Aminooxylated Carbohydrates: Synthesis and Applications.

Among other classes of biomolecules, carbohydrates and glycoconjugates are widely involved in numerous biological functions. In addition to addressing the related synthetic challenges, glycochemists have invested intense efforts in providing access to structures that can be used to study, activate, or inhibit these biological processes. Over the past few decades, aminooxylated carbohydrates have been found to be key building blocks for achieving these goals. This review provides the first in-depth overview covering several aspects related to the syntheses and applications of aminooxylated carbohydrates. After a brief introduction to oxime bonds and their relative stabilities compared to related C═N functions, synthetic aspects of oxime ligation and methodologies for introducing the aminooxy functionality onto both glycofuranosyls and glycopyranosyls are described. The subsequent section focuses on biological applications involving aminooxylated carbohydrates as components for the construcion of diverse architectures. Mimetics of natural structures represent useful tools for better understanding the features that drive carbohydrate-receptor interaction, their biological output and they also represent interesting structures with improved stability and tunable properties. In the next section, multivalent structures such as glycoclusters and glycodendrimers obtained through oxime ligation are described in terms of synthetic design and their biological applications such as immunomodulators. The second-to-last section discusses miscellaneous applications of oxime-based glycoconjugates, such as enantioselective catalysis and glycosylated oligonucleotides, and conclusions and perspectives are provided in the last section.

Absolute quantitation of acetaminophen-modified human serum albumin in acute liver failure patients by liquid chromatography/tandem mass spectrometry.

RATIONALE: Acetaminophen (APAP) is a well-known analgesic, deemed a very safe over-the-counter medication. However, it is also the main cause of acute liver failure (ALF) in the Western world, via the formation of its reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), and its covalent attachment to liver proteins. The aim of this study was to develop a sensitive and robust quantitative assay to monitor APAP-protein binding to human serum albumin (HSA) in patient samples. METHODS: A combination of isotope dilution, peptic digestion and solid-phase extraction coupled to liquid chromatography/multiple reaction monitoring (LC/MRM) was employed. An external calibration curve with surrogate modified protein spiked into blank serum was used for absolute quantitation. Samples were analyzed by LC/MRM to measure the modified active site peptide of HSA. The LC/MRM assay was validated and successfully applied to serum samples from patients suffering from APAP-induced ALF. RESULTS: Accuracy ranged from 83.8-113.3%, within-run coefficient of variation (CV) ranged from 0.3-6.9%, and total CVs from 1.6-10.6%. Patient samples ranged from 0.12-3.91 nmol/mL NAPQI-HSA; in-between the assay dynamic range of 0.11-50.13 nmol/mL serum. In vivo median concentrations were found to be 0.62 nmol/mL and 0.91 nmol/mL for non-spontaneous survivors (n = 25) and individuals with irreversible liver damage (n = 10), respectively (p-value = 0.028), demonstrating significant potential as a biomarker for ALF outcome. CONCLUSIONS: A fast and sensitive assay was developed to accurately quantify NAPQI-HSA as a biomarker for APAP-related covalent binding in human serum.

Effect of Dendrimer Generation and Aglyconic Linkers on the Binding Properties of Mannosylated Dendrimers Prepared by a Combined Convergent and Onion Peel Approach.

An efficient study of carbohydrate-protein interactions was achieved using multivalent glycodendrimer library. Different dendrimers with varied peripheral sugar densities and linkers provided an arsenal of potential novel therapeutic agents that could be useful for better specific action and greater binding affinities against their cognate protein receptors. Highly effective click chemistry represents the basic method used for the synthesis of mannosylated dendrimers. To this end, we used propargylated scaffolds of varying sugar densities ranging from 2 to 18 for the attachment of azido mannopyranoside derivatives using copper catalyzed click cycloaddition. Mannopyranosides with short and pegylated aglycones were used to evaluate their effects on the kinetics of binding. The mannosylated dendrons were built using varied scaffolds toward the accelerated and combined "onion peel" strategy These carbohydrates have been designed to fight E. coli urinary infections, by inhibiting the formation of bacterial biofilms, thus neutralizing the adhesion of FimH type 1 lectin present at the tip of their fimbriae against the natural multiantennary oligomannosides of uroplakin 1a receptors expressed on uroepithelial tissues. Preliminary DLS studies of the mannosylated dendrimers to cross- link the leguminous lectin Con A used as a model showed their high potency as candidates to fight the E. coli adhesion and biofilm formation.

Teaming up synthetic chemistry and histochemistry for activity screening in galectin-directed inhibitor design.

