A synthetic divalent cholera toxin glycocalix[4]arene ligand having higher affinity than natural GM1 oligosaccharide

A high-affinity ligand of cholera toxin, the divalent glycocalix[4]arene 1, was obtained by exploiting a combination of structure-based design of glycomimetic monovalent ligands and affinity enhancements by multivalent presentation through a calix[4]arene scaffold. It exhibits a slightly higher affinity for the toxin than its natural ligand, the GM1 oligosaccharide.     

Formation process of silver-polypyrrole coaxial nanocables synthesized by redox reaction between AgNO3 and pyrrole in the presence of poly(vinylpyrrolidone)

We have recently demonstrated a one-step process to fabricate silver-polypyrrole (PPy) coaxial nanocables (Chen, A.; Wang, H.; Li, X. Chem. Commun. 2005, 14, 1863). The formation process of silver-PPy coaxial nanocables is discussed in this article. It was found from the results of TEM and SEM images that large numbers of silver atoms were formed when AgNO3 was added to a pyrrole solution. Then silver atoms transform to silver-PPy nanosheets with regular morphology, which will connect together to be more stable. Silver-PPy nanocables will be able to grow at the expense of the silver-PPy nanosheets. Poly(vinylpyrrolidone) (PVP) plays crucial roles in this process: as a capping agent to form silver nanowires, and as a dispersant of pyrrole monomers, which can influence the site at which pyrrole monomer exists. On the basis of experimental analysis, the possible mechanism was proposed. Because of the effect of PVP, silver ions and pyrrole monomers are apt to be adsorbed at the [111] and [100] facets of silver nanosheets, respectively. Obvious polymerization will take place on the boundary of the [111] and [100] facets. The PPy layer stays stable on the [100] facets. Meanwhile, newly formed silver atoms and silver nanosheets will further ripen and grow on the [111] facets. In a word, the morphology of final products and the formation process are determined by the reaction site between AgNO3 and the pyrrole monomer, which is influenced by PVP.     

Development of a method for the identification of azaspiracid in shellfish by liquid chromatography-tandem mass spectrometry

Azaspiracid is the main toxin responsible for a number of recent human intoxications in Europe resulting from shellfish consumption. The first micro liquid chromatography-tandem mass spectrometry (micro-LC-MS-MS) method was developed for the determination of this novel shellfish poisoning toxin in mussels. The analyte was extracted from whole mussel meat with acetone and chromatographed on a C18 reversed-phase column (1.0 mm I.D.) by isocratic elution at 30 microl/min with acetonitrile-water (85:15, v/v), containing 0.03% trifluoroacetic acid. The toxin was ionised in an ionspray interface operating in the positive ion mode, where only the intact protonated molecule, [M+H]+, was generated at m/z 842. This served as precursor ion for collision-induced dissociation and three product ions, [M+H-nH2O]- with n=1-3, were identified for the unambiguous toxin confirmation by selected reaction monitoring LC-MS-MS analysis. A detection limit of 20 pg, based on a 3:1 signal-to-noise ratio, was achieved for the analyte. This LC-MS-MS method was successfully applied to determine azaspiracid in toxic cultivated shellfish from two regions of Ireland.     

Liquid chromatography-electrospray ionization mass spectrometry of the diarrhetic shellfish-poisoning toxins okadaic acid, dinophysistoxin-1 and pectenotoxin-6 in bivalves

Determination of diarrhetic shellfish-poisoning (DSP) toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-6 (PTX6) was carried out by liquid chromatography (LC) followed by on-line atmospheric pressure electrospray ionization-mass spectrometric (ESI-MS) detection with a heated capillary interface. Mass spectra of authentic OA, DTXI and PTX6 standards exhibited abundant [M-H] at m/z 803, 817 and 887, respectively. Linearity of peak area obtained by selected-ion monitoring (SIM) for [M-H]- of each toxin was confirmed over a wide range of concentrations from 10 pg to 30 ng. LC-ESI-MS analysis of OA, DTX1 and PTX6 in scallops and mussels, collected at the same site (Mutsu Bay, Japan), was carried out. Scallops and mussels collected at the same site showed different toxin profiles. Although PTX6 was detected from scallops, it was not detected from mussels.     

