Coumarin/Merocyanine Dyad Fluorophores:H₂S Sensing Behavior And Intracellular Imaging Application

Hydrogen sulfide(H2S),considered as the third endogenous signaling molecule after NO and CO,plays essential roles in different physiological processes.It was proposed as the cytoprotectant and gasotransmitter in many tissues.The most important function of hydrogen sulfide is mediating vascular tone in blood vessels as well as neuromodulation in the brain.The disorder of H2S was correlated to many diseases such as Alzheimer’s disease,Down’s syndrome,diabetes and cirrhosis of the liver.Therefore,understanding the exact physiological and pathological roles of H2S is attracting much more interests of scientists in the field of chemistry and biology,and the developing technique for quick and accurate detection of H2S in living systems are especially demanded by these studies.Fluorescence imaging of small biomolecules with the suitable sensor stands as one of the most reliable techniques to offer in situ information due to their high sensitivity,quick response and non-invasiveness.Therefore,the construction of fluorescent probes for H2S to realize the in situ detection of H2S in living systems is a hot topic in the field of sensing and imaging.Most reported probes for H2S function via a certain reaction between H2S and the probe,and the slow reaction makes these probes difficult to offer real time information of H2S.The construction of fluorescent probes of quick response and ratiometric sensing ability to realize the intracellular or even subcellular H2S imaging is still challenging.Our previous study has developed a coumarin/merocyanine hybrid fluorescent probe for H2S,CouMC.This probe displays not only the quick ratiometric sensing ability for H2S but also the fine mitochondria targeting ability.The latter modification of this probe found that the indole-N-substitution,such as indole-N-(N’-piperidinyl)propionamido-and indole-N-(N’-dodecyl)propionamido-groups,might lead to the distinct alteration of H2S sensing behavior.Focusing on this point,the following studies have been investigated in this thesis:Four coumarin/merocyanine dyad fluorophores of N-(N’-monoalkyl)propionamido-or N-(N’-dialkyl)propionamido-group,CM-N’1C2,CM-N’2C2,CM-N’1C12 and CM-N’2C12 were constructed.Absorption and emission spectroscopic study disclosed that CM-N’1C12 and CM-N’2C12 of the N-(N’-dodecyl)propionamido-or N-(N’-didodecyl)propionamido-group possess a quickly decreased emission band at 650 nm in the aqueous media,with CM-N’2C12 of the didodecyl group showing a more quick decrease.Therefore,the two compounds are not suitable for H2S sensing.However,CM-N’1C2 and CM-N’2C2 of the N-(N’-ethyl)propionamido-groups,display the stable emission and absorption spectra in aqueous media.This result implies that the longer alkyl chain in the N-(N’-alkyl)propionamido-group might lead to the quicker emission decrease at 650 nm of these dyad fluorophores.Both CM-N’1C2 and CM-N’2C2 display a quick hypsochromic emission shift from 650 to 500 nm upon H2S addition in 2 min.Their quick ratiometric sensing ability for H2S is not interfered by the other anions and nucleophiles in living systems.Confocal imaging demonstrated that CM-N’2C2 possesses the fine mitochondrial targeting ability,which CM-N’1C2 with only one ethyl group in the indole-N-substituent has no such a targeting ability.The ratiometric imaging results found both CM-N’1C2 and CM-N’2C2 are able to track the intracellular H2S deviation triggered by exogenous H2S addition.The above study implied that the lipophilicity of the indole-N-substituents might alter the H2S sensing behavior and intracellular distribution pattern of these coumarin/merocyanine dyad fluorophores.To remove the influence of N-propionamido-group,we simplify the model of these coumarin/merocyanine dyad fluorophores via replacing the indole-N-(N’-alkyl)propionamido-group with indole-N-alkyl group,and 7 dyad fluorophores were prepared.Spectroscopic study disclosed that CM-NC1,CM-NC4,CM-NC5,and CM-NC6 with the N-alkyl chain shorter than C8 all show the stable emission spectra in aqueous medium.Moreover,they all display the fine ratiometric sensing ability specifically for H2S in aqueous medium,similar to that of CM-N’1C2 and CM-N’2C2.This sensing behavior is also not interfered by the normal anions and nucleophiles in living systems,such as Cys,GSH and Hcy.Moreover,the longer N-alkyl chain will lead to the quicker sensing response to H2S.Among these probes,CM-NC6 of the N-hexyl group demonstrates the quickest response of 40s.The mitochondria co-localization indicated that the compound possesses the mitochondria targeting ability only when their N-alkyl chain attains to C4.All the four compounds are able to sense the intracellular H2S deviation triggered by the exogenous H2S addition via a dual channel ratiometric mode,and the related ratio enhancement factor can attain to 5-fold.When N-alkyl chain attains to C8,their emission band at 650 nm will decrease with the time in aqueous media,and the longer chain will lead to the quicker decrease.In fact,the emission band(650 nm)of CM-NC16 with the N-hexadecyl group disappears almost instantly in PBS buffer.The current study suggested that the instable emission band of these coumarin/merocyanine dyad flurophores of long alkyl chain might originated form the aggregation of the fluorophore in aqueous media via the intensive hydrophobic interaction between the long alkyl chains of these molecules.