Pentalenoic acid structural analogues, methods of making and using the same, and copper-catalyzed azide-yne cycloaddition reactions

Abstract

The application provides a pentenedioic acid structural analogue as well as a preparation method and application thereof and a kit, and belongs to the technical field of organic chemistry. The application provides a pentenedioic acid structural analogue. The pentenedioic acid structural analogue can be used in click chemistry, and the kind of raw materials used in click chemistry is enlarged. The data of the examples show that the Alk-GC can carry out copper catalysis azide-alkyne cycloaddition reaction, CD8 + T cells have dependent uptake on the Alk-GC, and the Alk-GC can modify PKM2.

Description

Technical Field

[0001] This invention relates to the field of organic chemistry, and in particular to a pentenedioic acid structural analogue, its preparation method and application, and copper-catalyzed azido-yne cycloaddition reaction. Background Technology

[0002] Click chemistry achieves efficient molecular construction by assembling small molecular modules using carbon-heteroatom bonds (CXC), with its core reaction being the copper-catalyzed azido-alkynyl cycloaddition reaction (CuAAC). This method is characterized by high yields and harmless byproducts, and can be applied in drug development, novel optical material preparation, and supramolecular assembly. The concept of click chemistry has made a significant contribution to the field of chemical synthesis, and has become one of the most useful and attractive synthetic concepts in many areas, including drug development and biomedical materials. It possesses the following characteristics: the reaction utilizes the concept of "combinatorial" synthesis, resulting in a wide range of applications; high yields; harmless byproducts; strong stereoselectivity; simple reaction conditions; readily available starting materials and reagents; and rapid synthesis, making it a highly suitable synthetic method.

[0003] The existing technology for click chemistry suffers from a limited variety of raw materials. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a pentenediaic acid structural analogue, its preparation method and application, and a copper-catalyzed azido-yne cycloaddition reaction. This invention provides a novel pentenediaic acid structural analogue for click chemistry.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a pentenediaic acid structural analog (Alk-GC) having the structure shown in Formula I: Formula I.

[0006] This invention also provides a method for preparing the pentenediaic acid structural analogue described in the above technical solution, comprising the following steps: The first reaction of cyclo(isopropyl)malonic acid with trimethyl orthocarbonate yields 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione. The 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione was reacted with hydrochloric acid to give 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione. The 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione was reacted with tert-butanol in a third reaction to give tert-butyl 3-oxopropionate. The tert-butyl 3-oxopropionate and tert-butyl triphenylphosphoacetate were subjected to a fourth reaction to obtain (E / Z)-pent-2-enedioic acid-5-tert-butyl ester-1-methyl ester. The (E / Z)-pent-2-enedioic acid-5-tert-butyl-1-methyl ester was reacted with trifluoroacetic acid in a fifth reaction to obtain (E / Z)-5-methoxy-5-oxopent-3-enoic acid. The (E / Z)-5-methoxy-5-oxopre-3-enoic acid was reacted with oxalyl chloride in a sixth reaction to give methyl (E / Z)-5-chloro-5-oxopre-2-enoic acid. The (E / Z)-5-chloro-5-oxopent-2-enoic acid methyl ester was reacted with propargylamine in a seventh reaction to obtain (E / Z)-5-oxo-5-(prop-2-yn-1-ylamino)pent-2-enoic acid methyl ester. The (E / Z)-5-oxo-5-(prop-2-yn-1-ylamino)pent-2-enoic acid methyl ester was reacted with lithium hydroxide in an eighth reaction to obtain the pentenediaic acid structural analog.

[0007] Preferably, the molar ratio of cyclo(isopropyl)malonic acid to trimethyl orthocarbonate is 1:2~4.

[0008] Preferably, the temperature of the first reaction is 80~90℃ and the time is 1.5~2.5h.

[0009] Preferably, the ratio of 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione to tert-butanol is (250~300) mmol:(30~35) mL.

[0010] Preferably, the temperature of the third reaction is 95~105℃ and the time is 2.5~3.5h.

[0011] Preferably, the ratio of (E / Z)-pent-2-enedioic acid-5-tert-butyl ester-1-methyl ester to trifluoroacetic acid is (60~70) mmol:(60~70) mL.

[0012] Preferably, the molar ratio of (E / Z)-5-methoxy-5-oxopram-3-enoic acid to oxalyl chloride is 1:2~4.

[0013] This invention also provides the application of the pentenediaic acid structural analogs described in the above technical solution in click chemistry.

