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Thiophenylazobenzene: an alternative photoisomerization controlled by lone‐pair⋅⋅⋅π interaction
(2019)
Azoheteroarene photoswitches have attracted attention due to their unique properties. We present the stationary photochromism and ultrafast photoisomerization mechanism of thiophenylazobenzene (TphAB). It demonstrates impressive fatigue resistance and photoisomerization efficiency, and shows favorably separated (E)‐ and (Z)‐isomer absorption bands, allowing for highly selective photoconversion. The (Z)‐isomer of TphAB adopts an unusual orthogonal geometry where the thiophenyl group is perfectly perpendicular to the phenyl group. This geometry is stabilized by a rare lone‐pair⋅⋅⋅π interaction between the S atom and the phenyl group. The photoisomerization of TphAB occurs on the sub‐ps to ps timescale and is governed by this interaction. Therefore, the adoption and disruption of the orthogonal geometry requires significant movement along the inversion reaction coordinates (CNN and NNC angles). Our results establish TphAB as an excellent photoswitch with versatile properties that expand the application possibilities of AB derivatives.
The nuclear farnesoid X receptor (FXR) and the enzyme soluble epoxide hydrolase (sEH) are validated molecular targets to treat metabolic disorders such as non‐alcoholic steatohepatitis (NASH). Their simultaneous modulation in vivo has demonstrated a triad of anti‐NASH effects and thus may generate synergistic efficacy. Here we report dual FXR activators/sEH inhibitors derived from the anti‐asthma drug Zafirlukast. Systematic structural optimization of the scaffold has produced favorable dual potency on FXR and sEH while depleting the original cysteinyl leukotriene receptor antagonism of the lead drug. The resulting polypharmacological activity profile holds promise in the treatment of liver‐related metabolic diseases.
Gram‐negative bacteria are intrinsically resistant against cytotoxic substances by means of their outer membrane and a network of multidrug efflux systems, acting in synergy. Efflux pumps from various superfamilies with broad substrate preferences sequester and pump drugs across the inner membrane to supply the highly polyspecific and powerful tripartite resistance–nodulation–cell division (RND) efflux pumps with compounds to be extruded across the outer membrane barrier. In Escherichia coli, the tripartite efflux system AcrAB–TolC is the archetype RND multiple drug efflux pump complex. The homotrimeric inner membrane component acriflavine resistance B (AcrB) is the drug specificity and energy transduction center for the drug/proton antiport process. Drugs are bound and expelled via a cycle of mainly three consecutive states in every protomer, constituting a flexible alternating access channel system. This review recapitulates the molecular basis of drug and inhibitor binding, including mechanistic insights into drug efflux by AcrB. It also summarizes 17 years of mutational analysis of the gene acrB, reporting the effect of every substitution on the ability of E. coli to confer resistance toward antibiotics (http://goethe.link/AcrBsubstitutions). We emphasize the functional robustness of AcrB toward single‐site substitutions and highlight regions that are more sensitive to perturbation.
The industry‐scale production of methylchloromonosilanes in the Müller–Rochow Direct Process is accompanied by the formation of a residue, the direct process residue (DPR), comprised of disilanes MenSi2Cl6‐n (n=1–6). Great research efforts have been devoted to the recycling of these disilanes into monosilanes to allow reintroduction into the siloxane production chain. In this work, disilane cleavage by using alkali and alkaline earth metal salts is reported. The reaction with metal hydrides, in particular lithium hydride (LiH), leads to efficient reduction of chlorine containing disilanes but also induces disproportionation into mono‐ and oligosilanes. Alkali and alkaline earth chlorides, formed in the course of the reduction, specifically induce disproportionation of highly chlorinated disilanes, whereas highly methylated disilanes (n>3) remain unreacted. Nearly quantitative DPR conversion into monosilanes was achieved by using concentrated HCl/ether solutions in the presence of lithium chloride.