A hallmark of endogenous lectins is their ability to select a few distinct glycoconjugates as counterreceptors for functional pairing from the natural abundance of cellular glycoproteins and glycolipids. As a consequence, assays to assess inhibition of lectin binding should necessarily come as close as possible to the physiological situation, to characterize an impact of a synthetic compound on biorelevant binding with pharmaceutical perspective. We here introduce in a proof-of-principle manner work with sections of paraffin-embedded tissue (jejunum, epididymis) and labeled adhesion/growth-regulatory galectins, harboring one (galectin-1 and galectin-3) or two (galectin-8) types of lectin domain. Six pairs of synthetic lactosides from tailoring of the headgroup (3'-O-sulfation) and the aglycone (ß-methyl to aromatic S- and O-linked extensions) as well as three bi- to tetravalent glycoclusters were used as test compounds. Varying extents of reduction in staining intensity by synthetic compounds relative to unsubstituted/free lactose proved the applicability and sensitivity of the method. Flanking cytofluorimetric assays on lectin binding to native cells gave similar grading, excluding a major impact of tissue fixation. The experiments revealed cell/tissue binding of galectin-8 preferentially via one domain, depending on the cell type so that the effect of an inhibitor in a certain context cannot be extrapolated to other cells/tissues. Moreover, the work with the other galectins attests that this assay enables comprehensive analysis of the galectin network in serial tissue sections to determine overlaps and regional differences in inhibitory profiles.

Cu0-Loaded organo-montmorillonite with improved affinity towards hydrogen: an insight into matrix-metal and non-contact hydrogen-metal interactions.

Copper-loaded organo-montmorillonite showed improved affinity towards hydrogen under ambient conditions. Clay ion exchange with a propargyl-ended cation followed by thiol-yne coupling with thioglycerol resulted in a porous structure with a 6 fold higher specific surface area, which dramatically decreased after copper incorporation. X-ray diffraction and photoelectron spectrometry, nuclear magnetic resonance (1H and 13C) and CO2-thermal programmed desorption revealed strong sulfur:Cu0 and oxygen:Cu0 interactions. This was explained in terms of structure compaction that 'traps' Cu0 nanoparticles (CuNPs) and reduces their mobility. Transmission electron microscopy showed predominant 1.0-1.5 nm CuNPs. Hydrogen capture appears to involve predominantly physical interaction, since differential scanning calorimetry measurements gave low desorption heat and almost complete gas release between 20 °C and 75 °C. Possible hydrogen condensation within the compacted structure should hinder gas diffusion inside CuNPs and prevent chemisorption. These results allow safe hydrogen storage with easy gas release to be envisaged even at room temperature under vacuum. The reversible capture of hydrogen can be even more attractive when using natural inorganic supports and commercial plant-derived dendrimers judiciously functionalized, even at the expense of porosity.

Protection against Streptococcus suis Serotype 2 Infection Using a Capsular Polysaccharide Glycoconjugate Vaccine.

Streptococcus suis serotype 2 is an encapsulated bacterium and one of the most important bacterial pathogens in the porcine industry. Despite decades of research for an efficient vaccine, none is currently available. Based on the success achieved with other encapsulated pathogens, a glycoconjugate vaccine strategy was selected to elicit opsonizing anti-capsular polysaccharide (anti-CPS) IgG antibodies. In this work, glycoconjugate prototypes were prepared by coupling S. suis type 2 CPS to tetanus toxoid, and the immunological features of the postconjugation preparations were evaluated in vivo In mice, experiments evaluating three different adjuvants showed that CpG oligodeoxyribonucleotide (ODN) induces very low levels of anti-CPS IgM antibodies, while the emulsifying adjuvants Stimune and TiterMax Gold both induced high levels of IgGs and IgM. Dose-response trials comparing free CPS with the conjugate vaccine showed that free CPS is nonimmunogenic independently of the dose used, while 25 µg of the conjugate preparation was optimal in inducing high levels of anti-CPS IgGs postboost. With an opsonophagocytosis assay using murine whole blood, sera from immunized mice showed functional activity. Finally, the conjugate vaccine showed immunogenicity and induced protection in a swine challenge model. When conjugated and administered with emulsifying adjuvants, S. suis type 2 CPS is able to induce potent IgM and isotype-switched IgGs in mice and pigs, yielding functional activity in vitro and protection against a lethal challenge in vivo, all features of a T cell-dependent response. This study represents a proof of concept for the potential of glycoconjugate vaccines in veterinary medicine applications against invasive bacterial infections.

