阅读说明：本技术 杯冠化合物及其用途 (Calixago compounds and uses thereof ) 是由 康仁哲 N·李 于 2020-03-16 设计创作，主要内容包括：本文提供的是新的杯冠化合物,例如式(I)化合物,其可用于包被固体基质例如蛋白质芯片、诊断试剂盒或蛋白质分离包。本文还提供的是用文中杯冠化合物包被的固体基质和固定的蛋白质检测蛋白质-蛋白质相互作用的方法。(Provided herein are novel cupule compounds, such as compounds of formula (I), which are useful for coating solid substrates such as protein chips, diagnostic kits, or protein separation packages. Also provided herein are methods for detecting protein-protein interactions using the solid matrix coated with the cupule compounds herein and the immobilized protein.)

1. A calix compound having formula I, II or III:

or a salt or ester thereof,

wherein

R¹And R³Independently represent hydrogen, -CH₂SH、—CH₂Cl、—CH₂CN、—CH₂CHO、—CH₂NH₂or-CH₂COOH；

R²And R⁴Independently represents-CH₂SH、—CH₂Cl、—CH₂CN、—CH₂CHO、—CH₂NH₂、—CH₂COOH, -CN, -CHO or-COOH; and is

X and Y independently represent hydrogen, C_1-4Alkyl, OH or C_1-4An alkoxy group.

2. The calixaprop compound of claim 1, or a salt or ester thereof, wherein R¹And R³Are all hydrogen.

3. The calix compound of claim 1 or 2, or a salt or ester thereof, wherein X and Y are both hydrogen.

4. The corolla compound of any one of claims 1 to 3, or a salt or ester thereof, wherein R²And R⁴Are all-COOH.

5. The calix compound of claim 1, or a salt or ester thereof, wherein the calix compound is characterized by the formula:

6. the calix compound of claim 1, or a salt or ester thereof, wherein the calix compound is characterized by IPS-Linker a or IPS-Linker B:

7. a method of immobilizing a protein on a solid substrate, the method comprising:

a) applying the cupola compound of any one of claims 1-6 to an inorganic or organic solid substrate to form a cupola compound coated solid substrate;

b) immersing the cupule compound-coated solid substrate in a solution comprising the protein.

8. The method of claim 7, wherein the solid substrate is an inorganic solid substrate.

9. The method of claim 8, wherein the solid substrate is a metallic solid substrate.

10. The method of claim 7, wherein the solid substrate is selected from the group consisting of gold, silver, glass, quartz crystal, mica, silicon, polystyrene, and polycarbonate.

11. The method of any one of claims 7-10, wherein the solution comprises the protein at a concentration of about 1nM to about 500 uM.

12. The method of any one of claims 7-10, wherein the protein is selected from a β_1-42Antibodies, enzymes, membrane-bound and non-membrane bound receptors, protein domains and motifs, and intracellular signaling proteins.

13. The method of any one of claims 7-12, wherein the corona calixate compound forms a monolayer on the solid substrate.

14. The method of any one of claims 7-13, wherein the solid substrate is a protein chip, a diagnostic kit, or a protein separation package.

15. A solid matrix with immobilized protein prepared by any one of claims 7-13.

16. A solid matrix coated with a corona-calixate compound according to any one of claims 1 to 6.

17. The solid substrate of claim 16, wherein the corona cupcompound forms a monolayer on the solid substrate.

18. The solid substrate of claim 16 or 17, wherein the solid substrate is an inorganic solid substrate.

19. The solid substrate of claim 18, wherein the solid substrate is a metallic solid substrate.

20. The solid substrate of claim 16 or 17, wherein the solid substrate is selected from the group consisting of gold, silver, glass, quartz crystal, mica, silicon, polystyrene, and polycarbonate.

21. A method of detecting protein-protein interactions, comprising:

a) immobilizing a first protein on the solid substrate of any one of claims 16-20 to form a solid substrate having the immobilized first protein;

b) incubating the solid substrate with the immobilized first protein with a solution containing a second protein; and

c) detecting an interaction between the immobilized first protein and second protein.

22. The method of claim 21, wherein detecting the antibody comprising the second protein contacts a solid substrate.

23. The method of claim 22, wherein detecting further comprises contacting a dye-labeled secondary antibody with the solid substrate, wherein secondary antibody binds to an antibody of the second protein.

24. The method of claim 23, wherein detecting further comprises measuring the level of dye-labeled secondary antibody associated with the solid substrate.

25. The method of any one of claims 21-24, wherein the solution comprising the second protein is from a biological fluid sample of the patient having a concentration of about 1aM (aM) to about 100 μ M.

26. The method of any one of claims 21-25, wherein the second protein is a biomarker for cancer, an inflammatory disease, a neurodegenerative disease, a metabolic disease, an allergic disease, an autoimmune disease, an infectious disease, or an endocrine disease.

27. The method of any one of claims 21-25, wherein the second protein is VEGF₁₆₅Antibodies, enzymes, membrane-bound and non-membrane bound receptors, protein domains and motifs, or intracellular signaling proteins.

28. The method of any one of claims 21-27, wherein the second protein is IL-17, IL-23, STING, or PD-1.

Technical Field

In various embodiments, the present invention relates broadly to novel cupule compounds and their use in bioassays.

Background

Immobilization of enzymes, antigens, antibodies, etc. on solid supports has become one of the most basic techniques in biotechnology and protein research such as immunochemistry and enzyme chemistry. For example, enzyme-linked immunosorbent assay (ELISA) is a widely used technique in biotechnology for analyzing a specific protein or a specific protein causing a certain disease in an experimental or clinical laboratory. The ELISA detection kit is commercially available. Development of protein chips requires improvement of a method for immobilizing proteins on a solid substrate, and recently, development of protein chips has received great attention in the field of biotechnology for further development of proteomics research in the post-genomic era.

