Site-specific glycosylation of an aglycosylated human IgG1-Fc antibody protein generates neoglycoproteins with enhanced function

A range of well-defined IgG glycoforms was prepared by employing a combination of synthetic carbohydrate chemistry and genetic engineering. The key aspect of this methodology is the coupling of thioaldoses with cysteine-containing proteins to give disulfide-linked neoglycoproteins. This technology was applied to the synthesis of a series of synthetic N-glycan thioaldoses which were coupled to an aglycosylated IgG1-Fc fragment, engineered to have Cys-297 in place of glycan-linked Asn (Deltah-Fc N297C). Analysis of the resulting Fc neoglycoproteins by mass spectrometry and trypsin digestion showed that the saccharides were site-selectively incorporated at Cys-297 to full occupancy without affecting other Fc protein disulfides. The neoglycoproteins were tested for their ability to interact with human FcgammaRI by inhibiting superoxide production by gamma-interferon-stimulated U937 cells. The neoglycoproteins displayed enhanced superoxide inhibition relative to aglycosylated Deltah-Fc N297C, where increased glycan size correlated positively with increased inhibition.     

Thermal behaviour of arsenic oxides (As2O5 and As2O3) and the influence of reducing agents (glucose and activated carbon)

In this paper the thermal behaviour of pure arsenic oxides (As₂O₅.aq and As₂O₃) and the influence of the presence of reducing agents (glucose or activated carbon) on the thermal behaviour of the arsenic oxides are studied through thermogravimetric (TG) analysis.The TG experiments with pure As₂O₅.aq reveal that the reduction reaction As₂O₅→As₂O₃+O₂ does not take place at temperatures lower than 500 °C. At higher temperatures decomposition is observed. Pure As₂O₃, however, is already released at temperatures as low as 200 °C. This release is driven by temperature dependent vapour pressures.Comparing these results with earlier observations concerning the thermal behaviour of chromated copper arsenate (CCA) treated wood, suggests that wood, char and pyrolysis vapours form a reducing environment that influences the thermal behaviour of arsenic oxides. Therefore, the influence of the presence of reducing agents on the thermal behaviour of As₂O₅.aq is studied. First, TG experiments are carried out with mixtures of As₂O₅ and glucose. The TG and DTG curves of the mixture are not a simple superposition of the curves of the two pure constituents. The interaction between As₂O₅.aq and glucose results in a faster decomposition of arsenic pentoxide. This effect is more pronounced if the purge gas nitrogen is mixed with oxygen. Second, TG experiments are performed with mixtures of As₂O₅ and activated carbon. The presence of activated carbon also promotes the volatilisation of arsenic for temperatures higher than 300 °C, probably through its reducing action.Extrapolation of the thermal behaviour of these model compounds to the real situation of pyrolysis of CCA treated wood confirms the statement that the reduction of pentavalent arsenic to trivalent arsenic is favoured by the reducing environment, created by the presence of wood, char and pyrolysis vapours.     

Solid-state NMR (19F and 13C) study of graphite monofluoride (CF)n: 19F spin-lattice magnetic relaxation and 19F/13C distance determination by Hartmann-Hahn cross polarization

Graphite monofluoride (CF)(n) was studied by solid-state NMR. (19)F spin-lattice relaxation time T(1) and second moment measurements of the (19)F line are presented. A "chair" conformation structure is found to be compatible with the experimental data. Relaxation is shown to be mainly due to paramagnetic oxygen. The presence of a molecular motion with an activation energy of 1.685 kJ.mol(-1) (202.7 K) is also evidenced. (19)F magic angle spinning (MAS) NMR and (13)C MAS NMR with (19)F to (13)C cross-polarization allows the determination of CF and CF(2) groups. Reintroduction of dipolar coupling by cross-polarization is used for C-F bond length determination (0.138 +/- 0.001 nm).     

Biosensor immunoassay for flumequine in broiler serum and muscle

Flumequine (Flu) is one of the fluoroquinolones most frequently applied for the treatment of broilers in The Netherlands. For the detection of residues of Flu in blood serum of broilers, a biosensor immunoassay (BIA) was developed which was fast (7.5 min per sample) and specific (no cross-reactivity with other (fluoro)quinolones). This inhibition assay was based on a rabbit polyclonal anti-Flu serum and a CM5 biosensor chip coated with Flu which could be detected in the range of 15-800 ng mL(-1). For the detection of Flu in muscle, an easy extraction procedure in buffer was selected and the measuring range was from 24 to 4000 ng g(-1). Average recoveries of 66 till 75% were found with muscle samples spiked at 0.5, 1 and 2 times the maximum residue limit (MRL in muscle = 400 ng g(-1)) and the decision limit (CCalpha) and the detection capability (CCbeta) were determined as 500 and 600 ng g(-1), respectively. Incurred muscle samples were analysed by the BIA and by LC-MS/MS and a good correlation was found (R2 = 0.998). Serum and muscle samples from with Flu treated broilers were analysed and the concentrations found in serum were always higher than those found in muscle (average serum/muscle ratio was 3.5) and this proved the applicability of the BIA in serum as predictor of the Flu concentration in muscle.     

