JP3220000B2 - SH group labeling reagent, method for producing the same, and labeling method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a reagent for labeling an SH group using an acridine derivative having a maleimide group, and more particularly to an SH group which is contained in a substance to be measured such as an amino acid or protein or can be easily introduced. The present invention relates to an SH group labeling reagent having an acridine derivative that has a maleimide group bonded to a group and emits chemiluminescence as a labeling substance, a method for producing the same, and a labeling method using the same.

[0002]

2. Description of the Related Art Acridinium ester, which is an acridine derivative, is useful as a chemiluminescent labeling substance because of its high luminous efficiency. The use of this acridinium ester as a chemiluminescent label in immunoluminescence assays in clinical tests is described, for example, in EP-A-82636 and US Pat. No. 47451.
No. 81 discloses it.

[0003] Among the known acridinium esters are:
Those having a sulfonium ion at the terminal (Japanese Unexamined Patent Publication No.
55264), those having a hydrazonium ion in the spacer portion (JP-A-5-255263) and those having a methyl group of acridinium replaced with a carboxyl group (JP-A-6-228102). Although it is known, these acridinium esters having a hydrophilic structure are intended to substantially label an amino group, and label an amino acid or a protein.
It is a compound suitable for use as a chemiluminescent label in immunoluminescence analysis in clinical tests.

By the way, most of the substances to be measured in immunoluminescence analysis in clinical tests are amino acids and proteins, but since these substances have many amino groups, the above-mentioned known acridinium compounds have been labeled. There is a great advantage in that it is easy.

[0005] However, conversely, as a result of labeling an amino group contained in an amino acid or a protein in a large amount, the number of sites to be labeled increases, resulting in problems of label uniformity, reproducibility, insolubilization of an object such as an antibody protein, and the problem of antibody. In the case of labeling, there has been a problem that a labeling compound binds to an amino group at an antigen recognition site, thereby reducing or losing the function as an antibody. When the substance to be labeled is a small molecule having a limited number of amino groups, the labeling reaction may be controlled and the labeling may be easily performed at a constant molar ratio. When the number of amino groups in a molecule cannot always be limited, there is also a disadvantage that the conditions for labeling at a fixed molar ratio must be preliminarily examined by trial and error.

On the other hand, in order for a certain compound to be put into practical use as a chemiluminescent labeling reagent, it is essential that the labeling reaction of the compound is easy and that the labeling conditions do not cause luminescence or decomposition. The partner to be labeled with acridinium ester is various, including low molecular weight compounds such as amino acids, and high molecular weight compounds such as enzymes or antibodies. As shown in the literature, an acridinium ester is often provided with an imide group and bonded to an amino group. Then, the reaction in the case of binding reaction with an amino group using this imide group proceeds efficiently with alkali, but in general, acridinium compound is an alkaline and unstable compound which emits light or decomposes, and therefore has luminescence activity. Efficient labeling is disadvantageous in that it requires conflicting reaction conditions.

[0007]

Accordingly, in a chemiluminescence labeling method used for immunoluminescence analysis or the like, it is possible to bind to a substance to be measured such as an amino acid or a protein under mild conditions.
In addition, there has been a demand for a method of finding a labeled compound having high specificity and sufficient binding force, and using the compound to stably and accurately detect a substance to be measured.

[0008]

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventor first found that a labeling group is not generally contained in an amino acid or a protein in place of an amino group conventionally used. And the use of SH groups having good reactivity. Then, they have found that an acridinium compound having a maleimide group introduced therein can be advantageously used as an SH group labeling reagent for labeling this SH group, and completed the present invention.

That is, an object of the present invention is to provide a compound represented by the general formula (I)

Embedded image (Wherein, R represents an alkyl or aryl group having 1 to 6 carbon atoms, X ⁻ represents an anion, and m represents an integer of 1 to 6). It is to provide. Another object of the present invention is to provide a compound of the formula (II) which is an intermediate of the acridine compound.

Embedded image (Wherein m has the meaning described above). Still another object of the present invention is to provide a method for producing acridine compounds represented by the above formulas (I) and (II). Still another object of the present invention is to provide a method for labeling a substance to be measured using the above formula (I) or (II).

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The compound represented by the general formula (I) of the present invention is a compound represented by the general formula (II) (hereinafter, referred to as “intermediate (II)”) according to the following reaction formula. )
It is obtained by reacting an alkylating agent (III) according to a conventional method.

