JP5219144B2 - Novel affinity labeling method and screening method using the labeling method - Google Patents

Description

  The present invention relates to a novel affinity labeling method for a target polymer compound, and relates to a novel affinity labeling method capable of labeling the polymer compound when a substance capable of interacting with the target polymer compound reacts. More specifically, the present invention relates to a novel affinity labeling method using a catalytic condensation reaction. The present invention also relates to a screening method for a target polymer compound and a substance capable of interacting by using the affinity labeling method.

  Macromolecular compounds such as functional proteins such as receptors, antibodies, and enzymes can interact with specific compounds (ligand compounds) and exhibit unique functions. As one of the methods to analyze what kind of polymer compound a specific compound interacts with and to which part of the polymer compound a specific compound binds and interacts, a ligand compound is labeled as a labeling substance. There is an affinity labeling method using a ligand derivative modified with the above. A target polymer compound can be screened from among many polymer compounds by an affinity labeling method in which a ligand derivative and a target polymer compound are cross-linked by a chemically stable covalent bond.

  In the case of labeling using a ligand derivative, if the ligand compound stably binds to the target polymer compound, labeling can be easily performed, but when the ligand compound interacts reversibly. Cannot perform stable labeling. Therefore, in order to perform stable labeling, a photocatalyst in which a photoreactive group is further introduced into the ligand derivative may be used. Since the photoreactive group can be stably bonded to the target polymer compound by light irradiation, the target polymer compound can be labeled stably. However, conventionally, (A) a ligand compound, (B) a labeling substance, and (C) a photoreactive group have to be the same molecule, but synthesis of a compound containing them has not been easy. In addition, the reactivity varies depending on the combination, but since the synthesis is not easy, the combination cannot be easily changed. For this reason, it is not easy to examine the optimal combination, and it has never been easy to screen the ligand compound or the target polymer compound, and to analyze to which site of the target polymer compound the ligand compound can bind.

  In the enzyme reaction, the substrate is taken into the substrate binding site of the active site and fixed at an appropriate position with respect to the catalytic group. In the artificial enzyme, various host compounds are used to capture the substrate. For example, artificial enzymes using cyclodextrin derivatives in which various functional groups are introduced into cyclodextrin have been reported (Non-Patent Documents 1 and 2). Here, a cyclodextrin derivative is used as an artificial enzyme, and 2-chloro-4,6, -dimethoxy-1,3,5-triazine (CDMT) is reacted with a cyclodextrin modified with a dimethylamino group, followed by dehydration condensation. It is disclosed that the reacted product is used as an artificial enzyme. By reacting the prepared artificial enzyme with a substrate modified with a carboxyl group, and further reacting various amines with the artificial enzyme, the reaction between the carboxyl group of the substrate which is easily included in the cyclodextrin and the amine proceeds. Thereby, the reaction of the substrate-specific artificial enzyme can be confirmed. However, there is no report of applying the above CDMT to the affinity labeling method.

  Water-soluble quaternary ammonium salt 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) obtained by replacing chlorine of CDMT, which is one of the above-mentioned triazine derivatives, with N-methylmorpholine (NMM) ) -4-Methylmorpholinium chloride (DMT-MM) has been reported (Patent Documents 1 and 2). Here, the water-soluble property of DMT-MM can be obtained by converting the fat-soluble CDMT into a non-volatile salt of DMT-MM, and a dehydrating condensing agent that is stable in water was obtained. However, there is no report that applies to the method of affinity labeling for the dehydrating condensing agent that is similarly stable in water obtained from the CDMT.

As described above, conventionally, a method for affinity labeling a polymer compound is not easy, and a simpler method has been desired.
International Publication WO2000 / 053544 Pamphlet International Publication WO2005 / 075442 Pamphlet J. Am. Chem. Soc., 123, pp.10760-10761 (2001) Angew. Chem. Int. Ed, 45, pp.1252-1255 (2006)

  The present invention relates to a novel affinity labeling method for a target polymer compound, and an object thereof is to provide a simple and effective labeling method. Furthermore, the present invention provides a screening method for a target polymer compound or a substance that can interact with the target polymer compound (hereinafter also simply referred to as “ligand compound”) by using the affinity labeling method. Let it be an issue.

  As a result of intensive studies to solve the above problems, the present inventors have reacted a ligand compound and a labeled compound together with a triazine-type dehydrating condensing agent to bind the labeled substance to the target polymer compound by a dehydrating condensation reaction. Thus, the present inventors have found that the target polymer compound can be effectively labeled when the target polymer compound reacts with the ligand compound, thereby completing the present invention.

The present invention comprises the following.
1. A method for affinity labeling of a target polymer compound comprising the following steps:
1) a step of modifying a compound (ligand compound) capable of interacting with the target polymer compound with a tertiary amine;
2) mixing the target polymer compound and the ligand compound modified with the tertiary amine;
3) a step of further reacting the 1,3,5-triazine compound with the target polymer compound;
4) A step of reacting an amine compound further containing a labeling site in the molecular structure.
2. The affinity labeling method according to item 1, wherein the 1,3,5-triazine compound is a compound represented by the following formula I:
[Wherein, R ¹ and R ² each independently represent a hydrogen atom, an alkyl group, a hydroxyalkyl group, a sulfo group, a phospho group, or —R ³ R ⁴ (where R ³ represents — (C _m H _2m O) _n and R ⁴ is a hydrogen atom, a sulfo group, a phospho group, or an alkyl group having an amino group, an ammonio group, a sulfuric acid group or a phosphoric acid group, and m is an integer of 1 to 30 And n is an integer of 1 to 120), and X is a chlorine, fluorine, bromine, iodine, trifluoromethanesulfonyloxy group or N-methylmorpholine (NMM). Is done. ]
3. The affinity labeling method according to item 1, wherein the 1,3,5-triazine compound is 2-chloro-4,6-dimethoxy-1,3,5-triazine compound (CMDT).
4). 4. The affinity labeling method according to any one of items 1 to 3, wherein the tertiary amine is a compound represented by the following formula II.
Wherein one or two of R ^5, R ⁶ and R ⁷ represents a substituent containing a functional group capable of interacting with the target polymer compound, and the remaining R ^5, R ⁶ and R ⁷ , Each independently an alkyl group, an alkenyl group, —CH ₂ COOR ⁸ , CH ₂ CONR ⁹ R ¹⁰ (where R ⁸ is a hydrogen atom or an alkyl group, R ⁹ is a hydrogen atom, an alkyl group, or — ( C _y H _2y O) _z , R ¹⁰ is a hydrogen atom, an alkyl group, an alkenyl group or an alkynyl group, y is an integer of 1 to 30, and z is an integer of 1 to 120)]
5. 5. The affinity labeling method according to any one of items 1 to 4, wherein the amine compound is represented by R ¹¹ R ¹² NH.
[One or two of R ¹¹ and R ¹² are a labeling functional group, and the rest are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms, an aryl group, an ammonio group, a sulfo group, An alkyl group having a sulfate group or a phosphate group. ]
6). 6. The affinity labeling method according to any one of 1 to 5 above, wherein the target polymer compound has a carboxyl group or is a compound modified with a carboxyl group.
7). 7. The affinity labeling method according to item 6 above, wherein the target polymer compound is a protein or peptide compound.
8). 8. A screening method for a target polymer compound using the affinity labeling method according to any one of 1 to 7 above.
9. 8. A screening method for a ligand compound using the affinity labeling method according to any one of 1 to 7 above.
10. A kit for affinity labeling of a target polymer compound comprising a 1,3,5-triazine compound and an amine compound containing a labeling site in the molecular structure.
11. 11. The kit for affinity labeling of a target polymer compound according to item 10, further comprising a ligand compound modified with a tertiary amine.

