Speeding up biomolecular interactions by molecular sledding

Numerous biological processes involve association of a protein with its binding partner, an event that is preceded by a diffusion-mediated search bringing the two partners together. Often hindered by crowding in biologically relevant environments, three-dimensional diffusion can be slow and result in long bimolecular association times. Similarly, the initial association step between two binding partners often represents a rate-limiting step in biotechnologically relevant reactions. We demonstrate the practical use of an 11-a.a. DNA-interacting peptide derived from adenovirus to reduce the dimensionality of diffusional search processes and speed up associations between biological macromolecules. We functionalize binding partners with the peptide and demonstrate that the ability of the peptide to one-dimensionally diffuse along DNA results in a 20-fold reduction in reaction time. We also show that modifying PCR primers with the peptide sled enables significant acceleration of standard PCR reactions.     

Diaryldichalcogenide radical cations

One-electron oxidation of two series of diaryldichalcogenides (C₆F₅E)₂ (13a–c) and (2,6-Mes₂C₆H₃E)₂ (16a–c) was studied (E = S, Se, Te). The reaction of 13a and 13b with AsF₅ and SbF₅ gave rise to the formation of thermally unstable radical cations [(C₆F₅S)₂]˙⁺ (14a) and [(C₆F₅Se)₂]˙⁺ (14b) that were isolated as [Sb₂F₁₁]^– and [As₂F₁₁]^– salts, respectively. The reaction of 13c with AsF₅ afforded only the product of a Te–C bond cleavage, namely the previously known dication [Te₄]²⁺ that was isolated as [AsF₆]^– salt. The reaction of (2,6-Mes₂C₆H₃E)₂ (16a–c) with [NO][SbF₆] provided the corresponding radical cations [(2,6-Mes₂C₆H₃E)₂]˙⁺ (17a–c; E = S, Se, Te) in the form of thermally stable [SbF₆]^– salts in nearly quantitative yields. The electronic and structural properties of these radical cations were probed by X-ray diffraction analysis, EPR spectroscopy, and density functional theory calculations and other methods.     

An inhibitor of ubiquitin conjugation and aggresome formation

Proteasome inhibitors have revolutionized the treatment of multiple myeloma, and validated the therapeutic potential of the ubiquitin proteasome system (UPS). It is believed that in part, proteasome inhibitors elicit their therapeutic effect by inhibiting the degradation of misfolded proteins, which is proteotoxic and causes cell death. In spite of these successes, proteasome inhibitors are not effective against solid tumors, thus necessitating the need to explore alternative approaches. Furthermore, proteasome inhibitors lead to the formation of aggresomes that clear misfolded proteins via the autophagy–lysosome degradation pathway. Importantly, aggresome formation depends on the presence of polyubiquitin tags on misfolded proteins. We therefore hypothesized that inhibitors of ubiquitin conjugation should inhibit both degradation of misfolded proteins, and ubiquitin dependent aggresome formation, thus outlining the path forward toward more effective anticancer therapeutics. To explore the therapeutic potential of targeting the UPS to treat solid cancers, we have developed an inhibitor of ubiquitin conjugation (ABP A3) that targets ubiquitin and Nedd8 E1 enzymes, enzymes that are required to maintain the activity of the entire ubiquitin system. We have shown that ABP A3 inhibits conjugation of ubiquitin to intracellular proteins and prevents the formation of cytoprotective aggresomes in A549 lung cancer cells. Furthermore, ABP A3 induces activation of the unfolded protein response and apoptosis. Thus, similar to proteasome inhibitors MG132, bortezomib, and carfilzomib, ABP A3 can serve as a novel probe to explore the therapeutic potential of the UPS in solid and hematological malignancies.     

Controlling the activity of quorum sensing autoinducers with light

Bacteria use a communication system, called quorum sensing (QS), to organize into communities and synchronize gene expression to promote virulence and secure survival. Here we report on a proof-of-principle for externally interfering with this bacterial communication system, using light. By employing photoswitchable small molecules, we were able to photocontrol the QS-related bioluminescence in an Escherichia coli reporter strain, and the expression of target QS genes and pyocyanin production in Pseudomonas aeruginosa.     

