A water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe and its preparation method and application

Abstract

The present invention discloses a water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe, its preparation method, and application. Luminol compounds and aza-fluoroboron fluorophore compounds are covalently linked to both ends of tetraphenylzinc porphyrin. The chemiluminescence of the luminol compound is transferred to the aza-fluoroboron fluorophore compound via the tetraphenylzinc porphyrin, achieving chemiluminescence in the near-infrared second region through two-step intramolecular energy transfer. Simultaneously, hydrophilic oligoethylene glycol monomethyl ether methacrylate is polymerized onto the fluorophore via atom transfer radical polymerization to obtain a water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe. The probe has a novel structure, a simple preparation method, and a high yield of the prepared product. During in vivo imaging, direct imaging is possible without the use of an external excitation light source, resulting in a higher imaging signal-to-noise ratio.

Description

Technical Field

[0001] The invention belongs to the technical field of amphiphilic fluorescent dyes, and particularly relates to a water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe and a preparation method and application thereof. Background Art

[0002] Trigger-activated optical sensing has become an indispensable technology for disease diagnosis and bioanalysis. Chemiluminescence (CL) detection relies on the emission of excited states formed by chemical reactions, rather than external light excitation, effectively reducing the autofluorescence and scattering caused by external light source excitation. Therefore, compared to traditional fluorescence imaging techniques that require external excitation light sources, CL exhibits higher sensitivity, millimeter-depth resolution, and an ultra-high imaging signal-to-noise ratio.

[0003] However, currently developed chemiluminescent probes are typically located in the visible light region of 400-650nm, with low resolution and penetration depth, limiting their further application in vivo. Optical imaging in the near-infrared (NIR-I) region of 650-900nm and the NIR-II region of 900-1700nm has attracted much attention due to its ability to reduce tissue autofluorescence interference and background scattering, thereby exhibiting higher spatial resolution and deeper tissue penetration depth.

[0004] Although some near-infrared chemiluminescent nanoprobes have been reported in the prior art, most of them are based on the intermolecular chemiluminescence resonance energy transfer (CRET) effect between a chemiluminescent donor and a fluorophore acceptor. For example, Angew.Chem.Int.Ed., 2020, 59, 18380–18385 reported a NIR-II chemiluminescent nanoprobe, which was assembled into a nanoprobe by assembling a chemical donor and two dye acceptors, and then using continuous chemiluminescence resonance energy transfer and fluorescence energy resonance transfer to achieve the transfer of chemical energy to NIR-II chemical emission. Because of the multi-step energy transfer involved, the intermolecular energy conversion efficiency is low, which in turn leads to a decrease in the NIR-II chemiluminescence intensity. In addition, the probe embedded in the nanoparticle may also have difficulties in approaching the target molecule. Moreover, these probes are generally highly hydrophobic and need to be prepared using liposomes or amphiphilic block polymers before they can become materials used in water (PNAS, 120(8), e2205186120). However, physically loaded dyes are easily leaked during blood circulation and have poor stability. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a water-soluble near-infrared II chemiluminescent macromolecular fluorescent probe, a preparation method and application thereof, and develops a single-molecule NIR chemiluminescent probe by covalently linking a chemiluminescent donor and an NIR-II fluorophore acceptor. The probe is also endowed with water solubility, so that it has the characteristics of near-infrared II chemiluminescence and water solubility, and can perform high signal-to-noise ratio imaging in a living inflammation model.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A water-soluble near-infrared second region chemiluminescent macromolecular fluorescent probe, the structural formula of the water-soluble near-infrared second region chemiluminescent macromolecular fluorescent probe is:

[0008] Here, n is an integer greater than or equal to 10.

[0009] The present invention also provides a method for preparing the water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe, which comprises the following steps:

[0010] (1) preparing a bifunctional azide-modified tetraphenylzinc porphyrin, wherein the bifunctional azide-modified tetraphenylzinc porphyrin has the structural formula:

[0011] (2) preparing an aza-fluoro-boron fluoride compound, wherein the aza-fluoro-boron fluoride compound has the structural formula:

[0012]

[0013] (3) Preparation of alkynyl-modified luminol compounds, the structural formula of which is:

