JP7113446B2 - A no-wash protein gel stain based on pyrene - Google Patents

Description

TECHNICAL FIELD The present invention relates to a gel stain using pyrene as a basic skeleton that does not require destaining and washing, and a protein staining detection method using the stain, for detecting a protein migration gel by electrophoresis.

BACKGROUND OF THE INVENTION Peptides and proteins are commonly detected and evaluated using gel electrophoresis, by solution quantification assays, or by detection on solid supports such as filter membranes. Minor proteins are not visible, but usually must be stained before they can be identified.

The two most common methods of staining proteins in gels are Coomassie Brilliant Blue (CBB) staining and silver staining. Silver staining is 100-1000 times more sensitive than CBB staining for certain proteins, but both methods have disadvantages. In contrast, for protein detection, the use of luminescent reagents, such as fluorescent, phosphorescent, or chemiluminescent reagents, greatly improves sensitivity and potentially increases linear quantification range. while simultaneously increasing the ease of use of the staining reagent.

A method using a fluorescent dye (fluorescence detection system) is widely used as a method for detecting a migration pattern in two-dimensional electrophoresis. For example, in a fluorescence detection system, it is known to stain electrophoresed proteins using Sypro (registered trademark) Ruby (Lonza Japan Co., Ltd.) as a gel staining reagent. Since the electrophoresis layer gel itself is also strongly stained, it is necessary to wash the gel once after staining with a fluorescent dye and perform a destaining operation. Usually, this washing and destaining operation takes 30 minutes or more. . On the other hand, fluorescent stains that can be detected by simply rinsing the gel surface after staining have also been reported (Non-Patent Documents 1 to 3), but rapid and simple detection methods that do not require washing or rinsing are desirable in research settings. It is rare.

Suzuki, Y.; Takagi, N.; Chimuro, T.; Shinohara, A.; Sakaguchi, N.; Suzuki, Y., Analytical Methods 2013, 5, 2174 Suzuki, Y.; Takagi, N.; Sano, T.; Chimuro, T., Electrophoresis 2013, 34, 2464-2472.

An object of the present invention is to provide a no-wash protein gel staining agent having pyrene as a basic skeleton, which can reduce background noise and obtain sufficient detection sensitivity in protein detection after electrophoresis.

The present inventors found that various pyrene derivatives do not cause staining of the gel itself in protein detection after electrophoresis compared to the use of conventional fluorescent staining agents, and that washing and destaining operations are unnecessary. and completed the present invention.

That is, the present invention is as follows.
[1] A no-wash protein gel stain containing a pyrene derivative.

[2] According to [1], wherein the pyrene derivative is selected from the group consisting of 1-pyrenemethanol, pyrenylmethyl ether derivatives, 1-pyrenecarboxylic acid derivatives, sugar-modified pyrenylmethyl derivatives, and pyrene-fluorescein conjugates. of no-wash protein gel stains.

[3] The pyrenyl methyl ether derivative has the following structure:

(In the formula,
R ₁ is selected from the group consisting of —OH, —C _1-6 alkyl, —OC _1-6 alkyl, —OTs, —N ₃ , —NH ₂ ; and m is an integer from 0 to 10)
The no-wash protein gel staining agent according to [2].

[4] The pyrenyl methyl ether derivative is the following:

The no-wash protein gel staining agent according to [3].

[5] The 1-pyrenecarboxylic acid derivative has the following structure:

(In the formula,
R ₂ is selected from the group consisting of —OH, —C _1-6 alkyl, —OC _1-6 alkyl, —OTs, —N ₃ , —NH ₂ ; and n is an integer from 0 to 10)
The no-wash protein gel staining agent according to [2].

[6] The 1-pyrenecarboxylic acid derivative is as follows:

The no-wash protein gel staining agent according to [2].

[7] The sugar-modified pyrenylmethyl derivative has the following:

The no-wash protein gel staining agent according to [2].

[8]
The pyrene-fluorescein conjugate is:

The no-wash protein gel staining agent according to [2].

[9] A staining solution containing the no-wash protein gel staining agent according to any one of [1] to [8], wherein the protein electrophoresed on the gel is fixed on the gel using a fixing solution. A protein staining detection method characterized by staining a protein immobilized on a gel using and obtaining a gel image after irradiating with ultraviolet (UV) light without destaining and washing after staining.

[10] A protein staining detection kit comprising the no-wash protein gel staining agent according to any one of [1] to [8].

According to the present invention, in protein detection after electrophoresis, various pyrene derivatives do not stain the gel itself as compared to the case of using a conventional fluorescent stain, and washing and destaining operations are unnecessary.

