Phosphorescent cellular probes and uptake indicators derived from cyclometalated iridium(III) bipyridine complexes appended with a glucose or galactose entity.

A series of phosphorescent cyclometalated iridium(III) polypyridine complexes appended with a ß-D-glucose moiety [Ir(N^C)2(bpy-TEG-ONCH3-ß-D-glc)](PF6) [bpy-TEG-ONCH3-ß-D-glc = 4-(10-N-methyl-N-(ß-D-glucopyranosyl)-amino-oxy-2,5,8-trioxa-dec-1-yl)-4'-methyl-2,2'-bipyridine; HN^C = 2-((1,1'-biphenyl)-4-yl)benzothiazole) (Hbt) (1a), 2-phenylpyridine (Hppy) (2a), 2-phenylquinoline (Hpq) (3a), 7,8-benzoquinoline (Hbzq) (4a)] has been synthesized and characterized. The D-galactose counterparts [Ir(N^C)2(bpy-TEG-ONCH3-ß-D-gal)](PF6) [bpy-TEG-ONCH3-ß-D-gal = 4-(10-N-methyl-N-(ß-D-galactopyranosyl)-amino-oxy-2,5,8-trioxa-dec-1-yl)-4'-methyl-2,2'-bipyridine; HN^C = Hbt (1b), Hppy (2b), Hpq (3b), Hbzq (4b)] and a sugar-free bt complex [Ir(bt)2(bpy-TEG-OMe)](PF6) [bpy-TEG-OMe = 4-(2,5,8,11-tetraoxa-dodec-1-yl)-4'-methyl-2,2'-bipyridine] (1c) have also been prepared. Upon photoexcitation, all the complexes displayed intense and long-lived triplet metal-to-ligand charge-transfer ((3)MLCT) [dπ(Ir) → π*(N^N)] or triplet intraligand ((3)IL) (π → π*) (N^C and N^N) emission. The lipophilicity, the cellular uptake efficiency, and cytotoxicity of the complexes toward human cervix epithelioid carcinoma cells (HeLa) have been examined. Temperature dependence and chemical inhibition experiments indicated that the transport of bt-glucose complex 1a across the cell membrane occurred through an energy-requiring process such as endocytosis, in additional to a pathway that was mediated by glucose transporters (GLUTs). Importantly, the cellular uptake efficiency of this complex was found to be strongly dependent on hormonal stimulation and inhibition, rendering it a new phosphorescent metabolic indicator. Additionally, laser-scanning confocal microscopy revealed that the complex was localized in the mitochondria and highly resistant to photobleaching compared to a fluorescent organic glucose derivative 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxy-d-glucose (2-NBDG).

Emissive behavior, cytotoxic activity, cellular uptake, and PEGylation properties of new luminescent rhenium(I) polypyridine poly(ethylene glycol) complexes.

