Synthesis of 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin: photoaffinity labeling of rat liver dicoumarol-sensitive NAD(P)H: quinone reductase.
A photoaffinity analog of 4-hydroxycoumarin containing an arylazido derivative at the 3-position has been synthesized and characterized. This compound, 3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin, serves as a strong competitive inhibitor of the dicoumarol-sensitive NAD(P)H: quinone reductase (DT-diaphorase) from rat liver, having an apparent inhibition constant of 4.2 10(-7) M. Irradiation of the reductase with ultraviolet light in the presence 10 microM of the photoprobe resulted in the covalent labeling of 2% of the reductase molecules. The enzyme is protected from labeling to greater than 99% by the inclusion of 3 microM dicoumarol, consistent with the specific labeling of the 4-hydroxycoumarin binding site of this enzyme. Furthermore, the quinone reductase was shown to specifically labeled by the probe even when contained within crude fractions rat liver cytosol.
Myszka
D G
DG
Department of Biochemistry, Ohio State University, Columbus 43210.
Swenson
R P
RP
D015110:4-Hydroxycoumarins / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D001728:Dicumarol / Q000494:pharmacology*; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D016660:NAD(P)H Dehydrogenase (Quinone); D010782:Photolysis; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013053:Spectrophotometry
D015110:4-Hydroxycoumarins; D000345:Affinity Labels; D001386:Azides; D007202:Indicators and Reagents; C066252:3-(4-azido-5-iodosalicylamido)-4-hydroxycoumarin; D001728:Dicumarol; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases