| Bioluminescence is a natural phenomenon that the chemical substance in the body is catalysed and oxidated under the action of enzymes to produce visible light.This process does not depend on the body's absorption of light,but transforms the biological energy into light energy directly.In the meanwhile,chemiluminescence only relies on chemical reactions to produce visible light in the absence of a specific enzyme.Bioluminescence is widely found in natural biological organisms,including bacteria,insects and marine organisms.Bioluminescent imaging(BLI)is a new imaging technique that can monitor the activity and behavior of luciferase-expressing cells or genes in living organisms by sensitive optical detection instruments with the advantages of quick and easy operation,high.sensitivity,real-time dynamic observation and non-invasive feature and therefore,it has an irreplaceable technological benefit in the areas of tumor growth monitoring and metastasis tracing,detection of target gene expression,protein-protein interaction,high-throughput screening and intracellular ATP level detection.As one of the common bioluminescence systems,renilla luciferase luminescence system requires renilla luciferase,coelenterazine and oxygen to produce a visible light.It should be noted that renilla luciferase system is too simple to need ATP,Mg2+ and other cofactors.Moreover,renilla luciferase can be expressed in mammalian cells with low cytotoxicity.However,if widely used,it is necessary to overcome the following shortcomings:firstly,the emission wavelength is so short(450-475 nm)that can easily be absorbed by the body organization,which is not conducive to animal imaging;secondly,the low stability of coelenterazine can lead to unnecessary chemiluminescence,thus raising the background signal.At present,even if scientists have modified the C-2,C-5,C-6 and C-8 positions of coelenterazine,there still are few reasonable substrates with superior properties,which may be mostly caused by that the modified coelenterazine analogues were not well recognized by the renilla luciferase.DeepBlueCTM is a commercially available coelenterazine analog that has been well used in bioluminescence resonance energy transfer studies with simple structure.Therefore,scientists often consider this compound as reference for structural modifications.Our research is divided into three parts:firstly,structural modification to obtain better coelenterazine analogues;secondly,developing the precursors of coelenterazine analogues to improve stability;and finally designing and synthesizing a bioluminescent and chemiluminescent probe for the detection of thiophenol.It needs to be known that our coherent study is very important for improving renilla bioluminescence imaging technology.Part 1(substrate modification):We performed structural modification for DeepBlueCTM to obtain the red-shifted and stable renilla luciferase bioluminescent substrates.We synthesized oxygen-containing and sulfur-containing coelenterazine analogs,because both oxygen and sulfur have similar properties to form p-?conjugation with the heterocyclic ring,which can lead to bathochromic emission wavelength.We then applied the purified renilla luciferase to study the bioactivity of these compounds in vitro and in cellulo.The experimental results demonstrated that such oxygen-containing coelenterazine analogue exhibited a greater red shift wavelength(63 nm)with decreased quantum yield.Moreover,oxygen-containing coelenterazine analogue at the cellular level exhibited a low luminous intensity,while sulfur-containing coelenterazine analog had a strong performance.Part 2(design of coelenterazine ester derivatives):To improve the stability of the coelenterazine analogue and prolong the luminescence time,we introduced the protecting groups at the 3-position carbonyl group by forming carboxylic ester or carbonic ester structures.Such a protecting group can be hydrolyzed by esterase,lipase,or nucleophilic group in the cell,thereby releasing the sulfur-containing coelenterazine analog,which reacts with intracellular renilla luciferase to produce light.Protected coelenterazine analogs can not be directly recognized by renilla luciferase,and therefore it can not produce light.We synthesized a total of 10 coelenterazine ester derivatives.Different blocking of the protective group leads to varied continuous luminescence time in the cell.In addition,we constructed a model of nude mice underarm transplantation tumor to conduct studies in vivo.The results showed that these compounds could prolong the time of bioluminescence.Part 3(design of thiophenol probe):Compared with fluorescence imaging,bioluminescence imaging has certain advantages:no need of light source,low background signal,good biocompatibility and high sensitivity.Therefore,probes based on bioluminescence have become a research hotspot.At present,most of the bioluminescent probes are based on the modification of the firefly luciferin structure,but the bioluminescent probes based on the renilla luciferase system are few.Previously,other research groups have developed a bioluminescent probe for the detection of ?-galactosidase,which introduced a glycosidic bond on the 3-position carbonyl group of coelenterazine.Based on the same principle,we designed and synthesized a bioluminescent and chemiluminescent probe for the detection of toxic substance thiophenols.We introduced 2,4-dinitrophenyl ether at the 3-position carbonyl group of the sulfur-containing coelenterazine analogue,which could be selectively removed by the strong nucleophilic thiophenol,then releasing the sulfur-containing coelenterazine analogue.Coelenterazine and its analogs not only produce bioluminescence,but also generate chemiluminescence in vitro when oxidized by some substances such as DMSO.The probe has high sensitivity and high selectivity,and can detect toxic substance thiophenol in aqueous solution,cells or plasma.So it is a potential bioluminescent and chemiluminescent probe.In summary,in order to find a high quantum yield and bathochromic renilla luciferase luminescent substrate,we introduced a sulfur atom in C-8 of the DeepBlueCTM,thus forming p-? conjugation.This change is very small,as far as possible to ensure the recognition of substrate and enzyme.Then we have demonstrated this idea through in vitro enzyme and cellular studies.The 3-position carbonyl group of coelenterazine is a key active site for its bioluminescence and chemiluminescence.If the carbonyl group is first protected and then released under specific conditions,the stability of the coelenterazine and its analogues can be improved.This idea is consistent with the prodrug principle of the pharmacy.Based on this principle,we designed 10 ester compounds capable of slowly releasing coelenterazine analog and a luminescent probe capable of detecting the toxic substance thiophenol,followed by in vitro and in vivo evaluation of these compounds.Luciferin-luciferase bioluminescent systems have been widely used in the field of chemical biology.However,as an important member of bioluminescence,renilla luciferase luminescence system requires further research and development.Moreover,such a strategy by introducting prodrug principle to bioluminescence is of significance for the application and promotion of renilla luciferase luminescence system... |
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