A class of fluorinated tags for protein cysteine ​​residue modification and its application

Abstract

The present invention belongs to the technical field of protein modification, and specifically discloses a type of fluorine-containing label for modifying protein cysteine ​​residues and its application. The present invention utilizes the specific reaction of sulfone compounds with thiol groups to construct a water-soluble 19 F tag, which can modify cysteine ​​residues under mild conditions to form stable carbon-sulfur bonds. The labeled protein effectively reduces the nuclear magnetic resonance detection concentration of the protein and can be used for intracellular research.

Description

Technical Field

[0001] The invention belongs to the technical field of protein chemical modification, and specifically relates to utilizing sulfone-based fluorine-containing compounds to specifically react with sulfhydryl groups in proteins, and utilizing nuclear magnetic resonance to study the structure, dynamics and interaction of polypeptides or proteins. Background Art

[0002] There is almost no free fluorine in the body, so fluorine-labeled proteins (or peptides) can be detected without background interference. At present, the biosynthesis of fluorinated amino acids (precursors) is still an effective method to obtain fluorine-labeled proteins. However, polyfluorine labeling may affect the folding process, expression efficiency or stability of proteins. Site-specific chemical labeling can be used as a beneficial supplement to biomarkers, and can provide a powerful labeling tool for proteins that are difficult to recombinantly express, have a very large molecular weight or are complex systems.

[0003] But currently used 19 There are still some problems with chemically labeled tags. On the one hand, these compounds are difficult to modify and are basically fat-soluble. They need to be greatly excessive or organic solvents added to promote solubilization to achieve efficient connection; on the other hand, the chemical bonds formed between proteins and tags are unstable or form isomers in the cell environment, affecting signal attribution. In response to the above problems, the present invention utilizes the specific reaction of sulfone groups and thiol groups to design fluorine-containing tags, which can not only achieve efficient connection with proteins under mild conditions, but also construct water-soluble tags through the modification of leaving groups. The results show that the labeled protein effectively reduces the nuclear magnetic resonance detection concentration of the protein and can be used for intracellular research. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a type of fluorinated label for modifying cysteine ​​residues in proteins and its application, and constructs a water-soluble fluorinated label by using the specific reaction of sulfone compounds with thiol groups. 19 F tag, which can modify cysteine ​​residues under mild conditions to form stable carbon-sulfur bonds. The labeled protein effectively reduces the nuclear magnetic resonance detection concentration of the protein and can be used for intracellular research.

[0005] In order to achieve the above object, the present invention adopts the following technical measures:

[0006] Fluorinated tags have the following structure:

[0007]

[0008] in, It is an electron-withdrawing aromatic ring containing a trifluoromethyl unit, including:

[0009]

[0010] The specific structure of the tag is as follows:

[0011]

[0012] The above-mentioned fluorine-containing label is used in the labeling of polypeptides or proteins: the polypeptides or proteins contain cysteine, and the fluorine-containing label reacts with the cysteine ​​residue to form a stable carbon-sulfur bond. The labeled polypeptides or proteins can be used for fluorine-19 nuclear magnetic resonance ( 19 In a specific embodiment of the present invention, fluorine-containing tags (tags 1-4) were used to label GB1 protein (streptococcal immune G protein B1 domain), and the mass spectrometry Q-TOF found that the connection efficiency of the four tags to the GB1 protein was close to 100%, especially tag 1 containing six equivalent fluorine atoms, which had a greater signal enhancement ability than other tags, and verified the universality of tag 1 in other protein labels (calmodulin, CaM; ubiquitin protein, UBQ; maltose binding protein, MBP), and can also be used for the detection of intracellular proteins.

[0013] Application of the above fluorine-containing labels in labeling sulfhydryl-containing small molecules: fluorine-containing labels can react with sulfhydryl groups, and the chemical shift of fluorine changes. In a specific embodiment of the present invention, fluorine-containing labels (labels 1-4) react with glutathione and can be used to detect sulfhydryl-containing small molecules through nuclear magnetic resonance fluorine spectroscopy.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] The present invention can achieve the modification of the water solubility of fluorine-containing labels, avoiding the influence of organic reagents or large amounts of labeling reagents on the protein structure during the labeling process; can achieve the modification of cysteine ​​residues under mild conditions to form stable carbon-sulfur bonds; can achieve the observation of proteins in mammalian cells, and reduce the detection concentration to micromolar or below, close to the physiological concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Comparison of the reaction rates of labels 1-4 with glutathione (GSH).

