Phosphonium compound, derivatization reagent kit, mass spectrometry method, and method for producing phosphonium compound

Abstract

To provide a novel technique for improving the ionization efficiency of a target compound.SOLUTION: Provided is a phosphonium compound represented by the following formula (I).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a phosphonium compound, a derivatization reagent kit, a mass spectrometry method, and a method for producing a phosphonium compound. [Background technology]

[0002] Liquid chromatography tandem mass spectrometry (LC-MS / MS) and matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) are extremely powerful analytical techniques for quantifying trace amounts of metabolites and other substances. These analytical techniques involve ionization of the target compound. However, in analyses where ionization of the target compound is performed, the ionization efficiency can be low for some target compounds, often resulting in detection failure or inability to ensure detection accuracy for quantification. Therefore, the target compound may be derivatized to increase ionization efficiency.

[0003] For example, Non-Patent Documents 1 and 2 below disclose that fat-soluble hormones (male hormones and female hormones) that do not contain nitrogen atoms are derivatized with a derivatizing reagent that contains a nitrogen atom. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] J. Chromatogr. B 2011, 879, 1159-1165. [Non-patent document 2] Biomed. Chromatogr. 2021, 35, e5036. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a new method for increasing the ionization efficiency of a target compound. [Means for solving the problem]

[0006] The present inventors have found that certain phosphonium compounds are suitable for derivatization to enhance ionization efficiency.

[0007] That is, the present invention is The following formula (I) [ka] [In formula (I), R 1 represents an alkyl group, and the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms or a cyclic alkyl group having 5 to 20 carbon atoms, R 2 , and R 3 each independently represents an alkyl group or an aryl group, The relevant The alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms or a cyclic alkyl group having 5 to 20 carbon atoms; The relevant The aryl group is a substituted or unsubstituted aryl group having 6 to 20 carbon atoms; X Ha, Chi a reactive group having an ester group; and Y - is an anion with an overall charge of −1, or Y - is absent] The present invention provides a phosphonium compound represented by the formula: In formula (I), the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 10 carbon atoms, or a cyclic alkyl group having 5 to 10 carbon atoms; The aryl group may be a substituted or unsubstituted aryl group having 6 to 10 carbon atoms. In formula (I), R 1 may represent either (a) or (b) below: R 2 、 and R 3 are, independently of each other, (a), (b), or (c) It may represent either one. [ka] [wherein m is an integer of 0 to 8, n is an integer of 0 to 5, and Z is H, NH2, COO - , COOM, SO3 - (sulfonic acid ion group), or SO3M (sulfonic acid group or sulfonate salt group), where M represents a hydrogen atom, a lithium atom, a sodium atom, or a potassium atom. ) [C3] [C4] In formula (I), When X is a reactive group having a thioester group, The reactive group is [ka] where V is H, NH2, SO3 - (sulfonic acid ion group), SO3M, COO - , COOM, or a hydrophilic tag, and M represents a hydrogen atom, a lithium atom, a sodium atom, or a potassium atom.

[0008] The present invention also provides The following formula (I) [ka] [In formula (I), R 1 , R 2 , and R 3 are the same or different hydrophobic hydrocarbon groups; X is a reactive group that reacts with an oxygen-containing functional group or a nitrogen-containing functional group; and Y - is a counter anion, or Y - is absent] Also provided is a phosphonium compound represented by the formula:

[0009] The present invention also provides The following formula (II) [ka] [In formula (II), R 1 represents an alkyl group, and the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms or a cyclic alkyl group having 5 to 20 carbon atoms, R 2 , and R 3 each independently represents an alkyl group or an aryl group, The relevant The alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms or a cyclic alkyl group having 5 to 20 carbon atoms; The relevant The aryl group is a substituted or unsubstituted aryl group having 6 to 20 carbon atoms; J is ,blood represents a group generated by the reaction of a reactive group having an ester group with a compound to be derivatized; and Y - is an anion with an overall charge of −1, or Y - is absent] Also provided is a phosphonium compound represented by the formula: J is [C8] [C9] [ka] (wherein Q3 is a group generated by reaction of an amino group contained in the compound to be derivatized with a thioester group).

[0010] The present invention also provides a derivatizing reagent comprising a phosphonium compound represented by formula (I). The reagent may be a reagent for derivatizing a female hormone.

[0011] The present invention also provides The following phosphine compounds [ka] (where R 1 , R 2 , and R 3each independently represents an alkyl group or an aryl group, the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms, or a cyclic alkyl group having 5 to 20 carbon atoms; The aryl group is a substituted or unsubstituted aryl group having 6 to 20 carbon atoms; blood a reactive compound having an ester group; Also provided is a derivatization reagent kit comprising:

[0012] The present invention also provides a method of mass spectrometry, which comprises derivatizing a compound to be analyzed by mass spectrometry with the phosphonium compound of formula (I), the derivatization reagent, or the derivatization reagent kit. The compound to be analyzed by mass spectrometry may be a female hormone.

[0013] [C12] [C13] [C14]

[0014] The present invention also provides 6. A method for producing a phosphonium compound according to claim 1, wherein X is a reactive group having a thioester group, The following phosphonium acetates [ka] (where, R 1 represents an alkyl group, and the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms or a cyclic alkyl group having 5 to 20 carbon atoms, R 2 、 and R 3 each independently represents an alkyl group or an aryl group, The relevant The alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms or a cyclic alkyl group having 5 to 20 carbon atoms; The relevant The aryl group is a substituted or unsubstituted aryl group having 6 to 20 carbon atoms; The following thiols and [ka] (where V is H, NH2, SO3 - (sulfonic acid ion group), SO3M, COO - , COOM, or a hydrophilic tag, and M represents a hydrogen atom, a lithium atom, a sodium atom, or a potassium atom) to produce the phosphonium compound. A method for making the same is also provided. [Effects of the Invention]

[0015] The present invention makes it possible to increase the ionization efficiency in an analytical method in which a target compound is ionized, such as a mass spectrometry method, thereby increasing the detection sensitivity of such an analytical method. 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]

[0016] [Figure 1-1] NMR spectrum data of Compound 1. [Figure 1-2] NMR spectrum data and ESI-MS data of Compound 1. [Figure 2-1] NMR spectrum data of compound 2. [Figure 2-2] NMR spectrum data and ESI-MS data of Compound 2. [Figure 3-1] NMR spectrum data of compound 3. [Figure 3-2] NMR spectrum data and ESI-MS data of Compound 3. [Figure 4-1] NMR spectrum data of compound 4. [Figure 4-2] NMR spectrum data and ESI-MS data of Compound 4. [Figure 5-1]NMR spectrum data of compound 5. [Figure 5-2] NMR spectrum data and ESI-MS data of Compound 5. [Figure 6-1] NMR spectrum data of compound 6. [Figure 6-2] NMR spectrum data and ESI-MS data of Compound 6. [Figure 7-1] NMR spectrum data of compound 7. [Figure 7-2] NMR spectrum data and ESI-MS data of Compound 7. [Figure 8-1] NMR spectrum data of compound 8. [Figure 8-2] NMR spectrum data and ESI-MS data of compound 8. [Figure 9-1] NMR spectrum data of compound 9. [Figure 9-2] NMR spectrum data and ESI-MS data of Compound 9. [Figure 10] FIG. 1 shows the structure of a phosphonium compound. [Figure 11] FIG. 1 shows a reaction scheme for testosterone derivatization. [Figure 12] FIG. 1 shows the chemical structures of testosterone derivatives. [Figure 13] FIG. 1 shows the chemical structures of Girard's reagents T and P. [Figure 14-1] This is an SRM chromatogram of the derivative produced. [Figure 14-2] This is an SRM chromatogram of the derivative produced. [Figure 14-3] This is an SRM chromatogram of the derivative produced. [Figure 14-4] This is an SRM chromatogram of the derivative produced. [Figure 14-5] This is an SRM chromatogram of the derivative produced. [Figure 14-6] This is an SRM chromatogram of the derivative produced. [Figure 15-1]1 is a graph showing measured peak areas. [Figure 15-2] 1 is a graph showing measured peak areas. [Figure 16] FIG. 1 shows a reaction scheme for estradiol derivatization. [Figure 17] NMR spectrum data of the reaction intermediate. [Figure 18-1] NMR spectrum data of derivative D11. [Figure 18-2] NMR spectrum data and ESI-MS data of derivative D11. [Figure 19] FIG. 1 shows the chemical structures of the estradiol derivatives produced. [Figure 20] FIG. 1 shows the chemical structures of the estradiol derivatives produced. [Figure 21] FIG. 1 shows the chemical structures of the estradiol derivatives produced. [Figure 22-1] This is an SRM chromatogram of the derivative produced. [Figure 22-2] This is an SRM chromatogram of the derivative produced. [Figure 22-3] This is an SRM chromatogram of the derivative produced. [Figure 22-4] This is an SRM chromatogram of the derivative produced. [Figure 22-5] This is an SRM chromatogram of the derivative produced. [Figure 22-6] This is an SRM chromatogram of the derivative produced. [Figure 22-7] This is an SRM chromatogram of the derivative produced. [Figure 23] 1 is a graph showing measured peak areas. [Figure 24] 1 is a graph showing measured peak areas. [Figure 25] 1 is a graph showing measured peak areas. [Figure 26-1] NMR spectrum data of compound 31. [Figure 26-2]1 shows MS spectrum data of compound 31. [Figure 27] FIG. 1 shows a reaction scheme for derivatization of oxytocin. [Figure 28] This is an SRM chromatogram of the derivative produced. [Figure 29] 1 is a graph showing measured peak areas. [Figure 30A] FIG. 1 shows a reaction scheme for testosterone derivatization. [Figure 30B] FIG. 1 shows a reaction scheme for estradiol derivatization. [Figure 30C] FIG. 1 shows a reaction scheme for derivatization of oxytocin. DETAILED DESCRIPTION OF THE INVENTION

[0017] 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.

