Ultrafast excited-state dynamics of DNA fluorescent intercalators: new insight into the fluorescence enhancement mechanism

The excited-state dynamics of the DNA bisintercalator YOYO-1 and of two derivatives has been investigated using ultrafast fluorescence up-conversion and time-correlated single photon counting. The free dyes in water exist in two forms: nonaggregated dyes and intramolecular H-type aggregates, the latter form being only very weakly fluorescent because of excitonic interaction. The excited-state dynamics of the nonaggregated dyes is dominated by a nonradiative decay with a time constant of the order of 5 ps associated with large amplitude motion around the monomethine bridge of the cyanine chromophores. The strong fluorescence enhancement observed upon binding of the dyes to DNA is due to both the inhibition of this nonradiative deactivation of the nonaggregated dyes and the dissociation of the aggregates and thus to the disruption of the excitonic interaction. However, the interaction between the two chromophoric moieties in DNA is sufficient to enable ultrafast hopping of the excitation energy as revealed by the decay of the fluorescence anisotropy. Finally, these dyes act as solvation probes since a dynamic fluorescence Stokes shift was observed both in bulk water and in DNA. Very similar time scales were found in bulk water and in DNA.     

Ultrafast excited-state dynamics of RNA and DNA C tracts

The excited-state dynamics of the RNA homopolymer of cytosine and of the 18-mer (dC)₁₈ were studied by steady-state and time-resolved absorption and emission spectroscopy. At pH 6.8, excitation of poly(rC) by a femtosecond UV pump pulse produces excited states that decay up to one order of magnitude more slowly than the excited states formed in the mononucleotide cytidine 5′-monophosphate under the same conditions. Even slower relaxation is observed for the hemiprotonated, self-associated form of poly(rC), which is stable at acidic pH. Transient absorption and time-resolved fluorescence signals for (dC)₁₈ at pH 6.8 are similar to ones observed for poly(rC) near pH 4, indicating that hemiprotonated structures are found in DNA C tracts at neutral pH. In both systems, there is evidence for two kinds of emitting states with lifetimes of 100 ps and slightly more than 1 ns. The former states are responsible for the bulk of emission from the hemiprotonated structures. Evidence suggests that slow electronic relaxation in these self-complexes is the result of vertical base stacking. The similar signals from RNA and DNA C tracts suggest a common base-stacked structure, which may be identical with that of i-motif DNA.     

Ultrafast excited-state dynamics of nanoscale near-infrared emissive polymersomes

Formed through cooperative self-assembly of amphiphilic diblock copolymers and electronically conjugated porphyrinic near-infrared (NIR) fluorophores (NIRFs), NIR-emissive polymersomes (50 nm to 50 microm diameter polymer vesicles) define a family of organic-based, soft-matter structures that are ideally suited for deep-tissue optical imaging and sensitive diagnostic applications. Here, we describe magic angle and polarized pump-probe spectroscopic experiments that: (i) probe polymersome structure and NIRF organization and (ii) connect emitter structural properties and NIRF loading with vesicle emissive output at the nanoscale. Within polymersome membrane environments, long polymer chains constrain ethyne-bridged oligo(porphinato)zinc(II) based supermolecular fluorophore (PZn n ) conformeric populations and disperse these PZn n species within the hydrophobic bilayer. Ultrafast excited-state transient absorption and anisotropy dynamical studies of NIR-emissive polymersomes, in which the PZn n fluorophore loading per nanoscale vesicle is varied between 0.1-10 mol %, enable the exploration of concentration-dependent mechanisms for nonradiative excited-state decay. These experiments correlate fluorophore structure with its gross spatial arrangement within specific nanodomains of these nanoparticles and reveal how compartmentalization of fluorophores within reduced effective dispersion volumes impacts bulk photophysical properties. As these factors play key roles in determining the energy transfer dynamics between dispersed fluorophores, this work underscores that strategies that modulate fluorophore and polymer structure to optimize dispersion volume in bilayered nanoscale vesicular environments will further enhance the emissive properties of these sensitive nanoscale probes.     

