Formaldehyde (FA) is a common environmental toxin that is also produced naturally in the body through a wide range of metabolic and epigenetic processes, motivating the development of new technologies to monitor this reactive carbonyl species (RCS) in living systems. Herein, we report a pair of first-generation chemiluminescent probes for selective formaldehyde detection. Caging phenoxy-dioxetane scaffolds bearing different electron-withdrawing groups with a general 2-aza-Cope reactive formaldehyde trigger provides chemiluminescent formaldehyde probes 540 and 700 (CFAP540 and CFAP700) for visible and near-IR detection of FA in living cells and mice, respectively. In particular, CFAP700 is capable of visualizing FA release derived from endogenous folate metabolism, providing a starting point for the use of CFAPs and related chemical tools to probe FA physiology and pathology, as well as for the development of a broader palette of chemiluminescent activity-based sensing (ABS) probes that can be employed from in vitro biochemical to cell to animal models.
Formaldehyde (FA) is a reactive carbonyl species (RCS) most commonly associated with being an external environmental pollutant, but it is also produced internally through a diverse array of biological processes.1, 2 This major one-carbon unit lies at the nexus of metabolism and epigenetics, participating in the synthesis of key biological molecules, including purines, amino acids, and neurotransmitters,3–5 as well as in the methylation status of a variety of nucleic acid, protein, and small-molecule metabolites.6–10
The small and transient nature of FA has motivated growing interest in developing new activity-based sensing (ABS) methods11–14 for selective FA detection,15, 16 including aza-Cope,17–24 aminal,25, 26 and formimine27–32 reaction-based approaches. Indeed, recent progress in developing fluorescent probes for FA detection in cells has elucidated more sophisticated biological roles for FA as both an exogenous toxin and an endogenous signaling molecule. Included is the discovery that FA is endogenously produced in the folate cycle through specific intermediates like tetrahydrofolate and 5,10-methylenetetrahydrofolate, but not others like 5-methyltetrahydrofolate,8 opening new doors to investigate the intriguing yet complex relationships between FA, methylation status, and carcinogenesis. Despite these advances in fluorescent FA detection, FA imaging in living mammals is limited to an aza-Cope-based PET tracer,19 which requires specialized equipment to implement. As such, we sought to develop a single, general platform for FA-selective ABS that would be applicable across a broader spectrum of biological models from in vitro biochemical to cell to in vivo animal systems. To this end, we turned our attention to chemiluminescent imaging, which offers an attractive approach in that it does not require external light irradiation, resulting in minimal background signal.33–37 Herein, we report first-generation chemiluminescent probes capable of visualizing changes in FA from in vitro to in vivo mouse models, offering a versatile approach for ABS of this central one-carbon molecule.
Our design relies upon a class of luminophores based on Schaap’s dioxetane,38, 39 which has been exploited to create chemiluminescent probes for analytes spanning reactive oxygen/sulfur species40–44 and enzyme activity.42, 45, 46 Recent work from one of our labs has shown that introducing electron-withdrawing groups at the ortho position of the phenol can result in a 1000-fold increase in chemiluminescence quantum efficiency and the ability to tune emission profiles,42 particularly to the near-IR region to enable better tissue penetration for in vivo imaging.47 Based on this scaffold, we created a set of chemiluminescent FA probes by caging this phenol with a 2-aza-Cope FA-reactive trigger (Scheme 1).20 Chemiluminescent formaldehyde probes 540 and 700 (CFAP540 and CFAP700) feature visible and near-IR emission profiles designed for in vitro cell and in vivo animal applications, respectively (see the Supporting Information for synthetic details). Reaction of FA at the homoallylamine, followed by immolation of the two-carbon linker through a β-elimination, yields the free phenoxy-dioxetane that subsequently decomposes through chemiexcitation to produce a photon.
