Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy.

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).

Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy.

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).

Emerging properties from mechanical tethering within a post-synthetically functionalised catenane scaffold.

Maintaining close spatial proximity of functional moieties within molecular systems can result in fascinating emergent properties. Whilst much work has been done on covalent tethering of functional units for myriad applications, investigations into mechanically linked systems are relatively rare. Formation of the mechanical bond is usually the final step in the synthesis of interlocked molecules, placing limits on the throughput of functionalised architectures. Herein we present the synthesis of a bis-azide [2]catenane scaffold that can be post-synthetically modified using CuAAC 'click' chemistry. In this manner we have been able to access functionalised catenanes from a common precursor and study the properties of electrochemically active, emissive and photodimerisable units within the mechanically interlocked system in comparison to non-interlocked analogues. Our data demonstrates that the greater (co-)conformational flexibility that can be obtained with mechanically interlocked systems compared to traditional covalent tethers paves the way for developing new functional molecules with exciting properties.

Unravelling structures of radicals of kynurenic acid formed in the photoinduced reactions with tryptophan and N-acetyl tyrosine.

Kynurenic acid (KNA) in the triplet state reacts with tryptophan (Trp) at neutral pH via proton-coupled electron transfer (PCET), which includes the stepwise transition of both electron and proton from Trp to triplet KNA. In the case of tyrosine (Tyr), the quenching reaction is H-transfer, a simultaneous transfer of electron and proton. In this work, we used the time-resolved chemically induced dynamic nuclear polarization (TR CIDNP) method to unveil the sites of H/H+ transfer within KNA. For this purpose, we obtained the values of 1H hyperfine coupling constants (HFCCs) and g-factors for different tautomeric forms of KNA radicals by the DFT method, then calculated CIDNP intensities using these g-factors and HFCCs according to the Adrian model. The calculated CIDNP intensities for different protons were correlated with their CIDNP intensities in the geminate spectra detected in the photoreactions of KNA with Trp, N-acetyl Trp, and N-acetyl Tyr. Best-fit proportionality relationships between calculated and experimental CIDNP intensities have shown that the KNA anion radical is present in two of the three possible tautomeric forms, which result from the H/H+ movement to the carbonyl oxygen of keto- and oxo-quinolinate forms of KNA, without any visible contribution of the H/H+ transfer to the nitrogen of the enol form. For 4-hydroxyquinoline (4HQN), being the chromophoric core of KNA and exhibiting the same PCET and H-transfer reactions with Trp and Tyr, a single possible tautomeric form of its radical has been revealed as H/H+ transfer to the carbonyl oxygen of the keto-form.

Visualising UV-A light-induced damage to plasma membranes of eye lens.

An eye lens is constantly exposed to the solar UV radiation, which is considered the most important external source of age-related changes to eye lens constituents. The accumulation of modifications of proteins and lipids with age can eventually lead to the development of progressive lens opacifications, such as cataracts. Though the impact of solar UV radiation on the structure and function of proteins is actively studied, little is known about the effect of photodamage on plasma membranes of lens cells. In this work we exploit Fluorescence Lifetime Imaging Microscopy (FLIM), together with viscosity-sensitive fluorophores termed molecular rotors, to study the changes in viscosity of plasma membranes of porcine eye lens resulting from two different types of photodamage: Type I (electron transfer) and Type II (singlet oxygen) reactions. We demonstrate that these two types of photodamage result in clearly distinct changes in viscosity - a decrease in the case of Type I damage and an increase in the case of Type II processes. Finally, to simulate age-related changes that occur in vivo, we expose an intact eye lens to UV-A light under anaerobic conditions. The observed decrease in viscosity within plasma membranes is consistent with the ability of eye lens constituents to sensitize Type I photodamage under natural irradiation conditions. These changes are likely to alter the transport of metabolites and predispose the whole tissue to the development of pathological processes such as cataracts.

Molecular rotors as reporters for viscosity of solutions of collagen like peptides.

