Stable isotope-labeled cysteinyldopa and analysis method

Abstract

[Problem] The purpose of the present invention is to provide a stable isotope-labeled compound that is usable for the detection or quantification of a metabolite related to melanin. [Solution] Provided is a stable isotope-labeled cysteinyldopa represented by formula (I). [In formula (I), X1, X3, X4, and X7 independently of one another represent C or 13C; X8 represents CH, CD, 13CH, or 13CD; X9 represents CH2, CHD, CD2, 13CH2, 13CHD, or 13CD2; X1' represents C or 13C; X2' represents CH, CD, 13CH, or 13CD; X3' represents CH2, CHD, CD2, 13CH2, 13CHD, or 13CD2; Y1 and Y1' independently of one another represent NH2 or 15NH2; Z1 represents O or 18O; Z2 to Z4 represent OH or 18OH; Z1' represents O or 18O; Z2' represents OH or 18OH; and n represents 1 or 2.]

Description

Stable isotope-labeled cysteinyldopa and analytical method

[0001] The present invention relates to stable isotope-labeled cysteinyldopa and an analytical method using the stable isotope-labeled cysteinyldopa.

[0002] Melanin is a pigment synthesized in melanocytes. There are two types of melanin: eumelanin and pheomelanin. During the biosynthesis of melanin, various metabolites are produced. Several methods have been proposed to measure these metabolites.

[0003] For example, Non-Patent Document 1 below discloses the analysis of urinary 5-S-cysteinyldopa (hereinafter also referred to as 5-S-CD) by electrochemical detection HPLC. Non-Patent Document 2 below discloses a mass spectrometric identification method for 5-S-cysteinyldopa (hereinafter also referred to as 5-S-CD), a precursor of submelanin, and Non-Patent Document 3 below discloses a quantitative analysis method for the indole metabolite 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C), a precursor of eumelanin, as a biomarker for melanoma progression. Furthermore, Non-Patent Document 4 below discloses a method for synthesizing 5-S-CD.

[0004] Automated high-performance liquid chromatographic determination of 5-S-cysteinyl-3,4-dihydroxyphenylalanine in urine. Kagedal, B.; Kallberg, M.; Arstrand, K.; Hansson, C. J. Chromatogr. 1989, 473, 359-370.Development of a mass spectrometry method for the determination of a melanoma biomarker, 5-S-cysteinyldopa, in human plasma using solid phase extraction for sample clean-up. J. Chromatogr. A 2007, 1156, 141-148.A method for measuring serum levels of melanin-associated indole metabolites using LC-MS / MS and its application to malignant melanoma. Takiwaki, M.; Umemura, H.; Kikutani, Y.; Fukuzawa, S.; Abe, K.; Fujino, K.; Sugihara, S.; Tachibana, K.; Morizane, S.; Satoh, M.; Nakayama, T.; Yamasaki, O. Clinica Chimica Acta 2024, 557, 11873.A convenient one step synthesis of 5-cystein-S-ylDOPA using ceric ammonium nitrate. Chioccara, F.; Novellino, E. Synth. Commun. 1986, 16, 967-971.

[0005] The various metabolites involved in melanin synthesis described above are thought to be useful as biomarkers for disease or nutritional status. For example, these metabolites could be used as tumor markers to understand the pathology of malignant melanoma, a type of skin cancer. LDH (lactate dehydrogenase) is used as a tumor marker for melanoma. However, LDH (lactate dehydrogenase) has low disease specificity and poor performance as a tumor marker, and 5-S-CD is considered promising. For example, Non-Patent Document 2 and Non-Patent Document 3 describe the use of 5-S-CD and 6H5MI2C, respectively, as biomarkers for melanoma, and HPLC quantification methods have been developed.

[0006] If such metabolites could be detected or quantified with high sensitivity and high resolution, their usefulness as biomarkers would be enhanced. Mass spectrometry, particularly LC-MS / MS, is a technique that provides high sensitivity and high resolution for compounds. To perform this analytical method, it is necessary to prepare a stable isotope-labeled compound of the target metabolite as an internal standard.

[0007] Therefore, an object of the present invention is to provide a stable isotope-labeled compound that can be used in the detection or quantification of melanin-related metabolites.

[0008] The present invention provides stable isotope-labeled cysteinyldopa, which can be used as an internal standard in mass spectrometry for the detection or quantification of melanin-related metabolites, for example.

[0009] That is, the present invention provides the following: [1] Stable isotope-labeled cysteinyldopa represented by the following formula (I): [In formula (I), X １ , X ３ , X ４ , and X ７ are independently C or １３ represents C, and X ８ , CH, CD, １３ CH, or １３ represents CD, and X ９ is CH２ , CHD, CD ２、 １３ CH ２ , １３ CHD, or １３ CD ２ represents X １ ' is C or １３ represents C, and X ２ ' is CH, CD, １３ CH, or １３ represents CD, and X ３ ' is CH ２ , CHD, CD ２ , １３ CH ２ , １３ CHD, or １３ CD ２ represents Y １ and Y １ ' are, independently of each other, NH ２ or １５ NH ２ represents Z １ is O or １８ represents O, and Z ２ ~Z ４ is OH or １８ represents OH; Z １ ' is O or １８ represents O, and Z ２ ' is OH or １８ OH, n=1 or 2, when n=1, X ２ , X ５ , X ６ Either one of the following is C or １３ C, and the rest are independently CH, CD, １３ CH, or １３ represents CD, and when n=2, X ２ , X ５ , X ６ Any two of these are C or １３ C, and the remaining one is CH, CD, １３ CH, or １３ [2] In the formula (I), D, １３ C, or １５[3] The stable isotope-labeled cysteinyldopa according to [1], having two or more N. [4] In the formula (I), １８ [4] The stable isotope-labeled cysteinyldopa according to [1], which has one or more O. １３ C, and １５ [5] The stable isotope-labeled cysteinyldopa according to [2], wherein the stable isotope-labeled cysteinyldopa has two or more isotope atoms selected from the group consisting of N, and the types of the isotope atoms contained in the formula (1) are the same or two or more types. １８ [6] The stable isotope-labeled cysteinyldopa according to [3], wherein the isotope atoms contained in the formula (1) are the same or two or more kinds. １３ C, or the above １５ [7] The stable isotope-labeled cysteinyldopa according to [2], wherein either or both of the L-dopa-derived skeleton and the cysteine-derived skeleton forming the formula (1) are １８ The stable isotope-labeled cysteinyldopa according to [3], having O. [8] An analytical method using the stable isotope-labeled cysteinyldopa according to [1]. [9] The analytical method according to [8], comprising carrying out mass spectrometry.