[0018] 1. Description of the Invention

[0019] Mass spectrometers such as the LC-MS / MS and MALDI-MS mentioned above are extremely powerful analytical methods for quantifying trace amounts of metabolites and other substances. However, depending on the compound, the ionization efficiency is low, so in many cases detection is impossible or detection is possible but not with sufficient accuracy to enable quantification. For samples that are available in a certain amount, such as foods and supplements, it is possible to compensate for the lack of sensitivity by increasing the sample amount. However, for such samples, it is desirable to be able to detect or quantify the target compound with a smaller sample amount, and improving the ionization efficiency is desirable. For example, when target compounds such as human metabolites are involved, it is often not possible to easily increase the sample volume containing the target compounds, and improving ionization efficiency is extremely important for such target compounds. To improve detection sensitivity, not only have instrumental improvements been made, such as increasing the sensitivity of mass spectrometers, but also modifications (derivatization) of target compounds to increase ionization efficiency have been widely used. Generally, biological metabolites are organic compounds primarily composed of carbon, hydrogen, oxygen, and nitrogen, and oxygen and nitrogen atoms with lone electron pairs are more likely to add hydrogen ions (protons), resulting in high ionization efficiency. Nitrogen atoms in particular have a higher proton-adding ability than oxygen atoms, and compounds containing nitrogen atoms tend to ionize more easily than compounds without them.

[0020] As mentioned above, fat-soluble hormones do not contain nitrogen atoms, so ionization efficiency has been improved by introducing nitrogen atoms through derivatization using nitrogen-containing derivatization reagents. On the other hand, peptides and proteins, which contain nitrogen atoms, generally tend to be more easily ionized than fat-soluble hormones, so derivatization is not necessarily a common method for peptides and proteins.

[0021] Although trace metabolites have already been analyzed using mass spectrometers, it is desirable to further improve the sensitivity for accurate quantification. In LC-MS / MS, the standard method for quantitative mass spectrometry, target compounds are ionized using electrospray ionization (ESI), and the ease with which the target compound can be ionized depends on the polarity of the target compound itself (ease of protonation) and the organic solvent composition of the mobile phase that evaporates during ionization. Different compounds have different structures and polarities, so optimizing the ionization efficiency to suit the compound is necessary to further increase detection sensitivity. Another important point is that LC-MS / MS enables more sensitive quantitative measurements through selective reaction detection (SRM), resulting in fragmentation with lower background.

[0022] The present inventors have found that a specific phosphonium compound can enhance the ionization efficiency in an analytical method in which a target compound is ionized. That is, the present invention provides a phosphonium compound having a group for controlling the hydrophobicity of the target compound and a group that reacts with the target compound.

[0023] The phosphonium compound contains a phosphorus atom, which is in the same group 15 as the nitrogen atom in the periodic table, but is located in the period below. Like the nitrogen atom, the phosphorus atom also has an unshared electron pair, making it easy to add protons. Additionally, because the phosphorus atom has an electron in its d orbital, it is softer than the nitrogen atom according to the HSAB (Hard and Soft Acids and Bases) rule, making it more susceptible to nucleophilic substitution reactions with alkyl or benzyl halides. Therefore, a wide variety of hydrophobic substituents can be easily introduced onto the phosphorus atom. Therefore, hydrophobic substituents can be attached to the phosphorus atom depending on the target compound, providing the optimal derivatization reagent for the target compound.

[0024] Peptides and proteins have multiple reactive sites, including not only the N-terminus but also the amino group on the side chain of lysine residues. Therefore, derivatization reactions via amino groups can produce multiple types of reaction products, which can adversely affect the sensitivity of quantitative analysis. The present invention also makes it possible to selectively derivatize peptides or proteins (e.g., peptide hormones such as oxytocin and vasopressin) that have a cysteine ​​residue at their N-terminus, for example, by native chemical ligation. The present invention can prevent the production of multiple types of reaction products and also suppress the derivatization of unintended compounds. This reduces background noise in mass spectrometry and improves detection sensitivity.

[0025] The phosphonium compounds according to the present invention can introduce charge and appropriate hydrophobicity into target compounds, thereby enabling mass spectrometry of the target compounds and optimizing the detection sensitivity of quantitative analysis. Furthermore, 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 for the quantification of trace metabolites by mass spectrometry to become widespread in the field of clinical testing, it is desirable that the same values ​​be obtained anytime, anywhere, and with any manufacturer's equipment. The improved detection sensitivity of the present invention can bridge the differences between models from different manufacturers, thereby supporting the widespread use of quantification of trace metabolites by mass spectrometry.

[0026] The present invention will now be described in more detail.

[0027] 2. First embodiment (phosphonium compound)

[0028] The present invention relates to a compound represented by the following formula (I): [ka] The present invention provides a phosphonium compound represented by the formula (I): Each component of formula (I) is described below.

[0029] In formula (I), R 1 , R 2 , and R 3 may each independently represent an alkyl group or an aryl group. The alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms or a cyclic alkyl group having 5 to 20 carbon atoms. The aryl group is a substituted or unsubstituted aryl group having 6 to 20 carbon atoms. R 1 , R 2 , and R 3 By this, hydrophobicity can be imparted to the target compound to be derivatized. 1 , R 2 , and R 3The type of may be appropriately selected by those skilled in the art depending on the degree of hydrophobicity to be imparted.

[0030] The alkyl group may be a substituted or unsubstituted, straight-chain or branched-chain alkyl group having 1 to 20 carbon atoms. The number of carbon atoms in the straight-chain or branched-chain alkyl group is preferably 1 to 18, and may be, for example, 1 to 16, 1 to 14, 1 to 12, or 1 to 10. This number of carbon atoms does not include the number of carbon atoms of substituents.

[0031] In one embodiment, the linear or branched alkyl group may be an unsubstituted alkyl group, ie, an alkyl group having only hydrogen atoms bonded to the carbon chain. In another embodiment, the linear or branched alkyl group may be a substituted alkyl group, i.e., one or more substituents may be attached to the carbon chain in addition to hydrogen. Examples of such substituents include, but are not limited to, alkoxy groups, aryl groups, hydroxy groups, amino groups, and halogen atoms. The positions and numbers of such substituents may be appropriately selected by those skilled in the art depending on various factors, such as the hydrophobicity to be introduced. Furthermore, when two or more substituents are present, they may be the same or different.

[0032] The alkyl group may be a substituted or unsubstituted cyclic alkyl group having 5 to 20 carbon atoms. The number of carbon atoms in the cyclic alkyl group is preferably 5 to 18, and may be, for example, 5 to 16, 5 to 14, 5 to 12, or 5 to 10. The number of carbon atoms does not include the number of carbon atoms in the substituent.

[0033] In one embodiment, the cyclic alkyl group may be an unsubstituted cyclic alkyl group, ie, an alkyl group having only hydrogen atoms attached to the carbon chain.

[0034] In another embodiment, the cyclic alkyl group may be a substituted alkyl group, i.e., one or more substituents may be attached to the carbon chain in addition to hydrogen. Such substituents include, but are not limited to, alkoxy groups, aryl groups, hydroxy groups, amino groups, and halogen atoms. The position and number of such substituents may be appropriately selected by those skilled in the art depending on various factors, such as the hydrophobicity to be introduced. Furthermore, when two or more substituents are present, they may be the same or different.

[0035] The aryl group is a substituted or unsubstituted aryl group having 6 to 20 carbon atoms. The number of carbon atoms in the aryl group is preferably 6 to 18, and may be, for example, 6 to 16, 6 to 14, 6 to 12, or 6 to 10. This number of carbon atoms does not include the number of carbon atoms in the substituent.

[0036] In one embodiment, the aryl group may be an unsubstituted aryl group, such as a phenyl group or a naphthyl group, in particular a phenyl group.

[0037] In another embodiment, the aryl group may be a substituted aryl group. Examples of the substituents in the substituted aryl group include an amino group, COO - (carboxylate ion group), COOM (carboxylate group or carboxylate salt group), SO3 - (sulfonic acid ion group), or SO3M (sulfonic acid group or sulfonate salt group), alkyl group, alkoxy group, aryl group, hydroxy group, and halogen atom. The number of the substituents may be one or more. When the substituted aryl group has two or more substituents, they may be the same or different. With respect to the COOM and SO3M, M may be a hydrogen atom or an alkali metal atom. The alkali metal atom may be, for example, a lithium atom, a sodium atom, or a potassium atom.

[0038] In a particularly preferred embodiment of the present invention, R in formula (I)1 , R 2 , and R 3 may, independently of one another, represent any of the following: [ka] [wherein m may be any integer from 0 to 8, particularly any integer from 0 to 6; n may be any integer from 0 to 5, for example, any integer from 0 to 4; Z may be H, NH2, COO - , COOM, SO3 - (sulfonic acid ion group), or SO3M (sulfonic acid group or sulfonate salt group), where M represents a hydrogen atom, a lithium atom, a sodium atom, or a potassium atom.

[0039] In formula (I), X is a reactive group having a hydrazide group, a halide group, a pseudohalide group, or a thioester group. The reactive group allows the phosphonium compound of formula (I) to bind to a target compound. The type of the reactive group may be appropriately selected by those skilled in the art depending on the type of target compound. The reactive group may be, for example, a reactive group that reacts with a functional group having an oxygen atom (e.g., carbonyl, phenol, or hydroxy), or a reactive group that reacts with a functional group having a nitrogen atom (e.g., amine).

[0040] In one embodiment, X in formula (I) may be a reactive group having a hydrazide group. In this embodiment, the reactive group may be a group that reacts with a functional group having an oxygen atom, or may be a group that reacts with a carbonyl. In this embodiment, it is particularly preferred that the reactive group is [ka] where o may be any integer from 0 to 6, for example, o may be any integer from 0 to 4, and particularly o may be 0, 1, or 2.

