Unravelling the mechanisms of drugs partitioning phenomena in micellar systems via NMR spectroscopy.

Despite extensive use of micelles in materials and colloidal science, their supramolecular organization as well as host-guest interactions within these dynamic assemblies are poorly understood. Small guest molecules in the presence of micelles undergo constant exchange between a micellar aggregate and the surrounding solution, posing a considerable challenge for their molecular level characterisation. In this work we reveal the interaction maps between small guest molecules and surfactants forming micelles via novel applications of NMR techniques supported with state-of-the-art analytical methods used in colloidal science. Model micelles composed of structurally distinct surfactants (block non-ionic polymer Pluronic® F-127, non-ionic surfactant Tween 20 or Tween 80, and ionic surfactant sodium lauryl sulphate, SLS) were selected and loaded with model small molecules of biological relevance (i.e. the drugs fluconazole, FLU or indomethacin, IMC) known to have different partition coefficients. Molecular level organization of FLU or IMC within hydrophilic and hydrophobic domains of micellar aggregates was established using the combination of NMR methods (1D 1H NMR, 1D 19F NMR, 2D 1H-1H NOESY and 2D 1H-19F HOESY, and the multifrequency-STD NMR) and corroborated with molecular dynamics (MD) simulations. This is the first application of multifrequency-STD NMR to colloidal systems, enabling us to elucidate intricately detailed patterns of drug/micelle interactions in a single NMR experiment within minutes. Importantly, our results indicate that flexible surfactants, such as block copolymers and polysorbates, form micellar aggregates with a surface composed of both hydrophilic and hydrophobic domains and do not follow the classical core-shell model of the micelle. We propose that the magnitude of changes in 1H chemical shifts corroborated with interaction maps obtained from DEEP-STD NMR and 2D NMR experiments can be used as an indicator of the strength of the guest-surfactant interactions. This NMR toolbox can be adopted for the analysis of broad range of colloidal host-guest systems from soft materials to biological systems.

Computational evidence of glycosyl cations.

Glycosyl cations are key intermediates in the glycosylation reactions taking place through a SN1-type mechanism. To obtain a reliable description of the glycosylation reaction mechanism a combination of computational studies and experimental data such as kinetic isotopic effects is needed. Computational studies have elucidated SN2-type glycosylation reaction mechanisms, but elucidation of mechanisms in which ion pairs can be formed presents some difficulties because of the recombination of the ions. Recent topological and dynamic studies open the door to the ultimate confirmation of the presence of glycosyl cations in the form of intimate ion pairs during certain glycosylation reactions. This review covers the state-of-the-art tools and applications of computational chemistry mainly developed during the last ten years to understand glycosylation reactions in which an oxocarbenium ion could be involved.

Structural basis of trehalose recognition by the mycobacterial LpqY-SugABC transporter.

The Mycobacterium tuberculosis (Mtb) LpqY-SugABC ATP-binding cassette transporter is a recycling system that imports trehalose released during remodeling of the Mtb cell-envelope. As this process is essential for the virulence of the Mtb pathogen, it may represent an important target for tuberculosis drug and diagnostic development, but the transporter specificity and molecular determinants of substrate recognition are unknown. To address this, we have determined the structural and biochemical basis of how mycobacteria transport trehalose using a combination of crystallography, saturation transfer difference NMR, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the synthesis of trehalose analogs. This analysis pinpoints key residues of the LpqY substrate binding lipoprotein that dictate substrate-specific recognition and has revealed which disaccharide modifications are tolerated. These findings provide critical insights into how the essential Mtb LpqY-SugABC transporter reuses trehalose and modified analogs and specifies a framework that can be exploited for the design of new antitubercular agents and/or diagnostic tools.

FUT8-Directed Core Fucosylation of N-glycans Is Regulated by the Glycan Structure and Protein Environment.

FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-ß-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

Self-acetylation at the active site of phosphoenolpyruvate carboxykinase (PCK1) controls enzyme activity.

Acetylation is known to regulate the activity of cytosolic phosphoenolpyruvate carboxykinase (PCK1), a key enzyme in gluconeogenesis, by promoting the reverse reaction of the enzyme (converting phosphoenolpyruvate to oxaloacetate). It is also known that the histone acetyltransferase p300 can induce PCK1 acetylation in cells, but whether that is a direct or indirect function was not known. Here we initially set out to determine whether p300 can acetylate directly PCK1 in vitro. We report that p300 weakly acetylates PCK1, but surprisingly, using several techniques including protein crystallization, mass spectrometry, isothermal titration calorimetry, saturation-transfer difference nuclear magnetic resonance and molecular docking, we found that PCK1 is also able to acetylate itself using acetyl-CoA independently of p300. This reaction yielded an acetylated recombinant PCK1 with a 3-fold decrease in kcat without changes in Km for all substrates. Acetylation stoichiometry was determined for 14 residues, including residues lining the active site. Structural and kinetic analyses determined that site-directed acetylation of K244, located inside the active site, altered this site and rendered the enzyme inactive. In addition, we found that acetyl-CoA binding to the active site is specific and metal dependent. Our findings provide direct evidence for acetyl-CoA binding and chemical reaction with the active site of PCK1 and suggest a newly discovered regulatory mechanism of PCK1 during metabolic stress.