[0014] (4) A bifunctional azide-modified tetraphenylzinc porphyrin and an aza-fluoroboron fluorine compound were subjected to a click chemistry reaction at a molar ratio of 1:1 using cuprous iodide and N,N-diisopropylethylamine as catalysts to obtain an intermediate product A; the intermediate product A has the structural formula:

[0015]

[0016] (5) The intermediate product A and the lumilol compound are subjected to a click chemistry reaction at a molar ratio of 1:1 to 1.2 using cuprous iodide and N,N-diisopropylethylamine as catalysts to obtain an intermediate product B; the structural formula of the intermediate product B is:

[0017] (6) The intermediate product B and oligoethylene glycol monomethyl ether methacrylate are reacted at a molar ratio of 1:10 to 150, using cuprous bromide and pentamethyldiethylenetriamine as catalysts, and subjected to polymerization reaction at 70 to 75° C. for 5 to 7 hours to obtain the water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe.

[0018] In step (4), the molar ratio of cuprous iodide to N,N-diisopropylethylamine is 1:1; the molar ratio of bifunctional azide-modified tetraphenylzinc porphyrin to the catalyst is 1:1.

[0019] In step (5), the molar ratio of cuprous iodide to N,N-diisopropylethylamine is 1:1; and the molar ratio of intermediate product A to the catalyst is 1:1.

[0020] In step (4) and step (5), the reaction solvent is dichloromethane.

[0021] In step (4) and step (5), the conditions for the click chemistry reaction are: reaction at room temperature for 6 to 8 hours.

[0022] In step (6), the molar ratio of cuprous bromide to pentamethyldiethylenetriamine is 1:1; and the molar ratio of intermediate product B to catalyst is 1:1.

[0023] The present invention also provides the use of the water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe in preparing a probe for in vivo imaging.

[0024] The water-soluble near-infrared region II chemiluminescent macromolecular fluorescent probe provided by the present invention comprises a luminol compound and an aza-fluoroborane compound covalently linked to either end of a tetraphenylzinc porphyrin. The chemiluminescence of the luminol compound is transferred to the aza-fluoroborane compound via the tetraphenylzinc porphyrin, achieving chemiluminescence in the near-infrared region II through a two-step intramolecular energy transfer. Simultaneously, hydrophilic oligoethylene glycol monomethyl ether methacrylate is polymerized onto the fluorophore via atom transfer radical polymerization, thereby obtaining a water-soluble near-infrared region II chemiluminescent macromolecular fluorescent probe.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The water-soluble near-infrared second-zone chemiluminescent macromolecular fluorescent probe provided by the present invention has a novel structure, a simple preparation method, and a high yield. It can directly image the site of inflammation in vivo without the need for an external excitation light source, and has a higher signal-to-noise ratio than fluorescence imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the H NMR spectrum of Alkyne-NBDP;

[0028] Figure 2is the C NMR spectrum of Alkyne-NBDP;

[0029] Figure 3 is the H NMR spectrum of TPP-2Br;

[0030] Figure 4 is the C NMR spectrum of TPP-2Br;

[0031] Figure 5 This is the H NMR spectrum of TPP(Zn)-2N3;

[0032] Figure 6 is the C NMR spectrum of TPP(Zn)-2N3;

[0033] Figure 7 is the H NMR spectrum of Alkyne-Luc;

[0034] Figure 8 (a) is the H NMR spectrum of CL912, and (b) is the tetrahydrofuran GPC result of CL912;

[0035] Figure 9 Matrix-assisted desorption ionization time-of-flight mass spectrometry of Alkyne-NBDP;

[0036] Figure 10 Matrix-assisted desorption ionization time-of-flight mass spectrometry of TPP-2Br;

[0037] Figure 11 It is the matrix-assisted desorption ionization time-of-flight mass spectrometry of TPP(Zn)-2Br;

[0038] Figure 12 It is the matrix-assisted desorption ionization time-of-flight mass spectrometry of TPP(Zn)-2N3;

[0039] Figure 13 Matrix-assisted desorption ionization time-of-flight mass spectrometry of Alkyne-NBDP-Br;

[0040] Figure 14 It is the matrix-assisted desorption ionization time-of-flight mass spectrometry of N3-TPP(Zn)-NBDP-Br;

[0041] Figure 15 Matrix-assisted desorption ionization time-of-flight mass spectrometry of Luc-TPP(Zn)-NBDP-Br;

