Method for producing derivative of cysteine, derivative agents for cysteine under acidic conditions, and reagents for analysis of cysteine

Abstract

To provide a novel method for producing a derivative of an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino.SOLUTION: A method for producing a derivative of an organic substance, the method including reacting an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino with an olefin compound under acidic conditions to obtain the derivative of the organic substance, wherein the olefin compound includes an ethylene structure having at least two electron-withdrawing groups, excluding halogen atoms.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a derivative of an organic substance and a method for analyzing a sample containing an organic substance. [Background technology]

[0002] Thiols play important roles in living organisms, such as maintaining the redox state in the body, regulating protein function, and detoxifying xenobiotics such as heavy metals. Therefore, attempts have been made to analyze thiols in samples such as biological samples. For example, there is a technique for analyzing thiols using an electrochemical detector that utilizes a redox reaction on the electrode surface (Non-Patent Documents 1 and 2). In addition, attempts have been made to convert thiols into derivatives and analyze them (Non-Patent Documents 3 to 8). Known reactions of thiols include reactions with carbon-carbon double bonds (Non-Patent Documents 9 and 10). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Anal.Biochem.,407(2010)151-159 [Non-patent document 2] Talanta,84(2011)789-801 [Non-patent document 3] Anal.Biochem.,27(1969)502-522 [Non-patent document 4] Biomed.Chromatogr.,20(2006)656-661 [Non-Patent Document 5] Chromatographia(1996)42:515 [Non-patent document 6] Journal of Chromatography A,Volume844,Issues1-2,4 June1999,Pages 361-369 [Non-Patent Document 7] Sci Rep,6(2016)21433 [Non-patent document 8] J Chromatogr B Analyt Technol Biomed Life Sci,1083(2018)12-19 [Non-Patent Document 9] European Journal of Organic Chemistry,Volume722, Issue 1,14.May 1969,Pages 222-224 [Non-Patent Document 10] ARKIVOC 2009(viii)187-198 Summary of the Invention [Problem to be solved by the invention]

[0004] However, in the analytical methods of Non-Patent Documents 1 and 2, thiols are analyzed without being derivatized, and therefore thiols are easily oxidized by dissolved oxygen in the sample or eluent used for analysis, making it difficult to obtain stable analytical results. Furthermore, electrochemical detectors do not have sufficient selectivity for thiols, and when measuring samples containing amino acids, sugars, and other complex impurities, the measurement may be hindered by the impurities. Furthermore, electrochemical detectors require maintenance such as cleaning of the electrodes, and their detection sensitivity easily fluctuates and becomes unstable with use, making them difficult to handle.

[0005] In the techniques of Non-Patent Documents 3 to 6, thiol derivatization is carried out under neutral to basic conditions. However, the present inventors have found that the analysis of thiols is difficult under neutral to basic conditions because oxidation reactions of thiols and exchange reactions between thiols and disulfide compounds readily proceed under these conditions. Therefore, accurate analysis of thiols may be difficult with the techniques of Non-Patent Documents 3 to 6, which involve the derivatization of thiols under neutral to basic conditions.

[0006] On the other hand, in the techniques of Non-Patent Documents 7 and 8, thiols are derivatized under acidic conditions (pH 3.5), but the reactivity is poor and special conditions such as a long reaction time or application of microwaves are required. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors have found that a derivative of an organic substance obtained by reacting a specific olefin compound with an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino under acidic conditions is useful for the analysis of organic substances containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, and have thus completed the present invention. The above prior art does not teach or suggest either (a) the reaction of a specific olefin compound with an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino under acidic conditions, or (b) the usefulness of a derivative of an organic substance obtained by such a reaction for the analysis of organic substances containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino.

[0008] That is, the present invention provides the following.

[0009] [1] A method for producing a derivative of an organic substance comprising reacting an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino with an olefin compound under acidic conditions, wherein the olefin compound contains an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms), and ... at least two electron-withdrawing groups (excluding halogen atoms), and wherein the olefin compound contains at least two electron-withdrawing groups. [2] The method for producing the derivative according to [1], wherein the ethylene structure has two electron-withdrawing groups. [3] The method for producing the derivative according to [1] or [2], wherein two electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure. [4] The method for producing a derivative according to any one of [1] to [3], wherein at least two electron-withdrawing groups are the same group. [5] The method for producing a derivative according to any one of [1] to [4], wherein the olefin compound is a compound represented by the following formula (I): [ka] (However, EWG 1 and EWG 2 each independently represents an electron-withdrawing group, and may form a ring together with the carbon atom to which they are attached. [6] The electron-withdrawing group, or EWG 1 and EWG 2 each independently represents -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR 3 )2, cyano, alkyl substituted with halogen atoms, carboxy, nitro, -S(=O)-R 4 , -C(=O)-R 5 or -C(=O)-NR 6 R 7 and where: R 1 , R 2 , R 3 , and R 4 each independently represents a monovalent hydrocarbon group or a monovalent heterocyclic group, which may have a substituent; R 5 , R 6 , and R 7 and each independently represent a hydrogen atom, a monovalent hydrocarbon group, or a monovalent heterocyclic group, which may have a substituent. The method for producing a derivative according to any one of [1] to [5]. [7] The method for producing a derivative according to any one of [1] to [6], wherein the acidic conditions are conditions with a pH of less than 6.0. [8] The method for producing a derivative according to any one of [1] to [7], wherein the organic substance is at least one selected from the group consisting of cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, cysteine persulfide, hypotaurine, glutathione persulfide, and peptidic compounds containing a cysteine residue. [9] The organic substance further contains an amino group, The method for producing the derivative according to any one of [1] to [8], comprising reacting the organic substance with the olefin compound under acidic conditions, and then further reacting them under neutral or basic conditions to obtain a derivative of the organic substance.

[10] (1) A sample containing an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino; an olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms); mixing under acidic conditions to obtain a treated sample containing a derivative of said organic material; and (2) A method for analyzing a sample containing an organic substance, comprising analyzing a derivative of the organic substance in the treated sample.

[11] The analysis in step (2) (2a) separating a derivative of the organic material from the treated sample; (2b) detecting a derivative of the separated organic material; The method for analyzing a sample containing an organic substance according to

[10] , comprising:

[12] The method for analyzing a sample containing an organic substance according to

[10] or

[11] , wherein the sample further contains a disulfide compound.

[13] The method for analyzing a sample containing an organic substance according to

[12] , wherein the disulfide compound is at least one selected from the group consisting of oxidized glutathione and cystine.

[14] The organic substance further contains an amino group; Step (1) is The method for analyzing a sample containing an organic substance according to any one of

[10] to

[13] , comprising: mixing the sample and the olefin compound under acidic conditions, and then further mixing under neutral or basic conditions to obtain a treated sample containing a derivative of the organic substance.

[15] A derivatizing agent for an organic substance containing at least one group selected from the group consisting of sulfanyl, selanyl, and sulfino, the derivatizing agent containing an olefin compound having an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms).

[16] A reagent for analyzing an organic substance containing at least one group selected from the group consisting of sulfanyl, selanyl, and sulfino, the reagent containing the derivatizing agent according to

[15] . [Advantages of the Invention]

[0010] According to the present invention, there are provided: a novel method for producing a derivative of an organic substance containing at least one group selected from the group consisting of sulfanyl (-SH), selanyl (-SeH), and sulfino (-S(=O)-OH); and a novel method for analyzing a sample containing such an organic substance. [Brief Description of the Drawings]

[0011]

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Embodiments for Carrying Out the Invention

[0012] Hereinafter, the present invention will be described in detail with reference to embodiments and examples. However, the present invention is not limited to the embodiments and examples shown below. Also, the "agent" may be a single substance or a composition composed of two or more substances.

[0013] [1. Method for Producing Derivatives of Organic Substances] The method for producing a derivative of an organic substance according to an embodiment of the present invention includes reacting an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino with an olefin compound under acidic conditions to obtain a derivative of the organic substance. Here, the olefin compound contains an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms).

[0014] [1.1.Organic substances] The organic material comprises one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, preferably one or more groups selected from the group consisting of sulfanyl and sulfino, and more preferably sulfanyl, where sulfanyl is —(S) n It is a group represented by -SH, selanyl is a group represented by -SeH, and sulfino is a group represented by -(S=O)-OH. Hereinafter, "one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino" may be referred to as "group A."

[0015] In the present invention, sulfanyl is -(S) n It is a group represented by —SH (n is an integer of 0 or more). According to the method for producing a derivative according to this embodiment, -(S) such as -S-SH and -SS-SH n Organic substances (hereinafter also referred to as persulfides) having a group represented by -SH (where n is an integer of 1 or more) can also be derivatized with olefin compounds. Such persulfides (for example, persulfated cysteine) play an important role in redox in vivo. According to the method for producing a derivative of this embodiment, persulfides can be derivatized while maintaining their redox state, making it possible to understand the redox state in vivo. From the viewpoint of the number of types and abundance of sulfanyl-containing organic substances (particularly natural organic substances in biological samples such as blood, saliva, urine, and feces), -(S) n In the group represented by —SH, n is preferably 0, 1, 2, or 3, more preferably 0, 1, or 2, even more preferably 0 or 1, and particularly preferably 0.

[0016] In the present invention, selanyl is a group represented by -SeH. The selenium atom belongs to Group 16 in the periodic table, just like the sulfur atom, and exhibits reactivity similar to that of the sulfur atom. Therefore, since the reactivity of the selenium atom in the organic substance with respect to the olefin compound is the same as the reactivity of the sulfur atom in the organic substance with respect to the olefin compound, the olefin compound having an ethylene structure described below, which can react well with the sulfur atom in the organic substance, can also react well with the selenium atom in the organic substance.

[0017] One or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino (-S(=O)-OH) may be contained in a plurality in one molecule of the organic substance. In addition, the organic substance may have one or a plurality (e.g., 2, 3, 4) of functional groups in addition to one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino. Examples of the functional group are not particularly limited, and include hydroxy, carboxy, amino, alkylamino, dialkylamino, alkyloxy, alkyloxycarbonyl, alkylcarbonyl, and alkylcarbonyloxy.

[0018] In one embodiment, the organic substance may contain an amino group in addition to group A. Here, the amino group that the organic substance may contain in addition to group A includes a mono-substituted amino group and a di-substituted amino group in addition to an unsubstituted amino group (-NH2). As the amino group that the organic substance may contain in addition to group A, one or more selected from the group consisting of an unsubstituted amino group and a mono-substituted amino group are preferable, and an unsubstituted amino group is more preferable. The organic substance containing group A may contain only 1 amino group in one molecule, or may contain a plurality of amino groups. The number of amino groups contained in one molecule of the organic substance may be 1, 2, or 3, preferably 1 or 2, and more preferably 1.

