CN113912525B - Probe for modifying protein cysteine residue and preparation method thereof - Google Patents

Abstract

The invention discloses a probe for modifying protein cysteine residue, which has the following structural general formula:the invention also provides a preparation method of the probe for modifying protein cysteine residue, which comprises the steps of adding substrate halogenated beta-carbonyl compound I and thioether II into a reaction container, wherein the structural formula of the halogenated beta-carbonyl compound I is as follows,the structural formula of the thioether II isThe product is directly separated out in a form of precipitation, or the reaction solvent is added with equal volume of diethyl ether to promote the separation of the product. Experiments prove that the protein cysteine probe III is very safe to cells, and can be used as a cysteine modified probe of living cells for development and exploration.

Description

A probe for modifying protein cysteine residues and its preparation method

technical field

The invention belongs to the field of biochemistry, and relates to a probe for modifying protein cysteine residues and a preparation method thereof, specifically a class of β-carbonylsulfide derivatives, which can be used for modifying protein cysteine acid residues.

Background technique

The chemical modification of proteins is of great significance to the basic research on the structure and function of the proteome, as well as the synthesis and modification of biomacromolecular drugs. Electrophilic groups targeting nucleophilic amino acid side chains have been used to create covalent ligands and drugs. Protein cysteine (Cys) is one of the essential amino acids for organisms, and plays a vital role in maintaining various physiological functions. The cysteine side chain residue as an amino group plays many important roles in protein function. The sulfhydryl side chain is a good nucleophilic group, and its chemical modification is expected to change the chemical structure of the protein to change its spatial structure, thereby improving its biological activity and function. Therefore, using chemical methods to modify cysteine in biological samples is an important means to study the three-dimensional structure and physiological functions of proteins, and it is also a method for directional modification of protein properties. It has broad application prospects in protein engineering and omics research.

Sulfonium salt compounds have good water solubility and are common active functional groups in living organisms. They do not need to use other organic solvents when interacting with living organisms, which is environmentally friendly and efficient. Using sulfonium salt as the key structural unit of the probe has better biocompatibility, lower biotoxicity, higher systemic and higher selectivity, and can solve other amino acid probes such as iodoacetamide as Due to the high toxicity of cysteine probes to cells, protein cysteine probes containing sulfonium salts have an important and very broad application space in chemical modification of protein side chains.

Contents of the invention

Aiming at the above-mentioned technical problems in the prior art, the present invention provides a probe for modifying protein cysteine residues and a preparation method thereof, the described probe for modifying protein cysteine residues The probe and its preparation method should solve the technical problem of high cytotoxicity of amino acid probes such as iodoacetamide used as cysteine probes in the prior art.

The present invention provides a probe for modifying protein cysteine residues, the general structural formula of which is as follows:

Wherein, R ^is selected from: hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkenyl, C2-C6 haloalkenyl, C2 -C6 alkynyl, C2-C6 haloalkynyl, hydroxyl, C3-C6 cycloalkyl, substituted alkylamino, substituted phenylamino, substituted piperidin-1-yl, substituted morpholine- 1-yl, substituted tetrahydropyrrol-1-yl, phenyl, halogen substituted phenyl, C1-C6 alkyl substituted phenyl, C1-C6 haloalkyl substituted phenyl, C3-C6 cycloalkyl Substituted phenyl, nitro substituted phenyl, C2-C6 alkenyl substituted phenyl, C2-C6 haloalkenyl substituted phenyl, C3-C6 cycloalkenyl substituted phenyl, C2-C6 alkynyl Substituted phenyl, C2-C6 haloalkynyl substituted phenyl, C3-C6 cycloalkynyl substituted phenyl, or pyridyl;

