CN119591663A

CN119591663A - A cross-linking agent targeting cell surface glycoprotein interaction and preparation method thereof

Info

Publication number: CN119591663A
Application number: CN202411815884.6A
Authority: CN
Inventors: 吴慈; 刘兵; 王欣月; 林瑶
Original assignee: Liaoning Normal University
Current assignee: Liaoning Normal University
Priority date: 2024-12-11
Filing date: 2024-12-11
Publication date: 2025-03-11

Abstract

The invention discloses a targeting cell surface glycoprotein interaction cross-linking agent and a preparation method thereof, wherein the prepared cross-linking agent simultaneously contains alkynyl groups reacting with azido sugar chains, photoreactive groups and biotin groups containing sulfonate groups. The method has the advantages of improving the targeting property of sugar chain crosslinking in glycoprotein interaction by utilizing alkynyl groups, further carrying out in-situ crosslinking on proteins interacting with the glycoprotein in cells by utilizing photoreactive groups, improving the glycoprotein crosslinking coverage, and reducing the interference of nonspecific binding of intracellular proteins by utilizing sulfonate-containing biotin enrichment groups to ensure that the crosslinking agent has high biocompatibility, cell membrane targeting property and high selective enrichment capability. Therefore, the deep coverage and the accurate analysis of the interaction between the cell surface glycosylated ligand and the receptor protein can be realized, and powerful technical support is provided for the research of the cell communication process.

Description

Targeting cell surface glycoprotein interaction cross-linking agent and preparation method thereof

Technical Field

The invention belongs to the technical field of cross-linking agents, and particularly relates to a targeting cell surface glycoprotein interaction cross-linking agent and a preparation method thereof.

Background

Glycoproteins are widely distributed on the cell surface and are involved in recognition and communication between cells. During embryonic development, interactions of cell surface glycoproteins are critical for cell differentiation, migration, and tissue formation. In addition, many glycoproteins are key components of the cell signaling pathway, regulating signal transmission and amplification by interaction with other proteins, thereby affecting physiological processes such as proliferation, differentiation, and apoptosis of cells (Fangxu S, suttipong S, ronghu W, chem Sci, 2021, 12, 2146). The abnormality of glycoprotein interaction is closely related to the occurrence and development of various diseases, glycoprotein can be used as a marker for disease diagnosis, and the glycoprotein can be used as a marker for disease diagnosis by detecting the change of glycoprotein interaction, thereby having great significance for improving the accuracy and sensitivity of disease diagnosis and discovering new drug targets. However, the low abundance nature of glycoproteins in biological systems and the transient dynamics of their interactions present a great challenge to the study of glycoprotein interactions.

The chemical crosslinking technology has the advantages of weak or transient protein interaction capture, high analysis sensitivity, high flux analysis and the like, and plays an extremely important role in the field of protein interaction analysis. At present, the research on glycoprotein interaction is reported that an azide group is mainly introduced onto a cell surface N-linked sialic acid sugar chain by a sugar metabolism labeling method, then covalent crosslinking is realized between the sugar chain and a polypeptide skeleton by using an alkynyl-azide click chemistry through a synthesized crosslinking agent, and a glycoprotein group analysis technology is combined, so that a plurality of pairs of interacted proteins （Wu H, Shajahan A, Yang J Y, Capota E, Wands A M, Arthur C M, Stowell S R, Moremen K W, Azadi P, Kohler J J, CCB, 2022, 29, 84）. on the cell surface can be successfully identified, but a reaction can be initiated by illumination at a specific time point by using a photocrosslinking technology, weak interaction or transient combination among proteins is quickly stabilized, transient interaction can be effectively captured, and the photocrosslinking group can react with residues of 20 amino acids, so that the crosslinking coverage （Yuwen C, Yuxin A, Zhongpeng D, Yi L, Zhen L, Qun Z, Lihua Z, Yukui Z, Anal Chim Acta, 2022, 1203, 339694）. is greatly improved.

Disclosure of Invention

The invention aims to solve the technical problems in the prior art and provides a targeting cell surface glycoprotein interaction cross-linking agent and a preparation method thereof.

The technical scheme of the invention is that a crosslinking agent for targeting interaction of glycoprotein on the surface of cells is characterized by having the following chemical structural formula:

Where n=1-5.