Previously, immobilization of proteins (e.g., antigens, antibodies or enzymes) has typically been achieved by physical adsorption of the protein onto high molecular weight biopolymers (e.g., various collagens, dextrans or cellulose derivatives). Covalent bonds between proteins formed by chemical reactions and the surface of a carrier have also been widely used as a method for protein immobilization. The method of protein immobilization by the "sandwich" technique (trimolecular layer) has been described in Zhang, X et al, Science 262: 1706-1708(1993), which describes a method for chemical bonding via a biotin-avidin (or streptavidin) interaction between a protein and a carrier surface. That is, biotin is attached to the surface of the carrier, and then avidin or streptavidin is attached to the biotin. Finally, the biotin-linked protein can be immobilized on a chemically modified support surface.

However, there are many problems in various methods of immobilizing proteins as follows.

1. Density of

The most critical problem with the protein immobilization methods used in the past has been the very small amount of protein immobilized on the surface of the substrate. When the density of proteins to be immobilized on the surface of the carrier is low, other proteins may form non-specific binding. Therefore, it is necessary to chemically treat the surface of the carrier to eliminate unwanted proteins bound to the surface of the carrier. However, the chemical treatment may inactivate or denature the immobilized protein molecules. Furthermore, even if a specific target protein is successfully immobilized on the surface of the carrier, only a very small amount of the protein can be captured, and therefore, it may be necessary to further confirm the test result by other analysis methods. The test method is simpler as more protein is immobilized per unit area on the carrier surface. In this regard, many studies have been conducted to develop a method of immobilizing a maximum amount of monolayer proteins on a carrier surface. However, satisfactory results have not been achieved.

2. Activity of

In the prior art methods of immobilizing proteins by chemical bonding or physical adsorption to a carrier surface, the activity of the immobilized protein is reduced compared to free protein in solution. It is well known that proteins immobilized on solid supports may lose their activity due to conformational changes or denaturation of the proteins, particularly around their active sites, because the proteins are tightly bound to the support surface by physical adsorption or chemical binding.

3. Direction of rotation

In existing methods for immobilizing proteins on a carrier surface, the active sites of the protein may become substantially directed towards the carrier surface, thereby masking the active sites, whereby the activity of the protein is lost. This phenomenon occurs in almost half of the immobilized proteins.

It has previously been shown that the calixarene compound can be used to form a monolayer of the calixarene compound on a solid substrate, which helps to immobilize the protein of interest, for example by recognizing cationic functional groups of amino acids, such as ammonium groups, on the surface of the protein. See, e.g., U.S. patent application No. 6,485,984B1 and Lee et al, Proteomics 3: 2289-. WO2009069980a2 also describes various uses of the cupule compounds in protein chips for determining kinase or phosphatase activity.

These previous generations of crown compounds may solve the above-mentioned problems to some extent. There is still a need for new protein chips, such as those with high sensitivity.

Brief description of the invention

In various embodiments, the present disclosure is based, at least in part, on the discovery that certain novel corona compounds can be used to coat solid substrates, and the coated solid substrates can be used to immobilize proteins and detect the presence of proteins (or protein-protein interactions) in a sample with high sensitivity.

In some embodiments, the disclosure relates to novel calix compounds having formula I, II or III as defined herein. In some particular embodiments, the present invention relates to compound 6(IPS-Linker A) or IPS-Linker B as defined herein.

In some embodiments, there is provided a solid substrate coated with a cupule compound of the present disclosure. In some embodiments, the solid substrate may be an inorganic or organic solid substrate, for example a substrate selected from gold, silver, glass, silicon, polystyrene, and polycarbonate. In some embodiments, the solid substrate is a protein chip, a diagnostic kit, or a protein isolation package.

In some embodiments, a solid substrate having an immobilized protein is provided, wherein the solid substrate is coated with a cupule compound of the present disclosure. In some embodiments, the solid substrate may be an inorganic or organic solid substrate, for example a substrate selected from gold, silver, glass, silicon, polystyrene, and polycarbonate. In some embodiments, the immobilized protein may be an antibody, an enzyme, membrane-bound and non-membrane-bound receptors, protein domains and motifs, and intracellular signaling proteins including modified proteins, such as bromodomain protein 4 (bromodomain-stabilizing protein 4), heparin-binding domains, Polo-Box domains, and the like.

In some embodiments, the present disclosure provides methods of immobilizing a protein on a solid substrate. In general, the method can include applying a cupola compound of the disclosure to an inorganic or organic solid substrate to form a cupola compound coated solid substrate; and subsequently immersing the cupule compound-coated solid matrix in a solution comprising the protein.

In some embodiments, methods of detecting protein-protein interactions are also provided. For example, in some embodiments, the method can comprise immobilizing a first protein on a solid substrate coated with a cupule compound of the present disclosure to form a solid substrate having the immobilized first protein; incubating the solid substrate with the immobilized first protein with a solution containing a second protein; and detecting an interaction between the immobilized first and second proteins. In some embodiments, the second protein is a biomarker for a disease.

Brief Description of Drawings

FIG. 1A shows the reaction of A.beta._1-42Fixation and use of different concentrations of VEGF₁₆₅Graph of the fluorescence signal detected from incubated ProLinker coated slides.

FIG. 1B shows the reaction of A.beta._1-42Fixation and use of different concentrations of VEGF₁₆₅Graph of the fluorescence signal detected from incubated IPS-Linker coated slides.

FIG. 2 detection of human IL-17 using IPS-Linker coated chips.

FIG. 3 shows the results of testing protein-protein interactions between IL-23 and its receptor using IPS-Linker-coated chips.

FIG. 4 shows the results of testing the protein-protein interaction between PD-1 and PD-L1 using an IPS-Linker-coated chip.

FIG. 5 shows the results of testing the protein-nucleotide interaction between STING and c-di-GMP using an IPS-Linker-coated chip.

Detailed Description

In various embodiments, the present disclosure provides novel cupule compounds and their use in biological assays.

Cupular crown compound

The calixarene compound was found to have ionophore properties for alkali and alkaline earth metal cations as well as tertiary amines. Their binding selectivity depends to a large extent on the number of oxygen atoms in the polyethylene glycol bridge, the nature of the substituents on the crown bridge, and the stereochemistry of the calixarene skeleton at the binding site. See generally Salorinne et al, J.Incl.Phenom.Macrocycle.chem.61: 11-27 (2008), incorporated herein by reference in its entirety.