[0017] Figure 2 After the reaction of labels 1-4 with GSH 19 F NMR spectrum.

[0018] Figure 3 This is the mass spectrum after the reaction of tags 1-4 with GB1 protein.

[0019] Figure 4 GB1 protein was simultaneously labeled with 3-fluorotyrosine biolabel and tags 1-4 in a buffer solution. 19F NMR spectrum.

[0020] Figure 5 GB1, CaM, UBQ and MBP labeled with tag 1 in buffer solution 19 F NMR spectrum.

[0021] Figure 6 GB1, CaM, UBQ and MBP labeled with Tag 1 in HeLa cells 19 F NMR spectrum. DETAILED DESCRIPTION

[0022] In order to better understand the content of the present invention, the content of the present invention is further described below in combination with the synthesis, verification and application of the water-soluble fluorine-containing label, but the protection content of the present invention is not limited to the following examples.

[0023] The raw materials used in the examples of the present invention can be purchased from the market, or can be synthesized using methods known in the art.

[0024] Example 1: Construction of water-soluble fluorinated tags

[0025] The tag structure is as follows

[0026]

[0027] Synthesis of Label 1

[0028]

[0029] Step 1: Under nitrogen protection, sodium 4-toluenesulfinate (TolSO 2 Na, 227 mg, 1.27 mmol), compound 4-bromo-2,6-bis(trifluoromethyl)pyridine (A1, 250 mg, 0.85 mmol) and tetrabutylammonium bromide (Bu 4 NBr, 82 mg, 0.26 mmol) was dissolved in 4 mL of N, N-dimethylacetamide (DMAc) and stirred at 100 ° C (600 rpm) for 20 hours. After the reaction solution was cooled to room temperature, 30 mL of deionized water was slowly added, filtered, and washed with water 3 times to obtain the product. The product was further separated and purified by silica gel chromatography (eluent: dichloromethane) and vacuum dried to obtain intermediate B1 (300 mg, yield: 95.6%). Characterization data of intermediate B1: 1 H NMR (CDCl 3 , 600MHz) δ (ppm): 8.29 (s, 2H), 7.90 (d, 2H, J = 8.4Hz), 7.43 (d, 2H, J = 8.4Hz), 2.47 (s, 3H); 13 C NMR (CDCl3 ,150MHz)δ(ppm):154.73,150.50(q,J CF =37.1Hz),146.86,134.99,130.88,128.60,120.09(q,J CF =273.9Hz),120.64,21.79; 19 F NMR (CDCl 3 ,564MHz)δ(ppm):-67.91(s,6F).

[0030] Step 2: Under nitrogen protection, compound B1 (280 mg, 0.76 mmol), N-bromosuccinimide (NBS, 148 mg, 0.83 mmol) and dibenzoyl peroxide (BPO, 9 mg, 0.04 mmol) were dissolved in 5 mL of carbon tetrachloride and stirred (600 rpm) and refluxed overnight. After the reaction was completed, saturated brine was added to the reaction solution, extracted with dichloromethane, the organic phase was collected and dried with anhydrous sodium sulfate, and the solvent was evaporated to obtain a crude product. The crude product was subjected to rapid column chromatography (eluent: dichloromethane / petroleum ether = 2:1) to obtain a mixture containing the raw material and the target product C1, which was not purified and directly used in the next step.

[0031] Step 3: Under nitrogen protection, dissolve the above mixture and anhydrous pyridine (120 mg, 1.52 mmol) in 5 mL of acetonitrile, and stir (600 rpm) and reflux overnight. After the reaction is completed, cool to room temperature and evaporate the solvent to obtain a crude product. Dissolve the crude product in as little methanol as possible, add a large amount of ether to precipitate the product, filter it, and wash it with ether several times to obtain a light yellow label 1 (210 mg, two-step yield of steps 2 and 3: 52.4%). Characterization data of label 1: 1 H NMR (MeOD-d 4 ,600MHz)δ(ppm):9.10(s,br,2H),8.67(t,1H,J=7.8Hz),8.59(s,2H),8.25(d,2H,J=8.4Hz),8.16(s,br,2H),7.76(s,br,2H),6.01(s,2H); 13 C NMR (MeOD-d 4 ,150MHz)δ(ppm):155.55,151.39(q,J CF =37.1Hz),147.80,146.48,141.93,141.14(s,br),131.56(s,br),131.03,129.97,122.82,120.17(q,J CF=272.9Hz),64.49; 19 F NMR (MeOD-d 4 ,564MHz)δ(ppm):-71.02(s,6F).