[0014] The present invention also provides the application of the pentenediaic acid structural analog described in the above technical solution in the detection of pyruvate kinase PKM2.

[0015] The present invention also provides a kit comprising the pentenediaic acid structural analogue, Cy3-azide, and copper catalyst described in the above technical solution.

[0016] This invention provides a pentenediaic acid structural analog.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The pentenic acid structural analogues of the present invention can be used in click chemistry, expanding the range of raw materials available for click chemistry. Data from the examples show that the Alk-GC of the present invention can carry out copper-catalyzed azide-alkyne cycloaddition reactions, CD8 + T cells exhibit Alk-GC-dependent uptake, and Alk-GC can modify PKM2.

[0018] The present invention also provides a method for preparing the pentenediaic acid structural analogues described in the above technical solution. The preparation method of the present invention is simple to operate and suitable for industrial application.

[0019] The present invention also provides the application of the pentenedia acid structural analogue described in the above technical solution in the detection of pyruvate kinase PKM2, which can perform quantitative and qualitative detection of pyruvate kinase PKM2.

[0020] The present invention also provides a kit comprising the pentenediaic acid structural analog, Cy3-azide and copper catalyst described in the above technical solution, for detecting pyruvate kinase PKM2. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the process for preparing pentenediaic acid analogues in Example 1; Figure 2 Chromatograms of pentenedioic acid structural analogs; Figure 3 Mass spectra of pentenic acid structural analogs; Figure 4 A schematic diagram illustrating the principle of copper-catalyzed azide-alkyne cycloaddition reaction for Alk-GC. Figure 5 The images are from flow cytometry analysis, where a is a peak distribution diagram of fluorescence intensity during flow cytometry, and b is a bar chart of quantitative statistical analysis of fluorescence intensity during flow cytometry. Figure 6 This is an immunofluorescence detection image; Figure 7 This is an immunoblot image of biotinylated immunoprecipitation-binding protein; Figure 8 Immunoblot image of tag-immunoprecipitation-binding proteins; Figure 9 This is a flowchart of an in vitro cell-free experiment; Figure 10This is a diagram from a validation experiment demonstrating click-through chemobinding immunoblotting. Detailed Implementation

[0022] This invention provides a pentenediaic acid structural analog (Alk-GC, (E / Z)-5-oxo-5-(prop-2-yn-1-ylamino)pent-2-enoic acid), having the structure shown in Formula I: Formula I.

[0023] This invention also provides a method for preparing the pentenediaic acid structural analogue described in the above technical solution, comprising the following steps: The first reaction of cyclo(isopropyl)malonic acid (compound 1) with trimethyl orthocarbonate (compound 2) yields 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (compound 3). The 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione was reacted with hydrochloric acid to give 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (compound 4). The 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione was reacted with tert-butanol in a third reaction to give tert-butyl 3-oxopropionate (compound 5). The tert-butyl 3-oxopropionate and tert-butyl triphenylphosphoacetate were subjected to a fourth reaction to obtain (E / Z)-pent-2-enedioic acid-5-tert-butyl ester-1-methyl ester (compound 6). The (E / Z)-pent-2-enedioic acid-5-tert-butyl-1-methyl ester was reacted with trifluoroacetic acid in a fifth reaction to give (E / Z)-5-methoxy-5-oxopent-3-enoic acid (compound 7). The (E / Z)-5-methoxy-5-oxoprene-3-enoic acid was reacted with oxalyl chloride in a sixth reaction to give methyl (E / Z)-5-chloro-5-oxoprene-2-enoic acid (compound 8). The (E / Z)-5-chloro-5-oxopent-2-enoic acid methyl ester was reacted with propargylamine (compound 9) in a seventh reaction to give (E / Z)-5-oxo-5-(prop-2-yn-1-ylamino)pent-2-enoic acid methyl ester (compound 10). The (E / Z)-5-oxo-5-(prop-2-yn-1-ylamino)pent-2-enoic acid methyl ester was reacted with lithium hydroxide in an eighth reaction to obtain the pentenediaic acid structural analog.

[0024] In this invention, compound 1 and compound 2 undergo a first reaction to obtain compound 3.

[0025] In this invention, the structures of compounds 1-3 are as follows: Compound 1 Compound 2 Compound 3.

[0026] In this invention, the molar ratio of cyclo(isopropyl)malonic acid to trimethyl orthocarbonate is preferably 1:2 to 4, and more specifically, it can be 1:3.

