WO2009128348A1

WO2009128348A1 - Phenylphosphorylcholine derivatives

Info

Publication number: WO2009128348A1
Application number: PCT/JP2009/056874
Authority: WO
Inventors: 睦廣伊逹; 諭狭場; 祐介中新井; 友和板井; 和仁谷本
Original assignee: 和光純薬工業株式会社
Priority date: 2008-04-14
Filing date: 2009-04-02
Publication date: 2009-10-22
Also published as: JP5614281B2; JPWO2009128348A1

Abstract

Phenylphosphorylcholine derivatives represented by general formula [1]; a method for calcium ion determination, characterized by mixing a derivative described above with phospholipase D in the presence of phosphomonoesterase to initiate a reaction, measuring the absorbance increase rate, and determining the quantity of calcium ion on the basis of the absorbance increase rate; reagents and kits for calcium ion determination, which contain the derivatives; and reagents for the determination of phospholipase D: [1] wherein R₁to R₄ are each independently hydrogen, halogeno, halogenated alkyl, or carboxy, with the proviso that at least one of R₁to R₄ is halogeno, halogenated alkyl, or carboxy.

Description

Phenyl phosphorylcholine derivative

The present invention relates to a novel substance serving as a substrate for phospholipase D, a method for measuring phospholipase D using the same, and a method for quantifying calcium ions.

Phospholipase D is an enzyme having a high enzyme affinity for the phosphorylcholine structure, and is generally known to require a divalent cation in the enzyme reaction. In recent years, using this property, a method for measuring the enzyme activity of phospholipase D using a commercially available paranitrophenyl phosphorylcholine as a chromogenic substrate and a method for measuring a divalent cation using phospholipase D have been studied. .

Examples of a method for measuring a divalent cation using a phosphorylcholine compound such as paranitrophenylphosphorylcholine as a substrate and phospholipase D are applied to a calcium measuring method (for example, Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5, etc.) are known. However, especially in the measurement system using paranitrophenylphosphorylcholine as a substrate, the dye pKa released by the enzymatic reaction is not in the optimum state in the measurement system, so the dye is not in a fully colored state, and the color stability However, there were problems such as poor sensitivity and insufficient sensitivity.

Japanese Patent Laid-Open No. 62-195297 Japanese Patent Laid-Open No. 4-187098 JP-A-4-23999 Japanese Unexamined Patent Publication No. 7-170999 JP 2002-238598 A

An object of the present invention is to provide a good phospholipase D substrate, and to maintain a substrate stability in the measurement of enzyme activity and divalent cation, while the released dye is high near the optimum pH of phospholipase D. An object of the present invention is to provide a chromogenic substrate that develops color, has excellent chromogenic stability, and has sufficient measurement sensitivity.

The present invention has been made for the purpose of solving the above-mentioned problems, and has the following configuration.

(1) The following formula [1]

[Wherein R ₁ to R ₄ each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R ₁ to R ₄ represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
A phenylphosphorylcholine derivative represented by:

(2) The following formula [1]

[Wherein R ₁ to R ₄ each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R ₁ to R ₄ represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
A reagent for measuring phospholipase D, comprising a phenylphosphorylcholine derivative represented by the formula:

(3) Test sample and the following formula [1]

[Wherein R ₁ to R ₄ each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R ₁ to R ₄ represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
And a phospholipase D is mixed with phospholipase D in the presence of phosphate monoesterase to start the reaction, and then the absorbance increase rate is measured, and this is performed based on the absorbance increase rate. A method for quantifying calcium ions.

(4) The following formula [1]

[Wherein R ₁ to R ₄ each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R ₁ to R ₄ represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
A reagent for quantifying calcium ions, comprising a phenylphosphorylcholine derivative represented by the formula:

(5) The following formula [1]

[Wherein R ₁ to R ₄ each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R ₁ to R ₄ represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
A kit for quantifying calcium ions, comprising a phenylphosphorylcholine derivative represented by the formula: phospholipase D and phosphate monoesterase as constituent components.

As a result of intensive studies to solve the above-described problems, the present inventors synthesized a phenylphosphorylcholine derivative of the present invention in which at least one electron-withdrawing group was introduced into paranitrophenylphosphorylcholine, and the derivative was released. It was found that the pKa of the dye is lower than that of the dye liberated from paranitrophenyl phosphorylcholine. As a result of further intensive research, it was found that the dye released from phenylphosphorischoline of the present invention produces a high color near neutral, which is the optimum pH of phospholipase D, and at a fixed measurement wavelength of 405 nm of an automatic analyzer. Thus, it was found that if the derivative was used as a substrate for phospholipase D and the calcium ion concentration was measured, the measurement could be performed with sufficient measurement sensitivity, and the present invention was completed.

The novel phenylphosphorylcholine derivative in which an electron withdrawing group is substituted on the dye skeleton of the present invention can be a good substrate of phospholipase D, and has excellent coloration stability in enzyme activity measurement and calcium ion measurement. It becomes a chromogenic substrate having measurement sensitivity.

6 is a graph showing the relationship between the substrate concentration (mM) of each substrate and the absorbance increase rate (ΔE / mim) obtained in Example 7. In FIG. 1, (a) shows the conventional paranitrophenylphosphorylcholine as a substrate for phospholipase D, (b) and (c) show the phenylphosphorylcholine derivative of the present invention as a substrate for phospholipase D, ie, compound 8a or compound 8c. The result when each is used is shown. It is the graph which showed the reaction time course at the time of measuring calcium ion concentration using each substrate obtained in Example 8, and plotted the light absorbency (ODx10000) in each photometry point (sec.). In FIG. 2, (a) is a conventional paranitrophenyl phosphorylcholine as a phospholipase D substrate, (b) to (e) are phospholipase D substrates, and the phenylphosphorylcholine derivative of the present invention, ie, compound 5b, compound 8a, The results when using Compound 8b and Compound 8c are shown. FIG. 9 is a calibration curve showing the relationship between the calcium concentration (analyte Ca amount) of the calcium solution obtained in Example 8 and the calcium concentration (measured value) of the calcium solution obtained from the calibration curve. In FIG. 3, (a) is a conventional paranitrophenyl phosphorylcholine as a substrate for phospholipase D, (b) to (e) are phenylphosphorylcholine derivatives of the present invention as substrates for phospholipase D, ie, compound 5b, compound 8a, The results when using Compound 8b and Compound 8c are shown. The graph which showed the result of having tested the temporal stability in the solution state of each substrate obtained in Example 9 and plotted the absolute absorbance at 405 nm obtained using each substrate solution after storage for each storage period. is there.