[0019] The organic substance may be derived from the sample described below. The organic substance may be a polymeric compound or a low molecular weight compound, and preferably is a low molecular weight compound. A low molecular weight compound refers to a compound with a molecular weight of 1500 or less. The molecular weight of a low molecular weight compound may be 1200 or less, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, or 300 or less. The molecular weight of a low molecular weight compound may also be 30 or more, 40 or more, or 50 or more. The low molecular weight compound may be an amino acid (eg, cysteine, selenocysteine), a peptide compound, or a salt thereof. Here, a peptide compound is a compound having a structure obtained by condensing two or more amino acid molecules. The organic substance may be a peptide compound, which is a polymer compound. Examples of the peptide compound include proteins such as albumin.

[0020] The organic substance may be a natural or synthetic compound.

[0021] Specific examples of organic substances include cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, persulfated cysteines (e.g., S-mercaptocysteine, S-disulfanylcysteine, S-trisulfanylcysteine), hypotaurine, persulfated glutathione, peptidic compounds containing a cysteine residue, allyl mercaptan, 2-furfurylthiol, 3-mercapto-3-methylbutylformate, 3-sulfanyl-1-hexanol, thiophen-2-ylmethanethiol, 1,6-hexanedithiol, 4-methyl-4-sulfanylpentan-2-one, 3-sulfanylpentan-2-one, thioterpineol, and 4-methoxy-2-methylbutane-2-thiol. The organic substance may be one kind alone or a combination of two or more kinds. The organic substance is preferably at least one selected from the group consisting of cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, persulfidated cysteine (e.g., S-mercaptocysteine, S-disulfanyl cysteine, S-trisulfanyl cysteine), hypotaurine, persulfidated glutathione, and peptide compounds containing cysteine residues. Here, the peptide compound containing a cysteine residue can be a low molecular compound (e.g., oligopeptide) or a high molecular compound (e.g., protein).

[0022] [1.2. Olefin Compound] The olefin compound contains an ethylene structure (ethene structure) having at least two electron-withdrawing groups, and is a compound capable of reacting with sulfanyl, selanyl, or sulfino in the organic substance via the ethylene structure. However, in this specification, the electron-withdrawing group does not include a halogen atom.

[0023] The number of electron-withdrawing groups in the ethylene structure is usually 2 or more and usually 4 or less, preferably 2 or more and 3 or less, and more preferably 2. When the number of electron-withdrawing groups is within the above range, the reactivity between the olefin compound and the organic substance is improved.

[0024] The electron-withdrawing group may be monovalent or divalent. The number of electron-withdrawing groups in the ethylene structure means the number of bonds between the ethylene structure and the electron-withdrawing groups. Therefore, when a certain electron-withdrawing group is a divalent group and the electron-withdrawing group is bonded to one ethylene structure using two bonds, the number of electron-withdrawing groups in the ethylene structure can be 2.

[0025] Preferably, in the olefin compound, two electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure. Thereby, in the ethylene structure, the reactivity of the carbon atom to which the two electron-withdrawing groups are bonded and the other carbon atom can be improved.

[0026] When two electron-withdrawing groups are bonded to the same carbon atom in the ethylene structure, and when electron-withdrawing groups are bonded to adjacent carbon atoms in the ethylene structure, the two electron-withdrawing groups may form a ring together with the carbon atoms to which they are bonded.

[0027] In the olefin compound, it is preferable that at least two of the electron-withdrawing groups possessed by the ethylene structure are the same group.

[0028] In the olefin compound, at least two of the electron-withdrawing groups in the ethylene structure are the same group, and it is more preferable that the two identical electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure.

[0029] It is more preferable that the olefin compound has an ethylene structure having two identical electron-withdrawing groups, the two identical electron-withdrawing groups being bonded to the same carbon atom constituting the ethylene structure, and two identical groups (which may be hydrogen atoms) being bonded to the carbon atom other than the carbon atom to which the two electron-withdrawing groups are bonded. This can prevent the organic substance from being converted into two diastereomeric derivatives due to the reaction between the olefin compound and the organic substance.

[0030] The ethylene structure may have a group other than the electron-withdrawing group. Examples of groups other than the electron-withdrawing group that the ethylene structure may have include monovalent hydrocarbon groups, preferably one or more types selected from the group consisting of monovalent chain hydrocarbons and monovalent aromatic hydrocarbon groups, and more preferably one or more types selected from the group consisting of alkyls and aryls. Preferably, the ethylene structure has no groups other than electron-withdrawing groups.

[0031] Examples of electron-withdrawing groups include, but are not limited to, -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR3 ) 2. Alkyl substituted with cyano or a halogen atom (e.g., perfluoroalkyl such as trifluoromethyl, perchloroalkyl such as trichloromethyl), carboxy, nitro, -S(=O)-R 4 , -C(=O)-R 5 , and -C(=O)-NR 6 R 7 may be mentioned. Here, R 1 , R 2 , R 3 , and R 4 each independently represents a monovalent hydrocarbon group or a monovalent heterocyclic group, which may or may not have substituents. R 5 , R 6 , and R 7 each independently represents a hydrogen atom, a monovalent hydrocarbon group, or a monovalent heterocyclic group, which may or may not have substituents.

[0032] Examples of the monovalent hydrocarbon group include a monovalent chain hydrocarbon group, a monovalent alicyclic hydrocarbon group, and a monovalent aromatic hydrocarbon group.

[0033] The monovalent chain hydrocarbon group means a hydrocarbon group composed only of a chain structure and does not include a cyclic structure in the main chain. However, the chain structure may be linear or branched. Examples of the monovalent chain hydrocarbon group include alkyl, alkenyl, and alkynyl. Alkyl, alkenyl, and alkynyl may be either linear or branched.

[0034] As alkyl, alkyl having 1 to 12 carbon atoms is preferable, alkyl having 1 to 6 carbon atoms is more preferable, and alkyl having 1 to 4 carbon atoms is still more preferable. The carbon atoms of the substituents are not included in the above carbon atom numbers. Examples of alkyl having 1 to 12 carbon atoms include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and dodecyl.

[0035] The alkenyl is preferably an alkenyl having 2 to 12 carbon atoms, more preferably an alkenyl having 2 to 6 carbon atoms, and even more preferably an alkenyl having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of alkenyl having 2 to 12 carbon atoms include vinyl, propenyl, and n-butenyl.

[0036] The alkynyl is preferably an alkynyl having 2 to 12 carbon atoms, more preferably an alkynyl having 2 to 6 carbon atoms, and even more preferably an alkynyl having 2 to 4 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of alkynyl having 2 to 12 carbon atoms include ethynyl, propynyl, and n-butynyl.

[0037] The monovalent chain hydrocarbon group is preferably an alkyl group.

[0038] A monovalent alicyclic hydrocarbon group refers to a hydrocarbon group that contains only alicyclic hydrocarbons as a ring structure and does not contain an aromatic ring, and the alicyclic hydrocarbon may be either monocyclic or polycyclic. However, it does not necessarily have to be composed only of alicyclic hydrocarbons, and may also contain a chain structure as part of it. Examples of monovalent alicyclic hydrocarbon groups include cycloalkyl, cycloalkenyl, and cycloalkynyl, which may be either monocyclic or polycyclic.

[0039] The cycloalkyl is preferably a cycloalkyl having 3 to 12 carbon atoms, more preferably a cycloalkyl having 3 to 6 carbon atoms, and even more preferably a cycloalkyl having 5 or 6 carbon atoms. The number of carbon atoms does not include the number of carbon atoms of substituents. Examples of cycloalkyl having 3 to 12 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0040] The cycloalkenyl is preferably a cycloalkenyl having 3 to 12 carbon atoms, more preferably a cycloalkenyl having 3 to 6 carbon atoms, and even more preferably a cycloalkenyl having 5 or 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of cycloalkenyl having 3 to 12 carbon atoms include cyclopropenyl, cyclobutenyl, cyclopentenyl, and cyclohexenyl.

[0041] The cycloalkynyl is preferably a cycloalkynyl having 3 to 12 carbon atoms, more preferably a cycloalkynyl having 3 to 6 carbon atoms, and even more preferably a cycloalkynyl having 5 or 6 carbon atoms. The number of carbon atoms in the above does not include the number of carbon atoms of substituents. Examples of cycloalkynyl having 3 to 12 carbon atoms include cyclopropynyl, cyclobutynyl, cyclopentynyl, and cyclohexynyl.

[0042] The monovalent alicyclic hydrocarbon group is preferably a cycloalkyl group.

[0043] A monovalent aromatic hydrocarbon group refers to a hydrocarbon group containing an aromatic ring structure. However, it does not have to be composed of only aromatic rings, and may partially contain a chain structure, an alicyclic hydrocarbon structure, or the like. Therefore, the monovalent aromatic hydrocarbon group can be an aralkyl. The aromatic ring may be either monocyclic or polycyclic. As the monovalent aromatic hydrocarbon group, an aryl having 6 to 12 carbon atoms is preferred, an aryl having 6 to 10 carbon atoms is more preferred, and an aryl having 6 carbon atoms is even more preferred. The number of carbon atoms does not include the number of carbon atoms of the substituent. Examples of aryl having 6 to 12 carbon atoms include phenyl and naphthyl.

[0044] The monovalent aromatic hydrocarbon group is preferably phenyl.

[0045] Among these, the monovalent hydrocarbon group is preferably alkyl, cycloalkyl, or aryl, and more preferably alkyl or aryl.

[0046] A monovalent heterocyclic group refers to a group obtained by removing one hydrogen atom from a cyclic compound containing a heterocycle. The heteroatom constituting the heterocyclic group preferably contains one or more heteroatoms selected from the group consisting of oxygen atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, boron atoms, and silicon atoms, and more preferably contains one or more heteroatoms selected from the group consisting of oxygen atoms, sulfur atoms, and nitrogen atoms. The monovalent heterocyclic group is a monovalent aromatic heterocyclic group or a monovalent non-aromatic heterocyclic group.

[0047] The monovalent aromatic heterocyclic group refers to a heterocyclic group containing a ring having aromaticity. As the monovalent aromatic heterocyclic group, an aromatic heterocyclic group having 1 to 15 carbon atoms is preferred, an aromatic heterocyclic group having 1 to 9 carbon atoms is more preferred, and an aromatic heterocyclic group having 1 to 6 carbon atoms is even more preferred. The number of carbon atoms in the above does not include the number of carbon atoms of the substituent. Examples of the monovalent aromatic heterocyclic group include pyrrolyl, furanyl, thiophenyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, indolyl, purinyl, anthraquinolyl, carbazonyl, fluorenyl, quinolinyl, isoquinolinyl, quinazolinyl, acridinyl, coumarinyl, xanthenyl, and phthalazinyl.

