Molecules for modifying proteins and / or peptides

Abstract

An object of the present invention is to provide a technology that allows other molecules / substances to be more selectively linked to the N-terminus in a simple and efficient manner even in native proteins, etc. This object is achieved by reacting a compound represented by formula (1) or a salt thereof, or a hydrate or solvate of the compound or its salt with a protein and / or peptide.

Description

Technical Field

[0001] The present invention relates to molecules for modifying proteins and / or peptides, among others. Background Art

[0002] Technologies for linking proteins and / or peptides (hereinafter referred to as "proteins") to other molecules / substances are important in the preparation of antibody-drug conjugates, fluorescent probe-labeled protein reagents, and inorganic materials for protein immobilization. For example, modifying proteins with azide groups is a technique that allows the introduction of various functional molecules through the alkyne-azide cycloaddition reaction (CuAAC). Due to its bioorthogonality, this technique has been widely used in fields such as bioimaging.

[0003] Citation List

[0004] Non-patent literature

[0005] Non-patent document 1: Metal-free and pH-controlled introduction of azides inproteins, Sanne Schoffelen, Mark B. van Eldijk, Bart Rooijakkers, Reinout Raijmakers, Albert JRHeck and Jan CMvan Hest, Chemical Science, 2011, 2,701.

[0006] Non-patent document 2: Selective N-terminal acylation of peptides and proteins with aGly-His tag sequence, MCMartos-Maldonado, CTHjuler, KKSorensen, MBThygesen, JERasmussen, K.Villadsen, SRMidtgaard, S.Kol, S.Schoffelen, KJJensen, Nature Communications, 2018, 9, 3307.

[0007] Non-patent document 3: Modification of N-Terminalα-Amino Groups of Peptides and Proteins Using Ketenes, AO-Y.Chan, C.-M.Ho, H.-C.Chong, Y.-C.Leung, J.-S.Huang, M.-K.Wong, C.-M.Che, Journal of the American Chemical Society, 2012, 134, 2589. Summary of the Invention

[0008] Technical issues

[0009] The present inventors focused on the following three points when studying the introduction position when linking other molecules / substances to proteins. The first point is that all monomeric proteins have only one N-terminal position, which is a universal modification base point. The second point is that the N-terminus rarely participates in protein active sites (molecular binding sites and catalytic reaction centers) or protein functional sites, and the impact of structural changes related to modification is believed to be small. The third point is that due to the pK a The N-terminus is considered less likely to compete with other amino acid residues (such as lysine, cysteine, and glutamine); while at the C-terminus, this a Therefore, the inventors focused on the N-terminus of proteins as the location for attaching other molecules / substances.

[0010] Various methods for linking other molecules / substances to the N-terminus of proteins, etc. have been reported (Non-Patent Documents 1 to 3). However, these methods are considered insufficient in terms of simplicity or N-terminal modification selectivity. For example, although chemical bonding methods or lipid modification enzyme methods that recognize specific amino acid sequences can specifically introduce other molecules / substances into the N-terminus, they require proteins, etc. in which specific amino acid sequences or specific amino acid residues are inserted. The preparation of such proteins, etc. is laborious, and these methods cannot be applied to natural proteins, etc. The amide bond formation reaction using activated esters or vinyl ketones is simple to operate and can be applied to natural proteins, etc.; however, it cannot specifically introduce other molecules / substances into the N-terminus because side reactions with lysine residues, etc. will occur.

[0011] Therefore, one object of the present invention is to provide a technology that allows other molecules / substances to be more selectively attached to the N-termini of all proteins and / or peptides in a simple and efficient manner.

[0012] Problem Solution

[0013] The present inventors have conducted extensive research to solve the above problems. They have discovered that these problems can be solved by reacting a compound represented by formula (1) or a salt thereof, or a hydrate or solvate of the compound or its salt with a protein and / or peptide. Based on this discovery, the present inventors have conducted further research and have completed the present invention.

[0014] Specifically, the present invention includes the following embodiments.

[0015] Item 1. A compound represented by formula (1) or a salt thereof, or a hydrate or solvate of the compound or a salt thereof,

[0016]

[0017] where R 1 and R 2 One of them means -N(-R 4 )-(where R 4 represents an organic group or a group derived from an inorganic material), the other represents =N-; and R 3 represents a hydrogen atom, an organic group, or a group derived from an inorganic material.

[0018] Item 2. The compound according to Item 1, or a salt thereof, or a hydrate or solvate of the compound or a salt thereof, wherein the compound is represented by formula (1Aa):

[0019]

[0020] where R 4 As defined above.

[0021] Item 3. The compound or salt thereof, or a hydrate or solvate of the compound or salt thereof according to Item 1 or Item 2, wherein the organic group is a group derived from an organic molecule or an organic molecule complex, and the organic molecule or organic molecule complex is a functional substance.

[0022] Item 4. The compound or salt thereof according to Item 3, or a hydrate or solvate of the compound or salt thereof, wherein the functional substance is a pharmaceutical compound, a luminescent molecule, a polymer compound, a ligand, a ligand-bound molecule, an antigenic protein, an antibody, a protein, a nucleic acid, a carbohydrate, a lipid, a cell, a virus, a label, a carbon electrode, a carbon nanomaterial, a linker, a spacer molecule, or a complex or connecting molecule thereof.

[0023] Item 5. The compound or salt thereof, or a hydrate or solvate of the compound or salt thereof according to any one of Items 1 to 4, wherein the inorganic material is an electrode material, metal fine particles, metal oxide fine particles, semiconductor particles, or magnetic particles.

[0024] Item 6. A reagent comprising the compound or a salt thereof according to any one of Items 1 to 5, or a hydrate or solvate of the compound or a salt thereof.

[0025] Item 7. The reagent according to Item 6, which is a reagent for protein and / or peptide modification.

[0026] Item 8. A method for preparing the compound or salt thereof, or a hydrate or solvate of the compound or salt thereof according to any one of Items 1 to 5, the method comprising:

[0027] The compound represented by formula (2) or a salt thereof is reacted with the compound represented by formula (3), the compound represented by formula (4) or the compound represented by formula (9),

[0028] R 4 -R 5 (2),

[0029] where R 4 represents an organic group or a group derived from an inorganic material, and R 5 represents -N3, -X (wherein X represents a halogen atom), -B(OH)2, -B(OR 51 )2(where each R 51 are the same or different and represent a hydrocarbon group, provided that the two R 51 Together with the adjacent oxygen atom, they can form a ring), or -N2 + ,

[0030]

[0031] where R 3 represents a hydrogen atom, an organic group, or a group derived from an inorganic material; R 6 、R 7 and R 8 are the same or different and each represents an alkyl group; and Y represents a reactive group.

[0032] Item 9. A method for preparing the compound or salt thereof, or a hydrate or solvate of the compound or salt thereof according to any one of Items 1 to 5, the method comprising:

[0033] The compound represented by formula (5) is reacted with the compound represented by formula (6):

[0034] R 4 -R 9 (5),

[0035] where R 4 represents an organic group or a group derived from an inorganic material; and R 9 Indicates -NR 9a R9b (where R 9a and R 9b are the same or different and each represents a hydrogen atom or an alkyl group),

[0036]

[0037] where R 10 Represents an electron-withdrawing group; R 11 Indicates -R 11a -R 12 (where R 11a represents a single bond or a linker; and R 12 represents a vector); n represents 0 or 1; and m represents an integer of 1 to 5.

[0038] Item 10. Compound represented by formula (6'):

[0039]

[0040] where R 10 Represents an electron-withdrawing group; R 11a Represents a single bond or a linker; R 12 represents a carrier; and m represents an integer of 1 to 5.

[0041] Item 11. A compound represented by formula (7) or a salt thereof, or a hydrate or solvate of the compound or a salt thereof,

[0042]

[0043] where R 1 and R 2 One of them means -N(-R 4 )-(where 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; a double line consisting of a dotted line and a solid line represents a single bond or a double bond; R 13 Indicates that the N-terminal amino acid residue and the adjacent -NH- group are excluded from the protein or peptide; R 14 Represents the side chain of the N-terminal amino acid residue of a protein or peptide.

[0044] Item 12. A method for preparing the compound or salt thereof according to Item 11, or a hydrate or solvate of the compound or salt thereof, which comprises reacting a protein and / or peptide with the compound or salt thereof according to any one of Items 1 to 5, or a hydrate or solvate of the compound or salt thereof.

[0045] Advantageous Effects of the Invention

[0046] The present invention can provide a technology that allows other molecules / substances to be more selectively linked to the N-terminus in a simple and efficient manner, even in native proteins and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The compound 1 1 H NMR spectrum (400 MHz, DMSO-d6).

[0048] Figure 2 The compound 2 1 H NMR spectrum (400 MHz, DMSO-d6).

[0049] Figure 3 The results show that compound 3 1 H NMR spectrum (400 MHz, DMSO-d6).

[0050] Figure 4 The results show that compound 4 1 H NMR spectrum (400 MHz, CDCl3).

[0051] Figure 5 The results show that compound 5 1 H NMR spectrum (400 MHz, DMSO-d6).

[0052] Figure 6 The compound 6 1 H NMR spectrum (400 MHz, DMSO-d6).

[0053] Figure 7 The results show that compound 7 1 H NMR spectrum (400 MHz, DMSO-d6).

[0054] Figure 8 The compound 8 1 H NMR spectrum (400 MHz, CDCl3).

[0055] Figure 9 The compound 9 1 H NMR spectrum (400 MHz, DMSO-d6).

[0056] Figure 10 The results show that compound 10 1 H NMR spectrum (400 MHz, DMSO-d6).

[0057] Figure 11 The results show that compound 13 1 H NMR spectrum (400 MHz, DMSO-d6).

[0058] Figure 12 The results show that compound 16 1 H NMR spectrum (400 MHz, DMSO-d6).

[0059] Figure 13 The results show that compound 17 1 H NMR spectrum (400 MHz, CD3CN).

[0060] Figure 14 The results show that compound 18 1 H NMR spectrum (400 MHz, CD3CN).

[0061] Figure 15 The results show that compound 19 1 H NMR spectrum (400 MHz, CD3CN).

[0062] Figure 16 The results show that compound 20 1 H NMR spectrum (400 MHz, CDCl3).

[0063] Figure 17 Compound 21 1 H NMR spectrum (400 MHz, CDCl3).

[0064] Figure 18 Compound 22 1 H NMR spectrum (400 MHz, CD3CN).

[0065] Figure 19 The reaction scheme and results of the N-terminal modification reaction of angiotensin I are shown (Example 8).

[0066] Figure 20 Compound 25 1 H NMR spectrum (400 MHz, CD3CN).

[0067] Figure 21 The reaction scheme and results of the N-terminal modification reaction of ribonuclease A are shown (Example 10).

[0068] Figure 22 The reaction scheme and results of modifying RNase A with biotin are shown (Example 11-2).

[0069] Figure 23 The reaction scheme and results of modifying ribonuclease A with a fluorescent dye are shown (Example 11-3).

[0070] Figure 24The reaction scheme and results of modifying ribonuclease A with an azide group are shown (Example 11-4).

[0071] Figure 25 Shown are the reaction schemes and results for modification with fluorescent dyes via strain-promoted alkyne-azide cycloaddition (Example 11-5).

[0072] Figure 26 The reaction scheme and results for modification of ribonuclease A with a strained alkyne moiety are shown (Example 11-6).

[0073] Figure 27 A summary of a method is shown (Example 12) in which various formaldehyde precursors are immobilized on a resin or solid material, the removal of by-products (aniline derivatives) generated after the reaction is simplified, and the resulting solution is directly used for N-terminal modification of proteins.

[0074] Figure 28 Compound 27 and a resin immobilized with Compound 27 (Example 12) are shown.

[0075] Figure 29 A synthetic scheme for 27 / PS resin is shown (Example 12-2).

[0076] Figure 30 The results of resin identification using infrared spectroscopy (Example 12-3) are shown. The right side is an enlarged view.

[0077] Figure 31 The results of LC / MS analysis of N-terminally modified ribonuclease A by sequential reactions using a resin (27 / PS resin) to which reactants were immobilized (Example 12-4) are shown.

[0078] Figure 32 A synthetic scheme for 27 / PS resin is shown (Example 12-2).

[0079] Figure 33 Compound 28 1 H NMR spectrum (400 MHz, DMSO-d6).

[0080] Figure 34 Compound 30 is shown 1 H NMR spectrum (400 MHz, DMSO-d6).

[0081] Figure 35 A scheme for N-terminal modification by sequential reactions using a resin (27 / PS resin) immobilized with reactants (Example 12-4) is shown (Example 12-6).

[0082] Figure 36 shows the results of LC / MS analysis of N-terminally modified RNase A using a resin (27 / PS resin) immobilized with reactants through sequential reactions (Example 12-6). Peaks corresponding to modified proteins are shown as "●" (filled circles), and peaks corresponding to unmodified proteins are shown as "○" (open circles).

[0083] Figure 37 A scheme for preparing triazole carboxaldehyde using the Dimrot rearrangement reaction in a homogeneous system and applying it to protein modification is shown (Example 13).

[0084] Figure 38 The reaction mechanism of the Dimrot rearrangement reaction is shown (Example 13-2).

[0085] Figure 39 The structure of the acid catalyst in the Dimrut rearrangement reaction using Compound 10 is shown (Example 13-2).

[0086] Figure 40 The LC-MS analysis results of the preparation of N-terminal modifiers by Dimrot rearrangement reaction and the continuous protein modification reaction (Example 13-3) are shown. The peak corresponding to the modified protein is shown as "●" (filled circle), and the peak corresponding to the unmodified protein is shown as "○" (open circle).

[0087] Figure 41 The preparation scheme of Bis-TA4C and the protein modification reaction (Example 14-2) are shown.

[0088] Figure 42 The results of LC-MS analysis of N-terminal modification of ribonuclease A using diamine as a precursor (Example 14-2) with Bis-TA4C are shown. The structure of the diamine as a precursor and the modification percentage calculated by LC-MS are shown.

[0089] Figure 43 A scheme for introducing a functional molecule into the N-terminus of a protein via oxime formation is shown (Example 14-3).

[0090] Figure 44 The results of LC-MS analysis of ribonuclease A modified with compound 37 are shown (Example 14-3).

[0091] Figure 45 The results of SDS-PAGE analysis of RNase A modified with Compound 38 are shown (Example 14-3).

[0092] Figure 46 Compound 39 1 H NMR spectrum (400 MHz, CDCl3).

[0093] Figure 47 Compound 40 1 H NMR spectrum (400 MHz, CDCl3).

[0094] Figure 48 A scheme of the Dimrut rearrangement reaction using Compound 40 (Example 15-2) is shown.

[0095] Figure 49 The yield of compound 7 in the Dimrut rearrangement reaction (Example 15-2) is shown. 1 Calculated from H NMR measurements.

[0096] Figure 50 The reaction scheme and results of the N-terminal modification reaction of human serum-derived albumin are shown (Example 16-2).

[0097] Figure 51 The reaction scheme and results of modification of cysteine ​​residues in human serum-derived albumin are shown (Example 17-2).

[0098] Figure 52 Shown are the reaction scheme and results of N-terminal modification of human serum-derived albumin, in which the cysteine ​​residue was modified (Example 17-3).

[0099] Figure 53 The LC / MS analysis results after the RNase-7 solution was left to stand for 24 hours are shown (Example 18-2).

[0100] Figure 54 Shown is the change in the release of the modifier in RNase-7 over time (Example 18-2).

[0101] Figure 55 The change in modifier release as a function of pH in RNase-7 is shown (Example 18-3).

[0102] Figure 56 Compound 42 1 H NMR spectrum (400 MHz, DMSO-d6).

[0103] Figure 57 Compound 43 1 H NMR spectrum (400 MHz, CDCl3).

[0104] Figure 58 Compound 44 1 H NMR spectrum (400 MHz, CDCl3).

