Metabolic Glycoengineering in hMSC-TERT as a Model for Skeletal Precursors by Using Modified Azide/Alkyne Monosaccharides

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac₄ManNAz) and N-alkyneacetylmannosamine (Ac₄ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac₄ManNAz was detectable for up to six days while Ac₄ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.     

Expanding the Genetic Code for a Dinitrophenyl Hapten

Haptens, such as dinitrophenyl (DNP) are small molecules that induce strong immune responses when attached to proteins or peptides and, as such, have been exploited for diverse applications. We engineered a Methanosarcina barkeri pyrrolysyl‐tRNA synthetase (mbPylRS) to genetically encode a DNP‐containing unnatural amino acid, N⁶‐(2‐(2,4‐dinitrophenyl)acetyl)lysine (DnpK). Although this moiety was unstable in Escherichia coli, we found that its stability was enhanced in mammalian HEK 293T cells and was able to induce selective interactions with anti‐DNP antibodies. The capability of genetically introducing DNP into proteins is expected to find broad applications in biosensing, immunology, and therapeutics.     

First Application of Secondary Phosphines as Supporting Ligands for the Palladium‐Catalyzed Heck Reaction: Efficient Activation of Aryl Chlorides

Secondary dialkylphosphines were successfully used for the first time as efficient supporting ligands for the palladium‐catalyzed Heck reaction of electron‐rich and electron‐poor aryl chlorides with olefins such as acrylate, ethylene, styrene, and n‐butyl vinyl ether. The yields with HP(t‐butyl)₂ and HP(adamantyl)₂ were comparable or better than those obtained with known systems of tertiary phosphines such as P(cyclohexyl)₃ and P(t‐butyl)₃, especially at a catalyst loading of <1 mol %. In comparison with tertiary phosphines, the secondary phosphines have the advantage of being readily available at low cost on a technical scale, and are comparable with respect to handling and oxygen sensitivity.     

Quantitative Standards of 4-O-Acetyl- and 9-O-Acetyl-N-Acetylneuraminic Acid for the Analysis of Plasma and Serum

N-Acetylneuraminic acid (sialic acid, Neu5Ac) is one of a large, diverse family of nine-carbon monosaccharides that play roles in many biological functions such as immune response. Neu5Ac has previously been identified as a potential biomarker for the presence and pathogenesis of cardiovascular disease (CVD), diabetes and cancer. More recent research has highlighted acetylated sialic acid derivatives, specifically Neu5,9Ac₂, as biomarkers for oral and breast cancers, but advances in analysis have been hampered due to a lack of commercially available quantitative standards. We report here the synthesis of 9-O- and 4-O-acetylated sialic acids (Neu5,9Ac₂ and Neu4,5Ac₂) with optimisation of previously reported synthetic routes. Neu5,9Ac₂ was synthesised in 1 step in 68 % yield. Neu4,5Ac₂ was synthesised in 4 steps in 39 % overall yield. Synthesis was followed by analysis of these standards via quantitative NMR (qNMR) spectroscopy. Their utilisation for the identification and quantification of specific acetylated sialic acid derivatives in biological samples is also demonstrated.     

One‐Step Derivatization of Reducing Oligosaccharides for Rapid and Live‐Cell‐Compatible Chelation‐Assisted CuAAC Conjugation

We report a new reagent for the functionalization of unprotected oligosaccharides with a picolyl azide group at the anomeric position for chelation‐assisted copper‐catalyzed alkyne–azide cycloaddition (CuAAC) glycoconjugation. We show that oligosaccharides functionalized with this moiety react with an apparent second‐order rate constant of 193 m ^?1 s^?1 and can be used to functionalize biomolecules bearing alkyne moieties introduced through metabolic labeling, including in live cells.     

