Targeted cell surface glycoprotein interaction cross-linking agent and preparation method thereof
By developing a targeted crosslinking agent containing alkynyl groups, photoreactive groups and biotin groups, the problem of insufficient targeting and coverage in the research on glycoprotein interactions in the prior art is solved, and efficient and precise capture and analysis of glycoprotein interactions on the cell surface is achieved.
Patent Information
- Application Number
- CN202411815884.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-03-11
AI Technical Summary
When studying the interactions of glycoproteins on the cell surface, the prior art has problems such as inability to enrich, lack of targeting, or inability to target the cell surface, which leads to difficult research and poor results.
A crosslinking agent targeting the interaction of glycoproteins on the surface of the cell is developed. The crosslinking agent contains alkynyl groups, photoreactive groups and sulfonate-containing biotin groups. Through azide-alkynyl click chemistry, photocrosslinking technology and biotin enrichment technology, targeted crosslinking and enrichment of cell surface glycoproteins interactions.
The targeting and coverage of sugar chain cross-linking in glycoprotein interactions is improved, the interference of non-specific binding of proteins in cells is reduced, and deep coverage and precise analysis of the interaction between glycosylated ligands and receptor proteins on the cell surface is achieved.
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Figure CN119591663A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of crosslinking agents, and in particular relates to a crosslinking agent targeting cell surface glycoprotein interaction and a preparation method thereof. Background Art
[0002] Glycoproteins are widely distributed on the cell surface and participate in intercellular recognition and communication. During embryonic development, the interaction of cell surface glycoproteins is essential for cell differentiation, migration, and tissue formation. In addition, many glycoproteins are key components of cell signaling pathways, regulating the transmission and amplification of signals by interacting with other proteins, thereby affecting physiological processes such as cell proliferation, differentiation, and apoptosis (Fangxu S, Suttipong S, Ronghu W, Chem Sci, 2021, 12, 2146). Abnormalities in glycoprotein interactions are closely related to the occurrence and development of a variety of diseases. Glycoproteins themselves can be used as markers for disease diagnosis. Detecting changes in glycoprotein interactions is of great significance for improving the accuracy and sensitivity of disease diagnosis and discovering new drug targets. However, the low abundance of glycoproteins in biological systems and the transient dynamics of their interactions have brought great challenges to the study of glycoprotein interactions.
[0003] Chemical cross-linking technology has the advantages of being able to capture weak or transient protein interactions, high analytical sensitivity, and high throughput analysis, and plays an extremely important role in the field of protein interaction analysis. At present, the research on glycoprotein interactions mainly uses the sugar metabolism labeling method to introduce azide groups into N-linked sialic acid sugar chains on the cell surface, and then uses synthetic cross-linkers and alkynyl-azide click chemistry to covalently cross-link sugar chains with polypeptide backbones. Combined with glycoproteome analysis technology, it can successfully identify multiple pairs of interacting proteins on the cell surface (Wu H, Shajahan A, Yang JY, Capota E, Wands AM, Arthur CM, Stowell SR, Moremen KW, Azadi P, Kohler JJ, CCB, 2022, 29, 84). However, the use of photocrosslinking technology can trigger the reaction by light at a specific time point, quickly stabilize the weak interaction or short-term binding between proteins, and effectively capture the transient interaction. In addition, the photocrosslinking group can react with the residues of 20 kinds of amino acids, greatly improving the coverage of the crosslinking reaction (Yuwen C, Yuxin A, Zhongpeng D, Yi L, Zhen L, Qun Z, Lihua Z, Yukui Z, AnalChim Acta, 2022, 1203, 339694). However, the above methods have problems such as inability to enrich, lack of targeting, or inability to target the cell surface. Summary of the invention
[0004] The present invention aims to solve the above technical problems existing in the prior art and provides a cross-linking agent targeting cell surface glycoprotein interaction and a preparation method thereof.
[0005] The technical solution of the present invention is: a cross-linking agent targeting the interaction between glycoproteins on the cell surface, characterized by the following chemical structure: Where n=1-5.
