Ignacio Pérez-Victoria - Academia.edu (original) (raw)
Uploads
Papers by Ignacio Pérez-Victoria
Journal of Peptide Science, 2005
Conformational investigations have been undertaken on oligomers (dimers, tetramers, hexamers) of ... more Conformational investigations have been undertaken on oligomers (dimers, tetramers, hexamers) of five closely related oxetane-based dipeptide isosteres. All the oligomers were subjected to a range of studies by NMR, FT-IR and CD spectroscopy. The oligomers derived from methyl 2,4-anhydro-5-azido-3-O-tert-butyldimethylsilyl-5-deoxy-L-rhamnonate ‘monomer’ all exhibited evidence of ordered conformations in chloroform and 2,2,2-trifluoroethanol (TFE) solution. 5-Acetamido and N-methylamide derivatives of the L-rhamnonate ‘monomer’, along with a ‘dimer’ lacking silyl protection at C-3, were synthesized to ascertain the role of intramolecular interactions. This led to the conclusion that, for the L-rhamnonate oligomers, steric interactions govern the conformational preference observed. The equivalent silyl-protected D-lyxonate oligomers gave ordered CD spectra in TFE solution, but NMR and FT-IR spectroscopy in chloroform solution suggested an irregular, non-hydrogen bonded system. The remaining silyl-protected 6-deoxy-L-altronate, 6-deoxy-D-gulonate and D-fuconate oligomers appear to be characterized by their lack of ordered conformation in TFE and chloroform solution. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.
Journal of Peptide Science, 2005
Routes to oligomers (dimers, tetramers, hexamers) of five oxetane-based dipeptide isosteres have ... more Routes to oligomers (dimers, tetramers, hexamers) of five oxetane-based dipeptide isosteres have been established. Methyl 2,4-anhydro-5-azido-5-deoxy-L-rhamnonate ‘monomer’ led, by coupling the corresponding carboxylic acid and amine, to a ‘dimer’. Reverse-aldol ring-opening occurred on attempted saponification of the dimer, so all further oligomerization was performed using TBDMS C-3 hydroxyl protection. The silyl protected L-rhamnonate monomer led in turn to the dimer (via the monomer acid and amine), the tetramer (via the dimer acid and amine) and finally the hexamer (via the tetramer acid and dimer amine). In each case the acids were obtained through saponification of the respective methyl esters and the amines were obtained by hydrogenation of the azides; coupling was TBTU-mediated. Essentially the same strategy was employed on equivalent D-lyxonate, 6-deoxy-L-altronate, 6-deoxy-D-gulonate and D-fuconate dipeptide isosteres to give the respective dimers, tetramers and hexamers. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.
Chemical Communications, 2009
Journal of Mass Spectrometry, 2008
Structural characterization and differentiation of a novel group of regioisomeric monolaurate est... more Structural characterization and differentiation of a novel group of regioisomeric monolaurate esters of the nonreducing trisaccharides raffinose and melezitose, and the nonreducing tetrasaccharide stachyose has been obtained using positive electrospray ionization (ESI) mass spectrometry with in-source fragmentation. The surfactant nature and high polarity of these compounds make them appropriate analytes for being studied by conventional ESI-MS. The position of the acyl chain in each particular regioisomer has been used as a reporter group that allows unambiguous rationalization of the fragmentation routes of the corresponding natriated molecular ions [M + Na]+. In all cases, the main fragment ions were produced through cleavage of the glycosidic bond involving two anomeric carbons, the C-1′ and C-2″ of the α-D-Glcp-(1–2)-β-D-Fruf bond, and it was observed that sodium cation retention occurred on the heavier mass fragment of the two formed fragments, (e.g. di- or trisaccharide type vs monosaccharide type). Our results may help to better understand the fragmentation behavior of nonreducing oligosaccharides (as sodium adducts) in positive ESI mass spectrometry. Copyright © 2008 John Wiley & Sons, Ltd.
