A pentenedioic acid small molecule compound with a tert-butyl protecting group and a preparation method thereof and a pentenedioyl-modified antigen, antibody

Abstract

The present application relates to the technical field of protein detection, and provides a pentenedioic acid small molecule compound with a tert-butyl protecting group, a preparation method of the pentenedioic acid small molecule compound, and a pentenedioic acid-modified antigen and antibody. The pentenedioic acid small molecule compound with the tert-butyl protecting group provided by the present application fully retains the structure of pentenedioic acid. After being combined with a carrier protein and deprotected, the obtained pentenedioic acid-modified antigen has high immunogenicity. After a host animal is immunized, the antibody produced by the host animal has high titer and high sensitivity, and thus efficient and specific recognition of the pentenedioic acid-modified protein is realized. The antibody of the present application proves that pentenedioic acid can induce PKM2 pentenedioylation, and realizes analysis of the PKM2 pentenedioylation level, which is beneficial to in-depth research on anti-tumor immune mechanisms and development of related drug targets.

Description

Technical Field

[0001] This invention relates to the field of protein detection technology, and in particular to a small molecule compound of pentenediaic acid with a tert-butyl protecting group, its preparation method, and pentenediacylation modification of antigens and antibodies. Background Technology

[0002] Tumor cells reshape their metabolic networks to create a tumor microenvironment (TME) conducive to tumor growth and immune escape. As tumors progress, the metabolic landscape within the TME continuously evolves dynamically, a process simultaneously regulated by tumor cells and their interactions with microenvironmental components. As the main force in anti-tumor therapy, immune cells within the TME constantly face challenges related to nutrient availability, metabolic preferences, and metabolic suitability, ultimately leading to immune dysfunction and weakened immunotherapy efficacy. Current research primarily focuses on tumor cells suppressing immune cells through nutrient competition and the secretion of immunosuppressive metabolites; however, the specific mechanisms by which tumors optimize metabolic synergy to achieve rapid proliferation and co-evolutionary immune escape remain unclear. Although tumor cells voraciously absorb nutrients to support anabolism, and amino acid catabolism is generally considered to enhance the suppression of T-cell immunity, our understanding of whether they actively restrict the use of specific nutrients and metabolic pathways to promote tumor progression remains very limited.

[0003] Amino acids are essential nutrients for the rapid proliferation of tumor cells, and their unavailability severely impairs T cell activation and function. Current research indicates the deprivation of non-essential amino acids (such as glutamine, arginine, and asparagine) and essential amino acids (such as methionine and tryptophan) in the tumor microenvironment (TME). In contrast, while lysine, an essential amino acid, plays a crucial role in biosynthesis, energy supply, and antioxidant activity, its dynamic changes in the TME and its specific impact on tumor progression and immune escape remain unclear. Limitations in this field include the incomplete elucidation of the lysine catabolism pathway and the unclear relationship between key catalytic enzymes and their corresponding metabolites, hindering our understanding of lysine metabolism in tumors and the development of related precision therapies. A few preliminary studies suggest that the lysine-catabolizing enzyme glutaryl-CoA dehydrogenase (GCDH) may promote the survival and chromatin stability of certain tumor cells (such as melanoma and glioblastoma stem cells). However, the interaction between tumor cells and tumor-infiltrating immune cells (especially cytotoxic CD8 cells) remains a significant concern. + How T cells dynamically utilize lysine and its catabolites to influence tumor progression remains poorly understood.

[0004] Previous studies have found that restricted lysine catabolism in tumor cells is a common feature in most cancer types. As tumors progress, tumor cells undergo a shift in arginine and lysine utilization preferences via the SLC7A1 / SLC7A2 transporter system. On the one hand, arginine uptake and mTORC1 activity are continuously enhanced; on the other hand, lysine catabolism is gradually downregulated, accompanied by a significant reduction in lysine accumulation and its catabolites in tumor interstitial fluid (TIF). Specifically, pentanoic acid produced by medium-chain acyl-CoA dehydrogenase (ACADM) can reactivate cytotoxic CD8 through glutaconylation of pyruvate kinase M2 (PKM2). + The anti-tumor effect of T cells. This research fills a gap in our understanding of how lysine metabolism influences tumor progression. Specifically, tumors evade immune attack by limiting the production of immune-activating metabolites, thus gaining a dual metabolic advantage that supports rapid growth and immune evasion.

[0005] However, there are currently no antibodies that specifically recognize pentenylated molecules, making it impossible to analyze the pentenylation level of PKM2, which restricts in-depth research on this anti-tumor immune mechanism and the development of related drug targets. Summary of the Invention

[0006] In view of this, the present invention provides a small molecule compound of pentenic acid with a tert-butyl protecting group, a method for preparing the same, and pentenylated modified antigens and antibodies. The small molecule compound of pentenic acid with a tert-butyl protecting group provided by the present invention fully retains the structure of pentenic acid. After binding to a carrier protein and removing the tert-butyl protecting group, the resulting pentenylated modified antigen exhibits high immunogenicity. After immunization with a host animal, it can generate antibodies with high titers and high sensitivity, thereby achieving efficient and specific recognition of pentenylated modified proteins.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A small molecule compound of pentenic acid with a tert-butyl protecting group has the structure shown in Formula A: Formula A.

