An activated alkyne-tetraphenylene sulfonic acid sodium salt molecule and preparation method and its application in high-throughput screening of antibiotics

Abstract

The present invention relates to a kind of activated alkyne tetraphenylene sulfonic acid sodium salt molecule and preparation method and its application in antibiotic high-throughput screening;Synthesize water-soluble fluorescent molecules containing active alkynes by six-step reaction;Use water-soluble, highly reactive fluorescent molecules, through metal-free catalytic click reaction, realize the fluorescence opening of bacterial lysate;Gram-negative bacteria and Gram-positive bacteria are incubated in liquid culture medium containing different antibiotic concentrations;By adding sodium hydroxide solution to crack Gram-negative bacteria / adding sodium hydroxide solution and heating to crack Gram-positive bacteria, the lysate is transferred to 96-well plates;Pre-prepared fluorescent molecules are added to well plates for incubation;Finally, fluorescence intensity is detected by microplate reader, and the relationship between the number of bacteria in the system and the antibiotic concentration is calculated by standard curve. The present invention can realize broad-spectrum, rapid, highly sensitive high-throughput antibiotic antibacterial sensitivity assessment.

Description

Technical Field

[0001] The present invention relates to a water-soluble fluorescent molecule containing active alkynes and a synthesis method, and also relates to the application of the water-soluble fluorescent molecule containing active alkynes in high-throughput antibiotic screening. In particular, an activated alkyne-tetraphenylene sulfonic acid sodium salt molecule and a preparation method and its application in high-throughput antibiotic screening are proposed. Background Art

[0002] According to statistics, 57 million people die every year worldwide, of which 15 million (about 26%) are directly caused by various infectious diseases caused by pathogenic bacteria. Therefore, bacterial infection is considered one of the most urgent and serious threats facing mankind. Due to the overuse and abuse of antibiotics, the problem of bacterial resistance is becoming increasingly serious. Various types of drug-resistant bacteria, especially some multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus, continue to emerge. These strains weaken the therapeutic effect of antibiotics and significantly increase the mortality rate of patients. Screening for new antibiotics is one of the effective ways to combat drug-resistant bacteria. The traditional antibiotic sensitivity test methods currently widely used include: paper diffusion method, broth dilution method and agar dilution method. These methods take a lot of time to achieve a resolution observable by the naked eye, and the experimental repeatability is very poor. Based on the latest development of technical means, nanovibration method, real-time fluorescence quantitative polynucleotide chain reaction, electrochemical detection and fluorescence test methods are also used in antibiotic screening. Among them, fluorescence detection is widely used in the imaging field due to its advantages such as high sensitivity and non-invasiveness. Researcher Zhu Chunlei and others have developed a new method for antimicrobial sensitivity evaluation and antibiotic screening based on the fluorescence resonance energy transfer (FRET) system composed of water-soluble conjugated polymers and fluorescein. In addition, the team of Academician Tang Benzhong reported a positively charged aggregation-induced emission (AIE) molecule, which binds to the bacterial surface through strong electrostatic interactions to induce aggregation and thus achieve fluorescence imaging of bacteria. Although fluorescence-based methods have made great achievements, they still have disadvantages such as long detection time and insufficient sensitivity. Therefore, it is very necessary to develop a broad-spectrum, sensitive, and rapid high-throughput antibiotic screening method.

[0003] In recent years, click reactions have been widely used in the construction of various types of bioconjugates. Among the different types of click reactions, metal-free catalytic click reactions based on active alkynes have attracted great interest among researchers. The team of Academician Tang Benzhong synthesized a series of active alkynes with AIE properties. Electron-deficient alkynes can undergo mild and rapid metal-free electrophilic addition reactions with amines, thiols, and hydroxyls in many biological targets (such as bacteria, antibodies, chitosan, and bovine serum albumin), thereby achieving simple fluorescence onset and functionalization of various biological systems. However, most of the reported active alkynes are hydrophobic in structure, which greatly limits their application in biological environments. Summary of the invention

[0004] In order to improve the bioconjugation efficiency of the molecule and reduce the background fluorescence signal in the water system, we considered synthesizing water-soluble AIE molecules containing active alkynes. For most fluorescence-based detection methods, the entire bacterium is considered to be an indivisible individual, and the detectable area is limited to the bacterial surface. Therefore, in the early days of antibiotic screening, it was difficult to accurately reflect the bacterial growth due to the small number of bacteria. We realized that there are a large number of clickable biological substrates inside the bacteria, and trying to release these components for analyte amplification through chemical and / or physical cleavage methods is expected to solve this problem. Further, through an efficient click reaction process, the clickable biological substrate is effectively labeled by the water-soluble AIE molecules containing active alkynes in a fluorescent-on manner. This cascade signal amplification strategy can improve detection sensitivity and shorten the time of drug sensitivity testing, thereby achieving broad-spectrum, sensitive, and rapid high-throughput antibiotic screening.

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a water-soluble fluorescent molecule based on active alkynes. At the same time, a large number of clickable biological substrates inside bacteria are released by chemical and / or physical cleavage methods for analyte amplification. Further, through an efficient click reaction process, the clickable biological substrate is effectively labeled by the water-soluble AIE molecules containing active alkynes in a fluorescently turned-on manner. This cascade signal amplification strategy can improve detection sensitivity, shorten the time of drug sensitivity testing, and thus achieve broad-spectrum, sensitive, and rapid high-throughput antibiotic screening.

[0006] The technical solution of the present invention is as follows:

[0007] An activated alkyne-tetraphenylene sulfonic acid sodium salt molecule, the structural formula of which is shown in the formula;

[0008]

[0009] Wherein, R1 is -H, -OCH3, -N(CH3)2, -NHCOCH3 or -OCOCH3.

[0010] The method for synthesizing the activated alkyne-tetraphenylene sulfonic acid sodium salt molecule of the present invention comprises the following steps:

[0011] 1) Weigh zinc powder, add tetrahydrofuran solution under argon protection, add titanium tetrachloride dropwise at -78°C to -50°C, and after the addition is complete, heat to reflux at 75°C to 80°C; add 4,4-dimethoxybenzophenone (I) and 4-bromobenzophenone (II) to the reaction system, heat to reflux at 75°C to 80°C for 3 to 4 hours, monitor by thin layer chromatography and purify by column chromatography to obtain 1-(4-bromophenyl)-2,2-di(4-methoxyphenyl)-1-phenylene compound (III);

[0012] 2) Compound (III) was dissolved in tetrahydrofuran, and n-butyl lithium solution was added dropwise under argon protection at -78°C to -50°C. After stirring for 2 to 3 hours, anhydrous N,N-dimethylformamide solution was added; the reaction system was slowly restored to room temperature and the reaction was continued for 3 to 5 hours. The reaction was monitored by thin layer chromatography and purified by column chromatography to obtain 4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)benzaldehyde compound (IV);

[0013] 3) Adding compound (IV) and boron tribromide to ultra-dry dichloromethane in an ice bath; reacting at room temperature for 3 to 5 hours after the addition is completed, monitoring by thin layer chromatography and purifying by column chromatography to obtain 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzaldehyde compound (V);

[0014] 4) adding compound (V) and ethynylmagnesium bromide to tetrahydrofuran in an ice bath; reacting at room temperature for 3 to 5 hours after the addition is completed, monitoring by thin layer chromatography and purifying by column chromatography to obtain 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzinol compound (VI);

[0015] 5) adding compound (VI) and sodium methoxide to anhydrous ethanol, stirring at room temperature for 1-2 hours; then adding 1,3-propane sultone, stirring at room temperature overnight for 12-16 hours, to obtain alkynol-tetraphenylethylene sulfonic acid sodium salt molecular compound (VII);

[0016] 6) Add compound (VII) and active manganese dioxide to a mixed solvent of DMSO and acetone, stir overnight at room temperature for 12 to 16 hours, and remove the metal oxide after the reaction by suction filtration to obtain activated alkyne-tetraphenylene sulfonate sodium salt molecule (VIII). 3. The method for synthesizing activated alkyne-tetraphenylene sulfonate sodium salt molecule as claimed in claim 2, characterized in that the molar ratio of zinc powder to titanium tetrachloride in step 1) is 2:1 to 3:1; the molar ratio of 4,4-dimethoxybenzophenone (I) to 4-bromobenzophenone (II) is 1:1.2 to 1:1.5; the molar ratio of zinc powder to 4,4-dimethoxybenzophenone (I) is 4:1 to 6:1; tetrahydrofuran is added as a solvent to completely dissolve compound (I) and compound (II) and the mass fraction is not less than 10%.

[0017] 4. The method for synthesizing the activated alkyne-tetraphenylene sulfonate sodium salt molecule as claimed in claim 2, characterized in that, in step 2), compound (III) is dissolved in tetrahydrofuran until it is completely dissolved and the mass fraction of compound (III) is not less than 10%; the molar ratio of compound (III) to n-butyl lithium is 1:1.5 to 1:2; the molar ratio of compound (III) to anhydrous N,N-dimethylformamide solution is 1:1 to 1:2.

[0018] 5. The method for synthesizing activated alkyne-tetraphenylethylene sulfonic acid sodium salt molecules as claimed in claim 2, characterized in that, in step 2), the molar ratio of compound (IV) to boron tribromide is 1:6 to 1:10; the compound (IV) is added to ultra-dry dichloromethane under an ice bath until it is completely dissolved and the mass fraction of compound (IV) is not less than 10%.

[0019] 6. The method for synthesizing activated alkyne-tetraphenylene sulfonic acid sodium salt molecules as claimed in claim 2, characterized in that, in step 4), the molar ratio of compound (V) to ethynyl magnesium bromide is 1:3 to 1:5; the compounds (V) are added to tetrahydrofuran until they are completely dissolved and the mass fraction of compound (V) is not less than 10%.

