We report a new class of ruthenium(II) polypyridine complexes functionalized with a nitrone group as phosphorogenic bioorthogonal probes. These complexes were very weakly emissive owing to rapid C=N isomerization of the nitrone moiety, but exhibited significant emission enhancement upon strain-promoted alkyne–nitrone cycloaddition (SPANC) reaction with bicyclo[6.1.0]nonyne (BCN)-modified substrates. The modification of nitrone with a dicationic ruthenium(II) polypyridine unit at the α-C-position and a phenyl ring at the N-position led to remarkably accelerated reaction kinetics, which are substantially greater (up to ≈278 fold) than those of other acyclic nitrone–BCN systems. Interestingly, the complexes achieved specific cell membrane/cytosol staining upon specific labeling of an exogenous substrate, BCN-modified decane (BCN-C10), in live cells. Importantly, the in situ generation of the more lipophilic isoxazoline adduct in the cytoplasm resulted in increased cytotoxicity, highlighting a novel approach to apply the SPANC labeling technique in drug activation.

The use of bioorthogonal probes that display fluorogenic or phosphorogenic properties is advantageous to the labeling and imaging of biomolecules in live cells and organisms. Herein we present the design of three iridium(III) complexes containing a nitrone moiety as novel phosphorogenic bioorthogonal probes. These probes were non-emissive owing to isomerization of the C=N group but showed significant emission enhancement upon cycloaddition reaction with strained cyclooctynes. Interestingly, the connection of the nitrone ligand to the cationic iridium(III) center led to accelerated reaction kinetics. These nitrone complexes were also identified as phosphorogenic bioorthogonal labels and imaging reagents for cyclooctyne-modified proteins. These findings contribute to the development of phosphorogenic bioorthogonal probes and imaging reagents.

The use of bioorthogonal probes that display fluorogenic or phosphorogenic properties is advantageous to the labeling and imaging of biomolecules in live cells and organisms. Herein we present the design of three iridium(III) complexes containing a nitrone moiety as novel phosphorogenic bioorthogonal probes. These probes were non-emissive owing to isomerization of the C=N group but showed significant emission enhancement upon cycloaddition reaction with strained cyclooctynes. Interestingly, the connection of the nitrone ligand to the cationic iridium(III) center led to accelerated reaction kinetics. These nitrone complexes were also identified as phosphorogenic bioorthogonal labels and imaging reagents for cyclooctyne-modified proteins. These findings contribute to the development of phosphorogenic bioorthogonal probes and imaging reagents.

The synthesis, characterization, photophysics, lipophilicity, and cellular properties of new phosphorescent ruthenium(II) polypyridine complexes functionalized with a dibenzocyclooctyne (DIBO) or amine moiety [Ru(N^N)₂(L)](PF₆)₂ are reported (L=4-(13-N-(3,4:7,8-dibenzocyclooctyne-5-oxycarbonyl) amino-4,7,10-trioxa-tridecanyl-aminocarbonyl-oxy-methyl)-4′-methyl-2,2′-bipyridine bpy-DIBO, N^N=2,2′-bipyridine bpy (1 a), 1,10-phenanthroline phen (2 a); L=4-(13-amino-4,7,10-trioxa-tridecanylaminocarbonyl-oxy-methyl)-4′-methyl-2,2′-bipyridine bpy-NH₂, N^N=bpy (1 b), phen (2 b)). The strain-promoted alkyne–azide cycloaddition (SPAAC) reaction of the DIBO complexes 1 a and 2 a with benzyl azide were studied. Also, the DIBO complexes 1 a and 2 a can selectively label N-azidoglycans located on the surface of CHO-K1 and A549 cells that were pretreated with 1,3,4,6-tetra-O-acetyl-N-azidoacetyl-D-mannosamine (Ac₄ManNAz). Additionally, the intracellular trafficking and localization of these biomolecules were monitored using laser-scanning confocal microscopy. Interestingly, the biolabeling and cellular uptake efficiency of the DIBO complexes 1 a and 2 a were cell-line dependent, as revealed by flow cytometry and ICP-MS. Furthermore, the complexes showed good biocompatibility toward the Ac₄ManNAz-pretreated cells in the dark, but exhibited photoinduced cytotoxicity due to the generation of singlet oxygen.