[0006] A method for preparing the above-mentioned cross-linking agent targeting cell surface glycoprotein interaction is carried out in the following steps: Step 1. Using Boc-L-glutamic acid as the starting material, DMSO (dimethyl sulfoxide) as the reaction solvent, EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) as the condensation agent, an esterification reaction is carried out with NHS (N-hydroxysuccinimide) to obtain compound Ⅰ—Boc-L-glutamic acid disuccinimide ester; Step 2. Compound I is used as a reaction raw material and DMSO is used as a reaction solvent. TEA (triethylamine) is added to react with 3-methyl-3-bis(aziridinyl)methylamine or a primary amine having a photoactive group to undergo a selective amidation reaction to obtain compound II—Boc-L-glutamic acid succinimide ester containing a photoactive group; Step 3. Using compound II as raw material, DMF ( N , N -dimethylformamide) as the reaction solvent, add TEA (triethylamine) to react with the alkynyl primary amine compound with a carbon chain length of n=1-5 to undergo an amidation reaction of one molecule, and then remove the Boc protecting group in TFA (trifluoroacetic acid) to obtain compound III - an L-glutamic acid compound containing a photoactive group and a sugar chain reactive group; Step 4. Compound III was used as the raw material, DMF as the reaction solvent, EDCI as the condensation agent, HoBt (1-hydroxybenzotriazole) as the racemization agent, and DIEA ( N , N -diisopropylethylamine) as an organic base, undergoes an amidation reaction with a compound Biotin-Sulfo-COOH to prepare a target cross-linking agent, wherein the Biotin-Sulfo-COOH is synthesized by reacting biotin-N-succinimidyl ester with 2-amino-3-sulfopropionic acid.
[0007] Preferably, the step 1 is to dissolve Boc-L-glutamic acid, EDCI and NHS in DMSO at a molar ratio of 1:1.1-1.5:2.0-3.0, react with stirring at 22-30°C for 10-24 h, wash with 80-150 mL of distilled water, add 25-35 mL of dichloromethane for extraction, and dry with anhydrous sodium sulfate to remove dichloromethane to obtain crude Boc-L-glutamic acid disuccinimide ester.
[0008] Preferably, the step 2 is to dissolve compound I in DMSO and add a small amount of TEA, then dissolve 3-methyl-3-bis(aziridinyl)methylamine or a primary amine having a photoactive group in DMSO and slowly dropwise add it to the solution of compound I, wherein the molar ratio of compound I to 3-methyl-3-bis(aziridinyl)methylamine or a primary amine having a photoactive group is 1:1; after the dropwise addition, stir at 22-30° C. for 1-4 h, separate and purify the reaction solution through a semi-preparative liquid phase system, and vacuum freeze-dry to obtain Boc-L-glutamate succinimide ester containing a photoactive group.
[0009] Preferably, the step 3 is to dissolve compound II in DMF and add a small amount of TEA, dissolve the alkynyl primary amine compound with a carbon chain length of n=1-5 in DMF and slowly drop it into the solution of compound II, wherein the molar ratio of compound II to the alkynyl primary amine compound with a carbon chain length of n=1-5 is 1:1.1-1.5; after the dropwise addition, stir at 22-30°C for 10-24 h, the reaction solution is separated and purified by a semi-preparative liquid phase system, and vacuum freeze-dried to obtain a Boc-L-glutamic acid compound containing a photoactive group and a sugar chain reaction group; the Boc-L-glutamic acid compound containing a photoactive group and a sugar chain reaction group is dissolved in dichloromethane (DCM), cooled to 0-5°C, TFA is added, and the reaction is stirred for 2-5h. After the reaction is completed, the dichloromethane is removed, and the crude product is further purified by column chromatography using dichloromethane and acetone in a volume ratio of 10-20:1 as a mobile phase to obtain L-glutamic acid succinimide ester containing a photoactive group.
