KR100804022B1 - Bisazacrownethercalix[4]arene linkers and method for synthesizing the same - Google Patents

Abstract

 The present invention relates to an immobilized linker of a protein capable of adsorbing a protein at a higher concentration and a method for preparing the same. More specifically, the present invention relates to bis-azacrownethercalix [4] arene as an immobilized linker of a protein. The present invention relates to a connector and a method of manufacturing the same.

In addition, the bis-azacrownethercalisarene linkage according to the present invention can form a self-assembled monomolecular membrane on a gold surface, and by confirming that the protein is immobilized at a high concentration with the formed membrane, it is possible to make a more excellent and various types of surfaces. In particular, it can be applied to a variety of uses, such as protein chips.

Calix [4] arene, azacrownether, self-assembled monolayer (SAM), linker, protein immobilized, bovine serum albumin (BSA), surface plasma resonator (SPR)

Description

Bis-Azacrown Ether-Calic Sarene Linkage and Its Manufacturing Method {BISAZACROWNETHERCALIX [4] ARENE LINKERS AND METHOD FOR SYNTHESIZING THE SAME}

1 shows a reaction scheme for synthesizing a bis-aza crown ether caxylene linker.

FIG. 2 shows data of the formation of a self-assembled monomolecular film on a gold surface of a commercially available prolinker and immobilization between the thus formed self-assembled monomolecular film and BSA protein by surface plasma resonance apparatus (SPR). will be.

3 is a SPR measurement of the formation of a self-assembled monomolecular membrane on the gold surface of the bis-azacrownethercalixylene linkage prepared according to the present invention and the immobilization between the self-assembled monomolecular membrane and bovine serum albumin (BSA) protein thus formed. Data is shown.

4 compares simulation data of commercially available prolinkers with data measured by experiments.

Figure 5 compares the data measured by the simulation data and the experimental data of the bis-a-crown ether calicsarene connector prepared according to the present invention.

The present invention relates to immobilized linkages of proteins capable of adsorbing proteins at higher concentrations and methods for their preparation. More specifically, the present invention relates to bis-azacrownethercalix [4] arene linkages and methods for preparing the same as immobilized linkages of proteins.

In addition, the bis-azacrownethercalisarene linkage according to the present invention can form a self-assembled monomolecular membrane on a gold surface, and by confirming that the protein is immobilized at a high concentration with the formed membrane, it is possible to make a more excellent and various types of surfaces. In particular, it can be applied to a variety of uses, such as protein chips.

More specifically, the present invention relates to a bis-azacrownethercalixylene derivative as a protein linker for immobilizing a protein by non-covalent bonds on a gold thin film surface for use as a protein chip, and a method for preparing the same. will be. More specifically, the present invention is to prepare a bis-aza crown ether calixar derivatives in excellent yield by using as a starting material (calix [4] arene), based on the bis-aza crown ether caxylene derivatives thus prepared By introducing a thiol (-SH) group selectively at the para position of the phenol group as a backbone, bis-azacrownethercalixylene linkages are obtained, and the new compound is used as a protein immobilization linkage to obtain a protein and a higher concentration. It relates to a method for producing a protein immobilized linker capable of binding.

Since the recent completion of the human genome map, many studies have focused on protein expression products, protein chips that can naturally be used for protein function analysis, and related biosensors, SPRs (surface plasma resonators, surfaces). Attention is focused on the development and development of various analytical equipment, including Plasmon Resonance, and the key area of this technology is the immobilization of proteins. In order to stably fix proteins on a substrate, the development of an immobilized linking layer is the most important core technology and the source technology.

The method of immobilizing proteins such as enzymes, antigens, antibodies, etc. on a solid substrate is the basis of the solid phase immunoassay (Solid Phase Immuno-assay), which is widely used for specific protein analysis, and many research and developments have been made. In particular, the ELISA (Enzyme Linked Immuno Sorbent Assay) method is widely used as an analytical method for detecting and diagnosing cancer or a specific disease, and various kinds of clinical diagnostic or research assay kits have been developed.

The immobilization reaction for attaching the protein to the surface of the solid substrate has been mainly used to physically adsorb the protein by coating a polymer surface or a polystyrene, nitrocellulose or polyvinyl chloride on the substrate surface. Recent chemical reactions have used surface immobilization reactions through covalent linkages between proteins and substrate surfaces, or biotin-avidin (or streptoavidin) reactions after biotin is deposited on solid substrates. A method of immobilizing proteins has been published.

