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

Abstract

Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij, L. and Aldrich, J. V. (2000) J. Peptide Res. 56, 80), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for δ opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal "message" sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar, V. and Aldrich, J. V. (2003) Org. Lett. 5, 613). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors.

Discovery of Dermorphin-Based Affinity Labels with Subnanomolar Affinity for Mu Opioid Receptors

“…Examples of peptide-based electrophilic affinity labels, selective for DOR, that have been reported include [D-Ala 2 ,Cys 6 ]enkephalin (DALCE),10 the chloromethyl ketone of [DAla 2 ,Leu 5 ]enkephalin,11 and isothiocyanate and bromoacetamide-containing derivatives of TIPP (Tyr-Tic-Phe-Phe) and other DOR opioid peptides discovered in our laboratory (see ref. 12). There have been very few reports of electrophilic peptide-based affinity labels selective for MOR.…”

“…The peptides were synthesized according to methods previously developed in our laboratory12, 19 (see Supporting Information). The side chains of Tyr and Ser were protected with the t Bu group, and the side chain of D-Orn or D-Lys was protected with the Aloc (allyloxycarbonyl) group.…”

“…Alkylation of the receptor by electrophilic affinity labels, on the other hand, depends on the selectivity and chemical reactivity of the electrophile, and thus is not subject to the receptor inactivation that can occur with photoaffinity labels. Examples of peptide-based electrophilic affinity labels, selective for DOR, that have been reported include [ d -Ala 2 ,Cys 6 ]enkephalin (DALCE), the chloromethyl ketone of [ d -Ala 2 ,Leu 5 ]enkephalin, and isothiocyanate and bromoacetamide-containing derivatives of TIPP (Tyr-Tic-Phe-Phe) and other DOR opioid peptides discovered in our laboratory (see ref ). There have been very few reports of electrophilic peptide-based affinity labels selective for MOR.…”

“…Solid phase synthesis of the peptides was carried out on the PAL-PEG-PS (peptide amide linker-poly(ethylene glycol)-polystyrene) resin using Fmoc (9-fluorenylmethoxycarbonyl)-protected amino acids, except for the N-terminal Tyr residue, which was protected with the Boc ( t -butyloxycarbonyl) group. The peptides were synthesized according to methods previously developed in our laboratory , (see Supporting Information). The side chains of Tyr and Ser were protected with the t Bu group, and the side chain of d -Orn or d -Lys was protected with the Aloc (allyloxycarbonyl) group.…”