Interaction of polypeptide neurotoxins with a receptor site associated with voltage-sensitive sodium channels

Anthopleurin A, a polypeptide toxin from the Pacific sea anemone Anthopleura xanthogrammica, enhances persistent activation of voltage-sensitive sodium channels by the alkaloid toxins veratridine and batrachotoxin with K0.5 = 20 nM. This effect is inhibited by depolarization. There is a close correlation between enhancement of sodium channel activation and block of [125I]scorpion toxin binding by unlabeled scorpion toxin, sea anemone toxin II from Anemonia sulcata, and Anthopleurin A, indicating that these three polypeptide toxins interact with a common receptor site in modifying sodium channel function. Photo-activable derivatives of scorpion toxin label a single Mr approximately 250,000 polypeptide chain at the polypeptide toxin receptor site. Labeling is blocked by unlabeled scorpion toxin or depolarization and is not observed in variant neuroblastoma clones, which lack sodium channels. These results identify a protein component of the polypeptide toxin receptor site of voltage-sensitive sodium channels.     

Synthesis of the EF-ring of ciguatoxin 3C based on the [2,3]-Wittig rearrangement and ring-closing olefin metathesis

The EF-ring segment of ciguatoxin 3C, a causative toxin of ciguatera fish poisoning, was synthesized in three major steps: 1,4-addition for the C20O-C27 bond connection, chirality transferring anti selective [2,3]-Wittig rearrangement for the construction of the anti-2-hydroxyalkyl ether part, and ring-closing olefin metathesis for the F-ring formation.     

Selective modification of cell surface proteins and thymidine transport in hamster cells exposed to cholera toxin.

The increased adherence and morphological response which occurs in Chinese hamster ovary cells as a result of exposure to cholera toxin is paralleled by modification in the relative exposure of outer proteins. Mild proteolysis treatment of the cells prelabeled with [3H] glucosamine reveals a markedly different kinetics of release of external glycopeptides as a result of exposure to cholera toxin. Selective alterations in external tyrosyl-rich proteins can also be detected by lactoperoxidase-catalyzed radioiodination. The above modifications are accompanied by a decrease in the rate of thymidine uptake by toxin-treated cells.     

Biosynthesis of 13-desmethyl spirolide C by the dinoflagellate Alexandrium ostenfeldii

Biosynthetic origins of the cyclic imine toxin 13-desmethyl spirolide C were determined by supplementing cultures of the toxigenic dinoflagellate Alexandrium ostenfeldii with stable isotope-labeled precursors [1,2-13C2]acetate, [1-13C]acetate, [2-13CD3]acetate, and [1,2-13C2,15N]glycine and measuring the incorporation patterns by 13C NMR spectroscopy. Despite partial scrambling of the acetate labels, the results show that most carbons of the macrocycle are polyketide-derived and that glycine is incorporated as an intact unit into the cyclic imine moiety. This work represents the first conclusive evidence that such cyclic imine toxins are polyketides and provides support for biosynthetic pathways previously defined for other polyether dinoflagellate toxins.     

Studies towards the synthesis of FCRR toxin: an expeditious entry into 7-5-6 ring systems via [5+2] oxidopyrylium-alkene cycloaddition

Synthetic studies towards the diterpene natural product FCRR toxin have been undertaken. An intermolecular [5+2] oxidopyrylium-alkene cycloaddition reaction was employed to construct the 7-5-6 tricyclic framework. The reaction proceeded with very high regio- and stereoselectivity and the bridging ether was reductively cleaved to unmask the carbocycle.     

Synthesis and cholera toxin binding properties of a lactose-2-aminothiazoline conjugate

[structure: see text] During the search for improved monovalent ligands for cholera toxin (CT), a new lactose-2-aminothiazoline conjugate was discovered. In a fluorescence binding assay the compound was found to be one of the strongest relatively simple CT ligands to date with a K(d) of 23 microM.     

Mass spectral studies on synthetic analogs of organophosphorus toxin occurring in dinoflagellate algae

A series of organophosphorus compounds related to PB-1 toxin [O,O-diphenyl N- cyclooctylphosphoramidate] occurring in dinoflagellate algae as fish toxin have been synthesized and subjected to mass spectral studies under electron ionization. The fragmentation pattern obtained for the compounds has been substantiated by performing tandem mass spectrometry experiments in product ion scan mode.     

Analysis of transmembrane dynamics of cholera toxin using photoreactive probes.

