Polymer with side chain containing tetraphenyl ethylene single-arm mercaptan mannose as well as preparation method and application of polymer

Abstract

The invention belongs to the technical field of glycopolymer, and particularly relates to a single-arm mercaptan mannose polymer with a side chain containing tetraphenyl ethylene as well as a preparation method and application of the single-arm mercaptan mannose polymer. The chemical structural formula of the mercaptan mannose polymer containing the tetraphenyl ethylene single arm on the side chain is shown as a formula (I). Wherein n is an integer greater than or equal to 2. The saccharide polymer has high biocompatibility and fluorescence recognition characteristics, and the tetraphenyl ethylene-containing single-arm mercaptan mannose polymer is prepared by innovatively applying efficient and rapid RAFT polymerization and click chemistry reaction.

Description

Technical Field

[0001] The present invention belongs to the technical field of sugar polymers, and in particular relates to a single-arm thiol mannose polymer containing tetraphenylethylene on the side chain, and a preparation method and application thereof. Background Art

[0002] Sugar-containing polymers have good biocompatibility, excellent water solubility and multivalent interactions with lectins. They show superior performance and broad application potential in biomedical fields such as modifying drugs to study pharmacokinetics, delivering drugs as carriers, treating various diseases as enzyme inhibitors, and simulating natural carbohydrates to study sugar-protein binding.

[0003] Reversible addition-fragmentation-chain transfer (RAFT) polymerization is the most versatile and convenient type of reversible deactivation radical polymerization (RDRP). The polymers obtained by this type of polymerization have controllable molecular weight, narrow molecular weight distributions (MWDs), high radical fidelity and functional group tolerance.

[0004] Click chemistry is an efficient organic reaction proposed by Sharpless. This type of click reaction is modular, simple, efficient, and has no by-products. In particular, the widely used Thiol-ene click reaction can not only synthesize sugar-containing derivatives (such as sugar amines) for post-modification of polymer scaffolds, but also synthesize functional sugar-containing monomers for direct polymerization. It has been proven to be an effective way to synthesize sugar-containing polymers.

[0005] Aggregation-induced emission (AIE) materials possess the unique optical property of emitting intense fluorescence in their aggregated state. Compared to materials with aggregation-induced quenching, they exhibit excellent photostability and a high signal-to-noise ratio, demonstrating their enormous market potential and scientific significance. Tetraphenylethylene (TPE), a typical AIE luminogen, has been widely used to construct fluorescent probes and imaging agents. Compared to other AIE-active materials, TPE offers advantages such as high emission efficiency, ease of synthesis, structural modification, and excellent stability. By conjugating TPE with specific carbohydrates, fluorescent glycopolymers that specifically bind to corresponding proteins or lectins can be prepared. TPE compounds with sugar moieties effectively mitigate the poor solubility of AIE molecules in water, reducing background fluorescence and biotoxicity, significantly expanding the application prospects of glycopolymers in the field of biomaterials. Glycopolymers containing TPE structures exhibit advantages such as moderate water solubility, good biocompatibility, excellent photostability, and a high signal-to-noise ratio. When combined with fluorescence techniques, they can be used for sensitive detection of a variety of lectins, toxins, pathogens, and enzymes. They have important applications in diverse scientific fields, including cell imaging, biosensing, and drug tracking, and are a crucial research tool for studying glycobiology. However, the current construction of sugar-containing monomers is relatively complex and direct polymerization is still difficult to achieve high yields. Some of these polymers lack biocompatibility, making them difficult to apply in medicine. Summary of the Invention

[0006] To address these issues, the present invention provides a single-armed mannose polymer containing tetraphenylethylene side chains, as well as its preparation and application. The glycopolymers described herein exhibit high biocompatibility and fluorescent recognition properties. Furthermore, they innovatively utilize the efficient and rapid RAFT polymerization and click chemistry reactions to prepare single-armed thiol-mannose polymers containing tetraphenylethylene side chains, providing a practical approach for preparing a series of single-armed thiol-mannose polymers containing tetraphenylethylene side chains.

[0007] The purpose of the present invention can be achieved by the following technical solutions: The first object of the present invention is to provide a thiol-mannose polymer containing a single arm of tetraphenylethylene on the side chain. The chemical structure of the thiol-mannose polymer containing a single arm of tetraphenylethylene on the side chain is shown in Formula (I):

[0008] Here, n is an integer ≥2.

[0009] The second object of the present invention is to provide a method for preparing a single-arm thiol-mannose polymer containing tetraphenylethylene in the side chain. The preparation method comprises the following steps: (1) 4-bromo-4'-methoxybenzophenone and diphenylmethane undergo a nucleophilic addition elimination reaction to obtain a first compound; (2) the first compound and boron tribromide undergo a demethylation reaction to obtain a second compound; (3) the second compound and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester undergo Suzuki coupling reaction to obtain a third compound; (4) esterifying the third compound with acryloyl chloride and anhydrous triethylamine to obtain a fourth compound; (5) The fourth compound is reacted with acetyl-protected thiol mannose and n-hexylamine through a Thiol-ene click chemistry reaction to obtain a fifth compound; (6) The fifth compound is reacted with trifluoroacetic acid to obtain a sixth compound; (7) Pentafluorophenol is reacted with triethylamine and acryloyl chloride to obtain the seventh compound; (8) The seventh compound is reacted with a chain transfer agent and an initiator through RAFT polymerization to obtain an eighth compound; (9) The eighth compound, the sixth compound and 4-diaminopyridine are subjected to an aminolysis reaction to obtain a ninth compound; (10) The ninth compound is reacted with sodium methoxide to obtain a single-arm thiol-mannose polymer containing tetraphenylethylene on the side chain; Among them, the chemical structures of the tetraphenylethylene-containing single-arm thiol mannose polymer, the first compound, the second compound, the third compound, the fourth compound, the fifth compound, the sixth compound, the seventh compound, the eighth compound, and the ninth compound are shown in Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X), respectively:

[0010] Wherein, n is an integer ≥2.

[0011] Preferably, during the nucleophilic reaction of step (1), the molar ratio of 4-bromo-4'-methoxybenzophenone to diphenylmethane is 1:(1.1-1.3); the reaction temperature is 0°C-100°C, and the reaction time is 3 h-24 h.

[0012] Preferably, during the demethylation reaction of step (2), the dosage ratio of the first compound to boron tribromide is 1 g: (2-3) mL; the reaction temperature is 10°C-30°C; and the reaction time is 18 h-30 h.

[0013] Preferably, during the Suzuki coupling reaction of step (3), the molar ratio of the second compound to N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester is 1:(1-3); the reaction temperature is 105°C-115°C, and the reaction time is 16 h-20 h.

[0014] Preferably, during the esterification reaction of step (4), the molar ratio of the third compound, acryloyl chloride and anhydrous triethylamine is 1:(1-3):(1-3); the reaction temperature is 0°C-25°C, and the reaction time is 18 h-24 h.

[0015] Preferably, during the Thiol-ene click chemistry reaction of step (5), the molar ratio of the fourth compound, acetyl-protected thiol mannose, and n-hexylamine is 1:(1-3):(0.1-0.15); the reaction temperature is 15°C-30°C, and the reaction time is 18 h-24 h.

[0016] Preferably, during the substitution reaction of step (6), the ratio of the fifth compound to trifluoroacetic acid is 1 mmol: (1-6) ml; the reaction temperature is 15°C-25°C; and the reaction time is 4 h-6 h.

[0017] Preferably, during the esterification reaction of step (7), the molar ratio of pentafluorophenol, triethylamine and acryloyl chloride is 1:(0.5~2):(0.5~2); the reaction temperature is 0℃~25℃, and the reaction time is 16 h~36 h.

[0018] Preferably, in the RAFT polymerization process of step (8), the molar ratio of the seventh compound, the chain transfer agent, and the initiator is (150-220): (7-10): 1; the chain transfer agent is trithiocarbonate, and the initiator is azobisisobutyronitrile; the reaction temperature is 70°C-90°C, and the reaction time is 20 h-30 h.

[0019] Preferably, during the aminolysis reaction of step (9), the molar ratio of the eighth compound, the sixth compound, and 4-diaminopyridine is (1-3):(2-5):1; the reaction temperature is 50°C-70°C, and the reaction time is 18 h-24 h.

[0020] Preferably, during the substitution reaction of step (10), the usage ratio of the ninth compound and sodium methoxide is 0.1 g: (30-60) mg; the reaction temperature is 15°C-30°C; and the reaction time is 1 h-3 h.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses diphenylmethane and 4-bromo-4'-methoxybenzophenone to generate tetraphenylethylene alcohol derivatives, which are then dehydrated with p-toluenesulfonic acid to generate tetraphenylethylene compounds. The acetyl-protected thiol-containing sugar compounds of the modified single-arm tetraphenylethylene are synthesized, providing a practical way to prepare sugar-containing polymers with functionalized tetraphenylethylene cores. The synthetic method is stable and efficient.

