Alkylating agent for polysulfide labeling and method for labeling polysulfides using the same

Abstract

The purpose of the present invention is to provide an alkylating agent that efficiently labels a polysulfide having an (S)n structure.　This problem can be solved by a functional group of the present invention, which is for labeling a polysulfide, represented by formula (1) (in the formula: R1 and R2 independently represent a hydrogen atom, an alkyl group having 1-6 carbon atoms, a substituted alkyl group having 1-6 carbon atoms, a cycloalkyl group having 3-7 carbon atoms, a substituted cycloalkyl group having 3-7 carbon atoms, an alkoxy group having 1-6 carbon atoms or a substituted alkoxy group having 1-6 carbon atoms, or R1 and R2 form, together with the carbon atom to which R1 and R2 are attached, a cycloalkyl group having 3-7 carbon atoms, a substituted cycloalkyl group having 3-7 carbon atoms, an aryl group having 6-12 carbon atoms, a substituted aryl group having 6-12 carbon atoms, a 3- to 7-membered heterocyclic group or a substituted 3- to 7-membered heterocyclic group; and X represents a halogen atom, a methanesulfonyloxy group, a trifluoromethanesulfonyloxy group or para-toluenesulfonyloxy group, provided that when R1 and R2 both represent a hydrogen atom, X represents a halogen atom other than an iodine atom, a methanesulfonyloxy group, a trifluoromethanesulfonyloxy group or para-toluenesulfonyloxy group).

Description

Technical Field

[0001] The present invention relates to an alkylating agent for polysulfide labeling and a method for labeling polysulfide using the same. According to the present invention, a polysulfide structure can be accurately labeled and detected.

Background Art

[0002] In recent years, it has been clarified that many molecular species containing a polysulfide structure such as persulfide, trisulfide, or tetrasulfide in which sulfur atoms are linked are present in vivo. For example, proteins containing cysteine having a polysulfide structure (-(S)n-SH) in which a plurality of sulfur atoms are added to a thiol (-SH) group or an oxidized structure (-S-(S)n-S-) thereof have been reported, and these proteins have been reported to exhibit high nucleophilicity or antioxidant activity. However, since polysulfides having complex chemical properties are difficult to detect, their physiological functions have not been sufficiently elucidated.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] A known method for detecting polysulfides involves modifying the thiol group with, for example, iodoacetamide (IAM) or β-(4-hydroxyphenyl)ethyliodoacetamide (HPE-IAM), and then detecting it by mass spectrometry (Non-Patent Documents 1 and 2). However, conventional alkylating agents for thiol group labeling have not been able to adequately detect (label) polysulfides. Therefore, an object of the present invention is to provide an alkylating agent that efficiently labels polysulfides having a (S)n structure. Another object is to provide a method for efficiently labeling polysulfides having a (S)n structure and a method for detecting polysulfides. [Means for solving the problem]

[0005] The inventors of the present invention diligently researched alkylating agents that efficiently label polysulfides having a (S)n structure, and surprisingly discovered that by modifying iodoacetamide, an alkylating agent capable of efficiently labeling polysulfides can be obtained. This invention is based on these findings. Therefore, the present invention is [1] The following formula (1): [ka] (In the formula, R1 and R2 are each independently a hydrogen atom, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a C3-C7 cycloalkyl group, a substituted C3-C7 cycloalkyl group, a C1-C6 alkoxy group, or a substituted C1-C6 alkoxy group, and R1 and R2 together with the carbon atoms to which they are bonded form a C3-C7 cycloalkyl group, a substituted C3-C7 cycloalkyl group, a C6-C12 aryl group, A polysulfide labeling functional group represented by a carbon-6 to carbon-12 aryl group having substitution, a 3 to 7-membered heterocyclic group, or a substituted 3 to 7-membered heterocyclic group, where X is a halogen atom, a methanesulfonyloxy group, a trifluoromethanesulfonyloxy group, or a p-toluenesulfonyloxy group, and when R1 and R2 are both hydrogen atoms, X is a halogen atom other than an iodine atom, a methanesulfonyloxy group, a trifluoromethanesulfonyloxy group, or a p-toluenesulfonyloxy group. [2][1] A polysulfide-labeling alkylating agent comprising a compound having the functional group described therein or a salt thereof, [3] A method for labeling a polysulfide, comprising the step of contacting the polysulfide with the alkylating agent described in [2], and A method for detecting polysulfide structures, comprising the step of analyzing a protein containing labeled polysulfide by mass spectrometry using the polysulfide labeling method of [4][3], Regarding. The inventors believe that conventional alkylating agents are unable to adequately detect (label) polysulfides because they are highly reactive and react sufficiently with thiol groups that do not contain polysulfides, making identification difficult, or because they may decompose the (S)n structure of the reduced polysulfide structure (-(S)n-SH). The alkylating agent for polysulfide labeling of the present invention is thought to be able to adequately label polysulfides by suppressing the reaction with thiol groups that do not contain polysulfides and suppressing the decomposition of the polysulfide structure. [Effects of the Invention]

[0006] The alkylating agent for polysulfide labeling of the present invention can efficiently label and detect polysulfides compared to conventional alkylating agents. Furthermore, by detecting polysulfides, it is possible to contribute to various biochemical studies, such as elucidating disease mechanisms related to polysulfide proteins, as well as oxidative stress research and signal transduction research. [Brief explanation of the drawing]

[0007] [Figure 1] Figure (A) shows the conventionally known thiol group (reduced form) and disulfide bond (oxidized form) in a cysteine ​​residue, and Figure (B) shows the thiol group (reduced form) and polysulfide bond (oxidized form) having a polysulfide structure. [Figure 2] This is a schematic diagram illustrating how conventional alkylating agents decompose persulfide or trisulfide structures. [Figure 3] This is a profile showing the mass spectrometry of NECFLQHK, the 99th-106th peptide of HSA. [Modes for carrying out the invention]

[0008] [1] Alkylating agent for polysulfide labeling The alkylating agent for polysulfide labeling of the present invention has a functional group suitable for labeling polysulfide structures. The functional group for polysulfide labeling is a group that can label polysulfide groups while suppressing reactions with thiol groups that do not contain polysulfide, and is also a group that suppresses excessive decomposition of the (S)n structure of the polysulfide structure (-(S)n-SH). By having the functional group for polysulfide labeling of the present invention, the alkylating agent for polysulfide labeling can efficiently label polysulfides and detect them.

[0009] Functional groups for polysulfide labeling The polysulfide labeling functional group of the present invention is of the following formula (1): [ka] The group is represented by [formula]. The polysulfide labeling functional group of the present invention is not limited to, but is for reduced polysulfide labeling or oxidized polysulfide labeling. R1 and R2 are not limited in that they do not have functional groups that are more reactive with the polysulfide structure compared to X. R1 and R2 are, for example, independently a hydrogen atom, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a C3-C7 cycloalkyl group, a substituted C3-C7 cycloalkyl group, a C1-C6 alkoxy group, or a substituted C1-C6 alkoxy group. C1-C6 alkyl groups are preferably C1-C3 alkyl groups, and more preferably C1-C3 linear alkyl groups. Specific examples of C1-C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secondary butyl, tertiary butyl, n-pentyl, isopentyl, neopentyl, tertiary pentyl, n-hexyl, and isohexyl groups. Cycloalkyl groups having 3 to 7 carbon atoms are preferably cycloalkyl groups having 4 to 6 carbon atoms. Specific examples of cycloalkyl groups having 3 to 7 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl groups. The alkoxy group having 1 to 6 carbon atoms is preferably an alkoxy group having 1 to 3 carbon atoms, and more preferably a linear alkoxy group having 1 to 3 carbon atoms. Specific examples of alkoxy groups having 1 to 6 carbon atoms include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, secondary butoxy, tertiary butoxy, n-pentyloxy, isopentyloxy, neopentyloxy, n-hexyloxy, or isohexyloxy.

