Labeling a protein of interest (POI) with a fluorescent reporter is a powerful strategy for studying protein structures and dynamics in their native environments. Compared to fluorescent proteins, synthetic dyes provide more choices in photophysical or photochemical attributes to microscopic characterizations. The specificity of bioorthogonal reactions in conjunction with the fidelity of subcellular destinations of genetically encoded protein tags can be employed to label POIs in live and fixed cells in a two-step process. In the present study the orthogonality of the strain-promoted azide–alkyne cycloaddition (SPAAC) and the inverse electron demand Diels–Alder (IEDDA) reaction is corroborated in concurrent labeling of two different intracellular targets. An azido group and a strained alkene are first installed at specific subcellular locations via orthogonal enzymatic reactions of the genetically incorporated SNAP- and CLIP-tags. The subsequent bioorthogonal reactions with fluorophores carrying matching reactive functionalities result in simultaneous dual labeling. The two-step “orthogonal-bioorthogonal” labeling process would increase the utilities of SNAP/CLIP-tags and, as a consequence, would expand the capability of decorating biological specimens with functionalities beyond fluorophores to potentially include spin labels, radioactive tracers, or catalysts.

Copper(II) acetate is a frequent empirical choice of the copper source in copper(II)‐mediated redox reactions. The effect of the acetate counterion appears crucial but has not been adequately investigated. Herein, we report that copper(II) acetate catalyzes the aerobic dehydrogenation of chelating aromatic secondary amines. The chemoselectivity of acetate and chelating amines in this reaction provides a unique opportunity for a mechanistic study. The progression of this homogeneous reaction is monitored by using electron paramagnetic resonance spectroscopy, UV/Vis absorption spectroscopy, and manometry. The kinetic dependence on the amine substrate, copper(II), and acetate counterion, together with the results of kinetic isotope and substituent effect experiments, suggests that acetate acts both as a bridging ligand of a dinuclear catalytic center for mediating two‐electron transfer steps and as a base in the turnover‐limiting C–H bond‐cleavage step. Upon including 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) as a surrogate base, DBU and acetate act in a complementary manner to enable a rapid, catalytic dehydrogenation reaction of a chelating secondary amine substrate. Finally, the contrasting reactivities between copper(II) acetate (promoting two‐electron transfer) and copper(II) perchlorate (promoting single‐electron transfer) underscores how a counterion could completely alter the mechanistic pathway of a copper‐mediated oxidation reaction.

SNAP-tag is a genetically encoded label for tracking proteins of interest that is known to utilize O 6 -benzylguanine derivatives as substrates. In this work, mass spectrometric analysis revealed that SNAP-tag also accepts O 6 -(5-pyridylmethyl)guanine derivatives as substrates. A fluorescently conjugated O 6 -(5-pyridylmethyl)guanine was synthesized and used to selectively label intracellular compartments. This compound also acts as a zinc(II) ion-sensitive fluorescent indicator and therefore can be used in organelle-specific zinc(II) imaging.

Genetically encoded protein or peptide tags and bioorthogonal chemistry are two growing classes of tools in cell biological research. A protein or peptide tag could be genetically fused to a protein of interest (POI) in the same manner as fluorescent proteins (FPs). An enzymatic ligation step would then follow to deliver a chemical entity, such as a dye or a probe with properties unattainable from FPs, to the tag, and consequently to the tethered POI. Bioorthogonal chemistry refers to a collection of (mostly) bimolecular conjugation reactions that are fine‐tuned to occur efficiently in a biological environment without the interference to or from the resident (bio)molecules or ions. These two areas of research have been developed independently and more or less concurrently. The combined use of them could expand the utilities of both tools, which is advocated in this Review. The mechanisms and utilities of protein or peptide tags and bioorthogonal chemistry are briefly reviewed separately in the first two sections, while in the third section the examples that utilize both protein or peptide tags and bioorthogonal chemistry are enumerated. Each tool and the strategy of their deployment carry their own advantages and disadvantages, and the selection of a particular tool depends on a given problem. The advances and combined use of the two topical areas are driven by the emerging challenges in illuminating the ever‐finer details of biological structures and actions.

Photoinduced electron transfer (PeT)-type fluorescent molecular switches are often applied in ion-selective sensors. Zinc-targeting sensors that contain an anilino-based electron donor (aka, the PeT 'switch') have multiple advantages over those with an aliphatic amino switch. In addition to the lower pK value of an aniline than that of a comparably substituted aliphatic amine, which reduces the interference of pH on the spectral properties of the attached fluorophore, the oxidation potentials of anilino groups are lower than those of aliphatic amino counterparts, which make them better electron donors in PeT. The effectiveness of anilino as a PeT switch is evaluated in a series of zinc-sensitive sensors that contain different fluorophores, zinc-binding ligands, and alkyl linkers between ligand and fluorophore. The abilities of these compounds to distinguish high and low intracellular zinc concentrations in living cells are demonstrated.