Ring flexibility within tricyclic antidepressant drugs

The internal flexibility of the central seven-membered ring of a series of tricyclic antidepressant drugs (TCAs), imipramine {l}, amitriptyline {2}, doxepin {3}, and dothiepin {4}, has been investigated by H-1 and C-13 nuclear magnetic (NMR) techniques. Two dynamic processes were examined: ring inversion and bridge flexing. H-1 NMR lineshape analysis was used to obtain ring inversion barriers for 2-4. These studies yielded energy barriers of 14.3, 16.7, and 15.7 +/- 0.6 kcal/mol for the hydrochloride salts of doxepin, dothiepin, and amitriptyline, respectively. The barriers for the corresponding free bases were lower by 0.6 kcal/mol on average. (CT1)-C-13 relaxation measurements were used to determine the degree of bridge flexing associated with the central seven-membered ring for all four compounds. By fitting the T-1 data to a two-state jump model, lifetimes and amplitudes of rapid bridge flexing motions were determined. The results show that imipramine has the fastest rate of bridge flexing, followed by amitriptyline, doxepin, and dothiepin. The pharmacological profiles of the TCAs are complex and they interact with many receptor sites, resulting in numerous side effects and a general lack of understanding of their precise mode of action in different anxiety-related disorders. They all have similar three-dimensional structures, which makes it difficult to rationalize their differing relative potency in different assays/clinical settings. However, the clear finding here that there are significantly different degrees of internal mobility suggests that molecular dynamics should be an additional factor considered when trying to understand the mode of action of this clinically important family of molecules. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:713-721, 2001.

The cystine knot motif in toxins and implications for drug design

The cystine knot structural motif is present in peptides and proteins from a variety of species, including fungi, plants, marine molluscs. insects and spiders. It comprises an embedded ring formed by two disulfide bonds and their connecting backbone segments which is threaded by a third disulfide bond. It is invariably associated with nearby beta-sheet structure and appears to be a highly efficient motif for structure stabilization. Because of this stability it makes an ideal framework for molecular engineering applications. In this review we summarize the main structural features of the cystine knot motif, focussing on toxin molecules containing either the inhibitor cystine knot or the cyclic cystine knot. Peptides containing these motifs are 26-48 residues long and include ion channel blockers, haemolytic agents, as well as molecules having antiviral and antibacterial activities. The stability of peptide toxins containing the cystine knot motif, their range of bioactivities and their unique structural scaffold can be harnessed for molecular engineering applications and in drug design. Applications of cystine knot molecules for the treatment of pain. and their potential use in antiviral and antibacterial applications are described. (C) 2000 Elsevier Science Ltd. All rights reserved.

The geochemistry of late Archaean microbial carbonate: Implications for ocean chemistry and continental erosion history

Trace element concentrations and combined Sr- and Nd-isotope compositions were determined on stromatolitic carbonates (microbialites) from the 2.52 Ga Campbellrand carbonate platform (South Africa). Shale-normalised rare earth element and yttrium patterns of the ancient samples are similar to those of modern seawater in having positive La and Y anomalies and in being depleted in light rare earth elements. In contrast to modem seawater (and microbialite proxies), the 2.52 Ga samples lack a negative Ce anomaly but possess a positive Eu anomaly. These latter trace element characteristics are interpreted to reflect anoxic deep ocean waters where, unlike today, hydrothermal Fe input was not oxidised, and scavenged and rare earth elements were not coprecipitated with Fe-oxyhydroxides. The persistence of a positive Eu anomaly in relatively shallow Campbellrand platform waters indicates a dramatic reversal from hydrothermally dominated (Archaean) to continental erosion-dominated (Phanerozoic) rare earth element flux ratio. The dominant hydrothermal input is also expressed in the initial Sr- and Nd-isotope ratios. There is collinear variation in Sr-Nd systematics, which range from primitive values (Sr-87/Sr-86 of 0.702386 and epsilon (Nd) of +2.1) to more evolved crustal ratios. Mixing calculations show that the range in trace element ratios (e.g., Y/Ho) and initial isotope ratios is not a result of contamination by trapped sediment, but that the chemical band isotopic variation reflects carbonate deposition in an environment where different water masses mixed. Calculated Nd flux ratios yield a hydrothermal input into the 2.52 Ga oceans one order of magnitude larger than continental input. Such a change in flux ratio most likely required substantially reduced continental inputs, which could, in turn, reflect a plate tectonic causation (e.g., reduced topography or expansion of epicontinental seas). Copyright (C) 2001 Elsevier Science Ltd.

