Free radical optical coupling method, kit for realizing optical coupling and application

Abstract

The invention belongs to the technical field of coupling, and relates to a free radical optical coupling method, a kit for realizing optical coupling and application. The free radical photo-coupling method comprises the following steps: modifying a to-be-coupled substance A with an o-nitrobenzene compound a with photosensitive activity to obtain a substance A-a with photosensitive activity; modifying an organic halide b on a to-be-coupled substance B to obtain a substance B-b; a-a and B-b trigger a reaction between a and b in the presence of a catalyst and under the illumination condition, and efficient and rapid coupling between the to-be-coupled substance A and the to-be-coupled substance B is achieved. Compared with the prior art, the method provided by the invention has the advantages that light-triggered rapid and efficient coupling can be realized, and the conversion efficiency (gt; therefore, the application problem of the free radical optical coupling technology is solved, and the application range of the free radical optical coupling technology is widened.

Description

Technical Field

[0001] The invention belongs to the technical field of coupling, and relates to a free radical photocoupling method, a kit for realizing photocoupling, and application. Background Art

[0002] Photocoupling technology refers to the process of coupling two substances to form a whole under light-triggered conditions. It has many applications in surface functionalization, polymer coupling, and modification and labeling of biomacromolecules. Thanks to the extremely high reactivity of free radicals, among the existing photocoupling technologies, the fastest coupling speed is achieved by free radicals reacting with double bonds (the reaction rate constant of the two can be as high as 10 4 M -1 s -1 ), the time required is the shortest. However, the high activity of free radicals also causes problems such as poor controllability and selectivity of the reaction, so the conversion efficiency of the reaction between free radicals and double bonds is generally low (the conversion rate is often less than 50% or even lower). Summary of the invention

[0003] In view of the shortcomings of the existing free radical photocoupling technology in terms of conversion efficiency, the present invention provides a free radical photocoupling method, a kit for realizing photocoupling and its application.

[0004] The present invention proposes a new free radical photocoupling method, in which a molecule a having photosensitivity and an organic halogenated compound b molecule are respectively connected to two substances to be coupled, and in the presence of a catalyst, the reaction between the molecule a and the molecule b is triggered by light to achieve efficient and rapid coupling of the two substances to be coupled.

[0005] The method provided by the present invention can realize fast and efficient light-triggered coupling and improve the conversion efficiency of free radical photocoupling reaction (>85%), thereby solving the application problems of free radical photocoupling technology and broadening the application scope of free radical photocoupling technology.

[0006] The present invention can be achieved through the following technical solutions:

[0007] The present invention provides a free radical photocoupling method, comprising the following steps:

[0008] Modifying the o-nitrobenzene compound a with photosensitizing activity on the substance to be coupled A to obtain the photosensitizing active substance Aa;

[0009] Modifying the organic halogenated compound b on the substance to be coupled B to obtain the substance Bb;

[0010] Aa and Bb trigger the reaction between a and b under the conditions of catalyst and light, achieving efficient and rapid coupling between the coupling substances A and B;

[0011] The o-nitrobenzene compound a refers to a compound containing a substituent group R at the ortho position of the nitro group on the benzene ring, and the o-nitrobenzene compound a is as shown in Formula I, wherein the substituent group R is ethyl, hydroxymethyl or 1-methylhydroxymethyl, and the other hydrogen atoms on the benzene ring in Formula I are allowed to be substituted by any number of other groups, wherein at least one substituent is an active group capable of connecting to the substance to be coupled A;

[0012] The organic halide b refers to an organic compound containing halogen and at least one active group capable of connecting with the substance to be coupled B, as shown in Formula II, wherein X is a halogen selected from Cl, Br or I.

[0013]

[0014] In one embodiment of the present invention, in the o-nitrobenzene compound a, the active group capable of connecting with the substance to be coupled A is selected from:

[0015] hydroxyl group, carboxyl group, amine group, ester group, mercapto group, aldehyde group, vinyl group, epoxy group, halogen group, isocyanate group, siloxane group, catechol group, o-phthalaldehyde group, or,

[0016] The terminal group is modified with an alkylene group or a modified alkylene group having a hydroxyl group, a carboxyl group, an amine group, an ester group, a mercapto group, an aldehyde group, a vinyl group, an epoxy group, a halogen group, an isocyanate group, a siloxane group, a catechol group or a phthalaldehyde group.

[0017] In one embodiment of the present invention, the modified alkylene group is obtained by replacing any carbon atom of the alkylene group with other heteroatoms.

[0018] In one embodiment of the present invention, preferably, the o-nitrobenzene compound a is selected from the following structures:

[0019]

[0020] Wherein, R1 is an active group connected to the substance A to be coupled.

[0021] The active group is selected from:

[0022] hydroxyl group, carboxyl group, amine group, ester group, mercapto group, aldehyde group, vinyl group, epoxy group, halogen group, isocyanate group, siloxane group, catechol group, o-phthalaldehyde group, or,

[0023] The terminal group is modified with an alkylene group or a modified alkylene group having a hydroxyl group, a carboxyl group, an amine group, an ester group, a mercapto group, an aldehyde group, a vinyl group, an epoxy group, a halogen group, an isocyanate group, a siloxane group, a catechol group or a phthalaldehyde group.

[0024] In one embodiment of the present invention, preferably, the organic halide b is selected from the following structures:

[0025]

[0026] Wherein, R2 is an active group connected to the substance B to be coupled.

[0027] The active group is selected from:

[0028] hydroxyl group, carboxyl group, amine group, ester group, mercapto group, aldehyde group, vinyl group, epoxy group, halogen group, isocyanate group, siloxane group, catechol group, o-phthalaldehyde group, or,

[0029] The terminal group is modified with an alkylene group or a modified alkylene group having a hydroxyl group, a carboxyl group, an amine group, an ester group, a mercapto group, an aldehyde group, a vinyl group, an epoxy group, a halogen group, an isocyanate group, a siloxane group, a catechol group or a phthalaldehyde group.

[0030] In one embodiment of the present invention, the substance to be coupled A and the substance to be coupled B are independently selected from small molecules, macromolecules, tissues, cells, organelles, exosomes, polymer materials, carbon materials, silicone materials, glass materials, ceramic materials, metal materials or hydrogel materials (hydrogel materials are cross-linked water-containing materials).

[0031] The polymer material is plastic, rubber, fiber, coating or adhesive and a composite material thereof.

[0032] In one embodiment of the present invention, the polymer is selected from one or more of polysaccharides, nucleic acids, proteins, polypeptides, and polyethylene glycol.

[0033] In one embodiment of the present invention, the cell organelle is selected from one or more of a cell nucleus, a mitochondria, a chloroplast, an endoplasmic reticulum, a Golgi apparatus, a lysosome, a vacuole, a ribosome or a centrosome.

[0034] In one embodiment of the present invention, when the substances A and B to be coupled are both small molecules or polymers, the method of modifying the o-nitrobenzene compound a with photosensitizing activity on the substance to be coupled to obtain the photosensitizing active substance Aa is: dissolving the a molecule and the molecule A to be coupled in a suitable solvent, achieving covalent bonding between the two by a selected chemical reaction method, and purifying and separating to obtain the product Aa; the method of modifying the organic halide b on the substance to be coupled to obtain the substance Bb is: dissolving the b molecule and the molecule B to be coupled in a suitable solvent, achieving covalent bonding between the two by a selected chemical reaction method, and purifying and separating to obtain Bb.

[0035] In one embodiment of the present invention, when the substance A to be coupled is a tissue, cell, organelle, exosome, polysaccharide, nucleic acid, protein, or polypeptide, the method of modifying the o-nitrobenzene compound a having photosensitizing activity on the substance to be coupled A to obtain the photosensitizing active substance Aa is as follows: the a molecule is introduced into the corresponding tissue, cell, organelle, exosome, polysaccharide, nucleic acid, protein, or polypeptide by conventional technical means in the field such as anti-metabolism antibody recognition technology, protein labeling technology, non-natural amino acid technology, chemical labeling technology, etc. to form Aa.

[0036] In one embodiment of the present invention, when the substance A to be coupled is a polymer material, a carbon material, an organosilicon material, a glass material, a ceramic material, a metal material or a hydrogel material, the method for modifying the o-nitrobenzene compound a having photosensitivity on the substance to be coupled to obtain the photosensitivity substance Aa is as follows: the a molecule is dissolved in a suitable solvent to form a solution, and based on conventional interfacial grafting technology such as siloxane coupling technology, catechol technology, and chemical modification technology, the a molecule is grafted onto the surface of the material A by spin coating, immersion, photomask or laser processing to form Aa.

[0037] In one embodiment of the present invention, the catalyst refers to a substance that can react with the organic halide b to generate free radicals.

[0038] In one embodiment of the present invention, the catalyst is preferably a transition metal or a complex of a transition metal and a ligand.

[0039] Further preferably, the catalyst is copper, ruthenium, iron, a complex of copper and a ligand, a complex of ruthenium and a ligand, or a complex of iron and a ligand.

[0040] In one embodiment of the present invention, the ligand is a polyamino compound containing multiple coordination sites.

[0041] In one embodiment of the present invention, the illumination condition is irradiation with a light source at a specific wavelength, a specific light intensity and a specific time. Preferably, the light wavelength is 254nm-420nm, and the light intensity is 10mW / cm 2 -10 W / cm 2 , the illumination time is from seconds to hours.

