WO2021221011A1 - Tyrosine residue modification method using highly reactive reagent - Google Patents

Abstract

The present invention provides, as a stable and easy-to-handle tyrosine modifier, a compound that is represented by general formula (I), (II), or (III) (in the formula, A represents a conjugated ring, L represents a linker or the like having at an end a functional group used for click reaction, R¹ represents a hydrogen atom or the like, R² represents methyl or the like, and X^- represents a halide ion).

Description

Method of modifying tyrosine residue with highly reactive reagent

The present invention relates to compounds that can be used to modify tyrosine. This compound is stable for a long time and can be used as an easy-to-use tyrosine modifier.

Modification of protein molecules is a very useful method in life science research. For example, by modifying a protein with a labeling substance, it becomes possible to identify molecules and binding sites that bind to the protein. In addition, modification of protein molecules is indispensable when an anticancer agent or the like is added to an antibody to prepare an antibody drug conjugate or when polyethylene glycol (PEG) is added to a peptide or the like to improve blood stability. It is a method.

Conventionally, modification of protein molecules has been performed by targeting nucleophilic amino acid residues (for example, lysine residues, cysteine residues, etc.) with electrophiles. However, since cysteine residues have a low abundance ratio and most of them form disulfide bonds in the protein molecule, they cannot be modified without reduction. On the other hand, the lysine residue has a high abundance ratio and does not have a problem like the cysteine residue, but when producing an antibody drug or the like, the abundance ratio is too high, so that site-specific modification is difficult. have. There is also a problem that the modification of the lysine residue does not proceed unless it is under basic conditions.

Recently, attention has been paid to a method for modifying a protein molecule that targets a tyrosine residue instead of the above-mentioned nucleophilic amino acid residue. For example, Barbas et al. Have reported a method of modifying a tyrosine residue using 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) (Non-Patent Document 1, Non-Patent). Document 2). The present inventors also reported a method of modifying a tyrosine residue using a luminol derivative in the presence of hydrogen peroxide and an iron catalyst (Patent Document 1) or by an electrochemical method (Patent Document 2). ing. Since tyrosine residues are often on the surface of proteins and their abundance ratio is not as high as lysine residues, this method is considered suitable for antibody modification. This method also has the advantage that it can be modified even under neutral conditions, unlike the method for modifying lysine residues.

Japanese Unexamined Patent Publication No. 2016-108266 Japanese Unexamined Patent Publication No. 2020-2095

As mentioned above, the method developed by Barbas et al. Is a useful method for modifying protein molecules, but there are some problems. For example, PTAD is an unstable substance, so it is difficult to store it for a long time. In addition, since the isocyanate generated by the decomposition of PTAD also reacts with a nucleophilic amino acid residue such as a lysine residue, there is a problem that the selectivity to a tyrosine residue is lowered.

An object of the present invention is to solve such a problem of the conventional method and to provide a stable and easy-to-use tyrosine modifier.

As a result of diligent studies to solve the above problems, the present inventor has found that the compound obtained by reacting 1-methyl-4-phenylurazole or an N-methylluminol derivative with N-bromoxinimide is stable. , It has been found that it can be used as an easy-to-use tyrosine modifier, and the present invention has been completed.

That is, the present invention provides the following (1) to (20).
(1) The following general formulas (I), (II), or (III)

[In the formula, A represents a conjugated ring and L represents a hydrogen atom, or a linker having a functional group used for a click reaction at the terminal or a linker having a labeling substance at the terminal (where L is on A). It may be present at any position), where R ¹ represents a hydrogen atom or represents a radioactive nuclei, a functional group used in the click reaction, an amino group, an acetamide group, a hydroxy group, an alkyl group, or an alkoxy group (representing a radioactive nuclei, an amino group, an acetamide group, a hydroxy group, or an alkoxy group ( However, R ¹ may be present at any position on A), R ² represents an alkyl group which may have a substituent or an aromatic group which may have a substituent, and X represents an aromatic group. ^- represents a halide ion. ]
The compound represented by.

(2) The compound according to (1), wherein A in the general formulas (I) and (II) is a benzene ring.

(3) The compound according to (1) or (2), wherein ^{R 2} in the general formulas (I), (II), and (III) is a methyl group.

A compound according to any one of the, characterized in that it is a bromide ion (1) to (3) ^- (4) In formula (I), (II), and X in (III).

(5) A tyrosine modifier containing the compound according to any one of (1) to (4).

(6) A tyrosine comprising a step of reacting the compound according to any one of (1) to (4) with a protein containing a tyrosine residue and binding the compound to the tyrosine residue of the protein. Modification method.

(7) Represented by the general formulas (I), (II), and (III), wherein the compound according to any one of (1) to (4) is stored in N, N-dimethylformamide. How to store the compound.

(8) The following general formulas (Ia), (IIa), or (IIIa)

[In the formula, A represents a conjugated ring and L represents a hydrogen atom, or a linker having a functional group used for a click reaction at the terminal or a linker having a labeling substance at the terminal (where L is on A). It may be present at any position), where R ¹ represents a hydrogen atom or represents a radioactive nuclei, a functional group used in the click reaction, an amino group, an acetamide group, a hydroxy group, an alkyl group, or an alkoxy group (representing a radioactive nuclei, an amino group, an acetamide group, a hydroxy group, or an alkoxy group ( However, R ¹ may be present at any position on A), and R ² represents an alkyl group which may have a substituent or an aromatic group which may have a substituent. ]
The compound represented by is reacted with N-halosuccinimide in a solvent, and the following general formula (I), (II), or (III)

Wherein, A, L, R ^1, and R ² are as defined above, X ^- represents a halide ion. ]
A method for producing a compound represented by the general formula (I), (II), or (III), which comprises a step of obtaining each of the compounds represented by.