The phenylphosphorylcholine derivative of the present invention is one in which at least one electron withdrawing group is introduced into paranitrophenylphosphorylcholine, and specifically, the following formula [1]

[Wherein R ₁ to R ₄ each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R ₁ to R ₄ represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
It has the structure shown by.

In the formula [1], R ₁ to R ₄ are each independently a hydrogen atom or an electron withdrawing group, and at least one of R ₁ to R ₄ is an electron withdrawing group. Examples of the electron withdrawing group include a halogen atom, a halogenated alkyl group, and a carboxyl group, and a halogen atom and a halogenated alkyl group are preferable.

Examples of the halogen atom include fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine, more preferably fluorine and chlorine.

The alkyl group of the halogenated alkyl group may be linear, branched or cyclic, and includes a lower alkyl group having a main chain length of up to 6 carbons, specifically, methyl group, ethyl group Group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, pentyl group, isopentyl group, tert-pentyl group, 1-methylpentyl group, n-hexyl group, isohexyl group, cyclopropyl Group, cyclopentyl group, cyclohexyl group, and the like. The main chain length is preferably 4 carbons or less, more preferably 2 carbons or less.

Examples of the halogen atom of the halogenated alkyl group include fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine, more preferably fluorine and chlorine.

Specific examples of the phenylphosphorylcholine derivative represented by the formula [1] include, for example,
O- (2-Chloro-4-Nitrophenylphosphoryl) choline,
O- (2-Fluoro-4-Nitrophenylphosphoryl) choline,
O- (3-Chloro-4-Nitrophenylphosphoryl) choline,
O- (3-Fluoro-4-Nitrophenylphosphoryl) choline,
O- (4-Nitro-3-Trifluoromethylphenylphosphoryl) choline,
O- (2-Carboxy-4-Nitrophenylphosphoryl) choline,
O- (3-Carboxy-4-Nitrophenylphosphoryl) choline,
Etc.

The method for synthesizing the phenylphosphorylcholine derivative of the present invention is not particularly limited. For example, phosphorylcholine is synthesized by esterification with paranitrophenol by dehydration condensation (S.uriKurioka, Journal of Biochemistry, 63 (5), (678 (1998) , A method of synthesizing phenylphosphodichloridate using paranitrophenol derivatives and various phosphorylating reagents and condensing it with a choline compound (B. Chesebro and H. Metzger, Biochemistry 11,1766 (1972), S. Kurioka and M. Matsuda, Analytical Biochemistry 75, 281-289 (1976), E. Barbar et.al., Biochemistry 35 (9), 2959 (1996)) and the like.

The following is the general formula [2]

(Wherein R ₁ to R ₄ are the same as above)
And a phosphorylating reagent to synthesize phenylphosphodichloridate using a paranitrophenol derivative (hereinafter sometimes abbreviated as “paranitrophenol derivative according to the present invention”) and condensing with a choline compound. An example of synthesizing the phenylphosphorylcholine derivative of the present invention according to is described.

The synthesis of the phenylphosphorylcholine derivative of the present invention includes (1) synthesis of a choline compound, and (2) phosphodichloridated paranitrophenol derivative according to the present invention (hereinafter referred to as “phenylphosphodichloridate according to the present invention”). And (3) condensation of the phenylphosphodichloridate according to the present invention with a choline compound.

(1) Synthesis of choline compound 1.0-2.0 moles of iodomethane with respect to dimethylaminoethanol are mixed with a solvent (for example, ethers such as diethyl ether, tetrahydrofuran, dioxane, anisole, ethylene glycol monoethyl ether, methanol, ethanol, propanol, Reaction in alcohols such as isopropanol, butanol, ethylene glycol, 1,4-butanediol) at -20 to 100 ° C (preferably 0 to 50 ° C) for 0.1 to 24 hours (preferably 1 to 12 hours). Thus, a choline compound is obtained.

(2) Synthesis of phenylphosphodichloridate according to the present invention 1.0-1.5 times mol of phosphorus oxychloride is added to a solvent (for example, diethyl ether, tetrahydrofuran, dioxane, anisole, ethylene glycol) with respect to the paranitrophenol derivative according to the present invention. In an ether such as monoethyl ether) at −80 to 0 ° C., preferably −78 to −50 ° C. for 0.1 to 24 hours, preferably 1 to 16 hours. Get a date.

The paranitrophenol derivative according to the present invention may be synthesized by a conventional method, but if a commercially available product is available, it may be used as it is.

(3) Condensation of phenylphosphodichloridate according to the present invention with a choline compound The choline compound 0.5 obtained according to (1) above with respect to the phenylphosphodichloridate according to the present invention obtained in (2) above -5.0 moles (preferably 1.0-2.0 moles) of base catalyst (eg pyridine, quinoline, triethylamine, N, N-dimethylaniline, piperidine, 4-dimethylaminopyridine, 1,5-diazabicyclo [4.3.0] non- In the presence of 5-ene, 1,8-diazabicyclo [5.4.0] undec-7-ene, organic amines such as tri-n-butylamine), if necessary, a solvent (eg acetonitrile, propionitrile, n-butyronitrile) Nitriles such as tetrahydrofuran, dioxane, anisole, ethers such as ethylene glycol monoethyl ether, N, N-dimethylformamide (DMF), N, N-dimethylacetate Amides such as amides (DMA), acetamide, N-methylpyrrolidone, etc.) at -20-100 ° C. (preferably 0-50 ° C.) for 0.1-24 hours (preferably 0.5-12 hours, more preferably 1-8) The target phenylphosphorylcholine derivative of the present invention can be obtained.

Examples of the reagent for measuring phospholipase D of the present invention include those containing the phenylphosphorylcholine derivative of the present invention.

To measure phospholipase D using the reagent for measuring phospholipase D of the present invention, a test sample and the phenylphosphorylcholine derivative of the present invention are mixed in the presence of phosphate monoesterase or a divalent metal ion to start the reaction. After that, the absorbance increase rate was measured, and the result was expressed in a calibration curve showing the relationship between the phospholipase D activity and the absorbance increase rate obtained using, for example, a sample containing phospholipase D having a known concentration. It can be done by applying.

Examples of the phosphate monoesterase used in the above method include alkaline phosphatase, neutral phosphatase, and acid phosphatase.

In order to carry out the method for quantifying calcium ions according to the present invention, for example, JP 2002-238598 A, JP 7-170999 A, and Japanese Patent Application Laid-Open No. H7-170999, except that the phenylphosphorylcholine derivative of the present invention is used as a substrate for phospholipase D. Measured in accordance with a known calcium measuring method using phospholipase D described in Japanese Patent Laid-Open No. 4-187098, Japanese Patent Laid-Open No. 4-23999, Japanese Patent Laid-Open No. 62-195297, etc. The color development (change in absorbance) of the paranitrophenol derivative may be measured.