[0042] Figure 16(a) Normalized chemiluminescence spectrum of Lumiq, absorption and emission spectra of TPP(Zn)-2N3, and absorption and emission spectra of Alkyne-NBDP-Br; (b) Absorption spectrum of probe CL912; (c) Chemiluminescence spectrum of CL912 probe in PBS solution after addition of H2O2 and HRP;

[0043] Figure 17 is the hydrated particle size of CL912;

[0044] Figure 18 (a) Imaging results of Alkyne-Luc, Alkyne-NBDP, and CL 912 probes in 4T1 tumors, where Alkyne-NBDP probe was excited by 808 nm laser, while Alkyne-Luc and CL 912 were not excited by external light source; (b) Signals of each group collected using 850 nm long-pass filter.

[0045] Figure 19 is the structural formula of amphiphilic zinc porphyrin fluorescent dye. DETAILED DESCRIPTION

[0046] The present invention is described in detail below with reference to the embodiments.

[0047] Example 1

[0048] Three functional small molecule precursors, bifunctional azide-modified tetraphenylzinc porphyrin (TPP(Zn)-2N3), aza-fluoroboron fluorine compound (Alkyne-NBDP-Br), and luminol compound (Alkyne-Luc), were synthesized. The synthesis route is as follows:

[0049]

[0050] (1) Synthesis of Alkyne-NBDP-Br, steps are as follows:

[0051] (1-1) Synthesis of Alkyne-NBDP: The aldehyde compound and the ketone carbonyl compound were dissolved in anhydrous ethanol at a molar ratio of 1:1, and then 4 equivalents of KOH were added. After ultrasonic dissolution, the reaction was carried out at room temperature overnight. After filtration and washing twice with cold ethanol, a yellow solid product was obtained with a yield of 50-70%. The above-obtained compound and nitromethane were dissolved in ethanol at a molar ratio of 1:10, and 4 equivalents of KOH were added. After the reaction was carried out at 70°C for 24 hours, the ethanol was dried and dichloromethane was added. After the reaction mixture was dissolved in n-butanol, 4 equivalents of ammonium acetate were added, and the mixture was reacted at 110° C. for 3-7 hours under a nitrogen atmosphere, and then the mixture was returned to room temperature. The solvent was dried, saturated brine was added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, concentrated and dried, and then the next step was directly carried out. The obtained product was dissolved in anhydrous dichloromethane, and three equivalents of N,N-diisopropylethylamine were added thereto at -20°C under N2 atmosphere. After 10 minutes, 5 equivalents of boron trifluoride ether were slowly added. The mixture was returned to room temperature and stirred for 2-5 hours. Saturated brine was slowly added, and the mixture was extracted twice with dichloromethane. The organic phase was dried over anhydrous magnesium sulfate, filtered, concentrated, and then subjected to column chromatography using dichloromethane and petroleum ether in a ratio of 1:1 to obtain a brown solid product with a yield of 15-25%. Its H NMR spectrum is as follows: Figure 1 As shown, the NMR carbon spectrum is Figure 2 As shown, the mass spectrum Figure 9 shown. 1 H NMR (400MHz, DMSO-d6) δ8.32–7.98(m,8H),7.32(s,2H),6.90(dd,J=39.3,8.4Hz,8H),4.30(s,4H),3.05(s,18H),2.86(d,J=9.1Hz,2H). 13 C NMR(101MHz,DMF-d7)δ155.99,151.59,150.97,145.01,141.68,131.62,130.83,121.25,120.97,115 .33,113.30,112.44,80.00,74.18,55.23,41.52,39.94,38.09.MALDI-TOF-MS(m / z):calcd.for[M+H] + ,718.6672,found,718.4567.

[0052] (1-2) Synthesis of Alkyne-NBDP-Br: Alkyne-NBDP (40 mg, 0.0556 mmol, 1.0 equiv) and 2-bromo-2-methylpropionic acid-2-azidoethyl ester (13 mg, 0.0556 mmol, 1.0 equiv) were dissolved in 5 mL of dichloromethane. Cuprous bromide (4.0 mg, 0.0278 mmol, 0.5 equiv), N,N-diisopropylethylamine (3.6 mg, 0.0278 mmol, 0.5 equiv), and CH3COOH (1.6 mg, 0.0278 mmol, 0.5 equiv) were added thereto. The mixture was reacted at room temperature for 8 h. The mixture was then eluted with a mixture of DCM and CH3OH in a volume ratio of 95:5 for column chromatography to obtain 35 mg of the final product with a yield of 66%. The mass spectrum thereof is shown in FIG. Figure 13 MALDI-TOF-MS (m / z): calcd.for [M+H] + ,955.7338,found,955.1243.

