Molecules for modifying proteins and/or peptides

文档序号：1850570 发布日期：2021-11-16 浏览：23次中文

阅读说明：本技术 用于修饰蛋白质和/或肽的分子 (Molecules for modifying proteins and/or peptides ) 是由林高史小野田晃井上望于 2020-02-28 设计创作，主要内容包括：本发明的一个目的是提供一种技术,该技术允许其他分子/物质以简单和有效的方式更选择性地连接到N端,甚至在天然蛋白质等中。该目的是通过使由式(1)表示的化合物或其盐或该化合物或其盐的水合物或溶剂合物与蛋白质和/或肽反应来实现。(It is an object of the present invention to provide a technique that allows other molecules/substances to be more selectively attached to the N-terminus, even in native proteins and the like, in a simple and efficient manner. 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 the salt, with a protein and/or a peptide.)

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

wherein R is¹And R²One of them represents-N (-R)⁴) - (wherein R)⁴Represents an organic group or a group derived from an inorganic material), and the other represents ═ N —; and R is³Represents a hydrogen atom, an organic group or a group derived from an inorganic material.

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

wherein R is⁴As defined above.

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

4. The compound or a salt thereof, or a hydrate or solvate of the compound or the salt thereof according to claim 3, wherein the functional substance is a pharmaceutical compound, a luminescent molecule, a polymeric compound, a ligand-binding molecule, an antigenic protein, an antibody, a protein, a nucleic acid, a saccharide, a lipid, a cell, a virus, a tag, a carbon electrode, a carbon nanomaterial, a linker, a spacer molecule, or a complex or a linker molecule thereof.

5. The compound or a salt thereof, or a hydrate or solvate of the compound or the salt thereof according to any one of claims 1 to 4, wherein the inorganic material is an electrode material, a metal fine particle, a metal oxide fine particle, a semiconductor particle, or a magnetic particle.

6. An agent comprising the compound or a salt thereof according to any one of claims 1 to 5, or a hydrate or solvate of the compound or the salt thereof.

7. The reagent according to claim 6, which is a reagent for protein and/or peptide modification.

8. A process for preparing a compound or a salt thereof, or a hydrate or solvate of the compound or the salt thereof according to any one of claims 1 to 5, which comprises the steps of:

reacting a compound represented by the formula (2) or a salt thereof with a compound represented by the formula (3), a compound represented by the formula (4) or a compound represented by the formula (9),

R⁴-R⁵(2)，

wherein R is⁴Represents an organic group or a group derived from an inorganic material, and R⁵represents-N₃-X (wherein X represents a halogen atom), -B (OH)₂，-B(OR⁵¹)₂(wherein each R is⁵¹Identical or different and represent a hydrocarbon radical, with the proviso that two R are⁵¹May form a ring together with the adjacent oxygen atom), or-N₂ ⁺，

Wherein R is³Represents a hydrogen atom, an organic group or a group derived from an inorganic material; r⁶、R⁷And R⁸Are the same or different and each represents an alkyl group; and Y represents a reactive group.

9. A process for preparing a compound or a salt thereof, or a hydrate or solvate of the compound or the salt thereof according to any one of claims 1 to 5, which comprises the steps of:

reacting a compound represented by formula (5) with a compound represented by formula (6):

R⁴-R⁹(5)，

wherein R is⁴Represents an organic group or a group derived from an inorganic material; and R is⁹represents-NR^9aR^9b(wherein R is^9aAnd R^9bThe same or different and each represents a hydrogen atom or an alkyl group),

wherein R is¹⁰Represents an electron withdrawing group; r¹¹represents-R^11a-R¹²(wherein R is^11aRepresents a single bond or a linker; and R is¹²Represents a carrier); n represents 0 or 1; and m represents an integer of 1 to 5.

10. A compound represented by formula (6'):

wherein R is¹⁰Represents an electron withdrawing group; r^11aRepresents a single bond or a linker; r¹²Represents a carrier; and m represents an integer of 1 to 5.

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

wherein R is¹And R²One of them represents-N (-R)⁴) - (wherein R)⁴Represents an organic group or a group derived from an inorganic material), and the other represents ═ N —; r³Represents a hydrogen atom, an organic group or a group derived from an inorganic material; the double line consisting of a dotted line and a solid line represents a single bond or a double bond; r¹³Represents a group in which the N-terminal amino acid residue and-NH-adjacent thereto are excluded from the protein or peptide; r¹⁴Represents the side chain of the N-terminal amino acid residue of a protein or peptide.

12. A method for producing the compound or a salt thereof according to claim 11, or a hydrate or solvate of the compound or the salt, which comprises reacting a protein and/or a peptide with the compound or the salt thereof according to any one of claims 1 to 5, or a hydrate or solvate of the compound or the salt.

Technical Field

The present invention relates to molecules for modifying proteins and/or peptides and the like.

Background

A technique for linking a protein and/or peptide (hereinafter, referred to as "protein or the like") to other molecules/substances is important in the preparation of antibody-drug conjugates, fluorescent probe-labeled protein reagents, protein-immobilized inorganic materials, and the like. For example, modification of a protein or the like with an azide group is a technique capable of introducing various functional molecules through an alkyne-azide cycloaddition reaction (CuAAC). Due to bio-orthogonality, the technology has been widely applied in the fields of bio-imaging and the like.

CITATION LIST

Non-patent document

Non-patent document 1: metal-free and pH-controlled introduction of azides in proteins, san ne Schoffelen, Mark B. van Eldijk, Bart Rooijakkers, Reinout Raijmakers, Albert J.R.heck and Jan C.M.van Hest, Chemical Science,2011,2,701.

Non-patent document 2: selective N-tertiary acyl of peptides with agy-His tag sequence, m.c. martos-Maldonado, c.t.hjuler, k.k.sorensen, m.b.thygesen, j.e.rasmussen, k.villardsen, s.r.midtgaard, s.kol, s.schoffeln, k.j.jensen, Nature Communications,2018,9,3307.

Non-patent document 3: modification of N-Terminal alpha-Amino Groups of Peptides and Proteins Using Ketenes, A.O. -Y.Chan, C. -M.Ho, H. -C.Chong, Y. -C.Leung, J. -S.Huang, M.Wong, C. -M.Che, Journal of the American Chemical Society,2012,134,2589.

Disclosure of Invention

Technical problem

The present inventors studied introduction sites for linking other molecules/substances to proteins and the likeWhen this happens, the following three points are focused on. The first point is that all monomeric proteins have only one N-terminal position, which is a universal base point for modification. The second point is that the N-terminus is rarely involved in the active site (molecular binding site and catalytic reaction center) of the protein or the functional site of the protein, and the influence of structural changes associated with modification is considered to be small. The third point is due to pK_aThe difference in (c), the N-terminus is considered to be less likely to compete with other amino acid residues (e.g., lysine, cysteine, and glutamine); at the C-terminal, the pK is based on_aThe reaction selectivity of (a) is considered to be difficult to achieve. Therefore, the inventors focused on the N-terminus of proteins and the like as a site for connecting other molecules/substances.

Various methods for linking other molecules/substances to the N-terminus of proteins and the like have been reported (non-patent documents 1 to 3). However, these methods are considered to be insufficient in terms of simplicity or selectivity of N-terminal modification. For example, although a chemical bonding method or a lipid modification enzyme method that recognizes a specific amino acid sequence can specifically introduce other molecules/substances into the N-terminus, they require a protein or the like into which a specific amino acid sequence or specific amino acid residues are inserted. The preparation of such proteins and the like is laborious, and these methods cannot be applied to natural proteins and the like. The amido bond formation reaction using the activated ester or ketene is simple to operate and can be applied to natural proteins and the like; however, it cannot specifically introduce other molecules/substances to the N-terminus because side reactions with lysine residues and the like occur.

It is therefore an object of the present invention to provide a technique that allows other molecules/substances to be more selectively attached to the N-terminus of all proteins and/or peptides in a simple and efficient manner.

Problem solving scheme

The present inventors have conducted extensive studies to solve the above problems. They found 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 the salt with a protein and/or a peptide. The present inventors have further studied based on this finding, and thus have completed the present invention.

Specifically, the present invention includes the following embodiments.

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

The compound according to item 1 or a salt thereof, or a hydrate or solvate of the compound or the salt thereof, wherein the compound is represented by formula (1 Aa):

wherein R is⁴As defined above.

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

The compound or a salt thereof according to item 3, or a hydrate or solvate of the compound or the salt thereof, wherein the functional substance is a pharmaceutical compound, a luminescent molecule, a polymeric compound, a ligand-binding molecule, an antigenic protein, an antibody, a protein, a nucleic acid, a saccharide, a lipid, a cell, a virus, a tag, a carbon electrode, a carbon nanomaterial, a linker, a spacer molecule, or a complex or a linker molecule thereof.

The compound or a salt thereof according to any one of claims 1 to 4, or a hydrate or solvate of the compound or the salt thereof, wherein the inorganic material is an electrode material, a metal fine particle, a metal oxide fine particle, a semiconductor particle, or a magnetic particle.

An agent 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.

The reagent according to item 6, which is a reagent for protein and/or peptide modification.

A process for producing the compound according to any one of items 1 to 5 or a salt thereof, or a hydrate or solvate of the compound or the salt thereof, which comprises:

R⁴-R⁵(2)，

A process for producing the compound according to any one of items 1 to 5 or a salt thereof, or a hydrate or solvate of the compound or the salt thereof, which comprises:

reacting a compound represented by formula (5) with a compound represented by formula (6):

R⁴-R⁹(5)，

Item 10. a compound represented by the formula (6'):

wherein R is¹⁰Represents an electron withdrawing group; r^11aRepresents a single bond or a linker; r¹²Represents a carrier; and m represents an integer of 1 to 5.

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

wherein R is¹And R²One of them represents-N (-R)⁴) - (wherein R)⁴Represents an organic group or a group derived from an inorganic material), and the other represents ═ N —; r³Represents a hydrogen atom, an organic group or a group derived from an inorganic material; the double line consisting of a dotted line and a solid line represents a single bond or a double bond; r¹³Represents an N-terminal amino acid residue and a group adjacent thereto in which-NH-is excluded from the protein or peptide; r¹⁴Represents the side chain of the N-terminal amino acid residue of a protein or peptide.

A method for producing the compound according to item 11 or a salt thereof, or a hydrate or solvate of the compound or the salt, which comprises reacting a protein and/or a peptide with the compound according to any one of items 1 to 5 or a salt thereof, or a hydrate or solvate of the compound or the salt.

Advantageous effects of the invention

The present invention can provide a technique that allows other molecules/substances to be more selectively attached to the N-terminus in a simple and efficient manner, even in native proteins and the like.

Drawings

FIG. 1 shows the preparation of Compound 1¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 2 shows the preparation of Compound 2¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 3 shows the preparation of Compound 3¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 4 shows the preparation of Compound 4¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 5 shows the preparation of Compound 5¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 6 shows the preparation of Compound 6¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 7 shows the preparation of Compound 7¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 8 shows the preparation of Compound 8¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 9 shows the preparation of Compound 9¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 10 shows the preparation of Compound 10¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 11 shows the preparation of Compound 13¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 12 shows the preparation of Compound 16¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 13 shows preparation of Compound 17¹H NMR Spectrum (400MHz, CD)₃CN)。

FIG. 14 shows preparation of Compound 18¹H NMR Spectrum (400MHz, CD)₃CN)。

FIG. 15 shows the preparation of Compound 19¹H NMR Spectrum (400MHz, CD)₃CN)。

FIG. 16 shows preparation of Compound 20¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 17 shows the preparation of Compound 21¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 18 shows preparation of Compound 22¹H NMR Spectrum (400MHz, CD)₃CN)。

Fig. 19 shows the reaction scheme and results of the N-terminal modification reaction of angiotensin I (example 8).

FIG. 20 shows preparation of Compound 25¹H NMR Spectrum (400MHz, CD)₃CN)。

FIG. 21 shows the reaction scheme and the results of N-terminal modification reaction of ribonuclease A (example 10).

FIG. 22 shows the reaction scheme and the results of modification of RNase A with biotin (example 11-2).

FIG. 23 shows the reaction scheme and the results of modifying RNase A with a fluorescent dye (example 11-3).

FIG. 24 shows the reaction scheme and the results of modifying ribonuclease A with an azido group (example 11-4).

FIG. 25 shows the reaction scheme and results of modification with fluorescent dyes by strain-promoted alkyne-azide cycloaddition reaction (examples 11-5).

FIG. 26 shows the reaction scheme and results of modification of RNase A with strained alkyne moiety (examples 11-6).

FIG. 27 shows a summary of the method for the simplified removal of the by-product (aniline derivative) generated after the reaction by immobilizing various formaldehyde precursors on a resin or solid material, and the solution after the reaction is directly used for N-terminal modification of proteins (example 12).

Fig. 28 shows compound 27 and a resin to which compound 27 is immobilized (example 12).

FIG. 29 shows a synthesis scheme of 27/PS resin (example 12-2).

FIG. 30 shows the resin identification results using infrared spectroscopy (example 12-3). The right side is an enlarged view.

FIG. 31 shows the results of LC/MS analysis of N-terminally modified ribonuclease A by sequential reactions using a reactant-immobilized resin (27/PS resin) (example 12-4).

FIG. 32 shows a synthesis scheme of 27/PS resin (example 12-2).

FIG. 33 shows preparation of Compound 28¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 34 shows the preparation of Compound 30¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 35 shows a scheme for N-terminal modification by sequential reactions (examples 12-6) using a resin immobilized with a reactant (27/PS resin) (example 12-4).