[0010] The substituents are not limited to but include alkyl groups having 1 to 6 carbon atoms, cycloalkyl groups having 3 to 7 carbon atoms, or aryl groups having 6 to 12 carbon atoms. The alkyl groups having 1 to 6 carbon atoms and cycloalkyl groups having 3 to 7 carbon atoms are as described above. Examples of aryl groups having 6 to 12 carbon atoms include phenyl groups or naphthyl groups. The substituted C1-C6 alkyl groups, substituted C3-C7 cycloalkyl groups, or substituted C1-C6 alkoxy groups are groups in which the hydrogen atoms of each group are replaced by substituents. Note that the C1-C6 in "substituted C1-C6 alkyl groups" does not include the number of carbon atoms of the substituents. The substituted C1-C6 alkyl group is preferably a substituted C1-C3 alkyl group, and more preferably a substituted C1-C3 linear alkyl group. Specific examples of substituted C1-C6 alkyl groups include branched alkyl groups, cycloalkylalkyl groups, or arylalkyl groups. The substituted C3-C7 cycloalkyl group is preferably a substituted C4-C6 cycloalkyl group. Specific examples of substituted C3-C7 cycloalkyl groups include alkylcycloalkyl groups, or saturated or unsaturated polycyclic hydrocarbon groups. The substituted C1-C6 alkoxy group is preferably a substituted C1-C3 alkoxy group, and more preferably a substituted C1-C3 linear alkoxy group. Specific examples of substituted C1-C6 alkoxy groups include alkylalkoxy groups, cycloalkylalkoxy groups, or arylalkoxy groups.

[0011] R1 and R2 may, for example, together with the carbon atoms to which R1 and R2 are bonded, form a cycloalkyl group having 3 to 7 carbon atoms, a cyclic alkyl group having 3 to 7 carbon atoms with a substituent, an aryl group having 6 to 12 carbon atoms with a substituent, a heterocyclic group having 3 to 7 members, or a heterocyclic group having 3 to 7 members with a substituent. The cycloalkyl group having 3 to 7 carbon atoms is preferably a cycloalkyl group having 4 to 6 carbon atoms, and more preferably a cycloalkyl group having 4 or 5 carbon atoms. Specific examples of cycloalkyl groups having 3 to 7 carbon atoms include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, or a cycloheptyl group. The substituents are not limited to but include alkyl groups having 1 to 6 carbon atoms, cycloalkyl groups having 3 to 7 carbon atoms, or aryl groups having 6 to 12 carbon atoms. The alkyl groups having 1 to 6 carbon atoms and cycloalkyl groups having 3 to 7 carbon atoms are as described above. Examples of aryl groups having 6 to 12 carbon atoms include phenyl groups or naphthyl groups. The substituted C3-C7 cycloalkyl group is preferably a substituted C4-C6 cycloalkyl group, and more preferably a substituted C4 or C5 cycloalkyl group. Specific examples of substituted C3-C7 cycloalkyl groups include alkylcycloalkyl groups, or saturated or unsaturated polycyclic hydrocarbon groups.

[0012] The 3- to 7-membered heterocyclic group is not particularly limited, but a 3- to 6-membered heterocyclic ring is preferred. Examples of heteroatoms included in the heterocyclic ring include oxygen (O), nitrogen (N), or sulfur (S). The number of heteroatoms included in the heterocyclic ring is also not limited, but the upper limit is preferably 4 or less, more preferably 3 or less, and even more preferably 2 or less. The heterocyclic group may be saturated or unsaturated. Specific examples of 3- to 7-membered heterocyclic rings include oxetane, oxetene, pyrrolidine, pyrrole, tetrahydrofuran, tetrahydrothiophene, imidazolidine, oxazolidine, dioxolane, piperidine, tetrahydropyran, pyran, thian, thiopyran, morpholine, oxazine, dioxane, dioxin, azepan, oxepan, and thiepan.

[0013] X is a halogen atom, a methanesulfonyloxy group (OSO2CH3), a trifluoromethanesulfonyloxy group (OSO2CF3), or a p-toluenesulfonyloxy group (OSO2C6H4CH3), and is preferably a halogen atom. Examples of halogen atoms include fluorine atoms, chlorine atoms, bromine atoms, or iodine atoms, but is preferably a chlorine atom or a bromine atom.

[0014] When both R1 and R2 are hydrogen atoms, X is a halogen atom other than an iodine atom, a methanesulfonyloxy group, a trifluoromethanesulfonyloxy group, or a p-toluenesulfonyloxy group, preferably a halogen atom other than an iodine atom, a methanesulfonyloxy group, or a p-toluenesulfonyloxy group, and more preferably a halogen atom other than an iodine atom.

[0015] A compound containing the polysulfide labeling functional group of the present invention is, for example, the following formula (2): [ka] (R1, R 2、 (and X is the same as in equation (1)) It is represented as follows. R3 is not limited as long as it does not have a functional group that is more reactive with the polysulfide structure than X. Furthermore, since R3 is far from X, which is the reactive group with polysulfide, it has little effect on the reaction between the functional group of the present invention and the polysulfide structure.Therefore, although it is not limited as long as it does not have a functional group that is more reactive with the polysulfide structure than X, examples include hydrocarbon groups (hydrocarbyl groups) that may contain heteroatoms having 1 to 100 carbon atoms.Examples of heteroatoms include sulfur atoms, oxygen atoms, or nitrogen atoms.The number of heteroatoms contained in R3 is not limited, but the number of sulfur atoms, oxygen atoms, or nitrogen atoms is 0 to 20, preferably 0 to 10, and more preferably 0 to 5, respectively.

[0016] As the R3 group, for example, a group containing biotin can be used. Since biotin has a high affinity for avidin, by using avidin beads, for example, only substances containing biotin structures can be bound to the avidin beads from a contaminating sample, and by releasing them, compounds containing biotin structures can be used in various analyses in a purified state. In other words, by using a biotin-containing group as the R3 group, it becomes possible to purify proteins containing polysulfide structures and their peptide digests, thereby improving analytical sensitivity.

[0017] As the R3 group, for example, a group containing an alkynyl group can be used. The alkynyl group, for example, undergoes a triazole cyclization reaction (Hüsgen cycloaddition reaction, or click chemistry) with an azide group in the presence of a copper catalyst, and is bonded. Therefore, any compound containing an azide group can be easily bonded. In other words, by using a group containing an alkynyl group as the R3 group, various substances can be bonded to the alkylating agent.

[0018] Furthermore, a group containing stable isotopes can be used as the R3 group. By replacing some elements of the R3 group with stable isotope elements (heavy elements), tag pairs with different mass numbers can be prepared with the same molecular structure as stable isotope-free labeling reagents (light elements). In this invention as well, by replacing some elements of the polysulfide-labeled molecule with stable isotope elements, it becomes possible to relatively quantify the polysulfide content of specific proteins and peptide digests. Heavy elements are used as stable isotope elements, specifically, 2 H, 13 C, 15 N, 18 O, or 34 Examples include S, but the list is not limited to these.

