Cephalosporin hybrid fluorescent probe and application thereof in identification of mycobacterium tuberculosis

Abstract

The invention belongs to the technical field of organic synthesis and medicine, and particularly relates to a cephalosporin hybrid fluorescent probe for specifically recognizing mycobacterium tuberculosis. Through a specific recognition strategy of biosynthesis of DprE1 enzyme for mycobacterium tuberculosis beta-lactamase and mycobacterium tuberculosis cell wall arabinogalactan, the probe designed by the invention couples a cephalosporin fragment, a fluorophore fragment and a mycobacterium tuberculosis cell wall recognition fragment to form the cephalosporin hybrid fluorescent probe. According to the cephalosporin heterozygous fluorescent probe developed by the invention, an electronic environment of a probe recognition fragment is created and innovated from a molecular level by changing an electronic effect, a steric-hindrance effect, a # imgabs0 # stacking effect, an action mode between a subject and an object and the like of a recognition center, and a brand-new three-dimensional structure of the recognition fragment is constructed; further, the fluorescent probe which is good in stability, high in solubility, high in luminous intensity and excellent in recognition performance compared with an existing mycobacterium tuberculosis fluorescent probe is prepared and screened.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis and medicine, and particularly relates to a cephalosporin hybrid fluorescent probe and application thereof in identifying Mycobacterium tuberculosis. Background Art

[0002] Early diagnosis of tuberculosis is extremely important for the treatment of patients. The current identification methods for Mycobacterium tuberculosis are: (1) Sputum smear method, which is the most commonly used and most basic method for diagnosing tuberculosis, but this method cannot distinguish the vital characteristics of Mycobacterium tuberculosis and has low sensitivity. (2) Sputum culture method, which can identify dead and live bacteria and has higher sensitivity. It is an irreplaceable method for live bacteria detection and is the "gold standard" for diagnosing tuberculosis. However, this method takes too long to culture and is cumbersome to operate, usually requiring 4-8 weeks, making it difficult to provide timely and effective evidence for clinical diagnosis. (3) Immunological diagnosis, among which, humoral immune detection, the heterogeneity of Mycobacterium tuberculosis antigens and the differences in patient antibody responses are significant, resulting in differences in the recognition of a single antigen, requiring multiple antigen joint detection to improve accuracy; cytological immunoassay, which cannot distinguish between latent infection, active tuberculosis or BCG vaccination reaction, and its sensitivity and specificity are easily affected by immune status and cross-reactions, requiring a second visit to interpret the results, and the interpretation of the results is subjective.

[0003] As a key tool, fluorescent probes play an important role in molecular biological diagnostic technology. They are expected to quickly fluorescently label Mycobacterium tuberculosis in a short period of time, and then specifically bind to Mycobacterium tuberculosis, identify and count them through fluorescence, and promote personalized treatment and prevention and control of drug-resistant tuberculosis.

[0004] Therefore, developing an effective method for rapid diagnosis of tuberculosis is of great significance to the prevention and treatment of tuberculosis. Summary of the Invention

[0005] like Figure 9 As shown, based on previous studies, the present invention uses a specific recognition strategy for Mycobacterium tuberculosis β-lactamase and the DprE1 enzyme of Mycobacterium tuberculosis cell wall arabinogalactan biosynthesis to design a probe that couples a cephalosporin fragment, a fluorescent group fragment, and a Mycobacterium tuberculosis cell wall recognition fragment to form a cephalosporin hybrid fluorescent probe. The reported Mycobacterium tuberculosis fluorescent probe based on β-lactamase recognition has poor water solubility and poor stability of the three-membered ring structure at the recognition end, making it difficult to store for a long time and clinically apply it (see ACS Central Science, Vol. 7, No. 5, April 21, 2021).

[0006] The cephalosporin hybrid fluorescent probe developed by the present invention changes the electronic effect, steric hindrance effect, Stacking effect, host-guest interaction mode, etc., and then create and innovate the electronic environment of the probe recognition fragment at the molecular level, construct a new three-dimensional structure of the recognition fragment, and then prepare and screen fluorescent probes that have better stability, higher solubility, higher luminescence intensity and excellent recognition performance than existing Mycobacterium tuberculosis fluorescent probes.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] A cephalosporin hybrid fluorescent probe, the structural formulas of which are shown in the following formulas (I) to (IX):

[0009]

[0010]

[0011]

[0012]

[0013] The present invention also protects the application of the cephalosporin hybrid fluorescent probe in the identification of Mycobacterium tuberculosis.

[0014] Furthermore, the cephalosporin hybrid fluorescent probe is used in identifying and detecting Mycobacterium tuberculosis β-lactamase.

[0015] Furthermore, the cephalosporin hybrid fluorescent probe is used to prepare a preparation for identifying Mycobacterium tuberculosis.

[0016] The present invention also provides a method for detecting Mycobacterium tuberculosis using the cephalosporin hybrid fluorescent probe. The detection method uses the cephalosporin hybrid fluorescent probe for detection, and the detection method comprises the following steps: providing a sample to be tested; mixing the sample to be tested and the fluorescent probe for incubation to obtain a mixed solution; and performing fluorescence spectrum detection or fluorescence imaging detection on the mixed solution.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] Through the specific recognition strategy of Mycobacterium tuberculosis β-lactamase and Mycobacterium tuberculosis cell wall arabinogalactan biosynthesis DprE1 enzyme, the probe designed by the present invention couples the cephalosporin fragment, the fluorescent group fragment and the Mycobacterium tuberculosis cell wall recognition fragment to form a cephalosporin hybrid fluorescent probe. The reported Mycobacterium tuberculosis fluorescent probe based on β-lactamase recognition has poor water solubility and poor stability of the three-membered ring structure at the recognition end, which makes it difficult to store it for a long time and use it in clinical practice. Compared with the reported ones, the cephalosporin hybrid fluorescent probe developed by the present invention changes the electronic effect, steric hindrance effect, Stacking effect, host-guest interaction mode, etc., and then create and innovate the electronic environment of the probe recognition fragment at the molecular level, construct a new three-dimensional structure of the recognition fragment, and then prepare and screen fluorescent probes with better stability, higher solubility, higher luminescence intensity and excellent recognition performance than the existing Mycobacterium tuberculosis fluorescent probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the fluorescence intensity of SO-NH2, SO-NHCO, and SO-E series probes;

[0020] Figure 2 is the fluorescence intensity of S-NH2, S-NHCO, and SE series probes;

[0021] Figure 3 is the fluorescence intensity of S-OMe-NH2, S-OMe-NHCO, and S-OMe-E series probes;

[0022] Figure 4 is the fluorescence intensity of the cephalosporin hybrid fluorescent probe;

[0023] Figure 5 are the fluorescence intensities of probe SE in the presence of excitation light and β-lactamase, respectively;

[0024] Figure 6 are the fluorescence intensities of the probe S-NH2 in the presence of excitation light and β-lactamase, respectively;

[0025] Figure 7 is the fluorescence intensity of the probe S-NHCO in the presence of excitation light and β-lactamase respectively;

[0026] Figure 8 is the fluorescence intensity of probe SE, S-NH2, and S-NHCO in the presence of β-lactamase;

