Search

Ultrafast spectroscopy and imaging have become tools utilized by a broad range of scientists involved in materials, energy, biological, and chemical sciences. Commercialization of ultrafast spectrometers including transient absorption spectrometers, vibrational sum frequency generation spectrometers, and even multidimensional spectrometers have put these advanced spectroscopy measurements into the hands of practitioners originally outside the field of ultrafast spectroscopy. There is now a technology shift occurring in ultrafast spectroscopy, made possible by new Yb-based lasers, that is opening exciting new experiments in the chemical and physical sciences. Amplified Yb-based lasers are not only more compact and efficient than their predecessors but also, most importantly, operate at many times the repetition rate with improved noise characteristics in comparison to the previous generation of Ti:sapphire amplifier technologies. Taken together, these attributes are enabling new experiments, generating improvements to long-standing techniques, and affording the transformation of spectroscopies to microscopies. This Account aims to show that the shift to 100 kHz lasers is a transformative step in nonlinear spectroscopy and imaging, much like the dramatic expansion that occurred with the commercialization of Ti:sapphire laser systems in the 1990s. The impact of this technology will be felt across a great swath of scientific communities. We first describe the technology landscape of amplified Yb-based laser systems used in conjunction with 100 kHz spectrometers operating with shot-to-shot pulse shaping and detection. We also identify the range of different parametric conversion and supercontinuum techniques which now provide a path to making pulses of light optimal for ultrafast spectroscopy. Second, we describe specific instances from our laboratories of how the amplified Yb-based light sources and spectrometers are transformative. For multiple probe time-resolved infrared and transient 2D IR spectroscopy, the gain in temporal span and signal-to-noise enables dynamical spectroscopy measurements from femtoseconds to seconds. These gains widen the applicability of time-resolved infrared techniques across a range of topics in photochemistry, photocatalysis, and photobiology as well as lower the technical barriers to implementation in a laboratory. For 2D visible spectroscopy and microscopy with white light, as well as 2D IR imaging, the high repetition rates of these new Yb-based light sources allow one to spatially map 2D spectra while maintaining high signal-to-noise in the data. To illustrate the gains, we provide examples of imaging applications in the study of photovoltaic materials and spectroelectrochemistry.

The spectroscopy and structural dynamics of a deep eutectic mixture (KSCN/acetamide) with varying water content is investigated from 2D IR (with the C–N stretch vibration of the SCN–anions as the reporter) and THz spectroscopy. Molecular dynamics simulations correctly describe the nontrivial dependence of both spectroscopic signatures depending on water content. For the 2D IR spectra, the MD simulations relate the steep increase in the cross-relaxation rate at high water content to the parallel alignment of packed SCN–anions. Conversely, the nonlinear increase of the THz absorption with increasing water content is mainly attributed to the formation of larger water clusters. The results demonstrate that a combination of structure-sensitive spectroscopies and molecular dynamics simulations provides molecular-level insights into the emergence of heterogeneity of such mixtures by modulating their composition.

The dynamics of peptide–protein binding and unbinding of a variant of the RNase S system has been investigated. To initiate the process, a photoswitchable azobenzene moiety has been covalently linked to the S-peptide, thereby switching its binding affinity to the S-protein. Transient fluorescence quenching was measured with the help of a time-resolved fluorometer, which has been specifically designed for these experiments and is based on inexpensive light-emitting diodes and laser diodes only. One mutant shows on–off behavior with no specific binding detectable in one of the states of the photoswitch. Unbinding is faster by at least 2 orders of magnitude, compared to that of other variants of the RNase S system. We conclude that unbinding is essentially barrier-less in that case, revealing the intrinsic dynamics of the unbinding event, which occurs on a time scale of a few hundred microseconds in a strongly stretched-exponential manner.

