Synthesis, Biological Evaluation, and Utility of Fluorescent Ligands Targeting the μ-Opioid Receptor

Abstract

Fluorescently labeled ligands are useful pharmacological research tools for studying receptor localization, trafficking, and signaling processes via fluorescence imaging. They are also employed in fluorescent binding assays. This study is centered on the design, synthesis, and pharmacological evaluation of fluorescent probes for the opioid receptors, for which relatively few non-peptidic fluorescent probes currently exist. The known μ-opioid receptor (MOR) partial agonist, buprenorphine, was structurally elaborated to include an amidoalkylamine linker moiety that was coupled with a range of fluorophores to afford new fluorescent probes. All compounds proved to be selective MOR antagonists. Confocal fluorescence microscopy studies revealed that the probe incorporating a sulfonated cyanine-5 fluorophore was the most appropriate for imaging studies. This ligand was subsequently employed in an automated fluorescence-based competition binding assay, allowing the pKi values of several well-known opioid ligands to be determined. Thus, this new probe will prove useful in future studies of MOR receptor pharmacology.

Development of novel fluorescent histamine H1-receptor antagonists to study ligand-binding kinetics in living cells

“…1b ). 6-(((4,4-Difluoro-5-(2-thienyl)-4-bora-3 a ,4 a -diaza- s -indacene-3-yl)styryloxy)acetyl)aminohexanoic acid (BODIPY630/650-X) was preferred due to previous success with this fluorophore for Class A GPCR fluorescent ligands 28 , 36 , 37 as it has favourable excitation/emission wavelength for planned techniques. BODIPY630/650 is known to have an increased quantum yield in a lipid environment 38 and as the modelling suggests the fluorophore would reside within the membrane bilayer this would increase its brightness for imaging studies and reduce background fluorescence in aqueous solution.…”

Fluorescently Labeled Morphine Derivatives for Bioimaging Studies

“…Sulfo-Cy5 was chosen as the fluorescent tag, as previous work by Schembri et al suggested that this particular fluorophore had the least potential for inducing non-specific binding to the cell membrane. 19 The functional handles on 11 and 15 were elongated with a simple Boc-protected diamine chain, followed by global deprotection and chemoselective conjugation to sulfo-Cy5 to give the final fluorescent morphine probes 18 and 21 (Schemes 4 and 5, respectively).…”

“…This new tool compound could also be used in conjunction with the previously described smallmolecule fluorescent opioid antagonists to study the differences in ligand-receptor complexes in natively expressing systems, rather than in transfected systems. 19 ¢ EXPERIMENTAL SECTION General Deprotection Procedure A. The protected compound was dissolved in dry DCM (typically 5 mL) under an N2 atmosphere.…”

“…In any case, a wash step is typically not included in confocal microscopy experiments, particularly where there is no significant background from extracellular ligand, as in this case. 19,29,30,31 To directly compare, experiments were conducted using the parent compound morphine as the ligand (Figure 7). Obviously the lack of a fluorescent tag prevents direct visualization of the ligand, but observation of the tagged receptor fluorescence is still of value, as it enables visual comparisons to be made between the behavior of our fluorescently tagged probe and its parent compound.…”

New Polyfluorinated Cyanine Dyes for Selective NIR Staining of Mitochondria

“…[6] The unique photophysical properties of cyanine dyes are used fort he photon upconversion in nanoparticles [7,8] or as contrasta gents in bioimaginga pplications. [9] Numerous examples of the utilization of cyanine fluorophores can be found in literature [2,[10][11][12][13][14][15] and their utilization is proven by thef luorescent contrasta gent indocyanine green (ICG, 1)a nd derivatives thereof (for example SIDAG [16] and cipate [17] ).…”