[0027] Figure 9 Schematic diagram of the structural design of the probe of the present invention. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention and comparative examples. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0030] Example 1: Synthesis route and specific implementation steps of cephalosporin hybrid fluorescent probe:

[0031] (1) Synthesis of fluorescent group fragment A5

[0032]

[0033]

[0034] Synthesis of compound A1

[0035]

[0036] 2-(tert-Butyloxycarbonylamino)ethyl bromide (29.10 g, 129.85 mmol) was added to a suspension of 4-bromo-3-methylphenol (20.24 g, 108.22 mmol) and potassium carbonate (22.43 g, 102.33 mmol) in N,N-dimethylformamide (87 mL). The mixture was then heated under reflux overnight. Ethyl acetate and water were added, the organic layer was separated, and the crude product was concentrated to dryness under vacuum for the next step. Hydrochloric acid-ethanol solution (54 mL) was added to the flask containing the crude product at 0°C, and the resulting mixture was stirred at room temperature for 2 hours. After concentration, the product was precipitated with diethyl ether to obtain Compound A1 (13.40 g, 47% total yield for two steps). 1 H NMR (400MHz, DMSO-d6) δ8.36–8.32(s,3H),7.53–7.45(d,J=8.8Hz,1H),7.04–6.99(d,J=3.1Hz,1H),6.82 –6.75(dd,J=8.7,3.1Hz,1H),4.22–4.15(t,J=5.2Hz,2H),3.21–3.14(t,J=5.2Hz,2H),2.34–2.30(s,3H). 13 C NMR (100MHz, DMSO-d6) δ157.67,138.78,133.12,117.81,115.61,114.88,64.85,38.59,23.05.

[0037] Synthesis of compound A2

[0038]

[0039] A1 (13.53 g, 50.67 mmol) and potassium carbonate (28.01 g, 202.68 mmol) were dissolved in N,N-dimethylformamide (844 mL) and stirred for 30 min. 3-Bromo-1-propene (10.96 mL, 126.68 mmol) was then added. The suspension was heated at 80°C overnight. The solvent was removed, and ethyl acetate and water were added. The organic layer was separated and concentrated using a rotary evaporator. The crude product was purified by flash column chromatography on silica gel to yield A2 (9.23 g, 59%). 1 H NMR(400MHz, CDCl3) δ7.31–7.23(d,J=8.8Hz,1H),6.71–6.66(d,J=3.2Hz,1H),6.54–6.47(dd,J=8.7,3.2Hz,1H),5.86–5.71(ddt,J=16.8,1 0.1, 6.5Hz, 1H), 5.16–5.03 (m, 5H), 3.94–3.86 (t, J = 6.1Hz, 2H), 3.13–3.07 (d, J = 6.5Hz, 4H), 2.80–2.73 (t, J = 6.1Hz, 2H), 2.27–2.23 (s, 3H). 13 C NMR (100MHz, CDCl3) δ158.07,138.75,135.55,132.77,117.76,117.15,115.40,113.55,77.40,77.09,76.77,66.70,57.75,51.88,23.14.

[0040] Synthesis of compound A3

[0041]

[0042] 2,2',4,4'-Tetrahydroxybenzophenone (4 g, 16.26 mmol) was dissolved in water (24 mL) and heated at Taiatsu The mixture was heated at 200° C. for 21 hours in a pressure tube. After cooling, the product was collected by filtration, washed with cold n-hexane and dried to give 2 as a white solid (3.7 g, yield 98%). 1 H NMR (400MHz, DMSO-d6) δ8.08–7.95 (d, J=8.7Hz, 2H), 6.90–6.84 (dd, J=8.7, 2.2Hz, 2H), 6.84–6.77 (d, J=2.2Hz, 4H). 13 C NMR (100MHz, DMSO-d6)) δ174.42,163.80,157.94,128.21,114.46,114.09,102.55.

[0043] Synthesis of compound A4

[0044]

[0045] A3 (3.7 g, 16.26 mmol) and imidazole (11.07 g, 162.65 mmol) were dissolved in N,N-dimethylformamide (65 mL) under nitrogen protection. After the reaction solution was cooled to 0°C, tert-butyldimethylsilyl chloride (14.708 g, 97.59 mmol) was added to the reaction flask in batches, and the mixture was stirred at room temperature for 5 hours to form an orange paste suspension. The reaction mixture was diluted with toluene (400 mL) and extracted three times with ethyl acetate. The organic phase was washed with water and saturated sodium chloride solution, dried over anhydrous magnesium sulfate, and evaporated to an orange residue, which was washed with ethanol and n-hexane to obtain an orange-red crystalline powder. (5.116 g, yield 69%) 1 HNMR (400MHz, CDCl3) δ8.09–8.04 (m, 2H), 6.88–6.43 (d, J = 7.8Hz, 4H), 0.88–0.87 (s, 18H), 0.18–0.11 (s, 12H). 13 C NMR (100MHz, CDCl3) δ175.75,161.40,157.78,128.22,117.60,116.47,107.37,77.36,77.04,76.72,25.58,18.29,-4.33.

[0046] Synthesis of compound A5

[0047]

[0048] Under nitrogen protection, anhydrous tetrahydrofuran (20 mL) was added to a flask containing A2 (3.10 g, 10 mmol) at -78 ° C, and sec-butyl lithium (1.6 M, 7.69 mL, 10 mmol) was added dropwise, and the reaction solution was stirred at this temperature for 30 minutes. A4 (4.57 g, 10 mmol) dissolved in anhydrous tetrahydrofuran (27 mL) was then slowly added to the reaction solution. The reaction mixture was reacted at room temperature for 1 hour, and 5 mL of 2M hydrochloric acid was added to quench the reaction. 100 ml of ethyl acetate was added, and the organic layer was washed twice with 20 ml of water. After concentration to dryness under vacuum, the crude product was purified by flash column chromatography to obtain product A5 (3.69 g, yield 87%). 1H NMR(400MHz, DMSO-d6)δ7.18–7.14(d,J=8.4Hz,1H),7.08–7.03(d,J=2.6Hz,1H),7. 01–6.94(dd,J=8.4,2.7Hz,1H),6.94–6.85(d,J=9.2Hz,1H),6.64–6.54(m,4H),5.95 –5.80(ddt,J=16.5,10.1,6.3Hz,2H),5.29–5.12(m,4H),4.18–4.10(t,J=6.1Hz,2H ),3.23–3.16(dt,J=6.4,1.5Hz,4H),2.89–2.82(t,J=6.1Hz,2H),2.01–1.97(s,3H). 13 C NMR (100MHz, DMSO-d6) δ159.64,156.87,150.22,137.86,136.38,130.89,124.77,118.02,116.80,112.71,103.91,66.55,57.25,51.90,19.92.

