Publications by authors named "Jesús Jiménez-Barbero"

375 Publications

Galectin-4 N-Terminal Domain: Binding Preferences Toward A and B Antigens With Different Peripheral Core Presentations.

Front Chem 2021 21;9:664097. Epub 2021 Apr 21.

CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Derio, Spain.

The tandem-repeat Galectin-4 (Gal-4) contains two different domains covalently linked through a short flexible peptide. Both domains have been shown to bind preferentially to A and B histo blood group antigens with different affinities, although the binding details are not yet available. The biological relevance of these associations is unknown, although it could be related to its attributed role in pathogen recognition. The presentation of A and B histo blood group antigens in terms of peripheral core structures differs among tissues and from that of the antigen-mimicking structures produced by pathogens. Herein, the binding of the N-terminal domain of Gal-4 toward a group of differently presented A and B oligosaccharide antigens in solution has been studied through a combination of NMR, isothermal titration calorimetry (ITC), and molecular modeling. The data presented in this paper allow the identification of the specific effects that subtle chemical modifications within this antigenic family have in the binding to the N-terminal domain of Gal-4 in terms of affinity and intermolecular interactions, providing a structural-based rationale for the observed trend in the binding preferences.
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http://dx.doi.org/10.3389/fchem.2021.664097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097242PMC
April 2021

Glycosyl Oxocarbenium Ions: Structure, Conformation, Reactivity, and Interactions.

Acc Chem Res 2021 Apr 30. Epub 2021 Apr 30.

CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Building 800, 48160 Derio, Spain.

ConspectusCarbohydrates (glycans, saccharides, and sugars) are essential molecules in all domains of life. Research on glycoscience spans from chemistry to biomedicine, including material science and biotechnology. Access to pure and well-defined complex glycans using synthetic methods depends on the success of the employed glycosylation reaction. In most cases, the mechanism of the glycosylation reaction is believed to involve the oxocarbenium ion. Understanding the structure, conformation, reactivity, and interactions of this glycosyl cation is essential to predict the outcome of the reaction. In this Account, building on our contributions on this topic, we discuss the theoretical and experimental approaches that have been employed to decipher the key features of glycosyl cations, from their structures to their interactions and reactivity.We also highlight that, from a chemical perspective, the glycosylation reaction can be described as a continuum, from unimolecular S1 with naked oxocarbenium cations as intermediates to bimolecular S2-type mechanisms, which involve the key role of counterions and donors. All these factors should be considered and are discussed herein. The importance of dissociative mechanisms (involving contact ion pairs, solvent-separated ion pairs, solvent-equilibrated ion pairs) with bimolecular features in most reactions is also highlighted.The role of theoretical calculations to predict the conformation, dynamics, and reactivity of the oxocarbenium ion is also discussed, highlighting the advances in this field that now allow access to the conformational preferences of a variety of oxocarbenium ions and their reactivities under S1-like conditions.Specifically, the ground-breaking use of superacids to generate these cations is emphasized, since it has permitted characterization of the structure and conformation of a variety of glycosyl oxocarbenium ions in superacid solution by NMR spectroscopy.We also pay special attention to the reactivity of these glycosyl ions, which depends on the conditions, including the counterions, the possible intra- or intermolecular participation of functional groups that may stabilize the cation and the chemical nature of the acceptor, either weak or strong nucleophile. We discuss recent investigations from different experimental perspectives, which identified the involved ionic intermediates, estimating their lifetimes and reactivities and studying their interactions with other molecules. In this context, we also emphasize the relationship between the chemical methods that can be employed to modulate the sensitivity of glycosyl cations and the way in which glycosyl modifying enzymes (glycosyl hydrolases and transferases) build and cleave glycosidic linkages in nature. This comparison provides inspiration on the use of molecules that regulate the stability and reactivity of glycosyl cations.
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http://dx.doi.org/10.1021/acs.accounts.1c00021DOI Listing
April 2021

Exploration of Galectin Ligands Displayed on Gram-Negative Respiratory Bacterial Pathogens with Different Cell Surface Architectures.

Biomolecules 2021 Apr 18;11(4). Epub 2021 Apr 18.

Instituto de Química Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain.

