A sulfonitroso near-infrared bioluminescent probe and a preparation method and application thereof

Abstract

This invention provides a near-infrared bioluminescent probe of thionitrite, its preparation method, and its application. The bioluminescent probe BF-5 has good selectivity, stability, biocompatibility, and a low detection limit (327 nM). It can detect the physiological level of HSNO in HCT116-luc cells and can be applied to bioluminescent imaging of HSNO in HCT116-luc colon cancer xenografts in nude mice. It 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.

Description

Technical Field

[0001] This invention belongs to the field of chemistry and analytical detection, specifically relating to a near-infrared bioluminescent probe of thionitrite, its preparation method, and its application. Background Technology

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

[0003] In recent years, increasing research has shown that hydrogen sulfide can inhibit or activate NOS activity. NO affects endogenous H2S production by inhibiting or inducing the activity or expression of cystathionine-β-synthase and cystathionine-γ-lyase. It exhibits a biphasic effect on cell proliferation and apoptosis, which is related to factors such as H2S / NO levels, duration of exposure, tumor cell type, and sensitivity. In colorectal 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 antitumor activity. In colorectal cancer, H2S participates in exogenous NO-mediated antitumor migration and invasion activities, suggesting a cross-talk mechanism mediated by the cGMP / VASP signaling pathway between H2S and NO. Therefore, the expression of H2S and NO, as well as their crosstalk, influence the development of colorectal cancer. HSNO is both a source of NO and a product of H2S-NO crosstalk; its level changes are closely related to the occurrence and development of colorectal cancer, but the specific mechanism remains unclear. There is an urgent need to develop accurate HSNO detection methods, which will help to better understand its physiological role and facilitate research on the relationship between colorectal cancer and changes in HSNO levels.

[0004] Existing methods for HSNO detection include Fourier transform infrared spectroscopy, 15 NMR and high-resolution mass spectrometry require the separation and extraction of cells or tissues and cannot provide real-time imaging. Fluorescent probe methods offer high selectivity and sensitivity, enabling real-time in-situ monitoring of HSNO. Currently, only two papers have reported on HSNO fluorescent probes, but these do not provide 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, and high sensitivity, suitable for in vivo detection of HSNO.

[0005] Bioluminescence technology generates measurable light signals through in vivo chemical reactions, exhibiting high sensitivity. Furthermore, bioluminescence does not rely on an external light source, thus avoiding background noise interference found in fluorescence detection and providing a higher signal-to-noise ratio. This technology also offers the advantages of being non-invasive and allowing for real-time monitoring, making it suitable for in vivo experiments. However, given the extremely low concentration of HSNO in vivo, traditional detection methods struggle to provide sufficient sensitivity and specificity. Summary of the Invention

[0006] The purpose of this invention is to synthesize a near-infrared bioluminescent probe, BF-5, based on existing technologies. This probe uses an akalumine luciferin analog as the recognition substrate for luciferase and a structural fragment containing o-phenylenediamine and 2-fluoro-5-nitrobenzoic acid as a novel specific recognition group for HSNO. BF-5 exhibits good selectivity, stability, biocompatibility, and a low detection limit (327 nM), enabling the detection of physiological levels of HSNO in HCT116-luc cells. It can be applied to bioluminescent imaging of HSNO in HCT116-luc colon cancer xenografts in nude mice, providing a visual detection tool for revealing the physiological and pathological mechanisms of HSNO. This is of great significance for studying diseases related to HSNO (such as cancer, cardiovascular diseases, and neurodegenerative diseases).

[0007] A second objective of this invention is to provide a method for preparing the aforementioned bioluminescent probe.

[0008] A third objective of this invention is to provide the application of the above-mentioned bioluminescent probe in the detection of thionitrous acid.

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

[0010] A near-infrared bioluminescent probe for thionitrite, the structural formula of which is shown below:

[0011]

[0012] The design concept of the near-infrared bioluminescent probe BF-5 of thionitrite provided by this invention is as follows: Based on the principle of luciferase-luciferin bioluminescence imaging, a structural fragment containing o-phenylenediamine and 2-fluoro-5-nitrobenzene ester is used as a novel and specific recognition group for HSNO, and a NIR luciferin analogue (BL) is used. 660 The probe BF-5 was constructed using luciferase as a recognition substrate.

[0013] The recognition mechanism of the near-infrared bioluminescent probe BF-5 of thionitrite provided by this invention is as follows: First, the thiol group of HSNO reacts with the -F group of 2-fluoro-5-nitrobenzene ester, resulting in an intramolecular cyclization reaction that removes p-nitrobenzodithione. Then, the N=O group in HSNO reacts with o-phenylenediamine to generate benzotriazole. Finally, benzotriazole is hydrolyzed to release BF-4, which is then recognized by luciferase and undergoes an enzymatic reaction to produce bioluminescence. High-resolution mass spectrometry verification of the product after the reaction of bioluminescent probe BF-5 with HSNO confirmed that the reaction of the probe with HSNO generates compound BF-4, demonstrating the reaction mechanism of the bioluminescent probe with HSNO.

