JPH0376709B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for the microquantification of certain compounds having a pyrimidine ring. In more detail,
The present invention relates to a method for microquantitative determination of a compound having a pyrimidine ring in which at least one nitrogen atom in the ring has no substituent. In living organisms, there are many compounds that have a pyrimidine ring in their chemical structure, including various pyrimidine bases, nucleotides, and nucleosides.
Most of them are known to play biologically and physiologically chemically important roles. Also,
The effects and roles of these substances have been rapidly elucidated in recent years, coupled with advances in biotechnology, and the importance of these substances has come to be increasingly recognized. Quantifying compounds with these pyrimidine rings present in various samples derived from living organisms is useful for diagnosing diseases caused by metabolic abnormalities or genetic information abnormalities in living organisms, or for using these compounds as medicines. It has important significance in relation to predicting and preventing side effects in treatment. In general. Since the amount of the above-mentioned compound having a pyrimidine ring present in the above-mentioned biological samples is extremely small, there is a strong demand in various fields to establish a method for quantifying the small amount thereof. As a result of intensive research into methods for quantifying trace amounts of compounds having a pyrimidine ring, the present inventors have provided, according to the present invention, a method for quantifying trace amounts of compounds in which at least one nitrogen atom in the pyrimidine ring is unsubstituted. succeeded in doing so. That is, the present invention provides a method for quantifying a small amount of a pyrimidine compound which has a pyrimidine ring in its chemical structure, and at least one nitrogen atom in the ring has no substituent. be. The method of the present invention is characterized in that a measurement sample is treated with 4-bromomethyl-7-methoxycoumarin in the presence of crown ethers, and the resulting product is subjected to fluorescent high performance liquid chromatography. be. The present invention will be explained in detail below. The compound to be measured in the method of the present invention is a pyrimidine compound having a pyrimidine ring in its chemical structure, and at least one nitrogen atom in the ring has no substituent. Examples include, first, bases of pyrimidine nucleic acids, nucleotides, and nucleosides. Specific examples of the individual compounds include, for example, uracil, thymine, cytosine,
-methylcytosine, 5,6-dihydrouracil,
3-methyluracil, 5,6-dihydrothymine,
5-hydroxymethylcytosine, 2'-0-methyluracil, 5-fluorouracil, 5,6-dihydro-5-fluorouracil, 1-hexylcarbamoyl-5-fluorouracil, 1-(tetrahydro-2-furanyl)-5-fluorouracil ,
1-(tetrahydro-2-furanyl)uracil,
Alloxan, uridine, thymidine, cytidine, 5
-Methylcytidine, N ⁴ -Methylcytidine, N ⁴ -
acetylcytidine, 2'-0-methylcytidine,
2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 5-trifluoro Methyl-2'-deoxyuridine, 6-azauridine, β-cytosine arabinoside, cytidine-
5'-monophosphate, cytidine phosphate, cytidine triphosphate, uridine-
5'-monophosphate, uridine diphosphate, uridine tulfophosphate, thymidine-
5′-monophosphate, thymidine phosphate, thymidine tulfophosphate, 2′-deoxycytidine-5′-monophosphate, 2′-deoxyuridine-5′-monophosphate, 2′- Deoxy-5-hydroxymethylcytidine-5'-monophosphate, 2'-deoxy-5-hydroxymethyluridine-5'-monophosphate, 5-
Fluorouridine-5'-monophosphate, 5
-Fluorouridine diphosphate, 5-fluorouridine triphosphate, 5-fluoro-2'-deoxyuridine-5'-monophosphate, 2'-deoxyuridine phosphate, 2'-
Examples include deoxyuridine triphosphate, 2'-deoxycytidine diphosphate, and 2'-deoxycytidine triphosphate. In the method of the present invention, in order to quantify these pyrimidine compounds, first, a measurement sample is mixed with 4-bromomethyl-7 in the presence of crown ethers.
