Radical photocoupling methods, kits for effecting photocoupling, and applications

Abstract

This invention belongs to the field of coupling technology, and relates to a free radical photocoupling method, a kit for achieving photocoupling, and its applications. The free radical photocoupling method includes the following steps: modifying a photosensitive o-nitrobenzene compound a onto a substance A to be coupled to obtain a photosensitive substance A-a; modifying an organohalogenated compound b onto a substance B to be coupled to obtain a substance B-b; under catalytic and light irradiation conditions, A-a and B-b trigger a reaction between a and b, achieving efficient and rapid coupling between substances A and B. Compared with existing technologies, the method provided by this invention can achieve rapid and efficient photo-triggered coupling, and can improve the conversion efficiency of the free radical photocoupling reaction (>85%), thereby solving the application problems of free radical photocoupling technology and broadening its application scope.

Description

Technical Field

[0001] This invention belongs to the field of coupling technology, and relates to a free radical optical coupling method, a reagent kit for realizing optical coupling, and its application. Background Technology

[0002] Optical coupling technology refers to the process by which two substances to be coupled are linked together to form a single entity under photo-triggered conditions. It has numerous applications in surface functionalization, polymer coupling, and the modification and labeling of biomolecules. Thanks to the extremely high reactivity of free radicals, among existing optical coupling technologies, coupling via the reaction of free radicals with double bonds is the fastest (the reaction rate constants can reach as high as 10⁻⁶). 4 M -1 s -1 It has the shortest reaction time. However, the high reactivity of free radicals also leads to problems such as poor reaction controllability and poor selectivity. Therefore, 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] To address the shortcomings of existing free radical photocoupling technologies in terms of conversion efficiency, this invention proposes a free radical photocoupling method, a reagent kit for achieving photocoupling, and its applications.

[0004] This invention proposes a novel free radical photocoupling method, which involves attaching a photosensitive molecule (a) and an organohalogenated molecule (b) to two substances to be coupled, respectively. In the presence of a catalyst, the reaction between the a and b molecules is triggered by light, thereby achieving efficient and rapid coupling of the two substances.

[0005] The method provided by this invention enables rapid and efficient photo-triggered coupling. It can improve the conversion efficiency of free radical photocoupling reactions (>85%), thereby solving the application problems of free radical photocoupling technology and broadening its application scope.

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

[0007] This invention provides a free radical optical coupling method, comprising the following steps:

[0008] A photosensitive o-nitrobenzene compound a was modified onto the substance to be coupled, resulting in a photosensitive substance Aa.

[0009] Organic halide b was modified onto the substance B to be coupled to obtain substance Bb;

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

[0011] The o-nitrobenzene compound a refers to a compound in which a substituent R is located at the ortho position of the nitro group on the benzene ring. The o-nitrobenzene compound a is shown in Formula I. The substituent R is ethyl, hydroxymethyl or 1-methylhydroxymethyl. Other hydrogen atoms on the benzene ring in Formula I are allowed to be replaced by any number of other groups. Among them, at least one substituent is an active group that can be connected to the substance A to be coupled.

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

[0013]

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

[0015] Hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, phthalaldehyde, or,

[0016] The end groups are modified with alkylene groups or modified alkylene groups, including hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, or phthalaldehyde groups.

[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] R1 is the active group that is attached to the substance A to be coupled.

[0021] The active groups are selected from:

[0022] Hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, phthalaldehyde, or,

[0023] The end groups are modified with alkylene groups or modified alkylene groups, including hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, or phthalaldehyde groups.

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

[0025]

[0026] R2 is the active group that is attached to the substance B to be coupled.

[0027] The active groups are selected from:

[0028] Hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, phthalaldehyde, or,

[0029] The end groups are modified with alkylene groups or modified alkylene groups, including hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, or phthalaldehyde groups.

[0030] In one embodiment of the present invention, the substances to be coupled, A and B, are independently selected from small molecules, polymers, tissues, cells, organelles, exosomes, polymeric materials, carbon materials, organosilicon materials, glass materials, ceramic materials, metallic materials, or hydrogel materials (hydrogel materials are cross-linked aqueous materials).

[0031] The polymeric material is a plastic, rubber, fiber, coating or adhesive, or 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 organelles are selected from one or more of the following: nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, lysosomes, vacuoles, ribosomes, or centrosomes.

[0034] In one embodiment of the present invention, when both the substances to be coupled, A and B, are small molecules or polymers, the method for modifying the photosensitive o-nitrobenzene compound a onto the substance to be coupled, to obtain the photosensitive substance Aa, is as follows: molecule a and the molecule to be coupled, A, are dissolved in a suitable solvent, and the two are covalently linked through a selected chemical reaction method, and then purified and separated to obtain product Aa; the method for modifying the organohalogenated compound b onto the substance to be coupled, to obtain substance Bb, is as follows: molecule b and the molecule to be coupled, B, are dissolved in a suitable solvent, and the two are covalently linked through a selected chemical reaction method, and then purified and separated to obtain Bb.

