Molecular Recognition of GalNAc in Mucin-Type O-Glycosylation.

Acetylgalactosamine (GalNAc)-type O-glycosylation is an essential posttranslational modification (PTM) that plays fundamental roles in biology. Malfunction of this PTM is exemplified by the presence of truncated O-glycans in cancer. For instance, the glycoprotein MUC1 is overexpressed in many tumor tissues and tends to carry simple oligosaccharides that allow for the presentation of different tumor-associated antigens, such as the Tn or sTn antigens (GalNAc-α-1-O-Thr/Ser and Neu5Acα2-6GalNAcα1-O-Ser/Thr, respectively). In other cases, such as tumoral calcinosis associated with O-glycosylation of the fibroblast growth factor 23, O-glycans are absent or less abundant. Significant progress has been made in determining the three-dimensional structures of biomolecules that recognize GalNAc, such as antibodies, lectins, mucinases, GalNAc-transferases, and other glycosyltransferases. Analysis of the complexes between these entities and GalNAc-containing glycopeptides, in most cases derived from crystallographic or NMR analysis, provides an understanding of the key structural elements that control molecular recognition of these glycopeptides. Here, we describe and compare the binding sites of these proteins in detail, focusing on how the GalNAc moieties interact selectively with them. We also summarize the differences and similarities in GalNAc recognition. In general, the recognition of GalNAc-containing glycopeptides is determined by hydrogen bonds between hydroxyl groups and the N-acetyl group of GalNAc with proteins, as well as CH-π contacts in which the hydrophobic α-face of the sugar and the methyl group of NHAc can be involved. The latter interaction usually provides the basis for selectivity. It is worth noting that binding of these glycopeptides depends primarily on recognition of the sugar moiety, with some exceptions such as a few anti-MUC1 antibodies that primarily recognize the peptide backbone and use the sugar to facilitate shape complementarity or to establish a limited number of interactions with the protein. Focusing specifically on the GalNAc moiety, we can observe that there is some degeneracy of interactions within the same protein families, likely due to substrate flexibility. However, when all studied proteins are considered together, despite the commonalities within each protein family, no pattern can be discerned between the different families, apart from the presence of common residues such as Tyr, His, or Asp, which are responsible for hydrogen bonds. The lack of a pattern can be anticipated, given the diverse functions of mucinases, glycosyltransferases, antibodies, and lectins. Finally, it is important to point out that the conformational differences observed in solution in glycopeptides bearing GalNAc-α-1-O-Ser or GalNAc-α-1-O-Thr also can be found in the bound state. This unique characteristic is exploited, for instance, by the enzyme C1GalT1 to broadly glycosylate both acceptor substrates. The findings summarized in this review may contribute to the rational structure-guided development of therapeutic vaccines, novel diagnostic tools for early cancer detection, and new cancer treatments for cancer with tailored anti-Tn or anti-STn antibodies or new drugs to inhibit GalNAc-T isoenzymes.

A Photochemical Strategy for the Conversion of Nitroarenes into Rigidified Pyrrolidine Analogues.

Bicyclic amines are important motifs for the preparation of bioactive materials. These species have well-defined exit vectors that enable accurate disposition of substituents toward specific areas of chemical space. Of all possible skeletons, the 2-azabicyclo[3.2.0]heptane framework is virtually absent from MedChem libraries due to a paucity of synthetic methods for its preparation. Here, we report a modular synthetic strategy that utilizes nitroarenes as flat and easy-to-functionalize feedstocks for the assembly of these sp3-rich materials. Mechanistically, this approach exploits two concomitant photochemical processes that sequentially ring-expand the nitroarene into an azepine and then fold it into a rigid bicycle pyrroline by means of singlet nitrene-mediated nitrogen insertion and excited-state-4π electrocyclization. A following hydrogenolysis provides, with full diastereocontrol, the desired bicyclic amine derivatives whereby the aromatic substitution pattern has been translated into the one of the three-dimensional heterocycle. These molecules can be considered rigid pyrrolidine analogues with a well-defined orientation of their substituents. Furthermore, unsupervised clustering of an expansive virtual database of saturated N-heterocycles revealed these derivatives as effective isosteres of rigidified piperidines. Overall, this platform enables the conversion of nitroarene feedstocks into complex sp3-rich heterocycles of potential interest to drug development.