We report here a new class of biological reagents derived from luminescent rhenium(I) polypyridine complexes modified with a poly(ethylene glycol) (PEG) pendant. The PEG-amine complexes [Re(N(^)N)(CO)(3)(py-PEG-NH(2))](PF(6)) (py-PEG-NH(2) = 3-amino-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine, MW(PEG) = 5000 Da, PDI(PEG) < 1.08; N(^)N = 1,10-phenanthroline (phen) (1-PEG-NH(2)), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me(4)-phen) (2-PEG-NH(2)), 4,7-diphenyl-1,10-phenanthroline (Ph(2)-phen) (3-PEG-NH(2))) and [Re(bpy-PEG)(CO)(3)(py-NH(2))](PF(6)) (bpy-PEG = 4-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine; py-NH(2) = 3-aminopyridine) (4-PEG-NH(2)) have been synthesized and characterized. The photophysical properties, lipophilicity, water solubility, cytotoxic activity, and cellular uptake properties of these complexes have been compared to those of their PEG-free counterparts [Re(N(^)N)(CO)(3)(py-Et-NH(2))](PF(6)) (py-Et-NH(2) = 3-amino-5-(N-(ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-Et-NH(2)), Me(4)-phen (2-Et-NH(2)), Ph(2)-phen (3-Et-NH(2))) and [Re(bpy-Et)(CO)(3)(py-NH(2))](PF(6)) (bpy-Et = 4-(N-(ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine) (4-Et-NH(2)). The PEG complexes exhibited significantly higher water solubility and lower cytotoxicity (IC(50) = 6.6 to 1152 µM) than their PEG-free counterparts (IC(50) = 3.6 to 159 µM), indicating that the covalent attachment of a PEG pendant to rhenium(I) polypyridine complexes is an effective way to increase their biocompatibility. The amine complexes 1-PEG-NH(2)-4-PEG-NH(2) have been activated with thiophosgene to yield the isothiocyanate complexes [Re(N(^)N)(CO)(3)(py-PEG-NCS)](PF(6)) (py-PEG-NCS = 3-isothiocyanato-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-PEG-NCS), Me(4)-phen (2-PEG-NCS), Ph(2)-phen (3-PEG-NCS)), and [Re(bpy-PEG)(CO)(3)(py-NCS)](PF(6)) (py-NCS = 3-isothiocyanatopyridine) (4-PEG-NCS) as a new class of luminescent PEGylation reagents. To examine their PEGylation properties, these isothiocyanate complexes have been reacted with a model substrate n-butylamine, resulting in the formation of the thiourea complexes [Re(N(^)N)(CO)(3)(py-PEG-Bu)](PF(6)) (py-PEG-Bu = 3-n-butylthioureidyl-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N(^)N = phen (1-PEG-Bu), Me(4)-phen (2-PEG-Bu), Ph(2)-phen (3-PEG-Bu)), and [Re(bpy-PEG)(CO)(3)(py-Bu)](PF(6)) (py-Bu = 3-n-butylthioureidylpyridine) (4-PEG-Bu). Additionally, bovine serum albumin (BSA) and poly(ethyleneimine) (PEI) have been PEGylated with the isothiocyanate complexes to yield bioconjugates 1-PEG-BSA-4-PEG-BSA and 1-PEG-PEI-4-PEG-PEI, respectively. Upon irradiation, all the PEGylated BSA and PEI conjugates exhibited intense and long-lived emission in aqueous buffer under ambient conditions. The DNA-binding and polyplex-formation properties of conjugate 3-PEG-PEI have been studied and compared with those of unmodified PEI. Furthermore, the in vivo toxicity of complex 3-PEG-NH(2) and its PEG-free counterpart 3-Et-NH(2) has been investigated using zebrafish embryos as an animal model. Embryos treated with the PEG complex at high concentrations revealed delayed hatching, which has been ascribed to hypoxia as a result of adhering of the complex to the external surface of the chorion.

Luminescent rhenium(I) polypyridine fluorous complexes as novel trifunctional biological probes.

We present the synthesis, characterization, and photophysical properties of three luminescent rhenium(I) polypyridine fluorous complexes [Re(Me(2)bpy)(CO)(3)(L)](PF(6)) (Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine; L = 3-amino-5-(N-((3-perfluorooctyl)propyl)aminocarbonyl)pyridine (py-Rf-NH(2)) (1), 3-isothiocyanato-5-(N-((3-perfluorooctyl)propyl)aminocarbonyl)pyridine (py-Rf-NCS) (2), 3-ethylthioureidyl-5-(N-((3-perfluorooctyl)propyl)aminocarbonyl)pyridine (py-Rf-TU-C(2)H(5)) (3)). The isothiocyanate complex 2 has been used to label bovine serum albumin (BSA) and glutathione (GSH). The photophysical properties of the resultant bioconjugates have been studied. The isolation of the luminescent fluorous rhenium-GSH conjugate from a mixture of 20 amino acids has been demonstrated using fluorous solid-phase extraction (FSPE). Additionally, the cytotoxicity of complexes 1 and 3 toward HeLa cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cellular uptake properties of complex 3 have also been investigated by laser-scanning confocal microscopy.

Phosphorescent biscyclometallated iridium(III) ethylenediamine complexes functionalised with polar ester or carboxylate groups as bioimaging and visualisation reagents.

