Synthesis of Peptides Containing C-Terminal Esters Using Trityl Side-Chain Anchoring: Applications to the Synthesis of C-Terminal Ester Analogs of the Saccharomyces cerevisiae Mating Pheromone a-Factor

Abstract

Peptides containing C-terminal esters are an important class of bioactive molecules that includes a-factor, a farnesylated dodecapeptide, involved in the mating of Saccharomyces 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. (1)H 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.

Figure 1

HPLC chromatograms of crude peptides (8a–8e) after cleavage from the trityl resin. A) 8a; B) 8b; C) 8c; D) 8d; E) 8e.

Figure 2

Fingerprint region of 700-MHz 2D ¹H–¹H TOCSY NMR spectrum of a-factor precursor peptides. A) Spectrum of L-Cys containing a-factor precursor peptide (8a); B) Superposition of the spectra of 8a (black) and 8b (red); C) D-Cys containing a-factor precursor peptide (8b) in DMSO-d6. Assignments are indicated adjacent to each cross peak.

Figure 3

Fingerprint region of 700-MHz 2D ¹H-¹H TOCSY NMR spectrum of a-factor (9a) in DMSO-d6. Assignments are labeled adjacent to each cross peak.

Figure 4

Biological assay of pheromones. Growth arrest in response to pheromone was determined for the a-factor responsive strain RC757 (left column; A, C, E, G, I) or the α-factor responsive strain LM102 (right column: B, D, F, H, J). A, B - wild type a-factor (9a), C, D – ethyl ester a-factor (9c), E, F – isopropyl ester a-factor (9d), G, H, I, J – benzyl ester a-factor (9e). The templates in the middle indicate the amount of synthetic a-factor and its analogs spotted on the plates. The benzyl ester a-factor analog was applied to the plates in two different sets of concentrations as indicated in the corresponding middle templates. At the top of each plate 2.5 μL of cells secreting α-factor (MATα) (on the left) or a-factor (MATa) were applied to the lawn as indicated in the templates.

Scheme 1

Loading of Fmoc-Cys-OR onto trityl-based resin. * is used to emphasize location of the stereogenic center of the cysteine α-carbon) producing a trityl side-chain anchored cysteine ester for SPPS.

Scheme 2

Synthesis of model tripeptides Gly-Phe-L-Cys-OMe and Gly-Phe-D-Cys-OMe. Reaction conditions: a) 20% piperidine in DMF; b) Fmoc SPPS; c) 20% piperidine in DMF; d) 5% TFA in CH₂Cl₂.

Scheme 3

Synthesis of a-factor (9a) and a-factor C-terminal ester analogs (9c–e) using the trityl side-chain anchoring strategy.