A Multi‐Component Sensor System for Detection of Amphiphilic Compounds

Many biologically important compounds are amphiphilic in character. Glycolipids, for example, represent a biologically important class of amphiphiles. Receptors and sensors for such compounds must also be amphiphilic making them a challenge to prepare. Here, a cucurbit[8]uril (CB[8])‐based sensor system has been prepared and tested for detection of amphiphilic compounds. This multi‐component system consists: a CB[8], which acts as a hydrophobic lipid receptor, a hydrophilic pyridinium‐based carbohydrate receptor, and a fluorescent indicator. The system self‐assembles in aqueous solution. The pyridinium quenches the fluorescence of the indicator giving a strong turn‐on response when it is displaced by the analyte. The sensor system was characterized by NMR, X‐ray crystallography, and fluorescence titrations.     

A Traveling Wave Ion Mobility Spectrometry (TWIMS) Study of the Robo1-Heparan Sulfate Interaction

Roundabout 1 (Robo1) interacts with its receptor Slit to regulate axon guidance, axon branching, and dendritic development in the nervous system and to regulate morphogenesis and many cell functions in the nonneuronal tissues. This interaction is known to be critically regulated by heparan sulfate (HS). Previous studies suggest that HS is required to promote the binding of Robo1 to Slit to form the minimal signaling complex, but the molecular details and the structural requirements of HS for this interaction are still unclear. Here, we describe the application of traveling wave ion mobility spectrometry (TWIMS) to study the conformational details of the Robo1-HS interaction. The results suggest that Robo1 exists in two conformations that differ by their compactness and capability to interact with HS. The results also suggest that the highly flexible interdomain hinge region connecting the Ig1 and Ig2 domains of Robo1 plays an important functional role in promoting the Robo1-Slit interaction. Moreover, variations in the sulfation pattern and size of HS were found to affect its binding affinity and selectivity to interact with different conformations of Robo1. Both MS measurements and CIU experiments show that the Robo1-HS interaction requires the presence of a specific size and pattern of modification of HS. Furthermore, the effect of N-glycosylation on the conformation of Robo1 and its binding modes with HS is reported.

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Combinatorial Probes for High‐Throughput Electrochemical Analysis of Circulating Nucleic Acids in Clinical Samples

The analysis of circulating tumour nucleic acids (ctNAs) provides a minimally invasive way to assess the mutational spectrum of a tumour. However, effective and practical methods for analyzing this emerging class of markers are lacking. Analysis of ctNAs using a sensor‐based approach has notable challenges, as it is vital to differentiate nucleic acids from normal cells from mutation‐bearing sequences emerging from tumours. Moreover, many genes related to cancer have dozens of different mutations. Herein, we report an electrochemical approach that directly detects genes with mutations in patient serum by using combinatorial probes (CPs). The CPs enable detection of all of the mutant alleles derived from the same part of the gene. As a proof of concept, we analyze mutations of the EGFR gene, which has more than 40 clinically relevant alterations that include deletions, insertions, and point mutations. Our CP‐based approach accurately detects mutant sequences directly in patient serum.     

Interaction of mineral surfaces with simple organic molecules by diffuse reflectance IR spectroscopy (DRIFT)

The adsorption of simple organic molecules to mineral surfaces was investigated by Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS) and by solution chemical analysis. Salicylic acid, myristic acid or octacosane were deposited from water or hexane onto gamma-alumina and kaolinite, and examined without evacuation or blending with KBr. The DRIFTS results replicated those from ATR-IR, where available, supporting the validity of DRIFTS as a versatile and convenient alternative to ATR. Results were obtained for coverage as low as 0.02 molecules/nm(2). Monolayer coverage on gamma-alumina was determined from spectra as 0.7 molecules/nm(2) for salicylic acid, confirmed by solution analysis, and 2 molecules/nm(2) for myristic acid. Spectral features of salicylic acid deposited on gamma-alumina from hexane were not distinguishable from those deposited from water. In almost all cases, spectral features associated with the carboxyl moiety were substantially altered, indicating a significant role in the adsorption mechanism. Adsorption of salicylic acid from hexane onto kaolinite was also as carboxylate, but myristic acid showed both carboxylate and carbonyl. The results from using hexane as solvent compared to water suggest that surface-adsorbed hydroxyl and molecular water, present on mineral oxides under ambient conditions, may be a key determinant of the adsorbate architecture.     