(Coumarin‐4‐yl)methyl (c4m) and p‐hydroxyphenacyl (pHP)‐based compounds are well known for their highly efficient photoreactions, but often show limited solubility in aqueous media. To circumvent this, we synthesized and characterized the two new c4m and pHP‐based photoacid generators (PAGs), 7‐[bis(carboxymethyl)amino]‐4‐(acetoxymethyl)coumarin (c4m‐ac) and p‐hydroxyphenacyl‐2,5,8,11‐tetraoxatridecan‐13‐oate (pHP‐t), and determined their solubilities, stabilities and photolysis in aqueous media. The two compounds showed high solubilities in water of 2.77 mmol L−1±0.07 mmol L−1 (c4m‐ac) and 124.66 mmol L−1±2.1 mmol L−1 (pHP‐t). In basic conditions at pH 9, solubility increased for c4m‐ac to 646.46 mmol L−1±0.63 mmol L−1, for pHP‐t it decreased to 34.68 mmol L−1±0.62 mmol L−1. Photochemical properties of the two PAGs, such as the absorption maxima, the maximum molar absorption coefficients and the quantum yields, were found to be strongly pH‐dependent. Both PAGs showed high stabilities s24h ≥95 % in water for 24 h, but decreasing stability with increasing pH value due to hydrolysis. The present study contributes to a clearer insight into the synthesis, solubilities, stabilities, and photolysis of c4m and pHP‐based PAGs for further photochemical applications when high PAG concentrations are required, such as in polymeric foaming.
Genetic code expansion facilitates position‐selective labeling of rna for biophysical studies
(2019)
Nature relies on reading and synthesizing the genetic code with high fidelity. Nucleic acid building blocks that are orthogonal to the canonical A‐T and G‐C base‐pairs are therefore uniquely suitable to facilitate position‐specific labeling of nucleic acids. Here, we employ the orthogonal kappa‐xanthosine‐base‐pair for in vitro transcription of labeled RNA. We devised an improved synthetic route to obtain the phosphoramidite of the deoxy‐version of the kappa nucleoside in solid phase synthesis. From this DNA template, we demonstrate the reliable incorporation of xanthosine during in vitro transcription. Using NMR spectroscopy, we show that xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7‐deaza‐xanthosine, which allows to site‐specifically modify transcribed RNA molecules with fluorophores or other labels.
De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the Type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT—malonyl‐/acetyltransferase) and the condensation (KS—β‐ketoacyl synthase) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate‐binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.
A novel method for the highly stereoselective synthesis of tetrahydropyrans is reported. This domino reaction is based on a twofold addition of enamides to aldehydes followed by a subsequent cyclization and furnishes fully substituted tetrahydropyrans in high yields. Three new σ‐bonds and five continuous stereogenic centers are formed in this one‐pot process with a remarkable degree of diastereoselectivity. In most cases, the formation of only one out of 16 possible diastereomers is observed. Two different stereoisomers can be accessed in a controlled fashion starting either from an E‐ or a Z‐configured enamide.
The fact that the interaction of oligonucleotides follows strict rules has been utilized to create two- or three-dimensional objects made of DNA. With computer-assisted design of DNA sequences, any arbitrary structure on the nanometer- to micrometer-scale can be generated just by hybridization of the needed strands. As astonishing these structures are, without any modification of the DNA strands involved no function can be assigned to them. Many different ways of functionalizing DNA-nanostructures have been developed with light-responsive nanostructures having a rather subordinated role. Almost all light responsive DNA-nanostructures involve the acyclic azobenzene-linking system tAzo based on D-threoninol which is known to work best at elevated temperatures to ensure optimal switching. As the structure of DNA-constructs is mainly maintained by hydrogen-bonding, variation of the temperature should be avoided in order to keep the structure intact.
