Compound, reagent for binding to protein, composition for NMR measurement, composition for chemically induced dynamic nuclear polarization, method for analyzing properties of protein, database, molecular design method, program, and methods for preventing or treating disease and alleviating symptoms thereof

Abstract

According to the present invention, a compound having a structure in which a dye for chemically induced dynamic nuclear polarization and a linking group having a functional group for binding to a protein are covalently bonded to each other is reacted with and linked to a protein to be analyzed, the nuclear spin of the protein is highly polarized by the chemically induced dynamic nuclear polarization, and NMR measurement is performed with high sensitivity.

Description

Compounds, protein-binding reagents, compositions for NMR measurements, compositions for chemically induced dynamic nuclear polarization, methods for analyzing protein properties, databases, molecular design methods, programs, and methods for preventing, treating, or alleviating symptoms of diseases

[0001] The present invention relates to a compound, a protein-binding reagent using the compound, a composition for NMR measurement, a composition for chemically induced dynamic nuclear polarization, and a method for analyzing the properties of proteins. The present invention also relates to a database, a molecular design method, a program, and a method for preventing, treating, or alleviating the symptoms of disease.

[0002] When atomic nuclei (nuclear spins) with magnetic moments are placed in a static magnetic field, they undergo precession. When electromagnetic waves of the same frequency as the precession are irradiated in this state, the nuclear spins resonate and absorb the energy of the electromagnetic field, resulting in the nuclear magnetic resonance (NMR) phenomenon. The resonant frequency of this NMR phenomenon varies depending on the nuclide and the chemical or magnetic environment in which the nuclei are placed. Therefore, in the fields of organic chemistry and biochemistry, NMR spectroscopy is widely used to analyze the molecular structure and physical properties of compounds by observing the frequency spectrum (chemical shift value) of the NMR signal, which converts the amount of energy absorbed by the resonance into an electrical signal. In addition, in the medical field, magnetic resonance imaging (MRI), which adds positional information to the NMR signal to create an image, is used for non-invasive examination of biological organs such as the brain. Here, when a static magnetic field is applied to such a collection of nuclear spins, for example, in the case of protons, they split into an energy state in which the spins are parallel to the magnetic field and an energy state in which the spins are antiparallel to the magnetic field. Here, the value obtained by dividing the difference in the number of spins (occupancy number) in each energy state by the total number of spins is called the polarization factor, and the intensity of the NMR signal is said to be proportional to this polarization factor. However, the polarization factor of nuclear spins is usually very low, less than one tens of thousands at room temperature, and this is the cause of the limitation of the sensitivity of NMR spectroscopy and MRI.

[0003] To address this issue, chemically induced dynamic nuclear polarization (CND) techniques have been developed to polarize nuclear spins. One of these techniques is the triplet-DNP technique, which involves polarizing the electron spins of a dye and then transferring the polarized state of the dye to the nuclear spins of the target molecule. Another technique is photoexcited CND, which involves irradiating a dye with excitation light to create an excited state, followed by electron transfer between the excited dye and the target molecule, resulting in dynamic nuclear polarization via radical pairs. Because photoexcited CND can easily polarize nuclear spins using only the simple operation of light irradiation, it has been widely applied in biochemistry, such as fragment screening for drug design and protein structural analysis.

[0004] JP 2017-15443 A

[0005] However, when the target of dynamic nuclear polarization is a protein, for example, simply mixing the target protein with a dye and performing the dynamic nuclear polarization process has the problem that the NMR signal of the protein cannot be sufficiently enhanced. Therefore, in order to solve this conventional problem, the present inventors have conducted extensive research with the aim of providing a reagent that can sufficiently enhance the NMR signal of a protein.

[0006] As a result of extensive research, the present inventors have discovered that by reacting a compound having a structure in which a dye is covalently bonded to a linking group having a functional group for binding to a protein with a protein, and then performing dynamic nuclear polarization, the NMR signal of the protein is enhanced, thereby improving the sensitivity of NMR measurements. They have also found that such compounds are useful as binding reagents for proteins in NMR measurements. The present invention has been proposed based on these findings and specifically has the following configurations: [1] A compound having a structure in which a dye for chemically induced dynamic nuclear polarization is covalently bonded to a linking group having a functional group for binding to a protein. [2] The compound according to [1], in which the functional group includes a maleimide structure or an acetamide structure. [3] The compound according to [1], in which the functional group is an isocyanate group or an activated ester group. [3-1] The compound according to [1], in which the functional group is a functional group that undergoes a click reaction. [3-2] The compound according to [3-1], in which the functional group includes an alkyne structure, optionally substituted at a carbon atom. [3-3] The compound according to [3-2], wherein the functional group comprises a 4,8-diazocyclononyne structure. [4] The compound according to any one of [1] to [3-3], wherein the functional group is bonded to the terminal of the linking group. [5] The compound according to any one of [1] to [4], wherein the number of atoms in the linking chain between the dye and the functional group in the linking group is 0 to 20. [6] The compound according to any one of [1] to [5], wherein the distance from the center of the dye to the terminal of the linking group is 8 to 35 Å. [7] The compound according to any one of [1] to [6], wherein the dye comprises a riboflavin structure, a lumiflavin structure, a flavin mononucleotide structure, a flavin structure, a fluorescein structure, a porphyrin structure, a 2,2'-bipyridyl structure, a benzophenone-3,3',4,4'-tetracarboxylic acid structure, or an eosin Y structure. [8] A binding reagent for proteins, comprising the compound according to any one of [1] to [7]. [9] The binding reagent according to [8], which is for highly polarizing the protein by chemically induced dynamic nuclear polarization by photoexciting the dye.

[10] The binding reagent according to [8] or [9], wherein the functional group reacts with an amino acid of the protein.

[11] The binding reagent according to [8] or [9], wherein the functional group reacts with a mercapto group of the protein. [11-1] The binding reagent according to [8] or [9], wherein the functional group reacts with an azide group of the protein.

[12] A compound having a structure in which a dye and a protein that becomes highly polarized by chemically induced dynamic nuclear polarization upon photoexcitation of the dye are covalently bonded via a linking group.

[13] The compound according to

[12] , wherein the protein is GB1, FKBP12, Hsf1, TDP-43, FUS, C9ORF72, tau, amyloid beta, α-synuclein, huntingtin, BRCA1 / BRCA2, nucleophosmin (NPM1), PML, or TFSBD.

[14] The compound according to

[13] , wherein the dye is a compound containing a riboflavin structure.

[15] The compound according to

[13] , wherein the dye is a compound containing a lumiflavin structure.

[16] The compound according to

[13] , wherein the dye is a compound containing a flavin mononucleotide structure.

[17] The compound according to

[13] , wherein the dye is a compound containing a flavin structure.

[18] The compound according to

[13] , wherein the dye is a compound containing a fluorescein structure.

[19] The compound according to

[13] , wherein the dye is a compound containing a porphyrin structure.

[20] The compound according to

[13] , wherein the dye is a compound containing a 2,2'-bipyridyl structure.

[21] The compound according to

[13] , wherein the dye is a compound containing a benzophenone-3,3',4,4'-tetracarboxylic acid structure.

[22] The compound according to

[13] , wherein the dye is a compound containing an eosin Y structure.

[23] A composition for NMR measurements, comprising the compound according to any one of

[12] to

[22] .

[24] A composition for chemically induced dynamic nuclear polarization, comprising the compound according to any one of

[12] to

[22] .

[25] A method for analyzing the properties of a protein, comprising: a first step of synthesizing a molecule in which a dye is bound to the protein; a second step of irradiating the dye with light to highly polarize the protein by chemically induced dynamic nuclear polarization; and a third step of performing NMR measurement of the molecule.

[26] The method according to

[25] , wherein in the first step, a plurality of molecules are synthesized in which the dye is bound to different sites on the protein, the second step is performed for each of the synthesized molecules, and the third step is performed for each of the molecules that have been subjected to the second step.

[27] The method according to

[25] or

[26] , wherein in the first step, the dye is bound to the protein via a linking group.

[28] A database storing information indicating the relationship between molecules obtained by carrying out the method according to any one of

[25] to

[27] and NMR measurement results.

