19F-NMR Unveils the Ligand-Induced Conformation of a Catalytically Inactive Twisted Homodimer of tRNA-Guanine Transglycosylase.

Understanding the structural arrangements of protein oligomers can support the design of ligands that interfere with their function in order to develop new therapeutic concepts for disease treatment. Recent crystallographic studies have elucidated a novel twisted and functionally inactive form of the homodimeric enzyme tRNA-guanine transglycosylase (TGT), a putative target in the fight against shigellosis. Active-site ligands have been identified that stimulate the rearrangement of one monomeric subunit by 130° against the other one to form an inactive twisted homodimer state. To assess whether the crystallographic observations also reflect the conformation in solution and rule out effects from crystal packing, we performed 19F-NMR spectroscopy with the introduction of 5-fluorotryptophans at four sites in TGT. The inhibitor-induced conformation of TGT in solution was assessed based on 19F-NMR chemical shift perturbations. We investigated the effect of C(4) substituted lin-benzoguanine ligands and identified a correlation between dynamic protein rearrangements and ligand-binding features in the corresponding crystal structures. These involve the destabilization of a helix next to the active site and the integrity of a flexible loop-helix motif. Ligands that either completely lack an attached C(4) substituent or use it to stabilize the geometry of the functionally competent dimer state do not indicate the presence of the twisted dimer form in the NMR spectra. The perturbation of crucial structural motifs in the inhibitors correlates with an increasing formation of the inactive twisted dimer state, suggesting these ligands are able to shift a conformational equilibrium from active C2-symmetric to inactive twisted dimer conformations. These findings suggest a novel concept for the design of drug candidates for further development.

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.

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.

Fragment Screening Hit Draws Attention to a Novel Transient Pocket Adjacent to the Recognition Site of the tRNA-Modifying Enzyme TGT.

Fragment-based lead discovery was applied to tRNA-guanine transglycosylase, an enzyme modifying post-transcriptionally tRNAs in Shigella, the causative agent of shigellosis. TGT inhibition prevents translation of Shigella's virulence factor VirF, hence reducing pathogenicity. One discovered fragment opens a transient subpocket in the preQ1-recognition site by pushing back an aspartate residue. This step is associated with reorganization of further amino acids structurally transforming a loop adjacent to the recognition site by duplicating the volume of the preQ1-recognition pocket. We synthesized 6-carboxamido-, 6-hydrazido-, and 4-guanidino-benzimidazoles to target the opened pocket, including a dihydro-imidazoquinazoline with a propyn-1-yl exit vector pointing into the transient pocket and displacing a conserved water network. MD simulations and hydration-site analysis suggest water displacement to contribute favorably to ligand binding. A cysteine residue, exclusively present in bacterial TGTs, serves as gatekeeper of the transient subpocket. It becomes accessible upon pocket opening for selective covalent attachment of electrophilic ligands in eubacterial TGTs.

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.

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.

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.

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.

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.

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.

Supramolecular Capsules: Strong versus Weak Chalcogen Bonding.

Resorcin[4]arene cavitands containing either 2,1,3-benzotelluradiazole or 2,1,3-benzothiadiazole motifs were dimerized to supramolecular capsules by chalcogen bonding. Their respective behavior varied depending on the interaction strength of the chalcogen bonds with Te forming strong interactions and S weak interactions. The tremendous strength of multiple 2Te-2N square interactions led to formation of a chalcogen-bonded dimeric capsule in all solvents, as shown by X-ray crystal structures with 16 short Te⋅⋅⋅N distances (≤2.9 Å) and confirmed by native electrospray ionization mass spectrometry (ESI-MS). With the S cavitand, solvent-dependent crystallization resulted in different arrangements: either a shifted 2S-2N square-bonded capsule or an interlocked 1D polymer with an infinite π-π stacking array. The association constant to form the dimeric capsule in [D8 ]THF at 283 K, solely based on weak 2S-2N square interactions, was determined as Ka =786 m-1 .

Hydrogen-Bonded Networks: Molecular Recognition of Cyclic Alcohols in Enantiopure Alleno-Acetylenic Cage Receptors.

