Hydrogen sulfide activated chemiluminescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of micromolecular chemiluminescent materials, and particularly relates to a hydrogen sulfide activated chemiluminescent probe as well as a preparation method and application thereof. The chemiluminescent probe disclosed by the invention is an adamantane-dioxetane chemical compound and has five types in total, and the five types are respectively marked as HS-CL, HS-CL-TCF, HS-CL-TCCH, HS-CL-QM or HS-CL-DCMC. The chemiluminescent probe can be used for detecting hydrogen sulfide, including measuring hydrogen sulfide generated by hydrolysis of beta-lactamase and beta-lactam drugs, distinguishing drug-resistant bacteria from drug-sensitive bacteria and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of small molecule chemiluminescent materials, and in particular relates to a hydrogen sulfide-activated chemiluminescent probe and a preparation method and application thereof. Background Art

[0002] The emergence and widespread spread of antimicrobial resistance in bacterial pathogens poses a significant threat to human health [Rudd, KE; Johnson, SC, et al. The Lancet 2020, 395(10219), 200-211; Denissen, J.; Reyneke, B.; Waso-Reyneke, M.; Havengga, B.; Barnard, T.; Khan, S.; Khan, W. Environ. Health 2022, 244, 114006]. Among various antibiotics, β-lactams are widely used due to their efficacy against a variety of bacterial infections. β-lactamases can hydrolyze β-lactamase drugs, thereby inactivating the drugs [Bush, K., Bradford PA. Nat Rev Microbiol. 2019, 17(5), 295-306]. Therefore, bacterial resistance caused by β-lactamases has a negative impact on the use of antibiotics and bacterial infections. Therefore, developing a detection method that can quickly identify bacterial resistance is an effective intervention measure to hinder the emergence and spread of antibiotic resistance. Studies have shown that β-lactamase produces hydrogen sulfide when hydrolyzing β-lactam drugs [Abdalla, MA; Fogg, AG; Burgess, C. Analyst. 1982, 107(1271), 213-217; Gholap, SP; Yao, C.; Green, O.; Babjak, M.; Jakubec, P.; Malatinsky, T.; Ihssen, J.; Wick, L.; Spitz, U.; Shabat, D. Bioconjugate Chem. 2021, 32(5), 991-1000.], so the development of a biosensor to detect hydrogen sulfide can indirectly detect bacterial resistance.

[0003] Chemiluminescence is the luminescence that accompanies a chemical reaction. Compared to fluorescence, chemiluminescence requires no external excitation light source, is unaffected by light scattering and autofluorescence, and exhibits a low background. In 1987, Professor Schaap developed a chemiluminescent probe based on an adamantane-dioxetane structure. However, the luminescence efficiency in aqueous solution was too low to be used for analysis in physiological environments [Schaap, AP; Handley, RS; Giri, BP, Tetrahedron Lett. 1987, 28, 935; Schaap, AP; Chen, T.-S.; Handley, RS; DeSilva, R.; Giri, BP, Tetrahedron Lett. 1987, 28, 1155.; Schaap, AP; Sandison, MD; Handley, RS, Tetrahedron Lett. 1987, 28, 1159]. Recent modifications have significantly improved the quantum yield in aqueous solution, and have good signal-to-noise ratio and application prospects [Fu A, Wang H, Huo T, et al. Anal Chem. 2021 Apr 27; 93(16): 6501-6507., Blau, R., Shelef, O., Shabat, D. et al. Nat. Rev. Bioeng., 2023, 1, 648-664]

[0004] In view of the characteristics of the chemiluminescence analysis method described above and the development of the adamantane-dioxetane chemiluminescence system, the present invention designs and synthesizes a hydrogen sulfide-activated multicolor chemiluminescent probe to address the problem of hydrogen sulfide detection. Summary of the Invention

[0005] The present invention aims to provide a multicolor chemiluminescent probe suitable for hydrogen sulfide activation, a preparation method and an application thereof.

[0006] The hydrogen sulfide-activated multicolor chemiluminescent probe provided by the present invention is an adamantane-dioxetane chemical compound, and its structural formula is shown in (I):

[0007]

[0008] Among them, R is CH3O-, C 10 H6N3O-(TCF), C 11 H 11 N2-(TCCH), C 14 H 10 N3-(QM) or C 12H5N2O-(DCMC), the corresponding probes are named HS-CL, HS-CL-TCF, HS-CL-TCCH, HS-CL-QM, and HS-CL-DCMC; they have different emission wavelengths and are all activated by hydrogen sulfide to release photons.

[0009] The present invention provides a method for preparing a hydrogen sulfide-activated multicolor chemiluminescent probe, comprising the following specific steps:

[0010] (1) The adamantane-dioxetane derivative precursor compound b and ethyl (triphenylphosphinimido) acetate were placed in a round-bottom flask, dichloromethane was added for dissolution, and the reaction was stirred for 15-45 minutes. The reaction solution was then washed with 0.8-1.2 M hydrochloric acid and extracted with dichloromethane. The organic phase was collected and rotary evaporated to dryness, and then purified by silica gel column chromatography to obtain a compound, which was recorded as CL.

