One-step enriched cell surface targeting cross-linking agent as well as preparation and application thereof

Abstract

The invention relates to development and application of a one-step enriched cell surface targeting cross-linking agent. The cross-linking agent is TMTC13NHS-SO3-, and the chemical structural formula of the cross-linking agent is shown as a formula I. The chemical cross-linking agent has the following functional characteristics that in-situ cross-linking capture of protein on the surface of a living cell can be realized, and the chemical cross-linking agent has high biological compatibility, water solubility and cell membrane impermeability due to a bis-succinimide ester-sulfonate reactive active group. The chemical cross-linking agent is applied to the field of proteomics and provides important technical support for analysis of living cell surface protein complexes. # imgabs0 #

Description

Technical Field

[0001] The invention relates to the preparation of a one-step enrichment type cell surface targeting cross-linking agent. The cross-linking agent of the invention has a trimethylpiperidinyl enrichment unit and a double-terminal succinimide ester-sulfonate reactive group. Background Art

[0002] Cell surface proteins play an important role in biological processes such as cell signaling, cell adhesion, cell recognition and immune response.

[0003] The in situ chemical cross-linking method can be used to fix the spatial position of cell surface proteins so that they maintain the original protein-protein interactions, thereby analyzing the interface site information of cell surface protein interactions, and then inferring the action relationship in the protein complex. The structure of cell surface protein complexes under physiological conditions can be analyzed in situ to obtain more realistic protein complex information. The high-dimensional signal transduction network assembled by protein complexes regulates various life processes of organisms (Curr. Opin. Biotechnol., 2020, 63, 48; Anal. Chem., 2018, 90, 144; Trends Biochem. Sci., 2018, 43, 157.).

[0004] Cell surface proteins play an important role in the occurrence and development of diseases. Cell surface proteins are considered to be key structures for identifying immune and targeted therapeutic targets. More than 60% of cell surface proteins have become targets of approved human disease drugs (Nat. Cancer, 2021, 12, 1406). Therefore, there is an urgent need to develop cell surface protein cell surface targeted cross-linking technology to better meet the application needs of more complex biological samples. Summary of the invention

[0005] Based on the research status and design principles of the above-mentioned chemical cross-linking agents, the present invention aims at the problem that the existing cross-linking agents have no enrichment groups or the enrichment steps are cumbersome, the losses are large, and the efficient identification of low-abundance cell surface proteins is not utilized. A cross-linking agent with a trimethylpiperidine group is developed, which can achieve one-step efficient enrichment of cross-linked peptides by specific binding with anti-TMT agarose beads, thereby improving the high coverage identification of in situ cross-linking information of cell surface proteins. At the same time, in view of the problem that the existing cross-linking agents are generally water-soluble and have poor biocompatibility, a succinimidyl ester-sulfonate reactive group is introduced to make it have very good water solubility, high biocompatibility and cell membrane impermeability, so as to achieve the analysis of the structure of protein complexes on the surface of living cells and the interaction information, so as to better meet the application needs of life sciences and clinical samples.

[0006] The one-step enrichment cell surface targeting cross-linking agent provided by the present invention has a structure as shown in the following figure:

[0007]

[0008] The present invention provides a method for preparing a cross-linking agent, and the specific steps are as follows:

[0009] In the first step, the reactant 3-bromopropionic acid is dissolved in a NaOH solution, the pH is adjusted to 13-14, and the temperature is lowered to 0-5°C. The starting material 2,6-dimethylpiperidine is added dropwise to the reaction solution, and the molar ratio of 2,6-dimethylpiperidine to 3-bromopropionic acid is 1:(1.5-2.0). After the dropwise addition, the reaction solution is heated to 25-30°C and stirred for 3-4 days. After the reaction is completed, the pH of the reaction solution is adjusted to 1-2, and dichloromethane is added to extract the product. Dichloromethane is removed, and the crude product is further purified by column chromatography with dichloromethane: methanol = 20:1 (volume ratio) as the fluidity to obtain 2,6-dimethylpiperidinyl acetic acid (Compound 1).

[0010] In the second step, compound 1, 1.2-1.5 molar equivalents of EDC, and 1.2-1.5 molar equivalents of NHS are dissolved in DMSO, and reacted at 25-30°C for 12-24 hours to prepare 2,6-dimethylpiperidinyl acetic acid succinimidyl ester intermediate; after the reaction is completed, 3-5 molar equivalents of triethylamine and 2.0-3.0 molar equivalents of 3-aminopropionic acid are added to the reaction solution, and the reaction is continued at 25-30°C for 1-2 hours. After the reaction is completed, the crude product has a fluidity of dichloromethane: methanol = 30:1 (volume ratio), and is further purified by column chromatography to obtain 2,6-dimethylpiperidinyl levulinic acid (compound 2).

