Solid-phase synthesis of nucleoside analogues

The synthesis of a 25 000 member library of nucleoside analogues as discrete compounds in milligram quantities is described. The use of the Nanokan technology developed by IRORI (Discovery Partners International) together with macroporous solid support allowed us to develop a highly reliable and practical synthetic route for the high-throughput derivatization of both the pyrimidine and purine nucleoside scaffold. A 2',3'-acetal linkage of the scaffolds to the solid support proved to be stable enough for the chemical transformations employed, yet labile enough for mild cleavage conditions to yield final products in high purity. The publication represents an example for combining synthetic organic chemistry on advanced scaffolds with the latest technologies of combinatorial chemistry in order to provide both industrial and academic institutions with compounds in high number and quality, thereby accelerating the search for novel biological targets and drug development.     

Oxidation and sulfonation of cellulosics

Bleached hardwood (HW) kraft pulp and derived nanocellulosic structures were modified by a periodate oxidation followed by treatment with sodium bisulfite to yield the corresponding C2/3 sulfonates. The impact of this oxidative–reductive protocol on the chemical and physical properties of cellulose was evaluated by determining physical dimensions, functional groups, and their water absorbency properties. It was found that the water absorbency of cellulosic material can be enhanced by 8.0–199.0% with this oxidation/sulfonation protocol. Distinct differences were observed between sulfonated pulp fibers and nanocellulosic structures, with the latter exhibiting relatively higher water retention values (WRV).     

Cyclization reactions involving palladium-catalyzed carbene insertion into aryl halides

Palladium is shown to catalyze the insertion of trimethylsilylmethylene into aryl halides, leading to benzylpalladium intermediates that cyclize to give indenylsilanes through carbopalladation of pendant alkenes or allenes. Allylsilanes generated through these processes are susceptible to protodesilylation in situ.     

Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3bromo-2-methylpropanoate and ethyl 3phenylbutyrate

Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons. Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate. Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E=17). Previous directed evolution identified mutants Asp158Asn and Leu181Gln that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3-phenylbutyrate. In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser). A homology model of PFE indicates that all mutations lie outside the active site, 12-14 A from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site. Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations. Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.     

Fast generation of an electron beam in a magnetron gun with a secondary-emission metallic cathode

The formation of an electron layer and the generation of an electron beam in magnetron guns where secondary emission is triggered by nanosecond pulses are studied. In the guns with small cross sizes, hollow electron beams with an outer diameter of 3–6 mm are generated. The beam current is 1–2 A, and the cathode voltage is 5–7 kV. Results obtained indicate that the generation of nanosecond beam-current pulses is a possibility.     

Structural Characterization and Comparison of Switchgrass Ball-milled Lignin Before and After Dilute Acid Pretreatment

To reduce the recalcitrance and enhance enzymatic activity, dilute H₂SO₄ pretreatment was carried out on Alamo switchgrass (Panicum virgatum). Ball-milled lignin was isolated from switchgrass before and after pretreatment. Its structure was characterized by ¹³C, HSQC, and ³¹P NMR spectroscopy. It was confirmed that ball-milled switchgrass lignin is of HGS type with a considerable amount of p-coumarate and felurate esters of lignin. The major ball-milled lignin interunit was the β-O-4 linkage, and a minor amount of phenylcoumarin, resinol, and spirodienone units were also present. As a result of the acid pretreatment, there was 36% decrease of β-O-4 linkage observed. In addition to these changes, the S/G ratio decreases from 0.80 to 0.53.     

Revised molecular basis of the promiscuous carboxylic acid perhydrolase activity in serine hydrolases

