Lysosomes-targetable And Near-infrared Probes Based On The Thiolysis Of NBD Amine And Developing Labelling Methods Of Tyr And His

Fluorescence-based methods have recently been emerged as a firstly preferred approach for visualizing living processes because of its high sensitivity,suitability for high throughput screening and capacity for cell imaging.Developing bio-fluorescence labelling technique is an attracting target for visualizing living processes.The general problem of this is to find and use efficient reaction with bio-selectivity and compatibility to fluorescence labelling the target biomolecule.It is essential to develop specific bio-fluorescence labelling technique because of a variety of molecules with different chemical property in living system.This thesis aims at developing new bio-fluorescence labelling reactions and methods for H₂S and protein:?1?We designed and synthesized a new FRET-based ratiomatric fluorescent probe for visualization of H₂S in lysosomes based on the thiolysis of NBD amine.The probe displayed good water solubility,a marked yellow to blue emission color change in response of H₂S and high H₂S selectivity.Moreover,the probe could be used to detect H₂S in lysosomes with good biocompatibility.The prformance of the probe indicates the design strategy for organelle-targetable FRET-based ratiometric H₂S probes is rational.?2?A new NIR fluorescence probe based on the thiolysis of NBD amine was developed for the detection of endogenous H₂S in colorectal cancer cells in mice.The probe displayed an 87-fold fluorescence enhancement at 796 nm?with excitation at 730 nm?when reacted with H₂S in a buffer?pH 7.4?.The probe was water-soluble,cell-membrane-permeable,had low cytotoxicity and high selectivity and sensitivity for H₂S.The properties of the probe enable its use in monitoring endogenous H₂S in living cells,tissues,and mice.Our bioimaging results indicated that?a?D-Cys could induce endogenous H₂S production in living cells and stimulate angiogenesis;?b?tail intravenous injection of the probe into mice generated strong fluorescence in the liver while intraperitoneal injection of D-Cys could further enhance fluorescence in the liver in vivo;?c?importantly,endogenous H₂S in colorectal cancer cells?HCT116,HT29?in vitro and in murine tumor models could be quickly and selectively detected by intratumoral injection of the probe.These results indicated that our new probe could serve as an efficient tool for the detection of cellular H₂S in living animals and even for cancer diagnosis.?3?We developed a series of bench-stable diazonium salt labelling reagents for the modification of Tyr by using hexafluorophosphate as a counterion to stabilize the diazonium cation.Ketone,alkyne and tetrazine groups could be introduced to Tyr residue respectively by coupling reaction with these reagents and the proteins and TMV could be labelled by the following relevant bioorthogonal reaction.Moreover,we designed a cross-linker with two diazonium cation at its terminal which was intended to be utilized for preparing TMV-based hydrogel.The result of EP and TEM showed that this linker could cross-link the TMV efficiently.In terms of the modification of His,the strategy was based on the first application of the Chan-Lam reaction for chemical labelling of proteins.With successful optimization of the Chan-Lam coupling in aqueous buffer and confirmation its selectivity of imidazole group,the single and dual fluorescence labelling of proteins were realized.