However, these protein immobilization methods have the following problems. The problem with the existing protein immobilization method is that the amount of protein immobilized on the surface is not sufficient. In this case, at low concentrations, the heterologous protein may react non-specifically to the surface of the solid substrate, which may cause serious errors in the experimental results. In addition, existing methods have difficulty in detecting and analyzing trace amounts of specific proteins because the amount or concentration of protein immobilized on the substrate surface is very low. Therefore, the greater the amount of protein immobilized on the surface per unit area, the more non-specific reaction can be minimized, thereby reducing the analytical error of the protein and improving the measurement sensitivity of the analyte protein. Thus, much research in the field of highly integrated protein chips is currently focused on the development of molecular linkages and analytical detection systems that serve as crosslinks between proteins and solid substrates in which the amount of immobilized protein can be maximized.

The problem with immobilizing a protein on a specific substrate is that the immobilized protein retains its biological activity intact. When proteins are immobilized on the substrate surface, they lose their activity more easily in the fluid phase and lose the specificity of protein interactions. This results in the modification and denaturation of the protein when the protein is immobilized by a chemical bond to a solid substrate, and the method of protein immobilization by physical adsorption loses the orientation of the adsorbed protein to inhibit the specific reaction of a specific analyte protein or other It causes a nonspecific reaction with the protein, resulting in poor selectivity. Therefore, heterogeneity of the modification and denaturation or orientation of the immobilized protein of the prior art has been shown to improve sensitivity, selectivity and reproducibility in the development of detection assays for analytical proteins using protein chips. There are many difficulties and limitations in raising.

On the other hand, there have been a lot of researches related to the manufacture of protein immobilized linkages. Among them, there have not been many attempts to develop the protein linkage layer using supramolecular system and apply it to the diagnostic protein chip. As a similar technique related to protein immobilization technology, Sweden's BlAcore uses cyclodextrin. However, cyclodextrin has difficulty in using in integrated protein chips due to its weak interaction force with protein. . In addition, there has been reported a new protein immobilizing material having a crown ether using a calixarene, a calixarene-crownether derivative (prolinker), and a self-assembled monomolecular membrane of a calixarene-crownether derivative formed on a gold surface. (Self-Assembeled Monolayer) and Bovine Serum Albumin (BSA), known as water-soluble standard protein, is known to be 175 ng / cm 2 (Kim, TS Proteogen, US Patent no. 6,485, 984 B1, Nov 26, 2002). .

In addition, European Patent Application No. 1110964 A1 discloses a calicarene derivative for immobilizing a protein, that is, a calicarene derivative capable of non-covalent interaction with a protein molecule based on calixarene. Contrary to the technique, the introduction of two groups of acetic acid groups to the phenol group succeeded in achieving a technical configuration capable of immobilizing a large amount of proteins non-covalently than the prior art, and thus led to the present invention.

Accordingly, the present inventors have succeeded in the production of a conjugated compound for immobilization of protein, which can adsorb a protein at a higher concentration by a non-covalent self-assembled monomolecular membrane formed on a gold surface in order to solve the problems as described above. It became.

In other words, the inventors of the present invention more specifically by confirming that the bis-azacrownethercalixylene linkage according to the present invention forms a self-assembled monomolecular membrane (SAM) on the gold surface and immobilizes the protein to a high concentration with the formed membrane. It can make excellent and various types of surface, and can be applied to various uses, especially protein chips. Thus, with the base of kalixarene, two azacrownether rings can form stronger hydrogen bonds with the ammonium ions coexisting at the charged site of the protein, and there are two hydroxy groups and strong hydrophilicity. This resulted in the recognition of stronger hydrogen bonds with proteins in aqueous solutions, thus completing the present invention.

Accordingly, an object of the present invention based on the above novel recognition is to provide a new bis-azacrownethercalixylene linker having a calixarene as a basic skeleton and a method for preparing the same.

In addition, the present invention is to provide a method for immobilizing a protein at a higher concentration with an effective protein immobilization linker using the bis-aza crown ether calixarene linker as described above.