[0010] Preferably, the step 5 is to dissolve compound III, Biotin-Sulfo-COOH, EDCI, HoBt and DIEA in DMF at a molar ratio of 1:1.1-1.4:1.4-2.0:1.4-2.0:2.0-4.0, and stir at 22-30°C for 10-24 h; after the reaction is completed, DMF is removed, and the crude product is further purified by column chromatography using dichloromethane and acetone in a volume ratio of 20-35:1 as the mobile phase to obtain the target cross-linking agent; the Biotin-Sulfo-COOH is prepared according to the following steps: 2-amino-3-sulfopropionic acid is dissolved in DMF and an appropriate amount of TEA is added and stirred evenly, biotin-N-succinimidyl ester is dissolved in DMF and slowly added dropwise to the 2-amino-3-sulfopropionic acid solution, the molar ratio of 2-amino-3-sulfopropionic acid to biotin-N-succinimidyl ester is 1.2-1.4, stirred at 25-30°C for 6-14 h, the reaction solution is poured into a large amount of distilled water, filtered and dried, and the crude product is further purified by column chromatography using dichloromethane and acetone in a volume ratio of 15-20:1 as the mobile phase to obtain Biotin-Sulfo-COOH.
[0011] The cross-linking agent of the present invention contains an alkynyl group that reacts with an azido sugar chain, a photoreactive group, and a biotin group containing a sulfonate group. By using the alkynyl group, a specific cross-linking reaction of glycoproteins is carried out with the help of copper-catalyzed azide-alkynyl click chemistry (CuAAC), which can improve the targeting of sugar chain cross-linking in glycoprotein interactions; by using the photoreactive group, the proteins that interact with glycoproteins in the cell are further cross-linked in situ to improve the coverage of glycoprotein cross-linking; by using the biotin enrichable group containing a sulfonate group, the cross-linking agent has a high degree of biocompatibility, cell membrane targeting, and high selective enrichment ability, so as to achieve targeted cross-linking and enrichment of cell surface glycoprotein interactions and reduce the interference of non-specific binding of intracellular proteins. In this way, deep coverage and precise analysis of the interaction between cell surface glycosylated ligands and receptor proteins can be achieved, providing strong technical support for the study of cell communication processes. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 Schematic diagram of the reaction route of the preparation method of the embodiment of the present invention.
[0013] Figure 2 This is a fluorescence confocal image of the chemical crosslinker in Experimental Example 1 of the present invention.
[0014] Figure 3 This is the mass spectrum of the chemical cross-linking agent of Experimental Example 2 of the present invention. DETAILED DESCRIPTION Example 1
[0015] The reaction route of the method for preparing the cross-linking agent targeting cell surface glycoprotein interaction of the present invention is as follows Figure 1 As shown, follow the steps below: Step 1. Using Boc-L-glutamic acid as the starting material, DMSO (dimethyl sulfoxide) as the reaction solvent, EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) as the condensation agent, an esterification reaction is carried out with NHS (N-hydroxysuccinimide) to obtain compound Ⅰ—Boc-L-glutamic acid disuccinimide ester; Specifically, Boc-L-glutamic acid (1.24 g, 5 mmol), EDCI (1.15 g, 6 mmol), and NHS (1.265 g, 11 mmol) were dissolved in 30 ml DMSO and stirred at 22°C for 16 h. 100 ml of distilled water was added to the reaction solution for washing, and then 30 ml of dichloromethane was added to extract the product. The extraction was performed three times, and the organic phases were combined and dried over anhydrous sodium sulfate. The desiccant was filtered out, and the dichloromethane was removed using a rotary evaporator to obtain compound I, i.e., crude Boc-L-glutamic acid disuccinimidyl ester (1.975 g, yield 89.6%).
[0016] Step 2. Compound I is used as a reaction raw material and DMSO is used as a reaction solvent. TEA (triethylamine) is added to react with 3-methyl-3-bis(aziridinyl)methylamine or a primary amine having a photoactive group to undergo a selective amidation reaction to obtain compound II—Boc-L-glutamic acid succinimide ester containing a photoactive group; Specifically, compound I (441 mg, 1 mmol) was dissolved in 15 ml DMSO, triethylamine (506 mg, 5 mmol) was added, 3-methyl-3-bis(aziridinyl)methylamine (85 mg, 1 mmol) was dissolved in 2 ml DMSO, and the mixture was slowly added dropwise to the solution of compound I. After the addition was completed, the mixed solution was stirred at 22° C. for 2 h, and the reaction solution was separated and purified by a semi-preparative liquid phase system; the semi-preparative liquid phase used phase A as 0.1% TFA / H2O, phase B as acetonitrile, the separation gradient was set to 0-40 min (10-50%, B), the flow rate was 40 mL / min, and the effluent was collected for 22-25 min; the product effluents were combined and freeze-dried at 20-30° C. for 12-24 h to obtain a light yellow oily product (131.5 mg, yield 32%), namely compound II.