[0008] This invention also provides a method for preparing the pentenediaic acid small molecule compound with a tert-butyl protecting group as described above, comprising the following steps: (1) The compound with the structure shown in Formula 1 and the compound with the structure shown in Formula 2 are mixed and reacted to obtain the compound with the structure shown in Formula 3; (2) The compound with the structure shown in Formula 3 is mixed with hydrochloric acid and reacted to obtain the compound with the structure shown in Formula 4; (3) The compound with the structure shown in Formula 4, tert-butanol and toluene are mixed and reacted to obtain a reaction solution containing the compound with the structure shown in Formula 5; (4) The reaction solution containing the compound with the structure shown in Formula 5 is mixed with tert-butyl triphenylphosphoacetate and reacted to obtain the compound with the structure shown in Formula 6; (5) The compound with the structure shown in Formula 6, an alcohol and lithium hydroxide are mixed and reacted to obtain a small molecule compound of pentenic acid with a tert-butyl protecting group with the structure shown in Formula A. Formula 1; Formula 2; Formula 3; Equation 4; Formula 5; Formula 6.

[0009] Preferably, in step (1), the molar ratio of the compound with the structure shown in Formula 1 to the compound with the structure shown in Formula 2 is 1:2~5; the reaction temperature is 80~90℃, and the reaction time is 1~5h.

[0010] Preferably, in step (2), the ratio of the compound with the structure shown in Formula 3 to hydrochloric acid is 1g:5~15mL; the concentration of the hydrochloric acid is 20~38wt%; and the reaction time is 1~5h.

[0011] Preferably, in step (3), the ratio of the compound with the structure shown in Formula 4, tert-butanol and toluene is 50 g: 30~50 mL: 400~600 mL; the reaction temperature is 80~120℃ and the reaction time is 1~5 h.

[0012] Preferably, in step (4), the molar ratio of the compound with the structure shown in Formula 5 to tert-butyl triphenylphosphoacetate is 1:1 to 1.2; the reaction temperature is 70 to 100 °C, and the reaction time is 1 to 5 h; In step (5), the molar ratio of the compound with the structure shown in Formula 6 to lithium hydroxide is 1:1 to 1.2; the reaction time is 10 to 20 h.

[0013] The present invention also provides a pentenylated modified polypeptide, comprising a polypeptide and a small molecule compound of the above scheme after detert-butyl protecting group coupled to the polypeptide; the sequence of the polypeptide is Cys-Lys.

[0014] The present invention also provides a pentenylated modified antigen, comprising a carrier protein and a pentenylated small molecule compound as described above, coupled to the carrier protein after the removal of a tert-butyl protecting group.

[0015] The present invention also provides a pentene diacylated antibody, which is obtained by immunizing a host animal with the antigen described in the above scheme.

[0016] The present invention also provides the application of the pentenediaic acid small molecule compound with tert-butyl protecting group, pentenediacylated modified antigen, or pentenediacylated antibody described in the above scheme in the detection of pentenediacylated molecules.

[0017] Preferably, the pentenylated molecule is a pentenylated modified protein.

[0018] This invention provides a small molecule compound of glutaconic acid with a tert-butyl protecting group, having the structure shown in Formula A. The compound provided by this invention fully retains the structure of glutaconic acid. After binding to a carrier protein, the resulting antigen exhibits high immunogenicity. Following immunization with a host animal, it can generate antibodies with high titers and high sensitivity, thereby achieving efficient and specific recognition of glutaconic acid-modified proteins. The antibody used in this invention has confirmed that glutaconic acid (GC) can induce PKM2 glutaconication, and the analysis of PKM2 glutaconication levels has been achieved, which is beneficial for in-depth research on anti-tumor immune mechanisms and the development of related drug targets. Attached Figure Description

[0019] Figure 1 This is a synthetic route diagram of the pentenediaic acid small molecule compound with a tert-butyl protecting group in Example 1 of the present invention; Figure 2 The results of ELISA titer assay (A) and Dot Blot assay (B) for pentene diacylated antibody are shown. Figure 3 The results of immunoprecipitation combined with Western blotting were used to detect the pentenylation modification of PKM2 protein, where A represents CD8+ modified by glutaric acid treatment. + In T cells, immunoprecipitation was performed using a pentenecylation antibody, followed by Western blotting with a PKM2 antibody to assess the results of PKM2 pentenecylation; B represents CD8 cells treated with glutaric acid. + In T cells, the results of PKM2 pentylation were evaluated by immunoprecipitation using PKM2 antibody and Western blotting using pentylation antibody. Figure 4 CD8 during tumor progression + Results of PKM2 pentylation level detection in T cells; Figure 5 This is the result of a specificity test for the pentene diacylated antibody. Detailed Implementation

[0020] This invention provides a small molecule compound of pentenic acid with a tert-butyl protecting group, having the structure shown in Formula A: Formula A.

