Label-free luminescence switch-on detection of hepatitis C virus NS3 helicase activity using a G-quadruplex-selective probe

A series of luminescent Ir(iii) complexes were synthesised and evaluated for their ability to act as luminescent G-quadruplex-selective probes. The Ir(iii) complex 9, [Ir(phq)₂(phen)]PF₆ (where phq = 2-phenylquinoline; phen = 1,10-phenanthroline), exhibited high luminescence in the presence of G-quadruplex DNA compared to dsDNA and ssDNA, and was employed to construct a label-free G-quadruplex-based assay for hepatitis C virus NS3 helicase activity in aqueous solution. Moreover, the application of the assay for screening potential helicase inhibitors was demonstrated. To our knowledge, this is the first G-quadruplex-based assay for helicase activity.     

Chemical assay-guided natural product isolation via solid-supported chemodosimetric fluorescent probe

As a new model of chemical assay-guided natural product isolation, an effective chemodosimetric assay system was devised. Our chemical assay system features a fluorogenic chemodosimeter immobilized on a solid support, which offers advantages in identifying the desired compounds in complex natural product mixtures. To isolate only compounds with the target functional groups, the click chemistry concept was adopted. The model system presented here was developed for natural products with a terminal alkyne. Using our newly designed alkyne sensing beads with the aforementioned features, we have readily identified, quantified, and isolated compounds with a terminal alkyne group from plant extracts.     

HOCl can appear in the mitochondria of macrophages during bacterial infection as revealed by a sensitive mitochondrial-targeting fluorescent probe

Macrophages, important cells of the innate immune system, can produce abundant HOCl in the cytoplasm to fight against bacteria. Recent studies suggest that mitochondria in macrophages play a role in antibacterial responses. During bacterial infection, however, it is uncertain whether HOCl is present in the mitochondria, mainly because of the lack of a suitable research method. Herein, by developing a new mitochondrial-targeting fluorescent HOCl probe, combined with confocal fluorescence imaging, we show for the first time that HOCl can appear in the mitochondria of macrophages (Raw264.7 cells) during bacterial infection, as confirmed with non-phagocytic cells and inhibitors as control experiments. Moreover, the developed probe exhibits an accurate mitochondrial-targeting ability, a fast response, and high selectivity and sensitivity (detection limit 9 nM), and is thus expected to be employed for further revealing the biological function of subcellular mitochondria.     

Stereocontrolled protein surface recognition using chiral oligoamide proteomimetic foldamers

The development of foldamers capable of selective molecular recognition of solvent exposed protein surfaces represents an outstanding challenge in supramolecular chemical biology. Here we introduce an oligoamide foldamer with well-defined conformation that bears all the hallmarks of an information rich oligomer. Specifically, the foldamer recognizes its target protein hDM2 leading to inhibition of its protein–protein interaction with p53 in a manner that depends upon the composition, spatial projection and stereochemistry of functional groups appended to the scaffold. Most significantly, selective inhibition of p53/hDM2 can be achieved against four other targets and the selectivity for p53/hDM2 inhibition versus Mcl-1/NOXA-B inhibition is critically dependent upon the stereochemistry of the helix mimetic.     

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.     

Controlled levels of protein modification through a chromatography-mediated bioconjugation

Synthetically modified proteins are increasingly finding applications as well-defined scaffolds for materials. In practice it remains difficult to construct bioconjugates with precise levels of modification because of the limited number of repeated functional groups on proteins. This article describes a method to control the level of protein modification in cases where there exist multiple potential modification sites. A protein is first tagged with a handle using any of a variety of modification chemistries. This handle is used to isolate proteins with a particular number of modifications via affinity chromatography, and then the handle is elaborated with a desired moiety using an oxidative coupling reaction. This method results in a sample of protein with a well-defined number of modifications, and we find it particularly applicable to systems like protein homomultimers in which there is no way to discern between chemically identical subunits. We demonstrate the use of this method in the construction of a protein-templated light-harvesting mimic, a type of system which has historically been difficult to make in a well-defined manner.     

Chemosensor properties of 7-hydroxycoumarin substituted cyclotriphosphazenes

The newly synthesized cyclotriphosphazene cored coumarin chemosensors 5, 6, and 7 were successfully characterized by ¹ H NMR, ³¹ P NMR, and MALDI-TOF mass spectrometry. Additionally, the photophysical and metal sensing properties of the targeted compounds were determined by fluorescence spectroscopy in the presence of various metals (Li ⁺ , Na ⁺ , K ⁺ , Cs ⁺ , Mg ²⁺ , Ca ²⁺ , Ba ²⁺ , Cr ³⁺ , Mn ²⁺ , Fe ³⁺ , Co ²⁺ , Al ³⁺ , Hg ⁺ , Cu ²⁺ , Zn ²⁺ , Ag ⁺ , and Cd ²⁺ ) . The fluorescence titration results showed that compounds 5, 6, and 7 could be employed as fluorescent chemosensors for Fe ³⁺ ions with high sensitivity. The complex stoichiometry between final cyclotriphosphazene chemosensors and Fe ³⁺ ions was also determined by Job’s plots.     

