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WO2009115288A1 - Sonde à base de thiostrepton - Google Patents

Sonde à base de thiostrepton Download PDF

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Publication number
WO2009115288A1
WO2009115288A1 PCT/EP2009/001949 EP2009001949W WO2009115288A1 WO 2009115288 A1 WO2009115288 A1 WO 2009115288A1 EP 2009001949 W EP2009001949 W EP 2009001949W WO 2009115288 A1 WO2009115288 A1 WO 2009115288A1
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Prior art keywords
radical
molecular probe
general formula
phenyl
probe according
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German (de)
English (en)
Inventor
Hans-Dieter Arndt
Sebastian Schoof
Sascha Baumann
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Technische Universitaet Dortmund
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Technische Universitaet Dortmund
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to compounds of the general formula (I)
  • Thiopeptide natural compounds such as thiostrepton, nosiheptide or nocathiacin are potent inhibitors of protein biosynthesis, which exert their effect by sub-nM binding at the so-called GTPase center of the ribosome.
  • GTPase center of the ribosome So far, all prokaryotic ribosomes, but also ribosomes from protozoa (eg plasmodia, toxoplasma) and the cell organelles of higher multicellular organisms (eg eukaryotic mitochondria) are known as thiopeptide-sensitive. but not the cytoplasmic ribosomes in eukaryotes. So far, no active ingredients in human therapy, which use this principle of action, as well as no methods known to find such agents specifically.
  • HTS high-throughput screening
  • HCS high-content screening
  • This object is achieved by providing the compounds listed below, as by attaching functional molecular entities such as e.g. fluorescent dyes to thiopeptides such as e.g. Thiostrepton, with the help of organic chemical synthesis, it is possible to produce compounds from thiostrepton, which show an exceptionally high affinity to the complex of ribosomal RNA and the ribosomal protein L11, which is essential for the effect.
  • Such compounds are stable, highly affine, and selective, and are particularly useful as molecular probes for a variety of molecular biology, biochemical, and biophysical test methods, i.a. for the discovery of antibiotic agents or for the characterization of ribosome complexes, in particular of eukaryotic ribosome complexes.
  • n is 0 or an integer equal to 1 or 2, preferably 1 or 2
  • Nitrogen atom is present as a ring member
  • R 1 and R 2 each independently of one another, represent a radical of the general formula (L) or (M)
  • X is a saturated or unsaturated, linear or branched, aliphatic hydrocarbon radical having 1 to 18 carbon atoms, which is unsubstituted or optionally with 1, 2, 3, 4 or 5 substituents, independently selected from the group consisting of -F 1 -Cl, -Br 1 -I 1 -CN 1 -NO 2 , -OH 1 -CH 2 -CN, -SH 1 -NH 2 , -S-CH 3 , -SC 2 H 5 , -S-phenyl, -S-CH 2 -phenyl, -O-CH 3 , -O-C 2 H 5 .
  • p is an integer from 1 to 8
  • n O or an integer from 1 to 8
  • C is hydrogen or an aliphatic carboxyl radical
  • FL stands for a fluorescent dye residue, their corresponding salts or solvates, their stereoisomers, racemates, enantiomers or mixtures of the enantiomers in any ratio,
  • B is a radical (BII) or A is a radical (All),
  • (hetero) cycloalkylene as used herein includes cyclic, saturated hydrocarbon chains.
  • suitable cycloalkylene radicals include cyclopropylene, cyclobutylene, cyclopentylene and cyclohexylene.
  • X is a hydrocarbon radical which is selected from the group consisting of "methylene, ethylene, n-propylene, / so-propylene, n-butylene, / so-butylene, 2-butylene, tert-butylene, pentylene, hexylene, heptylene , Octylene, nonylene and decylene, where the abovementioned hydrocarbon radicals are saturated or unsaturated, linear or branched, unsubstituted or optionally having 1, 2, 3, 4 or 5 substituents independently of one another selected from the group consisting of -F, -Cl, - Br, -I 1 -CN, -NO 2 , -OH, -CH 2 -CN, -SH 1 -NH 2 , -S-CH 3 , -SC 2 H 5 , -S-phenyl, -S-CH 2 phenyl, -0-CH3, -0-C 2 H 5, -O-C 3