A hexanuclear gold carbonyl cluster

The hexanuclear gold carbonyl cluster [PPh₄]₂[Au₆(CF₃)₆Br₂(CO)₂] (4) has been obtained by spontaneous self-assembly of the following independent units: CF₃AuCO (1) and [PPh₄][Br(AuCF₃)₂] (3). The cyclo-Au₆ aggregate 4, in which the components are held together by unassisted, fairly strong aurophilic interactions (Au···Au ∼310 pm), exhibits a cyclohexane-like arrangement with chair conformation. These aurophilic interactions also result in significant ν(CO) lowering: from 2194 cm^–1 in the separate component 1 to 2171 cm^–1 in the mixed aggregate 4. Procedures to prepare the single-bridged dinuclear component 3 as well as the mononuclear derivative [PPh₄][CF₃AuBr] (2) are also reported.     

Pattern-based detection of anion pollutants in water with DNA polyfluorophores

Many existing irrigation, industrial and chemical storage sites are currently introducing hazardous anions into groundwater, making the monitoring of such sites a high priority. Detecting and quantifying anions in water samples typically requires complex instrumentation, adding cost and delaying analysis. Here we address these challenges by development of an optical molecular method to detect and discriminate a broad range of anionic contaminants with DNA-based fluorescent sensors. A library of 1296 tetrameric-length oligodeoxyfluorosides (ODFs) composed of metal ligand and fluorescence modulating monomers was constructed with a DNA synthesizer on PEG-polystyrene microbeads. These oligomers on beads were incubated with Y^III or Zn^II ions to provide affinity and responsiveness to anions. Seventeen anions were screened with the library under an epifluorescence microscope, ultimately yielding eight chemosensors that could discriminate 250 μM solutions of all 17 anions in buffered water using their patterns of response. This sensor set was able to identify two unknown anion samples from ten closely-responding anions and could also function quantitatively, determining unknown concentrations of anions such as cyanide (as low as 1 mM) and selenate (as low as 50 μM). Further studies with calibration curves established detection limits of selected anions including thiocyanate (detection limit ∼300 μM) and arsenate (∼800 μM). The results demonstrate DNA-like fluorescent chemosensors as versatile tools for optically analyzing environmentally hazardous anions in aqueous environments.     

Conducting polyfurans by electropolymerization of oligofurans

Polyfurans have never been established as useful conjugated polymers, as previously they were considered to be inherently unstable and poorly conductive. Here, we show the preparation of stable and conducting polyfuran films by electropolymerization of a series of oligofurans of different chain lengths substituted with alkyl groups. The polyfuran films show good conductivity in the order of 1 S cm^–1, good environmental and electrochemical stabilities, very smooth morphologies (roughness 1–5 nm), long effective conjugation lengths, well-defined spectroelectrochemistry and electro-optical switching (in the Vis-NIR region), and have optical band-gaps in the range of 2.2–2.3 eV. A low oxidation potential needed for polymerization of oligofurans (compared to furan) is a key factor in achievement of improved properties of polyfurans reported in this work. DFT calculations and experiments show that polyfurans are much more rigid than polythiophenes, and alkyl substitution does not disturb backbone planarity and conjugation. The obtained properties of polyfuran films are similar or superior to the properties of electrochemically prepared poly(oligothiophene)s under similar conditions.     

Metal oxidation states in biological water splitting

A central question in biological water splitting concerns the oxidation states of the manganese ions that comprise the oxygen-evolving complex of photosystem II. Understanding the nature and order of oxidation events that occur during the catalytic cycle of five S_i states (i = 0–4) is of fundamental importance both for the natural system and for artificial water oxidation catalysts. Despite the widespread adoption of the so-called “high-valent scheme”—where, for example, the Mn oxidation states in the S₂ state are assigned as III, IV, IV, IV—the competing “low-valent scheme” that differs by a total of two metal unpaired electrons (i.e. III, III, III, IV in the S₂ state) is favored by several recent studies for the biological catalyst. The question of the correct oxidation state assignment is addressed here by a detailed computational comparison of the two schemes using a common structural platform and theoretical approach. Models based on crystallographic constraints were constructed for all conceivable oxidation state assignments in the four (semi)stable S states of the oxygen evolving complex, sampling various protonation levels and patterns to ensure comprehensive coverage. The models are evaluated with respect to their geometric, energetic, electronic, and spectroscopic properties against available experimental EXAFS, XFEL-XRD, EPR, ENDOR and Mn K pre-edge XANES data. New 2.5 K ⁵⁵Mn ENDOR data of the S₂ state are also reported. Our results conclusively show that the entire S state phenomenology can only be accommodated within the high-valent scheme by adopting a single motif and protonation pattern that progresses smoothly from S₀ (III, III, III, IV) to S₃ (IV, IV, IV, IV), satisfying all experimental constraints and reproducing all observables. By contrast, it was impossible to construct a consistent cycle based on the low-valent scheme for all S states. Instead, the low-valent models developed here may provide new insight into the over-reduced S states and the states involved in the assembly of the catalytically active water oxidizing cluster.     