We report the development of a series of rhenium(I) polypyridine complexes appended with an electron-rich diaminoaromatic moiety as phosphorogenic sensors for nitric oxide (NO). The diamine complexes [Re(N^N)(CO)₃(py-DA)][PF₆] (py-DA=3-(N-(2-amino-5-methoxyphenyl)aminomethyl)pyridine; N^N=1,10-phenanthroline (phen) (1 a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me₄-phen) (2 a), 4,7-diphenyl-1,10-phenanthroline (Ph₂-phen) (3 a)) have been synthesized and characterized. In contrast to common rhenium(I) diimines, these diamine complexes were very weakly emissive due to quenching of the triplet metal-to-ligand charge-transfer (³MLCT) emission by the diaminoaromatic moiety through photoinduced electron transfer (PET). Upon treatment with NO, the complexes were converted into the triazole derivatives [Re(N^N)(CO)₃(py-triazole)][PF₆] (py-triazole=3-((6-methoxybenzotriazol-1-yl)methyl)pyridine; N^N=phen (1 b), Me₄-phen (2 b), Ph₂-phen (3 b)), resulting in significant emission enhancement (I/I₀≈60). The diamine complexes exhibited high reaction selectivity to NO, and their emission intensity was found to be independent on pH. Also, these complexes were effectively internalized by HeLa cells and RAW264.7 macrophages with negligible cytotoxicity. Additionally, the use of complex 3 a as an intracellular phosphorogenic sensor for NO has been demonstrated.

We present a new class of phosphorescent cyclometalated iridium(III) bipyridyl–phenylenediamine complexes [Ir(N^C)₂(bpy-DA)](PF₆) (bpy-DA=4-(N-(2-amino-5-methoxyphenyl)aminomethyl)-4′-methyl-2,2′-bipyridine; HN^C=2-(2,4-difluorophenyl)pyridine (Hdfppy) (1 a), 2-phenylpyridine (Hppy) (2 a), 2-phenylquinoline (Hpq) (3 a), 2-phenylcinchoninic acid methyl ester (Hpqe) (4 a)) and their triazole counterparts [Ir(N^C)₂(bpy-T)](PF₆) (bpy-T=4-((6-methoxybenzotriazol-1-yl)methyl)-4′-methyl-2,2′-bipyridine; HN^C=Hdfppy (1 b), Hppy (2 b), Hpq (3 b), Hpqe (4 b)). Upon photoexcitation, the diamine complexes exhibited fairly weak green to red phosphorescence under ambient conditions whereas the triazole derivatives emitted strongly. The photophysical properties of complexes 2 a and 2 b have been studied in more detail. Upon protonation, the diamine complex 2 a displayed increased emission intensity, but the emission properties of its triazole counterpart complex 2 b were independent on the pH value of the solution. Also, complex 2 a was found to be readily converted into complex 2 b upon reaction with NO under aerated conditions, resulting in substantial emission enhancement of the solution. The reaction was highly specific toward NO over other reactive oxygen and nitrogen species (RONS) as revealed by spectroscopic analyses. The lipophilicity and cellular uptake efficiency of the diamine complexes have been examined and correlated to their molecular structures. Also, cell-based assays showed that these complexes were noncytotoxic toward human cervix epithelioid carcinoma (HeLa) cells (at 10 μM, 4 h, percentage survival ≈80–95 %). Additionally, the diamine complexes have been used to visualize intracellular NO generated both exogenously in HeLa cells and endogenously in RAW 264.7 murine macrophages by laser-scanning confocal microscopy.

