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 Km (6.6 μM) and Vmax (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.

Optimization of Metabolic Labeling with Alkyne-Containing Isoprenoid Probes

“…To improve understanding of the process of protein prenylation, as well as identify protein substrates that are lipidated through this process, synthetic small molecule analogues of the isoprenoids are being used. Photoactivatable [16,17] and fluorescent [18] isoprenoid analogues have been used to examine protein-protein interactions of the prenylated proteins and to develop convenient assays for prenyltransferase activity, respectively. In addition, there has been significant interest in identifying prenylated proteins via metabolic labeling using modified isoprenoid probes followed by enrichment and proteomic analysis.…”

Metabolic Labeling of Prenylated Proteins Using Alkyne‐Modified Isoprenoid Analogues

“…A number of these approaches rely on the use of chemically modified isoprenoid analogues that introduce bioorthogonal groups suitable for both in vitro and in vivo investigations (Wang & Distefano, 2016). Reports on functional groups used to modify isoprenoids include azides (Rose et al, 2005) and alkynes (Hosokawa et al, 2007) for copper-catalyzed cycloaddition (click) reactions, aldehydes for oxime ligation (Rashidian, Dozier, Lenevich, & Distefano, 2010) and photoactivatable benzophenones (Henry et al, 2009) and diazirines (Vervacke et al, 2014) for labeling of enzyme active sites. Some of these isoprenoid analogues have proved to be useful for biotechnology applications including enzymatic labeling for bioconjugation of proteins, DNA and nanostructures (Duckworth et al, 2007; Rashidian et al, 2013; Y.…”

Current advances of carbene-mediated photoaffinity labeling in medicinal chemistry

“…The greater yield of photocrosslinked His-Ste14p, coupled with the ease of analysis achieved with the probe, suggested that this new class of isoprenoid analogues should be more widely used in studies of enzymes that act on terpene-derived molecules. 181 …”