Type and location of fluorescent probes incorporated into the potent μ‐opioid peptide [Dmt1]DALDA affect potency,receptor selectivity and intrinsic efficacy*

Abstract: The dermorphin‐derived tetrapeptide H‐Dmt‐d ‐Arg‐Phe‐Lys‐NH₂ (Dmt = 2′,6′‐dimethyltyrosine) ([Dmt¹]DALDA) is a highly potent and selective μ‐opioid agonist capable of crossing the blood–brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt¹]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6‐dimethylamino‐2′‐naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea‐pig ileum and mouse vas deferens assays, and in μ‐, δ‐ and κ‐opioid receptor‐binding assays. The analogues showed various degrees of μ receptor‐binding selectivity, but all of them were less μ‐selective than the [Dmt¹]DALDA parent peptide. Most analogues retained potent, full μ‐agonist activity, except for one with fluorescein attached at the C‐terminus ( 3a ) (partial μ‐agonist) and one containing β‐(6′‐dimethylamino‐2′‐naphthoyl)alanine (aladan) in place of Phe³ ( 4 ) (μ‐ and κ‐antagonist). The obtained data indicate that the receptor‐binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence‐labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group ( 2a ) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy.