A near-infrared fluorogenic dimer enables background-free imaging of endogenous GPCRs in living mice.

Fluorescent probes are commonly used in studying G protein-coupled receptors in living cells; however their application to the whole animal receptor imaging is still challenging. To address this problem, we report the design and the synthesis of the first near-infrared emitting fluorogenic dimer with environment-sensitive folding. Due to the formation of non-fluorescent H-aggregates in an aqueous medium, the near-infrared fluorogenic dimer displays a strong turn-on response (up to 140-fold) in an apolar environment and exceptional brightness: 56% quantum yield and ≈444 000 M-1 cm-1 extinction coefficient. Grafted on a ligand of the oxytocin receptor, it allows the unprecedented background-free and target-specific imaging of the naturally expressed receptor in living mice.

Chemoselective Acylation of Hydrazinopeptides to Access Fluorescent Probes for Time-Resolved FRET Assays on GPCRs.

Fluorescence techniques represent a powerful tool to investigate dynamic and functional architecture of GPCRs. Thus, fluorescent GPCR ligands have found various applications in cellular imaging, in the development of binding assays as replacements for radioligands in the study of ligand-receptor but also in receptor-receptor interactions at the cell surface or in native tissues. To extend the applicability of these techniques, the design and the synthesis of fluorescent probes are critical steps. As there are numerous peptide receptors in the GPCR family, fluorescent peptide-based probes are of importance. Herein, we present a convenient method to facilitate the solution-phase fluorescent labeling of peptides which is based on the chemoselective acylation of α-hydrazinopeptides. This approach combines the advantages to use commercially available amine-reactive dyes and very mild conditions that are fully compatible with the chemical sensitivity of the dyes. It gives a rapid access to fluorescent peptidic probes compatible with the time-resolved fluorescence resonance energy transfer (TR-FRET) techniques.

[11C]PF-3274167 as a PET radiotracer of oxytocin receptors: Radiosynthesis and evaluation in rat brain.

INTRODUCTION: Oxytocin plays a major role in the regulation of social interactions in mammals by interacting with the oxytocin receptor (OTR) expressed in the brain. Furthermore, the oxytocin system appears as a possible therapeutic target in autism spectrum disorders and other psychiatric troubles, justifying current pharmacological researches. Since no specific PET radioligand is currently available to image OTR in the brain, the aim of this study was to radiolabel the specific OTR antagonist PF-3274167 and to evaluate [11C]PF-3274167 as a potential PET tracer for OTR in rat brains. METHODS: [11C]PF-3274167 was prepared via the O-methylation of its desmethyl precursor with [11C]methyl iodide. The lipophilicity of the radioactive compound was evaluated by measuring the n-octanol-buffer partition coefficient (logD). Autoradiography experiments were performed on rat brain tissue to evaluate the in vitro distribution of the [11C]PF-3274167. MicroPET experiments were conducted with and without pre-injection of ciclosporin in order to evaluate the influence of the P-glycoprotein (P-gp) on the brain uptake. RESULTS: [11C]PF-3274167 was synthesized with high radiochemical and chemical purities (>95%) and good specific activity. The measured logD was 1.93. In vitro, [11C]PF-3274167 did not show any evidence of specific binding to OTR. PET imaging showed that [11C]PF-3274167 uptake in rat brain was very low in basal conditions but increased significantly after the administration of ciclosporin, suggesting that it is a substrate of the P-gp. In the ciclosporin-pre-injected rat, however, [11C]PF-3274167 distribution did not match with the known distribution of OTR in rats. CONCLUSION: [11C]PF-3274167 is not a suitable tracer for imaging of OTR in rat brain, probably because of a too low affinity for this receptor in addition to a poor brain penetration.

Convenient Access to Fluorescent Probes by Chemoselective Acylation of Hydrazinopeptides: Application to the Synthesis of the First Far-Red Ligand for Apelin Receptor Imaging.

Herein, we develop a convenient method to facilitate the solution-phase fluorescent labelling of peptides based on the chemoselective acylation of α-hydrazinopeptides. This approach combines the advantages of using commercially available amine-reactive dyes and very mild conditions, which are fully compatible with the chemical sensitivity of the dyes. The usefulness of this approach was demonstrated by the labelling of apelin-13 peptide. Various fluorescent probes were readily synthesized, enabling the rapid optimization of their affinities for the apelin receptor. Thus, the first far-red fluorescent ligand with sub-nanomolar affinity for the apelin receptor was characterized and shown to track the receptor efficiently in living cells by fluorescence confocal microscopy.