[0017] Step 3. Using compound II as raw material, DMF ( N , N -dimethylformamide) as the reaction solvent, add TEA (triethylamine) to react with the alkynyl primary amine compound with a carbon chain length of n=1-5 to undergo an amidation reaction of one molecule, and then remove the Boc protecting group in TFA (trifluoroacetic acid) to obtain compound III - an L-glutamic acid compound containing a photoactive group and a sugar chain reactive group; Specifically, compound II (141 mg, 0.32 mmol) was dissolved in 20 ml DMF, triethylamine (162 mg, 1.6 mmol) and alkynylpropylamine (26.4 mg, 0.48 mmol) were dissolved in 2 ml DMF and slowly added to the compound II solution, and stirred at 22 ° C for 12 h. The reaction solution was separated and purified by a semi-preparative liquid phase system, wherein the semi-preparative liquid phase used phase A as H2O and phase B as acetonitrile, and the separation gradient was set to 0-30 min (20-50%, B), the flow rate was 50 mL / min, and the effluent of 25-27 min was collected. The product effluent was combined, placed in a vacuum freeze dryer, and freeze-dried at 20-30 ° C for 12-24 h to obtain a Boc-L-glutamic acid compound containing a photoactive group and a sugar chain reaction group (70.2 mg, yield 63%). The Boc-L-glutamic acid compound containing a photoactive group and a sugar chain reactive group was redissolved in 20 ml of dichloromethane, cooled to 0°C, TFA (2.0 g, 17.5 mmol) was added, and the reaction was stirred for 3 h. The dichloromethane was removed by rotary evaporation. The crude product was further purified by column chromatography using acetone: dichloromethane = 1: 15 (volume ratio) as the mobile phase. The organic phase was removed by rotary evaporation to obtain a light yellow oily product (42.82 mg, yield 85.3%), namely compound III.
[0018] Step 4. Compound III was used as the raw material, DMF as the reaction solvent, EDCI as the condensation agent, HoBt (1-hydroxybenzotriazole) as the racemization agent, and DIEA ( N , N -diisopropylethylamine) as an organic base, undergoes an amidation reaction with a compound Biotin-Sulfo-COOH to prepare a target cross-linking agent, wherein the Biotin-Sulfo-COOH is synthesized by reacting biotin-N-succinimidyl ester with 2-amino-3-sulfopropionic acid.
[0019] Specifically, compound III (42.82 mg, 0.17 mmol), Biotin-Sulfo-COOH (79 mg, 0.20 mmol), EDCI (46 mg, 0.24 mmol), HoBt (35 mg, 0.26 mmol) and DIEA (66 mg, 0.51 mmol) were dissolved in 30 ml of DMF, stirred at 25 °C for 14 h, and after the reaction was completed, DMF was removed by rotary evaporation. The crude product was further purified by column chromatography using acetone: dichloromethane = 1:20 (volume ratio) as the mobile phase, and the organic phase was removed by rotary evaporation to obtain the target cross-linking agent (36.3 mg, yield 34%). The Biotin-Sulfo-COOH is prepared according to the following steps: 2-amino-3-sulfopropionic acid is dissolved in DMF and an appropriate amount of TEA is added and stirred evenly, biotin-N-succinimidyl ester is dissolved in DMF and slowly added dropwise to the 2-amino-3-sulfopropionic acid solution, the molar ratio of 2-amino-3-sulfopropionic acid to biotin-N-succinimidyl ester is 1.3, and the mixture is stirred at 30° C. for 6 h. The reaction solution is poured into a large amount of distilled water, filtered and dried, and the crude product is further purified by column chromatography using dichloromethane and acetone in a volume ratio of 20:1 as the mobile phase to obtain Biotin-Sulfo-COOH.