[0019] The alkylating agent of the present invention may contain a salt of the compound. Specific examples of salts include salts with an inorganic base, an organic base, or a metal alkoxide. The salt can be produced by mixing the compound with an inorganic base, an organic base, or a metal alkoxide. Examples of the inorganic base capable of forming a salt include hydroxides, carbonates, hydrogencarbonates, acetates, or hydrides of alkali metals (for example, lithium, sodium, or potassium, etc.); hydroxides, or hydrides of alkaline earth metals (for example, magnesium, calcium, or barium), and the like. Examples of the organic base capable of forming a salt include dimethylamine, triethylamine, piperazine, pyrrolidine, piperidine, 2-phenylethylamine, benzylamine, ethanolamine, diethanolamine, pyridine, or collidine, etc.; examples of the metal alkoxide include sodium methoxide, potassium tert-butoxide, or magnesium methoxide, and the like.

[0020] The alkylating agent of the present invention can modify the thiol group (-SH group) of reduced polysulfide structures (R 4 -(S)n-SH) in proteins and the like. Specifically, X in the functional group for polysulfide labeling of the present invention and the hydrogen atom of the thiol group are eliminated as HX, and the thiol group is modified by the alkylating agent. Specific examples of the reduced polysulfide include hydrodisulfide (hydropersulfide: R 4 -S-S-H), hydrotrisulfide (R 4 -S-S-S-H), or hydrotetrasulfide (R 4 -S-S-S-S-H), and the like. Furthermore, the alkylating agent of the present invention can modify the polysulfide moiety (-(S)n-) of oxidized polysulfide structures (R 4 -S-(S)n-S-R 5 ). Specific examples of the oxidized polysulfide include trisulfide (R 4 -S-S-S-R 5 ), or tetrasulfide (R 4 -S-S-S-S-R 5 ), and the like.

[0021] <<Function>> Although the mechanism by which the alkylating agent of the present invention can efficiently label polysulfides compared to conventional alkylating agents has not been fully elucidated, it can be presumed as follows. However, the present invention is not limited by the following presumption. In the present invention, group X is a group that participates in the reaction of bonding with a thiol group. In conventional alkylating agents such as iodoacetamide (IAM) or β-(4-hydroxyphenyl)ethyliodoacetamide (HPE-IAM), this X is iodine (I). Generally, thiol groups (R) containing polysulfide structures 4 -(S)n-SH) is a thiol group (R) that does not contain polysulfide. 4 The negative common logarithm of the acid dissociation constant, pK, is better than -SH. a Because R is low, 4 -(S)n-SH is more reactive. On the other hand, alkylating agents where X is iodine(I) and R1 and R2 are hydrogen are originally R 4 -It is known to be highly reactive to SH, and exhibits high reactivity to thiol groups regardless of the presence or absence of polysulfide groups, R 4 It has low reaction selectivity to -(S)n-SH. 4 By substituting with a fluorine atom, chlorine atom, bromine atom, methanesulfonyloxy group, trifluoromethanesulfonyloxy group, or p-toluenesulfonyloxy group, which have low reactivity with -SH, R 4 Without impairing reactivity to -(S)n-SH, 4 It is presumed that reducing the reactivity with -SH can improve the reaction selectivity to the polysulfide structure. On the other hand, R1 and R2 are not groups that directly participate in the reaction with the thiol group, but are groups located near X. In conventional alkylating agents such as iodoacetamide (IAM) or β-(4-hydroxyphenyl)ethyliodoacetamide (HPE-IAM), R1 and R2 are hydrogen atoms (H). Because hydrogen atoms have a small molecular weight, R 4 - Without inhibiting the reaction between SH and X in terms of three-dimensional structure, R 4It also shows high reactivity to -SH. By substituting this hydrogen atom (H) with a group with a large molecular weight and causing steric hindrance, R 4 -It is presumed that this can suppress the reactivity between SH and X. Furthermore, while iodine(I) is highly reactive with thiol groups, its excessive reactivity can sometimes lead to the over-decomposition of the -(S)n- structure of polysulfides. It is hypothesized that substituting iodine(I) with fluorine, chlorine, bromine, methanesulfonyloxy, trifluoromethanesulfonyloxy, or p-toluenesulfonyloxy groups, which have low reactivity with thiol groups, can suppress the decomposition of the -(S)n- structure and efficiently label polysulfides. On the other hand, R1 and R2 are not groups that directly participate in the reaction with the thiol group, but are groups located close to X. In conventional alkylating agents such as iodoacetamide (IAM) or β-(4-hydroxyphenyl)ethyliodoacetamide (HPE-IAM), R1 and R2 are hydrogen atoms (H). Because hydrogen atoms have a small molecular weight, they do not inhibit the reaction between the thiol group and X in terms of stereochemistry. It is presumed that the reactivity between the thiol group and X can be suppressed by substituting these hydrogen atoms (H) with groups with a larger molecular weight, thereby causing steric hindrance. Furthermore, since R3 is geographically distant from X, it has almost no effect on the reaction between the thiol group and X. Therefore, it is presumed that a variety of groups can be used. X is a group that reacts with the hydrogen atom of the thiol group and is eliminated as XH, such as a halogen atom. Methanesulfonyloxy, trifluoromethanesulfonyloxy, and p-toluenesulfonyloxy groups also react with the hydrogen atom and are eliminated as XH, based on a similar reaction mechanism to that of halogen atoms.

[0022] [2] Method for labeling polysulfides The present invention provides a method for labeling polysulfides, which includes the step of contacting the polysulfide with the alkylating agent of the present invention. The polysulfide labeled by the labeling method of the present invention is, for example, a reduced thiol group (R) having the polysulfide structure shown in Figure 1B. 4 -(S)n-SH) and oxidized polysulfide structures (R 4 -S-(S)nSR 5 ) contains. Taking the reduced polysulfide as an example, the H of the thiol group reacts with the X of the alkylating agent (XZ) of the present invention to form HX, which is then eliminated and the thiol group is modified (R 4 -(S)nSZ). Conventional protein cysteine ​​residues were thought to be thiol groups having only one sulfur atom, as shown in the reduced thiol group (Cys-SH) in Figure 1A. Recently, it has been shown that polysulfide structures, as shown in Figure 1B, exist in living organisms. The labeling method of the present invention can label these polysulfide structures. Furthermore, the alkylating agent of the present invention also reacts with polysulfides contained in the oxidized cysteine ​​formation site, and the X of the alkylating agent (XZ) is removed in the reaction, resulting in R 4 -(S)nSZ or R 5 It is presumed to be labeled as -(S)nSZ. As shown in Figure 2, conventional alkylating agents are highly reactive and sometimes excessively decompose the -(S)n- structure of persulfide or trisulfide structures. Therefore, it was not possible to distinguish between, for example, a thiol group and a polysulfide group (persulfide or trisulfide structure) to which a sulfur atom has been further added. According to the polysulfide labeling method of the present invention, polysulfides can be efficiently labeled by using an alkylating agent that suppresses excessive reactions.

[0023] The reaction temperature in the polysulfide labeling method of the present invention is not particularly limited, but is, for example, 4 to 37°C, and preferably 25 to 37°C. The reaction time in the polysulfide labeling method of the present invention is not particularly limited, but is, for example, 30 minutes to 48 hours, and preferably 1 to 24 hours. The buffer used in the polysulfide labeling method of the present invention is not particularly limited as long as the labeling reaction proceeds, but for example, phosphate buffer, Tris-HCl buffer, HEPES buffer, or ammonium carbonate buffer can be used.

[0024] The substance labeled by the polysulfide labeling method of the present invention is not particularly limited as long as it is a substance containing polysulfides, but examples include proteins or peptide hormones. Specific samples are not limited as long as they contain a substance containing polysulfides, but examples include cells, cell extracts, cell supernatants, biopsy samples, organ extracts, blood, plasma, serum, saliva, cerebrospinal fluid, semen, or urine.