[0049] (2) Synthesis of cephalosporin derivatives

[0050]

[0051] Synthesis of compound B1

[0052]

[0053] To a 500mL round-bottom flask, add 7-aminocephalosporanic acid (7.45g, 64mmol), sodium carbonate (11.66g, 70.5mmol), 96mL of water, and 80mL of acetone. Cool the reaction mixture to -5°C and stir for 10 minutes to homogenize. At this temperature, add phenylacetyl chloride (8.72mL, 70.5mmol) dropwise. After the addition is complete, add 100mL of water and 30mL of acetone. Allow to react overnight at room temperature. After the reaction is complete, add 500mL of ethyl acetate and dropwise add 10% hydrochloric acid solution to adjust the pH of the reaction mixture to 3.0-3.5. Separate the ethyl acetate layer, extract the aqueous layer with ethyl acetate, and combine the organic phases, wash with 300mL of saturated NaCl solution, and dry over anhydrous sodium sulfate. Filter to remove the desiccant, concentrate under reduced pressure to dryness, add 300mL of petroleum ether (60-90°C), and stir at room temperature for 30 minutes. After filtration, the filter cake was dried under vacuum at 40°C to obtain a white solid B1 (25 g, yield 71%). 1H NMR(400MHz, DMSO-d6)δ14.05–12.60(s,1H),9.14–8.92(d,J=8.3Hz,1H),7.36–7.10(m,5H),5.73–5.65(dd,J=8.3,4.8Hz,1H),5.12–5.06(d, J=4.9Hz,1H),5.03–4.92(d,J=12.8Hz,1H),4.74–4.60(d,J=12.8Hz,1H),3.65–3.58(d,J=18.0Hz,1H),3.60–3.44(m,3H),2.06–1.99(s,3H). 13 C NMR(100MHz,DMSO-d6)δ171.44,170.68,165.26,163.32,136.27,129.48,1 28.70,126.97,126.85,123.79,63.18,59.57,57.90,42.05,26.01,21.05.

[0054] Synthesis of compound B3

[0055]

[0056] To a mixed solution of 40 mL of water and 30 mL of methanol cooled to -20°C was added B1 (7.81 g, 20 mmol). At this temperature, sodium hydroxide (1.2 g, 30 mmol) dissolved in 40 mL of aqueous solution was added over 40 minutes. The reaction mixture was stirred for 40 minutes. After complete conversion of the starting materials, 10% hydrochloric acid (3.5 mL) was added dropwise to neutralize the mixture to a pH of approximately 6 to obtain compound B1-1. A solution of diphenyldiazomethane (11.65 g, 60 mmol) dissolved in 20 mL of ethyl acetate was then added to the neutralized mixture. The mixture was stirred at 5°C for 3 hours. 8 mL of 10% hydrochloric acid was added dropwise to the mixture. Nitrogen evolution was observed during the reaction, and a precipitate gradually formed during stirring, forming a slurry. The slurry was stirred at the same temperature for another 2 hours until no nitrogen evolution was observed. The resulting precipitate was collected by filtration and washed twice with ethyl acetate to obtain the crude product. The dried crude product was dissolved in 45 mL of acetone and refluxed for 2 h to form a slurry. The slurry was cooled to room temperature and then filtered to obtain the target compound B3 (4.07 g, yield 40%). 1H NMR(400MHz, DMSO-d6)δ9.25–8.86(d,J=8.3Hz,1H),7.54–7.47(m,2H),7.44–7.39( m,2H),7.39–7.32(m,4H),7.32–7.19(m,7H),6.92–6.88(s,1H),5.78–5.64(dd,J=8. 4,4.7Hz,1H),5.19–5.14(t,J=5.7Hz,1H),5.12–5.08(d,J=4.8Hz,1H),4.28–4.14(m ,2H),3.65–3.59(s,2H),3.60–3.55(d,J=13.8Hz,1H),3.54–3.48(d,J=13.8Hz,1H). 13 C NMR(100MHz,DMSO-d6)δ171.59,165.83,161.48,140.62,140.56,136.40,135.30,129.60,129.43,129.09,128.96,1 28.89,128.79,128.65,128.41,128.34,127.34,127.15,127.06,122.52,78.92,60.33,59.48,58.30,42.18,26.18.

[0057] Synthesis of compound B4

[0058]

[0059] A suspension of calcium carbonate (3.7 g, 16.26 mmol), N,N-dimethylformamide (11.07 g, 162.65 mmol), and thionyl chloride in tetrahydrofuran was cooled to -20°C and then added portionwise to the reaction flask with B3 (14.708 g, 97.59 mmol). After stirring at -20°C for 30 minutes, an orange paste suspension was formed. The suspension was filtered through diatomaceous earth and the filter cake was washed with dichloromethane. After vacuum distillation, the crude product was purified by flash column chromatography to obtain product B4 (13.69 g, 66% yield). 1H NMR (400MHz, CDCl3) δ7.47–7.28(m,2H),7.28–7.08(m,13H),6.86–6.81(s,1H ),6.73–6.60(d,J=8.9Hz,1H),5.82–5.55(dd,J=8.9,4.9Hz,1H),5.01–4.60( d,J=4.9Hz,1H),4.25–4.21(s,2H),3.51–3.47(d,J=16.1Hz,1H),3.47–3.43( d,J=15.9Hz,1H),3.38–3.33(d,J=18.6Hz,1H),3.26–3.21(d,J=18.3Hz,1H). 13 C NMR (100MHz, CDCl3) δ171.59,165.09,160.54,139.16,139.03,134.00,129.48,129.08,128.69,128.5 7,128.35,128.24,127.73,127.68,127.59,126.98,125.51,79.93,59.23,57.83,43.16,43.13,27.19.

[0060] Synthesis of compound B6

[0061]

[0062] A mixture of B4 (17.5 g, 32.88 mmol), potassium carbonate (8.447 g, 65.76 mmol), sodium iodide (4.9 g, 150 mmol) and 4-hydroxybenzaldehyde (12.05 g, 122.12 mmol) was stirred in acetonitrile (657 mL) at room temperature for 6 h until the starting material 1 disappeared (monitored by thin layer chromatography (TLC)). The solvent was removed under reduced pressure, and the residue was dissolved in water and treated with ethyl acetate. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with aqueous sodium thiosulfate solution and brine, and then dried over magnesium sulfate. After purification by flash column chromatography on a silica gel column, a mixture of compound 5 and its isomer 5' was obtained. The mixture was dissolved in dichloromethane (300 mL). At 0°C, m-chloroperbenzoic acid (mCPBA, 68%, 4.14 g, 23.96 mmol) was added in several portions. After stirring at 0°C for 30 minutes to complete the reaction (monitored by TLC), the reaction mixture was diluted with dichloromethane, quenched with 10% sodium bisulfite solution, washed with aqueous sodium bicarbonate solution and brine. The crude product was purified by a short silica gel column to give compound B6 (8.29 g, 59% yield). 1H NMR (400MHz, CDCl3) δ9.89–9.84(s,1H),7.78–7.73(d,J=8.8Hz,2H),7.47–7.44(m,2H),7.42–7.22(m, 14H),6.97–6.94(s,1H),6.85–6.78(dd,J=9.4,7.5Hz,3H),6.15–6.07(dd,J=9.9,4.8Hz,1H),5.30–5. 23(d,J=13.9Hz,1H),4.78–4.72(d,J=13.9Hz,1H),4.49–4.42(dd,J=4.8,1.7Hz,1H),4.00–3.92(d,J= 13.9Hz,1H),3.69–3.63(d,J=15.6Hz,1H),3.63–3.57(d,J=15.6Hz,1H),3.32–3.20(d,J=13.9Hz,1H)). 13 C NMR (100MHz, CDCl3) δ190.62,171.36,164.27,162.42,159.95,138.91,138.87,133.67,132.06,130.72,129.40,129.0 7,128.73,128.64,128.37,127.63,127.50,127.06,124.67,122.29,114.79,80.49,67.33,66.87,59.10,45.38,43.37.

