KR101603038B1 - Disulfide-confined aryldiazonium salt compound and method for preparing the same - Google Patents

Abstract

The present invention relates to a disulfide-confined aryldiazonium(DSAD) salt compound, and to a producing method thereof and, more specifically, to a DSAD salt compound having a novel structure, and to a producing method which can efficiently synthesize the same. The DSAD salt compound enables the fixation of spatially-selective surface by simultaneously including a disulfide part activated by electrochemical oxidation and an aryldiazonium part which can be grafted on an electrode (ITO) or reduced graphene oxide (RGO), and can be helpfully used as a linker molecule for biomolecule fixation of an immunoassay and an immunological sensor platform.

Description

Disulfide-terminated aryldiazonium salt compounds and processes for their preparation [0002] DISULFIDE-CONFINED ARYLDIAZONIUM SALT COMPOUND AND METHOD FOR PREPARING THE SAME [0003]

Disclosed is a disulfide-confined aryldiazonium salt (DSAD) salt compound and a method for preparing the same. More particularly, the present invention relates to a disulfide-confined aryldiazonium salt (DSAD) By including the aryldiazonium moiety that can be grafted onto the pin oxide (RGO) simultaneously, it is possible to immobilize the surface-selective (surface-selective) surface, to effectively immobilize biomolecules such as antibodies, Disclosed is a novel disulfide-terminated aryldiazonium (DSAD) salt compound that can be usefully used as a linker molecule for immobilizing biomolecules on a sensor platform, and a method for efficiently synthesizing the same.

For chemical or biochemical sensors, adjusting the surface to have the desired final chemical functionality is a very important factor. There are many ways of modifying the surface, and well-known methods include forming a self-assembled monolayer (SAM) of an alkane thiol on gold, a technique of forming an indium tin oxide (ITO) Techniques for performing silanization of organosilanes, and techniques for electrochemically reducing diazonium salts on metals, carbon, or semiconductors.

Among these techniques, electrochemical grafting of diazonium salts offers advantages in terms of simplicity, stability and process time, and attracts a great deal of attention in terms of method diversity.

On the other hand, in order to obtain spatially-selective surface immobilization, it is important to design a highly reactive structure capable of forming a covalent bond between the surface and the molecule to be immobilized. For example, an activated form of disulfide, that is, a thiosulfinate group or a thiosulfonate group can form a covalent bond with a thiol (-SH) group of a biomolecule, Can be very advantageously applied. In this regard, the present inventors have recently reported on disulfide terminated silane molecules for multi-bio-functionalization.

However, there have been no studies on the disulfide-terminated aryldiazonium salt (DSAD) for use as a linker molecule. The disulfide-terminated aryldiazonium (DSAD) has excellent properties in relation to the surface grafting technology, and needs specific research and development.

On the other hand, reduced graphene oxide (RGO), a carbon layer of sp ² - hybrid single atom thickness, is attracting attention as a very interesting material with unique electrical, mechanical and optical properties.

Reduced graphene oxide (RGO) is a material with a very high potential range, but proper functionalization is required to use it in applications such as biosensors. Studies to functionalize graphene have been conducted, for example, to graft various diazoniums onto reduced graphene oxide (RGO).

However, a remarkable technique has not yet been developed that efficiently grafts the diazonium onto reduced graphene oxide (RGO) to functionalize the reduced graphene oxide (RGO) to suit the biosensor application.

In short, by simultaneously including an activatable disulfide moiety and an aryl diazonium moiety that can be effectively grafted on the electrode (ITO) to the reduced graphene oxide (RGO), spatially-selective surface immobilization (DSAD) salt compounds which can be usefully used as linker molecules for immunoassay and biosensor immobilization of biosensor platforms such as sandwich ELISA is required .

Korea Patent No. 10-0448880 Korean Patent Publication No. 10-1994-0011640

Disclosure of Invention Technical Problem [9] Accordingly, the present invention has been made to solve the above problems of the prior art, and it is an object of the present invention to provide a novel disulfide-terminated aryldiazonium (DSAD) salt compound suitable for immunoassay, its use as a linker for immobilizing biomolecules, The technical problem is to provide.

Disclosure of the Invention In order to accomplish the above object, the present invention provides a disulfide-terminated aryldiazonium (DSAD) salt compound represented by the following general formula (1).

[Chemical Formula 1]

(In the formula 1,

R is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,

Ar is an aryl group having 6 to 30 carbon atoms,

X is a halogen atom.)

Specifically, the present invention relates to a disulfide-terminated aryldiazonium (DSAD) salt compound represented by the following formula (2), 5- (1,2-dithiolan-3-yl) pentanamide- Chloride.

(2)

In another aspect of the present invention, there is provided a process for the preparation of tert-butyl 4- (4-fluorophenyl) diamine, comprising the steps of: a) reacting a lipoic acid with N-Boc- (5- (1,2-dithiolan-3-yl) pentanamide) phenylcarbamate; b) dissolving the compound of step a) in a mixture of dichloromethane (DCM) and trifluoroacetic acid (TFA) to form N- (4-aminophenyl) Synthesizing pentanamide; And c) dissolving the compound of step b) in hydrochloric acid (HCl) and adding sodium nitrite (NaNO ₂ ) to form 5- (1,2-dithiolan-3-yl) pentanamide-4-benzenediazonium chloride To obtain a disulfide-terminated aryldiazonium (DSAD) salt compound.

In another aspect of the present invention, the disulfide-terminated aryldiazonium (DSAD) salt compound is used as a linker for immobilizing a biomolecule on an immunoassay platform, for example, a living body of a sandwich immunoassay or a sandwich ELISA technique As a linker for molecular immobilization.

Disulfide-terminated aryldiazonium (DSAD) salt compounds according to the present invention can be used as efficient linker molecules for space-selective immobilization of biomolecules (proteins, antibodies, etc.) and for the production of graphene-based immunoassay platforms.

Specifically, when the disulfide-terminated aryldiazonium (DSAD) salt compound according to the present invention is used as a linker for space-selective immobilization of biomolecules, it is possible to manufacture biochips in various fields such as ultra fast detection or restricted release have.

Further, when the disulfide-terminated aryldiazonium (DSAD) salt compound according to the present invention is used as a linker for the preparation of a graphene-based immunoassay platform, graphene (e.g., RGO) is covalently functionalized, , Nucleic acids, and the like.

For example, a sandwich immunoassay (fluorescence detection) or sandwich ELISA using a disulfide-terminated aryldiazonium (DSAD) salt compound according to the present invention can efficiently and highly sensitively detect a target substance.

FIG. 1 is a schematic view illustrating a method of preparing a disulfide-terminated aryldiazonium (DSAD) salt compound according to an embodiment of the present invention.
FIG. 2 is a schematic view illustrating a method of spatially-selectively modifying the surface of an ITO electrode through a disulfide-terminal aryldiazonium salt (DSAD) salt compound of the present invention and a method for detecting a Rabbit antigen using the same.
FIG. 3 is a schematic view showing a method for functionalizing the ERGO surface using the disulfide-terminal aryldiazonium (DSAD) salt compound of the present invention and a mouse antigen detection method using the sandwich ELISA technique.

Hereinafter, the present invention will be described in detail.

Disulfide-confined aryldiazonium (DSAD) salt compounds of the present invention are represented by the following formula (1), more specifically represented by the following formula 2 (5- (1,2-dithiolane- 3-yl) pentanamide-4-benzenediazonium chloride).

[Chemical Formula 1]

(In the formula 1,

R is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,

Ar is an aryl group having 6 to 30 carbon atoms,

X is a halogen atom.)

(2)

The disulfide-terminated aryldiazonium salt (DSAD) salt compound according to the present invention synthesizes a carbamate-based compound containing a dithiolane moiety using a lipoic acid as a reactant; Removing the Boc moiety; And introducing a halogen anion.

In one embodiment, the disulfide-terminated aryldiazonium (DSAD) salt compound of Formula 2, according to the present invention,

a) reacting lipoic acid and N-Boc-1,4-phenylenediamine with triethylamine (TEA), hydroxybenzotriazole (HOBt) and EDCI (1-ethyl-3- [3- (dimethylamino) propyl] -carbodiimide hydrochloride to synthesize tert-butyl 4- (5- (1,2-dithiolan-3-yl) pentanamide) phenylcarbamate;

b) dissolving the compound of step a) in a mixture of dichloromethane (DCM) and trifluoroacetic acid (TFA) to form N- (4-aminophenyl) Synthesizing pentanamide; And

c) dissolving the compound of step b) in hydrochloric acid (HCl) and adding sodium nitrite (NaNO ₂ ) to give 5- (1,2-dithiolan-3-yl) pentanamide-4-benzenediazonium chloride (See Fig. 1).