[0011]

Embedded image (Wherein, X represents a leaving group that easily becomes an anion;
m has the meaning described above)

The alkylating agent (R-X) used in this reaction includes, for example, alkyl halides such as methyl iodide, ethyl bromide and ethyl iodide, methyl trifluoromethanesulfonate, methyl fluorosulfonate and methanesulfonate. And methyl p-toluenesulfonate. Therefore, X ⁻ in the above formula (I) is a halogen ion such as I ⁻ , Br ⁻ , CF ₃ SO ₃ ⁻ , FSO ₃
^-, CH ₃ SO ₃ ^- or _{p-CH 3 C 6 H 4} SO 3 - is the most representative, and R is an alkyl group or an aryl group having 1 to 6 carbon atoms such as methyl, ethyl. Examples of the solvent used in this reaction include ether, toluene, acetonitrile and the like.

The intermediate (II), which is a starting material for producing the compound (I), is an ω-aminoalkylenephenyl 9-acridine carboxylate (I
It is produced by reacting V) with maleic anhydride (V) in the presence of a base.

[0014]

Embedded image (Wherein, m has the meaning described above)

Specifically, for example, Clinical Chemistry (Clin. Chem. 1985, 31 (10), 16)
According to the method described in 64-1668), sodium acetate, potassium carbonate or the like is used as a base, and acetic acid or propionic acid is used as a solvent.
Compound (II) can be obtained by reacting compounds (IV) and (V) at a temperature of about ° C.

The intermediate (II) has a general formula described in Japanese Patent Application Laid-Open No. 62-69969. Since there is no description, it seems to be a novel compound in fact.

Next, a method of reacting the compound (I) of the present invention with an amino acid or an SH group in a protein to label these substances will be described.

For example, when labeling an antibody with the compound (I) of the present invention, first, the antibody is digested with pepsin and F
(Ab ') ₂ which is reduced under mild conditions to give Fa
Prepare b '. The Fab's SH were then added in solution.
The group can be labeled by reacting the maleimide group of the compound (I) of the present invention with the group.

In the reaction of Fab 'with the compound (I) of the present invention, the oxidation of the SH group by the dissolved enzyme in the reaction solution is protected by EDTA of about 1 to 5 mM, and the pH is 5 to 7, preferably 6 to 6. 5 in an aqueous solution at a temperature of 1 to 37 ° C., preferably 4 to 10 ° C., for 10 minutes to 72 hours, preferably for about 48 hours, at a molar ratio of Fab ′ to the compound (I) of the present invention of 1: 1. The reaction may be carried out at from 10 to 10.

The labeling with the compound (I) can be generally performed in an aqueous medium at a pH of 7 or less. it can. That is, water is added to a solution in which a substance to be labeled is dissolved in a small amount of an inert solvent that is well mixed with water (hereinafter, referred to as an “inactive solvent”) to form an aqueous solution of the substance to be labeled, and a compound ( The aqueous solution of I) may be mixed and the SH group and the maleimide group may be reacted at a pH of 7 or less. Since this reaction proceeds by the nucleophilic addition of the labeling substance to the unsaturated bond of the maleimide group of the compound (I) of the present invention, even when the labeling substance has an amino group in addition to the SH group, The compounds of the present invention can be used as reagents selective for SH groups in that the reaction at pH 7 or less suppresses the nucleophilicity of this amino group and does not produce a reaction product with the amino group. .

Here, the inert solvent used for labeling includes N, N-dimethylacetamide, dimethylsulfoxide, N, N-dimethylformamide (DMF), 1,4
-Dioxane or pyridine.

After the labeling, separation methods such as gel chromatography, ion exchange chromatography, affinity chromatography, and high performance liquid chromatography are used.
The target can be isolated. That is, labeling with a fixed molar ratio is possible by the above method.

When the substance to be labeled does not have an SH group, for example, a hydroxyl group or an amino group of the substance to be labeled is replaced with an S group.
-An SH group is introduced under mild conditions using acetylmercaptosuccinic anhydride or the like, and the labeling method of the present invention can be applied. In this case, the molar ratio between the substance to be labeled and the SH group can be estimated by quantifying the introduced SH group. Therefore, the substance to be measured that can be used in the labeling method of the present invention has an SH group,
Any substance can be used as long as it can introduce a group, and the compound (I) of the present invention can be labeled on mild molecules such as amino acids under mild conditions as described above.

