Growth of polystyrene films via gas‐phase polymerization with [Pd(CH3CN)4][BF4]2 thin film catalyst

The heterogeneous catalytic polymerization of styrene vapor with a tetrakis(acetonitrile)palladium(II) tetrafluoroborate, [Pd(CH₃CN)₄][BF₄]₂, thin film has been demonstrated. The catalyst is deposited by nebulization of dilute solutions onto a quartz crystal microbalance (QCM) and then exposed to styrene vapor in controlled environments. The use of QCM allows in situ monitoring of catalyst deposition and polymer growth kinetics. The polymerization process appears to involve the entire catalyst film rather than polymerization only at the catalyst film surface. The styrene vapor polymerization occurs rapidly after a short induction time needed for monomer dissolution and catalyst activation. The narrow molecular weight distribution of the produced polymer suggests that the deposited film acts as a single site catalyst. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1930–1934, 2005     

Enzymatic assay of marine bacterial phosphatases by capillary electrophoresis with laser-induced fluorescence detection

Microbial ectoenzyme activities in aquatic environments are important determinants of polymer hydrolysis and indicators of the state of microbial carbon, nitrogen, and phosphorus nutrition. Marine ectoenzymes are found on the cell surface or in the periplasmic space of gram-negative heterotrophic bacteria. Phosphatases, which remove phosphate groups from substrates, are one example of an ectoenzyme. Enzyme assays based on-capillary electrophoresis (CE) take advantage of CE's high-efficiency separation, extremely low sample volume requirements, and its ability to electrophoretically mix and separate zones of enzymes, substrates, and products all in one experimental run. CE has better resolving power and, when utilized with laser-induced fluorescence (LIF) detection, it is more sensitive than chromatography. CE-LIF is a promising tool for determining different phosphatases within a single microbial strain as well as the functional diversity between strains. In this study, four bacterial strains were studied (Shewanella sp., TW7, BB2AT2, and Vibrio alginolyticus) with each yielding at least one phosphatase that was kinetically characterized. K(m) values were calculated and found to be in the range of 0.0725-3.35 microM, whereas V(max) values ranged from 1.02 x 10(-3) to 1.05 x 10(-2) microM/min. The large range of values demonstrates differences among the phosphatases, suggesting different roles for each phosphatase not only between the species but also within a single bacterial species. This can have the important implications for organic matter processing in the sea.     

Enantiomer-specific binding of ruthenium(II) molecular wires by the amine oxidase of Arthrobacter globiformis

The copper amine oxidase from Arthrobacter globiformis (AGAO) is reversibly inhibited by molecular wires comprising a Ru(II) complex head group and an aromatic tail group joined by an alkane linker. The crystal structures of a series of Ru(II)-wire-AGAO complexes differing with respect to the length of the alkane linker have been determined. All wires lie in the AGAO active-site channel, with their aromatic tail group in contact with the trihydroxyphenylalanine quinone (TPQ) cofactor of the enzyme. The TPQ cofactor is consistently in its active ("off-Cu") conformation, and the side chain of the so-called "gate" residue Tyr296 is consistently in the "gate-open" conformation. Among the wires tested, the most stable complex is produced when the wire has a -(CH2)4- linker. In this complex, the Ru(II)(phen)(bpy)2 head group is level with the protein molecular surface. Crystal structures of AGAO in complex with optically pure forms of the C4 wire show that the linker and head group in the two enantiomers occupy slightly different positions in the active-site channel. Both the Lambda and Delta isomers are effective competitive inhibitors of amine oxidation. Remarkably, inhibition by the C4 wire shows a high degree of selectivity for AGAO in comparison with other copper-containing amine oxidases.     

Lancifodilactone G: insights about an unusually stable enol

From quantum mechanics calculations we confirm that the naturally occurring enol lancifodilactone G is stable over the keto form (by 2.6 kcal/mol in water), the only known stable aliphatic enol (devoid of conjugated or bulky aromatics and lacking a 1,3-diketone structural motif known to stabilize enols). We determine architectural elements responsible for the enol stabilization and find a mechanism for keto-enol conversion in solution. In addition, we correct previously reported computational results that were performed on the misinterpreted structure demonstrating that the enol form of this natural product is more stable than previously thought.     

Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL⁻¹. The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.     

