Synthesis of biotinylated and photoreactive probes for angiotensin receptors

Abstract

Three main types of bifunctional probe that can be obtained in a radiolabelled form and used for studying and purifying angiotensin receptors were developed: biotinyl-aminohexanoyl-[Tyr(3I)⁴, Phe(4N₃)⁸]angiotensin II, iminobiotinyl-glycyl-aminohexanoyl-[Ala¹, Tyr(3I)⁴, Phe(4N₃)⁸]angiotensin II, and biotinyl-ethyl-1,3′-dithiopropionyl-[Ala¹, Tyr(3I)⁴, Phe(4N₃)⁸]angiotensin II. Several improved, unequivocal synthetic pathways are described for these products, using both ‘stepwise’ methods and a ‘segment coupling’ strategy allowing preparation of L-Phe and D-Phe derivatives (agonists and antagonists, respectively) from the same intermediate. Complete attribution of the ¹H NMR signals is reported. The molecules showed no folding in NOESY and temperature-variation experiments, suggesting that they are capable of interacting simultaneously with the receptor and with avidin. They all show an affinity of the order of 10^–9 mol dm^–3 for angiotensin II receptors. After introduction of the photoactivatable group, covalent bonding of the probe with the receptor was obtained with a mean yield of 25%. The biotin or related residue allows specific detection of the receptors among membrane proteins. This procedure can be applied to other receptors bearing biotinylation and photolabelling sites.