Molecules for modifying proteins and / or peptides

Abstract

The present invention addresses the problem of providing a technology that allows more selective, easy and efficient bonding, to the N terminus of a natural protein and the like, of another molecule / substance. The problem is solved by reacting: a compound represented by general formula (1), a salt thereof, or a hydrate or a solvate of the compound or the salt thereof; and a protein and / or a peptide.

Description

[Technical field]

[0001] The present invention relates to molecules for modifying proteins and / or peptides, etc. [Background technology]

[0002] Techniques for linking other molecules / substances to proteins and / or peptides (hereinafter sometimes referred to as "proteins, etc.") are important techniques for producing antibody-drug conjugates, protein reagents labeled with fluorescent probes, protein-immobilized inorganic materials, etc. For example, azide group modification of proteins, etc. is a technique that enables the introduction of various functional molecules by alkyne-azide cycloaddition reaction (CuAAC), and is widely used in fields such as bioimaging because of its bioorthogonality. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Metal-free and pH-controlled introduction of azides in proteins, Sanne Schoffelen, Mark B. van Eldijk, Bart Rooijakkers, Reinout Raijmakers, Albert JR Heck and Jan CM van Hest, Chemical Science, 2011, 2, 701 [Non-Patent Document 2] Selective N-terminal acylation of peptides and proteins with a Gly-His tag sequence, MC Martos-Maldonado, CT Hjuler, KK Sorensen, MB Thygesen, JE Rasmussen, K. Villadsen, SR Midtgaard, S. Kol, S. Schoffelen, KJ Jensen, Nature Communication, 2018, 9, 3307. [Non-Patent Document 3] Modification of N-Terminal α-Amino Groups of Peptides and ProteinsUsing Ketenes, AO-Y. Chan, C.-M. Ho, H.-C. Chong, Y.-C. Leung, J.-S. Huang, M.-K. Wong, C.-M. Che, Journal of American Chemical Society, 2012, 134, 2589. Summary of the Invention [Problem to be solved by the invention]

[0004] In investigating the position of introduction when linking other molecules / substances to proteins, the present inventors focused on the following three points. First, the N-terminus is a position that all monomeric proteins have only one, making it a universal modification base point. Second, the N-terminus is rarely involved in protein active sites (molecular binding sites / catalytic reaction centers) or protein functional sites, and it is thought that the effects of structural changes accompanying modification are small. Third, the N-terminus has a pK a Due to the difference in pK, it is thought that the reaction is less likely to compete with other amino acid residues (lysine, cysteine, glutamine, etc.). aTherefore, the present inventors focused on the N-terminus of a protein or the like as a site for linking other molecules / substances.

[0005] Although various methods for linking other molecules / substances to the N-terminus of proteins and the like have been reported (Non-Patent Documents 1-3), these methods were considered to be insufficient in terms of their simplicity or N-terminus modification selectivity. For example, a chemical bonding method that recognizes a specific amino acid sequence or a lipid-modifying enzyme method can specifically introduce other molecules / substances to the N-terminus, but it is necessary to use a protein or the like into which a special amino acid sequence or a special amino acid residue has been inserted, so that the preparation requires a lot of effort and cannot be applied to natural proteins and the like. In addition, an amide bond formation reaction using an activated ester or ketene is easy to operate and can be applied to natural proteins and the like, but a side reaction with lysine residues and the like may proceed, so that it is not possible to specifically introduce other molecules / substances to the N-terminus.

[0006] Therefore, an objective of the present invention is to provide a technique that enables other molecules / substances to be linked simply and efficiently with higher selectivity to the N-terminus of any protein and / or peptide. [Means for solving the problem]

[0007] In view of the above problems, the present inventors have conducted intensive research and found that the above problems can be solved by reacting a compound represented by general formula (1), or a salt thereof, or a hydrate or solvate thereof, with a protein and / or a peptide. Based on this finding, the present inventors conducted further research and completed the present invention.

[0008] That is, the present invention includes the following aspects.

[0009] Section 1. General formula (1):

[0010] [ka]

[0011] [In the formula: R 1 and R 2 Of these, one is -N(-R 4 )-(R 4 represents an organic group or a group derived from an inorganic material. ) and the other represents =N-. R 3 represents a hydrogen atom, an organic group, or a group derived from an inorganic material. or a salt thereof, or a hydrate or solvate thereof.

[0012] Item 2. The compound is represented by general formula (1A)

[0013] [ka]

[0014] [In the formula: R 4 is the same as above.] Item 3. The compound according to item 1, which is represented by the following formula:

[0015] Item 3. The compound according to Item 1 or 2, or a salt thereof, or a hydrate or solvate thereof, wherein the organic group is a group derived from an organic molecule or an organic molecular complex, and the organic molecule or organic molecular complex is a functional substance.

[0016] Item 4. The compound according to Item 3, or a salt thereof, or a hydrate or solvate thereof, wherein the functional substance is a pharmaceutical compound, a luminescent molecule, a polymer compound, a ligand, a molecule to be bound to a ligand, an antigen protein, an antibody, a protein, a nucleic acid, a sugar, a lipid, a cell, a virus, a label, a carbon electrode, a carbon nanomaterial, a linker, or a spacer molecule, or a complex or linking molecule thereof.

[0017] Item 5. The compound according to any one of Items 1 to 4, or a salt thereof, or a hydrate or solvate thereof, wherein the inorganic material is an electrode material, metal fine particles, metal oxide fine particles, semiconductor particles, or magnetic particles.

[0018] Item 6. A reagent comprising the compound according to any one of items 1 to 5, or a salt thereof, or a hydrate or solvate thereof.

[0019] Item 7. The reagent according to Item 6, which is a reagent for modifying proteins and / or peptides.

[0020] Section 8. General formula (2): R 4 -R 5 (2) [In the formula: R 4 R represents an organic group or a group derived from an inorganic material. 5 -N 3 , -X (X represents a halogen atom), -B(OH) 2 , -B(OR 51 ) 2 (R 51 are the same or different and represent a hydrocarbon group. 51 may be bonded together to form a ring with the adjacent oxygen atom, or -N 2 + or a salt thereof, and a compound represented by general formula (3), a compound represented by general formula (4), or a compound represented by general formula (9):

[0021] [ka]

[0022] [In the formula: R 3 R represents a hydrogen atom, an organic group, or a group derived from an inorganic material. 6 , R 7 and R 8 are the same or different and each represents an alkyl group; Y represents a reactive group. 6. A method for producing the compound according to any one of items 1 to 5, or a salt thereof, or a hydrate or solvate thereof, comprising reacting

[0023] Section 9. General formula (5): R 4 -R 9 (5) [In the formula: R 4R represents an organic group or a group derived from an inorganic material. 9 -NR 9a R 9b (R 9a and R 9b are the same or different and represent a hydrogen atom or an alkyl group. ) and a compound represented by the general formula (6):

[0024] [ka]

[0025] [In the formula, R 10 R represents an electron-withdrawing group. 11 -R 11a -R 12 (R 11a R represents a single bond or a linker. 12 represents a carrier.) n represents 0 or 1. m represents an integer of 1 to 5.] 6. A method for producing the compound according to any one of items 1 to 5, or a salt thereof, or a hydrate or solvate thereof, comprising a step of reacting a compound represented by the following formula (1):

[0026] Section 10. General formula (6'):

[0027] [ka]

[0028] [In the formula, R 10 R represents an electron-withdrawing group. 11a R represents a single bond or a linker. 12 represents a carrier. m represents an integer of 1 to 5. A compound represented by the formula:

[0029] Section 11. General formula (7):

[0030] [ka]

[0031] [In the formula: R1 and R 2 Of these, one is -N(-R 4 )-(R 4 represents an organic group or a group derived from an inorganic material. ) and the other represents =N-. R 3 R represents a hydrogen atom, an organic group, or a group derived from an inorganic material. A double line consisting of a dotted line and a solid line represents a single bond or a double bond. 13 R represents a group obtained by removing the N-terminal amino acid residue and the adjacent -NH- from a protein or peptide. 14 represents the side chain of the N-terminal amino acid residue of the protein or peptide.] or a salt thereof, or a hydrate or solvate thereof.

[0032] Item 12. A method for producing the compound according to Item 11, or a salt thereof, or a hydrate or solvate thereof, comprising a step of reacting a protein and / or peptide with the compound according to any one of Items 1 to 5, or a salt thereof, or a hydrate or solvate thereof. Effect of the Invention

[0033] INDUSTRIAL APPLICABILITY The present invention can provide a technique that enables other molecules / substances to be linked simply and efficiently with high selectivity to the N-terminus of natural proteins and the like. [Brief description of the drawings]

[0034] [Figure 1] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 1 is shown. [Diagram 2] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 2 is shown. [Diagram 3] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 3 is shown. [Figure 4] The 1H NMR spectrum (400 MHz, CDCl3) of compound 4 is shown. [Diagram 5]The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 5 is shown. [Figure 6] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 6 is shown. [Figure 7] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 7 is shown. [Figure 8] The 1H NMR spectrum (400 MHz, CDCl3) of compound 8 is shown. [Figure 9] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 9 is shown. [Figure 10] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 10 is shown. [Figure 11] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 13 is shown. [Figure 12] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 16 is shown. [Figure 13] The 1H NMR spectrum (400 MHz, CD3CN) of compound 17 is shown. [Figure 14] The 1H NMR spectrum (400 MHz, CD3CN) of compound 18 is shown. [Figure 15] The 1H NMR spectrum (400 MHz, CD3CN) of compound 19 is shown. [Figure 16] The 1H NMR spectrum (400 MHz, CD3Cl) of compound 20 is shown. [Figure 17] The 1H NMR spectrum (400 MHz, CD3Cl) of compound 21 is shown. [Figure 18] The 1H NMR spectrum (400 MHz, CD3CN) of compound 22 is shown. [Figure 19] 1 shows a reaction scheme and results of an N-terminal modification reaction of angiotensin I (Example 8). [Figure 20] The 1H NMR spectrum (400 MHz, CD3CN) of compound 25 is shown. [Figure 21] 1 shows a reaction scheme and results of an N-terminal modification reaction of RNase A (Example 10). [Figure 22] 1 shows a reaction scheme and results of biotin modification of RNase A (Example 11-2). [Figure 23] The reaction scheme and results of modifying RNase A with a fluorescent dye are shown (Example 11-3). [Figure 24] 1 shows a reaction scheme and results of modification of RNase A with an azide group (Example 11-4). [Diagram 25] The reaction scheme and results of modification of a fluorescent dye by strain-promoted alkyne-azide cycloaddition reaction are shown (Example 11-5). [Figure 26] 1 shows the reaction scheme and results of modification of a strained alkyne site on RNase A (Example 11-6). [Figure 27] We outline a method in which various carbaldehyde precursors are immobilized on resins or solid materials, which simplifies the removal of by-products (aniline derivatives) generated after the reaction, and the post-reaction solution can be directly used for N-terminal modification of proteins (Example 12). [Figure 28] Compound 27 and a resin on which compound 27 is immobilized are shown (Example 12). [Figure 29] The synthesis scheme of 27 / PS resin is shown (Example 12-2). [Diagram 30] The results of resin identification using infrared spectroscopy are shown (Example 12-3). The right side shows an enlarged view. [Diagram 31] The results of LC / MS analysis of RNase A whose N-terminus was modified by a continuous reaction using a reactant immobilized resin (27 / PS resin) are shown (Example 12-4). [Diagram 32] The synthesis scheme of 27 / PS resin is shown (Example 12-5). [Diagram 33] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 28 is shown. [Diagram 34] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 30 is shown. [Diagram 35] The scheme of N-terminal modification by successive reactions using a reactant-immobilized resin (27 / PS resin) is shown (Example 12-6). [Diagram 36] The results of LC-MS analysis of RNase A that was N-terminally modified by successive reactions using a reagent-immobilized resin (27 / PS resin) are shown (Example 12-6). Peaks corresponding to modified proteins are indicated with ●, and peaks corresponding to unmodified proteins are indicated with ◯. [Figure 37] The scheme for the preparation of triazolecarbaldehyde using the Dimroth rearrangement reaction in a homogeneous system and its application to protein modification is shown (Example 13). [Figure 38] The reaction mechanism of the Dimroth rearrangement reaction is shown (Example 13-2). [Figure 39] The structure of the acid catalyst in the Dimroth rearrangement reaction using compound 10 is shown (Example 13-2). [Diagram 40] The results of LC-MS analysis of the preparation of an N-terminal modifier by the Dimroth rearrangement reaction and the subsequent protein modification reaction are shown (Example 13-3). Peaks corresponding to modified proteins are indicated by ●, and peaks corresponding to unmodified proteins are indicated by ◯. [Diagram 41] The preparation of Bis-TA4C and the protein modification reaction scheme are shown (Example 14-2). [Diagram 42] 1 shows the results of LC-MS analysis of the N-terminal modification of RNaseA by Bis-TA4C using a diamine as a precursor (Example 14-2). The structure of the precursor diamine and the modification rate calculated from LC-MS are shown. [Diagram 43] The following shows a scheme for introducing a functional molecule into the N-terminus of a protein via oxime formation (Example 14-3). [Diagram 44] The results of LC-MS analysis of RNase A modified with Compound 37 are shown (Example 14-3). [Diagram 45] 1 shows the results of SDS-PAGE analysis of RNase A modified with Compound 38 (Example 14-3). [Diagram 46]The 1H NMR spectrum (400 MHz, CDCl3) of compound 39 is shown. [Figure 47] The 1H NMR spectrum (400 MHz, CDCl3) of compound 40 is shown. [Figure 48] The Dimroth rearrangement reaction scheme using compound 40 is shown (Example 15-2). [Figure 49] The yield of compound 7 in the Dimroth rearrangement reaction is shown (Example 15-2). The yield was calculated from 1H NMR measurement. [Figure 50] 1 shows a reaction scheme and results of an N-terminal modification reaction of human serum-derived albumin (Example 16-2). [Figure 51] FIG. 17 shows a reaction scheme and results for modifying cysteine ​​residues in human serum-derived albumin (Example 17-2). [Figure 52] FIG. 1 shows a reaction scheme and results of N-terminal modification of cysteine ​​residue-modified human serum-derived albumin (Example 17-3). [Figure 53] The results of LC / MS analysis of the RNase_7 solution after standing for 24 hours are shown (Example 18-2). [Figure 54] 1 shows the change in release rate of a modifying agent over time for RNase_7 (Example 18-2). [Figure 55] 1 shows the change in release rate of a modifier associated with a change in pH of RNase_7 (Example 18-3). [Figure 56] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 42 is shown. [Figure 57] The 1H NMR spectrum (400 MHz, CDCl3) of compound 43 is shown. [Figure 58] The 1H NMR spectrum (400 MHz, CDCl3) of compound 44 is shown. [Figure 59] The 1H NMR spectrum (400 MHz, CDCl3) of compound 45 is shown. [Figure 60] The 1H NMR spectrum (400 MHz, CDCl3) of compound 46 is shown. [Figure 61]The 1H NMR spectrum (400 MHz, CDCl3) of compound 47 is shown. [Figure 62] The 1H NMR spectrum (400 MHz, CDCl3) of compound 48 is shown. [Figure 63] The 1H NMR spectrum (400 MHz, CDCl3) of compound 49 is shown. [Figure 64] The 1H NMR spectrum (400 MHz, CDCl3) of compound 50 is shown. [Figure 65] The 1H NMR spectrum (400 MHz, CDCl3) of compound 51 is shown. [Figure 66] The reaction scheme and results of N-terminal modification of angiotensin I are shown (Example 20). [Figure 67] 2 shows the reaction scheme and results of N-terminal modification of RNase A (Example 21). [Figure 68] The scheme and results of introducing an acetyl group and an azide group into RNase A are shown (Example 22-1). [Figure 69] The modification of a fluorescent dye by strain-promoted alkyne-azide cycloaddition reaction and the results are shown (Example 22-2). [Figure 70] The 1H NMR spectrum (400 MHz, DMSO-d6) of compound 52 is shown. [Figure 71] The 1H NMR spectrum (400 MHz, CDCl3) of compound 53 is shown. [Figure 72] The scheme and results of modifying RNase A with polyethylene glycol are shown (Example 23-2-2). [Figure 73] The scheme and results of the N-terminal modification reaction of HSA2 and the modification reaction of 26 are shown (Example 23-2-3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] In this specification, the expressions "contain" and "comprise" include the concepts of "contain", "include", "consist essentially of" and "consist only of".

[0036] 1. Protein and / or peptide modifying molecules In one aspect, the present invention provides a compound represented by general formula (1):

[0037] [ka]

[0038] [In the formula: R 1 and R 2 Of these, one is -N(-R 4 )-(R 4 represents an organic group or a group derived from an inorganic material. ) and the other represents =N-. R 3 represents a hydrogen atom, an organic group, or a group derived from an inorganic material. The present invention relates to a compound represented by the formula (I), a salt thereof, or a hydrate or solvate thereof (which may be collectively referred to as the "modified molecule of the present invention" in this specification). This will be described below.

