Water-soluble acridine sulfonamide carboxylic acid marker, preparation method, application thereof and kit

Abstract

The invention relates to the field of chemiluminescence immunodetection, in particular to a water-soluble acridine sulfonamide carboxylic acid marker, a preparation method, application of the water-soluble acridine sulfonamide carboxylic acid marker and a kit, in some embodiments, the water-soluble acridine sulfonamide carboxylic acid marker is formed by coupling acridine sulfonamide and glycine tripeptide with good water solubility through one-step reaction, synthesis is simple, and the yield is high. According to the water-soluble acridine sulfonamide carboxylic acid marker, the water solubility is increased, a connecting arm is prolonged, and the problem of water solubility of a protein marker is solved.

Description

Technical Field

[0001] The present application relates to a water-soluble acridine sulfonamide carboxylic acid marker, a preparation method, an application thereof and a kit, and belongs to chemiluminescent immunoassay. Background Art

[0002] Chemiluminescence immunoassay (CLIA) combines highly sensitive chemiluminescence assay technology with highly specific immune reactions. It can be divided into three categories according to the different markers, namely direct chemiluminescence immunoassay, chemiluminescence enzyme immunoassay and electrochemiluminescence immunoassay. Chemiluminescence immunoassay is mainly used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. It is the latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay. Summary of the invention

[0003] The purpose of the present application is to provide a water-soluble acridine sulfonamide carboxylic acid marker, a preparation method, its application and a kit, which solves the water solubility problem of protein markers and is easy to synthesize.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] In a first aspect, the present application provides a water-soluble acridinium sulfonamide carboxylic acid marker, wherein the water-soluble acridinium sulfonamide carboxylic acid marker has a structural formula shown in Formula 1:

[0006] (Formula 1)

[0007]

[0008] In a second aspect, the present application provides a method for preparing a water-soluble acridine sulfonamide carboxylic acid marker, which comprises the following steps:

[0009] Provides acridine sulfonamide and tripeptide glycine;

[0010] Adding an acridine sulfonamide solution to the weakly alkaline aqueous solution of the tripeptide glycine to carry out a reaction;

[0011] After the reaction is completed, the pH is adjusted and the product is separated and purified to obtain the water-soluble acridine sulfonamide carboxylic acid marker;

[0012] Wherein, the water-soluble acridine sulfonamide carboxylic acid marker has a structural formula shown in Formula 1, the acridine sulfonamide has a structural formula shown in Formula 2, and the tripeptide glycine has a structural formula shown in Formula 3:

[0013]

[0014] In one or more feasible embodiments, the step of adding an acridine sulfonamide solution to the weakly alkaline aqueous solution of the tripeptide glycine for reaction comprises:

[0015] Adding the tripeptide glycine and an alkaline substance into deionized water to obtain a weak alkaline aqueous solution of the tripeptide glycine;

[0016] adding the acridine sulfonamide into a water-soluble organic solvent to obtain the acridine sulfonamide solution;

[0017] The weak alkaline aqueous solution and the acridine sulfonamide solution were mixed and reacted at room temperature overnight.

[0018] In one or more feasible embodiments, after the reaction is completed, the pH is adjusted and the separation and purification are performed, comprising:

[0019] After the reaction is completed, an acidic substance is added to adjust the pH of the solution to acidic, and then liquid phase separation and purification is performed based on reverse phase distribution chromatography.

[0020] In one or more feasible embodiments, after the separation and purification, the method further comprises:

[0021] Subsequently, most of the solvent is evaporated and the mixture is freeze-dried to obtain the water-soluble acridinium sulfonamide carboxylic acid marker.

[0022] In one or more feasible embodiments, the alkaline substance is sodium carbonate, and the acidic substance is dilute hydrochloric acid.

[0023] In one or more feasible embodiments, the organic solvent is selected from any one or more of DMF, DCM, ethyl acetate or DMSO.

[0024] In a third aspect, the present application provides a water-soluble acridinium sulfonamide carboxylic acid marker or a stereoisomer / tautomer thereof prepared by the preparation method described in the second aspect.

[0025] In a fourth aspect, the present application provides a use of the water-soluble acridinium sulfonamide carboxylic acid marker or its stereoisomers / tautomers according to the third aspect in cancer detection.