(9) A method for producing a compound represented by the general formula (I), (II), or (III) according to (8), wherein the solvent is N, N-dimethylformamide.

(10) The general formula (I), according to (8) or (9), wherein A in the general formulas (Ia), (IIa), (I), and (II) is a benzene ring. A method for producing a compound represented by (II) or (III).

^{(11) R 2} in the general formulas (Ia), (IIa), (IIIa), (I), (II), and (III) is a methyl group (8) to (10). A method for producing a compound represented by the general formula (I), (II), or (III) according to any one of.

(12) N- halosuccinimide is a N- bromo succinimide, general formula (I), (II), and X in (III) ^-, characterized in that a bromide ion (8) to ( A method for producing a compound represented by the general formula (I), (II), or (III) according to any one of 11).

(13) The following general formulas (Ia), (IIa), or (IIIa)

[In the formula, A represents a conjugated ring and L represents a hydrogen atom, or a linker having a functional group used for a click reaction at the terminal or a linker having a labeling substance at the terminal (where L is on A). It may be present at any position), where R ¹ represents a hydrogen atom or represents a radioactive nuclei, a functional group used in the click reaction, an amino group, an acetamide group, a hydroxy group, an alkyl group, or an alkoxy group (representing a radioactive nuclei, an amino group, an acetamide group, a hydroxy group, or an alkoxy group ( However, R ¹ may be present at any position on A), and R ² represents an alkyl group which may have a substituent or an aromatic group which may have a substituent. ]
A tyrosine modification kit comprising the compound represented by and N-halosuccinimide.

(14) The tyrosine modification kit according to (13), wherein A in the general formulas (Ia) and (IIa) is a benzene ring.

(15) The tyrosine modification kit according to (13) or (14), wherein ^{R 2} in the general formulas (Ia), (IIa), and (IIIa) is a methyl group.

(16) The tyrosine modification kit according to any one of (13) to (15), wherein the N-halosuccinimide is N-bromosuccinimide.

(17) The following general formula (Ib), (IIb), or (IIIb)

[In the formula, A represents a conjugated ring, Lb represents a linker having a fluorescent dye or drug at the end (where Lb may be present at any position on A), and R ² has a substituent. represents an aromatic group optionally having also an alkyl group or a substituent are, R ^P represents a group derived from a protein containing tyrosine residues. ]
Fluorescent dye or drug binding protein represented by.

(18) The fluorescent dye or drug-binding protein according to (17), wherein A in the general formulas (Ib) and (IIb) is a benzene ring.

(19) The fluorescent dye or drug-binding protein according to (17) or (18), wherein ^{R 2} in the general formulas (Ib), (IIb), and (IIIb) is a methyl group.

(20) In formula (Ib), either of (IIb), and R ^P in (IIIb) is characterized in that it is a group derived from an antibody comprising a tyrosine residue (17) to (19) Fluorescent dye or drug binding protein according to.

This specification includes the contents described in the specification and / or drawings of the Japanese patent application, Japanese Patent Application No. 2020-080345, which is the basis of the priority of the present application.

The present invention provides a novel compound that can be used to modify tyrosine. This compound is stable for a long time and can be used as an easy-to-use tyrosine modifier.

Photographs showing the state immediately after the preparation of Compound 2 and PTAD and 16 hours after the preparation. FIG. 6 regarding modification of peptide by compound 2. FIG. 2 regarding modification of peptide by compound 2. FIG. 3 regarding modification of peptide by compound 2. The figure regarding the modification of a peptide by compound 3. The figure regarding the modification of a protein by compound 3. Fluorescence spectrum of the fluorescent label antibody prepared in Example 8. The concentration of the fluorescent label antibody (trastuzumab labeled with BODIPY-581) in PBS (pH 7.4) was 25 nM, the excitation wavelength was 585 nm, and the measurement was performed at room temperature.

Hereinafter, the present invention will be described in detail.
(1) Definition In the present invention, the "alkyl group having 1 to 20 carbon atoms" is a linear or branched alkyl group having 1 to 20 carbon atoms, for example, a methyl group, an ethyl group, or n-propyl. Group, iso-propyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, pentyl group, iso-pentyl group, neo-pentyl group, hexyl group, iso-hexyl group, heptyl group , Iso-Heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecil group, icosyl group and the like.

In the present invention, the "alkyl group having 1 to 10 carbon atoms" is a linear or branched alkyl group having 1 to 10 carbon atoms, for example, a methyl group, an ethyl group, an n-propyl group, or iso-. Propyl group, n-butyl group, iso-butyl group, sec-butyl group, tert-butyl group, pentyl group, iso-pentyl group, neo-pentyl group, hexyl group, iso-hexyl group, heptyl group, iso-heptyl Group, octyl group, nonyl group, decyl group and the like.

In the present invention, the "alkyl group having 1 to 3 carbon atoms" is a linear or branched alkyl group having 1 or more and 3 or less carbon atoms, and is, for example, a methyl group, an ethyl group, an n-propyl group, or iso-. Such as a propyl group.

In the present invention, the "alkoxy group having 1 to 20 carbon atoms" is a linear or branched alkoxy group having 1 to 20 carbon atoms, for example, a methoxy group, an ethoxy group, an n-propoxy group, or iso-. Propoxy group, n-butoxy group, iso-butoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, iso-pentyloxy group, neo-pentyloxy group, hexyloxy group, iso-hexyloxy group, heptyl Oxy group, iso-heptyloxy group, octyloxy group, nonyloxy group, decyloxy group, undecyloxy group, dodecyloxy group, tridecyloxy group, tetradecyloxy group, pentadecyloxy group, hexadecyloxy group, heptadecyl Oxy group, octadecyloxy group, nonadesyloxy group, icosyloxy group and the like.

