JP3057887B2 - Novel maleimide derivative and fluorescent labeling method of compound having thiol group using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel maleimide derivative useful as a water-soluble fluorescent labeling reagent for a compound having a thiol group, and the fluorescence of a compound having a thiol group using the compound as a water-soluble fluorescent labeling reagent. It relates to a labeling method.

[0002]

BACKGROUND OF THE INVENTION After a substance to be measured is labeled with a fluorescent substance,
The method of measuring the substance using the fluorescence is widely used as a method of measuring a trace substance containing only about nanomoles to picomoles in a sample.

However, conventionally used reagents for fluorescently labeling a substance to be measured, ie, fluorescently labeled reagents such as fluorescein isothiocyanate (FITC), eosin and coumarin isothiocyanate, have a water solubility of their own. Due to its low cost, it had various problems. That is, for example, when fluorescently labeling a trace substance contained in an aqueous solution, the fluorescent labeling reagent as described above must be once dissolved and used in a polar organic solvent, so that the fluorescent labeling operation is complicated. In addition, since the fluorescence-labeled substance prepared using the fluorescent-labeling reagent as described above has reduced water solubility of itself, for example, a fluorescent-labeled antibody prepared using the fluorescent-labeling reagent is used. In some cases, it may not be possible to prepare an aqueous solution having a required concentration when another substance is detected.

For the purpose of solving these problems, a water-soluble fluorescent labeling reagent such as dansyl chloride, lucifer yellow, o-phthalaldehyde and the like, into which a water-soluble group such as a sulfo group or a carboxyl group has been introduced, has been developed. . However, the fluorescent labeling substances prepared using these are:
Although it was improved in water solubility, it had a problem that it could not be stored after preparation because of its poor storage stability.

On the other hand, aminopyridines such as 2-aminopyridine and 3-aminopyridine are water-soluble and very stable fluorescent substances themselves, and substances labeled with the aminopyridine also have excellent storage stability. In addition, when the labeling substance is a water-soluble substance, the water-soluble property is maintained as it is, and therefore, it is a compound widely used recently as a fluorescent labeling substance for carbohydrates (Biochemical and Biophysical Reagent).
search Communications vol. 85, p. 257, 1978).

However, there has been a problem in using aminopyridine itself as a labeling substance for physiologically active substances such as antibodies, enzymes, hormones, various factors (eg, growth factors, growth factors, etc.), haptens and the like. That is, as a fluorescent labeling method using aminopyridine, a method of binding to an aldehyde group of a labeling target substance via a Schiff base is generally used. In order to perform binding in a yield and quantitatively, the labeling operation must be performed under non-aqueous conditions and at a high temperature of about 90 ° C. (Agri. Biol. Chem., Vol. 5).
4,2169, 1990), which is a problem in that, for example, antibody activity, enzyme activity, biological activity, etc. of a labeled physiologically active substance such as an antibody are often inactivated.

[0007] Therefore, development of a labeling reagent capable of introducing a pyridylamino group into a physiologically active substance such as an antibody, an enzyme, a hormone, various factors, or a hapten under mild conditions has been desired.

[0008]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned circumstances, and is intended to provide a compound having a thiol group under mild conditions in water or a polar organic solvent or a mixture thereof in water or a polar organic solvent. It is an object of the present invention to provide a labeling reagent that can be easily introduced into DNA, and a method for fluorescently labeling a compound having a thiol group using the labeling reagent.

[0009]

The present invention provides a compound represented by the following general formula (1):

Embedded image (Wherein R 1 represents a 2-pyridylamino group or a 3-pyridylamino group, and n represents an integer of 1 to 4) or a salt thereof, and a compound represented by the above general formula (1) or a compound thereof. It is an invention of a method for fluorescently labeling a compound having a thiol group, comprising reacting a salt with a compound having a thiol group in water or a polar organic solvent, or a mixed solvent thereof.