Ultrafast excited-state dynamics of tetraphenylethylene studied by semiclassical simulation

Detailed simulation study is reported for the excited-state dynamics of photoisomerization of cis-tetraphenylethylene (TPE) following excitation by a femtosecond laser pulse. The technique for this investigation is semiclassical dynamics simulation, which is described briefly in the paper. Upon photoexcitation by a femtosecond laser pulse, the stretching motion of the ethylenic bond of TPE is initially excited, leading to a significant lengthening of ethylenic bond in 300 fs. Twisting motion about the ethylenic bond is activated by the energy released from the relaxation of the stretching mode. The 90 degrees twisting about the ethylenic bond from an approximately planar geometry to nearly a perpendicular conformation in the electronically excited state is completed in 600 fs. The torsional dynamics of phenyl rings which is temporally lagging behind occurs at about 5 ps. Finally, the twisted TPE reverts to the initial conformation along the twisting coordinate through nonadiabatic transitions. The simulation results provide a basis for understanding several spectroscopic observations at molecular levels, including ultrafast dynamic Stokes shift, multicomponent fluorescence, viscosity dependence of the fluorescence lifetime, and radiationless decay from electronically excited state to the ground state along the isomerization coordinate.     

Ultrafast excited-state dynamics of strongly coupled porphyrin/core-substituted-naphthalenediimide dyads

The photophysics and excited-state dynamics of two dyads consisting of either a free-base or a zinc-tetraphenylporphyrin linked through a rigid bridge to a core-substituted naphthalenediimide (NDI) have been investigated by femtosecond-resolved spectroscopy. The absorption and fluorescence spectra differ substantially from those of the individual units, pointing to a substantial coupling and to a delocalisation of the excitation over the whole molecule, as confirmed by quantum chemistry calculations. A strong dependence of their excited-state dynamics on the solvent polarity has been observed. In toluene, the fluorescence quantum yield of the dyads is of the order of a few percent and the main decay channel of the emitting state is proposed as intersystem-crossing to the triplet state. However, in a medium polarity solvent like dichloromethane, the emitting state undergoes charge separation from the porphyrin to the NDI unit within 1-3 ps, and the ensuing charge-separated state recombines in about 10-20 ps. This solvent dependence can be explained by the weak driving force for charge separation in polar solvents and the large electronic coupling between the porphyrin and NDI moieties, making charge separation a solvent-controlled adiabatic process.     

Ab initio excited-state dynamics of the photoactive yellow protein chromophore

The photoisomerization mechanism of the neutral form of the photoactive yellow protein (PYP) chromophore is investigated using ab initio quantum chemistry and first-principles nonadiabatic molecular dynamics (ab initio multiple spawning or AIMS). We identify the nature of the two lowest-lying excited states, characterize the short-time behavior of molecules excited directly to S2, and explain the origin of the experimentally observed wavelength-dependent photoisomerization quantum yield.     

Ultrafast pump-probe study of excited-state charge-transfer dynamics in umecyanin from horseradish root

We have applied femtosecond pump-probe spectroscopy to investigate the excited-state dynamics of umecyanin from horseradish roots, by exciting its 600-nm ligand-to-metal charge-transfer band with a 15-fs pulse and probing over a broad range in the visible region. The decay of the pump-induced ground-state bleaching is modulated by clearly visible oscillations and occurs exponentially with a time constant depending on the observed spectral component of the transmission difference signal, ranging from 270 fs up to 700 fs. The slower decaying process characterizes the spectral component corresponding to the metal-to-ligand charge-transfer transition. The excited-state decay rate is significantly lower than in other blue copper proteins, probably because of the larger energy gap between ligand- and metal-based orbitals in umecyanin. Wavelength dependence of the recovery times could be due to either the excitation of several transitions or the occurrence of intramolecular vibrational relaxation within the excited state. We also find evidence of a hot ground-state absorption, at 700 nm, persisting for several picoseconds. The vibrational coherence induced by the ultrashort pump pulse allows vibrational activity to be observed, mainly in the ground state, as expected in a system with fast excited-state decay. However, we find evidence of a rapidly damped oscillation, which we assign to the excited state. Finally, the Fourier transform of the oscillatory component of the signal presents additional bands in the low-frequency region which are assigned to collective motions of the protein.     

