Thionitrous acid near-infrared bioluminescent probe as well as preparation method and application thereof

Abstract

The invention provides a thionitrous acid near-infrared bioluminescent probe and a preparation method and application thereof, the bioluminescent probe BF-5 has good selectivity, stability, biocompatibility and low detection limit (327nM), can detect physiological level HSNO in HCT116-luc cells, can be applied to bioluminescent imaging of HSNO in HCT116-luc colon cancer nude mouse transplanted tumor, and has good application prospects. The invention provides a visual detection tool for revealing the physiological and pathological mechanism of HSNO, and has important significance for researching diseases related to HSNO.

Description

Technical Field

[0001] The invention belongs to the field of chemistry and analytical detection, and specifically relates to a thionitrite near-infrared bioluminescent probe and a preparation method and application thereof. Background Art

[0002] Thionitrious acid (HSNO), as the smallest molecular weight S-nitrosothiol, is a key signal transduction molecule connecting H2S and NO signaling pathways. HSNO can not only regulate protein function through nitrosylation, but also convert NO and H2S into n , HNO and other important signal transduction molecules in the body are "transported" into the cells, thereby playing an important role in the cardiovascular, nervous and immune systems.

[0003] In recent years, more and more studies have shown that hydrogen sulfide can inhibit or activate the activity of NOS. NO affects the production of endogenous H2S by inhibiting or inducing the activity or expression of cystathionine-β-synthase and cystathionine-γ-lyase. There is a biphasic effect on cell proliferation and apoptosis, which is related to factors such as the level of H2S / NO, the duration of action, the type of tumor cells and sensitivity. In colon cancer, H2S and NO regulate cell proliferation through a bell-shaped concentration-response curve. Lower levels of endogenous H2S / NO promote tumor cell proliferation, while exposure to higher levels of exogenous H2S / NO shows anti-tumor activity. In colon cancer, H2S participates in the anti-tumor migration and invasion activity mediated by exogenous NO, suggesting that there is a cross-talk between H2S and NO mediated by the cGMP / VASP signaling pathway. It can be seen that the expression of H2S and NO and the crosstalk between them affect the development of colon cancer. HSNO is the source of NO and the product of the crosstalk between H2S and NO. Its level changes are closely related to the occurrence and development of colon cancer, but the specific mechanism is still unclear. It is urgent to develop an accurate HSNO detection method, which will help to better understand its physiological role and help study the relationship between colon cancer and HSNO level changes.

[0004] Existing HSNO detection methods include Fourier transform infrared spectroscopy, 15 N nuclear magnetic resonance method and high-resolution mass spectrometry require the separation and extraction of cells or tissues and cannot be imaged in real time. The fluorescent probe method has high selectivity and high sensitivity and can achieve real-time in situ monitoring of HSNO. At present, there are only two reports on HSNO fluorescent probes, but they cannot achieve non-invasive, real-time, and continuous in vivo detection of HSNO. Therefore, it is necessary to develop a molecular probe with strong tissue penetration, high spatiotemporal resolution, high sensitivity, and suitable for in vivo detection of HSNO.

[0005] Bioluminescence technology produces measurable light signals through chemical reactions in the body and has high sensitivity. In addition, bioluminescence does not rely on external light sources, thus avoiding background noise interference in fluorescence detection and providing a higher signal-to-noise ratio. This technology also has the advantages of non-invasiveness and real-time monitoring, making it suitable for in vivo experiments. Given that the concentration of HSNO in the body is extremely low, traditional detection methods are difficult to provide sufficient sensitivity and specificity. Summary of the invention

[0006] The purpose of the present invention is to synthesize a near-infrared bioluminescent probe BF-5 based on the prior art, using an akalumine luciferin analog as a recognition substrate of luciferase and a structural fragment containing o-phenylenediamine and 2-fluoro-5-nitrobenzoic acid as a specific new recognition group of HSNO. The bioluminescent probe BF-5 has good selectivity, stability, biocompatibility and a low detection limit (327nM), can detect physiological levels of HSNO in HCT116-luc cells, can be applied to bioluminescent imaging of HSNO in HCT116-luc colon cancer nude mouse transplanted tumors, and provides a visual detection tool for revealing the physiological and pathological mechanisms of HSNO, which is of great significance for studying diseases related to HSNO (such as cancer, cardiovascular disease and neurodegenerative diseases, etc.).

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned bioluminescent probe.

[0008] The third object of the present invention is to provide the use of the above-mentioned bioluminescent probe in detecting thionitrite.

[0009] The technical solution of the present invention is as follows:

[0010] A thionitrite near-infrared bioluminescent probe, the structural formula of the luminescent probe is as follows:

[0011]

[0012] The design concept of the thionitrite near-infrared bioluminescent probe BF-5 provided by the present invention is: based on the principle of luciferase-luciferin bioluminescent imaging, a structural fragment containing o-phenylenediamine and 2-fluoro-5-nitrobenzoate is used as a new specific recognition group for HSNO, and a NIR luciferin analog (BL 660 ) was used as the recognition substrate of luciferase to construct the probe BF-5.

[0013] The recognition mechanism of the thionitrite near-infrared bioluminescent probe BF-5 provided by the present invention is as follows: first, the sulfhydryl group of HSNO reacts with -F of 2-fluoro-5-nitrobenzoate, and then an intramolecular cyclization reaction occurs to remove the p-nitrobenzodithione, and then the N=O in HSNO reacts with o-phenylenediamine to generate benzotriazole, and finally the benzotriazole is hydrolyzed and removed to release BF-4, which is then recognized by luciferase to undergo an enzymatic reaction and generate bioluminescence. The product after the reaction of the bioluminescent probe BF-5 with HSNO is verified by high-resolution mass spectrometry. After the probe reacts with HSNO, the compound BF-4 is generated, which proves the reaction mechanism of the bioluminescent probe with HSNO.

[0014] The synthetic route of the thionitrite near-infrared bioluminescent probe is as follows:

[0015]

[0016]

[0017] The preparation method of thionitrite near-infrared bioluminescent probe comprises the following steps:

[0018] (1) In the presence of imidazole, p-hydroxybenzyl alcohol and tert-butyldimethylsilyl chloride react to prepare compound B-1;

[0019] (2) Compound B-1 is reacted with 2-fluoro-5-nitrobenzoic acid in the presence of 4-dimethylaminopyridine and EDCI to prepare compound B-2;

[0020] (3) Compound B-2 and p-toluenesulfonic acid react to prepare compound B-3;

[0021] (4) Compound B-3, K2CO3 and triphosgene are reacted to obtain an intermediate compound, which is then reacted with N-Boc-1,2-phenylenediamine and pyridine to prepare compound B-4;

[0022] (5) Compound B-4 reacts with trifluoroacetic acid to prepare compound B-5;

[0023] (6) Compound a reacts with triethyl 4-phosphonocrotonate and sodium hydride to prepare compound BF-1;

[0024] (7) hydrolyzing compound BF-1 in an alkaline solution, and then acidifying the resulting reaction solution with an acidic solution to prepare compound BF-2;

[0025] (8) In the presence of 4-dimethylaminopyridine and EDCI, compound BF-2 is chemically reacted with S-trityl-D-cysteine ​​methyl ester to prepare an intermediate compound, which is then chemically reacted with triphenylphosphine oxide and trifluoromethanesulfonic anhydride to prepare BF-3;

[0026] (9) hydrolyzing compound BF-3 in an acidic solution, and then acidifying the resulting reaction solution with an alkaline solution to prepare compound BF-4;

[0027] (10) In the presence of HBTU and HOBt, compound BF-4 and compound B-5 are chemically reacted, and the resulting reaction solution is then chemically reacted with DIPEA to prepare the bioluminescent probe BF-5.

[0028] In the present invention, in step (1), the molar ratio of p-hydroxybenzyl alcohol to tert-butyldimethylchlorosilane is 0.8-1.5:1, and may be but not limited to 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.4:1 or 1.5:1. In order to obtain better effects, the molar ratio of p-hydroxybenzyl alcohol to tert-butyldimethylchlorosilane is 1.0:1.

[0029] In step (1), the reaction temperature is 20-40°C, and may be but is not limited to 20°C, 25°C, 30°C, 35°C or 40°C. Preferably, the reaction temperature is 30°C.

[0030] For the present invention, in step (2), the molar ratio of compound B-1 to 2-fluoro-5-nitrobenzoic acid is 1:1.0-1.5, which can be but not limited to 1:1.0, 1:1.1, 1:1.2, 1:1.3 or 1:1.5. In order to obtain better effects, the molar ratio of compound B-1 to 2-fluoro-5-nitrobenzoic acid is 1:1.3.