[0042] In one embodiment of the present invention, in the free radical photocoupling method, the molar ratio of the o-nitrobenzene compound a to the organic halide b is selected as 1: (0.1 to 10000); the amount of the catalyst is selected as 0.001 to 2 times the molar content of the organic halide b. The preferred molar ratio of the o-nitrobenzene compound a to the organic halide b is 1: (0.1 to 100); the amount of the catalyst is selected as 0.01 to 1.5 times the molar content of the organic halide b.

[0043] The present invention further provides a kit for realizing photocoupling, wherein the kit is used to realize efficient and rapid coupling between substances A and B to be coupled, and the kit comprises:

[0044] The o-nitrobenzene compound a having photosensitizing activity is used to modify the substance A to be coupled to obtain the photosensitizing substance Aa;

[0045] Organic halide b: used to modify the substance to be coupled B to obtain substance Bb;

[0046] Aa and Bb trigger the reaction between a and b under the conditions of catalyst and light, achieving efficient and rapid coupling between the coupling substances A and B;

[0047] The o-nitrobenzene compound a refers to a compound containing a substituent group R at the ortho position of the nitro group on the benzene ring, and the o-nitrobenzene compound a is as shown in Formula I, wherein the substituent group R is ethyl, hydroxymethyl or 1-methylhydroxymethyl, and the other hydrogen atoms on the benzene ring in Formula I are allowed to be substituted by any number of other groups, wherein at least one substituent is an active group capable of connecting to the substance to be coupled A;

[0048] The organic halide b refers to an organic compound containing halogen and at least one active group capable of connecting with the substance to be coupled B, as shown in Formula II, wherein X is a halogen selected from Cl, Br or I.

[0049]

[0050] In one embodiment of the present invention, in the o-nitrobenzene compound a, the active group capable of connecting with the substance to be coupled A is selected from:

[0051] hydroxyl group, carboxyl group, amine group, ester group, mercapto group, aldehyde group, vinyl group, epoxy group, halogen group, isocyanate group, siloxane group, catechol group, o-phthalaldehyde group, or,

[0052] The terminal group is modified with an alkylene group or a modified alkylene group having a hydroxyl group, a carboxyl group, an amine group, an ester group, a mercapto group, an aldehyde group, a vinyl group, an epoxy group, a halogen group, an isocyanate group, a siloxane group, a catechol group or a phthalaldehyde group.

[0053] In one embodiment of the present invention, the modified alkylene group is obtained by replacing any carbon atom of the alkylene group with other heteroatoms.

[0054] In one embodiment of the present invention, preferably, the o-nitrobenzene compound a is selected from the following structures:

[0055]

[0056] Wherein, R1 is an active group connected to the substance A to be coupled.

[0057] In one embodiment of the present invention, preferably, the organic halide b is selected from the following structures:

[0058]

[0059] Wherein, R2 is an active group connected to the substance B to be coupled.

[0060] In one embodiment of the present invention, the kit further comprises a catalyst for realizing the reaction between Aa and Bb under light conditions, wherein the catalyst refers to a substance that can react with the organic halide b to generate free radicals.

[0061] In one embodiment of the present invention, the catalyst is preferably a transition metal or a complex of a transition metal and a ligand.

[0062] The present invention further provides an application of a kit for realizing optical coupling, the application comprising:

[0063] The kit is used to achieve chemical coupling between small molecules and small molecules, between macromolecules and macromolecules, and between macromolecules and small molecules;

[0064] The kit is used to achieve covalent linkage between small molecules or macromolecules and tissues, cells or organelles to achieve biomarkers;

[0065] The kit is used to achieve surface modification on the surfaces of different materials;

[0066] The kit is used to realize rapid, light-controllable cross-linking preparation of materials.

[0067] The reaction mechanism of the free radical photocoupling method of the present invention is: the organic halogenated compound b can reversibly generate carbon free radicals in the presence of a catalyst; when the o-nitrobenzene compound a is exposed to light, a nitrosobenzene structure is generated, and the nitroso group in the structure can react with the carbon free radical generated by the organic halogenated compound to form a coupling product. According to literature reports, the reaction rate constant of the nitroso-carbon free radical reaction is as high as 10 8 (CL Hawkins, MJ Davies, Biochim. Biophys. Acta, 2014, 1840, 708-721), which is significantly higher than the reaction rate constant (generally <10) of double bonds and carbon free radicals (reaction mechanism of existing free radical photocoupling technology). 5 ).

[0068] Therefore, compared with the prior art, the free radical photocoupling method of the present invention has a faster reaction rate than the existing free radical photocoupling method. At the same time, it is precisely because of the extremely high reaction rate and reaction activity that the nitroso-carbon free radical reaction can effectively reduce the side reactions of free radicals, such as self-quenching, oxygen inhibition, etc. Therefore, the free radical photocoupling method provided by the present invention has a higher conversion efficiency than the existing free radical photocoupling method.

[0069] In summary, the present invention realizes the organic combination of rapid kinetics and efficient conversion of free radical photocoupling method through reaction substrate design. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is the NMR image of the NMR titration conversion rate of formula 8 in Example 1.

[0071] Figure 2 The test curve diagram for calculating the reaction rate constant k1 for formulation 8 in Example 1.

[0072] Figure 3 The test curve diagram for calculating the reaction rate constant k2 for formulation 8 in Example 1.

[0073] Figure 4 This is a diagram of cell-polymer coupling using the free radical photocoupling method of the present invention as a biomarker method (left: control; right: free radical photocoupling method).

[0074] Figure 5 This is a diagram of the fluorescence modification of the surface of nano-silica spheres using the free radical photocoupling method of the present invention as a material surface modification method (left: control; right: free radical photocoupling method).

[0075] Figure 6 This is a graph showing the in-situ rheological test results of polyethylene glycol / polyethylene glycol photo-cross-linked hydrogel material constructed using the free radical photocoupling method of the present invention.

[0076] Figure 7 The tensile stress-strain curve diagram of the polyethylene glycol / polyethylene glycol photo-crosslinked hydrogel material constructed by the free radical photocoupling method of the present invention. DETAILED DESCRIPTION

[0077] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0078] Example 1: Determination of kinetic constants and conversion rates of free radical photocoupling method

[0079] Since the photosensitivity of o-nitrobenzene compounds is determined by their parent structure (P. Klán et al. Chemical Reviews 2013, 113, 119), when the parent structure of o-nitrobenzene compound a (the part other than R1) and the parent structure of organic halide b (the part other than R2) are determined, the kinetic constant and conversion rate of the reaction between the two are basically determined, that is, the selection of R1 and R2 groups will not significantly affect the kinetic constant and conversion rate of the reaction between the two. Based on this, in this embodiment, R1 in o-nitrobenzene compounds is selected as methyl, and R2 in organic halide is selected as ethyl, and the kinetic constant and conversion rate of the reaction between o-nitrobenzene compounds and organic halide are determined on this basis.

[0080] This embodiment uses the method of nuclear magnetic titration to determine the kinetic constants and conversion rate of the free radical photocoupling method, that is, a nuclear magnetic resonance spectrometer (400MHz) is used to determine the changes in the raw materials and products of o-nitrobenzene compounds over time. Taking the reaction of a2 and b8 as an example, a2 (1μM) and b8 (80μM) are dissolved in deuterated acetonitrile, copper powder (80μM) and N,N,N,N,N-pentamethyldiethylenetriamine (64μM) are added, and 3,4,5-trichloropyridine (1mM) is added as a nuclear magnetic calibration substance; after the solution is deoxygenated by nitrogen for 10 minutes, the adapted wavelength (intensity 1W / cm 2 The solution was illuminated by a light source of about 0s, 2s, 10s, 15s, and 20s, and then samples were taken for nuclear magnetic resonance detection (such as Figure 1 When b1 is in excess, the reaction can be regarded as a pseudo-first-order reaction, and the first-order rate constant k1 can be calculated by formula (1) (as shown in Figure 2 shown),

[0081]

[0082] Where a is a constant and e is the natural base.

[0083] Keep the concentration of a2 unchanged, change the concentration of b1, and calculate the k1 value at different concentrations.

[0084] Substitute different b1 concentrations [B] and corresponding k1 values ​​into the secondary rate constant calculation formula (2) to calculate the secondary rate constant k2 (such as Figure 3 shown),

[0085] ln[B]=c-k2x (2)

[0086] Where c is a constant.

[0087] Table 1 Reaction kinetic constants and conversion rates of o-nitrobenzene compounds and organic halides under different formulations

[0088]

[0089]

[0090] It can be seen from Table 1 that the reaction kinetic constants of the reaction between o-nitrobenzene compounds and organic halides under different formulations are as high as 10 4 M -1 s -1 , the reaction speed is extremely fast. More importantly, the conversion rate of the reaction of o-nitrobenzene compounds with organic halides under different formulas is more than 85%, and many formulas exceed 90%, which is much higher than the conversion efficiency of the reaction of double bonds with free radicals (usually <50% or even lower: Yu, Y. et al. Journal of the American Chemical Society 2018, 140, 6797-6800). It can be seen that the present invention realizes the organic combination of fast kinetics and efficient conversion of free radical photocoupling method through the reaction of o-nitrobenzene compounds with organic halides.

