CN114262356A - Method for modifying protein tryptophan residue - Google Patents
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- CN114262356A CN114262356A CN202111620984.XA CN202111620984A CN114262356A CN 114262356 A CN114262356 A CN 114262356A CN 202111620984 A CN202111620984 A CN 202111620984A CN 114262356 A CN114262356 A CN 114262356A
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 35
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- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
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- RGHHSNMVTDWUBI-UHFFFAOYSA-N 4-hydroxybenzaldehyde Chemical compound OC1=CC=C(C=O)C=C1 RGHHSNMVTDWUBI-UHFFFAOYSA-N 0.000 description 2
- GSSBOYWRKTVVQX-UHFFFAOYSA-N 4-prop-2-ynoxybenzaldehyde Chemical compound O=CC1=CC=C(OCC#C)C=C1 GSSBOYWRKTVVQX-UHFFFAOYSA-N 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical class S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
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- 235000018417 cysteine Nutrition 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
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- WKGZJBVXZWCZQC-UHFFFAOYSA-N 1-(1-benzyltriazol-4-yl)-n,n-bis[(1-benzyltriazol-4-yl)methyl]methanamine Chemical compound C=1N(CC=2C=CC=CC=2)N=NC=1CN(CC=1N=NN(CC=2C=CC=CC=2)C=1)CC(N=N1)=CN1CC1=CC=CC=C1 WKGZJBVXZWCZQC-UHFFFAOYSA-N 0.000 description 1
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- TXNLQUKVUJITMX-UHFFFAOYSA-N 4-tert-butyl-2-(4-tert-butylpyridin-2-yl)pyridine Chemical compound CC(C)(C)C1=CC=NC(C=2N=CC=C(C=2)C(C)(C)C)=C1 TXNLQUKVUJITMX-UHFFFAOYSA-N 0.000 description 1
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种修饰蛋白质色氨酸残基的方法，以缩硫醛类化合物或其衍生物为底物，在光氧化还原催化剂存在下，通过合适波长的可见光的照射，用于化学修饰蛋白质色氨酸残基。本发明可使用无毒的有机化合物或金属有机化合物为催化剂，可见光为光源，适合实验室和工业化蛋白质组学研究应用。
The invention discloses a method for modifying protein tryptophan residues, which uses thioacetal compounds or derivatives thereof as substrates, and is irradiated with visible light of suitable wavelength in the presence of photoredox catalysts for chemical modification. Protein tryptophan residues. The present invention can use non-toxic organic compounds or metal organic compounds as catalysts and visible light as light sources, and is suitable for laboratory and industrial proteomics research applications.
Description
Technical Field
The invention belongs to the field of biochemistry, relates to a protein tryptophan probe, and particularly relates to a method for modifying a protein tryptophan residue.
Background
Trp is an ideal bioconjugation residue because it is a rare (about 1% natural abundance) amino acid. It plays many important roles in protein function and has attracted increasing attention in recent years, but there is no robust modification method for tryptophan residues.
Molecules such as sulfonium and sulfonium salts containing tetravalent organic sulfur (IV) are very useful reactive functional groups in organic chemical synthesis. The sulfonium salt intermediate formed by photocatalytic desulfurization of mercaptal can generate S with an electron-rich indole ringNAr reacts to modify the side chain of tryptophan residue. The problem that the traditional protein amino acid probe is difficult to modify aromatic amino acid can be solved, so that the tryptophan probe containing the mercaptal structure has an important very wide application space in the aspect of protein side chain chemical modification.
Disclosure of Invention
The invention aims to provide a method for modifying protein tryptophan residues, which aims to solve the technical problem that the traditional protein amino acid probe in the prior art is difficult to modify aromatic amino acids.
