A Caspase-3 enzyme-responsive self-assembly probe and its synthesis method and application

Abstract

The present invention discloses a Caspase-3 enzyme-responsive self-assembly probe and a synthesis method and application thereof. The magnetic resonance signal of the probe of the present invention is weak in normal tissue because the probe is a small molecule compound when it does not respond to Caspase-3. 1 The H-MRI signal is weak, while 19 The F-MRI signal is shielded due to the paramagnetic relaxation enhancement effect. After the probe responds to Caspase-3, the DEVD sequence in the probe molecule will be cut off, thereby releasing two molecules, including CF3DEVD molecules and FFFK-GdDO3A molecules. The fluorine signal of the CF3DEVD molecule will be restored due to the disappearance of the shielding effect, while the FFFK-GdDO3A molecule will undergo intermolecular self-assembly due to the hydrophilic-hydrophobic effect, which will enhance the relaxation of the GdDO3A part, thereby achieving a double change in the magnetic resonance signal.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic resonance imaging, and in particular relates to a Caspase-3 enzyme-responsive self-assembly probe and a synthesis method and application thereof. Background Art

[0002] Caspase is a cysteine ​​protease that plays a key role in cell apoptosis. Caspase-3, as one of the most important members, is considered to be a key effector of apoptosis and a biomarker for detecting apoptosis. Therefore, designing molecular imaging probes that specifically respond to it to detect apoptosis signals during tumor treatment is an effective method for evaluating the efficacy of early tumor treatment, and is also an important means for monitoring the efficacy of drug treatment and evaluating different treatment methods. Therefore, many methods for detecting enzyme activity have been developed, among which optical methods are particularly common, such as fluorescence imaging (FI) and photoacoustic imaging (PAI). However, light has limited penetration in tissues and scatters, so it cannot provide three-dimensional tissue details. In contrast, MRI can provide high-quality three-dimensional soft tissue information and provide high spatial resolution images.

[0003] Among many imaging methods, magnetic resonance imaging has become an important disease diagnosis method due to its advantages such as non-invasiveness, no radiation, high tissue penetration and spatial resolution. Traditional magnetic resonance signals are derived from protons in water in the body and can provide structural images of tissues of interest. 19 Magnetic resonance imaging has no background signal interference in vivo, high contrast, and 19 The F element has a 100% natural abundance and a gyromagnetic ratio second only to that of protons. It is a new type of magnetic resonance imaging technology with potential for clinical application. Combining the two, it is possible to use fluorine-based imaging on the basis of proton imaging. 19 Magnetic resonance imaging performs hot spot imaging of areas of interest, and its imaging results are more accurate. Summary of the invention

[0004] Based on the above prior art, the present invention provides a Caspase-3 enzyme-responsive self-assembling probe and its synthesis method and application. The probe has 19 F-MRI signal and 1 H-MRI signal, after Caspase-3 response, 19 The F signal will be restored due to the paramagnetic enhancement effect (PRE) signal. 1 The H signal will change from weak to strong due to the self-assembly performance of phenylalanine and Gd-DOTA parts, thus realizing dual-modal activity monitoring of Caspase-3 enzyme.

[0005] The technical solution adopted to achieve the above-mentioned purpose of the present invention is:

[0006] A Caspase-3 enzyme-responsive self-assembling probe, the structural formula of which is as follows:

[0007]

[0008] A method for synthesizing a Caspase-3 enzyme-responsive self-assembly probe comprises the following steps:

[0009] S1. Using dichlororesin as a solid phase carrier, a polypeptide solid phase synthesis method is used to synthesize a polypeptide solid phase compound of formula (I), and the reaction formula is as follows:

[0010]

[0011] In the solid phase synthesis method of the peptide, eight amino acids and m-ditrifluoromethylbenzoic acid are grafted in sequence, the first amino acid is Fmoc-Lys(Dde)-OH, the second to fourth amino acids are all Fmoc-Phe-OH, the fifth amino acid is Fmoc-Asp(otBu)-OH, the sixth amino acid is Fmoc-Val-OH, the seventh amino acid is Fmoc-Glu(otBu)-OH, and the eighth amino acid is Fmoc-Asp(otBu)-OH;

