Enantioseparation, in vitro testing, and structural characterization of triple-binding reactivators of organophosphate-inhibited cholinesterases.

The enantiomers of racemic 2-hydroxyimino-N-(azidophenylpropyl)acetamide-derived triple-binding oxime reactivators were separated, and tested for inhibition and reactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibited with tabun (GA), cyclosarin (GF), sarin (GB), and VX. Both enzymes showed the greatest affinity toward the methylimidazole derivative (III) of 2-hydroxyimino-N-(azidophenylpropyl)acetamide (I). The crystal structure was determined for the complex of oxime III within human BChE, confirming that all three binding groups interacted with active site residues. In the case of BChE inhibited by GF, oximes I (kr = 207 M-1 min-1) and III (kr = 213 M-1 min-1) showed better reactivation efficiency than the reference oxime 2-PAM. Finally, the key mechanistic steps in the reactivation of GF-inhibited BChE with oxime III were modeled using the PM7R6 method, stressing the importance of proton transfer from Nε of His438 to Oγ of Ser203 for achieving successful reactivation.

Rapid chemical de-N-glycosylation and derivatization for liquid chromatography of immunoglobulin N-linked glycans.

Glycan analysis may result in exploitation of glycan biomarkers and evaluation of heterogeneity of glycosylation of biopharmaceuticals. For N-linked glycan analysis, we investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into alkaline de-N-glycosylation, we suppressed the degradation of released glycans and obtained a mixture of oximes, free glycans, and glycosylamines. The reaction was completed within 1 h, and the mixture containing oximes was easily tagged with 2-aminobenzamide by reductive amination. Here, we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, and examined whether this method could liberate glycans without degradation from apo-transferrin containing NeuAc and NeuGc and horseradish peroxidase containing Fuc α1-3 GlcNAc at the reducing end. Furthermore, we compared glycan recoveries between conventional enzymatic glycan release and this method. Increasing the reaction temperature and reaction duration led to degradation, whereas decreasing these parameters resulted in lower release. Considering this balance, we proposed to carry out the reaction at 80°C for 1 h for asialo glycoproteins from mammals and at 50°C for 1 h for sialoglycoproteins.

Bromotyrosine-derived metabolites from an Indonesian marine sponge in the family Aplysinellidae (Order Verongiida).

Seven new bromotyrosine-derived metabolites, purpuramine M-N (1-2), araplysillin VII-XI (3-7) and six known compounds (8-13) were isolated from an Indonesian sponge belonging to the family Aplysinellidae (Order Verongiida). The structures of the new compounds were determined by extensive NMR experiments and mass spectrometric measurements. These compounds were screened against BACE1 and five cancer cell lines.

Scent emission profiles from Darwin's orchid--Angraecum sesquipedale: Investigation of the aldoxime metabolism using clustering analysis.

The display of scent is crucial for plants in attracting pollinating insects to flowers and ensuring successful pollination and reproduction. The large number of aldoxime volatile species present in the scent of the Madagascan orchid Angraecum sesquipedale has been suggested to play a primary role in attracting the sphingid moth Xanthopan morgani praedicta. By solid phase micro-extraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS), we monitored the scent release from different flowers of a single orchid, day and night throughout the entire flowering period. In separate experiments, the diurnal release was monitored in 3h intervals and the tissue specific release from the different floral parts was tracked. Numerous novel compounds related to the aldoxime metabolism not previously detected in A. sesquipedale were identified and positioned into a proposed pathway for aldoxime metabolism. From the results, we hypothesize that (E/Z)-phenylacetaldoxime and its derivatives could be important attractants for the pollinating moth X. morgani praedicta. By applying an untargeted Partitioning Around Medoids (PAM) cluster analysis to the metabolite profiles in the scent, the proposed pathways for the formation of aldoximes were substantiated. With this study, we demonstrate the powerful utility of a bioinformatics tool to aid in the elucidation of the routes of formation for volatiles and provide a benchmark and guidelines for future detailed observations of hawkmoth pollination of Angraecum species, and in particular A. sesquipedale, in the wild.

