Gold nanoparticles coated with a pyruvated trisaccharide epitope of the extracellular proteoglycan of Microciona prolifera as potential tools to explore carbohydrate-mediated cell recognition

The species-specific cell adhesion in the marine sponge Microciona prolifera involves the interaction of an extracellular proteoglycan-like macromolecular complex, otherwise known as aggregation factor. In the interaction, two highly polyvalent functional domains play a role: a cell-binding and a self-interaction domain. The self-recognition has been characterized as a Ca(2+)-dependent carbohydrate-carbohydrate interaction of repetitive low affinity carbohydrate epitopes. One of the involved epitopes is the pyruvated trisaccharide beta-d-Galp4,6(R)Pyr-(1-->4)-beta-d-GlcpNAc-(1-->3)-l-Fucp. To evaluate the role of this trisaccharide in the proteoglycan-proteoglycan self-recognition, beta-d-Galp4,6(R)Pyr-(1-->4)-beta-d-GlcpNAc-(1-->3)-alpha-l-Fucp-(1-->O)(CH(2))(3)S(CH(2))(6)SH was synthesized, and partially converted into gold glyconanoparticles. These mimics are being used to explore the self-interaction phenomenon for the trisaccharide epitope, via TEM aggregation experiments (gold glyconanoparticles) and atomic force microscopy (AFM) experiments (self assembled monolayers; binding forces).     

Gold glyconanoparticles for mimics and measurement of metal ion-mediated carbohydrate-carbohydrate interactions

To mimic and measure calcium ion-mediated carbohydrate-carbohydrate interactions, four lactose derivatives have been synthesized for assembly on gold nanoparticles. The series of lactose derivatives varied by the length of the thiolated ethylene glycol anchor chain [O(CH2CH2O)(m)CH2CH2SH; where m = 0, 1, 2, and 3] used to self-assemble the carbohydrates to the preformed gold nanoparticles of ca. 16 nm diameter. Upon addition of calcium ions to the lactose-stabilized nanoparticles, rapid carbohydrate-carbohydrate interactions were visualized and subsequently measured using UV-visible spectrometry and transmission electron microscopy (TEM). The nanoparticle aggregates formed via metal-mediated carbohydrate-carbohydrate interactions could be readily redispersed through the addition of EDTA. Multiple reaggregation and redispersion cycles were achieved, confirming that the aggregation process was due to metal ion-mediated carbohydrate interactions rather than calcium chelation by residual citrate ions on the particle surface. The essential involvement of the lactose moiety in Ca2+ complexation was shown by control measurements on related D-glucose-derivatized nanoparticles, where a significantly reduced aggregation response was obtained only at high ion concentrations. Other group 2 metal ions with radii larger than that of calcium, viz., barium and strontium, were also shown to mediate the aggregation of the lactose-stabilized nanoparticles. The induced aggregation of the lactose nanoparticles was determined to be quantitatively dependent upon the calcium ion concentration. Furthermore, the analytical sensitivity of the calcium-induced aggregation and the linear dynamic range were dependent on the length of the ethylene glycol anchor chain. The shortest ethylene glycol chain (m = 0) gave the most sensitive response with the optimum limit of detection (0.8 mM Ca2+), whereas the longest ethylene glycol chain (m = 3) provides a measurement of calcium ion concentration over the largest linear dynamic range (10-35 mM Ca2+). This work has shown that the self-assembled deposition of lactose derivatives on gold nanoparticles provides multivalent carbohydrate surfaces that can be used as mimics for the measurement of biologically relevant carbohydrate-carbohydrate interactions. Additionally, this study has highlighted the importance of the structure and length of the ligand that anchors the carbohydrate sugar to the gold particle surface to facilitate such carbohydrate interactions and for "tuning" the analytical characteristics of bioassays developed using metal nanoparticle technology.     

Weak calcium-mediated interactions between Lewis X-related trisaccharides studied by NMR measurements of residual dipolar couplings

The Lewis X (LeX) determinant, a trisaccharide with the carbohydrate sequence Galbeta(1-->4)[Fucalpha(1-->3)]GlcNAcbeta, is believed to be responsible for Ca2+-mediated cell-cell interactions. In partly oriented phases composed of mixtures of penta(ethyleneglycol)monododecyl ether HO(CH2CH2O)5C12H25 and n-hexanol in the presence of Ca2+ ions, the variation of the residual dipolar couplings 1DCH of various CiHi vectors in LeX as a function of the concentration of the trisaccharide demonstrates the existence of very weak LeX-Ca2+-LeX complexes in solution. Synthetic 3-, 4-, and 6-deoxy-LeX variants were also shown to form complexes in the presence of calcium ions, despite the replacement of one of their hydroxyl groups by hydrogen atoms. This is the first direct observation in solution of a calcium-mediated interaction between LeX molecules.     