[29] A molecular design method for designing molecules based on information indicating the relationship between molecules obtained by carrying out the method according to any one of

[25] to

[27] and NMR measurement results.

[30] A program for carrying out the molecular design method of

[29] .

[31] A method for preventing, treating, or alleviating symptoms of a disease, comprising: carrying out the method according to any one of

[25] to

[27] for a protein involved in the disease to obtain information on the properties of the protein; and preventing, treating, or alleviating symptoms of the disease based on the information.

[32] The method for preventing, treating, or alleviating symptoms of a disease according to

[31] , wherein the disease is a neurological disease.

[33] The method for preventing, treating, or alleviating symptoms of a disease according to

[32] , wherein the disease is amyotrophic lateral sclerosis (ALS).

[34] The method for preventing, treating, or alleviating symptoms of a disease according to

[33] , wherein the method prevents, treats, or alleviates symptoms of muscle weakness, paralysis, or both.

[35] The method for preventing, treating, or alleviating symptoms of a disease according to

[33] or

[34] , wherein the protein is at least one selected from the group consisting of TDP-43 and FUS.

[36] The method for preventing, treating, or alleviating symptoms of a disease according to

[32] , wherein the disease is frontotemporal dementia (FTD).

[37] The method for preventing, treating, or alleviating symptoms of a disease according to

[36] , wherein the method prevents, treats, or alleviates symptoms of personality changes, cognitive decline, or both.

[38] The method for preventing, treating, or alleviating symptoms of a disease according to

[36] or

[37] , wherein the protein is TDP-43, FUS, or both.

[39] The method for preventing, treating, or alleviating symptoms of a disease according to

[32] , wherein the disease is Alzheimer's disease.

[40] The method for preventing, treating, or alleviating symptoms of a disease according to

[39] , which prevents, treats, or alleviates symptoms of memory loss, cognitive impairment, or both.

[41] The method for preventing, treating, or alleviating symptoms of a disease according to

[39] or

[40] , wherein the protein is tau, amyloid beta, or both.

[42] The method for preventing, treating, or alleviating symptoms of a disease according to

[32] , wherein the disease is Parkinson's disease.

[43] The method for preventing, treating, or alleviating symptoms of a disease according to

[42] , which prevents, treats, or alleviates one or more symptoms selected from the group consisting of tremor, rigidity, and bradykinesia.

[44] The method for preventing, treating, or alleviating symptoms of a disease according to

[42] or

[43] , wherein the protein is α-synuclein.

[45] The method for preventing, treating, or alleviating symptoms of a disease according to

[32] , wherein the disease is Huntington's disease.

[46] The method for preventing, treating, or alleviating symptoms of the disease according to

[45] , which prevents, treats, or alleviates symptoms of uncontrollable movement, cognitive decline, or both.

[47] The method for preventing, treating, or alleviating symptoms of the disease according to

[45] or

[46] , wherein the protein is huntingtin.

[48] The method for preventing, treating, or alleviating symptoms of the disease according to

[32] , wherein the disease is multiple system atrophy.

[49] The method for preventing, treating, or alleviating symptoms of the disease according to

[48] , which prevents, treats, or alleviates symptoms of movement disorders, autonomic failure, or both.

[50] The method for preventing, treating, or alleviating symptoms of the disease according to

[48] or

[49] , wherein the protein is α-synuclein.

[51] The method for preventing, treating, or alleviating symptoms of the disease according to

[31] , wherein the disease is cancer.

[52] The method for preventing, treating, or alleviating symptoms of the disease according to

[51] , wherein the disease is breast cancer.

[53] A method for preventing, treating, or alleviating symptoms of a disease according to

[52] , which prevents, treats, or alleviates symptoms of breast lumps, changes in breast shape, or both.

[54] A method for preventing, treating, or alleviating symptoms of a disease according to

[52] or

[53] , wherein the protein is BRCA1 / BRCA2, Hsf1, or both.

[55] A method for preventing, treating, or alleviating symptoms of a disease according to

[51] , wherein the disease is ovarian cancer.

[56] The method for preventing, treating, or alleviating symptoms of the disease according to

[55] , which prevents, treats, or alleviates symptoms of abdominal bloating, pelvic pain, or both.

[57] The method for preventing, treating, or alleviating symptoms of the disease according to

[55] or

[56] , wherein the protein is BRCA1 / BRCA2.

[58] The method for preventing, treating, or alleviating symptoms of the disease according to

[51] , wherein the disease is acute myeloid leukemia (AML).

[59] The method for preventing, treating, or alleviating symptoms of the disease according to

[58] , which prevents, treats, or alleviates symptoms of one or more selected from the group consisting of fatigue, frequent infections, and easy bruising.

[60] The method for preventing, treating, or alleviating symptoms of the disease according to

[58] or

[59] , wherein the protein is nucleophosmin (NPM1).

[61] The method for preventing, treating, or alleviating symptoms of the disease according to

[51] , wherein the disease is acute promyelocytic leukemia (APL).

[62] The method for preventing, treating, or alleviating symptoms of a disease according to

[61] , wherein the method prevents, treats, or alleviates both of one or more symptoms selected from the group consisting of fatigue, bleeding, and fever.

[63] The method for preventing, treating, or alleviating symptoms of a disease according to

[61] or

[62] , wherein the protein is PML.

[0007] The compounds of the present invention are useful as binding reagents to proteins for NMR measurements.

[0008] 1 is a schematic diagram showing the three-dimensional structure of a protein that can be used in the present invention. FIG. 2 is a schematic diagram for explaining the mechanism of photoexcited chemically induced dynamic nuclear polarization used in the present invention. FIG. 3 is a schematic diagram showing the dye-modified position of GB1 used in Example 1 and the target region of dynamic nuclear polarization. FIG. 4 is a schematic diagram showing the dye-modified position of the GB1 mutant used in Example 2 and the target region of dynamic nuclear polarization. FIG. 5 is a schematic diagram showing the dye-modified position of TFSBD used in Example 3 and the target region of dynamic nuclear polarization. FIG. 6 is a schematic diagram showing the three-dimensional structures of a complex of Compound 1 and GB1, a complex of Compound 2 and GB1, a complex of Compound 2 and a GB1 mutant, and a complex of Compound 2 and TFSBD. FIG. 7 shows absorption and emission spectra of a complex of Compound 2 and GB1, and Compound 2. FIG. 8 shows absorption and emission spectra of a mixture of Comparative Compound A and GB1, and Comparative Compound A. FIG. 9 is a schematic diagram showing the pulse sequence of the NMR measurement performed in this example. The complex of Compound 1 and GB1 was measured under light irradiation conditions and dark conditions. １ 1H-NMR spectra of the complex of Compound 2 and GB1 measured under light irradiation and dark conditions. １ 1H-NMR spectra of the complex of Compound 2 and the GB1 mutant measured under light irradiation and dark conditions. １ 1H-NMR spectra of the complex of Compound 2 and TFSBD measured under light irradiation and dark conditions. １ 1 H-NMR spectrum.

[0009] The present invention will be described in detail below. The following description of the constituent elements may be based on typical embodiments or specific examples of the present invention, but the present invention is not limited to such embodiments or specific examples. In this specification, a numerical range expressed using "to" means a range including the numerical values ​​before and after "to" as the lower and upper limits. In addition, the compound (H ２ O and D ２ The isotopes of hydrogen atoms present in the molecule (excluding O) are not particularly limited, and for example, １ H, or part or all of ２The "excitation light" in this specification refers to light that excites an object to cause it to emit light, and light having a wavelength that matches the absorption wavelength of the object can be used. The "room temperature" in this specification is 20°C.