Effect of Ganglioside GM3 Synthase Gene Knockout on the Glycoprotein N‐Glycan Profile of Mouse Embryonic Fibroblast

The structural and clinical significance of cellular glycoproteins and glycosphingolipids (GSLs) are often separately discussed. Considering the biosynthetic pathway of glycoconjugates, glycans of cell‐surface glycoproteins and GSLs might partially share functions in maintaining cellular homeostatis. The purpose of this study is to establish a general and comprehensive glycomics protocol for cellular GSLs and N‐glycans of glycoproteins. To test the feasibility of a glycoblotting‐based protocol, whole glycans released both from GSLs and glycoproteins were profiled concurrently by using GM3 synthase‐deficient mouse embryonic fibroblast GM3(?/?). GM3(?/?) cells did not synthesize GM3 or any downstream product of GM3 synthase. Instead, expression levels of o‐series gangliosides involving GM1‐b and GD1‐α increased dramatically, whereas a‐/b‐series gangliosides were predominantly detected in wild‐type (WT) cells. We also discovered that glycoprotein N‐glycan profiles of GM3(?/?) cells are significantly altered as compared to WT cells, although GM3 synthase is responsible only for GSLs synthesis and is not associated with glycoprotein N‐glycan biosynthesis. The present approach allows for high‐throughput profiling of cellular glycomes enriched by different classes of glycoconjugates, and our results demonstrated that gene knockout of the enzymes responsible for GSL biosynthesis significantly influences the N‐glycans of glycoproteins.     

Glycan Sequence‐Dependent Nod2 Activation Investigated by Using a Chemically Synthesized Bacterial Peptidoglycan Fragment Library

Nucleotide oligomerization domain‐containing protein 2 (Nod2), an innate immune receptor, recognizes bacterial cell‐wall peptidoglycan (PGN), the minimum ligand of which is muramyl dipeptide (MDP). Enzymatic digestion of PGN appears to be important for Nod2 recognition. PGN is degraded by muramidase or glucosamidase through a process that produces two types of glycan sequence; glycans containing GlcNAcβ(1→4)MurNAc or MurNAcβ(1→4)GlcNAc. In this report, a range of disaccharide or tetrasaccharide fragments of each sequence were chemically synthesized, and their activities in stimulating human Nod2 (hNod2) were investigated. The results reveal that hNod2 recognitions is dependent on the glycan sequence, as demonstrated by comparing the activities of glycans with the same peptide moieties. (MurNAcβ(1→4)GlcNAc)₂‐containing structures exhibited stronger activity than those containing (GlcNAcβ(1→4)MurNAc)₂. The results suggest that differences in the enzymatic degradation process affect the host's immunomodulation process.     

Genetics of Host Protection against Helicobacter pylori Infections

This narrative review discusses the genetics of protection against Helicobacter pylori (Hp) infection. After a brief overview of the importance of studying infectious disease genes, we provide a detailed account of the properties of Hp, with a view to those relevant for our topic. Hp displays a very high level of genetic diversity, detectable even between single colonies from the same patient. The high genetic diversity of Hp can be evaded by stratifying patients according to the infecting Hp strain. This approach enhances the power and replication of the study. Scanning for single nucleotide polymorphisms is generally not successful since genes rarely work alone. We suggest selecting genes to study from among members of the same family, which are therefore inclined to cooperate. Further, extending the analysis to the metabolism would significantly enhance the power of the study. This combined approach displays the protective role of MyD88, TIRAP, and IL1RL1 against Hp infection. Finally, several studies in humans have demonstrated that the blood T cell levels are under the genetic control of the CD39⁺ T regulatory cells (T_REGS).     

Glycidyl Azide Polymer Crosslinked Through Triazoles by Click Chemistry: Curing,Mechanical and Thermal Properties

Glycidyl azide polymer (GAP) was cured through “click chemistry” by reaction of the azide group with bispropargyl succinate (BPS) through a 1,3‐dipolar cycloaddition reaction to form 1,2,3‐triazole network. The properties of GAP‐based triazole networks are compared with the urethane cured GAP‐systems. The glass transition temperature (T_g), tensile strength, and modulus of the system increased with crosslink density, controlled by the azide to propargyl ratio. The triazole incorporation has a higher T_g in comparison to the GAP‐urethane system (T_g−20 °C) and the networks exhibit biphasic transitions at 61 and 88 °C. The triazole curing was studied using Differential Scanning Calorimetry (DSC) and the related kinetic parameters were helpful for predicting the cure profile at a given temperature. Density functional theory (DFT)‐based theoretical calculations implied marginal preference for 1,5‐addition over 1,4‐addition for the cycloaddition between azide and propargyl group. Thermogravimetic analysis (TG) showed better thermal stability for the GAP‐triazole and the mechanism of decomposition was elucidated using pyrolysis GC‐MS studies. The higher heat of exothermic decomposition of triazole adduct (418 kJ ⋅ mol⁻¹) against that of azide (317 kJ ⋅ mol⁻¹) and better mechanical properties of the GAP‐triazole renders it a better propellant binder than the GAP‐urethane system.     

