JP2000111480A - New labelling reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To generate a stable complex and at the same time achieve a fluorescent labeling method by utilizing the complex having a long fluorescent life and intense fluorescence by forming β-diketone structure shown by a specific formula for forming a rare earth ion and a complex in a molecule and a structure for containing a bonding group with a substance to be labeled and a hydrophilic group. SOLUTION: A reagent is composed by containing β-diketone structure that is shown by formula (in which, R2 and R3 indicate respectively β-diketone group that is bonded with the ortho or meta position of benzene ring) for forming at least a rare earth ion and a complex in a molecule, the bonding group with a substance to be labeled, and a hydrophilic group. At least two or four β- diketone structures are included and operate as a tetradentate or octadentate ligand for the rare earth ion. In the molecule, a hydrophilic part and a bonding group for labeling an organism substance are contained. The bonding group with the substance to be labeled reacts with a substituent to form a covalent bonding group. The hydrophilic group introduces hydrophilic property for easily adjusting, storing, or using a labeling reagent in a water solution.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel labeling reagent. Furthermore, the present invention relates to a fluorescent labeling agent containing a complex comprising the reagent and a lanthanoid metal ion (rare earth metal ion), and a fluorescent labeling method using the labeling agent.

[0002]

2. Description of the Related Art Hitherto, as a method of analyzing a trace substance in a biological sample, an immunoassay utilizing an antibody-antigen reaction, a DNA hybridization assay, and the like have been often used. In these analytical methods, it is necessary to use a labeling agent for labeling antibodies, antigens, DNA, DNA base derivatives, DNA oligonucleotides, etc. Labeling, labeling with enzymes and the like are often used.

[0003] Although labeling with radioactive isotopes is highly sensitive, it has drawbacks that involve danger in storage, use and processing. In addition, labeling with an enzyme (enzyme) has a problem that the enzyme has a large molecular weight, is easily affected by an external environment such as temperature, is unstable, has low reproducibility, and binds an enzyme labeling agent to a substance to be labeled. As a result, there is a disadvantage that the activities of the enzyme and the substance to be labeled are reduced.

[0004] Among the labeling methods using fluorescence, labels using organic fluorescent dyes (eg, fluorescein, rhodamine, dansyl chloride, umbelliferone, etc.) are known. However, there is a drawback that the background noise derived from the fluorescence of other coexisting substances present greatly hinders the fluorescence detection of the organic fluorescent dye, making high-sensitivity measurement difficult.

On the other hand, among fluorescent labeling methods, labeling with a complex containing an inorganic ion, particularly labeling with a rare earth ion complex, is known. In addition, rare earth ion complexes have a long fluorescence lifetime (compared to fluorescence lifetimes of several nanoseconds of ordinary fluorescent substances, rare earth fluorescent complexes have fluorescence lifetimes of tens or hundreds of microseconds or more), large Stokes shift, and sharpness. Fluorescent characteristic of having a high fluorescent peak.
Taking advantage of this property, a time-resolved fluorescence measurement method using a rare-earth fluorescent complex as a label has been developed. With such characteristics, interference with excitation light or short-lived background fluorescence derived from a biological sample is removed using time-resolved fluorescence measurement, and high-sensitivity measurement becomes possible.

Heretofore, β-diketone complexes and aromatic amine complexes have been known as fluorescent labeling agents using rare earth ion complexes. One of the rare earth fluorescent labeling agents already commercialized is "DEL" developed by WallacOy (Finland).
FIA "(Dissociation-Enhanced Lanthanide Fluoroimmun
oassay) system. In this DELFIA system, iso
thiocyanato-phenyl-EDTA or isothiocyanatopheny
l-DTTA (DTTA = diethylenetriaminetetracetic acid)
Using a complex of a rare earth and a label as a labeling agent, labeling a protein or nucleic acid, and adding a so-called fluorescence enhancement solution containing β-diketone-trioctylphosphine oxide (TOPO) -Triton X-100 before measuring fluorescence, This is a method of performing fluorescence measurement by releasing a rare earth metal ion from a non-fluorescent complex and generating a micellar solution of β-diketone-rare earth ion-TOPO ternary complex (E. Soini, T. Lovgre).
n, CRC Crit. Rev. Anal. Chem., 18, 105-154 (1987)). However, in this DELFIA system, excess β-diketone and T
Since OPO is present in the measurement solution, it reacts with rare earth metal ions from the environment, may emit strong fluorescence, and has a major drawback that it is very susceptible to contamination by rare earth metal ions. Furthermore, the DELFIA system requires the addition of a fluorescence enhancing solution, and thus has the disadvantage of requiring many reaction steps and the inability to perform solid-phase measurement.

One of the rare earth fluorescent labeling agents already commercialized is FIA developed by Diamandis et al. (Canada).
There is a labeling agent used in the GEN system (EPDiam
andis, Clin. Biochem., 21, 139-150 (1988); EFGDickso
n, A.Pollak, EPDiamandis, Pharmac.Ther., 66,207-235
(1995)). The FIAGEN system uses a fluorescent europium complex (4,7-bis (chlorosulfopop
henyl) -1,10-phenanthroline-2,9-dicarboxylic acid
(BCPDA) -Eu ³⁺ ). This system does not have the problem of europium contamination from the environment, and can also perform solid-phase measurement. However, the fluorescence intensity of the labeling agent of this system is weaker than the above-mentioned DELFIA system by more than two orders of magnitude, and the detection sensitivity is low, so that high-sensitivity analysis is difficult.

[0008] In addition to the above, several aromatic amine-type and β-diketone-type labeling agents are known (G. Mathis, Cli).
n.Chem., 39, 1953-1959 (1993); D. Horiguchi, K. Sasamot
o, H. Terasawa, H. Mochizuki, Y. Ohkura, Chem. Pharm. B
ull., 42, 972-975 (1994); Y.-X.Ci, X.-D. Yang, W.-B.
Chang, J. Immunol. Methods, 179, 233-241 (1995)). However, although these labeling agents have some improvement over conventional labeling agents, the complex has weak fluorescence, short excitation maximum wavelength, protein labeling reaction has many steps, and requires several kinds of cross-linking reagents Disadvantages are that several separations are required.

[0009]

Problems to be Solved by the Invention Japanese Patent Laid-Open No. 9-24123
No. 3 discloses a chlorosulfonylated tetradentate β-β-labeling agent capable of directly labeling a protein having an amino group as a labeling agent which has solved the disadvantages (insufficient fluorescence intensity) of the conventional fluorescent labeling agent using a rare earth ion complex described above. Diketone-type labeling agents are disclosed. In particular, 4,4'-bis (1 ", 1", 1 ", 2", 2 ", 3",
3 "-heptafluoro-4", 6 "-hexanedion-6" -yl) chlorosulfo-
o-terphenyl (BHHCT) can directly label proteins under mild conditions and forms a very stable fluorescent complex with rare earth ions. In the time-resolved fluorescence immunoassay using this as a labeling agent, 3 times as compared with the conventional method.
It is disclosed that a highly sensitive measurement method of an order of magnitude or more is possible (J. Yuan, K. Matsumoto, H. Kimura, Anal. Chem., 70, 5).
96-601 (1998); J. Yuan, G. Wang, H. Kimura, K. Matsumot
o, Anal.Biochem, 254, 283-287 (1997); J. Yuan, G. Wan
g, H. Kimura, K. Matsumoto, Anal.Sci., 14, 421-423 (199
8)).

On the other hand, as a labeling agent for labeling a biological substance, it is not enough that such a high fluorescence intensity is sufficient, but it is necessary to simultaneously satisfy the following conditions. That is, (1) the fluorescence is sufficiently strong, (2) it has a substituent capable of binding to the substance (or biological substance) to be labeled, (3) it is sufficiently stable in an aqueous solution, and (4) the fluorescence lifetime Is long.

A chlorosulfonylated 4-dentate β-diketone type labeling agent having a structure represented by the above-mentioned BHHCT is generally poor in water solubility, so that it can be used for a small biological substance (for example, a nucleic acid base having a small molecular weight having an amino group, Labeling other organic compounds) has the disadvantage that the water solubility of the labeled biological material is reduced and precipitates out of solution.

The present invention has been made in view of the above situation. It is easily soluble in an aqueous solution, has a binding group with a substance to be labeled (or a biological substance), easily forms a complex with a rare earth ion, and is sufficiently stable in an aqueous solution. An object of the present invention is to provide a novel labeling reagent characterized by having a high fluorescence intensity and a long fluorescence lifetime. Therefore, a novel labeling reagent according to the present invention is:
Proteins that are soluble in aqueous solution and have amino groups
Nucleic acid base derivatives, oligonucleotides, hormones and other organic compounds can be directly bound in an aqueous solution. Further, the labeled biological material also has sufficient water solubility.

The present invention also relates to a substance labeled with the novel labeling reagent according to the present invention (specifically, a protein, a peptide, a nucleic acid having an amino group or a mercapto group, etc.)
Nucleic acid bases and their derivatives, nucleotides, hormones and other organic compounds and a substance forming a covalent bond with the labeling reagent of the present invention) react with rare earth ions to form a very stable complex, Another object of the present invention is to provide a fluorescent labeling method utilizing a long fluorescence lifetime and strong fluorescence.