[0010] <First Aspect of the Compound> The compound of the first aspect of the present invention has a structure in which a dye for chemically induced dynamic nuclear polarization is covalently bonded to a linking group having a functional group for binding to a protein. In the present invention, the "dynamic nuclear polarization" of the "dye for chemically induced dynamic nuclear polarization" means that nuclear spins are highly polarized by the dye. Here, "polarization" or "spin polarization" refers to the fact that when a static magnetic field is applied to an aggregate of spins to cause Zeeman splitting, the number of spins occupying each energy level that is split is different from that of the other energy levels. Furthermore, when a combination of any two of the split energy levels is considered, the number of spins occupying each energy level, N, is the number of spins occupying each energy level that is split. １ and the number of spins in the other energy level, N ２ to the total number of spins, i.e., (N １ -N ２ ) / (N １ +N ２) is called the polarization rate. Here, a Zeeman-split energy level combination with a polarization rate of 0 is considered spin-unpolarized, while a Zeeman-split energy level combination with a polarization rate greater than 0 (positive or negative) is considered spin-polarized. The greater the polarization rate, the greater the spin polarization, indicating an excess of spin at one of the energy levels. In this specification, a "dye for chemically induced dynamic nuclear polarization" refers to a dye that can highly polarize the nuclear spin of a protein by reacting the functional group of the linking group attached to the dye with the protein via a covalent bond. For example, a dye that undergoes electron transfer with a protein upon photoexcitation can be used as a dye for chemically induced dynamic nuclear polarization. In one embodiment of the present invention, a dye that transfers electrons from a photoexcited triplet state to a protein is used. In another embodiment of the present invention, a dye that transfers electrons from a photoexcited singlet state to a protein is used. Examples of dyes for chemically induced dynamic nuclear polarization include compounds containing a riboflavin structure, a lumiflavin structure, a flavin mononucleotide structure, a flavin structure, a fluorescein structure, a porphyrin structure, a 2,2'-bipyridyl structure, a benzophenone-3,3',4,4'-tetracarboxylic acid structure, or an eosin Y structure. In these structures, at least one hydrogen atom may be substituted with a substituent, or one hydrogen atom may be removed and the remaining atom may serve as a bonding point with a linking group. The substituent may be selected from, for example, Substituent Group A, Substituent Group B, Substituent Group C, Substituent Group D, or Substituent Group E. Furthermore, the porphyrin structure may be substituted with a group containing a cationic or anionic group.

[0011] The porphyrin structure contained in the dye can be exemplified by the structure represented by the following general formula (1):

[0012] In general formula (1), R １１ ~R １８ Each independently represents a hydrogen atom or a substituent. １~Ar ４ represents a substituted or unsubstituted arylene group bonded to a linking group, and Ar １ ~Ar ４ The remainder of R each independently represents a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group. １１ ~R １８ The substituent in Ar may be selected from, for example, Substituent Group A, Substituent Group B, Substituent Group C, Substituent Group D, or Substituent Group E. １ ~Ar ４ The aryl group in the formula (I) is preferably substituted with a cationic group or an anionic group. The cationic group may be a cationic group or a group that becomes a cationic group by receiving a proton. Specific examples of the cationic group include a substituted or unsubstituted amino group and an ammonium group. The anionic group may be an anionic group or a group that becomes an anionic group by ionizing a proton. Specific examples of the anionic group include a carboxy group (-COOH), a phosphono group (-PO ３ H ２ ), a phosphonoxy group (—OPO ３ H ２ ), sulfo group (—SO ３ H), sulfoxy group (-OSO ３ Examples of the anionic group include a cation group (C), a hydroxy group (-H), a hydroxyl group (-OH), and an anionic group formed by ionization of a proton from these groups. The cationic group and the anionic group may form an ion pair with an anion and a cation, respectively. Examples of the anion include a halogen ion and a compound having the above-mentioned anionic group, and examples of the cation include an ammonium ion. Ar １ ~Ar ４The heteroaryl group in is preferably a cationic heteroaryl group such as a pyridinium group or an N-substituted pyridinium group. Examples of the substituent of the N-substituted pyridinium include a substituted or unsubstituted alkyl group (e.g., having 1 to 20 carbon atoms). The pyridinium group or the N-substituted pyridinium group may form an ion pair with an anion. Examples of the anion include a halogen ion and a compound having the above-mentioned anionic group. Ar １ ~Ar ４ The arylene group in the formula (I) is preferably a substituted or unsubstituted 1,4-phenylene group, and a linking group is covalently bonded to the 4-position.

[0013] Specific examples of dyes usable in the present invention are shown below. However, the dyes usable in the present invention should not be construed as being limited by the following specific examples. At least one hydrogen atom in the structural formula of the dye below may be substituted with a substituent, or one hydrogen atom of the dye may be removed and the remaining atom may serve as the point of attachment to the linking group. The substituent may be selected, for example, from Substituent Group A, Substituent Group B, Substituent Group C, Substituent Group D, or Substituent Group E. The position of the dye that serves as the point of attachment to the linking group is not particularly limited. For example, in a dye having a porphyrin structure, one of the cyclic groups bonded to the 5-, 10-, 15-, and 20-positions of the porphyrin skeleton may be replaced with a substituted or unsubstituted 1,4-phenylene group, and the 4-position of the phenylene group may serve as the point of attachment to the linking group.

[0014]

[0015]

[0016]

[0017] In the present invention, the term "linking group" refers to an atomic group that includes an atom serving as a point of attachment to the dye via a covalent bond and has a functional group for binding to a protein. Here, the point of attachment to the dye via a covalent bond may be an atom of the atomic group that constitutes the functional group, or an atom that constitutes the linking chain that links the dye and the functional group. The number of atoms in the linking chain of the linking group can be, for example, 0 to 20, preferably 0 to 14, more preferably 1 to 10, and even more preferably 2 to 8. Here, the "number of atoms in the linking chain" refers to the number of atoms (linking chain) that are linearly linked between the dye atom covalently bonded to the atom in the linking group and the functional group. For example, in the compound below, the terminal maleimide structure corresponds to the functional group, and -NH-CO-CH-CH- corresponds to the linking chain, and the number of atoms in the linking chain is 4.

[0018]

[0019] Note that a linking chain atom count of "0" means that no atoms exist between the dye and the functional group (the dye and the functional group are directly bonded). A linking chain atom count of "1" means that the dye and the functional group are connected by one atom. Note that another atom or atomic group may be bonded to the atom between the dye and the functional group. It is also preferable to select the number of linking chain atoms so that the distance from the center of the dye to the end of the linking group is 8 to 35 Å. The distance from the center of the dye to the end of the linking group can be calculated using density functional theory. An example of a linking group is a group represented by the following general formula (2). In general formula (2), * represents the bonding position with the dye, and ** represents the bonding position with the functional group. m represents an integer of 1 to 20, and may be an integer of 2 to 15 or 2 to 12.

[0020] General formula (2)

[0021] In the present invention, the term "functional group" refers to an atom or atomic group that can react with a protein to form a covalent bond and link the dye to the protein. The functional group in the present invention is preferably one that reacts with an amino acid of a protein to form a bond, and is also preferably one that reacts with a mercapto group of a protein to form a bond. Examples of functional groups include atomic groups containing a maleimide structure or an acetamide structure, an isocyanate group, or an activated ester group. At least one hydrogen atom in the maleimide structure or acetamide structure may be substituted with a substituent, or one hydrogen atom in these structures may be removed and the remaining atom may serve as the bonding point to the linking chain. The substituent may be selected, for example, from Substituent Group A, Substituent Group B, Substituent Group C, Substituent Group D, or Substituent Group E. The position of the functional group in the linking group is not particularly limited, but is preferably at the terminal of the linking group. That is, the functional group is preferably bonded to an atom at the terminal of the linking chain that constitutes the linking group.

[0022] Specific examples of functional groups used in the present invention are shown below. However, the functional groups that can be used in the present invention should not be construed as being limited by the following specific examples. In the structural formulas and reaction formulas of the functional groups below, R represents a dye or a linking chain to which a dye is bonded. That is, in the structural formulas shown as representing functional groups, the portion excluding R corresponds to the functional group. R ａ１ -CH(NH ２ ) COOH, R ａ２ Ha-CH ２ CH(NH ２ ) COOH, R ａ３ represents the phenylalanine structure. １ ~R ５ represents a hydrogen atom or a substituent, R ２ ~R ５One of the functional groups is a dye or a linking chain to which the dye is bonded. In addition, at least one hydrogen atom in the structural formula below may be substituted with a substituent. First, examples of functional groups include those containing a maleimide structure or an acetamide structure represented by the structural formula below.

[0023]

[0024] As shown in the structural formula below, these functional groups react with, for example, a mercapto group contained in a cysteine ​​residue of a protein to form a sulfide bond, thereby linking the dye to the protein.

