrum width, and excitation with various fluorescence wavelengths with one type of light source. Have. Therefore, it is a material that has been vigorously studied for bioimaging applications such as high-sensitivity detection and multicolor imaging.

  The optical properties of hydrophilic quantum dots vary greatly depending on the core semiconductor material. Examples of the semiconductor material include ZnS, ZnSe, CdS, CdSe, CdTe, PbS, InAs, and InP. For example, core-shell CdSe / ZnS (meaning CdSe particles coated with ZnS, the same applies hereinafter) with CdSe coated with ZnS as the core semiconductor has a high quantum yield and is suitable for flow cytometry. Since it shows fluorescence in the visible light region, it is suitable for the fluorescent probe of the present invention. InP / ZnS in which InP is coated with ZnS is also suitable.

  Core-shell structured particles are usually used with a particle size of 10 nm or less, preferably 2 to 8 nm, but the wavelength of the emitted fluorescence varies depending on the particle size of the core-shell structured particles used. For example, if the particle diameter of the core-shell structure is 5 to 8 nm, depending on it, it is possible to easily obtain quantum dots that emit fluorescence in the range of 500 to 800 nm. It is possible to synthesize a fluorescent probe showing For example, when considering application to flow cytometry, hydrophilic quantum dots that emit fluorescence at 625 nm or 655 nm with strong fluorescence intensity and little overlap of fluorescence wavelengths with FITC and PE, which are widely used in multicolor analysis, are suitable. It is. For application to biological imaging, hydrophilic quantum dots that are called biological windows and emit fluorescence in the near infrared region (650 to 1000 nm) with high biological permeability are suitable.

  Unlike conventional organic fluorescent dyes, hydrophilic quantum dots are characterized by emitting strong fluorescence as the wavelength becomes shorter. Therefore, when the fluorescent probe of the present invention is used in flow cytometry, it is preferable to excite with a UV laser (355 nm) or a violet laser (405 nm).

  Since the core-shell structured particles are highly hydrophobic and easily aggregate and precipitate in water, they are hydrophilized by various surface modification methods, and hydrophilic quantum dots subjected to hydrophilized surface treatment are used for the synthesis of fluorescent probes. Hydrophilic treatment is performed, for example, by coating the surface of the particle with a compound having a mercapto group and a hydrophilic group such as a carboxyl group and an amino group, specifically, mercaptoacetic acid, mercaptosuccinic acid, cysteine, dihydroriboic acid, and the like. A hydrophilic quantum dot can be synthesized by introducing a carboxylic acid by covalently coordinating a thiol group. In addition to this, there are a method of coating with an amphiphilic polymer or silica containing polyacrylate ester, polybutyl acrylate and polymethacrylic acid, a method of micellization using a surfactant such as phospholipid, and the like. is there.

Furthermore, it is preferable to modify the surface of the hydrophilic quantum dots with a polyether such as polyethylene glycol (PEG) because nonspecific adsorption can be suppressed. As such hydrophilic quantum dots, Qdot ^(R) nanocrystals, eFluor ^™ nanocrystals, and the like are commercially available from Invitrogen and Evident Technologies.

  In the fluorescent probe of the present invention, the core-shell structured particles are hydrophilized as described above, and appropriately non-specific adsorption suppression treatment is performed, usually 40 nm or less, preferably about 10 to 30 nm. Fluorescent nanoparticles having a particle size can be suitably used.

  In the present invention, a carboxylic acid group, an amino group, a thiol group, a maleimide group, a carbonate group, or an active ester group is formed on the surface of the fluorescent nanoparticle in order to bind the alkyne derivative of cyclooctane and the fluorescent nanoparticle. Is used, but those having an amino group or a carboxylic acid group introduced by the hydrophilization treatment can be used as they are.

2. Regarding Ring A and Ring A ′ Ring A in the fluorescent probe of the present invention of the formula (I) is a derivative of cyclooctane condensed with a triazole ring, for example, as shown in (a) to (f) of FIG. Such a thing is mentioned. In addition, ring A ′ in the intermediate of formula (IV) is an alkyne derivative of cyclooctane, and examples thereof include those shown in (a-1) to (f-1) of FIG.