[0031] In step (2), the molar ratio of compound B-1 to 4-dimethylaminopyridine is 6.0-8.0:1, which may be but is not limited to 6.0:1, 6.5:1, 7.0:1, 7.2:1, 7.3:1, 7.4:1, 7.5:1 or 8.0:1. In order to obtain better effect, the molar ratio of compound B-1 to 4-dimethylaminopyridine is 7.3:1.

[0032] In step (2), the molar ratio of compound B-1 to EDCI is 1:0.8-1.5, and can be but not limited to 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.4:1 or 1.5:1. In order to obtain better effects, the molar ratio of compound B-1 to EDCI is 1:1.0.

[0033] In step (2), the reaction temperature is 20-40°C, and may be but is not limited to 20°C, 25°C, 30°C, 35°C or 40°C. Preferably, the reaction temperature is 30°C.

[0034] For the present invention, in step (3), the molar ratio of compound B-2 to p-toluenesulfonic acid is 4.0-6.0:1, which can be but not limited to 4.0:1, 4.5:1, 5.0:1, 5.1:1, 5.2:1, 5.3:1, 5.5:1 or 6.0:1. In order to obtain better effect, the molar ratio of compound B-2 to p-toluenesulfonic acid is 5.2:1.

[0035] For the present invention, in step (4), the molar ratio of compound B-3 to K2CO3 is 1:3.0-5.0, which can be but not limited to 3.0:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4.0:1, 4.5:1 or 5.0:1. In order to obtain better effects, the molar ratio of compound B-3 to K2CO3 is 1:3.8.

[0036] In step (4), the molar ratio of compound B-3 to triphosgene is 1:0.8-1.5, and can be but not limited to 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.4:1 or 1.5:1. In order to obtain better results, the molar ratio of compound B-3 to triphosgene is 1:1.0.

[0037] In step (4), the molar ratio of compound B-3 to N-Boc-1,2-phenylenediamine is 1.5-2.5:1, and can be but not limited to 1.5:1, 1.8:1, 2.0:1, 2.1:1, 2.3:1, 2.4:1 or 2.5:1. In order to obtain better effect, the molar ratio of compound B-3 to N-Boc-1,2-phenylenediamine is 2.3:1.

[0038] In step (4), the molar ratio of compound B-3 to pyridine is 1:3.5-4.5, which may be but is not limited to 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4.0, 1:4.1, 1:4.2, 1:4.3, 1:4.4 or 1:4.5. In order to obtain better results, the molar ratio of compound B-3 to pyridine is 1:3.9.

[0039] For the present invention, in step (6), the molar ratio of compound a to triethyl 4-phosphonocrotonate is 1.0-2.0:1, preferably 1.6:1; it can be but not limited to 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2.0:1. In order to obtain better effect, the molar ratio of compound a to triethyl 4-phosphonocrotonate is 1.6:1.

[0040] In step (6), the molar ratio of compound a to sodium hydride is 1.0-2.0:1, preferably 1.6:1; it can be but not limited to 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2.0:1. In order to obtain better effect, the molar ratio of compound a to sodium hydride is 1.6:1.

[0041] In the present invention, in step (7), the alkaline solution is a sodium hydroxide solution, a potassium hydroxide solution or a sodium bicarbonate solution; and the acidic solution is a hydrochloric acid solution or a sulfuric acid solution.

[0042] For the present invention, in step (8), the molar ratio of compound BF-2 to S-trityl-D-cysteine ​​methyl ester is 1:0.8-1.5, and can be but not limited to 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.4:1 or 1.5:1. In order to obtain better effects, the molar ratio of compound BF-2 to S-trityl-D-cysteine ​​methyl ester is 1:1.1.

[0043] In step (8), the molar ratio of compound BF-2 to EDCI is 1:3.0-4.0, which may be but is not limited to 1:3.0, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9 or 1:4.0. In order to obtain better results, the molar ratio of compound BF-2 to EDCI is 1:3.4.

[0044] In step (8), the molar ratio of compound BF-2 to 4-dimethylaminopyridine is 1:2.0-3.0, and can be but not limited to 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9 or 1:3.0. In order to obtain better results, the molar ratio of compound BF-2 to 4-dimethylaminopyridine is 1:2.6.

[0045] In step (8), the molar ratio of compound BF-2 to triphenylphosphine oxide is 1:1.0-2.0, and can be but not limited to 1:1.0, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.0. In order to obtain better effect, the molar ratio of compound BF-2 to triphenylphosphine oxide is 1:1.5.

[0046] In step (8), the molar ratio of compound BF-2 to trifluoromethanesulfonic anhydride is 1:0.5-1.0, and can be but not limited to 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9 or 1:1.0. In order to obtain better effect, the molar ratio of compound BF-2 to trifluoromethanesulfonic anhydride is 1:0.7.

[0047] In the present invention, in step (9), the alkaline solution is a sodium hydroxide solution, a potassium hydroxide solution or a sodium bicarbonate solution; and the acidic solution is a hydrochloric acid solution or a sulfuric acid solution.

[0048] For the present invention, in step (10), the molar ratio of compound BF-4 to compound B-5 is 1:1.5-2.5, preferably 1:2.0, and can be but not limited to 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4 or 1:2.5. In order to obtain better effects, the molar ratio of compound BF-4 to compound B-5 is 1:2.0.

[0049] In step (10), the molar ratio of compound BF-4 to HBTU is 1:0.8-1.5, and can be but not limited to 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.4:1 or 1.5:1. To obtain better effects, the molar ratio of compound BF-4 to HBTU is 1:1.0.

[0050] In step (10), the molar ratio of compound BF-4 to HOBt is 1:0.8-1.5, and may be but not limited to 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.4:1 or 1.5:1. To obtain better effects, the molar ratio of compound BF-4 to HOBt is 1:1.0.

[0051] In step (10), the molar ratio of compound BF-4 to DIPEA is 1:0.8-1.5, and can be but not limited to 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.4:1 or 1.5:1. In order to obtain better effects, the molar ratio of compound BF-4 to DIPEA is 1:1.0.

[0052] Adopt the technical scheme of the present invention, the advantages are as follows:

[0053] The present invention provides a thionitrite near-infrared bioluminescent probe. The bioluminescent probe BF-5 has good selectivity, stability, biocompatibility and a low detection limit (327nM), can detect physiological levels of HSNO in HCT116-luc cells, can be applied to bioluminescent imaging of HSNO in HCT116-luc colon cancer nude mouse transplanted tumors, provides a visual detection tool for revealing the physiological and pathological mechanisms of HSNO, and is of great significance for studying diseases related to HSNO (such as cancer, cardiovascular diseases and neurodegenerative diseases, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is the recognition mechanism of the bioluminescent probe BF-5 to detect HSNO;

[0055] Figure 2 is compound B-1 1 H NMR spectrum; among them, 1 H NMR (400MHz, CDCl3) δ7.20-7.18(d,J=8.0Hz,2H),6.80-6.78(d,J=8.0Hz,2H),4.66(s,2H),0.93(s,9H),0.09(s,6H).

[0056] Figure 3 is compound B-2 1 H NMR spectrum; among them, 1 H NMR (400MHz, CDCl3) δ9.03-9.00 (dd, J=6.0, 3.0Hz, 1H), 8.51-8.47 (m, 1H), 7 .43-7.38(m,3H),7.22-7.19(m,2H),4.77(s,2H),0.95(s,9H),0.12(s,6H).

[0057] Figure 4 is compound B-3 1 H NMR spectrum; among them, 1 H NMR (400MHz, CDCl3) δ9.02-9.00 (dd, J=6.0, 3.0Hz, 1H), 8.52-8.48 (m, 1H), 7. 47-7.45(m,2H),7.43-7.39(t,J=9.2Hz,1H),7.25-7.23(m,2H),4.74(s,2H).

[0058] Figure 5is compound B-4 1 H NMR spectrum; among which, 1 H NMR (400MHz, DMSO-D6) δ8.95(s,1H),8.83(s,1H),8.62-8.60(d,J=8.4Hz,1H),8.51(s,1H),7.77-7.72(t,J=9. 2Hz,1H),7.57-7.51(m,4H),7.39-7.37(d,J=8.0Hz,2H),7.10-7.09(d,J=6.0Hz,2H),5.19(s,2H),1.46(s,9H).

