Intramolecular Singlet Fission Coupled with Intermolecular Triplet Separation as a Strategy to Achieve High Triplet Yields in Fluorene-Based Small Molecules.

In this work, detailed experimental proof and in-depth analysis of the singlet fission (SF) mechanism, operative in fluorene-based small molecules, are carried out by employing advanced time-resolved spectroscopies with nanosecond and femtosecond resolution. The investigation of the effect of solution concentration and solvent viscosity together with temperature and excitation wavelength demonstrates INTRAmolecular formation of the correlated triplet pair followed by INTERmolecular independent triplet separation via a "super-diffusional" triplet-triplet transfer process. This unconventional INTRA- to INTERmolecular SF may be considered an "ideal" mechanism. Indeed, intramolecular formation of the correlated triplet pair is here interestingly proved for small molecules rather than large multichromophoric systems, allowing easy synthesis and processability while maintaining good control over the SF process. On the other hand, the intermolecular triplet separation may be exploited to achieve high triplet quantum yields in these new SF small molecules.

Unveiling the double triplet nature of the 2Ag state in conjugated stilbenoid compounds to achieve efficient singlet fission.

In this investigation, the excited-state evolution in a series of all-trans stilbenoid compounds, displaying a low-lying dark singlet state of 2Ag-like symmetry nearly degenerate with the bright 1Bu state, was unveiled by employing advanced ultrafast spectroscopies while probing the effect of solvent polarizability. Together with the dual emission, femtosecond transient absorption and broadband fluorescence up-conversion disclosed the double nature of the 2Ag-like state showing both singlet features, a lifetime typical of a singlet and the ability to emit, and a triplet character, exhibiting a triplet-like absorption spectrum. The ultrafast formation (in hundreds of femtoseconds) from the non-relaxed upper singlet state led to the identification of 2Ag as the correlated triplet pair of singlet fission. The spectral difference obtained by comparison of transient absorption peaks of the 2Ag (1TT) and the triplet states was found to be in remarkable agreement with the observed triplet yield and the 1(TT) separation rate constant. Indeed, this spectral shift provided an experimental method to gain qualitative insight into the ease of separation of the 1(TT) and the relative SF efficiency. The highly conjugated polyene-like structures enable the ultrafast formation of the double triplet, but then the large binding energy prevents the triplet separation and thus the effective completion of singlet fission. Even though thermodynamically feasible for all the investigated stilbenoids according to TD-DFT calculations, singlet fission resulted to occur efficiently in the case of 1-(pyridyl-4-ylethenyl)-4-(p-nitrostyryl)benzene and nitro-styrylfuran with the triplet yield reaching 120% and 140%, respectively, triggered by their greatly enhanced intramolecular charge transfer character relative to the other compounds in the series.

Lighting-Up the Far-Red Fluorescence of RNA-Selective Dyes by Switching from Ortho to Para Position.

Fluorescence imaging is constantly searching for new far-red emitting probes whose turn-on response is selective upon the interaction with specific biological targets. Cationic push-pull dyes could indeed respond to these requirements due to their intramolecular charge transfer (ICT) character, by which their optical properties can be tuned, and their ability to interact strongly with nucleic acids. Starting from the intriguing results recently achieved with some push-pull dimethylamino-phenyl dyes, two isomers obtained by switching the cationic electron acceptor head (either a methylpyridinium or a methylquinolinium) from the ortho to the para position have been scrutinized for their ICT dynamics, their affinity towards DNA and RNA, and in vitro behavior. By exploiting the marked fluorescence enhancement observed upon complexation with polynucleotides, fluorimetric titrations were employed to evaluate the dyes' ability as efficient DNA/RNA binders. The studied compounds exhibited in vitro RNA-selectivity by localizing in the RNA-rich nucleoli and within the mitochondria, as demonstrated by fluorescence microscopy. The para-quinolinium derivative showed some modest antiproliferative effect on two tumor cell lines as well as improved properties as an RNA-selective far-red probe in terms of both turn-on response (100-fold fluorescence enhancement) and localized staining ability, attracting interest as a potential theranostic agent.

