The genetically encoded photo-cross-linkers promise to offer a temporally controlled tool to map transient and dynamic protein–protein interaction complexes in living cells. Here we report the synthesis of a panel of 2-aryl-5-carboxytetrazole-lysine analogs (ACTKs) and their site-specific incorporation into proteins via amber codon suppression in Escherichia coli and mammalian cells. Among five ACTKs investigated, N-methylpyrroletetrazole-lysine (mPyTK) was found to give robust and site-selective photo-cross-linking reactivity in E. coli when placed at an appropriate site at the protein interaction interface. A comparison study indicated that mPyTK exhibits higher photo-cross-linking efficiency than a diazirine-based photo-cross-linker, AbK, when placed at the same location of the interaction interface in vitro. When mPyTK was introduced into the adapter protein Grb2, it enabled the photocapture of EGFR in a stimulus-dependent manner. The design of mPyTK along with the identification of its cognate aminoacyl-tRNA synthetase makes it possible to map transient protein–protein interactions and their interfaces in living cells.

A series of red-shifted azobenzene amino acids were synthesized in moderate-to-excellent yields via a two-step procedure in which tyrosine derivatives were first oxidized to the corresponding quinonoidal spirolactones followed by ceric ammonium nitrate-catalyzed azo formation with the substituted phenylhydrazines. The resulting azobenzene–alanine derivatives exhibited efficient trans/cis photoswitching upon irradiation with a blue (448 nm) or green (530 nm) LED light. Moreover, nine superfolder green fluorescent protein (sfGFP) mutants carrying the azobenzene–alanine analogues were expressed in E. coli in good yields via amber codon suppression with an orthogonal tRNA/PylRS pair, and one of the mutants showed durable photoswitching with the LED light.

A series of naphthalimide–tetrazines were developed as bioorthogonal fluorogenic probes, which could produce significant fluorescence enhancement, notable aggregation-induced emission (AIE) characters and multicolor emissions after bioorthogonal reaction with strained dienophiles. Manipulating the π-bridge in the fluorophore skeleton allows fine-tuning of the emission wavelength and influences the AIE-active properties. With these probes, we succeeded in no-wash fluorogenic protein labeling and mitochondria-selective bioorthogonal imaging in live cells.

Tetrazine-based bio-orthogonally activated fluorogenic probes have drawn great attention due to their excellent performance in bioimaging; however, most of them suffer from aggregation-caused quenching (ACQ) problems. Herein, we developed a set of novel tetrazine-modified tetraphenylenes (TPEs) as bio-orthogonally activated aggregation-induced emission (AIE) fluorogenic probes. Both the fluorescence and AIE features are quenched by tetrazine, which is mediated by the through-bond energy-transfer (TBET) mechanism, and are activated upon converting tetrazine to pyridazine via the inverse electron-demand Diels–Alder (iEDDA) reaction. The activated cycloadducts displayed a notable fluorescence enhancement, a large Stokes shift, a high fluorescence quantum yield, and evident AIE-active features. Manipulating the length and position of the π-linker enables fine-tuning of the photophysical properties of the probes, while an overlong planar π-linker leads to AIE-to-ACQ transformation. We also designed bi-tetrazyl-substituted probes, which exhibited a higher turn-on ratio than the mono-tetrazyl analogs owing to the ‘double-quenched’ function. When they reacted with double-clickable linkers, fluorescent macrocycles were obtained because of the restriction of the free rotation of the phenyl rings of TPE. Using an organelle-pretargeting strategy, we succeeded in applying these probes for mitochondria-specific bio-orthogonal imaging in live cells under no-wash conditions, which is expected to provide a powerful tool for biomedical applications.