Synthesis, characterization and development of a high-throughput methodology for the discovery of botulinum neurotoxin a inhibitors

Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. Although only a few hundred cases of botulism are reported in the United States annually, there is growing interest in BoNTs attributable to their potential use as biological warfare agents. Neurotoxicity results from cleavage of the soluble NSF-attachment protein receptor complex proteins of the presynaptic vesicles by the BoNT light chain subunit, a Zn endopeptidase. Few effective inhibitors of BoNT/A LC (light chain) activity are known, and the discovery process is hampered by the lack of an efficient high-throughput assay for screening compound libraries. To alleviate this bottleneck, we have synthesized the peptide SNAPtide and have developed a robust assay for the high-throughput evaluation of BoNT/A LC inhibitors. Key aspects for the development of this optimized assay include the addition of a series of detergents, cosolvents, and salts, including 0.01% w/v Tween 20 to increase BoNT/A LC catalysis, stability, and ease of small molecule screening. To evaluate the effectiveness of the assay, a series of hydroxamate-based small molecules were synthesized and examined with BoNT/A LC. The methodology described is superior to other assays reported to date for the high-throughput identification of BoNT/A inhibitors.     

Development,optimization and application of an analytical methodology by ultra performance liquid chromatography–tandem mass spectrometry for determination of amanitins in urine and liver samples

Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the most potent poisons accounting for the hazardous effects on intestinal epithelium cells and hepatocytes, and probably the sole cause of fatal human poisoning.     

Direct,Enantioselective, and Nickel(II) Catalyzed Reactions of N-Azidoacetyl Thioimides with Trimethyl Orthoformate: A New Combined Methodology for the Rapid Synthesis of Lacosamide and Derivatives

A direct and highly enantioselective reaction of N-azidoacetyl-1,3-thiazolidine-2-thione with trimethyl orthoformate catalyzed by Tol-BINAPNiCl₂ in the presence of TESOTf and 2,6-lutidine is reported. The heterocyclic scaffold can be easily removed by addition of a wide array of amines to give the corresponding enantiomerically pure 2-azido-3,3-dimethoxypropanamides in high yields. Appropriate manipulation of the N-benzyl amide derivative provides an efficient access to the antiepileptic agent lacosamide through a new enantioselective C−C bond-forming process. DFT computational studies uncover clues for the understanding of the remarkable stereocontrol of the addition of a nickel(II) enolate to a putative oxocarbenium intermediate from trimethyl orthoformate.     

Ultrafast fluorescence dynamics of tryptophan in the proteins monellin and IIAGlc

The complete time-resolved fluorescence of tryptophan in the proteins monellin and IIA(Glc) has been investigated, using both an upconversion spectrophotofluorometer with 150 fs time resolution and a time-correlated single photon counting apparatus on the 100 ps to 20 ns time scale. In monellin, the fluorescence decay displays multiexponential character with decay times of 1.2 and 16 ps, and 0.6, 2.2, and 4.2 ns. In contrast, IIA(Glc) exhibited no component between 1.2 ps and 0.1 ns. For monellin, surprisingly, the 16 ps fluorescence component was found to have positive amplitude even at longer wavelengths (e.g., 400 nm). In conjunction with quantum mechanical simulation of tryptophan in monellin, the experimental decay associated spectra (DAS) and time-resolved emission spectra (TRES) indicate that this fluorescence decay time should be ascribed to a highly quenched conformer. Recent models (Peon, J.; et al. Proc. Natl.Acad. Sci. U.S.A. 2002, 99, 10964) invoked exchange-coupled relaxation of protein water to explain the fluorescence decay of monellin.     

Potential of mean force between hydrophobic solutes in the Jagla model of water and implications for cold denaturation of proteins

Using the Jagla model potential we calculate the potential of mean force (PMF) between hard sphere solutes immersed in a liquid displaying water-like properties. Consistent estimates of the PMF are obtained by (a) umbrella sampling, (b) calculating the work done by the mean force acting on the hard spheres as a function of their separation, and (c) determining the position dependent chemical potential after calculating the void space in the liquid. We calculate the PMF for an isobar along which cold denaturation of a model protein has previously been reported. We find that the PMF at contact varies non-monotonically, which is consistent with the observed cold denaturation. The Henry constant also varies non-monotonically with temperature. We find, on the other hand, that a second (solvent separated) minimum of the PMF becomes deeper as temperature decreases. We calculate the solvent-solvent pair correlation functions for solvents near the solute and in the bulk, and show that, as temperature decreases, the two pair correlation functions become indistinguishable, suggesting that the perturbation of solvent structure by the solute diminishes as temperature decreases. The solvent-solute pair correlation function at contact grows as the temperature decreases. We calculate the cavity correlation function and show the development of a solvent-separated peak upon decrease of temperature. These observations together suggest that cold denaturation occurs when the solvent penetrates between hydrophobic solutes in configurations with favorable free energy. Our results thus suggest that cold denatured proteins are structured and that cold denaturation arises from strong solvent-solute interactions, rather than from entropic considerations as in heat denaturation.     