[0014]

In order to solve the above-mentioned problems, a novel labeling reagent according to the present invention comprises a β-diketone structure capable of forming a complex with at least a rare earth ion in a molecule and a substance to be labeled ( (Including biological substances),
Further, it has a structure containing a hydrophilic group.

The β-diketone structure contains at least two or four β-diketone structures and acts as a tetradentate ligand or an octadentate ligand for rare earth ions. Further, in order to stabilize such a coordination mode, the two or four β-diketone structures have a specific steric configuration. Specifically, it is bonded at the ortho or meta position of the benzene ring, and therefore can be spatially arranged to facilitate the above-mentioned tetradentate or octadentate coordination. In addition, the β
The -diketone structure has a β-diketone structure characterized by an electron effect by bonding an electron-withdrawing alkyl group to an aromatic group. Such electron-withdrawing groups include, in particular, perfluoroalkyl groups.

The molecule has at least a hydrophilic portion and a binding group for labeling a biological substance.

A binding group to a substance to be labeled (including a biological substance, hereinafter sometimes referred to as a substance to be labeled) is a group capable of forming a covalent bonding group by reacting with a specific substituent of the substance to be labeled. is there.

Further, the hydrophilic group introduces hydrophilicity so that the novel labeling reagent of the present invention can be easily adjusted in an aqueous solution, stored, and used. Therefore, even if the substance to be labeled is a small molecule, it has sufficient water solubility even after labeling. Further, as the hydrophilic group, a carboxyl group, a fatty acid amide, a peptide, EDTA and DTPA, so as to be easily adjusted, stored and usable under a wide range of pH conditions and various coexisting components.
(diethylenetriamine pentaacetic acid) structure is selected.

More specifically, the present invention provides a labeling reagent represented by the general formula (1).

[0020]

Embedded image Here, R ₂ and R ₃ are β-diketone groups bonded to the ortho or meta position of the benzene ring, and are represented by the general formula (2).

[0021]

Embedded image Here, R ₄ is — (CH ₂ ) _n —, —NH (CH ₂ )
_{m -, - N (COCH 3} ) (CH 2) l -, or - (C
H ₂ ) _k NH—; n, m, k, and l each represent an integer of 0 to 10;

Ar ₁ is represented by the following formulas (3), (4), (5) and (6).

[0023]

Embedded image

Embedded image

Embedded image

Embedded image R ₅ represents a perfluoroalkyl group having 1 to 10 carbon atoms.

R ₁ is represented by the general formula (7).

[0025]

Embedded image Here, R ₆ represents —NH—, —NHNH—, —NHNH
—SO ₂ —, —NH (CH ₂ ) _p NH—, —NH (CH ₂ )
_{q represents} NH—SO ₂ — or a group represented by the formula (8) or (9).

[0026]

Embedded image

Embedded image R7 is represented by the general formula (10).

[0027]

Embedded image Here, R ₈ is -NCS, -NCO, -CO ₂ H, -C
_{OX, -CO 2 R 9, -N} 2 X, -N 3, -NH 2, -X,
_{-CH 2 X, -SH, -SO 2} X, -SO 3 H or, -
Represents SO ₃ CF ₃ , and R ₉ represents a group represented by the formula (11) or (12).

[0028]

Embedded image

Embedded image S represents an integer from 1 to 10. X is
It represents F, Cl, Br, I, -NH (CH 2) t -NCS or a represents a group represented by the formula (13), or (14). P, q, t, and u represent an integer of 1 to 10.

[0029]

Embedded image

Embedded image Further, the present invention provides a labeling reagent represented by the general formula (15).

[0030]

Embedded image Here, R ₁₀ and R ₁₁ , or R ₁₂ and R ₁₃ are β-diketone groups bonded to the ortho or meta positions of the respective benzene rings, and are represented by the general formula (16).

[0031]

Embedded image Here, R ₁₄ represents — (CH ₂ ) _n —, —NH (CH ₂ )
_{m -, - N (COCH 3} ) (CH 2) l -, or - (CH
₂₎ represents a _{k NH-, n, m, k} , l represents an integer of 0 to 10.

Ar ₂ represents a group represented by formulas (3), (4), (5) and (6).

[0033]

Embedded image

Embedded image

Embedded image

Embedded image R ₁₅ represents a perfluoroalkyl group having 1 to 10 carbon atoms.

R ₉ is represented by the general formula (17) or (18).

[0035]

Embedded image

Embedded image Here, R ₁₆ , R ₁₈ , R ₁₉ , and R ₂₁ represent —NH—, —NH
_{NH -, - NHNH-SO 2} -, - NH (CH 2) p NH
—, —NH (CH ₂ ) _q NH—SO ₂ —, or a group represented by the formula (8) or (9).

[0036]

Embedded image

Embedded image R ₁₇ and R ₂₀ are -NCS, -NCO, -CO ₂ H, -C
_{OX, -CO 2 R 21, -N} 2 X, -N 3, -NH 2, -X,
_{-CH 2 X, -SH, -SO 2} X, -SO 3 H or, -
Represents SO ₃ CF ₃ , and R ₂₁ represents a group represented by formulas (11) and (12).

[0037]

Embedded image

Embedded image S represents an integer from 1 to 10. X is
It represents F, Cl, Br, I, a group represented by -NH (CH _{2) t} -NCS, or formula (13) or (14). Where p,
q, t, and u represent an integer of 1 to 10.

[0038]

Embedded image

Embedded image The present invention also provides a complex comprising the above reagent and a rare earth metal ion. Preferably, it provides a complex with a lanthanoid ion, and particularly provides a complex with a europium ion. Such a rare earth ion complex is tetradentate or octadentate coordinated with the above β-diketone. Further, not only coordination in a solution but also those isolated as a complex are included.

Further, the present invention provides a fluorescent labeling agent for labeling the above-mentioned complex with a substance to be labeled.

The present invention also provides a fluorescent labeling method using the reagent or the fluorescent labeling agent according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS (Substances to be labeled) The substances to be labeled by the labeling reagent and the fluorescent labeling agent according to the present invention are not particularly limited, and are various biological substances and substances derived from biological substances. , Can be widely applied to synthetic materials. Furthermore, there is no limitation on the molecular size of the substance to be labeled, its form (solution or solid phase), whether it is a single substance or a composition. It is sufficient that at least one reactive group capable of covalently bonding to the labeling reagent or the fluorescent labeling agent of the present invention is present in a part of the substance to be labeled (or the reactive group can be introduced).

In particular, the biological substance or the biological substance derivative includes various enzymes, proteins, oligopeptides, sugars, glycoproteins, lipids, nucleic acids, nucleic acid derivatives, oligonucleotides, cells, fatty compounds, amino acids and the like. Is mentioned. In particular, proteins include antibodies and derivatives thereof, antigens and derivatives thereof, avidin (including streptavidin), serum albumin, various haptens, hormones, protein A, and protein G.

Examples of the synthetic substance include various chemical substances exhibiting a biological reaction activity. In particular,
Examples include synthetic antibiotics, drugs, pesticides, daily chemicals, chemical reagents, industrial synthetic chemicals, and the like.

(Structure of Labeling Reagent) The labeling reagent according to the present invention binds to a substance to be labeled through a covalent bond by a reaction under ordinary aqueous conditions, and converts the labeled substance into a complex with a rare earth ion. It becomes fluorescent by forming. Further, by measuring such fluorescence, fluorescent labeling is enabled. That is, at least (1) a β-diketone structure capable of forming a complex with a rare earth ion, (2) a structure containing a hydrophilic group, and (3) a substance to be labeled (including a biological substance) in a molecule. And a bonding group.
Hereinafter, each will be described in detail.

(1) β-diketone structure (tetradentate type) The β-diketone structure of the labeling reagent according to the present invention is:
Since it acts as a tetradentate ligand for rare earth ions to form a complex, two are included in one molecule. Further, in order to stabilize such a coordination mode, the two β
-A diketone structure is one that has a specific structure in the molecule to have a specific configuration.

Specifically, two β-dikent structures are
It has a structure bonded to the ortho or meta position of the benzene ring. Therefore, a spatial arrangement in which tetradentate coordination with rare earth ions can be easily formed becomes possible.

The β-diketone structure has a specific electron effect by bonding an electron-withdrawing alkyl group and an aromatic group to both ends. By such an electronic effect, the coordination with the rare earth ion is made extremely stable, and more intense fluorescence becomes possible. As such an electron withdrawing group, a fluorine-substituted alkyl group, a fluorine-substituted aryl group, and a fluorine-substituted aralkyl group are preferable, and a perfluoroalkyl group (having 1 to 10 carbon atoms, particularly preferably having 1 to 4 carbon atoms) is particularly preferable.

The preferred structure is more specifically one represented by R ₂ and R ₃ in the general formula (1). Note that R ₁
Represents a hydrophilic group described below.

