Luminescent Rhenium(I) and Iridium(III) Polypyridine Complexes as

Article pubs.acs.org/accounts

Luminescent Rhenium(I) and Iridium(III) Polypyridine Complexes as Biological Probes, Imaging Reagents, and Photocytotoxic Agents Kenneth Kam-Wing Lo* Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, P. R. China S Supporting Information *

CONSPECTUS: Although the interactions of transition metal complexes with biological molecules have been extensively studied, the use of luminescent transition metal complexes as intracellular sensors and bioimaging reagents has not been a focus of research until recently. The main advantages of luminescent transition metal complexes are their high photostability, long-lived phosphorescence that allows time-resolved detection, and large Stokes shifts that can minimize the possible self-quenching effect. Also, by the use of transition metal complexes, the degree of cellular uptake can be readily determined using inductively coupled plasma mass spectrometry. For more than a decade, we have been interested in the development of luminescent transition metal complexes as covalent labels and noncovalent probes for biological molecules. We argue that many transition metal polypyridine complexes display triplet charge transfer (3CT) emission that is highly sensitive to the local environment of the complexes. Hence, the biological labeling and binding interactions can be readily reflected by changes in the photophysical properties of the complexes. In this laboratory, we have modified luminescent tricarbonylrhenium(I) and bis-cyclometalated iridium(III) polypyridine complexes of general formula [Re(bpy-R1)(CO)3(py-R2)]+ and [Ir(ppyR3)2(bpy-R4)]+, respectively, with reactive functional groups and used them to label the amine and sulfhydryl groups of biomolecules such as oligonucleotides, amino acids, peptides, and proteins. Additionally, using a range of biological substrates such as biotin, estradiol, and indole, we have designed luminescent rhenium(I) and iridium(III) polypyridine complexes as noncovalent probes for biological receptors. The interesting results generated from these studies have prompted us to investigate the possible applications of luminescent transition metal complexes in intracellular systems. Thus, in the past few years, we have developed an interest in the cytotoxic activity, cellular uptake, and bioimaging applications of these complexes. Additionally, we and other research groups have demonstrated that many transition metal complexes have facile cellular uptake and organellelocalization properties and that their cytotoxic activity can be readily controlled. For example, complexes that can target the nucleus, nucleolus, mitochondria, lysosomes, endoplasmic reticulum, and Golgi apparatus have been identified. We anticipate that this selective localization property can be utilized in the development of intracellular sensors and bioimaging reagents. Thus, we have functionalized luminescent rhenium(I) and iridium(III) polypyridine complexes with various pendants, including molecule-binding moieties, sugar molecules, bioorthogonal functional groups, and polymeric chains such as poly(ethylene glycol) and polyethylenimine, and examined their potentials as biological reagents. This Account describes our design of luminescent rhenium(I) and iridium(III) polypyridine complexes and explains how they can serve as a new generation of biological reagents for diagnostic and therapeutic applications.

1. INTRODUCTION The interactions of luminescent transition metal complexes with biological molecules have been of long-standing interest, and the applications of these complexes to modify biomolecules have been widely investigated.1 However, the use of luminescent transition metal complexes as intracellular sensors and bioimaging reagents has not been a focus of research until recently.2−5 This interest is attributable to the following characteristics: (1) Many luminescent transition metal complexes have high photostability, which allows continuous exposure of the complexes to irradiation and enables real-time monitoring of the probes. (2) Since many transition metal complexes show long-lived phosphorescence (with lifetimes on the time scale of submicroseconds to microseconds), interference due to autofluorescence can be excluded using © XXXX American Chemical Society

time-gated detection. Also, the use of phosphorescence lifetime imaging microscopy (PLIM) offers very high sensitivity.5 (3) The triplet emissive states of transition metal complexes are associated with large Stokes shifts, which can minimize the possible self-quenching effect. Thus, a high local concentration of the probe due to multiple labeling of biomolecules or highly localized accumulation in live cells will not result in diminished brightness. (4) Transition metal complexes containing πconjugated ligands have been found to exhibit interesting twophoton absorption behavior, which is an attractive property for bioimaging applications because of the greater tissue-penetrating ability and excellent resolution. (5) The use of transition Received: April 16, 2015

A

DOI: 10.1021/acs.accounts.5b00211 Acc. Chem. Res. XXXX, XXX, XXX−XXX

Article

Accounts of Chemical Research

= Ir, Rh) (4),15,16 [Re(N^N)(CO)3(py-maleimide)]+ (5),17 and [Ir(N^C)2(phen-NHCOCH2I)]+ (6)13,14 (Figure 2). We

metal complexes means that the degree of cellular uptake can be readily determined using inductively coupled plasma mass spectrometry (ICP-MS). This quantitation method eliminates possible artifacts in microscopy since the amount of an intracellular probe may not always be proportional to its emission intensity, as in the case of probes that show highly environment-sensitive emission and those that carry a quencher. While the utilization of luminescent transition metal complexes in life sciences is emerging, two particular applications have received the most attention: (1) oxygen sensing based on the long-lived triplet states and (2) protein gel staining originating from the nonspecific lipophilic interactions between the complexes and proteins. For example, platinum(II) octaethylporphyrin6 and iridium(III) acetylacetonate7 complexes have been used as oxygen sensors in live cells. Also, ruthenium(II)8 and iridium(III)9 Ph2phen complexes modified with sulfonate groups have been utilized to stain protein gels for proteomic analyses. Additionally, ruthenium(II) complexes functionalized with N-hydroxysuccinimidyl ester have been developed as biomolecular labels.10 Some of these products are already commercially available. We have been interested in using luminescent tricarbonylrhenium(I) and bis-cyclometalated iridium(III) polypyridine complexes of general formula [Re(bpy-R1)(CO)3(py-R2)]+ and [Ir(ppy-R3)2(bpy-R4)]+, respectively (Figure 1), as biological reagents. Since the diimine ligands

Figure 2. Covalent labels 1−6.

have selected these complexes because they exhibit intense and long-lived triplet metal-to-ligand charge transfer (3MLCT) (dπ(Re or Ir) → π*(N^N or N^C)) or triplet intraligand (3IL) (π → π*) (N^N, N^C, or tpy) emission. As the isothiocyanate and aldehyde groups can react with primary amines and the maleimide and iodoacetamide groups with sulfhydryls, we have used these complexes to label amine- and sulfhydryl-modified oligonucleotides (e.g., M13 sequencing primers),11,13,14,17 amino acids (e.g., alanine),15 peptides (e.g., glutathione),17 and proteins (e.g., serum albumins and avidin).11−17 Importantly, the favorable photophysical properties of the labels and biological behavior of the biomolecules are both retained after bioconjugation.