[0027] In this invention, the temperature of the first reaction is preferably 80~90℃, specifically 85℃, and the time is preferably 1.5~2.5h, specifically 2h.

[0028] In this invention, compound 1 is preferably dissolved in orthomethyl trimethyl carbonate, the system is heated to 85°C and maintained at this temperature for 2 hours, and after the reaction is complete as monitored by TLC, it is concentrated to obtain compound 3.

[0029] After obtaining compound 3, the present invention reacts compound 3 with hydrochloric acid in a second reaction to obtain compound 4.

[0030] In this invention, the structure of compound 4 is as follows: .

[0031] In this invention, the temperature of the second reaction is preferably room temperature, and the time is preferably 3 hours.

[0032] In this invention, the preferred ratio of compound 3 to hydrochloric acid is 429.73 mmol: 800 mL, and the preferred concentration of hydrochloric acid is 20-38 wt, specifically 30 wt.

[0033] In this invention, compound 3 is preferably dissolved in hydrochloric acid, reacted at room temperature for 3 hours, and after the reaction is complete as monitored by TLC, the reaction solution is diluted with saturated sodium chloride, extracted with ethyl acetate, dried, and concentrated to obtain compound 4.

[0034] After obtaining compound 4, the present invention reacts compound 4 with tert-butanol in a third reaction to obtain compound 5.

[0035] In this invention, the structure of compound 5 is as follows: .

[0036] In this invention, the preferred ratio of compound 4 to tert-butanol is (250~300) mmol:(30~35) mL, specifically 290.46 mmol:32.5 mL.

[0037] In this invention, the temperature of the third reaction is preferably 95~105℃, specifically 100℃, and the time is preferably 2.5~3.5h, specifically 3h.

[0038] In this invention, compound 4 is preferably dissolved in tert-butanol and toluene, heated at 100°C for 3 hours, and after the reaction is complete as monitored by TLC, saturated sodium chloride is added to wash the reaction solution, and the mixture is dried without concentration to obtain compound 5.

[0039] After obtaining compound 5, the present invention reacts compound 5 with tert-butyl triphenylphosphoacetate in a fourth reaction to obtain compound 6.

[0040] In this invention, the structure of compound 6 is as follows: .

[0041] In this invention, the molar ratio of the triphenylphosphoacetic acid tert-butyl ester to compound 5 is preferably 1:1.

[0042] In this invention, the temperature of the fourth reaction is preferably 80°C, and the time is preferably 3 hours.

[0043] In this invention, tert-butyl triphenylphosphoacetate is added to the reaction solution of compound 5, heated at 80°C for 3 hours, and after the reaction is complete as monitored by TLC, the solution is concentrated and directly mixed and passed through a column to obtain compound 6.

[0044] In this invention, the eluent for column chromatography includes petroleum ether (P) and ethyl acetate (E), wherein the P:E ratio in the eluent is preferably 30:1 (volume ratio).

[0045] After obtaining compound 6, the present invention reacts compound 6 with trifluoroacetic acid in a fifth reaction to obtain compound 7.

[0046] In this invention, the structure of compound 7 is as follows: .

[0047] In this invention, the preferred ratio of compound 6 to trifluoroacetic acid (TFA) is (60~70) mmol:(60~70) mL, specifically 64.92 mmol:65 mL.

[0048] In this invention, the fifth reaction is preferably carried out in dichloromethane (DCM).

[0049] In this invention, the temperature of the fifth reaction is preferably room temperature, and the time is preferably 48 hours.

[0050] In this invention, compound 6 is preferably added to a DCM solution of TFA, reacted at room temperature for 48 hours, and after the reaction is complete as monitored by TLC, it is concentrated to obtain compound 7.

[0051] After obtaining compound 7, the present invention reacts compound 7 with oxalyl chloride in a sixth reaction to obtain compound 8.

[0052] In this invention, the structure of compound 8 is as follows: .

[0053] In this invention, the molar ratio of (E / Z)-5-methoxy-5-oxopram-3-enoic acid to oxaloyl chloride is preferably 1:2 to 4, and more specifically, it can be 1:3.

[0054] In this invention, the temperature of the sixth reaction is preferably room temperature, and the time is preferably 1 hour.

[0055] In this invention, compound 7 is preferably added to a DCM solution of N,N-dimethylformamide (DMF), the system temperature is adjusted to 0°C, oxalyl chloride is added dropwise, the reaction is carried out at room temperature for 1 hour, and after the reaction is complete as monitored by TLC, the mixture is concentrated to obtain compound 8.

