Sulfhydryl compound activated chemiluminescent probe as well as synthesis method and application thereof

Abstract

The invention discloses a sulfhydryl compound activated chemiluminescence probe as well as a synthesis method and application thereof, and belongs to the technical field of biological probes. The preparation method comprises the following steps: uniformly mixing prop-2-ene-1-yl N-[4-(hydroxymethyl) phenyl] carbamate with an acetonitrile mixture of NaI, then adding trimethylchlorosilane, stirring at room temperature in a dark place, diluting, washing and drying to obtain prop-2-ene-1-yl N-[4-(iodomethyl) phenyl] carbamate; and then adding 3-[2-hydroxy-4-(4-methoxy spiro [1, 2-dioxocyclobutane-3, 2 '-tricyclo [3.3. 1.13, 7] decyl]) phenyl]-2-acrylonitrile, mixing, reacting in a dark place, and purifying to obtain the sulfydryl compound activated chemiluminescent probe. The probe can detect a target spot in real time in a complex biological environment under the catalysis of a Ru catalyst, is used for detecting the activity of sulfhydryl-containing compounds, albumin, clinical blood copper ions and sulfhydryl-containing compounds in living cells, and is beneficial to the development of the BCR probe in the fields of biological application and optical imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of biological probes, and in particular relates to a thiol compound activated chemiluminescent probe and a synthesis method and application thereof. Background Art

[0002] "Bioorthogonal chemistry" was first proposed by Bertozzi and colleagues in 2003. It specifically refers to a type of high-yield chemical synthesis reaction that is not disturbed by the complex biological system environment and has the characteristics of rapidity and selectivity. At the same time, in contrast to bioorthogonal chemistry, bioorthogonal bond cleavage reactions (BCRs) refer to a type of biocompatible chemical decomposition reaction that cleaves bonds at target sites. It has the characteristics of rapidity and high selectivity and can be used in the field of bioassays; however, there are few studies on this type of reaction (based on 164 of 3096 articles in the PubMed database from 2003 to 2023), indicating that the potential clinical imaging value and application value of BCR-type probes in the field of biological detection are far underestimated.

[0003] Currently reported BCR-type probes are all labeled using chromophore and fluorophore methods ( Figure 1 ), after irradiation with external light, BCR-type probes can be used to quickly and easily detect the target site qualitatively and quantitatively by optical imaging. Nevertheless, the application of BCR-type probes in complex biological systems is limited due to the need for long-term irradiation. Therefore, providing a new and efficient real-time detection method based on BCR type can provide unprecedented opportunities for studying biological mechanisms in living systems. Summary of the invention

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a thiol compound activated chemiluminescent probe and a synthesis method and application thereof, so as to solve the technical problem that the current BCR probe requires continuous irradiation from an external light source.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention discloses a thiol compound activated chemiluminescent probe, the structural formula of which is as follows:

[0007]

[0008] The second aspect of the present invention discloses a method for synthesizing the above-mentioned thiol compound activated chemiluminescent probe, the steps of which are as follows:

[0009] 1) mixing prop-2-en-1-yl N-[4-(hydroxymethyl)phenyl]carbamate and a mixture of NaI and acetonitrile, then adding trimethylsilyl chloride, stirring at room temperature in the dark, diluting, washing, and drying to obtain prop-2-en-1-yl N-[4-(iodomethyl)phenyl]carbamate;

[0010] 2) Prop-2-en-1-yl N-[4-(iodomethyl)phenyl]carbamate is mixed with 3-[2-hydroxy-4-(4-methoxyspiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decane])phenyl]-2-acrylonitrile, reacted in the dark, purified, and obtained a thiol compound activated chemiluminescent probe.

[0011] Preferably, in step 1), the preparation method of prop-2-en-1-yl N-[4-(hydroxymethyl)phenyl]carbamate is: dissolving p-aminobenzyl alcohol in THF, NaHCO 3 and ddH 2 O, and then add allyl chloroformate to the mixed solution and react overnight to obtain prop-2-en-1-yl N-[4-(hydroxymethyl)phenyl]carbamate.

[0012] Preferably, in step 1), the equivalent ratio of prop-2-en-1-yl N-[4-(hydroxymethyl)phenyl]carbamate, the acetonitrile mixture of NaI and trimethylsilyl chloride is 1:5:5.

[0013] Preferably, in step 1), dilution is performed with EA.

[0014] Preferably, in step 1), the washing step is: saturated Na 2 S 2 O 3 Wash once, and then wash twice with saturated brine.

[0015] Preferably, in step 1), anhydrous Na 2 SO 4 The organic layer was dried.

[0016] Preferably, in step 2), the preparation method of 3-[2-hydroxy-4-(4-methoxyspiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decane])phenyl]-2-acrylonitrile is: 3-(methoxytricyclo〔3.3.1.13,7〕decane-2-ylidenemethyl)-phenol acrylic acid and anhydrous K 2 CO 3 Dissolve in dichloromethane, add methylene blue solution, and continue to pass O 2 , irradiate the reaction with light, freeze the reaction mixture, and wash with N 2 Fully replace the O in the reaction vessel 2, to obtain 3-[2-hydroxy-4-(4-methoxyspiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decane])phenyl]-2-propenenitrile.

[0017] The third aspect of the present invention discloses a BAC biosensor obtained by incubating the above-mentioned thiol compound-activated chemiluminescent probe with a Ru catalyst.

[0018] Preferably, the molar concentration ratio of the thiol compound activated chemiluminescent probe to the Ru catalyst is 10:1.

[0019] The fourth aspect of the present invention discloses the use of the above-mentioned thiol compound-activated chemiluminescent probe or the above-mentioned BAC biosensor in determining the content of thiol compounds.

[0020] The fifth aspect of the present invention discloses the use of the above-mentioned thiol compound-activated chemiluminescent probe or the above-mentioned BAC biosensor in determining the albumin content.