We have studied the suitability of using a molecular rotor-based steady-state fluorometric assay for evaluating changes in both the conformation and the viscosity of collagen-like peptide solutions. Our results indicate that a positive charge incorporated on the hydrophobic tail of the BODIPY molecular rotor favours the dye specificity as a reporter for viscosity of these solutions.

Kynurenic acid and its chromophoric core 4-hydroxyquinoline react with tryptophan via proton-coupled electron transfer, and with tyrosine via H-transfer.

Kynurenic acid (KNA) and 4-hydroxyquinoline (4HQN) are photochemically active products of tryptophan catabolism that readily react with tryptophan (Trp) and tyrosine (Tyr) after optical excitation. Recently, transient absorption experiments have shown that at neutral pH Trp reacts with triplet KNA via proton-coupled electron transfer (PCET), and not via electron transfer (ET) as it was suggested before. PCET includes the stepwise transition of both electrons and protons from Trp to triplet KNA. In this work, we confirmed that PCET is the reaction mechanism by the alternative method of time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP). Further studies by TR-CIDNP revealed hydrogen transfer as the mechanism of the reaction between triplet KNA and Tyr in neutral solutions and a transition of both PCET and H-transfer mechanisms to ET under acidic conditions. 4HQN, being the chromophoric core of KNA, exhibits different spectral and photophysical properties from KNA but employs the same mechanisms for the reactions of its triplet state with Trp and Tyr at neutral and acidic pH.

Influence of pH on radical reactions between kynurenic acid and amino acids tryptophan and tyrosine. Part II. Amino acids within the protein globule of lysozyme.

An acidosis, a decrease of pH within a living tissue, may alter yields of radical reactions if participating radicals undergo partial or complete protonation. One of photosensitizers found in the human eye lens, kynurenic acid (KNA-), possesses pKa 5.5 for its radical form that is close to physiological pH 6.89 for a healthy lens. In this work we studied the influence of pH on mechanisms and products of photoinduced radical reactions between KNA- and amino acids tryptophan (Trp) and tyrosine (Tyr) within a globule of model protein, Hen White Egg Lysozyme (HEWL). Our results show that the rate constant of back electron transfer from kynurenyl to HEWL• radicals with the restoration of initial reagents - the major decay pathway for these radicals - does not change in the pH 3-7. The quantum yield of HEWL degradation is also pH independent, however a shift of pH from 7 to 5 completely changes the outcome of photoinduced damage to HEWL from intermolecular cross-linking to oxygenation. HPLC-MS analysis has shown that four of six Trp and all Tyr residues of HEWL are modified in different extents at all pH, but the lowering of pH from 7 to 5 significantly changes the direction of main photodamage from Trp62 to Trp108 located at the entrance and bottom of enzymatic center, respectively. A decrease of intermolecular cross-links via Trp62 is followed by an increase in quantities of intramolecular cross-links Tyr20-Tyr23 and Tyr23-Tyr53. The obtained results point out the competence of cross-linking and oxygenation reactions for Trp and Tyr radicals within a protein globule and significant increase of oxygenation to the total damage of protein in the case of cross-linking deceleration by coulombic repulsion of positively charged protein globules.

Cyclopropyl Substituents Transform the Viscosity-Sensitive BODIPY Molecular Rotor into a Temperature Sensor.

A quantitative fluorescent probe that responds to changes in temperature is highly desirable for studies of biological environments, particularly in cellulo. Here, we report new cell-permeable fluorescence probes based on the BODIPY moiety that respond to environmental temperature. The new probes were developed on the basis of a well-established BODIPY-based viscosity probe by functionalization with cyclopropyl substituents at α and ß positions of the BODIPY core. In contrast to the parent BODIPY fluorophore, α-cyclopropyl-substituted fluorophore displays temperature-dependent time-resolved fluorescence decays showing greatly diminished viscosity dependence, making it an attractive sensor to be used with fluorescence lifetime imaging microscopy (FLIM). We performed theoretical calculations that help rationalize the effect of the cyclopropyl substituents on the photophysical behavior of the new BODIPYs. In summary, we designed an attractive new quantitative FLIM-based temperature probe that can be used for temperature sensing in live cells.

