Electronic Relaxation Dynamics of UV-Photoexcited 2-Aminopurine-Thymine Base Pairs in Watson-Crick and Hoogsteen Conformations.

The fluorescent analogue 2-aminopurine (2AP) of the canonical nucleobase adenine (6-aminopurine) base-pairs with thymine (T) without disrupting the helical structure of DNA. It therefore finds frequent use in molecular biology for probing DNA and RNA structures and conformational dynamics. However, detailed understanding of the processes responsible for fluorescence quenching remains largely elusive on a fundamental level. Although attempts have been made to ascribe decreased excited-state lifetimes to intrastrand charge-transfer and stacking interactions, possible influences from dynamic interstrand H-bonding have been widely ignored. Here, we investigate the electronic relaxation of UV-excited 2AP·T in Watson-Crick (WC) and Hoogsteen (HS) conformations. Although the WC conformation features slowed-down, monomer-like electronic relaxation in τ ∼ 1.6 ns toward ground-state recovery and triplet formation, the dynamics associated with 2AP·T in the HS motif exhibit faster deactivation in τ ∼ 70 ps. As recent research has revealed abundant transient interstrand H-bonding in the Hoogsteen motif for duplex DNA, the established model for dynamic fluorescence quenching may need to be revised in the light of our results. The underlying supramolecular photophysical mechanisms are discussed in terms of a proposed excited-state double-proton transfer as an efficient deactivation channel for recovery of the HS species in the electronic ground state.

Efficient intersystem crossing in 2-aminopurine riboside probed by femtosecond time-resolved transient vibrational absorption spectroscopy.

The photophysical dynamics of 2-aminopurine riboside (2APr) in CHCl3 have been studied following excitation at λpump = 310 nm by means of femtosecond transient vibrational absorption spectroscopy (TVAS) aided by quantum chemical density functional theory (DFT) and ab initio calculations. The experiments identified numerous vibrational marker bands in the regions of the NH2 stretch and the 2AP ring vibrations which could be assigned to the bleach of the S0 electronic ground state (GS) and to transient populations in the 1ππ* and 3ππ* excited electronic states. The temporal evolution of the transient vibrational bands shows that the decay of the 1ππ* population is accompanied by a partial recovery of the GS and a concurrent population of the 3ππ* state with a time constant of τ2 = 740 ± 15 ps. The ensuing electronic relaxation is concluded to proceed via the 1nπ* state as intermediate state. The absence of observable transient vibrational bands of this state hints at an upper limit for its lifetime of τ < 100 ps. The triplet quantum yield is found to be φT = 0.42 ± 0.07.

Ultrafast dynamics of the ESIPT photoswitch N-(3-pyridinyl)-2-pyridinecarboxamide.

Molecular switches based on proton transfer that are photochromic and can be interconverted by light at different wavelengths back and forth between two thermodynamically stable tautomeric states in solution at room temperature are rare to date. We report on a study of the ultrafast conversion of the bistable proton transfer switch N-(3-pyridinyl)-2-pyridinecarboxamide (NPPCA) to a corresponding iminol after photoexcitation at λpump ≈ 265 nm by means of femtosecond time-resolved broad-band and single-colour transient electronic absorption spectroscopy (TEAS), transient fluorescence spectroscopy (TFLS), and transient vibrational absorption spectroscopy (TVAS) in acetonitrile solution. The interpretation of the data was accompanied by ab initio quantum chemical calculations of the excited electronic states and the vibrational frequencies of the reactant and product in their ground electronic state. The TEAS experiments provided four time constants, τ1 = 0.09 ± 0.01 ps, τ2 = 0.61 ± 0.01 ps, τ3 = 5.10 ± 0.80 ps, and τ4 = 20.0 ± 1.0 ps. The first two agree well with the measured TFLS lifetimes, τ1,TFL < 0.18 ps and τ2,TFL = 0.50 ± 0.01 ps. τ1 is related to the relaxation of the initially excited Franck-Condon (FC) state of the pyridinecarboxamide, followed by the excited-state intramolecular proton transfer (ESIPT) step to the neighbouring pyridine. The subsequent return of the molecules to the electronic ground state takes place within τ2, mediated by a conical intersection (CI) at a twisted configuration of the pyridinecarboxamide moiety. The main components in all TEAS time profiles feature a rise with τ2 and a decay with τ4 and describe subsequent molecular transformations in the electronic ground state. τ3 is ascribed to vibrational cooling of the molecules. The final iminol exhibits a permanent UV absorption at λ = 247 nm, where its absorbance is stronger than that of the carboxamide reactant. The iminol structure is unambiguously identified by the TVA spectra, which show the build-up of corresponding vibrational bands with τ4,TVA = 23 ± 2 ps after the initial bleach of the reactant vibrational bands, in excellent agreement with the TEAS data. Its lifetime is >10 ns.