[0091] Example 2: High reaction selectivity of free radical photocoupling method

[0092] Different competitive reaction substrates (containing double bonds that can react with free radicals or thiol, amino, and sulfite that can react with nitroso groups) are added to the reaction system of o-nitrobenzene compounds and organic halides, and the effect of different reaction substrates on the reaction conversion rate of o-nitrobenzene compounds and organic halides is monitored by nuclear magnetic titration. Taking the reaction of a2 and b2 as an example, acetonitrile solutions of a2 (1μM) and b2 (80μM) are prepared, and copper powder (80μM) and N,N,N,N,N-pentamethyldiethylenetriamine (64μM) are added; styrene (1μM) is added; after the solution is deoxygenated by nitrogen for 10 minutes, the solution is titrated using an adapted wavelength (intensity 1W / cm 2 The light source (about ) was used for illumination for 0s, 2s, 10s, 15s, and 20s; samples were taken and the conversion rate of the reaction was calculated by nuclear magnetic titration.

[0093] Table 2 Effect of different substrates on the conversion rate of the reaction between o-nitrobenzene compounds and organic halides (R1 is methyl; R2 is ethyl)

[0094]

[0095]

[0096] It can be seen from Table 2 that different competitive reaction substrates have no significant effect on the conversion rate of the reaction between o-nitrobenzene compounds and organic halides. These results show that the free radical photocoupling method proposed in the present invention has extremely high reaction selectivity.

[0097] Example 3: Free radical photocoupling method as a chemical coupling method to achieve coupling between polymers (small molecules) and polymers (small molecules)

[0098] Experimental method: The substances A and B to be coupled are both small molecules or macromolecules. Method for connecting substance A to be coupled with molecule a: dissolve molecule a and molecule A to be coupled in a suitable solvent, achieve covalent connection between the two by a selected chemical reaction method, and purify and separate to obtain product Aa. Method for connecting molecule B to be coupled with molecule b: dissolve molecule b and molecule B to be coupled in a suitable solvent, achieve covalent connection between the two by a selected chemical reaction method, and purify and separate to obtain Bb. The grafting degree of the chain transfer agent molecule on the Bb molecule is ≤1.

[0099] Aa and Bb are mixed in a certain ratio to form a mixed solution, and a proper amount of catalyst is added. After light irradiation reaction, a coupling product of A and B is obtained.

[0100] The ratio of Aa to Bb is selected according to the molar ratio of a molecule to b molecule, preferably the molar ratio is 1:(0.1-100); the amount of the catalyst is adjusted according to the content of Bb, preferably 0.01-1.5 times the molar content of Bb; the mixed solution is preferably a deoxygenated mixed solution; the photoreaction conditions are preferably a light wavelength of 254nm-420nm and a light intensity of 10mW / cm 2 -10 W / cm 2 , reaction time 30s-60min.

[0101] Herein, macromolecule (small molecule) represents a macromolecule or a small molecule.

[0102] The specific embodiments are as follows:

[0103] Example 3-1: Free radical photocoupling method as a chemical coupling method to achieve the coupling of high molecular weight polyethylene glycol (PEG) and small molecule fluorescent dye fluorescein (FL)

[0104]

[0105] Photocoupling reaction of PEG-a3 (R1 is N-(2-aminoethyl)butyramide) and FL-b5 (R2 is hydroxyethyl): PEG-a3 (100 mg, 0.01 mmol) and FL-b5 (20.4 mg, 0.4 mmol) were dissolved in 100 mL of methanol, and copper powder (2.5 mg, 0.4 mmol) and N,N,N,N,N-pentamethyldiethylenetriamine (PMDETA, 5.5 mg, 0.32 mmol) were added. Argon was introduced for deoxygenation for 30 min, and the 405 nm (2 W / cm 2) After irradiation with a light source for 30 seconds, the system was swirled until it became viscous and poured into ether to obtain a rhodamine-modified PEG polymer, yielding 105 mg of product (yield 95%). The peak of 7.71 ppm belongs to the proton of the rhodamine group, which can be used to calculate the degree of modification (grafting degree), which is 0.97 (theoretical degree of modification is 1), which represents the average number of rhodamine molecules on a PEG molecule. In the figure, Y indicates that the compound may form different substitution groups at this position, but it does not affect the implementation and results of the free radical photocoupling method. The Y that appears below is the same as this case and will not be repeated. The above results verify the high efficiency and rapid reaction characteristics of the free radical photocoupling method of the present invention.

[0106]

[0107] Synthesis of compound a3-1: The synthesis was carried out according to the method disclosed by Nilotpal Sinha, Prasenjit Mistry, Sukanya Das, Tanmoy Datta, and Brindaban Roy. The Journal of Organic Chemistry 2023 88(13), 8969-8983. 1 H NMR (400MHz, CDCl3): δ = 8.32 (dd, 1H), 7.91 (d, 1H), 7.68 (dd, 1H), 3.16 (s, 1H). ESI-HRMS: [M+H] 184.0062.

[0108] Synthesis of compound a3: The synthesis was carried out according to the method disclosed in Yunlong Yang, Jieyuan Zhang, Zhen Liu, Qiuning Lin, Xiaolin Liu, Chunyan Bao, Yang Wang, Linyong Zhu. Adv. Mater., 2016, 28, 2724-2730. 1 H NMR (400MHz, CDCl3): δ=7.35-7.26(m,2H),7.06(d,1H),6.77(s,1H),4.80(dd,2H),3.94(t,2H),3.22(t,2H) ),3.05(t,2H),2.90(t,2H),2.27(s,3H),1.97(p,2H),1.74(t,1H),0.91(s,2H).ESI-HRMS:[M+H]314.1170.

[0109] Synthesis of PEG-a4: Dissolve the polyethylene glycol raw material (PEG-OH; Mw = 10000; 1 g, 0.1 mmol) with a methyl end cap, 4-dimethylaminopyridine (0.005 g, 0.05 mmol), triethylamine (0.101 g, 1.0 mmol), and 4-chloronitrobenzene (0.201 g, 1 mmol) in anhydrous dichloromethane (100 mL) and stir at room temperature for 5 hours. The solvent was evaporated to about half of its original volume under vacuum. The mixture was then poured into ether and the white precipitate formed was collected. The dried precipitate (7.8 g) was dissolved in dry DMF (50 mL) and a3 (0.327 g, 1 mmol) was added in the presence of TEA (0.1 mL). The resulting mixture was stirred at room temperature for another 6 hours, after which the solvent was removed under reduced pressure. Then add water to dissolve, dialyze with distilled water, and freeze-dry to obtain PEG-a3 (7.2 g) with a yield of 90%. The peak of 7.35-7.26 ppm belongs to the proton of the a3 group in the PEG-a3 molecule, which can be used to calculate the degree of modification (grafting degree), which is (1.8-1.9), which represents the average number of a3 molecules on one PEG molecule.

[0110]

[0111] Synthesis of compound b5: 2-bromopropionic acid (1.53 g, 10 mmol) and ethylene glycol (1.24 g, 20 mmol) were dissolved in anhydrous dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.751 g, 30 mmol) and 4-dimethylaminopyridine (61 mg, 0.5 mmol) were added. After stirring overnight, the system was extracted three times with water and saturated brine respectively, and the organic phase was dried over anhydrous sodium sulfate and the organic phase was spin-dried to obtain a crude product. The product (1.75 g, 90%) was purified by column chromatography. 1 H NMR (400MHz, CDCl3): δ = 4.85 (q, 1H), 4.45 (dd, 1H), 4.21 (dd, 1H), 3.98 (d, 1H), 3.85 (dd, 1H), 2.50 (t, 1H), 1.99 (d, 3H). MS (ESI): [M+H] 196.9808.

[0112] Synthesis of FL-b5: The synthesis was carried out by referring to the synthesis method of compound b5. 1H NMR (400MHz, CDCl3): δ=7.66(ddd,2H),7.57(td,1H),7.52-7.42(m,2H),7.03-6.93(m,2H),6.75(d,1H),6.65(dd,1H),6.22(d,1 H),5.88(s,1H),4.93(dt,1H),4.79(q,1H),4.55(dt,1H),3.86(ddd,1H),2.55(ddd,1H),1.86(d,3H).MS(ESI):[M+H]511.0387.

[0113] Example 3-2: Free radical photocoupling method as a chemical coupling method to achieve coupling of high molecular weight polyethylene glycol (PEG) and small molecule fluorescent dye rhodamine (RhB)

[0114]

[0115] Photocoupling reaction of HA-a4 (R1 is N-(2-aminoethyl)butyramide) and RhB-b2 (R2 is hydroxyethyl): HA-a4 (3.4 g, 0.01 mmol) and RhB-b2 (25.4 mg, 0.4 mmol) were dissolved in 100 mL of methanol, and copper powder (2.5 mg, 0.4 mmol) and N,N,N,N,N-pentamethyldiethylenetriamine (PMDETA, 5.5 mg, 0.32 mmol) were added. Argon was introduced for deoxygenation for 30 min, and the 405 nm (2 W / cm 2 ) After irradiation with a light source for 30 seconds, the system was swirled until it became viscous and poured into ether to obtain a rhodamine-modified PEG polymer, yielding 105 mg of the product (yield 95%). The peak of 7.97 ppm belongs to the proton of the rhodamine group, which can be used to calculate the degree of modification (grafting degree), which is 0.97 (theoretical degree of modification is 1), which represents the average number of rhodamine molecules on one PEG molecule. The above results verify the high efficiency and rapid reaction characteristics of the free radical photocoupling method of the present invention.

