A leucine aminopeptidase and glutathione dual-stimulus responsive probe, its preparation method and application

Abstract

The present invention discloses a leucine aminopeptidase and glutathione dual-stimuli responsive probe and its preparation method and application. The probe Ce6-Leu of the present invention undergoes a click condensation reaction under the stimulation of the tumor microenvironment, and then reassembles from nanoparticles into nanofibers. Specifically, under the stimulation of leucine aminopeptidase and glutathione overexpressed in tumor cells, the amino and mercapto groups originally in the cysteine structure of the probe Ce6-Leu are exposed, and then a click condensation reaction occurs to form an amphiphilic dimer, which reassembles to form nanofibers. The distance between Ce6 structures becomes larger, and the quenching between them weakens, so the fluorescence signal and the ability to generate ROS are enhanced, thereby realizing specific fluorescence imaging and photodynamic therapy of tumors, achieving the integration of tumor diagnosis and treatment, and having important scientific research and economic value.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor microenvironment-mediated reassembly technology, and relates to a leucine aminopeptidase and glutathione dual-stimulus-responsive probe, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer has become one of the diseases seriously endangering human health. Therefore, in recent years, molecular imaging technologies for cancer diagnosis and treatment have developed rapidly. The application of molecular imaging diagnostic technologies can diagnose cancer patients at an early stage, and provide important information for cancer classification, prognosis evaluation, and treatment plan selection, providing new opportunities for humans to overcome cancer.

[0003] Theranostics, as an emerging tumor diagnosis and treatment strategy that combines disease diagnosis, monitoring, and treatment, brings new hope for overcoming cancer; and the advantages of high efficiency and low toxicity and side effects of this strategy are expected to promote the rapid development of cancer diagnosis and treatment technologies. Currently, scientists have developed various contrast agents, among which nano-sized contrast agents, such as nanoemulsions, liposomes, dendrimers, and inorganic nano-multifunctional contrast agents designed based on heavy metal materials such as gold, tantalum, lanthanides, and bismuth. Such nanoparticle contrast agents have the advantages of long blood circulation time, low renal clearance rate, and low capillary leakage rate, and can passively accumulate in tumor sites through the enhanced permeation and retention effect (EPR) of solid tumors. However, most of these contrast agents are still in the preclinical research stage, lacking experimental evaluations of biotoxicity, pharmacokinetics, and in vivo distribution, and there is still a certain distance from clinical application. Summary of the Invention

[0004] In order to overcome the problems existing in the above-mentioned existing contrast agents, the present invention utilizes the stimulation of overexpressed leucine aminopeptidase and glutathione in the tumor microenvironment to design and develop a leucine aminopeptidase and glutathione dual-stimulus-responsive probe, realizing the specific response of the probe in the tumor, and then effectively improving the imaging and treatment effects of in vivo tumors; this method provides a new strategy and means for improving the disadvantages of traditional molecular probes, such as high imaging background noise and low diagnostic accuracy.

[0005] The present invention adopts the following technical solutions:

[0006] A leucine aminopeptidase and glutathione dual-stimulus-responsive probe has the following chemical structural formula:

[0007]

[0008] Use of the above-mentioned leucine aminopeptidase and glutathione dual-stimulus responsive probe in the preparation of tumor diagnostic and / or therapeutic reagents.

[0009] Preparation method of the above-mentioned leucine aminopeptidase and glutathione dual-stimulus responsive probe, comprising the following steps:

[0010] (1) Performing an amide condensation reaction between compound 1 and NH2-CBT to obtain compound 2;

[0011] (2) Removing the protecting group from compound 2 to obtain compound 3;

[0012] (3) Performing an amide condensation reaction between compound 3 and N-Fmoc-S-tert-butylthio-L-cysteine to obtain compound 4;

[0013] (4) Removing the protecting group from compound 4 to obtain compound 5;

[0014] (5) Reacting compound 5 with a photosensitizer to obtain compound 6;

[0015] (6) Removing the protecting group from compound 6 to obtain compound 7;

[0016] (7) Performing an amide condensation reaction between compound 7 and N-Boc-L-leucine to obtain compound 8;

[0017] (8) Removing the protecting group from compound 8 to obtain the leucine aminopeptidase and glutathione dual-stimulus responsive probe.

[0018] The leucine aminopeptidase and glutathione dual-stimulus responsive probe disclosed in the present invention self-assembles into spherical nanoparticles in vitro and the fluorescence is quenched; when the nanoparticles circulate to the tumor site, under the stimulation of overexpressed leucine aminopeptidase and glutathione in tumor cells, it selectively reassembles into nanofibers in tumor cells through an intermolecular CBT condensation reaction, completing the response of the near-infrared molecular probe in the tumor and the fluorescence recovery. Moreover, after the leucine aminopeptidase and glutathione dual-stimulus responsive probe responds at the tumor site, the ability to generate ROS is restored, and after irradiation with a 660 nm laser, the near-infrared small molecule photosensitizer is used for photodynamic therapy of tumors. The time point with the strongest fluorescence signal at the tumor site is 3 hours; when irradiated with a 660 nm laser, the irradiation intensity is 0.15 W / cm 2 , and the irradiation time is 10 minutes.

[0019] In the above technical solution, the leucine aminopeptidase and glutathione dual-stimuli-responsive probe is dissolved in a PBS (phosphate buffer, pH = 7.2 - 7.4) buffer solution (concentration: 100 μM), and reacted in an oscillator at 37 °C to self-assemble into spherical nanoparticles. Subsequently, it is injected into the tumor-bearing mice via the tail vein. After the probe circulates to the tumor site, under the stimulation of leucine aminopeptidase and glutathione overexpressed in tumor cells, it selectively reassembles into nanofibers through intermolecular CBT condensation reaction inside tumor cells, completing the response of the near-infrared molecular probe in the tumor and the fluorescence recovery. Moreover, after the probe circulates to the tumor site, under the stimulation of leucine aminopeptidase and glutathione overexpressed in tumor cells, it selectively reassembles into nanofibers through intermolecular CBT condensation reaction inside tumor cells, completing the response of the near-infrared molecular probe in the tumor, the fluorescence gradually recovers, and the ability to generate ROS gradually becomes stronger. When the fluorescence signal of the probe reaches the strongest at the tumor site, after irradiation with a 660 nm laser, the near-infrared small molecule photosensitizer is used for the treatment of tumors.