The cupola compounds of the present disclosure are typically cupola [4] crowns. The cup [4] crown may be 1, 3-bridged or 1, 2-bridged. The cup [4] crown of the present disclosure is typically 1, 3-bridged. Generally, the cupped compounds of the present disclosure can adopt a 1, 3-alternating conformation, although in some cases, partially tapered or pyramidal conformations are also possible.

The cupped crown compounds of the present disclosure can generally have a structure according to formula I, II or III. In some embodiments, the calix compounds herein can have amino and/or carboxylic acid groups, and the calix compounds can be present in the form of a salt. For the crown compounds herein having carboxylic acid functionality, esters thereof, e.g. C_1-4Alkyl esters are also novel compounds of the disclosure. For example, the esters can be used, for example, as intermediates for the preparation of compounds having the corresponding carboxylic acid functionality.

In some embodiments, the cupule compounds of the present disclosure can be characterized by formula I:

wherein:

R¹and R³Independently represent hydrogen, -CH₂SH、—CH₂Cl、—CH₂CN、—CH₂CHO、—CH₂NH₂or-CH₂COOH；

R²And R⁴Independently represents-CH₂SH、—CH₂Cl、—CH₂CN、—CH₂CHO、—CH₂NH₂、—CH₂COOH, -CN, -CHO or-COOH; and is

X and Y independently represent hydrogen, C_1-4Alkyl, OH or C_1-4An alkoxy group.

Typically, in formula I, R¹And R³Is hydrogen. However, in some embodiments, R¹And R³Can also be used independentlyAnd (ii) a moiety that has affinity for the surface of a solid substrate (e.g., gold). In some embodiments, R¹And R³May also independently be a group such as-CH₂SH、—CH₂CHO、—CH₂NH₂or-CH₂COOH。

Typically, in formula I, X and Y are hydrogen. However, in some embodiments, X and Y may also independently be a group such as C_1-4Radicals, OH or C_1-4An alkoxy group.

Typically, in formula I, R²And R⁴is-COOH. In some embodiments, R²And R⁴And may also independently be a group having affinity to the surface of a solid substrate (e.g., gold). In some embodiments, R²And R⁴May also independently be a group such as-CH₂SH、—CH₂CHO、—CH₂NH₂-CHO or-CH₂COOH。

In some embodiments, the calix compounds of formula I are compound 6(IPS-Linker A) or IPS-Linker B:

in some embodiments, the cupule compounds of the present disclosure can be characterized by formula II:

wherein:

R¹and R³Independently represent hydrogen, -CH₂SH、—CH₂Cl、—CH₂CN、—CH₂CHO、—CH₂NH₂or-CH₂COOH；

R²And R⁴Independently represents-CH₂SH、—CH₂Cl、—CH₂CN、—CH₂CHO、—CH₂NH₂、—CH₂COOH, -CN, -CHO or —COOH; and is

X and Y independently represent hydrogen, C_1-4Alkyl, OH or C_1-4An alkoxy group.

Typically, in formula II, R¹And R³Is hydrogen. However, in some embodiments, R¹And R³And may also independently be a group having affinity to the surface of a solid substrate (e.g., gold). In some embodiments, R¹And R³May also independently be a group such as-CH₂SH、—CH₂CHO、—CH₂NH₂or-CH₂COOH。

Typically, in formula II, X and Y are hydrogen. However, in some embodiments, X and Y may also independently be a group such as C_1-4Radicals, OH or C_1-4An alkoxy group.

Typically, in formula II, R²And R⁴is-COOH. In some embodiments, R²And R⁴And may also independently be a group having affinity to the surface of a solid substrate (e.g., gold). In some embodiments, R²And R⁴May also independently be a group such as-CH₂SH、—CH₂CHO、—CH₂NH₂-CHO or-CH₂COOH。

In some embodiments, the cupule compounds of the present disclosure can be characterized by formula III:

wherein:

R¹and R³Independently represent hydrogen, -CH₂SH、—CH₂Cl、—CH₂CN、—CH₂CHO、—CH₂NH₂or-CH₂COOH；

R²And R⁴Independently represents-CH₂SH、—CH₂Cl、—CH₂CN、—CH₂CHO、—CH₂NH₂、—CH₂COOH, -CN, -CHO or-COOH; and is

X and Y independently represent hydrogen, C_1-4Alkyl, OH or C_1-4An alkoxy group.

Typically, in formula III, R¹And R³Is hydrogen. However, in some embodiments, R¹And R³And may also independently be a group having affinity to the surface of a solid substrate (e.g., gold). In some embodiments, R¹And R³May also independently be a group such as-CH₂SH、—CH₂CHO、—CH₂NH₂or-CH₂COOH。

Typically, in formula III, X and Y are hydrogen. However, in some embodiments, X and Y may also independently be a group such as C_1-4Radicals, OH or C_1-4An alkoxy group.

Typically, in formula III, R²And R⁴is-COOH. In some embodiments, R²And R⁴And may also independently be a group having affinity to the surface of a solid substrate (e.g., gold). In some embodiments, R²And R⁴May also independently be a group such as-CH₂SH、—CH₂CHO、—CH₂NH₂-CHO or-CH₂COOH。

The cupola compounds disclosed herein can be readily prepared by one skilled in the art in light of the present disclosure. The calixarene compound can be readily prepared, for example, by reacting a calix [4] arene with the appropriate polyethylene glycol ditosylate in the presence of various bases. In the examples section, an example of the preparation of the cupola compound (compound 6) herein is also detailed.

Biological chip

The cupped compounds of the present disclosure are generally bifunctional compounds that allow them to bind to surfaces and recognize cationic functional groups. The cupola compounds of the present disclosure can also typically self-assemble on a solid matrix to form a monolayer, which can capture species with cationic functionality, such as proteins, through crown ether groups.