FIG. 36 shows the results of LC/MS analysis of N-terminal modified ribonuclease A by sequential reactions using a reaction immobilized resin (27/PS resin) (examples 12 to 6). The peak corresponding to the modified protein is shown as "●" (filled circles) and the peak corresponding to the unmodified protein is shown as ". smallcircle".

FIG. 37 shows a scheme for preparing triazolecarboxaldehyde using a Dimroth rearrangement reaction in a homogeneous system and applied to protein modification (example 13).

FIG. 38 shows the reaction mechanism of the Dimroth rearrangement reaction (example 13-2).

Fig. 39 shows the structure of an acid catalyst in a diemol rearrangement reaction using compound 10 (example 13-2).

FIG. 40 shows the LC-MS analysis results of the preparation of N-terminal modifier by Dimrot rearrangement reaction and the successive protein modification reactions (example 13-3). The peak corresponding to the modified protein is shown as "●" (filled circles) and the peak corresponding to the unmodified protein is shown as ". smallcircle".

FIG. 41 shows a scheme for the preparation of Bis-TA4C and a protein modification reaction (example 14-2).

FIG. 42 shows the results of LC-MS analysis of N-terminal modification of RNase A with Bis-TA4C using diamine as a precursor (example 14-2). The structure of the diamine as precursor and the percentage of modification calculated by LC-MS are shown.

FIG. 43 shows a scheme for introducing a functional molecule into the N-terminus of a protein by oxime formation (example 14-3).

FIG. 44 shows the results of LC-MS analysis of RNase A modified with compound 37 (example 14-3).

FIG. 45 shows the results of SDS-PAGE analysis of RNase A modified with compound 38 (example 14-3).

FIG. 46 shows preparation of Compound 39¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 47 shows preparation of Compound 40¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 48 shows a scheme for a Dimroth rearrangement reaction using compound 40 (example 15-2).

FIG. 49 shows the yield of Compound 7 in the Dimroth rearrangement reaction (example 15-2). Yield of¹H NMR measurement calculation.

FIG. 50 shows the reaction scheme and the results of the N-terminal modification reaction of human serum-derived albumin (example 16-2).

FIG. 51 shows a reaction scheme and results of cysteine residue modification of human serum-derived albumin (example 17-2).

FIG. 52 shows a reaction scheme and results of N-terminal modification of human serum-derived albumin in which cysteine residues are modified (example 17-3).

FIG. 53 shows the results of LC/MS analysis after leaving the RNase-7 solution for 24 hours (example 18-2).

FIG. 54 shows the release of the modifying agent in RNase-7 over time (example 18-2).

FIG. 55 shows the change in modifier release with pH change in RNase-7 (example 18-3).

FIG. 56 shows preparation of Compound 42¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 57 shows preparation of Compound 43¹H NMR Spectrum (400MHz, C)DCl₃)。

FIG. 58 shows preparation of Compound 44¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 59 shows preparation of Compound 45¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 60 shows preparation of Compound 46¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 61 shows the preparation of Compound 47¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 62 shows the preparation of Compound 48¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 63 shows the preparation of Compound 49¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 64 shows Compound 50¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 65 shows Compound 51¹H NMR Spectrum (400MHz, CDCl)₃)。

Fig. 66 shows the reaction scheme and results of N-terminal modification of angiotensin I (example 20).

FIG. 67 shows the reaction scheme and results of N-terminal modification of ribonuclease A (example 21).

FIG. 68 shows the scheme and the results of introducing an acetyl group and an azido group into ribonuclease A (example 22-1).

FIG. 69 shows the modification with fluorescent dye by strain-promoted alkyne-azide cycloaddition reaction and the results (example 22-2).

FIG. 70 shows preparation of Compound 52¹H NMR Spectrum (400MHz, DMSO-d)₆)。

FIG. 71 shows the preparation of Compound 53¹H NMR Spectrum (400MHz, CDCl)₃)。

FIG. 72 shows the scheme and the results of modifying ribonuclease A with polyethylene glycol (example 23-2-2).

FIG. 73 shows the scheme and the results of the reaction for N-terminal modification of HSA2 and the reaction for modification with Compound 26 (example 23-2-3).

Detailed Description

In this specification, the terms "comprising" and "including" include the concepts of "including", "comprising", "consisting essentially of … …" and "consisting of … …".

1. Protein and/or peptide modifying molecules

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 the salt thereof (in the present specification, these may be collectively referred to as "modified molecule of the present invention"):

wherein R is¹And R²One of them represents-N (-R)⁴) - (wherein R)⁴Represents an organic group or a group derived from an inorganic material), and the other represents ═ N-, R³Represents a hydrogen atom, an organic group or a group derived from an inorganic material.

The modified molecules of the invention are described below.

1-1. Compounds

Wherein R is¹And R²One of them represents-N (-R)⁴) - (wherein R)⁴Representing an organic group or a group derived from an inorganic material), and the other represents ═ N-, and the double line consisting of the dotted line and the solid line represents a single bond or a double bond, and whether it is a single bond or a double bond depends on R¹And R²Which one of them is-N (-R)⁴) -or ═ N-.

Specifically, when R is¹is-N (-R)⁴) -, and R²When is ═ N-, formula (1) is formula (1A):

wherein R is³And R⁴The definition of (1) is as above; and

when R is¹Is ═ N-, and R²is-N (-R)⁴) -when formula (1) is formula (1B):

wherein R is³And R⁴The definition of (A) is as above.

In the present invention, preferably, R¹is-N (-R)⁴) -and R²Is ═ N-; that is, formula (1) is formula (1A).

The organic group is not particularly limited as long as it 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. The organic molecule is not particularly limited and may be natural, synthetic or artificial. The organic molecule complex is not particularly limited, and examples include a complex (or organism) in which a plurality of molecules including one or more organic molecules are linked. The manner of attachment is not particularly limited, and examples include hydrogen bonds, electrostatic forces, van der waals forces, hydrophobic bonds, covalent bonds, coordination bonds, and the like. These bonds may be formed by linkers (see linkers described later for specific examples). The organic molecule or organic molecule complex is preferably a functional substance. Specific examples thereof include pharmaceutical compounds, luminescent molecules, macromolecular compounds, ligands, ligand-binding molecules, antigenic proteins, antibodies, proteins, nucleic acids, carbohydrates, lipids, cells, viruses, tags (e.g., radioisotope tags), carbon electrodes, carbon nanomaterials, linkers, spacer molecules (e.g., polyethylene glycol or derivatives thereof and peptides (e.g., peptides comprising an amino acid sequence cleaved by an enzyme in a cell)), and complexes and linker molecules thereof.

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

wherein R is³As defined above, and R³The same or different at each occurrence; r^4aRepresents a divalent organic group. R^4aMay comprise part of the structure of the modified molecule of the invention.

The inorganic material is a material containing or not containing one or more metal atoms, and is not particularly limited. Examples of the inorganic material include electrode materials, metal fine particles, metal oxide fine particles, semiconductor particles, magnetic particles, and the like. The inorganic material may retain organic molecules or organic molecular complexes.

The organic group or the group derived from an inorganic material may have a reactive group. In this case, the other substance may be further linked by a reactive group. Examples of reactive groups include ethynylene, vinyl, azido, epoxy, aldehyde, oxyamino, halogen, and the like. It is known that each of ethynylene and ethynylene undergoes a1, 3-dipolar cycloaddition reaction with azido to form a1, 2, 3-triazole ring. The vinyl group reacts with the thiol group to form a bond. The epoxy group reacts with the amino group or thiol group to form a bond. The aldehyde group reacts with the amino group to form a schiff base, which is reduced to form a bond. The oxyamino group reacts with a keto or aldehyde group to form an oxime. It is known that azide groups undergo a1, 3-dipolar cycloaddition reaction with alkynyl groups to form 1,2, 3-triazole rings.

R³Represents a hydrogen atom, an organic group or a group derived from an inorganic material. Examples of the organic group and the inorganic material include those described above as represented by R⁴Examples of the organic groups and inorganic material-derived groups are those shown. When R is³In the case of an organic group or an inorganic material-derived group, the organic group or the inorganic material-derived group preferably has a reactive group. In this case, the other species may be further linked by a reactive group, as described above.

In one embodiment of the invention, R³Is a hydrogen atom. In this case, formula (1) is, for example, formula (1a), (1Aa) or (1 Ba):

wherein R is¹、R²And R⁴As defined above.

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

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

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

1-2. Synthesis method 1

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

R⁴-R⁵(2)，

wherein R is⁴As defined above; r⁵represents-N₃-X (wherein X represents a halogen atom), -B (OH)₂、-B(OR⁵¹)₂(wherein each R is⁵¹Identical or different and represent a hydrocarbon radical, with the proviso that two R are⁵¹May form a ring with the adjacent oxygen atom), or-N₂ ⁺，

Wherein R is³As defined above; r⁶、R⁷And R⁸Identical or different, each represents an alkyl group; y represents a reactive group.

Examples of the halogen atom represented by X include fluorine, chlorine, bromine, iodine and the like. Preferably, the halogen atom represented by X is, for example, bromine.

From R⁵¹Examples of the hydrocarbon group represented include alkyl groups, cycloalkyl groups and the like.

From R⁵¹Alkyl groups represented include straight chain alkyl and branched chain alkyl groups. The number of carbon atoms in the alkyl group is not particularly limited, and is, for example, 1 to 8. Specific examples of alkyl groups 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.

R⁵¹The number of carbon atoms in the cycloalkyl group represented is not particularly limited, and is, for example, 3 to 10, preferably 4 to 10. Specific examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.

From two R⁵¹The ring formed together with the adjacent oxygen atom may be substituted with an alkyl group or the like.

R⁶、R⁷、R⁸The alkyl group represented is usually a straight-chain lower alkyl group, and preferably an ethyl group.

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

Details of each material used are described below.

1-2-1 case 1 (R)⁵is-N₃The case of using the compound represented by the formula (3)

The amount of the compound represented by the formula (2) used per mole of the compound represented by the formula (3) is usually preferably 0.1 to 5 moles, more preferably 0.3 to 2 moles, in terms of the number of moles of the functional group to be reacted (in this case, azide group), in terms of yield and the like.

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

The 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. In addition, reducing agents (e.g., hydroquinone or sodium ascorbate), ligands, and the like may also be used for the reaction.

The amount of the copper catalyst used is usually preferably 0.1 to 5 moles per mole of the azide-containing protein or peptide of the present invention in terms of yield and the like.

In addition to the above components, additives may be suitably used in the reaction as long as the progress of the reaction is not significantly impaired.

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 deg.c (particularly, 0 to 40 deg.c). The reaction time is not particularly limited, and may be usually 30 minutes to 3 hours, particularly 1 to 2 hours.

The progress of the reaction can be monitored by chromatography or other methods commonly used. After the reaction is completed, the solvent is distilled off, and the product can be isolated and purified by chromatography, recrystallization or other common methods as required. The structure of the product can be determined by elemental analysis, MS (ESI-MS) analysis, IR analysis, mass spectrometry, and the like,¹H-NMR、¹³C-NMR, etc.

1-2-2 case 2 (R)⁵is-N₃Other groups, the case of using the compound represented by the formula (3)

This case is the same as case 1 except that a catalyst is added to the reaction systemFor mixing R⁵A nitridizing agent converted into azide group.

Examples of the azide include inorganic azides such as sodium azide; sulfonyl azide; a silyl azide; a phosphoryl azide; alkyl ammonium azides; and the like.

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

1-2-3. case 3 (case of Using the Compound represented by formula (4))

In this case, the compound represented by formula (1) may be synthesized through several steps after the compound represented by formula (2) is reacted with the compound represented by formula (4). For example, in this case, the compound represented by formula (1) may be synthesized according to the following scheme.

Wherein R is¹、R²、R³、R⁴、R⁵And R⁸As defined above.

Step 1

The amount of the compound represented by the formula (2) to be used per mole of the compound represented by the formula (4) is usually preferably 0.1 to 5 moles, and more preferably 0.3 to 2 moles, in terms of the number of moles of the functional group to be reacted (in this case, an azide group), from the viewpoint of yield and the like.

The solvent used is not limited as long as it does not adversely affect the reaction. Examples include water, alcohol-based solvents (e.g., methanol, ethanol, isopropanol, N-butanol, trifluoroethanol, and ethylene glycol), ketone-based solvents (e.g., acetone and methyl ethyl ketone), ether-based solvents (e.g., tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane, and diglyme), ester-based solvents (e.g., methyl acetate and ethyl acetate), aprotic polar solvents (e.g., acetonitrile, N-dimethylformamide, and dimethyl sulfoxide), halogenated hydrocarbon solvents (e.g., dichloromethane and dichloroethane), and mixtures thereof. The solvent is preferably a mixture of water and an aprotic polar solvent, in particular dimethylformamide.

Step 1 is generally carried out in the presence of a base. The base may be, for example, an organic base. Examples of the organic base include trialkylamines such as trimethylamine, triethylamine and N, N-diisopropylethylamine, pyridine, quinoline, piperidine, imidazole, picoline, 4-dimethylaminopyridine, 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), and the like. When these bases are in liquid form, they can also be used as solvents. These bases may be used alone or in combination of two or more. The base is preferably piperidine.

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

The reaction temperature is not particularly limited. The reaction is usually carried out under cooling, at room temperature or under heating. The reaction may 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.

After the reaction is finished, the solvent is distilled off, 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, mass spectrometry, and the like,¹H-NMR、¹³C-NMR, etc.

Step 2

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.

Examples of reducing agents include sodium borohydride, zinc borohydride (Zn (BH)₄)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.

