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ABSTRACT: Here we present a new microparticle system for the selective detection and magnetic removal of bacteria from contaminated solutions. The novelty of this system lies in the combination of a biocompatible scaffold reducing unspecific interactions with high capacity for bacteria binding. We apply highly porous poly(ethylene glycol) (PEG) microparticles and functionalize them introducing both sugar ligands for specific bacteria targeting and cationic moieties for electrostatic loading of superparamagnetic iron oxide nanoparticles. The resulting magnetic, porous, sugar-functionalized (MaPoS) PEG microgels are able to selectively bind and discriminate between different strains of bacteria Escherichia coli. Furthermore, they allow for a highly efficient removal of bacteria from solution as their increased surface area can bind three times more bacteria than non-porous particles. All in all, MaPoS particles represent a novel generation of magnetic beads introducing for the first time a porous, biocompatible and easy to functionalize scaffold and show great potential for various biotechnological applications.
Biomacromolecules 04/2013; · 5.48 Impact Factor
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ABSTRACT: Automated carbohydrate synthesis breaks new grounds: The longest sugar chemically synthesized to date (a 30 mer) has been accessed. Key to the process is the use of a catch-release technique, which labels the saccharide, thus allowing it to be separated later through temporary attachement to magnetic particles.
Angewandte Chemie International Edition 04/2013; · 13.45 Impact Factor
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ABSTRACT: Glycosaminoglycans (GAGs) are important sulfated carbohydrates prevalent in the extracellular matrix. The synthesis of structurally defined GAGs requires laborious procedures, and incorporating defined sulfation patterns is challenging. The automated synthesis of defined sulfated chondroitin hexasaccharides on solid support has been achieved using a photolabile linker that is efficiently cleaved in a continuous-flow photoreactor.
Angewandte Chemie International Edition 04/2013; · 13.45 Impact Factor
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ABSTRACT: Carbohydrates on cell surfaces are critical components of the extracellular landscape and contribute to cell signalling, motility, adhesion and recognition. Multivalent effects are essential to these interactions that are inherently weak. Carbohydrate-functionalised surfaces meet an important need for studying the multivalent interactions between carbohydrates and other biomolecules. Innovations in nanomaterials are revolutionising how these carbohydrate interfaces are studied and underscore their importance in the cosmos of biochemical interactions.
Chemistry 03/2013; 19(12):3794-800. · 5.93 Impact Factor
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ABSTRACT: The first total synthesis of the O-antigen pentasaccharide repeating unit from Gram-negative bacteria Escherichia coli O111 was achieved starting from four monosaccharide building blocks. Key to the synthetic approach was a bis-glycosylation reaction to combine trisaccharide 10 and colitose 5. The colitose building block (5) was obtained de novo from non-carbohydrate precursors. The pentasaccharide was equipped at the reducing end with an amino spacer to provide a handle for subsequent conjugation to a carrier protein in anticipation of immunological studies.
Chemistry 03/2013; 19(12):3995-4002. · 5.93 Impact Factor
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ABSTRACT: Automated solid phase synthesis enables rapid access to the linear and branched arabinofuranoside oligosaccharides. A simple purification step is sufficient to provide the conjugation ready oligosaccharides in good yield.
Chemical Communications 01/2013; · 6.17 Impact Factor
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ABSTRACT: Poly/oligo(amidoamine)s (PAAs) have recently been recognised for their potential as well-defined scaffolds for multiple carbohydrate presentation and as multivalent ligands. Herein, we report two complimentary strategies for the preparation of such sequence-defined carbohydrate-functionalised PAAs that use photochemical thiolene coupling (TEC) as an alternative to the established azide-alkyne cycloaddition ("click") reaction. In the first approach, PAAs that contained multiple olefins were synthesised on a solid support from a new building block and subsequent conjugation with unprotected thio-carbohydrates. Alternatively, a pre-functionalised building block was prepared by using TEC and assembled on a solid support to provide a carbohydrate-functionalised PAA. Both methods rely on the use of a continuous flow photoreactor for the TEC reactions. This system is highly efficient, owing to its short path length, and requires no additional radical initiator. Performing the reactions at 254 nm in Teflon AF-2400 tubing provides a highly efficient TEC procedure for carbohydrate conjugation, as demonstrated in the reactions of O-allyl glycosides with thiols. This method allowed the complete functionalisation of all of the reactive sites on the PAA backbone in a single step, thereby obtaining a defined homogeneous sequence. Furthermore, reaction at 366 nm in FEP tubing in the flow reactor enabled the large-scale synthesis of an fluorenylmethyloxycarbonyl (Fmoc)-protected glycosylated building block, which was shown to be suitable for solid-phase synthesis and will also allow heterogeneous sequence control of different carbohydrates along the oligomeric backbone. These developments enable the synthesis of sequence-defined carbohydrate-functionalised PAAs with potential biological applications.
