CN113549125A - A photocatalytic method for chemical modification of biomacromolecules - Google Patents

Abstract

The invention discloses a method for photocatalytic chemical modification of biological macromolecules. The sulfur oxide Ylide compound is used as a substrate, and in the presence of an organic photocatalyst, a blue light source with a wavelength of 430 to 480 nm is used for light irradiation, which is used for chemical modification of polypeptides, biological macromolecules such as proteins and nucleic acids. The invention uses non-toxic organic compounds as catalysts and visible light as light sources, and is suitable for chemical modification and transformation of laboratory and industrialized polypeptides, proteins and nucleic acid drugs.

Description

Method for chemical modification of photo-catalytic biomacromolecule

Technical Field

The invention belongs to the field of biochemistry, relates to a sulfydryl-sulfur oxide Ylide click reaction, and particularly relates to a method for chemically modifying a photo-catalytic biological macromolecule.

Background

Chemical modification of biological macromolecules plays an important role in the research fields of chemical biology, molecular biology, drug research and development and the like. Among them, the coupling reaction of polypeptides, proteins and nucleic acids with small organic molecule drugs and probes is a powerful experimental tool for proteomics, medicinal chemistry and biotechnology research. For example, the successful development of antibody-drug conjugates has relied on the study of reliable bio-orthogonal reactions. Traditional chemical modification methods of proteins have focused mainly on chemical reactions directed to cysteine and lysine residues. In recent years, novel protein modification chemistry has focused on modification of amino acid residues, which has not been achieved under conventional conditions, or extremely efficient modification chemistry. For example, protein modification chemistry under mild conditions induced by visible light. This photo-redox catalytic mechanism provides a non-traditional strategy for biomacromolecule functionalization by creating free radicals to build unique chemical bonds.

The sulfur oxide YLide is a kind of electric amphoteric molecule, and can stably exist in water solution for a long time. In previous researches, noble metals or protonic acids are generally needed to catalyze the reaction of the noble metals or protonic acids with nucleophilic groups, and the problems of high cost, unsatisfactory reaction efficiency, harsh required reaction conditions and the like exist. Thus, the sulfur oxide Ylide active group is difficult to use for efficient, selective chemical modification of proteins under physiological conditions.

Disclosure of Invention

Aiming at the requirements of chemical selective modification technology and application of biological macromolecules, the invention provides a method for chemically modifying a photocatalytic biological macromolecule, and the method for chemically modifying the photocatalytic biological macromolecule aims to solve the technical problem that a sulfur oxide YLide active group is difficult to be used for efficient and selective chemical modification of proteins under physiological conditions in the prior art.

The invention provides a method for chemically modifying a photocatalytic biological macromolecule, which comprises the following reaction steps:

1) adding a reaction solvent into a container, wherein the reaction solvent is selected from any one of water, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerol, dimethyl sulfoxide or N, N-dimethylformamide or a mixed solvent of any two of the water, the acetonitrile, the methanol, the ethanol, the isopropanol, the tert-butanol, the ethylene glycol, the glycerol, the dimethyl sulfoxide or the N, N-dimethylformamide, and the pH range of the reaction solvent is 2 to 13;

2) then adding the modified biological macromolecules to dissolve the biological macromolecules in a reaction solvent; the biological macromolecule is any one of polypeptide, protein or nucleic acid;

3) adding a reaction substrate, namely a sulfur oxide Ylide compound and a derivative thereof, wherein the feeding amount of the sulfur oxide Ylide compound and the derivative thereof is 1 equivalent to 100 equivalents of the biomacromolecule; the structural formula of the sulfur oxide Ylide compound and the derivative thereof is as follows

4) Adding an organic photoredox catalyst, wherein the concentration of the catalyst in a reaction solvent is 10 micromole per liter to 100 micromole per liter; the organic photoredox catalyst is a riboflavin analogue; the structural formula of the riboflavin analogue organic photoredox catalyst is shown as follows;

5) adopting a blue light source for illumination, wherein the wavelength is 430-480 nm, and the power is 10-45W;

6) the reaction time is 1 minute to 1 hour, and the reaction temperature is 37 ℃; completing the chemical modification of the biological macromolecule.

Specifically, the acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerol, dimethyl sulfoxide and N, N-dimethylformamide are organic polar solvents.

The invention relates to a method for chemically modifying biological macromolecules by photocatalysis, which takes sulfur oxide Ylide compounds as substrates and is used for chemically modifying biological macromolecules such as polypeptides, proteins, nucleic acids and the like by irradiation of visible light with proper wavelength in the presence of an organic photocatalyst. The invention uses non-toxic organic compound as catalyst, visible light as light source, and is suitable for chemical modification of polypeptide, protein and nucleic acid medicine in laboratory and industrialization.