  The part of “—Y-ring A ′” in the intermediate of formula (IV) is, for example, (a-1-1), (a-1-2), (b-1-1) in FIG. (C-1-1), (d-1-1), (d-1-2), (e-1-1), and (f-1-1) alkyne derivatives of cyclooctane Formed by using. Furthermore, by reacting the alkyne moiety of the “—Y-ring A ′” with a compound having an azide group, the “—Y-ring A-” moiety in the fluorescent probe of formula (I) is formed.

  Here, alkyne is a generic term for hydrocarbons having only one carbon-carbon triple bond in the molecule. The raw material compound of the cyclooctane alkyne derivative that can be used in the present invention is not particularly limited. Among them, the 3-hydroxy described in (f-1-1) of FIG. -1,2: 5,6-Dibenzocycloocta-1,5,7-triene (DIB) (X. Ning et al., Angew. Chem. Int. Ed., 2008, 47, 2253) can be suitably used. Further, by introducing two fluorine atoms adjacent to the alkyne part of cyclooctane and improving the reactivity, (c-1-1) in FIG. The difluoroinated cyclooctyne derivatives described in (d-1-1) and (d-1-2) (JA Codelli, et al., JACS 2008, 130, 11486.) can also be suitably used.

3. Substance (Z) that can bind to fluorescently labeled object The fluorescently labeled object labeled with the fluorescent probe of the present invention includes cells, tissues, proteins, peptides, genes, sugar chains, etc., and is particularly limited is not. The substance (Z) that can bind to the fluorescent labeling target substance may be any substance that can bind to the fluorescent labeling target substance, and may be a low-molecular compound, protein, carbohydrate, nucleic acid, or the like. It may be a polymer material and is not particularly limited. For example, when the fluorescently labeled object is a tissue or a cell, Z may be a substance that can interact with a substance expressed on the surface of the tissue or cell. When the fluorescent labeling target is a protein, Z may be an antibody, a peptide that can specifically recognize the protein, a receptor for the protein, a low-molecular compound that can bind to the protein, or the like. When the product is a cell surface receptor such as a 7-transmembrane receptor (GPCR), Z is a compound that binds to the receptor, such as diphenhydramine or mepyramine that binds to the histamine receptor. Agents and the like. When the fluorescently labeled object is streptavidin, biotin can be used as Z.

4). Method for Producing Fluorescent Probe Synthesis Intermediate In order to produce the fluorescent probe of the present invention, a conjugate of fluorescent nanoparticles and an alkyne derivative of cyclooctane is produced. This fluorescent probe synthesis intermediate of the present invention can be prepared by a method including at least the following steps 1) to 3). The present invention extends to the fluorescent probe synthesis intermediate and its production method.

1) A step of preparing fluorescent nanoparticles into which a carboxylic acid group, amino group, thiol group, maleimide group, carbonate group, or active ester group has been introduced;
2) A step of preparing an alkyne derivative of cyclooctane into which functional groups capable of reacting with various functional groups introduced into the fluorescent nanoparticles in the step 1) are introduced;
3) A step of bonding the fluorescent nanoparticles into which various functional groups have been introduced in the steps 1) and 2) and an alkyne derivative of cyclooctane by a condensation reaction or a reaction via a linker.

  In the above, the order of the steps 1) and 2) may be any first. For example, when the step 2) is performed first, these functional groups are introduced into the alkyne derivative of cyclooctane by introducing a carboxylic acid group, amino group, thiol group, maleimide group, carbonate ester group, or active ester group. And a step of preparing fluorescent nanoparticles in which functional groups capable of reacting with various functional groups introduced into the cyclooctane alkyne derivative are introduced. Can be produced.

  In order to bind the fluorescent nanoparticle to the alkyne derivative of cyclooctane, it is possible to bond the alkyne derivative of cyclooctane and the fluorescent nanoparticle by introducing a combination of functional groups capable of reacting with each other and reacting them. . As the functional group, a carboxylic acid group, an amino group, a thiol group, a maleimide group, a carbonate ester group, an active ester group, and the like are suitable, and these functional groups can be introduced in combination. Such combinations include carboxylic acid groups and amino groups, maleimide groups and thiol groups, amino groups and carbonate groups, amino groups and active ester groups. For example, the combinations shown in Table 1 below can be considered. FIG. 2 schematically shows these reaction examples.