Figure 1. Tricarbonylrhenium(I) and bis-cyclometalated iridium(III) polypyridine complexes.

are usually involved in the emissive states of the complexes, modification of these ligands enables tuning of the photophysical properties. Thus, functionalization of the other ligands, such as the pyridine ligands of the rhenium(I) complexes and the cyclometalating ligands of the iridium(III) complexes, can endow the complexes with new biological properties. Concerning their photoredox behavior, rhenium(I) complexes generally have stronger photooxidizing properties compared with their cyclometalated iridium(III) counterparts. This Account describes our design of these complexes as biological probes, imaging reagents, and photocytotoxic agents. Readers are referred to other comprehensive reviews for related work.2−5

2.2. Noncovalent Probes

Many of our rhenium(I) and iridium(III) polypyridine complexes exhibit 3MLCT emission, which is very sensitive to the local environment of the complexes. For example, the emission quantum yields and lifetimes of these 3MLCT emitters increase with decreasing solvent polarity. Thus, after modification with biological substrates, the complexes are expected to show changes in their photophysical properties upon binding of the substrates to their biological receptors. In view of the importance of avidin−biotin recognition in chemical biology applications,18 we have developed luminescent rhenium(I), ruthenium(II), and iridium(III) polypyridine biotin complexes, including [Re(N^N)(CO)3(py-biotin)]+ (7),19,20 [Ru(N^N)2(bpy-biotin)]2+ (8),21 [Ir(N^C)2(bpy-biotin)]+ (9),22 [Ir(ppy-biotin)2(N^N)]+ (10),23,24 and [Ir(ppybiotin)2(bpy-biotin)]+ (11)24 (Figure 3). Importantly, unlike common organic biotin−fluorophores that suffer from selfquenching upon binding to avidin, these biotin complexes display a higher emission intensity and longer emission lifetime after binding to avidin as a result of the decreased polarity of their local environment. The avidin-cross-linking properties of the iridium(III) bis- and tris(biotin) complexes have also been examined.23,24

2. COVALENT LABELS AND NONCOVALENT PROBES FOR BIOMOLECULES 2.1. Covalent Labels

We have designed luminescent rhenium(I), iridium(III), and rhodium(III) polypyridine complexes that contain a reactive functional group such as isothiocyanate, aldehyde, maleimide, or iodoacetamide; examples include [Re(N^N)(CO)3(pyNCS)]+ (1),11 [Ir(tpy-R)(tpy-C6H4-NCS)]3+ (2),12 [Ir(N^C)2(phen-NCS)]+ (3),13,14 [M(ppy-CHO)2(N^N)]+ (M B


Article


Figure 4. Estradiol complexes 12−14 and indole complexes 15 and 16.

Figure 3. Biotin complexes 7−11.

The role of estrogen receptors (ERs) in hormone-dependent breast cancer is very important because the receptor content gives the most accurate index of the cancer.25 ERs are considered to be the target proteins of a number of endocrine disruptors, some of which are environmental pollutants and hazardous to humans and animals.26 Thus, the design of probes for these proteins has attracted much interest. We have exploited the luminescent transition metal estradiol complexes [Re(N^N)(CO)3(py-estradiol)]+ (12),27 [Ru(N^N)2(bpy-estradiol)]2+ (13),28 and [Ir(N^C)2(bpy-estradiol)]+ (14)29 (Figure 4) as probes for estrogen receptor α (ERα). Upon binding to the receptor, the complexes reveal emission enhancement and lifetime extension due to an increase in the hydrophobicity of their local environment (Figure 5). Using [Fe(CN)6]3− as an emission quencher for the unbound complexes, we have significantly increased the ERα-induced emission enhancement factor (I/I0) of the iridium(III) complexes to about 48.7.29 These estradiol complexes have a high potential to be developed as imaging reagents for ERpositive breast cancer cells. In other studies, we have examined the interactions of the luminescent transition metal polypyridine indole complexes [Re(N^N)(CO) 3 (py-indole)] + (15)30,31 and [Ir(N^C)2(bpy-indole)]+ (16)32 (Figure 4) with indole-binding proteins such as bovine serum albumin (BSA) and lysozyme.

Figure 5. Emission spectral traces of complex 14 (N^C = pq) (5 μM) in the presence of 0 to 375 nM ERα. The emission lifetimes increase from 0.25 to 1.45 μs. Adapted with permission from ref 29. Copyright 2007 Wiley-VCH.

types of iridium(III) polypyridine complexes that show dualemissive properties under ambient conditions. The first type is [Ir(ppy)2(dpq-CONH-R)]+ (R = nBu, biotin) (17) (Figure 6).33 The n-butyl complex binds to double-stranded DNA, giving rise to a structureless 3MLCT emission band at 602 nm (Figure 7 top), which is a typical feature of luminescent metallointercalators. Interestingly, although having the same luminophore, the biotin complex exhibits a vibronically structured emission band at 490 nm upon binding to avidin (Figure 7 bottom). The emission has been assigned to a 3IL (π → π*) (ppy or dpq-CONH-biotin) state, which is supported by the long emission lifetime (2.20 μs) and structured features. It is reasonable that intercalation of the dpq-CONH-nBu ligand into the base pairs of the DNA stabilizes the π*(N^N) levels

2.3. Dual-Emissive Probes

Dual-emissive probes are interesting reagents because they enable ratiometric sensing, which offers more reliable detection because the ratio of two emission intensities is measured instead of the intensity at one single wavelength, and thus, interference caused by other parameters such as the concentration of the probe can be minimized. During the course of designing biological probes, we have discovered two C


Article


Figure 7. (top) Emission spectra of complex 17 (N^N = dpqCONH-nBu) in the absence (blue) and presence (red) of doublestranded calf thymus DNA. (bottom) Emission spectra of complex 17 (N^N = dpq-CONH-biotin) in the absence (blue) and presence (red) of avidin. Adapted with permission from ref 33. Copyright 2006 WileyVCH.