[0020] 7. The method for synthesizing activated alkyne-tetraphenylene sulfonate sodium salt molecules as claimed in claim 2, characterized in that, in step 5), the molar ratio of compound (VI) to sodium methoxide is 1:3 to 1:5; the compound (VI) is added to anhydrous ethanol until it is completely dissolved and the mass fraction of compound (VI) is not less than 10%; the molar ratio of compound (VI) to 1,3-propane sultone is 1:3 to 1:5.

[0021] 8. The method for synthesizing activated alkyne-tetraphenylethylene sulfonic acid sodium salt molecules as claimed in claim 2, characterized in that, in step 6), the molar ratio of compound (VII) to active manganese dioxide is 1:25-1:30; and the volume ratio of DMSO to acetone is 1:6 so that compound (VII) is completely dissolved and the mass fraction is not less than 10%.

[0022] 9. A method for using the activated alkyne-tetraphenylene sulfonate sodium salt molecule of claim 1 as a water-soluble fluorescent molecule for high-throughput antibiotic screening, characterized in that it comprises the following steps

[0023] 1) Gram-negative bacteria / Gram-positive bacteria were incubated in liquid culture medium containing different antibiotic concentrations for more than 1.5 hours;

[0024] 2) After the incubation, the bacterial culture solution is added to a centrifuge tube and centrifuged (3500rpm-10000rpm) to discard the culture medium supernatant, leaving the bacterial precipitate, and then resuspended with the same volume of PBS solution; Gram-negative bacteria / Gram-positive bacteria bacterial suspensions are obtained respectively; for Gram-negative bacteria, 10M sodium hydroxide solution (volume ratio greater than 1:100) is added to the Gram-negative bacteria suspension and incubated in a 37°C constant temperature shaker for more than 10 minutes for lysis to obtain Gram-negative bacteria lysate; for Gram-positive bacteria, 10M sodium hydroxide solution (volume ratio greater than 1:100) is added to the Gram-positive bacteria suspension and heated in a 70°C-90°C oven for more than 10 minutes for lysis to obtain Gram-positive bacteria lysate;

[0025] 3) Add the pre-prepared activated alkyne-tetraphenylene sulfonic acid sodium salt molecule dimethyl sulfoxide solution (greater than 1 mM) to the Gram-negative bacteria or Gram-positive bacteria lysate and incubate in a constant temperature shaker at 37°C for more than 10 minutes (volume ratio greater than 1:20), detect the fluorescence intensity using an enzyme reader, and calculate the relationship between the number of bacteria in the system and the antibiotic concentration through the standard curve to achieve the antibacterial sensitivity evaluation of different antibiotics.

[0026] The water-soluble fluorescent molecules containing active alkynes of the present invention are used in high-throughput antibiotic screening. The water-soluble fluorescent molecules containing active alkynes are synthesized by six-step reactions; Gram-negative bacteria and Gram-positive bacteria are incubated in a liquid culture medium containing different antibiotic concentrations at 37°C for 1.5 hours; Gram-negative bacteria are lysed by adding sodium hydroxide solution / Gram-positive bacteria are lysed by adding sodium hydroxide solution and heating, and the lysate is transferred to a 96-well plate; the pre-prepared fluorescent molecules are added to the well plate and incubated at 37°C for 10 minutes; finally, the fluorescence intensity is detected by an ELISA instrument, and the relationship between the number of bacteria in the system and the antibiotic concentration is calculated by a standard curve. For most fluorescence-based detection methods, the entire bacterium is considered to be an indivisible individual, and the detectable area is limited to the surface of the bacteria. Therefore, in the early stages of antibiotic screening, it is difficult to accurately reflect the bacterial growth due to the small number of bacteria. As a result, the antibiotic screening time is too long (more than 4 hours) and the bacterial detection limit is high (10 4 -10 6 CFU / mL). In addition, most of the AIE molecules containing activated alkynes currently have poor water solubility and strong background fluorescence, which are not suitable for fluorescence detection and coupling reactions in physiological environments. We realize that there are a large number of clickable biological substrates inside bacteria, and attempts to release these components through chemical and / or physical cleavage methods for analyte amplification are expected to solve this problem. Furthermore, through an efficient click reaction process, the clickable biological substrate is effectively labeled with fluorescence-on by water-soluble AIE molecules containing active alkynes. The present invention utilizes water-soluble, highly reactive fluorescent molecules to achieve fluorescence on bacterial lysate through a metal-free click reaction. This cascade signal amplification strategy can improve detection sensitivity and shorten the time of drug sensitivity testing. Further, when used in conjunction with an enzyme reader, it can achieve broad-spectrum (including Gram-negative and Gram-positive bacteria) and sensitive (LOD value is 5-10×10 2 CFU / mL), rapid (less than 3 hours) high-throughput antibiotic screening.

[0027] The beneficial effects of the present invention are as follows:

[0028] (1) The present invention first synthesized a negatively charged AIE molecular probe, which has extremely strong water solubility. The probe hardly fluoresces in aqueous solution, but by restricting the movement of AIE molecules through click reactions, it shows obvious fluorescence on. Due to the weak background fluorescence, an extremely low bacterial detection limit was obtained at a higher AIEgen working concentration.

[0029] (2) Analyte amplification is achieved by releasing a large amount of clickable biological substrates inside the bacteria through chemical and / or physical lysis methods.

[0030] (3) The fluorescence intensity of the water-soluble fluorescent molecules in the present invention is proportional to the number of bacteria, and the antibacterial effects of different antibiotics on specific bacteria (such as IC 50 , MIC value) can be obtained by quantitative calculation through standard curve.

[0031] (4) The test method of the present invention is further combined with an ELISA instrument, which is expected to achieve broad-spectrum, high-sensitivity, and rapid high-throughput antibiotic screening. This invention will provide a new solution for evaluating bacterial resistance and antibiotic sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : Preparation and determination of the photophysical properties and click reaction ability of synthetic water-soluble fluorescent molecules containing active alkynes.

[0033] (A) Fluorescence emission graphs of pyone-TPE at different THF / water ratios.

[0034] (B) Fluorescence intensity of pyone-TPE at different THF contents. Inner panel: Photographs of pyone-TPE at 0% and 99% THF contents illuminated by a handheld UV lamp (365 nm).

[0035] (C) Schematic diagram of the click reaction between pyone-TPE and n-propylamine.

[0036] (D) Fourier transform infrared spectra of pyone-TPE and peone-TPE.

[0037] (E) The wave number of pyone-TPE and peone-TPE increased from 2600 cm -1 To 1800cm -1 Locally enlarged Fourier transform infrared spectrum.

[0038] Figure 2 :Preparation and application of synthetic water-soluble fluorescent molecules containing active alkynes in bacterial detection.

[0039] (A) Hand-held UV lamp (365nm) irradiation to treat methicillin-resistant Staphylococcus aureus (MRSA) in different ways

[0040] Photograph of the suspension.

[0041] (B) Fluorescence emission images of pyol-TPE and pyone-TPE after incubation with MRSA, respectively.

[0042] (C) Histogram of fluorescence intensity at 525 nm after incubation of pyol-TPE and pyone-TPE with MRSA.

[0043] Photos of pyol-TPE and pyone-TPE after incubation with MRSA, respectively, illuminated by a handheld UV lamp (365 nm).

[0044] (D) Photographs of wild-type Escherichia coli (WT E. coli) suspensions treated in different ways under irradiation with a handheld UV lamp (365 nm).

[0045] (E) Fluorescence emission images of pyol-TPE and pyone-TPE after incubation with WT E. coli, respectively.

[0046] (F) Histogram of fluorescence intensity at 525 nm after incubation of pyol-TPE and pyone-TPE with WT E. coli, respectively.

[0047] Inner image: Photos of pyol-TPE and pyone-TPE incubated with WT E.coli under irradiation of a handheld UV lamp (365nm).

[0048] Figure 3 :Prepared and synthesized water-soluble fluorescent molecules containing active alkynes were used to quantify the relationship between relative fluorescence intensity and bacterial concentration using a fluorimeter.

[0049] (A) Fluorescence emission images after incubation of pyone-TPE with different concentrations of MRSA.

[0050] (B) Images taken under a handheld UV lamp (365 nm) after pyone-TPE was incubated with PBS suspension containing / not containing MRSA.

[0051] (C) Standard curve of the relationship between fluorescence intensity and MRSA concentration.

[0052] (D) Fluorescence emission graph after incubation of pyone-TPE with different concentrations of WT E. coli.

[0053] (E) After incubation of pyone-TPE with PBS suspension containing / without WT E. coli, the cells were illuminated under a hand-held UV lamp (365 nm).

[0054] Pictures taken under illumination.

[0055] (F) Standard curve of the relationship between fluorescence intensity and WT E. coli concentration.

[0056] Figure 4 :Preparation and synthesis of water-soluble fluorescent molecules containing active alkynes combined with a fluorescence instrument for antibiotic screening.

[0057] (A) Graph showing the relationship between the number of MRSA and the concentrations of six antibiotics.

[0058] (B) Graph showing the relationship between the number of mutans Streptococcus (S.mutans) and the concentrations of six antibiotics.

[0059] (C) Graph showing the relationship between the number of Enterococcus faecalis and the concentrations of six antibiotics.

[0060] (D) Pseudomonas aeruginosa (P. aeruginosa) counts versus the concentrations of six antibiotics.

[0061] (E) Graph showing the relationship between the number of WT E. coli and the concentrations of six antibiotics.

[0062] (F) Ampicillin-resistant E. coli (WT E. coli Ampr ) as a function of the concentrations of six antibiotics.

[0063] Figure 5 : Preparation and synthesis of water-soluble fluorescent molecules containing active alkynes combined with a microplate reader for high-throughput antibiotic screening.

[0064] (A) Schematic diagram of microplate-assisted high-throughput antibiotic screening.

[0065] (B) Graph showing the relationship between the number of MRSA and the concentrations of six antibiotics.

[0066] (C) Graph showing the relationship between the number of S. mutans and the concentrations of six antibiotics.

[0067] (D) Graph showing the relationship between the number of E. faecalis and the concentrations of six antibiotics.