[0059] Figure 6 It is compound B-5 1 H NMR spectrum; among them, 1 H NMR (400MHz, DMSO-D6) δ8.86-8.62(m,3H),7.80-7.79(d,J=4.0Hz,1H),7.58(s,2H),7.42- 7.35(m,2H),7.23(s,1H),6.89(s,1H),6.70(s,1H),6.54(s,1H),5.20(s,2H),4.92(s,2H).

[0060] Figure 7 It is the compound BF-1 1 H NMR spectrum; among them, 1 H NMR (400MHz, CDCl3) δ7.48-7.41(dd,J=15.2,11.0Hz,1H),7.35-7.33(d,J=8.8Hz,2H),6.84-6.80(d,J=16.0Hz,1H),6.71-6.65(m,3H),5.8 8-5.84(d,J=16.0Hz,1H),4.24-4.18(q,J=7.0Hz,2H),3.41-3.36(q,J=7.0Hz,4H),1.32-1.29(t,J=7.0Hz,3H),1.20-1.16(t,J=8.0Hz,6H).

[0061] Figure 8 It is the compound BF-2 1 H NMR spectrum; among them, 1H NMR (400MHz, CD3OD) δ7.46-7.42(m,1H),7.39-7.34(m,2H),6.87-6.83(d,J=15.4Hz,1H),6.78-6.75(d,J=12.0H z,1H),6.72-6.66(m,2H),5.85-5.81(d,J=16.0Hz,1H),3.44-3.39(q,J=7.0Hz,4H),1.18-1.84(t,J=8.0Hz,6H).

[0062] Fig. 9 It is the compound BF-3 1 H NMR spectrum; among them, 1 H NMR (400MHz, CDCl3) δ7.28-7.26 (d, J=8.0Hz, 2H), 6.91-6.84 (dd, J=16.0, 12.0Hz, 1H), 6.71-6.58 (m, 4H), 6.48-6.44 (d, J=16. 0Hz,1H),5.11-5.07(t,J=8.0Hz,1H),3.75(s,3H),3.54-3.43(m,2H),3.34-3.29(q,J=7.0Hz,4H),1.13-1.09(t,J=8.0Hz,6H).

[0063] Fig.10 It is the compound BF-4 1 H NMR spectrum; among them, 1 H NMR (400MHz, CD3OD) δ7.36-7.35(d,J=6.4Hz,2H),7.11(s,1H),6.89-6.75(m,2H),6.66-6.64(d,J=6.4Hz,2H),6 .49(s,1H),5.04(s,1H),3.65-3.59(d,J=24.0Hz,2H),3.41-3.40(d,J=4.6Hz,4H),1.17-1.13(t,J=8.0Hz,6H).

[0064] Fig.11 The probe BF-5 1 H NMR spectrum; among them, 1H NMR (400MHz, DMSO-D6) δ8.94(s,1H),8.78-8.76(d,J=8.6Hz,1H),8.69(s,1H),8.36-8.34(d,J=8.8Hz,1H),8.08-8.07(d, J=6.8Hz,1H),7.93-7.91(d,J=8.2Hz,1H),7.87-7.83(t,J=8.0Hz,1H),7.62-7.58(t,J=7.6Hz,1H),7.48-7.46(d,J=6.6H z,2H),7.36-7.34(d,J=7.8Hz,2H),7.19(s,1H),7.14-7.12(d,J=8.8Hz,2H),6.85(s,2H),6.69-6.63(dd,J=14.4,7.6Hz, 3H), 6.53-6.51 (d, J=6.4Hz, 1H), 5.10 (s, 2H), 5.02-4.98 (t, J=7.0Hz, 1H), 3.49-3.44 (m, 2H), 1.11-1.07 (t, J=6.8Hz, 6H).

[0065] Fig.12 is the high-resolution mass spectrum of probe BF-5; where [MH] - calculated for C 39 H 36 FN5O7S,736.2247; found,736.2239.

[0066] Fig.13 is the linear relationship and detection limit of the reaction between probe BF-5 and HSNO; Fig.13 A: Probe BF-5 (10 μM) was incubated with different concentrations of HSNO (0-80 μM, 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM) in Tris-HCl buffer (50 mM, pH = 7.4, 1% DMSO, MgCl2 = 10 mM) at 37°C for 30 min, and then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added to measure the relative bioluminescence intensity; Fig.13 B shows the changes in bioluminescence intensity after the probe BF-5 was incubated with different concentrations of HSNO (0-80 μM) for 10 min; Fig.13 C is the linear relationship between bioluminescence intensity and HSNO concentration (0-9 μM); data are expressed as mean ± SD (n = 3);

[0067] Fig.14is the time gradient of BF-5 response to HSNO; where, Fig.14 A: BF-5 (10 μM) and HSNO (100 μM) were incubated in Tris-HCl buffer (50 mM, pH = 7.4, 1% DMSO, MgCl2 = 10 mM) at 37°C for 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80 and 90 min, respectively, and then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured; Fig.14 B is quantitative expression Fig.13 The total photon flux (p / sec / cm 2 / sr); data are expressed as mean±SD (n=3);

[0068] Fig.15 is the effect of pH on the reaction between BF-5 and HSNO; Fig.15 A: Probe BF-5 (10 μM) and HSNO (100 μM) were incubated in Tris-HCl buffer (50 mM, pH = 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.4; 8.0; 8.5; 9.0; 1% DMSO, MgCl2 = 10 mM) at different pH values ​​at 37°C for 30 min, and then ATP (2 mM, 45 μL) and luciferase (1 mg / mL, 5 μL) were added to measure the relative bioluminescence intensity; Fig.15 B is a quantitative representation of the total photon flux (p / sec / cm 2 / sr); data are expressed as mean±SD (n=3);

[0069] Fig.16 The effect of pH on the reaction of akalumine fluorescein and Luciferase; Fig.16 A is akalumine luciferin (10 μM) added to Tris-HCl buffer (50 mM, pH = 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.4; 8.0; 8.5; 9.0; 1% DMSO, MgCl2 = 10 mM) at different pH at 37°C, then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured; Fig.16 B is quantitative expression Fig.16 The total photon flux (p / sec / cm 2 / sr); data are expressed as mean±SD (n=3);

[0070] Fig.17 is the selectivity of probe BF-5 for HSNO; among them, Fig.17 A: BF-5 (10 μM) was incubated with HSNO (100 μM) and amino acids (blank; 1 mM Gly; 1 mM D-Ala; 1 mM Val; 1 mM Ile; 1 mM L-Cys; 1 mM L-Thr; 1 mM Asp; 1 mM L-Glu; 1 mM L-Arg; 1 mM L-Lys; 1 mM L-His; 1 mM Hcy) in Tris-HCl buffer (50 mM, pH = 7.4, 1% DMSO, MgCl2 = 10 mM) at 37°C for 30 min, and then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured; Fig.17 B is quantitative expression Fig.17 The total photon flux (p / sec / cm 2 / sr); 1. Blank; 2. HSNO (100 μM); 3. Gly (1 mM); 4. D-Ala (1 mM); 5. Val (1 mM); 6. Ile (1 mM); 7. L-Cys (1 mM); 8. L-Thr (1 mM); 9. Asp (1 mM); 10. L-Glu (1 mM); 11. L-Arg (1 mM); 12. L-Lys (1 mM); 13. L-His (1 mM); 14. Hcy (1 mM). Data are expressed as mean ± SD (n = 3);

[0071] Fig.18 is the selectivity of probe BF-5 for HSNO; among them, Fig.18 A is the reaction of BF-5 (10 μM) with HSNO (100 μM) and inorganic salt ions (blank; 1 mM Sn 2+ ; 1mM Cd 2+ ; 1mM Mn 2+ ; 1 mM Co 2+ ; 1 mM Cu 2+ ; 1mM Hg 2+ ; 1mM Zn 2+ ; 1mMFe 2+ ; 1mM Fe 3+ ; 1 mM Ca 2+ ; 1mM Al 3+ ; 1mM Ni 2+ ; 1 mM Mg 2+ ; 1mM Li + ; 1mM Na + ; 1mM K +; 1mM Ag + ) was incubated in Tris-HCl buffer (50 mM, pH = 7.4, 1% DMSO, MgCl2 = 10 mM) at 37°C for 30 min, and then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured; Fig.18 B is quantitative expression Fig.18 The total photon flux (p / sec / cm 2 / sr); 1.Blank; 2.HSNO (100μM); 3.Sn 2+ (1mM); 4.Cd 2+ (1mM); 5.Mn 2+ (1mM); 6.Co 2+ (1mM); 7.Cu 2+ (1mM); 8.Hg 2+ (1mM); 9.Zn 2+ (1mM); 10.Fe 2+ (1mM); 11.Fe 3+ (1mM); 12.Ca 2+ (1mM); 13.Al 3+ (1mM); 14.Ni 2+ (1mM); 15.Mg 2+ (1mM); 16.Li + (1mM); 17.Na + (1mM); 18.K + (1mM); 19.Ag + (1 mM); data are expressed as mean ± SD (n = 3);

