TRYPTOPHAN SELECTIVE MODIFIER, AND METHOD FOR PRODUCING Trp-S BOND-CONTAINING COMPOUND USING THE SAME

Abstract

To provide means capable of chemically modifying biomolecules such as peptides and proteins with high efficiency, without employing the harsh reaction conditions like conventional techniques.SOLUTION: A compound represented by chemical formula 1 or a salt thereof is employed as a tryptophan-selective modifier to selectively modify tryptophan residues in molecules, which contain the tryptophan residues, in the presence of a compound containing a thioether group. Each symbol in chemical formula 1 is as defined in the specifications.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a tryptophan-selective modifying agent and a method for producing a Trp-S bond-containing compound using the same. [Background technology]

[0002] Chemical modification of biomolecules such as peptides and proteins is an important fundamental technology that leads to the creation of pharmaceuticals and the elucidation of physiological functions. Therefore, the development of more efficient chemical modification techniques and the construction of new methodologies have the potential to contribute to the creation of revolutionary functional molecules in the fields of drug discovery and chemical biology research.

[0003] Common chemical modifications of biomolecules such as peptides and proteins are carried out by the active ester method using N-hydroxysuccinimide or the conjugate addition reaction method using maleimide derivatives for nucleophilic amino acid residues such as lysine and cysteine ​​residues, respectively. However, these residues often play important roles in the expression of physiological functions of biomolecules, and chemical modification may impair these functions. In addition, there are cases where multiple residues of both types exist in a protein, making it difficult to obtain highly homogeneous modifications.

[0004] On the other hand, chemical modification methods targeting tryptophan residues in biomolecules have been developed. Tryptophan is the rarest amino acid in eukaryotes, accounting for only about 1% of all amino acids (Non-Patent Document 1), but it is present in most proteins. Therefore, specific and selective reactions with respect to this residue may lead to the creation of promising chemical modification methods. To date, modification methods using "oxidation methods" using oxidizing agents such as N-bromosuccinimide and sulfoxide (Conventional Method 1; Non-Patent Document 2) and "CH activation method" of indole rings using heavy metal reagents such as palladium catalysts have been reported (Conventional Method 2; Non-Patent Document 3). However, unlike nucleophilic amino acids, tryptophan has low reactivity, so that modification of tryptophan using conventional methods requires harsh reaction conditions such as strong acidity, high temperature, and the use of harmful heavy metal reagents and organic solvents, and is often difficult to apply to biomolecules. In addition, when using oxidizing agents, the by-production of peroxides is also a problem.

[0005] In recent years, focusing on the fact that tryptophan has the highest molar absorptivity and photoionization ability among natural amino acids, electrochemical modification methods using photoinduced electron transfer have been reported (Non-Patent Document 4). Such methods can also be applied to tyrosine residues (Non-Patent Document 5). These electrochemical modification methods enable chemical modification under mild conditions (aqueous buffer, low concentration) for biomolecules, but it is necessary to consider the reaction efficiency when scaling up.

[0006] For these reasons, there is a need to develop unique and efficient chemical modification methods that differ from conventional methods. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] D. Gilis et al., Genome Biol., 2001, 2(11), research0049.1-0049.12. [Non-Patent Document 2] D. Manzanares et al., Biochemistry, 2007, 46, 5604-5615. [Non-Patent Document 3] J. Ruiz-Rodriguez et al., Chem. Eur. J., 2010, 16, 1124-1127. [Non-Patent Document 4] SJ Tower et al., J. Am. Chem. Soc., 2020, 142, 9112-9118. [Non-Patent Document 5] S. Sato et al., Angew. Chem. Int. Ed., 2013, 52, 8681-8684. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a means for achieving highly efficient chemical modification of biomolecules such as peptides and proteins without using harsh reaction conditions as in conventional methods. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems, and have surprisingly found that a compound having a specific chemical structure forms a covalent bond with a tryptophan residue in the presence of a thioether compound, thereby completing the present invention.

[0010] That is, according to one aspect of the present invention, there is provided a tryptophan selective modifier for selectively modifying a tryptophan residue in a molecule containing a tryptophan residue in the presence of a compound having a thioether group, the tryptophan selective modifier comprising a compound represented by the following chemical formula 1 or a salt thereof:

[0011] [ka]

[0012] In Chemical Formula 1, V is a nitrogen atom or a carbon atom; W together with other ring atoms forms a nitrogen-containing heterocycle selected from the group consisting of a pyridine ring, a pyrazine ring, an imidazole ring, an oxazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, a phenanthroline ring, a pteridine ring, and an azocine ring when V is a nitrogen atom, or forms an aromatic ring selected from the group consisting of a benzene ring, a 1H-pyrrole ring, a furan ring, a naphthalene ring, a benzo[h]quinoline ring, a pyrido[2,3-b]pyrazine ring, and a cycloocta-1,3,5,7-tetraene ring when V is a carbon atom; X is -O-, -NH- or a single bond; Y is a chlorine atom when X is a single bond, and is a group selected from the group consisting of a substituted or unsubstituted C1-C20 alkyl group, a substituted or unsubstituted C2-C20 alkenyl group, a substituted or unsubstituted C2-C20 alkynyl group, a substituted or unsubstituted C3-C20 cycloalkyl group, a substituted or unsubstituted C3-C20 cycloalkenyl group, a substituted or unsubstituted C6-C20 aryl group, a substituted or unsubstituted C3-C20 heteroaryl group, and a substituted or unsubstituted monovalent group derived from an electron-withdrawing aliphatic heterocycle when X is -O- or -NH-; Z represents a nitro group present on the nitrogen-containing heterocycle or the aromatic ring; p, q and r are each independently 0 or 1; s represents an integer from 0 to 10; L 0 and L 1 each independently represents a linker having a chemically stable structure, A a and A bare each independently a group selected from the group consisting of -CH=CH-, -C≡C-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, a C1 to C20 oxyalkylene group, a C1 to C20 alkyleneoxy group, -C(=O)-NH-, -NH-C(=O)-, -NH-C(=O)-NH-, hydrazine, triazole, sulfone, sulfoxide, sulfonic acid ester, sulfonamide, sulfinic acid ester, sulfinamide, piperidine, and dioxane, R is a hydrogen atom, a substituted or unsubstituted C1 to C20 alkyl group, a substituted or unsubstituted C2 to C20 alkenyl group, a substituted or unsubstituted C2 to C20 alkynyl group, a substituted or unsubstituted C3 to C20 cycloalkyl group, a substituted or unsubstituted C3 to C20 cycloalkenyl group, a substituted or unsubstituted C6 to C20 aryl group, a substituted or unsubstituted C3 to C20 heteroaryl group, an amino group, a hydroxyl group, a group G containing at its terminal a group capable of covalently bonding to a specific functional group possessed by an arbitrary compound, or a group FM derived from a functional substance.

[0013] According to another aspect of the present invention, there is provided a method for producing a Trp-S bond-containing compound, comprising contacting compound A having a tryptophan residue in the molecule with the tryptophan-selective modifier according to the above aspect in a reaction system in which compound B having a thioether group is present, thereby forming a covalent bond between a sulfur atom contained in the compound represented by chemical formula 1 and the tryptophan residue. Effect of the Invention

[0014] According to the present invention, it is possible to achieve chemical modification of tryptophan residues without using harsh reaction conditions as in conventional methods. By utilizing this, it is possible to selectively modify tryptophan residues located near methionine residues contained in molecules such as peptides and proteins. In addition, by utilizing the present invention, it is also possible to link various functional substances to tryptophan residues. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is an HPLC chart showing the time course of the reaction under the reaction conditions of Entry 1 when an Npys-modified tryptophan derivative was synthesized in the Examples. [Diagram 2] FIG. 2 shows a putative reaction mechanism of sulfenylation at tryptophan residues using the tryptophan-selective modifying agent of the present invention. [Diagram 3] FIG. 3 is an HPLC chart showing the time course of a sulfenylation reaction carried out on a peptide (Ac-Ala-Ala-Trp-Ala-Met-NH2) using the tryptophan-selective modifying agent of the present invention in an example. [Figure 4] FIG. 4 is an HPLC chart showing the time course of a sulfenylation reaction carried out on a peptide (Ac-neuromedin B) using the tryptophan-selective modifying agent according to the present invention in an example. [Diagram 5] FIG. 5 shows an HPLC chart illustrating the time course of a sulfenylation reaction carried out on a protein (lysozyme) using the tryptophan-selective modifying agent of the present invention in an example, and the results of mass spectrometry of compounds contained in the reaction mixture. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Hereinafter, an embodiment of the present invention will be described.

[0017] One aspect of the present invention is a tryptophan-selective modifier for selectively modifying a tryptophan residue in a molecule containing a tryptophan residue, comprising a compound represented by the following chemical formula 1 or a salt thereof in the presence of a compound having a thioether group:

[0018] [ka]

[0019] In Chemical Formula 1, V is a nitrogen atom or a carbon atom, and is preferably a nitrogen atom. When V is a nitrogen atom, W together with other ring atoms forms a nitrogen-containing heterocycle selected from the group consisting of a pyridine ring, a pyrazine ring, an imidazole ring, an oxazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, a phenanthroline ring, a pteridine ring, and an azocine ring, and when V is a carbon atom, forms an aromatic ring selected from the group consisting of a benzene ring, a 1H-pyrrole ring, a furan ring, a naphthalene ring, a benzo[h]quinoline ring, a pyrido[2,3-b]pyrazine ring, and a cycloocta-1,3,5,7-tetraene ring. That is, in a preferred embodiment, V is a nitrogen atom, and W together with other ring atoms forms the nitrogen-containing heterocycle. More preferably, V is a nitrogen atom, and W together with other ring atoms forms a pyridine ring.