Influence of pH on radical reactions between kynurenic acid and amino acids tryptophan and tyrosine. Part I. Amino acids in free state.

In the human eye lens the endogenous chromophores of UV-A light (315-400 nm) are able to sensitize radical reactions leading to protein modifications during normal aging and the cataract progression. Kynurenic acid (KNA-) is the most photochemically active dye of the human eye lens reported to date with pKa(KNAH2•) 5.5 for its radical form. Cataract is thought to develop under oxidative stress which could be accompanied by acidosis, an acidification of the intracellular environment. Protonation of kynurenyl radicals at mildly acidic conditions may change the outcome of radical reactions leading to additional damage to proteins. In this work we investigated the influence of pH on the degradation of initial reagents and the formation of products in photoinduced radical reactions between KNA- and amino acids tryptophan (Trp) and tyrosine (Tyr) in free states. Our results have shown that pH variation has minor influence on kinetics of reagent decay and accumulation of products in reactions between tyrosyl and kynurenic acid radicals. However in the case of Trp a two-fold decrease of the reagent degradation without visible changes in the composition of formed products was observed with pH decrease from 7 to 3. Time-resolved measurements have shown similar acidification-induced two-fold acceleration of decay of kynurenyl and tryptophanyl radicals via Back Electron Transfer (BET) with the restoration of initial reagents. Experiments with tryptophan derivatives with different pKa values for their radical forms point out the protonation of tryptophanyl radical as the driving force for BET acceleration at low pH. Our results demonstrate that the protonation of kynurenyl radical does not change its reactivity towards amino acids radicals but the total yield of radical photodamage decreases with the protonation of tryptophanyl radicals. It could be expected that radical induced damage to proteins will depend on the pKa of tryptophanyl radicals within a protein globule.

UV-A induced damage to lysozyme via Type I photochemical reactions sensitized by kynurenic acid.

In this work we studied the mechanisms of Type I photodamage to a model protein, hen egg white lysozyme (HEWL), sensitized by kynurenic acid (KNA) - one of the most efficient photosensitizers of the human eye lens present in trace amounts within tissue. The kynurenic acid radical, KNA•-, formed in the quenching of triplet KNA by HEWL, can be readily oxidized by molecular oxygen with the formation of superoxide anion radical O2•-. This leads to two ways of damage to proteins: either via the direct reactions between KNA•- and HEWL• radicals (Type Ia) or via the reactions between superoxide anion O2•- and HEWL• radicals (Type Ib). Our results demonstrate significant degradation of the protein during Type Ia photolysis with the formation of various oligomeric and oxygenated forms of HEWL and several deoxygenated products of KNA. Liquid chromatography-mass spectrometry analysis revealed the cross-linking of HEWL via tryptophan (Trp62) and tyrosine (Tyr23) residues and, for the first time, the covalent binding of KNA to protein via tryptophan (Trp62 and Trp123) residues. It was found that Type Ib reactions lead to substantially smaller damage to HEWL; the degradation quantum yields (Φdeg) of HEWL are 1.3 ± 0.3% and 0.12 ± 0.03% for Type Ia and Ib photolyses, respectively. Low Φdeg values for both types of photolysis indicate the Back Electron Transfer (BET) with the restoration of initial reagents as the main radical decay path with significantly higher BET efficiency in the case of Type Ib reactions. Therefore, in essentially oxygen-free tissues like the eye lens, the direct radical reactions via Type Ia mechanism could induce significantly larger damage to proteins, leading to their cross-linking and oxidation. The accumulation of these modifications can cause the development of various diseases, in particular, cataracts in the eye lens.

Competition of singlet and triplet recombination of radical pairs in photoreactions of carboxy benzophenones and aromatic amino acids.

