Biomolecules couple to their aqueous environment through a variety of noncovalent interactions. Local hydration structures at the surface of DNA and RNA are frequently determined by directed hydrogen bonds with water molecules, complemented by non-specific electrostatic and many-body interactions. I will present recent results from 2D-IR spectroscopy of sugar-phosphate backbone vibrations of native and artificial DNA and RNA, together with theoretical calculations of molecular couplings and molecular dynamics simulations. The results reveal the femtosecond fluctuation dynamics of the water shell, a short-range character of Coulomb interactions, and the strength and fluctuation amplitudes of interfacial electric fields [1,2]. Recent applications of phosphate vibrations [3,4,5] as probes for local hydration patterns and contact ion pair configurations hold strong potential for quantifying folding-induced changes of the ion distribution around DNA and RNA on a multitude of time scales.
References: 1. T. Siebert, B. Guchhait, Y. Liu, B. P. Fingerhut, T. Elsaesser, J. Phys. Chem. Lett. 7, 3131 (2016). 2. E. M. Bruening, J. Schauss, T. Siebert, B. P. Fingerhut, T. Elsaesser, J. Phys. Chem. Lett. 9, 583 (2018). 3. J. Schauss, F. Dahms, B. P. Fingerhut, T. Elsaesser, J. Phys. Chem. Lett. 10, 238 (2019). 4. A. Kundu, J. Schauss, B. P. Fingerhut, T. Elsaesser, J. Phys. Chem. B, 124, 2132 (2020). 5. J. Schauss, A. Kundu, B. P. Fingerhut, T. Elsaesser, J. Phys. Chem. B, 125, 740 (2021).

Water plays a vital role in many biological processes like enzyme activity, protein folding-refolding and denaturation. Interfacial water has a significant effect on the protein’s internal structure and dynamics. It is essential to know the time scales characteristic of both, the local protein rearrangements and water dynamics within the solvation shell to understand the protein-water interactions. Urea is a chaotropic agent and a well-known denaturant for proteins. The molecular picture of the interaction of urea with the water hydrogen bond network and thereby, the chemical denaturation of the proteins is still ambiguous. Time-resolved Optical Kerr effect (OKE) spectroscopy is a powerful spectroscopic technique to study the hydrogen-bonded structure and dynamics of complex aqueous systems, in the picosecond time scales. In this study, we have investigated the mechanism behind urea denaturation of three proteins of different hydrophobicities- lysozyme, BSA and trypsin. The OKE data reveals the effect that different concentrations of urea have on the aqueous protein solutions. The temporal profiles of the aqueous protein solutions are almost indistinguishable from that of pure water. Resolving the data in the frequency domain and subtracting the solvent contribution gives us a better picture of the water and water-urea interactions with the protein. The spectral density (SD) obtained contains the α relaxation at the lowest frequencies corresponding to the orientational diffusion of the molecules, linked by the stretched β relaxation to the intermolecular librational modes at terahertz frequencies. The shape of the SDs resembles that of urea solutions; the addition of protein brings down the contribution from the α relaxation. At lower urea concentrations, this change is even more apparent. The OKE spectra of urea have a huge α contribution compared to that of water which masks the faster β dynamics. Removing the diffusive contributions in the time domain itself leaves us with the Reduced Spectral Densities (RSD). Preliminary analysis of the RSDs shows the β relaxation timescales of water changes on the addition of urea and the subtracted spectra for the urea denatured lysozyme shows two distinct β processes characteristic of water and water-urea dynamics. A detailed analysis of the changes in the line shapes of the RSDs is required to elucidate the effect urea has on the water hydrogen bond network and to map out the structural changes occurring in three different proteins on the addition of urea.

The structure and dynamics of molecular liquids and their binary mixtures have been of interest for a long time. Azeotropes is a particular class of liquid mixture that boils at a constant temperature at a specific composition. Understanding of azeotropic systems at the molecular level is limited. In this study, we try to investigate this azeotropic non-ideality, using ultrafast Optical Kerr Effect (OKE) spectroscopy, broadband (1-10 THz) THz-Time domain spectroscopy (THz-TDS) along with temperature-dependent Nuclear Magnetic Resonance spectroscopy (NMR) of the azeotropic as well as mixtures of different compositions of benzene and methanol. Vibrational and NMR spectroscopic studies show that the formation of the methanol-benzene azeotrope weakens the hydrogen bond network. Intermolecular forces between the benzene molecules are also reduced significantly. Methanol disrupts the stacking in benzene which is also evident from the depression in the boiling points. Ultrafast OKE spectroscopy is a powerful tool to probe both, the collective orientational diffusion and the intermolecular dynamics in liquids. The spectral density (SD) obtained by the Fourier deconvolution of the OKE time transients gives us information about the structural relaxation occurring in the liquid at the terahertz and sub terahertz frequencies. With the increase of methanol in the mixtures, the α relaxation timescales become faster. An azeotropic composition mixture was prepared at room temperature without further distillation. The spectral densities of this mixture and the azeotrope at frequencies pertaining to collective intermolecular dynamics were quite different from each other. All of benzene’s entire rotational dynamics show up as molecular reorientation having contributions throughout the OKE spectra. The centre-of-mass translational part is only visible when it affects the interaction induced (I-I) part of the polarizabilityRyu, S.; Stratt, R. M. J. Phys. Chem. B 2004, 108, 6782. The addition of methanol decreases this contribution of the translations to the I-I term for benzene. A detailed analysis of the similarity of the azeotrope spectral density to benzene and how they differ from the other composition mixtures can give us useful insights into the structural fluctuations happening in the picosecond timescales in the benzene-methanol mixtures.

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Footnotes:

Ryu, S.; Stratt, R. M. J. Phys. Chem. B 2004, 108, 6782..

The acoustic spectra of furfural were studied in the frequency range from 0.1 MHz to 150 MHz and at temperatures 303 - 333 K by the coaxial resonator method using two different cells to cover the whole frequency range. The temperature in the cells was controlled with an accuracy of 0.05 degrees. The accuracy of the determination of the attenuation of acoustic waves in liquid furfural was approximately 5%. The studies were shown that the obtained experimental data can be explained with help of two relaxation processes. One relaxation process is observed in the low-frequency range of frequencies (  0.2 MHz), and the second relaxation process is observed in the frequency range of about 2 MHz. The process with a lower relaxation frequency can be associated with the internal rotation of the furfural molecules “X0-cis” and “X0-trans”. The kinetic analysis of acoustic data about conformation processes for a low-frequency relaxation process in pure furfural was carried out by using Eyring relations. The relaxation parameters and the velocity of acoustic waves, as well as the contribution of the different factors to the attenuation of acoustic waves have been determined from experimental results.

Polaritons are hybrid light-matter states that can be created by placing a molecule inside an optical cavity due to strong coupling between confined electromagnetic modes and optical transitions of the molecule, and can have different energies, coherence, and vibrational characteristics than the bare molecules outside of the cavity. The strong coupling results in new physicochemical properties of the hybrid system including their reactivity or charge and energy transfer characteristics and has recently gained significant research interest. We have used two-dimensional electronic spectroscopy (2DES) to study the dynamics of the copper phthalocyanine (CuPc) molecules placed inside an optical cavity. The 2DES is a powerful tool to investigate excited state dynamics that enables a direct correlation between excitation and detection energies of the system with ultrafast time resolution. We compare the dynamics of the CuPc molecules that are inside an optical cavity with that of the bare molecules outside the cavity under similar experimental conditions to understand how the optical cavity modifies the excited state dynamics of the molecules. The coupling between light and the optical transition of the material inside cavity can be controlled by changing the incident angle of the light. We demonstrate this by repeating 2DES experiments on a cavity sample at normal and 75 degree incidence of the pump pulse. The results show that the presence of an optical cavity can significantly modify the excited state dynamics of the molecules and enhance the coherence.

