Phenazine Cations as Anticancer Theranostics†.

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.

Phototoxicity of cyclometallated Ir(III) complexes bearing a thio-bis-benzimidazole ligand, and its monodentate analogue, as potential PDT photosensitisers in cancer cell killing.

Two novel cyclometallated iridium(III) complexes have been prepared with one bidentate or two monodentate imidazole-based ligands, 1 and 2, respectively. The complexes showed intense emission with long lifetimes of the excited state. Femtosecond transient absorption experiments established the nature of the lowest excited state as 3IL state. Singlet oxygen generation with good yields (40% for 1 and 82% for 2) was established by detecting 1O2 directly, through its emission at 1270 nm. Photostability studies were also performed to assess the viability of the complexes as photosensitizers (PS) for photodynamic therapy (PDT). Complex 1 was selected as a good candidate to investigate light-activated killing of cells, whilst complex 2 was found to be toxic in the dark and unstable under light. Complex 1 demonstrated high phototoxicity indexes (PI) in the visible region, PI > 250 after irradiation at 405 nm and PI > 150 at 455 nm, in EJ bladder cancer cells.

A Near-Infrared Luminescent Cr(III) N-Heterocyclic Carbene Complex.

Photoluminescent coordination complexes of Cr(III) are of interest as near-infrared spin-flip emitters. Here, we explore the preparation, electrochemistry, and photophysical properties of the first two examples of homoleptic N-heterocyclic carbene complexes of Cr(III), featuring 2,6-bis(imidazolyl)pyridine (ImPyIm) and 2-imidazolylpyridine (ImPy) ligands. The complex [Cr(ImPy)3]3+ displays luminescence at 803 nm on the microsecond time scale (13.7 µs) from a spin-flip doublet excited state, which transient absorption spectroscopy reveals to be populated within several picoseconds following photoexcitation. Conversely, [Cr(ImPyIm)2]3+ is nonemissive and has a ca. 500 ps excited-state lifetime.

Direct Determination of the Rate of Intersystem Crossing in a Near-IR Luminescent Cr(III) Triazolyl Complex.

A detailed understanding of the dynamics of photoinduced processes occurring in the electronic excited state is essential in informing the rational design of photoactive transition-metal complexes. Here, the rate of intersystem crossing in a Cr(III)-centered spin-flip emitter is directly determined through the use of ultrafast broadband fluorescence upconversion spectroscopy (FLUPS). In this contribution, we combine 1,2,3-triazole-based ligands with a Cr(III) center and report the solution-stable complex [Cr(btmp)2]3+ (btmp = 2,6-bis(4-phenyl-1,2,3-triazol-1-yl-methyl)pyridine) (13+), which displays near-infrared (NIR) luminescence at 760 nm (τ = 13.7 µs, ϕ = 0.1%) in fluid solution. The excited-state properties of 13+ are probed in detail through a combination of ultrafast transient absorption (TA) and femtosecond-to-picosecond FLUPS. Although TA spectroscopy allows us to observe the evolution of phosphorescent excited states within the doublet manifold, more significantly and for the first time for a complex of Cr(III), we utilize FLUPS to capture the short-lived fluorescence from initially populated quartet excited states immediately prior to the intersystem crossing process. The decay of fluorescence from the low-lying 4MC state therefore allows us to assign a value of (823 fs)-1 to the rate of intersystem crossing. Importantly, the sensitivity of FLUPS to only luminescent states allows us to disentangle the rate of intersystem crossing from other closely associated excited-state events, something which has not been possible in the spectroscopic studies previously reported for luminescent Cr(III) systems.

Ultrafast electronic, infrared, and X-ray absorption spectroscopy study of Cu(I) phosphine diimine complexes.