Ultrafast excited-state deactivation and energy transfer in guanine-cytosine DNA double helices

The DNA double helix poly(dGdC).poly(dGdC) is studied by fluorescence upconversion spectroscopy with femtosecond resolution. It is shown that the excited-state relaxation of the duplex is faster than that of the monomeric components dGMP and dCMP. This contrasts with the behavior of duplexes composed exclusively of adenine-thymine base pairs, for which an overall lengthening of the fluorescence lifetimes with respect to that of an equimolar mixture of dAMP and TMP was reported previously. Despite the difference in the excited-state deactivation rate between the two types of duplexes, the signature of ultrafast energy transfer is present in both of them. It is attested by the decrease of fluorescence anisotropy decay of the duplexes on the subpicosecond time scale, where molecular motions are inhibited, and is corroborated by the fact that their steady-state fluorescence spectra do not change with the excitation wavelength. Energy transfer involves excited states delocalized over at least two bases, whose existence is revealed by the UV absorption spectrum of the duplex, clearly different from that of an equimolar spectrum of dGMP and dCMP.     

Ultrafast excited-state dynamics in photochromic N-salicylideneaniline studied by femtosecond time-resolved REMPI spectroscopy

Ultrafast processes in photoexcited N-salicylideneaniline have been investigated with femtosecond time-resolved resonance-enhanced multiphoton ionization spectroscopy. The ion signals via the S(1)(n,pi( *)) state of the enol form as well as the proton-transferred cis-keto form emerge within a few hundred femtoseconds after photoexcitation to the first S(1)(pi,pi( *)) state of the enol form. This reveals that two ultrafast processes, excited-state intramolecular proton transfer (ESIPT) reaction and an internal conversion (IC) to the S(1)(n,pi( *)) state, occur on a time scale less than a few hundred femtoseconds from the S(1)(pi,pi( *)) state of the enol form. The rise time of the transient corresponding to the production of the proton-transferred cis-keto form is within 750 fs when near the red edge of the absorption is excited, indicating that the ESIPT reaction occurs within 750 fs. The decay time of the S(1)(pi,pi( *)) state of the cis-keto form is 8.9 ps by exciting the enol form at 370 nm, but it dramatically decreases to be 1.5-1.6 ps for the excitation at 365-320 nm. The decrease in the decay time has been attributed to the opening of an efficient nonradiative channel; an IC from S(1)(pi,pi( *)) to S(1)(n,pi( *)) of the cis-keto form promotes the production of the trans-keto form as the final photochromic products. The two IC processes may provide opposite effect on the quantum yield of photochromic products: IC in the enol form may substantially reduce the quantum yield, but IC in the cis-keto form increase it.     

Ultrafast excited-state isomerization dynamics of 1,1'-diethyl-2,2'-cyanine studied by four-wave mixing spectroscopy

Excited-state dynamics and solvent-solute interactions of 1,1'-diethyl-2,2'-cyanine iodine (1122C) in alcoholic solutions are investigated using time-integrated three-pulse photon-echo spectroscopy. 1122C serves as a model compound for ultrafast photoinduced isomerization-a key process in the light reception of plants, bacteria, and human vision. The photoreaction in 1122C is interrogated in dependence on solvent and excitation wavelength. The wavelength-dependent three-pulse photon-echo peak shift indicates strong alterations of the reaction pathways and points to the existence of a direct internal conversion channel in close proximity to the Franck-Condon point of absorption. The solvent-dependent S1-S0 internal conversion time does not follow conventional sheared viscosity dependence, suggesting that the solvent local friction has to be considered to account for the observed isomerization kinetics. The concerted discussion of transient grating and three-pulse photon-echo peak-shift data allows us to derive a complete picture of the solvent-solute interaction-controlled photoreaction. The results obtained are related to other work on reactive systems and are discussed in the framework of multilevel response functions.     