[10] The analytical method according to [9], wherein the stable isotope-labeled cysteinyldopa is used as an internal standard in the mass spectrometry.

[11] The analytical method according to [9], comprising detecting or quantifying cysteinyldopa in a biological sample by the mass spectrometry.

[12] The analytical method according to

[11] , wherein the biological sample is adsorbed to a solid phase carrier and subjected to a deproteinization treatment before carrying out the mass spectrometry.

[13] The analytical method according to

[11] , wherein the cysteinyldopa in the biological sample is a tumor marker for melanoma.

[14] One type of stable isotope-labeled cysteinyldopa represented by the following formula (I), or a mixture of two or more different types of cysteinyldopa: [In formula (I), X１ , X ３ , X ４ , and X ７ are independently C or １３ represents C, and X ８ , CH, CD, １３ CH, or １３ represents CD, and X ９ is CH ２ , CHD, CD ２、 １３ CH ２ , １３ CHD, or １３ CD ２ represents X １ ' is C or １３ represents C, and X ２ ' is CH, CD, １３ CH, or １３ represents CD, and X ３ ' is CH ２ , CHD, CD ２ , １３ CH ２ , １３ CHD, or １３ CD ２ represents Y １ and Y １ ' are, independently of each other, NH ２ or １５ NH ２ represents Z １ is O or １８ represents O, and Z ２ ~Z ４ is OH or １８ represents OH; Z １ ' is O or １８ represents O, and Z ２ ' is OH or １８ OH, n=1 or 2, when n=1, X ２ , X ５ , X ６ Either one of the following is C or １３ C, and the rest are independently CH, CD, １３ CH, or １３ represents CD, and when n=2, X ２ , X ５ , X ６Any two of these are C or １３ C, and the remaining one is CH, CD, １３ CH, or １３

[15] An analytical method using the mixture according to

[14] .

[16] A reagent kit for detecting or quantifying cysteinyldopa, comprising the stable isotope-labeled cysteinyldopa according to [1].

[17] A reagent kit for detecting or quantifying cysteinyldopa, comprising the mixture according to

[14] .

[0010] The present invention provides a standard substance that can be used, for example, in the detection or quantification of melanin-related metabolites. The compound according to the present invention can be used, for example, as an internal standard substance in mass spectrometry for the detection or quantification of melanin-related metabolites. This enables the detection or quantification of the metabolites with high sensitivity and high resolution. The effects of the present invention are not limited to those described herein, and may be any of the effects described herein.

[0011] FIG. 1 is a diagram showing the biosynthesis pathway of melanin. FIG. 2 is a diagram for explaining the method for producing stable isotope-labeled cysteinyldopa of the present invention. FIG. 3 is a diagram showing the reaction pathway for synthesizing stable isotope-labeled cysteinyldopa. FIG. 4 is a diagram showing the reaction pathway for synthesizing cysteinyldopa of formula (II) (5-S-CD-D) synthesized using L-DOPA as a raw material. ２ )of １ 1H NMR spectrum of cysteinyldopa (5-S-CD-D) of formula (II) synthesized from L-DOPA. ２ )of １３ 1 shows the C NMR spectrum of cysteinyldopa (5-S-CD-D) of formula (II) synthesized from L-DOPA. ２ ) is a diagram showing a mass spectrum of the result of LCMS analysis. １３ C ６　 Cysteinyldopa (5-S-CD-Ring) of formula (III) synthesized using 99% of the raw material １３ C ６ )of １ 1H NMR spectrum. １３ C ６　Cysteinyldopa (5-S-CD-Ring) of formula (III) synthesized using 99% of the raw material １３ C ６ )of １３ 1 shows the C NMR spectrum of L-DOPA (Ring １３ C ６　 Cysteinyldopa (5-S-CD-Ring) of formula (III) synthesized using 99% of the raw material １３ C ６ 1 shows the mass spectrum of L-DOPA(1-) analyzed by LCMS. １３ C, 99%) and cysteine ​​(3,3-D ２ , 98%) and cysteinyldopa (5-S-CD- １３ C, D ２ )of １ 1H NMR spectrum. １３ C, 99%) and cysteine ​​(3,3-D ２ , 98%) and cysteinyldopa (5-S-CD- １３ C, D ２ )of １３ 1 shows the C NMR spectrum of L-DOPA (1- １３ C, 99%) and cysteine ​​(3,3-D ２ , 98%) and cysteinyldopa (5-S-CD- １３ C, D ２ 1 shows the mass spectrum of L-DOPA(1-) analyzed by LCMS. １３ C, 99%) and cysteine ​​( １５ Cysteinyldopa (5-S-CD- １３ C. １５ N) １ 1H NMR spectrum. １３ C, 99%) and cysteine ​​( １５ Cysteinyldopa (5-S-CD- １３ C. １５ N) １３1 shows the C NMR spectrum of L-DOPA (1- １３ C, 99%) and cysteine ​​( １５ Cysteinyldopa (5-S-CD- １３ C. １５ N) １５ 1 shows the N NMR spectrum of L-DOPA (1- １３ C, 99%) and cysteine ​​( １５ Cysteinyldopa (5-S-CD- １３ C. １５ 5 shows the mass spectrum of the 5-S-CD-N) analyzed by LCMS. 5-S-CD and 5-S-CD-N) are shown as calibration curves. １３ C ６ FIG. 1 shows an SRM chromatogram of