Thus, in various embodiments, the present disclosure also provides solid substrates coated with the presently disclosed corona compounds, methods of making the same, and various related uses. In some embodiments, the solid substrate may be a protein chip, a diagnostic kit, or a protein separation package. In some embodiments, the solid matrix may also be a complete chip (well-on-a-chip), an array, or the like, which may be used, for example, for high throughput analysis/screening.

In some embodiments, the disclosure provides a solid substrate coated with a cupule compound (e.g., compound 6) of the disclosure. In some embodiments, the solid substrate is an inorganic or organic solid substrate. In some embodiments, the solid substrate is an inorganic solid substrate. Typically, the inorganic solid matrix may be a metal or glass matrix. For example, in some embodiments, the solid substrate may be a metal substrate such as gold, silver, platinum, and the like. In some embodiments, the solid matrix may also be a glass matrix. In some embodiments, the solid matrix may be a polymer-based matrix. Non-limiting examples include polystyrene and polycarbonate substrates. In some embodiments, the solid substrate may be selected from gold, silver, glass, silicon, polystyrene, and polycarbonate. In any of the embodiments described herein, the cupola compounds of the disclosure can form a monolayer on a solid substrate.

Described herein are methods of coating a solid substrate with the cupule compounds of the present disclosure. Typically, the method comprises applying the cupola compound of the disclosure to a solid substrate.

In some embodiments, the disclosure also provides a solid substrate having an immobilized protein, wherein the solid substrate is coated with a corona-capping compound of the disclosure (e.g., as described above, such as compound 6). In some embodiments, the solid substrate may be an inorganic or organic solid substrate, for example a substrate selected from gold, silver, glass, silicon, polystyrene, and polycarbonate. In some embodiments, the immobilized protein may be an antibody, an enzyme, membrane-bound and non-membrane bound receptors, protein domains and motifs, and intracellular signaling proteins including modified proteins, such as bromodomain protein 4, heparin binding domains, Polo-Box domains, and the like.

Methods of immobilizing proteins on solid substrates are described herein. For example, in some embodiments, the method can comprise applying a calixaprop compound of the disclosure (e.g., compound 6) to an inorganic or organic solid substrate to form a calixaprop compound-coated solid substrate; the solid matrix coated with the cupule compound is then immersed in a solution comprising the protein. Typically, the cupola compounds of the present disclosure form a monolayer on a solid substrate. The solution may be a buffer. After immersing the solid matrix coated with the cupola compound in the protein solution, the mixture can be incubated for a period of time to allow the protein to interact with the cupola compound. Typically, the solution contains the protein at a concentration of about 1nM to about 500uM, preferably about 1uM to about 500uM (e.g., about 50uM to about 250uM), or any concentration up to the solubility limit of the protein. Suitable solid matrices are described herein. After incubation with the protein solution, the solid matrix is typically washed, blocked to remove non-specific binding, and dried. The example section details an exemplary operation.

The solid matrix with immobilized protein herein can further be used for detecting protein-protein interactions or for detecting biomarkers. For example, in some embodiments, the present disclosure provides a method of detecting a protein-protein interaction, which method can comprise immobilizing a first protein on a solid substrate coated with a cupule compound of the present disclosure to form a solid substrate having the immobilized first protein; incubating the solid substrate with the immobilized first protein with a solution containing a second protein; and detecting an interaction between the immobilized first protein and the second protein. In some embodiments, the second protein may be a biomarker, for example, antibodies, enzymes, membrane-bound and non-membrane-bound receptors, protein domains and motifs, and intracellular signaling proteins including modified proteins, such as bromodomain protein 4, heparin-binding domains, Polo-Box domains, and the like. In some embodiments, the solution comprising the second protein is from a biological fluid sample of a patient (e.g., a human patient). In some embodiments, the patient may have cancer and the second protein is a biomarker for cancer. In some embodiments, the biological fluid sample may be a raw sample, a diluted sample, or other processed sample. In some embodiments, the solution can contain a concentration of the second protein in the range of about 1aM (atto) M to about 100 μ M, such as about 1fM (femto M) to about 10 μ M, about 10fM to about 10 μ M, about 50fM to about 2 μ M, about 100fM to about 1 μ M, about 250fM to about 1 μ M, and the like. For example, the protein can be detected by a colorimetric enzyme assay used in an ELISA system.

Typical ELISA system methods include:

preparation of the plates

The capture antibody was diluted to working concentration in PBS (1X) without carrier protein. The 96-well microplate was immediately coated with 100. mu.L/well of the diluted capture antibody. Plates were sealed and incubated overnight at room temperature. Each well was aspirated and washed with wash buffer (PBS 1X containing 0.05% tween 20), and this operation was repeated twice for a total of three washes. Washing was performed by filling each well with a washing buffer (400. mu.L) using a spray bottle, a manifold dispenser, or an automatic washing machine. Complete removal of liquid at each step is critical for good performance. After the last wash, the remaining wash buffer was removed by aspirating or inverting the plate and aspirating with a clean paper towel. The plate was blocked by adding 300 μ L reagent diluent (PBS 1X with 1% BSA) to each well. Incubate at room temperature for at least 1 hour. The suction/purge in step 2 is repeated. The plate is now ready for addition of the sample.

Testing method

To each well 100 μ L of sample or standard in a reagent diluent or appropriate diluent is added. Covered with tape and incubated at room temperature for 2 hours. The suction/washing in plate preparation step 2 was repeated. To each well 100 μ L of detection antibody was added and diluted in reagent diluent. Covered with fresh tape and incubated at room temperature for 2 hours. The suction/washing in plate preparation step 2 was repeated. To each well 100 μ L of streptavidin-HRP working dilution was added. Plates were covered and incubated at room temperature for 20 minutes. Avoid to beThe plate is placed under direct light. The pumping/purging in step 2 is repeated. To each well 100. mu.L of substrate solution (1: 1 mixture of developer A (H2O2) and developer B (tetramethylbenzidine)) (R)&D Systems, Minneapolis, MN). Incubate at room temperature for 20 minutes. Avoiding placing the board under direct light. Add 50. mu.L of stop solution (2N H) to each well₂SO₄(R&D Systems, Minneapolis, MN)). Gently tap the beater to ensure adequate mixing. The optical density of each well was immediately determined using a microplate reader set at 450 nm. If wavelength correction is available, it is set to 540nm or 570 nm. If wavelength correction is not available, the reading at 540nm or 570nm is subtracted from the reading at 450 nm. This subtraction will correct the optical defects in the plate. Readings taken directly at 450nm without correction may be higher and less accurate.

