JP2002145882A - Method for refining water-soluble dioxetane derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for refining by which unstabilized components can be removed and a stable water-soluble dioxetane derivative can be obtained. SOLUTION: This water-soluble dioxetane derivative is refined by bringing a solution containing the water-soluble dioxetane derivative into contact with a chelate resin and separately collecting the water-soluble dioxetane derivative.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying a water-soluble dioxetane derivative. Particularly, the present invention relates to a method useful for purifying a chemiluminescent water-soluble dioxetane derivative applicable to enzyme substrates or nucleic acid probes in chemiluminescence measurement.

[0002]

2. Description of the Related Art Water-soluble dioxetane derivatives are useful compounds, and their demand has been increasing in recent years. In particular, a water-soluble chemiluminescent dioxetane derivative is a very useful compound under conditions using a hydrophilic solvent such as for analysis of biological components. Water-soluble chemiluminescent dioxetane derivatives are generally stable, but are known to be decomposed by coexisting impurities. Therefore, attention must be paid to the temperature, light-shielding conditions, and the like. Regarding the removal of the impurities themselves,
For example, a method for purifying a water-soluble chemiluminescent dioxetane compound is described in Japanese Patent No. 2968811. That is, the 1,2-dioxetane water-soluble derivative containing impurities is purified by a reversed-phase chromatography method based on a base material having alkali stability. In Japanese Patent No. 2968811, a silica-based substrate which has been conventionally used is replaced with a reversed-phase chromatography method based on a substrate having alkali stability. As a result, the dioxetane compound can be separated and purified in a stable alkaline region, thereby preventing the decomposition of the 1,2-dioxetane derivative and effectively removing impurities.

[0003]

As a result of various studies, the present inventors have found that the use of a dioxetane derivative obtained by a conventional purification method results in high background or poor storage stability. Has been found to be a major problem. It has also been found that this is because in the purification of the water-soluble dioxetane derivative, there are destabilizing components that cannot be removed by only reversed-phase chromatography. Therefore, an object of the present invention is to provide a purification method capable of removing a destabilizing component and obtaining a stable water-soluble dioxetane derivative.

[0004]

Means for Solving the Problems To solve the above problems, the present inventors have made intensive studies and found the following effective solutions. That is, the present invention is a method for purifying a water-soluble dioxetane derivative, which comprises contacting a solution containing a water-soluble dioxetane derivative with a chelating resin, and then separating and recovering the water-soluble dioxetane derivative. Hereinafter, the present invention will be described in more detail.

The water-soluble dioxetane derivative to be purified in the present invention is not particularly limited as long as it is a water-soluble compound having a 1,2-dioxetane structure, and may have various functional groups such as a phosphate ester. . Preferably, 5-t-butyl-4,4-dimethyl-1- represented by the following formula:
(3′-phosphoryloxy) phenyl-2,6,7-trioxabicyclo [3.2.0] heptane disodium salt and the like are exemplified.

[0006]

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[0007] The chelate resin used in the present invention is not particularly limited as long as it has the ability to trap the metal ion by coordination with the metal ion. Preferably, the resin is a resin having iminodiacetic acid or the like as a functional group, and a styrene-divinylbenzene copolymer or the like is exemplified as the base matrix.

By bringing a solution containing a water-soluble dioxetane derivative into contact with the chelate resin and separating and recovering the water-soluble dioxetane derivative, a stable water-soluble dioxetane derivative from which a destabilizing component has been removed can be obtained.

Although the identity of the destabilizing component contained in the water-soluble dioxetane derivative has not been analyzed, the fact that a metal catalyst is used in the synthesis route of the water-soluble dioxetane derivative and that the organic solvent used is Since a trace amount of heavy metal is contained, the presence of heavy metal is considered as one of the destabilizing components, and it is possible to obtain a stable water-soluble dioxetane derivative by capturing and removing the heavy metal by a chelating resin. Conceivable.

[0010]

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to only these examples.

Example 1 Purification by Chelex resin 1) Purification by reverse phase chromatography of 5-membered ring dioxetane phosphate The above-mentioned 5-membered ring dioxetane phosphate (official name:
"5-t-butyl-4,4-dimethyl-1- (3'-phosphoryloxy) phenyl-2,6,7-trioxabicyclo [3.2.0] heptane disodium salt) was prepared by the following procedure. Purified, that is, JP-A-9-216887
According to the publication, a crude product (HPLC purity about 65%) after the completion of the 5-membered ring dioxetane phosphate synthesis step
6 mg was dissolved in eluent for preparative 1 (0.1% NaHCO ₃ aqueous solution: acetonitrile = 7: 3) to make 1.5 mL, and the following preparative 1 was performed.

1-1) Preparative 1 (salt exchange) Preparative column: YMC-Pack R & D P manufactured by YMC
polymer C18 300 × 20 mm l. D. Guard column: YMC-Guardpack manufactured by YMC
Polymer C18 50 × 20 mm l. D. Eluent: 0.1% NaHCO ₃ aqueous solution: acetonitrile = 7: 3 Flow rate: 5 mL / min The yield of preparative 1 was 85 mg. The same operation was repeated several times to finally obtain 152 mg of an intermediate purified product. This was dissolved in the eluent for preparative 2 and the following preparative 2 was performed.

