The hit-and-return system enables efficient time-resolved serial synchrotron crystallography.

We present a 'hit-and-return' (HARE) method for time-resolved serial synchrotron crystallography with time resolution from milliseconds to seconds or longer. Timing delays are set mechanically, using the regular pattern in fixed-target crystallography chips and a translation stage system. Optical pump-probe experiments to capture intermediate structures of fluoroacetate dehalogenase binding to its ligand demonstrated that data can be collected at short (30 ms), medium (752 ms) and long (2,052 ms) intervals.

Unraveling a Ligand-Induced Twist of a Homodimeric Enzyme by Pulsed Electron-Electron Double Resonance.

Mechanistic insights into protein-ligand interactions can yield chemical tools for modulating protein function and enable their use for therapeutic purposes. For the homodimeric enzyme tRNA-guanine transglycosylase (TGT), a putative virulence target of shigellosis, ligand binding has been shown by crystallography to transform the functional dimer geometry into an incompetent twisted one. However, crystallographic observation of both end states does neither verify the ligand-induced transformation of one dimer into the other in solution nor does it shed light on the underlying transformation mechanism. We addressed these questions in an approach that combines site-directed spin labeling (SDSL) with distance measurements based on pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. We observed an equilibrium between the functional and twisted dimer that depends on the type of ligand, with a pyranose-substituted ligand being the most potent one in shifting the equilibrium toward the twisted dimer. Our experiments suggest a dissociation-association mechanism for the formation of the twisted dimer upon ligand binding.

Molecular Recognition and Cocrystallization of Methylated and Halogenated Fragments of Danicalipin A by Enantiopure Alleno-Acetylenic Cage Receptors.

Enantiopure (P)4- and (M)4-configured alleno-acetylenic cage (AAC) receptors offer a highly defined interior for the complexation and structure elucidation of small molecule fragments of the stereochemically complex chlorosulfolipid danicalipin A. Solution (NMR), solid state (X-ray), and theoretical investigations of the formed host-guest complexes provide insight into the conformational preferences of 14 achiral and chiral derivatives of the danicalipin A chlorohydrin core in a confined, mostly hydrophobic environment, extending previously reported studies in polar solvents. The conserved binding mode of the guests permits deciphering the effect of functional group replacements on Gibbs binding energies ΔG. A strong contribution of conformational energies toward the binding affinities is revealed, which explains why the denser packing of larger apolar domains of the guests does not necessarily lead to higher association. Enantioselective binding of chiral guests, with energetic differences ΔΔG293 K up to 0.7 kcal mol-1 between diastereoisomeric complexes, is explained by hydrogen- and halogen-bonding, as well as dispersion interactions. Calorimetric studies (ITC) show that the stronger binding of one enantiomer is accompanied by an increased gain in enthalpy ΔH but at the cost of a larger entropic penalty TΔS stemming from tighter binding.

Stimuli-Responsive Resorcin[4]arene Cavitands: Toward Visible-Light-Activated Molecular Grippers.

Resorcin[4]arene cavitands, equipped with diverse quinone (Q) and [Ru(bpy)2 dppz]2+ (bpy=2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c]phenazine) photosensitizing walls in different configurations, were synthesized. Upon visible-light irradiation at 420 nm, electron transfer from the [Ru(bpy)2 dppz]2+ to the Q generates the semiquinone (SQ) radical anion, triggering a large conformational switching from a flat kite to a vase with a cavity for the encapsulation of small guests, such as cyclohexane and heteroalicyclic derivatives, in CD3 CN. Depending on the molecular design, the SQ radical anion can live for several minutes (≈10 min) and the vase can be generated in a secondary process without need for addition of a sacrificial electron donor to accumulate the SQ state. Switching can also be triggered by other stimuli, such as changes in solvent, host-guest complexation, and chemical and electrochemical processes. This comprehensive investigation benefits the development of stimuli-responsive nanodevices, such as light-activated molecular grippers.

The Quest for Molecular Grippers: Photo-Electric Control of Molecular Gripping Machinery.

The quest for nanoscale molecular machines has inspired the search for their close relatives, molecular grippers. This path was paved by the development of resorcin[4]arene cavitands and their quinone-based redox-active congeners. In this Concept article, the efforts to design and establish the control of quinone-functionalized resorcin[4]arenes by electronic and electromagnetic stimuli is described. This was achieved by relying on paramagnetic semiquinone radical anions formed electrochemically or by photoredox catalysis. The gripper-like motion of such species could not be studied by conventional NMR spectroscopy. Instead, an entirely different approach had to be developed that included various electroanalytical and spectroelectrochemical methods, including UV/Vis/NIR spectroelectrochemistry, pulsed EPR and Davies 1 H ENDOR spectroscopy, transient absorption spectroscopy, and time-resolved luminescence measurements, besides density functional theory calculations and X-ray crystallography. The conceptual breakthroughs are reviewed as well as the current state and future perspectives of photoredox-switchable molecular grippers.

Helicene Monomers and Dimers: Chiral Chromophores Featuring Strong Circularly Polarized Luminescence.