[0020] In Chemical Formula 1, X is -O-, -NH- or a single bond. Among these, X is preferably -O-.

[0021] In Chemical Formula 1, when X is a single bond, Y is a chlorine atom. Here, when V is a nitrogen atom (W forms a pyridine ring), an example of a compound in which X is a single bond and Y is a chlorine atom is 3-nitro-2-pyridinesulfenyl chloride (Npys-Cl). On the other hand, when X is -O- or -NH-, Y is a group selected from the group consisting of a substituted or unsubstituted C1-C20 alkyl group, a substituted or unsubstituted C2-C20 alkenyl group, a substituted or unsubstituted C2-C20 alkynyl group, a substituted or unsubstituted C3-C20 cycloalkyl group, a substituted or unsubstituted C3-C20 cycloalkenyl group, a substituted or unsubstituted C6-C20 aryl group, a substituted or unsubstituted C3-C20 heteroaryl group, and a substituted or unsubstituted monovalent group derived from an aliphatic heterocycle having electron-withdrawing properties. Here, the "monovalent group derived from an aliphatic heterocycle having electron-withdrawing properties" corresponds to an aliphatic heterocycle compound having an electron-withdrawing structure, and examples thereof include monovalent groups obtained by removing a hydroxyl group from an alcohol forming an active ester of a carboxylic acid in peptide synthesis. More specifically, the monovalent groups described in Tables 5.7 to 5.11 on pages 164 to 173 of "Continued Development of Pharmaceuticals (Vol. 14, Peptide Synthesis)" (Hirokawa Shoten) or monovalent groups derived from compounds described on the same pages are exemplified. Specific examples of these groups are as follows:

[0022] [ka]

[0023] Among these, the "monovalent group derived from an aliphatic heterocycle having electron-withdrawing properties" is preferably a substituted or unsubstituted succinimidyl group, maleimidyl group, phthalimidyl group, or 5-norbornene-2,3-dicarboximidyl group.

[0024] Y is preferably a substituted or unsubstituted C1-C20 alkyl group or a C6-C20 aryl group, more preferably a substituted or unsubstituted C6-C20 aryl group, even more preferably a substituted C6-C20 aryl group, still more preferably a substituted C6-C12 aryl group, particularly preferably a substituted phenyl group, and most preferably a phenyl group substituted with a halogen atom. An example of a compound in which Y is a phenyl group substituted with a halogen atom is 4-fluorophenyl 3-nitro-2-pyridinesulfenate (Npys-OPh(pF); see the Examples below).

[0025] In this specification, examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, a neopentyl group, a 1,2-dimethylpropyl group, an n-hexyl group, a 1,3-dimethylbutyl group, a 1-isopropylpropyl group, a 1,2-dimethylbutyl group, an n-heptyl group, a 1,4-dimethylpentyl group, a 2-methyl-1-isopropylpropyl group, a 1-ethyl-3-methylbutyl group, an n-octyl group, a 2-ethylhexyl group, a 3-methyl-1-isopropylbutyl group, a 2-methyl-1-isopropyl group, a 1-tert-butyl-2-methylpropyl group, an n-nonyl group, and a 3,5,5-trimethylhexyl group.

[0026] In this specification, examples of alkenyl groups include a vinyl group, an allyl group, an isopropenyl group, a 1-butenyl group, a 2-butenyl group, a 2-methyl-2-propenyl group, a 1-methyl-2-propenyl group, a 2-methyl-1-propenyl group, a pentenyl group, a 1-hexenyl group, and a 3,3-dimethyl-1-butenyl group.

[0027] In this specification, examples of alkynyl groups include an ethynyl group, a 1-propynyl group, a 2-propynyl group, a 1-butynyl group, a 2-butynyl group, a 3-butynyl group, a 3-methyl-1-propynyl group, a 2-methyl-3-propynyl group, a pentynyl group, a 1-hexynyl group, a 3-methyl-1-butynyl group, and a 3,3-dimethyl-1-butynyl group.

[0028] In this specification, examples of a cycloalkyl group include a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, and the like.

[0029] In this specification, examples of cycloalkenyl groups include a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, and the like.

[0030] In this specification, examples of the aryl group include a phenyl group, a 1-naphthyl group, a 2-naphthyl group, a 1-anthracenyl group, a 2-anthracenyl group, a 9-anthracenyl group, and the like.

[0031] In this specification, examples of heteroaryl groups include a 2-thienyl group, a 4-pyridyl group, a 3-pyridyl group, a 2-pyridyl group, a 1-pyridyl group, a 2-furyl group, a 2-pyrimidinyl group, a 2-benzothiazolyl group, a 1-imidazolyl group, a 1-pyrazolyl group, a benzotriazol-1-yl group, a 7-azabenzotriazol-1-yl group, and the like.

[0032] In this specification, examples of the azide group include a methyl azide group, an ethyl azide group, an n-propyl azide group, an i-propyl azide group, an n-butyl azide group, an s-butyl azide group, an i-butyl azide group, a t-butyl azide group, an n-pentyl azide group, an n-hexyl azide group, an n-heptyl azide group, and an n-octyl azide group.

[0033] In addition, in the present specification, when a group is "substituted", examples of the substituents that can substitute the group include halogen atoms such as fluorine, chlorine, bromine, and iodine, alkyl groups, aryl groups, alkoxy groups, aryloxy groups, alkoxycarbonyl groups, acyloxy groups, acyl groups, alkylsulfanyl groups, arylsulfanyl groups, alkylamino groups, dialkylamino groups, arylamino groups, hydroxy groups, carboxy groups, formyl groups, mercapto groups, sulfo groups, sulfinic acid groups, guanidino groups, carbamoyl groups, thiol groups, thioether groups, mesyl groups, p-toluenesulfonyl groups, amino groups, nitro groups, cyano groups, trifluoromethyl groups, trichloromethyl groups, trimethylsilyl groups, phosphinico groups, and phosphono groups. These substituents may also be further substituted with halogen atoms, alkyl groups, aryl groups, alkoxy groups, hydroxy groups, carboxy groups, amino groups, nitro groups, cyano groups, and the like. However, substitution in which the substituted group is included in the same definition as the group before the substitution is not considered.

[0034] In Chemical Formula 1, Z represents a nitro group present on the nitrogen-containing heterocycle or aromatic ring formed by W.

[0035] In Chemical Formula 1, p is 0 or 1. When p is 0, L 0 does not exist and A a Or A b or R is directly bonded to the ring formed by W, and when p is 1, L 0 In one preferred embodiment, p is 0.

[0036] In Chemical Formula 1, q is 0 or 1. When q is 0, L 1 does not exist, and when q is 1, L 1 exists.

[0037] In Chemical Formula 1, r is 0 or 1. When r is 0, A b does not exist, and when r is 1, A b exists.

[0038] In Chemical Formula 1, s is an integer of 0 to 10. When s is 0, [(A a )-(L 1 ) q ] does not exist and s is an integer between 1 and 10, then [(A a )-(L 1 ) q In addition, s is preferably 0 to 5, and more preferably 0 or 1.

[0039] In Chemical Formula 1, L 0 and L 1 When present, each independently represents a linker having a chemically stable structure. There is no particular limitation on the specific structure of such a linker, but examples thereof include a substituted or unsubstituted C1-C20 alkylene group, a substituted or unsubstituted C2-C20 alkenylene group, a substituted or unsubstituted C2-C20 alkynylene group, a substituted or unsubstituted C3-C20 cycloalkylene group, a substituted or unsubstituted C3-C20 cycloalkenylene group, a substituted or unsubstituted C6-C20 arylene group, a substituted or unsubstituted C3-C20 heteroarylene group, -NH-, -O-, -S-, -C(=O)-NH-, -NH-C(=O)-, -O-, -C(=O)-O-, -OC(=O)-, -S-, -C(=O)-, and a polyoxyalkylene group. In addition, L 0 and L 1 is a group represented by the following chemical formula (a):

[0040] [ka]

[0041] In this case, in the chemical formula (a), R a represents a substituted or unsubstituted C1 to C15 alkylene group, preferably a C1 to C8 alkylene group, more preferably a C1 to C4 alkylene group, particularly preferably a C1 to C2 alkylene group, and most preferably a C2 alkylene group (particularly an ethylene group). 0 and L 1Preferably, a C1 to C6 alkylene group (particularly an ethylene group), a polyoxyalkylene group having a molecular weight of 100 to 1000, or a group represented by the above chemical formula (a) is used as the alkyl group.