[0068] (E) Graph showing the relationship between the number of P. aeruginosa and the concentrations of six antibiotics.

[0069] (F) Graph showing the relationship between the number of WT E. coli and the concentrations of six antibiotics.

[0070] (G)WT E.coli Ampr Plot of the relationship between the number and the concentration of six antibiotics. DETAILED DESCRIPTION

[0071] The present invention will be further described below by means of specific examples.

[0072] The water-soluble fluorescent molecule containing active alkyne proposed by the present invention is an activated alkyne-tetraphenylene sulfonic acid sodium salt molecule, and the structural formula is shown in formula (VIII);

[0073]

[0074] Wherein, R1 is -H, -OCH3, -N(CH3)2, -NHCOCH3 or -OCOCH3.

[0075] The method for synthesizing the water-soluble fluorescent molecule activated alkyne-tetraphenylene sulfonate sodium salt molecule (VIII) containing active alkyne of the present invention comprises the following steps:

[0076] 1) Weigh zinc powder, add tetrahydrofuran solution under argon protection, add titanium tetrachloride dropwise at -78°C to -50°C (the molar ratio of zinc powder to titanium tetrachloride is between 2:1 and 3:1), and after the addition is completed, heat to reflux at 75°C to 80°C; add 4,4-dimethoxybenzophenone (I) and 4-bromobenzophenone (II) at a molar ratio of 1:1.2 to 1:1.5 to the reaction system, heat to reflux at 75°C to 80°C for 3 to 4 hours, monitor by thin layer chromatography and purify by column chromatography to obtain 1-(4-bromophenyl)-2,2-di(4-methoxyphenyl)-1-phenylene (III);

[0077] 2) Compound (III) is dissolved in tetrahydrofuran, and n-butyl lithium solution (the molar ratio of compound (III) to n-butyl lithium is between 1:1.5 and 1:2) is added dropwise under argon protection and -78°C to -50°C, and after stirring for 2 to 3 hours, an anhydrous N,N-dimethylformamide solution (the molar ratio of compound (III) to anhydrous N,N-dimethylformamide solution is between 1:1 and 1:2) is added; the reaction system is slowly restored to room temperature (25°C to 30°C) and the reaction is continued for 3 to 5 hours, and 4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)benzaldehyde (IV) is obtained by thin layer chromatography monitoring and column chromatography purification;

[0078] 3) Add compound (IV) and boron tribromide in a molar ratio of 1:6 to 1:10 to ultra-dry dichloromethane under ice bath. After the addition is completed, react at room temperature (25°C to 30°C) for 3 to 5 hours, monitor by thin layer chromatography and purify by column chromatography to obtain 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzaldehyde (V);

[0079] 4) Compound (V) and ethynylmagnesium bromide in a molar ratio of 1:3 to 1:5 are added to tetrahydrofuran in an ice bath. After the addition is completed, the mixture is reacted at room temperature (25°C to 30°C) for 3 to 5 hours, and 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzinol (VI) is obtained by thin layer chromatography monitoring and column chromatography purification;

[0080] 5) Add compound (VI) and sodium methoxide in a molar ratio of 1:3 to 1:5 to anhydrous ethanol, and stir at room temperature for 1 to 2 hours. Then add 1,3-propane sultone (the molar ratio of compound (VI) to 1,3-propane sultone is 1:3 to 1:5), and stir overnight at room temperature (25°C to 30°C) (12 to 16 hours) to obtain alkynol-tetraphenylethylene sulfonic acid sodium salt molecule (VII);

[0081] 6) Add compound (VII) with a molar ratio of 1:25 to 1:30 and pre-prepared active manganese dioxide to a mixed solvent of DMSO and acetone (volume ratio 1:6), stir overnight (12 to 16 hours) at room temperature (25°C to 30°C), and remove the metal oxide after the reaction by suction to obtain an activated alkyne-tetraphenylene sulfonic acid sodium salt molecule (VIII).

[0082] The reaction formula is as follows:

[0083]

[0084] Wherein, R1 is -H, -OCH3, -N(CH3)2, -NHCOCH3 or -OCOCH3.

[0085] The present invention discloses a method for using activated alkyne-tetraphenylethylene sulfonic acid sodium salt molecules as water-soluble fluorescent molecules for high-throughput antibiotic screening. The specific technical scheme is as follows:

[0086] 1) Gram-negative bacteria / Gram-positive bacteria were incubated in liquid culture medium containing different antibiotic concentrations for more than 1.5 hours;

[0087] 2) After the incubation, the bacterial culture solution is added to a centrifuge tube and centrifuged (3500rpm-10000rpm) to discard the culture medium supernatant, leaving the bacterial precipitate, and then resuspended with the same volume of PBS solution; Gram-negative bacteria / Gram-positive bacteria bacterial suspensions are obtained respectively; for Gram-negative bacteria, 10M sodium hydroxide solution (volume ratio greater than 1:100) is added to the Gram-negative bacteria suspension and incubated in a 37°C constant temperature shaker for more than 10 minutes for lysis to obtain Gram-negative bacteria lysate; for Gram-positive bacteria, 10M sodium hydroxide solution (volume ratio greater than 1:100) is added to the Gram-positive bacteria suspension and heated in a 70°C-90°C oven for more than 10 minutes for lysis to obtain Gram-positive bacteria lysate;

[0088] 3) Add the pre-prepared activated alkyne-tetraphenylene sulfonic acid sodium salt molecule dimethyl sulfoxide solution (greater than 1 mM) to the Gram-negative bacteria or Gram-positive bacteria lysate and incubate in a constant temperature shaker at 37°C for more than 10 minutes (volume ratio greater than 1:20), detect the fluorescence intensity using an enzyme reader, and calculate the relationship between the number of bacteria in the system and the antibiotic concentration through the standard curve to achieve the antibacterial sensitivity evaluation of different antibiotics.

[0089] The invention relates to an activated alkyne-tetraphenylene sulfonic acid sodium salt molecule and a preparation method thereof and application thereof in high-throughput screening of antibiotics.

[0090] The present invention synthesizes water-soluble fluorescent molecules containing active alkynes through six-step reactions; at 37°C, Gram-negative bacteria and Gram-positive bacteria are incubated in liquid culture media containing different antibiotic concentrations; Gram-negative bacteria are lysed by adding sodium hydroxide solution / Gram-positive bacteria are lysed by adding sodium hydroxide solution and heating, and the lysate is transferred to a 96-well plate; the pre-prepared fluorescent molecules are added to the well plate and continue to be incubated at 37°C; finally, the fluorescence intensity is detected by an ELISA instrument, and the relationship between the number of bacteria in the system and the antibiotic concentration is calculated by a standard curve. The present invention uses water-soluble, highly reactive fluorescent molecules to achieve fluorescence on bacterial lysate through a metal-free click reaction, and is further combined with an ELISA instrument to achieve a broad-spectrum, rapid, highly sensitive, high-throughput antibiotic antibacterial sensitivity assessment.

[0091] Example 1

[0092] Preparation of 1-(4-bromophenyl)-2,2-bis(4-methoxyphenyl)-1-phenylene (Compound III-1):

[0093] Zinc powder (7.584 g, 116 mmol) was added to a three-necked flask, and the system was ventilated and cooled at -78 °C. 50 mL of dry tetrahydrofuran solution was added, and the solution was cooled for 10 minutes, and then titanium tetrachloride (6.36 mL, 58 mmol) (the molar ratio of zinc powder to titanium tetrachloride was 2:1) was added. The solution was slowly restored to room temperature and then heated to 75 °C. After reacting for 2 hours, 4,4-dimethoxybenzophenone (Compound I) (4.846 g, 20 mmol) and 4-bromobenzophenone (Compound II-1) (6.26 g, 26 mmol) in a molar ratio of 1:1.2 were added to the flask at the same time, and the reaction was continued at 75 °C for 4 hours. The reaction was terminated by thin layer chromatography monitoring, and a saturated sodium bicarbonate solution was added, and the solution was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixture of petroleum ether and dichloromethane (volume ratio 4:1) to obtain compound III-1 with a yield of 31.5%.

[0094] The reaction formula is as follows:

[0095]

[0096] In the formula, when R1 of Ⅱ is -H, it is Ⅱ-1; when R1 of the product Ⅲ is -H, Ⅲ is Ⅲ-1.

[0097] Well enough:

[0098] When R1 of Ⅱ is -OCH3, it is Ⅱ-2;

[0099] When R1 of Ⅱ is -N(CH3)2, it is Ⅱ-3;

[0100] When R1 of Ⅱ is -NHCOCH3, it is Ⅱ-4;

[0101] When R1 of Ⅱ is -OCOCH3, it is Ⅱ-5;

[0102] The same as in this example, the corresponding compounds III-2 to III-5 can be obtained by reaction.

[0103] Example 2

[0104] Preparation of activated alkyne-tetraphenylene sulfonic acid sodium salt molecule (Compound VIII-1):

[0105] 1) Preparation of 4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)benzaldehyde (Compound IV-1):

[0106] Compound III-1 (0.471 g, 1 mmol) was added to the reaction bottle. After the system was ventilated, 3 mL of ultra-dry tetrahydrofuran solution was added and cooled at -78 °C for 10 to 20 minutes. Then n-butyl lithium solution (0.6 mL, 1.5 mmol) was slowly added to the above mixed solution (the molar ratio of compound III-1 to n-butyl lithium was 1:1.5). After the addition was completed, the reaction system was stirred at -78 °C for 2 hours. Then anhydrous N, N-dimethylformamide solution (0.08 mL, 1 mmol) was added (the molar ratio of compound III-1 to N, N-dimethylformamide solution was 1:1), and the reaction was carried out at room temperature (27 °C) for 5 hours. TLC monitoring, saturated ammonium chloride solution was added to quench the reaction, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixture of petroleum ether and dichloromethane (volume ratio 1:1) to obtain 4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)benzaldehyde (Compound IV-1) with a yield of 32.3%.