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

As the base, for example, an inorganic base can be used. Examples of the inorganic base include alkali metals (e.g., sodium and potassium), alkali metal bicarbonates (e.g., lithium bicarbonate, sodium bicarbonate, and potassium bicarbonate), alkali metal hydroxides (e.g., lithium hydroxide, sodium hydroxide, potassium hydroxide, and cesium hydroxide), alkali metal carbonates (e.g., lithium carbonate, sodium carbonate, potassium carbonate, and cesium carbonate), alkali metal lower (C)₁-C₄) Alkoxides (such as sodium methoxide, sodium ethoxide, and potassium tert-butoxide), alkali metal hydrides (such as sodium hydride and potassium hydride), and the like. These bases may be used alone or in combination of two or more. The base is preferably an alkali metal lower (C)₁-C₄) Alkoxides (especially sodium methoxide).

The solvent used is not limited as long as it does not adversely affect the reaction. Examples include water, alcohol-based solvents (e.g., methanol, ethanol, isopropanol, N-butanol, trifluoroethanol, and ethylene glycol), ketone-based solvents (e.g., acetone and methyl ethyl ketone), ether-based solvents (e.g., tetrahydrofuran), dioxane, diethyl ether, dimethoxyethane, and diglyme), ester-based solvents (e.g., methyl acetate and ethyl acetate), aprotic polar solvents (e.g., acetonitrile, N-dimethylformamide, and dimethyl sulfoxide), halogenated hydrocarbon solvents (e.g., dichloromethane and dichloroethane), and mixtures thereof. The solvent is preferably an alcohol-based solvent (particularly methanol).

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

Step 3

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

As the oxidizing agent, manganese dioxide, for example, can be used. Other examples of useful oxidizing agents include selenium dioxide, nitroxyl radicals such as 2,2,6, 6-tetramethylpiperidine 1-oxyl (TEMPO) and 2-azaadamantan-N-oxyl (AZADO), and the like. These oxidizing agents may be used alone or in combination of two or more.

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

The solvent used is not limited as long as it does not adversely affect the reaction. Examples include ketone-based solvents (e.g., acetone and methyl ethyl ketone), ether-based solvents (e.g., tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane and diglyme), ester-based solvents (e.g., methyl acetate and ethyl acetate), aprotic polar solvents (e.g., acetonitrile, N-dimethylformamide and dimethyl sulfoxide), halogenated hydrocarbon solvents (e.g., chloroform, dichloromethane and vinyl chloride), and mixtures thereof. The solvent is preferably a halogenated hydrocarbon solvent (particularly chloroform).

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

1-2-4 case 4 (R)⁵is-N₃The case of using the compound represented by the formula (9)

In this case, the compound represented by formula (1) may be synthesized through several steps after the compound represented by formula (2) is reacted with the compound represented by formula (9). For example, in this case, the compound represented by formula (1) may be synthesized according to the following scheme.

Step 4 may be performed according to or based on scenario 1 above. May be performed according to or based on step 3 aboveAnd 6. step 6. Step 5 may be performed by reacting a compound represented by formula (1 x) with a compound represented by formula (10): r³-Z reaction. Z is a reactive group that reacts with Y. When Y is halogen, for example, -B (OH) can be used₂As Z. The reaction conditions of step 5 can be determined according to the types of Y and Z based on known information.

1-3. Synthesis method 2

In addition to the above synthesis method 1, the compound represented by formula (1) may be synthesized by, for example, including reacting a compound represented by formula (5): r⁴-R⁹(5) Wherein R is⁴As defined above; r⁹represents-NR^9aR^9b(wherein R is^9aAnd R^9bThe same or different, each represents a hydrogen atom or an alkyl group) with a compound represented by the formula (6):

Further, the compound represented by the formula (1AA) can be synthesized by using the compound represented by the formula (5'): r⁹-R^4a-R⁹(5'), wherein R^4aAnd R^5aAs defined above, as a compound represented by the formula (5).

Examples of the alkyl group include, but are not particularly limited to, lower alkyl groups such as methyl and ethyl. R^9aAnd R^9bAre preferably hydrogen atoms.

Examples of electron withdrawing groups include, but are not particularly limited to, -NO₂、-F、CF₃CN, -COOMe, and the like. The position of the electron-withdrawing group is preferably para, e.g. in-NO₂In the case of (2), and preferably in ortho-and meta-positions, e.g. in-F and CF₃In the case of (1). In the latter case, more preferably, -CN is in para position.

When attracting electronsThe radical being, for example, -NO₂When m is 1, m is preferably. When the electron-withdrawing group is, for example, -F or CF₃When m is preferably from 4 to 5, more preferably all ortho-and meta-positions are replaced by R¹⁰. In the latter case, even more preferably, -CN is in para position.

when-F or CF₃(preferably-F) in all of the ortho-and meta-positions, and-CN in the para-position, the reaction with the compound represented by the formula (5) proceeds sufficiently even under relatively low temperature conditions (preferably 20 to 60 ℃ C., more preferably 25 to 40 ℃ C.).

The linker is not particularly limited as long as it can link the carrier to the benzene ring. Examples include a linker comprising any of the following partial structures in the backbone.

The number of atoms constituting the backbone of the linker is, for example, 1 to 100, 1 to 50, 1 to 20, or 1 to 10.

The carrier is not particularly limited.

The average particle size of the carrier particles is not particularly limited, but is preferably a size that is easily precipitated in a solution. The average particle diameter of the carrier particles is, for example, 1nm to 1mm, preferably 10nm to 100 μm.

The material of the carrier particles is not particularly limited, and examples thereof include metal particles of gold, silver, copper, iron, aluminum, nickel, manganese, titanium, and the like, and oxides thereof; resin particles such as polystyrene and latex; silica particles; and the like. The shape of the carrier particle is not particularly limited, and may be, for example, a sphere, a rectangular parallelepiped, a cube, a triangular pyramid, or the like. The carrier particles may have a substance on the surface that makes the binding of another substance (e.g., binding substance 2) easier and/or stronger. Examples of the substance include reactive group-containing substances such as epoxy group-containing substances, amino group-containing substances, carboxyl group-containing substances, azide group-containing substances; substances having affinity for other molecules, such as avidin, protein a and protein B; and the like. The carrier particles may further comprise 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.

The amount of the compound represented by formula (5) 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.

In this reaction, an acid catalyst is preferably used in view of yield and the like. Examples of the acid catalyst include, but are not particularly limited to, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, 2-morpholinoethanesulfonic acid (MES), 3-morpholinopropanesulfonic acid (MOPS), 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), and the like. Among them, from the viewpoint of not further inhibiting the subsequent protein modification reaction, Goodler buffers such as MES, MOPS, HEPES and TAPS are preferable.

The reaction temperature is not particularly limited. The reaction is usually carried out under cooling, at room temperature or under heating. The reaction may be preferably performed at a temperature of about 20 to 100 ℃ for 10 minutes to 30 hours.

When the compound represented by the formula (6) contains a carrier (i.e., when R¹¹is-R^11a-R¹²In this case), the purification operation can be carried out in a simple manner. Specifically, a compound represented by the formula (6x) which isBy-products of the reaction (aniline derivatives):

wherein R is^9a、R^9b、R¹⁰、R^11a、R¹²And m is as defined above, can be easily removed by a precipitation operation using a carrier or the like. Accordingly, in one embodiment of the present invention, the present invention relates to a compound represented by formula (6'):

wherein R is¹⁰、R¹¹、R¹²And m is as defined above.

1-4. use

The modified molecule of the present invention can be used for linking another molecule/substance to the N-terminus of a protein or peptide, for example, to produce a complex substance (a compound represented by formula (7) or a salt thereof, or a hydrate or solvate of the compound or the salt) of the present invention described later. Thus, the modified molecules of the invention may be suitable for use as reagents, in particular as active ingredients of protein and/or peptide modifying reagents. The reagent is not particularly limited as long as it contains the modified molecule of the present invention, and may contain other components 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, and the like.

2. Composite material

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 the salt thereof (in the present specification, these may be collectively referred to as "composite material of the present invention"):

wherein R is¹、R²、R³And R⁴As defined above; r¹³Represents a group in which the N-terminal amino acid residue and-NH-adjacent thereto are excluded from the protein or peptide; and R¹⁴Represents the side chain of the N-terminal amino acid residue of a protein or peptide. The composite material of the present invention is described below.

R¹³Denotes a group in which the N-terminal amino acid residue and-NH-adjacent thereto are excluded from the protein or peptide.

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

R¹⁴Represents 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 having basic side chains, such as lysine, arginine, and histidine; amino acid residues having acidic side chains, such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having a beta-branched side chain, such as threonine, valine, isoleucine; amino acid residues having aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine; and the like.

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

The protein or peptide may be chemically modified. The C-terminal of the protein or peptide may be carboxyl (-COOH), carboxylate (-COO)^-) Amide (-CONH)₂) Or an ester (-COOR). Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl or n-butyl_1-6An alkyl group; e.g. C_3-8Cycloalkyl groups such as cyclopentyl and cyclohexyl; e.g. C_6-12Aryl groups such as phenyl and α -naphthyl; for example, phenyl-C_1-2Alkyl groups such as benzyl and phenethyl; including alpha-naphthyl-C_1-2C7-14 aralkyl for alkyl, such as alpha-naphthylmethyl; pivaloyloxymethyl, and the like. In proteins or peptides, the carboxyl group (or carboxylate) other than the C-terminus may be amidated or esterified. In this case, for example, the C-terminal ester is used. In addition, proteins or peptides include those in which a substituent in an amino acid side chain in the molecule (e.g., -OH, -SH, amino, imidazolyl, indolyl, or guanidino) is protected with a suitable protecting group (e.g., including C)_1-6C of alkanoyl_1-6Acyl, such as formyl or acetyl) protected protein or peptide.

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

The protein or peptide may exist alone as a single molecule, or may be linked to another molecule to form a complex. For example, the protein or peptide may be a protein or peptide present on the surface of a cell, a protein or peptide in a cell disruption solution, or a protein or peptide supported on some substance. The linking means is not particularly limited, and may be, for example, a hydrogen bond, an electrostatic force, a van der waals force, a hydrophobic bond, a covalent bond, a coordinate bond, or the like.

Proteins or peptides can be cleaved by enzymatic or chemical reactions as long as the N-terminus is present. When the protein or peptide is modified at the N-terminus, the N-terminus may be modified by enzymatic or chemical reaction.

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

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

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

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. This reaction may be carried out after the synthesis reaction of the modified molecule of the present invention (particularly preferably the above "1-2. method of synthesis 2" reaction). In this case, for example, the reaction mixture obtained in the synthesis reaction of the modified molecule of the present invention may be diluted with the solvent used in the reaction, and then the reaction may be carried out.

The amount of the modified molecule of the present invention used is preferably 5 to 400 moles per mole of protein or peptide in terms of yield and the like.

The reaction is usually carried out in the presence of a reaction solvent. Examples of the reaction solvent include, but are not particularly limited to, water and the like. These solvents may be used alone or in combination of two or more. Further, it is preferable to add a buffer, for example, a phosphate buffer, to the solvent. When water is used, the pH of the reaction is preferably close to neutral, particularly preferably from 6 to 8.5, more preferably from 6.5 to 8, even more preferably from 7 to 7.5, in terms of N-terminal selectivity.

In addition to the above components, additives may be suitably used in the reaction as long as the progress of the reaction is not significantly impaired.

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

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:

wherein R is³、R^4a、R¹³And R¹⁴As defined above. In this case, an organic molecule or the like may be linked by further forming an oxime with hydroxylamine. Specifically, the compound represented by formula (1AA) may be reacted with a compound represented by formula (8): r⁴-O-NH₂(8) Wherein R is⁴As defined above, to synthesize a compound represented by formula (7 AAA):

wherein R is³、R^4a、R⁴、R¹³And R¹⁴As defined above. The conditions and the like of the synthesis reaction can be determined according to or based on the known reaction conditions for forming an oxime with hydroxylamine starting from an aldehyde. This reaction is also suitable for protein modification because it can be carried out even in water under mild conditions.

The complex substance of the present invention has a structure in which other substances are linked to a protein or a peptide. The complex substance of the present invention can be used, for example, as an antibody-drug conjugate, a labeled protein reagent, a protein-immobilized inorganic material, 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.

In one embodiment of the complex substance of the present invention, the complex substance may be designed such that the substance linked to the protein or peptide is gradually dissociated. Therefore, after administering the complex substance of the present invention, for example, in which a drug is linked to a protein or peptide, to an organism, the drug can be gradually released in the organism by utilizing the gradual dissociation of the drug of the complex substance of the present invention in the organism.

When the complex of the invention has reactive groups (e.g., when R³With a reactive group), the reactive group can be used to further attach another substance. For example, it can be used to link other species (e.g., organic molecules, organic molecular complexes, or inorganic materials) by reactions using azide groups (e.g., Huisgen cycloaddition reactions, strain-promoted azide-alkyne cycloaddition reactions, or Staudinger-Bertozzi linkages).

Examples

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

EXAMPLE 1 Synthesis of Compound 1 (method A)

1-1. apparatus used

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 measured solvent as an internal reference. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) was performed using a Bruker microOTOF focus III mass spectrometer, using methanol or acetonitrile (both HPLC grade) as 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 a diamond or gallium prism.

1-2. reagents, solvents, etc

Reagents and solvents used in the synthesis were used as they were in commercial products. The precursor azide used is synthesized with reference to published reports (y.zhao, p.gong, bioorg.med.chem.,2014,22, 6438-.

Synthesis of 1-3-1.1- (3-carboxyphenyl) -1H-1,2, 3-triazole-4-carbaldehyde (1)

Compound 1 was synthesized according to the following scheme.

Compound 1 was synthesized with reference to published reports (TA Bakka, MB Strom, JH Anderson, OR Gautun, bioorg. Med. chem. Lett.,2017,27, 1119-. Specific synthetic procedures and compound identification results are described below.