The present invention relates to immobilized linkages of proteins capable of adsorbing proteins at higher concentrations and methods for their preparation. More specifically, the present invention relates to a bis-azacrownethercalixylene linker and a method for preparing the same as an immobilized linker of a protein.

In addition, the present invention is to confirm that the protein can be immobilized to a high concentration through the bis-aza crown ether calicsarene linker to effectively fix the protein to a higher concentration with a bis-a-crown ether calic sarene linker It is about a method.

In addition, the bis-azacrownethercalixylene linkage according to the present invention forms a self-assembled monomolecular film on the gold surface, and by using the formed membrane to immobilize the protein at a high concentration, it is possible to make a more excellent and various types of surface, in particular It can be applied to various uses such as protein chips.

The novel bis-azacrownethercalixarenelinker of the present invention selectively connects 1-aza-18-crown-6 to the 1,3-position of the lower rim with the base of kalixaren, The thiol group (-SH) is selectively introduced at the para position of the benzene ring having a phenolic hydroxy group.

For the above purposes, the present invention is characterized in that symmetrically adding azacrown ether to calyxarene in the preparation of bis-azacrownethercalixylene derivatives, which are precursors in the preparation of bis-azacrownethercalixylene linkers. do. In addition, the quantitative introduction of a thiol group (-SH) to form a self-assembled monomolecular film to the bis-aza crown ether calicsarene derivatives prepared according to the present invention is characterized by a technical feature. In addition, the step of introducing a thiol group includes the step of reacting with a thiourea and then hydrolyzing the resulting salt into an aqueous alkali solution.

In addition, the novel compounds of the present invention form a self-assembled monomolecular film on a gold surface, and are used as protein immobilization linkers by adsorbing proteins at high concentration by non-covalent bonds on the formed self-assembled monomolecular film. It relates to the use of.

Hereinafter, the present invention will be described in more detail.

In the preparation of bis-azacrownethercalixylene derivatives, which are skeletal compounds of protein linkages, the intermediates thereof, which are represented by the following general formula (1), are converted to trimethylammonium carboxylic acid esters according to a known method. The hydroxide was obtained by hydrolysis with an aqueous solution.

Various activation reagents, such as carbodiimide, are possible to amidate amic an azacrownether, a secondary amine, to the calixarylene carboxylic acid of Formula 1, but the reaction cannot be successfully performed. Therefore, the more reactive chloride carboxylic acid derivative formula (2) was effectively prepared and amidated from azacrown ether.

In the step of converting Chemical Formula 1 into a carbonyl chloride group, oxalic acid chloride which can be reacted at a milder temperature than a reaction of Thionyl Chloride, which is a general reaction method, is used. Made with a high yield of 91%.

The reaction for introducing the chloromethyl group into the benzene ring of calixarene from the formula (3) requires a Lewis catalyst. However, the oxygen atom of the phenol functional group activates the reaction too much, causing excessive chloromethylation. Therefore, the reaction at low temperature is essential. The optimum reaction conditions were 1 hour in the ice bath and the reaction was increased to room temperature for 3 hours. In addition, the hydrogen of the phenolic hydroxy group bonds with the oxygen of the adjacent phenol ether group to reduce the electron density of the benzene ring of the phenol ether group, thereby reducing the activity, while the electron density of the phenolic benzene ring is relatively low. As the activity of the aromatic parent electron substitution reaction increases, the benzene ring can be selectively chloromethylated to selectively obtain the compound of formula (4).

The desired protein linker bis-azacrownethercalixylene can be prepared by thiolation from the above formula (4). Thiolation of the alkylhalogen compound is first reacted with thiourea and the resulting salt It can manufacture by hydrolyzing with aqueous alkali solution. When the thiolation reaction is applied from Chemical Formula 4, the compound is not soluble in ethanol, which is a general reaction solvent, and thus N, N -dimethylformamide (DMF) is used as a reaction solvent to complete the reaction with thiourea, and caustic. The following Formula 5 may be prepared by hydrolysis with an aqueous soda solution.

Formula 5 easily forms disulfide bonds between or within molecules, so that the structure of symmetry is not well observed in NMR. Therefore, the thiourea salt was separated and confirmed its symmetry as NMR, or sodium borohydride was added as a reducing agent in the purification process to confirm the symmetry in the NMR of the formula (5).