Molecules excited by strong-field laser pulses evolve through coupled nuclear and electronic motion over ultrafast timescales. Even simple molecules demonstrate a variety of characteristic dynamics, including vibrations, fragmentation, and ionization. The final products and their relative yields depend sensitively on details of these coupled processes that occur within the first few femtoseconds of interacting with the laser beam. Here, ultrafast pump-probe spectroscopy is applied to study the fragmentation and ionization dynamics of several molecules. Photodissociation of CO₂ proceeds via multiple pathways depending on available energy, but commonly into CO and O. Multiphoton excitation from a 35 fs pump laser impulsively transfers CO₂ from the ground to 1st excited state, preparing a bending vibrational wavepacket that influences its dissociation. Using mass spectrometry, we report changes in the fragmentation pattern as a function of time delay between the pump and probe laser pulses that reflect the vibrational motion. At well-defined time delays the dissociation oscillates between observable CO⁺ and O₂⁺ fragments. The state relaxes to the ground state through a conical intersection. Similar control over the fragmentation pathway and ion yields will be demonstrated for the excited state motions of ethanol and formic acid.

Nitrobenzene is often used as a model molecule for the study of the dissociation dynamics of nitroaromatic energetic materials. The potential energy surfaces for the ground cationic state D₀ and the first ten excited states D₁ through D₁₀ were calculated as a function of the C-NO₂ torsional angle using time-dependent density functional theory. These surfaces were employed in the prediction of the most efficient probe wavelength for femtosecond time-resolved mass spectrometry measurements. It was found that the D₀→ D₄ transition in nitrobenzene cation has a geometry-dependent oscillator strength, reaching a maximum at 90^° C-NO₂ torsional angle, with a corresponding energy gap of  ∼ 2 eV. These results are consistent with the experimental observation of a vibrational wave packet along the C-NO₂ torsional mode in nitrobenzene cation. Time-resolved measurements using a probe wavelength of 650 nm, nearly resonant with the strong D₀→ D₄ transition, result in enhanced ion yield oscillation amplitudes as compared to excitation with the nonresonant 800 nm wavelength. These results demonstrate that computational chemistry can predict the best choice of probe wavelength in time-resolved measurements of vibrational coherent states in molecular cations.

Multiply charged diatomic molecules are exotic gas-phase species and require high amounts of energy to be produced. Fundamental questions regarding the structures, stabilities, and bonding schemes of small multiply charged ions tests our concepts of bonding. Although uncommon in most chemical settings, the conditions found within the Earth’s ionosphere enable such species to be constantly produced, albeit for short periods of time, through the interaction between small atmospheric molecules and cosmic radiation in the extreme ultraviolet regime. We have performed laboratory experiments using intense laser pulses to investigate the stability and production of multiply charged ions from small atmospherically relevant molecules. I will present our recent work where femtosecond laser pulses are utilized to drive multiple ionization in molecular gas-phase formic acid dimers and studied using time-of-flight mass spectrometry. The interaction of formic acid dimer with 200 fs linearly polarized laser pulses of 400 nm with intensities up to 3.7x10¹⁵ W/cm² produces a carbon monoxide dication and trication. An enhanced ionization is observed in CO ions when the source analyte was switched from the molecular beam to formic acid clusters. Measurements of the kinetic energy release resulting from the Coulomb explosion of clusters are in excellent agreement with our simulations performed over the clusters observed in the mass spectra and suggest that almost no movement occurs during the ionization mechanism. Lastly, potential energy curves for COⁿ⁺, for n < 4, have been calculated using high level theory, confirming the existence of a metastable state with a large potential barrier with respect to dissociation of several eV.

Determining the mechanism by which Cytochrome P450s (P450s) can catalyze oxidation reactions of substrates with differing specificity and regioselectivity is crucial for aiding drug development and understanding drug metabolism. Cytochrome P450cam (P450cam), a model P450, catalyzes the hydroxylation of d-camphor to 5-exo-hydroxycamphor with high specificity and regioselectivity. P450cam can also act upon camphor-like analogs at the expense of regioselectivity. To investigate the contribution of conformational dynamics to varying regioselectivity of hydroxylation, we characterized specific locations throughout P450cam when the enzyme was in complex with camphor or norcamphor, substrates acted upon with 100% and 45% regioselectivity respectively. Linear and two-dimensional IR spectroscopy were applied to measure P450cam side-chain dynamics with high spatial and temporal resolution. Cyanophenylalanine was used as a vibrational probe and incorporated in five distinct locations of P450cam, three sites in the active site and two progressively distal from the active site. The results suggest norcamphor binding does not induce the same large-scale conformational change associated with the closed enzyme state found for the camphor-bound complex. Additionally, probes located in the active site of the enzyme report distinct, localized changes that, in some cases, can be directly correlated to hydroxylation product distribution. Overall, this study illustrates the utility of site-selective infrared spectroscopy to address questions of functional protein dynamics.