Light-Triggered Control of Glucocerebrosidase Inhibitors: Towards Photoswitchable Pharmacological Chaperones.

Piperidine-based photoswitchable derivatives have been developed as putative pharmacological chaperones for glucocerebrosidase (GCase), the defective enzyme in Gaucher disease (GD). The structure-activity study revealed that both the iminosugar and the light-sensitive azobenzene are essential features to exert inhibitory activity towards human GCase and a system with the correct inhibition trend (IC50 of the light-activated form lower than IC50 of the dark form) was identified. Kinetic analyses showed that all compounds are non-competitive inhibitors (mixed or pure) of GCase and the enzyme allosteric site involved in the interaction was identified by means of MD simulations. A moderate activity enhancement of mutant GCase assessed in GD patients' fibroblasts (ex vivo experiments) carrying the most common mutation was recorded. This promising observation paves the way for further studies to improve the benefit of the light-to-dark thermal conversion for chaperoning activity.

Silver-Free Gold-Catalyzed Heterocyclizations through Intermolecular H-Bonding Activation.

Modulable monosulfonyl squaramides have been shown to exert activation of gold(I) chloride complexes through H-bonding in an intermolecular way. Combinations of (PPh3)AuCl or IPrAuCl complexes and an optimal sulfonyl squaramide cocatalyst bearing two 3,5-bis(trifluoromethyl)phenyl groups efficiently catalyzed diverse heterocyclizations and a cyclopropanation reaction, avoiding in all cases undesired side reactions. Computational studies indicate that the Au-Cl bond breaks by transligation to the triple bond in a ternary complex formed by the actual AuCl···HBD catalyst and the substrate.

Organocatalytic Enantioselective Vinylcyclopropane-Cyclopentene (VCP-CP) Rearrangement.

We have demonstrated that the catalytic and enantioselective vinylcyclopropane-cyclopentene rearrangement can be carried out on (vinylcyclopropyl)acetaldehydes through activation via enamine intermediates. The reaction makes use of racemic starting materials that, upon ring opening facilitated by the catalytic generation of a donor-acceptor cyclopropane, deliver an acyclic iminium ion/dienolate intermediate in which all stereochemical information has been deleted. The final cyclization step forms the rearrangement product, showing that chirality transfer from the catalyst to the final compound is highly effective and leads to the stereocontrolled formation of a variety of structurally different cyclopentenes.

Computational studies of Brønsted acid-catalyzed transannular cycloadditions of cycloalkenone hydrazones.

The contribution to the energy barrier of a series of tethers in transannular cycloadditions of cycloalkenes with hydrazones has been computationally studied by using DFT. The Houk's distortion model has been employed to evaluate the influence of the tether in the cycloaddition reaction. That model has been extended to determine the contribution of each tether and, more importantly, the effect exerted between them. In addition to the distortion induced by the tethers, the entropy effects caused by them has also been studied. The analysis of the evolution of the electron localization function along the reaction revealed the highly concerted character of the reaction.

Acyl Group Migration in Pyranosides as Studied by Experimental and Computational Methods.

Acyl group migration affects the synthesis, isolation, manipulation and purification of all acylated organic compounds containing free hydroxyl groups, in particular carbohydrates. While several isolated studies on the migration phenomenon in different buffers have been reported, comprehensive insights into the overall migration process in different monosaccharides under similar conditions have been lacking. Here, we have studied the acyl migration in different monosaccharides using five different acyl groups by a combination of experimental, kinetic and theoretical tools. The results show that the anomeric configuration in the monosaccharide has a major influence on the migration rate, together with the relative configurations of the other hydroxyl groups and the nature of the migrating acyl group. Full mechanistic model, based on computations, demonstrates that the acyl migration proceeds through an anionic stepwise mechanism with linear dependence on the [OH- ] and the pKa of the hydroxyl group toward which the acyl group is migrating.

Brønsted Acid versus Phase-Transfer Catalysis in the Enantioselective Transannular Aminohalogenation of Enesultams.