[0025] The present invention provides a method for detecting polysulfides, comprising the step of analyzing a protein containing a polysulfide labeled by the polysulfide labeling method using mass spectrometry. The mass spectrometry method can be any mass spectrometry method commonly used in the art, except that a sample labeled by the polysulfide labeling method is used. For example, a protein containing a labeled polysulfide is digested with an enzyme (e.g., trypsin, endoproteinase Glu-C, Lys-C, pepsin, chymotrypsin, or clostripine). The digested protein is then analyzed using a mass spectrometer. [Examples]

[0026] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0027] Manufacturing Example 1 In this manufacturing example, alkylating agent 1 was produced in which R1 and R2 are hydrogen atoms and X is a chlorine atom (Cl). In a 10 mL flask, chloroacetic acid (105 mg, 1.13 mmol), tyramine (151 mg, 1.10 mmol), and N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (350 mg, 11.6 mmol) were added and dissolved in N,N-dimethylformamide (2 mL), and the mixture was stirred at room temperature. N,N-diisopropylethylamine (0.28 mL) was added, and the mixture was stirred at room temperature for 1 hour. Water was added to stop the reaction, and the mixture was extracted with ethyl acetate. The recovered ethyl acetate layer was washed twice with water and once with saturated brine, dried over sodium sulfate, and the solvent was removed by vacuum distillation. The resulting residue was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 50 / 50 → 25 / 75) to obtain the target compound (74.3 mg, yield 32.9%). HRMS-ESI (m / z): [M+Na] + calcd for C 10 H 12 ClNNaO2: 236.04488; found: 236.04391 (error: 1.0 mDa)

[0028] Manufacturing Example 2 In this manufacturing example, alkylating agent 2 was produced in which R1 is a hydrogen atom, R2 is a methyl group, and X is a bromine atom (Br). In a 10 mL flask, 2-bromopropionic acid (100 mg, 0.66 mmol), tyramine (100 mg, 0.72 mmol), and propylphosphonic anhydride (50% ethyl acetate solution, 500 μL) were added and dissolved in N,N-dimethylformamide (2 mL). The mixture was stirred at room temperature. N,N-diisopropylethylamine (172 μL) and 4-dimethylaminopyridine (22 mg) were added, and the mixture was stirred at room temperature for 2 hours. Water was added to stop the reaction, and the mixture was extracted with ethyl acetate. The recovered ethyl acetate layer was washed twice with water and once with saturated brine, dried over sodium sulfate, and the solvent was removed by vacuum distillation. The resulting residue was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 70 / 30 → 30 / 70) to obtain the target compound (151 mg, yield 84.4%). HRMS-ESI (m / z): [2M+H] +calcd for C 22 H 29 Br2N2O4: 545.04886; found: 545.04728 (error: 1.6 mDa)

[0029] 《Manufacturing Example 3》 In this manufacturing example, alkylating agent 3 was produced in which R1 is a hydrogen atom, R2 is a methyl group, and X is an iodine atom (I). The alkylating agent was obtained by repeating the procedure in Production Example 3, except that 2-iodopropionic acid was used instead of 2-bromopropionic acid as a raw material. HRMS-ESI (m / z): [M+Na] + calcd for C 11 H 14 INNaO2: 341.99614; found: 341.99548 (error: 0.7 mDa)

[0030] By using a method similar to the one described above, and reaction materials corresponding to the target product, alkylating agents having polysulfide labeling functional groups as shown in the table below can be produced.

[0031] [Table 1]

[0032] [Table 2]

[0033] [Table 3]

[0034] [Table 4]

[0035] [Table 5]

[0036] [Table 6]

[0037] [Table 7]

[0038] Furthermore, alkylating agents having the polysulfide labeling functional groups listed in the table below can be produced.

[0039] [Table 8]

[0040] Comparative Example 1 In this comparative example, the alkylating agent is the following formula (3): [ka] The polysulfides of polysulfide-modified human serum albumin (HSA) were analyzed using β-(4-hydroxyphenyl)ethyliodoacetamide (HPE-IAM) (Toronto Research Chemicals), in which R1 and R2 are hydrogen atoms and X is an iodine atom (I).

[0041] (Production of polysulfide-treated HSA) 4 mg of HSA was added to a 2 mL microcentrifuge tube and dissolved in 100 μL of ultrapure water. 2 μL of a pre-prepared 150 mM sodium disulfide aqueous solution was added, and the mixture was reacted at 37°C for 1 hour. This solution was purified using a PD SpinTrap G-25 column (Cytiva) according to the recommended protocol, removing excess sodium disulfide, and eluted with ultrapure water. Ultrapure water was added to this eluate to prepare a volume of 500 μL, and the solution was ultrafiltered using an Amicon Ultra-0.5 mL centrifugal filter (molecular weight cutoff: 3 kDa, Merck Millipore) (14,000 x g, 30 minutes). Ultrapure water was added to the concentrate to make a volume of 500 μL, and the ultrafiltration process was repeated to obtain polysulfide-conjugated HSA.

[0042] (Alkylation of polysulfide-modified HSA using alkylating agents and trypsin digestion) 10 μg of polysulfide-modified HSA was added to a 0.5 mL microtube, and diluted with 8 μL of 50 mM Tris-HCl buffer (pH 7.5). To this solution, 1 μL of 20 mM alkylating agent (ethanol solution) was added, followed by 1 μL of 0.1 μg / μL trypsin solution, and the mixture was allowed to stand at 37°C for 16 hours. 1 μL of 10% trifluoroacetic acid aqueous solution was added, and the mixture was allowed to stand at 37°C for 30 minutes to complete trypsin digestion. Desalting was then performed using a peptide desalting and concentration tip (GL-Tip SDB, GL Sciences Inc.), and the peptide fraction eluted from the tip with 80% acetonitrile and 0.1% trifluoroacetic acid aqueous solution was dried using a centrifugal concentrate. The dried material was dissolved in 2% acetonitrile and 0.1% formic acid aqueous solution, and this was used as the sample for mass spectrometry.

[0043] (Measurement by mass spectrometry LC-MS / MS) UltiMate for liquid chromatography (LC) TM The 3000 TSLCnano system (ThermoFisher Scientific) will be installed in the Mass Spectrometry (MS / MS) department at Q Exactive. TMPeptide analysis was performed using a nano ESI LC-MS / MS instrument equipped with a hybrid quadrupole-orbitrap mass spectrometer (ThermoFisher Scientific). For LC separation, an Acclaim PepMap 100, C18 (5 μm, 100 Å, inner diameter 300 μm x 5 mm, Thermo Fisher Scientific) was used as the trap column, and an NTCC-360 / 75-3-125 (C18, particle size 0.3 μm, inner diameter 0.075 mm x 125 mm, Nikkyo Technos) was used as the analytical column. The mobile phase consisted of a 0.1% formic acid aqueous solution (Solvent A) and a 0.1% formic acid acetonitrile solution (Solvent B), which were delivered at a flow rate of 0.3 μL / min to achieve the following solvent composition.

[0044] [Table 9]

[0045] A nanoESI was used as the ion source for the mass spectrometer, and ionization was performed with a spray voltage of 2.00kV, a capillary temperature of 275°C, and an S-lens RF level of 50.0V. The MS scan range was set to m / z 350-1500, and the AGC (automatic gain control) target was 3x10⁻¹⁶. 6 Set the parameters to Charges, acquire MS data in positive mode, then set NCE (Normalized Collision Energy) to 27, and AGC (automatic gain control) target to 1x10. 5 MS / MS data was acquired as Charges. The acquired data was analyzed using PEAKSXPro (Infocom) or Proteome Discoverer 2.4 SP1 (Thermo Fisher Scientific). Table 10 shows the percentage of detection intensities for peptides (-SZ, -SSZ, -SSSZ) in which an alkylating agent (Z) is labeled at cysteine ​​residues 34, 75, 101, 124, 177, 265, 392, 487, and 567 of HSA.

[0046] Example 1 In this example, the alkylating agent 1 obtained in Production Example 1 was used as the alkylating agent, and the polysulfide of polysulfide-modified human serum albumin (HSA) was analyzed. The procedure for comparative example 1 was repeated, except that alkylating agent 1 was used instead of HPE-IAM, and the polysulfides of polysulfide-modified HSA were analyzed. The results are shown in Table 10.

[0047] Example 2 In this example, the alkylating agent 2 obtained in Production Example 2 was used as the alkylating agent, and the polysulfide of polysulfide-modified human serum albumin (HSA) was analyzed. The procedure for comparative example 1 was repeated, except that alkylating agent 2 was used instead of HPE-IAM, and the polysulfides of polysulfide-modified HSA were analyzed. The results are shown in Table 10.

[0048] Example 3 In this example, the alkylating agent 3 obtained in Production Example 3 was used as the alkylating agent, and the polysulfide of polysulfide-modified human serum albumin (HSA) was analyzed. The polysulfide of polysulfide-modified HSA was analyzed by repeating the procedure of Comparative Example 1, except that alkylating agent 3 was used instead of HPE-IAM. The results are shown in Table 10.

[0049] [Table 10]

[0050] As shown in Table 10, the detection rate of polysulfides such as persulfides (-SSZ) and trisulfides (-SSSZ) was improved by substituting X from an iodine atom (I) to a chlorine atom (Cl). Furthermore, the detection rate of polysulfides was also improved by substituting R2 from a hydrogen atom to a methyl group. Furthermore, an example of peptides identified by mass spectrometry is shown in Figure 3. In NECFLQHK, the 99th-106th peptide of HSA, it was confirmed that alkylating agent 1 was labeled to the polysulfide-modified cysteine ​​residue at position 101.

[0051] Manufacturing Example 4 In this manufacturing example, a biotinylated alkylating agent containing a biotin residue represented by the following formula (4) was produced. [ka] The biotinylated alkylating agent was prepared according to the following reaction formula. [ka]

[0052] (Method for producing biotin-PEG3-amine) Biotin-PEG3-azide (103 mg) was added to a 30 mL flask and dissolved in methanol (10 mL). The mixture was degassed under reduced pressure and purged with argon, a catalytic amount of palladium-carbon was added, and the mixture was degassed under vacuum and purged with hydrogen gas. The mixture was stirred overnight at room temperature. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure to obtain the target compound (97.0 mg, 98.1%).

[0053] (Method for producing biotinylated alkylating agents) In a 10 mL flask, biotin-PEG3-amine (22.0 mg, 52.6 mmol), chloroacetic acid (8.6 mg, 113 mmol), and N,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (25.2 mg, 83.8 mmol) were added and dissolved in N,N-dimethylformamide (500 μL), and the mixture was stirred at room temperature. N,N-diisopropylethylamine (14 μL) was added, and the mixture was stirred further at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent: dichloromethane / methanol = 99 / 1 → 80 / 20) to obtain the target compound (19.1 mg, 73.5%). HRMS-ESI (m / z): [M+Na] + calcd for C 20 H 35 ClN4NaO6S: 517.18580; found: 517.18684 (error: 1.0 mDa)

[0054] Example 4 In this example, the polysulfides of human plasma proteins were analyzed using the biotinylated alkylating agent obtained in Production Example 4.

[0055] (Removal of albumin and IgG from human plasma samples) 50 μL of human plasma sample was diluted in 50 μL of PBS, and albumin and IgG were removed using an Albumin & IgG Depletion SpinTrap (Cytiva) according to the company's recommended protocol. In subsequent analytical experiments, both the sample with and without albumin / IgG removal were used.

[0056] (Biotinylation and trypsin digestion of human plasma proteins) Human plasma samples and albumin / IgG-removed samples were subjected to protein precipitation using the ProteoExtract® Protein Precipitation Kit (Merck Millipore) according to the company's recommended protocol. The precipitated proteins were redissolved in a 20 mM sodium acetate aqueous solution (pH 5.5, containing 6 M urea and 0.1% RapiGest (Waters)), and the protein concentration was measured. From these redissolved samples, solutions were taken so that each sample contained 50 μg of protein. 50 mM Tris-HCl aqueous solution (pH 7.5, containing 0.1% RapiGest (Waters)) was added to prepare the solution to a final protein concentration of 0.5 μg / μL and a final urea concentration of 1 M or less. To this solution, 1 μL of 2 mM biotinylated alkylating agent (dimethyl sulfoxide solution) was added, followed by 2 μL of 0.5 μg / μL trypsin solution. The mixture was allowed to stand at 37°C for 16 hours. 1 μL of 10% trifluoroacetic acid aqueous solution was added, and the mixture was allowed to stand at 37°C for 30 minutes to complete the reaction. This solution was purified using a cation exchange column (GL-Tip SCX, GL Sciences) according to the company's recommended protocol to remove unreacted biotinylated alkylating agent. Peptides were eluted using 100 μL of a 10 mM KH2PO4 aqueous solution (1 M KCl, 25% acetonitrile, pH 3.0) and used as a sample for avidin purification.

[0057] (Concentration and recovery of biotinylated peptides using avidin (NeutrAvidin) beads) 100 μL of peptide eluate was transferred to a 2 mL microcentrifuge tube, diluted with 150 μL of PBS, then 10 μL of avidin beads (NeutrAvidin® Agarose HC (Thermo Fisher Scientific)) were added, and the mixture was stirred at room temperature for 2 hours using a rotator. The beads were collected by centrifugation, the supernatant was removed, and the beads were washed four times with PBS (5% acetonitrile), three times with PBS, and three times with ultrapure water. 50 μL of 80% acetonitrile aqueous solution (containing 0.2% formic acid and 0.1% trifluoroacetic acid) was added to the beads, and the mixture was shaken at 70°C for 10 minutes to elute the biotinylated peptide. After returning to room temperature, the mixture was centrifuged, and the supernatant was collected. 50 μL of 80% acetonitrile aqueous solution (containing 0.2% formic acid and 0.1% trifluoroacetic acid) was added to the remaining beads, the supernatant was collected, and after mixing these supernatants, the mixture was dried using a centrifugal concentrate. The dried material was dissolved in a 2% acetonitrile 0.1% formic acid aqueous solution, and this was used as the sample for mass spectrometry.

[0058] The mass spectrometer and measurement conditions were the same as in Comparative Example 1, except that the mixing ratio of the mobile phase was changed as follows. The results are shown in Table 11.

[0059] [Table 11]

[0060] The results of the analysis using plasma proteins are shown below. In plasma proteins, 63 types of proteins were identified as containing persulfide sites, and of these, 7 types of proteins were identified as containing trisulfide structures. In other words, it is possible to concentrate the polysulfide structure using a biotinylated alkylating agent, improving the detection sensitivity of polysulfides.