The study aims to understand the role of the transient bonding in the interplay between the structural and electronic changes in heteroleptic Cu(I) diimine diphosphine complexes. This is an emerging class of photosensitisers which absorb in the red region of the spectrum, whilst retaining a sufficiently long excited state lifetime. Here, the dynamics of these complexes are explored by transient absorption (TA) and time-resolved infrared (TRIR) spectroscopy, which reveal ultrafast intersystem crossing and structural distortion occurring. Two potential mechanisms affecting excited state decay in these complexes involve a transient formation of a solvent adduct, made possible by the opening up of the Cu coordination centre in the excited state due to structural distortion, and by a transient coordination of the O-atom of the phosphine ligand to the copper center. X-ray absorption studies of the ground electronic state have been conducted as a prerequisite for the upcoming X-ray spectroscopy studies which will directly determine structural dynamics. The potential for these complexes to be used in bimolecular applications is confirmed by a significant yield of singlet oxygen production.

Conjugation-length dependence of regioregular oligo 3-alkyl(thienylene-vinylene)s demonstrates polyene-like behaviour with weak electron-electron correlations.

Poly(3-alkyl(thienylene-vinylene)) (P3TV) and its longer oligomers have negligibly low photoluminescence quantum yields, however, the reason for their low yields is currently debated. Here, we prepare a series of regioregular (3-dodecyl)thienylene-vinylene oligomers with n = 2-8 repeat units by iterative Horner-Wadsworth-Emmons reactions, and report their steady-state, transient absorption, and emission spectroscopy. The results presented here demonstrate that 3-alkyl(thienylene-vinylene) oligomers form part of the polyene family. The shortest (n = 2) oligomer emits from the bright 1Bu state, while fluorescence in oligomers with n = 3, 4 is from the formally dark 2Ag state, allowed via Herzberg-Teller vibronic coupling to the nearby bright 1Bu state as described for diphenyl-polyenes. Longer oligomers and the polymer are essentially non-emissive as the 2Ag state can no longer intensity-borrow from the 1Bu state. We demonstrate that the spectral shapes, photoluminescence quantum yield, and transient spectral behaviour can all be explained using a polyene model with weak electronic correlations.

Photocatalytic Reduction of CO2 to CO in Aqueous Solution under Red-Light Irradiation by a Zn-Porphyrin-Sensitized Mn(I) Catalyst.

This work demonstrates photocatalytic CO2 reduction by a noble-metal-free photosensitizer-catalyst system in aqueous solution under red-light irradiation. A water-soluble Mn(I) tricarbonyl diimine complex, [MnBr(4,4'-{Et2O3PCH2}2-2,2'-bipyridyl)(CO)3] (1), has been fully characterized, including single-crystal X-ray crystallography, and shown to reduce CO2 to CO following photosensitization by tetra(N-methyl-4-pyridyl)porphyrin Zn(II) tetrachloride [Zn(TMPyP)]Cl4 (2) under 625 nm irradiation. This is the first example of 2 employed as a photosensitizer for CO2 reduction. The incorporation of -P(O)(OEt)2 groups, decoupled from the core of the catalyst by a -CH2- spacer, afforded water solubility without compromising the electronic properties of the catalyst. The photostability of the active Mn(I) catalyst over prolonged periods of irradiation with red light was confirmed by 1H and 13C{1H} NMR spectroscopy. This first report on Mn(I) species as a homogeneous photocatalyst, working in water and under red light, illustrates further future prospects of intrinsically photounstable Mn(I) complexes as solar-driven catalysts in an aqueous environment.

Making the Right Link to Theranostics: The Photophysical and Biological Properties of Dinuclear RuII-ReI dppz Complexes Depend on Their Tether.