[0021] The sixth aspect of the present invention discloses the use of the above-mentioned thiol compound-activated chemiluminescent probe or the above-mentioned BAC biosensor in detecting the activity of thiol compounds in living cells.

[0022] The seventh aspect of the present invention discloses the use of the above-mentioned thiol compound-activated chemiluminescent probe or the above-mentioned BAC biosensor in the preparation of tumor diagnosis and imaging reagents.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a thiol compound activated chemiluminescent probe, the probe structure includes an acryl carbamate that can be specifically sheared by a thiol compound, a (adamantane)-1,2-dioxacyclic chemiluminescent group, and a self-cleavable connecting group. Compared with traditional probes, the probe in the present invention is designed with a connecting group having an allyl carbonyl group, so that the probe can achieve an efficient deprotection reaction through chemical excitation (Ru catalyst catalysis), release active ingredients and stimulate chemiluminescence, thereby achieving the purpose of detecting thiol compounds. The probe of the present invention is based on the reaction type of BCR and the chemiluminescent imaging of dioxacyclics, and accelerates the deprotection reaction through Ru catalysis, which significantly improves the detection efficiency and selectivity of the probe, and is particularly suitable for the detection of thiol compounds.

[0025] The present invention provides a method for synthesizing a thiol compound activated chemiluminescent probe. The raw materials used in the synthesis steps are common commodities that have been mass-produced or compounds that can be synthesized in one step from common commodities. The method has low hazard, mild and controllable reaction conditions, and simple operation. The method can solve the limitations of existing BCR imaging detection methods and make the BCR probe an effective tool for studying biological mechanisms in complex life environments. 2 It is the first designed and synthesized first-generation BCR-excited chemiluminescent probe. Because it can detect target sites in real time in complex biological environments, it can promote the application and development of high-efficiency chemiluminescent probes in biomedicine and imaging fields, and can also provide new synthetic ideas for a series of new detection methods based on BCR types to study biological mechanisms in life systems.

[0026] The present invention provides a BAC biosensor, which is formed by mixing and incubating a thiol compound-activated chemiluminescent probe and a Ru catalyst, and can be used to detect the content of thiol compounds. The working principle of the BAC biosensor is as follows: first, the Ru catalyst and the thiol compound-activated chemiluminescent probe undergo ligand conversion to form a Ru complex, followed by an oxidative addition reaction. During the Ru-catalyzed oxidation process, the Alloc protective group of the thiol compound-activated chemiluminescent probe is cleaved and decarboxylated to generate CO 2 , releasing a compound intermediate with an amino group. Secondly, the intermediate leaves through electron transfer to generate a PAD intermediate, which is then chemically excited to generate a molecule in a high-energy state, and then returns to the ground state and releases a photon. Finally, in the presence of R-SH with strong nuclear affinity, the Ru catalyst is regenerated and a new catalytic cycle is initiated, thereby promoting the chemiluminescence of the BAC probe. In this process, the thiol compound-activated chemiluminescent probe works synergistically with the Ru catalyst. Relying on the catalytic action of the Ru catalyst, the thiol compound-activated chemiluminescent probe releases photons through a catalytic reaction, enabling the biosensor to provide a higher signal-to-noise ratio without relying on an external light source, and to achieve high sensitivity and real-time detection in a complex biological environment. Compared with traditional biosensors, the advantage of BAC biosensors is that they can break through the limitations of traditional fluorescence imaging, avoid the interference of sample background fluorescence, and provide clearer cell-level images. They are suitable for the detection of target targets, determination of albumin content, clinical blood copper ion detection, monitoring of thiol compound activity in living cells, and visualization of H2S content in breast cancer mouse models. This technology will contribute to the development of BCR probes in the fields of biological applications and optical imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The following is a flow chart of the preparation process of the existing BCR type probe;

[0028] Figure 2 Probe A-CN-Ph-O synthesized in the present invention 2 Mass spectrum of

[0029] Figure 3 Probe A-CN-Ph-O synthesized in the present invention 2 High performance liquid chromatography (HPLC) diagram;

[0030] Figure 4 Probe A-CN-Ph-O synthesized in the present invention 2 Optical properties diagram; where a) is Probe A-CN-Ph-O 2 and the traditional chemiluminescent compound luminol-H 2 O 2 b) is the UV-visible spectrum detection result, c) is the fluorescence spectrum detection result, and d) is the chemiluminescence spectrum detection result;

[0031] Figure 5 Probe A-CN-Ph-O synthesized in the present invention 2 The best pH value detection experiment result diagram;

[0032] Figure 6 Probe A-CN-Ph-O synthesized in the present invention 2 Figure 1. Results of selective experiments on different proteins in the blood.

[0033] Figure 7 Probe A-CN-Ph-O synthesized in the present invention 2 Selectivity experiment diagram for different amino acids;

[0034] Figure 8 Probe A-CN-Ph-O synthesized in the present invention 2 Figure 1. Selectivity test results for different metal ions in blood.

[0035] Fig. 9 Probe A-CN-Ph-O synthesized in the present invention 2 The results of the selectivity experiments on different thiol compounds are shown in Fig.

[0036] Fig.10 Probe A-CN-Ph-O synthesized in the present invention 2 Figure 1. Results of cell activity experiments in EGC cells.