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.     

Harnessing selenocysteine reactivity for oxidative protein folding

Although oxidative folding of disulfide-rich proteins is often sluggish, this process can be significantly enhanced by targeted replacement of cysteines with selenocysteines. In this study, we examined the effects of a selenosulfide and native versus nonnative diselenides on the folding rates and mechanism of bovine pancreatic trypsin inhibitor. Our results show that such sulfur-to-selenium substitutions alter the distribution of key folding intermediates and enhance their rates of interconversion in a context-dependent manner.     

Accurate molecular weight determination of small molecules via DOSY-NMR by using external calibration curves with normalized diffusion coefficients

Determination of the aggregation and solvation numbers of organometallic complexes in solution is an important task to increase insight in reaction mechanisms. Thus knowing which aggregates are formed during a reaction is of high interest to develop better selectivity and higher yields. Diffusion-ordered spectroscopy (DOSY), which separates NMR signals according to the diffusion coefficient, finds increasing use to identify species in solution. However, there still is no simple relationship between diffusion coefficient and molecular weight (MW). Some methods have been developed to estimate the MW but still with a significant error of ±30%. Here we describe a novel development of MW-determination by using an external calibration curve (ECC) approach with normalized diffusion coefficients. Taking the shape of the molecules into account enables accurate MW-predictions with a maximum error of smaller than ±9%. Moreover we show that the addition of multiple internal references is dispensable. One internal reference (that also can be the solvent) is sufficient. If the solvent signal is not accessible, 16 other internal standards (aliphatics and aromatics) are available to avoid signal overlapping problems and provide flexible choice of analytes. This method is independent of NMR-device properties and diversities in temperature or viscosity and offers an easy and robust method to determine accurate MWs in solution.     

Ammonia decomposition catalysis using non-stoichiometric lithium imide

We demonstrate that non-stoichiometric lithium imide is a highly active catalyst for the production of high-purity hydrogen from ammonia, with superior ammonia decomposition activity to a number of other catalyst materials. Neutron powder diffraction measurements reveal that the catalyst deviates from pure imide stoichiometry under ammonia flow, with active catalytic behaviour observed across a range of stoichiometry values near the imide. These measurements also show that hydrogen from the ammonia is exchanged with, and incorporated into, the bulk catalyst material, in a significant departure from existing ammonia decomposition catalysts. The efficacy of the lithium imide–amide system not only represents a more promising catalyst system, but also broadens the range of candidates for amide-based ammonia decomposition to include those that form imides.     

Selective glycoprotein detection through covalent templating and allosteric click-imprinting

Many glycoproteins are intimately linked to the onset and progression of numerous heritable or acquired diseases of humans, including cancer. Indeed the recognition of specific glycoproteins remains a significant challenge in analytical method and diagnostic development. Herein, a hierarchical bottom-up route exploiting reversible covalent interactions with boronic acids and so-called click chemistry for the fabrication of glycoprotein selective surfaces that surmount current antibody constraints is described. The self-assembled and imprinted surfaces, containing specific glycoprotein molecular recognition nanocavities, confer high binding affinities, nanomolar sensitivity, exceptional glycoprotein specificity and selectivity with as high as 30 fold selectivity for prostate specific antigen (PSA) over other glycoproteins. This synthetic, robust and highly selective recognition platform can be used in complex biological media and be recycled multiple times with no performance decrement.     

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.     

Multiplexed detection of serological cancer markers with plasmon-enhanced Raman spectro-immunoassay