A Covalent Cysteine-Targeting Kinase Inhibitor of Ire1 Permits Allosteric Control of Endoribonuclease Activity.

The unfolded protein response (UPR) initiated by the transmembrane kinase/ribonuclease Ire1 has been implicated in a variety of diseases. Ire1, with its unique position in the UPR, is an ideal target for the development of therapies; however, the identification of specific kinase inhibitors is challenging. Recently, the development of covalent inhibitors has gained great momentum because of the irreversible deactivation of the target. We identified and determined the mechanism of action of the Ire1-inhibitory compound UPRM8. MS analysis revealed that UPRM8 inhibition occurs by covalent adduct formation at a conserved cysteine at the regulatory DFG+2 position in the Ire1 kinase activation loop. Mutational analysis of the target cysteine residue identified both UPRM8-resistant and catalytically inactive Ire1 mutants. We describe a novel covalent inhibition mechanism of UPRM8, which can serve as a lead for the rational design and optimization of inhibitors of human Ire1.

Glyconanosynthons as powerful scaffolds and building blocks for the rapid construction of multifaceted, dense and chiral dendrimers.

This review represents the first of its kind in that it is mainly devoted to the use of carbohydrates as both scaffolds and building blocks for the construction of a wide range of novel, dense, and chiral dendrimers. It deviates from several previous reviews describing solely carbohydrates as functional surface groups, mostly devoted to biological applications. A brief overview of the most recent synthetic strategies in dendrimer design will be presented for the purpose of comparing their differences, similitudes, and advantages. A particular emphasis will be devoted to the general family of core molecules or scaffolds possessing a large number of functional groups from which, carbohydrates clearly emanate for their wide structural diversities, abundant chiral centers, and the relative ease with which their functional groups can be selectively manipulated. This beneficial characteristic relies on the fact that carbohydrates exist in enantiomeric states, several conformations, anomeric configurations, range of functional groups, and as three to seven carbon units.

Synthesis of Dense and Chiral Dendritic Polyols Using Glyconanosynthon Scaffolds.

Most classical dendrimers are frequently built-up from identical repeating units of low valency (usually AB2 monomers). This strategy necessitates several generations to achieve a large number of surface functionalities. In addition, these typical monomers are achiral. We propose herein the use of sugar derivatives consisting of several and varied functionalities with their own individual intrinsic chirality as both scaffolds/core as well as repeating units. This approach allows the construction of chiral, dense dendrimers with a large number of surface groups at low dendrimer generations. Perpropargylated ß-D-glucopyranoside, serving as an A5 core, together with various derivatives, such as 2-azidoethyl tetra-O-allyl-ß-D-glucopyranoside, serving as an AB4 repeating moiety, were utilized to construct chiral dendrimers using "click chemistry" (CuAAC reaction). These were further modified by thiol-ene and thiol-yne click reactions with alcohols to provide dendritic polyols. Molecular dynamic simulation supported the assumption that the resulting polyols have a dense structure.

Defining the potential of aglycone modifications for affinity/selectivity enhancement against medically relevant lectins: synthesis, activity screening, and HSQC-based NMR analysis.

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N-linked-lactosides and of eight O-linked N-acetyllactosamines, each substituted with a 1,2,3-triazole unit, prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC). The totally regioselective ß-D-(1 → 4) galactosylation of a 6-O-TBDPSi-protected N-acetylglucosamine acceptor provided efficient access to the N-acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface-presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth-regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5-fold enhancement for prototype proteins, with further increases for galectins-3 and -4. Moreover, the availability of (15)N-labeled proteins and full assignments enabled (1)H, (15)N HSQC-based measurements for hu- man galectins-1, -3, and -7 against p-nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin-3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure-activity correlations at other bioinspired sites in glycans and beyond the tested lectin types.

Isotope-labeled differential profiling of metabolites using N-benzoyloxysuccinimide derivatization coupled to liquid chromatography/high-resolution tandem mass spectrometry.