Compared with the prior art, the invention has remarkable technical progress. The invention is suitable for chemical modification of biological macromolecules in vitro or in cells, and the reaction can be rapidly carried out 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 laboratory and industrial production, and are suitable for research and application of laboratory and industrial biomacromolecule.

Drawings

FIG. 1 general Synthesis of Sulfur oxide YLide Compounds.

FIG. 2 fluorescent chromogenic protein gel profile of ex vivo reactions of thionium oxide Ylide with different proteins.

FIG. 3 is a fluorescent chromogenic protein gel graph of sulfur oxide YLide reacted with protein at different times and concentration gradients.

FIG. 4 co-immunoprecipitated protein gel graph of the reaction of thiosulfide YLide with intracellular proteins.

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. Preparation of sulphur oxide Ylide

A round bottom flask was charged with 1.38g of 4-hydroxybenzoic acid and dissolved in 20mL of ethanol. Dissolving 1g of sodium hydroxide in waterTo 20mL of water was added the reaction system. After magnetic stirring for 10 minutes, 1.2g of 3-bromopropyne was added and the reaction was continued with stirring at room temperature for 24 hours. After the reaction was completed, the organic solvent was removed by rotary evaporation, and a 2M aqueous hydrochloric acid solution was used to adjust the pH to 3, whereby a large amount of white precipitate was precipitated. Vacuum filtration, water washing of the filter cake, and vacuum drying of the obtained white solid to obtain the product 4-propargyloxybenzoic acid (1.6g, yield 91%).¹H NMR(400MHz,Methanol-d₄)δ8.06–7.98(m,2H),7.12–7.05(m,2H),4.85(d,J＝2.3Hz,2H),3.03(t,J＝2.4Hz,1H).¹³C NMR(101MHz,Methanol-d₄)δ168.26,161.58,131.36,123.43,114.26,77.83,75.91,55.36.

Under nitrogen protection, 1.5g of 4-propargyloxybenzoic acid was added to a round-bottom flask and dissolved in 20mL of dichloromethane, 0.5mL of DMF was added as a catalyst, and the mixture was cooled in an ice bath. Subsequently, after 3.2g of oxalyl chloride was added dropwise using a syringe, the temperature of the reaction system was slowly raised to room temperature, and the reaction was continued for 3 hours. After the reaction is finished, the reaction solvent is distilled off under reduced pressure under the drying condition, and the acyl chloride intermediate is obtained for standby.

In a further round-bottomed flask was added 5.6g of trimethyl sulphoxide iodide and 5.7g of potassium tert-butoxide. Under nitrogen, 50mL of tetrahydrofuran was added, and the reaction mixture was heated to reflux for 3 hours. After the reaction was completed, the reaction system was cooled in an ice bath, and the above acid chloride intermediate was dissolved in 20mL of tetrahydrofuran, and the system was added dropwise, and the reaction was continued for 6 hours after warming to room temperature. After the reaction was complete, the solvent was removed by rotary evaporation and the resulting viscous mixture was diluted with 30mL of water. The aqueous solution was extracted with 30mL of 3 dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, concentrated by rotary evaporation and separated on a silica gel column to give the desired 4-propargyloxybenzoylthio A (1.7g, yield 80%).¹H NMR(400MHz,Chloroform-d)δ7.80–7.71(m,2H),6.99–6.91(m,2H),4.91(s,1H),4.71(d,J＝2.4Hz,2H),3.48(s,6H),2.52(t,J＝2.4Hz,1H).¹³C NMR(101MHz,Chloroform-d)δ181.79,159.75,132.58,128.44,114.44,78.34,75.95,67.67,55.94,42.72.

2. Reaction of Sulfur oxide YLIDE with Ex vivo proteins

Lysozyme (lysozyme) was dissolved in PBS at a concentration of 30uM, 10. mu.L was taken in a 0.6ml EP tube, and YLide A (100uM) and riboflavin phosphate sodium salt (10uM) as a catalyst were added, followed by 1 minute of blue light at 440 nm. Then, a 'click' reaction is utilized to mark a fluorescent label for the protein, and the specific method is to add CuSO into the system₄(1mM), TECP (1mM), TBTA (10mM), 5-Tamra-N3(100uM), the entire reaction system was incubated at 37 ℃ for 2 hours. After the reaction is finished, the reaction solution is fixed by using a Loading Buffer, and then is separated by using 15 percent Page gel electrophoresis, and fluorescence is observed at the emission wavelength of 546 nm. While the classical method IAA was used for comparison, the protein could be modified by incubating the protein with conventional IAA for 2 hours. The same procedure was used to modify BSA (bovine serum albumin). The results are shown in FIG. 2. Under the conditions of catalyst existence and illumination, the Lysozyme and BSA have obvious reaction bands, namely, the modification reaction is smoothly carried out. And compared with the traditional IAA modification method, the intensity of the reaction band of the sulfur Ylide photoreaction system is stronger. The method is proved to achieve better protein modification effect in shorter time, and simultaneously, blue light can be used as a switch to control the reaction. Protein modification can be performed as an alternative to a classical approach.