  According to the affinity labeling method of the present invention, a target polymer compound can be easily labeled without synthesizing a compound containing (A) a ligand compound, (B) a labeling substance, and (C) a photoreactive site as in the past. can do. The compounds containing (A), (B), and (C) are not only difficult to synthesize, but also difficult to change their combinations, and can only be labeled within a limited range. . According to the affinity labeling method of the present invention, the ligand compound and the labeling substance can be reacted with different molecules, and the above-described synthesis problem can be overcome. Thereby, it is possible to examine the labeling compounds in combination with each other and to perform labeling effectively.

  By the method of the present invention, a target polymer compound that interacts with a ligand compound can be easily screened from a sample in which various polymer compounds such as proteins are mixed. In addition, when various candidate compounds exist as ligand compounds with respect to the polymer compound, it interacts with the target polymer compound by reacting each candidate ligand compound individually by the affinity labeling method of the present invention. The ligand compound to be screened can be easily screened.

  Furthermore, by the method of the present invention, an acidic amino acid residue located spatially close to the ligand binding site of the target polymer compound can be identified. Further, by changing the modification site of the ligand compound, the chemical structure of the target polymer compound necessary for binding of the ligand compound can be known.

  In the present invention, the target polymer compound and the compound capable of interacting with the target polymer compound include, for example, a protein and a ligand compound, and more specifically, an enzyme and a substrate, a receptor and an agonist or an antagonist, and the like. Can be mentioned. The present invention is an affinity labeling method when a target polymer compound interacts with the above ligand compound. Affinity labeling can be performed by modifying a ligand compound with a tertiary amine, mixing the target polymer compound and the modified ligand compound, performing a catalytic condensation reaction, and mixing the labeled compound.

  The present invention relates to a method for affinity labeling a target polymer compound comprising the following steps 1) to 4). A schematic diagram as a representative example for carrying out the labeling method of the present invention is shown in FIGS. FIG. 3 shows a schematic diagram when the target polymer compound is a protein receptor on the cell membrane surface and is labeled when interacting with its ligand.

1) a step of modifying a compound (ligand compound) capable of interacting with a target polymer compound with a tertiary amine;
2) a step of mixing the target polymer compound and the ligand compound modified with the tertiary amine;
3) reacting the 1,3,5-triazine compound with the target polymer compound;
4) A step of reacting an amine compound containing a labeling site in the molecular structure.

  The 1,3,5-triazine compound used in the affinity labeling method of the present invention can be synthesized by a method known per se, for example, by the method described in Patent Document 1 or 2. The obtained 1,3,5-triazine compound can be separated and purified by means usually used by those skilled in the art. For example, after completion of the reaction, an organic solvent can be added to the reaction solution, and the resulting 1,3,5-triazine compound can be extracted into the organic layer or purified by a fractionation method known per se, such as a chromatograph.

In the present invention, the 1,3,5-triazine compound is preferably water-soluble, but may be amphiphilic or fat-soluble. Specifically, it is a compound represented by the following formula I.
[Wherein, R ¹ and R ² each independently represent a hydrogen atom, an alkyl group, a hydroxyalkyl group, a sulfo group, a phospho group, or —R ³ R ⁴ (where R ³ represents — (C _m H _2m O) _n and R ⁴ is a hydrogen atom, a sulfo group, a phospho group, or an alkyl group having an amino group, an ammonio group, a sulfuric acid group or a phosphoric acid group, and m is an integer of 1 to 30 And n is an integer of 1 to 120), and X is a chlorine, fluorine, bromine, iodine, trifluoromethanesulfonyloxy group or N-methylmorpholine (NMM). Is done. ]

  In the above, each of the alkyl group, hydroxyl group and alkoxyl group can have 1 to 30 carbon atoms, more preferably 1 to 20, and particularly preferably 1 to 6.

Preferably, R ¹ and R ² are each independently a hydrogen atom, a methyl group, an ethyl group, a hydroxyalkyl group having 2 to 5 carbon atoms, — (CH ₂ CH ₂ O) _n R ¹² (where n Is an integer of 1 to 120, and R ¹² is a hydrogen atom, a methyl group, an ethyl group, a propyl group, a sulfo group, or an alkyl group having an amino group, an ammonio group, a sulfo group, a sulfate group, a phosphate group, or the like. ), — (CH ₂ CH ₂ NR ¹³ ) _n H (where n is an integer from 1 to 120, and R ¹³ is an alkyl group having 2 to 5 carbon atoms, N, N-dialkylamino ethyl group, or a ^{_{- (CH 2 CH 2 N +}} (CH 3) a _{3), - CH 2 CH 2} SO 3 -, -CH 2 CH 2 N + (CH 3) is _3, X is chlorine, fluorine , Bromine, iodine, trifluoromethanesulfonyloxy group or N-methylmorpholine (NMM), most preferably 1,3,5-triazine compound is 2-chloro-4,6-dimethoxy-1,3 ,Five- Triazine compound (CMDT) or quaternary ammonium salt 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM). The 3,5-triazine compound can be synthesized by a method known per se, for example, by the method described in Patent Document 1 or 2.

  In the present invention, the tertiary amine is a compound represented by the following formula II. Tertiary amines are used to modify ligand compounds. Modification is by introducing a tertiary amine via a covalent bond at an appropriate site in the molecular structure of the ligand compound. When the ligand compound interacts with the target polymer compound, the 1,3,5-triazine compound bonded to the tertiary amine in the ligand compound reacts with the target polymer compound present nearby, The dehydration condensation reaction of the target polymer compound and the amine compound with the 1,3,5-triazine compound is effectively advanced.

Wherein one or two of R ^5, R ⁶ and R ⁷ represents a substituent containing a functional group capable of interacting with the target polymer compound, and the remaining R ^5, R ⁶ and R ⁷ , Each independently an alkyl group, an alkenyl group, —CH ₂ COOR ⁸ , CH ₂ CONR ⁹ R ¹⁰ (where R ⁸ is a hydrogen atom or an alkyl group, R ⁹ is a hydrogen atom, an alkyl group, or — ( C _y H _2y O) _z , R ¹⁰ is a hydrogen atom, an alkyl group, an alkenyl group or an alkynyl group, y is an integer of 1 to 30, and z is an integer of 1 to 120)]
Here, the alkyl group may be saturated or may be unsaturated having several double bonds or triple bonds.

Preferably, one or two of R ⁵ , R ⁶ and R ⁷ are substituents containing a functional group capable of interacting with the target polymer compound, and these are substituents of —CH ₂ COOR ¹³ or —CH ₂ CNMeR ¹³ It is preferably bonded to an amino group in the form. That is, R ¹³ is a ligand moiety), and the remaining R ⁵ , R ⁶ and R ⁷ are each independently a methyl group, —CH ₂ COOR ¹³ , or —CH ₂ CNR ⁸ R ¹³ (where R ⁸ is A hydrogen atom or an alkyl group). For example, when the ligand is not bonded to an amino group in the form of —CH ₂ COOR ¹³ or the like, it is preferable that these functional groups are contained. This is to make it difficult to become ammonium by lowering the basicity of the tertiary amine slightly, and as a result, to easily react with the 1,3,5-triazine compound.