[0063] Synthesis of compound B8

[0064]

[0065] To a solution of B5 (5.442 g, 8.57 mmol) in dichloromethane (190 mL) and methanol (190 mL) at 0°C, sodium borohydride (356.79 mg, 9.43 mmol) was added portionwise. The resulting mixture was stirred at 0°C until thin-layer chromatography (TLC) showed the disappearance of the starting material (approximately 0.5 hours). Water and 1N hydrochloric acid were then added to neutralize the reaction mixture, the organic layer was separated, and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over magnesium sulfate, and the solvent was removed by rotary evaporation to obtain crude product B6. Without further purification, 2,6-lutidine was added to a solution of crude product 8 in N,N-dimethylformamide (37 mL) at 0°C. After stirring for 10 minutes, methanesulfonyl chloride (3.53 mL, 13.94 mmol) was added dropwise. The resulting mixture was stirred at room temperature for four hours until thin-layer chromatography (TLC) showed the disappearance of the starting material. Ethyl acetate was added and the mixture was washed with water. The target compound B7 (5.74 g, 73% yield based on the consumption of starting material 7) was obtained by purification via silica gel flash chromatography. 1 H NMR (400MHz, DMSO) δ8.58–8.39 (d, J=8.4Hz, 1H), 7.57–7.50 (d, J=7.2Hz, 2H), 7.50–7.40 (d, J=7.5Hz, 2H), 6.82–6.66 (d, J=8.7Hz, 2H), 5.98–5.91 (dd, J=8.4, 4.8Hz, 1H), 4.98–4.97 (d, J=4.8Hz,1H),4.97–4.93(d,J=13.9Hz,1H),4.74–4.68(m,3H),4.09–4.02(d,J=19.1Hz,1H) ,3.74–3.70(d,J=14.1Hz,1H), 3.71–3.67f(d,J=19.1Hz,1H), 3.62–3.55(d,J=14.1Hz,1H). 13 C NMR (100MHz, DMSO) δ171.60,165.09,160.45,158.23,140.19,140.11,136.27,130.91,129.61,129.04,128.9 2,128.78,128.41,127.43,127.03,124.75,122.49,115.15,79.48,67.30,67.00,58.63,46.60,45.95,41.98.

[0066] (3) Synthesis of cephalosporin hybrid fluorescent probes SO-NH2, SO-NHCO, and SO-E

[0067]

[0068] Synthesis of compound SO-W

[0069]

[0070] A5 (6.971 g, 15.8 mmol), potassium bicarbonate (3.48 g, 31.6 mmol) and 18-crown ether-6 (4.177 g, 15.8 mmol) were placed in anhydrous N, N-dimethylformamide (50 mL) and stirred at room temperature for 10 minutes. Compound B7 was dissolved in anhydrous N, N-dimethylformamide (50 mL) and added dropwise to the above reaction solution. The reaction solution was stirred at room temperature under dark conditions for 4 days until thin layer chromatography (TLC) showed that the starting material disappeared. Ethyl acetate was added and washed with water. Purification was performed by silica gel flash chromatography to obtain pure compound SO-W (910 mg, yield 5.7%). 1 H NMR (500MHz, CDCL3) δ7.42–7.15(m,21H),7.07–7.01(m,4H),6.94–6.78(m,3H),6.69–6.63(d,J=8.4Hz,2H),6.63–6.59(d,J=1 0.2Hz,1H),6.52–6.37(m,3H),5.98–5.86(ddt,J=16.9,10.2,6.5Hz,2H),5.79–5.70(dd,J=10.1,4.6Hz,1H),5.32–5.15(m,4H ),5.05–4.95(s,2H),4.37–4.33(d,J=4.7Hz,1H),4.27–4.20(d,J=15.8Hz,1H),4.18–4.13(t,J=6.1Hz,2H),4.08–3.98(m,2H) ,3.96–3.90(d,J=14.8Hz,1H),3.57–3.53(s,2H),3.33–3.28(d,J=6.2Hz,4H),3.02–2.96(t,J=6.1Hz,2H),2.04–1.93(s,3H). 13C NMR (100MHz, CDCL3) δ185.91,171.42,166.55,164.68,163.81,159.59,159.44,157.62,156.63,154.87,151.45,146.74,138.5 6,138.48,137.94,134.00,131.78,131.25,130.47,129.93,129.48,129.41,129.11,129.01,128.98,128.91,128.79,128.69, 128.56,127.50,127.10,126.68,124.99,124.64,124.31,119.42,118.48,116.74,115.83,114.99,114.62,112.22,105.53,10 1.39,80.10,77.46,77.41,77.21,76.95,70.53,66.70,66.64,59.14,57.46,51.80,43.43,39.67,20.09.HRMS(ESI):Calc.for C 64 H 56 N3O 10 S - [MH] - 1058.3692,found 1058.3696.mp110-180℃.IR(neat):2925,2851,1736,1640,1595,1495,1454,137 9,1289,1241,1207,1176,1109,1030,909,805,731,701,676,665,649,641,616cm -

[0071] Synthesis of compound SO-NH2

[0072]

[0073] Under a nitrogen atmosphere, dichloromethane (7.2 mL) was added to a flask containing tetrakis(triphenylphosphine)palladium (37.35 mg, 15 mol%), 1,3-dimethylbarbituric acid (112.78 mg, 3.33 mmol) and compound SO-W (230 mg, 0.22 mmol). The resulting mixture was then reacted at 40 degrees Celsius for 3 hours. Dichloromethane and saturated sodium bicarbonate solution were then added, and the organic layer was separated, washed with brine, and dried over magnesium sulfate. Purification was performed by silica gel flash chromatography to obtain pure compound SO-NH2 (90.8 mg, 43% yield).1 H NMR(400MHz,DMSO)δ8.43–8.36(d,J=8.8Hz,1H),7.52–7.46(d,J=7.8Hz,2H),7.45–7.37(m,4H),7.28–7.21(dd,J=12.7,7.4Hz,6H),7.21–7.13(m,2H),7.07–6.97(d,J=12.8Hz,2H),6.97–6.89(q,J=8.4Hz,3H),6.67–6.58(t,J=8.7Hz,4H),6.50–6.43(d,J=10.4Hz,1H),6.28–6.22(s,1H),5.75–5.53(dd,J=8.7,4.7Hz,1H),5.26–5.12(s,2H),4.91–4.86(d,J=5.0Hz,1H),4.41–4.31(d,J=15.3Hz,1H),4.27–4.20(d,J=15.4Hz,1H),4.20–4.14(s,2H),4.01–3.92(d,J=14.9Hz,1H),3.92–3.86(d,J=15.0Hz,1H),3.74–3.65(d,J=14.3Hz,1H),3.63–3.56(d,J=14.3Hz,1H),3.19–3.03(s,2H),2.27–1.86(s,3H). 13 C NMR(100MHz,DMSO)δ184.47,171.40,166.74,164.99,163.64,159.45,158.89,157.72,157.01,154.41,149.55,143.37,140.06,139.86,137.91,136.12,132.50,131.99,131.06,130.91,130.16,129.70,129.27,129.20,129.16,129.05,128.93,128.75,128.68,128.56,128.51,127.04,126.99,126.70,124.87,124.30,121.29,118.22,116.89,115.51,115.00,114.75,114.63,112.86,105.19,101.96,79.63,70.38,66.89,66.28,65.79,59.16,42.15,38.37,19.89.HRMS(ESI):Calc.for C 58 H 48 N3O 10 S- [MH] + 978.3037,found 978.3078.mp141-187℃.IR(neat):1261,1095,1021,856,802,698