[0061] [Table 12]

[0062] [Table 13]

[0063] (Digestion of polysulfide-modified HSA using chymotrypsin and polysulfide analysis) Examples 5-7 and Comparative Example 2 In this example and comparative example, the polysulfides of polysulfide-modified human plasma proteins (HSAs) were analyzed using alkylating agents 1-3 from Production Examples 1-3 and the alkylating agent from Comparative Example 1. 10 μg of polysulfide-modified HSA was added to a 0.5 mL microtube, and diluted with 15.5 μL of 50 mM Tris-HCl buffer (pH 7.5) containing 1 M urea. To this solution, 2.0 μL of 10 mM Examples 1-3 or Comparative Example 1 (dimethyl sulfoxide solution) was added, followed by 2.0 μL of 100 mM calcium chloride aqueous solution and 0.5 μL of digestive enzyme solution (0.5 μg / μL chymotrypsin solution). The mixture was allowed to stand at 37°C for 4 hours, then diluted with 35 μL of 50 mM Tris-HCl buffer (pH 7.5), and allowed to stand at 37°C for 17 hours. After completing enzymatic digestion by adding a 10% trifluoroacetic acid aqueous solution, desalting was performed using a peptide desalting and concentration tip (GL-Tip SDB, GL Sciences Inc.). The peptide fraction eluted from the tip with 80% acetonitrile and 0.1% trifluoroacetic acid aqueous solution was dried using a centrifugal concentrate, and the dry material was dissolved in a 2% acetonitrile and 0.1% formic acid aqueous solution to prepare the sample for mass spectrometry. The procedure of Comparative Example 1 was repeated to analyze the polysulfide of polysulfidized HSA. The results are shown in Table 14.

[0064] [Table 14]

[0065] As shown in Table 14, even when the digestive enzyme was changed to chymotrypsin, the detection rate of polysulfides such as persulfide (-SSZ) and trisulfide (-SSSZ) in Examples 5-7 improved compared to Comparative Example 2. Furthermore, of these identified cysteine ​​residues, the 34th cysteine ​​residue (C34) has been reported to exist as reduced cysteine, and the 75th, 253rd, 265th, 316th, 392nd, 487th, and 514th cysteine ​​residues have been reported to be sites that form oxidized cysteine ​​(disulfide bonds) (disulfide bonds are formed in the combinations C75-C91, C245-C253, C265-C279, C316-C361, C392-C438, C476-C487, and C514-C559, respectively). In other words, the alkylating agent in the present invention can efficiently detect all of the polysulfide structures contained in these sites.

[0066] 《Manufacturing Example 5》 In this manufacturing example, alkylating agent 5 was produced, in which R1 is a hydrogen atom, R2 is an ethyl group, X is a bromine atom (Br), and R3 is a hydroxyphenylethyl group, as shown in formula (5) below. [ka] In a 2 mL tube, 2-bromobutyric acid (79 mg, 0.47 mmol), tyramine (65 mg, 0.47 mmol), and propylphosphonic anhydride (50% ethyl acetate solution, 450 μL) were added and dissolved in N,N-dimethylformamide (500 μL). The mixture was stirred at room temperature. N,N-diisopropylethylamine (165 μL) and 4-dimethylaminopyridine (4 mg) were added, and the mixture was stirred at room temperature for 45 minutes. After the reaction was complete, the mixture was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 80 / 20 → 50 / 50) to obtain the target compound (93.1 mg, yield 69.0%).

[0067] Manufacturing Comparison Example 1 In this comparative manufacturing example, comparative alkylating agent 1 was prepared in which R1 and R2 of the following formula (6) are hydrogen atoms, X is an iodine atom (I), and R3 is an alkynyl group. [ka] In a 10 mL round-bottom flask, iodoacetic acid (145 mg, 0.78 mmol) and propargylamine (43 mg, 0.47 mmol) were added, dissolved in N,N-dimethylformamide (1.5 mL), and stirred at room temperature. N,N-diisopropylethylamine (200 μL) and N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (252 mg, 0.84 mmol) were added, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was complete, the solvent was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 70 / 30 → 50 / 50) to obtain the target compound (107 mg, yield 61.5%). HRMS-ESI(m / z): [M+H] + calcd for C5H6INO:223.9567;found:223.9565(error:-0.2 mDa)

[0068] Manufacturing Example 6 In this manufacturing example, an alkylating agent 6 was produced in which R1 and R2 are hydrogen atoms, X is a chlorine atom (Cl), and R3 is an alkynyl group, as shown in formula (7) below. [ka] In a 10 mL round-bottom flask, chloroacetic acid (77 mg, 0.79 mmol) and propargylamine (43 mg, 0.47 mmol) were added and dissolved in N,N-dimethylformamide (1.5 mL), and the mixture was stirred at room temperature. N,N-diisopropylethylamine (200 μL) and N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (247 mg, 0.88 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the solvent was removed by vacuum distillation, and the resulting residue was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 80 / 20 → 50 / 50) to obtain the target compound (105 mg, yield 98.0%). HRMS-ESI(m / z): [M+H] + calcd for C5H6ClNO:132.0211;found:132.0211(error:0.0 mDa)

[0069] Manufacturing Example 7 In this manufacturing example, an alkylating agent 7 was produced in which R1 is a hydrogen atom, R2 is a methyl group, X is a bromine atom (Br), and R3 is an alkynyl group, as shown in formula (8) below. [ka] In a 10 mL round-bottom flask, 2-bromopropionic acid (119 mg, 0.78 mmol) and propargylamine (43 mg, 0.47 mmol) were added, dissolved in N,N-dimethylformamide (1 mL), and stirred at room temperature. N,N-diisopropylethylamine (200 μL) and COMU (366 mg, 0.86 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, the mixture was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 80 / 20 → 50 / 50) to obtain the target compound (102 mg, yield 68.6%). HRMS-ESI(m / z): [M+H] + calcd for C6H8BrNO: 189.9862;found:189.9863(error:0.1 mDa)

[0070] Manufacturing Example 8 In this manufacturing example, an alkylating agent 8 was produced in which R1 is a hydrogen atom, R2 is a methyl group, X is a bromine atom (Br), and R3 contains stable isotopes of carbon and nitrogen and an alkynyl group. In formula (9) below, the elements with asterisks are stable isotopes of carbon and nitrogen, respectively. 13 C and 15 This corresponds to N). [ka] The alkylating agent 8 was prepared according to the following reaction formula. [ka]

[0071] The compound of formula (10) below was synthesized. [ka] In a 10 mL round-bottom flask, add L-phenylalanine-N-FMOC( 13 C9,99%; 15 N,99%,CNLM-4362-H-PK, Cambridge Isotope Laboratories) (28.1 mg, 0.084 mmol) and Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (35.3 mg, 0.117 mmol) were added, and after drying thoroughly, the mixture was dissolved in N,N-dimethylformamide (300 μL), and propargylamine (8.6 mg, 0.47 mmol) and N,N-diisopropylethylamine (30 μL) were added, and the mixture was stirred at room temperature for 1 hour. The reaction solvent was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 70 / 30 → 30 / 70) to obtain the target compound Fmoc-Phe-alkyne( 13 C9, 15 N) was obtained (27.7 mg, yield 90.3%).

[0072] Next, the compound of formula (11) below was synthesized. [ka] In a 10 mL round-bottom flask, add Fmoc-Phe-alkyne( 13 C9, 15 N) (27.7 mg, 0.064 mmol), N,N-dimethylformamide (600 μL), and piperidine (150 μL) were added, and the mixture was stirred at room temperature for 30 minutes. The reaction solvent was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel chromatography (packing material: amino-modified type, eluent: hexane / ethyl acetate = 25 / 75 → 0 / 100), and the target compound H-Phe-alkyne ( 13 C9, 15 N) was obtained (13.6 mg, yield 100%).

