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Pavel Hrouzek - One of the best experts on this subject based on the ideXlab platform.
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Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC
Analytical and Bioanalytical Chemistry, 2017Co-Authors: José Cheel, Petra Urajova, Jan Hajek, Pavel Hrouzek, Marek Kuzma, Elodie Bouju, Karine Faure, Jiři KopeckýAbstract:Puwainaphycins are a recently described group of β-Amino Fatty Acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium ( Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n -hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min^−1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate–ethanol–water (5:1:5, v/v/v) system, afforded compounds 1 and 2 . The CCC separation of fraction II, with use of the n -hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system, afforded compounds 3 – 7 . In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min^−1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1 – 7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC–electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1 –7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1 , 2 , and 5 ) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass. Graphical Abstract Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Compounds 1 (12-hydroxy-4-methyl-Ahtea-Puw-F), 2 (11-chloro-4-methyl-Ahdoa-Puw-F), 3 (4-methyl-Ahdoa-Puw-F), 4 (4-methyl-Ahdoa-Puw-G), 5 (12-chloro-4-methyl-Ahtea-Puw-F), 6 (4-methyl-Ahtea-Puw-F) and 7 (4-methyl-Ahtea-Puw-G). Ahtea: 3-Amino-2-hydroxy tetradecanoic Acid. Ahdoa: 3-Amino-2-hydroxy dodecanoic Acid
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Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC.
Analytical and Bioanalytical Chemistry, 2016Co-Authors: José Cheel, Petra Urajova, Jan Hajek, Pavel Hrouzek, Marek Kuzma, Elodie Bouju, Karine Faure, Jiři KopeckýAbstract:Puwainaphycins are a recently described group of β-Amino Fatty Acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium (Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min−1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate–ethanol–water (5:1:5, v/v/v) system, afforded compounds 1 and 2. The CCC separation of fraction II, with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system, afforded compounds 3–7. In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min−1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1–7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC–electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1–7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1, 2, and 5) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass.
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a hybrid non ribosomal peptide polyketide synthetase containing Fatty acyl ligase faal synthesizes the β Amino Fatty Acid lipopeptides puwainaphycins in the cyanobacterium cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
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A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
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The cyanobacterial cyclic lipopeptides puwainaphycins F/G are inducing necrosis via cell membrane permeabilization and subsequent unusual actin relocalization.
Chemical Research in Toxicology, 2012Co-Authors: Pavel Hrouzek, Marek Kuzma, Jan Černý, Petr Novák, Radovan Fišer, Petr Šimek, Alena Lukešová, Jiři KopeckýAbstract:Puwainaphycins F and G, moderate cytotoxins, which cause necrotic cell death to mammalian cells, were isolated from the soil cyanobacterium Cylindrospermum alatosporum C24/89. Both compounds have been shown to be cyclic decapeptides containing unusual β-Amino Fatty Acid (2-hydroxy-3-Amino-4methyl tetradecanoic Acid). Described variants differ in the substitution of threonine by glutamine in the fourth position. Their structures differ from the known puwainaphycins in five Amino Acids positions as well as in the β-Amino Fatty Acid unit. The rapid interaction of these compounds with the plasma membrane of the mammal cell leads to an elevation of the concentration of intracellular Ca2+, with kinetics comparable to the well-established calcium ionophore ionomycin. Subsequently, the induction of tyrosine phosphorylation was observed to be followed by the unique transformation of the actin cytoskeleton into ring structures around the nuclei. All of these alterations in the cellular morphology and physiology res...
Petra Urajova - One of the best experts on this subject based on the ideXlab platform.
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Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC
Analytical and Bioanalytical Chemistry, 2017Co-Authors: José Cheel, Petra Urajova, Jan Hajek, Pavel Hrouzek, Marek Kuzma, Elodie Bouju, Karine Faure, Jiři KopeckýAbstract:Puwainaphycins are a recently described group of β-Amino Fatty Acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium ( Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n -hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min^−1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate–ethanol–water (5:1:5, v/v/v) system, afforded compounds 1 and 2 . The CCC separation of fraction II, with use of the n -hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system, afforded compounds 3 – 7 . In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min^−1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1 – 7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC–electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1 –7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1 , 2 , and 5 ) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass. Graphical Abstract Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Compounds 1 (12-hydroxy-4-methyl-Ahtea-Puw-F), 2 (11-chloro-4-methyl-Ahdoa-Puw-F), 3 (4-methyl-Ahdoa-Puw-F), 4 (4-methyl-Ahdoa-Puw-G), 5 (12-chloro-4-methyl-Ahtea-Puw-F), 6 (4-methyl-Ahtea-Puw-F) and 7 (4-methyl-Ahtea-Puw-G). Ahtea: 3-Amino-2-hydroxy tetradecanoic Acid. Ahdoa: 3-Amino-2-hydroxy dodecanoic Acid
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Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC.