Enantiopure (P)4 - and (M)4 -alleno-acetylenic cage (AAC) receptors form circular fourfold hydrogen-bonding networks in their closed cage conformation. Theoretical studies reveal a preferential clockwise (cw) orientation of the H-bonding array for (P)4 -configured and counterclockwise (ccw) for (M)4 -configured receptors (ΔEcw-ccw =-2.6 to -3.1 kcal mol-1 ). Solution and solid-state studies show how the H-bonding network of the receptor is expanded upon encapsulation of alcohol-containing guests. Topologies reminiscent of those found in isolated water clusters are observed: circular fourfold & docking, pentagonal, linear fivefold, and hexagonal boat-shaped. Expansion of the H-bonding network together with optimal space occupancy yields very high ligand affinities (ΔG293 K =-9.0 kcal mol-1 for endo-tropine). The H-bonding network in the complexes also contributes substantially to the enantioselective complexation of chiral diols, such as (R,R)- and (S,S)-trans-cyclohexane-1,2-diol.

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.

Aryl bis-sulfonamides bind to the active site of a homotrimeric isoprenoid biosynthesis enzyme IspF and extract the essential divalent metal cation cofactor.

Characterizing the mode of action of non-covalent inhibitors in multisubunit enzymes often presents a great challenge. Most of the conventionally used methods are based on ensemble measurements of protein-ligand binding in bulk solution. They often fail to accurately describe multiple binding processes occurring in such systems. Native electrospray ionization mass spectrometry (ESI-MS) of intact protein complexes is a direct, label-free approach that can render the entire distribution of ligand-bound states in multimeric protein complexes. Here we apply native ESI-MS to comprehensively characterize the isoprenoid biosynthesis enzyme IspF from Arabidopsis thaliana, an example of a homomeric protein complex with multiple binding sites for several types of ligands, including a metal cofactor and a synthetic inhibitor. While standard biophysical techniques failed to reveal the mode of action of recently discovered aryl-sulfonamide-based inhibitors of AtIspF, direct native ESI-MS titrations of the protein with the ligands and ligand competition assays allowed us to accurately capture the solution-phase protein-ligand binding equilibria in full complexity and detail. Based on these combined with computational modeling, we propose a mechanism of AtIspF inhibition by aryl bis-sulfonamides that involves both the competition with the substrate for the ligand-binding pocket and the extraction of Zn2+ from the enzyme active site. This inhibition mode is therefore mixed competitive and non-competitive, the latter exerting a key inhibitory effect on the enzyme activity. The results of our study deliver a profound insight into the mechanisms of AtIspF action and inhibition, open new perspectives for designing inhibitors of this important drug target, and demonstrate the applicability and value of the native ESI-MS approach for deep analysis of complex biomolecular binding equilibria.

Charge-Transfer Salts of 6,6-Dicyanopentafulvenes: From Topology to Charge Separation in Solution.

6,6-Dicyanopentafulvene derivatives and metallocenes with redox potentials appropriate for forming their radical anions form highly persistent donor-acceptor salts. The charge-transfer salts of 2,3,4,5-tetraphenyl-6,6-dicyanofulvene with cobaltocene (1⋅Cp2 Co) and 2,3,4,5-tetrakis(triisopropylsilyl)-6,6-dicyanofulvene with decamethylferrocene (2⋅Fc*) have been prepared. The X-ray structures of the two salts, formed as black plates, were obtained and are discussed herein. Compared with neutral dicyanopentafulvenes, the chromophores in the metallocene salts show substantial changes in bond lengths and torsional angles in the solid state. EPR, NMR, and optical spectroscopy, as well as superconducting quantum interference device (SQUID) measurements, reveal that charge-separation in the crystalline states and in frozen and fluid solutions depends on subtle differences of redox potentials, geometry, and on ion pairing. Whereas 1⋅Cp2 Co reveals paramagnetic character in the crystalline state and in solution, compound 2⋅Fc* shows a delicate balance between para- and diamagnetism, depending on the temperature and solvent characteristics.