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

[0036] In one embodiment of the present invention, when the substance to be coupled, A, is a polymer material, carbon material, organosilicon material, glass material, ceramic material, metal material, or hydrogel material, the method of modifying the substance to be coupled, a photosensitive o-nitrobenzene compound a, onto the substance to be coupled to obtain a photosensitive substance Aa is as follows: the a molecule is dissolved in a suitable solvent to form a solution, and based on conventional interface grafting techniques such as siloxane coupling technology, catechol technology, and chemical modification technology, the a molecule is grafted onto the surface of 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 capable of reacting with organohalogen 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] More preferably, the catalyst is a complex of copper, ruthenium, iron, copper and ligands, ruthenium and ligands, or iron and ligands.

[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 conditions are illumination by a light source at a specific wavelength, a specific light intensity, and a specific duration. Preferably, the light wavelength is 254nm-420nm, and the light intensity is 10mW / cm². 2 -10 W / cm 2 The duration of illumination ranges from seconds to hours.

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

[0043] The present invention further provides a kit for achieving optical coupling, the kit being used to achieve efficient and rapid coupling between substances A and B to be coupled, the kit comprising:

[0044] Photosensitive o-nitrobenzene compound a: used to modify the photosensitive substance Aa on the substance to be coupled A;

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

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

[0047] The o-nitrobenzene compound a refers to a compound in which a substituent R is located at the ortho position of the nitro group on the benzene ring. The o-nitrobenzene compound a is shown in Formula I. The substituent R is ethyl, hydroxymethyl or 1-methylhydroxymethyl. Other hydrogen atoms on the benzene ring in Formula I are allowed to be replaced by any number of other groups. Among them, at least one substituent is an active group that can be connected to the substance A to be coupled.

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

[0049]

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

[0051] Hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, phthalaldehyde, or,

[0052] The end groups are modified with alkylene groups or modified alkylene groups, including hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, or phthalaldehyde groups.

[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] R1 is the active group that is attached to the substance A to be coupled.

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

[0058]

[0059] R2 is the active group that is attached to the substance B to be coupled.

[0060] In one embodiment of the invention, the kit further includes a catalyst for realizing the reaction of Aa and Bb under light conditions, wherein the catalyst is a substance capable of reacting with organohalogen 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 applications of a reagent kit for achieving optical coupling, the applications including:

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

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

[0065] The kit is used to achieve surface modification of different material surfaces;

[0066] The kit is used to achieve rapid, photo-controlled crosslinking preparation of materials.

[0067] The reaction mechanism of the free radical photocoupling method of this invention is as follows: Organic halide b reversibly generates carbon free radicals in the presence of a catalyst; o-nitrobenzene compound a generates a nitrosobenzene structure upon exposure to light. The nitroso group in this structure can react with the carbon free radicals generated by the organic halide, thereby forming a coupling product. According to literature reports, the reaction rate constant of the nitroso-carbon free radical reaction is as high as 10. 8 (CLHawkins, MJDavies, Biochim. Biophys. Acta, 2014, 1840, 708-721), significantly higher than the reaction rate constant of double bonds and carbon radicals (the reaction mechanism of existing radical photocoupling technology) (generally <10). 5 ).

[0068] Therefore, compared with existing technologies, the free radical photocoupling method of the present invention has a faster reaction rate. Simultaneously, due to the extremely high reaction rate and reactivity, the nitroso-carbon free radical reaction can effectively reduce side reactions of free radicals, such as self-quenching and oxygen inhibition. Therefore, the free radical photocoupling method provided by the present invention has a higher conversion efficiency than existing free radical photocoupling methods.

[0069] In summary, this invention achieves an organic combination of rapid kinetics and efficient conversion in the radical photocoupling method through substrate design. Attached Figure Description

[0070] Figure 1 The NMR spectrum is the NMR conversion rate of formulation 8 in Example 1.

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

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

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

[0074] Figure 5 The images show fluorescence modification of the surface of nano-silica spheres using the free radical photocoupling method of this invention as a material surface modification method (left: control; right: free radical photocoupling method).

[0075] Figure 6 The figure shows the in-situ rheological test results of the polyethylene glycol / polyethylene glycol photocrosslinked hydrogel material constructed by the free radical photocoupling method of this invention.

[0076] Figure 7 The tensile stress-strain curve of the polyethylene glycol / polyethylene glycol photocrosslinked hydrogel material constructed by the free radical photocoupling method of this invention is shown. Detailed Implementation

[0077] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0078] Example 1: Determination of kinetic constants and conversion rate of the 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), once the parent structure of o-nitrobenzene compound a (excluding R1) and the parent structure of organohalogen b (excluding R2) are determined, the kinetic constants and conversion rates of their reactions are essentially determined. That is, the choice of R1 and R2 groups will not significantly affect the kinetic constants and conversion rates of their reactions. Based on this, in this embodiment, R1 in all o-nitrobenzene compounds was selected as methyl, and R2 in all organohalogens was selected as ethyl. The kinetic constants and conversion rates of the reactions between o-nitrobenzene compounds and organohalogens were determined based on this.

