| Bioluminescence is a common phenomenon for living organisms as producing luminescence and the essence of the process is caused by chemical reaction in vivo.Many kinds of creatures in nature can produce bioluminescence from protists to lower vertebrates,such as mica,bacteria,fireflies,etc.The firefly luciferase-luciferin system is one of the earliest and widely used among all bioluminescence systems.In the presence of O2,Mg2+and ATP,the luciferin can be catalyzed and oxidized by firefly luciferase,and then to emit yellow-green light at about 560 nm wavelength.Another kind of bioluminescence system that is widely studied and applied is the coelenterazine-luciferase system.Coelenterazine is a natural luminescent substrate of marine organisms,such as Jellyfish and sea pansy,which could be catalyzed and oxidized by renilla luciferase in the presence of O2 to emit blue light at around 480 nm wavelength.Using the visible bioluminescent signal produced by redox reactions of luciferase and luciferin,bioluminescent imaging(BLI)is a consistently sensitive,specifical,simple and non-invasive imaging technique that has been comprehensively applied for monitoring a myriad of life processes,including cell proliferation and migration,tumors growth,enzyme activities,protein-protein interactions and other uses.The current research is mainly divided into two parts:Firstly,we designed and synthesized three light-activated renilla luciferase substrates on the basis of the concept of photoactivatable molecules,then evaluated their biological activities.Secondly,we synthesized eight different luciferin derivatives based on different firefly luciferase substrates by using the prodrug design strategy,then evaluated their biological activities.Part I:We designed and synthesized three light-activated renilla luciferase substrates and systematically evaluated their bioactivity in buffer,cells and living animals.A kind of photoactivatable molecules are usually composed of photolabile groups and protected molecules.These protected molecules generally have vital biological functions and lose their activity by the introduction of light-sensitive protecting groups.However,photoactivatable molecules could release the protected molecules to exert its physiological effects when they are activated by appropriate wavelength of light.Light-directed technology,which has been developed by controlling photoactivatable molecules,has become an indispensable tool for a wide range of cellular and biological studies such as photo-activated proteins,neurotransmitters,mRNAs and nucleic acids.In this section,three photoactivatable molecules that cannot produce bioluminescence were designed and synthesized.All of them could release the free luciferin that could emit a bioluminescent signal in vitro and in vivo upon UV irradiation.As a result,they can be used as potential bioluminescent substrates and expand its application in bioluminescent imaging.Part ?:We designed and synthesized eight caged luciferin derivatives on the basis of three different firefly luciferase substrates and then investigated their activity.In order to prolong the luminescence time of luciferase substrates,we used the prodrug strategy to introduce protecting groups into the carboxy groups of D-luciferin,aminoluciferin and N-cyclobutylaminoluciferin respectively and then obtained the products.These caged luciferin derivatives cannot be recognized by firefly luciferase.However,they can hydrolyze in solution or in cells to release the free luciferin that could emit a realistic signal in the presence of firefly luciferase,ATP,Mg2+,and O2.Owing to the presence of protecting groups,these caged luciferin derivatives significantly prolonged the time of bioluminescence in cells.Moreover,the light signal of the compound of 110-50 could be detected after 6 h,and therefore,it is more suitable for stable and long-time imaging study.And our studies provide helpful information for further research and application on the bioluminescent system. |
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