Structural Characterization of Pyruvic Acid Dimers Formed inside Helium Nanodroplets by Infrared Spectroscopy and Ab Initio Study.

The structural arrangements of α-keto acid complexes hold significant interest across various fields of chemistry such as enzyme modeling, drug design, or polymer blending. Herein, we report mass-selective infrared (IR) spectra of pyruvic acid monomers and dimers in the range 1720-1820 cm-1 recorded in helium nanodroplets at 0.37 K. The monomer features IR bands at 1807.1 and 1734.5 cm-1, which are assigned to the carboxylic and ketonic C═O stretching vibrations, respectively. Furthermore, the pyruvic acid dimers generated inside the helium nanodroplets are characterized by carboxylic and ketonic C═O stretch vibrations appearing at 1799.2 and 1737.0 cm-1, respectively. This frequency shift of ±7 cm-1 for both C═O stretching bands from the monomer to the dimer demonstrates that the structural motif of the monomer is maintained upon dimer aggregation in helium nanodroplets. The structural assignments were supported by a comparison of the MP2/aug-cc-pVDZ-predicted harmonic vibrational spectra at the C═O stretching region with the experiments. The global minimum monomer structure with an intramolecular hydrogen bond and its dimer stabilized by both inter- and intramolecular hydrogen bonding interactions reproduce the experimental spectra from the monomer and dimer. This assigned dimer structure lies ca.11 kJ/mol above the corresponding global minimum and is favored in helium nanodroplets due to the long-range realignment of molecules via dipole-dipole interaction, followed by short-range stabilization upon intermolecular hydrogen bond formation. The barrier for reconfiguration of the precooled monomer conformer leading to the formation of the most stable dimer structure is around 58 kJ/mol, which is infeasible at 0.37 K.

Tuning Acid-Base Chemistry at an Electrified Gold/Water Interface.

Acid-base reactions are ubiquitous in solution chemistry, as well as in electrochemistry. However, macroscopic concepts derived in solutions, such as pKa and pH, differ significantly at electrified metal-aqueous interfaces due to specific solvation and applied voltage. Here, we measure the pKa values of an amino acid, glycine, at a gold/water interface under a varying applied voltage by means of spectroscopic titration. With the help of simulations, we propose a general model to understand potential-dependent shifts in pKa values in terms of local hydrophobicity and electric fields. These parameters can be tuned by adjusting the metal surface and applied voltage, respectively, offering promising, but still unexplored, paths to regulate reactivity. Our results change the focus with respect to common interpretations based on, for example, apparent local pH effects and open interesting perspectives for electrochemical reaction steering.

Bridging the Gap in Cryopreservation Mechanism: Unraveling the Interplay between Structure, Dynamics, and Thermodynamics in Cryoprotectant Aqueous Solutions.

Cryoprotectants play a crucial role in preserving biological material, ensuring their viability during storage and facilitating crucial applications such as the conservation of medical compounds, tissues, and organs for transplantation. However, the precise mechanism by which cryoprotectants modulate the thermodynamic properties of water to impede the formation and growth of ice crystals, thus preventing long-term damage, remains elusive. This is evident in the use of empirically optimized recipes for mixtures that typically contain DMSO, glycerol, and various sugar constituents. Here, we use terahertz calorimetry, Overhauser nuclear polarization, and molecular dynamics simulations to show that DMSO exhibits a robust structuring effect on water around its methyl groups, reaching a maximum at a DMSO mole fraction of XDMSO = 0.33. In contrast, glycerol exerts a smaller water-structuring effect, even at higher concentrations (Scheme 1). These results potentially suggest that the wrapped water around DMSO's methyl group, which can be evicted upon ligand binding, may render DMSO a more surface-active cryoprotectant than glycerol, while glycerol may participate more as a viscogen that acts on the entire sample. These findings shed light on the molecular intricacies of cryoprotectant solvation behavior and have potentially significant implications for optimizing cryopreservation protocols.

Entropy Tug-of-War Determines Solvent Effects in the Liquid-Liquid Phase Separation of a Globular Protein.

