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.
Department of Biochemistry, Ohio State University, Columbus 43210.
D G
DG
Myszka
R P
RP
Swenson
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