[0020] The cross-linking agent structural formula of the present invention is as follows: Example 2
[0021] The target cross-linking agent was obtained in the same manner as in Example 1, except that (4-azidophenyl)methanamine hydrochloride (148 mg, 1 mmol) was used instead of 3-methyl-3-bis(aziridinyl)methanamine (85 mg, 1 mmol) in step 2 to obtain a light yellow oily product (173 mg, yield 36.5%).
[0022] Embodiment 3: The target cross-linking agent was obtained in the same manner as in Example 1, except that 4-(aminomethyl)benzophenone (211 mg, 1 mmol) was used instead of 3-methyl-3-bis(aziridinyl)methylamine (85 mg, 1 mmol) in step 2 to obtain a light yellow oily product (150 mg, yield 28%).
[0023] Experimental Example 1: The crosslinker prepared in Example 1 was applied to A549 cells, and the cells were first labeled with N-azidoacetylmannosamine (ManNAz). The specific experimental method was to prepare a crosslinker stock solution with a final concentration of 5 mM, the solvent was PBS, added to A549 cells washed with PBS, and chemically crosslinked for 10 minutes. After the reaction was completed, 1 mL of 10% (v / v) formaldehyde solution was added to the cells, and the cells were fixed for 15 minutes. 1 mL of 0.1% triton X-100 was added to the cells and perforated for 60 minutes, incubated with 1 mL of avidin bonded with fluorescein for 1 hour, washed five times with PBS, and developed by fluorescence confocal fluorescence. The results are shown in Figure 2 .from Figure 2 It can be clearly observed that the cell surface is green fluorescence, while the blank group has no fluorescence, which indicates that the cross-linking agent of the present invention is successful and does not penetrate the cell membrane, providing a basis for the large-scale analysis of the in situ glycoprotein complex structure and its interaction on the cell membrane surface.
[0024] Experimental Example 2: The crosslinker prepared in Example 1 was applied to A549 cells. The specific experimental method was to dissolve the crosslinker in Example 1 in PBS to prepare a crosslinker master solution with a final concentration of 5 mM, add it to Hela cells washed with PBS, and incubate at room temperature for 10 min to complete the crosslinking reaction. 1 mL of 8 M urea and 50 mM TEAB lysis buffer were added to disrupt the cells, centrifuged at 12000 g, denatured and reduced alkylated proteins, digested by enzymes, enriched with streptavidin magnetic beads, desalted, and analyzed by Lumos Fusion LC-MS / MS. The mass spectrum is shown in Figure 1. Figure 3 The data were processed with pLink2 and 1% FDR, and finally 1578 cross-linked peptides of 457 cell surface proteins and 890 pairs of interacting glycoprotein cross-linking information formed by them were identified, proving that the cross-linking agent of the present invention can achieve in situ capture and high coverage identification of glycoproteins on the surface of living cells.
Claims
1. A cross-linking agent targeting cell surface glycoprotein interactions, characterized in that The chemical structure is as follows: Where n=1-5.
2. A method for preparing a cross-linking agent targeting cell surface glycoprotein interaction as claimed in claim 1, characterized in that Follow the steps below: Step 1. Using Boc-L-glutamic acid as the starting material, DMSO as the reaction solvent, and EDCI as the condensation agent, an esterification reaction is carried out with NHS to obtain compound Ⅰ—Boc-L-glutamic acid disuccinimidyl ester; Step 2. Compound I is used as a reaction raw material and DMSO is used as a reaction solvent, and TEA is added to react with 3-methyl-3-bis(aziridinyl)methylamine or a primary amine having a photoactive group to undergo a selective amidation reaction to obtain compound II—Boc-L-glutamic acid succinimide ester containing a photoactive group; Step 3. Compound II is used as a raw material and DMF is used as a reaction solvent. TEA is added to react with an alkynyl primary amine compound with a carbon chain length of n=1-5 to undergo an amidation reaction of one molecule, and then the Boc protecting group is removed in TFA to obtain compound III—an L-glutamic acid compound containing a photoactive group and a sugar chain reactive group; Step 4. Compound III is used as a raw material, DMF is used as a reaction solvent, EDCI is used as a condensing agent, HoBt is used as a racemizing agent, and DIEA is used as an organic base to undergo an amidation reaction with the compound Biotin-Sulfo-COOH to prepare a target cross-linking agent. The Biotin-Sulfo-COOH is synthesized by reacting biotin-N-succinimidyl ester with 2-amino-3-sulfopropionic acid.