[0041] In another embodiment, X in formula (I) may be a reactive group having a halide group or a pseudohalide group (e.g., a triflate group or a tosylate group), such as a sulfonyl halide group, an acetyl halide group, a sulfonyl triflate group, an acetyl triflate group, a sulfonyl tosylate group, or an acetyl tosylate group. X in formula (I) is particularly preferably a reactive group having a sulfonyl halide group, a sulfonyl triflate group, or a sulfonyl tosylate group, and more preferably a reactive group having a sulfonyl halide group or a sulfonyl triflate group. These groups are more excellent from the viewpoint of improving the sensitivity in mass spectrometry. In this embodiment, the reactive group may be a group that reacts with a functional group having an oxygen atom, and particularly may be a group that reacts with a phenol or a hydroxyl. In this embodiment, it is particularly preferred that the reactive group is [ka] where W represents a halogen atom (e.g., F, Cl, or Br) or a pseudohalogen (e.g., a triflate group (TfO) or a tosylate group). p may be an integer of 0 to 6, for example, p may be an integer of 0 to 4, particularly p may be 0, 1, or 2.

[0042] In yet another embodiment, X in formula (I) may be a reactive group having a thioester group. In this embodiment, the reactive group may be a group that reacts with a functional group having a nitrogen atom, particularly a group that reacts with an amine. In this embodiment, it is particularly preferred that the reactive group is [ka] where V is H, NH2, SO3 - (sulfonic acid ion group), SO3M, COO - , COOM, or a hydrophilic tag, and M represents a hydrogen atom, a lithium atom, a sodium atom, or a potassium atom.

[0043] In formula (I), Y - is an anion with an overall charge of −1, or Y - The anion may be, for example, a halogen ion (e.g., F - , Cl - , or Br - ) or a pseudohalogen ion (such as a triflate ion or a tosylate ion).

[0044] The phosphonium compounds having the structural features described above can impart a permanent positive charge to the target compound.

[0045] As described above, the phosphonium compound of the present invention contains a reactive group that reacts with a functional group having an oxygen atom or a nitrogen atom. When the reactive group contains a hydrazide, the target compound may be a compound having a carbonyl group, such as, but not limited to, a male hormone, particularly testosterone. When the reactive group contains a halide group or a pseudohalide group, the target compound may be a compound having a phenol group or a hydroxy group, such as, but not limited to, a female hormone, particularly estradiol. When the reactive group is a thioester, the target compound may be an amine-containing compound, such as a peptide or protein having an N-terminal cysteine ​​residue, including, but not limited to, oxytocin.

[0046] The target compound may be contained in a biological sample. The biological sample that may contain the target compound to be derivatized with the phosphonium compound of the present invention may be, for example, a body fluid or a sample derived from a body fluid, more specifically, any of serum, plasma, blood, urine, cerebrospinal fluid, and saliva. Such a biological sample may be subjected to a predetermined pretreatment according to each analytical method as necessary, and then subjected to a derivatization treatment with the phosphonium compound of the present invention, thereby enabling detection of the target compound.

[0047] By reacting the phosphonium compound of the present invention with a target compound, a positive charge is imparted to the target compound and the hydrophobicity of the target compound can be controlled (particularly, the hydrophobicity of the target compound can be increased or decreased). By imparting a positive charge to the target compound in mass spectrometry and controlling its hydrophobicity in this manner, the ionization efficiency of the target compound can be increased, which leads to improved detection sensitivity in mass spectrometry.

[0048] The reaction type between the phosphonium compound of the present invention and the target compound may be selected depending on the type of reactive group X possessed by the phosphonium compound. When the reactive group X is a hydrazide group, the reaction type may be a dehydration condensation reaction, in which case the derivatization is carried out as shown in Figure 30A. When the reactive group X is a halide group or a pseudohalide group, the reaction type may be an electrophilic substitution reaction. In this case, derivatization is performed as shown in Figure 30B. The upper reaction formula in the figure shows the derivatization reaction when the halide group is a sulfonyl halide group. The lower reaction formula in the figure shows the derivatization reaction when the halide group is an acetyl halide group. When the reactive group X is a thioester group, the reaction type may be native chemical ligation (also called NCL), in which case the derivatization is performed as shown in Figure 30C.

[0049] The present invention provides, for example, the following compounds, but is not limited to these compounds:1 , R 2 , R 3 , X, and Y may be appropriately selected depending on, for example, the type of target compound and the analytical method.

[0050] Examples of phosphonium compounds according to the invention in which X is a reactive group carrying a hydrazide group include: (2-hydrazinyl-2-oxoethyl)trimethylphosphonium bromide; (4-(hydrazinecarbonyl)benzyl)trimethylphosphonium bromide; Benzyl(2-hydrazinyl-2-oxoethyl)dimethylphosphonium bromide; (2-hydrazinyl-2-oxoethyl)triethylphosphonium bromide; Benzyl(2-hydrazinyl-2-oxoethyl)diethylphosphonium bromide; (2-hydrazinyl-2-oxoethyl)triphenylphosphonium bromide; Tributyl(4-(hydrazinecarbonyl)benzyl)phosphonium bromide; Tributyl(2-hydrazinyl-2-oxoethyl)phosphonium bromide; and (2-hydrazinyl-2-oxoethyl)diphenyl(3-sulfophenyl)phosphonium bromide Romid.

[0051] Examples of phosphonium compounds according to the invention in which X is a reactive group carrying a halide or pseudohalide group include: (4-(chlorosulfonyl)benzyl)trimethylphosphonium bromide; (4-(chlorosulfonyl)benzyl)triethylphosphonium bromide; (4-(chlorosulfonyl)benzyl)dimethylphenylphosphonium bromide; (4-(chlorosulfonyl)benzyl)diethylphenylphosphonium bromide; (4-(chlorosulfonyl)benzyl)diphenylmethylphosphonium bromide; (4-(chlorosulfonyl)benzyl)triphenylphosphonium bromide; (4-(chlorosulfonyl)benzyl)tributylphosphonium bromide; (4-(chlorosulfonyl)benzyl)tricyclohexylphosphonium bromide; (2-Bromo-2-oxoethyl)trimethylphosphonium bromide; (2-Bromo-2-oxoethyl)triethylphosphonium bromide; (2-bromo-2-oxoethyl)dimethylphenylphosphonium bromide; and (2-Bromo-2-oxoethyl)diethylphenylphosphonium bromide.

[0052] Examples of phosphonium compounds according to the invention in which X is a reactive group carrying a thioester group include: 2-(2-(4-((tributylphosphonio)acetyl)thio)phenyl)acetamido)ethane-1-sulfonate.

[0053] 3. Second embodiment (phosphonium compound)

[0054] The present invention also provides The following formula (I) [ka] [In formula (I), R 1 , R 2 , and R 3 are the same or different hydrophobic hydrocarbon groups; X is a reactive group that reacts with an oxygen-containing functional group or a nitrogen-containing functional group; and Y - is a counter anion, or Y - is absent] Also provided is a phosphonium compound represented by the formula:

[0055] The hydrophobic hydrocarbon group is a hydrocarbon group that controls the hydrophobicity of the target compound (for example, increases or decreases the hydrophobicity, particularly increases the hydrophobicity). 1 , R 2 , and R 3 The hydrophobic hydrocarbon group may be, but is not limited to, an alkyl group or an aryl group as described above. The hydrophobic hydrocarbon group may be another hydrocarbon group configured to control (e.g., increase or decrease, particularly increase) the hydrophobicity of the target compound. The other hydrocarbon group may be, for example, an alkenyl group. The alkenyl group may be linear or branched. The alkenyl group may have a substituent. The number of carbon atoms in the alkenyl group is preferably 1 to 20, more preferably 1 to 18, for example, 1 to 16, 1 to 14, 1 to 12, or 1 to 10. The number of carbon atoms does not include the number of carbon atoms in the substituent.

[0056] The reactive group that reacts with the oxygen atom-containing functional group or nitrogen atom-containing functional group is a reactive group that reacts with the functional group contained in the target compound to bond the phosphonium compound of the present invention to the target compound. The reactive group reactive with the oxygen-containing functional group may be, for example, a reactive group reactive with carbonyl, phenol, or hydroxy, including, but not limited to, the hydrazide group, halide group, or pseudohalide group described in relation to X in 2 above. The reactive group reactive with the nitrogen-containing functional group may be, for example, a reactive group reactive with an amine, such as, but not limited to, the thioester group described in relation to X in 2. above.

[0057] The counter anion may be an anion with an overall charge of −1. The anion may be, for example, a halogen ion (e.g., F - , Cl - , or Br -) or a pseudohalogen ion (e.g., triflate ion or tosylate ion). The counter anion may also be absent.

[0058] The phosphonium compound exhibits the effects described in 2 above. For example, the phosphonium compound can impart a permanent positive charge to a target compound. Furthermore, by reacting the phosphonium compound with a target compound, a positive charge is imparted to the target compound and the hydrophobicity of the target compound can be controlled (particularly, the hydrophobicity of the target compound can be increased or decreased).

[0059] 4. Third embodiment (phosphonium compound bound to target compound)

[0060] The present invention also provides a phosphonium compound obtained by bonding the phosphonium compound described in 2. or 3. above with a target compound. The phosphonium compound is a compound represented by the following formula (II): [ka]

[0061] In formula (II), R 1 , R 2 , and R 3 and Y - is as explained in the above 2. or 3. regarding formula (I), and the explanation also applies to this embodiment. 1 , R 2 , and R 3 and Y - The explanation of is omitted.

[0062] In formula (II), J represents a group generated by the reaction of a reactive group having a hydrazide group, a halide group, a pseudohalide group, or a thioester group with a compound to be derivatized. The reactive group is as explained in relation to formula (I) in 2. or 3. above, and the same explanation applies to this embodiment. The compound to be derivatized is the target compound explained in 2. above, and the same explanation applies to this embodiment. The combination of the reactive group and the compound to be derivatized is selected depending on the target compound, as explained in 2. above. A more specific example of J will be described below.

[0063] When the reactive group is a reactive group having a hydrazide group, J is [ka] Here, o may be an integer of 0 to 6, for example, an integer of 0 to 4, particularly 0, 1, or 2. Furthermore, Q1 may be a group generated by a reaction (particularly a dehydration condensation reaction) between a carbonyl group and a hydrazide group contained in the compound to be derivatized.

[0064] When the reactive group is a reactive group having a halide group or a pseudohalide group, J is [ka] Here, p may be any integer from 0 to 6, for example, p may be any integer from 0 to 4, particularly p may be 0, 1, or 2. Furthermore, Q2 is a group generated by a reaction (particularly an electrophilic substitution reaction) between a phenol group or a hydroxy group contained in the compound to be derivatized and a halide group or pseudohalide group.