[0012] Preferred embodiments of the present invention will be described below, however, the present invention is not limited to the following preferred embodiments and can be freely modified within the scope of the present invention.

[0013] 1. Description of the Invention

[0014] As mentioned above, various metabolites are produced during melanin biosynthesis. The melanin biosynthesis pathway is shown in Figure 1. As shown in the figure, eumelanin and pheomelanin share a common intermediate, dopaquinone, which is biosynthesized from tyrosine by tyrosinase in melanocytes. When dopaquinone is cysteine-conjugated, pheomelanin is produced via 5-S-CD, 2-S-CD, and 2,5-di-S-CD. When dopaquinone is not cysteine-conjugated, eumelanin is produced via DHICA. Some of these dopaquinone-derived metabolites leak into the bloodstream and are converted to 6H5MI2C or 5H6MI2C by O-methyltransferase (COMT) in the liver, ultimately being excreted in the urine.

[0015] It has been proposed that some of these melanin-related metabolites be used as tumor markers for melanoma. For example, 5-S-CD, which is also described in Non-Patent Document 1, is sometimes used clinically as a tumor marker. However, the method described in Non-Patent Document 2 uses α-methyldopa, which is obtained by removing cysteine ​​from 5-S-CD and introducing a methyl group to the α-carbon of dopa, as an internal standard. This is structurally completely different from 5-S-CD, and therefore has problems such as different recovery rates by solid-phase extraction and different retention times on chromatography.

[0016] The present inventors have developed a novel stable isotope-labeled cysteinyldopa that can be used in mass spectrometry to detect or quantify melanin-related metabolites. Mass spectrometry using this stable isotope-labeled cysteinyldopa as an internal standard enables detection or quantification of melanin-related metabolites with high sensitivity and high resolution. Furthermore, such detection or quantification of melanin-related metabolites with high sensitivity and high resolution contributes to improving the reliability of melanoma testing methods.

[0017] The above-mentioned Non-Patent Document 1 describes the detection of melanin-related metabolites by HPLC. However, the detection method described in this document detects the redox potential difference of catechol, and mass spectrometry is superior in terms of the reliability of analog separation and quantification. The compounds according to the present invention enable the quantification of melanin-related metabolites by mass spectrometry. Furthermore, mass spectrometry using the compounds according to the present invention allows for highly accurate quantification of melanin-related metabolites. Such accurate quantification contributes to improving the reliability of melanoma testing methods.

[0018] In Non-Patent Document 2, cysteinyldopa is quantified by mass spectrometry using an analogous compound as an internal standard, and therefore cannot be considered an accurate quantification method. Isotope dilution mass spectrometry, which uses a stable isotope-labeled compound as an internal standard, as in Non-Patent Document 3, is recognized as an accurate quantification method. That is, by synthesizing stable isotope-labeled cysteinyldopa according to the present invention, accurate quantification of cysteinyldopa is possible.

[0019] The present invention enables highly sensitive detection and even highly sensitive quantification of trace metabolites using mass spectrometry, for example, in the field of clinical testing. The present invention can be used regardless of the type of mass spectrometer. For example, in order to widely popularize the quantification of trace metabolites by mass spectrometry in the field of clinical testing, it is desirable that the same values ​​be obtained anytime, anywhere, and with any manufacturer's instrument. Cysteinyldopa according to the present invention can be used in mass spectrometry regardless of differences between models from different manufacturers. This can support the widespread use of quantification of trace metabolites by mass spectrometry.

[0020] The present invention will now be described in more detail.

[0021] 2. First embodiment (stable isotope-labeled cysteinyldopa)

[0022] (1) Stable isotope-labeled cysteinyldopa

[0023] The present invention relates to a compound represented by the following formula (I): The present invention provides a stable isotope-labeled cysteinyldopa represented by the formula (I): Each component of formula (I) is explained below.

[0024] In formula (I), n is an integer of 1 or 2. That is, the number of cysteines added to the cysteinyldopa of the present invention is one or two.

[0025] In formula (I), X １ , X ３ , X ４ , and X ７ are independently C or １３ represents C, and X ８ , CH, CD, １３CH, or １３ represents CD, and X ９ is CH ２ , CHD, or CD ２ represents X １ ' is C or １３ represents C, and X ２ ' is CH, CD, １３ CH, or １３ represents CD, and X ３ ' is CH ２ , CHD, or CD ２ When n=1, X ２ , X ５ , X ６ Either one of the following is C or １３ C, and the rest are independently CH, CD, １３ CH, or １３ represents CD, and when n=2, X ２ , X ５ , X ６ Any two of these are C or １３ C, and the remaining one is CH, CD, １３ CH, or １３ It represents CD. That is, X １ is C or １３ C. X ２ is C, １３ C, CH, CD, １３ CH, or １３ It's a CD. X ３ is C or １３ C. X ４ is C or １３ C. X ５ is C, １３ C, CH, CD, １３ CH, or １３ It's a CD. X ６ is C, １３ C, CH, CD, １３ CH, or １３ It's a CD. X ７ is C or １３ C. X ８ , CH, CD, １３ CH, or １３ It's a CD. X ９ is CH ２ , CHD, CD２、 １３ CH ２ , １３ CHD, or １３ CD ２ X １ ' is C or １３ C. X ２ ' is CH, CD, １３ CH, or １３ It's a CD. X ３ ' is CH ２ , CHD, CD ２ , １３ CH ２ , １３ CHD, or １３ CD ２ is.