Galectins bind various pathogens through recognition of distinct carbohydrate structures. In this work, we examined the binding of four human galectins to the Gram-negative bacteria (Kpn) and non-typeable (NTHi), which display different surface glycans. In particular, Kpn cells are covered by a polysaccharide capsule and display an O-chain-containing lipopolysaccharide (LPS), whereas NTHi is not capsulated and its LPS, termed lipooligosacccharide (LOS), does not contain O-chain. Binding assays to microarray-printed bacteria revealed that galectins-3, -4, and -8, but not galectin-1, bind to Kpn and NTHi cells, and confocal microscopy attested binding to bacterial cells in suspension. The three galectins bound to array-printed Kpn LPS. Moreover, analysis of galectin binding to mutant Kpn cells evidenced that the O-chain is the docking point for galectins on wild type Kpn. Galectins-3, -4, and -8 also bound the NTHi LOS. Microarray-assisted comparison of the binding to full-length and truncated LOSs, as well as to wild type and mutant cells, supported LOS involvement in galectin binding to NTHi. However, deletion of the entire LOS oligosaccharide chain actually increased binding to NTHi cells, indicating the availability of other ligands on the bacterial surface, as similarly inferred for Kpn cells devoid of both O-chain and capsule. Altogether, the results illustrate galectins' versatility for recognizing different bacterial structures, and point out the occurrence of so far overlooked galectin ligands on bacterial surfaces.
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http://dx.doi.org/10.3390/biom11040595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074145PMC
April 2021

Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins.

Glycobiology 2021 Apr 28. Epub 2021 Apr 28.

Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 du CNRS et Université de Lille, 50 Avenue Halley, 59650 Villeneuve d'Ascq, France.

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognise Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray. Among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose, and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a non-substituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.
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http://dx.doi.org/10.1093/glycob/cwab027DOI Listing
April 2021

Structural Insights into the Molecular Recognition Mechanism of the Cancer and Pathogenic Epitope, LacdiNAc by Immune-Related Lectins.

Chemistry 2021 Apr 7. Epub 2021 Apr 7.

UCIBIO, REQUIMTE, Departamento de Quimica, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516, Caparica, Portugal.

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcβ1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galβ1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.
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http://dx.doi.org/10.1002/chem.202100800DOI Listing
April 2021

Synthesis and chelation study of a fluoroionophore and a glycopeptide based on an aza crown iminosugar structure.

Carbohydr Res 2021 Mar 10;501:108258. Epub 2021 Feb 10.

Université de Poitiers, IC2MP, UMR CNRS 7285, Equipe "Synthèse Organique", Groupe Glycochimie, 4 rue Michel Brunet, 86073, Poitiers Cedex 9, France. Electronic address:

Capitalizing on a recently reported iminosugar-based aza-crown (ISAC) accessed by a double Staudinger azaWittig coupling reaction, we have expanded the structural diversity of this new family of sweet cyclam analogs. Replacement of the two secondary amines linking the iminosugar units by two amide bonds obtained a cyclodimerization by with BOP and DIPEA led to a macrocycle that did not demonstrate efficient Zn chelation unlike the parent ISAC. Introduction of two pyrene moieties on the secondary amines of the parent ISAC yielded a new fluoroionophore that selectively binds Hg in methanol.
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http://dx.doi.org/10.1016/j.carres.2021.108258DOI Listing
March 2021

Bacterial polysaccharides: conformation, dynamics and molecular recognition by antibodies.

Drug Discov Today Technol 2020 Dec 11;35-36:1-11. Epub 2020 Oct 11.

CIC bioGUNE, Basque Research Technology Alliance, BRTA, Bizkaia Technology Park, Building 800, 48160 Derio, Spain; Ikerbasque, Basque Foundation for Science, 48009 Bilbao, Spain; Department Organic Chemistry II, Faculty of Science and technology, UPV-EHU, 48940 Leioa, Spain.

Bacterial infections are the cause of different severe health conditions and new therapies to combat these pathogens have been widely investigated. Carbohydrates, being complex structures covering the surface of bacteria, are considered relevant targets for antibody and vaccine development. The biological activities in pathogenesis of bacterial capsular polysaccharides and lipopolisaccharides and their unique structures have boosted the study of the minimal antigenic binding epitopes and the structural details of antibody-carbohydrate recognition. This review describes the most recent advances on the field, examining the structure, conformation and dynamics of relevant bacterial carbohydrates and their complexes with antibodies. The understanding of key factors governing the recognition process is fundamental for the progress toward the development of specific and efficient bacterial therapeutics.
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http://dx.doi.org/10.1016/j.ddtec.2020.08.002DOI Listing
December 2020

Current Status on Therapeutic Molecules Targeting Siglec Receptors.

Cells 2020 12 15;9(12). Epub 2020 Dec 15.

Chemical Glycobiology Laboratory, CIC bioGUNE, Basque Research and Technology Alliance, BRTA, Bizkaia Technology Park, Building 800, 48160 Derio, Spain.

