Search

Ligand-centered functionalization reactions offer diverse strategies to prepare luminescent organometallic compounds. These compounds can have unique structures that are not accessible via traditional coordination chemistry and can possess enhanced or unusual photophysical properties. Here we show that bis-cyclometalated iridium bis-isocyanide complexes (

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., A.G.S. would like to thank the EU commission for a MSCA-IF postdoctoral fellowship. L.M.-J and L.D.-C thank the Ministerio de Economía , Industria y Competitividad and the Ministerio de Ciencia, Innovación y Universidades the for their FPI fellowships (BES-2014-070232 and BES-2017-080618). This investigation was supported by research grants from the Spanish Ministry of Science and Innovation (CTQ2016−79255-P, PID2019−107476GB-I00, BFU2017−89707-P, RTI2018−094862-B-100, RTI2018−099592−B-C21, PID2019−104090RB-100, RTI2018−094751−B-C22, and RTI2018−099592−B-C22), the Mizutani Foundation forGlycoscience (200077 and 170045), the Aragon Government (Groups 17R-34) and the computational resources from BIFI-ZCAM (Universidad de Zaragoza, Spain).

41 p.-6 fig., 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 sensitivity to Type VI secretion system peptidoglycan endopeptidases and recognition by the Drosophila innate immune system, suggesting relevant roles in the environmental adaptability of these bacteria. Collectively our findings demonstrate the discriminative power of chemometric tools on large cell wall-chromatographic data sets to discover novel peptidoglycan structural properties in bacteria., This work was supported by the Laboratory for Molecular Infection Medicine Sweden(MIMS), the Knut and Alice Wallenberg Foundation (KAW),and the Swedish Research Council to F. Cava, Kempe foundation scholarship to AE and UCMR Linneus professor- ship to MAP and grants CTQ2012-32025 and CTQ2015- 64597-C2 of Spanish Ministry of Economy and Competitive- ness to FJC.

Development of strong and selective binders from promiscuous lead compounds represents one of the most expensive and time-consuming tasks in drug discovery. We herein present a novel fragment-based combinatorial strategy for the optimization of multivalent polyamine scaffolds as DNA/RNA ligands. Our protocol provides a quick access to a large variety of regioisomer libraries that can be tested for selective recognition by combining microdialysis assays with simple isotope labeling and NMR experiments. To illustrate our approach, 20 small libraries comprising 100 novel kanamycin-B derivatives have been prepared and evaluated for selective binding to the ribosomal decoding A-Site sequence. Contrary to the common view of NMR as a low-throughput technique, we demonstrate that our NMR methodology represents a valuable alternative for the detection and quantification of complex mixtures, even integrated by highly similar or structurally related derivatives, a common situation in the context of a lead optimization process. Furthermore, this study provides valuable clues about the structural requirements for selective A-site recognition.

The increasing interest in the functional versatility of glycan epitopes in cellular glycoconjugates calls for developing sensitive methods to define carbohydrate conformation in solution and to characterize protein-carbohydrate interactions. Measurements of pseudocontact shifts in the presence of a paramagnetic cation can provide such information. In this work, the energetically privileged conformation of a disaccharide (lactose as test case) was experimentally inferred by using a synthetic carbohydrate conjugate bearing a lanthanide binding tag. In addition, the binding of lactose to a biomedically relevant receptor (the human adhesion/growth-regulatory lectin galectin-3) and its consequences in structural terms were defined, using Dy(3+), Tb(3+), and Tm(3+). The described approach, complementing the previously tested protein tagging as a way to exploit paramagnetism, enables to detect binding, even weak interactions, and to characterize in detail topological aspects useful for physiological ligands and mimetics in drug design.