To develop a light-responsive nanostructure model system with low-temperature operating azobenzene C-nucleosides, DNA-minicircles have been utilized. Those minicircles bear a lariat-like protrusion with a 10 base long single-stranded overhang, which is responsible for the dimerization with a ring bearing a complementary binding region. DNA-minicircles have been produced in a sequential manner by building and purifying the single stranded minicircle first by splint ligation and prepratative PAGE or RP-HPLC, followed by annealing it to the outer ring and subsequent purification by molecular-weight cut-off. Imaging of DNA-minicircles by atomic force microscopy (AFM) was possible with several methods of sample preparation leading to images of varying quality. With the help of AFM, qualitative analysis of the minicircles was possible. It could be shown, that theoretical and empirical size dimensions of the rings and their interactions were in great accordance. Designing the interaction site of the minicircles proved to be the main task in this project. The amount of C-nucleosidic modifications was identified by screening, followed by a screening of their optimal position and binding partners in the counterstrand. Two azobenzene C-nucleosides in a 10mer binding region and abasic sites opposing them appeared to give the best compromise between absolute dimerization ratio and photocontrolled change of it, as identified by native PAGE. In the following, the dimerization ratios of minicircles containing azobenzene C-nucleosides were compared with minicircles containing tAzo and unmodified minicircles. It could be shown, that the tAzo-modification leads to an elevated binding affinity compared to the unmodified minicircles, but the change upon irradiation is relatively humble compared to the C-nucleosides. For the C-nucleosidic modifications dimerization ratios reached a maximum of 40% in favored trans-state, but could be almost completely turned-off when switching into cis-state. In addition, arylazopyrazole-modified C-nucleosides could be switched into trans-state by irradiating at 530 nm, which is an improvement compared to standard azobenzene, as it shifts irradiation wavelength closer to the phototherapeutic window.
The utilization of DNA-analogous C-nucleosides bring two drawbacks with them: the ribose units include the flexibility of the sugar conformation and it is reasonable to think, that upon isomerization of the azobenzene, part of the steric stress generated is compensated by the sugar reconfiguration, which is lost for duplex
destabilization. In addition, the combination of the ribosidic linker end the end-to-end distance of trans-azobenzene causes the chromophore to penetrate deep into the base stack of the opposing strand, causing a serious destabilization even in favored trans-state. The goal was to find a linker system, that combines the benefits of the azobenzene C-nucleoside without the possibility to change sugar conformation and the strong destabilization in the trans-state. For this reason locked azobenzene C-nucleosides in analogy to LNA nucleosides have been synthesized. The synthesis of LNA analogous azobenzene C-nucleosides (LNAzo) was possible over a 16-step synthesis, with the critical step being the addition of in situ lithiated azobenzene to protected sugar aldehyde. Both anomers of LNAzo and mAzo as reference where incorporated into different oligonucleotide test systems by solid phase synthesis for thorough evaluation. It could be shown, that LNAzo β has a similar performance to mAzo in DNA with overall slightly increased TM- and ΔTM-values. Performance of LNAzo β was similar to mAzo even if steric stress is reduced by using abasic sites in the counterstrand opposing the azobenzene. Only in a RNA context, the true potential of LNAzo β could be observed. In a DNA/RNA duplex, photocontrol could be improved by almost 50%, in a RNA/RNA duplex even by over 100%. Although the primary goal was the improvement of the azobenzene C-nucleoside for a DNA-nanostructure context, LNAzo β proved not to give a sufficient improvement in regard to the cost-value ratio. Never the less, the invention of the locked azobenzene C-nucleoside was a huge success for reversible photoregulation of RNA hybridization. With this, a new way to regulate RNA hybridization has been found, which could be used to create RNA therapeutics in an antisense-approach.
As LNAzo β improved duplex stability only in a limited amount in DNA, further improvements on the backbone have been declared futile and focus shifted onto optimization of the chromophore. First, the azobenzene as it is installed on the ribosidic linker decreases duplex stability by forcing its distal aromat deep into opposing base stacking region. It would be an improvement, if in favored trans-state the distal aromat would be positioned in the less confined space of either major or minor groove and only upon isomerization would shift into base pairing region. Second, the azobenzene itself is not able to contribute to attractive interactions aside from relatively weak π-interactions to adjacent nucleobases, which could be improved, if it could partake in hydrogen bonding. For those apparent reasons, 2-phenyldiazenyl-modified purines have been selected as targets. They combine the ability to contribute to hydrogen bonding of nucleobases with the photochomicity of azobenzenes. Both 2’-deoxyadenosine- and 2’-deoxyguanosine-analogue photoswitches dAAzo and dGAzo have been synthesized and incorporated into 10mer DNA test systems by solid phase synthesis. It could be shown, that duplex stability could be increased compared to established azobenzene C-nucleoside. The improvement was stronger for dAAzo than for dGAzo as in the case for guanosine the amino function on the C2-position had to be replaced by the phenyldiazenyl function, reducing its ability to form hydrogen bonds. Unfortunately, photocontrol of duplex stability caused by 2-phenyldiazenyl purines was rather limited. A reason for this could be the positioning of the distal aromat within the duplex, which can be close to the opposing nucleobase (endo-helical) or in greater distance (exo-helical). The exo-helical conformation of the trans-isomer can only switch to the exo-P-cis-conformation, which relocates the distal aromat in the minor groove, without significant impact on duplex stability.