The sialic acid-binding immunoglobulin-type of lectins (Siglecs) are receptors that recognize sialic acid-containing glycans. In the majority of the cases, Siglecs are expressed on immune cells and play a critical role in regulating immune cell signaling. Over the years, it has been shown that the sialic acid-Siglec axis participates in immunological homeostasis, and that any imbalance can trigger different pathologies, such as autoimmune diseases or cancer. For all this, different therapeutics have been developed that bind to Siglecs, either based on antibodies or being smaller molecules. In this review, we briefly introduce the Siglec family and we compile a description of glycan-based molecules and antibody-based therapies (including CAR-T and bispecific antibodies) that have been designed to therapeutically targeting Siglecs.
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http://dx.doi.org/10.3390/cells9122691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7765293PMC
December 2020

Fluorinated Carbohydrates as Lectin Ligands: Simultaneous Screening of a Monosaccharide Library and Chemical Mapping by F NMR Spectroscopy.

J Org Chem 2020 12 1;85(24):16072-16081. Epub 2020 Dec 1.

Centro de Investigaciones Biológicas Margarita Salas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

Molecular recognition of carbohydrates is a key step in essential biological processes. Carbohydrate receptors can distinguish monosaccharides even if they only differ in a single aspect of the orientation of the hydroxyl groups or harbor subtle chemical modifications. Hydroxyl-by-fluorine substitution has proven its merits for chemically mapping the importance of hydroxyl groups in carbohydrate-receptor interactions. F NMR spectroscopy could thus be adapted to allow contact mapping together with screening in compound mixtures. Using a library of fluorinated glucose (Glc), mannose (Man), and galactose (Gal) derived by systematically exchanging every hydroxyl group by a fluorine atom, we developed a strategy combining chemical mapping and F NMR T filtering-based screening. By testing this strategy on the proof-of-principle level with a library of 13 fluorinated monosaccharides to a set of three carbohydrate receptors of diverse origin, i.e. the human macrophage galactose-type lectin, a plant lectin, agglutinin, and the bacterial Gal-/Glc-binding protein from , it became possible to simultaneously define their monosaccharide selectivity and identify the essential hydroxyls for interaction.
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http://dx.doi.org/10.1021/acs.joc.0c01830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773211PMC
December 2020

Structure of a protective epitope reveals the importance of acetylation of serogroup A capsular polysaccharide.

Proc Natl Acad Sci U S A 2020 11 6;117(47):29795-29802. Epub 2020 Nov 6.

Research and Development Centre, GlaxoSmithKline (GSK), 53100 Siena, Italy;

Meningococcal meningitis remains a substantial cause of mortality and morbidity worldwide. Until recently, countries in the African meningitis belt were susceptible to devastating outbreaks, largely attributed to serogroup A (MenA). Vaccination with glycoconjugates of MenA capsular polysaccharide led to an almost complete elimination of MenA clinical cases. To understand the molecular basis of vaccine-induced protection, we generated a panel of oligosaccharide fragments of different lengths and tested them with polyclonal and monoclonal antibodies by inhibition enzyme-linked immunosorbent assay, surface plasmon resonance, and competitive human serum bactericidal assay, which is a surrogate for protection. The epitope was shown to optimize between three and six repeating units and to be -acetylated. The molecular interactions between a protective monoclonal antibody and a MenA capsular polysaccharide fragment were further elucidated at the atomic level by saturation transfer difference NMR spectroscopy and X-ray crystallography. The epitope consists of a trisaccharide anchored to the antibody via the - and -acetyl moieties through either H-bonding or CH-π interactions. In silico docking showed that 3--acetylation of the upstream residue is essential for antibody binding, while -acetate could be equally accommodated at three and four positions of the other two residues. These results shed light on the mechanism of action of current MenA vaccines and provide a foundation for the rational design of improved therapies.
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http://dx.doi.org/10.1073/pnas.2011385117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703565PMC
November 2020

Targeting the CRD F-face of Human Galectin-3 and Allosterically Modulating Glycan Binding by Angiostatic PTX008 and a Structurally Optimized Derivative.

ChemMedChem 2021 Feb 1;16(4):713-723. Epub 2020 Dec 1.

Department of Biochemistry, Molecular Biology & Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA.

Calix[4]arene PTX008 is an angiostatic agent that inhibits tumor growth in mice by binding to galectin-1, a β-galactoside-binding lectin. To assess the affinity profile of PTX008 for galectins, we used N, H HSQC NMR spectroscopy to show that PTX008 also binds to galectin-3 (Gal-3), albeit more weakly. We identified the contact site for PTX008 on the F-face of the Gal-3 carbohydrate recognition domain. STD NMR revealed that the hydrophobic phenyl ring crown of the calixarene is the binding epitope. With this information, we performed molecular modeling of the complex to assist in improving the rather low affinity of PTX008 for Gal-3. By removing the N-dimethyl alkyl chain amide groups, we produced PTX013 whose reduced alkyl chain length and polar character led to an approximately eightfold stronger binding than PTX008. PTX013 also binds Gal-1 more strongly than PTX008, whereas neither interacts strongly, if at all, with Gal-7. In addition, PTX013, like PTX008, is an allosteric inhibitor of galectin binding to the canonical ligand lactose. This study broadens the scope for galectin targeting by calixarene-based compounds and opens the perspective for selective galectin blocking.
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http://dx.doi.org/10.1002/cmdc.202000742DOI Listing
February 2021

NMR of glycoproteins: profiling, structure, conformation and interactions.

