Optimization For Bioluminescence System:Design,Synthesis And Evaluation Of Novel Bioluminescent Substrates And Caged Bioluminescent Probes

Bioluminescence,is a natural phenomenon and a particular kind of chemiluminescence that emits visible light resulted from the corresponding luciferase luciferin biological reaction.Bioluminescent technology such as BRET,bioluminescent imaging,and dual-luciferase reporter,has catched more and more scientists' eyes owing to its broaden application in the investigation and imaging of biological process in vitro and in vivo.The firefly,coelenterazine and bacteria system are three main bioluminescence systems involved in bioluminescence technologies.Bioluminescence emission does not demand an excitation light source compared with fluorescence,which means that bioluminescence involved method has much lower background noise interference.Coelenterazine(CTZ)act as a famous widespread luciferin that is used by various marine luciferases from Renilla,Gaussia,Metridia,et al.Compared with firefly luciferin-luciferase system,bacterial bioluminescence system,CTZ-luciferase system is the simplest bioluminescent system.Coelenterazine involved bioluminescence mechanism is that biocatalysis of coelenterazine by luciferase yields strong peak emission around 480 nm without any co-factor.The Renilla luciferase is the best-known luciferase for coelenterazine.Besides,luminous bacteria are widespread in the marine surroundings and three major luminous bacteria are Photobacterium,Vibrio and Photorhabdus(Xenorhabdus).The bacterial bioluminescence reaction mechanism is that a long-chain aliphatic(C10-C12)aldehyde and reduced flavin are oxidized in the presence of luciferase.In the current study,in order to expand the application of bioluminescent methods and techniques,we intend to optimize the bioluminescence system by developing the novel analogs or caged probes.For coelenterazine-Renilla luciferase system,we designed and synthesized three series(B,A and T)of novel substrates with extended substituents at the C-6 site of the structural imidazopyrazinone core based on coelenterazine analog DeepBlueC and investigated their bioluminescence properties.After investigation in vitro and in vivo,some of them such as compound B2,B5,B12,B9,especially B2 display excellent bioluminescence properties such as stronger bioluminsencent intensity,longer bioluminescence emission,better kinetic characteristics and more red-shifed emission wavelength compared with DeepBlueC or coelenterazine,which means that these derivatives can become the most ideal luciferin candidates for Renilla system-involved bioluminescence techniques or further being modified to become probes to figure out some biological process in relevant fields.Series A and T do not have good preformence like series B.However,compounds A4 and A6 have brighter bioluminescence intensity and longer lasting emission in the cellular level than DeepBlueC.T series compounds do not have the properties to become the substrates of Renilla luciferase.In structure-activity relationship of series B compounds,the data demonstrated that the introduction of electron-rich functional groups with suitable molecular sizes into the C-6 site could improve the recognition and interaction with Renilla luciferase.What's more,it is important that the appropriate lipid-water partition coefficient guarantees beautiful bioluminescence imaging in cellulo and in vivo.In the case of series A,the introduction of the alkynyl bond could result in the enhancement of molecular rigidity so as to probably influence the entrance into the luciferase pocket and interaction with active sites of enzyme.The triazole as the bioisostere of benzene,was introduced in the position of C-6 of T series to weaken the bioluminescence.Coelenterazine is also the substrate for Gaussia luciferase.Therefore,we examined all three series of compounds'abilities to response to Gaussia luciferase.Overall,only a few of them have response to Gaussia luciferase.However,B series' performances are better than A and T series.Compound B2 is also the best one with higher bioluminescence than DeepBlueC,but it is not superior to coelenterzine.These interesting results indicated that most of them are not suitable to be substrates for Gaussia luciferase.For bacterial bioluminescence system,we designed and synthesized the first caged bioluminescent probe for mercury(?)discovery on the basis of the deprotection of the dithioacetal in the presence of mercury(?).The experimental results indicated this probe performed selective and sensitive "turn-on" response to mercury(?)over other ions.What's more,this bioluminescent probe has the ability of triggering the linear bioluminescence signal in response to the mercury(?)in the certain range of concentrations.The results suggested that it might become a novel method for mercury(?)bioluminescent imaging in biological area and environmental.What's more,we designed and synthesized several new substrates because of seldom work has been done in developing the new substrates for bacterial bioluminescence system.And we found these molecules 9a,9b,5a and 5b have less bioluminescence intensity but longer half decay time compared with origrial substrate decanal,which inditates these molecules are promising candidates for bacterial bioluminescence.In summary of structure-activity relationship,compound 5a and 5b behaved higher bioluminescent intensities and better kinetic properties than 9a and 9b.So these results suggested that the introduction of 1,8-naphthalimide group was more appropriate than 2,1,3-benzoxadiazol group.And 5b was always to exceed to 5a,and 9b seemed always to exceed than 9a.It is interesting that the results disclosure that 12 carbon aliphatic chain length was suitable to interact with the luciferase.In conclusion,herein we made modification of luciferin in coelenterazine-Renilla luciferase system and bacterial bioluminescence system in order to obtain high intensity,reasonable kinetic characteristics,long emission and red-shifted wavelength substrates with related luciferase.We believe that our compounds can enrich the chemodiversity of Coelenterazine-type substrates that are beneficial for the development of novel Coelenterazine-type molecules with superior bioluminescent properties.Moreover,we developed the first bioluminescence probe for the mercury(?)analysis on the basis of bacterial luciferin,thus providing valuable knowledge for further research and application in bioluminescence techniques.We further investigated the structural modification of the long-chain aliphatic aldehyde being as bacterial bioluminescent substrates,which interact with bacterial luciferase to emit obvious bioluminescence.In brief,our study will be helpful to guide the future development and application of novel bacterial bioluminescent substrates with superior bioluminescent properties.