968 resultados para solvent-free synthesis


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The alpha-conotoxin MII is a 16 amino acid long peptide toxin isolated from the marine snail, Conus magus. This toxin has been found to be a highly selective and potent inhibitor of neuronal nicotinic acetylcholine receptors of the subtype alpha3beta2. To improve the bioavailability of this peptide, we have coupled to the N-terminus of conotoxin MII, 2-amino-D,L-dodecanoic acid (Laa) creating a lipidic linear peptide which was then successfully oxidised to produce the correctly folded conotoxin MII construct.

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[GRAPHICS] The regioselective syntheses and structures are reported for two tris-macrocylic compounds, each possessing two antiparallel loops on a macrocyclic scaffold constrained by two oxazoles and two thiazoles. NMR solution structures show the loops projecting from the same face of the macrocycle. Such molecules are shown to be prototypes for mimicking multiple loops of proteins.

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Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

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A suite of allenic hydrocarbons, previously unknown as a molecular class from insects, has been characterized from several Australian melolonthine scarab beetles. The allenes are represented by the formula CH3(CH2)nCH=.=CH(CH2)(7)CH3 with n being 11-15, 17 and 19, and thus, all have Delta(9,10)-unsaturation. These structures have been confirmed by syntheses and comparisons of spectral and chromatographic properties with those of the natural components. The enantiomers of (+/-)-Delta(9,10)-tricosadiene and Delta(9,10)-pentacosadiene were separable on a modified beta-cyclodextrin column (gas chromatography), and the natural Delta(9,10)-tricosadiene (n = 11) and Delta(9,10)-pentacosadiene (n = 13) were shown to be of >85% ee. Syntheses of nonracemic allenes of known predominating chirality were acquired using both organotin chemistry and sulfonylhydrazine intermediates, and comparisons then demonstrated that the natural allenes were predominantly (R)-configured.

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Study design: Single-blind, placebo control, randomized, crossover, experimental Study with repeated measures, Objective: To determine the initial effects of a taping technique on grip strength and pain in individuals with lateral epicondylalgia. Background: Taping techniques are advocated for chronic musculoskeletal conditions such as lateral epicondylalgia, a prevalent disorder with significant impact on the individual and community. Little evidence exists supporting the effects of taping techniques on musculoskeletal pain. Methods and Measures: Sixteen participants (mean age +/- SD, 45.8 +/- 10.2 years) with chronic lateral epicondylalgia (rnean duration +/- SD, 13.1 +/- 9.9 months) participated in a placebo control study of an elbow taping technique. Outcome measures were pain-free grip strength and pressure pain threshold taken before, immediately after, and 30 minutes after application of tape. Results: The taping technique significantly improved pain-free grip strength by 24% from baseline (P = .028). The treatment effect was greater than that for placebo and control conditions. Changes in pressure pain threshold (19%), although positive, were not statistically significant. Conclusion: This preliminary study demonstrated an initial ameliorative effect of a taping technique for lateral epicondylalgia and suggests that it should be considered as an adjunct in the management of this condition.

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Background: In the presence of dNTPs, intact HIV-1 virions are capable of reverse transcribing at least part of their genome, a process known as natural endogenous reverse transcription (NERT). PCR analysis of virion DNA produced by NERT revealed that the first strand transfer reaction (1stST) was inefficient in intact virions, with minus strand (-) strong stop DNA (ssDNA) copy numbers up to 200 times higher than post-1stST products measured using primers in U3 and U5. This was in marked contrast to the efficiency of 1stST observed in single-round cell infection assays, in which (-) ssDNA and U3-U5 copy numbers were indistinguishable. Objectives: To investigate the reasons for the discrepancy in first strand transfer efficiency between intact cell-free virus and the infection process. Study design: Alterations of both NERT reactions and the conditions of cell infection were used to test whether uncoating and/or entry play a role in the discrepancy in first strand transfer efficiency. Results and Conclusions: The difference in 1stST efficiency could not be attributed simply to viral uncoating, since addition of very low concentrations of detergent to NERT reactions removed the viral envelope without disrupting the reverse transcription complex, and these conditions resulted in no improvement in 1stST efficiency. Virus pseudotyped with surface glycoproteins from either vesicular stomatitis virus or amphotrophic murine leukaemia virus also showed low levels of 1stST in low detergent NERT assays and equivalent levels of (-) ssDNA and 1stST in single-round infections of cells, demonstrating that the gp120-mediated infection process did not select for virions capable of carrying out 1stST. These data indicate that a post-entry event or factor may be involved in efficient HIV-1 reverse transcription in vivo. (C) 2002 Elsevier Science B.V. All rights reserved.