[0039] <1-1. Compound> R 1 and R 2 Of these, one is -N(-R 4 )-(R 4 represents an organic group or a group derived from an inorganic material. ) represents a ═N- group, and the other represents =N-. A double line consisting of a dotted line and a solid line represents a single bond or a double bond, and R 1 and R 2 Which of -N(-R 4 Depending on whether it is ═N— or ═N—, it is determined whether it is a single bond or a double bond.

[0040] That is, the general formula (1) is R 1 -N(-R 4 )- and R 2 When is =N-, the general formula (1A):

[0041] [ka]

[0042] [In the formula: R 3 and R 4 is the same as above.] and R 1 is =N- and R 2 -N(-R 4 )-, general formula (1B):

[0043] [ka]

[0044] [In the formula: R 3 and R 4 is the same as above.] It is.

[0045] In the present invention, R 1 -N(-R 4 )- and R 2 is preferably =N- (that is, general formula (1) is general formula (1A)).

[0046] The organic group is not particularly limited as long as it is a group derived from an organic molecule or an organic molecular complex, for example, a group obtained by removing one atom or multiple atoms from an organic molecule or an organic molecular complex. The organic molecule is not particularly limited and may be natural, synthetic, or artificial. The organic molecular complex is not particularly limited and may be, for example, a complex (or a living organism) formed by linking multiple molecules including organic molecules. The mode of the linkage is not particularly limited and may be, for example, a hydrogen bond, an electrostatic force, a van der Waals force, a hydrophobic bond, a covalent bond, a coordinate bond, etc. These bonds may be made via a linker (see the linker described below for specific examples). The organic molecule or organic molecule complex is preferably a functional substance, and specific examples thereof include medicinal compounds, luminescent molecules, polymer compounds, ligands, molecules to which ligands are bound, antigenic proteins, antibodies, proteins, nucleic acids, sugars, lipids, cells, viruses, labels (e.g., radioisotope labels), carbon electrodes, carbon nanomaterials, linkers, spacer molecules (e.g., polyethylene glycol or derivatives thereof, peptides (e.g., peptides containing an amino acid sequence that is cleaved by an enzyme within a cell), etc.), complexes thereof, linking molecules, etc.

[0047] In one embodiment of the present invention, one or more of the modifying molecules of the present invention may be linked as a partial structure (e.g., at the terminal) in the organic group. In this case, an example of the modifying molecule of the present invention is a compound represented by the general formula (1AA):

[0048] [ka]

[0049] [In the formula: R 3 is the same as above. 3 is the same or different in each occurrence. 4a represents a divalent organic group. Examples of the compound represented by R 4a may contain a partial structure of the modified molecule of the present invention.

[0050] The inorganic material may or may not contain metal atoms, and is not particularly limited. Examples of the inorganic material include electrode materials, metal particles, metal oxide particles, semiconductor particles, magnetic particles, etc. The inorganic material may hold organic molecules or organic molecule complexes.

[0051] The organic group or the group derived from an inorganic material may have a reactive group. In this case, other substances can be further linked via the reactive group. Examples of the reactive group include ethynyl group, ethynylene group, vinyl group, azide group, epoxy group, aldehyde group, oxylamino group, halogen atom, etc. It is known that ethynyl group and ethynylene group form 1,2,3-triazole ring by 1,3-dipolar cycloaddition reaction with azide group. Vinyl group reacts with thiol group to form bond. Epoxy group reacts with amino group or thiol group to form bond. Aldehyde group reacts with amino group to form Schiff base, which is reduced to form bond. Oxylamino group reacts with ketone group or aldehyde group to form oxime. Azide group is known to form 1,2,3-triazole ring by 1,3-dipolar cycloaddition reaction with alkynyl group.

[0052] R 3 represents a hydrogen atom, an organic group, or a group derived from an inorganic material. For organic groups and inorganic materials, refer to the above R 4 The same applies to organic groups and inorganic materials represented by R 3 When is an organic group or a group derived from an inorganic material, the organic group or the group derived from an inorganic material preferably has a reactive group. In this case, as described above, other substances can be further linked via the reactive group.

[0053] In one aspect of the invention, R 3 is a hydrogen atom. In this case, general formula (1) is, for example, general formula (1a), (1Aa), or (1Ba).

[0054] [ka]

[0055] [In the formula, R 1 , R 2 , and R 4 is the same as above.] The compound represented by the general formula (1) includes stereoisomers and optical isomers, and these are not particularly limited.

[0056] The salt of the compound represented by the general formula (1) is not particularly limited. The salt may be either an acid salt or a basic salt. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, perchlorate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine.

[0057] The compound represented by the general formula (1) can be converted into a hydrate or a solvate. Examples of the solvent include organic solvents (e.g., ethanol, glycerol, acetic acid, etc.).

[0058] <1-2. Synthesis method 1> The compound represented by the general formula (1) can be synthesized by various methods. For example, the compound represented by the general formula (1) can be synthesized by a method represented by the general formula (2): R 4 -R 5 (2) [In the formula: R 4 is the same as above. 5 -N 3 , -X (X represents a halogen atom), -B(OH) 2 , -B(OR 51 ) 2 (R 51are the same or different and represent a hydrocarbon group. 51 may be bonded together to form a ring with the adjacent oxygen atom, or -N 2 + or a salt thereof, a compound represented by general formula (3), a compound represented by general formula (4), or a compound represented by general formula (9):

[0059] [ka]

[0060] [In the formula: R 3 is the same as above. 6 , R 7 and R 8 are the same or different and each represents an alkyl group; Y represents a reactive group. The compound can be synthesized by a method comprising the step of reacting

[0061] Examples of the halogen atom represented by X include a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, etc., and preferably a bromine atom.

[0062] R 51 Examples of the hydrocarbon group represented by the formula (I) include an alkyl group and a cycloalkyl group.

[0063] R 51 The alkyl group represented by the formula (I) includes both linear and branched alkyl groups. The number of carbon atoms in the alkyl group is not particularly limited, and is, for example, 1 to 8. 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, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, a 3-methylpentyl group, an n-heptyl group, and an n-octyl group.

[0064] R 51The number of carbon atoms in the cycloalkyl group represented by the following formula is not particularly limited and is, for example, 3 to 10, and preferably 4 to 10. Specific examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group.

[0065] The Two R's 51 The ring formed by bonding together with the adjacent oxygen atom may be substituted with an alkyl group or the like.

[0066] R 6 , R 7 and R 8 As the alkyl group represented by the formula: embedded image, a straight-chain lower alkyl group is usually adopted, and an ethyl group is preferably adopted.

[0067] The reactive group represented by Y is the same as the reactive group that may be contained in the above-mentioned organic group or group derived from an inorganic material. Y is preferably a halogen atom (Y').

[0068] Below, we will provide a detailed explanation of the types of materials used.

[0069] <1-2-1. Case 1 (R 5 Ga-N 3 and when a compound represented by the general formula (3) is used) The amount of the compound represented by general formula (2) used is preferably 0.1 to 5 mol, more preferably 0.3 to 2 mol, per 1 mol of the compound represented by general formula (3) in terms of the mole number of the functional group to be reacted (in this case, the azide group), from the viewpoint of yield and the like.

[0070] This reaction is usually carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited, and examples thereof include water, methanol, tetrahydrofuran, dioxane, and dimethyl sulfoxide. The solvent may be used alone or in combination. It is also preferable to add a buffer such as a phosphate buffer to the solvent. When water is used, the pH of this reaction is preferably near neutral, specifically, preferably 6 to 8.5, more preferably 6.5 to 8, and even more preferably 7 to 7.5.

[0071] This reaction is preferably carried out in the presence of a suitable catalyst. Examples of the catalyst include copper catalysts. Examples of the copper catalyst include divalent copper such as copper sulfate, monovalent copper such as copper iodide, etc. In this reaction, a reducing agent (e.g., hydroquinone, sodium ascorbate), a ligand, etc. can also be used.

[0072] From the viewpoint of yield and the like, the amount of the copper catalyst used is usually preferably 0.1 to 5 moles per mole of the azide group-containing protein or peptide of the present invention.

[0073] In addition to the above-mentioned components, additives may also be used in this reaction as appropriate, provided that they do not significantly impair the progress of the reaction.

[0074] The reaction can be carried out under heating, at room temperature, or under cooling, and is usually preferably carried out at a temperature at which the azide group-containing protein or peptide of the present invention is not significantly denatured, for example, 0 to 45° C. (particularly, 0 to 40° C.) The reaction time is not particularly limited, and is usually 30 minutes to 3 hours, and particularly 1 to 2 hours.

[0075] The progress of the reaction can be monitored by a conventional method such as chromatography. After the reaction is completed, the solvent is removed by distillation, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization, if necessary. The structure of the product can be confirmed by elemental analysis, MS (ESI-MS) analysis, IR analysis, etc. 1 H-NMR, 13 It can be identified by C-NMR, etc.

[0076] <1-2-2. Case 2 (R 5 Ga-N 3 When the compound represented by the general formula (3) is used, the formula is other than the above. In this case, R 5 The procedure is the same as in Case 1, except that an azidation agent is added to convert the to an azido group.

[0077] Examples of the azidation agent include inorganic azides such as sodium azide, sulfonyl azide, silyl azide, phosphoric acid azide, and alkylammonium azide.

[0078] The amount of the azidation agent used is usually preferably 0.1 to 5 mol, more preferably 0.3 to 2 mol, per 1 mol of the compound represented by the general formula (2) from the viewpoint of yield and the like.

[0079] <1-2-3. Case 3 (When the compound represented by the general formula (4) is used)> In this case, after the reaction between the compound represented by the general formula (2) and the compound represented by the general formula (4), the compound represented by the general formula (1) can be synthesized through several steps. For example, in this case, the compound represented by the general formula (1) can be synthesized by the following scheme.

[0080] [ka]

[0081] [In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , and R 8 is the same as above.] (Process 1) The amount of the compound represented by general formula (2) used is preferably 0.1 to 5 moles, more preferably 0.3 to 2 moles, per mole of the compound represented by general formula (4) in terms of the mole number of the functional group to be reacted (in this case, the azide group) from the viewpoint of yield and the like.

[0082] The solvent may be any solvent that does not adversely affect the reaction, and examples thereof include water, alcohol solvents (e.g., methanol, ethanol, isopropanol, n-butanol, trifluoroethanol, ethylene glycol, etc.), ketone solvents (e.g., acetone, methyl ethyl ketone, etc.), ether solvents (e.g., tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane, diglyme, etc.), ester solvents (e.g., methyl acetate, ethyl acetate, etc.), aprotic polar solvents (e.g., acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, etc.), halogenated hydrocarbon solvents (e.g., methylene chloride, ethylene chloride, etc.), or mixed solvents thereof. A mixed solvent of water and an aprotic polar solvent (particularly dimethylformamide) is preferable.

[0083] Step 1 is usually carried out in the presence of a base. For example, an organic base can be used as the base. For example, trialkylamines (e.g., trimethylamine, triethylamine, N,N-diisopropylethylamine, etc.), pyridine, quinoline, piperidine, imidazole, picoline, 4-dimethylaminopyridine, N,N-dimethylaniline, N-methylmorpholine, 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 1,4-diazabicyclo[2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), etc. can be mentioned. In addition, when these bases are liquid, they can be used as a solvent. The base is used alone or in a mixture of two or more. The base is preferably piperidine.

[0084] When a base is used, the amount used is usually 0.05 to 1 mol, and preferably 0.1 to 0.5 mol, per 1 mol of the compound represented by formula (4).

[0085] The reaction temperature is not particularly limited, and the reaction is usually carried out under cooling, at room temperature, or under heating. The reaction is preferably carried out at a temperature of about 50 to 100° C., more preferably about 70 to 90° C., for 1 to 30 hours.

[0086] After the reaction is completed, the solvent is distilled off, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 It can be identified by C-NMR, etc.

[0087] (Process 2) In step 2, the compound represented by general formula (1″) is reacted in the presence of a reducing agent and an appropriate amount of base.

[0088] Examples of reducing agents include sodium borohydride and zinc borohydride (Zn(BH 4 ) 2 ), tetramethylammonium triacetoxyborohydride, lithium trisec-butylborohydride, borane, borane-THF complex, borane-dimethylsulfide complex, lithium aluminum hydride, diisobutylaluminum hydride, lithium borohydride, etc. These reducing agents are used alone or in combination of two or more. Sodium borohydride is preferred.

[0089] The amount of the reducing agent used is usually 1 to 15 mol, preferably 3 to 10 mol, per mol of the compound represented by formula (1'').

[0090] As the base, for example, an inorganic base can be used. As the organic base, for example, an alkali metal (e.g., sodium, potassium, etc.), an alkali metal hydrogen carbonate (e.g., lithium hydrogen carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, etc.), an alkali metal hydroxide (e.g., lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, etc.), an alkali metal carbonate (e.g., lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, etc.), an alkali metal lower (C1-C4) alkoxide (e.g., sodium methoxide, sodium ethoxide, potassium tertiary butoxide, etc.), an alkali metal hydride (e.g., sodium hydride, potassium hydride, etc.), etc. can be mentioned. The base is used alone or in combination of two or more kinds. The base is preferably an alkali metal lower (C1-C4) alkoxide (particularly, sodium methoxide).

[0091] The solvent may be any solvent that does not adversely affect the reaction, and examples thereof include water, alcohol solvents (e.g., methanol, ethanol, isopropanol, n-butanol, trifluoroethanol, ethylene glycol, etc.), ketone solvents (e.g., acetone, methyl ethyl ketone, etc.), ether solvents (e.g., tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane, diglyme, etc.), ester solvents (e.g., methyl acetate, ethyl acetate, etc.), aprotic polar solvents (e.g., acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, etc.), halogenated hydrocarbon solvents (e.g., methylene chloride, ethylene chloride, etc.), or mixed solvents thereof. Alcohol solvents (e.g., methanol, in particular) are preferred.

[0092] The reaction temperature is not particularly limited, and the reaction is usually carried out under cooling, at room temperature, or under heating. The reaction is preferably carried out at a temperature of 0 to 60° C., more preferably at about 10 to 40° C., for 1 to 30 hours.

[0093] After the reaction is completed, the solvent is distilled off, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 It can be identified by C-NMR, etc.

[0094] (Step 3) In step 3, the compound represented by general formula (1') is reacted in the presence of an oxidizing agent.

[0095] As the oxidizing agent, for example, manganese dioxide can be used. In addition, for example, selenium dioxide or nitroxyl radicals such as 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) or 2-azaadamantane-N-oxyl (AZADO) can also be used. The oxidizing agent can be used alone or in combination of two or more kinds.

[0096] The amount of the oxidizing agent used is usually 3 to 20 mol, preferably 5 to 15 mol, per mol of the compound represented by the general formula (1').

[0097] The solvent may be any solvent that does not adversely affect the reaction, and examples of the solvent include ketone solvents (e.g., acetone, methyl ethyl ketone, etc.), ether solvents (e.g., tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane, diglyme, etc.), ester solvents (e.g., methyl acetate, ethyl acetate, etc.), aprotic polar solvents (e.g., acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, etc.), halogenated hydrocarbon solvents (e.g., chloroform, methylene chloride, ethylene chloride, etc.), and mixtures of these solvents. Preferred are halogenated hydrocarbon solvents (particularly, chloroform).

[0098] The reaction temperature is not particularly limited, and the reaction is usually carried out under cooling, at room temperature, or under heating. The reaction is preferably carried out at a temperature of 0 to 60° C., more preferably at about 10 to 40° C., for 1 to 30 hours.

[0099] After the reaction is completed, the solvent is distilled off, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 It can be identified by C-NMR, etc.

[0100] <1-2-4. Case 4 (R 5 Ga-N 3 and when a compound represented by general formula (9) is used) In this case, after the reaction between the compound represented by the general formula (2) and the compound represented by the general formula (9), the compound represented by the general formula (1) can be synthesized through several steps. For example, in this case, the compound represented by the general formula (1) can be synthesized by the following scheme.

[0101] [ka]

[0102] Step 4 can be carried out according to or similar to the above-mentioned Case 1. Step 6 can be carried out according to or similar to the above-mentioned Step 3. Step 5 is a step of reacting a compound represented by the general formula (1**) with a compound represented by the general formula (10)R 3 This can be achieved by reacting a compound represented by -Z. Z is a reactive group that reacts with Y. When Y is a halogen, Z can be, for example, -B(OH) 2 The reaction conditions for step 5 can be set according to the types of Y and Z, based on publicly known information.

[0103] <1-3. Synthesis method 2> In addition to the synthesis method 1, the compound represented by the general formula (1) can be synthesized by, for example, a method represented by the general formula (5): R 4 -R 9 (5) [In the formula: R 4 is the same as above. 9 -NR 9a R9b (R 9a and R 9b are the same or different and represent a hydrogen atom or an alkyl group. ) and a compound represented by the general formula (6):

[0104] [ka]

[0105] [In the formula, R 10 R represents an electron-withdrawing group. 11 -R 11a -R 12 (R 11a R represents a single bond or a linker. 12 represents a carrier.) n represents 0 or 1. m represents an integer of 1 to 5.] The compound can be synthesized by a method including a step of reacting a compound represented by the formula:

[0106] In addition, the compound represented by the general formula (5) includes the compound represented by the general formula (5'): R 9 -R 4a -R 9 (5')[in formula:R 4a and R 9 is the same as above. By using a compound represented by the following formula (1AA), a compound represented by the following formula (1AA) can be synthesized.