[0011] (2) Knoevenagel condensation reaction was used for condensation. Compound b and the modification group TCF, TCCH, QM or DCMC were dissolved in acetonitrile, and then a catalyst was added and heated with stirring. The mixture was condensed and refluxed. The reaction was stopped after 6-8 hours. The reaction solution was washed with 1M hydrochloric acid and extracted with dichloromethane. The organic phase was collected and rotary evaporated to dryness, and then purified by silica gel column chromatography to obtain compounds, which were respectively designated as CL-TCF, CL-TCCH, CL-QM, and CL-DCMC;

[0012] (3) Compound CL in step (1) and the four compounds CL-TCF, CL-TCCH, CL-QM, and CL-DCMC in step (2) were dissolved in dichloromethane with o-iodobenzoyl chloride, and a basic catalyst was added dropwise. After the reaction was monitored by thin layer chromatography, the mixture was washed with aqueous solution and extracted with dichloromethane, and then dried over anhydrous Na2SO4 and rotary evaporated to dryness. After purification by silica gel column chromatography, the obtained products were respectively recorded as Compound 1, Compound 2, Compound 3, Compound 4, and Compound 5;

[0013] (4) Compound 1, Compound 2, Compound 3, Compound 4, and Compound 5 were dissolved in dichloromethane, methylene blue was added, oxygen was continuously introduced, and yellow light was irradiated to react, and the reaction was monitored by thin layer chromatography;

[0014] (5) After the reaction, the solvent was removed by vacuum rotary evaporation, and thin layer preparation and chromatography purification were performed to obtain the products, which were respectively designated as HS-CL, HS-CL-TCF, HS-CL-TCCH, HS-CL-QM, and HS-CL-DCMC.

[0015] Wherein, the structural formula of the compound b is:

[0016]

[0017] The structural formulas of the modifying groups TCF, TCCH, QM, and DCMC are:

[0018]

[0019] The name of the TCF compound is 2-dicyanomethylidene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran;

[0020] The name of the TCCH compound is 3-dicyanomethylimide-1,5,5-trimethylcyclohexene;

[0021] The name of the QM compound is 2-(1-ethyl-2-methylquinolin-4(1H)-ylidene)malononitrile;

[0022] The compound name of DCMC is 2-(2-methyl-4H-chrome-4-ylidene)malononitrile.

[0023] Further:

[0024] In step (1), the amount of compound b is 450-600 mg (1 equivalent), the amount of ethyl (triphenylphosphinimido) acetate is 510-650 mg (1 equivalent); the amount of 1M hydrochloric acid is 1-2 mL; and the elution conditions of silica gel column chromatography are petroleum ether:ethyl acetate = (20-15):1.

[0025] In step (2), the heating temperature is 75-90 ° C, the catalyst is piperidine, the amount is 5-50 μL, the amount of compound b is 40.0-110.0 mg (1 equivalent), the amount of the modifying group TCF is 30.0-45.0 mg (1 equivalent), the amount of TCCH is 22.0-33.0 mg (1 equivalent), the amount of QM is 77.0-115.5 mg (1 equivalent), the amount of DCMC is 35.0-54.0 mg (1 equivalent), and the amount of 1M hydrochloric acid is 5-10 mL; the elution conditions of silica gel column chromatography are petroleum ether: ethyl acetate = (15-10): 1.

[0026] In step (3), the alkaline catalyst is triethylamine, the amount is 2 drops, the molar ratio of the compound CL to o-iodobenzoyl chloride is 1: (1 to 1.3); the molar ratio of the compound CL-TCF to o-iodobenzoyl chloride is 1: (1.1 to 1.5); the molar ratio of the compound CL-TCCH to o-iodobenzoyl chloride is 1: (1 to 1.3); the molar ratio of the compound CL-QM to o-iodobenzoyl chloride is 1: (1 to 1.2); the molar ratio of the compound CL-DCMC to o-iodobenzoyl chloride is 1: (1.1 to 1.4);

[0027] In step (4), the amount of methylene blue used is 1-2 mg; the reaction time is 5-7 hours;

[0028] In step (5), the elution condition of thin layer chromatography is petroleum ether:ethyl acetate=(20-5):1.

[0029] The present invention also provides an application of the chemiluminescent probe in detecting hydrogen sulfide. Specifically, sodium sulfide solution is used as the hydrogen sulfide source, the hydrogen sulfide concentration is 5-1000 μM, and the concentrations of the five probes are all 20-100 μM.

[0030] The present invention also provides the use of the chemiluminescent probe HS-CL in the determination of β-lactamase, wherein the concentration of β-lactamase is 0.5-20 U / mL, and the β-lactam drug is cephalexin, ceftizoxime or cefazolin, and the concentration is 0.2-10 mM.

[0031] The present invention also provides the use of the chemiluminescent probe HS-CL in determining drug-resistant bacteria and drug-sensitive bacteria. The drug-resistant bacteria used are Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 35218), drug-resistant Staphylococcus aureus, and Klebsiella pneumoniae (ATCC 700603). The drug-sensitive bacteria are Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538). The bacterial concentration is 10 2 -10 5 CFU / mL.

