Novel enriched photo-crosslinking agent as well as preparation method and application thereof

Abstract

The invention provides a novel enriched photo-crosslinking agent, a preparation method of the novel enriched photo-crosslinking agent and application of the novel enriched photo-crosslinking agent in protein conformation and protein interaction analysis. The cross-linking agent can be subjected to a cross-linking reaction with protein at the moment of illumination, a cross-linked product can be enriched through click chemistry-biotin streptavidin, then steric hindrance of the cross-linked product is reduced by cutting azido biotin groups, and the mass spectrometric detection sensitivity and specificity of the cross-linked product are effectively improved.

Description

Novel photocrosslinking agents with enrichment capabilities, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of compounds, specifically relating to a novel photocrosslinking agent that can be enriched, its preparation method, and its application. Background Technology

[0002] As the primary executors of life activities, proteins function not only through their static three-dimensional structure but also through dynamic conformational changes and complex interaction networks under physiological conditions. Accurately elucidating the dynamic changes in proteins is crucial for revealing the molecular mechanisms of core life processes such as cell signal transduction and metabolic regulation, as well as the pathological mechanisms of disease development. Furthermore, in the field of innovative drug development, proteins and their complexes are key target molecules for drug action; elucidating their structure and interaction networks can provide crucial theoretical support for innovative drug design.

[0003] Currently, mainstream techniques for protein structure determination include X-ray crystallography, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy. These techniques can obtain static structural information at near atomic resolution, but they also have inherent limitations, such as stringent requirements for sample purity, homogeneity, and quantity; lengthy experimental procedures; poor biocompatibility; and difficulty in capturing conformational dynamics in solution or within cells. Although artificial intelligence-based computational prediction methods (such as the AlphaFold series) have achieved breakthroughs in predicting the static structure of proteins, their accuracy and reliability still need improvement in predicting flexible regions of multi-domain proteins, conformational dynamics, and the dynamic assembly processes of protein complexes.

[0004] Chemical cross-linking mass spectrometry (CCSS) is an emerging protein structure resolution technique. This technique introduces a chemical cross-linking agent to covalently link the side chains of two spatially close amino acids. Following enzymatic digestion, mass spectrometry identification, and bioinformatics analysis, the cross-linking sites are identified. Combined with spatial distance constraints and computer simulations, three-dimensional protein structure information can be provided. The significant advantages of this technique are low sample requirements, high throughput, and high biocompatibility. Currently, several commercially available cross-linking agents are available, primarily targeting amino, carboxyl, and thiol groups. Their specificity limits the resolution of protein structure resolution. Furthermore, the cross-linking reaction is time-consuming, making it difficult to accurately capture dynamic conformational changes in proteins. Simultaneously, the abundance and ionization efficiency of cross-linked products are weaker than those of non-cross-linked linear peptides, making them easily missed during detection, thus affecting the resolution and coverage of structure resolution.

[0005] In summary, there is an urgent need in this field for a new cross-linking agent that possesses the characteristics of rapid cross-linking reaction and enrichment of cross-linking products, in order to capture the dynamic conformation of proteins while improving the detection sensitivity of cross-linking products. Summary of the Invention

[0006] The purpose of this invention is to provide a novel photocrosslinking agent that can enrich proteins, as well as a method for preparing the crosslinking agent and its application. The crosslinking agent can undergo a crosslinking reaction with proteins instantaneously upon light exposure, and the crosslinking product can be enriched and purified by alkynyl-azide click chemistry combined with biotin-streptavidin purification, thereby improving the ability to resolve protein dynamic conformations and interaction networks.

[0007] The technical solution of this invention is as follows: The first objective of this invention is to provide a compound, the structural formula of which is: .

[0008] A second objective of this invention is to provide a method for preparing the aforementioned compound, wherein the reaction route is as follows: .

