The Design,Synthesis And Bioimaging Application Of Silicon-Substituted Coumarins

Fluorescence imaging has attracted tremendous attention in medical imaging,chemical detection,and other scientific fields with reliable sensitivity,good selectivity,and low cost.Fluorescent probes with excellent performance,as an indispensable part of fluorescence imaging technology,guarantee real-time,dynamic,sensitive,and accurate analysis of crucial biomolecules and structures using fluorescence imaging.Therefore,the design,development,and properties regulation of fluorescent probes has always been a hot spot in fluorescence imaging research.Molecular fluorescent probes have attracted extensive attention due to their specific structure,applicability,and facile property-regulation through structural modification.Among numerous methods,silicon-based substitution is an extraordinarily unique and rapidly developing strategy for regulating the properties of dyes.Especially with the extensive research and application of silicon-substituted xanthene dyes in recent years,many researchers have confirmed the significance of heteroatom introduction in dye performance modulation.Taking advantage of improving the properties of dyes by silicon atom substitution,this thesis introduces the silicon atom into the skeleton of coumarin,systematically explores the regulation of physicochemical properties of coumarin dyes by introducing a silicon atom.Based on the unique properties of Sicoumarin,a series of silicon-substituted coumarin dyes and fluorescent probes with characteristic properties have been constructed and successfully employed in the fluorescence imaging of various proteins and biological structures.The main contents of this thesis are as follows:1.A series of silicon-substituted coumarin(Si C)dyes were designed and synthesized.The Si Cs dyes were synthesized by a synthetic method,including lithiation reaction,olefin metathesis reaction,oxidation reaction,etc.Further,a series of Si C dye molecules Si C A-Si C F with tunable wavelength were obtained by different alkyl modifications on the Si C matrix.The introduction of a silicon atom significantly changes the electronic structure and physicochemical properties of dyes.Compared with traditional coumarin molecules,Si C is extremely sensitive to polarity.It is prone to form hydrogen bonds with hydrogen bond donor solvents,which further bathochromically shifted spectra,resulting in a 200 nm difference in the maximum fluorescence emission wavelength in various solvents.In polar,protic systems,the fluorescence spectra of some Si C molecules can be bathochromic-shifted to the nearinfrared region(NIR,670 nm),and show a ratiometric change with polarity and proticity.Based on the ultra-high sensitivity to the environment in Si C,we succeeded in tracking the formation of lipid droplets during adipocyte differentiation.2.To further investigate the influence of hydrogen bonds formed on the carbonyl oxygen atom on the optical properties of Si C,we designed a Si C derivative Si CBZA1 with intramolecular hydrogen bond and its analog Si C-BZA2 without the intramolecular hydrogen bonding structure.The single-crystal structure,NMR analysis,and theoretical calculation of Si C-BZA1 suggest the existence of an intramolecular hydrogen bond.Fluorescence lifetime,NMR characterization,and infrared spectroscopy proved that Si C-BZA1 also has strong intermolecular hydrogen bonds in protic solvents.Comparing the spectral properties of Si C-BZA1 and Si CBZA2,we realized that Si C-BZA1 with hydrogen bonding effect has the antisolvatochromic effect of hydrogen bond induced fluorescence enhancement and nano-aggregation effect in the aqueous solution.This provides an important theoretical basis for the subsequent design of Si C-based probes.3.Based on the unique physical and chemical properties of Si Cs,we designed seven fluorescent probes Si C-SAs for human carbonic anhydrase II(h CAII)using Si C as the fluorescent scaffold and benzenesulfonamide as the recognition group.The molecular modulation of Si C on h CAII fluorescence sensing was systematically and deeply explored in three aspects:(1)the alkylation modifications of the amino group at position 7 of the coumarin skeleton;(2)the existence of intramolecular hydrogen bond in the carbonyl group;(3)the alkyl linker between coumarin and h CAII inhibitor.The results suggest that the five-membered ring modification at the 7-position in coumarin leads to more red-shifted spectra and lower background fluorescence;the presence of a hydrogen bond donor structure at the carbonyl site can significantly enhance the fluorescence signal;the shorter length of the alkyl chain,the more enhanced fluorescence after recognition with the targets.Thus,the fluorescent probe Si C-SA3 with the five-membered ring at the 7-position and an amide bond connected to the recognition group has a strong fluorescence response to h CAII protein(the maximum fluorescence increase can be 17-fold)with excellent selectivity and sensitivity.4.Human serum albumin(HSA)is a protein widely distributed in the human circulatory system.Based on the unique structure and physicochemical properties of Si C,we designed 12 HSA fluorescent probes Si C-HSAs by directly connecting benzylamine derivatives through amide bonds with the five-membered ring Si C.Spectral analysis suggests that all Si C-HSAs possess antisolvatochromic effect and nano-aggregation effect.Moreover,they can bind to the warfarin sites in the HSA protein structure in a stoichiometric ratio of 1:1.Therefore,after the interaction of the Si C-HSA nano-aggregates in the aqueous solution of HSA,the nano-aggregates disassemble and enter the warfarin site,resulting in a 250-fold enhancement in the fluorescence due to the combined effects of the polar and protic microenvironment in the warfarin site in HSA protein cavity.5.To prove the generality of Si C in sensing proteins,we further designed several other Si C-based protein fluorescent probes,including anti-biotin protein probe Si CBiotin;SNAP-tag protein probes Si C-BG1 and Si C-BG2;and translocator protein(TSPO)probe Si C-PK.We systematically explored the physicochemical properties of the probes and successfully tracked the biological structures labeled by SNAP-tag proteins using Si C-BG1 in vivo.In addition,we located TSPO in C6 and MCF-7 cells and tracked the microglial cell activation by Si C-PK.Furthermore,the dynamic changes of microglia activation in brain injury tissues were monitored and imaged by Si C-PK.The results indicate that the Si C has excellent photophysical and chemical properties and can be used as a novel fluorescent strategy for sensing protein.This thesis introduces the silicon atom into the skeleton of coumarin,systematically explores the regulation of physicochemical properties of coumarin dyes by introducing a silicon atom.Based on the unique properties of Si-coumarin,a series of silicon-substituted coumarin dyes and fluorescent probes with characteristic properties have been constructed and successfully employed in the fluorescence imaging of various proteins and biological structures.