[0021] In this invention, the chemical name of the small molecule compound of pentenic acid with a tert-butyl protecting group shown in Formula A is (E / Z)-5-tert-butoxy-5-oxopent-2-enoic acid.

[0022] This invention also provides a method for preparing the pentenediaic acid small molecule compound with a tert-butyl protecting group as described above, comprising the following steps: (1) The compound with the structure shown in Formula 1 and the compound with the structure shown in Formula 2 are mixed and reacted to obtain the compound with the structure shown in Formula 3; (2) The compound with the structure shown in Formula 3 is mixed with hydrochloric acid and reacted to obtain the compound with the structure shown in Formula 4; (3) The compound with the structure shown in Formula 4, tert-butanol and toluene are mixed and reacted to obtain a reaction solution containing the compound with the structure shown in Formula 5; (4) The reaction solution containing the compound with the structure shown in Formula 5 is mixed with tert-butyl triphenylphosphoacetate and reacted to obtain the compound with the structure shown in Formula 6; (5) The compound with the structure shown in Formula 6, alcohol, water and lithium hydroxide are mixed and reacted to obtain a small molecule compound of pentenic acid with a tert-butyl protecting group with the structure shown in Formula A. Formula 1; Formula 2; Formula 3; Equation 4; Formula 5; Formula 6.

[0023] This invention involves reacting a compound with the structure shown in Formula 1 and a compound with the structure shown in Formula 2 to obtain a compound with the structure shown in Formula 3. In this invention, the compound with the structure shown in Formula 1 is cyclo(isopropyl)malonate, the compound with the structure shown in Formula 2 is trimethyl orthocarbonate, and the compound with the structure shown in Formula 3 is 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione; the molar ratio of the compound with the structure shown in Formula 1 to the compound with the structure shown in Formula 2 is 1:2 to 5, specifically 1:3; the preferred reaction temperature is 80 to 90°C, specifically 85°C, and the preferred reaction time is 1 to 5 hours, specifically 2 hours; in a specific embodiment of this invention, TLC monitoring is preferably used until the reaction is complete; after the reaction is complete, the resulting reaction solution is preferably concentrated to obtain a yellow oily substance, which is the crude product of the compound with the structure shown in Formula 3. The crude product does not require purification and can be directly used in the next reaction step.

[0024] After obtaining the compound with the structure shown in Formula 3, the present invention reacts the compound with hydrochloric acid to obtain the compound with the structure shown in Formula 4. In the present invention, the compound with the structure shown in Formula 4 is 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione; the preferred ratio of the compound with the structure shown in Formula 3 to hydrochloric acid is 1g:5~15mL, specifically 1g:10mL; the preferred concentration of the hydrochloric acid is 20~38wt%; the preferred reaction time is 1~5h, specifically 3h; the reaction temperature is room temperature; in a specific embodiment of the present invention, TLC monitoring is preferably used until the reaction is complete. After the reaction is complete, the present invention preferably dilutes the reaction solution with saturated sodium chloride, extracts the resulting diluted solution with ethyl acetate, dries the resulting organic phase, and concentrates it to obtain a yellow solid, which is the compound with the structure shown in Formula 4.

[0025] After obtaining the compound with the structure shown in Formula 4, the present invention mixes the compound with the structure shown in Formula 4, tert-butanol, and toluene to react and obtain a reaction solution containing the compound with the structure shown in Formula 5. In the present invention, the compound with the structure shown in Formula 5 is specifically tert-butyl 3-oxopropionate; the preferred ratio of the compound with the structure shown in Formula 4, tert-butanol, and toluene is 50g:30~50 mL:400~600 mL, specifically 50g:32.5mL:500mL; the preferred reaction temperature is 80~120℃, specifically 100℃; the preferred reaction time is 1~5h, specifically 3h; in a specific embodiment of the present invention, TLC monitoring is preferably used until the reaction is complete. After the reaction is complete, the present invention preferably washes the obtained reaction solution with saturated sodium chloride and then dries it to obtain a yellow reaction solution, which is the reaction solution containing the compound with the structure shown in Formula 5, and this reaction solution is directly used for the next reaction.

[0026] After obtaining the reaction solution containing the compound shown in Formula 5, the present invention mixes the reaction solution containing the compound shown in Formula 5 with tert-butyl triphenylphosphoacetate and reacts them to obtain the compound shown in Formula 6. In the present invention, the compound shown in Formula 6 is (E / Z)-pentan-2-enedia-5-tert-butyl ester-1-methyl ester; the molar ratio of the compound shown in Formula 5 to tert-butyl triphenylphosphoacetate is preferably 1:1 to 1.2, specifically 1:1; the reaction temperature is preferably 70 to 100 °C, specifically 80 °C, and the reaction time is preferably 1 to 5 h, specifically 3 h; in a specific embodiment of the present invention, TLC monitoring is preferably used until the reaction is complete. After the reaction is completed, the present invention preferably concentrates the obtained reaction solution and then performs column chromatography purification to obtain a colorless oily substance, which is the compound shown in Formula 6; the eluent used for column chromatography purification is a mixed solvent of petroleum ether and ethyl acetate, wherein the volume ratio of petroleum ether to ethyl acetate is preferably 30:1.