[0011] In the third step, compound 2, 1.1-1.3 molar equivalents of dimethyl ester amino hydrochloride, 1.5-2.0 molar equivalents of EDC, 1.5-2.0 molar equivalents of HoBt and 3.0-5.0 molar equivalents of DIEA are dissolved in DMF and reacted at 25-30° C. for 12-24 hours. After the reaction is completed, semi-preparative liquid phase separation and purification are performed to obtain 2,6-dimethylpiperidinyl dimethyl ester (compound 3).

[0012] In the fourth step, compound 3 is dissolved in MeOH, sodium hydroxide aqueous solution is added, the pH is adjusted to 13-14, and the reaction is carried out at 25-30° C. for 12-24 hours. After the reaction is completed, concentrated hydrochloric acid is added to adjust the pH to 2-3, and semi-preparative liquid phase separation and purification are performed to obtain 2,6-dimethylpiperidinyl dicarboxylic acid (compound 4).

[0013] The fifth step is to react compound 4 with 2.4-3.0 molar equivalents of NHS-SO3 relative to compound 4. -2.6-3.2 molar equivalents of EDC relative to compound 4 were dissolved in DMSO, reacted at 25-30°C for 12-24h, and subjected to semi-preparative liquid phase separation and purification and vacuum freeze-drying at a freeze-drying temperature of 20-25°C to obtain the target cross-linking agent TMTC13NHS-SO3 - .

[0014] The synthesis process of the target cross-linker is as follows:

[0015]

[0016] The cross-linking agent of the present invention is applied in the field of proteomics, and provides important technical support for realizing large-scale analysis of in situ protein complexes on the cell surface, large-scale analysis between cells and exosomes, analysis of target protein complexes between cells, spatial structure analysis of protein complexes, analysis of protein-protein interactions, and analysis of spatiotemporal dynamic changes of protein complexes.

[0017] Compared with the existing chemical cross-linking agents, the cross-linking agent of the present invention has the following advantages: it can solve the problem that the existing cross-linking agents have no enrichment groups or the enrichment steps are cumbersome, the loss is large, and the low-abundance cell surface proteins are not used for efficient identification. The cross-linking agent has a trimethylpiperidine group, which can achieve one-step efficient enrichment of cross-linked peptides by specifically binding to anti-TMT agarose beads, thereby improving the high coverage identification of in situ cross-linking information of cell surface proteins; the existing cross-linking agents generally have poor water solubility and biocompatibility. The cross-linking agent introduces a succinimidyl ester-sulfonate reactive group to make it have very good water solubility, high biocompatibility and cell membrane impermeability, which can achieve in situ cross-linking capture of living cell surface proteins while meeting the one-step enrichment requirements, thereby achieving in situ cross-linking high coverage identification of living cell surface proteins.

[0018] 1. It has a succinimidyl ester-sulfonate reactive group, which undergoes an amidation reaction with the terminal or N-terminal amino group of protein lysine residues under physiological conditions to achieve chemical cross-linking of spatially adjacent lysines in protein complexes;

[0019] 2. The sodium sulfonate group is introduced into the disuccinimide ester ring, which improves the hydrophilicity of the crosslinker on the one hand, and on the other hand, the crosslinker is negatively charged in the solution, which inhibits the membrane permeability of the crosslinker and makes the crosslinker localized on the cell surface for studying the structural information of cell surface proteins. At the same time, it has high biocompatibility and good water solubility, which enables the in-situ targeted crosslinking of surface proteins in the highly active state of cells;

[0020] 3. It has a trimethylpiperidinyl enrichment group, which can enrich cross-linked peptides by combining with anti-TMT agarose beads, improve the signal intensity of cross-linked peptides in mass spectrometry, reduce the background interference of conventional peptides, and realize high-throughput and high-sensitivity analysis of cross-linked peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the structural formula of chemical cross-linking agent.

[0022] Figure 2 The synthetic route for chemical cross-linking agents.

[0023] Figure 3 This is the NMR spectrum of the chemical cross-linking agent.

[0024] Figure 4 This is the mass spectrum of the chemical cross-linking agent.

[0025] Figure 5 This is a fluorescence confocal image of the chemical crosslinker in Example 2. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with specific embodiments.

