Dynamic three-dimensional structures of a metal-organic framework captured with femtosecond serial crystallography.

Crystalline systems consisting of small-molecule building blocks have emerged as promising materials with diverse applications. It is of great importance to characterize not only their static structures but also the conversion of their structures in response to external stimuli. Femtosecond time-resolved crystallography has the potential to probe the real-time dynamics of structural transitions, but, thus far, this has not been realized for chemical reactions in non-biological crystals. In this study, we applied time-resolved serial femtosecond crystallography (TR-SFX), a powerful technique for visualizing protein structural dynamics, to a metal-organic framework, consisting of Fe porphyrins and hexazirconium nodes, and elucidated its structural dynamics. The time-resolved electron density maps derived from the TR-SFX data unveil trifurcating structural pathways: coherent oscillatory movements of Zr and Fe atoms, a transient structure with the Fe porphyrins and Zr6 nodes undergoing doming and disordering movements, respectively, and a vibrationally hot structure with isotropic structural disorder. These findings demonstrate the feasibility of using TR-SFX to study chemical systems.

Capturing the generation and structural transformations of molecular ions.

Molecular ions are ubiquitous and play pivotal roles1-3 in many reactions, particularly in the context of atmospheric and interstellar chemistry4-6. However, their structures and conformational transitions7,8, particularly in the gas phase, are less explored than those of neutral molecules owing to experimental difficulties. A case in point is the halonium ions9-11, whose highly reactive nature and ring strain make them short-lived intermediates that are readily attacked even by weak nucleophiles and thus challenging to isolate or capture before they undergo further reaction. Here we show that mega-electronvolt ultrafast electron diffraction (MeV-UED)12-14, used in conjunction with resonance-enhanced multiphoton ionization, can monitor the formation of 1,3-dibromopropane (DBP) cations and their subsequent structural dynamics forming a halonium ion. We find that the DBP+ cation remains for a substantial duration of 3.6 ps in aptly named 'dark states' that are structurally indistinguishable from the DBP electronic ground state. The structural data, supported by surface-hopping simulations15 and ab initio calculations16, reveal that the cation subsequently decays to iso-DBP+, an unusual intermediate with a four-membered ring containing a loosely bound17,18 bromine atom, and eventually loses the bromine atom and forms a bromonium ion with a three-membered-ring structure19. We anticipate that the approach used here can also be applied to examine the structural dynamics of other molecular ions and thereby deepen our understanding of ion chemistry.

Single-Molecule X-ray Scattering Used to Visualize the Conformation Distribution of Biological Molecules via Single-Object Scattering Sampling.

Biological macromolecules, the fundamental building blocks of life, exhibit dynamic structures in their natural environment. Traditional structure determination techniques often oversimplify these multifarious conformational spectra by capturing only ensemble- and time-averaged molecular structures. Addressing this gap, in this work, we extend the application of the single-object scattering sampling (SOSS) method to diverse biological molecules, including RNAs and proteins. Our approach, referred to as "Bio-SOSS", leverages ultrashort X-ray pulses to capture instantaneous structures. In Bio-SOSS, we employ two gold nanoparticles (AuNPs) as labels, which provide strong contrast in the X-ray scattering signal, to ensure precise distance determinations between labeled sites. We generated hypothetical Bio-SOSS images for RNAs, proteins, and an RNA-protein complex, each labeled with two AuNPs at specified positions. Subsequently, to validate the accuracy of Bio-SOSS, we extracted distances between these nanoparticle labels from the images and compared them with the actual values used to generate the Bio-SOSS images. Specifically, for a representative RNA (1KXK), the standard deviation in distance discrepancies between molecular dynamics snapshots and Bio-SOSS retrievals was found to be optimally around 0.2 Å, typically within 1 Å under practical experimental conditions at state-of-the-art X-ray free-electron laser facilities. Furthermore, we conducted an in-depth analysis of how various experimental factors, such as AuNP size, X-ray properties, and detector geometry, influence the accuracy of Bio-SOSS. This comprehensive investigation highlights the practicality and potential of Bio-SOSS in accurately capturing the diverse conformation spectrum of biological macromolecules, paving the way for deeper insights into their dynamic natures.