[0026] In formula (I), Y １ and Y １ ' are, independently of each other, NH ２ or １５ NH ２ That is, Y １ is NH ２ or １５ NH ２ Y １ ' is NH ２ or １５ NH ２ is.

[0027] In formula (I), Z １ is O or １８ represents O, and Z ２ ~Z ４ is OH or １８ represents OH, and Z １ ' is O or １８ represents O, and Z ２ ' is OH or １８ OH. That is, Z １ is O or １８ It is O. Z ２ is OH or １８ OH. Z ３ is OH or １８ OH. Z ４ is OH or １８ OH. Z １ ' is O or１８ It is O. Z ２ ' is OH or １８ It's OH.

[0028] The cysteinyldopa of the present invention is a compound containing stable isotopes D (deuterium), １３ C and stable isotopes １５ In addition, the cysteinyldopa of the present invention may have two or more stable isotopes selected from the group consisting of D, １３ C, and １５ The compound of formula (1) may have two or more isotope atoms selected from the group consisting of D, N, and the types of isotope atoms contained in formula (1) may be the same or two or more types. １３ C, or the above １５ N may be included.

[0029] The number of stable isotopes D (deuterium) contained in the cysteinyldopa of the present invention is, for example, 9, preferably 8 or less, more preferably 7 or less, and even more preferably 6 or less. ２ , X ５ , X ６ , X ８ , X ９ , X ２ ', X ３ ' can exist as a component of '.

[0030] Stable isotopes contained in the cysteinyldopa of the present invention １３ The number of C is, for example, 12, preferably 10 or less, more preferably 8 or less, and even more preferably 7 or less. １３ C is the X mentioned above. １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ７ , X ８ , X ９ , X １ ', X ２ ', X ３' can exist as a component of '.

[0031] Stable isotopes contained in the cysteinyldopa of the present invention １５ The number of N is preferably 2 or less. １５ N is the Y mentioned above １ , Y １ ' can exist as a component of '.

[0032] The cysteinyldopa of the present invention may have, for example, two stable isotopes D (deuterium) as stable isotopes, or six stable isotopes D (deuterium) as stable isotopes. １３ C, and further, two stable isotopes D (deuterium) and one stable isotope １３ C, and one stable isotope １３ C and one stable isotope １５ N may be included.

[0033] Furthermore, the cysteinyldopa of the present invention contains stable isotopes D (deuterium), １３ C and stable isotopes １５ Instead of having two or more stable isotopes selected from the group consisting of N and １８ The cysteinyldopa of the present invention may have one or more O. １８ The isotope atoms contained in the formula (1) may be the same or may be two or more kinds. Furthermore, either or both of the L-dopa-derived skeleton and the cysteine-derived skeleton forming the formula (1) may be １８ O may be included.

[0034] Stable isotopes contained in the cysteinyldopa of the present invention １８ The number of O is preferably 6 or less. １８ O is the Z mentioned above. １ , Z ２ , Z ３ , Z ４ , Z １ ', Z ２ ' can exist as a component of '.

[0035] Preferably, the stable isotope-labeled cysteinyldopa of the present invention may be cysteinyldopa represented by the following formula (II), formula (III), formula (IV), or formula (V). In this specification, the compound of formula (II) is 5-S-CD-D as described below. ２ The compound of formula (III) is also called 5-S-CD-Ring １３ C ６ The compound of formula (IV) is also referred to as 5-S-CD- １３ C, D ２ The compound of formula (V) is also called 5-S-CD- １３ C. １５ Also called N.

[0036]

[0037]

[0038]

[0039] (2) Method for synthesizing stable isotope-labeled cysteinyldopa

[0040] The stable isotope-labeled cysteinyldopa of the present invention can be synthesized, for example, using stable isotope-labeled L-dopa (L-DOPA) as a starting material. The stable isotope-labeled cysteinyldopa of the present invention can be synthesized using stable isotope-labeled L-dopa by adding L-cysteine ​​to L-dopa using, for example, diammonium cerium (IV) nitrate. The synthesis can be carried out, for example, according to the method described in Non-Patent Document 4. Specifically, the synthesis may be carried out as shown in the Examples below.

[0041] Stable isotope-labeled L-dopa may be prepared by techniques known in the art or is commercially available. The synthesis of stable isotope-labeled L-dopa is described below.

[0042] L-DOPA can be synthesized, for example, from benzene, pyruvate, and ammonia according to the method described in, for example, Min, K.; Park, K.; Park, DH.; Yoo, YJ, "Overview on the biotechnological production of L-DOPA." Appl. Microbiol. Biotechnol. 2015, 99, 575-584.