[0012] S2. In the presence of a base and a condensation reagent, the compound of formula (I) undergoes an amide condensation reaction with tri-tert-butyl 2,2′,2″-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacyl)triacetate to generate a compound of formula (II), and the reaction formula is as follows:

[0013]

[0014] S3. In the presence of a deradical agent, the compound of formula (II) is hydrolyzed to remove the tert-butyl group to generate a compound of formula (III), and the reaction formula is as follows:

[0015]

[0016] S4. The compound of formula (III) reacts with trivalent gadolinium ions to generate the self-assembled probe responsive to the Caspase-3 enzyme. The reaction formula is as follows:

[0017]

[0018] Furthermore, the synthesis temperature of the polypeptide solid phase synthesis method is 20-40°C and the synthesis time is 1-3h.

[0019] Furthermore, the base is N,N-diisopropylethylamine.

[0020] Furthermore, the condensation reagent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0021] Furthermore, the reaction temperature of the amide condensation reaction is 20-40° C., and the reaction time is 1-3 h.

[0022] Furthermore, the deradicalizing agent is trifluoroacetic acid.

[0023] Furthermore, the hydrolysis reaction temperature is 25-30°C and the time is 12-24h.

[0024] Furthermore, the coordination reaction is carried out at a temperature of 20-30°C and for a time of 12-24 hours.

[0025] Furthermore, the trivalent gadolinium ion is selected from gadolinium trichloride or its hydrate.

[0026] Application of a Caspase-3 enzyme-responsive self-assembled probe in the preparation of magnetic resonance dual-modality contrast agents.

[0027] Further, the dual-modal agent is 19 F-MRI contrast agents and 1 H-MRI contrast agent.

[0028] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0029] 1. The magnetic resonance signal of the probe of the present invention is weak in normal tissues because the probe is a small molecule compound when it does not respond to Caspase-3. 1 H-MRI signal is weak, while 19 The F-MRI signal is shielded due to the paramagnetic relaxation enhancement effect. After the probe responds to Caspase-3 enzyme, the DEVD sequence in the probe molecule will be cut off, thereby releasing two molecules, including CF3DEVD molecules and FFFK-GdDO3A molecules. The fluorine signal of the CF3DEVD molecule will be restored due to the disappearance of the shielding effect, and the FFFK-GdDO3A molecule will undergo intermolecular self-assembly due to the hydrophilic-hydrophobic effect, which will enhance the relaxation of the GdDO3A part, thereby achieving a double change in the magnetic resonance signal.

[0030] 2. After the probe of the present invention responds to Caspase-3 enzyme, the FFFK-GdDO3A molecules in the response part will self-assemble into nanofibers, which have stronger tissue retention effect. In addition, the relaxation of the self-assembled molecules changes by more than 2 times, and the signal changes more significantly, which can greatly improve the sensitivity and accuracy of magnetic resonance imaging.

[0031] 3. Experiments show that the probe signal of the present invention reaches a peak value in 2 hours at the tumor site, and the proton signal is enhanced by 94%. 1 The H signal provides the structural image and distribution of overexpressed Caspase-3 in the tumor site, which can be used for qualitative analysis. 19 The F signal provides quantitative analysis of caspase activity without background interference.

[0032] 4. The probe of the present invention has good biosafety and water dispersibility, is suitable for in vivo MRI, and can better evaluate the therapeutic effect of early tumors.

[0033] 5. The probe preparation method of the present invention is simple, the raw materials are cheap and easily available, the synthesis conditions are relatively simple, the synthesis cost is relatively low, the yield is high, and it is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a TEM image of the Caspase-3 enzyme-responsive self-assembled probe prepared in Example 1.

[0035] Figure 2 This is a TEM image of the Caspase-3 enzyme-responsive self-assembled probe prepared in Example 1 after being treated with Caspase-3 enzyme.

[0036] Figure 3 The high performance liquid chromatograms of the Caspase-3 enzyme-responsive self-assembled probe prepared in Example 1 in response to different concentrations of Caspase-3 enzyme.