[0072] Fig.19 is the selectivity of probe BF-5 for HSNO; Fig.19A: BF-5 (10 μM) was incubated with HSNO (100 μM) and active sulfur (blank; 100 μM Na2S; 100 μM Na2S+100 μM DEA·NONOate; 100 μM Na2S2; 100 μM Na2S2+100 μM DEA·NONOate; 500 μM NaHS; 500 μM Na2SO3; 500 μM NaHSO3; 100 μM CH3SSSCH3; 1 mM GSSG, 500 μM S8; 10 mM GSH) in Tris-HCl buffer (50 mM, pH=7.4, 1% DMSO, MgCl2=10 mM) at 37°C for 30 min, and then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured; Fig.19 B is quantitative expression Fig.19 The total photon flux (p / sec / cm 2 / sr); 1. Blank; 2. HSNO (100 μM); 3. Na2S (100 μM); 4. Na2S (100 μM) + DEA·NONOate (100 μM); 5. Na2S2 (100 μM) + DEA·NONOate (100 μM); 6. Na2S2 (100 μM); 7. NaHS (500 μM); 8. Na2SO3 (500 μM); 9. NaHSO3 (500 μM); 10. CH3SSSCH3 (100 μM); 11. GSSG (1 mM); 12. S8 (500 μM); 13. GSH (10 mM); Data are expressed as mean ± SD (n = 3);

[0073] Fig. 20 is the selectivity of probe BF-5 for HSNO; among them, Fig. 20 A is the reaction of BF-5 (10 μM) with HSNO (100 μM) and ROS (blank; 100 μM H2O2; 100 μM ClO - ; 100μM t BuOOH; 100 μM · OH; 100 μM 1 O2; 100μM O2 ·- ; 100μM NO3 - ; 100μM NO2 - ; 100 μM ONOO -; 1 mM L-AA; 1 mM DHA; 1 mM MGO) were incubated in Tris-HCl buffer (50 mM, pH = 7.4, 1% DMSO, MgCl2 = 10 mM) at 37°C for 30 min, and then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured; Fig. 20 B is quantitative expression Fig. 20 The total photon flux (p / sec / cm 2 / sr); 1. Blank; 2. HSNO (100 μM); 3. H2O2 (100 μM); 4. ClO - (100μM); 5. t BuOOH (100 μM); 6. · OH(100μM);7. 1 O2(100μM);8.O2 ·- (100μM); 9. NO3 - (100μM); 10.NO2 - (100μM); 11.ONOO - (100 μM); 12. L-AA (1 mM); 13. DHA (1 mM); 14. MGO (1 mM); Data are expressed as mean ± SD (n = 3);

[0074] Fig.21 This is a study on the stability of probe BF-5 in Tris-HCl buffer; Fig.21 A: BF-5 (10 μM) was incubated in Tris-HCl buffer (50 mM, pH = 7.4, 1% DMSO, MgCl2 = 10 mM) at 37°C for 10, 20, 30, 40, 50, 60 min, and then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added to measure the relative bioluminescence intensity; Fig.21 B is a quantitative expression Fig.21 The total photon flux (p / sec / cm 2 / sr); data are expressed as mean±SD (n=3);

[0075] Fig. 22 The effect of probe BF-5 on cell survival rate; the effect of co-incubation of HCT116-luc cells with different concentrations of probe BF-5 (0, 5, 10, 25, 50 μM) for 48 h on cell survival rate; the data are expressed as mean ± standard deviation (n = 3);

[0076] Fig.23The effect of probe BF-5 on cell survival rate; the effect of co-incubation of HCT116-luc cells with the probe (final concentration 20 μM) for different time periods (0, 6, 12, 24, 48 h) on cell survival rate; the data are expressed as mean ± standard deviation (n = 3);

[0077] Fig.24 It is the bioluminescence imaging of HSNO detected by probe BF-5; among them, Fig.24 A is HCT116-luc cells; Fig.24 B: probe (20 μM) was incubated with HCT116-luc cells for 30 min; Fig.24 C is the probe (20 μM) co-incubated with HCT116-luc cells for 30 min, followed by the addition of 100 μM HSNO for 30 min; Fig.24 D is the probe (20 μM) co-incubated with HCT116-luc cells for 30 min, followed by the addition of 1 mM GSNO for another 30 min; Fig.24 E: The probe (20 μM) was co-incubated with HCT116-luc cells for 30 min, and then 150 μM Na2S was added for co-incubation for 30 min; Fig.24 F is quantitative expression Fig.24 The total photon flux (p / sec / cm2) that produced the bioluminescent signal in A, 24B, 24C, 24D, and 24E 2 / sr); data are expressed as mean±SD (n=3);

[0078] Fig.25 It is the bioluminescence imaging of HSNO detected by probe BF-5; Fig.25 A is the probe (20 μM) incubated with HCT116-luc cells for 30 min; Fig.25 B is the probe (20 μM) incubated with CBS siRNA-transfected HCT116 cells for 30 min; Fig.25 C, HCT116-luc cells were incubated with AOAA (20 μM) for 45 min and then with the probe (20 μM) for 30 min; Fig.25 D is quantitative expression Fig.25 A. Fig.25 B. Fig.25 The total photon flux (p / sec / cm 2 / sr), data are expressed as mean ± SD (n = 3); Fig.25 E and Fig.25F is the expression level of CBS protein in HCT116-luc cells and CBS siRNA-transfected HCT116-luc cells, respectively; data are expressed as mean ± SD (n = 3), compared with the control group cells, ****P < 0.0001;

[0079] Fig.26 The probe BF-5 was used to detect HSNO in HCT116-luc colon cancer nude mouse transplanted tumors; mice in the Saline group were intraperitoneally injected with normal saline (100 μL), and then injected with probe BF-5 (1.0 mM, 100 μL, DMSO: saline = 1:9) intratumorally 30 min later; mice in the HSNO group were intraperitoneally injected with exogenous HSNO (300 μM, 100 μL) in PBS solution (50 mM, pH = 7.4), and then injected with probe BF-5 (1.0 mM, 100 μL, DMSO: saline = 1:9) intratumorally 30 min later;

[0080] Fig. 27 It is a quantitative expression Fig.26 The total photon flux (p / sec / cm 2 / sr); data are expressed as mean±standard deviation (n=3). DETAILED DESCRIPTION

[0081] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0082] 1. Implementation Methods

[0083] 1 Materials and instruments

[0084] 1.1 Solution preparation

[0085] (1) Preparation of probe BF-5 stock solution: Dissolve BF-5 (7.40 mg, 0.01 mmol) in 1.0 mL of anhydrous DMSO to prepare a 10 mM probe solution. Dilute the stock solution to a 1.0 mM stock solution for later use. Store in a -20°C refrigerator away from light.

[0086] (2) Preparation of Tris-HCl buffer containing MgCl2 (10 mM): Dissolve MgCl2 (9.5 mg, 0.1 mmol) in Tris-HCl (50 mM, pH = 7.4, 10 mL) to prepare Tris-HCl buffer containing MgCl2 (10 mM).

[0087] (3) Preparation of luciferase stock solution: Luciferase (1.0 mg, 13.8 mg / mL) was dissolved in 927.5 μL of Tris-HCl buffer containing 1.0 mg / mL BSA to prepare a 1.0 mg / mL luciferase stock solution. The solution was stored in a -80°C refrigerator in the dark.

[0088] (4) Preparation of GSNO stock solution: Weigh GSNO (11.37 mg, 0.034 mmol), dissolve it in PBS buffer solution (50 mM, pH = 7.4, 33.8 mL) to prepare a 1 mM stock solution, and store it at -20°C.

[0089] (5) Preparation of Na2S stock solution: Weigh Na2S (3.21 mg, 0.041 mmol), dissolve it in PBS buffer solution (50 mM, pH = 7.4, 13.7 mL) to prepare a 3 mM stock solution, and then dilute the stock solution to a 300 μM solution for later use.