The compound represented by the general formula (1) of the present invention is
For example, it can be easily synthesized as follows. That is, first,
For example, 2- or 3-halopyridine such as 2-chloropyridine, 2-bromopyridine, 3-chloropyridine, or 3-bromopyridine, and usually 2 to 30 moles per mole of the 2- or 3-halopyridine.
A mole, preferably 10 to 20 moles of an alkylenediamine (eg, ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, etc.) is reacted with stirring at 80 to 120 ° C. for 6 to 48 hours. After the reaction, the reaction solution is concentrated under reduced pressure, the target substance is extracted with chloroform, dichloromethane, etc., and after the solvent is distilled off, the residue is recrystallized from an appropriate solvent (for example, a mixed solvent of benzene and petroleum ether) to give, for example, An aminoalkylaminopyridine such as 2- (2-aminoethyl) aminopyridine is obtained. Next, this is usually combined with 1 to 5 mol, preferably 1 to 3 mol, per mol of aminoalkylaminopyridine.
One mole of N-alkoxycarbonylmaleimide (eg, N-methoxycarbonylmaleimide, N-ethoxycarbomylmaleimide, etc.) is added to a weak alkaline aqueous solution (eg, an aqueous solution of sodium hydrogencarbonate, an aqueous solution of potassium hydrogencarbonate, etc.).
The reaction is carried out with stirring at 3030 ° C. for 2 to 6 hours. After the reaction, the precipitate is collected by filtration, washed with water, and if necessary, suspended in a solvent such as dioxane or tetrahydrofuran for the purpose of completing the reaction, and cooled to 0 to 10 ° C to 1 mol of aminoalkylaminopyridine under cooling. Then, 0.5 to 2 mol of a base such as triethylamine, N-methylmorpholine, N, N-diisopropylethylamine or the like is added, and the mixture is stirred and reacted at the same temperature for 1 to 2 hours. The reaction is carried out with stirring for hours. After the reaction, the solvent is distilled off, and the target substance is extracted from the residue with a solvent such as chloroform or dichloromethane. By evaporating the solvent from the extract and purifying the residue by recrystallization with a solvent such as diethyl ether or petroleum ether, the compound of the present invention represented by the general formula (1) is obtained. When this is further reacted with a mineral acid such as hydrochloric acid or sulfuric acid or an organic acid such as acetic acid, a mineral acid salt or an organic acid salt of the compound represented by the general formula (1) is obtained.

The N-alkoxycarbonylmaleimide used as the maleimidating reagent in the above reaction was prepared according to the method of Keller et al. (Helvetica Chimica Acta, vol. 58, 531, 19).
It is sufficient to use one synthesized according to 75).

The fluorescent labeling method of the present invention using the compound of the present invention as a fluorescent labeling reagent can be carried out, for example, by adding 1 to 10 equivalents, preferably 1 to 10 equivalents of a compound having a thiol group to 1 equivalent of the thiol group of the compound. 1 to 5 equivalents, more preferably 2 to 4 equivalents of the compound of the present invention is added to water or an alcohol such as methanol or ethanol, a polar organic solvent such as acetone, acetonitrile or dimethylformamide, or a polar organic solvent thereof. The reaction can be easily carried out by reacting the mixture in a mixed solvent of water and water (hereinafter collectively referred to as “aqueous solvent etc.”) at room temperature to slightly heated.

[0013] The pH of the reaction solution at the time of carrying out the above reaction is
Is not particularly limited as long as it does not affect the stability of the compound having a thiol group. However, considering the reactivity of the compound of the present invention, neutral to slightly alkaline (pH 6 to 8)
Degree) is desirable. In addition, the reaction solution does not have a thiol group such as a phosphate or a bicarbonate as long as it does not inhibit the reaction between the compound of the present invention as a water-soluble fluorescent labeling reagent and a thiol group. A buffer, a preservative, a surfactant, a stabilizer and the like may be present together.