[0037] Fig.11 Probe A-CN-Ph-O synthesized in the present invention 2The results of the cell apoptosis experiment in EGC cells; A is the flow cytometry scatter plot of EGC cells, B is the staining results based on Annexin V-FITC and PI, and C is the sample statistical results;

[0038] Fig.12 The schematic diagram of the state of EGC cells and the process after administration of Probe A-CN-Ph-O obtained by taking pictures under a fluorescence microscope of the present invention; wherein A is a flow chart of the system switching from the "OFF" state to the "ON" state, B is a fluorescence result diagram of the "OFF" state, the upper row is a bright field image and a blue light fluorescence channel, the lower row is a green fluorescence channel and a red fluorescence channel, C is a chemiluminescence imaging result diagram of the "OFF" state, D is a fluorescence result diagram of the "ON" state, the upper row is a bright field image and a blue light fluorescence channel, the lower row is a green fluorescence channel and a red fluorescence channel, and E is a chemiluminescence imaging result diagram of the "ON" state;

[0039] Fig.13 Probe A-CN-Ph-O synthesized in the present invention 2 Visualization results in breast cancer mice; a) is the use of BAC probe to detect H in breast cancer mice tumors 2 a) In vivo imaging of S levels, b) CL intensity of BAC biosensor in vivo at different time points, data are presented as mean ± SD (n = 3). DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0041] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0042] Herein, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0043] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0044] The present invention provides a thiol compound activated chemiluminescent probe (Probe A-CN-Ph-O 2 ), the structure is as follows:

[0045]

[0046] The present invention provides a method for synthesizing the above-mentioned thiol compound activated chemiluminescent probe, the steps of which are as follows:

[0047] 1. A mixture of 1.0 equivalent of prop-2-en-1-yl N-[4-(hydroxymethyl)phenyl]carbamate (Compound 5) and 5.0 equivalents of NaI in acetonitrile (MeCN) was stirred at 0°C for 5-15 min, then 5.0 equivalents of trimethylsilyl chloride (TMS-Cl) was added, stirred at room temperature in the dark for 45 min, diluted, washed and dried to obtain prop-2-en-1-yl N-[4-(iodomethyl)phenyl]carbamate (Compound 6).

[0048] 2. Compound 6 was mixed with 3-[2-hydroxy-4-(4-methoxyspiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decane])phenyl]-2-acrylonitrile (Compound 2), reacted in the dark for 2 h, and purified to obtain a thiol compound activated chemiluminescent probe (Compound 7; 3-[2-(hydroxymethylphenyl-N-formyl-prop-2-en-1-yl)-4-(4-methoxyspiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decane])phenyl]-2-acrylonitrile; Probe A-CN-Ph-O 2 );

[0049] Among them, the preparation method of compound 2 is synthesized by referring to the method of Chinese patent CN116410064A (Patent No.: CN202310397471.X, application date: 2023.04.13), first synthesizing compound 1 (3-(methoxytricyclo〔3.3.1.1 3,7〕dec-2-ylidenemethyl)-phenol acrylic acid, CAS: 2097411-05-5), and then synthesizing it by oxygen illumination, CAS: 2097410-92-7.

[0050] The present invention also provides a BAC biosensor based on the above-mentioned thiol compound activated chemiluminescent probe, which comprises Probe A-CN-Ph-O 2 The solution was mixed with the Ru catalyst at a molar concentration of 10:1 and then incubated;

[0051] Among them, Probe A-CN-Ph-O 2 The solution is Probe A-CN-Ph-O 2 It was dissolved in PB solution containing 10% DMSO.

[0052] The working principle of the above BAC biosensor is as follows: first, the Ru catalyst undergoes ligand conversion with the thiol compound activated chemiluminescent probe to form a Ru complex, followed by an oxidative addition reaction. During the Ru-catalyzed oxidation process, the Alloc protecting group of the thiol compound activated chemiluminescent probe is cleaved and decarboxylated to generate CO 2 , releasing a compound intermediate with an amino group. Secondly, the intermediate leaves by electron transfer to generate a PAD intermediate, which is then chemically excited to generate a molecule in a high-energy state, and then returns to the ground state and releases a photon. Finally, in the presence of R-SH with strong nuclear affinity, the Ru catalyst is regenerated and a new catalytic cycle is initiated, thereby promoting the chemiluminescence of the BAC probe.

[0053] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0054] In the following examples, conventional instruments and equipment in the art are used. All chemical reagents and solvents are purchased from Anhui Zesheng Technology Co., Ltd.'s Anaiji Chemical Brand, Shanghai Bid Pharmaceutical Technology Co., Ltd. and Shanghai McLean Biochemical Technology Co., Ltd. The analytical solvents and reagents are chromatographically pure, and conventional reagents are ultra-dry solvents or ordinary analytical pure and are purified by water removal. Leucine (Leu), alanine (Ala), glycine (Gly), glutamic acid (Glu), arginine (Arg), lysine (Lys), aspartic acid (Asp), valine (Val), threonine (Thr), proline (Pro), biothiols (Cys, Hcy, GSH) and dithiothreitol (DTT) are all analytical grade and do not require further purification. The experimental methods for the specific conditions not specified in the following examples are usually according to conventional conditions, or according to the conditions recommended by the manufacturer.

[0055] In the following examples, the nuclear magnetic spectra (1H NMR and 13C NMR) were measured by 400MHz JNM-ECZ400S / L1 Nuclear Magnetic Resonance Spectrometer, the solvent was CDCl3, the chemical shift (δ) was in ppm, the singlet, doublet, triplet, quartet, dd (doublet of doublets) peak, multiplet and broad peak were represented by s, d, t, q, dd, m and br respectively; the coupling constant (J) was in Hz; the number of hydrogen was determined by the integral value of the spectrum and marked as nH. The mass spectrum was measured by SHIMADZU LC-MS / MS 8040, and the mobile phase was methanol and water.

[0056] Regarding the synthetic reaction conditions in the preparation method provided by the present invention, those skilled in the art can understand that any condition that can achieve the conversion from reactants to products should be within the scope of protection of the present invention. The contribution of the inventor is first reflected in the fact that the BCR-based reaction type and the dioxocyclic CLI provide a first-generation thiol compound-activated chemiluminescent probe. Taking into account that synthesis technology is constantly improving, new synthesis technologies may be born in the future, such as using different BCR reaction types to synthesize corresponding dioxocyclic second-generation chemiluminescent probes, etc. As long as the above-mentioned conversion from reactants to products is achieved, it should be within the scope of protection of the present invention.