[0009] Specifically, the preparation method includes the following steps: S1, adding propargylamine, tert-butyl bromoacetate, and potassium carbonate to acetonitrile, stirring, and refluxing; extracting the reaction solution with water and ethyl acetate, combining the organic phases, drying with anhydrous sodium sulfate, and performing silica gel column chromatography to obtain compound 1; S2, dissolving compound 1 in a mixed solution of trifluoroacetic acid and dichloromethane, stirring at room temperature, and after the reaction is complete, evaporating the solvent to obtain crude 2,2'-(1-propynylazanediyl)diacetic acid; dissolving crude 2,2'-(1-propynylazanediyl)diacetic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1-hydroxybenzotriazole in N,N-dimethylformamide, stirring, adding 3-methyl-3H-bisacrylidine-3-ethylamine, stirring, extracting the reaction solution with water and ethyl acetate, combining the organic phases, drying with anhydrous sodium sulfate, and performing silica gel column chromatography to obtain the compound; the structural formula of compound 1 is as follows: .

[0010] A third objective of this invention is to provide the application of the aforementioned compound in the preparation of enrichable photocrosslinking agents for protein conformation or protein interactions, characterized in that the crosslinked product obtained after the compound crosslinks with a protein can be enriched based on the biotin-streptavidin purification reaction combined with alkynyl-azide click chemistry, thereby improving the detection sensitivity and specificity of the crosslinked product and obtaining protein conformation or protein interactions.

[0011] Furthermore, the application includes the following steps: (1) Cross-linking reaction: the compound is dissolved in DMSO and mixed with protein at a mass ratio of 1:1, and a cross-linking reaction occurs under laser irradiation; after enzymatic hydrolysis, a peptide mixture is obtained.

[0012] (2) Enrichment of cross-linked products: The peptide mixture obtained in (1) was reacted sequentially with biotinylated azide, copper sulfate, ascorbic acid, and tris(3-hydroxypropyltriazine)amine (THPTA) at 60°C for 2 h; the reacted system was incubated with streptavidin agarose resin at room temperature for 2 h, and finally the target component was eluted and detected using elution buffer; furthermore, the protein in (1) is a pure protein or a complex system including the protein.

[0013] Protein complex systems, such as proteins or protein complexes in cell or tissue lysates.

[0014] Furthermore, the final molar concentration ratio of each reagent in (2) is the compound: azide biotin: copper sulfate: THPTA: ascorbic acid = 1:1:2:1:5.

[0015] Azide biotin is N-[2-[2-[2-[2-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothiopheno[3,4-d]imidazol-4-yl]valerylamino]ethoxy]ethoxy]ethoxy]ethyl]-4-[[5-(2-azidoethyl)-2-hydroxyphenyl]azo]benzamide.

[0016] Furthermore, (2) the elution buffer is 300 mM Na2S2O4, 6 M urea and 2 M thiourea dissolved in 20 mM HEPES, pH 7.5.

[0017] Furthermore, in order to capture the transient conformation of proteins and the transient interactions between proteins, (1) also includes the step of rapidly freezing the sample before the cross-linking reaction occurs under laser irradiation.

[0018] Preferably, the quick-freezing method is liquid nitrogen quick-freezing.

[0019] Furthermore, the wavelength of the laser is 355 nm.

[0020] Furthermore, the laser irradiation time is 60 seconds. Beneficial effects

[0021] (1) The novel photocrosslinking agent used in this invention has no reactive selectivity to amino acid residues and can obtain high-resolution structural information.

[0022] (2) The photocrosslinking agent used in this invention has instantaneous crosslinking ability and can capture the instantaneous state of the protein or the protein conformational changes and protein interaction information at continuous time points.

[0023] (3) The cross-linked products obtained by the novel enrichable photocrosslinking agent used in this invention will generate reporter ions during the secondary mass spectrometry fragmentation process, which can improve the reliability of the identification of cross-linked products.

[0024] (4) The novel enrichable photocrosslinking agent used in this invention achieves enrichment of crosslinking products through highly selective chemical reaction, removes interference from non-crosslinked peptides, and improves detection sensitivity and identification reliability.

[0025] (5) After enrichment, the azide biotin group can be cleaved, reducing the steric hindrance of the cross-linked product, which is beneficial for the subsequent mass spectrometry detection of the cross-linked product. Attached Figure Description

[0026] Figure 1 shows the mass spectrum and corresponding secondary mass spectrum of the novel enrichable photocrosslinking agent, where: Figure 1A is the mass spectrum of the novel enrichable photocrosslinking agent; Figure 1B is the secondary mass spectrum of the novel enrichable photocrosslinking agent.