[0048]

Embedded image More specifically, R ₂ and R ₃ are preferably those represented by the general formula (2).

[0049]

Embedded image Here, as is R ₄ R ₄ are bonded via an aromatic ring Ar ₁ at one β- diketone skeleton _{- (CH 2) n -,} - NH
(CH ₂ ) _m —, —N (COCH ₃ ) (CH ₂ ) ₁ —, or — (CH ₂ ) _k NH— (n, m, k, l represent an integer from 0 to 10, preferably 1 Is an integer from to 4)
Is preferred. In addition, Ar ₁ is preferably a group represented by formulas (3), (4), (5), and (6) among various aromatic rings.

[0050]

Embedded image

Embedded image

Embedded image

Embedded image Further, on the other side of the β-diketone skeleton, as R ₅ , a fluorine-substituted alkyl group, a fluorine-substituted aryl group, or a fluorine-substituted aralkyl group is preferable, and a perfluoroalkyl group (having 1 to 10 carbon atoms, particularly 1 to 4 carbon atoms) is preferable. Particularly preferred).

(2) Hydrophilic group In the labeling reagent according to the present invention, a preferable structure of the hydrophilic group R ₁ is represented by the general formula (7).

[0052]

Embedded image Here, R ₁ is introduced in order to maintain sufficient solubility and stability in a solution under various pH conditions, and if necessary, further strengthen hydrophilicity as described below. It is also possible to introduce the group R ₇ . R ₆ is specifically —NH—, —NHNH—, —NHNH—SO _{2
-, - NH (CH 2)} p NH -, - NH (CH 2) q NH-
It is preferably SO ₂ — or a group represented by the formula (8) or (9).

[0053]

Embedded image

Embedded image Further, the R ₇ group for introducing stronger hydrophilicity and for introducing a bonding group to a labeling substance described below is specifically preferably represented by the general formula (10). That is, it has a structure having an appropriate number of consecutive amino acid structures. Based on the introduction of these groups, the labeling reagent according to the present invention can be stably and easily dissolved under a wide range of pH conditions, and can form a complex with a rare earth ion under various conditions. Things.

[0054]

Embedded image Here, s is an integer from 1 to 10 (particularly preferably 1
From 4 to 4).

[0055] (3) As explained above linking groups as the labeled substance, the labeled reagent of the present invention is to allow introduction of the linking group R ₈ for coupling with various of the labeled substance .

The preferred R ₈ depends on the structure of the substance to be labeled (that is, it preferably forms a share with the substance to be labeled). _{Specifically, -NCS, -NCO, -CO 2 H} ,
_{-COX, -CO 2 R 9, -N} 2 X, -N 3, -NH 2, -
_{X, -CH 2 X, -SH,} -SO 2 X, is -SO ₃ H or -SO ₃ CF _3, preferred. Further, as R ₉ ,
The groups represented by (11) and (12) are particularly preferred.

[0057]

Embedded image

Embedded image X is F, Cl, Br, I, or -NH
(CH ₂ ) _t -NCS or a group represented by the formula (13) or (14) is preferable. Also, p, q, t, and u are 1 to 1
Represents an integer of 0 (particularly preferably an integer of 1 to 4).

Embedded image

Embedded image (4) β-diketone structure (8-coordinated type) Another preferable structure of the β-diketone according to the present invention is that four β-diketones can be coordinated to one rare earth ion in one molecule so as to be eight-coordinated. It becomes possible to have a specific structure in which is introduced. In this case, a more stable complex can be formed as compared with the tetradentate coordination described in the above (1). Such a structure is not particularly limited, but a structure sandwiching a hydrophilic group described below is preferable. in this case,
From the degree of freedom of the whole molecule, octadentate coordination can be easily performed in the molecule with respect to the rare earth ion.

Specifically, those having a structure represented by the general formula (15) are preferable.

[0059]

Embedded image That is, R ₁₀ and R ₁₁ , or R ₁₂ and R ₁₃ are β-diketone groups bonded to the ortho or meta positions of the respective benzene rings, and are the same as those having the structure described in (1) above. .

Therefore, when R ₉ has a structure having a sufficient degree of freedom, eight β-diketones can be coordinated with one rare earth ion in the molecule by four β-diketones.

R ₉ holds the above-mentioned four β-diketones in a specific structure, introduces sufficient hydrophilicity, and further has a binding group to a substance to be labeled.
Specifically, those represented by the general formula (17) or (18) are preferable.

[0062]

Embedded image

Embedded image Here, R ₁₆ , R ₁₈ , R ₁₉ , and R ₂₁ represent —NH—, —NH
_{NH -, - NHNH-SO 2} -, - NH (CH 2) p NH
—, —NH (CH ₂ ) _q NH—SO ₂ —. Due to the structure of the aminocarboxylic acid, the labeling reagent of the present invention has sufficient hydrophilicity, reactivity over a wide range of pH,
Storage stability and the like are obtained.

In addition, R ₁₇ , R
_{As for 20,} a structure similar to the structure described in (3) above can be preferably selected.

(Synthesis of Labeling Reagent) The method for synthesizing the labeling reagent according to the present invention, starting materials, and the like are not particularly limited, but are preferably designed and synthesized in the following four steps. That is, the first step is to design and synthesize a β-diketone portion having the specific structure described above,
In the second step, a reactive group for introducing a hydrophilic group is introduced, and in the third step, a hydrophilic group having a specific structure is designed and synthetically introduced. Is introduced. Hereinafter, each step will be described specifically, but the present invention is not limited to these starting materials and synthesis methods. What is usually obtained based on a known design and synthesis method is also included in the present invention. Also, for the starting materials described below,
Selected for illustrative purposes only.

In the first step, a β-diketone compound is synthesized by a Claisen condensation reaction of an acetylated aromatic ring compound with ethyl perfluorocarboxylate or diethyl perfluorodicarboxylate. Specific examples of acetylated aromatic cyclic compounds include 4′-phenylacetophenone, 2-acetyldibenzothiophene (synthesized by the reaction of dibenzothiophene with acetyl chloride), methyl-4,4 ′,
4 ″ -terphenyl ketone (synthesized by reaction of 4,4 ′, 4 ″ -terphenyl ketone with acetyl chloride), 2-acetylthiophene, 2-acetylbenzothiophene (the reaction of benzothiophene with acetyl chloride) Synthesized by the reaction), 4,4′-diacetyl-o-
Terphenyl (synthesized by the reaction of o-terphenyl and acetyl chloride);

Specific examples of ethyl perfluorocarboxylates include ethyl trifluoroacetate, ethyl pentafluoropropionate, ethyl heptafluorobutyrate, ethyl perfluoropentanoate and the like. Specific examples of diethyl perfluorodicarboxylates include diethyl difluoromalonate,
Examples include diethyltetrafluorosuccinate, diethylhexafluoroglutarate, diethylperfluoroadipate, diethyldecafluoropimelate, diethyldodefluorosuberate and the like. Here, the catalyst is not particularly limited, but NaOCH ₃ is preferably used. Preferred solvents include ether. The product obtained is purified, if necessary, by recrystallization. It is preferable to use ethanol, 1,4-dioxane or a mixed solvent thereof as the recrystallization solvent.

Here, the case of a perfluoroalkyl group has been described as an example. However, the present invention is not limited to this, and the synthesis can be similarly performed using other suitable substituents described above.

In the second step, a chlorosulfonyl group (ClSO ₂ ⁻ ) is introduced into the aromatic ring of the β-diketone molecule by a chlorosulfonylation reaction between the synthesized β-diketone compound and chlorosulfuric acid. When the unreacted chlorosulfuric acid is hydrolyzed in cold water after the reaction between chlorosulfuric acid and β-diketone is completed, the chlorosulfonylated β-diketone does not dissolve in cold water and is obtained as a precipitate.

The third step is a step of further introducing a hydrophilic group. For example, the formation of a sulfonamide bond by the reaction of the amino group and the chlorosulfonyl group of the hydrophilic group shown above, or the formation of a sulfonhydrazine amide bond by the reaction of an acid anhydride with hydrazine and a chlorosulfonyl group. .

The fourth step is a step of further introducing a bonding group to the substance to be labeled, if necessary, and it is preferable to activate a specific substituent of the hydrophilic group. In particular,
Activating a carboxylic acid group present in the molecule to form an active ester group. Further, it is easy to introduce a substituent of a substituted methylamino group present in a hydrophilic group into various active groups by a substitution reaction.

(Rare earth (lanthanoid) metal ion complex) There is no structural limitation on the complex comprising the labeling reagent according to the present invention and the rare earth metal ion described above.
It is easy to select the type of the rare earth metal ion in consideration of the fluorescence intensity, the fluorescence wavelength, the fluorescence lifetime, and the like of the complex to be formed. Complexes with lanthanoid ions are preferred, and complexes with europium ions are particularly preferred. Such a rare earth ion complex can be reacted with the labeling reagent of the present invention to form
Or eight-coordinate configuration. Further, not only coordination in a solution but also those isolated as a complex are included.