[0022] (2) The present invention combines tetraphenylethylene and sugar units into one compound, which can be connected to the polymer chain through post-polymerization modification to form a sugar-containing polymer when used.

[0023] (3) Sugar-containing polymers can achieve molecular recognition with specific types of proteins through multivalent interactions, which provides a good foundation for drug carrier materials. The AIE effect of the tetraphenylethylene structure has a significant scientific role in imaging and tracing in vivo. It has a high responsiveness and large fluorescence changes, and can be used as a fluorescent probe. Therefore, the sugar-containing compound polymer precursor with a tetraphenylethylene structure designed and synthesized by the present invention can combine the properties of two individual components and has the potential to examine the protein recognition performance and drug release after polymerization. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the H NMR spectrum of the first compound in Example 1 of the present invention; Figure 2 is the C NMR spectrum of the first compound in Example 1 of the present invention; Figure 3 is the H NMR spectrum of the second compound in Example 1 of the present invention; Figure 4 is the C NMR spectrum of the second compound in Example 1 of the present invention; Figure 5 is the H NMR spectrum of the third compound in Example 1 of the present invention; Figure 6 is the C NMR spectrum of the third compound in Example 1 of the present invention; Figure 7 is the H NMR spectrum of the fourth compound in Example 1 of the present invention; Figure 8 is the C NMR spectrum of the fourth compound in Example 1 of the present invention; Figure 9 is the H NMR spectrum of the fifth compound in Example 1 of the present invention; Figure 10 is the NMR carbon spectrum of the fifth compound in Example 1 of the present invention; Figure 11 is the H NMR spectrum of the sixth compound in Example 1 of the present invention; Figure 12 is the H NMR spectrum of the sixth compound in Example 1 of the present invention; Figure 13 is the H NMR spectrum of the seventh compound in Example 1 of the present invention; Figure 14 is the H NMR spectrum of the eighth compound in Example 1 of the present invention; Figure 15 is the H NMR spectrum of the ninth compound in Example 1 of the present invention; Figure 16 This is the H NMR spectrum of the single-arm thiol-mannose polymer containing tetraphenylethylene in the side chain in Example 1 of the present invention; Figure 17 is a flow chart of an exemplary preparation method of the present invention; Figure 18 This is a fluorescence binding assay graph of a polymer containing a tetraphenylethylene mono-armed thiol mannose on the side chain of Example 1 of the present invention in a HEPES buffer at pH 7.4 with gradually increasing Con A concentrations; Figure 19 is a flow chart of an exemplary preparation method of Comparative Example 1; Figure 20 This is a diagram showing the recognition effect between the single-armed thiol-mannose polymer containing tetraphenylethylene on the side chain and Con A in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described through the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0026] The chemical structure of the tetraphenylethylene-containing single-arm thiol-mannose polymer is shown in Formula (I):

[0027] Wherein, n is an integer ≥ 2. Preferably, 2≤n≤40.

[0028] The present invention also provides a method for preparing the single-armed thiol-mannose polymer containing tetraphenylethylene in the side chain. First, diphenylmethane and 4-bromo-4'-methoxybenzophenone are reacted with n-butyl lithium to generate a tetraphenylethylene alcohol derivative, which is then dehydrated with p-toluenesulfonic acid to generate a tetraphenylethylene compound. This is then treated with boron tribromide to obtain a phenolic hydroxyl compound. A Suzuki coupling reaction is then performed to obtain an amino-protected homo-side hydroxyl group and a bromotetraphenylethylene compound. This compound is then esterified with acryloyl chloride. The resulting acrylate compound is then subjected to a Thiol-ene reaction between an olefin and a thiol with an α-D-pyranose mannothiolate. The tert-butyloxycarbonyl group is then removed to obtain a single-armed thiol-mannose derivative containing tetraphenylethylene. Finally, RAFT polymerization and post-polymerization modification are performed to prepare a polymer containing tetraphenylethylene and thiol-mannose in the side chain.

[0029] The following combination Figure 17 The preparation method of the single-arm thiol-mannose polymer containing tetraphenylethylene on the side chain is exemplified.

[0030] Step (1): 4-bromo-4'-methoxybenzophenone and diphenylmethane undergo a nucleophilic addition elimination reaction, and then undergo post-treatment to obtain a first compound. In one embodiment of the present invention, in step (1), the molar ratio of 4-bromo-4'-methoxybenzophenone, diphenylmethane, n-butyllithium, and p-toluenesulfonic acid is 1: (1.1-1.3): (1.3-1.5): (0.05-0.1). During the nucleophilic reaction, the reaction temperature is 0°C-100°C, and the reaction time is 3 h-24 h. In one embodiment of the present invention, in step (1), the post-treatment is neutralization, washing, drying, and purification.

[0031] Step (2): The first compound obtained in step (1) is subjected to a demethylation reaction with boron tribromide, followed by post-treatment to obtain a second compound. In one embodiment of the present invention, the first compound and boron tribromide are used in a ratio of 1 g: (2-3) mL; during the demethylation reaction, the reaction temperature is 10°C to 30°C, and the reaction time is 18 h to 30 h. In one embodiment of the present invention, in step (2), the post-treatment comprises extraction, drying, and purification.

[0032] Step (3): The second compound obtained in step (2) is subjected to a Suzuki coupling reaction with N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester, and post-processing to obtain a third compound. In one embodiment of the present invention, in step (3), the molar ratio of the first compound, N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester, anhydrous potassium phosphate, and X-Phos-Pd-G3 is 1: (1-3): (1-3): (0.05-0.1); during the Suzuki coupling reaction, the reaction temperature is 105°C to 115°C, and the reaction time is 16 h to 20 h. In one embodiment of the present invention, in step (3), the post-processing is washing, drying, and purification.

[0033] Step (4): The third compound obtained in step (3) is subjected to an esterification reaction with acryloyl chloride and anhydrous triethylamine, and then subjected to post-treatment to obtain a fourth compound. In one embodiment of the present invention, in step (4), the molar ratio of the third compound, acryloyl chloride, and anhydrous triethylamine is 1:(1-3):(1-3); during the esterification reaction, the reaction temperature is 0°C to 25°C, and the reaction time is 18 h to 24 h. In one embodiment of the present invention, in step (4), the post-treatment is washing, drying, and purification.

[0034] Step (5): The fourth compound obtained in step (4) is subjected to a Thiol-ene click chemistry reaction with acetyl-protected thiol mannose (α-D-pyranose mannothiolate) and n-hexylamine, followed by post-treatment to obtain a fifth compound. In one embodiment of the present invention, in step (5), the molar ratio of the fourth compound, α-D-pyranose mannothiolate, and n-hexylamine is 1:(1-3):(0.1-0.15); during the Thiol-ene click chemistry reaction, the reaction temperature is 15°C to 30°C, and the reaction time is 18h to 24h. In one embodiment of the present invention, in step (5), the post-treatment is washing, drying, and purification.

[0035] Step (6): The fifth compound obtained in step (5) is subjected to a substitution reaction with trifluoroacetic acid, followed by post-treatment to obtain a sixth compound. In one embodiment of the present invention, in step (6), the concentration ratio of the fourth compound to trifluoroacetic acid is 1 mmol: (1-6) ml; during the substitution reaction, the reaction temperature is 15°C to 25°C, and the reaction time is 4 h to 6 h. In one embodiment of the present invention, in step (6), the post-treatment is concentration.

[0036] Step (7): Pentafluorophenol is subjected to an esterification reaction with triethylamine and acryloyl chloride, and then post-treated to obtain a seventh compound. In one embodiment of the present invention, in step (7), the molar ratio of pentafluorophenol, triethylamine, and acryloyl chloride is 1:(0.5-2):(0.5-2); during the esterification reaction, the reaction temperature is 0°C-25°C, and the reaction time is 16 h-36 h. In one embodiment of the present invention, in step (7), the post-treatment is washing, drying, and purification.

[0037] Step (8): The seventh compound obtained in step (7) is subjected to a RAFT polymerization reaction with a chain transfer agent and an initiator, and then post-treated to obtain an eighth compound. In one embodiment of the present invention, in step (8), the molar ratio of the seventh compound, the chain transfer agent, and the initiator is (150-220): (7-10): 1; the chain transfer agent is trithiocarbonate, and the initiator is azobisisobutyronitrile; during the RAFT polymerization reaction, the reaction temperature is 70°C to 90°C, and the reaction time is 20 h to 30 h. In one embodiment of the present invention, in step (8), the post-treatment is washing, drying, and purification.