The preparation method of the cross-linking agent for targeting the interaction of the glycoprotein on the cell surface comprises the following steps in sequence:

step 1, taking Boc-L-glutamic acid as an initial raw material, taking DMSO (dimethyl sulfoxide) as a reaction solvent, taking EDCI (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) as a condensing agent, and carrying out esterification reaction with NHS (N-hydroxysuccinimide) to obtain a compound I-Boc-L-glutamic acid disuccinimide ester;

Step 2, using a compound I as a reaction raw material, using DMSO as a reaction solvent, adding TEA (triethylamine) and 3-methyl-3-biaziridinyl methylamine or primary amine with a photoactive group to perform selective amidation reaction to obtain a compound II, namely Boc-L-glutamic acid succinimide ester containing the photoactive group;

Step 3, using a compound II as a raw material, using DMF (N, N-dimethylformamide) as a reaction solvent, adding TEA (triethylamine) and an alkynyl primary amine compound with a carbon chain length of n=1-5 to perform amidation reaction of one molecule, and removing a Boc protecting group in TFA (trifluoroacetic acid) to obtain a compound III, namely an L-glutamic acid compound containing a photoactive group and a sugar chain reaction group;

And 4. Taking a compound III as a raw material, DMF as a reaction solvent, EDCI as a condensing agent, hoBt (1-hydroxybenzotriazole) as a racemizer, DIEA (N, N-diisopropylethylamine) as an organic base, carrying out amidation reaction with a compound Biotin-Sulfo-COOH, and preparing a target cross-linking agent, wherein the Biotin-Sulfo-COOH is synthesized by reacting Biotin-N-succinimidyl ester with 2-amino-3-sulfopropionic acid.

Preferably, in the step 1, boc-L-glutamic acid, EDCI and NHS are dissolved in DMSO according to a molar ratio of 1:1.1-1.5:2.0-3.0, and are stirred for reaction at 22-30 ℃ for 10-24 h, washed by 80-150mL of distilled water, extracted by adding 25-35mL of dichloromethane, and dried by anhydrous sodium sulfate to remove dichloromethane, so that the crude Boc-L-glutamic acid disuccinimide ester is obtained.

Preferably, in the step 2, the compound I is dissolved in DMSO and a small amount of TEA is added, then 3-methyl-3-bisaziridinyl methylamine or primary amine with a photoactive group is dissolved in DMSO and slowly added into the solution of the compound I in a dropwise manner, the molar ratio of the compound I to the 3-methyl-3-bisaziridinyl methylamine or primary amine with a photoactive group is 1:1, after the dropwise addition, the mixture is stirred at 22-30 ℃ for 1-4 h, the reaction solution is separated and purified through a semi-prepared liquid phase system, and the Boc-L-glutamic acid succinimide ester with the photoactive group is prepared through vacuum freeze drying.

The preferred step 3 is to dissolve the compound II in DMF and add a small amount of TEA, dissolve the alkynyl primary amine compound with the carbon chain length of n=1-5 in DMF and slowly drop into the solution of the compound II, the molar ratio of the compound II to the alkynyl primary amine compound with the carbon chain length of n=1-5 is 1:1.1-1.5, stir the mixture at 22-30 ℃ for 10-24 h, separate and purify the reaction solution by a semi-preparation liquid phase system, freeze-dry in vacuum to obtain the Boc-L-glutamic acid compound containing photoactive groups and sugar chain reaction groups, dissolve the Boc-L-glutamic acid compound containing photoactive groups and sugar chain reaction groups in Dichloromethane (DCM), cool to 0-5 ℃, add TFA, stir and react for 2-5h, remove dichloromethane after the reaction, and further purify the crude product by column chromatography with the volume ratio of dichloromethane to acetone as mobile phase, thus obtaining the L-succinimidyl ester containing photoactive groups.

The preferred step 5 is to dissolve a compound III and Biotin-Sulfo-COOH, EDCI, hoBt, DIEA in DMF according to the mol ratio of 1:1.1-1.4:1.4-2.0:1.4-2.0:2.0-4.0, stirring at 22-30 ℃ to obtain 10-24. 24 h, removing DMF after the reaction, taking the volume ratio of dichloromethane to acetone as a mobile phase, further purifying by column chromatography to obtain a target cross-linking agent, preparing the Biotin-Sulfo-COOH according to the following steps, dissolving 2-amino-3-sulfopropionic acid in DMF and adding a proper amount of TEA into the DMF for stirring uniformly, dissolving Biotin-N-succinimidyl ester in DMF and slowly dripping the DMF into 2-amino-3-sulfopropionic acid solution, stirring at the mol ratio of 2-amino-3-sulfopropionic acid to Biotin-N-succinimidyl ester of 1.2-1.4,25-30 ℃ to obtain h, pouring the reaction solution into the column chromatography with the volume ratio of the dichloromethane to the acetone as 20-35 ℃, and further purifying by column chromatography to obtain the crude product after the reaction phase, namely, drying the crude product after the reaction solution is subjected to the column chromatography to the volume of the reaction solution to the dry distillation to the dry solution.