We have studied the enantioselective transannular aminohalogenation reaction of unsaturated medium-sized cyclic benzosulfonamides by using both chiral Brønsted acid and phase-transfer catalysis. Under optimized conditions, a variety of bicyclic adducts can be obtained with good yields and high enantioselectivities. The mechanism of the reaction was also studied by using computational tools; we observed that the reaction involves the participation of a conformer of the nine-membered cyclic substrate with planar chirality in which the stereochemical outcome is controlled by the relative reactivity of the two pseudorotational enantiomers when interacting with the chiral catalyst.

Acetyl Group Migration in Xylan and Glucan Model Compounds as Studied by Experimental and Computational Methods.

It was recently demonstrated by us that acetyl groups in oligosaccharides can migrate not only within one saccharide unit but also between two different saccharide units. Kinetics of this phenomenon were previously investigated in both mannan model compounds and a naturally occurring polysaccharide. In addition to mannans, there are also several other naturally acetylated polysaccharides, such as xyloglucans and xylans. Both xyloglucans and xylans are some of the most common acetylated polysaccharides in nature, displaying important roles in the plant cells. Considering the various biological roles of natural polysaccharides, it could be hypothesized that the intramolecular migration of acetyl groups might also be associated with regulation of the biological activity of polysaccharides in nature. Consequently, a better understanding of the overall migration phenomenon across the glycosidic bonds could help to understand the potential role of such migrations in the context of the biological activity of polysaccharides. Here, we present a detailed investigation on acetyl group migration in the synthesized xylan and glucan trisaccharide model compounds by a combination of experimental and computational methods, showing that the migration between the saccharide units proceeds from a secondary hydroxyl group of one saccharide unit toward a primary hydroxyl group of the other unit.

Absence of Intermediates in the BINOL-Derived Mg(II)/Phosphate-Catalyzed Desymmetrizative Ring Expansion of 1-Vinylcyclobutanols.

The catalyzed desymmetrizative ring expansion of alkenylcyclobutanols promoted by halofunctionalization of the alkene moiety with N-bromosuccinimide has been experimentally and computationally studied. The reaction yields highly enantioenriched cyclopentanones bearing two all-carbon quaternary stereocenters, one of them being generated in the rearrangement of the cyclobutane ring and the other by enantioselective desymmetrization. The reaction is competitive with the formation of a spiroepoxide, but it turns completely selective toward the cyclopentanone when a chiral bisphosphonium magnesium salt is employed as a catalyst. Mechanistic studies support the formation of an ion pair leading to a complex with only a unit of phosphoric acid, which is the resting state of the catalytic cycle. Calculations reproduce in an excellent way the observed reactivity and predict the effect exerted by the substituents of the aromatic ring linked to the double bond. The computational studies also revealed the reaction as a highly asynchronous concerted process taking place as one kinetic step but in two stages: (i) halogenation of the double bond and (ii) rearrangement of the cyclobutane. No intermediates are present in the reaction as energy minima. The experimental scope of the reaction further confirms the predictions for the observed reactivity and selectivity.

The Essential Role of Water Molecules in the Reaction Mechanism of Protein O-Fucosyltransferase 2.

Protein O-fucosyltransferase 2 (PoFUT2) is an inverting glycosyltransferase (GT) that fucosylates thrombospondin repeats (TSRs) from group 1 and 2. PoFUT2 recognizes a large and diverse number of TSRs through a dynamic network of water-mediated interactions. By X-ray structural studies of C. elegans PoFUT2 complexed to a TSR of group 2, we demonstrate that this GT recognizes similarly the 3D structure of TSRs from both groups 1 and 2. Its active site is highly exposed to the solvent, suggesting that water molecules might also play an essential role in the fucosylation mechanism. We applied QM/MM methods using human PoFUT2 as a model, and found that HsPoFUT2 follows a classical SN 2 reaction mechanism in which water molecules contribute to a great extent in facilitating the release of the leaving pyrophosphate unit, causing the H transfer from the acceptor nucleophile (Thr/Ser) to the catalytic base, which is the last event in the reaction. This demonstrates the importance of water molecules not only in recognition of the ligands but also in catalysis.

Piperidine Azasugars Bearing Lipophilic Chains: Stereoselective Synthesis and Biological Activity as Inhibitors of Glucocerebrosidase (GCase).