- Treatment with methoxycoumarin. Through this treatment, the pyrimidine compound in the measurement sample reacts with 4-bromomethyl-7-methoxycoumarin to generate a fluorescent compound. This fluorescent compound has an unsubstituted nitrogen atom (7-methoxy-4-
coumarinyl) methyl group is engaged,
The method of the present invention attempts to quantify the pyrimidine compound in the measurement sample by quantifying this fluorescent compound by fluorescence high performance liquid chromatography using a fluorescence detector. Examples of crown ethers used in the method of the present invention include 18-crown-6, 15-crown-6, and 15-crown ethers.
Examples include crown-5, dicyclohexyl-18-crown-6, dicyclohexyl-24-crown-8, dibenzo-24-crown-8, dibenzo-18-crown-6, and the like. 18-crown-6 is a preferred example. The present invention is particularly useful as a microquantitative method for compounds having a pyrimidine ring present in biological components. Examples include samples derived from all kinds of tissues, including liquid tissues such as animal blood, urine, and lymph, organ tissues such as liver, kidney, lung, and fire extinguisher, and tissues such as skin and tumors. In addition, samples derived from cells of microorganisms such as bacteria, molds, viruses, etc. or their environmental systems can also be cited as samples to which the method of the present invention can be applied. By cleaning up these measurement samples, they can be made into more preferable measurement samples for the method of the present invention. Although the specific operations for the clean-up method vary depending on the type of biological component, it is usually possible to adopt a method used to extract and separate compounds having a pyrimidine ring from these biological components. It is possible. For example, in the case of body fluids, cells, etc., treatment with ultrasound or the like is performed as necessary. In addition, in the case of tissues, etc., homogenization is performed. The product thus obtained is subjected to protein removal treatment as desired, and as necessary, for example, centrifugation, ultrafiltration, salting out using barium hydroxide, sodium chloride, sodium laurate sulfate, Treatments such as dialysis against solutions of granidine salts, trichloroacetic acid, etc., as well as water or alcohols such as methanol, ethanol, butanol, various ethers, organic solvents such as acetone, chloroform, hexane, acetic acid, ethyl acetate, etc. Extraction operation using a mixture of these, or chromatography treatment, for example, ion exchange column chromatography using Celite 545, AG1-X4, AG1-X2, Amberlite XAD-2, etc., Cephadex LH-20, etc. Treatments using molecular sieve column chromatography, high performance liquid chromatography, thin layer chromatography, etc. are carried out in an appropriate combination. Through these treatments, biological components can be cleaned up and a more preferable measurement sample can be obtained. As a preferable example of the clean-up method, the following operation method can be mentioned. Add 0.1 ml of 4N hydrochloric acid and 0.4 g of sodium chloride to 1 ml of serum, and perform extraction three times with 3 ml of ethyl acetate. Separate the ethyl acetate layer, dry it under reduced pressure, add this to 1 ml of methanol three times while washing the inner wall of the device, and dry it again under reduced pressure. Dissolve the obtained residue in 0.1 ml of methanol,
This is subjected to thin layer chromatography using Kieselgel F ₂₅₄ as a thin layer and a mixture of trichloromethane:ethyl acetate:ethanol=5:24:6 as a developing solvent. A desired spot corresponding to a compound having a pyrimidine ring was collected, extracted three times with 10 ml of a 10% methanol-ethyl acetate mixture, and the organic solvent layer was separated.