[0037] Figure 4 The self-assembled probe responsive to Caspase-3 prepared in Example 1 at different time points under the Caspase-3 enzyme response 19 F NMR spectrum.

[0038] Figure 5 The self-assembled probe responsive to Caspase-3 prepared in Example 1 is used for the in vivo tumor under the action of Caspase-3. 1 H MRI image.

[0039] Figure 6 The self-assembled probe responsive to Caspase-3 prepared in Example 1 is used for the in vivo tumor under the action of Caspase-3. 19 F MRI image. DETAILED DESCRIPTION

[0040] The present invention is described in detail below with reference to specific embodiments.

[0041] Example 1

[0042] 1. Synthesis of compound of formula (I)

[0043] 1.1. Weigh dichlororesin (250 mg, 0.25 mmol) into a peptide synthesis tube, add 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide, and swell at room temperature and pressure for 2 hours. After draining the above solvents, the resin remains in the peptide synthesis tube. Add Fmoc-Lys(Dde)-OH (532.6 mg, 1 mmol), HATU (380.4 mg, 1 mmol) and DIPEA (368 μL, 2 mmol) to the peptide synthesis tube, then add 10 mL of N,N-dimethylformamide to dissolve, and then use nitrogen blowing to react. After 2 hours of reaction, the reaction is terminated. After draining the solution, wash the resin with 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide in turn. Add 20 wt% piperidine solution (solvent is N,N-dimethylformamide) into the peptide synthesis tube, and react by nitrogen blowing. After 0.5 hours, the reaction is terminated, the solution is drained, and the resin is washed with 5 mL of dichloromethane and 5 mL of N,N-dimethylformamide in turn.

[0044] 1.2. According to the operation and method of step 1.1, the second amino acid Fmoc-Phe-OH (644.7 mg, 1 mmol), the third amino acid Fmoc-Phe-OH (644.7 mg, 1 mmol), the fourth amino acid Fmoc-Phe-OH (644.7 mg, 1 mmol), the fifth amino acid Fmoc-Asp(otBu)-OH (411.4 mg, 1 mmol), the sixth amino acid Fmoc-Val-OH (339.4 mg, 1 mmol), the seventh amino acid Fmoc-Glu(otBu)-OH (425.4 mg, 1 mmol) and the eighth amino acid Fmoc-Asp(otBu)-OH (411.4 mg, 1 mmol) were grafted respectively.

[0045] 1.3. After the amino acid grafting is completed, m-ditrifluoromethylbenzoic acid (258.0 mg, 1 mmol), HATU (380.4 mg, 1 mmol) and DIPEA (368 μL, 2 mmol) are added to the peptide synthesis tube, and then 10 mL of N, N-dimethylformamide is added for dissolution. The reaction is terminated after the reaction is continued for 2 hours. After the solution is drained, the resin is washed with 5 mL of dichloromethane and 5 mL of N, N-dimethylformamide in sequence. Then, 10 wt% hydrazine hydrate solution is added to the resin to remove the Dde protecting group of the lysine side chain, and then the resin is washed with 5 mL of dichloromethane and 5 mL of N, N-dimethylformamide in sequence.

[0046] 1.4. Add 9-fluorenylmethyl-N-succinimidyl carbonate (337.3 mg, 1 mmol) and DIPEA (368 μL, 2 mmol) to the resin, then add 10 mL of N, N-dimethylformamide to dissolve, and terminate the reaction after 2 hours. After draining the solution, wash the resin with 5 mL of dichloromethane and 5 mL of N, N-dimethylformamide in sequence. Then add 10 mL of 1 wt% TFA trifluoroacetic acid dichloromethane solution to the resin to free the synthesized polypeptide from the resin. After filtering the resin to remove the solvent, spin dry to obtain a yellow oily liquid, add petroleum ether for precipitation, and precipitate 227 mg of white solid.