Using sodium dodecyl sulfate--polyacrylamide gel electrophoresis and autoradiography, we have shown that 125I-labeled cholera toxin binds to Newcastle disease virus. Pretreatment of Newcastle disease virus with "cold" cholera toxin (at 37 degrees C for 30 minutes) inhibits the binding of 125I-labeled toxin in a subsequent incubation (at 37 degrees C for 30 minutes). These results suggest that cholera toxin binds to Newcastle disease virus in a specific manner. The precise receptor for toxin is unknown in Newcastle disease virus but it is presumed to be the ganglioside GM1. We have previously shown that the photoreactive probe 12-(4-azido-2-nitrophenoxy)stearoylglucosamine[1-14C] labels the membrane proteins of Newcastle disease virus. Since the reactive group of the probe, ie, N3, resides within the membrane bilayer, studies were initiated to determined which, if any, of the subunits of cholera toxin cross the membrane of Newcastle disease virus and become radioactively labeled upon photoactivation of the probe at 360 nm. After a 15-minute incubation of cholera toxin with Newcastle disease virus containing the photoreactive probe, irradiation effected the 14C-labeling of the active A1 subunit of cholera toxin. Irradiation of cholera toxin in solution with an equivalent amount of probe but without virus resulted in no labeling of toxin subunits.     

Heterobifunctional multivalent inhibitor-adaptor mediates specific aggregation between Shiga toxin and a pentraxin

[reaction: see text] The first example of a multivalent heterofunctional inhibitor-adaptor, called "BAIT", is described. This multivalent inhibitor-adaptor is able to capture a "target" receptor (Shiga toxin) through its recognition of one ligand of a heterobivalent headgroup while the other ligand binds to an endogenous "trap" protein (serum amyloid P component, SAP). BAIT showed markedly enhanced inhibition of toxin activity. An efficient synthesis of this multivalent cluster containing heterobifunctional ligands was accomplished by chemical and chemoenzymatic approaches.     

Studies toward a marine toxin immunogen: enantioselective synthesis of the spirocyclic imine of (-)-gymnodimine

[reaction: see text] An enantioselective Diels-Alder reaction catalyzed by an Evans' copper-bis(oxazoline) complex was utilized to construct a highly functionalized spirolactam, a key intermediate in our projected total synthesis of the marine toxin, gymnodimine. Additional transformations, including a mild N-tosyl group deprotection, afforded a keto spirocyclic imine moiety, the proposed pharmacophore of gymnodimine. Thus, the prepared ketone is a potentially useful intermediate for conjugation to provide an immunogen for eventual monitoring of gymnodimine and congeners.     

Sensitive Liquid Chromatographic Determination of Neurotoxin β-ODAP and Non-protein Amino Acids in Lathyrus sativus by Precolumn Derivatization with Dansyl Chloride

Lathyrus sativus (L. sativus) is a highly drought resistant and protein-rich leguminous crop cultivated in Africa and Asia, where it is a major protein source for people in the lowest income groups. However, excessive ingestion of this pulse can lead to irreversible paralysis of the legs-a disease known as neurolathyrism or lathyrism. The causative agent was reported to be the nonprotein amino acid, 3-N-L-oxalyl-2,3-diaminopropionic acid (β-ODAP)^[1]. The α-isomer of ODAP has been shown to be nontoxic. L. sativus (shan li dou in China) shows good adaptation to the low rainfall conditions of northwestern China. Our group was exploring the breeding low or zero toxin varieties of L. sativus as grain crops for human consumption and as protein-rich feed for animals. Thus, it is necessary to develop a method to determine the toxin and other amino acids. Derivatization by 5-dimethylaminonaphthalene-l-sulfonyl chloride (DnsCl) as derivatization reagent was used for analysis of amino acids by Negro et al.^[3] Sanz^[4]. We have developed a HPLC method that can simultaneously determination the a- and|3-ODAP and other amino acids in L. sativus by dansylation. The method provides a simple accurate alternative to existing methods for plant screening purpose.     

A cornucopia of cycloadducts: theoretical predictions of the mechanisms and products of the reactions of cyclopentadiene with cycloheptatriene

The potential cycloaddition reactions between cyclopentadiene and cycloheptatriene have been explored theoretically. B3LYP/6-31G was used to locate the transition states, intermediates, and products for concerted pathways and stepwise pathways passing through diradical intermediates. Interconversions of various cycloadducts through sigmatropic shifts were also explored. CASPT2/6-31G single point calculations were employed to obtain independent activation energy estimates. MM3 was also used to compute reaction energetics. Several bispericyclic cycloadditions in which two cycloadducts are linked by a sigmatropic shift have been identified. B3LYP predicts, in line with frontier molecular orbital predictions, that the [6+4] cycloaddition is the favored concerted pathway, but an alternative [4+2] pathway is very close in energy. By contrast, CASPT2 predicts that a [4+2] cycloaddition is the preferred pathway. B3LYP predicts that the lowest energy path to many of the cycloadducts will involve diradical intermediates, whereas CASPT2 predicts that each of the products of orbital symmetry allowed reactions will be reached most readily by closed shell processes-concerted cycloadditions and sigmatropic shift rearrangements of cycloadducts.     

