The complex network of chemical processes that sustain life motivates the development of new synthetic tools to decipher biological mechanisms of action at a molecular level. In this context, fluorescent and related optical probes have emerged as useful chemical reagents for monitoring small-molecule and metal signals in biological systems, enabling visualization of dynamic cellular events with spatial and temporal resolution. In particular, metals occupy a central role in this field as analytes in their own right, while also being leveraged for their unique biocompatible reactivity with small-molecule substrates. This Viewpoint highlights the use of inorganic chemistry principles to develop activity-based sensing platforms mediated by metal reactivity, spanning indicators for metal detection to metal-based reagents for bioorthogonal tracking, and manipulation of small and large biomolecules, illustrating the privileged roles of metals at the interface of chemistry and biology.

Chemical probes that report on protein activity, rather than protein abundance, with spatial and temporal resolution can enable studies of their native function in biological contexts as well as provide opportunities for developing new types of biochemical reporters. Here we present a sensing platform, termed proximity-activated imaging reporter (PAIR), which combines activity-based methionine bioconjugation and antibody labeling with proximity-dependent oligonucleotide-based amplification to monitor dynamic changes of a given analyte in cells and animals through context-dependent methionine labeling of specific protein targets. We establish this PAIR method to develop sensors for imaging reactive oxygen species (ROS) and calcium ions through oxaziridine-directed labeling of reactive methionine residues on β-actin and calmodulin (CaM), respectively, where the extent of methionine bioconjugation on these protein targets can serve as an indicator of oxidative stress or calcium status. In particular, application of PAIR to activity-based CaM detection provides a method for imaging integrated calcium activity in both in vitro cell and in vivo zebrafish models. By relying on native protein biochemistry, PAIR enables redox and metal imaging without introduction of external small molecules or genetically encoded indicators that can potentially buffer the natural/existing pools. This approach can be potentially generalized to target a broader range of analytes by pairing appropriate activity-based protein probes with protein detection reagents in a proximity-driven manner, providing a starting point not only for designing new sensors but also for monitoring endogenous activity of specific protein targets in biological specimens with spatial and temporal fidelity.

The AlkB family of nonheme Fe(II)/2-oxoglutarate-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6-methyladenosine (m6A) but furnish different major products, N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a direct m6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for nonredundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.

Chemical probes that report on protein activity, rather than protein abundance, with spatial and temporal resolution can enable studies of their native function in biological contexts as well as provide opportunities for developing new types of biochemical reporters. Here we present a sensing platform, termed proximity-activated imaging reporter (PAIR), which combines activity-based methionine bioconjugation and antibody labeling with proximity-dependent oligonucleotide-based amplification to monitor dynamic changes of a given analyte in cells and animals through context-dependent methionine labeling of specific protein targets. We establish this PAIR method to develop sensors for imaging reactive oxygen species (ROS) and calcium ions through oxaziridine-directed labeling of reactive methionine residues on β-actin and calmodulin (CaM), respectively, where the extent of methionine bioconjugation on these protein targets can serve as an indicator of oxidative stress or calcium status. In particular, application of PAIR to activity-based CaM detection provides a method for imaging integrated calcium activity in both in vitro cell and in vivo zebrafish models. By relying on native protein biochemistry, PAIR enables redox and metal imaging without introduction of external small molecules or genetically encoded indicators that can potentially buffer the natural/existing pools. This approach can be potentially generalized to target a broader range of analytes by pairing appropriate activity-based protein probes with protein detection reagents in a proximity-driven manner, providing a starting point not only for designing new sensors but also for monitoring endogenous activity of specific protein targets in biological specimens with spatial and temporal fidelity.

Biological and heterogeneous catalysts for the electrochemical CO₂ reduction reaction (CO₂RR) often exhibit a high degree of electronic delocalization that serves to minimize overpotential and maximize selectivity over the hydrogen evolution reaction (HER). Here, we report a molecular iron(II) system that captures this design concept in a homogeneous setting through the use of a redox non-innocent terpyridine-based pentapyridine ligand (tpyPY2Me). As a result of strong metal-ligand exchange coupling between the Fe(II) center and ligand, [Fe(tpyPY2Me)]²⁺ exhibits redox behavior at potentials 640 mV more positive than the isostructural [Zn(tpyPY2Me)]²⁺ analog containing the redox-inactive Zn(II) ion. This shift in redox potential is attributed to the requirement for both an open-shell metal ion and a redox non-innocent ligand. The metal-ligand cooperativity in [Fe(tpyPY2Me)]²⁺ drives the electrochemical reduction of CO₂ to CO at low overpotentials with high selectivity for CO₂RR (>90%) and turnover frequencies of 100 000 s^-1 with no degradation over 20 h. The decrease in the thermodynamic barrier engendered by this coupling also enables homogeneous CO₂ reduction catalysis in water without compromising selectivity or rates. Synthesis of the two-electron reduction product, [Fe(tpyPY2Me)]⁰, and characterization by X-ray crystallography, Mössbauer spectroscopy, X-ray absorption spectroscopy (XAS), variable temperature NMR, and density functional theory (DFT) calculations, support assignment of an open-shell singlet electronic structure that maintains a formal Fe(II) oxidation state with a doubly reduced ligand system. This work provides a starting point for the design of systems that exploit metal-ligand cooperativity for electrocatalysis where the electrochemical potential of redox non-innocent ligands can be tuned through secondary metal-dependent interactions.