  The method for introducing the functional group is not particularly limited, and a method known per se or any method developed in the future can be applied. For example, the carboxylic acid can be introduced by a method in which a thiol group such as mercaptoacetic acid or dihydroriboic acid is covalently coordinated to the surface of the core-shell structured particle. Moreover, an amino group can be introduce | transduced into the surface of the particle | grains of a core shell structure by coat | covering with polymers, such as polyethyleneimine. In addition, a maleimide group can be introduced by condensation with an amino group on the surface of the core-shell structured particle using a crosslinking reagent such as 4-maleimidobutyric acid succinimidyl ester. Of course, the fluorescent nanoparticles into which an amino group, a carboxylic acid group or the like has been introduced by the above-described hydrophilization treatment can be used as they are.

  If the fluorescent nanoparticles and the alkyne derivative of cyclooctane have a functional group as shown in Table 1 or FIG. 2, they can be directly used for binding. For example, in the case of an alkyne derivative of cyclooctane having a hydroxy group such as the aforementioned DIB, a carbonate ester group can be introduced by condensation reaction of the alkyne derivative of cyclooctane with N, N′-disuccinimidyl carbonate. it can. Furthermore, an amino group can be introduced into the carbonate group by reacting a linker having an amino group at both ends, for example, a linker having an amino group via an alkyleneoxy group such as an alkylene group or PEG. Further, when a linker having an amino group at one end and a carboxylic acid group or thiol group at the other is reacted, a carboxylic acid group or thiol group can be introduced (see FIG. 2).

  The reaction between the fluorescent nanoparticles and the cyclooctane alkyne derivative is carried out by reacting an excess amount of the cyclooctane alkyne derivative with respect to 1 mol of the fluorescent nanoparticle, usually 20 to 500 mol, preferably 50 to 300 mol. . For the sake of simplicity, the reaction formula, general formula, and reaction schematic diagram shown in FIG. 2 are used to explain the reaction at one reaction point (amino group, etc.) on the surface of the fluorescent nanoparticle. However, there are usually a plurality of reaction points on the surface of the fluorescent nanoparticle of the present invention. For example, commercially available fluorescent nanoparticle surfaces, such as hydrophilic quantum dots, can be obtained having about 80 to 100 amino groups on the surface. Therefore, the present invention includes the case where a plurality of the following general formulas (VIII) and (IX) are bonded to X in the general formula (I) and the general formula (IV).

5). Method for Producing Fluorescent Probe The fluorescent probe of the present invention can be prepared by binding the “fluorescent probe synthesis intermediate” of the present invention and “substance (Z) that can bind to a fluorescently labeled object”. The fluorescent probe synthesis intermediate of the present invention can be prepared by a method including at least the following steps A) and B). The present invention extends to a method for producing a fluorescent probe.

A) a step of introducing an azide group into a substance (Z) that can bind to a fluorescently labeled object;
B) A step of reacting the azide group introduced in A) with an alkyne derivative of cyclooctane in the fluorescent probe synthesis intermediate.

A) Method for introducing an azide group into a substance (Z) that can bind to a fluorescently labeled object The method for introducing an azide group into a substance (Z) that can bind to a fluorescently labeled object of the present invention is a method known per se Alternatively, any method developed in the future can be applied and is not particularly limited. For example, by utilizing the high nucleophilicity of azide ions, it can be introduced by SN2 reaction with respect to alkyl halides, sulfonate esters and the like. If a compound having an azide group and an amino group in the same molecule is used, the compound can be introduced by a condensation reaction with a carboxylic acid in a low molecular compound. If a compound having a functional group such as carboxylic acid or aldehyde in the same molecule in addition to the amino group is used, an azide group can be introduced by reaction with an amino group in the low molecular compound.

  When an antibody is used as the substance Z, since the antibody has a disulfide bond in the molecule, the SH group obtained by reducing this can be used for labeling. Since the SH group thus obtained is unrelated to antigen recognition, there is an advantage that the specificity of the antibody is not impaired even if the SH group is used. First, Fab fragments are prepared by treating antibody molecules with proteolytic enzymes pepsin and papain. The Fab fragment thus prepared has an SH group and can be used for the next reaction. When a Fab fragment is treated with a crosslinking reagent having an azide group and a maleimide group in the molecule, the reaction between the SH group and the maleimide group in the Fab fragment proceeds selectively, and an azide Fab fragment can be synthesized. . By reacting this with an alkyne derivative of cyclooctane, a labeled antibody can be synthesized.