Is UV-Induced Electron-Driven Proton Transfer Active in a Chemically Modified A·T DNA Base Pair?

Transient electronic and vibrational absorption spectroscopies have been used to investigate whether UV-induced electron-driven proton transfer (EDPT) mechanisms are active in a chemically modified adenine-thymine (A·T) DNA base pair. To enhance the fraction of biologically relevant Watson-Crick (WC) hydrogen-bonding motifs and eliminate undesired Hoogsteen structures, a chemically modified derivative of A was synthesized, 8-(tert-butyl)-9-ethyladenine (8tBA). Equimolar solutions of 8tBA and silyl-protected T nucleosides in chloroform yield a mixture of WC pairs, reverse WC pairs, and residual monomers. Unlike previous transient absorption studies of WC guanine-cytosine (G·C) pairs, no clear spectroscopic or kinetic evidence was identified for the participation of EDPT in the excited-state relaxation dynamics of 8tBA·T pairs, although ultrafast (sub-100 fs) EDPT cannot be discounted. Monomer-like dynamics are proposed to dominate in 8tBA·T.

Ultrafast Electronic Deactivation Dynamics of Xanthosine Monophosphate.

Ultrafast energy dissipation is a crucial factor for the photostability of DNA and RNA, but even some of the key electronic deactivation pathways in monomeric nucleic acid building stones are still controversial. Here, we report on the excited-state dynamics of the rare nucleotide xanthosine monophosphate as a function of deprotonation state (XMP vs. XMP - ) and excitation wavelength ( λ pump = 278-243 nm) by femtosecond time-resolved fluorescence and absorption spectroscopy. We show that the predominating relaxation channel leads to a return of the photo-excited molecules to the electronic ground state in τ∼1 ps. The mechanism likely involves an out-of-plane deformation of the five-membered ring, different from the main electronic deactivation pathways in the canonical purine bases adenine and guanine. The results are discussed in terms of the structural and electronic differences of XMP compared to the canonical nucleotides.

Probing the excited state relaxation dynamics of pyrimidine nucleosides in chloroform solution.

Ultrafast transient electronic and vibrational absorption spectroscopy (TEAS and TVAS) of 2'-deoxy-cytidine (dC) and 2'-deoxy-thymidine (dT) dissolved in chloroform examines their excited-state dynamics and the recovery of ground electronic state molecules following absorption of ultraviolet light. The chloroform serves as a weakly interacting solvent, allowing comparisons to be drawn with prior experimental studies of the photodynamics of these nucleosides in the gas phase and in polar solvents such as water. The pyrimidine base nucleosides have some propensity to dimerize in aprotic solvents, but the monomer photochemistry can be resolved clearly and is the focus of this study. UV absorption at a wavelength of 260 nm excites a 1ππ* ← S0 transition, but prompt crossing of a significant fraction (50% in dC, 17% in dT) of the 1ππ* population into a nearby 1nπ* state is too fast for the experiments to resolve. The remaining flux on the 1ππ* state leaves the vertical Franck-Condon region and encounters a conical intersection with the ground electronic state of ethylenic twist character. In dC, the 1ππ* state decays to the ground state with a time constant of 1.1 ± 0.1 ps. The lifetime of the 1nπ* state is much longer in the canonical forms of both molecules: recovery of the ground state population from these states occurs with time constants of 18.6 ± 1.1 ps in amino-oxo dC and ∼114 ps in dT, indicating potential energy barriers to the 1nπ*/S0 conical intersections. The small fraction of the imino-oxo tautomer of dC present in solution has a longer-lived 1nπ* state with a lifetime for ground state recovery of 193 ± 55 ps. No evidence is found for photo-induced tautomerization of amino-oxo dC to the imino-oxo form, or for population of low lying triplet states of this nucleoside. In contrast, ∼8% of the UV-excited dT molecules access the long-lived T1 (3ππ*) state through the 1nπ* state. The primary influence of the solvent appears to be the degree to which it destabilizes the states of 1nπ* character, with consequences for the lifetimes of these states as well as the triplet state yields.