That is, the test sample, the phenylphosphorylcholine derivative of the present invention, and phospholipase D are mixed in the presence of phosphate monoesterase, and the rate of increase in absorbance is measured. There is a method of quantification by fitting to a calibration curve showing the relationship between the calcium ion concentration obtained using a sample containing ions and the rate of increase in absorbance.

For example, (1) a method in which a test sample is reacted with a test solution containing phospholipase D and phosphate monoesterase and then reacted with the phenylphosphorylcholine derivative of the present invention, and then the rate of increase in absorbance is measured; (2) Examples include a method of reacting a test sample with a test solution containing phospholipase D, then reacting a phosphate monoesterase with a test solution containing the phenylphosphorylcholine derivative of the present invention, and then measuring the rate of increase in absorbance.

The phenylphosphorylcholine derivative of the present invention used in the calcium ion quantification method according to the present invention and preferred specific examples thereof are as described above.

Among these, as preferred,
O- (2-Fluoro-4-Nitrophenylphosphoryl) choline,
O- (3-Chloro-4-Nitrophenylphosphoryl) choline,
O- (3-Fluoro-4-Nitrophenylphosphoryl) choline,
O- (4-Nitro-3-Trifluoromethylphenylphosphoryl) choline,
Is mentioned.

Particularly preferred are:
O- (2-Fluoro-4-Nitrophenylphosphoryl) choline,
O- (3-Fluoro-4-Nitrophenylphosphoryl) choline, and
O- (4-Nitro-3-Trifluoromethylphenylphosphoryl) choline,
Is mentioned.

The concentration of the phenylphosphorylcholine derivative of the present invention used in the quantification method of the present invention is usually 2 to 200 mM, preferably 4 to 40 mM as the concentration in the test solution, and the concentration in the final reaction solution is usually 0.5 to 50 mM, preferably 1 to 10 mM.

Examples of the phospholipase D used in the calcium ion quantification method according to the present invention include those requiring calcium ions for enzyme reaction. For example, those derived from plants and animals such as cabbage, carrots, peanuts, and porcine pancreas, those derived from microorganisms such as Streptomyces sp (Japanese Patent Laid-Open No. 000-270857) and Nocardia (Japanese Patent Laid-Open No. 60-164483) Etc. In view of stable supply, enzyme stability, and the like, microorganism-derived enzymes such as Streptomyces chromofuscus are preferred.

The concentration of phospholipase D used may be appropriately selected from the range usually used in this field. The concentration in the test solution is usually 0.075 to 15 U / ml, preferably 3 to 7.5 U / ml, and the concentration in the final reaction solution is usually 0.05 to 10 U / ml, preferably 2 ~ 5 U / ml.

As the phosphate monoesterase used in the calcium ion quantification method according to the present invention, alkaline phosphatase, neutral phosphatase or acid phosphatase can be used as microorganisms and various enzymes of animal origin. In view of the stability and availability of the enzyme, alkaline phosphatase is preferable, and among them, alkaline phosphatase derived from Escherichia coli having excellent stability is preferable.

The concentration of phosphate monoesterase used may be appropriately selected from the range usually used in this field. The concentration in the test solution is usually 0.4 to 80 U / ml, preferably 2 to 20 U / ml, and the concentration in the final reaction solution is usually 0.1 to 20 U / ml, preferably 0.8. 5-5 U / ml.

In addition, when measuring calcium ions using phospholipase D, it is known that the specificity for calcium ions increases when the measurement is performed in the presence of a chelating agent acting in addition to calcium ions (for example, Japanese Patent Laid-Open No. Hei 7). -170999, JP-A-2002-238598, etc.).

As the chelating agent used for this purpose, any agent that can eliminate the influence of divalent metal ions other than calcium ions can be used. For example, glycol ether diamine tetraacetic acid, diethylenetriaminepentaacetic acid and the like, and carboxylic acid compounds such as citric acid and oxalic acid can be mentioned. Although it is optional to use one or more of these chelating agents, it is preferable to use glycol ether diamine tetraacetic acid and diethylenetriaminepentaacetic acid in combination.

The concentration of the chelating agent used is 4 μM to 1.2 mM, preferably 40 to 400 μM as the concentration in the test solution, and 1 μM to 300 mM, preferably 10 to 100 μM, in the final reaction solution.

When glycol etherdiaminetetraacetic acid and diethylenetriaminepentaacetic acid are used in combination, the final concentration in the reaction solution is 5 to 200 μM for glycoletherdiaminetetraacetic acid, preferably 10 to 80 μM, and 1 to 50 μM for diethylenetriaminepentaacetic acid, preferably Is 5-20 μM.

Moreover, since phospholipase D used in the quantification method of the present invention is destabilized in the presence of a chelating agent, it is desirable to use magnesium ions as a stabilizer when a chelating agent is used. Moreover, since magnesium ion also becomes an activator of phosphate monoesterase, it is desirable to use magnesium ion also from this point.

As a method for allowing magnesium ions to coexist in the measurement system of the present invention, the method usually used in the form of a salt thereof is the simplest, but is not particularly limited to this method. The type of the salt used in this case is not particularly limited as long as it does not inhibit the stability of the reagent coexisting in the solution. For example, a salt with an inorganic acid such as sulfuric acid or nitric acid, for example, chlorine, Examples include salts (halides) with halogen atoms such as bromine and iodine, such as salts with organic acids such as acetic acid, citric acid, gluconic acid, propionic acid, and pantothenic acid.

The concentration is 1.5 to 450 mM, preferably 15 to 150 mM, as the concentration in the test solution, and 1 to 300 mM, preferably 10 to 100 mM, as the concentration in the final reaction solution.

Since the calcium ion quantification method according to the present invention is desirably performed within the optimum pH range of the enzyme to be used, the solvent for dissolving each reagent is preferably a buffer solution.

The preferred pH at the time of measurement in the quantification method of the present invention is pH 5 to 9, more preferably pH 6 to 7.5. As the buffer used for adjusting the pH to the above range, any buffer can be used as long as the enzyme activity is kept stable, the reagents are dissolved, and a predetermined pH is obtained. Examples of the buffer include dimethyl glutarate buffer. The concentration in the test solution is 7.5 mM to 2 M, preferably 30 to 400 mM, and the final concentration in the reaction solution is 5 to 500 mM, preferably 20 to 100 mM.