To a mixture of aqueous solution (7.5mL) of copper (II) pentahydrate (38mg,0.15mmol) and t-butanol (7.5mL) under nitrogen, azide (0.60mmol), propargylaldehyde diethylacetal (115. mu.L, 0.80mmol) and sodium ascorbate (60mg,0.30mmol) were added and the mixture was stirred at 70 ℃ for 24 h and at room temperature for 1h under air. The resulting suspension was diluted with a saturated aqueous sodium chloride solution (10mL), extracted with ethyl acetate (50 mL. times.3), and washed with a saturated aqueous sodium chloride solution (20 mL. times.2). The obtained organic layer was dried over sodium sulfate, and the solvent of the filtrate obtained by filtration was removed by distillation under the reduced pressure. The residue was purified by silica gel column chromatography to give compound 1 (yellow solid).FIG. 1 shows¹H NMR spectrum.

The yield is 48%;¹H 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)；¹³C NMR(100MHz,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) calculation of C₁₀H₇NaN₃O₃[M+Na]M/z of +240.04 to obtain 240.04; FT-IR (ATR mode, gallium prism), v cm^-1:3128,2923,1697,1263,1230,1184,757,673,647。

Synthesis of 1-3-2.1- (4-carboxyphenyl) -1H-1,2, 3-triazole-4-carbaldehyde (2)

Compound 2 (yellow solid) was synthesized as in example 1-3-1 using method a using 4-azidobenzoic acid as a precursor. FIG. 2 shows¹H NMR spectrum.

The yield is 63%;¹H NMR(400MHz,DMSO-d₆) Delta 10.13(s,1H),9.69(s,1H), 258.18-8.12 (m, 4H); ESI-TOF MS (Positive mode) calculation of C₁₀H₇NaN₃O₃[M+Na]M/z of +240.04 to obtain 240.04; FT-IR (ATR mode, gallium prism), v cm^-1:3110,1695,1683,1605,1429,1319,1294,1264,989,945,864,773,702,688。

Synthesis of 1-3-3.1- (3, 5-dicarboxyphenyl) -1H-1,2, 3-triazole-4-carbaldehyde (3)

Compound 3 (yellow solid) was synthesized as in example 1-3-1 using method a, using 5-azidoisophthalic acid as a precursor. FIG. 3 shows¹H NMR spectrum.

The yield is 10%;¹H NMR(400MHz,DMSO-d₆) δ 10.12(s,1H),9.81(s,1H),8.65(s,2H),8.56(s, 1H); FT-IR (ATR mode, gallium prism), v cm^-1:3133,1697,1296,1281,1247,1049,830,757,678,666,566,527。

Synthesis of 1-3-4.1- (4- (diethylamino) phenyl) -1H-1,2, 3-triazole-4-carbaldehyde (4)

Compound 4 (brown solid) was synthesized as in example 1-3-1 using method a using 4-azido-N, N-diethylaniline as a precursor. FIG. 4 shows¹H NMR spectrum.

The yield is 60%; 1H NMR (400MHz, CDCl)₃):δ10.12(s,1H),8.36(s,1H),7.52(d,J＝9.1Hz,2H),6.73(d,J＝9.1Hz,2H),3.42(q,J＝7.1Hz,4H),1.21(t,J＝7.1Hz,6H)；¹³C NMR (100MHz, DMSO-d6) delta 185.0,148.0,147.3,125.1,124.3,122.1,111.4,43.8, 12.3; ESI-TOF MS (Positive mode) calculation of C₁₃H₁₆NaN₄O[M+Na]267.13 was determined for m/z of + 267.12.

Synthesis of 1-3-5.1- (4-methoxyphenyl) -1H-1,2, 3-triazole-4-carbaldehyde (5)

Compound 5 (white solid) was synthesized as in example 1-3-1 using method a using 1-azido-4-methoxybenzene as a precursor. FIG. 5 shows¹H NMR spectrum.

The yield is 68 percent; 1H NMR (400MHz, DMSO-d6): δ 10.12(s,1H),8.43(s,1H),7.66(d, J ═ 8.9Hz,2H),7.06(d, J ═ 8.9Hz,2H),3.89(s, 3H);¹³c NMR (100MHz, DMSO-d6) delta 185.0,159.9,147.5,129.3,125.9,122.4,115.0, 55.7; ESI-TOF MS (Positive mode) calculation of C₁₀H₉NaN₃O-₂[M+Na]226.06 was determined for m/z of + 226.06.

Synthesis of 1-3-6.1-phenyl-1H-1, 2, 3-triazole-4-carbaldehyde (6)

Compound 6 (brown solid) was synthesized as in example 1-3-1 using method a using azidobenzene as a precursor. FIG. 6 shows¹H NMR spectrum.

The yield is 9%;¹H NMR(400MHz,DMSO-d₆₎:δ10.11(s,1H),9.57(s,1H),7.97(d,J＝8.0Hz,2H),7.66-7.54(m,3H).

EXAMPLE 2 Synthesis of Compound 2 (method B)

The equipment, reagents, solvents, etc. used were similar to those of example 1.

Synthesis of 2-1.1-benzyl-1H-1, 2, 3-triazole-4-carbaldehyde (7)

Compound 7 was synthesized with reference to published reports (j.t. fletcher, Tetrahedron lett.,2017,58, 4450-4454). Specific synthetic procedures and compound identification results are described below.

A mixture of copper sulfate pentahydrate 20(II) (47.8mg,0.19mmol) in water (7.5mL) and tert-butanol (7.5mL) was cooled to 0 deg.C and sodium azide (103mg,1.6mmol) was added to the mixture under nitrogen. After stirring at room temperature for 10 minutes, benzyl bromide (179. mu.L, 1.5mmol) and propargyl propionaldehyde diethylacetal (240. mu.L, 1.7mmol) were added to the mixture, and the mixture was stirred at 70 ℃ for 24 hours and at room temperature for 1 hour under air. The reaction solution was air-cooled to room temperature, and then diluted with saturated aqueous sodium chloride (5mL), followed by extraction with ethyl acetate (30 mL. times.3). The resulting organic layer was dried over magnesium sulfate. The filtrate from which the solid was filtered off was distilled under reduced pressure to give a crude product, which was purified by silica gel column chromatography (hexane: ethyl acetate ═ 2:1) to give compound 7 (white solid). FIG. 7 shows¹H NMR spectrum.

The yield is 46%;¹H NMR(400MHz,DMSO-d₆):δ10.00(s,1H),8.95(s,1H),7.41-7.32(m,5H),5.69(s,2H)；¹³C NMR(100MHz,DMSO-d₆) δ 185.0,147.0,135.3,128.9,128.4,128.3,128.1, 53.2; ESI-TOF MS (Positive mode) calculation of C₁₀H₉NaN₃O[M+Na]+210.06210.06 is obtained by the above method; FT-IR (ATR mode, gallium prism), v cm^-1:1694,1535,1237,1165,1052,877,796,767,714,701,565,556,543,532,515,505。

Synthesis of 2-2.1- (naphthalen-2-ylmethyl) -1H-1,2, 3-triazole-4-carbaldehyde (8)

Compound 8 (white solid) was synthesized as in example 2-1 using method B using 2- (bromomethyl) naphthalene as a precursor. FIG. 8 shows¹H NMR spectrum.

The yield is 20%;¹H-NMR(400MHz,CDCl₃) Delta 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) calculation of C₁₄H₁₁NaN₃O[M+Na]+260.08m/z to obtain 260.08; FT-IR (ATR mode, gallium prism), v cm^-1:3121,1709,1538,1239,1177,1052,1026,866,835,792,761,563,553,527,511。

EXAMPLE 3 Synthesis of Compound 3 (method C)

The equipment, reagents, solvents, etc. used were similar to those of example 1.

Synthesis of 3-1.1- (p-tolyl) -1H-1,2, 3-triazole-4-carbaldehyde (9)

Compound 9 was synthesized according to published reports (c. -z. tao, x. cui, j.li, ax. liu, l.liu, q. -x.guo, Tetrahedron lett.,2007,48, 3525-. Specific synthetic procedures and compound identification results are described below.

Pure water (5mL) containing copper (II) sulfate pentahydrate (47.8mg,0.19mmol) was added to a methanol solution (5mL) containing 4-methylphenylboronic acid (136mg, 1.0mmol) and sodium azide (98mg, 1.5mmol), and the mixture was stirred in the air for 5 hours. Subsequently, sodium ascorbate (79mg, 0.4mmol) and propargyl propionaldehyde diethyl acetal (286 μ L,1.7mmol) were added under nitrogen atmosphere, and the mixture was mixedThe mixture was stirred at 70 ℃ for 24 hours and at room temperature in air for 1 hour. The reaction solution was air-cooled to room temperature, and then diluted with saturated aqueous sodium chloride (40mL), followed by extraction with ethyl acetate (50 mL. times.3). The resulting organic layer was dried over magnesium sulfate. The filtrate from which the solid was filtered off was distilled under reduced pressure to give a crude product, which was dissolved in chloroform (3 mL). Pure water (3mL) and trifluoroacetic acid (3mL) were added, followed by vigorous stirring at room temperature. The mixture was diluted with saturated aqueous sodium chloride (30mL), and the organic layer was extracted with chloroform (30 mL. times.3). The resulting organic layer was dried over magnesium sulfate. The filtrate from which the solid was filtered off was distilled under reduced pressure to give a crude product, which was then purified by precipitation (hexane: chloroform) to give compound 9 (white solid). FIG. 9 shows¹H NMR spectrum.

The yield is 71%;¹H 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)；¹³C NMR(100MHz,CDCl₃) δ 185.3,148.2,140.3,134.0,130.7,123.2,120.9, 21.3; ESI-TOF MS (Positive mode) calculation of C₁₀H₉N₃ONa[M+Na]⁺210.06m/z, 210.06 was obtained.

EXAMPLE 4 Synthesis of Compound 4 (method D)

The equipment, reagents, solvents, etc. used were similar to those of example 1.

Synthesis of 4-1.1- (4-nitrophenyl) -1H-1,2, 3-triazole-4-carbaldehyde (10)

Reference is made to the published report for the synthesis of compound 10 (j.t. fletcher, j.e. reilly, Tetrahedron lett, 2011,52, 5512-. Specific synthetic procedures and compound identification results are described below.

A mixture of sodium azide (358mg,5.5mmol) in water (25mL) and tert-butanol (25mL) was cooled to 0 deg.C and nitrobenzene diazonium tetrafluoroborate (1.18g,5.0mmol) was added portionwise and then vigorously stirred for 1 hour. Subsequently, propargionaldehyde diethylacetal (783. mu.L, 5.5mmol) and sodium ascorbate (396 mg) were added under nitrogen2mmol) and then stirred at 70 ℃ overnight. The reaction solution was filtered, and then the filtrate was extracted with ethyl acetate (50 mL. times.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 reprecipitation (hexane: ethyl acetate) to obtain compound 10 (yellow solid). FIG. 10 shows¹H NMR spectrum.

The yield is 62%;¹H 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)；¹³C NMR(100MHz,DMSO-d₆) δ 185.0,147.8,147.4,140.3,126.9,125.6,121.5; ESI-TOF MS (Positive mode) calculation of C₉H₆N₄O₃Na[M+Na]⁺241.03m/z, 241.03 was obtained.

EXAMPLE 5 Synthesis of Compound 5 (method E)

The equipment, reagents, solvents, etc. used were similar to those of example 1.

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

The synthesis of compound 13 is described in reference to the published reports (J.Zhang, G.jin, S.Xiao, J.Wu, S.Guo, Tetrahedron 2013,69, 2352-.

Synthesis of ethyl 5-1, 5-methyl-1-phenyl-1H-1, 2, 3-triazole-4-carboxylate (11)

Ethyl acetoacetate (1.5mmol) and piperidine (20. mu.L, 0.2mmol) were added to a solution of azidobenzene (120mg,1.0mmol) in a mixture of dimethylformamide (10mL) and pure water (1mL), and the mixture was stirred at 80 ℃ for 24 hours. To the resulting solution was added water (20mL) to stop the reaction, followed by extraction with diethyl ether (20 mL. times.5). The resulting organic layer was washed with water (20 mL. times.2). The organic layer was dried over sodium sulfate, and the solvent of the filtrate obtained by filtration was removed by distillation under the reduced pressure. The resulting crude product was purified by silica gel column chromatography (hexane: ethyl acetate) to obtain compound 11 (white solid).

The yield is 27%;¹H 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)。

synthesis of (5-methyl-1-phenyl-1H-1, 2, 3-triazol-4-yl) methanol (12) sodium borohydride (29mg, 0.77mmol) was added to a solution of compound 11(0.12mmol) and sodium methoxide (1mg, 2 μmol) in methanol (1mL) under nitrogen atmosphere, followed by stirring at room temperature for 3 hours. An excess of 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 (5mL), followed by extraction with diethyl ether (20 mL. times.3) and washing with a saturated aqueous sodium chloride solution (5 mL. times.2). The resulting organic layer was dried over sodium sulfate. After that, filtration was performed, and the solvent of the filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (hexane: ethyl acetate ═ 2:1) to obtain compound 12 (white solid). FIG. 11 shows¹H NMR spectrum.

The yield is 37%;¹H 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)。

synthesis of 5-1-3.5-methyl-1-phenyl-1H-1, 2, 3-triazole-4-carbaldehyde (13)

Activated manganese dioxide (141mg, 1.6mmol) was added to a solution of compound 12(0.16mmol) in chloroform (5mL), and the mixture was stirred at room temperature under a nitrogen atmosphere 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).