The method of the present invention does not require the use of a polar organic solvent during the fluorescent labeling operation as in the conventional fluorescent labeling with dansyl chloride. Therefore, for example, antibodies, enzymes, hormones, various factors (for example, growth Factors, growth factors, etc.), haptens and the like can be easily labeled with a physiologically active substance which is easily denatured by an organic solvent.

The compound having a thiol group that can be fluorescently labeled by the method of the present invention is a compound having a property of being dissolved in an aqueous solvent or the like at a concentration of 1 picomolar or more,
And a compound having a thiol group in the molecule, or a compound capable of generating a free thiol group by reductive cleavage of a disulfide bond in the molecule with a reducing agent such as 2-mercaptoethanol or dithiothreitol. And natural and synthetic products without particular limitation. More specifically, for example, cysteine,
Amino acids such as N-acetylcysteine and cystine, for example, peptides such as glutathione, insulin A chain, insulin B chain, calcitonin, endothelin, oxytocin, vasopressin, for example, immunoglobulin, albumin, transferrin, luteinizing hormone (LH), follicle stimulation Hormone (FSH), thyroid stimulating hormone (TS)
H), glycoproteins (or proteins) such as human chorionic gonadotropin (hCG), and cysteines (or proteins) obtained by treating these glycoproteins (or proteins) with proteases such as trypsin, chymotrypsin, pepsin, papain, and V8 protease. Preferred examples include, but are not limited to, cystine) -containing glycopeptide fragments (or peptide fragments).

The type and number of sugar chains in the glycoprotein (or glycopeptide) having a thiol group which can be fluorescently labeled by the method of the present invention are not particularly limited. , Mannose, galactose, fucose, sialic acid, N-acetylglucosamine, N-
A sugar residue such as acetylgalactosamine, which has about 1 to 10 sugar residues, is preferred. There is no particular limitation on the type of bond between the sugar residues of the sugar chain. For example, α1 → 4, β1 → 4, α1 → 6, α2 →
3, β1 → 2, β1 → 3, α2 → 6, α1 → 3, α1 →
2, β1 → 6, etc. However, when a glycoprotein (glycopeptide) fluorescently labeled by the method of the present invention is used as an acceptor for glycosyltransferase or a substrate for glycolytic enzyme, the sugar chain of the glycoprotein (or glycopeptide) is Needless to say, it must be a substance that can serve as an acceptor for glycosyltransferase or a substrate for glycolytic enzyme.

Since the water-soluble fluorescent labeling reagent of the present invention has a property of emitting strong fluorescence only when bound to a thiol group, even if the unreacted labeling reagent coexists, the thiol group can be used. Since it has almost no effect on the detection of cysteine, it can be easily used for detecting and quantifying free thiol groups in samples, and for confirming the presence or absence of cysteine residues in fragments generated by protease treatment of proteins and peptides. It is.

Amino acids, peptides, proteins, glycopeptides, glycoproteins, and the like, which have been conventionally labeled with fluorescent labeling agents such as dansyl chloride, phenylthiohydantoin (PTH), and lucifer yellow, are stable upon storage. Because of poor properties, even a standard product had to be prepared before use. On the other hand, amino acids, peptides, proteins, glycopeptides, glycoproteins, and the like, which are fluorescently labeled using the compound of the present invention, have extremely high storage stability, and thus, those prepared in advance can be used as appropriate as standard products. It is. The detection sensitivity of amino acids, peptides, proteins, glycopeptides, glycoproteins and the like fluorescently labeled with the compound of the present invention is extremely high, several picomoles.

Other fluorescent labeling agents for thiol groups include
7-halo-2,1,3-benzoxadiazole derivatives such as 7-fluoro-4-sulfamoyl-2,1,3, -benzoxadiazole and 2,2′-dihydroxy-6,6′- Although there are disulfide compounds such as dinaphthyl sulfide, the former has a problem in terms of specificity to a thiol group, and the latter has a problem in that the labeling reaction takes time. On the other hand, when the compound of the present invention is used as a fluorescent labeling agent for a thiol group, all of these problems are solved.