Expanding discriminative dimensions for analysis and imaging

Eliminating the contribution of interfering compounds is a key step in chemical analysis. In complex media, one possible approach is to perform a preliminary separation. However purification is often demanding, long, and costly; it may also considerably alter the properties of interacting components of the mixture (e.g. in a living cell). Hence there is a strong interest for developing separation-free non-invasive analytical protocols. Using photoswitchable probes as labelling and titration contrast agents, we demonstrate that the association of a modulated monochromatic light excitation with a kinetic filtering of the overall observable is much more attractive than constant excitation to read-out the contribution from a target probe under adverse conditions. An extensive theoretical framework enabled us to optimize the out-of-phase concentration first-order response of a photoswitchable probe to modulated illumination by appropriately matching the average light intensity and the radial frequency of the light modulation to the probe dynamics. Thus, we can selectively and quantitatively extract from an overall signal the contribution from a target photoswitchable probe within a mixture of species, photoswitchable or not. This simple titration strategy is more specifically developed in the context of fluorescence imaging, which offers promising perspectives.     

Oxide-supported Ir nanodendrites with high activity and durability for the oxygen evolution reaction in acid PEM water electrolyzers

Reducing the noble-metal catalyst content of acid Polymer Electrolyte Membrane (PEM) water electrolyzers without compromising catalytic activity and stability is a goal of fundamental scientific interest and substantial technical importance for cost-effective hydrogen-based energy storage. This study presents nanostructured iridium nanodendrites (Ir-ND) supported on antimony doped tin oxide (ATO) as efficient and stable water splitting catalysts for PEM electrolyzers. The active Ir-ND structures exhibited superior structural and morphological properties, such as particle size and surface area compared to commercial state-of-art Ir catalysts. Supported on tailored corrosion-stable conductive oxides, the Ir-ND catalysts exhibited a more than 2-fold larger kinetic water splitting activity compared with supported Ir nanoparticles, and a more than 8-fold larger catalytic activity than commercial Ir blacks. In single-cell PEM electrolyzer tests, the Ir-ND/ATO outperformed commercial Ir catalysts more than 2-fold at technological current densities of 1.5 A cm^–2 at a mere 1.80 V cell voltage, while showing excellent durability under constant current conditions. We conclude that Ir-ND/ATO catalysts have the potential to substantially reduce the required noble metal loading, while maintaining their catalytic performance, both in idealized three-electrode set ups and in the real electrolyzer device environments.     

Azasugar inhibitors as pharmacological chaperones for Krabbe disease

Krabbe disease is a devastating neurodegenerative disorder characterized by rapid demyelination of nerve fibers. This disease is caused by defects in the lysosomal enzyme β-galactocerebrosidase (GALC), which hydrolyzes the terminal galactose from glycosphingolipids. These lipids are essential components of eukaryotic cell membranes: substrates of GALC include galactocerebroside, the primary lipid component of myelin, and psychosine, a cytotoxic metabolite. Mutations of GALC that cause misfolding of the protein may be responsive to pharmacological chaperone therapy (PCT), whereby small molecules are used to stabilize these mutant proteins, thus correcting trafficking defects and increasing residual catabolic activity in cells. Here we describe a new approach for the synthesis of galacto-configured azasugars and the characterization of their interaction with GALC using biophysical, biochemical and crystallographic methods. We identify that the global stabilization of GALC conferred by azasugar derivatives, measured by fluorescence-based thermal shift assays, is directly related to their binding affinity, measured by enzyme inhibition. X-ray crystal structures of these molecules bound in the GALC active site reveal which residues participate in stabilizing interactions, show how potency is achieved and illustrate the penalties of aza/iminosugar ring distortion. The structure–activity relationships described here identify the key physical properties required of pharmacological chaperones for Krabbe disease and highlight the potential of azasugars as stabilizing agents for future enzyme replacement therapies. This work lays the foundation for new drug-based treatments of Krabbe disease.     