The results of staining ability screening of pyrene derivatives (19 types) using a one-dimensional gel are shown. In the staining ability screening, an aqueous solution in which the pyrene derivative was suspended at 10 μM was used (except for compound 2, a 22 μM aqueous solution was used). FIG. 3 is a diagram showing the relationship between the staining ability and hydrophobicity (predicted Log value) of pyrene derivatives. The numbers in the figure correspond to the numbers of the compounds synthesized in Examples. The results of staining a two-dimensional electrophoresis gel are shown. A is a photographed image stained with Sypro Ruby (Sigma-Aldrich) solution as a control, and B and C are stained with methyl ether (compound 2) and 1-pyrenemethanol (compound 1), respectively. It is a photographed image.

According to the present invention there is provided a no-wash protein gel stain containing a pyrene derivative. The protein gel staining agent of the present invention is characterized in that it does not require a washing step, which is essential in ordinary gel staining, after staining the gel with a pyrene derivative.

1. Pyrene Derivatives Pyrene derivatives contained in the gel staining agent of the present invention include, but are not limited to, 1-pyrenemethanol, pyrenylmethyl ether derivatives, 1-pyrenecarboxylic acid derivatives, sugar-modified pyrenylmethyl derivatives, and pyrene-fluorescein conjugates. is mentioned. As these pyrene derivatives, commercially available ones may be used, or as described in Example 1 below, those synthesized by a conventional method may be appropriately used according to the use.

As used herein, "pyrenyl methyl ether derivative" refers to a derivative having a group --(O--CH.sub.2--CH.sub.2 _{) .sub.n--} at the ₁ -position of pyrene through a methylene group. Typically pyrenyl methyl ether derivatives have the following structures:

can have Here, in the above formula, m is an integer of 0-10, preferably 0-5, more preferably 0-3. R ₁ may also be, but is not limited to, a group selected from the group consisting of —OH, —C _1-6 alkyl, —OC _1-6 alkyl, —N ₃ and —NH ₂ . Also, R ₁ may be a group in which a protecting group (eg, a tosyl group (Ts)) is introduced into —OH.

A pyrenyl methyl ether derivative may typically have the structure shown below.

For synthesis of the above compound, refer to Example 1 described later.

As used herein, "1- _{pyrenecarboxylic} acid derivative" refers to a derivative having a group --(O--CH.sub.2--CH.sub.2) _.sub.n-- at the ₁ -position of pyrene via a carbonyl group. Typically, 1-pyrenecarboxylic acid derivatives have the structure:

can have Here, in the above formula, n is an integer of 0-10, preferably 0-5, more preferably 0-3. R ₁ may also be, but is not limited to, a group selected from the group consisting of —OH, —C _1-6 alkyl, —OC _1-6 alkyl, —N ₃ and —NH ₂ . Also, R ₁ may be a group obtained by protecting —OH with a protecting group (eg, tosyl group (Ts)).

Furthermore, the 1-pyrenecarboxylic acid derivative may have a structure having two pyrenes in one molecule, i.e., R ₂ may have the following structure:

may be

A 1-pyrenecarboxylic acid derivative may typically have the following structure.

For synthesis of the above compound, refer to Example 1 described later.

As used herein, "sugar-modified pyrenylmethyl derivative" refers to a derivative having a sugar at position 1 of pyrene via a methylene group. "Sugar" means an oxygen-retaining hydrocarbon-based compound containing at least 4 carbon atoms containing one or more alcohol functional groups which may have an aldehyde or ketone functional group. do. These sugars may be monosaccharides, oligosaccharides or polysaccharides. Sugars for modifying pyrene are preferably monosaccharides, such as glucose, mannose, and galactose.
A sugar-modified pyrenylmethyl derivative can typically have the structure shown below.

For synthesis of the above compound, refer to Example 1 described later.

In addition to the pyrene derivatives listed above, pyrene-fluorescein conjugates can also be used. Such pyrene-fluorescein conjugates are not limited, but may have the following structures.

2. Gel Staining Agent According to the present invention, the pyrene derivative described above is used as a staining agent that enables visual recognition of a biological sample separated after electrophoresis on a gel. As used in the present invention, "gel" refers to a molecular sieve material for separating and analyzing biological substances and chemical substances in the fields of biology, chemistry, medicine, pharmacy, bioscience, etc., and is preferably refers to a distribution support for sample components by electrophoresis. Materials for the gel are not particularly limited, but examples thereof include polyacrylamide and agarose. The gel used in the staining method of the present invention preferably includes a gel after one-dimensional or two-dimensional electrophoresis of proteins. Here, protein electrophoresis is not particularly limited, but conventional electrophoresis such as polyacrylamide gel electrophoresis (PAGE), polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE), Offerler's 2 Dimensional electrophoresis, RFHR two-dimensional electrophoresis, and the like. The size and shape of the gel are also not limited, and it may be in the form of a disc-type cylinder or a slab-type sheet. Devices and means for various electrophoresis using gels are well known to those skilled in the art.

When a pyrene derivative is used as a gel staining agent, it is preferably an aqueous solution in consideration of gel permeability. Such a gel staining agent can be obtained by dissolving the above-mentioned pyrene derivative in water or an aqueous buffer so as to be 1 to 100 μM, preferably 1 to 50 μM, more preferably 1 to 10 μM. Hereinafter, the aqueous solution of the pyrene derivative thus prepared may be simply referred to as "pyrene derivative aqueous solution".

3. Gel staining method and imaging According to the present invention, proteins electrophoresed on a gel are fixed on the gel using a fixing solution, and a staining solution containing the gel staining agent, that is, an aqueous pyrene derivative solution is used. can be used to stain proteins and gel images can be obtained after irradiation with ultraviolet (UV) light without post-staining destaining and washing. Here, fixatives that can be used as fixatives include chemicals used to protect biological samples from putrefaction. Such fixatives can prevent biological reactions occurring in the sample and increase the mechanical strength and stability of the sample. In one embodiment, various fixatives can be included in the fixative, such as methanol, ethanol, isopropanol, acetone, formaldehyde, glutaraldehyde, EDTA, detergents, metal salts, metal ions, urea, and amino acids. compound and the like. Also, the fixatives described above can be used under acidic conditions. More specifically, the immobilization of proteins on the gel after electrophoresis is not limited, but the gel is soaked in 40-70% methanol containing 1-10% aqueous acetic acid in a container for 5 minutes to 1 hour. A degree of shaking can be achieved. Furthermore, the immobilization step is not limited to one time and may be repeated several times.

After the proteins have been immobilized on the gel and before gel staining, the final wash is with water, especially distilled, demineralized, or deionized water (eg, purified with a Milli-Q system from Millipore). water, hereinafter referred to as "Milli-Q water") is preferably used. The final washing can be performed, for example, by immersing in deionized water and shaking for about 5 to 30 minutes. After that, the gel is dyed using the pyrene derivative aqueous solution. Gel staining can be performed by immersing the gel in an aqueous pyrene derivative solution and shaking overnight. According to the present invention, photographing and imaging can be performed without the need for washing and destaining steps after dyeing. On the other hand, when using a staining agent other than the pyrene derivative aqueous solution according to the present invention, generally, for example, washing and decoloring steps are required after staining, and an aqueous solution (eg, methanol + acetic acid aqueous solution) for that purpose is prepared separately. , the gel must be immersed in the aqueous solution and shaken for 30 minutes to several hours.

For visualization of proteins on a gel, the dye used can be appropriately selected according to its properties. According to the present invention, since a pyrene derivative is used as a staining agent, proteins can be detected by irradiation with UV light using, for example, a UV transilluminator. Further, the electrophoresis image obtained can be analyzed using image analysis software (eg, ImageJ) to calculate the signal intensity of the spots or bands.

4. Kit According to the present invention, a protein stain detection kit is provided. The protein staining detection kit can contain the pyrene derivative aqueous solution of the present invention as a staining agent, a fixing solution for fixing proteins, and a washing solution used before staining.

The present invention will be described in more detail with reference to the following examples, but the present invention is in no way limited by these examples.

Example 1 Synthesis of Pyrene Derivatives In this example, various pyrene derivatives were synthesized. The organic synthesis reagents used in the synthesis are from Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd., Aldrich Chemical Co., Ltd. , was purchased from Kanto Kagaku. In addition, tracking and confirmation of the organic synthesis reaction, and purification and synthesis confirmation of the desired pyrene derivative were carried out by silica gel thin layer chromatography, ¹ H NMR measurement, ¹³ C NMR measurement, and silica gel chromatography. More specifically, in silica gel thin-layer chromatography, Silica gel 60 F254 (Merck) is used, and for tracking and confirmation of the reaction, UV (254 nm), (365 nm) irradiation, or 1% Ce ( SO ₄ ) ₂ -1.5% (NH ₄ ) ₆ Mo ₇ O ₂₄ .4H ₂ O-10% H ₂ SO ₄ aqueous solution, colored with an electric heater, and detected. Silica Gel C-60, Wako Gel C-300, and Silica Gel 60N were used for isolation and purification. Furthermore, in confirmation of synthesis, OXFORD NMR AS500 (500 MHz) or JEOLJNX-EX-270 (270 MHz) was used for ¹ H NMR measurement. Chemical shifts are given as internal standard using the tetramethylsilane (δ ¹ H=0 ppm) solvent peak in CDCl ₃ .

(1) Synthesis of compound 2

To pyrenylmethyl bromide (201 mg, 0.680 mmol, 1 eq.) was added 3.4 mL of methanol and a solution of 1 M sodium methoxide in methanol (3.4 mL, 3.4 mmol, 5 eq.), and the mixture was stirred at room temperature for 2 hours. After that, 0.7 mL of DMF was added and stirred overnight. After completion of the reaction, water was added to quench. Ethyl acetate was added, and the mixture was washed twice with water and once with saturated brine. Then, it was dried with sodium sulfate, filtered, and distilled off under reduced pressure. Purification was performed by silica gel column chromatography (yield 64%, 107 mg).
Compound 2: Rf 0.54 (Hexane-EtOAc, 5:1) ¹ H NMR (500 MHz, Chloroform-d) δ 8.37 (d, J = 9.1 Hz, 1H), 8.20 (t, J = 7.7 Hz, 2H), 8.18-8.13 (m, 2H), 8.08-7.99 (m, 4H), 5.19 (s, 2H), 3.52 (s, 3H).