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Ageing results in a progressive, intrinsic and generalised imbalance of the control of regulatory systems. A key manifestation of this complex biological process includes the attenuation of the universal stress response. Here we provide the first global assessment of the ageing process as it affects the heat shock response, utilising human peripheral lymphocytes and cDNA microarray analysis. The genomic approach employed in our preliminary study was supplemented with a proteomic approach. In addition, the current study correlates the in vivo total antioxidant status with the age-related differential gene expression as well as the translational kinetics of heat shock proteins (hsps). Most of the genes encoding stress response proteins on the 4224 element microarray used in this study were significantly elevated after heat shock treatment of lymphocytes obtained from both young and old individuals albeit to a greater extent in the young. Cell signaling and signal transduction genes as well as some oxidoreductases showed varied response. Results from translational kinetics of induction of major hsps, from 0 to 24 It recovery period were broadly consistent with the differential expression of HSC 70 and HSP 40 genes. Total antioxidant levels in plasma from old individuals were found to be significantly lower by comparison with young, in agreement with the widely acknowledged role of oxidant homeostasis in the ageing process. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

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Free-space optical interconnects (FSOIs), made up of dense arrays of vertical-cavity surface-emitting lasers, photodetectors and microlenses can be used for implementing high-speed and high-density communication links, and hence replace the inferior electrical interconnects. A major concern in the design of FSOIs is minimization of the optical channel cross talk arising from laser beam diffraction. In this article we introduce modifications to the mode expansion method of Tanaka et al. [IEEE Trans. Microwave Theory Tech. MTT-20, 749 (1972)] to make it an efficient tool for modelling and design of FSOIs in the presence of diffraction. We demonstrate that our modified mode expansion method has accuracy similar to the exact solution of the Huygens-Kirchhoff diffraction integral in cases of both weak and strong beam clipping, and that it is much more accurate than the existing approximations. The strength of the method is twofold: first, it is applicable in the region of pronounced diffraction (strong beam clipping) where all other approximations fail and, second, unlike the exact-solution method, it can be efficiently used for modelling diffraction on multiple apertures. These features make the mode expansion method useful for design and optimization of free-space architectures containing multiple optical elements inclusive of optical interconnects and optical clock distribution systems. (C) 2003 Optical Society of America.

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Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-I, HIV-2) at nanomolar concentrations without cytotoxicity to uninfected cells below 10 mu M. Their activities against HIV-1 protease (K-i 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear cells (IC50 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC50 90 nM (1), 60 nM (2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to those of indinavir and amprenavir under identical conditions. There were significant differences in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55(gag) to p24 in virions derived from chronically infected cells, consistent with inhibition of the viral protease in cells. Crystal structures refined to 1.75 Angstrom (1) and 1.85 Angstrom (2) for two of the macrocyclic inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic components share the same space in the active site of the enzyme and make identical interactions with enzyme residues. This substrate-mimicking minimalist approach to drug design could have benefits in the context of viral resistance, since mutations which induce inhibitor resistance may also be those which prevent substrate processing.