The data of analyzing the new compounds of Chemical Formulas 3, 4, and 5 are described below using a nuclear magnetic resonance apparatus.

1.25,27-bis [(4,7,10,13,16- Pentaoxa -One- Azacyclooctade -One- Nylcarbonyl ) Methoxy] Calix [4] Aren  (Formula 3)

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04 (s, 2H, Ar-O H ), 6.99 (d, J = 7.5 Hz, 4H, Ar- H ), 6.88 (d, J = 7.6 Hz, 4H, Ar- H ), 6.72 (t, J = 7.5 Hz, 2H, Ar- H ), 6.58 (t, J = 7.6 Hz, 2H, Ar- H ), 4.89 (s, 4H, -OC H ₂ CO-) , 4.49 (d, J = 13.1 Hz, 4H, -C Ha Hb-), 3.79-3.73 (m, 48H, protons of crown ether moiety), 3.30 (d, J = 13.1 Hz, 4H, -CHa Hb -) ; ¹³ C NMR (100 MHz, CDCl ₃ ) δ 168.3, 153.5, 153.1, 133.6, 128.9, 128.3, 128.2, 125.2, 118.8, 73.8, 71.1, 70.77, 70.75, 70.69, 70.65, 70.64, 70.5, 70.4, 69.8, 69.5, 48.6, 47.0, 31.6

2. 25,27-bis [(4,7,10,13,16- Pentaoxa -One- Azacyclooctade -One- Nylcarbonyl ) Methoxy] -5,17-bis ( Chloromethyl ) Calix [4] Aren  (Formula 4)

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.99 (s, 4H, Ar- H ), 6.90 (d, J = 7.5 Hz, 4H, Ar- H ), 6.72 (t, J = 7.5 Hz, 2H, Ar -H ), 4.91 (s, 4H, -OC H ₂ CO-), 4.50 (d, J = 13.0 Hz, 4H, -C H _a H _b- ), 4.28 (s, 4H, -C H ₂ Cl) , 3.79-3.60 (m, 48H, protons of crown ether moiety), 3.33 (d, J = 13.0 Hz, 4H, -CH _a H _b- ); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 167.6, 152.5, 151.7, 132.6, 127.9, 127.3, 127.1, 126.9, 124.1, 73.7, 72.7, 70.0, 69.70 (double intensity), 69.64, 69.58, 69.46, 69.3, 68.7 , 68.4, 56.6, 47.5, 45.9, 30.6

3.25,27-bis [(4,7,10,13,16- Pentaoxa -One- Azacyclooctade -One- Nylcarbonyl ) Me Methoxy] -5,17-bis ( Mercaptomethyl ) Calix [4] Aren

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.90 (s, 4H, Ar- H ), 6.81 (d, J = 7.5 Hz, 4H, Ar- H ), 6.65 (t, J = 7.5 Hz, 2H, Ar -H ), 4.82 (s, 4H, -OC H ₂ CO-), 4.40 (d, J = 13.0 Hz, 4H, -C H _a H _b ), 4.22 (s, 4H, -C H ₂ SH), 3.71-3.53 (m, 48H, protons of crown ether moiety), 3.26 (d, J = 13.0 Hz, 4H, -CH _a H _b- ) ¹³ C NMR (100 MHz, CDCl ₃ ) δ 167.6, 152.4, 151.7, 132.4, 127.9, 127.3, 127.0, 124.5, 124.2, 73.7, 72.6, 70.0, 69.69, 69.64, 69.57, 69.4, 69.3, 68.6, 68.5, 56.6, 47.4, 45.9, 30.5, 29.3.

In addition, the present invention includes a step of forming a self-assembled monomolecular film on the gold surface of the bis-aza crown ether calixarene linkage obtained as described above, and thus the self-assembled monomolecular membrane formed comprises a method of effectively immobilizing proteins with non-covalent force. .

In the present invention, the concentration of the self-assembled monomolecular membrane of the bis-aza crown ether calixarene conjugate formed on the gold surface and the protein covalently adsorbed to the monomolecular membrane were obtained by the following equation.