[0105] Figure 59 Compound 45 1H NMR spectrum (400 MHz, CDCl3).

[0106] Figure 60 Compound 46 is shown 1 H NMR spectrum (400 MHz, CDCl3).

[0107] Figure 61 Compound 47 1 H NMR spectrum (400 MHz, CDCl3).

[0108] Figure 62 Compound 48 1 H NMR spectrum (400 MHz, CDCl3).

[0109] Figure 63 Compound 49 1 H NMR spectrum (400 MHz, CDCl3).

[0110] Figure 64 Compound 50 is shown 1 H NMR spectrum (400 MHz, CDCl3).

[0111] Figure 65 Compound 51 1 H NMR spectrum (400 MHz, CDCl3).

[0112] Figure 66 The reaction scheme and results for N-terminal modification of angiotensin I are shown (Example 20).

[0113] Figure 67 The reaction scheme and results of N-terminal modification of ribonuclease A are shown (Example 21).

[0114] Figure 68 The scheme and results of introducing an acetyl group and an azide group into ribonuclease A are shown (Example 22-1).

[0115] Figure 69 Modification with fluorescent dyes and the results via strain-promoted alkyne-azide cycloaddition are shown (Example 22-2).

[0116] Figure 70 Compound 52 1 H NMR spectrum (400 MHz, DMSO-d6).

[0117] Figure 71 Compound 53 1 H NMR spectrum (400 MHz, CDCl3).

[0118] Figure 72 The scheme and results of modifying ribonuclease A with polyethylene glycol are shown (Example 23-2-2).

[0119] FIG73 shows the scheme and results of the reaction for N-terminal modification of HSA2 and the modification reaction with Compound 26 (Example 23-2-3). DETAILED DESCRIPTION

[0120] In this specification, the terms “include” and “comprising” include the concepts of “including,” “comprising,” “consisting essentially of,” and “consisting of.”

[0121] 1. Protein and / or peptide modified molecules

[0122] In one embodiment, the present invention relates to a compound represented by formula (1) or a salt thereof, or a hydrate or solvate of the compound or its salt (in this specification, these may be collectively referred to as "the modified molecule of the present invention"):

[0123]

[0124] where R 1 and R 2 One of them means -N(-R 4 )-(where R 4 represents an organic group or a group derived from an inorganic material), the other represents =N-, R 3 represents a hydrogen atom, an organic group, or a group derived from an inorganic material.

[0125] The modified molecules of the present invention are described below.

[0126] 1-1. Compounds

[0127] where R 1 and R 2 One of them means -N(-R 4 )-(where R 4 represents an organic group or a group derived from an inorganic material), the other represents =N-, and a double line consisting of a dotted line and a solid line represents a single bond or a double bond, and whether it is a single bond or a double bond depends on R 1 and R 2 Which one is -N(-R 4 )-or=N-.

[0128] Specifically, when R 1 Yes-N(-R 4 )-, and R 2 When =N-, formula (1) becomes formula (1A):

[0129]

[0130] where R 3 and R 4 is as defined above; and

[0131] When R 1 =N-, and R 2 -N(-R 4 )-, formula (1) becomes formula (1B):

[0132]

[0133] where R 3 and R 4 Same definition as above.

[0134] In the present invention, preferably, R 1 Yes-N(-R 4 )-and R 2 =N-; that is, formula (1) is formula (1A).

[0135] As long as the organic group is a group derived from an organic molecule or an organic molecule complex, for example, a group obtained by removing one or more atoms from an organic molecule or an organic molecule complex, there is no particular limitation. The organic molecule is not particularly limited and can be natural, synthetic or artificial. The organic molecule complex is not particularly limited, and examples include a complex (or organism) in which multiple molecules including one or more organic molecules are connected. The mode of connection is not particularly limited, and examples include hydrogen bonds, electrostatic forces, van der Waals forces, hydrophobic bonds, covalent bonds, coordination bonds, etc. These bonds can be formed by a connector (specific examples are shown in the connector described later). The organic molecule or organic molecule complex is preferably a functional substance. Specific examples include pharmaceutical compounds, luminescent molecules, macromolecular compounds, ligands, ligand-bound molecules, antigenic proteins, antibodies, proteins, nucleic acids, carbohydrates, lipids, cells, viruses, labels (such as radioisotope labels), carbon electrodes, carbon nanomaterials, connectors, spacer molecules (for example, polyethylene glycol or its derivatives and peptides (for example, peptides comprising an amino acid sequence that is cleaved by an enzyme in a cell)) and their complexes and connecting molecules.

[0136] In one embodiment of the present invention, one or more modified molecules of the present invention may be attached as part of an organic group (e.g., at a terminal end). In this case, an example of a modified molecule of the present invention is a compound represented by formula (1AA):

[0137]

[0138] where R 3 As defined above, and R 3 Same or different at each occurrence; R 4aRepresents a divalent organic group. 4a It may contain a partial structure of the modified molecule of the present invention.

[0139] Inorganic materials are materials that may or may not contain one or more metal atoms and are not particularly limited. Examples of inorganic materials include electrode materials, metal particles, metal oxide particles, semiconductor particles, magnetic particles, etc. Inorganic materials may retain organic molecules or organic molecule complexes.

[0140] An organic group or a group derived from an inorganic material may have a reactive group. In this case, another substance can be further connected via the reactive group. Examples of reactive groups include ethynyl, ethynylene, vinyl, azido, epoxy, aldehyde, oxyamino, and halogen. It is known that ethynyl and ethynylene groups each undergo a 1,3-dipolar cycloaddition reaction with an azido group to form a 1,2,3-triazole ring. Vinyl groups react with thiol groups to form a bond. Epoxy groups react with amino or thiol groups to form a bond. Aldehyde groups react with amino groups to form a Schiff base, which is reduced to form a bond. Oxyamino groups react with ketone or aldehyde groups to form oximes. It is known that azido groups undergo a 1,3-dipolar cycloaddition reaction with alkynyl groups to form a 1,2,3-triazole ring.

[0141] R 3 R represents a hydrogen atom, an organic group or a group derived from an inorganic material. Examples of the organic group and the inorganic material include the above-mentioned 4 Those representing examples of organic groups and inorganic material-derived groups. 3 When it 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, another substance can be further linked via the reactive group, as described above.

[0142] In one embodiment of the present invention, R 3 In this case, the formula (1) is, for example, the formula (1a), (1Aa) or (1Ba):

[0143]

[0144] where R 1 、R 2 and R 4 As defined above.

[0145] The compound represented by formula (1) includes stereoisomers and optical isomers, and these isomers are not particularly limited.

[0146] The salt of the compound represented by formula (1) is not particularly limited. The salt may be an acid salt or a basic salt. Examples of acid salts include inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, nitrates, perchlorates and phosphates; and organic acid salts such as acetates, propionates, tartrates, fumarates, maleates, malates, citrates, methanesulfonates and p-toluenesulfonates. Examples of basic salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; organic amine salts such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine and tris(hydroxyalkyl)amine.

[0147] The compound represented by formula (1) may be a hydrate or a solvate. Examples of the solvent include organic solvents (eg, ethanol, glycerol, and acetic acid).

[0148] 1-2. Synthesis Method 1

[0149] The compound represented by formula (1) can be synthesized by various methods. For example, the compound represented by formula (1) can be synthesized by a method comprising reacting a compound represented by formula (2) or a salt thereof with a compound represented by formula (3), a compound represented by formula (4), or a compound represented by formula (9).

[0150] R 4 -R 5 (2),

[0151] where R 4 As defined above; R 5 represents -N3, -X (wherein X represents a halogen atom), -B(OH)2, -B(OR 51 )2(where each R 51 are the same or different and represent a hydrocarbon group, provided that the two R 51 With the adjacent oxygen atom can form a ring), or -N2 + ,

[0152]

[0153] where R 3 As defined above; R 6 、R 7 and R 8 are the same or different, and all represent an alkyl group; Y represents a reactive group.

[0154] Examples of the halogen atom represented by X include fluorine, chlorine, bromine, iodine, etc. Preferably, the halogen atom represented by X is, for example, bromine.

[0155] By R 51 Examples of the hydrocarbon group represented include an alkyl group, a cycloalkyl group, and the like.

[0156] By R 51 The alkyl group represented by includes a straight-chain alkyl group and a branched-chain alkyl group. 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 methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, n-hexyl, 3-methylpentyl, n-heptyl, n-octyl, and the like.

[0157] R 51 The number of carbon atoms in the cycloalkyl group is not particularly limited, and is, for example, 3 to 10, preferably 4 to 10. Specific examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.

[0158] By two R 51 The ring formed together with the adjacent oxygen atom may be substituted by an alkyl group or the like.

[0159] R 6 、R 7 、R 8 The alkyl group represented is usually a straight-chain lower alkyl group, preferably an ethyl group.

[0160] Examples of the reactive group represented by Y include those mentioned above as examples of the reactive group that may be contained in the organic group or the group derived from an inorganic material. Y is preferably a halogen atom (Y').

[0161] Details of each material used are described below.

[0162] 1-2-1. Case 1 (R 5 is -N3, when a compound represented by formula (3) is used)

[0163] In terms of yield etc., the amount of the compound represented by formula (2) used is preferably 0.1 to 5 moles, more preferably 0.3 to 2 moles, per mole of the compound represented by formula (3), in terms of the number of moles of the functional group to be reacted (in this case, the azide group).

[0164] The reaction is usually carried out in the presence of a reaction solvent. Examples of reaction solvents include, but are not particularly limited to, water, methanol, tetrahydrofuran, dioxane, dimethyl sulfoxide, and the like. These solvents can be used alone or in combination of two or more. Preferably, a buffer solution, such as a phosphate buffer, is added to the solvent. When water is used, the pH value of the reaction is preferably close to neutral, particularly preferably 6 to 8.5, more preferably 6.5 to 8, and even more preferably 7 to 7.5.

[0165] This reaction is preferably carried out in the presence of a suitable catalyst. The catalyst is, for example, a copper catalyst. Examples of copper catalysts include divalent copper, such as copper sulfate; monovalent copper, such as copper iodide; and the like. Furthermore, reducing agents (such as hydroquinone or sodium ascorbate) and ligands may also be used in this reaction.

[0166] In terms of yield and the like, the copper catalyst is preferably used in an amount of 0.1 to 5 moles per mole of the azide-containing protein or peptide of the present invention.

[0167] In addition to the above-mentioned components, additives may be appropriately used in this reaction as long as the progress of the reaction is not significantly impaired.

[0168] The reaction can be carried out under heating, room temperature, or cooling; and is generally preferably carried out at a temperature at which the azide-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 generally 30 minutes to 3 hours, particularly 1 to 2 hours.

[0169] The reaction process can be monitored by chromatography or other common methods. After the reaction is completed, the solvent is evaporated and the product can be separated and purified by chromatography, recrystallization or other common methods as needed. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 C-NMR etc.

[0170] 1-2-2. Case 2 (R 5 is a group other than -N3, and a compound represented by formula (3) is used)

[0171] This situation is the same as situation 1, except that a catalyst for converting R 5 An azidating agent that converts to an azide group.

[0172] Examples of the azidating agent include inorganic azides such as sodium azide; sulfonyl azide; silyl azide; phosphoryl azide; alkylammonium azide; and the like.

[0173] The amount of the azidating agent used is usually preferably 0.1 to 5 moles, more preferably 0.3 to 2 moles, per mole of the compound represented by formula (2) in terms of yield and the like.

[0174] 1-2-3. Case 3 (case of using a compound represented by formula (4))

[0175] In this case, the compound represented by formula (1) can be synthesized through several steps after reacting the compound represented by formula (2) with the compound represented by formula (4). For example, in this case, the compound represented by formula (1) can be synthesized according to the following scheme.

[0176]

[0177] where R 1 、R 2 、R 3 、R 4 、R 5 and R 8 As defined above.

[0178] Step 1

[0179] In terms of yield, etc., the amount of the compound represented by formula (2) used per mole of the compound represented by formula (4) is generally preferably 0.1 to 5 moles, more preferably 0.3 to 2 moles, based on the number of moles of the functional group to be reacted (in this case, the azide group).

[0180] The solvent used is not limited as long as it does not have an adverse effect on the reaction. Examples include water, alcohol-based solvents (such as methanol, ethanol, isopropanol, n-butanol, trifluoroethanol and ethylene glycol), ketone-based solvents (such as acetone and methyl ethyl ketone), ether-based solvents (such as tetrahydrofuran, dioxane, ether, dimethoxyethane and diglyme), ester-based solvents (such as methyl acetate and ethyl acetate), aprotic polar solvents (such as acetonitrile, N,N-dimethylformamide and dimethyl sulfoxide), halogenated hydrocarbon solvents (such as dichloromethane and dichloroethane) and mixtures thereof. The solvent is preferably a mixture of water and an aprotic polar solvent (particularly dimethylformamide).

[0181] Step 1 is usually carried out in the presence of a base. The base can be, for example, an organic base. Examples of organic bases include trialkylamines (e.g., trimethylamine, triethylamine, and N,N-diisopropylethylamine), 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. When these bases are in liquid form, they can also be used as solvents. These bases can be used alone or in combination of two or more. The base is preferably piperidine.

[0182] When a base is used, the amount of the base is usually 0.05 to 1 mole, preferably 0.1 to 0.5 mole, per mole of the compound represented by formula (4).

[0183] The reaction temperature is not particularly limited. The reaction is usually carried out under cooling, room temperature or heating. The reaction can be carried out at a temperature of preferably about 50 to 100° C., more preferably about 70 to 90° C., for 1 to 30 hours.

[0184] After the reaction is completed, the solvent is evaporated and the product can be separated and purified by chromatography, recrystallization or other common methods. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 C-NMR etc.

[0185] Step 2

[0186] In step 2, the compound represented by formula (1") is reacted in the presence of a reducing agent and an appropriate amount of a base.

[0187] Examples of reducing agents include sodium borohydride, zinc borohydride (Zn(BH4)2), tetramethylammonium triacetoxyborohydride, lithium tri-sec-butylborohydride, borane, tetrahydrofuran borane complex, borane-dimethyl sulfide complex, lithium aluminum hydride, diisobutylaluminum hydride, lithium borohydride, and the like. These reducing agents may be used alone or in combination of two or more. The reducing agent is preferably sodium borohydride.

[0188] The amount of the reducing agent used is usually 1 to 15 moles, preferably 3 to 10 moles, per mole of the compound represented by formula (1").

[0189] As the base, for example, an inorganic base can be used. The example of the inorganic base includes alkali metal (such as sodium and potassium), alkali metal bicarbonate (such as lithium bicarbonate, sodium bicarbonate and potassium bicarbonate), alkali metal hydroxide (such as lithium hydroxide, sodium hydroxide, potassium hydroxide and cesium hydroxide), alkali metal carbonate (such as lithium carbonate, sodium carbonate, potassium carbonate and cesium carbonate), alkali metal lower (C1-C4) alkoxide (such as sodium methoxide, sodium ethoxide and potassium tert-butoxide), alkali metal hydride (such as sodium hydride and potassium hydride) etc. These alkalis can be used alone or in combination of two or more. The alkali is preferably alkali metal lower (C1-C4) alkoxide (especially sodium methoxide).

[0190] The solvent used is not limited as long as it does not have a negative effect on reaction.Example includes water, alcohol-based solvents (such as methanol, ethanol, isopropanol, n-butanol, trifluoroethanol and ethylene glycol), ketone-based solvents (such as acetone and methyl ethyl ketone), ether-based solvents (such as tetrahydrofuran), dioxane, ether, dimethoxyethane and diglyme), ester-based solvents (such as methyl acetate and ethyl acetate), aprotic polar solvents (such as acetonitrile, N, N-dimethylformamide and dimethyl sulfoxide), halogenated hydrocarbon solvents (such as, methylene dichloride and ethylene dichloride), and mixtures thereof.Solvent is preferably an alcohol-based solvent (particularly methanol).