Resonance Raman and resonance hyper-Raman intensities: structure and dynamics of molecular excited states in solution

Resonance Raman scattering is discussed as a vibronic spectroscopy that can provide detailed information about the structure and dynamics of excited electronic states of molecules. The emphasis is on molecules in liquid solution. The theory of resonance Raman intensities and experimental and interpretive methods are discussed both in a historical context and in their present and future implementations. The related but much less developed technique of two-photon-resonant hyper-Raman scattering is also discussed in a similar context.     

Comparison of the quality of aqueous dispersions of single wall carbon nanotubes using surfactants and biomolecules

The use of single wall carbon nanotubes (SWCNTs) in current and future applications depends on the ability to process SWCNTs in a solvent to yield high-quality dispersions characterized by individual SWCNTs and possessing a minimum of SWCNT bundles. Many approaches for the dispersion of SWCNTs have been reported. However, there is no general assessment which compares the relative quality and dispersion efficiency of the respective methods. Herein we report a quantitative comparison of the relative ability of "wrapping polymers" including oligonucleotides, peptides, lignin, chitosan, and cellulose and surfactants such as cholates, ionic liquids, and organosulfates to disperse SWCNTs in water. Optical absorption and fluorescence spectroscopy provide quantitative characterization (amount of SWCNTs that can be suspended by a given surfactant and its ability to debundle SWCNTs) of these suspensions. Sodium deoxy cholate (SDOCO), oligonucleotides (GT)(15), (GT)(10), (AC)(15), (AC)(10), C(10-30), and carboxymethylcellulose (CBMC-250K) exhibited the highest quality suspensions of the various systems studied in this work. The information presented here provides a good framework for further study of SWCNT purification and applications.     

Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.     

Intramolecular energy transfer within butadiyne-linked chlorophyll and porphyrin dimer-faced, self-assembled prisms

The synthesis and photophysical properties of butadiyne-linked chlorophyll and porphyrin dimers in toluene solution and in several self-assembled prismatic structures are described. The butadiyne linkage between the 20-positions of the macrocycles results in new electronic transitions polarized along the long axes of the dimers. These transitions greatly increase the ability of these dimers to absorb the solar spectrum over a broad wavelength range. Femtosecond transient absorption spectroscopy reveals the relative rate of rotation of the macrocycles around the butadiyne bond joining them. Following addition of 3-fold symmetric, metal-coordinating ligands, both macrocyclic dimers self-assemble into prismatic structures in which the dimers comprise the faces of the prisms. These structures were confirmed by small-angle X-ray scattering experiments in solution using a synchrotron source. Photoexcitation of the prismatic assemblies reveals that efficient, through-space energy transfer occurs between the macrocyclic dimers within the prisms. The distance dependence of energy transfer between the faces of the prisms was observed by varying the size of the prismatic assemblies through the use of 3-fold symmetric ligands having arms with different lengths. These results show that self-assembly of discrete macrocyclic prisms provides a useful new strategy for controlling singlet exciton flow in antenna systems for artificial photosynthesis and solar cell applications.     

Spin-labeled analogs of CMP-NeuAc as NMR probes of the alpha-2,6-sialyltransferase ST6Gal I

Structural data on mammalian proteins are often difficult to obtain by conventional NMR approaches because of an inability to produce samples with uniform isotope labeling in bacterial expression hosts. Proteins with sparse isotope labels can be produced in eukaryotic hosts by using isotope-labeled forms of specific amino acids, but structural analysis then requires information from experiments other than nuclear Overhauser effects. One source of alternate structural information is distance-dependent perturbation of spin relaxation times by nitroxide spin-labeled analogs of natural protein ligands. Here, we introduce spin-labeled analogs of sugar nucleotide donors for sialyltransferases, specifically, CMP-TEMPO (CMP-4-O-[2,2,6,6-tetramethylpiperidine-1-oxyl]) and CMP-4carboxyTEMPO (CMP-4-O-[4-carboxy-2,2,6,6-tetramethylpiperidinine-1-oxyl]). An ability to identify resonances from active site residues and produce distance constraints is illustrated on a (15)N phenylalanine-labeled version of the structurally uncharacterized, alpha-2,6-linked sialyltransferase, ST6Gal I.