[0074] Synthesis of compound SO-NHCO

[0075]

[0076] SO-NH2 (90.8 mg, 0.09 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL). Triethylamine (55 μL, 0.40 mmol) was added to make the reaction solution pH>7, and then a solution of 3,5-dinitrobenzoyl chloride (35.67 mg, 0.15 mmol) dissolved in 1.5 mL of anhydrous tetrahydrofuran was added dropwise. The mixture was stirred at room temperature for 2 hours until thin layer chromatography (TLC) showed that the starting material disappeared. The solvent was concentrated and ether (7 mL) was added for precipitation. The solid was purified by silica gel column flash chromatography to obtain pure compound SO-NHCO (23 mg, yield 22%). 1 H NMR (400MHz, CDCl3) δ9.29–9.22(s,1H),9.20–9.15(s,1H),9.12–9.09(s,1H),7.38–7.28(m,17H),7.15–7.12(m,4H),7.1 1–7.03(m,3H),7.01–6.95(m,3H),6.95–6.91(m,2H),6.64–6.58(d,J=8.1Hz,1H),5.86–5.77(dd,J=7.8,4.5Hz,1H),5.18– 5.09(s,2H),4.45–4.42(d,J=4.3Hz,1H),4.39–4.31(m,2H),4.32–4.27(d,J=15.1Hz,1H),4.19–4.14(d,J=14.5Hz,1H),4 .14–4.11(dd,J=43.5,14.3Hz,1H),4.11–4.06(d,J=15.0Hz,1H),4.04–3.96(m,2H),3.70–3.45(s,2H),2.01–1.96(s,3H). 13CNMR (100MHz, CDCl3) δ184.48,171.10,166.28,164.70,164.26,163.46,160.92,159.64,159.33,157.47,155.14,149.59 ,148.75,148.43,145.59,138.35,138.22,138.03,133.82,133.19,132.07,130.39,129.78,129.52,129.37,129.06,128. 91,128.77,128.07,127.43,127.01,125.88,124.45,122.91,121.15,120.75,118.29,116.82,115.11,114.91,112.30,1 05.41,101.25,80.26,77.43,77.11,76.79,70.59,66.40,66.06,59.12,43.45,40.08,39.20,20.00.HRMS(ESI):Calc.for C 65 H 50 N5O 15 S - [MH] + 1172.3030,found1172.3036m.p.141-187℃.IR(neat):1542,1344,1257,1092,891,854,801,754,728,701

[0077] Synthesis of compound SO-E

[0078]

[0079] Compound SO-NHCO (54 mg, 0.05 mmol) was added to a 2 mL solution of dichloromethane, trifluoroacetic acid, triisopropylsilane, and water (volume ratio: 50:45:2.5:2.5). The resulting mixture was stirred at room temperature for 3 h until thin-layer chromatography (TLC) indicated the disappearance of the starting material. The solid was purified by silica gel flash chromatography to yield the target compound SO-E (23 mg, 50% yield). 1H NMR(400MHz,DMSO)δ9.89–9.48(d,J=9.9Hz,1H),9.20–9.02(m,3H),9.01–8.95(d,J=6.6Hz,1H),7.52–7.40(t,J=8.2Hz,2H),7.38–7.29(m,7H),7.29–7.15(m,6H),7.15–7.07(d,J=17.0Hz,1H),7.07–6.97(m,1H),6.90–6.76(d,J=9.9Hz,2H),5.75–5.69(m,1H),5.36–5.23(s,2H),5.02–4.85(d,J=4.6Hz,2H),4.68–4.58(d,J=12.1Hz,1H),4.38–4.26(s,2H),4.25–4.12(s,2H),3.90–3.74(s,2H),3.75–3.56(m,2H),3.56–3.47(m,2H),2.12–1.87(s,3H). 13 C NMR(100MHz,DMSO)δ179.51,171.50,167.23,166.15,165.42,163.01,162.04,159.99,159.46,158.90,158.46,156.81,149.27,148.86,148.69,138.09,137.24,136.62,136.33,133.77,132.66,131.13,130.89,130.64,130.45,130.00,129.81,129.56,129.51,129.13,128.86,128.81,128.73,128.49,128.02,126.98,126.40,124.32,123.47,122.08,121.39,118.14,117.65,116.97,116.50,115.79,115.64,114.72,112.78,107.48,104.08,103.21,101.86,79.12,71.14,68.05,67.51,66.42,58.60,46.06,42.00,41.57,19.97.HRMS(ESI):Calc.for C 52 H 41 N5NaO 15 S + [M+Na] +1030.2212,found 1030.2242mp126-173℃.IR(neat):1731,1673,1596,1541,1461,1344,1265,1240.12 03,1171,1116,1075,1024,912,860,833,800,759,729,718,681,672,660,626,610.

[0080] (4) Synthesis of cephalosporin hybrid fluorescent probes S-NH2, S-NHCO, SE

[0081]

[0082] Synthesis of compound SW

[0083]

[0084] To a mixed solution of SO-W (431.1 mg, 0.41 mmol) and sodium iodide (304.7 mg, 2.03 mmol) dissolved in anhydrous acetone (8 mL) at 0°C, trifluoroacetic anhydride (0.32 mL, 2.3 mmol) was added dropwise. The resulting mixture was stirred at 0°C for 1 hour, after which the solvent and HCl were removed under reduced pressure. The residue was dissolved in aqueous sodium bicarbonate and extracted with ethyl acetate. After purification by silica gel flash chromatography, the target compound SW (210 mg, 50% yield) was obtained. 1 H NMR (500MHz, CDCL3) δ7.35–7.28(m,3H),7.25–7.16(m,3H),7.05–7.02(d,J=8.9Hz,1H),6.99–6.96(m,1H),6.93–6.89(m ,1H),6.85–6.81(d,J=7.9Hz,1H),6.69–6.65(d,J=8.7Hz,1H),6.63–6.59(m,2H),6.51–6.47(m,2H),6.02–5.88(dt,J=16 .8,6.7Hz,1H),5.43–5.35(dd,J=8.6,4.2Hz,1H),5.33–5.18(t,J=14.8Hz,4H),5.13–4.98(s,2H),4.51–4.44(s,2H),4.2 3–4.17(m,2H),3.84–3.78(d,J=14.4Hz,1H),3.62–3.49(m,3H),3.39–3.31(m,4H),3.11–2.91(s,2H),2.06–1.88(s,3H).13 C NMR (100MHz, CDCL3) δ185.94,171.50,167.76,164.29,163.73,159.35,158.27,155.88,154.81,150.94,139.05,138.93 ,138.03,133.88,131.34,131.11,130.50,129.84,129.55,129.39,129.25,128.84,128.53,128.45,128.27,127.76,127 .34,126.93,126.71,125.26,124.91,122.96,118.57,116.70,115.22,115.12,114.94,112.23,105.61,101.46,79.40, 77.39,77.13,76.88,70.62,68.40,65.97,65.66,59.17,57.19,56.71,51.72,43.31,36.17,20.08.HRMS(ESI):Calc.for C 64 H 56 N3O9S - [MH] - 1042.3743,found 1042.3792.IR(neat):3398,930,1727,1641,1598,1512,1495,1453,1381,1243,1207,117,1109,1072,700,663,644,635,627,614,605.