Biochimica Et Biophysica Acta-biomembranes, 2011
Three families of non-reducing trisaccharide fatty acid monoesters bearing C10 to C18 acyl chains... more Three families of non-reducing trisaccharide fatty acid monoesters bearing C10 to C18 acyl chains have been prepared by enzymatic synthesis in organic media. Their critical micelle concentrations, determined by dye-inclusion measurements, cover a broad range from mM to μM. The new compounds are capable of dissolving phospolipid vesicles and have been characterized as detergents in membrane biochemistry. In a comparative screening test for solubilizing/extraction capacity under native conditions of an ABC transporter as model integral membrane protein, the novel detergents have shown an excellent behavior similar to other commercial carbohydrate-based detergents and in some cases even better than the commonly employed β-dodecylmaltoside. The new detergents are also efficient at extracting membrane proteins from different lipidic environments and are likewise compatible with common protein affinity chromatography purification. These compounds may also be used for the preparation of (proteo)liposomes by detergent removal, not only using the classical method of detergent adsorption on hydrophobic resins but also by enzyme-catalyzed hydrolysis of the ester bond. These results show the new detergents as promising tools to expand the arsenal for membrane protein studies.► The new surfactants, regioisomerically pure, cover CMCs from μM to mM. ► They solubilize membrane proteins independently of their lipidic environment. ► They keep the activity of solubilized proteins better than classical detergents. ► They are fully compatible with chromatographic protein purification methods. ► Large unilamellar (proteo)liposomes are produced by their enzymatic hydrolysis.
Protein Expression and Purification, 2006
The mature lipI gene, encoding the lipase I from Galactomyces geotrichum BT107, was obtained by P... more The mature lipI gene, encoding the lipase I from Galactomyces geotrichum BT107, was obtained by PCR from genomic DNA, sequenced and cloned into a Pichia pastoris expression vector. Clones containing multiple copies of lipI integrated in their genome were analyzed to achieve high-level expression of the recombinant lipase I. One strain with four or more copies of the expression cassette was able to produce more than 200 mg/L of extracellular heterologous protein. The lipase I was partially purified using anion exchange chromatography and its activity on monounsaturated (triolein) and polyunsaturated (triEPA) triglycerides was analyzed by a novel HPLC-MS assay.
Tetrahedron, 2006
The enzymatic transesterification of several tri- and tetrasaccharides with vinyl laurate is desc... more The enzymatic transesterification of several tri- and tetrasaccharides with vinyl laurate is described. The lipases from Candida antarctica B (Novozym 435) and Thermomyces lanuginosus (Lipozyme TL IM) and the alkaline protease from Bacillus licheniformis (subtilisin Carlsberg) have been used with each carbohydrate to obtain different regioisomers. By using the sugars in their amorphous form, complete solubility is achieved in the reaction media (tert-butanol/pyridine mixtures for the lipases and pyridine for the protease) and high isolated yields of the corresponding monoesters are obtained. Good to excellent regioselectivity is observed for all the enzymes, showing a final complementary picture respect to the primary hydroxyls of the oligosaccharides studied.Graphical Abstract
Journal of Molecular Catalysis B-enzymatic, 2007
The enzymatic transesterification of non-reducing trisaccharides raffinose and melezitose with di... more The enzymatic transesterification of non-reducing trisaccharides raffinose and melezitose with divinyl adipate using two lipases (Novozym 435, Lipozyme TL IM) and the protease subtilisin Carlsberg is described. Polymerizable vinyladipoyl sugar esters were obtained with good to excellent selectivity and high yields. Monoesters prepared with lipase catalysis have also been used as substrates for a second acylation step catalyzed by the protease. Diesters were obtained with high selectivity although the isolated yields were slightly lower than those obtained for the corresponding monoester derivatives. It is important to note that the preferential acylation positions for subtilisin are the same in the free sugar and in the corresponding used monoesters for both trisaccharides. This two-step enzymatic approach allows regioselective control in the incorporation of the sugar inside the polymer structure.