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

[0015]

[0016]

[0017] The preparation method of the near-infrared bioluminescent probe of thionitrous acid includes the following steps:

[0018] (1) Compound B-1 was prepared by chemically reacting p-hydroxybenzyl alcohol and tert-butyldimethylchlorosilane in the presence of imidazole;

[0019] (2) In the presence of 4-dimethylaminopyridine and EDCI, compound B-1 was chemically reacted with 2-fluoro-5-nitrobenzoic acid to prepare compound B-2;

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

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

[0022] (5) Compound B-4 and trifluoroacetic acid are reacted chemically to prepare compound B-5;

[0023] (6) Compound a was chemically reacted with triethyl 4-phosphonobutenoate and sodium hydride to prepare compound BF-1;

[0024] (7) Compound BF-1 was hydrolyzed in an alkaline solution, and the resulting reaction solution was then acidified in an acidic solution to prepare compound BF-2;

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

[0026] (9) Compound BF-3 was hydrolyzed in an acidic solution, and the resulting reaction solution was then acidified in an alkaline solution to prepare compound BF-4;

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

[0028] In this invention, in step (1), the molar ratio of p-hydroxybenzyl alcohol and tert-butyldimethylchlorosilane is 0.8-1.5:1, which may be, but is 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. For better results, the molar ratio of p-hydroxybenzyl alcohol and tert-butyldimethylchlorosilane is 1.0:1.

[0029] In step (1), the reaction temperature is 20-40℃, which can be but is not limited to 20℃, 25℃, 30℃, 35℃ or 40℃, and preferably, the reaction temperature is 30℃.

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

[0033] In step (2), the reaction temperature is 20-40℃, which can be but is not limited to 20℃, 25℃, 30℃, 35℃ or 40℃, and preferably, the reaction temperature is 30℃.

[0034] For the purposes of this invention, in step (3), the molar ratio of compound B-2 to p-toluenesulfonic acid is 4.0-6.0:1, which may be, but is 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. For better results, the molar ratio of compound B-2 to p-toluenesulfonic acid is 5.2:1.

[0035] For the purposes of this invention, in step (4), the molar ratio of compound B-3 to K2CO3 is 1:3.0-5.0, which may be, but is 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. To obtain better results, 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, which can be, but is 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. For 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-phenylene diamine is 1.5-2.5:1, which can be, but is 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. For better results, the molar ratio of compound B-3 to N-Boc-1,2-phenylene diamine 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. For better results, the molar ratio of compound B-3 to pyridine is 1:3.9.

[0039] For the purposes of this invention, in step (6), the molar ratio of compound a to triethyl 4-phosphonobutenoate is 1.0-2.0:1, preferably 1.6:1; it can be, but is 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. To obtain better results, the molar ratio of compound a to triethyl 4-phosphonobutenoate 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 is 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. For better results, the molar ratio of compound a to sodium hydride is 1.6:1.

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

[0042] For the purposes of this invention, in step (8), the molar ratio of compound BF-2 to S-triphenylmethyl-D-cysteine ​​methyl ester is 1:0.8-1.5, which may be, but is 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 results, the molar ratio of compound BF-2 to S-triphenylmethyl-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 can 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. For 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, which may be, but is 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. For 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, which can be, but is 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. For better results, 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, which can be, but is not limited to, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9 or 1:1.0. For better results, the molar ratio of compound BF-2 to trifluoromethanesulfonic anhydride is 1:0.7.

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

[0048] In this 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, but can be, but is 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. For better results, 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, which can be, but is 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. For better results, 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, which can be, but is 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. For better results, 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, which can be, but is 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. For better results, the molar ratio of compound BF-4 to DIPEA is 1:1.0.

[0052] The advantages of using the technical solution of this invention are as follows:

[0053] This invention provides a near-infrared bioluminescent probe for thionitrite. This bioluminescent probe, BF-5, exhibits good selectivity, stability, biocompatibility, and a low detection limit (327 nM). It can detect physiological levels of HSNO in HCT116-luc cells and can be applied to bioluminescent imaging of HSNO in HCT116-luc colon cancer xenografts in nude mice. This 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). Attached Figure Description

[0054] Figure 1 This describes the recognition mechanism of HSNO detection using the bioluminescent probe BF-5.

[0055] Figure 2 It is compound B-1 1 H NMR spectra; among which... 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 It is compound B-2 1 H NMR spectra; among which... 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 It is compound B-3 1 H NMR spectra; among which... 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 5It is compound B-4 1 H NMR spectra; 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 spectra; among which... 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 compound BF-1 1 H NMR spectra; among which... 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 compound BF-2 1 H NMR spectra; among which... 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] Figure 9 It is compound BF-3 1 H NMR spectra; among which... 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] Figure 10 It is compound BF-4 1 H NMR spectra; among which... 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] Figure 11 It's probe BF-5. 1 H NMR spectra; among which... 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] Figure 12 This is a high-resolution mass spectrum of probe BF-5; among which, [MH] - Calculated for C 39 H 36 FN5O7S,736.2247; found,736.2239.