The substance to be labeled thus obtained is caused to undergo chemiluminescence by the addition of H ₂ O ₂ or the like under a known acridinium ester luminescence condition, for example, an alkaline condition, and this luminescence is detected by a chemiluminescence detector. By detecting, it is possible to know its presence, and from the amount of chemiluminescence, it is possible to know the amount of the labeling substance. It can be applied to qualitative analysis and quantitative analysis in various fields.

Next, specific application examples of the labeling method using the compound (I) of the present invention will be described. As an application of the labeling method of the present invention, for example, as in a so-called chemiluminescent immunoassay sandwich method, a substance to be measured in a sample and a substance that specifically binds to the substance to be measured bound to a carrier are used. After capturing the substance to be measured by a binding reaction, a substance that specifically binds to the substance to be measured is bound to a substance labeled with the compound (I) of the present invention, and the luminescence intensity is measured. A method for measuring a substance is exemplified.

Also, as in the so-called chemiluminescent immunoassay competition method, the substance to be measured is labeled with the compound (I) of the present invention, and this is added at a predetermined concentration in advance.
Measuring the luminescence intensity using a reaction in which these analytes bind antagonistically to a substance that specifically binds to both the analyte and the labeled analyte in the sample Of the substance to be measured by the method described in US Pat. In these cases, examples of combinations of substances that specifically bind include antigens and antibodies, sequences complementary to nucleic acids, effector molecules and receptor molecules, enzymes and inhibitors, avidin and biotin, substances having sugar chains and corresponding lectins. And the like.

As another application, a substance to be measured is labeled with the compound (I) of the present invention, as in, for example, a so-called post-column high-performance liquid chromatography method, and is simply subjected to a separation method such as the above-mentioned chromatography method. A method of measuring the substance to be measured by using a chemiluminescence detector.

In the labeling method described above, the compound (I) of the present invention is required for insolubilization and biological activity of the labeled analyte, which are disadvantages of the conventionally known acridinium ester. Various problems such as the possibility of deactivation of amino groups by labeling, difficulty in binding at a fixed binding molar ratio, difficulty in obtaining reproducibility in the binding reaction, and emission or decomposition already occurring under the binding reaction conditions Can be overcome.

Further, another example of the application of the present invention includes the following method. That is, the present compound (I)
Intermediate (II), which is also used for synthesis, is reacted with a substance to be measured having an SH group in advance to bind (hereinafter, referred to as "pre-labeling"). Next, an appropriate N-alkylating agent is reacted, and further under an alkaline condition, an appropriate chemiluminescent agent such as H ₂ O _{2 is} allowed to act. The chemiluminescence is detected by a chemiluminescence detector, and the substance to be measured is measured. Method.

The acridine ester represented by the general formula (II) has a maleimide group at the terminal thereof having an activity of binding to an SH group and can be isolated as a stable compound. Can be combined.

The binding method in the pre-labeling reaction of the substance to be measured with the above-mentioned intermediate (II), the separation method of the obtained pre-labeled product, and the like can be performed in substantially the same manner as the method for compound (I). .

The pre-labeled product can be further converted to an acridinium ester-labeled product by reacting it with an alkylating agent (III) in a suitable solvent. As the alkylating agent (III) used, as described above,
For example, alkyl halides such as methyl iodide, ethyl iodide and ethyl bromide, methyl trifluoromethanesulfonate, methyl fluorosulfonate, methyl methanesulfonate and methyl p-toluenesulfonate can be used. As the solvent to be used, for example, in addition to the solvent used in the above pre-labeling reaction, tetrahydrofuran,
Acetonitrile and the like can be mentioned.

In the case of precipitation as crystals from the solvent used after the reaction, the acridinium ester-labeled product can be isolated by filtration, and in the case where precipitation does not occur, column chromatography is used. Can be isolated.

After the pre-labeling reaction with the intermediate (II), the chemiluminescence of the substance to be measured, which has been N-alkylated and further labeled with the alkylating agent (III), and the use thereof are also described for the compound (I). It is the same as

The labeling method using the intermediate (II) is as follows.
In view of the fact that the compound is labeled with the alkylating agent (III) after binding, it is not suitable for a substance to be measured having an active group in the alkylating agent other than the SH group. It is necessary to take measures such as protection with various protecting groups.