[0043] Among the compounds used in the synthesis of L-dopa, the benzene can be synthesized from, for example, acetylene. By using stable isotope-labeled acetylene as the acetylene, stable isotope-labeled benzene can be obtained. For example, Japanese Patent Application Laid-Open No. 2008-266149 discloses a method for synthesizing benzene using stable isotope-labeled acetylene. １３ A method for producing C-labeled benzene has been described, and such a method can be used to obtain stable isotope-labeled benzene. In this way, by using stable isotope-labeled acetylene, the six-membered ring moiety (X １ ~X ６ A stable isotope label can be introduced into X in formula (I). ２ , X ５ , X ６ Regarding , for example, as shown in FIG. 2, stable isotope-labeled deuterium D can be introduced by HD exchange using heavy water.

[0044] For example, as shown in Figure 2, stable isotope-labeled benzene can be synthesized by a reaction using stable isotope-labeled acetylene in the presence of a zeolite catalyst. The benzene is deuterium-labeled by subjecting it to an H-D exchange treatment using heavy water. An oxidation treatment of the benzene introduces a hydroxyl group, yielding catechol. Stable isotope-labeled L-dopa can be obtained by reacting the catechol with stable isotope-labeled pyruvic acid and stable isotope-labeled ammonia using β-tyrosinase, as shown in the figure.

[0045] Among the compounds used in the L-dopa synthesis, pyruvic acid and ammonia are commercially available, or stable isotope-labeled pyruvic acid and ammonia may be synthesized by a method known in the art. ８ ~X ９ A stable isotope label can be introduced into the Y １ A stable isotope label can be introduced into the

[0046] Stable isotope-labeled cysteinyldopa can be synthesized, for example, by adding L-cysteine ​​to L-dopa using diammonium cerium (IV) nitrate according to the reaction pathway shown in Figure 3. Regarding L-cysteine, stable isotope-labeled L-cysteine ​​is commercially available, or it may be synthesized by a method known in the art. The stable isotope-labeled L-cysteine ​​can be used to synthesize the X in formula (I). １ ’ ~X ３ ’ , Y １ ’ , Z １ ’ , Z ２ ’ In this way, the carbon or nitrogen forming the skeleton of the compound of formula (I) can be labeled with a stable isotope by appropriately using a stable isotope-labeled compound as a raw material compound corresponding to each element.

[0047] 3. Second embodiment (mixture)

[0048] The present invention also provides a mixture of one or two or more different stable isotope-labeled cysteinyldopa compounds according to the present invention described in the above section 2. That is, the present invention also provides a mixture of one or two or more different stable isotope-labeled cysteinyldopa compounds represented by the following formula (I): [In formula (I), X １, X ３ , X ４ , and X ７ are independently C or １３ represents C, and X ８ , CH, CD, １３ CH, or １３ represents CD, and X ９ is CH ２ , CHD, CD ２、 １３ CH ２ , １３ CHD, or １３ CD ２ represents X １ ' is C or １３ represents C, and X ２ ' is CH, CD, １３ CH, or １３ represents CD, and X ３ ' is CH ２ , CHD, CD ２ , １３ CH ２ , １３ CHD, or １３ CD ２ represents Y １ and Y １ ' are, independently of each other, NH ２ or １５ NH ２ represents Z １ is O or １８ represents O, and Z ２ ~Z ４ is OH or １８ represents OH; Z １ ' is O or １８ represents O, and Z ２ ' is OH or １８ OH, n=1 or 2, when n=1, X ２ , X ５ , X ６ Either one of the following is C or １３ C, and the rest are independently CH, CD, １３ CH, or １３ represents CD, and when n=2, X ２ , X ５ , X ６ Any two of these are C or１３ C, and the remaining one is CH, CD, １３ CH, or １３ represents CD.] The explanation regarding formula (I) in 2. above also applies to this embodiment.

[0049] The mixture can also be used as an internal standard in mass spectrometry, as described above. The one or more compounds contained in the mixture may differ only in the position of the cysteinyl group bond. In some cases, one or more cysteinyldopas in a biological sample, each having a similar chemical structure, do not need to be detected or quantified separately. In such cases, the internal standard may also be a mixture as described above to reduce costs.

[0050] For example, the mixture may be a mixture containing 5-S-cysteinyldopa (5-S-CD), 2-S-cysteinyldopa (2-S-CD), and 2,5-di-S-cysteinyldopa (2,5-di-S-CD) shown in FIG. 1, and in particular may contain only these two or three compounds.

[0051] 4. Third embodiment (analysis method)

[0052] The present invention also provides an analytical method using the cysteinyldopa described in the above section 2 or the mixture described in the above section 3. In the analytical method, the cysteinyldopa or the mixture may be used as a standard substance, more particularly as an internal standard substance.

[0053] The analytical method may be an analytical method using, as an analyte, cysteinyldopa, which is the same as the cysteinyldopa of the present invention described in 2. above, except that it is not labeled with a stable isotope. The analyte may be, for example, a stable isotope (D, １３ C, and １５ It may be cysteinyldopa with the corresponding atoms (H, C, and N) instead of the corresponding atoms (H, C, and N).

[0054] Even when the mixture is used, the analytical method may be an analytical method in which the analyte is the same cysteinyldopa as the cysteinyldopa of the present invention described in 2. above, except that it is not labeled with a stable isotope. The analytical method may particularly be a mass spectrometry method. When the mixture is used, the analyte may be any one or more of the cysteinyldopas according to the present disclosure that are the same as the cysteinyldopas constituting the mixture, except that it is not labeled with a stable isotope. For example, when the mixture contains 5-S-cysteinyldopa (5-S-CD), 2-S-cysteinyldopa (2-S-CD), and 2,5-di-S-cysteinyldopa (2,5-di-S-CD), the analyte may be at least one, two, or all selected from the group consisting of 5-S-cysteinyldopa (5-S-CD), 2-S-cysteinyldopa (2-S-CD), and 2,5-di-S-cysteinyldopa (2,5-di-S-CD). In this analytical method, 5-S-cysteinyldopa (5-S-CD), 2-S-cysteinyldopa (2-S-CD), and 2,5-di-S-cysteinyldopa (2,5-di-S-CD) may be analyzed separately.

