Design And Synthesis Of High Performance Two-photon/near-infrared Fluorescent Dyes And Probes For Bioimaging

As an intuitive and in-situ visual observation technique,fluorescence imaging has been widely used for functional molecular labeling and detection.Bio-molecular markers and fluorescent probes based on organic fluorescent dyes have been conveniently used for in-situ,real-time non-destructive detection of biomolecules and tracking biological processes,owing to the advantages of simple operation and good reproducibility.However,traditional small molecule organic fluorescent dyes still suffer from some disadvantages,such as small Stokes shift,large biological background fluorescence,and poor photobleaching resistance,which greatly reduce the signal-to-background ratio of imaging detection and limit its further in vivo application.On the other hand,two-photon fluorescence imaging,due to their deeper tissue penetration?up to 1,000?m?and smaller tissue absorption,together with reduced photodamage,photobleaching,and background fluorescence during sample imaging,has also been widely used for imaging and detection of biomolecules in biological systems.However,most of the developed two-photon dyes emit between420 and 550 nm,which is below the yellow fluorescence region.Within this regime,there is also serious autofluorescence in tissues,leading to significantly compromised signal-to-noise ratio.In order to better meet the needs of practical applications for bioanalysis,the design and development of organic fluorescent dyes/probes with excellent performance is imperative for current research.In order to provide new solutions of bottleneck problems for current fluorescence probes,based on the intersection of analytical chemistry,organic chemistry,material chemistry and biological science,a series of new functional small molecule dyes herein are designed and synthesized through the molecular luminescence mechanism and structural innovation.1)Most of the two-photon probes reported in the literature have short emission wavelengths and still suffer from strong background fluorescence in biological tissues.In the second chapter,inspired by anthocyanidin,we designed and developed a series of two-photon dyes?AC-Fluors?with large two-photon action cross section and far-red to near-infrared emission.Systematic study demonstrated that the emission wavelengths and quantum yields of AC-Fluor can be facilely and effectively conditioned by incorporating the electron donors to specific positions of the anthocyanidin rings.Their emission can be covering the whole visible light range,from 467 to 707 nm.Remarkably,AC-Fluors exhibit unprecedented environment-insensitive two-photon properties with a substantial cross section as large as 1139 GM in aqueous solution.To test AC-Fluor for bioimaging applications,we designed a mitochondria tag and a Cu²⁺turn-on probe,both through simple one-step chemistry.The experimental results showed that both Mito-tag and ACF27-Cu²⁺can be well applied to biological imaging research,especially the two-photon imaging study of deep tissue.We envision that our new dye scaffold AC-Fluor provides great potential for probe development and commercial labeling applications.2)For the problem of fluorescence quenching of traditional fluorescent dyes in the water environment,in the third chapter,we devised a novel class of D–A type green fluorescent protein?GFP?chromophore analogues?TPC?that form a hydrogen-bond network in water to improve the two-photon efficiency.Mechanism studies elaborated that TPC dyes bind to water molecules through multiple hydrogen bonds,achieving better solubility and higher stability of the ICT excited states,thus increasing the excited state oscillator and emitting strong fluorescence.Spectroscopic results show that our design results in TPC dyes with 0.80 quantum yield and large two-photon action cross section?210 GM?in water,which is very suitable to develop two-photon bioimaging probes.To demonstrate the improved in vivo imaging,we synthesized a H₂S selective probe based on the TPC scaffold and for the first time visualized H₂S generation and consumption in the cirrhotic liver tissue.3)In order to overcome the shortcomings of small Stokes shifts of traditional fluorescent dye,in the fourth chapter,we have developed a general method to significantly increase the Stokes shifts of common fluorophores.By simply appending a 1,4-diethyl-decahydro-quinoxaline?DQ?moiety onto the conjugated structure,we introduced a vibronic backbone that could facilely expand the Stokes shifts,emission wavelength,and photostability of 11 different fluorophores by more than 3-fold.Theoretical calculations showed that the introduction of strong electron donating groups DQ enhanced the vibration relaxation of traditional dyes in their excited states,thereby increasing their non-radiative transition and finally expanding their Stokes shifts and emission wavelengths.This generalizable method could significantly improve the imaging efficiency of commercial fluorophores.As a demonstration,we showed that the DQ derivative of hemicyanine generated 5-fold signal in mouse models over indocyanine green.Furthermore,the DQ-modified fluorophores could pair with their parent molecules to conduct one-excitation,multiple emission imaging,allowing us to study the cell behavior more robustly.4)Molecular fluorophores operating in the second near-infrared region?NIR-II,1000-1700 nm?has immensely attracted attention due to their deeper penetration?⁵-20 mm?in biological tissues,but an ongoing challenge is the control of their fluorescent property.In the fifth chapter,a series of new the NIR-II organic fluorescent dyes were designed and constructed.The experimental results show that NIR?-CS1-2has strong NIR-II emission,and their fluorescence can be easily cont rolled by the"on-off"of the spiral ring.In addition,NIR?-CS dyes also exhibit good anti-fluorescence quenching ability in aqueous solution,and are suitable for developing NIR? fluorescent probes for bioimaging.Based on this,three novel NIR? fluorescent probes NIR?-CS-pH,NIR?-CS-Hg and NIR?-CS-ATP with"zero background"are first time constructed.All these probes show good fluorescence response to their corresponding analytes with great sensitivity,indicating that NIR?-CS series dyes have great application potential in NIR? fluorescence imaging.