RMN y reconocimiento molecular. Interacciones carbohidrato-proteína

Molecular recognition by specific targets is at the heart of the life processes. In recent years, it has been shown that the interactions between proteins (lectins, enzymes, antibodies) and carbohydrates mediate a broad range of biological activities, from fertilization, embryogenesis, and tissue maturation, to pathological processes. The elucidation of the mechanisms that govern how sugars are accommodated in the binding sites of these receptors is currently a topic of interest. Thus, the determination of the structural and conformational factors and the physicochemical features which govern the molecular recognition of these molecules is of paramount importance. This presentation is focused on the application of standard and state-of-the-art NMR methods both from the ligand and receptor’s perspective (especially chemical shift perturbation analysis, Saturation Transfer Difference, and trNOESY experiments) to the study of molecular recognition processes between a variety of polypeptides of biomedical interest and carbohydrate-based molecules, drugs and inhibitors. Lectins, antibodies, and enzymes, both wild type and mutants, have been used as receptors with the final aim to know and to evaluate the relative importance of polar (hydrogen bonding, electrostatic interactions) and non polar (van der Waals, CH-π) forces in the molecular recognition process. As examples, structural and conformational details of glycan recognition by different domains will be shown, with special emphasis in the application of novel 19F- and paramagnetic-based NMR methodologiesUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Hamiltonian Gotay-Nester-Hinds analysis of the parametrized unimodular extension of the Holst action

We give a detailed account of the Hamiltonian Gotay-Nester-Hinds (GNH) analysis of the parametrized unimodular extension of the Holst action. The purpose of the paper is to derive, through the clear geometric picture furnished by the GNH method, a simple Hamiltonian formulation for this model and explain why it is difficult to arrive at it in other approaches. We will also show how to take advantage of the field equations to anticipate the simple form of the constraints that we find in the paper.This work has been supported by the Spanish Ministerio de Ciencia Innovación y Universidades-Agencia Estatal de Investigación/FIS2017-84440-C2-2-P grant. Bogar Díaz was partially supported by a DGAPA-UNAM postdoctoral fellowship and acknowledges support from the CONEX-Plus program funded by Universidad Carlos III de Madrid and the European Union’s Horizon 2020 program under the Marie Sklodowska-Curie grant agreement No. 801538. Juan Margalef-Bentabol is supported by the Eberly Research Funds of Penn State, by the NSFgrant PHY-1806356 and by the Urania Stott fund of Pittsburgh foundation UN2017-92945

The Flexibility of Oligosaccharides Unveiled Through Residual Dipolar Coupling Analysis

The intrinsic flexibility of glycans complicates the study of their structures and dynamics, which are often important for their biological function. NMR has provided insights into the conformational, dynamic and recognition features of glycans, but suffers from severe chemical shift degeneracy. We employed labelled glycans to explore the conformational behaviour of a β(1-6)-Glc hexasaccharide model through residual dipolar couplings (RDCs). RDC delivered information on the relative orientation of specific residues along the glycan chain and provided experimental clues for the existence of certain geometries. The use of two different aligning media demonstrated the adaptability of flexible oligosaccharide structures to different environments

Exploring multivalent carbohydrate–protein interactions by NMR

Nuclear Magnetic Resonance (NMR) has been widely employed to assess diverse features of glycan–protein molecular recognition events. Different types of qualitative and quantitative information at different degrees of resolution and complexity can be extracted from the proper application of the available NMR-techniques. In fact, affinity, structural, kinetic, conformational, and dynamic characteristics of the binding process are available. Nevertheless, except in particular cases, the affinity of lectin-sugar interactions is weak, mostly at the low mM range. This feature is overcome in biological processes by using multivalency, thus augmenting the strength of the binding. However, the application of NMR methods to monitor multivalent lectin–glycan interactions is intrinsically challenging. It is well known that when large macromolecular complexes are formed, the NMR signals disappear from the NMR spectrum, due to the existence of fast transverse relaxation, related to the large size and exchange features. Indeed, at the heart of the molecular recognition event, the associated free-bound chemical exchange process for both partners takes place in a particular timescale. Thus, these factors have to be considered and overcome. In this review article, we have distinguished, in a subjective manner, the existence of multivalent presentations in the glycan or in the lectin. From the glycan perspective, we have also considered whether multiple epitopes of a given ligand are presented in the same linear chain of a saccharide (i.e., poly-LacNAc oligosaccharides) or decorating different arms of a multiantennae scaffold, either natural (as in multiantennae N-glycans) or synthetic (of dendrimer or polymer nature). From the lectin perspective, the presence of an individual binding site at every monomer of a multimeric lectin may also have key consequences for the binding event at different levels of complexity.We thank generous funding by the European Research Council (RECGLYCANMR, Advanced Grant No. 788143), the Agencia Estatal de Investigación (Spain) for grant PDI2021-1237810B-C21, and CIBERES, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. We also thank Marie-Skłodowska-Curie actions (TN BactiVax, under grant agreement No. 860325)

Targeting Galectins With Glycomimetics

Among glycan-binding proteins, galectins, beta-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 detecthGal-1 andhGal-3.We thank the European Research Council (RECGLYCANMR, Advanced Grant no. 788143), ISCIII (Grant PRB3 IPT17/0019 to AG), and the Agencia Estatal de Investigacion (Spain) for Grants RTI2018-094751-B-C21, Ramon y Cajal contract to AA and the Severo Ochoa Excellence Accreditation (SEV-2016-0644)