Tuning the Photophysics of Two-Arm Bis[(dimethylamino)styryl]benzene Derivatives by Heterocyclic Substitution.

The identification of novel molecular systems with high fluorescence and significant non-linear optical (NLO) properties is a hot topic in the continuous search for new emissive probes. Here, the photobehavior of three two-arm bis[(dimethylamino)styryl]benzene derivatives, where the central benzene was replaced by pyridine, furan, or thiophene, was studied by stationary and time-resolved spectroscopic techniques with ns and fs resolution. The three molecules under investigation all showed positive fluorosolvatochromism, due to intramolecular charge-transfer (ICT) dynamics from the electron-donor dimethylamino groups, and significant fluorescence quantum yields, because of the population of a planar and emissive ICT state stabilized by intramolecular hydrogen-bond-like interactions. The NLO properties (hyperpolarizability coefficient and TPA cross-section) were also measured. The obtained results allowed the role of the central heteroaromatic ring to be disclosed. In particular, the introduction of the thiophene ring guarantees high fluorescent quantum yields irrespective of the polarity of the medium, and the largest hyperpolarizability coefficient because of the increased conjugation. An important and structure-dependent involvement of the triplet state was also highlighted, with the intersystem crossing being competitive with fluorescence, especially in the thiophene derivative, where the triplet was found to significantly sensitize molecular oxygen even in polar environment, leading to possible applications in photodynamic therapy.

Amphiphilicity-Controlled Localization of Red Emitting Bicationic Fluorophores in Tumor Cells Acting as Bio-Probes and Anticancer Drugs.

Small organic molecules arouse lively interest for their plethora of possible biological applications, such as anticancer therapy, for their ability to interact with nucleic acids, or bioimaging, thanks to their fluorescence emission. Here, a panchromatic series of styryl-azinium bicationic dyes, which have already proved to exhibit high water-solubility and significant red fluorescence in water, were investigated through spectrofluorimetric titrations to assess the extent of their association constants with DNA and RNA. Femtosecond-resolved transient absorption spectroscopy was also employed to characterize the changes in the photophysical properties of these fluorophores upon interaction with their biological targets. Finally, in vitro experiments conducted on tumor cell lines revealed that some of the bicationic fluorophores had a peculiar localization within cell nuclei exerting important antiproliferative effects, others were instead found to localize in the cytoplasm without leading to cell death, being useful to mark specific organelles in light of live cell bioimaging. Interestingly, this molecule-dependent behavior matched the different amphiphilicity featured by these bioactive compounds, which are thus expected to be caught in a tug-of-war between lipophilicity, ensured by the presence of aromatic rings and needed to pass cell membranes, and hydrophilicity, granted by charged groups and necessary for stability in aqueous media.

Acid-base strength and acido(fluoro)chromism of three push-pull derivatives of 2,6-distyrylpyridine.

The acidochromism and acid-base properties of 2,6-distyrylpyridine (2,6-DStP) derivatives bearing on the sides push/pull substituents (namely two dimethylamino, one nitro, and one methoxy and two nitro groups in the case of 2,6-bis[(E)-2-(4-dimetylaminophenyl)ethenyl]pyridine, 2-[(E)-2-(4-nitrophenyl)ethenyl],6-[(E)-2'-(4'-methoxyphenyl)ethenyl]pyridine and 2,6-bis[(E)-2-(4-nitrophenyl)ethenyl]pyridine, respectively) were investigated by stationary and time-resolved spectroscopies. The sensitivity of the absorption and emission spectrum to the medium acidity was found to enhance in the dimethylamino-derivative relative to the unsubstituted 2,6-DStP, also because of the second protonation by the N(CH3)2 group. Spectrophotometric titrations, also processed by a global fitting approach, gave pKa values, for the protonation of the central pyridine, higher in the derivatives with electron-donor unities and lower in compounds bearing electron-acceptor groups. A fluorometric titration was performed in the case of the dimethylamino-derivative thanks to non-negligible emission efficiencies for both neutral and protonated species, unveiling an attractive naked-eye acido(fluoro)chromism from green to yellow upon pyridine protonation, and then to purple with the second protonation involving the lateral N(CH3)2 substituent. Due to the extremely short excited-state lifetimes, as resulted from femtosecond transient absorption experiments, the pKa values for the excited state (pKa*) were estimated through the Förster cycle, revealing that the monoprotonated species of the dimethylamino-derivative would become upon excitation the only stable form in a wide range of pH.