[0020] In the above technical solution, in step (1), the molar ratio of compound 1 to NH2-CBT is 1:1.2; the amide condensation reaction is carried out in the presence of N-methylmorpholine and isobutyl chloroformate; the amide condensation reaction is a room temperature reaction for 15 - 24 hours.

[0021] In the above technical solution, in step (2), the deprotection of compound 2 is carried out in a mixed solvent of N,N-dimethylformamide / piperidine; the volume ratio of N,N-dimethylformamide to piperidine is 4:1.

[0022] In the above technical solution, in step (3), the molar ratio of compound 3 to N-fluorenylmethoxycarbonyl-S-tert-butylthio-L-cysteine is 1:1.2; the amide condensation reaction is carried out in the presence of 1-hydroxybenzotriazole, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate and diisopropylethylamine; the amide condensation reaction is a room temperature reaction for 2 - 4 hours.

[0023] In the above technical solution, in step (4), the deprotection of compound 4 is carried out in a mixed solvent of dichloromethane / trifluoroacetic acid; the volume ratio of dichloromethane to trifluoroacetic acid is 4:1.

[0024] In the above technical solution, in step (5), the molar ratio of compound 5 to the photosensitizer is 1.1:1; the photosensitizer is NHS-activated chlorin e6 (Ce6-NHS).

[0025] In the above technical solution, in step (6), the deprotection of compound 6 is carried out in a mixed solvent of N,N-dimethylformamide / piperidine; the volume ratio of N,N-dimethylformamide to piperidine is 4:1.

[0026] In the above technical solution, in step (7), the molar ratio of compound 7 to N-Boc-L-leucine is 1:1.2; the amide condensation reaction is carried out in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and diisopropylethylamine; the amide condensation reaction is a room temperature reaction for 8 to 12 hours.

[0027] In the above technical solution, in step (8), the deprotection of compound 8 is carried out in a dichloromethane / trifluoroacetic acid mixed solvent; the volume ratio of dichloromethane to trifluoroacetic acid is 4:1.

[0028] In the above technical solution, the chemical structural formulas of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, and compound 8 are as follows:

[0029]

[0030]

[0031]

[0032] The chemical structural formula of the NHS-activated photosensitizer chlorin e6 is as follows:

[0033]

[0034] In the present invention, after the deprotection of compound 8, it is separated and purified by semi-preparative high performance liquid chromatography to obtain a leucine aminopeptidase and glutathione dual-stimuli responsive probe. The product is a dark green solid powder, which is a conventional technique. Preferably, the high performance liquid chromatography separation method is: C18 column, 3.5 μm, 4.6×100 mm; mobile phase: A is water; B is acetonitrile; flow rate: 3 mL / min; linear gradient elution program: 0 min, A:B = 95:5; 13 min, A:B = 0:100.

[0035] The probe of the present invention is dual-stimulated by overexpressed leucine aminopeptidase and glutathione in the tumor microenvironment, so that the nanoparticle probe is reassembled into nanofibers, realizing the recovery of the fluorescence and ROS generation ability of the probe, thereby achieving specific fluorescence imaging and photodynamic therapy of tumors.

[0036] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0037] (1) In the present invention, 2-cyanobenzothiazole undergoes a rapid and efficient click condensation reaction with 1,2-aminothiol to form an amphiphilic dimer, and the nanoparticles are reassembled into nanofibers through the change of intermolecular forces.

[0038] (2) When the probe enters the tumor cells, under the stimulation of leucine aminopeptidase and glutathione overexpressed in the tumor cells, the original amino and mercapto groups in the cysteine structure are exposed, thus undergoing a click condensation reaction with the cyano group of 2-cyanobenzothiazole (CBT), and it is not affected by the external environment.

[0039] (3) The diagnostic and therapeutic functions of this tumor microenvironment-responsive intelligent probe can only be activated under the trigger of a special tumor microenvironment. Even if it is intercepted by normal tissues, its diagnostic and therapeutic functions will not be activated, so it will not interfere with the diagnosis and treatment of cancer. Therefore, the tumor microenvironment-responsive intelligent diagnostic and therapeutic reagent can effectively improve the accuracy of cancer diagnosis and the treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is the synthesis flow chart of the leucine aminopeptidase and glutathione dual-stimulus-responsive probe Ce6-Leu and the control probe Ce6-Ac in Example 1;

[0041] Figure 2 It is the high-resolution mass spectrometry characterization of the leucine aminopeptidase and glutathione dual-stimulus-responsive probe Ce6-Leu (a) and the control probe Ce6-Ac (b) in Example 2;

[0042] Figure 3 It is the TEM images of the leucine aminopeptidase and glutathione dual-stimulus-responsive probe Ce6-Leu before and after the reaction in the leucine aminopeptidase and glutathione solution in Example 3;

[0043] Figure 4 It is the change of ultraviolet absorption and fluorescence spectra of the leucine aminopeptidase and glutathione dual-stimulus-responsive probe Ce6-Leu before and after the reaction in the leucine aminopeptidase and glutathione solution in Example 4, a is the change of ultraviolet absorption, and b is the change of fluorescence signal;

[0044] Figure 5 It is the change of the ability to generate ROS of the leucine aminopeptidase and glutathione dual-stimulus-responsive probe Ce6-Leu before and after the reaction in the leucine aminopeptidase and glutathione solution in Example 5;

[0045] Figure 6 It is the change of the fluorescence intensity of the leucine aminopeptidase and glutathione dual-stimulus-responsive probe Ce6-Leu and the control probe Ce6-Ac in the tumor cells in Example 6;

[0046] Figure 7 Comparison of the ability of the dual-stimuli-responsive probes Ce6-Leu (leucine aminopeptidase and glutathione dual-stimuli-responsive probe) and the control probe Ce6-Ac to generate ROS in tumor cells in Example 7;

[0047] Figure 8 Comparison of the ability of the dual-stimuli-responsive probes Ce6-Leu (leucine aminopeptidase and glutathione dual-stimuli-responsive probe) and the control probe Ce6-Ac to photodynamically kill tumor cells in Example 8