[0027] After obtaining the compound with the structure shown in Formula 6, the present invention mixes the compound with the structure shown in Formula 6, an alcohol, and lithium hydroxide to react and obtain a small molecule compound of pentenic acid with a tert-butyl protecting group as shown in Formula A. In the present invention, the molar ratio of the compound with the structure shown in Formula 6 to lithium hydroxide is preferably 1:1 to 1.2, specifically 1:1; the lithium hydroxide is specifically lithium hydroxide monohydrate; the lithium hydroxide is preferably used in the form of an aqueous solution of lithium hydroxide, and the concentration of the aqueous solution of lithium hydroxide is preferably 0.05 to 0.06 g / mL; the alcohol is preferably methanol; the ratio of the amount of the compound with the structure shown in Formula 6 to the alcohol is preferably 13 g: 50 to 60 mL; the reaction time is preferably 10 to 20 h, specifically 12 h; the reaction temperature is room temperature; in a specific embodiment of the present invention, TLC is preferably used to monitor the reaction until it is complete. After the reaction is complete, the present invention preferably extracts the resulting reaction solution with ethyl acetate, and purifies the resulting aqueous phase using preparative high-performance liquid chromatography (HPLC). The mobile phase used in the preparative HPLC is preferably an aqueous formic acid solution, wherein the formic acid content in the aqueous formic acid solution is preferably 1 wt%. The pH of the aqueous phase is adjusted to acidic before injection. The purified white solid is a small molecule compound of pentenic acid with a tert-butyl protecting group, as shown in Formula A.

[0028] In this invention, the pentenediaic acid small molecule compound with tert-butyl protecting group can be used to prepare pentenediacylated modified antigens.

[0029] This invention also provides a pentenylated modified polypeptide, comprising a polypeptide and a small molecule compound as described above, coupled to the polypeptide after the removal of a tert-butyl protecting group. The small molecule compound after the removal of the tert-butyl protecting group is pentenic acid, wherein the carboxyl group at one end of the pentenic acid and the amino group on Cys are linked by an amide bond; the sequence of the polypeptide is Cys-Lys. Specifically, the small molecule compound after the removal of the tert-butyl protecting group is modified at the N-terminus (Cys end) of the polypeptide sequence.

[0030] In this invention, the pentenylated modified polypeptide is preferably synthesized using a solid-phase synthesis method, which may specifically include the following steps: According to the amino acid sequence of the polypeptide described in the above technical solution, Lys and Cys are sequentially coupled onto the resin in order from C-terminus to N-terminus. Then, the pentenediaic acid small molecule compound with tert-butyl protecting group and the amino group on Cys are reacted with a condensation reagent to obtain a peptide resin. The peptide resin is then cleaved with a cleavage reagent to obtain a crude peptide. The crude peptide is purified to obtain a pentenedialylated modified polypeptide. This invention does not have special requirements for the specific conditions of coupling Lys and Cys; any conditions well known to those skilled in the art can be used. The condensation reagent is preferably N,N′-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT), and the molar ratio of DIC to HBOT is preferably 2:1. In a specific embodiment of this invention, it is preferable to first dissolve the pentenediaic acid small molecule compound with tert-butyl protecting group and HOBT in DMF solvent, then add DIC dropwise for activation to obtain an activated solution, and then add the activated solution to the resin column for reaction. The lysis reagent is preferably a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water, with the mass ratio of TFA, TIS, and water preferably being 95:3:2. During the lysis process, the tert-butyl protecting group on the pentenediaic acid small molecule compound is simultaneously removed to obtain the crude peptide. The present invention does not have special requirements for the purification conditions, and those well known to those skilled in the art can be used.

[0031] The present invention also provides a pentenylated modified antigen, comprising a carrier protein and a pentenylated modified polypeptide coupled to the carrier protein; the carrier protein is preferably bovine serum albumin (BSA) or KLH protein; in a specific embodiment of the present invention, the thiol group on Cys in the pentenylated modified polypeptide is coupled to the primary amino group on the carrier protein via an SMCC reagent.

[0032] In this invention, the preferred method for preparing the pentenylated modified antigen includes: mixing a protein solution and an SMCC (4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester) solution for a first reaction, dialyzing the reaction solution to obtain a protein-SMCC solution; mixing a pentenylated modified peptide solution and a protein-SMCC solution for a second reaction to obtain the pentenylated modified antigen; the concentration of the SMCC solution is preferably 10 mg / mL, and the solvent is DMF; the concentration of the protein solution is preferably 15 mg / mL, and the solvent is PBS; the solvent of the pentenylated modified peptide solution is PBS; the temperature of the first reaction is room temperature, and the time is 1-2 h, specifically 1 h; the temperature of the second reaction is room temperature, and the time is 3-5 h, specifically 4 h; the mass ratio of SMCC to protein is preferably 1:5-7, specifically 1:6; the mass ratio of the protein-SMCC solution to the pentenylated modified peptide is preferably 2-3:3, specifically 2.5:3.