[0057] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The implementation methods and materials described herein are the optimal conditions explored by the applicant and are for demonstration purposes only.

[0058] The present invention is further described in detail below in conjunction with Formula 1 and the accompanying drawings.

[0059] 1. Synthesis of thiol-activated chemiluminescent probes

[0060]

[0061] 1. Synthesis of Compound 2

[0062] Compound 1 (3-(methoxytricyclo〔3.3.1.1 3,7〕dec-2-ylidenemethyl)-phenol acrylic acid; CAS: 2097411-05-5; 32 mg, 0.1 mmol, 1.0 equivalent) and anhydrous K 2 CO 3(70 mg, 0.5 mmol, 5.0 equivalents) was dissolved in 3 mL of dichloromethane (DCM) and stirred at room temperature for 15 min. 1 mg of methylene blue solution was added to the reaction solution and O was continuously introduced. 2 The mixture was then irradiated with 25W yellow LED light for 30 min. The reaction was monitored by LC-MS / MS during the reaction. After the reaction was completed, the reaction mixture was frozen with liquid nitrogen and then condensed with N 2 Fully replace the O in the reaction vessel 2 , to obtain compound 2 (3-[2-hydroxy-4-(4-methoxyspiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decane])phenyl]-2-acrylonitrile) for later use.

[0063] 2. Synthesis of Compound 5

[0064] Compound 4 (p-aminobenzyl alcohol; CAS: 623-04-1; 10.0 mmol, 1.0 equivalent) was dissolved in 20 mL of a mixed solution (2:2:1 THF / NaHCO 3 (sat.) / ddH 2 0 v / v) in a 100 mL round-bottom flask, stirred at room temperature for 15 min, then added compound 3 (allyl chloroformate; CAS: 2937-50-0; 1.17 mL, 11.0 mmol, 1.1 equivalent) within 5 min, and continued stirring at room temperature for 24 h. The reaction was monitored by TLC plate. After the reaction was completed, the reaction solution was diluted with ethyl acetate (EA) and NH 4 The organic layer was washed once with NaCl and twice with brine. 2 SO 4 After drying and evaporation under reduced pressure, the mixture was purified by column chromatography to give a white compound 5 (prop-2-en-1-yl N-[4-(hydroxymethyl)phenyl]carbamate; CAS: 914094-82-9) in a yield of 74%.

[0065] 1 H NMR(400MHz,Chloroform-d)δ7.37(d,J=8.3Hz,2H),7.30(d,J=8.5Hz,2H),6.73(s,1H),5.97(ddt,J=17.3,10.4, 5.7Hz, 1H), 5.36 (dq, J=17.2, 1.6Hz, 1H), 5.26 (dq, J=10.5, 1.3Hz, 1H), 4.66 (dt, J=5.7, 1.5Hz, 2H), 4.63 (s, 2H). 13C NMR(101MHz,Chloroform-d)δ153.33,137.36,136.13,132.47,128.06,118.90,118.41,65.99,65.02.MS / MS(ESI-):m / z calculated for C 11 H 13 NO 3 :207.09; found:206.05[MH] - .

[0066] 3. Synthesis of Compound 6

[0067] The mixture of compound 5 (prop-2-en-1-yl N-[4-(hydroxymethyl)phenyl]carbamate; 103.5 mg, 0.5 mmol, 1.0 equivalent) prepared in step 2 and NaI (375 mg, 2.5 mmol, 5.0 equivalent) in MeCN (ACN; 2 mL) was stirred at 0°C for 5 min, and then TMS-Cl (318 μL, 0.1 mmol, 5.0 equivalent) was added. The reaction solution was moved to room temperature (25°C) and stirred in the dark for 45 min. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was diluted with EA and saturated with Na 2 S 2 O 3 Wash once, and then wash twice with saturated brine. 2 SO 4 After drying and evaporation under reduced pressure, compound 6 (prop-2-en-1-yl N-[4-(iodomethyl)phenyl]carbamate) was obtained as a light yellow powder with a yield of 63.7%.

[0068] 1 H NMR(400MHz,Chloroform-d)δ7.32(s,4H),6.75(s,1H),5.96(ddt,J=16.3,10.7,5.6Hz, 1H), 5.36 (d, J = 17.2Hz, 1H), 5.26 (d, J = 10.4Hz, 1H), 4.66 (d, J = 5.8Hz, 2H), 4.45 (s, 2H). 13 C NMR (101MHz, Chloroform-d) δ153.08,137.42,134.27,132.31,129.59,118.89,118.41,65.98,5.85. 13C NMR(101MHz,Chloroform-d)δ156.06,146.71,141.87,139.17,135.21,129.18,122.03,120.3 3,119.26,116.49,96.58,58.20,39.16,39.02,36.96,32.45,30.61,28.12.MS / MS(ESI-):m / z calculated for C 11 H 12 NIO 2 :316.99; found:318.00[M+H] + .

[0069] 4. Synthesis of thiol-activated chemiluminescent probes

[0070] Compound 6 (95 mg, 0.3 mmol, 3.0 equivalents) dissolved in 2 mL of dichloromethane (DCM) was added to the compound 2 prepared in step 1, stirred for 2 h in the dark, and the reaction mixture was monitored by LC-MS / MS. After the reaction was completed, the mixture was evaporated under reduced pressure, quickly purified once with a short chromatography column (PE: EA = 10: 1), and then preparative high performance liquid chromatography (reverse phase column: NanoChrom ChromCore C18, 5 μm, 30 × 250 mm). The mobile phase (HPLC grade) was water / methanol (0-25 min, gradient elution: methanol increased from 70% to 95%, flow rate of 42.0 mL / min) to perform secondary purification of the product to obtain a thiol compound activated chemiluminescent probe (i.e., compound 7; 3-[2-(hydroxymethylphenyl-N-formyl-prop-2-en-1-yl)-4-(4-methoxyspiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decane])phenyl]-2-acrylonitrile; Probe A-CN-Ph-O 2 ), white powder, yield 22%, mass spectrum and HPLC chromatogram see Figure 2 and Figure 3 The results show that this method can be used to synthesize thiol compound-activated chemiluminescent probes.