[0036]

The labeling of the substance to be measured with the compound (I) or (II) of the present invention is a stable bond between the SH group and the maleimide group, which is carried out under mild conditions.
Then, as a result of labeling under mild conditions, for example, the antigen recognition site of the labeled antibody or the like is not damaged, and the acridine ester does not emit light to be consumed or decompose.

In the labeling method of the present invention, since the SH group, which is a relatively small group in amino acids and proteins, is used, a specific label can be applied to the substance to be measured. There are advantages such as that a bond can be obtained between the substance and the chemiluminescent substance at a fixed bond molar ratio.

For example, the labeling method of the present invention can
When applied to b ′, Fab ′ has one SH group, and thus compound (I) (or intermediate (II)) of the present invention is
Even when all of Fab's are labeled in addition to the ab's in a slight excess, they are at most only labeled at a molar ratio of 1: 1.
Furthermore, the SH group for this binding is independent of the Fab 'biologically active site, so that the antigen recognition site of the Fab' is not compromised.

Therefore, the qualitative and quantitative determination of the substance to be measured can be easily performed based on the amount of chemiluminescence, and in particular, the present invention can be applied to fields such as tracking the pharmacokinetics of pharmaceuticals requiring a very small amount of detection and diagnostic agents.

[0040]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 4- [2- (maleimido-1-yl) ethyl] phenyl 9-acridinecarboxylate (2) [intermediate (I
I)] Synthesis:

[0042]

Embedded image

4- (2-aminoethyl) phenyl 9-
0.71 g of acridine carboxylate (1) and 0.60 g of maleic anhydride, and 0.60 g of sodium acetate
Was added to 20 ml of acetic acid, and the mixture was heated under reflux for 3 hours. After cooling, acetic acid was distilled off under reduced pressure, water was added, and the mixture was extracted with CHCl ₃ . The extract is dried over anhydrous magnesium sulfate,
HCl ₃ was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (eluent: CHCl ₃ ) to obtain 0.14 g of the title compound (2) (9.5% yield). The analytical values of this substance were as follows.

NMR (CD ₃ OD) δ: 8.2-7.6
(M, 8H), 7.4 (s, 4H), 6.8 (s, 2H),
3.8 (t, J = 8.2H), 3.0 (t, J = 8.2H) MASS (FAB): 423 (M + 1)

Example 2 Synthesis of 4- [2- (maleimido-1-yl) ethyl] phenyl 10-methylacridinium-9-carboxylate (3) [Compound (I)]

[0046]

Embedded image

0.17 g of the above maleimide (2) was added to 20 m
Dissolved in 1 liter of toluene. To this solution, 0.10 g of methyl trifluoromethanesulfonate was added under stirring at room temperature, and the mixture was stirred at room temperature for 1 hour and left overnight. After standing, a yellow precipitate that precipitated was collected by filtration and washed with toluene. This was air-dried to give 0.12 g of the title compound (3).
Obtained (51% yield). The analytical values of this substance were as follows.

NMR (CD ₃ OD) δ: 8.9-8.0
(M, 8H), 7.4 (s, 4H), 6.8 (s, 2H),
5.1 (s, 2H), 3.8 (t, J = 8, 2H), 3.0
(T, J = 8.2H) MASS (FAB): 437 (M ⁺ )

EXAMPLE 3 Labeling of Anti-Human Hemoglobin Antibody: (1) 10 mg of purified anti-human hemoglobin rabbit polyclonal antibody was added to 3 ml of 0.1 M sodium acetate buffer (pH 4.5) containing 0.1 M NaCl. To this solution, 0.4 g of purified pepsin powder derived from porcine gastric juice was added and dissolved, and incubated at 37 ° C. for 48 hours to prepare rabbit I.
The Fc portion of gG was digested. Use this as eluent for 5m
0.1 M sodium phosphate buffer containing M EDTA (p
H6) [hereinafter referred to as pH6 buffer] and Superdex 200 column (Pharmacia) and fractionated by gel filtration using, F (ab 'in pH6 buffer 3 ml) ₂ 5
A solution containing mg was obtained.