[0048] The monovalent non-aromatic heterocyclic group refers to a heterocyclic group that does not contain an aromatic ring. As the monovalent non-aromatic heterocyclic group, a non-aromatic heterocyclic group having 2 to 15 carbon atoms is preferable, a non-aromatic heterocyclic group having 2 to 9 carbon atoms is more preferable, and a non-aromatic heterocyclic group having 2 to 6 carbon atoms is still more preferable. The carbon atoms of the substituents are not included in the number of carbon atoms. Examples of the monovalent non-aromatic heterocyclic group include, for example, oxiranyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, dihydrofuranyl, tetrahydrofuranyl, dioxolanyl, tetrahydrothiophenyl, pyrrolinyl, imidazolidinyl, oxazolidinyl, piperidinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, piperazinyl, dihydrooxazinyl, tetrahydrooxazinyl, dihydropyrimidinyl, and tetrahydropyrimidinyl.

[0049] Among these, as the monovalent heterocyclic group, a 5- or 6-membered heterocyclic group is preferable.

[0050] Examples of the substituent include a monovalent hydrocarbon group (which may be further substituted by a halogen atom), a monovalent heterocyclic group (which may be further substituted by a halogen atom), -O-R s1 (wherein R s1 represents a hydrogen atom or a monovalent hydrocarbon group.), -(C=O)-R s2 (wherein R s2 represents a hydrogen atom or a monovalent hydrocarbon group.), -(C=O)-O-R s3 (wherein R s3 represents a hydrogen atom or a monovalent hydrocarbon group.), -O-(C=O)-R s4 (wherein R s4 represents a hydrogen atom or a monovalent hydrocarbon group.), -N(R s5 )2 (wherein a plurality of R s5 each independently represents a hydrogen atom or a monovalent hydrocarbon group.), -(C=O)-N(R s6 )2(wherein, when there are a plurality of Rs, s6 each independently represents a hydrogen atom or a monovalent hydrocarbon group.). -N(R s7 )-(C=O)-R s8 (wherein, R s7 represents a hydrogen atom or a monovalent hydrocarbon group. R s8 represents a monovalent hydrocarbon group.). -SO2-R s9 (wherein, R s9 represents hydroxy or a monovalent hydrocarbon group.). -S(=O)-R s10 (wherein, R s10 represents hydroxy or a monovalent hydrocarbon group.). Nitro, Cyano, and halogen atom are exemplified. Here, examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, preferably a fluorine atom or a chlorine atom.

[0051] The substituent is preferably at least one selected from the group consisting of a monovalent hydrocarbon group (which may be further substituted by a halogen atom), nitro, cyano, and a halogen atom.

[0052] The electron-withdrawing group is preferably at least one selected from the group consisting of -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR 3 )2, cyano, alkyl substituted by a halogen atom, carboxy, nitro, -S(=O)-R 4 , -C(=O)-R 5 , and -C(=O)-NR 6 R 7 , and more preferably -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR 3)2. It is one or more selected from the group consisting of cyano, alkyl substituted with a halogen atom, and carboxy. More preferably, -C(=O)-OR 1 , -S(=O)2-R 2 , -P(=O)(-OR 3 )2, and one or more selected from the group consisting of cyano. Here, R 1 , R 2 , R 3 , R 4 , R 5 , R 6、 and R 7 are the same as defined above.

[0053] R 1 is preferably a monovalent hydrocarbon group, more preferably a monovalent chain hydrocarbon group, still more preferably alkyl, and particularly preferably alkyl having 1 to 4 carbon atoms. R 2 is preferably a monovalent hydrocarbon group, more preferably a monovalent aromatic hydrocarbon group, still more preferably aryl, and particularly preferably phenyl. R 3 is preferably a monovalent hydrocarbon group, more preferably a monovalent chain hydrocarbon group, still more preferably alkyl. R 4 is preferably a monovalent hydrocarbon group, more preferably a monovalent aromatic hydrocarbon group, still more preferably aryl. R 5 is preferably a monovalent hydrocarbon group, more preferably a monovalent aromatic hydrocarbon group, still more preferably aryl. R 6 is preferably a hydrogen atom or a monovalent hydrocarbon group, and more preferably a hydrogen atom. R 7 is preferably a hydrogen atom or a monovalent hydrocarbon group, more preferably a monovalent hydrocarbon group, still more preferably a monovalent chain hydrocarbon group or a monovalent aromatic hydrocarbon group, and particularly preferably alkyl or aryl.

[0054] The olefin compound is preferably a compound represented by the following formula (I) from the viewpoint of improving the reactivity with organic substances and the like.

[0055]

Chemical formula

[0056] In formula (I), EWG 1 and EWG 2 each independently represents an electron-withdrawing group, and together with the carbon atom to which they are attached, may form a ring.

[0057] Preferred examples of the electron-withdrawing group are the same as those described above. EWG 1 and EWG 2 are preferably the same group. This can prevent the derivative of the organic substance produced by the reaction of the olefin compound and the organic substance from becoming two derivatives that are diastereomers.

[0058] The olefin compound can be produced by a conventionally known method. Also, commercially available products can be used as the olefin compound.

[0059] [1.3. Reaction conditions] In the production method of the present embodiment, the organic substance and the olefin compound are reacted under acidic conditions. Thereby, the oxidation reaction of the group A possessed by the organic substance can be suppressed. Also, when an oxide such as a disulfide compound coexists in the reaction system, the reaction between the group A and the oxide can be suppressed. As a result, the organic substance can be efficiently converted into a derivative.

[0060] Under acidic conditions means that the reaction of the organic substance and the olefin compound is carried out in an acidic solution. The organic substance and the olefin compound do not necessarily have to be completely dissolved in the acidic solution, and the reaction may be carried out in a state where the organic substance and the olefin compound are dispersed in the acidic solution. The acidic solution generally has a pH of less than 7, preferably less than 6.5, more preferably less than 6.0, even more preferably less than 6.0, particularly preferably less than 5.5, 5.25, 5.0, 4.75, 4.5, or 4.25, and is preferably at least 0.0, more preferably at least 2.0. By controlling the pH of the acidic solution within the above range, decomposition of the derivative can be suppressed while side reactions such as oxidation of the organic substance and exchange reactions with the disulfide compound can be effectively suppressed. Here, the pH of the acidic solution and the pH of the neutral or basic solution described below can be measured at a temperature of 25°C using a pH meter using the glass electrode method.

[0061] The acidic solution is preferably a solution containing water. The acidic solution may contain an organic solvent in addition to water. The organic solvent is preferably a solvent that can be mixed with water in any ratio. Examples of such organic solvents include alcohol solvents such as methanol and ethanol; nitrile solvents such as acetonitrile; and aprotic polar solvents such as dimethyl sulfoxide, dimethylformamide, dioxane, and tetrahydrofuran. When the acidic solution is a solution containing water, the weight ratio of the organic solvent in the solution to the water is preferably 50% by weight or less, more preferably 10% by weight or less, even more preferably 5% by weight or less, and usually 0% by weight or more. More preferably, the acidic solution is an aqueous solution.

[0062] The acidic solution usually contains an acid. Examples of the acid include inorganic acids and organic acids. Examples of the inorganic acids include phosphoric acid, hydrochloric acid, sulfuric acid, and perchloric acid. Examples of the organic acids include formic acid, oxalic acid, acetic acid, trifluoroacetic acid, methanesulfonic acid, trifluoromethanesulfonic acid, toluenesulfonic acid, and cyanoacetic acid. As the acidic solution, a buffer solution whose pH has been adjusted to a desired range may be used.

[0063] The reaction temperature under acidic conditions is not particularly limited, but is preferably 50°C or lower, more preferably 45°C or lower, even more preferably 40°C or lower, and is preferably 5°C or higher, more preferably 10°C or higher, even more preferably 15°C or higher.

[0064] The reaction time under acidic conditions is not particularly limited, but is preferably 60 minutes or less, more preferably 30 minutes or less, even more preferably 15 minutes or less, and is usually 0 minutes or more.

[0065] The reaction under acidic conditions is usually carried out by mixing the organic substance and the olefin compound in an acidic solution. There are no particular limitations on the method for mixing an organic substance and an olefin compound in an acidic solution, and examples thereof include: 1) a method in which an acidic solution containing an organic substance is prepared, and an olefin compound is added to the solution and mixed therewith; and 2) a method in which a solution containing an organic substance is prepared, and a buffer solution or the like is added to the solution to adjust the pH to a desired range, and then the olefin compound is added and mixed therewith. From the viewpoint of suppressing the oxidation reaction of the organic substance, it is preferable to prepare an acidic solution containing the organic substance, and then add the olefin compound to the obtained solution and mix it in. The olefin compound may be added in its original form or in the form of a solution dissolved in a solvent (water, organic solvent).

[0066] The concentration of the organic substance in the acidic solution is not particularly limited, but may be, for example, 0.1 μmol / L to 100 mmol / L. The amount of the olefin compound relative to the organic substance is not particularly limited, but may be, for example, 1 to 100 in molar ratio.

[0067] When the organic substance contains an amino group in addition to group A, the organic substance may be reacted with the olefin compound under acidic conditions, and then further reacted under neutral or basic conditions (preferably basic conditions) to obtain a derivative of the organic substance. As a result, group A contained in the organic substance reacts with the olefin compound under acidic conditions and is converted into a substituent that is stable against side reactions such as oxidation. Next, under neutral or basic conditions, an amino group (which may be an unsubstituted amino group, a mono-substituted amino group, or a di-substituted amino group) contained in the organic substance reacts with the olefin compound to form a substituted amino group having a substituent derived from the olefin compound (when the amino group contained in the organic substance is a di-substituted amino group, a substituted ammonium group). By such a production method, derivatives in which both the group A contained in the organic substance and the amino group that the organic substance may contain react with the olefin compound and both the group A and the amino group are converted can be obtained.

[0068] Derivatives containing more substituted amino groups have advantages in terms of production and analysis compared to derivatives containing the amino group before substitution. Usually, a more substituted amino group has improved hydrophobicity compared to the amino group before substitution. Therefore, in liquid chromatography, it is often possible to easily set the separation conditions for derivatives of organic substances. On the other hand, derivatives containing amino groups with a low degree of substitution (e.g., derivatives containing unsubstituted amino groups such as cysteine and reduced glutathione) have high hydrophilicity, so they have weak retention in reverse-phase liquid chromatography and extremely strong retention in hydrophilic interaction chromatography (HILIC), and it may not be possible to easily set the separation conditions. In addition, derivatives containing more substituted amino groups often have higher detection sensitivity in a mass spectrometer compared to derivatives containing the amino group before substitution.

[0069] Neutral or basic conditions mean reacting an organic substance reacted under acidic conditions (hereinafter also referred to as an acidic-condition derivative) with an olefin compound in a neutral or basic solution. The acidic-condition derivative and the olefin compound do not necessarily have to be completely dissolved in the solution, and the reaction may be carried out in a state where the acidic-condition derivative and the olefin compound are dispersed in the solution.

[0070] The neutral or basic solution usually has a pH of 7 or higher, preferably a pH of 7.5 or higher, more preferably a pH of 8 or higher, even more preferably a pH of 8.5 or higher, and preferably a pH of 11 or lower, more preferably a pH of 10 or lower, even more preferably a pH of 9.5 or lower. By adjusting the pH of the neutral or basic solution to be within the above range, the reaction between the amino group that the organic substance may contain and the olefin compound can be promoted, and decomposition of the derivative can be suppressed.