We report a straightforward synthetic strategy for the preparation of trihydroxypiperidine azasugars decorated with lipophilic chains at both the nitrogen and the adjacent carbon as potential inhibitors of the lysosomal enzyme glucocerebrosidase (GCase), which is involved in Gaucher disease. The procedure relies on the preparation of C-erythrosyl N-alkylated nitrones 10 through reaction of aldehyde 8 and primary amines 13 followed by oxidation of the imines formed in situ with the methyltrioxorhenium catalyst and urea hydrogen peroxide. The addition of octylMgBr to nitrone 10e provided access to both epimeric hydroxylamines 21 and 22 with opposite configuration at the newly created stereocenter in a stereodivergent and completely stereoselective way, depending on the absence or presence of BF3·Et2O. Final reductive amination and acetonide deprotection provided compounds 14 and 15 from low-cost d-mannose in remarkable 43 and 32% overall yields, respectively, over eight steps. The C-2 R-configured bis-alkylated trihydroxypiperidine 15 was the best ligand for GCase (IC50 = 15 µM), in agreement with MD simulations that allowed us to identify the chair conformation corresponding to the best binding affinity.

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.

Biomarkers-based personalized follow-up in chronic heart failure improves patient's outcomes and reduces care associate cost.

BACKGROUND: Heart failure (HF) is a major and growing medical and economic problem, with high prevalence and incidence rates worldwide. Cardiac Biomarker is emerging as a novel tool for improving management of patients with HF with a reduced left ventricular ejection fraction (HFrEF). METHODS: This is a before and after interventional study, that assesses the impact of a personalized follow-up procedure for HF on patient's outcomes and care associated cost, based on a clinical model of risk stratification and personalized management according to that risk. A total of 192 patients were enrolled and studied before the intervention and again after the intervention. The primary objective was the rate of readmissions, due to a HF. Secondary outcome compared the rate of ED visits and quality of life improvement assessed by the number of patients who had reduced NYHA score. A cost-analysis was also performed on these data. RESULTS: Admission rates significantly decreased by 19.8% after the intervention (from 30.2 to 10.4), the total hospital admissions were reduced by 32 (from 78 to 46) and the total length of stay was reduced by 7 days (from 15 to 9 days). The rate of ED visits was reduced by 44% (from 64 to 20). Thirty-one percent of patients had an improved functional class score after the intervention, whereas only 7.8% got worse. The overall cost saving associated with the intervention was € 72,769 per patient (from € 201,189 to € 128,420) and €139,717.65 for the whole group over 1 year. CONCLUSIONS: A personalized follow-up of HF patients led to important outcome benefits and resulted in cost savings, mainly due to the reduction of patient hospitalization readmissions and a significant reduction of care-associated costs, suggesting that greater attention should be given to this high-risk cohort to minimize the risk of hospitalization readmissions.

A cost-effective IoT learning environment for the training and assessment of surgical technical skills with visual learning analytics.

