Mechanism-Dependent Modulation of Ultrafast Interfacial Water Dynamics in Intrinsically Disordered Protein Complexes.

The recognition of intrinsically disordered proteins (IDPs) is highly dependent on dynamics owing to the lack of structure. Here we studied the interplay between dynamics and molecular recognition in IDPs with a combination of time-resolving tools on timescales ranging from femtoseconds to nanoseconds. We interrogated conformational dynamics and surface water dynamics and its attenuation upon partner binding using two IDPs, IBB and Nup153FG, both of central relevance to the nucleocytoplasmic transport machinery. These proteins bind the same nuclear transport receptor (Importinß) with drastically different binding mechanisms, coupled folding-binding and fuzzy complex formation, respectively. Solvent fluctuations in the dynamic interface of the Nup153FG-Importinß fuzzy complex were largely unperturbed and slightly accelerated relative to the unbound state. In the IBB-Importinß complex, on the other hand, substantial relative slowdown of water dynamics was seen in a more rigid interface. These results show a correlation between interfacial water dynamics and the plasticity of IDP complexes, implicating functional relevance for such differential modulation in cellular processes, including nuclear transport.

Tuning Stilbene Photochemistry by Fluorination: State Reordering Leads to Sudden Polarization near the Franck-Condon Region.

Spontaneous polarization of a nonpolar molecule upon photoexcitation (the sudden polarization effect) earlier discussed for 90°-twisted alkenes is observed and calculated for planar ring-fluorinated stilbenes, trans-2,3,5,6,2',3',5',6'-octofluorostilbene (tF2356) and trans-2,3,4,5,6,2',3',4',5',6'-decafluorostilbene (tF23456). Due to the fluorination, Franck-Condon states S1FC and S2FC are dominated by the quasi-degenerate HOMO-1 → LUMO and HOMO-2 → LUMO excitations, while their interaction gives rise to a symmetry-broken zwitterionic S1 state. After optical excitation of tF2356, one observes an ultrafast (∼0.06 ps) evolution that reflects relaxation from initial nonpolar S3FC to long-lived (1.3 ns in n-hexane and 3.4 ns in acetonitrile) polar S1. The polarity of S1 is evidenced by a solvatochromic shift of its fluorescence band. The experimental results provide a sensitive test for quantum-chemical calculations. In particular, our calculations agree with the experiment, and raise concerns about the applicability of the common TDDFT approach to relatively simple stilbenic systems.

An Isosteric and Fluorescent DNA Base Pair Consisting of 4-aminophthalimide and 2,4-diaminopyrimidine as C-Nucleosides.

A 13mer DNA duplex containing the artificial 4-aminophthalimide:2,4-diaminopyrimidine (4AP:DAP) base pair in the central position was characterized by optical and NMR spectroscopy. The fluorescence of 4AP in the duplex has a large Stokes shift of Δλ=124 nm and a quantum yield of ΦF =24 %. The NMR structure shows that two interstrand hydrogen bonds are formed and confirms the artificial base pairing. In contrast, the 4-N,N-dimethylaminophthalimide moiety prefers the syn conformation in DNA. The fluorescence intensity of this chromophore in DNA is very low and the NMR structure shows no significant interaction with DAP. Primer-extension experiments with DNA polymerases showed that not only is the 4AP C nucleotide incorporated at the desired position opposite DAP in the template, but also that the polymerase is able to progress past this position to give the full-length product. The observed selectivity supports the NMR results.

ß-Carotene Revisited by Transient Absorption and Stimulated Raman Spectroscopy.

ß-Carotene in n-hexane was examined by femtosecond transient absorption and stimulated Raman spectroscopy. Electronic change is separated from vibrational relaxation with the help of band integrals. Overlaid on the decay of S1 excited-state absorption, a picosecond process is found that is absent when the C9 -methyl group is replaced by ethyl or isopropyl. It is attributed to reorganization on the S1 potential energy surface, involving dihedral angles between C6 and C9 . In Raman studies, electronic states S2 or S1 were selected through resonance conditions. We observe a broad vibrational band at 1770 cm(-1) in S2 already. With 200 fs it decays and transforms into the well-known S1 Raman line for an asymmetric C=C stretching mode. Low-frequency activity (<800 cm(-1) ) in S2 and S1 is also seen. A dependence of solvent lines on solute dynamics implies intermolecular coupling between ß-carotene and nearby n-hexane molecules.