Furthermore, in addition to these reagents, surfactants, various preservatives, stabilizers, activators, coexisting substance avoidance agents, and substances that are commonly used in clinical diagnostics may coexist. Needless to say. The concentration range of these reagents and the like may be selected by appropriately selecting and using a concentration range ordinarily used in a calcium ion quantification method using phospholipase D known per se. However, the calcium ion quantification according to the present invention is sufficient. It is desirable to select an enzyme that is highly stable within the optimum pH range of the enzymes used in the method and that does not inhibit the color development of the paranitrophenol derivative produced by the enzyme reaction.

As the spectrophotometer used for measuring the absorbance, any of those usually used in this field can be used without exception.

Of course, the measurement may be performed according to the method used, but it can also be applied to continuous measurement using an automatic analyzer often used in clinical laboratories.

There are no particular limitations on the combination of reagents used when performing measurement using a method or an automatic analyzer, and it may be performed as appropriate according to the environment of the automatic analyzer to be applied, other factors, and the like.

The change in absorbance may be obtained by single wavelength or two-wavelength photometry using a main wavelength and a sub wavelength.

The measurement wavelength for absorbance measurement may be appropriately selected depending on the type of phenylphosphorylcholine derivative used. When measuring at a single wavelength, the rate of increase in absorbance at an arbitrary wavelength of 380 to 450 nm may be measured. When measuring at two wavelengths, it may be measured near the main wavelength of 405 nm and the sub wavelength of 660 nm.

The test sample applied to the quantification method of the present invention includes blood, biological fluids such as plasma, serum or urine, etc., drainage, microbial culture fluids, animal and plant culture fluids, biological material extracts and the like.

An example of calcium measurement using the substrate of the present invention is shown below.
Reaction formula:
Phospholipase D, calcium ion + phenylphosphorylcholine derivative of the present invention + water → paranitrophenyl phosphate derivative + choline / phosphate monoesterase according to the present invention, paranitrophenyl phosphate derivative + water according to the present invention
→ Paranitrophenol derivative (yellow) according to the present invention + phosphoric acid

An example of the calcium ion quantification method according to the present invention includes, for example, a test sample, a reagent solution containing phospholipase D and phosphate monoesterase, and a reagent solution containing the phenylphosphorylcholine derivative of the present invention. In this case, the mixture is sequentially mixed in the presence of a chelating agent and magnesium ions, and is usually reacted at 10 to 50 ° C., preferably 20 to 40 ° C., usually for 2 to 10 minutes, preferably about 5 minutes. The color development derived from the paranitrophenol derivative according to the present invention produced is measured over time to obtain the rate of increase in absorbance. The obtained value is measured in the same manner using, for example, a calcium ion standard solution with a known concentration as a sample, and applied to a calibration curve showing the relationship between the prepared calcium ion concentration and the rate of increase in absorbance. The calcium ion concentration in the sample is determined.

As another example of the quantification method, for example, a test sample, a reagent solution containing phospholipase D, and a reagent solution containing the phenylphosphorylcholine derivative of the present invention and phosphate monoesterase are required. The mixture is sequentially mixed in the presence of a chelating agent and magnesium ions, and is usually reacted at 10 to 50 ° C., preferably 20 to 40 ° C., usually 2 to 10 minutes, preferably about 5 minutes. The color development derived from the paranitrophenol derivative according to the present invention produced is measured over time to obtain the rate of increase in absorbance. The obtained value is measured in the same manner using, for example, a calcium ion standard solution with a known concentration as a sample, and applied to a calibration curve showing the relationship between the prepared calcium ion concentration and the rate of increase in absorbance. The calcium ion concentration in the sample is determined.

The calcium ion measurement reagent of the present invention comprises the phenylphosphorylcholine derivative of the present invention, and preferred embodiments, specific examples, use concentrations, etc. are as described above.

The calcium ion measurement kit of the present invention only needs to comprise the phenylphosphorylcholine derivative of the present invention, phospholipase D, and phosphate monoesterase, and if necessary, a chelating agent and magnesium ions as constituent components. Preferred embodiments, specific examples, use concentrations and the like of each component are as described above.

Specific embodiments of the kit of the present invention include, for example, the following two-component configuration.

(1) a first test solution containing phospholipase D and phosphate monoesterase, and a second test solution containing the phenylphosphorylcholine derivative of the present invention,
(2) What consists of the 1st test solution containing phospholipase D and the 2nd test solution containing the phenylphosphorylcholine derivative of this invention, and a phosphate monoesterase.

Further, if necessary, the aforementioned chelating agent and / or magnesium ion may be contained in at least one of the first reagent solution and the second reagent solution.

In addition, each reagent of the kit may contain, for example, buffers, preservatives, surfactants, stabilizers, and the like that are usually used in this field, in a range normally used in this field. Furthermore, a calcium ion standard product may be combined with the kit as necessary.

In addition, when the kit is composed of a plurality of reagent solutions, each reagent solution also contains reagents necessary for measuring the component to be measured. These reagents are used when the reagents are mixed. In order to start the component measurement reaction, it may be appropriately dispersed in any of the test solutions. The use concentration of the reagents constituting these reagent solutions may be appropriately selected from the range usually used in this field.

Hereinafter, the present invention will be described in more detail with reference to Examples and Reference Examples, but the present invention is not limited thereto.

Experimental Example 1 Selection of Free Dye The pKa and color development rate of various paranitrophenol derivatives were measured by the following method, and a paranitrophenol derivative (free dye) that could be used for the calcium ion quantification method according to the present invention was selected. In addition, description of 3a, 3b etc. in the following description respond | corresponds to the compound name of following Table 1.
First, paranitrophenol was manufactured by Wako Pure Chemical Industries, Ltd. Compound 3a was synthesized by nitration of 2-Chloro phenol (manufactured by Wako Pure Chemical Industries, Ltd.) according to the method described in Synthetic Communication, 1996, 26 (20), 3783-3790.
Paranitrophenol, 5 types of paranitrophenol derivatives listed in Table 1, ie, compound 3a synthesized above, compound 3b (manufactured by Wako Pure Chemical Industries, Ltd.), compound 6a (manufactured by Wako Pure Chemical Industries, Ltd.) ), Compound 6b (manufactured by Wako Pure Chemical Industries, Ltd.), and Compound 6c (manufactured by Tokyo Chemical Industry Co., Ltd.) (concentration: 1.55 mM) were prepared. Next, 100 μl of each aqueous solution was dissolved in 3 mL of the following pH buffer solution (final concentration of each paranitrophenol derivative: 50 μM).
Buffer: 50 mM glycine-HCl buffer (pH 2-5, 30 ° C)
50 mM MES buffer (pH 5-8, 30 ° C)
50 mM CHES buffer (pH 8-11, 30 ° C)
About the obtained solution, an absorption curve was taken with a spectrophotometer (Hitachi U-3000 type), and OD value and pH at 405 nm and λmax were plotted to obtain pKa. Further, the color development rate at 405 nm and λmax (absorbance of paranitrophenol derivative in pH 7 buffer / 1 absorbance of paranitrophenol derivative in NaOH solution) was also determined.
The results are shown in Table 1.