When the compound of the present invention is used as a water-soluble fluorescent labeling reagent for a compound having a thiol group, the compound represented by the general formula (1)
In the above, the fluorescence when the compound in which n is 2 binds to the thiol group is the strongest. In the compound represented by the general formula (1), the compound in which n is 0 has low fluorescence intensity generated after the reaction with the thiol group, and both the excitation wavelength and the measurement wavelength at the time of fluorescence detection are on the short wavelength side. It is difficult to use for the purpose of the present invention because it has the property of being easily affected by coexisting substances due to migration.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[0022]

EXAMPLES Example 1. Synthesis of 2-pyridylaminoethylmaleimide hydrochloride 1) Synthesis of 2- (2-aminoethyl) aminopyridine 85 g of 2-chloropyridine and 450 g of ethylenediamine were mixed with 120 g of
The reaction was carried out at 0 ° C. for 8 hours with stirring. After completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the desired product was extracted with chloroform. Chloroform was distilled off from the extract, and the residue was recrystallized from benzene: petroleum ether (1: 5) to obtain 86 g of 2- (2-aminoethyl) aminopyridine as pale brown crystals. Yield = 83.7%. Melting point: 36-40C. IRνmax (neat) cm ^-1 : 3500-3100 (NH), 3000-2800 (-C
H2-), 780 (pyridine ring). ¹ H-NMR (60 MHz, CDCl ₃ ) δ ppm: 1.64 (s, 2H, NH ₂ ),
_{2.82~3.58 (m, 4H, -CH 2} CH 2 -), 6.40~8.24 (m, 4H, pyridine ring). MS: m / z 137 (M ^<+> ). 2) Synthesis of N-ethoxycarbonylmaleimide 29 g of maleimide, 33 ml of N-methylmorpholine and 1300 ml of ethyl acetate were stirred and mixed under ice cooling, and a mixture of 29 ml of ethyl chlorocarbonate and 200 ml of ethyl acetate was added dropwise under ice cooling. After that, the mixture was further stirred and reacted under ice cooling for 30 minutes. After the completion of the reaction, the precipitate was filtered off and the filtrate was concentrated under reduced pressure. The residue was recrystallized from ethyl acetate: diethyl ether (1:10), and the crystals were collected by filtration and washed with diethyl ether to obtain purple crystals of N-ethoxycarbonylmaleimide (37 g). Yield = 79.8%. Melting point: 57-59 [deg.] C. IRνmax (KBr) cm ^-1 : 3100 (CH), 1780 (ester), 171
0 (amide). ¹ H-NMR (60 MHz, CDCl ₃ ) δ ppm: 1.42 (t, 3H, -C
_{H 3), 4.47 (dd,} 2H, -CH 2 -), 6.86 (s, 2H, maleimide). MS: m / z 168 (M ^<+> ). 3) Synthesis of 2-pyridylaminoethylmaleimide hydrochloride 30 g of 2- (2-aminoethyl) aminopyridine obtained in 1 above was stirred in 1000 ml of a saturated aqueous solution of sodium hydrogen carbonate under ice-cooling,
After dissolving, 37 g of N-ethoxycarbonylmaleimide obtained in the above 2 was added, and the mixture was stirred and reacted under ice cooling for 1 hour. After completion of the reaction, the precipitated crystals were collected by filtration, washed with pure water, and the obtained crystals were suspended in 500 ml of dioxane. To this, 15 ml of triethylamine was added under ice-cooling, and the mixture was stirred and reacted for 1 hour. Then, the mixture was further stirred and reacted at room temperature for 1 hour. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the desired product was extracted from the obtained residue with chloroform. Next, the extract was concentrated again under reduced pressure, and the obtained residue was recrystallized from diethyl ether. The obtained crystals were dissolved in 3N-hydrochloric acid (100 ml), allowed to stand at room temperature for 8 hours, the reaction solution was dried under reduced pressure, and the residue was dissolved in 260 ml of a mixed solution of ethanol: methanol: chloroform = 20: 5: 1 by heating. After cooling the reaction solution, the precipitated crystals were collected by filtration to obtain 38 g of 2-pyridylaminoethylmaleimide hydrochloride as white crystals. Yield = 65.5%. Melting point: 200 DEG C .- (dec.). IRνmax (KBr) cm ^-1 : 3300 to 3000 (NH), 1700 (imide),
1600 (amide), 700 (pyridine ring). ¹ H-NMR (60 MHz, CDCl ₃ ): 3.55 (dd, 2H, N-CH _2- , J =
5.13), 3.78 (t, 2H , -CH 2 -N (CO) 2, J = 5.13), 6.40~8.05
(m, 6H, pyridine ring and maleimide). MS: m / z 264 (M ^<+> ).