[0055] The method of analysis may comprise carrying out mass spectrometry, in which the cysteinyldopa according to the invention or the mixture according to the invention may be used as a standard, in particular an internal standard, to detect or quantify the analyte.

[0056] The mass spectrometry may be used to detect or quantify cysteinyldopa in, for example, a biological sample. Biological samples include blood, serum, plasma, urine, sweat, cerebrospinal fluid, hair, nails, and feces, and can be detected or quantified directly or after appropriate pretreatment. As a pretreatment, the biological sample may be adsorbed onto a solid support to undergo deproteinization prior to mass spectrometry. In particular, the mass spectrometry may be used to detect or quantify cysteinyldopa that is not labeled with a stable isotope and is contained in the biological sample. The cysteinyldopa in the biological sample may be, for example, at least one, two, or all selected from the group consisting of 5-S-cysteinyldopa (5-S-CD), 2-S-cysteinyldopa (2-S-CD), and 2,5-di-S-cysteinyldopa (2,5-di-S-CD).

[0057] In one embodiment, cysteinyldopa in the biological sample may be a tumor marker for melanoma. For example, 5-S-cysteinyldopa (5-S-CD), 2-S-cysteinyldopa (2-S-CD), and 2,5-di-S-cysteinyldopa (2,5-di-S-CD) are promising tumor markers for melanoma. The analytical method of the present invention may comprise detecting or quantifying such melanoma tumor markers, and in particular may comprise detecting or quantifying such melanoma tumor markers by mass spectrometry.

[0058] Furthermore, the analytical method of the present invention may include generating, based on the results of the detection or quantification, data regarding the risk of melanoma in the human from whom the biological sample is derived, data regarding the presence or absence of melanoma in the human from whom the biological sample is derived, or data regarding the progression of melanoma in the human from whom the biological sample is derived.

[0059] The mass analysis may include ionizing the analyte, for example by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or matrix-assisted laser desorption ionization (MALDI), but may also be by other ionization techniques.

[0060] The mass analysis may be performed by a mass analyzer used in the art, such as a liquid chromatography tandem mass analyzer (LC-MS / MS) or a matrix-assisted laser desorption / ionization mass analyzer (MALDI-MS), but the apparatus for performing the mass analysis is not limited to these. Specific procedures for the mass analysis can be appropriately determined by those skilled in the art depending on, for example, the type of sample.

[0061] 5. Fourth embodiment (reagent kit for detecting or quantifying cysteinyldopa)

[0062] The present invention also provides a reagent kit for detecting or quantifying cysteinyldopa, which uses the cysteinyldopa described in the above section 2 or the mixture described in the above section 3. In the reagent kit for detection or quantification, the cysteinyldopa or the mixture may be used as a standard substance, more particularly as an internal standard substance.

[0063] The reagent kit for detecting or quantifying cysteinyldopa of the present invention may contain the stable isotope-labeled cysteinyldopa or the mixture.

[0064] 6. Working Example

[0065] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0066] [Example 1] 5-S-CD-D ２ Synthesis of

[0067] 5-S-CD-D ２ (i.e., cysteinyldopa of formula (II)) was synthesized by the following procedure. The reaction pathway is shown in Figure 3.

[0068] 40 mg of L-DOPA (Cambridge Isotope Laboratories, Inc. MA, USA, Compound i in the figure), 220 mg of diammonium cerium (IV) nitrate, 100 mg of cysteine ​​(3,3-D ２ ,98%) respectively 2M H ２ SO ４The L-DOPA solution was dissolved in 1 mL, 2 mL, and 1 mL of aqueous solution. While vigorously stirring, the diammonium cerium (IV) nitrate solution was added to the L-DOPA solution and reacted for about 15 seconds. Similarly, while vigorously stirring, cysteine ​​(3,3-D ２ A solution of 5-S-CD-D was added and the reaction was continued for several more minutes. The reaction solution was adsorbed onto a column (inner diameter 10 mm) packed with approximately 20 cm of strongly acidic cation exchange resin 50Wx2 200-400 mesh H type (Fujifilm Wako Pure Chemical Industries). While tracking the reaction using a UV-visible spectrophotometer or LC / MS, unreacted starting materials and the by-product Ce(III) were washed away with pure water and then 0.5 M hydrochloric acid, and then eluted with 3 M hydrochloric acid. The target product, 5-S-CD-D ２ The fractions containing 5-S-CD were collected and dried on a rotary evaporator. The resulting solid was dissolved in a small amount (approximately 1 mL) of hydrochloric acid and adsorbed onto a 50Wx2 200-400 mesh H-type resin (column length approximately 20 cm after equilibration) that had been previously equilibrated with 2 M hydrochloric acid. While analyzing with a UV-visible spectrophotometer or LCMS, the fractions containing 5-S-CD, free of by-products such as 2-S-CD, were collected and dried on a rotary evaporator to obtain white crystals (approximately 40 mg of the hydrochloride salt of the target compound) (Compound iv in the figure). These crystals were dissolved in heavy water and adsorbed onto a 50Wx2 200-400 mesh H-type resin (column length approximately 20 cm after equilibration). The elution was performed with 2 M hydrochloric acid while analyzing with a UV-visible spectrophotometer or LCMS. The fractions containing 5-S-CD, free of by-products such as 2-S-CD, were collected and dried on a rotary evaporator to obtain white crystals (approximately 40 mg of the hydrochloride salt of the target compound) (Compound iv in the figure). １ H NMR and １３ The NMR spectra obtained by C NMR measurement are shown in Figures 4 and 5. The mass spectrum obtained by analyzing this compound by LCMS is shown in Figure 6.