Characterization of structural transitions from aqueous solution to a lipid phase for α-MSH

Fluorescence spectroscopy results show that the α-melanocyte-stimulating hormone peptide (α-MSH) interacts with acidic lipid vesicles. Detectable structural changes are concomitant with the passage of a tryptophan residue from aqueous to lipidic media. The observed multiexponential decay of fluorescence, rationalized as originating from three rotameric populations of the tryptophan residue, has been used together with a matrix algorithm to find the most probable conformational families of α-MSH in water and lipid environments. A model is discussed in which the same conformational families occur in various phases, although with different probabilities. A conformational family in which χ₁ of the Trp9 side chain is in the trans-rotameric conformation is shown to have structural features highly appropriate to interact with negatively charged biological membranes, which are also in accordance with previous molecular dynamics simulations and with structures engineered in α-MSH analogs that show an increased potency in biological essays. The gauche minus and gauche plus side-chain conformations of Trp9, on the other hand, yield conformations more likely to predominate in aqueous solution. NMR spectroscopy measurements of α-MSH analogs indicate the existence in aqueous solution of a β strand in the vicinity of Trp9. A similar structural feature was found in the present conformational analysis for the gauche minus and gauche plus side-chain rotamers of Trp9. © 1995 John Wiley & Sons, Inc.     

Structure and magnetostructural correlation of ferrimagnetic meso-tetraphenylporphinatomanganese(III) dimethyl-N,N′-dicyanoquinone diiminide, [MnTPP]+[Me2DCNQI]·?

The structure [MnⅢ TPP][Me2 DCNQI] (TPP = tetraphenylporphinato; Me 2 DCNQI = 2,5-dimethyl-N,N′-dicyanoquinonediimine) has been determined from X-ray powder diffraction data. The nonsolvated structure is composed of linear (1-D) chains of alternating [MnⅢ TPP] + and μ-[Me 2 DCNQI] with intrachain Mn···Mn separations of 12.83 , and a Mn N DCNQI distance of 2.18. The dihedral angle between the mean Mn(N 4 ) TPP and [Me 2 DCNQI].- planes, and the Mn (N C) DCNQI angle are 84.18° and 143.6°, respectively. [MnⅢ TPP][Me 2 DCNQI] has a T c of 4.3 K from the 10 Hz χ"(T) data, 2-K coercivity of 5,600 Oe, and 6,300 emuOe/mol remnant magnetization that are reduced from that observed for related materials, and their inclusion extends the magnetostructural correlation between the intrachain coupling and both the dihedral angle between the mean Mn(N 4 ) TPP and [TCNE]*- (TCNE = tetracyanoethylene) planes and Mn (N C) TCNE angles. This is in accord with the intrachain coupling arising from the overlap of the MnⅢ d z 2 -like singly occupied molecular orbital (SOMO) and the z component of the [TCNE]*-π* (πz *) SOMO, which increases with decreasing dihedral angle between the mean Mn(N 4 ) TPP and [TCNE]*- planes and Mn (N C) TCNE angle.     

Comparative conformational analysis of peptide libraries

Six computer-based combinatorial libraries,including tetrapeptide sequences (generated with fiveamino acids) and conformations (generated with fivemain chain and three side chain rotamers), wereobtained and sequence-conformation probabilities werecalculated with a molecular and statistical mechanicsprocedure. The structural motifs -helix,-sheet, 3₁₀-helix, reverse turn I and-turn were focused in these calculations. Itis shown that sequence-conformation-probabilitysurfaces provide a broad view of structural changesaccompanying changes in sequence. Numerical indicesare defined to enable comparisons between frequenciesof occurrence of these structural motifs in peptidelibraries and in a database of low sequence identityprotein structures. Fine details ofsequence-conformation-probability surfaces show theeffect of point mutations. Broad comparisons betweendifferent regions of these surfaces indicate how toselect the occurrence of structural motifs in thecombinatorial synthesis of peptide chains.     

Secondary tritium isotope effects,a useful mechanistic probe for the study of acetoxyl participation and transfer

Tritium labelling on the acetoxyl methyl group has been used to probe the mode of incorporation of acetate into cinnamyl acetate in the solvolyes of cinnamyl chloride and cinnamylmercuric acetate, and in the allylic oxidation of allylbenzene with mercuric acetate.     

Thermal polymerization of levoglucosan

The uncatalyzed polymerization of neat levoglucosan (1,6-anhydro-β-D -glucopyranose) was studied at 190–210°C under argon, and the course of the reaction was followed by analyzing samples at various reaction times; the residual levoglucosan was determined by GLC, and the optical rotation of the mixture was also measured. The plots of percentage reaction versus time were sigmoid, indicating the occurrence of an autocatalytic reaction. A. kinetic equation of the form, dA/dt = k₂[A₁]² + k₁[A_t] ([A₀] ? [A_t]), where [A₀] and [A_t] denote the concentrations of levoglucosan at zero time and time t, was found to fit the results. A reaction scheme is proposed, consisting of a slower dimerization (k₂) followed by a faster reaction between levoglucosan and the dimers and other oligomers formed (k₁). This scheme results from the mechanistic assumption that axial hydroxyl groups in the unchanged levoglucosan are less reactive than equatorial hydroxyl groups present only in the oligomers, while the highest reactivity is ascribed to the primary hydroxyl at C₆, again only present in the oligomers. The relation between the mechanisms of the reactions occurring and the structure of the product are discussed.