Synthesis Of Enzyme-activated Fluorescent Probes And Its Application In Diagnosis Of Bacterial Infections

Bacterial infections are one of the leading causes of global disease and death.According to statistics,nearly a quarter of all deaths worldwide are caused by bacterial infections.However,in recent years,the widespread use(or even abuse)of antibiotics has led to the emergence of resistant bacteria and even super bacteria,which have re-spreaded infectious diseases that have been effectively contained.The diagnosis of bacterial infection is the most direct and effective way to inhibit the abuse of antibiotics.However,the diagnosis of bacterial infection is not the most important detection method in clinical medicine.This is because the detection sensitivity of bacterial infection is low at present,and the traditional detection method is difficult to distinguish between bacterial inflammation and sterile inflammation,so develop efficient technology.It is very urgent to diagnose bacterial infections.After years of rapid development,molecular imaging technology has become one of the important means of disease detection and diagnosis.It is a very urgent task to develop medical molecular probes with excellent performance,low price and independent intellectual property rights in China.An enzyme-activated fluorescent probe that catalyzes a fluorescent probe by an enzyme present in an organism,thereby enabling rapid fluorescence generation.Such probes have low background noise and high imaging signal-to-noise ratio for monitoring in vivo reactions.The process provides a common platform.The Cyanine(Cy)series of dyes have the advantages of a large molar extinction coefficient and a wide absorption wavelength range,making it possible to identify bacterial activation probes by fluorescence microscopy to diagnose bacterial infections.However,we have found that conventionally used Cy5 dyes are difficult to penetrate bacterial cell membranes.Therefore,the development of Cy series enzyme-activated fluorescent molecular probes capable of penetrating bacterial cell membranes is of great significance for the diagnosis of bacterial infections.In order to increase the permeability of the probe molecule to the bacterial cell membrane,we have proposed a strategy of introducing a lipophilic group into the probe molecule to enhance the membrane permeability of the probe molecule.The application of the nitroreductase-activated Cy series probe in the diagnosis of bacterial infection was realized.We designed and synthesized Cy5 series fluorescent probes and tested their optical properties and biological applications to identify bacterial nitroreductase(NTR).We introduced nitro-lipophilic groups into the Riand R2 positions of Cy5,respectively,and synthesized five fluorescent probes,in which probe 3 and probe 5 produced a rapid 10-fold fluorescence response after catalytic reduction by nitroreductase.In addition,these two probes can penetrate the bacterial cell membrane of Gram-negative bacteria as well as Gram-positive bacteria.These two probes exhibited the best enzyme activation at 37℃ and pH=7.4 and showed good selection specificity for nitroreductase.And when incubated with bacteria,the probe is time-dependent on the nitroreductase in the bacteria,and finally we successfully applied probe 3 and probe 5 to live cell imaging studies of various bacteria.