(Preparation of Rare-Earth Metal Ion Complex, Preparation of Fluorescent Labeling Agent) The method of forming the complex between the labeling reagent of the present invention and various rare-earth ions is not particularly limited, and the known starting methods are not particularly limited. Can be preferably used. Further, as an adjustment condition, an aqueous solution can be preferably used. If necessary, the complex can be isolated. Regarding the adjustment conditions in such a case, generally known methods can be preferably used. And its application to fluorescence analysis, Journal of the Spectrochemical Society of Japan, 19
1981, No.1, pp66-73).

Further, the solution containing the complex or the isolated complex is both a fluorescent labeling agent according to the present invention.

(Fluorescent Labeling Method) The fluorescent labeling method according to the present invention involves fluorescently labeling various substances to be labeled using the labeling reagent or fluorescent labeling agent obtained above.

When a labeling reagent is used, (i) a method of first reacting a substance to be labeled with a labeling reagent and then forming a complex with an appropriate rare earth ion to perform fluorescent labeling; And forming a complex with the compound to prepare a fluorescent labeling agent, and then reacting with a substance to be labeled to perform fluorescent labeling.

Specifically, the protein labeling reaction is carried out by an amide forming reaction between a chlorosulfonyl group and an amino group. At room temperature, carbonate buffer solution (pH = 9.0-9.
The reaction proceeds easily in 5). Examples of the immunoassay using the labeling reagent of the present invention include a time-resolved fluorescence immunoassay and a specific binding assay. Time-resolved fluorescence immunoassay refers to a long-lived fluorescent label (Eu-
Chelate, etc.), and refers to a high-sensitivity fluorescence immunoassay in which time-resolved fluorescence measurement of only the fluorescent signal of the label is performed after the background fluorescence having a short fluorescence lifetime has disappeared. The specific binding assay refers to an immunoassay utilizing an antigen-antibody reaction, an assay utilizing a receptor-acceptor binding reaction, an assay utilizing nucleic acid hybridization, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0078]

[Example] (Example 1): N, N'-diacetyl-N, N'-bis [4 ''
-(1 ''',1''', 1 ''',2''', 2 ''',3''', 3 '''-heptafluoro-
4 ''',6'''-hexanedion-6'''-yl)-4'-methylenebipheny
l] -3,5-diaminobenzoic acid (DBHDA) and its N-hydro
Active ester of xysulfosuccinimide (Sulfo-NHS) (NHS-
Synthesis of DBHDA) The synthesis reaction is shown in FIG.

(1) Reductive N- of 3,5-diaminobenzoic acid
Alkylation 20 mmol of 3,5-diaminobenzoic acid is dissolved in 100 ml of methanol, and p-phenylbenzaldehyde (40 m
mol) was added and the reaction occurred immediately. Here, quickly, sodium cyanoborohydride (NaBH ₃ CN) (40.5 mm
ol) was added and immediately reacted to generate hydrogen gas.
Thereafter, a large amount of precipitate was formed, and stirring became impossible, so 100 ml of methanol was further added. This was stirred overnight at room temperature. The resulting precipitate was separated by filtration, and the filtrate
The precipitate formed by adding 100 ml of water was further filtered and separated (the precipitate may be fine, and may be centrifuged). Acetic acid-containing water (about 30 ml of acetic acid and 50 ml of water)
, The color of the precipitate turned yellow (protonation was completed by this treatment). The resulting precipitate was filtered and washed well with water. Recrystallized from 2-butanone, N, N'-bis (4'-methyl
enebiphenyl) -3,5-diaminobenzoic acid (I) was obtained.
The yield after vacuum drying was 62.2%. 4 with AUTO-Mass
84 peaks were identified. _{C 33 H 28 N 2 O 2} (FW = 484.60).

(2) N, N'-bis (4'-methylenebiphenyl) -3,
Acetylation of 5-diaminobenzoic acid (I) 5 mmol of (I) was added to acetic anhydride (50 ml), and after stirring at room temperature for 2 hours, an orange solution was obtained (the acetylation of the amino group was completed). did). After that, acetic anhydride was distilled off under reduced pressure with a vacuum pump, and then acetic anhydride (10 mmol), dichlor
Omethane (50 ml) was added and dissolved. Further, the obtained solution was cooled to 0 ° C., anhydrous AlCl ₃ (40 mmol (8 eq.)) Was added, and the mixture was stirred for 1 hour. The color of the solution turned green and an insoluble black precipitate formed. This was returned to room temperature and stirred overnight to complete the reaction. The resulting solution was ice-cooled
About 50 ml of 15% HCl aqueous solution was added, and the mixture was stirred until the precipitate was dissolved (about 1, 2 hours). The dichloromethane phase was separated, washed with water, dried over Na ₂ SO ₄ and then reduced under reduced pressure.
e, and N, N'-diacetyl-N, N'-bis (4 ''-acetyl-4'-
methylenebiphenyl) -3,5-diaminobenzoic acid (II) was obtained. This was used for the next reaction without further purification. The yield after vacuum drying was 95% or more. FAB-Ma
A peak of 653 was confirmed by ss. C ₄₁ H ₃₆ N ₂ O ₆ (FW = 652.7
5) (3) N, N'-diacetyl-N, N'-bis (4 ''-acetyl-4'-methylen
Synthesis of β-diketone from ebiphenyl) -3,5-diaminobenzoic acid Methanol (20 mmol) was slowly added dropwise to 10 ml of anhydrous THF in which 20 mmol of NaH was suspended at room temperature. Aside from this,
(II) (2 mmol) was dissolved in 10 ml of anhydrous THF, and 8 mmol (4 eq.)
Of heptafluoro-n-butylic acid ethyl ester was added to prepare a solution. This solution was slowly dropped at room temperature into THF in which NaH was suspended. After a while, a pink precipitate formed. The reaction was further stirred overnight at room temperature to complete the reaction. While the reaction vessel was cooled with ice, 20 ml of 15% aqueous HCl was poured little by little, and a yellow precipitate was formed. THF was distilled off under reduced pressure, and the precipitate was separated by filtration and washed well with water. The obtained crude product was recrystallized from ethanol to give N, N'-diacetyl-
N, N'-bis [4``- (1 ''',1''', 1 ''',2''', 2 ''',3''', 3 '''-h
eptafluoro-4 ''',6'''-hexanedion-6'''-yl)-4'-methyl
enebiphenyl] -3,5-diaminobenzoic acid (DBHDA) (III)
Was obtained (72.4% yield).

Elemental analysis result: calc. (%) For C ₄₉ H ₃₄ N ₂ O ₈
F ₁₄ (FW = 1044.80): C: 56.33, H: 3.28, N: 2.68; fou
nd (%): C: 56.54, H: 3.37, N: 2.61. 1045 with FAB-Mass
Was confirmed.

(4) Active esterification of (III) —NHS-DBHDA
Synthesis of 150 mg (0.14 mmol) of (III) and 1 equivalent (31.2 mg) of N-hyd
roxysulfosuccinimide (Sulfo-NHS) was dissolved in 1 ml of DMF. While stirring at 0 ° C., 1 equivalent (29.6 mg) of N, N′-dicycloh
The reaction was completed by adding exyl-carbodiimide (DCC), returning to room temperature, and stirring overnight. The resulting white sediment (DCUrea) was removed by filtration with a membrane filter, and the solution was separated. The DMF of the obtained solution was distilled off under reduced pressure with a vacuum pump, and NHS-DBHDA
I got The obtained NHS-DBHDA could be used for protein labeling without purification.

Example 2 Synthesis of Gly ₃ (BHHCT), a Conjugate of Glycyl-Glycyl-Glycine and BHHCT, and Synthesis of Active Ester NHS-Gly ₃ (BHHCT) with Sulfo-NHS The synthesis method is shown in FIG. .

(1) Synthesis of BHHCT 3.0 g of NaOCH ₃ and 3.14 g (10 mmol) in 50 g of dry ether
4,4'-diacetyl-o-terphenyl and 20 m
mol C ₃ F ₇ COOC ₂ H ₅ was added, sealed at room temperature and stirred for 36 hours. Dry ether was removed by distillation and vacuum dried for 30 minutes. The product was neutralized with 100 ml of 15% sulfuric acid, the precipitate was filtered off and washed well with water.
The precipitate was dissolved in 200 ml of ethanol while heating, and insolubles were removed by filtration. About 20 ml of solution under reduced pressure
The solution was slowly added dropwise to 200 ml of petroleum ether with stirring. After sufficient stirring, a small amount of the deposited precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure to remove all organic solvents. After vacuum drying the obtained oil, a yellow powder was obtained. The yield was 6.70 g, and the yield was 94.9%. Elemental analysis result: Theoretical value: C% =
51.00, H% = 2.28 Measurement value: C% = 51.2
2. H% = 2.61 ¹ H NMR confirmed that the product was the target compound.

The resulting β-diketone was subjected to chlorosulfonylation as follows.