Ultraviolet Absorption Induces Hydrogen-Atom Transfer in G⋠C Watson-Crick DNA Base Pairs in Solution.

Ultrafast deactivation pathways bestow photostability on nucleobases and hence preserve the structural integrity of DNA following absorption of ultraviolet (UV) radiation. One controversial recovery mechanism proposed to account for this photostability involves electron-driven proton transfer (EDPT) in Watson-Crick base pairs. The first direct observation is reported of the EDPT process after UV excitation of individual guanine-cytosine (G⋅C) Watson-Crick base pairs by ultrafast time-resolved UV/visible and mid-infrared spectroscopy. The formation of an intermediate biradical species (G[-H]⋅C[+H]) with a lifetime of 2.9 ps was tracked. The majority of these biradicals return to the original G⋅C Watson-Crick pairs, but up to 10% of the initially excited molecules instead form a stable photoproduct G*⋅C* that has undergone double hydrogen-atom transfer. The observation of these sequential EDPT mechanisms across intermolecular hydrogen bonds confirms an important and long debated pathway for the deactivation of photoexcited base pairs, with possible implications for the UV photochemistry of DNA.

Ultrafast electronic deactivation dynamics of the inosine dimer--a model case for H-bonded purine bases.

The structural properties and ultrafast electronic deactivation dynamics of the inosine dimer in CHCl3 have been investigated by two-dimensional (1)H NMR and static FTIR spectroscopy and by femtosecond time-resolved transient absorption spectroscopy, respectively. The (1)H NMR and IR spectra show the formation of a well-defined, symmetric dimer with an association equilibrium constant of KI·I = 690 ± 100 M(-1). The excited-state dynamics after photoexcitation at λpump = 260 nm monitored by ultrafast absorption spectroscopy show great similarity with those of the monomer inosine in an aqueous solution and are governed by a decay time of τ = 90 ± 10 fs, which is one of the shortest electronic lifetimes of all nucleobases and nucleobase dimers studied so far. On the basis of these observations, the inosine dimer is expected to follow a similar relaxation pathway as the monomer, involving an out-of-plane deformation of the six-membered ring. The importance of the C(2) position for the electronic deactivation of hypoxanthine and guanine is discussed. The obtained well-determined structure and straightforward dynamics qualify the inosine dimer as an excellent reference case for more complicated systems such as the G·G dimer and the G·C and A·T Watson-Crick pairs.

Electronic deactivation of guanosine in extended hydrogen-bonded self-assemblies.

Guanosine (G) derivatives in nonpolar aprotic solvents self-assemble to intricate hydrogen-bonded supramolecular architectures, including dimers, ribbons, and cyclic quartets. Considerable interest exists in the nature of the excited electronic states, their lifetimes and the radiationless deactivation mechanisms of the molecules in those environments. Here, we report on the electronic relaxation of G in the extended H-bridged networks in solution in n-hexane. The resulting architectures were sampled by FTIR, UV, and CD spectroscopies. The dynamics after 260 nm photoexcitation were investigated by femtosecond fluorescence up-conversion, broadband UV-vis absorption, and single-color deep-UV measurements. The observed temporal profiles reveal a hierarchy of relaxation processes, with lifetimes τ1 = 0.63 ± 0.03 ps, τ2 = 5.9 ± 0.3 ps, and τ3 = 62 ± 7 ps. Moreover, about 10% of the photoexcited molecules transform to much longer-lived product states with lifetime τ4 ≈ 3.6 ± 1.0 ns. These excited-state lifetimes are much longer than in the G monomer or the G·G dimers studied previously, hinting at sizable energy shifts among the excited ππ* and nπ* states and trapping of excited-state population in the supramolecular networks by potential energy barriers along the optimal electronic deactivation pathways of the molecules.

N-H stretching vibrations of guanosine-cytidine base pairs in solution: ultrafast dynamics, couplings, and line shapes.

