Fluorescent Probes For β-Lactamases And Their Application To Detect Drug-resistant Pathogenic Bacteria

β-Lactam antibiotics play a critical role in the treating infection diseases:millions of lives have been saved by these small molecules.However,as these drugs were used extensively in clinical,antibiotic resistant pathogens emerge and spread all over the world rapidly,which is mainly due to the production of β-lactamases,threating severely public health.Rapid detection of antibiotic resistance is crucial for the disease control.In this thesis,we focus on the development of highly specific and sensitive fluorescent probes for the detection of β-lactamases.In these studies,with the core structures of p-lactam antibiotics as enzymatic recognition moieties and a range of fluorophores as reporters,we have designed and synthesized a series of fluorescent probes,which exhibit high specificity to differentβ-lactamases.These fluorescent probes include covalently labeling probes CFC-1 and CFC-2,extend-spectrum β-lactamases-specific fluorescent probe CTX-HN,carbapenemase-selective probes CPC-1,CB-1.Most of these probes were thoroughly characterized by detecting different β-lactamases.Further investigation of these probes on clinically important antibiotic-resistant bacteria demonstrates their high potential in clinical use.This dissertation consists of five chapters.The first chapter introduces the background of research,including antibiotic resistance,β-lactamases and the current design of fluorescent probes for β-lactamases detection.In the second chapter,we detail the development of two self-immobilizing and fluorogenic probes for the detection of β-lactamase activity.These imaging reagents can covalently link to the target enzymes or nearby proteins upon hydrolysis by P-lactamases and turn on fluorescent signal.The covalent labeling of proteins was further confirmed by means of SDS-PAGE and MALDI-TOF MS.This probe was further applied successfully to the fluorescent imaging of a range of p-lactamases-expressing bacteria.The third chapter is mainly about a sensor CTX-HN used to detect extend-spectrumβ-lactamases(ESBLs)and carbapenemase.CTX-HN with a 4-hydroxylnaphthalimide as a ratiometric signal group is based on cephalosporin with a methoxylimino side-chain.The probe is ultra-sensitive respond to carbapenemase and ESBLs.This compound achieves the efficient detection of live clinical carbapenemase-or ESBLs-expressing bacteria.In the forth chapter,we use carbapenem core structure as recognition site of carbapenemase and add a cleavable dye into 2’-position of carbapenem as a signal group.Several probes were designed and synthesized,and further explored about their basic properties.As a result,probe CPC-1 with 7-hydroxylcoumarin as a reporter detects carbapenemase efficiently.It can selectively detect carbapeneamse-expressing clinical bacteria.The last part is about fluorescent probes CNT-1 and CB-1 designed and synthesized according to the principle of varied conjugation of a probe along with its signal changes.We have systematically researched their optical properties and respond capacity to β-lactamases.Among them,probe CB-1 exhibits excellent performance and solves some application problems of probe CPC-1.Furthermore,CB-1 is a small molecule fluorescent reagent remarkably specific and sensitive towards carbapenemase.It was demonstrated that CB-1 selectively responds to carbapenemase-producing organisms(CPOs)over other prevalentβ-lactamases-containing bacteria.