Proton-Coupled Electron Transfer Ring Opening of Cycloalkanols Followed by a Giese Radical Addition Enabled by an Electron Donor-Acceptor Complex.

Herein, we describe the formation of an electron donor-acceptor (EDA) complex between electron-rich cycloalkanols and electron-deficient alkenes that triggers the proton-coupled electron transfer ring opening of strained and unstrained cycloalkanols without the need for an external photocatalyst. This activation generates a remote alkyl radical that undergoes a Giese reaction with the Michael acceptor in an efficient manner. Mechanistic investigations corroborate both the formation of the EDA complex and the consecutive Giese reaction.

Photodissociation of leucine-enkephalin protonated peptide: an experimental and theoretical perspective.

Understanding the competing processes that govern far ultraviolet photodissociation (FUV-PD) of biopolymers such as proteins is a challenge. Here, we report a combined experimental and theoretical investigation of FUV-PD of protonated leucine-enkephalin pentapeptide ([YGGFL + H]+) in the gas-phase. Time-dependent density functional theory (TD-DFT) calculations in combination with experiments and previous results for amino acids and shorter peptides help in rationalizing the evolution of the excited states. The results confirm that fragmentation of [YGGFL + H]+ results mainly from vibrationally excited species in the ground electronic state, populated after internal conversion. We also propose fragmentation mechanisms for specific photo-fragments such as tyrosine side chain loss (with an extra hydrogen) or hydrogen loss. In general, we observe the same mechanisms as for smaller peptides or protonated Tyr and Phe, that are not quenched by the presence of other amino acids. Nevertheless, we also found some differences, as for H loss, in part due to the fact that the charge is solvated by the peptide chain and not only by the COOH terminal group.

Ultrafast Formation of a Delocalized Triplet-Excited State in an Epigenetically Modified DNA Duplex under Direct UV Excitation.

Epigenetic modifications impart important functionality to nucleic acids during gene expression but may increase the risk of photoinduced gene mutations. Thus, it is crucial to understand how these modifications affect the photostability of duplex DNA. In this work, the ultrafast formation (<20 ps) of a delocalized triplet charge transfer (CT) state spreading over two stacked neighboring nucleobases after direct UV excitation is demonstrated in a DNA duplex, d(G5fC)9•d(G5fC)9, made of alternating guanine (G) and 5-formylcytosine (5fC) nucleobases. The triplet yield is estimated to be 8 ± 3%, and the lifetime of the triplet CT state is 256 ± 22 ns, indicating that epigenetic modifications dramatically alter the excited state dynamics of duplex DNA and may enhance triplet state-induced photochemistry.

Triplet Excimer Formation in a DNA Duplex with Silver Ion-Mediated Base Pairs.

The dynamics of excited electronic states in self-assembled structures formed between silver(I) ions and cytosine-containing DNA strands or monomeric cytosine derivatives were investigated by time-resolved infrared (TRIR) spectroscopy and quantum mechanical calculations. The steady-state and time-resolved spectra depend sensitively on the underlying structures, which change with pH and the nucleobase and silver ion concentrations. At pH ∼ 4 and low dC20 strand concentration, an intramolecularly folded i-motif is observed, in which protons, and not silver ions, mediate C-C base pairing. However, at the higher strand concentrations used in the TRIR measurements, dC20 strands associate pairwise to yield duplex structures containing C-Ag+-C base pairs with a high degree of propeller twisting. UV excitation of the silver ion-mediated duplex produces a long-lived excited state, which we assign to a triplet excimer state localized on a pair of stacked cytosines. The computational results indicate that the propeller-twisted motifs induced by metal-ion binding are responsible for the enhanced intersystem crossing that populates the triplet state and not a generic heavy atom effect. Although triplet excimer states have been discussed frequently as intermediates in the formation of cyclobutane pyrimidine dimers, we find neither computational nor experimental evidence for cytosine-cytosine photoproduct formation in the systems studied. These findings provide a rare demonstration of a long-lived triplet excited state that is formed in a significant yield in a DNA duplex, demonstrating that supramolecular structural changes induced by metal ion binding profoundly affect DNA photophysics.