[0107] 2) Preparation of 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzaldehyde (Compound V-1):

[0108] Compound IV-1 (0.211 g, 0.5 mmol) and boron tribromide solution (3.05 mL, 3.05 mmol) in a molar ratio of 1:6 were added to a 2 mL ultra-dry dichloromethane solution reaction bottle under 0°C ice bath conditions. The reaction was restored to room temperature (27°C) and the reaction was continued to stir for 3 hours. TLC monitoring, the reaction was quenched with pure water, extracted with saturated ammonium chloride and dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixed solution of methanol and dichloromethane (volume ratio 1:200) to obtain 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzaldehyde (compound V-1) with a yield of 89.3%.

[0109] 3) Preparation of 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzinol (Compound VI-1):

[0110] Compound VI-1 (0.98 g, 2.5 mmol) and ethynyl magnesium bromide (15 mL, 7.5 mmol) in a molar ratio of 1:3 were added to 3 mL of ultra-dry tetrahydrofuran solution under ice bath conditions at 0°C. The reaction was returned to room temperature (27°C) and continued for 3 hours. TLC monitoring, quenching the reaction with saturated ammonium chloride, extraction with dichloromethane, and drying the organic phase with anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixture of ethyl acetate and petroleum ether (volume ratio 1:10) to obtain 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzinol (compound VI-1) with a yield of 88.9%.

[0111] 1 H-NMR (400MHz, MeOD-d4): δ7.26 (d, J=8.0Hz, 2H), 7.07 (qd, J=7.6, 6.4, 3.6Hz, 3H), 6.98 (dd, J=7.8 ,5.3Hz,4H),6.81(t,J=8.6Hz,4H),6.54–6.45(m,4H),5.30(d,J=2.2Hz,1H),2.94(d,J=2.2Hz,1H). 13 C-NMR (100MHz, MeOD-d4): δ155.72,155.67,144.55,144.47,141.16,138.53,138.03,135.17,132.38,131. 35,131.17,131.11,127.37,125.86,125.77,125.73,114.18,114.07,83.68,73.97,63.19.HRMS(ESI,m / z,C 29 H 22 O3, [M–H]- ):calcd,417.14962; found,417.14896.

[0112] 4) Preparation of alkynol-tetraphenylene sulfonic acid sodium salt molecule (Compound VII-1):

[0113] Under argon protection, compound VII-1 (0.1 g, 0.24 mmol) and sodium methoxide (0.0375 g, 0.72 mmol) in a molar ratio of 1:3 were added to 1 mL of anhydrous ethanol and stirred at room temperature for 1 hour. Next, 1,3-propane sultone (0.72 mmol) dissolved in anhydrous ethanol was added dropwise to the mixed solution (the molar ratio of compound (VII-1) to 1,3-propane sultone was 1:3). The reaction system was vigorously stirred overnight at room temperature (27°C, 12 hours) until a yellow precipitate precipitated from the solution. After suction filtration and washing three times with anhydrous ethanol, a yellow solid product was obtained. The product was dissolved in 500 μL of dimethyl sulfoxide solution, and 5 mL of dichloromethane was added. The mixture was slurried under ultrasonic conditions, and a large amount of yellow flocculent precipitate was observed to precipitate from the solution. The crude product can be obtained by centrifugation (8000 rpm, 5 min). The pulping process is repeated 5 times to finally obtain the alkynol-tetraphenylethylene sulfonic acid sodium salt molecule (Compound VII-1) with a yield of 61.5%.

[0114] 1 H-NMR (400MHz, DMSO-d6): δ7.22(d,J=7.8Hz,2H),7.12(dt,J=17.3,7.9Hz,3H),6.94(d,J=7.8Hz,4H),6.84(dd,J=12.9,8.0Hz,4H),6 .66(t,J=9.6Hz,4H),5.96(d,J=6.2Hz,1H),5.26(d,J=6.1Hz,1H),3.95(d,J=6.0Hz,4H),1.94(d,J=9.9Hz,5H),1.76(t,J=7.8Hz,3H). 13 C-NMR (100MHz, DMSO-d6): δ157.62,144.38,143.65,140.46,139.99,138.72,138.59,136.01,132.48,131.21,131.04,128.34,1 27.12,126.71,126.40,114.16,114.06,85.89,76.28,69.54,67.77,66.91,62.51,48.86,48.33,26.14,25.72.HRMS(ESI,m / z,C 35 H 32Na2O9S2,[M–2Na] 2- ):calcd,330.07494; found,330.07418.

[0115] 5) Preparation of activated alkyne-tetraphenylene sulfonic acid sodium salt molecule (Compound VIII-1):

[0116] Compound Ⅶ-1 (0.1 g, 0.14 mmol) and active manganese dioxide (0.30 g, 3.5 mmol) in a molar ratio of 1:25 were added to a mixed solvent of 1 mL of dimethyl sulfoxide and acetone (volume ratio: 1:6). The mixed solution was stirred overnight at room temperature (27 ° C, 12 hours), and then the metal oxide after the reaction was removed by suction filtration. The crude product was dissolved in 500 μL of dimethyl sulfoxide solution, and 5 mL of dichloromethane was added. The pulping was performed under ultrasonic conditions, and a large amount of yellow flocculent precipitate was observed to precipitate from the solution. The crude product was obtained by centrifugation (8000 rpm, 5 min). The pulping process was repeated 5 times to finally obtain activated alkyne-tetraphenylethylene sulfonic acid sodium salt (compound Ⅷ-1) with a yield of 50.1%.

[0117] 1 H-NMR (400MHz, DMSO-d6): δ7.85(d,J=7.9Hz,1H),7.16(td,J=21.0,17.6,9.0Hz,5H),6.96(d,J=7.4Hz,3H),6.85(q,J =9.3,7.7Hz,4H),6.68(q,J=9.6Hz,4H),5.02(s,1H),3.96(d,J=7.0Hz,4H),1.95(t,J=6.9Hz,5H),1.84–1.72(m,3H). 13 C-NMR (100MHz, DMSO-d6): δ157.97,157.63,136.00,132.49,131.95,131.22,131.03,129.38,128.59,128.36,127.12,126.70, 126.40,114.32,114.15,114.06,85.72,76.27,69.58,66.94,62.52,58.17,55.39,48.88,48.34,26.17,25.75.HRMS(ESI,m / z,C 35 H 30 Na2O9S2,[M–2Na] 2- ):calcd,329.06711; found,329.06608.

[0118] The reaction formula is as follows:

[0119]

[0120] Example 3

[0121] Preparation of 1-(4-bromophenyl)-2,2-bis(4-methoxyphenyl)-1-phenylene (Compound III-1):

[0122] Zinc powder (9.48 g, 145 mmol) was added to a three-necked flask, and the system was ventilated and cooled at -78 °C. 50 mL of dry tetrahydrofuran solution was added, and the solution was cooled for 10 minutes, and then titanium tetrachloride (6.36 mL, 58 mmol) (the molar ratio of zinc powder to titanium tetrachloride was 2.5:1) was added. The solution was slowly restored to room temperature and then heated to 75 °C. After reacting for 2 hours, 4,4-dimethoxybenzophenone (Compound I) (4.846 g, 20 mmol) and 4-bromobenzophenone (Compound II-1) (6.79 g, 26 mmol) were added to the flask at a molar ratio of 1:1.3, and the reaction was continued at 75 °C for 4 hours. The reaction was terminated by thin layer chromatography monitoring, and a saturated sodium bicarbonate solution was added, and the solution was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixture of petroleum ether and dichloromethane (volume ratio 4:1) to obtain compound III-1 with a yield of 31.5%.

[0123] The reaction formula is as follows:

[0124]

[0125] In the formula, when R1 of Ⅱ is -H, it is Ⅱ-1; when R1 of the product Ⅲ is -H, Ⅲ is Ⅲ-1.

[0126] Well enough:

[0127] When R1 of Ⅱ is -OCH3, it is Ⅱ-2;

[0128] When R1 of Ⅱ is -N(CH3)2, it is Ⅱ-3;

[0129] When R1 of Ⅱ is -NHCOCH3, it is Ⅱ-4;

[0130] When R1 of Ⅱ is -OCOCH3, it is Ⅱ-5;

[0131] The same as in this example, the corresponding compounds III-2 to III-5 can be obtained by reaction.

[0132] Example 4

[0133] Preparation of activated alkyne-tetraphenylene sulfonic acid sodium salt molecule (Compound VIII-1):

[0134] 1) Preparation of 4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)benzaldehyde (Compound IV-1):

[0135] Compound III-1 (0.471 g, 1 mmol) was added to the reaction bottle. After the system was ventilated, 3 mL of ultra-dry tetrahydrofuran solution was added and cooled at -78 °C for 10 to 20 minutes. Then n-butyl lithium solution (0.7 mL, 1.75 mmol) was slowly added to the above mixed solution (the molar ratio of compound III-1 to n-butyl lithium was 1:1.75). After the addition was completed, the reaction system was stirred at -78 °C for 2 hours. Then anhydrous N, N-dimethylformamide solution (0.14 mL, 1.75 mmol) was added (the molar ratio of compound III-1 to N, N-dimethylformamide solution was 1:1.75), and the reaction was carried out at room temperature (27 °C) for 5 hours. TLC monitoring was performed, saturated ammonium chloride solution was added to quench the reaction, and the organic phase was extracted with ethyl acetate and dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixture of petroleum ether and dichloromethane (volume ratio 1:1) to obtain 4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)benzaldehyde (Compound IV-1) with a yield of 32.3%.

[0136] 2) Preparation of 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzaldehyde (Compound V-1):

[0137] Compound IV-1 (0.211 g, 0.5 mmol) and boron tribromide solution (4 mL, 4 mmol) in a molar ratio of 1:8 were added to a 2 mL ultra-dry dichloromethane solution reaction bottle under 0°C ice bath conditions. The reaction was restored to room temperature (27°C) and the reaction was continued to stir for 3 hours. TLC monitoring, the reaction was quenched with pure water, extracted with saturated ammonium chloride and dichloromethane, and the organic phase was dried with anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixed solution of methanol and dichloromethane (volume ratio 1:200) to obtain 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzaldehyde (compound V-1) with a yield of 89.3%.