Carbohydrate Research, 2007
A new LC–ESI-MS method for characterizing the regioisomeric distribution in carbohydrate monoeste... more A new LC–ESI-MS method for characterizing the regioisomeric distribution in carbohydrate monoesters with long-chain fatty acids is described. Sucrose monolaurate mixtures were used for development of the method. The surfactant nature and high polarity of these compounds make them appropriate analytes for being detected by electrospray-ionization mass spectrometry (ESI-MS). Despite the structural similarity of regioisomers, an excellent resolution of all regioisomers present in the different samples studied (sucrose monodecanoates, sucrose monolaurates, sucrose monopalmitates and melezitose monolaurates) is achieved. Reversed-phase liquid chromatography with isocratic acetonitrile–water mixtures was used for a proper separation of the analytes. Finally, the superiority of this chromatographic method for determining the regioselectivity in enzymatic carbohydrate acylation reactions, with respect to the typical methodology based on routine 13C NMR spectroscopy, is also discussed.
Biocatalysis and Biotransformation, 2008
... al. 1997; Ashok et al. 1999; Sharma et al. 2001). ... View larger version (37K), Figure 1. Co... more ... al. 1997; Ashok et al. 1999; Sharma et al. 2001). ... View larger version (37K), Figure 1. Construction of the lipI gene mutant library in a P. pastoris expression vector based on the site-specific recombination of phage lambda. Error ...
Cheminform, 2006
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.
Tetrahedron, 2006
Investigation of the acylation scope of carbohydrates by metalloprotease thermolysin immobilized ... more Investigation of the acylation scope of carbohydrates by metalloprotease thermolysin immobilized on Celite as biocatalyst has been carried out. The reactions were performed in DMSO, a good solvent for carbohydrates, where the enzyme has previously shown its activity in transesterifications of sucrose, maltose and maltose-containing oligosaccharides. Surprisingly, no reaction was observed for glucose or the glucose-containing disaccharides, trehalose and lactose. In contrast, laurate monoesters of several sucrose-containing tri- and tetrasaccharides were synthetized through a one step transesterification using vinyl laurate as the acylating agent. Enzyme regioselectivity was accurately determined by HPLC/MS and the structure of the main regioisomers was established by a combination of NMR experiments. The preferred position of acylation in all cases was the 2-OH of the α-d-glucopyranose moiety linked 1→2 to the β-d-fructofuranose unit. These results correlate with the regioselectivity observed in the case of the disaccharide sucrose. A general carbohydrate binding motif for catalysis by thermolysin is proposed.Graphical Abstract
Journal of Peptide Science, 2005
Conformational investigations have been undertaken on oligomers (dimers, tetramers, hexamers) of ... more Conformational investigations have been undertaken on oligomers (dimers, tetramers, hexamers) of five closely related oxetane-based dipeptide isosteres. All the oligomers were subjected to a range of studies by NMR, FT-IR and CD spectroscopy. The oligomers derived from methyl 2,4-anhydro-5-azido-3-O-tert-butyldimethylsilyl-5-deoxy-L-rhamnonate ‘monomer’ all exhibited evidence of ordered conformations in chloroform and 2,2,2-trifluoroethanol (TFE) solution. 5-Acetamido and N-methylamide derivatives of the L-rhamnonate ‘monomer’, along with a ‘dimer’ lacking silyl protection at C-3, were synthesized to ascertain the role of intramolecular interactions. This led to the conclusion that, for the L-rhamnonate oligomers, steric interactions govern the conformational preference observed. The equivalent silyl-protected D-lyxonate oligomers gave ordered CD spectra in TFE solution, but NMR and FT-IR spectroscopy in chloroform solution suggested an irregular, non-hydrogen bonded system. The remaining silyl-protected 6-deoxy-L-altronate, 6-deoxy-D-gulonate and D-fuconate oligomers appear to be characterized by their lack of ordered conformation in TFE and chloroform solution. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.
Journal of Peptide Science, 2005
Routes to oligomers (dimers, tetramers, hexamers) of five oxetane-based dipeptide isosteres have ... more Routes to oligomers (dimers, tetramers, hexamers) of five oxetane-based dipeptide isosteres have been established. Methyl 2,4-anhydro-5-azido-5-deoxy-L-rhamnonate ‘monomer’ led, by coupling the corresponding carboxylic acid and amine, to a ‘dimer’. Reverse-aldol ring-opening occurred on attempted saponification of the dimer, so all further oligomerization was performed using TBDMS C-3 hydroxyl protection. The silyl protected L-rhamnonate monomer led in turn to the dimer (via the monomer acid and amine), the tetramer (via the dimer acid and amine) and finally the hexamer (via the tetramer acid and dimer amine). In each case the acids were obtained through saponification of the respective methyl esters and the amines were obtained by hydrogenation of the azides; coupling was TBTU-mediated. Essentially the same strategy was employed on equivalent D-lyxonate, 6-deoxy-L-altronate, 6-deoxy-D-gulonate and D-fuconate dipeptide isosteres to give the respective dimers, tetramers and hexamers. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.