[0116]

[0117] Synthesis of compound a4: The synthesis was carried out according to the method disclosed in Yunlong Yang, Jieyuan Zhang, Zhen Liu, Qiuning Lin, Xiaolin Liu, Chunyan Bao, Yang Wang, Linyong Zhu. Adv. Mater., 2016, 28, 2724-2730. 1H NMR (400MHz, CDCl3): δ = 7.71 (s, 1H), 7.22 (s, 1H), 4.96 (s, 2H), 4.13 (t, 2H), 3.99 (s, 3H) ,3.32(dd,2H),2.82(t,2H),2.44(t,2H),2.26-2.17(m,3H).ESI-HRMS:[M+H]328.1507.

[0118] Synthesis of HA-a4: Dissolve hyaluronic acid (2 g, 340 kDa) in 100 mL 0.01 mol / L 2-(N-morpholine)ethanesulfonic acid MES buffer solution (pH = 5.2), stir until completely dissolved, weigh compound a4 (66 mg, 0.2 mmol), dissolve in 10 mL dimethyl sulfoxide DMSO and add to the above reaction solution, weigh 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride DMTMM (0.4 g, 1.5 mmol), dissolve in 3 mL MES buffer solution, add to the above reaction solution three times (every 1 h), and react at 35 ° C for 24 h. The reaction solution was then poured into a dialysis bag (MWCO 7000), dialyzed with deionized water for 2-3 days, and freeze-dried to obtain the photosensitive hyaluronic acid derivative HA-a4 (1.92 g). According to the H-NMR spectrum, the labeling rate of compound 3 was calculated to be approximately 3.29%.

[0119]

[0120] Synthesis of compound b2: The synthesis was carried out by referring to the synthesis method of compound b5. 1 H NMR (400MHz, CDCl3): δ = 4.23 (t, 2H), 3.62 (dt, 2H), 2.50 (t, 1H), 2.07 (s, 6H). MS (ESI): [M+H] 210.9964.

[0121] Synthesis of RhB-b2: The synthesis was carried out by referring to the synthesis method of compound b5. 1 H NMR (400MHz, CDCl3): δ=7.67(dd,1H),7.62-7.49(m,2H),7.38(td,1H),7.07(d,1H),6.81-6.74(m,2H),6.54-6.40(m,3H),4.50(s,3 H),4.49(d,1H),3.34(q,4H),1.99(s,3H),1.93(s,3H),1.58-1.49(m,3H),1.46-1.26(m,7H),1.10(t,6H).MS(ESI):[M+H]631.2115.

[0122] Example 4: Application of free radical photocoupling method as a biomarker method

[0123] Experimental method: The substance A to be coupled is tissue, cell, organelle, exosome, polysaccharide, nucleic acid, protein, or polypeptide; the substance B to be coupled is a small molecule or a macromolecule. The substance A to be coupled is connected to one of the molecules a or b, and the substance B to be coupled is connected to the other. The connected A and B are reacted under light in the presence of a catalyst to obtain a coupling product of A and B.

[0124] Optionally, the substance A to be coupled is connected to the a molecule, and the connection method is: the a molecule is introduced into the corresponding tissue, cell, organelle, exosome, polysaccharide, nucleic acid, protein, or polypeptide by conventional methods in the field, such as anti-metabolism antibody recognition method, protein labeling method, non-natural amino acid method, chemical labeling method, etc., to form Aa. The method for connecting the substance B to be coupled and the b molecule is: dissolving the substance B to be coupled and the b molecule in a suitable solvent, achieving covalent connection between the two by a selected chemical reaction method, and purifying and separating to obtain Bb. The grafting degree of the b molecule on the Bb molecule is ≤1.

[0125] Bb is dissolved in water to form a solution, which is added to a matrix containing Aa, and an appropriate amount of catalyst is added. After light irradiation reaction, tissues, cells, organelles, exosomes, polysaccharides, nucleic acids, proteins, or polypeptides A labeled with small molecules or polymers B are obtained.

[0126] The ratio of Aa to Bb is selected according to the molar ratio of a molecule to b molecule, preferably the molar ratio is 1:(0.1-100); the amount of the catalyst is adjusted according to the content of Bb, preferably 0.01-1.5 times the molar content of Bb; the photoreaction conditions are preferably a light wavelength of 365nm-420nm and a light intensity of 10mW / cm 2 -10W / cm 2 , reaction time is 5s-30min.

[0127] Example 4-1: Free radical photocoupling method as a biomarker method to achieve cell-polymer coupling

[0128] The polymer PNIPAM-RhB-b2 (R2 is hydroxyethyl) with narrow molecular weight distribution was constructed by atom transfer radical polymerization (ATRP), and the polymer was further used to label the cell surface through the membrane-localizing group DPPE modified with a8 (DPPE-a8, R1 is (methoxycarbonyl)glycine).

[0129]

[0130] Cell labeling: Human cervical cancer cells (HeLa S3) were cultured in DMEM medium (a medium containing various amino acids and glucose) containing high glucose (Life Technologies-Gibco, 11995073), fetal bovine serum (10% heat-inactivated, Omega Scientific, FB02), penicillin (100U / mL) and streptomycin (100μg / mL, LifeTechnologies-Gibco, 15140122). Cells were grown in an atmosphere containing 5% carbon dioxide (CO2) at 37°C. To promote cell adhesion during imaging, 96-well chamber slides pre-coated with 0.5% (w / v) poly-lysine were used for imaging. HeLa S3 cell lines in culture medium were incubated on 96-well plates at a density of 40% per well. After 24 hours, the cells were treated as follows: HeLa S3 cancer cells were placed in a PBS solution (containing 0.1% DMSO) with DPPE-a8 (200 μL, 60 nM) and incubated at 37°C for 60 minutes; the cells were washed with PBS solution to remove excess DPPE-a8; the washed cells were placed in a PBS solution containing PNIPAM-RhB-b2 (5 nM), copper powder (5 nM) and N,N,N,N,N-pentamethyldiethylenetriamine (4 nM). Finally, the cell solution was imaged using a light source (405 nm, 200 mW / cm 2 ) irradiated the cell solution for 60 seconds and then imaged it under a fluorescence microscope. The same solution without illumination was used as a control. The results are shown in Figure 4 As shown (left: control group; right: experimental group). Under the same conditions, the cells in the control group had almost no fluorescence, while the cell membrane fluorescence brightness of the experimental group was obvious. This result shows that the free radical photocoupling method can achieve cell-polymer coupling as a biomarker method, and also verifies the high efficiency and rapid response characteristics of the free radical photocoupling method of the present invention.

[0131]

[0132] Synthesis of polymer PNIPAM-RhB-b2: RhB-b2 (63.5 mg, 0.1 mmol), N,N-isopropylacrylamide (1.13 g, 10 mmol), and copper powder (7.0 mg, 0.11 mmol) were dissolved in 10 mL of anhydrous 1,4-dioxane. After freeze-drying three times, N,N,N,N,N-pentamethyldiethylenetriamine (13.9 mg, 0.08 mmol) was added and reacted for 24 h. After the reaction was completed, the system was vortexed until it became viscous and poured into ether to reprecipitate to obtain a purple-red solid product with a yield of 1 g (90%). The product was dissolved in DMF and GPC analysis (Mn, GPC =18kg / mol, Mw / Mn=1.2).

[0133]

[0134] Synthesis of compound a8-1: The synthesis was carried out according to the method disclosed by Bader TK, Xu F, Hodny MH, et al. Methoxy-substituted nitrodibenzofuran-based protecting group with an improved two-photon action cross-section for thiol protection in solid phase peptide synthesis [J]. The Journal of organic chemistry, 2019, 85(3): 1614-1625. 1 HNMR (400MHz, CDCl3): δ = 8.22 (d, 2H), 7.67 (d, 1H), 7.59 (d, 1H), 7.20 (dd, 1H), 4.81 (d, 2H), 1.53 (t, 1H). ESI-HRMS: [M+H] 272.0553.

[0135] Synthesis of compound a8-2: Compound a8-1 (5.4 g, 20 mmol) was dissolved in anhydrous dichloromethane (100 mL), anhydrous triethylamine (20.2 g, 20 mmol) was added, acetyl chloride (12.56 g, 16 mmol) was added dropwise under ice bath conditions, and stirred overnight at room temperature. After the reaction was completed, the solvent was dried, the system was dissolved in dichloromethane, extracted three times with water and saturated brine respectively, the organic phase was dried over anhydrous sodium sulfate, the organic phase was dried, and the crude product was purified by column chromatography to obtain a pure product as a yellow solid (5.4 g, 86%). 1 H NMR (400MHz, CDCl3): δ = 8.22 (d, 2H), 7.67 (d, 1H), 7.49 (d, 1H), 7.20 (dd, 1H), 5.18 (s, 2H), 2.24 (s, 3H). MS (ESI): [M+H] 314.0660.