[0107] The alkyl group is not particularly limited and may be, for example, a lower alkyl group such as a methyl group or an ethyl group. 9a and R 9b are preferably both hydrogen atoms.

[0108] The electron-withdrawing group is not particularly limited, but for example, -NO 2 , -F, CF 3 , -CN, -COOMe, etc. The position of the electron-withdrawing group is, for example, -NO 2 In the case of 3In the latter case, it is more preferable that the positions are -CN at the ortho and meta positions. In the latter case, it is more preferable that the para position is -CN.

[0109] m is, for example, an electron-withdrawing group such as -NO 2 In the case of 3 In the case of, 4 to 5 are preferred, and all of the ortho and meta positions are R 10 In the latter case, it is more preferred that the para position is also -CN.

[0110] All ortho and meta positions are -F or CF 3 When the alkyl group is (preferably -F) and the para position is -CN, the reaction with the compound represented by general formula (5) can proceed sufficiently even under relatively low temperature conditions (preferably 20 to 60°C, more preferably 25 to 40°C).

[0111] The linker is not particularly limited as long as it can link the carrier to the benzene ring, and examples thereof include linkers containing the following partial structures in the main chain:

[0112] [ka]

[0113] The number of atoms constituting the main chain of the linker is, for example, 1 to 100, 1 to 50, 1 to 20, or 1 to 10.

[0114] The carrier is not particularly limited.

[0115] The average particle size of the carrier particles is not particularly limited, but is preferably a size that allows easy precipitation in a solution, for example, 1 nm to 1 mm, and preferably 10 nm to 100 μm.

[0116] The material of the carrier particles is not particularly limited, and examples thereof include metal particles such as gold, silver, copper, iron, aluminum, nickel, manganese, titanium, and oxides thereof; resin particles such as polystyrene and latex; and silica particles. The shape of the carrier particles is not particularly limited, and examples thereof include spheres, rectangular parallelepipeds, cubes, triangular pyramids, and shapes similar thereto. The carrier particles may have a substance on the surface for making the binding of other substances (e.g., binding substance 2, etc.) easier and / or stronger. Examples of such substances include substances having reactive groups such as epoxy groups, amino groups, carboxy groups, and azide groups; substances having affinity for other molecules such as avidin, protein A, and protein B. The carrier particles may further contain a labeling substance. The carrier particles may be of only one type, or may be of a combination of two or more types.

[0117] The amount of the compound represented by formula (5) used is usually preferably 0.1 to 5 mol, more preferably 0.3 to 2 mol, per 1 mol of the compound represented by formula (6) from the viewpoint of yield and the like.

[0118] The solvent may be any solvent that does not adversely affect the reaction, and examples thereof include water, alcohol-based solvents (e.g., methanol, ethanol, isopropanol, n-butanol, trifluoroethanol, ethylene glycol, etc.), ketone-based solvents (e.g., acetone, methyl ethyl ketone, etc.), ether-based solvents (e.g., tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane, diglyme, etc.), ester-based solvents (e.g., methyl acetate, ethyl acetate, etc.), aprotic polar solvents (e.g., acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, etc.), halogenated hydrocarbon-based solvents (e.g., methylene chloride, ethylene chloride, etc.), or mixed solvents thereof. A mixed solvent of water and an alcohol-based solvent is preferable.

[0119] In this reaction, it is preferable to use an acid catalyst from the viewpoint of yield, etc. The acid catalyst is not particularly limited, but examples thereof include acetic acid, methanesulfonic acid, p-toluenesulfonic acid, 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), etc. Among these, Good's buffers such as MES, MOPS, HEPES, and TAPS are preferable from the viewpoint of less inhibition of the subsequent protein modification reaction.

[0120] The reaction temperature is not particularly limited, and the reaction is usually carried out under cooling, at room temperature, or under heating. The reaction is preferably carried out at a temperature of about 20 to 100° C. for 10 minutes to 30 hours.

[0121] After the reaction is completed, the solvent is distilled off, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 It can be identified by C-NMR, etc.

[0122] When the compound represented by the general formula (6) contains a carrier (i.e., R 11 -R 11a -R 12 In other words, the purification operation can be easily performed when the by-product (aniline derivative) of the reaction is represented by the general formula (6x):

[0123] [ka]

[0124] [In the formula: R 9a , R 9b , R 10 , R 11a , R 12 , and m are as defined above.] The compound represented by the general formula (6'): can be easily removed by precipitation using a carrier or the like. Therefore, in one aspect, the present invention provides a compound represented by the general formula (6'):

[0125] [ka]

[0126] [In the formula, R 10 , R 11 , R 12 , and m are as defined above.] The present invention relates to a compound represented by the formula:

[0127] <1-4.Application> The modifying molecule of the present invention is useful for linking other molecules / substances to the N-terminus of a protein or peptide, for example, for producing the composite substance of the present invention described below (a compound represented by general formula (7), or a salt thereof, or a hydrate or solvate thereof). For this reason, the modifying molecule of the present invention can be suitably used as a reagent, in particular as an active ingredient of a reagent for modifying proteins and / or peptides. The reagent is not particularly limited as long as it contains the modifying molecule of the present invention, and may further contain other components as necessary. The other components are not particularly limited as long as they are pharma- ceutically acceptable components, and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0128] 2. Composite substances In one aspect, the present invention provides a compound represented by general formula (7):

[0129] [ka]

[0130] [In the formula: R 1 , R 2 , R 3 , and R 4 is the same as above. 13R represents a group obtained by removing the N-terminal amino acid residue and the adjacent -NH- from a protein or peptide. 14 represents the side chain of the N-terminal amino acid residue of the protein or peptide.] The present invention relates to a compound represented by the formula (I), a salt thereof, or a hydrate or solvate thereof (which may be collectively referred to as the "composite substance of the present invention" in this specification). This will be described below.

[0131] R 13 represents the group obtained by removing the N-terminal amino acid residue and the adjacent --NH-- from a protein or peptide.

[0132] The protein or peptide is not particularly limited as long as it is a protein or peptide in which the N-terminal amino group is unmodified and the second amino acid residue from the N-terminus is an amino acid residue other than proline. The protein or peptide may be modified in various ways at a site other than the N-terminus (e.g., a cysteine ​​residue). Examples of such proteins or peptides include proteins or peptides represented by the general formula (7a).

[0133] [ka]

[0134] R 14indicates the side chain of the N-terminal amino acid residue of the protein or peptide. The amino acid residue may be either a natural amino acid residue or a synthetic amino acid residue, and examples thereof include amino acid residues having a basic side chain such as lysine, arginine, and histidine; amino acid residues having an acidic side chain such as aspartic acid and glutamic acid; amino acid residues having a non-charged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having a non-polar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having a β-branched side chain such as threonine, valine, and isoleucine; and amino acid residues having an aromatic side chain such as tyrosine, phenylalanine, tryptophan, and histidine.

[0135] The protein or peptide is not particularly limited, and may be a natural protein or peptide, or a synthetic or artificial protein or peptide.

[0136] The protein or peptide may be chemically modified. The protein or peptide may have a carboxyl group (-COOH) or a carboxylate (-COO - ), amide (-CONH 2 ) or ester (-COOR). Here, R in the ester may be, for example, C methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups; for example, C phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups: α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14Examples of the groups that can be used include aralkyl groups; pivaloyloxymethyl groups, etc. In the protein or peptide, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, the ester may be, for example, the C-terminal ester described above. Furthermore, in the protein or peptide, a suitable protecting group (e.g., C-terminal group such as formyl group, acetyl group, etc.) may be used to protect the substituents (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) on the side chains of amino acids in the molecule. 1-6 Alkanoyl group etc. 1-6 Also included are those protected with an aryl group (such as an acyl group).

[0137] The protein or peptide may be post-translationally modified, or may be post-translationally modified by artificial enzyme treatment or chemical modification. Examples of post-translational modifications include phosphorylation, N-glycosylation, O-glycosylation, C-glycosylation, phosphoglycosylation, glypiation, S-nitrosylation, methylation, N-acetylation, S-myristoylation, S-prenylation, and S-palmitoylation. The protein or peptide may be a protein or peptide such as a known protein tag or signal sequence, or a labeling substance may be added. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, and SPY tag. Examples of signal sequences include nuclear localization signals.

[0138] The protein or peptide may exist as a single molecule alone, or may be linked to other molecules to form a complex. For example, the protein or peptide may be a protein or peptide present on the cell surface, a protein or peptide in a cell lysate, or a protein or peptide supported by some substance. The mode of the linkage is not particularly limited, and examples thereof include hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, covalent bonds, and coordinate bonds.

[0139] The protein or peptide may be fragmented by an enzyme or chemical reaction as long as it has an N-terminus. In addition, if the protein or peptide is N-terminally modified, the N-terminus may be demodified by an enzyme or chemical reaction.

[0140] The compound represented by the general formula (7) includes stereoisomers and optical isomers, and these are not particularly limited.

[0141] The salt of the compound represented by the general formula (7) is not particularly limited. The salt may be either an acid salt or a basic salt. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, perchlorate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt; salts with ammonia; salts with organic amines such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine, and tri(hydroxyalkyl)amine.

[0142] The compound represented by the general formula (7) can be converted into a hydrate or a solvate. Examples of the solvent include organic solvents (e.g., ethanol, glycerol, acetic acid, etc.).

[0143] The compound represented by general formula (7) can be synthesized by various methods. As an example, the compound represented by general formula (7) can be produced by a method including a step of reacting a protein or peptide with the modified molecule of the present invention. This reaction may be a continuous reaction with the synthesis reaction of the modified molecule of the present invention (particularly preferably the reaction of "1-2. Synthesis method 2" above). In this case, for example, the reaction mixture obtained in the synthesis reaction of the modified molecule of the present invention can be diluted with the solvent used in this reaction before carrying out this reaction.

[0144] From the viewpoint of yield and the like, the amount of the modifying molecule of the present invention to be used is usually preferably 5 to 400 moles per mole of protein or peptide.

[0145] This reaction is usually carried out in the presence of a reaction solvent. The reaction solvent is not particularly limited, and examples thereof include water and the like. The solvent may be used alone or in combination. It is also preferable to add a buffer such as a phosphate buffer to the solvent. When water is used, the pH of this reaction is preferably near neutral from the viewpoint of N-terminal selectivity, specifically, 6 to 8.5 is preferable, 6.5 to 8 is more preferable, and 7 to 7.5 is even more preferable.

[0146] In addition to the above-mentioned components, additives may also be used in this reaction as appropriate, provided that they do not significantly impair the progress of the reaction.

[0147] The reaction can be carried out under heating, at room temperature, or under cooling, and is usually preferably carried out at a temperature at which the protein or peptide is not significantly denatured, for example, 0 to 45° C. (particularly, 0 to 40° C.) The reaction time is not particularly limited, and is usually 8 to 36 hours, and particularly 12 to 24 hours.

[0148] The progress of the reaction can be monitored by a conventional method such as chromatography. After the reaction is completed, the solvent is removed by distillation, and the product can be isolated and purified by a conventional method such as chromatography or recrystallization, if necessary. The structure of the product can be confirmed by elemental analysis, MS (ESI-MS) analysis, IR analysis, etc. 1 H-NMR, 13 It can be identified by C-NMR, etc.

[0149] When a compound represented by general formula (1AA) is used as the modifying molecule of the present invention, a compound represented by general formula (7AA):

[0150] [ka]

[0151] [In the formula: R 3 , R 4a , R 13 , and R 14 is the same as above.] In this case, an organic molecule or the like can be linked by further forming an oxime with hydroxylamine. That is, a compound represented by general formula (1AA) and a compound represented by general formula (8): R 4 -O-NH 2 (8) [In the formula: R 4 is the same as above.], to obtain a compound represented by the general formula (7AAA):

[0152] [ka]

[0153] [In the formula: R 3 , R 4a , R 4 , R 13 , and R 14 is the same as above.] The compound represented by the formula (1) can be synthesized. The conditions for this synthesis reaction can be determined according to or in accordance with the known reaction conditions for the formation of an oxime with hydroxylamine starting from an aldehyde. This reaction proceeds under mild conditions, even in water, and is therefore also applicable to protein modification.

[0154] The composite substance of the present invention has a structure in which another substance is linked to a protein or peptide, and can be used in a variety of fields depending on the substance to be linked, for example, as an antibody-drug conjugate, a labeled protein reagent, a protein-immobilized inorganic material, a fusion protein linked to a protein, a protein fused to a nucleic acid, etc.

[0155] In one embodiment of the composite substance of the present invention, the substance linked to the protein or peptide can be designed to dissociate gradually. Therefore, for example, after the composite substance of the present invention in which a drug is linked to a protein or peptide is administered to a living body, the drug can be gradually released in the living body by utilizing the gradual dissociation of the drug from the composite substance of the present invention in the living body.

[0156] When the composite material of the present invention has a reactive group (e.g., R 3 has a reactive group), it is possible to further link other substances using the reactive group. For example, it is useful for linking other substances (e.g., organic molecules, organic molecule complexes, inorganic materials, etc.) by reactions using azide groups (e.g., Huisgen cycloaddition reaction, strain-promoted azide-alkyne cycloaddition reaction, Staudinger-Bertozzi ligation). EXAMPLES

[0157] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0158] Example 1. Compound synthesis 1 (Method A) <1-1. Equipment used> Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE III HD nuclear magnetic resonance spectrometer, and chemical shifts were calculated using the residual signal of the measurement solvent as an internal standard. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) was performed on a Bruker micrOTOF focus III mass spectrometer, using methanol or acetonitrile (both HPLC grade) as the mobile phase. Fourier transform infrared (FT-IR) spectra were recorded on a Jasco FT / IR-4000 Fourier transform infrared spectrometer in ATR mode with a diamond or gallium prism.

[0159] <1-2. Reagents, solvents, etc.> The reagents and solvents used in the synthesis were commercially available products. The precursor azide compound was synthesized according to a previous report (Y. Zhao, P. Gong, Bioorg. Med. Chem., 2014, 22, 6438-6452).

[0160] <Synthesis of 1-3-1.1-(3-carboxyphenyl)-1H-1,2,3-triazole-4-carbaldehyde (1)> Compound 1 was synthesized according to the following scheme.

[0161] [ka]

[0162] Compound 1 was synthesized with reference to previous reports (TA Bakka, MB Strom, JH Anderson, OR Gautun, Bioorg. Med. Chem. Lett., 2017, 27, 1119-1123 and JT Fletcher, JA Cristensen, EM Villra, Tetrahedron Lett., 2017, 58, 4450-4454). The specific synthesis items and compound identification results are shown below.

[0163] A mixture of an aqueous solution (7.5 mL) of copper(II) sulfate pentahydrate (38 mg, 0.15 mmol) and t-butyl alcohol (7.5 mL) was added with the azide compound (0.60 mmol), propargylaldehyde diethyl acetal (115 μL, 0.80 mmol), and sodium ascorbate (60 mg, 0.30 mmol) under a nitrogen atmosphere, and the mixture was stirred at 70°C for 24 hours and at room temperature in air for 1 hour. The resulting suspension was diluted with saturated aqueous sodium chloride solution (10 mL), extracted with ethyl acetate (50 mL x 3), and washed with saturated aqueous sodium chloride solution (20 mL x 2). The resulting organic layer was dried over sodium sulfate, and the solvent of the filtrate was removed under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 1 (yellow solid). As shown in Figure 1, 1 The H NMR spectrum is shown.

[0164] Yield 48%; 1 H NMR (400 MHz, DMSO-d 6 ): δ10.12 (s, 1H), 9.67 (s, 1H), 8.48 (s, 1H), 8.16 (d, J = 7.8 Hz, 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.72 (t, J = 7.8 Hz, 1H); 13 C NMR (100 MHz, DMSO-d 6 ): δ184.9, 167.0, 147.6, 136.0, 130.1, 130.0, 126.6, 124.0, 121.3; ESI-TOF MS (positive mode) m / z calcd. for C 10 H 7 NaN 3 O 3 [M+Na] + 240.04, found 240.04; FT-IR (ATR mode, Ge prism), ν cm -1 : 3128, 2923, 1697, 1263, 1230, 1184, 757, 673, 647.

[0165] <1-3-2. Synthesis of 1-(4-carboxyphenyl)-1H-1,2,3-triazole-4-carbaldehyde (2)>

[0166] [ka]

[0167] Compound 2 (yellow solid) was synthesized using 4-azidobenzoic acid as a precursor in the same manner as in Example 1-3-1 using Method A. 1 The H NMR spectrum is shown.

[0168] Yield 63%; 1 H NMR (400 MHz, DMSO-d 6 ): δ10.13 (s, 1H), 9.69 (s, 1H), 8.18-8.12 (m, 4H); ESI-TOF MS (positive mode) m / z calcd. for C 10 H 7 NaN 3 O 3 [M+Na] + 240.04, found 240.04; FT-IR (ATR mode, Ge prism), ν cm -1 : 3110, 1695, 1683, 1605, 1429, 1319, 1294, 1264, 989, 945, 864, 773, 702, 688.