  Hereinafter, a substance that can bind to a fluorescently labeled object into which an azide group has been introduced is referred to as an “azide group-containing substance” for convenience.

B) Condensation reaction between the fluorescent probe synthesis intermediate and the azide group-containing substance The condensation reaction between the fluorescent probe synthesis intermediate and the azide group-containing substance is carried out by synthesizing the azide group and fluorescent probe introduced into the fluorescent labeling object in A) above. It can be carried out by condensation reaction with an alkyne derivative of cyclooctane in the intermediate. For the reaction of the alkyne derivative of cyclooctane and the azide group, a method known per se, for example, the method described in JACS 2004, 126, 15046 or Non-Patent Document 2, or any method developed in the future can be applied. Since the reaction between the alkyne derivative of cyclooctane and the azide group is very efficient, the fluorescent probe synthesis intermediate of the present invention and the azide group-containing substance can be subjected to a condensation reaction only by reacting at room temperature for several hours. A fluorescent probe can be easily prepared without using a reagent such as a condensing agent.

6). Description of Cell Fluorescent Labeling and Imaging Method The fluorescent probe of the present invention can be used for fluorescent labeling and imaging of cells, and highly sensitive detection of target molecules such as biosensors. For example, it can be used for high-sensitivity detection using a fluorescence plate reader or flow cytometry, in vitro imaging using a fluorescence microscope or confocal microscope, and in vivo imaging.

  Hereinafter, in order to deepen the understanding of the present invention, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples, and various modifications can be made without departing from the technical idea of the present invention. Can be applied.

Example 1 Production of Fluorescent Probe Synthesis Intermediate In this example, production of a fluorescent probe synthesis intermediate will be described with reference to the synthesis scheme of FIG.

i) Synthesis of Compound 1-3 Compound 1-3 was synthesized according to known literature (X. Ning et al., Angew. Chem. Int. Ed. 2008, 47, 2253).

ii) Synthesis of Compound 4 To a solution of Compound 3 (22 mg, 0.10 mmol) in dimethylformamide (DMF) (1 ml), 4-dimethylaminopyridine (12 mg, 0.10 mmol) and N, N'-disuccinimidyl carbonate ( 77 mg, 0.30 mmol) was added. After stirring at room temperature for 12 hours, a 1N hydrochloric acid solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic solvent layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by silica gel chromatography to obtain Compound 4 in 26 mg (yield 73%).

¹ H-NMR (CDCl ₃ , 400 MHz) δ: 7.61 (1H, d, J = 8.0 Hz), 5.53 (1H, br), 3.32 (1H, dd, J = 2.0, 15.6 Hz), 3.03 (1H, dd, J = 4.0, 15.6 Hz), 2.81 (4H, s).

iii) Synthesis of Compound 5 (Fluorescent Probe Synthesis Intermediate (QD-DIB)) In this example, Qdot ^(R) 655 ITK AMINO (PEG) (amino group on the surface ⁾ was used as the fluorescent nanoparticle constituting the fluorescent probe synthesis intermediate. CdSe / ZnS: Invitrogen) was used. To a PBS solution (100 μl) of fluorescent nanoparticles (25 μl, 0.20 nmol), a 10 mM DMSO solution (5 μl, 50 nmol) of the above compound 4 was added. After standing for 16 hours at room temperature, the PBS of (400 [mu] l) was added to the reaction solution was transferred to a centrifuge for filtration devices (10K Nanosep® ^(R) filter (manufactured by Paul)). A washing operation by centrifugation at 4 ° C. and 7000 rpm for 10 minutes was performed 3 times to remove excess compound 4. A solution of the fluorescent probe synthesis intermediate (compound 5) was recovered from the device for centrifugal filtration, and an appropriate amount of PBS was added to prepare 50 μl.

Example 2 Production of Fluorescent Probe (QD-Biotin) In this example, referring to the synthesis scheme of FIG. 4, fluorescence obtained by binding a substance (biotin) that can bind to a fluorescent labeling target to a fluorescent probe synthesis intermediate. The production of the probe will be described.

