Scheme 2 depicts the synthetic route used to prepare the Mitochondrial Ratiometric Formaldehyde Probe (MitoRFAP) dyes described in this manuscript. First, compound 1 was acylated via a titanium-catalyzed Friedel-Crafts reaction to yield the alkyne-functionalized coumarin 2 in 63% yield, in accordance with a published procedure. Dye 2 was then formylated, using the Vilsmeier-Haack formylation, to isolate the corresponding coumarin aldehyde 3 in 74% yield. Aldehyde 3 was then transformed by amino-prenylation by treatment with ammonia followed by 3methyl-2-butenylboronic acid pinacol ester to achieve the modular RFAP-alkyne synthon 4. RFAP alkyne 4 was then ligated to the azide-TPP targeting agent via copper-catalyzed click chemistry. showing an approximately 5.5-fold change in excitation fluorescence ratios to FA within 2 hours as measured at lem = 510 nm with lex = 470 nm for product and lex = 420 nm for reactant (Figure ). This observed change in ratio is based on the FA-dependent, aza-Cope-driven conversion of an electron-donating homoallylamine group into an electron-withdrawing aldehyde, which leads to a red shift in the dye excitation profile by increasing favorability of internal charge transfer in this push- We then applied MitoRFAP-2 to a broader range of cell lines with co-staining using organelle markers. In each cell line, Mito-RFAP-2 showed faithful mitochondrial localization, suggesting that this probe can be used in live-cell mitochondrial FA imaging applications derived from a range of tissue types (Figure , S5, S5). To ensure that MitoRFAP-2 is sensitive to changes in mitochondrialFA levels in a live-cell context, we loaded the probe in HEK-293T and HeLa cells with subsequent exogenous additions of small amounts of FA. Additions of exogenous FA resulted in a dosedependent increase in the lex = 488 nm (product)/lex = 405 nm (reactant) ratio of the MitoRFAP-2 reagent (Figure ).