A role for hydrophobicity in a Diels–Alder reaction catalyzed by pyridyl-modified RNA

New classes of RNA enzymes or ribozymes have been obtained by in vitro evolution and selection of RNA molecules. Incorporation of modified nucleotides into the RNA sequence has been proposed to enhance function. DA22 is a modified RNA containing 5-(4-pyridylmethyl) carboxamide uridines, which has been selected for its ability to promote a Diels–Alder cycloaddition reaction. Here, we show that DA_TR96, the most active member of the DA22 RNA sequence family, which was selected with pyridyl-modified nucleotides, accelerates a cycloaddition reaction between anthracene and maleimide derivatives with high turnover. These widely used reactants were not used in the original selection for DA22 and yet here they provide the first demonstration of DA_TR96 as a true multiple-turnover catalyst. In addition, the absence of a structural or essential kinetic role for Cu²⁺, as initially postulated, and nonsequence-specific hydrophobic interactions with the anthracene substrate have led to a reevaluation of the pyridine modification''s role. These findings broaden the catalytic repertoire of the DA22 family of pyridyl-modified RNAs and suggest a key role for the hydrophobic effect in the catalytic mechanism.     

Synthesis of polysubstituted β-amino cyclohexane carboxylic acids via Diels–Alder reaction using Ni(II)-complex stabilized β-alanine derived dienes

This paper describes the design and synthesis of a new class of β-alanine derived dienes stabilized by Ni(II)-complex. Preliminary study of their Diels–Alder cycloaddition reactions with several types of dienophiles demonstrates their significant synthetic potential for the preparation of various polyfunctional β-aminocyclohexane carboxylic acids.     

Reverse-docking study of the TADDOL-catalyzed asymmetric hetero-Diels–Alder reaction

The reverse-docking of a TADDOL catalyst to rigid transition-state (TS) representations of an asymmetric hetero-Diels–Alder reaction is described. The resulting docking poses represent a simplified geometric model of the TS for the catalyzed reaction. The conformational space of the catalyst in proximity to the catalyst-free TS models is sampled stochastically and the energetically favored poses are subjected to a clustering procedure to highlight structural attributes compatible with organocatalysis. Each pose is scored and ranked based on its molecular-mechanics docking energy. The reverse-docking procedure reveals a clear energetic trend in favor of the experimentally preferred product enantiomers. Analysis of the best poses suggests a geometric model that is consistent with principles of molecular recognition, catalysis, and experimental data.      

Diels–Alder reactivity of benzannulated isobenzofurans as assessed by density functional theory

Density functional theory (DFT) calculations at the B3LYP/6-31G* level for isobenzofuran 1 and eleven benzannulated derivatives of types 2 and 3 have been performed in order to compare their relative reactivities as dienes in Diels–Alder reactions. The transition state (TS) energies for their reactions with ethylene have been determined and shown to form a linear correlation between activation energies and structure count (SC) ratios. TS energies as a method for comparison of diene reactivities can be applied to IBFs bearing substituents on the ring as well as those containing heteroatoms, for which the SC ratio method failed. Different measures of aromaticity of benzannulated IBFs indicated a decrease in aromaticity going from 4 to 14, which is also reflected in their reactivity as a dienes in Diels–Alder reaction. Figure Isobenzofuran 1 and benzannulated isobenzofurans 2 and 3      

Extended Parker–Sochacki method for Michaelis–Menten enzymatic reaction model

In this article, a new approach—namely, the extended Parker–Sochacki method (EPSM)—is presented for solving the Michaelis–Menten nonlinear enzymatic reaction model. The Parker–Sochacki method (PSM) is combined with a new resummation method called the Sumudu–Padé resummation method to obtain approximate analytical solutions for the model. The obtained solutions by the proposed approach are compared with the solutions of PSM and the Runge–Kutta numerical method (RKM). The comparison proves the practicality, efficiency, and correctness of the presented approach. It serves as a basis for solving other nonlinear biochemical reaction models in the future.     