  Biotin (244 mg, 1.0 mmol) and 14-azido-3,6,9,12-tetraoxatetradecan-1-amine (14-azido-3,6,9,12-tetraoxatetradecan-1-amine) To a mixed solution of (524 mg, 2.0 mmol) in MeOH-MeCN (1: 3) (12 ml), EDC (N-ethyl-N'-3- (dimethylaminopropyl) carbodiimide) (288 mg, 1.5 mmol) was added. . After stirring at room temperature for 42 hours, the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by silica gel chromatography to obtain Compound 6 (493 mg, yield 100%).

¹ H-NMR (CDCl ₃ , 400 MHz) δ: 6.63 (1H, brt, J = 4.8 Hz), 5.86 (1H, s), 4.98 (1H, s), 4.50-4.45 (1H, m), 4.32- 4.28 (1H, m), 3.70-3.56 (14H, m), 3.54 (2H, t, J = 4.8 Hz), 3.43 (2H, t, J = 4.8 Hz), 3.37 (2H, t, J = 4.8 Hz) ), 3.14-3.10 (1H, m), 2.90 (1H, dd, J = 4.8, 13.2 Hz), 2.71 (1H, d, J = 13.2 Hz), 2.21 (2H, dt, J = 2.8, 7.6 Hz) , 1.78-1.60 (4H, m), 1.43 (2H, quin, J = 7.6 Hz)

A 10 mM DMSO solution (5 μl, 50 nmol) of the compound 6 prepared above was added to a PBS solution (100 μl) of the compound 5 (10 μl, 0.040 nmol) prepared in Example 1. After leaving still at room temperature for 2 hours, PBS (400 microliters) was added to the reaction liquid, and it moved to the device for centrifugal filtration (10K Nanosep ^(R) filter (made by Paul)). Washing operation by centrifugation at 4 ° C. and 7000 rpm for 10 minutes was performed 3 times to remove excess compound 6. A solution of the fluorescent probe (compound 7) was recovered from the centrifugal filtration device, and an appropriate amount of PBS was added to prepare 50 μl.

Example 3 Production of Fluorescent Probe (QD-NP) In this example, referring to the synthesis scheme of FIG. 5, a substance (nitrophenylacetyl: NP) that can bind to a fluorescently labeled target substance with a fluorescent probe synthesis intermediate The production of a fluorescent probe bound with is described.

4-hydroxy-3-nitrophenylacetic acid (197 mg, 1.0 mmol) and 14-azido-3,6,9,12-tetraoxatetradecan-1-amine (14-azido -3,6,9,12-tetraoxatetradecan-1-amine) (524 mg, 2.0 mmol) in CH ₂ Cl ₂ (10 ml) and N, N-dimethyl-4-aminopyridine (N, N-dimethyl) -4-aminopyridine: DMAP) (183 mg, 1.5 mmol) and EDC (288 mg, 1.5 mmol) were added. After stirring at room temperature for 3 hours, a 1N hydrochloric acid solution was added to the reaction mixture, and the mixture was extracted with CH ₂ Cl ₂ . The organic solvent layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by silica gel chromatography to obtain Compound 8 in 512 mg (yield 100%).

¹ H-NMR (CDCl ₃ , 400 MHz) δ: 10.5 (1H, s), 8.01 (1H, d, J = 2.0 Hz), 7.55 (1H, dd, J = 2.0, 8.8 Hz), 7.11 (1H, d, J = 8.8 Hz), 6.42 (1H, br), 3.68-3.56 (14H, m), 3.53 (2H, t, J = 5.2 Hz), 3.50 (2H, s), 3.43 (2H, t, J = 5.2 Hz), 3.36 (2H, t, J = 5.2 Hz)

To a solution of Compound 5 (10 μl, 0.040 nmol) prepared in Example 1 in PBS (100 μl), 10 mM DMSO solution (5 μl, 50 nmol) of Compound 8 prepared above was added. After leaving still at room temperature for 2 hours, PBS (400 microliters) was added to the reaction liquid, and it moved to the device for centrifugal filtration (10K Nanosep ^(R) filter (made by Paul)). Washing operation by centrifugation for 10 minutes at 4 ° C. and 7000 rpm was performed three times to remove excess compound 8. A solution of the fluorescent probe (Compound 9) was collected from the centrifugal filtration device, and an appropriate amount of PBS was added to prepare 50 μl.