[0032] Synthesis of Label 2

[0033]

[0034] Step 1: Under nitrogen protection, sodium 4-toluenesulfinate (345 mg, 1.94 mmol), compound 2-chloro-6-trifluoromethylnicotinonitrile (A2, 200 mg, 0.97 mmol) and tetrabutylammonium bromide (94 mg, 0.29 mmol) were dissolved in 3 mL of N,N-dimethylacetamide and stirred at 100 ° C (600 rpm) for 20 hours. After the reaction solution was cooled to room temperature, 30 mL of deionized water was slowly added, filtered, and washed with water 3 times to obtain the product. The product was further separated and purified by silica gel chromatography (eluent: dichloromethane) and vacuum dried to obtain intermediate B2 (137 mg, yield: 43.4%). Characterization data of intermediate B2: 1 H NMR (CDCl 3 , 600MHz) δ (ppm): 8.40 (d, 1H, J = 7.8Hz), 8.05 (d, 2H, J = 8.4Hz), 7.94 (d, 1H, J = 8.4Hz), 7.41 (d, 2H, J = 8.4Hz), 2.46 (s, 3H); 13 C NMR (CDCl 3 ,150MHz)δ(ppm):160.98,150.32(q,J CF =37.4Hz),146.77,145.76,133.71,130.31,130.03,123.07,119.92(q,J CF =274.2Hz),113.08,110.96,21.97; 19 F NMR (CDCl 3 ,564MHz)δ(ppm):-68.32(s,3F).

[0035] Step 2: Under nitrogen protection, compound B2 (130 mg, 0.40 mmol), N-bromosuccinimide (78 mg, 0.44 mmol) and dibenzoyl peroxide (5 mg, 0.02 mmol) were dissolved in 5 mL of carbon tetrachloride and stirred (600 rpm) and refluxed overnight. After the reaction was completed, saturated brine was added to the reaction solution, extracted with dichloromethane, the organic phase was collected and dried with anhydrous sodium sulfate, and the solvent was evaporated to obtain a crude product. The crude product was subjected to rapid column chromatography (eluent: dichloromethane / petroleum ether = 2:1) to obtain a mixture containing the raw material and the target product C2, which was not purified and directly used in the next step.

[0036] Step 3: Under nitrogen protection, the above mixture and anhydrous pyridine (63 mg, 0.8 mmol) were dissolved in 4 mL of acetonitrile, and stirred (600 rpm) and refluxed overnight. After the reaction was completed, it was cooled to room temperature and the solvent was evaporated to obtain a crude product. The crude product was dissolved in as little methanol as possible, and a large amount of ether was added to precipitate the product, filtered, and washed with ether several times to obtain a light yellow label 2 (85 mg, two-step yield of steps 2 and 3: 43.9%). Characterization data of label 2: 1 H NMR (MeOD-d 4 ,500MHz)δ(ppm):9.21(d,2H,J=5.8Hz),8.79(d,1H,J=8.1Hz),8.70(t,1H,J=7.8Hz),8.2 6(d,1H,J=8.1Hz),8.21(m,2H),8.14(d,2H,J=8.1Hz),7.86(d,2H,J=8.1Hz),6.13(s,2H); 13 C NMR (MeOD-d 4 ,126MHz)δ(ppm):160.40,150.49(q,J=37.0Hz),148.81,147.80,146.44,142.03,139 .14,131.73,131.11,130.01,125.58,121.36(q,J=274.6Hz),114.18,112.00,64.49; 19 F NMR (MeOD-d 4 ,471MHz)δ(ppm):-69.73(s,3F).

[0037] Synthesis of Tag 3

[0038]

[0039] Step 1: Under nitrogen protection, sodium 4-toluenesulfinate (296 mg, 1.66 mmol), compound 2-trifluoromethyl-4-bromopyridine (A3, 250 mg, 1.11 mmol) and tetrabutylammonium bromide (107 mg, 0.33 mmol) were dissolved in 3 mL of N,N-dimethylacetamide and stirred at 100 ° C (600 rpm) for 20 hours. After the reaction solution was cooled to room temperature, 30 mL of deionized water was slowly added, filtered, and washed with water 3 times to obtain the product. The product was further separated and purified by silica gel chromatography (eluent: dichloromethane) and vacuum dried to obtain intermediate B3 (330 mg, yield: 98.7%). Characterization data of intermediate B3: 1 H NMR (CDCl 3 , 500MHz) δ (ppm): 8.92 (d, 1H, J = 4.8Hz), 8.11 (s, 1H), 7.96 (d, 1H, J = 4.6Hz), 7.87 (d, 2H, J = 8.1Hz), 7.39 (d, 2H, J = 8.0Hz), 2.45 (s, 3H); 13 C NMR (CDCl 3 ,126MHz)δ(ppm):152.53,151.85,150.18(q,J=36.0Hz),146.42,136.11,1 30.86,128.68,123.76,121.02(q,J=274.8Hz),117.99(q,J=2.8Hz),22.01; 19 F NMR (CDCl 3 ,471MHz)δ(ppm):-69.99(s,3F).

[0040] Step 2: Under nitrogen protection, compound B3 (250 mg, 0.83 mmol), N-bromosuccinimide (162 mg, 0.91 mmol) and dibenzoyl peroxide (10 mg, 0.04 mmol) were dissolved in 5 mL of carbon tetrachloride and stirred (600 rpm) and refluxed overnight. After the reaction was completed, saturated brine was added to the reaction solution, extracted with dichloromethane, the organic phase was collected and dried with anhydrous sodium sulfate, and the solvent was evaporated to obtain a crude product. The crude product was subjected to rapid column chromatography (eluent: dichloromethane / petroleum ether = 2:1) to obtain a mixture containing the raw material and the target product C3, which was directly used in the next step without purification.

[0041] Step 3: Under nitrogen protection, the above mixture and anhydrous pyridine (131 mg, 1.66 mmol) were dissolved in 3 mL of acetonitrile, and stirred (600 rpm) and refluxed overnight. After the reaction was completed, it was cooled to room temperature and the solvent was evaporated to obtain a crude product. The crude product was dissolved in as little methanol as possible, and a large amount of ether was added to precipitate the product, filtered, and washed with ether several times to obtain a light yellow label 3 (169 mg, two-step yield of steps 2 and 3: 44.4%). Characterization data of label 3: 1 H NMR (MeOD-d 4 , 600MHz) δ (ppm): 9.10 (d, 2H, J = 5.9Hz), 8.99 (d, 1H, J = 5.0Hz), 8.65 (t, 1H, J = 7.8Hz), 8.26 (s, 1H), 8.17 (m, 5H), 7.76 (d, J = 8.2Hz, 2H), 6.01 (s, 2H); 13 C NMR (MeOD-d 4 ,150MHz)δ(ppm):153.50,152.95,150.72(q,J CF =36.2Hz),147.78,146.47,141.76,141.55,131.46,130.68,129.97,125.72,122.26(q,J CF =273.7Hz),118.92,64.50; 19 F NMR (MeOD-d 4 ,564MHz)δ(ppm):-69.54(s,3F).

[0042] Synthesis of Tag 4

[0043]