[0038] Step (9): The eighth compound obtained in step (8) is subjected to an aminolysis reaction with the sixth compound obtained in step (6) and 4-diaminopyridine, and then subjected to post-treatment to obtain a ninth compound. In one embodiment of the present invention, in step (9), the molar ratio of the eighth compound, the sixth compound, and 4-diaminopyridine is (1-3): (2-5): 1; during the aminolysis reaction, the reaction temperature is 50°C to 70°C, and the reaction time is 18 h to 24 h. In one embodiment of the present invention, in step (9), the post-treatment is washing, drying, and purification.

[0039] Step (10): The ninth compound (sugar-containing polymer) obtained in step (9) is subjected to a substitution reaction with sodium methoxide, and then post-treated to obtain a single-arm thiol-mannose polymer containing tetraphenylethylene on the side chain. In one embodiment of the present invention, in step (10), the amount ratio of the ninth compound to sodium methoxide is 0.1 g: (30-60) mg; during the substitution reaction, the reaction temperature is 15°C to 30°C, and the reaction time is 1 h to 3 h. In one embodiment of the present invention, in step (10), the post-treatment is purification.

[0040] Among them, the chemical structures of the tetraphenylethylene-containing single-arm thiol mannose polymer, the first compound, the second compound, the third compound, the fourth compound, the fifth compound, the sixth compound, the seventh compound, the eighth compound, and the ninth compound are shown in Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X), respectively:

[0041] Here, n is an integer ≥2.

[0042] In the above preparation process, n-butyl lithium is first used to react diphenylmethane and 4-bromo-4'-methoxybenzophenone to generate a tetraphenylethylene alcohol derivative, which is then dehydrated with p-toluenesulfonic acid to generate a tetraphenylethylene compound, which is treated with boron tribromide to obtain a phenolic hydroxyl compound, and then a Suzuki coupling reaction is performed to obtain an amino-protected homolateral hydroxyl group and a bromotetraphenylethylene compound. This compound is esterified with acryloyl chloride, and the obtained acrylate compound is subsequently subjected to a Thiol-ene reaction between an olefin and a thiol with an α-D-pyranose mannothiol compound, and then the tert-butyloxycarbonyl group is removed to obtain a homolateral single-armed thiolmannose derivative containing tetraphenylethylene. A pentafluorophenol active ester polymer is prepared by RAFT polymerization, and a substitution reaction is performed to obtain a single-armed thiolmannose polymer derivative containing tetraphenylethylene in the side chain. Finally, the acetyl protection is removed under sodium methoxide conditions to obtain a thiolmannose polymer containing tetraphenylethylene in the side chain.

[0043] Therefore, the present invention innovatively uses efficient and fast RAFT polymerization and click chemistry reactions to prepare tetraphenylethylene-containing single-arm thiol-mannose polymers with high biocompatibility and fluorescent recognition properties, providing a practical approach for preparing functionalized tetraphenylethylene-containing single-arm thiol-mannose polymers.

[0044] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. It should also be understood that the following embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific numerical values ​​exemplified below.

[0045] In the following examples, the sources of the reagents used are as follows: acetylthiol mannose (α-Man-OAc-SH) was synthesized according to the literature (Herzberger J, Leibig D, Langhanki J, Moers C, Opatz T, Frey h. “Clickable PEG” via anionic copolymerization of et hylene oxide and glycidylpropargyl et her. Polymer Chemistry 2017, 8 (12): 1882-1887.); diphenylmethane, 4-bromo-4'-methoxybenzophenone, N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester, and X-Phos-Pd-G3 were purchased from Shanghai Shaoyuan Reagent Co., Ltd.; anhydrous potassium phosphate (≥98%) was purchased from Shanghai Haohong Biopharmaceutical Technology Co., Ltd.; anhydrous titanium tetrachloride, trifluoroacetic acid, n-butyllithium (2.5 M The solution was in n-hexane, safely sealed) was purchased from Anaiji Chemical; boron tribromide, pentafluorophenol, acryloyl chloride, triethylamine, azobisisobutyronitrile, and tri-generation carbonate were purchased from Bid Pharmaceuticals; anhydrous potassium carbonate (>99%), sodium bicarbonate (>99.5%), and other reagents not mentioned were purchased from Shanghai Runjie Technology Development Co., Ltd.

[0046] In addition, in the following examples, unless otherwise specified, the raw materials, reagents or processing techniques are all conventional commercially available raw materials or conventional processing techniques in the art.

[0047] In the following examples, the characterization instruments are as follows: a nuclear magnetic resonance spectrometer, model AS400 400 MHz, manufactured by Wuhan Zhongke Co., Ltd.; a high-resolution mass spectrometer, model Bruker SolariX 7.0, manufactured by Bruker Corporation; a fluorescence spectrophotometer, model FS5, manufactured by Edinburgh Instruments; and a gel permeation chromatograph, model Agilent 1260, manufactured by Agilent.

[0048] Unless otherwise specified in the present invention, room temperature or normal temperature refers to 10-30°C.

[0049] Unless otherwise specified in the present invention, the eluent ratio refers to the volume ratio.

[0050] Example 1

[0051] This embodiment provides a single-arm thiol-mannose polymer containing tetraphenylethylene in the side chain and a preparation method thereof.

[0052] (1) Preparation of the first compound Diphenylmethane (1.75 g, 10.4 mmol) was added to a reaction flask, followed by 50 mL of anhydrous tetrahydrofuran. Stirring was performed in an ice bath under nitrogen. 5 mL of n-butyllithium (2.5 M solution in n-hexane, securely sealed) was slowly added, resulting in an orange-red color. Stirring was continued in an ice bath for 1 h. 4-Bromo-4'-methoxybenzophenone (2.5 g, 8.63 mmol) was dissolved in 40 mL of anhydrous tetrahydrofuran and slowly added to the mixture via syringe. The reaction was continued at room temperature for 24 h. After completion of the reaction, the reaction was quenched with saturated ammonium chloride solution, washed with saturated brine, and the organic layer was extracted with dichloromethane, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was dissolved in 50 mL of toluene, and p-toluenesulfonic acid (0.104 mmol) was added. Once completely dissolved, the product was reacted at 100°C for 3 h. After the reaction, the mixture was cooled to room temperature, extracted with dichloromethane and saturated brine, dried over anhydrous magnesium sulfate, and purified by silica gel column chromatography with petroleum ether / ethyl acetate (30:1) to obtain 1.92 g of a light yellow oil with a yield of 50%.

[0053] The H-NMR spectrum and C-NMR spectrum of the first compound are shown in Figure 1 and Figure 2 shown.

[0054] The chemical structure of the first compound is shown below:

[0055] The NMR data of the first compound are shown below: 1 H NMR (401 MHz, CDCl3) δ 7.27 (d, J = 8.5 Hz, 2H), 7.22 – 7.12 (m,6H), 7.11 – 7.03 (m, 4H), 7.01 – 6.92 (m, 4H), 6.70 (d, J = 8.8 Hz, 2H), 3.79(s, 3H).

[0056] 13 C NMR (101 MHz, CDCl3) δ 158.27, 143.70, 143.03, 140.74, 139.27, 135.63, 133.11, 132.58, 131.32, 130.86, 127.86, 126.53, 120.41, 113.21, 55.16.

[0057] (2) Preparation of the second compound The first compound (1.2 g, 2.71 mmol) was dissolved in 20 mL of anhydrous dichloromethane and purged three times with nitrogen. The mixture was then placed in an ice bath to allow complete dissolution. BBr₃ (3.6 mL) was then slowly added to the mixture using a syringe. The reaction was continued at room temperature for 24 hours. After TLC analysis confirmed the complete reaction, the mixture was extracted three times with dichloromethane and ice water. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography using petroleum ether / ethyl acetate (20:1) to obtain the second compound in an 86% yield.

[0058] The H-NMR spectrum and C-NMR spectrum of the second compound are shown in Figure 3 and Figure 4 shown.

[0059] The chemical structure of the second compound is shown below:

[0060] The NMR data of the second compound are shown below: 1 H NMR (401 MHz, CDCl3) δ 7.25 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 2.7Hz, 6H), 7.08 – 7.00 (m, 4H), 6.92 (dd, J = 15.6, 8.5 Hz, 4H), 6.61 (d, J =8.5 Hz, 2H).

[0061] 13 C NMR (101 MHz, CDCl3) δ 154.40, 143.66, 142.93, 140.75, 139.21, 135.71, 133.08, 132.76, 131.30, 130.84, 127.84, 126.53, 120.41, 114.76.