The photochemistry of a molecular pentad composed of a central anthraquinone (AQ) acceptor flanked by two Ru(bpy)32+photosensitizers and two peripheral triarylamine (TAA) donors was investigated by transient IR and UV–vis spectroscopies in the presence of 0.2 M p-toluenesulfonic acid (TsOH) in deaerated acetonitrile. In ∼15% of all excited pentad molecules, AQ is converted to its hydroquinone form (AQH2) via reversible intramolecular electron transfer from the two TAA units (τ = 65 ps), followed by intermolecular proton transfer from TsOH (τ ≈ 3 ns for the first step). Although the light-driven accumulation of reduction equivalents occurs through a sequence of electron and proton transfer steps, the resulting photoproduct decays via concerted PCET (τ = 4.7 μs) with an H/D kinetic isotope effect of 1.4 ± 0.2. Moreover, the reoxidation of AQH2seems to take place via a double electron transfer step involving both TAA+units rather than sequential single electron transfer events. Thus, the overall charge-recombination reaction seems to involve a concerted proton-coupled two-electron oxidation of AQH2. The comparison of experimental data obtained in neat acetonitrile with data from acidic solutions suggests that the inverted driving-force effect can play a crucial role for obtaining long-lived photoproducts resulting from multiphoton, multielectron processes. Our pentad provides the first example of light-driven accumulation of reduction equivalents stabilized by PCET in artificial molecular systems without sacrificial reagents. Our study provides fundamental insight into how light-driven multielectron redox chemistry, for example the reduction of CO2or the oxidation of H2O, can potentially be performed without sacrificial reagents.

A series of photoswitchable, α-helical peptides were studied using two-dimensional infrared spectroscopy (2D-IR). Single-isotope labeling with 13C18O at various positions in the sequence was employed to spectrally isolate particular backbone positions. We show that a single 13C18O label can give rise to two bands along the diagonal of the 2D-IR spectrum, one of which is from an amide group that is hydrogen-bonded internally, or to a solvent molecule, and the other from a non-hydrogen-bonded amide group. The photoswitch enabled examination of both the folded and unfolded state of the helix. For most sites, unfolding of the peptide caused a shift of intensity from the hydrogen-bonded peak to the non-hydrogen-bonded peak. The relative intensity of the two diagonal peaks gives an indication of the fraction of molecules hydrogen-bonded at a certain location along the sequence. As this fraction varies quite substantially along the helix, we conclude that the helix is not uniformly folded. Furthermore, the shift in hydrogen bonding is much smaller than the change of helicity measured by CD spectroscopy, indicating that non-native hydrogen-bonded or mis-folded loops are formed in the unfolded ensemble.

Increased long-term performance was found for photocatalytic H2production in a homogeneous combination of [Re(NCS)(CO)3bipy] (1; bipy = 2,2′-bipyridine), [Co(dmgH)2] (dmgH2= dimethylglyoxime), triethanolamine (TEOA), and [HTEOA][BF4] in N,N-dimethylformamide, achieving TONReup to 6000 (H/Re). The system proceeded by reductive quenching of *1by TEOA, followed by fast (k1= 1.3 × 108M−1s−1) electron transfer to [CoII(dmgH)2] and subsequent protonation (K2) and elimination (k3, second-order process in cobalt) of H2. Observed quantum yields were up to ∼90% (H produced per absorbed photon). The type of acid had a substantial effect on the long-term stability. A decomposition pathway involving cobalt is limiting the long-term performance. Time-resolved infrared (IR) spectroscopy confirmed that photooxidized TEOA generates a second reducing equivalent, which can be transferred to 1(70%, k2e−= 3.3 × 108M−1s−1) if no [CoII(dmgH)2] is present.