[0033] This invention also provides a pentene-acylated antibody, obtained by immunizing a host animal with the antigen described in the above-described scheme. In this invention, the pentene-acylated antibody is a polyclonal antibody, and the host animal is preferably a white rabbit, specifically a New Zealand white rabbit. This invention does not have special requirements for the immunization method; any method well-known to those skilled in the art can be used. In a specific embodiment of this invention, it is preferred to immunize the host animal with the antigen 4-6 times, and then perform serum testing on the immunized host animal using the ELISA method. When the serum parameters meet the requirements, blood is collected to collect antiserum, which is then purified until the titer reaches the required level, thus obtaining the pentene-acylated antibody of this invention. In this invention, the titer of the antiserum is preferably >1:32000, and the titer of the purified antibody is preferably greater than 1:64000.

[0034] This invention also provides the application of the pentenylic acid small molecule compound with a tert-butyl protecting group, the pentenylated modified polypeptide, the pentenylated modified antigen, or the pentenylated antibody described above in the detection of pentenylated molecules; wherein the pentenylated molecule is a pentenylated modified protein; the structure of the pentenylated modified protein is shown below: .

[0035] In a specific embodiment of the present invention, the pentenylated modified protein is pentenylated modified PKM2.

[0036] The present invention does not have any special requirements for the method used to detect pentenylated molecules; any method well known to those skilled in the art can be used.

[0037] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0038] Example 1: Synthesis of small pentenic acid compounds with tert-butyl protecting groups (1) Step 1: Synthesis of 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (a compound with the structure shown in Formula 3) The compound with the structure shown in Formula 1 (cyclo(isopropyl)malonate, 80 g, 555.05 mmol) was dissolved in the compound with the structure shown in Formula 2 (trimethyl orthocarbonate, 176.7 g, 1665.16 mmol). The system was heated to 85 °C and maintained at this temperature for 2 hours. After the reaction was completed by TLC monitoring, the mixture was concentrated to give a yellow oil (80 g, 429.73 mmol), which is compound 3. The crude product was not calculated in yield and was used directly in the next step.

[0039] (2) Second step: Synthesis of 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (compound 4) The compound with the structure shown in Formula 3 (80 g, 429.73 mmol) was dissolved in hydrochloric acid (800 mL) and reacted at room temperature for 3 hours. After the reaction was completed by TLC monitoring, the reaction solution was diluted with saturated sodium chloride, extracted with ethyl acetate, dried, and concentrated to give a yellow solid (50 g, 290.46 mmol, two-step yield 52.63%), which is compound 4.

[0040] (3) Third step: Synthesis of tert-butyl 3-oxopropionate (the compound with the structure shown in Formula 5) The compound with the structure shown in Formula 4 (50 g, 290.46 mmol) was dissolved in tert-butanol (32.5 mL) and toluene (500 mL), and heated at 100 °C for 3 hours. After the reaction was complete as monitored by TLC, the reaction solution was washed with saturated sodium chloride, dried, and without concentration, a yellow reaction solution containing the compound with the structure shown in Formula 5 (41.86 g, 290.35 mmol) was obtained. The crude product was not calculated in yield and was used directly in the next step.

[0041] (4) Fourth step: Synthesis of (E / Z)-pent-2-enic acid-5-tert-butyl ester-1-methyl ester (the compound with the structure shown in Formula 6) 109.29 g (290.35 mmol) of tert-butyl triphenylphosphoacetate was added to a reaction solution containing a compound with the structure shown in Formula 5 (41.86 g, 290.35 mmol), and the mixture was heated at 80 °C for 3 hours. After the reaction was completed by TLC monitoring, the mixture was concentrated, and the concentrate was directly mixed with a sample for column chromatography purification. The eluent used for column chromatography purification was a mixture of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 30:1. The purified product was a colorless oil (13 g, 64.92 mmol, two-step yield 22.36%), denoted as the compound with the structure shown in Formula 6.

[0042] (5) Fifth step: Synthesis of (E / Z)-5-tert-butoxy-5-oxopent-2-enoic acid (pentenylated small molecule compound with the structure shown in Formula A) The compound with the structure shown in Formula 6 (13 g, 64.92 mmol) was added to methanol (50 mL), followed by an aqueous solution of lithium hydroxide (2.72 g, 64.92 mmol) (50 mL). The reaction was carried out at room temperature for 12 hours. After the reaction was completed by TLC monitoring, the mixture was back-extracted twice with ethyl acetate, and the aqueous phase was purified by preparative high-performance liquid chromatography (using a 1 wt% formic acid aqueous solution as the mobile phase, which was adjusted for acidity during preparation) to give a white solid (4 g, 21.48 mmol, 33.11%).