(2) Synthesis of compound 4

3 mL of dichloromethane was used as a solvent, and ethylene glycol (345 mg, 3.25 mmol, 10 equivalents) and 1-pyrenecarboxylic acid (50 mg, 0.325 mmol, 1 equivalent) were added. Then EDC (75 mg, 0.39 mmol, 1.2 eq) was added and DMAP (40 mg, 0.325 mmol, 1.2 eq) was added and stirred at room temperature for 16 hours. After quenching with water, it was diluted with ethyl acetate and washed twice with water and once with saturated brine. Then, it was dried with sodium sulfate, filtered, and distilled off under reduced pressure. Purification was performed by silica gel column chromatography (yield 34%, 109 mg).
Compound 4: Rf 0.17 (Hexane-EtOAc, 1:1) ¹ H NMR (500 MHz, Chloroform-d) δ 9.26 (d, J = 9.4 Hz, 1H), 8.65 (d, J = 8.1 Hz, 1H), 8.30-8.22 (m, 3H), 8.20-8.16 (m, 2H), 8.12-8.04 (m, 2H), 4.72-4.67 (m, 2H), 4.00-3.96 (m, 2H), 3.84-3.79 (m , 2H), 3.75-3.72 (m, 2H), 2.10 (s, 1H).

(3) Synthesis of compound 5

Diethylene glycol (16 mg, 0.155 mmol, 1 eq) was suspended in 3 mL of dichloromethane and 1-pyrenecarboxylic acid (50 mg, 0.325 mmol, 2.1 eq) was added. Then EDC (89 mg, 0.465 mmol, 3.0 eq) was added and DMAP (38 mg, 0.31 mmol, 2.0 eq) was added and stirred at room temperature for 24 hours. After quenching by adding water, it was diluted with dichloromethane and washed twice with water and once with saturated brine. Then, it was dried with sodium sulfate, filtered, and distilled off under reduced pressure. Purification was performed by silica gel column chromatography (yield 41%, 36 mg).
Compound 5: Rf 0.50 (Hexane-EtOAc, 1:1) ¹ H NMR (500 MHz, Chloroform-d) δ 9.12 (d, J = 9.5 Hz, 2H), 8.50 (d, J = 8.1 Hz, 2H), 8.10 (d, J = 7.6 Hz, 2H), 8.06 (d, J = 7.6 Hz, 2H), 8.01 (d, J = 9.4 Hz, 2H), 7.96- 7.91 (m, 4H), 7.77 (d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 4.74 (t, J = 4.6 Hz, 4H), 4.12-4.08 (m, 4H).

(4) Synthesis of compound 6

Ethylene glycol (9.2 mg, 0.148 mmol, 1 eq) was dissolved in dichloromethane and 1-pyrenecarboxylic acid (50 mg, 0.325 mmol, 2.2 eq) was added. Then EDC (85 mg, 0.444 mmol, 3.0 eq) was added and DMAP (1.8 mg, 0.0148 mmol, 0.1 eq) was added and stirred at room temperature for 29 hours. After quenching with water, it was diluted with dichloromethane and washed twice with water and once with saturated brine. Then, it was dried with sodium sulfate, filtered, and distilled off under reduced pressure. After purification by silica gel column chromatography, production of compound 6 was confirmed as a by-product (yield 46%, 32 mg).
Compound 6: Rf 0.73 (Hexane-EtOAc, 1:1) ¹ H NMR (500 MHz, Chloroform-d) δ 9.54 (d, J = 9.4 Hz, 2H), 8.92 (d, J = 8.2 Hz, 2H), 8.40-8.33 (m, 6H), 8.30-8.24 (m, 4H), 8.18-8.11 (m, 4H).

(5) Synthesis of compound 7

THF 3 mL was added to sodium hydride (24 mg, 1.02 mmol, 1.5 equivalents) and stirred under ice cooling, diethylene glycol (719 mg, 6.78 mmol, 10 equivalents) dissolved in THF 3 mL was added and stirred for 30 minutes. . After that, 1-(bromomethyl)pyrene (200 mg, 0.678 mmol, 1.0 equivalent) dissolved in 3 mL of THF was added and reacted overnight at room temperature. After completion of the reaction, water was added to quench the sodium hydride. Ethyl acetate was added, and the mixture was washed twice with water and once with saturated brine. Then, it was dried with sodium sulfate, filtered, and distilled off under reduced pressure. Purification was performed by silica gel column chromatography (yield 84%, 183 mg).
Compound 7: Rf 0.38 (EtOAc) ¹ H NMR (500 MHz, Chloroform-d) δ 8.40 (d, J = 9.2 Hz, 1H), 8.23-8.13 (m, 4H), 8.09-7.99 (m, 4H), 5.29 (s, 2H), 3.78-3.70 (m, 6H
), 3.61 (t, J = 4.5 Hz, 2H), 2.30 (s, 1H).