[0191] The reaction temperature is not particularly limited. The reaction is usually carried out under cooling, room temperature or heating. It can be carried out at a temperature of preferably about 0°C to 60°C, more preferably about 10°C to 40°C, for 1 to 30 hours.

[0192] After the reaction is completed, the solvent is evaporated and the product can be separated and purified by chromatography, recrystallization or other common methods. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 C-NMR etc.

[0193] Step 3

[0194] In step 3, the compound represented by formula (1') is reacted in the presence of an oxidizing agent.

[0195] As an oxidizing agent, for example, manganese dioxide can be used. Other examples of usable oxidizing agents include selenium dioxide, nitroxyl radicals such as 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO) and 2-azaadamantane-N-oxyl (AZADO). These oxidizing agents can be used alone or in combination of two or more.

[0196] The amount of the oxidizing agent used is usually 3 to 20 moles, preferably 5 to 15 moles, per mole of the compound represented by formula (1').

[0197] The solvent used is not limited as long as it does not have a negative effect on the reaction.Examples include ketone-based solvents (for example, acetone and methyl ethyl ketone), ether-based solvents (for example, tetrahydrofuran, dioxane, ether, dimethoxyethane and diglyme), ester-based solvents (for example, methyl acetate and ethyl acetate), aprotic polar solvents (for example, acetonitrile, N,N-dimethylformamide and dimethyl sulfoxide), halogenated hydrocarbon solvents (for example chloroform, methylene chloride and vinyl chloride), and mixtures thereof.Solvent is preferably a halogenated hydrocarbon solvent (particularly chloroform).

[0198] The reaction temperature is not particularly limited. The reaction is usually carried out under cooling, room temperature or heating. The reaction can be carried out at a temperature preferably of about 0°C to 60°C, more preferably of about 10°C to 40°C, for 1 to 30 hours.

[0199] After the reaction is completed, the solvent is evaporated and the product can be separated and purified by chromatography, recrystallization or other common methods. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 C-NMR etc.

[0200] 1-2-4. Case 4 (R 5is -N3, and a compound represented by formula (9) is used)

[0201] In this case, the compound represented by formula (1) can be synthesized through several steps after reacting the compound represented by formula (2) with the compound represented by formula (9). For example, in this case, the compound represented by formula (1) can be synthesized according to the following scheme.

[0202]

[0203] Step 4 can be performed according to or based on the above-mentioned case 1. Step 6 can be performed according to or based on the above-mentioned step 3. Step 5 can be performed by reacting a compound represented by formula (1**) with a compound represented by formula (10): R 3 The reaction of step 5 can be carried out by reacting -Z. Z is a reactive group that reacts with Y. When Y is a halogen, for example, -B(OH)2 can be used as Z. The reaction conditions of step 5 can be determined based on known information and the types of Y and Z.

[0204] 1-3. Synthesis Method 2

[0205] In addition to the above-mentioned synthesis method 1, the compound represented by formula (1) can be prepared by, for example, including a compound represented by formula (5): R 4 -R 9 (5), where R 4 As defined above; R 9 Indicates -NR 9a R 9b (where R 9a and R 9b The synthesis method is to react alkyl groups (the same or different, each representing a hydrogen atom or an alkyl group) with a compound represented by formula (6):

[0206]

[0207] where R 10 Represents an electron-withdrawing group; R 11 Indicates -R 11a -R 12 (where R 11a represents a single bond or a linker; and R 12 represents a vector); n represents 0 or 1; and m represents an integer of 1 to 5.

[0208] Furthermore, the compound represented by formula (1AA) can be synthesized by using the compound represented by formula (5'): R 9 -R 4a -R 9 (5'), where R 4a and R 5a As defined above, the compound represented by formula (5).

[0209] Examples of the alkyl group include, but are not particularly limited to, lower alkyl groups such as methyl and ethyl. 9a and R 9b Both are preferably hydrogen atoms.

[0210] Examples of electron withdrawing groups include, but are not particularly limited to, -NO , -F, CF , -CN, -COOMe, and the like. The position of the electron withdrawing group is preferably para, such as in the case of -NO , and preferably ortho and meta, such as in the case of -F and CF . In the latter case, more preferably, -CN is located in the para position.

[0211] When the electron withdrawing group is, for example, -NO2, m is preferably 1. When the electron withdrawing group is, for example, -F or CF3, m is preferably 4 to 5, more preferably all ortho and meta positions are replaced by R 10 In the latter case, even more preferably, the -CN is located in the para position.

[0212] When -F or CF3 (preferably -F) is at all ortho and meta positions, and -CN is at para position, the reaction with the compound represented by formula (5) can proceed sufficiently even at relatively low temperature conditions (preferably 20-60°C, more preferably 25-40°C).

[0213] The linker is not particularly limited as long as it can connect the carrier to the benzene ring. Examples include linkers containing any of the following partial structures in the main chain.

[0214]

[0215] 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.

[0216] The carrier is not particularly limited.

[0217] The average particle size of the carrier particles is not particularly limited, but is preferably a size that allows easy precipitation in a solution. The average particle size of the carrier particles is, for example, 1 nm to 1 mm, preferably 10 nm to 100 μm.

[0218] The material of the carrier particles is not particularly limited, and examples include metal particles such as gold, silver, copper, iron, aluminum, nickel, manganese, titanium, and their oxides; resin particles such as polystyrene and latex; silica particles; and the like. The shape of the carrier particles is not particularly limited, and for example, it may be a sphere, a cuboid, a cube, a triangular pyramid, and the like. The carrier particles may have a substance on the surface that makes the binding of another substance (for example, binding substance 2) easier and / or stronger. Examples of the substance include substances containing reactive groups, such as substances containing epoxy groups, substances containing amino groups, substances containing carboxyl groups, substances containing azide groups; substances with affinity for other molecules, such as avidin, protein A, and protein B; and the like. The carrier particles may further contain a labeling substance. Only one type of carrier particles may be used, or two or more types of carrier particles may be used in combination.

[0219] The amount of the compound represented by formula (5) to be used is usually preferably 0.1 to 5 moles, and more preferably 0.3 to 2 moles, per mole of the compound represented by formula (6) in terms of yield and the like.

[0220] The solvent used is not limited as long as it does not have an adverse effect on the reaction. Examples include water, alcohol-based solvents (such as methanol, ethanol, isopropanol, n-butanol, trifluoroethanol and ethylene glycol), ketone-based solvents (such as acetone and methyl ethyl ketone), ether-based solvents (such as tetrahydrofuran, dioxane, ether, dimethoxyethane and diglyme), ester-based solvents (such as methyl acetate and ethyl acetate), aprotic polar solvents (such as acetonitrile, N,N-dimethylformamide and dimethyl sulfoxide), halogenated hydrocarbon solvents (such as dichloromethane and dichloroethane) and mixtures thereof. The solvent is preferably a mixture of water and an alcoholic solvent.

[0221] In this reaction, an acid catalyst is preferably used from the perspective of yield, etc. Examples of acid catalysts include, but are not particularly limited to, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, 2-morpholineethanesulfonic acid (MES), 3-morpholinepropanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), etc. Among them, from the perspective of not interfering with subsequent protein modification reactions, Goode buffers such as MES, MOPS, HEPES, and TAPS are preferred.

[0222] The reaction temperature is not particularly limited. The reaction is usually carried out under cooling, room temperature or heating. The reaction can be preferably carried out at a temperature of about 20°C to 100°C for 10 minutes to 30 hours.

[0223] After the reaction is completed, the solvent is evaporated and the product can be separated and purified by chromatography, recrystallization or other common methods. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1H-NMR, 13 C-NMR etc.

[0224] When the compound represented by formula (6) contains a carrier (ie, when R 11 -R 11a -R 12 Specifically, the compound represented by formula (6x) is a by-product (aniline derivative) produced by the reaction:

[0225]

[0226] Among them, R 9a 、R 9b 、R 10 、R 11a 、R 12 and m are as defined above, and can be easily removed by precipitation operation using a carrier, etc. Therefore, in one embodiment of the present invention, the present invention relates to a compound represented by formula (6'):

[0227]

[0228] where R 10 、R 11 、R 12 and m are as defined above.

[0229] 1-4. Application

[0230] The modified molecule of the present invention can be used to connect another molecule / substance to the N-terminus of a protein or peptide, for example, to produce the composite substance of the present invention described later (a compound represented by formula (7) or a salt thereof, or a hydrate or solvate of the compound or a salt thereof). Therefore, the modified molecule of the present invention can be suitable for use as a reagent, in particular as an active ingredient of a protein and / or peptide modification reagent. The reagent is not particularly limited as long as it contains the modified molecule of the present invention, and may also contain other ingredients as needed. The other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients. Examples of other ingredients include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, wetting agents, colorants, fragrances, chelating agents, etc.

[0231] 2. Composite substances

[0232] In one embodiment of the present invention, the present invention relates to a compound represented by formula (7) or a salt thereof, or a hydrate or solvate of the compound or a salt thereof (in this specification, these may be collectively referred to as "the composite substance of the present invention"):

[0233]

[0234] where R 1 、R 2 、R 3 and R 4 As defined above; R 13 represents a group in which the N-terminal amino acid residue and the adjacent -NH- are excluded from the protein or peptide; and R 14 represents the side chain of the N-terminal amino acid residue of a protein or peptide. The composite substance of the present invention is described below.

[0235] R 13 It represents a group in which the N-terminal amino acid residue and the adjacent -NH- are excluded from the protein or peptide.

[0236] The protein or peptide is not particularly limited as long as the N-terminal amino group is unmodified and the second amino acid residue at the N-terminus is an amino acid residue other than proline. In the protein or peptide, various modifications can be made at sites other than the N-terminus (e.g., cysteine ​​residues). Examples of such proteins or peptides include proteins or peptides represented by formula (7a).

[0237]

[0238] R 14 Represents the side chain of the N-terminal amino acid residue of a protein or peptide. The amino acid residue may be a natural amino acid residue or a synthetic amino acid residue. Examples include amino acid residues with basic side chains, such as lysine, arginine, and histidine; amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with uncharged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with nonpolar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine; and the like.

[0239] The protein or peptide is not particularly limited and may be natural, synthetic, or artificial.

[0240] Proteins or peptides can be chemically modified. The C-terminus of a protein or peptide can be a carboxyl group (-COOH), a carboxylate (-CO - ), amide (-CONH2) or ester (-COOR). Here, R in the ester is, for example, a C group such as methyl, ethyl, n-propyl, isopropyl or n-butyl. 1-6 Alkyl; for example, C 3-8 Cycloalkyl groups such as cyclopentyl and cyclohexyl; for example, C 6-12Aryl, such as phenyl and α-naphthyl; for example, phenyl-C 1-2 Alkyl groups, such as benzyl and phenethyl; including α-naphthyl-C 1-2 C7-14 aralkyl of alkyl, such as α-naphthylmethyl; pivaloyloxymethyl, etc. In proteins or peptides, carboxyl groups (or carboxylates) other than the C-terminus may be amidated or esterified. As esters in this case, for example, the above-mentioned C-terminal esters are used. In addition, proteins or peptides include those in which the substituents (e.g., -OH, -SH, amino, imidazolyl, indolyl or guanidino) in the amino acid side chains in the molecules are protected by suitable protecting groups (e.g., including C 1-6 Alkanoyl C 1-6 Acyl groups, such as formyl or acetyl) protected proteins or peptides.

[0241] Protein or peptide can be post-translationally modified, or can be post-translationally modified by artificial enzyme treatment or chemical modification.Examples of post-translationally modified include phosphorylation, N-glycosylation, O-glycosylation, C-glycosylation, phosphoglycosylation, glycosylphosphatidylinositolization, S-nitrosylation, methylation, N-acetylation, S-myristoylation, S-prenylation, S-palmitoylation etc.Protein or peptide can be added protein or peptide, for example known protein tag or signal sequence, or protein or peptide of marker substance.Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, SPY tag etc.Examples of signal sequences include nuclear localization signal etc.

[0242] A protein or peptide can exist alone as a single molecule, or can be linked to another molecule to form a complex. For example, the protein or peptide can be present on the cell surface, in a cell lysate, or loaded onto a substance. The linkage method is not particularly limited and may include, for example, hydrogen bonding, electrostatic forces, van der Waals forces, hydrophobic bonds, covalent bonds, coordination bonds, and the like.

[0243] As long as the N-terminus exists, the protein or peptide can be cleaved by enzymes or chemical reactions. When the protein or peptide is modified at the N-terminus, the N-terminus can be modified by enzymes or chemical reactions.

[0244] The compound represented by formula (7) includes stereoisomers and optical isomers, and these isomers are not particularly limited.

[0245] The salt of the compound represented by formula (7) is not particularly limited. The salt may be an acid salt or a basic salt. Examples of acid salts include inorganic acid salts such as hydrochlorides, hydrobromides, sulfates, nitrates, perchlorates and phosphates; and organic acid salts such as acetates, propionates, tartrates, fumarates, maleates, malates, citrates, methanesulfonates and p-toluenesulfonates. Examples of basic salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; ammonium salts; organic amine salts such as morpholine, piperidine, pyrrolidine, monoalkylamine, dialkylamine, trialkylamine, mono(hydroxyalkyl)amine, di(hydroxyalkyl)amine and tris(hydroxyalkyl)amine.

[0246] The compound represented by formula (7) may be a hydrate or a solvate. Examples of the solvent include organic solvents (eg, ethanol, glycerol, and acetic acid).

[0247] The compound represented by formula (7) can be synthesized by various methods. For example, the compound represented by formula (7) can be produced by a method comprising reacting a protein or peptide with the modified molecule of the present invention. The reaction can be carried out after 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 the reaction, and then the reaction is carried out.

[0248] In terms of yield and the like, the amount of the modified molecule of the present invention to be used is preferably generally 5 to 400 mol per mol of protein or peptide.

[0249] The reaction is usually carried out in the presence of a reaction solvent. Examples of reaction solvents include, but are not particularly limited to, water. These solvents can be used alone or in combination of two or more. In addition, a buffer, such as a phosphate buffer, is preferably added to the solvent. When water is used, the pH of the reaction is preferably close to neutral in terms of N-terminal selectivity, particularly preferably 6 to 8.5, more preferably 6.5 to 8, and even more preferably 7 to 7.5.

[0250] In addition to the above-mentioned components, additives may be appropriately used in this reaction as long as the progress of the reaction is not significantly impaired.

[0251] The reaction can be carried out under heating, room temperature or cooling. The reaction is usually preferably carried out at a temperature at which the protein or peptide does not significantly denature, for example, 0°C to 45°C (particularly 0°C to 40°C). The reaction time is not particularly limited and can generally be 8 to 36 hours, particularly 12 to 24 hours.

[0252] The reaction process can be monitored by chromatography or other common methods. After the reaction is completed, the solvent is evaporated and the product can be separated and purified by chromatography, recrystallization or other common methods as needed. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, 1 H-NMR, 13 C-NMR etc.

[0253] When the compound represented by formula (1AA) is used as the modified molecule of the present invention, the compound represented by formula (7AA) is obtained by the above reaction:

[0254]

[0255] where R 3 、R 4a 、R 13 and R 14 As defined above. In this case, the organic molecules etc. can be linked by further forming an oxime with hydroxylamine. Specifically, the compound represented by formula (1AA) can react with the compound represented by formula (8): R 4 -O-NH2(8), wherein R 4 As defined above, the compound represented by the synthetic formula (7AAA):

[0256]

[0257] where R 3 、R 4a 、R 4 、R 13 and R 14 As defined above. The conditions of the synthesis reaction can be determined according to or based on the known reaction conditions for forming oximes starting from aldehydes and hydroxylamine. This reaction is also suitable for protein modification because it can be carried out even in water under mild conditions.