A modified version of the digestion–ligation cloning method for more efficient molecular cloning

Here we describe a modified version of the digestion–ligation approach for efficient molecular cloning. In comparison with the original method, the modified method has the additional steps of gel purification and a second ligation after the first ligation of the linearized vector and DNA insert. During this process, the efficiency and reproducibility could be significantly improved for both stick-end cloning and blunt-end cloning. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology.     

A Lagrangian particle method for reaction–diffusion systems on deforming surfaces

Reaction–diffusion processes on complex deforming surfaces are fundamental to a number of biological processes ranging from embryonic development to cancer tumor growth and angiogenesis. The simulation of these processes using continuum reaction–diffusion models requires computational methods capable of accurately tracking the geometric deformations and discretizing on them the governing equations. We employ a Lagrangian level-set formulation to capture the deformation of the geometry and use an embedding formulation and an adaptive particle method to discretize both the level-set equations and the corresponding reaction–diffusion. We validate the proposed method and discuss its advantages and drawbacks through simulations of reaction–diffusion equations on complex and deforming geometries.     

Characterization of very dense mineral oxide–gel composites for fluidized-bed adsorption of biomolecules

Efficient design of fluidized-bed biomolecule adsorption from crude feed stock requires particles with elevated density, large adsorption capacity and broad chemical stability. Moreover, combinations of small particle diameters with high densities allow for high fluidization velocities while preserving a rapid mass transfer.This approach has been implemented by combining stable porous mineral oxide of high density (2.2, 4.7, 5.7, 9.4 g/ml) with functionalized hydrogels. The cross-linked hydrogel derivative fills the internal porosity of the beads and provides a high equilibrium binding capacity.Various porous mineral oxides (silica, titania, zirconia and hafnia) have been characterized in term of fluidization behavior, surface reactivity and chemical resistance to harsh CIP procedures. Porous zirconia particles were also modified into ion-exchangers by suitable surface modification and intraparticle polymerization of functionalized stable derivatives of acrylic monomers. Back-mixings in fluidized bed columns were analyzed by residence time distribution analysis of inert tracers. 328 and 218 mixing plates per meter were found for respectively, bed expansions of 1.7 and 2.9. The dynamic protein adsorption behaviors of zirconia-based polymeric anion-exchange sorbents were obtained in fluidized-bed, using BSA as model protein. A dynamic binding capacity of 62 mg/ml was observed at a fluidizing velocity of 320 cm/h. These investigations substantiate the favorable physical and chemical characteristics anticipated for dense composite beads for use as fluidized bed adsorbents.     

Revised method for routine determination of urinary dialkyl phosphates using gas chromatography–mass spectrometry

Among urinary organophosphorus pesticide (OP) metabolites, dialkyl phosphates (DAPs) have been most often measured as a sensitive biomarker in non-occupational and occupational OP exposure risk assessment. In our conventional method, we have employed a procedure including simple liquid–liquid extraction (diethyl ether/acetonitrile), derivatization (pentafluorobenzylbromide, PFBBr) and clean-up (multi-layer column) for gas chromatography–mass spectrometry (GC–MS) analysis starting from 5-mL urine samples. In this study, we introduce a revised analytical method for urinary DAPs; its main modification was aimed at improving the pre-derivatization dehydration procedure. The limits of detection were approximately 0.15 μg/L for dimethylphosphate (DMP), 0.07 μg/L for diethylphosphate (DEP), and 0.05 μg/L for both dimethylthiophosphate (DMTP) and diethylthiophosphate (DETP) in 2.5-mL human urine samples. Within-run precision (percent of relative standard deviation, %RSD) at the DAP levels varying in the range of 0.5–50 μg/L was 6.0–19.1% for DMP, 3.6–18.3% for DEP, 8.0–25.6% for DMTP and 9.6–27.8% for DETP. Between-run precision at 5 μg/L was below 15.7% for all DAPs. The revised method proved to be feasible to routine biological monitoring not only for occupational OP exposure but also for environmental background levels in the general population. Compared to our previous method, the revised method underscores the importance of adding pre-derivatization anhydration for higher sensitivity and precision.     