In the present invention, the amine compound refers to a compound represented by R ¹¹ R ¹² NH.
One or two of R ¹¹ and R ¹² are a labeling functional group, and the rest are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms, an aryl group, an ammonio group, a sulfo group, a sulfuric acid group Or an alkyl group having a phosphate group or the like.
Preferably, R ¹¹ is a labeling compound, R ¹² is hydrogen, and the labeling compound is most preferably a fluorescent compound from the viewpoint of sensitivity and handling.

In the present invention, the labeling compound may be any compound that can be contained in the molecular structure of the amine compound, and a known compound can be used. For example, a fluorescent compound, a luminescent compound, a radioactive compound, etc. are mentioned. Specific examples of fluorescent compounds include Alexa Fluor, BODIPY FL, Cascade Blue, FITC, Oregon Green, RITC, Texas Red, TRITC, Coumarin Maleimide, Cy Dye, Dansyl Chloride, Dansyl Hydrazine, etc. ³ H, ¹⁴ C, ¹¹ C, ¹²³ I, ²⁰¹ TI, ⁶⁷ Ga and the like.

  In the present invention, the target polymer compound is a compound having a carboxyl group or an acidic amino acid side chain having a carboxyl group such as an aspartic acid or glutamic acid residue, such as a protein or peptide, or a carboxyl terminal. It is necessary to be. In the present invention, labeling of the target polymer compound is performed, for example, to select a ligand compound or to select a target polymer compound that interacts with the ligand compound.

  When there are multiple types of candidate compounds that can be ligands, if any of the candidate compounds interacts with the target polymer compound and is labeled, it can interact by detecting the signal from the labeling. Compounds, so-called ligand compounds can be selected. In addition, when a plurality of polymer target compounds are present with respect to the ligand compound, the polymer target compound that can interact with the ligand compound can also be detected. Furthermore, it is possible to specify an acidic amino acid residue that is spatially adjacent to the ligand compound binding site of the target polymer compound. Therefore, by changing the modification site of the ligand compound, the chemical structure of the polymer target compound necessary for binding of the ligand compound can be known.

  Hereinafter, the steps 1) to 4) in the method for affinity labeling a target polymer compound of the present invention will be described in detail. In the above, further necessary processing steps may be provided before and after each step. In addition, steps 3) and 4) may be reversed in order as necessary, or may be performed simultaneously.

1) Step of modifying the target polymer compound and the ligand compound with a tertiary amine In the present invention, the interaction of the ligand compound with the target polymer compound means a reversible and binding non-covalent interaction, It may be a case where it binds as a ligand. The affinity labeling of the present invention is effectively performed when the ligand compound interacts with the target polymer compound.

  In the present invention, a compound obtained by modifying a ligand compound with a tertiary amine is hereinafter referred to as “modified ligand compound”. In the modification of the ligand compound with a tertiary amine, the introduction site and the introduction method are appropriately determined depending on the chemical structure.

2) Step of mixing the target polymer compound and the modified ligand compound In the present invention, the step of mixing the target polymer compound and the modified ligand compound is performed by mixing the modified ligand compound in the target polymer compound aqueous solution, This is due to a certain period of time by stirring, standing, incubation, etc. until the equilibrium state of bond dissociation is reached. The solvent of the aqueous solution, the concentration ratio of the target polymer compound and the modified ligand compound, the temperature, and the time can be appropriately determined for each compound. Here, it is preferable to add one type of modified ligand compound to one container.

  For example, when a target polymer compound that can interact with a certain ligand compound is mixed with multiple types of polymer compounds, the modified polymer compound is reacted with the target polymer compound in the same process in the same step. be able to. On the other hand, when screening and selecting a ligand compound capable of binding to a target polymer compound from a plurality of candidate ligand compounds, each ligand compound is individually modified and subjected to the above steps in each reaction vessel. A modified ligand compound can be reacted with a target polymer compound. For each container, a more effective ligand compound can be selected by measuring a signal due to labeling.

3) Step of reacting 1,3,5-triazine compound In the present invention, by the step of reacting 1,3,5-triazine compound, 1,3,5-triazine compound is a tertiary compound in the modified ligand compound. Bonds to amine to become a dehydration condensing agent. When the carboxylic acid in the target compound is present in the vicinity of the 1,3,5-triazine, the triazine ring is attacked, and the tolidine is transferred from the tertiary amine to the carboxylic acid. As a result, the carboxylic acid is activated in the form of an active ester (referred to as acyloxytriazine, triazino ester, or triazinyl ester), and finally an amine compound containing a labeling site in the molecular structure attacks the active ester. And an amide bond.

  This step is by adding a triazine compound aqueous solution or a methanol solution containing a triazine compound to the aqueous solution obtained by the above step 2) or 4) below. The concentration of the triazine compound, the reaction temperature, and the reaction time can be appropriately determined. This step may be performed after the following step 4) as necessary, or the step 4) and this step may be performed simultaneously.

4) A step of reacting an amine compound containing a labeling site in the molecular structure.
In the present invention, the step of reacting the amine compound containing the labeling site in the molecular structure is by adding the aqueous amine compound solution to the aqueous solution obtained by the above step 2) or 3). The concentration of the amine compound, the reaction temperature, and the reaction time can be appropriately determined. This step may be performed after the step 2) as necessary, or the step 3) and this step may be performed simultaneously.

  The target polymer compound is a 1,3,5-triazine compound containing a carboxyl group on the target polymer compound, an amine on the modified ligand compound, and a labeling substance when the modified ligand compound interacts. The dehydration condensation reaction is used to bind the carboxyl group on the target polymer compound and the amine compound containing the labeling substance, and the target polymer compound is labeled. By the above series of reactions, affinity labeling of the target polymer compound of the present invention is achieved.

  In the present invention, the method for detecting the labeled target polymer compound is not particularly limited. For example, it can be detected by measuring a signal (for example, fluorescence intensity in the case of a fluorescent label) for each reaction container. In addition, when multiple types of target polymer compounds are present in the same sample, the target polymer compound is analyzed by a method known per se, such as gel filtration, and the signal of each obtained fraction is measured. Thus, it can be detected.

  The present invention also includes a kit used for the affinity labeling method of the target polymer compound. Specifically, a kit for affinity labeling of a target polymer compound containing a reagent containing a 1,3,5-triazine compound and an amine compound containing a labeling substance site in the molecular structure can be mentioned. The kit may contain a reagent for modifying the ligand compound with a tertiary amine. Furthermore, a ligand compound modified with a tertiary amine may be included. The kit of the present invention can also contain other reagents and instruments necessary for labeling.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Synthetic Scheme of Modified Ligand Compound (Modified Catalytic Ligand) Modification of a tertiary amine of a ligand compound for affinity labeling of a target polymer compound will be specifically illustrated and described. Here, the target polymer compound is avidin, the ligand compound is biotin, and the tertiary amine is dimethylglycine.

  By the following method, 1- (N, N-dimethylamino) acetic acid 5-aminopentyl (Compound 3) was synthesized using Compound 1 as a starting material. Next, biotin was modified to synthesize biotinyl-N-hydroxysuccinimide (Compound 5). Compounds 3 and 5 were amidated to synthesize a modified ligand compound (Compound 6).