As described in the examples section, the cupola compounds of the disclosure (e.g., compound 6) are coated and subsequently immobilized protein (A β) when compared to a control solid substrate coated with the prior generation cupola compound ProLinker_1-42) The coated solid matrix can detect VEGF in obviously lower concentration₁₆₅. See U.S. Pat. Nos. 6,485,984B1 and Lee et al, Proteomics 3:2289 and 2304(2003), each of which is incorporated by reference in its entirety. WO2009069980A2 also describes various uses of the cupule compounds in protein chips for determining kinase or phosphatase activity, which are incorporated herein by reference in their entirety. Thus, the use of the cupholder compounds of the present disclosure can improve the detection limit and sensitivity, which is more advantageous than the prior art.

The second protein captured on the solid substrate can be detected by various methods. For example, in some embodiments, the detecting comprises contacting an antibody to the second protein with a solid substrate. In some embodiments, the detecting further comprises contacting the dye-labeled secondary antibody with a solid substrate, wherein the secondary antibody binds to an antibody of the second protein. In some embodiments, the detecting further comprises measuring the level of dye-labeled secondary antibody associated with the solid substrate. Any suitable dye may be used. Typically, the dye is a fluorescent dye and the measuring comprises measuring a fluorescent signal. For example, in some embodiments, the dye may be Cy 5.

Definition of

It is to be understood that for the formulae herein, all groups and combinations thereof maintain the appropriate valence state.

It is to be understood that a particular embodiment of a variable group herein may be the same or different from another particular embodiment having the same identifier.

Where applicable, the appropriate groups of variables in the compounds of formula I, II or III are independently selected. The embodiments of the invention may be combined. Such combinations are contemplated and are within the scope of the present invention. For example, the definition of a variable may be combined with any definition of any other variable in formula I, II or III.

As used herein, the term "calixaprop compound of the present disclosure" and the like refers to any compound described herein according to formula I, II or III, or compound 6, isotopically-labeled compounds thereof, tautomers thereof, conformational isomers thereof, salts thereof (e.g., base addition salts such as Na salts), and/or esters thereof (e.g., C salts)_1-4Alkyl esters).

As used herein, the term "alkyl" (by itself or when used as part of another group) refers to a straight or branched chain aliphatic hydrocarbon, typically containing 1-20 carbons. In some embodiments, alkyl is straight chain C_1-6An alkyl group. In other embodiments, the alkyl group is a branched chain C_3-6An alkyl group. In other embodiments, alkyl is straight chain C_1-4An alkyl group. As understood by those skilled in the art, alkyl groups are saturated. C as used herein_1-4Alkyl means methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl or isobutyl. Also as understood by those skilled in the art, "alkylene" refers to a divalent group derived from the corresponding alkyl group. For example, C as used herein_1-4Alkylene group means methylene, ethylene, propylene, isopropylene, butylene, sec-butylene, tert-butylene or isobutylene.

As used herein, the term "alkoxy" (when used by itself OR as part of another group) refers to the formula OR^a1Radical (I)Wherein R is^a1Is an alkyl group.

The term "patient" as used herein refers to an animal, e.g., a mammal, non-human or human, who is the object of treatment, observation or experiment.

Examples

EXAMPLE 1 preparation of the Calycan Compound 6

1) Synthesis of Compound 1 (tetraethylene glycol Dimethylbenzenesulfonate)

Tetraethylene glycol (4mL, 23mmol) was dissolved in anhydrous chloroform (30 mL). The solution was cooled to-20 ℃ in a sodium chloride ice bath. P-toluenesulfonyl chloride (13g, 69mmol) and anhydrous pyridine (24mL) were added sequentially while maintaining the solution temperature below 0 ℃. After reaction at-20 ℃ for 5h, chloroform and pyridine were removed under reduced pressure, ice water (250ml) was added, and the solution was taken with CH₂Cl₂(200ml each time) three times. The combined organic phases were washed twice with HCl (2N, 250mL) and water (200mL) in succession. After drying the organic layer over sodium sulfate, the solvent was removed under reduced pressure. The residue was chromatographed on silica gel using ethyl acetate/hexane (ethyl acetate to hexane ratio 2:8 to 5:5) to give the product as a colourless oil (9.45g), yield: 725. r_f0.31 (silica gel, 1:1, ethyl acetate/hexane).¹H NMR(400MHz,CDCl₃)δppm 2.45(s,6H),3.68(t,J＝4.7Hz,4H),4.16(t,J＝4.7Hz,4H),7.35(d,J＝8.2Hz,4H),7.80(d,J＝8.2Hz,4H)；¹³C NMR(400MHz,CDCl₃)δppm 21.6,68.7,69.3,70.5,70.7,128.0,129.8,133.0,144.8.

2) Synthesis of Compound 2

2-1) p-tert-butylphenol (Compound 1) (150g,1mol) and NaOH (1.8g,45mmol) were dissolved in 37% formaldehyde (100.7g,1.24 mol). The reaction mixture was refluxed at 120 ℃ for 12 hours. After the solution was cooled to room temperature, H was removed in vacuo₂O, then phenyl ether (450mL) and toluene (150mL) were added. The reaction mixture was again refluxed at 250 ℃. The color of the reaction mixture turned dark brown. The crude product is then taken up in ethyl acetate (3)00mL) and washed with acetic acid (100 mL). White crystalline solid 2, yield 56.79%.¹H NMR(400MHz,CDCl₃)δppm 10.36(s,4H),7.04(s,8H),4.25(d,4H),3.49(d,4H),1.21(s,36H).

