Highly selective photoaffinity labeling of mu and delta opioid receptors.

Abstract

We report the synthesis and photolabeling properties of two highly selective ligands for ,u and 8 opioidbinding sites: Tyr-D-Ala-Gly-MePhe(pN3)-Gly-ol (AZ-DAMGE) and Tyr-D-Thr-Gly-Phe(pN3)-Leu-Thr (AZ-DTLET). An irreversible inhibition of the electrically induced contractions of mouse vas deferens is caused by irradiation (at 254 nm) of the muscle strip in the presence of AZ-DTLET (1 nM). This phenomenon is antagonized only at large concentrations (10 FM) of naloxone, in accordance with the well-known lower selectivity of naloxone for 8 sites. Competition experiments with [3H]DAMGE and [3HJDTLET on crude rat brain membranes showed that the azido photoprobes display a similar (AZ-DAMGE) and even a better (AZ-DTLET) selectivity than their respective parent compounds DAMGE and DTLET. Up to 25 nM, AZ-DTLET irreversibly and selectively photolabels the 6 sites of crude rat brain homogenates. Due to its lower affinity AZ-DAMGE provides similar selective photolabeling of the jA sites but at higher concentrations (=0.3 jiM). When

An affinity label for δ‐opioid receptors derived from [d‐Ala2]deltorphin I*

“…The only affinity label derivatives of agonists modified in the ‘message’ sequence reported to date have been photoaffinity labels that are derivatives of the enkephalins [i.e. [Phe( p ‐N 3 ) 4 ]DTLET (12) and [Phe( p ‐N 3 ) 4 ]‐DAMGO (12)]. Thus by determining the attachment point of this affinity label derivative to δ receptors, [ d ‐Ala 2 ,Phe( p ‐NCS) 3 ]deltorphin I can provide information on receptor–ligand interactions of the ‘message’ sequence of this amphibian peptide with δ receptors.…”

“…Because of the potential differences in binding modes between peptides and non‐peptide ligands (3), peptide‐based affinity labels may provide complementary structural information about the binding sites of opioid receptors. Derivatives of opioid peptides containing photoaffinity labels have been reported (12), but the use of these agents has been limited because of the inactivation of opioid receptors by the short wavelength UV light used to generate the reactive intermediates (13). Incorporation of a chloromethylketone at the C‐terminus of [ d ‐Ala 2 ,Leu 5 ]enkephalin produced [ d ‐Ala 2 ,Leu 5 ]enkephalin chloromethylketone (DALECK), which was the first reported opioid peptide‐based electrophilic affinity label (14,15).…”

Phosphoramidate‐peptide synthesis by solution‐ and solid‐phase Staudinger‐phosphite reactions

“…In an alternative strategy, Pap can be conveniently installed into peptides by diazotization of p ‐aminophenylalanine (Apa) in solution after cleavage from the solid support, which has been demonstrated to proceed in the presence of various functional groups. The efficiency of the transformation of Apa into Pap seems to be sequence dependent, yet the specific amino acids responsible for decreasing the conversion could not be identified based on the available literature reports24–33. Furthermore, when Met is present in the peptide sequence, the thioether is expected to undergo oxidation during the diazotization reaction in water24.…”

Subcellular Compartmentation of Opioid Receptors: Modulation by Enkephalin and Alkaloids

“…Yet, the synthesis of enkephalin in NG108-15 cells (Glaser et al, 1981) and the abundance of opioid peptides in the brain should be taken into account when the mechanism for receptor internalization under normal physiological conditions is proposed. Recently developed ligands that bind covalently to the receptors (Klee et al, 1982;Newman and Barnard, 1984;Garbay-Jaureguiberry et al, 1984) should be useful for further analysis of the receptor fate and the possibility of its recycling.…”