The crosslinking agent of the invention contains alkynyl groups which react with azido sugar chains, photoreactive groups and biotin groups containing sulfonate groups. The method comprises the steps of carrying out specific crosslinking reaction on glycoprotein by utilizing alkynyl groups and copper-catalyzed azide-alkynyl click chemistry (CuAAC), improving the targeting property of sugar chain crosslinking in glycoprotein interaction, further carrying out in-situ crosslinking on protein interacted with glycoprotein in cells by utilizing photoreactive groups, improving the crosslinking coverage of glycoprotein, and utilizing sulfonate-containing biotin enrichment groups to enable the crosslinking agent to have high biocompatibility, cell membrane targeting property and high selective enrichment capability, so that the targeting crosslinking and enrichment on cell surface glycoprotein interaction are realized, and the interference of nonspecific binding of intracellular proteins is reduced. Therefore, the deep coverage and the accurate analysis of the interaction between the cell surface glycosylated ligand and the receptor protein can be realized, and powerful technical support is provided for the research of the cell communication process.

Drawings

FIG. 1 is a schematic illustration of the reaction scheme of the preparation method according to the embodiment of the invention.

FIG. 2 is a fluorescence confocal image of the chemical cross-linking agent of experimental example 1 of the present invention.

FIG. 3 is a mass spectrum of the chemical cross-linking agent of experimental example 2 of the present invention.

Detailed Description

Example 1

The reaction route of the preparation method of the crosslinking agent for targeting cell surface glycoprotein interaction of the present invention is shown in fig. 1, and the preparation method is sequentially carried out according to the following steps:

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 30ml of DMSO and reacted for 16 hours under 22 ℃ stirring, 100ml of distilled water was added to the reaction solution to wash, 30ml of dichloromethane was added to extract the product, extraction was performed three times, the organic phases were combined, dried over anhydrous sodium sulfate, and the drying agent was removed by filtration, and dichloromethane was removed by a rotary evaporator to obtain Compound I, namely crude Boc-L-glutamic acid disuccinimide ester (1.975 g, yield 89.6%).

The preparation method comprises the steps of dissolving a compound I (441 mg, 1 mmol) in 15ml of DMSO, adding triethylamine (506-mg, 5 mmol), dissolving 3-methyl-3-bisaziridylmethylamine (85 mg, 1-mmol) in 2ml of DMSO, slowly dropwise adding the solution into the solution of the compound I, stirring the mixed solution at 22 ℃ for reaction for 2H after the dropwise addition, separating and purifying the reaction solution by a semi-preparation liquid phase system, wherein the semi-preparation liquid phase adopts 0.1% of TFA/H2O as an A phase, acetonitrile as a B phase, the separation gradient is set to 0-40 min (10-50%, B) and the flow rate is 40 mL/min, collecting 22-25-min effluent, and combining the effluent and freeze-drying the effluent at 20-30 ℃ for 12-24-H to obtain a pale yellow oily product (131.5 mg, the yield 32%), namely the compound II.

Specifically, compound II (141 mg, 0.32 mmol) was dissolved in 20ml DMF, triethylamine (162 mg, 1.6 mmol), alkynylpropylamine (26.4 mg, 0.48 mmol) was dissolved in 2ml DMF and slowly added dropwise to compound II solution, and the reaction was stirred at 22 ℃ for 12h. The reaction liquid is separated and purified by a semi-preparation liquid phase system, wherein the semi-preparation liquid phase uses H2O as a phase A, acetonitrile as a phase B, the separation gradient is set to be 0-30 min (20-50 percent, B), the flow rate is 50 mL/min, and 25-27 min effluent liquid is collected. The product effluent is combined and placed in a vacuum freeze dryer to freeze-dry 12-24 h at 20-30 ℃ to obtain the Boc-L-glutamic acid compound containing photoactive groups and sugar chain reaction groups (70.2 mg, yield 63%). The Boc-L-glutamic acid compound containing photoactive group and sugar chain reaction group is redissolved in 20ml of dichloromethane, cooled to 0 ℃, TFA (2.0 g, 17.5 and mmol) is added, stirred and reacted for 3 hours, dichloromethane is removed by rotary evaporation, the crude product takes acetone: dichloromethane=1:15 (volume ratio) as mobile phase, the further purification is carried out by column chromatography, and the organic phase is removed by rotary evaporation, thus obtaining pale yellow oily product (42.82 mg, yield 85.3%), namely compound III.