Chemical Communications, 2009
Journal of Mass Spectrometry, 2008
Structural characterization and differentiation of a novel group of regioisomeric monolaurate est... more Structural characterization and differentiation of a novel group of regioisomeric monolaurate esters of the nonreducing trisaccharides raffinose and melezitose, and the nonreducing tetrasaccharide stachyose has been obtained using positive electrospray ionization (ESI) mass spectrometry with in-source fragmentation. The surfactant nature and high polarity of these compounds make them appropriate analytes for being studied by conventional ESI-MS. The position of the acyl chain in each particular regioisomer has been used as a reporter group that allows unambiguous rationalization of the fragmentation routes of the corresponding natriated molecular ions [M + Na]+. In all cases, the main fragment ions were produced through cleavage of the glycosidic bond involving two anomeric carbons, the C-1′ and C-2″ of the α-D-Glcp-(1–2)-β-D-Fruf bond, and it was observed that sodium cation retention occurred on the heavier mass fragment of the two formed fragments, (e.g. di- or trisaccharide type vs monosaccharide type). Our results may help to better understand the fragmentation behavior of nonreducing oligosaccharides (as sodium adducts) in positive ESI mass spectrometry. Copyright © 2008 John Wiley & Sons, Ltd.
Biochimica Et Biophysica Acta-biomembranes, 2011
Three families of non-reducing trisaccharide fatty acid monoesters bearing C10 to C18 acyl chains... more Three families of non-reducing trisaccharide fatty acid monoesters bearing C10 to C18 acyl chains have been prepared by enzymatic synthesis in organic media. Their critical micelle concentrations, determined by dye-inclusion measurements, cover a broad range from mM to μM. The new compounds are capable of dissolving phospolipid vesicles and have been characterized as detergents in membrane biochemistry. In a comparative screening test for solubilizing/extraction capacity under native conditions of an ABC transporter as model integral membrane protein, the novel detergents have shown an excellent behavior similar to other commercial carbohydrate-based detergents and in some cases even better than the commonly employed β-dodecylmaltoside. The new detergents are also efficient at extracting membrane proteins from different lipidic environments and are likewise compatible with common protein affinity chromatography purification. These compounds may also be used for the preparation of (proteo)liposomes by detergent removal, not only using the classical method of detergent adsorption on hydrophobic resins but also by enzyme-catalyzed hydrolysis of the ester bond. These results show the new detergents as promising tools to expand the arsenal for membrane protein studies.► The new surfactants, regioisomerically pure, cover CMCs from μM to mM. ► They solubilize membrane proteins independently of their lipidic environment. ► They keep the activity of solubilized proteins better than classical detergents. ► They are fully compatible with chromatographic protein purification methods. ► Large unilamellar (proteo)liposomes are produced by their enzymatic hydrolysis.
Protein Expression and Purification, 2006
The mature lipI gene, encoding the lipase I from Galactomyces geotrichum BT107, was obtained by P... more The mature lipI gene, encoding the lipase I from Galactomyces geotrichum BT107, was obtained by PCR from genomic DNA, sequenced and cloned into a Pichia pastoris expression vector. Clones containing multiple copies of lipI integrated in their genome were analyzed to achieve high-level expression of the recombinant lipase I. One strain with four or more copies of the expression cassette was able to produce more than 200 mg/L of extracellular heterologous protein. The lipase I was partially purified using anion exchange chromatography and its activity on monounsaturated (triolein) and polyunsaturated (triEPA) triglycerides was analyzed by a novel HPLC-MS assay.