[0258] The composite substance of the present invention has a structure in which another substance is linked to a protein or peptide. Depending on the substance to be linked, the composite substance of the present invention can be used, for example, as an antibody-drug conjugate, a reagent for labeling a protein, an inorganic material for immobilizing a protein, a fusion protein in which a protein is linked, or a protein having a nucleic acid fused thereto, in various fields depending on the substance to be linked.

[0259] In one embodiment of the composite substance of the present invention, the composite substance can be designed so that the substance linked to the protein or peptide gradually dissociates. Therefore, after the composite substance of the present invention, for example, a drug linked to a protein or peptide, is administered to a living organism, the drug can be gradually released in the living organism by utilizing the gradual dissociation of the drug from the composite substance of the present invention in the living organism.

[0260] When the composite material of the present invention has a reactive group (for example, when R 3 When the azide group has a reactive group, the reactive group can be used to further connect another substance. For example, it can be used to connect other substances (such as organic molecules, organic molecule complexes, or inorganic materials) by a reaction using the azide group (for example, Huisgen cycloaddition reaction, strain-promoted azide-alkyne cycloaddition reaction, or Staudinger-Bertozzi ligation).

[0261] Example

[0262] Examples are given below to illustrate the present invention in more detail; however, the present invention is not limited to these examples.

[0263] Example 1. Synthesis of Compound 1 (Method A)

[0264] 1-1. Equipment used

[0265] Nuclear magnetic resonance (NMR) spectra were measured using a Bruker AVANCE III HD NMR spectrometer, and chemical shifts were calculated using the residual signal of the measurement solvent as an internal reference. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) was performed using a Bruker micrOTOF focus III mass spectrometer, using methanol or acetonitrile (both HPLC grade) as the mobile phase. Fourier transform infrared absorption (FT-IR) spectra were measured using a Jasco FT / IR-4000 Fourier transform infrared spectrophotometer in ATR mode using either diamond or gallium prisms.

[0266] 1-2. Reagents, solvents, etc.

[0267] Commercially available reagents and solvents were used as they were in the synthesis. The precursor azide used was synthesized with reference to a published report (Y. Zhao, P. Gong, Bioorg. Med. Chem., 2014, 22, 6438-6452).

[0268] 1-3-1. Synthesis of 1-(3-carboxyphenyl)-1H-1,2,3-triazole-4-carboxaldehyde (1)

[0269] Compound 1 was synthesized according to the following scheme.

[0270]

[0271] Compound 1 was synthesized with reference to published 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 process and compound identification results are described below.

[0272] Under a nitrogen atmosphere, azide (0.60 mmol), propargylaldehyde diethyl acetal (115 μL, 0.80 mmol) and sodium ascorbate (60 mg, 0.30 mmol) were added to a mixture of an aqueous solution (7.5 mL) of copper (II) sulfate pentahydrate (38 mg, 0.15 mmol) and tert-butanol (7.5 mL). The mixture was stirred at 70 ° C for 24 hours and stirred in air at room temperature for 1 hour. The resulting suspension was diluted with a saturated aqueous sodium chloride solution (10 mL), extracted with ethyl acetate (50 mL × 3), and washed with a saturated aqueous sodium chloride solution (20 mL × 2). The obtained organic layer was dried over sodium sulfate, and the solvent of the filtrate obtained by filtration was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography to obtain compound 1 (yellow solid). Figure 1 Shows 1 H NMR spectrum.

[0273] Yield: 48% 1 H NMR (400MHz, DMSO-d6): δ10.12(s,1H),9.67(s,1H),8.48(s,1H),8.16(d,J=7.8Hz,1H),8.09(d,J=7.8Hz,1H),7.72(t,,J=7.8Hz,1H); 13 C NMR (100 MHz, DMSO-d6): δ 184.9, 167.0, 147.6, 136.0, 130.1, 130.0, 126.6, 124.0, 121.3; ESI-TOF MS (positive mode) calculated for C 10 The m / z of H7NaN3O3[M+Na]+240.04 was found to be 240.04; FT-IR (ATR mode, gallium prism), νcm -1 :3128,2923,1697,1263,1230,1184,757,673,647.

[0274] 1-3-2. Synthesis of 1-(4-carboxyphenyl)-1H-1,2,3-triazole-4-carboxaldehyde (2)

[0275]

[0276] Using 4-azidobenzoic acid as a precursor, compound 2 (yellow solid) was synthesized using method A as in Example 1-3-1. Figure 2 Shows 1 H NMR spectrum.

[0277] Yield: 63% 1 H NMR (400 MHz, DMSO-d6): δ 10.13 (s, 1H), 9.69 (s, 1H), 25 8.18-8.12 (m, 4H); ESI-TOF MS (positive mode) calculated for C 10 The m / z of H7NaN3O3[M+Na]+240.04 was found to be 240.04; FT-IR (ATR mode, gallium prism), νcm -1 :3110,1695,1683,1605,1429,1319,1294,1264,989,945,864,773,702,688.

[0278] 1-3-3. Synthesis of 1-(3,5-dicarboxyphenyl)-1H-1,2,3-triazole-4-carboxaldehyde (3)

[0279]

[0280] Compound 3 (yellow solid) was synthesized as in Example 1-3-1 using 5-azidoisophthalic acid as a precursor by using Method A. Figure 3 Shows 1 H NMR spectrum.

[0281] Yield 10%; 1 H NMR (400 MHz, DMSO-d6): δ10.12 (s, 1H), 9.81 (s, 1H), 8.65 (s, 2H), 8.56 (s, 1H); FT-IR (ATR mode, gallium prism), ν cm -1 :3133,1697,1296,1281,1247,1049,830,757,678,666,566,527.

[0282] 1-3-4. Synthesis of 1-(4-(diethylamino)phenyl)-1H-1,2,3-triazole-4-carboxaldehyde (4)

[0283]

[0284] Using 4-azido-N,N-diethylaniline as a precursor, compound 4 (brown solid) was synthesized using method A as in Example 1-3-1. Figure 4 Shows 1 H NMR spectrum.

[0285] Yield 60%; 1H NMR (400 MHz, CDCl 3 ): δ 10.12 (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-d6): δ 185.0, 148.0, 147.3, 125.1, 124.3, 122.1, 111.4, 43.8, 12.3; ESI-TOFMS (positive mode) calculated for C 13 H 16 The m / z of NaN4O[M+Na]+267.12 was found to be 267.13.

[0286] 1-3-5. Synthesis of 1-(4-methoxyphenyl)-1H-1,2,3-triazole-4-carbaldehyde (5)

[0287]

[0288] Compound 5 (white solid) was synthesized as in Example 1-3-1 by using Method A using 1-azido-4-methoxybenzene as a precursor. Figure 5 Shows 1 H NMR spectrum.

[0289] Yield 68%; 1H NMR (400 MHz, DMSO-d6): δ 10.12 (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-d6): δ 185.0, 159.9, 147.5, 129.3, 125.9, 122.4, 115.0, 55.7; ESI-TOF MS (positive mode) calculated for C 10 The m / z of H9NaN3O-2[M+Na]+226.06 was found to be 226.06.

[0290] 1-3-6. Synthesis of 1-phenyl-1H-1,2,3-triazole-4-carboxaldehyde (6)

[0291]

[0292] Compound 6 (brown solid) was synthesized as in Example 1-3-1 by using method A using benzene azide as a precursor. Figure 6 Shows 1 H NMR spectrum.

[0293] Yield 9%; 1 H NMR (400 MHz, DMSO-d 6) : δ10.11(s,1H),9.57(s,1H),7.97(d,J=8.0Hz,2H),7.66-7.54(m,3H).

[0294] Example 2. Synthesis of Compound 2 (Method B)

[0295] The equipment, reagents, solvents, etc. used are similar to those in Example 1.

[0296] 2-1. Synthesis of 1-benzyl-1H-1,2,3-triazole-4-carboxaldehyde (7)

[0297]

[0298] Compound 7 was synthesized based on a previously published report (JT Letcher, Tetrahedron Lett., 2017, 58, 4450-4454). The specific synthesis process and compound identification results are described below.

[0299] A mixture of copper 20(II) sulfate pentahydrate (47.8 mg, 0.19 mmol) in water (7.5 mL) and tert-butanol (7.5 mL) was cooled to 0°C, and sodium azide (103 mg, 1.6 mmol) was added to the mixture 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 then in air at room temperature for 1 hour. The reaction solution was air-cooled to room temperature, then diluted with saturated sodium chloride aqueous solution (5 mL), and extracted with ethyl acetate (30 mL x 3). The resulting organic layer was dried over magnesium sulfate. The solid was filtered and the resulting filtrate was distilled under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography (hexane:ethyl acetate = 2:1) to obtain compound 7 (white solid). Figure 7 Shows 1 H NMR spectrum.

[0300] Yield: 46% 1 H NMR (400MHz, DMSO-d6): δ10.00(s,1H),8.95(s,1H),7.41-7.32(m,5H),5.69(s,2H); 13C NMR (100 MHz, DMSO-d6): δ 185.0, 147.0, 135.3, 128.9, 128.4, 128.3, 128.1, 53.2; ESI-TOF MS (positive mode) calculated for C 10 The m / z of H9NaN3O[M+Na]+210.06 was found to be 210.06; FT-IR (ATR mode, gallium prism), νcm -1 :1694,1535,1237,1165,1052,877,796,767,714,701,565,556,543,532,515,505.

[0301] 2-2. Synthesis of 1-(naphthalen-2-ylmethyl)-1H-1,2,3-triazole-4-carboxaldehyde (8)

[0302]

[0303] Compound 8 (white solid) was synthesized as in Example 2-1 by using Method B using 2-(bromomethyl)naphthalene as a precursor. Figure 8 Shows 1 H NMR spectrum.

[0304] Yield 20%; 1 H-NMR (400 MHz, CDCl3): δ 10.13 (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) calculated for C 14 H 11 NaN3O[M+Na]+260.08m / z, found 260.08; FT-IR (ATR mode, gallium prism), νcm -1 :3121,1709,1538,1239,1177,1052,1026,866,835,792,761,563,553,527,511.

[0305] Example 3. Synthesis of Compound 3 (Method C)

[0306] The equipment, reagents, solvents, etc. used are similar to those in Example 1.

[0307] 3-1. Synthesis of 1-(p-Tolyl)-1H-1,2,3-triazole-4-carboxaldehyde (9)

[0308]

[0309] Compound 9 was synthesized with reference to a published report (C.-Z. Tao, X. Cui, J. Li, AX. Liu, L. Liu, Q.-X. Guo, Tetrahedron Lett., 2007, 48, 3525-3529). The specific synthesis process and compound identification results are described below.

[0310] Pure water (5 mL) containing copper (II) sulfate pentahydrate (47.8 mg, 0.19 mmol) was added to a methanol solution (5 mL) containing 4-methylphenylboronic acid (136 mg, 1.0 mmol) and sodium azide (98 mg, 1.5 mmol), and the mixture was stirred in air for 5 hours. Subsequently, sodium ascorbate (79 mg, 0.4 mmol) and propargylaldehyde diethyl acetal (286 μL, 1.7 mmol) were added 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 reaction solution was air-cooled to room temperature, then diluted with saturated sodium chloride aqueous solution (40 mL), and then extracted with ethyl acetate (50 mL×3). The resulting organic layer was dried over magnesium sulfate. The filtrate from which the solid was filtered out 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 then stirred vigorously at room temperature. The mixture was diluted with saturated aqueous sodium chloride (30 mL), and the organic layer was extracted with chloroform (30 mL x 3). The resulting organic layer was dried over magnesium sulfate. The filtrate from which the solid was filtered was distilled under reduced pressure to obtain a crude product, which was then purified by reprecipitation (hexane:chloroform) to obtain compound 9 (white solid). Figure 9 Shows 1 HNMR spectrum.

[0311] Yield: 71% 1 H NMR (400MHz, CDCl3): δ10.2 (s, 1H), 8.48 (s, 1H), 7.64 (d, J = 8.3Hz, 2H), 7.37 (d, J = 8.3Hz, 2H), 2.45 (s, 3H); 13 C NMR (100 MHz, CDCl3): δ 185.3, 148.2, 140.3, 134.0, 130.7, 123.2, 120.9, 21.3; ESI-TOF MS (positive mode) calculated for C 10 H9N3ONa[M+Na] + 210.06m / z, found 210.06.

[0312] Example 4. Synthesis of Compound 4 (Method D)

[0313] The equipment, reagents, solvents, etc. used are similar to those in Example 1.

[0314] Synthesis of 4-1.1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carboxaldehyde (10)

[0315]

[0316] The synthesis of compound 10 was based on a previously published report (JT Letcher, JE Reilly, Tetrahedron Lett., 2011, 52, 5512-5515). The specific synthesis process and compound identification results are described below.

[0317] A mixture of an aqueous solution (25 mL) of sodium azide (358 mg, 5.5 mmol) and tert-butanol (25 mL) was cooled to 0°C, and nitrobenzenediazonium tetrafluoroborate (1.18 g, 5.0 mmol) was added portionwise, followed by vigorous stirring for 1 hour. Subsequently, 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 at 70°C overnight. The reaction solution was filtered, and the filtrate was extracted with ethyl acetate (50 mL × 3). The organic layer was dried over sodium sulfate, and the solvent was removed by distillation under reduced pressure. The crude product was purified by reprecipitation (hexane:ethyl acetate) to obtain compound 10 (yellow solid). Figure 10 Shows 1 H NMR spectrum.

[0318] Yield: 62% 1 H NMR (400MHz, DMSO-d6): δ10.15(s,1H),9.78(s,1H),8.49(d,J=9.2Hz,2H),8.32(d,J=9.2Hz,2H); 13 C NMR (100 MHz, DMSO-d6): δ 185.0, 147.8, 147.4, 140.3, 126.9, 125.6, 121.5; ESI-TOF MS (positive mode) calculated for C9H6N4O3Na[M+Na] + 241.03m / z, obtained as 241.03.

[0319] Example 5. Synthesis of Compound 5 (Method E)

[0320] The equipment, reagents, solvents, etc. used are similar to those in Example 1.

[0321] Synthesis of 5-1,5-methyl-1-phenyl-1H-1,2,3-triazole-4-carbaldehyde (13)

[0322] The synthesis of compound 13 was based on published 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, S. M. A. Lam, MSK Halmuthur, Arch. Pharm. Chem. Life Sciences, 2015, 348, 796-807).

[0323]

[0324] Synthesis of 5-1-1,5-methyl-1-phenyl-1H-1,2,3-triazole-4-carboxylic acid ethyl ester (11)

[0325] Ethyl acetoacetate (1.5 mmol) and piperidine (20 μL, 0.2 mmol) were added to a solution of benzene azide (120 mg, 1.0 mmol) in a mixture of 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 resulting solution to stop the reaction, followed by extraction with ether (20 mL × 5). The resulting organic layer was washed with water (20 mL × 2). The organic layer was dried over sodium sulfate, and the solvent of the filtrate obtained by filtration was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography (hexane: ethyl acetate) to give compound 11 (white solid).

[0326] Yield 27%; 1 H NMR (400MHz, CDCl3): δ7.61-7.55(m,3H), 7.47-7.44(m,2H), 4.47(q,J=7.1Hz,2H), 2.60(s,3H), 1.46(t,J=7.1Hz,3H).

[0327] 5-1-2. Synthesis of (5-methyl-1-phenyl-1H-1,2,3-triazol-4-yl)methanol (12) Under a nitrogen atmosphere, sodium borohydride (29 mg, 0.77 mmol) was added to a solution of sodium methoxide (1 mg, 2 μmol) in compound 11 (0.12 mmol) and methanol (1 mL), and then stirred at room temperature for 3 hours. Excess methanol was added to the reaction solution to stop the reaction. The reaction solution was concentrated under reduced pressure, and the residue was diluted with a saturated aqueous sodium chloride solution (5 mL), followed by extraction with diethyl ether (20 mL×3) and washed with a saturated aqueous sodium chloride solution (5 mL×2). The obtained organic layer was dried over sodium sulfate. Afterwards, the crude product obtained by concentrating the solvent of the filtrate under reduced pressure was purified by silica gel column chromatography (hexane: ethyl acetate = 2:1) to obtain compound 12 (white solid). Figure 11 Shows 1 H NMR spectrum.

