Cyclopropanated iminosugars have a locked conformation that may enhance the inhibitory activity a... more Cyclopropanated iminosugars have a locked conformation that may enhance the inhibitory activity and selectivity against different glycosidases. We show the synthesis of new cyclopropane-containing piperidines bearing five stereogenic centers from natural amino acids l-serine and l-alanine. Those prepared from the latter amino acid may mimic l-fucose, a natural-occurring monosaccharide involved in many molecular recognition events. Final compounds prepared from l-serine bear S configurations on the C5 position. The synthesis involved a stereoselective cyclopropanation reaction of an α,β-unsaturated piperidone, which was prepared through a ring-closing metathesis. The final compounds were tested as possible inhibitors of different glycosidases. The results, although, in general, with low inhibition activity, showed selectivity, depending on the compound and enzyme, and in some cases, an unexpected activity enhancement was observed.
Combining genetic, biochemical and computational approaches, we elucidated the molecular mechanis... more Combining genetic, biochemical and computational approaches, we elucidated the molecular mechanisms underlying the recognition ofListeriawall teichoic acid by bacteriophage-encoded SH3b repeats.
Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bac... more Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bacteria via their cell wall-binding domains (CBDs). The molecular basis for selective recognition of surface carbohydrate ligands by CBDs remains elusive. Here, we describe, in atomic detail, the interaction between the Listeria phage endolysin domain CBD500 and its cell wall teichoic acid (WTA) ligands. We show that 3’ O-acetylated GlcNAc residues integrated into the WTA polymer chain are the key epitope recognized by a CBD binding cavity located at the interface of tandem copies of beta-barrel, pseudo-symmetric SH3b-like repeats. This cavity consists of multiple aromatic residues making extensive interactions with two GlcNAc acetyl groups via hydrogen bonds and van der Waals contacts, while permitting the docking of the diastereomorphic ligands. The multidisciplinary approach described here delineates a previously unknown recognition mechanism by which a phage endolysin specifically reco...
Background: The interest for finding novel b-glucosidases that can improve the yields to produce ... more Background: The interest for finding novel b-glucosidases that can improve the yields to produce second-generation (2G) biofuels is still very high. One of the most desired features for these enzymes is glucose tolerance, which enables their optimal activity under high-glucose concentrations. Besides, there is an additional focus of attention on finding novel enzymatic alternatives for glycoside synthesis, for which a mutated version of glycosidases, named glycosynthases, has gained much interest in recent years. Results: In this work, a glucotolerant β-glucosidase (BGL-1) from the ascomycete fungus Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris , purified, and characterized. The enzyme showed good efficiency on p NPG ( K m =3.36 ± 0.7 mM, k cat =898.31 s -1 ), but its activity on cellooligosaccharides, the natural substrates of these enzymes, was much lower, which could limit its exploitation in lignocellulose degradation applications. Interestingly, w...
Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the ide... more Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease w...
Glycan‐protein interactions play an important role in a broad range of physiological processes, r... more Glycan‐protein interactions play an important role in a broad range of physiological processes, raising interest to elucidate the structural interplay. Yet, their dynamic nature limits the analysis by crystallography, whereas NMR spectroscopy suffers from the low 1H dispersion of glycans. Therefore, their sparse fluorination and NMR screening by 1D Saturation Transfer Difference with relay to 19F (STDreF) was previously proposed to exploit the superior dispersion in 19F NMR spectroscopy. A new 2D STD‐TOCSYreF experiment is presented here that enables comprehensive epitope mapping of fluorinated glycans by combining the spectral resolution of 19F with the spatial resolution and coverage of 1H. For an illustration, the 2‐deoxy‐2‐fluoro derivative of the N‐glycan core trimannoside was synthesised and its recognition of Pisum sativum agglutinin by either of the two terminal mannose residues was confirmed. Going beyond the crystallographic information, the 2D STD‐TOCSYreF spectrum moreov...
Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenov... more Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple β-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutag...