Dry under reduced pressure. The inner wall of the instrument used was washed with ethanol in the same manner as in the above procedure, and this was added to the above and dried again under reduced pressure. The obtained residue is dissolved in 0.25 ml of 0.05 M monoammonium phosphate buffer (PH3.5), filtered using a Millipore filter (type HA, 0.45 μm), and this liquid is used as a measurement sample. The above procedure also applies when using urine, homogenates of various tissues, etc. In this case, cleanup can be carried out conveniently by using optionally modified methods. A preferable measurement sample obtained in this way can be prepared using, for example, 4-bromomethyl-7-methoxycoumarin in an organic solvent in the presence of crown ethers according to the same procedure as in Example 1 described below. will be processed. This produces a fluorescent compound, which can be subjected to fluorescent high performance liquid chromatography using a detector such as a nitrogen laser fluorescence detector, with or without thin layer chromatography. A trace amount of a compound having a pyrimidine ring present in a measurement sample can be determined. Examples of the above organic solvents include ketones such as acetone and methyl ethyl ketone, ethers such as isopropyl ether and dioxane, benzene,
Examples include aromatic hydrocarbons such as toluene and acetonitrile. The aforementioned reactions are preferably conveniently carried out in anhydrous conditions. The present inventors investigated the proportion of 4-bromomethyl-7-methoxycoumarin or crown ethers used in compounds having a pyrimidine ring, and found that 4-bromomethyl-7- Methoxycoumarin can be used in a range of about 3 to 15 times, preferably around 10 times the mole, and crown ethers can be used in an amount of about 1 mole or less per mole of 4-bromomethyl-7-methoxycoumarin. It has been found that it can be used within a range, and that it is preferable to use around 0.2 times the mole. In addition, pyrimidine compounds and 4-bromomethyl-
In order for the reaction with 7-methoxycoumarin to proceed smoothly, it is necessary that cations, particularly metal ions, be present in the reaction system, which can be captured by crown ethers and enable complex formation. Therefore, it is preferable to add salts, especially inorganic salts, which can generate cations such as potassium, lithium, sodium, barium, and calcium in the reaction system, if desired. In particular, the combination use of 18-crown-6 and potassium carbonate gives good results. The reaction temperature is usually 45
The purpose can be achieved at a temperature of about 15°C to 95°C and a reaction time of about 15 minutes to 3 hours. It is suitable to carry out the reaction at a reaction temperature of about 75° C. for about 30 minutes in order to obtain a product with preferable fluorescent properties in the method of the present invention. After the reaction is completed, an aliphatic carboxylic acid is added to the reaction system, if desired, and the mixture is subsequently heated to reflux. This operation makes it possible to avoid interference with fluorescence measurement caused by 4-bromomethyl-7-methoxycoumarin remaining in the reaction system. Particularly preferred as the aliphatic carboxylic acid is n-valeric acid. A heating and refluxing time of about 5 minutes is sufficient. The aliphatic carboxylic acid mentioned above reacts rapidly with 4-bromomethyl-7-methoxycoumarin, and the reaction product has a retention time on the chromatogram that is significantly different from that of the fluorescent compound having a pyrimidine ring. Therefore, the two can be easily separated. The reaction mixture thus obtained is then subjected to fluorescent high performance liquid chromatography. It is sufficient that such chromatographic treatment is carried out in accordance with the procedure normally adopted for separating compounds having a pyrimidine ring, and the treatment procedure is not specified. For example, a commercially available high-performance liquid chromatography device is used, the separation column is filled with a resin such as octadecyl silica (trade name: Nagel's product), etc. in a conventional manner, and a methanol-water mixed solvent is used. Expand by. The method for measuring the fluorescence of the produced fluorescent compound can be generally carried out according to a commonly used method, and the means are not specified. For example, using a fluorescence detector whose excitation light source is a commercially available low-pressure to medium-pressure mercury lamp used in ordinary fluorescent high-performance liquid chromatography,
By adopting a fluorescence wavelength of around 360 nm and a fluorescence wavelength of around 410 nm, it is possible to measure the fluorescence of the above-mentioned reaction product in the method of the present invention. Furthermore, when carrying out the method of the present invention, fluorescence measurement can also be carried out using a laser fluorescence detector using a laser as a light source. As a light source for laser fluorescence in such a case, it is preferable to use a wavelength variable dye laser excited by a pulsed laser such as a nitrogen laser. Generally, when laser fluorescence is used, measurement sensitivity increases significantly, but blind fluorescence and Raman scattered light due to solvent also become stronger, resulting in a poor S/N (signal/noise) ratio. There are many things. However, since the fluorescent compound produced in the method of the present invention has excellent fluorescent properties,
This method does not cause such inconvenience and can be said to be suitable for measurement using a laser beam detector. In order to perform the quantitative determination using the method of the present invention by fluorescence measurement, a calibration curve is prepared using a standard concentration of the substance to be measured, and the pyrimidine compound in the measurement sample is determined using the calibration curve. Examples of the preparation of a calibration curve are listed below, but the procedure for quantifying a pyrimidine compound in a measurement sample by the method of the present invention may be performed in accordance with these examples. Example 1 0.5 mg of 5-fluorodeoxyuridine and the same amount of 5-fluorouracil in 20 ml of acetone,
Dissolve uridine, deoxyuridine, and thymidine, and add 5.0 mg of 4-bromomethyl-7-methoxycoumarin, 1 mg of 18-crown-6, and 25 mg of thymidine.