The photophysics of protonated cytidine and hemiprotonated cytidine base pair: A computational study.

We here study the effect that a lowering of the pH has on the excited state processes of cytidine and a cytidine/cytidine pair in solution, by integrating time-dependent density functional theory and CASSCF/CASPT2 calculations, and including solvent by a mixed discrete/continuum model. Our calculations reproduce the effect of protonation at N3 on the steady-state infrared and absorption spectra of a protonated cytidine (CH+ ), and predict that an easily accessible non-radiative deactivation route exists for the spectroscopic state, explaining its sub-ps lifetime. Indeed, an extremely small energy barrier separates the minimum of the lowest energy bright state from a crossing region with the ground electronic state, reached by out-of-plane motion of the hydrogen substituents of the CC double bond, the so-called ethylenic conical intersection typical of cytidine and other pyrimidine bases. This deactivation route is operative for the two bases forming an hemiprotonated cytidine base pair, [CH·C]+ , the building blocks of I-motif secondary structures, whereas interbase processes play a minor role. N3 protonation disfavors instead the nπ* transitions, associated with the long-living components of cytidine photoactivated dynamics.

Effect of the DNA Polarity on the Relaxation of Its Electronic Excited States.

The DNA polarity, i.e., the order in which nucleobases are connected together via the phosphodiester backbone, is crucial for several biological processes. But, so far, there has not been experimental evidence regarding its effect on the relaxation of DNA electronic excited states. Here we examine this aspect for two dinucleotides containing adenine and guanine: 5'-dApdG-3' and 5'-dGpdA-3' in water. We used two different femtosecond transient absorption setups: one providing high temporal resolution and broad spectral coverage (330-650 nm) between 30 fs and 50 ps, and the other recording decays at selected wavelengths until 1.2 ns. The transient absorption spectra corresponding to the minima in the potential energy surface of the first excited state were computed by quantum chemistry methods. Our results show that the excited charge transfer state in 5'-dGpdA-3' is formed with a ∼75% higher quantum yield and exhibits slower decay (170 ± 10 ps vs 112 ± 12 ps) compared to 5'-dApdG-3'.

Curved Nanographenes: Multiple Emission, Thermally Activated Delayed Fluorescence, and Non-Radiative Decay.

The intriguing and rich photophysical properties of three curved nanographenes (CNG 6, 7, and 8) are investigated by time-resolved and temperature-dependent photoluminescence (PL) spectroscopy. CNG 7 and 8 exhibit dual fluorescence, as well as dual phosphorescence at low temperature in the main PL bands. In addition, hot bands are detected in fluorescence as well as phosphorescence, and, in the narrow temperature range of 100-140 K, thermally activated delayed fluorescence (TADF) with lifetimes on the millisecond time-scale is observed. These findings are rationalized by quantum-chemical simulations, which predict a single minimum of the S1 potential of CNG 6, but two S1 minima for CNG 7 and CNG 8, with considerable geometric reorganization between them, in agreement with the experimental findings. Additionally, a higher-lying S2 minimum close to S1 is optimized for the three CNG, from where emission is also possible due to thermal activation and, hence, non-Kasha behavior. The presence of higher-lying dark triplet states close to the S1 minima provides mechanistic evidence for the TADF phenomena observed. Non-radiative decay of the T1 state appears to be thermally activated with activation energies of roughly 100 meV and leads to disappearance of phosphorescence and TADF at T > 140 K.

Peptide Bond Formation in the Protonated Serine Dimer Following Vacuum UV Photon-Induced Excitation.

Possible routes for intra-cluster bond formation (ICBF) in protonated serine dimers have been studied. We found no evidence of ICBF following low energy collision-induced dissociation (in correspondence with previous works), however, we do observe clear evidence for ICBF following photon absorption in the 4.6-14 eV range. Moreover, the comparison of photon-induced dissociation measurements of the protonated serine dimer to those of a protonated serine dipeptide provides evidence that ICBF, in this case, involves peptide bond formation (PBF). The experimental results are supported by ab initio molecular dynamics and exploration of several excited state potential energy surfaces, unraveling a pathway for PBF following photon absorption. The combination of experiments and theory provides insight into the PBF mechanisms in clusters of amino acids, and reveals the importance of electronic excited states reached upon UV/VUV light excitation.