(Experimental example 1) Evaluation of fluorescent probe (QD-Biotin) (compound 7) by biotin-streptavidin interaction In this experimental example, the fluorescent probe was confirmed by confirming the binding of the fluorescent probe to the streptavidin-coated plate. The function of (QD-Biotin) was evaluated. A 96-well plate coated with streptavidin (Thermo) was washed with PBS, and then blocked with 2% BSA / PBS solution (100 μl). Thereafter, 2 μl of the solution containing the fluorescent probe (QD-Biotin) prepared in Example 2 was added and allowed to act at room temperature for 60 minutes. After washing twice with 0.1% Tween-20 / PBS, fluorescence measurement was performed with a multiplate reader (FlexStation ^™ (Molecular Devices)).

  As a result, in order to evaluate the labeling method utilizing a fluorescent probe (QD-Biotin), analysis was performed using an assay system utilizing biotin-streptavidin interaction. When the streptavidin-coated plate was treated with a fluorescent probe (QD-Biotin), it showed a high fluorescence intensity. On the other hand, even when the solution prepared in Example 1 was treated with a fluorescent probe synthesis intermediate (compound 5) diluted 5-fold, the fluorescence intensity was hardly shown (see FIG. 6). That is, the method for producing a fluorescent probe of the present invention succeeded in introducing a biotin molecule, which is a substance capable of binding to a fluorescently labeled object, on the surface of a fluorescent nanoparticle.

(Experiment 2) Evaluation of fluorescent probe (QD-NP) by flow cytometry 1
In this experimental example, a fluorescent probe (QD-NP) (compound 9) was evaluated using A20 cells (A20 / BCR) that highly express immunoglobulin IgG1 that specifically recognizes NP (nitrophenylacetyl) as an antigen. I did it. A20 cells (3 x 10 ⁵ cells) were suspended in a 2% FBS / PBS solution (200 μl), and a fluorescent probe (QD-NP) (2 μl) diluted 10-fold with the solution prepared in Example 3 was added. Allowed to act on ice for 30 minutes. As a control, a fluorescent probe synthesis intermediate (compound 5) obtained by similarly diluting the solution prepared in Example 1 50 times was allowed to act. After washing twice with PBS, the cells were filtered with a cell strainer (Becton Dickinson (BD), which is a flow cytometry tube, and measured with a flow cytometer (FACS Canto ^™ II (BD)). For detection of specific binding, a similar experiment was performed with the addition of unlabeled NP (100 μM), and specific binding was calculated by subtracting nonspecific binding from the total binding value.

The A20 cells (A20 / BCR) were prepared as follows according to a conventional method. That is, RNA was extracted from splenic B cells of mice immunized with NPized chicken γ globulin, and the variable region of the immunoglobulin locus (VH186.2) was cloned by PCR. Further, the full-length λ1 light chain and the constant region of the γ1 heavy chain constituting the antibody were similarly cloned by the PCR method. Thereafter, the cloned VH186.2 variable region was connected to the γ1 constant region by PCR to obtain a full-length γ1 heavy chain gene that recognizes the NP hapten. The genes of λ1 light chain and full-length γ1 heavy chain obtained in A20 which is a mouse B cell tumor cell constitutively expressing γ2b heavy chain were introduced by retrovirus method. Reconstitution of the antigen receptor that recognizes the NP hapten was confirmed by flow cytometry (Facs ARIA ^™ (manufactured by BD)) by staining with phycoerythrin bovine serum albumin bound to the NP hapten.

  As a result of FACS analysis by flow cytometry for the cells treated with the fluorescent probe (QD-NP) and the control, when treated with the fluorescent probe (QD-NP) compared to the fluorescent probe synthesis intermediate, the fluorescence intensity is the control Increased by about 35 times. From the above, it was confirmed that the fluorescent probe (QD-NP) produced by the method of the present invention is a fluorescent probe that can be used for cell labeling (see FIG. 7).