[0027] Example 1

[0028] This embodiment discloses a one-step enrichment cell surface targeting cross-linking agent ( Figure 1 )TMTC13NHS-SO3 - The preparation method comprises five reaction steps, and the preparation method is as follows:

[0029]

[0030] The first step is the preparation of 2,6-dimethylpiperidinyl acetic acid (Compound 1). The reactant 3-bromopropionic acid (3.31 g, 24 mmol) was dissolved in 10 ml of sodium hydroxide aqueous solution to obtain a mixed solution with a pH value of 14, and the temperature was lowered to 0°C. Weigh 2,6-dimethylpiperidine (1.36 g, 12 mmol) and slowly drop it into the reaction solution, and continue stirring for 1 hour. Restore to 25°C and continue stirring for 3 days. After the reaction is completed, the mass concentration is 37% concentrated hydrochloric acid to adjust the pH to 2.0, and 30 ml of dichloromethane is added to extract the product. Extract three times, combine the organic phases, and remove dichloromethane by rotary evaporation to obtain a pale yellow oily crude product. The crude product has a fluidity of dichloromethane: methanol = 20:1 (volume ratio), and is further purified by column chromatography. The separation column is C18 filler silica ball (10μm, ), 50mm id×250mm, and the organic phase was removed by rotary evaporation to obtain 2.08g of light yellow oily product with a yield of 60.8%. HR-MS (C9H 17 NO2): theoretical value: 171.1259 measured value [M+H]: 1721589. The molecular weight determined by LTQ-MS mass spectrometry is shown in the reaction process compound 1.

[0031] Step 2, preparation of 2,6-dimethylpiperidinyl levulinic acid (Compound 2). Dissolve compound 1 (855 mg, 5 mmol), NHS (690 mg, 6 mmol) and EDCI (1.15 g, 6 mmol) in 30 ml DMSO and react at 25 ° C for 18 h to obtain 2,6-dimethylpiperidinyl acetic acid succinimidyl ester intermediate. Continue to add triethylamine (2.5 g, 25 mmol) and 3-aminopropionic acid (890 mg, 10 mmol) to the reaction solution and react at 25 ° C for 1 h. After the reaction is completed, the crude product has a fluidity of dichloromethane: methanol = 30: 1 (volume ratio), and is further purified by column chromatography (the separation column is the same as the first step) to obtain 1.09 g of light yellow oil with a yield of 90%. HR-MS (C 12 H 22 N2O3): theoretical value: 242.1630, measured value [M+H]: 243.1813. The molecular weight determined by LTQ-MS mass spectrometry is shown in the reaction process compound 2.

[0032] Step 3, preparation of 2,6-dimethylpiperidinyl heptane dimethyl ester (Compound 3). Compound 2 (1.21 g, 5 mmol), dimethyl 7-aminomalonate hydrochloride (1.32 g, 5.5 mmol), EDCI (1.44 g, 7.5 mmol), HoBt (1350 mg, 10 mmol) and DIEA (2.5 ml, 15 mmol) were dissolved in 30 ml DMF and reacted at 25 °C for 24 h. After the reaction was completed, semi-preparative liquid phase separation and purification were performed, and the preparative column was C18 filler silica spheres (10 μm, ), 50mm id×250mm, gradient method (A phase 0.1% v / v TFA / H2O, B phase acetonitrile (ACN); B phase from 10%-30%, 40min), collect 24-32min effluent, vacuum freeze-dry, and obtain 1.97g of colorless oil, yield 92.3%. HR-MS (C 21 H 37 N3O6): Theoretical value: 427.2682, measured value [M+H]: 428.3813. The molecular weight determined by LTQ-MS mass spectrometry is shown in the reaction process compound 3.

[0033] Step 4, preparation of 2,6-dimethylpiperidinyl heptanediol (Compound 4). Dissolve compound 3 (2.36 g, 5 mmol) in 10 ml MeOH, add 10 ml sodium hydroxide aqueous solution, adjust the pH to 14, and react at 25°C for 12 h. After the reaction is completed, add 37% concentrated hydrochloric acid to adjust the pH to 2, and perform semi-preparative liquid phase separation and purification. The preparative column is C18 filler silica spheres (10 μm, ), 50mm id×250mm, gradient method (phase A 0.1% TFA / H2O, phase B ACN; phase B from 5%-20%, 20min), collect the effluent from 17-20min, vacuum freeze-dry, and obtain 1.87g of colorless oil, with a yield of 93.5%. HR-MS (C 19 H 33 N3O6): Theoretical value: 399.2369, measured value [M+H]: 400.6712. The molecular weight determined by LTQ-MS mass spectrometry is shown in the reaction process compound 4.