[0044] Step 1: Under nitrogen protection, sodium 4-toluenesulfinate (388 mg, 2.18 mmol), 4-bromo-2,8-bis(trifluoromethyl)quinoline (A4, 500 mg, 1.45 mmol) and tetrabutylammonium bromide (140 mg, 0.44 mmol) were dissolved in 5 mL of N,N-dimethylacetamide and stirred at 100 ° C (600 rpm) for 20 hours. After the reaction solution was cooled to room temperature, 30 mL of deionized water was slowly added, filtered, and washed with water 3 times to obtain the product. The product was further separated and purified by silica gel chromatography (eluent: dichloromethane) and vacuum dried to obtain intermediate B4 (500 mg, yield: 82.2%). Characterization data of intermediate B4: 1 H NMR (CDCl 3, 600MHz) δ (ppm): 8.99 (d, 1H, J = 8.7Hz), 8.54 (s, 1H), 8.23 ​​(d, 1H, J = 7.2Hz), 7. 90(d,2H,J=8.2Hz),7.85(t,1H,J=8.0Hz),7.37(d,2H,J=8.1Hz),2.42(s,3H); 13 C NMR (CDCl 3 ,150MHz)δ(ppm):148.58,148.41(q,J CF =37.2Hz),146.26,145.00,136.25,130.63,130.09(q,J CF =5.3Hz),129.97(q,J CF =30.6Hz),129.62,128.66,128.37,123.73,123.17(q,J CF =274.3Hz),120.68(q,J CF =274.4Hz),118.05,21.82; 19 F NMR (CDCl 3 ,564MHz)δ(ppm):-60.29(s,3F),-67.79(s,3F).

[0045] Step 2: Under nitrogen protection, compound B4 (300 mg, 0.72 mmol), N-bromosuccinimide (140 mg, 0.79 mmol) and dibenzoyl peroxide (9 mg, 0.04 mmol) were dissolved in 5 mL of carbon tetrachloride and stirred (600 rpm) and refluxed overnight. After the reaction was completed, saturated brine was added to the reaction solution, extracted with dichloromethane, the organic phase was collected and dried with anhydrous sodium sulfate, and the solvent was evaporated to obtain a crude product. The crude product was subjected to rapid column chromatography (eluent: dichloromethane / petroleum ether = 2:1) to obtain a mixture containing the raw material and the target product C4, which was not purified and directly used in the next step.

[0046] Step 3: Under nitrogen protection, the above mixture and anhydrous pyridine (114 mg, 1.44 mmol) were dissolved in 5 mL of acetonitrile, and stirred (600 rpm) and refluxed overnight. After the reaction was completed, it was cooled to room temperature and the solvent was evaporated to obtain a crude product. The crude product was dissolved in as little methanol as possible, and a large amount of ether was added to precipitate the product, filtered, and washed with ether several times to obtain a brown solid label 4 (88 mg, two-step yield of steps 2 and 3: 21.2%). Characterization data of label 4: 1 H NMR (MeOD-d 4,600MHz)δ(ppm):9.11(d,2H,J=5.1Hz,),8.99(d,1H,J=8.7Hz),8.68(s,1H),8.63(t,1H,J=7.8Hz),8.34(d, 1H,J=7.2Hz),8.21(d,2H,J=8.3Hz),8.14(m,2H),8.00(t,1H,J=8.0Hz),7.76(d,2H,J=7.9Hz,),6.02(s,2H); 13 C NMR (MeOD-d 4 ,150MHz)δ(ppm):149.21(q,J CF =36.4Hz),148.99,1 47.72,146.44,145.89,141.73,141.61,131.70(q,J CF =5.3Hz),131.59,131.44,130.49,130.36(q,J CF =31.6Hz),129.93,129.74,124.73,124.55(q,J CF =271.6.4Hz),122.00(q,J CF =273.5Hz),119.64,64.30; 19 F NMR (MeOD-d 4 ,564MHz)δ(ppm):-61.50(s,3F),-69.28(s,3F).

[0047] Example 2: Reaction of fluorinated tags with thiols

[0048] The experiment used glutathione as an example to verify its reactivity. At room temperature, glutathione (GSH) was quickly mixed with labels 1-4 in a phosphate buffer solution (pH = 7.4, containing 10% heavy water). The concentrations of glutathione and labels were 10.0mM and 1.0mM, respectively. The reaction solution was immediately placed in a nuclear magnetic resonance spectrometer to collect the nuclear magnetic resonance fluorine spectrum and record the time. The reactivity of the label was compared based on the change in the label integral intensity. The results are shown in Figure 1 As shown, these tags can react with thiol groups, but their activities are different. The reaction rate under this condition is: tag 2 ≈ tag 4> tag 1> tag 3. The chemical shift of fluorine changes before and after the reaction, indicating that tags 1-4 can be used for peptide or small molecule thiol detection. The results are shown in Figure 2 Shown (spectrum 180 minutes after mixing).