[0047]

[0048] 1 H NMR (500MHz, DMSO) δ12.63(s,1H),9.27(d,J=7.8Hz,1H),8.50(s,2H),8.33(d,J=17.2Hz,1H),8.21(dd,J=13.1,7.8Hz,3H),8.13(d,J=8.0Hz,1H),8. 04(d,J=8.0Hz,1H),7.88(d,J=7.5Hz,2H),7.69(dd,J=14.4,8.0Hz,4H),7. 40(t,J=7.4Hz,2H),7.36–7.07(m,18H),4.85(td,J=8.5,5.5Hz,1H),4.66–

[0049] 4.48(m,3H),4.43(dd,J=12.5,7.9Hz,1H),4.35–4.24(m,3H),4.18(dd,J=14.7, 8.3Hz,3H),3.06(dt,J=19.5,9.9Hz,1H),3.02–2.94(m,3H),2.91–2.59(m,6H),2 .54(dd,J=8.6,6.0Hz,1H),2.41–2.08(m,3H),1.94–1.81(m,2H),1.79–1.66(m,2 H),1.66–1.53(m,1H),1.46–1.38(m,2H),1.37–1.13(m,29H),0.79–0.66(m,6H).

[0050] 13 C NMR(126MHz,DMSO)δ173.43(s),171.75(s),170.90(s),170.81–

[0051] 170.42(m),170.21(s),170.04(s),169.83(s),169.26(s),169.09(s),163.46 (s),156.09(s),143.94(s),140.74(s),137.57(d,J=3.0Hz),137.31(s),136. 19(s),130.88(s),130.62(s),130.35(s),130.09(s),129.47–128.99(m),128 .30(s),128.06–127.77(m),127.58(s),127.04(s),126.21(dd,J=22.8,13.0Hz ),125.14(s),124.19(s),122.02(s),120.10(s),119.85(s),80.17(d,J=9.9H z),79.49(s),65.19(s),57.07(s),53.58(s),51.99(d,J=4.1Hz),50.53(s),49 .47(s),46.78(s),37.51(d,J=19.0Hz),31.28(s),30.88(d,J=15.8Hz),29.06 (s), 27.60 (d, J = 2.3Hz), 27.09 (s), 22.69 (s), 19.19 (s), 17.68 (s), -18.79 (s).

[0052] HRMS: m / z=1676.7356[M+H]+.

[0053] 2. Synthesis of the compound of formula (II)

[0054] Weigh a white solid (167.5 mg, 0.1 mmol) and tri-tert-butyl 2,2′,2″-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacyl)triacetate (92.1 mg, 0.15 mmol) in a 10 mL round-bottom flask, add HATU (38.0 mg, 0.1 mmol) and DIPEA (36.8 μL, 2 mmol) to the round-bottom flask, then add 5 mL of N,N-dimethylformamide to dissolve, and react at room temperature and pressure for 2 hours. After the reaction is completed, the obtained mixed product is used to remove the solvent by a rotary evaporator, and the residue is a yellow oily liquid. After adding petroleum ether, a light yellow solid is precipitated, which is directly used for the next step without purification.

[0055]

[0056] HRMS: m / z=1676.7356[M+H]+ .

[0057] 3. Synthesis of compound of formula (III)

[0058] Weigh a light yellow solid (227 mg, 0.1 mmol) into a 50 mL round-bottom flask, then add 10 mL of trifluoroacetic acid, and stir the reaction at room temperature and pressure overnight. After the reaction, remove the solvent from the obtained mixed product under reduced pressure, and dissolve the residue in pure acetonitrile, perform preparative liquid chromatography separation (phase A: water, phase B: acetonitrile), and freeze-dry to obtain a white solid (158.8 mg, yield 81.9%).