Inhibition of F1Fo ATP synthases by bacterial
virulence factors and photoswitchable azopolyphenols
(2019)
F1Fo ATP synthases are important membrane-embedded nano-machines which are conserved among all three kingdoms of life. They use a proton or sodium gradient across the membrane to drive ATP synthesis, which is the major source of energy for the cell. As ATP synthases are essential for pathogens such as mycobacteria, they are important drug targets for the treatment of infectious diseases. In this work, structural studies on the E. coli ATP synthase are performed. Furthermore, bacterial virulence MgtC proteins are investigated. Additionally, photo-switches are used to spatiotemporally control yeast ATPase activity...
Advanced colorectal carcinoma is currently incurable, and new therapies are urgently needed. We report that phosphotyrosine-dependent Eph receptor signaling sustains colorectal carcinoma cell survival, thereby uncovering a survival pathway active in colorectal carcinoma cells. We find that genetic and biochemical inhibition of Eph tyrosine kinase activity or depletion of the Eph ligand EphrinB2 reproducibly induces colorectal carcinoma cell death by autophagy. Spautin and 3-methyladenine, inhibitors of early steps in the autophagic pathway, significantly reduce autophagy-mediated cell death that follows inhibition of phosphotyrosine-dependent Eph signaling in colorectal cancer cells. A small-molecule inhibitor of the Eph kinase, NVP-BHG712 or its regioisomer NVP-Iso, reduces human colorectal cancer cell growth in vitro and tumor growth in mice. Colorectal cancers express the EphrinB ligand and its Eph receptors at significantly higher levels than numerous other cancer types, supporting Eph signaling inhibition as a potential new strategy for the broad treatment of colorectal carcinoma.
High-resolution cryo-EM structures of respiratory complex I: Mechanism, assembly, and disease
(2019)
Respiratory complex I is a redox-driven proton pump, accounting for a large part of the electrochemical gradient that powers mitochondrial adenosine triphosphate synthesis. Complex I dysfunction is associated with severe human diseases. Assembly of the one-megadalton complex I in the inner mitochondrial membrane requires assembly factors and chaperones. We have determined the structure of complex I from the aerobic yeast Yarrowia lipolytica by electron cryo-microscopy at 3.2-Å resolution. A ubiquinone molecule was identified in the access path to the active site. The electron cryo-microscopy structure indicated an unusual lipid-protein arrangement at the junction of membrane and matrix arms that was confirmed by molecular simulations. The structure of a complex I mutant and an assembly intermediate provide detailed molecular insights into the cause of a hereditary complex I-linked disease and complex I assembly in the inner mitochondrial membrane.