Curr Opin Struct Biol 2020 Oct 28;68:9-17. Epub 2020 Oct 28.

Molecular Recognition and Host-Pathogen Interactions, CIC bioGUNE, Basque Research and Technology Alliance, BRTA, Bizkaia Technology Park, Building 800, 48162 Derio, Bizkaia, Spain. Electronic address:

In glycoproteins, carbohydrates are responsible for the selective interaction and tight regulation of cellular processes, constituting the main information transducer interface in protein-glycoprotein interactions. Increasing experimental and computational evidence suggest that such interactions often induce allosteric changes in the host protein, underlining the importance of studying intact glycoproteins. Technical issues have precluded such studies for years but, nowadays, a promising era is emerging where NMR spectroscopy, among other techniques, allows the characterization of the composition, structure and segmental dynamics of glycoproteins. In this review, we discuss such advances and highlight some selected examples. This novel technology unravels multiple new functional mechanisms, subtly hidden within the sugar code.
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http://dx.doi.org/10.1016/j.sbi.2020.09.009DOI Listing
October 2020

Insight into the Ferrier Rearrangement by Combining Flash Chemistry and Superacids.

Angew Chem Int Ed Engl 2021 01 23;60(4):2036-2041. Epub 2020 Nov 23.

IC2MP UMR CNRS 7285, Equipe "Synthèse Organique", Université de Poitiers, 4 rue Michel Brunet, 86073, Poitiers cedex 9, France.

The transformation of glycals into 2,3-unsaturated glycosyl derivatives, reported by Ferrier in 1962, is supposed to involve an α,β unsaturated glycosyl cation, an elusive ionic species that has still to be observed experimentally. Herein, while combination of TfOH and flow conditions failed to observe this ionic species, its extended lifetime in superacid solutions allowed its characterization by NMR-based structural analysis supported by DFT calculations. This allyloxycarbenium ion was further exploited in the Ferrier rearrangement to afford unsaturated nitrogen-containing C-aryl glycosides and C-alkyl glycosides under superacid and flow conditions, respectively.
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http://dx.doi.org/10.1002/anie.202010175DOI Listing
January 2021

Single-Step Glycosylations with C-Labelled Sulfoxide Donors: A Low-Temperature NMR Cartography of the Distinguishing Mechanistic Intermediates.

Chemistry 2021 Jan 23;27(6):2030-2042. Epub 2020 Nov 23.

Glycochemistry and Molecular Recognition group, Dpt. Bio-Organic Chemistry, Instituto de Química Orgánica General (IQOG-CSIC), Juan de la Cierva 3., 28006, Madrid, Spain.

Glycosyl sulfoxides have gained recognition in the total synthesis of complex oligosaccharides and as model substrates for dissecting the mechanisms involved. Reactions of these donors are usually performed under pre-activation conditions, but an experimentally more convenient single-step protocol has also been reported, whereby activation is performed in the presence of the acceptor alcohol; yet, the nature and prevalence of the reaction intermediates formed in this more complex scenario have comparatively received minimal attention. Herein, a systematic NMR-based study employing both C-labelled and unlabelled glycosyl sulfoxide donors for the detection and monitoring of marginally populated intermediates is reported. The results conclusively show that glycosyl triflates play a key role in these glycosylations despite the presence of the acceptor alcohol. Importantly, the formation of covalent donor/acceptor sulfonium adducts was identified as the main competing reaction, and thus a non-productive consumption of the acceptor that could limit the reaction yield was revealed.
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http://dx.doi.org/10.1002/chem.202003850DOI Listing
January 2021

Mono- and Di-Fucosylated Glycans of the Parasitic Worm S. mansoni are Recognized Differently by the Innate Immune Receptor DC-SIGN.

Chemistry 2020 Dec 22;26(67):15605-15612. Epub 2020 Oct 22.

Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Bijvoet Center for Biomolecular Research, Utrecht University, Universiteitsweg 99, 3584, CG, Utrecht, Netherlands.

The parasitic worm, Schistosoma mansoni, expresses unusual fucosylated glycans in a stage-dependent manner that can be recognized by the human innate immune receptor DC-SIGN, thereby shaping host immune responses. We have developed a synthetic approach for mono- and bis-fucosylated LacdiNAc (LDN-F and LDN-DF, respectively), which are epitopes expressed on glycolipids and glycoproteins of S. mansoni. It is based on the use of monosaccharide building blocks having carefully selected amino-protecting groups, facilitating high yielding and stereoselective glycosylations. The molecular interaction between the synthetic glycans and DC-SIGN was studied by NMR and molecular modeling, which demonstrated that the α1,3-fucoside of LDN-F can coordinate with the Ca -ion of the canonical binding site of DC-SIGN allowing for additional interactions with the underlying LDN backbone. The 1,2-fucoside of LDN-DF can be complexed in a similar manner, however, in this binding mode GlcNAc and GalNAc of the LDN backbone are placed away from the protein surface resulting in a substantially lower binding affinity. Glycan microarray binding studies showed that the avidity and selectivity of binding is greatly enhanced when the glycans are presented multivalently, and in this format Le and LDN-F gave strong responsiveness, whereas no binding was detected for LDN-DF. The data indicates that S. mansoni has developed a strategy to avoid detection by DC-SIGN in a stage-dependent manner by the addition of a fucoside to a number of its ligands.
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http://dx.doi.org/10.1002/chem.202002619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894523PMC
December 2020

Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins.

Angew Chem Int Ed Engl 2020 12 22;59(52):23763-23771. Epub 2020 Oct 22.

CIC bioGUNE, Basque Research and Technology Alliance, BRTA, Bizkaia Technology Park, 48162, Derio, Spain.

The glycan structures of the receptor binding domain of the SARS-CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD C-labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, N-labelled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.
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http://dx.doi.org/10.1002/anie.202011015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894318PMC
December 2020

Synthesis of long-chain alkyl glucosides via reverse hydrolysis reactions catalyzed by an engineered β-glucosidase.

Enzyme Microb Technol 2020 Oct 22;140:109591. Epub 2020 May 22.

Department of Biochemistry, Faculty of Science, and Center for Advanced Studies in Tropical Natural Resources, NRU-KU, Kasetsart University, Bangkok 10900, Thailand. Electronic address:

Long-chain alkyl glucosides, such as octyl and decyl β-d-glucopyranosides (OG and DG, respectively), are regarded as a new generation of biodegradable, non-ionic surfactants. Previously, the mutants of Dalbergia cochinchinensis Pierre dalcochinase showed potential in the synthesis of oligosaccharides and alkyl glucosides. In this study, the N189F dalcochinase mutant gave the highest yields of OG and DG synthesis under reverse hydrolysis conditions. The optimized yield of OG (57.5 mol%) was obtained in the reactions containing 0.25 M glucose and 0.3 units of the N189 F mutant in buffer-saturated octanol at 30 °C. The identity of OG and DG products was confirmed by high resolution mass spectrometry (HRMS) and NMR. Consistent with its capability for synthesis, the reactivation kinetics and ITC analysis revealed that the aglycone binding pocket of the N189F mutant was more favorable for long-chain alkyl alcohols than the wild-type dalcochinase, while their glycone binding pockets showed similar affinity for the glucosyl moiety. STD NMR revealed higher interactions at the aglycone sites than the glycone sites. Our results demonstrated a promising potential of the N189F dalcochinase mutant in the future commercial production of long-chain alkyl glucosides via reverse hydrolysis reactions.
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http://dx.doi.org/10.1016/j.enzmictec.2020.109591DOI Listing
October 2020

Targeting Galectins With Glycomimetics.

Front Chem 2020 7;8:593. Epub 2020 Aug 7.

CIC bioGUNE, Basque Research Technology Alliance, Derio, Spain.

Among glycan-binding proteins, galectins, β-galactoside-binding lectins, exhibit relevant biological roles and are implicated in many diseases, such as cancer and inflammation. Their involvement in crucial pathologies makes them interesting targets for drug discovery. In this review, we gather the last approaches toward the specific design of glycomimetics as potential drugs against galectins. Different approaches, either using specific glycomimetic molecules decorated with key functional groups or employing multivalent presentations of lactose and N-acetyl lactosamine analogs, have provided promising results for binding and modulating different galectins. The review highlights the results obtained with these approximations, from the employment of S-glycosyl compounds to peptidomimetics and multivalent glycopolymers, mostly employed to recognize and/or detect Gal-1 and Gal-3.
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http://dx.doi.org/10.3389/fchem.2020.00593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426508PMC
August 2020

Complete Dynamic Reconstruction of C, C, and (CN) Encapsulation into an Adaptable Supramolecular Nanocapsule.

J Am Chem Soc 2020 09 3;142(37):16051-16063. Epub 2020 Sep 3.

Institut de Quı́mica Computacional i Catàlisi (IQCC) and Departament de Quı́mica, Universitat de Girona, Campus Montilivi, Girona E-17003, Catalonia, Spain.

