Preparation method and application of novel fluorescence probe activated by o-dithiol protein

Abstract

The invention belongs to the technical field of medicines, and discloses a red fluorescent probe molecule activated by vicinal dithiol proteins (VDPs), which is used for monitoring the phenomenon of reduction-state VDPs content downloading in the brain of an ischemia reperfusion injury model mouse for the first time. The fluorescent probe has great advantages in the aspects of biological small molecule identification, protein marking and disease diagnosis, and is one of the most convenient and most popular tools for exploring a complex biological process. According to the invention, methacrylamide is taken as a recognition site to connect a fluorophore with an a-hydroxymethyl unsaturated ketone part, so that the fluorescent probe molecule capable of releasing red fluorescence after being activated by VDPs is obtained. The fluorescent probe molecule disclosed by the invention can be specifically combined with VDPs in a living body, so that the purposes of monitoring VDPs and diagnosing diseases are achieved.

Description

Technical Field

[0001] The present invention relates to the fields of organic synthesis and molecular medicine, and in particular to a preparation method and application of a novel fluorescent probe activated by vicinal dithiol proteins. Background Art

[0002] VDPs are widely present in various proteins and are essential elements for maintaining protein structure and post-translational modification of proteins. These proteins are characterized by the presence of at least one pair of spatially adjacent dithiols / disulfides that can be converted into each other. This process enriches the spatial diversity and functional range of proteins, giving them a wider range of biological roles. More and more studies have shown that the dysregulation of VDPs is closely related to many diseases, such as inflammation, neurodegenerative diseases, stroke, cancer, etc. Therefore, it is of great significance to develop a high-performance fluorescent probe to monitor VDP levels. Fluorescent probes have great advantages in the identification of small biological molecules, protein labeling, and disease diagnosis, and are one of the most convenient and popular tools for exploring complex biological processes. In recent years, a variety of fluorescent probes with high specificity and excellent optical properties have been developed for sensing vicinal dithiols of proteins. Based on this, our research group has developed a new fluorescent probe for detecting VDPs. The probe uses methacrylamide as a recognition site, which can perform real-time fluorescence imaging of stroke mice and monitor the changes of VDPs in their bodies. These findings provide valuable insights into the early detection and treatment of stroke and contribute to the advancement of clinical diagnosis and treatment strategies. Summary of the invention

[0003] The first purpose of the present invention is to provide a fluorescent probe molecule which has a simple structure, is easy to synthesize and has good performance, and has high application value.

[0004] The second purpose of the present invention is to provide a method for preparing a fluorescent probe molecule, wherein a fluorophore with an isocyanate group is connected to a corresponding α-hydroxymethyl unsaturated ketone to obtain a probe molecule with a fast reaction rate and high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 This is a table showing the reactivity test results of compounds 1-6 provided in Experimental Example 1 of the present invention with bovine serum albumin (rBSA) and GSH;

[0006] Figure 2 It is the spectrum response and high performance liquid chromatography result diagram of the compound VDP-red provided in Experimental Example 1 of the present invention to rBSA;

[0007] Figure 3 1 is a graph showing the fluorescence spectrum response of the compound VDP-red provided in Experimental Example 1 of the present invention to rBSA at different pH values ​​and the test result of the selectivity of VDP-red to rBSA;

[0008] Figure 4 This is an in vivo imaging diagram of the compound VDP-red provided in Experimental Example 1 of the present invention in healthy mice;

[0009] Figure 5 This is an imaging diagram of the compound VDP-red provided in Experimental Example 1 of the present invention in the brain of mice with cerebral ischemia-reperfusion injury (CIRI);

[0010] Example 1

[0011] The present invention discloses a fluorescent probe molecule, the structural formula of which is:

[0012]

[0013] The synthetic route of the fluorescent probe molecule of the present invention is as follows:

[0014]

[0015] Synthesis of compound FR-NH2: A mixture of 4-(diethylamino) salicylaldehyde (1.03 mmol, 1.0 eq.) and 6-amino-3,4-dihydro-1(2H)-naphthalenone (1.13 mmol, 1.1 eq.) in methanesulfonic acid was stirred at 90°C for 5 hours. The reaction mixture was poured into an ice-water mixture (200 mL). Perchloric acid (70%; 7 mL) was then slowly added to the reaction mixture. After filtration, a black solid product FR-NH2 (76%) was obtained. Synthesis of compound FR-NCO: FR-NH2 (0.28 mmol, 1.0 eq.) was dissolved in 10 mL of dry DCM. Triphosgene (0.14 mmol, 0.5 eq.) and triethanolamine (0.34 mmol, 1.2 eq.) were then added. The reaction mixture was stirred at 45°C for 4 hours. The organic layer was concentrated to obtain a crude product FR-NCO.