[0138] 3) Preparation of 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzinol (Compound VI-1):

[0139] Compound VI-1 (0.98 g, 2.5 mmol) and ethynyl magnesium bromide (20 mL, 10 mmol) in a molar ratio of 1:4 were added to 5 mL of ultra-dry tetrahydrofuran solution under 0°C ice bath conditions. The reaction was returned to room temperature (27°C) and the reaction was continued for 3 hours. TLC monitoring, the reaction was quenched with saturated ammonium chloride, extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixture of ethyl acetate and petroleum ether (volume ratio 1:10) to obtain 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzinol (compound VI-1) with a yield of 88.9%.

[0140] 1 H-NMR (400MHz, MeOD-d4): δ7.26 (d, J=8.0Hz, 2H), 7.07 (qd, J=7.6, 6.4, 3.6Hz, 3H), 6.98 (dd, J=7.8 ,5.3Hz,4H),6.81(t,J=8.6Hz,4H),6.54–6.45(m,4H),5.30(d,J=2.2Hz,1H),2.94(d,J=2.2Hz,1H). 13 C-NMR (100MHz, MeOD-d4): δ155.72,155.67,144.55,144.47,141.16,138.53,138.03,135.17,132.38,131. 35,131.17,131.11,127.37,125.86,125.77,125.73,114.18,114.07,83.68,73.97,63.19.HRMS(ESI,m / z,C 29 H 22 O3, [M–H] - ):calcd,417.14962; found,417.14896.

[0141] 4) Preparation of alkynol-tetraphenylene sulfonic acid sodium salt molecule (Compound VII-1):

[0142] Under argon protection, compound VII-1 (0.1 g, 0.24 mmol) and sodium methoxide (0.05 g, 0.96 mmol) in a molar ratio of 1:4 were added to 1 mL of anhydrous ethanol and stirred at room temperature for 1 hour. Next, 1,3-propane sultone (0.96 mmol) dissolved in anhydrous ethanol was added dropwise to the mixed solution (the molar ratio of compound (VII-1) to 1,3-propane sultone was 1:4). The reaction system was vigorously stirred overnight at room temperature (27°C, 12 hours) until a yellow precipitate precipitated from the solution. After suction filtration and washing three times with anhydrous ethanol, a yellow solid product was obtained. The product was dissolved in 500 μL of dimethyl sulfoxide solution, and 5 mL of dichloromethane was added. The mixture was slurried under ultrasonic conditions, and a large amount of yellow flocculent precipitate was observed to precipitate from the solution. The crude product can be obtained by centrifugation (8000 rpm, 5 min). The pulping process is repeated 5 times to finally obtain the alkynol-tetraphenylethylene sulfonic acid sodium salt molecule (Compound VII-1) with a yield of 61.5%.

[0143] 1 H-NMR (400MHz, DMSO-d6): δ7.22(d,J=7.8Hz,2H),7.12(dt,J=17.3,7.9Hz,3H),6.94(d,J=7.8Hz,4H),6.84(dd,J=12.9,8.0Hz,4H),6 .66(t,J=9.6Hz,4H),5.96(d,J=6.2Hz,1H),5.26(d,J=6.1Hz,1H),3.95(d,J=6.0Hz,4H),1.94(d,J=9.9Hz,5H),1.76(t,J=7.8Hz,3H). 13 C-NMR (100MHz, DMSO-d6): δ157.62,144.38,143.65,140.46,139.99,138.72,138.59,136.01,132.48,131.21,131.04,128.34,1 27.12,126.71,126.40,114.16,114.06,85.89,76.28,69.54,67.77,66.91,62.51,48.86,48.33,26.14,25.72.HRMS(ESI,m / z,C 35 H 32 Na2O9S2,[M–2Na] 2- ):calcd,330.07494; found,330.07418.

[0144] 5) Preparation of activated alkyne-tetraphenylene sulfonic acid sodium salt molecule (Compound VIII-1):

[0145] Compound Ⅶ-1 (0.1 g, 0.14 mmol) and active manganese dioxide (0.33 g, 3.78 mmol) in a molar ratio of 1:27 were added to a mixed solvent of 1 mL of dimethyl sulfoxide and acetone (volume ratio: 1:6). The mixed solution was stirred overnight at room temperature (27 ° C, 12 hours), and then the metal oxide after the reaction was removed by suction filtration. The crude product was dissolved in 500 μL of dimethyl sulfoxide solution, and 5 mL of dichloromethane was added. The pulping was performed under ultrasonic conditions, and a large amount of yellow flocculent precipitate was observed to precipitate from the solution. The crude product was obtained by centrifugation (8000 rpm, 5 min). The pulping process was repeated 5 times to finally obtain activated alkyne-tetraphenylethylene sulfonic acid sodium salt (compound Ⅷ-1) with a yield of 50.1%.

[0146] 1 H-NMR (400MHz, DMSO-d6): δ7.85(d,J=7.9Hz,1H),7.16(td,J=21.0,17.6,9.0Hz,5H),6.96(d,J=7.4Hz,3H),6.85(q,J =9.3,7.7Hz,4H),6.68(q,J=9.6Hz,4H),5.02(s,1H),3.96(d,J=7.0Hz,4H),1.95(t,J=6.9Hz,5H),1.84–1.72(m,3H). 13 C-NMR (100MHz, DMSO-d6): δ157.97,157.63,136.00,132.49,131.95,131.22,131.03,129.38,128.59,128.36,127.12,126.70, 126.40,114.32,114.15,114.06,85.72,76.27,69.58,66.94,62.52,58.17,55.39,48.88,48.34,26.17,25.75.HRMS(ESI,m / z,C 35 H 30 Na2O9S2,[M–2Na] 2- ):calcd,329.06711; found,329.06608.

[0147] The reaction formula is as follows:

[0148]

[0149] Example 5

[0150] Preparation of 1-(4-bromophenyl)-2,2-bis(4-methoxyphenyl)-1-phenylene (Compound III-1):

[0151] Zinc powder (11.376 g, 174 mmol) was added to a three-necked flask, and the system was ventilated and cooled at -78 °C. 50 mL of dry tetrahydrofuran solution was added, and the solution was cooled for 10 minutes, and then titanium tetrachloride (6.36 mL, 58 mmol) (the molar ratio of zinc powder to titanium tetrachloride was 3:1) was added. The solution was slowly restored to room temperature and then heated to 75 °C. After reacting for 2 hours, 4,4-dimethoxybenzophenone (Compound I) (4.846 g, 20 mmol) and 4-bromobenzophenone (Compound II-1) (7.83 g, 30 mmol) were added to the flask at a molar ratio of 1:1.5, and the reaction was continued at 75 °C for 4 hours. The reaction was terminated by thin layer chromatography monitoring, and a saturated sodium bicarbonate solution was added, and the solution was extracted with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixture of petroleum ether and dichloromethane (volume ratio 4:1) to obtain compound III-1 with a yield of 31.5%.

[0152] The reaction formula is as follows:

[0153]

[0154] In the formula, when R1 of Ⅱ is -H, it is Ⅱ-1; when R1 of the product Ⅲ is -H, Ⅲ is Ⅲ-1.

[0155] Well enough:

[0156] When R1 of Ⅱ is -OCH3, it is Ⅱ-2;

[0157] When R1 of Ⅱ is -N(CH3)2, it is Ⅱ-3;

[0158] When R1 of Ⅱ is -NHCOCH3, it is Ⅱ-4;

[0159] When R1 of Ⅱ is -OCOCH3, it is Ⅱ-5;

[0160] The same as in this example, the corresponding compounds III-2 to III-5 can be obtained by reaction.

[0161] Example 6

[0162] Preparation of activated alkyne-tetraphenylene sulfonic acid sodium salt molecule (Compound VIII-1):

[0163] 1) Preparation of 4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)benzaldehyde (Compound IV-1):

[0164] Compound III-1 (0.471 g, 1 mmol) was added to the reaction bottle. After the system was ventilated, 3 mL of ultra-dry tetrahydrofuran solution was added and cooled at -78 °C for 10 to 20 minutes. Then n-butyl lithium solution (0.8 mL, 2 mmol) was slowly added to the above mixed solution (the molar ratio of compound III-1 to n-butyl lithium was 1:2). After the addition was completed, the reaction system was stirred at -78 °C for 2 hours. Then anhydrous N, N-dimethylformamide solution (0.16 mL, 2 mmol) was added (the molar ratio of compound III-1 to N, N-dimethylformamide solution was 1:2) and reacted at room temperature (27 °C) for 5 hours. TLC monitoring, saturated ammonium chloride solution was added to quench the reaction, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixture of petroleum ether and dichloromethane (volume ratio 1:1) to obtain 4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)benzaldehyde (Compound IV-1) with a yield of 32.3%.

[0165] 2) Preparation of 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzaldehyde (Compound V-1):

[0166] Compound IV-1 (0.211 g, 0.5 mmol) and boron tribromide solution (5 mL, 5 mmol) in a molar ratio of 1:10 were added to a 2 mL ultra-dry dichloromethane solution reaction bottle under 0°C ice bath conditions. The reaction was restored to room temperature (27°C) and the reaction was continued to stir for 3 hours. TLC monitoring, the reaction was quenched with pure water, extracted with saturated ammonium chloride and dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixed solution of methanol and dichloromethane (volume ratio 1:200) to obtain 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzaldehyde (compound V-1) with a yield of 89.3%.

[0167] 3) Preparation of 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzinol (Compound VI-1):

[0168] Compound VI-1 (0.98 g, 2.5 mmol) and ethynyl magnesium bromide (25 mL, 12.5 mmol) in a molar ratio of 1:5 were added to 5 mL of ultra-dry tetrahydrofuran solution under 0°C ice bath conditions. The reaction was restored to room temperature (27°C) and continued for 3 hours. TLC monitoring, quenching the reaction with saturated ammonium chloride, extraction with dichloromethane, and drying the organic phase with anhydrous sodium sulfate. After removing the solvent, the crude product was purified by silica gel column chromatography and eluted with a mixture of ethyl acetate and petroleum ether (volume ratio 1:10) to obtain 4-(2,2-bis(4-hydroxyphenyl)-1-phenylvinyl)benzinol (compound VI-1) with a yield of 88.9%.