[0062] (3) Preparation of the third compound The second compound (500 mg, 1.17 mmol) was dissolved in 10 mL of 1,4-dioxane. N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (905 mg, 1.755 mmol), X-Phos-Pd-G3 (198 mg, 0.117 mmol), and anhydrous potassium phosphate (625 mg, 1.755 mmol) were added sequentially. The nitrogen atmosphere was then replaced three times, and the reaction was continued at 105°C for 16 h. After completion of the reaction, the mixture was extracted three times with ethyl acetate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography with petroleum ether / ethyl acetate (10:1) to obtain 460 mg of the third compound (a 73% yield).

[0063] The H-NMR spectrum and C-NMR spectrum of the third compound are shown as follows: Figure 5 and Figure 6 shown.

[0064] The chemical structure of the third compound is shown below:

[0065] The NMR data of the third compound are shown below: 1 H NMR (400 MHz, CDCl3) δ 7.10 (dd, J = 11.8, 5.2 Hz, 8H), 7.04 – 7.00(m, 4H), 6.98 (d, J = 8.3 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 6.56 (d, J = 8.5Hz, 2H), 6.00 (s, 1H), 4.04 (s, 2H), 3.59 (t, J = 5.5 Hz, 2H), 2.45 (s, 2H), 1.50 – 1.45 (m, 9H).

[0066] 13 C NMR (101 MHz, CDCl3) δ 154.46, 144.01, 142.98, 140.09, 138.13, 135.96, 132.72, 131.40, 127.67, 126.24, 123.86, 114.65, 79.77, 60.44, 28.49.

[0067] HRMS (ESI): m / z calc. for C 36 H 35 NO3(M+Na+) calc.for: 552.26306; found: 552.28011.

[0068] (4) The fourth compound The third compound (400 mg, 0.755 mmol) was added to a reaction flask, followed by 5 mL of dichloromethane and triethylamine (0.21 mL, 1.51 mmol). The mixture was stirred evenly on ice. Acryloyl chloride (0.13 mL, 1.51 mmol) was slowly added dropwise to the resulting mixture. Stirring was continued on ice for 2.5 hours, followed by reaction at room temperature for 24 hours. The organic layer was extracted with saturated brine and dichloromethane, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography with petroleum ether / ethyl acetate (15:1) to obtain 223 mg of the fourth compound (51% yield).

[0069] The H NMR spectrum and C NMR spectrum of the fourth compound are as follows: Figure 7 and Figure 8 shown.

[0070] The chemical structural formula of the fourth compound is shown below:

[0071] The NMR data of the fourth compound are shown below: 1 H NMR (400 MHz, CDCl3) δ 7.16 – 7.07 (m, 8H), 7.05 – 6.96 (m, 8H), 6.88 (d, J = 8.6 Hz, 2H), 6.56 (d, J = 17.3 Hz, 1H), 6.27 (dd, J = 17.3, 10.4Hz, 1H), 5.99 (t, J = 9.7 Hz, 2H), 4.04 (s, 2H), 3.59 (t, J = 5.4 Hz, 2H), 2.46 (s, 2H), 1.48 (s, 9H).

[0072] 13C NMR (101 MHz, CDCl3) δ 164.35, 149.01, 143.59, 142.44, 141.33, 139.54, 138.40, 132.41, 131.58 – 131.02, 128.00, 127.77, 126.56, 124.01, 120.66, 79.65, 60.41, 28.50.

[0073] HRMS (ESI): m / z calc. for C 39 H 37 NO4(M+Na+) calc.for: 606.27159; found: 606.27224.

[0074] (5) Preparation of the fifth compound The fourth compound (630 mg, 1.08 mmol) obtained through multiple syntheses and α-Man-OAc-SH (882 mg, 2.16 mmol) were added to a reaction flask. 5 mL of ultra-dry dichloromethane was added. After complete dissolution, n-hexylamine (0.108 mmol) was added and the mixture was allowed to react at room temperature for 18 hours. After TLC plate analysis confirmed complete reaction, the mixture was extracted three times with dichloromethane and saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography with petroleum ether / ethyl acetate (3:1) to obtain 670 mg of the fifth compound (a 64% yield).

[0075] The H-NMR spectrum and C-NMR spectrum of the fifth compound can be found in Figure 9 and Figure 10 shown.

[0076] The chemical structural formula of the fifth compound is shown below:

[0077] The NMR data of the fifth compound are shown below: 1 H NMR (401 MHz, CDCl3) δ 7.18 – 7.09 (m, 8H), 7.02 (dd, J = 17.2,6.9 Hz, 8H), 6.87 (d, J = 7.1 Hz, 2H), 6.03 (s, 1H), 5.34 (t, J= 12.2 Hz,1H), 5.28 (s, 1H), 4.89 (s, 1H), 4.31 (dd, J = 12.0, 4.9 Hz, 1H), 4.16 (dd, J = 7.1, 1.3 Hz, 1H), 4.07 (s, 2H), 3.89 (dd, J = 15.8, 7.0 Hz, 1H), 3.73 (dd, J = 10.0, 5.3 Hz, 1H), 3.62 (s, 2H), 2.96 (t, J = 7.0 Hz, 2H), 2.90 – 2.79(m, 4H), 2.48 (s, 2H), 2.18 (s, 3H), 2.12 (s, 3H), 2.06 (s, 3H), 2.01 (s,3H), 1.50 (s, 9H).

[0078] 13 C NMR (101 MHz, CDCl3) δ 171.22, 170.70, 170.00, 149.00, 143.57, 142.43, 141.38, 139.49, 138.40, 132.38, 132.08 – 130.97 , 127.79 ,126.58 , 124.01 , 120.70 , 97.65 , 79.67 , 69.51 , 69.03 , 68.79 , 68.11 ,66.04 , 62.49 , 60.44 , 35.03, 31.44, 28.51 , 27.57 , 21.26 – 20.61.

[0079] HRMS (ESI): m / z calc. for C 54 H 59 NO 14 S(M+Na+) calc.for: 1000.37552; found: 1000.37720.

[0080] (6) Preparation of the Sixth Compound Compound 5 (600 mg, 0.625 mmol) was added to a reaction flask and completely dissolved in 5 mL of dichloromethane. Then, 3 mL of trifluoroacetic acid was added and stirred at room temperature for 6 h. After TLC analysis confirmed complete reaction, the reaction solution was diluted with dichloromethane, dried by evaporation, dissolved in dichloromethane, and concentrated to yield 520 mg of compound 6 in a 96% yield.

[0081] The H-NMR spectrum and C-NMR spectrum of the sixth compound can be found in Figure 11 and Figure 12 shown.

[0082] The chemical structural formula of the sixth compound is shown below:

[0083] The NMR data of the sixth compound are shown below: 1 H NMR (401 MHz, CDCl3) δ 7.14 (s, 8H), 7.05 (d, J = 7.6 Hz, 8H),6.89 (d, J = 8.3 Hz, 2H), 6.00 (s, 1H), 5.40 – 5.33 (m, 1H), 5.30 (s, 1H), 4.94 – 4.85 (m, 1H), 4.32 (dd, J = 12.3, 5.1 Hz, 1H), 4.15 (d, J = 10.9 Hz,3H), 3.95 – 3.82 (m, 1H), 3.79 – 3.67 (m, 1H), 3.35 (d, J = 104.1 Hz, 2H), 3.01 – 2.81 (m, 6H), 2.76 (s, 2H), 2.22 – 1.99 (m, 12H).

[0084] 13C NMR (101 MHz, CDCl3) δ 170.44 – 169.54, 149.04, 143.32, 141.97, 132.38, 131.69, 131.28, 127.85, 126.76, 124.26, 120.82, 97.62, 70.17 – 69.58, 69.31, 68.74, 68.12, 66.04, 62.57, 53.51, 35.01, 31.44, 27.57, 20.81.

[0085] HRMS (ESI): m / z calc. For C 49 H 51 NO 12 S(M+Na+) calc.for: 900.31012; found: 900.32155.

[0086] (7) Preparation of the seventh compound Pentafluorophenol (10.0 g, 54.33 mmol) was dissolved in 250 mL of anhydrous dichloromethane. Ultra-dry triethylamine (7.53 mL, 54.33 mmol) was added dropwise in an ice bath. After stirring for 0.5 h, acryloyl chloride (4.42 mL, 54.33 mmol) was added dropwise. The reaction was then maintained at 0°C and allowed to continue. The mixture was then brought to room temperature overnight. After completion of the reaction, the mixture was extracted with dichloromethane and saturated brine, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether) to obtain 8.6 g of a light yellow liquid (the seventh compound) in a 66% yield.

[0087] The H NMR spectrum of the seventh compound can be found in Figure 13 shown.