Cerium Photocatalyst in Action: Structural Dynamics in the Presence of Substrate Visualized via Time-Resolved X-ray Liquidography.

[Ce(III)Cl6]3-, with its earth-abundant metal element, is a promising photocatalyst facilitating carbon-halogen bond activation. Still, the structure of the reaction intermediate has yet to be explored. Here, we applied time-resolved X-ray liquidography (TRXL), which allows for direct observation of the structural details of reaction intermediates, to investigate the photocatalytic reaction of [Ce(III)Cl6]3-. Structural analysis of the TRXL data revealed that the excited state of [Ce(III)Cl6]3- has Ce-Cl bonds that are shorter than those of the ground state and that the Ce-Cl bond further contracts upon oxidation. In addition, this study represents the first application of TRXL to both photocatalyst-only and photocatalyst-and-substrate samples, providing insights into the substrate's influence on the photocatalyst's reaction dynamics. This study demonstrates the capability of TRXL in elucidating the reaction dynamics of photocatalysts under various conditions and highlights the importance of experimental determination of the structures of reaction intermediates to advance our understanding of photocatalytic mechanisms.

Photoactivation of triosmium dodecacarbonyl at 400 nm probed with time-resolved X-ray liquidography.

The photoactivation mechanism of Os3(CO)12 at 400 nm is examined with time-resolved X-ray liquidography. The data reveal two pathways: the vibrational relaxation following an internal conversion to the electronic ground state and the ligand dissociation to form Os3(CO)11 with a ligand vacancy at the axial position.

Determining the charge distribution and the direction of bond cleavage with femtosecond anisotropic x-ray liquidography.

Energy, structure, and charge are fundamental quantities characterizing a molecule. Whereas the energy flow and structure change in chemical reactions are experimentally characterized, determining the atomic charges of a molecule in solution has been elusive, even for a triatomic molecule such as triiodide ion, I3-. Moreover, it remains to be answered how the charge distribution is coupled to the molecular geometry; which I-I bond, if two I-I bonds are unequal, dissociates depending on the electronic state. Here, femtosecond anisotropic x-ray solution scattering allows us to provide the following answers in addition to the overall rich structural dynamics. The analysis unravels that the negative charge of I3- is highly localized on the terminal iodine atom forming the longer bond with the central iodine atom, and the shorter I-I bond dissociates in the excited state, whereas the longer one in the ground state. We anticipate that this work may open a new avenue for studying the atomic charge distribution of molecules in solution and taking advantage of orientational information in anisotropic scattering data for solution-phase structural dynamics.

Structural Dynamics of C2F4I2 in Cyclohexane Studied via Time-Resolved X-ray Liquidography.

The halogen elimination of 1,2-diiodoethane (C2H4I2) and 1,2-diiodotetrafluoroethane (C2F4I2) serves as a model reaction for investigating the influence of fluorination on reaction dynamics and solute-solvent interactions in solution-phase reactions. While the kinetics and reaction pathways of the halogen elimination reaction of C2H4I2 were reported to vary substantially depending on the solvent, the solvent effects on the photodissociation of C2F4I2 remain to be explored, as its reaction dynamics have only been studied in methanol. Here, to investigate the solvent dependence, we conducted a time-resolved X-ray liquidography (TRXL) experiment on C2F4I2 in cyclohexane. The data revealed that (ⅰ) the solvent dependence of the photoreaction of C2F4I2 is not as strong as that observed for C2H4I2, and (ⅱ) the nongeminate recombination leading to the formation of I2 is slower in cyclohexane than in methanol. We also show that the molecular structures of the relevant species determined from the structural analysis of TRXL data provide an excellent benchmark for DFT calculations, especially for investigating the relevance of exchange-correlation functionals used for the structural optimization of haloalkanes. This study demonstrates that TRXL is a powerful technique to study solvent dependence in the solution phase.

Optical Kerr Effect of Liquid Acetonitrile Probed by Femtosecond Time-Resolved X-ray Liquidography.