3) Synthesis of Compound 3

2-2) adding p-tert-butyl cup [4]]Aromatic hydrocarbon (compound 2) (10.00g, 13.5mmol), toluene (100mL) and phenol (1.75g, 18.60mmol) were added to the flask, and the solution was stirred under an argon atmosphere for 10 minutes. Aluminum trichloride (10.00g, 75.0mmol) was added with vigorous mechanical stirring. The mixture was stirred at room temperature for 5 hours. The mixture was poured into a 500mL beaker containing crushed ice (200g) and was washed with CH₂Cl₂(400mL) was extracted. The organic layer was washed with 1N HCl (3X 100mL) and water (2X 100mL), and with NaSO₄And (5) drying. The solvent was evaporated in vacuo. To the orange oily residue was added diethyl ether (50mL) and the heterogeneous mixture was kept at-15 ℃ for 1 h. The precipitated solid was filtered and triturated with ether (100 mL). The mixture was held at-15 ℃ for 1 hour and filtered to provide 5.54g of a pale yellow powder (90%).¹H NMR(400MHz,CDCl₃):δ10.20(s,4H),7.04(d,8H,J＝7.6Hz),6.73(t,4H,J＝7.6Hz),4.24(br s,4H),3.54(br s,4H).

3) Synthesis of Compound 4

To a 2000mL three-necked flask containing a mixture of NaH (5.00eq,2.16g,90.0mmol) and DMF (1300mL) under nitrogen over 1 hour was added 25,26,27, 28-tetrahydroxy cup [4]]A solution of aromatic hydrocarbon (Compound 3) (1.00eq,7.64g,18.0mmol) in DMF (100 mL). The mixture was stirred for an additional 1 hour. Over 1 hour, tetraethylene glycol ditosylate (2.20eq,13.88g,39.6mmol) in DMF (100mL) was added. The mixture was stirred at 50 ℃ for 72 hours. Addition of H₂The reaction was quenched with O (50mL) and the solution was quenched with CH₂Cl₂Extracted three times (50 mL). The combined organic phases were washed twice with HCl (3N,50mL) and water (200mL) in succession. The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was subjected to silica gel chromatography using ethyl acetate/hexane (ethyl acetate to hexane ratio 1: 4-1: 2) to give a pale yellow powdery product (2.34g) in 45% yield. R_fNot 0.55 (silica gel, 1:1, ethyl acetate/hexane)。¹H NMR(400MHz,CDCl₃):δ8.34(s,2H),7.13-6.87(m,8H),6.80(t,J＝7.5Hz,2H),6.60(t,J＝7.5Hz,2H),4.62-4.41(m,12H),4.41-4.28(m,2H),4.11(t,J＝9.0Hz,2H),4.06-3.88(m,6H),3.87-3.65(m,4H),3.39(d,J＝12.3Hz,1H),3.36(d,J＝13.5Hz,2H),3.32(d,J＝3.8Hz,1H).

4) Synthesis of Compound 5

In a round bottom flask, compound 4(1g, 1.77mmol) was dissolved in dry acetonitrile. Potassium carbonate (1.17g, 8.41mmol) and tert-butyl bromoacetate (0.90g, 4.61mmol) were added and the mixture was refluxed for 24 hours. After completion, the solvent was evaporated, water (100ml) was added and extracted with dichloromethane (2 × 100 ml). The organic layer was separated, dried over sodium sulfate, filtered and concentrated. Water (250mL) was added and the solution was taken up in CH₂Cl₂Three extractions (200mL each). The residue was subjected to silica gel chromatography using ethyl acetate/hexane (ethyl acetate to hexane ratio 1:3) to give a pale yellow powder product (1.1g) in 90% yield. R_f0.6 (silica gel, 1:2, ethyl acetate/hexane).¹H NMR(400MHz,CDCl₃):δ6.59(m,12H),4.64(d,4H),4.21(m,2H),3.98(m,6H),3.18(m,4H),1.45(s,18H).

5) Synthesis of Compound 6(IPS linker A)

An aqueous solution of NaOH (15%) (252mL) was added to a solution of Compound 5(1.16g,1.68mmol) in ethanol (70 mL). The reaction mixture was heated to reflux for 24 hours. The reaction was cooled to room temperature and the solvent was removed by rotary evaporation. Then, 50mL of cold water was added to the solid material, and HCl (5N) was added dropwise with vigorous stirring until the pH of the solution reached 7. The solution is treated with CH₂Cl₂Extracted three times (50 mL). The combined organic phases were washed twice with water (50 mL). The organic layer was dried over sodium sulfate, and the solvent was removed under reduced pressure. The residue was chromatographed on silica gel using methanol/dichloromethane (methanol to dichloromethane ratio 1:9-1:1) to give the product as a pale yellow powder (0.7g) in 60% yield. R_f0.3 (silica gel, 3:1, methanol: dichloromethane).¹H NMR(300MHz,CDCl₃):7.08(m,8H),6.80(s,4H),4.43(m,4H),4.22-3.66(m,16H),3.35(m,4H).

Example 2 preparation of protein chips coated with Calycor Compound 6

The slide glass was placed in 60mL of a washing solution (methanol: 35% hydrogen chloride ═ 1:1) and washed for 30 minutes. The slide glass was immersed in Piranha solution (sulfuric acid: hydrogen peroxide ═ 3:1) and washed with distilled water for 30 minutes. Slides dried with nitrogen were soaked in 60ml of aminated solution (3% (3-aminopropyl) triethoxysilane in ethanol) for 2 hours in the dark. It was washed three times with ethanol, then repeatedly washed with distilled water, and finally washed with ethanol. The slide was dried with nitrogen and reacted at 100 ℃ for 2 hours. It was soaked with 60ml of solution A (10mg of IPS-linker (calixaprop 6) in DMF, 5mg of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC-HCl), 3mg of 1-hydroxybenzotriazole hydrate (HOBt), 1mg of 4-N, N-Dimethylaminopyridine (DMAP)) and incubated at room temperature for 12 hours. The slides were washed 3 times with 70ml DMF for 10 min and then repeated 2 more times. Slides were dried with nitrogen and stored under vacuum at room temperature until use.