  • p is an integer from 1 to 3
  • n is 0 or an integer from 1 to 3
  • C is hydrogen or an acyl radical, preferably hydrogen
  • FL stands for a fluorescent dye residue.
  • Particularly preferred compounds of the general formulas (I) - (V) given above are compounds in which R 1 and R 2 , in each case independently of one another, represent a radical of the formula (L1) or (M1)
  • fluorescent dye moiety is selected from the group comprising fluorescein, Alexa Fluor 488, eosin yellowish, eosin bluish, phloxine, erythrosine, rose bengal, rhodamine , Rhodamine Green, Hydroxycumarin, Benzofuran, Texas Red, Oregon Green 488, Biman, NBD, dansyl, Dabsyl, Bodipy, Phycoerythrin, Cyanine Fluorophore Cy3 TM, Cy5 TM, Xanten and a dye moiety of Formula Z
  • Such fluorescent dyes are generally known to the person skilled in the art and are commercially available.
  • Another aspect of the present invention relates to a probe of the above-mentioned general formulas (I) - (V) 1 preferably for use as listed below.
  • Ribosomes are complexes of proteins and ribonucleic acids (RNA), which among other things in the cytoplasm of the cells of living things as well as in cell organelles such as the Mitochondria occur. Ribosomes are composed of two subunits, a large subunit that links the amino acids to the chain in protein biosynthesis (peptidyltransferase activity), and a small subunit responsible for mRNA recognition. Both prokaryotes and eukaryotes have ribosomes, but these differ from each other.
  • the molecular probes according to the invention have a high affinity for both prokaryotic and eukaryotic ribosome complexes, which is why such molecular probes allow a characterization and / or a diagnosis of ribosomes in prokaryotes and / or in eukaryotes, wherein a characterization and / or a diagnosis of ribosomes in eukaryotes is particularly preferred.
  • Characteristics of the molecular probes according to the invention are further allowed to characterize and / or diagnose ribosome subtypes in eukaryotes, in particular mitochondrial, cytoplasmic or pre-ribosomes.
  • Terms such as “mitochondrial ribosomes”, “cytoplasmic ribosomes” or “pre-ribosomes” are generally known and familiar to the person skilled in the art.
  • substance testing methods are used to find drugs, including the testing of large molecule libraries to find those substances that later serve as a lead for further development.
  • substance testing methods are preferably cellular, biochemical, molecular biological or biophysical and, according to the number of substances to be tested, under the terms high-throughput (HTS), medium throughput (MTS), low-throughput (LTS), and / or high-content screening (HCS).
  • HTS high-throughput
  • MTS medium throughput
  • LTS low-throughput
  • HCS high-content screening
  • HTS HTS, MTS and LTS
  • biochemical, molecular biological, cellular and biophysical test procedures are usually performed in microtiter plates in a few micro / nanoliter sample volumes which, with a desired biological effect, result in a color change or fluorescence which can be rapidly and accurately quantified.
  • the technique of HTS, MTS and LTS is well known to those skilled in the art.
  • HCS testing methods include, for example, ImageXpress ULTRA (Molecular Devices, Union City, USA), Pathway 855 and 435 (BD Biosciences), OPERA (Evotec Technologies, Hamburg, DE), Incell 1000 and 3000 ( GE / Amersham Biosciences,shire, UK), Arrayscan (Cellomics), Scanalyzer (Scanalyzer LemnaTec) and ImageXpress MICRO (Molecular Devices).
  • the HCS substance testing methods are preferably carried out with the molecular probe according to the invention on at least one of these devices.
  • the term "High Content Screening" is well known to those skilled in the art.
  • biochemical, cellular, molecular biological or biophysical substance testing methods for finding active substances, as well as in substance test methods for finding active ingredients in high molecular weight.
