Synthesis of biotinylated and photoreactive probes for angiotensin receptors

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.