[0053] (2) Synthesis of TPP(Zn)-2N3:

[0054] First, TPP-2Br was synthesized by dissolving 4-(2-bromoethoxy)benzaldehyde (2.28 g, 10 mmol, 1.0 equivalent), benzaldehyde (1.06 g, 10 mmol, 1.0 equivalent), and purified pyrrole (6.7 g, 100 mmol, 10.0 equivalent) in 100 mL of acetic acid. The mixture was reacted at 130°C for 2 h and then returned to room temperature. The acetic acid was removed by rotary evaporation with an oil pump, and the mixture was redispersed in dichloromethane. The mixture was washed three times with saturated brine, and the organic phase was collected, dried over anhydrous sodium sulfate, and then subjected to column chromatography to obtain 250 mg of a dark brown solid product with a yield of 5.8%. Its H NMR spectrum was as follows: Figure 3 As shown, the NMR carbon spectrum is Figure 4 As shown, the mass spectrum Figure 10 MALDI-TOF-MS (m / z): calcd.for [M+H] + ,862.2531,found,862.6411.

[0055] The solid powder obtained above (86 mg, 0.1 mmol, 1.0 equivalent) was dissolved in a 1:1 mixed solvent of dichloromethane and N,N-dimethylformamide (5 mL in total), and zinc acetate (36.6 mg, 0.2 mmol, 2.0 equivalent) was added thereto. The mixture was reacted at 70°C for 4 h, and the solvent was removed by rotary evaporation. The mixture was dissolved in dichloromethane and washed twice with saturated brine. Column chromatography gave 55 mg of the product with a yield of 60%. The mass spectrum of the product is shown as follows: Figure 11MALDI-TOF-MS (m / z): calcd.for [M+H] + ,925.0263,found,925.2192.

[0056] The above-obtained product (50 mg, 0.054 mmol, 1.0 equivalent) was dissolved in 5 mL of N,N-dimethylformamide, and sodium azide (35 mg, 0.54 mmol, 10.0 equivalent) was added. The mixture was reacted at 90°C for 4 h, and then returned to room temperature. 50 mL of dichloromethane was added, and the mixture was extracted three times with saturated brine. The organic phase was concentrated and dried to obtain the final product, 40 mg, with a yield of 88%. Its H NMR spectrum is as follows: Figure 5 As shown, the NMR carbon spectrum is Figure 6 As shown, the mass spectrum Figure 12 MALDI-TOF-MS (m / z): calcd.for [M+H] + ,849.2667,found,849.2585.

[0057] (3) Synthesis of Alkyne-Luc:

[0058] First, TMS-Alkyne-Luc was synthesized. Aminolumilol (2.0 g, 11.3 mmol, 1.0 equivalent) was dissolved in 5 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (1.75 g, 13.56 mmol, 1.2 equivalents) was added thereto. Under stirring in an ice-water bath, 3-(trimethylsilyl)propioyl chloride (2.0 g, 12.43 mmol, 1.1 equivalents) was slowly added thereto. The reaction was then carried out at room temperature for 6 h. After the reaction was completed, 50 mL of dichloromethane was added thereto. The mixture was extracted three times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, and then column chromatography was performed to obtain 1.2 g of the product. The obtained product was dissolved in 20 mL of methanol, potassium carbonate (1.1 g, 2 equivalents) was added thereto, and the mixture was stirred at room temperature for 2 h, filtered, concentrated, and subjected to column chromatography. A mixture of DCM and CH3OH in a volume ratio of 95:5 was used as the eluent to obtain 600 mg of the product with a yield of 66.5%. The H NMR spectrum was as follows: Figure 7 shown. 1 HNMR (400MHz, DMSO-d6) δ13.52(s,1H),11.98(s,2H),8.74(dd,J=8.3,1.0Hz,1H),7.90(t,J=8.1Hz,1H),7.69(dd,J=8.0,1.0Hz,1H),4.58(s,1H).