BACKGROUND: Surgeons need to train and certify their technical skills. This is usually done with the intervention of experts who monitor and assess trainees. Nevertheless, this is a time-consuming task that is subject to variations among evaluators. In recent decades, subjectivity has been significantly reduced through 1) the introduction of standard curricula, such as the Fundamentals of Laparoscopic Surgery (FLS) program, which measures students' performance in specific exercises, and 2) rubrics, which are widely accepted in the literature and serve to provide feedback about the overall technical skills of the trainees. Although these two elements reduce subjectivity, they do not, however, eliminate the figure of the expert evaluator, and so the process remains time consuming. OBJECTIVES: The objective of this work is to automate those parts of the work of the expert evaluator that the technology can measure objectively, using sensors to collect evidence, and visualizations to provide feedback. We designed and developed 1) a cost-effective IoT (Internet of Things) learning environment for the training and assessment of surgical technical skills and 2) visualizations supported by the literature on visual learning analytics (VLA) to provide feedback about the exercises (in real time) and overall performance (at the end of the training) of the trainee. METHODS: A hybrid approach was followed based on previous research for the design of the sensor based IoT learning environment. Previous studies were used as the basis for getting best practices on the tracking of surgical instruments and on the detection of the force applied to the tissue, with a focus on reducing the costs of data collection. The monitoring of the specific exercises required the design of sensors and collection mechanisms from scratch as there is little existing research on this subject. Moreover, it was necessary to design the overall architecture to collect, process, synchronize and communicate the data coming from the different sensors to provide high-level information relevant to the end user. The information to be presented was already validated by the literature and the focus was on how to visualize this information and the optimal time for its presentation to end users. The visualizations were validated with 18 VLA experts assessing the technical aspects of the visualizations and 4 medical experts assessing their functional aspects. RESULTS: This IoT learning environment amplifies the evaluation mechanisms already validated by the literature, allowing automatic data collection. First, it uses IoT sensors to automatically correct two of the exercises defined in the FLS (peg transfer and precision cutting), providing real-time visualizations. Second it monitors the movement of the surgical instruments and the force applied to the tissues during the exercise, computing 6 of the high-level indicators used by expert evaluators in their rubrics (efficiency, economy of movement, hand tremor, depth perception, bimanual dexterity, and respect for tissue), providing feedback about the technical skills of the trainee using a radar chart with these six indicators at the end of the training (summative visualizations). CONCLUSIONS: The proposed IoT learning environment is a promising and cost-effective alternative to help in the training and assessment of surgical technical skills. The system shows the trainees' progress and presents new indicators about the correctness of each specific exercise through real-time visualizations, as well as their general technical skills through summative visualizations, aligned with the 6 more frequent indicators in standardized scales. Early results suggest that although both types of visualizations are useful, it is necessary to reduce the cognitive load of the graphs presented in real time during training. Nevertheless, an additional evaluation is needed to confirm these results.

Enantio- and Diastereoselective Nucleophilic Addition of N-tert-Butylhydrazones to Isoquinolinium Ions through Anion-Binding Catalysis.

A highly enantio- and diastereoselective thiourea-catalyzed dearomatization of isoquinolines employing N-tert-butylhydrazones as neutral α-azo carbanions and masked acyl anion equivalents has been developed. Experimental and computational data supports the generation of highly ordered complexes wherein the chloride behaves as a template for the catalyst, the hydrazone reagent, and the isoquinolinium cation, providing excellent stereocontrol in the formation of two contiguous stereogenic centers. The ensuing selective and high-yielding transformations provide appealing dihydroisoquinoline derivatives.

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.

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

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 ß-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 ß-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 ß-close ion pair (ß-CIP) species, the formation of the α-glycoside is necessarily preceded by a bimolecular α → ß 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 ß-glycosyl triflates against neutral and anionic acceptors.

A Modular Experimentation Methodology for 5G Deployments: The 5GENESIS Approach.

The high heterogeneity of 5G use cases requires the extension of the traditional per-component testing procedures provided by certification organizations, in order to devise and incorporate methodologies that cover the testing requirements from vertical applications and services. In this paper, we introduce an experimentation methodology that is defined in the context of the 5GENESIS project, which aims at enabling both the testing of network components and validation of E2E KPIs. The most important contributions of this methodology are its modularity and flexibility, as well as the open-source software that was developed for its application, which enable lightweight adoption of the methodology in any 5G testbed. We also demonstrate how the methodology can be used, by executing and analyzing different experiments in a 5G Non-Standalone (NSA) deployment at the University of Malaga. The key findings of the paper are an initial 5G performance assessment and KPI analysis and the detection of under-performance issues at the application level. Those findings highlight the need for reliable testing and validation procedures towards a fair benchmarking of generic 5G services and applications.

Enantioselective Synthesis of Tropanes: Brønsted Acid Catalyzed Pseudotransannular Desymmetrization.

The enantioselective synthesis of tropanols has been accomplished through chiral phosphoric acid catalyzed pseudotransannular ring opening of 1-aminocyclohept-4-ene-derived epoxides. The reaction proceeds together with the desymmetrization of the starting material and leads to the direct formation of the 8-azabicyclo[3.2.1]octane scaffold with excellent stereoselectivity. The synthetic applicability of the reaction was demonstrated by the enantioselective synthesis of the two natural products (-)-α-tropanol and (+)-ferruginine.