[0059]

[0060] 1H NMR (500 MHz, DMSO) δ 9.23 (d, J = 7.2 Hz, 1H), 8.52 (s, 2H), 8.42 (s, 1H), 8.33 (s, 1H), 8.27 (d, J = 7.7 Hz, 1H), 8.19 (d, J = 7.4 Hz, 1H), 8.08–7.94 (m, 3H), 7.88 (d, J = 7.5 Hz, 2H), 7.81 (s, 1H), 7.75 (d, J = 7.3 Hz, 1H), 7.67 (d, J = 7.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 1H), 7.41 (t, J = 7.4 Hz, 2H), 7.31 (dd, J = 10.8, 4.1 Hz, 2H), 7.25 (t, J = 6.5 Hz, 5H), 7.22–7.06 (m, 11H), 4.82 (ddd, J = 9.4, 7.5, 4.7 Hz, 2H), 4.56 (ddd, J = 21.1, 13.5, 7.8 Hz, 3H), 4.47 (dd, J = 12.8, 8.2 Hz, 1H), 4.39 (dd, J = 12.6, 7.6 Hz, 2H), 4.30 (dd, J = 14.7, 7.8 Hz, 3H), 4.22–3.96 (m, 6H), 3.84 (s, 2H), 3.63 (d, J = 4.2 Hz, 3H), 3.36 (s, 6H), 3.10 (d, J = 21.6 Hz, 10H), 2.99–2.93 (m, 3H), 2.91–2.67 (m, 6H), 2.60 (dd, J = 16.6, 5.1 Hz, 1H), 2.45 (dd, J = 16.7, 8.3 Hz, 1H), 2.32–2.13 (m, 2H), 2.00–1.85 (m, 2H), 1.82–1.72 (m, 1H), 1.65 (s, 1H), 1.52 (d, J = 9.3 Hz, 1H), 1.39 (d, J = 6.6 Hz, 2H), 1.33–1.18 (m, 2H), 0.73 (dd, J = 17.1, 6.7 Hz, 6H).

[0061] HRMS: m / z = 1936.7856 [M + H]+.

[0062] 4. Synthesis of Self-Assembled Probes

[0063] Weigh the white solid (193.5 mg, 0.1 mmol) and dissolve it in a mixed solvent of acetonitrile and water (5 mL / 5 mL). Slowly drip 1 M sodium hydroxide solution to adjust the system pH to 5.5-6.0. During this period, 1 M hydrochloric acid solution can be used to adjust the system pH to 5.5-6.0. Add gadolinium trichloride hexahydrate (388.6 mg, 0.1 mmol) while stirring, and then continue to stir and react for 6 hours. During the reaction, the xylenol orange test is used to monitor the reaction until the solution color stabilizes in light yellow. After the reaction is completed by LC-MS monitoring, it is purified by high-performance liquid preparative chromatography (mobile phase: phase A is water, phase B is acetonitrile) to obtain the final product with a yield of 72.4%. The purity of the final product is analyzed by high-performance liquid analytical chromatography, and the purity is 95%. The molecular weight of the final product identified by LC-MS is 2092.1990 [M+H]

[0064] The final product prepared in this example (Caspase-3 enzyme-responsive self-assembled probe dispersed in a solvent) (dispersed in a 5% DMSO aqueous solution) was scanned using a transmission electron microscope, and the resulting TEM image is shown in FIG. Figure 1 As shown, from Figure 1 It can be seen from the figure that the self-assembled probe prepared in this example is in an irregular spherical shape.

[0065] The final product (self-assembled probe responsive to Caspase-3 enzyme) prepared in this example was scanned by a transmission electron microscope after being responsive to Caspase-3 enzyme (dispersed in a 5% DMSO aqueous solution). The obtained TEM image is shown in FIG. Figure 2 As shown, from Figure 2 It can be seen from the figure that the self-assembling probe prepared in this example self-assembles into a rod shape after the Caspase-3 enzyme response.

[0066] Test 1. Caspase-3 enzyme response test of the self-assembled probe responsive to Caspase-3 of the present invention

[0067] Test method:

[0068] 1. Weigh 10 mg of the self-assembling probe molecule prepared in Example 1 and dissolve it in 10 mL of PBS (5 v / v% DMSO) to prepare a 1 mg / mL probe solution.

[0069] 2. The 200 μg / mL Caspase-3 stock solution was graded diluted with PBS to obtain 100 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL, and 10 μg / mL Caspase-3 solutions, respectively.

[0070] 3. Take 100 μL of each Caspase-3 solution, then add 100 μL of probe solution to each Caspase-3 solution, and then react at 37°C for 2 hours. After the reaction is completed, use high performance liquid chromatography (phase A: water; phase B: acetonitrile; gradient: phase B 5-95%, 30 min) for analysis and testing.