Mechanistic twist of the [8+2] cycloadditions of dienylisobenzofurans and dimethyl acetylenedicarboxylate: stepwise [8+2] versus [4+2]/[1,5]-vinyl shift mechanisms revealed through a theoretical and experimental study

Recently, it was reported that both dienylfurans and dienylisobenzofurans could react with dimethyl acetylenedicarboxylate (DMAD) to give [8+2] cycloadducts. Understanding these [8+2] reactions will aid the design of additional [8+2] reactions, which have the potential for the synthesis of 10-membered and larger carbocycles. The present Article is aimed to understand the detailed mechanisms of the originally reported [8+2] cycloaddition reaction between dienylisobenzofurans and alkynes at the molecular level through the joint forces of computation and experiment. Density functional theory calculations at the (U)B3LYP/6-31+G(d) level suggest that the concerted [8+2] pathway between dienylisobenzofurans and alkynes is not favored. A stepwise reaction pathway involving formation of a zwitterionic intermediate for the [8+2] reactions between dienylisobenzofurans that contain electron-donating methoxy groups present in their diene moieties and DMAD has been predicted computationally. This pathway is in competition with a Diels-Alder [4+2] reaction between the furan moieties of dienylisobenzofurans and DMAD. When there is no electron-donating group present in the diene moieties of dienylisobenzofurans, the [8+2] reaction occurs through an alternative mechanism involving a [4+2] reaction between the furan moiety of the tetraene and DMAD, followed by a [1,5]-vinyl shift. This computationally predicted novel mechanism was supported experimentally.     

From dimerization, to cycloaddition, to atom transfer cyclization: the further chemistry of TMM diradicals

We describe [a] the first examples of intramolecular cycloaddition of a TMM diyl to a remotely tethered aldehyde, [b] the effect of a Lewis acid upon the course of TMM chemistry, [c] examples of exclusive intramolecular cycloaddition, competitive cycloaddition and ATC, and exclusive ATC, and [d] a set of predictive guidelines with which to assess whether cycloaddition or ATC will be the preferred path, and when the two processes will be competitive. Remarkably, a wide variety of structures can be obtained simply by varying the length of the tether within the diazenes investigated. DFT calculations were used to probe the energy surfaces for both atom transfer and cycloaddition. The transition structure for atom transfer involving the captodative system indicates that it occurs earlier along the reaction coordinate than for a system having only one radical stabilizing group. This is consistent with the existence of an exothermic process leading from the initial diyl to the captodatively stabilized distonic diyl. Gratifyingly, theory agrees with observation and provides substantial insight into the chemistry.     

4-羟基脯氨酸立体异构对α-芋螺毒素溶液构象的影响

在α-芋螺毒素及其它家族的芋螺毒素中, 脯氨酸的羟基化是非常普遍的后转录修饰方式. 在天然芋螺毒素中脯氨酸的羟基常采用反式构型, 且该残基对芋螺毒素的结构与生物活性产生了重要的影响, 而顺式构型的羟脯氨酸对α-芋螺毒素的折叠与生物活性的影响还鲜有研究. 本工作通过二维(2D)溶液核磁共振方法测定了经过化学修饰的三个含有反式或顺式羟脯氨酸的α-芋螺毒素的溶液结构, 它们是α4/7亚家族芋螺毒素肽[γ15E]Sr1B、[O7O'/γ15E]Sr1B和[O6O'/γ14E]Vc1A. 研究表明, 羟基顺反异构化学修饰对芋螺毒素的结构影响显著. 羟脯氨酸羟基的反式到顺式修饰导致α-芋螺毒素肽明显的溶液构象变化, 这些变化包括二级结构的改变、关键残基的侧链取向变化以及氢键性质的改变. [O7O'/γ15E]Sr1B与[γ15E]Sr1B相比, 典型的α-芋螺毒素ω弯曲结构发生形变. 而[O6O'/γ14E]Vc1A不同于Vc1A的是末端转角结构的缺失. 本工作加深了对α-芋螺毒素肽的化学修饰法的理解, 该方法是阐明α-芋螺毒素结构-生物活性关系的有用工具.     

Synthesis of imidazolo analogues of the oxidation-reduction cofactor pyrroloquinoline quinone (PQQ)

Parallel syntheses of 2-hydro-, 2-methyl-, and 2-methoxycarbonylimidazo-7,9-dimethoxycarbonyl analogues of the oxidation-reduction cofactor pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid] have been developed. The properties of the imidazolo analogues in relation to the corresponding pyrrole analogues will be important in assessing the origins of catalysis and biological activity in the cofactor, which has recently been shown to be a vitamin.