Observations of probe dependence of the solvation dynamics in ionic liquids.

Solvation and rotational dynamics of 4-aminophthalimide (4AP) in four ionic liquids (ILs) are measured using a combination of fluorescence upconversion spectroscopy and time-correlated single photon counting. These data are compared with previously reported data for coumarin 153 (C153) to investigate the probe dependence of solvation dynamics. No fast component (<15 ps) in the fluorescence anisotropy is observed with 4AP. The differences between the solvation response functions of 4AP and C153 are significant in all four ILs, but these differences can be reduced by applying a correction for solute rotation using measured emission anisotropies. Response functions of other probes available in the literature are used to further examine the validity of this correction. The corrected data are also compared to predictions of dielectric continuum models of solvation. By replacing the measured static conductivity of the ILs with an estimated value, such predictions show good agreement with the observed spectral response functions, especially when the anion size is small.

Synthesis of 4-aminophthalimide and 2,4-diaminopyrimidine C-nucleosides as isosteric fluorescent DNA base substitutes.

The 4-aminophthalimide C-nucleoside 1 was designed as an isosteric DNA base surrogate, and a synthetic route to nucleoside 1 together with the 2,4-diaminopyrimidine-C-nucleoside 2 as a potential counterbase was worked out. The key steps in both synthetic routes represent a stereoselective Heck-type palladium-catalyzed cross-coupling with 2'-deoxyribofuranoside glycal followed by stereoselective reduction with NaBH(OAc)3. The nucleoside 1 shows a solvatofluorochromic behavior and significantly red-shifted fluorescence in solvents of high polarity and with hydrogen bonding capabilities. Both nucleosides 1 and 2 can be further processed to the corresponding phosphoramidite as DNA building blocks that allow incorporation of these chromophores as artificial DNA bases by automated DNA synthesis. The combination of the poor stacking properties of 1 and the hydrogen bonding interface at the phthalimide functionality that does not fit to any of natural DNA bases in the counterstrand yields destabilization of the duplex by 4-11 °C. The fluorescence of 1 in a representative double stranded DNA is characterized by a large Stokes' shift and a quantum yield of approximately 12%. These are remarkable optical properties considering the very small size of the chromophore and indicate a high potential of these nucleoside analogues for fluorescent DNA analytics and imaging.

Barrierless photoisomerisation of the "simplest cyanine": joining computational and femtosecond optical spectroscopies to trace the full reaction path.

The photoisomerisation of 1,1'-diethyl-2,2'-pyridocyanine, regarded by Brooker as the simplest cyanine, is examined in methanol by time-resolved experiments and PCM/TD-CAM-B3LYP calculations. Femtosecond transient absorption, fluorescence upconversion, and stimulated Raman scattering, all with broadband coverage, provide a panoramic view of the photoreaction. On the computational side, evolving distributions on an S(1) minimum-energy path are obtained by solving the Smoluchowski equation for drift and diffusion of torsional motion. Absorption and fluorescence bandshapes are calculated and compared to the observations; near-quantitative agreement implies that the entire S(1) path has been observed. Most importantly the global S(1) minimum, i.e. the perpendicular "phantom state" P*, can be identified and characterized in this way. Internal conversion of P* (3.7 ps), assisted by solvent equilibration, leads to the hot ground state. Within 5 ps, vibrational bands of cis and trans isomers are recognized with the help of calculated Raman spectra. The differences between observed and simulated spectra are discussed.

Photoisomerization around a fulvene double bond: coherent population transfer to the electronic ground state?