The thus prepared disulfide-terminated aryldiazonium (DSAD) salt compound of the present invention can be very usefully used as a linker for immobilizing a biomolecule in various immune assay platforms and biosensors for detection of target substances . For example, when the disulfide-terminated aryldiazonium (DSAD) salt compound is electrochemically oxidized, the disulfide moiety is activated with a thiosulfinate group or a thiosulfonate group, The biomolecule can be efficiently fixed by forming a covalent bond with the thiol (-SH) group.

The disulfide-terminated aryldiazonium (DSAD) salt compounds of the present invention can be broadly applied to an immunoassay platform in the following two ways.

First, the above-mentioned disulfide-terminated aryldiazonium (DSAD) salt compound is applied in a spatially-selective manner for electrodeposition, cathodic deposition and activation on a patterned microelectrode array To be used as a linker for biomolecule immobilization of a fluorescence-based sandwich immunoassay. This approach can be applied specifically for the detection of, for example, Rabbit antigens.

Specifically,

(1) electrodepositing the DSAD salt compound onto an indium tin oxide (ITO) microelectrode array through a cyclic voltammetry (CV);

(2) electrochemically oxidizing the DSAD salt compound electrodeposited on some ITO microelectrodes to convert the disulfide group on the surface to a thiosulfinate group (R ¹ -S-SO-R ² ) or a thiosulfonate group (R ¹ -S- SO _{2 -} R ² );

(3) spatially-selectively immobilizing the biomolecule only in the activated portion by exposing the biomolecule having the thiol group (primary antibody) (for example, Anti-Rabbit antigen);

(4) exposing and immobilizing the target material (target antigen) (e.g., Rabbit antigen);

(5) exposing and immobilizing a secondary antibody (for example, Anti-Rabbit antigen) labeled with TRITC (Tetramethylrhodamine isothiocyanate);

(6) observing the surface with a fluorescence microscope;

The target substance can be fluorescence-detected (see FIG. 2).

Secondly, the disulfide-terminated aryldiazonium (DSAD) salt compound is reacted with reduced graphene oxide (RGO), such as electrochemically reduced graphene oxide (ERGO (Electrochemical grafting) by electrodepositing (electrochemically grafting) the electrochemical catalytic activity on the surface of the substrate, and the electrochemical catalytic activity is maintained or increased by mutual bonding. The electrochemical immunoassay using the sandwich ELISA technique as a linker for immobilizing biomolecules will be. This approach can be applied specifically for electrochemical detection of, for example, mouse antigens.

Specifically,

(1) An ITO electrode washed with a Piranha solution was washed with water again to form a negative charge on the electrode surface, and the positive electrode was charged with a positive charge of polyethylenimine (PEI) (ERGO / PEI / ITO) by electrochemically reducing GO by forming a graphene oxide (GO) through electrostatic interaction on the obtained surface after pretreatment with an aqueous solution. ;

(2) electrodepositing the DSAD salt compound onto ERGO to obtain a DSAD functionalized ERGO surface (DSAD / ERGO / PEI / ITO);

(3) The DSAD salt compound of DSAD / ERGO / PEI / ITO is electrochemically oxidized to convert the disulfide group on the surface to a thiosulfinate group (R ¹ -S-SO-R ² ) or a thiosulfonate group (R ¹ -S- SO ₂ -R ² ) and activating (ODSAD / ERGO / PEI / ITO surface) to produce an electrochemical immunosensor;

(4) exposing and fixing a primary antibody (e.g., Anti-Mouse antigen) having a thiol group on the ODSAD / ERGO / PEI / ITO surface;

(5) exposing and immobilizing a target antigen (e. G., Mouse antigen);

(6) exposing and immobilizing a secondary antibody labeled with HRP (e.g., Anti-Mouse antigen);

(7) Exposure of an electrolyte (for example, hydrogen peroxide and hydroquinone solution) causes a chemical catalytic reaction, and then the amount of a substance (for example, benzoquinone) generated through electrochemical reaction is detected by using a cyclic voltammetry Expelling step;

The target antigen can be electrochemically detected by a sandwich ELISA method (see FIG. 3).

