Stable isotope-labeled cysteinyldopa and analysis method

Abstract

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 the following formula (I).SELECTED DRAWING: None

Description

[Technical Field]

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

[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. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Automated high-performance liquid chromatographic determination of 5-S-cysteinyl-3,4-dihydroxyphenylalanine in urine. Kagedal, B.; Kallberg, M.; Arstrand, K.; Hansson, CJ Chromatogr. 1989, 473, 359-370. [Non-patent document 2] 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. [Non-patent document 3] 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. [Non-patent document 4] A convenient one step synthesis of 5-cystein-S-ylDOPA using ceric ammonium nitrate. Chioccara, F.; Novellino, E. Synth. Commun. 1986, 16, 967-971. Summary of the Invention [Problem to be solved by the invention]

[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 offers high sensitivity and high resolution for compounds. To perform this analytical method, stable isotope-labeled compounds of the target metabolites must be prepared as internal standards.

[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. [Means for solving the problem]

[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] A stable isotope-labeled cysteinyldopa represented by the following formula (I): [ka] [In formula (I), X1, X3, X4, and X7 are each independently C or 13 represents C, X8 is CH, CD, 13 CH, or 13 represents CD, X9 is CH2, CHD, CD 2、 13 CH2, 13 CHD, or 13 represents CD2, X1' is C or 13 represents C, X2' is CH, CD, 13 CH, or 13 represents CD, X3' is CH2, CHD, CD2, 13 CH2, 13 CHD, or 13 represents CD2, Y1 and Y1' are independently NH2 or 15 represents NH2, Z1 is O or 18 represents O, Z2 to Z4 are OH or 18 represents OH, Z1' is O or 18 represents O, Z2' is OH or 18 represents OH, n=1 or 2, When n=1, one of X2, X5, and X6 is C or 13 C, and the rest are independently CH, CD, 13 CH, or 13 represents CD, When n=2, any two of X2, X5, and X6 are C or 13 C, and the remaining one is CH, CD, 13 CH, or 13 It represents a CD.] [2] In the formula (I), D, 13 C, or 15 A stable isotope-labeled cysteinyldopa according to [1], having two or more Ns. [3] In the formula (I), 18 A stable isotope-labeled cysteinyldopa according to [1], which has one or more O. [4] In the formula (I), D, 13 C, and 15 A stable isotope-labeled cysteinyldopa according to [2], which has two or more isotope atoms selected from the group consisting of N, and the types of isotope atoms contained in the formula (1) are the same or two or more types. [5] In the formula (I), 18 The stable isotope-labeled cysteinyldopa according to [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 13 C, or the above 15 Stable isotope-labeled cysteinyldopa according to [2], having N. [7] Either or both of the L-dopa-derived skeleton and the cysteine-derived skeleton forming the formula (1) are 18 A stable isotope-labeled cysteinyldopa having O as described in [3]. [8] An analytical method using stable isotope-labeled cysteinyldopa according to [1]. [9] [8] The analytical method according to [8], comprising performing 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 onto a solid phase carrier and subjected to a deproteinization treatment before carrying out the mass spectrometry.

[13] The analytical method according to

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

[14] A stable isotope-labeled cysteinyldopa compound represented by the following formula (I), or a mixture of two or more different cysteinyldopa compounds: [ka] [In formula (I), X1, X3, X4, and X7 are each independently C or 13 represents C, X8 is CH, CD, 13 CH, or 13 represents CD, X9 is CH2, CHD, CD 2、 13 CH2, 13 CHD, or 13 represents CD2, X1' is C or 13 represents C, X2' is CH, CD, 13 CH, or 13 represents CD, X3' is CH2, CHD, CD2, 13 CH2, 13 CHD, or 13 represents CD2, Y1 and Y1' are independently NH2 or 15 represents NH2, Z1 is O or 18 represents O, Z2 to Z4 are OH or 18 represents OH, Z1' is O or 18 represents O, Z2' is OH or 18 represents OH, n=1 or 2, When n=1, one of X2, X5, and X6 is C or 13 C, and the rest are independently CH, CD, 13 CH, or13 represents CD, When n=2, any two of X2, X5, and X6 are C or 13 C, and the remaining one is CH, CD, 13 CH, or 13 It represents a CD.]