[0056] After obtaining compound 8, the present invention performs a seventh reaction with compound 9 to obtain compound 10.

[0057] In this invention, the structures of compounds 9 and 10 are as follows: Compound 9 Compound 10.

[0058] In this invention, the molar ratio of compound 8 to compound 9 is preferably 1:1 to 3, and more specifically, it can be 1:2.

[0059] In this invention, the temperature of the seventh reaction is preferably room temperature, and the time is preferably 1 hour.

[0060] In this invention, propargylamine and triethylamine are preferably added to DCM, the system temperature is adjusted to 0°C, the DCM solution of compound 8 is added dropwise, the reaction is carried out at room temperature for 1 hour, the reaction is monitored by TLC until it is complete, the mixture is concentrated, and the sample is directly mixed and passed through a column to obtain compound 10.

[0061] In this invention, the eluent used for column chromatography includes P and E, wherein the P:E ratio is preferably 4:1 (volume ratio).

[0062] After obtaining compound 10, the present invention reacts compound 10 with lithium hydroxide in an eighth reaction to obtain the pentenediaic acid structural analog.

[0063] In this invention, the molar ratio of compound 10 to lithium hydroxide is preferably 1:1 to 3, and more specifically, it can be 1:2.

[0064] In this invention, the temperature of the eighth reaction is preferably room temperature, and the time is preferably 12 hours.

[0065] In this invention, compound 10 is preferably added to methanol, followed by an aqueous solution of lithium hydroxide. The reaction is carried out at room temperature for 12 hours. After the reaction is complete as monitored by TLC, the mixture is concentrated, back-extracted twice with ethyl acetate, and the pH is adjusted to 3 with 1M HCl. The mixture is then extracted with ethyl acetate again, concentrated, and directly mixed with pure DCM for column chromatography to obtain the pentenediaic acid analogue.

[0066] This invention also provides the application of the pentenediaic acid structural analogs described in the above technical solution in click chemistry.

[0067] In this invention, the click chemistry preferably includes the interaction between pentenoic acid and protein, as well as the glutamate modification of protein (such as PKM2).

[0068] In this invention, the click chemistry is preferably an experiment to screen pentenedioic acid-mutant proteins and an experiment to screen proteins that have undergone pentenedioylation modification.

[0069] This invention also provides a copper-catalyzed azide-alkyne cycloaddition reaction, comprising the following steps: The pentenic acid analogue, Cy3-azide, and copper catalyst described in the above technical solution are subjected to a copper-catalyzed azido-yne cycloaddition reaction.

[0070] The present invention does not impose any special limitations on the specific parameters of the copper-catalyzed azido-alkyne cycloaddition reaction, and any method known to those skilled in the art can be used.

[0071] The present invention also provides the application of the pentenediaic acid structural analog described in the above technical solution in the detection of pyruvate kinase PKM2.

[0072] The present invention also provides the application of the pentenediaic acid structural analogs described in the above technical solution in the preparation of antitumor drugs.

[0073] The present invention also provides a kit comprising the pentenediaic acid structural analogue, Cy3-azide, and copper catalyst described in the above technical solution.

[0074] The present invention does not have any particular limitation on the type of copper catalyst, and any type known to those skilled in the art can be used.

[0075] 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. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0076] Example 1 Preparation of pentenic acid analogues. Figure 1 A schematic diagram of the process for preparing pentenediaic acid analogues, including the following steps: Step 1: Synthesis of 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione Compound 1, cycloisopropyl malonate (555.05 mmol), was dissolved in trimethyl carbonate (1665.16 mmol). The system was heated to 85 °C and maintained at this temperature for 2 h. After the reaction was completed by TLC monitoring, the solution was concentrated to give compound 3 (80 g, 429.73 mmol), a yellow oil. The crude product was not calculated in yield and was used directly in the next step.

[0077] Step 2: Synthesis of 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione Compound 3 (429.73 mmol) was dissolved in hydrochloric acid (800 mL, 30 wt%) and reacted at room temperature for 3 h. After the reaction was complete as monitored by TLC, the reaction solution was diluted with saturated sodium chloride, extracted with ethyl acetate, dried, and concentrated to give compound 4 (50 g, 290.46 mmol, two-step yield 52.63%) as a yellow solid.