Several serine hydrolases catalyze a promiscuous reaction: perhydrolysis of carboxylic acids to form peroxycarboxylic acids. The working hypothesis is that perhydrolases are more selective than esterases for hydrogen peroxide over water. In this study, we tested this hypothesis, and focused on L29P-PFE (Pseudomonas fluorescens esterase), which catalyzes perhydrolysis of acetic acid 43-fold faster than wild-type PFE. This hypothesis predicts that L29P-PFE should be approximately 43-fold more selective for hydrogen peroxide than wild-type PFE, but experiments show that L29P-PFE is less selective. The ratio of hydrolysis to perhydrolysis of methyl acetate at different concentrations of hydrogen peroxide fit a kinetic model for nucleophile selectivity. L29P-PFE (β(0)=170 M(-1)) is approximately half as selective for hydrogen peroxide over water than wild-type PFE (β(0)=330 M(-1)), which contradicts the working hypothesis. An alternative hypothesis is that carboxylic acid perhydrolases increase perhydrolysis by forming the acyl-enzyme intermediate faster. Consistent with this hypothesis, the rate of acetyl-enzyme formation, measured by (18)O-water exchange into acetic acid, was 25-fold faster with L29P-PFE than with wild-type PFE, which is similar to the 43-fold faster perhydrolysis with L29P-PFE. Molecular modeling of the first tetrahedral intermediate (T(d)1) suggests that a closer carbonyl group found in perhydrolases accepts a hydrogen bond from the leaving group water. This revised understanding can help design more efficient enzymes for perhydrolysis and shows how subtle changes can create new, unnatural functions in enzymes.     

Shaken, not stirred: collapsing a peptoid monolayer to produce free-floating, stable nanosheets

Two-dimensional nanomaterials play a critical role in biology (e.g., lipid bilayers) and electronics (e.g., graphene) but are difficult to directly synthesize with a high level of precision. Peptoid nanosheet bilayers are a versatile synthetic platform for constructing multifunctional, precisely ordered two-dimensional nanostructures. Here we show that nanosheet formation occurs through an unusual monolayer intermediate at the air-water interface. Lateral compression of a self-assembled peptoid monolayer beyond a critical collapse pressure results in the irreversible production of nanosheets. An unusual thermodynamic cycle is employed on a preparative scale, where mechanical energy is used to buckle an intermediate monolayer into a more stable nanosheet. Detailed physical studies of the monolayer-compression mechanism revealed a simple preparative technique to produce nanosheets in 95% overall yield by cyclical monolayer compressions in a rotating closed vial. Compression of monolayers into stable, free-floating products may be a general and preparative approach to access 2D nanomaterials.     

Structural changes in switchgrass lignin and hemicelluloses during pretreatments by NMR analysis

Switchgrass is currently being developed as a sustainable bio-energy crop due to its broad adaptability, high mass yield and low agricultural input. Its current conversion to biofuels is detrimentally impacted by its native recalcitrance which is typically addressed using chemical and/or biological pretreatments. In this study, extractives free switchgrass was pretreated with steam, dilute H₂SO₄ and lime at 160 °C for 1 h. The degradation and impact of pretreatment was estimated semi-quantitatively by ¹³C–¹H HSQC (heteronuclear single quantum coherence) NMR analysis of ball milled untreated and pretreated switchgrass samples in perdeuterated pyridinium chloride–DMSO-d₆ solvent system. As a result of steam pretreatment the resulting switchgrass was depleted of xylan and a slight degradation of lignin were observed. This was confirmed by the relative decrease of cross peak intensity for β-aryl ether, phenylcoumaran, resinol and dibenzodioxocin units. Significant structural changes observed due to the lime pretreatment of switchgrass was deacetylation/dissolution of hemicellulose and the extent of delignification was less however, a preferential removal p-hydroxy of benzoyl ester, ferulate and coumarate type linkages were notified from the HSQC studies. Finally the most significant degradation resulted in acid pretreatment involving ∼90% loss of hemicellulose and a substantial degradation of various lignin sub-units. These results are further supported by the composition analysis of the respective switchgrass samples.     

Mechanisms controlling the sensitivity of amperometric biosensors in flow injection analysis systems

This paper numerically investigates the sensitivity of an amperometric biosensor acting in the flow injection mode when the biosensor contacts an analyte for a short time. The analytical system is modelled by non-stationary reaction-diffusion equations containing a non-linear term related to the Michaelis-Menten kinetics of an enzymatic reaction. The mathematical model involves three regions: the enzyme layer where enzymatic reaction as well as the mass transport by diffusion takes place, a diffusion limiting region where only the diffusion takes place, and a convective region. The biosensor operation is analysed with a special emphasis to the conditions at which the biosensor sensitivity can be increased and the calibration curve can be prolonged by changing the injection duration, the permeability of the external diffusion layer, the thickness of the enzyme layer and the catalytic activity of the enzyme. The apparent Michaelis constant is used as a main characteristic of the sensitivity and the calibration curve of the biosensor. The numerical simulation was carried out using the finite difference technique.