Optical Kerr effect (OKE) spectroscopy is a method that measures the time-dependent change of the birefringence induced by an optical laser pulse using another optical laser pulse and has been used often to study the ultrafast dynamics of molecular liquids. Here we demonstrate an alternative method, femtosecond time-resolved X-ray liquidography (fs-TRXL), where the microscopic structural motions related to the OKE response can be monitored using a different type of probe, i.e., X-ray solution scattering. By applying fs-TRXL to acetonitrile and a dye solution in acetonitrile, we demonstrate that different types of molecular motions around photoaligned molecules can be resolved selectively, even without any theoretical modeling, based on the anisotropy of two-dimensional scattering patterns and extra structural information contained in the q-space scattering data. Specifically, the dynamics of reorientational (libration and orientational diffusion) and translational (interaction-induced motion) motions are captured separately by anisotropic and isotropic scattering signals, respectively. Furthermore, the two different types of reorientational motions are distinguished from each other by their own characteristic scattering patterns and time scales. The measured time-resolved scattering signals are in excellent agreement with the simulated scattering signals based on a molecular dynamics simulation for plausible molecular configurations, providing the detailed structural description of the OKE response in liquid acetonitrile.

Filming ultrafast roaming-mediated isomerization of bismuth triiodide in solution.

Roaming reaction, defined as a reaction yielding products via reorientational motion in the long-range region (3 - 8 Å) of the potential, is a relatively recently proposed reaction pathway and is now regarded as a universal mechanism that can explain the unimolecular dissociation and isomerization of various molecules. The structural movements of the partially dissociated fragments originating from the frustrated bond fission at the onset of roaming, however, have been explored mostly via theoretical simulations and rarely observed experimentally. Here, we report an investigation of the structural dynamics during a roaming-mediated isomerization reaction of bismuth triiodide (BiI3) in acetonitrile solution using femtosecond time-resolved x-ray liquidography. Structural analysis of the data visualizes the atomic movements during the roaming-mediated isomerization process including the opening of the Bi-Ib-Ic angle and the closing of Ia-Bi-Ib-Ic dihedral angle, each by ~40°, as well as the shortening of the Ib···Ic distance, following the frustrated bond fission.

Uncovering the Conformational Distribution of a Small Protein with Nanoparticle-Aided Cryo-Electron Microscopy Sampling.

Here, we introduce the nanoparticle-aided cryo-electron microscopy sampling (NACS) method to access the conformational distribution of a protein molecule. Two nanogold particles are labeled at two target sites, and the interparticle distance is measured as a structural parameter via cryo-electron microscopy (cryo-EM). The key aspect of NACS is that the projected distance information instead of the global conformational information is extracted from each protein molecule. This is possible because the contrast provided by the nanogold particles is strong enough to provide the projected distance, while the protein itself is invisible due to its low contrast. We successfully demonstrate that various protein conformations, even for small or disordered proteins, which generally cannot be accessed via cryo-EM, can be captured. The demonstrated method with the potential to directly observe the conformational distribution of such systems may open up new possibilities in studying their dynamics at a single-molecule level.

Ultrafast coherent motion and helix rearrangement of homodimeric hemoglobin visualized with femtosecond X-ray solution scattering.

Ultrafast motion of molecules, particularly the coherent motion, has been intensively investigated as a key factor guiding the reaction pathways. Recently, X-ray free-electron lasers (XFELs) have been utilized to elucidate the ultrafast motion of molecules. However, the studies on proteins using XFELs have been typically limited to the crystalline phase, and proteins in solution have rarely been investigated. Here we applied femtosecond time-resolved X-ray solution scattering (fs-TRXSS) and a structure refinement method to visualize the ultrafast motion of a protein. We succeeded in revealing detailed ultrafast structural changes of homodimeric hemoglobin involving the coherent motion. In addition to the motion of the protein itself, the time-dependent change of electron density of the hydration shell was tracked. Besides, the analysis on the fs-TRXSS data of myoglobin allows for observing the effect of the oligomeric state on the ultrafast coherent motion.

Gold Nanoparticle Formation via X-ray Radiolysis Investigated with Time-Resolved X-ray Liquidography.

