Fig. 4. 4ax-mediated bioorthogonal decaging on Fmoc-PABK and fLuc within living cells. (A) Reaction scheme between Fmoc-PABK and 4ax to generate Fmoc-lysine in H2O/DMSO (v/v = 1 : 1) at 37 °C. (B) Time-course study of the decaging efficiency of Fmoc-PABK's side chain in the presence of 5 equivalents 4ax as monitored by LC-MS. The peaks of both the reactant Fmoc-PABK (red) and the product Fmoc-Lys (blue) are shown. (C) Schematic representation of fLuc reactivation via the PABK/4ax bioorthogonal cleavage pair. The fLuc's activity which was initially caged by PABK can be rescued via a 4ax-mediated decaging reaction, which regenerates the wild-type fLuc activity in catalyzing luciferin oxidation with bright bioluminescence (PDB 4G36). (D) and (E) The dose- and time-dependent rescue of PfLuc activity by varying 4ax concentration at a constant incubation time (1.5 h) (D) or by varying time points with a constant 4ax concentration (500 µM) (E). fLuc was fused with a myc tag. BL: bioluminescence; BF: bright field. The histograms show bioluminescence intensity from three independent experiments. Error bars represent ±1 SEM.