Kinetic Studies of Acetyl Group Migration between the Saccharide Units in an Oligomannoside Trisaccharide Model Compound and a Native Galactoglucomannan Polysaccharide

Acyl group migration is a fundamental phenomenon in carbohydrate chemistry, recently shown to take place also between two non-adjacent hydroxyl groups, across the glycosidic bond, in a beta-(1 -> 4)-linked mannan trisaccharide model compound. With the central mannoside unit containing acetyl groups at the O2 and O3 positions, the O2-acetyl was in the earlier study shown to migrate to O6 of the reducing end. Potential implications of the general acyl migration process on cell signaling events and plant growth in nature are intriguing open questions. In the present work, migration kinetics in this original trisaccharide model system were studied in more detail together with potential interactions of the model compound and the migration products with DC-SIGN lectin. Furthermore, we demonstrate here for the first time that similar migration may also take place in native polysaccharides, here represented by galactoglucomannan from Norway spruce.The authors acknowledge the European Research Council for financial support (ERC-2017-AdG, project number 788143-RECGLYCA NMR) and the Agencia Estatal de Investigacion (Spain) for project RTI2018-094751-B-C21 (to JJB

Molecular Recognition in C-Type Lectins: The Cases of DC- SIGN, Langerin, MGL, and L-Sectin

Carbohydrates play a pivotal role in intercellular communication processes. In particular, glycan antigens are key for sustaining homeostasis, helping leukocytes to distinguish damaged tissues and invading pathogens from healthy tissues. From a structural perspective, this cross-talk is fairly complex, and multiple membrane proteins guide these recognition processes, including lectins and Toll-like receptors. Since the beginning of this century, lectins have become potential targets for therapeutics for controlling and/or avoiding the progression of pathologies derived from an incorrect immune outcome, including infectious processes, cancer, or autoimmune diseases. Therefore, a detailed knowledge of these receptors is mandatory for the development of specific treatments. In this review, we summarize the current knowledge about four key C-type lectins whose importance has been steadily growing in recent years, focusing in particular on how glycan recognition takes place at the molecular level, but also looking at recent progresses in the quest for therapeutics.We thank the European Research Council (RECGLYCA NMR, advanced grant no. 788143), and the Agencia Estatal de Investigacion (Spain) for grants RTI2018-094751-B-C21 and B-C22, Ramon y Cajal contract to A.A. and the Severo Ochoa Excellence Accreditation (SEV-2016-0644)

Biomimetic Tweezers for N-Glycans: Selective Recognition of the Core GlcNAc(2) Disaccharide of the Sialylglycopeptide SGP

In recent years, glycomics have shown how pervasive the role of carbohydrates in biological systems is and how chemical tools are essential to investigate glycan function and modulate carbohydrate-mediated processes. Biomimetic receptors for carbohydrates can carry out this task but, although significant affinities and selectivities toward simple saccharides have been achieved, targeting complex glycoconjugates remains a goal yet unattained. In this work we report the unprecedented recognition of a complex biantennary sialylglycopeptide (SGP) by a tweezers-shaped biomimetic receptor, which selectively binds to the core GlcNAc2 disaccharide of the N-glycan with an affinity of 170 μM. Because of the simple structure and the remarkable binding ability, this biomimetic receptor can represent a versatile tool for glycoscience, opening the way to useful applications.We thank MIUR-Italy “Progetto Dipartimenti di Eccellenza 2018–2022” allocated to Department of Chemistry Ugo Schiff, COST Action (CA18132), MIUR-Italy PRIN2017 (2017XZ2ZBK) for granting a fellowship to F.M. and Ente Cassa di Risparmio di Firenze (Italy) is acknowledged for granting an ITC nanocalorimeter and a high-field NMR spectrometer. Open Access funding provided by Università degli Studi di Firenze within the CRUI-CARE Agreement

Novel Dextran-Supported Biological Probes Decorated with Disaccharide Entities for Investigating the Carbohydrate–Protein Interactions of Gal-3

The quest for novel natural-like biomolecular probes that can be used to gain information on biological recognition events is of topical interest to several scientific areas. In particular, the recognition of carbohydrates by proteins modulates a number of important biological processes. These molecular recognition events are, however, difficult to study by the use of naturally occurring oligosaccharides and polysaccharides owing to their intrinsic structural heterogeneity and to the many technical difficulties encountered during the isolation of sufficient quantities of pure material for detailed structural and biological studies. Therefore, the construction of homogenous biomolecular probes that can mimic both the biophysical properties of polysaccharide backbones and the properties of bioactive oligosaccharide fragments are highly sought after. Herein, synthetic methodology for the construction of well-defined bioconjugates consisting of biologically relevant disaccharide fragments grafted onto a dextran backbone is presented, and a preliminary NMR spectroscopy study of their interactions with galectin-3 as a model lectin is conducted.Peer reviewe

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

Conspectus We also highlight that, from a chemical perspective, the glycosylation reaction can be described as a continuum, from unimolecular SN1 with naked oxocarbenium cations as intermediates to bimolecular SN2-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 SN1-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