In the present invention, the "alkoxy group having 1 to 10 carbon atoms" is a linear or branched alkoxy group having 1 to 10 carbon atoms, for example, a methoxy group, an ethoxy group, an n-propoxy group, or iso-. Propoxy group, n-butoxy group, iso-butoxy group, sec-butoxy group, tert-butoxy group, pentyloxy group, iso-pentyloxy group, neo-pentyloxy group, hexyloxy group, iso-hexyloxy group, heptyl Oxy group, iso-heptyloxy group, octyloxy group, nonyloxy group, decyloxy group and the like.

In the present invention, the "alkoxy group having 1 to 3 carbon atoms" is a linear or branched alkoxy group having 1 or more and 3 or less carbon atoms, and is, for example, a methoxy group, an ethoxy group, an n-propoxy group, or iso-. It is a propoxy group.

In the present invention, the "alkyl group which may have a substituent" means an alkyl group which does not have a substituent or an alkyl group which has at least one substituent. The substituent may be any, and can be selected from the group consisting of, for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a hydroxy group, a methoxy group and the like. Further, the substituent may be a radionuclide or a functional group used in the click reaction.

In the present invention, the "aromatic group which may have a substituent" means an aromatic group which does not have a substituent or an aromatic group which has at least one substituent. Here, the "aromatic group" refers to a group obtained by removing one hydrogen atom from an aromatic compound, for example, a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a pyridine-2-yl group, and a pyridine-. 3-yl group, pyridine-4-yl group, pyrimidine-2-yl group, pyrimidine-4-yl group, pyrimidine-5-yl group, pyrazine-2-yl group, pyrazine-3-yl group, pyridazine-3 -Il group, pyridazine-4-yl group, furan-2-yl group, furan-3-yl group, thiophen-2-yl group, thiophen-3-yl group, pyrrol-1-yl group, pyrrol-2- Il group, pyrrol-3-yl group, pyrazole-1-yl group, pyrazole-3-yl group, pyrazole-4-yl group, pyrazole-5-yl group, imidazol-1-yl group, imidazol-2-yl group Groups, imidazol-4-yl groups, imidazol-5-yl groups, etc. The substituent may be any, and can be selected from the group consisting of, for example, a methyl group, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a hydroxy group, a methoxy group and the like. Further, the substituent may be a radionuclide or a functional group used in the click reaction. A suitable example of an aromatic group having at least one substituent can be a phenyl group having at least one substituent, and specific examples thereof include a 2-methylphenyl group. 3-Methylphenyl group, 4-Methylphenyl group, 2,3-dimethylphenyl group, 2,4-dimethylphenyl group, 2,5-dimethylphenyl group, 2,6-dimethylphenyl group, 3,4-dimethylphenyl Group, 3,5-dimethylphenyl group, 2-fluorophenyl group, 3-fluorophenyl group, 4-fluorophenyl group, 2,3-difluorophenyl group, 2,4-difluorophenyl group, 2,5-difluorophenyl Group, 2,6-difluorophenyl group, 3,4-difluorophenyl group, 3,5-difluorophenyl group, 2-chlorophenyl group, 3-chlorophenyl group, 4-chlorophenyl group, 2,3-dichlorophenyl group, 2, 4-Dichlorophenyl group, 2,5-dichlorophenyl group, 2,6-dichlorophenyl group, 3,4-dichlorophenyl group, 3,5-dichlorophenyl group, 2-bromophenyl group, 3-bromophenyl group, 4-bromophenyl group , 2,3-Dibromophenyl group, 2,4-dibromophenyl group, 2,5-dibromophenyl group, 2,6-dibromophenyl group, 3,4-dibromophenyl group, 3,5-dibromophenyl group, 2 -Iodophenyl group, 3-iodophenyl group, 4-iodophenyl group, 2,3-diiodophenyl group, 2,4-diiodophenyl group, 2,5-diiodophenyl group, 2,6-diiode Phenyl group, 3,4-diiodophenyl group, 3,5-diiodophenyl group, 2-hydroxyphenyl group, 3-hydroxyphenyl group, 4-hydroxyphenyl group, 2-methoxyphenyl group, 3-methoxyphenyl group , 4-Methenyl phenyl group and the like.

In the present invention, the "functional group used in the click reaction" is, for example, an azide group or an ethynyl group.

In the present invention, the "conjugated ring" means a ring having a conjugated double bond. The conjugated ring may be an aromatic ring or a non-aromatic ring. Further, the conjugated ring may be a ring composed of only carbon atoms or a heterocycle containing atoms other than carbon, for example, atoms such as nitrogen, oxygen and sulfur. Specific examples of the conjugated ring include a 6-membered ring such as a benzene ring, a 1,3-cyclohexadiene ring, a pyridine ring, a pyrimidine ring, a pyrazine ring, a pyridazine ring, and a triazine ring, a cyclopentadiene ring, a furan ring, a thiophene ring, and a pyrazole. Examples thereof include a 5-membered ring such as a ring, a pyrazole ring, and an imidazole ring.

In the present invention, the term "radionuclide" means, for example, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ⁶² Cu, ⁶⁸ Ga, ⁷⁶ Br, ^{99 m} Tc, ¹¹¹ In, ⁶⁷ Ga, ²⁰¹ Tl, ¹²³ I, ¹³³ Xe. And so on. ^{Among these, 18} F can be mentioned as a suitable radionuclide.

In the present invention, the "labeled substance" means a substance that enables detection of the protein by directly or indirectly binding to the protein, for example, interacting with a fluorescent substance, a radioactive nuclei, or a specific substance. It is a substance that does. Examples of the fluorescent substance include fluorescein, fluorescein isothiocyanate (FITC), and rodamine. Examples of the substance that interacts with a specific substance include antigens of low molecular weight antibodies such as biotin and dinitrophenyl groups. Examples thereof include substances having a structure capable of forming a covalent bond, such as HaloTag (registered trademark) and SNAP-tag (registered trademark), and substances having a molecular structure capable of forming a covalent bond.