[0027] Figure 2 shows the cross-linking mass spectrum of the novel enrichable photocrosslinking agent and the model peptide, as well as the corresponding secondary mass spectrum. In Figure 2A, the cross-linking mass spectrum of the novel enrichable photocrosslinking agent and the model peptide; and Figure 2B, the secondary mass spectrum of the cross-linking of the novel enrichable photocrosslinking agent and the model peptide.

[0028] Figure 3 is a schematic diagram of the reaction of the novel enrichable photocrosslinking agent.

[0029] Figure 4 shows the click reaction of the novel enrichable photocrosslinking agent and the elution mass spectra after streptavidin enrichment, where: Figure 4A is the mass spectrum of the click reaction of the novel enrichable photocrosslinking agent; Figure 4B is the mass spectrum of streptavidin enrichment and elution after the click reaction of the novel enrichable photocrosslinking agent.

[0030] Figure 5 shows the 3D structural diagrams of the crosslinking results obtained before and after enrichment of the crosslinking product mapped to BSA, where: Figure 5A is the crosslinking result obtained before enrichment; Figure 5B is the crosslinking result obtained after enrichment. Detailed Implementation

[0031] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0032] Experimental methods not specifying specific conditions in the embodiments of this disclosure are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, conventional reagents.

[0033] Q-TOF mass spectrometry (Waters, SYNAPT G2-Si) is used to detect the reaction products of model peptides and cross-linking agents.

[0034] The Orbitrap Eclipse Tribrid mass spectrometer is used to detect protein cross-linking products.

[0035] The laser instrument used has the following parameters and model: wavelength: 355 nm, 120 μJ, Beijing Laserwave Optoelectronics Technology Co., Ltd. Example

[0036] .

[0037] (1) Synthesis of compound 1: Propylene acetate (55 mg, 1 mmol), tert-butyl bromoacetate (429 mg, 2.2 mmol) and potassium carbonate (276 mg, 2 mmol) were added to a 25 mL round-bottom flask, followed by 10 mL of acetonitrile. The mixture was stirred and refluxed overnight. The reaction mixture was extracted with water (10 mL) and ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain compound 1.

[0038] (2) Synthesis of Compound 2: Compound 1 (283 mg, 1 mmol) was dissolved in 10 mL of a mixed solution of trifluoroacetic acid and dichloromethane (volume ratio 1:2). The mixture was stirred at room temperature and monitored by TLC until the reaction was complete. The solvent was evaporated, and 6 mL × 3 mL of dichloromethane was added to dissolve the compound. The solvent was then evaporated again to obtain crude 2,2'-(1-propynylazanediyl)diacetic acid. Crude 2,2'-(1-propynylazanediyl)diacetic acid (1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (288 mg, 1.5 mmol), and 1-hydroxybenzotriazole (203 mg, 1.5 mmol) were dissolved in 2 mL of N,N-dimethylformamide and stirred for 15 min. 3-methyl-3H-bisacrylidine-3-ethylamine (217 mg, 2.2 mmol) was added and the mixture was stirred at room temperature for 18 h. The reaction mixture was extracted with water (10 mL) and ethyl acetate (10 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the photocrosslinking agent was obtained by silica gel column chromatography. The corresponding molecular weight and structural information were verified by mass spectrometry, as shown in Figures 1A and 1B. Example

[0039] The model peptide (amino acid sequence: SSTNVG) was precisely weighed, and a 2 mM peptide stock solution was prepared using deionized water and allowed to stand at room temperature for 15 h. The novel photocrosslinking agent was precisely weighed, and a 30 mM stock solution was prepared using DMSO. After mixing with the peptide (peptide:crosslinking agent = 1:10, molar ratio), the mixture was transferred to an opaque glass inner tube, flash-frozen in liquid nitrogen, and then irradiated with laser for 60 s. The reaction products were detected using Q-TOF mass spectrometry (Waters, SYNAPT G2-Si). The main mass spectrometry parameters were set as follows: positive ion mode; capillary voltage 3 kV; cone voltage 40 V; compensation voltage 80 V; desolvation temperature 450 °C; ion source temperature 100 °C; scan time 0.5 s; mass range of m / z 50-2000.

