Translocation of single-stranded DNA through the α-hemolysin protein nanopore in acidic solutions

The effect of acidic pH on the translocation of single-stranded DNA through the α-hemolysin pore is investigated. Two significantly different types of events, i.e. deep blockades and shallow blockades, are observed at low pH. The residence times of the shallow blockades are not significantly different from those of the DNA translocation events obtained at or near physiological pH, whereas the deep blockades have much larger residence times and blockage amplitudes. With a decrease in the pH of the electrolyte solution, the percentage of the deep blockades in the total events increases. Furthermore, the mean residence time of these long-lived events is dependent on the length of DNA, and also varies with the nucleotide base, suggesting that they are appropriate for use in DNA analysis. In addition to being used as an effective approach to affect DNA translocation in the nanopore, manipulation of the pH of the electrolyte solution provides a potential means to greatly enhance the sensitivity of nanopore stochastic sensing.     

Evaluation of α-pyrones and pyrimidones as photoaffinity probes for affinity-based protein profiling

α-Pyrones and pyrimidones are common structural motifs in natural products and bioactive compounds. They also display photochemistry that generates high-energy intermediates that may be capable of protein reactivity. A library of pyrones and pyrimidones was synthesized, and their potential to act as photoaffinity probes for nondirected affinity-based protein profiling in several crude cell lysates was evaluated. Further "proof-of-principle" experiments demonstrate that a pyrimidone tag on an appropriate scaffold is equally capable of proteome labeling as a benzophenone.     

Application of Isocyanides Derived from α-Amino Acids as Substrates for the Ugi Reaction

A systematic study on the influence of Lewis acid, solvent, and temperature on the stereochemical course of the Ugi reaction was performed to find conditions in which the isocyanides derived from enantiopure α-amino acids do not racemize. After a series of experiments, dichloromethane was used as a solvent and BF₃ · OEt₂ as a Lewis acid catalyst. This combination allowed us to obtain a product in a model reaction with 99% diastereoisomeric ratio (dr), with the structure motif of a group of cathepsin K inhibitors.     

Diphenylparabanic acid as a synthon for the synthesis of α-diketones and α-ketocarboxylic acids

Diphenylparabanic acid was found to react with >2 equiv of organolithiums at -78 °C to effectively give the corresponding symmetrical α-diketones. However, upon treatment with 1 equiv of organolithium, the parabanic acid gave mainly 5-substituted 5-hydroxyimidazolidine-2,4-diones. On the other hand, Grignard reagents were less reactive toward the parabanic acid at low temperature, and selectively gave the corresponding 5-hydroxyimidazolidine-2,4-diones even if more than 1 equiv of the reagents was used. A tandem process in which the parabanic acid was first reacted with a Grignard reagent and then reacted in one-pot with an organolithium effectively gave the unsymmetrical α-diketone. 5-Substituted 5-hydroxyimidazolidine-2,4-diones were useful as versatile precursors for preparing α-ketocarboxylic acids as well as unsymmetrical α-diketones.     

Magnetic Bead Cellulose as a Suitable Support for Immobilization of α-Chymotrypsin

Magnetic bead cellulose was prepared by a suspension method from the mixture of viscose and magnetite using thermal sol?Cgel transition and regeneration of cellulose. The prepared magnetic particles after their activation with divinyl sulfone were shown to be suitable magnetic carrier for immobilization of ??-chymotrypsin and for its application in proteomic studies. The specific activity of the immobilized proteinase was high; its activity did not change in the course of storage. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH and temperature dependence of the activity, self-cleavage activity, and possibility of repeated use. ??-Chymotrypsin immobilized to magnetic bead cellulose was used for the proteolytic digestion of porcine pepsin A and human gastric juice and a possibility of direct use of enzyme reaction products for matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis was shown.     

Application of α-alkoxy bridgehead radical for coupling of oxygenated carbocycles

A new coupling methodology for assembly of highly oxygenated carbocycles was developed. An α-alkoxy bridgehead radical was employed as the key reactive intermediate due to its potent reactivity, minimum steric interaction, and predestined stereochemical outcome. The radical of the trioxadamantane structure, generated from the O,Se-acetal, was reacted with electron-deficient cyclic olefins of various ring sizes. Intermolecular formation of sterically congested linkages between two tetrasubstituted carbons and application of the method to a three-component coupling were two significant achievements.     

A stereoselective synthesis of an α-substituted α-amino acid as a substructure for the construction of myriocin

A synthetic route to the protected quaternary α-amino acid 2 with a hydroxylated side chain has been achieved. The key transformations are the diastereoselective substrate-controlled epoxidation of allylic alcohol 4; a highly stereoselective oxidation–reduction protocol, and the excellent regioselective isomerization of the oxazolidinone ring to give an oxazinanone skeleton in derivative 3. The carboxylic acid 2 obtained represents the polar substructure, which is present in myriocin 1.     