Photophysical Characterization of Isoguanine in a Prebiotic-Like Environment.

It is intriguing how a mixture of organic molecules survived the prebiotic UV fluxes and evolved into the actual genetic building blocks. Scientists are trying to shed light on this issue by synthesizing nucleic acid monomers and their analogues under prebiotic Era-like conditions and by exploring their excited state dynamics. To further add to this important body of knowledge, this study discloses new insights into the photophysical properties of protonated isoguanine, an isomorph of guanine, using steady-state and femtosecond broadband transient absorption spectroscopies, and quantum mechanical calculations. Protonated isoguanine decays in ultrafast time scales following 292 nm excitation, consistently with the barrierless paths connecting the bright S1 (ππ*) state with different internal conversion funnels. Complementary calculations for neutral isoguanine predict similar photophysical properties. These results demonstrate that protonated isoguanine can be considered photostable in contrast to protonated guanine, which exhibits 40-fold longer excited state lifetimes.

Effect of the Thermal Fluctuations of the Photophysics of GC and CG DNA Steps: A Computational Dynamical Study.

Here we refine and assess two computational procedures aimed to include the effect of thermal fluctuations on the electronic spectra and the ultrafast excited state dynamics of multichromophore systems, focusing on DNA duplexes. Our approach is based on a fragment diabatization procedure that, from a given Quantum Mechanical (QM) reference method, can provide the parameters (energy and coupling) of the reference diabatic states on the basis of the isolated fragments, either for a purely electronic excitonic Hamiltonian (FrDEx) or a linear vibronic coupling Hamiltonian (FrD-LVC). After having defined the most cost-effective procedure for DNA duplexes on two smaller fragments, FrDEx is used to simulate the absorption and Electronic Circular Dichroism (ECD) spectra of (GC)5 sequences, including the coupling with the Charge Transfer (CT) states, on a number of structures extracted from classical Molecular Dynamics (MD) simulations. The computed spectra are close to the reference TD-DFT calculations and fully consistent with the experimental ones. We then couple MD simulations and FrD-LVC to simulate the interplay between local excitations and CT transitions, both intrastrand and interstrand, in GC and CG steps when included in a oligoGC or in oligoAT DNA sequence. We predict that for both sequences a substantial part of the photoexcited population on G and C decays, within 50-100 fs, to the corresponding intrastrand CT states. This transfer is more effective for GC steps that, on average, are more closely stacked than CG ones.

Nucleic Acids as a Playground for the Computational Study of the Photophysics and Photochemistry of Multichromophore Assemblies.