The dynamic adaptability of tetragonal prismatic nanocapsule in the selective separation of fullerenes and endohedral metallofullerenes (EMFs) remains unexplored. Therefore, the essential molecular details of the fullerene recognition and binding process into the coordination capsule and the origins of fullerene selectivity remain elusive. In this work, the key steps of fullerene recognition and binding processes have been deciphered by designing a protocol which combines H-H exchange spectroscopy (2D-EXSY) NMR experiments, long time-scale Molecular Dynamics (MD) and accelerated Molecular Dynamics (aMD) simulations, which are combined to completely reconstruct the spontaneous binding and unbinding pathways from nanosecond to second time-range. On one hand, binding (') and unbinding () rate constants were extracted from H-H exchange spectroscopy (EXSY) NMR experiments for both C and C. On the other hand, MD and aMD allowed monitoring the molecular basis of the encapsulation and guest competition processes at a very early stage under nonequilibrium conditions. The receptor capsule displays dynamical adaptability features similar to those observed in the process of biomolecular recognition in proteins. In addition, the encapsulation of bis-aza[60]fullerene (CN) within a supramolecular coordination capsule has been studied for the first time, showcasing the pros and cons of the dumbbell-shaped guest in the dynamics of the encapsulation process and in the stability of the final bound adduct. The powerful combination of NMR, MD, and aMD methodologies allows to obtain a precise picture of the subtle events directing the encapsulation and is thus a predictive tool for understanding host-guest encapsulation and interactions in numerous supramolecular systems.
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http://dx.doi.org/10.1021/jacs.0c07591DOI Listing
September 2020

Oral Treatment with Iododiflunisal Delays Hippocampal Amyloid-β Formation in a Transgenic Mouse Model of Alzheimer's Disease: A Longitudinal in vivo Molecular Imaging Study1.

J Alzheimers Dis 2020 ;77(1):99-112

CIC biomaGUNE, Basque Research and Technology Alliance (BRTA), San Sebastián, Guipúzcoa, Spain.

Background: Transthyretin (TTR) is a tetrameric, amyloid-β (Aβ)-binding protein, which reduces Aβ toxicity. The TTR/Aβ interaction can be enhanced by a series of small molecules that stabilize its tetrameric form. Hence, TTR stabilizers might act as disease-modifying drugs in Alzheimer's disease.

Objective: We monitored the therapeutic efficacy of two TTR stabilizers, iododiflunisal (IDIF), which acts as small-molecule chaperone of the TTR/Aβ interaction, and tolcapone, which does not behave as a small-molecule chaperone, in an animal model of Alzheimer's disease using positron emission tomography (PET).

Methods: Female mice (AβPPswe/PS1A246E/TTR+/-) were divided into 3 groups (n = 7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100 mg/Kg/day) was started at 5 months of age. Treatment efficacy assessment was based on changes in longitudinal deposition of Aβ in the hippocampus (HIP) and the cortex (CTX) and determined using PET-[18F]florbetaben. Immunohistochemical analysis was performed at age = 14 months.

Results: Standard uptake values relative to the cerebellum (SUVr) of [18F]florbetaben in CTX and HIP of non-treated animals progressively increased from age = 5 to 11 months and stabilized afterwards. In contrast, [18F]florbetaben uptake in HIP of IDIF-treated animals remained constant between ages = 5 and 11 months and significantly increased at 14 months. In the tolcapone-treated group, SUVr progressively increased with time, but at lower rate than in the non-treated group. No significant treatment effect was observed in CTX. Results from immunohistochemistry matched the in vivo data at age = 14 months.

Conclusion: Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aβ deposition in certain brain regions.
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http://dx.doi.org/10.3233/JAD-200570DOI Listing
January 2020

Unravelling the Time Scale of Conformational Plasticity and Allostery in Glycan Recognition by Human Galectin-1.

Chemistry 2020 Dec 29;26(67):15643-15653. Epub 2020 Oct 29.

Molecular Recognition and Host-Pathogen Interactions, CIC bioGUNE, Basque Research and Technology Alliance, BRTA, Bizkaia Technology Park, Building 800, 48162, Derio, Bizkaia, Spain.

The interaction of human galectin-1 with a variety of oligosaccharides, from di-(N-acetyllactosamine) to tetra-saccharides (blood B type-II antigen) has been scrutinized by using a combined approach of different NMR experiments, molecular dynamics (MD) simulations, and isothermal titration calorimetry. Ligand- and receptor-based NMR experiments assisted by computational methods allowed proposing three-dimensional structures for the different complexes, which explained the lack of enthalpy gain when increasing the chemical complexity of the glycan. Interestingly, and independently of the glycan ligand, the entropy term does not oppose the binding event, a rather unusual feature for protein-sugar interactions. CLEANEX-PM and relaxation dispersion experiments revealed that sugar binding affected residues far from the binding site and described significant changes in the dynamics of the protein. In particular, motions in the microsecond-millisecond timescale in residues at the protein dimer interface were identified in the presence of high affinity ligands. The dynamic process was further explored by extensive MD simulations, which provided additional support for the existence of allostery in glycan recognition by human galectin-1.
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http://dx.doi.org/10.1002/chem.202003212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756784PMC
December 2020

Dissection of the key steps of amyloid-β peptide 1-40 fibrillogenesis.