2,5-Furandicarboxylic acid is a renewable building block for the production of polyfurandicarboxy... more 2,5-Furandicarboxylic acid is a renewable building block for the production of polyfurandicarboxylates, which are biodegradable polyesters expected to substitute their classical counterparts derived from fossil resources. It may be produced from bio-based 5-hydroxymethylfurfural or 5-methoxymethylfurfural, both obtained by the acidic dehydration of biomass-derived fructose. 5-Methoxymethylfurfural, which is produced in the presence of methanol, generates less by-products and exhibits better storage stability than 5-hydroxymethylfurfural being, therefore, the industrial substrate of choice. In this work, an enzymatic cascade involving three fungal oxidoreductases has been developed for the production of 2,5-furandicarboxylic acid from 5-methoxymethylfurfural. Aryl-alcohol oxidase and unspecific peroxygenase act on 5-methoxymethylfurfural and its partially oxidized derivatives yielding 2,5-furandicarboxylic acid, as well as methanol as a by-product. Methanol oxidase takes advantage of...
Glycans play a key role as recognition elements in the communication of cells and other organisms... more Glycans play a key role as recognition elements in the communication of cells and other organisms. Thus, the analysis of carbohydrate-protein interactions has gained significant importance. In particular, nuclear magnetic resonance (NMR) techniques are considered powerful tools to detect relevant features in the interaction between sugars and their natural receptors. Here, we present the results obtained in the study on the molecular recognition of different mannose-containing glycans by Pisum sativum agglutinin. NMR experiments supported by Corcema-ST analysis, isothermal titration calorimetry (ITC) experiments, and molecular dynamics (MD) protocols have been successfully applied to unmask important binding features and especially to determine how a remote branching substituent significantly alters the binding mode of the sugar entity. These results highlight the key influence of common structural modifications in natural glycans on molecular recognition processes and underscore th...
Journal of the American Chemical Society, Jul 27, 2016
Peptidoglycan is a fundamental structure for most bacteria. It contributes to the cell morphology... more Peptidoglycan is a fundamental structure for most bacteria. It contributes to the cell morphology and provides cell wall integrity against environmental insults. While several studies have reported a significant degree of variability in the chemical composition and organization of peptidoglycan in the domain Bacteria, the real diversity of this polymer is far from fully explored. This work exploits rapid ultraperformance liquid chromatography and multivariate data analysis to uncover peptidoglycan chemical diversity in the Class Alphaproteobacteria, a group of Gram negative bacteria that are highly heterogeneous in terms of metabolism, morphology and life-styles. Indeed, chemometric analyses revealed novel peptidoglycan structures conserved in Acetobacteria: amidation at the α-(l)-carboxyl of meso-diaminopimelic acid and the presence of muropeptides cross-linked by (1-3) l-Ala-d-(meso)-diaminopimelate cross-links. Both structures are growth-controlled modifications that influence se...
Chemistry (Weinheim an der Bergstrasse, Germany), Jan 10, 2015
Sugar function, structure and dynamics are intricately correlated. Ring flexibility is intrinsica... more Sugar function, structure and dynamics are intricately correlated. Ring flexibility is intrinsically related to biological activity; actually plasticity in L-iduronic rings modulates their interactions with biological receptors. However, the access to the experimental values of the energy barriers and free-energy difference for conformer interconversion in water solution has been elusive. Here, a new generation of fluorine-containing glycomimetics is presented. We have applied a combination of organic synthesis, NMR spectroscopy and computational methods to investigate the conformational behaviour of idose- and glucose-like rings. We have used low-temperature NMR spectroscopic experiments to slow down the conformational exchange of the idose-like rings. Under these conditions, the exchange rate becomes slow in the (19) F NMR spectroscopic chemical shift timescale and allows shedding light on the thermodynamic and kinetic features of the equilibrium. Despite the minimal structural di...
Cyclopropanated iminosugars have a locked conformation that may enhance the inhibitory activity a... more Cyclopropanated iminosugars have a locked conformation that may enhance the inhibitory activity and selectivity against different glycosidases. We show the synthesis of new cyclopropane-containing piperidines bearing five stereogenic centers from natural amino acids l-serine and l-alanine. Those prepared from the latter amino acid may mimic l-fucose, a natural-occurring monosaccharide involved in many molecular recognition events. Final compounds prepared from l-serine bear S configurations on the C5 position. The synthesis involved a stereoselective cyclopropanation reaction of an α,β-unsaturated piperidone, which was prepared through a ring-closing metathesis. The final compounds were tested as possible inhibitors of different glycosidases. The results, although, in general, with low inhibition activity, showed selectivity, depending on the compound and enzyme, and in some cases, an unexpected activity enhancement was observed.
