Photoaffinity radioligand for NADH:ubiquinone oxidoreductase: [S-C3H2](trifluoromethyl)diazirinyl-pyridaben

Abstract

Pyridaben is a new and very potent insecticide and miticide that acts by inhibiting the activity of NADH:ubiquinone oxidoreductase (the most complex of all the respiratory enzymes). The binding site, presumed to be the same as that of rotenone and fenazaquin, resides at an unknown location within the 43-polypeptide-subunit Complex I. To define the structure of the pyridaben-inhibition site(s), we prepared [S-C3H2](trifluoromethyl)diazirinyl-pyridaben as a photoaffinity probe. Tritium was incorporated by reducing 4-(trifluoromethyl)diazirinylphenylacetyl fluoride to the benzyl alcohol with freshly prepared LiB3H4 at 97% tritium enrichment. The tritium-labeled alcohol was converted to the benzyl bromide derivative and coupled to 2-tert-butyl-4-chloro-5-mercapto-3(2H)-pyridazinone to obtain the photoaffinity probe (56 Ci/mmol) with an IC50 of 3.0 nM for NADH:ubiquinone oxidoreductase activity of bovine heart electron transport particles. [S-C3H2](Trifluoromethyl)diazirinyl-pyridaben is an improved photoaffinity radioligand combining outstanding potency for inhibiting NADH:ubiquinone oxidoreductase activity, high specific activity close to the theoretical value, and a preferred photolabile substituent (known to combine high reactivity and generation of a carbene species at wavelengths not damaging to proteins). © 1998 John Wiley & Sons, Ltd.