[0088] The chemical structure of the seventh compound is shown below:

[0089] The NMR data of the seventh compound are shown below: 1 H NMR (400 MHz, CDCl3) δ 6.74 (d, J = 16.2 hz, 1 H), 6.40 (dd, J =16.8, 10.5 hz, 1 H), 6.19 (d, J = 11.7 hz, 1 H).

[0090] (8) Preparation of the eighth compound The seventh compound (3 g, 12.4 mmol) was dissolved in 5 mL of anhydrous 1,4-dioxane. Trithiocarbonate (210 mg, 0.55 mmol) and azobisisobutyronitrile (AIBN) (6 mg, 0.069 mmol) were then added. The atmosphere was purged with nitrogen three times, and the mixture was reacted at 85°C for 24 hours. After the reaction, the solution was cooled to room temperature, dissolved in dichloromethane, and precipitated in methanol three times. The mixture was then dried to yield 1.22 g of a pale yellow powder (i.e., the eighth compound: pPFPA).

[0091] The H NMR spectrum of pPFPA can be found in Figure 14 shown.

[0092] The chemical structure of pPFPA is shown below:

[0093] Here, n is 36.

[0094] The NMR data of pPFPA are shown below: 1 H NMR (400 M Hz, CDCl3) δ 3.09 (s, 8 H), 2.87 (s, 1 H), 2.50 (s, 4H), 2.11 (s, 8 H), 1.96 (s, 4 H), 1.38 (s, 1 H), 1.25 (s, 1 H).

[0095] 19 F NMR (470 MHz, CDCl3): δ = -153.21 (bs, 2F, ortho), -156.76 (bs,1F, para), -162.22 (bs, 2F, meta).

[0096] (9) Preparation of the ninth compound Under a nitrogen atmosphere, pPFPA (90.365 mg, 0.38 mmol) was dissolved in 3 mL of DMF. Compound 6 (400 mg, 0.456 mmol) and DMAP (23.22 mg, 0.19 mmol) were added, and the mixture was reacted at 65°C for 24 h. After the reaction, the mixture was centrifuged three times in diethyl ether and then dried to obtain 320 mg of a dark yellow-brown powder (Compound 8) in a 65% yield.

[0097] The NMR spectrum of the ninth compound can be found in Figure 15 shown.

[0098] The chemical structure of the ninth compound is shown below:

[0099] Here, n is 36.

[0100] The NMR data of the ninth compound are as follows: 1 H NMR (401 MHz, CDCl3) δ 7.14 (s, 8H), 7.05 (d, J = 7.6 Hz, 8H), 6.89(d, J = 8.3 Hz, 2H), 6.00 (s, 1H), 5.40 – 5.33 (m, 1H), 5.30 (s, 1H), 4.94 –4.85 (m, 1H), 4.32 (dd, J = 12.3, 5.1 Hz, 1H), 4.15 (d, J = 10.9 Hz, 3H), 3.95 – 3.82 (m, 1H), 3.79 – 3.67 (m, 1H), 3.35 (d, J = 104.1 Hz, 2H), 3.01 –2.81 (m, 6H), 2.76 (s, 2H), 2.22 – 1.99 (m, 12H), 1.29 (s, 6H).

[0101] (10) Preparation of single-arm thiol-mannose polymers containing tetraphenylethylene on the side chain The ninth compound (130 mg, 0.1235 mmol) was dissolved in 1 mL of a mixture of anhydrous methanol and anhydrous dichloromethane (MeOH:DCM, volume ratio = 1:1). 1 mL of sodium methoxide in dichloromethane (40 mg, 0.741 mmol) was added dropwise at 25°C. The mixture was then allowed to react for 1 h at room temperature. After the reaction, the solvent was rotary evaporated, and the residue was dissolved in 1 mL of DMF. The polymer solution was transferred to a MWCO of 3500 Da and dialyzed against methanol for 24 h. After dialysis, the final homopolymer was lyophilized to obtain 92 mg of a light yellow powder (i.e., a single-arm thiol-mannose polymer containing tetraphenylethylene side chains) in an 82% yield.

[0102] The H NMR spectrum of the single-arm thiol-mannose polymer containing tetraphenylethylene on the side chain can be found in Figure 16 shown.

[0103] The chemical structure of the tetraphenylethylene-containing single-arm thiol-mannose polymer is shown below:

[0104] Here, n is 36.

[0105] The NMR data of the single-arm thiol-mannose polymer containing tetraphenylethylene on the side chain are shown below: 1 H NMR (400 MHz, DMSO) δ 6.78 (dd, J = 181.6, 75.2 Hz, 18H), 6.06 (s,1H), 4.98 (d, J = 268.2 Hz, 3H), 3.48 (dd, J = 60.5, 38.7 Hz, 8H), 2.75 (t, J = 59.4 Hz, 8H), 1.98 (s, 2H), 1.24 (s, 6H).

[0106] Table 1 shows gel permeation chromatography (GPC) characterization of polymers containing tetraphenylethylene monostyrene-armed thiol-mannose side chains. Test conditions: 1 mL / min mobile phase (HPLC grade DMF containing 50 mmol / L LiBr); 35°C temperature; Waters 4.6 x 30 mm guard column; three Waters WAT054466, WAT044226, and WAT044223 columns; instrument calibration using 10 PMMA standards.

[0107] The gel permeation chromatography (GPC) characterization is shown in Table 1. It can be seen that the dispersion width of the prepared side chain-containing tetraphenylethylene single-arm thiol mannose polymer is 1.08, and the dispersion degree is relatively small.

[0108] Table 1 GPC characterization of single-arm thiol-mannose polymers containing tetraphenylethylene on the side chain

[0109] The fluorescence emission graph of the glycopolymer containing a tetraphenylethylene-armed thiol-mannose polymer on the side chain obtained by fluorescence binding assay is shown in the following figure: Figure 18The test method was based on the literature (Xiaoying Gao, Duo Mao, Xingang Zuo, et al. Specific Targeting, Imaging and Ablation of Tumor-Associated Macrophages by Theranostic Mannose-AIEgenConjugates. Analytical Chemistry. 2019, 91 (10): 6836-6843.).

[0110] It can be seen that the polymer containing tetraphenylethylene mono-armed thiol mannose on the side chain obtained in this example has good biocompatibility and a significant concentration-dependent fluorescence increase characteristic, and can be used as an AIE sugar-containing fluorescent probe.

[0111] Example 2

[0112] This embodiment provides a single-arm thiol-mannose polymer containing tetraphenylethylene in the side chain and a preparation method thereof.

[0113] (1) Preparation of the first compound Diphenylmethane (1.75 g, 10.4 mmol) was added to a reaction flask, followed by 50 mL of anhydrous tetrahydrofuran. Stirring was continued in an ice bath under nitrogen. 5 mL of n-butyllithium (2.5 M solution in n-hexane, securely sealed) was slowly added, resulting in an orange-red color. Stirring was continued in an ice bath for 1 h. 4-Bromo-4'-methoxybenzophenone (2.5 g, 8.63 mmol) was dissolved in 50 mL of anhydrous tetrahydrofuran and slowly added to the mixture via syringe. The reaction was continued at room temperature for 24 h. After completion, the reaction was quenched with saturated ammonium chloride solution, washed with saturated brine, and the organic layer was extracted with dichloromethane, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was dissolved in 50 mL of toluene, and p-toluenesulfonic acid (0.863 mmol) was added. Once completely dissolved, the product was reacted at 100°C for 3 h. After the reaction, the mixture was cooled to room temperature, extracted with dichloromethane and saturated brine, and dried over anhydrous magnesium sulfate. The product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (30:1) to obtain 2.1 g of a light yellow oil with a yield of 55%.

[0114] (2) Preparation of the second compound The first compound (1.2 g, 2.71 mmol) was dissolved in 20 mL of anhydrous dichloromethane and purged three times with nitrogen. The mixture was then placed in an ice bath to allow complete dissolution. BBr₃ (3.6 mL) was then slowly added to the resulting mixture via syringe, and the reaction was continued at room temperature for 24 h. After TLC analysis confirmed the complete reaction, the mixture was extracted three times with dichloromethane and ice water. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography using petroleum ether / ethyl acetate (20:1) to yield compound 0.95 (the second compound) in 82% yield.

[0115] (3) Preparation of the third compound The second compound (500 mg, 1.17 mmol) was dissolved in 10 mL of 1,4-dioxane. N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (905 mg, 1.755 mmol), X-Phos-Pd-G3 (198 mg, 0.117 mmol), and anhydrous potassium phosphate (625 mg, 1.755 mmol) were added sequentially. The nitrogen atmosphere was then replaced three times, and the reaction was continued at 105°C for 16 h. After completion of the reaction, the mixture was extracted three times with ethyl acetate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography with petroleum ether / ethyl acetate (10:1) to obtain 465 mg of the third compound (75% yield).