Int J Biol Macromol 2020 Dec 6;164:2240-2246. Epub 2020 Aug 6.

i3S - Instituto de Investigação e Inovação em Saúde, Rua Alfredo Allen, 208, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular Universidade do Porto, Rua Alfredo Allen, 208, Porto, Portugal; ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Rua de Jorge Viterbo Ferreira 228, Porto, Portugal. Electronic address:

The aggregation kinetics of Aβ1-40 peptide was characterized using a synergistic approach by a combination of nuclear magnetic resonance, thioflavin-T fluorescence, transmission electron microscopy and dynamic light scattering. A major finding is the experimental detection of high molecular weight oligomers (HMWO) that converts into fibrils nuclei. Our observations are consistent with a mechanism of Aβ1-40 fibrillogenesis that includes the following key steps: i) slow formation of HMWO (Rh ~ 20 nm); ii) conversion of the HMWO into more compact Rh ~ 10 nm fibrils nuclei; iii) fast formation of additional fibrils nuclei through fibril surface catalysed processes; and iv) growth of fibrils by addition of soluble Aβ species. Moreover, NMR diffusion experiments show that at 37 °C soluble Aβ1-40 remains intrinsically disordered and mostly in monomeric form despite evidences of the presence of dimers and/or other small oligomers. A mathematical model is proposed to simulate the aggregation kinetics of Aβ1-40.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.08.023DOI Listing
December 2020

An Assay for Screening Potential Drug Candidates for Alzheimer's Disease That Act as Chaperones of the Transthyretin and Amyloid-β Peptides Interaction.

Chemistry 2020 Dec 19;26(72):17462-17469. Epub 2020 Nov 19.

Institut de Química Avançada de Catalunya (I.Q.A.C.-C.S.I.C.), Jordi Girona 18-26, 08034, Barcelona, Spain.

The protein transthyretin (TTR) modulates amyloid-β (Aβ) peptides deposition and processing and this physiological effect is further enhanced by treatment with iododiflunisal (IDIF), a small-molecule compound (SMC) with TTR tetramer stabilization properties, which behaves as chaperone of the complex. This knowledge has prompted us to design and optimize a rapid and simple high-throughput assay that relies on the ability of test compounds to form ternary soluble complexes TTR/Aβ/SMC that prevent Aβ aggregation. The method uses the shorter Aβ(12-28) sequence which is cheaper and simpler to use while retaining the aggregation properties of their parents Aβ(1-40) and Aβ(1-42). The test is carried out in 96-plate wells that are UV monitored for turbidity during 6 h. Given its reproducibility, we propose that this test can be a powerful tool for efficient screening of SMCs that act as chaperones of the TTR/Aβ interaction that may led to potential AD therapies.
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http://dx.doi.org/10.1002/chem.202002933DOI Listing
December 2020

The Interaction of Fluorinated Glycomimetics with DC-SIGN: Multiple Binding Modes Disentangled by the Combination of NMR Methods and MD Simulations.

Pharmaceuticals (Basel) 2020 Aug 4;13(8). Epub 2020 Aug 4.

CIC bioGUNE, Basque Research Technology Alliance, BRTA, Bizkaia Technology park, Building 800, 48160 Derio, Spain.

Fluorinated glycomimetics are frequently employed to study and eventually modulate protein-glycan interactions. However, complex glycans and their glycomimetics may display multiple binding epitopes that enormously complicate the access to a complete picture of the protein-ligand complexes. We herein present a new methodology based on the synergic combination of experimental F-based saturation transfer difference (STD) NMR data with computational protocols, applied to analyze the interaction between DC-SIGN, a key lectin involved in inflammation and infection events with the trifluorinated glycomimetic of the trimannoside core, ubiquitous in human glycoproteins. A novel 2D-STD-TOCSYreF NMR experiment was employed to obtain the experimental STD NMR intensities, while the Complete Relaxation Matrix Analysis (CORCEMA-ST) was used to predict that expected for an ensemble of geometries extracted from extensive MD simulations. Then, an in-house built computer program was devised to find the ensemble of structures that provide the best fit between the theoretical and the observed STD data. Remarkably, the experimental STD profiles obtained for the ligand/DC-SIGN complex could not be satisfactorily explained by a single binding mode, but rather with a combination of different modes coexisting in solution. Therefore, the method provides a precise view of those ligand-receptor complexes present in solution.
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http://dx.doi.org/10.3390/ph13080179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463913PMC
August 2020

Structural Basis of Noscapine Activation for Tubulin Binding.