[0136] Synthesis of compound a8-3: The synthesis was carried out according to the method disclosed in Yunlong Yang, Jieyuan Zhang, Zhen Liu, Qiuning Lin, Xiaolin Liu, Chunyan Bao, Yang Wang, Linyong Zhu. Adv. Mater., 2016, 28, 2724-2730. 1H NMR (400MHz, CDCl3): δ = 8.24 (s, 1H), 7.74-7.64 (m, 2H), 7.49 (d, 1H), 7.20 (dd, 1H) ),5.18(s,2H),4.80(d,2H),2.24(s,3H),1.49(t,1H).ESI-HRMS:[M+H]316.0816.

[0137] Synthesis of compound a8-4: Compound a8-3 (8.7 g, 27.4 mmol), tert-butyldimethylsilyl chloride (6.2 g, 41.1 mmol), imidazole (2.8 g, 41.1 mmol) in DMF solution (100 mL) was stirred at room temperature for 24 hours, and DMF was removed by evaporation under reduced pressure. The mixture was purified by column chromatography to obtain compound a8-4 (11.4 g, 94%). 1 H NMR (400MHz, CDCl3): δ=7.65(d,1H),7.48(d,1H),7.38(d,2H),7.23(dd,1H),5.18(s ,2H),5.05(s,2H),2.24(s,3H),1.08(s,9H),0.08(s,6H).ESI-HRMS:[M+H]430.1680.

[0138] Synthesis of compound a8-5: Compound a8-4 (8.6 g, 20 mmol) was dissolved in methanol (100 mL), and an aqueous solution of sodium hydroxide (NaOH, 10% by mass) was added dropwise, and the reaction was detected by thin layer chromatography until the reaction was completed. After the reaction was completed, the solvent was dried, the system was dissolved with dichloromethane, extracted three times with water and saturated brine, respectively, the organic phase was dried with anhydrous sodium sulfate, the organic phase was dried, and the crude product was purified by column chromatography to obtain a pure product as a yellow solid (7.1 g, 92%). 1 H NMR (400MHz, CDCl3): δ = 8.22 (s, 1H), 7.71-7.63 (m, 2H), 7.53 (d, 1H), 7.21 (dd, 1H), 5.05 (s,2H),4.81(d,2H),1.52(t,1H),1.07(s,10H),0.08(s,7H).ESI-HRMS:[M+H]388.1575.

[0139] Synthesis of compound a8-6: Compound a8-5 (3.9 g, 10 mmol), 4-dimethylaminopyridine (0.061 g, 0.5 mmol), triethylamine (2.5 g, 25 mmol), 4-chloroformic acid nitrobenzene (4.1 g, 20 mmol) were dissolved in anhydrous dichloromethane (100 mL) and stirred at room temperature for 10 min. The organic phase was dried, the crude product was purified by column chromatography, dissolved in anhydrous dichloromethane (100 mL), triethylamine (2.5 g, 25 mmol) and glycine (1.5 g, 20 mmol), and stirred at room temperature for 30 min. After the reaction was completed, the solvent was dried, the system was dissolved in dichloromethane, extracted three times with water and saturated brine, the organic phase was dried with anhydrous sodium sulfate, the organic phase was dried, the crude product was purified by column chromatography, and the pure product was a yellow solid (4.4 g, 90%). 1 H NMR (400MHz, CDCl3): δ = 8.23 ​​(s, 1H), 7.73-7.66 (m, 2H), 7.52 (d, 1H), 7.23 (dd, 1H), 6.05 (s, 1H) ),5.17(s,2H),5.05(s,2H),3.90(s,2H),1.08(s,9H),0.08(s,6H).ESI-HRMS:[M+H]489.1688.

[0140] Synthesis of compound a8: Add tetrabutylammonium fluoride (5.2 g, 20 mmol) to a stirred THF solution (100 mL) of compound a8-6 (4.9 g, 10 mmol). Stir at room temperature for 8 hours and evaporate in vacuo. The resulting mixture is purified by column chromatography to obtain a yellow solid product a8 (4.4 g, 90%). 1 H NMR (400MHz, CDCl3): δ = 8.25 (s, 1H), 7.74-7.66 (m, 2H), 7.52 (d, 1H), 7.23 (dd, 1H), 5.17 (s,2H),5.00(s,1H),4.80(d,2H),3.90(s,2H),1.50(t,1H).ESI-HRMS:[M+H]375.0823.

[0141] Synthesis of compound DPPE-a8: Compound a8 (15 mg, 0.04 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.4 mg, 0.08 mmol) and N-hydroxysuccinimide (9.2 mg, 0.08 mmol) were dissolved in 20 mL of anhydrous N,N-dimethylformamide. After reacting for 30 min, 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (34.6 mg, 0.05 mmol) was added. After reacting overnight, the solvent was dried and the crude product was purified by column chromatography to obtain the product (30 mg, 80%). 1 H NMR (400MHz, CDCl3): δ = 8.23 ​​(d, 2H), 7.74-7.64 (m, 2H), 7.21 (dt, 1H), 5.79 (dt, 1H), 5.63 (ddd, 1H),5.17(d,1H),4.82(dd,1H),4.70-4.56(m,2H),4.49(d,1H),4.44-4.32(m,2H),4.30-4.01( m,4H),3.33(dt,1H),2.64(t,1H),2.37(dddd,2H),2.30-2.09(m,3H),1.82-1.42(m,12H),1.46 -1.16(m,13H),1.20-1.10(m,2H),1.15-1.02(m,2H),0.89(t,8H).ESI-HRMS:[M+H]1047.5797.

[0142] Example 4-2: Free radical photocoupling method as a biomarker method to achieve protein-polymer coupling

[0143] A polymer P(PEGA)-FL-b1 (R2 is hydroxyethyl) of methoxy polyethylene glycol acrylate (Mw=400) with a narrow molecular weight distribution was constructed by atom transfer radical polymerization (ATRP), and the protein was further labeled by the polymer by modifying N-hydroxysuccinimide with a7 (NHS-a7, R1 is 4-ethoxy-4-oxobutyric acid).

[0144]

[0145] Protein labeling: Dissolve bovine serum albumin (0.3 g, 10 mmol) in 4 mL phosphate buffer (PH = 7.4), add NHS-a7 (3.9 g, 10 mmol) and stir at room temperature overnight. Pour the system into anhydrous ethanol to precipitate, centrifuge to obtain a7-labeled bovine serum albumin, and the a7 labeling rate is 130% (molar ratio) by UV detection. Dissolve a7-labeled bovine serum albumin (200 μL, 0.4 mmol) in phosphate buffer (PH = 7.4), add P(PEGA)-FL-b1 (5 nM), copper powder (5 nM) and N,N,N,N,N-pentamethyldiethylenetriamine (4 nM) in PBS. Finally, image the cell solution using a light source (420 nm, 200 mW / cm 2 ) irradiated the cell solution for 30 seconds, and the system was dialyzed and freeze-dried to obtain the polymer-labeled bovine serum protein Protein-P(PEGA)-FL. The labeling rate of the polymer was 90% calculated by ultraviolet. This result shows that the free radical photocoupling method can achieve cell-polymer coupling as a biolabeling method, and also verifies the high efficiency and rapid response characteristics of the free radical photocoupling method of the present invention.

[0146]

[0147] Synthesis of compound b5: refer to the synthesis method of compound b5. 1 H NMR (400MHz, CDCl3): δ=7.81(dd,1H),7.66-7.52(m,2H),7.52-7.40(m,2H),7.02-6.93(m,2H),6.76-6.63 (m,2H),6.18(d,1H),5.91(s,1H),4.54-4.45(m,2H),4.50(s,2H),4.26(s,2H).MS(ESI):[M+H]497.0230.

[0148] Synthesis of polymer PNIPAM-RhB-b2: Synthesis was performed according to the synthesis method of polymer PNIPAM-RhB-b2. GPC analysis (Mn, GPC =16kg / mol, Mw / Mn=1.28).

[0149]

[0150] Synthesis of compound a7-1: 4-bromo-N-methylaniline (1.9 g, 10 mmol), 2-bromoethyl acetate (3.3 g, 20 mmol) and potassium carbonate (4.0 g, 30 mmol) were dissolved in anhydrous N, N-dimethylformamide (50 mL) and stirred overnight at 90°C. After the reaction was completed, the solvent was dried, the system was dissolved in dichloromethane, extracted three times with water and saturated brine respectively, the organic phase was dried over anhydrous sodium sulfate, the organic phase was dried, and the obtained crude product was purified by column chromatography to obtain a pure product as a white solid (2.3 g, 86%). 1 H NMR (400MHz, CDCl3): δ = 7.19-7.12 (m, 2H), 6.52-6.45 (m, 2H), 4.31 (t, 2H), 3.88 (t, 2H), 2.94 (s, 3H), 2.02 (s, 3H). ESI-HRMS: [M+H] 272.0281.

[0151] Synthesis of compound a7-2: The synthesis was carried out according to the method disclosed by Madalina T. Mihai, Benjamin D. Williams, and Robert J. Phipps. Journal of the American Chemical Society 2019 141(39), 15477-15482. 1 H NMR (400MHz, CDCl3): δ = 7.98 (d, 2H), 7.76-7.70 (m, 1H), 4.80 (dd, 2H), 1.55 (t, 1H), 1.40 (s, 14H). ESI-HRMS: [M+H] 280.1350.