[0116] (4) The fourth compound The third compound (400 mg, 0.755 mmol) was added to a reaction flask, followed by 5 mL of dichloromethane and triethylamine (0.21 mL, 1.51 mmol). The mixture was stirred evenly in an ice bath. Acryloyl chloride (0.13 mL, 1.51 mmol) was slowly added dropwise to the resulting mixture. Stirring was continued in an ice bath for 2.5 hours, followed by reaction at room temperature for 24 hours. The organic layer was extracted with saturated brine and dichloromethane, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography with petroleum ether / ethyl acetate (15:1) to obtain 268 mg of the fourth compound (a 61% yield).

[0117] (5) Preparation of the fifth compound The fourth compound (630 mg, 1.08 mmol) obtained through multiple syntheses and α-Man-OAc-SH (882 mg, 2.16 mmol) were added to a reaction flask. 5 mL of ultra-dry dichloromethane was added. After complete dissolution of the starting materials, n-hexylamine (0.108 mmol) was added and the mixture was allowed to react at room temperature for 18 hours. After TLC plate analysis confirmed complete reaction, the mixture was extracted three times with dichloromethane and saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography with petroleum ether / ethyl acetate (3:1) to obtain 695 mg of the fifth compound (a 65% yield).

[0118] (6) Preparation of the Sixth Compound Compound 5 (600 mg, 0.625 mmol) was added to a reaction flask and completely dissolved in 5 mL of dichloromethane. Then, 3 mL of trifluoroacetic acid was added and stirred at room temperature for 6 h. After TLC analysis of the reaction, the reaction solution was diluted with dichloromethane, dried, dissolved in dichloromethane, and concentrated to yield 524 mg of compound 6 (94% yield).

[0119] (7) Preparation of the seventh compound Pentafluorophenol (10.0 g, 54.33 mmol) was dissolved in 250 mL of anhydrous dichloromethane. Ultra-dry triethylamine (7.53 mL, 54.33 mmol) was added dropwise in an ice bath. After stirring for 0.5 h, acryloyl chloride (4.42 mL, 54.33 mmol) was added dropwise. The reaction was then maintained at 0°C and allowed to continue. The mixture was then brought to room temperature overnight. After completion of the reaction, the mixture was extracted with dichloromethane and saturated brine, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether) to obtain 8.5 g of a light yellow liquid (Compound 7) in a 64% yield.

[0120] (8) Preparation of the eighth compound The seventh compound (3 g, 12.4 mmol) was dissolved in 5 mL of anhydrous 1,4-dioxane. Trithiocarbonate (210 mg, 0.55 mmol) and azobisisobutyronitrile (AIBN) (6 mg, 0.069 mmol) were then added. The atmosphere was purged with nitrogen three times, and the mixture was reacted at 85°C for 24 hours. After the reaction, the solution was cooled to room temperature, dissolved in dichloromethane, and precipitated in methanol three times. The mixture was then dried to yield 1.21 g of a pale yellow powder (i.e., the eighth compound: pPFPA).

[0121] (9) Preparation of the ninth compound Under a nitrogen atmosphere, pPFPA (90.365 mg, 0.38 mmol) was dissolved in 3 mL of DMF. Compound 6 (400 mg, 0.456 mmol) and DMAP (23.22 mg, 0.19 mmol) were added, and the mixture was reacted at 65°C for 24 h. After the reaction, the mixture was centrifuged three times in diethyl ether and then dried to obtain 284 mg of a dark yellow-brown powder (compound 9) with a yield of 58%.

[0122] (10) Preparation of single-arm thiol-mannose polymers containing tetraphenylethylene on the side chain The ninth compound (130 mg, 0.1235 mmol) was dissolved in 1 mL of a mixture of anhydrous methanol and anhydrous dichloromethane (MeOH:DCM, volume ratio = 1:1). 1 mL of sodium methoxide in dichloromethane (40 mg, 0.741 mmol) was added dropwise at 25°C, followed by reaction at room temperature for 1 h. After completion of the reaction, the solvent was rotary evaporated, and the residue was dissolved in 1 mL of DMF. The polymer solution was transferred to a MWCO of 3500 Da and dialyzed against methanol for 24 h. After dialysis, the final homopolymer P3 was lyophilized to obtain 96 mg of a light yellow powder (i.e., a single-arm thiol-mannose polymer containing tetraphenylethylene side chains) with a yield of 85%.

[0123] Example 3

[0124] This embodiment provides a single-arm thiol-mannose polymer containing tetraphenylethylene in the side chain and a preparation method thereof.

[0125] (1) Preparation of the first compound Diphenylmethane (1.75 g, 10.4 mmol) was added to a reaction flask, followed by 50 mL of anhydrous tetrahydrofuran. Stirring was performed in an ice bath under a nitrogen atmosphere. 5 mL of n-butyllithium (2.5 M solution in n-hexane, securely sealed) was slowly added, causing the reaction solution to turn orange-red. Stirring was continued in an ice bath for 1 h. 4-Bromo-4'-methoxybenzophenone (2.5 g, 8.63 mmol) was dissolved in 40 mL of anhydrous tetrahydrofuran and slowly added to the resulting mixture via syringe. The reaction was continued at room temperature for 24 h. After completion, the reaction was quenched with saturated ammonium chloride solution, followed by washing with saturated brine. The organic layer was extracted with dichloromethane, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was dissolved in 50 mL of toluene, and p-toluenesulfonic acid (0.104 mmol) was added. Once completely dissolved, the product was reacted at 100°C for 3 h. After the reaction, the mixture was cooled to room temperature, extracted with dichloromethane and saturated brine, and dried over anhydrous magnesium sulfate. The product was purified by silica gel column chromatography with petroleum ether / ethyl acetate (30:1) to obtain 2.3 g of a light yellow oil with a yield of 61%.

[0126] (2) Preparation of the second compound The first compound (1.2 g, 2.71 mmol) was dissolved in 20 mL of anhydrous dichloromethane and purged three times with nitrogen. The mixture was then placed in an ice bath to allow complete dissolution. BBr₃ (3.6 mL) was then slowly added to the mixture using a syringe. The reaction was continued at room temperature for 24 hours. After TLC analysis confirmed the complete reaction, the mixture was extracted three times with dichloromethane and ice water. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography using petroleum ether / ethyl acetate (20:1) to obtain compound 1.05 (the second compound) in a 93% yield.

[0127] (3) Preparation of the third compound The second compound (500 mg, 1.17 mmol) was dissolved in 10 mL of 1,4-dioxane. N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (905 mg, 1.755 mmol), X-Phos-Pd-G3 (198 mg, 0.117 mmol), and anhydrous potassium phosphate (625 mg, 1.755 mmol) were added sequentially. The nitrogen atmosphere was then replaced three times, and the reaction was continued at 105°C for 16 h. After completion of the reaction, the mixture was extracted three times with ethyl acetate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography with petroleum ether / ethyl acetate (10:1) to obtain 483 mg of the third compound (a 78% yield).

[0128] (4) The fourth compound The third compound (400 mg, 0.755 mmol) was added to a reaction flask, along with 5 mL of dichloromethane and triethylamine (0.21 mL, 1.51 mmol). The mixture was stirred evenly in an ice bath. Acryloyl chloride (0.13 mL, 1.51 mmol) was slowly added dropwise to the resulting mixture. Stirring was continued in an ice bath for 2.5 hours, followed by reaction at room temperature for 24 hours. The organic layer was extracted with saturated brine and dichloromethane, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography with petroleum ether / ethyl acetate (15:1) to obtain 255 mg of the fourth compound (58% yield).

[0129] (5) Preparation of the fifth compound The fourth compound (630 mg, 1.08 mmol) obtained through multiple syntheses and α-Man-OAc-SH (882 mg, 2.16 mmol) were added to a reaction flask. 5 mL of ultra-dry dichloromethane was added. After complete dissolution, n-hexylamine (0.162 mmol) was added and the mixture was allowed to react at room temperature for 20 h. After TLC plate analysis confirmed complete reaction, the mixture was extracted three times with dichloromethane and saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by silica gel column chromatography with petroleum ether / ethyl acetate (3:1) to obtain 645 mg of the fifth compound (a 60% yield).

[0130] (6) Preparation of the Sixth Compound Compound 5 (600 mg, 0.625 mmol) was added to a reaction flask and completely dissolved in 5 mL of dichloromethane. Then, 3 mL of trifluoroacetic acid was added and stirred at room temperature for 6 h. After TLC analysis confirmed complete reaction, the reaction solution was diluted with dichloromethane, dried by spin drying, dissolved in dichloromethane, and concentrated to yield 513 mg of compound 6 (compound 6) in a 92% yield.