We report the cellular properties of a luminescent cyclometalated iridium(III) complex, [Ir(pq)₂(phen-ITC)](PF₆) (Ir-ITC; Hpq=2-phenylquinoline, phen-ITC=5-isothiocyanate-1,10-phenanthroline), that efficiently and specifically labels mitochondria in living mammalian cells. Ir-ITC can be covalently conjugated to its protein targets, and its luminescence survived cell lysis, protein extraction, and gel electrophoresis under denaturing conditions. The conjugation of Ir-ITC with live-cell proteins is rapid and highly selective; the process requires active cellular metabolism, as the conjugation is abolished at nonphysiological temperature or in the presence of sodium azide. Based on measurements of the luminescence intensity, we have devised a biochemical fractionation procedure that allows the enrichment of the conjugated proteins, and their subsequent separation by two-dimensional gel electrophoresis (2DGE). Luminescent protein spots were picked from the gel and analyzed by mass spectrometry; this resulted in the identification of 46 proteins. Many of the strongly luminescently labeled proteins are mitochondrial proteins. One of the targets is VDAC1 (voltage-dependent anion channel 1). Consistent with known phenotypes of VDAC1 deregulation, prolonged exposure of cells to Ir-ITC led to significant mitochondrial shortening and fragmentation. As far as we know, this is the first report on the molecular characterization of the interactions of a luminescent dye with its biological targets. As many biological dyes exhibit specific intracellular staining patterns, the identification of their molecular targets can help elucidate the mechanisms behind their staining specificities and cytotoxicity. We believe our biochemical approach can be applied to identify the targets of a wide range of fluorescent and luminescent probes.

A new class of phosphorescent cyclometalated iridium(III)–polyamine complexes [{Ir(N^C)₂}_n(bPEI)](PF₆)_n (bPEI=branched poly(ethyleneimine), average M_w=25 kDa, n=15.6–27.4; HN^C=2-phenylpyridine Hppy (1 a), 2-((1,1′-biphenyl)-4-yl)pyridine Hpppy (2 a), 2-phenylquinoline Hpq (3 a), 2-phenylbenzothiazole Hbt (4 a), 2-(1-naphthyl)benzothiazole Hbsn (5 a)) and [Ir(N^C)₂(en)](PF₆) (en=ethylenediamine; HN^C=Hppy (1 b), Hpppy (2 b), Hpq (3 b), Hbt (4 b), Hbsn (5 b)) have been synthesized and characterized. The X-ray crystal structure of complex 5 b was also determined. All of these complexes showed a reversible iridium(IV/III) oxidation couple at +1.01 to +1.26 V and a quasi-reversible ligand-based reduction couple at −1.54 to −2.08 V (versus SCE). Upon photoexcitation, the complexes displayed intense and long-lived green to orange–red emission in fluid solutions at room temperature and in low-temperature glass. Lipophilicity measurements indicated that bPEI played a dominant role in the polar nature of complexes 1 a–5 a, thus rendering them very soluble in aqueous solutions. Inductively coupled plasma–mass spectrometry (ICP-MS) and confocal laser scanning microscopy (CLSM) data indicated that an energy-requiring process, such as endocytosis, was involved in the cellular uptake of all of the complexes. In addition, the cytotoxicity of the complexes toward human cervix epithelioid carcinoma (HeLa) and human embryonic kidney 293T (HEK293T) cell-lines has been evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. The DNA-binding properties of complex 5 a have been investigated by gel-retardation assays and the polyplexes that were formed from this complex with plasmid DNA (pDNA) were studied by zeta-potential measurements and particle-size estimation. Furthermore, complex 5 a was grafted with poly(ethylene glycol) (PEG, average M_w=2 kDa) to different extents, thereby yielding the phosphorescent copolymers PEG_12.3-g-5 a, PEG_25.4-g-5 a, and PEG_62.1-g-5 a. Interestingly, these copolymers showed enhanced transfection activity, as revealed by in vitro transfection experiments with tissue-culture-based luciferase assays.

We report the use of an organo-iridium dye conjugated with a water-soluble copolyethylenimine polymer, allowing the hybrid material to be used in combination with thioacid-coated CdTe quantum dots in an aqueous medium. When they are combined, hot carrier cooling observed in the pure quantum-dot case is heavily suppressed indicating fast (ps) electron transfer on a timescale that competes with non-radiative (Auger) relaxation.

