Modular development of organelle-targeting fluorescent probes for imaging formaldehyde in live cells.

Formaldehyde (FA) is endogenously generated via fundamental biological processes in living systems. Aberrant FA homeostasis in subcellular microenvironments is implicated in numerous pathological conditions. Fluorescent probes for detecting FA in specific organelles are thus of great research interest. Herein, we present a modular strategy to construct diverse organelle-targeting FA probes by incorporating selective organelle-targeting moieties into the scaffold of a 1,8-naphthalimide-derived FA fluorescent probe. These probes react with FA through the 2-aza-Cope arrangement and exhibit highly selective fluorescence increases for detecting FA in aqueous solutions. Moreover, these organelle-targeting probes, i.e., FFP551-Nuc, FFP551-ER, FFP551-Mito, and FFP551-Lyso, allow selective localization and imaging of FA in the nucleus, endoplasmic reticulum, mitochondria, and lysosomes of live mammalian cells, respectively. Furthermore, FFP551-Nuc has been successfully employed to monitor changes of endogenous FA levels in the nucleus of live mammalian cells. Overall, these probes should represent new imaging tools for studying the biology and pathology associated with FA in different intracellular compartments.

Systematic investigation of the aza-Cope reaction for fluorescence imaging of formaldehyde in vitro and in vivo.

Increasing evidence has highlighted the endogenous production of formaldehyde (FA) in a variety of fundamental biological processes and its involvement in many disease conditions ranging from cancer to neurodegeneration. To examine the physiological and pathological relevance and functions of FA, fluorescent probes for FA imaging in live biological samples are of great significance. Herein we report a systematic investigation of 2-aza-Cope reactions between homoallylamines and FA for identification of a highly efficient 2-aza-Cope reaction moiety and development of fluorescent probes for imaging FA in living systems. By screening a set of N-substituted homoallylamines and comparing them to previously reported homoallylamine structures for reaction with FA, we found that N-p-methoxybenzyl homoallylamine exhibited an optimal 2-aza-Cope reactivity to FA. Theoretical calculations were then performed to demonstrate that the N-substituent on homoallylamine greatly affects the condensation with FA, which is more likely the rate-determining step. Moreover, the newly identified optimal N-p-methoxybenzyl homoallylamine moiety with a self-immolative ß-elimination linker was generally utilized to construct a series of fluorescent probes with varying excitation/emission wavelengths for sensitive and selective detection of FA in aqueous solutions and live cells. Among these probes, the near-infrared probe FFP706 has been well demonstrated to enable direct fluorescence visualization of steady-state endogenous FA in live mouse brain tissues and elevated FA levels in a mouse model of breast cancer. This study provides the optimal aza-Cope reaction moiety for FA probe development and new chemical tools for fluorescence imaging and biological investigation of FA in living systems.