[0169] <1-3-3. Synthesis of 1-(3,5-dicarboxyphenyl)-1H-1,2,3-triazole-4-carbaldehyde (3)>

[0170] [ka]

[0171] Compound 3 (yellow solid) was synthesized using 5-azidoisophthalic acid as a precursor in the same manner as in Example 1-3-1, using Method A. 1 The H NMR spectrum is shown.

[0172] Yield 10%; 1 H NMR (400 MHz, DMSO-d 6 ): δ10.12 (s, 1H), 9.81 (s, 1H), 8.65 (s, 2H), 8.56 (s, 1H); FT-IR (ATR mode, Ge prism), ν cm -1 : 3133, 1697, 1296, 1281, 1247, 1049, 830, 757, 678, 666, 566, 527.

[0173] <1-3-4. Synthesis of 1-(4-(diethylamino)phenyl)-1H-1,2,3-triazole-4-carbaldehyde (4)>

[0174] [ka]

[0175] Compound 4 (brown solid) was synthesized using 4-azido-N,N-diethylaniline as a precursor by using Method A in the same manner as in Example 1-3-1. 1 The H NMR spectrum is shown.

[0176] Yield 60%; 1 H NMR (400 MHz, CDCl 3 ): δ10.12 (s, 1H), 8.36 (s, 1H), 7.52 (d, J = 9.1 Hz, 2H), 6.73 (d, J = 9.1 Hz, 2H), 3.42 (q, J = 7.1 Hz, 4H), 1.21 (t, J = 7.1 Hz, 6H); 13 C NMR (100 MHz, DMSO-d 6): δ185.0, 148.0, 147.3, 125.1, 124.3, 122.1, 111.4, 43.8, 12.3; ESI-TOF MS (positive mode) m / z calcd. for C 13 H 16 NaN 4 O [M+Na] + 267.12, found 267.12.

[0177] <1-3-5. Synthesis of 1-(4-methoxyphenyl)-1H-1,2,3-triazole-4-carbaldehyde (5)>

[0178] [ka]

[0179] Compound 5 (white solid) was synthesized using 1-azido-4-methoxybenzene as a precursor in the same manner as in Example 1-3-1, using Method A. 1 The H NMR spectrum is shown.

[0180] Yield 68%; 1 H NMR (400 MHz, DMSO-d 6 ): δ10.12 (s, 1H), 8.43 (s, 1H), 7.66 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 3.89 (s, 3H); 13 C NMR (100 MHz, DMSO-d 6 ): δ185.0, 159.9, 147.5, 129.3, 125.9, 122.4, 115.0, 55.7; ESI-TOF MS (positive mode) m / z calcd. for C 10 H 9 NaN 3 O- 2 [M+Na] + 226.06, found 226.06.

[0181] <1-3-6. Synthesis of 1-phenyl-1H-1,2,3-triazole-4-carbaldehyde (6)>

[0182] [ka]

[0183] Compound 6 (brown solid) was synthesized using azidobenzene as a precursor in the same manner as in Example 1-3-1, using Method A. 1 The H NMR spectrum is shown.

[0184] Yield 9%; 1 H NMR (400 MHz, DMSO-d 6 ): δ10.11 (s, 1H), 9.57 (s, 1H), 7.97 (d, J = 8.0 Hz, 2H), 7.66-7.54 (m, 3H).

[0185] Example 2. Compound synthesis 2 (Method B) The equipment, reagents, solvents, etc. used were the same as in Example 1.

[0186] <Synthesis of 2-1.1-benzyl-1H-1,2,3-triazole-4-carbaldehyde (7)>

[0187] [ka]

[0188] Compound 7 was synthesized based on a previous report (JT Fletcher, Tetrahedron Lett., 2017, 58, 4450-4454). The specific synthesis and compound identification results are shown below.

[0189] A mixture of copper(II) sulfate pentahydrate (47.8 mg, 0.19 mmol) in water (7.5 mL) and t-butyl alcohol (7.5 mL) was cooled to 0 °C, and sodium azide (103 mg, 1.6 mmol) was added under a nitrogen atmosphere. After stirring at room temperature for 10 minutes, benzyl bromide (179 μL, 1.5 mmol) and propargylaldehyde diethyl acetal (240 μL, 1.7 mmol) were added to the mixture, and the mixture was stirred at 70 °C for 24 hours and at room temperature in air for 1 hour. The reaction solution was cooled to room temperature, diluted with saturated sodium chloride aqueous solution (5 mL), and extracted with ethyl acetate (30 mL x 3). The organic layer obtained was dried over magnesium sulfate, the solid was filtered off, and the filtrate was distilled under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1) to obtain compound 7 (white solid). Figure 7 shows 1 The H NMR spectrum is shown.

[0190] Yield 46%; 1 H NMR (400 MHz, DMSO-d 6 ): δ10.00 (s, 1H), 8.95 (s, 1H), 7.41-7.32(m, 5H), 5.69 (s, 2H); 13 C NMR (100 MHz, DMSO-d 6 ): δ185.0, 147.0, 135.3, 128.9, 128.4, 128.3, 128.1, 53.2; ESI-TOF MS (positive mode) m / z calcd. for C 10 H 9 NaN 3 O [M+Na] + 210.06, found 210.06; FT-IR (ATR mode, Ge prism), νcm -1 : 1694, 1535, 1237, 1165, 1052, 877, 796, 767, 714, 701, 565, 556, 543, 532, 515, 505.

[0191] <Synthesis of 2-2.1-(naphthalen-2-ylmethyl)-1H-1,2,3-triazole-4-carbaldehyde (8)>

[0192] [ka]

[0193] Compound 8 (white solid) was synthesized using 2-(bromomethyl)naphthalene as a precursor in the same manner as in Example 2-1, using Method B. 1 The H NMR spectrum is shown.

[0194] Yield 20%; 1 H-NMR (400 MHz, CDCl 3 ): δ10.13 (s, 1H), 8.02 (s, 1H), 7.90-7.80 (m, 4H), 7.57-7.53 (m, 2H), 7.38-7.35 (m, 1H) 5.75 (s, 2H); ESI-TOF MS (positive mode) m / z calcd. for C 14 H 11 NaN 3 O [M+Na] + 260.08, found 260.08; FT-IR (ATR mode, Ge prism), νcm -1 : 3121, 1709, 1538, 1239, 1177, 1052, 1026, 866, 835, 792, 761, 563, 553, 527, 511.

[0195] Example 3. Compound synthesis 3 (Method C) The equipment, reagents, solvents, etc. used were the same as in Example 1.

[0196] <Synthesis of 1-(p-tolyl)-1H-1,2,3-triazole-4-carbaldehyde (9)>

[0197] [ka]

[0198] Compound 9 was synthesized with reference to a previous report (C.-Z. Tao, X. Cui, J. Li, AX. Liu, L. Liu, Q.-X. Guo, Tetrahedron Lett., 2007, 48, 3525-3529). The specific synthesis items and compound identification results are shown below.

[0199] A methanol solution (5 mL) containing 4-methylphenylboronic acid (136 mg, 1.0 mmol) and sodium azide (98 mg, 1.5 mmol) was added with pure water (5 mL) containing copper(II) sulfate pentahydrate (47.8 mg, 0.19 mmol) and stirred in air for 5 hours. Sodium ascorbate (79 mg, 0.4 mmol) and propargylaldehyde diethyl acetal (286 μL, 1.7 mmol) were added under a nitrogen atmosphere and stirred at 70°C for 24 hours and at room temperature in air for 1 hour. The reaction solution was cooled to room temperature, diluted with saturated aqueous sodium chloride solution (40 mL), and extracted with ethyl acetate (50 mL x 3). The organic layer was dried over magnesium sulfate, the solid was filtered off, and the filtrate was distilled under reduced pressure to obtain a crude product, which was dissolved in chloroform (3 mL), pure water (3 mL) and trifluoroacetic acid (3 mL) were added, and the mixture was stirred vigorously at room temperature. The mixture was diluted with saturated aqueous sodium chloride solution (30 mL), and the organic layer was extracted with chloroform (30 mL x 3). The organic layer was dried over magnesium sulfate, and the solid was filtered off. The filtrate was evaporated under reduced pressure, and the crude product was purified by reprecipitation (hexane:chloroform) to obtain compound 9 (white solid). 1 The H NMR spectrum is shown.

[0200] Yield 71%; 1 H NMR (400 MHz, CDCl 3): δ10.2 (s, 1H), 8.48 (s, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.3 Hz, 2H), 2.45 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ): δ185.3, 148.2, 140.3, 134.0, 130.7, 123.2, 120.9, 21.3; ESI-TOF MS (positive mode) m / z calcd. for C 10 H 9 N 3 ONa [M+Na] + 210.06, found 210.06.

[0201] Example 4. Synthesis of Compound 4 (Method D) The equipment, reagents, solvents, etc. used were the same as in Example 1.

[0202] <Synthesis of 4-1.1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde (10)>

[0203] [ka]

[0204] Compound 10 was synthesized based on a previous report (JT Fletcher, JE Reilly, Tetrahedron Lett., 2011, 52, 5512-5515). The specific synthesis and compound identification results are shown below.

[0205] A mixture of an aqueous solution (25 mL) of sodium azide (358 mg, 5.5 mmol) and t-butyl alcohol (25 mL) was cooled to 0 °C, and nitrobenzenediazonium tetrafluoroborate (1.18 g, 5.0 mmol) was added in small portions, followed by vigorously stirring for 1 hour. Next, propargylaldehyde diethyl acetal (783 μL, 5.5 mmol) and sodium ascorbate (396 mg, 2 mmol) were added under a nitrogen atmosphere, and the mixture was stirred overnight at 70 °C. The reaction solution was filtered, and the filtrate was extracted with ethyl acetate (50 mL x 3). The organic layer was dried over sodium sulfate, the solvent was removed under reduced pressure, and the crude product was purified by reprecipitation (hexane:ethyl acetate) to obtain compound 10 (yellow solid). Figure 10 shows the reaction mixture. 1 The H NMR spectrum is shown.

[0206] Yield 62%; 1 H NMR (400 MHz, DMSO-d 6 ): δ10.15 (s, 1H), 9.78 (s, 1H), 8.49 (d, J = 9.2 Hz, 2H), 8.32 (d, J = 9.2 Hz, 2H); 13 C NMR (100 MHz, DMSO-d 6 ): δ185.0, 147.8, 147.4, 140.3, 126.9, 125.6, 121.5; ESI-TOF MS (positive mode) m / z calcd. for C 9 H 6 N 4 O 3 Na [M+Na] + 241.03, found 241.03.

[0207] Example 5. Synthesis of Compound 5 (Method E) The equipment, reagents, solvents, etc. used were the same as in Example 1.

[0208] <Synthesis of 5-1.5-methyl-1-phenyl-1H-1,2,3-triazole-4-carbaldehyde (13)> Compound 13 was synthesized according to the following scheme based on previous reports (J. Zhang, G. Jin, S. Xiao, J. Wu, S. Guo, Tetrahedron 2013, 69, 2352-2356, I. Ibnusaud, B. Singaram, J. Org. Chem., 2018, 83, 1431-1440, and T. Ismail, S. Shafi, I. Hyder, T. Sidiq, A. Khajuria, SM Alam, MSK Halmuthur, Arch. Pharm. Chem. Life Sci., 2015, 348, 796-807).

[0209] [ka]

[0210] <Synthesis of 5-1-1.ethyl 5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylate (11)> Ethyl acetoacetate (1.5 mmol) and piperidine (20 μL, 0.2 mmol) were added to a mixture of phenyl azide (120 mg, 1.0 mmol) in dimethylformamide (10 mL) and pure water (1 mL), and the mixture was stirred at 80°C for 24 hours. Water (20 mL) was added to the solution to stop the reaction, and the mixture was extracted with diethyl ether (20 mL x 5), and the resulting organic layer was washed with water (20 mL x 2). The organic layer was dried over sodium sulfate, filtered, and the solvent of the filtrate was distilled off under reduced pressure. The resulting crude product was purified by silica gel column chromatography (hexane: ethyl acetate) to obtain compound 11 (white solid).

[0211] Yield 27%; 1 H NMR (400 MHz, CDCl 3): δ7.61-7.55 (m, 3H), 7.47-7.44 (m, 2H), 4.47 (q, J = 7.1 Hz, 2H), 2.60 (s, 3H), 1.46 (t, J = 7.1 Hz, 3H).

[0212] <5-1-2.Synthesis of (5-methyl-1-phenyl-1H-1,2,3-triazol-4-yl)methanol (12)> Sodium borohydride (29 mg, 0.77 mmol) was added to a solution of compound 11 (0.12 mmol) and sodium methoxide (1 mg, 2 μmol) in methanol (1 mL) under a nitrogen atmosphere, and the mixture was stirred at room temperature for 3 hours. Excess methanol was added to the reaction solution to stop the reaction, and the mixture was concentrated under reduced pressure, and the residue was diluted with saturated aqueous sodium chloride solution (5 mL). This was extracted with diethyl ether (20 mL x 3) and washed with saturated aqueous sodium chloride solution (5 mL x 2). The organic layer obtained was dried over sodium sulfate and then filtered. The solvent of the filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (hexane: ethyl acetate = 2:1) to obtain compound 12 (white solid). As shown in Figure 11, 1 The H NMR spectrum is shown.

[0213] Yield 37%; 1 H NMR (400 MHz, CDCl 3 ): δ7.58-7.52 (m, 3H), 7.48-7.46 (m, 2H), 4.83 (d, J = 6.0 Hz, 2H), 2.37 (s, 2H), 1.96 (t, J = 6.0 Hz, 1H).

[0214] <Synthesis of 5-1-3.5-methyl-1-phenyl-1H-1,2,3-triazole-4-carbaldehyde (13)> Activated manganese dioxide (141 mg, 1.6 mmol) was added to a solution of compound 12 (0.16 mmol) in chloroform (5 mL) and stirred at room temperature under a nitrogen atmosphere for 24 hours. The reaction mixture was filtered, and the solvent in the filtrate was distilled off under reduced pressure to obtain compound 13 (white solid).

[0215] Yield 89%; 1 H NMR (400 MHz, DMSO-d 6 ): δ7.58-7.52 (m, 3H), 7.48-7.46 (m, 2H), 4.83 (d, J = 6.0 Hz, 2H), 2.37 (s, 2H), 1.96 (t, J = 6.0 Hz, 1H); 13 C NMR (100 MHz, DMSO-d 6 ): δ186.1, 143.3, 138.7, 134.7, 130.3, 129.8, 125.3, 9.3.

[0216] <Synthesis of 5-1-4.ethyl 1-phenyl-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxylate (14)>

[0217] [ka]

[0218] Compound 14 (yellow oil) was synthesized in the same manner as in Example 5-1-1 using Ethyl 4,4,4-Trifluoroacetoacetate as a precursor.

[0219] Yield 35%; 1 H NMR (400 MHz, CDCl 3 ): δ7.63-7.57 (m, 3H), 7.47 (d, J = 7.3 Hz, 2H), 4.51 (q, J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H).

[0220] <5-1-5.Synthesis of (1-phenyl-5-(trifluoromethyl)-1H-1,2,3-triazol-4-yl)methanol (15)>

[0221] [ka]

[0222] Compound 15 (white solid) was synthesized using compound 14 as a precursor in the same manner as in Example 5-1-2.

[0223] Yield 83%; 1 H NMR (400 MHz, CDCl 3 ): δ7.61-7.55 (m, 3H), 7.47 (d, J = 7.2 Hz, 2H), 4.96 (d, J = 6.0 Hz, 2H).

[0224] <5-1-6.Synthesis of (1-phenyl-5-(trifluoromethyl)-1H-1,2,3-triazol-4-yl)methanol (16)>

[0225] [ka]

[0226] Compound 16 (white solid) was synthesized using compound 15 as a precursor in the same manner as in Example 5-1-3. 1 The H NMR spectrum is shown.

[0227] Yield 75%; 1 H NMR (400 MHz, DMSO-d 6 ): δ10.21 (s, 1H), 7.74-7.66 (m, 5H).

[0228] Example 6. Synthesis of Compound 6 (Method F) The equipment, reagents, solvents, etc. used were the same as in Example 1.

[0229] <Synthesis of 6-1.1-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)-1H-1,2,3-triazole-4-carbaldehyde (17)>

[0230] [ka]

[0231] Compound 10 (109 mg, 0.5 mmol) and 2-(2-(2-propynyloxy)ethoxy]ethylamine (72 μL, 0.5 mmol) were dispersed in a mixture of pure water (1 mL) and t-butyl alcohol (1 mL) and stirred overnight at 60°C under a nitrogen atmosphere. After cooling the reaction solution to 0°C, 0.1 M HCl aq. (50 mL) was added to stop the reaction, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and the crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 0 to 60%) to obtain compound 17 (yellow oil). Figure 13 shows the elution product. 1 The H NMR spectrum is shown.

[0232] Yield 70%; 1 H NMR (400 MHz, CD 3 CN): δ10.04 (s, 1H), 8.42 (s, 1H), 4.59 (t, J = 5.1 Hz, 2H), 4.11 (d, J = 2.4 Hz, 2H), 3.87 (t, J = 5.1 Hz, 2H), 3.60-3.56 (m, 4H), 2.69 (t, J = 2.4 Hz, 1H); 13 C NMR (100 MHz, CD 3 CN): δ185.7, 148.4, 128.6, 80.8, 75.7, 70.6, 69.7, 69.5, 58.7, 51.3; ESI-TOF MS (positive mode) m / z calcd. for C 10 H 13 N3 O 3 Na [M+Na] + 246.08, found 246.08.