[0080] This embodiment uses nuclear magnetic resonance (NMR) titration to determine the kinetic constants and conversion rates of the free radical photocoupling method. Specifically, it uses an NMR spectrometer (400 MHz) to measure the changes in the reactants and products of o-nitrobenzene compounds over time. Taking the reaction of a2 and b8 as an example, a2 (1 μM) and b8 (80 μM) are dissolved in deuterated acetonitrile, copper powder (80 μM) and N,N,N,N,N-pentamethyldiethylenetriamine (64 μM) are added, and 3,4,5-trichloropyridine (1 mM) is added as an NMR calibrator. After purging the solution with nitrogen for 10 min to remove oxygen, the NMR is measured using a suitable wavelength (intensity 1 W / cm). 2 The solution was illuminated by a light source (approximately 0s, 2s, 10s, 15s, and 20s), followed by sampling and NMR analysis (e.g., ...). Figure 1 (As shown). In the case of excess b1, 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). Figure 2 (as shown),

[0081]

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

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

[0084] Substitute different b1 concentrations [B] and their corresponding k1 values ​​into the second-order rate constant calculation formula (2) to calculate the second-order rate constant k2 (e.g., Figure 3 (as 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 reacting with organohalides under different formulations.

[0088]

[0089]

[0090] As shown in Table 1, the reaction kinetic constants of the reactions between o-nitrobenzene compounds and organohalides under different formulations are all as high as 10. 4 M -1 s -1 The reaction rate is extremely fast. More importantly, the conversion rates of o-nitrobenzene compounds reacting with organohalides under different formulations all reach over 85%, with many formulations exceeding 90%, far exceeding the conversion efficiency of double bond-free radical reactions (usually <50% or even lower: Yu, Y. et al. Journal of the American Chemical Society 2018, 140, 6797-6800). Therefore, this invention achieves an organic combination of rapid kinetics and efficient conversion in the free radical photocoupling method through the reaction of o-nitrobenzene compounds with organohalides.

[0091] Example 2: High Reaction Selectivity of the Free Radical Photocoupling Method

[0092] Different competing substrates (containing double bonds capable of reacting with free radicals or thiol, amino, and sulfite groups capable of reacting with nitrosyl groups) were added to the reaction system of o-nitrobenzene compounds and organohalides. The effect of different substrates on the reaction conversion rate of o-nitrobenzene compounds and organohalides was monitored by NMR titration. Taking the reaction of a2 and b2 as an example, acetonitrile solutions of a2 (1 μM) and b2 (80 μM) were prepared, and copper powder (80 μM) and N,N,N,N,N-pentamethyldiethylenetriamine (64 μM) were added; styrene (1 μM) was added; after the solution was deoxygenated by purging with nitrogen for 10 min, the reaction was performed using an appropriate wavelength (intensity 1 W / cm). 2 Irradiation was performed using a light source (left and right) for 0s, 2s, 10s, 15s, and 20s; samples were taken and the conversion rate of the reaction was calculated using nuclear magnetic titration.

[0093] Table 2. Effect of different substrates on the conversion rate of o-nitrobenzene compounds with organohalides (R1 is methyl; R2 is ethyl)

[0094]

[0095]

[0096] As shown in Table 2, none of the different competing reaction substrates significantly affected the conversion rate of the reaction between o-nitrobenzene compounds and organohalides. These results indicate that the radical photocoupling method proposed in this invention has extremely high reaction selectivity.

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

[0098] Experimental Methods: Both substances A and B to be coupled are small molecules or polymers. Method for linking substance A to molecule a: Molecule a and molecule A to be coupled are dissolved in a suitable solvent, and covalently linked through a selected chemical reaction method. The product Aa is then purified and separated. Method for linking molecule B to molecule b: Molecule b and molecule B to be coupled are dissolved in a suitable solvent, and covalently linked through a selected chemical reaction method. The product Bb is then purified and separated. The grafting degree of the chain transfer reagent molecule on the Bb molecule is ≤1.

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

[0100] The ratio of Aa to Bb is selected based on the molar ratio of a molecules to b molecules, preferably 1:(0.1-100); the amount of 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 / cm². 2 -10 W / cm 2 The reaction time is 30 seconds to 60 minutes.

[0101] Among them, "high molecular weight (small molecular weight)" refers to either high molecular weight polymers or small molecular weight polymers.

[0102] The specific implementation method is as follows:

[0103] Example 3-1: Free radical photocoupling method as a chemical coupling method to achieve 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 gas was introduced for deoxygenation for 30 min, and the reaction was carried out at 405 nm (2 W / cm²). 2After irradiation with a light source for 30 seconds, the system spun to a viscous consistency and was then poured into diethyl ether for recrystallization to obtain rhodamine-modified PEG polymer, yielding 105 mg of product (95% yield). The peak at 7.71 ppm belongs to the proton of the rhodamine group and can be used to calculate the degree of modification (grafting degree), which is 0.97 (the theoretical degree of modification is 1), representing the average number of rhodamine molecules on a PEG molecule. Y in the figure indicates that the compound may form different substituent groups at this position, but this does not affect the implementation and results of the free radical photocoupling method. The same applies to subsequent Y occurrences and will not be repeated. The above results verify the high efficiency and rapid reaction characteristics of the free radical photocoupling method of this invention.