[0065] When the reactive group is a reactive group having a thioester group, J is [ka] Here, Q3 is a group generated by a reaction (native chemical ligation) between an amino group and a thioester group contained in the compound to be derivatized.

[0066] 5. Fourth embodiment (derivatization reagent)

[0067] The present invention also provides a derivatization reagent comprising the phosphonium compound of formula (I) described in 2. or 3. above. The derivatization reagent may be a reagent used to derivatize the target compound described in 2. above. More specifically, the derivatization reagent may be a reagent used to impart a positive charge to the target compound, particularly a reagent used to impart a positive charge to the target compound and control the hydrophobicity of the target compound.

[0068] The derivatization reagent may contain a solvent in addition to the phosphonium compound. That is, in the derivatization reagent, the phosphonium compound may be present in a solvent. The solvent may be an organic solvent, for example, but is not limited to, an alcohol such as methanol, ethanol, or propanol. Alternatively, the solvent may be water or a mixed solvent of water with the alcohol or acetonitrile.

[0069] 6. Fifth embodiment (derivatization reagent kit)

[0070] The present invention also provides a derivatization reagent kit, which is used to produce the compound represented by formula (II) described in 4. above.

[0071] In the derivatization of the target compound using the phosphonium compound described in 2. or 3. above, R 1 , R 2 , and R 3 A phosphonium compound having the formula (I) and a reactive group for binding to a target compound is prepared, and the target compound is derivatized with the phosphonium compound. To derivatize a target compound, the target compound is first reacted with a reactive group-containing compound to obtain a conjugate, and then the conjugate is derivatized with R 1, R 2 , and R 3 In this manner, the target compound may be sequentially reacted with the reactive group-containing compound and the phosphine compound to derivatize the target compound. The present invention also provides a reagent kit used for such derivatization. In addition, for the derivatization of a target compound, a target compound, a reactive group-containing compound, and R 1 , R 2 , and R 3 The target compound may be derivatized by reacting it with a phosphine compound having the formula: In this way, the reactive group-containing compound and the phosphine compound may be bonded to the target compound in a single reaction treatment.

[0072] That is, the derivatization reagent kit of the present invention comprises: The following phosphine compounds [ka] (where R 1 , R 2 , and R 3 each independently represents an alkyl group or an aryl group, the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms, or a cyclic alkyl group having 5 to 20 carbon atoms; The aryl group is a substituted or unsubstituted aryl group having 6 to 20 carbon atoms; a reactive compound having a hydrazide group, a halide group or a pseudohalide group, or a thioester group; may include:

[0073] When a target compound is derivatized using the derivatization reagent kit of the present invention, first, a reactive compound having a hydrazide group, a halide group, a pseudohalide group, or a thioester group is reacted with the target compound to obtain a conjugate of the reactive compound and the target compound, and then, the phosphine compound is bound to the conjugate to derivatize the target compound.

[0074] R contained in the phosphine compound 1 , R 2 , and R 3 may be as explained in the above 2. regarding the compound of formula (I), and the explanation also applies to this embodiment. The reactive compound is a compound having a reactive group X and an anion Y as described in the above section 2. for the compound of formula (I). - and XY, The compound may be represented by: The reactive group X is as explained in the above 2. for the compound of formula (I), and the explanation also applies to this embodiment. Y is an atom of the anion explained in the above 2. for the compound of formula (I). In one preferred embodiment, X is a reactive group having a halide group or a pseudohalide group, or a reactive group having a thioester group, as described in the above section 2. Regarding the derivatization of a target compound using these reactive groups, the efficiency of producing the derivative may be increased by derivatizing the target compound using the kit according to this embodiment rather than by derivatizing the target compound using the phosphonium compound described in the above section 2.

[0075] 7. Sixth Embodiment (Mass Spectrometric Method)

[0076] The present invention also provides a mass spectrometry method including a derivatization step of derivatizing a compound to be analyzed by mass spectrometry using the phosphonium compound described in 2. or 3. above, the derivatization reagent described in 5. above, or the derivatization reagent kit described in 6. above. The compound to be analyzed by mass spectrometry may be the target compound described in 2 above. The derivatized compound to be analyzed by mass spectrometry may be the phosphonium compound described in 4 above. The derivatization can improve the ion efficiency of the compound to be analyzed by mass spectrometry, thereby increasing the detection sensitivity in mass spectrometry.

[0077] The mass analysis method includes, after the derivatization step, a mass analysis step including ionizing the compound to be analyzed by mass analysis. The ionization may be performed by, for example, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or matrix-assisted laser desorption ionization (MALDI) for more efficient ionization, but may also be performed by other ionization methods. By ionizing the derivatized target compound according to the present invention using these ionization methods, it is possible to measure or observe the quantification of the target compound and the distribution (imaging) of the target compound within tissues or cells more accurately and with higher resolution. The mass analysis step may be performed by, for example, a liquid chromatography tandem mass spectrometer (LC-MS / MS) or a matrix-assisted laser desorption / ionization mass spectrometer (MALDI-MS), but the device for performing the mass analysis step is not limited thereto. The specific procedures in the mass analysis step can be appropriately determined by those skilled in the art depending on, for example, the ionization method employed and the apparatus used.

[0078] 8. Seventh embodiment (method for producing phosphonium compound)

[0079] The present invention also provides a method for producing the phosphonium compound of formula (I) described in 2. or 3. above.

[0080] In the case where X in formula (I) is a reactive group having a hydrazide group, the production method includes the following steps: The following phosphine compounds [ka] the following halogenated ethyl carboxylates: [ka] to produce the following compound: [ka] a first reaction step to produce and a second reaction step of reacting the compound produced in the first reaction step with hydrazine to produce the phosphonium compound.

[0081] With respect to the phosphine compound used in the first reaction step, R 1 , R 2 , and R 3 may be as explained in 2. above. Furthermore, Y of the halogenated ethyl carboxylate used in the first reaction step may also be Y as explained in 2. above.

[0082] When X in formula (I) is a reactive group having a thioester group, the production method includes the steps of: The following phosphonium acetates [ka] The following thiols and [ka] to produce the phosphonium compound.

[0083] R of the phosphonium acetate 1 , R 2 , and R 3 may be as explained in 2. above. Furthermore, V of the thiol may be V as explained in 2. above. [Example]

[0084] 9. Working Example The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0085] [Example 1] Synthesis of a phosphonium compound containing a hydrazide group ((2-hydrazinyl-2-oxoethyl)trimethylphosphonium bromide) Ethyl bromoacetate (64 μL, 0.6 mmol) was dissolved in 2 mL of acetonitrile, and a trimethylphosphine-tetrahydrofuran solution (3 M, 0.24 mL, trimethylphosphine amount: 0.72 mmol) was added to obtain a mixture. A white precipitate formed in the mixture immediately after the addition. The mixture was stirred at room temperature for 1 hour, and then methanol was added to the mixture to dissolve the precipitate, followed by concentration on a rotary evaporator. The residue obtained by the concentration was dissolved in 2 mL of ethanol, and hydrazine monohydrate (80% purity, 59 μL, 0.96 mmol) was added thereto. The mixture was stirred at room temperature for 1 hour, and then stirred at 50°C for an additional 1.5 hours. After stirring, the mixture was concentrated on a rotary evaporator to obtain the reaction product (2-hydrazineyl-2-oxoethyl)trimethylphosphonium bromide, hereinafter also referred to as "Compound 1") (139 mg, 0.6 mmol). For Compound 1, 1 H-NMR measurement, 13 C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H-NMR (CD3OD) δ 3.56 (d, 2 J PH = 15.0 Hz, 2H), 2.07 (d, 2 J PH = 12.0 Hz, 9H) (A in Figure 1-1); 13 C-NMR (CD3OD) δ 165.9, 31.5, 9.8 (Fig. 1-1B); 31 P-NMR (CD3OD) δ 31.1 (C in Figure 1-2); ESI-MS calculated for C5H 14 N2OP + [M + ] 149.08383, found 149 (D in Figure 1-2). The structure of compound 1 is shown by reference numeral 1 in FIG.

[0086] [Example 2] Synthesis of a phosphonium compound containing a hydrazide group ((4-(hydrazinecarbonyl)benzyl)trimethylphosphonium bromide) 4-Methoxycarbonylbenzyl bromide (1 g, 4.4 mmol) was dissolved in 8 mL of acetonitrile, and trimethylphosphine-tetrahydrofuran solution (3 M 1.7 mL, trimethylphosphine amount: 5.2 mmol) was added and stirred at room temperature. After stirring for 1.5 hours, a white precipitate formed. The precipitate was filtered, and the white solid was washed with chloroform and dried to obtain (4-(methoxycarbonyl)benzyl)trimethylphosphonium bromide (1.5 g, 4.4 mmol). (4-(Methoxycarbonyl)benzyl)trimethylphosphonium bromide (115 mg, 0.37 mmol) was dissolved in 2 mL of methanol, and hydrazine monohydrate (80% purity, 27 μL, 0.45 mmol) was added. The mixture was stirred under ice-cooling for 30 minutes, and then concentrated using a rotary evaporator to obtain the reaction product ((4-(hydrazinecarbonyl)benzyl)trimethylphosphonium bromide), hereinafter also referred to as "Compound 2") (114 mg, 0.37 mmol). For compound 2, 1 H-NMR measurement, 13 C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H-NMR (CD3OD) δ 8.04 (d, J = 7.6 Hz, 2H), 7.53 (d, J = 7.6 Hz, 2H), 3.98 (d, 2 J PH = 16.8 Hz, 2H), 1.93 (d, 2 J PH = 14.8 Hz, 9H) (A in Figure 2-1); 13 C-NMR (CD3OD) δ 168.6, 136.4, 135.0, 132.2, 130.6, 53.6, 8.7 (B in Figure 2-1); 31P-NMR (CD3OD) δ 28.0 (C in Figure 2-2); ESI-MS calculated for C 11 H 18 N2OP + [M + ] 225.11513, found 225 (D in Figure 2-2). The structure of compound 2 is shown by reference numeral 2 in FIG.