[15]

[14] An analytical method using the mixture described in

[14] .

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

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

[14] . [Effects of the Invention]

[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 here, and may be any of the effects described in this specification. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the melanin biosynthetic pathway. [Figure 2] FIG. 1 is a diagram illustrating the method for producing stable isotope-labeled cysteinyldopa of the present invention. [Figure 3] FIG. 1 shows a reaction pathway for the synthesis of stable isotope-labeled cysteinyldopa. [Figure 4] FIG. 1 shows the H NMR spectrum of cysteinyldopa (5-S-CD-D2) of formula (II) synthesized using L-DOPA as a starting material. [Figure 5]FIG. 1 shows the C NMR spectrum of cysteinyldopa (5-S-CD-D2) of formula (II) synthesized using L-DOPA as a starting material. [Figure 6] FIG. 1 shows a mass spectrum obtained by LCMS analysis of cysteinyldopa (5-S-CD-D2) of formula (II) synthesized using L-DOPA as a starting material. [Figure 7] FIG. 1 shows the H NMR spectrum of cysteinyldopa (5-S-CD-Ring 13C6) of formula (III) synthesized using L-DOPA (Ring13C6 99%) as a starting material. [Figure 8] FIG. 1 shows the C NMR spectrum of cysteinyldopa (5-S-CD-Ring 13C6) of formula (III) synthesized using L-DOPA (Ring 13C6 99%) as a starting material. [Figure 9] FIG. 1 shows a mass spectrum obtained by LCMS analysis of cysteinyldopa (5-S-CD-Ring 13C6) of formula (III) synthesized using L-DOPA (Ring13C6 99%) as a starting material. [Figure 10] FIG. 1 shows the H NMR spectrum of cysteinyldopa (5-S-CD-13C,D2) of formula (IV) synthesized using L-DOPA (1-13C,99%) and cysteine ​​(3,3-D2,98%) as raw materials. [Figure 11] FIG. 1 shows the C NMR spectrum of cysteinyldopa (5-S-CD-13C,D2) of formula (IV) synthesized using L-DOPA (1-13C,99%) and cysteine ​​(3,3-D2,98%) as raw materials. [Figure 12] FIG. 1 shows the mass spectrum of cysteinyldopa (5-S-CD-13C,D2) of formula (IV) synthesized using L-DOPA (1-13C,99%) and cysteine ​​(3,3-D2,98%) as starting materials, analyzed by LCMS. [Figure 13] FIG. 1 shows the H NMR spectrum of cysteinyldopa (5-S-CD-13C,15N) of formula (V) synthesized using L-DOPA (1-13C,99%) and cysteine ​​(15N,98%) as raw materials. [Figure 14] FIG. 1 shows the C NMR spectrum of cysteinyldopa (5-S-CD-C,N) of formula (V) synthesized using L-DOPA (1-C,99%) and cysteine ​​(N,98%) as raw materials. [Figure 15] FIG. 1 shows the 15N NMR spectrum of cysteinyldopa (5-S-CD-13C,15N) of formula (V) synthesized using L-DOPA (1-13C,99%) and cysteine ​​(15N,98%) as raw materials. [Figure 16] FIG. 1 shows the mass spectrum of cysteinyldopa (5-S-CD-13C,15N) of formula (V) synthesized using L-DOPA (1-13C,99%) and cysteine ​​(15N,98%) as starting materials, analyzed by LCMS. [Figure 17] FIG. 1 shows a calibration curve for 5-S-CD. [Figure 18] FIG. 1 shows SRM chromatograms of 5-S-CD and 5-S-CD-13C6 in pooled serum. DETAILED DESCRIPTION OF THE INVENTION

[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 blood and are converted to 6H5MI2C or 5H6MI2C by O-methyltransferase (COMT) in the liver, and are ultimately 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 for the detection or quantification of melanin-related metabolites. Mass spectrometry using this stable isotope-labeled cysteinyldopa as an internal standard enables the detection or quantification of melanin-related metabolites with high sensitivity and high resolution. Furthermore, such highly sensitive and highly resolvable detection or quantification of melanin-related metabolites will also contribute 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 analogue 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. In other words, accurate quantification of cysteinyldopa is possible by synthesizing stable isotope-labeled cysteinyldopa according to the present invention.