Add finely powdered potassium carbonate and raise the water bath temperature to 75°C.
℃ and refluxed for 30 minutes. Then 0.2 ml of n
- Valeric acid was added and refluxed for a further 5 minutes. Approximately 3μ of the reaction mixture thus obtained
The mixture is separated and stored for separate thin layer chromatography. Acetone was added to the remaining reaction mixture to give a dilution of 10 ² to 10 ⁵ times. The acetone solution obtained in this way was analyzed using a high-performance liquid chromatography device (Shimadzu LC-
4A) was used to perform the separation operation. As a separation column, a Shimadzu stainless steel tube FCF-04E (manufactured by Shimadzu Corporation) with an inner diameter of 4 mm and a length of 200 mm was used, which was filled with Nucleosil 5C ₁₈ (manufactured by Nagel) with a particle size of 5 μm by a slurry method. For development, use a 54% (volume ratio) methanol-water mixture from 0 to 20 minutes, a 65% (volume ratio) methanol-water mixture from 20 to 25 minutes, and 100% methanol-water mixture after 35 minutes. % methanol, respectively flow rate 0.6
It was performed stepwise at ml/min. For fluorescence measurement, use Fluorescence Spectromonitor RF-530
(manufactured by Shimadzu Corporation) at an excitation wavelength of 360 nm and a fluorescence wavelength of 410 nm, and the results were reported using a Unicoder U.
-228 (manufactured by Nippon Denshi Kagaku). FIG. 1 shows a chromatogram obtained by the above-described high-performance fluid chromatography operation. Further, FIG. 2 shows calibration curves for various compounds having a pyrimidine ring obtained in this example. Identification of the fluorescent compounds present in the reaction mixture obtained above and each peak separated by high performance liquid chromatography was performed using separately produced 5-fluorodeoxyuridine, 5-fluorouracil, uridine, and deoxyuridine. Thin layer chromatography was carried out as follows using fluorescent compounds of thymidine and thymidine as standards. That is, Kieselgel 60F ₂₅₄ (manufactured by Merck & Co., Ltd.) was used as a thin layer, and the developing solvent was a mixed solvent (solvent) of methyl acetate: ether: 10% formic acid = 9:1:1 (volume ratio) or ethyl acetate (solvent). Expansion was carried out using conventional methods. Fluorescent spots excited at 360 nm were detected using Superlight (manufactured by Irie Seisakusho) as a fluorescence detector. The fluorescent spots obtained from the sample in the reaction mixture and each peak separated by high performance liquid chromatography and those obtained from the standard product showed good correspondence. Table 1 shows the R _f values of various fluorescent compounds.