[0026] In one or more feasible embodiments, it is used to detect carbohydrate antigens; or, it is used to detect carbohydrate antigens 199.

[0027] In a fifth aspect, the present application provides a kit comprising the water-soluble acridinium sulfonamide carboxylic acid marker or a stereoisomer / tautomer thereof as described in the third aspect.

[0028] According to the embodiments of the present application, the water-soluble acridinium sulfonamide carboxylic acid marker of the present application is prepared by coupling acridinium sulfonamide and a glycine tripeptide with good water solubility in a one-step reaction, and the synthesis is simple and the yield is high. The water-soluble acridinium sulfonamide, which originally has poor water solubility and large steric hindrance, is synthesized into the water-soluble acridinium sulfonamide carboxylic acid marker of the present application, which not only increases the water solubility, but also extends the connecting arm.

[0029] According to the embodiments of the present application, when the water-soluble acridine sulfonamide carboxylic acid marker of the present application is used to label protein, it has good water solubility and small steric hindrance, which solves the water solubility problem of the protein marker to the greatest extent.

[0030] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a H NMR spectrum of the water-soluble acridinium sulfonamide carboxylic acid marker shown in one embodiment of the present application;

[0032] Figure 2 is a mass spectrum of a water-soluble acridinium sulfonamide carboxylic acid marker as shown in one embodiment of the present application;

[0033] Figure 3 This is a concentration-luminescence value curve of the water-soluble acridine sulfonamide carboxylic acid marker in the detection of carbohydrate antigen 199 shown in an embodiment of the present application;

[0034] Figure 4 This is a concentration-luminescence value curve of a common acridine sulfonamide marker in the detection of carbohydrate antigen 199 shown in an embodiment of the present application. DETAILED DESCRIPTION

[0035] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0036] The terms "include", "comprises", "have", etc. are used interchangeably and have the same meaning. Similarly, "includes", "includes", "have", etc. are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "include" under ordinary U.S. patent law, so each term can be understood as an open term, which means "at least the following" and can also be interpreted as not excluding additional features, limitations, aspects, etc. Therefore, for example, "a device having components a, b and c" means that the device includes at least components a, b and c. Similarly, the phrase: "a method involving steps a, b and c" means that the method includes at least steps a, b and c. In addition, although the steps and processes may be outlined in a particular order herein, those skilled in the art will recognize that the order steps and processes may vary.

[0037] "Derivative" is used in its ordinary sense in chemistry and biology to refer to a compound that is structurally similar to another compound (the so-called "reference" compound) but has a different composition (e.g., an atom is replaced by an atom of a different element, or a specific functional group is present, or one functional group is replaced by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound is different). Thus, an analog is a compound that is similar or equivalent in function and appearance to a reference compound but has a different structure or origin.

[0038] As used herein, the term "antibody" refers to immunoglobulins or immunoglobulin-like molecules, including, for example, but not limited to: IgA, IgD, IgE, IgG and IgM and combinations thereof; similar molecules produced during the immune response of any vertebrate (e.g., in mammals such as humans, goats, rabbits and mice, etc.); and antibody fragments that specifically bind to a target molecule (or a group of highly similar target molecules) and substantially exclude binding to other molecules. "Antibody" further refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region, which specifically recognizes and binds to an epitope of an antigen. An antibody can be composed of a heavy chain and a light chain, each of which has a variable region, referred to as a variable heavy chain (VH) region and a variable light chain (VL) region. The VH region and the VL region are jointly responsible for binding to the antigen recognized by the antibody. The term "antibody" also includes complete immunoglobulins and variants and portions thereof well known in the art.

[0039] The titles of this application are for convenience only and are not used to interpret the scope or meaning of the disclosed embodiments of the invention.

[0040] Direct chemiluminescent immunoassay is to directly label antibodies (antigens) with acridinium esters. After an immune reaction occurs with the corresponding antigen (antibody) in the test specimen, a solid-phase coated antibody-test antigen-acridinium ester labeled antibody complex is formed. At this time, only oxidants (H2O2) and NaOH need to be added to create an alkaline environment. When the molecules are attacked by hydrogen peroxide, unstable ethylene dioxide is generated. At this time, ethylene dioxide decomposes into CO2 and electronically excited N-methylacridone. When it returns to the ground state, it emits photons with a maximum emission wavelength of 430nm. Acridinium ester decomposes and emits light without the need for a catalyst.