Coprismycins A and B, neuroprotective phenylpyridines from the dung beetle-associated bacterium, Streptomyces sp.

Two new phenylpyridines, named coprismycins A and B (1 and 2), and previously reported dipyridines (3-6) were isolated from a culture of Streptomyces sp. associated with the dung beetle, Copris tripartitus. The structures of the coprismycins (1 and 2) were elucidated by the analysis of 1D and 2D NMR spectra and mass, UV, and IR spectra. Coprismycins A-B (1 and 2) and dipyridines (5 and 6) displayed comparable neuroprotective effects against MPP(+) (1-methyl-4-phenylpyrimidium)-induced neurotoxicity in neuroblastoma SH-SY5Y cells.

Partition of bispyridinium oximes (trimedoxime and K074) administered in therapeutic doses into different parts of the rat brain.

The penetration of acetylcholinesterase reactivators (oximes) into the central nervous system is typically restricted by the blood-brain barrier. Although oximes are highly hydrophilic compounds, some contradictory results confirming permeation into the brain exist. The aim of this study is to verify the penetration of oximes through the blood-brain barrier and to detect their levels achieved in different brain regions 60 min after the administration. It was confirmed that oximes are able to penetrate into the brain after injection of therapeutic doses corresponding with 5% of LD(50). The level in whole brain was 0.58% for trimedoxime and 0.85% for the experimental drug oxime K074 as the percentage of their plasma concentration. The highest concentration was found in frontal cortex (trimedoxime 2.27%; oxime K074 0.95%) and lowest in basal ganglia (trimedoxime 0.86%; oxime K074 0.42%). Entry of oximes into the brain is minimal, but some low reactivation effect should be expected. The reactivation potency of oximes might be higher or lower, depending on the real oxime concentration in a given area.

Diarylheptanoid hirsutenoxime inhibits toll-like receptor 4-mediated NF-κB activation regulated by Akt pathway in keratinocytes.

Microbial products, including lipopolysaccharides, may be involved in the pathogenesis of inflammatory skin diseases. We examined the effect of hirsutenoxime on the Toll-like receptor 4-mediated activation of Akt and nuclear factor (NF)-κB in lipopolysaccharide-stimulated keratinocytes. Hirsutenoxime, a cell signaling Akt inhibitor, and Bay 11-7085, an inhibitor of NF-κB activation, attenuated the lipopolysaccharide-induced expression of Toll-like receptor 4, activation of NF-κB and Akt, and the production of chemokines and reactive oxygen/nitrogen species. Hirsutenoxime may reduce the Toll-like receptor 4 expression-mediated NF-κB activation, which is regulated by the Akt pathway in keratinocytes exposed to lipopolysaccharides. This effect may reduce the skin inflammatory response.

Chromatographic analysis of toxic phosphylated oximes (POX): a brief overview.

Poisoning with organophosphorus compounds (OP), e.g. pesticides and nerve agents, causes inhibition of acetylcholinesterase (AChE) by phosphylation of the active site serine residue. Consequently, accumulation of stimulating acetylcholine in the synaptic cleft induces cholinergic crisis which ultimately may lead to death. For standard causal therapy, enzyme reactivators are administered representing oxime derivatives of quarternary pyridinium compounds, e.g. pralidoxime (2-PAM), obidoxime and HI 6. The mechanism of action includes removal of the phosphyl moiety by a nucleophilic attack of the oximate molecule substituting the enzyme and forming a phosphylated oxime (POX). POX is produced in stoichiometric amounts of reactivated enzyme and exhibits a significantly enhanced toxicity (inhibition rate constant) when compared to the parent OP. However, stability of POX under physiological conditions appears to be highly limited. Nevertheless, the presence of POX reveals a potential critical issue for both therapeutic efficacy in vivo and pharmacokinetic and pharmacodynamic (PK-PD) modelling based on cholinesterase activity data. Detailed characterization represents an important need for elaboration of the entire oxime pharmacology.Nevertheless, reports on POX toxicity and analysis are quite rare and may therefore be indicative of the challenge of POX analysis. This review provides a concise overview of chromatographic approaches applied to POX separation. Chromatography represents the key technology for POX purification and quantification in kinetic in vitro studies using buffers and biological fluids. Applications based on reversed-phase chromatography (RPC), ion pair chromatography (IPC) and an affinity approach as well as thin layer chromatography (TLC) are discussed and novel applications and data are presented.