Push-pull dioxaborine as fluorescent molecular rotor: far-red fluorogenic probe for ligand-receptor interactions.

Fluorescent solvatochromic dyes and molecular rotors increase their popularity as fluorogenic probes for background-free detection of biomolecules in cellulo in no-wash conditions. Here, we introduce a push-pull boron-containing (dioxaborine) dye that presents unique spectroscopic behavior combining solvatochromism and molecular rotor properties. Indeed, in organic solvents, it shows strong red shifts in the absorption and fluorescence spectra upon increase in solvent polarity, typical for push-pull dyes. On the other hand, in polar solvents, where it probably undergoes Twisted Intramolecular Charge Transfer (TICT), the dye displays strong dependence of its quantum yield on solvent viscosity, in accordance to Förster-Hoffmann equation. In comparison to solvatochromic and molecular rotor dyes, dioxaborine derivative shows exceptional extinction coefficient (120,000 M-1 cm-1), high fluorescence quantum yields and red/far-red operating spectral range. It also displays much higher photostability in apolar media as compared to Nile Red, a fluorogenic dye of similar color. Its reactive carboxy derivative has been successfully grafted to carbetocin, a ligand of the oxytocin G protein-coupled receptor. This conjugate exhibits >1000-fold turn on between apolar 1,4-dioxane and water. It targets specifically the oxytocin receptor at the cell surface, which enables receptor imaging with excellent signal-to-background ratio (>130). We believe that presented push-pull dioxaborine dye opens a new page in the development of fluorogenic probes for bioimaging applications.

Imaging and manipulating proteins in live cells through covalent labeling.

The past 20 years have witnessed the advent of numerous technologies to specifically and covalently label proteins in cellulo and in vivo with synthetic probes. These technologies range from self-labeling proteins tags to non-natural amino acids, and the question is no longer how we can specifically label a given protein but rather with what additional functionality we wish to equip it. In addition, progress in fields such as super-resolution microscopy and genome editing have either provided additional motivation to label proteins with advanced synthetic probes or removed some of the difficulties of conducting such experiments. By focusing on two particular applications, live-cell imaging and the generation of reversible protein switches, we outline the opportunities and challenges of the field and how the synergy between synthetic chemistry and protein engineering will make it possible to conduct experiments that are not feasible with conventional approaches.

Selective nonpeptidic fluorescent ligands for oxytocin receptor: design, synthesis, and application to time-resolved FRET binding assay.

The design and the synthesis of the first high-affinity fluorescent ligands for oxytocin receptor (OTR) are described. These compounds enabled the development of a TR-FRET based assay for OTR, readily amenable to high throughput screening. The validation of the assay was achieved by competition experiments with both peptide and nonpeptide OTR ligands as competitors. These probes represent the first selective fluorescent ligands for the oxytocin G protein-coupled receptor.

Fluorogenic squaraine dimers with polarity-sensitive folding as bright far-red probes for background-free bioimaging.

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.

Red fluorescent turn-on ligands for imaging and quantifying G protein-coupled receptors in living cells.

Classical fluorescence-based approaches to monitor ligand-protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn-on probes for a G protein-coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor-specific turn-on response. The new ligand was successfully applied for background-free imaging and quantification of oxytocin receptors in living cells.

Rational design of a solvatochromic fluorescent uracil analogue with a dual-band ratiometric response based on 3-hydroxychromone.

Fluorescent nucleoside analogues with strong and informative responses to their local environment are in urgent need for DNA research. In this work, the design, synthesis and investigation of a new solvatochromic ratiometric fluorophore compiled from 3-hydroxychromones (3HCs) and uracil fragments are reported. 3HC dyes are a class of multi-parametric, environment-sensitive fluorophores providing a ratiometric response due to the presence of two well-resolved bands in their emission spectra. The synthesized conjugate demonstrates not only the preservation but also the improvement of these properties. The absorption and fluorescence spectra are shifted to longer wavelengths together with an increase of brightness. Moreover, the two fluorescence bands are better resolved and provide ratiometric responses across a broader range of solvent polarities. To understand the photophysical properties of this new fluorophore, a series of model compounds were synthesized and comparatively investigated. The obtained data indicate that uracil and 3HC fragments of this derivative are coupled into an electronic conjugated system, which on excitation attains strong charge-transfer character. The developed fluorophore is a prospective label for nucleic acids. Abstract in Ukrainian: .