Synthesis of poly(methyl methacrylate‐g‐glycidyl azide) graft copolymers using N,N‐dithiocarbamate‐mediated iniferters

A glycidyl azide polymer with pendent N, N‐diethyl dithiocarbamate groups (GAP‐DDC) was prepared by the reaction of poly(epichlorohydrin) (PECH) with pendent N, N‐diethyl dithiocarbamate groups (PECH‐DDC) and sodium azide (NaN₃) in dimethylformamide (DMF). It was then used as a macro‐photoinitiator for the graft polymerization of methyl methacrylate (MMA). Photopolymerization was carried out in a photochemical reactor at a wavelength greater than 300 nm. Conversion was determined gravimetrically and first‐order time conversion plot for the polymerization system showed linear increase with the polymerization time indicating that polymerization proceed in controlled fashion. The molecular weight distribution (M_w/M_n) was in the range of 1.4–1.6 during polymerization. The formation of poly(methyl methacrylate‐g‐glycidyl azide) (PMMA‐g‐GAP) graft copolymer was characterized by gel permeation chromatography, FT‐IR spectroscopy, Thermogravimetric analysis, and differential scanning calorimetry. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Drug Screening Boosted by Hyperpolarized Long‐Lived States in NMR

Transverse and longitudinal relaxation times (T_1ρ and T₁) have been widely exploited in NMR to probe the binding of ligands and putative drugs to target proteins. We have shown recently that long‐lived states (LLS) can be more sensitive to ligand binding. LLS can be excited if the ligand comprises at least two coupled spins. Herein we broaden the scope of ligand screening by LLS to arbitrary ligands by covalent attachment of a functional group, which comprises a pair of coupled protons that are isolated from neighboring magnetic nuclei. The resulting functionalized ligands have longitudinal relaxation times T₁(¹H) that are sufficiently long to allow the powerful combination of LLS with dissolution dynamic nuclear polarization (D‐DNP). Hyperpolarized weak “spy ligands” can be displaced by high‐affinity competitors. Hyperpolarized LLS allow one to decrease both protein and ligand concentrations to micromolar levels and to significantly increase sample throughput.     

Caulerpin Mitigates Helicobacter pylori-Induced Inflammation via Formyl Peptide Receptors

The identification of novel strategies to control Helicobacter pylori (Hp)-associated chronic inflammation is, at present, a considerable challenge. Here, we attempt to combat this issue by modulating the innate immune response, targeting formyl peptide receptors (FPRs), G-protein coupled receptors that play key roles in both the regulation and the resolution of the innate inflammatory response. Specifically, we investigated, in vitro, whether Caulerpin—a bis-indole alkaloid isolated from algae of the genus Caulerpa—could act as a molecular antagonist scaffold of FPRs. We showed that Caulerpin significantly reduces the immune response against Hp culture filtrate, by reverting the FPR2-related signaling cascade and thus counteracting the inflammatory reaction triggered by Hp peptide Hp(2–20). Our study suggests Caulerpin to be a promising therapeutic or adjuvant agent for the attenuation of inflammation triggered by Hp infection, as well as its related adverse clinical outcomes.     

Evidence of Strict Stereospecificity in the Structure of sn‐1,2‐Diacyl‐3‐Acetyl‐Glycerols from Euonymus maximowiczianus Seeds Using Nuclear Magnetic Resonance Spectroscopy

Asymmetric, optically active sn‐1,2‐diacyl‐3‐acetyl‐glycerols (AcDAG) have been known to scientists for several decades. However, to date, the problem of their structure has not been definitely resolved, which has led to a vast diversity of terms used for their designation in the literature. Using two‐dimensional nuclear magnetic resonance, we have investigated AcDAG from the mature seeds of Euonymus maximowiczianus, from which we have been able to both identify a correlation of the methyl group in acetic acid residue with protons at the carbon atom at sn‐3 position in the glycerol residue of the AcDAG molecule and, for the first time, demonstrate that this correlation is observed exclusively with one carbon atom at the α‐position, but not with two as would have been expected in case of a racemic mixture. Moreover, results of our analysis of AcDAG isolated from the seeds of E. maximowiczianus directly confirm that diacylglycerol‐3‐acetyl‐transferase is responsible for their biosynthesis, which reveals a strict specificity not only to acetyl‐CoA as one of the substrates but also to the sn‐3‐position of the glycerol residue in sn‐1,2‐diacylglycerol during their biosynthesis.     

Polymer‐Supported N‐Phenylsulfonyloxaziridine (Davis Reagent): A Versatile Oxidant

An efficient preparation of a new recyclable polymer‐supported oxidizing agent based on N‐phenylsulfonyloxaziridine has been developed. This new polymer‐supported oxidant was shown to effect a clean and selective conversion of a range of sulfides, selenides, amines, phosphines and enolates to the corresponding sulfoxides, selenoxides, N‐oxides, phosphine oxides and α‐hydroxy carbonyl compounds. It also enabled tetrahydrobenzimidazoles to be oxidatively rearranged to spiro‐fused 5‐imidazolones. Recycling of the polymer‐supported oxidant is possible with minimal loss of activity after several reoxidations.     

New Antimicrobial Hexapeptides: Synthesis,Antimicrobial Activities,Cytotoxicity, and Mechanistic Studies

Synthetic antimicrobial peptides have recently emerged as promising candidates against drug‐resistant pathogens. We identified a novel hexapeptide, Orn‐D ‐Trp‐D ‐Phe‐Ile‐D ‐Phe‐His(1‐Bzl)‐NH₂, which exhibits broad‐spectrum antifungal and antibacterial activity. A lead optimization was undertaken by conducting a full amino acid scan with various proteinogenic and non‐proteinogenic amino acids depending on the hydrophobic or positive‐charge character of residues at various positions along the sequence. The hexapeptide was also cyclized to study the correlation between the linear and cyclic structures and their respective antimicrobial activities. The synthesized peptides were found to be active against the fungus Candida albicans and Gram‐positive bacteria such as methicillin‐resistant Staphylococcus aureus and methicillin‐resistant Staphylococcus epidermidis, as well as the Gram‐negative bacterium Escherichia coli; MIC values for the most potent structures were in the range of 1–5 μg mL^?1 (IC₅₀ values in the range of 0.02–2 μg mL^?1). Most of the synthesized peptides showed no cytotoxic effects in an MTT assay up to the highest test concentration of 200 μg mL^?1. A tryptophan fluorescence quenching study was performed in the presence of negatively charged and zwitterionic model membranes, mimicking bacterial and mammalian membranes, respectively. The results of the fluorescence study demonstrate that the tested peptides are selective toward bacterial over mammalian cells; this is associated with a preferential interaction between the peptides and the negatively charged phospholipids of bacterial cells.     

Recognition and Conformational Properties of an Alternative Antithrombin Binding Sequence Obtained by Chemoenzymatic Synthesis

Heparin is a highly sulfated glycosaminoglycan (GAG) of natural origin used as an anticoagulant and antithrombotic drug. These properties are principally based on the binding and activation of antithrombin (AT) through the pentasaccharide sequence GlcNAc/NS,6S‐GlcA‐GlcNS,3,6S‐IdoA2S‐GlcNS,6S (AGA*IA). Literature data show that the population of the ²S₀ ring conformation of the 2‐O‐sulfo‐α‐l ‐iduronic acid (IdoA2S) motif correlates with the affinity and activation of AT. It was recently demonstrated that two synthetic AGA*IA‐containing hexasaccharides (one G unit added at the reducing end), differing in the degree of sulfation of the IdoA unit, show comparable affinity and ability to activate AT, despite a different conformation of the IdoA residue. In this paper, the binding of these two glycans to AT was studied by isothermal titration microcalorimetry (ITC), transferred (tr‐) NOESY, saturation transfer difference (STD) NMR spectroscopy and molecular dynamics (MD) simulations. Results indicated that both the IdoA2S and the IdoA units assume a ²S₀ conformation when bound with AT, and so present a common binding epitope for the two glycans, centred on the AGA*IA sequence.     

α‐Thiophene end‐capped styrene copolymer containing fullerene pendant moieties: Synthesis,characterization, and gas sensing properties

We report the synthesis, characterization, and gas sensing properties of a styrene copolymer bearing α‐thiophene end group and fullerene (C₆₀) pendant moieties P(S‐co‐CMS‐C₆₀). First, the copolymer of styrene (S) and chloromethylstyrene (CMS) monomers was prepared in bulk via a bimolecular nitroxide‐mediated radical polymerization (NMP) technique using benzoyl peroxide (BPO) as the radical initiator and nitroxy‐functional thiophene compound (Thi‐TEMPO) as the co‐radical and this gave α‐thiophene end‐capped copolymer P(S‐co‐CMS). The chloromethylstyrene units of P(S‐co‐CMS) allowed further side‐chain functionalization onto P(S‐co‐CMS). The obtained P(S‐co‐CMS) was then reacted with sodium azide (NaN₃) and this led to the copolymer with pendant azide groups, P(S‐co‐CMS‐N₃), and then grafted with electron‐acceptor C₆₀ via the reaction between N₃ and C₆₀. The final product was characterized by using NMR, FTIR, and UV–vis methods. Electrical characterization of P(S‐co‐CMS‐C₆₀) thin film was also investigated at between 30 and 100 °C as the ramps of 10 °C. Temperature dependent electrical characterization results showed that P(S‐co‐CMS‐C₆₀) thin film behaves like a semiconductor. Furthermore, P(S‐co‐CMS‐C₆₀) was employed as the sensing layer to investigate triethylamine (TEA), hydrogen (H₂), acetone, and ethanol sensing properties at 100 °C. The results revealed that P(S‐co‐CMS‐C₆₀) thin film has a sensing ability to H₂. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43641.     

Solvent‐free diazotization–azidation of aryl amine using a polymer‐supported azide ion

Crosslinked poly (4‐vinylpyridine)‐supported azide ion was used as an effective azidating agent for deazodination of stable arenediazonium salts under solvent‐free conditions in high yields. The diazotization of aromatic amines was prepared by grinding the combination of an aromatic amine, sodium nitrite (NaNO₂), p‐toluene sulfonic acid (p‐TsOH), and 0.2 mL H₂O in a mortar. Grinding was continued for deazodination–azidation of the obtained relatively stable diazonium salts, with addition of crosslinked poly (4‐vinylpyridine) supported azide ion to obtain the corresponding aryl azides. The spent polymeric reagents can usually be removed and regenerated. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Temperature and pH responsive hydrogels based on polyethylene glycol analogues and poly(methacrylic acid) via click chemistry

A novel temperature responsive copolymer, poly[2‐(2‐methoxyethoxy)ethyl methacrylate‐co‐oligo(ethylene glycol)methacrylate‐co‐N‐hydroxymethyl acrylamide] [P(MEO₂MA‐co‐OEGMA‐co‐HMAM)], was synthesized by atom transfer radical polymerization. pH responsive poly(methacrylic acid) (PMAA) was synthesized by reversible addition‐fragmentation chain transfer polymerization. After the hydroxyl groups on P(MEO₂MA‐co‐OEGMA‐co‐HMAM) were transformed into azide groups and the carboxyl groups on PMAA were transformed into alkyne groups respectively, a novel temperature and pH responsive hydrogel was fabricated by click chemistry between the azide‐P(MEO₂MA‐co‐OEGMA‐co‐HMAM) and alkyne‐PMAA in the presence of CuSO₄ and sodium ascorbate in aqueous solution. The rheological kinetics of gel formation demonstrated that gelation had commenced within 5 min at 25 °C, since then the storage modulus (G′) was higher than the loss modulus (G″). SEM images of hydrogel morphology and the swelling ratios of hydrogel at different temperatures and pH proved that the formed hydrogel had temperature and pH sensitivities. Bovine serum albumin was used as a model to evaluate the sustained release of the hydrogel; the results indicated that the hydrogel was a promising candidate for controlling protein drug delivery. © 2015 Society of Chemical Industry