[0169] 1 H-NMR (400MHz, MeOD-d4): δ7.26 (d, J=8.0Hz, 2H), 7.07 (qd, J=7.6, 6.4, 3.6Hz, 3H), 6.98 (dd, J=7.8 ,5.3Hz,4H),6.81(t,J=8.6Hz,4H),6.54–6.45(m,4H),5.30(d,J=2.2Hz,1H),2.94(d,J=2.2Hz,1H). 13 C-NMR (100MHz, MeOD-d4): δ155.72,155.67,144.55,144.47,141.16,138.53,138.03,135.17,132.38,131. 35,131.17,131.11,127.37,125.86,125.77,125.73,114.18,114.07,83.68,73.97,63.19.HRMS(ESI,m / z,C 29 H 22 O3, [M–H] - ):calcd,417.14962; found,417.14896.

[0170] 4) Preparation of alkynol-tetraphenylene sulfonic acid sodium salt molecule (Compound VII-1):

[0171] Under argon protection, compound VII-1 (0.1 g, 0.24 mmol) and sodium methoxide (0.0625 g, 1.2 mmol) in a molar ratio of 1:5 were added to 1 mL of anhydrous ethanol and stirred at room temperature for 1 hour. Next, 1,3-propane sultone (1.2 mmol) dissolved in anhydrous ethanol was added dropwise to the mixed solution (the molar ratio of compound (VII-1) to 1,3-propane sultone was 1:3). The reaction system was vigorously stirred overnight at room temperature (27°C, 12 hours) until a yellow precipitate precipitated from the solution. After suction filtration and washing three times with anhydrous ethanol, a yellow solid product was obtained. The product was dissolved in 500 μL of dimethyl sulfoxide solution, and 5 mL of dichloromethane was added. The mixture was slurried under ultrasonic conditions, and a large amount of yellow flocculent precipitate was observed to precipitate from the solution. The crude product can be obtained by centrifugation (8000 rpm, 5 min). The pulping process is repeated 5 times to finally obtain the alkynol-tetraphenylethylene sulfonic acid sodium salt molecule (Compound VII-1) with a yield of 61.5%.

[0172] 1 H-NMR (400MHz, DMSO-d6): δ7.22(d,J=7.8Hz,2H),7.12(dt,J=17.3,7.9Hz,3H),6.94(d,J=7.8Hz,4H),6.84(dd,J=12.9,8.0Hz,4H),6 .66(t,J=9.6Hz,4H),5.96(d,J=6.2Hz,1H),5.26(d,J=6.1Hz,1H),3.95(d,J=6.0Hz,4H),1.94(d,J=9.9Hz,5H),1.76(t,J=7.8Hz,3H). 13 C-NMR (100MHz, DMSO-d6): δ157.62,144.38,143.65,140.46,139.99,138.72,138.59,136.01,132.48,131.21,131.04,128.34,1 27.12,126.71,126.40,114.16,114.06,85.89,76.28,69.54,67.77,66.91,62.51,48.86,48.33,26.14,25.72.HRMS(ESI,m / z,C 35 H 32 Na2O9S2,[M–2Na] 2- ):calcd,330.07494; found,330.07418.

[0173] 5) Preparation of activated alkyne-tetraphenylene sulfonic acid sodium salt molecule (Compound VIII-1):

[0174] Compound Ⅶ-1 (0.1 g, 0.14 mmol) and active manganese dioxide (0.37 g, 4.24 mmol) in a molar ratio of 1:30 were added to a mixed solvent of 1 mL of dimethyl sulfoxide and acetone (volume ratio: 1:6). The mixed solution was stirred overnight at room temperature (27 ° C, 12 hours), and then the metal oxide after the reaction was removed by suction filtration. The crude product was dissolved in 500 μL of dimethyl sulfoxide solution, and 5 mL of dichloromethane was added. The pulping was performed under ultrasonic conditions, and a large amount of yellow flocculent precipitate was observed to precipitate from the solution. The crude product was obtained by centrifugation (8000 rpm, 5 min). The pulping process was repeated 5 times, and finally the activated alkyne-tetraphenylethylene sulfonic acid sodium salt (compound Ⅷ-1) was obtained with a yield of 50.1%.

[0175] 1 H-NMR (400MHz, DMSO-d6): δ7.85(d,J=7.9Hz,1H),7.16(td,J=21.0,17.6,9.0Hz,5H),6.96(d,J=7.4Hz,3H),6.85(q,J =9.3,7.7Hz,4H),6.68(q,J=9.6Hz,4H),5.02(s,1H),3.96(d,J=7.0Hz,4H),1.95(t,J=6.9Hz,5H),1.84–1.72(m,3H). 13 C-NMR (100MHz, DMSO-d6): δ157.97,157.63,136.00,132.49,131.95,131.22,131.03,129.38,128.59,128.36,127.12,126.70, 126.40,114.32,114.15,114.06,85.72,76.27,69.58,66.94,62.52,58.17,55.39,48.88,48.34,26.17,25.75.HRMS(ESI,m / z,C 35 H 30 Na2O9S2,[M–2Na] 2- ):calcd,329.06711; found,329.06608.

[0176] The reaction formula is as follows:

[0177]

[0178] Example 7

[0179] Photochemical and click reaction properties of the activated alkyne-tetraphenylene sulfonic acid sodium salt synthesized in Examples 2, 4 and 6.

[0180] The activated alkyne-tetraphenylene sulfonate sodium salt (pyone-TPE) synthesized in Examples 2, 4 and 6 has strong water solubility and exhibits obvious aggregation-induced emission properties in the presence of a poor solvent (tetrahydrofuran), that is, as the proportion of the poor solvent increases, the fluorescence gradually increases ( Figure 1 A, B). In order to further verify the click reaction ability of the molecule, n-propylamine was selected to react with pyone-TPE at room temperature. The reaction equation is as follows: Figure 1 C. The Fourier infrared spectrum shows that C≡C at 2095cm -1 The characteristic absorption at the wave number of 1.37 nm completely disappeared in the generated peone-TPE, proving that pyone-TPE containing active alkynes can undergo mild metal-free click reactions ( Figure 1 D, E).

[0181] Example 8

[0182] The lysis method of Gram-negative bacteria and Gram-positive bacteria and the specific click fluorescence turn-on process of the activated alkyne-tetraphenylethylene sulfonic acid sodium salt synthesized in Examples 2, 4 and 6.

[0183] We used methicillin-resistant Staphylococcus aureus (MRSA) and wild-type Escherichia coli (WT E.coli) as examples to establish an efficient bacterial lysis method that is applicable to both Gram-positive and Gram-negative bacteria. Due to the presence of a thick layer of peptidoglycan, NaOH and high-temperature incubation (70°C, 10 minutes) are required to effectively lyse Gram-positive bacteria. Since Gram-negative bacteria have a thinner peptidoglycan layer, they can be effectively lysed by incubation with NaOH at physiological temperature (37°C, 10 minutes). Therefore, for an unknown strain, NaOH and high-temperature incubation are required to ensure the most sensitive fluorescence response. Under UV light, the presence of MRSA and WT E.coli is easily displayed by the yellow-green fluorescence of pyone-TPE ( Figure 2 A and D). In addition, the fluorescent turn-on labeling effect of pyridine ketone in pyone-TPE was confirmed by comparison with the control molecule pyol-TPE. Figure 2 As shown in B, C, E, and F, the presence of pyone-TPE significantly enhances the relative emission intensity of MRSA and WT E. coli, and the intensity is about 4.3 times and 4.8 times that of pyol-TPE, respectively, indicating that the system has the AIE phenomenon of chemical bond enhancement. This experiment further proves the click labeling ability of pyone-TPE for biological systems.

[0184] Example 9

[0185] The activated alkyne-tetraphenylene sulfonic acid sodium salt synthesized in Examples 2, 4 and 6 was applied to the fluorescence spectrophotometer to test the antibiotic antibacterial sensitivity (Gram-negative bacteria)

[0186] 1) Standard curve determination:

[0187] Sodium hydroxide solution (10M, 10μL) was added to 900μL of PBS buffer containing different concentrations of Gram-negative bacteria. Incubate in a 37°C shaking incubator (170rpm) for 10 minutes, add pyone-TPE (1mM, 50μL) synthesized in Examples 2, 4 and 6 to the mixed solution, continue to incubate in a 37°C shaking incubator (170rpm) for 10 minutes, and then use a fluorescence spectrophotometer to measure the fluorescence intensity of the solution at 525nm (Ex=405nm). Finally, a standard curve was obtained by establishing the relationship between bacterial concentration and fluorescence enhancement ((I-I0) / I0). (I0 is OD 600 = 0 fluorescence intensity).

[0188] 2) Drug sensitivity test:

[0189] First, add lysate broth (LB) liquid medium into a 24-well plate and increase the concentration of the antibiotic to be screened from 256 μg mL -1 Serially double dilute to 1 μg mL -1 The final volume of each well was 900 μL. Next, Gram-negative bacteria (10 μL, OD 600 =1.0) to a final concentration of approximately 1×10 6 CFU mL -1 . After the 24-well plate was incubated in a 37°C shaker (170 rpm) for 1.5 h, the bacterial solution in each well was taken into a 1.5 ml centrifuge tube, centrifuged at 10,000 rpm for 5 minutes, the supernatant was discarded, and the suspension was resuspended with 900 μL PBS buffer. Sodium hydroxide solution (10 M, 10 μL) was added to the above suspension, incubated in a 37°C shaking incubator (170 rpm) for 10 minutes, pyone-TPE (1 mM, 50 μL) synthesized in Examples 2, 4 and 6 was added to the above mixed solution, and after continuing to incubate in a 37°C shaking incubator (170 rpm) for 10 minutes, the fluorescence intensity of the solution at 525 nm (Ex = 405 nm) was measured using a fluorescence spectrophotometer. Finally, the relationship between the number of bacteria in the system and the concentration of different antibiotics was quantified using a standard curve.

