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WO2009128348A1 - Dérivés de phénylphosphorylcholine - Google Patents

Dérivés de phénylphosphorylcholine Download PDF

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Publication number
WO2009128348A1
WO2009128348A1 PCT/JP2009/056874 JP2009056874W WO2009128348A1 WO 2009128348 A1 WO2009128348 A1 WO 2009128348A1 JP 2009056874 W JP2009056874 W JP 2009056874W WO 2009128348 A1 WO2009128348 A1 WO 2009128348A1
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compound
phenylphosphorylcholine
present
derivative
phospholipase
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Japanese (ja)
Inventor
睦廣 伊逹
諭 狭場
祐介 中新井
友和 板井
和仁 谷本
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Fujifilm Wako Pure Chemical Corp
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Wako Pure Chemical Industries Ltd
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Priority to JP2010508169A priority Critical patent/JP5614281B2/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/12—Esters of phosphoric acids with hydroxyaryl compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G01N2333/92—Triglyceride splitting, e.g. by means of lipase

Definitions

  • 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.
  • 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. .
  • 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.
  • 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.
  • 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:
  • 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 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.
  • 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:
  • 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 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.
  • FIG. 6 is a graph showing the relationship between the substrate concentration (mM) of each substrate and the absorbance increase rate ( ⁇ E / mim) obtained in Example 7.
  • FIG. 1 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.).
  • ODx10000 light absorbency
  • 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.
  • 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.
  • 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.
  • 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.
  • halogen atom examples 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.
  • halogen atom of the halogenated alkyl group examples include fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine, more preferably fluorine and chlorine.
  • 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.
  • 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 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.
  • 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).
  • 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
  • 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.
  • Examples of the reagent for measuring phospholipase D of the present invention include those containing the phenylphosphorylcholine derivative 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.
  • 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.
  • the color development (change in absorbance) of the paranitrophenol derivative may be measured.
  • 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.
  • rate of increase in absorbance 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.
  • (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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • alkaline phosphatase 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.
  • any agent that can eliminate the influence of divalent metal ions other than calcium ions can be used.
  • glycol ether diamine tetraacetic acid, diethylenetriaminepentaacetic acid and the like, and carboxylic acid compounds such as citric acid and oxalic acid can be mentioned.
  • carboxylic acid compounds such as citric acid and oxalic acid
  • the concentration of the chelating agent used is 4 ⁇ M to 1.2 mM, preferably 40 to 400 ⁇ M as the concentration in the test solution, and 1 ⁇ M to 300 mM, preferably 10 to 100 ⁇ M, in the final reaction solution.
  • the final concentration in the reaction solution is 5 to 200 ⁇ M for glycoletherdiaminetetraacetic acid, preferably 10 to 80 ⁇ M, and 1 to 50 ⁇ M for diethylenetriaminepentaacetic acid, preferably Is 5-20 ⁇ M.
  • magnesium ions are used as a stabilizer when a chelating agent is used.
  • magnesium ion also becomes an activator of phosphate monoesterase, it is desirable to use magnesium ion also from this point.
  • 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.
  • 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.
  • 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.
  • any buffer can be used as long as the enzyme activity is kept stable, the reagents are dissolved, and a predetermined pH is obtained.
  • 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.
  • 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.
  • concentration range ordinarily used in a calcium ion quantification method using phospholipase D known per se 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.
  • 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.
  • any of those usually used in this field can be used without exception.
  • 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.
  • 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.
  • the rate of increase in absorbance at an arbitrary wavelength of 380 to 450 nm may be measured.
  • it may be measured near the main wavelength of 405 nm and the sub wavelength of 660 nm.
  • 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 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.
  • 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.
  • a test sample 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.
  • kit of the present invention include, for example, the following two-component configuration.
  • 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.
  • 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.
  • a calcium ion standard product may be combined with the kit as necessary.
  • each reagent solution 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.
  • 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 ⁇ max were plotted to obtain pKa. Further, the color development rate at 405 nm and ⁇ max (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.
  • 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.
  • the fixed wavelength used is often around 405 nm.
  • the conventional paranitrophenol has a low coloration rate near 405 nm near neutrality.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • purified water 5 ml
  • pyridine 25 ml
  • 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.
  • 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).
  • 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 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.
  • 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 ⁇ M glycol ether diamine tetraacetic acid, 13.3 ⁇ M 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.
  • FIG. 1 shows the relationship between the obtained substrate concentration (mM) and the rate of increase in absorbance ( ⁇ E / min).
  • (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.
  • Km was calculated
  • Example 8 Quantification of calcium ions Calcium ions were quantified using the phenylphosphorylcholine derivatives of the present invention synthesized in the above examples.
  • Test solution 1 4 U / ml phospholipase D (T-07 manufactured by Asahi Kasei Corporation, derived from Streptomyces chromofuscus ), 53.3 ⁇ M glycol ether diamine tetraacetic acid, 13.3 ⁇ M 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).
  • 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.
  • 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.
  • (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.
  • 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.
  • (a) is a conventional paranitrophenyl phosphorylcholine as a substrate for phospholipase D
  • (b) to (e) are phenylphosphorylcho
  • the calcium concentration was measured using the phenylphosphorylcholine derivative of the present invention under the optimum conditions of the conventional substrate paranitrophenylphosphorylcholine.
  • 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).
  • 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).
  • sample solution 1 was mixed with 180 ⁇ L, sample solution 2 was mixed with 60 ⁇ L, 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.
  • FIGS. 4 (a) to (c) show 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).
  • 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)).
  • 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.

