Synthesis Of Sialidase Targeting Probe And Its Biological Application

Sialosides,typically located at the termini of cell surface oligosaccharides,participate in diverse physiological processes including cell recognition,cell adhesion and intercellular signalling,and viral infection.Relative to cell sialylation,sialidase(also known as sialic acid hydrolase)-mediated desialylation mediates a set of distinct biological processes,ranging from inflammation to tumor invasion,and to sepsis.As such,chemical tools allowing real time imaging/tracking of sialidases activity in living cells are of significance to investigate the patho-physiological roles of sialidases.This dissertation is dedicated to development and evaluation of sialidase probes for imaging intracellular sialidases.The specific topics include:Chapter one introduces the biological roles of sialic acids and sialidase.s,and existing strategies for design of glycosidase probes.In the second chapter,we designed an enzyme inhibitor-based pH-responsive polymeric probe(Rb-CS@ZA),which exploits Zanamivir,a commercial drug against influenza sialidases,to target sialidases on mammalian cell surfaces.Rb-CS@ZA consists of a polymer backbone of poly(styrene-alter-maleic acid),a Zanamivir derivative(ZA),a red rhodamibe-B fluorophore(RB-Pip),and a pH-responsive near-infrared profluorophore(CS-EDA).The aim to achieve multivalent interactions between Rb-CS@ZA and cell surface sialidases,and thus to increasing probe affinity for sialidase-expressing cells.RB-Pip exhibits "always-on"bright red fluorescence,which is used for long-term tracking of the polymeric probe whereas CS-EDA allows sesnsing of acidic pH during probe internalization into cell lysosomes.This probe is currently being evaluated for imaging of cell surface sialidases on a number of cancer cells via sialidase-mediated binding,cell endocytosis and fluorescence activatiob inside lysosomesIn chapter 3,we constructed a substrate-based small molecule probe of sialidases.termed Sia-RQ.The probe includes a sialoside moiety for sialidase recognition,a fluorescence quencher BHQ,and a moiety of rhodmaine-X(Rox)with bright red fluorescence.Non-fluorescent Sia-RQ releases BHQ moiety upon sialidase hydrolyis,giving rise to a dye-labelled chemically reactive quinone methide which is covalently captured by sialidases and proteins in vicinity.The fluorescence labelling of sialidases and nearby proteins allow fluorescence-on imaging of sialidases in live cells.With Sia-RQ,the levels of sialidase in cell senescence are successfully imaged.These findings clearly established the use of over-expressed sialidase-1(NEU-1)as a new biomarker for senescent cells.In summary,we synthesized two sets of sialidase-targeting probes,which would be of broad utility for interrogating myriad patho-physiological events associated with sialidases such as inflammation,tumor development and viral infection.