JPS5951270A - 2-oxabenzo-1,3-diazole derivative and composition containing it for measuring thiol - Google Patents

Abstract

NEW MATERIAL:7-Fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid shown by the formula I and its salt. USE:A fluorescent agent. It is reacted with a specimen to give a fluorescent derivative, whose intensity is measured to determine a thiol group. PROCESS:7-Fluoro-2-oxabenzo-1,3-diazole shown by the formula II is reacted with a sulfonating agent such as sulfuric acid, fuming sulfuric acid, sulfuryl chloride, etc. in a solvent such as acetone, THF, benzene, ethyl acetate, etc. at 100-140 deg.C for 1-24hr, to give a compound shown by the formula I .

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel compound or a salt thereof useful for detecting and quantifying a thiol group, a composition for quantifying a thiol group containing the compound or a salt thereof as a constituent component, and a composition for quantifying a thiol group, which is useful for detecting and quantifying a thiol group. The present invention relates to a method for quantifying thiol groups, which comprises using thiol group or a salt thereof. As a fluorescent reagent for detecting and quantifying thiol groups, N-substituted maleimide (Machida et al.
, Pharm.

Bull, (Tokyo) 25.1678 (19
77), Nara et al. Agric.

Biol, Ohem, 42.793 (1978) containing dansyl acyridine (Fah, ey R, O, etc.
1. Biocbem, 107 + 1 (1980
) + bimain (Lan1anyr B, P, etc. J,
Ohromatogr, 2228249 (1981)
and ammonium 7-chlorotetrabenzo-2-oxa-1
, 3-diazole-4-sulfo and *-) (hereinafter r8I) D-0β ammonium salt JJIG
5) (Andrews, J.L., et al.Arc
h, Biochem, Biophys, 214.

386 (1982)) are known.

Among these, 5BD-CI! , is '774 for water
Compared to other fluorescent assays, it has excellent Wl properties and good stability of the complex with thiol groups, and is therefore more suitable for biological purposes such as detection and quantification of thiol groups in proteins. Its wide applicability has been previously mentioned. (Antlrews
or, L., etc. Arch, Biochem, 13
iopbys, 214 r 38 G (1982)
) However, the reactivity of 5Bn-ai with respect to thiol groups is low, and the inventors have found that 5Bn-ai has a low reactivity with respect to thiol groups, and the present inventors have found that 5Bn-ai has a low reactivity with respect to thiol groups, and as a result, it has been found that
Furthermore, even if the reaction was carried out at room temperature for 24 hours, the reaction rate with thiol groups was only a few percent, and therefore it could not be used for the determination of low v'2 thiol groups.

Under such current circumstances, the present inventor surprisingly found that 8B
A new compound in which the chloro group at position 7 of D-CQ is replaced with a fluoro group has extremely high reactivity with thiol groups,
Therefore, it was discovered that a very small amount of thiol group can be used,
The present invention was completed based on this knowledge.

That is, the present invention provides 7-fluorobenzo-2-oxa-1,3 represented by the following borrowing formula:
- A composition for quantifying a thiol group containing diazole-4-sulfonic acid (hereinafter abbreviated as rsnD-FJ) or a salt thereof and the 8BD-F or a salt thereof as a fluorescent agent, further comprising the 5I3D-F or a salt thereof. This is a method for quantifying thiol groups, which consists of adding a salt to a sample, causing a reaction, and measuring the fluorescence intensity of the resulting fluorescent M2S substance.

The compound of the present invention, SBD-F, is a new compound that has not been described in any literature, and can be obtained, for example, by sulfonating 7-fluoro-2-oxabenzo-1,3-diazole as shown in the reaction diagram below.

Examples include sulfur chloride, dioxane adduct, chlorsulfur, sulfuryl chloride, etc., but oleum is preferable.

Chemicals that can be used in the reaction are acetone, tetrahydrofuran, benzene, diethyl ether, dimethylformamide, pyridine, and acetonitrile.

Inert 4'SS such as dioxane, chloroform, dichloromethane, dichloroethane, ethyl acetate? jrI
J% is used. Among these, hydrophilic solvents can also be used in combination with water.

The reaction is usually carried out under heating. That is, usually 50
-150°C, preferably 100-1
The temperature is 40°C.

The reaction time varies depending on the reaction temperature, the compound used in the reaction, the solvent, etc., but is generally 5 to 48 hours.
Preferably, the time period is appropriately selected within the range of 1 to 24 hours.

Isolation and purification of the target product from the reaction mixture can be easily carried out according to conventional methods. For example, extraction with organic solvents such as dichloromethane, polyproform, and ethyl acetate;
Or activated carbon, silica gel.