FtsZ is the key protein of bacterial cell-division and target for new antibiotics. Selective inhibition of FtsZ polymerization without impairing the assembly of the eukaryotic homologue tubulin was demonstrated with C8-substituted guanine nucleotides. By combining NMR techniques with biochemical and molecular modeling procedures, we have investigated the molecular recognition of C8-substituted-nucleotides by FtsZ from Methanococcus jannaschii (Mj-FtsZ) and Bacillus subtilis (Bs-FtsZ). STD epitope mapping and trNOESY bioactive conformation analysis of each nucleotide were employed to deduce differences in their recognition mode by each FtsZ species. GMP binds in the same anti conformation as GTP, whereas 8-pyrrolidino-GMP binds in the syn conformation. However, the anti conformation of 8-morpholino-GMP is selected by Bs-FtsZ, while Mj-FtsZ binds both anti- and syn-geometries. The inhibitory potencies of the C8-modified-nucleotides on the assembly of Bs-FtsZ, but not of Mj-FtsZ, correlate with their binding affinities. Thus, MorphGTP behaves as a nonhydrolyzable analog whose binding induces formation of Mj-FtsZ curved filaments, resembling polymers formed by the inactive forms of this protein. NMR data, combined with molecular modeling protocols, permit explanation of the mechanism of FtsZ assembly impairment by C8-substituted GTP analogs. The presence of the C8-substituent induces electrostatic remodeling and small structural displacements at the association interface between FtsZ monomers to form filaments, leading to complete assembly inhibition or to formation of abnormal FtsZ polymers. The inhibition of bacterial Bs-FtsZ assembly may be simply explained by steric clashes of the C8-GTP-analogs with the incoming FtsZ monomer. This information may facilitate the design of antibacterial FtsZ inhibitors replacing GTP. © 2013 American Chemical Society., MINECO; Comunidad de Madrid, Fundación Severo Ochoa; European Comission (BM1003, CM1102 COST)

The current surge in defining glycobiomarkers by applying lectins rekindles interest in definition of the sugar-binding sites of lectins at high resolution. Natural complex-type N-glycans can present more than one potential binding motif, posing the question of the actual mode of interaction when interpreting, for example, lectin array data. By strategically combining N-glycan preparation with saturation-transfer difference NMR and modeling, we illustrate that epitope recognition depends on the structural context of both the sugar and the lectin (here, wheat germ agglutinin and a single hevein domain) and cannot always be predicted from simplified model systems studied in the solid state. We also monitor branch-end substitutions by this strategy and describe a three-dimensional structure that accounts for the accommodation of the α2,6-sialylated terminus of a biantennary N-glycan by viscumin. In addition, we provide a structural explanation for the role of terminal α2,6-sialylation in precluding the interaction of natural N-glycans with lectin from Maackia amurensis . The approach described is thus capable of pinpointing lectin-binding motifs in natural N-glycans and providing detailed structural explanations for lectin selectivity.

The identification of MUC1 tumor-associated Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.

Sugar-oligoamides have been designed and synthesized as structurally simple carbohydrate-based ligands to study carbohydrate-DNA interactions. The general design of the ligands 1-3 has been done as to favor the bound conformation of Distamycin-type gamma-linked covalent dimers which is a hairpin conformation. Indeed, NMR analysis of the sugar-oligoamides in the free state has indicated the presence of a percentage of a hairpin conformation in aqueous solution. The DNA binding activity of compounds 1-3 was confirmed by calf thymus DNA (ct-DNA) NMR titration. Interestingly, the binding of the different sugar-oligoamides seems to be modulated by the sugar configuration. Semiquantitative structural information about the DNA ligand complexes has been derived from NMR data. A competition experiment with Netropsin suggested that the sugar-oligoamide 3 bind to DNA in the minor groove. The NMR titrations of 1-3 with poly(dA-dT) and poly(dG-dC) suggested preferential binding to the ATAT sequence. TR-NOE NMR experiments for the sugar-oligoamide 3-ct-DNA complex both in D(2)O and H(2)O have confirmed the complex formation and given information on the conformation of the ligand in the bound state. The data confirmed that the sugar-oligoamide ligand is a hairpin in the bound state. Even more relevant to our goal, structural information on the conformation around the N-glycosidic linkage has been accessed. Thus, the sugar asymmetric centers pointing to the NH-amide and N-methyl rims of the molecule have been characterized.