Photoisomerization around a central fulvene-type double bond is known to proceed through a conical intersection at the perpendicular geometry. The process is studied with an indenylidene-dihydropyridine model compound, allowing the use of visible excitation pulses. Transient absorption shows that 1) stimulated emission shifts to the red and loses oscillator strength on a 50 fs timescale, and 2) bleach recovery is highly nonexponential and not affected by solvent viscosity or methyl substitution at the dihydropyridine ring. Quantum-chemical calculations are used to explain point 1 as a result of initial elongation of the central C=C bond with mixing of S(2) and S(1) states. From point 2 it is concluded that internal conversion of S(1)→S(0) does not require torsional motion to the fully perpendicular state. The S(1) population appears to encounter a sink on the torsional coordinate before the conical intersection is reached. Rate equations cannot model the observed ground-state recovery adequately. Instead the dynamics are best described with a strongly damped oscillatory contribution, which could indicate coherent S(1)-S(0) population transfer.

Polar solvation dynamics in water and methanol: search for molecularity.

Time-dependent Stokes shifts (TDSS) were measured for diverse polarity probes in water, heavy water, methanol, and benzonitrile, by broadband fluorescence up-conversion with 85 fs time resolution. In water the spectral dynamics is solute-independent and quantitatively described by simple dielectric continuum theory of solvation. In methanol the slower part of the TDSS is solute-dependent. A correlation with anisotropy decay suggests that methanol solvation dynamics is modulated by orientational solute diffusion. An empirical power law which links the solvation relaxation function of a mobile solute to that of an immobile solute is experimentally verified. Activation energies for the average relaxation rate are also given. Solvation dynamics in H(2)O and D(2)O are identical at and above 20 °C but diverge below.

Ultrafast internal conversion in a model anthocyanin-polyphenol complex: implications for the biological role of anthocyanins in vegetative tissues of plants.

The red flavylium cations of anthocyanins form ground-state charge-transfer complexes with several naturally occurring electron-donor copigments, such as hydroxylated flavones and hydroxycinnamic or benzoic acids. Excitation of the 7-methoxy-4-methyl-flavylium-protocatechuic acid complex results in ultrafast (240 fs) internal conversion to the ground state of the complex by way of a low-lying charge-transfer state. Thus, both uncomplexed anthocyanins, whose excited state decays by fast (5-20 ps) excited-state proton transfer, and anthocyanin-copigment complexes have highly efficient mechanisms of deactivation that are consistent with the proposed protective role of anthocyanins against excess solar radiation in the vegetative tissues of plants.

Dynamic polar solvation is reported by fluorescing 4-aminophthalimide faithfully despite H-bonding.

Solvation dynamics of 4-aminophthalimide (4AP) in methanol is measured by broadband upconversion of the fluorescence band. The peak emission frequency nu(t) is determined from 100 fs onward with 85 fs time resolution. Polar solvation based on simple continuum theory, including solute polarizability, describes the temporal shape of nu(t) quantitatively. Extrapolation nu(t-->0) points to an initial emission frequency which agrees with the result from stationary spectroscopy in a nonpolar solvent. The extent (4300 cm(-1)) of the dynamic Stokes shift is largely due (50%) to H-bonding, however. The observations imply that H-bonds with 4AP adiabatically follow the dielectric relaxation of the methanol network. The stimulated emission band is also used to measure solvation dynamics. The evolving band is monitored by transient absorption spectroscopy of supercontinuum probe pulses. But the excited-state absorption spectrum, its relative amplitude, and its evolution are needed to extract nu(t) from such measurements. These key data are obtained by comparison with the upconversion results. Thus calibrated photometrically, 4AP transient absorption can be used to monitor solvation dynamics in any solvent. The excited-state absorption spectrum is assigned with the help of time-dependent density-functional calculations. Fluorescence excitation and double-resonance spectroscopy of isolated 4AP, cooled in a supersonic jet, is used to determine optically active modes. An intramolecular reorganization energy is inferred which is consistent with the value in 2-methylbutane (2025 cm(-1)). The crystal structure is also provided.

Synthesis and photophysical properties of 7-deaza-2'-deoxyadenosines bearing bipyridine ligands and their Ru(II)-complexes in position 7.

The synthesis of the title 7-deazaadenine 2'-deoxyribonucleosides bearing bipyridine, phenanthroline or terpyridine ligands linked to position 7 via an acetylene or phenylene spacer is reported based on aqueous cross-coupling reactions of unprotected 7-iodo-7-deaza-2'-deoxyadenosine with ligand-functionalized acetylenes or boronic acids. The aqueous cross-coupling with acetylene or boronate building blocks containing the Ru(bpy)(3)-type of complex gave the corresponding Ru-containing nucleosides. Photophysical and electrochemical properties were studied and the most efficient type of complex was selected for future luminescent and redox labelling of DNA. The title nucleosides also showed some cytostatic and anti-HCV activities.