[0042] In Chemical Formula 1, A a and A b When present, each independently represents a group selected from the group consisting of -CH=CH-, -C≡C-, -C(=O)-, -C(=O)-O-, -OC(=O)-, -O-, C1-C20 oxyalkylene, C1-C20 alkyleneoxy, -C(=O)-NH-, -NH-C(=O)-, -NH-C(=O)-NH-, hydrazine, triazole, sulfone, sulfoxide, sulfonic acid ester, sulfonamide, sulfinic acid ester, sulfinamide, piperidine, and dioxane. a and A b Preferably, -C(=O)-, -C(=O)-O-, -O-, C1 to C20 oxyalkylene, C1 to C20 alkyleneoxy, -OC(=O)-, -C(=O)-NH- or -NH-C(=O)- is used as the alkyl group.

[0043] In Chemical Formula 1, R is a hydrogen atom, a substituted or unsubstituted C1-C20 alkyl group, a substituted or unsubstituted C2-C20 alkenyl group, a substituted or unsubstituted C2-C20 alkynyl group, a substituted or unsubstituted C3-C20 cycloalkyl group, a substituted or unsubstituted C3-C20 cycloalkenyl group, a substituted or unsubstituted C6-C20 aryl group, a substituted or unsubstituted C3-C20 heteroaryl group, an amino group, a hydroxy group, a group G containing at its terminal a group capable of covalently bonding to a specific functional group possessed by an arbitrary compound, or a group FM derived from a functional substance.

[0044] The term "group G having at its terminal a group capable of covalently bonding with a specific functional group of an arbitrary compound" as R means a functional group capable of performing a 1,3-dipole cycloaddition reaction utilizing click chemistry. A "1,3-dipole" is a 4π-electron chemical species consisting of three atoms, which undergoes a cycloaddition reaction with a compound having a dipolarophile such as an alkenyl group or an alkynyl group to form a five-membered heterocyclic compound. Therefore, when R has a "1,3-dipole", a specific functional group of an arbitrary compound has a "dipophile", and conversely, when R has a "dipophile", a specific functional group of an arbitrary compound can have a "1,3-dipole".

[0045] Here, examples of the "1,3-dipole" applicable to R include an azomethine ylide group, a nitrone group, a nitrile ylide group, a diazomethyl group, an azide group, and a nitrile oxide group. Examples of the "dipolephile" applicable to R include an alkenyl group and an alkynyl group. For example, when R is a group containing a nitrone group at its terminal, it undergoes a cycloaddition reaction with a compound having an alkenyl group to form an isoxazole ring. When R is a group containing an azide group at its terminal, it undergoes a cycloaddition reaction with a compound having an alkynyl group to form a triazole ring (Husgen cycloaddition reaction). When R is a group containing a nitrile oxide group at its terminal, it undergoes a cycloaddition reaction with a compound having an alkenyl group to form an isoxazole ring. In the above example, the case where R has a "1,3-dipole" and any compound has a "dipolephile" is illustrated, but the same reaction can be carried out even when R has a "dipolephile" and any compound has a "1,3-dipole". Among them, R is preferably a group containing an alkynyl group or an azide group at the end, and more preferably an unsubstituted or substituted linear or branched alkynyl group having 2 to 8 carbon atoms, or an azide group having a linear or branched alkyl group having 1 to 8 carbon atoms. From the viewpoint of maintaining high hydrophilicity, it is particularly preferable that R is a linear or branched alkynyl group having 2 to 3 carbon atoms, or an azide group having a linear alkyl group having 1 to 2 carbon atoms.

[0046] The "functional substance-derived group FM" as R is a monovalent group derived from any substance having various functions. Examples of functional substances include drugs, radioactive substances, fluorescent chemical substances, luminophores, fluorescent proteins, luminescent proteins, magnetic substances, enzymes, coenzymes, biologically active substances, DNA, RNA, PEG, and other substances having specific functions.

[0047] Here, for example, when a radioactive substance, a fluorescent chemical substance, a luminophore, a fluorescent protein, a luminescent protein, a magnetic substance, or the like is adopted as the functional substance, since these substances can emit a signal, it is possible to detect a molecule bound to the tryptophan selective modifier of the present invention by detecting the signal. It can also be used for imaging, diagnosis, etc. of specific diseases such as cancer.

[0048] Furthermore, when drugs, enzymes, coenzymes, biologically active substances, etc. are used as functional substances, the tryptophan selective modifier of the present invention can deliver these functional substances to specific sites, and the tryptophan selective modifier of the present invention can be used as a drug delivery carrier for DDS. Furthermore, when a radioactive substance is used as a functional substance, the radioactive substance can be used as a radiotherapy agent for cancer.

[0049] Furthermore, when DNA or RNA is employed as the functional substance, it is possible to further bind to other substances or capture other substances through the DNA or RNA. For example, it can be used as a biosensor or a microarray by binding to a substrate through DNA or RNA. Furthermore, when DNA or RNA is employed as the functional substance, it is also possible to introduce DNA or RNA in a cell-specific manner. In this case, examples of the DNA or RNA to be introduced include RNA strands such as dsRNA that cause RNAi (RNA interference), antisense RNA strands, and DNA strands and antisense DNA strands that express these RNAs, and in this case, the transcription and expression of a specific gene can be suppressed in the cell. Furthermore, a transgene for transformation can be introduced using the tryptophan selective modifying agent according to the present invention.

[0050] Furthermore, when PEG (polyethylene glycol) is used as a functional substance, it is possible to impart new functions to the tryptophan selective modifying agent of the present invention, such as improved stability and a longer half-life in the body.

[0051] (Use of the compound of the present invention) The present inventors have surprisingly found that the compound of the present invention (or a salt thereof) functions as a tryptophan selective modifier that selectively reacts with and bonds to a tryptophan residue in the presence of a compound having a thioether group. The use of the compound of the present invention as a tryptophan selective modifier has the excellent effect of enabling organic compounds such as peptides and proteins to be selectively modified by a simple treatment in a short time and by a chemically stable method without using harsh reaction conditions as in conventional methods. That is, according to another aspect of the present invention, a method for producing a Trp-S bond-containing compound is also provided. This production method is characterized in that it includes contacting a compound having a tryptophan residue in the molecule (hereinafter also referred to as "compound A") with the tryptophan selective modifier according to one aspect of the present invention in the presence of a compound having a thioether group (hereinafter also referred to as "compound B") to form a covalent bond between a sulfur atom contained in the compound represented by the chemical formula 1 and the tryptophan residue.

[0052] Here, the structure of the "compound having a tryptophan residue in the molecule (compound A)" ​​to be modified by the tryptophan selective modifying agent is not particularly limited. Examples of compound A include compounds containing amino acid residues such as amino acids, peptides (oligopeptides, polypeptides), and proteins, as well as polymer compounds, low molecular compounds, and derivatives containing isotopes thereof. Of these, compound A is preferably a peptide or a protein.

[0053] When compound A is a peptide or a protein, the peptide or protein may be naturally derived or artificially synthesized. When the peptide or protein is artificially synthesized, there is no particular limitation on the synthesis method, and conventionally known knowledge may be appropriately referred to. As an artificial synthesis method of peptides, "solid phase synthesis" and "liquid phase synthesis" are known, and in the case of solid phase synthesis, the Fmoc method and the Boc method are further known. When compound A is a peptide, the peptide may be synthesized by any method. In addition, as a method of synthesizing a protein, a method using a transformant transformed to synthesize a desired protein can be mentioned. In one example, the transformant is a bacterium (such as Escherichia coli), a yeast (such as Saccharomyces cerevisiae, oleaginous yeast), an insect, an animal, or a plant. The synthesized protein can be isolated from the host cell or outside the cell (such as a medium) and purified as a substantially pure and homogeneous protein. For the separation and purification of the protein, a separation method and a purification method used in the separation and purification of a normal protein may be used.

[0054] Compound A, the target of tryptophan residue modification by the above-mentioned method, is, for example, a "compound having one tryptophan residue in the molecule." In this embodiment, a Trp-S bond can be selectively formed with the tryptophan residue contained in compound A, and a desired Trp-S bond-containing compound can be produced in high yield and high purity.

[0055] Here, when compound A, the target of tryptophan residue modification, is a peptide, compound A may contain two or more tryptophan residues in the molecule. Therefore, in such a case, when it is desired to selectively modify only a specific tryptophan residue, it is necessary to protect the tryptophan residue that is not to be modified with a protecting group. When compound A is a peptide, the introduction of such a protecting group can be achieved by introducing an amino acid with a protected tryptophan residue at a desired position during the synthesis of the above-mentioned peptide. In addition, examples of the protecting group of the tryptophan residue in the "amino acid with a protected tryptophan residue" used during the synthesis of a peptide in such a method include, for example, a Boc protecting group, an Ac protecting group, a benzyloxycarbonyl protecting group, a 2,4-dichlorobenzyloxycarbonyl protecting group, a 2-chlorobenzyloxycarbonyl protecting group, a 2,2,2-trichloroethoxycarbonyl protecting group, a formyl protecting group, a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group, a mesitylenebenzenesulfonyl protecting group, and the like for the amino group of the side chain.