[0073] Next, the following procedure was performed to obtain alkylating agent 8 of formula (8) above. In a 10 mL round-bottom flask, H-Phe-alkyne( 13 C9, 15 N) (13.6 mg, 0.064 mmol), Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (38.6 mg, 0.128 mmol) were added, and after drying thoroughly, the mixture was dissolved in N,N-dimethylformamide (500 μL). (R)-(+)-2-bromopropionic acid (19.5 mg, 0.094 mmol) and N,N-diisopropylethylamine (28 μL) were added, and the mixture was stirred at room temperature for 2.5 hours. The reaction solvent was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 70 / 30 → 50 / 50) to obtain the target compound. (19.7 mg, yield 89.1%) HRMS-ESI(m / z): [M+H] + calcd for C9[ 13 C]9H 17 BrN[ 15 N]O2:347.0819;found:347.0818(error: -0.1 mDa)

[0074] 《Manufacturing Example 9》 In this production example, a compound was synthesized that does not use the stable isotope of alkylating agent 8 obtained in production example 8. Specifically, alkylating agent 9 was produced that contains a hydrogen atom as R1, a methyl group as R2, a bromine atom (Br) as X, and an alkynyl group as R3 in the following formula (12), and does not use a stable isotope. [ka] The alkylating agent 9 was prepared according to the following reaction formula. [ka]

[0075] The compound of formula (13) below was synthesized. [ka] In a 10 mL round-bottom flask, L-phenylalanine-N-FMOC (30.4 mg, 0.078 mmol) and tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (35.3 mg, 0.117 mmol) were added. After drying thoroughly, the mixture was dissolved in N,N-dimethylformamide (300 μL), and propargylamine (8.6 mg, 0.47 mmol) and N,N-diisopropylethylamine (30 μL) were added. The mixture was stirred at room temperature for 1 hour. The reaction solvent was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 80 / 20 → 50 / 50) to obtain the target compound Fmoc-Phe-alkyne (32.5 mg, yield 97.6%). HRMS-ESI(m / z): [M+H] + calcd for C 27 H 24 N2O3: 425.1860;found:425.1860(error:0.0 mDa)

[0076] Next, the compound of formula (14) below was synthesized. [ka] In a 10 mL round-bottom flask, Fmoc-Phe-alkyne (32.5 mg, 0.077 mmol), N,N-dimethylformamide (200 μL), and piperidine (50 μL) were added, and the mixture was stirred at room temperature for 30 minutes. The reaction solvent was removed by reduced pressure, and the resulting residue was purified by silica gel chromatography (packing material: amino-modified, eluent: hexane / ethyl acetate = 30 / 70 → 0 / 100) to obtain the target compound H-Phe-alkyne (9.5 mg, yield 61.3%).

[0077] Next, the alkylating agent 9 of formula (12) was obtained by performing the following procedure. In a 10 mL round-bottom flask, H-Phe-alkyne (9.5 mg, 0.047 mmol) and Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (30.0 mg, 0.100 mmol) were added, and after drying thoroughly, the mixture was dissolved in N,N-dimethylformamide (200 μL). (R)-(+)-2-bromopropionic acid (14.3 mg, 0.094 mmol) and N,N-diisopropylethylamine (20 μL) were added, and the mixture was stirred at room temperature for 2.5 hours. The reaction solvent was removed by distillation under reduced pressure, and the resulting residue was purified by silica gel chromatography (eluent: hexane / ethyl acetate = 70 / 30 → 50 / 50) to obtain the target compound. (15.1 mg, yield 95.6%) HRMS-ESI(m / z): [M+H] + calcd for C 15 H 17 BrN2O2:337.0546;found:337.0538(error:0.8 mDa)

[0078] Examples 8-12, Comparative Example 3 In this example and comparative example, the polysulfides of human plasma proteins (HSAs) were analyzed using alkylating agents 5-9 and comparative alkylating agent 1. 10 μg of polysulfide-containing HSA was added to a 0.5 mL microtube, and diluted with 3.6 μL of 50 mM Tris-HCl buffer (pH 7.5) containing 6 M urea. To this solution, 0.5 μL of either 40 mM alkylating agent 6-10 or comparative alkylating agent 1 (dimethyl sulfoxide solution) was added, followed by 0.4 μL of digestive enzyme solution (0.5 μg / μL trypsin-LysC mixed solution). The mixture was allowed to stand at 37°C for 4 hours, then diluted with 35 μL of 50 mM Tris-HCl buffer (pH 7.5), and allowed to stand at 37°C for 20 hours. After adding 1 μL of 50% trifluoroacetic acid aqueous solution to complete the enzymatic digestion, the peptide was desalted using a peptide desalting and concentration tip (GL-Tip SDB, GL Sciences Inc.). The peptide fraction eluted from the tip with 80% acetonitrile and 0.1% trifluoroacetic acid aqueous solution was dried using a centrifugal concentrate, and the dried material was dissolved in 2% acetonitrile and 0.1% formic acid aqueous solution to obtain the sample for mass spectrometry.

[0079] Mass spectrometry was performed in the same manner as in Comparative Example 1. The results are shown in Table 15.

[0080] [Table 15]

[0081] As shown in Table 15, the detection rate of polysulfides such as persulfide (-SSZ) and trisulfide (-SSSZ) was also improved in the alkylating agent with an ethyl group for R2 (Example 8). Furthermore, in compounds containing an alkynyl group for R3, the detection rate of polysulfides such as persulfide (-SSZ) and trisulfide (-SSSZ) was improved by substituting X from an iodine atom (I) to a chlorine atom (Cl) (Example 9). In addition, the detection rate of polysulfides was also improved by substituting R2 from a hydrogen atom to a methyl group and X from an iodine atom (I) to a bromine atom (Br) (Example 10). Furthermore, comparing Examples 10, 11, and 12, it was found that there was no significant difference in the detection rate of polysulfides in compounds containing alkynyl groups and stable isotopes, provided that the structures of X and R1 were the same.

[0082] Examples 13-15 In this example, the alkylating agent 7 obtained in Production Example 7 was reacted with polysulfide-modified HSA, and then the alkynyl group of the alkylating agent was bonded to biotin having an azide group. The polysulfide of the HSA was analyzed by mass spectrometry of the resulting product. 50 μg of polysulfide-modified HSA was added to a 0.5 mL microtube, and diluted with 18 μL of 50 mM Tris-HCl buffer (pH 7.5) containing 6 M urea. To this solution, 2.5 μL of 40 mM alkylating agent 8 (dimethyl sulfoxide solution) obtained in Preparation Example 7 was added, followed by 2 μL of digestive enzyme solution (0.5 μg / μL trypsin-LysC mixed solution). The mixture was allowed to stand at 37°C for 4 hours, then diluted with 140 μL of 50 mM Tris-HCl buffer (pH 7.5), and allowed to stand at 37°C for 18 hours. This solution was dispensed into two 0.5 mL microcentrifuge tubes, 32 μl each. 4 μl of 10 mM biotin-PEG3-azide (Example 13; dimethyl sulfoxide solution) was added to one tube, and 4 μl of 10 mM Biotin-PC-azide (Example 14; Sigma-Aldrich) was added to the other. 1.6 μL of 25 mM tris(3-hydroxypropyltriazolylmethyl)amine aqueous solution and 1.6 μL of 25 mM copper sulfate aqueous solution were added to each tube, followed by 0.8 μL of 100 mM sodium ascorbate. The mixture was shaken at room temperature for 1 hour. After adding 1 μL of 50% trifluoroacetic acid aqueous solution, the mixture was desalted using a peptide desalting and concentration tip (GL-Tip SDB, GL Sciences). The peptide fraction eluted from the chip with an 80% acetonitrile, 0.1% trifluoroacetic acid aqueous solution was dried using a centrifugal concentrate, and the dried material was dissolved in a 2% acetonitrile, 0.1% formic acid aqueous solution to prepare the sample for mass spectrometry. For the sample to which Biotin-PC-azide was bound, a portion of the desalted sample was injected and irradiated with ultraviolet light (365 nm) for 1 hour. Mass spectrometry was then used to confirm whether the linker portion had been cleaved by photoreaction (Example 15). The following shows the scheme for the process described above. Note that biotin-PEG3-azide is not described. [ka] In peptide digests containing cysteine ​​groups 75, 101, 177, 265, 392, and 487 of albumin, peptides were identified that took into account the mass number of alkyne-containing tag molecules and azide compounds bound to the polysulfide group. Specifically, selective labeling of the polysulfide group by the alkyne-containing label molecule and the hysgen cycloaddition reaction proceeded. In addition, in peptides bound to biotin-PC-azide, mass number changes reflecting molecular structural changes due to light irradiation were confirmed by mass spectrometry, making it possible to apply biotin compounds containing linkers that cleave upon stimulation to polysulfide labeling reagents.