[0190] Example 10

[0191] The activated alkyne-tetraphenylene sulfonic acid sodium salt synthesized in Examples 2, 4 and 6 was applied to the fluorescence spectrophotometer to test the antibiotic antibacterial sensitivity (Gram-positive bacteria)

[0192] 1) Standard curve determination:

[0193] Sodium hydroxide solution (10M, 10μL) was added to 900μL of PBS buffer containing different concentrations of Gram-positive bacteria. After incubation in a 70°C oven for 10 minutes, pyone-TPE (1mM, 50μL) synthesized in Examples 2, 4 and 6 was added to the above mixed solution, and after further incubation in a 37°C shaking incubator (170rpm) for 10 minutes, the fluorescence intensity of the solution at 525nm (Ex=405nm) was measured using a fluorescence spectrophotometer. Finally, a standard curve was obtained by establishing the relationship between bacterial concentration and fluorescence enhancement ((I-I0) / I0). (I0 is OD 600 = 0 fluorescence intensity).

[0194] 2) Drug sensitivity test:

[0195] First, tryptic soy casein (TSB) liquid medium was added to a 24-well plate, and the antibiotic to be screened was adjusted from 256 μg mL -1 Serially double dilute to 1 μg mL -1 The final volume of each well was 900 μL. Next, Gram-positive bacteria (10 μL, OD 600 =1.0) to a final concentration of approximately 1×10 6 CFU mL -1 . After the 24-well plate was incubated in a 37°C shaker (170 rpm) for 1.5 h, the bacterial solution in each well was taken into a 1.5 ml centrifuge tube, centrifuged at 10,000 rpm for 5 minutes, the supernatant was discarded, and the suspension was resuspended with 900 μL PBS buffer. Sodium hydroxide solution (10 M, 10 μL) was added to the above suspension, incubated in a 70°C oven for 10 minutes, pyone-TPE (1 mM, 50 μL) synthesized in Examples 2, 4 and 6 was added to the above mixed solution, and after continuing to incubate in a 37°C shaking incubator (170 rpm) for 10 minutes, the fluorescence intensity of the solution at 525 nm (Ex = 405 nm) was measured using a fluorescence spectrophotometer. Finally, the relationship between the number of bacteria in the system and the concentration of different antibiotics was quantified using a standard curve.

[0196] Based on the preliminary conclusions obtained from the above experiments, the fluorescence signal was quantitatively linked to the number of bacteria using the methods of Examples 9 and 10, and standard curves for MRSA and WT E. coli based on a fluorescence spectrophotometer were established. Figure 3As shown in A and D, the fluorescence emission intensity is proportional to the bacterial concentration, and the fluorescence intensity increases with the increase of the bacterial OD value. Since the presence of bacteria can be effectively labeled by the fluorescence of the pyone-TPE synthesized in Examples 2, 4 and 6, MRSA and WT E.coli can be easily observed by the naked eye under ultraviolet light ( Figure 3 B and E). Based on Figure 3 Based on the results shown in A and D, we further plotted standard curves between the relative fluorescence emission intensity of MRSA and WT E.coli and the bacterial concentration. The minimum detection limits (LOD) of MRSA and WT E.coli were 7.6×10 2 and 6.7×10 2 CFU / mL( Figure 3 C and F). To demonstrate the universality of this method, standard curves for four other bacteria were also established, including two Gram-positive bacteria (Streptococcus mutans (S. mutans) and Enterococcus faecalis (E. faecalis)) and two Gram-negative bacteria (Pseudomonas aeruginosa (P. aeruginosa) and ampicillin-resistant Escherichia coli (E. coli Ampr )). We then proposed a method to link the number of bacteria in the system to the efficacy of antibiotics for antimicrobial susceptibility testing. Effective drugs inhibit the exponential proliferation of bacteria, while ineffective drugs have no effect on bacterial growth. After bacterial lysis, the ineffective drug group produced a large amount of clickable biomass relative to the effective drug group, which resulted in very strong fluorescence emission after incubation with pyone-TPE. The number of bacteria in the system can be easily calibrated by the relative emission intensity based on the pre-established standard curve. To verify this argument, we evaluated the efficacy of six antibiotics, including chloramphenicol (CHL), ampicillin sodium (AMP), kanamycin sulfate (KAN), streptomycin sulfate (STM), erythromycin (ERM), and vancomycin hydrochloride (VAN), against the above six bacteria. Taking MRSA as an example, the number of bacteria in the CHL, AMP, and VAN treatment groups decreased in a concentration-dependent manner ( Figure 4 A) MIC and IC of CHL according to the fitting function 50 The MIC and IC of AMP were 2 and 10 μg / mL, respectively. 50 The MIC and IC of VAN were 9 and 15 μg / mL, respectively. 50 The MIC values ​​were consistent with those of the traditional broth dilution method. Figure 4BF and Table 1. It is noteworthy that the results obtained by our proposed new method are very consistent with those of the broth dilution method, but the overall operation time is shortened to less than 3 h, and the antibiotic concentration at any inhibition rate can be obtained at one time, which is impossible with the traditional method.

[0197] Embodiment 11

[0198] Application of the activated alkyne-tetraphenylene sulfonic acid sodium salt synthesized in Examples 2, 4 and 6 to high-throughput antibiotic screening (Gram-positive bacteria)

[0199] 1) Standard curve determination:

[0200] Sodium hydroxide solution (10M, 1μL) was added to 90μL of PBS buffer containing different concentrations of Gram-positive bacteria. Incubate in a 70°C oven for 10 minutes, add pyone-TPE (1mM, 5μL) synthesized in Examples 2, 4 and 6 to the mixed solution, and continue to incubate in a 37°C shaking incubator (170rpm) for 10 minutes, and then use a microplate reader to measure the fluorescence intensity of the solution at 525nm with an excitation wavelength of 405nm. Finally, a standard curve was obtained by establishing the relationship between bacterial concentration and fluorescence enhancement ((I-I0) / I0). (I0 is OD 600 = 0 fluorescence intensity).

[0201] 2) High-throughput antibiotic screening:

[0202] First, TSB liquid medium was added to a 24-well plate and the antibiotic to be screened was diluted from 256 μg mL -1 Serially double dilute to 1 μg mL -1 The final volume of each well was 900 μL. Next, Gram-negative bacteria (10 μL, OD 600 =1.0) to a final concentration of approximately 1×10 6 CFU mL -1. After the 24-well plate was incubated in a 37°C shaker (170 rpm) for 1.5 h, the bacterial solution in each well was taken into a 1.5 ml centrifuge tube, centrifuged at 10,000 rpm for 5 minutes, the supernatant was discarded, and the suspension was resuspended with 900 μL PBS buffer. 90 μL of the suspension was taken into a 96-well plate (three parallel wells were set for each concentration), sodium hydroxide solution (10 M, 1 μL) was added to the well, incubated in a 37°C shaking incubator (170 rpm) for 10 minutes, pyone-TPE (1 mM, 5 μL) synthesized in Examples 2, 4 and 6 was added to the above mixed solution, and continued to incubate in a 37°C shaking incubator (170 rpm) for 10 minutes, and the fluorescence intensity of the solution at 525 nm was measured using an ELISA instrument with an excitation wavelength of 405 nm. Finally, the relationship between the number of bacteria in the system and the concentration of different antibiotics was quantified by a standard curve. As the concentration of effective antibiotics increased, the number of bacteria in the system showed a significant downward trend. For ineffective antibiotics, as their concentration increases, the number of bacteria in the system decreases slowly or even remains unchanged. Figure 5 B, MRSA was sensitive to CHL, AMP, and VAN, with MIC values ​​of 11, 15, and 3 μg / mL, respectively, and was resistant to KAN, STM, and ERM, with MIC values ​​of >256, >256, and >256 μg / mL, respectively; Figure 5 C, S. mutans was sensitive to CHL, AMP, ERM, and VAN, with MIC values ​​of 8, 4, <1, and 4 μg / mL, respectively, and was resistant to KAN and STM, with MIC values ​​of 226 and 245 μg / mL, respectively; Figure 5 D, E. faecalis was sensitive to CHL, AMP, ERM and VAN, with MIC values ​​of 15, 3, <1 and 4 μg / mL, respectively, and was resistant to KAN and STM, with MIC values ​​of 203 and 177 μg / mL, respectively.

[0203] Example 12

[0204] Application of the activated alkyne-tetraphenylene sulfonic acid sodium salt synthesized in Examples 2, 4 and 6 to high-throughput antibiotic screening (Gram-negative bacteria)

[0205] 1) Standard curve determination:

[0206] Sodium hydroxide solution (10M, 1μL) was added to 90μL of PBS buffer containing different concentrations of Gram-negative bacteria. Incubate in a 37°C shaking incubator (170rpm) for 10 minutes, add pyone-TPE (1mM, 5μL) synthesized in Examples 2, 4 and 6 to the mixed solution, and continue to incubate in a 37°C shaking incubator (170rpm) for 10 minutes, then use a microplate reader to measure the fluorescence intensity of the solution at 525nm with an excitation wavelength of 405nm. Finally, a standard curve was obtained by establishing the relationship between bacterial concentration and fluorescence enhancement ((I-I0) / I0). (I0 is OD 600 = 0 fluorescence intensity).