[0048] Figure 9 Fluorescence imaging photographs (a) and corresponding fluorescence signal values (b) of mice after tail vein injection of the dual-stimuli-responsive probes Ce6-Leu (leucine aminopeptidase and glutathione dual-stimuli-responsive probe) and the control probe Ce6-Ac at different times in Example 9;

[0049] Figure 10 Study on the photodynamic therapy effect of the dual-stimuli-responsive probes Ce6-Leu (leucine aminopeptidase and glutathione dual-stimuli-responsive probe) and the control probe Ce6-Ac in Example 10

[0050] Figure 11 Comparison of the survival times of mice in each group. Detailed implementation manners

[0051] The present invention has developed a dual-responsive intelligent molecular probe for leucine aminopeptidase and glutathione that integrates fluorescence imaging and photodynamic therapy, which has great research and application value. This contrast agent can reassemble from spherical nanoparticles into nanofibers under the stimulation of the tumor microenvironment, and the fluorescence and the ability to generate ROS are restored, thereby realizing specific fluorescence imaging and photodynamic therapy of solid tumors.

[0052] Specifically, the method provided by the present invention comprises the following steps:

[0053] (1) Construct and synthesize the dual-stimuli-responsive probe:

[0054] According to the designed synthesis steps: First, compound 1 undergoes an amide condensation reaction with NH2-CBT, and then the protecting group Fmoc is removed using 20% piperidine (N,N-dimethylformamide:piperidine = 4:1, v / v); then it undergoes an amide condensation reaction with N-fluorenylmethoxycarbonyl-S-tert-butylthio-L-cysteine, and subsequently the Boc protecting group of the intermediate compound is removed using 20% trifluoroacetic acid (dichloromethane:trifluoroacetic acid = 4:1, v / v); then it reacts with the photosensitizer chlorin e6 that has been activated with NHS. The resulting intermediate compound is then treated with 20% piperidine (N,N-dimethylformamide:piperidine = 4:1, v / v) to remove the protecting group Fmoc. The resulting intermediate reacts with N-tert-butoxycarbonyl-L-leucine to obtain the product, and then the Boc protecting group is removed using 20% trifluoroacetic acid (dichloromethane:trifluoroacetic acid = 4:1, v / v) to obtain the final probe Ce6-Leu. The structural formula is as follows:

[0055]

[0056] (2)Response of the leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu in tumor cells:

[0057] The leucine aminopeptidase and glutathione dual-stimuli responsive probe obtained in step (1) is dissolved in cell culture medium and added to a HepG2 cell culture dish (concentration: 20 μM), and then placed in an incubator for cultivation. Under the stimulation of overexpressed leucine aminopeptidase and glutathione in tumor cells, the molecular probe reassembles into nanofibers through an intermolecular CBT condensation reaction, completing the response of the near-infrared molecular probe in tumor cells and restoring fluorescence, which is beneficial for realizing specific imaging of tumor cells.

[0058] (3)Responsive fluorescence imaging of the leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu in solid tumors:

[0059] The leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu obtained in step (1) is dissolved in PBS buffer (concentration: 100 μM) to self-assemble into nanoparticles, and then the probe is injected into female nude mice bearing HepG2 liver cancer tumors via tail vein injection. After the nanoparticles circulate to the tumor site, they selectively respond to leucine aminopeptidase and glutathione in tumor cells and reassemble into nanofibers through an intermolecular CBT condensation reaction, completing the responsive fluorescence imaging of the near-infrared molecular probe in tumors, thereby realizing specific fluorescence imaging of in vivo tumors.

[0060] (4)Leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu for photodynamic therapy of tumors in vivo:

[0061] Dissolve the leucine aminopeptidase and glutathione dual-stimuli responsive probe obtained in step (1) in PBS buffer (concentration: 200 μM), and inject the probe into female nude mice bearing HepG2 liver cancer tumors by tail vein injection. After the probe circulates to the tumor site and responds to enzymes and glutathione, it selectively reassembles into nanofibers in tumor cells through intermolecular CBT condensation reaction, completing the response of the near-infrared molecular probe in the tumor, the fluorescence gradually recovers, and the ability to generate ROS gradually becomes stronger. When the fluorescence signal of the probe reaches the strongest at the tumor site, after irradiating with a 660 nm laser (0.15 W / cm 2 ) for 10 minutes, the treatment of tumors with near-infrared small molecule photosensitizers is completed.

[0062] The present invention will be further elaborated below in conjunction with the drawings and specific embodiments. It should be understood that these embodiments are only used to explain and illustrate the technical solutions in the present invention, and are not intended to limit the scope of the present invention. In addition, unless otherwise specified, the materials, reagents, instruments, etc. used in the following embodiments can be obtained by commercial means.

[0063] Example 1: Synthesis and characterization of leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu and control probe Ce6-Ac

[0064] (1) Dissolve compound 1 (400 mg, 0.85 mmol) in 10 mL of tetrahydrofuran, then dropwise add N-methylmorphine (130 mg, 1.28 mmol), and then place the round-bottom flask in an ice-salt bath and cool to 0 o °C. Subsequently, dropwise add isobutyl chloroformate (175 mg, 1.28 mmol), activate for half an hour, and then add 2-amino-6-cyanobenzothiazole (NH2-CBT, 179 mg, 1.00 mmol) dissolved in dry tetrahydrofuran, and keep the reaction at 0 o °C for 1 hour, and then stir at room temperature overnight. After the reaction is completed, evaporate the solvent by a rotary evaporator, then redissolve the residual solid in ethyl acetate (50 mL), and extract it three times with an aqueous solution of sodium bicarbonate. The organic phase is dried with Na2SO4 and then filtered by suction, and the solvent is evaporated. Using petroleum ether (PE) and ethyl acetate (EA) = 2:1 by volume as the eluent, purify the crude product with a silica gel chromatography column to obtain intermediate 1 (the structure is as shown in Figure 1 compound 2 therein) (531.88 mg, yield: 85%); 1¹H NMR (600 MHz, Methanol-d4) δ 8.59 (s,1H), 8.04 (d, J J = 9.0 Hz, 1H), 7.74 (d, J J = 7.6 Hz, 2H), 7.63 (dt, J J = 14.8,5.0 Hz, 3H), 7.33 (t, J J = 7.5 Hz, 2H), 7.26 (t, J J = 7.7 Hz, 2H), 4.36 (d, J J =6.9 Hz, 2H), 4.24 (dd, J J = 9.0, 5.4 Hz, 1H), 4.17 (t, J J = 7.0 Hz, 1H), 3.02(dt, J J = 9.8, 4.5 Hz, 2H), 1.83 (dp, J J = 15.1, 5.4 Hz, 1H), 1.73 (tq, J J =11.7, 7.9, 6.3 Hz, 1H), 1.48 (p, J J = 9.4, 8.3 Hz, 2H), 1.37 (s, 9H), 1.30–1.18 (m, 2H). 13 ¹³C NMR (151 MHz, Methanol-d4) δ 172.33, 157.18, 148.38, 143.84,143.70, 141.80, 141.14, 139.09, 136.58, 135.25, 127.33, 126.72, 124.76,124.49, 120.82, 119.49, 112.63, 111.40, 78.44, 66.51, 55.84, 47.00, 39.57,31.51, 29.20, 27.36, 22.80;