We present two-dimensional infrared (2D IR) spectra of the OD stretch vibration of isotope diluted water (HOD/H2O) from ambient conditions (293 K) down to the metastable supercooled regime (260 K). We observe that spectral diffusion slows down from 700 fs to 2.6 ps as we lower the temperature. A comparison between measurements performed at the magic angle with those at parallel polarization shows that the 2D IR line shape is affected by the frequency-dependent anisotropy decay in the case of parallel polarization, altering the extracted correlation decay. A fit within the framework of an Arrhenius law reveals an activation energy of Ea= 6.2 ± 0.2 kcal/mol and a pre-exponential factor of 1/A= 0.02 ± 0.01 fs. Alternatively, a power law fit results in an exponent γ = 2.2 and a singularity temperature Ts= 221 K. We tentatively conclude that the power law provides the better physical picture to describe the dynamics of liquid water around the freezing point.

We explore the capability of the non-natural amino acid azidohomoalanine (AHA) as an IR label to sense relatively small structural changes in proteins with the help of 2D IR difference spectroscopy. To that end, we AHA-labeled an allosteric protein (the PDZ2 domain from human tyrosine-phosphatase 1E) and furthermore covalently linked it to an azobenzene-derived photoswitch as to mimic its conformational transition upon ligand binding. To determine the strengths and limitations of the AHA label, in total six mutants have been investigated with the label at sites with varying properties. Only one mutant revealed a measurable 2D IR difference signal. In contrast to the commonly observed frequency shifts that report on the degree of solvation, in this case we observe an intensitychange. To understand this spectral response, we performed classical MD simulations, evaluating local contacts of the AHA labels to water molecules and protein side chains and calculating the vibrational frequency on the basis of an electrostatic model. Although these simulations revealed in part significant and complex changes of the number of intraprotein and water contacts upon trans–cisphotoisomerization, they could not provide a clear explanation of why this one label would stick out. Subsequent quantum-chemistry calculations suggest that the response is the result of an electronic interaction involving charge transfer of the azido group with sulfonate groups from the photoswitch. To the best of our knowledge, such an effect has not been described before.

We present systematic kinetic studies of the interaction of a rhenium-based photosensitizer with a cobalt(II) tetrapyridyl water reduction catalyst coadsorbed on ZrO2by transient IR and visible spectroscopies. The study focuses on the competition between the reduction of the excited photosensitizer by an electron donor in solution and nonproductive quenching between the photosensitizer and the catalyst, either by Dexter energy transfer or by electron transfer followed by ultrafast geminate recombination. The implications of both interactions for the charge transfer reactions on the surface are investigated. We find that the kinetics of the system as a whole and the achievable yield of reduced photosensitizer are determined by the inhomogeneous distribution of next neighbor distances between photosensitizers and the water reduction catalysts at the nanoscale level. This provides insight for rational design of heterogeneous water splitting systems with coimmobilized photosensitizers and catalysts.

Hydridocarbonyl complexes, a class of industrially relevant catalysts, contain both the M–H and M–CO moieties. Here, using two-dimensional infrared spectroscopy, we examine the coupling of the typically weak M–H stretching mode and the intense M(C≡O) mode. By studying a series of Ir(I)- and Ir(III)-based hydridocarbonyl complexes, we show that the arrangement of the H and CO ligands in a transconfiguration leads to strong vibrational coupling and mode delocalization. In contrast, a cisarrangement leads to no coupling, with the localized M–H mode having a much larger anharmonicity. These results highlight a promising strategy for enhancing the M–H vibration by intensity borrowing from the strong C≡O modes in a transconfiguration, allowing for direct determination by infrared spectroscopy of both the oxidation (by frequency shifts) and the protonation state (via vibrational coupling) of the complex, in mechanistic studies of proton-coupled electron transfer reactions.

To employ molecular water reduction catalysts (WRC) in a heterogeneous setup, a stable, macrocyclic CoIII–polypyridyl WRC containing two phosphonic acid groups was anchored on TiO2to investigate photo- and electrocatalytic proton reduction. Photocatalytic investigations included kinetic studies of the electron transfer from the reduced photosensitizer to the WRC as well as H2-evolution measurements. Linear sweep voltammetry (LSV) performed on the immobilized WRC on a TiO2-coated FTO-glass electrode showed an onset potential of −0.6 V vs.SHE at pH = 5 for proton reduction, while operando UV/VIS confirmed the reduced CoI-species as the key catalytic intermediate. Finally, chronoamperometric investigations combined with XPS studies and ICP-MS studies of electrode and electrolyte revealed stable binding of the WRC on the electrode under catalytic conditions and constant H2-formation over the period of two days.

In the wider context of artificial photosynthesis, this work aims to explore the functionality and characteristics of rhenium tricarbonyl complexes as photosensitizers in heterogeneous water reduction systems. To that end, the reductive quenching of an excited ReICl(2,2′-bipyridine-4,4′-bisphosphonic acid)(CO)3adsorbed on a redox-neutral scaffold by phenothiazine was observed by transient IR spectroscopy. From the spectroscopic and time response, the full reaction cycle could be elucidated and the intrinsic lifetime of the excited Re complex, together with rates for reductive quenching, cage escape, as well as geminate recombination and secondary back electron transfer could be determined. Three different scenarios have been explored, which decrease the mobility of the reactants in the system in a stepwise manner: Starting from a reference system with all compounds in solution, first the Re complex was immobilized on the surface, and in a second step also the quencher. The overall reaction cycle for all reactants in solution is preserved as long as the quencher is in solution, with relatively minor changes of the rates of the individual reaction steps. The overall cage escape yield was found to be larger on the surface. As soon as the quencher is coadsorbed alongside the Re complex, however, the reaction cycle changes completely. Electron transfer occurred only from quencher molecules that sit next to an excited Re complex, and there is no possibility of cage escape. Varying the ratio of quencher molecules and Re complexes, it is concluded that molecules do not cluster on the surface and that excitation energy migration is not a very efficient process.

An azobenzene-derived photoswitch has been covalently cross-linked to two sites of the S-peptide in the RNase S complex in a manner that the α-helical content of the S-peptide reduces upon cis-to-transisomerization of the photoswitch. Three complementary experimental techniques have been employed, isothermal titration calorimetry, circular dichroism spectroscopy and intrinsic tyrosine fluorescence quenching, to determine the binding affinity of the S-peptide to the S-protein in the two states of the photoswitch. Five mutants with the photoswitch attached to different sites of the S-peptide have been explored, with the goal to maximize the change in binding affinity upon photoswitching, and to identify the mechanisms that determine the binding affinity. With regard to the first goal, one mutant has been identified, which binds with reasonable affinity in the one state of the photoswitch, while specific binding is completely switched off in the other state. With regard to the second goal, accompanying molecular dynamics simulations combined with a quantitative structure activity relationship revealed that the α-helicity of the S-peptide in the binding pocket correlates surprisingly well with measured dissociation constants. Moreover, the simulations show that both configurations of all S-peptides exhibit quite well-defined structures, even in apparently disordered states.

The noncanonical amino acid azidohomoalanine (Aha) is known to be an environment-sensitive infrared probe for the site-specific investigation of protein structure and dynamics. Here, the capability of that label is explored to detect protein–ligand interactions by incorporating it in the vicinity of the binding groove of a PDZ2 domain. Circular dichroism and isothermal titration calorimetry measurements reveal that the perturbation of the protein system by mutation is negligible, with minimal influence on protein stability and binding affinity. Two-dimensional infrared spectra exhibit small (1–3 cm–1) but clearly measurable red shifts of the Aha vibrational frequency upon binding of two different peptide ligands, while accompanying molecular dynamics simulations suggest that these red shifts are induced by polar contacts with side chains of the peptide ligands. Hence, Aha is a versatile and minimally invasive vibrational label that is not only able to report on large structural changes during, e.g., protein folding, but also on very subtle changes of the electrostatic environment upon ligand binding.