Reference: Known pKa
4-nitrophenol: 7.1, 2-fluoro-4-nitrophenol: 7.1, 3-4-nitrophenol: 5.3,
2,3-difluoro-4-nitrophenol: 4.7, 2,5-difluoro-4-nitrophenol: 4.7,
3,5-difluoro-4-nitrophenol: 4.4, 4-nitro-2,3,6-trifluorophenol: 3.5,
4-nitro-2,3,5,6-tetrafluorophenol: 2.9, 2-chloro-4-nitrophenol: 5.5.

The optimum pH of phospholipase D used for the quantification of calcium ions is around neutral. Therefore, usually, when calcium ions are quantified using phospholipase D, the pH of the reaction solution is set near neutral in order to allow the reaction to proceed efficiently. In addition, when measuring paranitrophenol, which is the final product of a conventional measurement system, with an automatic analyzer, the fixed wavelength used is often around 405 nm. However, as apparent from Table 1 above, the conventional paranitrophenol has a low coloration rate near 405 nm near neutrality.
Furthermore, as apparent from Table 1, the p-nitrophenol derivative according to the present invention has a lower pKa than that of the conventional para-nitrophenol, and is highly colored near neutrality, that is, the color development rate is increased. It can be seen that the color stability is improved.
From the above results, it was found that phenylphosphorylcholine derivatives that liberate these paranitrophenol derivatives are more suitable for quantification of calcium ions using phospholipase D.
Therefore, phenylphosphorylcholine derivatives that liberate these paranitrophenol derivatives were synthesized according to the following reaction scheme.

[Reaction Scheme A]

[Reaction Scheme B]

[Reaction Scheme C]

[Reaction Scheme D]

[Reaction Scheme E]

Experimental Example 2. Choline compound (synthesis of compound (2))
Diethylaminoethanol (25 ml, 0.249 mmol) and iodomethane (50 g, 0.352 mmol) were added to diethyl ether (500 ml) and stirred at 0 ° C. overnight. After completion of the reaction, the precipitated salt was washed with diethyl ether (300 ml) to obtain the desired choline compound (Compound 2 of Reaction Scheme A) (56.7 g, yield 99%).

Experimental Example 3. Synthesis of Phenylphosphodichloridate (Compound 4a) According to the Present Invention To diethyl ether (200 ml), phosphorus oxychloride (10 g, 65.2 mmol), compound 3a synthesized in Experimental Example 1 (in compound 3 of reaction scheme B, R = Cl compound) (11.3 g, 65.2 mmol) and triethylamine (6.6 g, 65.2 mmol) were added and stirred at −78 ° C. overnight. After completion of the reaction, the deposited salt is filtered, and the solvent is distilled off under reduced pressure to obtain the target phenylphosphodichloridate according to the present invention (compound 4a, compound 4 of reaction scheme B, R = Cl compound). Obtained (15.0 g, yield 79%).

Example 1. Synthesis of phenylphosphorylcholine derivative of the present invention (Compound 5a) Compound 4a (15 g, 51.7 mmol) obtained in Experimental Example 3 was dissolved in acetonitrile (100 ml), and Compound 2 (11.9 g, obtained in Experimental Example 2) was dissolved therein. 51.7 mmol) and quinoline (6.7 g, 51.7 mmol) were added and stirred at 0 ° C. for 6 hours. Thereafter, purified water (5 ml) and pyridine (23 ml) were added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was neutralized with saturated aqueous sodium bicarbonate (400 ml), purified with Sephadex G-10 (eluent: water), and the phenylphosphorylcholine derivative of the present invention (O- (2-Chloro-4-Nitrophenylphosphoryl) ) choline, Compound 5a, Compound 5 of Reaction Scheme B, R = Cl) was obtained (5.2 g, yield 30%).
Mass (posi = 339)
NMR (D ₂ O, 400 MHz) δ: 8.38 (d, 1H, J = 2.8), 8.13-8.16 (dd, 1H, J = 2.8,9.2), 7.48 (d, 1H, J = 9.2), 4.42 (t , 2H, J = 4.4), 3.65 (t, 2H, J = 4.4), 3.13 (s, 9H).

Experimental Example 4 Synthesis of phenylphosphodichloridate according to the present invention (compound 4b) Diethyl ether (200 ml) was mixed with phosphorus oxychloride (10 g, 65.2 mmol), compound 3b (compound 3 of reaction scheme B, R = F compound, Mitsui Pure Chemical Industries, Ltd. (10.2 g, 65.2 mmol) and triethylamine (6.6 g, 65.2 mmol) were added, and the mixture was stirred at -78 ° C overnight. After completion of the reaction, the precipitated salt is filtered, and the solvent is distilled off under reduced pressure to obtain the desired phenylphosphodichloridate according to the present invention (compound 4b, (compound 4 of reaction scheme B, R = F compound)). (14.4 g, 81% yield) was obtained.

Example 2 Synthesis of phenylphosphorylcholine derivative of the present invention (Compound 5b) Compound 4b (14.4 g, 52.6 mmol) obtained in Experimental Example 4 was dissolved in acetonitrile (100 ml), and Compound 2 (12.2 g obtained in Experimental Example 2) was dissolved therein. , 52.6 mmol) and quinoline (6.8 g, 52.6 mmol) were added and stirred at 0 ° C. for 6 hours. Thereafter, purified water (5 ml) and pyridine (25 ml) were added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was distilled off under reduced pressure and purified by silica gel column chromatography (eluent: methanol) and recrystallization (solvent: methanol, acetone) to obtain the phenylphosphorylcholine derivative (O- (2-Fluoro) of the present invention. -4-Nitrophenylphosphoryl) choline, Compound 5b, Compound 5 of Reaction Scheme B, R = F) was obtained (6.5 g, yield 38%).
Mass (posi = 323)
NMR (D ₂ O, 400 MHz) δ: 8.02-8.10 (m, 2H), 7.41-7.45 (dd, 1H, J = 8.0, 16.8), 4.39 (t, 2H, J = 4.4), 3.63 (t, 2H , J = 4.4), 3.13 (s, 9H).