Chemistry 01/2013; · 5.93 Impact Factor
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ABSTRACT: Automated oligosaccharide assembly requires suitable linkers to connect the first monosaccharide to a solid support. A new hydrogenolysis-labile linker that is stable under both acidic and basic conditions was designed, synthesized and coupled to different resins. Glycosylation and cleavage efficiencies on these functionalized solid supports were investigated, and restrictions for the choice of solid support for oligosaccharide synthesis were found.
Beilstein Journal of Organic Chemistry 01/2013; 9:97-105. · 2.52 Impact Factor
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ABSTRACT: The glycoproteins hemagglutinin (HA) and neuraminidase are the major determinants of host range and tissue tropism of the influenza virus. HA is the most abundant protein in the virus particle membrane and represents the basis of most influenza vaccines. It has been reported that influenza virus HA N-glycosylation markedly depends on the host cell line used for virus production. However, little is known about how differential glycosylation affects immunogenicity of the viral proteins. This is of importance for virus propagation in chicken eggs as well as for innovative influenza vaccine production in mammalian cell lines. In this study, we investigated the impact of the differential N-glycosylation patterns of two influenza A virus PR/8/34 (H1N1) variants on immunogenicity. Madin-Darby canine kidney cell-derived and Vero cell-derived glycovariants were analyzed for immunogenicity in a TCR-HA transgenic mouse model. Next-generation pyrosequencing validated the congruence of the potential HA N-glycosylation sites as well as the presence of the HA peptide recognized by the TCR-HA transgenic T cells. We show that differential HA N-glycosylation markedly affected T cell activation and cytokine production in vitro and moderately influenced IL-2 production in vivo. Cocultivation assays indicated that the difference in immunogenicity was mediated by CD11c(+) dendritic cells. Native virus deglycosylation by endo- and exoglycosidases dramatically reduced cytokine production by splenocytes in vitro and markedly decreased HA-specific Ab production in vivo. In conclusion, this study indicates a crucial importance of HA N-glycosylation for immunogenicity. Our findings have implications for cell line-based influenza vaccine design.
The Journal of Immunology 12/2012; · 5.79 Impact Factor
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ABSTRACT: A chemoenzymatic synthesis of sialic acid from inexpensive N-acetyl-d-glucosamine is described. In a three-step Wittig-protection-ozonolysis strategy manno-configured aldehydes are obtained. Treatment with oxaloacetate in the presence of macrophomate synthase affords the signature α-keto-γ-hydroxy acid moiety with high diastereoselectivity.
Chemical Communications 11/2012; · 6.17 Impact Factor
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ABSTRACT: Glycosylphosphatidylinositols (GPIs) are complex glycolipids that are covalently linked to the C-terminus of proteins as a posttranslational modification. They anchor the attached protein to the cell membrane and are essential for normal functioning of eukaryotic cells. GPI-anchored proteins are structurally and functionally diverse. Many GPIs have been structurally characterized but comprehension of their biological functions, beyond the simple physical anchoring, remains largely speculative. Work on functional elucidation at a molecular level is still limited. This Review focuses on the roles of GPI unraveled by using synthetic molecules and summarizes the structural diversity of GPIs, as well as their biological and chemical syntheses.