The yield is 89%;¹H 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)；¹³C NMR(100MHz,DMSO-d₆):δ186.1,143.3,138.7,134.7,130.3，129.8，125.3，9.3。

synthesis of ethyl 5-1-4.1-phenyl-5- (trifluoromethyl) -1H-1,2, 3-triazole-4-carboxylate (14)

Compound 14 (yellow oil) was synthesized in the same manner as in example 5-1-1 using ethyl 4,4, 4-trifluoroacetoacetate as a precursor.

The yield is 35%;¹H 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)。

synthesis of (1-phenyl-5- (trifluoromethyl) -1H-1,2, 3-triazol-4-yl) methanol (15)

Compound 15 (white solid) was synthesized by the same method as in example 5-1-2 using Compound 14 as a precursor.

The yield is 83%;¹H NMR(400MHz,CDCl₃):δ7.61-7.55(m,3H),7.47(d,J＝7.2Hz,2H),4.96(d,J＝6.0Hz,2H)。

synthesis of (1-phenyl-5- (trifluoromethyl) -1H-1,2, 3-triazol-4-yl) methanol (16)

Compound 16 (white solid) was synthesized by the same method as in example 5-1-3 using Compound 15 as a precursor. FIG. 12 shows¹H NMR spectrum.

The yield is 75%;¹H NMR(400MHz,DMSO-d₆):δ10.21(s,1H),7.74-7.66(m,5H)。

EXAMPLE 6 Synthesis of Compound 6 (method F)

The equipment, reagents, solvents, etc. used were similar to those of example 1.

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

Compound 10(109mg,0.5mmol) and 2- (2- (2-propynyloxy) ethoxy]Ethylamine (72 μ L,0.5mmol) was dispersed in a mixture of pure water (1mL) and tert-butanol (1mL), and the mixture was stirred at 60 ℃ overnight under a nitrogen atmosphere, the reaction solution was cooled to 0 ℃, then 0.1M aqueous HCl (50mL) was added to stop the reaction, followed by extraction with ethyl acetate (20mL × 3), the organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (hexane: ethyl acetate ═ 0 to 60%) to give compound 17 (yellow oil). FIG. 13 is a drawing showing¹H NMR spectrum.

The yield is 70%;¹H 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)；¹³C NMR(100MHz,CD₃CN) delta 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) calculation of C₁₀H₁₃N₃O₃Na[M+Na]⁺246.08m/z, 246.08 was obtained.

Example 7 compound synthesis 7: synthesis of functional molecules

The equipment, reagents, solvents, etc. used were similar to those of example 1. The reaction precursors used are either commercially available or appropriately synthesized.

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

Using N- (2- (2- (2- (2-azidoethoxy) 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 (method a) as in example 1-3-1. FIG. 14 shows¹H NMR spectrum.

The yield is 23%;¹H NMR(400MHz,CD₃CN+DMSO-d₆(one drop)). delta.10.0 (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 ═ 5.6Hz,2H),3.27(t, J ═ 5.6Hz,2H),3.17-3.12(m,1H),2.88(dd, J ═ 5.0,12.6, 1H),2.63(d, 12.6, 1H), 3.17-3.12(m, 1H); ESI-TOF MS (Positive mode) calculation of C₂₁H₃₄N₆O₆Na[M+Na]⁺521.1, found 521.1.

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

With 5-azido-3 ', 6 ' -dihydroxy-3H-spiro [ isobenzofuran-1, 9' -xanthene]-3-ketone as a precursor, and Compound 19 (orange solid) was synthesized by the same method as in example 1-3-1 (method A). FIG. 15 shows¹H NMR spectrum.

The yield is 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)；¹³C NMR(100MHz,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) calculation of C₂₃H₁₄N₃O₆[M+H]⁺428.09m/z, 428.10 was obtained.

7-3 Synthesis of polyethylene glycol tethered triazole-4-carbaldehyde (20) (MW. -4 kDa)

Polyethylene glycol monomethyl ether having a molecular weight of about 4kDa (manufactured by Tokyo chemical Co., Ltd.)Starting materials, compound 20 (brown solid) was synthesized using the same method as in example 1-3-1, with reference to published reports (MB van Eldijk, FCM Smit, N.Vermue, MF Debets, S.Schoffelen, JCM van Hest, Biomacromolecules,2014,15, 2751-. Purification was carried out by reprecipitation with diethyl ether. FIG. 16 shows¹H NMR spectrum.

The yield is 50%;¹H NMR(400MHz,CDCl₃):δ10.14(s,1H),8.41(s,1H),4.63(t,J＝4.8Hz,1H),3.91-3.45(m,366H)。

synthesis of 7-4.1- (2- (2- (2- (2-azidoethoxy) ethoxy) ethyl) -1H-1,2, 3-triazole-4-carbaldehyde (21)

Compound 21 (yellow oil) was synthesized in the same manner as in example 1-3-1 (method A) using 1-azido-2- (2- (2- (2-azidoethoxy) ethoxy) ethane as a precursor. FIG. 17 shows¹H NMR spectrum.

The yield is 27%; 1H NMR (400MHz, CDCl)₃):δ10.1(s,1H),8.41(s,1H),4.62(t,J＝4.8Hz,2H),3.90(t,J＝4.8Hz,2H),3.68(m,4H),3.65-3.63(m,6H),3.38(t,J＝5.0Hz,2H)；13C NMR(100MHz,CDCl₃) δ 185.4,148.0,126.9,70.7(two signal we summed), 70.7(two signals combined), 70.2,69.0,50.8, 50.7; ESI-TOF MS (Positive mode) calculation of C₁₁H₁₈N₆O₄Na[M+Na]⁺321.1m/z, 321.1 was obtained.

7-5 Synthesis of Compound 22

Using the same procedure as in example 6 (method F), N- (3-aminopropionyl) -5, 6-dihydro-11, 12-didehydrodibenzo [ b, F)]Azacyclooctatetraine was used as a precursor for the synthesis of compound 22 (yellow solid). FIG. 18 shows¹H NMR spectrum.

The yield is 75%;¹H NMR(400MHz,CD₃CN):δ9.89(s,1H),7.88(s,1H),7.62(d,J＝7.2Hz,1H),7.45-7.43(m,4H),7.39-7.32(m,2H),7.22(dd,J＝1.4,7.4Hz,1H),5.06(d,J＝14Hz,1H),4.45(m,2H),3.66(d,J＝14Hz,1H),2.90(td,J＝6.0,17Hz,1H),2.35(td, J ═ 6.0,17Hz, 1H); ESI-TOF MS positive mode) to calculate C₁₁H₁₈N₆O₄Na[M+Na]⁺321.1m/z, 321.1 was obtained.

Example 8N-terminal modification of peptides 1

8-1. reagents, solvents, etc

The ultrapure water used was obtained by purification using Millipore Integral 3. As the other reagents and solvents, commercially available products can be used as they are.

8-2. peptide N-terminal modification

The method is directed to the N-terminus of the peptide. The peptide that can be targeted is a peptide in which the N-terminal amino group is unmodified and the second amino acid residue at the N-terminal is an amino acid other than proline. As a specific example, the N-terminal modification of angiotensin I is as follows.

The amino acid sequence of angiotensin I is shown below.

DRVYIHPFHL(SEQ ID NO:1)

N-terminal modifications of peptides were performed with reference to published reports (j.i.macdonald, h.k.munch, t.moore, m.b.francis, nat.chem.biol.2015,11, 326-331). The specific experimental procedure is described below.

A solution (200mM, 1. mu.L, 0.2. mu. mol, final concentration: 10mM) of Compound 7 in dimethyl sulfoxide (DMSO) was diluted with potassium phosphate buffer (10mM, pH 7.5, 17. mu.L). An aqueous peptide solution (1mM, 2. mu.L, 2nmol, final concentration: 100. mu.M) was added thereto, and the mixture was shaken at 37 ℃ for 4 hours. The percent modification (═ modified peptide/(total peptide amount)) was evaluated from the peak intensity in the mass spectrum using LC/MS. Fig. 19 shows the results. The percent modification 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 modifiers.

Example 9 peptide N-terminal modification 2

The reagents, equipment, solvents, etc. used were similar to those of 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.

9-1. peptide N-terminal modification

Structural analysis of the product of 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.

A solution (200. mu.L) of compound 24(44mg,0.4mmol) in Dimethylformamide (DMF) was added to compound 23(11mg,0.04mmol) in potassium phosphate buffer (10mM, pH 7.5,1.8mL), and the mixture was shaken at 37 ℃ for 16 hours. The solvent was distilled off under reduced pressure. The resulting crude product was purified by silica gel column chromatography (ethyl acetate: acetonitrile: water: 95:5:0 to 0:95:5) to give compound 25.

9-2. of Compound 25¹H NMR analysis

FIG. 20 shows the reaction of Compound 25 in deuterated acetonitrile¹And (5) attributing the results of the H NMR spectrum.

Two peaks at 5.7ppm were assigned to the 2-position specific proton H of the 4-imidazolidinone ring_eThis reaction was confirmed to form a 4-imidazolidinone ring at the N-terminus of the peptide. These two peaks are from isomers in which the asymmetric point is the 2-position of the 4-imidazolidinone ring.

Example 10N-terminal modification of protein 1

10-1. reagents, solvents, etc

Ribonuclease A (RNase) from bovine pancreas was purchased from Roche. The ultrapure water used was obtained by purification using Millipore Integral 3. As the other reagents and solvents, commercially available products can be used as they are.

10-2. protein modification

The method is directed to the N-terminus of the protein. The protein that can be targeted is a protein in which the N-terminal amino group is not modified and the second amino acid residue at the N-terminal is an amino acid other than proline. As a specific example, N-terminal modification of ribonuclease A (RNase) derived from bovine pancreas is described below.

The amino acid sequence of RNase (PDB: 1FS3) was as follows.

N-terminal modification of proteins was performed with reference to published reports (J.I.MacDonald, H.K.Munch, T.Moore, M.B.Francis, nat.chem.biol.2015,11, 326-331). The specific experimental procedure is described below.

A solution of Compound 7 (200mM, 2.5. mu.L, 0.5. mu. mol, final concentration: 10mM) in dimethyl sulfoxide (DMSO) was diluted with potassium phosphate buffer (10mM, pH 7.5, 45. mu.L). To this was added an ultrapure aqueous solution of RNase (1mM, 2.5. mu.L, 2.5nmol, final concentration: 50. mu.M), and the mixture was shaken at 37 ℃ for 16 hours. The percent modification (═ modified RNase/(total RNase amount)) was assessed from the peak intensity in the mass spectrum using LC/MS. Fig. 21 shows the results. The percent modification under the reaction conditions was calculated to be 88%. The product after the reaction was purified by size exclusion chromatography as needed.

Example 11 modification of the N-terminus of a protein with a functional molecule

11-1. reagents, solvents, etc

11-2 modification of the N-terminus of the protein with Biotin

The modification of the N-terminus of the protein with biotin was carried out in the same manner as in example 10-2 using compound 18 as a modifier. FIG. 22 shows the results of LC/MS analysis of the product. The percentage modification of the biotin moiety was calculated to be 79%.

11-3, modifying protein N-terminal with fluorescent dye

The N-terminus of the protein was modified with a fluorescent dye (a fluorescein molecule herein) by the same method as in example 10-2 using compound 19 as a modifying agent. FIG. 23 shows the results of LC/MS analysis of the product. The percentage of modification with respect to the fluorescent dye moiety was calculated to be 73%.

11-4 modification of the N-terminus of the protein with azido

The N-terminus of the protein was modified with an azide group by the same method as in example 10-2 using Compound 21 as a modifying agent. FIG. 24 shows the results of LC/MS analysis of the product. The percentage modification of azido groups was calculated to be 79%.

11-5 modification with functional molecules, starting from the modification of the azido group at the N-terminus of the protein

The azido-modified RNase prepared in example 11-4 was modified with a fluorescent dye by a strain-promoted alkyne-azide cycloaddition reaction. Compound 26, fluorescein having a dibenzocyclooctyne moiety as the alkyne substrate, was used in the reaction. Fig. 25 shows the protocol and results.

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

11-6 modification of the N-terminus of proteins with strained alkyne moieties

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. FIG. 26 shows the results of LC/MS analysis of the product. The percent modification of the cyclooctyne moiety was calculated to be 40%.

EXAMPLE 12 Synthesis of a resin immobilized with a reactant for heterogeneous reaction

The construction of a new N-terminal selectivity modifier by 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. Thus, fig. 27 shows a method of simplifying the removal of by-products (aniline derivatives) generated after the reaction by immobilizing various formaldehyde precursors on a resin or a solid material and directly performing N-terminal modification of proteins using the reacted solution.

12-1. equipment, reagents, solvents, etc. used

The equipment, reagents and solvents used were the same as in examples 1 and 8. The aminomethyl polystyrene resin used was purchased from Tokyo chemical industries, Ltd.

12-2 Synthesis of reactant-immobilized resin

As a specific example, fig. 28 shows the synthesis of a polystyrene resin having compound 27 immobilized thereon. The synthesis was performed according to the scheme shown in figure 29.

After an aminomethyl polystyrene resin (aminomethyl/PS resin, 200mg) was dispersed in DMF (30mL), the resin was swelled by shaking for 10 minutes. 5-azido-2-nitrobenzoic acid (208mg,1.0mmol), 1-hydroxybenzotriazole monohydrate (200mg,1.3mmol) and N, N' -diisopropylcarbodiimide (156. mu.L, 1.0mmol) were added to the resin dispersion and the mixture was shaken overnight. The resin after the reaction was filtered, washed with dimethylformamide-pure water-chloroform and acetone in this order, and dried under reduced pressure. Subsequently, in order to inactivate the unreacted amino group, the resulting resin was treated with a mixed solution of acetic anhydride (1mL) and chloroform (4mL) for 30 minutes, washed with chloroform-methanol and acetone, and dried under reduced pressure to give an azido-modified polystyrene resin (azido/PS resin).