1) Synthesis of Compound 1 As Compound 1, Nt-butoxycarbonyl-5-amino-1-pentanol was synthesized by the following method.

Di-tert-butyl carbonate in a solution of 5-Amino-1-pentanol (Tokyo Chemical) (3.0 g, 29.1 mmol) in CH ₂ Cl ₂ (29 mL) at 0 ° C (di-tert-butyl dicarbonate) (7.6 g, 34.9 mmol) was added dropwise. After stirring at room temperature for 1 hour, the reaction mixture was separated as it was by silica gel column chromatography (leaving with CH ₂ Cl ₂ solution and eluted with hexane (hexane: AcOEt = 1: 1)), and further distilled under reduced pressure (2 mmHg, 220 And compound 1 was obtained. Colorless oil (5.9 g, 60% yield).

¹ H NMR (CDCl ₃ ): δ1.33-1.63 (m, 6H), 1.44 (s, 9H), 3.12 (td, 2H, J = 6.2, 6.4Hz), 3.64 (t, 2H, J = 6.4Hz ), 4.64 (s, 1H).
LRMS (ESI): 204 (M + H) ⁺ , 226 (M + Na) ⁺
IR: ν 3469, 2932, 1678, 1644, 1536, 1169 cm ^-1

2) Synthesis of Compound 2 As compound 2, 1- (N, N-dimethylamino) acetic acid Nt-Butoxycarbonyl-5-aminopentyl 1- (N, N-dimethylamino) was prepared by the following method. ) acetate) was synthesized.

Compound 1 (2.2 g, 10.8 mmol), N, N-dimethylglycine hydrochloride (Tokyo Kasei) (1.5 g, 10.8 mmol), N-methylmorpholine (1.1 g, 10.8 mmol) and 4-dimethylaminopyridine (WAKO) (0.7 g, 5.37 mmol) in CH ₂ Cl ₂ (32 mL) in 1-ethyl-3- (3-dimethylaminopropyl ) 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (manufactured by Tokyo Chemical Industry) (EDC-HCl; 3.1 g, 16.2 mmol) was added and reacted at 0 ° C. The ice bath was removed and the mixture was stirred overnight. The reaction mixture was extracted with CH ₂ Cl ₂ , and the organic layer was washed with saturated sodium carbonate (Na ₂ CO ₃ ) and then with saturated brine, dried over magnesium sulfate, and concentrated. And purified by silica gel column chromatography (AcOEt) to obtain Compound 2. Colorless oil (2.4 g, yield 78%).

¹ H NMR (CDCl ₃ ): δ1.33-1.56 (m, 4H), 1.44 (s, 9H), 1.62-1.72 (m, 2H), 2.35 (s, 6H), 3.12 (q, 2H, J = 6.4Hz), 3.16 (s, 2H), 4.13 (t, 2H, J = 6.8Hz).
LRMS (ESI): 289 (M + H) ⁺ , 311 (M + Na) ⁺
IR: ν 3441, 2938, 1692, 1641, 1166 cm ^-1

3) Synthesis of Compound 3 As compound 3, 1- (N, N-dimethylamino) acetic acid 5-aminopentyl (5-Aminopentyl 1- (N, N-dimethylamino) acetate) was synthesized by the following method.

To a solution of compound 2 (1.0 g, 3.47 mmol) in CH ₂ Cl ₂ (6.3 mL) was added trifluoroacetic acid (TFA) (3.9 g, 34.7 mmol) under a nitrogen atmosphere, and the mixture was stirred at room temperature for 3 hours. CH ₂ Cl ₂ was distilled off under reduced pressure, CH ₂ Cl ₂ (about 6 mL) was added to the residue, and the residue was again distilled off under reduced pressure. This operation was repeated two more times. After drying under reduced pressure with a pump overnight, the obtained oily substance was decanted with AcOEt-Et ₂ O while being cooled with dry ice. The obtained oily substance was dried under reduced pressure to obtain the desired product. Colorless crystals (1.4 g, 100% yield salt of TFA)

bp: 51-55 ℃
¹ H NMR (methanol-d ₄ ): δ1.42-1.54 (m, 2H), 1.63-1.82 (m, 4H), 2.93 (t, 2H, J = 7.7Hz), 2.97 (s, 6H), 4.15 (s, 2H), 4.28 (t, 2H, J = 6.6Hz).
LRMS (ESI): 189 (M + H) ⁺ , 95 (M + 2H) ²⁺ / 2
IR (KBr): ν 3452, 2955,1742, 1676, 1468, 1425, 1202, 1136 cm ^-1

4) Synthesis of Compound 4 As compound 4, biotinyl-N-hydroxysuccinimide was synthesized by the following method.

(+)-Biotin (manufactured by WAKO) (1.0 g, 4.1 mmol) was added to N, N-dimethylformamide (DMF) (20 mL) under a nitrogen atmosphere and dissolved by heating to 80 ° C. N, N'-carbonyldiimidazole (0.67 g, 4.1 mmol) was added at the same temperature and stirred. After the generation of CO ₂ stopped, the heating was terminated, and the mixture was further heated at room temperature for 2 hours. Stir. When N-hydroxysuccinimide (manufactured by WAKO) was added to the resulting white suspension, white sediment disappeared and the reaction was allowed to proceed for 24 hours. The solid obtained by distilling off the solvent under reduced pressure was recrystallized from 2-propanol to obtain Compound 4. Colorless crystals (985 mg, yield 70%). Compound 4 is known and commercially available products such as those sold by Sigma may be used.

mp: 138-140 ℃
¹ H NMR (DMSO-d ₆ ): δ1.35-1.75 (m, 6H), 2.58 (d, 1H, J = 12.4Hz), 2.67 (t, 2H, J = 7.3Hz), 2.81 (s, 4H ), 2.83 (dd, 1H, J = 5.0, 12.4Hz), 3.06-3.15 (m, 1H), 4.11-4.19 (m, 1H), 4.31 (dd, 1H, J = 5.0, 7.4Hz), 6.35 ( s, 1H), 6.41 (s, 1H)
LRMS (ESI):
IR (KBr): ν 3223, 2943, 2912, 2876, 1820, 1789, 1746, 1695 cm ^-1

5) Synthesis of Compound 5 As compound 5, N-Biotinyl-6-aminohexanoic acid was synthesized by the following method.