[0032] The present invention also provides the use of the chemiluminescent probe HS-CL-DCMC in determining drug-resistant bacteria and drug-sensitive bacteria. The drug-resistant bacteria used are Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 35218), drug-resistant Staphylococcus aureus, and Klebsiella pneumoniae (ATCC 700603). The drug-sensitive bacteria are Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538). The bacterial concentration is 10 3 -10 6 CFU / mL.

[0033] This study designed and synthesized five hydrogen sulfide-activated chemiluminescent probes with different emission wavelengths. These probes can be used to detect hydrogen sulfide produced by the hydrolysis of drug-resistant bacteria and β-lactam drugs, thereby distinguishing drug-resistant from drug-sensitive bacteria. The development of adamantane-dioxetane chemiluminescent probes with multiple emission wavelengths represents a significant technological breakthrough, and further expanding their application will be a key development direction in clinical diagnostics and drug discovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The chemiluminescent signals of five chemiluminescent probes to hydrogen sulfide.

[0035] Figure 2 Fluorescence spectra of five chemiluminescent groups.

[0036] Figure 3 The linear curves of five chemiluminescent probes for the determination of hydrogen sulfide at different concentrations.

[0037] Figure 4 This is the chemiluminescence curve and quantification diagram of the probe HS-CL for the determination of β-lactamase.

[0038] Figure 5 The probe HS-CL is used to detect drug-resistant and drug-sensitive bacteria.

[0039] Figure 6 The probe HS-CL-DCMC was used to determine drug-resistant and drug-sensitive bacteria. DETAILED DESCRIPTION

[0040] The present invention will be further described below through embodiments with reference to the accompanying drawings.

[0041] Example 1: Synthesis of multi-color adamantane-dioxetane chemiluminescent probes of hydrogen sulfide HS-CL, HS-CL-TCF, HS-CL-TCCH, HS-CL-QM and HS-CL-DCMC.

[0042] (1) Synthesis of compound HS-CL, the synthetic route is:

[0043]

[0044] All abbreviations represent compound names: dichloromethane (DCM), thin layer chromatography (TLC), petroleum ether (PE), ethyl acetate (EA).

[0045] The specific steps of synthesis are:

[0046] (1) Synthesis of compound b:

[0047] Compound a (800 mg, 2.625 mmol, 1 eq) was placed in a round-bottom flask with paraformaldehyde (1.89 g, 21 mmol, 8 eq) and anhydrous magnesium chloride (549 mg, 5.77 mmol, 2.2 eq). 5 mL of anhydrous acetonitrile was added for dissolution. After adding triethylamine (664 μL, 5.77 mmol, 2.2 eq), the mixture was heated to 85°C, refluxed under condensation, and stirred for reaction. After overnight reaction, the reaction was complete. Dichloromethane was added for dilution, and a small amount of 1 M hydrochloric acid was added for washing. The organic phase was extracted with dichloromethane twice and collected. The mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography to obtain a white solid b (yield 85%). MS (ESI - ):m / z C 19 H 21 ClO3: 332.12 (theoretical value), visible 331.22 [MH] - . 1 H NMR (600MHz, CDCl3) δ 11.62 (s, 1H), 9.90 (d, J = 5.2Hz, 1H), 7.47 (d, J = 6.0Hz, 1H), 6.99 (d, J = 6.5Hz, 1H), 3.28 (s, 3H), 2.17-1.67 (m, 14H).

[0048] (2) Synthesis of compound CL:

[0049] Compound b (545 mg, 1.64 mmol, 1 eq) and ethyl (triphenylphosphinimido) acetate (603.3 mg, 1.8 mmol, 1.1 eq) were dissolved in an appropriate amount of DCM and stirred for 30 min before stopping. A small amount of 1 M hydrochloric acid was added for washing and extraction with DCM. The mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (PE:EA=15:1) to obtain a white solid (yield 80%). MS (ESI + ):m / zC 22 H 25 ClO4: 388.14 (theoretical value), visible 389.23 [M+H] + . 1 H NMR(600MHz, CDCl3) δ7.86(d,J=16.2Hz,1H),7.61(d,J=8.2Hz,1H),7.50(d,J=8.1Hz,1H) ,6.47(d,J=16.2Hz,1H),3.80(s,3H),3.23(s,3H),1.94-1.70(m,7H),1.52-1.17(m,7H).

[0050] (3) Synthesis of Compound 1:

[0051] Compound CL (0.081 mmol, 50 mg, 1 eq) and o-iodobenzoyl chloride (0.081 mmol, 22 mg, 1 eq) were dissolved in 10 mL of DCM. Two drops of triethylamine were added and the reaction progress was monitored by TLC. After completion of the reaction, the mixture was washed with water and extracted with DCM, dried over anhydrous sodium sulfate, and rotary evaporated to obtain crude compound 1.