The invention provides a method for modifying protein tryptophan residues, which comprises the following steps:
1) taking a mercaptal compound or a derivative thereof as a reaction substrate; the structural formula of the mercaptal compound or the derivative thereof is as follows,
2) R is
Or
Or
Or
R' is
Or
Adding a protein to be modified, wherein the chemical modification site is a protein tryptophan residue;
3) adopting a photo-oxidation-reduction catalyst which is an organic or metal photo-oxidation-reduction catalyst; the structural formula of the organic or metal photoredox catalyst is as follows;
(4,4 '-di-tert-butyl-2, 2' -bipyridine) bis [ (2-pyridyl) phenyl group]Iridium (III) hexafluorophosphate or
Tris (2, 2' -bipyridine) ruthenium (II) hexafluorophosphate
4) Adopting illumination, wherein the used light source is blue light, the wavelength is 430-480 nm, and the power is 10-45W;
5) the reaction solvent is a polar organic solvent, and the pH range is 4-10; the solvent is selected from any one of water, acetonitrile, methanol, ethanol, isopropanol, tert-butyl alcohol, ethylene glycol, glycerol, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide or N, N-dimethylformamide or a mixed solvent of any two of the above solvents;
6) the reaction time is 1 to 4 hours, and the modification of the protein tryptophan residue is completed.
Further, in the step 2), the charging amount of the mercaptal compound or the derivative substrate thereof is 10 to 200 equivalents, preferably 10 equivalents, of the protein.
Further, in the step 3), the catalyst was used in a concentration of 0.1. mu. mol per liter in the reaction system.
Further, in the step 6), the reaction temperature is 37 ℃.
The invention relates to a method for modifying protein tryptophan residue, which is a high-efficiency selective chemical modification method of protein tryptophan residue with mercaptal as an active functional group. The invention can use nontoxic organic compounds or metal organic compounds as catalysts, and visible light as a light source, and is suitable for laboratories and industrial proteomics research and application.
Compared with the prior art, the invention has remarkable technical progress. The invention is suitable for chemical modification of protein tryptophan residues, and realizes rapid reaction in a short time through photocatalysis. The reaction substrate and the photocatalyst used in the invention are easy to obtain and have low toxicity, and are suitable for laboratories and industrial production.
Drawings
FIG. 1 shows a general synthesis of mercaptal compounds.
FIG. 2 shows the reaction and characterization of mercaptal compounds with polypeptides.
FIG. 3 shows a fluorescent chromogenic protein gel profile of the reaction of a mercaptal probe with an ex vivo protein.
FIG. 4 is a graph of co-immunoprecipitated protein gels of the reaction of mercaptal probes with cell lysates.
FIG. 5 shows that a 10-fold single amount of TAA probe was able to react well with the protein and that the reaction reached saturation after 1 hour.
FIG. 6 shows that TAA is not capable of labeling cysteine
FIG. 7 shows TAA labeling on a non-lysine residue.
Detailed Description
The following examples serve to illustrate the invention in further detail, but the invention is by no means restricted thereto.
Example 1
1. Synthesis of mercaptal substrates
A round-bottom flask was charged with 12.2g of 4-hydroxybenzaldehyde and 11.8g of bromopropyne and dissolved with 300mL of ethanol. 13.8g of anhydrous potassium carbonate was added to the reaction system, and the reaction was refluxed with heating in an oil bath for 6 hours. After the reaction was complete, the organic solvent was removed by rotary evaporation and the resulting viscous mixture was diluted with 500mL of water. The aqueous solution was extracted with 100mL of X3 ethyl acetate, the organic phases were combined, washed with 100mL of X20.1M dilute hydrochloric acid and 100mL of X2 saturated brine, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The crude product was recrystallized from petroleum ether and ethyl acetate, and the resulting white solid was filtered and dried to give the product 4-propargyloxybenzaldehyde (13.9g, yield 87%).
1H NMR(300MHz,Chloroform-d)δ9.84(s,1H),7.95–7.67(m,2H),7.16–6.94(m,2H),4.73(d,J＝2.4Hz,2H),2.57(t,J＝2.4Hz,1H).13C NMR(75MHz,CDCl3)δ190.85,162.34,131.87,130.48,115.14,77.55,76.44,55.91。
A round bottom flask was charged with thiol (4 equivalents) and 4-propargyloxybenzaldehyde (1 equivalent), and dichloromethane was added to dissolve the starting materials. A catalytic amount of N-bromosuccinimide (NBS, 5 mol%) was added to the mixture. The solution was stirred at room temperature for 2 hours. After completion of the reaction, the organic phase was washed twice with saturated brine, dried over anhydrous sodium sulfate, and concentrated on a rotary evaporator. The crude product was purified by flash column chromatography using the eluent petroleum ether/ethyl acetate. The product structure, i.e., nuclear magnetic characterization data, is as follows.