[0059] Example 2

[0060] A water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe (CL912) has the structural formula:

[0061]

[0062] Its synthetic route is as follows:

[0063]

[0064] The preparation steps are as follows:

[0065] (1) Synthesis of N3-TPP(Zn)-NBDP-Br: TPP(Zn)-2N3 (8.5 mg, 0.01 mmol, 1.0 equivalent), Alkyne-NBDP-Br (9.6 mg, 0.01 mmol, 1.0 equivalent), cuprous iodide (1.0 mg, 0.005 mmol, 0.5 equivalent), and N,N-diisopropylethylamine (0.6 mg, 0.005 mmol, 0.5 equivalent) were dissolved in 5 mL of dichloromethane and reacted at room temperature for 6 h. After concentration, the mixture was eluted with a mixture of DCM and PE in a volume ratio of 1:1 and column chromatography was performed to obtain 10 mg of the product with a yield of 55%. The mass spectrum thereof is shown in FIG. Figure 14 MALDI-TOF-MS (m / z): calcd.for [M+H] + ,1802.9925,found,1802.1012.

[0066] (2) Synthesis of Luc-TPP(Zn)-NBDP-Br: N3-TPP(Zn)-NBDP-Br (9 mg, 0.005 mmol, 1.0 equivalent) and Alkyne-Luc (1.3 mg, 0.006 mmol, 1.2 equivalent) were dissolved in 5 mL of dichloromethane, to which 0.5 equivalent of cuprous iodide and 0.5 equivalent of N,N-diisopropylethylamine were added. The mixture was reacted at room temperature for 6 h. After concentration, the mixture was eluted with a mixture of DCM and CH3OH in a volume ratio of 95:5 for column chromatography to obtain 5 mg of the product with a yield of 49%. The mass spectrum thereof is shown in FIG. Figure 15 MALDI-TOF-MS (m / z): calcd.for [M+H] + ,2032.1842,found,2031.8580.

[0067] (3) Synthesis of CL912: Luc-TPP(Zn)-NBDP-Br (4 mg, 0.002 mmol, 1.0 equiv.) was added to a sealed tube, to which oligoethylene glycol monomethyl ether methacrylate (100 mg, 0.2 mmol, 100 equiv.), 0.5 equiv. of cuprous bromide, and 0.5 equiv. of pentamethyldiethylenetriamine were added, and 100 μL of dioxane was added as a solvent. After sealing the tube, the product was polymerized at 70°C for 6 h, frozen with liquid nitrogen, and the tube was opened. The product was precipitated with ether three times to obtain an oily product. The H NMR spectrum was as follows: Figure 8 a, and the molecular weight and molecular weight distribution of the polymer were characterized using tetrahydrofuran GPC, as shown in Figure 8 As shown in b, the average molecular weight of the obtained probe is 22000 Da, and the molecular weight distribution is 1.3.

[0068] The UV absorption and fluorescence spectra of TPP(Zn)-2N3, Alkyne-NBDP-Br and Alkyne-Luc are as follows: Figure 16 As shown in Figure a, it can be seen that the chemiluminescence of lumilol overlaps well with the absorption of TPP(Zn)-2N3, and the emission of TPP(Zn)-2N3 overlaps greatly with the absorption of Alkyne-NBDP-Br, thus ensuring an effective energy transfer process. The UV absorption spectrum of CL912 is shown in Figure 5. Figure 16 As shown in Figure b, it can be seen that the molecule couples the absorption spectrum characteristics of TPP(Zn)-2N3 and Alkyne-NBDP-Br. After adding H2O2, the chemiluminescence spectrum of this polymer chemiluminescent probe is as follows Figure 16 As shown in c, its chemiluminescence peak position is at 912nm, which is consistent with Figure 16 The fluorescence emission peak position of Alkyne-NBDP-Br in a is consistent.

[0069] The CL912 probe was dissolved in PBS solution at pH 7.4 to a final probe concentration of 1 mg / mL, and its hydrated particle size was characterized using dynamic light scattering, as shown in Figure 2. Figure 17 As shown in Figure 2, the hydrated particle size is 147 nm. H2O2 and 10 U of horseradish peroxidase were added to the nanoprobe at a final concentration of 100 μM, and the intensity of the near-infrared second region chemiluminescence was tested. The experimental results are shown in Figure 2. Figure 16 As shown in Figure c, it can be seen from the figure that its chemiluminescence position is at 912nm, further confirming the near-infrared second region luminescence characteristics of the probe.