The existence of stabilizing carbohydrate-aromatic interactions is demonstrated from both the theoretical and experimental viewpoints. The geometry of experimentally based galactose-lectin complexes has been properly accounted for by using a MP2/6-31G(d,p) level of theory and by considering a counterpoise correction during optimization. In this case, the stabilizing interaction energy of the fucose-benzene complex amounts to 3.0 kcal/mol. The theoretical results obtained herein indicate that the carbohydrate-aromatic interactions are stabilizing interactions with an important dispersive component and that electronic density between the sugar hydrogens and the aromatic ring indeed exists, thus giving rise to three so-called nonconventional hydrogen bonds. Experimental evidence of the intrinsic tendency of aromatic moieties to interact with certain sugars has also been shown by simple NMR experiments in water solution. Benzene and phenol specifically interact with the clusters of C-H bonds of the alpha face of methyl beta-galactoside, without requiring the well-defined three-dimensional shape provided by a protein receptor, therefore resembling the molecular recognition features that are frequently observed in many carbohydrate-protein complexes.

The conformational behavior of different aza-C-glycosides synthesized as glycosidase inhibitors has been studied using a combination of NMR spectroscopy (J and NOE data) and time-averaged restrained molecular dynamics calculations. The obtained results show that the population distribution of conformers around their pseudoglycosidic linkages is mainly controlled by 1,3-syn-diaxial interactions. Electrostatic effects slightly modulate the conformational equilibrium. This result is in contrast with that observed for O-glycosides. For these natural compounds, the conformational behavior around the glycosidic linkage Φ is mainly governed by the exo-anomeric effect. Experimentally based energy values for both the 1,3-syn-diaxial interactions and the stereoelectronic effect have been deduced. Finally, the inhibitory activity of these compounds has been tested again a variety of glycosidase enzymes.

The C-glycoside analogue of lactose harbors a pronounced flexibility in water with three conformers in equilibrium. The bound conformation of C-lactose to bovine heart galectin-1 in solution has been determined by NMR spectroscopy. It is demonstrated that the lectin selects the syn conformation of the structural analogue of natural lactose and not the global minimum, anti conformation. The bound conformer resembles those found in the crystal structures of complexes of galectin-1/N-acetyllactosamine-containing oligosaccharides and in solution for an avian galectin. Docking of the analogue within the galectin's binding site furnishes explanations, in structural terms, for the exclusive recognition of the syn conformer.

The conformational behavior of the synthetic glycosidase inhibitor C-lactose (1) has been studied in different solvents (water, N,N-dimethylformamide, dimethyl sulfoxide, pyridine) using NMR spectroscopy and molecular mechanics calculations. The obtained results have been compared to those previously obtained for its natural analogue, methyl α-lactoside (2). It is shown that the conformational behavior of C- and O-lactoses is only similar around the glycosidic bond, but not around the aglyconic bond. In addition, the extent of flexibility around the β(1→4) linkage is much larger for C-lactose (1) than for methyl α-lactoside, about 23% of the complete potential energy surface of 1 is appreciably populated, and several energy minima coexist in solution. The obtained results indicate that β-linked C-glycosides are fairly flexible compounds and that even variations of the solvent may heavily affect their conformational behavior. Finally, we report on the use of 2D transferred NOE experiments to study the reco...

We show that the conformational features of the molecular complexes of E. coli beta-galactosidase and O-glycosides may differ from those formed with closely related compounds in their chemical nature, such as C- and S-glycosyl analogues. In the particular case presented here, NMR and ab initio quantum mechanical results show that the 3D-shapes of the ligand/inhibitor within the enzyme binding site depend on the chemical nature of the compounds. In fact, they depend on the relative size of the stereoelectronic barriers for chair deformation or for rotation around Phi glycosidic linkage.