Fluorescent analogues of the potent and highly selective δ-opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) containing the exceptionally environmentally sensitive fluorescent amino acid β-(6′-dimethylamino-2′-naphthoyl)alanine (Aladan [Ald]) in place of Phe3 were synthesized. The Ald3- and D-Ald 3 analogues of TIPP and TIP all retained δ-opioid antagonist properties. The most potent analogue, [Ald3]TIPP, showed a K e value of 2.03 nM in the mouse vas deferens assay and five times higher δ vs. μ selectivity (Kiμ/K iδ = 7930) than the TIPP parent peptide in the opioid receptor binding assays. Theoretical conformational analyses of [Ald 3]TIPP and [Ald3]TIP using molecular mechanics calculations resulted in a number of low-energy conformers, including some showing various patterns of aromatic ring stacking and others with the Aid side chain and a carbonyl group (fluorescence quencher) in close proximity. These ensembles of low-energy conformers are in agreement with the results of steady-state fluorescence experiments (fluorescence emission maxima and quantum yields) and fluorescence decay measurements (fluorescence lifetime components), which indicated that the fluorophore was either engaged in intramolecular hydrophobic interactions or in proximity of a fluorescence quencher (e.g., a carbonyl group). These fluorescent TIP(P) δ-opioid antagonists represent valuable pharmacological tools for various applications, including studies on membrane interactions, binding to receptors, cellular uptake and intracellular distribution, and tissue distribution.
- Fluorescence decay measurements
- Fluorescent TIP(P) analogues
- Opioid activity profiles in vitro
- Steady-state fluorescence spectroscopy
- TIP(P) δ-opioid antagonists
- Theoretical conformational analysis