Combining genetic, biochemical and computational approaches, we elucidated the molecular mechanis... more Combining genetic, biochemical and computational approaches, we elucidated the molecular mechanisms underlying the recognition ofListeriawall teichoic acid by bacteriophage-encoded SH3b repeats.
Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bac... more Endolysins are bacteriophage-encoded peptidoglycan hydrolases targeting the cell wall of host bacteria via their cell wall-binding domains (CBDs). The molecular basis for selective recognition of surface carbohydrate ligands by CBDs remains elusive. Here, we describe, in atomic detail, the interaction between the Listeria phage endolysin domain CBD500 and its cell wall teichoic acid (WTA) ligands. We show that 3’ O-acetylated GlcNAc residues integrated into the WTA polymer chain are the key epitope recognized by a CBD binding cavity located at the interface of tandem copies of beta-barrel, pseudo-symmetric SH3b-like repeats. This cavity consists of multiple aromatic residues making extensive interactions with two GlcNAc acetyl groups via hydrogen bonds and van der Waals contacts, while permitting the docking of the diastereomorphic ligands. The multidisciplinary approach described here delineates a previously unknown recognition mechanism by which a phage endolysin specifically reco...
Background: The interest for finding novel b-glucosidases that can improve the yields to produce ... more Background: The interest for finding novel b-glucosidases that can improve the yields to produce second-generation (2G) biofuels is still very high. One of the most desired features for these enzymes is glucose tolerance, which enables their optimal activity under high-glucose concentrations. Besides, there is an additional focus of attention on finding novel enzymatic alternatives for glycoside synthesis, for which a mutated version of glycosidases, named glycosynthases, has gained much interest in recent years. Results: In this work, a glucotolerant β-glucosidase (BGL-1) from the ascomycete fungus Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris , purified, and characterized. The enzyme showed good efficiency on p NPG ( K m =3.36 ± 0.7 mM, k cat =898.31 s -1 ), but its activity on cellooligosaccharides, the natural substrates of these enzymes, was much lower, which could limit its exploitation in lignocellulose degradation applications. Interestingly, w...
Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the ide... more Dynamic combinatorial chemistry (DCC) has proven its potential in drug discovery speeding the identification of modulators of biological targets. However, the exchange chemistries typically take place under specific reaction conditions, with limited tools capable of operating under physiological parameters. Here we report a catalyzed protein-directed DCC working at low temperatures that allows the calcium sensor NCS-1 to find the best ligands in situ. Ultrafast NMR identifies the reaction intermediates of the acylhydrazone exchange, tracing the molecular assemblies and getting a real-time insight into the essence of DCC processes at physiological pH. Additionally, NMR, X-ray crystallography and computational methods are employed to elucidate structural and mechanistic aspects of the molecular recognition event. The DCC approach leads us to the identification of a compound stabilizing the NCS-1/Ric8a complex and whose therapeutic potential is proven in a Drosophila model of disease w...
Glycan‐protein interactions play an important role in a broad range of physiological processes, r... more Glycan‐protein interactions play an important role in a broad range of physiological processes, raising interest to elucidate the structural interplay. Yet, their dynamic nature limits the analysis by crystallography, whereas NMR spectroscopy suffers from the low 1H dispersion of glycans. Therefore, their sparse fluorination and NMR screening by 1D Saturation Transfer Difference with relay to 19F (STDreF) was previously proposed to exploit the superior dispersion in 19F NMR spectroscopy. A new 2D STD‐TOCSYreF experiment is presented here that enables comprehensive epitope mapping of fluorinated glycans by combining the spectral resolution of 19F with the spatial resolution and coverage of 1H. For an illustration, the 2‐deoxy‐2‐fluoro derivative of the N‐glycan core trimannoside was synthesised and its recognition of Pisum sativum agglutinin by either of the two terminal mannose residues was confirmed. Going beyond the crystallographic information, the 2D STD‐TOCSYreF spectrum moreov...
Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenov... more Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple β-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutag...
2,5-Furandicarboxylic acid is a renewable building block for the production of polyfurandicarboxy... more 2,5-Furandicarboxylic acid is a renewable building block for the production of polyfurandicarboxylates, which are biodegradable polyesters expected to substitute their classical counterparts derived from fossil resources. It may be produced from bio-based 5-hydroxymethylfurfural or 5-methoxymethylfurfural, both obtained by the acidic dehydration of biomass-derived fructose. 5-Methoxymethylfurfural, which is produced in the presence of methanol, generates less by-products and exhibits better storage stability than 5-hydroxymethylfurfural being, therefore, the industrial substrate of choice. In this work, an enzymatic cascade involving three fungal oxidoreductases has been developed for the production of 2,5-furandicarboxylic acid from 5-methoxymethylfurfural. Aryl-alcohol oxidase and unspecific peroxygenase act on 5-methoxymethylfurfural and its partially oxidized derivatives yielding 2,5-furandicarboxylic acid, as well as methanol as a by-product. Methanol oxidase takes advantage of...
Glycans play a key role as recognition elements in the communication of cells and other organisms... more Glycans play a key role as recognition elements in the communication of cells and other organisms. Thus, the analysis of carbohydrate-protein interactions has gained significant importance. In particular, nuclear magnetic resonance (NMR) techniques are considered powerful tools to detect relevant features in the interaction between sugars and their natural receptors. Here, we present the results obtained in the study on the molecular recognition of different mannose-containing glycans by Pisum sativum agglutinin. NMR experiments supported by Corcema-ST analysis, isothermal titration calorimetry (ITC) experiments, and molecular dynamics (MD) protocols have been successfully applied to unmask important binding features and especially to determine how a remote branching substituent significantly alters the binding mode of the sugar entity. These results highlight the key influence of common structural modifications in natural glycans on molecular recognition processes and underscore th...
Journal of the American Chemical Society, Jul 27, 2016
Peptidoglycan is a fundamental structure for most bacteria. It contributes to the cell morphology... more Peptidoglycan is a fundamental structure for most bacteria. It contributes to the cell morphology and provides cell wall integrity against environmental insults. While several studies have reported a significant degree of variability in the chemical composition and organization of peptidoglycan in the domain Bacteria, the real diversity of this polymer is far from fully explored. This work exploits rapid ultraperformance liquid chromatography and multivariate data analysis to uncover peptidoglycan chemical diversity in the Class Alphaproteobacteria, a group of Gram negative bacteria that are highly heterogeneous in terms of metabolism, morphology and life-styles. Indeed, chemometric analyses revealed novel peptidoglycan structures conserved in Acetobacteria: amidation at the α-(l)-carboxyl of meso-diaminopimelic acid and the presence of muropeptides cross-linked by (1-3) l-Ala-d-(meso)-diaminopimelate cross-links. Both structures are growth-controlled modifications that influence se...
Chemistry (Weinheim an der Bergstrasse, Germany), Jan 10, 2015
Sugar function, structure and dynamics are intricately correlated. Ring flexibility is intrinsica... more Sugar function, structure and dynamics are intricately correlated. Ring flexibility is intrinsically related to biological activity; actually plasticity in L-iduronic rings modulates their interactions with biological receptors. However, the access to the experimental values of the energy barriers and free-energy difference for conformer interconversion in water solution has been elusive. Here, a new generation of fluorine-containing glycomimetics is presented. We have applied a combination of organic synthesis, NMR spectroscopy and computational methods to investigate the conformational behaviour of idose- and glucose-like rings. We have used low-temperature NMR spectroscopic experiments to slow down the conformational exchange of the idose-like rings. Under these conditions, the exchange rate becomes slow in the (19) F NMR spectroscopic chemical shift timescale and allows shedding light on the thermodynamic and kinetic features of the equilibrium. Despite the minimal structural di...
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Papers by F Javier Cañada