(6) Synthesis of compound 8 and compound 9

The corresponding raw material alcohols (precursor of compound 8, 13 mg, 0.428 mmol, 1 equivalent; precursor of compound 9, 147 mg, 0.403 mmol, 1 equivalent) were dissolved in 4 mL of pyridine, and p-toluenesulfonyl chloride (compound 8 Synthesis of Compound 9, 122 mg, 0.641 mmol, 1.5 eq.; Synthesis of Compound 9, 115 mg, 0.605 mmol, 1.5 eq.) were added and stirred at room temperature for 16 hours under an argon atmosphere. Water was added to quench, and the solvent was distilled off under reduced pressure. Then, it was diluted with ethyl acetate and washed twice with saturated saline. Then, it was dried with sodium sulfate, filtered, and distilled off under reduced pressure. Purification was performed by silica gel column chromatography. (Yields, compound 8: 51%, 103 mg; compound 9: 52%, 109 mg)
Compound 8: Rf 0.15 (Hexane-EtOAc, 4:1) ¹ H NMR (500 MHz, Chloroform-d) δ 8.37 (d, J = 9.3 Hz, 1H), 8.23-8.18 (m, 2H), 8.17-8.12 (m, 2H), 8.08-7.99 (m, 4H), 7.78-7.74 (m, 2H), 7.22 (d, J = 8.0 Hz, 2H), 5.25 (s, 2H), 4.19-4.15 (m, 2H ), 3.72-3.68 (m, 4H), 3.67-3.63 (m, 2H), 2.33 (s, 3H).
Compound 9: Rf 0.70 (Hexane-EtOAc, 1:4) ¹ H NMR (500 MHz, Chloroform-d) δ 8.40 (d, J = 9.1 Hz, 1H), 8.23-8.17 (m, 2H), 8.17-8.12 (m, 2H), 8.08-7.99 (m, 4H), 7.75 (d, J = 8.3 Hz, 2H), 7.25-7.23 (m, 2H, with CHCl ₃ ), 5.28 (s, 2H), 4.13-4.09 (m, 2H), 3.76-3.72 (m, 2H), 3.69-3.63 (m, 4H), 3.61-3.55 (m, 4H), 2.36 (s, 3H).

(7) Synthesis of compound 10 and compound 11

The corresponding starting materials (compound 8, 98 mg, 0.207 mmol; compound 9, 91 mg, 0.175 mmol) were dissolved in 2 mL of DMF and sodium azide (synthesis of compound 10, 16.3 mg, 0.248 mmol, 1.2 eq; Synthesis of Compound 11, 13.7 mg, 0.211 mmol, 1.2 eq.) was added and stirred at 60° C. for 17 hours. Water was added to quench. Then, it was diluted with ethyl acetate and washed twice with water and once with saturated brine. Then, it was made to dry with sodium sulfate, filtration was performed, and the solvent was depressurizingly distilled. Purification was performed by silica gel column chromatography. (Yields, compound 10: 93%, 66 mg; compound 11: 96%, 66 mg)
Compound 10: Rf 0.56 (Hexane-EtOAc, 2:1) ¹ H NMR (500 MHz, Chloroform-d) δ 8.41 (d, J = 9.2 Hz, 1H), 8.20 (t, J = 8.2 Hz, 2H), 8.17-8.13 (m, 2H), 8.08-7.99 (m, 4H), 5.30 (s, 2H), 3.80-3.76 (m, 2H), 3.75-3.71 (m, 2H), 3.70-3.66 (m, 2H) ), 3.39 (t, J = 5.1 Hz, 2H).
Compound 11: Rf 0.40 (Hexane-EtOAc, 2:1) ¹ H NMR (500 MHz, Chloroform-d) δ 8.41 (dd, J = 9.3, 1H), 8.20 (t, J = 7.7 Hz, 2H), 8.17 -8.13 (m, 2H), 8.08-7.99 (m, 4H), 5.30 (s, 2H), 3.79-3.75 (m, 2H), 3.74-3.70 (m, 2H), 3.69-3.61 (m, 6H) , 3.32 (t, J = 5.1 Hz, 2H).