Figure 6. Dual-emissive complexes 17, 18, and 18a.

and thereby promotes the 3MLCT emission. The reason for the switch to a 3IL transition in the avidin adduct of the biotin complex is not fully understood. However, the amide group could play an important role; for example, it may establish hydrogen-bonding interactions with amino acid residues of the protein, leading to a change in the electronic structure of the complex and the population of an emissive 3IL state. The second system is an iridium(III) polypyridine complex containing a secondary amine in the ppy ligand, [Ir(ppyCH2NH-C4H9)2(bpy-CONH-C2H5)]+ (18) (Figure 6).34 Remarkably, the complex shows a high-energy (HE) structured band at ca. 490 nm (τo = 2−3 μs) and a low-energy (LE) band/ shoulder at ca. 570−610 nm (τo < 1 μs) in CH2Cl2. In more polar solvents such as CH3CN and phosphate buffer, the spectra are dominated by the HE band (Figure 8). On the basis of the photophysical data and time-dependent density functional theory calculations, we have assigned the HE structured band to a 3IL emissive state and the LE broad band to a state of high 3MLCT parentage that may also be mixed with some triplet amine-to-ligand charge transfer (3NLCT) (amine → π*(N^N)) character. These interesting results have prompted us to design dual-emissive iridium(III) complexes containing a biotin, estradiol, or octadecyl unit, [Ir(ppy-CH 2 NHC4H9)2(bpy-CONH-R)]+ (R = biotin, estradiol, C18H37) (18a), as sensors for avidin, ERα, and the lipid-binding protein human serum albumin (HSA), respectively.

Figure 8. Normalized emission spectra of complex 18 in degassed CH2Cl2 (red), CH3CN (green), and phosphate buffer (blue) at 298 K. Adapted with permission from ref 34. Copyright 2008 Wiley-VCH.

3. CELLULAR UPTAKE AND LOCALIZATION STUDIES 3.1. Biotinylation Reagents and Multibiotin Complexes

Modification of biomolecules with biotin (biotinylation) is widely applied in the purification and recognition of biomolecules because of the rapid and strong avidin−biotin interactions.18 In 2007 we reported rhenium(I) complexes containing a biotin unit and an isothiocyanate moiety, [Re(N^N)(CO)3(py-biotin-NCS)]+ (19) (Figure 9), as the first luminescent biotinylation reagents.35 These complexes D


Article

Accounts of Chemical Research 3.2. Nitric Oxide Sensors

Nitric oxide (NO) plays a key role in the signaling processes of the immune, cardiovascular, and nervous systems.37 Since unregulated NO production may be associated with cancer and other diseases,38 the development of NO sensors is of paramount importance. Fluorometric detection of NO has basically relied on two strategies. The first one is the exploitation of a paramagnetic metal ion such as copper(II) as a quencher for a fluorescent ligand.39 After the metal ion is reduced by NO and displaced from the ligand, the fluorescence is restored. The second strategy involves photoinduced electron transfer (PeT) quenching of a fluorophore by an electron-rich diaminoaromatic moiety, which also acts as an NO sensing unit. 40 Emission enhancement is observed when the diaminoaromatic moiety is converted to an electron-deficient benzotriazole derivative by NO. We believe that the second strategy can be applied to luminescent transition metal complexes because their emission is readily quenched by a diaminoaromatic moiety. Thus, we have designed the luminescent rhenium(I) polypyridine diamine complexes [Re(N^N)(CO)3(py-diamine)]+ (21) (Figure 11).41 The Figure 9. Biotinylation reagents 19 and 20 and thiourea complex 19a.

have been reacted with ethylamine and BSA, resulting in the formation of the thiourea complexes [Re(N^N)(CO)3(pybiotin-TU-Et)]+ (19a) (Figure 9) and rhenium−BSA conjugates, respectively. These luminescent thiourea complexes and bioconjugates show strong binding affinity toward avidin. Additionally, the cellular uptake of one of the thiourea complexes has been investigated; fluorescence microscopy reveals that the complex is localized in the Golgi apparatus (Figure 10). In another study, we have developed the cyclometalated iridium(III) and rhodium(III) bis(pyridylbenzaldehyde) biotin complexes [Ir(ppy-CHO)2(bpybiotin)]+ (M = Ir, Rh) (20) (Figure 9) as luminescent biotinylation reagents.36 We have biotinylated BSA with the iridium(III) complex, and the resultant conjugate displays intense emission and avidin-binding properties. The lipophilicity, cytotoxicity, cellular uptake, and localization properties of the aforementioned iridium(III) multibiotin complexes 10 and 11 (Figure 3) have been investigated.24 Since biotin is a relatively polar compound, iridium(III) complexes containing two or more biotin pendants are less lipophilic than their biotin-free counterparts. As a result, the multibiotin complexes exhibit less efficient uptake by live cells and negligible cytotoxicity.

Figure 11. Nitric oxide sensors 21, 21a, and 22.

emission quantum yields and lifetimes of the complexes are substantially lower and shorter than those of common rhenium(I) diimine complexes as a result of PeT. Addition of the NO-releasing agent NOC-7 into a solution of the Ph2phen−diamine complex results in significant emission enhancement (I/I0 = 31), which is attributed to the conversion of the diamine complex to its triazole counterpart. To increase the reactivity of the diaminoaromatic unit, we have used a methoxy substituent to enrich its electron density, leading to the complexes [Re(N^N)(CO)3{py-CH2NHC6H3(NH2)(OCH3)}]+ (21a) (Figure 11).42 Addition of NOC-7 to a solution of the phen−diamine complex leads to substantial emission enhancement (I/I0 = 64). Also, the complex displays selectivity toward NO over other biologically relevant species such as HO·, H2O2, O2−, OCl−, ONOO−, NO2−, and NO3−. No significant emission is observed upon incubation of HeLa cells with the Ph2phen−diamine complex.