[0106]

[0107] Synthesis of compound a3-1: The synthesis was carried out according to the method disclosed in 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, Zhenzhen 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: A methyl-terminated polyethylene glycol (PEG-OH; Mw = 10000; 1 g, 0.1 mmol), 4-dimethylaminopyridine (0.005 g, 0.05 mmol), triethylamine (0.101 g, 1.0 mmol), and nitrobenzene 4-chloroformate (0.201 g, 1 mmol) were dissolved in anhydrous dichloromethane (100 mL) and stirred at room temperature for 5 hours. The solvent was then rotary evaporated under vacuum to approximately half its original volume. The mixture was then poured into diethyl ether, and the resulting white precipitate 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. Dissolve in water, dialyze by distilled water, and freeze-dry to obtain PEG-a3 (7.2 g), yield 90%. The peak at 7.35-7.26 ppm belongs to the protons of the a3 group in the PEG-a3 molecule and can be used to calculate the degree of modification (grafting degree), which is (1.8-1.9), representing the average number of a3 molecules on a 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 mixture was extracted three times with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, and the crude product was obtained by rotary evaporation. The crude 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 following the method used for 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 gas was introduced for deoxygenation for 30 min, and the reaction was carried out at 405 nm (2 W / cm²). 2 After irradiation with a light source for 30 seconds, the system spun to a viscous consistency and was then poured into diethyl ether for recrystallization to obtain rhodamine-modified PEG polymer, yielding 105 mg of product (95% yield). The peak at 7.97 ppm belongs to the protons of the rhodamine group and can be used to calculate the degree of modification (grafting degree), which is 0.97 (the theoretical degree of modification is 1), representing the average number of rhodamine molecules on a PEG molecule. These results verify the high efficiency and rapid reaction characteristics of the free radical photocoupling method of this invention.

[0116]

[0117] Synthesis of compound a4: The synthesis was carried out according to the method disclosed in Yunlong Yang, Jieyuan Zhang, Zhenzhen 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: Hyaluronic acid (2g, 340kDa) was dissolved in 100mL of 0.01mol / L 2-(N-morpholine)ethanesulfonic acid MES buffer solution (pH=5.2) and stirred until completely dissolved. Compound a4 (66mg, 0.2mmol) was weighed and dissolved in 10mL of dimethyl sulfoxide (DMSO) and added to the above reaction solution. 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) (0.4g, 1.5mmol) was weighed and dissolved in 3mL of MES buffer solution and added to the above reaction solution in three portions (every 1h). The reaction was carried out at 35℃ for 24h. 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 (1.92g). Based on the 1H NMR spectrum, the labeling rate of compound 3 can be 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 following the same method as 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 the free radical photocoupled method as a biological marker method

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

[0124] Optionally, the substance to be coupled, A, is linked to molecule a. The linking method involves introducing molecule a into the corresponding tissue, cell, organelle, exosome, polysaccharide, nucleic acid, protein, or polypeptide using conventional methods in this field, such as antigenic antibody recognition, protein tagging, non-natural amino acid methods, or chemical labeling methods, to form Aa. The linking method between the substance to be coupled, B, and molecule b involves dissolving both substances in a suitable solvent, achieving covalent linking through a selected chemical reaction method, and then purifying and separating to obtain Bb. The grafting degree of molecule b on the Bb molecule is ≤1.

[0125] Bb is dissolved in water to form a solution, which is then added to a matrix containing Aa. An appropriate amount of catalyst is added, and after photoreaction, tissues, cells, organelles, exosomes, polysaccharides, nucleic acids, proteins, or polypeptides labeled with small or large molecules of B are obtained.

[0126] The ratio of Aa to Bb is selected based on the molar ratio of a molecules to b molecules, preferably a molar ratio of 1:(0.1 to 100); the amount of catalyst is adjusted according to the content of Bb, preferably 0.01 to 1.5 times the molar content of Bb; the preferred photoreaction conditions are a light wavelength of 365 nm to 420 nm and a light intensity of 10 mW / cm². 2 -10W / cm 2 The reaction time is 5 seconds to 30 minutes.

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

[0128] The polymer PNIPAM-RhB-b2 (R2 being hydroxyethyl) with a narrow molecular weight distribution was constructed by atom transfer radical polymerization (ATRP), and the polymer was further labeled on the cell surface by the membrane-directing group DPPE (DPPE-a8, R1 being (methoxycarbonyl)glycine) modified by a8.