[0090] (6) Preparation of HSNO stock solution: In a dark environment, at room temperature and under nitrogen protection, freshly prepared 1 mM GSNO solution and 0.3 mM Na2S solution were placed in a PBS buffer solution (50 mM, pH = 7.4) to obtain a 300 μM HSNO stock solution. Next, the stock solution was diluted to a 100 μM solution and used immediately.

[0091] (7) Preparation of glutathione (GSH) stock solution: Add GSH (15.36 mg, 0.05 mmol) to deionized water (10.0 mL) to prepare a 5.0 mM stock solution. Then dilute the stock solution into 1.0 mM and 10.0 μM solutions for later use.

[0092] (8) Preparation of L-cysteine ​​(L-Cys) stock solution: Cys (6.05 mg, 0.05 mmol) was added to deionized water (10.0 mL) to prepare a 5.0 mM stock solution. The stock solution was then diluted to 1.0 mM and 10.0 μM solutions for later use.

[0093] (9) Preparation of DEA·NONOate stock solution (as a source of NO): DEA·NONOate (15.5 mg, 0.1 mmol) was added to 0.01 M sodium hydroxide solution (10 mL) to prepare a 10.0 mM stock solution. The stock solution was then diluted to 1.0 mM and 10.0 μM solutions for later use.

[0094] (10) Preparation of H2O2 stock solution: Add 1 mL of H2O2 (30%) to deionized water (9.0 mL). Determine the concentration of the H2O2 stock solution by measuring the absorbance at 240 nm. The molar extinction coefficient is 43.6 M -1 cm -1 , the calculation formula is c=A / (bε).

[0095] (11)ClO - Preparation of stock solution: Add NaClO (10%) to deionized water and then measure the absorbance at 209 nm. The molar extinction coefficient is 350 M -1 cm -1 , determination of ClO - The concentration of the stock solution is calculated using the formula c = A / (bε).

[0096] (12) t Preparation of BuOOH stock solution: Commercial products t BuOOH (5 mM).

[0097] (13) · Preparation of OH solution: Add ferrous sulfate (15.20 mg, 0.10 mmol) to hydrogen peroxide solution (10 mL) and mix to prepare 10 mM · OH stock solution.

[0098] (14) 1 Preparation of O2 stock solution: methylene blue (31.98 mg, 0.10 mmol) was added with 5 mL of NaClO4 (1 mM) solution and 5 mL of H2O2 (30%) solution under LED light to prepare a 10 mM stock solution for immediate use.

[0099] (15)O2 ·- Preparation of stock solution: Under nitrogen protection, add KO2 (7.10 mg, 0.10 mmol) to anhydrous DMSO (10 mL) to prepare a 10 mM stock solution for immediate use.

[0100] (16) NO3 - Preparation of stock solution: Add NaNO3 (8.50 mg, 0.1 mmol) to deionized water (10 mL) to prepare a NO stock solution with a concentration of 10 mM. 3- Stock solution.

[0101] (17) NO2 - Preparation of stock solution: Add NaNO2 (6.90 mg, 0.1 mmol) to deionized water (10 mL) to prepare a NO solution with a concentration of 10 mM. 2- Stock solution.

[0102] (18)ONOO - Preparation of stock solution: NaNO2 (0.6 M, 10 mL) and H2O2 (0.7 M, 10 mL) were added to deionized water and stirred vigorously. HCl (0.6 M, 10 mL) was added at 0 °C, and then NaOH solution (1.5 M, 20 mL) was quickly added. ONOO was determined by UV analysis. - The molar extinction coefficient is 1670M -1 cm -1 , the calculation formula is c=A / (bε).

[0103] In addition to the above substances, other stock solutions are prepared, such as amino acids (Gly, D-Ala, Val, Ile, L-Thr, Asp, L-Glu, L-Arg, L-Lys, L-His, Hcy); inorganic salts (Sn 2+ 、Cd 2+ , Mn 2+ 、Co 2+ , Cu 2+ , Hg 2+ 、Zn 2+ , Fe 2+ , Fe 3 + , Ca 2+ 、Al 3+ 、Ni 2+ Mg 2+ , Li + 、Na + , K + 、Ag + ); active sulfur (Na2S2, NaHS, Na2SO3, NaHSO3, CH3SSSCH3, CysSSCys, GSSG, S8) were directly dissolved in deionized water for preparation.

[0104] 1.2 Cells

[0105] Species and strain: HCT116-luc (human colon cancer luciferase-expressing cell line) Source: Zhejiang Nobo Biological Products Co., Ltd.

[0106] 1.3 Experimental animals

[0107] The species and strain were healthy male Balb / C nude mice, weighing between 20 and 25 g, which were obtained from Changzhou Cavens Experimental Animal Co., Ltd.

[0108] 2. Methods

[0109] 2.1 Synthesis of bioluminescent probe BF-5

[0110] Compound B-1 is obtained by substitution reaction of compound p-hydroxybenzyl alcohol and tert-butyldimethylsilyl chloride, compound B-1 and 2-fluoro-5-nitrobenzoic acid are obtained by esterification reaction, compound B-2 is then desilylated by p-toluenesulfonic acid to obtain compound B-3, compound B-3 and N-Boc-1,2-phenylenediamine are condensed by triphosgene to obtain compound B-4, compound B-4 is de-Bocated by trifluoroacetic acid to obtain compound B-5. The specific synthesis route is as follows.

[0111] Compound a (4-diethylaminobenzaldehyde) and triethyl 4-phosphonobutylene acid ester are reacted through nucleophilic addition reaction to obtain compound BF-1, compound BF-1 is hydrolyzed to obtain compound BF-2, compound BF-2 and S-trityl-D-cysteine ​​methyl ester are reacted through amide condensation reaction and cyclization reaction to obtain compound BF-3, compound BF-3 is hydrolyzed to obtain compound BF-4, and compound BF-4 and compound B-5 are reacted through amide condensation reaction to obtain bioluminescent probe BF-5.

[0112] Synthesis of compound B-1: Compound p-hydroxybenzyl alcohol (1.00 g, 8.06 mmol) was dissolved in 20 mL DMF, and the resulting mixed solution was added with imidazole (0.55 g, 8.06 mmol) and tert-butyldimethylsilyl chloride (1.20 g, 8.06 mmol) at 0 ° C. After stirring evenly, the mixture was reacted at 30 ° C for 1 h. The resulting reaction solution was poured into a saturated NH4Cl aqueous solution (200 mL) for extraction and washed with ether (3×50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a white solid, which was purified by silica gel column chromatography (silica, PE: EtOAc, 6: 1 v / v) to obtain 1.80 g of white solid compound B-1, with a yield of 93.78%. R f =0.6, TLC (silica, PE:EtOAc, 2:1v / v).

[0113] Synthesis of compound B-2: Dissolve compound 2-fluoro-5-nitrobenzoic acid (1.84 g, 9.95 mmol), 4-dimethylaminopyridine (125.00 mg, 1.03 mmol), and EDCI (1.90 g, 9.91 mmol) in 25 mL of dichloromethane and stir in an ice-water bath for 5 min. Add compound B-1 (1.80 g, 7.56 mmol) to the resulting mixture and stir in the dark at 30 ° C for 12 h. After the reaction is complete, the resulting reaction solution is concentrated under reduced pressure to obtain a white solid, which is purified by silica gel column chromatography (silica, PE: EtOAc, 4: 1 v / v) to obtain 1.84 g of white solid compound B-2, with a yield of 60.07%. f=0.3, TLC (silica, PE:EtOAc, 2:1v / v).

[0114] Synthesis of compound B-3: Compound B-2 (1.84 g, 4.54 mmol) and p-toluenesulfonic acid (151.80 mg, 0.87 mmol) were dissolved in 6 mL of methanol, and the resulting mixed solution was stirred under argon protection at 20-30°C for 4 h. After the reaction was complete, the resulting reaction solution was concentrated under reduced pressure to obtain a white solid, which was purified by silica gel column chromatography (silica, DCM: MeOH, 60: 1 v / v) to obtain 1.10 g of white solid compound B-3, with a yield of 83.25%. f =0.4, TLC (silica, DCM:MeOH, 30:1v / v).

