Tetra-fluorinated aromatic azide for highly efficient bioconjugation in living cells

We developed a fast strain-promoted azide–alkyne cycloaddition reaction (SPAAC) by tetra-fluorinated aromatic azide with a kinetic constant of 3.60 M−1 s−1, which is among the fastest SPAAC ligations reported so far. We successfully employed the reaction for covalent labelling of proteins with high efficiency and for bioimaging of mitochondria in living cells. The reaction could be a generally useful toolbox for chemical biology and biomaterials.


Reaction kinetics of this SPAAC
Fluorescent kinetics analysis: 2 µL 5 (2 mM) in DMSO and 2-12 µL 4 (10 mM) in DMSO was added to 2 mL PBS (50 mM, pH 7.4, containing 70% CH 3 CN).The progress of the reaction was monitored by fluorescence spectrophotometry with excitation at 473 nm.The time-dependent emission at 511 nm was shown in Fig. S2.The pseudo-first-order rate, k obs was determined by fitting the fluorescence intensity data with single exponential function.The linear fitting between k obs and concentrations of 4 gives the reaction rate (k 2 ).The reaction product of 4 and 5 was confirmed by HRMS (Figure 3S).

Fluorescence labelling of BSA and lysozyme
100 μM BSA and lysozyme were treated with 0.5 mM 7 in 50 mM PBS (pH 8.5, contain 10% DMSO) for 2 h at room temperature respectively.Then the free 7 need to be removed by a desalting column (Zeba Spin Desalting Columns, 0.5 mL, Thermo scientific).7-labelled protein was placed in the center of the resin bed after equilibrating column.Then the sample was placed in new microcentrifuge collection tube and centrifuged at 1500 × g for two minutes.Subsequently, the 7-labelled protein was incubated with 1 mM 5 for another 2 h.The control group was also treated with 2 mM Na 2 S for 10 minutes at room temperature.The labeled protein suspension was mixed with loading buffer and directly used for electrophoresis in 15% SDS-PAGE.The gel was firstly imaged under UV lamp to detect whether the protein was fluorescently labelled.Then, the gel was stained by Coomassie brilliant blue and imaged.

Fluorescence labelling of mitochondria in living cells
Cell culture: HEK-293 cells were cultured at 37 o C, CO 2 (5%) air environment in high glucose DMEM (GIBICO) supplemented with FBS (10%), penicillin (100 μg/ml), streptomycin (100 μg/ml) and l-glutamine (4 mm).The cells were maintained in exponential growth phase, and then seeded in a glass-bottom 35 mm plate (~ 2x10 4 cells per well).Cells were passaged every 2-3 days and used between passages 3 and 10.Confocal imaging experiments: Cells were imaged on an FV1000 inverted fluorescence confocal microscope (Olympus, Japan) with a UPLSAPO 40 x objective lens.All images were analyzed with Olympus FV1000-ASW software.Co-localization: HEK 293 cells were incubated with 8 (10 μM) for 20 min and washed with 1mL PBS three times.Then the cells were incubated with 5 (5μM) for another 30 min and washed with PBS again.Finally, the medium was replaced by PBS, and cells were imaged via the green channels (lex= 488 nm, lem=500-600 nm).Control cells were treated with just 8 (10 μM) for 20 min.

Fig. S2
Fig. S2 The time-dependent fluorescence intensity at 511 nm of 5 (2 μM) in the presence of different concentrations of 4.

Fig. S3
Fig. S3 High resolution mass spectrum of the reaction product for 4 and 5.