Experimental Example 5. Synthesis of phenylphosphodichlorolide (compound 7a) according to the present invention Diethyl ether (200 ml) was mixed with phosphorus oxychloride (6.8 g, 44.4 mmol), compound 6a (compound 6 of reaction scheme C, R = Cl compound, Wako Pure Chemical Industries, Ltd. (7.7 g, 44.4 mmol) and triethylamine (4.5 g, 44.4 mmol) were added, and the mixture was stirred at −78 ° C. overnight. After completion of the reaction, the deposited salt is filtered and the solvent is distilled off under reduced pressure to obtain the desired phenylphosphodichloridate according to the present invention (compound 7a, compound 7 in reaction scheme C, R = Cl compound). Obtained (12.1 g, 95% yield).

Example 3 Synthesis of phenylphosphorylcholine derivative of the present invention (Compound 8a) Compound 7a (12.1 g, 41.7 mmol) obtained in Experimental Example 5 was dissolved in acetonitrile (100 ml), and Compound 2 (9.6 g, obtained in Experimental Example 2) was dissolved. 41.7 mmol) and quinoline (5.4 g, 41.7 mmol) were added and stirred at 0 ° C. for 6 hours. Thereafter, purified water (5 ml) and pyridine (25 ml) were added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was distilled off under reduced pressure and purified by silica gel column chromatography (eluent: methanol) and recrystallization (solvent: methanol, acetone) to obtain the phenylphosphorylcholine derivative (O- (3-Chloro) of the present invention. -4-Nitrophenylphosphoryl) choline, Compound 8a, and Compound 8 of Reaction Scheme C, R = Cl compound) was obtained (5.1 g, yield 36%).
Mass (posi = 339)
NMR (D ₂ O, 400 MHz) δ: 8.01 (dd, 1H, J = 0.8,8.8), 7.38 (dd, 1H, J = 1.2,2.4), 7.19-7.22 (m, 1H), 4.35 (t, 2H , J = 4.4), 3.61 (t, 2H, J = 4.4), 3.12 (s, 9H).

Experimental Example 6. Synthesis of Phenylphosphodichloridate (Compound 7b) According to the Present Invention Diethyl ether (200 ml) was added to phosphorus oxychloride (10 g, 65.2 mmol), compound 6b (compound 6 of reaction scheme C, R = F compound, Mitsui Pure Chemical Industries, Ltd. (10.2 g, 65.2 mmol) and triethylamine (6.6 g, 65.2 mmol) were added, and the mixture was stirred at -78 ° C overnight. After completion of the reaction, the deposited salt is filtered and the solvent is distilled off under reduced pressure to obtain the target phenylphosphodichloridate according to the present invention (compound 7b, compound 7 in reaction scheme C, R = F compound). Obtained (14.4 g, 81% yield).

Example 4 Synthesis of phenylphosphorylcholine derivative of the present invention (Compound 8b) Compound 7b (14.4 g, 52.6 mmol) obtained in Experimental Example 6 was dissolved in acetonitrile (100 ml), and Compound 2 (12.2 g, obtained in Experimental Example 2) was dissolved. 52.6 mmol) and quinoline (6.8 g, 52.6 mmol) were added and stirred at 0 ° C. for 6 hours. Thereafter, purified water (5 ml) and pyridine (25 ml) were added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was distilled off under reduced pressure and purified by silica gel column chromatography (eluent: methanol) and recrystallization (solvent: methanol, acetone) to obtain the phenylphosphorylcholine derivative (O- (3-Fluoro) of the present invention. -4-Nitrophenylphosphoryl) choline, Compound 8b, Compound 8 of Reaction Scheme C, R = F) was obtained (6.6 g, 39% yield).
Mass (posi = 323)
NMR (D ₂ O, 400 MHz) δ: 8.11 (dd, 1H, J =, 8.8, 18.0), 7.07-7.15 (m, 2H), 4.35 (t, 2H, J = 4.4), 3.61 (t, 2H, J = 4.4), 3.12 (s, 9H).

Experimental Example 7. Synthesis of phenylphosphodichloridate according to the present invention (compound 7c) Diethyl ether (200 ml) was mixed with phosphorus oxychloride (7.0 g, 45.6 mmol), compound (6c) (in compound 6 of reaction scheme C, R = CF ₃ (Tokyo Chemical Industry Co., Ltd.) (9.5 g, 45.6 mmol) and triethylamine (4.6 g, 45.6 mmol) were added, and the mixture was stirred at −78 ° C. overnight. After completion of the reaction, the deposited salt is filtered, the solvent is distilled off under reduced pressure, and the target phenylphosphodichloridate according to the present invention (compound 7c, compound 7 in reaction scheme C, R = CF ₃ compound) (12.5 g, 85% yield) was obtained.

Example 5 FIG. Synthesis of phenylphosphorylcholine derivative of the present invention (Compound 8c) Compound 7c (12.5 g, 38.6 mmol) obtained in Experimental Example 7 was dissolved in acetonitrile (100 ml), and Compound 2 (8.9 g, obtained in Experimental Example 2) was dissolved. 38.6 mmol) and quinoline (5.0 g, 38.6 mmol) were added and stirred at 0 ° C. for 6 hours. Thereafter, purified water (5 ml) and pyridine (25 ml) were added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was distilled off under reduced pressure and purified by silica gel column chromatography (eluent: methanol) and recrystallization (solvent: methanol, acetone) to obtain the phenylphosphorylcholine derivative (O- (4-Nitro) of the present invention. -3-Trifluoromethylphenylphosphoryl) choline, compound 8c, in the compound 8 reaction scheme C, to give the compound of _{R = CF 3) (3.3g,} 23% yield).
Mass (posi = 373)
NMR (D ₂ O, 400 MHz) δ: 8.05 (d, 1H, J =, 8.8), 7.63 (d, 1H, J = 1.6), 7.51 (dd, 1H, J = 2.0, 9.2), 4.36 (t, 2H, J = 4.4), 3.61 (t, 2H, J = 4.4), 3.12 (s, 9H).

Experimental Example 8.3 Synthesis of 5-difluoro-4-nitrophenol 3,5-difluorophenol (Compound 9 of Reaction Scheme D, manufactured by Wako Pure Chemical Industries, Ltd.) (25.0 g, 0.19 mol) in dichloromethane (500 ml) ) Was dissolved, 70% concentrated nitric acid (13.5 ml, 0.19 mol) was slowly added at 0 ° C., and the mixture was stirred for 3 hours. After completion of the reaction, the reaction solution was poured into ice water, extracted (CH ₂ Cl ₂ ), dried (MgSO ₄ ), and purified by silica gel column chromatography (eluent; ethyl acetate: hexane = 1: 9). The desired 3,5-difluoro-4-nitrophenol (Compound 10 of Reaction Scheme D) was obtained (12.8 g, 38% yield).