[0328] Yield: 37% 1 H NMR (400MHz, CDCl3): δ7.58-7.52 (m, 3H), 7.48-7.46 (m, 2H), 4.83 (d, J = 6.0Hz, 2H), 2.37 (s, 2H), 1.96 (t, J = 6.0Hz, 1H).

[0329] Synthesis of 5-1-3.5-Methyl-1-phenyl-1H-1,2,3-triazole-4-carbaldehyde (13)

[0330] Activated manganese dioxide (141 mg, 1.6 mmol) was added to a chloroform (5 mL) solution of compound 12 (0.16 mmol), and the mixture was stirred at room temperature under nitrogen for 24 hours. The reaction mixture was filtered, and the solvent of the filtrate was distilled off under reduced pressure to obtain compound 13 (white solid).

[0331] Yield: 89%; 1 H NMR (400MHz, DMSO-d6): δ7.58-7.52(m,3H),7.48-7.46(m,2H),4.83(d,J=6.0Hz,2H),2.37(s,2H),1.96(t,J=6.0Hz,1H); 13 C NMR (100MHz, DMSO-d6): δ186.1, 143.3, 138.7, 134.7, 130.3, 129.8, 125.3, 9.3.

[0332] 5-1-4. Synthesis of ethyl 1-phenyl-5-(trifluoromethyl)-1H-1,2,3-triazole-4-carboxylate (14)

[0333]

[0334] Compound 14 (yellow oil) was synthesized using ethyl 4,4,4-trifluoroacetoacetate as a precursor by the same method as in Example 5-1-1.

[0335] Yield 35%; 1 H NMR (400MHz, CDCl3): δ7.63-7.57 (m, 3H), 7.47 (d, J = 7.3Hz, 2H), 4.51 (q, J = 7.1Hz, 2H), 1.45 (t, J = 7.1Hz, 3H).

[0336] 5-1-5. Synthesis of (1-phenyl-5-(trifluoromethyl)-1H-1,2,3-triazol-4-yl)methanol (15)

[0337]

[0338] Compound 15 (white solid) was synthesized using compound 14 as a precursor by the same method as in Example 5-1-2.

[0339] Yield: 83% 1 H NMR (400MHz, CDCl3): δ7.61-7.55 (m, 3H), 7.47 (d, J = 7.2 Hz, 2H), 4.96 (d, J = 6.0 Hz, 2H).

[0340] 5-1-6. Synthesis of (1-phenyl-5-(trifluoromethyl)-1H-1,2,3-triazol-4-yl)methanol (16)

[0341]

[0342] Compound 16 (white solid) was synthesized using compound 15 as a precursor by the same method as in Example 5-1-3. Figure 12 Shows 1 H NMR spectrum.

[0343] Yield 75%; 1 H NMR (400MHz, DMSO-d6): δ10.21 (s, 1H), 7.74-7.66 (m, 5H).

[0344] Example 6. Synthesis of Compound 6 (Method F)

[0345] The equipment, reagents, solvents, etc. used are similar to those in Example 1.

[0346] 6-1. Synthesis of 1-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethyl)-1H-1,2,3-triazole-4-carbaldehyde (17)

[0347]

[0348] 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 tert-butanol (1 mL), and the mixture was stirred at 60°C overnight under a nitrogen atmosphere. After the reaction solution was cooled to 0°C, 0.1 M HCl aqueous solution (50 mL) was added to stop the reaction, and then extracted with ethyl acetate (20 mL × 3). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting crude product was purified by silica gel column chromatography (hexane:ethyl acetate = 0-60%) to obtain compound 17 (yellow oil). Figure 13 for 1 H NMR spectrum.

[0349] Yield 70%; 1 H NMR (400MHz, CD3CN): δ10.04(s,1H),8.42(s,1H),4.59(t,J=5.1Hz,2H),4.11(d ,J=2.4Hz,2H),3.87(t,J=5.1Hz,2H),3.60-3.56(m,4H),2.69(t,J=2.4Hz,1H); 13 C NMR (100 MHz, CD3CN): δ 185.7, 148.4, 128.6, 80.8, 75.7, 70.6, 69.7, 69.5, 58.7, 51.3; ESI-TOF MS (positive mode) calculated for C 10 H 13 N3O3Na[M+Na] + 246.08m / z, found 246.08.

[0350] Example 7. Compound Synthesis 7: Synthesis of Functional Molecules

[0351] The equipment, reagents, solvents, etc. used are similar to those in Example 1. The reaction precursors used are purchased or appropriately synthesized.

[0352] 7-1. Synthesis of N-(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)

[0353]

[0354] 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, compound 18 (white solid) was synthesized using the same method as Example 1-3-1 (Method A). Figure 14 Shows 1 H NMR spectrum.

[0355] Yield 23%; 1 H NMR(400MHz,CD3CN+DMSO-d6(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=5.0Hz,2H ),4.42-4.38(m,1H),4.24-4.21(m,1H),3.88(t,J=5.0Hz,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, 12.6 Hz, 1H), 2.63 (d, J = 5.0, 12.6 Hz, 1H), 2.12 (t, J = 7.3 Hz, 2H), 1.63-1.49 (m, 4H), 1.41-1.34 (m, 2H); ESI-TOF MS (positive mode) calculated for C 21 H 34 N6O6Na[M+Na] + 521.1, we get 521.1.

[0356] 7-2. Synthesis of 1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthene]-5-yl)-1H-1,2,3-triazole-4-carboxaldehyde (19)

[0357]

[0358] Compound 19 (orange solid) was synthesized using 5-azido-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthene]-3-one as a precursor by the same method as in Example 1-3-1 (Method A). Figure 15 Shows 1 H NMR spectrum.

[0359] Yield: 47% 1H NMR (400MHz, DMSO-d6): δ10.19(s,2H),10.15(s,1H),9.80(s,1H),8.60(d,J=1.7Hz,1H),8.43 (dd,J=2.0,8.3Hz,1H),7.57(d,J=8.3Hz,1H),6.71-6.67(m,4H),6.57(dd,J=2.2,8.7Hz,2H); 13 C NMR (100 MHz, CDCl3): δ 185.0, 167.5, 159.7, 152.6, 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) calculated for C 23 H 14 N3O6[M+H] + 428.09m / z, found 428.10.

[0360] 7-3. Synthesis of polyethylene glycol-tethered triazole-4-carboxaldehyde (20) (MW. ~4 kDa)

[0361]

[0362] Compound 20 (brown solid) was synthesized using polyethylene glycol monomethyl ether (Tokyo Chemical Industry Co., Ltd.) with a molecular weight of approximately 4 kDa as a starting material using the same method as in Example 1-3-1, with reference to a published report (MB van Eldijk, FCM Smit, N. Vermue, MF Debets, S. Schoffelen, JCM van Hest, Biomacromolecules, 2014, 15, 2751-2759). Purification was performed by reprecipitation with diethyl ether. Figure 16 Shows 1 H NMR spectrum.

[0363] Yield 50%; 1 H NMR (400MHz, CDCl3): δ10.14 (s, 1H), 8.41 (s, 1H), 4.63 (t, J = 4.8Hz, 1H), 3.91-3.45 (m, 366H).

[0364] 7-4. Synthesis of 1-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazole-4-carbaldehyde (21)

[0365]

[0366] Compound 21 (yellow oil) was synthesized using the same method as in Example 1-3-1 (Method A) except that 1-azido-2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane was used as a precursor. Figure 17 Shows 1 H NMR spectrum.

[0367] Yield 27%; 1H NMR (400 MHz, CDCl3): 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 = 5.0 Hz, 2H); 13C NMR (100 MHz, CDCl3): 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) calculated for C 11 H 18 N6O4Na[M+Na] + 321.1m / z, found to be 321.1.

[0368] 7-5. Synthesis of Compound 22

[0369] Compound 22 (yellow solid) was synthesized using the same method as in Example 6 (Method F) using N-(3-aminopropionyl)-5,6-dihydro-11,12-didehydrodibenzo[b,f]azepine as a precursor. Figure 18 Shows 1 H NMR spectrum.

[0370] Yield 75%; 1 H NMR (400 MHz, CD3CN): 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), 5.06 (d, J = 14 Hz, 1H), 4.45 (m, 2H), 3.66 (d, J = 14 Hz, 1H), 2.90 (td, J = 6.0, 17 Hz, 1H), 2.35 (td, J = 6.0, 17 Hz, 1H); ESI-TOF MS positive mode) calculated for C 11 H 18 N6O4Na[M+Na] + 321.1m / z, found to be 321.1.

[0371] Example 8. Peptide N-terminal modification 1

[0372] 8-1. Reagents, solvents, etc.

[0373] The ultrapure water used was obtained by purification using Millipore Integral 3. As other reagents and solvents, commercially available products were used as they are.

[0374] 8-2. Peptide N-terminal modification

[0375] This method targets the N-terminus of a peptide. A peptide that can be targeted 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, the N-terminus of angiotensin I is modified as follows.

[0376] The amino acid sequence of angiotensin I is shown below.

[0377] DRVYIHPFHL (SEQ ID NO: 1)

[0378] The N-terminal modification of the peptide was performed with reference to a published report (JI MacDonald, HK Munch, T. Moore, MB Francis, Nat. Chem. Biol. 2015, 11, 326-331). The specific experimental procedure is described below.

[0379] A solution of compound 7 in dimethyl sulfoxide (DMSO) (200 mM, 1 μL, 0.2 μmol, final concentration: 10 mM) was diluted with potassium phosphate buffer (10 mM, pH 7.5, 17 μL). An aqueous peptide solution (1 mM, 2 μL, 2 nmol, final concentration: 100 μM) was added thereto, and the mixture was shaken at 37° C. for 4 hours. The percentage of modification (=modified peptide / (total peptide amount)) was assessed by using LC / MS from the peak intensity in the mass spectrum. Figure 19 The results are shown. The modification percentage under the reaction conditions was calculated to be 89%. The above results confirm that the compounds according to the present invention are useful as protein modifying agents.

[0380] Example 9. Peptide N-terminal modification 2

[0381] The reagents, equipment, solvents, etc. used were similar to those in Example 8. As Compound 24, a compound synthesized according to a published report (H. Hagiwara, S. Okada, Chem. Commun., 2016, 52, 815-818) was used.

[0382] 9-1. Peptide N-terminal modification

[0383] The structural analysis of the product in this modification reaction was performed using a model peptide as a substrate. As a specific example, the reaction shown in the following scheme was performed.

[0384]

[0385] A solution of compound 24 (44 mg, 0.4 mmol) in dimethylformamide (DMF) (200 μL) was added to a potassium phosphate buffer (10 mM, pH 7.5, 1.8 mL) of compound 23 (11 mg, 0.04 mmol), and the mixture was shaken at 37 ° C for 16 hours. The solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate: acetonitrile: water = 95:5:0 to 0:95:5) to obtain compound 25.

[0386] 9-2. Compound 25 1 H NMR analysis

[0387] Figure 20 The results show that compound 25 in deuterated acetonitrile 1 H NMR spectrum assignment results.

[0388] The two peaks at 5.7 ppm are attributed to the specific proton H at the 2-position of the 4-imidazolidinone ring. e , confirming that the reaction forms a 4-imidazolidinone ring at the N-terminus of the peptide. The two peaks are derived from isomers, in which the asymmetric point is the 2-position of the 4-imidazolidinone ring.

[0389] Example 10. Protein N-terminal modification 1

[0390] 10-1. Reagents, solvents, etc.

[0391] Ribonuclease A (RNase) from bovine pancreas was purchased from Roche. Ultrapure water used was purified using Millipore Integral 3. Other reagents and solvents were used as they are.

[0392] 10-2. Protein modification

[0393] This method targets the N-terminus of proteins. Proteins that are candidates for this approach are those whose N-terminal amino group is unmodified and whose second amino acid residue is an amino acid other than proline. As a specific example, the following describes the N-terminal modification of ribonuclease A (RNase) from bovine pancreas.

[0394] The following is the amino acid sequence of RNase (PDB: 1FS3).

[0395]

[0396] The N-terminal modification of proteins was performed with reference to a published report (JI MacDonald, HK Munch, T. Moore, MB Francis, Nat. Chem. Biol. 2015, 11, 326-331). The specific experimental procedures are described below.

[0397] Compound 7 solution (200mM, 2.5 μ L, 0.5 μ mol, final concentration: 10mM) is diluted with potassium phosphate buffer (10mM, pH 7.5, 45 μ L).To it, add the ultrapure water solution (1mM, 2.5 μ L, 2.5nmol, final concentration: 50 μ M) of RNase, the mixture is shaken at 37 ℃ for 16 hours.Modification percentage (=modified RNase / (total RNase amount)) is by using LC / MS from the peak intensity assessment in mass spectrum. Figure 21 The results are shown. The modification percentage under these reaction conditions was calculated to be 88%. The product after the reaction was purified by size exclusion chromatography as needed.

[0398] Example 11. Modification of the N-terminus of a protein with a functional molecule

[0399] 11-1. Reagents, solvents, etc.

[0400] Ribonuclease A (RNase) from bovine pancreas was purchased from Roche. Ultrapure water used was purified using Millipore Integral 3. Other reagents and solvents were used as they are.

[0401] 11-2. Modification of the N-terminus of proteins with biotin

[0402] Compound 18 was used as a modifying agent and the N-terminus of the protein was modified with biotin using the same method as in Example 10-2. Figure 22 The results of LC / MS analysis of the product are shown. The modification percentage of the biotin moiety was calculated to be 79%.

[0403] 11-3. Modification of protein N-terminus with fluorescent dye

[0404] Using compound 19 as a modifying agent, the N-terminus of the protein was modified with a fluorescent dye (here, a fluorescein molecule) by the same method as in Example 10-2. Figure 23 The results of LC / MS analysis of the product are shown. The percentage of modification with respect to the fluorescent dye moiety was calculated to be 73%.

[0405] 11-4. Modification of the N-terminus of proteins with an azide group

[0406] Using Compound 21 as a modifying agent, the N-terminus of the protein was modified with an azide group by the same method as in Example 10-2. Figure 24 The results of LC / MS analysis of the product are shown. The modification percentage of the azide group was calculated to be 79%.

[0407] 11-5. Modification with functional molecules, starting with the modification of the azido group at the N-terminus of the protein

[0408] The azide-modified RNase prepared in Example 11-4 was modified with a fluorescent dye via a strain-promoted alkyne-azide cycloaddition reaction. Compound 26, a fluorescein having a dibenzocyclooctyne moiety as an alkyne substrate, was used in the reaction. Figure 25 The scenarios and results are shown.

[0409] A solution of compound 26 in dimethyl sulfoxide (DMSO) (10 mM, 1 μL, final concentration: 100 μM) was added to a phosphate buffer containing RNase-21 (RNase-21 concentration: 120 μM, final concentration: 20 μM, buffer concentration: 10 mM, pH 7.5, 82.3 μL), and the mixture was allowed to stand at 4° C. for 16 hours. The modification percentage with respect to the fluorescent dye moiety was calculated to be 55%.

[0410] 11-6. Modification of the N-terminus of proteins with strained alkyne moieties

[0411] The N-terminus of the protein was modified with a strained alkyne moiety by the same method as in Example 10-2 using Compound 22 as a modifying agent. Figure 26 The results of LC / MS analysis of the product are shown. The percentage of modification of the cyclooctyne moiety was calculated to be 40%.

[0412] Example 12. Synthesis of Resins Immobilized with Reactants for Heterogeneous Reactions

[0413] The construction of a new N-terminal selective modifier by the rearrangement reaction between compound 10 and the amine precursor shown in Example 6 can be used as a clean reaction without the use of a copper catalyst. Figure 27 A method is shown in which various formaldehyde precursors are immobilized on a resin or solid material to simplify the removal of by-products (aniline derivatives) produced after the reaction and to directly use the post-reaction solution for protein N-terminal modification.