Tetrahedron, 2006
The enzymatic transesterification of several tri- and tetrasaccharides with vinyl laurate is desc... more The enzymatic transesterification of several tri- and tetrasaccharides with vinyl laurate is described. The lipases from Candida antarctica B (Novozym 435) and Thermomyces lanuginosus (Lipozyme TL IM) and the alkaline protease from Bacillus licheniformis (subtilisin Carlsberg) have been used with each carbohydrate to obtain different regioisomers. By using the sugars in their amorphous form, complete solubility is achieved in the reaction media (tert-butanol/pyridine mixtures for the lipases and pyridine for the protease) and high isolated yields of the corresponding monoesters are obtained. Good to excellent regioselectivity is observed for all the enzymes, showing a final complementary picture respect to the primary hydroxyls of the oligosaccharides studied.Graphical Abstract
Journal of Molecular Catalysis B-enzymatic, 2007
The enzymatic transesterification of non-reducing trisaccharides raffinose and melezitose with di... more The enzymatic transesterification of non-reducing trisaccharides raffinose and melezitose with divinyl adipate using two lipases (Novozym 435, Lipozyme TL IM) and the protease subtilisin Carlsberg is described. Polymerizable vinyladipoyl sugar esters were obtained with good to excellent selectivity and high yields. Monoesters prepared with lipase catalysis have also been used as substrates for a second acylation step catalyzed by the protease. Diesters were obtained with high selectivity although the isolated yields were slightly lower than those obtained for the corresponding monoester derivatives. It is important to note that the preferential acylation positions for subtilisin are the same in the free sugar and in the corresponding used monoesters for both trisaccharides. This two-step enzymatic approach allows regioselective control in the incorporation of the sugar inside the polymer structure.
Carbohydrate Research, 2007
A new LC–ESI-MS method for characterizing the regioisomeric distribution in carbohydrate monoeste... more A new LC–ESI-MS method for characterizing the regioisomeric distribution in carbohydrate monoesters with long-chain fatty acids is described. Sucrose monolaurate mixtures were used for development of the method. The surfactant nature and high polarity of these compounds make them appropriate analytes for being detected by electrospray-ionization mass spectrometry (ESI-MS). Despite the structural similarity of regioisomers, an excellent resolution of all regioisomers present in the different samples studied (sucrose monodecanoates, sucrose monolaurates, sucrose monopalmitates and melezitose monolaurates) is achieved. Reversed-phase liquid chromatography with isocratic acetonitrile–water mixtures was used for a proper separation of the analytes. Finally, the superiority of this chromatographic method for determining the regioselectivity in enzymatic carbohydrate acylation reactions, with respect to the typical methodology based on routine 13C NMR spectroscopy, is also discussed.
Biocatalysis and Biotransformation, 2008
... al. 1997; Ashok et al. 1999; Sharma et al. 2001). ... View larger version (37K), Figure 1. Co... more ... al. 1997; Ashok et al. 1999; Sharma et al. 2001). ... View larger version (37K), Figure 1. Construction of the lipI gene mutant library in a P. pastoris expression vector based on the site-specific recombination of phage lambda. Error ...
Cheminform, 2006
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.
Tetrahedron, 2006
Investigation of the acylation scope of carbohydrates by metalloprotease thermolysin immobilized ... more Investigation of the acylation scope of carbohydrates by metalloprotease thermolysin immobilized on Celite as biocatalyst has been carried out. The reactions were performed in DMSO, a good solvent for carbohydrates, where the enzyme has previously shown its activity in transesterifications of sucrose, maltose and maltose-containing oligosaccharides. Surprisingly, no reaction was observed for glucose or the glucose-containing disaccharides, trehalose and lactose. In contrast, laurate monoesters of several sucrose-containing tri- and tetrasaccharides were synthetized through a one step transesterification using vinyl laurate as the acylating agent. Enzyme regioselectivity was accurately determined by HPLC/MS and the structure of the main regioisomers was established by a combination of NMR experiments. The preferred position of acylation in all cases was the 2-OH of the α-d-glucopyranose moiety linked 1→2 to the β-d-fructofuranose unit. These results correlate with the regioselectivity observed in the case of the disaccharide sucrose. A general carbohydrate binding motif for catalysis by thermolysin is proposed.Graphical Abstract