[0041] For example, in the acridinium ester and acridinium sulfonamide shown in the following formula, R, R', and R" are alkyl, alkoxy, aryl, and other substituents; X, X', and X" are coupling groups used to couple antigens or antibodies and increase the solubility of the compound.

[0042]

[0043] Its luminescence mechanism is:

[0044]

[0045] Acridinium ester luminescent reagents have mild reaction conditions, simple luminescent systems, no catalytic process, and no enhancer, thereby reducing background luminescence, improving signal-to-noise ratio, and reducing interference. At the same time, light release is rapid and concentrated, with high luminescence efficiency and high luminescence intensity. However, the acridinium ester structure has poor stability and is easy to decompose. Based on this, the present application provides a water-soluble acridinium sulfonamide carboxylic acid marker based on acridinium sulfonamide, and the water-soluble acridinium sulfonamide carboxylic acid marker has a structural formula shown in Formula 1:

[0046] (Formula 1)

[0047]

[0048] In one embodiment, the present application provides a method for preparing a water-soluble acridine sulfonamide carboxylic acid marker, as shown in the following formula:

[0049]

[0050] It includes the following steps:

[0051] Provides acridine sulfonamide and tripeptide glycine;

[0052] Adding an acridine sulfonamide solution to the weakly alkaline aqueous solution of the tripeptide glycine to carry out a reaction;

[0053] After the reaction is completed, the pH is adjusted and the product is separated and purified to obtain the water-soluble acridine sulfonamide carboxylic acid marker;

[0054] Wherein, the water-soluble acridine sulfonamide carboxylic acid marker has a structural formula shown in Formula 1, the acridine sulfonamide has a structural formula shown in Formula 2, and the tripeptide glycine has a structural formula shown in Formula 3:

[0055]

[0056] In one embodiment, the preparation method specifically comprises:

[0057] Adding the tripeptide glycine and an alkaline substance into deionized water to obtain a weak alkaline aqueous solution of the tripeptide glycine;

[0058] adding the acridine sulfonamide into a water-soluble organic solvent to obtain the acridine sulfonamide solution;

[0059] Mixing the weak alkaline aqueous solution and the acridine sulfonamide solution, and reacting at room temperature overnight;

[0060] After the reaction is completed, an acidic substance is added to adjust the pH of the solution to be acidic, and then liquid phase separation and purification are performed based on reverse phase distribution chromatography, and then most of the solvent is evaporated and freeze-dried to obtain the water-soluble acridine sulfonamide carboxylic acid marker.

[0061] As an example but not a limitation, the organic solvent is selected from any one or more of DMF, DCM, ethyl acetate or DMSO. The alkaline substance is sodium carbonate, and the acidic substance is dilute hydrochloric acid.

[0062] In the examples, unless otherwise specified, the reagents used were purchased from the market.

[0063] In the examples of the present application, acridine sulfonamide and tripeptide glycine are coupled by a one-step reaction to obtain the above-mentioned water-soluble acridine sulfonamide carboxylic acid marker, which has a simple synthesis and a high yield.

[0064] Example 1: Tripeptide glycine (0.08 mmol, 16 mg, 1.5 eq) and sodium carbonate (3 eq, 0.11 mmol, 9 mg) were added to deionized water (0.4 ml) to dissolve, and then acridine sulfonamide (50 mg, 0.073 mmol, 1 eq) was added to DMF (0.8 ml), and then the water and DMF phases were mixed and reacted at room temperature overnight.

[0065] An appropriate amount of dilute hydrochloric acid was added to adjust the pH to acidic. Then, the product was separated and purified using reverse phase C18 preparative liquid phase. Most of the solvent was evaporated off using a rotary evaporator, and then freeze-dried to obtain the target product with a yield of 72.1%.

[0066] Example 2: Tripeptide glycine (0.16mmol, 32mg, 1.5eq) and sodium carbonate (3eq, 0.22mmol, 18mg) were added to deionized water (0.8ml) to dissolve, and then acridine sulfonamide (100mg, 0.146mmol, 1eq) was added to DMF (1.6ml), and then the water and DMF phases were mixed and reacted at room temperature overnight.