[0025]

[0026] Further examples of functional groups include those represented by the following structural formula: These selectively react with lysine residues in proteins to link the dye to the protein.

[0027] The reaction formula in which these functional groups react with lysine is shown below. In the reaction formula below, the compound to the right of the "+" is lysine, and the compound to the right of the arrow is a complex of the dye and lysine produced by the reaction. In a protein containing a lysine residue, these functional groups react with the lysine residue to form a linkage structure similar to that shown to the right of the arrow in the reaction formula below, and the dye is linked to the protein.

[0028]

[0029] Furthermore, functional groups that undergo a click reaction with a functional group introduced into a protein to form a bond can also be used. Examples of functional groups that undergo a click reaction include alkyne structures in which carbon atoms may be substituted, particularly medium-sized ring alkyne structures in which carbon atoms may be substituted. For example, functional groups that undergo a click reaction include those having a DACN (4,8-diazacyclononyne) structure represented by the following structural formula:

[0030]

[0031] The functional group having the DACN structure is an azide group (-N ３The protein undergoes a click reaction without a catalyst to form a linking structure having a triazole structure. In the following structural formula, Pro represents the protein.

[0032]

[0033] In the present invention, examples of click reactions that can be used to react functional groups with proteins are listed below: In the reaction scheme below, the compound on the left side of the "+" is an unnatural amino acid, the compound on the right side of the "+" is a dye linked to a functional group that undergoes a click reaction, and the compound on the right side of the arrow is a conjugate of the dye and unnatural amino acid produced by the reaction.

[0034]

[0035] Unnatural amino acids that can be used for the above-mentioned Click reaction also include compounds represented by the following structural formulas:

[0036]

[0037] By attaching a linking group having a functional group that undergoes a Click reaction to a dye and modifying a protein so that it contains one of the unnatural amino acids listed above as an amino acid residue, the dye can be linked to the protein using the Click reaction.

[0038] Specific examples of the first aspect of the compound are shown below, but the compounds that can be used in the present invention should not be construed as being limited by these specific examples.

[0039]

[0040] <Protein-Binding Reagent> The protein-binding reagent of the present invention includes a compound having a structure in which a dye for chemically induced dynamic nuclear polarization is covalently bonded to a linking group having a functional group for binding to a protein. For an explanation of the "compound" used in the present invention, please refer to the description in the "First Aspect of Compound" section above. In the compound used in the present invention, the linking group covalently bonded to the dye has a functional group for binding to a protein. Therefore, when the compound is contacted with a protein, the functional group reacts with the protein to form a covalent bond, thereby producing a compound (dye-protein complex) having a structure in which the dye and protein are bound via the linking group. In the complex thus produced, the dye enters a triplet excited state upon irradiation with excitation light, forming a radical pair with the protein and highly polarizing the nuclear spin of the protein. This increases the NMR signal intensity of the protein and improves the sensitivity of NMR measurements. Therefore, the protein-binding reagent of the present invention can be effectively used as a binding reagent for chemically induced dynamic nuclear polarization or a binding reagent for NMR measurements, which highly polarize proteins by chemically induced dynamic nuclear polarization through photoexcitation of the dye. The mechanism of chemically induced dynamic nuclear polarization used in the present invention is shown in Figure 2. Chemically induced dynamic nuclear polarization consists of a dye excitation step and a high-polarization step. [1] Dye excitation step: In this step, the dye is irradiated with excitation light to transition to an excited triplet state. Irradiation with excitation light causes the dye to transition from the ground singlet state to an excited singlet state, as shown in Figure 2, and then intersystem crossing from the excited singlet state occurs to transition to an excited triplet state. [2] High-polarization step: In this step, electron transfer occurs between the dye in the triplet excited state and the protein, resulting in the formation of a triplet radical pair. The formed triplet radical pair transitions to a singlet radical pair through spin-selective intersystem crossing, at which time the nuclear spins become highly polarized. Proteins with highly polarized nuclear spins can produce high-intensity NMR signals, enabling high measurement sensitivity in NMR spectroscopy and MRI. In addition, the above steps [1] and [2] may be performed such that step [2] is performed after step [1] is performed, or step [1] and step [2] may be performed simultaneously.

[0041] The protein to which the binding reagent of the present invention binds (the target protein) is not particularly limited, and examples thereof include proteins containing an amino acid residue having a mercapto group (e.g., a cysteine ​​residue or a homocysteine ​​residue), proteins containing a lysine residue, and modified proteins containing a non-natural amino acid residue into which a functional group such as an azide group has been introduced. For the bond (linking structure) formed when a functional group of a compound reacts with a protein, the description in the "First Aspect of the Compound" section above can be referenced. In one aspect of the present invention, a covalent bond is formed when a functional group of a compound reacts with an amino acid of a protein. In another aspect of the present invention, a sulfide bond is formed when a functional group of a compound reacts with a mercapto group of a protein. For specific examples of the target protein to which the binding reagent of the present invention binds, the description in the "Second Aspect of the Compound" section below can be referenced.

[0042] <Second Aspect of the Compound> The compound of the second aspect of the present invention has a structure in which a dye and a protein that are highly polarized by chemically induced dynamic nuclear polarization due to photoexcitation of the dye are covalently bonded via a linking group. In the following description, a compound having a structure in which a dye and a protein are covalently bonded via a linking group may be referred to as a "dye-protein complex" or a "complex." For a description of the dye and linking group used in the present invention, please refer to the descriptions of "Dye for Chemically Induced Dynamic Nuclear Polarization" and "Linking Group" in the description of the first aspect of the compound above. However, the "linking group" in the second aspect has a linking structure that includes a covalent bond formed by the reaction of a functional group with a protein, instead of a functional group. For the linking structure, please refer to the description of the link between a dye and a protein in the description of the first aspect of the compound above. The linking structure may include, for example, a sulfide bond, an amide bond, or a triazole structure. Specific examples of proteins that can be used in the present invention include GB1, FKBP12, Hsf1, TDP-43, FUS, tau, amyloid beta, α-synuclein, huntingtin, BRCA1 / BRCA2, nucleophosmin (NPM1), PML, and TFSBD. Furthermore, the proteins used in the present invention may be mutants of these proteins in which the reactive region with a compound or the target region for dynamic nuclear polarization has been altered, or may be modified proteins in which a functional group such as an azide group has been introduced. The three-dimensional structures of GB1, FKBP12, Hsf1, and TFSBD are shown in Figure 1 as representative examples. However, the proteins that can be used in the present invention should not be construed as being limited by these specific examples.

[0043] In the compound of the second aspect, the dye enters a triplet excited state upon photoexcitation, and the nuclear spins of proteins can be highly polarized by chemically induced dynamic nuclear polarization, thereby increasing the NMR signal intensity of the protein and improving the sensitivity of NMR measurements. Therefore, the compound of the second aspect can be effectively used as a component of a composition for NMR measurements and a composition for chemically induced dynamic nuclear polarization. For the mechanism of chemically induced dynamic nuclear polarization, please refer to the description in the above section on "Reagents for binding to proteins."

[0044] <Method of synthesizing compounds of the first and second aspects> The compounds of the first and second aspects described above are novel compounds. These compounds can be synthesized using known reactions. For specific conditions and procedures for the synthesis methods, please refer to the descriptions in the Examples section.