[0071] 1 H NMR (600 MHz, DMSO-d 6)δ9.78(s,1H),7.75(d,J=15.1Hz,1H),7.74–7.71(m,2H),7.66(dd,J=5.7,3.3Hz,1H),7.46(d,J=8.2Hz ,2H),7.36(d,J=8.1Hz,2H),6.52(d,J=16.5Hz,1H),5.97(ddt,J=17.3,10.6,5.4Hz,1H),5.36–5.33(dt ,1H),5.31(d,J=12.4Hz,1H),5.25(d,J=12.4Hz,1H),5.23–5.21(dt,1H),5.06(s,2H),4.60(dt,J=5.5, 1.6Hz,2H),4.22(t,J=6.6Hz,3H),4.03(q,J=7.1Hz,2H),3.10(s,3H),2.85(s,1H),1.67-1.45(m,13H). 13 C NMR (151 MHz, DMSO-d 6 )δ170.80,167.42,156.63,145.36,139.39,138.60,133.73,130.57,129.17,129.14,128.70,119.48,118.65,117.91,95.19 ,70.02,65.50,65.16,65.08,60.21,50.23,31.47,30.47,29.50,25.79,25.55,21.23,19.12,14.54,14.00.MS / MS(ESI+):m / z calculated for C 32 H 34 N 2 O6:542.24; found:542.99[M+H] + .

[0072] 2. Preparation of BAC Biosensors

[0073] Probe A-CN-Ph-O 2 Dissolved in PB solution containing 10% DMSO to obtain Probe A-CN-Ph-O with a pH value of 7.4 2 Solution.

[0074] 40 μM Probe A-CN-Ph-O 2 The solution and 4 μM Ru catalyst (Annaiji, CAS#: 92390-26-6) were mixed and incubated at 37° C. for 5 min to obtain a BAC biosensor.

[0075] 3. Performance Exploration

[0076] 1. Optical property detection

[0077] like Figure 4 As shown in a, by comparing the thiol compound activated chemiluminescent probe (Probe A-CN-Ph-O 2 ), BAC biosensors and traditional chemiluminescent compounds luminol-H 2 O 2 The optical properties of the (Luminol) system were explored by ProbeA-CN-Ph-O 2 , the differences between BAC biosensors and traditional chemiluminescent probes.

[0078] 100 μM Probe A-CN-Ph-O 2 (200 μL, 10% DMSO in PB, pH 7.4) The solution was gently shaken to mix and incubated at 37°C for 5 min to obtain the Probe only group;

[0079] A 100 μM Luminol (200 μL, 10% DMSO in PB, pH 7.4) solution was gently shaken to mix and incubated at 37°C for 5 min to obtain the Luminol group;

[0080] 100 μM Probe A-CN-Ph-O 2 (200 μL, 10% DMSO in PB, pH 7.4) solution and 40 μM Ru catalyst were mixed and incubated at 37°C for 5 min to obtain the Probe + 10% Ru group;

[0081] 100 μM Probe A-CN-Ph-O 2 (200 μL, 10% DMSO in PB, pH 7.4) solution, 40 μM Ru catalyst and 400 μM thiol were mixed and incubated at 37° C. for 5 min to obtain the Probe+10% Ru+R-SH group.

[0082] 1) UV-visible spectrum detection

[0083] The absorbance data of each solution in the wavelength range of 200-900 nm were recorded using a UV-Vi spectrophotometer (Purkinje TU 1810PC). Each sample was measured three times and the average value was taken. 2 The absorption peaks in the UV to visible range and their relationship with H 2 S (400μM) response before and after changes.

[0084] 2) Detection of fluorescence spectrum

[0085] The fluorescence spectrophotometer (SHIMADZU RF-6000) was set to the absorption wavelength mode, and the emission wavelengths were set to 235 nm, 280 nm, 330 nm, and 370 nm, respectively, to detect the fluorescence photometric values ​​of the Probe only group and Luminol group solutions, respectively, to explore the effect of Probe A-CN-Ph-O 2 Fluorescence emission intensity at a specific excitation wavelength and its relationship with H 2 The luminescence changes after S reaction.

[0086] 3) Detection of chemiluminescence spectrum

[0087] The fluorescence spectrophotometer (SHIMADZU RF-6000) was set to the absorption wavelength mode, and the emission wavelength was set to 0 nm with an open filter. The chemiluminescence optical properties of the Probe only group, Luminol group, Probe + 10% Ru group, and Probe + 10% Ru + R-SH group solutions were detected to explore the chemiluminescence optical properties of Probe A-CN-Ph-O 2 In H 2 The intensity of the light signal and its luminescent wavelength range generated by the chemiluminescent reaction under the action of S.

[0088] The results of UV-visible spectroscopy, fluorescence spectroscopy and chemiluminescence spectroscopy are shown in Figure 2. Figure 4 As shown in b to c, the results show that Probe A-CN-Ph-O 2 It has higher sensitivity and longer continuous luminescence time.

[0089] 2. pH value detection

[0090] To detect Probe A-CN-Ph-O 2 The chemiluminescence intensity of the probe under different pH conditions was detected to determine whether it can emit light normally in a physiological environment. 2 The samples were dripped onto a black 96-well plate containing 10% dimethyl sulfoxide (DMSO) at pH values ​​of 3, 4, 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10, respectively. Chemiluminescent images were acquired using a small animal in vivo imager (LVIS SPECTR) every 3 min for 2 h. The chemiluminescent signal of the probe was then measured under different pH conditions. Each experiment was repeated three times, and each sample was measured three times to take the average value.