Biosynthetic insights provided by unusual sesterterpenes from the medicinal herb Aletris farinosa

A series of novel sesterterpenes (2–6) have been isolated from the roots of Aletris farinosa and structurally characterized by MS, NMR, and X-ray crystallography in conjunction with computational modeling. Their structures provide new insights into the mechanisms of sesterterpene biosynthesis. Specifically, we propose with support from density functional theory computations that the configuration at a single stereocenter determines the fate of a key tetracyclic carbocationic intermediate, derived from an oxidogeranylfarnesol precursor. Whereas one epimer of the carbocation undergoes H⁺ elimination to give 6, the other undergoes a spectacular cascade of seven 1,2-methyl and hydride migrations leading to the previously unreported carbon skeleton of 5. Theoretical calculations suggest that the cascade is triggered by substrate preorganization in the enzyme active site.     

A macrocycle-assisted nanoparticlization process for bulk Ag2S

We report herein a new nanoparticlization process for the bulk-to-nano transformation of Ag₂S by incorporating both top-down and bottom-up approaches. Bulk Ag₂S was dissolved in solution with the assistance of a macrocyclic ligand, hexamethylazacalix[6]pyridine (Py[6]), to produce polynuclear silver sulfide cluster aggregates. All Ag–S cluster aggregates obtained in three crystalline complexes were protected by Py[6] macrocycles. Removing the protective Py[6] macrocycles by protonation led to the generation of unconventional Ag–S nanoparticles with a large energy gap. Theoretical calculations by a hybrid DFT method demonstrated that the silver sulfide clusters with high Ag/S ratio exhibited more localized HOMO–LUMO orbitals, which consequently enlarged their band gap energies. These experimental and theoretical studies broaden our understanding of the fabrication of nanomaterials by virtue of the advantages of both bottom-up and top-down methods and meanwhile provide a viable means of adjusting the band gap of binary nanomaterials independent of their size.     

N-Heterocyclic carbene catalysed redox isomerisation of esters to functionalised benzaldehydes

N-Heterocyclic carbene catalysed redox isomerisation with reduction about the carbonyl has been developed in the transformation of trienyl esters to tetrasubstituted benzaldehydes. The reaction proceeds in good to excellent yield, and in cases that provide 2,2′-biaryls, enantioselectivity is observed. Mechanistic studies demonstrate the intermediacy of a cyclohexenyl β-lactone, while implicating formation of the homoenolate as turnover limiting.     

Experiences with a researcher-centric ELN

Electronic Laboratory Notebooks (ELNs) are progressively replacing traditional paper books in both commercial research establishments and academic institutions. University researchers require specific features from ELNs, given the need to promote cross-institutional collaborative working, to enable the sharing of procedures and results, and to facilitate publication. The LabTrove ELN, which we use as our exemplar, was designed to be researcher-centric (i.e., not only aimed at the individual researcher''s basic needs rather than to a specific institutional or subject or disciplinary agenda, but also able to be tailored because it is open source). LabTrove is being used in a heterogeneous set of academic laboratories, for a range of purposes, including analytical chemistry, X-ray studies, drug discovery and a biomaterials project. Researchers use the ELN for recording experiments, preserving data collected, and for project coordination. This perspective article describes the experiences of those researchers from several viewpoints, demonstrating how a web-based open source electronic notebook can meet the diverse needs of academic researchers.     

Cytotoxicity of guanine-based degradation products contributes to the antiproliferative activity of guanine-rich oligonucleotides

Guanine-rich oligonucleotides (GROs) have attracted considerable attention as anticancer agents, because they exhibit cancer-selective antiproliferative activity and can form G-quadruplex structures with higher nuclease resistance and cellular uptake. Recently, a GRO, AS1411 has reached phase II clinical trials for acute myeloid leukemia and renal cell carcinoma. The antiproliferative activity of GROs has been associated with various protein targets; however the real mechanisms of action remain unclear. In this study, we showed evidence that antiproliferative activity of GROs (including AS1411) is mainly contributed by the cytotoxicity of their guanine-based degradation products, such as monophosphate deoxyguanosine (dGMP), deoxyguanosine (dG) and guanine. The GROs with lower nuclease resistance exhibited higher antiproliferative activity. Among nucleotides, nucleosides and nucleobases, only guanine-based compounds showed highly concentration-dependent cytotoxicity. Our results suggest that it is necessary to reconsider the cancer-selective antiproliferative activity of GROs. Since guanine-based compounds are endogenous substances in living organisms, systematic studies of the cytotoxicity of these compounds will provide new information for the understanding of certain diseases and offer useful information for drug design.     