Time-resolved chemically induced dynamic nuclear polarization (CIDNP) and transient absorption (TA) were applied to reveal the branching ratio of the singlet and triplet recombination channels in the reaction of short-lived radicals of carboxy benzophenones and the aromatic amino acids histidine, tryptophan, and tyrosine in neutral aqueous solution. It was established that the share of triplet recombination increases with increasing number of carboxylic groups: no triplet recombination was found for 4-carboxy benzophenone, whereas ∼13% of radicals of 4,4'-dicarboxy benzophenone (DCBP) and ∼27% of radicals of 3,3',4,4'-tetracarboxy benzophenone (TCBP) react with histidine radicals from the triplet state of radical pairs. The main idea is that the protonated (π,π*) triplet state of TCBP or DCBP is populated via back electron transfer from the ketyl radical of TCBP or DCBP to the radical of the amino acid. The protonated triplet state of the ketone decays with the formation of a metastable hydroxylated product, which is detected by TA. Taking into account triplet recombination provides excellent coincidence between experimental data and the simulated CIDNP kinetics.

Determination of Hyperfine Coupling Constants of Fluorinated Diphenylacetylene Radical Anions by Magnetic Field-Affected Reaction Yield Spectroscopy.

Magnetic field-affected reaction yield (MARY) spectroscopy is a spin chemistry technique for detecting short-lived radical ions. Having sensitivity to transient species with lifetimes as short as nanoseconds, MARY spectroscopy usually does not provide detailed information on their magnetic resonance parameters, except for simple systems with equivalent magnetic nuclei. In this work, the radical anions of two fluorinated diphenylacetylene derivatives with nonequivalent magnetic nuclei and unknown hyperfine coupling constants ( AHF) were investigated by MARY spectroscopy. The MARY spectra were found to be resolved and have resonance lines in nonzero magnetic fields, which are determined by the AHF values. Simple relationships between the positions of resonance MARY lines and the AHF values were established from the analysis of the different Hamiltonian block contributions to the MARY spectrum. The obtained experimental AHF values are in agreement with the results of quantum chemical calculations at the density functional theory level.

Crystal packing control of a trifluoromethyl-substituted furan/phenylene co-oligomer.

Furan/phenylene co-oligomer single crystals are considered as future materials for organic optoelectronics. Here, the effects of trifluoromethyl substituents on the crystallization, structure and optical properties of furan/phenylene co-oligomer 1,4-bis{5-[4-(trifluoromethyl)phenyl]furan-2-yl}benzene are studied systematically. The solution growth methods and physical vapor transport result in the formation of three polymorphs depending on the growth method and the solvent. Single-crystal X-ray analysis reveals the crystal structures to correspond to H-, J- or mixed aggregates. All obtained crystals exhibit high photoluminescence efficiency and have optical properties which strongly depend on the crystal packing. Variable-temperature X-ray powder diffraction analysis shows the thermal transition of two forms (H- and J-aggregates) into a third one (mixed aggregate). Terminal trifluoromethyl groups induce weak intermolecular interactions which control the crystal packing and optical properties of co-oligomer single crystals.

Ultrafast excited state decay of natural UV filters: from intermolecular hydrogen bonds to a conical intersection.

Kynurenines (KNs) are natural UV filters of the human eye lens, protecting the eye tissues from solar UV radiation. Key points of their effective protection are the intramolecular charge transfer (ICT) in the excited state and the fast dissipation of absorbed light energy into heat via the intermolecular H-bonds. Herein we report that the introduction of an unsaturated double bond in the amino acid side chain, operating as an ICT-enhancing electron donor group, drastically accelerates the internal conversion (IC) due to a conical intersection (CI) between the potential energy surfaces of the excited and ground states. Our photophysical study of a deaminated KN (carboxyketoalkene, CKA), an intrinsic product of KN thermal decomposition, demonstrates an unusually fast excited state decay in a broad range of solvents of different polarity and proticity. The detailed analysis of interactions in the excited state by different computational techniques revealed that the changes in molecular structure - the twist of the carbonyl group from the plane of the aromatic ring followed by the formation of two mutually orthogonal conjugated substructures - are responsible for the CI of the excited and ground state potential energy surfaces. Intermolecular H-bonds facilitate the transition to the CI, but do not play a crucial role in the fast decay of the excited state. An extremely fast and efficient IC in CKA opens the way for the design of new types of organic UV filters and their applications in material science, cosmetics and medicine.