  • Throughput, medium throughput, low-throughput, and / or high-content screening assays where the term "assay” in the sense of the description is to be understood as meaning a standardized reaction sequence for detecting a substance with a specific method.
  • the molecular probe according to the invention binds with high affinity to the ribosomal GTPase center, it is possible with the aid of this probe to perform a quantification of binding parameters of ligands of the ribosomal GTPase center using appropriate measurement methods.
  • a further aspect of the present invention relates to the use of a molecular probe as described above for the quantification of binding parameters of ligands of the ribosomal GTPase center.
  • the molecular probe of the present invention binds to ribosome complexes with high affinity
  • another aspect of the present invention relates to its use for finding agents that are inhibitors of protein biosynthesis, in particular for finding antibacterial agents and / or unicellular parasitic agents, such as for example, Plasmodium falciparum or Toxoplasma ghondii, and generally against bacterial pathogens, such as S. aureus, M. tuberculosis, Enterococci, streptococci (all Gram-positive), pseudomonads, Acitenobacter or Klebsiella (gram-negative).
  • Ribosome staining reagents are not yet commercially available, but have a wide range of uses in basic biological and clinical research (cell and tissue microscopy) as well as a large application potential in clinical diagnostics. Because of their exceptionally high affinity for ribosome complexes, the molecular probes according to the invention are particularly suitable for selectively staining thiopeptide-sensitive ribosomes, preferably of eukaryotes, in cells and tissues.
  • a further aspect of the present invention therefore relates to the use of a molecular probe according to the invention as described above as a fluorescence marker of ribosome complexes, for detecting the inhibition of the ribosomal GTPase center, as well as in fluorescence microscopy methods.
  • a further aspect of the present invention relates to a process for the preparation of the compounds of the abovementioned general formula (I) - (V), comprising the steps:
  • a suitable solvent preferably an organic solvent, more preferably a polar, organic solvent, and then adding an amine, preferably an aliphatic amine, preferably at a temperature between -8O 0 C and 100 0 C, more preferably from -2O 0 C to 2O 0 C and then removing the solvent and optionally cleaning / isolating the compound obtained;
  • radicals FL and X have the abovementioned meaning.
  • the starting compounds in particular the fluorescent dyes, are commercially available or can be prepared by the customary processes known to the person skilled in the art.
  • the intermediate and end products obtained according to the above-described reactions can each be purified and / or isolated, if desired and / or required, by customary methods known to those skilled in the art. Suitable purification methods are, for example, extraction methods and chromatographic methods, such as column chromatography or preparative chromatography.
  • RNA The synthesis of 58 nt fragments (1051-1108) of the Escherichia coli wild-type or mutated 23S rRNA was carried out by means of in vitro transcription (T7 RNA polymerase; MEGAscript Kit, Ambion).
  • the carrier vector for the DNA template was the pUC 19 vector system.
  • RNA products were purified by PAGE and gel filtration. PAGE and gel filtration are standard methods of molecular biology. The 2'-O-methyl-A1067 fragment was obtained from Dharmacon.
  • the DNA ORF from Thermus thermophilus and Escherichia coli coding for the L11 protein was heterologously overexpressed in E. coli using the pQE30 Xa vector system as N-terminal His 6 fusion protein.
  • the purification was carried out by means of Ni 2+ -NTA affinity chromatography and subsequent gel filtration. Such methods for cloning, protein expression and purification are well known to those skilled in the art.
