Construction Of Photo-Controlled Ratiometric Fluorescent Probe For Application In Spatio-Temporally Resolved Imaging Of Tyrosinase

Tyrosinase has a unique catalytic function,which can hydroxylate monophenols(such as tyrosine)to catechol,which is then oxidized to the corresponding phthaloquinone product with the assistance of molecular oxygen.As a key factor in melanin synthesis in vivo,its abnormal activity can lead to an imbalance in melanin metabolism,thereby increasing the risk of diseases such as melanoma,epilepsy,and vitiligo.Therefore,it is of great significance to develop a molecular tool that can accurately detect endogenous tyrosinase.In recent years,more and more ratiometric fluorescent probes have been developed for the imaging analysis of tyrosinases in cells and living organisms,but the enzyme recognition sites are exposed,thus leading to a decrease in detection accuracy and the inability to achieve spatial and temporal artificially controlled monitoring of tyrosinases at specific locations.To address this aspect,this thesis designs and synthesizes a class of light-controlled tyrosinase ratiometric fluorescent probes based on a light-controlled molecular recognition strategy for the imaging analysis of endogenous tyrosinase activity in cells,zebrafish,and melanosomes.Two specific works were carried out as follows:(1)Photo-controlled tyrosinase ratiometric fluorescent probe imaging study.A light-controlled ratiometric tyrosinase fluorescent probe SRFP was designed and synthesized by coupling the light-controlled group 2-nitrobenzyl to the tyrosinase recognition site 3-hydroxybenzoxy as a response unit and introducing it to the water-soluble aminotriglycol monomethyl ether at both ends of the naphthoylimide fluorophore with ratiometric properties.When no light treatment is performed,the probe cannot recognize tyrosinase due to the site-blocking effect,and only when the substrate is released after light exposure can the probe respond to tyrosinase.This design can avoid false-positive signals due to early activation of the probe during entry into the cell,and achieve light-controlled identification and precise imaging analysis of tyrosinase in cells and zebrafish,and is expected to be a useful tool for diagnosis and drug screening of diseases related to tyrosinase concentration abnormalities.(2)Melanosome-targeted light-controlled fluorescent probes and their application to in situ monitoring of tyrosinase.The melanosome is the only place where melanin is synthesized,and tyrosinase is one of the necessary factors in its synthesis.Therefore,based on the good results of the above probe for endogenous tyrosinase photocontrol recognition imaging,we introduced the morpholine moiety with melanosome targeting ability into it and further designed a photocontrol tyrosinase fluorescent probe MTFP targeting melanosomes,and successfully applied it for in situ monitoring imaging of tyrosinase activity on cells and melanosomes.