[0085] Synthesis of compound S-NH2

[0086]

[0087] The synthesis steps of SO-NH2 are similar to those described above: under a nitrogen atmosphere, dichloromethane (14 mL) is added to a flask containing tetrakis(triphenylphosphine)palladium (68.66 mg, 15 mol%), 1,3-dimethylbarbituric acid (211.62 mg, 3.33 mmol) and compound SW (425 mg, 0.40 mmol). The resulting mixture is then reacted at 40 degrees Celsius for 3 hours. Dichloromethane and saturated sodium bicarbonate solution are then added, and the organic layer is separated, washed with brine, and dried over magnesium sulfate. Purification is performed by flash chromatography on a silica gel column to obtain pure compound S-NH2 (180 mg, 41% yield). 1H NMR(500MHz,DMSO-D6)δ9.22–9.10(d,J=6.6Hz,1H),7.39–7.35(d,J=7.6Hz,2H),7.32–7.21(qd,J=15.6,8.3Hz,9H),7.02–6.97(d,J=8.3Hz,2H),6.92–6.89(d,J=9.0Hz,1H),6.87–6.82(d,J=9.6Hz,1H),6.80–6.76(s,1H),6.75–6.70(d,J=9.1Hz,3H),6.61–6.56(d,J=8.2Hz,2H),6.44

[0088] –6.38(d,J=9.8Hz,1H),6.23–6.17(s,1H),5.32–5.24(m,1H),5.18–5.13(s,1H),5.11–5.07(d,J=4.2Hz,1H),4.60–4.53(d,J=11.7Hz,1H),4.53–4.48(d,J=12.1Hz,1H),4.22–4.17(t,J=5.2Hz,2H),3.79–3.69(d,J=14.9Hz,1H),3.63–3.56(d,J=14.2Hz,1H),3.53–3.49(d,J=13.9Hz,1H),3.23–3.17(t,J=5.3Hz,2H),3.10–3.03(d,J=11.7Hz,1H),2.09–1.81(s,3H). 13 C NMR(100MHz,DMSO-D6)δ210.29,184.56,171.83,167.95,164.63,163.77,159.16,158.96,158.69,158.40,156.80,154.51,149.63,140.50,140.17,138.03,136.33,131.54,131.02,130.28,129.65,129.16,129.13,128.76,128.55,127.22,127.02,126.75,126.37,125.21,124.99,118.95,118.25,116.96,116.57,115.47,115.12,114.87,114.64,113.00,105.23,102.03,79.04,70.53,68.66,65.38,60.15,56.62,41.95,35.54,19.94HRMS(ESI):Calc.for C 58 H 48N3O9S - [MH] - 962.3117, found962.3120.mp131-145℃.IR(neat):2358.96,2339.95,1452.42,1175.02,1103.48,856.41,755.78,721.08,696.15,669.66.

[0089] Synthesis of compound S-NHCO

[0090]

[0091] Similar to the SO-NHCO synthesis step described above, S-NH2 (120 mg, 0.13 mmol) was dissolved in anhydrous tetrahydrofuran (4 mL). Triethylamine (75 μL, 0.54 mmol) was added to make the reaction solution pH>7, and then a solution of 3,5-dinitrobenzoyl chloride (47.96 mg, 0.21 mmol) dissolved in 2 mL of anhydrous tetrahydrofuran was added dropwise. The mixture was stirred at room temperature for 2 hours until thin layer chromatography (TLC) showed that the starting material disappeared. The solvent was concentrated and ether (7 mL) was added for precipitation. The solid was purified by silica gel flash chromatography to obtain pure compound S-NHCO (41 mg, 32% yield) 1 H NMR(400MHz, CDCl3)δ9.34–9.26(s,1H),9.21–9.08(m,2H),7.39–7.30(m,8H),7.25–7.18(m,2H),7.05–6.95(m,2H),6.91 –6.88(s,1H),6.79–6.73(d,J=8.7Hz,1H),6.60–6.56(s,1H),6.17–6.09(d,J=8.6Hz,1H),5.50–5.41(dd,J=8.6,4.3Hz,1H ),5.21–5.17(s,2H),5.16–5.13(d,J=4.3Hz,1H),4.61–4.55(d,J=12.7Hz,0H),4.55–4.51(d,J=12.1Hz,1H),4.36–4.32(m ,3H),4.02–3.98(m,2H),3.95–3.87(d,J=14.5Hz,1H),3.82–3.76(d,J=14.8Hz,1H),3.66–3.62(s,2H),2.01–1.96(s,3H). 13C NMR (100MHz, CDCl3) δ171.44,167.88,167.45,166.23,164.77,163.58,16 1.18,160.19,159.62,158.36,156.58,149.30,148.91,148.48,138.89,13 8.77,138.12,137.90,134.14,133.31,133.12,132.51,132.06,131.63,1 31.01,130.57,129.92,129.73,129.53,129.06,128.96,128.91,128.88,1 28.65,128.53,128.25,127.54,127.41,126.92,126.54,123.97,123.10, 122.51,120.80,118.10,117.10,115.87,115.11,112.54,104.63,101.26, 79.56,77.42,77.37,77.17,76.91,71.32,68.55,68.24,66.36,65.41,64. 97,59.21,56.88,43.16,38.78,36.31,23.07,20.21.HRMS(ESI):Calc.for C 64 H 56 N3O 10 S + [M+H] + 1158.3226,found 1158.3225mp126-173℃.IR(neat):1652,1395,1476,1094,854,800,715,659.