General method for purification of α-amino acid-n-carboxyanhydrides using flash chromatography

We describe the application of flash column chromatography on silica gel as a rapid and general method to obtain pure α-amino acid-N-carboxyanhydride (NCA) monomers, the widely used precursors for the synthesis of polypeptides, without the need for recrystallization. This technique was effective at removing all common impurities from NCAs and was found to work for a variety of NCAs, including those synthesized using different routes, as well as those bearing either hydrophilic or hydrophobic side chains. All chromatographed NCAs required no further purification and could be used directly to form high molecular weight polypeptides. This procedure is especially useful for the preparation of highly functional and low melting NCAs that are difficult to crystallize and, consequently, to polymerize. This method solves many long-standing problems in NCA purification and provides rapid access to NCAs that were previously inaccessible in satisfactory quality for controlled polymerization. This method is also practical in that it requires less time than recrystallization and often gives NCAs in improved yields.     

An efficient method for the selective synthesis of 2-deoxy-2-iodo-glycosides by O-glycosidation of D-glucal using I₂-Cu(OAc)₂

An efficient and convenient method for the synthesis of 2-deoxy-2-iodo-O-glycosides from tri-O-acetyl-d-glucal with various alcohols by using I₂-Cu(OAc)₂ is described. The 21 examples of corresponding glycosides were obtained in high yields, with good anomeric selectivity.     

A simple method using β-globin polymerase chain reaction for the species identification of animal cell lines—A progress report

Continuous cell lines are widely used in cell biology and serve as model systems in basic and applied research. Fundamental requirements for the use of cell lines are a well-identified origin and the exclusion of cross-contamination by prokaryotic or eukaryotic cells. Because the cross-contamination of one cell line with another cell line may occur in a concealed manner, special emphasis must be taken to (1) prevent such an "accident" and (2) monitor regularly the identity of the cell line(s) in use. Apart from human cell lines, mouse-, rat-, and hamster-derived cell lines are used in basic cell culture and biotechnology. We established a polymerase chain reaction (PCR) assay to detect and confirm the species origin for these species and to detect interspecies cross-contamination. Our PCR method is based on oligonucleotide primers annealing to specific sequences in the beta-globin gene, which were designed to amplify one deoxyribonucleic acid (DNA) segment only per analyzed sample. We confirmed the species identity of 82 cell lines as human, mouse, rat, and Syrian hamster by beta-globin PCR. The DNAs from eight additional cell lines of less frequently used species were not amplified with the primers chosen. Cross-contamination of 5-10% of either mouse or rat DNA was detectable. One species-specific primer pair was sufficient for confirmation of the expected species, and for identification of an unknown cell line the combination of two or more primer pairs is suggested. Our PCR assay represents a powerful, fast, easy, robust, and inexpensive method for speciation and does not need any elaborate sequencing or computer-based analysis system.     

Synthesis of novel chromeno-annulated cis-fused pyrano[3,4-c]benzopyran and naphtho pyran derivatives via domino aldol-type/hetero Diels–Alder reaction and their cytotoxicity evaluation

New chromeno-annulated cis-fused pyrano[3,4-c]benzopyran and naphtho pyran derivatives have been synthesized by domino aldol-type reaction/hetero Diels–Alder reaction generated from o-quinone methide in situ from 7-O-prenyl derivatives of 8-formyl-2,3-disubstituted chromenones with resorcinols/naphthols in the presence of 20 mol % ethylenediamine diacetate (EDDA), triethylamine (2 mL) as co-catalyst in CH₃CN under reflux conditions in good yields. The structures were established based on spectroscopic data, and further confirmed by X-ray diffraction analysis. The results showed that compounds 4h and 4j exhibited very potent cytotoxicity against human cervical cancer cell line (HeLa). Compound 4h displayed good inhibitory activity against both breast cancer cell lines, MDA-MB-231 and MCF-7. Further, the compound 4i exhibited good cytotoxicity against only MDA-MB-231, and compound 4j showed promising activity against human lung cancer cell line, A549 with IC₅₀ value of 2.53 ± 0.07 μM, which was comparable to the standard doxorubicin (IC₅₀ = 1.21 ± 0.1 μM).     