RATIONALE: An isotopic labeling strategy based on derivatizing amine-containing metabolites has been developed using light ((12) C6 ) and heavy ((13) C6 ) N-benzoyloxysuccinimide reagents for semi-targeted metabolomic applications. METHODS: Differentially labeled samples were combined and analyzed simultaneously by liquid chromatography/high-resolution tandem mass spectrometry (LC/HR-MS/MS) to compare relative amounts of amine-containing metabolites. The selectivity of the reaction was determined with model metabolites and was shown to also be applicable to thiol and phenol moieties. The potential for relative quantitation was evaluated in cell extracts and the method was then applied to quantify metabolic perturbations occurring in human cultured cells under normal vs. oxidative stress conditions. RESULTS: A total of 279 derivatized features were detected in HL60 cell extracts, 77 of which yielded significant concentration changes upon oxidative stress treatment. Based on accurate mass measurements and MS/MS spectral matching with reference standard solutions, 10 metabolites were clearly identified. Derivatized compounds were found to have diagnostic fragment ions from the reagent itself, as well as structurally informative ions useful for metabolite identification. CONCLUSIONS: This simple derivatization reaction can be applied to the relative quantitation of amine-, thiol- and phenol-containing compounds, with improved sensitivity and chromatographic peak shapes due to the increased hydrophobicity of polar metabolites not readily amenable to reversed-phase LC/MS analysis.

Absolute quantitation of NAPQI-modified rat serum albumin by LC-MS/MS: monitoring acetaminophen covalent binding in vivo.

Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding.

Aromatic thioglycoside inhibitors against the virulence factor LecA from Pseudomonas aeruginosa.

Three small families of hydrolytically stable thioaryl glycosides were prepared as inhibitors of the LecA (PA-IL) virulence factor corresponding to the carbohydrate binding lectin from the bacterial pathogen Pseudomonas aeruginosa. The monosaccharidic arylthio ß-d-galactopyranosides served as a common template for the major series that was also substituted at the O-3 position. Arylthio disaccharides from lactose and from melibiose constituted the other two series members. In spite of the fact that the natural ligand for LecA is a glycolipid of the globotriaosylceramide having an α-d-galactopyranoside epitope, this study illustrated that the ß-d-galactopyranoside configuration having a hydrophobic aglycon could override the requirement toward the anomeric configuration of the natural sugar. The enzyme linked lectin assay together with isothermal titration microcalorimetry established that naphthyl 1-thio-ß-d-galactopyranoside () gave the best inhibition with an IC50 twenty-three times better than that of the reference methyl α-d-galactopyranoside. In addition it showed a KD of 6.3 µM which was ten times better than that of the reference compound. The X-ray crystal structure of LecA with was also obtained.

Diazo transfer and click chemistry in the solid phase syntheses of lysine-based glycodendrimers as antagonists against Escherichia coli FimH.

Uropathogenic Escherichia coli infections, ultimately leading to cystitis and pyelonephritis, are initially mediated by the adhesion of the bacterial FimH to the transmembrane glycoprotein uroplakin-1a present at the surface of urothelial cells. The adhesion is based on the recognition and high avidity binding between the high-mannose glycans of the uroplakin and the FimH, a mannose-specific lectin located at the tip of type 1 fimbriae. We found that synthetic multiantennary mannopyranosides glycodendrons, harboring triazole functionality at the anomeric position, were potent hemagglutination inhibitors of guinea pig erythrocytes and E. coli. A mannosylated dendrimer exposing up to sixteen sugar residues showed an HAI titer of 1 µM and was thus 500-fold more potent than the corresponding monovalent methyl α-d-mannopyranoside. The synthesis of the glycodendrons involved highly efficient solid-phase synthesis of branched l-lysine scaffolds, diazo transfer reaction on the terminal amine residues, and 1,3-dipolar copper-catalyzed azide-alkyne cycloaddition using propargyl α-d-mannopyranoside.

Palladium-catalyzed Ullmann-type reductive homocoupling of iodoaryl glycosides.

A catalytic synthesis of novel biaryl-linked divalent glycosides was achieved using an electroreductive palladium-catalyzed iodoaryl-iodoaryl coupling reaction. This new method was optimized for the synthesis of divalent biaryl-linked mannopyranosides that was subsequently generalized toward several carbohydrate substrates with yields up to 96%.

Multivalent presentation of mannose on hyperbranched polyglycerol and their interaction with concanavalin A lectin.

We describe the synthesis of multivalent mannose derivatives by using hyperbranched polyglycerols (hPG) as a scaffold with different linker structures. Grafting of protected mannose (Man) units is achieved by using Cu(I) -catalyzed Huisgen click chemistry with either an anomeric azide or propargyl ether onto complementarily functionalized alkyne or azido polymer surfaces. NMR spectroscopy, dynamic light scattering (DLS), IR spectroscopy, size-exclusion chromatography (SEC), and elemental analysis have been used to characterize the hPG-Man compounds. The surface availability and bioactivity of Man-modified polymers were evaluated by using a competitive surface plasmon resonance (SPR)-based binding assay by interactions of the glycopolymers with concanavalin A (Con A), a lectin that binds mannose containing molecules. The results indicated that the novel glycoarchitectures presented in this work are efficient inhibitors of Con A-mannose recognition and resulted in inhibitor concentrations (mean IC(50)) from the micro- to the nanomolar range, whereas the corresponding monovalent mannoside (methyl-Man) requires millimolar concentrations. The results provide an interesting structure-activity relationship for libraries of materials that differ in the linkage of the sugar moiety presented on a biocompatible polyglycerol scaffold.