[0071] Test results:

[0072] The high performance liquid chromatogram of the self-assembled probe prepared in Example 1 under the action of Caspase-3 is as shown in the figure below: Figure 3 As shown by Figure 3 It can be seen that as the concentration of added Caspase-3 gradually increases, the peak area proportions at the peak times of 17 minutes and 21.7 minutes gradually increase. The compound at the peak time of 17 minutes was identified by mass spectrometry as the fragment CF3DEVDNH2 after the molecular probe was cleaved by Caspase-3, and the compound at 21.7 minutes was the fragment FFFK(Fmoc)-GdDOTA after the molecular probe was cleaved by Caspase-3. When the Caspase-3 concentration reached 100 μg / mL, the generated cleavage fragments CF3DEVDNH2 and FFFK(Fmoc)-GdDOTA accounted for the largest proportion, indicating that the cleavage efficiency was the highest.

[0073] Experiment 2: Self-assembled probes responsive to Caspase-3 enzyme of the present invention 19 F NMR experiments

[0074] Test method:

[0075] 1. Weigh 20 mg of the self-assembling probe molecule prepared in Example 1 and dissolve it in 2 mL of PBS to prepare a 10 mg / mL probe solution.

[0076] 2. Dilute the 200 μg / mL Caspase-3 stock solution with PBS to make a 100 μg / mL Caspase-3 solution.

[0077] 3. Add 500 μL of 10 mg / mL probe solution to the EP tube, then add 500 μL of 100 μg / mL Caspase-3 solution, and then react at 37°C. At different time points (0 min, 10 min, 20 min, 30 min, 60 min, 120 min), take 300 μL of the reaction solution into the NMR tube and perform NMR on the Bruker Ascend WB 500 MHz spectrometer. 19 F NMR experiments.

[0078] Test results:

[0079] The self-assembling probe prepared in Example 1 is activated by Caspase-3. 19 The F NMR spectrum changes with time Figure 4 As shown by Figure 4 It can be seen that after adding Caspase-3 enzyme, as the reaction time gradually increases, the probe molecules 19 The F NMR signal was recovered and detected at -62.5 ppm 19 F NMR signal generation, at this time 19 The F NMR signal changes from OFF to ON. When the reaction lasts for 120 minutes, 19 The F NMR signal can be restored to its maximum value.

[0080] Experiment 3: In vivo self-assembly probes of the Caspase-3 enzyme response of the present invention 1 H MRI test

[0081] Test method:

[0082] 1. Weigh 3 mg of the self-assembled probe prepared in Example 1 and dissolve it in 500 μL PBS to prepare a 6 mg / mL contrast agent.

[0083] 2. Take subcutaneous tumor model mice (Babl / c mice) and inject 4T1 cells subcutaneously in the right foreleg, about 1×10 6 The subcutaneous tumor model mice were anesthetized with isoflurane, and 300 μL of contrast agent was injected into the subcutaneous tumor model mice through the tail vein. Then, the subcutaneous tumor model mice were detected by 9.4T nuclear magnetic imaging at different time points (10 min, 30 min, 60 min, 2 h, 4 h, 6 h, 12 h). 1 H MRI images, 1 H MRI images were acquired by the RARE method with the following parameters: TR = 500 ms, TE = 6 ms, FOV = 4 × 4 cm, slice thickness = 30 mm, acquisition time was 1 minute 16 seconds, RARE factor was 4, matrix size was 96*96, and average times was 4.

[0084] Test results:

[0085] The self-assembled probe prepared in Example 1 was activated by Caspase-3 in the tumor area to reduce the number of tumors in the subcutaneous tumor model mice. 1 The change of H MRI intensity with contrast agent injection time is shown in the figure Figure 5 As shown by Figure 5 It can be seen that the probe has spread throughout the mouse body through blood circulation at the initial time of 10 minutes, and 1 The H magnetic resonance signal reaches its peak at 12 hours.1 The H magnetic resonance signal is still strong, indicating that the subassembly probe of the present invention self-assembles at the mouse tumor site, thereby prolonging the retention time and imaging time of the probe at the mouse tumor site.