After swelling the azido/PS resin (220mg) with a mixed solution of DMF/water (6:1,7mL), copper (II) sulfate pentahydrate (25mg,0.1mmol), sodium ascorbate (40mg,0.2mmol) and propargyl propionaldehyde diethylacetal (356. mu.L, 2.5mmol) were added to the resin dispersion and the mixture was shaken at room temperature for 12 hours. The reacted resin was filtered, and washed with dimethylformamide-pure water-chloroform, methanol and acetone in this order. Subsequently, the resin was treated with a 12% aqueous ammonia solution (5mL) for 10 minutes, filtered, and washed with pure water to remove the copper catalyst remaining on the surface of the resin. Finally, the obtained resin was treated with a mixed solution of hydrochloric acid aqueous solution/tetrahydrofuran (1:1, 5mL) 3 times for 2 minutes each, filtered, and the deprotected acetal protecting group was washed with pure water and acetone to obtain a resin (27/PS resin) having compound 27 immobilized thereon.

12-3. identification of resins

The chemical species on the surface of the resin (27/PS resin) on which the compound 27 was immobilized was determined by infrared spectroscopy. The results are shown in FIG. 30. In the azido-modified resin (azido/PS resin), at 2115cm^-1The telescopic vibration characteristic of the azide group was observed.In the resin (27/PS resin) after the triazole ring was formed by the CuAAC reaction, the azide stretching vibration peak disappeared, and 1699cm due to the aldehyde stretching vibration was newly observed^-1Is absorbed by the skin. The above results support the modification with aldehyde groups by the CuAAC reaction, indicating that 27/PS resin was prepared.

12-4 protein N-terminal modification

The prepared resin immobilized with a reactant (27/PS resin) was used for the preparation of N-terminal modifier and the subsequent N-terminal modification of protein. As a specific example, the N-terminal modification of ribonuclease A (RNase A) is as follows.

27/PS resin (5mg) was added to a dimethylsulfoxide solution (100mM, 50. mu.L, 5. mu. mol) of benzylamine, and the mixture was heated at 100 ℃ for 90 minutes using a heating block. After the resulting mixture was allowed to air cool to room temperature, the supernatant (10. mu.L, 1. mu. mol) was diluted with phosphate buffer (10mM, pH 7.5, 85. mu.L). To this was added RNase A solution (1mM, 5. mu.L, 5nmol), and the mixture was shaken at 37 ℃ for 16 hours. After the reaction, modification evaluation was performed using LC/MS. Even in the case of using the resin immobilized with a reactant (27/PS resin), a modification percentage (75%) equivalent to that in the case of using the N-terminal modifier isolated and purified was obtained. This result demonstrates a method of N-terminal modification of a protein by a subsequent reaction using a resin immobilized with a reactant (27/PS resin). Fig. 31 shows the results.

12-5. improvement of synthesis method of resin fixed with reactant

In example 12-3, in the sample obtained by protein modification, a product which seems to add oxygen to the protein was observed. This is considered to be because the copper catalyst used for resin synthesis remains on the resin. Thus, 27/PS resin was prepared under a new synthetic scheme and applied to protein modification. Specifically, a 27/PS resin was prepared according to the scheme shown in FIG. 32.

Synthesis of 12-5-1.5-azido-2-nitrobenzoic acid (28)

Sodium nitrite (727mg,10.5mmol) in water (6mL) was added to concentrated HCl/EtOH/H containing 5-amino-2-nitrobenzoic acid (1.60g,8.9mmol)₂O solvent mixture (2:1:2, 43mL total) and the mixture was stirred at 0 ℃ for 1 hour. Subsequently, sodium azide (868mg, 13.4mmol) was added in portions, and the solution was stirred vigorously at 0 ℃ for 1 hour, and then at room temperature for 1 hour.

The reaction solution was diluted with ultrapure water, filtered, and then washed with ultrapure water (20 mL. times.2). Drying under reduced pressure gave compound 28 (light yellow solid). FIG. 33 shows¹H NMR spectrum.

The yield is 37%;¹H NMR(400MHz,DMSO-d₆):δ,8.1(d,J＝9.4Hz,3H),7.43-7.41(m,2H)；¹³C NMR(100MHz,DMSO-d₆) δ,165.8,145.3,143.4,130.8,126.2,121.7,119.6; ESI-TOF MS (Positive mode) calculation of C₇H₄NaN₄O₄[M+Na]⁺231.012m/z, 231.011 was obtained.

Synthesis of 12-5-2.2, 5-dioxopyrrolidin-1-yl-5- (4-formyl-1H-1, 2, 3-triazol-1-yl) -2-nitrobenzoate (30)

Compound 28(1.75g,8.4mmol) and 3, 3-diethoxyprop-1-yne (1.2mL,1.08g,8.4mmol) were dispersed in a mixture of pure water (21mL) and tert-butanol (21 mL). Copper (II) sulfate pentahydrate (419mg, 1.68mmol, 20 mol%) and sodium ascorbate (666mg, 3.36mmol, 40 mol%) were added and stirred at 70 ℃ under nitrogen overnight. The reaction mixture was cooled to room temperature, and then saturated brine was added to stop the reaction, followed by extraction with ethyl acetate (20 mL. times.3). The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product containing compound 29, which was used directly for the next reaction without purification.

N-hydroxysuccinimide (764mg, 6.64mmol) was added to a DMF solution (22mL) containing the crude product, and the mixture was stirred at room temperature for 10 min. Subsequently, N' -dicyclohexylcarbon is addedDiimine (1.14g,5.5mmol) and the reaction mixture is stirred overnight. The precipitate was filtered off and the filtrate was dried under reduced pressure to remove the solvent. The residue was dissolved in ethyl acetate (50mL) and the resulting precipitate was filtered off again. The obtained filtrate was washed with saturated brine (20mL × 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. Purification by column chromatography on silica gel afforded compound 30 (pale yellow solid). FIG. 34 shows¹H NMR spectrum.

Yield 18% (after two steps);¹H 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)；¹³C NMR(100MHz,DMSO-d₆) Delta, 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) calculation of C₁₄H₉NaN₅O₇[M+Na]⁺382.038) m/z, to obtain 382.039.

12-5-3.27/PS resin Synthesis 2

After an aminomethyl polystyrene resin (aminomethyl/PS resin, 200mg) was dispersed in DMF (5mL), the resin was swelled by shaking for 1 hour. Compound 30(150mg,0.42mmol) and N, N' -diisopropylcarbodiimide (146. mu.L, 0.84mmol) were added to the resin dispersion and the mixture was shaken overnight. The resin after the reaction was filtered, washed with dimethylformamide-pure water-chloroform and acetone in this order, and dried under reduced pressure. Subsequently, in order to inactivate the unreacted amino group, the resulting resin was treated with a mixed solution of acetic anhydride (1mL) and chloroform (4mL) for 30 minutes, washed with chloroform-methanol and acetone, and dried under reduced pressure to give a 27/PS resin.

12-6 protein N-terminal modification

Preparation of the N-terminal modifier and subsequent N-terminal modification of the protein were carried out using a freshly prepared resin (27/PS resin) to which the reactant was immobilized. As a specific example, the N-terminal modification of ribonuclease A (RNase A) is as follows. This scheme is shown in figure 35.

27/PS resin (5mg) was added to a dimethylsulfoxide solution (100mM, 40. mu.L, 4. mu. mol) of benzylamine, and the mixture was heated at 100 ℃ for 90 minutes using a heating block. After the resulting mixture was allowed to air cool to room temperature, the supernatant (10. mu.L, 1. mu. mol) was diluted with phosphate buffer (10mM, pH 7.5, 85. mu.L). To this was added RNase A solution (1mM, 5. mu.L, 5nmol), and the mixture was shaken at 37 ℃ for 16 hours. After the reaction, modification evaluation was performed using LC/MS. Fig. 36 shows the results. Even in the case of using the resin immobilized with a reactant (27/PS resin), a modification percentage (82%) equivalent to the case of using the N-terminal modifier isolated and purified was obtained. In addition, it was demonstrated that the N-terminus of the protein can be specifically modified by the same method for various functional amine compounds.

Example 13 Synthesis of protein modifiers and subsequent proteins Using the Dimroth rearrangement reaction in a homogeneous System Texture modification

Construction of a novel N-terminal selective modifier by rearrangement reaction between compound 10 and the amine precursor shown in example 6 can be used as a clean reaction without the use of copper catalysts. Example 12 shows a diemol rearrangement reaction in a heterogeneous system to simplify the removal of by-products. On the other hand, it is also assumed that the presence of aniline derivatives as by-products does not cause problems in protein modification. Thus, this section shows the synthesis of formaldehyde derivatives and subsequent protein modification in a disco rearrangement reaction in a homogeneous system. Fig. 37 shows this scheme.

13-1. reagents, solvents, etc

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. The ultrapure water used was purified by Millipore Integral 3.

13-2 optimization of Dimroth rearrangement reaction conditions

Fig. 38 shows the reaction mechanism of the disco rearrangement reaction. Since the reaction includes imine formation as a first step, it is expected that an acid catalyst is added to improve the reaction efficiency. Thus, the diemroth rearrangement reaction with compound 10 was carried out using benzylamine as a model substrate in the presence of various acid catalysts.

A solution of compound 10 in dimethyl sulfoxide (200mM, 20. mu.L, 4. mu. mol) and an acidic aqueous solution (200mM or 400mM, 1. mu.L, 5 mol% or 10 mol%) were added to a solution of benzylamine in dimethyl sulfoxide (200mM, 20. mu.L, 4. mu. mol), and the mixture was heated at 100 ℃ for 30 minutes using a heating block. After air-cooling to room temperature, 1. mu.L of the reaction solution was diluted with a phosphate buffer (100mM, pH 7.0, 200. mu.L) and transferred to a 96-well plate for UV-visible absorption measurement. The conversion of the reaction was calculated from a standard curve drawn based on the absorption intensity at 380nm, which is characteristic of p-nitroaniline (a product). Fig. 39 shows the structure of the acid catalyst used, and table 1 shows the conversion obtained.

In Table 1, the conversion rates^aCalculated according to the absorption intensity of the by-product p-nitroaniline.^bThe amount of MOPS added was set to 10 mol%.

TABLE 1

The above results show that the addition of an acid catalyst improves the conversion. It is suggested that the addition of sulfonic acid is particularly effective. In addition, various sulfonates known for use as Goodpastel buffers have been found to improve the efficiency of the reaction. In general, since these sulfonates have no significant effect on the function or structure of the protein, the mixed solution of the diemol rearrangement reaction can be directly used for the subsequent protein modification reaction. 3-morpholinopropanesulfonic acid (MOPS) was selected as the acid catalyst for subsequent experiments.

13-3 protein N-terminal modification

A solution of compound 10 in dimethyl sulfoxide (200mM, 20. mu.L, 4. mu. mol) and an aqueous MOPS solution (200mM or 400mM, 1. mu.L, 5 mol% or 10 mol%) were added to a solution of benzylamine in dimethyl sulfoxide (200mM, 20. mu.L, 4. mu. mol), and the mixture was heated at 100 ℃ for 30 minutes using a heating block. After air cooling to room temperature, the reaction mixture (5. mu.L) was diluted with phosphate buffer (10mM, pH 7.5, 42.5. mu.L). To this was added RNase A solution (1mM, 2.5. mu.L, 5nmol), and the mixture was shaken at 37 ℃ for 16 hours. After the reaction, modification evaluation was performed using LC-MS. FIG. 40 shows the results of LC-MS analysis.

Various functional amine precursors (e.g., alkynes, azides, and fluorescent dyes) are used to prepare protein modifiers and to effect the introduction of the N-terminus of the protein. Further, even if the preparation of the N-terminal modifier and the protein modification reaction are continuously performed, the modification percentage is comparable to the case of using the N-terminal modifier which is isolated and purified. The results demonstrate a method for modifying the N-terminus of a protein by sequential reactions using a Dimroth rearrangement reaction in a homogeneous system.

Example 14 modification of the N-terminus of a protein Using the Bis-TA4C molecule and introduction of functional molecules by oxime formation

In the discorote rearrangement using a diamine having amino groups at both terminals as a precursor, a molecule having TA4C moieties introduced at both terminals (hereinafter referred to as "Bis-TA 4C") was obtained. In the modification of the N-terminus of the protein using this Bis-TA4C, an aldehyde moiety may be introduced into the N-terminus of the protein. One 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, functional molecules can also be removed as needed. Therefore, by using various diamines as precursors, a protein modification reaction was performed by performing a disco rearrangement reaction using compound 10.

14-1. reagents, solvents, etc

14-2. preparation of Bis-TA4C and protein modification reaction

A solution of compound 10 in dimethyl sulfoxide (200mM, 20. mu.L, 4. mu. mol) and an acidic aqueous solution (200mM or 400mM, 1. mu.L, 5 mol% or 10 mol%) were added to separate solutions of each of the diamines 31-36 in dimethyl sulfoxide (100mM, 20. mu.L, 2. mu. mol), and the mixtures were heated separately at 90 ℃ for 60 minutes using a heating block. After air cooling to room temperature, each reaction mixture (5. mu.L) was diluted with phosphate buffer (10mM, pH 7.5, 42.5. mu.L). To this was added RNase A solution (1mM, 2.5. mu.L, 5nmol), followed by shaking at 37 ℃ for 16 hours. After the reaction, modification evaluation was performed using LC-MS. FIG. 41 shows a scheme, and FIG. 42 shows the results of protein modification.