Experimental Example 9. Synthesis of phenylphosphodichloridate according to the present invention (Compound 10) In diethyl ether (250 ml), phosphorus oxychloride (10.7 g, 69.7 mmol), compound 10 obtained in Experimental Example 8 (12.2 g, 69.7 mmol), Triethylamine (7.1 g, 69.7 mmol) was added and stirred at −78 ° C. overnight. After completion of the reaction, the precipitated salt was filtered and the solvent was distilled off under reduced pressure to obtain the target phenylphosphodichloridate according to the present invention (Compound 11 of Reaction Scheme D) (14.3 g, yield 70%). ).

Example 6 Synthesis of phenylphosphorylcholine derivative of the present invention (Compound 12) Compound 11 (14.2 g, 48.6 mmol) obtained in Experimental Example 9 was dissolved in acetonitrile (100 ml), and Compound 2 (11.2 g, obtained in Experimental Example 2) was dissolved. 48.6 mmol) and quinoline (6.3 g, 48.6 mmol) were added and stirred at 0 ° C. for 6 hours. Thereafter, purified water (6 ml) and pyridine (21 ml) were added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the reaction solution was distilled off under reduced pressure and purified by silica gel column chromatography (eluent: methanol) and recrystallization (solvent: methanol, acetone) to obtain the phenylphosphorylcholine derivative of the present invention (compound 12 of Reaction Scheme D). (3.2 g, yield 20%).
Mass (posi = 339)
NMR (D ₂ O, 400 MHz) δ: 6.99 (d, 2H, J = 10.8), 4.36 (t, 2H, J = 4.0), 3.62 (t, 2H, J = 4.0), 3.14 (s, 9H).

Example 7 Substrate specificity test (1) Preparation of reagent solution Each reagent solution having the following composition was prepared.
Test solution 1: 4 U / ml phospholipase D (T-07 manufactured by Asahi Kasei Corporation, derived from Streptomyces chromofuscus ), 53.3 μM glycol ether diamine tetraacetic acid, 13.3 μM diethylenetriaminepentaacetic acid, 33.3 mM magnesium chloride, 0.13% Triton X 1.1 mM PIPES-NaOH buffer (pH 7.3) containing -100.
Reagent 2: 0-100 mM paranitrophenylphosphorylcholine, or the phenylphosphorylcholine derivative of the present invention (compound 8a or compound 8c in Reaction Scheme C) synthesized in Examples 3 and 4 above, 5 mM PIPES containing 5 U / ml alkaline phosphatase -NaOH buffer (pH 7.2).
Calcium ion solution: Calcium chloride (manufactured by Wako Pure Chemical Industries, Ltd., special grade) was diluted with purified water to prepare a 100 mg / dL aqueous solution as calcium ions to obtain a calcium ion solution.

(2) Reaction rate measurement Using Hitachi automatic analyzer (model 7170), sample solution 1 is mixed with 180 μL, sample solution 2 is mixed with 60 μL, and calcium solution is 4 μL, and reacted at 37 ° C., with a main wavelength of 405 nm and a subwavelength of 660 nm. Was measured over time to obtain a reaction time course. From the obtained reaction time course, the absorbance increase rate was calculated.
Subsequently, the rate of increase in absorbance with respect to the concentration of each substrate used (paranitrophenylphosphorylcholine or the phenylphosphorylcholine derivative of the present invention) was plotted to obtain an inverse graph.

(3) Results FIG. 1 shows the relationship between the obtained substrate concentration (mM) and the rate of increase in absorbance (ΔE / min).
In FIG. 1, (a) shows the conventional paranitrophenylphosphorylcholine as a substrate for phospholipase D, (b) to (c) show the phenylphosphorylcholine derivative of the present invention as a substrate for phospholipase D, that is, compound 8a or compound 8c, respectively. The results when used are shown.
Moreover, Km was calculated | required from the approximate expression of each reciprocal graph obtained based on FIG.
The results are shown in Table 2.

As is clear from the results in Table 2, it was found that Compound 8a and Compound 8c had a significantly lower Km than paranitrophenyl phosphorylcholine, and the substrate specificity of phospholipase D was significantly improved.

Example 8 FIG. Quantification of calcium ions Calcium ions were quantified using the phenylphosphorylcholine derivatives of the present invention synthesized in the above examples.

(1) Preparation of reagent solution Each reagent solution having the following composition was prepared.
Test solution 1: 4 U / ml phospholipase D (T-07 manufactured by Asahi Kasei Corporation, derived from Streptomyces chromofuscus ), 53.3 μM glycol ether diamine tetraacetic acid, 13.3 μM diethylenetriaminepentaacetic acid, 33.3 mM magnesium chloride, 0.13% Triton X 61.1 mM PIPES-NaOH buffer (pH 7.3) containing -100.
Test solution 2: 16 mM paranitrophenylphosphorylcholine, or the phenylphosphorylcholine derivative of the present invention synthesized in Examples 2, 3, 4 and 5 above (compound 5b in reaction scheme B, compound 8a in reaction scheme C, compound 8b, compound 8c) ) 5mMPIPES-NaOH buffer (pH 7.2) containing 5 U / ml alkaline phosphatase.
Calcium ion standard solution: Multicalibrator A (Wako Pure Chemical Industries, Ltd.) 10 mg / dL.
Calcium ion solution: Prepare a Ca: 20 mg / dL solution using [Calcium chloride, special grade, manufactured by Wako Pure Chemical Industries, Ltd.], dilute this product with purified water, and prepare an aqueous solution of each concentration (1 mg / dL 2 mg / dL, 4 mg / dL, 10 mg / dL, 20 mg / dL).

(2) Measurement of calcium ion concentration Using Hitachi automatic analyzer (model 7170), mix sample solution 1 with 180 μL, sample solution 2 with 60 μL, and calcium ion solution with each concentration of 4 μL, react at 37 ° C., main wavelength Absorbance at 405 nm and subwavelength 660 nm was measured over time at each photometric point to obtain a reaction time course.
The results are shown in FIGS. 2 (a) to (e).
In FIG. 2, (a) is a conventional paranitrophenyl phosphorylcholine as a phospholipase D substrate, (b) to (e) are phospholipase D substrates, and the phenylphosphorylcholine derivative of the present invention, ie, compound 5b, compound 8a, The results when using Compound 8b and Compound 8c are shown.
The concentration of each reagent in the final reaction solution is about 4 mM for the phenylphosphorylcholine derivative of the present invention, about 3 U / mL for phospholipase D, and about 1.25 U / mL for alkaline phosphatase.
Further, the absorbance increase rate was calculated from the obtained reaction time course.
Separately, except that purified water or calcium ion standard solution (10 mg / dL) is used in place of the calcium ion solution, the reaction is carried out in the same manner as described above, the absorbance is measured over time, and the rate of increase in absorbance from the obtained reaction time course. And a calibration curve showing the relationship between the calcium ion concentration and the rate of increase in absorbance was prepared.
Next, the absorbance increase rate obtained by performing measurement for each concentration of calcium solution obtained above was applied to a calibration curve to determine the concentration of each calcium solution.