Solvation oscillations and excited-state dynamics of 2-amino- and 2-hydroxy-7-nitrofluorene and its 2'-deoxyriboside.

Push-pull substituted fluorenes are considered for use as dynamic solvation probes in polynucleotides. Their fluorescence band is predicted (by simulations) to show weak spectral oscillations on the subpicosecond time scale depending on the nucleotide sequence. The oscillations reflect the local far-infrared spectrum of the environment around the probe molecule. A connection is provided by the continuum theory of polar solvation which, however, neglects molecular aspects. We examine the latter using acetonitrile solution as a test case. A collective librational solvent mode at 100 cm(-1) is observed with 2-amino-7-nitrofluorene, 2-dimethylamino-7-nitrofluorene, 2-hydroxy-7-nitrofluorene, and its 2'-deoxyriboside. Different strengths of the oscillation indicate that rotational friction of nearby acetonitrile molecules depends on the solute structure or that H bonding is involved in launching the librational coherence. Polar solvation in methanol is used for comparison. With hydroxynitrofluorenes, the observation window is limited by intersystem crossing for which rates are reported. A prominent excited-state absorption band of nitrofluorenes at 430 nm can be used to monitor polar solvation. Structural and electronic relaxation pathways are discussed with the help of quantum chemical calculations.

Effect of a Tertiary Butyl Group on Polar Solvation Dynamics in Aqueous Solution: Femtosecond Fluorescence Spectroscopy.

We monitor the time-dependent Stokes shift (TDSS) of fluorescence from the zwitterionic probe N-methyl-6-oxyquinolinium betaine in water. A spectral relaxation time τsolv = 0.57 ps (at 20.5 °C) is attributed to a solvation process involving water in the hydration layer. In this article we show that a tertiary butyl group, when attached to the chromophore, slows the dynamics to τsolv = 0.76 ps and increases the corresponding activation energy by 5 kJ/mol. In a companion paper (10.1021/acs.jpcb.7b05039), simulations suggest that the observed slow-down indicates coupling of solute vibrations to hydration water. Thus, a new angle on a thoroughly researched topic, solvation dynamics, has been opened.

Femtosecond broadband fluorescence upconversion spectroscopy: Spectral coverage versus efficiency.

Sum frequency mixing of fluorescence and ∼1300 nm gate pulses, in a thin ß-barium borate crystal and non-collinear type II geometry, is quantified as part of a femtosecond fluorimeter [X.-X. Zhang et al., Rev. Sci. Instrum. 82, 063108 (2011)]. For a series of fixed phasematching angles, the upconversion efficiency is measured depending on fluorescence wavelength. Two useful orientations of the crystal are related by rotation around the surface normal. Orientation A has higher efficiency (factor ∼3) compared to B at the cost of some loss of spectral coverage for a given crystal angle. It should be used when subtle changes of an otherwise stationary emission band are to be monitored. With orientation B, the fluorescence range λF > 420-750 nm is covered with a single setting of the crystal and less gate scatter around time zero. The accuracy of determining an instantaneous emission band shape is demonstrated by comparing results from two laboratories.

Perpendicular State of an Electronically Excited Stilbene: Observation by Femtosecond-Stimulated Raman Spectroscopy.

In the photoisomerization path of stilbene, a perpendicular state P on the S1 potential energy surface is expected just before internal conversion through a conical intersection S1/S0. For decades the observation of P was thwarted by a short lifetime τP in combination with slow population flow over a barrier. But these limitations can be overcome by ethylenic substitution. Following optical excitation of trans-1,1'-dicyanostilbene, P is populated significantly (τP = 27 ps in n-hexane) and monitored by an exited-state absorption band at 370 nm. Here we report stimulated Raman lines of P. The strongest, at 1558 cm-1, is attributed to stretching vibrations of the phenyl rings. Transient electronic states, resonance conditions, and corresponding Raman signals are discussed.