Analytical and Bioanalytical Chemistry, 2016Co-Authors: José Cheel, Petra Urajova, Jan Hajek, Pavel Hrouzek, Marek Kuzma, Elodie Bouju, Karine Faure, Jiři KopeckýAbstract:Puwainaphycins are a recently described group of β-Amino Fatty Acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium (Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min−1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate–ethanol–water (5:1:5, v/v/v) system, afforded compounds 1 and 2. The CCC separation of fraction II, with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system, afforded compounds 3–7. In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min−1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1–7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC–electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1–7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1, 2, and 5) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass.
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a liquid chromatography mass spectrometric method for the detection of cyclic β Amino Fatty Acid lipopeptides
Journal of Chromatography A, 2016Co-Authors: Petra Urajova, Jan Hajek, Matti Wahlsten, Jouni Jokela, Tomas Galica, David P Fewer, Andreja Kust, Eliska Zapomělovakozlikova, Kateřina Delawska, Kaarina SivonenAbstract:Abstract Bacterial lipopeptides, which contain β-Amino Fatty Acids, are an abundant group of bacterial secondary metabolites exhibiting antifungal and/or cytotoxic properties. Here we have developed an LC-HRMS/MS method for the selective detection of β-Amino Fatty Acid containing cyclic lipopeptides. The method was optimized using the lipopeptides iturin A and puwainaphycin F, which contain Fatty Acids of similar length but differ in the Amino Acid composition of the peptide cycle. Fragmentation energies of 10–55 eV were used to obtain the Amino Acid composition of the peptide macrocycle. However, fragmentation energies of 90–130 eV were used to obtain an intense fragment specific for the β-Amino Fatty Acid (C n H 2 n +2 N + ). The method allowed the number of carbons and consequently the length of the β-Amino Fatty Acid to be estimated. We identified 21 puwainaphycin variants differing in Fatty Acid chain in the crude extract of cyanobacterium Cylindrospermum alatosporum using this method. Analogously 11 iturin A variants were detected. The retention time of the lipopeptide variants showed a near perfect linear dependence ( R 2 = 0.9995) on the length of the Fatty Acid chain in linear separation gradient which simplified the detection of minor variants. We used the method to screen 240 cyanobacterial strains and identified lipopeptides from 8 strains. The HPLC-HRMS/MS method developed here provides a rapid and easy way to detecting novel variants of cyclic lipopeptides.
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a hybrid non ribosomal peptide polyketide synthetase containing Fatty acyl ligase faal synthesizes the β Amino Fatty Acid lipopeptides puwainaphycins in the cyanobacterium cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
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A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
Jan Hajek - One of the best experts on this subject based on the ideXlab platform.
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Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC
Analytical and Bioanalytical Chemistry, 2017Co-Authors: José Cheel, Petra Urajova, Jan Hajek, Pavel Hrouzek, Marek Kuzma, Elodie Bouju, Karine Faure, Jiři KopeckýAbstract:Puwainaphycins are a recently described group of β-Amino Fatty Acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium ( Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n -hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min^−1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate–ethanol–water (5:1:5, v/v/v) system, afforded compounds 1 and 2 . The CCC separation of fraction II, with use of the n -hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system, afforded compounds 3 – 7 . In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min^−1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1 – 7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC–electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1 –7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1 , 2 , and 5 ) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass. Graphical Abstract Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Compounds 1 (12-hydroxy-4-methyl-Ahtea-Puw-F), 2 (11-chloro-4-methyl-Ahdoa-Puw-F), 3 (4-methyl-Ahdoa-Puw-F), 4 (4-methyl-Ahdoa-Puw-G), 5 (12-chloro-4-methyl-Ahtea-Puw-F), 6 (4-methyl-Ahtea-Puw-F) and 7 (4-methyl-Ahtea-Puw-G). Ahtea: 3-Amino-2-hydroxy tetradecanoic Acid. Ahdoa: 3-Amino-2-hydroxy dodecanoic Acid
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Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC.
Analytical and Bioanalytical Chemistry, 2016Co-Authors: José Cheel, Petra Urajova, Jan Hajek, Pavel Hrouzek, Marek Kuzma, Elodie Bouju, Karine Faure, Jiři KopeckýAbstract:Puwainaphycins are a recently described group of β-Amino Fatty Acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium (Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min−1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate–ethanol–water (5:1:5, v/v/v) system, afforded compounds 1 and 2. The CCC separation of fraction II, with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system, afforded compounds 3–7. In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min−1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1–7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC–electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1–7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1, 2, and 5) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass.
