Diazirine-containing photoactivatable isoprenoid: synthesis and application in studies with isoprenylcysteine carboxyl methyltransferase

Abstract

Photoaffinity labeling is a useful technique employed to identify protein-ligand and protein-protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones. Diazirines are intrinsically smaller than benzophenones and generate carbenes upon photolysis that react with a broader range of amino acid side chains compared with the benzophenone-derived diradical; this makes diazirines potentially more general photoaffinity-labeling agents. In this article, we describe the development and application of a new isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an a-factor-derived peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with K(m) (6.6 μM) and V(max) (947 pmol min(-1) mg(-1)) values comparable or better than a-factor peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with observably higher efficiency than benzophenone-containing a-factor peptides.

Figure 1

Photoactivatable isoprenoid analogues. Left: farnesol, diazirine 1, benzophenone 2, and DATFP 3. Right: space-filling models of the isoprenoid analogues. Hydrogen atoms are omitted for clarity. Color scheme: carbon, green; oxygen, red; nitrogen, blue; and fluorine, white. Spheres are shown at 0.6 van der Waals radii for clarity.

Figure 2

a-Factor-based peptides for the in vitro study of Icmt.

Scheme 1. Synthesis of a Diazirine-Containing Analogue of a Farnesyl Group

Scheme 2. Synthesis of Prenylated a-Factor Precursor Peptide 16

Figure 3

Analysis of cross-linking reactions containing purified His-Ste14p and different photoactive probes. (A) Fluorescent imaging, (B) immunoblot analysis with NeutrAvidin HRP, and (C) immunoblot analysis with α-Ste14 . Experiments were performed with 16, 18, and 19. For this experiment, purified His-Ste14p (0.25 μg) was incubated with the probes indicated (50 μM) and irradiated on ice for 30 min followed by fractionation via SDS-PAGE. The resulting gel was visualized using (A) a fluorescence scanner or transferred to a nitrocellulose membrane and visualized using (B) NeutrAvidin HRP or (C) an α-Ste14 antibody. The data shown is from one of three replicates of this experiment.

Figure 4

Competition of photolabeling of His-Ste14p by 16 using a biotinylated a-factor precursor peptide 17. For this experiment, His-Ste14p crude membrane protein (100 μg) was mixed with 16 and 17 at the concentrations indicated and irradiated on ice for 30 min followed by enrichment with neutravidin-agarose beads. The samples were then eluted and resolved via SDS-PAGE. The resulting gel was blotted to a nitrocellulose membrane and visualized using an α-Ste14 antibody. The data shown is from one of two replicates of this experiment.