[0087] [Example 3] Synthesis of a phosphonium compound containing a hydrazide group (benzyl(2-hydrazinyl-2-oxoethyl)dimethylphosphonium bromide) Dimethylphenylphosphine (103 μL, 0.72 mmol) and ethyl bromoacetate (76 μL, 0.72 mmol) were added to 1 mL of toluene, resulting in a cloudy mixture. Adding 1 mL of acetonitrile to the mixture immediately produced a white precipitate. Adding 1 mL of methanol to the mixture rapidly dissolved the precipitate. After dissolution, the mixture was stirred at room temperature for 1.5 hours and then concentrated on a rotary evaporator. The residue obtained by this concentration was dissolved in 3 mL of ethanol, and hydrazine monohydrate (80% purity, 71 μL, 1.1 mmol) was added. The mixture was stirred at room temperature for 1 hour and then concentrated on a rotary evaporator to obtain the reaction product (benzyl(2-hydrazineyl-2-oxoethyl)dimethylphosphonium bromide, hereinafter also referred to as "Compound 3") (99 mg, 0.69 mmol). For compound 3, 1 H-NMR measurement, 13 C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H‐NMR (CDCl3) δ 7.68 (br. s, 2H), 7.47 (br. s, 3H), 3.40 (br. s, 2H), 1.69 (d, 2 J PH = 13.2 Hz, 6H) (A in Figure 3-1); 13C-NMR (CDCl3) δ 170.8, 135.0, 134.0, 131.6, 129.5, 128.7, 50.3, 17.9 (B in Figure 3-1); 31 P-NMR (CDCl3) δ 35.1 (C in Figure 3-2); ESI-MS calculated for C 11 H 18 N2OP + [M + ] 225.11513, found 225 (D in Figure 3-2). The structure of compound 3 is shown in FIG.

[0088] [Example 4] Synthesis of a phosphonium compound containing a hydrazide group ((2-hydrazinyl-2-oxoethyl)triethylphosphonium bromide) Ethyl bromoacetate (64 μL, 0.6 mmol) was dissolved in 1 mL of acetonitrile, and triethylphosphine-tetrahydrofuran solution (1 M, 1 mL, triethylphosphine amount: 1 mmol) was slowly added to the resulting solution. The mixture was stirred at room temperature for 30 minutes and then concentrated on a rotary evaporator. The residue obtained by this concentration was dissolved in 2 mL of ethanol, and hydrazine monohydrate (80% purity, 59 μL, 0.96 mmol) was added and the mixture was heated to reflux for 1 hour. After heating to reflux, the mixture was concentrated on a rotary evaporator to obtain the reaction product ((2-hydrazineyl-2-oxoethyl)triethylphosphonium bromide, hereinafter also referred to as "Compound 4") (139 mg, 0.51 mmol). For compound 4, 1 H-NMR measurement, 13 C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H-NMR (CDCl3) δ 3.93 (d, 2 J PH = 14.4 Hz, 6H), 2.45 (dq, 2 J PH= 13.2 Hz, J = 7.2 Hz, 6H), 1.30 (dt, 3 J PH = 18.8 Hz, J = 7.6 Hz, 9H) (A in Figure 4-1); 13 C-NMR (CDCl3) δ 162.8, 50.6, 12.9, 5.9 (B in Figure 4-1); 31 P-NMR (CDCl3) δ 38.9 (C in Figure 4-2); ESI-MS calculated for C8H 20 N2OP + [M + ] 191.13078, found 191 (D in Figure 4-2). The structure of compound 4 is shown by reference numeral 4 in FIG.

[0089] [Example 5] Synthesis of a phosphonium compound containing a hydrazide group (benzyl(2-hydrazinyl-2-oxoethyl)diethylphosphonium bromide) Diethylphenylphosphine (105 μL, 0.6 mmol) and ethyl bromoacetate (64 μL, 0.6 mmol) were added to 2 mL of acetonitrile, and the resulting solution was stirred at room temperature for 1 hour. The solution was concentrated using a rotary evaporator, and the residue obtained by this concentration was dissolved in 2 mL of toluene. To the resulting solution, hydrazine monohydrate (80% purity, 59 μL, 1.2 mmol) was added and heated to reflux for 4.5 hours. After heating to reflux, the mixture was concentrated using a rotary evaporator to obtain the reaction product (benzyl(2-hydrazineyl-2-oxoethyl)diethylphosphonium bromide, hereinafter also referred to as "Compound 5") (195 mg, 0.6 mmol). For compound 5, 1 H-NMR measurement, 13 C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1H‐NMR (CDCl3) δ 10.16 (br. s, 1H), 7.95 (m, 2H), 7.57 (m, 3H), 6.77 (br. s, 2H), 4.23 (d, 2 J PH = 14.4 Hz, 2H), 2.79 (m, 4H), 1.16 (m, 6H) (A in Figure 5-1); 13 C-NMR (CDCl3) δ 162.5, 134.4, 132.3, 131.3, 130.3, 130.0, 128.4, 22.0, 14.4, 5.6 (B in Figure 5-1); 31 P-NMR (CDCl3) δ 33.3 (C in Figure 5-2); ESI-MS calculated for C 12 H 20 N2OP + [M + ] 239.13078, found 239 (D in Figure 5-2). The structure of compound 5 is shown in FIG.

[0090] [Example 6] Synthesis of a phosphonium compound containing a hydrazide group ((2-hydrazinyl-2-oxoethyl)triphenylphosphonium bromide) Triphenylphosphine (91 mg, 0.35 mmol) was dissolved in 2 mL of toluene to obtain a solution. Ethyl bromoacetate (41 μL, 0.38 mmol) was slowly added to the solution, and the mixture was stirred at room temperature for 30 minutes. After stirring, the mixture was heated to reflux for 1 hour, resulting in the precipitation of a white precipitate. After the precipitation, the mixture was concentrated using a rotary evaporator. The residue obtained by the concentration was dissolved in 2 mL of ethanol to obtain a solution. Hydrazine monohydrate (80% purity, 21 μL, 0.28 mmol) was added to the solution, and the mixture was heated to reflux for 1 hour. After heating to reflux, the solution was concentrated using a rotary evaporator to obtain the reaction product ((2-hydrazineyl-2-oxoethyl)triphenylphosphonium bromide, hereinafter also referred to as "Compound 6") (140 mg, 0.34 mmol). Regarding compound 6, 1 H-NMR measurement, 13C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H-NMR (CDCl3) δ 10.36 (br. s, 1H), 7.6 (m, 15H), 5.01 (d, 2 J PH = 14.0 Hz, 2H) (A in Figure 6-1); 13 C-NMR (CDCl3) δ 161.6, 135.1, 134.0, 132.9, 132.0, 130.2, 128.5, 50.6 (B in Figure 6-1); 31 P-NMR (CDCl3) δ 30.0 (C in Figure 6-2); ESI-MS calculated for C 20 H 20 N2OP + [M + ] 335.13078, found 335 (D in Figure 6-2). The structure of compound 6 is shown in FIG.

[0091] [Example 7] Synthesis of a phosphonium compound containing a hydrazide group (tributyl(4-(hydrazinecarbonyl)benzyl)phosphonium bromide) 4-Methoxycarbonylbenzyl bromide (100 mg, 0.44 mmol) was dissolved in 2 mL of acetonitrile to obtain a solution. Tributylphosphine (123 mL, 0.52 mmol) was added to the solution and stirred at room temperature for 1 hour. After stirring, the solution was concentrated using a rotary evaporator. The residue obtained by the concentration was dissolved in 3 mL of toluene-acetonitrile (2:1 (v / v)), and hydrazine monohydrate (80% purity, 102 μL, 1.7 mmol) was added. The mixture was heated under reflux for 1 hour, and then concentrated using a rotary evaporator to obtain the reaction product (tributyl(4-(hydrazinecarbonyl)benzyl)phosphonium bromide, hereinafter also referred to as "Compound 7") (206 mg, 0.44 mmol). For compound 7, 1 H-NMR measurement, 13C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H-NMR (CDCl3) δ 7.95 (d, J = 8.0 Hz, 2H), 7.56 (dd, J = 8.0, 2.4 Hz, 2H), 4.46 (d, 2 J PH = 15.6 Hz, 2H), 3.43 (br. s, 1H), 2.36 (m, 6H), 1.41 (m, 12H), 0.87 (t, J = 7.2 Hz, 9H) (A in Figure 7-1); 13 C-NMR (CDCl3) δ 166.3, 134.1, 134.0, 130.39, 130.37, 130.3, 130.2, 52.3, 27.2, 23.8, 18.7, 13.4 (B in Figure 7-1); 31 P-NMR (CDCl3) δ 32.5 (C in Figure 7-2); ESI-MS calculated for C 20 H 36 N2OP + [M + ] 351.25598, found 351 (D in Figure 7-3). The structure of compound 7 is shown in FIG.