[0078] Step 3: Synthesis of tert-butyl 3-oxopropionate Compound 4 (290.46 mmol) was dissolved in tert-butanol (32.5 mL) and toluene (500 mL) and heated at 100 °C for 3 h. After the reaction was complete as monitored by TLC, the reaction solution was washed with saturated sodium chloride, dried, and without concentration, yielded a yellow liquid compound 5 (41.86 g, 290.35 mmol). The crude product was used directly in the next step without yield calculation.

[0079] Step 4: Synthesis of (E / Z)-pentan-2-enedioic acid-5-tert-butyl ester-1-methyl ester 290.35 mmol of tert-butyl triphenylphosphoacetate was added to the reaction solution of compound 5 (290.35 mmol), and the mixture was heated at 80 °C for 3 h. After the reaction was completed by TLC monitoring, the mixture was concentrated and directly passed through a column with a P:E ratio of 30:1 (volume ratio) to obtain a colorless oily compound 6 (13 g, 64.92 mmol, two-step yield 22.36%).

[0080] Step 5: Synthesis of (E / Z)-5-methoxy-5-oxopram-3-enoic acid Compound 6 (64.92 mmol) was added to a solution of TFA (65 mL) and DCM (130 mL), and the reaction was carried out at room temperature for 48 h. After the reaction was completed by TLC monitoring, the mixture was concentrated to give a yellow oily compound 7 (12 g, 83.25 mmol). The yield of the crude product was not calculated.

[0081] Step 6: Synthesis of (E / Z)-5-chloro-5-oxopram-2-enoic acid methyl ester Compound 7 (83.25 mmol) was added to a DCM solution of DMF (120 mL), and the system temperature was adjusted to 0 °C. Oxaloyl chloride (249.77 mmol) was added dropwise. The reaction was allowed to proceed at room temperature for 1 h. After the reaction was confirmed to be complete by TLC, the solution was concentrated to obtain compound 8 (13.53 g, 83.22 mmol). The crude product was not calculated in yield and was directly added to the next step.

[0082] Step 7: Synthesis of (E / Z)-5-oxo-5-(prop-2-yn-1-ylamino)pent-2-enoic acid methyl ester Propyleneamine (166.45 mmol) and triethylamine (249.67 mmol) were added to DCM (200 mL), and the system temperature was adjusted to 0 °C. A DCM solution of compound 8 (83.22 mmol) was then added dropwise. After the reaction was complete, the mixture was concentrated and directly passed through a column (P:E = 4:1) to obtain a blue oily compound 10 (6 g, 33.11 mmol, two-step yield 39.81%).

[0083] Step 8: Synthesis of (E / Z)-5-oxo-5-(prop-2-yn-1-ylamino)pent-2-enoic acid Compound 10 (33.11 mmol) was added to methanol (60 mL), followed by an aqueous solution of lithium hydroxide (66.22 mmol) (30 mL). The mixture was reacted at room temperature for 12 h. After the reaction was completed by TLC monitoring, the mixture was concentrated, back-extracted twice with ethyl acetate, and the pH was adjusted to 3 with 1 M HCl. The mixture was then extracted with ethyl acetate, concentrated, and directly passed through a column of pure DCM to obtain a white solid (2.8 g, 16.75 mmol, two-step yield 45.23%).

[0084] The structures of the synthesized pentenedioic acid analogues were characterized. Figure 2 The chromatogram of the pentenediaic acid structural analog shows that the purity of the obtained pentenediaic acid structural analog is 98 wt%.

[0085] Figure 3 This is a mass spectrum of a pentenedioic acid structural analog.

[0086] The 1H NMR spectra of pentenediic acid analogues are as follows: 1 HNMR (400 MHz, Chloroform-d) δ 6.79 (dt, J = 10.0, 3.7 Hz, 1H), 6.30(dt, J = 10.1. 2.1 Hz, 1H). 4.61 (d, J = 2.5 Hz. 2H). 3.48 (dd, J = 3.7, 2.0 Hz, 2H). 2.15 (t, J =2.5 Hz, IH).

[0087] Pyruvate kinase PKM2 can undergo pentenylation modification. Using PKM2 as a target, we aim to verify the function of Alk-GC and conduct copper-catalyzed azide-alkyne cycloaddition reactions. Figure 4 A schematic diagram of the copper-catalyzed azido-alkyne cycloaddition reaction for Alk-GC.