Structure and properties of two glucose-based deep eutectic systems.

Continued industrialization and increasing environmental problems have highlighted the need to research new eco-friendly solvents, also known as deep eutectic solvents (DESs). To implement these solvents in industrial processes, the knowledge of their molecular organization and thermophysical properties must be enhanced. In this work, two DESs have been characterized: d-glucose:choline chloride:water (GCH) and d-glucose:citric acid:water (GCiH). NMR techniques were used to analyse both the supramolecular structure and the role of water and to calculate the diffusion coefficients. Moreover, seven thermophysical properties at several temperatures were evaluated. As a second aim, the solubility of quercetin was determined. NMR studies showed a stronger supramolecular structure of GCH and a high ratio of ß-glucose in both DESs. Based on the thermophysical results, the solvent with choline chloride had the most compact fluid structure. Finally, the solubility of quercetin in the DESs was higher than in water, especially for GCH.

Dissecting the Structural and Chemical Determinants of the "Open-to-Closed" Motion in the Mannosyltransferase PimA from Mycobacteria.

The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) α-helix-to-ß-strand and ß-strand-to-α-helix transitions and (ii) an "open-to-closed" motion between the two Rossmann-fold domains, a conformational change that is necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such an activation mechanism, we analyzed a series of chemical derivatives, including mannose phosphate (Man-P) and mannose pyrophosphate-ribose (Man-PP-RIB), and additional GDP derivatives, such as pyrophosphate ribose (PP-RIB) and GMP, by the combined use of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry, and small angle X-ray scattering methods. Although the ß-phosphate is present, we found that the mannose ring, covalently attached to neither phosphate (Man-P) nor PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the "open-to-closed" motion, with the ß-phosphate group providing the high-affinity binding to PimA. Altogether, the experimental data contribute to a better understanding of the structural determinants involved in the "open-to-closed" motion not only observed in PimA but also visualized and/or predicted in other glycosyltransfeases. In addition, the experimental data might prove to be useful for the discovery and/or development of PimA and/or glycosyltransferase inhibitors.

Characterization of xylitol or citric acid:choline chloride:water mixtures: Structure, thermophysical properties, and quercetin solubility.

The industrial implementation of new eco-friendly solvents has highlighted the need to analyse both the structures and thermophysical properties of these solvents. Here, two deep eutectic solvents (DESs) used in the agro-food field were studied: xylitol:choline chloride:water (1:2:3 M ratio), XoCH, and citric acid:choline chloride:water (1:1:6 M ratio), CiCH. The H-bond network between the components of each DES was evaluated and the diffusion coefficients at 298.15 K were calculated using NMR spectroscopy. In addition, seven thermophysical properties were determined from 278.15 to 338.15 K. Also, the solubility of quercetin in water and in the two eutectic mixtures was measured and the interactions between components were studied. NMR experiments revealed the presence of water within the supramolecular structure of XoCH, but CiCH is a "DES-in-water" solution. Based on the results, XoCH is the most compact mixture. Finally, quercetin was remarkably more soluble in the studied DESs than in pure water.

Synthesis, Biological and In Silico Evaluation of Pure Nucleobase-Containing Spiro (Indane-Isoxazolidine) Derivatives as Potential Inhibitors of MDM2-p53 Interaction.

Nucleobase-containing isoxazolidines spiro-bonded to an indane core have been synthesized in very good yields by regio- and diastereoselective 1,3-dipolar cycloaddition starting from indanyl nitrones and N-vinylnucleobases by using environmentally benign microwave technology. The contemporary presence of various structural groups that are individually active scaffolds of different typology of drugs, has directed us to speculate that these compounds may act as inhibitors of MDM2-p53 interaction. Therefore, both computational calculations and antiproliferative screening against A549 human lung adenocarcinoma cells and human SH-SY5Y neuroblastoma cells were carried out to support this hypothesis.

Native Quercetin as a Chloride Receptor in an Organic Solvent.

The binding properties of quercetin toward chloride anions were investigated by means of ¹H-NMR, 13C-NMR, and electrospray ionization mass spectrometry (ESI-MS) measurements, as well as computational calculations. The results indicate that quercetin behaves primarily as a ditopic receptor with the binding site of the B ring that exhibits stronger chloride affinity compared to the A ring. However, these sites are stronger receptors than those of catechol and resorcinol because of their conjugation with the carbonyl group located on the C ring. The 1:1 and 1:2 complexation of this flavonoid with Cl- was also supported by ESI mass spectrometry.