[0065] (2)Dissolve intermediate 1 (500 mg, 0.80 mmol) in 8 mL of DMF, then place the reaction flask in an ice-water bath, and subsequently add dropwise 2 mL of piperidine, and keep the reaction at 0 o °C for 5 minutes. After the reaction is completed, remove the solvent and piperidine by rotary evaporation. Use a silica gel chromatography column to purify the crude product with an eluent of dichloromethane (DCM) and methanol (MeOH) in a volume ratio of 80:1 to obtain intermediate 2 (whose structure is asFigure 1 The compound shown in Compound 3) (290.18 mg, yield: 90%); 1 H NMR (600 MHz, Methanol-d4) δ 8.59–8.45 (m, 1H), 8.07–7.93 (m, 1H), 7.73–7.54 (m, 1H), 3.47 (q, J J = 6.7 Hz, 1H), 3.06–2.97 (m, 2H), 1.85–1.58 (m, 2H), 1.49 (h, J J = 7.1 Hz, 3H),1.37 (d, J J = 5.9 Hz, 9H), 1.26 (d, J J = 3.5 Hz, 1H). 13 C NMR (151 MHz, Methanol-d4) δ 174.81, 161.86, 157.07, 149.04, 141.79, 137.45, 137.07,123.84, 119.90, 112.02, 78.37, 55.30, 39.62, 34.65, 29.43, 27.32, 22.52;

[0066] (3) Add intermediate 2 (250 mg, 0.62 mmol) to a 20 mL round-bottom flask, then dissolve it with dry DMF. Subsequently, add HBTU (282.15 mg, 0.74 mmol), HOBT (100.44 mg, 0.74 mmol) and DIPEA (213.68 μL). After stirring for 15 minutes, add compound N-Fmoc-S-tert-butylthio-L-cysteine (321.04 mg, 0.74 mmol). Continue stirring and react at room temperature for 2 hours. After the reaction is completed, remove the solvent by rotary evaporation, then add 25 mL of ethyl acetate to redissolve the crude product. Subsequently, wash the organic phase with 25 mL of ultrapure water, saturated sodium bicarbonate, and sodium chloride aqueous solution once each. Dry the organic phase with anhydrous sodium sulfate and then remove the solvent by rotary evaporation. Use a silica gel chromatography column to purify the crude product with a volume ratio of petroleum ether (PE) and ethyl acetate (EA) = 2:1 as the eluent to obtain intermediate 3 (the structure of which is as shown in Figure 1 Compound 4 in) (405.26 mg, yield: 80%); 1 H NMR (600 MHz, Methanol-d4) δ 8.37 (d, J J = 2.1 Hz,1H), 7.84 (d, J= 9.0 Hz, 1H), 7.71 (dd, J = 9.0, 2.1 Hz, 1H), 7.58 (d, J =7.5 Hz, 1H), 7.52 (dd, J = 11.0, 7.6 Hz, 2H), 7.45 (d, J = 7.5 Hz, 1H), 7.38–7.27 (m, 1H), 7.22 (q, J = 7.1 Hz, 2H), 7.16 (t, J = 7.4 Hz, 1H), 4.52 (dd, J = 9.9, 4.5 Hz, 1H), 4.42 (t, J = 7.3 Hz, 1H), 4.28 (qd, J = 10.6, 7.5 Hz,2H), 4.08 (q, J = 7.1 Hz, 1H), 3.18–3.02 (m, 2H), 3.02 (s, 2H), 1.99 (s, 1H),1.77–1.68 (m, 1H), 1.53–1.42 (m, 2H), 1.38 (s, 9H), 1.34 (s, 11H). 13 C NMR(151 MHz, Methanol-d4) δ 172.13, 171.21, 157.15, 157.05, 148.27, 143.49,143.32, 141.02, 140.78, 138.75, 136.27, 135.19, 127.25, 126.78, 124.74,124.18, 120.92, 119.34, 112.64, 111.50, 78.43, 66.92, 55.08, 54.09, 46.76,40.65, 39.75, 37.46, 30.87, 29.03, 28.86, 27.39, 22.96. MS (ESI) Calcd for:C 41 H 48 N6O6S3 ([M+H] + ) : 817.2800, found: 817.2865;

[0067] (4) Add intermediate 3 to a 20 mL DMF solution containing 20% (volume ratio) trifluoroacetic acid. After reacting at room temperature for 1 hour, remove the solvent and trifluoroacetic acid by rotary evaporation to obtain intermediate 4 (whose structure is as shown in Figure 1 compound 5 in). Intermediate 4 is not further purified. Accurately weigh 40 mg of intermediate 4 (0.0558 mmol), dissolve it in 20 mL of anhydrous DMF solution until clear, then add 45.71 mg of Ce6-NHS (0.05 mmol) and 7.76 mg of DIPEA (0.06 mmol). After stirring at room temperature for 2 hours, separate and purify by HPLC, and collect the fraction with an absorption spectrum at 400 nm to obtain intermediate 5 (whose structure is as shown in Figure 1 compound 6 in) (45.9 mg, yield: 71%); MS (MALDI-TOF) Calcd for: C 70 H 75 N 10 O9S3 ([M+H] + ): 1295.48, found: 1295.736;