Various types of chromatography using ion exchange resins, dextran crosslinked polymers, porous polymers of styrene or acrylic esters, etc. can be applied.

Starting material 7-fluoro-2-oxabenzo-1,3-
Diazoles are known compounds, for example the method of Nunno et al. (J, Ohem, 5oc(c) 1433.1970
).

Since SBD-1i' thus obtained is usually an oily substance, it can be obtained as a crystalline salt of 5BD-F by a conventional method, that is, by treating the sulfonic acid group with an equimolar amount of a base. .

If the base does not affect the measurement system, it will ionically combine with the sulfonic acid group in 5BD-F to form a salt. salts with alkaline earth metals such as calcium and magnesium, salts with inorganic or organic bases such as ammonium salts, salts with amines such as ethanolamine, triethylamine, and dicyclohexylamine. I can do something.

Is the compound of the present invention 5BD-F itself? It does not show any phosphorescence, but shows remarkable fluorescence after selectively reacting with thiol groups. Therefore, if the intensity of the fluorescence is investigated, it is possible to detect and determine the thiol in the sample. can do. When quantifying thiol groups using a composition containing the compound of the present invention, SBD-F or a salt thereof, or a composition containing SBD-F or a salt thereof as a fluorescent agent, good reactivity can be obtained. The preferred conditions for this are that the amount of SBD-F added to the sample is preferably 10 mol or more, particularly preferably 102 to 104 mol, per 1 mol of thiol group, and I"' is preferably alkaline. 9~
11. Reaction temperature is 20°C or higher, preferably 30-70°C,
In order to maintain the pH during the reaction time, it is preferable to use a diluted solution, and examples of the diluted solution include sulfuric acid solution, glycine sedative solution, Tris-HCl buffer, etc.
Preliminary solution of Hou Iakj2 is preferable. The excitation wavelength and fluorescence wavelength used to excite the fluorescent derivative obtained by bonding with a thiol group and measure the resulting fluorescence intensity are 370 to 390 thnt%, preferably 380 nv.
The wavelength of the t- and gif light can be selected from 490 to 530 nm, preferably 515π7.

Although the compound of the present invention, 5I3D-F, has many characteristics,
They are listed as follows.

1) Due to high reactivity with thiol groups, the detection limit can be significantly lowered.

2) Since the blind value is low, the measurement sensitivity is good.

3) High solubility in water or biological fluids.

4) High stability in solution state. In the liquid state, it is stable for more than a week at room temperature.

5) High stability of derivatives with thiol groups. pH9.5
It is stable for more than a week in the refrigerator under these conditions.

The compound of the present invention, 8BD-F, has the above-mentioned characteristics.
Not only can it be applied to the fluorescence quantitative analysis of thiol compounds in quantities of 10 to 1O-8ffi, but it can also be applied to a wide range of biological samples.
'F, camellia, histological examination to detect thiol groups in fields and mountains, quantification of proteins and peptides containing thimer, research on the function of thimer in enzymes, +1! , I.G.
, Thin Ji::+1,111 Detection of thiol groups in living organisms of 1 x cotton and t of these living organisms'+7'j precepts,! , 7-zo and (n I4' II)
Secretion of ii? 1 other fi((, i bed nl(blasphemy)
The basics of automation of clinical analysis and their automation 4J, biochemistry.

It can be applied to a wide range of non-'1:i studies, including chemistry, basic science, and clinical research.

1 (3 is preferably in the form of a salt, and facing the salt &J',
'Itt solid objects that are processed because they are solid when wet;
For example, it can be used in the form of tablets, dry descabber products, etc. together with other necessary ingredients such as chelating agents.

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples unless the gist thereof is exceeded.

Example 1 Synthesis of 5BD-F 0.51 of 7-fluoro-2-oxabenzo-1,3-diazole was added with 60% smoke-emitting solution: 1
Heat and reflux at 30-140°C for 4 hours. After the reaction is complete,
Cool the reaction solution in a water bath. 4N sodium hydroxide 1
'i', add liquid to make 1u113~4 and benzene 500
Add v and e and extract. 11 layers with branches 141:
After drying over sodium sodium chloride and filtering, the lachrymal fluid was strained under regional pressure to obtain an oily substance (0.47).

IRνKBY (oil): 1235 r 1190a
x Example S 13 D -F Ammonium salt! ! Construction example 1
7-sulfo0-2-oxabenzo-1,3- obtained in
Diazo-/I/-4-7, IL'rhnvJf2
After dissolving 4g in water, add 2g while cooling in water.
Add 8% ammonia water to adjust pH to 3.

Ethanol lβ is added to the n゛f wave and filtered, and the precipitate is washed with a small amount of ethanol, and the washing liquid and added liquid are combined with polystyrene 4y? fli'i'L S 110 P
Column chromatography (1, Fic + ηX 50 cm
) and let it come out with water.