[0034] Step 5: 2,6-dimethylpiperidinyl heptadiosuccinimide ester (crosslinking agent TMTC13NHS-SO3 - ) was prepared by mixing compound 4 (399 mg, 1 mmol) and NHS-SO3 - (456 mg, 3 mmol) and EDCI (575 mg, 3 mmol) were dissolved in 30 ml DMSO and reacted at 25 °C for 24 h. The mixture was purified by semi-preparative liquid phase separation using a C18 silica ball (10 μm, ), 50mm id×250mm, gradient method (A phase 0.1% trifluoroacetic acid (TFA) / H2O, B phase ACN; B phase from 10%-30%, 40min), collect 23-24min effluent, vacuum freeze-dry, and obtain 347mg of colorless oil, which is the target cross-linking agent. NMR detection is as follows Figure 3 As shown, the yield is 60%. HR-MS theoretical value: 835.8 Found value [M+H]: 836.43. (as Figure 4 shown).

[0035] Example 2

[0036] The cross-linking agent prepared in Example 1 was applied to Hela cells. A cross-linking agent mother solution with a final concentration of 5 mM was prepared, the solvent was PBS, and it was added to 1E4 Hela cells washed with PBS, and chemical cross-linking was performed 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 for 60 minutes, incubated with 1 mL of anti-TMT protein bound to fluorescein for 1 hour, and washed five times with PBS. After fluorescence confocal fluorescence color development, green fluorescence can be clearly observed on the cell surface, while there is no fluorescence in the blank group (attached Figure 5 ), indicating that the cross-linking agent of the present invention is successful and does not penetrate the cell membrane. This experiment provides a basis for the large-scale analysis of the in situ protein complex structure and its interaction on the cell membrane surface.

[0037] Example 3

[0038] The cross-linking agent prepared in Example 1 was applied to Hela human cells. The cross-linking agent in Example 1 was dissolved in PBS to prepare a cross-linking agent mother solution with a final concentration of 5mM, and added to 1E4 Hela cells washed with PBS. Incubate at room temperature for 10 minutes to complete the cross-linking reaction. Add 1mL of ammonium bicarbonate solution to extinguish the fire, add 1mL of RIPA lysis buffer to break the cells, centrifuge at 12000g, extract the cytoplasmic membrane, add ionic liquid, extract membrane proteins, denature and reduce alkylation of proteins, enzyme digestion, streptavidin magnetic ball enrichment, sodium dithionite release cross-linked peptides, desalt, Lumos Fusion liquid mass tandem mass spectrometry analysis, data processing using pLink2, 1% FDR, and finally identified 408 cross-linked peptides of 85 cell surface proteins, as well as 230 pairs of interacting protein cross-linking information formed by them, proving the TMTC13NHS-SO3 protected by this patent. - Cross-linkers enable in situ capture and high-coverage identification of proteins on the surface of living cells.

Claims

1. A one-step enrichment cell surface targeting cross-linking agent, characterized in that: Its chemical structure is:

2. A method for preparing the crosslinking agent according to claim 1, characterized in that The specific process is as follows: Step 1: Using 2,6-dimethylpiperidine and 3-bromopropionic acid as starting materials, a nucleophilic substitution reaction occurs in a sodium hydroxide aqueous solution, and a molecule of hydrogen bromide is removed to prepare 2,6-dimethylpiperidinyl acetic acid (Compound 1); Step 2: Compound 1 is used as a reaction raw material, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) is used as a condensation agent, and dimethyl sulfoxide (DMSO) is used as a reaction solvent to undergo an esterification reaction with N-hydroxysulfonylsuccinimide (NHS) to prepare a 2,6-dimethylpiperidinyl acetate succinimide ester intermediate; triethylamine and 3-aminopropionic acid are then added to undergo an amidation reaction to prepare 2,6-dimethylpiperidinyl levulinic acid (Compound 2); Step 3: Compound 2 is used as a raw material, EDCI is used as a condensing agent, 1-hydroxybenzotriazole (HoBt) is used as a racemizing agent, diisopropylethylamine (DIEA) is used as an organic base, and N,N-dimethylformamide (DMF) is used as a reaction solvent to undergo an amidation reaction with aminotridecanedioic acid methyl ester to prepare 2,6-dimethylpiperidinyl dimethyl ester (Compound 3); Step 4: using compound 3 as a raw material and methanol as an organic solvent, a hydrolysis reaction is carried out in a sodium hydroxide aqueous solution to obtain a carboxylic acid sodium salt; and then adjusting the pH to 2-3 with concentrated hydrochloric acid to prepare 2,6-dimethylpiperidinyl dicarboxylic acid (compound 4); Step 5: Compound 4 is used as a raw material, EDCI is used as a condensation agent, and DMSO is used as a reaction solvent. - ) undergoes esterification reaction to prepare the target chemical crosslinking agent TMTC13NHS-SO3 - .