Embodiment 2 FIG. Fluorescent labeling of N-acetylcysteine (Operation method) To a solution of 1.63 g of N-acetylcysteine in 10 ml of distilled water, 2.65 g of 2-pyridylaminoethylmaleimide hydrochloride obtained in Example 1 above was added, and the mixture was added at room temperature. After stirring and reacting for 10 hours, 10 μl of the reaction solution was analyzed by high performance liquid chromatography (HPLC). The HPLC measurement conditions are as follows. -Equipment: SHIMADSU LC-6A (manufactured by Shimadzu Corporation). -Column: Wakopak 5C18 (4.6 x 150 mm) (manufactured by Wako Pure Chemical Industries, Ltd.). -Eluent A: 0.1 M ammonium acetate buffer (pH 4.0). Eluent B: 0.1 M ammonium acetate buffer (pH 4.0) containing 50% acetonitrile.・ Gradient: A → B 0 → 15 minutes (linear gradient) ・ Eluent flow rate: 1.5 ml / min. -Column temperature: 55 ° C. -Fluorescence detection wavelength: excitation wavelength 320nm and fluorescence wavelength 400nm. (Results) The analysis results by HPLC are shown in FIG. In the figures, 1 is the analysis result by HPLC obtained by using a solution obtained by reacting N-acetylcysteine and 2-pyridylaminoethylmaleimide hydrochloride as a sample, and 2 is the distillation result containing only 2-pyridylaminoethylmaleimide hydrochloride. The analysis results by HPLC obtained using water as a sample are shown respectively. As is clear from FIG. 1, 2-pyridylaminoethylmaleimide itself elutes at 9.22 minutes and forms an extremely weak peak.
It can be seen that the minute peak disappears and a new fluorescent peak appears at 8.89 minutes instead. From this result, the compound of the present invention reacted with the thiol group promptly and at a high conversion,
It can be seen that the obtained reaction product emits strong fluorescence.