Carbohydrate-based templates for synthetic vaccines and drug delivery

Methyl tetra-O-allyl, and tetra-O-[2-(tetrahydro-2H-pyranyl)oxy.-3-oxapentyl glucosides, and tetra-O-(cyanoethyl)galactosyl azide were converted into derivatives containing linkers with terminal carboxylic acid functionalities at the anomeric position and bearing four arms with phthaloyl- or BOC-protected terminal amino groups. These molecules were suitable for use in solid-phase peptide synthesis and for the preparation of dendrimers, containing multiple copies of peptides. (C) 2001 Elsevier Science Ltd. All rights reserved.

Structural effects of lipophilic methotrexate conjugates on model phospholipid biomembranes

Lipophilic conjugates of the antitumor drug methotrexate (MTX) with lipoamino acids (LAAs) have been previously described as a tool to enhance MTX passive entrance into cells, overcoming a form of transport resistance which makes tumour cells insensitive to the antimetabolite. A knowledge of the mechanisms of interaction of such lipophilic derivatives with cell membranes could be useful for planning further lipophilic MTX derivatives with an optimal antitumour activity. To this aim, a calorimetric study was undertaken using a biomembrane model made from synthetic 1,2-dipalmitoyl-glycero-3-phosphocholine (DPPC) multilamellar liposomes. The effects of MTX and conjugates on the phase transition of liposomes were investigated using differential scanning calorimetry. The interaction of pure MTX with the liposomes was limited to the outer part of the phospholipid bilayers, due to the polar nature of the drug. Conversely, its lipophilic conjugates showed a hydrophobic kind of interaction, perturbing the packing order of DPPC bilayers. In particular, a reduction of the enthalpy of transition from the gel to the liquid crystal phase of DPPC membranes was observed. Such an effect was related to the structure and mole fraction of the conjugates in the liposomes. The antitumour activity of MTX conjugates was evaluated against cultures of a CCRF-CEM human leukemic T-cell line and a related MTX resistant sub-line. The in vitro cell growth inhibitory activity was higher for bis(tetradecyl) conjugates than for both the other shorter- and longer-chain derivatives. The biological effectiveness of the various MTX derivatives correlated very well with the thermotropic effects observed on the phase transition of DPPC biomembranes. (C), 2001 Elsevier Science B.V All rights reserved.

A novel method for solid-phase synthesis of oligosaccharides using the N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker

The application of the N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker for the solid-phase synthesis of oligosaccharides is described. The oligosaccharide products can be cleaved from the resin by hydrazine, ammonia or primary amines, but the linker is stable under the conditions of oligosaccharide synthesis. The first sugar can be attached to the resin linker via a vinylogous amide bond, or by ether linkage using a p-aminobenzyl alcohol converter. (C) 2001 Elsevier Science Ltd. All rights reserved.