Figure 10. (left) Bright-field microscopy, (middle) overlay, and (right) fluorescence microscopy images of HeLa cells incubated with complex 19a (N^N = Ph2phen). Scale bars = 25 μm. Adapted with permission from ref 35. Copyright 2008 American Chemical Society. E


Article


Figure 12. Confocal microscopy images of RAW264.7 cells upon incubation with complex 21a (N^N = Ph2phen) without pretreatment (left), pretreated with LPS (middle), and pretreated with both LPS and LNNA (right). Scale bars = 25 μm. Adapted with permission from ref 42. Copyright 2014 Wiley-VCH.

suggesting the involvement of glucose transporters (GLUTs) in the uptake.45 To investigate the role of GLUTs, we have synthesized the iridium(III) glucose and galactose complexes [Ir(N^C)2(bpy-TEG-ONCH3-β-D-sugar)]+ (sugar = glucose, galactose) (23a) (Figure 13).46 Temperature-dependent and chemical inhibition experiments indicate that the uptake of the bt−glucose complex occurs through an energy-requiring process such as endocytosis in addition to a pathway that is mediated by GLUTs. We have also synthesized the related luminescent rhenium(I) glucose complexes [Re(N^N)(CO)3(py-3-β-D-glucose)]+ (24) (Figure 13).47 Importantly, the uptake of one of these glucose complexes by the transformed cell lines HeLa and human breast adenocarcinoma (MCF-7) is higher than that by two nontransformed cell lines, human embryonic kidney cells (HEK293T) and mouse embryonic fibroblasts (NIH/3T3), suggestive of GLUT-mediated uptake since the transformed cell lines are known to overexpress these transporters. The ICP-MS data show that the concentration of the complex (0.94 ± 0.12 mM) in HeLa cells is much higher than the incubation concentration (100 μM), indicating the enrichment of the complex through efficient cellular uptake. The most important observation is the inhibition of uptake of the complex by Dglucose and 2-deoxy-D-glucose but not by L-glucose. Confocal microscopy indicates that the complex is localized in the mitochondria (Figure 14), which is a result of its cationic and lipophilic nature. The substrates of GLUTs are not limited to glucose; for example, GLUT5 selectively facilitates the uptake of fructose.48 This transporter is overexpressed in breast cancer tissues, but its expression in other cancer cells and normal breast tissues is very limited. We have designed the luminescent iridium(III) fructose complex [Ir(N^C)2(bpy-fructose)]+ (25)49 and the rhenium(I) fructose complex [Re(Ph2phen)(CO)3(py-fructose)]+ (26)50 (Figure 13). Notably, competitive experiments involving the breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231), two nonbreast cancer cell lines (A549 and HepG2), and two nontransformed cell lines (NIH/3T3 and HEK293T) indicate that the uptake of the complexes is much more efficient with the breast cancer cells than with the other cells and that the uptake is inhibited by unmodified fructose (Figure 15). This highlights the possible use of these fructose complexes as imaging reagents for breast cancer cells and luminescent fructose-uptake indicators.

However, after the cells are further treated with NOC-7, intense intracellular emission is detected. We have applied this complex to murine macrophage RAW264.7 cells, which are known to generate high levels of NO upon stimulation by endotoxins or cytokines. Treatment of RAW264.7 cells with the complex gives only very weak emission. However, after stimulation by the endotoxin lipopolysaccharide (LPS), intense intracellular emission is observed (Figure 12). The increase in emission intensity is suppressed by the NO synthase inhibitor NG-nitro-L-arginine (LNNA). Furthermore, we have developed the related iridium(III) diamine complexes [Ir(N^C)2{bpyCH2NH-C6H3(NH2)(OCH3)}]+ (22) (Figure 11), which can also detect exogenous and endogenous NO in HeLa and RAW264.7 cells, respectively.43 3.3. Sugar Conjugates

We have designed iridium(III) complexes appended with an Nmethylaminooxy group, [Ir(N^C)2(bpy-ONHCH3)]+ (23) (Figure 13).44 The complexes are used to label monosaccharides (D-glucose and D-galactose) and disaccharides (Dlactose and D-maltose), and the products are obtained exclusively as the pyranose form. Interestingly, the β-glucose conjugate is taken up by HeLa cells more effectively than its βgalactose, α-galactose, β-lactose, and β-maltose counterparts,

3.4. Bioorthogonal Probes

Bioorthogonal chemistry has emerged as a versatile method to image biomolecules in their native settings.51 In a typical

Figure 13. Sugar label 23 and sugar conjugates 23a and 24−26. F


Article


Figure 14. Confocal microscopy images of a HeLa cell upon incubation successively with MitoTracker Deep Red FM (633 nm) and complex 24 (N^N = Ph2phen) (405 nm). Scale bars = 5 μm. Adapted with permission from ref 47. Copyright 2011 Wiley-VCH.

Figure 15. Confocal microscopy images of six different types of cells upon incubation of complex 26 in the presence of 0 and 50 mM fructose. Scale bars = 25 μm. Adapted with permission from ref 50. Copyright 2013 American Chemical Society.

procedure, a substrate modified with a chemical reporter is incorporated into live cells or organisms, where it is then recognized by a bioorthogonal probe that carries the complementary functionality. Among the chemical reporters, azide is particularly useful because of its small size, non-native nature, and inertness toward biomolecules. Its strain-promoted alkyne−azide cycloaddition (SPAAC) with cyclooctynes such as dibenzocyclooctyne (DIBO) is an attractive strategy for bioimaging.52 We have synthesized the luminescent iridium(III) DIBO complexes [Ir(N^C)2(bpy-C6-DIBO)]+ (27)53 (Figure 16) and investigated their reactions with the model compound benzyl azide. Unfortunately, the cellular uptake of these lipophilic DIBO complexes is so efficient that they cannot label membrane-bound azidoglycans of Chinese hamster ovary (CHO) cells treated with 1,3,4,6-tetra-O-acetyl-N-azidoacetylD-mannosamine (Ac4ManNAz). Thus, to lower the lipophilicity and decrease the cellular uptake rate, we have installed two carboxyl groups to obtain the complex [Ir(ppy-COOH)2(bpyTEG-DIBO)]+ (27a) (Figure 16). Interestingly, incubation of Ac4ManNAz-treated CHO cells with 27a leads to intense emission from the cell membrane. In contrast, the Ac4ManNAzuntreated cells do not show any emission, highlighting the bioorthogonal labeling characteristics of the complex. Among different bioorthogonal reactions, the inverse electron-demand Diels−Alder cycloaddition of tetrazine with dienophiles such as alkenes and alkynes has attracted much attention.54 A remarkable advantage is that many fluorophore− tetrazine conjugates experience emission quenching due to Förster resonance energy transfer (FRET) and hence display emission “turn-on” upon reaction with a dienophile as a result of the conversion of tetrazine into a nonquenching molecule.55 We have designed new phosphorogenic bioorthogonal probes derived from the mononuclear and binuclear rhenium(I)