[0071] The neutral or basic solution is preferably a solution containing water. The neutral or basic solution may contain an organic solvent in addition to water. The organic solvent is preferably a solvent that can be mixed with water in any ratio. Examples of such organic solvents include the organic solvents exemplified in the description of the acidic solution. When the neutral or basic solution is a solution containing water, the weight range of the organic solvent in the solution relative to the water may be the same as the preferred range mentioned above in the description of the acidic solution. More preferably, the neutral or basic solution is an aqueous solution.

[0072] A basic solution usually contains a base. Examples of the base include inorganic bases and organic bases. Examples of the inorganic base include metal hydroxides (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide), metal carbonates (e.g., sodium carbonate, sodium bicarbonate, potassium carbonate), sodium phosphate, potassium phosphate, ammonia, sodium borate, and potassium borate. Examples of the organic base include amines (e.g., trimethylamine, triethylamine). As the neutral or basic solution, a buffer solution with a pH adjusted to a desired range may be used.

[0073] The reaction temperature under neutral or basic conditions is not particularly limited, but is preferably 50°C or lower, more preferably 45°C or lower, even more preferably 40°C or lower, and is preferably 5°C or higher, more preferably 10°C or higher, even more preferably 15°C or higher.

[0074] The reaction time under neutral or basic conditions is not particularly limited, but is preferably 60 minutes or less, more preferably 30 minutes or less, even more preferably 15 minutes or less, and is usually 0 minutes or more.

[0075] The reaction under neutral or basic conditions is usually carried out by mixing the derivative and the olefin compound under acidic conditions in a neutral or basic solution.

[0076] The derivative under acidic conditions may be isolated from the acidic solution and then mixed with the olefin compound in a neutral or basic solution, or the pH of the acidic solution may be adjusted to a desired neutral or basic pH without isolating the derivative under acidic conditions from the acidic solution, and then mixed with the olefin compound in a neutral or basic solution.

[0077] When the derivative is mixed with the olefin compound in a solution under acidic conditions without isolation as described above, the olefin compound may be newly added to the solution. Alternatively, an excess amount of the olefin compound may be added during the reaction under acidic conditions, and the excess olefin compound may be used for the reaction under neutral or basic conditions.

[0078] The production of the organic substance derivative can be confirmed by, for example, liquid chromatography, mass spectrometry, or the like.

[0079] The method for producing a derivative according to the present embodiment may include any step other than the reaction step of reacting an organic substance with an olefin compound. Examples of the optional step include a step of diluting the reaction solution containing the derivative after the reaction step, and a step of isolating the derivative from the reaction solution.

[0080] In the derivatives of organic substances produced by the above method, group A is converted to a group that is more stable to oxidation, and therefore the derivatives of organic substances can be analyzed more accurately than when the organic substances are analyzed directly.

[0081] [2. Analytical methods for samples containing organic substances] According to one embodiment of the present invention, a method for analyzing a sample containing an organic substance includes: (1) a sample containing an organic substance having one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino; an olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms); mixing under acidic conditions to obtain a treated sample containing a derivative of said organic material; and (2) analyzing the derivatives of the organic material in the treated sample.

[0082] [2.1. Process (1)] In step (1), a sample containing an organic substance containing one or more groups (group A) selected from the group consisting of sulfanyl, selanyl, and sulfino is mixed with an olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms) under acidic conditions to obtain a treated sample containing a derivative of the organic substance.

[0083] Examples and preferred examples of organic substances include those mentioned above. A sample may contain multiple types of organic substances. Examples of samples containing organic substances include, but are not limited to, biological samples derived from living organisms, synthetic samples obtained by organic synthesis reactions, and non-biological samples such as environmental samples present in the natural environment. Examples of organisms from which biological samples are derived include mammals (e.g., humans, monkeys, mice, rats, rabbits, cows, pigs, horses, goats, and sheep), birds, and other animals, insects, mollusks, microorganisms, and plants, with mammals being preferred. Examples of biological samples include blood samples (e.g., whole blood, serum, and plasma), saliva, urine, feces, bile, sweat, tears, cerebrospinal fluid, culture media used in cell or microbial culture, and tissue and cell extracts. Examples of synthetic samples include reaction products obtained by organic synthesis reactions that produce organic substances, and culture media used in cell or microbial culture. Examples of environmental samples include samples derived from soil, seawater, and freshwater.

[0084] The concentrations of the organic substance and the olefin compound in the sample are not particularly limited as long as they react to produce a derivative of the organic substance and the product can be analyzed. The concentrations of the organic substance and the olefin compound in the sample may be, for example, 0.001 μmol / L to 1000 mmol / L, preferably 0.01 μmol / L to 100 mmol / L, more preferably 0.1 μmol / L to 10 mmol / L, and even more preferably 1 μmol / L to 1 mmol / L.

[0085] The sample may be a sample that has been subjected to pretreatment, such as deproteinization. In the analytical method of this embodiment, the sample is treated under acidic conditions. Therefore, after performing an acidic pretreatment such as protein removal, the pH of the sample does not need to be adjusted to neutral or basic. The sample can be mixed with an olefin compound while maintaining its acidic pH. Under acidic conditions, side reactions such as the oxidation reaction of group A and the exchange reaction between group A and disulfide are unlikely to proceed. Therefore, side reactions can be suppressed from proceeding between the pretreatment and the analysis. As a result, organic substances having group A can be analyzed more accurately.

[0086] The sample may contain disulfide compounds. Disulfide compounds are compounds containing a group represented by -SS-. Disulfide compounds may be generated by oxidation of thiols. For example, information on the ratio of thiols (eg, cysteine, reduced glutathione) to disulfide compounds (eg, cystine, oxidized glutathione) in a biological sample is useful for determining the redox state of the biological sample. In the analytical method of this embodiment, a group A, such as sulfanyl, contained in an organic substance, which is easily oxidized, is reacted with an olefin compound to convert the organic substance into a derivative, and then the organic substance is analyzed. Furthermore, in the analytical method of this embodiment, the sample is treated under acidic conditions. As described above, under acidic conditions, the exchange reaction between thiol and disulfide compounds is less likely to occur. Therefore, the amount of organic substance present in the sample can be accurately determined.

[0087] Examples of disulfide compounds that may be contained in the sample include compounds in which sulfanyl groups that may be contained in the organic substances are converted into disulfides through intermolecular reactions, and more specifically, examples thereof include dimethyl disulfide, diethyl disulfide, oxidized glutathione, and cystine. The disulfide compound that may be contained in the sample may contain an amino group. Here, the amino group that may be contained in the disulfide compound includes not only an unsubstituted amino group (—NH2) but also a mono-substituted amino group and a di-substituted amino group. The amino group that may be contained in the disulfide compound is preferably one or more selected from the group consisting of an unsubstituted amino group and a mono-substituted amino group, and more preferably an unsubstituted amino group. The disulfide compound may contain only one amino group or multiple amino groups per molecule. The number of amino groups that may be contained in one molecule of the disulfide compound may be 1, 2, 3, or 4, preferably 1, 2, or 3, and more preferably 1 or 2. In one embodiment, the sample comprises one or more selected from the group consisting of oxidized glutathione and cystine.

[0088] Examples and preferred examples of the olefin compound include the same examples as those mentioned above.

[0089] A sample containing an organic substance is mixed with an olefin compound under acidic conditions to obtain a treated sample containing a derivative of the organic substance. Preferably, the sample containing the organic substance is mixed with the olefin compound in an acidic solution, the type and pH of which may be the same as those described in the preferred examples in [1.3. Reaction Conditions].

[0090] The mixing method is not particularly limited, and examples include: 1) a method in which a solution containing a sample containing an organic substance and an acidic solution having a desired pH value is prepared, and an olefin compound is added to this and mixed; and 2) a method in which a sample containing an organic substance is diluted with dilute hydrochloric acid, a solvent, etc. to form a solution, a buffer solution, etc. is added to this to adjust the pH to a desired range, and then an olefin compound is added and mixed.

[0091] The treatment temperature and treatment time are not particularly limited, but can be the same as the preferred examples of the reaction temperature and reaction time described in [1.3. Reaction conditions].

[0092] When the organic substance further contains an amino group in group A, step (1) preferably includes step (1'). In step (1'), the sample and the olefin compound are mixed under acidic conditions, and then further mixed under neutral or basic conditions to obtain a treated sample containing a derivative of the organic substance. As described above, group A contained in the organic substance in the sample reacts with the olefin compound under acidic conditions and is converted into a substituent that is stable against side reactions such as oxidation. Next, under neutral or basic conditions, the amino group (which may be an unsubstituted amino group, a monosubstituted amino group, or a disubstituted amino group) contained in the organic substance reacts with the olefin compound and is converted into a substituted amino group (a substituted ammonio group if the amino group contained in the organic substance is a disubstituted amino group), thereby obtaining a derivative of the organic substance. As described above, a more substituted amino group has improved hydrophobicity, and therefore, derivatives in which a lightly substituted amino group has been converted into a more substituted amino group can be easily separated by easily setting separation conditions in typical liquid chromatography. Furthermore, derivatives in which a lightly substituted amino group has been converted into a more substituted amino group typically exhibit increased detection sensitivity in mass spectrometry.

[0093] When a sample contains compounds with low-substituted amino groups (e.g., disulfide compounds with unsubstituted amino groups), the compounds are highly hydrophilic, resulting in weak retention in reversed-phase liquid chromatography and extremely strong retention in hydrophilic interaction chromatography (HILIC), making it difficult to set separation conditions. Even if a sample contains compounds containing lightly substituted amino groups, such as disulfide compounds containing unsubstituted amino groups, the amino groups react with the olefin compound under neutral or basic conditions to convert them to more substituted amino groups (or to substituted ammonio groups if the amino groups are disubstituted amino groups). Similar to derivatives of organic substances, compounds such as disulfide compounds in which amino groups have been converted to more substituted amino groups can be easily separated by standard liquid chromatography, allowing for easy setup of separation conditions. Furthermore, the detection sensitivity of standard mass spectrometers is increased.

[0094] For example, an analytical method will be described for the case where the organic substance that may be contained in a sample is cysteine and the disulfide compound that may be contained in the sample is cystine, which is a dimer of cysteine. A sample treated under acidic conditions, obtained by mixing the olefin compound with the sample under acidic conditions, is thought to contain a derivative of cysteine (first derivative) and cystine, which are obtained by reaction of the sulfanyl group of cysteine with the olefin compound.

[0095] Cysteine derivatives have one unsubstituted amino group, while cystine has two unsubstituted amino groups. This results in a large difference in hydrophobicity between cysteine derivatives and cystine derivatives. Therefore, when analyzing by liquid chromatography, it may not be easy to set conditions for simultaneously analyzing cysteine derivatives and cystine.