[0066] Figure 13 This describes the linear relationship between the probe BF-5 and HSNO, as well as the detection limit; among which, Figure 13 A is probe BF-5 (10 μM) and 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. Then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured. Figure 13 B represents the change in bioluminescence intensity of probe BF-5 after incubation with different concentrations of HSNO (0-80 μM) for 10 min; Figure 13 C represents the linear relationship between bioluminescence intensity and HSNO concentration (0-9 μM); data are expressed as mean ± SD (n = 3);

[0067] Figure 14It is the time gradient of BF-5 response to HSNO; where, Figure 14 A was prepared by incubating BF-5 (10 μM) and HSNO (100 μM) 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. Then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured. Figure 14 B represents quantitative expression. Figure 13 Total photon flux (p / sec / cm) of bioluminescent signals generated in A 2 / sr); data are expressed as mean ± SD (n = 3);

[0068] Figure 15 The effect of pH on the reaction between BF-5 and HSNO; among which, Figure 15 A. Probe BF-5 (10 μM) and HSNO (100 μM) were incubated at 37°C for 30 min in Tris-HCl buffers at different pH values ​​(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). Then, ATP (2 mM, 45 μL) and luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured. Figure 15 B represents the total photon flux (p / sec / cm) that generates bioluminescent signals in Figure A. 2 / sr); data are expressed as mean ± SD (n = 3);

[0069] Figure 16 The effect of pH on the reaction between akalumine luciferin and Luciferase; among which, Figure 16 A was akalumine fluorescein (10 μM) added to Tris-HCl buffers at different pH values ​​(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 37 °C, followed by the addition of ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL), and the relative bioluminescence intensity was measured. Figure 16 B represents quantitative expression. Figure 16 Total photon flux (p / sec / cm) of bioluminescent signals generated in A 2 / sr); data are expressed as mean ± SD (n = 3);

[0070] Figure 17 It is the selectivity of probe BF-5 for HSNO; among which, Figure 17 A is BF-5 (10 μM) and 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 M-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, then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured. Figure 17 B represents quantitative expression. Figure 17 Total photon flux (p / sec / cm) of bioluminescent signals generated in A 2 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] Figure 18 It is the selectivity of probe BF-5 for HSNO; among which, Figure 18 A represents BF-5 (10 μM), HSNO (100 μM), and inorganic salt ions (blank; 1 mM Sn). 2+ 1mM Cd 2+ ;1mM Mn 2+ 1mM Co 2+ 1mM Cu 2+ 1mM Hg 2+ 1mM Zn 2+ ;1mMFe 2+ 1mM Fe 3+ 1mM Ca 2+ 1mM Al 3+ 1mM Ni 2+ 1mM Mg 2+ 1mM Li + 1mM Na + 1mM K +1mM Ag + The sample 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. The relative bioluminescence intensity was then measured. Figure 18 B represents quantitative expression. Figure 18 Total photon flux (p / sec / cm) of bioluminescent signals generated in A 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 + (1mM); data are expressed as mean ± SD (n = 3);

[0072] Figure 19 It is the selectivity of probe BF-5 for HSNO; Figure 19A was BF-5 (10 μM) and 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 MDEA·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. Then ATP (2 mM, 45 μL) and Luciferase (1 mg / mL, 5 μL) were added, and the relative bioluminescence intensity was measured. Figure 19 B represents quantitative expression. Figure 19 Total photon flux (p / sec / cm) of bioluminescent signals generated in A 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] Figure 20 It is the selectivity of probe BF-5 for HSNO; among which, Figure 20 A represents BF-5 (10 μM), HSNO (100 μM), and ROS (blank; 100 μM H2O2; 100 μM ClO). - 100μM t BuOOH; 100μM · OH; 100μM 1 O2; 100μM O2 ·- 100μM NO3 - 100μM NO2 - 100μM ONOO -1 mM L-AA, 1 mM DHA, and 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. The relative bioluminescence intensity was then measured. Figure 20 B represents quantitative expression. Figure 20 Total photon flux (p / sec / cm) of bioluminescent signals generated in A 2 / sr); 1. Blank; 2. HSNO (100μM); 3. H2O2 (100μM); 4. ClO - (100μM); 5. t BuOOH (100 μM); 6. · OH (100 μM); 7. 1 O2 (100μM); 8.O2 ·- (100μM); 9.NO3 - (100μM); 10 NO2 - (100μM); 11.ONOO - 12. L-AA (1mM); 13. DHA (1mM); 14. MGO (1mM); Data are expressed as mean ± SD (n = 3);

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

[0075] Figure 22 The study investigated the effect of probe BF-5 on cell viability; specifically, 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 viability; data are expressed as mean ± standard deviation (n = 3).

[0076] Figure 23The study investigated the effect of probe BF-5 on cell viability; specifically, the effect of co-incubating HCT116-luc cells with the probe (final concentration 20 μM) for different times (0, 6, 12, 24, 48 h) on cell viability; data are expressed as mean ± standard deviation (n = 3).

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

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

[0079] Figure 26 The probe BF-5 was used to detect HSNO in HCT116-luc colon cancer xenografts in nude mice. Mice in the Saline group were injected intraperitoneally with physiological saline (100 μL), and 30 min later, probe BF-5 (1.0 mM, 100 μL, DMSO:saline = 1:9) was injected into the tumor. Mice in the HSNO group were injected intraperitoneally with exogenous HSNO (300 μM, 100 μL) in PBS solution (50 mM, pH = 7.4), and 30 min later, probe BF-5 (1.0 mM, 100 μL, DMSO:saline = 1:9) was injected into the tumor.