[0045] In the present specification, the term "alkyl group" refers to a group having a chemical structure that may be linear, branched, or cyclic. The alkyl group may also contain two or more of the linear, cyclic, and branched groups. The alkyl group may have, for example, one or more, two or more, or four or more carbon atoms. The alkyl group may have 30 or less, 20 or less, 10 or less, 6 or less, or 4 or less carbon atoms. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, an n-hexyl group, an isohexyl group, a 2-ethylhexyl group, an n-heptyl group, an isoheptyl group, an n-octyl group, an isooctyl group, an n-nonyl group, an isononyl group, an n-decanyl group, an isodecanyl group, a cyclopentyl group, a cyclohexyl group, and a cycloheptyl group. In this specification, the term "aryl group" refers to a group consisting of a monocyclic aromatic ring, a fused ring in which two or more aromatic rings are fused, or a linked ring in which two or more aromatic rings are linked. When two or more aromatic rings are linked, they may be linked in a linear or branched manner. The number of carbon atoms in the aromatic ring constituting the aryl group is preferably 6 to 22, more preferably 6 to 18, even more preferably 6 to 14, and even more preferably 6 to 10. Specific examples of the aryl group include a phenyl group, a naphthalenyl group, and a biphenyl group. In this specification, the term "arylene group" refers to a group consisting of a monocyclic aromatic ring, a fused ring in which two or more aromatic rings are fused, or a linked ring in which two or more aromatic rings are linked. When two or more aromatic rings are linked, they may be linked in a linear or branched manner. The number of carbon atoms in the aromatic ring constituting the arylene group is preferably 6 to 22, more preferably 6 to 18, even more preferably 6 to 14, and even more preferably 6 to 10. Specific examples of the arylene group include a phenylene group and a naphthylene group. In this specification, the "heteroaryl group" may be composed of a single ring or may be composed of a fused ring in which one or more heterocycles are fused with an aromatic ring or a heterocycle.The number of carbon atoms in the heterocyclic ring constituting the heteroaryl group is preferably 3 to 40, more preferably 5 to 22, even more preferably 5 to 18, still more preferably 5 to 14, and particularly preferably 5 to 10. Examples of heteroatoms constituting the heterocyclic ring include a nitrogen atom, an oxygen atom, and a sulfur atom. Specific examples of the heterocyclic ring include a pyridine ring, a pyridazine ring, a pyrimidine ring, a triazole ring, a benzotriazole ring, and a pyridinium ring.

[0046] In the present specification, when a "substituent" or "substituted or unsubstituted" is used, the substituent may be selected, for example, from the following Substituent Group A, Substituent Group B, Substituent Group C, Substituent Group D, or Substituent Group E. In the present specification, "Substituent Group A" refers to a hydroxyl group, a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, an iodine atom), an alkyl group (e.g., having 1 to 40 carbon atoms), an alkoxy group (e.g., having 1 to 40 carbon atoms), an alkylthio group (e.g., having 1 to 40 carbon atoms), an aryl group (e.g., having 6 to 30 carbon atoms), an aryloxy group (e.g., having 6 to 30 carbon atoms), an arylthio group (e.g., having 6 to 30 carbon atoms), a heteroaryl group (e.g., having 5 to 30 atoms constituting the ring skeleton), a heteroaryloxy group (e.g., having 5 to 30 atoms constituting the ring skeleton ... It means one group or a combination of two or more groups selected from the group consisting of a heteroarylthio group (for example, having 5 to 30 atoms constituting the ring skeleton), an acyl group (for example, having 1 to 40 carbon atoms), an alkenyl group (for example, having 1 to 40 carbon atoms), an alkynyl group (for example, having 1 to 40 carbon atoms), an alkoxycarbonyl group (for example, having 1 to 40 carbon atoms), an aryloxycarbonyl group (for example, having 1 to 40 carbon atoms), a heteroaryloxycarbonyl group (for example, having 1 to 40 carbon atoms), a silyl group (for example, a trialkylsilyl group having 1 to 40 carbon atoms), and a nitro group. In the present specification, "substituent group B" refers to one group or a combination of two or more groups selected from the group consisting of alkyl groups (e.g., having 1 to 40 carbon atoms), alkoxy groups (e.g., having 1 to 40 carbon atoms), aryl groups (e.g., having 6 to 30 carbon atoms), aryloxy groups (e.g., having 6 to 30 carbon atoms), heteroaryl groups (e.g., having 5 to 30 ring atoms), heteroaryloxy groups (e.g., having 5 to 30 ring atoms), and diarylamino groups (e.g., having 0 to 20 carbon atoms). In the present specification, "substituent group C" refers to one group or a combination of two or more groups selected from the group consisting of alkyl groups (e.g., having 1 to 20 carbon atoms), aryl groups (e.g., having 6 to 22 carbon atoms), heteroaryl groups (e.g., having 5 to 20 ring atoms), and diarylamino groups (e.g., having 12 to 20 carbon atoms).As used herein, "substituent group D" refers to one group or a combination of two or more groups selected from the group consisting of alkyl groups (e.g., having 1 to 20 carbon atoms), aryl groups (e.g., having 6 to 22 carbon atoms), and heteroaryl groups (e.g., having 5 to 20 ring skeleton atoms). As used herein, "substituent group E" refers to one group or a combination of two or more groups selected from the group consisting of alkyl groups (e.g., having 1 to 20 carbon atoms) and aryl groups (e.g., having 6 to 22 carbon atoms).

[0047] <Method for Analyzing Protein Properties> The method for analyzing protein properties of the present invention (hereinafter sometimes referred to as the "protein property analysis method") is characterized by comprising: a first step of synthesizing a molecule having a dye bound to a protein; a second step of irradiating the dye with light to highly polarize the protein using chemically induced dynamic nuclear polarization; and a third step of performing NMR measurement of the molecule. For an explanation of the "molecule having a dye bound to a protein" in the present invention, please refer to the description in the "Second Aspect of Compound" above. For an explanation of "chemically induced dynamic nuclear polarization" and "NMR measurement," please refer to the corresponding descriptions in the "Reagent Binding to Protein" above. Furthermore, "NMR measurement" can be performed using any known method. In the protein property analysis method of the present invention, it is preferable that in the first step, multiple molecules having dyes bound to different sites on the protein are synthesized, the second step is performed for each of the synthesized molecules, and the third step is performed for each molecule that has been subjected to the second step. It is also preferable that in the first step, the dye is bound to the protein via a linking group.

[0048] By using the protein characterization method of the present invention, a database can be obtained that stores information showing the relationship between molecules obtained by carrying out the method and NMR measurement results. Furthermore, a molecular design method can be constructed that performs molecular design based on information showing the relationship between molecules obtained by carrying out the protein characterization method of the present invention and NMR measurement results, and a program for carrying out such a molecular design method can be realized.

[0049] Furthermore, by performing the protein characterization method of the present invention on disease-related proteins to obtain information on their properties, disease prevention, treatment, or symptom alleviation can be performed based on that information. In the following description, a protein whose properties are analyzed using the protein characterization method of the present invention is referred to as a "target protein." Examples of target diseases for prevention, treatment, or symptom alleviation include neurological disorders such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple system atrophy. In amyotrophic lateral sclerosis (ALS), symptoms such as muscle weakness and paralysis can be targeted for prevention, treatment, or symptom alleviation. In this case, the target protein can be, for example, TDP-43, FUS, or both. In frontotemporal dementia (FTD), symptoms such as personality changes and cognitive decline can be targeted for prevention, treatment, or symptom alleviation. In this case, the target protein can be, for example, TDP-43, FUS, or both. In Alzheimer's disease, symptoms such as memory loss and cognitive impairment can be prevented, treated, or alleviated. In this case, the target protein can be, for example, tau, amyloid beta, or both. In Parkinson's disease, symptoms such as tremor, rigidity, and bradykinesia can be prevented, treated, or alleviated. In this case, the target protein can be, for example, α-synuclein. In Huntington's disease, symptoms such as uncontrollable movements and cognitive decline can be prevented, treated, or alleviated. In this case, the target protein can be, for example, huntingtin. In multiple system atrophy, symptoms such as movement disorders and autonomic failure can be prevented, treated, or alleviated. In this case, the target protein can be, for example, α-synuclein.

[0050] Examples of diseases for which prevention, treatment, or symptom alleviation is desired include cancers such as breast cancer, ovarian cancer, acute myeloid leukemia (AML), and acute promyelocytic leukemia (APL). For breast cancer, symptoms such as breast lumps and changes in breast shape can be prevented, treated, or alleviated. In this case, the target protein can be, for example, BRCA1 / BRCA2, Hsf1, or both. For ovarian cancer, symptoms such as abdominal bloating and pelvic pain can be prevented, treated, or alleviated. In this case, the target protein can be, for example, BRCA1 / BRCA2. For acute myeloid leukemia (AML), symptoms for which prevention, treatment, or symptom alleviation can be desired include one or more symptoms selected from the group consisting of fatigue, frequent infections, and easy bruising. In this case, the target protein can be, for example, nucleophosmin (NPM1). For acute promyelocytic leukemia (APL), symptoms for which prevention, treatment, or symptom alleviation can be desired include one or more symptoms selected from the group consisting of fatigue, bleeding, and fever. In this case, for example, PML can be used as the protein to be analyzed.