[0414] 12-1. Equipment, reagents, solvents, etc. used

[0415] The equipment, reagents and solvents used were the same as those in Examples 1 and 8. The aminomethyl polystyrene resin used was purchased from Tokyo Chemical Industry Co., Ltd.

[0416] 12-2. Synthesis of Resin Immobilizing Reactants

[0417] As a specific example, Figure 28 The synthesis of a polystyrene resin having compound 27 immobilized thereon is shown. Figure 29 The synthesis was performed according to the scheme shown.

[0418] After dispersing aminomethyl polystyrene resin (aminomethyl / PS resin, 200 mg) in DMF (30 mL), the resin was swollen by shaking for 10 minutes. 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 the mixture was shaken overnight. The reacted resin was filtered, washed with dimethylformamide-pure water-chloroform and acetone in sequence, and dried under reduced pressure. Subsequently, to deactivate unreacted amino groups, the resulting 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 an azido-modified polystyrene resin (azido / PS resin).

[0419] After swell-forming the azido / PS resin (220 mg) with a mixture of DMF / water (6:1, 7 mL), 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 the mixture was shaken at room temperature for 12 hours. The reacted resin was filtered and washed sequentially with dimethylformamide-pure water-chloroform, methanol, and acetone. Subsequently, the resin was 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 resulting resin was treated with a mixture of aqueous hydrochloric acid / tetrahydrofuran (1:1, 5 mL) for 2 minutes each time, filtered, and washed with pure water and acetone to deprotect the acetal protecting group, thereby obtaining a resin immobilized with compound 27 (27 / PS resin).

[0420] 12-3. Identification of resin

[0421] The chemical species on the surface of the resin (27 / PS resin) on which compound 27 was immobilized were identified by infrared spectroscopy. Figure 30 The results are shown. In the azide-modified resin (azide / PS resin), the -1 In the resin (27 / PS resin) after the triazole ring was formed by CuAAC reaction, the azide stretching vibration peak disappeared, and a new peak at 1699 cm caused by the stretching vibration of the aldehyde group was observed. -1 The above results support the modification with aldehyde groups via CuAAC reaction, indicating the preparation of 27 / PS resin.

[0422] 12-4. Protein N-terminal modification

[0423] The prepared resin (27 / PS resin) immobilized with reactants was used to prepare N-terminal modifiers and subsequently modify the N-terminus of proteins. As a specific example, the N-terminus modification of ribonuclease A (RNase A) is described as follows.

[0424] 27 / PS resin (5 mg) was added to a dimethyl sulfoxide solution of benzylamine (100 mM, 50 μL, 5 μmol), and the mixture was heated at 100 ° C for 90 minutes using a heating block. After the resulting mixture was air-cooled to room temperature, the supernatant (10 μL, 1 μmol) was diluted with phosphate buffer (10 mM, pH 7.5, 85 μL). RNase A solution (1 mM, 5 μL, 5 nmol) was added thereto, and the mixture was shaken at 37 ° C for 16 hours. After the reaction, modification evaluation was performed using LC / MS. Even in the case of using a resin (27 / PS resin) fixed with a reactant, a modification percentage (75%) comparable to that of the isolated and purified N-terminal modifier was obtained. This result demonstrates a method for N-terminal modification of proteins by subsequent reactions using a resin (27 / PS resin) fixed with a reactant. Figure 31 The results are displayed.

[0425] 12-5. Improvement of the Synthesis Method of Resins Immobilizing Reactants

[0426] In Example 12-3, in the sample obtained by protein modification, a product that appeared to have added oxygen to the protein was observed. This was believed to be because the copper catalyst used for resin synthesis remained on the resin. Therefore, 27 / PS resin was prepared under a new synthesis protocol and applied to protein modification. Specifically, according to Figure 32 The protocol shown was used to prepare 27 / PS resin.

[0427] Synthesis of 1,2-5-1,5-azido-2-nitrobenzoic acid (28)

[0428]

[0429] An aqueous solution (6 mL) of sodium nitrite (727 mg, 10.5 mmol) was added to a concentrated HCl / EtOH / H2O solvent mixture (2:1:2, 43 mL in total) containing 5-amino-2-nitrobenzoic acid (1.60 g, 8.9 mmol), and the mixture was stirred at 0°C for 1 hour. Subsequently, sodium azide (868 mg, 13.4 mmol) was added portionwise, and the solution was vigorously stirred at 0°C for 1 hour, and then at room temperature for 1 hour.

[0430] The reaction solution was diluted with ultrapure water, filtered, washed with ultrapure water (20 mL x 2), and dried under reduced pressure to obtain Compound 28 (pale yellow solid). Figure 33 Shows 1 H NMR spectrum.

[0431] Yield: 37% 1 H NMR (400MHz, DMSO-d6): δ, 8.1 (d, J = 9.4Hz, 3H), 7.43-7.41 (m, 2H); 13 C NMR (100 MHz, DMSO-d6): δ, 165.8, 145.3, 143.4, 130.8, 126.2, 121.7, 119.6; ESI-TOFMS (positive mode) calculated for C7H4NaN4O4 [M+Na] + 231.012m / z, obtained as 231.011.

[0432] Synthesis of 12-5-2,2,5-dioxopyrrolidin-1-yl-5-(4-formyl-1H-1,2,3-triazol-1-yl)-2-nitrobenzoate (30)

[0433]

[0434] 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 tert-butanol (21 mL). Copper (II) sulfate pentahydrate (419 mg, 1.68 mmol, 20 mol%) and sodium ascorbate (666 mg, 3.36 mmol, 40 mol%) were added, and the mixture was stirred overnight at 70°C under a nitrogen atmosphere. After cooling the reaction solution to room temperature, saturated brine was added to terminate the reaction, followed by extraction with ethyl acetate (20 mL x 3). The organic layer was dried over sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain a crude product containing compound 29. Purification was omitted and the crude product was used directly in the next reaction.

[0435] N-hydroxysuccinimide (764 mg, 6.64 mmol) was added to a DMF solution (22 mL) containing the crude product, and the mixture was stirred at room temperature for 10 minutes. Subsequently, N, N'-dicyclohexylcarbodiimide (1.14 g, 5.5 mmol) was added, and the reaction mixture was stirred overnight. The precipitate was filtered out, 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 out again. The resulting filtrate was washed with saturated brine (20 mL × 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 purified by silica gel column chromatography. Figure 34 Shows 1 H NMR spectrum.

[0436] Yield 18% (after two steps); 1 H NMR (400MHz, DMSO-d6): 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-d6): 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) calculated for C 14 H9NaN5O7[M+Na] + 382.038)m / z, and found 382.039.

[0437] 12-5-3.27 / PS resin synthesis 2

[0438] After dispersing aminomethyl polystyrene resin (aminomethyl / PS resin, 200 mg) in DMF (5 mL), the resin was swollen by shaking for 1 hour. Compound 30 (150 mg, 0.42 mmol) and N,N'-diisopropylcarbodiimide (146 μL, 0.84 mmol) were added to the resin dispersion, and the mixture was shaken overnight. The reacted resin was filtered, washed with dimethylformamide-pure water-chloroform and acetone in sequence, and dried under reduced pressure. Subsequently, in order to deactivate the unreacted amino groups, the obtained 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.

[0439] 12-6. Protein N-terminal modification

[0440] The N-terminal modification agent and subsequent protein N-terminal modification were prepared using a freshly prepared resin (27 / PS resin) immobilized with reactants. As a specific example, ribonuclease A (RNase A) N-terminal modification was described below. Figure 35 The scheme is shown.

[0441] 27 / PS resin (5 mg) was added to a dimethyl sulfoxide solution of benzylamine (100 mM, 40 μL, 4 μmol), and the mixture was heated at 100 ° C for 90 minutes using a heating block. After the resulting mixture was air-cooled to room temperature, the supernatant (10 μL, 1 μmol) was diluted with phosphate buffer (10 mM, pH 7.5, 85 μL). RNase A solution (1 mM, 5 μL, 5 nmol) was added thereto, and the mixture was shaken at 37 ° C for 16 hours. After the reaction, modification evaluation was performed using LC / MS. Figure 36 shows the results. Even when using a resin (27 / PS resin) fixed with the reactant, a modification percentage (82%) comparable to that using an isolated and purified N-terminal modifier was obtained. In addition, for various functional amine compounds, it was demonstrated that the protein N-terminus can be specifically modified by the same method.

[0442] Example 13. Synthesis of protein modifiers and subsequent protein synthesis using the Dimrot rearrangement reaction in a homogeneous system Quality Modification

[0443] The construction of a new N-terminus-selective modifier via a rearrangement reaction between compound 10 and the amine precursor shown in Example 6 can be used as a clean reaction without the use of a copper catalyst. Example 12 demonstrates a Dimrut rearrangement reaction in a heterogeneous system to simplify byproduct removal. On the other hand, it is also speculated that there are cases where the presence of aniline derivatives as byproducts does not pose a problem for protein modification. Therefore, this section demonstrates the synthesis of formaldehyde derivatives and subsequent protein modification in a Dimrut rearrangement reaction in a homogeneous system. Figure 37 The scheme is shown.

[0444] 13-1. Reagents, solvents, etc.

[0445] The equipment, reagents, and solvents used were similar to those in Examples 1 and 8. Ribonuclease A (RNase) from bovine pancreas was purchased from Roche. Ultrapure water used was purified using Millipore Integral 3.

[0446] 13-2. Optimization of Dimrot rearrangement reaction conditions

[0447] Figure 38The reaction mechanism of the Dimrut rearrangement reaction is shown. Since this reaction involves imine formation as the first step, the addition of an acid catalyst can be expected to improve the reaction efficiency. Therefore, the Dimrut rearrangement reaction with compound 10 was carried out using benzylamine as a model substrate in the presence of various acid catalysts.

[0448] A solution of compound 10 in dimethyl sulfoxide (200 mM, 20 μL, 4 μmol) and an acidic aqueous solution (200 mM or 400 mM, 1 μL, 5 mol% or 10 mol%) were added to a solution of benzylamine in dimethyl sulfoxide (200 mM, 20 μL, 4 μmol), and the mixture was heated at 100 ° C for 30 minutes using a heating block. After air cooling to room temperature, 1 μL of the reaction solution was diluted with phosphate buffer (100 mM, pH 7.0, 200 μL) and transferred to a 96-well plate for UV-visible absorption measurement. The reaction conversion was calculated based on a standard curve drawn based on the absorption intensity at 380 nm, which is characteristic of p-nitroaniline (a product). Figure 39 The structures of the acid catalysts used are shown, and Table 1 shows the conversions obtained.

[0449] In Table 1, the conversion rate a It is calculated based on the absorption intensity of the by-product p-nitroaniline. b The addition amount of MOPS was set to 10 mol%.

[0450] Table 1

[0451]

[0452] The above results indicate that the addition of an acid catalyst improves conversion. It is suggested that the addition of sulfonic acid is particularly effective. Furthermore, various sulfonates known to function as Gould buffers have been found to improve reaction efficiency. Generally speaking, since these sulfonates have no significant effect on protein function or structure, the mixed solution from the Dimrot rearrangement reaction can be used directly in subsequent protein modification reactions. 3-Morpholinopropanesulfonic acid (MOPS) was selected as the acid catalyst for subsequent experiments.

[0453] 13-3. Protein N-terminal modification

[0454] A solution of compound 10 in dimethyl sulfoxide (200mM, 20μL, 4μmol) and a MOPS aqueous solution (200mM or 400mM, 1μL, 5mol% or 10mol%) were added to a solution of benzylamine in dimethyl sulfoxide (200mM, 20μL, 4μmol), and the mixture was heated at 100°C for 30 minutes using a heating block. After air cooling to room temperature, the reaction mixture (5μL) was diluted with phosphate buffer (10mM, pH 7.5, 42.5μL). RNase A solution (1mM, 2.5μL, 5nmol) was added thereto, and the mixture was shaken at 37°C for 16 hours. After the reaction, modification evaluation was performed using LC-MS. Figure 40 The results of LC-MS analysis are shown.

[0455] Protein modifiers were prepared using various functional amine precursors (e.g., alkynes, azides, and fluorescent dyes) and introduced into the N-terminus of proteins. Furthermore, even when the preparation of the N-terminal modifier and the protein modification reaction were performed sequentially, the modification percentage was comparable to that using isolated and purified N-terminal modifiers. The results demonstrate a method for modifying the N-terminus of proteins using sequential reactions of the Dimrot rearrangement reaction in a homogeneous system.

[0456] Example 14. Modification of the N-terminus of a protein using Bis-TA4C molecules and introduction of functional molecules via oxime formation

[0457] In the Dimrut rearrangement using a diamine having amino groups at both ends as a precursor, a molecule having a TA4C moiety introduced at both ends (hereinafter 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 into the N-terminus of the protein. An example of a chemical modification reaction starting from an aldehyde includes the formation of an oxime with hydroxylamine. This reaction is also suitable for protein modification because it can be carried out even in water under mild conditions. In addition, since the oxime bond is a dynamic bond, the functional molecule can also be removed as needed. Therefore, by using various diamines as precursors, a protein modification reaction is carried out by carrying out a Dimrut rearrangement reaction using compound 10.

[0458] 14-1. Reagents, solvents, etc.

[0459] The equipment, reagents, and solvents used were similar to those in Examples 1 and 8. Ribonuclease A (RNase) from bovine pancreas was purchased from Roche. Ultrapure water used was purified using Millipore Integral 3.

[0460] 14-2. Preparation of Bis-TA4C and Protein Modification Reaction

[0461] Compound 10 solution (200mM, 20 μL, 4 μmol) and acidic aqueous solution (200mM or 400mM, 1 μL, 5mol% or 10mol%) are added to each of diamines 31-36 in dimethyl sulfoxide (100mM, 20 μL, 2 μmol) in a separate solution, and the mixture is heated separately at 90 ° C for 60 minutes using a heating block. After air cooling to room temperature, each reaction mixture (5 μL) is diluted with phosphate buffer (10mM, pH 7.5, 42.5 μL). RNase A solution (1mM, 2.5 μL, 5nmol) is added thereto, followed by shaking at 37 ° C for 16 hours. After the reaction, LC-MS is used to perform modification evaluation. Figure 41 Shows the plan, Figure 42 Results for protein modifications are shown.

[0462] When Bis-TA4C with an alkyl chain or oligoethylene glycol chain as a linker was used, a good amount of modified protein (60% to 82%) was obtained. In contrast, Bis-TA4C containing an aromatic backbone such as aniline did not undergo modification. This is believed to be due to the very low solubility of Bis-TA4C. Therefore, the introduction of a moiety that improves water solubility is expected to increase the reaction yield.

[0463] 14-3. Introduction of functional molecules into proteins modified with Bis-TA4C through oxime formation

[0464] The oxime formation reaction starts from the aldehyde moiety introduced into the N-terminus of the protein with Bis-TA4C. Specifically, the reaction is carried out according to Figure 43 The procedure shown in was performed with reference to a published report (M. Rashidian, MM Mahmoodi, R. Shah, JK Dozier, CR Wagner, MD Distefano, Bioconjugate Chem. 2013, 24, 333-342). As an example, modification using fluorescent dyes and polyethylene glycol is described below.

[0465] Bis-TA4C modified RNase A (60 μM, 8.3 μL, 0.5 nmol) prepared using compound 35 as a precursor was diluted with phosphate buffer (50 mM, pH 7.0, 35.7 μL), and a dimethyl sulfoxide solution containing hydroxylamine 37 (5 mM, 1 μL, 5 nmol) and an aqueous solution of m-phenylenediamine (m-PDA) as a catalyst (50 mM, 5 μL, 0.25 μmol) were added thereto, followed by shaking at 4°C for 6 hours. After the reaction, modification was evaluated using LC-MS. Figure 44The results of protein modification are shown. Although some adducts of the catalyst m-PDA were observed, conversion to the oxime adduct was confirmed in good yield.