[0056] There is also no particular limitation on the structure of the "compound having a thioether group (compound B)." The "thioether group" is a group in which the oxygen atom of an ether group is replaced with a sulfur atom.Specific examples of compound B include dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, diethyl sulfide, dipentyl sulfide, tert-butyl methyl sulfide, divinyl sulfide, diphenyl sulfide, diphenyl disulfide, diphenyl trisulfide, thioanisole, bis(hydroxyphenyl)sulfide, ditolyl sulfide, ditolyl disulfide, bis(nitrophenyl)sulfide, bis(aminophenyl)sulfide, bis(chlorophenyl)sulfide, bis(meth)phenylsulfide, and bis(phenyl)sulfide. methoxyphenyl) sulfide, dibenzoyl sulfide, dibenzoyl disulfide, dibenzyl sulfide, dibenzyl disulfide, dibenzyl trisulfide, dibenzyl tetrasulfide, dibenzyl pentasulfide, dibenzyl hexasulfide, dibenzyl heptasulfide, dibenzyl octasulfide, dicyclohexyl sulfide, dicyclohexyl disulfide, methylcyclohexyl sulfide, methyl phenyl sulfide, methyl allyl sulfide, methyl benzyl sulfide, phenyl allyl sulfide, phenyl vinyl sulfide, phenyl benzyl sulfide, phenyl styryl sulfide, phenyl cinnamyl sulfide, phenyl benzoyl sulfide, phenyl phenacyl sulfide, phenyl cyclohexyl sulfide, tolyl benzyl sulfide, benzyl allyl sulfide, trimethylene sulfide, trimethylene disulfide, tetramethylene sulfide, tetramethylene disulfide, pentamethylene sulfide, thiophene, 4H-thiine, benzothiophene, dibenzothiophene, thiachlorobenzene, Mann, thiaxanthene, diethylthioacetal, α-phenyl-diethylthioacetal, 1,1-bis(ethylthio)propane, 1,1-bis(ethylthio)propene, 1,4-bis(methyldithio)cyclohexane, 1,3-dithiolane, 1,3-dithiane, 1,4-dithiane, 1,4-dithiene, 1,4-dithiadiene, thianthrene, 1,3,5-trithiane, polyphenylene sulfide, methionine; methionine derivatives such as ethionine, N-formylethionine, and S-adenosylmethionine. These compounds may be used alone or in combination of two or more.

[0057] Furthermore, the "compound having a thioether group (compound B)" may be a peptide or protein having a methionine (Met) residue. In this case, compound B may be a molecule different from compound A, which is the target of modification, but compound B is preferably the same molecule as compound A (i.e., compound A and compound B are a single compound), and the thioether group provided by compound B is preferably a group derived from a methionine residue present in the vicinity of the tryptophan residue to be modified in the peptide or protein. According to such an embodiment, there is an advantage that selective modification of tryptophan residues can be performed by a very simple operation of mixing only the tryptophan selective modifying agent according to one embodiment of the present invention and the compound (peptide or protein) to be modified in a solvent. Note that the position of the methionine residue "in the vicinity" of the tryptophan residue is not particularly limited as long as these residues are present at the position where the above-mentioned modification reaction proceeds. For example, the distance between these residues in the primary structure of the amino acid sequence of a peptide or protein is, for example, 1 to 20 amino acid residues, preferably 1 to 15 amino acid residues, more preferably 1 to 10 amino acid residues, even more preferably 1 to 5 amino acid residues, particularly preferably 1 to 3 amino acid residues, and most preferably 1 amino acid residue (the above residues are linked via a peptide bond). However, it is often the case that a methionine residue and a tryptophan residue that are far apart in the primary structure of the amino acid sequence are present in "proximity" due to the three-dimensional structure such as the tertiary structure of a peptide or protein. In the present invention, such cases are of course also included in the technical scope.

[0058] Here, when the target to be modified using the tryptophan selective modifying agent according to the present invention is a peptide, the peptide may not contain a tryptophan residue and / or a methionine residue. In such a case, the amino acid sequence of the peptide may be modified so that a tryptophan residue and / or a methionine residue is introduced at a desired position during the synthesis of the peptide, thereby introducing a tryptophan residue and / or a methionine residue into the peptide to be modified to obtain compound A. In this case, the site at which the tryptophan residue and / or the methionine residue is introduced is preferably a site that does not adversely affect the expression of the activity of the peptide.

[0059] Furthermore, when the target of modification using the tryptophan-selective modifying agent of the present invention is a protein, it is possible to modify tryptophan residues more selectively and efficiently by modifying the amino acid sequence of the protein using genetic recombination techniques.

[0060] For example, when compound A is a protein, the protein may be one in which a tryptophan residue has been inserted at a desired position by genetic recombination, or an amino acid residue at a desired position may be modified to a tryptophan residue. This method may be used for the purpose of introducing a modifiable tryptophan residue onto the surface of a protein particle when the tertiary structure of the protein does not have a modifiable tryptophan residue on the surface of the protein particle.

[0061] In addition, when compound A is a protein, the protein may be one in which a tryptophan residue at a desired position has been deleted or the tryptophan residue at a desired position has been modified to another amino acid residue by genetic recombination. This method may be used for the purpose of removing a tryptophan residue that is not desired to be modified when a plurality of tryptophan residues that can be modified are present on the surface of a protein particle in the tertiary structure of the protein.

[0062] Furthermore, when compound A is a protein, the protein may be one in which a methionine residue has been inserted at a desired position by genetic recombination, or an amino acid residue at a desired position may be modified to a methionine residue. This method may be used for the purpose of introducing a methionine residue near a tryptophan residue on the surface of a protein particle when the tertiary structure of the protein does not have a methionine residue near the tryptophan residue.

[0063] In addition, when compound A is a protein, the protein may be one in which a methionine residue at a desired position has been deleted or the methionine residue at a desired position has been modified to another amino acid residue by genetic recombination. This method may be used for the purpose of removing a methionine residue near a tryptophan residue that is not to be modified when a plurality of tryptophan residues that can be modified are present on the surface of a protein particle together with the methionine residues nearby in the tertiary structure of the protein.

[0064] Incidentally, some of the compounds represented by Chemical Formula 1 according to the present invention have the function of forming a disulfide bond between the two (or more) reduced thiol groups by contacting them with a compound (e.g., a peptide or a protein) having two (or more) reduced thiol groups in the molecule (i.e., they act as disulfidation reagents) (International Publication No. 2017 / 200109). Therefore, when compound A, which is the target of modification using the tryptophan-selective modifying agent according to the present invention, is a peptide or protein, and contains two or more structures (e.g., cysteine ​​residues) containing reduced thiol groups that can form disulfide bonds with the modifying agent (i.e., when compound A is a peptide or protein having two or more cysteine ​​residues having reduced thiol groups), it is preferable to protect the reduced thiol group contained in compound A using a protecting group before contacting compound A with the tryptophan-selective modifying agent, and then contact compound A with the protected reduced thiol group with the tryptophan-selective modifying agent, and then deprotect the reduced thiol group. By adopting such a configuration, it becomes possible to prevent the compound represented by Chemical Formula 1 according to the present invention from acting as a disulfidation reagent and to allow it to function solely as a tryptophan-selective modifying agent.

[0065] In the method for producing a Trp-S bond-containing compound according to the present embodiment, compound A is contacted with the tryptophan selective modifier according to the present invention in a reaction system in the presence of compound B. As a result, a covalent bond is formed between the sulfur atom contained in the compound represented by chemical formula 1 and the tryptophan residue contained in compound A, thereby obtaining a Trp-S bond-containing compound.

[0066] There is no particular limitation on the specific method of contacting compound A with compound B, but compound A and compound B are usually contacted in a solvent. There is no particular limitation on the solvent, but it is preferable that the solvent contains water. In addition, a buffer solution can be used instead of water as the solvent, and any of water, a buffer solution, and an organic solvent may be used in combination. On the other hand, when an organic solvent is used in combination, an organic solvent that is miscible with water is preferable, and examples of the organic solvent include acetonitrile, dimethylformamide, acetone, dimethyl sulfoxide, alcohol, tetrahydrofuran, and 1,4-dioxane, and acetonitrile is preferable as the organic solvent.

[0067] From the viewpoint of improving the yield of the reaction, it is preferable that an acid is further present in the reaction system. The acid dissociation constant (pKa) of this acid is preferably 6 or less, more preferably 5 or less, even more preferably 4 or less, even more preferably 3 or less, particularly preferably 2 or less, and most preferably 1 or less. Examples of acids having a pKa of 6 or less include acetic acid, sulfonic acid, hydrochloric acid, nitric acid, phosphoric acid, phosphoric acid monoester, phosphoric acid diester, phosphorous acid, phosphorous acid ester, hypophosphorous acid, halogenated alkyl carboxylic acids such as trifluoroacetic acid, chloroacetic acid, dichloroacetic acid, and trichloroacetic acid, and halogenated aryl carboxylic acids such as pentafluorobenzoic acid and 2,4,6-trifluoromethylbenzoic acid. Among them, acetic acid or halogenated alkyl carboxylic acids are preferable, halogenated alkyl carboxylic acids are more preferable, and trifluoroacetic acid is even more preferable. There is no particular limitation on the concentration of the acid in the reaction system, but it is preferably 5 to 85% by volume, more preferably 20 to 80% by volume, and even more preferably 30 to 80% by volume.

