Synthesis of a-factor peptide from Saccharomyces cerevisiae and photoactive analogues via Fmoc solid phase methodology

Abstract

a-Factor from Saccharomyces cerevisiae is a farnesylated dodecapeptide involved in mating. The molecule binds to a G-protein coupled receptor and hence serves as a simple system for studying the interactions between prenylated molecules and their cognate receptors. Here, we describe the preparation of a-factor and two photoactive analogues via Fmoc solid-phase peptide synthesis using hydrazinobenzoyl AM NovaGel™ resin; the structure of the synthetic a-factor was confirmed by MS-MS analysis and NMR; the structures of the analogues were confirmed by MS-MS analysis. Using a yeast growth arrest assay, the analogues were found to have activity comparable to a-factor itself.

Figure 1

Structures of a-factor (1) and photoactive analogues with 5-carbon (2) and 10-carbon (3) isoprenoid spacers.

Figure 2

HPLC of crude reaction mixtures from the preparation of a-factor after oxidative resin cleavage Panel A: HPLC of crude reaction mixture of 1 after oxidative cleavage of 6 as described in Scheme 1. The arrow indicates the peak containing 1. Panel B: HPLC of crude reaction mixture of 9 after oxidative cleavage and Reagent K treatment as shown in Scheme 2. The arrow indicates the peak containing 9.

Figure 3

HPLC analysis of starting material and crude reaction mixture from the alkylation of pure 9 to yield a-factor (1). Panel A: HPLC of purified starting material 9. Panel B: HPLC of crude alkylation reaction mixture containing 9 and farnesyl bromide in the presence of Zn(OAc)₂ to yield 1.

Figure 4

ESI-MS-MS analysis of a-factor (1) produced by Fmoc SPPS. -f: loss of farnesyl (loss of C₁₅H₂₅).

Figure 5

ESI-MS-MS analysis of a-factor analogue 2 incorporating a photoactive isoprenoid with a 5-carbon spacer. BP: benzophenone fragment, (3-benzoylphenyl)methylium, (C₁₄H₁₁O⁺); -d: loss of (3-(hydroxymethyl)phenyl)(phenyl)menthanone (C₁₄H₁₃O₂).

Figure 6

ESI-MS-MS analysis of a-factor analogue 3 incorporating a photoactive isoprenoid with a 10-carbon spacer. BP: benzophenone fragment, (3-benzoylphenyl)methylium, (C₁₄H₁₁O⁺); BPH₂O⁺: (3-benzoylbenzyl)-oxonium, (C ₁₄H₁₃O₂⁺); -d: loss of (3-(hydroxymethyl)phenyl)(phenyl)menthanone (C₁₄H₁₃O₂); -e: loss of (3-(((2E,6E)-2,6-dimethylocta-2,6-dienyloxy)methyl)phenyl)(phenyl)methanone, (loss of C₂₄H₂₈O₂).

Figure 7

Fingerprint region from a 2D ¹H-¹H TOCSY NMR spectrum of a-factor (1) in DMSO-d₆. Assignments are indicated adjacent to each cross peak. A peak arising from exchange of the amide proton of Ile 2 with residual H₂O is marked with an asterisk in the spectrum. The peak corresponding to Ala 11 could be detected at a three-fold lower contour level as shown in the inset.

Figure 8

Biological assay of pheromones. Growth arrest in response to pheromone was determined for the a-factor responsive strain RC757 (A–D) or the α-factor responsive strain LM102 (E–H). The upper template indicates the amount of control synthetic a-factor that was spotted on plates A and E. A disk impregnated with α-factor (0.2 μg) was applied to the bottom of the plate E. Compounds were applied to the remaining plates as indicated in the lower template: B & F, synthetic a-factor (1), C & G 3, D & H, 2. At the top of each plate (A–H) cells secreting a-factor (MATa) or α-factor (MATα) were applied to the lawn as indicated in the templates.

Scheme 1

Initial strategy for the synthesis of a-factor peptide precursor by oxidative resin cleavage of an unprotected peptide.

Scheme 2

Successful strategy for the synthesis of a-factor peptide (1) precursor by oxidative resin cleavage of a fully protected peptide.