Ultrafast excited-state dynamics in vitamin B12 and related cob(III)alamins

Femtosecond transient IR and visible absorption spectroscopies have been employed to investigate the excited-state photophysics of vitamin B12 (cyanocobalamin, CNCbl) and the related cob(III)alamins, azidocobalamin (N3Cbl), and aquocobalamin (H2OCbl). Excitation of CNCbl, H2OCbl, or N3Cbl results in rapid formation of a short-lived excited state followed by ground-state recovery on time scales ranging from a few picoseconds to a few tens of picoseconds. The lifetime of the intermediate state is influenced by the sigma-donating ability of the axial ligand, decreasing in the order CNCbl > N3Cbl > H2OCbl, and by the polarity of the solvent, decreasing with increasing solvent polarity. The peak of the excited-state visible absorption spectrum is shifted to ca. 490 nm, and the shape of the spectrum is characteristic of weak axial ligands, similar to those observed for cob(II)alamin, base-off cobalamins, or cobinamides. Transient IR spectra of the upper CN and N3 ligands are red-shifted 20-30 cm(-1) from the ground-state frequencies, consistent with a weakened Co-upper ligand bond. These results suggest that the transient intermediate state can be attributed to a corrin ring pi to Co 3d(z2) ligand to metal charge transfer (LMCT) state. In this state bonds between the cobalt and the axial ligands are weakened and lengthened with respect to the corresponding ground states.     

Solvent effect on the excited-state dynamics of analogues of the photoactive yellow protein chromophore

We previously reported that two analogues of the Photoactive Yellow Protein chromophore, trans-p-hydroxycinnamic acid (pCA(2-)) and its amide derivative (pCM-) in their deprotonated forms, undergo a trans-cis photoisomerization whereas the thioester derivative, trans-p-hydroxythiophenyl cinnamate (pCT-), does not. pCT- is also the only one to exhibit a short-lived intermediate on its excited-state deactivation pathway. We here further stress the existence of two different relaxation mechanisms for these molecules and examine the reaction coordinates involved. We looked at the effect of the solvent properties (viscosity, polarity, solvation dynamics) on their excited-state relaxation dynamics, probed by ultrafast transient absorption spectroscopy. Sensitivity to the solvent properties is found to be larger for pCT- than for pCA(2-) and pCM-. This difference is considered to reveal that either the relaxation pathway or the reaction coordinate is different for these two classes of analogues. It is also found to be correlated to the electron donor-acceptor character of the molecule. We attribute the excited-state deactivation of analogues bearing a weaker acceptor group, pCA(2-) and pCM-, to a stilbene-like photoisomerization mechanism with the concerted rotation of the ethylenic bond and one adjacent single bond. For pCT-, which contains a stronger acceptor group, we consider a photoisomerization mechanism mainly involving the single torsion of the ethylenic bond. The excited-state deactivation of pCT- would lead to the formation of a ground-state intermediate at the "perp" geometry, which would return to the initial trans conformation without net isomerization.     

Ultrafast dynamics of photoactive yellow protein via the photoexcitation and emission processes

Pump-dump fluorescence spectroscopy was performed for photoactive yellow protein (PYP) at room temperature. The effect of the dump pulse on the population of the potential energy surface of the electronic excited state was examined as depletion in the stationary fluorescence intensity. The dynamic behavior of the population in the electronic excited state was successfully probed in the various combinations of the pump-dump delay, the dump-pulse wavelength, the dump-pulse energy and the observation wavelength. The experimental results were compared with the results obtained by the femtosecond time-resolved fluorescence spectroscopy.     

Ultrafast energy transfer and structural dynamics in DNA

Ultrafast structural dynamics concomitant to excitation energy transfer in DNA has been studied using a pair of pyrene-labeled DNA bases. The temporal evolution of the femtosecond pump-probe spectra reveals the existence of two electronic coupling pathways, through-base stack and through-space, which lead to excitation energy transfer and excimer formation even when the labeled DNA bases are separated by one AT base pair. The electronic coupling which mediates through-base stack energy transfer is so strong that a new absorption band arises in the excited-state absorption spectrum within 300 fs. From the analysis of time-dependent spectral shifts due to through-space excimer formation, the local structural dynamics and flexibility of DNA are characterized on the picosecond and nanosecond time scale.     

Ultrafast excited-state intermolecular proton transfer of cyanine fluorochrome dyes

Steady-state and time-resolved emission spectroscopy techniques were employed to study the excited-state proton transfer (ESPT) to water and D(2)O from QCy7, a recently synthesized near-infrared (NIR)-emissive dye with a fluorescence band maximum at 700 nm. We found that the ESPT rate constant, k(PT), of QCy7 excited from its protonated form, ROH, is ~1.5 × 10(12) s(-1). This is the highest ever reported value in the literature thus far, and it is comparable to the reciprocal of the longest solvation dynamics time component in water, τ(S) = 0.8 ps. We found a kinetic isotope effect (KIE) on the ESPT rate of ~1.7. This value is lower than that of weaker photoacids, which usually have KIE value of ~3, but comparable to the KIE on proton diffusion in water of ~1.45, for which the average time of proton transfer between adjacent water molecules is similar to that of QCy7.     