  • Example Compound 1 (Compound 1) shown above in a reaction buffer (10 mM MOPS pH 8.0, 100 mM KCl, 5 mM MgCl 2 , 2 % TFE) with varying amounts of RNA (10 ⁇ M max.) And fluorescence anisotropy change after 15 min. Incubation determined.
  • RNA / protein / example compound 1 is taken into account.
  • the following formula was used to determine the probe affinity:
  • Example Compound 1 To determine the affinity of Example Compound 1, the same experimental conditions were used as in point I.2. selected. In contrast to the point 1.2. however, the corresponding protein from E. coli was used instead of T. thermophilus.
  • Example compound 1 also binds to E.coli 23S rRNA complexes with E. coli L11 protein, the K 0 being about 540 pM (see fit of the above equation).
  • 10 nM example compound 1 10 nM RNA and 0.6 uM protein are for about 10 min. in reaction buffer (10 mM MOPS pH 8.0, 100 mM KCl, 5 mM MgCl 2 , 2% TFE), then titrated with varying concentrations of the thiopeptide natural product and the fluorescence anisotropy change after 15 min. Incubation determined. Results:
  • Nosiheptide binds 2 times more to the complex than thiostrepton and slightly higher
  • Example Compound 1 shows the affinity of Example Compound 1 for the 23S rRNA fragment / L11 protein complex (plot of anisotropy versus RNA concentration, see item l.2.c) and I.2.C 1 ))
  • reaction buffer 10 mM MOPS pH 8.0, 100 mM KCl, 5 mM MgCl 2 , 2% TFE. They are then titered with various concentrations of RNA, and after 15 minutes of incubation, the change in fluorescence anisotropy is determined.
  • Example Compound 1 Titration of Example Compound 1 in the presence of thiostrepton or nosiheptide gives a significant shift of the binding curve to the right.
  • the binding curve can be used to determine the binding constants using the standard methods of numerical simulation and the known target affinity of the fluorescent probe.
  • Example Compound 1 shows the KD values of Example Compound 1 as well as thiostrepton, nosiheptide and micrococcin with respect to a 23S rRNA (Eco //) / L11 protein (T. thermophilus) complex.
  • the measured physical quantity is the fluorescence polarization / anisotropy with an excitation wavelength of 475 nm and an emission wavelength of 520 nm.
  • the duration of the measurement of a full microtiter plate with 50 measurements per well was 5 min.
  • Retaining purine base has no effect on binding.
  • the 2'O-methyl-A1067 fragment (the natural defense mechanism of thiopeptide-producing Streptomyces strains) has the highest K D , a 3000-fold decrease in affinity compared to wild-type RNA.
  • BSC-1 cells (ATCC No. CCL 26) are incubated in D-MEM medium (Gibco) supplemented with 4500 mg / L glucose, pyruvate, glutamine, nonessential amino acids and 10% FCS (Gibco) at 37 ° C and 5% CO 2 cultured. In the present case, only cells are used after 5-25 passages.
  • the cells are washed with 0.25% trypsin and 1 mM EDTA in HBSS (8 g / L NaCl, 0.4 g / L KCl, 60 mg / L KH 2 PO 4 , 1 g / L glucose, 48 mg / L Na 2 HPO 4 , 98 mg / L MgSO 4 , 140 mg / L CaCl 2 , 350 mg / L Na 2 CO 3 ) and in a density of 20,000 cells per slide in D-MEM with 4500 mg / L glucose, pyruvate, glutamine, not Essential amino acids and 10% FCS plated.
  • HBSS 8 g / L NaCl, 0.4 g / L KCl, 60 mg / L KH 2 PO 4 , 1 g / L glucose, 48 mg / L Na 2 HPO 4 , 98 mg / L MgSO 4 , 140 mg / L CaCl 2 , 350 mg / L Na 2 CO 3
  • the fixation is 10 min. by means of a fixative solution containing 37% formaldehyde, 100 mM KPipes, 10 mM EGTA, 1 mM MgCl, 0.1% TX-100 (Sigma) in water.
  • the fixed cells are washed with TBS-T and blocked with 2% BSA (Sigma) in TBS-T for 1 h and washed again in TBS-T.
  • the chemicals and solvents used were obtained commercially from the conventional suppliers (Acros, Avocado, Aldrich, Bachern, Fluka, Lancaster, Maybridge, Merck, Sigma, TCI, etc.) or synthesized according to methods known to those skilled in the art.
  • the concentrated reaction mixture is taken up in CHCl 3 / MeOH 2% and separated by means of a silica column.
  • the eluent used is CHCl 3 / MeOH 2-5%.