[Table] As is clear from the results shown in Figure 1, the fluorescent compounds produced by the method of the present invention showed an extremely excellent separation state by high performance liquid chromatography, and the results shown in Figure 2 As is clear from the above, the method of the present invention has an excellent measurement range with an excellent detection limit and a good linear relationship over a wide range. For example, when comparing the detection limit between the method of the present invention and the method of detecting at a wavelength of 265 nm using an ultraviolet detector, Table 2 shows that the method of the present invention is 20 to 250 times superior in sensitivity. It is clear that

[Table] Next, a method for producing a fluorescent compound of 5-fluorodeoxyuridine used as a standard product, and the structure and physical properties of the produced compound are shown as a reference example. Reference example: 1.0g of 5-fluoro in 100ml of dry acetone
2'-deoxyuridine, 1.42 g (7-methoxy-4-coumarinyl)methyl bromide and 2.0 g
Finely powdered anhydrous potassium carbonate was suspended and refluxed for 1.5 hours on a water bath (bath temperature: 75°C). Insoluble matter was removed, and the liquid was concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (elution solvent:
The target fractions were collected and concentrated under reduced pressure. The resulting solid residue was recrystallized from water-ethanol (1:2) to yield 1.18 g (67.0%) of 3-(7-methoxy-
4-Coumarinyl)methyl-5-fluoro-2'-deoxyuridine was obtained as colorless needles. Melting point: 160-163℃. UVλ ^EtOH _nax nm: 217 (sh), 276, 322 NMRδ (ppm, DMSO-d ₆ ): Uridine moiety 2.21
(2H, broad t, J=6Hz, C _2' -H), 3.5-
3.7 (2H, m, C ₅ ′-H), 3.7-3.9 (1H, m,
C ₄ ′-H), 4.2-4.4 (1H, m, C ₃ ′-H), 5.1-
5.3 (2H, m, OH x 2, disappeared by addition of D ₂ O), 6.16
(1H, broad t, J=6Hz, C _1' -H), 8.42
(1H, d, J = 7Hz, C ₆ -H); coumarin part,
3.88 (3H, s, OCH ₃ ), 5.17 (2H, s, CH ₂ ),
6.05 (1H, s, _C3 -H), 6.9-7.1 (1H, m, _C6
-H), 7.05 (1H, s, C ₈ -H), 7.85 (1H, d,
J _{=10Hz, C5-H) Elemental analysis value: C20H19FN2O8 Calculated value (} %): C, 55.30; H, 4.41; N, 6.45 Actual value (%): C, 55.43 _; H , 4.46; N, 6.53 The retention time of the peak of the standard product in the chromatogram obtained by performing fluorescent high performance liquid chromatography using the standard product thus obtained in the same manner as in Example 1 above. is 3 in Figure 1.
It showed good agreement with that of the peak corresponding to . The method for quantifying a small amount of a compound having a pyrimidine ring according to the present invention is extremely useful in the diagnosis or treatment of diseases, and is also extremely useful in the production of compounds having a unique pyrimidine ring based on genetic engineering technology. Useful.

[Brief explanation of drawings]

FIG. 1 shows the high performance liquid chromatogram obtained in Example 1 above. In FIG. 1, 1 indicates the presence of each fluorescent compound derived from uridine, 2 from deoxyuridine, 3 from 5-fluorodeoxyuridine, 4 from thymidine, and 5 from 5-fluorouracil. In addition, A is thought to be due to impurities mixed in the reagent used,
B is due to a fluorescent compound derived from n-valeric acid. FIG. 2 shows calibration curves of various compounds having a pyrimidine ring prepared in Example 1 above. In this figure, 1 shows the respective calibration curves for 5-fluorouracil, 2 for deoxyuridine, 3 for 5-fluorodeoxyuridine, 4 for uridine, and 5 for thymidine.

Claims (1)

[Scope of Claims] 1. A method for quantifying a trace amount of a pyrimidine compound having a pyrimidine ring in which at least one nitrogen atom does not have a substituent, the method comprising: Below, a method for quantifying a trace amount of a compound having a pyrimidine ring, which comprises subjecting a product obtained by treatment with 4-bromomethyl-7-methoxycoumarin to fluorescent high-performance liquid chromatography. 2. The pyrimidine compound having a pyrimidine ring according to claim 1, wherein the pyrimidine compound in which at least one nitrogen atom in the ring has no substituent is a nucleotide or a nucleoside. Microquantification methods. 3. The method for quantifying a small amount of a compound having a pyrimidine ring according to claim 1, wherein the crown ether is 18-crown-6.