[0069] [Example 2] 5-S-CD-Ring １３ C ６ Synthesis of

[0070] 5-S-CD-Ring １３ C ６ (i.e., cysteinyldopa of formula (III)) was synthesized by the following procedure.

[0071] 0.1g of L-DOPA (Ring １３ C ６　 99%), 0.55 g of diammonium cerium(IV) nitrate, and 0.25 g of cysteine ​​were dissolved in 2 M H ２ SO ４The solution was dissolved in 2.5 mL, 5 mL, and 2.5 mL of aqueous solution. １３ C ６ A diammonium cerium (IV) nitrate solution was added to the (1,99%) solution and allowed to react for about 15 seconds. Similarly, a cysteine ​​solution was added with vigorous stirring and allowed to react for a few more minutes. The subsequent procedures were the same as in Example 1, yielding white crystals (approximately 0.1 g of the hydrochloride salt of the target compound). The crystals were dissolved in heavy water and １ H NMR and １３ The NMR spectra obtained by C NMR measurement are shown in Figures 7 and 8. The mass spectrum obtained by analyzing this compound by LCMS is shown in Figure 9.

[0072] [Example 3] 5-S-CD- １３ C, D ２ Synthesis of

[0073] 5-S-CD- １３ C, D ２ (i.e., cysteinyldopa of formula (IV)) was synthesized by the following procedure.

[0074] 40 mg of L-DOPA (1- １３ C, 99%), 220 mg diammonium cerium (IV) nitrate, 100 mg cysteine ​​(3,3-D ２ , 98%) to 2MH ２ SO ４ The L-DOPA solution was added with cerium (IV) diammonium nitrate solution under vigorous stirring and reacted for about 15 seconds. Similarly, cysteine ​​(3,3-D ２ A solution of 1,000 sucrose (98%) was added and the mixture was allowed to react for several minutes. The subsequent procedures were carried out in the same manner as in Example 1 to obtain white crystals (about 40 g of the hydrochloride salt of the target compound). The crystals were dissolved in heavy water and １ H NMR and １３ The NMR spectra obtained by C NMR measurement are shown in Figures 10 and 11. The mass spectrum obtained by analyzing this compound by LCMS is shown in Figure 12.

[0075] [Example 4] 5-S-CD- １３ C. １５ Synthesis of N

[0076] 5-S-CD- １３ C. １５ N (i.e., cysteinyldopa of formula (V)) was synthesized by the following procedure.

[0077] 40 mg of L-DOPA (1- １３ C, 99%), 220 mg diammonium cerium nitrate, 100 mg cysteine ​​( １５ N, 98%) to 2MH ２ SO ４ The L-DOPA solution was dissolved in 1 mL, 2 mL, and 1 mL of aqueous solution. While vigorously stirring, a diammonium cerium (IV) nitrate solution was added to the L-DOPA solution, and the reaction was allowed to proceed for approximately 15 seconds. Similarly, while vigorously stirring, a cysteine ​​(15N, 98%) solution was added, and the reaction was allowed to proceed for several more minutes. The subsequent procedures were carried out in the same manner as in Example 1, yielding white crystals (approximately 40 g of the hydrochloride salt of the target compound). The crystals were dissolved in heavy water, and the resulting mixture was then cooled to room temperature. １ H NMR, １３ C NMR, １５ The NMR spectra obtained by N NMR measurement are shown in Figures 13, 14, and 15. The mass spectrum obtained by analyzing this compound by LCMS is shown in Figure 16.

[0078] [Example 5] LC-MS / MS analysis of 5-S-CD in pooled serum

[0079] (1) Preparation of calibrators

[0080] A 5-S-CD sample was added to 0.2 M hydrochloric acid containing ascorbic acid (100 mg / L) to prepare a solution with the concentrations shown in Table 1 below.

[0081]

[0082] (2) Preparation of IS (internal standard) solution

[0083] 5-S-CD- １３ C ６ The sample was added to a 200 mM ammonium formate solution containing 4% phosphoric acid to prepare a concentration of 5 ng / mL.

[0084] (3) Sample pretreatment

[0085] Sample pretreatment was performed using the following procedure: 1. SPE (Solid Phase) Extraction 1) 100 μL of serum was mixed with 400 μL of IS solution. 2) 500 μL of the serum / IS mixed solution was loaded onto an Oasis PRiME MCX 96-well μElution Plate (Waters). 3) 200 μL of 5% methanol was added for washing. 4) 100 μL of a methanol / concentrated aqueous ammonia (90 / 10, v / v) solution was added for elution. This process was repeated twice. 2. Drying: Nitrogen spray or centrifugal concentration for approximately 200 minutes. 3. 50 μL of distilled water was added to prepare the sample for LC-MS / MS analysis.

[0086] (4) LC analysis conditions

[0087] The LC analysis conditions were as follows: Apparatus: Waters ACQUITY UPLC I-Class Analytical column: Waters ACQUITY UPLC BEH C18 1.7 mm ID × 50 mm Elution conditions: Flow rate 0.4 mL / min Solvent A: 0.1% formic acid - water B: 0.1% formic acid - acetonitrile The details of the elution conditions are shown in Table 2 below.