[0070] Example 3

[0071] Application of Water-Soluble Near-Infrared Second Region Chemiluminescent Macromolecular Fluorescent Probe CL912 in In Vivo Imaging

[0072] The 4T1 tumor-bearing mouse model was established by inoculating 4T1 cells (5×10 6 ) were injected subcutaneously into the right leg abdomen of three groups of BALB / c mice. After 7 days, when the tumor reached 150 mm 3 20 μL of 5 mg / mL LPS solution was injected intratumorally to induce the production of H2O2 in vivo. 8 h later, 10 μL of 100 μM Alkyne-Luc solution in DMSO was injected into the tumor of the first group, 10 μL of 100 μM Alkyne-NBDP solution in DMSO was injected into the tumor of the second group, and 10 μL of 100 μM CL 912 solution in PBS was injected into the tumor of the third group. Imaging was performed 30 min later. The first and third groups did not use external laser excitation, while the second group used 808 nm laser with a power of 1.0 W / cm 2 The fluorescence was excited by laser and collected using an 850 nm long-pass filter.

[0073] Imaging results and signal-to-noise ratio Figure 18 As shown in Figures a) and b), no signal is detected in the Alkyne-Luc sample using an 850nm longpass filter within the tumor. However, the signal-to-noise ratio of Alkyne-NBDP within the tumor is 4.9, while that of CL 912 reaches 22.4. This indicates that the signal-to-noise ratio of CL 912's near-infrared chemiluminescence is 4.5 times that of Alkyne-NBDP.

[0074] The detailed description of a water-soluble near-infrared second-zone chemiluminescent macromolecular fluorescent probe, its preparation method and application with reference to the above examples is illustrative rather than restrictive. Several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe, characterized in that: The structural formula of the water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe is: , where n is an integer greater than 10.

2. The method for preparing the water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Preparing a bifunctional azide-modified tetraphenylzinc porphyrin, wherein the bifunctional azide-modified tetraphenylzinc porphyrin has the structural formula: ; (2) Preparing an aza-fluoro-boron fluoride compound, wherein the aza-fluoro-boron fluoride compound has the structural formula: ； (3) preparing a luminol compound having the structural formula: ; (4) A bifunctional azide-modified tetraphenylzinc porphyrin and an aza-fluoroborane fluoride compound were reacted in a molar ratio of 1:1 with cuprous iodide and N,N-diisopropylethylamine as catalysts to undergo a click chemistry reaction to obtain intermediate product A; (5) Intermediate product A and a lumilol compound are reacted in a molar ratio of 1:1 to 1.2 using cuprous iodide and N,N-diisopropylethylamine as catalysts to undergo a click chemistry reaction to obtain intermediate product B; (6) The intermediate product B and oligoethylene glycol monomethyl ether methacrylate are mixed at a molar ratio of 1:10-150, using cuprous bromide and pentamethyldiethylenetriamine as catalysts, and polymerized at 70-75°C for 5-7 h to obtain the water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe.

3. The preparation method according to claim 2, characterized in that In step (4), the molar ratio of cuprous iodide to N,N-diisopropylethylamine is 1:1; the molar ratio of bifunctional azide-modified tetraphenylzinc porphyrin to the catalyst is 1:

1.

4. The preparation method according to claim 2, characterized in that In step (5), the molar ratio of cuprous iodide to N,N-diisopropylethylamine is 1:1; the molar ratio of intermediate product A to the catalyst is 1:

1.

5. The preparation method according to any one of claims 2 to 4, characterized in that In step (4) and step (5), the reaction solvent is dichloromethane.

6. The preparation method according to any one of claims 2 to 4, characterized in that: In step (4) and step (5), the conditions for the click chemistry reaction are: reaction at room temperature for 6 to 8 hours.

7. The preparation method according to any one of claims 2 to 4, characterized in that: In step (6), the molar ratio of cuprous bromide to pentamethyldiethylenetriamine is 1:

1.

8. The preparation method according to any one of claims 2 to 4, characterized in that: In step (6), the molar ratio of the intermediate product B to the catalyst is 1:

1.

9. Use of the water-soluble near-infrared second-region chemiluminescent macromolecular fluorescent probe according to claim 1 in preparing a probe for in vivo imaging.

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