[0051] The features of the present invention will be explained in more detail below with reference to examples. The materials, processing details, processing procedures, etc. shown below can be modified as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below. NMR signals were measured using an NMR device (Bruker: Ultra-shield Plus 500 MHz).

[0052] (Synthesis Example 1) Synthesis of Compounds 1 to 3 Synthesis of Compound 1a

[0053] Pyrrole (5.4 mL, 72 mmol) was added to a propionic acid (180 mL) solution containing 4-pyridinecarboxaldehyde (4.9 mL, 52 mmol) and 4-acetamidobenzaldehyde (3.55 g, 21.5 mmol). This mixed solution was heated to 160°C and reacted for 2 hours. After completion of the reaction, the mixed solution was cooled to room temperature, and the solvent was distilled off under reduced pressure. The resulting residue was subjected to silica gel column chromatography (developing solvent: dichloromethane / methanol = 20 / 1), and the product was recrystallized in methanol to give Compound 1a (805 mg, yield 6.9%) as purple crystals. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 9.06-9.05 (m, 6H), 8.94 (d, 2H), 8.85-8.82 (m 6H), 8.17-8.15 (m, 8H), 7.94 (d, 2H), 2.38 (s. 3H), -2.88 (s, 2H), MS (MALDI-TOF) m / z 675 [M+H] +

[0054] Synthesis of Compound 1b

[0055] Compound 1a (420.6 mg, 0.62 mmol) was added to an aqueous hydrochloric acid solution (6 M, 150 mL) and heated to reflux at 100°C for 4 hours. The reaction solution was cooled to room temperature, and the solvent was removed under reduced pressure. The resulting residue was dissolved in dichloromethane / triethylamine (9 / 1, 300 mL) and washed three times with water (150 mL). The organic layer was separated, dried over anhydrous sodium sulfate, and then filtered to recover the solution. After removing the solvent under reduced pressure, the residue was recrystallized in methanol to obtain compound 1b (367 mg, yield 93%) as purple crystals. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 9.05-9.01 (m, 8H), 8.84-8.80 (m, 6H), 8.82 (m, 6H), 8.00 (d, 2H), 7.09 (d, 2H), 4.09 (s, 2H), -2.85 (s, 2H) MS (MALDI-TOF) m / z 633 [M+H] +

[0056] Synthesis of Compound 3d

[0057] 12-Aminododecanoic acid (646 mg, 3 mmol) and maleic anhydride (353 mg, 3.6 mmol) were dissolved in acetic acid (20 mL). The resulting solution was heated at 120°C for 6 hours and then cooled to room temperature. Water (50 mL) was then added, and the precipitated solid was collected by filtration and washed with water. The resulting crude product was purified by flash column chromatography (developing solvent: ethyl acetate / hexane = 1 / 5) to give compound 3d (437 mg, yield 49%) as a white powder. 1 H-NMR (400 MHz, DMSO): δ (ppm) 11.95 (s, 1H), 7.00 (s, 2H), 3.39-3.36 (t, 2H), 2.20-2.16 (t, 2H), 1.48-1.45 (m, 4H), 1.22 (m, 14H)

[0058] Synthesis of Compounds 1d and 2d Compounds 1d and 2d were synthesized using 3-aminopropanoic acid or 6-aminohexanoic acid instead of 12-aminododecanoic acid.

[0059] Synthesis of Compounds 1e to 3e

[0060] 3-Maleimidopropionic acid (1b, 25.4 mg, 0.15 mmol) and N,N-dimethylformamide (catalytic amount, 1 drop) were dissolved in anhydrous dichloromethane (2 mL). Oxalyl chloride (38.6 μL, 0.45 mmol) was added dropwise to this solution in an ice bath, followed by stirring for 10 minutes and then at room temperature for 30 minutes. Volatile components were then removed under reduced pressure, and the residue was redissolved in anhydrous dichloromethane (2 mL). Compound 1b (31.7 mg, 0.05 mmol) and triethylamine (70 μL, 0.5 mmol) were added to the resulting solution in an ice bath, followed by stirring for 10 minutes and then at room temperature overnight. The reaction mixture was diluted with dichloromethane and washed with water. The organic layer was collected, dried over anhydrous sodium sulfate, and filtered to obtain a solution. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (developing solvent: dichloromethane / methanol=20 / 1) to give Compound 1e as purple crystals (30.8 mg, yield 79%). 1 H-NMR (400 MHz, CDCl3): δ (ppm) 10.49 (s, 1H), 9.05 (d, 6H), 8.94 (m, 2H), 8.90 (m, 6H), 8.28 (m, 6H), 8.15 (d, 2H), 8.02 (d, 2H) 7.11 (s, 2H), 3.88-84 (t, 2H), 2.81-77 (t, 2H), -3.01 (s, 2H) MS (MALDI-TOF) m / z 784 [M+H] +

[0061] Compounds 2e and 3e were synthesized in the same manner using compounds 2d and 3d instead of compound 1d. NMR analysis results for compound 2e 1H-NMR (400 MHz, CDCl3): δ (ppm) 9.09-9.07 (m, 6H), 8.97 (d, 2H), 8.88-8.84 (m, 6H), 8.20-8.18 (m, 8H), 7.98 (d, 2H), 6.76 (s, 2H), 3.65 (t, 2H), 2.58 (t, 2H), 1.95 (q, 2H), 1.77 (q, 2H), -2.86 (s, 2H) (2H of alkyl chain was missed due to the overlapping with water signal.) MS(MALDI-TOF) m / z 826 [M+H] + NMR analysis results of compound 3e 1 H-NMR (400 MHz, CDCl3): δ (ppm) 9.09-9.07 (m, 6H), 8.97 (d, 2H), 8.88-8.84 (m, 6H), 8.20-8.18 (m, 8H), 7.98 (d, 2H), 6.70 (s, 2H), 3.55 (t, 2H), 2.58 (t, 2H), 1.90 (q, 2H), 1.34-27 (m, 16H), -2.86 (s, 2H) MS(MALDI-TOF) m / z 910 [M+H] +

[0062] Synthesis of Compounds 1 to 3

[0063] Compound 1e (23.9 mg, 0.03 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 mL), and iodomethane (350 μL) was added dropwise to this solution. The mixture was then stirred at 45°C for 6 hours. After the reaction mixture was cooled to room temperature, diethyl ether was added, and the precipitated solid was collected by filtration. The resulting solid was washed with diethyl ether and dried to give Compound 1 (23.0 mg, yield 62%) as a purple solid. 1H-NMR (400 MHz, DMSO): δ (ppm) 10.52 (s, 1H), 9.46 (d, 6H), 9.13 (m, 4H), 9.05 (m, 4H), 9.00-8.97 (m, 6H), 8.15 (d, 2H), 8.06 (d, 2H) 7.12 (s, 2H), 4.71 (s, 9H), 3.88-84 (t, 2H), 2.82-79 (t, 2H), -3.01 (s, 2H)

[0064] Compounds 2 and 3 were synthesized in the same manner using compounds 2e and 3e instead of compound 1e. 1 H-NMR (400 MHz, DMSO): δ (ppm) 10.38 (s, 1H), 9.47 (d, 6H), 9.14 (s, 4H), 9.07-05 (m, 4H), 9.01-8.98 (m, 6H), 8.17-8.10 (q, 4H), 7.07 (s, 2H), 4.72 (s, 9H), 3.50-3.47 (t, 2H), 1.78-1.70 (q, 2H), 1.66-1.59 (q, 2H), 1.43-1.35 (q, 2H), -3.00 (s, 2H) (2H of alkyl chain was missed due to the overlapping with DMSO signal.) NMR analysis results of compound 3 1 H-NMR (400 MHz, DMSO): δ (ppm) 10.38 (s, 1H), 9.47 (d, 6H), 9.13 (s, 4H), 9.05 (m, 4H), 9.0 (m, 6H), 8.16-8.10 (q, 4H), 6.98 (s, 2H), 4.71 (s, 9H), 1.75-1.71 (m, 2H), 1.51-1.48 (m, 2H), 1.41-1.20 (m,br), -3.00 (s, 2H) (some alkyl chain was missed due to the overlapping with DMSO signal.)