We report the generation of gold nanoparticles (AuNPs) from the aqueous solution of chloro(2,2',2″-terpyridine)gold(III) ion ([Au(tpy)Cl]2+) through X-ray radiolysis and optical excitation at a synchrotron. The original purpose of the experiment was to investigate the photoinduced structural changes of [Au(tpy)Cl]2+ upon 400 nm excitation using time-resolved X-ray liquidography (TRXL). Initially, the TRXL data did not show any signal that would suggest structural changes of the solute molecule, but after an induction time, the TRXL data started to show sharp peaks and valleys. In the early phase, AuNPs with two types of morphology, dendrites, and spheres, were formed by the reducing action of hydrated electrons generated by the X-ray radiolysis of water, thereby allowing the detection of TRXL data due to the laser-induced lattice expansion and relaxation of AuNPs. Along with the lattice expansion, the dendritic and spherical AuNPs were transformed into smaller, raspberry-shaped AuNPs of a relatively uniform size via ablation by the optical femtosecond laser pulse used for the TRXL experiment. Density functional theory calculations confirm that the reduction potential of the metal complex relative to the hydration potential of X-ray-generated electrons determines the facile AuNP formation observed for [Au(tpy)Cl]2+.

Mapping the emergence of molecular vibrations mediating bond formation.

Fundamental studies of chemical reactions often consider the molecular dynamics along a reaction coordinate using a calculated or suggested potential energy surface1-5. But fully mapping such dynamics experimentally, by following all nuclear motions in a time-resolved manner-that is, the motions of wavepackets-is challenging and has not yet been realized even for the simple stereotypical bimolecular reaction6-8: A-B + C â†’ A + B-C. Here we track the trajectories of these vibrational wavepackets during photoinduced bond formation of the gold trimer complex [Au(CN)2-]3 in an aqueous monomer solution, using femtosecond X-ray liquidography9-12 with X-ray free-electron lasers13,14. In the complex, which forms when three monomers A, B and C cluster together through non-covalent interactions15,16, the distance between A and B is shorter than that between B and C. Tracking the wavepacket in three-dimensional nuclear coordinates reveals that within the first 60 femtoseconds after photoexcitation, a covalent bond forms between A and B to give A-B + C. The second covalent bond, between B and C, subsequently forms within 360 femtoseconds to give a linear and covalently bonded trimer complex A-B-C. The trimer exhibits harmonic vibrations that we map and unambiguously assign to specific normal modes using only the experimental data. In principle, more intense X-rays could visualize the motion not only of highly scattering atoms such as gold but also of lighter atoms such as carbon and nitrogen, which will open the door to the direct tracking of the atomic motions involved in many chemical reactions.

Protein folding from heterogeneous unfolded state revealed by time-resolved X-ray solution scattering.

One of the most challenging tasks in biological science is to understand how a protein folds. In theoretical studies, the hypothesis adopting a funnel-like free-energy landscape has been recognized as a prominent scheme for explaining protein folding in views of both internal energy and conformational heterogeneity of a protein. Despite numerous experimental efforts, however, comprehensively studying protein folding with respect to its global conformational changes in conjunction with the heterogeneity has been elusive. Here we investigate the redox-coupled folding dynamics of equine heart cytochrome c (cyt-c) induced by external electron injection by using time-resolved X-ray solution scattering. A systematic kinetic analysis unveils a kinetic model for its folding with a stretched exponential behavior during the transition toward the folded state. With the aid of the ensemble optimization method combined with molecular dynamics simulations, we found that during the folding the heterogeneously populated ensemble of the unfolded state is converted to a narrowly populated ensemble of folded conformations. These observations obtained from the kinetic and the structural analyses of X-ray scattering data reveal that the folding dynamics of cyt-c accompanies many parallel pathways associated with the heterogeneously populated ensemble of unfolded conformations, resulting in the stretched exponential kinetics at room temperature. This finding provides direct evidence with a view to microscopic protein conformations that the cyt-c folding initiates from a highly heterogeneous unfolded state, passes through still diverse intermediate structures, and reaches structural homogeneity by arriving at the folded state.

Estimating signal and noise of time-resolved X-ray solution scattering data at synchrotrons and XFELs.