[0096] On the other hand, when a sample and the olefin compound are mixed under acidic conditions and then further mixed under neutral or basic conditions to obtain a treated sample under neutral or basic conditions, the treated sample is likely to contain a cysteine derivative (second derivative) obtained by reaction of the sulfanyl group and amino group with the olefin compound, and a cystine derivative obtained by reaction of the amino group with the olefin compound.

[0097] Since the amino groups of cysteine and cystine react with the olefin compound, the difference in hydrophobicity between the cysteine derivative (second derivative) and the cystine derivative becomes small, and it becomes relatively easy to set conditions for simultaneously analyzing the cysteine derivative and cystine during liquid chromatography analysis.

[0098] Therefore, by including step (1') in step (1), organic substances containing an amino group in addition to group A in a sample can be analyzed accurately and easily.

[0099] In step (1'), the sample containing the organic substance and the olefin compound are preferably mixed in an acidic solution, and then further mixed in a neutral or basic solution. The type and pH of the neutral or basic solution may be the same as those described in the preferred examples in [1.3. Reaction Conditions].

[0100] The method of mixing in a neutral or basic solution is not particularly limited. For example, a method of mixing a sample and the olefin compound in an acidic solution, then adding a basic solution to the resulting mixed solution (sample treated under acidic conditions) to adjust the pH of the mixed solution to a desired neutral or basic pH, and further mixing the sample and the olefin compound in the neutral or basic solution can be mentioned; or a method of adding the mixed solution to a prepared neutral or basic solution and mixing them.

[0101] The treatment temperature and treatment time under neutral or basic conditions are not particularly limited, but can be the same as the preferred examples of the reaction temperature and reaction time under neutral or basic conditions described in [1.3. Reaction conditions].

[0102] A sample containing an organic substance and the olefin compound are mixed in an acidic solution, and then further mixed in a neutral or basic solution, whereby a treated sample containing a derivative of the organic substance can be obtained.

[0103] [2.2. Process (2)] In step (2), the derivatives of the organic substances in the treated sample are analyzed. Step (2) may include step (2a) and step (2b) in this order. [2.2.1. Step (2a)] In step (2a), derivatives of organic substances are separated from the processed sample in step (1). The separation can be carried out by any method by which substances other than the derivatives contained in the processed sample (for example, disulfide compounds) and the derivatives can be separated. Examples of the separation method are not particularly limited, but include liquid chromatography, supercritical fluid chromatography (SFC), capillary electrophoresis (CE), and gas chromatography (GC). The separation method may be appropriately selected according to the types and properties of impurities contained in the sample and the types and properties of the derivatives of the organic substances. As the separation method, liquid chromatography is preferable, and high performance liquid chromatography (HPLC) is more preferable. These separation methods can be carried out by previously known methods. These separation methods may be used alone or in combination.

[0104] Examples of liquid chromatography include reverse phase liquid chromatography, normal phase liquid chromatography, hydrophilic interaction chromatography (HILIC), ion exchange chromatography, and size exclusion chromatography.

[0105] Examples of the stationary phase of reverse phase liquid chromatography are not particularly limited, but include silica gel modified with a hydrophobic compound such as octadecylsilane. Examples of the mobile phase are not particularly limited, but include organic solvents, aqueous solutions, and mixed solvents thereof. Preferable examples of the organic solvent include acetonitrile, methanol, ethanol, isopropanol, etc. Preferable examples of the aqueous solution include water, aqueous formic acid solution, aqueous ammonium formate solution, aqueous trifluoroacetic acid solution, aqueous acetic acid solution, aqueous ammonium acetate solution, aqueous ammonium bicarbonate solution, buffer solution, etc.

[0106] Examples of stationary phases for normal-phase liquid chromatography include, but are not particularly limited to, silica gel and alumina. Examples of mobile phases include, but are not particularly limited to, hexane, ethyl acetate, methylene chloride, isopropanol, ethanol, methanol, tetrahydrofuran, and mixed solvents thereof.

[0107] Examples of stationary phases for hydrophilic interaction chromatography include, but are not particularly limited to, silica gel and silica gel modified with aminopropyl, amide, diol, or cyano. Examples of mobile phases include, but are not particularly limited to, organic solvents, aqueous solutions, and mixed solvents thereof. Preferred examples of organic solvents include acetonitrile, methanol, ethanol, isopropanol, etc. Preferred examples of aqueous solutions include water, aqueous formic acid solution, aqueous ammonium formate solution, aqueous trifluoroacetic acid solution, aqueous acetic acid solution, aqueous ammonium acetate solution, aqueous ammonium bicarbonate solution, buffer solutions, etc.

[0108] Hydrophilic interaction chromatography is preferably used when separating highly hydrophilic targets with weak retention on the stationary phase of reverse-phase chromatography. For example, disulfide compounds such as cystine and oxidized glutathione that are not derivatized with olefin compounds and thiol derivatives such as cysteine derivatives and reduced glutathione derivatives can be simultaneously analyzed by hydrophilic interaction chromatography.

[0109] In any liquid chromatography, by using a column packed with a chiral packing material (chiral column), chiral compounds can be separated when the organic substance is a mixture of chiral compounds. For example, derivatives of D-cysteine (which has a low natural abundance) and derivatives of L-cysteine can be separated by a chiral column.

[0110] [2.2.2. Step (2b)] In step (2b), the derivative of the separated organic substance is detected. Examples of detection methods include, but are not limited to, mass spectrometry using a mass spectrometer, ultraviolet absorption detection (UV), photodiode array detection (PDA), fluorescence detection (FL), visible light absorption detection, inductively coupled plasma optical emission spectroscopy (ICP), corona charged particle detection, refractive index detection (RI), and evaporative light scattering detection (ELSD). The detection method may be selected appropriately depending on the type and properties of impurities contained in the sample and the type and properties of the derivative of the organic substance. Preferred detection methods include mass spectrometry, ultraviolet absorption detection, fluorescence detection, visible light absorption detection, and inductively coupled plasma optical emission spectrometry, and more preferred are mass spectrometry and ultraviolet absorption detection. These detection methods may be used alone or in combination. These separation methods can be carried out by known methods.

[0111] The mass spectrometry method is not particularly limited, and a mass spectrometry method that combines various ionization methods, various ion separation methods, and various detectors may be used. Specific examples of mass spectrometers used in mass spectrometry include triple quadrupole mass spectrometers, time-of-flight mass spectrometers (TOF-MS), and ion trap mass spectrometers (eg, Orbitrap (registered trademark) mass spectrometers).

[0112] [2.3. Optional steps] The analysis method of this embodiment may include any step other than the steps (1) and (2). An example of an optional step is a step of derivatizing an amino acid with an amino acid derivatizing reagent such as ninhydrin, o-phthalaldehyde, etc., when a sample containing an amino acid is used as the sample. The step of derivatizing an amino acid is preferably carried out after the step (1) and before the step (2). By including such an optional step, the following advantages can be obtained, for example. For example, cysteine and cystine are usually difficult to analyze simultaneously using an amino acid analyzer. This is because when cysteine and cystine are derivatized with an amino acid derivatizing agent, their retention times usually become similar. However, by performing step (1), only cysteine among cysteine and cystine reacts with the olefin compound and is converted into a derivative, so the retention time of cysteine after derivatization with the amino acid derivatizing agent is usually different from that of cystine. Therefore, by the analysis method of the present embodiment, cysteine and cystine can be analyzed simultaneously using an amino acid analyzer.

[0113] [3. Derivatizing Agent for Organic Substances under Acidic Conditions] The agent according to an embodiment of the present invention is a derivatizing agent for an organic substance containing at least one group selected from the group consisting of sulfanyl, selanyl, and sulfino under acidic conditions, and includes an olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms).

[0114] Examples and preferred examples of the olefin compound are the same as those described above. Also, examples and preferred examples of the organic substance are the same as those described above. The conditions for producing the derivative using the derivatizing agent are acidic. Preferably, the derivatization of the organic substance is carried out in an acidic solution containing the derivatizing agent. The type of the acidic solution and the pH of the acidic solution can be the same as the preferred examples described in [1.3. Reaction Conditions].

[0115] The concentration of the olefin compound in the derivatizing agent depends on the dilution rate of the derivatizing agent, but is, for example, 0.001 μmol / L to 1000 mol / L, preferably 0.01 μmol / L to 100 mol / L, more preferably 0.1 μmol / L to 10 mol / L, and even more preferably 1 μmol / L to 1 mol / L.

[0116] [4. Reagent for Analyzing Organic Substances] The reagent according to an embodiment of the present invention is a reagent for analyzing an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, and includes the derivatizing agent.

[0117] Examples and preferred examples of the olefin compound are the same as those described above. Also, examples and preferred examples of the organic substance are the same as those described above.

[0118] The concentration of the olefin compound in the analytical reagent depends on the dilution ratio of the analytical reagent, and is, for example, 0.001 μmol / L to 1000 mol / L, preferably 0.01 μmol / L to 100 mol / L, more preferably 0.1 μmol / L to 10 mol / L, and even more preferably 1 μmol / L to 1 mol / L.

[0119] The analytical reagent is preferably used in a method for analyzing a sample, including the above steps (1) and (2).

Examples

[0120] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples. The following operations were carried out at normal temperature under atmospheric pressure unless otherwise specified.

[0121] [Explanation of Abbreviations] In the following, the abbreviations represent the following meanings. (Olefin Compound (Derivatizing Agent)) EMM: Diethyl methylenemalonate BPSE: 1,1 - Bis(phenylsulfonyl)ethylene BEPE: 1,1 - Bis(diethoxyphosphoryl)ethylene NEM: N - Ethylmaleimide

[0122]

Chemical Formula

[0123] In the above chemical formula, "Ph" represents phenyl and "Et" represents ethyl.

[0124] (Organic substances (including chalcogen compounds)) · Thiols Cys: Cysteine Cys2: Cystine GSH: Glutathione - reduced form GSSG: Glutathione - oxidized form gEC: γ - Glutamylcysteine CG: Cysteinylglycine Hcy: Homocysteine NAC: N - Acetylcysteine

[0125] · Persulfide cysteines Cys - SH: S - mercaptocysteine Cys - SSH: S - disulfanylcysteine

[0126]

Chem.

[0127] (Apparatus, analysis method, etc.) LC - MS / MS: Liquid chromatograph - tandem mass spectrometer HILIC: Hydrophilic Interaction Chromatography (Hydrophilic interaction chromatography)

[0128] [Example A: Example using EMM and NEM] [Example A1] Reaction of EMM, NEM with cysteine (Cys) under acidic conditions In this example, the reactivity of EMM and NEM under acidic conditions was estimated from the amount of residual Cys after the reaction. Also, the EMM - derivatized Cys peak was confirmed by LC - MS / MS.

[0129]

Chem.