[0086] Experiment 4: In vivo self-assembly probes responsive to Caspase-3 enzyme of the present invention 19 F MRI test

[0087] Test method:

[0088] 1. Weigh 10 mg of the self-assembled probe prepared in Example 1 and dissolve it in 1 mL of PBS to prepare a 10 mg / mL contrast agent.

[0089] 2. Take subcutaneous tumor model mice (Babl / c mice) and inject 4T1 cells subcutaneously in the right foreleg, about 1×10 6 The subcutaneous tumor model mice were anesthetized with isoflurane, and 100 μL of contrast agent was injected into the tumor area of ​​the subcutaneous tumor model mice by intravenous injection in situ. After 2 hours, the tumor was detected by 9.4T nuclear magnetic imaging. 19 F MRI images, 19 F MRI images were acquired by the RARE method with the following parameters: TR = 4000 ms, TE = 3 ms, FOV = 4.5 × 4.5 cm, TR = 500 ms, TE = 6 ms, FOV = 4 × 4 cm, slice thickness = 30 mm, acquisition time was 1 minute 16 seconds, RARE factor was 4, matrix size was 96 × 96, and the number of averages was 4.

[0090] Test results:

[0091] The self-assembled probe prepared in Example 1 was exposed to Caspase-3 enzyme in the tumor area for 2 hours, and the tumor area of ​​the subcutaneous tumor model mouse was 19 F MRI images are as follows Figure 6 As shown by Figure 6 It can be seen that 19 F MRI signal area and 1 The H MRI signal region and the tumor region are highly overlapped, indicating that the subassembly probe of the present invention can recognize the Caspase-3 enzyme in the tumor region in vivo.

Claims

1. A Caspase-3 enzyme-responsive self-assembling probe, characterized in that Its structural formula is as follows:

2. A method for synthesizing a Caspase-3 enzyme-responsive self-assembling probe as claimed in claim 1, characterized in that The steps include: S1. Using dichlororesin as a solid phase carrier, the peptide solid phase synthesis method is used to synthesize the compound of formula (I), and the reaction formula is as follows: S2. In the presence of N,N-diisopropylethylamine and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, the compound of formula (I) undergoes an amide condensation reaction with tri-tert-butyl 2,2',2"-(10-(2-((2-aminoethyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacyl)triacetate to produce a compound of formula (II), and the reaction formula is as follows: S3. In the presence of trifluoroacetic acid, the compound of formula (II) is hydrolyzed to remove the tert-butyl group to generate a compound of formula (III), and the reaction formula is as follows: S4. The compound of formula (III) is subjected to coordination reaction with gadolinium trichloride or its hydrate to generate the self-assembled probe responsive to the Caspase-3 enzyme, and the reaction formula is as follows:

3. The method for synthesizing the Caspase-3 enzyme-responsive self-assembling probe according to claim 2, characterized in that: The synthesis temperature of the polypeptide solid phase synthesis method is 20-40°C and the synthesis time is 1-3h.

4. The method for synthesizing the Caspase-3 enzyme-responsive self-assembly probe according to claim 2, characterized in that: The reaction temperature of the amide condensation reaction is 20-40°C, and the reaction time is 1-3h.

5. The method for synthesizing the Caspase-3 enzyme-responsive self-assembling probe according to claim 2, characterized in that: The hydrolysis reaction temperature is 25-30°C and the time is 12-24h.

6. The method for synthesizing the Caspase-3 enzyme-responsive self-assembling probe according to claim 2, characterized in that: The temperature of the coordination reaction is 20-30°C and the time is 12-24h.

7. Use of the Caspase-3 enzyme-responsive self-assembly probe according to claim 1 in the preparation of a magnetic resonance dual-modality contrast agent.

8. Use of the Caspase-3 enzyme-responsive self-assembly probe according to claim 7 in the preparation of a magnetic resonance dual-modality contrast agent, characterized in that: The dual-modal agent is 19 F-MRI contrast agents and 1 H-MRI contrast agent.

Images