[0067] An appropriate amount of dilute hydrochloric acid was added to adjust the pH to acidic. Then, the product was separated and purified using reverse phase C18 preparative liquid phase. Most of the solvent was evaporated off using a rotary evaporator, and then freeze-dried to obtain the target product with a yield of 72.7%.

[0068] Example 3: Tripeptide glycine (0.24mmol, 48mg, 1.5eq) and sodium carbonate (3eq, 0.33mmol, 27mg) were added to deionized water (1.2ml) to dissolve, and then acridine sulfonamide (150mg, 0.219mmol, 1eq) was added to DMF (2.4ml), and then the water and DMF phases were mixed and reacted at room temperature overnight.

[0069] An appropriate amount of dilute hydrochloric acid was added to adjust the pH to acidic. Then, the product was separated and purified using reverse phase C18 preparative liquid phase. Most of the solvent was evaporated off using a rotary evaporator, and then freeze-dried to obtain the target product with a yield of 72.4%.

[0070] The H NMR spectrum and mass spectrum of the obtained product are shown as follows: Figure 1 and Figure 2 As shown in the figure, it can be seen that the compound shown in formula 1 is obtained, and its M=755.8.

[0071] The applicant has found that the water-soluble acridine sulfonamide carboxylic acid marker of the embodiment of the present application can be used in cancer detection. Specifically, it can be used to detect CA199 based on the magnetic microparticle chemiluminescence immunoassay. CA199, or carbohydrate antigen 199, is a carbohydrate antigen associated with gastrointestinal cancer. Clinically, the detection of CA199 serum concentration can be used as an auxiliary diagnostic indicator for malignant tumors such as pancreatic cancer and gallbladder cancer. Its chemiluminescence detection method is sandwich detection, biotin-labeled capture antibody, acridine sulfonamide-labeled detection antibody, and then incubated with the antigen to be detected CA199, wash off the excess detection antibody, and react with acridine sulfonamide in hydrogen peroxide under alkaline NaOH conditions to emit fluorescence. The strength of the fluorescence signal is detected to detect the concentration of the antigen to be detected.

[0072] Specifically, in one embodiment of detecting CA199, a water-soluble acridinium sulfonamide carboxylic acid label (1 eq) was added to DMF, Hosu (1.2 eq) was added, and then EDC.HCl (1.2 eq) was added, and the reaction was carried out at room temperature for more than 2 hours to obtain an active ester solution. An appropriate amount of the active ester solution was added to the detection antibody, and the labeling reaction was carried out at room temperature for more than 1.5 hours. No precipitation was formed, and the detection antibody labeled with a water-soluble acridinium salt was obtained by purification by dialysis, and the labeling effect was good.

[0073] Comparative test: Use common acridine sulfonamide to carry out the antibody labeling experiment. Dissolve acridine sulfonamide in DMF, take the solution with the same molar amount as the water-soluble acridine sulfonamide active ester in the above example and add it to the same amount of detection antibody as in the above example. After reacting at room temperature for more than 1.5 hours, it is found that antibody precipitates in the solution. The reason is that after labeling the antibody, the water solubility of the antibody becomes poor, thus causing precipitation.

[0074] The test results of the water-soluble acridine sulfonamide labeled antibody reagent on the chemiluminescence instrument are as follows Figure 3 And the results shown in Table 1:

[0075] Table 1

[0076]

[0077]

[0078] Depend on Figure 3 As shown in Table 1, the concentration and luminescence value have a good correlation, and the correlation coefficient R 2 It reaches 0.99, indicating that the luminescence value increases with the increase of the concentration of antigen CA199. The synthesized water-soluble acridine sulfonamide carboxylic acid marker is very suitable as a luminescent marker for magnetic particle chemiluminescent immunoassay.