Post-source decay fragmentation analyses of linkage isomers of Lewis-type oligosaccharides in curved-field reflectron matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: combined in-source decay/post-source decay experiments and relative ion abundance analysis

Linkage isomers of Lewis(X) trisaccharide (Le(X)) and Lewis(a) trisaccharide (Le(a)) were distinguished by the post-source decay (PSD) fragment spectra obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) without permethylation. Both Y- and Z-type fragmentations were observed at the C-3 position of N-acetylhexosamine. beta-Elimination at C-3 of the reducing-end N-acetylglucosamine in Le(X) formed a double bond, which conjugated to an N-acetyl group, making the chemical species stable. In contrast, the double bond formed in the reducing end glucose of 3-fucosyllactose was unstable owing to the lack of a conjugated system. Therefore, beta-elimination of N-acetylglucosamine occurred predominantly rather than that of hexose in MALDI-PSD fragmentation. The measurements of the PSD fragment mass spectra using pseudo precursor ions originating from in-source decay were useful for the analyses of the fragmentation mechanisms and for the assignments of the chemical species of the fragment ions. The combined in-source decay/post-source decay experiments revealed the formation of a double bond between C-2 and C-3 in N-acetylglucosamine of Le(X). Abundance analysis of the PSD ions indicated that the 1-3 glycosyl linkage cleaves more easily than does the 1-4 linkage in MALDI-PSD fragmentation. Ion abundance analyses were useful in estimating the degree of Y- and Z-type fragmentation at the C-3 position of hexose and N-acetylhexosamine. The analysis of the relative ion abundances was a powerful tool for the assignments of the chemical species of the PSD ions.     

Insights into molecular recognition of Lewis(X) mimics by DC-SIGN using NMR and molecular modelling

In this work, we have studied in detail the binding of two α-fucosylamide-based mimics of Lewis(X) to DC-SIGN ECD (ECD = extracellular domain) using STD NMR and docking. We have concluded that the binding mode occurs mainly through the fucose moiety, in the same way as Lewis(X). Similarly to other mimics containing mannose or fucose previously studied, we have shown that both compounds bind to DC-SIGN ECD in a multimodal fashion. In this case, the main contact is the interaction of two hydroxyl groups one equatorial and the other one axial (O3 and O4) of the fucose with the Ca(2+) as Lewis(X) and similarly to mannose-containing mimics (in this case the interacting groups are both in the equatorial position). Finally, we have measured the K(D) of one mimic that was 0.4 mM. Competitive STD NMR experiments indicate that the aromatic moiety provides additional binding contacts that increase the affinity.     

Structural basis for the activation of platelet integrin αIIbβ3 by calcium- and integrin-binding protein 1

Calcium and integrin binding protein 1 (CIB1) is a specific binding partner for the cytoplasmic domain of the αIIb subunit of the highly abundant platelet integrin αIIbβ3. This protein has been suggested to be involved in the regulation of the activation of αIIbβ3, a process leading to platelet aggregation and blood coagulation. In this work, the solution structure of the deuterated Ca(2+)-CIB1 protein complexed with an αIIb peptide was first determined through modern RDC-based NMR methods. Next, we generated a complex structure for CIB1 and the αIIb domain (Ca(2+)-CIB1/αIIb) using the program Haddock, which is based on experimental restraints obtained for the protein-peptide interface from cross-saturation NMR experiments. In this data-driven complex structure, the N-terminal α-helix of the cytoplasmic domain of αIIb is buried in the hydrophobic pocket of the C-lobe of Ca(2+)-CIB1. The C-terminal acidic tail of αIIb remains unstructured and likely interacts with several positively charged residues in the N-lobe of Ca(2+)-CIB1. A potential molecular mechanism for the CIB1-mediated activation of the platelet integrin could be proposed on the basis of the model structure of this protein complex. Another feature of this work is that, in the NMR cross-saturation experiments, we applied the selective radio frequency irradiation to the smaller binding partner (the αIIb peptide), and successfully detected the binding interface on the larger binding partner Ca(2+)-CIB1 through its selectively protonated methyl groups. This 'reverse' methodology has a broad potential to be employed to many other complexes where synthetic peptides and a suitably isotope-labeled medium- to large-sized protein are used to study protein-protein interactions.     

Preparation of biotinylated glyconanoparticles via a photochemical process and study of their bioconjugation to streptavidin

We report here the preparation of novel biotinylated glyconanoparticles from well-defined biotinylated glycopolymers and poly(N-isopropylacrylamide) (PNIPAAm) synthesized via the reversible addition fragmentation chain transfer (RAFT) polymerization process. The in situ reduction of the biotinylated glycopolymers, PNIPAAm, poly(ethylene glycol), and HAuCl4 via a photochemical process resulted in the formation of biotinylated gold nanoparticles. The multifunctional biotinylated glyconanoparticles were then evaluated for their bioconjugation toward streptavidin using UV-vis spectroscopy and surface plasmon resonance (SPR). The biotinylated nanoparticles underwent aggregation in the presence of streptavidin as revealed by spectrophotometry, which indicates the accessibility of the biotin for conjugation. These results were further confirmed by surface plasmon resonance even in the case of surface-immobilized streptavidin.     

Hg2+-mediated aggregation of gold nanoparticles for colorimetric screening of biothiols

In this work, we report a colorimetric assay for the screening of biothiols including glutathione (GSH), cysteine (Cys), and homocysteine (Hcys) based on Hg(2+)-mediated aggregation of gold nanoparticles (AuNPs). Hg(2+) can induce aggregation of thiol-containing naphthalimide (1) capped AuNPs due to the cross-linking interactions from the resulting "thymine-Hg(2+)-thymine" (T-Hg(2+)-T) analogous structure. When Hg(2+) is firstly treated with biothiols, followed by mixing with 1-capped AuNPs suspension, AuNPs undergo a transformation from an aggregation to a dispersion state depending on the concentration of biothiols. This anti-aggregation or re-dispersion of AuNPs is due to the higher affinity of Hg(2+) for biothiols relative to compound 1. The corresponding color variation in the process of anti-aggregation of AuNPs can be used for the quantitative screening of biothiols through UV-vis spectroscopy or by the naked eye. Under optimized conditions, a good linear relationship in the range of 0.025-2.28 μM is obtained for GSH, 0.035-1.53 μM for Cys, and 0.040-2.20 μM for Hcys. The detection limits of this assay for GSH, Cys, and Hcys are 17, 9, and 18 nM, respectively. This colorimetric assay exhibits a high selectivity and sensitivity with tunable dynamic range. The proposed method has been successfully used in the determination of total biothiol content in human urine samples.     

Design and synthesis of C3-symmetric Lewis(x) antigen

[structure: see text] C(3)-Symmetric glycoconjugates carrying three equivalent Lewis(X) antigens or beta-lactosides were synthesized from p-nitrophenyl glycosides and trimesic acid via regio- and stereocontrolled glycosylation reactions. An (1)H NMR study has shown that the C(3)-symmetric glycoconjugates soluble in water provide useful probes to investigate the Ca(2+)-dependent Lewis(X)-Lewis(X) association.     

Glyconanoparticles for biomedical applications

Over the past two decades, glycosylated nanoparticles (i.e., glyconanoparticles having sugar residues on the surface) received much attention for biomedical applications such as bioassays and targeted drug delivery. This minireview focuses on three aspects: (1) glycosylated gold nanoparticles, (2) glycosylated quantum dots, and (3) glyconanoparticles self-assembled from amphiphilic glycopolymers. The synthetic methods and the multivalent interactions between glyconanoparticles and lectins is shortly illustrated.     

Gold and gold-iron oxide magnetic glyconanoparticles: synthesis, characterization and magnetic properties

The preparation, characterization and the magnetic properties of gold and gold-iron oxide glyconanoparticles (GNPs) are described. Glyconanoparticles were prepared in a single step procedure in the presence of aqueous solution of thiol functionalized neoglycoconjugates and either gold salts or both gold and iron salts. Neoglycoconjugates of lactose and maltose disaccharides with different linkers were used. Iron-free gold or gold-iron oxide GNPs with controlled gold-iron ratios were obtained. The average core-size diameters are in the range of 1.5-2.5 nm. The GNPs are fully characterized by (1)H NMR spectrometry, transmission electron microscopy (TEM), and UV-vis and X-ray absorption (XAS) spectroscopies. Inductive plasma-atomic emission spectrometry (ICP) and elemental analysis gave the average number of neoglycoconjugates per cluster. The magnetic properties were measured in a SQUID magnetometer. The most remarkable results was the observation of a permanent magnetism up to room temperature in the iron-free gold GNPs, that was not present in the corresponding gold-iron oxide GNPs.     