When Bis-TA4C having an alkyl chain or an oligoethylene glycol chain as a linker was used, a good modified protein (60% to 82%) was obtained. In contrast, Bis-TA4C having an aromatic skeleton such as aniline did not undergo a modification reaction. This is believed to be due to the very low solubility of Bis-TA 4C. Therefore, introduction of a moiety that increases water solubility is expected to increase reaction yield.

14-3 introduction of functional molecules into proteins modified with Bis-TA4C by oxime formation

The oxime-forming reaction was carried out starting from the aldehyde moiety introduced into the N-terminus of the protein having Bis-TA 4C. Specifically, the reaction was carried out according to the procedure shown in FIG. 43, with reference to published reports (M.Rashidian, MM Mahomodii, R.Shah, JK Dozier, CR Wagner, MD Distefano, Bioconjugate chem.2013,24, 333-. As an example, modifications using fluorescent dyes and polyethylene glycol are described below.

Bis-TA4℃ -modified RNase A (60. mu.M, 8.3. mu.L, 0.5. mu.L) prepared using compound 35 as a precursor was diluted with a phosphate buffer (50mM, pH 7.0, 35.7. mu.L) and a dimethylsulfoxide solution (5mM, 1. mu.L, 5. mu.L) containing hydroxylamine 37 and an aqueous solution (50mM, 5. mu.L, 0.25. mu.L) of M-phenylenediamine (M-PDA) as a catalyst were added thereto, followed by shaking at 4 ℃ for 6 hours. After the reaction, modification evaluation was performed using LC-MS. FIG. 44 shows the results of protein modification. Although some adducts of m-PDA were observed as catalysts, it was confirmed that the conversion to the oxime adduct was achieved in good yield.

Bis-TA4C modified RNase A (60. mu.M, 8.3. mu.L, 0.5nmol) prepared using compound 35 as a precursor was diluted with phosphate buffer (50mM, pH 7.0, 35.7. mu.L); and thereto were added hydroxylamine 38(2mg, 500nmol) and an aqueous solution (50mM, 5. mu.L, 0.25. mu. mol) of m-phenylenediamine (m-PDA) as a catalyst, followed by shaking at room temperature for 16 hours. After the reaction, the modification was evaluated by SDS-PAGE. FIG. 45 shows the results of protein modification.

For comparison, experiments were also performed using unmodified protein or polyethylene glycol having a hydroxyl group as a substrate. Band shift on SDS-PAGE was observed only in the combination of aldehyde group at the N-terminus of the protein and hydroxylamine moiety in polyethylene glycol, confirming that polyethylene glycol was introduced by oxime formation.

Example 15 Dimroth rearrangement under Mild conditions

The preparation of N-terminal modifiers by the Dimrot rearrangement reaction in homogeneous systems as shown in examples 13 and 14 requires high reaction temperatures of 90 ℃ to 100 ℃. Therefore, further improvements in precursor structure and reaction conditions are needed to accommodate substrates with low heat resistance. In order to achieve the reaction at a lower temperature, the structure of compound 10 as a precursor was modified. In particular, a compound in which a nitrophenyl moiety is changed to a substituent having a higher electron-withdrawing property can be expected to be used as a useful precursor because the intermediate is stable and the reverse reaction is unlikely to proceed. This section describes the synthesis of compounds according to this strategy and examples of applications to the disco rearrangement reaction.

15-1 Synthesis of Compounds

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

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

A solution of pentafluorobenzonitrile (850mg, 541. mu.L, 4.4mmol) and sodium azide (260mg, 4.0mmol) in acetonitrile (10mL) was stirred at 60 ℃ for 16 hours under a nitrogen atmosphere. Subsequently, propargionaldehyde diethylacetal (570 μ L, 4.0mmol) and copper (I) iodide (76mg, 0.4mmol) were added under nitrogen atmosphere, and the mixture was stirred at room temperature for 16 hours. The reaction solution was air-cooled to room temperature, and then diluted with saturated aqueous sodium chloride (40mL), followed by extraction with ethyl acetate (50 mL. times.3). Sulfur is used for the obtained organic layerMagnesium is dried, and the filtrate of which the solid is filtered out is 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). FIG. 46 shows¹H NMR spectrum.

The yield is 86%;¹H-NMR(400MHz,CDCl₃):δ7.97(s,1H),5.82(s,1H),3.79-3.64(m,4H),1.28(t,J＝7.1Hz,6H)。

synthesis of 15-1-12.2,3,5, 6-tetrafluoro-4- (4-formyl-1H-1, 2, 3-triazol-1-yl) benzonitrile (40)

Compound 39(688mg, 2.0mmol) was dissolved in chloroform (4 mL). Trifluoroacetic acid (2mL) was added thereto, and the mixture was stirred at room temperature for 16 hours. The solvent and trifluoroacetic acid were distilled off under reduced pressure to give the crude product. The crude product was purified by reprecipitation (hexane: chloroform) to obtain compound 40 (white solid). FIG. 47 shows¹H NMR spectrum.

The yield is 78%;¹H-NMR(400MHz,CDCl₃):δ10.26(s,1H),8.50(s,1H)。

15-2 Dimroth rearrangement reaction using Compound 40 as a precursor

To evaluate the reactivity of compound 40, a disco rearrangement reaction was performed using benzylamine as a substrate.

Compound 40 in dimethylsulfoxide (100mM, 40. mu.L, 4. mu. mol) and MOPS in water (200mM, 2. mu.L, 10 mol%) were added to benzylamine in dimethylsulfoxide (100mM, 40. mu.L, 4. mu. mol), and the mixture was heated at 40 ℃ for 12 hours using a heating block. After air cooling to room temperature, the reaction mixture was diluted with deuterated dimethyl sulfoxide (310 μ L). As an internal standard, a solution of 1,3, 5-trimethoxybenzene in deuterated dimethyl sulfoxide (400mM, 10. mu.L, 4mol) was added and the reaction was carried out¹H NMR measurement. The yield was calculated from the integrated value of the peak corresponding to the proton at the benzylic position of compound 7 as a product based on the integrated value of the internal standard. FIG. 48 shows the scheme, and FIG. 49 shows the result.

The results show that the diemol rearrangement reaction proceeds at low temperature with good yield when compound 40 is used, compared to the case of compound 10. This indicates that the introduction of an electron-withdrawing substituent at the N1 position of the triazole ring contributes significantly to the improvement of reactivity of the diemol rearrangement reaction.

Example 16N-terminal modification of proteins 2

Human Serum Albumin (HSA), which is commonly used as a protein substrate, was subjected to an N-terminal modification reaction.

16-1. reagents, solvents, etc

Human Serum Albumin (HSA) was purchased from Merck. The ultrapure water used was obtained by purification using Millipore Integral 3. As the other reagents and solvents, commercially available products can be used as they are. As HSA, a mixture with HSA that has undergone modification in a living body is used. Published reports have identified modified HSAs (a. kawakami, k.kubota, n.yamada, u.tagami, k.takehana, i.sonaka, e.suzuki, k.hirayama, FEBS j.,2006,273, 3346-.

16-2 protein modification

The method is directed to the N-terminus of the protein. The protein that can be a target is a protein in which the amino group at the N-terminus is unmodified and the second amino acid residue at the N-terminus is an amino acid other than proline. As specific examples, N-terminal modifications of human serum derived albumin (HSA) are described below.

The amino acid sequence of HSA (PDB: 1AO6) is as follows.

The N-terminal modification of the protein was carried out in the same manner as in example 10-2, using Compound 7 as a modifier and HSA as a target protein. FIG. 50 shows the results of LC/MS analysis of the product. The modified protein is referred to as "HSA 1". The percent modification under the reaction conditions was calculated to be 99% or higher. The product after the reaction was purified by size exclusion chromatography as needed.

Example 17 Dual modification of proteins targeting the N-terminus and cysteine residues of proteins

17-1. reagents, solvents, etc

The equipment, reagents, solvents, etc. used were similar to those of example 16.

17-2 cysteine residue modification in proteins

Cysteine residue modification in proteins is carried out with reference to published reports (X.Chen, H.Wu, C. -M.park, T.H.Poole, G.Keceli, N.O.Devrarie-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 procedure is described below.

A solution of Compound 41 (25mM, 40. mu.L, 1. mu. mol, final concentration: 500. mu.M) in a DMSO/water (1:1) mixture was diluted with phosphate buffer (100mM, pH 7.0, 1.86 mL). To this was added an ultrapure aqueous solution of HSA (1mM, 100. mu.L, 100nmol, final concentration: 50. mu.M), and the mixture was allowed to stand at 4 ℃ for 12 hours. After the reaction, modification evaluation was performed using LC/MS. Fig. 51 shows the results. The modified protein is referred to as "HSA 2". The percent modification under the reaction conditions was calculated to be 99% or higher. The product after the reaction was purified by size exclusion chromatography as needed.

17-3. N-terminal modification of proteins in which cysteine residues are modified

HSA2 prepared in example 17-2 was modified at the N-terminus of the protein using Compound 7. Fig. 52 shows the protocol and results.

A solution (200mM, 1. mu.L, final concentration: 10mM) of Compound 7 in dimethyl sulfoxide (DMSO) was added to a phosphate buffer solution (HSA2 concentration: 200. mu.M, final concentration: 50. mu.M, buffer solution concentration: 10mM, pH 7.5, 19. mu.L) containing HSA2, and the mixture was shaken at 37 ℃ for 16 hours. The modified protein is referred to as "HSA 3". The percent modification under the reaction conditions was calculated to be 72%.

Example 18 stability of proteins modified at the N-terminus

18-1. reagents, solvents, etc

The equipment, reagents, solvents, etc. used were similar to those of example 10.

18-2 stability of modified proteins over time

The phosphate buffer containing RNase-7 (RNase-7 concentration: 100. mu.M, final concentration: 10. mu.M, buffer solution concentration: 100mM, pH 7.0, 10. mu.L) was diluted with phosphate buffer (100mL, pH 7.0, 90. mu.L) and allowed to stand at 37 ℃ for 12 hours, 24 hours and 48 hours. For example, FIG. 53 shows the results of LC/MS analysis after 24 hours of standing.

The modification reaction involving the formation of a 4-imidazolinone ring at the N-terminus is an equilibrium reaction. From the results of LC/MS analysis, it is considered that the unmodified protein and the modifying agent are regenerated by 4-imidazolidinone ring hydrolysis (which is a reverse reaction) in the N-terminally modified protein. Fig. 54 shows a graph whose vertical axis represents the release amount calculated from the intensity of peaks in the mass spectrum (═ 1- (modified RNase/total RNase amount after standing)/(modified RNase/total RNase amount before standing)); the horizontal axis represents the time during which RNase was allowed to stand.

18-3 stability of modified proteins with changes in pH

Phosphate buffer containing RNase-7 (RNase-7 concentration: 100. mu.M, final concentration: 10. mu.M, buffer solution concentration: 100mM, pH 7.0, 10. mu.L) was diluted with buffer (100mL, 90. mu.L) to pH 4,5, 6, 7, 8, and left at 37 ℃ for 12 hours. The dilution was performed using acetate buffer for the conditions of pH 4 and 5, and phosphate buffer for the conditions of pH 6, 7 and 8. Fig. 55 shows the results of the release.

EXAMPLE 19 Synthesis of Compound 8 (Synthesis of triazolecarboxaldehyde having substituents at 1-and 5-positions)

Reagents and solvents used in the synthesis were used as they were in commercial products. Azides, acetylenes and pinacolborates as precursors are reported in the published literature (L.S.C. -Verduyn, L.Mirfeizi, R.A.Dierckx, P.H.Elsinga, B.L.Feringga, chem.Commun.,2009,16, 2139. J. 2140; E.Jahnke, J.Weiss, S.Neuhaus, T.N.Hoheisel H.Frauendrath, chem.Eur.J.,2009,15, 388. 404; J.C.Pieck, D.Kuch, F.Grolle, U.Linne, C.Haas, T.Carell, J.Amm.Chem.Soc., 2006,128,1404-, J.midt, M.Roster, T.iser, Wittin, J.Am, J.Chem.Chem.R.R.R.R.R.R.D.J., Pluronic, R.R.R.D.D.E.D.D.D.E.D.D.H.E.E.S.S.S.E.S.S.E.E.S.E.S.E.R.D.S.E.E.E.S.S.E.E.S.S.S. K.E.E.E.E.E.E.S.E.C. P.S.S.S.S. P.S. C. P.S. No. Pat. No. Pat. No. 7, J. Pat. No. As, J. Pat. D. Pat. D. Pat. No. D. Pat. No. D. Pat. D. No. D. Pat. No. D. No. Pat. D. Pat. No. Pat. No. D. No. D. Pat. D..

Synthesis of 19-1.1-benzyl-5-phenyl-1H-1, 2, 3-triazole-4-carbaldehyde (44)

Compound 44 was synthesized according to the following scheme, with reference to published reports (K.Yamamoto, T.Bruun, J.Y.Kim, L.Zhang, M.Lautens, org.Lett.,2016,18, 2644-.

Synthesis of 19-1-1.1-benzyl-5-iodo-1H-1, 2, 3-triazol-4-yl) methanol (42)

THF (89mL) was added to a mixture of benzyl azide (0.67g,5.0mmol), 3-iodoprop-2-ynyl-1-ol (0.91g,5.0mmol), copper (I) iodide (95mg,0.50mmol), TBTA (0.27g,0.50mmol) and potassium acetate (1.5g,15mmol) under a nitrogen atmosphere and the mixture was stirred at room temperature overnight. The solvent of the reaction mixture was distilled off under reduced pressure, diluted with water (30mL) and ethyl acetate (30mL), and then extracted with ethyl acetate (30 mL. times.3). The resulting organic layer was washed with saturated brine (30mL × 2) and dried over sodium sulfate, and the solvent of the filtrate was removed by filtration by distillation under the reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate) to give compound 42 (white solid). FIG. 56 shows¹H NMR spectrum.