The synthesis of new dinuclear complexes containing linked RuII(dppz) and ReI(dppz) moieties is reported. The photophysical and biological properties of the new complex, which incorporates a N,N'-bis(4-pyridylmethyl)-1,6-hexanediamine tether ligand, are compared to a previously reported RuII/ReI complex linked by a simple dipyridyl alkane ligand. Although both complexes bind to DNA with similar affinities, steady-state and time-resolved photophysical studies reveal that the nature of the linker affects the excited state dynamics of the complexes and their DNA photocleavage properties. Quantum-based DFT calculations on these systems offer insights into these effects. While both complexes are live cells permeant, their intracellular localizations are significantly affected by the nature of the linker. Notably, one of the complexes displayed concentration-dependent localization and possesses photophysical properties that are compatible with SIM and STED nanoscopy. This allowed the dynamics of its intracellular localization to be tracked at super resolutions.

Solvent-Mediated Activation/Deactivation of Photoinduced Electron-Transfer in a Molecular Dyad.

Herein is presented a molecular dyad comprised of a [Ru(bpy)3]2+ photosensitizer and an anthraquinone (AQ) acceptor coupled by an ethynyl linker ([Ru(bpy)2(bpy-cc-AQ)]2+) in which activation/deactivation of photoinduced electron-transfer from the [Ru(bpy)3]2+ photosensitizer to the AQ acceptor is achieved and characterized as a function of the dielectric constant and hydrogen-bond donating ability of the solvent used. It is demonstrated that the rate of photoinduced electron-transfer can be modulated over several orders of magnitude (105-1011 s-1) by choice of solvent. Nanosecond transient absorption spectra are dominated by MLCT signals and exhibit identical decay kinetics to the corresponding emission signals. Ultrafast transient absorption and time-resolved infrared spectroscopies provide direct evidence for the formation of the charge-separated (CS) state and rapid (on the order of a few picoseconds) establishment of an excited-state pseudoequilibrium.

Unequivocal Experimental Evidence of the Relationship between Emission Energies and Aurophilic Interactions.

In this paper, we describe experimental evidence of a change in the emission energy as a function of the Au-Au distance. We have employed a luminescent complex exhibiting an aurophilic interaction, which is weak enough to allow its length to be modified by external pressure but rigid enough to confer structural stability on the complex. By determining the crystal structures and emission characteristics over a range of pressures, we have identified an exponential relationship between the energy of the emitted light and the metal-metal distances under pressure. This result can be indirectly related to the repulsive branch of the fitted function representing the energy of the system in the ground state at different Au-Au distances. The relativistic nature of gold appears to play an important role in the behavior of this complex.

Photophysics of Cage/Guest Assemblies: Photoinduced Electron Transfer between a Coordination Cage Containing Osmium(II) Luminophores, and Electron-Deficient Bound Guests in the Central Cavity.

A heterometallic octanuclear coordination cage [Os4Zn4(Lnap)12]X16 (denoted Os•Zn; X = perchlorate or chloride) has been prepared (Lnap is a bis-bidentate bridging ligand containing two pyrazolyl-pyridine chelating units separated by a 1,5-naphthalenediyl spacer group). The {Os(NN)3}2+ units located at four of the eight vertices of the cube have a long-lived, phosphorescent 3MLCT excited state which is a stronger electron donor than [Ru(bipy)3]2+. The chloride form of Os•Zn is water-soluble and binds in its central cavity the hydrophobic electron-accepting organic guests 1,2,4,5-tetracyanobenzene, 1,4-naphthoquinone and 1-nitronaphthalene, with binding constants in the range 103-104 M-1, resulting in quenching of the phosphorescence arising from the Os(II) units. A crystal structure of an isostructural Co8 cage containing one molecule of 1,2,4,5-tetracyanobenzene as a guest inside the cavity has been determined. Ultrafast transient absorption measurements show formation of a charge-separated Os(III)/guest•- state arising from cage-to-guest photoinduced electron transfer; this state is formed within 13-21 ps, and decays on a time scale of ca. 200 ps. In the presence of a competing guest with a large binding constant (cycloundecanone) which displaces each electron-accepting quencher from the cage cavity, the charge-separated state is no longer observed. Further, a combination of mononuclear {Os(NN)3}2+ model complexes with the same electron-accepting species showed no evidence for formation of charge-separated Os(III)/guest•- states. These two control experiments indicate that the {Os(NN)3}2+ chromophores need to be assembled into the cage structure to bind the electron-accepting guests, and for PET to occur. These results help to pave the way for use of photoactive coordination cages as hosts for photoredox catalysis reactions on bound guests.