The interaction between light and multichromophoric assemblies (MCAs) is the primary event of many fundamental processes, from photosynthesis to organic photovoltaics, and it triggers dynamical processes that share remarkable similarities at the molecular scale: light absorption, energy and charge transfer, internal conversions, emission, and so on. Those events often involve many chromophores and different excited electronic states that are coupled on an ultrafast time scale. This Account aims to discuss some of the chemical physical effects ruling these processes, a fundamental step toward their control, based on our experience on nucleic acids.In the last 15 years, we have, indeed, studied the photophysics and photochemistry of DNA and its components. By combining different quantum mechanical methods, we investigated the molecular processes responsible for the damage of the genetic code or, on the contrary, those preventing it by dissipating the excess energy deposited in the system by UV absorption. Independently of its fundamental biological role, DNA, with its fluctuating closely stacked bases stabilized by weak nonbonding interactions, can be considered a prototypical MCA. Therefore, it allows one to tackle within a single system many of the conceptual and methodological challenges involved in the study of photoinduced processes in MCA.In this Account, by using the outcome of our studies on oligonucleotides as a guideline, we thus highlight the most critical modellistic issues to be faced when studying, either experimentally or computationally, the interaction between UV light and DNA and, at the same time, bring out their general relevance for the study of MCAs.We first discuss the rich photoactivated dynamics of nucleobases (the chromophores), highlighting the main effects modulating the interplay between their excited states and how the latter can affect the photoactivated dynamics of the polynucleotides, either providing effective monomer-like nonradiative decay routes or triggering reactive processes (e.g., triplet generation).We then tackle the reaction paths involving multiple bases, showing that in the DNA duplex the most important ones involve two stacked bases, forming a neutral excimer or a charge transfer (CT) state, which exhibit different spectral signatures and photochemical reactivity. In particular, we analyze the factors affecting the dynamic equilibrium between the excimer and CT, such as the fluctuations of the backbone or the rearrangement of the solvent.Next, we highlight the importance of the effects not directly connected to the chromophores, such as the flexibility of the backbone or the solvent effect. The former, affecting the stacking geometry of the bases, can determine the preference between different deactivation paths. The latter is particularly influential for CT states, making very important an accurate treatment of dynamical solvation effects, involving both the solvent bulk and specific solute-solvent interactions.In the last section, we describe the main methodological challenges related to the study of polynucleotide excited states and stress the benefits derived by the integration of complementary approaches, both computational and experimental. Only exploiting different point of views, in our opinion, it is possible to shed light on the complex phenomena triggered by light absorption in DNA, as in every MCA.

Thienoguanosine, a unique non-perturbing reporter for investigating rotational dynamics of DNA duplexes and their complexes with proteins.

Time-resolved fluorescence anisotropy (TRFA) provides key information on the dynamics of biomolecules and their interaction with ligands. However, since natural nucleosides are almost non-fluorescent, its application to DNA duplexes (dsDNA) requires fluorescent labels, which can alter dsDNA stability, hinder protein binding, and complicate interpretation of TRFA experiments due to their local motion. As shown here, thienoguanosine (thG), a fluorescent analogue of guanosine, overcomes all these limitations. We recorded the TRFA decays of thG-labelled dsDNA of different lengths. thG behaved as a rigid, non-perturbing reporter, since no fast correlation time was recorded for any tested dsDNA. Due to its perfect stacking, only two correlation times, instead of the typical three, describe thG-labelled dsDNA rotational dynamics. Thanks to these features, we provided a complete description of the tumbling of the different dsDNA and their complexes with the Set and Ring Associated (SRA) domain of UHRF1, a key epigenetic regulator, obtaining values in excellent agreement with theoretical predictions. Moreover, thG was also found sensitive to SRA-induced base flipping of neighboring nucleobases. In the DNA label toolbox, thG thus stands out as a unique reporter for investigating the rotational dynamics of dsDNA and protein/dsDNA complexes.

2-Oxopurine Riboside: A Dual Fluorescent Analog and Photosensitizer for RNA/DNA Research.

There is significant interest in developing suitable nucleoside analogs exhibiting high fluorescence and triplet yields to investigate the structure, dynamics, and binding properties of nucleic acids and promote selective photosensitized damage to DNA/RNA, respectively. In this study, steady-state, laser flash photolysis, time-resolved IR luminescence, and femtosecond broad-band transient absorption spectroscopies are combined with quantum chemical calculations to elucidate the excited-state dynamics of 2-oxopurine riboside in aqueous solution and to investigate its prospective use as a fluorescent or photosensitizer analog. The Franck-Condon population in the S1 (ππ*) state decays through a combination of solvent and conformational relaxation to its minimum in 1.9 ps. The population trapped in the 1ππ* minimum bifurcates to either fluoresce or intersystem cross to the triplet manifold within ca. 5 ns, while another fraction of the population decays nonradiatively to the ground state. It is demonstrated that 2-oxopurine riboside exhibits both high fluorescent (48%) and significant triplet (between 10% and 52%) yields, leading to a yield of singlet oxygen generation of 10%, making this nucleoside analog a dual fluorescent and photosensitizer analog for DNA and RNA research.