[0068] There is no particular restriction on the reaction conditions, and the reaction temperature is, for example, 10 to 40°C, preferably 20 to 30°C, and the reaction time is, for example, 0.5 to 96 hours, preferably 2 to 72 hours, more preferably 5 to 60 hours, and further preferably 10 to 54 hours. There is no restriction on the reaction pressure, and the reaction may be carried out under normal pressure (atmospheric pressure). The amount of the tryptophan selective modifier according to the present invention and compound B used in the reaction may be increased or decreased depending on the amount of compound A. For example, the amount of the tryptophan selective modifier according to the present invention and compound B is preferably 1 equivalent to an excess amount, more preferably 1 equivalent to 2 equivalents, relative to 1 equivalent of compound A. The completion of the reaction may be determined based on the consumption of compound A in the solution based on a general analytical method. For example, applicable analytical methods include HPLC, NMR, TLC, IR, MS spectrum, titration, and the like.

[0069] After the reaction, the tryptophan-selective modifying agent, the target product Trp-S bond-containing compound, unreacted compound A and compound B, and compounds that are converted from compound A and / or compound B as the reaction proceeds can be purified by a general purification method.

[0070] When the tryptophan selective modifier used in the production method according to the present embodiment has a group FM derived from a functional substance as R in chemical formula 1, the Trp-S bond-containing compound produced by the production method also has the group FM. In this way, it is possible to impart various functions resulting from the group FM to the raw material compound A. As described above, it is possible to use groups derived from a very wide range of functional substances as the group FM. Therefore, the present invention provides a highly advantageous technology that can significantly increase the variety of compounds by imparting a wide range of functions to various compounds.

[0071] The following embodiments are also included within the scope of the present invention: the modifying agent according to claim 1 having the characteristics of claim 2; the modifying agent according to claim 1 or 2 having the characteristics of claim 3; the modifying agent according to any one of claims 1 to 3 having the characteristics of claim 4; the modifying agent according to any one of claims 1 to 4 having the characteristics of claim 5; the modifying agent according to any one of claims 1 to 5 having the characteristics of claim 6; the modifying agent according to any one of claims 1 to 6 having the characteristics of claim 7; the manufacturing method according to claim 9 using the modifying agent according to any one of claims 1 to 8; the manufacturing method according to claim 9 having the characteristics of claim 10; the manufacturing method according to claim 9 or 10 having the characteristics of claim 11; the manufacturing method according to any one of claims 9 to 11 having the characteristics of claim 12; the manufacturing method according to any one of claims 9 to 12 having the characteristics of claim 13; and the manufacturing method according to any one of claims 9 to 13 having the characteristics of claim 14. EXAMPLES

[0072] The present invention will be described below with reference to examples, but the scope of the present invention is not limited to these examples.

[0073] [1.General information] Nuclear magnetic resonance (NMR) spectra were measured using a Bruker AVANCE-III (400 MHz). Mass spectrometry (HRMS (ESI) m / z) was measured using a micromass LCT. 1 In the H-NMR data, in the NMR signal pattern, s means singlet, d means doublet, t means triplet, q means quartet, m means multiplet, J means coupling constant, Hz means Hertz, CDCl3 means deuterated chloroform, and CD3OD means deuterated methanol. 1 In H-NMR data, protons of hydroxyl groups (OH), amino groups (NH2), and carboxyl groups (COOH) are not recorded if they are broadband and cannot be confirmed. In HRMS (ESI) m / z data, M is the molecular weight, [M+H] + , [M+Na] + means the molecular ion peak.

[0074] [2.Synthesis of Npys derivatives] First, Npys-OPh(pF) was synthesized based on the description in the paper (J. Pept. Sci., 2018, 24, e3070.).

[0075] [3. Solvent considerations for the synthesis of Npys-modified tryptophan derivatives]

[0076] [ka]

[0077] Ethyl sulfide (1.0 equivalent) and Npys-OPh(pF) (1.0 equivalent) were added to a solvent (Table 1, Entries 1 to 7 below) containing Cbz-Trp-OH (1.0 equivalent) at room temperature, and the mixture was stirred at the same temperature for 48 hours. A portion of the reaction solution was collected, and the resulting Npys-modified Trp derivative P1 was analyzed by HPLC. The HPLC yield was calculated based on the calibration curve of compound P1. The results are shown in Table 1 below.

[0078] [Table 1]

[0079] When the reaction solvent was a 75% aqueous acetic acid solution, compound P1 was obtained in a yield of 55% (entry 1). In addition, when lithium chloride (Org. Biomol. Chem., 2020, 18, 7094-7097.) was used as an additive as in entry 2, a slight improvement in yield was observed (yield 60%). At an acid concentration of 37.5%, it was shown that the reaction proceeds more efficiently when an acid with a low dissociation constant (pKa) (pKa = 0.23) such as trifluoroacetic acid (TFA) is used compared to acetic acid (entry 4: yield 95%). On the other hand, it was also found that the yield was low when acids such as hexafluoro-2-propanol (HFIP) or 2,2,2-trifluoroethanol (TFE) were used as in entries 5 to 7, or when no acid was used.

[0080] Here, the HPLC chart showing the reaction over time under the reaction conditions in Entry 1 is shown in Figure 1. Here, in Figure 1, * indicates the peak corresponding to 4-fluorophenol, and ** indicates the peak corresponding to the Npys dimer. From the chart shown in Figure 1, it was confirmed that after 1 hour of reaction, pyrroloindoline derivatives P2 and P3 were first produced, and then converged to the Npys-modified Trp derivative P1 over time. From these results, it is believed that this sulfenylation reaction proceeds according to the reaction mechanism shown in Figure 2.

[0081] [4. Synthesis of Npys modified tryptophan derivative ((S)-2-(((benzyloxy)carbonyl)amino)-3-(2-((3-nitropyridin-2-yl)thio)-1H-indol-3-yl)propanoic acid, P1)]

[0082] [ka]

[0083] Ethyl sulfide (15.9μL, 148μmol) and Npys-OPh(pF) (59.0mg, 222μmol) were added to an aqueous solution of Cbz-Trp-OH (50.0mg, 148μmol) in acetic acid (3:1, v / v, 1.5mL) at room temperature, and the mixture was stirred at the same temperature for 72 hours. The reaction solution was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and then dried over magnesium sulfate. After filtration, the mother liquor was distilled under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform:methanol:acetone=70:1:1) to obtain the Npys-modified tryptophan derivative (P1) as a yellow solid (23.9mg, 48.5μmol, yield 33%).

[0084] [ka]

[0085] In order to identify the chemical structures of the pyrroloindoline derivatives P2 and P3 described above, these compounds were synthesized and various instrumental data were obtained.

[0086] [5. Synthesis of pyrroloindoline derivative ((2S)-1-((benzyloxy)carbonyl)-3a-((3-nitropyridin-2-yl)thio)-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid, P2)]

[0087] [ka]

[0088] Ethyl sulfide (31.8μL, 296μmol) and Npys-OPh(pF) (78.7mg, 296μmol) were added to a 75% aqueous acetic acid solution (1.5mL) of Cbz-Trp-OH (100.0mg, 296μmol) at room temperature, and the mixture was stirred at 4℃ for 42 hours. The reaction solution was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and then dried over magnesium sulfate. After filtration, the mother liquor was distilled under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform:methanol:acetone=70:1:1) to obtain the pyrroloindoline derivative (P2) as a yellow solid (15.5mg, 31.5μmol, yield 11%).

[0089] [ka]

[0090] Synthesis of pyrroloindoline derivative ((2S)-1-((benzyloxy)carbonyl)-3a-((3-nitropyridin-2-yl)thio)-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid, P3)

[0091] [ka]

[0092] Ethyl sulfide (15.9μL, 148μmol) and Npys-OPh(pF) (59.0mg, 222μmol) were added to an aqueous solution of Cbz-Trp-OH (50.0mg, 148μmol) in acetic acid (3:1, v / v, 1.5mL) at room temperature, and the mixture was stirred at the same temperature for 1 hour. The reaction solution was extracted with ethyl acetate. The organic layer was washed with water and saturated saline, and then dried over magnesium sulfate. After filtration, the mother liquor was distilled under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform:methanol:acetone=70:1:1) and HPLC to obtain the pyrroloindoline derivative (P3) as a yellow solid (26.8mg, 54.3μmol, yield 37%).

[0093] [ka]

[0094] [6. Reaction selectivity for amino acids]

[0095] [ka]

[0096] Npys-OPh(pF) (1 equivalent) was added to an aqueous solution of Cbz-protected amino acid derivative (1 equivalent) in acetic acid (3:1, v / v, 100 mM) at room temperature. Ethyl sulfide (1 equivalent) was then added and stirred at the same temperature for 1.5 hours. A portion of the reaction solution was diluted, and the molecular weight of the Npys-modified amino acid derivative contained in the solution was analyzed by mass spectrometry (HRMS). The results are shown in Table 2 below.