[0092] Synthesis of compound SE

[0093]

[0094] Similar to the synthesis of SO-E described above, S-NHCO (50 mg, 0.04 mmol) was added to 1.6 mL of a solution of dichloromethane, trifluoroacetic acid, triisopropylsilane, and water (50:45:2.5:2.5 by volume). The resulting mixture was stirred at room temperature for 3 h until thin-layer chromatography (TLC) indicated the disappearance of the starting material. The solid was purified by silica gel flash chromatography to afford the target compound SO-E (17 mg, 50% yield). 1H NMR(500MHz,DMSO-D6)δ9.60–9.47(t,J=5.4Hz,2H),9.14–9.11(t,J=2.1Hz,1H),9.10–9.09(d,J=2.1Hz,1H),9.08–9.05(d,J=2.1Hz,1H),8.96–8.91(t,J=2.1Hz,1H),7.40–7.10(m,15H),6.64–6.54(d,J=8.2Hz,1H),5.42–5.33(dd,J=8.0,4.2Hz,1H),5.12–5.07(d,J=4.2Hz,1H),4.29–4.22(t,J=5.6Hz,2H),4.15–4.10(d,J=16.8Hz,1H),3.95–3.90(d,J=12.2Hz,1H),3.79–3.74(q,J=5.4Hz,2H),3.57–3.51(m,1H),3.50–3.44(m,1H),3.37–3.33(d,J=7.0Hz,1H),3.33–3.31(d,J=7.0Hz,1H),3.24–3.17(d,J=4.5Hz,1H),3.13–3.08(d,J=10.4Hz,1H),1.96–1.92(s,3H). 13 C NMR(100MHz,DMSO-D6)δ172.48,171.56,170.10,163.08,161.80,160.45,160.26,159.08,158.91,155.54,149.37,148.87,148.76,141.81,141.54,138.19,137.28,136.32,132.98,131.45,131.27,130.19,130.06,129.62,129.44,129.20,128.93,128.76,128.72,128.08,127.61,127.03,126.76,126.46,125.28,124.61,124.21,123.68,121.47,121.33,117.05,116.77,116.05,115.51,115.02,114.37,112.79,103.14,70.31,66.50,65.45,60.59,60.39,54.59,42.19,41.62,40.50,40.34,40.17,40.00,39.84,39.67,39.50,34.97,34.17,31.82,22.62,20.09.HRMS(ESI):Calc.for C 52H 41 N5NaO 14 S + [M+Na] + 1014.2263,found1014.2270.IR(neat):1640,1593,1541,1463,1381,1343, 1287,1242,1206,1172,1151,1109,1073,1025,908,853,816,728,720,670.

[0095] (5) Synthesis of cephalosporin hybrid fluorescent probes S-OMe-NH2, S-OMe-NHCO, and S-OMe-E

[0096]

[0097]

[0098] Synthesis of compound S-Ome-W

[0099]

[0100] Under an argon atmosphere, to a solution of lithium methoxide (39.08 mg, 1.1 mmol) dissolved in anhydrous tetrahydrofuran (4 mL) and anhydrous methanol (1.54 mL) at -78 ° C, a solution of compound 3 (179 mg, 0.17 mmol) dissolved in anhydrous tetrahydrofuran (1.5 mL) was added dropwise. Then, tert-butyl hypochlorous acid (78 μL, 0.68 mmol) was added dropwise, and the mixture was stirred at the same temperature for half an hour. The reaction solution was poured into an aqueous ammonium chloride solution at one time and extracted with ethyl acetate. It was then purified by flash chromatography on a silica gel column to obtain the target compound S-OMe-W (81 mg, 41% yield). 11H NMR (400 MHz, CDCl3) δ 7.37–6.43 (m, 28H), 6.40–6.34 (d, J = 2.0 Hz, 2H), 5.92–5.78 (ddt, J = 16.6, 10.0, 6.4 Hz, 2H), 5.22–5.10 (m, 4H), 5.05–4.99 (d, J = 4.5 Hz, 2H), 4.34–4.29 (d, J = 11.6 Hz, 1H), 4.29–4.24 (d, J = 11.9 Hz, 1H), 4.10–4.04 (t, J = 5.9 Hz, 2H), 3.90–3.81 (d, J = 15.3 Hz, 1H), 3.61–3.52 (dd, J = 10.4, 3.7 Hz, 2H), 3.44–3.36 (d, J = 14.9 Hz, 1H), 3.28–3.24 (s, 3H), 3.22–3.16 (d, J = 6.6 Hz, 4H), 2.91–2.86 (t, J = 6.1 Hz, 2H), 2.14–1.75 (s, 3H). 13 13C NMR (126 MHz, CHLOROFORM-D) δ 185.94, 172.62, 163.60, 159.56, 159.25, 158.18, 154.78, 150.49, 147.42, 139.04, 137.98, 133.91, 131.01, 130.48, 130.16, 129.80, 129.49, 129.29, 129.18, 128.86, 128.79, 127.78, 127.35, 127.II, 12X.71, 118.70, 116.68, 115.07, 114.36, 112.20, 105.70, 101.37, 93.85, 79.57, 77.41, 77.16, 76.90, 70.56, 68.09, 66.17, 62.15, 57.47, 53.20, 51.76, 43.56, 35.76, 34.33, 32.00, 29.79, 29.62, 29.41, 29.33, 27.30, 25.09, 22.78, 20.08.

[0101] Synthesis of Compound S-OMe-NH2

[0102]

[0103] Similar to the SO-NH2 synthesis steps described above: Under a nitrogen atmosphere, dichloromethane (14 mL) was added to a flask containing tetrakis(triphenylphosphine)palladium (68.66 mg, 15 mol%), 1,3-dimethylbarbituric acid (211.62 mg, 3.33 mmol) and compound S-Ome-W (0.40 mmol). The resulting mixture was then reacted at 40 degrees Celsius for 3 hours. Dichloromethane and saturated sodium bicarbonate solution were then added, and the organic layer was separated, washed with brine, and dried over magnesium sulfate. Purification was performed by silica gel column flash chromatography to obtain pure compound S-Ome-NH2 with a yield of 73%. Synthesis of compound S-OMe-NHCO

[0104]

[0105] Similar to the SO-NHCO synthesis procedure described above, S-OMe-NH2 (0.13 mmol) was dissolved in anhydrous tetrahydrofuran (4 mL). Triethylamine (75 μL, 0.54 mmol) was added to raise the reaction solution to pH > 7, and then a solution of 3,5-dinitrobenzoyl chloride (47.96 mg, 0.21 mmol) dissolved in 2 mL of anhydrous tetrahydrofuran was added dropwise. The mixture was stirred at room temperature for 2 hours until thin-layer chromatography (TLC) showed the disappearance of the starting material. The solvent was concentrated and ether (7 mL) was added for precipitation. The solid was purified by flash chromatography on a silica gel column to obtain pure compound S-OMe-NHCO (yield 41%).

[0106] Synthesis of compound S-OMe-E

[0107]

[0108] Similar to the synthesis of SO-E described above, S-OMe-NHCO (0.04 mmol) was added to 1.6 mL of a solution of dichloromethane, trifluoroacetic acid, triisopropylsilane, and water (50:45:2.5:2.5 by volume). The resulting mixture was stirred at room temperature for 3 h until thin-layer chromatography (TLC) showed the disappearance of the starting material. The solid was purified by silica gel flash chromatography to afford the target compound, SO-OMe-E, in 52% yield.

[0109] Application Example Probe Fluorescence Intensity and Its Identification of Mycobacterium Tuberculosis:

[0110] 1. Method for measuring the fluorescence intensity of the probe:

[0111] (1) Preparation of mother liquor

[0112] Buffer: PBS buffer pH = 7.4 (phosphate buffer solution);

[0113] The probe compounds were dissolved in 1% DMSO solution by volume to prepare 1 mM stock solution for use;

[0114] All solutions were prepared freshly for use.