[0052] (4) Synthesis of compound HS-CL:

[0053] Compound 1 was dissolved in 15 mL of DCM, 1 mg of methylene blue was added, oxygen was continuously introduced, and yellow light was irradiated for 0.5 to 7 h. The reaction was monitored by TLC. After completion of the reaction, the compound HS-CL was purified by silica gel column chromatography (PE:EA=5:1) to obtain compound HS-CL (yield 92%). MS (ESI + ):m / z C 29 H 28 ClIO7: 650.06 (theoretical value), observed 651.21 [M+H] + . 1 H NMR (600MHz, CDCl3) δ8.23(d,1H),8.12-8.07(m,2H),7.81(d,J=16.0Hz,1H),7.72(d,J=8.4Hz,1H),7.55- 7.49(m,1H),7.29(d,J=1.7Hz,1H),6.55(d,J=16.0Hz,1H),3.79(s,3H),3.24(s,3H),1.79-1.50(m,14H).

[0054] (II) Synthesis of compound HS-CL-TCF, the synthetic route is:

[0055]

[0056] The specific steps of synthesis are:

[0057] (1) Compound b (50 mg, 0.151 eq, 1 eq) and 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (30 mg, 0.151 mmol, 1 eq) were dissolved in 5 mL of acetonitrile, 5 μL of piperidine was added, and the mixture was heated to 85°C, refluxed under condensation, and stirred for reaction. After overnight reaction, the mixture was washed with 1 M hydrochloric acid, extracted twice with dichloromethane, and the organic phase was collected. The mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography (PE:EA=10:1) to obtain an orange solid (yield 86%). MS (ESI - ):m / z C 30 H 28ClN3O3: 513.18 (theoretical value), visible 512.21 [MH] - . 1 H NMR (600MHz, CDCl3) δ7.67(d,J=8.1Hz,1H),7.49(d,J=7.9Hz,1H),7.36(d,J=8 .1Hz,1H),7.34(d,J=7.9Hz,1H),5.30(s,1H),3.34(s,3H),1.93-1.65(m,20H).

[0058] (2) Synthesis of Compound 2:

[0059] Compound CL-TCF (14 mg, 0.0271 mmol, 1 eq) and o-iodobenzoyl chloride (8 mg, 0.0298 mmol, 1,1 eq) were dissolved in 5 mL of DCM. Two drops of triethylamine were added and stirred for reaction. The reaction was complete within 5 minutes and the mixture was rotary evaporated to obtain the crude product, Compound 2.

[0060] (3) Synthesis of compound HS-CL-TCF:

[0061] Compound 2 was dissolved in an appropriate amount of DCM, 2 mg of methylene blue was added, oxygen was introduced into the reaction solution, and the reaction was allowed to proceed under yellow light for 0.5 to 7 h. The reaction was monitored by TLC. After the reaction was complete, the product was rotary evaporated and purified by silica gel column chromatography (PE:EA=9:1) to obtain compound HS-CL-TCF (yield 90%). MS (ESI + ):m / z C 37 H 31 ClN3O6: 775.09 (theoretical value), observed 777.13 [M+H] + . 1 HNMR (400MHz, CDCl3) δ8.23(m,1H),8.11(m,1H),8.09(m,1H),7.81(d,J=16.0Hz,1H),7.71(d,J=8 .4Hz,1H),7.54(m,1H),7.51(d,J=3.1Hz,1H),6.55(d,J=16.1Hz,1H),3.79(s,3H),1.43(s,20H).

[0062] (III) Synthesis of compound HS-CL-TCCH, the synthetic route is:

[0063]

[0064] The specific steps of synthesis are:

[0065] (1) Compound b (40 mg, 1.2 mmol, 1 eq) and 3-dicyanomethylimine-1,5,5-trimethylcyclohexene (22.4 mg, 1.2 mmol, 1 eq) were dissolved in 10 mL of acetonitrile, 18 μL of piperidine was added, and the mixture was heated to 85°C, refluxed under condensation, and stirred for reaction. The reaction was monitored by TLC. After the reaction was completed, the mixture was diluted with dichloromethane and washed with 1 M hydrochloric acid. The organic phase was extracted with dichloromethane three times and then dried over anhydrous sodium sulfate. Purification was performed by silica gel column chromatography (PE:EA=10:1) to obtain a yellow solid (yield 80%). MS (ESI + ):m / z C 31 H 33 ClN2O2: 500.22 (theoretical value), visible 501.44 [M+H] + . 1 H NMR (600MHz, CDCl3) δ8.26(d,J=16.1Hz,1H),7.57(d,J=8.1Hz,1H),7.14(d,J=8 .1Hz,1H),6.92(d,J=16.2Hz,1H),6.82(s,1H),3.38(s,3H),1.57-1.27(m,24H).

[0066] (2) Synthesis of compound 3:

[0067] Compound CL-TCCH (16.3 mg, 0.0326 mmol, 1 eq) and o-iodobenzoyl chloride (10 mg, 0.0358 mmol, 1.1 eq) were dissolved in 20 mL of DCM. Two drops of triethylamine were added and stirred for reaction. The reaction was complete after 5 minutes and the mixture was rotary evaporated to obtain the crude product, Compound 3.