Swapping Interface Contacts in the Homodimeric tRNA-Guanine Transglycosylase: An Option for Functional Regulation.

The enzyme tRNA-guanine transglycosylase, a target to fight Shigellosis, recognizes tRNA only as a homodimer and performs full nucleobase exchange at the wobble position. Active-site inhibitors block the enzyme function by competitively replacing tRNA. In solution, the wild-type homodimer dissociates only marginally, whereas mutated variants show substantial monomerization in solution. Surprisingly, one inhibitor transforms the protein into a twisted state, whereby one monomer unit rotates by approximately 130°. In this altered geometry, the enzyme is no longer capable of binding and processing tRNA. Three sugar-type inhibitors have been designed and synthesized, which bind to the protein in either the functionally competent or twisted inactive state. They crystallize with the enzyme side-by-side under identical conditions from the same crystallization well. Possibly, the twisted inactive form corresponds to a resting state of the enzyme, important for its functional regulation.

Sugar Acetonides are a Superior Motif for Addressing the Large, Solvent-Exposed Ribose-33 Pocket of tRNA-Guanine Transglycosylase.

The intestinal disease shigellosis caused by Shigella bacteria affects over 120 million people annually. There is an urgent demand for new drugs as resistance against common antibiotics emerges. Bacterial tRNA-guanine transglycosylase (TGT) is a druggable target and controls the pathogenicity of Shigella flexneri. We report the synthesis of sugar-functionalized lin-benzoguanines addressing the ribose-33 pocket of TGT from Zymomonas mobilis. Ligand binding was analyzed by isothermal titration calorimetry and X-ray crystallography. Pocket occupancy was optimized by variation of size and protective groups of the sugars. The participation of a polycyclic water-cluster in the recognition of the sugar moiety was revealed. Acetonide-protected ribo- and psicofuranosyl derivatives are highly potent, benefiting from structural rigidity, good solubility, and metabolic stability. We conclude that sugar acetonides have a significant but not yet broadly recognized value in drug development.

Unconventional Synthesis of a CuI Rotaxane with a Superacceptor Stopper: Ultrafast Excited-State Dynamics and Near-Infrared Luminescence.

A CuI bis-phenanthroline rotaxane was prepared by using the [2+2] cycloaddition-retroelectrocyclization (CA-RE) reaction to graft a bulky dicyanoquinodimethane (DCNQ) stopper. The electronic properties were investigated with electrochemical and photophysical techniques, in parallel with three reference compounds, namely, the DCNQ derivative alone, the DCNQ-based phenanthroline ligand, and an analogue CuI complex lacking the DCNQ moiety. In all the systems containing the DCNQ unit, the lowest electronic excited states are centered thereon, with the singlet level (S1 ) located at about 1.0 eV, as suggested by TDDFT calculations. Accordingly, in the DCNQ-equipped rotaxane, the typical metal-to-ligand charge-transfer luminescence of the CuI center is totally quenched. Ultrafast transient absorption and emission studies show that, in the rotaxane, the final sink of photoinduced processes is the lowest singlet state of the DCNQ moiety (S1 ), which exhibits strong charge-transfer character and a lifetime of 0.4 ps. Its deactivation leads to population of another excited state with a lifetime of 1.3 ps, which can be the related triplet state (T1 ) or a vibrationally hot level (hot-S0 ). Notably, S1 also shows stimulated fluorescence in the near-infrared (NIR) region between 1100 and 1500 nm, corroborating the TDDFT prediction. This unusual finding opens up the study of ultrashort-lived NIR luminescence in organic donor-acceptor systems.

Biological Evaluation and X-ray Co-crystal Structures of Cyclohexylpyrrolidine Ligands for Trypanothione Reductase, an Enzyme from the Redox Metabolism of Trypanosoma.

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure-based design. The best inhibitors were freely soluble and showed competitive inhibition constants (Ki ) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half-maximal inhibitory concentration, IC50 ) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X-ray co-crystal structures confirmed the binding of the ligands to the hydrophobic wall of the "mepacrine binding site" with the new, solubility-providing vectors oriented toward the surface of the large active site.