Hyper‐Assembly of Self‐Assembled Glycoclusters Mediated by Specific Carbohydrate–Carbohydrate Interactions

Hybridization of a self‐assembled, spherical complex with oligosaccharides containing Lewis X, a functional trisaccharide displayed on various cell surfaces, yielded well‐defined glycoclusters. The self‐assembled glycoclusters exhibited homophilic hyper‐assembly in aqueous solution in a Ca²⁺‐dependent manner through specific carbohydrate–carbohydrate interactions, offering a structural scaffold for functional biomimetic systems.     

Interpretation of the magnetic structures of Cu2Te2O5X2 (X = Cl,Br) and Ca3.1Cu0.9RuO6 on the basis of electronic structure considerations: cases for strong super-superexchange interactions involving Cu2+ ions

For super-superexchange interactions between Cu(2+) ions, a qualitative rule was formulated to assess their strengths based on the geometrical parameters of the exchange paths. Spin dimer analysis was carried out for Cu(2)Te(2)O(5)X(2) (X = Cl, Br) and Ca(3.1)Cu(0.9)RuO(6) to evaluate the relative strengths of their superexchange and super-superexchange interactions. The strongest antiferromagnetic interactions in Cu(2)Te(2)O(5)X(2) (X = Cl, Br) are given by the super-superexchange interactions involving the most linear Cu-X.X-Cu paths between tetrahedral clusters Cu(4)O(8)X(4) along the (a +/- b)-directions. The adjacent CuRuO(6) chains of Ca(3.1)Cu(0.9)RuO(6) are antiferromagnetically coupled through the most linear Cu-O.O-Cu paths along the direction perpendicular to the plane of the CuRu zigzag chain. The spin lattices of Cu(2)Te(2)O(5)X(2) and Ca(3.1)Cu(0.9)RuO(6) deduced from our spin dimer analysis are consistent with the available magnetic data. The spin lattice of a magnetic solid should be determined on the basis of appropriate electronic structure considerations.     

Synthesis of a dimeric Lewis antigen and the evaluation of the epitope specificity of antibodies elicited in mice

The Lewis(y)-Lewis(x) heptasaccharide, modified by an artificial aminopropyl spacer, was synthesized by an approach that employed two orthogonally protected lactosamine building blocks. A p-(benzoyl)-benzyl glycoside was used as a novel anomeric protecting group, which could be selectively removed at a late stage in the synthesis, thus offering the benefit of enhanced flexibility. The artificial aminopropyl moiety was modified by a thioacetyl group, which allowed an efficient conjugation to keyhole limpet hemocyanin (KLH) that had been activated with electrophilic 3-(bromoacetamido)-propionyl groups. Mice were immunized with the Le(y)Le(x)-BrAc-KLH antigen. Analysis of the sera by ELISA established that a strong helper T-cell immune response was raised against the Le(y)Le(x) saccharide. Further ELISA analysis showed that the titer for monomeric Le(y) tetrasaccharide was tenfold lower whereas recognition of the Le(x) trisaccharide was negligible.     

Luminescent gold(I) and silver(I) complexes of 2-(diphenylphosphino)-1-methylimidazole (dpim): characterization of a three-coordinate Au(I)-Ag(I) dimer with a short metal-metal separation

The Au(I) and Ag(I) closed-shell metal dimers of 2-(diphenylphosphino)-1-methylimidazole, dpim, were investigated. dpim formed the discreet binuclear species [Ag2(dpim)2(CH3CN)2](2+) (1) when reacted with appropriate Ag(I) salts. Likewise, [Au2(dpim)2](2+) (3) and [AuAg(dpim)3](2+) (4) were produced via reactions with (tht)AuCl, tht is tetrahydrothiophene, and Ag(I). Compound 3 exhibits an intense blue luminescence (lambdamax=483 nm) in the solid state. However, upon initial formation of 3, a small impurity of Cl- was present giving rise to an orange emission (lambdamax=548 nm). Attempts to form [Au2(dpim)2]Cl2 yielded only (dpim)AuCl (2), which is not visibly emissive. The rare three-coordinate heterobimetallic complex [AuAg(dpim)3](2+) (4) exhibits intense luminescence in the solid-state resembling that of 3. The crystal structures of 1-4 were determined, revealing strong intramolecular aurophilic and argentophilic interactions in the dimeric compounds. Compound 1 has an Ag(I)-Ag(I) separation of 2.9932(9) A, while compound 3 has a Au(I)-Au(I) separation of 2.8174(10) A. Compound 4 represents the first example of a three-coordinate Au(I)-Ag(I) dimer and has a metal-metal separation of 2.8635(15) A. The linear Au(I) monomer, 2, has no intermolecular Au(I)-Au(I) interactions, with the closest separation greater than 6.8 A.     