[0080] Figure 27 It is a quantitative expression Figure 26 Total photon flux (p / sec / cm) that generates bioluminescent signals 2 / sr); data are expressed as mean ± standard deviation (n=3). Detailed Implementation

[0081] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0082] I. 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 solution 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 freezer protected from light.

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

[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. Store in a -80°C freezer protected from light.

[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℃.

[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: Under light-protected, room temperature, and nitrogen protection conditions, place freshly prepared 1 mM GSNO solution and 0.3 mM Na2S solution in PBS buffer (50 mM, pH = 7.4) to obtain a 300 μM HSNO stock solution. Then, dilute the stock solution to 100 μM and use 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 to 1.0 mM and 10.0 μM solutions for later use.

[0092] (8) Preparation of L-cysteine ​​(L-Cys) stock solution: Add Cys (6.05 mg, 0.05 mmol) to deionized water (10.0 mL) to prepare a 5.0 mM stock solution. Then dilute the stock solution 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 10 mL of 0.01 M sodium hydroxide solution 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), and 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 the 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 t BuOOH (5mM).

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

[0098] (14) 1 Preparation of O2 stock solution: Methylene blue (31.98 mg, 0.10 mmol) was irradiated with an LED lamp, and 5 mL of NaClO4 (1 mM) solution and 5 mL of H2O2 (30%) solution were added to prepare a 10 mM stock solution, which was prepared and used immediately.

[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, which should be prepared and used immediately.

[0100] (16) NO3 - Preparation of stock solution: Add NaNO3 (8.50 mg, 0.1 mmol) to deionized water (10 mL) to prepare a 10 mM NO solution. 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 10 mM NO solution. 2- Stock solution.

[0102] (18)ONOO - Preparation of the stock solution: Add NaNO₂ (0.6 M, 10 mL) and H₂O₂ (0.7 M, 10 mL) to deionized water and stir vigorously. Add HCl (0.6 M, 10 mL) at 0 °C, then quickly add NaOH solution (1.5 M, 20 mL). Determine the ONOO₂ concentration by UV analysis. - The concentration of , with a molar extinction coefficient of 1670 M -1 cm -1 The calculation formula is c = A / (bε).

[0103] In addition to the substances mentioned above, other stock solutions may be prepared using 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 prepared by directly dissolving them in deionized water.

[0104] 1.2 Cells

[0105] Species and strain: HCT116-luc (human colon cancer luciferase expression cell line) Source: Zhejiang Nuobo Biotechnology Co., Ltd.

[0106] 1.3 Laboratory Animals

[0107] The strain is a healthy male Balb / C nude mouse, weighing between 20-25g, sourced from Changzhou Cavens Laboratory Animal Co., Ltd.

[0108] 2. Method

[0109] 2.1 Synthesis of bioluminescent probe BF-5

[0110] Compound B-1 was obtained by substitution reaction of p-hydroxybenzyl alcohol and tert-butyldimethylchlorosilane. Compound B-1 and 2-fluoro5-nitrobenzoic acid were then esterified to obtain compound B-2. Compound B-2 was then deprotected with p-toluenesulfonic acid to obtain compound B-3. Compound B-3 and N-Boc-1,2-phenylene diamine were then condensed with triphosgene to obtain compound B-4. Compound B-4 was deBoc-treated with trifluoroacetic acid to obtain compound B-5. The specific synthetic route is as follows.

[0111] Compound a (4-diethylaminobenzaldehyde) and triethyl 4-phosphonobutenoate were reacted via nucleophilic addition to yield compound BF-1. Compound BF-1 was then hydrolyzed to yield compound BF-2. Compound BF-2 and S-triphenylmethyl-D-cysteine ​​methyl ester were reacted via amide condensation and cyclization to yield compound BF-3. Compound BF-3 was then hydrolyzed to yield compound BF-4. Compound BF-4 and compound B-5 were reacted via amide condensation to yield the bioluminescent probe BF-5.

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

[0113] Synthesis of compound B-2: 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) were dissolved in 25 mL of dichloromethane and reacted with the solution in an ice-water bath for 5 min. Compound B-1 (1.80 g, 7.56 mmol) was then added to the resulting mixture and stirred in the dark at 30 °C for 12 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, PE:EtOAc, 4:1 v / v) to give 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. The resulting mixture was stirred at 20-30 °C for 4 h under argon protection. 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 give 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. The resulting solution was stirred at 0 °C for 1 h under nitrogen protection. Then, anhydrous THF solution (5 mL) of compound B-3 (1.10 g, 3.78 mmol) was added dropwise to the resulting mixture, and the mixture was stirred at 30 °C in the dark for 12 h. After the reaction was complete, the mixture was filtered and the filtrate was collected. The organic phase was concentrated under reduced pressure to obtain a white intermediate.