[0068] (5) Dissolve intermediate 5 (45 mg, 0.035 mmol) in 8 mL of DMF, then place the reaction flask in an ice-water bath, and then dropwise add 2 mL of piperidine, maintaining a reaction at 0 o °C for 5 minutes. After the reaction is completed, separate and purify by HPLC, and collect the fraction with an absorption spectrum at 400 nm to obtain intermediate 6 (whose structure is as shown in Figure 1 compound 7 in) (26.3 mg, yield: 70%); MS (MALDI-TOF) Calcd for: C 55 H 65 N 10 O7S3 ([M+H] + ): 1073.36, found: 1073.687;

[0069] (6) Add intermediate 6 (26 mg, 0.025 mmol) to a 10 mL round-bottom flask, dissolve it in 5 mL of anhydrous DMF until clear, then add NHS-activated N-Boc-L-leucine (9.85 mg, 0.03 mmol). After reacting at room temperature for 2 hours, remove the solvent by rotary evaporation, and use semi-preparative high-performance liquid chromatography for separation and purification to obtain intermediate 7 (whose structure is as shown in Figure 1 compound 8 in) (19.3 mg, yield: 60%); MS (MALDI-TOF) Calcd for: C 66 H 84 N 11 O10 S3 ([M+H] + ): 1286.64, found: 1286.896;

[0070] (7) Add intermediate 7 (19 mg, 0.015 mmol) to 5 mL of dichloromethane solution containing 20% trifluoroacetic acid. After reacting at room temperature for 1 hour, remove the solvent and trifluoroacetic acid by rotary evaporation. Use semi-preparative high-performance liquid chromatography for separation and purification to obtain the experimental group probe Ce6-Leu (12.25 mg, yield: 70%); MS (MALDI-TOF) Calcd for: C 61 H 76 N 11 O8S3([M+H] + ): 1186.520, found: 1186.788。

[0071] (8) Add intermediate 6 (26 mg, 0.025 mmol) to a 10 mL round-bottom flask, dissolve it with 5 mL of anhydrous DMF, then add acetic anhydride (3.06 mg, 0.03 mmol). After stirring at room temperature for 2 hours, remove the solvent by rotary evaporation. Purify and separate by HPLC to obtain the control group probe Ce6-Ac (22.3 mg, yield: 80%); MS (MALDI-TOF) Calcd for: C 57 H 67 N 10 O8S3 ([M+H] + ): 1115.40, found: 1115.525。

[0072] Example 2: High-resolution mass spectrometry characterization of the leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu and the control probe Ce6-Ac

[0073] Dilute the leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu and the control probe Ce6-Ac prepared in Example 1 with methanol to a concentration of 5 μM, and then determine the molecular weight of the probe by high-resolution mass spectrometry.

[0074] As Figure 2 shown in a, the theoretical m / z of the leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu is 1186.520, and the m / z obtained from the actual high-resolution mass spectrometry spectrum is 1186.788. The two are in agreement, indicating the desired compound; Figure 2b shows that the theoretical m / z of the control probe Ce6-Ac is 1115.40, and the m / z obtained from the high-resolution mass spectrometry is 1115.525, which are in agreement, and the compound is the desired one.

[0075] Example 3: Reassembly of the dual-stimuli-responsive probe Ce6-Leu from nanoparticles into nanofibers mediated by leucine aminopeptidase and glutathione

[0076] The dual-stimuli-responsive probe Ce6-Leu prepared in Example 1 was added to a PBS buffer solution containing 10 mM glutathione and 50 U / mL leucine aminopeptidase, and reacted at 37 °C for 24 hours. The morphological changes of the probe before and after the reaction were observed by TEM.

[0077] As Figure 3 shown in a, the pure probe Ce6-Leu forms nanoparticles with a particle size of approximately 79.8 ± 9.3 nm in PBS solution, and reassembles into nanofibers (3b) under the stimulation of leucine aminopeptidase and glutathione.

[0078] Example 4: Changes in the ultraviolet absorption spectra and fluorescence spectra of the dual-stimuli-responsive probe Ce6-Leu and the control probe Ce6-Ac before and after reaction in leucine aminopeptidase and glutathione solutions

[0079] The dual-stimuli-responsive probe Ce6-Leu and the control probe Ce6-Ac prepared in Example 1 were respectively added to the systems of PBS (pH = 7.2) and PBS (50 U / mL LAP enzyme, 10 mM GSH) to make the final concentration of the probe 20 μM, and then placed in an oscillator at 37 °C for 24 hours. The ultraviolet absorption spectra and fluorescence spectra of the probes were measured using an ultraviolet-visible absorption spectrometer and a steady-state / transient fluorescence spectrometer, respectively.

[0080] As Figure 4 shown, the probe Ce6-Leu undergoes a CBT condensation reaction and reassembles into nanofibers under the dual stimulation of leucine aminopeptidase and glutathione. The distance between Ce6 structures becomes larger, the ultraviolet absorption changes from J-aggregates to monomers, the ultraviolet absorption increases and undergoes a blue shift, and the fluorescence signal is enhanced. The control probe Ce6-Ac does not have enzyme responsiveness and cannot undergo a condensation reaction. Even in the PBS (50 U / mL LAP enzyme, 10 mM GSH) solution, it still exists in the form of aggregated nanoparticles, so there are no obvious changes in the ultraviolet absorption and fluorescence signals.

[0081] Example 5: Changes in the ability of the leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu to generate ROS before and after reaction in leucine aminopeptidase and glutathione solutions

[0082] The leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu prepared in Example 1 was added to systems of PBS (pH = 7.2) and PBS (50 U / mL LAP enzyme, 10 mM GSH) respectively, such that the final concentration of the probe was 20 μM, and then it was placed in an oscillator at 37 o °C and reacted for 24 hours. After the reaction ended, the singlet oxygen indicator ABDA was added to the solution, and then it was irradiated with a 660 nm laser (0.15 W / cm 2 ²). After ABDA was oxidized by singlet oxygen, its ultraviolet absorption peak at 380 nm would disappear. Therefore, it could be detected using a UV-visible spectrophotometer.

[0083] As Figure 5 shown, compared with the PBS solution of the original probe Ce6-Leu (5a), the ability of the solution to generate singlet oxygen became significantly stronger after the response (5b). This was because during the reassembly process of the nanoparticles, the distance between the Ce6 structures became larger, and the quenching between them weakened, thus restoring the ability to generate singlet oxygen.