[0092] [Example 8] Synthesis of a phosphonium compound containing a hydrazide group (tributyl(2-hydrazinyl-2-oxoethyl)phosphonium bromide) Tributylphosphine (118 μL, 0.5 mmol) was dissolved in 2 mL of toluene, and ethyl bromoacetate (55 μL, 0.5 mmol) was slowly added to the resulting solution. After the addition, the solution was heated under reflux for 1 hour and then concentrated using a rotary evaporator. The residue obtained by the concentration was purified by silica gel column chromatography (column size: diameter 1.5 cm x length 5 cm, 10% (v / v) methanol-chloroform) to give tributyl(2-ethoxy-2-oxoethyl)phosphonium bromide (171 mg, 0.46 mmol). Tributyl(2-ethoxy-2-oxoethyl)phosphonium bromide (151 mg, 0.41 mmol) was dissolved in 2 mL of ethanol, and the resulting solution was stirred under ice-cooling. Hydrazine monohydrate (80% purity, 20 μL, 0.33 mmol) was added to the solution, and the mixture was stirred under ice-cooling for 30 minutes, followed by an additional 40 minutes of stirring at room temperature. Hydrazine monohydrate (80% purity, 20 μL, 0.33 mmol) was then added to the solution, and the mixture was stirred at room temperature for 1 hour, followed by heating to reflux for 15 minutes. After heating to reflux, the solution was cooled to room temperature, and then additional hydrazine monohydrate (80% purity, 20 μL, 0.33 mmol) was added and the mixture was stirred at room temperature for 1 hour. Subsequently, additional hydrazine monohydrate (80% purity, 20 μL, 0.33 mmol) was added to the solution, and the mixture was heated and stirred at 50°C overnight, followed by concentration on a rotary evaporator. The residue obtained by the concentration was purified by silica gel column chromatography (column size: diameter 1.5 cm × length 5 cm, 10% (v / v) methanol-chloroform) to obtain the reaction product (tributyl(2-hydrazineyl-2-oxoethyl)phosphonium bromide, also referred to as "compound 8") (157 mg, 0.41 mmol). For compound 8, 1 H-NMR measurement, 13 C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H‐NMR (CDCl3) δ 11.0 (br. s, 1H), 4.11 (d, 2 J PH = 14.0 Hz, 2H), 2.30 (m, 8H), 1.45 (m, 12H), 0.88 (m, 9H) (A in Figure 8-1); 13 C-NMR (CDCl3) δ 159.2, 50.1, 27.1, 23.7, 19.4, 13.4 (B in Figure 8-1); 31 P-NMR (CDCl3) δ 33.7 (C in Figure 8-2); ESI-MS calculated for C 14 H 32 N2OP+ [M + ] 275.22468, found 275 (D in Figure 8-2). The structure of compound 8 is shown in FIG.

[0093] [Example 9] Synthesis of a phosphonium compound containing a hydrazide group ((2-hydrazinyl-2-oxoethyl)diphenyl(3-sulfophenyl)phosphonium bromide) Ethyl bromoacetate (29 μL, 0.27 mmol) was dissolved in 1 mL of toluene, and sodium diphenylphosphinobenzene-3-sulfonate (100 mg, 0.27 mmol) was added, followed by 1 mL of acetonitrile to obtain a suspension. 1 mL of methanol was added to the suspension to completely dissolve the mixture, and the resulting solution was heated under reflux for 1 hour. After heating under reflux, the solution was concentrated using a rotary evaporator, and the residue obtained by the concentration was dissolved in 3 mL of ethanol to obtain a solution. Hydrazine monohydrate (80%, 27 μL, 0.44 mmol) was added to the solution, and the mixture was heated under reflux for 2 hours. After the heating under reflux, hydrazine monohydrate (purity 80%, 27 μL, 0.44 mmol) was further added to the solution, and the mixture was heated under reflux for 2 hours. The mixture was then concentrated using a rotary evaporator to obtain a reaction product ((2-hydrazineyl-2-oxoethyl)diphenyl(3-sulfophenyl)phosphonium bromide, hereinafter also referred to as "Compound 9") (122 mg, 0.25 mmol). For compound 9, 1 H-NMR measurement, 13 C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H-NMR (CD3OD) δ 8.18 (ddd, J = 12.0, 1.2, 1.2 Hz, 2H), 8.10 (ddd, J = 12, 1.2, 1.2 Hz, 2H), 7.64 (m, 8H), 7.55 (m, 4H) (A in Figure 9-1); 13C-NMR (CD3OD) δ 173.3, 147.6, 135.4, 134.7, 133.7, 133.6, 130.9, 130.7, 120.3, 119.5, 21.4 (B in Figure 9-1); 31 P-NMR (CD3OD) δ 32.1 (C in Figure 9-2); ESI-MS calculated for C 20 H 20 N2O4PS + [M + ] 415.08759, found 415 (D in Figure 9-2). The structure of compound 9 is shown in FIG.

[0094] [Example 10] Derivatization of testosterone with phosphonium compounds bearing a hydrazide group. A 10 μL sample (testosterone standard, pooled serum extract, or acetonitrile (for background verification)) was placed in a 2 mL microtube, and 10 μL of a methanol solution (1 mg / mL) of a phosphonium compound and 50 μL of a 10% (v / v) acetic acid solution in methanol were added to the microtube. The tube was then heated at 50°C for 45 minutes in a heat block. This resulted in testosterone being derivatized with the phosphonium compound. The above derivatization treatment was performed on each of Compounds 1 to 9 obtained in Examples 1 to 9. The testosterone derivatives obtained by the derivatization treatment with Compounds 1 to 9 are hereinafter also referred to as Derivatives D1 to D9, respectively. The reaction formula for the derivatization of testosterone with a phosphonium compound having a hydrazide group is shown in Figure 11. As shown in the figure, testosterone is derivatized with the phosphonium compound of formula (I). The chemical structures of derivatives D1 to D9 are shown in Figure 12. In Figure 12, the upper row shows the testosterone portion of the derivative, and the lower row shows the portion bound to testosterone by derivatization. Using chiral reagents T and P as the comparison substances (the structures of these reagents are shown in Fig. 13), the same derivatization treatment was carried out. The testosterone derivatives obtained by the derivatization treatment with chiral reagents T and P are hereinafter also referred to as derivatives DT and DP, respectively. The structures of derivatives DT and DP are also shown in Fig. 12. The solution after heating by the heat block was centrifugally concentrated, and the product obtained by the centrifugal concentration was redissolved in 100 μL of an aqueous acetonitrile solution of 50% (v / v). Using 10 μL of the solution obtained by this redissolution, LC-MS / MS analysis was performed as described in Example 11 below.

[0095] [Example 11] LC-MS / MS Analysis of Testosterone Derivatives Under the following LC analysis conditions and MS / MS analysis conditions, LC-MS / MS analysis was performed on the testosterone derivatives (derivatives D1 to D9 and derivatives DT and DP) obtained by the derivatization treatment described in Example 10. Also, LC-MS / MS analysis was performed on testosterone that had not been derivatized in the same manner.

[0096] <LC Analysis Conditions> Apparatus High Performance Liquid Chromatograph LC-20A (Shimadzu Corporation) Analysis Column YMC Triart C18 (Column size: inner diameter 2.1 mm x length 100 mm, WMC Co., Ltd.) Elution Conditions Flow rate 0.3 mL / min Solvent A: 0.1% (v / v) formic acid-water B: methanol

[0097] [Table 1]

[0098] <MS / MS Analysis Conditions> Apparatus Liquid Chromatograph Mass Spectrometer (LCMS8040 triple quadrupole mass spectrometer, Shimadzu Corporation) Ionization Conditions ESI positive ion mode SRM parameters (as shown in Table 2 below) The SRM chromatograms (testosterone 100 pg) in the analysis of the derivatives D1 to D9 and the derivatives DT and DP are shown in Figures 14-1 to 14-6.

[0099] [Table 2]

[0100] <Analysis results> Figure 15-1 shows the analytical results for the background, testosterone standard (10 pg testosterone), and pooled serum extract. In this figure, the vertical axis represents the peak area value. The measured peak area values ​​are also shown in Table 3 below.

[0101] [Table 3]

[0102] The analytical results showed that the peak areas of the testosterone standard and pooled serum extract for all of compounds 1 to 9 were much higher than the peak areas of the background. This indicates that testosterone can be appropriately detected by derivatization using compounds 1 to 9. Furthermore, the above analytical results show that when any of compounds 1 to 9 was used, testosterone derivatives were detected with sensitivity comparable to or higher than that of the conventionally used reagents T and P. Furthermore, without derivatization, the peak area value for 10 pg of testosterone was close to the peak area value for the background. Therefore, it can be seen that without derivatization, testosterone cannot be properly detected. Based on this result, it can be seen that when derivatized according to the present invention, testosterone can be detected with higher sensitivity than when not derivatized.

[0103] In the above analysis, a sample containing 10 pg of testosterone was analyzed. A similar analysis was performed for samples containing smaller amounts of testosterone. The analysis results are shown in Figure 15-2. The measured peak area values ​​are shown in Table 4 below. As shown in the figure, the testosterone amounts of the samples analyzed were 100 fg and 1 pg. As shown by these results, all of the compounds according to the present invention can detect 1 pg of testosterone with high sensitivity. Furthermore, when the amount of testosterone is 100 fg, if it is not derivatized, the difference from the background is almost zero and measurement is impossible, whereas compounds 1, 3, and 4 according to the present invention have a sufficient difference from the background, that is, these compounds can detect 100 fg of testosterone with high sensitivity. Furthermore, for Girard's reagent P and the non-derivatized case, the peak area values ​​at 100 fg of testosterone were comparable to the background area values, indicating that quantification was impossible in these cases. On the other hand, for the compound according to the present invention, the peak area values ​​at 100 fg of testosterone were higher than the background area values. Furthermore, a comparison of Reagent T and Compound 4 when the amount of testosterone was 100 fg reveals that the present invention can improve sensitivity by about two times compared to when the conventional Girard's Reagent T was used.

[0104] [Table 4]

[0105] [Example 12] Derivatization of estradiol (11) with 4-bromomethylbenzenesulfonyl chloride and trimethylphosphine (synthesis of estradiol-3-(4-trimethylphosphoniummethyl)benzenesulfonate) Estradiol (100 mg, 0.37 mmol) and 4-bromomethylbenzenesulfonyl chloride (99 mg, 0.37 mmol) were placed in a 30 mL recovery flask, and 3 mL of acetone was added to dissolve the mixture. The mixture was then stirred at room temperature. Sodium bicarbonate (46 mg, 0.55 mmol) was dissolved in 1 mL of water to obtain an aqueous solution. This aqueous solution was added dropwise to the reaction mixture in the recovery flask and stirred at 60°C for 24 hours. After stirring, the reaction mixture was cooled to room temperature, diluted with 4 mL of ethyl acetate, washed with 2 mL of saturated brine, and concentrated on a rotary evaporator. The residue obtained by this concentration was purified by silica gel column chromatography (column size: 1.5 cm x 10 cm, 50% (v / v) ethyl acetate-hexane) and concentrated on a rotary evaporator. After concentration, the mixture was dried under reduced pressure to obtain estradiol-3-(4-bromomethyl)benzenesulfonate (hereinafter also referred to as "reaction intermediate 1") (79 mg, 0.16 mmol). The structures of estradiol and reaction intermediate 1 are shown in the left and center of Figure 16, respectively. Regarding reaction intermediate 1, 1 H-NMR measurement, 13 C-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H-NMR (CDCl3) δ 7.81 (d, J = 8.8 Hz, 2H), 7.5 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 1H), 6.69 (s, 1H), 6.68 (d, J = 8.8 Hz, 1H), 4.60 (s, 2H), 4.47 (s, 1H), 3.69 (t, J = 8.0 Hz, 1H), 2.73 (m, 2H), 2.24 (m, 1H), 2.14 (m, 1H), 2.07 (m, 1H), 1.91 (dt, J = 12.1, 3.4 Hz, 1H), 1.83 (m, 1H), 1.65 (m, 1H), 1.34 (m, 8H), 0.74 (s, 3H) (Figure 17A); 13C-NMR (CDCl3) δ 147.1, 143.7, 139.5, 138.7, 135.4, 129.5, 129.0, 128.9, 128.8, 126.5, 122.1, 118.9, 81.6, 49.9, 44.6, 44.0, 43.1, 38.2, 36.5, 30.4, 29.3, 26.8, 26.0, 23.0, 11.0 (Figure 17B).