We report the synthesis, characterisation and photophysical properties of new phosphorescent biscyclometallated iridium(III) ethylenediamine (en) complexes functionalised with polar ester or carboxylate groups [Ir(N^C)2(en)](n)(X) (n = +1, X = Cl(-), HN^C = methyl 4-(2-pyridyl)benzoate Hppy-COOMe (1a), methyl 2-phenyl-4-quinolinecarboxylate Hpq-COOMe (2a); n = -1, X = Li(+), HN^C = 4-(2-pyridyl)benzoate Hppy-COO(-) (1b), 2-phenyl-4-quinolinecarboxylate Hpq-COO(-) (2b)). In aqueous solutions, the carboxylate complexes 1b and 2b displayed emission quenching (ca. 7 and 74 fold, respectively) and lifetime shortening upon protonation, and their pKa values were determined to be 5.13 and 3.46, respectively. The pq complexes 2a and 2b exhibited hypsochromic shifts in their emission maxima and a significant increase in emission intensity (ca. 84 and 15 fold, respectively) upon nonspecific binding to the protein bovine serum albumin (BSA). Inductively coupled plasma-mass spectroscopy (ICP-MS) and laser-scanning confocal microscopy (LSCM) results revealed that the ester complexes 1a and 2a were efficiently internalised by the human cervix epithelioid carcinoma (HeLa) cells through energy-requiring pathways and subsequently localised in endosomes and mitochondria, respectively. They showed good biocompatibility in the dark, but became significantly cytotoxic upon photoirradiation due to the generation of singlet oxygen. In contrast, in aqueous solutions of physiological pH, the carboxylate complexes 1b and 2b existed as the anionic form and hardly entered cells due to limited membrane permeability, as evidenced by the intense emission surrounding the plasma membrane of the cells. They showed negligible cytotoxicity and the cell viability remained over 95% for an incubation period of 24 hours. In view of the low cytotoxicity and strongly emissive nature of the hydrophilic ppy-COO(-) complex 1b in an aqueous medium, the potential application of the complex as a visualisation reagent has been demonstrated using zebrafish (Danio rerio) as an animal model.

Mitochondria-targeting cyclometalated iridium(III)-PEG complexes with tunable photodynamic activity.

We present a new class of phosphorescent cyclometalated iridium(III) polypyridine poly(ethylene glycol) (PEG) complexes [Ir(N(^)C)2(bpy-CONH-PEG)](PF6) (bpy-CONH-PEG = 4-(N-(2-(ω-methoxypoly-(1-oxapropyl))ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine, number average molecular weight (Mn) = 5272.23, weight average molecular weight (Mw) = 5317.38, polydispersity index (PDI) = 1.009; HN(^)C = 2-phenylpyridine, Hppy (1a), 2-((1,1'-biphenyl)-4-yl)pyridine, Hpppy (2a), 2-phenylquinoline, Hpq (3a), 2-phenylbenzothiazole, Hbt (4a), 2-(1-naphthyl)benzothiazole, Hbsn (5a)). The photophysical, photochemical, and biological properties of these complexes have been compared with those of their PEG-free counterparts [Ir(N(^)C)2(bpy-CONH-Et)](PF6) (bpy-CONH-Et = 4-(N-ethylaminocarbonyl)-4'-methyl-2,2'-bipyridine; HN(^)C = Hppy (1b), Hpppy (2b), Hpq (3b), Hbt (4b), Hbsn (5b)). Upon irradiation, all the complexes exhibited intense and long-lived green to orange-red emission under ambient conditions. The emission was phosphorescence in nature and can be quenched by O2 with the generation of singlet oxygen ((1)O2). The quantum yields for (1)O2 production of the complexes in aerated DMSO (0.24-0.83) were found to be dependent on the excited-state lifetimes of the complexes, which can be altered using different cyclometalating ligands (N(^)C). Cell-based assays indicated that the PEG complexes were noncytotoxic in the dark (IC50 > 300 µM); however, most of them became significantly cytotoxic upon irradiation (IC50 = 3.4 - 23.2 µM). Laser-scanning confocal microscopy images revealed localization of complex 3a in the mitochondrial region of HeLa cells and the induction of rapid necrotic cell death upon light activation. Additionally, the lack of dark toxicity and potential application of the PEG complexes as a visualizing reagent have been demonstrated using zebrafish (Danio rerio) as an animal model.

Luminescent cyclometallated iridium(III) bis(quinolylbenzaldehyde) diimine complexes--synthesis, photophysics, electrochemistry, protein cross-linking properties, cytotoxicity and cellular uptake.