[0233] Example 7. Synthesis of Compounds 7: Synthesis of Functional Molecules The equipment, reagents, solvents, etc. used were the same as in Example 1. The reaction precursors used were either purchased or appropriately synthesized.

[0234] <7-1.Synthesis of N-(2-(2-(2-(2-(4-formyl-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-5-((4S)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (18)>

[0235] [ka]

[0236] Compound 18 (white solid) was synthesized using the same method (Method A) as in Example 1-3-1, using N-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-5-((4S)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide as a precursor. 1 The H NMR spectrum is shown.

[0237] Yield 23%; 1 H NMR (400 MHz, CD 3 CN+DMSO-d 6(one drop)): δ10.0 (s, 1H), 8.47 (s, 1H), 6.49 (s, 1H), 5.17 (s, 1H), 4.98 (s, 1H), 4.60 (t, J = 5.0 Hz, 2H), 4.42-4.38 (m, 1H), 4.24-4.21 (m, 1H), 3.88 (t, J = 5.0 Hz, 2H), 3.59-3.55 (m, 2H), 3.54 (m, 6H), 3.45 (t, J = 5.6 Hz, 2H), 3.27 (t, J = 5.6 Hz, 2H), 3.17-3.12 (m, 1H), 2.88 (dd, J = 5.0, ESI-TOF MS (positive mode) m / z calcd. for C 21 H 34 N 6 O 6 Na [M+Na] + 521.1, found 521.1.

[0238] <Synthesis of 7-2.1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)-1H-1,2,3-triazole-4-carbaldehyde (19)>

[0239] [ka]

[0240] Compound 19 (orange solid) was synthesized using 5-azido-3',6'-dihydroxy-3H-spiro[isobenzofuran-1,9'-xanthen]-3-one as a precursor in the same manner as in Example 1-3-1 (Method A). 1 The H NMR spectrum is shown.

[0241] Yield 47%; 1 H NMR (400 MHz, DMSO-d 6 ): δ10.19 (s, 2H), 10.15 (s, 1H), 9.80 (s, 1H), 8.60 (d, J = 1.7 Hz, 1H), 8.43 (dd, J = 2.0, 8.3 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 6.71-6.67 (m, 4H), 6.57 (dd, J = 2.2, 8.7 Hz, 2H); 13 C NMR (100 MHz, CDCl 3 ): δ185.0, 167.5, 159.7, 152.6, 151.9, 147.7, 137.4, 129.2, 128.0, 127.9, 126.8, 126.0, 116.7, 112.7, 108.9, 102.3, 83.6; ESI-TOF MS (positive mode) m / z calcd. for C 23 H 14 N 3 O 6 [M+H] + 428.09, found 428.10。

[0242] <Synthesis of 7-3.Polyethylene glycol-tethered triazole-4-carbaldehyde (MW. ~ 4 kDa) (20)>

[0243]

Chem.

[0244] Compound 20 (brown solid) was synthesized using a method similar to that of Example 1-3-1, with reference to a previous report (MB van Eldijk, FCM Smit, N. Vermue, MF Debets, S. Schoffelen, JCM van Hest, Biomacromolecules, 2014, 15, 2751-2759) and polyethylene glycol monomethyl ether (Tokyo Chemical Industry Co., Ltd.) with a molecular weight of about 4 kDa as the starting material. Purification was performed by reprecipitation with diethyl ether. As shown in FIG. 1 The H NMR spectrum is shown.

[0245] Yield 50%; 1 H NMR (400 MHz, CDCl 3 ): δ10.14 (s, 1H), 8.41 (s, 1H), 4.63 (t, J = 4.8 Hz, 1H), 3.91-3.45 (m, 366H).

[0246] <Synthesis of 7-4.1-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazole-4-carbaldehyde (21)>

[0247] [ka]

[0248] Compound 21 (yellow oil) was synthesized using 1-azido-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane as a precursor in the same manner as in Example 1-3-1 (Method A). 1 The H NMR spectrum is shown.

[0249] Yield 27%; 1 H NMR (400 MHz, CDCl 3): δ10.1 (s, 1H), 8.41 (s, 1H), 4.62 (t, J = 4.8 Hz, 2H), 3.90 (t, J = 4.8 Hz, 2H), 3.68 (m, 4H), 3.65-3.63 (m, 6H), 3.38 (t, J = 5.0Hz, 2H); 13 C NMR (100 MHz, CDCl 3 ): δ185.4, 148.0, 126.9, 70.7 (two signals were merged), 70.7 (two signals were merged), 70.2, 69.0, 50.8, 50.7; ESI-TOF MS (positive mode) m / z calcd. for C 11 H 18 N 6 O 4 Na [M+Na] + 321.1, found 321.1.

[0250] <7-5. Synthesis of compound 22>

[0251] [ka]

[0252] Compound 22 (yellow solid) was synthesized using the same method (Method F) as in Example 6, with N-(3-aminopropionyl)-5,6-dihydro-11,12-didehydrodibenz[b,f]azocine as a precursor. 1 The H NMR spectrum is shown.

[0253] Yield 75%; 1 H NMR (400 MHz, CD 3CN): δ9.89 (s, 1H), 7.88 (s, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.45-7.43 (m, 4H), 7.39-7.32 (m, 2H), 7.22 (dd, J = 1.4, 7.4 Hz, 1H), ESI-TOF MS (positive mode) m / z calcd. for C 11 H 18 N 6 O 4 Na [M+Na] + 321.1, found 321.1.

[0254] Example 8. Peptide N-terminal modification 1 <8-1. Reagents, solvents, etc.> Ultrapure water was purified using Millipore Integral 3. Other reagents and solvents were commercially available and used as is.

[0255] <8-2. N-terminal modification of peptides> In this method, the N-terminus of a peptide is targeted. The target peptide is one in which the N-terminal amino group is unmodified and the second amino acid residue from the N-terminus is an amino acid other than proline. As a specific example, modification of the N-terminus of angiotensin I is shown.

[0256] The amino acid sequence of angiotensin I is shown. DRVYIHPFHL (sequence number 1).

[0257] The peptide N-terminal modification was performed with reference to a previous report (JI MacDonald, HK Munch, T. Moore, MB Francis, Nat. Chem. Biol. 2015, 11, 326-331). The specific experimental procedures are described below.

[0258] A dimethyl sulfoxide (DMSO) solution of compound 7 (200 mM, 1 μL, 0.2 μmol, final concentration 10 mM) was diluted with potassium phosphate buffer (10 mM, pH 7.5, 17 μL), to which an aqueous peptide solution (1 mM, 2 μL, 2 nmol, final concentration 100 μM) was added, and the mixture was shaken at 37°C for 4 hours. Using LC / MS, the modification rate [= modified peptide / (total peptide amount)] was evaluated from the peak intensity in the mass spectrum. The results are shown in Figure 19. The modification rate under these reaction conditions was calculated to be 89%. These results confirmed that this compound group functions as a protein modification agent.

[0259] Example 9. Peptide N-terminal modification 2 The reagents, equipment, solvents, etc. used were the same as in Example 8. Compound 24 was synthesized according to a previous report (H. Hagiwara, S. Okada, Chem. Commun., 2016, 52, 815-818).

[0260] <9-1. N-terminal modification of peptides> Using a model peptide as a substrate, the structure of the product in this modification reaction was analyzed. As a specific example, the reaction shown in the following scheme was carried out.

[0261] [ka]

[0262] Compound 24 (44 mg, 0.4 mmol) in dimethylformamide (DMF) (200 μL) was added to compound 23 (11 mg, 0.04 mmol) in potassium phosphate buffer (10 mM, pH 7.5, 1.8 mL) and the mixture was shaken at 37° C. for 16 hours. The solvent was removed under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (ethyl acetate: acetonitrile: water = 95: 5: 0 to 0: 95: 5) to obtain compound 25.

[0263] <9-2. Compound 25 1 H NMR analysis> Compound 25 in deuterated acetonitrile 1 The H NMR spectrum assignment results are shown in FIG.

[0264] The two peaks at 5.7 ppm are the protons specific to the 2-position of the 4-imidazolidinone ring. e It was confirmed that the reaction proceeded to form a 4-imidazolidinone ring at the N-terminus of the peptide. The two peaks were derived from isomers with the 2-position of the 4-imidazolidinone ring as an asymmetric center.

[0265] Example 10. Protein N-terminal modification 1 <10-1. Reagents, solvents, etc.> Ribonuclease A (RNase) derived from bovine pancreas was purchased from Roche. Ultrapure water purified using Millipore Integral 3 was used. Other reagents and solvents were commercially available and used as is.

[0266] <10-2. Protein modification> This method targets the N-terminus of a protein. Target proteins are those whose N-terminal amino group is unmodified and whose second amino acid residue from the N-terminus is an amino acid other than proline. As a specific example, we will show the modification of the N-terminus of bovine pancreatic ribonuclease A (RNase).

[0267] The amino acid sequence of RNase is shown (PDB: 1FS3). KETAAAKFER QHMDSSTSAA SSSNYCNQMM KSRNLTKDRC KPVNTFVHE SLADVQAVCS QKNVACKNGQ TNCYQSYSTM SITDCRETGS SKYPNCAYKT TQANKHIIVA CEGNPYVPVH FDASV (SEQ ID NO:2).

[0268] Protein N-terminal modification was performed with reference to a previous report (JI MacDonald, HK Munch, T. Moore, MB Francis, Nat. Chem. Biol. 2015, 11, 326-331). Specific experimental procedures are shown below.

[0269] A dimethyl sulfoxide (DMSO) solution of compound 7 (200 mM, 2.5 μL, 0.5 μmol, final concentration 10 mM) was diluted with potassium phosphate buffer (10 mM, pH 7.5, 45 μL), to which an ultrapure RNase solution (1 mM, 2.5 μL, 2.5 nmol, final concentration 50 μM) was added, and the mixture was shaken at 37°C for 16 hours. The modification rate [= modified RNase / (total RNase amount)] was evaluated from the peak intensity in the mass spectrum using LC / MS. The results are shown in Figure 21. The modification rate under these reaction conditions was calculated to be 88%. If necessary, the product after the reaction was purified by size exclusion chromatography.

[0270] Example 11. Functional molecule modification to the N-terminus of a protein <11-1. Reagents, solvents, etc.> Ribonuclease A (RNase) derived from bovine pancreas was purchased from Roche. Ultrapure water purified using Millipore Integral 3 was used. Other reagents and solvents were commercially available and used as is.

[0271] <11-2. Biotin modification to protein N-terminus> The N-terminus of the protein was modified with biotin using the same method as in Example 10-2, but using compound 18 as a modifying agent. The results of LC / MS analysis of the product are shown in Figure 22. The modification rate of the biotin site was calculated to be 79%.

[0272] <11-3. Modification of protein N-terminus with fluorescent dye> Compound 19 was used as a modifying agent to modify the N-terminus of the protein with a fluorescent dye (here, a fluorescein molecule) in the same manner as in Example 10-2. The results of LC / MS analysis of the product are shown in Figure 23. The modification rate of the fluorescent dye site was calculated to be 73%.

[0273] <11-4. Modification of protein N-terminus with azide group> Compound 21 was used as a modifying agent to modify the N-terminus of the protein with an azide group in the same manner as in Example 10-2. The results of LC / MS analysis of the product are shown in Figure 24. The modification rate of the azide group was calculated to be 79%.

[0274] <11-5. Functional molecule modification starting from azide group modified at the N-terminus of a protein> The RNase modified with the azide group prepared in Example 11-4 was modified with a fluorescent dye by strain-promoted alkyne-azide cycloaddition reaction. Compound 26, a fluorescein having a dibenzocyclooctyne moiety, was used as the alkyne substrate in the reaction. The scheme and results are shown in Figure 25.

[0275] A dimethyl sulfoxide (DMSO) solution of compound 26 (10 mM, 1 μL, final concentration 100 μM) was added to a phosphate buffer solution containing RNase_21 (RNase_21 concentration 120 μM, final concentration 20 μM, buffer solution concentration 10 mM, pH 7.5, 82.3 μL) and left to stand at 4 °C for 16 hours. The modification rate of the fluorescent dye site was calculated to be 55%.

[0276] <11-6. Modification of protein N-terminus with distorted alkyne moiety> Compound 22 was used as a modifying agent to modify the strained alkyne moiety at the protein N-terminus in the same manner as in Example 10-2. The results of LC / MS analysis of the product are shown in Figure 26. The modification rate of the cyclooctyne moiety was calculated to be 40%.

[0277] Example 12. Synthesis of reactant immobilized resin for heterogeneous reaction The construction of a new N-terminal selective modifier by the rearrangement reaction between compound 10 and an amine precursor shown in Example 6 is useful as a clean reaction that does not require a copper catalyst. Therefore, by immobilizing various carbaldehyde precursors on a resin or solid material, it is possible to easily remove the by-product (aniline derivative) generated after the reaction, and the solution after the reaction can be directly used for protein N-terminal modification. Figure 27 shows a method.

[0278] <12-1. Equipment, reagents, solvents, etc.> The equipment, reagents, and solvents used were the same as those in Examples 1 and 8. The aminomethylpolystyrene resin used was purchased from Tokyo Chemical Industry Co., Ltd.

[0279] <12-2. Synthesis of reactant immobilized resin> As a specific example, the synthesis of polystyrene resin immobilized with compound 27 is shown in Figure 28. The synthesis was carried out according to the scheme shown in Figure 29.

[0280] Aminomethyl polystyrene resin (Aminomethyl / PS resin, 200 mg) was dispersed in DMF (30 mL) and shaken for 10 minutes to swell. 5-azido-2-nitrobenzoic acid (208 mg, 1.0 mmol), 1-hydroxybenzotriazole monohydrate (200 mg, 1.3 mmol), and N,N'-diisopropylcarbodiimide (156 μL, 1.0 mmol) were added to the resin dispersion and shaken overnight. After the reaction, the resin was filtered, washed with dimethylformamide, pure water, chloroform, and acetone, and dried under reduced pressure. To inactivate the unreacted amino groups, the resin was treated with a mixed solution of acetic anhydride (1 mL) and chloroform (4 mL) for 30 minutes, washed with chloroform, methanol, and acetone, and dried under reduced pressure to obtain polystyrene resin modified with azido groups (Azido / PS resin).

[0281] Azido / PS resin (220 mg) was swollen in a DMF / water mixture (6:1, 7 mL), and then copper(II) sulfate pentahydrate (25 mg, 0.1 mmol), sodium ascorbate (40 mg, 0.2 mmol), and propargylaldehyde diethyl acetal (356 μL, 2.5 mmol) were added to the resin dispersion and shaken at room temperature for 12 hours. After the reaction, the resin was filtered and washed with dimethylformamide, pure water, chloroform, methanol, and acetone, in that order. It was then treated with 12% aqueous ammonia solution (5 mL) for 10 minutes, filtered, and washed with pure water to remove the copper catalyst remaining on the resin surface. Finally, the resin was treated with a mixture of aqueous hydrochloric acid and tetrahydrofuran (1:1, 5 mL) for 2 min each time, three times in total, and then filtered and washed with pure water and acetone to deprotect the acetal protecting group, yielding a resin (27 / PS resin) on which compound 27 was immobilized.

[0282] <12-3. Identification of resin> The chemical species on the surface of the resin (27 / PS resin) to which compound 27 was immobilized were identified by infrared spectroscopy. The results are shown in Figure 30. The azide group-modified resin (Azido / PS resin) showed an IR spectrum of 2115 cm -1 Next, in the resin (27 / PS resin) after the formation of the triazole ring by the CuAAC reaction, the stretching vibration peak of the azide group disappeared, and a new peak at 1699 cm resulting from the stretching vibration of the aldehyde group was observed. -1 The above results support the modification of aldehyde groups by the CuAAC reaction, and it became clear that 27 / PS resin was successfully prepared.

[0283] <12-4. Protein N-terminal modification> Using the prepared reagent-immobilized resin (27 / PS resin), we prepared an N-terminal modifier and performed consecutive protein N-terminal modification. As a specific example, we will show the N-terminal modification of ribonuclease A (RNase A).

[0284] 27 / PS resin (5 mg) was added to a dimethyl sulfoxide solution of benzylamine (100 mM, 50 μL, 5 μmol) and heated at 100 °C for 90 minutes using a heat block. After cooling to room temperature, the supernatant (10 μL, 1 μmol) was diluted with phosphate buffer solution (10 mM, pH 7.5, 85 μL), to which RNase A solution (1 mM, 5 μL, 5 nmol) was added, and the mixture was shaken at 37 °C for 16 hours. After the reaction, modification was evaluated using LC / MS. Even when using the reactant-immobilized resin (27 / PS resin), the modification rate (75%) was similar to that when using the isolated and purified N-terminal modifier. From the above, we have demonstrated a method for modifying protein N-terminals by continuous reactions using the reactant-immobilized resin (27 / PS resin). The results are shown in Figure 31.

