Bioinspired vesicle restraint and mobilization using a biopolymer scaffold

Biology employs vesicles to package molecules (e.g., neurotransmitters) for their targeted delivery in response to specific spatiotemporal stimuli. Biology is also capable of employing localized stimuli to exert an additional control on vesicle trafficking; intact vesicles can be restrained (or mobilized) by association with (or release from) a cytoskeletal scaffold. We mimic these capabilities by tethering vesicles to a biopolymer scaffold that can undergo (i) stimuli-responsive network formation (for vesicle restraint) and (ii) enzyme-catalyzed network cleavage (for vesicle mobilization). Specifically, we use the aminopolysaccharide chitosan as our scaffold and graft a small number of hydrophobic moieties onto its backbone. These grafted hydrophobes can insert into the bilayer to tether vesicles to the scaffold. Under acidic conditions, the vesicles are not restrained by the hydrophobically modified chitosan (hm-chitosan) because this scaffold is soluble. Increasing the pH to neutral or basic conditions allows chitosan to form interpolymer associations that yield a strong, insoluble restraining network. Enzymatic hydrolysis of this scaffold by chitosanase cleaves the network and mobilizes intact vesicles. Potentially, this approach will provide a controllable means to store and liberate vesicle-based reagents/therapeutics for microfluidic/medical applications.     

High-capacity binding of proteins by poly(acrylic acid) brushes and their derivatives

Polymeric coatings with high protein-binding capacities are important for increasing the output of affinity-based protein purification and decreasing the detection limits of antibody microarrays. This report describes the use of thick poly(acrylic acid) (PAA) brushes to immobilize as much as 80 monolayers of protein. The brushes were prepared using a recently developed procedure that allows polymerization of 100-nm-thick poly(tert-butyl acrylate) films from a surface in just 5 min along with hydrolysis of these films to PAA in 15 min. Covalent binding of bovine serum albumin (BSA) to PAA brushes that were activated using standard coupling agents, however, resulted in immobilization of less than two monolayers of BSA because of competitive hydrolysis of the esters in the activated film. In contrast, derivatization of PAA with nitrilotriacetate (NTA)-Cu2+ complexes yielded films capable of binding many monolayers of protein via metal-ion affinity interactions. For example, derivatization of 55-nm-thick PAA films with NTA-Cu2+ allowed immobilization of about 15 monolayers (5.8 microg/cm2 or 58 nm) of BSA. The binding capacity was even higher for myoglobin (7.7 microg/cm2) and anti-IgG (9.6 microg/cm2). Remarkably, electrostatic adsorption of lysozyme in 55-nm-thick, underivatized PAA resulted in as much as 80 monolayers (16.2 microg/cm2 or 162 nm) of adsorbed protein. Polymer synthesis, derivatization, and swelling, as well as BSA immobilization kinetics and thermodynamics were characterized using reflectance FT-IR spectroscopy, ellipsometry, and protein assays.     

Modification of TiO2 for enhanced surface properties: finite Ostwald ripening by a microwave hydrothermal process

The effect of microwave modification of colloidal TiO2 suspensions under extended periods of treatment is presented. The nanoparticulate TiO2 is compared and contrasted to similar convection hydrothermally treated TiO2 and a commercial titania product, namely Degussa P25. Microwave-treated samples were analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy to determine their physicochemical characteristics. Comparative surface area analyses were performed by N2 adsorption and calculated from a Brunauer-Emmett-Teller (BET) isotherm. The complementary techniques of XRD and TEM showed good correlation between observed and calculated particle sizes (by application of the Scherrer equation), with the material being highly crystalline anatase TiO2, as identified by XRD and Raman. This investigation identified that extended periods of microwave hydrothermal treatment do not greatly enhance the crystallinity and primary grain size. Treatment of >180 min has a negative effect on crystallite growth; however, treatment up to this time had a significant effect on the material's surface area. The limiting regime of Ostwald ripening for hydrothermal treatment is discussed in relation to the mechanism of microwave treatment, that is, rapid heating to temperature and extremely rapid rates of crystallization. The effect of these property modifications are further discussed in relation to photocatalytic and photoelectrochemical applications of TiO2 nanoparticles.     

1H and 13C NMR assignments for two new xanthones from the endophytic fungus Xylaria sp. FRR 5657

The complete 1H and 13C NMR assignments are reported for the new natural products, 7-hydroxy-3-(hydroxymethyl)-1-methoxy-9H-xanthen-9-one (1) and 2,5-dihydroxy-8-methoxy-6-methyl-9-oxo-9H-xanthene -1-carboxylic acid (2). Both of these secondary metabolites were isolated from the fermentation culture of the endophytic fungus Xylaria sp. FRR 5657. 1D and 2D NMR experiments that included 1H, gCOSY, gHSQC and gHMBC were used for the determination of the structure and assignment of these xanthones.     

MALDI-TOF MS as a label-free approach to rapid inhibitor screening

Mass Spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays. These measurements are typically performed by time-consuming LC-MS to eliminate buffer salts that interfere with electrospray ionization MS. However, matrix-assisted laser desorption ionization, time-of-flight MS (MALDI-TOF MS) offers a label-free and direct readout of substrate and product, a fast sampling rate, and is tolerant of many buffer salts, reagents, and compounds that are typically found in enzyme reaction mixtures. In this report, a demonstration of how MALDI-TOF MS can be used to directly measure ratios of substrates and products to produce IC(50) curves for rapid enzyme assays and compound screening is provided. Typical reproducibility parameters were <7% RSD-a value comparable to ESI MS quantitative assays and well within the acceptable limits for screening assays. The speed of the MALDI readout is currently about 10 s per sample, thus allowing for over 7500 samples/day. From a simplicity standpoint, the enzymatic reaction mixtures are prepared by liquid handling robots, the reactions are stopped by addition of a 10 times volume of acidic matrix solution, and the samples are simultaneously transferred to MALDI target plate for analysis. Importantly, the ratios of substrate to product are of sufficient reproducibility to eliminate the need for internal standards and, thus, minimize the cost and increasing the speed of assay development.     