[0131] (7) Preparation of the seventh compound Pentafluorophenol (10.0 g, 54.33 mmol) was dissolved in 250 mL of anhydrous dichloromethane. Ultra-dry triethylamine (7.53 mL, 54.33 mmol) was added dropwise in an ice bath. After stirring for 0.5 h, acryloyl chloride (4.42 mL, 54.33 mmol) was added dropwise. The reaction was then maintained at 0°C and allowed to continue. The mixture was then brought to room temperature overnight. After completion of the reaction, the mixture was extracted with dichloromethane and saturated brine, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography (petroleum ether) to obtain 8.7 g of a light yellow liquid (Compound 7) in a 67% yield.

[0132] (8) Preparation of the eighth compound The sixth compound (3 g, 12.4 mmol) was dissolved in 5 mL of anhydrous 1,4-dioxane. Trithiocarbonate (210 mg, 0.55 mmol) and azobisisobutyronitrile (AIBN) (6 mg, 0.069 mmol) were then added. The atmosphere was purged with nitrogen three times, and the mixture was reacted at 85°C for 24 hours. After the reaction, the solution was cooled to room temperature, dissolved in dichloromethane, and precipitated in methanol three times. The mixture was then dried to yield 1.25 g of a pale yellow powder (i.e., the eighth compound: pPFPA).

[0133] (9) Preparation of the ninth compound Under a nitrogen atmosphere, pPFPA (90.365 mg, 0.38 mmol) was dissolved in 3 mL of DMF. Compound 6 (400 mg, 0.456 mmol) and DMAP (23.22 mg, 0.19 mmol) were added, and the mixture was reacted at 65°C for 24 h. After the reaction, the mixture was centrifuged three times in diethyl ether and then dried to obtain 304 mg of a dark yellow-brown powder (compound 9) in a 62% yield.

[0134] (10) Preparation of single-arm thiol-mannose polymers containing tetraphenylethylene on the side chain Compound 9 (130 mg, 0.1235 mmol) was dissolved in 1 mL of a mixture of anhydrous methanol and anhydrous dichloromethane (MeOH:DCM, volume ratio = 1:1). 1 mL of sodium methoxide in dichloromethane (40 mg, 0.741 mmol) was added dropwise at 25°C, followed by reaction at room temperature for 1 h. After the reaction, the solvent was rotary evaporated, and the residue was dissolved in 1 mL of DMF. The polymer solution was transferred to a MWCO of 3500 Da and dialyzed against methanol for 24 h. After dialysis, the final homopolymer P3 was lyophilized to obtain 100 mg of a light yellow powder (i.e., a single-arm thiol-mannose polymer containing tetraphenylethylene side chains) in a 90% yield.

[0135] Comparative Example 1 Figure 19 This is a flow chart of an exemplary preparation method for comparative example 1. The specific preparation process is as follows:

[0136] Step 1: To a mixture of 4-(1-(4-bromophenyl)-2,2-diphenylvinyl)phenol (0.5 g, 1.13 mmol) in 1.4-dioxane (50 mL) and water (5 mL) at 25°C were added N-BOC-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (0.7 g, 2.56 mmol), potassium phosphate (3.59 g, 1.69 mmol), X-Phos-Pd-G3 (20 mg, 0.03 mmol), SPHOS (20 mg, 0.05 mmol), and tetrabutylammonium bromide (10 mg, 0.03 mmol). The resulting mixture was stirred at 105°C under a nitrogen atmosphere for 16 hours. After the reaction, 50 mL of dichloromethane was added to the reaction solution for dilution and filtration. The filtrate was washed with brine (50 mL x 3), and the organic phase was dried and concentrated. The crude product was purified by forward column chromatography (n-hexane / ethyl acetate = 60:40) to obtain white compound 2 (0.52 g, yield 85.4%).

[0137] 1 H NMR (400 MHz, DMSO) δ 9.34 (s, 1H), 7.20 (d, J = 8.3 Hz, 2H), 7.17– 7.06 (m, 6H), 6.98 – 6.90 (m, 6H), 6.73 (d, J = 8.5 Hz, 2H), 6.50 (d, J =8.6 Hz, 2H), 6.11 (s, 1H), 3.96 (s, 2H), 3.48 (t, J = 5.5 Hz, 2H), 2.37 (d, J = 5.7 Hz, 2H), 1.41 (s, 9H).

[0138] MS (ESI): m / z calc. for C 36 H 34 NO3 (MH)-: 528.3; found: 528.4.

[0139] Step 2: To a mixture of compound 2 (0.50 g, 0.95 mmol) in acetonitrile (50 mL) at 0°C, potassium carbonate (0.39 g, 2.83 mmol) and propyne bromide (0.34 mL, 2.83 mmol) were added. The resulting mixture was stirred at 75°C under a nitrogen atmosphere for 12 hours. After completion of the reaction, the reaction mixture was diluted with 50 mL of dichloromethane and washed with brine (50 mL x 3). The organic phase was dried and concentrated, and the crude product was purified by a normal column chromatography (hexane / ethyl acetate = 70:30) to afford compound 3 (0.45 g, 71.79% yield) as a yellow solid.

[0140] 1 H NMR (400 MHz, DMSO) δ 7.21 (d, J = 8.4 Hz, 2H), 7.17 – 7.08 (m,6H), 7.00 – 6.91 (m, 6H), 6.90 – 6.84 (m, 2H), 6.74 (d, J = 8.8 Hz, 2H), 6.12(s, 1H), 4.71 (d, J = 2.3 Hz, 2H), 3.96 (s, 2H), 3.54 (t, J = 2.3 Hz, 1H),3.48 (t, J= 5.5 Hz, 2H), 2.37 (s, 2H), 1.41 (s, 9H).

[0141] MS (ESI): m / z calc. for C 39 H 38 NO3 (M+H) + : 568.3; found: 567.5.

[0142] Step 3: To a solution of compound 3 (250 mg, 0.441 mmol) and azidoacetylmannose (Man-OAc-N3) (180 mg, 0.485 mmol) in anhydrous DCM and MeOH (1 v:1 v, 5 mL) were added sodium L-ascorbate (174.6 mg, 0.882 mmol) and copper sulfate pentahydrate (110 mg, 0.441 mmol). The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 12 hours. After completion of the reaction, the reaction mixture was filtered and dried, and the crude product was purified by forward column chromatography (hexane / ethyl acetate = 30:70) to afford compound 4 (0.37 g, 89.37% yield) as a white solid.

[0143] 1 H NMR (400 MHz, DMSO-d6) δ 8.41 (s, 1H), 7.21 (d, J = 8.3 Hz, 2H),7.20 – 7.05 (m, 7H), 6.97 (d, J = 6.6 Hz, 4H), 6.91 (dd, J = 17.5, 8.5 Hz,4H), 6.88 – 6.75 (m, 3H), 6.45 (d, J = 1.8 Hz, 1H), 6.12 (s, 1H), 5.81 (dd, J = 9.4, 3.8 Hz, 1H), 5.76 (s, 1H), 5.25 (t, J = 9.4 Hz, 1H), 5.12 (s, 2H), 4.23 (dd, J = 12.6, 4.9 Hz, 1H), 4.02 (dt, J = 9.4, 4.6 Hz, 2H), 3.96 (s,2H), 3.90 – 3.82 (m, 1H), 3.49 (t, J= 5.2 Hz, 2H), 2.38 (s, 2H), 2.15 (s,3H), 2.04 (s, 3H), 1.99 (d, J = 3.1 Hz, 7H), 1.41 (s, 9H).

[0144] MS (ESI): m / z calc. for C 53 H 56 N4O 12 (M+H) + : 941.4; found: 941.3.

[0145] Step 4: Compound 4 (370 mg, 0.0.397 mmol) was dissolved in anhydrous DCM (4 mL) and trifluoroacetic acid (2 mL) was added under an ice bath. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 hours. After completion of the reaction, the reaction solution was diluted with ethyl acetate and neutralized with saturated sodium bicarbonate solution to a weak base. The mixture was extracted three times with ethyl acetate, washed with brine, and the organic phase was dried over anhydrous sodium sulfate, filtered, and dried. The crude product was purified by a normal column chromatography (hexane / ethyl acetate = 60:40) to afford compound 5 (0.27 g, 81.82% yield) as a white solid.

[0146] 1 H NMR (400 MHz, DMSO) δ 8.42 (s, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.20– 7.06 (m, 6H), 6.98 (dd, J = 9.1, 4.8 Hz, 5H), 6.89 (d, J = 8.8 Hz, 2H),6.83 (d, J = 8.9 Hz, 2H), 6.45 (d, J = 2.1 Hz, 1H), 6.16 (s, 1H), 5.81 (dd, J = 9.4, 3.9 Hz, 1H), 5.76 (s, 1H), 5.25 (t, J = 9.4 Hz, 1H), 5.12 (s, 2H), 4.23 (dd, J = 12.6, 5.0 Hz, 1H), 4.07 – 3.96 (m, 1H), 3.90 – 3.80 (m, 1H), 3.71 (s, 2H), 3.27 (t, J= 6.0 Hz, 3H), 2.58 (s, 2H), 2.15 (s, 3H), 2.04 (s,3H), 1.99 (d, J = 3.2 Hz, 6H).