3. The method for preparing a crosslinking agent according to claim 2, characterized in that: In step 1, the raw material 3-bromopropionic acid is dissolved in a sodium hydroxide solution to make the pH value 13-14, and the temperature is lowered to 0-5°C; the raw material 2,6-dimethylpiperidine is added dropwise to the reaction solution, and the molar ratio of 2,6-dimethylpiperidine to 3-bromopropionic acid is 1:(1.5-2.0). After the addition, the reaction solution is heated to 25-30°C and stirred for 3-4 days; after the reaction is completed, the reaction solution is adjusted to a pH value of 1-2 with concentrated hydrochloric acid having a mass concentration of 30-36%, and dichloromethane is added to extract the product; dichloromethane is removed, and the crude product is further purified by column chromatography using dichloromethane:methanol=(10-20):1 (volume ratio) as the mobile phase to obtain 2,6-dimethylpiperidinyl acetic acid (Compound 1).

4. The method for preparing a crosslinking agent according to claim 2, characterized in that: In step 2, compound 1, 1.2-1.5 molar equivalents of EDCI relative to compound 1, and 1.2-1.5 molar equivalents of NHS relative to compound 1 are dissolved in DMSO, and reacted at 25-30° C. for 12-24 hours to prepare a 2,6-dimethylpiperidinyl acetic acid succinimidyl ester intermediate; after the reaction is completed, 3.0-5.0 molar equivalents of triethylamine and 2.0-3.0 molar equivalents of 3-aminopropionic acid relative to compound 1 are added to the reaction solution, and the reaction is continued at 25-30° C. for 1-2 hours; after the reaction is completed, the crude product is further purified by column chromatography using dichloromethane: methanol = (20-30): 1 (volume ratio) as the mobile phase to obtain 2,6-dimethylpiperidinyl levulinic acid (compound 2).

5. The method for preparing a chemical crosslinking agent according to claim 2, characterized in that: In step 3, compound 2, 1.1-1.3 molar equivalents of 7-aminomalonate hydrochloride relative to compound 2, 1.5-2.0 molar equivalents of EDCI relative to compound 2, 1.5-2.0 molar equivalents of HoBt relative to compound 2, and 3.0-5.0 molar equivalents of DIEA relative to compound 2 are dissolved in DMF and reacted at 25-30° C. for 12-24 hours; after the reaction is completed, separation and purification are performed by reverse phase liquid chromatography to obtain 2,6-dimethylpiperidinyl dimethyl ester (compound 3).

6. The method for preparing a chemical crosslinking agent according to claims 2-5, characterized in that: In step 4, compound 3 is dissolved in methanol, and sodium hydroxide aqueous solution is added to adjust the pH to 13-14, and the reaction is carried out at 25-30° C. for 12-24 hours. After the reaction is completed, concentrated hydrochloric acid with a mass concentration of 30-36% is added to adjust the pH to 2-3, and the mixture is separated and purified by reverse phase liquid chromatography to obtain 2,6-dimethylpiperidinyl dicarboxylic acid (compound 4).

7. The method for preparing a chemical crosslinking agent according to claims 2-6, characterized in that: In step 5, compound 4 and 2.4-3.0 molar equivalents of NHS-SO3 relative to compound 4 are added. - 2.6-3.2 molar equivalents of EDC relative to compound 4 were dissolved in DMSO, reacted at 25-30°C for 12-24h, and subjected to semi-preparative liquid phase separation and purification and vacuum freeze-drying at a freeze-drying temperature of 20-25°C to obtain the target cross-linking agent TMTC13NHS-SO3 - .

8. An application of the cross-linking agent according to claim 1, wherein the cross-linking agent can be applied to in situ cross-linking and large-scale analysis of protein complexes on the surface of living cells, and analysis of protein-protein interactions on the surface of cells.

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