J Med Chem 2020 08 29;63(15):8495-8501. Epub 2020 Jul 29.

Centro de Investigaciones Biológicas Margarita Salas-CSIC, Ramiro de Maeztu, 9, C.P. 28040 Madrid, Spain.

Noscapine is a natural alkaloid that is used as an antitussive medicine. However, it also acts as a weak anticancer agent in certain models through a mechanism that is largely unknown. Here, we performed structural studies and show that the cytotoxic agent 7A--demethoxy-amino-noscapine (7A-aminonoscapine) binds to the colchicine site of tubulin. We suggest that the 7A-methoxy group of noscapine prevents binding to tubulin due to a steric clash of the compound with the T5-loop of α-tubulin. We further propose that the anticancer activity of noscapine arises from a bioactive metabolite that binds to the colchicine site of tubulin to induce mitotic arrest through a microtubule cytoskeleton-based mechanism.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00855DOI Listing
August 2020

Scientific Response to the Coronavirus Crisis in Spain: Collaboration and Multidisciplinarity.

ACS Chem Biol 2020 07 25;15(7):1722-1723. Epub 2020 Jun 25.

CIC bioGUNE, Basque Research Technology Alliance, BRTA, Bizkaia Technology Park, Building 800, 48160 Derio, Spain.

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http://dx.doi.org/10.1021/acschembio.0c00496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323048PMC
July 2020

Dissecting the Essential Role of Anomeric β-Triflates in Glycosylation Reactions.

J Am Chem Soc 2020 07 6;142(28):12501-12514. Epub 2020 Jul 6.

Instituto de Quı́mica Orgánica (IQOG-CSIC), 28006 Madrid, Spain.

Glycosylations promoted by triflate-generating reagents are widespread synthetic methods for the construction of glycosidic scaffolds and glycoconjugates of biological and chemical interest. These processes are thought to proceed with the participation of a plethora of activated high energy intermediates such as the α- and β-glycosyl triflates, or even increasingly unstable glycosyl oxocarbenium-like species, among which only α-glycosyl triflates have been well characterized under representative reaction conditions. Interestingly, the remaining less accessible intermediates, yet to be experimentally described, seem to be particularly relevant in α-selective processes, involving weak acceptors. Herein, we report a detailed analysis of several paradigmatic and illustrative examples of such reactions, employing a combination of chemical, NMR, kinetic and theoretical approaches, culminating in the unprecedented detection and quantification of the true β-glycosyl triflate intermediates within activated donor mixtures. This achievement was further employed as a stepping-stone for the characterization of the triflate anomerization dynamics, which along with the acceptor substitutions, govern the stereochemical outcome of the reaction. The obtained data conclusively show that, even for highly dissociative reactions involving β-close ion pair (β-CIP) species, the formation of the α-glycoside is necessarily preceded by a bimolecular α → β triflate interconversion, which under certain circumstances becomes the rate-limiting step. Overall, our results rule out the prevalence of the Curtin-Hammett fast-exchange assumption for most glycosylations and highlight the distinct reactivity properties of α- and β-glycosyl triflates against neutral and anionic acceptors.
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http://dx.doi.org/10.1021/jacs.0c05525DOI Listing
July 2020

Fluorinated carbohydrates as chemical probes for molecular recognition studies. Current status and perspectives.

Chem Soc Rev 2020 Jun;49(12):3863-3888

CIC bioGUNE, Basque Research and Technology Alliance (BRTA), 48160 Derio, Spain. and Ikerbasque, Basque Foundation for Science, Maria Diaz de Haro 3, 48013 Bilbao, Spain and Department of Organic Chemistry II, Faculty of Science and Technology, UPV/EHU, 48940 Leioa, Spain.

This review provides an extensive summary of the effects of carbohydrate fluorination with regard to changes in physical, chemical and biological properties with respect to regular saccharides. The specific structural, conformational, stability, reactivity and interaction features of fluorinated sugars are described, as well as their applications as probes and in chemical biology.
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http://dx.doi.org/10.1039/c9cs00099bDOI Listing
June 2020

An Epoxide Intermediate in Glycosidase Catalysis.

ACS Cent Sci 2020 May 16;6(5):760-770. Epub 2020 Apr 16.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria 3010, Australia.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 -α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope () conformation. Kinetic isotope effects (/) for anomeric-H and anomeric-C support an oxocarbenium ion-like transition state, and that for C2-O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
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http://dx.doi.org/10.1021/acscentsci.0c00111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256955PMC
May 2020