Two cobalt(III) mono-dimethylglyoximates isolated from one reaction.

The reaction of cobalt(II) nitrate hexahydrate with dimethylglyoxime (DMGH(2)) and 1,10-phenanthroline (phen) in a 1:1:2 molar ratio results in two Co(III) mono-dimethylglyoximates having two chelating phen ligands in cis positions and the Co(III) atom coordinated by six N atoms in a distorted octahedral coordination geometry. The isolated products differ in the deprotonation state of the DMGH(2) ligand. In [mu-hydrogen bis(N,N'-dioxidobutane-2,3-diimine)]tetrakis(1,10-phenanthroline)cobalt(III) trinitrate ethanol disolvate 1.87-hydrate, [Co(2)(C(4)H(6)N(2)O(2))(C(4)H(7)N(2)O(2))(C(12)H(8)N(2))(4)](NO(3))(3).2C(2)H(6)O.1.87H(2)O, (I), the C(2)-symmetric cation is formed with the coordination [Co(DMG)(phen)(2)](+) cations aggregating via a very strong O(-)...H(+)...O(-) hydrogen bond with an O...O distance of 2.409 (4) A. Crystals of (I) exhibit extensive disorder of the solvent molecules, the nitrate anions and one of the phen ligands. Compound (I) is a kinetic product, not isolated previously from similar systems, that transforms slowly into (N-hydroxy-N'-oxidobutane-2,3-diimine)bis(1,10-phenanthroline)cobalt(III) dinitrate ethanol monosolvate 0.4-hydrate, [Co(C(4)H(7)N(2)O(2))(C(12)H(8)N(2))(2)](NO(3))(2).C(2)H(6)O.0.40H(2)O, (II), with the DMGH(-) ligand hydrogen bonded to one of the nitrate anions. In (II), the solvent molecules and one of the nitrate anions are disordered.

Organic-solvent-free extraction method for determination of carbamate and carbamoyloxime pesticides in soil and sediment samples.

This study evaluated the usefulness of monochloroacetic acid buffer (MCAAB) for extracting several carbamate/carbamoyloxime pesticides from a silt-loam soil and sediment, and an organic clay soil. The MCAAB extraction method, relative to acetonitrile and methanol extractants, was more accurate and precise for extraction of aldicarb, aldicarb sulfoxide, aldicarb sulfone, oxamyl, methomyl, carbofuran, 3-hydroxy-carbofuran, and propoxur; with recoveries ranging from 78.8% to 121.1%. Recoveries of carbaryl and methiocarb ranged from 0% to 64.1%. The MCCAB extraction method did not perform well for extraction of most compounds from the organic clay soil, with recoveries ranging from 0% to 66.7%.

Chromatographic separation and NMR characterization of the isomers of MMB-4, a bis-(pyridiniumaldoxime).

1,1'-Methylenebis{4-[(hydroxyimino)methyl]pyridinium) dichloride (MMB-4), a promising antidote for organophosphate poisoning, has been shown by chromatography and NMR to be a mixture of geometric isomers, predominantly the E/E form. The chromatographically separated isomers have been isolated, directly characterized by NMR to be E/E and E/Z isomers of high purity, and shown by HPLC and NMR to re-equilibrate in solution to the isomeric mixture found in bulk MMB-4. These findings clearly show that a minor component in MMB-4 is not an impurity, but a geometric isomer of the principal component and demonstrate the need to understand equilibrium processes for drug characterizations and isomer distributions of chemicals proposed for animal and human clinical trials. Evidence for the presence of the Z/Z isomer could not be found.

TLC analysis of intermediates arising during the preparation of oxime HI-6 dimethanesulfonate.