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Abstract

L'invention porte sur des dérivés de phénylphosphorylcholine représentés par la formule générale [1] ; sur un procédé pour le dosage des ions calcium, caractérisé par le mélange d'un dérivé décrit ci-dessus avec de la phospholipase D en présence d'une phosphomonoestérase pour amorcer une réaction, et par la mesure de la vitesse d'augmentation de l'absorbance et la détermination de la quantité d'ion calcium sur la base de la vitesse d'augmentation de l'absorbance ; sur des réactifs et des kits pour le dosage de l'ion calcium qui contiennent les dérivés, et sur des réactifs pour le dosage de la phospholipase D : [1] dans laquelle R1 à R4 représentent chacun indépendamment l'hydrogène, l'halogéno, l'alkyle halogéné ou le carboxy, à la condition qu'au moins l'un de R1 à R4 soit l'halogéno, l'alkyle halogéné ou le carboxy.
PCT/JP2009/056874 2008-04-14 2009-04-02 Dérivés de phénylphosphorylcholine Ceased WO2009128348A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012242277A (ja) * 2011-05-20 2012-12-10 Rohm Co Ltd マイクロチップ、ならびに、それを用いた測定システムおよび測定方法
WO2016060096A1 (fr) * 2014-10-15 2016-04-21 日油株式会社 Composé contenant un groupe phosphorylcholine et complexe de phosphorylcholine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS58501357A (ja) * 1981-08-28 1983-08-18 バクスター　インターナショナル　インコーポレーテッド 生物学的液体の酵素分析方法
JPH0284199A (ja) * 1988-09-20 1990-03-26 Wako Pure Chem Ind Ltd 酸性ホスフアターゼ活性測定用試液
JP2002238598A (ja) * 2001-02-15 2002-08-27 Asahi Kasei Corp カルシウムイオン測定用組成物および測定方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS58501357A (ja) * 1981-08-28 1983-08-18 バクスター　インターナショナル　インコーポレーテッド 生物学的液体の酵素分析方法
JPH0284199A (ja) * 1988-09-20 1990-03-26 Wako Pure Chem Ind Ltd 酸性ホスフアターゼ活性測定用試液
JP2002238598A (ja) * 2001-02-15 2002-08-27 Asahi Kasei Corp カルシウムイオン測定用組成物および測定方法

Non-Patent Citations (1)

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Title
KAZUHITO TANIMOTO: "Kosoho ni yoru Shinki Calcium Sokutei Shiyaku no Kaihatsu", RINSHO KAGAKU, vol. 37, no. 1, 29 August 2008 (2008-08-29), pages 173 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012242277A (ja) * 2011-05-20 2012-12-10 Rohm Co Ltd マイクロチップ、ならびに、それを用いた測定システムおよび測定方法
WO2016060096A1 (fr) * 2014-10-15 2016-04-21 日油株式会社 Composé contenant un groupe phosphorylcholine et complexe de phosphorylcholine
US9850266B2 (en) 2014-10-15 2017-12-26 Nof Corporation Phosphorylcholine group-containing compound and phosphorylcholine complex

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