PAMAM-mediated labeling of small molecule compounds and their synthesis and applications

Abstract

The present invention belongs to the field of immunochemiluminescence technology and relates to PAMAM-mediated labeled small molecule compounds, their synthesis methods, and applications. The PAMAM-mediated labeled small molecule compounds are composed of a small molecule, PAMAM, and a label. The small molecule has an active group or a derivative group capable of reacting with an amino group under certain activation conditions to form a covalent bond; the label has a succinimide group. The present invention minimizes the structural differences between the coupling product and the small molecule through PAMAM mediation. PAMAM usage is minimal and stable, and its molecular weight is more uniform than PEG. This significantly reduces nonspecific binding due to hydrophobic interactions during immune responses, and the product does not require purification to obtain a good signal and separation.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunochemiluminescence, and relates to a PAMAM (Polyamidoamine)-mediated labeled small molecule compound, a synthesis method, and an application thereof. Background Art

[0002] In the development of immunochemiluminescent reagents, the detection of small molecule antigens is often achieved using competitive assays. Relevant assays include testosterone, progesterone, estradiol, vitamin D, vitamin B12, T3, and T4. During the assay, the small molecule antigen in the sample and the small molecule antigen in the reagent system compete for binding sites on the antibody in the reaction system. In some reagent systems, the small molecule antigen can be linked to a labeling molecule, such as alkaline phosphatase, acridinium ester, or ruthenium terpyridine; the antibody is attached to a solid-phase magnetic bead. This completes the reaction pathway of solid-phase magnetic bead-antibody-small molecule+label, forming a complex that generates a signal. The more small molecule antigen in the sample, the fewer complexes formed by the small molecule+label and antibody-bead reaction. Conversely, the less antigen, the more complexes formed. This quantitative relationship allows the concentration of the small molecule antigen to be determined.

[0003] The connection of small molecules to markers can be roughly divided into two categories. In alkaline phosphatase systems, small molecules can be labeled on the enzyme protein; in acridinium ester or electrochemical systems, small molecules can be directly linked to acridinium esters or ruthenium terpyridine. Taking acridinium esters as an example, conventionally, small molecules can be directly linked to acridinium esters by derivatizing carboxyl groups (-COOH) or amino groups (-NH2). However, due to the structure of small molecules, they generally do not have many reactive groups, which ultimately limits the proportion of acridinium esters that can be coupled to the small molecule. Fewer acridinium esters inevitably result in lower luminescence signal values, which cannot meet the requirements of reagent development. A solution derived from this is to first couple the small molecule to the protein and then couple the acridinium ester to the protein; however, this approach can lead to structural imbalance between the small molecule in the sample and the small molecule conjugate in the reagent, mismatch in competitive kinetics, or require a higher antibody concentration to bind to the small molecule on the protein; and this structure requires optimization of the small molecule feed ratio and the acridinium ester feed ratio during the production process, because small molecules and acridinium esters are often connected using amino groups, and the preparation process is relatively long.

[0004] The applicant previously utilized a multi-arm polyethylene glycol structure, with each arm containing an amino group at the top. Estradiol was first attached to one arm, followed by an acridinium ester to the other arms, thereby achieving a structure in which one estradiol was coupled to multiple markers. This structure achieved coupling of the small molecule to be tested and the marker, representing a certain improvement over conventional protein-mediated coupling structures. However, the structure was still relatively large, with the final marker molecular weight ranging from 5 to 12 kD depending on the molecular weight of the multi-arm polyethylene glycol. This still presented challenges with competitive kinetics, and the background was high in some projects. Summary of the Invention

[0005] In view of the above technical problems, the purpose of the present invention is to provide a PAMAM-mediated labeled small molecule compound and its synthesis method and application.

[0006] The technical solution adopted by the present invention to achieve the technical purpose is:

[0007] The present invention provides a PAMAM-mediated labeled small molecule compound, which is composed of a small molecule, PAMAM and a label. The small molecule has an active group or a derivative group that can react with an amino group to form a covalent bond under certain activation conditions; the label has a succinimide group.