R ² and R ³ are respectively selected from: C1-C6 alkyl, C1-C6 haloalkyl, C2-C6 alkenyl, C2-C6 haloalkenyl, C2-C6 alkynyl, C2-C6 haloalkynyl, benzene Halogen substituted phenyl, C1-C6 alkyl substituted phenyl, C1-C6 haloalkyl substituted phenyl, C3-C6 cycloalkyl substituted phenyl, nitro substituted phenyl, C2-C6 alkene C2-C6 haloalkenyl substituted phenyl, C3-C6 cycloalkenyl substituted phenyl, C2-C6 alkynyl substituted phenyl, C2-C6 haloalkynyl substituted phenyl , C3-C6 cycloalkynyl substituted phenyl, or pyridyl;

The halogen is fluorine, chlorine, bromine or iodine;

The alkyl, alkenyl, and alkynyl are linear or branched alkyl; the alkyl itself or as part of other substituents is selected from methyl, ethyl, propyl, butyl, pentyl, hexyl or isomers selected from isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl or tert-amyl;

The haloalkyl group is selected from groups containing one or more identical or different halogen atoms, and the haloalkyl group is selected from CH ₂ Cl, CHCl ₂ , CCl ₃ , CH ₂ F, CHF ₂ , CF ₃ , CF ₃ CH ₂ , CH ₃ CF ₂ , CF ₃ CF ₂ or CCl ₃ CCl ₂ ;

The cycloalkyl itself or as part of other substituents is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;

The alkenyl itself or as part of other substituents is selected from vinyl, allyl, 1-propenyl, buten-2-yl, buten-3-yl, penten-1-yl, penten- 3-yl, hexen-1-yl or 4-methyl-3-pentenyl;

The alkynyl itself or as part of other substituents is selected from ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, 1-methyl- 2-butynyl, hexyn-1-yl or 1-ethyl-2-butynyl.

X is selected from: alkanoate, substituted alkanoate, halogen and its oxo acid, phosphate, sulfate, sulfonate or borate.

The alkyl acid group, the substituted alkyl acid group is selected from C1-C6 alkyl acid group or C1-C6 halogenated alkyl acid group;

The halogen and its oxyacids are selected from fluorine, chlorine, bromine or iodine, hypochlorite, chlorite, chlorate, perchlorate, hypobromite, bromite, bromate, perbromate, iodine acid, iodate, iodate or periodate;

The phosphate is selected from monohydrogen phosphate, dihydrogen phosphate, pyrophosphate, metaphosphate, hypophosphite, phosphite, polyphosphate, phosphate or hexafluorophosphate;

The sulfate group is selected from sulfide, bisulfate, sulfate, bisulfite, sulfite, pyrosulfate, dithionite, thiosulfate, dithionite or persulfate;

The sulfonate is selected from trifluoromethanesulfonate, methanesulfonate, phenylsulfonate or p-toluenesulfonate;

The borate is selected from borate or tetrafluoroborate.

Specifically, the R ¹ is preferably selected from: phenyl, substituted phenyl, substituted alkylamino, substituted aniline; R ² and R ³ are respectively selected from: C1-C6 alkyl, C3-C6 cycloalkyl , Substituted phenyl; X is preferably selected from: bromine or tetrafluoroborate.

The present invention also provides a method for preparing a probe for modifying protein cysteine residues, comprising the steps of:

Add substrate halogenated β-carbonyl compound I and thioether II in a reaction vessel, the structural formula of described halogenated β-carbonyl compound I is,

The structural formula of the thioether II is /> The reaction solvent is selected from: dichloromethane, toluene, acetonitrile, diethyl ether, tetrahydrofuran, the product is precipitated directly in the form of precipitation, or adding an equal volume of diethyl ether to the reaction solvent to promote the precipitation of the product, the yield is 82%-96%; compound III prepared The amount and the volume of the reaction vessel are expanded or reduced in proportion.

Specifically, the thioether II is selected from: dimethyl sulfide, tetrahydrothiophene, methyl phenyl sulfide, diphenyl sulfide or dibenzyl sulfide.

The synthetic method of protein cysteine probe III of the present invention is as follows:

The present invention also provides the above-mentioned β-carbonylsulfonium salt compounds or derivatives thereof.