(8) Synthesis of compound 12 and compound 13

The corresponding various starting materials (compound 10, 52 mg, 0.15 mmol; compound 11, 55 mg, 0.141 mmol, 1 eq) were dissolved in 2 mL of THF and triphenylphosphine (synthesis of compound 12, 50.3 mg, 0.191 mmol, Synthesis of Compound 13, 41.3 mmol, 0.16 mmol, 1.1 eq.) were added to each starting material and stirred at room temperature for 25 hours. Water was added, and the reaction was allowed to proceed at room temperature for an additional 7.5 hours, followed by stirring at 50° C. for 15 hours. After completion of the reaction, distillation under reduced pressure was performed. Purification was performed by silica gel column chromatography. (Yields, compound 12: 92%, 44 mg; compound 13: 94%, 48 mg)
Compound 12: Rf 0.05 (DCM-MeOH, 95:5) ¹ H NMR (500 MHz, Chloroform-d) δ 8.41 (d, J = 9.2 Hz, 1H), 8.20 (t, J = 7.6 Hz, 2H), 8.17-8.12 (m, 2H), 8.08-7.98 (m, 4H), 5.29 (s, 2H), 3.77-3.73 (m, 2H), 3.70-3.64 (m, 2H), 3.49 (t, J = 5.2 Hz, 2H), 2.84 (t, J = 5.2, 2H).
Compound 13: Rf 0.05 (DCM-MeOH, 95:5) ¹ H NMR (500 MHz, Chloroform-d) δ 8.41 (d, J = 9.2 Hz, 1H), 8.20 (t, J = 7.6 Hz, 2H), 8.17-8.12 (m, 2H), 8.08-7.98 (m, 4H), 5.29 (s, 2H), 3.79-3.74 (m, 2H), 3.74-3.69 (m, 2H), 3.68-3.63 (m, 2H) ) 3.63-3.58 (m, 2H), 3.46 (t, J = 5.2 Hz, 2H), 2.84-2,76 (m, 2H).

(9) Synthesis of compound 15 and compound 16

The corresponding amino forms of various starting materials (compound 12, 37 mg, 0.114 mmol, 1.0 equivalents; compound 13, 40 mg, 0.111 mmol, 1.0 equivalents) were dissolved in 1 mL of DMF, and TEA (synthesis of compound 15 , 79 μL, 0.57 mmol; Synthesis of Compound 16, 77 μL, 0.56 mmol, all 5 eq) was added and FITC (Synthesis of Compound 15, 49 mg, 0.125 mmol, 1.1 eq; Synthesis of Compound 16, 48 mg, 0.122 mmol, 1.1 eq.) was added and stirred overnight at room temperature. After completion of the reaction, methanol was added to quench. After that, the solvent was distilled off under reduced pressure. Purification was performed by silica gel column chromatography (yield, compound 15: 63 mg, 78%; compound 16: 74%, 62 mg).
Compound 15: Rf 0.51 (DCM-MeOH, 9:1) ¹ H NMR (500 MHz, DMSO-d6) δ 10.4-9.94 (m, 3H), 8.39 (d, J = 9.2 Hz, 1H), 8.31-8.21. (m, 5H), 8.18-8.03 (m, 5H), 7.69 (d, J = 8.2 Hz, 1H), 7.13 (d, J = 8.3 Hz, 1H), 6.66 (d, J = 2.3 Hz, 2H) , 6.57 (d, J = 8.8 Hz 2H), 6.55-6.51 (m, 2H), 5.23 (s, 2H), 3.81-3.76 (m, 2H), 3.74-3.62 (m, 6H).
Compound 16: Rf 0.44 (DCM-MeOH, 9:1) ¹ H NMR (500 MHz, DMSO-d6) δ 10.1(s, 2H), 10.0 (s, 1H), 8.38 (d, J = 9.2 Hz, 1H ), 8.32-8.21 (m, 5H), 8.16 (s, 2H), 8.11-8.04 (m, 3H), 7.69 (d, J = 8.3 Hz, 1H), 7.14 (d, J = 8.3 Hz, 1H) , 6.66 (d, J = 2.3 Hz, 2H), 6.57 (d, 8.7 Hz, 2H), 6.53 (dd, J = 8.7 Hz, 2.3 Hz, 2H), 5.20 (s, 2H), 3.75-3.70 (m , 2H), 3.70-3.55 (m, 10H).

In addition, the following compounds 14 and 17 were used as comparative compounds.

Compound 14 was synthesized as follows, and compound 17 (FITC) was a commercially available product. The starting amino form (2-(2-aminoethoxy)ethanol, 41 mg, 0.386 mmol, 1.5 equivalents) was dissolved in 3 mL of DMF, TEA (180 μL, 1.29 mmol, 5 equivalents) was added, and FITC (100 mg , 0.257 mmol, 1.0 equiv) was added and stirred overnight at room temperature. After completion of the reaction, methanol was added to quench. After that, the solvent was distilled off under reduced pressure. Purification was performed by silica gel column chromatography (yield, 40%, 51 mg).
Compound 14: ¹ H NMR (500 MHz, DMSO-d6) δ 10.10 (s, 1H), 8.23 (s, 1H), 8.15 (s, 1H), 7.72 (d, J = 8.3 Hz, 1H), 7.15 ( d, J = 8.3 Hz, 1H), 6.64-6.59 (m, 4H), 6.53 (dd, J = 8.7, 2.3 Hz, 2H), 3.61-3.57 (m, 2H), 3.54-3.51 (m, 2H) , 3.50-3.36 (m, 4H).

