A new cysteine anchoring method was developed for the synthesis of peptides containing C-terminal cysteine methyl esters. This method consists of attachment of Fmoc-Cys-OCH3 to either 2-ClTrt-Cl or Trt-Cl resins (via the side-chain thiol) followed by preparation of the desired peptide using Fmoc-based SPPS. We applied this method to the synthesis of the mating pheromone a-factor and a 5-FAM labeled a-factor analog. The peptides were obtained with high yield and purity and were shown to be bioactive in a growth arrest assay.

Protein prenylation is a post-translational modification that involves the addition of one or two isoprenoid groups to the C-terminus of selected proteins using either farnesyl diphosphate or geranylgeranyl diphosphate. Three crucial enzymatic steps are involved in the processing of prenylated proteins to yield the final mature product. The farnesylated dodecapeptide, a-factor, is particularly useful for studies of protein prenylation because it requires the identical three-step process to generate the same C-terminal farnesylated cysteine methyl ester substructure present in larger farnesylated proteins. Recently, several groups have developed isoprenoid analogs bearing azide and alkyne groups that can be used in metabolic labeling experiments. Those compounds have proven useful for profiling prenylated proteins and also show great promise as tools to study how the levels of prenylated proteins vary in different disease models. Herein, we describe the preparation and use of prenylated a-factor analogs, and precursor peptides, to investigate two key questions. First, a-factor analogues containing modified isoprenoids were prepared to evaluate whether the non-natural lipid group interferes with the biological activity of the a-factor. Second, a-factor-derived precursor peptides were synthesized to evaluate whether they can be efficiently processed by the yeast proteases Rce1 and Ste24 as well as the yeast methyltransferase Ste14 to yield mature a-factor analogues. Taken together, the results reported here indicate that metabolic labeling experiments with azide- and alkyne-functionalized isoprenoids can yield prenylated products that are fully processed and biologically functional. Overall, these observations suggest that the isoprenoids studied here that incorporate bio-orthogonal functionality can be used in metabolic labeling experiments without concern that they will induce undesired physiological changes that may complicate data interpretation.

Peptides containing C-terminal esters are an important class of bioactive molecules that includes a-factor, a farnesylated dodecapeptide, involved in the mating of S. cerevisiae. Here, results that expand the scope of solid phase peptide synthetic methodology that uses trityl side-chain anchoring for the preparation of peptides with C-terminal cysteine alkyl esters are described. In this method, Fmoc-protected C-terminal cysteine esters are anchored to trityl chloride resin and extended by standard solid phase procedures followed by acidolytic cleavage and HPLC purification. Analysis using a Gly-Phe-Cys-OMe model tripeptide, revealed minimal epimerization of the C-terminal cysteine residue under basic conditions used for Fmoc deprotection. 1H-NMR analysis of the unfarnesylated a-factor precursor peptide confirmed the absence of epimerization. The side-chain anchoring method was used to produce wild type a-factor that contains a C-terminal methyl ester along with ethyl-, isopropyl- and benzyl-ester analogs in good yield. Activity assays using a yeast-mating assay demonstrate that while the ethyl and isopropyl esters manifest near-wild type activity, the benzyl ester-containing analog is ca. 100-fold less active. This simple method opens the door to the synthesis of a variety of C-terminal ester modified peptides that should be useful in studies of protein prenylation and other structurally related biological processes.