[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): [ka] 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), X1, X3, X4, and X7 are each independently C or 13 C, and X8 represents CH, CD, 13 CH, or 13 X9 represents CH2, CHD or CD2; X1' represents C or 13 C, and X2' represents CH, CD, 13 CH, or 13 X3' represents CH2, CHD, or CD2. When n=1, one of X2, X5, and X6 is C or 13 C, and the rest are independently CH, CD, 13 CH, or 13 represents CD, and when n=2, any two of X2, X5, and X6 are C or 13 C, and the remaining one is CH, CD, 13 CH, or 13 represents CD. That is, X1 is either C or 13 C. X2 is C, 13 C, CH, CD, 13 CH, or 13 CD. X3 is C or 13 C. X4 is either C or 13 C. X5 is C, 13 C, CH, CD, 13 CH, or 13 CD. X6 is C, 13 C, CH, CD, 13 CH, or13 CD. X7 is C or 13 C. X8 is CH, CD, 13 CH, or 13 It's a CD. X9 is CH2, CHD, CD 2、 13 CH2, 13 CHD, or 13 CD2. X1' is C or 13 C. X2' is CH, CD, 13 CH, or 13 CD. X3' is CH2, CHD, CD2, 13 CH2, 13 CHD, or 13 It's CD2.

[0026] In formula (I), Y1 and Y1' are each independently NH2 or 15 NH2. That is, Y1 represents NH2 or 15 NH2. Y1' is NH2 or 15 It is NH2.

[0027] In formula (I), Z1 is O or 18 O, and Z2 to Z4 are OH or 18 represents OH, Z1' represents O or 18 O, Z2' represents OH or 18 Represents OH. That is, Z1 is O or 18 O. Z2 is OH or 18 Z3 is OH or 18 Z4 is OH or 18 OH. Z1' is O or 18 O. Z2' 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 15In 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 and N, and the types of isotope atoms contained in formula (1) may be the same or two or more types. Furthermore, either or both of the L-dopa-derived skeleton and the cysteine-derived skeleton forming formula (1) may contain the D, 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. The stable isotope D can exist as a component of the above-mentioned X2, X5, X6, X8, X9, X2', and X3'.

[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 can be present as a component of X1, X2, X3, X4, X5, X6, X7, X8, X9, X1', X2', and X3' described above.

[0031] Stable isotopes contained in the cysteinyldopa of the present invention 15 The number of N is preferably 2 or less. 15 N can be present as a component of Y1 and Y1' described above.

[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 isotope13 C and one stable isotope 15 N may be included.

[0033] In addition, the cysteinyldopa of the present invention is a compound containing 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 It may have O.

[0034] Stable isotopes contained in the cysteinyldopa of the present invention 18 The number of O is preferably 6 or less. 18 O can be present as a component of Z1, Z2, Z3, Z4, Z1', and Z2' described above.

[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 also referred to as 5-S-CD-D2, as described below, and the compound of formula (III) is a cysteinyldopa having a 5-S-CD-Ring. 13 Also referred to as C6, the compound of formula (IV) is 5-S-CD- 13 Compounds of formula (V), also known as C,D2, are prepared by the reaction of 5-S-CD- 13 C, 15 Also called N. [ka]

[0036] [ka]

[0037] [ka]

[0038] [ka]

[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, it may be carried out as shown in the Examples below.

[0041] Stable isotope-labeled L-dopa may be prepared by methods 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, stable isotope labels can be introduced into the six-membered ring moiety (X1 to X6) in formula (I) by using stable isotope-labeled acetylene. Furthermore, stable isotope-labeled deuterium D can be introduced into X2, X5, and X6 in formula (I) by HD exchange using heavy water, as shown in Figure 2.

[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 HD exchange using heavy water. An oxidation process is then performed on the benzene to introduce 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 techniques known in the art. The pyruvic acid can be used to introduce stable isotope labels into X8 and X9 in formula (I). The ammonia can be used to introduce stable isotope labels into Y1 in formula (I).

