The development of efficient bioorthogonal reactions for sensing of endogenous biomolecules and for bioconjugation should be of paramount importance in the field of chemical biology. In this work, the o,o'-difluorinated aromatic azide was firstly employed to develop a new fast-response fluorescent probe 1 for HS detection and for bioorthogonal reactions. Compared with non- and mono-fluorinated probes, 1 showed faster reaction toward HS, the third gasotransmitter, in buffer (pH 7.4), implying that the reaction rate could be enhanced by the dual-fluorine groups. Furthermore, such enhanced reaction rates of 1 were also observed in the Staudinger reaction and strain-promoted azide-alkyne cycloaddition (SPAAC) based on the comparison studies of the non-fluorinated probe. Our results firstly highlight that the o,o'-difluorinated aromatic azide group should be useful for fast bioorthogonal reactions and HS detection.

The development of highly efficient bioorthogonal reactions is of paramount importance for the research fields of biomaterials and chemical biology. We found that the o,o'-difluorinated aromatic azide was able to react with triphenylphosphine to produce water-stable phosphanimine. To further improve the efficiency of this kind of nonhydrolysis Staudinger reaction, a tetrafluorinated aromatic azide was employed to develop a faster nonhydrolysis Staudinger reaction with a rate of up to 51 m s , as revealed by high-performance liquid chromatography (HPLC) analysis and fluorescence kinetics. As a proof-of-concept study, the highly efficient Staudinger reaction was successfully used for chemoselective fluorescence labeling of proteins and nucleic acids (DNA and RNA) as well as for protein polyethyleneglycol (PEG)ylation. We believe that this bioorthogonal reaction can provide a broadly useful tool for various bioconjugations.

The one-pot nonhydrolysis Staudinger reaction and Staudinger or SPAAC ligation were used for producing a FRET-based dyad in living cells as a proof-of-concept study.

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A fast strain-promoted azide–alkyne cycloaddition based on tetra-fluorinated aromatic azide was developed and applied to label proteins and living cells with high efficiency.