  • the thiazoline ring of the thiostrepton skeleton can be selectively oxidized to the corresponding thiazole ring. This modification can be inserted both to thiostrepton itself, as well as to the shortened variants V1 and V2.
  • the thiostrepton derivative V1ox is obtained by oxidation of the thiazoline ring of the singly truncated thiostrepton variant V1.
  • the product is purified by preparative HPLC at a gradient of 40 to 75%.
  • Thiostrepton (50 mg, 0.03 mmol) is dissolved in dry THF (2ml) and agitt to -10 0 C in an inert atmosphere.
  • NaBH 3 CN (3 eq) is dissolved in dry THF (0.5 ml) and added slowly to the thiostrepton solution dropwise.
  • the reaction mixture is after 1 No longer cooled and then stirred at room temperature for 15 hours.
  • the volatiles are removed under reduced pressure and the residue dissolved in trifluoroethanol.
  • the solution is purified by preparative HPLC (C4-RP column) in a water-acetonitrile gradient or by means of a silica gel column (CHCVMethonal), chromatographically.
  • Thiostrepton derivatives of general structure D1 can be obtained by reaction of singly truncated thiostrepton derivative V1 (see above) with various functionalized thiols (LX), where the radicals FL and X have the abovementioned meaning [Scheme 1].
  • Analogous addition products D2 can be obtained by reaction of the trisubstituted thiostrepton derivative V2 with various functionalized thiols (LX), where the radicals FL and X have the abovementioned meaning [Scheme 2].
  • double adducts D3 which, starting from thiostrepton derivative V1, are formed by addition of two functionalized thiols (LX), where the radicals FL and X have the abovementioned, if appropriate, different meanings [Scheme 3].
  • Functionalized thiols with the rest of the general formula (L1) can be obtained starting from diamine A1, trityl-protected mercaptopropionic acid A2 and acylating reagent A3.
  • the amine A4 is obtained by an amide linkage, which is converted in a further step by means of acylating reagent A3 in the presence of a fluorescent dye A5 to the functionalized (and protected) thiol (L1a).
  • Trityl deprotection leads to functional thiol with the remainder (L1), [Scheme 4], which has the abovementioned meaning.
  • Trt-mercaptopropionic acid 1 mmol
  • DMF dimethyl methoxysulfoxide
  • HBTU hydroxybenzyl sulfonate
  • this mixture is slowly added dropwise to a solution of 2.19 ml of PEG (10 eq) in 10 ml of DCM at 0 ° C. with constant stirring. After 30 minutes, the cold bath is removed and stirred overnight at room temperature.
  • the product is purified by silica flash chromatography with a solvent gradient of CHCl 3 / MeOH 5-9%.
  • the product is purified by silica flash chromatography with a solvent gradient of ethyl acetate / MeOH 5-10%.
  • the concentrated reaction mixture is resumed in 5 ml of TFE / 1 ml of 50 mM sodium carbonate buffer pH 10.
  • the pH of the solution is adjusted to pH 8 with saturated NaHCO 3 solution.
  • To this solution is added 66 mg of singly truncated thiostrepton derivative V1.
  • the reaction is carried out under Ar protective gas atmosphere. After 4 days, the reaction solution is concentrated under reduced pressure and purified [Scheme 7].
  • the product is purified by preparative HPLC at a gradient of 10 to 40%.
  • UV-Vis 30 ⁇ M in 10% TFE in 50 mM sodium phosphate buffer pH 9. ⁇ max 496 nm, 283 nm,
  • the fluorescein compound (90 mg, 0.127 mmol) and triethylamine (30 ⁇ l, 1.1 eq) obtained according to 11.9.2 are dissolved in undiluted trifluoroacetic acid (1.5 ml) and stirred for 20 minutes. The volatiles are removed and it is evaporated 3 times with toluene.
  • Example Compound 2 The remaining residue is dissolved in trifluoroethanol (4 ml) and treated with sodium phosphate buffer (50 mmol, pH 9, 1 ml) and then treated with the thiostreptone derivative V1 (1-fold shortened thiostrepton) and diethylamine (88 ul, 5 eq). The reaction mixture is stirred under an inert gas atmosphere for 16 hours. Thereafter, the probe shown above (Example Compound 2) is obtained by HPLC purification.