[0115] (2) Test conditions

[0116] All UV spectroscopic measurements were performed at room temperature using a wavelength range of 200-600 nm, a mid-range scan speed (240 nm / min), a spectral bandwidth of 2 nm, and baseline correction using a blank PBS solution. The sample chamber was scanned three times consecutively and the average value was used to eliminate instrument fluctuations. The sample cell was a 10.0 mm wide quartz cuvette. The excitation wavelength of the fluorescence spectrometer was determined by the maximum absorption wavelength of the different probes. The wavelength range was 490-600 nm, and baseline calibration was performed before testing.

[0117] (3) Study on the UV absorption spectral properties of the probe

[0118] Accurately pipette 30 μL of the probe stock solution, add PBS solution (pH=7.4) and dilute to 3 mL to prepare a 10 μM standard solution. Obtain the UV absorption spectrum of the probe.

[0119] (4) Probe fluorescence intensity study

[0120] Accurately measure 30 μL of the probe stock solution and dilute it to 3 mL with PBS solution (pH 7.4) to prepare a 10 μM standard solution. Perform a spectral scan to obtain the fluorescence spectrum of the probe.

[0121] Take another 30 μL of probe stock solution and add 30 μL of β-lactamase (10 4 UmL -1 ) was used as a cephalosporin hydrolyzate, and the total volume was adjusted to 3 mL with PBS buffer. The final concentrations of the components were 10 μM probe and 100 U / mL β-lactamase, respectively. After mixing evenly, the mixture was placed on a fluorescence spectrometer for testing (the excitation wavelength was set to λex = 490 nm) and scanned every 1 minute for a total of 10 minutes.

[0122] 2. Probe recognition of Mycobacterium tuberculosis β-lactamase

[0123] (1) Preparation of mother liquor

[0124] Buffer: PBS buffer PH=7.4 (phosphate buffer solution) b. The probe S compounds were dissolved in 1% by volume DMSO solution to prepare 100 μM stock solutions for use.

[0125] β-lactamase is configured as 10 5 U / mL PBS solution for later use.

[0126] All solutions were prepared freshly for use.

[0127] (2) Test method

[0128] The enzyme titration test was performed at room temperature on a multifunctional microplate reader.

[0129] Take 10 6 The enzyme stock solution was diluted 2-fold to prepare 9 concentration points. 20 μL of 100 μM probe solution and 10 μL of enzyme solution with different concentrations after gradient dilution were added to the 96-well plate in sequence. 70 μL of PBS buffer was added to adjust the total volume of each well to 100 μL. The final concentrations of each component were 10 4 , 5000, 2500, 625, 312.5, 156.3, 78.13, 39.06, and 19.53 U / mL enzyme solution, and 20 μM probe solution. After uniform mixing, the 96-well plate was quickly placed on a microplate reader and scanned every 20 seconds for 30 minutes (excitation wavelength 490 nm, emission wavelength 521 nm). The experiment was repeated three times.

[0130] 3. Results and structure-activity relationship:

[0131] (1) The present invention uses a specific recognition strategy for Mycobacterium tuberculosis β-lactamase and the DprE1 enzyme that biosynthesizes arabinogalactan in the cell wall of Mycobacterium tuberculosis to design a fluorescent probe that couples a cephalosporin fragment, a fluorescent group fragment, and a Mycobacterium tuberculosis cell wall recognition fragment to form a cephalosporin hybrid fluorescent probe. The reported Mycobacterium tuberculosis fluorescent probe based on β-lactamase recognition has poor water solubility, and the three-membered ring structure at position 7 of the cephalosporin recognition end is unstable, making it difficult to store for a long time and use it clinically. The cephalosporin hybrid fluorescent probe developed by the present invention abandons the three-membered strained ring at position 7 and synthesizes the SO-NH2, SO-NHCO, and SO-E series fluorescent probes. By reducing the sulfoxide structure at position 1 of the cephalosporin, the electronic effect, conjugation effect, host-guest interaction mode, etc. of the probe recognition end are changed to synthesize the S-NH2, S-NHCO, and SE series fluorescent probes. A methoxy group was further introduced at position 6 of the cephalosporin recognition end to further change the electronic effect and steric effect of the probe, and the S-OMe-NH2, S-OMe-NHCO, and S-OMe-E series of fluorescent probes were synthesized.

[0132] (2) The maximum ultraviolet absorption wavelength of the fluorescent probe was used as the excitation wavelength of the probe fluorescence spectrum to test the fluorescence spectrum of the probe. The results showed that: ① SO-NH2, SO-NHCO, SO-E series probes, SO-E has the highest fluorescence intensity, which is 4.5 times higher than SO-NH2 and one order of magnitude higher than SO-NHCO. Figure 1② The fluorescence intensity of the S-NH2, S-NHCO, and SE series probes increased due to changes in the electronic environment. Among them, the fluorescence intensity of S-NH2 was the highest, comparable to that of SE. The fluorescence intensity of both was about 50% higher than that of S-NHCO. Figure 2 ③S-OMe-NH2, S-OMe-NHCO, S-OMe-E series probes, the fluorescence intensity of S-OMe-NHCO probe is 3 times higher than that of S-OMe-NH2 and 5 times higher than that of S-OMe-E, as shown. Figure 3 ④Comparison of the three types of fluorescent probes, the fluorescence intensity of the S-NH2, S-NHCO, SE series probes is higher than that of the S-OMe-NH2, S-OMe-NHCO, S-OMe-E series probes, and higher than that of the SO-NH2, SO-NHCO, SO-E series probes, as shown in Figure 4. Figure 4 shown.

[0133] (3) Based on the fluorescence intensity characteristics of the three types of probes, the S-NH2, S-NHCO, and SE series probes have the highest fluorescence intensity and have the potential for development. Therefore, the present invention tested the ability of the S-NH2, S-NHCO, and SE series probes to identify Mycobacterium tuberculosis. The results showed that all three probes in this series can effectively identify β-lactamase of Mycobacterium tuberculosis. Under the same conditions, after the reaction with β-lactamase, the fluorescence intensity increased by about 100 times (2 orders of magnitude), as shown in Figure 2. Figures 5 to 7 As shown. Among them, SE has the highest fluorescence intensity after reacting with β-lactamase, followed by S-NH2. Both probes are higher than S-NHCO ( Figure 8 ). This indicates that SE probes have potential application prospects.

[0134] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. A cephalosporin hybrid fluorescent probe, characterized in that: The structural formula is shown in any one of the following formulas I to IX:

2. Use of the cephalosporin hybrid fluorescent probe according to claim 1 in identifying Mycobacterium tuberculosis.

3. The use according to claim 2, characterized in that The cephalosporin hybrid fluorescent probe is used in identifying and detecting Mycobacterium tuberculosis beta-lactamase.

4. The use according to claim 2 or 3, characterized in that The cephalosporin hybrid fluorescent probe is used for preparing a preparation for identifying Mycobacterium tuberculosis.

5. A method for detecting Mycobacterium tuberculosis using the cephalosporin hybrid fluorescent probe according to claim 1, characterized in that: The detection method adopts a cephalosporin hybrid fluorescent probe for detection, and the detection method comprises the following steps: providing a sample to be detected; mixing the sample to be detected and the fluorescent probe for incubation to obtain a mixed solution; and performing fluorescence spectrum detection or fluorescence imaging detection on the mixed solution.

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