[0083] Example 16 (Biotinylation and trypsin digestion of human plasma proteins) Human plasma samples and albumin / IgG-removed samples were subjected to protein precipitation using the ProteoExtract® Protein Precipitation Kit (Merck Millipore) according to the company's recommended protocol. The precipitated proteins were redissolved in a 20 mM sodium acetate aqueous solution (pH 6.0, 6 M urea), and the protein concentration was measured. From these redissolved samples, solutions were taken so that each sample contained 70 μg of protein. 25 mM Tris-HCl aqueous solution (pH 7.5, 6 M urea) was added, followed by 7 μL of 40 mM alkylating agent 7 (acetonitrile solution) obtained in Preparation Example 7. 1.4 μL of digestive enzyme solution (0.5 μg / μL trypsin-LysC mixed solution) was added to prepare the solution to a final protein concentration of 1.0 μg / μL. The solutions were allowed to stand at 37°C for 5 hours, then diluted with 350 μL of 25 mM Tris-HCl buffer (pH 7.5), and allowed to stand at 37°C for 14 hours. To this solution, 42 μL of 10 mM Biotin-PC-azide (Example 12; Sigma-Aldrich) was added, and the mixture was diluted with 75.6 μL of ultrapure water. 5.6 μL of 100 mM tris(3-hydroxypropyltriazolylmethyl)amine aqueous solution and 5.6 μL of 100 mM copper sulfate aqueous solution were added, followed by 11.2 μL of 100 mM sodium ascorbate. The mixture was shaken at room temperature for 1.5 hours. The reaction mixture was diluted with 490 μL of 30% acetonitrile and 0.1% trifluoroacetic acid aqueous solution, and 1 μL of 50% trifluoroacetic acid aqueous solution was added. The mixture was then purified using a cation exchange column (Monospin® SCX, GL Sciences) according to the company's recommended protocol to remove unreacted alkylating agents and biotin compounds. Peptides were eluted using 100 μL each of a 500 mM ammonium acetate aqueous solution (30% acetonitrile, 4% trifluoroacetic acid) and a 500 mM ammonium acetate aqueous solution (30% acetonitrile). The samples were dried using a centrifugal concentrate and used as samples for avidin purification.

[0084] (Concentration and recovery of biotinylated peptides using avidin (NeutrAvidin) beads) The peptide eluate was dissolved in 70 μL of PBS (2.5% acetonitrile), the pH was adjusted to 7 using 1 M Tris-HCl aqueous solution (pH 8.0), and PBS (2.5% acetonitrile) was added to make a total volume of 140 μL. This was dispensed in 120 μL portions, then 15 μL of avidin beads (NeutrAvidin® Agarose HC (Thermo Fisher Scientific)) were added, and PBS (2.5% acetonitrile) was added to make a total volume of 300 μL. The mixture was stirred at room temperature for 2 hours using a rotator. The beads were collected by centrifugation, the supernatant was removed, and the beads were washed three times with PBS (2.5% acetonitrile) and twice with ultrapure water. These beads were suspended in 50 μL of 50 mM Tris-HCl aqueous solution (2.5% acetonitrile, pH 7.5), irradiated with ultraviolet light (365 nm) for 1 hour, the suspension was filtered, and the filtrate was desalted using a peptide desalting and concentration tip (GL-Tip SDB, GL Sciences Inc.). The peptide fraction eluted from the tip was dried using a centrifugal concentrate with 80% acetonitrile and 0.1% trifluoroacetic acid aqueous solution, and the dried material was dissolved in 2% acetonitrile and 0.1% formic acid aqueous solution, which was used as the sample for mass spectrometry.

[0085] Proteome analysis by mass spectrometry identified 150 peptides containing persulfide (-SSZ), 25 peptides containing trisulfide (-SSSZ), and 6 peptides containing tetrasulfide (-SSSSZ) in plasma proteins, and identified 50 types of proteins containing polysulfides. Here, Z indicates the molecular structure produced by attaching Biotin-PC-azide and irradiating with ultraviolet light (Example 14). In other words, selective labeling of polysulfide groups with alkyne-containing labeled molecules, the hysgen cyclization reaction of biotin-PC-azide, concentration with avidin beads, and linker cleavage by ultraviolet irradiation enabled the concentration of polysulfide structures, improving the detection sensitivity of polysulfides.

[0086] Furthermore, upon identifying polysulfide sites in specific proteins, we detected polysulfide structures at cysteine ​​residues 99, 125, 148, 201, 289, 416, 500, 511, 582, and 591 in albumin (Protein accession: P02768, Albumin precursor) (cysteine ​​residues 75, 101, 124, 177, 265, 392, 476, 487, 558, and 567 in the sequence after signal peptide and propeptide cleavage). Many of these cysteine ​​residues are known to form oxidized structures (disulfide bonds). In addition, we detected a polysulfide structure at cysteine ​​residue 256 in α1-antitrypsin (Protein accession: P01009, Alpha-1-antitrypsin precursor). This cysteine ​​residue is known to form a reduced structure. In other words, we succeeded in detecting the polysulfide modification structure contained in the above structure even in endogenous proteins. [Industrial applicability]

[0087] The alkylating agent of the present invention can be used to detect the polysulfide structure of polysulfide proteins.

Claims

1. The following formula (2): 【Chemistry 1】 (In the formula, R １ and R ２ Each of these is independently a hydrogen atom, a C1-C6 alkyl group, a substituted C1-C6 alkyl group, a C3-C7 cycloalkyl group, a substituted C3-C7 cycloalkyl group, a C1-C6 alkoxy group, or a substituted C1-C6 alkoxy group. R １ and R ２ R １ and R ２ Together with the bonded carbon atom, it forms a cycloalkyl group having 3 to 7 carbon atoms, a cycloalkyl group having 3 to 7 carbon atoms with a substituent, an aryl group having 6 to 12 carbon atoms, an aryl group having 6 to 12 carbon atoms with a substituent, a heterocyclic group having 3 to 7 members, or a heterocyclic group having 3 to 7 members with a substituent. The substituent is an alkyl group having 1 to 6 carbon atoms, a cycloalkyl group having 3 to 7 carbon atoms, or an aryl group having 6 to 12 carbon atoms. X is a halogen atom, R １ and R ２ If all of them are hydrogen atoms, then X is a fluorine atom, a chlorine atom, and a bromine atom. A polysulfide-labeling alkylating agent comprising a compound having a polysulfide-labeling functional group represented by R3 (where R3 is a functional group with lower reactivity with the polysulfide structure compared to X), or a salt thereof.

2. A method for labeling a polysulfide, comprising the step of contacting the polysulfide with the alkylating agent described in claim 1 in vitro.

3. A method for detecting a polysulfide structure, comprising the step of analyzing a protein containing a polysulfide labeled by the polysulfide labeling method described in claim 2 using mass spectrometry.

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