[0043] The synthetic route of the pentenediaic acid small molecule compound with tert-butyl protecting group in Example 1 is as follows: Figure 1 As shown.

[0044] Example 2 Preparation of pentenylated modified peptides A pentenylated modified peptide with the sequence Cys-Lys and N-terminal (Cys terminus) modification with pentenic acid was prepared using a solid-phase synthesis method. The specific preparation method is as follows: 1. Using 2-Cl resin with a substitution degree of 0.5 mmol / g, and employing the Fmoc process, amino acids are sequentially condensed from the C-terminus to the N-terminus (from right to left) according to the above peptide sequence until peptide chain condensation is complete. The specific steps are as follows: (1) Take 1.0g of 2-Cl resin and add it to the cleaned reactor. Add 10mL of DCM, soak and swell for 20min and then stop. Filter to remove the liquid.

[0045] (2) Accurately weigh 0.5 mmol Fmoc-Lys(Dde)-OH, pour it into the reactor, add 10 mL DMF as solvent, add 0.5 mL DIEA dropwise, stop the reaction after 2 h, add 8 mL methanol, react for 15 min, filter to remove the liquid, and wash with DMF 5 times.

[0046] (3) Removal of FMOC: Add 20 mL of 20% hexahydropyridine / DMF solution, stir for 10 minutes, remove the liquid, add 20% hexahydropyridine / DMF solution again, stir for 5 minutes, remove the liquid, wash with DMF 5 times; take a sample for ninhydrin colorimetric detection, and record the detection color (blue).

[0047] (4) Condensation reaction: Add materials in an amount three times the molar amount of resin substitution. Weigh 1.5 mmol Boc-Cys(trt)-OH and 1.5 mmol HoBt, add 16 mL DMF to dissolve, add 0.5 mmol DIC dropwise for 10 minutes to activate, and then add to the reaction column. React for 1.5 h (no color development is detected by ninhydrin colorimetric method) and remove. Wash 5 times with DMF.

[0048] (5) Removal of Dde group: Add 20 mL of 3% hydrazine hydrate / DMF solution, stir for 10 minutes, remove the liquid, add 20 mL of 3% hydrazine hydrate / DMF solution again, stir for 10 minutes, remove the liquid, wash with DMF 5 times; take a sample for ninhydrin colorimetric detection, and record the detection color (blue).

[0049] (6) Pentenedylation modification: Add 0.75 mmol of pentenic acid small molecule compound with tert-butyl protecting group and 1.0 mmol of HoBt according to 1.5 times the molar amount of resin substitution degree. Dissolve in 12 mL of DMF, activate with 0.5 mmol of DIC for 10 minutes, and then add to the reaction column. React for 1.5 h (no color development when detected by ninhydrin colorimetric method) and remove. Wash 3 times with DMF and 2 times with methanol, and dry under vacuum.

[0050] 2. Peptide cleavage Preparation of lysis reagent: Calculate the amount of lysis reagent needed based on 15 mL of lysis buffer per 1 g of resin peptide. Add the required lysis reagents (H2O, TIS, and TFA) sequentially to the lysis reaction flask according to a mass ratio of TFA:TIS:H2O = 95:3:2, maintaining the temperature of the lysis reagent at 0–10 °C. Add the lysis reagent to the peptide resin while stirring, and after the system temperature stabilizes, continue stirring at 20–25 °C for 2 hours. Filter the lysis buffer and precipitate it with 10 times the volume of filtrate in ice-cold diethyl ether. Wash four times by centrifugation, and dry the precipitate under reduced pressure at room temperature to obtain 0.15 g of crude product.

[0051] 3. Purification A liquid chromatography system was prepared using 0.1% TFA aqueous solution as mobile phase A and 0.1% TFA acetonitrile as mobile phase B. A 10 μm reversed-phase C18 (50 × 250 mm) packing material was used, with a UV detector set to 220 nm and a flow rate of 40 mL / min. The system was equilibrated with 10% acetonitrile for 10 minutes. 0.15 g of crude peptide was ground and crushed, dissolved in 10% acetonitrile aqueous solution, microwaved until completely dissolved, filtered through a 0.45 μm filter, and injected. Elution was performed using a gradient of 1% (0.01 min) to 10% (40 min) acetonitrile concentration at room temperature. The absorbance was monitored, and impurity peaks were removed. The target product was collected. Analysis yielded a purified solution with >95% purity. Lyophilization yielded 18 mg of the target product (white powder). After lyophilization, purity and MS analysis were performed again. The results showed a product purity of 95.598%, an MS molecular weight of 361.2, and a calculated yield of 10.0%.

[0052] Example 3 Preparation of Pentylated Modified Antigen Dissolve 20 mg of SMCC (the amount used to conjugate 40 peptides) in 2 mL of DMF to obtain an SMCC solution; add 0.8 mL of KLH protein to a round-bottom flask, and add 1×PBS (pH 7.2) to bring the final protein concentration to 15 mg / mL to obtain a KLH protein solution.