[0115] Synthesis of compound B-4: First, K2CO3 (2.02 g, 14.46 mmol) and triphosgene (1.10 g, 3.72 mmol) were dissolved in 15 mL of anhydrous THF, and the resulting solution was stirred at 0°C for 1 h under nitrogen protection. Then, an anhydrous THF solution (5 mL) of compound B-3 (1.10 g, 3.78 mmol) was added dropwise to the resulting mixed solution, and the mixture was stirred at 30°C in the dark for 12 h. After the reaction was complete, the filtrate was collected by suction, and the organic phase was concentrated under reduced pressure to obtain a white intermediate.

[0116] N-Boc-1,2-phenylenediamine (330.00 mg, 1.65 mmol) and pyridine (1.21 mL, 14.74 mmol) were dissolved in 15 mL of anhydrous dichloromethane, and stirred at 30 ° C for 30 min under nitrogen protection. Then, an anhydrous dichloromethane solution (5 mL) containing the white intermediate was added dropwise to the above-obtained reaction solution, and stirred at 20-30 ° C for 12 h. After the reaction was complete, the obtained reaction solution was concentrated under reduced pressure to obtain a light yellow oil, which was purified by silica gel column chromatography (silica, DCM: MeOH, 90: 1 v / v) to obtain 794.90 mg of light yellow solid compound B-4, with a yield of 40.04%. R f =0.5, TLC (silica, DCM:MeOH, 30:1v / v).

[0117] Synthesis of compound B-5: Compound B-4 (794.90 mg, 1.52 mmol) and trifluoroacetic acid (1 mL) were dissolved in 5 mL of anhydrous dichloromethane solution and stirred at 20-30°C for 1 h. The resulting reaction solution was concentrated under reduced pressure to obtain a light yellow solid, which was purified by silica gel column chromatography (silica, DCM: MeOH, 100: 1 v / v) to obtain 514.76 mg of light yellow solid compound B-5, with a yield of 79.67%. f =0.4, TLC (silica, DCM:MeOH, 50:1v / v).

[0118] Synthesis of compound BF-1: Compound a (5.00 g, 25.90 mmol) was dissolved in triethyl 4-phosphonobutenoate (4.45 mL, 25.90 mmol) and stirred at 120° C. for 1 h. The resulting reaction solution was distilled under reduced pressure to obtain an orange-yellow oil (used directly in the next step without purification).

[0119] The orange-yellow oil obtained in the first step was slowly added to 10 mL of anhydrous THF containing sodium hydride (727.80 mg, 30.50 mmol), and the reaction was stirred at 0 ° C for 20 min. Then a 5 mL anhydrous THF solution of compound a (2.69 g, 15.18 mmol) was slowly added to the above reaction solution, and the reaction was stirred at 30 ° C for 3 h. The resulting reaction solution was transferred to a saturated NaCl aqueous solution (200 mL) for quenching and extracted with ethyl acetate (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a yellow oil, which was purified by silica gel column chromatography (silica, PE: EtOAc, 25: 1 v / v) to obtain 2.04 g of yellow solid compound BF-1, with a yield of 49.19%. R f =0.5, TLC (silica, PE:EtOAc, 5:1v / v).

[0120] Synthesis of compound BF-2: 1M NaOH solution (0.55mL) was added to a 15mL isopropanol suspension containing BF-1 (2.04g, 7.47mmol), and refluxed at 90°C for 8h. After the reaction was complete, the reaction solution was concentrated under reduced pressure to obtain a yellow oil, which was then added to a 1M HCl solution (30mL). The acidified mixture was poured into a saturated NaCl aqueous solution (200mL) and extracted with chloroform (3×50mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an orange-yellow oil, which was purified by silica gel column chromatography (silica, DCM: MeOH, 50: 1v / v) to obtain 1.74g of an orange-yellow solid compound BF-2 with a yield of 95.02%. Rf =0.3, TLC (silica, DCM:MeOH, 25:1v / v).

[0121] Synthesis of compound BF-3: Compound BF-2 (1.74 g, 7.10 mmol), S-trityl-D-cysteine ​​methyl ester (2.95 g, 7.84 mmol), EDCI (4.58 g, 23.90 mmol) and DMAP (2.22 g, 18.20 mmol) were dissolved in 20 mL of anhydrous DMF and stirred at 20-30 ° C for 24 h under nitrogen protection. The resulting reaction solution was transferred to a saturated aqueous NaCl solution (200 mL) for quenching and extracted with ethyl acetate (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an orange-red oil (no purification required, directly used in the next step).

[0122] Under nitrogen protection, triphenylphosphine oxide (2.96g, 10.64mmol) was dissolved in 14mL of anhydrous dichloromethane solution, and then trifluoromethanesulfonic anhydride (900.00μL, 5.32mmol) was slowly added to the resulting solution, and the reaction was stirred at 0°C for 30min. Subsequently, 10mL of anhydrous dichloromethane solution containing the orange-red oil was slowly added, and the reaction was stirred at 0°C for 10min. The resulting reaction solution was transferred to a saturated NaHCO3 aqueous solution (200mL) for quenching and extracted with dichloromethane (3×50mL). The resulting organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain an orange-red oil, which was purified by silica gel column chromatography (silica, PE: EtOAc, 25: 1v / v) to obtain 1.59g of an orange-red solid compound BF-3, with a yield of 65.07%. R f =0.4, TLC (silica, PE:EtOAc, 5:1v / v).

[0123] Synthesis of compound BF-4: Compound BF-3 (1.59 g, 4.62 mmol) was added to 4M HCl aqueous solution (10 mL) and stirred at 20-30°C for 24 h. The resulting reaction solution was transferred to a saturated NaHCO3 aqueous solution (100 mL) for neutralization and extracted with dichloromethane (3×50 mL). The resulting organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a dark red solid, which was purified by silica gel column chromatography (silica, DCM: MeOH, 10: 1 v / v) to obtain 610.00 mg of dark red solid compound BF-4, with a yield of 39.99%. f =0.3, TLC (silica, DCM:MeOH, 10:1v / v).

[0124] Synthesis of compound BF-5: Compound BF-4 (610.00 mg, 1.85 mmol), B-5 ​​(1.60 g, 3.77 mmol), HBTU (701.60 mg, 1.85 mmol) and HOBt (250.00 mg, 1.85 mmol) were dissolved in 10 mL of anhydrous DMF and stirred at 20-30 ° C for 5 min under nitrogen protection. DIPEA (322.00 μL, 1.85 mmol) was then added to the resulting reaction solution and stirred at 20-30 ° C for 12 h. The resulting reaction solution was transferred to a saturated NaCl aqueous solution (100 mL) for quenching and extracted with ethyl acetate (3×50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a light yellow oil, which was purified by silica gel column chromatography (silica, DCM:MeOH, 100:1 v / v) to obtain 559.00 mg of light yellow solid compound BF-5, with a yield of 40.98%. f =0.4, TLC (silica, DCM:MeOH, 40:1v / v).

[0125] 2.2 Mechanism verification of the recognition of HSNO by probe BF-5

[0126] Under light-proof conditions, probe BF-5 (final concentration 20 μM) and HSNO (final concentration 300 μM) were dissolved in Tris-HCl buffer (50 mM, pH = 7.4, 1% DMSO, MgCl2 = 10 mM) and incubated at 37°C for 30 min. The mixture was extracted with ethyl acetate (3 × 1 mL), the organic phase was concentrated under reduced pressure, the product was separated, and then the reaction product was confirmed by high-resolution mass spectrometry to verify the mechanism of probe BF-5 recognizing HSNO.

[0127] 2.3 Study on the detection performance of bioluminescent probe BF-5 for HSNO

[0128] 2.3.1 In vitro detection performance study

[0129] The mixed system of probe BF-5 and HSNO solution was added to a black 96-well cell culture plate and incubated at 37°C for 30 minutes. ATP (2mM, 45μL) and luciferase (1mg / mL, 5μL) were then added to each well. The blank control group did not add HSNO solution. Imaging was performed using the LB983 NightOWL IILB983 small animal live imager. The bioluminescence mode was selected and the exposure time was set to 120s. Finally, indiGo software was used for image and data analysis. Each data was measured at least 3 times in parallel, and the results were presented as mean±SD.

[0130] 2.3.2 Cellular bioluminescence imaging

[0131] The cells were divided into two groups, group A was HCT116-luc cells, and group B was HCT116-luc cells transfected with CBS siRNA. The above cells were inoculated in a black 96-well plate and cultured in an incubator at 37°C, 5% CO2 for 48 hours. The probe BF-5 (final concentration 20 μM) was added and incubated for 30 minutes before imaging, and gently washed with PBS buffer before imaging.

