PHOTOAFFINITY RADIOLIGAND FOR NADH-UBIQUINONE OXIDOREDUCTASE - [S-(CH2)-H-3](TRIFLUOROMETHYL)DIAZIRINYL-PYRIDABEN

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 t o be the same as that of rotenone and fenazaquin, resides at an unknow n location within the 43-polypeptide-subunit Complex I. To define the structure of the pyridaben-inhibition site(s), we prepared [S-C3H2](tr ifluoromethyl)diazirinylphenylacetyl as a photoaffinity probe. Tritium was incorporated by reducing 4-(trifluoromethyl)diazirinylphenylacety l fluoride to the benzyl alcohol with freshly prepared (LiBH4)-H-3 at 97% tritium enrichment. The tritium-labeled alcohol was converted to t he benzyl bromide derivative and coupled to -tert-butyl-4-chloro-5-mer capto-3(2H)-pyridazinone to obtain the photoaffinity probe (56 Ci/mmol ) with an IC50 of 3.0 nM for NADH:ubiquinone oxidoreductase activity o f bovine heart electron transport particles. [S-C3H2](Trifluoromethyl) diazirinyl-pyridaben is an improved photoaffinity radioligand combinin g outstanding potency for inhibiting NADH:ubiquinone oxidoreductase ac tivity, high specific activity close to the theoretical value, and a p referred photolabile substituent (known to combine high reactivity and generation of a carbene species at wavelengths not damaging to protei ns).