At room temperature, 2 mmol of the β-diketone obtained above were slowly added to 3.5 ml of chlorosulfuric acid with stirring. After stirring at room temperature for 7 hours, the reaction solution was carefully added dropwise to 150 ml of stirring water-ice (cooled externally with ice-water). The precipitate formed was quickly centrifuged, washed with cold water (about 5 ° C.) and centrifuged twice. The precipitate was transferred to a glass filter with a small amount of cold water and the water was removed by suction filtration. 4 at room temperature
The resulting chlorosulfonylated β-diketone was dried under vacuum for more than 8 hours. The yield was 77%. Elemental analysis result: Theoretical value: C% = 44.76, H% = 1.88 Measurement value: C% = 44.50, H% = 1.92 ¹ H NMR confirmed that the product was the target compound did.

(2) Synthesis of Gly ₃ (BHHCT) 217.2 mg (1 mmol) of glycyl-glycyl-glycine ethyl este
After r was dissolved in 3 ml of dry DMF and 1.0 g of N (C ₂ H ₅ ) ₃ , a 3 ml DMF solution containing 805 mg (1 mmol) of BHHCT was gradually added dropwise with stirring. After stirring at room temperature for 1 hour, the reaction solution was gradually dropped into 100 ml of 10-fold diluted hydrochloric acid while stirring, and stirred at room temperature for 15 minutes. The precipitate was separated by filtration and washed well with pure water. The resulting precipitate was dissolved in 50 ml THF-20 ml water-5 ml concentrated HCl solution, refluxed overnight, and then THF was removed by distillation under reduced pressure. After the insoluble product was separated and dissolved in 5 ml of acetone, the acetone solution was added dropwise with stirring to an aqueous HCl solution diluted 10 times to 100 ml with stirring. The precipitate was separated by filtration, washed well with water, and recrystallized with a mixed solvent of acetone and water. The obtained crystals were dried in vacuo (610 mg, 63.8% yield).
Elemental _{analysis:. (C 36 H 25 N} 3 O 10 F 14 S), Calc (%), C = 45.
15, H = 2.63, N = 4.38. Found (%), C = 45.45, H = 2.48, N
= 4.12. Further, the structure of the product was confirmed by ¹ H NMR.

Gly3 (BHHCT) was soluble in an aqueous solution having a pH of 8.5 or more, and had a solubility of about 10 mM in a 0.05 M Tris-HCL solution having a pH of 9.0.

(3) Synthesis of NHS-Gly ₃ (BHHCT) 191.5 mg (0.2 mmol) of Gly ₃ (BHHCT) and 43.4 mg (0.2 m
mol) of N-hydroxysulfosuccinimide (Sulfo-NHS) in 2 ml
After dissolving in dry DMF, stirring at room temperature, 41.3 mg
(0.2 mmol) N, N'-dicyclohexyl-carbodiimide (DCC) was added. After stirring at room temperature for 20 hours, the resulting white precipitate (DCUre
a) was filtered off and the filtrate was separated. After DMF was distilled off from the filtrate under reduced pressure by a vacuum pump, the obtained product was dried under vacuum. The product obtained here could be used for labeling reactions of proteins and nucleic acids without further purification.

(Example 3): 4-amino-butyric acid and BHH
Synthesis of CT Conjugate BA (BHHCT) and Active Ester NHS-BA (BHHCT) with Sulfo-NHS The synthesis method is shown in FIG.

(1) Synthesis of BA (BHHCT) 167.6 mg (1 mmol) of ethyl 4-aminobutyrate hydrochloride,
After 1 g of N (C ₂ H ₅ ) ₃ was added to 2 ml of dry DMF, a solution of 805 mg (1 mmol) of BHHCT in 3 ml of dry DMF was gradually added dropwise to the obtained solution. After the resulting solution was stirred at room temperature for 1 hour, it was gradually added dropwise to 100 ml of 10-fold diluted hydrochloric acid, and further stirred at room temperature for 15 minutes. The resulting precipitate was separated by filtration and washed well with pure water. The obtained precipitate was dissolved in 50 ml of THF, 20 ml of water and 5 ml of concentrated hydrochloric acid, refluxed overnight, and then THF was removed by distillation under reduced pressure. After the insoluble matter was separated and dissolved in 5 ml of acetone, the acetone solution was gradually added dropwise to 100 ml of 10-fold diluted hydrochloric acid with stirring. The precipitate was separated by filtration, washed well with water, recrystallized from a mixed solvent of acetone and water, and the product was dried in vacuo (550 mg, 63.1% yield). Elemental _{analysis:. (C 34 H 23 NO} 8 F 14 S), Calc (%), C = 46.85, H = 2.66,
N = 1.60. Found (%), C = 46.67, H = 2.51, N = 1.48. Further
¹ H NMR confirmed the product structure.

BA (BHHCT) can be dissolved in an aqueous solution having a pH of 8.7 or higher, and has a solubility of about 1 mM in a 0.05 M Tris-HCl solution having a pH of 9.0.

(2) Synthesis of NHS-BA (BHHCT) 174.3 mg (0.2 mmol) of BA (BHHCT) and 43.4 mg (0.2 mmo
l) N-hydroxysulfosuccinimide (Sulfo-NHS) was dissolved in 2 ml of dry DMF, and stirred at room temperature while stirring at 41.3 mg (0.2
mmol) of N, N'-dicyclohexyl-carbodiimide (DCC). After stirring at room temperature for 20 hours, the resulting white precipitate (DCUrea) was filtered off and the filtrate was separated. DMF was distilled off from the obtained filtrate under reduced pressure by a vacuum pump to obtain a product. The product could be used without further purification for labeling reactions of proteins and nucleic acids.

Example 4 DTPA (BHHCT) ₂ and Sulfo
Synthesis of active ester NHS-DTPA (BHHCT) ₂ with -NHS The synthesis method is shown in FIG.

(1) Synthesis of DTPA (BHHCT) ₂ 4 ml of dry DMF, 1.5 g of NEt ₃ and 32 mg of NH ₂ NH ₂
(1 mmol), while stirring, 179 mg of DT
PA dianhydride (0.5 mmol) was added, and the mixture was stirred at room temperature for 2 hours. To this solution, a solution of 805 mg (1 mmol) of BHHCT-4 ml DMF was gradually added dropwise, followed by stirring at room temperature for 1 hour. 100 ml of hydrochloric acid (adjusted from 90 ml of H ₂ O and 10 ml of concentrated hydrochloric acid) was added to the product obtained by removing the organic solvent from the resulting solution by distillation under reduced pressure, and the mixture was further stirred at room temperature for 30 minutes. The precipitate was filtered off and washed well with pure water. The product was vacuum-dried and then recrystallized twice from a mixed solvent (composed of 10 ml of EtOH and 1.5 ml of H ₂ O), and the obtained crystal was vacuum-dried (288 mg, 29.4% yield). ). Elemental analysis: C ₇₄ H ₅₅ N ₇ O ₂ 0F
₂₈ S ₂ , Calcd. (%), C = 45.38, H = 2.83, N = 5.00; Found
(%), C = 46.00, H = 2.98, N = 4.66.

DTPA (BHHCT) 2 was soluble in an aqueous solution having a pH of 8.5 or more, and had a solubility of about 5 mM in a 0.05 M Tris-HCl solution having a pH of 9.0.

DTPA (BHHCT) ₂ was added to 0.05 M Tris-HCl at pH 9.0.
Dissolve in a buffer solution, add europium chloride, and add DTPA (B
The fluorescence spectrum and fluorescence lifetime of the (HHCT) ₂ -Eu ³⁺ solution were measured. FIG. 5 shows the fluorescence spectrum. The fluorescence lifetime of this solution was 350 μs.

(2) Synthesis of NHS-DTPA (BHHCT) ₂ 0.1 mmol (195.8 mg) of DTPA (BHHC) was added to 1.5 ml of dry DMF.
T) ₂ and 0.15 mmol (32.6 mg) of Sulfo-NHS were added and dissolved by stirring, and then 0.15 mmol of DCC (31 mg) was further added. After sealing the obtained reaction solution and stirring at room temperature for 20 hours, the filtrate was separated by removing a white precipitate generated by filtration. The organic solvent was removed from the filtrate by distillation under reduced pressure, and the obtained product was dried under vacuum to obtain NHS-DTPA.
(BHHCT) ₂ was obtained. NHS-DTPA (BHHCT) ₂ could be used for labeling reaction without purification.

(Example 5): NHS-DBHDA and NHS-DTPA (BH
HCT) of proteins using ₂ labeled (1) labeled 13.8 mg of BSA with 1.8 ml of 0.1 M carbonate buffer solution of bovine serum albumin with NHS-DBHDA (BSA) (pH =
9.2) and dissolved in 0.2 ml of DMF with stirring.
0.0 mg of NHS-DBHDA was added dropwise (to the amino group of BSA
2.4 times). After stirring at room temperature for 4.5 hours, 1.0
An aliquot was taken and subjected to gel filtration with Sephadex G-50. The column was 1 × 35 cm, and the eluent used was 50 mM NH ₄ HCO ₃ (pH = 8.0). Fractions were collected every 1 ml and the absorbance at 350 nm was measured. When the molar extinction coefficient of the labeling agent at 335 nm was measured using the remaining labeling agent solution, ε = 4.80 × 10 ⁴
M ⁻¹ cm ⁻¹ . When the labeling ratio was determined, n = 27 was obtained.