[0008] Preferably, the molecular weight of the PAMAM is 517D, 1430D, 3256D and 6909D, corresponding to different numbers of surface amino groups of 4, 8, 16 and 32. If constructing a small molecule marker, a smaller molecular weight PAMAM is preferred to retain similar competitive kinetics to the small molecule itself.

[0009] Preferably, the small molecules with active groups include but are not limited to progesterone, testosterone, estradiol, vitamin D or vitamin B12, etc.

[0010] Preferably, the label includes but is not limited to acridinium ester, acridinium sulfonamide, terpyridine ruthenium or FITC.

[0011] The present invention also provides a method for synthesizing a PAMAM-mediated labeled small molecule compound, comprising: first reacting a small molecule with PAMAM in a certain ratio, and then reacting the label with the remaining amino group to obtain the PAMAM-mediated labeled small molecule compound.

[0012] Preferably, when the small molecule is estradiol E2-NHS carrying an NHS group activation structure, the synthesis method comprises: reacting according to a molar ratio of E2-NHS:PAMAM-4NH2=0.9:1, reacting for 1 hour, adding a label acridinium ester or terpyridine ruthenium AE / Ru according to a molar ratio of AE / Ru:PAMAM=3:1, and using a glycine buffer to provide 4 times the molar molecular weight of PAMAM free amino -NH2 to terminate the reaction.

[0013] More preferably, when conducting the reaction, PAMAM-4NH2 is diluted to 1 mg / ml with PBS buffer at pH 7.5-8.5, E2-NHS is dissolved in DMF (dimethylsilane) to a concentration of 5 mg / ml, and acridinium ester or terpyridine ruthenium AE / Ru is dissolved in DMF (dimethylsilane) to a concentration of 5 mg / ml.

[0014] The present invention further provides a method for characterizing any of the above-mentioned PAMAM-mediated labeled small molecule compounds or PAMAM-mediated labeled small molecule compounds synthesized by any of the above-mentioned synthesis methods in immunochemiluminescence detection.

[0015] The present invention further provides an immunochemiluminescence detection kit comprising any of the above-mentioned PAMAM-mediated labeled small molecule compounds or a PAMAM-mediated labeled small molecule compound synthesized by any of the above-mentioned synthesis methods.

[0016] Compared with existing methods using inert proteins (such as BSA) or PEG as mediators, PAMAM mediation has the following advantages:

[0017] 1. Minimize the differences between the coupling product (small molecule-PAMAM-luminescent molecule) and the small molecule structure. BSA has a molecular weight of 67,000 Da, PEG has different molecular weights but generally ranges from 5,000 to 10,000 Da and does not provide many active groups. However, PAMAM can provide four amino groups for subsequent reactions with a minimum molecular weight of 517 Da.

[0018] 2.PAMAM is a relatively cheap, inactive synthetic compound that is used in very small amounts and is stable. Its molecular weight is more uniform than that of PEG.

[0019] 3. PAMAM contains a large number of highly polar chemical bonds such as -NH and C=O bonds, which can easily form hydrogen bonds with water to maintain hydrophilicity, which will greatly reduce nonspecific binding caused by hydrophobic effects during immune responses.

[0020] 4. The required small molecular weight is less, because it is necessary to maintain the advantage of the number of amino groups to ensure that most molecules can be coupled, so the product does not need to be purified to obtain good signal and separation. Of course, in theory, purification can obtain better signal and separation and more uniform structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The relationship between the E2-PAMAM-AE dilution ratio and the signal value in Test Example 1 is shown. DETAILED DESCRIPTION

[0022] In order to more clearly illustrate the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention. Example

[0023] 1. Taking estradiol (E2) as an example, the structures that can be selected include β-estradiol-6-one 6-(O-carboxymethyloxime) (CAS: 35048-47-6), the carboxyl group of which can be converted to an NHS group through reaction; or activated structures (Steraloids, E0982) directly carrying NHS groups can directly react with the primary amine group of PAMAM. This application selects the activated form directly carrying NHS groups (hereinafter abbreviated as E2-NHS). This small molecule structure can be expanded to other possible detection targets, such as progesterone, testosterone, vitamin D, vitamin B12, etc.

