Convenient Synthesis of a [1‐¹⁴C]Diazirinylbenzoic Acid as a Photoaffinity Label for Binding Studies of V‐ATPase Inhibitors

Abstract

Diazirine‐tagged systems are considered reliable compounds for photoaffinity labeling (PAL) in biochemical studies as they enable investigation and understanding of biological mechanisms through covalent bonding to the target and subsequent detection. 14C‐labeled 4‐(3‐trifluoromethyl‐3H‐diazirin‐3‐yl)benzoic acid (11) was prepared by a lithium‐bromide exchange on the bis‐silylated 4‐bromophenyldiaziridine 19 with subsequent transformations with electrophiles as key steps of the synthesis. Using 14CO2, which was generated from rather inexpensive Ba14CO3, the desired diaziridinylbenzoic acid 21 was obtained in 78 % yield based on the employed radioactive material. Oxidation under mild conditions then yielded diazirine 11 in a 100 mg scale. This versatile photoaffinity label was selectively attached to the tetrahydropyrane ring of bafilomycin A1 (2) and concanamycin A‐derived 3, which both specifically inhibit the V‐ATPases. Inhibition assays were performed and revealed that the inhibitory capacities of the labeled derivatives are suitable for PAL studies on this important group of enzymes to elucidate the as yet unknown binding sites. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

The Binding Site of the V-ATPase Inhibitor Apicularen Is in the Vicinity of Those for Bafilomycin and Archazolid

“…17. Based on previous evaluation of apicularen analogues, we favored position C3 to attach the diazirinyl moiety (7,24).…”

“…However, with respect to the mutational analysis and modeling of the binding site within the c-ring, this was a strong indication that position C9 of concanamycin may be deeply buried between two adjacent c subunits. In this study, we used derivatives of bafilomycin and concanamycin modified with the newly developed 14 C-labeled 4-(3-trifluoromethyl-diazirin-3-yl)benzoic acid (17). By repositioning the diazirinyl moiety to the opposite side of the plecomacrolide structures ( Fig.…”

“…Yet it was not possible to elucidate where apicularen binds within the V O complex. The development of the 14 C-labeled 4-(3-trifluoromethyldiazirin-3-yl)benzoic acid mentioned above now provided a convenient way to prepare an apicularen derivative that irreversibly cross-links to the protein upon UV exposure and therefore could be used to identify the interacting V-ATPase subunit(s) (17).…”

Inhibitors of V-ATPases: old and new players

“…For those inhibitors where the binding site is less understood or completely unknown, new techniques are emerging such as designing cross-linkable derivatives of the antibiotics to make responsible subunits detectable (Bender et al, 2007;Biasotti et al, 2003;Mayer and Maier, 2007;Shen et al, 2005). But as long as a high resolution structure of the complete V-ATPase or at least the V O complex is not available, a major part of this interaction will remain enigmatic.…”

“…They have various functions including the energization of transport processes across membranes and the regulation of the intracellular or intraorganellar pH (Beyenbach and Wieczorek, 2006;Forgac, 2007). V-ATPases are heteromultimeric enzymes consisting of a cytosolically oriented catalytic V 1 complex, composed of the subunits A 3 B 3 CDE 2 FG 2 H (numbers are indicating the putative stoichiometry of the subunits) and a membrane bound proton translocating V O complex, composed of the subunits ac n de with the possible c isoforms cЈ and cЉ.…”