The present invention also provides the use of the above-mentioned probe or its intermediate in modifying protein cysteine.

The present invention also provides a method for modifying protein cysteine residues using the above-mentioned probe, characterized in that the protein to be modified is added, and the chemical modification site is a protein cysteine residue; protein Cysteine Probe III was fed in the range of 0.1 to 200 equivalents of protein.

Further, the reaction solvent is water or a polar organic solvent: the solvent is selected from water, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerin, trifluoroethanol, hexafluoro Any one of isopropanol, dimethyl sulfoxide or N,N-dimethylformamide or a mixed solvent of any two of them.

Further, the reaction time is from 0.1 hour to 100 hours.

Further, the reaction temperature is -20 to 50 degrees Celsius.

The present invention is aimed at the requirement of chemoselective modification technology and application of protein cysteine residues, and provides a method for chemically modifying protein cysteine with high efficiency. Efficient and selective chemical modification of protein cysteine residues as reactive functional groups.

The beneficial effect of the present invention is: the protein cysteine probe III has been synthesized, and the chemical biological activity of the protein cysteine probe III has been explored, including protein chemical modification and chemical proteomics research wait. Compared with the prior art (mainly including iodoacetamide compounds, etc.), the invention has obvious advantages in terms of water solubility, cell penetration and cytotoxicity.

Description of drawings

Figure 1 shows the reaction of Protein Cysteine Probe III with bovine serum albumin.

Figure 2 shows the reaction of Protein Cysteine Probe III with cell lysates and the toxicity of commercial probes.

Detailed ways

The present invention more specifically illustrates the synthesis and biological application of protein cysteine probe III through specific preparation and chemical biological examples. It is an example to illustrate, rather than limit the patent, the specific implementation is as follows:

Embodiment 1: the preparation of compound III-1:

Add 1.2 g of I and 1.5 mL of tetrahydrothiophene to a 100 mL one-necked round bottom flask. 30 ml of reaction solvent dichloromethane was added. After the completion of the reaction monitored by TLC, the product was directly precipitated in the form of a precipitate, the product was 1.4 g of white powder, and the yield was 88%. The NMR data of this compound are as follows: ¹ H NMR (400MHz, D ₂ O) δ7.91(d, J=8.9Hz, 2H), 7.12–7.05(m, 2H), 4.81(d, J=2.3Hz, 2H ), 3.62(dt, J=13.2, 6.5Hz, 2H), 3.46(dt, J=12.5, 5.5Hz, 2H), 2.90(t, J=2.3Hz, 1H), 2.27(dhept, J=14.0, 6.4,5.7Hz,4H).

Synthesis and characterization data of similar compounds:

White powder, yield 85%. ¹ H NMR (400MHz, D ₂ O) δ7.96–7.89 (m, 2H), 7.73–7.64 (m, 1H), 7.52 (t, J=7.9Hz, 2H), 2.94(s,6H).

White powder, yield 89%. ¹ H NMR (400MHz, D ₂ O) δ7.92(d, J=7.5Hz, 2H), 7.69(t, J=7.4Hz, 1H), 7.52(t, J= 7.7Hz, 2H), 3.64(dt, J=13.1, 6.0Hz, 2H), 3.48(h, J=5.6, 4.5Hz, 2H), 2.28(pd, J=13.3, 12.7, 4.8Hz, 4H).

White powder, yield 81%. ¹ H NMR (400MHz, D ₂ O) δ7.97–7.89 (m, 2H), 7.74–7.64 (m, 1H), 7.55–7.47 (m, 2H), 3.59–3.48 (m,2H),3.25(ddd,J=12.8,9.1,3.1Hz,2H),2.10(dtt,J=14.8,7.3,3.4Hz,2H),1.90(dtt,J=15.7,9.0,3.4Hz ,2H),1.77–1.54(m,2H).

White powder, yield 74%. ¹ H NMR (400MHz, D ₂ O) δ7.41–7.30 (m, 4H), 7.26–7.14 (m, 1H), 2.93 (s, 6H).