[0091] The results are as follows Figure 5As shown in the figure, the luminescence intensity of the probe is the highest when the pH value is 7.4, indicating that Probe A-CN-Ph-O 2 It is feasible to conduct in vitro and in vivo experiments.

[0092] 3. Selective experimental testing

[0093] In order to investigate Probe A-CN-Ph-O 2 The selectivity of the probe was studied to investigate the chemiluminescence intensity of different analytes. Briefly, each well of a black 96-well plate contained 10% Ru catalyst (4 μM) and PB (10% DMSO) as a solution with a pH value of 7.4 (total volume 200 μL). Amino acids, proteins, metal ions, sulfur compounds and other metabolites (400 μM) abundant in blood were added to the 96-well plate. Each well was incubated at 37°C for 5 minutes, and then 40 μM Probe A-CN-Ph-O 2 After the probe was added, the chemiluminescent signal was immediately acquired every 30 seconds using a small animal living imager (LVIS SPECTR) for 180 minutes. The intensity of each well of the 96-well plate was quantified using the LivingImage 4.4 software ROI detection method, and the obtained data was plotted as a kinetic curve according to the chemiluminescent intensity vs. time. The total light emission quantum number of various metabolites was measured and the time required to emit half a photon was calculated to further evaluate the Probe A-CN-Ph-O 2 The half-life of different metabolites (t 1 / 2 ). Each experiment was repeated three times, each sample was measured three times, and the average value was taken. The specific steps are as follows:

[0094] 1) Detection of probes in kinetic experiments on different proteins

[0095] BAC biosensors (composed of 40 μM Probe A-CN-Ph-O 2 solution and 4 μM Ru catalyst were mixed and incubated), or 40 μM Luminol group solution was added, and only Probe A-CN-Ph-O 2The solution was used as the control group (Blank), and then the chemiluminescence image was immediately acquired every 30 seconds on a 96-well black plate using a small animal living imager (LVISSPECTR) for 180 minutes. The intensity of each well of the 96-well plate was quantified using the LivingImage 4.4 software ROI detection method, and the obtained data was plotted as a kinetic curve according to the chemiluminescence intensity vs. time. The total number of light emission quanta in the presence of thiol was measured and the time required to emit half a photon was calculated to further evaluate the performance of Probe A-CN-Ph-O 2 The half-life of different proteins (t 1 / 2 Each experiment was repeated three times, each sample was measured three times, and the average value was taken.

[0096] The experimental results are as follows Figure 6 As shown in the figure, the luminescence intensity of human serum albumin and bovine serum albumin increased by 50.9 and 53.3 times compared with the blank control group and other metabolites, respectively, indicating that Probe A-CN-Ph-O 2 Probe A-CN-Ph-O has excellent selectivity for albumin. 2 It can be used to determine the content of albumin.

[0097] 2) Probe detection of amino acid kinetics experiments

[0098] Probe A-CN-Ph-O 2 It has good selectivity for albumin. In order to explore the reasons why albumin is sensitive to Probe A-CN-Ph-O 2 The BAC biosensor (composed of 40 μM Probe A-CN-Ph-O 2 solution and 4 μM Ru catalyst were mixed and incubated), or 40 μM Luminol group solution was added, and only Probe A-CN-Ph-O 2The solution with BSA was used as control group 1 (Blank), and the solution with BSA plus the corresponding probe was used as control group 2 (BSA). The chemiluminescent images were immediately acquired every 30 seconds on a 96-well black plate using a small animal living imager (LVIS SPECTR) for 180 minutes. The intensity of each well of the 96-well plate was quantified using the LivingImage 4.4 software ROI detection method, and the obtained data was plotted as a kinetic curve according to the chemiluminescence intensity vs. time. The half-life (t 1 / 2 Each experiment was repeated three times, each sample was measured three times, and the average value was taken.

[0099] The experimental results are as follows Figure 7 As shown, the probe Probe A-CN-Ph-O 2 In addition to a strong response to bovine serum albumin (BSA), it also has good selectivity for cysteine ​​(Cys), an amino acid with a sulfhydryl structure on its side chain (one of the main amino acid components of albumin), indicating that Probe A-CN-Ph-O 2 It can be used to determine the content of thiol compounds.

[0100] 3) Probe detection of kinetic experiments on different metal ions

[0101] Probe A-CN-Ph-O 2 The probe has good selectivity for the metal catalyst Ru. In order to expand the application scenarios of the probe, the kinetic experiments of the probe for different metal ions were designed and tested. 2 + , Fe 2+ , Fe 3+ , Sr 2+ , Ca 2+ 、Ni 2+ 、Zn 2+ Cr 3+ , Ba 2+ ) were added to black 96-well plates. 2 solution and 4 μM Ru catalyst were mixed and incubated), or 40 μM Luminol group solution was added, and only Probe A-CN-Ph-O 2The solution was used as the control group (Blank), and then the chemiluminescence image was immediately acquired every 15 seconds on a 96-well black plate using a small animal living imager (LVIS SPECTR) for 180 minutes. The intensity of each well of the 96-well plate was quantified using the LivingImage 4.4 software ROI detection method, and the obtained data was plotted as a kinetic curve according to the chemiluminescence intensity vs. time. The half-life (t 1 / 2 Each experiment was repeated three times, each sample was measured three times, and the average value was taken.

[0102] The experimental results are as follows Figure 8 As shown, the probe Probe A-CN-Ph-O 2 For Cu 2+ It has excellent selectivity, indicating that the probe can be used for the detection of blood copper ions in clinical practice.