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a liquid chromatography mass spectrometric method for the detection of cyclic β Amino Fatty Acid lipopeptides
Journal of Chromatography A, 2016Co-Authors: Petra Urajova, Jan Hajek, Matti Wahlsten, Jouni Jokela, Tomas Galica, David P Fewer, Andreja Kust, Eliska Zapomělovakozlikova, Kateřina Delawska, Kaarina SivonenAbstract:Abstract Bacterial lipopeptides, which contain β-Amino Fatty Acids, are an abundant group of bacterial secondary metabolites exhibiting antifungal and/or cytotoxic properties. Here we have developed an LC-HRMS/MS method for the selective detection of β-Amino Fatty Acid containing cyclic lipopeptides. The method was optimized using the lipopeptides iturin A and puwainaphycin F, which contain Fatty Acids of similar length but differ in the Amino Acid composition of the peptide cycle. Fragmentation energies of 10–55 eV were used to obtain the Amino Acid composition of the peptide macrocycle. However, fragmentation energies of 90–130 eV were used to obtain an intense fragment specific for the β-Amino Fatty Acid (C n H 2 n +2 N + ). The method allowed the number of carbons and consequently the length of the β-Amino Fatty Acid to be estimated. We identified 21 puwainaphycin variants differing in Fatty Acid chain in the crude extract of cyanobacterium Cylindrospermum alatosporum using this method. Analogously 11 iturin A variants were detected. The retention time of the lipopeptide variants showed a near perfect linear dependence ( R 2 = 0.9995) on the length of the Fatty Acid chain in linear separation gradient which simplified the detection of minor variants. We used the method to screen 240 cyanobacterial strains and identified lipopeptides from 8 strains. The HPLC-HRMS/MS method developed here provides a rapid and easy way to detecting novel variants of cyclic lipopeptides.
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a hybrid non ribosomal peptide polyketide synthetase containing Fatty acyl ligase faal synthesizes the β Amino Fatty Acid lipopeptides puwainaphycins in the cyanobacterium cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
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A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
Jiři Kopecký - One of the best experts on this subject based on the ideXlab platform.
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Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC
Analytical and Bioanalytical Chemistry, 2017Co-Authors: José Cheel, Petra Urajova, Jan Hajek, Pavel Hrouzek, Marek Kuzma, Elodie Bouju, Karine Faure, Jiři KopeckýAbstract:Puwainaphycins are a recently described group of β-Amino Fatty Acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium ( Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n -hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min^−1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate–ethanol–water (5:1:5, v/v/v) system, afforded compounds 1 and 2 . The CCC separation of fraction II, with use of the n -hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system, afforded compounds 3 – 7 . In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min^−1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1 – 7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC–electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1 –7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1 , 2 , and 5 ) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass. Graphical Abstract Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC. Compounds 1 (12-hydroxy-4-methyl-Ahtea-Puw-F), 2 (11-chloro-4-methyl-Ahdoa-Puw-F), 3 (4-methyl-Ahdoa-Puw-F), 4 (4-methyl-Ahdoa-Puw-G), 5 (12-chloro-4-methyl-Ahtea-Puw-F), 6 (4-methyl-Ahtea-Puw-F) and 7 (4-methyl-Ahtea-Puw-G). Ahtea: 3-Amino-2-hydroxy tetradecanoic Acid. Ahdoa: 3-Amino-2-hydroxy dodecanoic Acid
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Separation of cyclic lipopeptide puwainaphycins from cyanobacteria by countercurrent chromatography combined with polymeric resins and HPLC.
