Study On Novel PH Fluorescent Probes And Their Application In Cells Imaging

Chapter 1 This chapter reviewed the typical mechanism of recognition, functions and monitoring significance of intracellular pH (pHi), methods for delivering fluorescent dyes into cells, research progress of pHi fluorescent probes. Based on the summary of previous work, we presented our research in this thesis.Chapter 2:Two styrylcyanine-based pH fluorescent probes (SCY-1 and SCY-2) were synthesized from (benzo)indole derivatives and 4-(diphenylamino)benzaldehyde using one-step condensation reaction. The probes were non-fluorescent in neutral media but significantly fluorescent in acidic environments with vis-NIR fluorescent emission (λem= 655 nm and 646 nm), which could effectively avoid native fluorescence from biological system. The pKa values were 4.40 (for SCY-1) and 4.71 (for SCY-2), respectively, making them favourable for detecting H+ in the acidic organelles. Their significant colocalizations of SCY-1 and SCY-2 with LysoTracker Green DND-26 in C6 cells suggested that the probes had good cell membrane permeability, and could be used as novel vis-NIR pH probes for effectively imaging pH changes in acidic organelles (i.e. lysosome).Chapter 3:A styrylcyanine-based vis-NIR neutral pH probe ESCY was synthesized based on ICT mechanism. When the pH was decreased from 10.00 to 5.00, ESCY displayed a remarkable emission enhancement in the vis-NIR fluorescence region (λem= 650 nm) with pKa value of 7.38. In addition, the probe exhibited strong pH-dependent behavior and responded linearly and rapidly to minor pH fluctuations within the range of 6.50-8.20, which was valuable for near-neutral cytosolic pH research. The pH response mechanism was confirmed by 1H NMR titration and theoretical calculations. The fluorescent images of SW480 cells indicated that ESCY could visualize near-neutral pH fluctuations in live cells with negligible autofluorescence. It provides a new analytical method for monitoring near-neutral range pH changes in live cells.Chapter 4:Two simple benzothiazole-based pH fluorescent probes (PBT and MBT) were synthesized by improving the literature. The compounds were used to synthesize of organic electroluminescent materials. In this chapter we developed their applications in terms of the pH fluorescent probes. The pH titration indicated that the probes were non-fluorescent in neutral media with the pH value more than 6, but significantly fluorescent in extremely acidic media (pH<2.2) with Ⅲ0 more than 50-fold. The pKa values were 4.40 (for PBT) and 4.71 (for MBT), respectively. In particular, their high sensitivity and high quantum yields in extremely acidic conditions could effectively reduce the interference of background fluorescence, and they were also confirmed by fluorescence imaging control experiments. In double staining experiments, PBT and MBT had excellent colocalizations with LysoTracker Red DND-99, and could be used as potential pH fluorescent probes to visualize extremely acidic pH fluctuations. They broaden the types of extreme acidic intracellular pH fluorescent probes.Chapter 5:The pH response performances of a series of C-6’substituted C-8’ oxime-appended spirobenzopyrans (5-la-d) were studied by UV-vis and fluorescence spectroscopy, respectively. Under the acidic conditions, the closed spirobenzopyrans forms transformed to their ring-opened merocyanine derivatives, which can facilitated the subsequent enol-keto tautomerization giving dual emissive peaks via the excited state ESIPT process. The pH response range was 3.0-9.0, the pKa values were between 5.26 and 6.32. The influences the different substituents appended at the C-61 of spirobenzopyrans to the pH sensitivity and pKa values were analyzed. It will provide some clues of the chemical structure designs to improve the pH response spectral properties of these spirobenzopyrans-based compounds. The cross interference caused by common metal ions except Cu2+ to the pH probes was negligible. These results suggested that these compounds have potential application in detecting intracellular pH quantitatively.