UDP-GlcNAc Analogues as Inhibitors of O-GlcNAc Transferase (OGT): Spectroscopic, Computational, and Biological Studies.

A series of glycomimetics of UDP-GlcNAc, in which the ß-phosphate has been replaced by either an alkyl chain or a triazolyl ring and the sugar moiety has been replaced by a pyrrolidine ring, has been synthesized by the application of different click-chemistry procedures. Their affinities for human O-GlcNAc transferase (hOGT) have been evaluated and studied both spectroscopically and computationally. The binding epitopes of the best ligands have been determined in solution by means of saturation transfer difference (STD) NMR spectroscopy. Experimental, spectroscopic, and computational results are in agreement, pointing out the essential role of the binding of ß-phosphate. We have found that the loss of interactions from the ß-phosphate can be counterbalanced by the presence of hydrophobic groups at a pyrroline ring acting as a surrogate of the carbohydrate unit. Two of the prepared glycomimetics show inhibition at a micromolar level.

Inhibitors against Fungal Cell Wall Remodeling Enzymes.

Fungal ß-1,3-glucan glucanosyltransferases are glucan-remodeling enzymes that play important roles in cell wall integrity, and are essential for the viability of pathogenic fungi and yeasts. As such, they are considered possible drug targets, although inhibitors of this class of enzymes have not yet been reported. Herein we report a multidisciplinary approach based on a structure-guided design using a highly conserved transglycosylase from Sacharomyces cerevisiae, that leads to carbohydrate derivatives with high affinity for Aspergillus fumigatus Gel4. We demonstrate by X-ray crystallography that the compounds bind in the active site of Gas2/Gel4 and interact with the catalytic machinery. The topological analysis of noncovalent interactions demonstrates that the combination of a triazole with positively charged aromatic moieties are important for optimal interactions with Gas2/Gel4 through unusual pyridinium cation-π and face-to-face π-π interactions. The lead compound is capable of inhibiting AfGel4 with an IC50 value of 42 µm.

The small molecule luteolin inhibits N-acetyl-α-galactosaminyltransferases and reduces mucin-type O-glycosylation of amyloid precursor protein.

Mucin-type O-glycosylation is the most abundant type of O-glycosylation. It is initiated by the members of the polypeptide N-acetyl-α-galactosaminyltransferase (ppGalNAc-T) family and closely associated with both physiological and pathological conditions, such as coronary artery disease or Alzheimer's disease. The lack of direct and selective inhibitors of ppGalNAc-Ts has largely impeded research progress in understanding the molecular events in mucin-type O-glycosylation. Here, we report that a small molecule, the plant flavonoid luteolin, selectively inhibits ppGalNAc-Ts in vitro and in cells. We found that luteolin inhibits ppGalNAc-T2 in a peptide/protein-competitive manner but not promiscuously (e.g. via aggregation-based activity). X-ray structural analysis revealed that luteolin binds to the PXP motif-binding site found in most protein substrates, which was further validated by comparing the interactions of luteolin with wild-type enzyme and with mutants using 1H NMR-based binding experiments. Functional studies disclosed that luteolin at least partially reduced production of ß-amyloid protein by selectively inhibiting the activity of ppGalNAc-T isoforms. In conclusion, our study provides key structural and functional details on luteolin inhibiting ppGalNAc-T activity, opening up the way for further optimization of more potent and specific ppGalNAc-T inhibitors. Moreover, our findings may inform future investigations into site-specific O-GalNAc glycosylation and into the molecular mechanism of luteolin-mediated ppGalNAc-T inhibition.

Racemic hemiacetals as oxygen-centered pronucleophiles triggering cascade 1,4-addition/Michael reaction through dynamic kinetic resolution under iminium catalysis. Development and mechanistic insights.

2-Hydroxydihydropyran-5-ones behave as excellent polyfunctional reagents able to react with enals through oxa-Michael/Michael process cascade under the combination of iminium and enamine catalysis. These racemic hemiacetalic compounds are used as unconventional O-pronucleophiles in the initial oxa-Michael reaction, also leading to the formation of a single stereoisomer under a dynamic kinetic resolution (DKR) process. Importantly, by using ß-aryl or ß-alkyl substituted α,ß-unsaturated substrates as initial Michael acceptors either kinetically or thermodynamically controlled diastereoisomers were formed with high stereoselection through the careful selection of the reaction conditions. Finally, a complete experimental and computational study confirmed the initially proposed DKR process during the catalytic oxa-Michael/Michael cascade reaction and also explained the kinetic/thermodynamic pathway operating in each case.

New mechanistic interpretations for nitrone reactivity.