[0285] <12-5. Improvement of the synthesis method of reactive agent immobilized resin> In Example 12-3, in the sample that underwent protein modification, a product that resembled the addition of oxygen to the protein was observed. This is thought to be due to the copper catalyst used in the resin synthesis remaining on the resin. Therefore, 27 / PS resin was created based on a new synthesis scheme and applied to protein modification. Specifically, 27 / PS resin was prepared according to the scheme shown in Figure 32.

[0286] <Synthesis of 12-5-1.5-Azido-2-nitrobenzoic Acid (28)>

[0287] [ka]

[0288] 5-amino-2-nitrobenzoic acid (1.60 g, 8.9 mmol) in concentrated HCl / EtOH / H 2To the O mixed solvent (2:1:2, total 43 mL) was added an aqueous solution (6 mL) of sodium nitrite (727 mg, 10.5 mmol) and stirred for 1 h at 0 °C. Sodium azide (868 mg, 13.4 mmol) was then added in small portions, and the solution was then vigorously stirred for 1 h at 0 °C and then at room temperature for 1 h.

[0289] The reaction solution was diluted with ultrapure water, filtered, and then washed with ultrapure water (20 mL × 2). Compound 28 (light yellow solid) was obtained by drying under reduced pressure. 1 The H NMR spectrum is shown.

[0290] Yield 37%; 1 H NMR (400 MHz, DMSO-d 6 ): δ, 8.1 (d, J = 9.4 Hz, 3H), 7.43-7.41 (m, 2H); 13 C NMR (100 MHz, DMSO-d 6 ): δ, 165.8, 145.3, 143.4, 130.8, 126.2, 121.7, 119.6; ESI-TOF MS (positive mode) m / z calcd. for C 7 H 4 NaN 4 O 4 [M+Na] + 231.012, found 231.011.

[0291] <Synthesis of 12-5-2.2,5-Dioxopyrrolidin-1-yl-5-(4-formyl-1H-1,2,3-triazol-1-yl)-2-nitrobenzoate (30)>

[0292] [ka]

[0293] Compound 28 (1.75 g, 8.4 mmol) and 3,3-diethoxyprop-1-yne (1.2 mL 1.08 g, 8.4 mmol) were dispersed in a mixture of pure water (21 mL) and t-butyl alcohol (21 mL), and copper(II) sulfate pentahydrate (419 mg, 1.68 mmol, 20 mol%) and sodium ascorbate (666 mg, 3.36 mmol, 40 mol%) were added and stirred overnight at 70 °C under a nitrogen atmosphere. After cooling the reaction solution to room temperature, saturated saline was added to stop the reaction, and the mixture was extracted with ethyl acetate (20 mL x 3). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product containing compound 29. The product was used as it was in the next reaction without purification.

[0294] N-hydroxysuccinimide (764 mg, 6.64 mmol) was added to the DMF solution (22 mL) containing the crude product, and the mixture was stirred at room temperature for 10 minutes. Next, N,N'-dicyclohexylcarbodiimide (1.14 g, 5.5 mmol) was added, and the reaction mixture was stirred overnight. The precipitate was filtered off, and the filtrate was dried under reduced pressure to remove the solvent. The residue was dissolved in ethyl acetate (50 mL), and the resulting precipitate was filtered off again. The filtrate was washed with saturated saline (20 mL x 2), the organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product containing compound 29. Compound 30 (light yellow solid) was obtained by purification using silica gel column chromatography. As shown in Figure 34, 1 The H NMR spectrum is shown.

[0295] Yield 18% (after 2 steps); 1 H NMR (400 MHz, DMSO-d 6 ): δ, 10.15 (s, 1H), 9.91 (s, 1H), 8.64-8.61 (m, 2H), 8.57-8.55 (m, 1H), 2.91 (s, 4H); 13 C NMR (100 MHz, DMSO-d 6): δ, 184.9, 169.7, 160.1, 147.8, 146.8, 139.3, 127.5, 127.4, 126.0, 123.0, 121.9, 25.6; ESI-TOF MS (positive mode) m / z calcd. for C 14 H 9 NaN 5 O 7 [M+Na] + 382.038, found 382.039.

[0296] <Synthesis of 12-5-3.27 / PS resin 2> Aminomethyl polystyrene resin (Aminomethyl / PS resin, 200 mg) was dispersed in DMF (5 mL) and shaken for 1 hour to allow swelling. 30 (150 mg, 0.42 mmol) and N,N'-diisopropylcarbodiimide (146 μL, 0.84 mmol) were added to the resin dispersion and shaken overnight. After the reaction, the resin was filtered, washed with dimethylformamide, pure water, chloroform, and acetone, and dried under reduced pressure. To inactivate the unreacted amino groups, the resin was treated with a mixed solution of acetic anhydride (1 mL) and chloroform (4 mL) for 30 minutes, washed with chloroform, methanol, and acetone, and dried under reduced pressure to obtain 27 / PS resin.

[0297] <12-6. Protein N-terminal modification> Using the newly prepared reactant immobilized resin (27 / PS resin), we prepared an N-terminal modifier and performed consecutive protein N-terminal modification. As a specific example, we will show the N-terminal modification of ribonuclease A (RNase A). The scheme is shown in Figure 35.

[0298] 27 / PS resin (5 mg) was added to a dimethyl sulfoxide solution of benzylamine (100 mM, 40 μL, 4 μmol) and heated at 100 °C for 90 minutes using a heat block. After cooling to room temperature, the supernatant (10 μL, 1 μmol) was diluted with phosphate buffer solution (10 mM, pH 7.5, 85 μL), to which RNase A solution (1 mM, 5 μL, 5 nmol) was added, and the mixture was shaken at 37 °C for 16 hours. After the reaction, modification was evaluated using LC-MS. The results are shown in Figure 36. Even when using the reagent-immobilized resin (27 / PS resin), the modification rate (82%) was similar to that when using the isolated and purified N-terminal modifier. Furthermore, it was demonstrated that the same method can be used to modify the N-terminus of proteins with various functional amine compounds.

[0299] Example 13. Synthesis of protein modifiers using the Dimroth rearrangement reaction in homogeneous systems and sequential protein modification The construction of a new N-terminal selective modifier by the rearrangement reaction between compound 10 and an amine precursor shown in Example 6 is useful as a clean reaction that does not use a copper catalyst. In Example 12, a Dimroth rearrangement reaction in a heterogeneous system was shown to simplify the removal of by-products. On the other hand, in protein modification, there are also cases where the presence of aniline derivatives as by-products is not a problem. Therefore, in this section, the synthesis of carbaldehyde derivatives by the Dimroth rearrangement reaction in a homogeneous system and the subsequent protein modification are shown. The scheme is shown in Figure 37.

[0300] <13-1. Reagents, solvents, etc.> The equipment, reagents, and solvents used were the same as those in Examples 1 and 8. Ribonuclease A (RNase) derived from bovine pancreas was purchased from Roche. Ultrapure water purified by Millipore Integral 3 was used.

[0301] <13-2. Optimization of Dimroth rearrangement reaction conditions> The reaction mechanism of the Dimroth rearrangement is shown in Figure 38. Since this reaction involves imine formation in the first step, the addition of an acid catalyst is expected to improve the reaction efficiency. Therefore, the Dimroth rearrangement reaction was carried out with compound 10 using benzylamine as a model substrate in the presence of various acid catalysts.

[0302] A dimethyl sulfoxide solution of compound 10 (200 mM, 20 μL, 4 μmol) and an acid aqueous solution (200 mM or 400 mM, 1 μL, 5 mol% or 10 mol%) were added to a dimethyl sulfoxide solution of benzylamine (200 mM, 20 μL, 4 μmol), and the mixture was heated at 100 °C for 30 minutes using a heat block. After cooling to room temperature, 1 μL of the reaction mixture was diluted with a phosphate buffer solution (100 mM, pH 7.0, 200 μL), transferred to a 96-well plate, and UV-visible absorption measurements were performed. The conversion rate of the reaction was calculated from a standard curve created based on the absorption intensity at 380 nm characteristic of the product p-Nitroaniline. Figure 39 shows the structure of the acid catalyst used, and the conversion rates obtained are shown in Table 1.

[0303] In Table 1, a The conversion rate was calculated based on the absorption intensity of the by-product p-Nitroaniline. b The amount of MOPS added was 10 mol %.

[0304] [Table 1]

[0305] It was shown that the conversion rate improved with the addition of an acid catalyst. It was suggested that the addition of sulfonic acid was particularly effective. In addition, it was revealed that various sulfonates, which are known to function as a Good's buffer, also improved the reaction efficiency. In general, these sulfonates do not significantly affect protein function or structure, so it is possible to directly use the Dimroth rearrangement reaction mixture for the subsequent protein modification reaction. 3-Morpholinopropanesulfonic acid (MOPS) was selected as the acid catalyst for the subsequent experiments.

[0306] <13-3. Protein N-terminal modification> A dimethyl sulfoxide solution of compound 10 (200 mM, 20 μL, 4 μmol) and an aqueous MOPS solution (200 mM or 400 mM, 1 μL, 5 mol% or 10 mol%) were added to a dimethyl sulfoxide solution of benzylamine (200 mM, 20 μL, 4 μmol), and the mixture was heated at 100 °C for 30 minutes using a heat block. After cooling to room temperature, the reaction mixture (5 μL) was diluted with a phosphate buffer solution (10 mM, pH 7.5, 42.5 μL), to which an RNase A solution (1 mM, 2.5 μL, 5 nmol) was added, and the mixture was shaken at 37 °C for 16 hours. After the reaction, modification evaluation was performed using LC-MS. The results of the LC-MS analysis are shown in Figure 40.

[0307] Using various functional amine precursors such as alkynes, azides, and fluorescent dyes, we prepared protein modification reagents and achieved their introduction into the N-terminus of proteins. In addition, even when the preparation of the N-terminal modification reagent and the protein modification reaction were performed consecutively, the modification rate was comparable to that when an isolated and purified N-terminal modification reagent was used. Thus, we have demonstrated a method for protein N-terminal modification by consecutive reactions using the Dimroth rearrangement reaction in a homogeneous system.

[0308] Example 14. Modification of protein N-terminus using Bis-TA4C molecule and introduction of functional molecule by oxime formation In the Dimroth rearrangement using a diamine with amino groups at both ends as a precursor, a molecule with TA4C moieties introduced at both ends (hereafter referred to as "Bis-TA4C") is obtained. In the N-terminal modification of a protein using this Bis-TA4C, an aldehyde moiety can be introduced to the protein's N-terminus. An example of a chemical modification reaction starting from an aldehyde is the formation of an oxime with hydroxylamine. This reaction proceeds under mild conditions, even in water, and is therefore applicable to protein modification. In addition, since the oxime bond is a dynamic bond, it is possible to remove the functional molecule as necessary. Therefore, a Dimroth rearrangement reaction was performed using compound 10 as a precursor to various diamines, and a protein modification reaction was carried out.

[0309] <14-1. Reagents, solvents, etc.> The equipment, reagents, and solvents used were the same as those in Examples 1 and 8. Ribonuclease A (RNase) derived from bovine pancreas was purchased from Roche. Ultrapure water purified by Millipore Integral 3 was used.

[0310] <14-2. Preparation of Bis-TA4C and protein modification reaction> A dimethyl sulfoxide solution of compound 10 (200 mM, 20 μL, 4 μmol) and an acid aqueous solution (200 mM or 400 mM, 1 μL, 5 mol% or 10 mol%) were added to a dimethyl sulfoxide solution of diamines 31-36 (100 mM, 20 μL, 2 μmol), and the mixture was heated at 90 °C for 60 minutes using a heat block. After cooling to room temperature, the reaction mixture (5 μL) was diluted with a phosphate buffer solution (10 mM, pH 7.5, 42.5 μL), to which an RNase A solution (1 mM, 2.5 μL, 5 nmol) was added, and the mixture was shaken at 37 °C for 16 hours. After the reaction, the modification was evaluated using LC-MS. The scheme is shown in Figure 41, and the results of the protein modification are shown in Figure 42.

[0311] Bis-TA4C, which has an alkyl chain or an oligoethylene glycol chain as a linker, gave a good protein modification yield of 60-82%. In contrast, the modification reaction did not proceed with Bis-TA4C that contains an aromatic skeleton such as aniline. This is thought to be due to the extremely low solubility of Bis-TA4C. Therefore, it is expected that the reaction yield will be improved by introducing a moiety that improves water solubility.

[0312] <14-3. Introduction of functional molecules into proteins modified with Bis-TA4C via oxime formation> An oxime formation reaction was carried out starting from the aldehyde moiety introduced to the N-terminus of the protein by Bis-TA4C. Specifically, the reaction was carried out according to the procedure shown in Figure 43, with reference to a previous report (M. Rashidian, MM Mahmoodi, R. Shah, JK Dozier, CR Wagner, MD Distefano, Bioconjugate Chem. 2013, 24, 333-342). As examples, fluorescent dye and polyethylene glycol modification are shown.

[0313] RNase A (60 μM, 8.3 μL, 0.5 nmol) modified with Bis-TA4C prepared from compound 35 as a precursor was diluted with phosphate buffer solution (50 mM, pH 7.0, 35.7 μL), to which was added a dimethyl sulfoxide solution (5 mM, 1 μL, 5 nmol) containing hydroxylamine 37 and an aqueous solution of m-phenylenediamine (m-PDA) (50 mM, 5 μL, 0.25 μmol) as a catalyst, and the mixture was shaken at 4°C for 6 hours. After the reaction, the modification was evaluated using LC-MS. Figure 44 shows the results of protein modification. Although some adducts of the catalyst m-PDA were observed, it was clear that the protein was converted to an oxime adduct in good yield.

[0314] RNase A (60 μM, 8.3 μL, 0.5 nmol) modified with Bis-TA4C prepared from compound 35 as a precursor was diluted with phosphate buffer solution (50 mM, pH 7.0, 35.7 μL), to which hydroxylamine 38 (2 mg, 500 nmol) and m-phenylenediamine (m-PDA) aqueous solution (50 mM, 5 μL, 0.25 μmol) as a catalyst were added, and the mixture was shaken at room temperature for 16 hours. After the reaction, modification was evaluated using SDS-PAGE. Figure 45 shows the results of protein modification.

[0315] For comparison, we also performed experiments using unmodified protein or polyethylene glycol with a hydroxyl group as the substrate. A band shift was observed on SDS-PAGE only in the combination of an aldehyde group at the N-terminus of the protein and a hydroxylamine site in the polyethylene glycol, confirming the introduction of polyethylene glycol through oxime formation.

[0316] Example 15. Dimroth rearrangement reaction under mild conditions In the preparation of N-terminal modifiers by the Dimroth rearrangement reaction in a homogeneous system shown in Examples 13 and 14, high reaction temperatures of 90 to 100 °C are required. Therefore, further improvements in the precursor structure and reaction conditions are required to adapt to substrates with low heat resistance. Therefore, in order to achieve reactions at lower temperatures, the structure of the precursor, compound 10, was modified. In particular, a compound in which the nitrophenyl group moiety has been changed to a more electron-withdrawing substituent is expected to function as a useful precursor because it stabilizes the intermediate and makes the reverse reaction less likely to proceed. In this section, we will show the synthesis of compounds based on this strategy and examples of application to the Dimroth rearrangement reaction.

[0317] <15-1. Compound synthesis> As a specific structural example, the synthesis of triazolecarbaldehyde 40 having a 4-cyanotetrafluorophenyl group is shown below.

[0318] [ka]

[0319] <Synthesis of 15-1-1.4-(4-(diethoxymethyl)-1H-1,2,3-triazol-1-yl)-2,3,5,6-tetrafluorobenzonitrile (39)> A solution of pentafluorobenzonitrile (850 mg, 541 μL, 4.4 mmol) and sodium azide (260 mg, 4.0 mmol) in acetonitrile (10 mL) was stirred at 60 °C under nitrogen atmosphere for 16 hours. Then, under nitrogen atmosphere, propargylaldehyde diethyl acetal (570 μL, 4.0 mmol) and copper(I) iodide (76 mg, 0.4 mmol) were added and stirred at room temperature for 16 hours. After cooling the reaction solution to room temperature, it was diluted with saturated aqueous sodium chloride solution (40 mL) and extracted with ethyl acetate (50 mL x 3). The organic layer obtained was dried over magnesium sulfate, and the solid was filtered off. The filtrate obtained was evaporated under reduced pressure to obtain a crude product. Compound 39 (white solid) was obtained by purification using silica gel column chromatography. As shown in Figure 46, 1 The H NMR spectrum is shown.

[0320] Yield 86%; 1 H-NMR (400 MHz, CDCl 3 ): δ 7.97 (s, 1H), 5.82 (s, 1H), 3.79-3.64 (m, 4H), 1.28 (t, J = 7.1 Hz, 6H).

[0321] <Synthesis of 15-1-2.2,3,5,6-tetrafluoro-4-(4-formyl-1H-1,2,3-triazol-1-yl)benzonitrile (40)> Compound 39 (688 mg, 2.0 mmol) was dissolved in chloroform (4 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at room temperature for 16 hours. The solvent and trifluoroacetic acid were removed by distillation under reduced pressure to obtain a crude product. Compound 40 (white solid) was obtained by reprecipitation (hexane:chloroform). 1 The H NMR spectrum is shown.