[0079] In the comparative experiment, the results of the test of the common acridine sulfonamide labeled antibody reagent on the chemiluminescence instrument are as follows: Figure 4 As shown in Table 2:

[0080] Table 2

[0081] Concentration (ng / ml) Luminescence value (RLU) C0 0IU / ml 2543 C1 6.95IU / ml 3653 C2 27.43IU / ml 3982 C3 52.93IU / ml 4100 C4 232.27IU / ml 4508 C5 976.69IU / ml 4646

[0082] Depend on Figure 4 As shown in Table 2, the linear relationship between concentration and luminescence value is poor, and the correlation coefficient R 2 It is only 0.36, indicating that the luminescence value does not change much with the increase of the concentration of antigen CA199.

[0083] Of course, in other embodiments, the stereoisomers or tautomers of the water-soluble acridinium sulfonamide carboxylic acid labeling substance shown in Formula 1 also have the above-mentioned effects, and those skilled in the art can perform corresponding tests according to the disclosure of this application.

[0084] The present application also discloses a chemiluminescent immunoassay kit, which comprises the above-mentioned water-soluble acridine sulfonamide carboxylic acid marker or its stereoisomer / tautomer.

[0085] In summary, the water-soluble acridinium sulfonamide carboxylic acid marker of the present application is prepared by coupling acridinium sulfonamide and a glycine tripeptide with good water solubility in a one-step reaction, and the synthesis is simple and the yield is high. The water-soluble acridinium sulfonamide, which originally has poor water solubility and large steric hindrance, is synthesized into the water-soluble acridinium sulfonamide carboxylic acid marker of the present application, which not only increases the water solubility but also extends the connecting arm.

[0086] When the water-soluble acridinium sulfonamide carboxylic acid marker of the present application is used to label protein, it has good water solubility and small steric hindrance, thus solving the water solubility problem of the protein marker to the greatest extent.

[0087] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0088] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A water-soluble acridine sulfonamide carboxylic acid label, characterized in that: The water-soluble acridine sulfonamide carboxylic acid marker has a structural formula shown in Formula 1: (Formula 1) 2. A method for preparing a water-soluble acridine sulfonamide carboxylic acid marker, characterized in that: The following steps are involved: Provides acridine sulfonamide and tripeptide glycine; Adding an acridine sulfonamide solution to the weakly alkaline aqueous solution of the tripeptide glycine to carry out a reaction; After the reaction is completed, the pH is adjusted and the product is separated and purified to obtain the water-soluble acridine sulfonamide carboxylic acid marker; Wherein, the water-soluble acridine sulfonamide carboxylic acid marker has a structural formula shown in Formula 1, the acridine sulfonamide has a structural formula shown in Formula 2, and the tripeptide glycine has a structural formula shown in Formula 3:

3. The preparation method according to claim 2, characterized in that: The step of adding an acridine sulfonamide solution to the weakly alkaline aqueous solution of the tripeptide glycine to carry out a reaction comprises: Adding the tripeptide glycine and an alkaline substance into deionized water to obtain a weak alkaline aqueous solution of the tripeptide glycine; adding the acridine sulfonamide into a water-soluble organic solvent to obtain the acridine sulfonamide solution; The weak alkaline aqueous solution and the acridine sulfonamide solution were mixed and reacted at room temperature overnight.

4. The preparation method according to claim 2 or 3, characterized in that: After the reaction is completed, the pH is adjusted and the separation and purification are performed, including: After the reaction is completed, an acidic substance is added to adjust the pH of the solution to acidic, and then liquid phase separation and purification is performed based on reverse phase distribution chromatography.

5. The preparation method according to claim 4, characterized in that: After the separation and purification, the method further comprises: Subsequently, most of the solvent is evaporated and the mixture is freeze-dried to obtain the water-soluble acridinium sulfonamide carboxylic acid marker.

6. The preparation method according to claim 4, characterized in that: The alkaline substance is sodium carbonate, and the acidic substance is dilute hydrochloric acid.

7. A water-soluble acridinium sulfonamide carboxylic acid label or a stereoisomer / tautomer thereof obtained by the preparation method according to any one of claims 2 to 6.

8. Use of the water-soluble acridinium sulfonamide carboxylic acid label or its stereoisomers / tautomers according to claim 7 in cancer detection.

9. The use according to claim 7, characterized in that Used to detect carbohydrate antigens; or, used to detect carbohydrate antigen 199.

10. A kit, characterized in that: It comprises the water-soluble acridinium sulfonamide carboxylic acid labeling substance or its stereoisomer / tautomer as described in claim 7.

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