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) are dissolved in 30ml of DMF, stirred and reacted for 14 hours at 25 ℃, after the reaction is finished, DMF is removed by rotary evaporation, acetone: dichloromethane=1:20 (volume ratio) is taken as a mobile phase of a crude product, the crude product is further purified by column chromatography, and an organic phase is removed by rotary evaporation, so that the target cross-linking agent (36.3 mg, yield 34%);

The Biotin-Sulfo-COOH is prepared by dissolving 2-amino-3-Sulfo-propionic acid in DMF, adding a proper amount of TEA, stirring uniformly, dissolving Biotin-N-succinimidyl ester in DMF, slowly dropwise adding the solution into the 2-amino-3-Sulfo-propionic acid, stirring 6h at a molar ratio of 2-amino-3-Sulfo-propionic acid to Biotin-N-succinimidyl ester of 1.3,30 ℃, pouring the reaction solution into a large amount of distilled water, filtering and drying, and further purifying the crude product by column chromatography with a volume ratio of dichloromethane to acetone of 20:1 as a mobile phase to obtain the Biotin-Sulfo-COOH.

The structural formula of the cross-linking agent is as follows:

Example 2

The title cross-linker was obtained in example 1 except that (4-azidophenyl) methylamine hydrochloride (148 mg,1 mmol) was used instead of 3-methyl-3-bisaziridinyl methylamine (85 mg,1 mmol) in step 2 to give the product as a pale yellow oil (173 mg, yield 36.5%).

Example 3:

The title cross-linker was obtained in example 1 except that 4- (aminomethyl) benzophenone (211 mg,1 mmol) was used instead of 3-methyl-3-bisaziridinyl methylamine (85 mg,1 mmol) in step 2 to give the product as a pale yellow oil (150 mg, yield 28%).

Experimental example 1:

The crosslinker prepared in example 1 was applied to a549 cells, which were first labeled with N-azidoacetaminophen (ManNAz). The specific experimental method is to prepare a cross-linking agent mother solution with the final concentration of 5 mM, add PBS into the A549 cells washed by PBS, and chemically cross-link 10 min. After the reaction, 1mL of 10% (v/v) formaldehyde solution was added to the cells, and the cells were fixed 15 min. 1mL of 0.1% triton X-100 cells were added and punched 60 min, incubated with 1mL fluorescein-conjugated avidin for 1 h, washed five times with PBS, and developed by fluorescence confocal fluorescence, as seen in FIG. 2. The green fluorescence on the cell surface was clearly observed from FIG. 2, whereas the blank group did not have any fluorescence. The cross-linking agent provided by the invention is successful and does not permeate cell membranes, and provides basis for large-scale analysis of the in-situ glycoprotein complex structure and interaction of the in-situ glycoprotein complex structure on the surface of the cell membranes.

Experimental example 2:

The crosslinker prepared in example 1 was applied to a549 cells. The specific experimental method is that the cross-linking agent in the embodiment 1 is dissolved in PBS to prepare cross-linking agent mother liquor with the final concentration of 5mM, the cross-linking agent mother liquor is added into the HeLa cells washed by PBS, and the cross-linking reaction is completed after incubation for 10min under the room temperature condition. 1mL 8M urea was added, 50mM TEAB lysate was subjected to cell disruption, 12000 g centrifugation, protein denaturation reduction alkylation, digestion, streptavidin magnetic sphere enrichment, desalting, lumos Fusion liquid mass tandem mass spectrometry, and a mass spectrum is shown in FIG. 3. The data were processed with pLink <2 >, 1% FDR, and finally 1578 cross-linked peptide fragments of 457 cell surface proteins and 890 pairs of interaction glycoprotein cross-linking information formed by the same were identified, which demonstrates that the cross-linking agent of the invention can achieve in situ capture and high coverage identification of living cell surface glycoproteins.

Claims

1. A cross-linking agent for targeting cell surface glycoprotein interactions, characterized by the following chemical structural formula:

Where n=1-5.