[0084] Example 6: Changes in the fluorescence intensity of the leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu and the control probe Ce6-Ac in tumor cells HepG2

[0085] The HepG2 cells in the logarithmic growth phase were digested, centrifuged, and after discarding the supernatant, fresh medium was added again and blown into a cell suspension. After the cells were counted, they were inoculated into 8-well confocal dishes, with 5000 HepG2 cells added to each well. Subsequently, the 8-well confocal dishes were placed in an incubator and cultured for another 24 hours. After 24 hours, the original medium was discarded, and 200 μL of the probes Ce6-Leu and Ce6-Ac (20 μM) dissolved in medium were added to each well respectively, and then placed in the incubator and cultured for different times (1 h, 2 h, 4 h, 8 h, 12 h). After the culture ended, the original medium in the wells was discarded, and it was washed three times with PBS, shaken for 5 minutes each time (to wash away the unreacted materials). Then the nuclei were stained with Hoechst33342 solution and incubated at room temperature in the dark for 15 minutes, and then washed with PBS again. Finally, fresh medium was added and the fluorescence in HepG2 cells was observed using a laser confocal microscope.

[0086] As Figure 6As shown, within different incubation times, the fluorescence signal of the probe Ce6-Leu in HepG2 cells was significantly stronger than that of Ce6-Ac in HepG2 cells, indicating that the ability of the experimental group probe to undergo responsive condensation and reassembly in tumor cells was significantly superior to that of the control group probe, and thus the fluorescence was significantly stronger than that of the control group.

[0087] Example 7: Comparison of the ability of the leucine aminopeptidase- and glutathione-dual-stimuli-responsive probe Ce6-Leu and the control probe Ce6-Ac to generate ROS in tumor cells

[0088] Take HepG2 cells in the logarithmic growth phase. After discarding the original medium, digest them with trypsin, centrifuge, add fresh medium and pipette them into a suspension. Then count them with a counting plate and inoculate them into confocal dishes, with the final number of cells per well being 4×10 5 , and continue to culture them in an incubator for 12 hours. After 12 hours, discard the original medium, and then add 1.5 mL of medium solutions of the Ce6-Leu and Ce6-Ac probes with the same concentration (20 μM), and set up a blank control group (control group: no probe added), and place them in the incubator and continue to incubate for 8 hours. When the incubation time ends, discard the original medium, wash 3 times with PBS for 5 minutes each time, and then add 1.5 mL of fresh medium containing the ROS indicator DCFH-DA to each well, and continue to culture for half an hour. When the incubation time ends, discard the original medium, wash with PBS, and then add 1.5 mL of fresh medium to each well. Subsequently, irradiate them with a 660 nm laser for 5 minutes (control group + λ 660 nm : no probe added, irradiated with a 660 nm laser alone; Ce6-Leu + λ 660 nm : after incubating with the Ce6-Leu probe for 8 hours, irradiated with a 660 nm laser; Ce6-Ac + λ 660 nm : after incubating with the Ce6-Ac probe for 8 hours, irradiated with a 660 nm laser), and the laser power is 0.15 W / cm 2 , and at the same time set up experimental control groups (control group: no probe added, no 660 nm laser irradiation; Ce6-Leu: incubated with the Ce6-Leu probe for 8 hours, but no 660 nm laser irradiation; Ce6-Ac: incubated with the Ce6-Ac probe for 8 hours, but no 660 nm laser irradiation). The above 6 groups of cells that have undergone different experimental operations (control group, Ce6-Ac, Ce6-Leu, control group + λ 660 nm , Ce6-Ac + λ 660 nm , Ce6-Leu + λ 660 nm)The nuclei were stained with Hoechst 33342 solution and incubated in the dark at room temperature for 15 minutes. Then, they were washed three times with PBS, shaken for 5 minutes each time. Finally, fresh medium was added, and the green fluorescence (DCF) in HepG2 cells was observed using a laser confocal microscope. DCFH-DA, as a fluorescent probe for detecting reactive oxygen species, has no fluorescence itself and can freely cross the cell membrane. After entering the cell, it will be hydrolyzed by intracellular esterase into DCFH. DCFH cannot penetrate the cell membrane. In the presence of reactive oxygen species, DCFH is oxidized to the fluorescent substance DCF, and the intensity of the green fluorescence is proportional to the level of reactive oxygen species in the cell.

[0089] As Figure 7 shown, compared with the control group cells, although the cells in the control group + λ 660 nm group were irradiated with 660 nm laser, no ROS was generated in the cells, indicating that simple 660 nm laser irradiation could not induce ROS production in cells. In addition, incubating cells with pure Ce6-Ac and Ce6-Leu alone could not induce ROS production in cells. Comparing the cells in the Ce6-Ac + λ 660 nm group, only a small amount of ROS was observed in the cells, indicating that the Ce6-Ac probe still existed in an aggregated state in the cells, so the amount of ROS produced in the cells was very small. Compared with the cells in the Ce6-Leu + λ 660 nm group, the Ce6-Leu probe has the ability of responsive self-assembly. Stimulated by overexpressed leucine aminopeptidase and glutathione in tumor cells, the probe can undergo an intermolecular condensation reaction and reassemble into nanofibers. The distance between Ce6 structures becomes larger, and the quenching between them weakens, so the ability to generate singlet oxygen is restored. Therefore, after irradiation with 660 nm laser, a large amount of ROS is produced in the cells, and obvious green fluorescence (DCF) can be detected.