White powder, yield 82%. ¹ H NMR (400MHz, D ₂ O) δ7.42–7.31(m,4H),7.26–7.15(m,1H),3.67–3.42(m,4H),2.38–2.16 (m,4H).

White powder, yield 90%. ¹ H NMR (400MHz, D ₂ O) δ4.37(dd, J=5.0, 2.8Hz, 1H), 4.25(q, J=7.1Hz, 2H), 2.94(s, 6H), 1.23(t, J=7.1Hz, 3H).

White powder, yield 91%. ¹ H NMR (400MHz, D ₂ O) δ4.33–4.20(m,3H),3.69–3.57(m,2H),3.57–3.46(m,2H),2.39–2.18 (m,4H),1.24(t,J=7.2Hz,3H).

White powder, yield 89%. ¹ H NMR (400MHz, D ₂ O) δ7.93–7.84(m,2H),7.06–6.97(m,2H),3.83(s,3H),3.67–3.56(m ,2H),3.52–3.37(m,2H),2.37–2.17(m,4H).

Pale yellow powder, yield 88%. ¹ H NMR (400MHz, D ₂ O) δ8.35–8.27 (m, 2H), 8.15–8.07 (m, 2H), 3.73–3.62 (m, 2H), 3.57– 3.46(m,2H),2.41–2.20(m,4H).

Pale yellow powder, yield 81%. ¹ H NMR (400MHz, D ₂ O) δ8.19 (dd, J=8.3, 1.1Hz, 1H), 7.85 (td, J=7.6, 1.2Hz, 1H), 7.75 (ddd, J=8.3,7.6,1.5Hz,1H),7.59(dd,J=7.6,1.5Hz,1H),3.74–3.63(m,2H),3.58–3.47(m,2H),2.39–2.22 (m,4H).

White powder, yield 92%. ¹ H NMR (400MHz, D ₂ O) δ8.01–7.91(m,2H),7.26–7.16(m,2H),3.61(dt,J＝13.6,6.9Hz,2H ), 3.45(dt, J＝11.8, 5.7Hz, 2H), 2.39–2.15(m, 4H).

White powder, yield 94%. ¹ H NMR (400MHz, D ₂ O) δ7.90–7.82 (m, 2H), 7.55–7.47 (m, 2H), 3.62 (dt, J＝13.6, 6.8Hz, 2H ), 3.46(dt, J＝13.8, 6.6Hz, 2H), 2.35–2.16(m, 4H).

White powder, yield 90%. ¹ H NMR (400MHz, D ₂ O) δ7.85–7.78 (m, 2H), 6.92–6.84 (m, 2H), 3.60 (dt, J＝13.9, 6.7Hz, 2H ), 3.43(dt, J＝13.7, 5.9Hz, 2H), 2.35–2.15(m, 4H).

White powder, yield 83%. ¹ H NMR (400MHz, D ₂ O) δ7.96(dd, J=5.0, 1.1Hz, 1H), 7.90(dd, J=3.9, 1.1Hz, 1H), 7.21( dd,J＝5.0,4.0Hz,1H),3.68–3.55(m,2H),3.54–3.43(m,2H),2.38–2.17(m,4H).

White powder, yield 80%. ¹ H NMR (400MHz, D ₂ O) δ7.96–7.88 (m, 2H), 7.13–7.05 (m, 2H), 4.81 (d, J=2.2Hz, 2H), 2.92(s,6H),2.89(t,J=2.4Hz,1H).

White powder, yield 74%. ¹ H NMR (400MHz, D ₂ O) δ7.54–7.46(m,2H),7.46–7.37(m,2H),4.51(s,2H),3.46(s,1H ),2.97(s,6H).

White powder, yield 75%. ¹ H NMR (400MHz, D ₂ O) δ7.53–7.47(m,2H),7.43–7.37(m,2H),4.42(s,2H),3.67–3.60(m ,2H),3.52(dt,J=12.0,5.9Hz,2H),3.46(s,1H),2.38–2.24(m,4H).