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A series of 'pellicular' type supports were fabricated by direct gamma-radiation-mediated graft polymerisation of styrene onto polypropylene, followed by aminomethylation. Raman spectroscopy was used for measuring the level of penetration of polystyrene graft into polypropylene, and other structural features such as density of graft and depth of functionalisation. The kinetics of the coupling of fluorenylmethylcarbamate (Fmoc)-labelled amino acids, to the aminomethylated polystyrene grafts have been measured by UV absorption followed cleavage of the Fmoc chromophore. The Raman spectroscopy results showed that for this series of experiments the calculated rate coefficient for coupling of Fmoc-labelled amino acids was primarily dependent on graft thickness, but was also influenced by the proportion of polystyrene graft to polypropylene. In general, it was also shown that with increasing loading capacity of support the calculated rate coefficient for amino-acid coupling decreased correspondingly. In addition, a support that had both a high rate coefficient and a high loading capacity was prepared from polypropylene base material with a co-continuous porous structure (high surface area). (C) 2003 Society of Chemical Industry.

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New mono- and bis-chelated zinc(II) and cadmium(II) complexes of formula, [M(dpksbz)NCS] (dpksbz = anionic form of the di-2-pyridylketone Schiff base of S-benzyldithiocarbazate) and [M(dpksbz)(2)] (M = Zn-II, Cd-II) have been prepared and characterized. The structure of the bis-ligand complex, [Zn(dpksbZ)(2)] has been determined by X-ray diffraction. The complex has a distorted octahedral geometry in which the ligands are coordinated to the zinc(II) ion as uninegatively charged tridentate chelates via the thiolate sulfur atoms, the azomethine nitrogen atoms and the pyridine nitrogen atoms. The distortion from a regular octahedral geometry is attributed to the restricted bite angles of the Schiff base ligands. X-ray structural analysis shows that the [Cd(dpksbz)NCS](2) complex is a centrosymmetric dimer in which each of the cadmium(II) ions adopts a five-coordinate, approximately square-pyramidal configuration with the Schiff base acting as a tetradentate chelating agent coordinating a cadmium(II) ion via one of the pyridine nitrogen atoms, the azomethine nitrogen atom and the thiolate sulfur atom; the second pyridine nitrogen atom is coordinated to the other cadmium(II) ion of the dimer. The fifth coordination position around each cadmium(II) is occupied by an N-bonded thiocyanate ligand. (C) 2003 Elsevier Science Ltd. All rights reserved.

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delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

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Purpose. As reductions in dermal clearance increase the residence time of solutes in the skin and underlying tissues we compared the topical penetration of potentially useful vasoconstrictors (VCs) through human epidermis as both free bases and ion-pairs with salicylic acid (SA). Methods. We determined the in vitro epidermal flux of ephedrine, naphazoline, oxymetazoline, phenylephrine, and xylometazoline applied as saturated solutions in propylene glycol: water (1: 1) and of ephedrine, naphazoline and tetrahydrozoline as 10% solutions of 1: 1 molar ratio ion-pairs with SA in liquid paraffin. Results. As free bases, ephedrine had the highest maximal flux, Jmax = 77.4 +/- 11.7 mug/cm(2)/h, being 4-fold higher than tetrahydrozoline and xylometazoline, 6-fold higher than phenylephrine, 10-fold higher than naphazoline and 100-fold higher than oxymetazoline. Stepwise regression of solute physicochemical properties identified melting point as the most significant predictor of flux. As ion-pairs with SA, ephedrine and naphazoline had similar fluxes (11.5 +/- 2.3 and 12.0 +/- 1.6 mug/cm(2)/h respectively), whereas tetrahydrozoline was approximately 3-fold slower. Corresponding fluxes of SA from the ion-pairs were 18.6 +/- 0.6, 7.8 +/- 0.8 and 1.1 +/- 0.1 respectively. Transdermal transport of VC's is discussed. Conclusions. Epidermal retention of VCs and SA did not correspond to their molar ratio on application and confirmed that following partitioning into the stratum corneum, ion-pairs separate and further penetration is governed by individual solute characteristics.

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This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography-mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18-23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential. (C) 2003 Elsevier B.V All rights reserved.