YIV: Add 1' of ethanol to the liquid and add 100 ml of ethanol.
After azeotroping at ~110°C, the residual oil was dissolved in a mixture of acetonitrile and ethanol (9:1) and filtered. The furnace liquid was reduced by r% under reduced pressure to obtain 0.4 g of white needle-like crystals.

Decomposition point 290~300℃ Elemental analysis Calculated value as 06H5N304SF (season 0,
30.64 t 11 r 2-57 p N s l
7.87 Actual value (phantom 0, 30.80; H, 2, 52;
Pe, 13.14N MR (DMSO-Drs, 10
0MHz) δ (Ign+) Near, 816 (S, OH)
, 7.767CB, 0I-1> , 7.74(S, 0
IZ), 7.691 (S, 0) I), 7.505 (8,
0)I).

7.429 (S, OH), 7.400 (S, OH),
7.324 (S, 0) (), 7.108 (S, NH4)
(3,1:lXl03), 283(3,17X10
3), 313 (4,15X10) Example 3゜EDTA 2Na (2mA4), glutathione (10 μM). I M yh @ Loose can liquid (
pH 9,5) 2 ml and 5BD-F ammonium salt or 5J3D-On ammonium salt 1. Of? 0.1 M Hounse buffer (pH (9,5) 2
Two groups of specimen samples are prepared by mixing ml. These two groups of specimens F1 were reacted for various fixed times in a constant temperature bath set at 60±1°C, and after the reaction was completed, each specimen sample was cooled with ice water, and then heated using a Hitachi spectrophotometer 650-108. The fluorescence intensity was determined at t(l) using an excitation wavelength of 380 nm and a fluorescence wavelength of 515 μm.

The results are shown in Figure 1.

Example 4゜ The detection limits were determined for the following six types of substances. .

・Geltadeone Coenzyme Human - EDTA 2Na (1@
M) Contains 0.1. Prepare M borate buffer (pH 9,5).

Add 5BD-'f' ammonium salt (21:re) to each sample sample diluted to an appropriate degree with the same buffer solution.
0.1 M borax slow solution (PI
(9,5)2? 1 of the mixture was added, and the mixture was allowed to react for 1 hour in a constant temperature &V adjusted to 60±1°C.

After the reaction was completed, each test sample was cooled in ice water at °C, and the excitation wavelength was 380 nm using a fluorophotometer as in Example 2.
The fluorescence intensity was measured at a fluorescence wavelength of 5155 m.

Note that the detection limit was set to twice that of a blind test. Furthermore, in parallel to the above experiment, a control experiment using 5 μM of alanine and proline was conducted. The results are shown in Table 1.

Table I Rice: Not detected Example 5 Preparation of calibration cotton FiDTA pH 9°5 containing 2 mM 5BD-F ammonium salt 0, IM boric acid pickle solution containing 2mM 5BD-F ammonium salt (hereinafter referred to as t(s) (abbreviated as sample) are provided.

Next, each W, q, Y: pH containing % glutathione
9.5 ED'I'A 2Na (1,t+tM)-containing 0.1M boric acid buffer solution was applied to each sample 1 me k- + each ■ sample ] rye was added, and the fluorescence intensity was increased to 1!l using the same method as in Example 3.
'ii:Z L/ta.

In addition, in parallel to the above fruit 1, p which does not contain glutathione
H9,5 EDTA 0.1M containing 2Na (1?nM)
Hou r;i! A blind test was conducted using only the Rissho liquid. Table 11 shows the results.
In addition, the plot diagram (calibration curve) is shown in Figure 2.

Table■ Excitation wavelength 380 nm Fluorescent wavelength 51? l’1lL

[Brief explanation of the drawing]

Figure 1 shows the reactivity with glutathione. In the figure, -0- indicates the results for 8BD-F, and -0- indicates the results for 5BD-OJ2. FIG. 2 shows the calibration curve of Example 5. Applicant: Imai - Western mail, figure 1 time (phantom mail, figure 2 L)

Claims (3)

[Claims] (1) Represented by the following formula: 7-fluorobenzo 2-oxa = 1,3
-Diazole-4-sulfonic acid or its temperature. (2) 7-Fluotetrabenzo 2-oxa-1,3 represented by the following I/f formula
- A composition for quantifying thiol groups containing diazole-4-sulfonic acid or a salt thereof as a fluorescent agent. (3) 7-fluorobenzo-2-oxa-1,3 represented by the following formula
- A method for quantifying thiol groups, which comprises adding diazole-4-sulfonic acid or a salt thereof to a sample specimen, causing a reaction, and measuring the fluorescence intensity of the resulting fluorescent derivative.