Synthetic probes for the α‐factor receptor

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Abstract
The binding of the tridecapeptide yeast mating phermone, α‐factor, to its receptor represents an excellent model for the investigation of peptide hormone–receptor interactions. In this paper we present a number of strategies to probe the binding site of the α‐factor receptor, and discuss the synthesis of probes containing radioactive and affinity tags. Preferential acylation of the α‐ or ε‐amine in [Nle12]‐α‐factor was accomplished using 3‐[3,5‐diiodo‐4‐hydroxyphenyl] propanoic acid hydroxysuccinimide ester (diiodo Bolton‐Hunter reagent). At pH 8.0 in a N‐N‐dimethylformamide/water mixture the ratio of ε‐ to α‐acylation was 2.15 to 1, whereas at pH 6.5 in a 1,2‐dimethyoxyethane/water mixture α‐acylation was favored by more than 3 to 1. The product distribution was sound to depend on pH, organic cosolvent, and the ratio of organic solvent and aqueous buffer. Product distributions were followed using analytical high performance liquid chromatography and the products were characterized enzymatically and by mass spectrometry. Citraconic anhydride preferentially α‐acylated [Nle12]‐α‐factor and served as a temporary masking group during the synthesis of ε‐Bolton–Hunter acylated phermone. Biotin or diiodo Bolton–Hunter reagents were also directly incorporated into [Nle12]‐α‐factor or Lys[Nle12]‐α‐factor during peptide synthesis. The peptides were assembled on a chloromethyl polystyrene resin or on a (phenylace‐tamido)methyl resin, and cleaved using anhydrous hydrogen fluoride (HF). Probes were inserted on amino groups either prior (biotin) or subsequent (Bolton–Hunter reagent) to HF cleavage. The biological activity of the synthetic peptides was characterized using growth arrest assays. These studies provide insights into the structural requirements for binding of α‐factor to its receptor and form a basis for detailed investigation of the receptor using biochemical procedures.