The Development Of Bioorthogonal Photoclick Reactions And Their Chemical Biological Applications

Bioorthogonal reaction is a kind of reaction that can occur under physiological conditions and don't interfere with other biochemical reactions occurring simultaneously in vivo,nor cause damage to the organism and the target molecule Photocatalytic bioorthogonal reaction is a kind of important reaction in bioorthogonal chemistry.It is an addition reaction induced by light irradiation.Photocatalytic reaction exhibited mild reaction conditions and high reaction efficiency.Photoclick reaction based on tetrazole and alkenyl is a bioorthogonal reaction commonly used for biomacromolecular labeling,and this reaction plays a very important role in chemical biology research.In the first part of the paper,we demonstrated the first example of using the biocompatible photoclick reaction to develop photoclickable miRNAs for intracellular target identification.The 3'-modification of miRNAs with tetrazole as pretag did not affect the intracellular biological function of miRNAs to repress target gene expression,which is superior to the direct miRNA modification with biotin tag.The rapid intermolecular photoclick reaction between the tetrazole tag and MF-Biotin allowed postlabeling of the miRNAs in the extracted cell lysate with biotin for further enrichment,isolation,and identification.With the probe of photoclickable miRNAs,we validated their ability to isolate confirmed target genes of miR-106a and miR-122.Then we identified several unknown target genes of miR-122 and revealed a new pathway in HepG2 cells that miR-122 promotes cell apoptosis by targeting PEG10.The use of different photoclickable miRNA probes in combination with high-throughput screening techniques such as microarray or proteomics may reveal more unknown miRNA-involved cellular regulation pathways.Simultaneous use of miRNAs modified with different bio-orthogonal functional groups for sequential identification of target genes for different miRNAs is now underway in our groupIn the secend part of the paper,we have developed a novel photo-clickable miRNA that allowed the in situ activation of fluorescence in living cells without impairing the intracellular function of the corresponding miRNA.The photo-clickable Tet?-miR122 under mild light irradiation induced a rapid intramolecular photoclick reaction and led to the formation of Pyr?-miR122 with bright fluorescence emission under one-photon or two-photon excitation.HepG2 cells transfected with Tet?-miR122 were labeled with green fluorescence in a spatial and temporal manner through mild light irradiation.This photo-clickable miRNA thus o ffers a practical way to track and investigate intracellular miRNA in a spatiotemporal manner.Further exploration of this photo-clickable miRNA in imaging of miRNA in deep tissues is now underway in our groupSo far,only a limited number of reactions have been widely used and have demonstrated high biocompatibility and selectivity.Find a new biological orthogonal reaction is an exciting thing that has far-reaching significance and also has considerable challenges.In the third part of the dissertation,we developed a new type of bio-orthogonal reaction based on LED white light induction for the first time.The reaction can induce[4+2]cycloaddition of phenanthrenequinone and vinyl ether under the irradiation of LED white light.This reaction can react rapidly under physiological conditions.Its secondary reaction kinetic constant is k2=2.76M-1S-1,This reaction also has a highly efficient covalent labeling effect of protein.We successfully use this reaction to covalently label the BSA protein with a fluorescent dye under physiological conditions.It is worth noting that because the reaction is induced by LED white light,it is harmless to cells and animals and other organisms,which is also an advantage compared to the UV-induced bioorthogonal reaction.This LED lingt induced bioorthogonal reaction will be widely used in the fields of chemical and biological research.