Anion-Mediated Photophysical Behavior in a C60 Fullerene [3]Rotaxane Shuttle.

By addressing the challenge of controlling molecular motion, mechanically interlocked molecular machines are primed for a variety of applications in the field of nanotechnology. Specifically, the designed manipulation of communication pathways between electron donor and acceptor moieties that are strategically integrated into dynamic photoactive rotaxanes and catenanes may lead to efficient artificial photosynthetic devices. In this pursuit, a novel [3]rotaxane molecular shuttle consisting of a four-station bis-naphthalene diimide (NDI) and central C60 fullerene bis-triazolium axle component and two mechanically bonded ferrocenyl-functionalized isophthalamide anion binding site-containing macrocycles is constructed using an anion template synthetic methodology. Dynamic coconformational anion recognition-mediated shuttling, which alters the relative positions of the electron donor and acceptor motifs of the [3]rotaxane's macrocycle and axle components, is demonstrated initially by 1H NMR spectroscopy. Detailed steady-state and time-resolved UV-vis-IR absorption and emission spectroscopies as well as electrochemical studies are employed to further probe the anion-dependent positional macrocycle-axle station state of the molecular shuttle, revealing a striking on/off switchable emission response induced by anion binding. Specifically, the [3]rotaxane chloride coconformation, where the ferrocenyl-functionalized macrocycles reside at the center of the axle component, precludes electron transfer to NDI, resulting in the switching-on of emission from the NDI fluorophore and concomitant formation of a C60 fullerene-based charge-separated state. By stark contrast, in the absence of chloride as the hexafluorophosphate salt, the ferrocenyl-functionalized macrocycles shuttle to the peripheral NDI axle stations, quenching the NDI emission via formation of a NDI-containing charge-separated state. Such anion-mediated control of the photophysical behavior of a rotaxane through molecular motion is unprecedented.

Photophysical and Cellular Imaging Studies of Brightly Luminescent Osmium(II) Pyridyltriazole Complexes.

The series of complexes [Os(bpy)3- n(pytz) n][PF6]2 (bpy = 2,2'-bipyridyl, pytz = 1-benzyl-4-(pyrid-2-yl)-1,2,3-triazole, 1 n = 0, 2 n = 1, 3 n = 2, 4 n = 3) were prepared and characterized and are rare examples of luminescent 1,2,3-triazole-based osmium(II) complexes. For 3 we present an attractive and particularly mild preparative route via an osmium(II) η6-arene precursor circumventing the harsh conditions that are usually required. Because of the high spin-orbit coupling constant associated with the Os(II) center the absorption spectra of the complexes all display absorption bands of appreciable intensity in the range of 500-700 nm corresponding to spin-forbidden ground-state-to-3MLCT transitions (MLCT = metal-to-ligand charge transfer), which occur at significantly lower energies than the corresponding spin-allowed 1MLCT transitions. The homoleptic complex 4 is a bright emitter (λmaxem = 614 nm) with a relatively high quantum yield of emission of ∼40% in deoxygenated acetonitrile solutions at room temperature. Water-soluble chloride salts of 1-4 were also prepared, all of which remain emissive in aerated aqueous solutions at room temperature. The complexes were investigated for their potential as phosphorescent cellular imaging agents, whereby efficient excitation into the 3MLCT absorption bands at the red side of the visible range circumvents autofluorescence from biological specimens, which do not absorb in this region of the spectrum. Confocal microscopy reveals 4 to be readily taken up by cancer cell lines (HeLa and EJ) with apparent lysosomal and endosomal localization, while toxicity assays reveal that the compounds have low dark and light toxicity. These complexes therefore provide an excellent platform for the development of efficient luminescent cellular imaging agents with advantageous photophysical properties that enable excitation and emission in the biologically transparent region of the optical spectrum.