[0129]

[0130] Cell labeling: Human cervical cancer cells (HeLa S3) were cultured in DMEM medium (a medium containing various amino acids and glucose) with high glucose (Life Technologies-Gibco, 11995073), fetal bovine serum (10% heat-inactivated, Omega Scientific, FB02), penicillin (100 U / mL), and streptomycin (100 μg / mL, Life Technologies-Gibco, 15140122). Cells were grown at 37°C in a 5% carbon dioxide (CO2) atmosphere. To promote cell adhesion during imaging, 96-well slides pre-coated with 0.5% (w / v) polylysine were used for imaging. HeLa S3 cell lines were incubated in the medium at a density of 40% per well in 96-well plates. Twenty-four hours later, the cells were treated as follows: HeLa S3 cancer cells were placed in 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 then placed in 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 / cm²). 2 The cell solution was irradiated for 60 seconds, and then imaged under a fluorescence microscope, with an unilluminated identical solution used as a control. The results are as follows: Figure 4 As shown (left: control group; right: experimental group). Under the same conditions, the cells in the control group showed almost no fluorescence, while the cell membranes of the experimental group exhibited significantly bright fluorescence. This result demonstrates that the free radical photocoupling method, as a biomarker, can achieve cell-polymer coupling, and also verifies the high efficiency and rapid response characteristics of the free radical photocoupling method of this 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 three freeze-drying cycles, N,N,N,N,N-pentamethyldiethylenetriamine (13.9 mg, 0.08 mmol) was added and the mixture was reacted for 24 h. After the reaction was complete, the system was vortexed until viscous and then reprecipitated in diethyl ether to give a purple-red solid product with a yield of 1 g (90%). The product was dissolved in DMF and analyzed by GPC (Mn, GPC =18 kg / mol, Mw / Mn = 1.2).

[0133]

[0134] Synthesis of compound a8-1: The synthesis was carried out according to the method disclosed in 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), and anhydrous triethylamine (20.2 g, 20 mmol) was added. Acetyl chloride (12.56 g, 16 mmol) was added dropwise under ice bath conditions, and the mixture was stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated, the system was dissolved in dichloromethane, and extracted three times with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, and the crude product was purified by column chromatography. The pure product was 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, Zhenzhen 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: A DMF solution (100 mL) of compound a8-3 (8.7 g, 27.4 mmol), tert-butyldimethylchlorosilane (6.2 g, 41.1 mmol), and imidazole (2.8 g, 41.1 mmol) was stirred at room temperature for 24 hours, and the DMF was removed by evaporation under reduced pressure. The mixture was purified by column chromatography to give 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 sodium hydroxide (NaOH, 10% by mass) aqueous solution was added dropwise. The reaction was monitored by thin-layer chromatography until completion. After the reaction was complete, the solvent was evaporated, the system was dissolved in dichloromethane, and extracted three times with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, and the crude product was purified by column chromatography. The pure product was 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), and nitrobenzene 4-chloroformate (4.1 g, 20 mmol) were dissolved in anhydrous dichloromethane (100 mL) and the mixture was stirred at room temperature for 10 min. The organic phase was evaporated to dryness, and the crude product was purified by column chromatography. The crude product was then dissolved in anhydrous dichloromethane (100 mL), and triethylamine (2.5 g, 25 mmol) and glycine (1.5 g, 20 mmol) were added. The mixture was stirred at room temperature for 30 min. After the reaction was complete, the solvent was evaporated to dryness, and the mixture was dissolved in dichloromethane. The solution was extracted three times with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, and the crude product was purified by column chromatography. The purified 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: Tetrabutylammonium fluoride (5.2 g, 20 mmol) was added to 100 mL of stirred THF solution containing compound a8-6 (4.9 g, 10 mmol). After stirring at room temperature for 8 hours, the mixture was evaporated under vacuum. The resulting mixture was purified by column chromatography to give 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-propanetriol-3-phosphoethanolamine (34.6 mg, 0.05 mmol) was added. After reacting overnight, the solvent was evaporated, 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 for protein-polymer coupling

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

[0144]

[0145] Protein labeling: Bovine serum albumin (0.3 g, 10 mmol) was dissolved in 4 mL of phosphate buffer (pH 7.4), and NHS-a7 (3.9 g, 10 mmol) was added. The mixture was stirred overnight at room temperature, and the system was reprecipitated in anhydrous ethanol and centrifuged to obtain a7-labeled bovine serum albumin. The a7 labeling rate was 130% (molar ratio) as determined by UV detection. A7-labeled bovine serum albumin (200 μL, 0.4 mmol) was dissolved in phosphate buffer (pH 7.4), and P(PEGA)-FL-b1 (5 nM), copper powder (5 nM), and N,N,N,N,N-pentamethyldiethylenetriamine (4 nM) in PBS solution were added. Finally, the cell solution was imaged using a light source (420 nm, 200 mW / cm²). 2 The cell solution was irradiated for 30 seconds, and the system was dialyzed and lyophilized to obtain polymerically labeled bovine serum albumin Protein-P(PEGA)-FL. The labeling rate of the polymer was calculated to be 90% by UV analysis. This result indicates that the free radical photocoupling method can achieve cell-polymer coupling as a biolabeling method, and also verifies the high efficiency and rapid reaction characteristics of the free radical photocoupling method of this invention.