[0132] The LB983 NightOWL II small animal in vivo imager was used for imaging, and the Luminescence mode was selected with an exposure time of 120 s. Finally, indiGo software was used for image and data analysis. Each data was measured at least 3 times in parallel, and the results were presented as mean ± SD. 2.3.3 In vivo bioluminescence imaging

[0133] The mice were divided into two groups. (1) Nude mice were injected intratumorally with probe BF-5 (1.0 mM, 100 μL, DMSO: saline = 1:9). (2) Nude mice were injected intraperitoneally with exogenous HSNO (300 μM, 100 μL) in PBS solution (50 mM, pH = 7.4) and incubated for 30 min, and then injected intratumorally with probe BF-5 (1.0 mM, 100 μL, DMSO: saline = 1:9).

[0134] The imaging was performed using the LB983 NightOWL IILB983 small animal living imager. Imaging conditions: Luminescence mode was selected, and the exposure time was 120s for imaging. The Living Image software was used for image and data analysis.

[0135] 2.3.4 Data processing

[0136] Data were expressed as mean ± SD and analyzed using SPSS software. The data between the two groups were compared using the student's t-test, and the data between multiple groups were compared using one-way ANOVA with a completely randomized design. P < 0.05 indicated a statistically significant difference.

[0137] 2. Results and Discussion

[0138] 1. Study on the detection performance of bioluminescent probe BF-5 for HSNO

[0139] 1.1 Linear relationship and detection limit of the reaction between probe BF-5 and HSNO

[0140] The present invention investigates the response of probe BF-5 to HSNO. Fig.13 As shown, the probe BF-5 itself has no bioluminescent signal. When the probe BF-5 was incubated with different concentrations of HSNO (0-80μM), the bioluminescent signal gradually increased. In the HSNO concentration range of 0-9μM, the fluorescence intensity showed a good linear relationship with the HSNO concentration. In addition, in Tris-Cl buffer, the probe detection limit was 327nM, indicating that the probe BF-5 has a high detection sensitivity.

[0141] 1.2 Response time of bioluminescent probe BF-5 to HSNO

[0142] In order to evaluate the detection ability of probe BF-5 for HSNO, BF-5 was incubated with HSNO for different time periods, and then ATP and luciferase were added to explore the relationship between the change of bioluminescence intensity over time. Fig.14 As shown in the figure, the response of the probe to HSNO reached a peak at about 30 minutes, and the bioluminescent signal could be stable for 90 minutes. The above results show that the bioluminescent signal generated by BF-5 and HSNO has good stability.

[0143] 1.3 Effect of reaction system pH on the reaction between probe BF-5 and HSNO

[0144] In order to study the effect of different pH environments on the reaction of BF-5 and HSNO, the probe BF-5 (10 μM) and HSNO (100 μM) were incubated at 37°C for 30 min in Tris-HCl buffer (50 mM, pH = 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.4; 8.0; 8.5; 9.0; 1% DMSO, MgCl2 = 10 mM) at different pH values, and then ATP (2 mM, 45 μL) and luciferase (1 mg / mL, 5 μL) were added to start the reaction, and then the relative bioluminescence intensity was measured. Fig.15 As shown, at pH 7.4, the bioluminescence intensity reaches the maximum value; in the pH range of 6.5-7.4 and pH 8.0-9.0, the bioluminescence intensity decreases significantly; in the pH range of 4.0-6.5, the bioluminescence is quenched. This shows that the catalytic activity of luciferase is affected by pH, resulting in changes in the bioluminescence signal. The above results show that the probe BF-5 is suitable for the detection of HSNO under physiological conditions (pH = 7.4).

[0145] 1.4 Effect of reaction system pH on the reaction between akalumine and luciferase

[0146] In order to study the effect of different pH environments on the reaction of akalumine luciferase and luciferase, akalumine luciferase (10 μM) was added to Tris-HCl (50 mM, pH = 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.4; 8.0; 8.5; 9.0; 1% DMSO, MgCl2 = 10 mM) buffer at different pH at 37°C, and then ATP (2 mM, 45 μL) and luciferase (1 mg / mL, 5 μL) were added to start the reaction, and then the relative bioluminescence intensity was measured. Fig.16 As shown in the figure, the bioluminescence intensity reaches the maximum at pH 7.4; in the pH 6.5-7.4 and pH 8.0-9.0 ranges, the bioluminescence intensity decreases significantly; in the pH 4.0-6.5 range, the bioluminescence is almost completely quenched. This shows that the performance of the bioluminescence of akalumine fluorescein in Tris-HCl buffer at different pH values ​​is consistent with the results of probe BF-5 ( Fig.15 ), reflecting that pH can significantly affect the catalytic efficiency and stability of luciferase.

[0147] 1.5 Study on the selectivity of bioluminescent probe BF-5 for HSNO

[0148] Considering the complex environment in the body, the high selectivity of the probe for the analyte is the key to achieve accurate detection. In order to test the selectivity of probe BF-5 for HSNO, the probe BF-5 was firstly mixed with the following interfering substances: amino acids (Gly, D-Ala, Val, Ile, L-Cys, L-Thr, Asp, L-Glu, L-Arg, L-Lys, L-His, Hcy), inorganic salts (Sn 2+ 、Cd 2+ , Mn 2+ 、Co 2+ , Cu 2+ , Hg 2+ 、Zn 2+ , Fe 2+ , Fe 3+ , Ca 2+ 、Al 3+ 、Ni 2+ Mg 2+ , Li + 、Na + , K + 、Ag + ), active sulfur (Na2S, Na2S2, NaHS, Na2SO3, NaHSO3, CH3SSSCH3, GSSG, S8, GSH), active oxygen (H2O2, ClO -, t BuOOH, · OH, 1 O2, O2 ·- ), active nitrogen (NO3 - 、NO2 - 、ONOO - , DEA-NONOate) and other interfering substances (L-AA, DHA, MGO) were incubated for 30 min, and then luciferase was added to start the reaction, and then the bioluminescence intensity was detected. Figure 17-20 As shown, the probe BF-5 has good selectivity and is not interfered by other substances in the body. It will only produce a strong bioluminescent signal when reacting with HSNO, thereby achieving specific detection of HSNO.

[0149] 1.6 Study on the stability of probe BF-5 in Tris-HCl buffer

[0150] Maintaining the stability of the probe in Tris-HCl buffer effectively avoids false positive signals, which is a key prerequisite for accurate detection. In order to study the stability of the probe BF-5, BF-5 was placed in Tris-HCl buffer (50mM, pH=7.4, 1% DMSO, MgCl2=10mM) and incubated at 37°C for 10, 20, 30, 40, 50, and 60 minutes, and then ATP (2mM, 45μL) and luciferase (1mg / mL, 5μL) were added to start the reaction, and then the relative bioluminescence intensity was measured. Fig.21 As shown in the figure, the probe has good stability in Tris-HCl buffer and does not cause nonspecific signals.

[0151] 2. Cellular bioluminescence imaging

[0152] 2.1 Cytotoxicity assay

[0153] The safety of probe BF-5 in cells was investigated using the CCK8 method. Human colon cancer cells stably transfected with luciferase (HCT116-luc) were selected and incubated with different concentrations of probe BF-5 (0, 5, 10, 25, 50 μM) for 48 hours. The results are shown in Fig. 22 As shown, the cell viability was always maintained above 90%, indicating that the probe had low cytotoxicity.

[0154] In order to observe the changes in cell activity over time after the cells were incubated with the probe, the cell activity was further detected after the probe (final concentration 20 μM) was incubated with cells for different time periods (0, 6, 12, 24, and 48 h). Fig.23As shown, after incubation with the probe for 48 h, the cell viability was still above 90%, indicating that the probe had low toxicity to cells and had little effect on cell viability.

[0155] 2.2 Bioluminescence imaging of exogenous HSNO in cells

[0156] The in vitro experiment successfully verified that the probe BF-5 has good imaging performance for HSNO. The present invention further explores the bioluminescent imaging performance of the probe for HSNO in cells. No bioluminescent signal was observed when only HCT116-luc cells were cultured ( Fig.24 A). The probe BF-5 was co-incubated with HCT116-luc cells for 30 min, and a weak bioluminescent signal was observed ( Fig.24 B). When exogenous 100 μM HSNO (a mixture of freshly prepared 0.3 mM GSNO and 0.1 mM Na2S dissolved in PBS buffer was used as the source of HSNO) was introduced into cells co-incubated with the probe, the bioluminescence signal was significantly enhanced ( Fig.24 C), indicating that the probe can effectively detect exogenous HSNO. In order to exclude the interference of GSNO and Na2S on the bioluminescent signal of group C, the probe was first incubated with cells, and then co-incubated with Na2S (150μM) or GSNO (1mM), and no significant enhancement of bioluminescent signal was observed in either group ( Fig.24 D and Fig.24 E) This demonstrates that the probe can specifically detect exogenous HSNO in living cells.

