Design,Synthesis And Evaluation Of Renilla Luciferase Bioluminescent Probes

Bioluminescence is a special type of chemiluminescence that exists in living or-ganisms.It doesn't rely on excitation light;under the catalysis of enzymes chemical substances convert the chemical energy into light energy efficiently.The biolumines-cence imaging has been widely applied in various fields due to its advantages of non-invasiveness,highly sensitivity and selectivity,visualization,and real-time mon-itoring.Based on bioluminescence,many kinds of analyzing tools have gradually been developed and some of them have been successfully applied to actual produc-tion.The current bioluminescence system mainly includes firefly luciferin system,coelenterazine luciferase system and bacteria LuxAB system.The coelenterazine sub-stratesare widely distributed and can be catalyzed by various luciferases to emit blue light.It is worth noted that such asystem doesn't require other cofactors.The coelen-terazine luciferase system is relatively simple,well biocompatiblility,low background,and less interference,and all of which is favorable to monitor physiological and pathological processes in vivo.The most studied coelenterazine luciferase is Renilla luciferase.Under oxygen conditions,the coelenterazine substrate is oxidized and cy-clized to form an unstable four-membered ring,which is rapidly cleaved to produce CO2 and excited amide derivatives.In such a process,oxidized luciferin can be trans-ited from the excited state to the ground state so that the excess energy can release in the form of photons.In this project,two kinds of bioluminescent probes,including nitroreductase probes and hydrogen sulfide probes were well designed based on the coelenterazine analogues.This thesis mainly includes four parts,including preface,preparation of Renilla luciferase substrates,development of nitroreductase probe,and design,syn-thesis and evaluation of hydrogen sulfide probe.Hypoxia usually leads to increasing nitroreductase in tumor.In the absence of oxygen,reduced nicotinamide adenine dinucleotide(NADH)acts as an electron donor.Nitroreductase can reduce a variety of exogenous nitroaromatic compounds,which are formed hydroxylamines or amino groups.Based on this reduction behavior,we designed nitroreductase bioluminescent probes to detect hypoxia state in solid tumors.We introduced 4-nitrobenzyl or 2-nitrobenzyl at carbonyl group to cage substrates.When nitro group is reduced to amino group,rearrangement elimination reaction fol-lowed,the substrates were released and catalyzed to emit light.Nitroreductase was detected by bioluminescence imaging.The results depicted that the bioluminescence intensity of probes(Al,A2,and A5)exhibited a good linear relationship with the concentration of nitroreductase.When the concentration of other revelent substances is over 100-fold than that of nitroreductase,the bioluminescence intensity of probes(A1,A2 and A5)is three times higher than that of other revelent substances.As a result,these bioluminescent probes have highly selectivity and can detect nitroreductase-qualitatively and quantitatively in vitro.Among all probes,probe A5 performed good biocompatibility and could be applied to detect endogenous nitroreductase in cells.Compared to the control group,the tumor bioluminescence intensity increased 7-fold for A5.Therefore,our designed nitroreductase probes can be suitable for detecting tumor in vivo.Hydrogen sulfide plays an important rolein regulating blood vessels,nerves,and the immune system as a signal molecule.Once cells couldn't adjust hydrogen sulfide concentration into normal level,various diseases will occur.In order to understand the production,transport,and metabolism of hydrogen sulfide,we developed a new method of detecting hydrogen sulfide,in which coelenterazine substrates were caged by 4-azidobenzyl group.When the azide group was reduced by hydrogen sulfide formed amino group,rearrangement elimination reaction occurred,luciferasecata-lyzed substrates to emit light.At the molecular level,the probe showed excellent se-lectivity and sensitivity.Under the same conditions,the bioluminescence intensity of hydrogen sulfide probes is over 3 folds than that of other revelent substances.The probes also showed good detection performance in cell.In vivo,the bioluminescence intensity of C1 was 4 times than that of the control group.Therefore,our designed bioluminescent hydrogen sulfide probes provide a new method of detecting hydrogen sulfide.This is the first approach that nitroreductase and hydrogen sulfide probe have been designed based on the coelenterazine luciferase system.The probes have been successfully applied for detecting nitroreductase and hydrogen sulfide in solution,cells and organisms.Overall,they can provide a new method for studying the related physiological and pathological processes.