Biomolecular interaction monitoring of autoantibodies by scanning surface plasmon resonance microarray imaging

A new commercial surface plasmon resonance (SPR) imaging analysis system with a novel SPR dip angle scanning principle allows the measurement, without the need for labeling, of the exact SPR dip angle. With this system hundreds of biomolecular interactions can be monitored on microarrays simultaneously and with great precision. The potency of this system is demonstrated by automatically monitoring the interactions between citrullinated peptides and serum autoantibodies of 50 rheumatoid arthritis (RA) patients and 29 controls in a single step. The smallest antibody concentration that could be measured in this experimental setup was 0.5 pM.     

Linear solvation energy relationships of anionic dimeric surfactants in micellar electrokinetic chromatography II. Effect of the length of a hydrophilic spacer

Anionic dimeric surfactants with hydrophilic spacers containing two to six oxygen atoms were synthesized and applied as pseudostationary phases in micellar electrokinetic chromatography. Their selectivity was determined via linear solvation energy relationships. There were no differences in cohesiveness, polarizability or dipolarity with increasing spacer length, but there was a clear trend in increasing hydrogen bond accepting ability, and a concomitant decrease in hydrogen bond donating ability. The different selectivity of these dimeric surfactants compared to sodium dodecylsulfate can be useful for optimizing separations of mixtures of solutes for which these types of interactions are important. Their critical micelle concentrations were in the range of 0.2-0.3mM, except for the surfactant with the shortest spacer (<0.03 mM), and are much lower than those of conventional surfactants used in micellar electrokinetic chromatography.     

Multifunctional Helicene-Based Ytterbium Coordination Polymer Displaying Circularly Polarized Luminescence,Slow Magnetic Relaxation and Room Temperature Magneto-Chiral Dichroism**

The combination of physical properties sensitive to molecular chirality in a single system allows the observation of fascinating phenomena such as magneto-chiral dichroism (MChD) and circularly polarized luminescence (CPL) having potential applications for optical data readout and display technology. Homochiral monodimensional coordination polymers of Yb^III were designed from a 2,15-bis-ethynyl-hexahelicenic scaffold decorated with two terminal 4-pyridyl units. Thanks to the coordination of the chiral organic chromophore to Yb(hfac)₃ units (hfac⁻=1,1,1,5,5,5-hexafluoroacetylaconate), efficient NIR-CPL activity is observed. Moreover, the specific crystal field around the Yb^III induces a strong magnetic anisotropy which leads to a single-molecule magnet (SMM) behaviour and a remarkable room temperature MChD. The MChD-structural correlation is supported by computational investigations.     

Assembly of a Complex Branched Oligosaccharide by Combining Fluorous‐Supported Synthesis and Stereoselective Glycosylations using Anomeric Sulfonium Ions

There is an urgent need to develop reliable strategies for the rapid assembly of complex oligosaccharides. This paper presents a set of strategically selected orthogonal protecting groups, glycosyl donors modified by a (S)‐phenylthiomethylbenzyl ether at C‐2, and a glycosyl acceptor containing a fluorous tag, which makes it possible to rapidly prepare complex branched oligosaccharides of biological importance. The C‐2 auxiliary controlled the 1,2‐cis anomeric selectivity of the various galactosylations. The orthogonal protecting groups, 2‐naphthylmethyl ether (Nap) and levulinic ester (Lev), made it possible to generate glycosyl acceptors and allowed the installation of a crowded branching point. After the glycosylations, the chiral auxiliary could be removed using acidic conditions, which was compatible with the presence of the orthogonal protecting groups Lev and Nap, thereby allowing the efficient installation of 1,2‐linked glycosides. The light fluorous tag made it possible to purify the compounds by a simple filtration method using silica gel modified by fluorocarbons. The set of building blocks was successfully employed for the preparation of the carbohydrate moiety of the GPI anchor of Trypanosoma brucei, which is a parasite that causes sleeping sickness in humans and similar diseases in domestic animals.     

Polymer networks via cationic ring-opening polymerization

Three methods for the formation of polymer networks from bifunctionally growing polymers obtained by cationic ring-opening polymerization are described. The first method is based on the irreversible inter-molecular termination reaction of thietane polymerizations. Starting from bifunctionally living poly(THF) a new kind of polymer structure consisting of ABA block copolymers with cross-linked A-segments is obtained. The second method is the direct coupling of active species with primary amines or ammonia. The third method consists in transformation of the living end groups of poly(THF) into triethoxysilane end groups, followed by cross-linking by addition of water and a trace of acid.