Γ: surface concentration of adsorbed molecules,

d: thickness of the adsorbed molecular layer,

n _b = Refractive index of the buffer,

n: refractive index of the adsorbed molecular layer,

r (the specific refractivity of BSA): 0.234 mL / g

v (the partial specific volume of BSA): 0.729 mL / g

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1> 25,27-bis [(4,7,10,13,16- Pentaoxa -One- Keep it up - Cyclooctade -One- Nylcarbonyl ) Methoxy ] Calix [4] Aren's  Produce

To 30 ml of acetonitrile, 1.7 g of Kalix [4] arene, 0.68 g of anhydrous potassium carbonate and 1.40 g of ethyl bromoacetate were added and stirred under reflux for 24 hours. Recrystallization with a dichloromethane / methanol solvent separated the calixarene ester derivative from the reaction mixture in a yield of 81%.

2.75 g of the calicsarene ester derivative obtained as described above was dissolved in 80 ml of tetrahydrofuran (THF), 40 ml of tetramethylammonium hydroxide (25% aqueous solution) and 80 ml of distilled water were added and stirred under reflux for 24 hours. The thus obtained calixarylene carboxylic acid derivative was obtained in a yield of 91%.

The solution obtained by dissolving 134 mg of the calixarethylene carboxylic acid derivative obtained in 10 ml of tetrahydrofuran was cooled in an ice bath, and 116 mg of oxalic chloride and one drop of N, N -dimethylformamide were stirred for 40 minutes. 131 mg of 1-aza-18-crown-6, 5 ml of tetrahydrofuran, and 80 µl of triethylamine were added to the obtained calciric acid chloride derivatives. Stir again. Column chromatography (silica gel, methanol / dichloromethane) was isolated in 91% yield.

< Example  2> 25,27-bis [(4,7,10,13,16- Pentaoxa -One- Azacyclooctade -One- Nylcarbonyl ) Methoxy ] -5,17-bis ( Chloromethyl ) Calix [4] Aren's  Produce

In an ice bath, add 0.78 ml of tin (II) chloride (1M dichloromethane solution) and 125 mg of chloromethylmethyl ether in 15 ml of chloroform, and after 15 minutes, add 40 mg of bis-azacrownethercalixylene derivative dissolved in 10 ml of chloroform. After stirring for an hour, the mixture was stirred for 3 hours while raising to room temperature. Column chromatography (silica gel, methanol / dichloromethane) was isolated in 45% yield.

< Example  3> 25,27-bis [(4,7,10,13,16- Pentaoxa -One- Azacyclooctade -One- Nylcarbonyl ) Methoxy ] -5,17-bis ( Mercaptomethyl ) Calix [4] Aren's  Produce

25,27-bis [(4,7,10,13,16-pentaoxa-1-azacyclooctade-1-ylcarbonyl) methoxy] -5,17-bis (chloromethyl) calix [4 ] Add 81 mg of arene, 33 mg of thiourea, and 7 ml of N, N -dimethylformamide , stir the reaction overnight, remove the solvent, dissolve again in 10 ml of tetrahydrofuran, and add 5 ml of aqueous sodium hydroxide solution (0.3 M aqueous solution). After stirring for 10 minutes, the solvent was again removed by rotary evaporator, and 80 mg of sodium borohydride was added to a solution of the residue dissolved in 5 ml of dichloromethane. The mixture was washed with saturated aqueous ammonium chloride solution and dried over anhydrous magnesium sulfate. The filtrate was concentrated by evaporation to give a solid product in 99% yield.

< Example  4> Available on Gold Surface With pro linker  Developed Bis-Aza Crown Connected  The formation of self-assembled monolayers SPR  Measure

The gold surface was washed with methanol and again with a mixed solution of chloroform / methanol (1: 99 v / v). A 0.1 mM solution is prepared using a mixture of chloroform / methanol (1: 99 v / v) as a prolinker and a bis-aza crown ether caxylene linker, respectively. The washed gold substrate was immersed and the solution was flowed at 5 ml / min for 5 hours to form a self-assembled monomolecular film, and the surface plasma resonance device (Surface Plasmon Resonance) to fix the process of the resonance angle of the SPR Observation was made until there was no change. After the immobilization process, the mixture was washed sequentially with methanol and distilled water.