[0207] 2) High-throughput antibiotic screening:

[0208] First, LB liquid culture medium was added to a 24-well plate and the antibiotic to be screened was diluted from 256 μg mL -1 Serially double dilute to 1 μg mL -1 The final volume of each well was 900 μL. Next, Gram-negative bacteria (10 μL, OD 600 =1.0) to a final concentration of approximately 1×10 6 CFU mL -1 . After the 24-well plate was incubated in a 37°C shaker (170 rpm) for 1.5 h, the bacterial solution in each well was taken into a 1.5 ml centrifuge tube, centrifuged at 10,000 rpm for 5 minutes, the supernatant was discarded, and the suspension was resuspended with 900 μL PBS buffer. 90 μL of the suspension was taken into a 96-well plate (three parallel wells were set for each concentration), sodium hydroxide solution (10 M, 1 μL) was added to the well, incubated in a 37°C shaking incubator (170 rpm) for 10 minutes, pyone-TPE (1 mM, 5 μL) synthesized in Examples 2, 4 and 6 was added to the above mixed solution, and continued to incubate in a 37°C shaking incubator (170 rpm) for 10 minutes, and the fluorescence intensity of the solution at 525 nm was measured using an ELISA instrument with an excitation wavelength of 405 nm. Finally, the relationship between the number of bacteria in the system and the concentration of different antibiotics was quantified by a standard curve. As the concentration of effective antibiotics increased, the number of bacteria in the system showed a significant downward trend. For ineffective antibiotics, as their concentration increases, the number of bacteria in the system decreases slowly or even remains unchanged. Figure 5 E, P. aeruginosa was sensitive to CHL, AMP, KAN, STM, ERM, and VAN, with MIC values ​​of 2, 2, 26, 30, <1, and <1 μg / mL, respectively; Figure 5F, WT E. coli was sensitive to CHL with an MIC value of 26 μg / mL and resistant to AMP, KAN, STM, ERM, and VAN with MIC values ​​of >256, >256, >256, >256, and 240 μg / mL; Figure 5 G, E. coli Ampr It was sensitive to CHL and KAN, with MIC values ​​of 4 and 15 μg / mL, respectively; it was resistant to AMP, STM, ERM and VAN, with MIC values ​​of >256, >256, 196 and 142 μg / mL, respectively.

[0209] In order to facilitate large-scale antibiotic screening and appropriately reduce the total experimental volume, a high-throughput antimicrobial susceptibility test was performed using a microplate according to the method of Examples 11 and 12. Figure 5 As shown in A, samples incubated with different concentrations of antibiotics were placed in the microplate wells for single-point signal readout. A high fluorescence emission intensity indicates a poor antibacterial effect. Similar to the experimental method of the fluorescence spectrophotometer, a standard curve based on the microplate test for six bacteria was established. Next, the relative fluorescence emission intensity of different bacteria treated with different antibiotics was recorded to establish the relationship between the number of bacteria and the antibiotic concentration ( Figure 5 BG). According to the test results (MIC and IC 50 value), it is easy to screen out effective antibiotics against specific strains.

[0210] The present invention develops a cascade signal amplification strategy for ultrafast and high-throughput antibiotic screening. This approach is implemented by a new type of water-soluble AIE fluorescent molecule containing an active alkyne group, which can undergo fluorescence turn-on and metal-free click reactions with biological species under physiological conditions. Through efficient bacterial lysis, a large amount of clickable biological substances are released from the bacteria, resulting in significant amplification of the analyte. Using fluorescence turn-on and metal-free click bioconjugation technology, cascade signal amplification for bacterial labeling is achieved, laying the foundation for ultrafast and sensitive antimicrobial susceptibility detection. In our experiments, the entire process took less than 3 hours, and the measured LOD value was as low as 5-10×10 2 CFU / mL. It is worth noting that this method is applicable to both Gram-positive and Gram-negative bacteria, and can obtain antibiotic concentrations at any inhibition rate at one time. Most importantly, this method can be integrated with microplates for high-throughput antibiotic screening, which has great potential in the field of new antibiotic development.

[0211] Table 1 MIC and IC of different antibiotics tested by fluorescence spectrophotometer 50 value.

[0212]

[0213]

[0214] The technical solutions disclosed and proposed by the present invention can be realized by those skilled in the art by referring to the contents of this article and appropriately changing the conditions, routes and other links. Although the methods and preparation techniques of the present invention have been described through preferred embodiments, relevant technicians can obviously modify or re-combine the methods and technical routes described herein without departing from the content, spirit and scope of the present invention to realize the final preparation technology. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention.

Claims

1. An activated alkyne-tetraphenylene sulfonic acid sodium salt molecule, characterized in that: The structural formula is shown in the formula; （Ⅷ）； Wherein, R1 is -H, -OCH3, -N(CH3)2, -NHCOCH3 or -OCOCH3.

2. The synthetic method of the activated alkyne-tetraphenylene sulfonic acid sodium salt molecule of claim 1, characterized in that: The steps include: 1) Weigh zinc powder, add tetrahydrofuran solution under argon protection, add titanium tetrachloride dropwise at -78°C~-50°C, and after the addition is complete, heat under reflux at 75°C~80°C; add 4,4-dimethoxybenzophenone I and compound II to the reaction system, heat under reflux at 75°C~80°C for 3~4 hours, monitor by thin layer chromatography and purify by column chromatography to obtain olefin compound III; 2) Compound III was dissolved in tetrahydrofuran, and n-butyl lithium solution was added dropwise under argon protection at -78°C to -50°C. After stirring for 2 to 3 hours, anhydrous N,N-dimethylformamide solution was added. The reaction system was returned to room temperature and the reaction was continued for 3 to 5 hours. Compound IV was obtained by thin layer chromatography monitoring and column chromatography purification. 3) Compound IV and boron tribromide were added to ultra-dry dichloromethane in an ice bath; after the addition was completed, the mixture was reacted at room temperature for 3 to 5 hours, and the mixture was monitored by thin layer chromatography and purified by column chromatography to obtain compound V; 4) Compound V and ethynylmagnesium bromide were added to tetrahydrofuran in an ice bath; after the addition was completed, the mixture was reacted at room temperature for 3 to 5 hours, and the mixture was monitored by thin layer chromatography and purified by column chromatography to obtain compound VI; 5) Add compound VI and sodium methoxide to anhydrous ethanol and stir at room temperature for 1-2 hours; Then, 1,3-propane sultone was added, and the mixture was stirred at room temperature overnight for 12 to 16 hours to obtain compound VII; 6) Compound VII and active manganese dioxide are added to a mixed solvent of DMSO and acetone, stirred overnight at room temperature for 12 to 16 hours, and the metal oxide after the reaction is removed by filtration to obtain activated alkyne-tetraphenylethylene sulfonic acid sodium salt molecule VIII; The general reaction formula is as follows: ； R1 is defined as in claim 1.

3. The synthetic method of activated alkyne-tetraphenylene sulfonic acid sodium salt molecule as claimed in claim 2, characterized in that, In step 1), the molar ratio of zinc powder to titanium tetrachloride is 2:1-3:1; the molar ratio of 4,4-dimethoxybenzophenone I to compound II is 1:1.2-1:1.5; the molar ratio of zinc powder to 4,4-dimethoxybenzophenone (I) is 4:1-6:

1.

4. The synthetic method of activated alkyne-tetraphenylene sulfonic acid sodium salt molecule as claimed in claim 2, characterized in that: Step 2) The molar ratio of compound III to n-butyl lithium is 1:1.5 to 1:2; the molar ratio of compound III to anhydrous N,N-dimethylformamide solution is 1:1 to 1:

2.

5. The synthetic method of activated alkyne-tetraphenylene sulfonic acid sodium salt molecule as claimed in claim 2, characterized in that: Step 3) The molar ratio of compound IV to boron tribromide is 1:6 to 1:

10.

6. The method for synthesizing the activated alkyne-tetraphenylene sulfonic acid sodium salt molecule according to claim 2, characterized in that: Step 4) The molar ratio of compound V to ethynylmagnesium bromide is 1:3 to 1:

5.

7. The method for synthesizing the activated alkyne-tetraphenylene sulfonate sodium salt molecule according to claim 2, characterized in that: Step 5) The molar ratio of compound VI to sodium methoxide is 1:3 to 1:5; the molar ratio of compound VI to 1,3-propane sultone is 1:3 to 1:

5.

8. The method for synthesizing the activated alkyne-tetraphenylene sulfonic acid sodium salt molecule according to claim 2, characterized in that: Step 6) The molar ratio of compound VII to active manganese dioxide is 1:25-1:30; the mixed solvent of DMSO and acetone completely dissolves compound VII with a mass fraction of not less than 10%, and the volume ratio of DMSO and acetone is 1:

6.

9. A method for using the activated alkyne-tetraphenylene sulfonate sodium salt molecule of claim 1 as a water-soluble fluorescent molecule for high-throughput antibiotic screening, characterized in that: The following steps are included 1) Gram-negative bacteria / Gram-positive bacteria were incubated in liquid culture medium containing different antibiotic concentrations for more than 1.5 hours; 2) After the incubation, the bacterial culture solution is added to a centrifuge tube, centrifuged and the culture medium supernatant is discarded, leaving the bacterial precipitate, and then resuspended with the same volume of PBS solution; Gram-negative bacteria / Gram-positive bacteria bacterial suspensions are obtained respectively, and the centrifugal speed is 3500rpm~10000rpm; for Gram-negative bacteria, 10M sodium hydroxide solution is added to the Gram-negative bacteria suspension and incubated in a 37°C constant temperature shaker for more than 10 minutes to lyse, to obtain Gram-negative bacteria lysate; for Gram-positive bacteria, 10M sodium hydroxide solution is added to the Gram-positive bacteria suspension and heated in a 70°C~90°C oven for more than 10 minutes to lyse, to obtain Gram-positive bacteria lysate; 3) Add the pre-prepared activated alkyne-tetraphenylene sulfonic acid sodium salt molecule dimethyl sulfoxide solution to the Gram-negative bacteria or Gram-positive bacteria lysate and incubate them in a constant temperature shaker at 37°C for more than 10 minutes. Use an ELISA reader to detect the fluorescence intensity, and calculate the relationship between the number of bacteria in the system and the antibiotic concentration through the standard curve to achieve the antibacterial sensitivity evaluation of different antibiotics.

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