[0046] Stable isotope-labeled cysteinyldopa can be synthesized by adding L-cysteine ​​to L-dopa using diammonium cerium (IV) nitrate according to the reaction pathway shown in Figure 3. Stable isotope-labeled L-cysteine ​​is commercially available, or it can be synthesized by a method known in the art. The stable isotope-labeled L-cysteine ​​can be used to convert X1 in formula (I) into ’ ~X3 ’ , Y1 ’ , Z1 ’ , Z2 ’ A stable isotope label can be introduced into the 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 one or a mixture of two or more different stable isotope-labeled cysteinyldopas according to the present invention described in 2. above. That is, the present invention also provides one or a mixture of two or more different stable isotope-labeled cysteinyldopa compounds represented by the following formula (I): [ka] [In formula (I), X1, X3, X4, and X7 are each independently C or 13 represents C, X8 is CH, CD, 13 CH, or 13 represents CD, X9 is CH2, CHD, CD 2、 13 CH2, 13 CHD, or 13 represents CD2, X1' is C or 13 represents C, X2' is CH, CD, 13 CH, or 13 represents CD, X3' is CH2, CHD, CD2, 13 CH2, 13 CHD, or 13 represents CD2, Y1 and Y1' are independently NH2 or 15 represents NH2, Z1 is O or 18 represents O, Z2 to Z4 are OH or 18 represents OH, Z1' is O or 18 represents O, Z2' is OH or 18 represents OH, n=1 or 2, When n=1, one of X2, X5, and X6 is C or 13 C, and the rest are independently CH, CD, 13 CH, or 13 represents CD, and when n=2, any two of X2, X5, and X6 are C or 13 C, and the remaining one is CH, CD, 13 CH, or 13 It represents a 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 at which the cysteinyl group is bonded. In some cases, it may not be necessary to separately detect or quantify one or more cysteinyldopas in a biological sample because they have similar chemical structures. 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 Figure 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 2. above or the mixture described in 3. above. 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 that make up 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 before 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 include detecting or quantifying such melanoma tumor markers, particularly 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, such as 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, for example, 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 thereto. The specific procedure for mass spectrometry 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 2. above or the mixture described in 3. above. In the reagent kit for detecting or quantifying cysteinyldopa, 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] Synthesis of 5-S-CD-D2

[0067] 5-S-CD-D2 (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, and 100 mg of cysteine ​​(3,3-D2, 98%) were dissolved in 1 mL, 2 mL, and 1 mL of 2 M H2SO4, respectively. The diammonium cerium(IV) nitrate solution was added to the L-DOPA solution with vigorous stirring and allowed to react for approximately 15 seconds. Similarly, the cysteine ​​(3,3-D2, 98%) solution was added with vigorous stirring and allowed to react for several minutes. The reaction solution was then loaded onto a column (10 mm internal diameter) packed with approximately 20 cm of strongly acidic cation exchange resin, 50W x 2, 200-400 mesh, H type (Fujifilm Wako Pure Chemical Industries). While monitoring 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 0.5M hydrochloric acid, followed by elution with 3M hydrochloric acid. Fractions containing the target compound, 5-S-CD-D2, 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 50W x 2 200-400 mesh H-type resin (column length approximately 20 cm after equilibration) that had been previously equilibrated with 2M hydrochloric acid. While analyzing the reaction using a UV-visible spectrophotometer or LC / MS, 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 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 LCMS analysis of this compound is shown in Figure 6.

[0069] [Example 2] 5-S-CD-Ring 13 Synthesis of C6

[0070] 5-S-CD-Ring 13 C6 (i.e., cysteinyldopa of formula (III)) was synthesized by the following procedure.

[0071] 0.1g of L-DOPA (Ring 13 0.55 g of cerium(IV) diammonium nitrate, and 0.25 g of cysteine ​​were dissolved in 2.5 mL, 5 mL, and 2.5 mL of 2 M H2SO4 aqueous solution, respectively. 13 A diammonium cerium (IV) nitrate solution was added to the C6 (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, and white crystals (approximately 0.1 g of the hydrochloride salt of the target compound) were obtained. These 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 LCMS analysis of this compound is shown in Figure 9.

