Synthesis, Properties, and Live-Cell Imaging Studies of Luminescent Cyclometalated Iridium(III) Polypyridine Complexes Containing Two or Three Biotin Pendants

Abstract

Three luminescent cyclometalated iridium(III) bis-biotin complexes [Ir(N(wedge)C)(2)(N(wedge)N)](PF(6)) (HN(wedge)C = 2-(4-(N-(6-(biotinamido)hexyl)aminomethyl)phenyl)pyridine, HppyC6B, N(wedge)N = 2,2'-bipyridine, bpy (1); HN(wedge)C = 2-phenylpyridine, Hppy, N(wedge)N = 4,4'-bis((2-(biotinamido)ethyl)aminocarbonyl)-2,2'-bipyridine, bpyC2B2 (2); HN(wedge)C = Hppy, N(wedge)N = 4,4'-bis((2-((6-(biotinamido)hexanoyl)amino)ethyl)aminocarbonyl)-2,2'-bipyridine, bpyC2C6B2 (3)) and one tris-biotin complex [Ir(ppyC6B)(2)(bpyC6B)](PF(6)) (bpyC6B = 4-((6-(biotinamido)hexyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine) (4) have been synthesized and characterized. The biotin-free complex [Ir(ppy)(2)(bpyC4)](PF(6)) (bpyC4 = 4,4'-bis(n-butylaminocarbonyl)-2,2'-bipyridine) (5) has also been prepared for comparison studies. Upon photoexcitation, all the complexes displayed intense and long-lived greenish-yellow to red triplet metal-to-ligand charge-transfer ((3)MLCT) (dpi (Ir) --> pi*(N(wedge)N)) emission in fluid solutions at room temperature and in low-temperature glass. Cyclic voltammetric studies revealed iridium(IV/III) oxidation at about +1.21 to + 1.29 V and diimine-based reductions at about -1.07 to -1.39 V versus SCE. The interactions of the bis-biotin and tris-biotin complexes with avidin have been studied by 4'-hydroxyazobenzene-2-carboxylic acid (HABA) assays, emission titrations, and dissociation assays. The possibility of these complexes as cross-linkers for avidin has been examined by microscopy studies using avidin-conjugated green fluororescent microspheres and size-exclusion HPLC analysis. Utilization of these luminescent iridium(III) biotin complexes in signal amplification has been demonstrated using avidin-coated nonfluorescent microspheres and complex 3 as an example. Additionally, the lipophilicity of all the complexes has been determined by reversed-phase HPLC. The cytotoxicity of these iridium(III) complexes toward the human cervix epithelioid carcinoma (HeLa) cell line has been evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assays. Furthermore, the cellular uptake of the complexes has been examined by ICP-MS, laser-scanning confocal microscopy, and flow cytometry.