In the present invention, the "halide ion" is, for example, a fluoride ion, a chloride ion, a bromide ion, an iodide ion, and an asstatide ion.

In the present invention, "N-halosuccinimide" is, for example, N-iodosuccinimide, N-bromosuccinimide, N-chlorosuccinimide.

(2) Compound The compound of the present invention is represented by the following general formula (I), (II) or (III).

In general formulas (I) and (II), A represents a conjugated ring. A may be a conjugated ring, but is preferably a benzene ring.

In the general formulas (I) and (II), R ¹ represents a hydrogen atom, or represents a radioactive nuclei, a functional group used in the click reaction, an amino group, an acetamide group, a hydroxy group, an alkyl group, or an alkoxy group. When R ¹ represents a radioactive nuclei or a functional group used in the click reaction, R ¹ is a single group located at any position on A, and R ¹ is an amino group, an acetamide group, a hydroxy group, an alkyl group. , Or when representing an alkoxy group, R ¹ is one or two groups present at any position on A. If R ¹ there are two on A, they may be the same or different. Also, the two R ¹ may be present in any position on the conjugated ring. The number of carbon atoms of the alkyl group and the alkoxy group is not particularly limited, but the number of carbon atoms is preferably 1 to 20, more preferably 1 to 10 carbon atoms, and particularly preferably 1 to 3 carbon atoms. preferable. R ¹ may be the above-mentioned group, but is preferably a hydrogen atom or one amino group, an acetamide group, or a methoxy group, and more preferably a hydrogen atom or one methoxy group. ..

In the general formulas (I), (II), and (III), R ² represents an alkyl group which may have a substituent or an aromatic group which may have a substituent. Here, the number of carbon atoms of the alkyl group is not particularly limited, but the number of carbon atoms is preferably 1 to 20, more preferably 1 to 10 carbon atoms, and particularly preferably 1 to 3 carbon atoms. preferable. R ² may be any of the above-mentioned groups, but is preferably a methyl group or a phenyl group, and more preferably a methyl group.

In the general formulas (I), (II), and (III), L represents a hydrogen atom, or a linker having a functional group used for a click reaction at the terminal or a linker having a labeling substance at the terminal. L may be at any position on A. The linker may have any structure as long as it does not lose the binding property of the functional group used in the click reaction and the function of the labeling substance, but is preferably an alkylene group. However, one or more -CH _2- of the alkylene group may be substituted with -O-, -S-, -NH-, or -CO-. The number of carbon atoms of the alkylene group is not particularly limited, but is preferably 4 to 20, and more preferably 4 to 10. _{Even when -CH 2-} in the alkylene group is replaced with -O-, -S-, or -NH-, it is assumed that these groups have one carbon, and the above-mentioned "number of carbon atoms of the alkylene group" is assumed. Included in.

Formula (I), (II), the and (III) X ^- represents a halide ion. X ^- is but may be a halide ion, is preferably a bromide ion.

Since the compound of the present invention specifically binds to a tyrosine residue in a protein, it can be used as a tyrosine modifier. Further, as will be described later, the compound of the present invention can be easily prepared from a precursor of the compound of the present invention (a compound represented by the general formula (Ia), (IIa), or (IIIa)) and N-halosuccinimide. Therefore, the precursor of the compound of the present invention and N-halosuccinimide can also be used as a tyrosine modification kit.

(3) Method for modifying tyrosine The method for modifying tyrosine of the present invention includes a step of reacting the compound of the present invention with a protein containing a tyrosine residue and binding the compound of the present invention to the tyrosine residue of the protein. It is a feature.

The protein to be used is not particularly limited as long as it contains a tyrosine residue, and a protein having a short chain length that is generally recognized as a "peptide" may be used instead of a "protein". As the protein, a receptor protein, an antibody, a protein having pharmacological activity (peptide) and the like can be used, and among these, an antibody is preferably used. An antibody-drug conjugate can be easily prepared by binding a compound of the present invention having a functional group used in a click reaction to an antibody, and further binding a small molecule drug to the functional group used in the click reaction. Because it can be done. As the antibody, an antibody used in an antibody-drug conjugate, for example, trastuzumab, which is an anti-HER2 antibody, faretsuzumab, which is an anti-FRα antibody, and the like can be used.

The amount of the compound of the present invention to be used is not particularly limited, but is usually 1 to 200 mol, preferably 3 to 100 mol, with respect to 1 mol of the protein to be modified.

The method for modifying tyrosine of the present invention is usually carried out in a solvent. The solvent used is not particularly limited as long as it does not inhibit the reaction, and water, acetonitrile, methanol, ethanol, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), dioxane and the like can be used. The pH of the solvent is not particularly limited, but is usually 6 to 8.5, preferably 7 to 8.

The temperature at the time of reaction is not particularly limited, but is usually 0 to 40 ° C, preferably 0 to 25 ° C. The reaction time is also not particularly limited, but is usually 1 to 60 minutes, preferably 1 to 10 minutes.

The compound of the present invention binds one or two to one tyrosine residue in a protein. The binding site is presumed to be a carbon atom adjacent to the carbon atom to which the hydroxy group on the benzene ring of the side chain of tyrosine is bonded.

The method for modifying tyrosine of the present invention has the following advantages.
1) PTAD, which is a known tyrosine modifier, is an unstable compound and causes a side reaction with an amino group other than a tyrosine residue, and the tyrosine modifying ability decreases with the passage of time. On the other hand, the compound used in the method for modifying tyrosine of the present invention is stable, and there is almost no decrease in tyrosine modifying ability or side reaction with the passage of time.
2) Tyrosine can be modified in a shorter time than the tyrosine modification methods described in JP-A-2016-108266 and JP-A-2020-2095.
3) The tyrosine modification method described in JP-A-2016-108266 uses hydrogen peroxide which may damage proteins, but the tyrosine modification method of the present invention does not require hydrogen peroxide.
4) The tyrosine modification method described in JP-A-2020-2095 requires a special device for modifying tyrosine by an electrochemical method, but the tyrosine modification method of the present invention does so. No equipment is required.

