Boosting the Efficiency of Red Thermally Activated Delayed Fluorescence via Conjugation Enhancement in Push-Pull Naphthalimide Derivatives.

In this work, we synthesize a series of push-pull compounds bearing naphthalimide as the electron acceptor and tetraphenylethylene (TPE)/triphenylamine (TPA)/phenothiazine (PTZ) as the electron rich/electron donor units. These moieties are arranged in highly conjugated quadrupolar structures. The structure-property relationships are investigated through a joint experimental time-resolved spectroscopic and computational TD-DFT study. The femtosecond transient absorption and fluorescence up-conversion experiments reveal ultrafast photoinduced intramolecular charge transfer. This is likely the key factor leading to efficient spin-orbit CT-induced intersystem crossing for the TPA- and PTZ-derivatives as well as to small singlet-to-triplet energy gap. Consequently, evidence for a delayed fluorescence component is found together with the main prompt emission in the fluorescence kinetics both in solution and in thin film. The weight of the Thermally Activated Delayed Fluorescence (TADF) is greatly enhanced when these fluorophores are used as guests in solid-state host matrices. TADF is interestingly revealed in the orange-red region of the visible. Such long wavelength emission is here observed with surprisingly large fluorescence quantum yields, thanks to the conjugation enhancement achieved in these newly synthesized structures relative to previous studies. Our findings may be thus promising for the future development of efficient third generation TADF-based OLEDs.

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.

Room-Temperature Phosphorescence and Cellular Phototoxicity Activated by Triplet Dynamics in Aggregates of Push-Pull Phenothiazine-Based Isomers.

In this study, we report a comprehensive time-resolved spectroscopic investigation of the excited-state deactivation mechanism in three push-pull isomers characterized by a phenothiazine electron donor, a benzothiazole electron acceptor, and a phenyl π-bridge where the connection is realized at the relative ortho, meta, and para positions. Spin-orbit charge-transfer-induced intersystem crossing takes place with high yield in these all-organic donor-acceptor compounds, leading also to efficient production of singlet oxygen. Our spectroscopic results give clear evidence of room-temperature phosphorescence not only in solid-state host-guest matrices but also in highly biocompatible aggregates of these isomers produced in water dispersions, as rarely reported in the literature. Moreover, aggregates of the isomers could be internalized by lung cancer and melanoma cells and display bright luminescence without any dark cytotoxic effect. On the other hand, the isomers showed significant cellular phototoxicity against the tumor cells due to light-induced reactive oxygen species generation. Our findings strongly suggest that nanoaggregates of the investigated isomers are promising candidates for imaging-guided photodynamic therapy.

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.

Design, Synthesis and Characterization of a Visible-Light-Sensitive Molecular Switch and Its PEGylation Towards a Self-Assembling Molecule.

HBDI-like chromophores represent a novel set of biomimetic switches mimicking the fluorophore of the green fluorescent protein that are currently studied with the hope to expand the molecular switch/motor toolbox. However, until now members capable of absorbing visible light in their neutral (i. e. non-anionic) form have not been reported. In this contribution we report the preparation of an HBDI-like chromophore based on a 3-phenylbenzofulvene scaffold capable of absorbing blue light and photoisomerizing on the picosecond timescale. More specifically, we show that double-bond photoisomerization occurs in both the E-to-Z and Z-to-E directions and that these can be controlled by irradiating with blue and UV light, respectively. Finally, as a preliminary applicative result, we report the incorporation of the chromophore in an amphiphilic molecule and demonstrate the formation of a visible-light-sensitive nanoaggregated state in water.

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.

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.

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.

Xanthopsin-Like Systems via Site-Specific Click-Functionalization of a Retinoic Acid Binding Protein.