An aqueous solution of 6-aminohexanoic acid (376 mg, 2.87 mmol) dissolved in 0.1M NaHCO ₃ (12 mL) was added to a suspension of compound 4 (980 mg, 2.87 mmol) in DMF (9 mL) and stirred for 4 hours. . After the solvent was distilled off under reduced pressure, citric acid (100 g / L) was added to the obtained crystals and stirred, and the crystals collected by suction filtration were washed 4-5 times with cold water. The crystal was dried in a desiccator containing tetraphosphorus decaoxide (P ₄ O ₁₀ ) (45 ° C., 1 day) to obtain the desired product. Colorless crystals (937 mg, yield 91%). Compound 5 is known, and commercially available products such as those sold by Sigma may be used.

mp: 218-220 ℃
¹ H NMR (DMSO-d ₆ ): δ1.18-1.68 (m, 12H), 2.04 (t, 2H, J = 7.4Hz), 2.19 (t, 2H, J = 7.4Hz), 2.58 (d, 1H , J = 12.4Hz), 2.82 (dd, 1H, J = 5.1, 12.4Hz), 3.00 (td, 2H, J = 6.0, 6.7Hz), 3.05-3.15 (m, 1H), 4.09-4.17 (m, 1H), 4.30 (dd, 1H, J = 5.2, 7.4Hz), 6.34 (s, 1H), 6.40 (s, 1H), 7.73 (m, 1H).
LRMS (ESI): 358 (M + H) ⁺ , 380 (M + Na) ⁺
IR (KBr): ν 3417, 3377, 3287, 2938, 2877, 1711, 1698, 1633, 1541 cm ^-1

6) Synthesis of modified ligand compound by amidation of compounds 3 and 5 (compound 6)
Compounds 3 and 5 are amidated and N, N-dimethyl-15- (biotinylamide) -2,10-dioxo-3-oxa-9-azapentadecanamine (N, N-dimethyl-15) is prepared by the following method. -(Biotinylamido) -2,10-dioxo-3-oxa-9-azapentadecanamine) (Compound 6) was synthesized as a modified ligand compound.
A solution of compound 3 (346 mg, 0.83 mmol), compound 5 (300 mg, 0.83 mmol), and N-methylmorpholine (NMM: 252 mg, 2.49 mmol) in MeOH (21 mL) was added to a quaternary ammonium salt 4- (4,6-Dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride (DMT-MM: 574 mg, 2.07 mmol) was added, and the mixture was stirred overnight at room temperature.

After partially distilling off methanol (MeOH) as a solvent under reduced pressure, ether (Et ₂ O) was added to precipitate crystals. The crystals were collected by suction filtration, washed with Et ₂ O, and then separated by silica gel column chromatography (eluted with MeOH → MeOH 1% Et ₃ N) to obtain the desired product as a trifluoroacetate salt (TFA salt). The obtained salt was dissolved in MeOH, neutralized with 0.05 M KOH, the solvent was distilled off under reduced pressure and then dissolved in CHCl ₃ , and insoluble matters were removed by filtration. The solid obtained from the filtrate was recrystallized (MeOH-Et ₂ O). Colorless crystals (262.8 mg, yield 60%).

bp: 124-133 ℃
¹ H NMR (MeOH-d ₄ ): δ1.25-1.80 (m, 18H), 2.18 (t, 2H, J = 7.4Hz), 2.19 (t, 2H, J = 7.3Hz), 2.33 (s, 6H ), 2.70 (d, 1H, J = 12.7Hz), 2.93 (dd, 1H, J = 4.9, 12.7Hz), 3.11-3.24 (m, 7H), 4.13 (t, 2H, J = 6.6Hz), 4.30 (dd, 1H, J = 4.5, 7.8Hz), 4.49 (dd, 1H, J = 4.9, 7.8Hz).
¹³ C NMR (MeOH-d4): δ 23.3, 25.7, 25.9, 26.6, 28.3, 28.5, 28.8, 29.0, 29.1, 35.8, 36.0, 39.1, 39.2, 40.0 (CH ₂ -S), 44.2 (2CH ₃ ) , 56.0 (CH-S), 59.3 (CH ₂ -N), 60.6 (CH-N), 62.4 (CH-N), 64.9 (CH ₂ -O), 165.1 (N-CO-N), 170.1 (CO -O), 174.9 (CO-NH), 175.0 (CO-NH)
LRMS (ESI): 528 (M + H) ⁺ , 550 (M + Na) ⁺
HRMS (ESI-TOF): calcd for C ₂₅ H ₄₆ N ₅ O ₅ S (M + H) ⁺ 528.3214; found 528.3211.
IR (KBr): ν 3302, 2924, 2853, 1738, 1701, 1638, 1560 cm ^-1

(Example 2) Avidin-specific affinity label (fluorescent label)
In this example, biotin as a ligand compound was detected from a solution containing avidin and γ-globulin by an affinity labeling method. Biotin as a ligand compound was modified with a tertiary amine by the method described in Example 1 to obtain a modified ligand compound. These were affinity labeled by the following method.

1. Materials Each of avidin, γ-globulin, modified ligand compound, fluorescent agent and CMDT used in this experimental example was dissolved in 50 mM phosphate buffer (pH 8.0). As the phosphate buffer, sodium dihydrogen phosphate dihydrate (39.0 g, 0.25 mol) was dissolved in an appropriate amount of water, and a 2.5 M NaOH solution was gradually added thereto to adjust the pH to 8.0. This solution was transferred to a volumetric flask, and water was added to make a total volume of 500 mL.

a) Avidin solution: 1 mg of egg white-derived avidin (SIGMA) was dissolved in 100 μL of 50 mM phosphate buffer (pH 8.0).
b) γ-globulin solution: 21 mg of γ-globulin (SIGMA) was dissolved in 1 mL of 50 mM phosphate buffer (pH 8.0).
c) Modified ligand compound solution: 1.2 mg of the modified ligand compound prepared in Example 1 was dissolved in 2 mL of 50 mM phosphate buffer (pH 8.0).
d) Labeling agent (fluorescent agent) solution: 3.5 mg of fluorescent amine (Cascade Blue ^(R) (Invitrogen)) was dissolved in 1 mL of 50 mM phosphate buffer (pH 8.0).
e) CDMT solution: 2.0 mg of CDMT was dissolved in 1 mL of 50 mM phosphate buffer (pH 8.0).

2. Experimental procedure 1) 40 μL of avidin solution, 40 μL of γ-globulin solution, and 20 μL of modified ligand compound solution were added to a test tube (Eppendorf ^™ tube) and left at room temperature for 2 hours.
2) Thereafter, 40 μL of the fluorescent agent solution and 20 μL of the CDMT solution were added, and left at room temperature for 24 hours.
3) Thereafter, gel filtration analysis was performed under the following conditions to separate the two proteins, and the fluorescence of each fraction and the absorbance at UV (wavelength 280 nm) were measured.
4) Gel filtration conditions ^{gel: Sephadex (R) G-100} superfine (GE Healthcare Inc.)
Mobile phase: 50 mM phosphate buffer (pH 8.0, 0.1 M NaCl)
Column size: 1.5 × 40cm
Flow rate: 2 mL / hr
Fraction volume: 1.0 mL
Fluorescence: Quantitative measurement at excitation maximum wavelength of 399 nm and fluorescence maximum wavelength of 423 nm
UV: Fixed wavelength measurement at 280 nm

3. Results The results of gel filtration analysis of γ-globulin and avidin are shown in FIGS. Each eluted protein was quantified by UV absorbance (FIG. 4), and the fluorescence intensity at each peak was measured (FIG. 5). As a result, there was a significant difference in the fluorescence intensity of the fraction of γ-globulin and avidin, and it can be said that avidin was selectively labeled. That is, by the affinity labeling method of the present invention, among γ-globulin and avidin, avidin was screened and confirmed to be a substance capable of interacting with biotin. FIG. 6 shows a reaction schematic diagram relating to the above-described detection method for labeling with avidin and biotin.

(Example 3) Avidin-specific affinity label and quantification of labeling rate In this example, a solution containing avidin or γ-globulin and a modified ligand compound (modified biotin) synthesized by the method of Example 1 were mixed. Affinity labeling was performed by the method described above, and the labeling rate was quantified.

