Desorption/ionization on porous silicon mass spectrometry (DIOS-MS) is a novel method for generating and analyzing gas-phase ions that employs direct laser vaporization. The structure and physicochemical properties of the porous silicon surfaces are crucial to DIOS-MS performance and are controlled by the selection of silicon and the electrochemical etching conditions. Porous silicon generation and DIOS signals were examined as a function of silicon crystal orientation, resistivity, etching solution, etching current density, etching time, and irradiation. Pre-and postetching conditions were also examined for their effect on DIOS signal as were chemical modifications to examine stability with respect to surface oxidation. Pore size and other physical characteristics were examined by scanning electron microscopy and Fourier transform infrared spectroscopy, and correlated with DIOS-MS signal. Porous silicon surfaces optimized for DIOS response were examined for their applicability to quantitative analysis, organic reaction monitoring, post-source decay mass spectrometry, and chromatography.
KO-42, a polypeptide with 42 amino acid residues has been designed to fold into a hairpin helix−loop−helix motif that dimerizes and forms a four-helix bundle. The solution structure of the folded KO-42 dimer has been determined by NMR and CD spectroscopy and ultracentrifugation. On the surface of the folded polypeptide a reactive site has been engineered that is capable of catalyzing acyl-transfer reactions of reactive esters. The reactive site of KO-42 contains six histidine residues with perturbed pK a values. The pK as of His-15, His-30, and His-34 are close to 5, whereas those of His-11, His-19, and His-26 are close to 7, with nonideal titration curves. The second-order rate constant for the KO-42 catalyzed hydrolysis of mono-p-nitrophenyl fumarate at pH 4.1 and 290 K is 0.1 M-1 s-1, which is 1140 times larger than that of the 4-methylimidazole (4-MeIm) catalyzed reaction, 8.8 × 10-5 M-1 s-1. The second-order rate constant for the KO-42 catalyzed transesterification of mono-p-nitrophenyl fumarate to form the corresponding trifluoroethyl ester in 10 vol % trifluoroethanol at pH 4.1 and 290 K is 0.052 M-1 s-1 which is 620 times larger than that of the 4-MeIm catalyzed reaction, 8.4 × 10-5 M-1 s-1. KO-42 catalyzes the corresponding reactions of other p-nitrophenyl esters with similar rate enhancements. At pH 4.1 in aqueous solution where the rate constant ratio k 2(KO-42)/k 2(4-MeIm) is larger than 103 the predominant reactive species of KO-42 have unprotonated histidines flanked by protonated histidines. The kinetic solvent isotope effect at pH 4.7 is 2.0 which shows that isotopic fractionation occurs in the transition state. The kinetic solvent isotope effect at pH 6.1 is 1.1 which shows that there is neither general acid−general base catalysis nor strong hydrogen bonding in the transition state of the rate-limiting reaction step at that pH. The results suggest that at low pH the dominant catalytic species functions through a mechanism where unprotonated nucleophilic histidines are flanked by protonated histidines that bind to one or both of the ester oxygens in the transition state.
Epoxy resins can be prepared from numerous chemical compositions. Until recently, alternatives to epoxy resins based on diglycidyl ethers of bisphenol A (DGEBA) or bisphenol F (DGEBF) monomers have not received commercial interest, but are presently doing so, as epoxy resins with various properties are desired. Epoxy resin systems are known to cause allergic contact dermatitis because of contents of uncured monomers, reactive diluents, and hardeners. Reactive diluents, for example, glycidyl ethers, which also contain epoxide moieties, are added to reduce viscosity and improve polymerization. We have investigated the contact allergenic properties of a series of six analogues to phenyl glycidyl ether (PGE), all with similar basic structures but with varying carbon chain lengths and degrees of saturation. The chemical reactivity of the compounds in the test series toward the hexapeptide H-Pro-His-Cys-Lys-Arg-Met-OH was investigated. All epoxides were shown to bind covalently to both cysteine and proline residues. The percent depletion of nonreacted peptide was also studied resulting in 88% depletion when using PGE and 46% when using butyl glycidyl ether (5) at the same time point, thus revealing a large difference between the fastest and the slowest reacting epoxide. The skin sensitization potencies of the epoxides using the murine local lymph node assay (LLNA) were evaluated in relation to the observed physicochemical and reactivity properties. To enable determination of statistical significance between structurally closely related compounds, a nonpooled LLNA was performed. It was found that the compounds investigated ranged from strong to weak sensitizers, congruent with the reactivity data, indicating that even small changes in chemical structure result in significant differences in sensitizing capacity.