The synthesis and chiroptical properties of a series of enantiomerically pure, C2 -symmetrical carbo[6]helicene dimers are reported. Two helicene cores are connected through a buta-1,3-diyne-1,4-diyl linker or a heteroaromatic bridge and bear arylethynyl substituents at their 15-positions. This ensures the possibility of extended electronic communication throughout the whole molecule. The new chromophores exhibit intense ECD spectra with strong bands in the UV/Vis region well above 400 nm. The anisotropy factor gabs (defined as Δϵ/ϵ) reaches values up to 0.047, which are unusually large for single organic molecules. They also display blue fluorescence, with good quantum yields (Φf ∼0.25). The emitted light is circularly polarized to an outstanding extent: in some cases, the luminescence dissymmetry factor glum =2(IL -IR )/(IL +IR ) attains values of |0.025|. To the best of our knowledge, such values are among the highest ever reported for non-aggregated organic fluorophores.

Chalcogen Bonding "2S-2N Squares" versus Competing Interactions: Exploring the Recognition Properties of Sulfur.

Chalcogen bonding (CB) is the focus of increased attention for its applications in medicinal chemistry, materials science, and crystal engineering. However, the origin of sulfur's recognition properties remains controversial, and experimental evidence for supporting theories is still emerging. Here, a comprehensive evaluation of sulfur CB interactions is presented by investigating 2,1,3-benzothiadiazole X-ray crystallographic structures gathered from the Cambridge Structure Database (CSD), Protein Data Bank (PDB), and own laboratory findings. Through the systematic analysis of substituent effects on a subset library of over thirty benzothiadiazole derivatives, the competing interactions have been categorized into four main classes, namely 2S-2N CB square, halogen bonding (XB), S⋅⋅⋅S, and hydrogen-bonding (HB). A geometric model is employed to characterize the 2S-2N CB square motifs and discuss the role of electrostatic, dipole, and orbital contributions toward the interaction.

Targeting a Large Active Site: Structure-Based Design of Nanomolar Inhibitors of Trypanosoma brucei Trypanothione Reductase.

Trypanothione reductase (TR) plays a key role in the unique redox metabolism of trypanosomatids, the causative agents of human African trypanosomiasis (HAT), Chagas' disease, and leishmaniases. Introduction of a new, lean propargylic vector to a known class of TR inhibitors resulted in the strongest reported competitive inhibitor of Trypanosoma (T.) brucei TR, with an inhibition constant Ki of 73 nm, which is fully selective against human glutathione reductase (hGR). The best ligands exhibited in vitro IC50 values (half-maximal inhibitory concentration) against the HAT pathogen, T. brucei rhodesiense, in the mid-nanomolar range, reaching down to 50 nm. X-Ray co-crystal structures confirmed the binding mode of the ligands and revealed the presence of a HEPES buffer molecule in the large active site. Extension of the propargylic vector, guided by structure-based design, to replace the HEPES buffer molecule should give inhibitors with low nanomolar Ki and IC50 values for in vivo studies.

Molecular Recognition with Resorcin[4]arene Cavitands: Switching, Halogen-Bonded Capsules, and Enantioselective Complexation.

The development of synthetic host-guest chemistry to investigate and quantify weak, non-covalent interactions has been key to unraveling the complexity of molecular recognition in chemical and biological systems. Macrocycles have shown great utility in the design of receptors, enabling the development of highly preorganized structures. Among macrocycles, resorcin[4]arene-based cavitands have become privileged scaffolds due to their synthetic tunability, which allows access to structures with precisely defined geometries, as well as receptors that display conformational switching between two distinct states with a large difference in guest-binding properties. Here, we highlight three case studies demonstrating redox- and photoredox-controlled switching of molecular recognition properties, the formation of guest-binding supramolecular capsules based solely on halogen-bonding interactions, and enantioselective encapsulation of chiral, substituted cyclohexanes by enantiopure cage compounds as a result of perfect shape complementarity, dispersion interactions, and halogen bonding. The high geometrical and conformational control that can be achieved with resorcin[4]arene-derived host systems will continue to be a powerful resource in future molecular recognition studies.

Selective Extraction of C70 by a Tetragonal Prismatic Porphyrin Cage.

Along with the advent of supramolecular chemistry, research on fullerene receptors based on noncovalent bonding interactions has attracted a lot of attention. Here, we present the design and synthesis of a cationic molecular cage: a cyclophane composed of two tetraphenylporphyrins, bridged face-to-face by four viologen units in a rhomboid prismatic manner. The large cavity inside the cage, as well as the favorable donor-acceptor interactions between the porphyrin panels and the fullerene guests, enables the cage to be an excellent fullerene receptor. The 1:1 host-guest complexes formed between the cage and both C60 and C70 were characterized in solution by HRMS and NMR, UV-vis and fluorescence spectroscopies, and confirmed in the solid state by single-crystal X-ray diffraction analyses. The results from solution studies reveal that the cage has a much stronger binding for C70 than for C60, resulting in a selective extraction of C70 from a C60-enriched fullerene mixture (C60/C70 = 10/1), demonstrating the potential of the cage as an attractive receptor for fullerene separation.