Figure 16. Bioorthogonal probes 27, 27a, 28, and 28a.

tetrazine complexes [Re(N^N)(CO)3(py-Tz)]+ (28) and [{Re(N^N)(CO)3}2(μ-py-Tz-py)]2+ (28a) (Figure 16).56 Upon photoexcitation, the complexes exhibit only very weak 3 MLCT emission because of FRET from the rhenium(I) polypyridine unit to the tetrazine moiety. The complexes undergo facile reaction with the model substrates 5-norbornen2-ol (NBO) and (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN), leading to significant emission enhancement (up to 181.1-fold) (Figure 17). We have concluded that these G


Article


generate different reactive oxygen species such as singlet oxygen.59 Although photoactivated biological activity has been observed in many transition metal complexes, most of these complexes still display high cytotoxicity in the dark. We believe that the attachment of PEG pendants will minimize these unfavorable interactions. Thus, we have developed luminescent iridium(III) complexes with nondiscrete PEG, [Ir(N^C)2(bpyPEG)]+ (MWPEG = ca. 5 kDa) (29a) (Figure 18).60 Similar to their discrete PEG counterparts, these iridium(III) PEG complexes exhibit negligible cytotoxicity in the dark. However, they display interesting cytotoxic activity upon irradiation due to the generation of singlet oxygen, with photodynamic indices (IC50,dark/IC50,light) ranging from 12.9 to 88.2. The application of the complexes as a visualizing reagent has been demonstrated using zebrafish (Danio rerio) as an animal model (Figure 19). Related rhenium(I) PEG complexes 30 (Figure 18) also show enhanced water solubility and reduced cytotoxicity.61

Figure 17. Emission spectra of complex 28a (N^N = Ph2phen) (10 μM) in the absence (black) and presence (red) of BCN (150 μM). Inset: comparison of the emission of the complex (left) and its pyridazine product (right). Adapted with permission from ref 56. Copyright 2015 Royal Society of Chemistry.

tetrazine complexes display excellent phosphorogenic properties and that modification of the tetrazine moiety with the positively charged rhenium(I) polypyridine units significantly increases its reactivity. 3.5. Poly(ethylene glycol) Complexes and Photocytotoxic Agents

In the design of biological probes using transition metal complexes, we have been confronted with two problems: the low water solubility and high cytotoxicity of transition metal complexes. We envisage that the attachment of poly(ethylene glycol) (PEG) pendants to the metal complexes will circumvent these problems.57 In the first study, we have designed luminescent cyclometalated iridium(III) complexes containing discrete PEG units, [Ir(N^C)2(bpy-PEGdiscrete)]+ (29) (Figure 18).58 Modification of the complexes with PEG significantly enhances their solubility in aqueous solution (>3 mM) and considerably reduces their cytotoxicity (IC50 = 287−1180 μM), which is much lower than that of their ethyl counterparts (IC50 = 4.1−14.6 μM). The excited states of many luminescent transition metal complexes are effectively quenched by molecular oxygen to

Figure 19. Biodistribution of complex 29a (N^C = pq) in zebrafish larva through intravascular loading at 48 hpf. (left) The complex moves from the point of injection (white) to notochord, spinal cord (yellow), and brain ventricle (red). (right) The complex gradually accumulates in the yolk sac and cardiac cavity (yellow) and the head space (red) 24 h after loading. Scale bars = 200 μm. Adapted with permission from ref 60. Copyright 2013 Elsevier.

Since the binuclear iridium(III) complexes [Ir2(N^C)4Cl2] react with neutral and anionic bidentate ligands such as diimines, picolinates, and acetylacetonates to form stable complexes, we anticipate that they will also react with polyethylenimine (PEI), which possesses the NCH2CH2N chelating unit. Thus, we have synthesized the family of iridium(III) PEI conjugates 31 (Figure 18) that show blue to red emission upon excitation.62 Because of their polyamine nature, these conjugates are very polar (log Po/w = −1.38 to −0.37). After cellular uptake, they are enclosed in endosomes and subsequently delivered to lysosomes. Remarkably, these conjugates form polyplexes with plasmid DNA, and the uptake and trafficking of the polyplexes have been studied by confocal microscopy.

Figure 18. PEG and PEI complexes 29, 29a, 30, and 31. H



■

4. CONCLUSION The presence of d-block metal centers enables transition metal complexes to establish new electronic states, which give rise to characteristic photophysical and photochemical properties that are significantly different from those of fluorescent organic dyes, lanthanide chelates, and quantum dots. We have demonstrated that these properties, which include high photostability, long emission lifetimes, large Stokes shifts, inter/intramolecular energy/electron transfer, and the photogeneration of reactive oxygen species, make transition metal complexes useful candidates as photofunctional biological reagents. Current challenges in the applications of luminescent transition metal complexes as biosensors and imaging reagents include reversible binding to analytes (i.e., non-reaction-based), which would realize continuous detection; emission intensities and lifetimes that are more sensitive to the hydrophobicity of the local environment of the complexes, which would allow monitoring of intracellular biological events at the molecular level; and the incorporation of magnetically active and/or radioactive atoms, which would facilitate the development of multimodal imaging reagents. For these goals to be achieved, advanced molecular design based on a thorough investigation of the ground- and excited-state natures of the complexes is required. We believe that new luminescent transition metal complexes will continue to serve as functional reagents for diagnostic and therapeutic applications.

