Synthesis And Application Of Targeted Protein Rhodamine-based PH Fluorescent Probes

Currently,fluorescent probes can be used to detect cell microenvironment,active small molecule,anions,cations,etc.,but most fluorescent probes have some disadvantages,such as subcellular targeting instability,poor pH sensitivity and pKa inappropriate ect.Therefore,it is particularly important to develop fluorescent probes that are suitable for targeting neutral and acidic subcellular organelles.This article designs and synthesizes rhodamine-based fluorescent probes targeting the nuclear near-neutral pH based on the Halo-tag protein,and rhodamine-based fluorescent probes targeting nucleic acid pH values based on Halo-tag and Clip-tag proteins.This paper studys the optical physical properties of these fluorescent probes and their applications in cell fluorescence imaging.As follows:(1)Explore the design ideas of rhodamine-based pH fluorescent probes.The rhodamine B fluorophore was used as the parent,then the reduced rhodamine B aldehyde group was connected with phenylhydrazine,4-hydroxybenzoylhydrazone and o-phenylenediamine,respectively,finally the three fluorescent dyes were synthesized via the “Schiff base” reaction,Rhs-1,Rhs-2 and Rhs-3.Spectral properties show that the fluorescence intensity of Rhs-1 is not affected by pH and is not sensitive to pH;the pKa of Rhs-2,which is alkaline,is not suitable for detecting the pH of subcellular organelles;the pKa of Rhs-3 is acidic,it can be suitable for detecting the pH of acidic subcellular organelles.It lays the foundation for the next step in the design of probes for the detection of pH by the synthesis of a targeted protein acidic subcellular organelle.(2)The detection of near-neutral pH of targeted nucleus was achieved.The intermediate AMR is synthesized by the reaction and reductive of rhodamine B and propynylamine,followed by the "Click" reaction to connect with Halo-AD to synthesize the fluorescent probe AMR-CA(chloroalkane)that can target the Halo protein.AMR-CA is extremely sensitive to pH and has a response range of only one pH unit from pH 6 to pH 7.It is extremely sensitive to pH and is suitable for targeted detection of sub-cellar organelles with near-neutral pH.The water solubility of the molecule AMR-CA is 3 ?M,cell imaging tests show that the probe can quickly locate the Halo fusion protein in the nucleus of living cells through the cell membrane,and the specificity is very high.The probe AMR-CA and Halo-tag protein are stably present as covalent bonds.(3)The detection of acid pH of targeted nucleus was achieved.An intermediate TBM was synthesized by linking bromo tetramethylrhodamine with o-phenylenediamine,followed by condensation,followed by reaction with Halo-PY and Clip-PY through a “sonogashira coupling” reaction to finally synthesize TBM-CA and TBM-BC that can target the fusion protein.Spectroscopic properties indicate that the pKa of the molecules TBM-CA and TBM-BC are all acidic,are suitable for the detection of acidic targeting fusion protein organelles.In cell imaging,living cells were fixed with formaldehyde,and the transfected Halo protein and TBM-CA were stably present as covalent bounds in the presence of PBS at pH =3.5,as well as the nucleus Clip protein and TBM-BC can also stably exist in the form of covalent bonds,suitable for the detection of acidic subcellular pH,high specificity.