Three structurally very different antifreeze proteins (AFPs) are studied, addressing the question as to what extent the hypothesized preordering-binding mechanism is still relevant in the second solvation layer of the protein and beyond. Assuming a two-state model of water, the solvation layers are analyzed with the help of molecular dynamics simulations together with a Markov state model, which investigates the local tedrahedrality of the water hydrogen-bond network around a given water molecule. It has been shown previously that this analysis can discriminate the high-entropy, high-density state of the liquid (HDL) from its more structured low-density state (LDL). All investigated proteins, regardless of whether they are an AFP or not, have a tendency to increase the amount of HDL in their second solvation layer. The ice binding site (IBS) of the antifreeze proteins counteracts that trend, with either a hole in the HDL layer or a true excess of LDL. The results correlate to a certain extent with recent experiments, which have observed ice-like vibrational (VSFG) spectra for the water atop the IBS of only a subset of antifreeze proteins. It is concluded that the preordering-binding mechanism indeed seems to play a role but is only part of the overall picture.

Ultrafast vibrational dynamics of monolayers from a rhenium carbonyl complex at solid–liquid interfaces is investigated with the help of 2D ATR IR spectroscopy in dependence of the solvation environment. By changing the solvent from methanol to acetonitrile, we observe a structural transition between two states and find that this transition is fully reversible. The two coexisting states are identified by distinct but closely spaced (∼15 cm–1) vibrational bands in the region of the A′(1) symmetric carbonyl stretch vibration. Surface-dilution experiments suggest that the newly formed state in acetonitrile is related to aggregation of molecules at the surface. The vibrational energy transfer rate between the two species (∼30 ps) is used as a structural measure, based on which the morphology is characterized as partially ordered dimers in an otherwise disordered but closely packed monolayer.

The structural and dynamical properties of water are known to be affected by ion solvation. However, a consistent molecular picture that describes how and to what extent ions perturb the water structure is still missing. Here we apply 2D Raman–terahertz spectroscopy to investigate the impact of monatomic cations on the relaxation dynamics of the hydrogen-bond network in aqueous salt solutions. The inherent ability of multidimensional spectroscopy to deconvolute heterogeneous relaxation dynamics is used to reveal the correlation between the inhomogeneity of the collective intermolecular hydrogen-bond modes and the viscosity of a salt solution. Specifically, we demonstrate that the relaxation time along the echo direction t1 = t2correlates with the capability of a given cation to ‘structure’ water. Moreover, we provide evidence that the echo originates from the water–water modes, and not the water–cation modes, which implies that cations can structure the hydrogen-bond network to a certain extent.

Several ways to electronically synchronize different types of amplified femtosecond laser systems are presented based on a single freely programmable electronics hardware: arbitrary-detuning asynchronous optical sampling (ADASOPS), as well as actively locking two femtosecond laser oscillators, albeit not necessarily to the same round-trip frequency. They allow us to rapidly probe a very wide range of timescales, from picoseconds to potentially seconds, in a single transient absorption experiment without the need to move any delay stage. Experiments become possible that address a largely unexplored aspect of many photochemical reactions, in particular in the context of photo-catalysis as well as photoactive proteins, where an initial femtosecond trigger very often initiates a long-lasting cascade of follow-up processes. The approach is very versatile and allows us to synchronize very different lasers, such as a Ti:Sa amplifier and a 100 kHz Yb-laser system. The jitter of the synchronization, and therewith the time-resolution in the transient experiment, lies in the range from 1 to 3 ps, depending on the method. For illustration, transient IR measurements of the excited state solvation and decay of a metal carbonyl complex as well as the full reaction cycle of bacteriorhodopsin are shown. The pros and cons of the various methods are discussed, with regard to the scientific question one might want to address, and also with regard to the laser systems that might be already existent in a laser lab.