[0016] Synthesis of compound 1-6: To a solution of crude FR-NCO (0.28 mmol, 1.0 eq.) in dry DCM (2 mL) at 0°C, α-hydroxyene ketone (1a-6a) (0.28 mmol, 1.0 eq.) and triethanolamine (0.34 mmol, 1.2 eq.) were added. The reaction mixture was stirred for 10-120 minutes. The residue was purified by silica gel flash chromatography (CH2Cl2 / MeOH=100 / 1) to give compound 1-6.

[0017] Compound 1: 11H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.66 (s, 1H), 8.20 (d, J = 8.7 Hz, 1H), 7.93 (d, J = 9.4 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.57 (s, 1H), 7.45 (d, J = 9.4 Hz, 1H), 7.28 (s, 1H), 6.33 (s, 1H), 6.03 (s, 1H), 4.87 (s, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.69 (q, J = 7.0 Hz, 4H), 3.12 (d, J = 7.3 Hz, 4H), 1.24 (t, J = 7.0 Hz, 9H). 13 13C NMR (151 MHz, DMSO-d6) δ 165.19, 163.13, 158.53, 155.64, 153.15, 148.70, 145.57, 144.29, 135.96, 132.26, 128.75, 127.87, 121.16, 120.80, 118.52, 118.28, 117.74, 117.43, 96.11, 63.38, 61.21, 55.38, 45.86, 27.10, 25.00, 14.50. HRMS: + calculated for C 28 H 31 N2O5 + : 476.2306; found: 476.2272.

[0018] Compound 2: 1 1H NMR (600 MHz, DMSO-d6) δ 10.47 (s, 1H), 10.07 (s, 1H), 8.66 (s, 1H), 8.19 (d, J = 8.7 Hz, 1H), 7.93 (d, J = 9.4 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.7 Hz, 1H), 7.58 (s, 1H), 7.45 (dd, J = 9.4, 2.5 Hz, 1H), 7.33 (dd, J = 8.5, 7.3 Hz, 2H), 7.30 - 7.26 (m, 1H), 7.09 (t, J = 7.4 Hz, 1H), 6.14 (s, 1H), 5.90 (s, 1H), 4.96 (s, 2H), 3.69 (q, J = 7.1 Hz, 4H), 3.03 (s, 4H), 1.24 (t, J = 7.1 Hz, 6H). 13C NMR(151MHz,DMSO-d6)δ165.18,163.16,158.52,155.63,153.27,148.70,145.65,144.30,140.05,139.25,132.26,129.10,12 7.86,124.21,123.16,121.16,120.76,120.60,118.51,118.27,117.74,117.42,96.11,64.44,45.86,27.10,25.00.HRMS:[M] + calculated for C 32 H 32 N3O4 + :523.2466; found:523.2426.

[0019] Compound 3: 1 H NMR(600MHz,DMSO-d6)δ10.47(s,1H),8.78(t,J=6.1Hz,1H),8.66(s,1H),8.20( d,J=8.7Hz,1H),7.93(d,J=9.3Hz,1H),7.67(d,J=8.1Hz,1H),7.58(s,1H),7.45( dd,J=9.4,2.4Hz,1H),7.34-7.21(m,6H),6.03(s,1H),5.75(s,1H),4.89(s,2H), 4.37(d,J=6.0Hz,2H),3.69(q,J=7.1Hz,4H),3.04(s,4H),1.25(t,J=7.2Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ166.04,163.19,158.53,155.63,153.27,148.70,145.71,144.31,139.89,139.70,132.27,128.76,127.8 8,127.63,127.24,122.01,121.17,120.73,118.50,118.26,117.73,117.42,96.12,64.38,45.86,42.68,27.11,25.01.HRMS:[M] + calculated for C 33 H 34 N3O4 + :536.2549; found:536.2547.