Dynamics and couplings of N-H stretching vibrations of chemically modified guanosine-cytidine (G·C) base pairs in chloroform are investigated with linear infrared spectroscopy and ultrafast two-dimensional infrared (2D-IR) spectroscopy. Comparison of G·C absorption spectra before and after H/D exchange reveals significant N-H stretching absorption in the region from 2500 up to 3300 cm(-1). Both of the local stretching modes ν(C)(NH(2))(b) of the hydrogen-bonded N-H moiety of the cytidine NH(2) group and ν(G)(NH) of the guanosine N-H group contribute to this broad absorption band. Its complex line shape is attributed to Fermi resonances of the N-H stretching modes with combination and overtones of fingerprint vibrations and anharmonic couplings to low-frequency modes. Cross-peaks in the nonlinear 2D spectra between the 3491 cm(-1) free N-H oscillator band and the bands centered at 3145 and 3303 cm(-1) imply N-H···O═C hydrogen bond character for both of these transitions. Time evolution illustrates that the 3303 cm(-1) band is composed of a nearly homogeneous band absorbing at 3301 cm(-1), ascribed to ν(G)(NH(2))(b), and a broad inhomogeneous band peaking at 3380 cm(-1) with mainly guanosine carbonyl overtone character. Kinetics and signal strengths indicate a <0.2 ps virtually complete population transfer from the excited ν(G)(NH(2))(b) mode to the ν(G)(NH) mode at 3145 cm(-1), suggesting lifetime broadening as the dominant source for the homogeneous line shape of the 3301 cm(-1) transition. For the 3145 cm(-1) band, a 0.3 ps population lifetime was obtained.

Sequential photoisomerisation dynamics of the push-pull azobenzene Disperse Red 1.

The ultrafast dynamics of the push-pull azobenzene Disperse Red 1 following photoexcitation at λ(pump) = 475 nm in solution in 2-fluorotoluene have been probed by broadband transient absorption spectroscopy and fluorescence up-conversion spectroscopy. The measured two-dimensional spectro-temporal absorption map features a remarkable "fast" excited-state absorption (ESA) band at λ ≈ 570 nm appearing directly with the excitation laser pulse and showing a sub-100 fs lifetime with a rapid spectral blue-shift. Moreover, its ultrafast decay is paralleled by rising distinctive ESA at other wavelengths. Global fits to the absorption-time profiles using a consecutive kinetic model yielded three time constants, τ(1) = 0.08 ± 0.03 ps, τ(2) = 0.99 ± 0.02 ps, and τ(3) = 6.0 ± 0.1 ps. Fluorescence-time profiles were biexponential with time constants τ(1)' = 0.12 ± 0.06 ps and τ(2)' = 0.70 ± 0.10 ps, close to the absorption results. Based on the temporal evolution of the transient spectra, especially the "fast" excited-state absorption band at λ ≈ 570 nm, and on the global kinetic analysis of the time profiles, τ(1) is assigned to an ultrafast transformation of the optically excited ππ* state to an intermediate state, which may be the nπ* state, τ(2) to the subsequent isomerisation and radiationless deactivation time to the S(0) electronic ground state, and τ(3) to the eventual vibrational cooling of the internally "hot" S(0) molecules.

Dynamics and couplings of N-H stretching excitations of guanosine-cytidine base pairs in solution.

N-H stretching vibrations of hydrogen-bonded guanosine-cytidine (G·C) base pairs in chloroform solution are studied with linear and ultrafast nonlinear infrared (IR) spectroscopy. Assignment of the IR-active bands in the linear spectrum is made possible by combining structural information on the hydrogen bonds in G·C base pairs with literature results of density functional theory calculations, and empirical relations connecting frequency shifts and intensity of the IR-active vibrations. A local mode representation of N-H stretching vibrations is adopted, consisting of ν(G)(NH(2))(f) and ν(C)(NH(2))(f) modes for free NH groups of G and C, and of ν(G)(NH(2))(b), ν(G)(NH), and ν(C)(NH(2))(b) modes associated with N-H stretching motions of hydrogen-bonded NH groups. The couplings and relaxation dynamics of the N-H stretching excitations are studied with femtosecond mid-infrared two-dimensional (2D) and pump-probe spectroscopy. The N-H stretching vibrations of the free NH groups of G and C have an average population lifetime of 2.4 ps. Besides a vibrational population lifetime shortening to subpicosecond values observed for the hydrogen-bonded N-H stretching vibrations, the 2D spectra reveal vibrational excitation transfer from the ν(G)(NH(2))(b) mode to the ν(G)(NH) and/or ν(C)(NH(2))(b) modes. The underlying intermode vibrational couplings are on the order of 10 cm(-1).