[0088] Add 1-3 mM of Alk-GC to CD8 + After culturing T cells in culture medium for 6 hours, Cy3-azide was added for click chemistry. Flow cytometry and immunofluorescence both detected dose-dependent uptake of Alk-GC by the cells (see...). Figures 5-6 ), Figure 5 Image a shows the peak distribution of fluorescence intensity from flow cytometry, and image b shows the quantitative statistical bar chart of fluorescence intensity from flow cytometry. Figure 6 The scale bars are all 20 μm. (Activation of CD8) + In T cells and HEK293 cells overexpressing Flag-PKM2, the effects of different concentrations of Alk-GC on the molecular modification level of PKM2 were investigated using a biotinylation immunoprecipitation assay combined with Western blot analysis. The effect of different concentrations of Alk-GC on the biotinylation modification of the Flag-labeled target protein PKM2 was verified using IP-Flag combined with Western blot analysis. A dose-dependent Alk-GC modification of PKM2 was observed in both assays. Figure 7 and8 ), Figure 7 This is an immunoblot image of biotinylated immunoprecipitation-binding protein. Figure 8 This is an immunoblot image of the tag-immunoprecipitation binding protein.

[0089] A cell-free enzymatic reaction system was established in vitro to catalyze Alk-GC modification of PKM2, indicating that this modification was also validated in a cell-free system. Figure 9 and 10 ), Figure 9 This is a flowchart of an in vitro cell-free experiment used to investigate the effects of Alk-GC on the glutarylation modification and catalytic function of PKM2 protein. Figure 10 The image shows the verification experiment of click-through chemical binding immunoblotting, indicating that Alk-GC successfully biotinylated the PKM2 protein.

[0090] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles 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 pentenediaic acid structural analog, characterized in that, It has the structure shown in Equation I: Equation I.

2. The method for preparing the pentenediaic acid structural analogue according to claim 1, characterized in that, Includes the following steps: The first reaction of cyclo(isopropyl)malonic acid with trimethyl orthocarbonate yields 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione. The 5-(methoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione was reacted with hydrochloric acid to give 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione. The 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione was reacted with tert-butanol in a third reaction to give tert-butyl 3-oxopropionate. The tert-butyl 3-oxopropionate and tert-butyl triphenylphosphoacetate were subjected to a fourth reaction to obtain (E / Z)-pent-2-enedioic acid-5-tert-butyl ester-1-methyl ester. The (E / Z)-pent-2-enedioic acid-5-tert-butyl-1-methyl ester was reacted with trifluoroacetic acid in a fifth reaction to obtain (E / Z)-5-methoxy-5-oxopent-3-enoic acid. The (E / Z)-5-methoxy-5-oxopre-3-enoic acid was reacted with oxalyl chloride in a sixth reaction to give methyl (E / Z)-5-chloro-5-oxopre-2-enoic acid. The (E / Z)-5-chloro-5-oxopent-2-enoic acid methyl ester was reacted with propargylamine in a seventh reaction to obtain (E / Z)-5-oxo-5-(prop-2-yn-1-ylamino)pent-2-enoic acid methyl ester. The (E / Z)-5-oxo-5-(prop-2-yn-1-ylamino)pent-2-enoic acid methyl ester was reacted with lithium hydroxide in an eighth reaction to obtain the pentenediaic acid structural analog.

3. The preparation method according to claim 2, characterized in that, The molar ratio of cyclo(isopropyl)malonic acid to trimethyl carbonate is 1:2~4.

4. The preparation method according to claim 2 or 3, characterized in that, The temperature of the first reaction is 80~90℃, and the time is 1.5~2.5h.

5. The preparation method according to claim 2, characterized in that, The ratio of 5-(hydroxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione to tert-butanol is (250~300) mmol:(30~35) mL.

6. The preparation method according to claim 2 or 5, characterized in that, The temperature of the third reaction is 95~105℃, and the time is 2.5~3.5h.

7. The preparation method according to claim 2, characterized in that, The ratio of (E / Z)-pent-2-enedioic acid-5-tert-butyl ester-1-methyl ester to trifluoroacetic acid is (60~70) mmol:(60~70) mL.

8. The application of the pentenediaic acid structural analogue of claim 1 in click chemistry.

9. The application of the pentenediaic acid structural analogue according to claim 1 in the detection of pyruvate kinase PKM2.

10. A reagent kit, characterized in that, It includes the pentenediaic acid structural analogue as described in claim 1, Cy3-azide, and copper catalyst.

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