1-2) Preparative 2 (desalting) Preparative column: YMC-Pack R & D P manufactured by YMC
polymer C18 300 × 20 mm l. D. Guard column: YMC-Guardpack manufactured by YMC
Polymer C18 50 × 20 mm l. D. Eluent: 0.1% NaHCO ₃ aqueous solution: acetonitrile = 8: 2 Flow rate: 5 mL / min The 5-membered ring dioxetane phosphate portion of the fraction was recovered and freeze-dried. As a result, 125 mg of white powder was finally obtained. Obtained.

FIG. 1 shows an HPLC chart of the fraction after fractionation. As is clear from FIG. 1, no impurities were confirmed from the reverse phase chromatography chart.

Analysis column: YMC-Pack R & D Polymer C18 manufactured by YMC 300 × 4.6 mm l. D. Eluent: A solution 0.1% NaHCO ₃ aqueous solution: acetonitrile = 9: 1 B solution 0.1% NaHCO ₃ aqueous solution: acetonitrile = 2: 8 Gradient: A: B = 100: 0 (1 minute hold) → 15 minutes → 0: 100 (1 minute hold) Flow rate: 1 mL / min.

2) Chelex resin (Chelex 10)
Pretreatment of 0): Chelex 100 is a chelating resin manufactured by Bio-Rad Co., and has a styrene-divinylbenzene copolymer as a base matrix and iminodiacetic acid as a functional group. Washing of this Chelex resin with pure water was repeated in advance. Take 50 μl of the washed Chelex resin and filter the centrifuge tube ULTRAFREE manufactured by Millipore.
-MC (0.22 μm) and using a small centrifugal filter CFM-200 manufactured by IWAKI (6000 rpm, 5 rpm).
Min) and excess water was removed by centrifugation.

3) Chelex treatment Next, 2 mg of the 5-membered ring dioxetane phosphate obtained in Preparative Step 2 was taken and dissolved in 200 μl of pure water. ULTRAFR filled with 100 μl of Chelex resin
After being added to EE-MC and mixed, a 5-membered ring dioxetane phosphate aqueous solution was separated and recovered from the Chelex resin by centrifugation using CFM-200.

Example 2 Investigation of Contribution of Kilex Resin Treatment to Substrate Performance Aqueous 5-membered dioxetane phosphate aqueous solution recovered from the Kilex treatment and untreated 5-membered ring dioxetane phosphate aqueous solution were subjected to the following alkaline phosphatase Nega. . Or Posi. (1 × 10 ⁻¹⁸ mol / ml) was used in an amount of 10 μl, and the amount of luminescence was measured. A luminometer LB96V manufactured by Berthold was used for the luminescence measurement, and the photometric conditions were measured for 1 second after 5 minutes. Table 1 shows the results.

Nega. : 50 mM Tris-HCl
(0.1% gelatin hydrolyzate, 1 mM MgCl ₂ ,
0.15 M NaCl, and 0.05% NaN ₃ ) Posi. : Nega alkaline phosphatase to a concentration of 10 ^-18 mol / ml. Adjusted with solution.

[0020]

[Table 1]

As shown in Table 1, when the Kilex treatment was performed, the high background luminescence (Nega.) Observed in the case of no treatment was reduced, and the luminescence in the presence of alkaline phosphatase (Posi. ) Was not affected.

Next, an untreated 5-membered ring dioxetane phosphate solution (10 mg / ml) or a chelex-treated 5-membered ring dioxetane phosphate solution (10 m / ml)
g / ml) was placed in a storage container and subjected to an accelerated deterioration test in an incubator under light-shielded conditions at 40 ° C for 4 days. Specifically, (1) a treated or untreated 5-membered ring dioxetane phosphate solution was stored in a light-shielded incubator at 40 ° C., and (2) after 4 days, each solution was taken out and an enhancer solution (Emerald from Tropix) was obtained.
-II), (3) alkaline phosphatase Posi. (Containing 1 × 10 ⁻¹⁸ mol / ml) was measured as a sample, and (4) the residual activity was determined by comparing the amount of luminescence with the measurement on day 0, and the stability was compared. Table 2 shows the results.

[0023]

[Table 2]

As is evident from the results in Table 2, the activity was reduced to 67% in the case of the untreated one, whereas the activity was hardly changed in the case of the Kilex treatment, and the stability was maintained. I understand. From this, it is considered that the storage stability of the 5-membered ring dioxetane phosphate was improved by the Kirex treatment.

[0025]

According to the purification method of the present invention, it has become possible to remove a destabilizing substance mixed in a water-soluble dioxetane derivative which could not be removed by a conventional method. As a result, when the purified water-soluble dioxetane derivative according to the present invention is used as a chemiluminescent substrate, various performances such as a reduction in background luminescence and an improvement in storage stability can be improved.

[Brief description of the drawings]

FIG. 1 is a diagram showing a chart of HPLC performed in Example 1.

Continued on the front page F term (reference) 2G054 AA06 AB07 BA03 BB01 BB02 BB04 BB05 BB11 BB12 BB13 BB20 CA22 CA28 CB02 CD01 CD04 CE02 EA01 EB05 FA22 FA36 JA09 JA10 4C071 AA01 BB01 CC13 EE03 FF15 GG03 BB10

Claims (2)

[Claims] 1. A method for purifying a water-soluble dioxetane derivative, comprising bringing a solution containing a water-soluble dioxetane derivative into contact with a chelating resin, and then separating and recovering the water-soluble dioxetane derivative. 2. The purification method according to claim 1, wherein the water-soluble dioxetane derivative has a phosphate ester.