[0090] Example 8: Comparison of the ability of the leucine aminopeptidase and glutathione dual-stimuli responsive probe Ce6-Leu and the control probe Ce6-Ac to kill tumor cells by photodynamic therapy

[0091] HepG2 cells in the logarithmic growth phase were taken. After discarding the original medium, they were digested with trypsin, centrifuged, resuspended in fresh medium after adding it, then counted using a counting plate and seeded in a 12-well plate. Finally, the number of cells in each well was 4×10 5, continue to incubate in the incubator for 12 hours. After 12 hours, discard the original culture medium, and then add 1 mL of the culture medium solution of Ce6-Leu and Ce6-Ac probes with the same concentration (20 μM). Set up a blank control group (control group: no probe added), and place it in the incubator to continue incubating for 8 hours. After the incubation time ends, discard the original culture medium, wash it 3 times with PBS for 5 minutes each time, then add 1 mL of fresh culture medium to each well, and irradiate it with a 660 nm laser for 5 minutes (control group + λ 660 nm : no probe added, irradiated with a 660 nm laser alone; Ce6-Ac + λ 660 nm : after incubating with the Ce6-Ac probe for 8 hours, irradiate with a 660 nm laser; Ce6-Leu + λ 660 nm : after incubating with the Ce6-Leu probe for 8 hours, irradiate with a 660 nm laser), and the laser power is 0.15 W / cm 2 . At the same time, set up an experimental control group (control group: no probe added, no 660 nm laser irradiation; Ce6-Ac: incubated with the Ce6-Ac probe for 8 hours, but no 660 nm laser irradiation; Ce6-Leu: after incubating with the Ce6-Leu probe for 8 hours, but no 660 nm laser irradiation). Place the above 6 groups of cells (control group, control group + λ 660 nm , Ce6-Ac, Ce6-Ac + λ 660 nm , Ce6-Leu, Ce6-Leu + λ 660 nm ) that have undergone different experimental operations into the incubator to continue culturing for 24 hours. Then, collect the culture medium and cells respectively, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend the cells with PBS and centrifuge, take the precipitate, repeat this operation twice, and then blow the cells into a single-cell suspension. Add Annexin V-FITC / PI double-staining apoptosis detection solution (for specific operations, see the kit instructions). After the culture ends, use a flow cytometer to detect the apoptosis of tumor cells.

[0092] As Figure 8 shown, compared with the cells in the control group, the cells in the control group + λ 660 nm Although irradiated with a 660 nm laser, the survival of the cells is hardly affected, indicating that the simple 660 nm laser irradiation has little effect on cell viability. In addition, incubating Ce6-Ac and Ce6-Leu with cells for 8 hours hardly affects cell viability, indicating that the two probes have little toxicity to cells at the experimental concentration and will not cause cell death. Then, compare Ce6-Ac + λ 660 nmFor the cells, only a small amount of cell death was observed, indicating that the Ce6-Ac probe still existed in an aggregated form inside the cells, could only generate trace amounts of ROS, and could not achieve the goal of killing tumor cells. Compared with Ce6-Leu + λ 660 nm For the cells, the Ce6-Leu probe has the ability of responsive self-assembly. In the microenvironment of tumor cells, the probe can undergo an intermolecular condensation reaction and reassemble into nanofibers under the stimulation of leucine aminopeptidase and glutathione. The distance between Ce6 structures becomes larger, and the quenching between them weakens, so the ability to generate singlet oxygen is restored. Therefore, after irradiation with a 660 nm laser, a large amount of ROS is generated inside the cells, resulting in the death of a large number of cells.

[0093] Example 9: Fluorescence imaging photos of mice and corresponding fluorescence signal values at different time points after intravenous injection of the dual-stimulus-responsive probe Ce6-Leu and the control probe Ce6-Ac

[0094] Two groups of female nude mice bearing HepG2 tumors (3 mice in each group) were taken. After gas anesthesia, they were placed in a small animal IVIS Lumina XRMS in vivo imaging system for mouse autofluorescence imaging. Then, one group (experimental group) of mice was injected with 200 μL of a PBS buffer solution containing 100 μM Ce6-Leu via the tail vein, and the other group (control group) of mice was injected with 200 μL of a PBS buffer solution containing 100 μM Ce6-Ac via the tail vein. After that, the two groups of mice were placed in the above-mentioned small animal in vivo imaging system for imaging at different time points (1, 2, 3, 6, 10, 12, and 24 h). After imaging, image processing was performed using the IVIS in vivo imaging analysis software, and the fluorescence intensity of the tumor sites of the two groups of mice at each time point was calculated.

[0095] As Figure 9 shown, the probe Ce6-Leu has a good fluorescence enhancement effect in mouse tumors. The fluorescence imaging image (a) shows that the peak of the fluorescence signal is reached at 3 hours, and Figure 9 (b) shows that the signal intensity of the experimental group (Ce6-Leu) is significantly greater than that of the control group (Ce6-Ac). This indicates that the probe Ce6-Leu has good responsiveness to the tumor microenvironment and can undergo a responsive intermolecular condensation reaction and reassemble into nanofibers under the stimulation of leucine aminopeptidase and glutathione overexpressed in tumor cells. The distance between Ce6 structures becomes larger, and the quenching between them weakens, resulting in fluorescence recovery.

[0096] Example 10: Study on the in vivo tumor photodynamic therapy of the dual-stimulus-responsive probe Ce6-Leu and the control probe Ce6-Ac

[0097] Thirty-six female nude mice weighing about 18 g and bearing HepG2 tumors in their hind legs were divided into 6 groups (6 mice in each group), namely Group 1, Group 2, Group 3, Group 4, Group 5, and Group 6:

[0098] Group 1: mice were injected with 200 μL PBS solution through the tail vein;

[0099] Group 2: Mice were injected with 200 μL PBS solution through the tail vein. Three hours after the injection, the power was 0.15 W / cm 2 The 660 nm laser was irradiated at the tumor site for 10 minutes;

[0100] Group 3: Mice were injected with 200 μL of PBS buffer solution containing 200 μM Ce6-Ac via tail vein injection;

[0101] Group 4: The mice were injected with 200 μL of PBS buffer solution with a concentration of 200 μM Ce6-Ac through the tail vein. Three hours after the injection, the probe Ce6-Ac was injected with a power of 0.15 W / cm 2 The 660 nm laser was irradiated at the tumor site for 10 minutes;

[0102] Group 5: The mice were injected with 200 μL of PBS buffer solution containing 200 μM probe Ce6-Leu via tail vein injection;

[0103] Group 6: The mice were injected with 200 μL of PBS buffer solution with a concentration of 200 μM Ce6-Leu via tail vein injection. Three hours after the injection, the cells were injected with a power of 0.15 W / cm 2 The 660 nm laser was irradiated at the tumor site for 10 minutes;

[0104] Before photodynamic therapy, mice were anesthetized by intraperitoneal injection of 120 μL of 4% chloral hydrate aqueous solution. After the treatment, the six groups of mice were placed under the same conditions for feeding. After 24 hours, one mouse from each group was taken to remove the tumor, sliced, stained with HE and TUNEL, and observed for apoptosis of tumor cells. The mice were then photographed and observed every two days, and the mice were weighed and the tumor volume was measured (tumor volume Vm = length × width 2 / 2), after 14 days of observation, the mice were euthanized and the tumors were removed and photographed.

