Design, Synthesis And Evaluation Of Bioluminescent Probes And Substrates Of Firefly Luciferase

Bioluminescence is a special kind of chemiluminiscence in living organism, which is a high efficient process that converts biochemical energy to light energy without absorption of external light. Because of being noninvasive, high sensitivity, visualization and real-time in detection, the bioluminescence imaging has been widely used in various fields, which is becoming one of the most imaging technologies in vivo.As the most studied bioluminescent system, the firefly luciferin-luciferase system is widely used in bioluminescence imaging. Owing to the good high-solubility, efficient response and strong bioluminescent intensity, firefly luciferin(D-lucferin) is one of the most important probe in luciferin-luciferase system. D-lucferin can be oxidated by firefly luciferase in the presence of ATP, Mg2+ and oxygen, subsequently to release oxidizing luciferin, AMP, CO2 as well as to produce visible light.Based on the structure of firefly luciferin, this topic contains two parts:1. One is the design, synthesis and evaluation of three species small active molecules which are hydrogen peroxide, hydrogen sulfide and fluorine ion bioluminescence probes. These small active molecules often have significant roles in mediating redox modifications and other essential physiological processes. Considering that H2O2 is a substantial member of ROS, detection and quantitation of H2O2 undertakes important but urgent responsibility. Fluorine ion is the smallest anion in the natural world. It is one of the essential microelement for human body, which can prevent dental caries and treat osteoporosis as a drug. However, one of the reasons of the teeth and bone poisoning is high fluorine concentration; hydrogen sulfide is one of gas signal molecules for the regulation in Blood vessels, nerves, immune system. These caged luciferin probes reported herein include two species with self-immolative linker or without. In addition, all probes contain two common parts:a recognized grope and a bioluminescent luciferin moiety. These probes are some potential bioluminescent sensors for small active molecules since it can afford a unique technology for real-time detection of basal levels of small active molecules generated in vitro and in vivo.2. In order to enhance the bioluminescence sensitvity, a series of N-cycloalkylaminoluciferins (cLucs) are well developed by introducing lipophilic N-cycloalkylated substitution. The experimental results evidently demonstrate that these cLucs are competent substrates for native Fluc that can demonstrate strong bioluminescent signal in vitro, in cellulo and in vivo. In addition, the red-shifted bioluminescent emission, longer emit detectable bioluminescence time, capacity of accessing blood brain barrier and increasing cell permeability of cLucs are of significance for penetrating tissues in live animal imaging. These novel firefly luciferase substrates will expand the imaging toolkit and inspire new applications of bioluminescence technology.