Thienoguanosine brightness in DNA duplexes is governed by the localization of itsππ* excitation in the lowest energy absorption band.

Thienoguanosine (thG) is an isomorphic fluorescent guanosine (G) surrogate, which almost perfectly mimics the natural G in DNA duplexes and may therefore be used to sensitively investigate for example protein-induced local conformational changes. To fully exploit the information given by the probe, we carefully re-investigated the thG spectroscopic properties in 12-bp duplexes, when the Set and Ring Associated (SRA) domain of UHRF1 flips its 5' flanking methylcytosine (mC). The SRA-induced flipping of mC was found to strongly increase the fluorescence intensity of thG, but this increase was much larger when thG was flanked in 3' by a C residue as compared to an A residue. Surprisingly, the quantum yield and fluorescence lifetime values of thG were nearly constant, regardless of the presence of SRA and the nature of the 3' flanking residue, suggesting that the differences in fluorescence intensities might be related to changes in absorption properties. We evidenced that thG lowest energy absorption band in the duplexes can be deconvoluted into two bands peaking at ∼350 nm and ∼310 nm, respectively red-shifted and blue-shifted, compared to the spectrum of thG monomer. Using quantum mechanical calculations, we attributed the former to a nearly pureππ* excitation localized on thG and the latter to excited states with charge transfer character. The amplitude of thG red-shifted band strongly increased when its 3' flanking C residue was replaced by an A residue in the free duplex, or when its 5' flanking mC residue was flipped by SRA. As only the species associated with the red-shifted band were found to be emissive, the highly unusual finding of this work is that the brightness of thG in free duplexes as well as its changes on SRA-induced mC flipping almost entirely depend on the relative population and/or absorption coefficient of the red-shifted absorbing species.

Ultrafast Excited-State Decay Mechanisms of 6-Thioguanine Followed by Sub-20 fs UV Transient Absorption Spectroscopy.

Understanding the primary steps following UV photoexcitation in sulphur-substituted DNA bases (thiobases) is fundamental for developing new phototherapeutic drugs. However, the investigation of the excited-state dynamics in sub-100 fs time scales has been elusive until now due to technical challenges. Here, we track the ultrafast decay mechanisms that lead to the electron trapping in the triplet manifold for 6-thioguanine in an aqueous solution, using broadband transient absorption spectroscopy with a sub-20 fs temporal resolution. We obtain experimental evidence of the fast internal conversion from the S2(ππ*) to the S1(nπ*) states, which takes place in about 80 fs and demonstrates that the S1(nπ*) state acts as a doorway to the triplet population in 522 fs. Our results are supported by MS-CASPT2 calculations, predicting a planar S2(ππ*) pseudo-minimum in agreement with the stimulated emission signal observed in the experiment.

Molecular Identification of the Transient Species Mediating the Deactivation Dynamics of Solvated Guanosine and Deazaguanosine.

Small structural alterations of the purine/pyrimidine core have been related to important photophysical changes, such as the loss of photostability. Similarly to canonical nucleobases, solute-solvent interactions can lead to a change in the excited state lifetimes and/or to the interplay of different states in the photophysics of these modified nucleobases. To shed light on both effects, we here report a complete picture of the absorption spectra and excited state deactivation of deoxyguanosine and its closely related derivative, deoxydeazaguanosine, in water and methanol through the mapping of the excited state potential energy surfaces and molecular dynamics simulations at the TD-DFT level of theory. We show that the N by CH exchange in the imidazole ring of deoxyguanosine translates into a small red-shift of the bright states and slightly faster dynamics. In contrast, changing solvent from water to methanol implies the opposite, i.e., that the deactivation of both systems to the ground state is significantly hindered.

The Excited State Dynamics of a Mutagenic Cytidine Etheno Adduct Investigated by Combining Time-Resolved Spectroscopy and Quantum Mechanical Calculations.