[0152] Synthesis of compound a7-3: A mixture of compound a7-1 (5.4 g, 20 mmol) and compound a7-2 (5.6 g, 20 mmol), potassium carbonate (4.0 g, 30 mmol), tetrakis(triphenylphosphine)palladium (1.1 g, 1 mmol) (catalyst) was heated overnight at 150° C. in a mixed solution of EtOH (10 mL) and water (5 mL), water (100 mL) was added, and the aqueous phase was extracted with ethyl acetate (200 mL). The organic phase was spin-dried, and the obtained crude product was purified by column chromatography to obtain a pure product as a yellow solid (3.0 g, 86%). 1H NMR (400MHz, CDCl3): δ=8.10-7.99(m,2H),7.78(dd,1H),7.34-7.27(m,2H),6.90-6.83(m,2H),4.80 (dd,2H),4.31(t,2H),3.88(t,2H),2.94(s,3H),2.02(s,3H),1.56(t,1H).MS(ESI):[M+H]345.1445.

[0153] Synthesis of compound a7-4: The synthesis was carried out according to the synthesis method of compound a8-4. 1 H NMR (400MHz, CDCl3): δ=7.63-7.44(m,2H),7.29-7.20(m,3H),6.79-6.71(m,2H),5.05(d,2H),4.31(t,2H), 3.88(t,2H),2.84(s,3H),2.30(s,3H),2.02(s,3H),1.07(s,10H),0.08(s,6H).ESI-HRMS:[M+H]459.2310.

[0154] Synthesis of compound a7-5: The synthesis was carried out according to the synthesis method of compound a8-5. 1 H NMR (400MHz, CDCl3): δ=8.02(d,1H),7.90(dd,1H),7.76(dd,1H),7.30-7.23(m,2H),6.79-6.72(m,2H),5.0 5(d,2H),3.85-3.75(m,1H),3.79(s,2H),2.94(s,3H),1.05(s,9H),0.08(s,6H).ESI-HRMS:[M+H]417.2204.

[0155] Synthesis of compound a7-6: Compound a7-5 (8.6 g, 20 mmol) was dissolved in anhydrous dichloromethane (100 mL), succinic anhydride (8.6 g, 20 mmol) was added, and the reaction was detected by thin layer chromatography until the reaction was completed. After the reaction was completed, the system was extracted three times with water and saturated brine, the organic phase was dried over anhydrous sodium sulfate, the organic phase was spin-dried, and the crude product was purified by column chromatography to obtain a pure product as a yellow solid (7.1 g, 92%). 1H NMR (400MHz, CDCl3): δ=7.60-7.51(m,2H),7.24(t,3H),6.80-6.72(m,2H),5.05(d,2H),4.31(t,2H),3.88(t,2H),2 .97(s,3H),2.67-2.57(m,2H),2.57-2.47(m,2H),2.32(s,3H),1.11(s,9H),0.08(s,6H).ESI-HRMS:[M+H]517.2365.

[0156] Synthesis of compound a7: The synthesis was carried out according to the synthesis method of compound a8. 1 H NMR (400MHz, CDCl3): δ=8.08(d,1H),7.93(dd,1H),7.78(dd,1H),7.32-7.24(m,2H),6.82-6.74(m,2H),4.80(dd,2 H),4.31(t,2H),4.05(t,1H),3.88(t,2H),2.98(s,3H),2.62(t,2H),2.56-2.48(m,2H).ESI-HRMS:[M+H]403.1500.

[0157] Synthesis of compound NHS-a7: Compound a7 (15 mg, 0.04 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (15.4 mg, 0.08 mmol) and N-hydroxysuccinimide (9.2 mg, 0.08 mmol) were dissolved in 50 mL of anhydrous dichloromethane. After reacting overnight, the solvent was dried and the crude product was purified by column chromatography to obtain the product (30 mg, 80%). 1 H NMR (400MHz, CDCl3): δ=8.31(d,1H),7.89-7.78(m,2H),7.25-7.17(m,2H),6.83-6.75(m,2H),4.80(dd,2H),4.31(t, 2H),3.88(t,2H),2.91(s,3H),2.80(s,4H),2.62(td,2H),2.56-2.47(m,2H),1.45(t,1H).ESI-HRMS:[M+H]500.1664.

[0158] Example 5: Application of free radical photocoupling method as a material surface modification method

[0159] Experimental scheme: The substance A to be coupled is a polymer material, a carbon material, an organic silicon material, a glass material, a ceramic material, a metal material or a hydrogel material, and the substance B to be coupled is a small molecule or a polymer. The substance A to be coupled is connected to one of the molecules a or b, and the substance B to be coupled is connected to the other. The connected A and B react under light in the presence of a catalyst to obtain a coupling product of A and B.

[0160] Optionally, the substance A to be coupled is connected with the a molecule, and the connection method is: the a molecule is dissolved in a suitable solvent to form a solution, and based on a conventional interface grafting method such as a siloxane coupling method, a catechol method, a chemical modification method, the a molecule is grafted onto the surface of the material A by spin coating, immersion, photomask or laser processing to form Aa. The method for connecting the substance B to be coupled with the b molecule is: the substance B to be coupled and the b molecule are dissolved in a suitable solvent, the two are covalently connected by a selected chemical reaction method, and Bb is purified and separated.

[0161] The material Aa is immersed in the solution formed by Bb, and a proper amount of catalyst is added. After light irradiation reaction, the material is taken out from the solution and rinsed to obtain the material A with the surface modified with small molecules or polymer B.

[0162] The ratio of Aa to Bb is selected according to the molar ratio of a molecule to b molecule, and the preferred molar ratio is 1:(0.1-100); the amount of the catalyst is adjusted according to the content of Bb, and is preferably 0.01-1.5 times the molar content of Bb; the photoreaction conditions are preferably a light wavelength of 254nm-420nm, a light intensity of 10mW / cm2-10W / cm2, and a reaction time of 30s-60min.

[0163] Example 5-1: Free radical photocoupling method as a material surface modification method to achieve fluorescence modification of the surface of nano-silica spheres

[0164] RhB-b2 (R2 is hydroxyethyl) is obtained by reacting rhodamine B with an organic halide b4, and the dye is further used to mark the surface of nano-silica through an a10-modified silane coupling agent (Si-a10, R1 is butyric acid).

[0165]

[0166] A10 marking on the surface of nano-silica spheres: Nano-silica spheres (0.1 g) were dispersed in anhydrous toluene (10 mL), Si-a10 (47.3 mg, 0.1 mmol) was added, and refluxed at 120°C overnight. After the reaction was completed, centrifuged and washed three times with toluene. The washed and centrifuged solution was fixed to 100 mL, and the amount of unreacted Si-a4 was detected by ultraviolet absorption, so that the grafting amount of Si-a4 on the surface of the nano-silica spheres was calculated to be 33% (mass ratio).

[0167] The free radical photocoupling method was used to realize the fluorescent labeling on the surface of nano-silica spheres: the nano-silica spheres labeled with Si-a10 (10 mg) were dispersed in deionized water (100 mL), RhB-b4 (189.0 mg, 0.4 mmol, 0.1% DMSO), copper powder (12.7 mg, 0.4 mmol) and N,N,N,N,N-pentamethyldiethylenetriamine (28 mg, 0.32 mmol) were added, and argon was introduced for deoxygenation for 30 min. A light source (405 nm, 2 W / cm 2 ) After irradiating the system for 60 seconds, centrifugation was performed, and the system was washed three times with ethanol. The system was observed under a confocal microscope, and a non-irradiated group treated under the same conditions was used as a control. Figure 5 As shown, there is almost no fluorescence on the surface of the nano-silica spheres in the control group, while there is obvious fluorescence on the surface of the nano-silica spheres in the experimental group. This result shows that the free radical photocoupling method of the present invention can achieve fluorescence modification of the surface of nano-silica spheres as a material surface modification method, and also verifies the high efficiency and rapid reaction characteristics of the free radical photocoupling method of the present invention.

[0168]

[0169] Synthesis of a4-1: The synthesis was carried out according to the method disclosed in Ossipov DA, Romero AB, Ossipova E. Light-activatable prodrugs based on hyaluronic acid biomaterials [J]. Carbohydrate polymers, 2018, 180: 145-155. 1H NMR (400MHz, CDCl3): δ=7.53(s,1H),7.43(d,1H),5.12-5.01(m,1H),4.98(td,1H),4.88(d,1H),4.26-4.16(m,1H),3.90(s,3H),3 .24-3.10(m,2H),2.91-2.72(m,3H),2.61-2.51(m,1H),2.05(tq,1H),1.99-1.85(m,1H),1.47(d,3H).ESI-HRMS:[M+H]397.1242.

[0170] Synthesis of Si-a4: The synthesis was carried out by referring to the synthesis method of compound DPPE-a8. 1 H NMR (400MHz, CDCl3): δ = 7.77 (s, 1H), 7.45 (d, 1H), 6.69 (s, 1H), 5.33-5.21 (m, 1H) ),4.88(d,1H),4.54-4.42(m,1H),4.15(dddt,1H),3.93-3.77(m,6H),3.52(dq,4 H),3.30(dt,1H),2.92(ddt,,1H),2.42-2.31(m,1H),2.06-1.88(m,2H),1.71(dd dd,2H),1.49(d,3H),1.13(t,9H),0.51-0.42(m,2H).ESI-HRMS:[M+H]503.2420.

