Preparation And Properties Of PH Fluorescent Probes Based On Phenoxazine Derivatives

pH is an important parameter in clinical analysis,chemical control processes,food production,waste water treatment procedures,environmental and life sciences and so on.In cell biology,intracellular hydrogen ion plays a vital role in various physiological and pathological processes,such as receptor-mediated signaling,enzyme activity,cell proliferation and apoptosis,drug resistance,muscle contraction,swallowing,ion migration,internal environment steady state,inflammation and tumor growth,and so on.When the pH value exceeds the normal range,the acid-base steady state will be destroyed,leading to cell dysfunction and affects the body's physiological mechanisms and cause serious diseases such as neuronal degenerative diseases,cancer,Alzheimer's disease and so on.Therefore,it is a very meaningful task to develop reliable techniques to monitor the pH fluctuation inside and outside the cell.Compared with microelectrode,nuclear magnetic resonance(NMR),absorption spectroscopy and other methods,pH fluorescent probe has high sensitivity and selectivity,rapid response,easy operation,real-time in situ monitoring and other advantages,the use of pH fluorescent probe has become a monitoring of intracellular a powerful tool for detecting pH changes.At present,there are two typical types of pH fluorescent probes:one is near neutral pH probe,the other is weak acid pH probe,and very acidic(pH<4.0)pH probe is relatively rare.Different types of pH probes are suitable for different subcellular regions to work,because each subcellular region has its own unique function,it is important to study pH fluorescent probes,which are suitable for different subcellular regions.In this paper,the pH probes based on phenoxazine derivatives were designed and synthesized,and their optical properties and applications were studied.Mainly divided into the following three parts:1.Two pH fluorescent probes 5a-b based on phenoxazine were designed and synthesized,all of which were reversible and lysosomal-targeted pH probes.With the decrease of pH from 7.4 to 4.4,the fluorescence maximum emission peak intensity of the fatty amine probe 5a was increased about 2.3 times at 665 nm;the maximum emission peak of aromatic amine probe 5b was located at 679 nm,the fluorescence intensity was very weak at pH=7.4,the fluorescence intensity reached the maximum at the pH=4.2.Its fluorescence intensity was increased about 20-fold,OFF-ON type of emission can be almost obtained.The pK_a values of probes 5a-b are both 5.5;In addition,the optical properties of the probes 5a-b and the PET mechanism have been explained by the(TD)DFT theory.Cell experiments of the oxazine 1 and 5a-b showed that the classical dye oxazine 1 could not overlap with the commercial lysosomal marker and the probes 5a-b could overlap well with each other,which indicating that the probes 5a-b could act as lysosomes biomarkers.Probes can enter lysosomes mainly dominated by acid-dominated rather than OFF-ON-type.2.A pH fluorescence probe 7 based on phenoxazine derivatives was designed and synthesized for pH detection under extreme acid conditions.With the decrease of pH=3.2to 0.2,the maximum emission peak of probe 7 was located at 679 nm,the fluorescence intensity was gradually weakened,and the emission finally closed to the ON-OFF type,the pK_a value was calculated about 1.5,which indicated that probe 7 could be used to detect pH under strong acid condition.In cell imaging experiments,probe 7 did not overlap well with lysosomal marker,it was just a normal pH probe in strong acid condition.3.Three phenoxazine dyes 8a-c with near-infrared emission were selected,their reversible absorption and emission responses under extreme acidic and basic conditions were studied.Dyes 8a and 8c showed a color change under strong acid conditions(pH<2.0),while dye 8b did not show color change under the same conditions;in strong alkaline conditions(pH>7.6),dyes 8a-c also show color change.Fluorescence pH titration showed that near-ON-OFF and OFF-ON emission was obtained when dye 8a-c are excited with 633 nm under extremely acid and alkaline conditions,and their maximum emission peaks located at 678,652 and 665 nm;The fluorescence intensity of the dye 8a-c did not change significantly under sub-acidic and neutral conditions.In addition,the absorption and emission of dyes 8a-c were reversible to different pH under extremely acid and alkaline conditions.