Graphene oxide as an enzyme inhibitor: modulation of activity of α-chymotrypsin

We have investigated the efficacy of graphene oxide (GO) in modulating enzymatic activity. Specifically, we have shown that GO can act as an artificial receptor and inhibit the activity of α-chymotrypsin (ChT), a serine protease. Most significantly, our data demonstrate that GO exhibits the highest inhibition dose response (by weight) for ChT inhibition compared with all other reported artificial inhibitors. Through fluorescence spectroscopy and circular dichroism studies, we have shown that this protein-receptor interaction is highly biocompatible and conserves the protein's secondary structure over extended periods (>24 h). We have also explored GO-enzyme interactions by controlling the ionic strength of the medium, which attenuates the host-guest electrostatic interactions. These findings suggest a new generation of enzymatic inhibitors that can be applied to other complex proteins by systematic modification of the GO functionality.     

Time-dependent nanogel aggregation for naked-eye assays of α-amylase activity

This work designs an enzyme-stimulated nanogel aggregation system for the naked-eye assays of α-amylase activity. The visible aggregation of the starch-stabilized CdTe nanogels may be accelerated by α-amylase through its efficient cleavage of glycosidic bonds in the starch network, which has been verified by the evidences from transmission electron microscopy and dynamic light scattering spectra. The required aggregation time, as validated by both the theoretical deduction and the experimental results, is inversely proportional to the enzymatic activity. Therefore a facile method has been proposed for the detection of enzyme activity, with an excellent linear range and a low detection limit. This nanogel-based protocol can be successfully applied in the fast and accurate assays of α-amylase activity in saliva samples with a satisfactory correlation with the standard protocol, suggesting its promising applications in the biomedical and clinical fields, especially in point-of-care testing.     

Application of the Intramolecular α-Amido-alkylation Reaction for the Synthesis of Dibenzo /a,h/ Quinolizines

5,6,9-Trihydro-8H-dibenzo [a,h] quinolizin-8-ones 6 are obtained from adducts of 3,4-dihydroiso-quinolines 1 and phenylacetyl chlorides as a result of an intramolecular α-amidoalkylation reaction.     

Engineering of a bis-chelator motif into a protein α-helix for rigid lanthanide binding and paramagnetic NMR spectroscopy

Attachment of two nitrilotriacetic acid-based ligands to a protein α-helix in an i, i + 4 configuration produces an octadentate chelating motif that is able to bind paramagnetic lanthanide ions rigidly and with high affinity, leading to large pseudocontact shifts and residual dipolar couplings in the NMR spectrum.     

N-Chlorosuccinimide as a versatile reagent for the sulfenylation of ketones: a facile synthesis of α-ketothioethers

The sulfenylation of ketones having α-hydrogens has been achieved using N-chlorosuccinimide (NCS) under mild reaction conditions to produce α-ketothioethers in excellent yields with high selectivity. The use of NCS makes this method quite simple, convenient and practical.     

Diaminomethylenemalononitrile as a Chiral Single Hydrogen Bond Catalyst: Application to Enantioselective Conjugate Addition of α-Branched Aldehydes

An improved diaminomethylenemalononitrile organocatalyst, bearing a N,N-disubstituted structure, promoted enantioselective conjugate addition reaction of α-branched aldehydes with vinyl sulfone, affording adducts with excellent enantioselectivities (up to 96% ee). Mechanistic studies revealed that the diaminomethylenemalononitrile motif holds the vinyl sulfone substrate using a single hydrogen bond accompanied by multiple weak interactions, including electrostatic C−H⋅⋅⋅O interactions.     

A new potentiometric sensor for the determination of α-amylase activity

A platinum redox sensor for the direct potentiometric determination of α-amylase concentration has been described. The sensor measured the amount of triiodide released from a starch-triiodide complex, which was correlated with the α-amylase activity after biocatalytic starch degradation. The composition and stability of the potassium triiodide solution was optimized. The starch-triiodide complex was characterized potentiometrically at variable starch and triiodide concentrations. The response mechanism of the platinum redox sensor towards α-amylase was proposed and the appropriate theoretical model was elaborated. The results obtained using the redox sensor exhibited satisfactory accuracy and precision and good agreement with a standard spectrophotometric method and high-sensitive fully automated descret analyser method. The sensor was tested on pure α-amylase (EC 3.2.1.1, Fluka, Switzerland), industrial granulated α-amylase Duramyl 120 T and an industrial cogranulate of protease and α-amylase Everlase/Duramyl 8.0 T/60 T. The detection limit was found to be 1.944 mU for α-amylase in the range of 0-0.54 U (0-15 μg), 0.030 mKNU for Duramyl 120 T in the range of 0-9.6 mKNU (0-80 μg) and 0.032 mKNU for Everlase/Duramyl 8.0 T/60 T in the range of 0-9.24 mKNU (0-140 μg).     