In this method, the aryl radical of the disulfide-terminated aryldiazonium (DSAD) salt compound during the electrodeposition process forms a covalent bond with the sp ² carbon atom of RGO. In addition, in the presence of hydrogen peroxide (H ₂ O ₂ ), HRP (Horseradish peroxidase) catalyzes the conversion of hydroquinone (HQ) into benzoquinone (BQ) resulting in a signal current proportional to the amount of target antigen . ELISA is a sensitive diagnostic tool for antigen detection and a technique used for quality control in various industries. An electric signal is generated by an enzyme reaction product linked to a detection reagent, and accurate measurement is possible. And depends on the amplification of such electrical signals. The biosensor platform using the disulfide-terminated aryldiazonium salt (DSAD) salt compound according to the present invention showed excellent sensitivity and specificity to the target substance, particularly mouse antigen, when applied to the electrochemical ELISA technique.

The disulfide-terminated aryldiazonium (DSAD) salt compounds of the present invention can be prepared by reacting the aryldiazonium moieties with a high reactivity (e.g., high reactivity) to aryldiazonium moieties on a variety of solid surfaces (e.g. carbonaceous materials such as graphene, graphite and diamond; gold; platinum; , It can be used as a highly useful linker molecule with respect to the modification of functional nanomaterial surface through covalent immobilization.

Hereinafter, the present invention will be described more specifically by way of examples. However, these examples are provided only for the understanding of the present invention, and the scope of the present invention is not limited to these examples in any sense.

Example : Disulfide -end Aryldiazonium ( DSAD ) Preparation of salt compounds

(1) reagents and devices

(LA), N-Boc-1,4-phenylenediamine, triethylamine (TEA), hydroxybenzotriazole (HOBt), EDCl, tetrabutylammonium perchlorate (TBAP) ) phosphine (TCEP), the graphite powder (<20μm), sulfuric acid (H ₂ SO _4), hydrogen peroxide (H ₂ O _2), hydrochloric acid (HCl), sodium nitrite (NaNO _2), sodium chloride (NaCl), Rabbit antigen, Mouse antigen, Anti-Rabbit antigen, Anti-Mouse antigen, Anti-Rabbit antigen TRITC and Anti-Mouse antigen HRP were obtained from Sigma Aldrich.

The phosphate buffered saline (PBS) solution was composed of 0.01 M phosphate and 0.15 M NaCl, and PBST was composed of PBS and 0.05% (w / w) Tween-20.

All chemicals were analytical grade and were used without further purification.

All buffers and aqueous solutions were prepared with 18.2 MΩ pure water.

Fluorescence imaging, cyclic voltammetry (CV) and X-ray photoelectron spectroscopy (XPS) were performed using the same instrument as reported previously in Haque et al., 2012 Respectively.

(2) Rat -Butyl 4- (5- (1,2- Dithian -3 days) Pentanamide ) Phenylcarbamate  Synthesis: (1a)

N-Boc-1,4-phenylenediamine, triethylamine (TEA), hydroxybenzotriazole (HOBt), EDCl and additional triethylamine (TEA) were successively added to the liposic acid solution dissolved in dichloromethane .

The reaction mixture was then stirred at 25 &lt; 0 &gt; C overnight and diluted with water.

The product was extracted with dichloromethane (DCM), dried over magnesium sulfate, filtered and concentrated under reduced pressure.

The resulting reddish brown powder was suspended in diethyl ether, followed by filtration and rinsing with ether to give a pale pink powder (yield: 80.5%).

1 H-NMR (CDCl ₃ , 400 MHz):? Ppm = 1.25 (s, 1H, CH ₂ ); 1.91 (s, 9 H, BOC); 1.61 (s, 1 H, CH ₂ ); _{1.68-1.78 (m, 4H, CH 2} ); 1.91-1.94 (t, 1H, -S- CH 2); _{2.32-2.36 (t, 2H, CH 2} ); 2.43-12.48 (q, 1H, -S- CH 2); _{3.10-3.18 (m, 2H, CH 2} ), 3.56-3.59 (t, 1H, -S-CH-); 6.46 (s, 1H, CONH); 7.18 (s, 1H, CONH-BOC); 7.26-7.31 (d, 2H, arom); 7.41-7.43 (d, 2H, arom.).