Exploring a new class of singlet fission fluorene derivatives with high-energy triplets.

In this study, we report strong experimental evidence for singlet fission (SF) in a new class of fluorene-based molecules, exhibiting two-branched donor-acceptor structures. The time-resolved spectroscopic results disclose ultrafast formation of a double triplet state (occurring in few picoseconds) and efficient triplet exciton separation (up to 145% triplet yield). The solvent polarity effect and the role of intramolecular charge transfer (ICT) on the SF mechanism have been thoroughly investigated with several advanced spectroscopies. We found that a stronger push-pull character favors SF, as long as the ICT does not act as a trap by opening a competitive pathway. Within the context of other widely-known SF chromophores, the unconventional property of generating high-energy triplet excitons (ca. 2 eV) via SF makes these materials outstanding candidates as photosensitizers for photovoltaic devices.

An Efficient Aequorea victoria Green Fluorescent Protein for Stimulated Emission Depletion Super-Resolution Microscopy.

In spite of their value as genetically encodable reporters for imaging in living systems, fluorescent proteins have been used sporadically for stimulated emission depletion (STED) super-resolution imaging, owing to their moderate photophysical resistance, which does not enable reaching resolutions as high as for synthetic dyes. By a rational approach combining steady-state and ultrafast spectroscopy with gated STED imaging in living and fixed cells, we here demonstrate that F99S/M153T/V163A GFP (c3GFP) represents an efficient genetic reporter for STED, on account of no excited state absorption at depletion wavelengths <600 nm and a long emission lifetime. This makes c3GFP a valuable alternative to more common, but less photostable, EGFP and YFP/Citrine mutants for STED imaging studies targeting the green-yellow region of the optical spectrum.

The dependence of the spectroscopic properties of orcein dyes on solvent proticity: insights from theory and experiments.

The electronic spectral properties of α-hydroxy-orcein (α-HO), one of the main components of the orcein dye, have been extensively investigated in solvents of different proticity through UV-Vis spectrophotometry combined with DFT and TDDFT calculations. The results highlight the occurrence of an acid-base equilibrium between the neutral (absorption maximum at 475 nm) and the monoanionic (absorption maximum at 578 nm) forms of the molecule. The position of this equilibrium was found to be sensitively dependent on solvent proticity, solution concentration and pH. Quantum mechanical calculations support the rationalization of the experimental data, confirming the key role of the protic solvent in shifting the acid-base equilibrium, through the establishment of hydrogen bond interactions on specific functional groups of the dye. Both deprotonation and dye coordination with protic solvent molecules determine the reduction of the HOMO-LUMO energy gap (0.71 eV), that can be related with the bathochromic effect envisaged both experimentally (0.59 eV) and theoretically (0.50 eV).

The role of twisting in driving excited-state symmetry breaking and enhanced two-photon absorption in quadrupolar cationic pyridinium derivatives.