Analytical and Bioanalytical Chemistry, 2016Co-Authors: José Cheel, Petra Urajova, Jan Hajek, Pavel Hrouzek, Marek Kuzma, Elodie Bouju, Karine Faure, Jiři KopeckýAbstract:Puwainaphycins are a recently described group of β-Amino Fatty Acid cyclic lipopeptides of cyanobacterial origin that possess interesting biological activities. Therefore, the development of an efficient method for their isolation from natural sources is necessary. Following the consecutive adsorption of the crude extract on Amberlite XAD-16 and XAD-7 resins, countercurrent chromatography (CCC) was applied to separate seven puwainaphycin variants from a soil cyanobacterium (Cylindrospermum alatosporum CCALA 988). The resin-enriched extract was first fractionated by CCC into fractions I and II with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system at a flow rate of 2 mL min−1 and a rotational speed of 1400 rpm. The CCC separation of fraction I, with use of the ethyl acetate–ethanol–water (5:1:5, v/v/v) system, afforded compounds 1 and 2. The CCC separation of fraction II, with use of the n-hexane–ethyl acetate–ethanol–water (1:5:1:5, v/v/v/v) system, afforded compounds 3–7. In both cases, the lower phases were used as mobile phases at a flow rate of 1 mL min−1 with a rotational speed of 1400 rpm and a temperature of 28 °C. The CCC target fractions obtained were repurified by semipreparative high-performance liquid chromatography (HPLC), leading to compounds 1–7 with purities of 95 %, 95 %, 99 %, 99 %, 95 %, 99 %, and 90 % respectively, as determined by HPLC–electrospray ionization high-resolution mass spectrometry (ESI-HRMS). The chemical identity of the isolated puwainaphycins (compounds 1–7) was confirmed by ESI-HRMS and NMR analyses. Three new puwainaphycin variants (compounds 1, 2, and 5) are reported for the first time. This study provides a new approach for the isolation of puwainaphycins from cyanobacterial biomass.
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a hybrid non ribosomal peptide polyketide synthetase containing Fatty acyl ligase faal synthesizes the β Amino Fatty Acid lipopeptides puwainaphycins in the cyanobacterium cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
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A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
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The cyanobacterial cyclic lipopeptides puwainaphycins F/G are inducing necrosis via cell membrane permeabilization and subsequent unusual actin relocalization.
Chemical Research in Toxicology, 2012Co-Authors: Pavel Hrouzek, Marek Kuzma, Jan Černý, Petr Novák, Radovan Fišer, Petr Šimek, Alena Lukešová, Jiři KopeckýAbstract:Puwainaphycins F and G, moderate cytotoxins, which cause necrotic cell death to mammalian cells, were isolated from the soil cyanobacterium Cylindrospermum alatosporum C24/89. Both compounds have been shown to be cyclic decapeptides containing unusual β-Amino Fatty Acid (2-hydroxy-3-Amino-4methyl tetradecanoic Acid). Described variants differ in the substitution of threonine by glutamine in the fourth position. Their structures differ from the known puwainaphycins in five Amino Acids positions as well as in the β-Amino Fatty Acid unit. The rapid interaction of these compounds with the plasma membrane of the mammal cell leads to an elevation of the concentration of intracellular Ca2+, with kinetics comparable to the well-established calcium ionophore ionomycin. Subsequently, the induction of tyrosine phosphorylation was observed to be followed by the unique transformation of the actin cytoskeleton into ring structures around the nuclei. All of these alterations in the cellular morphology and physiology res...
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a hybrid non ribosomal peptide polyketide synthetase containing Fatty acyl ligase faal synthesizes the β Amino Fatty Acid lipopeptides puwainaphycins in the cyanobacterium cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
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A Hybrid Non-Ribosomal Peptide/Polyketide Synthetase Containing Fatty-Acyl Ligase (FAAL) Synthesizes the β-Amino Fatty Acid Lipopeptides Puwainaphycins in the Cyanobacterium Cylindrospermum alatosporum
PLOS ONE, 2014Co-Authors: Jan Mares, Petra Urajova, Jan Hajek, Jiři Kopecký, Pavel HrouzekAbstract:A putative operon encoding the biosynthetic pathway for the cytotoxic cyanobacterial lipopeptides puwainphycins was identified in Cylindrospermum alatosporum. Bioinformatics analysis enabled sequential prediction of puwainaphycin biosynthesis; this process is initiated by the activation of a Fatty Acid residue via Fatty acyl-AMP ligase and continued by a multidomain non-ribosomal peptide synthetase/polyketide synthetase. High-resolution mass spectrometry and nuclear magnetic resonance spectroscopy measurements proved the production of puwainaphycin F/G congeners differing in FA chain length formed by either 3-Amino-2-hydroxy-4-methyl dodecanoic Acid (4-methyl-Ahdoa) or 3-Amino-2-hydroxy-4-methyl tetradecanoic Acid (4-methyl-Ahtea). Because only one puwainaphycin operon was recovered in the genome, we suggest that the Fatty acyl-AMP ligase and one of the Amino Acid adenylation domains (Asn/Gln) show extended substrate specificity. Our results provide the first insight into the biosynthesis of frequently occurring β-Amino Fatty Acid lipopeptides in cyanobacteria, which may facilitate analytical assessment and development of monitoring tools for cytotoxic cyanobacterial lipopeptides.