[0065] (Synthesis Example 2) Synthesis of Compound 4 Synthesis of Compound 4a

[0066] Compound 4a was synthesized in the same manner as in the synthesis of compound 1a, except that methyl 4-formylbenzoate was used instead of 4-acetamidobenzaldehyde. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 9.06 (d, 6H), 8.86-8.83 (m, 8H), 8.46 (d, 2H), 8.29 (d, 2H), 8.16 (d, 6H), 4.12 (s, 3H), -2.88 (s, 2H)

[0067] Synthesis of Compound 4b

[0068] Compound 4a (570 mg, 0.84 mmol) was dissolved in ethanol (100 mL), and an aqueous solution (10 mL) of potassium hydroxide (1.15 g, 20.4 mmol) was added to this solution. The resulting mixed solution was stirred at 80°C overnight and then cooled to room temperature. The solvent was removed under reduced pressure, and the residue was neutralized with 1 M aqueous hydrochloric acid (25 mL). The precipitated solid was collected by filtration, washed with methanol, and dried to obtain compound 4b as a purple solid. 1 H-NMR (400 MHz, DMSO): δ (ppm) 9.05 (d, 6H), 8.90 (d, 8H), 8.39 (d, 2H), 8.33 (d, 2H), 8.27 (d, 6H), -3.02 (s, 2H)

[0069] Synthesis of Compound 4d

[0070] Compound 4b (16.6 mg, 25 μmol), compound 4c (7.2 mg, 30 μmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 11.5 mg, 60 μmol), 4-dimethylaminopyridine (DMAP, 7.3 mg, 60 μmol), and triethylamine (10 μL) were dissolved in anhydrous N,N-dimethylformamide. The resulting reaction mixture was stirred overnight at room temperature. After the reaction, the solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (developing solvent: 4% methanol in chloroform). After purification, the solvent was removed under reduced pressure to obtain compound 4d (11.9 mg, yield 56.4%) as a purple solid. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 9.09 (d, 6H), 8.89 (m, 8H), 8.31 (d, 2H), 8.19 (d, 6H), 7.92-7.86 (m, 2H), 4.65 (s, 0.6H), 4.48 (s, 1.2H), 4.12 (s, 2H), 4.00-3.95 (m, 2H), 3.63-3.58 (m, 2H), 2.96 (s, 3H), 2.48 (s, 2H), -2.87 (s, 2H)

[0071] Synthesis of Compound 4

[0072] Compound 4 was synthesized in the same manner as in the synthesis of Compound 1, except that Compound 4d was used in place of Compound 1e. 1 H-NMR (400 MHz, DMSO): δ (ppm) 9.47 (d, 6H), 9.14-8.98 (m, 14H), 8.30 (d, 2H), 7.92 (d, 2H), 4.71 (s, 9H), 4.56-4.51 (m, 2H), 4.10-4.06 (m, 2H), 3.81 (m, 2H), 3.03-3.00 (m, 3H), 2.20 (m, 1.3H), 2.04 (m, 0.7H), -3.03 (s, 2H)

[0073] (Synthesis Example 3) Synthesis of Compound 5 Synthesis of Compound 5a

[0074] Compound 4c (14.3 mg, 0.06 mmol) and glutaric anhydride (7.5 mg, 0.66 mmol) were dissolved in anhydrous dichloromethane (2 mL) containing triethylamine (20.2 mg, 0.2 mmol) and stirred at room temperature for 1 hour. 1M aqueous hydrochloric acid solution (2 mL) was added to the reaction mixture, followed by extraction with dichloromethane. The organic layer was separated, dried over anhydrous sodium sulfate, and filtered to recover the solution. The solvent was removed under reduced pressure to give compound 5a (15.5 mg, 82.0% yield) as a white solid. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 4.18 (d, 2H), 3.94 (d, 2H), 3.50 (t, 2H), 3.22 (m, 2H), 2.94 (d, 3H), 2.36-2.32 (m, 2H), 2.26-2.23 (m, 2H), 1.94-1.88 (m, 2H), 1.74-1.68 (m, 2H)

[0075] Synthesis of Compound 5

[0076] Compound 5b was synthesized using the same procedure as in the synthesis of compound 4d, except that compound 1b was used instead of compound 4b and compound 5a was used instead of compound 4c. Subsequently, compound 5 was synthesized using the same procedure as in the synthesis of compound 1, except that compound 5b was used instead of compound 1e.

[0077] (Synthesis Example 4) Synthesis of Compounds 6 to 8 Synthesis of Compound 6a

[0078] Compound 6a was synthesized in the same manner as in the synthesis of compound 1a, except that benzaldehyde was used instead of 4-pyridinecarboxaldehyde. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 8.86-8.84 (m, 8H), 8.22-8.16 (d, 8H), 7.90 (d, 2H), 7.78-7.74 (m, 10H), 2.37 (s. 3H), -2.78 (s, 2H)

[0079] Synthesis of Compound 6b

[0080] Compound 6b was synthesized in the same manner as in the synthesis of compound 1b, except that compound 6a was used instead of compound 1a. 1 H-NMR (400 MHz, CDCl3): δ (ppm) 8.93 (d, 2H), 8.83 (d, 6H), 8.21 (d, 6H), 8.00 (d, 2H), 7.77-7.74 (m, 10H), 7.07 (d, 2H), 4.04 (s. sH), -2.76 (s, 2H)

[0081] Synthesis of Compound 6c

[0082] Compound 6b (365 mg, 0.58 mmol) was added to concentrated sulfuric acid (8 mL) and stirred at 100°C for 10 hours. After cooling the reaction mixture to room temperature, saturated aqueous sodium bicarbonate solution was added dropwise in an ice bath to neutralize the solution until the color of the solution changed to red. The solvent was then removed under reduced pressure, methanol was added to the residue, and insoluble matter was removed by filtration. This procedure was repeated, and the solvent was then removed under reduced pressure to obtain compound 6c as a purple solid.

[0083] Synthesis of Compounds 6-8

[0084] Compounds 6 to 8 were synthesized in the same manner as in the synthesis of compounds 1c to 3c, except that compound 6c was used instead of compound 1b.

[0085] Synthesis of Compound 9 Compound 9 was synthesized in the same manner as in the synthesis of Compound 1.

[0086] Example 1: Synthesis of conjugates of compounds 1 to 8 and GB1 (protein) In this example, conjugates of compounds 1 to 8 and GB1 were synthesized. However, for the synthesis of conjugates of compounds 4 and 5 and GB1, modified GB1 in which an azide group was introduced into the phenylalanine residue (Ph) was used. The three-dimensional structure of GB1 is shown in Figure 3. In the three-dimensional structure on the right side of Figure 3, the "T25C dye-modified position" is the region where an SH group exists as a binding site, and the "K28Y target" is the target region where nuclear spins are highly polarized in the "CIDNP evaluation" described below. In the three-dimensional structure on the left side of Figure 3, the "T25AzPhe dye-modified position" is the region where an azide group exists as a binding site.

[0087] A mixed solution was prepared by adding a dimethyl sulfoxide solution (10 mM) of compound 1 in an equimolar amount to the protein to a solution (protein concentration: 20-30 μM) of reduced GB1 dissolved in phosphate-buffered saline. This mixed solution was reacted for 3 hours in the dark at room temperature, and then 2-mercaptoethanol was added to terminate the reaction. The progress of the addition reaction was confirmed by mass spectrometry and NMR measurement. The resulting reaction mixture was purified by anion exchange column chromatography using 50 mM Tris buffer (pH 8.0), and the solvent was then replaced with a measurement buffer (a heavy water solution containing 50 mM sodium phosphate buffer, pH 7.0, 100 mM sodium chloride, and 0.05% sodium azide) and concentrated to 500 μL to obtain a complex sample of compound 1 and GB1. Furthermore, complex samples of compounds 2-8 and GB1 were prepared using the same procedure, using compounds 2-8 instead of compound 1.