Ultrafast excited-state dynamics of eosin B: a potential probe of the hydrogen-bonding properties of the environment

The photophysics of two dyes from the xanthene family, eosin B (EB), and eosin Y (EY) has been investigated in various solvents by femtosecond transient absorption spectroscopy, first, to clarify the huge disparity of the EB fluorescence lifetimes reported in literature, and, second, to understand the mechanism responsible for the ultrafast excited-state deactivation of EB in water. The excited-state lifetime of EB was found to be much shorter in water and in other protic solvents, due to the occurrence of hydrogen-bond assisted nonradiative deactivation. This mechanism is associated with the hydrogen bonds between the solvent molecules and the nitro groups of EB, which become stronger upon optical excitation due to the charge-transfer character of the excited-state. This process is not operative with EY, where the nitro groups are replaced by bromine atoms. Therefore, the excited-state lifetime of EB in solution is directly related to the strength of the solvent as a hydrogen-bond donor, offering the possibility to build a corresponding scale based on the fluorescence quantum yield or lifetime of EB. This scale of hydrogen-bonding strength could be especially useful for studies of liquid interfaces by time-resolved surface second harmonic generation.     

Ultrafast excited-state dynamics of adenine and monomethylated adenines in solution: implications for the nonradiative decay mechanism

The DNA base adenine and four monomethylated adenines were studied in solution at room temperature by femtosecond pump-probe spectroscopy. Transient absorption at visible probe wavelengths was used to directly observe relaxation of the lowest excited singlet state (S(1) state) populated by a UV pump pulse. In H(2)O, transient absorption signals from adenine decay biexponentially with lifetimes of 0.18 +/- 0.03 ps and 8.8 +/- 1.2 ps. In contrast, signals from monomethylated adenines decay monoexponentially. The S(1) lifetimes of 1-, 3-, and 9-methyladenine are similar to one another and are all below 300 fs, while 7-methyladenine has a significantly longer lifetime (tau = 4.23 +/- 0.13 ps). On this basis, the biexponential signal of adenine is assigned to an equilibrium mixture of the 7H- and 9H-amino tautomers. Excited-state absorption (ESA) by 9-methyladenine is 50% stronger than by 7-methyladenine. Assuming that ESA by the corresponding tautomers of adenine is unchanged, we estimate the population of 7H-adenine in H(2)O at room temperature to be 22 +/- 4% (estimated standard deviation). To understand how the environment affects nonradiative decay, we performed the first solvent-dependent study of nucleobase dynamics on the ultrafast time scale. In acetonitrile, both lowest energy tautomers of adenine are present in roughly similar proportions as in water. The lifetimes of the 9-substituted adenines depend somewhat more sensitively on the solvent than those of the 7-substituted adenines. Transient signals for adenine in H(2)O and D(2)O are identical. These solvent effects strongly suggest that excited-state tautomerization is not an important nonradiative decay pathway. Instead, the data are most consistent with electronic energy relaxation due to state crossings between the optically prepared (1)pipi* state and one or more (1)npi* states and the electronic ground state. The pattern of lifetimes measured for the monomethylated adenines suggests a special role for the (1)npi* state associated with the N7 electron lone pair.     

Ultrafast vibrational dynamics of adenine-thymine base pairs in DNA oligomers

N-H stretching excitations of DNA oligomers containing 23 alternating adenine-thymine base pairs are studied in femtosecond two-color pump-probe experiments. For a DNA film in a zero relative humidity atmosphere, transient vibrational spectra and their time evolution up to 10 ps demonstrate negligible spectral diffusion and allow for discerning different N-H stretching bands and the O-H stretching absorption of residual water molecules. Lifetimes on the order of 0.5 ps are found for both N-H and O-H stretching modes. The time-dependent pump-probe anisotropies of the different N-H excitations point to a pronounced coupling among them, whereas the O-H stretching anisotropy remains essentially constant.