[0466] Bis-TA4C-modified RNase A (60 μM, 8.3 μL, 0.5 nmol) prepared using compound 35 as a precursor was diluted with phosphate buffer (50 mM, pH 7.0, 35.7 μL); hydroxylamine 38 (2 mg, 500 nmol) and a m-phenylenediamine (m-PDA) aqueous solution (50 mM, 5 μL, 0.25 μmol) as a catalyst were added, and then shaken at room temperature for 16 hours. After the reaction, modification was evaluated using SDS-PAGE. Figure 45 Displays the results of protein modifications.

[0467] For comparison, experiments were also performed using unmodified proteins or polyethylene glycol with a hydroxyl group as substrates. Band shifts on SDS-PAGE were observed only for combinations of the aldehyde group at the protein N-terminus and the hydroxylamine moiety in the polyethylene glycol, confirming the incorporation of the polyethylene glycol via oxime formation.

[0468] Example 15. Dimrut rearrangement reaction under mild conditions

[0469] The preparation of N-terminal modifiers via the Dimrut rearrangement reaction in the homogeneous system shown in Examples 13 and 14 requires high reaction temperatures of 90°C to 100°C. Therefore, further improvements in the precursor structure and reaction conditions are needed to accommodate substrates with low heat resistance. In order to achieve the reaction at lower temperatures, the structure of compound 10, which serves as a precursor, was modified. In particular, compounds in which the nitrophenyl moiety is changed to a substituent with higher electron-withdrawing properties are expected to serve as useful precursors because the intermediates are stable and the reverse reaction is less likely to proceed. This section describes examples of the synthesis of compounds according to this strategy and its application to the Dimrut rearrangement reaction.

[0470] 15-1. Compound Synthesis

[0471] As a specific structural example, the synthesis of triazole carboxaldehyde 40 having a 4-cyanotetrafluorophenyl group is described below.

[0472]

[0473] Synthesis of 15-1-1.4-(4-(diethoxymethyl)-1H-1,2,3-triazol-1-yl)-2,3,5,6-tetrafluorobenzonitrile (39)

[0474] An acetonitrile solution (10 mL) containing pentafluorobenzonitrile (850 mg, 541 μL, 4.4 mmol) and sodium azide (260 mg, 4.0 mmol) was stirred at 60 ° C for 16 hours under a nitrogen atmosphere. Subsequently, propargylaldehyde diethyl acetal (570 μL, 4.0 mmol) and copper (I) iodide (76 mg, 0.4 mmol) were added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 16 hours. The reaction solution was air-cooled to room temperature, then diluted with saturated sodium chloride aqueous solution (40 mL), and then extracted with ethyl acetate (50 mL×3). The obtained organic layer was dried over magnesium sulfate, and the filtrate from which the solid was filtered out was distilled under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography to give compound 39 (white solid). Figure 46 Shows 1 H NMR spectrum.

[0475] Yield: 86%; 1 H-NMR (400MHz, CDCl3): δ7.97 (s, 1H), 5.82 (s, 1H), 3.79-3.64 (m, 4H), 1.28 (t, J = 7.1Hz, 6H).

[0476] 15-1-12. Synthesis of 2,3,5,6-tetrafluoro-4-(4-formyl-1H-1,2,3-triazol-1-yl)benzonitrile (40)

[0477] Compound 39 (688 mg, 2.0 mmol) was dissolved in chloroform (4 mL). Trifluoroacetic acid (2 mL) was added thereto, 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. The crude product was purified by reprecipitation (hexane: chloroform) to obtain compound 40 (white solid). Figure 47 Shows 1 H NMR spectrum.

[0478] Yield: 78%; 1 H-NMR (400MHz, CDCl3): δ10.26 (s, 1H), 8.50 (s, 1H).

[0479] 15-2. Dimrot rearrangement reaction using compound 40 as a precursor

[0480] To evaluate the reactivity of compound 40, a Dimrot rearrangement reaction was performed using benzylamine as a substrate.

[0481] 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 heating block. After air cooling to room temperature, the reaction mixture was diluted with deuterated dimethyl sulfoxide (310 μL). As an internal standard, a deuterated dimethyl sulfoxide solution of 1,3,5-trimethoxybenzene (400 mM, 10 μL, 4 mol) was added and the mixture was heated to 40 ° C for 12 hours. 1 H NMR measurement: The yield was calculated from the integrated value of the peak corresponding to the proton at the benzyl position of the product compound 7 based on the integrated value of the internal standard. Figure 48 Indicates the plan, Figure 49 The results are shown.

[0482] The results showed that the Dimrut rearrangement reaction proceeded at a low temperature with good yield when using compound 40, compared with the case of using compound 10. This suggests that the introduction of an electron-withdrawing substituent at the N1 position of the triazole ring significantly contributes to the improved reactivity of the Dimrut rearrangement reaction.

[0483] Example 16. Protein N-terminal modification 2

[0484] Human serum albumin (HSA), which is commonly used as a protein substrate, was subjected to N-terminal modification reaction.

[0485] 16-1. Reagents, solvents, etc.

[0486] Human serum albumin (HSA) was purchased from Merck. The ultrapure water used was purified using a Millipore Integral 3. Commercially available products were used as other reagents and solvents. A mixture of HSA with HSA that had been modified in vivo was used as HSA. Modified HSA has been identified in published reports (A. Kawakami, K. Kubota, N. Yamada, U. Tagami, K. Takehana, I. Sonaka, E. Suzuki, K. Hirayama, FEBS J., 2006, 273, 3346-3357).

[0487] 16-2 Protein modification

[0488] This method targets the N-terminus of proteins. Proteins that can be targeted 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, the N-terminal modification of human serum-derived albumin (HSA) is described below.

[0489] The following is the amino acid sequence of HSA (PDB: 1006).

[0490]

[0491] Compound 7 was used as the modifying agent and HSA was used as the target protein. The protein N-terminus was modified using the same method as in Example 10-2. Figure 50 The results of LC / MS analysis of the product are shown. The modified protein is designated "HSA1." The modification percentage under these reaction conditions was calculated to be 99% or greater. The product after the reaction was purified by size exclusion chromatography, as needed.

[0492] Example 17. Dual modification of proteins targeting the N-terminus and cysteine ​​residues

[0493] 17-1. Reagents, solvents, etc.

[0494] The equipment, reagents, solvents, etc. used are similar to those in Example 16.

[0495] 17-2. Modification of Cysteine ​​Residues in Proteins

[0496] The cysteine ​​residues in proteins were modified with reference to a published report (X. Chen, H. Wu, C.-M. Park, T. H. Poole, G. Keceli, N. O. Devarie-Baez, A. W. Tsang, W. T. Lowther, L. B. Poole, S. B. King, M. Xian, C. M. Furdui, ACS Chem. Biol., 2017, 12, 2201-2208). The specific experimental procedures are described below.

[0497] A solution of compound 41 in a DMSO / water (1:1) mixture (25 mM, 40 μL, 1 μmol, final concentration: 500 μM) was diluted with phosphate buffer (100 mM, pH 7.0, 1.86 mL). An ultrapure aqueous solution of HSA (1 mM, 100 μL, 100 nmol, final concentration: 50 μM) was added thereto, and the mixture was allowed to stand at 4°C for 12 hours. After the reaction, modification evaluation was performed using LC / MS. Figure 51 The results are shown. The modified protein is designated "HSA2." The modification percentage under these reaction conditions was calculated to be 99% or higher. The product after the reaction was purified by size exclusion chromatography as needed.

[0498] 17-3. N-terminal modification of proteins in which cysteine ​​residues are modified

[0499] Compound 7 was used to modify the N-terminus of HSA2 prepared in Example 17-2. Figure 52 The scenarios and results are shown.

[0500] A solution of compound 7 in dimethyl sulfoxide (DMSO) (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 the mixture was shaken at 37°C for 16 hours. The modified protein is designated "HSA3." The percentage of modification under these reaction conditions was calculated to be 72%.

[0501] Example 18. Stability of proteins modified at the N-terminus

[0502] 18-1. Reagents, solvents, etc.

[0503] The equipment, reagents, solvents, etc. used are similar to those in Example 10.

[0504] 18-2. Temporal Stability of Modified Proteins

[0505] 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 allowed to stand at 37°C for 12 hours, 24 hours, and 48 hours. For example, Figure 53 The results of LC / MS analysis after standing for 24 hours are shown.

[0506] This modification reaction involving the formation of a 4-imidazolidinone ring at the N-terminus is an equilibrium reaction. From the results of LC / MS analysis, it can be assumed that the unmodified protein and the modifying agent are regenerated by hydrolysis of the 4-imidazolidinone ring in the N-terminus-modified protein, which is the reverse reaction. Figure 54 A graph is shown, wherein the vertical axis represents the release amount calculated from the peak intensity in the mass spectrum (=1-(modified RNase after standing / total RNase amount) / (modified RNase before standing / total RNase amount)); the horizontal axis represents the standing time of the RNase.

[0507] 18-3. Stability of modified proteins with pH changes

[0508] 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 adjust the pH to 4, 5, 6, 7, or 8 and incubated at 37°C for 12 hours. For pH 4 and 5, dilutions were performed using acetate buffer, while for pH 6, 7, and 8, dilutions were performed using phosphate buffer. Figure 55 The results of the release are shown.

[0509] Example 19. Synthesis of Compound 8 (Synthesis of Triazole Carboxaldehyde with Substitution at the 1- and 5-Positions)

[0510] The reagents and solvents used in the synthesis were directly commercially available. The azide compound, alkyne compound, and boronate pinacol ester used as precursors were obtained from published reports (LSC-Verduyn, L. Mirfeizi, RA Dierckx, PH Elsinga, BL Feringa, Chem. Commun., 2009, 16, 2139-2140; E. Jahnke, J. Weiss, S. Neuhaus, TN Hoheisel et al ... H.Frauenrath,Chem.Eur.J.,2009,15,388-404;JCPieck,D.Kuch,F.Grolle,U.Linne,C.Haas,T.Carell,J.Am.Ch em.Soc.,2006,128,1404-1405; 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,GJPrice,S.Schiffers,PRRaithby,PKPlucinski,CGFrost,Tetrahedron Letters, 2010, 51, 3913-3917).

[0511] Synthesis of 19-1.1-Benzyl-5-phenyl-1H-1,2,3-triazole-4-carbaldehyde (44)

[0512] Compound 44 was synthesized according to the following scheme, with reference to published reports (K. Yamamoto, T. Bruun, J. Y. 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).

[0513]

[0514] Synthesis of 19-1-(1,1-benzyl-5-iodo-1H-1,2,3-triazol-4-yl)methanol (42)

[0515] Under a nitrogen atmosphere, THF (89 mL) was added to a mixture of benzyl azide (0.67 g, 5.0 mmol), 3-iodoprop-2-ynyl-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), and the mixture was stirred at room temperature overnight. The solvent of the reaction mixture was removed by distillation 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 × 3). The resulting organic layer was washed with saturated brine (30 mL × 2) and dried over sodium sulfate, and the solvent of the filtrate was removed by distillation under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate) to obtain compound 42 (white solid). Figure 56 Shows 1 H NMR spectrum.

[0516] Yield 82%: 1 H NMR (400MHz, DMSO-d6): δ,7.42-7.33(m,3H),7.24-7.22(m,2H),5.65(s,2H),5.27(t,J=5.6Hz,1H),4.47(d,J=5.6Hz,2H): 13 C NMR (100 MHz, DMSO-d6): δ, 151.30, 135.52, 128.89, 128.20, 127.54, 83.79, 54.99, 53.25: ESI-TOF MS (positive mode) calculated for C 10 H 10 IN3NaO[M+Na] + 337.976m / z, obtained as 337.977.

[0517] 19-1-2. Synthesis of (1-benzyl-5-phenyl-1H-1,2,3-triazol-4-yl)methanol (43)

[0518] Pure water (2.4 mL) was added 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). Thereafter, THF (9.6 mL) was added under a nitrogen atmosphere, and the mixture was stirred at 70 ° C overnight. The solvent of the reaction mixture was distilled off under reduced pressure, diluted with water (30 mL) and ethyl acetate (30 mL), and then extracted with ethyl acetate (30 mL×3). The obtained organic layer was washed with saturated brine (30 mL×2) and dried over sodium sulfate, and the solvent of the filtrate obtained after filtration was distilled off under reduced pressure. The residue was purified by flash column chromatography (hexane: ethyl acetate) to obtain compound 43 (light yellow solid). Figure 57 Shows 1 H NMR spectrum.

[0519] Yield: 54% 1 H NMR (400 MHz, CDCl3): 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) calculated for C 16 H 15 N3NaO[M+Na] + 288.111m / z, found 288.112.

[0520] 19-1-3. Synthesis of 1-benzyl-5-phenyl-1H-1,2,3-triazole-4-carbaldehyde (44)

[0521] Activated manganese dioxide (163 mg, 1.9 mmol) was added to a solution (10 mL) of compound 43 (50 mg, 0.19 mmol) in 1,4-dioxane, and the mixture was stirred at room temperature overnight. The reaction mixture was filtered and the solvent of the filtrate was removed by distillation under reduced pressure. Afterwards, the crude product was purified by flash column chromatography (hexane: ethyl acetate) to give compound 44 (oil). Figure 58 Shows 1 H NMR spectrum.

[0522] Yield 91%; 1H NMR (400 MHz, CDCl3): 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) calculated C 16 H13 NaN3O[M+Na] + 286.095m / z, obtained as 286.093.

[0523] 19-1-4. Synthesis of (1-benzyl-5-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methanol (45)

[0524]

[0525] Compound 45 (light yellow solid) was synthesized by the same method as in Example 19-1-2 using 4-methoxyphenylboronic acid as a precursor. Figure 59 Shows 1 H NMR spectrum.

[0526] Yield 97%; 1 H NMR (400MHz, CDCl3): δ, 7.29-7.27 (m, 3H), 7.17 (d, J = 8.8Hz, 2H), 7.08-7.07 (m, 2H), 6.95 (d,J=8.8Hz,2H),5.46(s,2H),4.67(d,J=6.0Hz,2H),3.85(s,3H),2.09(t,J=6.0Hz,1H): 13 C NMR (100 MHz, CDCl3): 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) calculated for C 17 H 17 N3NaO2[M+Na] + 318.121m / z, obtained as 318.121.

[0527] Synthesis of 19-1-5.1-Benzyl-5-(4-methoxyphenyl)-1H-1,2,3-triazole-4-carbaldehyde (46)

[0528]

[0529] Compound 46 (oil) was synthesized using compound 45 as a precursor by the same method as in Example 19-1-3. Figure 60 Shows 1 H NMR spectrum.

[0530] Yield 91%: 1H NMR (400MHz, CDCl3): δ, 10.14 (s, 1H), 7.32-7.30 (m, 3H), 7.23 (d, J = 8.8Hz, 2H), 7.10-7.08 (m, 2H), 6.99 (d, J = 8.8Hz, 2H), 5.49 (s, 2H), 3.87 (s, 3H): 13 C NMR (100 MHz, CDCl3): 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) calculated for C 17 H 15 N3NaO2[M+Na] + 316.106m / z, obtained as 316.104.

[0531] 19-1-6. Synthesis of (1-benzyl-5-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)methanol (47)

[0532]

[0533] Compound 47 (brown solid) was synthesized by the same method as in Example 19-1-2 using 4-nitrophenylboronic acid as a precursor. Figure 61 Shows 1 H NMR spectrum.

[0534] Yield 58%: 1 H NMR (400MHz, CDCl3): δ, 8.28 (d, J = 8.8Hz, 2H), 7.46 (d, J = 8.8Hz, 2H), 7.31-7.29 (m ,3H),7.05-7.03(m,2H),5.52(s,2H),4.69(d,J=6.0Hz,2H),2.14(t,J=6.0Hz,1H): 13 C NMR (100 MHz, CDCl3): 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) calculated for C 16 H 14 N4NaO3[M+Na] + 333.096m / z, obtained 333.095.

