title: Development of a General Aza-Cope Reaction Trigger Applied to Fluorescence Imaging of Formaldehyde in Living Cells.
abstract: Formaldehyde (FA) is a reactive signaling molecule that is continuously produced through a number of central biological pathways spanning epigenetics to one-carbon metabolism. On the other hand, aberrant, elevated levels of FA are implicated in disease states ranging from asthma to neurodegenerative disorders. In this context, fluorescence-based probes for FA imaging are emerging as potentially powerful chemical tools to help disentangle the complexities of FA homeostasis and its physiological and pathological contributions. Currently available FA indicators require direct modification of the fluorophore backbone through complex synthetic considerations to enable FA detection, often limiting the generalization of designs to other fluorophore classes. To address this challenge, we now present the rational, iterative development of a general reaction-based trigger utilizing 2-aza-Cope reactivity for selective and sensitive detection of FA in living systems. Specifically, we developed a homoallylamine functionality that can undergo a subsequent self-immolative β-elimination, creating a FA-responsive trigger that is capable of masking a phenol on a fluorophore or any other potential chemical scaffold for related imaging and/or therapeutic applications. We demonstrate the utility of this trigger by creating a series of fluorescent probes for FA with excitation and emission wavelengths that span the UV to visible spectral regions through caging of a variety of dye units. In particular, Formaldehyde Probe 573 (FAP573), based on a resorufin scaffold, is the most red-shifted and FA sensitive in this series in terms of signal-to-noise responses and enables identification of alcohol dehydrogenase 5 (ADH5) as an enzyme that regulates FA metabolism in living cells. The results provide a starting point for the broader use of 2-aza-Cope reactivity for probing and manipulating FA biology.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Bruemmer
authors.forename: Ryan R
authors.initials: RR
authors.lastname: Walvoord
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Brewer
authors.affiliation: MRC Laboratory of Molecular Biology , Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Burgos-Barragan
authors.affiliation: MRC Laboratory of Molecular Biology , Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Niek
authors.initials: N
authors.lastname: Wit
authors.affiliation: MRC Laboratory of Molecular Biology , Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
authors.forename: Lucas B
authors.initials: LB
authors.lastname: Pontel
authors.affiliation: MRC Laboratory of Molecular Biology , Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.|Department of Medicine, Addenbrooke's Hospital, University of Cambridge , Cambridge CB2 2QQ, United Kingdom.
authors.forename: Ketan J
authors.initials: KJ
authors.lastname: Patel
authors.forename: Christopher J
authors.identifier: 0000-0001-5732-9497
authors.initials: CJ
authors.lastname: Chang
mesh_terms: D001372:Aza Compounds / Q000737:chemistry*; D002470:Cell Survival; D005557:Formaldehyde / Q000032:analysis* / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D015394:Molecular Structure; D061848:Optical Imaging
chemical_list: D001372:Aza Compounds; D005557:Formaldehyde