[0088]

[0089] (5) MS / MS analysis conditions

[0090] The MS / MS analysis conditions were as follows: Apparatus: Waters Xevo TQ-XS triple quadrupole mass spectrometer Ionization conditions: ESI negative ion mode The SRM parameters were as shown in Table 3 below.

[0091]

[0092] Analytical Results The calibration curve for 5-S-CD is shown in Figure 17. The calibration curve for 5-S-CD and 5-S-CD- in pooled serum is shown in Figure 18. １３ C ６The SRM chromatogram of the above is shown. Quantitative Value of 5-S-CD in Pooled Serum Based on the above analytical results, the 5-S-CD concentration in pooled serum was quantified to be 0.13 ng / mL. Thus, 5-S-CD can be quantified by mass spectrometry using stable isotope-labeled cysteinyldopa according to the present invention. Furthermore, although 5-S-CD is present in extremely trace amounts in biological samples, mass spectrometry using stable isotope-labeled cysteinyldopa according to the present invention can detect and quantitate such trace amounts of melanin-related metabolites.

Claims

1. Stable isotope-labeled cysteinyldopa represented by the following formula (I): [In formula (I), X １ , X ３ , X ４ , and X ７ are independently C or １３ represents C, and X ８ , CH, CD, １３ CH, or １３ represents CD, and X ９ is CH ２ , CHD, CD ２、 １３ CH ２ , １３ CHD, or １３ CD ２ represents X １ ' is C or １３ represents C, and X ２ ' is CH, CD, １３ CH, or １３ represents CD, and X ３ ' is CH ２ , CHD, CD ２ , １３ CH ２ , １３ CHD, or １３ CD ２ represents Y １ and Y １ ' are, independently of each other, NH ２ or １５ NH ２ represents Z １ is O or １８ represents O, and Z ２ ~Z ４ is OH or １８ represents OH; Z １ ' is O or １８ represents O, and Z ２ ' is OH or １８ OH, n=1 or 2, when n=1, X ２ , X ５ , X ６ Either one of the following is C or １３ C, and the rest are independently CH, CD, １３ CH, or １３ represents CD, and when n=2, X ２ , X ５ , X ６ Any two of these are C or １３ C, and the remaining one is CH, CD, １３ CH, or １３ Represents a CD.] 2. In the formula (I), D, １３ C, or １５ The stable isotope-labeled cysteinyldopa of claim 1, having two or more Ns.

3. In the formula (I), １８ The stable isotope-labeled cysteinyldopa of claim 1, having one or more O.

4. In the formula (I), D, １３ C, and １５ 3. A stable isotope-labeled cysteinyldopa according to claim 2, which has two or more isotope atoms selected from the group consisting of N, and the types of isotope atoms contained in formula (1) are the same or two or more types.

5. In the formula (I), １８ 4. The stable isotope-labeled cysteinyldopa according to claim 3, which has one or more O and the types of isotope atoms contained in the formula (1) are the same or two or more types.

6. Either or both of the L-dopa-derived skeleton and the cysteine-derived skeleton forming the formula (1) are selected from the group consisting of D, １３ C, or the above １５ The stable isotope-labeled cysteinyldopa of claim 2, having N.

7. Either or both of the L-dopa-derived skeleton and the cysteine-derived skeleton forming the formula (1) are １８ The stable isotope-labeled cysteinyldopa of claim 3, having O.

8. An analytical method using the stable isotope-labeled cysteinyldopa described in claim 1.

9. The analytical method of claim 8, comprising performing mass spectrometry.

10. The analytical method according to claim 9, wherein the stable isotope-labeled cysteinyldopa is used as an internal standard in the mass spectrometry.

11. The analytical method according to claim 9, which comprises detecting or quantifying cysteinyldopa in a biological sample by mass spectrometry.

12. The analytical method according to claim 11, wherein the biological sample is adsorbed onto a solid phase carrier and subjected to a deproteinization treatment before carrying out the mass spectrometry.

13. The analytical method according to claim 11, wherein cysteinyldopa in the biological sample is a tumor marker for melanoma.

14. One type of stable isotope-labeled cysteinyldopa represented by the following formula (I), or a mixture of two or more different types of cysteinyldopa: [In formula (I), X １ , X ３ , X ４ , and X ７ are independently C or １３ represents C, and X ８ , CH, CD, １３ CH, or １３ represents CD, and X ９ is CH ２ , CHD, CD ２、 １３ CH ２ , １３ CHD, or １３ CD ２ represents X １ ' is C or １３ represents C, and X ２ ' is CH, CD, １３ CH, or １３ represents CD, and X ３ ' is CH ２ , CHD, CD ２ , １３ CH ２ , １３ CHD, or １３ CD ２ represents Y １ and Y １ ' are, independently of each other, NH ２ or １５ NH ２ represents Z １ is O or １８ represents O, and Z ２ ~Z ４ is OH or １８ represents OH; Z １ ' is O or １８ represents O, and Z ２ ' is OH or １８ OH, n=1 or 2, when n=1, X ２ , X ５ , X ６ Either one of the following is C or １３ C, and the rest are independently CH, CD, １３ CH, or １３ represents CD, and when n=2, X ２ , X ５ , X ６ Any two of these are C or １３ C, and the remaining one is CH, CD, １３ CH, or １３ Represents a CD.] 15. An analytical method using the mixture of claim 14.

16. A reagent kit for detecting or quantifying cysteinyldopa, comprising the stable isotope-labeled cysteinyldopa described in claim 1.

17. A reagent kit for detecting or quantifying cysteinyldopa, comprising the mixture of claim 14.

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