The use of light-responsive proteins to control both living or synthetic cells, is at the core of the expanding fields of optogenetics and synthetic biology. It is thus apparent that a richer reaction toolbox for the preparation of such systems is of fundamental importance. Here, we provide a proof-of-principle demonstration that Morita-Baylis-Hillman adducts can be employed to perform a facile site-specific, irreversible and diastereoselective click-functionalization of a lysine residue buried into a lipophilic binding pocket and yielding an unnatural chromophore with an extended π-system. In doing so we effectively open the path to the in vitro preparation of a library of synthetic proteins structurally reminiscent of xanthopsin eubacterial photoreceptors. We argue that such a library, made of variable unnatural chromophores inserted in an easy-to-mutate and crystallize retinoic acid transporter, significantly expand the scope of the recently introduced rhodopsin mimics as both optogenetic and "lab-on-a-molecule" tools.

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.

Tuning the Fluorescence and the Intramolecular Charge Transfer of Phenothiazine Dipolar and Quadrupolar Derivatives by Oxygen Functionalization.

A series of new naphthalimide and phenothiazine-based push-pull systems (NPI-PTZ1-5), in which we structurally modulate the oxidation state of the sulfur atom in the thiazine ring, i.e., S(II), S(IV), and S(VI), was designed and synthesized by the Pd-catalyzed Sonogashira cross-coupling reaction. The effect of the sulfur oxidation state on the spectral, photophysical, and electrochemical properties was investigated. The steady-state absorption and emission results show that oxygen functionalization greatly improves the optical (absorption coefficient and fluorescence efficiency) and nonlinear optical (hyperpolarizability) features. The cyclic voltammetry experiments and the quantum mechanical calculations suggest that phenothiazine is a stronger electron donor unit relative to phenothiazine-5-oxide and phenothiazine-5,5-dioxide, while the naphthalimide is a strong electron acceptor in all cases. The advanced ultrafast spectroscopic measurements, transient absorption, and broadband fluorescence up conversion give insight into the mechanism of photoinduced intramolecular charge transfer. A planar intramolecular charge transfer (PICT) and highly fluorescent excited state are populated for the oxygen-functionalized molecules NPI-PTZ2,3 and NPI-PTZ5; on the other hand, a twisted intramolecular charge transfer (TICT) state is produced upon photoexcitation of the oxygen-free derivatives NPI-PTZ1 and NPI-PTZ4, with the fluorescence being thus significantly quenched. These results prove oxygen functionalization as a new effective synthetic strategy to tailor the photophysics of phenothiazine-based organic materials for different optoelectronic applications. While oxygen-functionalized compounds are highly fluorescent and promising active materials for current-to-light conversion in organic light-emitting diode devices, oxygen-free systems show very efficient photoinduced ICT and may be employed for light-to-current conversion in organic photovoltaics.

The Role of the Core Attachment Positioning in Triggering Intramolecular Singlet Exciton Fission in Perylene Diimide Tetramers.

Previous studies have proposed that the presence of a flexible π-bridge linker is crucial in activating intramolecular singlet exciton fission (iSEF) in multichromophoric systems. In this study, we report the photophysical properties of three analogous perylene diimide (PDI) dendritic tetramers showing flexible/twisted π-bridged structures with α- and ß-substitutions and a rigid/planar structure with a ß-fused ring (ßC) connection to a benzodithiophene-thiophene (BDT-Th) core. The rigidity and enhanced planarity of ßC lead to significant intramolecular charge transfer and triplet formation via an intersystem crossing pathway. Steady-state spectroscopic measurements reveal similar absorption and emission spectra for the α-tetramer and the parent PDI monomer. However, their fluorescence quantum yield is significantly different. The negligible fluorescence yield of the α-tetramer (0.04%) is associated with a competitive nonradiative decay pathway. Indeed, for this twisted compound in a high polar environment, a fast and efficient iSEF with a triplet quantum yield of 124% is observed. Our results show that the α-single-bond connections in the α compound are capable of interrupting the coupling among the PDI units, favoring iSEF. We propose that the formation of the double triplet (1[TT]) state is through a superposition of singlet states known as [S1S0][TT]CT, which has been suggested previously for pentacene derivatives. Using steady-state and time-resolved spectroscopic experiments, we demonstrate that the conformational flexibility of the linker itself is necessary but not sufficient to allow iSEF. For the case of the other twisted tetramer, ß, the strong π-π co-facial interactions between the adjacent PDI units in its structure lead to excimer formation. These excimer states trap the singlet excitons preventing the formation of the 1[TT] state, thus inhibiting iSEF.