< Experimental Example  1>

With pro linker Bis-Azacrown Ether Calixen Connected  Bovine serum albumin on self-assembled monolayers ( BSA A) protein immobilization and SPR  Measure

The self-assembled monolayers of the pro-linker and bis-azacrownethercalisarene linkages were examined for protein immobilization using BSA, known as a water-soluble standard protein. The immobilization concentration of BSA was prepared and applied at a concentration of 5 mM in phosphate buffer solution (0.01 M, pH 7.4), and the immobilization process was continuously measured until the saturation time of the SPR resonance no longer occurred.

result

As a result of the experiment according to the <Example>, it was observed that the SPR resonance angle increases and saturates within 5 hours as the self-assembled monomolecular film of the prolinker is formed on the gold surface. In this case, the change in the resonance angle was 0.08 °, and it was found that a self-assembled monomolecular film was formed on the gold surface. It was observed that the change in resonance angle due to BSA protein immobilization on the self-assembled monomolecular membrane of the prolinker thus formed occurred at 0.56 degrees (FIG. 2).

In addition, as a result of the SPR experiment of the bis-aza crown ether calixarene conjugate prepared according to the <Example>, as the self-assembled monomolecular film is formed on the gold surface, the change of the resonance angle is 0.17 ° and the self-assembled stage on the gold surface. It was observed that a molecular film was formed. Immobilization of the BSA protein on the self-assembled monomolecular membrane of the thus formed bis-aza crown ether calicsarene conjugate was measured by SPR, and it was found that the immobilization occurred at 0.41 ° (FIG. 3).

In order to obtain the concentration of the self-assembled monomolecular membrane formed on the gold surface and the adsorption concentration between the self-assembled monomolecular membrane and the BSA protein from Equation 1 from the SPR test results of the prolinker, the optical constant obtained by the simulation is required. On the basis of the validity of, it is necessary to compare the curves obtained from the simulations with those obtained from the experimental results. As a result, the concentration of the self-assembled monolayer of prolinker formed on the gold surface was 260 pmole / cm ² , and the concentration of BSA protein adsorbed on the self-assembled monolayer was 175 ng / cm ² . In addition, the curves obtained from the simulations and the curves obtained from the experimental results are very consistent, showing the validity of this experiment (FIG. 4).

In order to obtain the concentrations of the self-assembled monomolecular film formed on the gold surface and the adsorption concentration of BSA protein on the self-assembled monomolecular membrane from Equation 1, the optical constants obtained by simulation It is also necessary to compare the curves obtained from the simulation results with those obtained from the experimental results on the basis of the validity of the experimental results. As a result, the concentration of the self-assembled monomolecular film of bis-aza crown ether calixarene conjugate formed on the gold surface was 235 pmole / cm ² The concentration of BSA protein adsorbed on the self-assembled monomolecular membrane was 197 ng / cm ² (FIG. 5).

The low concentration of the monomolecular membrane of the protein linkages formed on the gold surface is due to the size of the molecular plane of the biszacrownether linker (MW 1123.37) being larger than that of the prolinker (MW 702.92). However, the amount of BSA protein immobilized on the self-assembled monomolecular membrane of the bis-azacrownethercalixylene linker was shown to be about 11% better than the amount of protein immobilization by the prolinker protein linker. The curves obtained from the simulations and the curves obtained from the experimental results are very similar to show the validity of this experiment.

In the present invention, by confirming that the protein can be immobilized to a high concentration through the bis-aza crown ether caxylene linker, a method for immobilizing the protein at a higher concentration with an effective protein immobilization link using the bis-aza crown ether caxylene linker to provide.

As described above, the bis-azacrownethercalisarene linkage of the present invention can form a self-assembled monomolecular film on a gold surface, and the surface of the bis-aza crown ether-calixylene linker can be modified to have a better and more diverse shape by immobilizing proteins at a high concentration. By using this, it can be applied to various uses such as protein chip.

Claims (11)

delete A compound characterized by the following formula.

3. A compound according to claim 2, wherein said compound forms a self-assembled monomolecular film on the surface of gold. delete 4. A compound according to claim 3, wherein said compound immobilizes the protein. delete 6. A compound according to claim 5, wherein said compound is used in a protein chip. (1) preparing a bis-azacrownethercalixylene derivative represented by the following chemical formula by adding azacrown ether using Calix [4] arene as a starting material; and (2) a method for producing a novel compound, comprising introducing a thiol group at a para position of a benzene ring having a phenolic hydroxy group as a base skeleton of the derivative.

delete delete The method for producing a compound according to claim 8, wherein the compound is represented by the following formula.

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