Stereospecific synthesis of a carbene-generating angiotensin II analogue for comparative photoaffinity labeling: improved incorporation and absence of methionine selectivity.
A stereospecific convergent synthesis of N-[(9-fluorenyl)methoxycarbonyl]-p-[3-(trifluoromethyl)-3H-diazirin-3-yl]-l-phenylalanine (Fmoc-12, Fmoc-Tdf) and its incorporation into the C-terminal position of the angiotensin II (AngII) peptide to form (125)I[Sar(1),Tdf(8)]AngII ((125)I-13) is presented. This amino acid photoprobe is a highly reactive carbene-generating diazirine phenylalanine derivative that can be used for photoaffinity labeling. Using model receptors, we compared the reactivity and the Met selectivity of 12 to that of the widely used and reputedly Met-selective p-benzoyl-l-phenylalanine (Bpa) photoprobe. Wild-type and mutant AngII type 2 receptors, a G protein-coupled receptors, were photolabeled with (125)I-13 as well as with (125)I[Sar(1),Bpa(8)]AngII ((125)I-14), and the respective incorporation yields were assessed. The carbene-generating 12 was more reactive toward inert residues and was not Met-selective compared to the biradical ketone-generating Bpa, allowing for more precise determination of ligand contact points in peptidergic receptors.
Department of Pharmacology, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4.
Dany
D
Fillion
Maud
M
Deraët
Brian J
BJ
Holleran
Emanuel
E
Escher
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D006801:Humans; D006838:Hydrocarbons / Q000737:chemistry; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008697:Methane / Q000031:analogs & derivatives* / Q000737:chemistry; D008715:Methionine / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D019920:Photoaffinity Labels / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D011869:Radioligand Assay; D044139:Receptor, Angiotensin, Type 2 / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D006838:Hydrocarbons; D007457:Iodine Radioisotopes; C509925:N-((9-fluorenyl)methoxycarbonyl)-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenylalanine; D019920:Photoaffinity Labels; D044139:Receptor, Angiotensin, Type 2; C509926:angiotensin II, iodo-Sar(1)-Tdf(8)-; D000804:Angiotensin II; C030011:carbene; D010649:Phenylalanine; D003978:Diazomethane; D008715:Methionine; D008697:Methane