Embodiment 2: the preparation of compound III-2:

1.2 g of I, 2.5 ml of methyl phenyl sulfide and 1.9 g of silver tetrafluoroborate AgBF ₄ were added to a 100 ml one-necked round bottom flask. 30 ml of reaction solvent dichloromethane was added. After TLC monitors that the reaction is complete, use diatomaceous earth to filter under reduced pressure, combine the organic phases and concentrate under reduced pressure to remove excess solvent, and the residue is purified by 100-200 mesh silica gel column chromatography to obtain compound III, and the eluent is dichloromethane:methanol , the volume ratio is 30:1, the product is 1.7 g of colorless oil, and the yield is 44%. The NMR data of the compound are as follows: ¹ H NMR (400MHz, Methanol-d ₄ )δ8.10(d, J=7.8Hz, 2H), 8.06–8.00(m, 2H), 7.81–7.71(m, 3H), 7.20–7.12(m,2H),5.11(s,2H),3.35(s,3H),2.83(s,1H).

Synthesis and characterization data of similar compounds:

White powder, yield 61%. ¹ H NMR (400MHz, MeOD) δ8.18–8.11(m,2H),8.11–8.03(m,2H),7.89–7.70(m,4H),7.60(t,J =7.8Hz,2H),3.41(s,3H).

White powder, yield 71%. ¹ H NMR (400MHz, MeOD) δ8.17-8.08 (m, 6H), 7.88-7.72 (m, 7H), 7.67-7.59 (m, 2H).

Embodiment 3: the reaction of protein cysteine probe III of the present invention and isolated protein:

In order to verify the reactivity of the protein cysteine probe covalently bound to cysteine at the protein labeling level, the protein was dissolved in distilled water and prepared into a protein solution of 5 μM to 100 μM. Accurately weigh the protein cysteine probe III and use PBS buffer solution or distilled water to prepare the corresponding concentration of the reaction test solution. The protein solution and the probe were incubated in PBS solution at 37°C. Then use the "click" reaction to label the protein with a fluorescent tag. The specific method is to add CuSO ₄ , TECP, TBTA, 5-TAMRA-N ₃ to the reaction system, incubate at 25°C, and the reaction ends. Finally, run SDS-PAGE protein gel to observe the fluorescence in the gel. The protein may be bovine serum albumin (BSA), horse serum albumin (HSA).

From the labeling experiment of bovine serum albumin in Figure 1a, 1b, we observed that the sulfur salt probe has strong fluorescence, CP1, CP2 and CP3 all show strong fluorescent labeling ability, especially the high fluorescent labeling ability of CP2 exceeds Commercial cysteine probe IAA-alkyne, so we choose CP2 for subsequent protein labeling reaction. From Fig. 1c, it is found that the CP2 probe has a certain fluorescence intensity after reacting with the protein for 10 minutes, and simultaneously with the prolongation of the reaction time, the reaction fluorescence intensity of the protein increases, which proves that the protein cysteine probe III of the present invention and bovine serum albumin The reaction speed is faster. In terms of the reaction dose (Fig. 1d), when the concentration of the probe is 5 μM, the reaction with bovine serum albumin will show a certain amount of fluorescence. At the same time, the fluorescence will gradually increase with the increase of the concentration of CP2. have an important impact.

Commercial cysteine probe IAA-alkyne was used as a control to study the specific labeling of cysteine by the probe of the present invention. IAA is a commercially available blocking reagent for cysteine, and CP-B is a probe used for blocking cysteine in the present invention. From Figure 1e, it was found that the reaction between bovine serum albumin and CP2 probe could be competed by IAA and CP-B. As the concentration of IAA and CP-B increased, the fluorescence was gradually blocked, indicating that the label could be commercialized semi- Blocked by cystine blocking reagent, and the fluorescence change trend of the probe CP2 of the present invention is consistent with that of the commercial cysteine probe IAA-alkyne, which proves the selectivity of the protein cysteine probe III in this study on protein cysteine residues.