Characterization and control of lipid layer fluidity in hybrid bilayer membranes

The main gel-to-liquid-crystal (LC) phase transition temperature, T(m), of the distal lipid layer in hybrid bilayer membranes (HBMs) under water was investigated using vibrational sum frequency spectroscopy (VSFS). VSFS has unique sensitivity to order/disorder transitions in the lipid acyl chains and can determine T(m) for the lipid monolayers in HBMs. We recently reported the observation that T(m) is raised and the transition width is broadened for the overlying phospholipid monolayer in HBM systems formed on densely packed crystalline self-assembled monolayers (SAMs) as compared to that of vesicles in solution. In this report, we establish that T(m) for the lipid layer of HBMs can be controlled by proper choice of the SAM underlayer. The SAM underlayer of the HBM was systematically altered by using an alkane thiol, a saturated thiolipid, a mixed SAM of a saturated lipid-pyridine disulfide, and finally a mixed SAM of an unsaturated lipid-pyridine disulfide. T(m) was measured for two different chain length saturated phosphatidylcholine lipid overlayers on these SAMs. The values obtained show that Tm of the lipid layer of HBMs is sensitive to the composition and/or packing density of molecules in the underlying SAM.     

Porous Liquids: A Promising Class of Media for Gas Separation

A porous liquid containing empty cavities has been successfully fabricated by surface engineering of hollow structures with suitable corona and canopy species. By taking advantage of the liquid‐like polymeric matrices as a separation medium and the empty cavities as gas transport pathway, this unique porous liquid can function as a promising candidate for gas separation. Moreover, such a facile synthetic strategy can be further extended to the fabrication of other types of nanostructure‐based porous liquid, opening up new opportunities for preparation of porous liquids with attractive properties for specific tasks.     

Immobilization of proteins on carboxylic acid functionalized nanopatterns

The immobilization of proteins on nanopatterned surfaces was investigated using in situ atomic force microscopy (AFM) and ex situ infrared reflectance–absorption spectroscopy (IRAS). The AFM-based lithography technique of nanografting provided control of the size, geometry, and spatial placement of nanopatterns within self-assembled monolayers (SAMs). Square nanopatterns of carboxylate-terminated SAMs were inscribed within methyl-terminated octadecanethiolate SAMs and activated using carbodiimide/succinimide coupling chemistry. Staphylococcal protein A was immobilized on the activated nanopatterns before exposure to rabbit immunoglobulin G. In situ AFM was used to monitor changes in the topography and friction of the nanopatterns in solution upon protein immobilization. Complementary studies with ex situ IRAS confirmed the surface chemistry that occurred during the steps of SAM activation and subsequent protein immobilization on unpatterned samples. Since carbodiimide/succinimide coupling chemistry can be used for surface attachment of different biomolecules, this protocol shows promise for development of other aqueous-based studies for nanopatterned protein immobilization.     

Study of the Air-Water Interfacial Properties of Biodegradable Polyesters and Their Block Copolymers with Poly(ethylene glycol)

It has been reported that the surface pressure-area isotherm of poly(d,l-lactic acid-ran-glycolic acid) (PLGA) at the air-water interface exhibits several interesting features: (1) a plateau at intermediate compression levels, (2) a sharp rise in surface pressure upon further compression, and (3) marked surface pressure-area hysteresis during compression-expansion cycles. To investigate the molecular origin of this behavior, we conducted an extensive set of surface pressure and AFM imaging measurements with PLGA materials having several different molecular weights and also a poly(d,l-lactic acid-ran-glycolic acid-ran-caprolactone) (PLGACL) material in which the caprolactone monomers were incorporated as a plasticizing component. The results suggest that (i) the plateau in the surface pressure-area isotherm of PLGA (or PLGACL) occurs because of the formation (and collapse) of a continuous monolayer of the polymer under continuous compression; (ii) the PLGA monolayer becomes significantly resistant to compression at high compression because under that condition the collapsed domains become large enough to become glassy (such behavior was not observed in the nonglassy PLGACL sample); and (iii) the isotherm hysteresis is due to a coarsening of the collapsed domains that occurs under high-compression conditions. We also investigated the monolayer properties of PEG-PLGA and PEG-PLGACL diblock copolymers. The results demonstrate that the tendency of PLGA (or PLGACL) to spread on water allows the polymer to be used as an anchoring block to form a smooth biodegradable monolayer of block copolymers at the air-water interface. These diblock copolymer monolayers exhibit protein resistance.