Elucidating the structural dynamics of small molecules and proteins in the liquid solution phase is essential to ensure a fundamental understanding of their reaction mechanisms. In this regard, time-resolved X-ray solution scattering (TRXSS), also known as time-resolved X-ray liquidography (TRXL), has been established as a powerful technique for obtaining the structural information of reaction intermediates and products in the liquid solution phase and is expected to be applied to a wider range of molecules in the future. A TRXL experiment is generally performed at the beamline of a synchrotron or an X-ray free-electron laser (XFEL) to provide intense and short X-ray pulses. Considering the limited opportunities to use these facilities, it is necessary to verify the plausibility of a target experiment prior to the actual experiment. For this purpose, a program has been developed, referred to as S-cube, which is short for a Solution Scattering Simulator. This code allows the routine estimation of the shape and signal-to-noise ratio (SNR) of TRXL data from known experimental parameters. Specifically, S-cube calculates the difference scattering curve and the associated quantum noise on the basis of the molecular structure of the target reactant and product, the target solvent, the energy of the pump laser pulse and the specifications of the beamline to be used. Employing a simplified form for the pair-distribution function required to calculate the solute-solvent cross term greatly increases the calculation speed as compared with a typical TRXL data analysis. Demonstrative applications of S-cube are presented, including the estimation of the expected TRXL data and SNR level for the future LCLS-II HE beamlines.

Fate of transient isomer of CH2I2: Mechanism and origin of ionic photoproducts formation unveiled by time-resolved x-ray liquidography.

Diiodomethane, CH2I2, in a polar solvent undergoes a unique photoinduced reaction whereby I2 - and I3 - are produced from its photodissociation, unlike for other iodine-containing haloalkanes. While previous studies proposed that homolysis, heterolysis, or solvolysis of iso-CH2I-I, which is a major intermediate of the photodissociation, can account for the formation of I2 - and I3 -, there has been no consensus on its mechanism and no clue for the reason why those negative ionic species are not observed in the photodissociation of other iodine-containing chemicals in the same polar solvent, for example, CHI3, C2H4I2, C2F4I2, I3 -, and I2. Here, using time-resolved X-ray liquidography, we revisit the photodissociation mechanism of CH2I2 in methanol and determine the structures of all transient species and photoproducts involved in its photodissociation and reveal that I2 - and I3 - are formed via heterolysis of iso-CH2I-I in the photodissociation of CH2I2 in methanol. In addition, we demonstrate that the high polarity of iso-CH2I-I is responsible for the unique photochemistry of CH2I2.

Structural Dynamics of Bismuth Triiodide in Solution Triggered by Photoinduced Ligand-to-Metal Charge Transfer.

Bismuth triiodide, BiI3, is one of the simplest bismuth halides, which have recently attracted considerable attention because of their promising properties. Here, we investigate the structural dynamics of a photoinduced reaction of BiI3 in solution phase using time-resolved X-ray liquidography (TRXL) and density functional theory (DFT) and time-dependent DFT (TDDFT) calculations. The photoreaction was initiated by excitation at 400 nm, which corresponds to the ligand-to-metal charge-transfer transition. The detailed structures and kinetic profiles of all relevant intermediate species from the TRXL data show that the trigonal planar structure of BiI3, which is predicted to be the most stable structure of the lowest excited state by TDDFT calculation, was not observed, and the photoreaction proceeds via two parallel pathways within the time resolution of 100 ps: (i) isomer formation to produce iso-BiI2-I, which relaxes back to the ground-state structure, and (ii) dissociation into BiI2· and I· radicals, which nongeminately recombine to generate ground-state BiI3 and I2.

Direct Observation of a Transiently Formed Isomer During Iodoform Photolysis in Solution by Time-Resolved X-ray Liquidography.

Photolysis of iodoform (CHI3) in solution has been extensively studied, but its reaction mechanism remains elusive. In particular, iso-iodoform (iso-CHI2-I) is formed as a product of the photolysis reaction, but its detailed structure is not known, and whether it is a major intermediate species has been controversial. Here, by using time-resolved X-ray liquidography, we determined the reaction mechanism of CHI3 photodissociation in cyclohexane as well as the structure of iso-CHI2-I. Both iso-CHI2-I and CHI2 radical were found to be formed within 100 ps with a branching ratio of 40:60. Iodine radicals (I), formed during the course of CHI3 photolysis, recombine nongeminately with either CHI2 or I. Based on our structural analysis, the I-I distance and the C-I-I angle of iso-CHI2-I were determined to be 2.922 ± 0.004 Å and 133.9 ± 0.8°, respectively.