A series of potential affinity label derivatives of the amphibian opioid peptide [D-Ala2]deltorphin I were prepared by incorporation at the para position of Phe3 (in the 'message' sequence) or Phe5 (in the 'address' sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc-Phe (p-NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the δ-opioid receptor affinity, selected analogs retained nanomolar affinity for δ receptors. [D-Ala2,Phe(p-NCS)3]deltorphin I exhibited moderate affinity (IC50 = 83 nM) and high selectivity for δ receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in δ-receptor affinity (80- and >1200-fold, respectively, compared with [D-Ala2]deltorphin I). In the 'address' sequence, the Phe(p-NH2)5 derivative showed the highest δ-receptor affinity (IC50 = 32 nM), while the Phe(p-NHCOCH 2Br)5 and Phe(p-NCS)5 peptides displayed four- and tenfold lower δ-receptor affinities, respectively. [D-Ala 2,Phe(p-NCS)3]deltorphin I exhibited wash-resistant inhibition of [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) binding to δ receptors at a concentration of 80 nM. [D-Ala 2, Phe(p-NCS)3]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the 'message' sequence of the peptide.
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