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

Abstract

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 delta-opioid receptor affinity, selected analogs retained nanomolar affinity for delta receptors. [D-Ala2,Phe(p-NCS)3]deltorphin I exhibited moderate affinity (IC50=83 nM) and high selectivity for delta receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in delta-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 delta-receptor affinity (IC50=32 nM), while the Phe(p-NHCOCH2Br)5 and Phe(p-NCS)5 peptides displayed four- and tenfold lower delta-receptor affinities, respectively. [D-Ala2,Phe(p-NCS)3]deltorphin I exhibited wash-resistant inhibition of [3H][D-Pen2,D-Pen5]enkephalin (DPDPE) binding to delta receptors at a concentration of 80 nM. [D-Ala2, 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.

Analogues of deltorphin I containing conformationally restricted amino acids in position 2: structure and opioid activity

“…As reported in Table , the affinity of analogues containing ( R ) or ( S )‐α‐methyl‐β‐azidoalanine in position 2 to δ‐receptors strongly depends on the chirality of the α,α‐disubstituted residue. Peptide II , containing ( S )‐α‐methyl‐β‐azidoalanine, displays slightly lower δ‐receptor affinity than the parent peptide but higher δ‐receptor selectivity, whereas the ( R ) isomer is less potent and δ‐selective. It should be noted that the topographical location of the methyl group in ( S )‐α‐methyl‐β‐azidoalanine is related to the methyl group of D‐Ala, whose position is crucial for chemical activity.…”

Solid Phase Synthesis and Application of Labeled Peptide Derivatives: Probes of Receptor-Opioid Peptide Interactions

“…One of these derivatives [Phe( p -NHCOCH 2 Br) 4 ]TIPP, exhibits 85% WRIB at a concentration of only 2.5 nM (Kumar et al, 2002). We have also identified the first electrophilic affinity label derivative of one of the potent and selective amphibian opioid peptides, [D-Ala 2 , Phe( p -NCS) 4 ]deltorphin I (Aldrich et al, 2004); this peptide is also the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the “message” sequence of the peptide. Recently we have successfully identified our first peptide-based affinity labels for MOR (manuscript in preparation).…”

“…For example, while substitution with a para-isothiocyanate on the phenyl ring of Phe 3 in the amphibian peptides is tolerated by DOR (Aldrich et al, 2004), this functionality causes large decreases in MOR affinity (Choi et al, 2003a). In the case of the dermorphin derivative, this resulted in a switch in receptor selectivity from MOR to DOR (Choi et al, 2003a).…”

Dual Labeled Peptides as Tools to Study Receptors: Nanomolar Affinity Derivatives of TIPP (Tyr-Tic-Phe-Phe) Containing an Affinity Label and Biotin as Probes of δ Opioid Receptors

“…Our research focuses on the synthesis of labeled derivatives of opioid peptides as tools to study opioid receptors. We have been particularly successful in identifying electrophilic affinity label derivatives of several peptides selective for δ opioid receptors (DOR) (8)(9)(10)(11), including analogues of the potent and selective DOR antagonist TIPP (Tyr-Tic-Phe-PheOH, where Tic ) 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) 1 (9,10). The [Phe(p-X) 4 ]TIPP derivatives (where X is an isothiocyanate or bromoacetamide group) have high affinity for DOR (IC 50 ) 5 nM) and inhibit binding in a wash-resistant manner (9,10).…”

“…Our research focuses on the synthesis of labeled derivatives of opioid peptides as tools to study opioid receptors. We have been particularly successful in identifying electrophilic affinity label derivatives of several peptides selective for δ opioid receptors (DOR) − , including analogues of the potent and selective DOR antagonist TIPP (Tyr-Tic-Phe-PheOH, where Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) 1 Abbreviations: Alloc, allyloxycarbonyl; CHO, Chinese hamster ovary; DAMGO, [ d -Ala 2 ,NMePhe 4 ,glyol 5 ]enkephalin; DIC, N,N -diisopropylcarbodiimide; DIEA, N,N -diisopropylethylamine; DMA, N,N -dimethylacetamide; Dmt, 2′,6′-dimethyltyrosine; DPDPE, cyclo [ d -Pen 2 , d -Pen 5 ]enkephalin; Fmoc, fluorenylmethoxycarbonyl; HOBt, hydroxybenzotriazole; HRP, horseradish peroxidase; PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; TCDI, thiocarbonyl diimidazole; TFA, trifluoracetic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; TIPP, Tyr-Tic-Phe-PheOH. , . The [Phe( p -X) 4 ]TIPP derivatives (where X is an isothiocyanate or bromoacetamide group) have high affinity for DOR (IC 50 = 5 nM) and inhibit binding in a wash-resistant manner , .…”