Cyanobacteriochromes are bistable photoreceptor proteins with desirable photochemical properties for biotechnological applications, such as optogenetics or fluorescence microscopy. Here, we investigate Slr1393-g3, a cyanobacteriochrome that reversibly photoswitches between a red-absorbing (Pr) and green-absorbing (Pg) form. We applied advanced IR spectroscopic methods to track the sequence of intermediates during the photocycle over many orders of magnitude in time. In the conversion from Pg to Pr, we have revealed a new intermediate with distinct spectroscopic features in the IR, which precedes Pr formation using transient IR spectroscopy. In addition, stationary and transient 2D IR experiments measured the vibrational couplings between different groups of the chromophore and the protein in these intermediate states, as well as their structural disorder. Anharmonic QM/MM calculations predict spectra in good agreement with experimental 2D IR spectra of the initial and final states of the photocycle. They facilitate the assignment of the IR spectra that serve as a basis for the interpretation of the spectroscopic results and suggest structural changes of the intermediates along the photocycle.

The binding of small molecules (e.g., natural ligands, metabolites, and drugs) to proteins governs most biochemical pathways and physiological processes. Here, we use molecular dynamics to investigate the unbinding of dimethyl sulfoxide (DMSO) from two distinct states of a small rotamase enzyme, the FK506-binding protein (FKBP). These states correspond to the FKBP protein relaxed with and without DMSO in the active site. Since the time scale of ligand unbinding (2–20 ns) is faster than protein relaxation (100 ns), a novel methodology is introduced to relax the protein without having to introduce an artificial constraint. The simulation results show that the unbinding time is an order of magnitude longer for dissociation from the DMSO-bound state (holo-relaxed). That is, the actual rate of unbinding depends on the state of the protein, with the protein having a long-lived memory. The rate thus depends on the concentration of the ligand as the apo and holo states reflect low and high concentrations of DMSO, respectively. Moreover, there are multiple binding modes in the apo-relaxed state, while a single binding mode dominates the holo-relaxed state in which DMSO acts as hydrogen bond acceptor from the backbone NH of Ile56, as in the crystal structure of the DMSO/FKBP complex. The solvent relaxes very fast (∼1 ns) close to the NH of Ile56 and with the same time scale of the protein far away from the active site. These results have implications for high-throughput docking, which makes use of a rigid structure of the protein target.

We investigate surface enhancement in two-dimensional attenuated total reflectance infrared (2D ATR IR) spectroscopy from organic monolayers (MLs) at metal–liquid interfaces. We consider MLs from both aromatic and aliphatic organic samples equipped with nitrile and azide functional groups, both of which are widely used as local vibrational probes in ultrafast spectroscopy. Polarization-dependent 2D ATR IR spectroscopy indicates the excitation of local hot spots formed between gold (Au) nanoparticles as the dominant origin of signal enhancement. The highest enhancement factors (∼50) are observed in the case of aromatic nitrile MLs, whereas modest values (<10) are found for aliphatic azide and nitrile groups. Different contributions to signal enhancement are evaluated systematically and indicate the presence of both electromagnetic enhancement and contributions from molecular properties. The obtained enhancement factors are promising to allow 2D ATR IR spectroscopy to become applicable as a versatile technique for the detection of ultrafast structural dynamics in even low-absorbing organic MLs at solid–liquid interfaces.

A new method is presented for the combination of spectro-electrochemistry and femtosecond 2D IR spectroscopy. The key concept is based on ultrathin (∼nm) conductive layers of noble metals and indium–tin oxide (ITO) as working electrodes on a single-reflection attenuated total reflectance (ATR) element in conjunction with ultrafast, multidimensional ATR spectroscopy. The ATR geometry offers prominent benefits in ultrafast spectro-electrochemistry, that is, surface sensitivity for studying electrochemical processes directly at the solvent–electrode interface as well as the application of strongly IR-absorbing solvents such as water due to a very short effective path length of the evanescent wave at the interface. We present a balanced comparison between usable electrode materials regarding their performance in the ultrafast ATR setup. The electrochemical performance is demonstrated by vibrational Stark-shift spectroscopy of carbon monoxide (CO) adsorbed to platinum-coated, ultrathin ITO electrodes. We furthermore measure vibrational relaxation and spectral diffusion of the stretching mode from surface-bound CO dependent on the applied potential to the working electrode and find a negligible impact of the electrode potential on ultrafast CO dynamics.