[0106] Reaction intermediate 1 was placed in a 30 mL recovery flask, and 2 mL of acetone was added to dissolve the intermediate. The mixture was stirred at room temperature. A trimethylphosphine-tetrahydrofuran solution (1 mL of 3 M solution, trimethylphosphine amount: 3 mmol) was added dropwise to the solution in the recovery flask. The mixture was stirred at room temperature for 5 minutes and then heated to 60°C in an oil bath for 5 minutes while stirring. This resulted in the formation of a white precipitate. The solution containing the precipitate was concentrated using a rotary evaporator and dried under vacuum to obtain the reaction product (estradiol-3-(4-trimethylphosphoniummethyl)benzenesulfonate, hereinafter also referred to as "derivative D11") (90 mg, 0.16 mmol). The structure of derivative D11 is shown on the right side of Figure 17. Regarding derivative D11, 1 H-NMR measurement, 13 C-NMR measurement, 31 P-NMR measurement and mass spectrometry were carried out, and the results of these measurements were as follows: 1 H‐NMR (CD3OD) δ 7.87 (d, J = 8.0 Hz, 2H), 7.63 (dd, J = 8.0 Hz, 4 J PH = 1.4 Hz, 2H), 7.18 (d, J = 9.2 Hz, 1H), 6.69 (d, J = 9.2 Hz, 1H), 6.68 (s, 1H), 3.97 (d, 2 J PH= 16.8 Hz, 2H), 3.69 (t, J = 8.8 Hz, 1H), 2.70 (m, 2H), 2.24 (br. d, J = 13.2 Hz, 1H), 2.11 (m, 1H), 2.00 (m, 1H), 1.92 (m, 1H), 1.91 (d, 2 J PH = 16.8 Hz, 6H), 1.83 (m, 1H), 1.65 (m, 1H), 1.55 (d, 2 J PH = 16.8 Hz, 3H), 1.31 (m, 8H) (A in Figure 18-1); 13 C-NMR (CD3OD) δ 149.4, 141.8, 140.8, 138.3, 137.5, 133.0, 131.4, 131.3, 130.8, 128.6, 124.0, 121.0, 83.1, 52.0, 46.2, 45.1, 40.6, 38.6, 31.5, 28.8, 28.1, 24.8, 18.3, 17.6, 12.5, 8.60 (B in Figure 18-1); 31 P-NMR (CD3OD) δ 28.5 (C in Figure 18-2); ESI-MS calculated for C 28 H 38 O4PS + [M + ] 501.22229, found 501 (D in Figure 18-2).

[0107] [Example 13] Derivatization of estradiol with 4-(bromomethyl)benzenesulfonyl chloride and various phosphines A 10 μL sample (estradiol standard or acetonitrile (for background verification)) was placed in a 2 mL microtube, 50 μL of 4-(bromomethyl)benzenesulfonyl chloride-acetone solution (1 mg / mL) and 50 μL of sodium bicarbonate aqueous solution (100 mM) were added, and the mixture was heated at 60°C for 5 minutes in a heat block. After heating, 50 μL of a 1% (v / v) phosphine-acetonitrile solution was added to the sample in the microtube, and the mixture was further heated at 60°C for 2 minutes in a heat block. This produced estradiol derivatives. In addition to the trimethylphosphine used in Example 12, seven other phosphines were also used. This produced eight estradiol derivatives (also referred to as derivatives D11 to D18). The structures of the derivatives produced are shown in Figure 19. The upper part of the figure shows the structure common to these derivatives, while the lower part shows the structure resulting from the phosphine used. After heating, the mixture was centrifuged and concentrated, and the product obtained by centrifugal concentration was redissolved in 100 μL of 50% (v / v) acetonitrile aqueous solution. 10 μL of the solution obtained by redissolution was used for LC-MS / MS analysis.

[0108] [Comparative Example 14] Derivatization of estradiol with dansyl chloride. 10 μL of sample (estradiol standard or acetonitrile (for background verification)) was placed in a 2 mL microtube, 50 μL of dansyl chloride-acetone solution (1 mg / mL) and 50 μL of aqueous sodium bicarbonate solution (100 mM) were added, and the mixture was heated in a heat block at 60°C for 5 minutes. After heating, the mixture was centrifuged to concentrate, and the product obtained by centrifugal concentration was redissolved in 100 μL of 50% (v / v) acetonitrile solution in water. 10 μL of the solution obtained by redissolution was analyzed by LC-MS / MS (the structure of the derivative produced is shown in Figure 20. This derivative is also referred to as derivative DD). The dansyl form was the substance to be compared.

[0109] [Example 15] Derivatization of Estradiol with Bromoacetyl Bromide and Various Phosphines Place 10 μL of the sample (estradiol standard or acetonitrile (for background verification)) in a 2 mL microtube, add 50 μL of bromoacetyl bromide - acetone solution (1 mg / mL) and 50 μL of triethylamine - acetonitrile solution (100 mM), and heat in a heat block at 60 °C for 5 minutes. After this heating, add 50 μL of 1% (v / v) phosphine - acetonitrile solution to the sample in the microtube and further heat in a heat block at 60 °C for 2 minutes. As the phosphine, not only trimethylphosphine but also three other kinds of phosphines were used. As a result, four kinds of estradiol derivatives (also referred to as derivatives D21 - D24 respectively) were generated. The structures of the generated derivatives are shown in Figure 21 respectively. On the left of the figure, the structure common to these derivatives is shown. On the right of the figure, the structure resulting from the phosphine used is shown. Perform centrifugal concentration after heating with the heat block, and redissolve the product obtained by this centrifugal concentration in 100 μL of 50% (v / v) aqueous acetonitrile solution. Perform LC - MS / MS analysis using 10 μL of the solution obtained by this redissolution.

[0110] [Example 16] LC - MS / MS Analysis of Estradiol Derivatives Under the following LC analysis conditions and MS / MS analysis conditions, the LC - MS / MS analyses described in Example 13, Comparative Example 14, and Example 15 were performed.

[0111] <LC Analysis Conditions> Apparatus High - performance liquid chromatograph LC - 20A (Shimadzu Corporation) Analysis column YMC Triart C18 (column size: inner diameter 2.1 mm x length 100 mm, WMC Co., Ltd.) Elution conditions Flow rate 0.3 mL / min Solvent A: 0.1% (v / v) formic acid - water B: methanol

[0112]

Table 5

[0113] <MS / MS Analysis Conditions> Apparatus Liquid chromatograph mass spectrometer (LCMS8040 triple quadrupole mass spectrometer, Shimadzu Corporation) Ionization conditions ESI positive ion mode SRM parameters (as shown in Table 6 below)

[0114]

Table 6

[0115] Note that the SRM chromatograms of each derivative (for derivatives 11 - 18 and derivative DD, estradiol 10 pg; for derivatives 21 - 24, estradiol 10 ng) are shown in Figures 22 - 1 to 22 - 7.

[0116] <Analysis Results> Figure 23 shows the analysis results regarding derivatives 11 - 18 and derivative DD, and the background and testosterone standard (testosterone 10 pg). In the figure, the vertical axis indicates the peak area. Also, the measured peak area values are shown in Table 7 below.

[0117]

Table 7

[0118] From the above analysis results, for any of derivatives 11 - 18 according to the present invention, the peak area in the case of the estradiol standard was much higher than the peak area in the case of the background. Therefore, it can be seen that estradiol can be appropriately detected by derivatizing estradiol according to the present invention. Furthermore, the above analytical results show that when any of the derivatives 11 to 18 was used, the estradiol derivative was detected with a sensitivity comparable to or higher than that of the conventionally used derivative DD (dansyl derivative).

[0119] In the above analysis, a sample containing 10 pg of estradiol was analyzed. Similar analyses were also performed using samples containing smaller amounts of estradiol. The analysis results are shown in Figure 24. The measured peak area values ​​are shown in Table 8 below. As shown in the figure, the estradiol amounts of the samples analyzed were 10 fg and 100 fg.

[0120] [Table 8]

[0121] The results shown in the figure show that when the amount of estradiol was 100 fg, the peak area values ​​for all derivatives were much higher than the background. Therefore, it can be seen that estradiol can be detected even at 100 fg, which is even lower than 10 pg. Furthermore, the analytical results show that for derivative DD (comparative example), the peak area value when the estradiol amount is 10 fg is slightly higher than the area value in the background. On the other hand, for the other derivatives according to the present invention, the peak area value when the estradiol amount is 10 fg is significantly higher than the area value in the background. Therefore, it can be seen that the present invention can improve sensitivity by about 10 times compared to when the conventional reagent dansyl chloride is used.

[0122] Figure 25 shows the analytical results of the background and testosterone standard (10 ng of testosterone) for derivatives D21 to D24. In this figure, the vertical axis represents the peak area. Note that because the background peak area is small, the bars for these peaks are almost invisible in the figure.