[0116] N-Boc-1,2-phenylene diamine (330.00 mg, 1.65 mmol) and pyridine (1.21 mL, 14.74 mmol) were dissolved in 15 mL of anhydrous dichloromethane and reacted under nitrogen protection at 30 °C for 30 min with stirring. Then, an anhydrous dichloromethane solution (5 mL) containing a white intermediate was added dropwise to the above reaction solution, and the reaction was stirred at 20-30 °C for 12 h. After the reaction was complete, the resulting reaction solution was concentrated under reduced pressure to obtain a pale yellow oil, which was purified by silica gel column chromatography (silica, DCM:MeOH, 90:1 v / v) to give 794.90 mg of pale yellow solid compound B-4, with a yield of 40.04%. 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 the mixture was stirred at 20-30 °C for 1 h. The resulting reaction solution was concentrated under reduced pressure to obtain a pale yellow solid, which was purified by silica gel column chromatography (silica, DCM:MeOH, 100:1 v / v) to give 514.76 mg of pale 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 the mixture was stirred at 120 °C for 1 h. The resulting reaction solution was distilled under reduced pressure to obtain an orange-yellow oil (no purification required, used directly in the next step).

[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 mixture was stirred at 0 °C for 20 min. Then, 5 mL of anhydrous THF solution containing compound a (2.69 g, 15.18 mmol) was slowly added to the above reaction solution, and the mixture 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 give 2.04 g of yellow solid compound BF-1, with a yield of 49.19%. f =0.5, TLC (silica, PE:EtOAc, 5:1v / v).

[0120] Synthesis of compound BF-2: 0.55 mL of 1 M NaOH solution was added to 15 mL of isopropanol suspension containing BF-1 (2.04 g, 7.47 mmol), and the mixture was refluxed at 90 °C for 8 h. After the reaction was complete, the resulting reaction solution was concentrated under reduced pressure to obtain a yellow oil, which was then added to 30 mL of 1 M HCl solution. The acidified mixture was poured into 200 mL of saturated NaCl aqueous solution and extracted with chloroform (3 × 50 mL). The resulting 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, 50:1 v / v) to give 1.74 g of orange-yellow solid compound BF-2, with a yield of 95.02%.f =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-triphenylmethyl-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 reacted under nitrogen protection at 20–30 °C for 24 h with stirring. 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 give an orange-red oil (no purification required, used directly in the next step).

[0122] Triphenylphosphine oxide (2.96 g, 10.64 mmol) was dissolved in 14 mL of anhydrous dichloromethane under nitrogen protection. Trifluoromethanesulfonic anhydride (900.00 μL, 5.32 mmol) was then slowly added dropwise to the resulting solution, and the mixture was stirred at 0 °C for 30 min. Subsequently, 10 mL of the above-mentioned anhydrous dichloromethane solution containing the orange-red oil was slowly added dropwise, and the mixture was stirred at 0 °C for 10 min. The resulting reaction solution was transferred to a saturated NaHCO3 aqueous solution (200 mL) for quenching and extracted with dichloromethane (3 × 50 mL). 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, 25:1 v / v) to give 1.59 g of the orange-red solid compound BF-3, with a yield of 65.07%. 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 10 mL of 4 M HCl aqueous solution and stirred at 20-30 °C for 24 h. The resulting reaction solution was transferred to 100 mL of saturated NaHCO3 aqueous solution 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 give a dark red solid. Purification was performed by silica gel column chromatography (silica, DCM:MeOH, 10:1 v / v) to give 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: Compounds 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 reacted under nitrogen protection at 20–30 °C for 5 min with stirring. Then, DIPEA (322.00 μL, 1.85 mmol) was added to the resulting reaction solution, and the reaction was 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 give a pale yellow oil. Purification was performed by silica gel column chromatography (silica, DCM:MeOH, 100:1 v / v) to give 559.00 mg of a pale 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 BF-5 probe for recognizing HSNO

[0126] Under light-protected 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. Extraction was performed with ethyl acetate (3 × 1 mL), and the organic phase was concentrated under reduced pressure. The products were then separated, and high-resolution mass spectrometry was used to confirm the reaction products, thereby verifying the mechanism by which probe BF-5 recognizes HSNO.

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

[0128] 2.3.1 Study on in vitro detection performance

[0129] The mixture of probe BF-5 and HSNO solution was added to black 96-well cell culture plates and incubated at 37°C for 30 min. Then, ATP (2 mM, 45 μL) and luciferase (1 mg / mL, 5 μL) were added to each well. The blank control group did not receive HSNO solution. Imaging was performed using an LB983 NightOWL II LB983 small animal in vivo imaging system. Bioluminescence mode was selected, and the exposure time was set to 120 s. Finally, image and data analysis were performed using indiGo software. Each data point was measured at least three times, and results are presented as mean ± SD.

[0130] 2.3.2 Cell-level bioluminescence imaging

[0131] Cells were divided into two groups: Group A consisted of HCT116-luc cells, and Group B consisted of HCT116-luc cells transfected with CBS siRNA. The cells were seeded in black 96-well plates and cultured at 37°C with 5% CO2 for 48 hours. After incubation with probe BF-5 (final concentration 20 μM) for 30 minutes, imaging was performed. Cells were gently washed with PBS buffer before imaging.