[0146]

[0147] Synthesis of compound b5: The synthesis of compound b5 was carried out according to the method for synthesizing 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: The polymer PNIPAM-RhB-b2 was synthesized according to the same method. GPC analysis (Mn, GPC =16 kg / 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 complete, the solvent was evaporated to dryness, the system was dissolved in dichloromethane, and extracted three times with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, and the crude product was purified by column chromatography to obtain 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 in 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), and 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 evaporated to dryness, and the crude product was purified by column chromatography to obtain a pure 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 method used for the synthesis 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 method used for the synthesis 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), and succinic anhydride (8.6 g, 20 mmol) was added. The reaction was monitored by thin-layer chromatography until completion. After the reaction was complete, the system was extracted three times with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by column chromatography, and the pure product was 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 using the same method as that used for 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 evaporated, 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 optical coupling method as a material surface modification method

[0159] Experimental Procedure: Substance A to be coupled is a polymer, carbon material, organosilicon material, glass material, ceramic material, metal material, or hydrogel material; substance B to be coupled is a small molecule or polymer. Substance A is coupled to either molecule a or molecule b, while substance B is coupled to the other molecule. The coupled A and B react under light in the presence of a catalyst to obtain the coupling product of A and B.

[0160] Optionally, the coupling substance A is connected to molecule a. The connection method is as follows: molecule a is dissolved in a suitable solvent to form a solution. Based on conventional interfacial grafting methods such as siloxane coupling, catechol coupling, and chemical modification, molecule a is grafted onto the surface of material A through spin coating, immersion, photomask, or laser processing to form Aa. The coupling substance B is connected to molecule b. The coupling substances B and b are dissolved in a suitable solvent. They are covalently linked through a selected chemical reaction method, and then purified and separated to obtain Bb.

[0161] Material Aa is immersed in a solution formed by Bb, and an appropriate amount of catalyst is added. After photo-irradiation reaction, the material is removed from the solution, rinsed, and material A with surface-modified small molecules or polymers B is obtained.

[0162] The ratio of Aa to Bb is selected based on the molar ratio of a molecules to b molecules, preferably a molar ratio of 1:(0.1 to 100); the amount of catalyst is adjusted according to the content of Bb, preferably 0.01 to 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: Fluorescence modification of nano-silica spheres using free radical photocoupling as a material surface modification method.

[0164] RhB-b2 (R2 being hydroxyethyl) was obtained by reacting Rhodamine B with an organohalogenated compound b4, and the dye was further used to label the surface of nano-silica by a silane coupling agent modified with a10 (Si-a10, R1 being butyric acid).

[0165]

[0166] a10 labeling on the surface of nano-silica spheres: 0.1 g of nano-silica spheres were dispersed in anhydrous toluene (10 mL), and Si-a10 (47.3 mg, 0.1 mmol) was added. The mixture was refluxed overnight at 120 °C. After the reaction was complete, the mixture was centrifuged and washed three times with toluene. The washed and centrifuged solutions were brought to a final volume of 100 mL. The amount of unreacted Si-a4 was detected by UV absorption, and the Si-a4 grafting amount on the surface of the nano-silica spheres was calculated to be 33% (mass ratio).

[0167] Fluorescent labeling of silica nanospheres was achieved using a free radical photocoupling method: 10 mg of Si-a10 labeled silica nanospheres were dispersed in 100 mL of deionized water. 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 the mixture was deoxygenated by purging with argon gas for 30 min. A light source (405 nm, 2 W / cm²) was used. 2 After irradiating the system for 60 seconds, it was centrifuged, washed three times with ethanol, and observed using a confocal microscope. The un-illuminated group, treated under the same conditions, served as a control. Results are as follows: Figure 5 As shown, the control group's nano-silica spheres showed almost no fluorescence on their surface, while the experimental group's nano-silica spheres exhibited significant fluorescence. This result demonstrates that the free radical photocoupling method of this invention can achieve fluorescence modification of the nano-silica sphere surface, and also verifies the high efficiency and rapid response characteristics of the free radical photocoupling method of this invention.

[0168]

[0169] Synthesis of a4-1: The synthesis was performed 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 crosslinking method

[0172] Experimental procedure: Substance A to be coupled is a small molecule or a polymer, and substance B to be coupled is a small molecule or a polymer. One of the molecules, a or b, is grafted onto substance A, and the other is grafted onto substance B, with a grafting degree ≥ 2 in both cases. The grafted A and B undergo a photochemical reaction to obtain the coupling product of A and B.

[0173] Optionally, molecule a is grafted onto substance A to be coupled. The grafting method is as follows: molecule a and substance A to be coupled are dissolved in a suitable solvent, the grafting reaction is carried out through a selected chemical reaction method, and the product Aa is purified and separated. The grafting degree of molecule a on the molecule A to be coupled is ≥2. Similarly, molecule b is grafted onto substance B to be coupled. The method is as follows: molecule b and substance B to be coupled are dissolved in a suitable solvent, the grafting reaction is carried out through a selected chemical reaction method, and the product Bb is purified and separated. The grafting degree of molecule b on the molecule 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 an appropriate amount of catalyst is added. The mixture is then photocrosslinked or photocrosslinked after the solvent is removed to obtain a crosslinked material of A and B.