[0123] The above analysis results show that for all of the derivatives D21 to D24 according to the present invention, the peak area in the case of the estradiol standard was much higher than the peak area in the case of the background. Therefore, it is clear that estradiol can be appropriately detected by derivatizing estradiol according to the present invention.

[0124] [Example 17] Synthesis of a phosphonium compound bearing a thioester group (2-(2-(4-((tributylphosphonio)acetyl)thio)phenyl)acetamido)ethane-1-sulfonate) Trityl chloride (0.83 g, 3.0 mmol) was dissolved in 3 mL of chloroform, and 4-mercaptophenylacetic acid (500 mg, 3.0 mmol) was added to the resulting solution and stirred for 1 hour. The solution was then neutralized with aqueous sodium hydroxide (1 M, 4.5 mL). After neutralization, ethyl acetate was added. After the addition, the mixture was washed with saturated brine, and the organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain an oily residue. The resulting residue was purified on silica gel to obtain 4-(tritylthio)phenylacetic acid (hereinafter also referred to as the "trityl form") (0.81 g, 2.0 mmol). Taurine (0.46 g, 3.7 mmol) and triethylamine (0.51 mL, 3.7 mmol) were dissolved in 15 mL of water, and 24 mL of a solution of the trityl derivative (0.76 g, 1.9 mmol) and 1-hydroxybenzotriazole (0.50 g, 3.7 mmol) in N-methylpyrrolidone was added. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.39 g, 2.0 mmol) was then added and stirred for 2 days. The reaction mixture was purified on silica gel to give 2-(2-(4-(tritylthio)phenyl)acetamido)ethane-1-sulfonic acid (hereinafter also referred to as "taurine conjugate") (1.2 g, 2.3 mmol). The taurine conjugate (0.96 g, 1.9 mmol) and triethylsilane (0.32 mL, 2.0 mmol) were dissolved in 14 mL of chloroform, 4.1 mL of trifluoroacetic acid was added, and the mixture was stirred for 30 minutes. The reaction mixture was concentrated to give an oily residue, 2-(2-(4-mercaptophenyl)acetamido)ethane-1-sulfonic acid (hereinafter also referred to as "thiol") (1.7 g, 1.9 mmol). Tributylphosphine (0.52 mL, 2.1 mmol) and tert-butyl bromoacetate (0.29 mL, 2.0 mmol) were dissolved in 10 mL of toluene and stirred for 3 hours. The reaction mixture was diluted with toluene, washed with water, and then concentrated under reduced pressure. The resulting residue was dissolved in 8.8 mL of 90% (v / v) aqueous trifluoroacetic acid and stirred for 1.5 hours. The reaction mixture was concentrated under reduced pressure to give an oily residue, tributylphosphonium acetate (hereinafter also referred to as "carboxylic acid") (0.89 g, 2.0 mmol). The thiol (0.70 g, 2.5 mmol) and the carboxylic acid (0.19 g, 0.56 mmol) were dissolved in 2 mL of N,N'-dimethylformamide. To the resulting solution, diisopropylethylamine (0.14 mL, 0.80 mmol) and 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (0.42 g, 0.80 mmol) were added. The pH was adjusted to approximately 7 with N,N'-diisopropylethylamine and then stirred for 2 hours. The reaction mixture was diluted with 0.1% (v / v) aqueous trifluoroacetic acid and purified by reverse-phase HPLC. The fractions containing the target product were lyophilized to obtain the thioester (2-(2-(4-((tributylphosphonio)acetyl)thio)phenyl)acetamido)ethane-1-sulfonate, hereafter referred to as "Compound 31") as a powder (80 mg, 0.15 mmol). For compound 31, 1 H-NMR measurement and mass spectrometry were carried out, and the results were as follows: 1 H-NMR (CD3OD) (Figure 26-1); ESI-MS calculated for C 24 H 41NO5PS2 + [M +H + ] 518.21583, found 518 (Figure 26-2).

[0125] [Example 18] Derivatization of oxytocin using the NCL method Put 100 μL of 100 mM phosphate buffer (pH 8.0) into a 1.5 mL AMR Proteo Save tube (TPX). 10 μL of an aqueous solution of acetic acid and acetonitrile (acetic acid concentration 1% (v / v) and acetic acid and acetonitrile concentration 10% (v / v)) containing oxytocin at a concentration of 100 ng / mL was added thereto. 5 μL of freshly prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (100 mg / mL) and 5 μL of 50% acetonitrile aqueous solution containing compound 31 at a concentration of 0.2 mg / mL were added to the tube, stirred, and allowed to react for 15 minutes at room temperature. After the reaction, 5 μL of MES aqueous solution was added, stirred, and allowed to react for 15 minutes at room temperature for derivatization. The derivatization reaction scheme is shown in Figure 27.

[0126] [Example 19] Post-treatment and LC-MS / MS analysis of oxytocin derivatives To remove the large amount of salts that are present during derivatization and enable the concentration of the derivatives, Empore TM A disk-shaped solid phase of 2215-C18 Octadecyl (CDS Analytical) was used. TMPacked to the tip of -H Tip (Shimadzu Corporation) and created and used a self-made microcolumn. After passing 50 μL of acetonitrile through the microcolumn, 50 μL of an aqueous solution of acetic acid and acetonitrile (acetic acid concentration 1% (v / v) and acetonitrile concentration 10% (v / v)) was passed through for equilibration, and then the entire amount of the derivatized sample in Example 18 was loaded. 50 μL of the aqueous solution of acetic acid and acetonitrile was passed through for washing, and eluted with 120 μL of an aqueous solution of acetic acid and acetonitrile (acetic acid concentration 0.1% (v / v) and acetonitrile concentration 50% (v / v)), and the eluate was used as a sample for LC-MS / MS analysis. The conditions for LC-MS / MS analysis were as follows.

[0127] <LC analysis conditions> Instrument Agilent 1290 Infinity Analysis column Waters ACQUITY UPLC Peptide BEH C18 2.1mm x 50 mm Elution conditions Flow rate 0.5 mL / min Solvent A: 0.05% (v / v) acetic acid - water B: 0.05% (v / v) acetic acid - acetonitrile The elution conditions were as shown in Table 9 below.

[0128]

Table 9

[0129] <MS / MS analysis conditions><--- Instrument SCIEX QTRAP 4500 Ionization conditions ESI positive ion mode The SRM parameters were as shown in Table 10 below.

[0130]

Table 10

[0131] <Analysis results> The analytical results are shown in Figures 28 and 29. In Figure 29, the vertical axis represents the peak area. The analytical results show that when oxytocin was derivatized according to the present invention, the detection intensity was approximately eight times higher than when it was not derivatized. This demonstrates that the present invention can improve the detection sensitivity of oxytocin.

[0132] 1 illustrates another aspect of the present technology. ·〔1〕 The following formula (I) [ka] [In formula (I), R 1 、R 2 , and R 3 each independently represents an alkyl group or an aryl group, the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms, or a cyclic alkyl group having 5 to 20 carbon atoms; The aryl group is a substituted or unsubstituted aryl group having 6 to 20 carbon atoms; X is a reactive group having a hydrazide group, a halide group or a pseudohalide group, or a thioester group; and Y - is an anion with an overall charge of −1, or Y - is absent] A phosphonium compound represented by the formula: ·〔2〕 In formula (I), the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 10 carbon atoms, or a cyclic alkyl group having 5 to 10 carbon atoms; The aryl group is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms. The phosphonium compound according to [1] above. ·〔3〕 In formula (I), R 1 、R 2 , and R 3 represent, independently of one another, one of the following: [ka] [wherein m is an integer of 0 to 8, n is an integer of 0 to 5, Z is H, NH 2 , COO- , COOM, SO 3 - (sulfonic acid ion group), or SO 3 M (sulfonic acid group or sulfonate group), and M represents a hydrogen atom, a lithium atom, a sodium atom, or a potassium atom. The phosphonium compound according to [1] above. ·〔4〕 In formula (I), When X is a reactive group having a hydrazide group, The reactive group is

change

change

change

change

change

change

change

change

change

change

change

change

change

change

Claims

1. The following formula (I) 【Chemical 1】 [In formula (I), R １ represents an alkyl group, and the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 20 carbon atoms or a cyclic alkyl group having 5 to 20 carbon atoms; R ２ , and R ３ each independently represents an alkyl group or an aryl group, the alkyl group being a substituted or unsubstituted linear or branched alkyl group having 1 to 20 carbon atoms or a cyclic alkyl group having 5 to 20 carbon atoms, and the aryl group being a substituted or unsubstituted aryl group having 6 to 20 carbon atoms; X is a reactive group having a thioester group, and the reactive group is 【Chemistry 2】 wherein V is H, NH 2 , SO 3 − (sulfonic acid ion group), SO 3 M, COO − , COOM, or CH 2 CONHCH 2 CH 2 SO 3 − , and M represents a hydrogen atom, a lithium atom, a sodium atom, or a potassium atom; and Y － is an anion with an overall charge of −1, or Y － is absent] A phosphonium compound represented by the formula:

2. In formula (I), the alkyl group is a substituted or unsubstituted, linear or branched alkyl group having 1 to 10 carbon atoms, or a cyclic alkyl group having 5 to 10 carbon atoms; The aryl group is a substituted or unsubstituted aryl group having 6 to 10 carbon atoms. The phosphonium compound according to claim 1.

3. In formula (I), R １ represents either (a) or (b) below: R ２ , and R ３ represent, independently of one another, any of the following (a), (b), or (c): 【Chemistry 3】 [wherein m is an integer of 0 to 8, n is an integer of 0 to 5, and Z is H, NH ２ , COO － , COOM, SO ３ － (sulfonic acid ion group), or SO ３ M (sulfonic acid group or sulfonate salt group), and M represents a hydrogen atom, a lithium atom, a sodium atom, or a potassium atom. The phosphonium compound according to claim 1.

4. A derivatization reagent comprising the phosphonium compound according to any one of claims 1 to 3.

5. A mass spectrometry method, comprising derivatizing a compound to be subjected to mass spectrometry using the phosphonium compound according to any one of claims 1 to 3 and the derivatization reagent according to claim 4.

Images