To Be Based On β-Lactamase Probe Of Lactamase Used In Resistant Bacteria Study

Objective:To investigate effective small molecule photosensitizers against drug-resistant bacteria with highβ-lactamase expression,we expect to screen 1-2photosensitizers with high selectivity,good water solubility,good biocompatibility and high antibacterial activity to provide new ideas to tackle the global problem of bacterial drug resistance.Methods:（1）Design,preparation and characterization of OHOA-CA photosensitizer probe.Firstly,the cephalosporin was iodinated,so as to enhance the reactivity of cephalosporin.Then the reaction product reacts with OHOA to generate intermediate 1,and then the intermediate 1 is oxidized to generate intermediate 2,thus eliminating isomers and making it more stable.Finally,the intermediate 2 was deprotected to produce the final product OHOA-CA photosensitizer probe.Subsequently,intermediate 1,intermediate 2 and the final product OHOA-CA photosensitizer probe were detected by low resolution mass spectrometry,¹H NMR and ¹³C NMR to determine the final structure of OHOA-CA photosensitizer probe.（2）Photophysical properties of OHOA-CA photosensitizer probe.OHOA-CA photosensitizer probe with a fixed concentration andβ-lactamase with a fixed concentration were reacted at a fixed temperature for a period of time,The absorbance,absorption and emission spectra of OHOA-CA photosensitizer probe in aqueous solution were detected by ultraviolet spectrophotometer and fluorescence spectrophotometer;Reacting OHOA-CA photosensitizer probe with different concentrations ofβ-lactamase at a fixed temperature for a period of time,and recording the changes of fluorescence spectrum of OHOA-CA photosensitizer probe,so as to obtain the spectrum of the reaction ability of OHOA-CA photosensitizer probe withβ-lactamase;The fixed concentration of OHOA-CA photosensitizer probe is mixed with different potential interferents,such as inorganic salts,amino acids,oxidation/reduction small molecules,esterase,trypsin,metal matrix protease,etc.,and then the change of fluorescence intensity of OHOA-CA photosensitizer probe is detected by fluorescence spectrophotometer,so as to obtain the anti-interference ability data of OHOA-CA photosensitizer probe;The stability data of OHOA-CA photosensitizer probe were obtained by reacting OHOA-CA photosensitizer probe withβ-lactamase at fixed concentration at different temperatures and different p H values for a period of time,and recording the fluorescence spectrum changes of OHOA-CA photosensitizer probe;9,10-anthracenediamine malonic acid（ABDA）with a fixed concentration was compared with OHOA-CA photosensitizer probe with and withoutβ-lactamase,and the reaction was irradiated with 500 nm laser for a period of time,and the ultraviolet spectrum was detected by ultraviolet spectrophotometer,so as to detect singlet oxygen produced by the reaction of OHOA-CA photosensitizer probe withβ-lactamase.（3）Hydrolysis and spectral change mechanism of OHOA-CA photosensitizer probe.The affinity between OHOA-CA photosensitizer probe andβ-lactamase was verified by docking calculation with software Autodock 4.2 and Discovery Studio 2021;The spectral changes of the reaction between OHOA-CA photosensitizer probe andβ-lactamase were evaluated by time-dependent density generalized function theory calculation（TD-DFT）.（4）Bioimaging of OHOA-CA photosensitizer probe in vitro.Taking mouse fibroblast epithelial cell line（L929）as the experimental object,cell proliferation-toxicity test kit（CCK-8）was used to study the cytotoxicity and phototoxicity of OHOA-CA photosensitizer probe.Methicillin-resistant Staphylococcus aureus（MRSA）producingβ-lactamase was selected as the experimental object,Staphylococcus aureus（S.aureus）and Escherichia coli（E.coli）as the control,respectively,were incubated with OHOA-CA photosensitizer probe at a fixed concentration for a certain period of time,and photographed by confocal microscope to verify the specific biological imaging of OHOA-CA photosensitizer probe in MRSA.（5）In vitro antibacterial activity of OHOA-CA photosensitizer probe.Firstly,the dark toxicity of OHOA-CA photosensitizer probes to MRSA was verified.The OHOA-CA photosensitizer probes with different concentrations were incubated with MRSA in a volume of 1:1 for a period of time,placed in a shaker at 37℃for 2 hours,then diluted and coated with a certain concentration,placed in a shaker,and counted the bacterial colonies after 15 hours.Then,the photodynamic antibacterial effect was tested.OHOA-CA photosensitizer probes with different concentrations were incubated with MRSA,irradiated with 500 nm LED for a certain period of time,placed in a shaker at 37℃for 2 hours,diluted and coated with a certain concentration,placed in a shaker,and counted the number of bacterial colonies after 15 hours.The OHOA-CA photosensitizer probe with a fixed concentration was incubated with MRSA in a volume of 1:1 for a period of time,irradiated with 500 nm LED for different times,placed in a shaker at 37℃for 2 hours,diluted and coated with a certain concentration,placed in a shaker,and counted the bacterial colonies after 15hours.Results:（1）After OHOA-CA photosensitizer probe reacts withβ-lactamase,the fluorescence mother nucleus is released,and the fluorescence intensity is obviously enhanced.The ultraviolet also has obvious red shift.（2）The reaction between OHOA-CA photosensitizer probe andβ-lactamase is time-dependent,and the fluorescence intensity increases obviously with the extension of time.（3）With the increase ofβ-lactamase concentration,the fluorescence intensity of the reaction between OHOA-CA photosensitizer probe andβ-lactamase increased obviously.（4）OHOA-CA photosensitizer probe showed good specificity and stability forβ-lactamase.（5）OHOA-CA photosensitizer probe has good affinity withβ-lactamase.（6）When OHOA-CA photosensitizer probes are incubated withβ-lactamase,singlet oxygen will be produced under the irradiation of 500 nm LED.（7）OHOA-CA photosensitizer probe has good compatibility with normal tissues and will not damage normal tissues and cells.（8）OHOA-CA photosensitizer probe can specifically recognize MRSA expressingβ-lactamase and produce green fluorescence.OHOA-CA photosensitizer probe has good photodynamic antibacterial effect.Conclusion:（1）OHOA-CA photosensitizer probe can specifically detect MRSA expressingβ-lactamase.（2）OHOA-CA photosensitizer probe has selective photodynamic antibacterial effect on MRSA.