(I) Evaluation of fluorescence characteristics of BSA-europium solution labeled with DBHBA: the concentration of the labeling reagent was 3.0 × 10 ^−6.
Excitation / fluorescence spectra were measured at M and Eu concentrations of 1.0 × 10 ⁻⁵ M. The excitation maximum wavelength was 334 nm, and the fluorescence maximum wavelength was 612 nm. Furthermore, the effects of the fluorescence properties on the buffer solution and pH were investigated.

(Ii) Effect of buffer solution: B labeled with DBHBA
The effect of the buffer solution on the fluorescence properties of the SA-Europium solution was examined. The results are shown in Table 1.

[0102]

[Table 1]

From the above results, although phosphoric acid is optimal as a buffer solution, the fluorescence intensity decreases rapidly as the concentration of the complex decreases.

(Iii) Effect of pH: The effect was examined by changing the pH using 0.1 M Tris-HCl as a buffer solution. Labeling reagent concentration is 3.0 x 10 ^-6 M, Eu concentration is 1.0 x 10 ^-5 M
Met. At around pH = 9.3, both the fluorescence intensity and the fluorescence lifetime reached a maximum.

(Iv) Detection limit of novel labeling reagent: The DBHBA-labeled BSA solution was serially diluted with various buffer solutions, and the detection limit was determined by time-resolved fluorescence measurement. At that time, the concentration of europium was adjusted to 1 x 10 ^-6 M, 5 x 10 ^-7 M, 1 x 10 ^-7 M, 5
x 10 ^-8 M, and the effect on the detection limit was examined. The device used was Arcus 1232.

As a buffer solution, 0.1 M Tris-HCl (pH = 9.0
)), When the concentration of europium was 1.0 × 10 ⁻⁷ M, a detection limit of 3.2 × 10 ⁻¹² M was obtained in the concentration of the labeling reagent. At this time, the concentration of BSA was 1.2 × 10 ⁻¹³ M.

0.1 containing 1.0 × 10 ⁻⁵ M TOPO and 0.05% SDS
When M NaHCO ₃ is used, the concentration of europium is also optimally 1.0 × 10 ⁻⁷ M, and the concentration of the labeling reagent is 1.5 × 10 7
A value of 5.6 x 10 ^-15 M was obtained at a concentration of ^-13 M and BSA.

(2) Labeling of bovine serum albumin with NHS-DTPA (BHHCT) ₂ : 10.0 g of BSA was added to 5 ml of 0.1 M at pH 9.1.
Dissolve in carbonate buffer and stir with 20 mg NHS-DTPA (B
A 0.25 ml DMF solution of (HHCT) ₂ was slowly added dropwise. 2 at room temperature
After stirring for an hour, labeled BSA was separated by Sephadex G-50 gel filtration.
And unreacted labeling agent were separated (1 x 28 cm column, pH =
8.0 in 0.05 M NH ₄ HCO ₃ solution). Fractions were collected in 1 ml portions, the absorbance of the fraction at 280 nm was measured, and the labeled BSA solution was collected. Molar extinction coefficient of DTPA (BHHCT) ₂ at 330 nm (7.88 × 10 ⁴ M ⁻¹ .cm ^−
The labeling ratio was determined from ¹ ) and the absorbance of the labeled BSA solution, and a labeling ratio of n = 24 was obtained. 0.1 g of NaN ₃ was added to the labeled BSA solution, the pH of the solution was adjusted to 6.5 with 1 M HCl, and the solution was stored at 4 ° C.

(I) Fluorescence characteristics of BSA [DTPA (BHHCT) ₂ ] _24- Eu ³⁺ solution: Influence of buffer solution: Tris-HC of BSA [DTPA (BHHCT) ₂ ] _24- Eu ³⁺
l, carbonate and phosphate buffer solutions were prepared, and the fluorescence spectrum, fluorescence intensity and fluorescence lifetime were measured. The Tris-HCl solution showed an excitation maximum wavelength of 329 nm and a fluorescence maximum wavelength of 612 nm, and the carbonate and phosphate buffer solutions showed an excitation maximum wavelength of 327 nm and a fluorescence maximum wavelength of 611 nm, respectively. 0.1 M Na
The solution of HCO ₃ -0.05% SDS-1 × 10 ^-5 M TOPO showed an excitation maximum wavelength of 332 nm and a fluorescence maximum wavelength of 613 nm. The fluorescence intensity and fluorescence lifetime in these buffer solutions are shown in Table 2.

[0110]

[Table 2]

Effect of pH: Using a 0.05 M Tris-HCl buffer solution, the effect of pH on the fluorescence characteristics of BSA [DTPA (BHHCT) ₂ ] ₂₄ -Eu ³⁺ solution was examined. The results are shown in Table 3. . As can be seen from Table 3, the fluorescence intensity and the fluorescence lifetime increase as the pH increases, and when the pH reaches about 9, the fluorescence intensity and the fluorescence lifetime reach maximum values (Table 3).

[0112]

[Table 3]

Detection Limit of New Labeling Reagent: DTPA (BHHCT) _2- labeled BSA solution was serially diluted with various buffer solutions, and the detection limit was determined by time-resolved fluorescence measurement. At that time, the concentration of europium was adjusted to 1 x 10 ^-6 M, 5 x 10 ^-7 M, 1 x 10 ^-7 M, 5
x 10 ^-8 M, and the effect on the detection limit was examined. The device used was Arcus 1232.

As a buffer solution, 0.1 M Tris-HCl (pH = 9.0
)), When the concentration of europium was 1.0 × 10 ⁻⁷ M, a detection limit of 3.6 × 10 ⁻¹³ M was obtained in the concentration of the labeling reagent. At this time, the concentration of BSA was 1.5 × 10 ⁻¹⁴ M.

0.1 M containing 1 × 10 ⁻⁵ M TOPO and 0.05% SDS
When NaHCO ₃ is used, the optimal concentration of europium is also 1.0 × 10 ⁻⁷ M, and the concentration of the labeling reagent is 8.6 × 10 7
A value of 3.6 x 10 ^-15 M was obtained at a concentration of ^-14 M and BSA.

Table 4 shows the measurement sensitivity when DTPA (BHHCT) ₂ and BHHCT were used as labeling reagents. Tris-HCl and TOPO-SDS-N
In the aHCO ₃ buffer solution, the sensitivity of the measurement using DTPA (BHHCT) ₂ is about 2-3 times higher than the measurement sensitivity using BHHCT, and at the same time, DTPA (BHHCT) ₂ is easily soluble in water.
It has excellent features not found in HCT (Table 4).

[0117]

[Table 4]

Example 6 Labeling of Streptavidin-Bovine Serum Albumin Conjugate Using NHS-DTPA (BHHCT) ₂ : To label streptavidin (SA) with more labeling agent, use SA-BSA conjugate A body was made and this was labeled with a labeling reagent.

5 mg of BSA and 5 mg of SA were added to 2 ml of 0.1 ml of pH 7.1.
After dissolving in 1 M phosphate buffer, add 0.1 ml of 1% glutaraldehyde, react at 4 ° C overnight, and add 2 mg of NaBH
₄ was added and left at room temperature for 4 hours. This was dialyzed twice against 4 L of physiological saline and pure water. 8 ml of pure water and 84 mg of NaHC
After addition of O _3, to label the NHS-DTPA (BHHCT) _2,
The pH of the solution was adjusted to 9.1 with 1 M NaOH. 150 μ under stirring
15 mg of NHS-DTPA (BHHCT) ₂ dissolved in 1 l of DMSO was gradually added dropwise, and the mixture was stirred at room temperature for 1 hour. The unreacted labeling reagent and the labeled protein were separated by gel filtration of the reaction solution. 1.0 x 35 cm Sephadex G-50 column, 0.05
Developed with M NH ₄ HCO ₃ solution. 330 of labeled protein solution
When the labeling ratio was calculated from the absorbance at nm, SA (B
SA) [DTPA (BHHCT) ₂ ] ₂₅ solution was obtained. After adding 20 mg of NaN ₃ as a preservative and 50 mg of BSA to prevent adsorption of the labeled protein to the container, the pH of the solution was adjusted to about 6.5 with 1 M HCl to prevent hydrolysis of the labeling reagent. Adjust, -20 ℃
Saved in.

Example 7: Using NHS-BA (BHHCT)
(3-amino) allyl deoxyuridine 5'-triphosphate (AA-dU
5 mg of AA-dUTP sodium salt was added to 1 ml of 0.1 M pH 9.1
Dissolve in carbonate buffer and stir with 15 mg of NHS-BA (BH
HCT) in 0.15 ml DMSO was slowly added dropwise. After stirring at room temperature for 2 hours, labeled AA-dUTP and unreacted labeling reagent were separated on a Sephadex G-50 column. The developing solvent is 0.01MT at pH 7.5.
ris-HCl. Labeled AA-dUTP fractions were collected and stored at -20 ° C until use.