[0322] Yield 78%; 1 H-NMR (400 MHz, CDCl 3 ): δ 10.26 (s, 1H), 8.50 (s, 1H).

[0323] <15-2. Dimroth rearrangement reaction using compound 40 as a precursor> To evaluate the reactivity of compound 40, the Dimroth rearrangement reaction was carried out using benzylamine as a substrate.

[0324] A dimethyl sulfoxide solution of compound 40 (100 mM, 40 μL, 4 μmol) and an aqueous MOPS solution (200 mM, 2 μL, 10 mol%) were added to a dimethyl sulfoxide solution of benzylamine (100 mM, 40 μL, 4 μmol), and the mixture was heated at 40 °C for 12 hours using a heat block. After cooling to room temperature, the reaction mixture was diluted with deuterated dimethyl sulfoxide (310 uL), and a deuterated dimethyl sulfoxide solution of 1,3,5-trimethoxybenzene (400 mM, 10 μL, 4 mol) was added as an internal standard. 1 H NMR measurement was performed. The yield was calculated from the integral value of the peak corresponding to the proton at the benzyl position of the product, Compound 7, based on the integral value of the internal standard. The scheme is shown in FIG. 48, and the results are shown in FIG. 49.

[0325] It was revealed that the Dimroth rearrangement reaction proceeded in good yield at low temperatures when using compound 40, compared with the case of using compound 10. Therefore, it was shown that the introduction of an electron-withdrawing substituent at the N1 position of the triazole ring greatly contributes to improving the reactivity in the Dimroth rearrangement reaction.

[0326] Example 16. Protein N-terminal modification 2 We carried out an N-terminal modification reaction on human serum albumin (HSA), which is a commonly used protein substrate.

[0327] <16-1. Reagents, solvents, etc.> Human serum albumin (HSA) was purchased from Merck. Ultrapure water was purified using Millipore Integral 3. Other reagents and solvents were used as they were. HSA was used as a mixture with HSA that had already been modified in vivo. Modifications of HSA have been identified in a previous report (A. Kawakami, K. Kubota, N. Yamada, U. Tagami, K. Takehana, I. Sonaka, E. Suzuki, K. Hirayama, FEBS J., 2006, 273, 3346-3357.).

[0328] <16-2. Protein modification> This method targets the N-terminus of proteins. Target proteins are those whose N-terminal amino group is unmodified and whose second amino acid residue from the N-terminus is an amino acid other than proline. As a specific example, we will show the modification of the N-terminus of human serum albumin (HSA).

[0329] The amino acid sequence of HSA is shown (PDB: 1AO6). DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH EAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE RQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK ADDKETCFAE EGKKLVAASQ AALGL (SEQ ID NO: 3).

[0330] Compound 7 was used as a modifying agent and HSA was used as a target protein, and protein N-terminal modification was performed in the same manner as in Example 10-2. The results of LC / MS analysis of the product are shown in Figure 50. The modified protein is referred to as HSA1. The modification rate under these reaction conditions was calculated to be 99% or more. If necessary, the product after the reaction was purified by size exclusion chromatography.

[0331] Example 17. Dual modification of proteins targeting the protein N-terminus and cysteine ​​residues <17-1. Reagents, solvents, etc.> The equipment, reagents, solvents, etc. used were the same as in Example 16.

[0332] <17-2. Modification of protein cysteine ​​residues> Cysteine ​​residue modification of proteins was performed with reference to a previous report (X. Chen, H. Wu, C.-M. Park, TH Poole, G. Keceli, NO Devarie-Baez, AW Tsang, WT Lowther, LB Poole, SB King, M. Xian, CM Furdui, ACS Chem. Biol., 2017, 12, 2201-2208). The specific experimental procedures are described below.

[0333] A DMSO / water (1:1) mixed solution of compound 41 (25 mM, 40 μL, 1 μmol, final concentration 500 μM) was diluted with phosphate buffer (100 mM, pH 7.0, 1.86 mL), to which was added an ultrapure HSA solution (1 mM, 100 μL, 100 nmol, final concentration 50 μM), and the mixture was allowed to stand at 4°C for 12 hours. After the reaction, modification was evaluated using LC / MS. The results are shown in Figure 51. The modified protein is referred to as HSA2. The modification rate under these reaction conditions was calculated to be 99% or more. If necessary, the product after the reaction was purified by size exclusion chromatography.

[0334] <17-3. N-terminal modification of proteins with modified cysteine ​​residues> The HSA2 prepared in Example 17-2 was subjected to N-terminal modification of the protein with compound 7. Figure 52 shows the scheme and results.

[0335] A dimethyl sulfoxide (DMSO) solution of compound 7 (200 mM, 1 μL, final concentration 10 mM) was added to a phosphate buffer solution containing HSA2 (HSA2 concentration 200 μM, final concentration 50 μM, buffer solution concentration 10 mM, pH 7.5, 19 μL) and shaken at 37 °C for 16 hours. The modified protein is referred to as HSA3. The modification rate under these reaction conditions was calculated to be 72%.

[0336] Example 18. Stability of N-terminally modified proteins <18-1. Reagents, solvents, etc.> The equipment, reagents, solvents, etc. used were the same as in Example 10.

[0337] <18-2. Stability of modified proteins over time> Phosphate buffer containing RNase_7 (RNase_7 concentration 100 μM, final concentration 10 μM, buffer solution concentration 100 mM, pH 7.0, 10 μL) was diluted with phosphate buffer (100 mL, pH 7.0, 90 μL) and left at 37 °C for 12 hours, 24 hours, and 48 hours, respectively. As an example, the results of LC / MS analysis after leaving it for 24 hours are shown in Figure 53.

[0338] This modification reaction, which involves the formation of a 4-imidazolidinone ring at the N-terminus, is an equilibrium reaction. The results of LC / MS analysis suggest that the unmodified protein and modifier are regenerated by the reverse reaction, hydrolysis of the 4-imidazolidinone ring, of the protein modified at the N-terminus. Figure 54 shows a graph in which the release rate calculated from the peak intensity in the mass spectrum [=1-(modified RNase after standing / total RNase amount) / (modified RNase / total RNase amount before standing)] is on the vertical axis and the standing time is on the horizontal axis.

[0339] <18-3. Stability of modified proteins with pH change> Phosphate buffer containing RNase_7 (RNase_7 concentration 100 μM, final concentration 10 μM, buffer solution concentration 100 mM, pH 7.0, 10 μL) was diluted with buffer (100 mL, 90 μL) to pH 4, 5, 6, 7, and 8, respectively, and left at 37°C for 12 hours. Dilution was performed with acetate buffer for pH 4 and 5 conditions, and with phosphate buffer for pH 6, 7, and 8 conditions. The results of the release rate are shown in Figure 55.

[0340] Example 19. Compound synthesis 8 (synthesis of triazolecarbaldehyde having substituents at 1- and 5-positions) The reagents and solvents used in the synthesis were commercially available and used as they were. The precursors, azides, alkynes, and pinacol boronates, have been reported (LSC-Verduyn, L. Mirfeizi, RA Dierckx, PH Elsinga, BL Feringa, Chem. Commun., 2009, 16, 2139-2140; E. Jahnke, J. Weiss, S. Neuhaus, TN Hoheisel, H. Frauenrath, Chem. Eur. J., 2009, 15, 388-404; JC Pieck, D. Kuch, F. Grolle, U. Linne, C. Haas, T. Carell, J. Am. Chem. Soc., 2006, 128, 1404-1405; and J. Schmidt, M. Rotter, T. Weiser, S. Wittmann, L. Weizel, A. Kaiser, J. Heering, T. Goebel, C. Angioni, M. Wurglics, A. Paulke, G. Geisslinger, A. Kahnt, D. Steinhilber, E. Proschak, D. Merk, J. Med. Chem., 2017, 60, 7703-7724 and JR White, GJ Price, S. Schiffers, PR Raithby, PK Plucinski, CG Frost, Tetrahedron Letters, 2010, 51, 3913-3917).

[0341] <Synthesis of 19-1.1-benzyl-5-phenyl-1H-1,2,3-triazole-4-carbaldehyde (44)> Compound 44 was synthesized according to the following scheme, with reference to previous reports (K. Yamamoto, Ta Bruun, JY Kim, L. Zhang, M. Lautens, Org. Lett., 2016, 18, 2644-2647 and J. Deng, Y.-M. Wu, Q.-Y. Chen, Synthesis, 2005, 16, 2730-2738).

[0342] [ka]

[0343] <19-1-1.Synthesis of (1-benzyl-5-iodo-1H-1,2,3-triazol-4-yl)methanol (42)> To a mixture of benzyl azide (0.67 g, 5.0 mmol), 3-iodoprop-2-yn-1-ol (0.91 g, 5.0 mmol), copper(I) iodide (95 mg, 0.50 mmol), TBTA (0.27 g, 0.50 mmol) and potassium acetate (1.5 g, 15 mmol), THF (89 mL) was added under nitrogen atmosphere and stirred at room temperature overnight. The solvent of the reaction mixture was distilled off under reduced pressure, and the mixture was diluted with water (30 mL) and ethyl acetate (30 mL), and then extracted with ethyl acetate (30 mL x 3). The obtained organic layer was washed with saturated saline (30 mL x 2), dried over sodium sulfate, and the solvent of the filtrate was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate) to obtain compound 42 (white solid). As shown in Figure 56, 1 The H NMR spectrum is shown.

[0344] Yield 82%: 1 H NMR (400 MHz, DMSO-d 6): δ, 7.42-7.33 (m, 3H), 7.24-7.22 (m, 2H), 5.65 (s, 2H), 5.27 (t, J = 5.6 Hz, 1H), 4.47 (d, J = 5.6 Hz, 2H): 13 C NMR (100 MHz, DMSO-d 6 ): δ, 151.30, 135.52, 128.89, 128.20, 127.54, 83.79, 54.99, 53.25: ESI-TOF MS (positive mode) m / z calcd. for C 10 H 10 IN 3 NaO [M+Na] + 337.976, found 337.977.

[0345] <19-1-2.Synthesis of (1-benzyl-5-phenyl-1H-1,2,3-triazol-4-yl)methanol (43)> To a mixture of compound 42 (0.24 g, 0.75 mmol), phenylboronic acid (0.18 g, 1.5 mmol), potassium carbonate (0.21 g, 1.5 mmol), and palladium acetate (17 mg, 0.075 mmol), pure water (2.4 mL) was added, followed by THF (9.6 mL) under a nitrogen atmosphere and stirring at 70°C overnight. The solvent of the reaction mixture was removed under reduced pressure, and the mixture was diluted with water (30 mL) and ethyl acetate (30 mL), and then extracted with ethyl acetate (30 mL x 3). The obtained organic layer was washed with saturated saline (30 mL x 2), dried over sodium sulfate, and the solvent of the filtrate was removed under reduced pressure. The residue was purified by flash column chromatography (hexane:ethyl acetate) to obtain compound 43 (light yellow solid). As shown in Figure 57, 1 The H NMR spectrum is shown.

[0346] Yield 54%: 1 H NMR (400 MHz, CDCl 3): δ, 7.49-7.42 (m, 3H), 7.29-7.23 (m, 5H), 7.06-7.04 (m, 2H), 5.48 (s, 2H), 4.68 (d, J = 6.0 Hz, 2H), 2.16 (t, J = 6.0 Hz, 1H): ESI-TOF MS (positive mode) m / z calcd. for C 16 H 15 N 3 NaO [M+Na] + 288.111, found 288.112.

[0347] <Synthesis of 19-1-3.1-benzyl-5-phenyl-1H-1,2,3-triazole-4-carbaldehyde (44)> Activated manganese dioxide (163 mg, 1.9 mmol) was added to a 1,4-dioxane solution (10 mL) of compound 43 (50 mg, 0.19 mmol) and the mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the solvent of the obtained filtrate was distilled off under reduced pressure. The crude product was purified by flash column chromatography (hexane:ethyl acetate) to obtain compound 44 (oil). 1 The H NMR spectrum is shown.

[0348] Yield 91%: 1 H NMR (400 MHz, CDCl 3 ): δ, 10.14 (s, 1H), 7.56-7.46 (m, 3H), 7.31-7.27 (m, 5H), 7.07-7.05 (m, 2H), 5.49 (s, 2H): ESI-TOF MS (positive mode) m / z calcd. for C 16 H 13 NaN 3 O [M+Na] + 286.095, found 286.093.

[0349] <19-1-4.Synthesis of (1-benzyl-5-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methanol (45)>

[0350] [ka]

[0351] Compound 45 (light yellow solid) was synthesized in the same manner as in Example 19-1-2 using 4-methoxyphenylboronic acid as a precursor. 1 The H NMR spectrum is shown.

[0352] Yield 97%: 1 H NMR (400 MHz, CDCl 3 ): δ, 7.29-7.27 (m, 3H), 7.17 (d, J = 8.8 Hz, 2H), 7.08-7.07 (m, 2H), 6.95 (d, J = 8.8 Hz, 2H), 5.46 (s, 2H), 4.67 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H), 2.09 (t, J = 6.0 Hz, 1H): 13 C NMR (100 MHz, CDCl 3 ): δ, 160.75, 144.90, 135.95, 135.63, 131.16, 128.95, 128.32, 127.43, 118.45, 114.62, 56.09, 55.53, 52.07: ESI-TOF MS (positive mode) m / z calcd. for C 17 H 17 N 3 NaO 2 [M+Na] + 318.121, found 318.121.

[0353] <Synthesis of 19-1-5.1-benzyl-5-(4-methoxyphenyl)-1H-1,2,3-triazole-4-carbaldehyde (46)>

[0354] [ka]

[0355] Compound 46 (oil) was synthesized using compound 45 as a precursor in the same manner as in Example 19-1-3. 1 The H NMR spectrum is shown.

[0356] Yield 91%: 1 H NMR (400 MHz, CDCl 3 ): δ, 10.14 (s, 1H), 7.32-7.30 (m, 3H), 7.23 (d, J = 8.8 Hz, 2H), 7.10-7.08 (m, 2H), 6.99 (d, J = 8.8 Hz, 2H), 5.49 (s, 2H), 3.87 (s, 3H): 13 C NMR (100 MHz, CDCl 3 ): δ, 184.68, 161.42, 143.49, 140.76, 134.72, 131.23, 129.04, 128.59, 127.44, 116.48, 114.54, 55.49, 51.85: ESI-TOF MS (positive mode) m / z calcd. for C 17 H 15 N 3 NaO 2 [M+Na] + 316.106, found 316.104.

[0357] <19-1-6.Synthesis of (1-benzyl-5-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methanol (47)>

[0358] [ka]

[0359] Compound 47 (brown solid) was synthesized using 4-nitrophenylboronic acid as a precursor in the same manner as in Example 19-1-2. 1 The H NMR spectrum is shown.

[0360] Yield 58%: 1 H NMR (400 MHz, CDCl 3 ): δ, 8.28 (d, J = 8.8 Hz, 2H), 7.46 (d, J = 8.8 Hz, 2H), 7.31-7.29 (m, 3H), 7.05-7.03 (m, 2H), 5.52 (s, 2H), 4.69 (d, J = 6.0 Hz, 2H), 2.14 (t, J = 6.0 Hz, 1H): 13 C NMR (100 MHz, CDCl 3 ): δ, 148.56, 145.90, 134.85, 134.12, 133.18, 130.84, 129.22, 128.77, 127.25, 124.22, 55.83, 52.74: ESI-TOF MS (positive mode) m / z calcd. for C 16 H 14 N 4 NaO 3 [M+Na] + 333.096, found 333.095.

[0361] <Synthesis of 19-1-7.1-benzyl-5-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde (48)>

[0362] [ka]

[0363] Compound 48 (yellow solid) was synthesized using compound 47 as a precursor in the same manner as in Example 19-1-3. 1 The H NMR spectrum is shown.

[0364] Yield 93%:1 H NMR (400 MHz, CDCl 3 ): δ, 10.20 (s, 1H), 8.31 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 7.33-7.28 (m, 3H), 7.04-7.02 (m, 2H), 5.52 (s, 2H): 13 C NMR (100 MHz, CDCl 3 ): δ, 185.02, 149.11, 144.27, 137.67, 133.99, 131.05, 129.37, 129.15, 127.45, 124.07, 52.64: ESI-TOF MS (positive mode) m / z calcd. for C 16 H 12 N 4 NaO 3 [M+Na] + 331.080, found 331.082.

[0365] <Synthesis of 19-1-8.1-(4-((4-(hydroxymethyl)-5-iodo-1H-1,2,3-triazol-1-yl)methyl)phenyl)ethan-1-one (49)>

[0366] [ka]

[0367] Compound 49 (white solid) was synthesized using 1-(4-(azidomethyl)phenyl)ethan-1-one as a precursor in the same manner as in Example 19-1-1. 1 The H NMR spectrum is shown.

[0368] Yield 40%: 1 H NMR (400 MHz, CDCl 3): δ, 7.94 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 5.65 (s, 2H), 4.74 (s, 2H), 2.59 (s, 3H), 2.15 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ): δ, 197.5, 151.5, 139.1, 137.4, 129.1, 128.1, 78.9, 56.8, 53.9, 26.8: ESI-TOF MS (positive mode) m / z calcd. for C 12 H 12 IN 3 NaO 2 [M+Na] + 379.987, found 379.987.