[0053] The dissolved SMCC solution was slowly added dropwise to 120 mg of KLH protein solution, and the reaction was stirred at room temperature for 1 h. Then, the mixture was dialyzed against 1 L of 1×PBS (pH 7.4) at 4°C for 6 hours to remove free SMCC. The dialyzed reaction solution was poured into a 50 mL centrifuge tube, and its volume was determined by the graduations. The concentration of the dialyzed protein was calculated based on the amount of KLH protein added before the reaction. Then, based on this concentration, 2.5 mg of KLH-SMCC solution was transferred to a 5 mL centrifuge tube.

[0054] 3.0 mg of pentenylated modified peptide (prepared in Example 2) was dissolved in 0.6 mL of 1×PBS (pH 7.2). The thiol groups in the peptide were detected using Ellman's reagent: 100 μl of Ellman's reagent stock solution was added to a 96-well plate, followed by 10 μl of the peptide solution. The UV absorbance was measured at λ=412 nm using a Nano spectrophotometer. If the OD value was >0.15, proceed to the next step; if the OD value was <0.15 but >0.05, more peptide was added until the requirement was met; if the OD value was <0.05, the peptide synthesis step was returned for quality control again.

[0055] Add the peptide solution dropwise into a KLH-SMCC tube and mix it with a vertical mixer at room temperature for 4 hours.

[0056] Detection of thiol groups in peptides using Ellman's reagent: Add 100 μl of Ellman's reagent stock solution to a 96-well plate, followed by 10 μl of the cross-linked peptide solution. Measure the UV absorbance at λ=412 nm using a Nano spectrophotometer. An OD value <0.03 indicates that the cross-linking rate between the peptide and KLH protein has reached over 80%; an OD value >0.03 requires further addition of SMCC-activated KLH protein for continued cross-linking. If Ellman's reagent turns yellow, it indicates incomplete coupling between the peptide and KLH protein; if Ellman's reagent does not turn yellow, it indicates complete coupling between the peptide and KLH protein.

[0057] Example 4 Preparation of rabbit polyclonal antibodies Two New Zealand white rabbits were immunized six times with the antigen prepared in Example 3. The antiserum titer after the 3rd, 4th, 5th, and 6th immunizations was detected by indirect ELISA, and the antiserum titer was required to be >1:32000. If the antiserum titer was greater than 1:32000, blood was collected to obtain antiserum, and antibody purification was performed (purification method was antigen-specific affinity purification).

[0058] The purified antibody titer must be greater than 1:64000 and the Dot Blot result must be positive.

[0059] Example 5: Antibody titer and function detection First, the titer of the pentene-modified antibody prepared in Example 4 was tested: The antibody was coated onto plates with both the pentene-modified antigen (carrier protein was BSA protein, prepared according to the method in Example 3, except that the KLH protein was replaced with BSA protein) and the control antigen (BSA protein). ELISA was performed using the antibody at different dilution ratios (1:1K, 1:4K, 1:16K, 1:64K, 1:256K, 1:1024K, and 1:4096K). The results are as follows: Figure 2 As shown in Figure A. According to... Figure 2 As can be seen from Figure A, the pentenylated antibody prepared in this invention can specifically recognize pentenylated modified peptides, while the affinity for the control antigen is extremely low, and its binding decreases accordingly with increasing antibody dilution ratio, indicating that the pentenylated antibody prepared in this invention has the characteristics of high sensitivity and specificity. Furthermore, Dot Blot immunohybridization reactions were performed using different concentrations of control antigen and pentenylated modified antigen to achieve specific and sensitive detection of the antibody. The results are as follows... Figure 2 As shown in Figure B, the results obtained are consistent with those obtained from the ELISA experiment.

[0060] Given that previous studies have found CD8 infiltration during tumor progression +The PKM2 receptor on T cells can undergo pentenylation modification, a finding validated by protein modification mass spectrometry and click chemistry experiments. Therefore, this invention further validated this phenomenon using a pentenylation antibody. This antibody was used on activated CD8 cells. + T cells were cultured with 5 mM pentenoic acid for 6 hours, then collected and lysed. Immunoprecipitation enrichment of PKM2 protein followed by Western blotting further confirmed that PKM2 in T cells can indeed undergo pentenoylation modification. Figure 3 As shown, Figure 3 The middle one is CD8 treated with glutaric acid. + In T cells, immunoprecipitation was performed using a pentenecylation antibody, followed by Western blotting with a PKM2 antibody to assess the results of PKM2 pentenecylation; B represents CD8 cells treated with glutaric acid. + In T cells, the results of PKM2 pentenylation were evaluated by immunoprecipitation using PKM2 antibody and Western blotting using pentenylation antibody. Based on... Figure 3 It can be seen that the pentenecylated antibody developed in this invention can indeed specifically detect the pentenecylation modification of PKM2. This experiment verifies that pentenecylic acid can indeed modify PKM2 by pentenecylation, and most importantly, it fully demonstrates the feasibility of the pentenecylated antibody prepared in this invention from the perspective of functional verification.