(3) Results FIGS. 3A to 3E show the relationship between the calcium ion concentration (analyte Ca amount) of each calcium solution and the calcium ion concentration (measured value) of the calcium solution obtained from the calibration curve.
In FIG. 3, (a) is a conventional paranitrophenyl phosphorylcholine as a substrate for phospholipase D, (b) to (e) are phenylphosphorylcholine derivatives of the present invention as substrates for phospholipase D, ie, compound 5b, compound 8a, The results when using Compound 8b and Compound 8c are shown.
As is clear from FIGS. 3 (b) to 3 (e), when the calcium ion was quantified using the phenylphosphorylcholine derivative of the present invention, the conventional paranitrophenylphosphorylcholine was used (FIG. 3 (a)). Similarly, a good calibration curve proportional to the calcium ion concentration is obtained, and it can be seen that it can be used for quantification of calcium ions using phospholipase D.
In addition, the actual calcium concentration of the calcium solution used for the measurement and the calcium concentration of the same calcium solution obtained from the calibration curve correlate well, and the reliability of the measurement values obtained by this measurement system is high. Recognize.

In this example, the calcium concentration was measured using the phenylphosphorylcholine derivative of the present invention under the optimum conditions of the conventional substrate paranitrophenylphosphorylcholine. However, the optimum measurement and composition conditions of the phenylphosphorylcholine derivative of the present invention are different from those of paranitrophenylphosphorylcholine. This is easily inferred from the fact that the phenylphosphorylcholine derivative of the present invention has a significantly different Km value from that of paranitrophenylphosphorylcholine (Example 7). In the compounds 8a and 8c, the measured values slightly deviate from the calibration curve within the high calcium ion concentration range (FIGS. 3 (c) and (e)). This is probably because the measurement was not performed under the conditions. If the measurement is performed under the optimum measurement / composition conditions for each of the compounds 8A and 8c, the measurement result is presumed to be on the calibration curve even at a high concentration of calcium.

Example 9 Stability of substrate over time (1) Preparation of test solution Test solution 1 and test solution 2 having the following composition were prepared.
Reagent 1: Same as Example 8.
Test solution 2: 16 mM paranitrophenylphosphorylcholine, or the phenylphosphorylcholine derivative of the present invention (compound 8b or compound 8c) synthesized in Examples 4 and 5 above, 5 mM PIPES-NaOH buffer containing 5 U / ml alkaline phosphatase (PH 7.2).

(2) Measurement of absorbance during dissolution and storage Using a Hitachi automated analyzer (model 7170), sample solution 1 was mixed with 180 μL, sample solution 2 was mixed with 60 μL, and physiological saline, reacted at 37 ° C., and absorbance at 405 nm was measured. It was measured.
Thereafter, the test solution 2 was stored under refrigerated (7 ° C.) and harsh (25 ° C.) conditions, and the same measurement was performed using physiological saline as a sample after 2 weeks and 4 weeks.

(3) Results The results are shown in FIGS. 4 (a) to (c).
In FIG. 4, (a) shows the results of using conventional paranitrophenylphosphorylcholine, (b) and (c) show the results of using the nitrophenylphosphorylcholine derivative of the present invention, ie, compound 8b or compound 8c, respectively.
4 (a) to 4 (c),-□-indicates the results when stored under refrigeration (7 ° C), and-■-indicates the results when stored under severe conditions (25 ° C).
As is clear from FIGS. 4 (b) to (c), the phenylphosphorylcholine derivatives according to the present invention (compounds 8b and 8c) hardly change in absolute absorbance even when stored at 7 ° C. or 25 ° C. for 4 weeks. It can be seen that the stability is comparable to that of conventional paranitrophenyl phosphorylcholine (FIG. 4 (a)).

In FIGS. 4 (a) to (c),-□-indicates the results when stored under refrigeration (7 ° C), and-■-indicates the results when stored under severe conditions (25 ° C).

Claims

Following formula [1]

[Wherein R 1 to R 4 each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R 1 to R 4 represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
A phenylphosphorylcholine derivative represented by:
The phenylphosphorylcholine derivative according to claim 1, wherein R 2 is a fluorine atom, and R 1 , R 3 and R 4 are hydrogen atoms.
The phenylphosphorylcholine derivative according to claim 1, wherein R 1 is a fluorine atom, and R 2 , R 3 and R 4 are hydrogen atoms.
The phenylphosphorylcholine derivative according to claim 1, wherein R 2 is a trifluoromethyl group, and R 1 , R 3 and R 4 are hydrogen atoms.
Following formula [1]

[Wherein R 1 to R 4 each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R 1 to R 4 represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
A reagent for measuring phospholipase D, comprising a phenylphosphorylcholine derivative represented by the formula:
Test sample and following formula [1]

[Wherein R 1 to R 4 each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R 1 to R 4 represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
And a phospholipase D is mixed with phospholipase D in the presence of phosphate monoesterase to start the reaction, and then the absorbance increase rate is measured, and this is performed based on the absorbance increase rate. A method for quantifying calcium ions.
The quantification method according to claim 6, wherein the phosphate monoesterase is alkaline phosphatase or neutral phosphatase.
Following formula [1]

[Wherein R 1 to R 4 each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R 1 to R 4 represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
A reagent for quantifying calcium ions, comprising a phenylphosphorylcholine derivative represented by the formula:
Following formula [1]

[Wherein R 1 to R 4 each independently represents a hydrogen atom, a halogen atom, a halogenated alkyl group, or a carboxyl group. However, at least one of R 1 to R 4 represents a halogen atom, a halogenated alkyl group, or a carboxyl group. ]
A kit for quantifying calcium ions, comprising a phenylphosphorylcholine derivative represented by the formula: phospholipase D and phosphate monoesterase as constituent components.
The kit according to claim 9, wherein the phosphate monoesterase is alkaline phosphatase or neutral phosphatase.