[0097] [Table 2]

[0098] [7. Modification of tryptophan residues in peptides] (7-1. Peptide synthesis) Synthesis of Ac-Ala-Ala-Trp-Ala-Ala-NH2 (peptide a; Ac-AAWAA-NH2; SEQ ID NO: 1) A 20% piperidine / DMF solution was added to Fmoc-Rink-amide resin (90.9 mg, 40.0 mmol) and the mixture was shaken at room temperature. After removing the reaction solution by filtration, the peptide chain was extended by Fmoc-solid phase peptide synthesis using Fmoc-amino acid derivatives (3 equivalents), 1-hydroxybenzotriazole (HOBt·H2O, 3 equivalents) and N,N'-diisopropylcarbodiimide (DIPCI, 3 equivalents). DMF, acetic anhydride (3 equivalents) and DIPEA (3 equivalents) were added to the obtained H-Ala-Ala-Trp(Boc)-Ala-Ala-NH-resin (SEQ ID NO: 2) and the mixture was shaken at room temperature for 60 minutes to perform Ac-labeling of the N-terminus of the peptide. The obtained resin (Ac-Ala-Ala-Trp(Boc)-Ala-Ala-NH-resin; SEQ ID NO: 3) was washed with DMF, methanol and diethyl ether and then dried. TFA:H2O (95:5, v / v, 5 mL) was added to the dried resin and stirred at room temperature for 3 hours. The reaction solution was filtered and the reaction solution was evaporated by blowing nitrogen. Ice-cold diethyl ether was added to the resulting residue to precipitate the peptide. The peptide was further washed twice with ice-cold diethyl ether and dried. The crude product was purified by HPLC (gradient: milliQ (0.1% TFA) / CH3CN (0.1% TFA) = 76:24 to 60:40 over 16 min, flow late 5 mL / min, UV: 230 nm, column: Sunfire TM Prep C18 OBD TM The target peptide (peptide a) was obtained by purifying the product using a 5 μm, 19×150 mm column (12.5 mg, 23.7 μmol, 59% yield).

[0099] [ka]

[0100] Synthesis of Ac-Ala-Ala-Trp-Ala-Met-NH2 (peptide b; Ac-AAWAM-NH2; SEQ ID NO: 4) Dry resin Ac-Ala-Ala-Trp(Boc)-Ala-Met-NH-resin (SEQ ID NO:5) was obtained using Fmoc-Rink-amide resin (90.9 mg, 40.0 mmol) by the same synthesis as peptide a. TFA:H2O (95:5, v / v, 5 mL) was added to the dry resin obtained and stirred at room temperature for 3 hours. The reaction solution was filtered and the reaction solution was distilled off by blowing nitrogen. Ice-cold diethyl ether was added to the residue obtained to precipitate the peptide. It was further washed twice with ice-cold diethyl ether and dried. The crude product was purified by HPLC (gradient: milliQ (0.1% TFA) / CH3CN (0.1% TFA) = 73:27 to 60:40 over 13 min, flow rate 5 mL / min, UV: 230 nm, column: COSMOSIL 5C4-AR-300, Packed Column 4.6 mm I.D. x 250 mm) to obtain the desired peptide (peptide b) (12.4 mg, 21.0 μmol, yield 52%).

[0101] [ka]

[0102] Synthesis of Ac-neuromedin B (Ac-Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH2; SEQ ID NO: 6) Using Fmoc-Rink-amide resin (681 mg, 0.30 mmol), the same synthesis as for peptide a was carried out to obtain dried resin H-Gly-Asn(Trt)-Leu-Trp(Boc)-Ala-Thr(t-Bu)-Gly-His(Trt)-Phe-Met-NH-resin (SEQ ID NO: 7) (1168 mg). Of this, 584 mg was subjected to Ac-conjugation of the N-terminus of the peptide in the same manner as for the synthesis of peptide Ac-Ala-Ala-Trp-Ala-Ala-NH2. The obtained dried resin Ac-Gly-Asn(Trt)-Leu-Trp(Boc)-Ala-Thr(t-Bu)-Gly-His(Trt)-Phe-Met-NH-resin (SEQ ID NO: 8) was added to TFA: triisopropylsilane: 1,3-dimethoxybenzene: 1,2-ethanedithiol (90:2.5:5.0:2.5, v / v / v / v, 5 mL) and stirred at room temperature for 3 hours. The reaction solution was filtered and the reaction solution was distilled off by blowing nitrogen. Ice-cold diethyl ether was added to the obtained residue to precipitate the peptide. It was further washed twice with ice-cold diethyl ether and dried. The crude product was purified by HPLC (gradient: milliQ (0.1% TFA) / CH3CN (0.1% TFA) = 70:30 to 55:45 over 15 min, flow rate 5mL / min, UV: 230nm, column: Sunfire TM Prep C18 OBD TM The target Ac-neuromedin B was obtained by purifying the fraction using a 5 μm, 19×150 mm column (37.9 mg, 32.3 μmol, 22% yield).

[0103] [ka]

[0104] Glucagon synthesis Using Fmoc-Thr(t-Bu)-Trt-PEG-resin (0.20 mmol / g, 500 mg, 0.10 mmol), the peptide was synthesized in the same manner as peptide a, to obtain the dried resin H-His(Trt)-Ser(t-Bu)-Gln(Trt)-Gly-Thr(t-Bu)-Phe-Thr(t-Bu)-Ser(t-Bu)-Asp(Ot-Bu)-Tyr(t-Bu)- Ser(t-Bu)-Lys(Boc)-Tyr(t-Bu)-Leu-Asp(Ot-Bu)-Ser(t-Bu)-Arg(Pbf)-Arg(Pbf)-Ala-Gln(Trt)-Asp(Ot-Bu)-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Met-Asn(Trt)-Thr(t-Bu)-O-resin (SEQ ID NO: 9) was obtained. TFA:H2O:triisopropylsilane:1,2-ethanedithiol (94:2.5:1.0:2.5, v / v / v / v, 10 mL) was added to the obtained dried resin, and the mixture was stirred at room temperature for 3 hours. The reaction solution was filtered, and the reaction solution was distilled off by blowing nitrogen. Ice-cold diethyl ether was added to the obtained residue to precipitate the peptide. The mixture was further washed twice with ice-cold diethyl ether and dried. The crude product was purified by HPLC (gradient: milliQ (0.1% TFA) / CH3CN (0.1% TFA) = 70:30 to 55:45 over 15 min, flow rate 5 mL / min, UV: 230 nm, column: Sunfire TM Prep C18 OBD TM The desired glucagon was obtained by purification using a 5 μm, 19 x 150 mm column (105 mg, 26.7 μmol, 27%).

[0105] [ka]

[0106] Synthesis of melanin-concentrating hormone (MCH) MCH was synthesized based on the description in the paper (Eur. J. Org. Chem., 2021, 956-963.).

[0107] (7-2. Examination of chemical modifications to peptides) To investigate the sulfenylation reaction of peptides, peptide b (Ac-Ala-Ala-Trp-Ala-Met-NH2) was used as a Trp / Met residue-containing peptide. As shown in entry 1 of Table 3 below, the above peptide was sulfenylated with Npys-OPh(pF) (1 equivalent) in 75% aqueous acetic acid, and the HPLC yield of the Npys-modified product after 72 hours was 91%. The HPLC chart showing the time course of this reaction is shown in Figure 3.

[0108] As in the sulfenylation reaction of Z-Trp-OH described above, the peaks converged over time in this experiment. Therefore, even in the case of peptides, it is considered that the reaction converged to the Npys-modified peptide P3 via pyrroloindoline-type modifications P1 and P2. On the other hand, when peptide a (Ac-Ala-Ala-Trp-Ala-Ala-NH2) without a Met residue was used, the reaction did not proceed and no Npys-modified product was produced (entry 2). This result suggests that the sulfenylation of Trp residues in peptides also proceeds with the side chain thioether structure of the Met residue as a catalyst.

[0109] Next, we investigated Npys modification in biologically active peptides with more complex structures. For glucagon (30 residues) with an a-helical structure, the yield was 60% (74% when 2 equivalents of Npys-OPh(pF) were used) (entry 3), and for MCH (19 residues, disulfide bond between the 7th and 16th Cys residues), the modification was confirmed with an HPLC yield of 74% (entry 4).

[0110] [Table 3]

[0111] Here, the experimental methods for each entry shown in Table 3 above are described below.

[0112] Chemical modification of Ac-AAWAM-NH2 (peptide b) (entry 1 in Table 3) Ac-AAWAM-NH2 (peptide b) in AcOH:H2O (3:1, v / v) solution (1 equivalent, 1 mM) was added to Npys-OPh(pF) in AcOH:H2O (3:1, v / v) solution (1 equivalent, 1 mM) at room temperature and stirred at the same temperature for 72 hours (final peptide concentration: 0.5 mM). The reaction was tracked by diluting a portion of the reaction solution and analyzing it by HPLC (HPLC conditions: linear concentration gradient from 5% CH3CN to 75% CH3CN in 0.1% TFA aqueous solution (35 minutes), flow rate 1.0 mL / min, detection at 230 nm, Column: COSMOSIL Packed Column 5C 18 -AR-II 4.6IDx150mm). After 72 hours, the HPLC yield of Npys-modified peptide P3 was 91%.

[0113] [ka]

[0114] Chemical modification of Ac-AAWAA-NH2 (peptide a) (entry 2 in Table 3) A solution of Ac-AAWAA-NH2 (peptide a) in AcOH:H2O (3:1, v / v) (1 equivalent, 1 mM) was added to a solution of Npys-OPh(pF) in AcOH:H2O (3:1, v / v) (1 equivalent, 1 mM) at room temperature, and the mixture was stirred at the same temperature for 72 hours (final peptide concentration: 0.5 mM). The reaction was tracked by diluting a portion of the reaction solution and analyzing it by HPLC. When the reaction solution after 72 hours was subjected to mass spectrometry, the mass of the Npys-modified peptide was not detected.