The structure of IPS-linker (Compound 6) is as follows:

preparation of ProteoChip coated with Prolinker

The slide glass was immersed in a washing solution (methanol: HCl ═ 1:1) for 30 minutes and in Piranha solution for 10 minutes (room temperature). The slides were thoroughly washed with sterile d.w. and dried. The slides were immersed in the amination solution for 6 hours at room temperature. After washing with toluene, the mixture was washed with anhydrous ethanol. Incubate at 100 ℃ for 1 hour. The aminated slides were immersed in 10mM Prolinker chloroform solution at room temperature for 3 hours. After washing with chloroform, the mixture was washed with anhydrous ethanol. After washing with sterile d.w., thoroughly dried.

Diluting the buffer (30% glycerol)PBS solution, pH 7.4) of Abeta_1-42Spotting was performed on a ProteoChip (Protogen, Korea, ProLinker-coated chip) or an IPS chip (Innopharmascreen, Korea, IPS-linker-coated chip), and the chips were incubated overnight in a humidity chamber at 4 ℃. The prolinker structure of the ProteoChip is as follows:

the chip was rinsed twice in PBST (0.5% PBS solution of Tween-20), held for 10 minutes, and incubated with 3% BSA containing 0.05% Tween-20 solution at room temperature to block non-specific binding. After a large amount of washing, Abeta_1-42Microarrays can be used for detection of Abeta_1-42Protein interaction of (2).

Example 3 protein interaction assays for VEGF-A β binding were performed using protein chips.

A beta prepared in example 2_1-42VEGF for microarray applications₁₆₅The mixture (Vexxon, Korea) was spotted. After washing with PBST and DW, rabbit-anti-VEGF (A20) (Santacruz, Germany) was diluted to 1:10 with PBS containing 3% BSA and 30% glycerol, spotted to recognize the presence of A.beta._1-42Bound VEGF₁₆₅. After washing with PBST and DW, an anti-rabbit secondary antibody labeled with Cy5(Invitrogen, USA) (diluted 1:100 with PBS containing 3% BSA and 30% glycerol) was applied to the chip for 1 hour at 30 ℃. After rinsing with PBST and DW, at N₂The chips were dried in a stream of air. Protein-protein interactions were determined by measuring the relative fluorescence intensity of the mixture spots to the control spots (VEGF only).

Detection and data analysis: the chip was scanned using a Genetix aQuireTM scanner (Genetix, UK) and saved as a TIFF file. The scanned images were analyzed using GenePix Pro 6.0(Axon Instruments, CA, USA), and data were analyzed using Excel (Microsoft, Redmond, WA) and Origin 6.1(Origin, MA, USA).

As a result: a β microarrays were constructed for analysis of A β 1-42-VEGF165 interactions. To construct an A.beta.microarray, soluble A.beta.1-42 (50mg/ml) was immobilized as capture molecules on each protein chip substrate. The A β 1-42 microarray on the chip interacted with VEGF165 at different concentrations ranging from 0.25 μ g/mL to 250.0 μ g/mL (FIGS. 1A and 1B). The results show that in both chip systems, A.beta.1-42 interacts well with VEGF165 in a dose-dependent manner. As shown, IPS-CHIP coated with IPS-Linker was able to detect low levels (0.25mg/ml) of VEGF protein compared to Prolinker (approximately 3.9mg/ml) coated ProteoChip. In summary, this finding indicates that the IPS-Linker-coated chip is more sensitive in detecting proteins than the Prolinker-coated chip.

Example 4 detection of human IL-17 Using IPS-Linker-coated chips

Capturing IL-17 antibody (R)&D systems, USA) were immobilized on IPS-Linker-coated chips overnight at 4 ℃. The chip was rinsed twice in wash solution for 10 minutes each, and then blocked with blocking solution for 1 hour. Blocked IL-17 antibody chips were washed in washing solution and DW (distilled water) and then in N₂Drying under air flow. After washing the reaction solution in a large amount, the recombinant IL-17 protein (R) in the reaction solution was washed in a humidity chamber at 37 deg.C&D systems, USA) were spotted on IL-17 antibody chips for 2 hours. The chip was rinsed in washing solution and DW, then in N₂Drying under air flow. After washing a large amount of the reaction solution, an IL-17 detection antibody (R) was added to the reaction solution&D systems, USA) were spotted on the chip and incubated in a humidity chamber at 37 ℃ for 1 hour. Then, the chip was rinsed in the washing solution and DW, followed by N₂Drying under air flow. After extensive washing, Cy 5-labeled streptavidin (GE Healthcare, USA) in the reaction solution was spotted on the chip and incubated in a humidity chamber at 37 ℃ for 1 hour. After washing with PBST (PBS-Tween 20) and DW, the chip was mounted in N₂Dried under air flow and the fluorescence intensity was measured using a fluorescence scanner.

Detection and data analysis: the chip was scanned using a GenePix 4000B scanner (Molecular Devices, USA) and saved as a TIFF file. The scanned images were analyzed using GenePix Pro 6.0(Molecular Devices, USA), and the data were analyzed using Excel (Microsoft, WA) and Origin 6.1(Origin, USA).

As a result: an Abeta microarray was constructed for quantitative analysis of human IL-17. To construct an IL-17 detection CHIP, a capture IL-17 antibody (0.1mg/ml) was immobilized on each IPS-CHIP plate. The IL-17 detection chip interacted with different concentrations of IL-17 protein from 0.06pg/ml to 60000pg/ml, and then detected by detection of IL-17 antibody and Cy 5-labeled streptavidin (FIG. 2). Human IL-17 protein was detected at low levels (0.06pg/ml) by IPS-CHIP coated with IPS-Linker compared to 96-well plate based ELISA (15.6 pg/ml). Taken together, this finding indicates that the IPS-Linker-coated chip is more sensitive in detecting proteins than the 96-well plate-based ELISA.