[0068] (3) Synthesis of compound HS-CL-TCCH:

[0069] Compound 3 was dissolved in an appropriate amount of DCM, 1 mg of methylene blue was added, oxygen was introduced into the reaction solution, and the reaction was allowed to proceed under yellow light for 0.5-7 h. The reaction was monitored by TLC. After the reaction was complete, the product was evaporated and purified by silica gel column chromatography (PE:EA=10:1) to obtain compound HS-CL-TCCH (yield 89%). MS (ESI + ):m / z C 38 H 36 ClN2O5: 762.14 (theoretical value), visible 785.18 [M+Na] + . 1H NMR (600MHz, CDCl3) δ8.24(d,J=7.7Hz,1H),8.12(d,J=8.0Hz,1H),8.10(d,J=16.3Hz,1H),7.76(d,J=8.6Hz,1H),7.59-7.53 (m,1H),7.34(d,J=9.0Hz,1H),7.30(d,J=9.1Hz,1H),7.08(d,J=16.2Hz,1H),6.86(s,1H),3.26(s,3H),1.52-1.28(m,24H).

[0070] (IV) Synthesis of compound HS-CL-QM, the synthetic route is:

[0071]

[0072] The specific steps of synthesis are:

[0073] (1) Compound b (110 mg, 0.331 mmol, 1 eq) and 2-(1-ethyl-2-methylquinolin-4(1H)-ylidene)malononitrile (77.3 mg, 0.033 mmol, 1 eq) were dissolved in 10 mL of acetonitrile, 50 μL of piperidine was added, and the mixture was heated to 85°C, refluxed under condensation, and stirred for reaction. The reaction was monitored by TLC. After the reaction was completed, the mixture was diluted with DCM and washed with 1 M hydrochloric acid. The organic phase was extracted with DCM three times and then dried over anhydrous sodium sulfate. Purification was performed by silica gel column chromatography (PE:EA=3:1) to obtain an orange solid (yield 82%). MS (ESI - ):m / z C 34 H 32 ClN3O2: 549.22 (theoretical value), visible 548.30 [MH] - . 1 H NMR (600MHz, CDCl3) δ9.16(d,J=1.4Hz,1H),7.76(m,1H),7.62(d,J=8.8Hz,1H),7.48(d,1H),7.45(d,J=5.8Hz ,2H),7.38(d,J=7.9Hz,1H),6.93(d,J=7.9Hz,1H),5.30(s,1H),4.39(m,2H),3.34(s,3H),1.94-1.65(m,17H).

[0074] (2) Synthesis of compound 4:

[0075] Compound CL-QM (20 mg, 0.0364 mmol, 1 eq) and o-iodobenzoyl chloride (9.7 mg, 0.0364 mmol, 1 eq) were dissolved in 5 mL of DCM, and 2 drops of triethylamine were added. The mixture was stirred and reacted for 2 hours until the reaction was complete. The mixture was then rotary evaporated to obtain the crude product, Compound 4.

[0076] (3) Synthesis of compound HS-CL-QM:

[0077] Compound 4 was dissolved in an appropriate amount of DCM, 2 mg of methylene blue was added, oxygen was introduced into the reaction solution, and the reaction was allowed to proceed under yellow light for 0.5 to 7 h. The reaction was monitored by TLC. After the reaction was complete, the product was rotary evaporated and purified by silica gel column chromatography (PE:EA=5:1) to obtain compound HS-CL-QM (yield 85%). MS (ESI - ):m / z C 41 H 35 ClN3O5: 811.13 (theoretical value), observed 811.25 [MH] - . 1 HNMR (600MHz, CDCl3) δ8.09 (d, J=4.7Hz, 1H), 7.58-7.55 (m, 1H), 7.53 (d, J= 8.6Hz,2H),7.49(d,J=8.5Hz,2H),7.43-7.36(m,1H),7.36-7.35(m,1H),7.1 6(d,J=2.4Hz,1H),7.14(d,J=2.4Hz,1H),7.13(d,J=2.5Hz,1H),7.12(d,J=2 .5Hz,1H),5.04(s,1H),3.72-3.72(m,2H),3.36(s,3H),1.28-1.28(m,17H).

[0078] (V) Synthesis of compound HS-CL-DCMC, the synthetic route is:

[0079]

[0080] The specific steps of synthesis are:

[0081] (1) Compound b (50 mg, 0.151 mmol, 1 eq) and 2-(2-methyl-4H-chromium-4-ylidene)malononitrile (31.5 mg, 0.151 mmol, 1 eq) were dissolved in 5 mL of acetonitrile, 23 μL of piperidine was added, and the mixture was heated to 85°C, refluxed under condensation, and stirred for reaction. The reaction was monitored by TLC. After the reaction was completed, the mixture was diluted with DCM and washed with 1 M hydrochloric acid. The organic phase was extracted with DCM three times and then dried over anhydrous sodium sulfate. Purification was performed by silica gel column chromatography (PE:EA=7:1) to obtain a red solid (yield 78%). MS (ESI+ ):m / zC 32 H 27 ClN2O3: 522.17 (theoretical value), visible 523.68 [M+H] + . 1 H NMR (600MHz, CDCl3) δ8.92(dd,J=8.6,4.1Hz,3H),7.86(d,J=16.1Hz,1H),7.71(d,J=8.5Hz,1H),7.60(d,J =8.4Hz,1H),7.05(d,J=16.1Hz,1H),6.93(d,J=7.9Hz,1H),6.35(s,1H),3.34(s,3H),2.67-2.37(m,14H).