13C NMR(75MHz,CDCl3)δ166.84,144.64,133.06,127.91,127.43,79.59,71.65,52.57,34.28,29.67,22.46,13.47.
2. Reaction of mercaptal probes with polypeptides
To a colorless, clear 1mL glass vial equipped with a magnetic stir bar was added mercaptal TA-2(2mg), polypeptide (0.5mg), and scarlet sodium salt (10 mol%) and 200. mu.L of trifluoroethanol solvent. The vial was then sealed and placed on a magnetic stirrer about 2cm from a 10W blue LED lamp and the reaction stirred for 1 hour. The reaction solvent was lyophilized and dissolved using a mixture of 200 μ l acetonitrile/water 1/1 and filtered, followed by direct separation by HPLC to give the desired product and identification of the polypeptide product structure using secondary mass spectrometry. The results are shown in FIG. 2, where the side chain of the tryptophan residue is the reactive site.
3. Reaction of mercaptal probes with isolated proteins
To verify the reactivity of covalent binding of mercaptal to tryptophan (His) at the protein tag level, 10 μ M commercial BSA (bovine serum albumin) was incubated with 100 μ M TAA in PBS solution at different pHs with 5% tiger red sodium salt catalyst at 37 ℃ under blue light (440nm) for 3 hours. Then, a 'click' reaction is used for marking a fluorescent label on the protein, and the specific method is to add CuSO into the reaction system4(1mM)，TECP(1mM)，TBTA(100μM)，5-TAMRA-N3(100. mu.M), run SDS-PAGE protein gel last, observe fluorescence in the gel. It was found from FIG. 3 that the TAA probe showed better labeling efficiency under the medium alkaline condition. To further explore the kinetics and stoichiometry of the TAA probe labeling reactions, BSA was compared to the TAA probe at different timesThe reaction occurred with an intermediate gradient and a concentration gradient, and FIG. 4 and FIG. 5 show that 10-fold protein equivalent of TAA probe was able to react well with the protein, and that the reaction reached saturation after 1 hour.
4. Reaction of mercaptal probes with cell lysates
For further use of the thioacetal probe in tryptophan labeling, there was no change in fluorescence intensity throughout the lane pretreated with IAA, indicating that TAA was not able to label cysteine (fig. 6). Furthermore, the NHS-Ace pretreated lanes showed strong marker fluorescence, confirming that TAA labeling also did not occur on lysine residues (FIG. 7).
Claims (4)
1. A method for modifying tryptophan residues in a protein, comprising the following reaction steps:
1) taking a mercaptal compound or a derivative thereof as a reaction substrate; the structural formula of the mercaptal compound or the derivative thereof is as follows,
or
R is
Or
R' is
Or
(without R');
2) adding a protein to be modified, wherein the chemical modification site is a protein tryptophan residue;
3) adding a photo-oxidation-reduction catalyst, wherein the used photo-oxidation-reduction catalyst is an organic or metal photo-oxidation-reduction catalyst; the structural formula of the organic or metal photoredox catalyst is as follows;
or
4) Adopting illumination, wherein the used light source is blue light, the wavelength is 430-480 nm, and the power is 10-45W;
5) the reaction solvent is a polar organic solvent, and the pH range is 4-10; the solvent is selected from any one of water, acetonitrile, methanol, ethanol, isopropanol, tert-butyl alcohol, ethylene glycol, glycerol, trifluoroethanol, hexafluoroisopropanol, dimethyl sulfoxide or N, N-dimethylformamide or a mixed solvent of any two of the above solvents;
6) the reaction time is 1 to 4 hours, and the modification of the protein tryptophan residue is completed.
2. The method of claim 1, wherein the tryptophan residues of the protein are modified by: in the step 2), the feeding amount of the mercaptal compound or the derivative substrate thereof is 10 equivalent of the protein.
3. The method of claim 1, wherein the tryptophan residues of the protein are modified by: in step 3), the concentration of the catalyst used in the reaction system was 0.1. mu. mol per liter.
4. The method of claim 1, wherein the tryptophan residues of the protein are modified by: in the step 6), the reaction temperature is 37 ℃.
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| CN114262356B (en) | 2023-10-24 |
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