[Fe(Ox)3]3- complex as a photodegradation agent at neutral pH: Advances and limitations.

In the present work advances and limitations in the application of Fe(III)-oxalate complexes (namely, [Fe(Ox)3]3-) to the photodegradation of a model persistent organic contaminant - 2,4-dichlorophenoxybutanoic acid (2,4-DB) in neutral aqueous solutions were systematically investigated for the first time. It has been shown that the efficiency of [Fe(Ox)3]3- system greatly depends on the initial concentrations of oxalate ion due to the fast consumption of the ligand during photodegradation process leading to the formation of photochemically less active Fe(III) species. Efficiency of Fe(Ox)33- system normalized to UVA absorption at the excitation wavelength is practically independent on [Fe(III)]. Thus, it is highly probable that concentrations of Fe(III) as low as < 10-5 M could be applied in water treatment procedures using reactors with very long optical path. The system also keeps high efficiency at low concentration of pollutant (<10-5 M) though this results in higher relative consumption rate of Fe(III) and oxalate ions.

Photooxidation of herbicide amitrole in the presence of fulvic acid.

Fulvic acid (Henan ChangSheng Corporation) photoinduced degradation of non-UVA-absorbing herbicide amitrole (3-amino-1,2,4-triazole, AMT) as a way for its removal from polluted water was investigated in details. It was shown that the main primary species generated by fulvic acid under UVA radiation, triplet state and hydrated electron, are not directly involved in the herbicide degradation. AMT decays in reactions with secondary intermediates, reactive oxygen species, formed in reactions of the primary ones with dissolved oxygen. Singlet oxygen is responsible for 80% of herbicide oxidation, and •OH and O2-• radicals-for the remaining 20% of AMT. It was found that quantum yield of AMT photodegradation (ϕ 365nm) decreases linearly from 2.2 × 10-3 to 1.2 × 10-3 with the increase of fulvic acid concentration from 1.1 to 30 mg L-1. On the contrary, the increase of AMT concentration from 0.8 to 25 mg L-1 leads to practically linear growth of ϕ 365nm value from 1.8 × 10-4 to 4 × 10-3. Thus, the fulvic acid exhibits a good potential as UVA photooxidizer of organic pollutants sensitive to the singlet oxygen (ϕ 532nm(1O2) = 0.025 at pH 6.5).

X-ray Generated Recombination Exciplexes of Substituted Diphenylacetylenes with Tertiary Amines: A Versatile Experimental Vehicle for Targeted Creation of Deep-Blue Electroluminescent Systems.

Customizable and technology-friendly functional materials are one of the mainstays of emerging organic electronics and optoelectronics. We show that recombination exciplexes of simple substituted diphenylacetylenes with tertiary amines can be a convenient source of tunable deep-blue emission with possible applications in organic electroluminescent systems. The optically inaccessible exciplexes were produced via recombination of radiation-generated radical ion pairs in alkane solution, which mimics charge transport and recombination in the active layer of practical organic light-emitting diodes in a simple solution-based experiment. Despite varying and rather poor intrinsic emission properties, diphenylacetylene and its prototypical methoxy (donor) or trifluoromethyl (acceptor) monosubstituted derivatives readily form recombination exciplexes with N,N-dimethylaniline and other tertiary amines that produce emission with maxima ranging from 385 to 435 nm. The position of emission band maximum linearly correlates with readily calculated gas-phase electron affinity of the corresponding diphenylacetylene, which can be used for fast computational prescreening of the candidate molecules, and various substituted diphenylacetylenes can be synthesized via relatively simple and universal cross-coupling reactions of Sonogashira and Castro. Together, the simple solution-based experiment, computationally cheap prescreening method, and universal synthetic strategy may open a very broad and chemically convenient class of compounds to obtain OLEDs and OLED-based multifunctional devices with tunable emission spectrum and high conversion efficiency that has yet not been seriously considered for these purposes.

