One-carbon metabolism plays a central role in regulating cellular metabolism and epigenetics, but aberrations in one-carbon cycles are associated with the progression of serious diseases, including diabetes, chronic liver and heart diseases, neurodegenerative diseases, and cancer. In this context, formate is a primary one-carbon unit that is produced, utilized, and regulated within one-carbon metabolic cycles and is essential for sustaining amino acid (serine, glycine, methionine) and nucleotide (purine/thymidylate) biosynthesis, as well as redox, energy, and epigenetic homeostasis. In mammals, formate concentrations normally reside in range of 10-100 µM in blood and can rise to above 1 mM within cells; 6 moreover, its levels can fluctuate greatly between healthy and diseased states. Indeed, increased formate overflow is a potential biomarker for neurodegenerative Alzheimer's disease and a hallmark of oxidative stress in cancer. In one striking clinical study, formate was posited as a potential biomarker for esophageal cancer progression, with the observation that formate levels progressively increased in esophageal tumors relative to normal mucosae from Stage I (8.45x) to Stage II (13.51x) to Stage III (14.84x) to Stage IV (21.48x). Despite its significant contributions to the fundamental chemistry of the cell and potential translational applications to medicine, methods for monitoring formate within intact, living biological specimens remain underdeveloped. Along these lines, current methods for formate analysis in biological samples include its derivatization through amide bond formation to formyl-2-nitrophenyl-hydrazide for detection by LC-MS, enzyme-mediated colorimetric detection in post-lysis specimens, and NMR analysis, all of which enable formate quantification but only in simple biochemical mixtures and/or with extensive sample processing/destruction. We envisioned that activity-based sensing (ABS), which leverages the intrinsic chemical reactivity of an analyte for its selective and sensitive detection, could present a complementary strategy for formate analysis that is compatible with live cells and can provide spatiotemporal information regarding one-carbon metabolic flux. As part of a growing field to discover and decipher the chemical roles that dynamic one-carbon metabolites play at a cellular level, our laboratory and others have developed ABS methods for carbon monoxide, formaldehyde, and carbon dioxide, as well as related carbon signaling molecules such as ethylene. Here, we report an organometallic ABS method for detection of biological formate using a two-component system, where formate-mediated iridium transfer hydrogenation (TH) catalysis enables aldehyde-to-alcohol reduction on fluorophore scaffolds accompanied by a fluorescence response (Figure ). Owing to its selectivity and sensitivity for this key one-carbon unit, this ABS system can monitor changes in endogenous formate levels in living cells, including in cell-based models of cancer with altered one-carbon metabolism. Activitybased sensing (ABS) methods achieve selectivity by judicious exploitation of the intrinsic reactivity of an analyte of interest for its detection, rather than traditional binding-based approaches that rely on lock-and-key molecular recognition. This strategy is particularly attractive for detection of reactive small molecules in biological systems, such as reactive carbon species, as these species are often transient and can interconvert between competing biological analytes of similar shape and size. In this regard, we recognized that formate is a hydride donor (∆Gº=24.1 kcal/mol in H2O). This reactivity is illustrated by the Eschweiler-Clarke reaction, its widespread use in transfer hydrogenation (TH) catalysis, and its biochemical roles in regeneration of NADPH from NADP + via ALDH1L1/2 activity. Therefore, we selected transfer hydrogenation catalysis as an ABS strategy for monitoring cellular formate fluxes (Figure ). To this end, we sought to develop a two-component system for activity-based formate sensing comprised of a transfer hydrogenation catalyst that uses formate as a reductant along with a fluorescent dye substrate that generates a fluorescence response upon its reduction. To achieve this goal of formate ABS, we sought to solve the following synthetic methods challenges: 1) develop an appropriate fluorophore platform where reductive aldehyde-to-alcohol conversion would generate a measurable fluorescence response, 2) identify a transition metal catalyst for formate-mediated transfer hydrogenation that could operate with sufficiently high activity and robustness in the complex milieu of living cells, and 3) tune the catalyst/fluorophore pair so as to achieve biologically-relevant selectivity for formate over other potential cellular hydride sources (e.g, NAD(P)H, 5,10-Me-THF, and FADH2) and selectivity for the aldehydic fluorophore over other hydride acceptors, including electrophiles such as NAD(P) + , carbonyls, etc., and oxidants such as O2, H2O2, etc. We discuss below how comprehensive screening experiments led to the selection and use of fluorophore F-7 and complex 9 as a two-component system for enabling visualization of formate fluxes in living cells using confocal microscopy (Figure ). Fluorophore Development and Selection for Turn-On and Ratiometric Responses to Formate. At the outset of our investigations, we sought to identify an aldehyde-containing fluorescent dye platform where formate-mediated reduction to the corresponding alcohol fluorophore would give an intensitybased turn-on response. In this context, known aldehyde-containing fluorophores reported to