A histidine-based two-residue reactive site for the catalysis of hydrolysis and transesterification reactions of p-nitrophenyl esters has been engineered into a helix in a designed helix−loop−helix motif, and it has been shown to function through cooperative nucleophilic and general-acid catalysis. The two-residue site has been expanded by the incorporation of two arginine residues in the neighboring helix, and the arginines have been found to provide further transition state binding of the anionic transition state. The second-order rate constant in aqueous solution at pH 5.1 and 290 K for the peptide with the most efficient two-residue site is 0.054 M-1 s-1, the pK a value of both the histidine residues, His-30 and His-34, is 5.6, and the kinetic solvent isotope effect is 1.5. The introduction of Arg-11 and Arg-15 increases the second-order rate constant to 0.105 M-1 s-1. The second-order rate constant of a peptide with a two-residue site of His-26 and His-30 is 0.010 M-1 s-1, and the pK a values of the two His residues are 6.8 and 5.6, respectively. The difference in reactivity between the two peptides is consistent with a model where the His with the higher number in the sequence is the nucleophile and the His with the lower number is a general-acid catalyst. The results are incompatible with a model where the histidine residue with the lower number is the nucleophile.
Identification of settlement cues for marine fouling organisms opens up new strategies and methods for biofouling prevention, and enables the development of more effective antifouling materials. To this end, the settlement behaviour of zoospores of the green alga Ulva linza onto cationic oligopeptide self-assembled monolayers (SAMs) has been investigated. The spores interact strongly with lysine- and arginine-rich SAMs, and their settlement appears to be stimulated by these surfaces. Of particular interest is an arginine-rich oligopeptide, which is effective in attracting spores to the surface, but in a way which leaves a large fraction of the settled spores attached to the surface in an anomalous fashion. These 'pseudo-settled' spores are relatively easily detached from the surface and do not undergo the full range of cellular responses associated with normal commitment to settlement. This is a hitherto undocumented mode of settlement, and surface dilution of the arginine-rich peptide with a neutral triglycine peptide demonstrates that both normal and anomalous settlement is proportional to the surface density of the arginine-rich peptide. The settlement experiments are complemented with physical studies of the oligopeptide SAMs, before and after extended immersion in artificial seawater, using infrared spectroscopy, null ellipsometry and contact angle measurements.
Small change: Nanoparticles can induce a functional helix from an unstructured peptide (see picture). The ability to generate stable, well‐defined structures on surfaces opens up the possibility of creating nanosystems with a variety of functionalities, which is demonstrated by the introduction of a catalytic site for ester hydrolysis.
Mass spectrometry and fluorescent probes have provided direct evidence that alkylating agents permeate the protein capsid of naked viruses and chemically inactivate the nucleic acid. N-acetylaziridine and a fluorescent alkylating agent, dansyl sulfonate aziridine, inactivated three different viruses, flock house virus, human rhinovirus-14, and foot and mouth disease virus. Mass spectral studies as well as fluorescent probes showed that alkylation of the genome was the mechanism of inactivation. Because particle integrity was not affected by selective alkylation (as shown by electron microscopy and sucrose gradient experiments), it was reasoned that the dynamic nature of the viral capsid acts as a conduit to the interior of the particle. Potential applications include fluorescent labeling for imaging viral genomes in living cells, the sterilization of blood products, vaccine development, and viral inactivation in vivo.A ntiviral agents usually attack the viral life cycle by inhibiting intracellular expression of viral enzymes or by interfering with extracellular steps such as interaction of the virus with the cellular receptor (1-5). Viral protease and replicase inhibitors are highly specific, but their efficacy can be significantly reduced by the emergence of viral mutants. A more general approach to disarming viruses is through chemical modification of the virus particles, such as with N-acetyl-aziridine (Fig. 1), as used in the production of killed-virus vaccines (6, 7). Here we report how alkylating agents inactivate viruses, and we introduce a versatile molecular design for viral inactivants.Two possible mechanisms exist for viral inactivation with alkylating agents. One mechanism involves the modification of proteins, which would cause inhibition of viral cell entry or the release of the genome. The second mechanism allows the alkylating reagents direct access to the viral genome through a mobile protein capsid (8-12). The recent findings (8-11) that the protein capsids of viruses in solution have a much higher degree of dynamics than their crystallized counterparts suggested that the second mechanism might be the means of inactivation. Focusing on this latter idea, the ability of small alkylating agents to react with either the capsid or encapsidated nucleic acid was investigated initially by using flock house virus (FHV), an RNA-containing model virus used in previous studies (8, 9).The alkylating agent N-acetyl-aziridine is a virus inactivant that has been used in vaccine preparation since the 1950s, yet no direct evidence for its mechanism of inactivation has been determined, making it a suitable starting point for this investigation. The chemistry of aziridines is dominated by ring strain, thus leading to enhanced reactivity in reactions where the strain is relieved. The tendency of aziridines to undergo ring-opening reactions with nucleophiles such as the nitrogen atoms in adenine and guanine make them natural alkylating agents of nucleotides. MethodsCompounds. The synthesis of N-acetyl-aziridine was ...
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