To this solution, 24 mg of 2-mercaptoethylamine was added and incubated at 37 ° C. for 24 hours to reduce F (ab ′) ₂ to Fab ′. Subsequently, a Sephadex G25 column (manufactured by Pharmacia, eluent: p
Gel filtration with H6 buffer, pH 6 buffer 3 ml
A solution containing 2 mg of Fab ′ was obtained.

Using a part of this solution, the amount of 4-mercaptopyridine obtained as a result of quantitative reaction of 4,4′-dithiopyridine with the SH group of Fab ′ was measured at an absorbance of 324 nm to determine the SH group. Quantification was performed.
As a result, the number of SH groups per Fab ′ molecule was one.

(2) Next, p containing 0.5 mg of Fab '
To 0.75 ml of the H6 buffer, 0.5 ml of a pH 6 buffer containing 0.04 mg of the compound (3) synthesized in Example 2 was added. That is, Fab 'and compound (3) were added such that the molar ratio of the binding reaction was 1: 5, and the mixture was incubated at 4 ° C for 48 hours, and Fab' was labeled with compound (3).
After labeling, the mixture was subjected to gel filtration using a Sephadex G25 column (manufactured by Pharmacia, eluent pH 6 buffer) to separate the compound (3) added in excess, and a solution containing 0.5 mg of the labeled antibody in 3 ml of pH 6 buffer was used. (Labeled antibody A).

This was quantified by the molar extinction coefficient of E _{280 of} Fab ′ and E ₂₆₀ of compound (3).
The resulting labeled antibody A had Fab 'and compound (3) bound at a molar ratio of 1: 1.

Example 4 Assay of human hemoglobin with labeled antibody: 0.15
A solution containing 5 μg of purified anti-human hemoglobin mouse monoclonal antibody in 0.1 ml of 0.05 M phosphate buffer (hereinafter abbreviated as “PBS”) at pH 7 containing M NaCl was prepared. This solution was dispensed in 0.1 ml portions into each well of a microplate, and allowed to stand at 4 ° C. for 24 hours to allow the monoclonal antibody to be adsorbed on the inner surface of the well and immobilized.

This antibody-immobilized plate was subjected to a blocking treatment with 0.1 ml of PBS containing 1 mg of bovine serum albumin. Separately, purified human hemoglobin was added to each of 0,
0.2, 1, 5, 25, and 125 μg / ml were dissolved in PBS.
1 was dispensed into each well of the microplate, and allowed to stand at 4 ° C. for 24 hours to allow human hemoglobin in the sample to be captured by the immobilized antibody. Each well is then washed with PBS,
After the unbound human hemoglobin was separated, PBS containing 5 μg of the labeled antibody A prepared in Example 3 was added to each of the cells.
1 ml was dispensed into each well, and left at 4 ° C. overnight to react.

Each well was washed with PBS, and the unbound labeled antibody A was separated.
1 and 0.5% H ₂ O ₂ were added to emit light, and the measurement was carried out using Luminous CT-9000D (manufactured by Diamondtron) which is a kind of chemiluminescence measurement microplate reader. The light emission amount was an integrated value for 2 seconds. Table 1 summarizes the above results.

[0057]

As shown in the table, the amount of chemiluminescence derived from the labeled antibody A increased according to the concentration of human hemoglobin.
That is, it is shown to be useful for quantification of human hemoglobin. that's all

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ identification code FI G01N 33/532 G01N 33/532 B (58) Investigated field (Int.Cl. ⁷ , DB name) C07D 401/12 C09B 15 / 00 C09K 11/07 G01N 31/22 G01N 33/52 G01N 33/532 CA (STN) REGISTRY (STN)

Claims (4)

(57) [Claims] 1. A compound of the general formula (I) (Wherein, R represents an alkyl or aryl group having 1 to 6 carbon atoms, X ⁻ represents an anion, and m represents an integer of 1 to 6). . 2. A compound of the general formula (II) (Wherein m represents an integer of 1 to 6). An SH group labeling reagent containing an acridine compound represented by the following formula: 3. A compound of the general formula (I) (Wherein, R represents an alkyl or aryl group having 1 to 6 carbon atoms, X ⁻ represents an anion, and m represents an integer of 1 to 6). A method for labeling a substance to be measured, characterized by reacting with a group. 4. A compound of the general formula (II) (Wherein m represents an integer of 1 to 6). A method for prelabeling a substance to be measured, which comprises reacting an acridine compound represented by the following formula with an SH group in the substance to be measured.