Two symmetric quadrupolar cationic push-pull compounds with a central electron-acceptor (N+-methylpyrydinium, A+) and different lateral electron-donors, (N,N-dimethylamino and N,N-diphenylamino, D) in a D-π-A+-π-D arrangement, were investigated together with their dipolar counterparts (D-π-A+) for their excited-state dynamics and NLO properties. As for the quadrupolar compounds, attention was focused on excited-state symmetry breaking (ESSB), which leads to a relaxed dipolar excited state. Both electron charge displacements and structural rearrangements were recognized in the excited-state dynamics of these molecules by resorting to femtosecond-resolved broadband fluorescence up-conversion experiments and advanced data analysis, used as a valuable alternative approach for fluorescent molecules compared to time-resolved IR spectroscopy, only suitable for compounds bearing IR markers. Specifically, intramolecular charge transfer (ICT) was found to be guided by ultrafast inertial solvation, while diffusive solvation can drive the twisting of lateral groups to originate twisted-ICT (TICT) states on a picosecond time scale. Yet still, only the bis-N,N-diphenylamino-substituted compound undergoes ESSB, in both highly and sparingly polar solvents, provided that it can experience large amplitude motions to a fully symmetry-broken TICT state. Besides well-known solvation effects, this structural requirement proved to be a necessary condition for these quadrupolar cations to undergo ESSB. In fact, a more efficient uncoupling between the out-of-plane D and A+ groups in the TICT state allows a greater stabilization gained through solvation, relative to the bis-N,N-dimethylamino-substituted derivative, which instead maintains its symmetry. This different behavior parallels the two-photon absorption (TPA) ability, which is greatly enhanced in the case of the bis-N,N-diphenylamino-substituted compound, paving the way for cutting-edge bio-imaging applications.

Competition between fluorescence and triplet production ruled by nitro groups in one-arm and two-arm styrylbenzene heteroanalogues.

The competition between excited state deactivation processes in mono and double-arm push-pull systems bearing pyridine, furan or thiophene (electron donors) and nitro groups (electron acceptors) was investigated in several solvents through nanosecond and femtosecond transient absorption spectroscopy. Triplet population is the main deactivation pathway for the mono-arm compounds. The large triplet production is mainly ascribed to 3(n,π*) states almost isoenergetic to S1, introduced by nitro groups, as predicted by TD-DFT calculations. The large triplet population may indeed be exploited to produce long-lived excitons for photovoltaic and optoelectronic applications. Two-arm furan and thiophene derivatives instead undergo strong ultrafast intramolecular charge transfer (ICT), which is responsible for their appreciable two-photon absorption cross-sections. In this case, significant fluorescence and singlet oxygen quantum yields are obtained, making these two compounds interesting as potential traceable photosensitizers in photodynamic therapy.

Quaternized styryl-azinium fluorophores as cellular RNA-binders.

The capability of three quaternized styryl-azinium iodides to bind cellular RNA has been tested by means of Fluorescence Confocal Microscopy imaging of stained MCF-7 cells treated with RNase. Their association constants have been estimated through spectrophotometric and fluorimetric titrations with tRNA and compared to their affinity toward DNA. Transient absorption spectroscopy with femtosecond resolution confirmed the binding of the investigated compounds with tRNA and shed new light on the excited state dynamics of their complexes, by revealing a significant lengthening of the lifetime of S1 upon complexation, which parallels the fluorescence quantum yield enhancement.

Fine structural tuning of styryl-based dyes for fluorescence and CD-based sensing of various ds-DNA/RNA sequences.

A set of styryl- and bis-styryl dyes, varying in length, aromatic surface, net positive charge and steric positioning or bulkiness of substituents, was tested for interactions with various ds-DNA or ds-RNA. Most of the compounds showed strong affinity toward ds-DNA/RNA, directly correlated to the synergistic contribution of the aromatic-conjugated surface and net positive charge. The volume or positioning of terminal aromatic substituents directly controlled the binding mode of the core structure, shifting between DNA/RNA groove binding or DNA/RNA intercalation. Consequently, upon binding to DNA/RNA the fluorimetric and induced CD (ICD) response varied for different compounds, for instance one derivative showed specific fluorescence increase with AT-DNA, while another derivative showed specific ICD response with AU-RNA. Preliminary screening on human tumour cell lines revealed an efficient cellular uptake for all dyes. Only mono-styryl-quinoline derivatives showed a strong antiproliferative activity combined with efficient fluorescent localisation, thus showing promising theragnostic potential, while other compounds were negligibly cytotoxic but still efficient fluorescent markers of cytoplasmic organelles.