Four new luminescent cyclometallated iridium(III) bis(quinolylbenzaldehyde) diimine complexes [Ir(qba)(2)(N⁁N)](PF(6)) (Hqba = 4-(2-quinolyl)benzaldehyde, N⁁N = 2,2'-bipyridine, bpy (1); 1,10-phenanthroline, phen (2); 3,4,7,8-tetramethyl-1,10-phenanthroline, Me(4)-phen (3); 4,7-diphenyl-1,10-phenanthroline, Ph(2)-phen (4)) have been synthesised and characterised, and their electronic absorption, emission and electrochemical properties investigated. The X-ray crystal structures of complexes 1 and 2 have been determined. Upon irradiation, complexes 1-4 exhibited intense and long-lived orange-yellow emission in fluid solutions at 298 K and in alcohol glass at 77 K. The emission has been assigned to a triplet intra-ligand ((3)IL) excited state associated with the qba ligand, probably with mixing of some triplet metal-to-ligand charge-transfer ((3)MLCT) (dπ(Ir) →π*(qba)) character. Reductive amination reactions of complexes 1-4 with the protein bovine serum albumin (BSA) afforded the bioconjugates 1-BSA-4-BSA, respectively. Upon photoexcitation, these bioconjugates displayed intense and long-lived (3)MLCT (dπ(Ir) →π*(N⁁C)) emission in aqueous buffer at 298 K. The cross-linked nature of the Ir-BSA bioconjugates has been verified by SDS-PAGE. Additionally, the cytotoxicity of the complexes towards human cervix epithelioid carcinoma (HeLa) cells has been examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the cellular uptake of complex 4 has been investigated by laser-scanning confocal microscopy and flow cytometry.

Rhenium(I) polypyridine biotin isothiocyanate complexes as the first luminescent biotinylation reagents: synthesis, photophysical properties, biological labeling, cytotoxicity, and imaging studies.

We report here the design of the first class of luminescent biotinylation reagents derived from rhenium(I) polypyridine complexes. These complexes [Re(N-N)(CO)(3)(py-biotin-NCS)](PF(6)) (py-biotin-NCS = 3-isothiocyanato-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine; N-N = 1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me(4)-phen) (2a), 4,7-diphenyl-1,10-phenanthroline (Ph(2)-phen) (3a)), containing a biotin unit and an isothiocyanate moiety, have been synthesized from the precursor amine complexes [Re(N-N)(CO)(3)(py-biotin-NH(2))](PF(6)) (py-biotin-NH(2) = 3-amino-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine; N-N = phen (1c), Me(4)-phen (2c), Ph(2)-phen (3c)). To investigate the amine-specific reactivity of the isothiocyanate complexes 1a-3a, they have been reacted with a model substrate ethylamine, resulting in the formation of the thiourea complexes [Re(N-N)(CO)(3)(py-biotin-TU-Et)](PF(6)) (py-biotin-TU-Et = 3-ethylthioureidyl-5-(N-((2-biotinamido)ethyl)aminocarbonyl)pyridine; N-N = phen (1b), Me(4)-phen (2b), Ph(2)-phen (3b)). All the rhenium(I) complexes have been characterized, and their photophysical properties have been studied. The avidin-binding properties of the thiourea complexes 1b-3b have been examined by the 4'-hydroxyazobenzene-2-carboxylic acid (HABA) assay. Titration results indicated that the complexes exhibited emission enhancement by ca. 1.4-1.5-fold upon binding to avidin, and the lifetimes were elongated to ca. 0.8-2.0 micros. Additionally, we have biotinylated bovine serum albumin (BSA) with the isothiocyanate complexes. All the resultant rhenium-BSA bioconjugates displayed intense and long-lived orange-yellow to greenish-yellow emission upon irradiation in aqueous buffer under ambient conditions. The avidin-binding properties of the bioconjugates have been investigated using the HABA assay. Furthermore, the cytotoxicity of the thiourea complexes 1b-3b toward the HeLa cells has been examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC50 values were determined to be ca. 17.5-28.5 microM, which are comparable to that of cisplatin (26.7 microM) under the same conditions. The cellular uptake of complex 3b has been investigated by fluorescence microscopy, and the results showed that the complex was localized in the perinuclear region after interiorization.