Biologically active aromatic retinoids bearing azido photoaffinity-labeling groups and their binding to cellular retinoic acid-binding protein

Abstract Retinoids bearing azido photoaffinity-labeling groups (azidoretinoids) have potential as probes for investigating the molecular mechanisms of action of all-trans-retinoic acid (RA) as mediated by its cellular retinoic acid-binding protein (CRABP) and nuclear receptor proteins. Two new azidoretinoids, 3-azido-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1 E -propen-1-yl]-benzoic acid and 4-(4-azido-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid were synthesized, and evaluated for their in vitro biological potency, and binding affinity for CRABP. Like RA, these aromatic azides had significant activity in modulating cell differentiation in retinoid-deficient hamster tracheal organ culture (ED 50 0.02 nM and 0.03 nM, respectively) and in the inhibition of the induction of ornithine decarboxylase in mouse epidermis (ED 50 7.0 nmol and 0.5 nmol, respectively). They also possessed high binding affinity for CRABP (ID 50 0.9 μM and 0.85 μM, respectively). The tritiated aromatic azides were further evaluated for their ability to bind covalently to CRABP after photolysis. On photolysis at −78°C, the two radiolabeled azidoretinoids formed stable adducts with CRABP. Treatment of the adducts with either RA or p -chloromercuriphenylsulfonic acid (CMPS) and subsequent dialysis did not cause any dissociation, indicating the formation of a covalent bond. In contrast, treatment of the unirradiated complexes with RA or CMPS led to dissociation of the complex. Synthesis of affinity labels and characterization of CRABP-retinoid complexes should provide useful information on the ligand-binding regions and insights into the mechanism of action of RA.