1. Experimental operation:
Each of avidin, γ-globulin, modified ligand compound, fluorescent agent and CMDT used in this experimental example was dissolved in 50 mM phosphate buffer (pH 8.0). In the same manner as described in Example 2, the phosphate buffer was prepared by dissolving sodium dihydrogen phosphate dihydrate (39.0 g, 0.25 mol) in an appropriate amount of water, and adding a 2.5 M NaOH solution little by little thereto. The one adjusted to pH 8.0 was used. This solution was transferred to a volumetric flask, and water was added to make a total volume of 500 mL.

a) Avidin solution: Egg white-derived avidin (SIGMA) was dissolved in 100 μL (pH 8.0) of 50 mM phosphate buffer to obtain a 0.15 mM avidin solution.
b) γ-globulin solution: γ-globulin (SIGMA) was dissolved in 100 μL of 50 mM phosphate buffer (pH 8.0) to obtain a 0.15 mM γ-globulin solution.
c) Modified ligand compound solution: The modified ligand compound prepared in Example 1 was dissolved in 100 μL of 50 mM phosphate buffer (pH 8.0) to obtain a 1.2 mM modified ligand compound solution.
d) Labeling agent (fluorescent agent) solution: Cascade Blue ^(R) (Invitrogen) was dissolved in 100 μL of 50 mM phosphate buffer (pH 8.0) to obtain a 6.0 mM labeling agent solution.
e) CDMT solution: CDMT was dissolved in 100 μL (pH 8.0) of 50 mM phosphate buffer to obtain a 12 mM CDMT solution.

To a 0.15 mM avidin solution or 0.15 mM γ-globulin solution, 5 μL of a 1.2 mM modified ligand compound solution was added, stirred with vortex ^(R) , and allowed to stand at room temperature for 30 minutes.

Next, 30 μL of 50 mM phosphate buffer (pH 8.0), 20 μL of 6.0 mM labeling agent solution, and 10 μL of 12 mM CDMT solution were added, stirred with vortex ^(R) , and allowed to stand at room temperature for 8 hours. The reaction solution was subjected to gel filtration chromatography was separated by ^{(Sephadex (R) G-50} (GE Healthcare Corp.), 50 mM phosphate buffer (pH 8.0), eluted with 0.1 M NaCl), was determined labeling rate from UV spectra .

  The initial concentration of each compound in the reaction solution (75 μL) was avidin: 20 μM; modified ligand compound: 80 μM; labeling agent: 1.6 mM; CDMT: 1.6 mM. Further, in the control experiment in the avidin system, N, N-dimethylglycine ethyl ester without biotin was used as a catalyst instead of the modified ligand compound. Avidin specificity was evaluated using equimolar amounts of γ-globulin. The biotin specificity was evaluated by adding 25 equivalents of natural biotin having no catalytic ability to the catalyst.

2. UV measurement and calculation of labeling rate The molar extinction coefficients at specific absorption wavelengths of avidin and cascade blue ^(R) were as follows.

a) Cascade Blue ^(R) molar extinction coefficient: ε (399 nm) = 26400 M ^-1 cm ^-1 , ε (280 nm) = 18792 M ^-1 cm ^-1
b) Avidin molar extinction coefficient: ε (280 nm) = 93960 M ^-1 cm ^-1
Each UV spectrum is shown in FIGS. Each concentration was calculated from the following formula. Since the cell length was 1 cm, it was omitted from the calculation formula.
Cascade blue ^(R) (cb) concentration C _cb (M): Absorbance A _cb399 (actual value) at 399 nm,
C _cb = A _cb399 / 26400
Avidin protein concentration C _av (M): Absorbance A ₂₈₀ (measured value) at 280 nm is the sum of absorption at the same wavelength of Avidin and Cascade Blue (A _av280 + A _cb280 )
A _av280 = A ₂₈₀ − A _cb280 = A ₂₈₀ − 18792 × C _cb
Therefore,
C _av = (A ₂₈₀ − 18792 × C _cb ) / 93960
Therefore, the labeling rate L _av (%) was obtained by substituting the above values into the following equation.
L _av = 100 × C _cb / C _av (%)

3) Results The above results are shown in Table 1 and FIG.

From the above results, the following can be considered.
(1) Since avidin is a tetramer of the same monomer subunit, it was possible to simply label at 4 sites per molecule, indicating that almost half of them were labeled under this condition.
(2) As a result of using a modified ligand compound having biotin as a catalyst, it is specifically labeled with respect to avidin, and not with γ-globulin or avidin control (system using a catalyst without biotin). It was.
(3) About 10% of labeling was observed due to the non-specific reaction under these conditions.
(4) Since the specific reaction was completely inhibited by the addition of an excessive amount of biotin, it was confirmed that the labeling was based on the interaction between avidin and biotin. Since it was inhibited by biotin that specifically binds to avidin, the reaction with the modified ligand compound having biotin can be said to be a specific reaction based on the binding of avidin / biotin.

(Example 4) Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) In the labeling experiment performed according to Example 2, the protein labeled by SDS-PAGE was analyzed. According to a conventional method, 4% concentrated gel and 12% separation gel were used and electrophoresed at a constant voltage of 200 V (electrophoresis buffer: 0.025 M Tris-HCI, pH 8.3, 0.192 M glycine, 0.1% SDS). Samples were added to 10 μL of 1 μg / μL solution, 10 μL of sample buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-Mercaptethanol, 0.02% bromophenol blue), and vortexed ^{(R ) And} then denatured by heating at 100 ° C. for 30 minutes in a water bath. After the electrophoresis, the gel was subjected to fluorescence detection, and further stained with CBB (Coommassie Brilliant Blue). As a result, it was revealed that avidin was fluorescently labeled through a covalent bond only when a modified ligand was used (FIG. 11: lanes 1 and 4).

The description of the symbols in FIG. 11 is as follows.
Lane 1: avidin + compound 6
Lane 2: avidin + compound 6 + biotin (25eq)
Lane 3: avidin ++ N, N-dimethylglycine ethyl ester Lane 4: avidin + compound 6 + γ-globulin Lane 5: avidin + γ-globulin + N, N-dimethylglycine ethyl ester Lane 6: molecular marker
a: Avidin subunit monomer
b: light chain of γ-globulin
c: Avidin subunit dimer
d: heavy chain of γ-globulin

(Example 5) Identification of labeled amino acid by mass spectrometry (mass) analysis From the analysis result of electrophoresis by SDS-PAGE of Example 4, a gel of the monomer subunit of avidin was cut out and digested with trypsin in the gel according to a conventional method. The resulting peptide fragment mixture was analyzed by reverse phase HPLC and electrospray ionization mass spectrometry (HPLC-ESI-MS).

1. Experimental operation:
Labeling agent: Cascade Blue ^(R) has three anionic sulfo groups, and mass analysis becomes difficult due to the ionization. Therefore, 4-bromobenzylamine was used for labeling for this experiment. Since the labeled compound has a primary amino group, labeling is expected to proceed in the same manner as Cascade Blue ^(R) . This compound is not charged by itself, and the isotopes of bromine (79 and 81 are almost the same number) give a characteristic peak pattern in the mass spectrum, so it is considered that the analysis is easy.
The labeling experiment was carried out in the same manner as in Examples 2 and 3.

2. HPLC-ESI-MS conditions
(1) HPLC: HP1100 nanoLC ^TM (Bruker Daltonics)
Column: Capillary HPLC column, MonoCap ^(R) for fast flow (GL Science)
Column size 0.05x150mm;
HPLC column (Zorbax ^TM 30SB C18 (Agilent)
Column size 0.3x5cm
Mobile phase: Solvent A: 0.1% formic acid aqueous solution; Solvent B: 0.1% formic acid acetonitrile solution Flow rate: 0.3 μL / min
Gradient: 0 min / solvent A: B = 95: 5, 40 min / solvent A: B = 10: 90
(2) ESI-MS: micrOTOF-Q ^TM II w / online nanosprayer (Bruker Daltonics)

3. result
It was revealed that Asp-108 (the 108th aspartic acid) was specifically labeled among the 128 amino acid monomer subunits. Interestingly, it was found that Asp was also present in the neighboring structures 105 and 109 in the primary structure, but their carboxyl groups were not modified.

  Based on the X-ray data of the complex of avidin and biotin derivative (N-Biotinyl-6-aminohexanoic acid) downloaded from NCBI (National Center for Biotechnology Information), the surface amino acids around the binding site (Asp with carboxyl group or Of Glu), it was confirmed that Asp-108 was considered to be present at the position closest to the reaction site of the modified ligand compound. On the other hand, in Asp of No. 105 and No. 109, the carboxyl groups seem to face the opposite side with respect to the catalyst (FIG. 12). Thereby, it was confirmed that the reaction site of avidin and a biotin derivative is labeled.

Example 6 Specific Labeling of Acetylcholine Receptor
Focusing on hexamethonium, a competitive inhibitor of nicotinic acetylcholine receptor, we extended the side chain of one of the methyl groups and introduced dimethylglycine as a catalytic site at the end. If one or two quaternary ammoniums are present and the distance is appropriately maintained, the binding ability to the receptor is expected to be maintained. Since the binding site exists in the hydrophilic part of the membrane-bound receptor, it is considered that target aspartic acid and glutamic acid are sufficiently present on the hydrophilic protein surface in the vicinity thereof.

1. Design of modified ligand compounds:
Actually, a modified ligand compound as shown in the figure was synthesized, and the labeling reaction was examined on the membrane fraction obtained from the electric organ of American American rayray.

1) Synthesis of [6- (Dimethylamino) hexyl] trimethylammonium iodide ([6- (Dimethylamino) hexyl] trimethylammonium iodide)

N, N, N ', N'-Tetramethyl-1,6-hexanediamine (N, N, N', N'-tetramethyl-1,6-hexanediamine) (1.72 g, 10 mmol) in CH ₃ CN ( 40 mL) A solution of methyl iodide (1.42 g, 10 mmol) in CH ₃ CN (10 mL) was added dropwise to the solution at room temperature under nitrogen. The reaction mixture was stirred at room temperature for 2 hours, the resulting precipitate was removed by filtration, and the filtrate was concentrated. The obtained solid was recrystallized from 2-propanol / ethyl ether to obtain a white solid.

1.74 g, yield 55.4%. mp 142-144 ° C.
¹ H NMR (400 MHz, DMSO-d6) δ: 1.41 (m, 4H), 1.53 (m, 2H), 1.80 (m, 2H), 2.24 (s, 6H), 2.32 (t, J = 7.62 Hz, 2H), 3.13 (s, 9H), 3.35 (m, 2H).
¹³ C NMR (100 MHz, DMSO-d6) δ: 22.0, 25.6, 26.3, 26.8, 45.2, 52.2, 58.9, 65.3. IR (KBr) cm ^-1 : 3493, 2934, 2811, 2775, 1482. HRESI-MS m / z: Calcd for C ₁₁ H ₂₇ N ₂ ⁺ [M] ⁺ 187.2169, found 187.2169.

2) Synthesis of 6-Bromohexyl 2- (dimethylamino) acetate

To a solution of N, N-Dimethylglycine hydrochloride (1.39 g, 10 mmol) in CH ₂ Cl ₂ (40 mL), add 4- (dimethylamino) pyridine (4- (dimethylamino) pyridine). (DMAP; 1.46 g, 12 mmol) and EDC · HCl (2.30 g, 12 mmol) were added at 0 ° C. under nitrogen, followed by stirring at the same temperature for 1 hour. After raising the reaction temperature to room temperature, 6-bromo-1-hexanol (1.81 g, 10 mmol) of CH ₂ Cl ₂ (10 mL) was added dropwise, and further at room temperature 3 Stir for hours. The reaction mixture was extracted with CH ₂ Cl ₂ and concentrated. The residue was purified by silica gel column chromatography [EtOAc-Hexand (1: 1)] to obtain the desired product as a colorless oil.

2.01 g, 76% yield.
¹ H NMR (400 MHz, CDCl3) δ: 1.32-1.52 (m, 4H), 1.62-1.71 (quint J = 6.72 Hz, 2H), 1.83-1.91 (quint J = 6.78 Hz, 2H), 2.35 (s, 6H), 3.16 (s, 2H), 3.53 (t, J = 6.78 Hz, 2H), 4.13 (t, J = 6.72 Hz, 2H).
¹³ C NMR (100 MHz, CDCl ₃ ) δ: 25.0, 27.7, 28.4, 32.5, 33.6, 45.2, 60.4, 64.3, 170.6. IR (neat) cm ^-1 : 2937, 1747. HRESI-MS m / z: Calcd for C ₁₀ H ₂₀ BrNO ₂ [M + H] ⁺ 266.0750, found 266.0741.

3) Synthesis of dimethyl [6- (2-dimethylaminoacetoxy) hexyl]

6- (Trimethylammonio) hexylammonium bromide iodide [6- (dimethylamino) hexyl] trimethylammonium iodide synthesized on [6- (trimethylammonio) hexylammonium bromide iodide) iodide) (314 mg, 1 mmol) in CH ₃ CN (10 mL) solution of 6-bromohexyl 2- (dimethylamino) acetate (400 mg, 1.5 mmol) in CH ₃ CN (5 mL) solution was added dropwise at room temperature, and the mixture was stirred at room temperature for 72 hours. Ethyl ether (50 mL) was added to the reaction solution to precipitate the target product as a white precipitate, and the supernatant was removed. The obtained solid was recrystallized by recrystallization (CH ₃ CN / ethyl ether) to obtain the desired product as a white solid.

280 mg, Yield 48% mp 98-103 ° C.
¹ H NMR (400 MHz, CD ₃ OD) δ: 1.40-1.50 (m, 8H), 1.65-1.92 (m, 8H), 2.34 (s, 6H), 3.11 (s, 6H), 3.17 (s, 9H ) <3.22 (s, 2H), 3.25-3.45 (m, 6H), 4.10-4.18 (t, J = 6.35 Hz, 2H).
¹³ C NMR (100 MHz, CD ₃ OD) δ: 23.4, 23.5, 23.7, 26.5, 26.66, 26.67, 27.0, 29.5, 45.4, 51.3, 53.73, 60.7, 65.3, 65.5, 67.6, 171.7. IR (KBr) cm ^-1 : 3470, 2945, 1740, 1482, 1203. HRESI-MS m / z: Calcd for C ₂₁ H ₄₇ N ₃ O ₂ ⁺² [M] ⁺² 186.6828, found 186.6898.