[0082] (2) Synthesis of Compound 5:

[0083] Compound CL-DCMC (16.6 mg, 0.0317 mmol, 1 eq) and o-iodobenzoyl chloride (9.3 mg, 0.0348 mmol, 1.1 eq) were dissolved in 5 mL of DCM, and 2 drops of triethylamine were added. The mixture was stirred and reacted. After 30 minutes, the reaction was complete and the mixture was evaporated to give the crude product Compound 5.

[0084] (3) Synthesis of compound HS-CL-DCMC:

[0085] Compound 5 was dissolved in an appropriate amount of DCM, 1 mg of methylene blue was added, oxygen was introduced into the reaction solution, and the reaction was allowed to proceed under yellow light for 0.5 to 7 h. The reaction was monitored by TLC. After the reaction was complete, the mixture was rotary evaporated and purified by silica gel column chromatography (PE:EA=10:1) to obtain compound HS-CL-QM (yield). MS (ESI - ):m / z C 39 H 30 ClN2O6: 784.08 (theoretical value), visible 819.39 [M+Cl] - . 1 HNMR (600MHz, CDCl3) δ8.93-8.88(m,1H),8.28-8.24(m,1H),8.19-8.12(m,2H),7.80(d,J=8.4Hz,1H),7.73(d,J=16.0Hz,1H),7.60-7.5 6(m,1H),7.51-7.47(m,1H),7.47-7.44(m,1H),7.36-7.30(m,2H),6.95(d,J=16.0Hz,1H),5.30(s,1H),3.27(s,3H),1.28-1.25(m,14H).

[0086] Example 2: Five probes were used to measure hydrogen sulfide.

[0087] Sodium sulfide solution was prepared. Sodium sulfide solution and normal saline were respectively placed in the wells of a black 96-well plate. 100 μL of five chemiluminescent probes at a concentration of 40 μM were added to each well. The probes were detected by the Fluoroskan Ascent Fluorescence instrument and quantitatively analyzed by the instrument's built-in software. The results were as follows: Figure 1 All five probes respond to hydrogen sulfide, and the luminescence signal is essentially absent in saline. Among the five probes, HS-CL has the highest signal-to-noise ratio. The signal-to-noise ratios of the other four probes are ranked in descending order: HS-CL-TCF, HS-CL-DCMD, HS-CL-TCCH, and HS-CL-QM.

[0088] Example 3: Determination of the fluorescence spectra of five probes.

[0089] After the five chemiluminescent groups, namely CL, CL-TCF, CL-TCCH, CL-QM, and CL-DCMC, were prepared into a 40 μM physiological fluid aqueous solution containing 1% DMSO, 100 μL was taken and the fluorescence spectra of the five chemiluminescent groups were measured using a SpectraMax i3x microplate reader. The results are as follows: Figure 2 The excitation wavelength of CL is 420 nm and the emission wavelength is 535 nm. The excitation wavelength of CL-TCF is 440 nm and the emission wavelength is 525 nm. The excitation wavelength of CL-TCCH is 450 nm and the emission wavelength is 530 nm. The excitation wavelength of CL-QM is 450 nm and the emission wavelength is 615 nm. The excitation wavelength of CL-DCMC is 590 nm and the emission wavelength is 660 nm.

[0090] Example 4: Five probes were used to measure hydrogen sulfide at different concentrations.

[0091] After configuring hydrogen sulfide concentrations to 1000μM, 800μM, 600μM, 400μM, 200μM, 100μM, 80μM, 60μM, 40μM, 20μM, 10μM, and 5μM, 100μL of hydrogen sulfide solutions of different concentrations were added to the wells of a black 96-well plate. 100μL of five chemiluminescent probes at a concentration of 40μM was added to each well. The samples were detected by the Fluoroskan Ascent Fluorescence instrument and quantitatively analyzed by the instrument's built-in software. The results are shown in Figure 2. Figure 3As shown. The luminescence signals of the five probes showed an upward trend with the increase of hydrogen sulfide concentration. The luminescence signal of probe HS-CL had a good linear relationship with the hydrogen sulfide concentration of 5-100μM; the luminescence signal of probe HS-CL-TCF had a good linear relationship with the hydrogen sulfide concentration of 5-200μM; the luminescence signal of probe HS-CL-TCCH had a linear relationship with the hydrogen sulfide concentration of 10-200μM; the luminescence signal of probe HS-CL-QM had a good linear relationship with the hydrogen sulfide concentration of 5-200μM; the luminescence signal of probe HS-CL-DCMC had a good linear relationship with the hydrogen sulfide concentration of 5-200μM.