[0072] [Example 3] 5-S-CD- 13 Synthesis of C and D2

[0073] 5-S-CD- 13 C and D2 (i.e., cysteinyldopa of formula (IV)) were synthesized by the following procedure.

[0074] 40 mg of L-DOPA(1- 13 C, 99%), 220 mg of diammonium cerium (IV) nitrate, and 100 mg of cysteine ​​(3,3-D2, 98%) were dissolved in 1 mL, 2 mL, and 1 mL of 2M H2SO4 aqueous solution, respectively. The diammonium cerium (IV) nitrate solution was added to the L-DOPA solution with vigorous stirring and allowed to react for about 15 seconds, and then the cysteine ​​(3,3-D2, 98%) solution was added with similar vigorous stirring and allowed to react for a few more minutes. The following procedures were carried out in the same manner as in Example 1 to obtain white crystals (approximately 40 g of the hydrochloride salt of the target compound). These 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 LCMS analysis of this compound 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 The L-DOPA solution was dissolved in 1 mL, 2 mL, and 1 mL of 2M H2SO4 aqueous solution. While vigorously stirring, a diammonium cerium (IV) nitrate solution was added to the L-DOPA solution and allowed to react for about 15 seconds. Similarly, while vigorously stirring, a cysteine ​​(15N, 98%) solution was added and allowed to react for a few more minutes. The following procedures were carried out in the same manner as in the examples to obtain white crystals (approximately 40 g of the hydrochloride salt of the target compound). These crystals were dissolved in heavy water and 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 LCMS analysis of this compound 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 preparation was added to 0.2 M hydrochloric acid containing ascorbic acid (100 mg / L) to prepare the concentration shown in Table 1 below.

[0081] [Table 1]

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

[0083] 5-S-CD-13 A C6 preparation 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] The sample was pretreated according to 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 mixture was loaded onto an Oasis PRiME MCX 96-well μElution Plate (Waters). 3) 200 μL of 5% methanol was added and washed. 4) 100 μL of methanol / concentrated aqueous ammonia (90 / 10, v / v) solution was added for elution. This was repeated twice. 2. Drying: Nitrogen spray or centrifugal concentration, approximately 200 minutes 3. 50 μL of distilled water was added to prepare a sample for LC-MS / MS analysis.

[0086] (4)LC analysis conditions

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

[0088] [Table 2]

[0089] (5)MS / MS analysis conditions

[0090] The MS / MS analysis conditions were as follows: Equipment: 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] [Table 3]

[0092] ·Analysis results The calibration curve for 5-S-CD is shown in FIG. Figure 18 shows the results of 5-S-CD and 5-S-CD- 13 The SRM chromatogram of C6 is shown. Quantitative value of 5-S-CD in pooled serum Based on the above analytical results, the 5-S-CD concentration in the pooled serum was determined 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 small 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. A stable isotope-labeled cysteinyldopa represented by the following formula (I): 【Chemistry 1】 [In formula (I), X １ , X ３ , X ４ , and X ７ are independently C or １３ represents C, X ８ , CH, CD, １３ CH, or １３ represents a CD, 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, X ２ ' is CH, CD, １３ CH, or １３ represents a CD, 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, Z ２ ~Z ４ is OH or １８ represents OH, Z １ ' is O or １８ represents O, Z ２ ' is OH or １８ represents 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 a CD, 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 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 according to claim 1.

9. 10. The 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, comprising 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 the mass spectrometry is performed.

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

14. A stable isotope-labeled cysteinyldopa compound represented by the following formula (I), or a mixture of two or more different cysteinyldopa compounds: 【Chemistry 2】 [In formula (I), X １ , X ３ , X ４ , and X ７ are independently C or １３ represents C, X ８ , CH, CD, １３ CH, or １３ represents a CD, 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, X ２ ' is CH, CD, １３ CH, or １３ represents a CD, 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, Z ２ ~Z ４ is OH or １８ represents OH, Z １ ' is O or １８ represents O, Z ２ ' is OH or １８ represents 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 a CD, 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 of claim 1.

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

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