Rational coupled dynamics network manipulation rescues disease-relevant mutant cystic fibrosis transmembrane conductance regulator

Many cellular functions necessary for life are tightly regulated by protein allosteric conformational change, and correlated dynamics between protein regions has been found to contribute to the function of proteins not previously considered allosteric. The ability to map and control such dynamic coupling would thus create opportunities for the extension of current therapeutic design strategy. Here, we present an approach to determine the networks of residues involved in the transfer of correlated motion across a protein, and apply our approach to rescue disease-causative mutant cystic fibrosis transmembrane regulator (CFTR) ion channels, ΔF508 and ΔI507, which together constitute over 90% of cystic fibrosis cases. We show that these mutations perturb dynamic coupling within the first nucleotide-binding domain (NBD1), and uncover a critical residue that mediates trans-domain coupled dynamics. By rationally designing a mutation to this residue, we improve aberrant dynamics of mutant CFTR as well as enhance surface expression and function of both mutants, demonstrating the rescue of a disease mutation by rational correction of aberrant protein dynamics.     

Hyperpolarization of amino acid derivatives in water for biological applications

We report on the successful synthesis and hyperpolarization of N-unprotected α-amino acid ethyl propionate esters and extensively, on an alanine derivative hyperpolarized by PHIP (4.4 ± 1.0% ¹³C-polarization), meeting required levels for in vivo detection. Using water as solvent increases biocompatibility and the absence of N-protection is expected to maintain biological activity.     

Catalysis of water oxidation in acetonitrile by iridium oxide nanoparticles

Water oxidation catalysed by iridium oxide nanoparticles (IrO₂ NPs) in water–acetonitrile mixtures using [Ru^III(bpy)₃]³⁺ as oxidant was studied as a function of the water content, the acidity of the reaction media and the catalyst concentration. It was observed that under acidic conditions (HClO₄) and at high water contents (80% (v/v)) the reaction is slow, but its rate increases as the water content decreases, reaching a maximum at approximately equimolar proportions (≈25% H₂O (v/v)). The results can be rationalized based on the structure of water in water–acetonitrile mixtures. At high water fractions, water is present in highly hydrogen-bonded arrangements and is less reactive. As the water content decreases, water clustering gives rise to the formation of water-rich micro-domains, and the number of bonded water molecules decreases monotonically. The results presented herein indicate that non-bonded water present in the water micro-domains is considerably more reactive towards oxygen production. Finally, long term electrolysis of water–acetonitrile mixtures containing [Ru^II(bpy)₃]²⁺ and IrO₂ NPs in solution show that the amount of oxygen produced is constant with time demonstrating that the redox mediator is stable under these experimental conditions.     

Synthesis and structural analysis of nonstoichiometric ternary fulleride K 1.5 Ba 0.25 CsC 60

The existence of cation-vacancy sites in fullerides might lead to long-range ordering and generate a new vacancy-ordered superstructure. The purpose of this work is to search whether or not long-range ordering of vacant tetrahedral sites, namely superstructure emerges in nonstoichiometric K _1.5 Ba _0.25 CsC ₆₀ fulleride. Therefore, K _1.5 Ba _0.25 CsC ₆₀ with cation-vacancy sites is synthesized using a precursor method to avoid inadequate stoichiometry control and formation of impurity phases within the target composition. For this purpose, first, phase-pure K ₆ C ₆₀ , Ba ₆ C ₆₀ and Cs ₆ C ₆₀ precursors are synthesized. Stoichiometric quantities of these precursors are used for further reaction with C ₆₀ to afford K _1.5 Ba _0.25 CsC ₆₀ . Rietveld analysis of the high-resolution synchrotron X-ray powder diffraction data of the precursors and K _1.5 Ba _0.25 CsC ₆₀ confirms that K ₆ C ₆₀ , Ba ₆ C ₆₀ and Cs ₆ C ₆₀ are single-phase and they crystallize in a body-centered-cubic structure ( Im 3) as reported in the literature. The analysis also shows that K _1.5 Ba _0.25 CsC ₆₀ phase can be perfectly modeled using a face-centered cubic structure. No new peaks appear which could have implied the appearance of a superstructure. This suggests that there is no long-range ordered arrangement of vacant tetrahedral sites in K _1.5 Ba _0.25 CsC ₆₀ .