(10) Synthesis of compound 18 and compound 19 Compound 18 and compound 19 were synthesized according to the following synthesis scheme.

1-Pyrenemethanol (630 mg, 2.71 mmol, 1.6 eq) was dissolved in anhydrous dichloromethane and silver carbonate (813 mg, 2.95 mmol, 1.7 eq) was added followed by bromo 2,3,4,6- O-tetraacetyl-α-D-glucopyranoside (700 mg, 1.70 mmol, 1 eq) was added and allowed to react for 5 days. After completion of the reaction, the reaction mixture was filtered through celite and then distilled off under reduced pressure. Silica gel column chromatography was used for purification. After that, the compound was dissolved in a mixed solution of 5 mL of THF, 0.5 mL of AcOH, and 0.5 mL of H ₂ O and stirred overnight. Then, it was purified using silica gel column chromatography to obtain the desired product (yield 36%, 347 mg).
Compound 19: Rf 0.55 (Hexane-EtOAc, 1:1) ¹ H NMR (500 MHz, Chloroform-d) δ 8.32 (d, J = 9.3 Hz, 1H), 8.22 (d, J = 7.7 Hz, 2H), 8.17-8.01 (m, 5H), 7.96 (d, J = 7.8 Hz, 1H), 5.64 (d, J = 12.3 Hz), 5.31 (d, J = 12.4 Hz, 1H), 5.16-5.01 (m, 3H ), 4.53 (d, J = 7.6 Hz, 1H), 4.33 (dd, J = 12.2, 4.6 Hz, 1H), 4.25 (dd, J = 12.2, 2.5 Hz, 1H), 3.68-3.63 (m, 1H) , 2.17 (s, 3H), 2.00 (s, 3H), 1.94 (s, 3H), 1.70 (s, 3H).

Compound 19 (347 mg, 0.62 mmol) was dissolved in methanol, 0.5 mL of 1M sodium methoxide was added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, the solvent was distilled off under reduced pressure, and purification was performed using silica gel column chromatography to obtain the desired product 18 (yield 91%, 223 mg).
Compound 18: Rf 0.70 (DCM-MeOH, 4:1) ¹ H NMR (500 MHz, DMSO-d6) δ 8.45 (d, J = 9.2 Hz, 1H), 8.34-8.16 (m, 7H), 8.08 (t , J = 7.6 Hz, 1H), 5.57 (d, J = 12.1 Hz, 1H), 5.30 (d, J = 12.1 Hz, 1H), 5.11 (d, J = 4.8 Hz, 1H), 5.00-4.93 (m , 2H), 4.68-4.63 (m, 1H), 4.40 (d, J = 7.6 Hz, 1H), 3.81-3.75 (m, 1H), 3.58-3.51 (m, 1H), 3.46-3.39 (m, 1H ), 3.21-3.06 (m, 4H).

Example 2: Verification in one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (1) Preparation of various solutions Preparation of 30% acrylamide monomer solution: acrylamide monomer 9.93 g, NN'-methylenebisacrylamide 0 .27 g, Milli-Q water was added to make a total of 34 mL of solution. Preparation of 10% acrylamide monomer solution: 33 mL of 30% acrylamide monomer, 16 mL of Milli-Q water, 50 mL of 0.75 M TrisHCl and 1 mL of 10% SDS were each added to prepare a total of 100 mL of solution. A concentrated gel solution was prepared by adding 7.5 mL of 30% acrylamide monomer, 29 mL of Milli-Q water, 37.5 mL of 0.25 M TrisHCl (pH 6.8), and 0.75 mL of 10% SDS to make a total of 75 mL of solution.

(2) One-dimensional SDS-PAGE and Gel Staining/Photographing Method To 20 mL of 10% acrylamide monomer solution, 67 μL of 25% APS and 16 μL of TEMED were added, and the mixed solution was poured onto a glass plate for electrophoresis to gel. After 30 minutes, 2 mL of Milli-Q water was added to wash the gel plate. Next, 7.5 mL of concentrated gel solution, 25 μL of 25% APS, and 6 μL of TEMED were added, and the concentrated gel was poured onto the above gel layer as a concentrated gel to gel. Next, as a sample for gel electrophoresis, a mixed solution of 100 μL of BSA (1 mg/mL) and 100 μL of 2× loading buffer was prepared, heated at 99° C. for 5 minutes, and used as a sample for SDS-PAGE. . 10 μL (5 μg of BSA) was introduced into each lane, and gel electrophoresis was performed under conditions of 150 V and about 100 minutes. Thereafter, the gel was removed from the electrophoresis glass plate, strips were cut from each lane, immersed in a 10 μM, approximately 10 mL (1% DMSO) pyrene derivative staining agent aqueous solution, and shaken for 1 hour. However, in compound 2, the concentration was set to 22 μM. After shaking, electrophoresis images were taken without washing.