Sensitivity analysis of thermodynamic properties of liquid water: a general approach to improve empirical potentials

A sensitivity analysis of bulk water thermodynamics is presented in an effort to understand the relation between qualitative features of molecular potentials and properties that they predict. The analysis is incorporated in molecular dynamics simulations and investigates the sensitivity of the Helmholtz free energy, internal energy, entropy, heat capacity, pressure, thermal pressure coefficient, and static dielectric constant to components of the potential rather than the parameters of a given functional form. The sensitivities of the properties are calculated with respect to the van der Waals repulsive and the attractive parts, plus short- and long-range Coulomb parts of three four site empirical water potentials: TIP4P, Dang-Chang and TTM2R. The polarization sensitivity is calculated for the polarizable Dang-Chang and TTM2R potentials. This new type of analysis allows direct comparisons of the sensitivities for different potentials that use different functional forms. The analysis indicates that all investigated properties are most sensitive to the van der Waals repulsive, the short-range Coulomb and the polarization components of the potentials. When polarization is included in the potentials, the magnitude of the sensitivity of the Helmholtz free energy, internal energy, and entropy with respect to this part of the potential is comparable in magnitude to the other electrostatic components. In addition similarities in trends of observed sensitivities for nonpolarizable and polarizable potentials lead to the conclusion that the complexity of the model is not of critical importance for the calculation of these thermodynamic properties for bulk water. The van der Waals attractive and the long-range Coulomb sensitivities are relatively small for the entropy, heat capacity, thermal pressure coefficient and the static dielectric constant, while small changes in any of the potential contributions will significantly affect the pressure. The analysis suggests a procedure for modification of the potentials to improve predictions of thermodynamic properties and we demonstrate this general approach for modifying potentials for one of the potentials.     

A synthetic approach toward nitiol: construction of two 1,22-dihydroxynitianes

Synthetic work toward the total synthesis of nitiol has culminated in the construction of two epimeric hydroxylated derivatives, the 1,22-dihydroxynitianes. Key stereodefining steps in the construction of the A-ring fragment (13) were the use of a siloxy-epoxide rearrangement reaction, a Pauson-Khand reaction, a Norrish 1 photochemical cleavage reaction, and a highly regio- and stereoselective hydrostannylation reaction of an ynoate. The stereochemistry of the synthetically challenging C-ring fragment (20) was established using an Ireland-Claisen reaction and a Grubbs ring-closing metathesis process as key steps. The 12-membered B-ring of the nitiane skeleton was constructed using a copper-promoted Stille cross-coupling and a Kishi-Hiyama-Nozaki carbonyl addition reaction. Unfortunately, the carbonyl addition reaction produced hydroxyl functionality that could not be selectively removed. Consequently, a synthesis of epimeric 1,22-dihydroxynitianes, which are compounds that are structural hybrids of two natural products, nitiol and variculanol, was completed.     

Mn-mediated coupling of alkyl iodides and chiral N-acylhydrazones: optimization, scope, and evidence for a radical mechanism

Stereoselective radical additions have excellent potential as mild, nonbasic carbon-carbon bond constructions for direct asymmetric amine synthesis. Efficient intermolecular radical addition to C=N bonds with acyclic stereocontrol has previously been limited mainly to secondary and tertiary radicals, a serious limitation from the perspective of synthetic applications. Here, we provide full details of the use of photolysis with manganese carbonyl to mediate stereoselective intermolecular radical addition to N-acylhydrazones. Photolysis (300 nm) of alkyl halides and hydrazones in the presence of Mn2(CO)10 and InCl(3) as a Lewis acid led to reductive radical addition; diastereomer ratios ranged from 93:7 to 98:2 at ca. 35 degrees C. The reaction tolerates additional functionality in either reactant, enabling subsequent transformations as shown in an efficient asymmetric synthesis of coniine. A series of hydrazones bearing different substituents on the oxazolidinone auxiliary were compared; consistently high diastereocontrol revealed that the identity of the substituent had little practical effect on the diastereoselectivity. Further mechanistic control experiments confirmed the intermediacy of radicals and showed that independently prepared alkyl- or acylmanganese pentacarbonyl compounds do not undergo efficient addition to the N-acylhydrazones under thermal or photolytic (300 nm) conditions. These Mn-mediated conditions avoid toxic tin reagents and enable stereoselective intermolecular radical additions to C=N bonds with the broadest range of alkyl halides yet reported, including previously ineffective primary alkyl halides.     

State-resolved thermalization of singlet and mixed singlet-triplet states of CH2

The role of mixed states in the collision-induced thermalization, intersystem crossing, and reactive loss of CH(2) (~a (1)A1) has been monitored using Doppler-resolved transient frequency modulation absorption spectroscopy. Singlet CH(2) is produced in a hot initial distribution of translation and rotational energy states in the 308 nm photodissociation of ketene in a large excess of argon. Collisions with Ar and ketene cool the translational and rotational degrees of freedom, while depleting the total singlet CH(2) population through reaction and intersystem crossing. Direct monitoring of the time-dependent populations of rotational levels containing mixed singlet and triplet character reveals a rapid interconversion between the two components, but no discernable difference between the kinetics of the pure singlet and mixed states at longer times.