Design And Imaging Of Two-photo Fluorescence Probe For Hydrogen Sulfide And The Related Enzyme Activity

Hydrogen sulfide(H2S),as an important gaseous transmitter molecule,plays an important role in the life activity.The production of H2S in organisms can occue mainly through enzymatic reaction.The procedure is cysteine sulfur ether beta synthase(CBS)or cysteine sulfur ether gamma lyase(CSE)catalyzed the L-cysteine.Howerer,once the activity of CBS/CSE changes,which would seriously impact the physiological concentration of H2S,will lead to all kinds of diseases.Therefore,it is becoming very important to accurately monitor the concentration of H2S and the relative physiological mechanisms.This requires the researchers not only to precisely track the physiological concentrations changes of H2S,it remain unsolved that real-time monitoring the activity of CBS/CSE.The chemists have developed a variety of fluorescent probes for monitoring the H2S in recent years.However,most of the reported H2S probes were designed based on single photon method,can not realize their application in complex system.Though two-photon(TP)fluorescent probes are favorable as powerful molecular tools for imaging in biology,they display a delayed response time,always more than 30 min,to H2S,which is not suitable for real-time imaging of quick H2S-related biological processes since metabolism of H2S is very fast.Besides,developing a probe used to detect the activity of CBS/CSE for further understanding of the physiological mechanism of H2S has important value,as the H2S has obvious relevance to the activity of CSE and CBS.But,the methods for detecting the CBS/CSE is mainly focused on the isotope labeling,colorimetric and spectrophotometric,which have some inherent defects,such as cumbersome,complex procedures and low sensitivity,etc.,just for in vitro detection as it can not be readily able to realize real-time,non-destructive and in situ detection.Therefore,it is very important to develop a method that can achieve the real-time and non-destructive detection of the activity of CBS/CSE in vivo.In view of this above problems and based on the obvious advantages,such as high resolution and tissue penetration,of two photon fluorescence analysis method,the detailed works are listed as below.(1)Given the excellent two-photon absorption action cross sections of the coumarin derivative,a new type of two-photon fluorescent probe TPP-H2S was developed.In order to improve the reaction rate of the TPP-H2S with H2S,NBD-C1 group is introduced.As the strong electron withdrawing of NBD-C1,the linker group O possesses partial positive charge which provides a good nucleophilic sites.The nucleophilic aromatic substitutions reaction of TPP-H2S and H2S can be completed quickly within two minutes because of the strong nucleophilic of H2S.In addition,Based on the good internal charge transfer of NBD-C1 to the coumarin,TPP-H2S gives silent fluorescence.The hydroxyl is released and coumarin shows both one-photon and two photon excited fluorescence enhancement only when the ether bond is cut off by H2S.Cell experiment further confirmed TPP-H2S have excellent membrane permeability and successfully applied to cells and fast visualized the endogenous H2S in living cells and applied to measure the endogenous H2S level in different viscera by vivisection for the first time.(2)Based on chloro-coumarin-hemicyanine,a new two-photon fluorescent probe which can be response to H2S and Cys simultaneously and detect the activity of CBS/CSE is designed.To distinguish the fluorescence emission spectra of with H2S and Cys,taking into the molecular characteristics of H2S and Cys account,two reactive sites of a chloro-coumarin and the double bonds are designed-When the probe react with H2S or Cys,the former product is thio-coumarin-hemicyanine,the latter is amino-coumarin-hemicyanine because of the nucleophilic groups-NH2 of Cys during the nucleophilic substitution reaction followed rearrangement.Subsequently,an intramolecular cyclization between the thiol group and the double bond lead to six membered ring and seven membered ring with different ? electron structure,what selective discrimination of the two fluorescence emission spectra.As the Cys can be transformed into H2S under the catalysis of CBS/CSE,it can realize the analysis of CBS/CSE activity based on the relative changes of the products of the reaction between the probe and H2S or Cys.Through this two works,the rapid detection the concentration of H2S and successfully applied to measure the endogenously produced H2S levels in different fresh mouse tissues slice was initial realized.Most importantly,this work have realized the analysis of CBS/CSE activity,which can chang H2S content.Based on this above works,this study expects provide potential value in physiological mechanisms which can affect the concentration of H2S,not only the surface phenomenon occurs,which will help us have a comprehensive detection the dynamic changes of H2S and provide more effective means for the diagnosis and treatment of some physiological diseases.