Joint femtosecond fluorescence upconversion experiments and theoretical calculations provide a hitherto unattained degree of characterization and understanding of the mutagenic etheno adduct 3,N4-etheno-2'-deoxycytidine (εdC) excited state relaxation. This endogenously formed lesion is attracting great interest because of its ubiquity in human tissues and its highly mutagenic properties. The εdC fluorescence is modified with respect to that of the canonical base dC, with a 3-fold increased lifetime and quantum yield at neutral pH. This behavior is amplified upon protonation of the etheno ring (εdCH+). Quantum mechanical calculations show that the lowest energy state ππ*1 is responsible for the fluorescence and that the main nonradiative decay pathway to the ground state goes through an ethene-like conical intersection, involving the out-of-plane motion of the C5 and C6 substituents. This conical intersection is lower in energy than the ππ* state (ππ*1) minimum, but a sizable energy barrier explains the increase of εdC and εdCH+ fluorescence lifetimes with respect to that of dC.

Early steps of oxidative damage in DNA quadruplexes are position-dependent: Quantum mechanical and molecular dynamics analysis of human telomeric sequence containing ionized guanine.

Guanine radical cation (G•+) is a key intermediate in many oxidative processes occurring in nucleic acids. Here, by combining mixed Quantum Mechanical/Molecular Mechanics calculations and Molecular Dynamics (MD) simulations, we study how the structural behaviour of a tract GGG(TTAGGG)3 (hereafter Tel21) of the human telomeric sequence, folded in an antiparallel quadruple helix, changes when one of the G bases is ionized to G•+ (Tel21+). Once assessed that the electron-hole is localized on a single G, we perform MD simulations of twelve Tel21+ systems, differing in the position of G•+ in the sequence. When G•+ is located in the tetrad adjacent to the diagonal loop, we observe substantial structural rearrangements, which can decrease the electrostatic repulsion with the inner Na+ ions and increase the solvent exposed surface of G•+. Analysis of solvation patterns of G•+ provides new insights on the main reactions of G•+, i.e. the deprotonation at two different sites and hydration at the C8 atom, the first steps of the processes producing 8oxo-Guanine. We suggest the main structural determinants of the relative reactivity of each position and our conclusions, consistent with the available experimental trends, can help rationalizing the reactivity of other G-quadruplex topologies.

Electron Holes in G-Quadruplexes: The Role of Adenine Ending Groups.

The study deals with four-stranded DNA structures (G-Quadruplexes), known to undergo ionization upon direct absorption of low-energy UV photons. Combining quantum chemistry calculations and time-resolved absorption spectroscopy with 266 nm excitation, it focuses on the electron holes generated in tetramolecular systems with adenine groups at the ends. Our computations show that the electron hole is placed in a single guanine site, whose location depends on the position of the adenines at the 3' or 5' ends. This position also affects significantly the electronic absorption spectrum of (G+)● radical cations. Their decay is highly anisotropic, composed of a fast process (<2 µs), followed by a slower one occurring in ~20 µs. On the one hand, they undergo deprotonation to (G-H2)● radicals and, on the other, they give rise to a reaction product absorbing in the 300-500 nm spectral domain.

The Structural Duality of Nucleobases in Guanine Quadruplexes Controls Their Low-Energy Photoionization.

Guanine quadruplexes are four-stranded DNA/RNA structures composed of a guanine core (vertically stacked guanine tetrads) and peripheral groups (dangling ends and/or loops). Such a dual structural arrangement of the nucleobases favors their photoionization at energies significantly lower than the guanine ionization potential. This effect is important with respect to the oxidative DNA damage and for applications in the field of optoelectronics. Photoionization quantum yields, determined at 266 nm by nanosecond transient absorption spectroscopy, strongly depend on both the type and position of the peripheral nucleobases. The highest value (1.5 × 10-2) is found for the tetramolecular structure (AG4A)4 in which adenines are intermittently stacked on the adjacent guanine tetrads, as determined by nuclear magnetic resonance spectroscopy. Quantum chemistry calculations show that peripheral nucleobases interfere in a key step preceding electron ejection: charge separation, initiated by the population of charge transfer states during the relaxation of electronic excited states.