[0020] Compound 4: 11H NMR (600 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.67 (s, 1H), 8.29 - 8.14 (m, 2H), 7.93 (d, J = 9.4 Hz, 1H), 7.67 (dd, J = 8.7, 2.1 Hz, 1H), 7.58 (s, 1H), 7.45 (dd, J = 9.4, 2.5 Hz, 1H), 7.29 (s, 1H), 5.94 (s, 1H), 5.69 (s, 1H), 4.85 (s, 2H), 3.69 (q, J = 7.0 Hz, 4H), 3.20 - 3.12 (m, 2H), 3.03 (s, 4H), 1.24 (t, J = 7.0 Hz, 6H), 1.06 (t, J = 7.2 Hz, 3H). 13 13C NMR (151 MHz, DMSO-d6) δ 165.71, 163.16, 158.51, 155.62, 153.26, 148.67, 145.70, 144.28, 139.86, 132.25, 127.85, 121.42, 121.13, 120.70, 118.49, 118.25, 117.70, 117.40, 96.11, 64.34, 45.86, 34.14, 27.10, 25.00, 15.13. HRMS: + calculated for C 28 H 32 N3O4 + : 474.2387; found: 474.2895.

[0021] Compound 5: 1 1H NMR (600 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.66 (s, 1H), 8.28 - 8.14 (m, 2H), 7.93 (d, J = 9.4 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.58 (s, 1H), 7.44 (d, J = 9.3 Hz, 1H), 7.28 (s, 1H), 5.95 (s, 1H), 5.68 (s, 1H), 4.84 (s, 2H), 3.68 (q, J = 7.1 Hz, 4H), 3.12 (t, J = 6.4 Hz, 2H), 3.03 (s, 4H), 1.44 (p, J = 7.4 Hz, 2H), 1.30 (q, J = 7.5 Hz, 2H), 1.24 (t, J = 7.1 Hz, 6H), 0.87 (t, J = 7.4 Hz, 3H). 1313C NMR (151 MHz, DMSO-d6) δ 165.88, 163.13, 158.49, 155.61, 153.27, 148.67, 145.72, 144.25, 139.87, 132.25, 127.83, 121.31, 121.10, 120.66, 118.48, 118.23, 117.68, 117.40, 96.12, 64.34, 45.86, 31.61, 29.49, 27.11, 25.00, 22.57, 20.10, 14.19. HRMS: [M] + calculated for C 30 H 36 N3O4 + : 502.2700; found: 502.3238.

[0022] Compound 6: 1 1H NMR (600 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.65 (s, 1H), 8.18 (d, J = 8.7 Hz, 1H), 7.92 (d, J = 9.4 Hz, 1H), 7.64 (d, J = 9.3 Hz, 1H), 7.57 (s, 1H), 7.44 (d, J = 9.4 Hz, 1H), 7.27 (s, 1H), 5.48 (s, 1H), 5.29 (s, 1H), 4.82 (s, 2H), 3.68 (q, J = 7.1 Hz, 4H), 3.35 - 3.27 (m, 4H), 3.02 (s, 4H), 1.24 (t, J = 7.1 Hz, 7H), 1.11 - 1.03 (m, 6H). 13 13C NMR (151 MHz, DMSO-d6) δ 168.45, 163.07, 158.49, 155.63, 153.17, 148.67, 145.55, 144.23, 140.83, 132.25, 127.81, 121.11, 120.76, 118.51, 118.26, 117.76, 117.46, 116.28, 96.10, 65.46, 45.87, 27.08, 24.98. HRMS: [M] + calculated for C 30 H 36 N3O4 + : 502.2700; found: 502.3215.

[0023] Experimental Example 2

[0024] This experimental example provides a table of reactivity test results of compounds 1-6 with bovine serum albumin (rBSA) and GSH;

[0025] Experimental method: Compounds 1-6 were prepared with rBSA or GSH in a PBS buffer solution at 37°C to form solutions of appropriate concentrations and incubated for 30 minutes. The fluorescence intensities were then measured and the fluorescence enhancement ratio (F / F0) was calculated.

[0026] Experimental results: As shown in the table, compound 3 (VDP-red) increased its reaction to rBSA by 31 times, but had almost no reaction to GSH. It has the best performance among all compounds, and the subsequent experimental examples are only conducted on compound 3.

[0027] Experimental Example 3

[0028] This experimental example provides the spectral response of VDP-red to rBSA and the HPLC result graph;

[0029] Experimental method: VDP-red was added to PBS buffer solution to prepare a solution of appropriate concentration, and then an appropriate amount of rBSA was added, and then its absorption spectrum, fluorescence spectrum and high performance liquid chromatography were measured.