[0115] Chemical modification of glucagon (Entry 3 in Table 3) A solution of Npys-OPh(pF) in AcOH:HO (3:1, v / v) (1 equivalent, 1 mM) was added to a solution of glucagon in AcOH:HO (3:1, v / v) (1 equivalent, 1 mM) at room temperature and stirred at the same temperature for 72 hours (final peptide concentration: 0.5 mM). The reaction was tracked by diluting a portion of the reaction solution and analyzing it by HPLC. The HPLC yield of the Npys-modified peptide after 72 hours was 60%. The HPLC yield of the Npys-modified peptide when 2 equivalents of Npys-OPh(pF) were used was 74%.

[0116] [ka]

[0117] Chemical modification of melanin-concentrating hormone (MCH) (entry 4 in Table 3) A solution of Npys-OPh(pF) in AcOH:HO (3:1, v / v) (1 equivalent, 1 mM) was added to a solution of melanin-concentrating hormone in AcOH:HO (3:1, v / v) (1 equivalent, 1 mM) at room temperature, and the mixture was stirred at the same temperature for 72 hours (final peptide concentration: 0.5 mM). The reaction was tracked by diluting a portion of the reaction solution and analyzing it by HPLC. The HPLC yield of the Npys-modified peptide after 72 hours was 74%.

[0118] [ka]

[0119] (7-3. Examination of reaction solvents for chemical modification of peptides) A solution of Npys-OPh(pF) (1 equivalent, 1 mM) was added to a solution of Ac-neuromedin B (1 equivalent, 1 mM) at room temperature and stirred at the same temperature (final peptide concentration: 0.5 mM). In this case, the reaction solvent and reaction time were changed as shown in Table 4 below. The reaction was tracked by diluting a part of the reaction solution and analyzing it by HPLC (HPLC conditions: linear concentration gradient from 5% CH3CN to 75% CH3CN in 0.1% TFA aqueous solution (35 minutes), flow rate 1.0 mL / min, detection at 230 nm, Column: HITACHI LaChrom II C 18 -(5μm) P / N 889-0911 4.6ID×150mm). The HPLC yield of the Npys-modified peptide P3 thus obtained is shown in Table 4 below. The HPLC chart showing the time course of the reactions of entries 1 to 3 in Table 4 is shown in Figure 4. Here, the * in Figure 4 indicates the peak corresponding to the monooxygenated form of Ac-neuromedin B.

[0120] [ka]

[0121] [Table 4]

[0122] As shown in entries 1 to 3 in Table 4, when the reaction was carried out in a 75% AcOH solution, the HPLC yields of Npys-modified peptide P3 at 24, 48, and 72 hours were 73, 82, and 86%, respectively. As shown in FIG. 4, this reaction also first produced pyrroloindoline-type modified peptides P1 and P2, which converged to Npys-modified peptide P3 over time. Next, even if the concentration of acetic acid contained in the reaction solvent was reduced from 75% to 50%, 20%, 5%, and 1%, it was possible to obtain Npys-modified peptide P3 in yields of 82%, 75%, 71%, 65%, and 55%, respectively.

[0123] 8. Modification of tryptophan residues in proteins (8-1. Synthesis of Npys derivative (compound 3))

[0124] [ka]

[0125] First, 6-(benzylthio)-5-nitronicotinic acid (compound 1) was synthesized based on the description in the paper (Asian J. Org. Chem., 2015, 4, 1030-1033.).

[0126] Synthesis of N-(3-azidopropyl)-6-(benzylthio)-5-nitronicotinamide (compound 2) To a DMF solution (13 mL) of compound 1 (1.41 g, 4.93 mmol), 3-azidopropan-1-amine (0.99 g, 9.86 mmol), HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (2.25 g, 5.92 mmol) and DIPEA (diisopropylethylamine) (1.02 mL, 5.92 mmol) were added at room temperature, and the mixture was stirred at the same temperature for 2 hours and 30 minutes. The reaction solution was distilled under reduced pressure, and the resulting residue was dissolved in ethyl acetate, washed with 10% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, water, and saturated saline, and dried over Na2SO4. After filtration, the mother liquor was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane:ethyl acetate=1:1) to obtain a yellow solid (compound 2) (1.62 g, 4.35 mmol, yield 88%).

[0127] [ka]

[0128] Synthesis of N-(3-azidopropyl)-6-((4-fluorophenoxy)thio)-5-nitronicotinamide (compound 3) To a mixture of dichloromethane and 1,2-dichloroethane (1:1, v / v, 3 mL) containing compound 2 (109 mg, 0.293 mmol), sulfuryl chloride (52.0 μL, 0.644 mmol) and pyridine (11.8 μL, 0.147 mmol) were added at room temperature, and the mixture was stirred at the same temperature for 1 hour. The reaction solution was distilled under reduced pressure and azeotroped with hexane. The resulting residue was used in the next reaction without purification. The resulting residue was dissolved in a mixture of dichloromethane and 1,2-dichloroethane (1:1, v / v, 3 mL), and p-fluorophenol (39.4 mg, 0.352 mmol) and DIPEA (101 μL, 0.586 mmol) were added under ice-cooling and stirring, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was evaporated under reduced pressure, and the resulting residue was dissolved in chloroform, washed with 10% aqueous citric acid, water, and saturated saline, and dried over Na2SO4. After filtration, the mother liquor was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane:ethyl acetate=2:1) ​​to give a yellow solid (compound 3) (82.9 mg, 0.211 mmol, 72% yield for two steps).

[0129] [ka]

[0130] (8-2. Synthesis of fluorescent molecule-Npys derivatives)

[0131] [ka]

[0132] First, tert-butyl 20-azido-3,6,9,12,15,18-hexaoxaicosanoate (compound 4) was synthesized based on the description in the papers (J. Am. Chem. Soc., 2006, 128, 1414-1415. and ACS Chem. Biol., 2008, 3, 567-576.).

[0133] On the other hand, compound 5 having a fluorescent group was synthesized based on the description in the paper (J. Am. Chem. Soc., 2016, 138, 10798-10801.).

[0134] Synthesis of tert-butyl 20-(4-(((9-(2-(methoxycarbonyl)phenyl)-3-oxo-3H-xanthen-6-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18-hexaoxaicosanoate (compound 6) Compound 5 (211 mg, 0.55 mmol) was added to a mixture of compound 4 (231 mg, 0.55 mmol) in t-BuOH:H2O:DMF (3:1:1, v / v / v, 16 mL) at 65°C and stirred at the same temperature for 1 hour. CuSO4·5H2O (6.87 mg, 0.028 mmol) and sodium ascorbate (55.5 mg, 0.28 mmol) were then added at the same temperature and stirred for 24 hours. The reaction solution was evaporated under reduced pressure, and the resulting residue was dissolved in ethyl acetate, washed with water and saturated saline, and dried over Na2SO4. After filtration, the mother liquor was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform:methanol = 25:1) to obtain a brown solid (compound 6) (297.24 mg, 0.37 mmol, yield 67%).

[0135] [ka]

[0136] Synthesis of 20-(4-(((9-(2-(methoxycarbonyl)phenyl)-3-oxo-3H-xanthen-6-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18-hexaoxaicosanoic acid (compound 7) Compound 6 (281.81 mg, 0.35 mmol) was added with 4N HCl / dioxane (6 mL) at room temperature and stirred at the same temperature for 24 hours. The reaction solution was distilled under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform:methanol=10:1) to obtain a brown solid (compound 7) (176.22 mg, 0.24 mmol, yield 67%).

[0137] [ka]

[0138] Synthesis of methyl 2-(6-((1-(1-((5aS,6R,6aR)-1-(3-(6-((4-fluorophenoxy)thio)-5-nitronicotinamido)propyl)-1,4,5,5a,6,6a,7,8-octahydrocyclopropa[5,6]cycloocta[1,2-d][1,2,3]triazol-6-yl)-3,14-dioxo-2,7,10,16,19,22,25,28,31-nonaoxa-4,13-diazatritriacontan-33-yl)-1H-1,2,3-triazol-4-yl)methoxy)-3-oxo-3H-xanthen-9-yl)benzoate (compound 10) To a solution of compound 7 (59.98 mg, 0.080 mmol) in DMF (2 mL), HOBt·H2O (1-hydroxybenzotriazole monohydrate) (18.38 mg, 0.120 mmol), EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride) (19.94 mg, 0.104 mmol), compound 8 (25.92 mg, 0.080 mmol) and DIPEA (26.5 μL, 0.160 mmol) were added in sequence at room temperature, and the mixture was stirred overnight under light shielding at the same temperature. The reaction solvent was distilled off under reduced pressure, and the resulting residue was dissolved in chloroform, washed with water and saturated saline, and dried over Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure to obtain compound 9 (82.61 mg, 0.078 mmol). Compound 9 was used in the next reaction without purification.