H2C[P(Ph)2AUX]2和HC[P(Ph)2AUX]3(X=I,CI)型配合物的构象和Au (I)-Au(I)相互作用的从头算研究

根据配合物H₂C[P(Ph)₂AUX]₂(X=I,CI)和HC[P(Ph)₂AUX]₃(X=I,CI)的晶体结构对它们进行了从头算研究,在MP2近似水平下得到绕C-P旋转所产生构象的势能曲线,从而揭示AU(I)－AU(I)相互作用. 计算结果表明,在所研究的四个配合物中均存在AU(I)-AU(I)相互作用,该作用较弱,约为10. 0～16. 5kJ/mol,与Schmibaur的实验估计值和Pyykko等对其它模型配合物的计算结果接近.     

Activation of nicotinic acetylcholine receptor prevents the production of reactive oxygen species in fibrillar beta amyloid peptide (1-42)-stimulated microglia

Recent studies have reported that the cholinergic anti-inflammatory pathway regulates peripheral inflammatory responses via alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-alpha and prostaglandin E2 in microglial cultures. In a previous study we showed that ATP released by beta-amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X(7) receptor (P2X(7)R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar beta amyloid peptide (1-42) (fAbeta1-42)-induced ROS production by modulating ATP efflux-mediated Ca(2+) influx through P2X(7)R. Nicotine inhibited ROS generation in fAbeta(1-42)-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and a-bungarotoxin, a selective alpha7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca(2+) influx in fAbeta(1-42)-stimulated microglia. Moreover, ATP release from fAbeta(1-42)-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca(2+) influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X(7)R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fAbeta1-42-stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X(7)R.     

乳糖和麦芽糖水解反应速率常数的毛细管电泳测定方法

利用具有高分辩率和高灵敏度的毛细管电泳－电化学检测方法测定乳糖、麦芽糖及其水解产物的浓度随反应时间变化的规律。在H     

Binding of the two-valence-electron metal ions Sc(+), Ti(2+), and V(3+) to the second-row hydrides BeH(2), BH(3), NH(3), OH(2), and FH: a computational study

We report results from a computational study of the binding in complexes formed from one of the transition-metal ions Sc(+), Ti(2+), or V(3+), each of which has two valence electrons outside an argon core, and one of the second-row hydrides FH, OH(2), NH(3), BH(3), or BeH(2). The complexes that involve the electron-rich ligands FH, OH(2), and NH(3) have strong ion-dipole components to their binding. There are large stabilization energies for sigma-interactions that transfer charge from occupied lone-pair natural bond orbitals on the F, O, or N atom of the (idealized) Lewis structure into empty non-Lewis orbitals on the metal ions; these interactions effectively increase electron density in the bonding region between the metal ion and liganded atom, and the metal ions in these complexes act in the capacity of Lewis acids. The complexes formed from the electron-poor hydrides BH(3) and BeH(2) consistently incorporate bridging hydrogen atoms to support binding, and there are large stabilization energies for interactions that transfer charge from the Be-H or B-H bonds into the region between the metal ion and liganded atom. The metal ions in Sc(+)-BeH(2), Ti(2+)-BeH(2), Ti(2+)-BH(3), and V(3+)-BH(3) act in the capacity of Lewis acids, whereas the scandium ion in Sc(+)-BH(3) acts as a Lewis base.     

Synthesis of 2-aminoethyl 3′-(N-glycolylneuraminyl)-β-actoside and neoglycoconjugates based thereon — a prototype of a cancer vaccine and artificial antigens for the control of immune response

Russian Chemical Bulletin - The 2-aminoethyl β-glycoside of the trisaccharide 3′-(N-glycolylneuraminyl)lactose was synthesized in the framework of studies of approaches to the...