[0030] Experimental results: As shown in the figure, the excitation wavelength of VDP-red is 580nm, and the emission wavelength is 625nm. After adding rBSA to the probe, the fluorescence emission intensity stabilizes within 10 minutes, which is 31 times that of the beginning. And the probe has a higher fluorescence release when the rBSA concentration is about 1μM. It can be seen from high-performance liquid chromatography that when VDP-red reacts with rBSA, the carbamate bond in the probe molecule is broken to generate FR-NH2. When the reaction reaches 10 minutes, the reaction is basically completed.

[0031] Experimental Example 4

[0032] This experiment provides the fluorescence spectrum response of VDP-red to rBSA at different pH and the results of VDP-red selectivity test to rBSA;

[0033] Experimental method: VDP-red and rBSA were added to buffer solutions of different pH values ​​to prepare solutions of appropriate concentrations and incubated for a certain period of time, and then their fluorescence spectra were measured. VDP-red and various test substances were then added to PBS buffer solutions to prepare solutions of appropriate concentrations and incubated for a certain period of time, and then their fluorescence spectra were measured. Finally, VDP-red, rBSA and different concentrations of phenylarsine oxide (PAO) were added to PBS buffer solutions to prepare solutions of appropriate concentrations and incubated for a certain period of time, and then their fluorescence spectra were measured.

[0034] Experimental results: As shown in the figure, after adding rBSA, the fluorescence signal intensity increased significantly and the relative intensity of the fluorescence signal remained unchanged in the pH range of 7.0-11.0, indicating that the probe is suitable for physiological environments. The selectivity test results showed that only reduced VDPs showed good reactivity with the probe, while other substances did not react with the probe. Figure 3 In C, it can be observed that the reactivity of VDP-red with rBSA decreases with the increase of PAO concentration, which further indicates that the reaction of VDP-red with VDPs is selective. Figure 3 In D, compared with the fluorescence enhancement caused by reduced E-Trx, the fluorescence of the reduced E-TrxC35S (Cys35 mutated to Ser, from adjacent dithiol to monothiol) reaction is extremely weak, which further illustrates the selectivity of VDP-red for VDPs.

[0035] Experimental Example 5

[0036] This experiment provides images of VDP-red in healthy mice;

[0037] Experimental method: VDP-red was dissolved in PBS buffer to prepare a solution of appropriate concentration (containing 5% ethanol). After the mice were anesthetized with isoflurane, an appropriate amount of VDP-red was injected into the abdomen of the mice, and the mice were imaged at different time points. After that, another healthy mouse was taken and VDP-red was injected into the tail vein of the mouse. After 2 hours and 12 hours, the heart, liver, spleen, lung and kidney of the mouse were imaged respectively.

[0038] Experimental results: As time went by, the fluorescence signal in the mouse body gradually increased. It can be found that esterase is mainly distributed in the liver, lungs and kidneys of mice. And after 12 hours, there is still a lot of fluorescence in the mouse lungs. This shows that the probe has good stability in the body and VDPs mainly accumulate in the mouse lungs.

[0039] Experimental Example 6

[0040] This experimental example provides images of VDP-red in the brain of mice with cerebral ischemia-reperfusion injury (CIRI);

[0041] Experimental methods: The bilateral common carotid artery ligation (BCAL) ischemia model reported in the literature was used to induce global ischemia in the mouse brain. After 60 minutes of ischemia, reperfusion was induced. After preparing the ischemia-reperfusion model mice, the probe was introduced into the brain, and the brain tissue was dissected and imaged under a fluorescence microscope.

[0042] Experimental results: Compared with the red fluorescence of normal brain, the fluorescence of CIRI brain is weaker, which shows that the probe VDP-red can specifically respond to VDPs in vivo and monitor its changes.

Claims

1. A fluorescent probe molecule, characterized in that: Its structural formula is: Among them, the R group is ethoxy, phenylamino, benzylamino, ethylamino, butylamino, and diethylamino.

2. The fluorescent probe molecule according to claim 1, characterized in that The compound is any one of the following:

3. The new use of the fluorescent probe molecule according to claim 1 or 2, characterized in that: Monitoring VDPs in living organisms.

Images