[0157] 2.3 Bioluminescence imaging of HSNO in HCT116-luc cells transfected with CBS-siRNA

[0158] Thionitrite is an important crosstalk product between H2S and NO, and its level changes are usually regulated by H2S and NO. Studies have shown that endogenous H2S in cells can react with exogenous NO donors to generate HSNO in situ. This indicates that H2S plays a key role in the generation of endogenous HSNO in cells. When the CBS gene is knocked out, the intracellular H2S level is significantly reduced, which in turn leads to a decrease in the endogenous HSNO level. The present invention uses CBS-siRNA to transfect HCT116-luc cells to reduce the expression of CBS protein, thereby reducing the endogenous HSNO level. Fig.25 As shown in E and 25F, the expression of CBS protein in HCT116-luc cells transfected with CBS-siRNA was significantly reduced compared with the control group. Fig.25 A and Fig.25 As shown in B, compared with the control group, the bioluminescence intensity of CBS-siRNA transfected cells was significantly reduced, indicating that the intracellular HSNO level was reduced.

[0159] Studies have shown that aminooxyacetic acid (AOAA) is an inhibitor of the H2S synthase cystathionine β-synthase (CBS), which can reduce endogenous H2S levels. In this experiment, AOAA (20μM) was used to pretreat cells for 45 minutes to reduce the production of endogenous H2S, thereby inhibiting the reaction of endogenous H2S with NO and reducing HSNO levels. Subsequently, the probe was incubated and the results showed that the bioluminescence signal was significantly reduced ( Fig.25 C) Description Fig.25 The bioluminescent signal in A is caused by the physiological level of HSNO in cells. The above results show that the probe can detect endogenous and physiological levels of HSNO with high sensitivity.

[0160] 3. Animal-level HSNO bioluminescence imaging

[0161] The present invention continues to investigate the imaging performance of probe BF-5 in the HCT116-luc colon cancer nude mouse transplant tumor model. First, the animals were randomly divided into two groups: Saline group and HSNO group. Two groups of tumor-bearing mice were injected with saline (100 μL) and HSNO (300 μM, 100 μL) by intraperitoneal injection, and then probe BF-5 (1.0 mM, 100 μL) was injected into the tumor 30 minutes later for in vivo imaging. The results are as follows Fig.26 As shown in the figure, no bioluminescent signal was observed in the Saline group. After intraperitoneal injection of exogenous HSNO, the bioluminescent signal at the tumor site of the mouse gradually increased (318 times), reaching a peak at around 30 minutes, and there was still a strong signal at 120 minutes ( Fig. 27 ). It can be seen that the probe BF-5 has good stability and is suitable for detecting HSNO in mice.

[0162] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that it is still possible to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A thionitrite near-infrared bioluminescent probe, the structural formula of which is as follows:

2. A method for preparing the thionitrite near-infrared bioluminescent probe according to claim 1, characterized in that: Its synthetic route is as follows:

3. The method for preparing the thionitrite near-infrared bioluminescent probe according to claim 2, characterized in that: The steps include: (1) In the presence of imidazole, p-hydroxybenzyl alcohol and tert-butyldimethylsilyl chloride react to prepare compound B-1; (2) Compound B-1 is reacted with 2-fluoro-5-nitrobenzoic acid in the presence of 4-dimethylaminopyridine and EDCI to prepare compound B-2; (3) Compound B-2 and p-toluenesulfonic acid react to prepare compound B-3; (4) Compound B-3, K2CO3 and triphosgene are reacted to obtain an intermediate compound, which is then reacted with N-Boc-1,2-phenylenediamine and pyridine to prepare compound B-4; (5) Compound B-4 reacts with trifluoroacetic acid to prepare compound B-5; (6) Compound a reacts with triethyl 4-phosphonocrotonate and sodium hydride to prepare compound BF-1; (7) hydrolyzing compound BF-1 in an alkaline solution, and then acidifying the resulting reaction solution with an acidic solution to prepare compound BF-2; (8) In the presence of 4-dimethylaminopyridine and EDCI, compound BF-2 is chemically reacted with S-trityl-D-cysteine ​​methyl ester to prepare an intermediate compound, which is then chemically reacted with triphenylphosphine oxide and trifluoromethanesulfonic anhydride to prepare BF-3; (9) hydrolyzing compound BF-3 in an acidic solution, and then acidifying the resulting reaction solution with an alkaline solution to prepare compound BF-4; (10) In the presence of HBTU and HOBt, compound BF-4 and compound B-5 are chemically reacted, and the resulting reaction solution is then chemically reacted with DIPEA to prepare the bioluminescent probe BF-5.

4. The method for preparing the thionitrite near-infrared bioluminescent probe according to claim 3, characterized in that: In step (1), the molar ratio of p-hydroxybenzyl alcohol to tert-butyldimethylsilyl chloride is 0.8-1.5:1, preferably 1:1.0; the molar ratio of p-hydroxybenzyl alcohol to imidazole is 0.8-1.5:1, preferably 1.0:1; and the reaction temperature is 20-40°C, preferably 30°C.

5. The method for preparing the thionitrite near-infrared bioluminescent probe according to claim 3, characterized in that: In step (2), the molar ratio of compound B-1 to 2-fluoro-5-nitrobenzoic acid is 1:1.0-1.5, preferably 1:1.3; the molar ratio of compound B-1 to 4-dimethylaminopyridine is 6.0-8.0:1, preferably 7.3:1; the molar ratio of compound B-1 to EDCI is 1:0.8-1.5, preferably 1:1.0; The reaction temperature is 20-40°C, preferably 30°C.

6. The method for preparing the thionitrite near-infrared bioluminescent probe according to claim 3, characterized in that: In step (3), the molar ratio of compound B-2 to p-toluenesulfonic acid is 4.0-6.0:1, preferably 5.2:1; in step (4), the molar ratio of compound B-3 to K2CO3 is 1:3.0-5.0, preferably 1:3.8; the molar ratio of compound B-3 to triphosgene is 1:0.8-1.5, preferably 1:1.0; the molar ratio of compound B-3 to N-Boc-1,2-phenylenediamine is 1.5-2.5:1, preferably 2.3:1; the molar ratio of compound B-3 to pyridine is 1:3.5-4.5, preferably 1:3.

9.

7. The method for preparing the thionitrite near-infrared bioluminescent probe according to claim 3, characterized in that: In step (6), the molar ratio of compound a to triethyl 4-phosphonocrotonate is 1.0-2.0:1, preferably 1.6:1; the molar ratio of compound a to sodium hydride is 1.0-2.0:1, preferably 1.3:1; in step (7) or step (9), the alkaline solution is sodium hydroxide solution, potassium hydroxide solution or sodium bicarbonate solution; The acidic solution is a hydrochloric acid solution or a sulfuric acid solution.

8. The method for preparing the thionitrite near-infrared bioluminescent probe according to claim 3, characterized in that: In step (8), the molar ratio of compound BF-2 to S-trityl-D-cysteine ​​methyl ester is 1:0.8-1.5, preferably 1:1.1; the molar ratio of compound BF-2 to EDCI is 1:3.0-4.0, preferably 1:3.4; the molar ratio of compound BF-2 to 4-dimethylaminopyridine is 1:2.0-3.0, preferably 1:2.6; the molar ratio of compound BF-2 to triphenylphosphine oxide is 1:1.0-2.0, preferably 1:1.5; the molar ratio of compound BF-2 to trifluoromethanesulfonic anhydride is 1:0.5-1.0, preferably 1:0.

7.

9. The method for preparing the thionitrite near-infrared bioluminescent probe according to claim 3, characterized in that: In step (10), the molar ratio of compound BF-4 to compound B-5 is 1:1.5-2.5, preferably 1:2.0; the molar ratio of compound BF-4 to HBTU is 1:0.8-1.5, preferably 1:1.0; the molar ratio of compound BF-4 to HOBt is 1:0.8-1.5, preferably 1:1.0; the molar ratio of compound BF-4 to DIPEA is 1:0.8-1.5, preferably 1:1.

0.

10. Use of the thionitrite near-infrared bioluminescent probe according to claim 1 in detecting thionitrite.

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