Metal Complexes for Two-Photon Photodynamic Therapy: A Cyclometallated Iridium Complex Induces Two-Photon Photosensitization of Cancer Cells under Near-IR Light.

Photodynamic therapy (PDT) uses photosensitizers (PS) which only become cytotoxic upon light-irradiation. Transition-metal complexes are highly promising PS due to long excited-state lifetimes, and high photo-stabilities. However, these complexes usually absorb higher-energy UV/Vis light, whereas the optimal tissue transparency is in the lower-energy NIR region. Two-photon excitation (TPE) can overcome this dichotomy, with simultaneous absorption of two lower-energy NIR-photons populating the same PS-active excited state as one higher-energy photon. We introduce two low-molecular weight, long-lived and photo-stable iridium complexes of the [Ir(N^C)2 (N^N)]+ family with high TP-absorption, which localise to mitochondria and lysosomal structures in live cells. The compounds are efficient PS under 1-photon irradiation (405 nm) resulting in apoptotic cell death in diverse cancer cell lines at low light doses (3.6 J cm-2 ), low concentrations, and photo-indexes greater than 555. Remarkably 1 also displays high PS activity killing cancer cells under NIR two-photon excitation (760 nm), which along with its photo-stability indicates potential future clinical application.

Directly Coupled Versus Spectator Linkers on Diimine PtII Acetylides-Change the Structure, Keep the Function?

Modification of light-harvesting units with anchoring groups for surface attachment often compromises light-harnessing properties. Herein, a series of [donor-acceptor-anchor] platinum(II) diimine (bis-)acetylides was developed in order to systematically compare the effect of conjugated versus electronically decoupled modes of attachment of protected anchoring groups on the photophysical properties of light-harvesting units. The first examples of "decoupled" phosphonate diimine PtII complexes are reported, and their properties are compared and contrasted to those of carboxylate analogues studied by a diversity of methods. Ultrafast time-resolved IR and transient absorption spectroscopy revealed that all complexes have a charge-transfer (CT) lowest excited state with lifetimes between 2 and 14 ns. Vibrational signatures and dynamics of CT states were identified; the assignment of electronic states and their vibrational origin was aided by TDDFT calculations. Ultrafast energy redistribution accompanied by structural changes was directly captured in the CT states. A significant difference between the structures of the electronic ground and CT excited states, as well as differences in the structural reorganisation in the complexes bearing directly attached or electronically decoupled anchoring groups, was discovered. This work demonstrates that decoupling of the anchoring group from the light-harvesting core by a saturated spacer is an easy approach to combine surface attachment with high reduction potential and ten times longer lifetime of the CT excited state of the light-absorbing unit, and retain electron-transfer photoreactivity essential for light-harvesting applications.

Multimodal Probes: Superresolution and Transmission Electron Microscopy Imaging of Mitochondria, and Oxygen Mapping of Cells, Using Small-Molecule Ir(III) Luminescent Complexes.

We describe an Ir(III)-based small-molecule, multimodal probe for use in both light and electron microscopy. The direct correlation of data between light- and electron-microscopy-based imaging to investigate cellular processes at the ultrastructure level is a current challenge, requiring both dyes that must be brightly emissive for luminescence imaging and scatter electrons to give contrast for electron microscopy, at a single working concentration suitable for both methods. Here we describe the use of Ir(III) complexes as probes that provide excellent image contrast and quality for both luminescence and electron microscopy imaging, at the same working concentration. Significant contrast enhancement of cellular mitochondria was observed in transmission electron microscopy imaging, with and without the use of typical contrast agents. The specificity for cellular mitochondria was also confirmed with MitoTracker using confocal and 3D-structured illumination microscopy. These phosphorescent dyes are part of a very exclusive group of transition-metal complexes that enable imaging beyond the diffraction limit. Triplet excited-state phosphorescence was also utilized to probe the O2 concentration at the mitochondria in vitro, using lifetime mapping techniques.