[0171] Example 6: Application of free radical photocoupling method as a material cross-linking method

[0172] Experimental scheme: The substance A to be coupled is a small molecule or a macromolecule, and the substance B to be coupled is a small molecule or a macromolecule. One of the a molecule or the b molecule is grafted onto the substance A to be coupled, and the other is grafted onto the substance B to be coupled, and the grafting degree is ≥ 2. The grafted A and B react through light to obtain the coupling product of A and B.

[0173] Optionally, the a molecule is grafted onto the substance A to be coupled, and the grafting method thereof is: dissolving the a molecule and the substance A to be coupled in a suitable solvent, carrying out a grafting reaction by a selected chemical reaction method, and purifying and separating to obtain a product Aa, wherein the grafting degree of the a molecule onto the molecule of the substance A to be coupled is ≥ 2. The b molecule is grafted onto the substance B to be coupled by: dissolving the b molecule and the substance B to be coupled in a suitable solvent, carrying out a grafting reaction by a selected chemical reaction method, and purifying and separating to obtain a product Bb, wherein the grafting degree of the b molecule onto the molecule of the substance B to be coupled is ≥ 2.

[0174] Aa and Bb are mixed in a certain ratio or dissolved in a solvent to form a mixed solution, and a proper amount of catalyst is added, and light cross-linking is performed or the solvent is removed and light cross-linking is performed to obtain a cross-linked material of A and B.

[0175] The ratio of Aa to Bb is selected according to the molar ratio of a molecule to b molecule, and the preferred molar ratio is 1:(0.1-100); the amount of the catalyst is adjusted according to the content of Bb, and is preferably 0.01-1.5 times the molar content of Bb; the photoreaction conditions are preferably a light wavelength of 254nm-420nm, a light intensity of 10mW / cm2-10W / cm2, and a reaction time of 30s-60min.

[0176] Example 6-1: Free radical photocoupling method as a material cross-linking method to achieve the construction of polyethylene glycol / polyethylene glycol photocross-linked hydrogel

[0177]

[0178] Preparation of hydrogel: Dissolve the modified a4 four-arm polyethylene glycol (4A-PEG-a4, R1 is N-(2-aminoethyl) butyramide, Mn = 40kD, 100mg) and modified b4 four-arm polyethylene glycol (4A-PEG-b4, R2 is hydrogen, Mn = 40kD, 100mg), add copper powder (0.16mg) and N,N,N,N,N-pentamethyldiethylenetriamine (0.43mg), and prepare a hydrogel precursor solution. Lead it into a mold and pass it through a light source (405nm, 200mW / cm 2 ) was irradiated for 60 s to obtain a photo-crosslinked hydrogel. Figure 6 The results of photocuring rheological tests show that the hydrogel precursor solution can undergo cross-linking after only a few seconds of light exposure. Figure 7 The tensile test results show that the cross-linked hydrogel material has deformation ability and certain strength.

[0179]

[0180] Synthesis of 4A-PEG-a4: Synthesized by referring to the synthesis method of PEG-a3. 1 H NMR (400 MHz, D2O): δ3.72 (s, ~3636H), 7.36 (s, ~4H), 7.86 (s, ~4H), 4.99 (s, ~8H), 2.43 (q, ~8H), 2.12 (m, ~8H). The peak of 7.3-7.8 ppm belongs to the proton of the a4 end group of PEG-a4, which can be used to calculate the degree of modification (grafting degree) (3.6-3.8), which represents the average number of a4 molecules on one PEG molecule.

[0181]

[0182] Synthesis of 4A-PEG-b2: 2-bromo-2-methylpropionic acid (0.17 g, 1 mmol) and four-arm polyethylene glycol (20 g, 0.5 mmol; Mw = 40K) were dissolved in anhydrous dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.58 g, 3 mmol) and 4-dimethylaminopyridine (6.1 mg, 0.05 mmol) were added. After stirring overnight, the system was dried by spin drying, dissolved in water, dialyzed for 3 days, and freeze-dried to obtain 19 g (95%) of the product. 1 H NMR (400 MHz, D2O): δ = 3.72 (s, ~3636H), 7.31 (s, ~8H), 6.35 (s, ~8H), 1.60 (s, ~24H). The peak at 6.3-7.4 ppm belongs to the protons of the RAFT end group of PEG-RAFT and can be used to calculate the degree of modification (grafting degree) (3.6-3.8), which represents the average number of b2 molecules on one PEG molecule.

[0183] Example 6-2: Free radical photocoupling method as a material cross-linking method to achieve the construction of sodium hyaluronate / sodium hyaluronate photocross-linked hydrogel

[0184]

[0185] Preparation of hydrogel: Sodium hyaluronate modified with a9 (HA-a9, R1 is N-(2-aminoethyl)butyramide, Mn=340kD, 100mg) and sodium hyaluronate modified with b10 (HA-b10, R2 is aminoethyl, Mn=340kD, 100mg) were dissolved, copper powder (0.16mg) and N,N,N,N,N-pentamethyldiethylenetriamine (0.43mg) were added to prepare a hydrogel precursor solution. The solution was placed in a mold and illuminated by a light source (405nm, 200mW / cm 2 ) was irradiated for 60 s to obtain a photo-crosslinked hydrogel.

[0186]

[0187] Synthesis of compound a9-1: The synthesis was carried out according to the method disclosed in Hu X, Shi J, Thomas S W. Photolabile ROMP gels using ortho-nitrobenzyl functionalized crosslinkers [J]. Polymer Chemistry, 2015, 6 (27): 4966-4971. 1H NMR (400MHz, CDCl3): δ = 7.77 (d, 1H), 7.17 (dd, 1H), 6.93 (dd, 1H), 5.19-5.07 (m, 1H), 5.11 (s, 1H), 4.88 (d, 1H), 1.49 (d, 3H). ESI-HRMS: [M+H] 184.0604.

[0188] Synthesis of compound a9: The synthesis was carried out according to the method disclosed in Yunlong Yang, Jieyuan Zhang, Zhen Liu, Qiuning Lin, Xiaolin Liu, Chunyan Bao, Yang Wang, Linyong Zhu. Adv. Mater., 2016, 28, 2724-2730. 1 H NMR (400MHz, CDCl3): δ=7.33(dd,1H),7.13(d,1H),6.88(dd,1H),6.07(s,1H ),4.97-4.86(m,1H),4.41-4.29(m,1H),3.88-3.68(m,3H),3.21(ddd,1H),2 .93(td,1H),2.81(ddd,1H),2.72-2.59(m,1H),2.47-2.32(m,2H),2.28(s,3 H),2.02-1.86(m,1H),1.44(d,3H),1.04(s,2H).ESI-HRMS:[M+H]312.1554.

[0189] Synthesis of HA-a9: The synthesis was carried out by referring to the synthesis method of HA-a4. According to the H-NMR spectrum, the labeling rate of compound a9 was calculated to be approximately 4.21%.

[0190]

[0191] Synthesis of compound a10-1: α-bromophenylacetic acid (2.2 g, 10 mmol) and N-(tert-butyloxycarbonyl)ethanolamine (3.2 g, 20 mmol) were dissolved in anhydrous dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.751 g, 30 mmol) and 4-dimethylaminopyridine (61 mg, 0.5 mmol) were added. After stirring overnight, the system was extracted three times with water and saturated brine respectively, and the organic phase was dried over anhydrous sodium sulfate and the organic phase was spin-dried to obtain a crude product. The product (3.2 g, 90%) was purified by column chromatography. 1H NMR (400MHz, CDCl3): δ=7.60(ddd,2H),7.42-7.25(m,3H),5.56(t,1H),5.34(s,1H),4.70(dd d,1H),4.21(ddd,1H),3.57(ddd,1H),3.11(ddd,1H),1.44(s,9H).ESI-HRMS:[M+H]358.0648.

[0192] Synthesis of compound a10: Compound a10-1 (3.6 g, 10 mmol) was dissolved in anhydrous dichloromethane (100 mL), 10 mL of trifluoroacetic acid was added, and the reaction was detected by thin layer chromatography until the reaction was completed. After the reaction was completed, the solvent was dried, and the obtained crude product was purified by column chromatography to obtain a pure product as a solid (2.1 g, 86%). 1 H NMR (400MHz, CDCl3): δ=7.62-7.54(m,2H),7.41-7.25(m,3H),5.52(t,1H),4.55(ddd,1H ),3.87(ddd,1H),3.18(ddd,1H),2.73(ddd,1H),1.53(s,2H).ESI-HRMS:[M+H]258.0124.

[0193] Synthesis of HA-b10: The synthesis was carried out by referring to the synthesis method of HA-a4. According to the H-NMR spectrum, the labeling rate of compound a9 was calculated to be approximately 4.19%.

[0194] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A free radical photocoupling method, characterized in that: The following steps are involved: Modifying the o-nitrobenzene compound a with photosensitizing activity on the substance to be coupled A to obtain the photosensitizing active substance Aa; Modifying the organic halogenated compound b on the substance to be coupled B to obtain the substance Bb; Aa and Bb trigger the reaction between a and b under the conditions of catalyst and light, achieving efficient and rapid coupling between the coupling substances A and B; The o-nitrobenzene compound a refers to a compound containing a substituent group R at the ortho position of the nitro group on the benzene ring, and the o-nitrobenzene compound a is as shown in Formula I, wherein the substituent group R is ethyl, hydroxymethyl or 1-methylhydroxymethyl, and the other hydrogen atoms on the benzene ring in Formula I are allowed to be substituted by any number of other groups, wherein at least one substituent is an active group capable of connecting to the substance to be coupled A; The organic halide b refers to an organic compound containing halogen and at least one active group capable of connecting with the substance to be coupled B, as shown in Formula II, wherein X is a halogen selected from Cl, Br or I.