(3) N- (4- Aminophenyl ) -5- (1,2- Dithian -3 days) Pentanamide  Synthesis: (2a)

Compound 1a (210.97 mg, 0.532 mM) was dissolved in 6.666 mL dry DCM: TFA (10: 1) mixture, stirred overnight at room temperature, then washed with water and extracted with DCM.

The resulting solution was dried over magnesium sulfate and concentrated under reduced pressure.

Purification was then performed on a silica-gel column (mobile phase 5% methanol in DCM) to give a beige solid (21% yield).

1 H-NMR (CDCl ₃ , 400 MHz):? Ppm = 0.83-0.85 (d, 1H, CH ₂ ); _{1.23-1.26 (d, 1H, CH 2} ); _{1.36-1.40 (t, 2H, CH 2} ); _{1.56-1.59 (t, 2H, CH 2} ); _{1.68-1.70 (m, 1H, CH 2} ); 1.84-1.89 (m, 1 H, CH ₂ ); 2.20-2.23 (t, 1 H, CH ₂ ); 2.39 - 2.45 (m, 1H, CH ₂ ); _{3.09-3.21 (m, 2H, CH 2} ); 3.59-3.66 (m, 1H, -S-CH-); 4.87 (s, 2H, NH ₂ ); 6.46-6.49 (d, 2H, arom); 7.19-7.21 (d, 2H, arom); 9.45 (s, 1H, CONH).

(4) 5- (1,2- Dithian -3 days) Pentanamide Benzene Diazonium  Synthesis of chloride

Compound 2a (0.889 mg) was dissolved in 1.9 mL of 0.1 M HCl and cooled in an ice bath to prepare 2 mL of a 1.5 mM solution.

Then, 100 μL of cold NaNO ₂ (0.23 mg in 0.1 mL H ₂ O) was added dropwise with stirring.

Stirring was continued for a further 30 minutes to finally obtain a solution of the disulfide-terminated aryldiazonium (DSAD) salt compound.

Claims (8)

Disulfide-confined aryldiazonium (DSAD) salt compound represented by the following formula 1:
[Chemical Formula 1]

In Formula 1,
R is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
Ar is an aryl group having 6 to 30 carbon atoms,
X is a halogen atom.
The method according to claim 1,
The disulfide-terminated aryldiazonium (DSAD) salt compound is represented by the following general formula (2)
(2)

.
a) reacting lipoic acid with N-Boc-1,4-phenylenediamine in the presence of triethylamine (TEA), hydroxybenzotriazole (HOBt) Olan-3-yl) pentanamide) phenylcarbamate;
b) dissolving the compound of step a) in a mixture of dichloromethane (DCM) and trifluoroacetic acid (TFA) to form N- (4-aminophenyl) Synthesizing pentanamide; And
c) dissolving the compound of step b) in hydrochloric acid (HCl) and adding sodium nitrite (NaNO ₂ ) to give 5- (1,2-dithiolan-3-yl) pentanamide-4-benzenediazonium chloride ;
Lt; RTI ID = 0.0 &gt; (DSAD) &lt; / RTI &gt;
3. The method according to claim 1 or 2,
The disulfide-terminated aryldiazonium (DSAD) salt compound is used as a linker for immobilizing biomolecules on an immunoassay platform.
5. The method of claim 4,
The disulfide-terminated aryldiazonium (DSAD) salt compound is used as a linker for biomolecule immobilization of a sandwich immunoassay.
6. The method of claim 5,
The disulfide-terminated aryldiazonium (DSAD) salt compound is formed on the microelectrode array, and a compound formed on some microelectrodes is activated to spatially-selectively immobilize biomolecules Disulfide-terminated aryldiazonium (DSAD) salt compound.
5. The method of claim 4,
The disulfide-terminated aryldiazonium (DSAD) salt compound is used as a linker for biomolecule immobilization of the sandwich ELISA technique.
8. The method of claim 7,
The disulfide-terminated aryldiazonium (DSAD) salt compound is bonded to the surface of electrochemically reduced graphene oxide (ERGO) formed on the electrode. The disulfide-terminated aryldiazonium DSAD) salt compound.

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