(3) Imaging of one-dimensional gel electrophoresis image and calculation of staining ability (evaluation)
Using a UV transilluminator MD-25 (λ _ex 312 nm), UV light was irradiated from the underside of the gel, and gel images were taken using a digital camera. Using ImageJ (image analysis software), the electrophoresis image obtained was used to calculate the signal intensity of the BSA band. Dyeing experiments were performed on compounds 1 to 19, and compounds 1, 2, and 7 were found to exhibit good dyeing ability (Fig. 1).

(4) Relationship between staining ability and hydrophobicity (predicted Log value) of pyrene derivative In order to examine the correlation between hydrophobicity and staining ability of the pyrene derivative used in this example, the Log P value, which is a hydrophobicity parameter, was analyzed using ChemDraw. was obtained as a predicted value using For compounds 2, 1, and 7, which gave good results in staining ability, the predicted Log P value was in the range of about 3.5 to 4.5, and there was a certain correlation between hydrophobicity and staining ability. was suggested (Fig. 2).

Example 3: Verification in two-dimensional electrophoresis (1) Electrophoresis sample preparation E. coli cell samples disrupted with CelLyicB (Sigma-Aldrich) were desalted using a 2D cleanup kit (GE Healthcare), and then subjected to 2D Protein amounts were quantified using the Quant kit (GE Healthcare). A 50 μg portion thereof was used for one two-dimensional electrophoresis.

(2) Two-dimensional electrophoresis The first dimension is isoelectric focusing using Immobiline Dry Strip (GE Healthcare), and the second dimension is NuPAGE 4-12% Bis-Tris ZOOM gel (Thermo Fisher Scientific).

(3) Immobilization of protein spots on two-dimensional electrophoresis gel After electrophoresis, the gel was immersed in a 50% methanol/7% acetic acid aqueous solution and shaken for 30 minutes. This operation was repeated twice. After that, it was immersed in Milli-Q water and shaken for 10 minutes.

(4) Staining and Imaging of Two-Dimensional Electrophoresis Gel The gel after fixation was immersed in 40 mL of Sypro Ruby (Sigma-Aldrich) solution or 10 μM pyrene derivative aqueous solution and shaken overnight for staining. Pyrene derivatives used were pyrenyl methyl methyl ether (compound 2) and 1-pyrenemethanol (compound 1). After that, only Sypro Ruby for comparison was immersed in 10% methanol and 7% acetic acid aqueous solution and shaken for 30 minutes for washing and decolorization, and then immersed in Milli-Q water and shaken for 30 minutes to decolorize. . As described above, after staining, the Sypro Ruby for comparison was photographed after destaining, while the pyrene derivative stain was photographed without washing. The images were scanned using a Typhoon FLA9500 (GE Healthcare) with an excitation wavelength of 473 nm and LPG (575 nm long-pass filter). The results are shown in FIG. Furthermore, when the number of spots was counted using Image Master, the control Sypro Ruby-stained image had 1,247 spots, whereas the pyrenyl methyl methyl ether (Compound 2) was used. When 1-pyrenemethanol (compound 1) was used, the number was 683. From these results, it was found that using the pyrene derivative of the present invention, two-dimensional electrophoresis gels could be stained without washing and destaining operations.

By using the pyrene derivative of the present invention, in protein detection by electrophoresis, protein spots can be detected with high sensitivity without performing washing and destaining operations after gel staining, so a more rapid and simple protein detection method. can be provided.

All publications and patent documents cited herein are hereby incorporated by reference in their entirety. Although specific embodiments of the invention have been described herein for purposes of illustration, it will be appreciated by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. will be easily understood.

Claims (4)

A no-wash protein gel stain containing a pyrene derivative, said pyrene derivative having the following structure:

(In the formula,
R ₁ is selected from the group consisting of —OH, —C _1-6 alkyl, and —OC _1-6 alkyl; and m is an integer from 0 to 3)
The above no-wash protein gel stain. The pyrene derivative is 1-pyrenemethanol ,

The no-wash protein gel stain according to claim 1, which is The protein electrophoresed on the gel is fixed on the gel using a fixing solution, and the staining solution containing the no-wash protein gel staining agent according to claim 1 or 2 is used to fix the protein on the gel. 1. A protein staining detection method comprising staining a protein and obtaining a gel image after irradiating with ultraviolet (UV) light without destaining and washing after staining. A protein stain detection kit comprising the no-wash protein gel stain according to claim 1 or 2 .

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