(Experimental example 3) Evaluation of fluorescent probe (QD-NP) by fluorescence microscope imaging A20 cells (1 x 10 ⁶ cells) highly expressing BCR used in Experimental example 2 in 2% FBS / PBS solution (800 μl) A fluorescent probe (QD-NP) solution (8 μl), which was suspended and diluted 10-fold from the solution prepared in Example 3, was added and allowed to act on ice for 30 minutes. After washing twice with PBS, 2% formaldehyde / PBS solution (200 μl) was added and fixed for 10 minutes. A 2% FBS-PBS solution (8 μl) was allowed to act at room temperature for 10 minutes and then washed twice with PBS. Next, the obtained A20 cells (2 x 10 ⁴ cells) are seeded on a slide glass (manufactured by Matsunami) using a cell collection centrifuge (Cytospin (manufactured by Thermo)), sealed under a cover glass with glycerol, and observed with a fluorescence microscope (Carl Zeiss).

  As a result of conducting a fluorescence microscope experiment on cells treated with a fluorescent probe (QD-NP) (compound 9) and a fluorescent probe synthetic intermediate (compound 5) diluted 50-fold with the solution prepared in Example 1 as a control, Compound 9 stained A20 / BCR cells, while compound 5 did not. That is, it was found that the molecular probe synthesized by this method has little background such as nonspecific adsorption and can be effectively used for cell imaging research (see FIG. 8).

(Experimental example 4) Evaluation of fluorescent probe (QD-NP) by flow cytometry 2
For A20 cells (A20 / BCR) highly expressing BCR and A20 cells not expressing BCR (A20 (-)) used in Experimental Example 2, 3 x 10 ⁵ cells were each 2% FBS / PBS solution ( A fluorescent probe (QD-NP) solution (2 μl) suspended in 200 μl) and diluted 10-fold from the solution prepared in Example 3 was added and allowed to act on ice for 30 minutes. After washing twice with PBS, the flow cytometry tube cell strainer (filtered by Becton Dickinson (BD), measured by flow cytometry (FACS Canto ^TM II (Becton Dickinson)) was analyzed. .

  As a result of imaging experiments on A20 / BCR and A20 (-) cells using a fluorescent probe (QD-NP), A20 / BCR cells expressing high affinity BCR using a fluorescent probe (QD-NP) It was confirmed that it was selectively labeled (see FIG. 9). That is, it was found that the molecular probe produced by the method of the present invention can be applied not only to fluorescence microscope imaging but also to FACS imaging.

(Experimental example 5) Evaluation of fluorescent probe (QD-NP) by flow cytometry 3
A20 cells (A20 / BCR) (3 × 10 ⁵ cells) highly expressing BCR used in Experimental Example 2 were suspended in 2% FBS / PBS solution (200 μl) and diluted to a concentration of 0.16-20 μM ( NP) or non-antagonist (2,4-dinitrophenol (DNP)) (2 μl). Subsequently, a fluorescent probe (QD-NP) solution (2 μl) diluted 10-fold was added and allowed to act on ice for 30 minutes. After washing twice with PBS, the flow cytometry tube cell strainer (filtered by Becton Dickinson (BD), measured by flow cytometry (FACS Canto ^TM II (Becton Dickinson)) and analyzed .

  The biggest concern when synthesizing molecular probes is that labeling impairs specificity for target molecules such as receptors. Therefore, in order to confirm the specificity of the molecular probe synthesized by this method, a competitive inhibition experiment in which an antagonist was added was performed. When unlabeled NP was added as an antagonist, the fluorescence intensity decreased depending on the concentration of the added unlabeled NP. On the other hand, even when DNP having no affinity (non-antagonistic) DNP was added to the NP high affinity BCR, no significant change was observed in the fluorescence intensity (see FIG. 10). That is, it was confirmed that the fluorescent probe (QD-NP) is a BCR-specific fluorescent probe. From the above, it can be said that the method for producing a fluorescent probe of the present invention is an excellent method that is simple and versatile.