[0130] (Production of A1-1 Derivative) L-cysteine hydrochloride (manufactured by Sigma) was dissolved in a 0.1% formic acid solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., prepared by diluting with pure water) to a concentration of 10 mmol / L. The derivatizing agent EMM (manufactured by Asta Tech) was dissolved in acetonitrile (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to a concentration of 10 μL / mL, and the derivatizing agent NEM (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in acetonitrile to a concentration of 10 mg / mL. 10 μL of the derivatizing agent solution (EMM or NEM) or 10 μL of acetonitrile was added to 10 μL of the cysteine solution, and the mixture was left at room temperature for 10 minutes. After the reaction, 980 μL of 0.1% formic acid was added and stirred well to obtain a sample solution.

[0131] (Analysis of A1-2 Derivative) The sample solution was analyzed by LC-MS / MS under the following conditions. For LC, an Agilent 1200 series (manufactured by Agilent) was used, and for the tandem mass spectrometer, a 3200 QTRAP system (manufactured by Sciex) was used.

[0132] (LC Conditions) · Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile · Gradient conditions: 5 - 80% B (0 - 8 min), 80% B (8 - 10 min), 80 - 5% B (10 - 10.5 min), 5% B (10.5 - 15 min) · Column: XBridge C18 3.5 μm, 2.1×100 mm (manufactured by Waters) · Column temperature: 40 °C · Flow rate: 0.4 mL / min · Injection volume: 1 μL

[0133] (MS / MS Conditions) The analysis was carried out under the conditions shown in the following table. In the following table, "NEM-Cys" and "EMM-Cys" represent Cys derivatized by NEM and Cys derivatized by EMM, respectively.

[0134]

Table 1

[0135] (Analysis results) The peak of non-derivatized Cys was confirmed at 1.36 min, the NEM-Cys peaks were confirmed at 2.00 and 2.14 min respectively (two peaks were observed due to the formation of diastereomers), and the EMM-Cys peak was confirmed at 8.23 min. Each peak was integrated. The measurement was performed twice, and the result was the average value of the two measurements. The derivatization rate was calculated by the following formula (1).

[0136]

Equation

[0137] The analysis results are shown in the following table. Also, an example of the chromatogram is shown in FIGS. 1-3. FIG. 1 is an example of a chromatogram showing the peak of cysteine. FIG. 2 is an example of a chromatogram showing the peak of NEM-Cys. FIG. 3 is an example of a chromatogram showing the peak of EMM-Cys.

[0138]

Table 2

[0139] According to the above results, it can be seen that under acidic conditions and mild temperature conditions (room temperature), Cys can be derivatized with a high reaction rate by NEM or EMM. In particular, when EMM was used, a higher reaction rate was shown compared to NEM. Also, when Cys, a chiral compound, was derivatized with NEM, diastereomers were formed and the NEM-Cys peak split into two. On the other hand, when Cys was derivatized with EMM, no diastereomers were formed and no peak splitting occurred.

[0140] [Example A2] Simultaneous Detection and Quantification of EMM-Derivatized Thiols (Separation by Reverse-Phase LC) In this example, six thiol compounds derivatized with EMM under acidic conditions were simultaneously separated and detected using LC-MS / MS.

[0141] (A2-1. Preparation of Derivatives) A 0.01 mol / L hydrochloric acid standard thiol mixed solution (diluted with pure water from FUJIFILM Wako Pure Chemical Corporation) containing 25 μmol / L each of L-cysteine hydrochloride, glutathione-reduced (GSH) (manufactured by FUJIFILM Wako Pure Chemical Corporation), D,L-homocysteine (Hcy) (manufactured by Sigma), cysteinylglycine (CG) (manufactured by Bachem), γ-glutamylcysteine (gEC) (manufactured by Sigma), and N-acetylcysteine (NAC) (manufactured by Junsei Chemical Co., Ltd.) was prepared. To 60 μL of 20 mmol / L sodium phosphate buffer (pH 2.5) (diluted with pure water from Nacalai Tesque), 10 μL of the above standard thiol solution was added, 10 μL of an EMM acetonitrile solution (10 μL / mL) was added, stirred, and left at room temperature for 10 minutes. 220 μL of 0.1% formic acid solution was added and stirred well to obtain a sample solution.

[0142] (A2-2. Analysis of Derivatives) The sample solution was analyzed by LC-MS / MS. For LC, Nexera (manufactured by Shimadzu Corporation) was used, and for the tandem mass spectrometer, Triple Quad 6500 system (manufactured by Sciex) was used.

[0143] (LC Conditions) · Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile · Gradient conditions: 15 - 20% B (0 - 2.5 min), 20 - 90% B (2.5 - 4.0 min), 90% B (4.0 - 6.0 min), 90 - 15% B (6.0 - 6.2 min), 15% B (6.2 - 8.0 min) · Column: L-Column ODS, 3 μm, 2.1×50 mm (manufactured by the National Institute of Advanced Industrial Science and Technology), with Inertsil ODS-3, 3 μm, 1.5×10 mm Guard column for UHPLC (manufactured by GL-Science) attached as a guard column · Column temperature: 40 °C · Flow rate: 0.4 mL / min · Injection volume: 1 μL

[0144] (MS / MS conditions) Performed under the conditions shown in the following table.

[0145]

Table 3

[0146] (Analysis results) The analysis results are shown in the following table.

[0147]

Table 4

[0148] According to the above results, it can be seen that various thiols can be derivatized by EMM, the derivatized thiols can be separated all at once by reversed-phase LC, and detected by MS / MS.

[0149] [Example A3] Analysis of Sulfinic Acid In this example, it was confirmed that not only thiol (-SH) but also sulfinic acid (-SO2H) has nucleophilicity and can be derivatized by an EMM agent for analysis.

[0150]

Chemical formula

[0151] (A3-1. Production of Derivative) Hypotaurine (Sigma) was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 1 mmol / L. 10 μL of the standard hypotaurine solution was added to 60 μL of 20 mmol / L sodium phosphate buffer (pH 2.5), and 10 μL of EMM acetonitrile solution (10 μL / mL) was added. The mixture was stirred and left at room temperature for 10 minutes. 920 μL of 0.1% formic acid solution was added and stirred well to prepare the sample solution.

[0152] (A3-2. Analysis of Derivatives) The sample solution was analyzed by LC-MS / MS under the following conditions: Agilent 1200 series (Agilent) was used for LC, and 3200QTRAP system (Sciex) was used as the tandem mass spectrometer.

[0153] (LC conditions) Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile Gradient conditions: 15-40%B (0-3.0 min), 40-90%B (3.0-3.2 min), 90%B (3.2-5.5 min), 90-15%B (5.5-5.8 min), 15%B (5.8-9.5 min) Column: L-Column ODS, 3 μm, 2.1 × 50 mm (Chemicals Evaluation and Research Institute, Japan) equipped with Inertsil ODS-3, 3 μm, 1.5 × 10 mm Guard column for UHPLC (GL-Science) as a guard column Column temperature: 40℃ ·Flow rate: 0.4mL / min Injection volume: 10 μL

[0154] (MS / MS conditions) The test was carried out under the conditions shown in the table below.

[0155] [Table 5]

[0156] (Analysis results) The analysis results are shown in the following table. An example of a chromatogram is shown in Fig. 4. Fig. 4 is an example of a chromatogram showing the peak of derivatized hypotaurine by EMM.

[0157] [Table 6]

[0158] According to the above results, it can be seen that under acidic conditions, EMM can derivatize and analyze sulfinic acids such as hypotaurine.

[0159] [Example A4] Analysis of Persulfide In this example, it was confirmed that the derivatization and analysis of persulfide were possible. Cysteine persulfide (Cys-SH and Cys-SSH) was used as the persulfide. Cysteine persulfide has an important role in redox in living organisms. Therefore, in the present invention, if derivatization can be carried out while maintaining the redox state of persulfide, it is useful for grasping the redox state in living organisms.

[0160] (A4-1. Production of Derivative) To 70 μL of an ammonium formate solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with a pH of 6.7, 10 μL of a 100 mmol / L cysteine solution, 10 μL of an aqueous solution of 100 mmol / L sodium sulfide (diluted with pure water from Fujifilm Wako Pure Chemical Industries, Ltd.), and 10 μL of a 0.1% hydrogen peroxide aqueous solution (diluted with pure water from Kanto Chemical Co., Inc.) were mixed and reacted at 30 °C for 30 minutes to generate cysteine persulfide in the system. To 10 μL of the above solution, 60 μL of a 20 mmol / L sodium phosphate buffer (pH 2.5) and 10 μL of an EMM acetonitrile solution (10 μL / mL) were added, stirred, and left at room temperature for 10 minutes. 420 μL of a 0.1% formic acid solution was added and stirred well to obtain a sample solution.

[0161] (A4-2. Analysis of Derivative) The sample solution was analyzed by LC-MS / MS under the following conditions. An Agilent 1200 series (manufactured by Agilent) was used for LC, and a 3200 QTRAP system (manufactured by Sciex) was used for the tandem mass spectrometer.

[0162] (LC conditions) · Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile · Gradient conditions: 15 - 40% B (0 - 3.0 min), 40 - 90% B (3.0 - 3.2 min), 90% B (3.2 - 5.5 min), 90 - 15% B (5.5 - 5.8 min), 15% B (5.8 - 9.5 min) · Column: L-Column ODS, 3 μm, 2.1×50 mm (manufactured by the National Institute of Advanced Industrial Science and Technology), with an Inertsil ODS-3, 3 μm, 1.5×10 mm Guard column for UHPLC (manufactured by GL-Science) attached as a guard column · Column temperature: 40 °C · Flow rate: 0.4 mL / min · Injection volume: 10 μL

[0163] (MS / MS conditions) Performed under the conditions shown in the following table.

[0164]

Table 7

[0165] (Analysis results) The analysis results are shown in the following table. Also, an example of the chromatogram is shown in Figure 5. Figure 5 is an example of a chromatogram showing the peaks of cysteine and persulfidated cysteine derivatized by EMM.

[0166]

Table 8

[0167] According to the above results, it can be seen that under acidic conditions, EMM can derivatize and analyze persulfides such as cysteine persulfide.

[0168] [Example A5] Simultaneous analysis of Cys, Cys2, and amino acids using an amino acid analyzer In this example, it was confirmed that Cys, Cys2, and amino acids can be simultaneously analyzed using an amino acid analyzer. (Outline of analysis method) The sample was treated with EMM to derivatize Cys and Cys2, then the amino acids were separated from the sample using an ion exchange column, and then the amino acids were derivatized with ninhydrin by post-column derivatization method to simultaneously analyze Cys, Cys2, and amino acids. Note that in an amino acid analyzer, Cys and Cys2 usually elute from the column simultaneously, so it is usually difficult to analyze Cys and Cys2 simultaneously.