Application of Mn(Ⅱ) as a Mimetic Enzyme of Horseradish Peroxidase

HRP is a most commonly used enzyme in H2O2 and glucose detection1, owing to its 2strong activity and quite good selectivity. However, natural HRP has manyshortcomings, such as its expensiveness and instability; the experimental conditions andstorage environment are inconvenient. Therefore, the search for a replacement3-6 forHRP has become an important and interesting work. This paper describes a catalytic spectrophotometry method for the determination ofH2O2 based on …     

Bifunctional compounds for controlling metal-mediated aggregation of the aβ42 peptide

Abnormal interactions of Cu and Zn ions with the amyloid β (Aβ) peptide are proposed to play an important role in the pathogenesis of Alzheimer's disease (AD). Disruption of these metal-peptide interactions using chemical agents holds considerable promise as a therapeutic strategy to combat this incurable disease. Reported herein are two bifunctional compounds (BFCs) L1 and L2 that contain both amyloid-binding and metal-chelating molecular motifs. Both L1 and L2 exhibit high stability constants for Cu(2+) and Zn(2+) and thus are good chelators for these metal ions. In addition, L1 and L2 show strong affinity toward Aβ species. Both compounds are efficient inhibitors of the metal-mediated aggregation of the Aβ(42) peptide and promote disaggregation of amyloid fibrils, as observed by ThT fluorescence, native gel electrophoresis/Western blotting, and transmission electron microscopy (TEM). Interestingly, the formation of soluble Aβ(42) oligomers in the presence of metal ions and BFCs leads to an increased cellular toxicity. These results suggest that for the Aβ(42) peptide-in contrast to the Aβ(40) peptide-the previously employed strategy of inhibiting Aβ aggregation and promoting amyloid fibril dissagregation may not be optimal for the development of potential AD therapeutics, due to formation of neurotoxic soluble Aβ(42) oligomers.     

Molecular basis for antifreeze activity difference of two insect antifreeze protein isoforms

The insect spruce budworm(Choristoneura fumiferana) produces antifreeze protein(AFP) to assist in the protection of the over-wintering larval stage and contains multiple isoforms. Structures for two isoforms,known as CfAFP-501 and CfAFP-337,show that both possess similar left-handed β-helical structure,although thermal hysteresis activity of the longer isoform CfAFP-501 is three times that of CfAFP-337. The markedly enhanced activity of CfAFP-501 is not proportional to,and cannot be simply accounted for,by the increased ice-binding site resulting from the two extra coils in CfAFP-501. In or-der to investigate the molecular basis for the activity difference and gain better understanding of AFPs in general,we have employed several different computational methods to systematically study the structural properties and ice interactions of the AFPs and their deletion models. In the context of intact AFPs,a majority of the coils in CfAFP-501 has better ice interaction and causes stronger ice lattice disruption than CfAFP-337,strongly suggesting a cooperative or synergistic effect among β-helical coils. The synergistic effect would play a critical role and make significant contributions to the anti-freeze activity β-helical antifreeze proteins. This is the first time that synergistic effect and its implica-tion for antifreeze activity are reported for β-helical antifreeze proteins.     

Computer-aided design and discovery of protein–protein interaction inhibitors as agents for anti-HIV therapy

Protein–protein interactions (PPI) are involved in most of the essential processes that occur in organisms. In recent years, PPI have become the object of increasing attention in drug discovery, particularly for anti-HIV drugs. Although the use of combinations of existing drugs, termed highly active antiretroviral therapy (HAART), has revolutionized the treatment of HIV/AIDS, problems with these agents, such as the rapid emergence of drug-resistant HIV-1 mutants and serious adverse effects, have highlighted the need for further discovery of new drugs and new targets. Numerous investigations have shown that PPI play a key role in the virus’s life cycle and that blocking or modulating them has a significant therapeutic potential. Here we summarize the recent progress in computer-aided design of PPI inhibitors, mainly focusing on the selection of the drug targets (HIV enzymes and virus entry machinery) and the utilization of peptides and small molecules to prevent a variety of protein–protein interactions (viral–viral or viral–host) that play a vital role in the progression of HIV infection.