Dimerization and oxidation of tryptophan in UV-A photolysis sensitized by kynurenic acid.

Photoinduced generation of radicals in the eye lens may play an important role in the modification of proteins leading to their coloration, aggregation, and insolubilization. The radicals can be formed via the reactions of photoexcited endogenous chromophores of the human lens with lens proteins, in particular with tryptophan residues. In the present work we studied the reactions induced by UV-A (315-400nm) light between kynurenic acid (KNA), an effective photosensitizer present in the human lens, and N-acetyl-L-tryptophan (NTrpH) under aerobic and anaerobic conditions. Our results show that the reaction mechanism strongly depends on the presence of oxygen in solution. Under aerobic conditions, the generation of singlet oxygen is the major channel of the effective NTrpH oxidation. In argon-bubbled solutions, the quenching of triplet KNA by NTrpH results in the formation of KNA•- and NTrp• radicals. Under laser pulse irradiation, when the radical concentration is high, the main pathway of the radical decay is the back electron transfer with the restoration of initial reagents. Other reactions include (i) the radical combination yielding NTrp dimers and (ii) the oxygen atom transfer from KNA•- to NTrp• with the formation of oxidized NTrp species and deoxygenated KNA products. In continuous-wave photolysis, even trace amounts of molecular oxygen are sufficient to oxidize the majority of KNA•- radicals with the rate constant of (2.0 ± 0.2) × 109M-1s-1, leading to the restoration of KNA and the formation of superoxide radical O2•-. The latter reacts with NTrp• via either the radical combination to form oxidized NTrp (minor pathway), or the electron transfer to restore NTrpH in the ground state (major pathway). As the result, the quantum yields of the starting compound decomposition under continuous-wave anaerobic photolysis are rather low: 1.6% for NTrpH and 0.02% for KNA. The photolysis of KNA with alpha-crystallin yields the same deoxygenated KNA products as the photolysis of KNA with NTrpH, indicating the similarity of the photolysis mechanisms. Thus, inside the eye lens KNA can sensitize both protein photooxidation and protein covalent cross-linking with the minor self-degradation. This may play an important role in the lens protein modifications during the normal aging and cataract development.

Spin-state-correlated optical properties of copper(ii)-nitroxide based molecular magnets.

Molecular magnets based on copper(ii) ions and stable nitroxide radicals exhibit promising switchable behavior triggered by a number of external stimuli; however, their spin-state-correlated optical properties vital for photoinduced switching have not been profoundly investigated to date. Herein, the electronic absorption spectra of single crystals of three representatives of this unique family are studied experimentally and theoretically in the visible and near-IR regions. We established that the color of the complexes is mainly determined by optical properties of the nitroxide radicals, whereas the Cu(hfac)2 fragment contributes to the near-IR range with the intensity smaller by an order of magnitude. The thermochromism of these complexes evident upon thermal spin state switching is mainly caused by a spectral shift of the absorption bands of the nitroxides. The vibrational progression observed in the visible range for single crystals as well as for solutions of pure nitroxides is well reproduced by DFT calculations, where the C-C stretching mode governs the observed progression. The analysis of the spectra of single crystals in the near-IR region reveals changes in the energy and in the intensity of the copper(ii) d-d transitions, which are well reproduced by SOC-NEVPT2 calculations and owe to the flip of the Jahn-Teller axis in the coordination environment of copper. Further strategies for designing bidirectional magnetic photoswitches using these appealing compounds are discussed.