3. The method for preparing a target for cell surface glycoprotein interaction according to claim 2, characterized in that: The step 1 is to dissolve Boc-L-glutamic acid, EDCI and NHS in DMSO at a molar ratio of 1:1.1-1.5:2.0-3.0, stir and react at 22-30° C. for 10-24 h, wash with 80-150 mL of distilled water, add 25-35 mL of dichloromethane for extraction, and dry with anhydrous sodium sulfate to remove dichloromethane, so as to obtain crude Boc-L-glutamic acid disuccinimide ester.
4. The method for preparing a target for cell surface glycoprotein interaction according to claim 3, characterized in that: The step 2 is to dissolve compound I in DMSO and add TEA, then dissolve 3-methyl-3-bis(aziridinyl)methylamine or a primary amine having a photoactive group in DMSO and slowly dropwise add the solution of compound I, wherein the molar ratio of compound I to 3-methyl-3-bis(aziridinyl)methylamine or a primary amine having a photoactive group is 1:1; after the dropwise addition, stir at 22-30° C. for 1-4 h, separate and purify the reaction solution through a semi-preparative liquid phase system, and vacuum freeze-dry to obtain Boc-L-glutamic acid succinimide ester containing a photoactive group.
5. The method for preparing a target for cell surface glycoprotein interaction according to claim 4, characterized in that: The step 3 is to dissolve compound II in DMF and add TEA, dissolve an alkynyl primary amine compound with a carbon chain length of n=1-5 in DMF and slowly drip it into the solution of compound II, wherein the molar ratio of compound II to the alkynyl primary amine compound with a carbon chain length of n=1-5 is 1:1.1-1.5; after the dripping, stir at 22-30°C for 10-24 hours, separate and purify the reaction solution through a semi-preparative liquid phase system, and freeze-dry in vacuum to obtain a Boc-L-glutamic acid compound containing a photoactive group and a sugar chain reaction group; dissolve the Boc-L-glutamic acid compound containing a photoactive group and a sugar chain reaction group in dichloromethane, cool to 0-5°C, add TFA, stir and react for 2-5 hours, terminate the reaction, remove dichloromethane, and further purify the crude product through column chromatography using dichloromethane and acetone in a volume ratio of 10-20:1 as the mobile phase to obtain L-glutamic acid succinimide ester containing a photoactive group.
6. The method for preparing a target for cell surface glycoprotein interaction according to claim 5, characterized in that: The step 5 is to dissolve compound III, Biotin-Sulfo-COOH, EDCI, HoBt, and DIEA in DMF at a molar ratio of 1:1.1-1.4:1.4-2.0:1.4-2.0:2.0-4.0, and stir at 22-30° C. for 10-24 h; after the reaction is completed, DMF is removed, and the crude product is further purified by column chromatography using dichloromethane and acetone in a volume ratio of 20-35:1 as the mobile phase to obtain the target cross-linking agent; The Biotin-Sulfo-COOH is prepared according to the following steps: 2-amino-3-sulfopropionic acid is dissolved in DMF and TEA is added and stirred evenly, biotin-N-succinimidyl ester is dissolved in DMF and slowly added dropwise to the 2-amino-3-sulfopropionic acid solution, the molar ratio of 2-amino-3-sulfopropionic acid to biotin-N-succinimidyl ester is 1.2-1.4, stirring at 25-30° C. for 6-14 h, pouring the reaction solution into distilled water, filtering and drying, and further purifying the crude product by column chromatography using dichloromethane and acetone in a volume ratio of 15-20:1 as the mobile phase to obtain Biotin-Sulfo-COOH.