[0169] (A5-1. Production of derivative) To 100 μL of an amino acid mixed standard solution, H type (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) (a solution containing 2.5 mmol / L each of L-aspartic acid, L-threonine, L-serine, L-glutamic acid, L-proline, glycine, L-alanine, L-cystine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-lysine, L-histidine, ammonium chloride, and L-arginine), 25 μL of a 10 mmol / L aqueous solution of cysteine hydrochloride and 125 μL of pure water were added to prepare a mixed standard solution. To 100 μL of this product, 200 μL of a 20 mmol / L sodium phosphate buffer solution (pH 2.5) and 50 μL of an EMM acetonitrile solution (5 μL / mL) were added, stirred, and left at room temperature for 10 minutes. After the reaction, 650 μL of 0.01 mol / L hydrochloric acid was added to obtain a sample solution.

[0170] (A5-2. Analysis of derivative) The sample solution was analyzed using the following analytical instrument. · Analytical instrument: L-8900 type high-speed amino acid analyzer (manufactured by Hitachi) · Analytical method: The "biofluid analysis method" in the method package was used with an analysis time of "148 minutes".

[0171] (Analysis results) The cysteine derivative eluted at a retention time of 40.3 minutes, and Cys2 eluted at a retention time of 48.8 minutes. The other amino acids eluted at their retention times in the analysis method with the normal "biofluid analysis method" analysis time of "148 minutes". An example of the chromatogram is shown in Fig. 6. Fig. 6 is an example of a chromatogram showing the simultaneous analysis results of Cys, Cys2, and amino acids using an amino acid analyzer. From the above results, it can be seen that by treating a sample containing Cys, Cys2, and various amino acids under acidic conditions and analyzing it with a normal amino acid analyzer, Cys, Cys2, and various amino acids can be analyzed simultaneously.

[0172] [Example B: Example using BPSE] [Example B1] Derivatization and analysis with BPSE In this example, it was confirmed that thiols can be derivatized and analyzed with BPSE. The following shows a reaction example of BPSE and thiol.

[0173] [Chemical formula]

[0174] (B1-1. Production of derivative) Standard thiol solutions were prepared for each compound, with six types of standard thiol solutions made by preparing a 0.01 mol / L hydrochloric acid standard thiol solution containing 10 mmol / L of L-Cys, GSH, Hcy, CG, gEC, and NAC. To 60 μL of a 20 mmol / L sodium phosphate buffer (pH 2.5), 10 μL of the above standard thiol solution was added, 10 μL of a BPSE acetonitrile solution (10 mg / mL) was added, stirred, and left at room temperature for 10 minutes. 420 μL of a 0.1% formic acid aqueous solution was added and stirred well to obtain a sample solution.

[0175] (Analysis of the derivative) The sample solution was analyzed by LC under the following conditions. Peaks were detected with an HPLC-UV detector. (LC conditions) · Mobile phase A: 20 mmol / L sodium phosphate buffer (pH 2.5) · Mobile phase B: Acetonitrile · Gradient conditions: 30% B (0 - 10 min), 30 - 90% B (10 - 11 min), 90% B (11 - 16 min), 90 - 30% B (16 - 16.5 min), 30% B (16.5 - 20 min) · Column: Triart C18, 3 μm, 150 × 4.6 mm (manufactured by WMC) · Column temperature: 40 °C · Flow rate: 0.8 mL / min · Injection volume: 5 μL (Detection conditions) · Detection wavelength: 260 nm (Analysis results) The analysis results are shown in the table below. Also, an example of the chromatogram is shown in Figure 7. Figure 7 is an example of the chromatogram of the thiol derivative by BPSE.

[0176]

Table 9

[0177] From the above results, it can be seen that under acidic conditions, thiol can be derivatized by BPSE, separated by column chromatography, and detected by a UV detector. Amino acids having sulfanyl are expected to be converted into highly hydrophobic amino acids by derivatization and to be well separated from other less hydrophobic amino acids by column chromatography using an ODS (C18) column and detected.

[0178] [Example B2] Separation and detection of chiral thiol In this example, it was confirmed that chiral thiols can be derivatized by BPSE and separated and detected by column chromatography using a chiral column.

[0179] (B2-1. Production of derivatives) A D,L-cysteine mixed standard solution containing 5 mmol / L of L-Cys and D-Cys (manufactured by Sigma) each was prepared using 0.1 mol / L hydrochloric acid. To 60 μL of 20 mmol / L sodium phosphate buffer (pH 2.5), 10 μL of the above standard thiol solution was added, 10 μL of a BPSE acetonitrile solution (10 mg / mL) was added, stirred, and left at room temperature for 10 minutes. 420 μL of 20 mmol / L sodium phosphate buffer (pH 2.5) was added and stirred well. Further, this solution was diluted 10-fold with the mobile phase to obtain a sample solution.

[0180] (B2-2. Analysis of derivatives) The sample solution was analyzed by LC under the following conditions. Peaks were detected by an HPLC-UV detector. (LC conditions) · Mobile phase: Methanol / acetonitrile / water (49 / 49 / 2 (v / v / v)) to which formic acid and diethylamine were added at concentrations of 50 mmol / L and 25 mmol / L, respectively, and used under isocratic conditions · Column: CHIRAL PAK (registered trademark) ZWIX, 3 μm, 150×3 mm (manufactured by Daicel) · Column temperature: 25 °C · Flow rate: 0.4 mL / min · Injection volume: 10 μL (Detection conditions) · Detection wavelength: 260 nm

[0181] (Analysis results) The analysis results are shown in the table below. Also, an example of the chromatogram is shown in Fig. 8. Fig. 8 is an example of the chromatogram of derivatized chiral thiols.

[0182]

Table 10

[0183] These results demonstrate that chiral thiols can be derivatized with BPSE under acidic conditions, separated on a chiral column, and detected. Therefore, the method of the present invention demonstrates that D-Cys, which is naturally occurring in low abundance, can be efficiently detected by separating it from L-Cys.

[0184] [Examples and Reference Example C: Evaluation of reactivity of olefin compounds] In this Example and Reference Example, the reactivity of olefin compounds (derivatizing agents) including EMM and BPSE with Cys was evaluated. The olefin compounds used in the Examples and Reference Examples and their abbreviations are shown below.

[0185] [ka]

[0186] (C-1. Sample preparation and derivative production) L-Cys hydrochloride monohydrate was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 1 mmol / L. To 10 μL of this Cys solution, 60 μL of buffer solutions of various pH values and 10 μL of derivatizing agent solution (or acetonitrile) were added, and the mixture was left to stand at room temperature for 10 minutes. After the reaction, 920 μL of 0.1% formic acid solution was added to prepare the sample solution. Acetonitrile solutions of the derivatizing agent were prepared at a concentration of 10 mg / mL (solid) or 10 μL / mL (liquid). A 20 mM sodium phosphate buffer solution at pH 2.5 or 7.0 was used as the buffer. For derivatization with EMM, 200 mM sodium citrate at pH 3.25 or 4.25, or 20 mM sodium phosphate buffer solution at pH 6.0 was also used as the buffer.

[0187] (C-2. Analysis of Derivatives) The sample solution was analyzed by LC-MS / MS under the following conditions. For LC, an Agilent 1200 series (manufactured by Agilent) was used, and for the tandem mass spectrometer, a 3200 QTRAP system (manufactured by Sciex) was used. The measurement was performed in the selected ion detection (SIM) mode. (LC conditions) · Mobile phase A: 0.1% aqueous formic acid solution · Mobile phase B: acetonitrile · Gradient conditions: 10% B (0 - 1.0 min), 10 - 90% B (1.0 - 5.0 min), 90% B (5.0 - 6.5 min), 90 - 10% B (6.5 - 7.0 min), 10% B (7.0 - 10.0 min) · Column: L-Column ODS, 3 μm, 2.1×50 mm (manufactured by the National Institute of Technology and Evaluation) · Column temperature: 40 °C · Flow rate: 0.4 mL / min · Injection volume: 10 μL

[0188] (MS conditions) The following conditions were used. DP (V): 46 EP (V): 8 CEP (V): 14 The detection conditions and retention times of Cys and Cys derivatized with various derivatizing agents are shown in the following table. In the following table, the notation X-Cys means Cys derivatized with the derivatizing agent X. In addition, for the detection of EBzM-Cys, two peaks were observed as diastereomers, and their respective retention times are listed in the following table.

[0189] [Table 11]

[0190] (Analysis results) The analysis results are shown in the following table. The Cys reaction rate was calculated by the above formula (1). In addition, the items marked with "*" indicate that no data is available.

[0191] [Table 12]

[0192] At pH 7.0, derivatives were generated and detected with all agents. Under acidic conditions of pH 2.5, derivatives were generated when olefin compounds containing an ethylene structure with two electron-withdrawing groups (EMM, BPSE, Acry-CF3A, Acry-CF3E, EiPrM, EBzM, BEPE, CA) were used. In particular, when the olefin compounds represented by formula (I) (EMM, BPSE, Acry-CF3A, Acry-CF3E, BEPE, CA) were used, derivatives were generated at a high reaction rate.

[0193] In addition, the results when the reaction was carried out in various buffer solutions using EMM as the derivatizing agent are shown in the table below.

[0194] [Table 13]

[0195] From the above results, it can be seen that at pH 6.0 or higher, the derivative Cys peak area value decreases.

[0196] [Reference Example D: Behavior of Organic Substances under Various pH Conditions] In this reference example, the stability of organic substances under acidic conditions is evaluated by observing the behavior of organic substances under various pH conditions.

[0197] (D-1. Observation of Thiol Exchange Reaction) (Sample Preparation) L-Cysteine hydrochloride monohydrate was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 10 mmol / L, and GSSG was dissolved to a concentration of 50 mmol / L. To 50 μL of the Cys solution (10 mmol / L), 850 μL of various sodium phosphate buffers (pH 2.5, 6.0, 7.0, or 8.0) and 100 μL of the GSSG solution (50 mmol / L) were added to prepare a mixed solution. (Finally, 0.5 mmol / L of Cys and 5 mmol / L of GSSG were present in the solution.) Sampling was carried out over time, derivatization was performed with BPSE, and analysis was performed by LC under the following conditions to measure the area values of Cys and GSH. The peak was detected with an HPLC-UV detector.

[0198] (Sampling and Derivatization) To 10 μL of the mixed solution, 10 μL of a 10% (w / v) aqueous trichloroacetic acid solution, 80 μL of a sodium phosphate buffer at pH 2.5, and 20 μL of a BPSE acetonitrile solution (5 mg / mL) were added, and the mixture was allowed to stand at room temperature for 10 minutes. After the reaction, 100 μL of a sodium phosphate buffer at pH 2.5 was added to obtain a sample solution.

