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

Bioluminescence is a phenomenon that the chemical energy of chemical substances can be converted into light energy under the action of internal enzymes,which does not require external light sources.Bioluminescence exists widely in the biological organisms of nature,including bacteria,insects and marine organisms,and firely luciferase system is the most widely used.Bioluminescent imaging(BLI)is a new imaging technology in vivo based on the visible light produced by the reaction between luciferase and substrate.BLI technology has the advantages of simple operation,intuitionistic results,high specificity,high sensitivity and small damage to the body.It should be noted that this technique has been widely used in a number of important fields,including medicine,biochemistry,molecular biology and microbiology,so that plays a crucial role in tracing tumor growth and drug metastasis,high-throughput screening,gene therapy,target gene expressiondetection,stem cell labeling and protein-protein interaction and other fields.This thesis is mainly divided into three parts:the first part is to further explore the naphthenic hydrocarbon size on the activity,in which we synthesized two macrocyclic cycloalkylation substrate with D-Luc and aminoluciferin as the control group to detect substrate activity.In the second part,a bioluminescent probe was prepared for the detection of endogenous hypochlorite in an inflammatory model.In the third part,five bioluminescence probes for the detection of tyrosinase were well designed and synthesized.The first part focuses the macrocyclic cycloalkylation substrate.Based on the amino naphthenic reaction before the laboratory,two macrocyclic cycloalkylation substrates were further synthesized.The effect of naphthenic substrate on activity was investigated and compared with natural luciferin and aminoluciferin at the enzyme level and cell level in vitro.The results displayed that substrates 079-045 and 079-066 in cells level remained lower luminous intensity than substrates aminoluciferin and D-luc,and among which,the best substrate 079-066 in 20 ?M is 1/4 luminous intensity of aminoluciferin,1/8 luminous intensity of D-luc,and in vitro level the light intensity of novel substrate and control substrates have a bigger gap.The second part is the design of hypochlorite probes,two different recognition groups are used:thiocarbamate and aminophenol,in which the former one is based on the affinity of Cl+ to sulfur,and the latter one is proved to be a recognition group for detecting hypochlorite.In the natural luciferin,it is better to introduce the recognition group to the 6-hydroxyl group.It is proposed that these two probes can selectively release hydroxyl luciferin through the breakdown of hypochlorite to produce bioluminescence under the condition of firefly luciferase.After experiment,the thiocarbamate as recognition group in vitro on hypochlorite demonstrated high selectivity and sensitivity,and in a inflammation mouse model that can increase hypochlorite in vivo,this probe achieves the sensitive detection of endogenous hypochlorite.The third part is the design of tyrosinase bioluminescent probe.Compared with fluorescence imaging,bioluminescence imaging has no need of excitation light,low background and feasible animal imaging.Herein,we designed two different recognition groups to cage different position of luciferin,so that it can be hydrolyzed by tyrosinase to release the substrate luciferin.It is found that probe 079-067 has high sensitivity and selectivity in vitro so that it is a poten bioluminescent probe for detecting tyrosinase in cells.