[0132] Imaging was performed using an LB983 NightOWL II small animal in vivo imager in Luminescence mode with an exposure time of 120 s. Finally, image and data analysis was performed using indiGo software. Each data point was measured at least three times in parallel, and results are presented as mean ± SD. 2.3.3 In vivo bioluminescence imaging

[0133] 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), incubated for 30 min, and then injected intratumorally with probe BF-5 (1.0 mM, 100 μL, DMSO:saline = 1:9).

[0134] Imaging was performed using the LB983 NightOWL II LB983 small animal live imaging system. Imaging conditions: Luminescence mode was selected, and the exposure time was 120 seconds. Living Image software was used for image and data analysis.

[0135] 2.3.4 Data Processing

[0136] Data are expressed as mean ± standard deviation (mean ± SD), and statistical analysis was performed using SPSS software. Student's t-test was used to compare data between two groups, and one-way ANOVA with a completely randomized design was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0137] II. Results and Discussion

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

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

[0140] This invention examines the response of probe BF-5 to HSNO. For example... Figure 13 As shown, probe BF-5 itself does not exhibit a bioluminescent signal. However, the bioluminescent signal gradually increases after incubation with different concentrations of HSNO (0-80 μM). Within the HSNO concentration range of 0-9 μM, the fluorescence intensity shows a good linear relationship with the HSNO concentration. Furthermore, the detection limit of the probe in Tris-Cl buffer is 327 nM, indicating that probe BF-5 possesses high detection sensitivity.

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

[0142] To evaluate the detection capability of probe BF-5 for HSNO, BF-5 was incubated with HSNO for different times, followed by the addition of ATP and luciferase, and the relationship between bioluminescence intensity and time was investigated. Figure 14 As shown, the probe and HSNO response reached their peak at approximately 30 minutes, and the bioluminescent signal remained stable for 90 minutes. These results indicate that the bioluminescent signal generated by BF-5 and HSNO exhibits good stability.

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

[0144] To investigate the effect of different pH environments on the reaction between BF-5 and HSNO, probe BF-5 (10 μM) and HSNO (100 μM) were incubated at 37°C for 30 min in Tris-HCl buffers at different pH values ​​(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). Then, ATP (2 mM, 45 μL) and luciferase (1 mg / mL, 5 μL) were added to initiate the reaction, and the relative bioluminescence intensity was measured. Figure 15 As shown, the bioluminescence intensity reaches its maximum at pH 7.4; it significantly decreases within the pH ranges of 6.5–7.4 and 8.0–9.0; and it is quenched within the pH range of 4.0–6.5. This indicates that the catalytic activity of luciferase is affected by pH, leading to changes in the bioluminescence signal. These results demonstrate that probe BF-5 is suitable for the detection of HSNO under physiological conditions (pH = 7.4).

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

[0146] To investigate the effect of different pH environments on the reaction between akalumine luciferin and luciferase, akalumine luciferin (10 μM) was added to Tris-HCl buffers at different pH values ​​(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 37°C. Then, ATP (2 mM, 45 μL) and luciferase (1 mg / mL, 5 μL) were added to initiate the reaction, and the relative bioluminescence intensity was subsequently measured. Figure 16 As shown, the bioluminescence intensity reached its maximum at pH 7.4; it decreased significantly in the pH ranges of 6.5–7.4 and 8.0–9.0; and was almost completely quenched in the pH range of 4.0–6.5. This indicates that the bioluminescence performance of akalumine luciferin in Tris-HCl buffer at different pH values ​​is consistent with the results of probe BF-5. Figure 15 This reflects that pH significantly affects the catalytic efficiency and stability of luciferase.

[0147] 1.5 Selectivity study of bioluminescent probe BF-5 for HSNO

[0148] Considering the complex environment within organisms, high selectivity of probes for analytes is crucial for accurate detection. To test the selectivity of probe BF-5 for HSNO, probe BF-5 was first reacted 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) and 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 + ), reactive sulfur (Na2S, Na2S2, NaHS, Na2SO3, NaHSO3, CH3SSSCH3, GSSG, S8, GSH), reactive oxygen (H2O2, ClO) -, t BuOOH、 · OH、 1 O2, O2 ·- ), reactive nitrogen (NO3) - NO2 - ONOO - The mixture was incubated with DEA-NONOate and other interfering substances (L-AA, DHA, MGO) for 30 minutes, then luciferase was added to initiate the reaction, followed by detection of bioluminescence intensity. Figure 17-20 As shown, probe BF-5 has good selectivity and is not affected by other substances in the body. It only produces a strong bioluminescent signal when it reacts with HSNO, thus achieving specific detection of HSNO.

[0149] 1.6 Stability study of probe BF-5 in Tris-HCl buffer

[0150] Maintaining probe stability in Tris-HCl buffer effectively avoids false positive signals, a crucial prerequisite for accurate detection. To investigate the stability of probe BF-5, BF-5 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, and 60 min. Then, ATP (2 mM, 45 μL) and luciferase (1 mg / mL, 5 μL) were added to initiate the reaction, followed by measurement of relative bioluminescence intensity. Figure 21 As shown, the probe exhibits good stability in Tris-HCl buffer and does not induce nonspecific signals.