[0103] 4) Probe detection of kinetic experiments on different thiol compounds

[0104] Probe A-CN-Ph-O 2 The probe has good selectivity for amino acid cysteine ​​(Cys). In order to explore the reaction mechanism of the probe, a kinetic experiment of the probe against sulfhydryl compounds was designed and tested. 3 PhSH、PhCH 2 BAC biosensors (40 μM Probe A-CN-Ph-O 2 solution and 4 μM Ru catalyst were mixed and incubated), or Luminol group solution was added, and only Probe A-CN-Ph-O 2 The solution was used as the control group (Blank), and then the chemiluminescent images were immediately acquired every 30 seconds on a 96-well black plate using a small animal living imager (LVISSPECTR) for 180 minutes. The intensity of each well of the 96-well plate was quantified using the LivingImage 4.4 software ROI detection method, and the obtained data was plotted as a kinetic curve according to the chemiluminescent intensity vs. time. The half-life (t 1 / 2 Each experiment was repeated three times, each sample was measured three times, and the average value was taken.

[0105] The experimental results are as follows Fig. 9As shown, the probe Probe A-CN-Ph-O 2 It has good selectivity for cysteine ​​(Cys) and amino acids with thiol structures on their side chains, and can be used to determine the content of active thiol compounds, especially methylmercaptobenzene and cysteine.

[0106] The above results show that the probe Probe A-CN-Ph-O synthesized by this method 2 There is a significant chemiluminescent signal for sulfur-containing metabolites, including amino acids.

[0107] 4. Cell culture experiments

[0108] Rat enteric glial cells (EGC; CRL-2690, ATCC) were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin / streptomycin (Gibco). All cells were maintained at 37°C with 5% CO 2 Culture in a humidified incubator.

[0109] 1) Toxicity test

[0110] In order to verify whether the probe has toxicity and side effects in vivo and in vitro and thus affects cells, the toxicity experiment of the probe was continued. The cytotoxicity test was detected using a standard cell counting kit (CCK-8). EGC cells were seeded on a 96-well plate (8000 cells / well) and incubated at 37°C for 24 hours. 2 (0, 10, 20, 40, 80, 160, 320 μM) were incubated with cells at 37°C for 1 h, and then 10 μL of CCK-8 solution was added to each well and incubated for another 4 h. The absorbance of live cells at 450 nm was measured using an ELISA reader to determine cell viability. The cell viability calculation formula is: Cell viability = (OD Treated -OD Blank / OD Control -OD Blank )×100%. Each concentration was measured in six groups and the results were averaged.

[0111] The experimental results are as follows Fig.10 As shown, the probe Probe A-CN-Ph-O 2 The probe has little effect on cell activity in the concentration range of 0 to 160 μM, further demonstrating the feasibility of conducting in vitro and in vivo experiments.

[0112] 2) Cell apoptosis detection

[0113] In order to further verify whether the probe would affect cells in vivo and in vitro, apoptosis experiments were performed. Annexin-V / APC and DAPI-based flow cytometry was used to distinguish the apoptotic population from the healthy population. For EGC cells, about 5×10 cells were seeded in a 6-well plate one day before administration. 4 The cells were washed with PB (pH 7.4) and then treated with 40 μM Probe A-CN-Ph-O 2 Incubate for 2 h. Next, digest the cells with trypsin solution (without EDTA), collect the suspension and centrifuge. Subsequently, remove the supernatant and collect the cell pellet. Stain the cells with Annexin V-fluoresceinisothiocyanate (FITC) as directed. Co-stain the cells with PI (Invitrogen) 5 min before loading, and finally, the stained cells are immediately measured with ACEA NovoCyte and analyzed with Novoexpress software (8,000 events collected for each sample).

[0114] The experimental results are as follows Fig.11 As shown, Fig.11 A in the figure shows the flow cytometric scatter plot of EGC cells, which is used to distinguish different cell populations, among which the P1 region is the selected cell population, accounting for 44.1% of the total cells. Fig.11 Middle B shows the staining results based on Annexin V-FITC and PI, which further divide the apoptosis and necrosis status of P1 cells: Q2-1 region (2.92%): represents early apoptotic cells (Annexin V positive, PI negative), Q2-2 region (0.71%): represents late apoptotic or necrotic cells (Annexin V and PI double positive), Q2-3 region (96.32%): represents surviving cells (Annexin V and PI double negative), Q2-4 region (0.05%): represents necrotic cells (Annexin V negative, PI positive). Fig.11 Figure C summarizes the number of cells (Count), percentage (% Parent), and median values ​​(Median X and Median Y) of the fluorescence signal in each region, providing quantitative data support for different cell states. These results show that the 40 μM probe does not cause premature cell apoptosis at this concentration, further demonstrating that the probe is feasible for in vitro and in vivo experiments.

[0115] 3) Luminescence imaging of living cells

[0116] The previous experimental results show that the probe is feasible for in vitro and in vivo experiments. In this experiment, the probe and 10% equivalent of Ru were incubated with cells for 1 hour and the cell status was photographed under a fluorescence microscope.

[0117] like Fig.12 As shown in Figure A, EGC cells (250,000 cells / well) were seeded in a 6-well transparent bottom plate one day before treatment. The culture medium was removed, the cells were washed 3 times with PB, and Probe A-CN-Ph-O 2 (40 μM, 10% DMSO, pH = 7.4) for 1 h, and the system was in the "OFF" state. A Nikon Eclipse Ts2 fluorescence microscope and a Nikon DS-Ri2 camera were used to capture luminescence images, including bright field, fluorescence field (blue light excitation), fluorescence field (green light excitation), fluorescence field (red light excitation), and representative chemiluminescent cell images in the luminescence field. The detection results are shown in Fig.12 As shown in B and C, no obvious signal is shown.