[0199] (LC Conditions) · Mobile phase A: 20 mmol / L sodium phosphate buffer (pH 2.5) · Mobile phase B: Acetonitrile · Gradient conditions: 25% B (0 - 8.0 min), 25 - 90% B (8.0 - 9.0 min), 90% B (9.0 - 11.0 min), 90 - 25% B (11.0 - 12.0 min), 25% B (12.0 - 15.0 min) · Column: Triart C18, 3 μm, 150 × 4.6 mm (manufactured by Wako Chemicals) · Column temperature: 40 °C · Flow rate: 1.0 mL / min · Injection volume: 10 μL Under these conditions, Cys elutes at 6.3 minutes and GSH elutes at 6.8 minutes. (Detection Conditions) · Detection wavelength: 260 nm

[0200] (Analysis Results) The analysis results are shown in the following table and Figures 9 to 12. Figures 9, 10, 11, and 12 are graphs respectively representing the change over time of the peak area values of Cys and GSH at pH 8.0, pH 7.0, pH 6.0, and pH 2.5. Note that the data at 0 minutes for pH 6.0, 7.0, and 8.0 were considered to be those without buffer treatment and were made common with the data at 0 minutes for pH 2.5.

[0201] [Table 14]

[0202] [Table 15]

[0203] [Table 16]

[0204] [Table 17]

[0205] According to the above results, it can be seen that under conditions of pH 7.0 or higher, Cys and GSSG react rapidly, and GSH generated by the exchange reaction is detected, while Cys decreases. On the other hand, under conditions of pH 6.0 or lower, the reaction between Cys and GSSG hardly proceeds. In particular, under the condition of pH 2.5, it can be seen that GSH, which is the product of the exchange reaction, is not detected even after 1 hour. These results indicate that even in a sample where a disulfide compound and a thiol compound coexist, the thiol compound can be accurately analyzed under acidic conditions.

[0206] (Observation of Thiol Oxidation Reaction) (Sample Preparation) L-Cysteine hydrochloride monohydrate was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 10 mmol / L, and copper(II) sulfate pentahydrate was dissolved to a concentration of 0.1 mmol / L. To 50 μL of the Cys solution (10 mmol / L), 850 μL of various sodium phosphate buffers (pH 2.5, 6.0, 7.0, or 8.0) and 100 μL of an aqueous copper sulfate solution (0.1 mmol / L) were added to prepare a mixed solution. (Finally, 0.5 mmol / L of Cys and 0.01 mmol / L of copper sulfate were present in the solution.) Sampling was carried out over time, derivatization was performed with BPSE, and analysis was performed by LC under the same conditions as in (D-1. Observation of thiol exchange reaction), and the area value of Cys was measured. The same methods as in (D-1. Observation of thiol exchange reaction) were used as the analytical method and the derivatization method.

[0207] (Analysis results) The analysis results are shown in the following table and Fig. 13. Fig. 13 is a graph showing the change over time in the peak area value of Cys at pH 8.0, pH 7.0, pH 6.0, and pH 2.5. Note that the data at 0 minutes for pH 6.0, 7.0, and 8.0 were considered to be those without buffer treatment and were made common with the data at 0 minutes for pH 2.5.

[0208] [Table 18]

[0209] According to the above results, it can be seen that under conditions of pH 7 or higher, Cys rapidly decreases and Cys is rapidly oxidized. On the other hand, under conditions of pH 6.0 or lower, it can be seen that Cys hardly decreases and the oxidation reaction of Cys hardly proceeds. These results indicate that under acidic conditions, the oxidation reaction of the thiol compound is suppressed and the thiol compound can be accurately analyzed.

[0210] [Example E] Derivatization of sulfanyl group and amino group In this example, after cysteine or cystine was mixed with EMM under acidic conditions, the pH of the solution was further adjusted to basic, and it was confirmed that the amino group (-NH2) of cysteine or cystine reacted with EMM to derivatize cysteine or cystine. That is, after the sulfanyl group (-SH) of cysteine reacted with EMM under acidic conditions and was converted to a substituent that is stable against oxidation, the amino group of cysteine reacted with EMM under basic conditions to obtain a derivative. Under acidic conditions, amino groups generally do not react with EMM. Therefore, under acidic conditions, disulfide compounds such as cystine that have an amino group but no sulfanyl group generally do not react with EMM and are not derivatized. However, it was confirmed that even such disulfide compounds can be derivatized by the reaction of the amino group with EMM under basic conditions. By reacting not only the sulfanyl group but also the amino group with EMM, the hydrophobicity of disulfide compounds that are not derivatized under acidic conditions can be increased, facilitating separation by liquid chromatography. In addition, the detection sensitivity of the derivative by a mass spectrometer can be increased.

[0211] [Chemical formula]

[0212] (E-1. Production of derivative) Cysteine or cystine (manufactured by Sigma) was dissolved in 0.01 mol / L hydrochloric acid to a concentration of 250 μmol / L to prepare a standard sample. 20 μL of this standard sample, 20 μL of 10% trichloroacetic acid aqueous solution, and 40 μL of 0.1 mol / L hydrochloric acid were mixed and stirred well. 50 μL of this solution was mixed with 300 μL of 20 mmol / L sodium phosphate buffer (pH 2.5), 50 μL of EMM acetonitrile solution (10 μL / mL) was added, stirred, and allowed to stand at 40 °C for 5 minutes. 100 μL of this solution was taken, mixed with 200 μL of 100 mmol / L sodium phosphate buffer (pH 9.0), further stirred, and allowed to stand at 40 °C for 5 minutes. 15 μL of this solution was taken, 50 μL of 0.3% formic acid solution was added and stirred well to prepare an analytical sample.

[0213] (Analysis of E-2 derivative) The sample solution was analyzed by LC-MS / MS under the following conditions. For LC, an Agilent 1290 Infinity II series (manufactured by Agilent) was used, and for the tandem mass spectrometer, a QTRAP 5500 system (manufactured by Sciex) was used.

[0214] (LC conditions) · Mobile phase A: 0.1% formic acid, Mobile phase B: acetonitrile · Gradient conditions: 30 - 60% B (0 - 6.0 min), 60 - 90% B (6.0 - 7.5 min), 90% B (7.5 - 9.0 min), 90 - 30% B (9.0 - 9.1 min), 30% B (9.1 - 10.0 min) · Column: L-Column ODS, 3 μm, 2.1×50 mm (manufactured by National Institute of Advanced Industrial Science and Technology), with an Inertsil ODS-3, 3 μm, 1.5×10 mm Guard column for UHPLC (manufactured by GL-Science) attached as a guard column · Column temperature: 40 °C · Flow rate: 0.4 mL / min · Injection volume: 1 μL

[0215] (MS / MS conditions) The analysis was performed under the conditions shown in the following table.

[0216]

Table 19

[0217] (Analysis results) The analysis results are shown in the table below. Also, an example of a chromatogram is shown in Fig. 14. In the table below and Fig. 14, “3EMM-Cysteine” and “3EMM-Cys” mean derivatives obtained by reacting 1 molecule of cysteine with 3 molecules of EMM. “4EMM-Cystine” and “4EMM-Cys2” mean derivatives obtained by reacting 1 molecule of cystine with 4 molecules of EMM. Fig. 14 is an example of a chromatogram showing the peak of derivatized cysteine or the peak of derivatized cystine by EMM.

[0218]

Table 20

[0219] According to the above results, it can be seen that by reacting cysteine with EMM under acidic conditions, the sulfanyl group reacts with EMM, and then by making the solution basic, the amino group reacts with 2 molecules of EMM, resulting in the derivatization of cysteine. Cystine is derivatized by reacting 2 amino groups with 4 molecules of EMM by making the reaction solution basic. That is, the above results indicate that cysteine can be analyzed by LC-MS / MS as a compound reacted with 3 molecules of EMM, and cystine can be analyzed as a compound reacted with 4 molecules of EMM.

Claims

1. reacting an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino with an olefin compound under acidic conditions to obtain a derivative of the organic substance, wherein the olefin compound contains an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms), a method for producing a derivative of an organic substance.

2. The method for producing a derivative according to claim 1, wherein the ethylene structure has two electron-withdrawing groups.

3. The method for producing a derivative according to claim 1 or 2, wherein the two electron-withdrawing groups are bonded to the same carbon atom constituting the ethylene structure.

4. The method for producing a derivative according to any one of claims 1 to 3, wherein at least two electron-withdrawing groups are the same group.

5. The method for producing a derivative according to any one of claims 1 to 4, wherein the olefin compound is a compound represented by the following formula (I). 【Chemical 1】 (However, EWG １ and EWG ２ each independently represents an electron withdrawing group, and together with the carbon atom to which they are attached, may form a ring.)

6. the electron-withdrawing group, or EWG １ and EWG ２ are each independently, -C(=O)-OR １ , -S(=O) ２ -R ２ , -P(=O)(-OR ３ ) ２ , alkyl substituted with a cyano, halogen atom, carboxy, nitro, -S(=O)-R ４ , -C(=O)-R ５ , or -C(=O)-NR ６ R ７ wherein wherein, R １ 、R ２ 、R ３ 、and R ４ each independently represents a monovalent hydrocarbon group or a monovalent heterocyclic group, which may have substituents, R ５ , R ６ , and R ７ each independently represents a hydrogen atom, a monovalent hydrocarbon group, or a monovalent heterocyclic group, and these may have substituents. A method for producing a derivative according to any one of claims 1 to 5.

7. The method for producing a derivative according to any one of claims 1 to 6, wherein the acidic condition is a condition with a pH of less than 6.

0.

8. The method for producing a derivative according to any one of claims 1 to 7, wherein the organic substance is one or more selected from the group consisting of cysteine, reduced glutathione, γ-glutamylcysteine, cysteinylglycine, homocysteine, N-acetylcysteine, persulfidated cysteine, hypotaurine, persulfidated glutathione, and peptidic compounds containing cysteine residues.

9. the organic substance further contains an amino group, The method for producing a derivative according to any one of claims 1 to 8, further comprising reacting the organic substance with the olefin compound under acidic conditions and then reacting under neutral or basic conditions to obtain a derivative of the organic substance.

10. (1) A sample containing an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino and an olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms) are mixed under acidic conditions to obtain a treated sample containing a derivative of the organic substance, and (2) analyzing the derivative of the organic substance in the treated sample, an analysis method for a sample containing an organic substance.

11. The analysis in the step (2) is (2a) separating the derivative of the organic substance from the treated sample, Detecting a derivative of the separated organic substance; The method for analyzing a sample containing an organic substance according to claim 10, comprising:

12. The method for analyzing a sample containing an organic substance according to claim 10 or 11, wherein the sample further contains a disulfide compound.

13. The method for analyzing a sample containing an organic substance according to claim 12, wherein the disulfide compound is one or more selected from the group consisting of oxidized glutathione and cystine.

14. The organic substance further contains an amino group, Step (1) is (1') mixing the sample and the olefin compound under acidic conditions, and then further mixing under neutral or basic conditions to obtain a treated sample containing a derivative of the organic substance, the method for analyzing a sample containing an organic substance according to any one of claims 10 to 13.

15. A derivatizing agent for an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino under acidic conditions, comprising an olefin compound containing an ethylene structure having at least two electron-withdrawing groups (excluding halogen atoms).

16. A reagent for analyzing an organic substance containing one or more groups selected from the group consisting of sulfanyl, selanyl, and sulfino, comprising the derivatizing agent according to claim 15.

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