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Photochemically prepared transition-metal complexes are known to be effective at cleaving the strong C–H bonds of organic molecules in room temperature solutions. There is also ample theoretical evidence that the two-way, metal to ligand (MLCT) and ligand to metal (LMCT), charge-transfer between an incoming alkane C–H group and the transition metal is the decisive interaction in the C–H activation reaction. What is missing, however, are experimental methods to directly probe these interactions in order to reveal what determines reactivity of intermediates and the rate of the reaction. Here, using quantum chemical simulations we predict and propose future time-resolved valence-to-core resonant inelastic X-ray scattering (VtC-RIXS) experiments at the transition metal L-edge as a method to provide a full account of the evolution of metal–alkane interactions during transition-metal mediated C–H activation reactions. For the model system cyclopentadienyl rhodium dicarbonyl (CpRh(CO)2), we demonstrate, by simulating the VtC-RIXS signatures of key intermediates in the C–H activation pathway, how the Rh-centered valence-excited states accessible through VtC-RIXS directly reflect changes in donation and back-donation between the alkane C–H group and the transition metal as the reaction proceeds via those intermediates. We benchmark and validate our quantum chemical simulations against experimental steady-state measurements of CpRh(CO)2 and Rh(acac)(CO)2 (where acac is acetylacetonate). Our study constitutes the first step towards establishing VtC-RIXS as a new experimental observable for probing reactivity of C–H activation reactions. More generally, the study further motivates the use of time-resolved VtC-RIXS to follow the valence electronic structure evolution along photochemical, photoinitiated and photocatalytic reactions with transition metal complexes.

Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster (6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.

Transition metal reactivity toward carbon-hydrogen (C-H) bonds hinges on the interplay of electron donation and withdrawal at the metal center. Manipulating this reactivity in a controlled way is difficult because the hypothesized metal-alkane charge-transfer interactions are challenging to access experimentally. Using time-resolved x-ray spectroscopy, we track the charge-transfer interactions during C-H activation of octane by a cyclopentadienyl rhodium carbonyl complex. Changes in oxidation state as well as valence-orbital energies and character emerge in the data on a femtosecond to nanosecond timescale. The x-ray spectroscopic signatures reflect how alkane-to-metal donation determines metal-alkane complex stability and how metal-to-alkane back-donation facilitates C-H bond cleavage by oxidative addition. The ability to dissect charge-transfer interactions on an orbital level provides opportunities for manipulating C-H reactivity at transition metals.

Optical-domain transient grating (TG) spectroscopy is a versatile background-free four-wave-mixing technique that is used to probe vibrational, magnetic and electronic degrees of freedom in the time domain1. The newly developed coherent X-ray free-electron laser sources allow its extension to the X-ray regime. X-rays offer multiple advantages for TG: their large penetration depth allows probing the bulk properties of materials, their element specificity can address core excited states, and their short wavelengths create excitation gratings with unprecedented momentum transfer and spatial resolution. Here, we demonstrate TG excitation in the hard X-ray range at 7.1 keV. In bismuth germanate (BGO), the non-resonant TG excitation generates coherent optical phonons detected as a function of time by diffraction of an optical probe pulse. This experiment demonstrates the ability to probe bulk properties of materials and paves the way for ultrafast coherent four-wave-mixing techniques using X-ray probes and involving nanoscale TG spatial periods.

The structural changes of water upon deep supercooling were studied through wide-angle x-ray scattering at SwissFEL. The experimental setup had a momentum transfer range of 4.5 Å-1, which covered the principal doublet of the x-ray structure factor of water. The oxygen-oxygen structure factor was obtained for temperatures down to 228.5 ± 0.6 K. Similar to previous studies, the second diffraction peak increased strongly in amplitude as the structural change accelerated toward a local tetrahedral structure upon deep supercooling. We also observed an anomalous trend for the second peak position of the oxygen-oxygen structure factor (q2). We found that q2 exhibits an unprecedented positive partial derivative with respect to temperature for temperatures below 236 K. Based on Fourier inversion of our experimental data combined with reference data, we propose that the anomalous q2 shift originates from that a repeat spacing in the tetrahedral network, associated with all peaks in the oxygen-oxygen pair-correlation function, gives rise to a less dense local ordering that resembles that of low-density amorphous ice. The findings are consistent with that liquid water consists of a pentamer-based hydrogen-bonded network with low density upon deep supercooling., QC 20220125

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.

Azidohomoalanine (Aha) is an unnatural amino acid containing an infrared active azido side chain group that can, through frequency shifts of the azido stretch vibration, act as a probe of local structure. To realize the potential of such structural probes for protein science, we have developed a two-dimensional infrared spectrometer employing fast mechanical scanning and intrinsic phasing of the resulting spectra, leading to a lower sensitivity limit of similar to 100 mu OD level samples. Using this approach, we quantify the biomolecular recognition between a PDZ2 domain and two Aha-mutated peptides. It is shown that this method can distinguish different binding modes and that the energetics of binding can be determined.

We show that it is possible to phase multidimensional infrared spectra generated by a boxcars geometry four-wave mixing spectrometer directly from the signal generated by the molecular vibration of interest, without the need for auxiliary phasing measurements. For isolated vibrations, the phase profile of the 2D response smoothly varies between fixed phase limits, allowing for a general target for phasing independent of the degree of anharmonicity exhibited between the ground and excited state. As a proof of principle, the 2D response of the similar to 2155 cm(-1) thiocyanate stretch vibration of MeSCN, a system exhibiting anharmonicity such that the 0-1 and 1-2 transitions are spectrally isolated, is successfuly phased directly from the experimental spectra. The methodology is also applied to correctly phase extremely weak signals of the unnatural amino acid azidohomoalanine following background subtraction.

2D-IR spectroscopy has matured to a powerful technique to study the structure and dynamics of peptides, but its extension to larger proteins is still in its infancy, the major limitations being sensitivity and selectivity. Site-selective information requires measuring single vibrational probes at sub-millimolar concentrations where most proteins are still stable, which is a severe challenge for conventional (FT)IR spectroscopy. Besides its ultrafast time-resolution, a so far largely underappreciated potential of 2D-IR spectroscopy lies in its sensitivity gain. The present paper sets the goals and outlines strategies how to use that sensitivity gain together with properly designed vibrational labels to make IR spectroscopy a versatile tool to study a wide class of proteins.