■

REFERENCES

(1) For example, see: Terpetschnig, E.; Szmacinski, H.; Lakowicz, J. R. Long-lifetime Metal-ligand Complexes as Probes in Biophysics and Clinical Chemistry. Methods Enzymol. 1997, 278, 295−321. (2) Fernández-Moreira, V.; Thorp-Greenwood, F. L.; Coogan, M. P. Application of d6 Transition Metal Complexes in Fluorescence Cell Imaging. Chem. Commun. 2010, 46, 186−202. (3) Zhao, Q.; Huang, C.; Li, F. Phosphorescent Heavy-metal Complexes for Bioimaging. Chem. Soc. Rev. 2011, 40, 2508−2524. (4) Lo, K. K.-W.; Choi, A. W.-T.; Law, W.-H. Applications of Luminescent Inorganic and Organometallic Transition Metal Complexes as Biomolecular and Cellular Probes. Dalton Trans. 2012, 41, 6021−6027. (5) Baggaley, E.; Weinstein, J. A.; Williams, J. A. G. Lighting the Way to See inside the Live Cell with Luminescent Transition Metal Complexes. Coord. Chem. Rev. 2012, 256, 1762−1785. (6) Koo, Y.-E. L.; Cao, Y.; Kopelman, R.; Koo, S. M.; Brasuel, M.; Philbert, M. A. Real-time Measurements of Dissolved Oxygen inside Live Cells by Organically Modified Silicate Fluorescent Nanosensors. Anal. Chem. 2004, 76, 2498−2505. (7) Zhang, S.; Hosaka, M.; Yoshihara, T.; Negishi, K.; Iida, Y.; Tobita, S.; Takeuchi, T. Phosphorescent Light-emitting Iridium Complexes Serve as a Hypoxia-sensing Probe for Tumor Imaging in Living Animals. Cancer Res. 2010, 70, 4490−4498. (8) Rabilloud, T.; Strub, J. M.; Luche, S.; van Dorsselaer, A.; Lunardi, J. A Comparison between Sypro Ruby and Ruthenium(II) Tris(bathophenanthroline Disulfonate) as Fluorescent Stains for Protein Detection in Gels. Proteomics 2001, 1, 699−704. (9) Jia, J.; Fei, H.; Zhou, M. Luminescent Iridium(III) Complexes as Novel Protein Staining Agents. Electrophoresis 2012, 33, 1397−1401. (10) Geisser, B.; Ponce, A.; Alsfasser, R. pH-Dependent Excited-state Dynamics of [Ru(bpy)3]2+-modified Amino Acids: Effects of an Amide Linkage and Remote Functional Groups. Inorg. Chem. 1999, 38, 2030−2037. (11) Lo, K. K.-W.; Ng, D. C.-M.; Hui, W.-K.; Cheung, K.-K. Luminescent Rhenium(I) Polypyridine Complexes with an Isothiocyanate Moiety − Versatile Labeling Reagents for Biomolecules. J. Chem. Soc., Dalton Trans. 2001, 2634−2640. (12) Lo, K. K.-W.; Chung, C.-K.; Ng, D. C.-M.; Zhu, N. Synthesis, Characterization and Photophysical Studies of Novel Biological Labeling Reagents Derived from Luminescent Iridium(III) Terpyridine Complexes. New J. Chem. 2002, 26, 81−88. (13) Lo, K. K.-W.; Ng, D. C.-M.; Chung, C.-K. First Examples of Luminescent Cyclometalated Iridium(III) Complexes as Labeling Reagents for Biological Substrates. Organometallics 2001, 20, 4999− 5001. (14) Lo, K. K.-W.; Chung, C.-K.; Lee, T. K.-M.; Lui, L.-H.; Tsang, K. H.-K.; Zhu, N. New Luminescent Cyclometalated Iridium(III) Diimine Complexes as Biological Labeling Reagents. Inorg. Chem. 2003, 42, 6886−6897. (15) Lo, K. K.-W.; Chung, C.-K.; Zhu, N. Synthesis, Photophysical and Electrochemical Properties, and Biological Labeling Studies of Cyclometalated Iridium(III) Bis(pyridylbenzaldehyde) Complexes: Novel Luminescent Cross-linkers for Biomolecules. Chem. - Eur. J. 2003, 9, 475−483. (16) Lo, K. K.-W.; Li, C.-K.; Lau, K.-W.; Zhu, N. Luminescent Cyclometalated Rhodium(III) Bis(pyridylbenzaldehyde) Complexes with Long-lived Excited States. Dalton Trans. 2003, 4682−4689. (17) Lo, K. K.-W.; Hui, W.-K.; Ng, D. C.-M.; Cheung, K.-K. Synthesis, Characterization, Photophysical Properties, and Biological Labeling Studies of a Series of Luminescent Rhenium(I) Polypyridine Maleimide Complexes. Inorg. Chem. 2002, 41, 40−46. (18) Wilchek, M.; Bayer, E. A. The Avidin-biotin Complex in Bioanalytical Applications. Anal. Biochem. 1988, 171, 1−32. (19) Lo, K. K.-W.; Hui, W.-K.; Ng, D. C.-M. Novel Rhenium(I) Polypyridine Biotin Complexes That Show Luminescence Enhancement and Lifetime Elongation upon Binding to Avidin. J. Am. Chem. Soc. 2002, 124, 9344−9345.

ASSOCIATED CONTENT

S Supporting Information *

Photophysical data and biological applications of selected complexes. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/ acs.accounts.5b00211.

■

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biography Kenneth Kam-Wing Lo obtained his B.Sc. and Ph.D. degrees at The University of Hong Kong in 1993 and 1997, respectively. He then worked as a Croucher Foundation Postdoctoral Research Fellow at the Inorganic Chemistry Laboratory of the University of Oxford. In 1999, he joined the Department of Biology and Chemistry of City University of Hong Kong as an Assistant Professor and has been a Professor since 2011. He received an APA Prize for Young Scientist from the Asian and Oceanian Photochemistry Association in 2005, a Distinguished Lectureship Award from the Chemical Society of Japan in 2011, and a Croucher Senior Research Fellowship from the Croucher Foundation in 2015. His research interest is the utilization of luminescent transition metal complexes as biological probes.

■

ACKNOWLEDGMENTS We thank the Hong Kong Research Grants Council and City University of Hong Kong for financial support. K.K.-W.L. thanks his collaborators and research group members, whose names appear in the reference list. He is grateful to the Croucher Foundation for the award of a Croucher Senior Research Fellowship. I


