| Biorthogonal chemistry has emerged as a kind of new important tool for studying the process of biomolecules,which is carried out efficiently under physiological conditions.Chemical labeled probes based on biorthogonal reaction have realized specific imaging and labeling of nucleic acid,protein,sugar and other biological macromolecules in vivo.Until now,researchers have been devoloping varietys of biorthogonal reaction and reagents,which including Staudinger,Aldol ketone condensation,SPAAC,CuAAC,IEDDA etc.Among them,IEDDA reaction has been widely used as a kind of biological orthogonal reaction due to its advantages of fast reaction rate and no need of catalyst.In this thesis,we focused on the cycloaddition reaction of methylcyclopropene derivatives and near-infrared（NIR）s-tetrazine fluorescent probe,systematically explored the photophysical properties,chemical reactivity and fluorescence imaging ability of the probe at the cell level,and achieved some research results.The three sections are summarized as follow:Section 1:we designed and synthesized near infrared s-tetrazine cyanine fluorescent dyes:103,106 and 107.After the reaction with strain olefin TCO,the fluorescence is turned on,and the fluorescence intensity increases about 4 times before and after the reaction.Through theoretical computational studies,we have shown tetrazine-based nearinfrared cyanine dyes 103,106 and 107 have limited fluorescence turn-on ratios.We also demonstrated that the fluorogenic nature of these dyes results from the energy transfer to a lowlying dark-state centered in the tetrazine moiety.Based on the literature reports,its have shown the emissions from the dark n-π*excitation of the s-tetrazine moiety spans from-570 to～710 nm.Therefore,it is thus not surprising that the s-tetrazine moiety cannot function as fluorescence quencher for near-infrared fluorophores with emission larger than 710 nm.However,the dark state quenching mechanism of s-tetrazine compounds is not suitable for developing fluorescent dyes with emission wavelengths longer than 710 nm,because the bright state energy level of the fluorophore is lower than that of the dark state.Although the characteristical of the developed s-tetrazine NIR fluorescent probe is poor,the results of theoretical chemistry computational provide an important guiding significance for the rational to design of long wavelength s-tetrazine based NIR fluorescent dyes.Section 2:cyclopropene nucleic acid derivatives 175,178,179 and 183 were synthesized efficiently using methylcyclopropanol as building block;Cyclopropene amino acid derivatives 174 and 177 for protein unnatural amino acid labeling;And cyclopropene PEG derivatives 180,181,182 and 184 can be used in ADC and protacs.The experimental results of kinetics,stability and biorthogonality showed that these cyclopropene derivatives had good biorthogonality,stability and reactivity.Among them,the cyclopropene uracil nucleoside derivative 175 had the highest second-order kinetic constant,which is reached 17.5 ± 0.1 M-1 s-1.Section 3:we developed a methylcyclopropene probes for nucleic acid labeling.Methylcyclopropene uracil nucleoside probe 175 and methylcyclopropene guanine nucleoside probe 179 were used for nucleic acid labeling imaging.The IEDDA cycloaddition reaction of cyclopropene probe 175 with 5-TAMRA-Tz fluorescent dye in pure PBS system was carried out at a rate of 56.6 ± 1.94 M-1 s-1（It is 103 times faster than that reported in the literature of VdU nucleoside probe）.The cyclopropene probes 175 and 179 achieved the staining and imaging of nucleolus and cytoplasm of HeLa cells at low concentration. |