(Experimental Example 6 and Comparative Example) Evaluation when Fluorescein Derivative Fluorescent Probe is Used In order to confirm the usefulness of the fluorescent probe of the present invention, a fluorescent probe (fluorescent NP) using an organic fluorescent dye and the present invention A comparative experiment using a fluorescent probe (QD-NP) was conducted. Using a fluorescein derivative, which is a typical organic fluorescent dye, NP was introduced through a linker to synthesize fluorescent NP. Next, according to the experimental method shown in Experimental Example 4, FACS analysis using A20 / BCR cells was performed. As a result, when treated with the fluorescent probe of the comparative example (fluorescent NP), the fluorescence intensity does not decrease with or without the addition of 100 μM antagonist (unlabeled NP), and very high non-specific binding is observed. It was. In contrast, in the case of QD-NP of the present invention, a clear difference in fluorescence intensity was observed depending on the presence or absence of an antagonist (see FIG. 11). That is, it was confirmed that the fluorescent probe (QD-NP) of the present invention is superior in that it can suppress non-specific adsorption, which is a major obstacle with conventional fluorescent probes using organic fluorescent dyes. .

  As described in detail above, the fluorescent probe of the present invention can be said to be an excellent fluorescent probe with extremely high specificity, in which non-specific binding is suppressed. The fluorescent probe of the present invention can be used for fluorescent labeling and imaging of cells, and highly sensitive detection of target molecules such as biosensors. For example, it can be used for high-sensitivity detection using a fluorescent plate reader or flow cytometry, in vitro imaging using a fluorescence microscope or a confocal microscope, and in vivo in vivo. Furthermore, according to the method for producing a fluorescent probe of the present invention, the fluorescent probe of the present invention can be easily and efficiently produced without using a reagent such as a condensing agent. A solid phase fluorescent probe such as a bead can also be easily produced. Moreover, since the selectivity of the reaction is high and the reaction efficiency is good, the target fluorescent probe can be easily purified.

Claims (8)

A fluorescent probe represented by the following general formula (I).
[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
Ring A represents a derivative of cyclooctane, and the cyclooctane moiety may be further condensed with an aromatic ring;
R represents an alkyl group, an alkoxy group or a halogen atom, and n is an integer of 0 to 2;
Z represents a substance that can bind to the fluorescently labeled object;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]
The fluorescent probe according to claim 1, which is represented by the following general formula (III).
[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
Z represents a substance that can bind to the fluorescently labeled object;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]
The fluorescent probe according to claim 2, wherein Y is -NH-CO-O- in the fluorescent probe represented by the general formula (III). A fluorescent probe synthesis intermediate represented by the following general formula (IV).
[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
Ring A ′ represents an alkyne derivative of cyclooctane, and the cyclooctane moiety may be further condensed with an aromatic ring;
R represents an alkyl group, an alkoxy group or a halogen atom, and n is an integer of 0 to 2;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]
The fluorescent probe synthesis intermediate according to claim 4, which is represented by the following general formula (V).
[Wherein X represents a fluorescent nanoparticle;
Y is represented by -NH-CO-W-, -W-CO-NH-, or the following general formula (II), in which W is an oxygen atom, an alkylene group, an alkyleneoxy group, an alkylenecarbonyl group. Or -Ph-B-, Ph represents a phenylene group, B represents an alkylene group or an alkyleneoxy group, p is an integer of 1 to 6, and q is an integer of 0 to 6;
NH and CO in Y may be directly bonded or may be bonded via a linker. ]
The fluorescent probe synthesis intermediate according to claim 5, wherein Y is -NH-CO-O- in the fluorescent probe synthesis intermediate represented by the general formula (V). The method for producing a fluorescent probe synthesis intermediate according to any one of claims 4 to 6, comprising the following steps:
1) A step of preparing fluorescent nanoparticles into which a carboxylic acid group, amino group, thiol group, maleimide group, carbonate group, or active ester group is introduced;
2) A functional group capable of reacting with various functional groups introduced into the fluorescent nanoparticles in the step 1) is introduced into the cyclooctane alkyne derivative, and the cyclooctane alkyne derivative into which the functional group is introduced is obtained. Preparing step;
3) A step of binding the fluorescent nanoparticles into which various functional groups prepared in the steps 1) and 2) are introduced and an alkyne derivative of cyclooctane. The manufacturing method of the fluorescent probe of any one of Claims 1-3 including the following processes:
A) a step of introducing an azide group into a substance (Z) that can bind to a fluorescently labeled object;
B) A step of binding the alkyne derivative of cyclooctane in the fluorescent probe synthesis intermediate according to any one of claims 4 to 6 to the azide group introduced in the step A).