[0040] The product obtained after crosslinking the crosslinking agent with the model peptide is shown in Figure 2A. The mass spectrometry signal of the peptide (m / z 564.26) and the corresponding crosslinking product (m / z 841.45) were detected, with a molecular weight difference of 277.19 Da, consistent with the molecular weight of the designed crosslinking agent after the loss of diacaridine at both ends. Secondary mass spectrometry analysis of m / z 841.45 (Figure 2B) revealed a highly abundant fragment ion (m / z 278.19), the same molecular weight as the residue after the loss of diacaridine in the crosslinking agent, presumably due to the breakage of the crosslinking agent. The crosslinking results indicate that the designed crosslinking agent has transient crosslinking capability, and the resulting crosslinked product can generate a reporter ion (m / z 278.19) in secondary mass spectrometry. Other fragment ions (m / z 337.22, 353.22, 452.26, 667.35, 766.42) all match the theoretical fragmentation ions. Example

[0041] (1) Alkyne-azide click chemistry: 2 mM crosslinking agent, 20 mM biotin azide, and 100 mM THPTA were prepared using DMSO. 100 mM copper sulfate and 100 mM ascorbic acid were prepared using ultrapure water. 200 μL of the crosslinking agent stock solution was taken, and biotin azide was added sequentially to a final concentration of 2 mM, copper sulfate to a final concentration of 4 mM, THPTA to a final concentration of 2 mM, and ascorbic acid to a final concentration of 10 mM. After vortex mixing, the mixture was reacted at 60℃ for 2 h. Biotin azide and the alkynyl group of the crosslinking agent were linked via a copper-catalyzed azide-alkynyl cycloaddition reaction.

[0042] (2) After the reaction, the sample was incubated with streptavidin resin (manufacturer: Thermo Scientific, trade name: Pierce high-capacity streptavidin agarose) at room temperature for 2 hours. After incubation, the sample was eluted twice for 15 minutes each time with elution buffer to cleave the nitrogen-nitrogen double bonds in azide biotin. The elution buffer consisted of 300 mM Na2S2O4, 6 M urea, and 2 M thiourea dissolved in 20 mM HEPES, pH 7.5.

[0043] The reaction products were detected using Q-TOF mass spectrometry (Waters, SYNAPT G2-Si), under the same mass spectrometry conditions as in Example 2.

[0044] The product structure after the crosslinking agent undergoes a click chemistry reaction is shown in Figure 3. A mass spectrometry signal of m / z 1045.53 can be detected, which is consistent with the molecular weight after the crosslinking agent reacts with biotin azide, indicating that the click chemistry reaction was successful. The results are shown in Figure 4A.

[0045] After enrichment and purification with streptavidin and elution with buffer to cleave azide-biotin, the structure of the obtained product is shown in Figure 3. The mass spectrometry signal m / z 512.30 matches the molecular weight of the enriched and cleaved product, indicating that this method successfully applies the click chemistry and streptavidin enrichment and purification strategy. The results are shown in Figure 4B. Example

[0046] This embodiment uses BSA protein as an example. After cross-linking, it is mixed with HeLa cell lysate to simulate a complex system, and the feasibility of enriching the cross-linked product obtained after cross-linking the protein with the photocrosslinking agent in the complex system is investigated.

[0047] HeLa cell lysis buffer was chosen as a complex system. HeLa cells were cultured in MEM medium containing 10% fetal bovine plasma in a cell culture incubator at 37°C and 5% CO2. After trypsin digestion, digestion was terminated with medium containing 10% fetal bovine serum, cells were aspirated, and centrifuged at 800 g for 5 min to obtain cell pellet. The cell pellet was resuspended in PBS, and then sonicated to obtain HeLa cell lysis buffer, followed by centrifugation at 13000 g for 10 min to remove cell debris.

[0048] Prepare a 75 μM BSA protein solution and a 30 mM cross-linking agent stock solution using DMSO. Mix the protein and cross-linking agent at a ratio of 1:1 (mass ratio) until homogeneous, transfer to an opaque glass inner tube, flash freeze in liquid nitrogen, and irradiate under a 355 nm laser for 60 s. After cross-linking, mix with the HeLa lysis buffer obtained above. The protein obtained by acetone precipitation is then subjected to enzymatic hydrolysis followed by click chemistry and enrichment purification elution, following the same steps as in Example 3.