Example 8 Labeling of Streptavidin-Bovine Serum Albumin Conjugate Using NHS-DBHDA and Time-Resolved Fluorescent Immunoassay of CEA (Carcinoembryonic Antigen) Using Labeled SA-BSA (i) Labeling: 5 2 mg of BSA and 5 mg of SA 0.1 ml of pH 7.1
Dissolved in phosphate buffer solution. To this, add 0.1 ml of 1% glutaraldehyde, react at 4 ° C overnight, and add 2 mg of glutaraldehyde.
NaBH ₄ was added, and the mixture was left at room temperature for 4 hours. This was dialyzed twice against 4 L of physiological saline and pure water. 13 ml of pure water and 126
After addition of 1 mg NaHCO ₃ , 1 M
The pH of the solution was adjusted to 9.1 with NaOH. 160 μl of DMS under stirring
15.5 mg of NHS-DBHDA dissolved in O was gradually added dropwise, and the mixture was stirred at room temperature for 1 hour. The unreacted labeling agent and the labeled protein were separated by gel filtration of the reaction solution. 1.
It was developed with a 0.05 M NH ₄ HCO ₃ solution on a 0 × 35 cm Sephadex G-50 column. When the labeling ratio was calculated from the absorbance at 335 nm of the labeled protein solution, SA (BSA) (DBHDA
) ₃₈ solutions were obtained. 20 mg of NaN ₃ as a preservative,
50 mg BSA to prevent labeled proteins from adsorbing to the container
After addition of 1 M HCl to prevent hydrolysis of the labeling reagent.
The pH of the solution was adjusted to about 6.5 with and stored at -20 ° C.

(Ii) Coating of 96-well microtiter plate: After diluting goat anti-human CEA polyclonal antibody to 7.5 μg / ml with 0.1 M carbonate buffer solution at pH 9.6,
Dispense 100 μl each into a 96-well transparent polystyrene microtiter plate (FluoroNunc plate),
For 24 hours. The wells were washed once with fixation carbonate buffer. 5% BSA and 0.05% in each well
A 0.1 M Tris-HCl buffer solution (Buffer 1) containing NaN ₃ and 0.9% NaCl and having a pH of 7.8 was dispensed in 100 μl portions, and allowed to stand at room temperature for 30 min
This was incubated. Afterwards, the plate is
0.1 M Tri at pH 7.8 containing 0.05% NaN ₃ and 0.9% NaCl
The plate was washed with an s-HCl buffer solution (Buffer 2) and stored tightly at -20 ° C.

The above operations were coating and blocking of the plate, and the obtained plate could be stored at −20 ° C. for one month.

(Iii) Biotinylation of antibody: 0.5 ml (2.4 mg
/ ml) Rabbit anti-human CEA antibody solution (Dako-immunoglobulin
s, Denmark) was dialyzed twice against 3 L of physiological saline at 4 ° C. for 24 hours, and 0.5 ml of pure water was added. In the resulting solution, 8.4 mg of NaHCO ₃ and 6 mg of sulfosuccinimidyl-6- (b
iotinamido) hexanoate (NHS-LC-Biotin, Pierce Chem.
Co.), and the mixture was stirred at room temperature for 1 hour, and then incubated at 4 ° C. for 24 hours. The reaction solution was mixed with 3 L of 0.1 M NaHCO ₃ −
After dialysis twice against a 0.25 g NaN ₃ solution at 4 ° C. for 24 hours, 10 mg of BSA was added, and the mixture was stored at −20 ° C. until use in an immunoassay.

(Iv) CEA immunoassay: A standard solution for calibration curve measurement was prepared by diluting a human CEA standard solution (purchased from Seikagaku Corporation) with Buffer 1. Serum samples were diluted twice in the same buffer and used for measurement.

A CEA solution (standard solution or serum sample) was dispensed in 50 μl aliquots into the coated and blocked wells, and incubated at 37 ° C. for 2 hours.
0.1 M Tris-HCl (pH 7.8) containing 5% Tween 20 (Buffer
3) twice, 0.1 M Tris-HCl, pH 7.8 without Tween 20
The plate was washed once with (Buffer 4).

50 μl of biotinylated rabbit anti-human CEA antibody diluted 100-fold with Buffer 2 was dispensed into each well, and incubated at 37 ° C. for 1 hour. Buffer 3 (twice) and Buffe
The plate was washed with r4 (once).

DBH diluted with Buffer 2 to 2.5 μg / ml
Dispense 50 μl of DA-labeled SA-BSA solution into wells at 37 ° C
After incubating for 50 minutes with 0.05% Tween 20
Wash wells 5 times with 0.05 M Tris-HCl, pH 9.1 (Buffer 4), then add 0.1 M Tris-HCl, pH 9.1 without Tween 20
The wells were washed twice with (Buffer 5).

The washed plate was used as it was for time-resolved fluorescence measurement on a solid phase.

The time-resolved fluorescence measuring device used for this measurement was Ar
cus 1232, and the intensity of fluorescence at 615 nm was measured using a xenon lamp pulse light source of 340 nm as excitation light. Measurement conditions: Delay time = 200 μs, Window time = 400 μs.
The calibration curve obtained by the above immunoassay is shown in FIG.

Assuming that the background + 3σ is the detection limit, the detection limit in this method was 6 pg / ml. This value was 1000 times more sensitive than a commercially available radioimmunoassay or enzyme immunoassay.

FIG. 7 shows a comparison between a calibration curve when a serum sample was diluted and a calibration curve when a standard sample was diluted. The figure showed a very good correlation. This indicates that correct results can be obtained regardless of the dilution ratio of the serum.

To determine the accuracy of this assay, a spike recovery test was performed. This is a method of adding a known amount of a standard substance to a serum sample and measuring the concentration. Table 5 shows the results. 90 for all samples
A good value of -120% was obtained.

To further examine the precision of the assay, the same sample was added to five wells in the same plate, and the coefficient of variation (coefficient of variation,
CV) (Intra-assay). Similarly, add the same sample to four different plates (one sample
Was measured in one well) and the coefficient of variation was determined (Inte
r-assay). Table 6 shows the results. Intra-assay 3
-7% and 6-9% at Inter-assay, which also gave good results.

This assay and an existing enzyme immunoassay (Enyme linked immonosorbent assay, ELIS
The result of examining the correlation with A) is shown in FIG. As a result of measuring 30 samples, the correlation coefficient was 0.98, indicating a good correlation.

[0136]

[Table 5]

[0137]

[Table 6]

[0138] Labeling of (Example 9) NHS-DTPA (BHHCT) AFP of time-resolved fluorescence immunoassay antibodies using anti-human a-fetoprotein (AFP) antibody labeled and the labeled antibody using a _2: 0.4 ml of goat anti A human AFP antibody solution (specific antibody concentration = 0.5 mg / ml, protein concentration = 6.3 mg / ml, Japan Biotest Laboratory) was added to 3 L of physiological saline at 4 ° C for 2 hours.
After dialysis twice for 4 hours each, it was diluted with pure water to 4 ml.

34 mg NaHCO ₃ was added and the solution was added with 0.1 M NaOH.
After adjusting the pH to 9.1, with stirring, 5 mg NHS-DTPA (BHH
CT) was gradually added dropwise to ₂ -0.4 ml ethanol solution. After stirring at room temperature for 1 hour, the labeled protein and the unreacted labeling reagent were separated by Sephadex G-50 gel filtration.

The labeled protein solution was collected, the absorbance at 330 nm was measured, and the concentration of the labeling reagent in the labeled protein solution was calculated (the molar extinction coefficient of DTPA (BHHCT) ₂ at 330 nm was 7.88 × 10 ⁴ M ^−1. cm ^-1 ). 20 for labeled protein solution
After adding mg BSA and 10 mg NaN _3, the pH of a solution at 1 M HCl
Adjusted to 6.4 and stored at -20 ° C. In addition, it was difficult to calculate the labeling rate of the specific antibody because the antibody coexists with another protein in the labeled antibody solution.

When using the labeled antibody solution for the immunoassay, 7.0 × 10 ⁻⁷ M EuCl ₃ -0.2% BSA-0.1% NaN ₃ -0.9% NaC
After diluting 200-fold with 0.05 M Tris-HCl at pH 7.8 containing
It was used (the concentration of the labeling reagent DTPA (BHHCT) _{2 in} the solution after dilution was about 3.5 × 10 ⁻⁷ M).

For comparison, a BHHCT-labeled anti-AFP antibody was prepared using BHHCT in the same manner as described above.

AFP immunoassay : Anti-human AFP monoclonal antibody (Biostrid) in 0.1 M carbonate buffer at pH 9.6.
e, Inc.) to 5 μg / ml.
100 μl was dispensed into each well of an r plate (Fluoro Nunc) and incubated at 4 ° C. for 24 hours.

0.05 M Tri at pH 7.8 containing 0.05% Tween 20
After washing the wells twice with s-HCl solution (buffer 1),
The wells were further washed once with 7.8 of 0.05 M Tris-HCl solution (buffer 2) once. The plate coated as above
Stored at -20 ° C until use (can be stored within one month).