[0092] Example 5: Determination of β-lactamase using probe HS-CL.

[0093] 10 U / mL of β-lactamase dissolved in 50 mM PBS buffer (pH 7.0) and three β-lactam drugs (cephalexin, ceftizoxime, and cefazolin) dissolved in ultrapure water at a concentration of 1 mM were placed in equal volumes in a 1.5 mL EP tube and incubated at 37°C for 1 hour. 200 μL of the mixture was added to a black 96-well plate, followed by 100 μL of 40 μM probe HS-CL. The results were detected by a Fluoroskan Ascent Fluorescence instrument and analyzed by the instrument's built-in software. Figure 4 As shown, the drug-treated group showed a clear signal, while the untreated group showed essentially no luminescent signal. The ceftizoxime group produced the highest chemiluminescent signal. These results indicate that the HS-CL probe can successfully detect hydrogen sulfide produced by the hydrolysis of β-lactamase and β-lactam drugs.

[0094] Example 6: Probe HS-CL detects hydrogen sulfide produced by β-lactamase and β-lactam drugs.

[0095] Put 10 4 CFU / mL of drug-resistant bacteria, including Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC35218), drug-resistant Staphylococcus aureus, Klebsiella pneumoniae (ATCC 700603) and 10 4CFU / mL of drug-sensitive bacteria, including Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538), and 1mM β-lactam drug ceftizoxime were added to a 1.5L EP tube and incubated at 37°C for 3 hours. After that, 200μL of the mixture was added to a black 96-well plate, followed by 100μL of 40μM probe HS-CL. The results were detected by Fluoroskan Ascent Fluorescence instrument and analyzed by the instrument's built-in software. Figure 5 As shown, the chemiluminescence signals of drug-resistant Acinetobacter baumannii (a, e), drug-resistant Staphylococcus aureus (b, f), drug-resistant Escherichia coli (c, g), and drug-resistant Klebsiella pneumoniae (d, h) showed significant differences between the drug-treated and untreated groups. However, there was no significant difference in the chemiluminescence signals of drug-sensitive Staphylococcus aureus (i, j) and drug-sensitive Escherichia coli (k, l). These results demonstrate that the surface probe HS-CL can detect and distinguish drug-resistant bacteria from drug-sensitive bacteria.

[0096] Example 7: Probe HS-CL-DCMC was used to measure hydrogen sulfide produced by β-lactamase and β-lactam drugs.

[0097] Put 10 5 CFU / mL of drug-resistant bacteria, including Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC35218), drug-resistant Staphylococcus aureus, Klebsiella pneumoniae (ATCC 700603) and 10 4 CFU / mL of drug-sensitive bacteria, including Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538), and 1 mM of the β-lactam drug cefuroxime were added to a 1.5 L EP tube and incubated at 37 °C for 3 hours. After that, 200 μL of the mixture was added to a black 96-well plate, followed by 100 μL of the probe HS-CL-DCMC at a concentration of 40 μM. The results were detected by the Fluoroskan Ascent Fluorescence instrument and analyzed by the instrument's own software. Figure 6 As shown in the figure, the chemiluminescent signals of drug-resistant Acinetobacter baumannii (a, e), drug-resistant Staphylococcus aureus (b, f), drug-resistant Escherichia coli (c, g), and drug-resistant Klebsiella pneumoniae (d, h) showed significant differences between the drug-treated and untreated groups, while the chemiluminescent signals of drug-sensitive Staphylococcus aureus (i, j) and drug-sensitive Escherichia coli (k, j) did not differ significantly between the drug-treated and untreated groups. These results demonstrate that the surface probe HS-CL-DCMC is capable of detecting and distinguishing drug-resistant and drug-sensitive bacteria.

Claims

1. A hydrogen sulfide activated chemiluminescent probe, characterized in that: It is an adamantane-dioxetane chemical compound, and its structural formula is shown in (I): Among them, R is CH3O-, C 10 H6N3O-(TCF), C 11 H 11 N2-(TCCH), C 14 H 10 N3-(QM) or C 12 H5N2O-(DCMC), the corresponding probes are named HS-CL, HS-CL-TCF, HS-CL-TCCH, HS-CL-QM, and HS-CL-DCMC; they have different emission wavelengths and can all be activated by hydrogen sulfide to release photons.

