Design,Synthesis And Application Of Near-infrared Excitated/Bioluminescent Molecular Probes

The detection of biologically relevant molecules plays a pivotal role in the development of life sciences.Among various detection methods and techniques,optical imaging based on non-ionizing radiation plays an important role in biology and medicine.However,conventional optical imaging is mainly concentrated in the ultraviolet-visible region.In this region,biological tissues have strong scattering and absorption of light;in addition,there is significant tissue autofluorescence.Therefore,conventional optical imaging faces the challenge of relatively low tissue penetration and poor signal-to-noise ratio.Two-photon,near-infrared fluorescence imaging uses a near-infrared laser as the excitation light,with deeper tissue penetration depth and less autofluorescence interference.In addition,since the bioluminescence-based probe does not require excitation light,the disadvantages caused by the excitation light can be avoided.Therefore,the design and development of new two-photon,near-infrared,and bioluminescent probes are of great significance for promoting the development of life sciences.Based on this,we designed and synthesized a series of functional molecular probes based on two-photon,near-infrared and bioluminescence,which were applied to the detection and imaging of biologically active species in cells and in vivo.The specific works are as follows:（1）One-photon fluorescent probes in the ultraviolet-visible region have some shortcomings,such as photodamage,photobleaching and autofluorescence.Compared to one-photon imaging,two-photon imaging uses two near-infrared photons to excite fluorescent probes.Therefore,two-photon imaging has less photobleaching,phototoxicity,autofluorescence,better three-dimensional spatial localization,and deeper tissue penetration depth than one-photon imaging.Based on this,in Chapter 2,we designed and synthesized a two-photon probe FATP1 for formaldehyde detection and two-photon imaging in live cells and tissues.By introducing homoallylic amine as a formaldehyde recognition unit on the naphthalene derivative and nitrobenzyl group as a quencher,FATP1 shows weak fluorescence due to the d-PeT process.When FATP1 reacts with formaldehyde,it undergoes 2-aza-Cope rearrangement and hydrolysis to form a two-photon fluorescent dye,and the fluorescence enhanced.FATP1 has high selectivity and sensitivity for formaldehyde detection,with linear detection range of 1.0μM to 50μM,and detection limits of 0.2μM.In addition,FATP1 has low cytotoxicity and good photostability and can be used for two-photon fluorescence imaging of exogenous and endogenous formaldehyde in live cells.Furthermore,in two-photon imaging of mouse liver tissue,FATP1 showed a deeper tissue penetration depth（40-170μm）.（2）Although two-photon fluorescence imaging has deeper tissue penetration depth,reduced autofluorescence interference,and higher spatial resolution than one-photon imaging in the conventional visible region,two-photon fluorescence imaging still has certain limitations in the application of in vivo imaging.For example,the high requirements on the instrument,it is necessary to equip pulse laser with the scanning speed to the femtosecond level.In addition,the emission wavelength of most two-photon fluorophores is still in the visible region,hindering deeper tissue imaging.In order to further increase the penetration depth of the emitted light,we developed a fluorescent probe with emission wavelengths in the near-infrared region for detection and imaging in vivo.In Chapter 3,we we designed and synthesized a near-infrared fluorescent probe NC1 for fast and highly selective detection of Cys.The response of NC1 to Cys is not only a significant enhancement of near-infrared fluorescence,but also a significant change in absorption in the near-infrared region,accompanied by visible color chang es.At the same time,NC1has low cytotoxicity and good membrane permeability.Based on this,NC1 was successfully applied to detection and imaging of Cys in live cells and in vivo.（3）In the field of optical imaging,fluorescent probes that require excitation light still have some photobleaching and autofluorescence during imaging,and may cause singlet oxygen and damage samples.Bioluminescence imaging does not require excitation light.So the probe is not interfered by autofluorescence during detection and imaging,and there is no phototoxicity or photobleaching.In order to overcome the interference caused by the excitation light,in Chapter 4,we designed and synthesized a bioluminescent probe BP-PN for highly selective detection of ONOO^-.Upon modification of theα-ketoamide group on the luciferase substrate,BP-PN is capable of reacting specifically with ONOO^-and releasing aminoluciferin,and then producing bioluminescence in the presence of firefly luciferase and coenzyme.BP-PN showed high signal-to-noise ratio imaging of ONOO^-in live cell and mouse tumor models.In addition,BP-PN was successfully applied to bioluminescence imaging of endogenous ONOO^-in a mouse model of inflammation.（4）In order to verify the wider application of the bioluminescent system,i n Chapter5,we designed and synthesized the first bioluminescent probe BP-PS for detecting H₂S_n.By protecting D-luciferin with a H₂S_n recognition moiety,BP-PS shows weak bioluminescence.When reacting with H₂S_n,BP-PS is converted to free D-luciferin and emits photons in the presence of luciferase and coenzyme.BP-PS exhibits high selectivity and sensitivity to H₂S_n with a linear response range of 0.1 to 2μM and detection limits as low as 30 nM.In addition,BP-PS has excellent biocompatibility and can be used for bioluminescence imaging of endogenous H ₂S_n in LPS and bacterial infection-induced mouse inflammation models.