Embodiment 3 FIG. Fluorescence labeling of glutathione and its quantification (Fluorescent labeling reagent solution) The 2-pyridylaminoethylmaleimide hydrochloride obtained in Example 1 was dissolved in distilled water to give 0.1 M
The solution was used as a fluorescent labeling reagent. (Reaction buffer) Sodium dihydrogen phosphate dihydrate 15.6
g was dissolved in 90 ml of distilled water, the pH was adjusted to 7.0 with a 1N sodium hydroxide solution, and the total volume was adjusted to 100 ml, which was used as a reaction buffer. (Sample) Glutathione (manufactured by Wako Pure Chemical Industries, Ltd.) is appropriately dissolved in distilled water, and 1, 5, 10, 50, 100 or 200 μg / ml
Was prepared and used as a sample. (Operation method) 20 µl of a fluorescent labeling reagent, 60 µl of a reaction buffer, and 20 µl of a sample having a predetermined concentration were mixed and reacted at 37 ° C for 3 hours. After the reaction is completed, take 10 μl of the reaction solution, add 200 ml of distilled water
μl was added and mixed well, and 100 μl thereof was analyzed by HPLC. The HPLC measurement conditions are as follows. -Equipment: SHIMADSU LC-6A (manufactured by Shimadzu Corporation).・ Column: Wakopak 5C18 (4.6 x 150 mm, Wako Pure Chemical Industries)
Co., Ltd.). Eluent A: 0.1 M ammonium acetate buffer (pH 4.0). Eluent B: 0.1 M ammonium acetate buffer (pH 4.0) containing 60% acetonitrile.・ Gradient: A 0 → 5 minutes A → B 5 → 20 minutes (linear gradient) B 20 → 25 minutes ・ Eluent flow rate: 1.5 ml / min. -Column temperature: 55 ° C. -Fluorescence detection wavelength: excitation wavelength 314 nm and fluorescence wavelength 386 nm. (Results) FIG. 2 is a graph showing the relationship between the glutathione concentration and the fluorescence intensity of the fluorescence-labeled glutathione.
As a result of analysis by LC, it was found that the reaction product of 2-pyridylaminoethylmaleimide and glutathione eluted at 17.6 minutes. ). FIG. 2 shows that the graph showing the relationship between the glutathione concentration in the sample and the fluorescence intensity of the fluorescence-labeled glutathione obtained using the sample shows good linearity. From the above results, it can be seen that the compound of the present invention reacts quantitatively with glutathione and that the amount of glutathione in a sample can be quantified with high sensitivity by fluorescently labeling glutathione using the compound of the present invention as a fluorescent labeling reagent. I understood.

As described above, the present invention provides a water-soluble fluorescent label which can easily introduce a pyridylamino group, which is extremely effective for fluorescent labeling of a trace substance, into a compound having a thiol group in an aqueous solvent or the like. The present invention provides a method for introducing a pyridylamino group into a compound having a thiol group using the same, and according to the present invention, antibodies, enzymes, hormones, various factors, haptens, etc. It has an excellent effect in that it is possible to introduce a pyridylamino group, which was difficult to introduce into a physiologically active substance while retaining these activities, while allowing these activities to be retained under mild conditions. It is a great invention that contributes to the industry.

[Brief description of the drawings]

FIG. 1 shows the results of high-performance liquid chromatography (HPLC) analysis of N-acetylcysteine fluorescently labeled with the compound of the present invention, obtained in Example 2.

FIG. 2 is a graph showing the relationship between the concentration (μg / ml) of glutathione fluorescently labeled with the compound of the present invention and the fluorescence intensity (10-3 μV) obtained in Example 3.

[Explanation of symbols]

Each symbol in FIG. 1 indicates the following. Analysis results by HPLC obtained using a solution obtained by reacting 1 N-acetylcysteine with 2-pyridylaminoethylmaleimide hydrochloride as a sample. 2 Analysis results by HPLC obtained using distilled water containing only 2-pyridylaminoethylmaleimide hydrochloride as a sample.

────────────────────────────────────────────────── ─── Continuing from the front page Examiner Tamotsu Tominaga (58) Field surveyed (Int. Cl. ⁷ , DB name) C07D 401/12 C09K 11/06 G01N 33/533 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] (1) The following general formula (1): (Wherein, R 1 represents a 2-pyridylamino group or a 3-pyridylamino group, and n represents an integer of 1 to 4) or a salt thereof. (2) The following general formula (1): Wherein R 1 and n are as defined above, or a salt thereof, and a compound having a thiol group in water or a polar organic solvent, or a mixed solvent thereof. Fluorescent labeling of a compound having a thiol group. 3. The compound having a thiol group is an amino acid,
3. The fluorescent labeling method according to claim 2, which is a peptide, protein, glycopeptide or glycoprotein.