[0369] <Synthesis of 19-1-9.1-(4-((5-(4-(azidomethyl)phenyl)-4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)ethan-1-one (50)>

[0370] [ka]

[0371] Compound 50 (light yellow solid) was synthesized in the same manner as in Example 19-1-2 using compound 49 and 2-(4-(azidomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as precursors. 1 The H NMR spectrum is shown.

[0372] Yield 71%: 1 H NMR (400 MHz, CDCl 3): δ, 7.87 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 5.54 (s, 2H), 4.69 (d, J = 5.8 Hz, 2H), 4.42 (s, 2H), 2.57 (s, 3H), 2.17 (t, J = 5.8 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ): δ, 197.5, 145.4, 140.3, 137.5, 137.1, 135.6, 130.2, 129.1, 128.9, 127.5, 126.3, 55.9, 54.4, 51.9, 26.8: ESI-TOF MS (positive mode) m / z calcd. for C 19 H 18 N 6 NaO 2 [M+Na] + 385.138, found 385.138.

[0373] <Synthesis of 19-1-10.1-(4-acetylbenzyl)-5-(4-(azidomethyl)phenyl)-1H-1,2,3-triazole-4-carbaldehyde (51)>

[0374] [ka]

[0375] Compound 51 (transparent, oily) was synthesized using compound 50 as a precursor in the same manner as in Example 19-1-3. 1 The H NMR spectrum is shown.

[0376] Yield 85%: 1 H NMR (400 MHz, CDCl 3): δ, 10.17 (s, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 5.55 (s, 2H), 4.45 (s, 2H), 2.58 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ): δ, 197.4, 184.7, 143.9, 140.0, 139.3, 138.6, 137.4, 130.1, 129.1, 128.7, 127.7, 124.6, 54.3, 51.7, 26.8: ESI-TOF MS (positive mode) m / z calcd. for C 19 H 16 N 6 NaO 2 [M+Na] + 383.123, found 383.123.

[0377] Example 20. Peptide N-terminal modification 3 The peptide N-terminal modification was performed with reference to a previous report (JI MacDonald, HK Munch, T. Moore, MB Francis, Nat. Chem. Biol. 2015, 11, 326-331). The specific experimental procedures are shown below.

[0378] A dimethyl sulfoxide (DMSO) solution of compound 44 (200 mM, 2 μL, 0.4 μmol, final concentration 10 mM) was diluted with phosphate buffer (10 mM, pH 7.5, 34 μL), and an aqueous peptide solution (1 mM, 4 μL, 4 nmol, final concentration 100 μM) was added thereto and shaken at 37°C for 16 hours. The modification rate [= (amount of modified peptide) / (total amount of peptide)] was evaluated from the peak intensity in the mass spectrum using LC / MS. The results are shown in Figure 66. The modification rate under these reaction conditions was calculated to be >99%.

[0379] Example 21. Protein N-terminal modification 4 The N-terminal modification of proteins was performed with reference to a previous report (JI MacDonald, HK Munch, T. Moore, MB Francis, Nat. Chem. Biol. 2015, 11, 326-331). The specific experimental procedures are shown below.

[0380] A dimethyl sulfoxide (DMSO) solution of compound 44 (200 mM, 5 μL, 1.0 μmol, final concentration 10 mM) was diluted with phosphate buffer (10 mM, pH 7.5, 90 μL), to which an ultrapure RNase solution (1 mM, 5 μL, 5 nmol, final concentration 50 μM) was added, and the mixture was shaken at 37°C for 16 hours. The modification rate [= (modified RNase / (total amount of RNase)] was evaluated from the peak intensity in the mass spectrum using LC / MS. The results are shown in Figure 67. The modification rate under these reaction conditions was calculated to be 48%.

[0381] Example 22. Functional molecule modification to protein N-terminus <22-1. Dual modification of protein N-terminus with acetyl and azide groups> Compound 51 was used as a modifying agent to perform double modification of the protein N-terminus with an acetyl group and an azide group in the same manner as in Example 21. The results of LC / MS analysis of the product are shown in Figure 68. The modification rate of the acetyl group and the azide group was calculated to be 28%. <22-2. Functional molecule modification starting from azide group modified at the N-terminus of protein> The RNase modified with the azide group prepared in Example 22-1 was modified with a fluorescent dye by strain-promoted alkyne-azide cycloaddition reaction. Compound 26, a fluorescein having a dibenzocyclooctyne moiety, was used as the alkyne substrate in the reaction. Figure 69 shows the scheme and results.

[0382] A dimethyl sulfoxide (DMSO) solution of compound 26 (8 mM, 1 μL, final concentration 400 μM) was added to a phosphate buffer solution containing RNase_51 (RNase_51 concentration 76 μM, final concentration 20 μM, buffer concentration 10 mM, pH 7.5, 19 μL) and left at room temperature for 6 hours. The modification rate of the fluorescent dye site was calculated to be >99%.

[0383] Example 23. Introduction of polymer molecules into the N-terminus of a protein <23-1. Synthesis of polyethylene glycol having triazolecarbaldehyde moiety> We synthesized polyethylene glycol 54, which has a triazole carbaldehyde moiety. The synthesis was carried out according to the scheme shown below, with reference to previous reports (L. Rocard, A. Berezin, F. De Leo, D. Bonifazi, Angew. Chem. Int. Ed., 2015, 54, 15739-15743 and MB van Eldijk, FCM Smit, N. Vermue, MF Debets, S. Schoffelen, JCM van Hest, Biomacromolecules, 2014, 15, 2751-2759).

[0384] [ka]

[0385] <Synthesis of 23-1-1.4-((4-formyl-1H-1,2,3-triazol-1-yl)methyl)benzoic acid (52)> Compound 52 (white solid) was synthesized using (4-azidomethyl)benzoic acid as a precursor in the same manner as in Example 1-3-1 (Method A). 1 The H NMR spectrum is shown.

[0386] Yield 71%; 1 H NMR (400 MHz, DMSO-d 6): δ, 10.02 (s, 1H), 9.00 (s, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 5.79 (s, 2H); 13 C NMR (100 MHz, DMSO-d 6 ): δ, 185.0, 166.9, 147.1, 140.1, 130.7, 129.9, 128.7, 128.2, 52.8; ESI-TOF MS (positive mode) m / z calcd. for C 11 H 9 N 3 O 3 Na [M+Na] + 254.054, found 254.053.

[0387] <Synthesis of 23-1-2.2,5-dioxopyrrolidin-1-yl 4-((4-formyl-1H-1,2,3-triazol-1-yl)methyl)benzoate (53)> N-hydroxysuccinimide (138 mg, 1.2 mmol) and N,N′-dicyclohexylcarbodiimide (206 mg, 1.0 mmol) were added to a THF solution (10 mL) containing 52 (231 mg, 1.0 mmol), and the mixture was stirred at 0° C. under nitrogen atmosphere for 24 hours. After filtering off the precipitate, the filtrate was diluted with ethyl acetate (50 mL) and washed with saturated aqueous sodium bicarbonate (20 mL × 2) and saturated saline (20 mL × 2). The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure to obtain the crude product. Compound 53 (white solid) was obtained by purification using silica gel column chromatography. As shown in Figure 71, 1 The H NMR spectrum is shown.

[0388] Yield 71%; 1 H NMR (400 MHz, CDCl 3): δ, 10.15 (s, 1H), 8.16 (d, J = 8.5 Hz, 2H), 8.07 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H), 5.70 (s, 2H), 2.91 (s, 4H); 13 C NMR (100 MHz, CDCl 3 ): δ, 185.0, 169.1, 161.3, 148.4, 140.5, 131.7, 128.5, 126.3, 125.4, 54.1, 25.8; ESI-TOF MS (positive mode) m / z calcd. for C 15 H 12 N 4 O 5 Na [M+Na] + 351.070, found 351.069.

[0389] <23-1-2.Synthesis of Polyethylene glycol-tethered triazole-4-carbaldehyde (54)> To methylene chloride (15 mL) containing 53 (53 mg, 0.16 mmol), polyethylene glycol (molecular weight: approximately 4000, 400 mg, 0.1 mmol) with an amino group at the end and triethylamine (45 L, 0.32 mmol) were added and stirred under a nitrogen atmosphere for 24 hours. After the solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography. The resulting oily crude product was purified by reprecipitation (diethyl ether / methylene chloride) to obtain compound 54 (white solid).

[0390] <23-2. Protein N-terminal modification using compound 54> The N-terminus of a protein was modified with polyethylene glycol using compound 54. An example in which ribonuclease was used as a substrate is shown below.

[0391] <23-2-1. Reagents, solvents, etc.> Ribonuclease A (RNase) derived from bovine pancreas was purchased from Roche. Ultrapure water purified using Millipore Integral 3 was used. Other reagents and solvents were commercially available and used as is.

[0392] <23-2-2. Modification of protein N-terminus with polyethylene glycol> Compound 54 was used as a modifying agent to modify the N-terminus of the protein with polyethylene glycol in the same manner as in Example 10-2. The results of SDS-PAGE analysis of the product are shown in Figure 72. The modification rate of the polyethylene glycol site was calculated to be 50%.

[0393] <23-2-3. Polymer modification to the N-terminus of a protein with modified cysteine ​​residues> Compound 54 was used as a modifying agent to modify the N-terminus of HSA2 protein with polyethylene glycol in the same manner as in Example 17-3. The HSA obtained by the reaction, in which an azide group was attached to the cysteine ​​residue and polyethylene glycol was attached to the N-terminus (hereinafter referred to as HSA4), was modified with a fluorescent dye by strain-promoted alkyne-azide cycloaddition reaction using compound 26. The modified protein is referred to as HSA5.

[0394] A dimethyl sulfoxide (DMSO) solution of compound 26 (6 mM, 1 μL, final concentration 300 μM) was added to a phosphate buffer solution containing HSA4 (HSA4 concentration 62 μM, final concentration 10 μM, buffer solution concentration 10 mM, pH 7.5, 19 μL) and left to stand at 4 °C for 6 hours. The same procedure was performed for HSA and HSA2, and the products were analyzed using SDS-PAGE. The scheme and results are shown in Figure 73.

[0395] Fluorescence was observed only in the lanes of HSA2 and HSA4 modified with compound 41 and containing an azide group when compound 26 was reacted. In addition, a new band associated with an increase in molecular weight appeared only in the lane of HSA4 modified with compound 54, a polyethylene glycol derivative. These results confirmed that the polyethylene glycol modification and fluorescent dye modification of the protein had progressed.

Claims

1. General formula (1): 【Chemistry 1】 [In the formula: R 1 and R 2 Of these, one is -N (-R 4 ) - (R 4 represents an organic group or a group derived from an inorganic material. ) and the other represents =N-. R 3 represents a hydrogen atom, an organic group, or a group derived from an inorganic material. or a salt thereof, or a hydrate or solvate thereof.

2. The compound is represented by the general formula (1Aa) 【Chemistry 2】 [In the formula: R 4 is the same as above.] The reagent according to claim 1, which is a compound represented by the formula:

3. The reagent according to claim 1 or 2, wherein the organic group is a group derived from an organic molecule or an organic molecular complex, and the organic molecule or the organic molecular complex is a functional substance.

4. The reagent according to claim 3, wherein the functional substance is a pharmaceutical compound, a luminescent molecule, a polymer compound, a ligand, a molecule to be bound to a ligand, an antigen protein, an antibody, a protein, a nucleic acid, a sugar, a lipid, a cell, a virus, a label, a carbon electrode, a carbon nanomaterial, a linker, or a spacer molecule, or a complex or linked molecule thereof.

5. The reagent according to any one of claims 1 to 4, wherein the inorganic material is an electrode material, a metal particle, a metal oxide particle, a semiconductor particle, or a magnetic particle.

6. General formula (5): R 4 -R 9 (5) [In the formula: R 4 R represents an organic group or a group derived from an inorganic material. 9 Ha-NH 2 The organic group is a group obtained by removing one atom or multiple atoms from a pharmaceutical compound, a luminescent molecule, a polymer compound, a ligand, a molecule to be bound to a ligand, an antigen protein, an antibody, a protein, a nucleic acid, a sugar, a lipid, a cell, a virus, a label, a carbon electrode, a carbon nanomaterial, a linker, a spacer molecule, or a complex or linking molecule thereof. The group derived from an inorganic material is a group containing an electrode material, a metal fine particle, a metal oxide fine particle, a semiconductor particle, or a magnetic particle.] and a compound represented by the general formula (6): 【Chemistry 3】 [In the formula, R 10 R represents an electron-withdrawing group. 11 Ha-R 11a -R 12 (R 11a R represents a single bond or a linker. 12 represents a carrier. n represents 0 or 1. m represents an integer of 1 to 5. 【Chemistry 4】 [In the formula: R 1 and R 2 Of these, one is -N (-R 4 ) - (R 4 represents an organic group or a group derived from an inorganic material. ) and the other represents =N-. R 3 represents a hydrogen atom, an organic group, or a group derived from an inorganic material. The organic group is a group obtained by removing one atom or multiple atoms from a pharmaceutical compound, a luminescent molecule, a polymer compound, a ligand, a molecule to be bound to a ligand, an antigen protein, an antibody, a protein, a nucleic acid, a sugar, a lipid, a cell, a virus, a label, a carbon electrode, a carbon nanomaterial, a linker, a spacer molecule, or a complex or linking molecule thereof. The group derived from an inorganic material is a group containing an electrode material, a metal fine particle, a metal oxide fine particle, a semiconductor particle, or a magnetic particle.] or a salt thereof, or a hydrate or solvate thereof.

7. R 4 is a group formed by removing one atom or multiple atoms from an antibody, a protein, a nucleic acid, a sugar, a lipid, or a complex or linked molecule thereof, R 3 is a hydrogen atom or a group obtained by removing one atom or multiple atoms from an antibody, a protein, a nucleic acid, a sugar, a lipid, or a complex or linked molecule thereof; The method according to claim 6.

8. General formula (6'): 【Chemistry 5】 [In the formula, R 10 are the same or different, -NO 2 , -F, CF 3 , -CN, or -COOMe. 11a R represents a single bond or a linker. 12 represents a carrier. m represents an integer of 1 to 5. A compound represented by the formula:

9. General formula (7): 【Chemistry 6】 [In the formula: R 1 and R 2 Of these, one is -N (-R 4 ) - (R 4 represents an organic group or a group derived from an inorganic material. ) and the other represents =N-. R 3 R represents a hydrogen atom, an organic group, or a group derived from an inorganic material. A double line consisting of a dotted line and a solid line represents a single bond or a double bond. 13 R represents a group obtained by removing the N-terminal amino acid residue and the adjacent -NH- from a protein or peptide. 14 indicates the side chain of the N-terminal amino acid residue of the protein or peptide. The organic group is a group obtained by removing one atom or multiple atoms from a pharmaceutical compound, a luminescent molecule, a polymer compound, a ligand, a molecule to be bound to a ligand, an antigen protein, an antibody, a protein, a nucleic acid, a sugar, a lipid, a cell, a virus, a label, a carbon electrode, a carbon nanomaterial, a linker, a spacer molecule, or a complex or linking molecule thereof. The group derived from an inorganic material is a group containing an electrode material, a metal fine particle, a metal oxide fine particle, a semiconductor particle, or a magnetic particle.] or a salt thereof, or a hydrate or solvate thereof.

10. R 4 is a group formed by removing one atom or multiple atoms from an antibody, a protein, a nucleic acid, a sugar, a lipid, or a complex or linked molecule thereof, R 3 is a hydrogen atom or a group obtained by removing one atom or multiple atoms from an antibody, a protein, a nucleic acid, a sugar, a lipid, or a complex or linked molecule thereof; 10. The compound according to claim 9, or a salt thereof, or a hydrate or solvate thereof.

11. A protein and / or peptide and a compound of the general formula (1): 【Chemistry 7】 [In the formula: R 1 and R 2 Of these, one is -N (-R 4 ) - (R 4 represents an organic group or a group derived from an inorganic material. ) and the other represents =N-. R 3 represents a hydrogen atom, an organic group, or a group derived from an inorganic material. The organic group is a group obtained by removing one atom or multiple atoms from a pharmaceutical compound, a luminescent molecule, a polymer compound, a ligand, a molecule to be bound to a ligand, an antigen protein, an antibody, a protein, a nucleic acid, a sugar, a lipid, a cell, a virus, a label, a carbon electrode, a carbon nanomaterial, a linker, a spacer molecule, or a complex or linking molecule thereof. The group derived from an inorganic material is a group containing an electrode material, a metal fine particle, a metal oxide fine particle, a semiconductor particle, or a magnetic particle.] or a salt thereof, or a hydrate or solvate thereof.

12. R 4 is a group formed by removing one atom or multiple atoms from an antibody, a protein, a nucleic acid, a sugar, a lipid, or a complex or linked molecule thereof, R 3 The method according to claim 11, wherein is a hydrogen atom or a group formed by removing one atom or multiple atoms from an antibody, a protein, a nucleic acid, a saccharide, a lipid, or a complex or linked molecule thereof.

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