The cover picture shows the numerous opportunities luminescent rhenium(I) tricarbonyl polypyridine complexes offer regarding the development of probes for biological applications. Since the spectroscopic and luminescence properties of rhenium(I) tricarbonyl polypyridine complexes were reported in the 1970s, a number of sensors and probes have been derived from these complexes. K. K.-W. Lo et al. describe in their Microreview on p. 3551 ff the fundamental emission characteristics of these complexes and explain why they hold promise for use as luminescent sensors. Additionally, the recent design of rhenium(I) tricarbonyl polypyridine complexes as biomolecular and cellular probes is summarized. Emphasis is placed on the structure–property relationships, bioconjugation, biomolecular binding, cellular uptake, cytotoxicity, and bioimaging studies of these complexes. Mr. Michael Wai-Lun Chiang is acknowledged for the cover picture design.

The interesting emission properties of rhenium(I) tricarbonyl polypyridine complexes have been exploited in the development of various sensors and probes for analytes. Luminescent probes targeting biomolecules have also been developed. Additionally, there has been a fast-growing interest in the cellular uptake properties of these complexes with a focus on their potential as cellular imaging reagents. In this Microreview, we describe the fundamental emission characteristics of luminescent rhenium(I) tricarbonyl polypyridine complexes and explain why they hold promise for use as luminescent sensors. Additionally, we summarize the recent design of these complexes as biomolecular and cellular probes, with an emphasis on studies of their structure–property relationships, bioconjugation, biomolecular binding, cellular uptake, cytotoxicity, and bioimaging applications.

We report the synthesis, characterization, and photophysical properties of a new class of luminescent cyclometalated iridium(III) polypyridine poly(ethylene glycol) (PEG) complexes [Ir(N^C)₂(N^N)](PF₆) (HN^C=Hppy (2-phenylpyridine), N^N=bpyCONHPEG1 (bpy=2,2′-bipyridine; 1 a), bpyCONHPEG3 (1 b); HN^C=Hpq (2-phenylquinoline), N^N=bpyCONHPEG1 (2 a), bpyCONHPEG3 (2 b); HN^C=Hpba (4-(2-pyridyl)benzaldehyde), N^N=bpyCONHPEG1 (3)) and their PEG-free counterparts (N^N=bpyCONHEt, HN^C=Hppy (1 c); HN^C=Hpq (2 c)). The cytotoxicity and cellular uptake of these complexes have been investigated by the MTT assay, ICPMS, laser-scanning confocal microscopy, and flow cytometry. The results showed that the complexes supported by the water-soluble PEG can act as biological probes and labels with considerably reduced cytotoxicity. Because the aldehyde groups of complex 3 are reactive toward primary amines, the complex has been utilized as the first luminescent PEGylation reagent. Bovine serum albumin (BSA) and poly(ethyleneimine) (PEI) have been PEGylated with this complex, and the resulting conjugates have been isolated, purified, and their photophysical properties studied. The DNA-binding and gene-delivery properties of the luminescent PEI conjugate 3-PEI have also been investigated.

Two novel luminescent polypyridinerhenium(I) bis-biotin complexes [Re(NN)(CO)₃(pyridine)](CF₃SO₃) {NN = 4,4′-bis{[2-(biotinamido)ethyl]aminocarbonyl}-2,2′-bipyridine, bpyC2B2 (1), 4,4′-bis[(2-{[6-(biotinamido)hexanoyl]amino}ethyl)aminocarbonyl]-2,2′-bipyridine, bpyC2C6B2 (2)} and their biotin-free counterpart [NN = 4,4′-bis(n-butylaminocarbonyl)-2,2′-bipyridine, bpyC4 (3)] have been synthesized and characterized. Upon irradiation, all the complexes exhibited triplet metal-to-ligand charge-transfer (³MLCT) emission in fluid solutions at room temperature and alcohol glass at 77 K. The avidin-binding properties of the bis-biotin complexes 1 and 2 have been studied by 4′-hydroxyazobenzene-2-carboxylic acid (HABA) assays, emission titrations, and dissociation assays. The potential use of the complexes as signal amplifiers for heterogeneous recognition assays has been demonstrated using avidin-coated microspheres and one of the complexes. Additionally, the cytotoxicity of these rhenium(I) complexes towards 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 laser-scanning confocal microscopy. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)