[0147] MS (ESI): m / z calc. for C 48 H 48 N4O 10 (M+H) + : 841.3; found: 841.3.

[0148] Step 5: Compound 5 (270 mg, 0.321 mmol), pPFPA (70 mg, 0.2922 mmol), and 4-dimethylaminopyridine (23.2 mg, 0.1894 mmol) were added to a reaction flask. Anhydrous DMF (3 mL) was added under a nitrogen atmosphere. The resulting mixture was subjected to RAFT polymerization at 65°C under a nitrogen atmosphere for 20 hours. After completion of the reaction, the reaction solution was decanted three times (3 times with 45 mL of diethyl ether), and the resulting solid was lyophilized to obtain polymer (OAc) (220 mg, 81% yield).

[0149] 1 H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.02 (d, J = 36.8 Hz, 16H), 6.65 (s, 2H), 6.01 – 5.86 (m, 3H), 5.37 (t, J = 8.9 Hz, 1H), 5.30 (s, 1H), 5.04 (s, 2H), 4.33 (s, 1H), 4.04 (s, 1H), 3.88 (s, 1H), 3.48 (q, J = 7.0 Hz,1H), 2.92 (d, J = 29.2 Hz, 1H), 2.16 (s, 3H), 2.05 (s, 9H), 1.21 (t, J = 7.0 Hz, 2H).

[0150] Step 6: OAc-P1 (220 mg) was dissolved in a mixed solution of dichloromethane and methanol (v / v = 1 / 1). After dissolution, an excess of sodium methoxide was added and the pH of the solution was monitored. The mixture was stirred at room temperature for two hours. After being spin-dried, it was dissolved in a small amount of DMF solution and dialyzed in methanol for 48 h. The mixture was lyophilized to obtain P1 as a white solid (65 mg, yield, 46%).

[0151] 1 H NMR (400 MHz, DMSO) δ 8.27 (s, 1H), 7.21 – 6.65 (m, 18H), 5.91 (s,1H), 5.28 (s, 1H), 5.00 (s, 3H), 4.61 (s, 1H), 4.41 (s, 1H), 3.84 (s, 1H), 3.60 (s, 3H), 3.33 (s, 9H), 1.24 (s, 1H).

[0152] Turbidimetry is a method for determining the concentration of suspended matter by measuring the intensity of light transmitted through a suspended particle medium. Ultraviolet spectrophotometry can be used to qualitatively analyze the recognition effect between glycopolymers and Con A. Since the initial rate of change in absorbance is related to the binding rate between glycopolymers and lectins, the initial slope of the curve (K i , Abs / s) to compare the binding rates of the polymers.

[0153] Figure 20 This is a diagram showing the recognition effect between the tetraphenylethylene-containing side chain single-arm thiol mannose polymer and Con A in Example 1 of the present invention. It can be seen that the turbidity (absorbance) changes after mixing the TPE mannose polymer and Con A. The initial rate of change in the absorbance curves of the TPE mannose polymers P1 (n = 36) and P2 (P2 is the compound in Example 1) is very large, but gradually stabilizes. The binding rate of P1 is K i = 0.01668; the binding rate of P2 is K i = 0.02776. Dendrimers containing flexible thioether linkages via a thiol-ene reaction exhibited superior protein recognition compared to those containing rigid triazole linkages via a CuAAC reaction. This is because the flexible geometry of the polymer side chains provides more space for Con A binding; on the other hand, the flexible linkages may also have a synergistic effect on Con A recognition by the glycopolymers.

[0154] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. 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 side chain-containing tetraphenylethylene single-arm thiol mannose polymer, characterized in that: The chemical structure of the tetraphenylethylene-containing single-arm thiol-mannose polymer is shown in Formula (I): ； Here, n is an integer ≥2.

2. A method for preparing a single-arm thiol-mannose polymer containing tetraphenylethylene in the side chain according to claim 1, characterized in that: The following steps are involved: (1) 4-bromo-4'-methoxybenzophenone and diphenylmethane undergo nucleophilic addition elimination reaction to obtain the first compound; (2) the first compound and boron tribromide undergo a demethylation reaction to obtain a second compound; (3) the second compound and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester are subjected to Suzuki coupling reaction to obtain a third compound; (4) esterifying the third compound with acryloyl chloride and anhydrous triethylamine to obtain a fourth compound; (5) The fourth compound reacts with acetyl-protected thiol mannose and n-hexylamine through a Thiol-ene click chemistry reaction to obtain a fifth compound; (6) The fifth compound is reacted with trifluoroacetic acid to obtain a sixth compound; (7) Pentafluorophenol is reacted with triethylamine and acryloyl chloride to obtain the seventh compound; (8) The seventh compound is reacted with a chain transfer agent and an initiator through RAFT polymerization to obtain an eighth compound; (9) The eighth compound, the sixth compound and 4-diaminopyridine are subjected to an aminolysis reaction to obtain a ninth compound; (10) The ninth compound is reacted with sodium methoxide to obtain a single-arm thiol-mannose polymer containing tetraphenylethylene on the side chain; Among them, the chemical structures of the tetraphenylethylene-containing single-arm thiol mannose polymer, the first compound, the second compound, the third compound, the fourth compound, the fifth compound, the sixth compound, the seventh compound, the eighth compound, and the ninth compound are shown in Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), Formula (IX), and Formula (X), respectively: ； Here, n is an integer ≥2.

3. The preparation method according to claim 2, wherein During the nucleophilic addition elimination reaction of step (1), the molar ratio of 4-bromo-4'-methoxybenzophenone to diphenylmethane is 1:(1.1-1.3); the reaction temperature is 0°C-100°C, and the reaction time is 3 h-24 h.

4. The preparation method according to claim 2, wherein During the demethylation reaction of step (2), the dosage ratio of the first compound to boron tribromide is 1 g: (2-3) mL; the reaction temperature is 10°C-30°C, and the reaction time is 18 h-30 h.

5. The preparation method according to claim 2, wherein During the Suzuki coupling reaction of step (3), the molar ratio of the second compound to N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester is 1:(1~3); the reaction temperature is 105℃~115℃, and the reaction time is 16 h~20 h.

6. The preparation method according to claim 2, wherein During the esterification reaction of step (4), the molar ratio of the third compound, acryloyl chloride and anhydrous triethylamine is 1:(1-3):(1-3); the reaction temperature is 0°C-25°C, and the reaction time is 18 h-24 h.

7. The preparation method according to claim 2, wherein In the Thiol-ene click chemistry reaction of step (5), the molar ratio of the fourth compound, acetyl-protected thiol mannose, and n-hexylamine is 1:(1-3):(0.1-0.15); the reaction temperature is 15°C-30°C, and the reaction time is 18 h-24 h.

8. The preparation method according to claim 2, wherein During the substitution reaction of step (6), the ratio of the fifth compound to trifluoroacetic acid is 1 mmol: (1-6) ml; the reaction temperature is 15°C-25°C; and the reaction time is 4 h-6 h.

9. The preparation method according to claim 2, wherein During the esterification reaction of step (7), the molar ratio of pentafluorophenol, triethylamine, and acryloyl chloride is 1:(0.5~2):(0.5~2); the reaction temperature is 0 ℃~25 ℃, and the reaction time is 16 h~36 h.

10. The preparation method according to claim 2, wherein During the RAFT polymerization reaction of step (8), the molar ratio of the seventh compound, the chain transfer agent, and the initiator is (150-220): (7-10): 1; the chain transfer agent is trithiocarbonate, and the initiator is azobisisobutyronitrile; the reaction temperature is 70°C-90°C, and the reaction time is 20 h-30 h.

11. The preparation method according to claim 2, wherein During the aminolysis reaction of step (9), the molar ratio of the eighth compound, the sixth compound, and 4-diaminopyridine is (1-3):(2-5):1; the reaction temperature is 50°C-70°C, and the reaction time is 18 h-24 h.

12. The preparation method according to claim 2, wherein During the substitution reaction of step (10), the usage ratio of the ninth compound and sodium methoxide is 0.1 g: (30-60) mg; the reaction temperature is 15°C-30°C, and the reaction time is 1 h-3 h.

13. Use of the single-arm thiol-mannose polymer containing tetraphenylethylene in the side chain according to claim 1 in preparing a fluorescent probe.

Images