Article

Accounts of Chemical Research (20) Lo, K. K.-W.; Hui, W.-K. Design of Rhenium(I) Polypyridine Biotin Complexes as a New Class of Luminescent Probes for Avidin. Inorg. Chem. 2005, 44, 1992−2002. (21) Lo, K. K.-W.; Lee, T. K.-M. Luminescent Ruthenium(II) Polypyridine Biotin Complexes: Synthesis, Characterization, Photophysical and Electrochemical Properties, and Avidin-binding Studies. Inorg. Chem. 2004, 43, 5275−5282. (22) Lo, K. K.-W.; Chan, J. S.-W.; Lui, L.-H.; Chung, C.-K. Novel Luminescent Cyclometalated Iridium(III) Diimine Complexes That Contain a Biotin Moiety. Organometallics 2004, 23, 3108−3116. (23) Lo, K. K.-W.; Lau, J. S.-Y. Cyclometalated Iridium(III) Diimine Bis(biotin) Complexes as the First Luminescent Biotin-based Crosslinkers for Avidin. Inorg. Chem. 2007, 46, 700−709. (24) Zhang, K. Y.; Lo, K. K.-W. Synthesis, Properties, and Live-cell Imaging Studies of Luminescent Cyclometalated Iridium(III) Polypyridine Complexes Containing Two or Three Biotin Pendants. Inorg. Chem. 2009, 48, 6011−6025. (25) Gelbfish, G. A.; Davidson, A. L.; Kopel, S.; Schreibman, B.; Gelbfish, J. S.; Degenshein, G. A.; Herz, B. L.; Cunningham, J. N. Relationship of Estrogen and Progesterone Receptors to Prognosis in Breast Cancer. Ann. Surg. 1988, 207, 75−79. (26) Colborn, T. Environmental Estrogens: Health Implications for Humans and Wildlife. Environ. Health Persp. 1995, 103, 135−136. (27) Lo, K. K.-W.; Tsang, K. H.-K.; Zhu, N. Luminescent Tricarbonylrhenium(I) Polypyridine Estradiol Conjugates: Synthesis, Crystal Structure, and Photophysical, Electrochemical, and Proteinbinding Properties. Organometallics 2006, 25, 3220−3227. (28) Lo, K. K.-W.; Lee, T. K.-M.; Lau, J. S.-Y.; Poon, W.-L.; Cheng, S.-H. Luminescent Biological Probes Derived from Ruthenium(II) Estradiol Polypyridine Complexes. Inorg. Chem. 2008, 47, 200−208. (29) Lo, K. K.-W.; Zhang, K. Y.; Chung, C.-K.; Kwok, K. Y. Synthesis, Photophysical and Electrochemical Properties, and Proteinbinding Studies of Luminescent Cyclometalated Iridium(III) Bipyridine Estradiol Conjugates. Chem. - Eur. J. 2007, 13, 7110−7120. (30) Lo, K. K.-W.; Tsang, K. H.-K.; Hui, W.-K.; Zhu, N. Luminescent Rhenium(I) Diimine Indole Conjugates − Photophysical, Electrochemical and Protein-binding Properties. Chem. Commun. 2003, 2704−2705. (31) Lo, K. K.-W.; Tsang, K. H.-K.; Hui, W.-K.; Zhu, N. Synthesis, Characterization, Crystal Structure, and Electrochemical, Photophysical, and Protein-binding Properties of Luminescent Rhenium(I) Diimine Indole Complexes. Inorg. Chem. 2005, 44, 6100−6110. (32) Lau, J. S.-Y.; Lee, P.-K.; Tsang, K. H.-K.; Ng, C. H.-C.; Lam, Y.W.; Cheng, S.-H.; Lo, K. K.-W. Luminescent Cyclometalated Iridium(III) Polypyridine Indole Complexes − Synthesis, Photophysics, Electrochemistry, Protein-binding Properties, Cytotoxicity, and Cellular Uptake. Inorg. Chem. 2009, 48, 708−718. (33) Lo, K. K.-W.; Chung, C.-K.; Zhu, N. Nucleic Acid Intercalators and Avidin Probes Derived from Luminescent Cyclometalated Iridium(III)−Dipyridoquinoxaline and −Dipyridophenazine Complexes. Chem. - Eur. J. 2006, 12, 1500−1512. (34) Lo, K. K.-W.; Zhang, K. Y.; Leung, S.-K.; Tang, M.-C. Exploitation of the Dual-emissive Properties of Cyclometalated Iridium(III)−Polypyridine Complexes in the Development of Luminescent Biological Probes. Angew. Chem., Int. Ed. 2008, 47, 2213−2216. (35) Lo, K. K.-W.; Louie, M.-W.; Sze, K.-S.; Lau, J. S.-Y. Rhenium(I) Polypyridine Biotin Isothiocyanate Complexes as the First Luminescent Biotinylation Reagents − Synthesis, Photophysical Properties, Biological Labeling, Cytotoxicity, and Imaging Studies. Inorg. Chem. 2008, 47, 602−611. (36) Leung, S.-K.; Kwok, K. Y.; Zhang, K. Y.; Lo, K. K.-W. Design of Luminescent Biotinylation Reagents Derived from Cyclometalated Iridium(III) and Rhodium(III) Bis(pyridylbenzaldehyde) Complexes. Inorg. Chem. 2010, 49, 4984−4995. (37) Murad, F. Discovery of Some of the Biological Effects of Nitric Oxide and Its Role in Cell Signaling. Angew. Chem., Int. Ed. 1999, 38, 1856−1868.

