Water-soluble acridine sulfonamide carboxylic acid markers, methods of making, uses thereof, and kits

Abstract

The application relates to the field of chemiluminescence immunoassay, in particular to a water-soluble acridine sulfonamide carboxylic acid label, a preparation method, application and kit, wherein in some embodiments, the water-soluble acridine sulfonamide carboxylic acid label is coupled by one-step reaction of acridine sulfonamide and a water-soluble glycine tripeptide, and the synthesis is simple and the yield is high. The water-soluble acridine sulfonamide carboxylic acid label not only increases water solubility, but also prolongs a connecting arm, and solves the water solubility problem of a protein label.

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 immunoassays (CLIA) combine highly sensitive chemiluminescence technology with highly specific immune responses. Based on the specific markers used, they can be divided into three categories: direct chemiluminescence immunoassay, chemiluminescent enzyme immunoassay, and electrochemiluminescent immunoassay. CLIA is primarily 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 this 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 the 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 acridinium sulfonamide carboxylic acid marker, comprising 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] The water-soluble acridine sulfonamide carboxylic acid label has the structural formula shown in Formula 1, the acridine sulfonamide has the structural formula shown in Formula 2, and the tripeptide glycine has the 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 to carry out the reaction comprises:

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

[0016] adding the acridine sulfonamide to 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 are performed based on reverse phase partition 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 product is freeze-dried to obtain the water-soluble acridinium sulfonamide carboxylic acid labeling substance.

[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 label or a stereoisomer / tautomer thereof prepared according to 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 label 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 label or its stereoisomers / tautomers described in the third aspect.

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

[0029] According to the embodiments of the present application, when the water-soluble acridinium sulfonamide carboxylic acid marker of the present application is used to label proteins, it has good water solubility and low 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 with reference to 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 described in one embodiment of the present application;

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

[0034] Figure 4 This is a concentration-luminescence value curve of a common acridinium sulfonamide label 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 through specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed 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 unless they conflict.

[0036] The terms "including," "comprising," and "having" are used interchangeably and have the same meaning. Likewise, "including," "comprising," and "having" are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "including" under ordinary U.S. patent law, so that each term can be understood as an open term meaning "at least the following" and can also be interpreted as not excluding additional features, limitations, aspects, etc. Thus, 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. Furthermore, although steps and processes may be outlined herein in a particular order, those skilled in the art will recognize that the order steps and processes may vary.

[0037] The term "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 (for example, in mammals such as humans, goats, rabbits and mice); 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 uses acridinium ester to directly label antibodies (antigens). 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. The 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 luminescence systems, and no catalytic process or enhancer is required, thereby reducing background luminescence, improving the 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 is unstable and easily decomposed. Based on this, the present application provides a water-soluble acridinium sulfonamide carboxylic acid marker based on acridinium sulfonamide. The water-soluble acridinium sulfonamide carboxylic acid marker has the 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] The water-soluble acridine sulfonamide carboxylic acid label has the structural formula shown in Formula 1, the acridine sulfonamide has the structural formula shown in Formula 2, and the tripeptide glycine has the 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 weakly alkaline aqueous solution of the tripeptide glycine;

[0058] adding the acridine sulfonamide to 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 acidic, and then liquid phase separation and purification are performed based on reverse phase partition chromatography. Subsequently, 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 through a one-step reaction to obtain the above-mentioned water-soluble acridine sulfonamide carboxylic acid labeling agent, 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) and dissolved. Acridine sulfonamide (50 mg, 0.073 mmol, 1 eq) was then added to DMF (0.8 ml). The water and DMF phases were then mixed and reacted at room temperature overnight.

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

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

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

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

[0069] An appropriate amount of dilute hydrochloric acid was added to adjust the pH to acidic. The product was then separated and purified using a reverse phase C18 preparative liquid phase. The purified liquid was first evaporated using a rotary evaporator to remove most of the solvent 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, the compound of formula 1 is obtained, and its M=755.8.

