title: Fluorogenic squaraine dimers with polarity-sensitive folding as bright far-red probes for background-free bioimaging.
abstract: Polarity-sensitive fluorogenic dyes raised considerable attention because they can turn on their fluorescence after binding to biological targets, allowing background-free imaging. However, their brightness is limited, and they do not operate in the far-red region. Here, we present a new concept of fluorogenic dye based on a squaraine dimer that unfolds on changing environment from aqueous to organic and thus turns on its fluorescence. In aqueous media, all three newly synthesized dimers displayed a short wavelength band characteristic of an H-aggregate that was practically nonfluorescent, whereas in organic media, they displayed a strong fluorescence similar to that of the squaraine monomer. For the best dimer, which contained a pegylated squaraine core, we obtained a very high turn-on response (organic vs aqueous) up to 82-fold. Time-resolved studies confirmed the presence of nonfluorescent intramolecular H-aggregates that increased with the water content. To apply these fluorogenic dimers for targeted imaging, we grafted them to carbetocin, a ligand of the oxytocin G protein-coupled receptor. A strong receptor-specific signal was observed for all three conjugates at nanomolar concentrations. The probe derived from the core-pegylated squaraine showed the highest specificity to the target receptor together with minimal nonspecific binding to serum and lipid membranes. The obtained dimers can be considered as the brightest polarity-sensitive fluorogenic molecules reported to date, having ∼660,000 M(-1) cm(-1) extinction coefficient and up to 40% quantum yield, whereas far-red operation region enables both in vitro and in vivo applications. The proposed concept can be extended to other dye families and other membrane receptors, opening the route to new ultrabright fluorogenic dyes.
authors.affiliation: Laboratoire d'Innovation Thérapeutique, UMR 7200 CNRS/Université de Strasbourg, Labex MEDALIS , Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France.
authors.forename: Iuliia A
authors.initials: IA
authors.lastname: Karpenko
authors.forename: Mayeul
authors.initials: M
authors.lastname: Collot
authors.forename: Ludovic
authors.initials: L
authors.lastname: Richert
authors.forename: Christel
authors.initials: C
authors.lastname: Valencia
authors.forename: Pascal
authors.initials: P
authors.lastname: Villa
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
authors.forename: Marcel
authors.initials: M
authors.lastname: Hibert
authors.forename: Dominique
authors.initials: D
authors.lastname: Bonnet
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Klymchenko
mesh_terms: D003503:Cyclobutanes / Q000138:chemical synthesis / Q000737:chemistry*; D019281:Dimerization; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D015394:Molecular Structure; D010636:Phenols / Q000138:chemical synthesis / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry
chemical_list: D003503:Cyclobutanes; D005456:Fluorescent Dyes; D010636:Phenols; D012997:Solvents; C480596:squaraine