The reactivity of nitrones in cycloadditions and related reactions is revisited by introducing a topological perspective. In particular, the study of electron localization function (ELF) along a reaction pathway allows evaluating bond reorganization showing that in several cases the bonds are formed in a sequential way, the second one being formed once the first one is already formed. Both classical 1,3-dipolar cycloadditions and Mannich-type reactions revealed unexpected features often underestimated in classical mechanistic studies.

Regioselectivity Change in the Organocatalytic Enantioselective (3+2) Cycloaddition with Nitrones through Cooperative Hydrogen-Bonding Catalysis/Iminium Activation.

The reaction of nitrones with enals through iminium activation can be modulated by using cooperative hydrogen-bonding catalysis to induce the participation of a nitrone ylide (C-N-C) instead of the classical C-N-O dipole. As a consequence, N-hydroxypyrrolidines are obtained, rather than the expected isoxazolidines. The reaction proceeds smoothly and high enantioselectivities are observed in all cases. By using the appropriate substrate, polysubstituted pyrrolidines incorporating quaternary stereocenters can be efficiently prepared.

Azomethine Ylides from Nitrones: Using Catalytic nBuLi for the Totally Stereoselective Synthesis of trans-2-Alkyl-3-oxazolines.

The cycloaddition of azomethine ylide N-oxides (nitrone ylides) with aldehydes provides 3-oxazolines in a completely stereoselective manner in the presence of a catalytic amount of n-butyllithium. The process involves an initial nucleophilic attack on the aldehyde, followed by intramolecular oxygen addition to the nitrone moiety and lithium-assisted elimination of water, regenerating the catalytic species. Various Li-based catalytic systems are possible and the in situ generated water is required for continuing the catalytic cycle. The best results are observed with 20 mol % of n-butyllithium, whereas the use of stoichiometric amounts inhibit the rate of catalysis. Experimental, spectroscopic, and computational mechanistic studies have provided evidence of lithium-ion catalysis and rationalized several competing catalytic pathways.

Glycomimetics Targeting Glycosyltransferases: Synthetic, Computational and Structural Studies of Less-Polar Conjugates.

The Leloir donors are nucleotide sugars essential for a variety of glycosyltransferases (GTs) involved in the transfer of a carbohydrate to an acceptor substrate, typically a protein or an oligosaccharide. A series of less-polar nucleotide sugar analogues derived from uridine have been prepared by replacing one phosphate unit with an alkyl chain. The methodology is based on the radical hydrophosphonylation of alkenes, which allows coupling of allyl glycosyl compounds with a phosphate unit suitable for conjugation to uridine. Two of these compounds, the GalNAc and galactose derivatives, were further tested on a model GT, such as GalNAc-T2 (an important GT widely distributed in human tissues), to probe that both compounds bound in the medium-high micromolar range. The crystal structure of GalNAc-T2 with the galactose derivative traps the enzyme in an inactive form; this suggests that compounds only containing the ß-phosphate could be efficient ligands for the enzyme. Computational studies with GalNAc-T2 corroborate these findings and provide further insights into the mechanism of the catalytic cycle of this family of enzymes.

Rational Design of Glycomimetic Compounds Targeting the Saccharomyces cerevisiae Transglycosylase Gas2.

The transglycosylase Saccharomyces cerevisiae Gas2 (ScGas2) belongs to a large family of enzymes that are key players in yeast cell wall remodeling. Despite its biologic importance, no studies on the synthesis of substrate-based compounds as potential inhibitors have been reported. We have synthesized a series of docking-guided glycomimetics that were evaluated by fluorescence spectroscopy and saturation-transfer difference (STD) NMR experiments, revealing that a minimum of three glucose units linked via a ß-(1,3) linkage are required for achieving molecular recognition at the binding donor site. The binding mode of our compounds is further supported by STD-NMR experiments using the active site-mutants Y107Q and Y244Q. Our results are important for both understanding of ScGas2-substrate interactions and setting up the basis for future design of glycomimetics as new antifungal agents.

Highly diastereoselective 1,3-dipolar cycloadditions of chiral non-racemic nitrones to 1,2-diaza-1,3-dienes: an experimental and computational investigation.

Asymmetric 1,3-dipolar cycloadditions between 1,2-diaza-1,3-dienes and chiral non-racemic nitrones to give 3-substituted-5-diazenyl isoxazolidines were studied both experimentally and theoretically. Whereas cyclic nitrones provide complete selectivity for the cycloaddition reaction (only one isomer is obtained), acyclic nitrones derived from D-glyceraldehyde and D-galactose lead to 1 : 1 mixtures of two isomers. A DFT analysis based on reactivity indices correctly predicts the regiochemistry of the reaction in agreement with the high electron-withdrawing character of the diazenyl group. The same theoretical studies considering solvent effects (PCM model) based on transition state theory are in qualitative agreement with the observed experimental results.