[0175] The ratio of Aa to Bb is selected based on the molar ratio of a molecules to b molecules, preferably a molar ratio of 1:(0.1 to 100); the amount of catalyst is adjusted according to the content of Bb, preferably 0.01 to 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: Construction of polyethylene glycol / polyethylene glycol photocrosslinked hydrogels using free radical photocoupling as a material crosslinking method.

[0177]

[0178] Hydrogel preparation: A four-arm polyethylene glycol modified with a4 (4A-PEG-a4, R1 is N-(2-aminoethyl)butyramide, Mn = 40 kD, 100 mg) and a four-arm polyethylene glycol modified with b4 (4A-PEG-b4, R2 is hydrogen, Mn = 40 kD, 100 mg) were dissolved. Copper powder (0.16 mg) and N,N,N,N,N-pentamethyldiethylenetriamine (0.43 mg) were added to prepare a hydrogel precursor solution. This solution was then placed in a mold and exposed to a light source (405 nm, 200 mW / cm²). 2 Irradiation for 60 seconds yielded a photocrosslinked hydrogel. Figure 6 Photocuring rheological tests showed that the hydrogel precursor solution could crosslink after only a few seconds of light exposure. Figure 7 Tensile test results show that the cross-linked hydrogel material has deformation capacity and a certain strength.

[0179]

[0180] Synthesis of 4A-PEG-a4: The synthesis was performed following the same method as PEG-a3. 1 ¹H NMR (400MHz, D₂O): δ 3.72 (s, ~3636H), 7.36 (s, ~4H), 7.86 (s, ~4H), 4.99 (s, ~8H), 2.43 (q, ~8H), 2.12 (m, ~8H). The peaks in the 7.3-7.8 ppm range belong to the protons of the a4 terminal group of PEG-a4 and can be used to calculate the degree of modification (grafting degree) (3.6-3.8), which represents the average number of a4 molecules on a PEG molecule.

[0181]

[0182] Synthesis of 4A-PEG-b2: 2-Bromo-2-methylpropionic acid (0.17 g, 1 mmol) and tetra-arm polyethylene glycol (20 g, 0.5 mmol; Mw = 40 K) were dissolved in anhydrous dichloromethane. 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 evaporated to dryness, dissolved in water, dialyzed for 3 days, and lyophilized to give 19 g (95%) of the product. 1 ¹H NMR (400MHz, D₂O): δ = 3.72 (s, ~3636H), 7.31 (s, ~8H), 6.35 (s, ~8H), 1.60 (s, ~24H). The peaks in the range of 6.3-7.4 ppm belong to the protons of the RAFT end groups 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 a PEG molecule.

[0183] Example 6-2: Construction of sodium hyaluronate / sodium hyaluronate photocrosslinked hydrogels using free radical photocoupling as a material crosslinking method.

[0184]

[0185] Hydrogel preparation: 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, and copper powder (0.16mg) and N,N,N,N,N-pentamethyldiethylenetriamine (0.43mg) were added to prepare a hydrogel precursor solution. This solution was then placed in a mold and subjected to a light source (405nm, 200mW / cm²). 2 Irradiation for 60 seconds yielded a photocrosslinked hydrogel.

[0186]

[0187] Synthesis of compound a9-1: The synthesis was carried out according to the method disclosed by 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, Zhenzhen 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 1H NMR spectrum, the labeling rate of compound a9 can be calculated to be approximately 4.21%.

[0190]

[0191] Synthesis of compound a10-1: α-Bromophenylacetic acid (2.2 g, 10 mmol) and N-(tert-Butoxycarbonyl)ethanolamine (3.2 g, 20 mmol) were dissolved in anhydrous dichloromethane. 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 mixture was extracted three times with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, and the crude product was obtained by rotary evaporation. The crude 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), and 10 mL of trifluoroacetic acid was added. The reaction was monitored by thin-layer chromatography until completion. After the reaction was complete, the solvent was evaporated, and the crude product was purified by column chromatography. The pure product was 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 1H NMR spectrum, the labeling rate of compound a9 can be calculated to be approximately 4.19%.

[0194] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any 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 invention should be within the protection scope of the present invention.

Claims

1. A free radical optical coupling method, characterized in that, Includes the following steps: A photosensitive o-nitrobenzene compound a was modified onto the substance to be coupled, resulting in a photosensitive substance Aa. Organic halide b was modified onto the substance B to be coupled to obtain substance Bb; Under catalytic and light conditions, Aa and Bb trigger a reaction between a and b, achieving efficient and rapid coupling between the substances to be coupled, A and B, with a conversion efficiency of >85% for the free radical photocoupling reaction; The o-nitrobenzene compound a is selected from the following structures: ； Wherein, R1 is the active group connected to the substance A to be coupled; The organohalogenated compound b is selected from the following structures: ； Wherein, R2 is the active group connected to the substance B to be coupled; The active groups are selected from: Hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, phthalaldehyde, or, The terminal groups are modified with hydroxyl, carboxyl, amino, ester, mercapto, aldehyde, vinyl, epoxy, halogen, isocyanate, siloxane, catechol, or phthalaldehyde groups, or alkylene or modified alkylene groups. The modified alkylene group is obtained by replacing any carbon atom of the alkylene group with other heteroatoms; The catalyst refers to a substance that can react with organohalogenated compound b to produce carbon free radicals; Organohalogenated compound b reversibly generates carbon free radicals in the presence of a catalyst; o-nitrobenzene compound a generates nitrosobenzene structure when exposed to light, and the nitroso group in this structure can react with the carbon free radicals generated by the organohalogenated compound to form coupling products.