[0118] like Fig.12 As shown in A, H is added to the system in the "OFF" state. 2 S and 10% equivalent of Ru activated ProbeA-CN-Ph-O 2 , switch the system to the "ON" state. Use a Nikon Eclipse Ts2 fluorescence microscope and a Nikon DS-Ri2 camera to capture luminescence images, including bright field, fluorescence field (blue light excitation), fluorescence field (green light excitation), fluorescence field (red light excitation), and representative chemiluminescent cell images in the luminescence field. The detection results are shown in Fig.12 As shown in D and E, obvious luminescence signals can be seen.

[0119] The above results show that the probe A-CN-Ph-O 2 It has high sensitivity and selectivity, and can obtain clearer images than fluorescence imaging when realizing cell-level detection, which proves that the chemiluminescent probe ProbeA-CN-Ph-O 2 The synergistic effect with Ru catalyst can be used for the chemiluminescence of thiol-containing compounds in living cells, further demonstrating the feasibility of the probe for in vivo experiments.

[0120] 5. Animal experiments

[0121] 1. Cell Culture and Preparation

[0122] SKBR-3 breast cancer cells were cultured in a sterile environment using McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS) and 5 μg / mL mycoplasma scavenger. The cells were cultured to the logarithmic growth phase and the cell morphology was observed to be normal (adherence, vigorous growth, no contamination). The cells were then gently digested with trypsin (0.25% Trypsin-EDTA), centrifuged and washed twice with PBS to remove the residual culture medium. The cells were resuspended in serum-free medium or saline and the cell concentration was adjusted to 1×10 7 cells / mL, collect the cells and set aside.

[0123] 2. Construction of Breast Cancer Mouse Model

[0124] BALB / c-nu immunodeficient mice weighing 18-22 g and aged 6-8 weeks were selected and kept in an SPF (specific pathogen-free) animal room for one week to acclimate to the environment and ensure that there was no infection. The mice were placed in a lightly anesthetized state by inhalation of isoflurane, and then 100 μL of the prepared cell suspension (about 1×10 6 cells) and injected into the subcutaneous tissue of the mouse abdomen to obtain a breast cancer mouse model.

[0125] 3. Visual Verification

[0126] Breast cancer mice were randomly divided into a control group and an experimental group. The control group mice were intraperitoneally injected with 150 μL PB containing 10% DMSO after anesthesia, and the experimental group mice were intraperitoneally injected with 150 μL BAC biosensor (composed of 40 μM Probe A-CN-Ph-O 2 After incubation with a mixed solution and 4 μM Ru catalyst, representative chemiluminescence (CL) images of each group of mice were taken at different time points (including 0, 10, 20, 30, 40, 50, and 60 min) in the fluorescence mode: excitation filter = 430 nm, emission filter = 540 nm, for visualization of H 2 S level.

[0127] In vivo imaging results Fig.13 As shown in Figure a), after the injection of the BAC biosensor, the CL signal at the tumor site of the mouse gradually increased, reaching a peak at 20 to 30 minutes, and the signal stabilized after 60 minutes. However, there was no obvious signal at the tumor site of the control group of mice that were not injected with the BAC biosensor, indicating that the BAC biosensor has high specificity and low background interference. The statistical results of CL intensity are shown in Figure 2. Fig.13As shown in (b), the CL signal of the BAC biosensor group was significantly higher than that of the control group, and reached the maximum intensity at 20 minutes, and then gradually decreased. The signal of the control group always maintained a low level, further indicating the high sensitivity and specificity of the BAC biosensor response. The above results show that the BAC biosensor can quickly and real-time detect endogenous H in breast cancer. 2 S, the signal dynamic changes are clear and suitable for tumor diagnosis and imaging research.

[0128] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A thiol compound activated chemiluminescent probe, characterized in that: The structural formula is as follows:

2. The method for synthesizing the thiol compound activated chemiluminescent probe according to claim 1, characterized in that: Here are the steps: 1) mixing prop-2-en-1-yl N-[4-(hydroxymethyl)phenyl]carbamate and a mixture of NaI and acetonitrile, then adding trimethylsilyl chloride, stirring at room temperature in the dark, diluting, washing, and drying to obtain prop-2-en-1-yl N-[4-(iodomethyl)phenyl]carbamate; 2) Prop-2-en-1-yl N-[4-(iodomethyl)phenyl]carbamate is mixed with 3-[2-hydroxy-4-(4-methoxyspiro[1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]decane])phenyl]-2-acrylonitrile, reacted in the dark, purified, and obtained a thiol compound activated chemiluminescent probe.

3. The method for synthesizing the thiol compound activated chemiluminescent probe according to claim 2, characterized in that: In step 1), the equivalent ratio of prop-2-en-1-yl N-[4-(hydroxymethyl)phenyl]carbamate, the acetonitrile mixture of NaI and trimethylsilyl chloride is 1:5:

5.

4. The method for synthesizing a thiol compound-activated chemiluminescent probe according to claim 2, wherein: In step 1), dilute with EA.

5. The method for synthesizing the thiol compound activated chemiluminescent probe according to claim 2, characterized in that: In step 1), the washing steps are: washing once with saturated Na2S2O3, and then washing twice with saturated brine.

6. A BAC biosensor, characterized in that: The chemiluminescent probe activated by the thiol compound according to claim 1 is mixed with a Ru catalyst and then incubated.

7. Use of the thiol compound-activated chemiluminescent probe according to claim 1 or the BAC biosensor according to claim 6 in determining the content of thiol-containing compounds.

8. Use of the thiol compound-activated chemiluminescent probe according to claim 1 or the BAC biosensor according to claim 6 in determining albumin content.

9. Use of the thiol compound-activated chemiluminescent probe according to claim 1 or the BAC biosensor according to claim 6 in detecting the activity of thiol compounds in living cells.

10. Use of the thiol compound-activated chemiluminescent probe according to claim 1 or the BAC biosensor according to claim 6 in the preparation of tumor diagnosis and imaging reagents.

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