Yield 82%:¹H 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):¹³C NMR(100MHz,DMSO-d₆) Delta, 151.30,135.52,128.89,128.20,127.54,83.79,54.99,53.25 ESI-TOF MS (Positive mode) calculation of C₁₀H₁₀IN₃NaO[M+Na]⁺337.976m/z, 337.977 was obtained.

Synthesis of (1-benzyl-5-phenyl-1H-1, 2, 3-triazol-4-yl) methanol (43)

Pure water (2.4mL) was added to a mixture of compound 42(0.24g,0.75mmol), phenylboronic acid (0.18g,1.5mmol), potassium carbonate (0.21g,1.5mmol), and palladium acetate (17mg,0.075 mmol). Thereafter, THF (9.6mL) was added under a nitrogen atmosphere, and the mixture was stirred at 70 ℃ overnight. The solvent of the reaction mixture was distilled off under reduced pressure, and water (30mL) and ethyl acetate (30)mL) was diluted, followed by extraction with ethyl acetate (30mL × 3). The obtained organic layer was washed with saturated brine (30mL × 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 give compound 43 (pale yellow solid). FIG. 57 shows¹H NMR spectrum.

The yield is 54%;¹H NMR(400MHz,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 ═ 6.0Hz,2H),2.16(t, J ═ 6.0Hz,1H) ESI-TOF MS (positive mode) calculation of C₁₆H₁₅N₃NaO[M+Na]⁺288.111m/z, 288.112 was obtained.

Synthesis of 19-1-3.1-benzyl-5-phenyl-1H-1, 2, 3-triazole-4-carbaldehyde (44)

Activated manganese dioxide (163mg, 1.9mmol) was added to a solution (10mL) of compound 43(50mg, 0.19mmol) in 1, 4-dioxane, and the mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the solvent of the resulting filtrate was distilled off under reduced pressure. Thereafter, the resulting crude product was purified by flash column chromatography (hexane: ethyl acetate) to obtain compound 44 (oil). FIG. 58 shows¹H NMR spectrum.

The yield is 91%; 1H NMR (400MHz, CDCl)₃) δ,10.14(s,1H),7.56-7.46(m,3H),7.31-7.27(m,5H),7.07-7.05(m,2H),5.49(s,2H) ESI-TOF MS (Positive mode) calculation of C₁₆H₁₃NaN₃O[M+Na]⁺286.095m/z, 286.093 was obtained.

Synthesis of (1-benzyl-5- (4-methoxyphenyl) -1H-1,2, 3-triazol-4-yl) methanol (45)

Compound 45 (pale yellow solid) was synthesized in the same manner as in example 19-1-2, using 4-methoxyphenylboronic acid as a precursor. FIG. 59 shows¹H NMR spectrum.

The yield is 97%;¹H 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):¹³C NMR(100MHz,CDCl₃) Delta, 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) calculation of C₁₇H₁₇N₃NaO₂[M+Na]⁺318.121m/z, 318.121 was obtained.

Synthesis of 19-1-5.1-benzyl-5- (4-methoxyphenyl) -1H-1,2, 3-triazole-4-carbaldehyde (46)

Compound 46 (oil) was synthesized by the same method as in example 19-1-3 using Compound 45 as a precursor. FIG. 60 shows¹H NMR spectrum.

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):¹³C NMR(100MHz,CDCl₃) Delta 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) calculation of C₁₇H₁₅N₃NaO₂[M+Na]⁺316.106m/z, 316.104 was obtained.

Synthesis of (1-benzyl-5- (4-nitrophenyl) -1H-1,2, 3-triazol-4-yl) methanol (47)

Compound 47 (brown solid) was synthesized by the same method as in example 19-1-2 using 4-nitrophenylboronic acid as a precursor. FIG. 61 shows¹H NMR spectrum.

Yield 58%:¹H 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):¹³C NMR(100MHz,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) calculation of C₁₆H₁₄N₄NaO₃[M+Na]⁺333.096m/z, 333.095 was obtained.

Synthesis of 19-1-7.1-benzyl-5- (4-nitrophenyl) -1H-1,2, 3-triazole-4-carbaldehyde (48)

Compound 48 (yellow solid) was synthesized in the same manner as in example 19-1-3 using Compound 47 as a precursor. FIG. 62 shows¹H NMR spectrum.

Yield 93%:¹H 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):¹³C NMR(100MHz,CDCl₃) Delta, 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) calculation of C₁₆H₁₂N₄NaO₃[M+Na]⁺331.080m/z, 331.082 was obtained.

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

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. FIG. 63 shows¹H NMR spectrum.

Yield 40%:¹H 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(100MHz,CDCl₃) Delta 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) calculation of C₁₂H₁₂IN₃NaO₂[M+Na]⁺379.987m/z, to obtain 379.987。

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

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-dioxaborane as precursors. FIG. 64 shows¹H NMR spectrum.

Yield 71%:¹H 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(100MHz,CDCl₃) Delta 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) calculation of C₁₉H₁₈N₆NaO₂[M+Na]⁺385.138m/z, 385.138 was obtained.

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

Compound 51 (clear, oily) was synthesized in the same manner as in example 19-1-3, using Compound 50 as a precursor. FIG. 65 shows¹H NMR spectrum.

Yield 85%:¹H NMR(400MHz,CDCl₃):δ,10.17(s,1H),7.88(d,J＝8.0Hz,2H),7.44(d,J＝8.0Hz,2H),7.28(d,J＝8.0Hz,2H),7.14(d,J＝8.0Hz,2H),5.55(s,2H),4.45(s,2H),2.58(s,3H)；13C NMR(100MHz,CDCl₃) Delta 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) calculation of C₁₉H₁₆N₆NaO₂[M+Na]⁺383.123m/z, 383.123 was obtained.

Example 20N-terminal modification of peptides 3

The N-terminus of the peptide was modified with reference to published reports (J.I.MacDonald, H.K.Munch, T.Moore, M.B.Francis, nat.chem.biol.2015,11, 326-331). The specific experimental procedure is described below.

A solution of compound 44 in dimethyl sulfoxide (DMSO) (200mM, 2. mu.L, 0.4. mu. mol, final concentration: 10mM) was diluted with phosphate buffer (10mM, pH 7.5, 34. mu.L). An aqueous peptide solution (1mM, 4. mu.L, 4nmol, final concentration: 100. mu.M) was added thereto, and the mixture was shaken at 37 ℃ for 16 hours. The percent modification (═ modified peptide amount)/(total peptide amount)) was evaluated from the peak intensity in the mass spectrum using LC/MS. Fig. 66 shows the results. The percent modification under the reaction conditions was calculated to be > 99%.

Example 21N-terminal modification of proteins 4

Protein N-terminal modifications were made with reference to published reports (J.I.MacDonald, H.K.Munch, T.Moore, M.B.Francis, nat.chem.biol.2015,11, 326-331). The specific experimental procedure is described below.

A solution (200mM, 5. mu.L, 1.0. mu. mol, final concentration: 10mM) of compound 44 in dimethyl sulfoxide (DMSO) was diluted with a phosphate buffer (10mM, pH 7.5, 90. mu.L). To this was added an ultrapure aqueous RNase solution (1mM, 5. mu.L, 5nmol, final concentration: 50. mu.M) and the mixture was shaken at 37 ℃ for 16 hours. The percent modification (═ modified RNase)/(total RNase amount)) was estimated from the peak intensity in the mass spectrum by using LC/MS. Fig. 67 shows the results. The percent modification under the reaction conditions was calculated to be 48%.

Example 22 modification of the N-terminus of a protein with a functional molecule

22-1, double modification of N-terminal of protein by acetyl and azide

Using compound 51 as a modifier, the N-terminus of the protein was double-modified with acetyl group and azide group by the same method as in example 21. FIG. 68 shows the results of LC/MS analysis of the product. The percentage modification of acetyl and azido groups was calculated to be 28%.

22-2. modification with functional molecule, starting from modification of the azido group at the N-terminus of the protein

The azido-modified RNase prepared in example 22-1 was modified with a fluorescent dye by a strain-promoted alkyne-azide cycloaddition reaction. Compound 26, fluorescein having a dibenzocyclooctyne moiety as the alkyne substrate, was used in the reaction. Fig. 69 shows the protocol and results.

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

Example 23 introduction of Polymer molecules into the N-terminus of proteins

23-1 Synthesis of polyethylene glycol with triazole Formaldehyde moiety

Polyethylene glycol 54 having a triazole formaldehyde moiety incorporated therein was synthesized. The synthesis was carried out according to the following scheme with reference to published reports (L.Rocard, A.Berezin, F.De Leo, D.Bonifaizi, Angew.chem.int.Ed.,2015,54, 15739-.

Synthesis of 23-1-1.4- ((4-formyl-1H-1, 2, 3-triazol-1-yl) methyl) benzoic acid (52)

Compound 52 (white solid) was synthesized by using the same method (method a) as in example 1-3-1, using (4-azidomethyl) benzoic acid as a precursor. FIG. 70 shows¹H NMR spectrum.

The yield is 71%;¹H 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)；¹³C NMR(100MHz,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) calculation of C₁₁H₉N₃O₃Na[M+Na]⁺254.054m/z, 254.053 was obtained.

Synthesis of 23-1-2.2, 5-dioxopyrrolidin-1-yl-4- ((4-formyl-1H-1, 2, 3-triazol-1-yl) methyl) benzoate (53)

N-hydroxysuccinimide (138mg, 1.2mmol) and N, N' -dicyclohexylcarbodiimide (206mg, 1.0mmol) were added to a THF solution (10mL) containing compound 52(231mg, 1.0mmol), and the mixture was stirred at 0 ℃ for 24 hours under a nitrogen atmosphere. The precipitate was filtered off, and the filtrate was diluted with ethyl acetate (50mL) and washed with saturated aqueous sodium bicarbonate (20 mL. times.2) and saturated brine (20 mL. times.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 give compound 53 (white solid). FIG. 71 shows¹H NMR spectrum.

The yield is 71%;¹H 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)；¹³C NMR(100MHz,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) calculation of C₁₅H₁₂N₄O₅Na[M+Na]⁺351.070m/z, 351.069 was obtained.

23-1-2. Synthesis of polyethylene glycol tethered triazole-4-carbaldehyde (54)

Amino-terminated polyethylene glycol (molecular weight: about 4000, 400mg, 0.1mmol) and triethylamine (45L, 0.32mmol) were added to dichloromethane (15mL) containing compound 53(53mg, 0.16mmol), and the mixture was stirred under nitrogen for 24 hours. After the solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography. The resulting oily crude product was purified by reprecipitation (diethyl ether/dichloromethane) to yield compound 54 (white solid).

23-2 modification of the N-terminus of the protein with Compound 54

The N-terminus of the protein was modified with polyethylene glycol using compound 54. The following are examples using ribonucleases as substrates.

23-2-1. reagents, solvents, etc

23-2-2 polyethylene glycol modified protein N-terminal

The N-terminus of the protein was modified with polyethylene glycol in the same manner as in example 10-2 using compound 54 as a modifying agent. FIG. 72 shows the result of SDS-PAGE analysis of the product. The percentage modification of the polyethylene glycol moiety was calculated to be 50%.

23-2-3 modification of the N-terminus of the protein with a polymer in which the cysteine residue is modified

The N-terminus of HSA2 protein was modified with polyethylene glycol by the same method as in example 17-3 using compound 54 as a modifier. HSA (abbreviated as "HSA 4") obtained by the reaction, in which a cysteine residue is modified with an azide group and the N-terminus is modified with polyethylene glycol, is modified with a fluorescent dye by promoting alkyne-azide cycloaddition reaction by strain using compound 26. The modified protein is referred to as "HSA 5".

A dimethyl sulfoxide (DMSO) solution (6mM, 1. mu.L, final concentration: 300. mu.M) of compound 26 was added to a phosphate buffer solution (HSA4 concentration: 62. mu.M, final concentration: 10. mu.M, buffer concentration: 10mM, pH 7.5, 19. mu.L) containing HSA4, and the mixture was allowed to stand at 4 ℃ for 6 hours. The same procedure was also performed for HSA and HSA2, and the product was analyzed using SDS-PAGE. Fig. 73 shows the protocol and results.

When reacted with compound 26, fluorescence was observed only in lanes of HSA2 and HSA4 (both modified with compound 41 to introduce an azide group). In addition, a new band with increased molecular weight, which is a polyethylene glycol derivative, appears only in lane HSA4 modified with compound 54. These results confirm that polyethylene glycol modification and fluorescent dye modification were performed in the protein.

Sequence listing

<110> national university Council 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> Intelligent people

<400> 1

Asp Arg Val Tyr Ile His Pro Phe His Leu

1 5 10

<210> 2

<211> 124

<212> PRT

<213> cattle

<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> Intelligent people

<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 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 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

Ser Ala Lys Gln Arg Leu Lys Cys Ala 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 Lys Leu Val Thr Asp Leu Thr Lys

225 230 235 240

Val His Thr Glu Cys Cys His Gly Asp Leu Leu 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

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 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 Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

385 390 395 400

Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys

420 425 430

Val Gly Ser Lys Cys 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

119页详细技术资料下载

上一篇：一种医用注射器针头装配设备

下一篇：一种CDK激酶抑制剂及其应用

Molecules for modifying proteins and/or peptides

相关技术

网友询问留言