Applications And Development Of Alkene Associated Bioorthogonal Reactions

The development of bioorthogonal reactions makes biological study more convenient, and there are series biorthogonal reactions for use to label biomolecules. At the same time, various unnatural amino acids (UAAs) can be incorporated into proteins with the development of PyIRS/tRNACUA pairs. Now the combination of the two powerful tools can enrich strategies for chemical biology.With the successful incorporation of photo-activable ONBK into OspF, photo-activation of OspF in cells was achieved in this study. After the attempts to capture OspF substrates, peptides bearing dehydroalanine, which was formed with treatment of OspF, can be captured by tetrazole. The final product of tetrazole labeled peptide was identified with LC-MS/MS. However tetrazoles had poor reaction activity towards dehydrobutyrine and crotonyllysine, which suggests tetrazoles can capture α,β-unsaturated terminal double bond rather than α,β-unsaturated un-terminal double bond in biomolecules. Further investigation showed although tetrazoles had well fluorescent labeling ability, which was achieved by the fluorogenic property, they had poor chemical selectivity. All the proteins can be labeled with tetrazoles without fluorescence. The poor chemical selectivity hindered applications of tetrazoles for the capture of OspF substrates.Inspired by photo-induced [4+2] cycloaddition of bicyclopropylidene (BCP), BCP bearing UAA BCPK was successfully recognized by an MwPylRS mutant and incorporated into proteins. In the exploring of BCPK for bioorthogonal reaction applications, Heck reation catalyzed with Pd nanoparticles was introduced to achieve high labeling efficiency. Compared with BCPK and other alkene, StyK bearing styrene had best reaction activity. And StyK can be further introduced into protein labeling in live cells and showed better labeling efficiency comparing with "Cu catalyzed click chemistry".To utilize the property of Carbon-Carbon bond of Heck reaction and the conjugated double bond of StyK, co-designed reaction between StyK and fluorescence quenched BrBDP was developed. This new labeling strategy can realize fluorescence turn on and red shift after the labeling, which can increase the signal noise ratio of fluorescent labeling. Compared with phenylacetylene bearing UAA KPhK, which can also form Carbon-Carbon bond and expand conjugated system after reaction with BrBDP via Sonogashira coupling, StyK had better fluorescence turn on ability. Heck reaction was further utilized in labeling of Dha bearing proteins, and labeled protein band can be observed easily compared with tetrazoles. Successful labeling of Dha made Heck reaction a promise strategy for chemical biology study in the future.