[0049] Example 3: Labeling GB1 protein with fluorine-containing tags

[0050] The experiment uses the model protein GB1 (T11C) as an example. At room temperature, the 3-fluorotyrosine biolabeled proteins were mixed with tags 1-4 in a Tris buffer solution (pH = 7.4), with the concentrations of protein and tag being 0.1mM and 0.3mM, respectively, and reacted overnight. The mixed solution was ultrafiltered and centrifuged five times to remove the tags and other small molecules. It can be seen from the mass spectrometry Q-TOF that the molecular ion peak of unreacted GB1 is almost unobservable, indicating that the connection efficiency of all tags is close to 100%. The results are as follows Figure 3 As shown in (proteins have multiple charges, the calculation results and predicted values ​​have been marked). The labeled GB1 was collected with a fluorine spectrum, and the result is as follows Figure 4 As shown, these tags can react with proteins and the signal intensity is significantly enhanced compared with 3-fluorotyrosine biomarkers.

[0051] Example 4: Fluorine-containing tag 1 labels GB1, CaM, UBQ and MBP

[0052] From the perspective of protein signal enhancement, label 1 contains six equivalent fluorine atoms, which has greater signal enhancement ability than other labels, so label 1 is used as an example. At room temperature, proteins containing mutant cysteine ​​(streptococcal immune G protein B1 domain GB1, calmodulin CaM, ubiquitin protein UBQ, maltose binding protein MBP) were mixed with label 1 in Tris buffer solution (pH = 7.4), with the concentrations of protein and label being 0.1mM and 0.3mM respectively, and reacted overnight. The mixed solution was subjected to multiple ultrafiltration centrifugation to remove the label and other small molecules, and the fluorine spectrum was collected. The results are shown in Figure 2. Figure 5 As shown, it is illustrated that label 1 can be applied to label proteins of different molecular weights.

[0053] Example 5 Label 1 can be used for detection of intracellular proteins

[0054] After the protein in Example 4 was labeled with Tag 1, it was introduced into HeLa cells by electroporation. The specific method is as follows: HeLa cells were digested and eluted with 0.25% trypsin, centrifuged at 200×g for 5 minutes at room temperature, and washed once with PBS buffer solution. The target protein was dissolved in the electroporation buffer solution EPB (100mM Na 3 PO 4 ,5mM KCl,15mM MgCl 2 , 15mM HEPES, 5mM ATP, 5mM GSH, pH 7.0) to a final concentration of 0.3-0.6mM. 7The cells were concentrated and suspended in 100 μL of the above EPB buffer solution containing the target protein and transferred to a 100 μL electroporation cell (model: Nucleocuvette TM Vessel) and use the CN114 program of the electroporation instrument (model Amaxa 4D-Nucleofector) to transfer proteins, which generally requires 3-4 electric shocks. The cells were then transferred to a culture dish containing 2 mL of cell culture medium and cultured for about 5 hours. The attached cells were washed again with PBS 3 times, digested and eluted with 0.125% trypsin, centrifuged at 200 × g for 5 minutes at room temperature, the cells were collected, and washed once with PBS buffer solution. The cells were resuspended in the culture medium for nuclear magnetic signal acquisition (5mM HEPES, pH 7.2, 90mM D-glucose, 10% D 2 O) 300 μL. 19 F NMR spectrum, the results are as follows Figure 6 As shown, it is shown that the connection mode between label 1 and protein is stable and can be used for the detection of intracellular protein. By adding fluorinated internal standard (3-fluorotyrosine), the integrated intensity is compared to confirm that the concentration of protein in cells is between 0.5-3μM, which is equivalent to its physiological concentration.

Claims

1. A fluorine-containing label, characterized in that: It has the following structure: in, is an electron-withdrawing aromatic ring containing a trifluoromethyl unit, wherein the structure of the electron-withdrawing aromatic ring containing a trifluoromethyl unit is 2. Use of the fluorine-containing label according to claim 1 in peptide or protein labeling, characterized in that: The polypeptide or protein contains cysteine.

3. Use of the fluorine-containing label according to claim 1 in labeling small molecules containing thiol groups.

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