2. A free radical photocoupling method according to claim 1, characterized in that: In the o-nitrobenzene compound a, the active group capable of connecting with the substance to be coupled A is selected from: hydroxyl group, carboxyl group, amine group, ester group, mercapto group, aldehyde group, vinyl group, epoxy group, halogen group, isocyanate group, siloxane group, catechol group, o-phthalaldehyde group, or, The terminal group is modified with an alkylene or modified alkylene group having a hydroxyl group, a carboxyl group, an amine group, an ester group, a thiol group, an aldehyde group, a vinyl group, an epoxy group, a halogen group, an isocyanate group, a siloxane group, a catechol group or a phthalaldehyde group; The modified alkylene group is obtained by replacing any carbon atom of the alkylene group with other heteroatoms.

3. A free radical photocoupling method according to claim 1, characterized in that: The o-nitrobenzene compound a is selected from the following structures: Wherein, R1 is an active group connected to the substance A to be coupled; The active group is selected from: hydroxyl group, carboxyl group, amine group, ester group, mercapto group, aldehyde group, vinyl group, epoxy group, halogen group, isocyanate group, siloxane group, catechol group, o-phthalaldehyde group, or, The terminal group is modified with an alkylene or modified alkylene group having a hydroxyl group, a carboxyl group, an amine group, an ester group, a thiol group, an aldehyde group, a vinyl group, an epoxy group, a halogen group, an isocyanate group, a siloxane group, a catechol group or a phthalaldehyde group; The modified alkylene group is obtained by replacing any carbon atom of the alkylene group with other heteroatoms.

4. A free radical photocoupling method according to claim 1, characterized in that: The organic halide b is selected from the following structures: Wherein, R2 is an active group connected to the substance B to be coupled; The active group is selected from: hydroxyl group, carboxyl group, amine group, ester group, mercapto group, aldehyde group, vinyl group, epoxy group, halogen group, isocyanate group, siloxane group, catechol group, o-phthalaldehyde group, or, The terminal group is modified with an alkylene or modified alkylene group having a hydroxyl group, a carboxyl group, an amine group, an ester group, a thiol group, an aldehyde group, a vinyl group, an epoxy group, a halogen group, an isocyanate group, a siloxane group, a catechol group or a phthalaldehyde group; The modified alkylene group is obtained by replacing any carbon atom of the alkylene group with other heteroatoms.

5. A free radical photocoupling method according to claim 1, characterized in that: The substance to be coupled A and the substance to be coupled B are independently selected from small molecules, macromolecules, tissues, cells, organelles, exosomes, polymer materials, carbon materials, organosilicon materials, glass materials, ceramic materials, metal materials or hydrogel materials; The polymer material is plastic, rubber, fiber, coating or adhesive and a composite material thereof.

6. A free radical photocoupling method according to claim 5, characterized in that: The macromolecule is selected from one or more of polysaccharides, nucleic acids, proteins, polypeptides, and polyethylene glycol; The cell organelle is selected from one or more of a cell nucleus, a mitochondria, a chloroplast, an endoplasmic reticulum, a Golgi apparatus, a lysosome, a vacuole, a ribosome or a centrosome.

7. A free radical photocoupling method according to claim 6, characterized in that: When the substances A and B to be coupled are both small molecules or macromolecules, the method of modifying the o-nitrobenzene compound a with photosensitizing activity on the substance A to be coupled to obtain the photosensitizing active substance Aa is as follows: dissolving the a molecule and the molecule A to be coupled in a solvent, achieving covalent bonding between the two by a chemical reaction method, and purifying and separating to obtain the product Aa; the method of modifying the organic halogenated compound b on the substance B to be coupled to obtain the substance Bb is as follows: dissolving the b molecule and the molecule B to be coupled in a solvent, achieving covalent bonding between the two by a chemical reaction method, and purifying and separating to obtain Bb; When the substance A to be coupled is a tissue, cell, organelle, exosome, polysaccharide, nucleic acid, protein or polypeptide, the method of modifying the o-nitrobenzene compound a having photosensitizing activity on the substance A to be coupled to obtain the photosensitizing active substance Aa is as follows: introducing the a molecule into the corresponding tissue, cell, organelle, exosome, polysaccharide, nucleic acid, protein or polypeptide to form Aa; When the substance A to be coupled is a polymer material, a carbon material, an organosilicon material, a glass material, a ceramic material, a metal material or a hydrogel material, the method for modifying the o-nitrobenzene compound a having photosensitivity on the substance A to obtain the photosensitivity substance Aa is as follows: the a molecule is dissolved in a solvent to form a solution, and based on the interface grafting technology, the a molecule is grafted onto the surface of the material A to form Aa.

8. A free radical photocoupling method according to claim 1, characterized in that: The catalyst refers to a substance that can react with the organic halide b to generate free radicals, and the catalyst is a transition metal or a complex of a transition metal and a ligand.

9. A free radical photocoupling method according to claim 8, characterized in that: The catalyst is copper, ruthenium, iron, a complex of copper and a ligand, a complex of ruthenium and a ligand, or a complex of iron and a ligand, and the ligand is a polyamino compound containing multiple coordination sites.

10. A free radical photocoupling method according to claim 1, characterized in that: The illumination conditions are: light wavelength of 254nm-420nm, light intensity of 10mW / cm 2 -10 W / cm 2 .

11. A free radical photocoupling method according to claim 1, characterized in that: In the free radical photocoupling method, the molar ratio of the o-nitrobenzene compound a to the organic halide b is selected to be 1:(0.1-10000), preferably 1:(0.1-100); The amount of the catalyst is selected to be 0.001 to 2 times, preferably 0.01 to 1.5 times, the molar content of the organic halide b.

12. A kit for realizing photocoupling, wherein the kit is used to realize efficient and rapid coupling between substances A and B to be coupled, characterized in that: The kit comprises: The o-nitrobenzene compound a having photosensitizing activity is used to modify the substance A to be coupled to obtain the photosensitizing substance Aa; Organic halide b: used to modify the substance to be coupled B to obtain substance Bb; Aa and Bb trigger the reaction between a and b under the conditions of catalyst and light, achieving efficient and rapid coupling between the coupling substances A and B; The o-nitrobenzene compound a refers to a compound containing a substituent group R at the ortho position of the nitro group on the benzene ring, and the o-nitrobenzene compound a is as shown in Formula I, wherein the substituent group R is ethyl, hydroxymethyl or 1-methylhydroxymethyl, and the other hydrogen atoms on the benzene ring in Formula I are allowed to be substituted by any number of other groups, wherein at least one substituent is an active group capable of connecting to the substance to be coupled A; The organic halide b refers to an organic compound containing a halogen and at least one active group capable of connecting with the substance to be coupled B, as shown in Formula II, wherein X is a halogen selected from Cl, Br or I; 13. The kit for realizing photocoupling according to claim 12, characterized in that: The o-nitrobenzene compound a is selected from the following structures: Wherein, R1 is an active group connected to the substance A to be coupled; The active group is selected from: hydroxyl group, carboxyl group, amine group, ester group, mercapto group, aldehyde group, vinyl group, epoxy group, halogen group, isocyanate group, siloxane group, catechol group, o-phthalaldehyde group, or, The terminal group is modified with an alkylene or modified alkylene group having a hydroxyl group, a carboxyl group, an amine group, an ester group, a thiol group, an aldehyde group, a vinyl group, an epoxy group, a halogen group, an isocyanate group, a siloxane group, a catechol group or a phthalaldehyde group; The modified alkylene group is obtained by replacing any carbon atom of the alkylene group with other heteroatoms.

14. The kit for realizing photocoupling according to claim 12, characterized in that: The organic halide b is selected from the following structures: Wherein, R2 is an active group connected to the substance B to be coupled; The active group is selected from: hydroxyl group, carboxyl group, amine group, ester group, mercapto group, aldehyde group, vinyl group, epoxy group, halogen group, isocyanate group, siloxane group, catechol group, o-phthalaldehyde group, or, The terminal group is modified with an alkylene or modified alkylene group having a hydroxyl group, a carboxyl group, an amine group, an ester group, a thiol group, an aldehyde group, a vinyl group, an epoxy group, a halogen group, an isocyanate group, a siloxane group, a catechol group or a phthalaldehyde group; The modified alkylene group is obtained by replacing any carbon atom of the alkylene group with other heteroatoms.

15. The kit for realizing photocoupling according to claim 12, characterized in that: The kit also includes a catalyst for achieving the reaction between Aa and Bb under light conditions, wherein the catalyst refers to a substance that can react with the organic halide b to generate free radicals, and the catalyst is a transition metal or a complex of a transition metal and a ligand.

16. Use of the kit for realizing photocoupling according to claim 12, characterized in that: The applications include: The kit is used to achieve chemical coupling between small molecules and small molecules, between macromolecules and macromolecules, and between macromolecules and small molecules; The kit is used to achieve covalent linkage between small molecules or macromolecules and tissues, cells or organelles to achieve biomarkers; The kit is used to achieve surface modification on the surfaces of different materials; The kit is used to realize rapid, light-controllable cross-linking preparation of materials.

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