[0151] 2. Cell-level bioluminescence imaging

[0152] 2.1 Cytotoxicity assay

[0153] The safety of probe BF-5 in cells was investigated using the CCK8 assay. Human colon cancer cells stably transfected with luciferase (HCT116-luc) were co-incubated with different concentrations of probe BF-5 (0, 5, 10, 25, 50 μM) for 48 h. Results are as follows: Figure 22 As shown, the cell survival rate remained above 90%, indicating that the probe had low cytotoxicity.

[0154] To observe the changes in cell viability over time after incubation with the probe, cell viability was further examined after incubation with the probe (final concentration 20 μM) for different times (0, 6, 12, 24, 48 h). Figure 23As shown, after 48 hours of incubation with the probe, the cell survival rate was still above 90%, indicating that the probe had low toxicity to cells and had little impact on cell survival.

[0155] 2.2 Bioluminescent imaging of exogenous HSNO in cells

[0156] In vitro experiments successfully verified that the probe BF-5 has good imaging performance for HSNO. This invention further explores the probe's bioluminescent imaging performance for intracellular HSNO. No bioluminescent signal was observed when HCT116-luc cells were cultured alone. Figure 24 A). When probe BF-5 was co-incubated with HCT116-luc cells for 30 min, a weak bioluminescent signal was observed. Figure 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 as the source of HSNO) was introduced into cells that had been co-incubated with the probe, a significant enhancement of bioluminescent signal was observed. Figure 24 C) indicates that the probe can effectively detect exogenous HSNO. To eliminate the interference of GSNO and Na2S on the bioluminescence signal of group C, the probe was first incubated with cells, and then co-incubated with Na2S (150 μM) or GSNO (1 mM), respectively. The results showed that no significantly enhanced bioluminescence signal was observed in either group. Figure 24 D and Figure 24 E). This demonstrates that the probe can specifically detect exogenous HSNO in living cells.

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

[0158] Thionidium thionitrite, as a significant crosstalk product of H2S and NO, is typically regulated by both 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 crucial role in the generation of endogenous HSNO in cells. When the CBS gene is knocked out, intracellular H2S levels are significantly reduced, leading to a decrease in endogenous HSNO levels. This invention uses CBS-siRNA transfection of HCT116-luc cells to reduce CBS protein expression, thereby reducing endogenous HSNO levels. Figure 25 As shown in E and 25F, CBS protein expression was significantly reduced in HCT116-luc cells transfected with CBS-siRNA compared to the control group. Figure 25 A and Figure 25 As shown in Figure B, compared with the control group, the bioluminescence intensity of CBS-siRNA transfected cells was significantly reduced, suggesting a decrease in intracellular HSNO levels.

[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, cells were pretreated with AOAA (20 μM) for 45 min to reduce the production of endogenous H2S, thereby inhibiting the reaction between endogenous H2S and NO and reducing HSNO levels. Subsequent incubation with a probe showed a significant reduction in bioluminescent signal. Figure 25 C). Explanation Figure 25 The bioluminescent signal in A was induced by HSNO at the physiological level of the cell. These results indicate that the probe can detect endogenous and physiological levels of HSNO with high sensitivity.

[0160] 3. Animal-level HSNO bioluminescence imaging

[0161] This invention further investigates the imaging performance of probe BF-5 in a nude mouse xenograft model of HCT116-luc colon cancer. Animals were first randomly divided into two groups: a Saline group and an HSNO group. Both groups of tumor-bearing mice were injected intraperitoneally with saline (100 μL) and HSNO (300 μM, 100 μL), respectively. Thirty minutes later, probe BF-5 (1.0 mM, 100 μL) was injected intratumorally for in vivo imaging. Results are as follows: Figure 26 As shown, no bioluminescent signal was observed in the Saline group. Following intraperitoneal injection of exogenous HSNO, the bioluminescent signal at the tumor site in mice gradually increased (318-fold), reaching a peak at approximately 30 minutes, and still exhibiting a strong signal at 120 minutes. Figure 27 Therefore, the BF-5 probe exhibits 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, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications may still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions may be made to some of the technical features. Such modifications or substitutions 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 near-infrared bioluminescent probe for thionitrous acid, the structural formula of which is shown below:

2. A method for preparing the near-infrared bioluminescent probe of thionitrous acid as described in claim 1, characterized in that, Its synthetic route is as follows:

3. The method for preparing the near-infrared bioluminescent probe of thionitrous acid according to claim 2, characterized in that, Includes the following steps: (1) Compound B-1 was prepared by chemically reacting p-hydroxybenzyl alcohol and tert-butyldimethylchlorosilane in the presence of imidazole; (2) In the presence of 4-dimethylaminopyridine and EDCI, compound B-1 was chemically reacted with 2-fluoro-5-nitrobenzoic acid to prepare compound B-2; (3) Compound B-2 and p-toluenesulfonic acid were chemically reacted to prepare compound B-3; (4) Compound B-3, K2CO3 and triphosgene are chemically reacted to obtain an intermediate compound, which is then chemically reacted with N-Boc-1,2-phenylene diamine and pyridine to prepare compound B-4. (5) Compound B-4 and trifluoroacetic acid are reacted chemically to prepare compound B-5; (6) Compound a was chemically reacted with triethyl 4-phosphonobutenoate and sodium hydride to prepare compound BF-1; (7) Compound BF-1 was hydrolyzed in an alkaline solution, and the resulting reaction solution was then acidified in an acidic solution to prepare compound BF-2. (8) In the presence of 4-dimethylaminopyridine and EDCI, compound BF-2 was chemically reacted with S-triphenylmethyl-D-cysteine ​​methyl ester to prepare an intermediate compound, which was then chemically reacted with triphenylphosphine oxide and trifluoromethanesulfonic anhydride to prepare BF-3. (9) Compound BF-3 was hydrolyzed in an acidic solution, and the resulting reaction solution was then acidified in an alkaline solution to prepare compound BF-4; (10) In the presence of HBTU and HOBt, compound BF-4 and compound B-5 undergo a chemical reaction, and the resulting reaction solution is then chemically reacted with DIPEA to prepare the bioluminescent probe BF-5.