Rhodopsin-based voltage imaging tools for use in muscles and neurons of Caenorhabditis elegans
(2019)
Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of all-trans retinal (ATR), while in electrochromic FRET (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. In recent years, such tools have become widely used in mammalian cells but are less commonly used in invertebrate systems, mostly due to low fluorescence yields. We systematically assessed Arch(D95N), Archon, QuasAr, and the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in the nematode Caenorhabditis elegans ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs), in the pharynx, the feeding organ [where Arch(D95N) showed approximately 128% ΔF/F increase per 100 mV], and in neurons, integrating circuit activity. ATR fluorescence is very dim, yet, using the retinal analog dimethylaminoretinal, it was boosted 250-fold. eFRET sensors provided sensitivities of 45 to 78% ΔF/F per 100 mV, induced by BWM action potentials, and in pharyngeal muscle, measured in simultaneous optical and sharp electrode recordings, MacQ-mCitrine showed approximately 20% ΔF/F per 100 mV. All sensors reported differences in muscle depolarization induced by a voltage-gated Ca2+-channel mutant. Optogenetically evoked de- or hyperpolarization of motor neurons increased or eliminated action potential activity and caused a rise or drop in BWM sensor fluorescence. Finally, we analyzed voltage dynamics across the entire pharynx, showing uniform depolarization but compartmentalized repolarization of anterior and posterior parts. Our work establishes all-optical, noninvasive electrophysiology in live, intact C. elegans.
Remote control of the synthesis of a [2]rotaxane and its shuttling via metal‐ion translocation
(2019)
Remote control in an eight‐component network commanded both the synthesis and shuttling of a [2]rotaxane via metal‐ion translocation, the latter being easily monitored by distinct colorimetric and fluorimetric signals. Addition of zinc(II) ions to the red colored copper‐ion relay station rapidly liberated copper(I) ions and afforded the corresponding zinc complex that was visualized by a bright sky blue fluorescence at 460 nm. In a mixture of all eight components of the network, the liberated copper(I) ions were translocated to a macrocycle that catalyzed formation of a rotaxane by a double‐click reaction of acetylenic and diazide compounds. The shuttling frequency in the copper‐loaded [2]rotaxane was determined to k298=30 kHz (ΔH≠=62.3±0.6 kJ mol−1, ΔS≠=50.1±5.1 J mol−1 K−1, ΔG≠298=47.4 kJ mol−1). Removal of zinc(II) ions from the mixture reversed the system back generating the metal‐free rotaxane. Further alternate addition and removal of Zn2+ reversibly controlled the shuttling mode of the rotaxane in this eight‐component network where the ion translocation status was monitored by the naked eye.
Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, Cα, Cβ, C′) resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.
Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in‐cell NMR spectroscopy experiments. We are able to monitor real‐time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer‐based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.
Current metabolomics approaches utilize cellular metabolite extracts, are destructive, and require high cell numbers. We introduce here an approach that enables the monitoring of cellular metabolism at lower cell numbers by observing the consumption/production of different metabolites over several kinetic data points of up to 48 hours. Our approach does not influence cellular viability, as we optimized the cellular matrix in comparison to other materials used in a variety of in‐cell NMR spectroscopy experiments. We are able to monitor real‐time metabolism of primary patient cells, which are extremely sensitive to external stress. Measurements are set up in an interleaved manner with short acquisition times (approximately 7 minutes per sample), which allows the monitoring of up to 15 patient samples simultaneously. Further, we implemented our approach for performing tracer‐based assays. Our approach will be important not only in the metabolomics fields, but also in individualized diagnostics.
To date, in-cell NMR has elucidated various aspects of protein behaviour by associating structures in physiological conditions. Meanwhile, current studies of this method mostly have deduced protein states in cells exclusively based on ‘indirect’ structural information from peak patterns and chemical shift changes but not ‘direct’ data explicitly including interatomic distances and angles. To fully understand the functions and physical properties of proteins inside cells, it is indispensable to obtain explicit structural data or determine three-dimensional (3D) structures of proteins in cells. Whilst the short lifetime of cells in a sample tube, low sample concentrations, and massive background signals make it difficult to observe NMR signals from proteins inside cells, several methodological advances help to overcome the problems. Paramagnetic effects have an outstanding potential for in-cell structural analysis. The combination of a limited amount of experimental in-cell data with software for ab initio protein structure prediction opens an avenue to visualise 3D protein structures inside cells. Conventional nuclear Overhauser effect spectroscopy (NOESY)-based structure determination is advantageous to elucidate the conformations of side-chain atoms of proteins as well as global structures. In this article, we review current progress for the structure analysis of proteins in living systems and discuss the feasibility of its future works.
Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection.
Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator cavity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.