Design And Synthesis Of β-gal-targeted ¹⁹F NMR/fluorescent Bimodal ESIPT Molecular Probes And Their Imaging In Tumor Cells

Objective β-galactosidase(β-gal)is a hydrolase present in cytosol and widely involved in a variety of important physiological and pathological processes in living organisms.Meanwhile,it is overexpressed in primary ovarian cancer and is considered as a typical biomarker in ovarian cancer detection.We designed a series of molecular probes which can selectively bind and react with β-gal,using the HBI molecule with ESIPT effect as the basic structure of the probe,and explored the possible influence of different substituents on fluorescence at the same position by modifying the molecular structure.We innovatively combined the fluorescence imaging method with 19 F NMR,expecting that this new attempt can detect β-galactosidase/ tumor with high sensitivity,and the fluorescence enhanced by ESIPT effect can accurately obtain the location of β-galactosidase/ tumor and its size change.Methods On this purpose,we designed a series of HBI compounds with F-atom substitution at the fifth/third position and modified the fourth position with different substituents.Then the various properties of these new compounds were tested,compared and screened.A total of 52 compounds were synthesized,purified and identified,and 41 new compounds were successfully developed,of which 15 were the target of galactoside molecules,all show enzymatic activity as β-gal substrates.After detailed evaluation,we screened and got a total of 8 ideal β-gal fluorescent molecular probes with significant changes in 19 F NMR chemical shifts before and after enzymatic hydrolysis.Results After cytotoxic activity testing,x fluorescent probes with low cyotoxicity were screened and further evaluated(He La,MCF-7,SKOV-3,OVCAR-3)for intracellular imaging.The test results showed that H3-1 is ideal 19 F NMR/fluorescent dual-modality molecular probe,which is expected to be used for subsequent imaging exploration in vivo and provide a practical template for the application of new mechanism.Conclusions 1.The enhanced fluorescence probe H-Br shows up to 120-fold enhancement with extremely low background signal,but exhibits a tendency to decay,limiting its long-term imaging.2.H-1 and H3-F4 are suitable for long-term imaging and enzyme concentration quantification with the help of the properties of ratio fluorescent probes,and each property is ideal for the detection of β-gal in living cells.3.H3-1 has a fast response to β-gal,relatively low background fluorescence,up to 70-fold growth,and good selectivity for various interferents,successfully targeting β-gal imaging in ovarian cancer cells.It also has a great 19 F chemical shift change(9.80 ppm),which lays the foundation for a new mechanism of 19 F NMR/fluorescence imaging dual-modality for early detection and therapeutic monitoring of ovarian cancer.4.In the course of the project,we compared the basic properties of the probes and discussed the relationships of the probe fluorescence with the conformation of compounds.The introduction of the 19 F atom at position three exhibited a larger 19 F NMR shift compared to position five,but the fluorescence performance was slightly inferior to the probe with 19 F atom at position five.The introduction of different substituents at position four of the HBI backbone had a significant effect on the fluorescence of the probe,in which the bromine atom being the most obvious.