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognostication and monitoring of therapeutic response. Emerging data, however, suggest that single markers are inadequate in describing complex pathologic transformations. Architecting assays capable of parallel measurements of multiple biomarkers can help achieve the desired clinical sensitivity and specificity while conserving patient specimen and reducing turn-around time. Here we describe a plasmon-enhanced Raman spectroscopic assay featuring nanostructured biomolecular probes and spectroscopic imaging for multiplexed detection of disseminated breast cancer markers cancer antigen (CA) 15-3, CA 27-29 and cancer embryonic antigen (CEA). In the developed SERS assay, both the assay chip and surface-enhanced Raman spectroscopy (SERS) tags are functionalized with monoclonal antibodies against CA15-3, CA27-29 and CEA, respectively. Sequential addition of biomarkers and functionalized SERS tags onto the functionalized assay chip enable the specific recognition of these biomarkers through the antibody-antigen interactions, leading to a sandwich spectro-immunoassay. In addition to offering extensive multiplexing capability, our method provides higher sensitivity than conventional immunoassays and demonstrates exquisite specificity owing to selective formation of conjugated complexes and fingerprint spectra of the Raman reporter. We envision that clinical translation of this assay may further enable asymptomatic surveillance of cancer survivors and speedy assessment of treatment benefit through a simple blood test.     

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.     

Rapid access to phospholipid analogs using thiol-yne chemistry

Phospholipids and glycolipids constitute an essential part of biological membranes, and are of tremendous fundamental and practical interest. Unfortunately, the preparation of functional phospholipids, or synthetic analogs, is often synthetically challenging. Here we utilize thiol-yne click chemistry methodology to gain access to phospho- and glycolipid analogs. Alkynyl hydrophilic head groups readily photoreact with numerous thiol modified lipid tails to yield the appropriate dithioether phospho- or glycolipids. The resulting structures closely resemble the structure and function of native diacylglycerolipids. Dithioether phosphatidylcholines (PCs) are suitable for forming giant unilamellar vesicles (GUV), which can be used as vessels for cell-free expression systems. The unnatural thioether linkages render the lipids resistant to phospholipase A2 hydrolysis. We utilize the improved stability of these lipids to control the shrinkage of GUVs composed of a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and dioleyl-dithioether PC, concentrating encapsulated nanoparticles. We imagine that these readily accessible lipids could find a number of applications as natural lipid substitutes.     

Reaction of a diaryldigermyne with ethylene

Reaction of the stable digermyne BbtGeGeBbt (Bbt = 2,6-[CH(SiMe₃)₂]₂-4-[C(SiMe₃)₃]-C₆H₂) with ethylene initially afforded the corresponding 1,2-digermacyclobutene. Depending on the reaction conditions applied, further reaction of this 1,2-digermacyclobutene with ethylene furnished two different reaction products: a 1,4-digermabicyclo[2.2.0]hexane or a bis(germiranyl)ethane. Combined experimental and theoretical results suggested that the 1,4-digermabicyclo[2.2.0]hexane and the bis(germiranyl)ethane are the thermodynamic and kinetic reaction products, respectively. A reaction mechanism in agreement with these results was proposed.     

Highly enantioselective sulfa-Michael addition reactions using N-heterocyclic carbene as a non-covalent organocatalyst

We report the first asymmetric sulfa-Michael addition (SMA) reactions using a chiral N-heterocyclic carbene (NHC) as a non-covalent organocatalyst. We demonstrate that a triazolium salt derived NHC functions as an excellent Brønsted base to promote enantioselective carbon–sulfur bond formation. The reaction is applicable to a wide range of thiols and electrophilic olefins. Notably, quaternary chiral centers bearing both an S atom and a CF₃ group were synthesized with excellent asymmetric control. Mechanistic studies suggest that the facial discrimination is likely to be guided by non-covalent interactions: hydrogen bonding and π–π stacking.     

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 .     

A recyclable polyoxometalate-based supramolecular chemosensor for efficient detection of carbon dioxide

A new type of supramolecular chemosensor based on the polyoxometalate (POM) Na₉DyW₁₀O₃₆ (DyW₁₀) and the block copolymer poly(ethylene oxide-b-N,N-dimethylaminoethyl methacrylate) (PEO₁₁₄-b-PDMAEMA₁₆) is reported. By taking advantage of the CO₂ sensitivity of PDMAEMA blocks to protonate the neutral tertiary amino groups, CO₂ can induce the electrostatic coassembly of anionic DyW₁₀ with protonated PDMAEMA blocks, and consequently trigger the luminescence chromism of DyW₁₀ due to the change in the microenvironment of Dy³⁺. The hybrid complex in dilute aqueous solution is very sensitive to CO₂ content and shows rapid responsiveness in luminescence. The luminescence intensity of the DyW₁₀/PEO-b-PDMAEMA complex increases linearly with an increasing amount of dissolved CO₂, which permits the qualitative and quantitative detection of CO₂. The complex solution also shows good selectivity for CO₂, with good interference tolerance of CO, N₂, HCl, H₂O and SO₂. The supramolecular chemosensor can be recycled through disassembly of the hybrid complex by simply purging with inert gases to remove CO₂.