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.

Activating intramolecular singlet exciton fission by altering π-bridge flexibility in perylene diimide trimers for organic solar cells.

In this study, two analogous perylene diimide (PDI) trimers, whose structures show rotatable single bond π-bridge connection (twisted) vs. rigid/fused π-bridge connection (planar), were synthesized and investigated. We show via time resolved spectroscopic measurements how the π-bridge connections in A-π-D-π-A-π-D-π-A multichromophoric PDI systems strongly affect the triplet yield and triplet formation rate. In the planar compound, with stronger intramolecular charge transfer (ICT) character, triplet formation occurs via conventional intersystem crossing. However, clear evidence of efficient and fast intramolecular singlet exciton fission (iSEF) is observed in the twisted trimer compound with weaker ICT character. Multiexciton triplet generation and separation occur in the twisted (flexible-bridged) PDI trimer, where weak coupling among the units is observed as a result of the degenerate double triplet and quintet states, obtained by quantum chemical calculations. The high triplet yield and fast iSEF observed in the twisted compound are due not only to enthalpic viability but also to the significant entropic gain allowed by its trimeric structure. Our results represent a significant step forward in structure-property understanding, and may direct the design of new efficient iSEF materials.

Ground-State Structural Disorder and Excited-State Symmetry Breaking in a Quadrupolar Molecule.

The influence of torsional disorder around the ethynyl π-bridges of a linear D-π-A-π-D molecule on the nature of its S1 excited state was investigated using ultrafast time-resolved infrared spectroscopy. By tuning the pump wavelength throughout the S1 ← S0 absorption band, subpopulations with different extents of asymmetry could be excited. In nonpolar solvents, the equilibrated S1 state is symmetric and quadrupolar independently of the initial degree of distortion. Photoexcitation of distorted molecules is followed by planarization and symmetrization of the S1 state. Excited-state symmetry breaking is only observed in polar environments, where the equilibrated S1 state has a strong dipolar character. However, neither the extent nor the rate of symmetry breaking are enhanced in an initially distorted molecule. They are only determined by the polarity and the dynamic properties of the solvent.

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.

Long-Range Energy Transfer in Protein Megamolecules.

In this investigation, we report evidence for energy transfer in new protein-based megamolecules with tunable distances between donor and acceptor fluorescent proteins. The megamolecules used in this work are monodisperse oligomers, with molecular weights of ∼100-300 kDa and lengths of ∼5-20 nm, and are precisely defined structures of fusion protein building blocks and covalent cross-linkers. Such structures are promising because the study of energy transfer in protein complexes is usually difficult in this long length regime due to synthetic limitations. We incorporated fluorescent proteins into the megamolecule structure and varied the separation distance between donor and acceptor by changing the length of the cross-linker in dimer conjugates and inserting nonfluorescent spacer proteins to create oligomers. Two-photon absorption measurements demonstrated strong coupling between donor and acceptor dipoles in the megamolecules. For the dimer systems, no effect of the cross-linker length on energy transfer efficiency was observed with the steady-state fluorescence investigation. However, for the same dimer conjugates, energy transfer rates decreased upon increasing cross-linker length, as evaluated by fluorescence up-conversion. Molecular dynamics simulations were used to rationalize the results, providing quantitative agreement between measured and calculated energy transfer lengths for steady-state results, and showing that the differences between the time-resolved and steady-state measurements arise from the long time scale for large-scale fluctuations in the megamolecule structure. Our results show an increase in energy transfer length with increasing megamolecule size. This is evidence for long-range energy transfer in large protein megamolecules.

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.