Example 5 detection of protein-protein interactions of IL-23 with its receptor Using IPS-Linker-coated chips

100 ug/ml of recombinant human IL-23 receptor Fc chimeric protein (R)&D systems, USA) were immobilized in a fixed buffer onto IPS-CHIP. After washing with PBST and DW, each well was blocked with 3% BSA in PBS. Wash blocked IPS-CHIP with PBST and DW, then at N₂Drying in air flow. After the chip was completely dried, IL-23 ligand labeled with Cy5 diluted to various concentrations in PBS containing 1% BSA and 30% glycerol was spotted on the chip and incubated in a humidity chamber at 37 ℃ for 1 hour. In PBST and DW and in N₂After drying under flow, the fluorescence intensity was measured to confirm the protein-protein interaction.

Detection and data analysis: the chip was scanned using a GenePix 4000B scanner (Molecular Devices, USA) and saved as a TIFF file. The scanned images were analyzed using GenePix Pro 6.0(Molecular Devices, USA), and the data were analyzed using Excel (Microsoft, WA) and Origin 6.1(Origin, USA).

As a result: mu.l of protein was spotted in wells as described previously. On each well, 100ng of recombinant human IL-23 receptor Fc chimera was immobilized and incubated with different concentrations (12.8pg-20ng) of IL-23 ligand, respectively (FIG. 3). As shown, the IL-23 receptor binds to IL-23 ligand in a dose-dependent manner, and only 1.6ng of IL-23 ligand is sufficient for detection. In summary, IPS-Linker coated IPS-CHIPIPS is a sensitive and cost-effective method for detecting protein interactions.

Example 6 detection of protein interaction between PD-1 and PD-L1 Using IPS-Linker-coated chips

Recombinant human PD-1Fc chimeric protein (ACRO Biosystems, USA) was diluted to a working concentration of 1.6. mu.g/mL-400. mu.g/mL with 30% glycerol in PBS. PD-1 protein was immobilized on an IPS-Linker-coated chip at 4 ℃ for 24 hours. Then, it was washed twice with 50mL of PBST solution (10 mM PBS containing 0.1% tween 20, pH 7.8) and dried under the surface of a gas stream. The chips were blocked with 0.005% tween 20 and 3% BSA in PBS for 1 hour at room temperature. Washing was performed in the same manner as in the previous operation. Biotinylated recombinant human PD-L1 protein (R & D Systems, USA) was diluted to a working concentration of 0.4. mu.g/mL-100. mu.g/mL in 30% glycerol in PBS and added to each PD-1 protein spot on IPS-CHIP and incubated at 37 ℃ for 1 hour. Then, washing was performed in the same manner as in the previous operation. 10 μ g/mL Cy 5-conjugated streptavidin (GE Healthcare, USA) was added to each spot and incubated for 1 hour at 37 ℃. Washing was performed in the same manner as in the previous operation.

Detection and data analysis: the chip was scanned using a GenePix 4000B scanner (Molecular Devices, USA) and saved as a TIFF file. The scanned images were analyzed using GenePix Pro 6.0(Molecular Devices, USA), and the data were analyzed using Excel (Microsoft, WA) and Origin 6.1(Origin, USA).

As a result: different concentrations of PD-1 protein from 1.6. mu.g/mL to 400. mu.g/mL interacted with different concentrations of PD-L1 protein from 0.4. mu.g/mL to 100. mu.g/mL (FIG. 4). The results show that PD-1 and PD-L1 proteins interact in a dose-dependent manner. As shown, with the IPS-Linker-coated chip, the detection limit sensitivities of PD-1 and PD-L1 were 1.6. mu.g/mL and 10. mu.g/mL, respectively.

Example 7 detection of protein-nucleotide interactions of STING and c-di-GMP binding Using IPS-Linker-coated chips

For concentration-dependent binding analysis between the STING protein and its known ligand, c-di-GMP, His X6 antibody (Thermo Fisher Scientific, USA) diluted with 30% glycerol was immobilized at 100. mu.g/ml concentration on IPS-CHIP for 3h at 4 ℃. The chip was washed with PBST (10 mM PBS containing 0.1% Tween 20, pH 7.8) and dried with nitrogen. Then, recombinant proteins of His-tagged STING (Active motif, USA) in 30% glycerol were dispensed to each well of the chip at a series of concentrations of 0 μ M, 5 μ M, 10 μ M, and the reaction was continued overnight at 4 ℃. The chip was then washed with PBST and dried with nitrogen. After blocking with 5% BSA for 1h at room temperature, the chip was washed with PBST and dried with nitrogen. To test the binding capacity of the STING protein to its ligand, 2'[ DY-547] -AHC-c-di GMP (BioLog, germany) called CDN ligand, 2' [ DY-547] -AHC-c-di GMP in 30% glycerol was dispensed in a series of concentrations 0 μ M, 3.9 μ M, 15.62 μ M, 62.25 μ M, 250 μ M into each well on the chip and incubated at 37 ℃ for 1 hour. The chip was washed with PBST and dried with nitrogen.

Detection and data analysis: the chip was scanned using a GenePix 4000B scanner (Molecular Devices, USA) and saved as a TIFF file. The scanned images were analyzed using GenePix Pro 6.0(Molecular Devices, USA), and the data were analyzed using Excel (Microsoft, WA) and Origin 6.1(Origin, USA).

As a result: the interaction of 5. mu.M-10. mu.M STING protein with 3.9. mu.M-250. mu.M c-di-GMP was shown on the chip (FIG. 5). Furthermore, the interaction between STING and c-di-GMP is dose dependent. In conclusion, as shown in the figure, the IPS-Linker coated chip is suitable for detecting the interaction between protein (STING) and nucleotide (c-di-GMP), down to 5. mu.M of STING protein and 3.9. mu. Mc-di-GMP.

It is to be understood that the detailed description section, and not the summary and abstract sections, is intended to be used to interpret the claims. The summary and abstract sections may set forth one or more, but not all exemplary embodiments of the present invention as contemplated by the inventors, and are therefore not intended to limit the present invention and the appended claims in any way.

The invention has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. Boundaries of the functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

With respect to aspects of the invention described as generic, all individual species are individually considered as separate aspects of the invention. If an aspect of the invention is described as "comprising" a feature, embodiments are also considered to be "consisting of …" or "consisting essentially of …" features.

The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

All of the various aspects, embodiments and options described herein may be combined in any and all variations.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. To the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition of the term in this document shall govern.