[0071] The applicant has discovered 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, namely 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 a sandwich detection method, in which the capture antibody is labeled with biotin and the detection antibody is labeled with acridine sulfonamide. Then, they are incubated together with the antigen CA199 to be detected, the excess detection antibody is washed away, and acridine sulfonamide undergoes a chemical reaction 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 of the embodiments for detecting CA199, the water-soluble acridine sulfonamide carboxylic acid label (1 eq) is added to DMF, and then Hosu (1.2 eq) and EDC.HCl (1.2 eq) are added, and the reaction is carried out at room temperature for more than 2 h to obtain an active ester solution. An appropriate amount of the active ester solution is added to the detection antibody, and the labeling reaction is carried out at room temperature for more than 1.5 h without the formation of precipitates. After dialysis and purification, the water-soluble acridine salt-labeled detection antibody is obtained, and the labeling effect is good.

[0073] In a comparative experiment, the detection antibody is labeled with a common acridine sulfonamide. The acridine sulfonamide is dissolved in DMF, and a solution of the same molar amount of the water-soluble acridine sulfonamide active ester as in the above example is added to the same amount of detection antibody as in the above example, and the reaction is carried out at room temperature for more than 1.5 h. It is found that the antibody precipitates in the solution, which is caused by the poor water solubility of the labeled antibody, resulting in precipitation.

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

[0075] Table 1

[0076]

[0077]

[0078] From Figure 3 and Table 1, it can be seen that the correlation between the concentration and the luminescence value is good, and the correlation coefficient R 2 reaches 0.99, indicating that the luminescence value increases with the increase of the concentration of the antigen CA199. The synthesized water-soluble acridine sulfonamide carboxylic acid label is very suitable as a luminescent label for magnetic particle chemiluminescence immunoassay.

[0079] The test results of the reagent prepared from the water-soluble acridine sulfonamide-labeled antibody on the chemiluminescence instrument are as shown in Table 1. Figure 4 and the results shown in Table 1:

[0080] Table 2

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

[0082] From Figure 4 and Table 2, it can be seen that the linear relationship between the concentration and the luminescence value is poor, and the correlation coefficient R 2 is only 0.36, indicating that the luminescence value does not change much with the increase of the concentration of the antigen CA199.

[0083] Of course, in other embodiments, stereoisomers or tautomers of the water-soluble acridine sulfonamide carboxylic acid marker shown in formula I also have the above effects, and those skilled in the art can make corresponding tests according to the disclosure of the present application.

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

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

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

[0087] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description simple, all possible combinations of the technical features in the above-mentioned embodiments are not described, however, as long as the combinations of the technical features do not contradict, they should be considered as the scope of the present application.

[0088] The above-mentioned embodiments only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. A water-soluble acridine sulfonamide carboxylic acid label, characterized in that: The water-soluble acridine sulfonamide carboxylic acid marker has the 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; The water-soluble acridine sulfonamide carboxylic acid label has the structural formula shown in Formula 1, the acridine sulfonamide has the structural formula shown in Formula 2, and the tripeptide glycine has the structural formula shown in Formula 3: 。 3. The preparation method according to claim 2, wherein The step of adding an acridine sulfonamide solution to the weakly alkaline aqueous solution of the tripeptide glycine to carry out the reaction comprises: adding the tripeptide glycine and an alkaline substance into deionized water to obtain a weakly alkaline aqueous solution of the tripeptide glycine; adding the acridine sulfonamide to 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, wherein After the reaction is completed, the pH is adjusted and the separation and purification are carried out, comprising: 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 are performed based on reverse phase partition chromatography.

5. The preparation method according to claim 4, wherein After the separation and purification, the method further comprises: Subsequently, most of the solvent is evaporated and the product is freeze-dried to obtain the water-soluble acridinium sulfonamide carboxylic acid labeling substance.

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

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