[0024] 2. PAMAM: Different test items require consideration of different numbers of amino groups. For estradiol, we chose PAMAM containing 4 amino groups, hereinafter abbreviated as PAMAM-4NH2, with the structural formula shown below: ;

[0025] 3. Labels include acridinium ester (AE, Cas 199293-83-9) and terpyridine ruthenium (Ru, CAS: 136724-73-7), both of which carry NHS groups that can directly react with amino groups;

[0026] 4. The synthesis of E2-PAMAM-AE begins with the reaction of E2-NHS and PAMAM-4NH2. PAMAM-4NH2 is diluted to 1 mg / ml in PBS (phosphate buffer, pH 7.5-8.5). E2-NHS is dissolved in DMF (dimethylformamide) to a concentration of 5 mg / ml. The reaction ratio is 0.9:1 molar ratio of E2-NHS:PAMAM-4NH2. Considering the four NH2 groups on PAMAM, this ratio ensures that most of the E2-NHS is coupled to the PAMAM-4NH2.

[0027] 5. After 1 hour of reaction, add the marker. Dissolve acridinium ester (AE) in DMF (dimethylaminomethyl sulfoxide) at 5 mg / ml. The reaction ratio is AE:PAMAM = 3:1. This ratio allows for the majority of the marker to be coupled and reduces background adsorption.

[0028] 6. Use glycine buffer to provide 4 times the molar molecular weight of PAMAM free amino groups -NH2 to terminate the reaction.

[0029] It is worth noting that the overall reaction ensures that most of the added E2 and markers are captured by the amino groups on the PAMAM, but a portion of free substances still remains, and the reaction product will be a heterocomplex. The number of E2 or markers on each PAMAM is not fixed.

[0030] To verify the effect of this structure, we designed a simple reaction model to demonstrate its binding efficiency.

[0031] The test was performed using an immunochemiluminescence instrument (Shenzhen Yingkai, FP2910).

[0032] Test Example 1

[0033] The kit consists of the following:

[0034] Streptavidin-coated 2.8 μm magnetic beads (SA magnetic beads, Dynal, 0.70 mg / ml), Mes buffer (25 mM Mes, 1% BSA, pH 6.5), and biotin-labeled anti-estradiol antibody (Bioventix, 2C7, 0.1 μg / ml) were used. E2-PAMAM-AE was diluted in Mes buffer at the appropriate ratio (see Table 1 for dilution information) and added to the reaction system as the sample. The reaction parameters were 50 μl of E2-PAMAM-AE dilution + 50 μl of Mes buffer, incubated for 9 minutes, followed by the addition of 50 μl of biotinylated antibody and 50 μl of SA magnetic beads, and incubated again for 9 minutes. This resulted in a solid-phase magnetic bead-SA-biotin+antibody-E2-PAMAM-AE structure. Luminescence values were positively correlated with E2-PAMAM-AE concentration; data are shown in Table 1.

[0035] Table 1. Luminescence detection results at different E2-PAMAM-AE concentrations when antibodies were added

[0036]

[0037] like Figure 1 As shown in the figure, we can roughly see that between dilutions 2 and 5, the dilution ratio and signal value show a roughly linear relationship, indicating that the signal is indeed related to the synthesized structure. Dilution 1 is too high and there are kinetic reasons, so it does not show a linear relationship with the subsequent dilutions.

[0038] Test Example 2

[0039] If we remove the antibody (replace the antibody component with buffer), and the other configurations are the same as in Test Example 1, then theoretically the same sample will produce no meaningful signal or only background signal under the same system. The data are shown in Table 2.

[0040] Table 2. Luminescence detection results at different E2-PAMAM-AE concentrations without antibody

[0041]

[0042] It can be seen that the signal value of the same sample decreased significantly after the antibody was removed from the reaction system, indicating that the antibody indeed linked to E2-PAMAM-AE and magnetic beads to generate the signal; the signal generated in the absence of the antibody is presumably caused by the nonspecific adsorption of E2-PAMAM-AE and magnetic beads.