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Abstract

Composés de formule générale (I), procédé de préparation de ces composés, sondes moléculaires basées sur des composés de ce type, leur utilisation pour caractériser et/ou diagnostiquer des ribosomes dans des procaryotes et/ou des eucaryotes, plus particulièrement des sous-types de ribosomes dans des eucaryotes, leur utilisation en tant qu'outils pharmacologiques pour des procédés biochimiques, cellulaires, de biologie moléculaire ou biophysiques de test de substances, destinés à la recherche de substances actives, leur utilisation dans des procédés de test de substances, destinés à la recherche de substances actives dans des essais de criblage à haut débit, à moyen débit, à faible débit et/ou à haut contenu, en tant que marqueurs de fluorescence de complexes ribosomiques, pour la quantification de paramètres de liaison de ligands du centre GTPasique ribosomique, destinés à la recherche d'inhibiteurs de la biosynthèse protéique, de substances à action antibactérienne et/ou de substances contre des parasites unicellulaires, ainsi qu'au diagnostic clinique.
PCT/EP2009/001949 2008-03-17 2009-03-17 Sonde à base de thiostrepton Ceased WO2009115288A1 (fr)

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JP2013503624A (ja) * 2009-09-02 2013-02-04 ピラマル・ライフ・サイエンシーズ・リミテッド 抗生物質化合物
EP3685846A1 (fr) * 2019-01-24 2020-07-29 Genethon Utilisation de thiostrepton ou de ses dérivés pour le traitement de maladies génétiques liées à un trouble conformationnel de protéine
US20220315624A1 (en) * 2019-05-13 2022-10-06 Yale University Thiostrepton analogs and methods of making and using same

Citations (2)

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WO2001044505A2 (fr) * 1999-12-16 2001-06-21 Ribotargets Limited Procedes et kits permettant de decouvrir des antimicrobiens se liant a l'arn
WO2005036169A2 (fr) * 2003-10-03 2005-04-21 Cumbre Inc. Sondes fluorescentes pour ribosomes et leur procede utilisation

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WO2002102400A1 (fr) * 2001-06-18 2002-12-27 Genoprot Co., Ltd. Utilisation de composes peptidiques macrocycliques en tant qu'agents anticancereux et methode de diagnostic du cancer
WO2007127136A2 (fr) * 2006-04-24 2007-11-08 Merck & Co., Inc. Composés antibiotiques

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WO2001044505A2 (fr) * 1999-12-16 2001-06-21 Ribotargets Limited Procedes et kits permettant de decouvrir des antimicrobiens se liant a l'arn
WO2005036169A2 (fr) * 2003-10-03 2005-04-21 Cumbre Inc. Sondes fluorescentes pour ribosomes et leur procede utilisation

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Title
SCHOOF SEBASTIAN ET AL: "A fluorescent probe for the 70 S-ribosomal GTPase-associated center.", CHEMBIOCHEM : A EUROPEAN JOURNAL OF CHEMICAL BIOLOGY 26 JAN 2009, vol. 10, no. 2, 26 January 2009 (2009-01-26), pages 242 - 245, XP002529583, ISSN: 1439-7633 *
YAN KANG ET AL: "Fluorescence polarization method to characterize macrolide-ribosome interactions", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON, DC, US, vol. 49, no. 8, 1 August 2005 (2005-08-01), pages 3367 - 3372, XP002381573, ISSN: 0066-4804 *

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