Embodiment 4: the reaction of protein cysteine probe III of the present invention and cell lysate:

The harvested cells were lysed by ultrasound to determine the concentration. Accurately weigh the protein cysteine probe III and use PBS buffer solution or distilled water to prepare the corresponding concentration of the reaction test solution. Dilute the cell lysate to an appropriate concentration and incubate with the probe in PBS solution at 37°C. Then use the "click" reaction to label the protein with a fluorescent tag. The specific method is to add CuSO ₄ , TECP, TBTA, 5-TAMRA-N ₃ to the reaction system, incubate at 25°C, and the reaction ends. Finally, run SDS-PAGE protein gel to observe the fluorescence in the gel. Cells can be A549, 293T, Hela, MCF-7, etc.

From the labeling experiments of cell lysates in Figure 2a and 2b, we observed that the carbonyl sulfide salt probe CP2 has a good fluorescent labeling effect on cells, and the CP2 probe has a certain fluorescence intensity after reacting with the cell lysate for 10 minutes, and at the same time With the prolongation of the reaction time and the enhancement of the fluorescence intensity of the protein, the fluorescence tends to balance at 3-6h. In terms of the reaction dose (Figure 2b), when CP2 is 5 μM, it shows a certain amount of fluorescence when it reacts with the cell lysate. With the increase of CP2 concentration, the fluorescence gradually increases, and the probe can have a large Good marking effect.

The commercial cysteine probe IAA-alkyne was also used as a control in the cell lysate, the commercial cysteine blocking reagent IAA was used for blocking cysteine, and the CP-B of the present invention was used for cysteine Acid-blocked probes. From Figure 2d, it is found that the reaction of the cell lysate and the CP2 probe can be competed by IAA and CP-B, and as the concentration of IAA and CP-B increases, the fluorescence is gradually blocked, and simultaneously the probe CP2 of the present invention has an effect on the cells. The fluorescent labeling effect of the lysate is consistent with the trend of the commercial cysteine probe IAA-alkyne, and the cell lysate also shows that the cysteine probe III in this study can selectively act on protein cysteine acid residues.

Example 5: Toxicity study of the protein cysteine probe III of the present invention:

The protein cysteine probe III and cytotoxicity assay of the present invention, the specific steps are: in 96-well plates, about 5000 cells per well, the protein lysine probe III and the cells are cultivated for 20 hours, and then added MTT reagent continued to incubate for 4 hours. Remove the medium, add DMSO to dissolve water-insoluble blue-purple crystalline formazan, detect the absorbance value with a microplate reader, and calculate the relative cell viability compared with the control.

At the same time, compared with the commercial probe IAA-alkyne, the CP2 probe is safer to cells. From Figure 2c, as the concentration of the drug increases, the survival rate of the cells treated with CP2 decreases slowly, but the survival rate of the cells treated with the commercial probe IAA-alkyne decreases rapidly. It is close to 80%, while the cell survival rate of commercial IAA-alkyne is only 30% at this concentration, which proves that the protein cysteine probe III of the present invention is very safe to cells and can be used as a cysteine modification probe for living cells. Needle development exploration.

Claims (6)

1. A probe for modifying a cysteine residue of a protein, characterized by any one of the following structural formulas:

。

2. use of the probe of claim 1 for protein cysteine modification.

3. A method for modifying a protein cysteine residue using the probe of claim 1, wherein a protein to be modified is added, and the chemical modification site is a protein cysteine residue; the probe charge is 0.1 to 200 equivalents of protein.

4. A method according to claim 3, wherein the reaction solvent is selected from any one of water, acetonitrile, methanol, ethanol, isopropanol, t-butanol, ethylene glycol, glycerol, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide, N-dimethylformamide or a mixed solvent of any two thereof.

5. A process according to claim 3, wherein the reaction time is from 0.1 hours to 100 hours.

6. A method according to claim 3, wherein the reaction temperature is from-20 to 50 degrees celsius.