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.

Mechanosensitive Oligodithienothiophenes: Transmembrane Anion Transport Along Chalcogen‐Bonding Cascades

“…Namely, with DOPC membranes in L d phase at 55 °C and 25 °C, the excitation maxima were nearly identical. Moreover, the emission maximum λ em = 575 nm was essentially insensitive to changes from S o to L d phase, confirming that contributions from other mechanisms like solvatochromism do not account for the observed changes in excitation ( Figure ,f ). According to lessons learned from dimeric flipper probes including DA homolog 3 , the response in excitation of the elongated trimeric DDA probe 2 to increasing order in the surrounding membrane originates from planarization in the ground state, i.e ., mechanosensitivity ( Figure ,c ).…”

“…Mechanical planarization of the twisted push‐pull probe 3 in the ground state causes the excitation maxima to shift to the red. This differs conceptually from other membrane probes that operate by deplanarization (molecular rotors), polarization (solvatochromism) and electron or proton transfer in the excited state and thus report in emission rather than excitation . The resulting flipper probes 3 are important because they provide access to the so far elusive imaging of forces such as membrane tension in living cells (unpublished).…”

Recent Development of Chemosensors Based on Cyanine Platforms

“…To further explore its potential as bioimaging agent, they coupled 221 with an oxytocin receptor ligand (Lys 8 -carbetocin) to obtain conjugate 222. 266 The foldamer showed low nonspecific binding in the presence of lipids or proteins (BSA was selected as an example), because PEG groups from both ends of the squaraines prevent the molecule from interacting with BSA and thus maintaining the form of nonfluorescent H-aggregates. However, it was able to image the oxytocin receptor of HEK293 cells due to the specificbinding induced polarity change.…”

Twisted Push‐Pull Probes with Turn‐On Sulfide Donors

“…The application of this more complex lesson from nature to fluorescent probes has received little attention so far. However, a combination of planarization and polarization in the ground state appeared most intriguing, particularly with regard to fluorescent probes that could report on key characteristics of biomembranes such as fluidity, potential as well as the so far elusive membrane tension …”