[0061] Given that previous studies have found CD8 infiltration during tumor progression + This invention also addresses the decreased PKM2 pentenylation level in T cells and the infiltration of CD8+ in the tumor microenvironment during tumor progression. + The level of PKM2 pentenylation in T cells was detected. Subcutaneous tumorigenesis of MC38 cells was performed in mice, and tumors were collected on days 14 and 21 of growth. Infiltrating CD8+ cells were then sorted. + T cells were lysed, and PKM2 protein was enriched by immunoprecipitation. Then, PKM2 pentanoylation was detected by Western blotting combined with a pentanoylation antibody. The results are as follows: Figure 4 As shown, the results indicate that CD8 is involved in tumor progression. + The level of PKM2 pentylation in T cells decreases as the tumor progresses.

[0062] Previous studies identified the major pentenylation modification sites of PKM2 as lysine residues at positions 336 and 337 using protein modification mass spectrometry. To further validate this finding and reaffirm the specificity of the pentenylated antibody, this invention constructed an overexpression plasmid with lysine mutations at positions 336 and 337 of PKM2. The plasmid was transfected into 293T cells, and the cells were cultured with pentenic acid for 6 hours. The cells were then harvested and lysed. Flag-PKM2 was enriched using immunoprecipitation with Flag magnetic beads, and the pentenylation modification status of PKM2 was detected by Western blotting. The experimental results are as follows: Figure 5 As shown, when lysine at positions 336 and 337 is mutated to arginine, the level of pentene acylation modification of PKM2 is significantly reduced, which once again verifies the previous research results and further verifies the specificity of the pentene acylation antibody through point mutation experiments.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A small molecule compound of pentenic acid with a tert-butyl protecting group, characterized in that, It has the structure shown in Equation A: Formula A.

2. The method for preparing the pentenic acid small molecule compound with a tert-butyl protecting group according to claim 1, characterized in that, Includes the following steps: (1) The compound with the structure shown in Formula 1 and the compound with the structure shown in Formula 2 are mixed and reacted to obtain the compound with the structure shown in Formula 3; (2) The compound with the structure shown in Formula 3 is mixed with hydrochloric acid and reacted to obtain the compound with the structure shown in Formula 4; (3) The compound with the structure shown in Formula 4, tert-butanol and toluene are mixed and reacted to obtain a reaction solution containing the compound with the structure shown in Formula 5; (4) The reaction solution containing the compound with the structure shown in Formula 5 is mixed with tert-butyl triphenylphosphoacetate and reacted to obtain the compound with the structure shown in Formula 6; (5) The compound with the structure shown in Formula 6, an alcohol and lithium hydroxide are mixed and reacted to obtain a small molecule compound of pentenic acid with a tert-butyl protecting group with the structure shown in Formula A. Formula 1; Formula 2; Formula 3; Equation 4; Formula 5; Formula 6.

3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of the compound with the structure shown in Formula 1 to the compound with the structure shown in Formula 2 is 1:2~5; the reaction temperature is 80~90℃ and the reaction time is 1~5h.

4. The preparation method according to claim 2, characterized in that, In step (2), the ratio of the compound with the structure shown in Formula 3 to hydrochloric acid is 1g:5~15mL; the concentration of the hydrochloric acid is 20~38wt%; and the reaction time is 1~5h.

5. The preparation method according to claim 2, characterized in that, In step (3), the ratio of the compound with the structure shown in Formula 4, tert-butanol, and toluene is 50 g: 30~50 mL: 400~600 mL; the reaction temperature is 80~120℃, and the reaction time is 1~5 h. In step (4), the molar ratio of the compound with the structure shown in Formula 5 to tert-butyl triphenylphosphoacetate is 1:1 to 1.2; the reaction temperature is 70 to 100 °C, and the reaction time is 1 to 5 h. In step (5), the molar ratio of the compound with the structure shown in Formula 6 to lithium hydroxide is 1:1 to 1.2; the reaction time is 10 to 20 h.

6. A pentenylated modified polypeptide, characterized in that, The small molecule compound of claim 1 includes a polypeptide and a detert-butyl protecting group conjugated to the polypeptide; the polypeptide has the sequence Cys-Lys.

7. A pentenylated modified antigen, characterized in that, This includes a carrier protein and a pentenylated modified polypeptide coupled to the carrier protein.

8. A pentenylated antibody, characterized in that, It is obtained by immunizing a host animal with the antigen described in claim 8.

9. The use of the pentenic acid small molecule compound with a tert-butyl protecting group as described in claim 1, the pentenyl acylated modified polypeptide as described in claim 6, the pentenyl acylated modified antigen as described in claim 7, or the pentenyl acylated antibody as described in claim 8 in the detection of pentenyl acylated molecules.

10. The application according to claim 9, characterized in that, The pentene diacylated molecule is a protein modified with pentene diacylation.

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