"Active-latent" thioglycosyl donors and acceptors in oligosaccharide syntheses.

The fine tuning of thioglycosides used as glycosyl donors occurs through careful manipulations of the aglycon's nucleofugality, for example, by using "active-latent" principles. In the first section, the control of the relative leaving group abilities will be discussed in terms of electronic factors, including electron-donating/withdrawing substituents. In the second section, the nucleofugality will be adjusted by steric factors. Quantitative reactivity relationships will then be documented followed by presentation of other controlling elements including locked conformations, solvents, and promoters that will be illustrated throughout.

Enantioselective de novo synthesis of 4-deoxy-D-hexopyranoses via hetero-Diels-Alder cycloadditions: total synthesis of ezoaminuroic acid and neosidomycin.

The de novo synthesis of carbohydrates constitutes an important aspect of organic chemistry, and its application toward deoxy sugars is particularly noteworthy in targeting biologically active compounds. The enantioselective preparation of 4-deoxy-D-ribo-, 4-deoxy-D-lyxo-, and 4-deoxy-D-xylo-hexopyranosides, along with their uronate counterparts has been successfully accomplished using hetero-Diels-Alder reactions as the key step. Jacobsen chromium(III) catalyst and a titanium-binaphthol complex have been used to successfully catalyze diene and aldehyde cycloadditions, leading to optically active dihydropyran templates. 6-Hydroxydesosamine, orthogonally protected ezoaminuroic acid, and neosidomycin were synthesized using a comparative study. Also, a novel chiron approach to 4-deoxy-lyxo-hexopyranosiduronic acid methyl ester derivatives was efficiently accomplished starting from readily accessible starting materials. This work represents a systematic and comprehensive study toward a de novo synthesis of 4-deoxy-hexopyranoses via enantioselective hetero-Diels-Alder reactions.

Organic chemistry and immunochemical strategies in the design of potent carbohydrate-based vaccines.

A brief overview of carbohydrate antigens processing and uptakes involved in the adaptive immune system is highlighted. To counter balance the poor immunogenicity and T-cell independent characteristics of carbohydrate antigens, chemists have developed original hybrid molecules aimed at targeting specific competent immune cell receptors. Amongst several potential vaccine candidates dedicated against diseases, this short report will focused on those most advance and state of the art organic chemistry involved therein. One case has led to the first example of a commercial vaccine entirely prepared from a synthetic carbohydrate antigen against infections caused by the Gram-negative bacteria Haemophilus influenza type b responsible for pneumonia and acute bacterial meningitis in infants. Other commendable examples will illustrate the immunochemical strategies engaged in the development of anticancer carbohydrate-based vaccines.

Aberrant glycosylation patterns on cancer cells: Therapeutic opportunities for glycodendrimers/metallodendrimers oncology.

Despite exciting discoveries and progresses in drug design against cancer, its cure is still rather elusive and remains one of the humanities major challenges in health care. The safety profiles of common small molecule anti-cancer therapeutics are less than at acceptable levels and limiting deleterious side-effects have to be urgently addressed. This is mainly caused by their incapacity to differentiate healthy cells from cancer cells; hence, the use of high dosage becomes necessary. One possible solution to improve the therapeutic windows of anti-cancer agents undoubtedly resides in modern nanotechnology. This review presents a discussion concerning multivalent carbohydrate-protein interactions as this topic pertains to the fundamental aspects that lead glycoscientists to tackle glyconanoparticles. The second section describes the detailed properties of cancer cells and how their aberrant glycan surfaces differ from those of healthy cells. The third section briefly describes the immune systems, both innate and adaptative, because the numerous displays of cell surface protein receptors necessitate to be addressed from the multivalent angles, a strength full characteristic of nanoparticles. The next chapter presents recent advances in glyconanotechnologies, including glycodendrimers in particular, as they apply to glycobiology and carbohydrate-based cancer vaccines. This was followed by an overview of metallodendrimers and how this rapidly evolving field may contribute to our arsenal of therapeutic tools to fight cancer. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.