In this study, several thin-layer chromatography (TLC) methods are described for the identification of quaternary and non-quaternary compounds (parent compounds, intermediates, by-products, and products) arisen within the synthesis of the acetylcholinesterase reactivator HI-6, at present the most promising antidote in the case of nerve agent poisonings. Using the TLC technique, particular E and Z isomers of this compound on the oxime group are separated. These TLC methods could be of high interest as quick purity control for those who are interested in development of new acetylcholinesterase reactivators and the synthesis of HI-6 in laboratories or in large-scale production.

Novel inhibitors of the mitochondrial respiratory chain: oximes and pyrrolines isolated from Penicillium brevicompactum and synthetic analogues.

The capacity of inhibition of the mammalian mitochondrial respiratory chain of brevioxime 5a, a natural insecticide compound isolated from Penicillium brevicompactum culture broth, and another 15 analogue compounds, other oximes 5b and 5c; two diastereomeric pyrrolidines 1c' and 1c' '; five pyrrolines 3c', 3c' ' (diastereomers between them), 3a, 3b, and 6; two oxazines 4c' and 4c' ' (also diastereomers between them); and four pyrrol derivatives 7-10, are analyzed in this paper. Compounds 3b, 3c', 3c' ', 4c', 4c' ', 5b, 5c, 6, and 10 were found to be inhibitors of the integrated electron transfer chain (NADH oxidase activity) in beef heart submitochondrial particles (SMP), establishing that all of them except compound 3b and 6 only affected to complex I of the mitochondrial respiratory chain. The most potent product was 5b, with an IC50 of 0.27 microM, similar to the IC50 values of other known complex I inhibitors. The diastereomeric pairs 1c'/1c' ', 3c'/3c' ', 4c'/4c' ', and 5c have not been previously described. Chemical characterization, on the basis of spectral data, is also shown.

Determination of the diastereoisomeric purity of D,L- and meso-HM-PAO by 13C-NMR spectroscopy.

D,L-hexamethylpropyleneamine oxime (HM-PAO) is well known to be the effective isomer when HM-PAO is used as a radiopharmaceutical. Its diastereoisomeric purity is of great importance because meso-impurity decreases the concentration of the 99mTc-complex in the brain. The described investigation shows that 13C-NMR spectroscopy is a suitable analytical method for the determination of the diastereoisomeric purity of HM-PAO. It also can be used for assessment of the relative ratio of both isomers in diastereoisomeric mixtures. It is important to note that the patterns of behaviour of both isomers in 13C-NMR spectra are the same in all solvents tested. The method is simple, fast and explicit.

Purification and stabilization of 99mTc-d,l-HMPAO: role of organic extractants.

99mTc-d,l-HMPAO, an important SPECT agent for imaging cerebral perfusion, suffers from the disadvantage of an inherent instability and its shelf life has been reported to be 30 min. The latter is a harsh constraint and not compatible with Centralized Radiopharmacy procedures. During the attempts to improve upon the stability of 99mTc-d,l-HMPAO, preservation of product as an organic extract into suitable solvents like diethylether, ethylacetate, methylethylketone, chloroform was tried out. Chloroform extraction (yield: >90%) resulted in a product having stability not less than 5 hours. Gentle drying of the chloroform extract and reconstitution in normal saline resulted in quantitative recovery of 99mTc-d,l-HMPAO with acceptable radiochemical purity (>90%). This finding is thus of much significance, especially in the context of centralized large hospital radiopharmacy setting, by rendering convenience and flexibility in scheduling patients.

Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium.

Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a key objective in the treatment of OP poisoning. This study with native, wild-type, and mutant recombinant DNA-expressed AChEs, each inhibited by representative OP compounds, establishes a relationship between edrophonium acceleration of oxime-induced reactivation of OP-AChE conjugates and phosphoryl oxime inhibition of the reactivated enzyme that occurs during reactivation by pyridinium oximes LüH6 and TMB4. No such recurring inhibition could be observed with HI-6 as the reactivator due to the extreme lability of the phosphoryl oximes formed by this oxime. Phosphoryl oximes formed during reactivation of the ethoxy methylphosphonyl-AChE conjugate by LüH6 and TMB4 were isolated for the first time and their structures confirmed by (31)P NMR. However, phosphoryl oximes formed during the reactivation of the diethylphosphoryl-AChE conjugate were not sufficiently stable to be detected by (31)P NMR. The purified ethoxy methylphosphonyl oximes formed during the reactivation of ethoxy methylphosphonyl-AChE conjugate with LüH6 and TMB4 are 10- to 22-fold more potent than MEPQ as inhibitors of AChE and stable for several hours at pH 7.2 in HEPES buffer. Reactivation of both ethoxy methylphosphonyl- and diethylphosphoryl-AChE by these two oximes was accelerated in the presence of rabbit serum paraoxonase, suggesting that organophosphorus hydrolase can hydrolyze phosphoryl oxime formed during the reactivation. Our results emphasize that certain oximes, such as LüH6 and TMB4, if used in the treatment of OP pesticide poisoning may cause prolonged inhibition of AChE due to formation of phosphoryl oximes.

Evaluation and separation of steroid-bovine serum albumin conjugates by high-performance liquid chromatography.

The conventional methods for characterization of steroid immunogens are based on the determination of the total amount of hapten bound to the protein carrier either by the UV spectroscopy or titration of unsubstituted amino groups. These methods do not allow more detailed insight into the immunogen composition. HPLC of the immunogen combined with UV detection is a relatively rapid and convenient method enabling determination of the hapten content in each fraction and, eventually, separation of individual fractions differing in the hapten content or purification of crude product.

Solid-phase extraction coupled with electrochemical detection for the determination of the herbicide bromofenoxim in water samples at low- and sub-microgram l-1 levels.

The application of solid-phase extraction (SPE) as a preconcentration and clean-up step with subsequent off-line flow injection amperometric (FI-AD) or batch square-wave voltammetric (SWV) detection of the herbicide bromofenoxim was developed. The selection of an appropriate organic eluent, some parameters influencing the efficiency of the SPE and the electrochemical detection of bromofenoxim in the organic effluent solution were thoroughly investigated. Undiluted acetonitrile with SWV and acetonitrile-water (80 + 20) with FI-AD, both containing 0.1 mol l-1 LiClO4, were chosen as the most appropriate SPE eluents. The addition of LiClO4 as supporting electrolyte to the eluent and acidification of a water sample to 1 x 10(-3) mol l-1 HClO4 (pH 3) prior to the SPE procedure were found to improve greatly the current response on mercury drop (SWV) and mercury film (FI-AD) electrodes. Subsequent to the SPE procedure, the effluents were transferred to the voltammetric cell or injected into the flow injection system without any further treatment. The calibration plots obtained for bromofenoxim in pure water samples were linear over the ranges 0.2-12.0 micrograms l-1 and 3.0-120 micrograms l-1, with calculated detection limits of 0.05 and 1.5 micrograms l-1 (100 ml samples), for the SPE-SWV and SPE-FI-AD procedures, respectively. The actual detection capabilities of the proposed methods depend on the water sample volumes applied to the extraction cartridges. The recoveries of the over-all procedures, applying spiked tap water samples, and the corresponding RSDs were 92% and 6% (n = 6) and 121% and 9% (n = 7) for SPE-SWV and SPE-FI-AD, respectively. The practical applicability of the proposed methods for the analysis of ground and tap water samples was confirmed via an inter-laboratory test on a tap water sample containing five common pesticides including bromofenoxim.

Isolation and structure of hemibastadinols 1-3 from the Papua New Guinea marine sponge Ianthella basta.

Further investigation of the Bismarck Archipelago (Papua New Guinea) marine sponge Ianthella basta for biologically active constituents has led to the isolation of hemibastadins 1 (2), 2 (3), and 3 (4) and the new brominated tyrosine derivatives hemibastadinols 1-3 (9, 13, and 14). Isolation and structure elucidation of the monomethyl ether derivatives (7 and 8) of hemibastadins 1 and 2 and the 3-bromotyramine amide of oxalic acid amide (1a) concluded our chemical investigation of I. basta. The hemibastadins and hemibastadinols represent important biosynthetic links to a series of bromotyrosine tetramers collectively known as the bastadins. The antimicrobial activity of the bastadins, hemibastadins, and hemibastadinols is summarized.