have the highest intensitybased turn-on responses upon aldehyde-to-alcohol reduction in the literature are SI-1, a push-pull fluorophore, which provides a ~10x increase in emission intensity with aldehyde-toalcohol reduction; however, this dye scaffold requires high-energy ultraviolet excitation at 300 nm. In terms of shifting optical responses to the visible excitation/emission region, which minimizes cellular autofluorescence and photodamage, the BODIPY fluorophore SI-2 achieves excitation at 480 nm but shows only a ~5x increase upon reduction (Table ). We thus turned our attention to fluorescein-like xanthenone scaffolds, as rational principles for modulation of their fluorescence properties have been elucidated by Nagano and Urano. In particular, we sought to employ a donor photoinduced electron transfer (dPeT) strategy to develop aldehyde dyes that would exhibit significant turn-on responses upon reduction to their alcohol congeners at lower energy visible excitation wavelengths (λex ~500 nm). Based on the aforementioned reasoning, we calculated LUMO energy values for selected substituted benzaldehydes and then correlated these values with fluorescence quantum yields measured experimentally in previous reports. This data-driven process identified aldehyde F-1 and its corresponding alcohol F-9 as a candidate pair for a turn-on dPeT fluorescence response (Figure ). We then made two other considerations in selecting F-1. First, we placed the activity-based sensing aldehyde group at the meta position of the aromatic bottom ring to mitigate potential undesirable reactivity with cellular aldehyde dehydrogenases, which can react with para benzaldehydes. Second, our calculations indicated that the hydrate form of F-1, designed as F-8, is also expected to be bright (Figure ), so an analysis correlating LUMO energy values with experimentally measured equilibrium values for substituted benzaldehyde-hydrate pairs was also conducted (Figure ). This analysis predicted a 98.5 to 1.5 ratio between F-1 and F-8 in pH 7 water -sufficiently in favor of the aldehyde form to motivate synthetic efforts. Milligram quantities of fluorophore F-1 were obtained in 7 steps from compound 20 (Figure ). Gratifyingly, we found that F-9 provides an ~11-fold increase in emission intensity relative to F-1 upon excitation at 500 nm under physiologically relevant aqueous conditions in vitro (pH 7.4 phosphate-buffered saline at 37 ºC, Figure ). We employed fluorophore F-1 for catalyst evaluation and selectivity optimization studies in vitro, but this intensity-based scaffold did not provide reliable turn-on responses to formate in preliminary confocal imaging experiments (vide infra). Instead, we observed time-and formate-dependent decreases in fluorescence intensity, which we speculated may be due to fluorophore export and/or degradation from formate-stimulated production of ROS, 52 among other possibilities. As several attempts overcome this problem were ineffective, we sought to replace the intensity-responsive fluorophore F-1 with a ratiometric fluorophore. We reasoned that this alternative approach would enable us to determine changes in formate concentrations based on the relative ratio of aldehyde/alcohol fluorophore species via two distinct excitation wavelengths with an internal self-calibration, rather than rely on the absolute intensity of emission of a mixture of fluorophores at a single wavelength. Indeed, twocolor ratiometric measurements can minimize artifacts that can arise from variations in sample illumination and/or collection, non-homogeneous probe loading, changes in pH and probe localization, and fluorophore export and/or degradation. 18 Evaluation of several types of dyes led us to aldehyde F-7 and its corresponding alcohol form, F-10 (Figure ), which display suitable excitation scan profiles (λex = 400-500 nm respectively and λem = 510 nm) and sufficiently low steric hindrance about the aldehyde functional group to facilitate reduction by various transfer hydrogenation catalysts, including Catalyst 9 (Figure ). By comparison, aldehyde F-2 required ultraviolet excitation at 300 nm, which is incompatible with live-cell imaging. Likewise, reduction of F-3 resulted in a turn-off response, while dyes F-4 and F-5 not exhibit suitable ratiometric behavior. Finally, aldehyde F-6 underwent reduction more slowly than F-7 suitable catalyst system for formate-mediated reduction of this aldehyde dye to its alcohol congener. Of the thousands of known transition metal TH catalysts, we focused on a subset of iridium, osmium, and ruthenium complexes reported to engage in transfer hydrogenation under aqueous conditions (Figure ). Most notable among these systems, the Do laboratory has reported an unprotected half-sandwich iridium complex, which can reduce the aldehydic BODIPY fluorophore SI-2 (Table The one exception was iridium catalyst 3, which is known to reduce molecular oxygen to hydrogen peroxide and did not reduce F-1. Therefore, we focused our efforts on screening iridium complexes bearing bidentate N-N chelating ligands. We were gratified to find that iridium complexes bearing either pyridine amide and sulfonamide amine ligands reduce fluorophore F-1 at 10 mM concentrations of F-1 and metal complex in a formate-dependent manner, consistent with the previously observed first order rate dependence 65 on all three reactants. To evaluate selectivity between biologically relevant hydride sources, representative iridium catalysts 4 and 15 were initially surveyed in vitro for reduction of aldehyde F-1 with NADH, NADPH, FADH2 and 5,10-Me-THF. These pilot experiments indicated that NAD(P)H, but not FADH2 or ).