The human AFP standard solution series is 5% BSA-0.1% N
A concentrated human AFP standard solution (Dakopatts, Denmark) was diluted with a 0.05 M Tris-HCl solution at pH 7.8 containing aN ₃ -0.9% NaCl and adjusted.

[0146] 50 µl of the AFP standard solution was dispensed into the coated wells, and incubated at 37 ° C for 1 hour.
Washed twice with 1 and once with buffer 2. Dispense 50 μl of the diluted labeled anti-AFP antibody solution into each well, incubate at 37 ° C. for 1 hour, and add 0.05% Tween 20
After washing four times with a 0.05 M Tris-HCl solution of 9.1, a time-resolved fluorescence measurement on a solid phase was performed as it was. The measuring device is Arcus
1234, Delay time = 0.2 ms, Window time = 0.4 m
s.

For comparison purposes, exactly the same as the above method,
A time-resolved fluorescence immunoassay of AFP was also performed using an anti-AFP antibody labeled with BHHCT-Eu ³⁺ .

AFP immunoassay results: FIG. 9 shows BHHCT-
The calibration curve for the measurement of AFP in human serum by a time-resolved fluorescence immunoassay using an anti-AFP antibody labeled with Eu ³⁺ and an anti-AFP antibody labeled with DTPA (BHHCT) ₂ -Eu ³⁺ was shown.

When 3 SD of the background signal was determined as the detection limit, the measurement using the anti-AFP antibody labeled with BHHCT-Eu ³⁺ gave a detection limit of 0.07 ng / ml.
(BHHCT) measurement using anti-AFP antibody labeled with _2- Eu ³⁺ is 0.03
A detection limit of 6 ng / ml was obtained. As this limit of detection indicates, the sensitivity of the latter is apparently about twice as high as the former.

Table 7 shows the signal and background results for both assays. As can be seen from Table 7, DTPA (BHHC
Fluorescence intensity measurements using anti-AFP antibody labeled with T) ₂ -Eu ³⁺ whereas than 4 times higher fluorescence intensity measurements using anti-AFP antibody labeled with BHHCT-Eu ^3+, back The ground was just 2.5 times higher.

[0151]

[Table 7]

[0152]

According to the present invention, the compound is easily soluble in an aqueous solution, has a binding group to a substance to be labeled (or a biological substance), easily forms a complex with a rare earth ion, and A novel labeling reagent is obtained, which is characterized in that the complex is sufficiently stable in an aqueous solution and has a sufficient fluorescence intensity and a long fluorescence lifetime. Therefore, by using the novel labeling reagent according to the present invention, it is easily soluble in an aqueous solution and can directly bind proteins, nucleobase derivatives, oligonucleotides, hormones and other organic compounds having an amino group or the like in an aqueous solution. It is. Further, the labeled biological substance also has sufficient water solubility.

Further, according to the present invention, a substance labeled by using the novel labeling reagent according to the present invention (specifically, a protein, a peptide, a nucleic acid, a nucleic acid base having an amino group, a mercapto group, etc. Derivatives, nucleotides, hormones, and other organic compounds form covalent bonds) with rare earth ions to form a very stable complex, which has a very long fluorescence lifetime and strong fluorescence. This allows a labeling method.

[Brief description of the drawings]

FIG. 1 is a diagram showing a method for synthesizing NHS-DBHDA according to an example of the present invention.

FIG. 2 is a diagram showing a method for synthesizing Gly ₃ (BHHCT) and its active ester NHS-Gly ₃ (BHHCT) according to an example of the present invention.

FIG. 3 is a diagram showing a method for synthesizing BA (BHHCT) and its active ester NHS-BA (BHHCT) according to an example of the present invention.

FIG. 4 shows DTPA (BHHCT) ₂ and NHS- according to an embodiment of the present invention.
FIG. 3 is a view showing a method for synthesizing DTPA (BHHCT) ₂ .

FIG. 5 shows DTPA (BHHCT) ₂ -Eu ^{3+ according} to an embodiment of the present invention.
FIG. 4 is a diagram showing an excitation (Ex) and emission (Em) spectrum of a solution. [DTPA (BHHCT) ₂ ] = 3.6 x 10 ^-7 M, [Eu ³⁺ ] = 1.0 x
10 ^-6 M. 0.05 M Tris-HCl buffer of pH 9.1.

FIG. 6 is a diagram showing a calibration curve obtained by an immunoassay according to an example of the present invention.

FIG. 7 is a diagram showing a comparison between a calibration curve when a serum sample is diluted and a calibration curve when a standard sample is diluted according to an example of the present invention.

FIG. 8 is a diagram showing the results of examining the correlation between an assay method and an existing enzyme immunoassay (ELISA) according to an example of the present invention.

FIG. 9: Anti-AFP antibody labeled with BHHCT-Eu ³⁺ and NHS-DTPA (BH
FIG. 4 is a diagram showing a calibration curve of AFP measurement in human serum by a time-resolved fluorescence immunoassay using an anti-AFP antibody labeled with (HCT) ₂ -Eu ³⁺ .

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) C07D 207/416 C07D 207/416 C07F 5/00 C07F 5/00 DF term (reference) 2G054 AA06 CA21 CA22 CA23 CE01 CE10 EA03 4C069 AC31 AC32 BB02 BB17 BB34 BC12 BC18 4H006 AA03 AB92 4H048 AA03 AB92 VA11 VA20 VA30 VA32 VA40 VA42 VA70 VB10

Claims (4)

[Claims] 1. A labeling reagent represented by the general formula (1). Embedded image Here, R ₂ and R ₃ are β-diketone groups bonded to the ortho or meta position of the benzene ring, and are represented by the general formula (2). Embedded image Here, R ₄ is — (CH ₂ ) _n —, —NH (CH ₂ )
_{m -, - N (COCH 3} ) (CH 2) l -, or - (CH
₂₎ represents a _{k NH-, n, m, k} , l represents an integer of 0 to 10. Ar ₁ is a group represented by the following formulas (3), (4), (5), and (6). Embedded image Embedded image Embedded image Embedded image R ₅ represents a perfluoroalkyl group having 1 to 10 carbon atoms. R ₁ is a group represented by the general formula (7). Embedded image Here, R ₆ represents —NH—, —NHNH—, —NHNH
—SO ₂ —, —NH (CH ₂ ) _p NH—, —NH (CH ₂ )
_{q represents} NH—SO ₂ — or a group represented by the formula (8) or (9). Embedded image Embedded image R7 is a group represented by the general formula (10). Embedded image Here, R ₈ is -NCS, -NCO, -CO ₂ H, -C
_{OX, -CO 2 R 9, -N} 2 X, -N 3, -NH 2, -X,
_{-CH 2 X, -SH, -SO 2} X, -SO 3 H or, -
Represents SO ₃ CF ₃ , and R ₉ is a group represented by the formula (11) or (12). Embedded image Embedded image S represents an integer from 1 to 10. X is
It represents F, Cl, Br, I, -NH (CH 2) t -NCS or, a group represented by the formula (13), or (14). Also,
p, q, t, and u represent an integer of 1 to 10. Embedded image Embedded image 2. A labeling reagent represented by the general formula (15). Embedded image Here, R ₁₀ and R ₁₁ , or R ₁₂ and R ₁₃ are β-diketone groups bonded to the ortho or meta positions of the respective benzene rings, and are represented by the general formula (16). Embedded image Here, R ₁₄ represents — (CH ₂ ) _n —, —NH (CH ₂ )
_{m -, - N (COCH 3} ) (CH 2) l -, or - (CH
₂₎ represents a _{k NH-, n, m, k} , l represents an integer of 0 to 10. Ar ₂ represents a group represented by formulas (3), (4), (5), and (6). Embedded image Embedded image Embedded image Embedded image R ₁₅ represents a perfluoroalkyl group having 1 to 10 carbon atoms. R ₉ is a group represented by the general formula (17) or (18). Embedded image Embedded image Here, R ₁₆ , R ₁₈ , R ₁₉ , and R ₂₁ represent —NH—, —NH
_{NH -, - NHNH-SO 2} -, - NH (CH 2) p NH
—, —NH (CH ₂ ) _q NH—SO ₂ —, or a group represented by the formula (8) or (9). Embedded image Embedded image R ₁₇ and R ₂₀ are -NCS, -NCO, -CO ₂ H, -C
_{OX, -CO 2 R 21, -N} 2 X, -N 3, -NH 2, -X,
_{-CH 2 X, -SH, -SO 2} X, -SO 3 H or, -
Represents SO ₃ CF ₃ , and R ₂₁ represents a group represented by formulas (11) and (12). Embedded image Embedded image S represents an integer from 1 to 10. X is
It represents F, Cl, Br, I, a group represented by -NH (CH _{2) t} -NCS, or formula (13) or (14). Where p,
q, t, and u represent an integer of 1 to 10. Embedded image Embedded image 3. A fluorescent labeling agent comprising a complex comprising the reagent according to claim 1 and a rare earth metal ion. 4. A fluorescent labeling method using the reagent according to claim 1 and a rare earth metal ion.