[0043] Based on the above two tests, we determined that the structure of E2-PAMAM-AE was successful and had the potential to be applied to actual sample detection. We constructed a serum estradiol detection system based on this molecular structure.

[0044] Application Examples

[0045] The kit consists of 2.8 μm streptavidin-coated magnetic beads (SA beads, Dynal, M280, 0.70 mg / ml), an appropriate concentration of E2-PAMAM-AE diluted in Mes buffer (25 mM Mes, 1% BSA, pH 6.5), and a biotinylated anti-estradiol antibody (Bioventix, 2C7, 0.1 μg / ml, supplemented with 100 ng / ml of norgestrel as a dissociator). Serum samples containing varying estradiol concentrations were measured using a Roche E411 electrochemical detector and added to the reaction system as samples. The reaction parameters are: 50ul of sample is first incubated with 50ul of antibody for 9 minutes, followed by the addition of 50ul of E2-PAMAM-AE and 50ul of SA magnetic beads, and incubation for another 9 minutes. E2-PAMAM-AE binds to sites on the antibody not bound by E2, and the biotin on the antibody forms a structure of solid-phase magnetic beads-SA-biotin+antibody-E2-PAMAM-AE. The luminescence value is negatively correlated with the concentration of estradiol in the serum sample; data are shown in Table 3.

[0046] Table 3. Luminescence value detection results of serum samples with different concentrations of estradiol

[0047]

[0048] The signal-to-noise ratio was calculated by dividing the signal at 3.2 pg / ml by the signal value at each concentration point.

[0049] As can be seen from the data in the above table, E2 in the sample and E2-PAMAM-AE in the reagent compete with each other. The development of competitive detection reagents for estradiol can be further carried out on this basis. E2-PAMAM-AE is an effective structure for constructing competitive detection reagents for estradiol.

[0050] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A PAMAM-mediated labeling of a small molecule compound, characterized in that: The invention is composed of a small molecule, PAMAM and a marker, wherein the small molecule is estradiol E2-NHS carrying an NHS group activation structure; the molecular weight of the PAMAM is 517D, and the corresponding number of surface amino groups is 4; the marker is an acridinium ester, and the labeled small molecule compound is synthesized by the following method: PAMAM-4NH2 is diluted to 1 mg / ml with a PBS buffer of pH 7.5-8.5, E2-NHS is dissolved to a concentration of 5 mg / ml with DMF, and acridinium ester AE is dissolved to 5 mg / ml with DMF; the reaction is carried out at a molar ratio of E2-NHS:PAMAM-4NH2=0.9:1, and after reacting for 1 hour, the marker acridinium ester AE is added at a molar ratio of AE:PAMAM=3:1, and a glycine buffer is used to provide free amino groups -NH2 with a molar molecular weight 4 times that of PAMAM to terminate the reaction.

2. A PAMAM-mediated synthesis method of labeled small molecule compounds, characterized in that: include: First, the small molecule estradiol E2-NHS is reacted with PAMAM, and then the labeling substance acridinium ester AE is reacted with the remaining amino groups to obtain a PAMAM-mediated labeled small molecule compound. The molecular weight of the PAMAM is 517D, and the number of corresponding surface amino groups is 4; The synthesis method comprises: carrying out a reaction at a molar ratio of E2-NHS:PAMAM-4NH2=0.9:1, reacting for 1 hour, adding a marker acridinium ester AE at a molar ratio of AE:PAMAM=3:1, using a glycine buffer to provide a free amino group -NH2 with a molar molecular weight 4 times that of PAMAM to terminate the reaction, and during the reaction, diluting PAMAM-4NH2 to 1 mg / ml with a PBS buffer at pH 7.5-8.5, dissolving E2-NHS to a concentration of 5 mg / ml with DMF, and dissolving acridinium ester AE to 5 mg / ml with DMF.

3. An immunochemiluminescence detection kit, characterized in that: The invention comprises the PAMAM-mediated labeled small molecule compound according to claim 1 or the PAMAM-mediated labeled small molecule compound synthesized by the synthesis method according to claim 2.

Images