[0049] After proteomic sample processing, crosslinking products were detected using Orbitrap Eclipse Tribrid mass spectrometry, and crosslinked peptides were identified using xiSEARCH (version 1.7.6.7). The parameters were set as follows: crosslinker mass was set to 290.1994 Da, the error range for primary precursor ions and secondary fragment ions was 10 ppm, the enzyme was selected as Trypsin, the fixed modification was set to Carbamidomethylation (C), the variable modification was set to Oxidation (M), the minimum peptide contained 6 amino acids, the maximum peptide mass was 5000, the maximum values ​​of PSM FDR, Peptidepair FDR, Residuepair FDR and Proteinpairs FDR were all set to 5, and the boost was selected as Residue pairs.

[0050] The enrichment results of the cross-linking products are shown in Figure 5. Without enrichment treatment, only 5 cross-linking products could be detected in the compound of this invention (Figure 5A). After enrichment and purification, the number of detectable cross-linking products increased to 16, a significant improvement (Figure 5B).

[0051] The above results indicate that the novel photocrosslinking agent of the present invention, based on achieving instantaneous crosslinking, can specifically enrich crosslinking products by using a click chemistry reaction combined with streptavidin purification strategy, eliminating interference from non-crosslinked peptides, and thus improving the ability to identify low-abundance crosslinking products.

[0052] 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 compound, characterized in that, The structural formula of the compound is: 。 2. The method for preparing the compound according to claim 1, characterized in that, The preparation method includes the following steps: S1, adding propargylamine, tert-butyl bromoacetate, and potassium carbonate to acetonitrile, stirring, and refluxing; extracting the reaction solution with water and ethyl acetate, combining the organic phases, drying with anhydrous sodium sulfate, and obtaining compound 1 by silica gel column chromatography; S2, dissolving compound 1 in a mixed solution of trifluoroacetic acid and dichloromethane, stirring at room temperature, and drying the solvent after the reaction to obtain crude 2,2'-(1-propargylazanediyl)diacetic acid; dissolving crude 2,2'-(1-propargylazanediyl)diacetic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1-hydroxybenzotriazole in N,N-dimethylformamide, stirring, adding 3-methyl-3H-bisacrylidine-3-ethylamine, stirring, extracting the reaction solution with water and ethyl acetate, combining the organic phases, drying with anhydrous sodium sulfate, and obtaining the compound by silica gel column chromatography; the structural formula of compound 1 is as follows. 。 3. The use of the compound of claim 1 in the preparation of photocrosslinking agents that can enrich protein conformations or protein interactions, characterized in that, The cross-linked products obtained after the compound cross-links with the protein can be enriched based on the biotin-streptavidin purification reaction using alkynyl-azide click chemistry to obtain protein conformation or protein-protein interactions.

4. The application according to claim 3, characterized in that, The application The steps include: (1) Cross-linking reaction: The compound described in claim 1 is dissolved in DMSO and mixed with protein at a mass ratio of 1:1, and a cross-linking reaction occurs under laser irradiation; After enzymatic hydrolysis, a peptide mixture was obtained; (2) enrichment of cross-linked products: the peptide mixture obtained in (1) was reacted sequentially with azide biotin, copper sulfate, ascorbic acid, tris(3-hydroxypropyltriazine)amine (THPTA) at 60°C for 2 h; the reaction system was incubated with streptavidin agarose resin at room temperature for 2 h, and finally the target components were eluted and detected using elution buffer.

5. The application according to claim 4, characterized in that, (1) The protein is a pure protein or a complex system including the protein.

6. The application according to claim 4, characterized in that, (2) The ratio of the final molar concentrations of each reagent is the compound described in claim 1: azide biotin: copper sulfate: THPTA: ascorbic acid = 1:1:2:1:

5.

7. The application according to claim 4, characterized in that, (2) The elution buffer is 300 mM Na2S2O4, 6 M urea and 2 M thiourea dissolved in 20 mM HEPES, pH 7.

5.

8. The application according to claim 4, characterized in that, (1) It also includes the step of quick-freezing the sample before the cross-linking reaction occurs under laser irradiation; preferably, the quick-freezing method is liquid nitrogen quick-freezing.

9. The application according to claim 4, characterized in that, The wavelength of the laser is 355 nm.

10. The application according to claim 4, characterized in that, The laser irradiation time is 60 seconds.

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