[0139] [ka]

[0140] Next, compound 3 (30.60 mg, 0.078 mmol) was added to a CH3CN:H2O mixed solution (3:1, v / v, 2.6 mL) containing compound 9 (82.61 mg, 0.078 mmol) at room temperature, and the mixture was stirred in a dark place at room temperature for 1 hour and 45 minutes. The reaction solvent was distilled off under reduced pressure, and the resulting residue was dissolved in chloroform, washed with water and saturated saline, and dried over Na2SO4. After filtration, the mother liquor was distilled off under reduced pressure, and the resulting residue was purified by silica gel chromatography (chloroform:methanol = 10:1) and HPLC (gradient: milliQ (0.1% TFA) / CH3CN (0.1% TFA) = 60:40 to 40:60 over 20 min, flow late 5 mL / min, UV: 365 nm, column: Sunfire TM Prep C18 OBD TM The residue was purified using a 5 μm, 19×150 mm column to obtain a yellow solid (Compound 10) (23.61 mg, 16.30 μmol, yield 21%).

[0141] [ka]

[0142] (8-3. Modification of Lysozyme) A solution of compound 10 (2 mg, 1.3 μmol) in acetic acid was added to a 50% aqueous solution of lysozyme (3.95 mg, 0.2 μmol) in acetic acid to prepare a 75% aqueous solution of acetic acid. This reaction solution was shaken at room temperature for 19 hours (final concentration of lysozyme: 0.5 mM; final concentration of compound 10: 3.25 mM). The reaction was tracked by diluting a portion of the reaction solution and analyzing it by HPLC (HPLC conditions: linear concentration gradient from 5% CH3CN to 95% CH3CN in 0.1% TFA aqueous solution (40 minutes), flow rate 1.0 mL / min, detection at 230 nm, Column: COSMOSIL Packed Column 5C 18-AR-II 4.6IDx150mm). In addition, mass spectrometry of the compounds contained in the reaction solution 19 hours after the start of the reaction was performed using MALDI-TOF MASS, and the production of a modified product with one molecule of Npys added (Npys modified product) was confirmed (found: 15653.86). The reaction solution was purified by HPLC to obtain the Npys modified product. Figure 5 shows an HPLC chart showing the reaction over time when a sulfenylation reaction was performed on lysozyme, and the results of mass spectrometry of the compounds contained in the reaction mixture.

Claims

1. A tryptophan-selective modifying agent for selectively modifying a tryptophan residue in a molecule containing a tryptophan residue, comprising a compound represented by the following chemical formula 1 or a salt thereof, in the presence of a compound having a thioether group: 【Chemical 1】 In Chemical Formula 1, V is a nitrogen atom or a carbon atom; W together with other ring atoms forms, when V is a nitrogen atom, a nitrogen-containing heterocycle selected from the group consisting of a pyridine ring, a pyrazine ring, an imidazole ring, an oxazole ring, a quinoline ring, an isoquinoline ring, a quinoxaline ring, a phenanthroline ring, a pteridine ring, and an azocine ring; when V is a carbon atom, W forms, together with other ring atoms, an aromatic ring selected from the group consisting of a benzene ring, a 1H-pyrrole ring, a furan ring, a naphthalene ring, a benzo[h]quinoline ring, a pyrido[2,3-b]pyrazine ring, and a cycloocta-1,3,5,7-tetraene ring; X is —O—, —NH— or a single bond; Y is a chlorine atom when X is a single bond, and when X is -O- or -NH-, it is a group selected from the group consisting of a substituted or unsubstituted C1 to C20 alkyl group, a substituted or unsubstituted C2 to C20 alkenyl group, a substituted or unsubstituted C2 to C20 alkynyl group, a substituted or unsubstituted C3 to C20 cycloalkyl group, a substituted or unsubstituted C3 to C20 cycloalkenyl group, a substituted or unsubstituted C6 to C20 aryl group, a substituted or unsubstituted C3 to C20 heteroaryl group, and a substituted or unsubstituted monovalent group derived from an electron-withdrawing aliphatic heterocycle; Z represents a nitro group present on the nitrogen-containing heterocycle or the aromatic ring; p, q, and r are each independently 0 or 1; s represents an integer of 0 to 10; L ０ and L １ each independently represents a linker having a chemically stable structure, A ａ and A ｂ are each independently a group selected from the group consisting of -CH=CH-, -C≡C-, -C(=O)-, -C(=O)-O-, -O-C(=O)-, -O-, a C1 to C20 oxyalkylene group, a C1 to C20 alkyleneoxy group, -C(=O)-NH-, -NH-C(=O)-, -NH-C(=O)-NH-, hydrazine, triazole, sulfone, sulfoxide, sulfonic acid ester, sulfonamide, sulfinic acid ester, sulfinamide, piperidine, and dioxane; R is a hydrogen atom, a substituted or unsubstituted C1 to C20 alkyl group, a substituted or unsubstituted C2 to C20 alkenyl group, a substituted or unsubstituted C2 to C20 alkynyl group, a substituted or unsubstituted C3 to C20 cycloalkyl group, a substituted or unsubstituted C3 to C20 cycloalkenyl group, a substituted or unsubstituted C6 to C20 aryl group, a substituted or unsubstituted C3 to C20 heteroaryl group, an amino group, a hydroxy group, a group G containing at its terminal a group capable of covalently bonding to a specific functional group of an arbitrary compound, or a group FM derived from a functional substance.

2. The tryptophan-selective modulating agent according to claim 1 , wherein V is a nitrogen atom, and W, together with other ring member atoms, forms the pyridine ring.

3. The tryptophan-selective modulating agent according to claim 1 or 2, wherein X is —O— and Y is a substituted or unsubstituted C6 to C20 aryl group.

4. L ０ and L １ are each independently a substituted or unsubstituted C1 to C20 alkylene group, a substituted or unsubstituted C2 to C20 alkenylene group, a substituted or unsubstituted C2 to C20 alkynylene group, a substituted or unsubstituted C3 to C20 cycloalkylene group, a substituted or unsubstituted C3 to C20 cycloalkenylene group, a substituted or unsubstituted C6 to C20 arylene group, a substituted or unsubstituted C3 to C20 heteroarylene group, —NH—, —O—, —S—, —C(═O)—NH—, —NH-C(═O)—, —O—, —C(═O)—O—, —O-C(═O)—, —S—, —C(═O)—, a polyoxyalkylene group, and a group represented by the following chemical formula (a): 【Chemistry 2】 In chemical formula (a), R ａ represents a substituted or unsubstituted C1 to C15 alkylene group, (wherein these alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, arylene groups and heteroarylene groups may have a substituent), the tryptophan-selective modulating agent according to claim 1 or 2, wherein the alkylene group, alkenylene group, alkynylene group, cycloalkylene group, cycloalkenylene group, arylene group and heteroarylene group are selected from the group consisting of:

5. A ａ is a group selected from the group consisting of —C(═O)—, —C(═O)—O—, —O—C(═O)—, —O—, —C(═O)—NH— and —NH—C(═O)—.

6. The tryptophan-selective modulating agent according to claim 1 or 2, wherein p is 0.

7. 3. The tryptophan-selective modifier according to claim 1, wherein R is a group G containing at its terminal a group capable of covalently bonding to a specific functional group of any compound, and the group G contains one or more groups selected from the group consisting of an azomethine ylide group, a nitrone group, a nitrile ylide group, a diazomethyl group, an azide group, a nitrile oxide group, an alkenyl group, and an alkynyl group.

8. 3. The tryptophan-selective modifier according to claim 1, wherein R is a group FM derived from a functional substance, and the functional substance comprises one or more selected from the group consisting of a drug, a radioactive substance, a fluorescent chemical substance, a luminophore, a fluorescent protein, a luminescent protein, a magnetic material, an enzyme, a coenzyme, a physiologically active substance, DNA, RNA, and PEG.

9. 3. A method for producing a Trp-S bond-containing compound, comprising contacting compound A having a tryptophan residue in the molecule with the tryptophan-selective modifying agent of claim 1 or 2 in a reaction system in which compound B having a thioether group is present, thereby forming a covalent bond between a sulfur atom contained in the compound represented by chemical formula 1 and the tryptophan residue.

10. The method for producing a Trp-S bond-containing compound according to claim 9, wherein an acid is further present in the reaction system.

11. The method for producing a Trp-S bond-containing compound according to claim 9, wherein compound A is a peptide or a protein.

12. 10. The method for producing a Trp-S bond-containing compound according to claim 9, wherein compound A and compound B are a single compound, and the thioether group is a group derived from a methionine residue present in the vicinity of the tryptophan residue to be modified in the peptide or protein.

13. The compound A is a protein, and the protein is produced by genetic recombination. A tryptophan residue has been inserted at the desired position, The amino acid residue at the desired position is modified to a tryptophan residue, The tryptophan residue at the desired position is deleted, or The tryptophan residue at the desired position is modified to another amino acid residue, a methionine residue inserted at the desired position, The amino acid residue at the desired position is modified to a methionine residue, a deletion of the methionine residue at the desired position, or The methionine residue at the desired position is changed to another amino acid residue. The method for producing the Trp-S bond-containing compound according to claim 9.

14. the compound A is a peptide or protein having two or more cysteine ​​residues having a reduced thiol group, protecting the reduced thiol group with a protecting group prior to contacting Compound A with the tryptophan-selective modifying agent; The method for producing a Trp-S bond-containing compound according to claim 9, further comprising contacting compound A, in which the reduced thiol group has been protected, with the tryptophan-selective modifying agent, and then deprotecting the reduced thiol group.

Images