Transient absorption spectroscopic studies on 4-nitroquinoline N-oxide: From femtoseconds to microseconds timescale.

The singlet excited state of 4-nitroquinoline N-oxide (1NQNO*) has been characterized by different spectroscopic techniques, combining transient absorption with steady state and time-resolved emission spectroscopy. The energy of 1NQNO* has been established as 255 kJ/mol from the fluorescence spectrum, whereas its lifetime has been found to be 10 ps in the femto-laser flash photolysis (LFP) experiments, where a characteristic S1Sn absorption band with maximum centered at 425 nm is observed. In a first stage, the triplet excited state of NQNO (3NQNO*) has also been characterized by emission spectroscopy in solid matrix, at low temperature. Thus, from the steady-state phosphorescence spectrum the triplet energy has been estimated as 183 kJ/mol, whereas the setup with time resolution has allowed us to determine the phosphorescence lifetime as 3 ms. Formation of 3NQNO* by intersystem crossing in solution at room temperature, has been monitored by femto-LFP, which shows the appearance of a band with maximum at 560 nm (T1-Tn). It increases with the decreasing intensity of its precursor 425 nm (S1Sn) band, giving rise to an isosbestic point at 500 nm. The characterization of 3NQNO* has been completed by nano-LFP, using xanthone as photosensitizer and oxygen as well as ß-carotene as quenchers. In addition, quenching of 3NQNO* by electron donors (DABCO) is also observed in aprotic solvents, leading to the radical anion of NQNO (-NQNO). If there is a proton source in the medium (Et3N as electron donor or MeCN:H2O/4:1 as solvent system) protonation of the radical anion results in formation of the neutral radical of NQNO (NQNOH). In general, all processes are slower in protic solvents because of the solvation sphere. Overall, this information provides a deeper insight into the formation and behavior of excited states and radical ionic species derived from the title molecule NQNO.

New Styryl Phenanthroline Derivatives as Model D-π-A-π-D Materials for Non-Linear Optics.

Four novel push-pull systems combining a central phenanthroline acceptor moiety and two substituted benzene rings, as a part of the conjugated π-system between the donor and the acceptor moieties, have been synthetized through a straightforward and efficient one-step procedure. The chromophores display high fluorescence and a peculiar fluorosolvatochromic behaviour. Ultrafast investigation by means of state-of-the-art femtosecond-resolved transient absorption and fluorescence up-conversion spectroscopies allowed the role of intramolecular charge transfer (ICT) states to be evidenced, also revealing the crucial role played by both, the polarity and proticity of the medium on the excited state dynamics of the chromophores. The ICT processes, responsible for the solvatochromism, also lead to interesting non-linear optical (NLO) properties: namely great two photon absorption cross-sections (hundreds of GM), investigated by the Two Photon Excited Fluorescence (TPEF) technique, and large second order hyperpolarizability coefficients, estimated through a convenient solvatochromic method.

Photoinduced Intramolecular Charge Transfer and Hyperpolarizability Coefficient in Push-Pull Pyridinium Salts with Increasing Strength of the Acceptor Group.

The synthesis of three push-pull cationic dyes is reported here together with a photophysical study carried out by stationary and ultrafast spectroscopies. The hyperpolarizability (ß) values of the three molecules have been estimated through a simple solvatochromic method based on conventional, low-cost equipment, which had been tested previously with success in our laboratory. The investigated pyridinium salts showing strong negative solvatochromism bear the same piperidine ring as a strong electron-donor group and the same thiophenes as electron-rich π-linkers, but differ in terms of the N-substituent on the electron-acceptor pyridinium unit, namely N-methyl in compound A, N-pyrimidin-2yl in B and N-2,4-dinitrophenyl in C. The derived ß values were found to increase (in the order A

Understanding the interplay between the solvent and nuclear rearrangements in the negative solvatochromism of a push-pull flexible quinolinium cation.