Liquid-liquid phase separation (LLPS) plays a key role in the compartmentalization of cells via the formation of biomolecular condensates. Here, we combined atomistic molecular dynamics (MD) simulations and terahertz (THz) spectroscopy to determine the solvent entropy contribution to the formation of condensates of the human eye lens protein γD-Crystallin. The MD simulations reveal an entropy tug-of-war between water molecules that are released from the protein droplets and those that are retained within the condensates, two categories of water molecules that were also assigned spectroscopically. A recently developed THz-calorimetry method enables quantitative comparison of the experimental and computational entropy changes of the released water molecules. The strong correlation mutually validates the two approaches and opens the way to a detailed atomic-level understanding of the different driving forces underlying the LLPS.

Extreme ultraviolet time-resolved photoelectron spectroscopy of adenine, adenosine and adenosine monophosphate in a liquid flat jet.

Time-resolved photoelectron spectroscopy using an extreme-ultraviolet (XUV) probe pulse was used to investigate the UV photoinduced dynamics of adenine (Ade), adenosine (Ado), and adenosine-5-monophosphate (AMP) in a liquid water jet. In contrast to previous studies using UV probe pulses, the XUV pulse at 21.7 eV can photoionize all excited states of a molecule, allowing for full relaxation pathways to be addressed after excitation at 4.66 eV. This work was carried out using a gas-dynamic flat liquid jet, resulting in considerably enhanced signal compared to a cylindrical jet. All three species decay on multiple time scales that are assigned based on their decay associated spectra; the fastest decay of ∼100 fs is assigned to ππ* decay to the ground state, while a smaller component with a lifetime of ∼500 fs is attributed to the nπ* state. An additional slower channel in Ade is assigned to the 7H Ade conformer, as seen previously. This work demonstrates the capability of XUV-TRPES to disentangle non-adiabatic dynamics in an aqueous solution in a state-specific manner and represents the first identification of the nπ* state in the relaxation dynamics of adenine and its derivatives.

Hydration water drives the self-assembly of guanosine monophosphate.

Guanosine monophosphate (GMP) is a nucleotide that can self-assemble in aqueous solution under certain conditions. An understanding of the process at the molecular level is an essential step to comprehend the involvement of DNA substructures in transcription and replication, as well as their relationship to genetic diseases such as cancer. We present the temperature-dependent terahertz (1.5-12 THz, 50-400 cm-1) absorptivity spectra of aqueous Na2 GMP solution in comparison with the aqueous solutions of other RNA nucleotides. Distinct absorption features were observed in the spectrum of GMP, which we attribute to the intramolecular modes of the self-assemblies (i.e., G-complexes) that, at 1 M, start to form at 313 K and below. Changes in broad-band features of the terahertz spectrum were also observed, which we associate with the release of hydration water in the temperature-dependent formation of guanine quadruplexes. Using a state-of-the-art THz calorimetry approach correlating spectroscopic to thermodynamic changes, we propose a molecular mechanism of hydrophilic hydration driving GMP self-assembly as a function of temperature. The free energy contribution of hydrophilic hydration is shown as a decisive factor in guanine-quadruplex formation. Our findings spotlight the role of hydration in the formation of macromolecular structures and suggest the potential of hydration tuning for regulating DNA transcription and replication.

Studying Local Electrostatics by Terahertz Spectroscopy Using Amines as a Probe.

In a previous study[1] we could show that a large amplitude mode of the zwitterion glycine can serve as a sensitive probe for protonation and allows to deduce local pKa values. Here we show that the underlying concept is more general: We present the results of a pH dependent measurement of Terahertz-FTIR (THz-FTIR) spectra of solvated amines, i. e. Diethylamine (DEA), Triethylamine (TEA), and Diisopropylamine (DiPA). We show that amines serve as a sensitive, label free probe for local protonation. Protonation of the amines yield intensity changes which can be quantified by precise THz spectroscopy (30 cm-1 -450 cm-1 ). A detailed analysis allows us to correlate the titration spectra of solvated amines in the THz range with pKa values. This demonstrates the potential of THz spectroscopy to probe the charge state of biomolecules in water in a label free manner.

Energy Dissipation into the Solvent during Proton Transfer Occurs via Acoustic Phonons.

In photochemistry, rapid energy dissipation into the solvent is mandatory to prevent radiation damages. By optical pump THz spectroscopy, we are able to follow the details of the energy dissipation mechanism upon photoexcitation of the photoacid to the hydrogen-bonded network of water: Based on the frequency maps subsequent to photoexcitation, we propose that energy transfer takes place via propagation of an acoustic phonon. The dissipation into the solvent can be rationalized by an initial first hydration shell response followed by energy dissipation via an acoustic phonon. Surprisingly, for the first 10 ps, the propagation in the water network can be described by a wave packet with a constant group velocity, indicating a long-range correlation. After 300 ps, thermalization in the liquid jet is reached and the THz spectrum reflects a Boltzmann population, corresponding a temperature increase of ΔT = 0.5 °C.

From Local Hydration Motifs in Aqueous Acetic Acid Solutions to Macroscopic Mixing Thermodynamics: A Quantitative Connection from THz-Calorimetry.

We report the results of THz measurements (30-440 cm-1) of aqueous acetic acid solutions over the full mixing range (XAcAc = 0-1). We recorded spectroscopic observables as a function of the acetic acid concentration in the frequency range of the intermolecular stretch at 150 cm-1 and of the librational modes at 350-440 cm-1. This allowed us to unravel changes in hydrophobic and hydrophilic hydration motifs, respectively. By means of a novel THz-calorimetry approach, we quantitatively correlated these changes in local hydration motifs to excess mixing entropy and enthalpy. We find that ΔHmix is determined by both hydrophobic and hydrophilic solvation contributions. In contrast, ΔSmix is governed by hydrophobic cavity formation. Our results further suggest that acetic acid-water mixtures are systems at the edge of phase separation due to endothermic contributions from both hydrophilic and hydrophobic solvation in a large portion of the miscibility range. Our work establishes a quantitative relationship between the balance of local hydrophobic and hydrophilic solvation motifs and the macroscopic mixing thermodynamic properties.

Hydration makes a difference! How to tune protein complexes between liquid-liquid and liquid-solid phase separation.

Understanding how protein rich condensates formed upon liquid-liquid phase separation (LLPS) evolve into solid aggregates is of fundamental importance for several medical applications, since these are suspected to be hot-spots for many neurotoxic diseases. This requires developing experimental approaches to observe in real-time both LLPS and liquid-solid phase separation (LSPS), and to unravel the delicate balance of protein and water interactions dictating the free energy differences between the two. We present a vibrational THz spectroscopy approach that allows doing so from the point of view of hydration water. We focus on a cellular prion protein of high medical relevance, which we can drive to undergo either LLPS or LSPS with few mutations. We find that it is a subtle balance of hydrophobic and hydrophilic solvation contributions that allows tuning between LLPS and LSPS. Hydrophobic hydration provides an entropic driving force to phase separation, through the release of hydration water into the bulk. Water hydrating hydrophilic groups provides an enthalpic driving force to keep the condensates in a liquid state. As a result, when we modify the protein by a few mutations to be less hydrophilic, we shift from LLPS to LSPS. This molecular understanding paves the way for a rational design of proteins.

Local solvation structures govern the mixing thermodynamics of glycerol-water solutions.

Glycerol is a major cryoprotective agent and is widely used to promote protein stabilization. By a combined experimental and theoretical study, we show that global thermodynamic mixing properties of glycerol and water are dictated by local solvation motifs. We identify three hydration water populations, i.e., bulk water, bound water (water hydrogen bonded to the hydrophilic groups of glycerol) and cavity wrap water (water hydrating the hydrophobic moieties). Here, we show that for glycerol experimental observables in the THz regime allow quantification of the abundance of bound water and its partial contribution to the mixing thermodynamics. Specifically, we uncover a 1 : 1 connection between the population of bound waters and the mixing enthalpy, which is further corroborated by the simulation results. Therefore, the changes in global thermodynamic quantity - mixing enthalpy - are rationalized at the molecular level in terms of changes in the local hydrophilic hydration population as a function of glycerol mole fraction in the full miscibility range. This offers opportunities to rationally design polyol water, as well as other aqueous mixtures to optimize technological applications by tuning mixing enthalpy and entropy based on spectroscopic screening.

Real-time measure of solvation free energy changes upon liquid-liquid phase separation of α-elastin.

Biological condensates are known to retain a large fraction of water to remain in a liquid and reversible state. Local solvation contributions from water hydrating hydrophilic and hydrophobic protein surfaces were proposed to play a prominent role for the formation of condensates through liquid-liquid phase separation (LLPS). However, although the total free energy is accessible by calorimetry, the partial solvent contributions to the free energy changes upon LLPS remained experimentally inaccessible so far. Here, we show that the recently developed THz calorimetry approach allows to quantify local hydration enthalpy and entropy changes upon LLPS of α-elastin in real time, directly from experimental THz spectroscopy data. We find that hydrophobic solvation dominates the entropic solvation term, whereas hydrophilic solvation mainly contributes to the enthalpy. Both terms are in the order of hundreds of kJ/mol, which is more than one order of magnitude larger than the total free energy changes at play during LLPS. However, since we show that entropy/enthalpy mostly compensates, a small entropy/enthalpy imbalance is sufficient to tune LLPS. Theoretically, a balance was proposed before. Here we present experimental evidence based on our spectroscopic approach. We finally show that LLPS can be steered by inducing small changes of solvation entropy/enthalpy compensation via concentration or temperature in α-elastin.

Caught in the act: real-time observation of the solvent response that promotes excited-state proton transfer in pyranine.

Photo-induced excited-state proton transfer (ESPT) reactions are of central importance in many biological and chemical processes. Identifying mechanistic details of the solvent reorganizations that facilitate proton transfer however, is challenging for current experimental and theoretical approaches. Using optical pump THz probe (OPTP) spectroscopy and molecular dynamics simulations, we were able to elucidate the ultrafast changes in the solvation environment for three derivatives of pyranine: the photoacid HPTS, the methoxy derivative MPTS, and the photobase OPTS. Experimentally, we find damped oscillations in the THz signal at short times and our simulations enable their assignment to vibrational energy transfer beatings between the photoexcited chromophore and nearby solvent molecules. The simulations of HPTS reveal strikingly efficient sub-ps energy transfer into a particular solvent mode, that is active near 4 THz, and which can provide the requisite energy required for solvent reorganization promoting proton transfer. Similar oscillations are present in the THz signal for all three derivatives, however the signal is damped rapidly for HPTS (within 0.4 ps) and more slowly for MPTS (within 1.4 ps) and OPTS (within 2.0 ps). For HPTS, we also characterize an additional phonon-like propagation of the proton into the bulk with a 140 ps period and an 83 ps damping time. Thermalization of the solvent occurs on a time scale exceeding 120 ps.

Liquid-Liquid Phase Separation? Ask the Water!

Water is more than an inert spectator during liquid-liquid phase separation (LLPS), the reversible compartmentalization of protein solutions into a protein-rich and a dilute phase. We show that LLPS is driven by changes in hydration entropy and enthalpy. Tuning LLPS by adjusting experimental parameters, e.g., addition of co-solutes, is a major goal for biological and medical applications. This requires a general model to quantify thermodynamic driving forces. Here, we develop such a model based on the measured amplitudes of characteristic THz-features of two hydration populations: "Cavity-wrap" water hydrating hydrophobic patches is released during LLPS leading to an increase in entropy. "Bound" water hydrating hydrophilic patches is retained since it is enthalpically favorable. We introduce a THz-phase diagram mapping these spectroscopic/thermodynamic changes. This provides not only a precise understanding of hydrophobic and hydrophilic hydration driving forces as a function of temperature and concentration but also a rational means to tune LLPS.

The birth and evolution of solvated electrons in the water.

The photo-induced radiolysis of water is an elementary reaction in biology and chemistry, forming solvated electrons, OH radicals, and hydronium cations on fast time scales. Here, we use an optical-pump terahertz-probe spectroscopy setup to trigger the photoionization of water molecules with optical laser pulses at ~400 nm and then time-resolve the transient solvent response with broadband terahertz (THz) fields with a ~90 fs time resolution. We observe three distinct spectral responses. The first is a positive broadband mode that can be attributed to an initial diffuse, delocalized electron with a radius of (22 ± 1) Å, which is short lived (<200 fs) because the absorption is blue-shifting outside of the THz range. The second emerging spectroscopic signature with a lifetime of about 150 ps is attributed to an intermolecular mode associated with a mass rearrangement of solvent molecules due to charge separation of radicals and hydronium cations. After 0.2 ps, we observe a long-lasting THz signature with depleted intensity at 110 cm-1 that is well reproduced by ab initio molecular dynamics. We interpret this negative band at 110 cm-1 as the solvent cage characterized by a weakening of the hydrogen bond network in the first and second hydration shells of the cavity occupied by the localized electron.

Highly Altered State of Proton Transport in Acid Pools in Charged Reverse Micelles.

Transport mechanisms of solvated protons of 1 M HCl acid pools, confined within reverse micelles (RMs) containing the negatively charged surfactant sodium bis(2-ethylhexyl) sulfosuccinate (NaAOT) or the positively charged cetyltrimethylammonium bromide (CTABr), are analyzed with reactive force field simulations to interpret dynamical signatures from TeraHertz absorption and dielectric relaxation spectroscopy. We find that the forward proton hopping events for NaAOT are further suppressed compared to a nonionic RM, while the Grotthuss mechanism ceases altogether for CTABr. We attribute the sluggish proton dynamics for both charged RMs as due to headgroup and counterion charges that expel hydronium and chloride ions from the interface and into the bulk interior, thereby increasing the pH of the acid pools relative to the nonionic RM. For charged NaAOT and CTABr RMs, the localization of hydronium near a counterion or conjugate base reduces the Eigen and Zundel configurations that enable forward hopping. Thus, localized oscillatory hopping dominates, an effect that is most extreme for CTABr in which the proton residence time increases dramatically such that even oscillatory hopping is slow.

Observation of dissipating solvated protons upon hydrogel formation.

Aqueous hyaluronan solutions form an elastic hydrogel within a narrow pH range, around pH 2.4, making this a model system to study the conformational changes of the hydrogen bond network upon gelation. This pH-dependent behavior allows us to probe water surrounding a biologically relevant molecule in different environments (liquid versus elastic state) which change due to an environmental stimulus. Here, we use Terahertz (THz) reflection absorption spectroscopy in attenuated total reflection (ATR) geometry as a tool to study gelation. THz spectroscopy is sensitive to changes in the hydrogen-bonded water network, and here we show that we can correlate changes in macroscopic properties to changes in the solvation of hyaluronan. Above and below the gelation pH, solvated protons are present in the solutions, however, this spectral signature is completely absent between pH 2.4-2.8, which is the pH at which hyaluronan forms a hydrogel. We propose that solvated protons are forming ion pairs with hyaluronan in this pH range. Adding urea or glucose to hyaluronan solutions changes their elasticity, in which an increase or decrease in elasticity can be linked to the formation and destruction of these ion pairs, respectively.

Nanoconfinement effects on water in narrow graphene-based slit pores as revealed by THz spectroscopy.

The properties of water at interfaces have long been known to differ from those of bulk water in many distinctive ways. More recently, specific confinement effects different from mere interfacial effects have been discovered upon enclosing water in very narrow cylindrical pores and planar surfaces as offered by nanotubes and slit pores, respectively. Using experimental and theoretical THz spectroscopy, we elucidate nanoconfinement effects on the H-bond network of stratified water lamellae that are hosted within graphene-based two-dimensional pores. Characteristic confinement-induced changes of the THz response are traced back to the level of structural dynamics, notably distinct resonances due to intralayer and interlayer H-bonds at correspondingly low and high intermolecular stretching frequencies and impact of dangling (free) OH bonds at the water-graphene interface that enormously broaden the librational band in sufficiently narrow pores. The interplay of these molecular effects causes characteristic changes of the THz lineshape upon nanoconfining water.

Reaction of lithium hexamethyldisilazide (LiHMDS) with water at ultracold conditions.

Alkali metal amides are highly reactive reagents that are broadly applied as strong bases in organic synthesis. Here, we use a combined helium nanodroplet IR spectroscopic and theoretical (DFT calculation) study to show that the reaction of the model compound lithium hexamethyldisilazide (LiHMDS) with water is close to barrierless even at ultra-cold conditions. Upon complex formation of dimeric (LiHMDS)2 with water in helium nanodroplets as ultra-cold nano-reactors (0.37 K) we observed the reaction product (LiOH)2(HMDS)2. This can be rationalized as aggregation induced reation upon stepwise addition of water. With increasing water partial pressure, only the product (LiOH)2(HMDS)2 is observed experimentally. This implies that the large interaction energy (69 kJ mol-1) of (LiHMDS)2 with water is sufficient to overcome the follow-up reaction barriers, in spite of the rapid cooling rates in He nanodroplets.