We study the dynamics of a system searching for minutes to hours to establish a population of nuclei, which can then go on to create a phase change, following the rise of an exciton in thin films of solid ammonia with deposition temperatures T_d = 48 K, 50 K and 52 K. This behaviour is tracked by following the growth of the exciton, using vacuum ultraviolet absorption spectra of ices at 194.4 nm in the ùA₂" ← ~X¹A' band. Absorbance is observed to increase through an order of magnitude between Td = 48K to 52K, through greater flexing of the solid state structure, as the size of crystallites expands from an average of 10± 2 unit cells at 48K to 34± 8 at 52K. Time delays, associated with nucleation, are encountered before the appearance of exciton absorption, varying between 7870 seconds at 48K to 120 seconds at 52K, with rates of subsequent exciton absorbance growth between 1.49 x 10⁻⁶ s⁻¹ and 1.19 x 10⁻⁴ s⁻¹. Activation energies of 21.7± 0.2 kJ mol⁻¹ for the nucleation process and 22.8± 0.2 kJ mol⁻¹ for the phase change are derived, corresponding to the breaking of two to three hydrogen bonds. Results demonstrate a new means to track nucleation and recrystallization rates in polymorphic systems.

Multidimensional nonlinear spectroscopic methods are powerful tools to investigate ultrafast vibrational dynamics and coherences. Several techniques that have recently been applied to excited electronic states require three or more ultrashort pulses to measure the nonlinear optical response. The additional beamsplitters, mirrors and optical delay stages in these techniques complicates their application in the extreme ultraviolet and X-ray regime, where reflectance is low except at grazing incidence. Optical Kerr effect spectroscopy is a traditional two-pulse technique to measure the 3^rd-order nonlinear optical susceptibility of molecules in their ground electronic state. Ultrafast transient polarization spectroscopy (UTPS) is an extension of this technique to excited electronic states, enabling coupled electronic and nuclear dynamics to be investigated by the 3^rd-order nonlinear optical susceptibility. In UTPS, a pump pulse excites a population, then a Kerr gate and probe pulse monitor the 3^rd-order nonlinear response of excited-state molecules, as a function of delay between each of the three pulses. Two-photon absorption of femtosecond pump pulses populate the S₁ excited-state of nitrobenzene. We were able to extract a dephasing time that oscillates as function of the time delay after excitation. This dephasing time and supporting theoretical calculation allows for distinguishing between an intersystem crossing conical intersection and an internal conversion conical intersection on the same excited-state potential energy surface.

Molecules with ultrafast dynamics inherently have broad spectral features and modeling ultrafast spectroscopy data is a complex art. The complexity is reduced in the gas phase, however most ultrafast spectroscopies are limited to high-density samples such as liquids. By using a frequency comb laser and optical cavities, we present a technique for performing ultrafast optical spectroscopy with high sensitivity, enabling work in dilute gas-phase molecules and clustersM. A. R. Reber, Y. Chen, T. K. Allison, Optica 3, 311 (2016) This sensitivity enables ultrafast spectroscopy in dilute molecular beams, where cold isolated designer molecules, radicals, and clusters can be produced that do not exist in solution. This new technique enables direct comparisons to well-established ultrafast techniques which can provide additional insight for theoretical models and molecular dynamics. In this talk we will introduce our broadband cavity-enhanced r0ptFigure transient absorption spectrometerM. C. Silfies, G. Kowzan, N. Lewis, and T. K. Allison, arXiv:2102.04981 (2021) which has a UV pump at 355 nm and a probe tunable from 450 to 700 nm, and discuss measurements of chemical dynamics of molecules in seeded molecular beams. Example systems discussed will include proton transfer in 1’-hydroxy 2’-acetonapthone and the effects of argon clustering on the internal conversion of salicylidene aniline.

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Footnotes:

M. A. R. Reber, Y. Chen, T. K. Allison, Optica 3, 311 (2016). M. C. Silfies, G. Kowzan, N. Lewis, and T. K. Allison, arXiv:2102.04981 (2021),