[0088] Example 2 Synthesis of a Conjugate of Compound 2 and a GB1 Mutant In this example, a GB1 mutant having a dye modification position of D36C and a target of Y33 was used to prepare a conjugate sample of Compound 2 and the GB1 mutant. The three-dimensional structure of the GB1 mutant is shown in Figure 4. The conjugate sample was prepared in the same manner as in Example 1, except that a reduced GB1 mutant was used instead of reduced GB1, and Compound 2 was used instead of Compound 1.

[0089] Example 3 Synthesis of Complexes of Compounds 1 to 8 with TFSBD (Protein) In this example, complex samples of Compounds 1 to 8 with the protein TFSBD were prepared. The three-dimensional structure of TFSBD is shown in Figure 5. In Figure 5, the "E383C dye-modified position" is the region where an SH group exists as a binding site, and the "Y378 target" is the target region where nuclear spins are highly polarized in the "CIDNP evaluation" described below. The complex samples were prepared using reduced TFSBD instead of reduced GB1, following the same procedure as in Example 1.

[0090] Of the conjugates synthesized in each Example, the three-dimensional structures of the conjugate between Compound 1 and GB1, the conjugate between Compound 2 and GB1, the conjugate between Compound 2 and a GB1 mutant, and the conjugate between Compound 2 and TFSBD are shown in Figure 6.

[0091] (Comparative Example 1) Preparation of a mixture of comparative compound A and GB1 The following comparative compound A (a dye having no linking group with a functional group) and GB1 were dissolved in equimolar amounts in a measurement buffer to prepare a mixture of comparative compound A and GB1.

[0092]

[0093] The complex samples prepared in each Example and the mixture prepared in Comparative Example 1 were mixed with a solution of 8 μM glucose oxidase and 50 mM D-glucose (solvent: deuterium solution containing 50 mM sodium phosphate buffer, pH 7.0, 100 mM sodium chloride, and 0.05% sodium azide) to prepare measurement samples containing final concentrations of 20 to 50 μM complex, 0.2 μM glucose oxidase, and 2.5 mM D-glucose. These samples were used for evaluating optical properties and chemically induced dynamic nuclear polarization (CIDNP). The mixture measurement samples were prepared so that the final concentrations of Compound 2 and Comparative Compound A were 20 to 50 μM. Furthermore, the measurement samples used for CIDNP evaluation further contained 3-(trimethylsilyl)-1-propanesulfonic acid (DSS) as an internal standard to a final concentration of 100 μM.

[0094] [Evaluation of Optical Properties] The results of measuring the absorption and emission spectra of the composite of Compound 2 and GB1, and Compound 2 are shown in Figure 7, and the results of measuring the absorption and emission spectra of the mixture of Comparative Compound A and GB1, and Comparative Compound A are shown in Figure 8. The excitation light wavelength when measuring the emission spectra was 500 nm. The fluorescence lifetimes calculated from the transient decay curves of emission measured with 405 nm excitation light were 6.73 nanoseconds for Compound 2, 3.26 nanoseconds for the composite of Compound 2 and GB1, 3.99 nanoseconds for Comparative Compound A, and 4.02 nanoseconds for the mixture of Comparative Compound A and GB1.

[0095] [Evaluation of Chemically Induced Dynamic Nuclear Polarization (CIDNP)] Each prepared measurement sample was placed in a 5 mm NMR tube and subjected to photo-CIDNP measurement. Specifically, a measurement device connected to a laser light source and an NMR spectrometer, capable of synchronously controlling the timing of light irradiation and NMR signal acquisition, was used. A 525 nm continuous wave laser (1.25 W) was introduced into the measurement sample in the NMR tube, and 500 MHz was measured. １ The H NMR spectrum was measured. In this example, the pulse sequence shown in FIG. 9 was used under light irradiation conditions (Light). １ After measuring the H NMR spectrum, １ The H NMR spectrum was measured. The pulse width was 16 microseconds. １ The H NMR spectrum of the complex of Compound 2 and GB1 is shown in FIG. １ The H NMR spectrum of the complex of Compound 2 with the GB1 mutant is shown in FIG. １ The H NMR spectrum of the complex of Compound 2 and TFSBD is shown in FIG. １ The H NMR spectrum is shown in Figure 13. In each figure, "Light" indicates measurements under light irradiation conditions. １ H NMR spectrum, "Dark" indicates measurements taken under dark conditions. １ H NMR spectrum is shown, and "Difference" was measured under dark conditions. １ H NMR spectrum and photoirradiation conditions were measured. １ The difference in intensity between the H NMR spectrum and the H NMR spectrum is shown. Looking at the difference in each figure, a valley is observed at 6.6-6.7 ppm for the complex of compound 1 and GB1 and the complex of compound 2 and GB1, two valleys are observed at 6.5-7.0 ppm for the complex of compound 2 and the GB1 mutant, and a valley is observed at 6.9-7.0 ppm for the complex of compound 2 and TFSBD. The valleys in the difference are due to the enhanced sensitivity caused by the induction of highly polarized nuclear spins of the protein by the excited dye under light irradiation conditions. These results confirmed that dyes covalently bonded to a functional linking group are useful as binding reagents for proteins in MMR measurements.

[0096] By using the protein-binding reagent of the present invention, the NMR signal intensity due to chemically induced dynamic nuclear polarization of the protein can be increased. Because proteins are involved in various diseases, information about the properties of proteins obtained from strong NMR signals can be effectively used to prevent, treat, or alleviate symptoms of the diseases. Therefore, the present invention has high industrial applicability.

Claims

1. A compound having a structure in which a dye for chemically induced dynamic nuclear polarization is covalently bonded to a linking group having a functional group for binding to a protein.

2. The compound according to claim 1, wherein the functional group comprises a maleimide structure or an acetamide structure.

3. The compound according to claim 1, wherein the functional group is an isocyanate group or an active ester group.

4. The compound according to any one of claims 1 to 3, wherein the functional group is bonded to the terminal of the linking group.

5. The compound according to any one of claims 1 to 4, wherein the linking group has a linking chain between the dye and the functional group having 0 to 20 atoms.

6. The compound according to any one of claims 1 to 5, wherein the distance from the center of the dye to the end of the linking group is 8 to 35 Å.

7. The compound of any one of claims 1 to 6, wherein the dye comprises a riboflavin structure, a lumiflavin structure, a flavin mononucleotide structure, a flavin structure, a fluorescein structure, a porphyrin structure, a 2,2'-bipyridyl structure, a benzophenone-3,3',4,4'-tetracarboxylic acid structure, or an eosin Y structure.

8. A protein binding reagent comprising a compound according to any one of claims 1 to 7.

9. The binding reagent of claim 8 for hyperpolarizing said protein by chemically induced dynamic nuclear polarization by photoexciting said dye.

10. The binding reagent of claim 8 or 9, wherein the functional group reacts with an amino acid of the protein.

11. The binding reagent according to claim 8 or 9, wherein the functional group reacts with a sulfhydryl group of the protein.

12. A compound having a structure in which a dye and a protein that becomes highly polarized by chemically induced dynamic nuclear polarization due to photoexcitation of the dye are covalently bonded via a linking group.

13. A composition for NMR measurements, comprising the compound according to claim 12.

14. A composition for chemically induced dynamic nuclear polarization, comprising the compound of claim 12.

15. A method for analyzing the properties of a protein, comprising: a first step of synthesizing a molecule in which a dye is bound to the protein; a second step of irradiating the dye with light to highly polarize the protein by chemically induced dynamic nuclear polarization; and a third step of performing NMR measurements of the molecule.

16. The method according to claim 15, wherein in the first step, a plurality of molecules are synthesized in which the dye is bound to different sites of the protein, the second step is carried out for each of the synthesized molecules, and the third step is carried out for each of the molecules that have been subjected to the second step.

17. A database storing information indicating the relationship between molecules obtained by carrying out the method according to claim 15 or 16 and NMR measurement results.

18. A molecular design method for designing a molecule based on information indicating the relationship between a molecule and NMR measurement results obtained by carrying out the method according to claim 15 or 16.

19. A program for carrying out the molecular design method according to claim 18.

20. A method for preventing, treating or alleviating symptoms of a disease, comprising performing the method described in claim 16 or 16 on a protein related to the disease to obtain information on the properties of the protein, and preventing, treating or alleviating symptoms of the disease based on that information.

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