2. Affinity labeling reaction for membrane fraction containing acetylcotylin receptor Membrane fraction was prepared from an electric organ of Torpedo californica according to methods known in the literature (J. Elliott, SG Blanchard, W. Wu J. Miller, CD Strader, P. Hartig, H.-P. Moore, J. Racs, MA Raftery, Biochem. J., 1980, 185, 667).
To a receptor fraction containing 217 mg of protein, a modified ligand compound having a hexamethonium structure (0.44 mM), Cascade Blue ^(R) (1.1 mM), and CDMT (1.1 mM) are added (50 containing 0.1 M NaC). mM phosphate buffer pH 8.0; total volume 0.20 mL; reaction concentration in parentheses), vortex ^(R) and then stirred at 4 ° C. for 24 hours. After centrifuging the reaction solution, the supernatant was removed, and the same buffer was added again to the precipitate, followed by centrifugation to remove the supernatant. After repeating this operation five times, the precipitate was subjected to SDS-PAGE in the same manner as in Example 4 to detect fluorescence by CBB (Coomasie Brilliant Blue) and labeling of the present invention. The results are shown in FIG.

The reference numerals in FIG. 13 are described as follows.
Lane 1: Molecular marker Lane 2: ARM
Lane 3: alkali M
Lane 4: ARM (affinity labeling)
Lane 5: alkali M (affinity labeling)
ARM (AChR-ruch membrane)
alkali M (alkali treatment ARM)

As described in detail above, the target polymer compound can be effectively labeled by the method of the present invention.
For example, when there are multiple types of candidate compounds that can be ligand compounds, if the candidate compound is labeled when it interacts with the target polymer compound, the ligand compound is selected by detecting the signal due to the labeling, Can be screened. In addition, when a plurality of types of polymer target compounds are present, a polymer target compound that can interact with a ligand compound can also be detected and screened. This makes it possible to easily confirm the reaction between the enzyme and the substrate, and the interaction between the receptor and the agonist or antagonist. For example, when reversible reaction has not been possible to label easily and detection has been easy However, according to the method of the present invention, a substance that can interact easily can be detected.
Furthermore, the method of the present invention can identify an acidic amino acid residue located spatially close to the ligand binding site of the target polymer compound, and binding of the ligand compound by changing the modification site of the ligand compound The chemical structure of the target polymer compound required for

  As described above, for example, in the development of a new drug, it is easy to apply the affinity labeling method of the present invention to those that could not be screened conventionally regarding the interaction between the target polymer compound and the candidate substance. Can be screened. Furthermore, the most effective candidate substance can be designed by knowing the chemical structure necessary for the binding of the ligand compound for the target polymer compound.

It is a schematic diagram which shows an example of reaction of the affinity labeling method of this invention. It is a schematic diagram which shows an example of reaction of the affinity labeling method of this invention. It is a schematic diagram which shows an example of reaction of the affinity labeling method of this invention. It is a figure which shows the HPLC analysis result of avidin and gamma globulin in a sample. (Example 2) It is a figure which shows the measurement result of the label intensity | strength (fluorescence intensity) about the HPLC analysis fraction of avidin and gamma globulin in a sample. (Example 2) 4 is a reaction schematic diagram of an affinity labeling method in Example 2. FIG. It is a figure which shows the UV spectrum of avidin. (Example 3) It is a figure which shows the UV spectrum of cascade blue ^(R) . (Example 3) It is a figure which shows the UV spectrum of labeled avidin. (Example 3) It is a figure which shows the labeling (labeling) rate of avidin, avidin control, (gamma) -globulin, and biotin. (Example 3) It is a figure which shows the result of fluorescence detection and CBB dyeing | staining by avidin specific labeling. Example 4 It is a figure which shows the reaction site with biotin and the position of aspartic acid (Asp) in avidin in the X-ray structure of the avidin monomer. (Example 5) It is a figure which shows the result of the fluorescence detection and CBB dyeing | staining by acetylcholine specific labeling. (Example 6)

Claims (8)

A method for affinity labeling a target polymer compound comprising the following steps 1) to 4) :
The target polymer compound has a carboxyl group or is a compound modified with a carboxyl group,
A ligand compound is a compound capable of interacting with the target polymer compound,
A method for affinity labeling a target polymer compound, wherein the tertiary amine comprises a compound represented by the following formula (II):
1) modifying the ligand compound with a tertiary amine;
2) mixing the target polymer compound and the ligand compound modified with the tertiary amine;
3) a step of further reacting the 1,3,5-triazine compound with the target polymer compound;
4) A step of reacting an amine compound further containing a labeling site in the molecular structure.
Wherein one or two of R ^5, R ⁶ and R ⁷ are methyl groups and the remaining R ^5, R ⁶ and R ⁷ are each independently an alkyl group having 1 to 6 carbon atoms , An alkenyl group having 1 to 6 carbon atoms, —CH ₂ COOR ⁸ , CH ₂ CONR ⁹ R ¹⁰ (where R ⁸ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R ⁹ is a hydrogen atom, carbon An alkyl group having 1 to 6 carbon atoms, and R ¹⁰ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, or an alkynyl group having 1 to 6 carbon atoms)] The affinity labeling method according to claim 1, wherein the 1,3,5-triazine compound is a compound represented by the following formula I.

[Wherein, R ¹ and R ² each independently represent a hydrogen atom, an alkyl group, a hydroxyalkyl group, a sulfo group, a phospho group, or —R ³ R ⁴ (where R ³ represents — (C _m H _2m O) _n and R ⁴ is a hydrogen atom, a sulfo group, a phospho group, or an alkyl group having an amino group, an ammonio group, a sulfuric acid group or a phosphoric acid group, and m is an integer of 1 to 30 And n is an integer of 1 to 120), and X is a chlorine, fluorine, bromine, iodine, trifluoromethanesulfonyloxy group or N-methylmorpholine (NMM). Is done. ] The affinity labeling method according to claim 1, wherein the 1,3,5-triazine compound is a 2-chloro-4,6-dimethoxy-1,3,5-triazine compound (CMDT). The affinity labeling method according to any one of claims 1 to 3 , wherein the amine compound is represented by R ¹¹ R ¹² NH.
[One or two of R ¹¹ and R ¹² are a labeling functional group, and the rest are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms, an aryl group, an ammonio group, a sulfo group, An alkyl group having a sulfate group or a phosphate group. ] The affinity labeling method according to any one of claims 1 to 4 , wherein the target polymer compound is a protein or peptide compound. The screening method of the target polymer compound using affinity labeling method according to any one of claims 1-5. The screening method of a ligand compound using the affinity labeling method of any one of Claims 1-6 . 1,3,5-triazine compounds, comprises the following ligand compounds modified with tertiary amine comprising a compound represented by the formula (II), and an amine compound containing a labeling portion in its molecular structure, according to claim 1 A kit for affinity labeling of a target polymer compound used in the affinity labeling method according to any one of -5 .
Wherein one or two of R ^5, R ⁶ and R ⁷ are methyl groups and the remaining R ^5, R ⁶ and R ⁷ are each independently an alkyl group having 1 to 6 carbon atoms , An alkenyl group having 1 to 6 carbon atoms, —CH ₂ COOR ⁸ , CH ₂ CONR ⁹ R ¹⁰ (where R ⁸ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R ⁹ is a hydrogen atom, carbon An alkyl group having 1 to 6 carbon atoms, and R ¹⁰ is a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, or an alkynyl group having 1 to 6 carbon atoms)]