[0535] 19-1-7. Synthesis of 1-benzyl-5-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde (48)

[0536]

[0537] Compound 48 (yellow solid) was synthesized using compound 47 as a precursor by the same method as in Example 19-1-3. Figure 62 Shows 1 H NMR spectrum.

[0538] Yield 93%: 1 H NMR (400MHz, CDCl3): δ, 10.20 (s, 1H), 8.31 (d, J = 8.8Hz, 2H), 7.43 (d, J = 8.8Hz, 2H), 7.33-7.28 (m, 3H), 7.04-7.02 (m, 2H), 5.52 (s, 2H): 13 C NMR (100 MHz, CDCl3): 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) calculated for C 16 H 12 N4NaO3[M+Na] + 331.080m / z, obtained as 331.082.

[0539] 19-1-8. Synthesis of 1-(4-((4-(hydroxymethyl)-5-iodo-1H-1,2,3-triazol-1-yl)methyl)phenyl)ethan-1-one (49)

[0540]

[0541] Compound 49 (white solid) was synthesized by the same method as in Example 19-1-1 using 1-(4-(azidomethyl)phenyl)ethan-1-one as a precursor. Figure 63 Shows 1 H NMR spectrum.

[0542] Yield 40%: 1 H NMR (400MHz, CDCl3): δ, 7.94 (d, J = 8.3Hz, 2H), 7.33 (d, J = 8.3Hz, 2H), 5.65 (s, 2H), 4.74 (s, 2H), 2.59 (s, 3H), 2.15 (s, 3H); 13C NMR (100 MHz, CDCl3): 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) calculated for C 12 H 12 IN3NaO2[M+Na] + 379.987m / z, obtained as 379.987.

[0543] 19-1-9. Synthesis of 1-(4-((5-(4-(azidomethyl)phenyl)-4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)ethan-1-one (50)

[0544]

[0545] Compound 50 (pale yellow solid) was synthesized by the same method 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. Figure 64 Shows 1 H NMR spectrum.

[0546] Yield 71%: 1 H NMR (400MHz, CDCl3): δ,7.87(d,J=8.0Hz,2H),7.40(d,J=8.0Hz,2H),7.26(d,J=8.0Hz,2H),7.13(d,J =8.0Hz,2H),5.54(s,2H),4.69(d,J=5.8Hz,2H),4.42(s,2H),2.57(s,3H),2.17(t,J=5.8Hz,1H); 13C NMR (100 MHz, CDCl3): 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) calculated for C 19 H 18 N6NaO2[M+Na] + 385.138m / z, obtained as 385.138.

[0547] 19-1-10. Synthesis of 1-(4-acetylbenzyl)-5-(4-(azidomethyl)phenyl)-1H-1,2,3-triazole-4-carbaldehyde (51)

[0548]

[0549] Compound 51 (clear, oily) was synthesized using compound 50 as a precursor by the same method as in Example 19-1-3. Figure 65 Shows 1 H NMR spectrum.

[0550] Yield 85%: 1 H NMR (400 MHz, CDCl3): 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); 13C NMR (100 MHz, CDCl3): 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) calculated for C 19 H 16 N6NaO2[M+Na] + 383.123m / z, obtained as 383.123.

[0551] Example 20. Peptide N-terminal modification 3

[0552] The N-terminus of the peptide was modified with reference to a published report (JI MacDonald, HK Munch, T. Moore, MB Francis, Nat. Chem. Biol. 2015, 11, 326-331). The specific experimental process is described below.

[0553] A solution of compound 44 in dimethyl sulfoxide (DMSO) (200 mM, 2 μL, 0.4 μmol, final concentration: 10 mM) was diluted with phosphate buffer (10 mM, pH 7.5, 34 μL). An aqueous peptide solution (1 mM, 4 μL, 4 nmol, final concentration: 100 μM) was added thereto, and the mixture was shaken at 37°C for 16 hours. The percentage of modification (=(modified peptide amount) / (total peptide amount)) was assessed by using LC / MS from the peak intensity in the mass spectrum. Figure 66 The results are shown. The percent modification under these reaction conditions was calculated to be >99%.

[0554] Example 21. Protein N-terminal modification 4

[0555] Protein N-terminal modification was performed with reference to a published report (JI MacDonald, HK Munch, T. Moore, MB Francis, Nat. Chem. Biol. 2015, 11, 326-331). The specific experimental procedure is described below.

[0556] Compound 44 solution (200mM, 5 μ L, 1.0 μ mol, final concentration: 10mM) is diluted with phosphate buffer (10mM, pH 7.5, 90 μ L). An ultrapure aqueous solution of RNase (1mM, 5 μ L, 5nmol, final concentration: 50 μ M) is added thereto and shaken at 37°C for 16 hours. The peak intensity from mass spectrometry is assessed using LC / MS for percentage modification (=(RNase modified) / (total RNase amount)). Figure 67 The results are shown. The percentage of modification under these reaction conditions was calculated to be 48%.

[0557] Example 22. Modification of the N-terminus of a protein with a functional molecule

[0558] 22-1. Double modification of protein N-termini with acetyl and azide groups

[0559] The N-terminus of the protein was doubly modified with an acetyl group and an azide group by the same method as in Example 21 using Compound 51 as a modifying agent. Figure 68 The LC / MS analysis results of the product are shown. The modification percentage of the acetyl and azide groups was calculated to be 28%.

[0560] 22-2. Modification with functional molecules, starting with the modification of the azido group at the N-terminus of the protein

[0561] The azide-modified RNase prepared in Example 22-1 was modified with a fluorescent dye via a strain-promoted alkyne-azide cycloaddition reaction. Compound 26, a fluorescein having a dibenzocyclooctyne moiety as an alkyne substrate, was used in the reaction. Figure 69 The scenarios and results are shown.

[0562] A solution of compound 26 in dimethyl sulfoxide (DMSO) (8 mM, 1 μL, final concentration: 400 μM) was added to a phosphate buffer containing RNase-51 (RNase-51 concentration: 76 μM, final concentration: 20 μM, buffer solution concentration: 10 mM, pH 7.5, 19 μL), and the mixture was allowed to stand at room temperature for 6 hours. The modification percentage of the fluorescent dye moiety was calculated to be >99%.

[0563] Example 23. Introducing polymer molecules into the N-terminus of proteins

[0564] 23-1. Synthesis of polyethylene glycol with triazole carboxaldehyde moiety

[0565] Polyethylene glycol 54, into which a triazole carboxaldehyde moiety was introduced, was synthesized according to the following scheme with reference to published reports (L. Rocard, A. Berezin, F. De Leo, D. Bonifazi, Angew. Chem. Int. Ed., 2015, 54, 15739-15743; and M.B. van Eldijk, FCM Smit, N. Vermue, M.F. Debets, S. Schoffelen, J.C.M. van Hest, Biomacromolecules, 2014, 15, 2751-2759).

[0566]

[0567] Synthesis of 2,3-1-(4-formyl-1H-1,2,3-triazol-1-yl)methyl)benzoic acid (52)

[0568] Compound 52 (white solid) was synthesized by using the same method as in Example 1-3-1 (Method A) using (4-azidomethyl)benzoic acid as a precursor. Figure 70 Shows 1 H NMR spectrum.

[0569] Yield: 71% 1 H NMR (400MHz, DMSO-d6): δ, 10.02 (s, 1H), 9.00 (s, 1H), 7.94 (d, J = 8.0Hz, 2H), 7.43 (d, J = 8.0Hz, 2H), 5.79 (s, 2H); 13 C NMR (100 MHz, DMSO-d6): δ, 185.0, 166.9, 147.1, 140.1, 130.7, 129.9, 128.7, 128.2, 52.8; ESI-TOF MS (positive mode) calculated for C 11 H9N3O3Na[M+Na] + 254.054m / z, obtained as 254.053.

[0570] Synthesis of 2,3-1-2,2,5-dioxopyrrolidin-1-yl-4-((4-formyl-1H-1,2,3-triazol-1-yl)methyl)benzoate (53)

[0571] 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 compound 52 (231 mg, 1.0 mmol), and the mixture was stirred at 0°C under a nitrogen atmosphere for 24 hours. The precipitate was filtered off, and the filtrate was diluted with ethyl acetate (50 mL) and washed with saturated sodium bicarbonate aqueous solution (20 mL x 2) and saturated brine (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. The crude product was purified by silica gel column chromatography to obtain compound 53 (white solid). Figure 71 Shows 1 H NMR spectrum.

[0572] Yield: 71% 1 H NMR (400MHz, CDCl3): δ, 10.15 (s, 1H), 8.16 (d, J = 8.5Hz, 2H), 8.07 (s, 1H), 7.42 (d, J = 8.5Hz, 2H), 5.70 (s, 2H), 2.91 (s, 4H); 13 C NMR (100 MHz, CDCl3): 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) calculated for C 15 H 12 N4O5Na[M+Na] + 351.070m / z, found 351.069.

[0573] 23-1-2. Synthesis of polyethylene glycol-tethered triazole-4-carboxaldehyde (54)

[0574] Amino-terminated polyethylene glycol (molecular weight: approximately 4000, 400 mg, 0.1 mmol) and triethylamine (45 L, 0.32 mmol) were added to dichloromethane (15 mL) containing compound 53 (53 mg, 0.16 mmol), and the mixture was stirred under a nitrogen atmosphere for 24 hours. After the solvent was removed by distillation under reduced pressure, the residue was purified by silica gel column chromatography. The resulting crude oil was purified by reprecipitation (ether / dichloromethane) to obtain compound 54 (white solid).

[0575] 23-2. Modification of protein N-terminus using compound 54

[0576] The N-terminus of the protein was modified with polyethylene glycol using compound 54. The following is an example using ribonuclease as a substrate.

[0577] 23-2-1. Reagents, solvents, etc.

[0578] Ribonuclease A (RNase) from bovine pancreas was purchased from Roche. Ultrapure water used was purified using Millipore Integral 3. Other reagents and solvents were used as they are.

[0579] 23-2-2. Polyethylene glycol modification of protein N-terminus

[0580] Compound 54 was used as a modifying agent and the N-terminus of the protein was modified with polyethylene glycol by the same method as in Example 10-2. Figure 72 The results of SDS-PAGE analysis of the products are shown. The modification percentage of the polyethylene glycol moiety was calculated to be 50%.

[0581] 23-2-3. Modification of the N-terminus of a protein with a polymer, wherein the cysteine ​​residue is modified

[0582] The N-terminus of HSA2 was modified with polyethylene glycol using compound 54 as a modifier, using the same method as in Example 17-3. The resulting HSA (abbreviated as "HSA4"), in which the cysteine ​​residue was modified with an azide group and the N-terminus was modified with polyethylene glycol, was then modified with a fluorescent dye via a strain-promoted alkyne-azide cycloaddition reaction using compound 26. The modified protein was designated "HSA5."

[0583] A dimethyl sulfoxide (DMSO) solution of compound 26 (6 mM, 1 μL, final concentration: 300 μM) was added to a phosphate buffer containing HSA4 (HSA4 concentration: 62 μM, final concentration: 10 μM, buffer concentration: 10 mM, pH 7.5, 19 μL), and the mixture was allowed to stand at 4°C for 6 hours. The same operation was performed on HSA and HSA2, and the products were analyzed using SDS-PAGE. Figure 73 shows the scheme and results.

[0584] When reacted with compound 26, fluorescence was observed only in the lanes for HSA2 and HSA4 (both modified with compound 41 to introduce an azide group). Furthermore, a new band with increased molecular weight, representing a polyethylene glycol derivative, appeared only in the lane for HSA4 modified with compound 54. These results confirm that both polyethylene glycol and fluorescent dye modification have occurred in the protein. Sequence Listing <110> National University Corporation Osaka University <120> Molecules for modifying proteins and / or peptides <130> P19-297WO <150> JP 2019-035340 <151> 2019-02-28 <160> 3 <170> PatentIn Version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Asp Arg Val Tyr Ile His Pro Phe His Leu 1 5 10 <210> 2 <211> 124 <212> PRT <213> Bos taurus <400> 2 Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser Ser 1 5 10 15 Thr Ser Ala Ala Ser Ser Ser Asn Tyr Cys Asn Gln Met Met Lys Ser 20 25 30 Arg Asn Leu Thr Lys Asp Arg Cys Lys Pro Val Asn Thr Phe Val His 35 40 45 Glu Ser Leu Ala Asp Val Gln Ala Val Cys Ser Gln Lys Asn Val Ala 50 55 60 Cys Lys Asn Gly Gln Thr Asn Cys Tyr Gln Ser Tyr Ser Thr Met Ser 65 70 75 80 Ile Thr Asp Cys Arg Glu Thr Gly Ser Ser Lys Tyr Pro Asn Cys Ala 85 90 95 Tyr Lys Thr Thr Gln Ala Asn Lys His Ile Ile Val Ala Cys Glu Gly 100 105 110 Asn Pro Tyr Val Pro Val His Phe Asp Ala Ser Val 115 120 <210> 3 <211> 584 <212> PRT <213> Homo sapiens <400> 3 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys His Gly Asp Leads To Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Ser 290,295,300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn With Lys Gln Asn Cys Glu To Phe Glu Gln To Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Val Pro 405 410 415 Gln Will Be Thr Pro Thr Leu Val Glu Will Be Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Lys His Glu Ala Lys Arg Met Pro Cys Ala 435 440 445 Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu 450 455 460 Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu 465 470 475 480 Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr 485 490 495 Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile 500 505 510 Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu 515 520 525 Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys 530 535 540 Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala 545 550 555 560 Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala 565 570 575 Ala Ser Gln Ala Ala Leu Gly Leu 580

Claims

1. Use of a reagent for non-disease diagnosis and treatment purposes for selectively modifying the N-terminus of a protein and / or peptide, wherein the reagent comprises a compound represented by formula (1) or a salt thereof, where R 1 Indicates -N(-R 4 )-, where R 4 Represents an organic group, R 2 =N-; and R 3 represents a hydrogen atom or an organic group; wherein the organic group is a group derived from an organic molecule or an organic molecule complex, and the organic molecule or organic molecule complex is a functional substance; The functional substance is a pharmaceutical compound, a luminescent molecule, a ligand, a ligand-bound molecule, an antigenic protein, an antibody, a nucleic acid, a carbohydrate, a lipid, a cell, a virus, a label, a carbon electrode, a carbon nanomaterial, a linker, a spacer molecule, or a complex thereof.

2. The method according to claim 1, wherein the compound is represented by formula (1Aa): where R 4 As defined above.

3. A method for preparing a compound represented by formula (7) or a salt thereof for non-disease diagnosis and treatment purposes, where R 1 Indicates -N(-R 4 )-, where R 4 Represents an organic group, R 2 =N-;R 3 represents a hydrogen atom or an organic group; a double line consisting of a dotted line and a solid line represents a single bond or a double bond; R 13 represents a group of proteins or peptides in which the N-terminal amino acid residue and the adjacent -NH- are excluded; R 14 Represents the side chain of the N-terminal amino acid residue of a protein or peptide; The method comprises reacting a protein and / or peptide with a compound represented by formula (1) or a salt thereof, where R 1 Indicates -N(-R 4 )-, where R 4 Represents an organic group, R 2 =N-; and R 3 represents a hydrogen atom or an organic group; wherein the organic group is a group derived from an organic molecule or an organic molecule complex, and the organic molecule or organic molecule complex is a functional substance; The functional substance is a pharmaceutical compound, a luminescent molecule, a ligand, a ligand-bound molecule, an antigenic protein, an antibody, a nucleic acid, a carbohydrate, a lipid, a cell, a virus, a label, a carbon electrode, a carbon nanomaterial, a linker, a spacer molecule, or a complex thereof.

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