Homo- and Heteroleptic Phototoxic Dinuclear Metallo-Intercalators Based on RuII (dppn) Intercalating Moieties: Synthesis, Optical, and Biological Studies.

Using a new mononuclear "building block," for the first time, a dinuclear RuII (dppn) complex and a heteroleptic system containing both RuII (dppz) and RuII (dppn) moieties are reported. The complexes, including the mixed dppz/dppn system, are 1 O2 sensitizers. However, unlike the homoleptic dppn systems, the mixed dppz/dppn complex also displays a luminescence "switch on" DNA light-switch effect. In both cisplatin sensitive and resistant human ovarian carcinoma lines the dinuclear complexes show enhanced uptake compared to their mononuclear analogue. Thanks to a favorable combination of singlet oxygen generation and cellular uptake properties all three of the new complexes are phototoxic and display potent activity against chemotherapeutically resistant cells.

Probing and Exploiting the Interplay between Nuclear and Electronic Motion in Charge Transfer Processes.

The Born-Oppenheimer approximation refers to the assumption that the nuclear and electronic wave functions describing a molecular system evolve and can be determined independently. It is now well-known that this approximation often breaks down and that nuclear-electronic (vibronic) coupling contributes greatly to the ultrafast photophysics and photochemistry observed in many systems ranging from simple molecules to biological organisms. In order to probe vibronic coupling in a time-dependent manner, one must use spectroscopic tools capable of correlating the motions of electrons and nuclei on an ultrafast time scale. Recent developments in nonlinear multidimensional electronic and vibrational spectroscopies allow monitoring both electronic and structural factors with unprecedented time and spatial resolution. In this Account, we present recent studies from our group that make use of different variants of frequency-domain transient two-dimensional infrared (T-2DIR) spectroscopy, a pulse sequence combining electronic and vibrational excitations in the form of a UV-visible pump, a narrowband (12 cm(-1)) IR pump, and a broadband (400 cm(-1)) IR probe. In the first example, T-2DIR is used to directly compare vibrational dynamics in the ground and relaxed electronic excited states of Re(Cl)(CO)3(4,4'-diethylester-2,2'-bipyridine) and Ru(4,4'-diethylester-2,2'-bipyridine)2(NCS)2, prototypical charge transfer complexes used in photocatalytic CO2 reduction and electron injection in dye-sensitized solar cells. The experiments show that intramolecular vibrational redistribution (IVR) and vibrational energy transfer (VET) are up to an order of magnitude faster in the triplet charge transfer excited state than in the ground state. These results show the influence of electronic arrangement on vibrational coupling patterns, with direct implications for vibronic coupling mechanisms in charge transfer excited states. In the second example, we show unambiguously that electronic and vibrational movement are coupled in a donor-bridge-acceptor complex based on a Pt(II) trans-acetylide design motif. Time-resolved IR (TRIR) spectroscopy reveals that the rate of electron transfer (ET) is highly dependent on the amount of excess energy localized on the bridge following electronic excitation. Using an adaptation of T-2DIR, we are able to selectively perturb bridge-localized vibrational modes during charge separation, resulting in the donor-acceptor charge separation pathway being completely switched off, with all excess energy redirected toward the formation of a long-lived intraligand triplet state. A series of control experiments reveal that this effect is mode specific: it is only when the high-frequency bridging C≡C stretching mode is pumped that radical changes in photoproduct yields are observed. These experiments therefore suggest that one may perturb electronic movement by stimulating structural motion along the reaction coordinate using IR light. These studies add to a growing body of evidence suggesting that controlling the pathways and efficiency of charge transfer may be achieved through synthetic and perturbative approaches aiming to modulate vibronic coupling. Achieving such control would represent a breakthrough for charge transfer-based applications such as solar energy conversion and molecular electronics.