Synthesis of alpha-factor analogues containing photoactivatable and labeling groups

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing both p-benzoyl phenylalanine (Bpa), a photoactivatable group, and 3-(mono- or di-iodo-4-hydroxyphenyl)propanoic acid (iodinated HPP) or biotin as a tag, were synthesized using solid-phase methodologies on a [phenylacetamido]-methyl (PAM) resin. Bpa was introduced into the peptides using Bpa-hydroxybenzotriazole active ester during peptide chain assembly. Biotinylated alpha-factor analogues were prepared by assembling the desired peptide on the resin, and then reacting a specific amino group either with the symmetrical anhydride of biotin or with biotin using BOP as the activating agent prior to anhydrous hydrogen fluoride cleavage. Iodinated HPP was incorporated by acylating free peptides with Bolton-Hunter reagent (3-[diiodo-4-hydroxyphenyl]propanoic acid hydroxysuccinimide ester) in N,N-dimethylformamide and borate buffer (pH 8.0) solutions. Purification of all peptides to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase mu-Bondapak C18 column with acetonitrile/water/trifluoroacetic acid as the mobile phase. All products were characterized by amino acid analysis and fast atom bombardment mass spectrometry. Two analogues, alpha-(diiodotyrosine)-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr-O H, and epsilon-(diiodo-HPP)-Lys-His-Bpa-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Nle-Tyr -OH, were one twentieth to one-fortieth as active as a alpha-factor, and exhibited approximately one order of magnitude lower affinity to the alpha-factor receptor. The results suggest that these two analogues are alpha-factor agonists and that they can be used as probes of the alpha-factor receptor.