(4) Method for producing a compound The method for producing a compound of the present invention is the following general formula (Ia), (IIa), or (IIIa).

[In the formula, A, L, R ¹ , and R ² have the same meaning as above. ]
The compound represented by is reacted with N-halosuccinimide in a solvent, and the following general formula (I), (II), or (III)

[In the formula, A, L, R ¹ , R ² , and X ^- have the same meaning as above. ]
It is characterized by including a step of obtaining each of the compounds represented by.

The N-halosuccinimide used is not particularly limited, but it is preferable to use N-bromosuccinimide.

The solvent used is not particularly limited as long as it does not inhibit the reaction and does not destabilize the compound represented by the general formula (I), (II) or (III), and N, N-dimethylformamide, Acetonitrile, dioxane and the like can be used. Of these, it is preferable to use N, N-dimethylformamide.

The amount of the compound represented by the general formula (Ia), (IIa), or (IIIa) to be used is not particularly limited, but is usually 1 to 10 mol, preferably 1 to 2 mol, based on 1 mol of N-halosuccinimide. Is.

The temperature at the time of reaction is not particularly limited, but is usually 0 to 40 ° C, preferably 0 to 25 ° C. The reaction time is also not particularly limited, but is usually 0.5 to 20 minutes, preferably 1 to 10 minutes.

The produced compounds represented by the general formulas (I), (II), or (III) can be stored for a long time in a suitable solvent. As the solvent, N, N-dimethylformamide, acetonitrile, dioxane and the like can be used. Of these, it is preferable to use N, N-dimethylformamide. Further, storage is preferably carried out under low temperature conditions, for example, at -80 to 4 ° C, preferably -20 to 4 ° C.

(5) Fluorescent dye or drug-binding protein The fluorescent dye or drug-binding protein of the present invention is represented by the following general formula (Ib), (IIb), or (IIIb).

Wherein, Lb represents a linker having a fluorescent dye or drug at the end, R ^P represents a group derived from a protein containing tyrosine residues, A and R ² have the same meanings as described above. ]

Here, the "group derived from a protein containing a tyrosine residue" means, for example, a monovalent group obtained by removing one hydrogen atom in a protein molecule containing a tyrosine residue. The hydrogen atom to be removed is usually a hydrogen atom adjacent to the hydroxy group on the benzene ring contained in the tyrosine residue.

The "fluorescent dye" in Lb is an antibody sensor called "Q-body" (R. Abe, H. Ohashi, I. Iijima, M. Ihara, H. Takagi, T. Hohsaka and H. Ueda J. Am. It is preferable to use the fluorescent dye used in Chem. Soc. 133, 17386-17394 (2011)). Many fluorescent dyes that can be used for Q-body are known. For example, the fluorescent dyes described in WO2013 / 065314 and WO2020 / 026733 can be used. Specifically, rhodamine, coumarin, Cy, EvoBlue, oxazine, Carbopyronin, naphthalene, biphenyl, anthracene, phenenthrene, pyrene, carbazole, BODIPY (4,4-difluoro-4-bora-3a, 4a-diaza-s-indacene) ) And the like as a basic skeleton, and derivatives of the fluorescent dye can be exemplified. Specific examples of the fluorescent dye include CR110: carboxylrhodamine 110: Rhodamine Green (trade name), TAMRA: carbocytotremethlrhodamine: TMR, Carboxyrhodamine 6G: CR6G, ATTO655 (trade name), BODIPY FL (trade name): 4,4-difluoro- 5,7-dimethyl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 493/503 (trademark): 4,4-difluoro-1,3,5,7-tetramethyl- 4-bora-3a, 4a-diaza-s-indancene-8-propionicacid, BODIPY R6G (trade name): 4,4-difluoro-5- (4-phenyl-1,3-butadienyl) -4-bora-3a , 4a-diaza-s-indancene-3-propionic acid, BODIPY 558/568 (trade name): 4,4-difluoro-5- (2-thienyl) -4-bora-3a, 4a-diaza-s-indancene -3-propionic acid, BODIPY 564/570 (trademark): 4,4-difluoro-5-styryl-4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 576/589 (trademark) Name): 4,4-difluoro-5- (2-pyrrolyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY 581/591 (trade name): 4,4-difluoro -5- (4-phenyl-1, 3-butadienyl) -4-bora-3a, 4a-diaza-s-indancene-3-propionic acid, BODIPY TMR (trademark), Cy3 (trademark), Cy3B (trademark) Name), Cy3.5 (trademark name), Cy5 (trademark name), Cy5.5 (trademark name), EvoBlue10 (trademark name), EvoBlue30 (trademark name), MR121, ATTO390 (trademark name), ATTO425 (trademark name) Name), ATTO465 (trademark), ATTO488 (trademark), ATTO495 (trademark), ATTO520 (Trademark name), ATTO532 (Trademark name), ATTORho6G (Trademark name), ATTO550 (Trademark name), ATTO565 (Trademark name), ATTORho3B (Trademark name), ATTORho11 (Trademark name), ATTORho12 (Trademark name) Trademark name), ATTOThio12 (trademark name), ATTO610 (trademark name), ATTO611X (trademark name), ATTO620 (trademark name), ATTORho14 (trademark name), ATTO633 (trademark name), ATTO647 (trademark name) Name), ATTO647N (trademark name), ATTO655 (trademark name), ATTOOxa12 (trademark name), ATTO700 (trademark name), ATTO725 (trademark name), ATTO740 (trademark name), Alexa Fluor350 (trademark name) Name), Alexa Fluor 405 (trademark), Alexa Fluor 430 (trademark), Alexa Fluor 488 (trademark), Alexa Fluor 532 (trademark), Alexa Fluor 546 (trademark), Alexa Fluor 555 (trademark) , Alexa Fluor 568 (trademark), Alexa Fluor 594 (trademark), Alexa Fluor 633 (trademark), Alexa Fluor 647 (trademark), Alexa Fluor 680 (trademark), Alexa Fluor 700 (trademark), Alexa Fluor 750 (trademark name), Alexa Fluor 790 (trademark name), Rhodamine Red-X (trademark name), Texas Red-X (trademark name), 5 (6) -TAMRA-X (trademark name), 5TAMRA (trademark name) ), SFX (trade name) can be mentioned. Among these, CR110 and TAMRA, which are rhodomine-based fluorescent dyes, ATTO655, which is an oxazine-based fluorescent dye, and BODIPY FL, BODIPY R6G, BODIPY 558/568, BODIPY 581/591, and BODIPY TMR, which are BODIPY-based fluorescent dyes, are particularly preferable. It can be exemplified as a fluorescent dye.