Cellulase immobilized by sol–gel entrapment for efficient hydrolysis of cellulose

Cellulase from Trichoderma reesei (Celluclast 1.5 L, Novozyme) was immobilized by sol–gel encapsulation, using binary or ternary mixtures of tetramethoxysilane (TMOS) with alkyl- or aryl-substituted trimethoxysilanes as precursors. Optimization of immobilization conditions resulted in 92 % recovery of total enzymatic activity in the best immobilized preparate. The immobilized cellulase exhibiting the highest activity, obtained from tetramethoxysilane and methyltrimethoxysilane precursors at 3:1 molar ratio, was investigated in the hydrolysis reaction of microcrystalline cellulose (Avicel PH101). Although the optimal values did not change significantly, both temperature and pH stabilities of the sol–gel entrapped cellulase improved compared to the native enzyme. Immobilization also conferred superior resistance against the inactivation effect of glucose. Reuse of the sol–gel entrapped cellulase showed 40 % retention of the initial activity after five batch hydrolysis cycles, demonstrating the potential of this biocatalyst for large-scale application.     

Polymerase chain reaction–hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation

In this article, we discuss the polymerase chain reaction (PCR)–hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA–BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase–streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR–hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR–hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications.     

Palladium nanoparticles enzyme aggregate (PANEA) as efficient catalyst for Suzuki–Miyaura reaction in aqueous media

Palladium nanoparticles enzyme aggregate (PANEA) were prepared from Candida antarctica B lipase and palladium salt by precipitation and subsequent in situ Pd nanoparticle formation. This heterogeneous catalyst was successfully used for the Suzuki–Miyaura cross-coupling reaction between bromobenzene with different phenylboronic acid derivatives under mild reaction conditions and using low Pd amount. The nanocatalyst exhibited the highest catalytic activity in a mixture of methanol/water (1:1), obtaining good to excellent product yields from the cross-coupling reaction. A variety of functional groups were accepted and the catalyst was recycled 4 times without activity loss.     

Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag™ Technology

Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 μl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple, potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies.     

Stationary multiple spots for reaction–diffusion systems

In this paper, we review analytical methods for a rigorous study of the existence and stability of stationary, multiple spots for reaction–diffusion systems. We will consider two classes of reaction–diffusion systems: activator–inhibitor systems (such as the Gierer–Meinhardt system) and activator–substrate systems (such as the Gray–Scott system or the Schnakenberg model). The main ideas are presented in the context of the Schnakenberg model, and these results are new to the literature. We will consider the systems in a two-dimensional, bounded and smooth domain for small diffusion constant of the activator. Existence of multi-spots is proved using tools from nonlinear functional analysis such as Liapunov–Schmidt reduction and fixed-point theorems. The amplitudes and positions of spots follow from this analysis. Stability is shown in two parts, for eigenvalues of order one and eigenvalues converging to zero, respectively. Eigenvalues of order one are studied by deriving their leading-order asymptotic behavior and reducing the eigenvalue problem to a nonlocal eigenvalue problem (NLEP). A study of the NLEP reveals a condition for the maximal number of stable spots. Eigenvalues converging to zero are investigated using a projection similar to Liapunov–Schmidt reduction and conditions on the positions for stable spots are derived. The Green’s function of the Laplacian plays a central role in the analysis. The results are interpreted in the biological, chemical and ecological contexts. They are confirmed by numerical simulations.      

Interpolation method for accurate affinity ranking of arrayed ligand–analyte interactions

The values of the affinity constants (k_d, k_a, and K_D) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (k_d and k_a) and dissociation equilibrium (K_D) constants for various ligand densities and analyte concentrations are exponentially interpolated to the K_D at R_max = 100 RU response level (K_D^R100).