[0105] like Figure 10 The HE and TUNEL staining results in a were shown.2 ) The apoptosis of tumor cells in the mice injected with the probe Ce6-Leu (Group6) was significantly higher than that in the mice injected with the Ce6-Ac probe (Group4) after being irradiated with 660 nm laser for the same time (10 minutes). However, no obvious apoptosis was observed in the tumor cells of Group1, Group2, Group3, and Group5. The experimental results demonstrated that the probe Ce6-Leu had good responsiveness at the tumor site and could effectively treat tumors through its excellent ROS generation ability. From Figure 10 b, it can be seen that the body weights of the six groups of mice all increased slightly during the observation period but showed no obvious differences, indicating that the probe had low toxicity and the photodynamic therapy caused little damage, without causing damage to the mouse body. And from Figure 10 d, it can be seen that in the excised tumors of the mice 14 days after treatment, the tumors of the control group, control group + λ 660 nm , Ce6-Ac, Ce6-Ac + λ 660 nm , and Ce6-Leu still existed and had relatively large volumes, while in the Ce6-Leu + λ 660 nm group, most of the tumors of the mice were well cured. Even if there was recurrence, it was significantly smaller compared with the other five groups, which was consistent with Figure 10 the results of the in-vivo tumor measurement and the photos of the mice taken in c and 10e. Figure 11 Figure shows the comparison of the survival times of the mice in each group. The above experiments all verified that the probe Ce6-Leu was an excellent photodynamic therapy reagent.

[0106] The present invention belongs to the technical field of tumor microenvironment-mediated reassembly, and relates to a dual-stimuli responsive probe responsive to leucine aminopeptidase and glutathione, and a preparation method and application thereof. It mainly uses leucine aminopeptidase and glutathione overexpressed in solid tumors to stimulate the reassembly of spherical nanoparticles into nanofibers, so as to improve the specificity and accuracy of the probe in tumor cell and in vivo imaging detection, realize specific fluorescence imaging of tumors, and effectively improve the photodynamic therapy effect of tumors. As an emerging tumor diagnosis and treatment strategy that combines the diagnosis, monitoring and treatment of diseases, the integration of diagnosis and treatment brings new hope for humans to overcome cancer; and the advantages of high efficiency and low toxicity and side effects of this strategy are expected to promote the rapid development of cancer diagnosis and treatment technologies. In order to overcome the shortcomings of traditional diagnostic and therapeutic molecular probes, a tumor microenvironment-responsive near-infrared molecular probe is constructed, which uses leucine aminopeptidase and glutathione overexpressed in tumor cells to trigger a condensation reaction, and then undergoes reassembly, so that the fluorescence and the ability to generate ROS of the probe are specifically restored at the tumor site, thereby effectively improving the imaging and treatment effects of tumors. It has the following advantages: First, the condensation reaction is efficient, mild, fast and highly selective; Second, when the probe enters tumor cells, under the stimulation of leucine aminopeptidase and glutathione overexpressed in tumor cells, the original amino and mercapto groups in the cysteine structure are exposed, thus a click condensation reaction occurs, which is not affected by the external environment. Therefore, the near-infrared photosensitizer probe has further applications in the biological and other fields.

Claims

1. A leucine aminopeptidase and glutathione dual-stimulus responsive probe, characterized in that, The leucine aminopeptidase and glutathione dual-stimuli responsive probe has the following chemical structural formula: 。 2. Use of the leucine aminopeptidase and glutathione dual-stimulus responsive probe according to claim 1 in the preparation of tumor diagnosis and / or treatment reagents.

3. The use according to claim 2, characterized in that, The leucine aminopeptidase and glutathione dual-stimuli responsive probe is in the form of nanoparticles outside the tumor and nanofibers inside the tumor.

4. The use according to claim 2, characterized in that, The treatment is photodynamic therapy.

5. A preparation method of the leucine aminopeptidase and glutathione dual-stimulus responsive probe according to claim 1, characterized in that, It includes the following steps: (1) Compound 1 reacts with NH2-CBT to obtain Compound 2; (2) Compound 2 removes the protecting group to obtain Compound 3; (3) Compound 3 reacts with N-fluorenylmethoxycarbonyl-S-tert-butylthio-L-cysteine to obtain Compound 4; (4) Compound 4 removes the protecting group to obtain Compound 5; (5) Compound 5 reacts with a photosensitizer to obtain Compound 6; (6) Compound 6 removes the protecting group to obtain Compound 7; (7) Compound 7 reacts with N-tert-butoxycarbonyl-L-leucine to obtain Compound 8; (8) Compound 8 removes the protecting group to obtain the leucine aminopeptidase and glutathione dual-stimuli responsive probe; The chemical structural formulas of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, and Compound 8 are as follows: 。 6. The preparation method according to claim 5, characterized in that, The molar ratio of Compound 1 to NH2-CBT is 1:1.2; the removal of the protecting group of Compound 2 is carried out in a mixed solvent of N,N-dimethylformamide / piperidine; the molar ratio of Compound 3 to N-fluorenylmethoxycarbonyl-S-tert-butylthio-L-cysteine is 1:1.

2.

7. The preparation method according to claim 5, characterized in that, The removal of the protecting group of Compound 4 is carried out in a mixed solvent of dichloromethane / trifluoroacetic acid; the molar ratio of Compound 5 to the NHS-activated photosensitizer is 1.1:

1.

8. The preparation method according to claim 5, characterized in that, The photosensitizer is chlorin e6.

9. The preparation method according to claim 5, characterized in that, The removal of the protecting group of Compound 6 is carried out in a mixed solvent of N,N-dimethylformamide / piperidine; the molar ratio of Compound 7 to N-tert-butoxycarbonyl-L-leucine is 1:1.2; the removal of the protecting group of Compound 8 is carried out in a mixed solvent of dichloromethane / trifluoroacetic acid.

10. The preparation method according to claim 5, characterized in that, The reaction is carried out at room temperature.

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