As the "drug" in Lb, it is preferable to use a drug used in an antibody drug conjugate (ADC), for example, a cytotoxic drug (such as emtancin).

The "protein" in R ^P, antibodies are preferred. The antibody to be used is not particularly limited, and when Lb is a linker having a fluorescent dye at the end, the antibody used for Q-body is preferable, and when Lb is a linker having a drug at the end, it is used for ADC. Antibodies are preferred.

Specific examples of the antibody used in the Q-body include rituximab, which is an anti-CD20 antibody, trastuzumab, which is an anti-HER2 antibody, an antibody against a substance that is a marker of a disease, and an antibody against a low molecular weight compound. Here, specific examples of low molecular weight compounds include stimulants and drugs such as amphetamine, methanephetamine, morphine, heroine, and codeine, aflatoxin, sterigmatocystin, neosolaniol, nivalenol, fumonicin, okratoxin, and endophyte production. Mold poisons such as toxins, sex hormones such as testosterone and estradiol, additives used illegally in feeds such as clembuterol and lactopamine, harmful substances such as PCB, gosipole, histamine, benzpyrene, melamine, acrylamide, dioxin, acetamiprid, imidacloprid, Residual pesticides such as chlorphenapir, malathion, carbalyl, clothianidin, triflumizole, chlorotalonyl, spinosad, lannate, methamidophos, chlorpyrifos, and environmental hormones such as bisphenol A.

Specific examples of the antibody used in the ADC include the above-mentioned rituximab and trastuzumab.

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

[Example 1] Preparation of modifier solution

1: 1 DMF solution (concentration: 200 mM) of 1-methyl-4-allylurazole (MAUra) or N-methylluminol derivative (N-Me-Lumi) and DMF solution of NBS (concentration: 200 mM) The mixture was mixed at a ratio, stirred with a vortex mixer, and allowed to stand at room temperature for 5 minutes to prepare a solution containing a modifier (Compound 1 or Compound 2 above). With the formation of the azo structure, the reaction solution changed from transparent to yellow. The resulting yellow modifier solution was stored on ice until use.

[Example 2] Stability of modifier The modifier (compound 2) solution prepared in Example 1 was allowed to stand at room temperature for 16 hours, and the state after standing was observed. No change was seen (Fig. 1, bottom). For comparison, the DMF solution of PTAD was allowed to stand at room temperature for 16 hours, and the state after standing was observed. The color of the solution changed significantly from red to brown (Fig. 1, top).

[Example 3] Peptide modification Peptide Angiotensin II (sequence: DRVYIHPF) was dissolved in 50 mM Tris buffer (pH 7.4) at a concentration of 100 μM, and the modifier immediately after preparation (Compound 2) was shaken with a tube shaker. ) Was added to a final concentration of 1 mM (10 eq) (from 100 mM DMF solution). After allowing to stand at room temperature for 20 minutes, trifluoroacetic acid was added to a final concentration of 0.1%. The product was analyzed by MALDI-TOF MS (Fig. 2). As shown in FIG. 2, a peak of Angiotensin II in which tyrosine was modified by a modifier was detected around 1400 m / z. It is presumed that two modifiers are attached at positions adjacent to the hydroxy group on the benzene ring of the side chain of tyrosine.

[Example 4] Peptide modification (elapsed time from preparation)
In addition to the modifier (Compound 2) immediately after preparation, a modifier after standing at room temperature for 3 hours or 16 hours after preparation is used, and the others are reacted with the peptide Angiotensin II in the same manner as in Example 3 to MALDI. -The product was analyzed by TOF MS (Fig. 3). For comparison, the product was analyzed in the same manner using PTAD instead of the modifier (Fig. 3).

As shown in FIG. 3, a large peak was detected around 1400 m / z when the modifier immediately after preparation was used, and 1220 m / z when the modifier after standing for 3 hours or standing for 16 hours was used. A large peak was detected in the vicinity. The peak near 1400 m / z indicates Angiotensin II (double mod. (Y)) in which two modifiers are bound, and the peak near 1220 m / z indicates Angiotensin II (single mod. (Y)) in which one modifier is bound. ) Is presumed to be shown.

On the other hand, when PTAD was used, large peaks were detected near 1220 m / z and around 1050 m / z, but the peaks around 1220 m / z became smaller in PTAD after standing for 3 hours, and after standing for 16 hours. It almost disappeared in PTAD. The peak near 1220 m / z indicates Angiotensin II (single mod. (Y)) with one PTAD bound, and the peak near 1050 m / z indicates Angiotensin II (angiotensin II (SM)) without PTAD. It is presumed. In addition, peaks presumed to indicate Angiotensin II with PTAD bound to sites other than tyrosine (“single mod. (Y) + PhNCO mod. (NH ₂ )” and “PhNCO mod. (NH ₂ )” in the figure. ) Was also detected.