Biosynthesis and insecticidal properties of plant cyclotides: The cyclic knotted proteins from Oldenlandia affinis

Several members of the Rubiaceae and Violaceae families produce a series of cycloticles or macrocyclic peptides of 29-31 amino acids with an embedded cystine knot. We aim to understand the mechanism of synthesis of cyclic peptides in plants and have isolated a cDNA clone that encodes the cyclotide kalata Ell as well as three other clones for related cycloticles from the African plant Olden-landia affinis. The cDNA clones encode prepropeptides with a 20-aa signal sequence, an N-terminal prosequence of 46-68 amino acids and one, two, or three cyclotide domains separated by regions of about 25 aa. The corresponding cycloticles have been isolated from plant material, indicating that the cyclotide domains are excised and cyclized from all four predicted precursor proteins. The exact processing site is likely to lie on the N-terminal side of the strongly conserved GlyLeuPro or SerLeuPro sequence that flanks both sides of the cyclotide domain. Cyclotides have previously been assigned an antimicrobial function; here we describe a potent inhibitory effect on the growth and development of larvae from the Lepidopteran species Helicoverpa punctigera.

A comparison of low-storage strategies for spectral analysis in dissipative systems: filter diagonalisation in the Lanczos representation and harmonic inversion of the Chebychev-order-domain autocorrelation function

We compare the performance of two different low-storage filter diagonalisation (LSFD) strategies in the calculation of complex resonance energies of the HO2, radical. The first is carried out within a complex-symmetric Lanczos subspace representation [H. Zhang, S.C. Smith, Phys. Chem. Chem. Phys. 3 (2001) 2281]. The second involves harmonic inversion of a real autocorrelation function obtained via a damped Chebychev recursion [V.A. Mandelshtam, H.S. Taylor, J. Chem. Phys. 107 (1997) 6756]. We find that while the Chebychev approach has the advantage of utilizing real algebra in the time-consuming process of generating the vector recursion, the Lanczos, method (using complex vectors) requires fewer iterations, especially for low-energy part of the spectrum. The overall efficiency in calculating resonances for these two methods is comparable for this challenging system. (C) 2001 Elsevier Science B.V. All rights reserved.

Time evolution in the unimolecular master equation at low temperatures: full spectral solution with scalable iterative methods and high precision

A new method is presented to determine an accurate eigendecomposition of difficult low temperature unimolecular master equation problems. Based on a generalisation of the Nesbet method, the new method is capable of achieving complete spectral resolution of the master equation matrix with relative accuracy in the eigenvectors. The method is applied to a test case of the decomposition of ethane at 300 K from a microcanonical initial population with energy transfer modelled by both Ergodic Collision Theory and the exponential-down model. The fact that quadruple precision (16-byte) arithmetic is required irrespective of the eigensolution method used is demonstrated. (C) 2001 Elsevier Science B.V. All rights reserved.

Subspace wavepacket evolution with Newton polynomials

Time-dependent wavepacket evolution techniques demand the action of the propagator, exp(-iHt/(h)over-bar), on a suitable initial wavepacket. When a complex absorbing potential is added to the Hamiltonian for combating unwanted reflection effects, polynomial expansions of the propagator are selected on their ability to cope with non-Hermiticity. An efficient subspace implementation of the Newton polynomial expansion scheme that requires fewer dense matrix-vector multiplications than its grid-based counterpart has been devised. Performance improvements are illustrated with some benchmark one and two-dimensional examples. (C) 2001 Elsevier Science B.V. All rights reserved.

A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS-MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues, The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-mul amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C-18 (150X4.6 mm, 5 mum) column at 35 degreesC. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored. (C) 2001 Elsevier Science B.V. All rights reserved.

Structure and absolute configuration of mycobactin J

Mycobactin J 1 is a commercially available siderophore isolated from Mycobacterium avium subsp, paratuberculosis. There are discrepancies between previous reports of its structure and none have addressed its absolute configuration. We report here the complete structure and stereochemistry of mycobactin J, along with methodology to enable the determination of the absolute configuration of other mycobactins on a small scale. (C) 2001 Elsevier Science Ltd. All rights reserved.

Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from rhesus macaque leukocytes

Most mammalian defensins are cationic peptides of 29-42 amino acids long, stabilized by three disulfide bonds. However, recently Tang et al. (1999, Science 286, 498-502) reported the isolation of a new defensin type found in the leukocytes of rhesus macaques. In contrast to all the other defensins found so far, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue [Tang et al. (1999) Science 286, 498-502]. To elucidate the three-dimensional structure of RTD-1 and its open chain analogue, both peptides were synthesized using solid-phase peptide synthesis and tert-butyloxycarbonyl chemistry. The structures of both peptides in aqueous solution were determined from two-dimensional H-1 NMR data recorded at 500 and 750 MHz. Structural constraints consisting of interproton distances and dihedral angles were used as input for simulated-annealing calculations and water refinement with the program CNS. RTD-1 and its open chain analogue oRTD-1 adopt very similar structures in water. Both comprise an extended beta -hairpin structure with turns at one or both ends. The turns are well defined within themselves and seem to be flexible with respect to the extended regions of the molecules. Although the two strands of the beta -sheet are connected by three disulfide bonds, this region displays a degree of flexibility. The structural similarity of RTD-1 and its open chain analogue oRTD-1, as well as their comparable degree of flexibility, support the theory that the additional charges at the termini of the open chain analogue rather than overall differences in structure or flexibility are the cause for oRTD-1's lower antimicrobial activity. In contrast to numerous other antimicrobial peptides, RTD-1 does not display any amphiphilic character, even though surface models of RTD-1 exhibit a certain clustering of positive charges. Some amide protons of RTD-1 that should be solvent-exposed in monomeric beta -sheet structures show low-temperature coefficients, suggesting the possible presence of weak intermolecular hydrogen bonds.

Characterization of activated carbons using liquid phase adsorption

A modification of the Dubinin-Radushkevich pore filling model by incorporation of the repulsive contribution to the pore potential, and of bulk non-ideality, is proposed in this paper for characterization of activated carbon using liquid phase adsorption. For this purpose experiments have been performed using ethyl propionate, ethyl butyrate, and ethyl isovalerate as adsorbates and the microporous-mesoporous activated carbons Filtrasorb 400, Norit ROW 0.8 and Norit ROX 0.8 as adsorbents. The repulsive contribution to the pore potential is incorporated through a Lennard-Jones intermolecular potential model, and the bulk-liquid phase non-ideality through the UNIFAC activity coefficient model. For the characterization of activated carbons, the generalized adsorption isotherm is utilized with a bimodal gamma function as the pore size distribution function. It is found that the model can represent the experimental data very well, and significantly better than when the classical energy-size relationship is used, or when bulk non-ideality is neglected. Excellent agreement between the bimodal gamma pore size distribution and DFT-cum-regularization based pore size distribution is also observed, supporting the validity of the proposed model. (C) 2001 Elsevier Science Ltd. All rights reserved.

Application of heterogeneous vacancy solution theory to characterization of microporous solids

The vacancy solution theory of adsorption is re-formulated here through the mass-action law, and placed in a convenient framework permitting the development of thermodynamic ally consistent isotherms. It is shown that both the multisite Langmuir model and the classical vacancy solution theory expression are special cases of the more general approach when the Flory-Huggins activity coefficient model is used, with the former being the thermodynamically consistent result. The improved vacancy solution theory approach is further extended here to heterogeneous adsorbents by considering the pore-width dependent potential along with a pore size distribution. However, application of the model to numerous hydrocarbons as well as other adsorptives on microporous activated carbons shows that the multisite model has difficulty in the presence of a pore size distribution, because pores of different sizes can have different numbers of adsorbed layers and therefore different site occupancies. On the other hand, use of the classical vacancy solution theory expression for the local isotherm leads to good simultaneous fit of the data, while yielding a site diameter of about 0.257 nm, consistent with that expected for the potential well in aromatic rings on carbon pore surfaces. It is argued that the classical approach is successful because the Flory-Huggins term effectively represents adsorbate interactions in disguise. When used together with the ideal adsorbed solution theory the heterogeneous vacancy solution theory successfully predicts binary adsorption equilibria, and is found to perform better than the multisite Langmuir as well as the heterogeneous Langmuir model. (C) 2001 Elsevier Science Ltd. All rights reserved.