4. The method for preparing the near-infrared bioluminescent probe of thionitrous acid according to claim 3, characterized in that, In step (1), the molar ratio of p-hydroxybenzyl alcohol to tert-butyldimethylchlorosilane is 0.8-1.5:1; the molar ratio of p-hydroxybenzyl alcohol to imidazole is 0.8-1.5:1; and the reaction temperature is 20-40℃.

5. The method for preparing the near-infrared bioluminescent probe of thionitrous acid according to claim 4, characterized in that, In step (1), the molar ratio of p-hydroxybenzyl alcohol to tert-butyldimethylchlorosilane is 1:1.0; the molar ratio of p-hydroxybenzyl alcohol to imidazole is 1.0:1; and the reaction temperature is 30℃.

6. The method for preparing the near-infrared bioluminescent probe of thionitrous acid 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; the molar ratio of compound B-1 to 4-dimethylaminopyridine is 6.0-8.0:1; the molar ratio of compound B-1 to EDCI is 1:0.8-1.5; and the reaction temperature is 20-40℃.

7. The method for preparing the near-infrared bioluminescent probe of thionitrous acid according to claim 6, characterized in that, In step (2), the molar ratio of compound B-1 to 2-fluoro-5-nitrobenzoic acid is 1:1.3; the molar ratio of compound B-1 to 4-dimethylaminopyridine is 7.3:

1. The molar ratio of compound B-1 to EDCI was 1:1.0; the reaction temperature was 30℃.

8. The method for preparing the near-infrared bioluminescent probe of thionitrous acid 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; in step (4), the molar ratio of compound B-3 to K2CO3 is 1:3.0-5.0; the molar ratio of compound B-3 to triphosgene is 1:0.8-1.5; the molar ratio of compound B-3 to N-Boc-1,2-phenylene diamine is 1.5-2.5:1; and the molar ratio of compound B-3 to pyridine is 1:3.5-4.

5.

9. The method for preparing the near-infrared bioluminescent probe of thionitrous acid according to claim 8, characterized in that, In step (3), the molar ratio of compound B-2 to p-toluenesulfonic acid is 5.2:1; in step (4), the molar ratio of compound B-3 to K2CO3 is 1:3.8; the molar ratio of compound B-3 to triphosgene is 1:1.0; the molar ratio of compound B-3 to N-Boc-1,2-phenylene diamine is 2.3:1; and the molar ratio of compound B-3 to pyridine is 1:3.

9.

10. The method for preparing the near-infrared bioluminescent probe of thionitrous acid according to claim 3, characterized in that, In step (6), the molar ratio of compound a to triethyl 4-phosphonobutenoate is 1.0-2.0:1; the molar ratio of compound a to sodium hydride is 1.0-2.0:

1.

11. The method for preparing the near-infrared bioluminescent probe of thionitrous acid according to claim 10, characterized in that, In step (6), the molar ratio of compound a to triethyl 4-phosphonobutenoate is 1.6:1; the molar ratio of compound a to sodium hydride is 1.3:

1.

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

0.

13. The method for preparing the near-infrared bioluminescent probe of thionitrous acid according to claim 12, characterized in that, In step (8), the molar ratio of compound BF-2 to S-triphenylmethyl-D-cysteine ​​methyl ester is 1:1.1; the molar ratio of compound BF-2 to EDCI is 1:3.4; the molar ratio of compound BF-2 to 4-dimethylaminopyridine is 1:2.6; the molar ratio of compound BF-2 to triphenylphosphine oxide is 1:1.5; and the molar ratio of compound BF-2 to trifluoromethanesulfonic anhydride is 1:0.

7.

14. The method for preparing the near-infrared bioluminescent probe of thionitrous acid 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; the molar ratio of compound BF-4 to HBTU is 1:0.8-1.

5. The molar ratio of compound BF-4 to HOBt is 1:0.8-1.5; the molar ratio of compound BF-4 to DIPEA is 1:0.8-1.

5.

15. The method for preparing the near-infrared bioluminescent probe of thionitrous acid according to claim 14, characterized in that, In step (10), the molar ratio of compound BF-4 to compound B-5 is 1:2.0; the molar ratio of compound BF-4 to HBTU is 1:1.

0. The molar ratio of compound BF-4 to HOBt is 1:1.0; the molar ratio of compound BF-4 to DIPEA is 1:1.

0.

16. The application of the near-infrared bioluminescent probe for thionitrous acid as described in claim 1 in the detection of thionitrous acid.

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