[Example 5] Peptide modification (equivalent)
Change the equivalent of the modifier (Compound 2) (1 equivalent, 3 equivalents, or 10 equivalents), react the modifier with the peptide Angiotensin II, and analyze the product with MALDI-TOF MS, as in Example 3. (Fig. 4). As shown in FIG. 4, a large peak was detected near 1050 m / z when 1 equivalent of the modifier was added, and around 1050 m / z, around 1300 m / z, and around 1300 m / z when 3 equivalents of the modifier were added. A large peak was detected near 1400 m / z, and when 10 equivalents of the modifier was added, a large peak was detected only around 1400 m / z. The peak near 1050 m / z indicates Angiotensin II with no modifier bound, the peak near 1300 m / z indicates Angiotensin II with one modifier bound, and the peak near 1400 m / z shows two modifiers. Presumed to indicate combined Angiotensin II.

[Example 6] Peptide modification (modifier having an azide group)
The following compound (Compound 3) in which an azide group is bonded to Compound 2 via a linker instead of Compound 2.

Was used as a modifier, and the modifier was reacted with the peptide Angiotensin II in the same manner as in Example 3, and the product was analyzed by MALDI-TOF MS (Fig. 5). As shown in FIG. 5, in addition to the peak near 1050 m / z, which is presumed to indicate Angiotensin II to which the modifier is not bound, a large peak was detected near 1440 m / z. This peak is presumed to indicate Angiotensin II bound to one modifier. Further, it is presumed that the modifier is bonded to a position adjacent to the hydroxy group on the benzene ring of the side chain of tyrosine, and a bromine atom is bonded to the other adjacent position.

[Example 7] Modification of protein The protein was dissolved in 50 mM Tris buffer (pH 7.4) at a concentration of 5 μM, and the modifier (Compound 3) was added to a final concentration of 300 μM while shaking with a tube shaker. Was added (from a 30 mM DMF solution). Eight kinds of proteins, BSA, SAv, peanut agglutinin, wheat germ agglutinin, Concanavalin A, Carbonic anhydrase, Beta-lactoglobulin A, and RNAase A, were used. After allowing to stand at room temperature for 20 minutes, a small molecule compound component derived from a modifier other than protein was removed using a Sephadex G-25 gel column (GE Healthcare) (elution conditions from gel: 2000 × g, 4 min). DBCO-Cy3 was added to a final concentration of 10 μM, shaken at 37 ° C for 30 minutes, and then unreacted DBCO-Cy3 was removed using a Sephadex G-25 gel column (GE Healthcare) (from the gel). Dissolution conditions: 2000 × g, 4 min). Modification was confirmed by separating the protein by SDS-PAGE and detecting Cy3 bound to the protein (Fig. 6).

As shown in FIG. 6, fluorescence of Cy3 was detected in all the proteins used in the experiment, and the binding of the modifier to the protein was confirmed.

[Example 8] Preparation of HER2 antibody Q-body (A) Method (1) Preparation of DBCO-BODIPY581

1 μL of 10 mM NHS-BODIPY581, 2 μL of 10 mM DBCO-amine, 2 μL of 10 mM DIEA, and 5 μL of DMF were mixed and stirred at room temperature for 1 hour while shading.

(2) Activation of MAUra-N ₃

2 μL of 100 mM MAUra-N ₃ (DMSO solution) and 2 μL of 100 mM N-bromosuccinimide (DMF solution) were mixed and incubated at room temperature for 5 minutes. 2.6 μL of DMF was added to bring the final concentration of _{MAUra-N 3 to 30 mM.}

(3) Fluorescent Labeling of Antibodies 1 μL of 30 mM MAUra-N ₃ prepared in (2) was added to 100 μL of 5 μM trastuzumab (50 mM Tris-HCl buffer, pH 7.4) and incubated for 20 minutes at room temperature. The unreacted small molecules were then removed on a Sephadex G-25 column. To the obtained azide antibody, 1 μL of 1 mM DBCO-BODIPY581 prepared in (1) was added, and the mixture was shaken at 37 ° C. for 30 minutes. The unreacted fluorescent dye was then removed on a Sephadex G-25 column.

(4) Antibody-antibody reaction and fluorescence measurement The 10 μL 5 μM fluorescent label antibody prepared in (3), SKBR3 cell disruption solution, and PBS were mixed so that the final volume was 2 mL, and the fluorescence spectrum was measured with a fluorometer. SKBR cells were prepared to a final concentration of 37.5 or 75 μg / mL. The conditions for the fluorescence spectrophotometer are as follows.
Cell: Polystyrene Disposable Cell Excited Bandwidth: 5 nm
Fluorescent bandwidth: 5 nm
Response: 50msec
Sensitivity: Medium
Measuring range: 570-650nm
Data acquisition interval: 0.2nm
Excitation wavelength: 585.0 nm
Scanning speed: 50 nm / min
Number of repetitions: 1

(B) Results The fluorescence spectrum of the fluorescent label antibody prepared in (3) is shown in FIG. As shown in FIG. 7, the fluorescence intensity was increased by the addition of the SKBR3 cell (cell that highly expresses HER2, which is the target of trastuzumab) disruption solution. (Fluorescence intensity in the presence of cell disruption solution / fluorescence intensity in the absence of cell disruption solution) at 594 nm was 4.4 (cell disruption solution concentration: 37.5 μg / mL) or 5.9 (cell disruption solution concentration: 75 μg / mL). rice field. This result indicates that the quenching was canceled by the addition of the antigen, and suggests that the fluorescent label antibody functioned as a Q-body.