(38) Calabrese, V.; Bates, T. E.; Stella, A. M. G. NO Synthase and NO-dependent Signal Pathways in Brain Aging and Neurodegenerative Disorders: the Role of Oxidant/Antioxidant Balance. Neurochem. Res. 2000, 25, 1315−1341. (39) Lim, M. H.; Xu, D.; Lippard, S. J. Visualization of Nitric Oxide in Living Cells by a Copper-based Fluorescent Probe. Nat. Chem. Biol. 2006, 2, 375−380. (40) Nagano, T.; Yoshimura, T. Bioimaging of Nitric Oxide. Chem. Rev. 2002, 102, 1235−1270. (41) Choi, A. W.-T.; Poon, C.-S.; Liu, H.-W.; Cheng, H.-K.; Lo, K. K.-W. Rhenium(I) Polypyridine Complexes Functionalized with a Diaminoaromatic Moiety as Phosphorescent Sensors for Nitric Oxide. New J. Chem. 2013, 37, 1711−1719. (42) Choi, A. W.-T.; Yim, V. M.-W.; Liu, H.-W.; Lo, K. K.-W. Rhenium(I) Polypyridine Diamine Complexes as Intracellular Phosphorogenic Sensors: Synthesis, Characterization, Emissive Behavior, Biological Properties, and Nitric Oxide Sensing. Chem. - Eur. J. 2014, 20, 9633−9642. (43) Law, W. H.-T.; Leung, K.-K.; Lee, L. C.-C.; Poon, C.-S.; Liu, H.W.; Lo, K. K.-W. Cyclometalated Iridium(III) Bipyridyl−Phenylenediamine Complexes with Multicolor Phosphorescence: Synthesis, Electrochemistry, Photophysics, and Intracellular Nitric Oxide Sensing. ChemMedChem 2014, 9, 1316−1329. (44) Liu, H.-W.; Zhang, K. Y.; Law, W. H.-T.; Lo, K. K.-W. Cyclometalated Iridium(III) Bipyridine Complexes Functionalized with an N-Methylamino-oxy Group as Novel Phosphorescent Labeling Reagents for Reducing Sugars. Organometallics 2010, 29, 3474−3476. (45) Medina, R. A.; Owen, G. I. Glucose Transporters: Expression, Regulation and Cancer. Biol. Res. 2002, 35, 9−26. (46) Law, W. H.-T.; Lee, L. C.-C.; Louie, M.-W.; Liu, H.-W.; Ang, T. W.-H.; Lo, K. K.-W. Phosphorescent Cellular Probes and Uptake Indicators Derived from Cyclometalated Iridium(III) Bipyridine Complexes Appended with a Glucose or Galactose Entity. Inorg. Chem. 2013, 52, 13029−13041. (47) Louie, M.-W.; Liu, H.-W.; Lam, M. H.-C.; Lam, Y.-W.; Lo, K. K.-W. Luminescent Rhenium(I) Polypyridine Complexes Appended with an α-D-Glucose Moiety as Novel Biomolecular and Cellular Probes. Chem. - Eur. J. 2011, 17, 8304−8308. (48) Burant, C. F.; Takeda, J.; Brot-Laroche, E.; Bell, G. I.; Davidson, N. O. Fructose Transporter in Human Spermatozoa and Small Intestine Is GLUT5. J. Biol. Chem. 1992, 267, 14523−14526. (49) Lo, K. K.-W.; Law, W. H.-T.; Chan, J. C.-Y.; Liu, H.-W.; Zhang, K. Y. Photophysical and Cellular Uptake Properties of Novel Phosphorescent Cyclometalated Iridium(III) Bipyridine D-Fructose Complexes. Metallomics 2013, 5, 808−812. (50) Zhang, K. Y.; Tso, K. K.-S.; Louie, M.-W.; Liu, H.-W.; Lo, K. K.W. A Phosphorescent Rhenium(I) Tricarbonyl Polypyridine Complex Appended with a Fructose Pendant That Exhibits Photocytotoxicity and Enhanced Uptake by Breast Cancer Cells. Organometallics 2013, 32, 5098−5102. (51) Sletten, E. M.; Bertozzi, C. R. Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality. Angew. Chem., Int. Ed. 2009, 48, 6974−6998. (52) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. Rapid Cu-free Click Chemistry with Readily Synthesized Biarylazacyclooctynones. J. Am. Chem. Soc. 2010, 132, 3688−3690. (53) Lo, K. K.-W.; Chan, B. T.-K.; Liu, H.-W.; Zhang, K. Y.; Li, S. P.Y.; Tang, T. S.-M. Cyclometalated Iridium(III) Polypyridine Dibenzocyclooctyne Complexes as the First Phosphorescent Bioorthogonal Probes. Chem. Commun. 2013, 49, 4271−4273. (54) Devaraj, N. K.; Weissleder, R. Biomedical Applications of Tetrazine Cycloadditions. Acc. Chem. Res. 2011, 44, 816−827. (55) Meimetis, L. G.; Carlson, J. C. T.; Giedt, R. J.; Kohler, R. H.; Weissleder, R. Ultrafluorogenic Coumarin−Tetrazine Probes for Realtime Biological Imaging. Angew. Chem., Int. Ed. 2014, 53, 7531−7534. (56) Choi, A. W.-T.; Tso, K. K.-S.; Yim, V. M.-W.; Liu, H.-W.; Lo, K. K.-W. Modification of 1,2,4,5-Tetrazine with Cationic Rhenium(I) Polypyridine Units to Afford Phosphorogenic Bioorthogonal Probes J


Article

Accounts of Chemical Research with Enhanced Reaction Kinetics. Chem. Commun. 2015, 51, 3442− 3445. (57) Thompson, M. S.; Vadala, T. P.; Vadala, M. L.; Lin, Y.; Riffle, J. S. Synthesis and Applications of Heterobifunctional Poly(ethylene oxide) Oligomers. Polymer 2008, 49, 345−373. (58) Li, S. P.-Y.; Liu, H.-W.; Zhang, K. Y.; Lo, K. K.-W. Modification of Luminescent Iridium(III) Polypyridine Complexes with Discrete Poly(ethylene glycol) (PEG) Pendants: Synthesis, Emissive Behavior, Intracellular Uptake, and PEGylation Properties. Chem. - Eur. J. 2010, 16, 8329−8339. (59) Abdel-Shafi, A. A.; Bourdelande, J. L.; Ali, S. S. Photosensitized Generation of Singlet Oxygen from Rhenium(I) and Iridium(III) Complexes. Dalton Trans. 2007, 24, 2510−2516. (60) Li, S. P.-Y.; Lau, C. T.-S.; Louie, M.-W.; Lam, Y.-W.; Cheng, S. H.; Lo, K. K.-W. Mitochondria-targeting Cyclometalated Iridium(III)−PEG Complexes with Tunable Photodynamic Activity. Biomaterials 2013, 34, 7519−7532. (61) Choi, A. W.-T.; Louie, M.-W.; Li, S. P.-Y.; Liu, H.-W.; Chan, B. T.-N.; Lam, T. C.-Y.; Lin, A. C.-C.; Cheng, S.-H.; Lo, K. K.-W. Emissive Behavior, Cytotoxic Activity, Cellular Uptake, and PEGylation Properties of New Luminescent Rhenium(I) Polypyridine Poly(ethylene glycol) Complexes. Inorg. Chem. 2012, 51, 13289− 13302. (62) Li, S. P.-Y.; Tang, T. S.-M.; Yiu, K. S.-M.; Lo, K. K.-W. Cyclometalated Iridium(III)−Polyamine Complexes with Intense and Long-lived Multicolor Phosphorescence: Synthesis, Crystal Structure, Photophysical Behavior, Cellular Uptake, and Transfection Properties. Chem. - Eur. J. 2012, 18, 13342−13354.

K


Luminescent Rhenium(I) and Iridium(III) Polypyridine Complexes as

Recommend Documents