2. The free radical optical coupling method according to claim 1, characterized in that, The substances to be coupled, A and B, are independently selected from small molecules, polymers, tissues, cells, organelles, exosomes, polymeric materials, carbon materials, organosilicon materials, glass materials, ceramic materials, metallic materials, or hydrogel materials. The polymer is selected from one or more of polysaccharides, nucleic acids, proteins, polypeptides, and polyethylene glycol; The polymeric material is a plastic, rubber, fiber, coating or adhesive, or a composite material thereof.

3. The free radical optical coupling method according to claim 2, characterized in that, The organelles are selected from one or more of the following: nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, lysosomes, vacuoles, ribosomes, or centrosomes.

4. The free radical optical coupling method according to claim 3, characterized in that, When both the substances to be coupled, A and B, are small molecules or macromolecules, the method for modifying the photosensitive o-nitrobenzene compound a onto the substance to be coupled, to obtain the photosensitive substance Aa, is as follows: molecule a and the molecule to be coupled, A, are dissolved in a solvent, and covalently linked through a chemical reaction, followed by purification and separation to obtain product Aa; the method for modifying the organohalogenated compound b onto the substance to be coupled, to obtain substance Bb, is as follows: molecule b and the molecule to be coupled, B, are dissolved in a solvent, and covalently linked through a chemical reaction, followed by purification and separation to obtain Bb; When the substance to be coupled, A, is a tissue, cell, organelle, exosome, polysaccharide, nucleic acid, protein, or polypeptide, the method of modifying the substance to be coupled, A, with a photosensitive o-nitrobenzene compound a, to obtain a photosensitive substance Aa is as follows: the a molecule is introduced into the corresponding tissue, cell, organelle, exosome, polysaccharide, nucleic acid, protein, or polypeptide to form Aa. When the material to be coupled, A, is a polymer, carbon, organosilicon, glass, ceramic, metal, or hydrogel material, the method of modifying the material to be coupled, A, with a photosensitive o-nitrobenzene compound a, to obtain a photosensitive 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 material A to form Aa.

5. The free radical optical coupling method according to claim 1, characterized in that, The catalyst is a transition metal or a complex of a transition metal and a ligand.

6. The free radical optical coupling method according to claim 5, characterized in that, The catalyst is a complex of copper, ruthenium, iron, copper and ligands, ruthenium and ligands, or iron and ligands, wherein the ligand is a polyamino compound containing multiple coordination sites.

7. The free radical optical coupling method according to claim 1, characterized in that, The illumination conditions are: light wavelength of 254 nm-420 nm and light intensity of 10 mW / cm². 2 -10 W / cm 2 .

8. The free radical optical coupling method according to claim 1, characterized in that, In the free radical photocoupling method, the molar ratio of o-nitrobenzene compound a to organohalogenated compound b is selected as 1:(0.1~10000). The amount of catalyst is selected to be 0.001 to 2 times the molar content of organohalogen b.

9. The free radical optical coupling method according to claim 8, characterized in that, In the free radical photocoupling method, the molar ratio of o-nitrobenzene compound a to organohalogenated compound b is selected as 1:(0.1~100). The amount of catalyst is selected to be 0.01 to 1.5 times the molar content of organohalogen b.

10. A kit for achieving optical coupling, the kit being used to achieve efficient and rapid coupling between substances A and B to be coupled, characterized in that, The kit includes: Photosensitive o-nitrobenzene compound a: used to modify the photosensitive substance Aa on the substance to be coupled A; Organic halogenated compound b: used to modify the substance B to be coupled to obtain substance Bb; Under the conditions of catalyst and light, Aa and Bb trigger a reaction between a and b, achieving efficient and rapid coupling between substances A and B to be coupled; The o-nitrobenzene compound a is as defined in claim 1, and the organohalogenated compound b is as defined in claim 1.

11. The reagent kit for achieving optical coupling according to claim 10, characterized in that, The kit also includes a catalyst for enabling the reaction of Aa and Bb under light conditions. The catalyst is a substance capable of reacting with organohalogen b to generate free radicals. The catalyst is a transition metal or a complex of a transition metal and a ligand.

12. The application of the reagent kit for achieving optical coupling as described in claim 10, characterized in that, The applications include: The kit is used to achieve chemical coupling between small molecules, between polymers, and between polymers and small molecules; The kit is used to achieve covalent linkage between small molecules or macromolecules and tissues, cells or organelles to achieve biomarking; The kit is used to achieve surface modification of different material surfaces; The kit is used to achieve rapid, photocontrolled crosslinking preparation of materials.

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