All publications, patents and patent applications cited in this specification shall be incorporated herein by reference as is.

Since the present invention is useful for producing pharmaceuticals such as antibody drug conjugates, it can be used in the industrial field related to the production of such pharmaceuticals.

Claims (20)

1. The following general formula (I), (II), or (III)
   

   

   [In the formula, A represents a conjugated ring and L represents a hydrogen atom, or a linker having a functional group used for a click reaction at the terminal or a linker having a labeling substance at the terminal (where L is on A). It may be present at any position), where R ¹ represents a hydrogen atom or represents a radioactive nuclei, a functional group used in the click reaction, an amino group, an acetamide group, a hydroxy group, an alkyl group, or an alkoxy group (representing a radioactive nuclei, an amino group, an acetamide group, a hydroxy group, or an alkoxy group ( However, R ¹ may be present at any position on A), R ² represents an alkyl group which may have a substituent or an aromatic group which may have a substituent, and X represents an aromatic group. ^- represents a halide ion. ]
   The compound represented by.
2. The compound according to claim 1, wherein A in the general formulas (I) and (II) is a benzene ring.
3. The compound according to claim 1 or 2, wherein ^{R 2} in the general formulas (I), (II), and (III) is a methyl group.
4. General formula (I), (II), X in and (III) ^- A compound according to any one of claims 1 to 3, characterized in that the bromide ion.
5. A tyrosine modifier containing the compound according to any one of claims 1 to 4.
6. A method for modifying tyrosine, which comprises a step of reacting the compound according to any one of claims 1 to 4 with a protein containing a tyrosine residue and binding this compound to the tyrosine residue of the protein.
7. The compound represented by the general formulas (I), (II), and (III), which comprises storing the compound according to any one of claims 1 to 4 in N, N-dimethylformamide. Preservation method.
8. The following general formulas (Ia), (IIa), or (IIIa)
   

   

   [In the formula, A represents a conjugated ring and L represents a hydrogen atom, or a linker having a functional group used for a click reaction at the terminal or a linker having a labeling substance at the terminal (where L is on A). It may be present at any position), where R ¹ represents a hydrogen atom or represents a radioactive nuclei, a functional group used in the click reaction, an amino group, an acetamide group, a hydroxy group, an alkyl group, or an alkoxy group (representing a radioactive nuclei, an amino group, an acetamide group, a hydroxy group, or an alkoxy group ( However, R ¹ may be present at any position on A), and R ² represents an alkyl group which may have a substituent or an aromatic group which may have a substituent. ]
   The compound represented by is reacted with N-halosuccinimide in a solvent, and the following general formula (I), (II), or (III)
   

   

   Wherein, A, L, R ^1, and R ² are as defined above, X ^- represents a halide ion. ]
   A method for producing a compound represented by the general formula (I), (II), or (III), which comprises a step of obtaining each of the compounds represented by.
9. The method for producing a compound represented by the general formula (I), (II), or (III) according to claim 8, wherein the solvent is N, N-dimethylformamide.
10. The general formula (I), (II), or the general formula (II), according to claim 8 or 9, wherein A in the general formulas (Ia), (IIa), (I), and (II) is a benzene ring. A method for producing the compound represented by (III).
11. Any one of claims 8 to 10, wherein ^{R 2} in the general formulas (Ia), (IIa), (IIIa), (I), (II), and (III) is a methyl group. A method for producing a compound represented by the general formula (I), (II), or (III) described in 1.
12. N- halosuccinimide is N- bromo are succinimides, formula (I), X in (II), and (III) ^- is any one of claims 8 to 11, characterized in that the bromide ion A method for producing a compound represented by the general formula (I), (II), or (III) described in item 1.
13. The following general formulas (Ia), (IIa), or (IIIa)
    

    

    [In the formula, A represents a conjugated ring and L represents a hydrogen atom, or a linker having a functional group used for a click reaction at the terminal or a linker having a labeling substance at the terminal (where L is on A). It may be present at any position), where R ¹ represents a hydrogen atom or represents a radioactive nuclei, a functional group used in the click reaction, an amino group, an acetamide group, a hydroxy group, an alkyl group, or an alkoxy group (representing a radioactive nuclei, an amino group, an acetamide group, a hydroxy group, or an alkoxy group ( However, R ¹ may be present at any position on A), and R ² represents an alkyl group which may have a substituent or an aromatic group which may have a substituent. ]
    A tyrosine modification kit comprising the compound represented by and N-halosuccinimide.
14. The tyrosine modification kit according to claim 13, wherein A in the general formulas (Ia) and (IIa) is a benzene ring.
15. The tyrosine modification kit according to claim 13 or 14, wherein ^{R 2} in the general formulas (Ia), (IIa), and (IIIa) is a methyl group.
16. The tyrosine modification kit according to any one of claims 13 to 15, wherein N-halosuccinimide is N-bromosuccinimide.
17. The following general formula (Ib), (IIb), or (IIIb)
    

    

    [In the formula, A represents a conjugated ring, Lb represents a linker having a fluorescent dye or drug at the end (where Lb may be present at any position on A), and R ² has a substituent. represents an aromatic group optionally having also an alkyl group or a substituent are, R ^P represents a group derived from a protein containing tyrosine residues. ]
    Fluorescent dye or drug binding protein represented by.
18. The fluorescent dye or drug-binding protein according to claim 17, wherein A in the general formulas (Ib) and (IIb) is a benzene ring.
19. The fluorescent dye or drug-binding protein according to claim 17 or 18, wherein ^{R 2} in the general formulas (Ib), (IIb), and (IIIb) is a methyl group.
20. Formula (Ib), according to R ^P is, any one of claims 17 to 19, characterized in that a group derived from an antibody comprising a tyrosine residue in (IIb), and (IIIb) Fluorescent dye or drug binding protein.

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