And meanwhile, the Lysozyme is used as a template to screen the concentration of the catalyst and the illumination time of 440 nanometers, and the reaction time and the concentration of the protein modification process are optimized. The results are shown in FIG. 3. The reaction band becomes saturated already at 1 minute and becomes stronger with increasing concentration. The result proves that the blue light of 440 nm can reach good modification effect after being irradiated for 1 minute, and the reaction effect is better when the concentration of the YLide is higher.

3. Reaction of Sulfur oxide Ylide with intracellular proteins

The method selects the Hela cell which is commonly used as the model cell. Hela cells were cultured in 10cM diameter culture dish in 10% DMEM medium at an initial density of 30% for 12 hours at 37 ℃. YLide (1mM) was dissolved in 10% DMEM medium together with riboflavin (Catalyst A,100uM), riboflavin phosphate sodium salt (Catalyst B,100uM), and riboflavin tetrabutyl ester (Catalyst C,100uM), respectively, and the cells were replaced with this medium, cultured at 37 ℃ for 12 hours, and then irradiated with 440 nm blue light for 1 minute.

The collected cells were resuspended in PBS, the cells were lysed by sonication, the supernatant was taken by ultracentrifugation, and the BCA concentration was determined. Taking lysate containing 10 micrograms of protein, and marking a protein with a Biotin label by utilizing click reaction, wherein the specific method is to add CuSO into a system₄(1mM), TECP (1mM), TBTA (10mM), Biotin-N3(100uM), the entire reaction system was incubated at 37 ℃ for 2 hours. After the reaction is finished, fixing the mixture by using Loading Buffer, then carrying out electrophoretic separation by using 10% Page gel, transferring the protein in the Page gel to a PC membrane, dissolving skim milk with the volume percentage concentration of 5% in TBST to block the PC membrane for 1 hour, then incubating the PC membrane with Anti-biotin for 12 hours at 4 ℃, and detecting by using a gel imager. The results are shown in FIG. 4. In the presence of three different catalysts, reaction bands appear, and the reaction of the sulfur Ylide reaction system in living cells is proved to be successful. Wherein, the reaction is more obvious in the presence of riboflavin sodium phosphate and riboflavin tetrabutyl ester serving as catalysts.

The experimental results are combined, and the photocatalytic sulfur oxide YLide click reaction system only needs 1 minute of illumination reaction time to achieve the ideal effect of the traditional protein cysteine modification method. Therefore, the novel reaction system is a more efficient protein modification technology compared with the traditional method. And the reaction condition in the cells also shows more biotin co-immunoprecipitation bands, which proves the good biocompatibility and application prospect of the reaction system.

Claims (1)

1. A method for chemically modifying a biological macromolecule by photocatalysis is characterized by comprising the following reaction steps:

1) adding a reaction solvent into a container, wherein the reaction solvent is selected from any one of water, acetonitrile, methanol, ethanol, isopropanol, tert-butanol, ethylene glycol, glycerol, dimethyl sulfoxide or N, N-dimethylformamide or a mixed solvent of any two of the water, the acetonitrile, the methanol, the ethanol, the isopropanol, the tert-butanol, the ethylene glycol, the glycerol, the dimethyl sulfoxide or the N, N-dimethylformamide, and the pH range of the reaction solvent is 2 to 13;

2) then adding the modified biological macromolecules to dissolve the biological macromolecules in a reaction solvent; the biological macromolecule is any one of polypeptide, protein or nucleic acid;

3) adding a reaction substrate, namely a sulfur oxide Ylide compound and a derivative thereof, wherein the feeding amount of the sulfur oxide Ylide compound and the derivative thereof is 1 equivalent to 100 equivalents of the biomacromolecule; the structural formula of the sulfur oxide Ylide compound and the derivative thereof is as follows

The R' substituent is hydrogen, oxyl, alkylamino or halogen; the position of the N atom is at the 2-, 3-or 4-position; x is an S atom, an O atom or an N-methyl group; r' is alkyl, alkenyl, alkynyl or an amino acid residue;

4) adding an organic photoredox catalyst, wherein the concentration of the catalyst in a reaction solvent is 10 micromole per liter to 100 micromole per liter; the organic photoredox catalyst is a riboflavin analogue; the structural formula of the organic photoredox catalyst is as follows;

5) adopting a blue light source for illumination, wherein the wavelength is 430-480 nm, and the power is 10-45W;

6) the reaction time is 1 minute to 1 hour, and the reaction temperature is 37 ℃; completing the chemical modification of the biological macromolecule.

Images