2. The method for preparing a chemiluminescent probe according to claim 1, wherein The specific steps are: (1) Adamantane-dioxetane derivative precursor compound b and ethyl (triphenylphosphinimido) acetate were placed in a round-bottom flask, dichloromethane was added for dissolution, and the reaction was stirred for 15-45 minutes. The reaction solution was then washed with 0.8-1.2 M hydrochloric acid and extracted with dichloromethane. The organic phase was collected and rotary evaporated to dryness, and then purified by silica gel column chromatography to obtain a compound, which was recorded as CL. (2) Knoevenagel condensation reaction was used for condensation. Compound b and the modification group TCF, TCCH, QM or DCMC were dissolved in acetonitrile, and then a catalyst was added and heated with stirring. The mixture was condensed and refluxed. The reaction was stopped after 6-8 hours. The reaction solution was washed with 1M hydrochloric acid and extracted with dichloromethane. The organic phase was collected and rotary evaporated to dryness, and then purified by silica gel column chromatography to obtain compounds, which were respectively designated as CL-TCF, CL-TCCH, CL-QM, and CL-DCMC; (3) Compound CL in step (1) and the four compounds CL-TCF, CL-TCCH, CL-QM, and CL-DCMC in step (2) were dissolved in dichloromethane with o-iodobenzoyl chloride, and a basic catalyst was added dropwise. After the reaction was monitored by thin layer chromatography, the mixture was washed with aqueous solution and extracted with dichloromethane, and then dried over anhydrous Na2SO4 and rotary evaporated to dryness. After purification by silica gel column chromatography, the obtained products were respectively recorded as Compound 1, Compound 2, Compound 3, Compound 4, and Compound 5; (4) Compound 1, Compound 2, Compound 3, Compound 4, and Compound 5 were dissolved in dichloromethane, methylene blue was added, oxygen was continuously introduced, and yellow light was irradiated to react, and the reaction was monitored by thin layer chromatography; (5) After the reaction, the solvent was removed by vacuum rotary evaporation, and thin layer preparation and chromatography purification were performed to obtain the products, which were respectively designated as HS-CL, HS-CL-TCF, HS-CL-TCCH, HS-CL-QM, and HS-CL-DCMC; Wherein, the structural formula of the compound b is: The structural formulas of the modifying groups TCF, TCCH, QM and DCMC are: The name of the TCF compound is 2-dicyanomethylidene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran; The name of the TCCH compound is 3-dicyanomethylimide-1,5,5-trimethylcyclohexene; The name of the QM compound is 2-(1-ethyl-2-methylquinolin-4(1H)-ylidene)malononitrile; The compound name of DCMC is 2-(2-methyl-4H-chrome-4-ylidene)malononitrile.

3. The preparation method according to claim 2, characterized in that In step (1), the amount of compound b is 450-600 mg, the amount of ethyl (triphenylphosphinimido) acetate is 510-650 mg; the amount of 1M hydrochloric acid is 1-2 mL; and the elution conditions of silica gel column chromatography are petroleum ether:ethyl acetate = (20-15):

1.

4. The preparation method according to claim 2, characterized in that In step (2), the heating temperature is 75-90°C, the catalyst is piperidine in an amount of 5-50 μL, the amount of compound b is 40-110 mg (1 equivalent), the amount of the modifying group TCF is 30.0-45.0 mg, the amount of TCCH is 22.0-33.0 mg, the amount of QM is 77.0-115.5 mg, the amount of DCMC is 35.0-54.0 mg, and the amount of 1M hydrochloric acid is 5-10 mL; the elution conditions for silica gel column chromatography are petroleum ether:ethyl acetate = (15-10):

1.

5. The preparation method according to claim 2, characterized in that In step (3), the alkaline catalyst is triethylamine, the amount of which is 2 to 5 drops, the molar ratio of the compound CL to o-iodobenzoyl chloride is 1:(1 to 1.3); the molar ratio of the compound CL-TCF to o-iodobenzoyl chloride is 1:(1.1 to 1.5); the molar ratio of the compound CL-TCCH to o-iodobenzoyl chloride is 1:(1 to 1.3); the molar ratio of the compound CL-QM to o-iodobenzoyl chloride is 1:(1 to 1.2); and the molar ratio of the compound CL-DCMC to o-iodobenzoyl chloride is 1:(1.1 to 1.4).

6. The preparation method according to claim 2, characterized in that In step (4), the amount of methylene blue used is 1-2 mg; and the reaction time is 5-7 hours.

7. The preparation method according to claim 2, characterized in that In step (5), the elution condition of thin layer chromatography is petroleum ether:ethyl acetate=(20-5):

1.

8. Use of the chemiluminescent probe according to claim 1 in detecting hydrogen sulfide; the concentration of hydrogen sulfide is 5-1000 μM, and the concentrations of the five probes are all 20-100 μM.

9. Use of the chemiluminescent probe HS-CL according to claim 1 in the determination of β-lactamase; the concentration of β-lactamase is 0.5-20 U / mL, and the β-lactam drug is cephalexin, ceftizoxime or cefazolin, and the concentration is 0.2-10 mM.

10. The use of the chemiluminescent probe HS-CL or HS-CL-DCMC as claimed in claim 1 in determining drug-resistant bacteria and drug-sensitive bacteria, wherein the drug-resistant bacteria used are Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 35218), drug-resistant Staphylococcus aureus, and Klebsiella pneumoniae (ATCC 700603), and the drug-sensitive bacteria are Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538); for the chemiluminescent probe HS-CL, the bacterial concentration is 10 2 -10 5 CFU / mL; chemiluminescent probe HS-CL-DCMC, bacterial concentration 10 3 -10 6 CFU / mL.

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