Fret-based PH Sensitive Fluorescent Probe:Design,Synthesis And PH_i Imaging Of Fungal Cells

The intracellular pH(pH_i)and pH homeostasis are critical for the control of growth and metabolity of eukaryotic cells.The pH_i of fungi cells regulates the nutrient transportation through the plasma membrane,the activity of metabolic enzymes and synthesis of macromolecules.The pH_i of fungi cells is also thought to be a trigger for the other cell responses,including the induction of heat shock proteins?And the action mechanism of some marketed drugs showing antifungal activity such as the proton pump inhibitor omeprazole and the antiarrhythmic drug amiodarone is also connected with a drop in pH_i.To study the pH_i related regulation system and action mode of antifungal drugs in vivo,measurement for accurate pH_i is very important.The currently aviliable methods for determination of pH_i in fungi cells were pH-sensitive fluorescent proteins and small molecular fluorescent probes.Despite its non-invasive,using ratiometric fluorescent proteins for measuring pH_i is complicated,time-consuming and high-cost.In contrast,small molecular fluorescent probe is rather simple and time saving.Unfortunately,there are very few small molecular probes applied in fungi,and most of them are commercial dyes with limitations: 1.hardly incorporated into cells,and should be transformed to its non-fluorescent acetoxymethyl ester which are membrane permeable and hydrolyzed in the cytoplasm by esterases to result in accumulation of the fluorescent forms.The insufficient hydrolysis of dyes in some fungi cells also goes against the visualization and accurate measurement of p Hi.2.the pKa of some dyes is rather high and not optimal for the measurement of pH_i in fungi cells.The aim in this paper is to design and synthesize novel pH-sensitive small molecular probes for the visualization and determinization of pH_i of fungi cells.Spiroboronate R-B is a novel pH sensitive fluorophore through a reversible ring-opening process triggered by H+ and synthsized firstly by our research group.The pKa of this probe was 9.46,which means its limit in the visualization and determinization of physiological pH_i of fungi cells.In order to improve the property of pKa of R-B,we considered to design a derivative,which was a ratiometric probe based on FRET.Namely,we choose this fluorophore as an energy donor,coumarin as the corresponding energy receptor,piperidine ring as a linker,and designed and synthesized a novel FRET based pH sensitive probe R-BC.Through evaluation of the properties of this probe,R-BC(pKa = 6.25)could meet the requirement of pKa for measurement pH_i and pH_i acidification in fungi cells.In the meanwhile,this probe exhibits good reversibility and selectivity for sensing H+.On the other hand,R-BC can easily go through the cell membrane of fungi and has no significant cytotoxicity,exhibiting nice biocompatibility of fungi cells.Through in situ calibration,there was a linear correlation between the fluorescence intensity ratio of the probe R-BC at two channels Ired/Iblue and the pH_i(pH = 5.0-8.0)of fungi cells,which means its accurate determination of pH_i in this physiological range.Then this probe was used to monitor changes of pH_i of C.albicans exposed to amiodarone.It was found that amiodarone could also induce the intracellular acidification of C.albicans,which was considered as a cause of the antifungal activity of this drug.Small molecular probes R-BN-1 and R-BC-1 separately containing acetyl and coumarin acetyl were also designed synthesized to investigate preliminarily the influencing of structure modification to the pKa of spiroboronate fluorophore.Through studying their spectroscopic properties and comparing the p Ka,it was suggested that introducing a coumarin fluorophore lead the moving of pKa to the acidic range while adding a methylene to the linker urge the further moving of pKa to the acidic range.Nevertheless,the corresponding efficacy of FRET was lowered because of the increasing of the spatial distance of two fluorophores and the weakened rigidity of the linker..FRET based small molecular fluorescent probes have been widely appied in mammal cells.However,to the best of our knowledge,there is no report on the visualization of intracellular pH of fungal cells with a small FRET based fluorescent probe.In this paper,we designed and synthesized a novel FRET based pH-sensitive probe R-BC and use it to visualize and determine the pH_i in fungi cells.Compared with the commercial dyes,probe R-BC can be easily incorporated into fungi cells and determine pH_i in a wide range,and became a powerful tool for better understanding p Hi regulation systems of fungi cells and pH_i relevant action mode of drugs with antifungal activity.