2. A method of preparing a crosslinker for targeting cell surface glycoprotein interactions according to claim 1, characterized by the following steps in sequence:

step 1, taking Boc-L-glutamic acid as an initial raw material, taking DMSO as a reaction solvent, taking EDCI as a condensing agent, and carrying out esterification reaction with NHS to obtain a compound I-Boc-L-glutamic acid disuccinimide ester;

Step 2, using a compound I as a reaction raw material, using DMSO as a reaction solvent, adding TEA and 3-methyl-3-biaziridinyl methylamine or primary amine with a photoactive group to perform selective amidation reaction to obtain a compound II, namely Boc-L-glutamic acid succinimide ester with a photoactive group;

step 3, using a compound II as a raw material, using DMF as a reaction solvent, adding TEA and an alkynyl primary amine compound with a carbon chain length of n=1-5 to perform amidation reaction of one molecule, and removing a Boc protecting group in the TFA to obtain a compound III, namely an L-glutamic acid compound containing a photoactive group and a sugar chain reaction group;

and 4. Taking the compound III as a raw material, DMF as a reaction solvent, EDCI as a condensing agent, hoBt as a racemization agent, DIEA as an organic base, carrying out 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.

3. The method for preparing the targeted cell surface glycoprotein interaction according to claim 2, wherein the step 1 is characterized in that Boc-L-glutamic acid, EDCI and NHS are dissolved in DMSO according to a molar ratio of 1:1.1-1.5:2.0-3.0, stirred and reacted at 22-30 ℃ for 10-24 h, washed with 80-150mL of distilled water, extracted with 25-35mL of dichloromethane, and dried over anhydrous sodium sulfate to remove dichloromethane, thereby obtaining crude Boc-L-glutamic acid disuccinimide ester.

4. The method for preparing the targeted cell surface glycoprotein interaction according to claim 3, wherein the step 2 is to dissolve the compound I in DMSO and add TEA, dissolve 3-methyl-3-bisaziridinyl methylamine or primary amine with photoactive group in DMSO and slowly drop-add the compound I into the solution, the molar ratio of the compound I to the 3-methyl-3-bisaziridinyl methylamine or primary amine with photoactive group is 1:1, stir the mixture at 22-30 ℃ for 1-4 h after dropping, separate and purify the reaction solution by a semi-preparative liquid phase system, and freeze-dry the reaction solution in vacuum to obtain Boc-L-glutamic acid succinimidyl ester with photoactive group.

5. The method for preparing the targeted cell surface glycoprotein interaction according to claim 4, wherein the step 3 is characterized in that a compound II is dissolved in DMF and TEA is added, an alkynyl primary amine compound with a carbon chain length of n=1-5 is dissolved in DMF and slowly added into a solution of the compound II in a dropwise manner, the molar ratio of the compound II to the alkynyl primary amine compound with the carbon chain length of n=1-5 is 1:1.1-1.5, the dropwise ratio of the compound II to the alkynyl primary amine compound with the carbon chain length of n=1-5 is 10-24 h, the reaction solution is separated and purified by a semi-preparative liquid phase system, the mixture is lyophilized in vacuum, the Boc-L-glutamic acid compound containing photoactive groups and sugar chain reactive groups is prepared, the Boc-L-glutamic acid compound containing photoactive groups and sugar chain reactive groups is dissolved in dichloromethane, the mixture is cooled to 0-5 ℃, the TFA is added, the reaction is completed, the dichloromethane is removed, the volume ratio of the dichloromethane to the acetone is 10-20:1 as a mobile phase, and the crude product is further purified by column chromatography, and the photoactive group containing succinimidyl glutamate is prepared.

6. The method for preparing the targeted cell surface glycoprotein interaction according to claim 5, wherein the step 5 comprises dissolving a compound III and Biotin-Sulfo-COOH, EDCI, hoBt, DIEA in DMF according to a molar ratio of 1:1.1-1.4:1.4-2.0:1.4-2.0:2.0-4.0, stirring at 22-30 ℃ for 10-24: 24h, removing DMF after the reaction, and further purifying the crude product by column chromatography with a volume ratio of dichloromethane to acetone of 20-35:1 as a mobile phase to obtain the target cross-linking agent;

The Biotin-Sulfo-COOH is prepared by dissolving 2-amino-3-Sulfo-propionic acid in DMF and adding TEA, stirring uniformly, dissolving Biotin-N-succinimidyl ester in DMF and slowly dripping the solution into the 2-amino-3-Sulfo-propionic acid solution, stirring 6-14 h at the molar ratio of the 2-amino-3-Sulfo-propionic acid to the Biotin-N-succinimidyl ester of 1.2-1.4,25-30 ℃, pouring the reaction solution into distilled water, filtering and drying, and further purifying the crude product by column chromatography with the volume ratio of dichloromethane to acetone of 15-20:1 as a mobile phase to obtain the Biotin-Sulfo-COOH.