A detailed computational characterization of the one-photon absorption spectrum of a 2-((E)-2-[2,2']-bithiophenyl-5-yl-vinyl)-1-methyl-quinolinium cation in acetonitrile solution is presented. The main physico-chemical effects (solvation, vibronic progression) affecting the band position and shape are progressively introduced in the computational model, highlighting their relative role in the spectral profile. The reported results underline how an accurate reproduction of the experimental spectrum can only be obtained by going beyond oversimplified methods. Moreover, the deep interplay between the solvent effects and nuclear rearrangements permits the negative solvatochromism exhibited by hypsochromic molecules to be explained. This illustrates the potential of the computational investigation, which can shed light on the information hidden in experimental spectra.

Photoexcitation and relaxation kinetics of molecular systems in solution: towards a complete in silico model.

In this study, we have modelled, through a theoretical-computational approach based on classical molecular dynamics simulations and quantum-chemical calculations, the complete relaxation process of a photo-excited ionic stilbene-like compound termed as DASPMI in solution. Starting from the absorption spectrum we have reconstructed the entire process of the excited-state relaxation involving the intramolecular charge-transfer and eventually leading to the charge-recombination regenerating the ground state. The results obtained, well reproducing the experimental time-resolved emission spectra and kinetic observables, show that the relaxation process is essentially driven by the internal conformational transitions of the chromophore with the solvent almost instantaneously relaxed for each chromophore conformation. This study represents the first attempt, carried out using our theoretical-computational approach, of modelling a complete experiment involving the overposition of relaxation kinetics ranging from hundreds of femtoseconds to nanoseconds on the time scale.

The Role of pH in Modulating the Electronic State Properties of Minocycline Drug and Its Inclusion within Micellar Carriers.

A detailed investigation of the spectral and photophysical properties of minocycline (MC) in water at different pHs, solvents of different polarity, and micellar surfactant solutions was carried out in this study. An unusual behavior was highlighted with respect to other tetracyclines due to the presence of an additional dimethylamino group in the MC molecular structure. In particular, four equilibrium constants associated with mono-deprotonation reactions were characterized by steady-state spectroscopy. Femtosecond time-resolved pump-probe and fluorescence up-conversion measurements allowed the dynamics of the lowest excited singlet state of the five different acid-base species of MC to be characterized in terms of lifetimes and transient spectra. Two emissive species associated with keto-enol tautomerism resulting from excited-state intramolecular proton transfer (ESIPT) were revealed with time constants of a few and tens of picoseconds. TD-DFT quantum mechanical calculations were also performed to define the state order and nature of the differently protonated species, together with their absorption spectra. The role of pH proved to be fundamental in modulating the drug charge and therefore the interaction with cationic micelles where the neutral form of MC, that is the biologically active one, resulted efficiently included.

Efficient Excited-State Symmetry Breaking in a Cationic Quadrupolar System Bearing Diphenylamino Donors.

We report a joint experimental and theoretical investigation of a quadrupolar D-π-A(+) -π-D system, the electron donors being diphenylamino groups and the electron acceptor being a methylpyridinium, in comparison with the dipolar D-π-A(+) system. The emission spectra of the two compounds overlap in all the investigated solvents. This finding could be rationalized by TD-DFT calculations: the LUMO-HOMO molecular orbitals involved in the emission transition are localized on the same branch of the quadrupolar structure that becomes the fluorescent portion, corresponding to that of the single-arm compound. Excited-state symmetry breaking has been rarely observed for quadrupolar systems showing negative solvatochromism and is here surprisingly revealed, even in low polarity solvents. Femtosecond transient absorption measurements revealed that an efficient photoinduced intramolecular charge transfer takes place in the quadrupolar chromophore, more efficient than in its dipolar analogue. This result is promising in view of the application of these compounds as novel two-photon absorbing materials.