Angewandte Chemie International Edition 10/2012; · 13.45 Impact Factor
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ABSTRACT: Toxoplasma gondii is the causative agent of toxoplasmosis, one of the most widespread infections in humans and animals, and is a major opportunistic pathogen in immunocompromized patients. T. gondii is unique as it can invade virtually any nucleated cell, although the mechanisms are not completely understood. Parasite attachment to the host cell is a prerequisite for reorientation and penetration and likely requires recognition of molecules at the host cell surface. It has been reported that the affinity of tachyzoites, the invasive form of T. gondii, for host cells can be inhibited by a variety of soluble sulfated GAGs, such as heparan sulfate. Using heparin-functionalized zeolites in the absence of host cells we visualized heparin binding sites on the surface of tachyzoites by confocal and atomic force microscopy. Furtheremore, we report that protein components of the parasite rhoptry, dense granule and surface bind GAGs. In particular, the proteins ROP2 and ROP4 from the rhoptry, GRA2 from the dense granules, and the surface protein SAG1 were found to bind heparin. The binding specificities and affinities of individual parasite proteins for natural heparin and heparin oligosaccharides were determined by a combination of heparin oligosaccharide microarrays and surface plasmon resonance. Our results suggest that interactions between sulfated GAGs and parasite surface antigens contribute to T. gondii attachment to host cell surfaces as well as initiating the invasion process, while rhoptries and dense granule organelles may play an important role during the establishment of the infection and during the life of the parasite inside the parasitophorous vacuole.
Glycobiology 09/2012; · 3.58 Impact Factor
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ABSTRACT: Styrene is the classical monomer obeying zero-one kinetics in radical emulsion polymerization. Accordingly, particles that are less than 100 nm in diameter contain either one or no growing radical(s). We describe a unique photoinitiated polymerization reaction accelerated by snowballing radical generation in a continuous flow reactor. Even in comparison to classical emulsion polymerization, these unprecedented snowballing reactions are rapid and high-yielding, with each particle simultaneously containing more than one growing radical. This is a consequence of photoinitiator incorporation into the nascent polymer backbone and repeated radical generation upon photo-irradiation.
Macromolecular Rapid Communications 07/2012; 33(20):1770-4. · 4.60 Impact Factor
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ABSTRACT: Continuous synthesis meets continuous purification to produce pure products from crude reaction mixtures. In the nucleophilic aromatic substitution of 2,4-difluoronitrobenzene with morpholine the desired monosubstituted product can be continuously separated from the byproducts in a purity of over 99 % by coupling a flow reactor to a simulated moving bed (SMB) chromatography module.
Angewandte Chemie International Edition 06/2012; 51(28):7028-30. · 13.45 Impact Factor
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Toshiaki K. Shibata,
Fumiko Matsumura,
Ping Wang,
ShinYi Yu,
Chi-Chi Chou,
Kay-Hooi Khoo,
Kazuko Kitayama,
Tomoya O. Akama,
Kazuhiro Sugihara,
Naohiro Kanayama,
Kyoko Kojima-Aikawa, Peter H. Seeberger,
Minoru Fukuda,
Atsushi Suzuki,
Daisuke Aoki,
Michiko N. Fukuda
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ABSTRACT: A humanized monoclonal antibody raised against human ovarian cancer RMG-I cells and designated as HMOCC-1 (Suzuki, N., Aoki,
D., Tamada, Y., Susumu, N., Orikawa, K., Tsukazaki, K., Sakayori, M., Suzuki, A., Fukuchi, T., Mukai, M., Kojima-Aikawa, K.,
Ishida, I., and Nozawa, S. (2004) Gynecol. Oncol. 95, 290–298) was characterized for its carbohydrate epitope structure. Specifically, a series of co-transfections was performed
using mammalian expression vectors encoding specific glycosyltransferases and sulfotransferases. These experiments identified
one sulfotransferase, GAL3ST3, and one glycosyltransferase, B3GNT7, as required for HMOCC-1 antigen formation. They also suggested
that the sulfotransferase CHST1 regulates the abundance and intensity of HMOCC-1 antigen. When HEK293T cells were co-transfected
with GAL3ST3 and B3GNT7 expression vectors, transfected cells weakly expressed HMOCC-1 antigen. When cells were first co-transfected
with GAL3ST3 and B3GNT7 and then with CHST1, the resulting cells strongly expressed HMOCC-1 antigen. However, when cells were
transfected with a mixture of GAL3ST3 and CHST1 before or after transfection with B3GNT7, the number of antigen-positive cells
decreased relative to the number seen with only GAL3ST3 and B3GNT7, suggesting that CHST1 plays a regulatory role in HMOCC-1
antigen formation. Because these results predicted that HMOCC-1 antigens are SO3→3Galβ1→4GlcNAcβ1→3(±SO3→6)Galβ1→4GlcNAc, we chemically synthesized mono- and disulfated and unsulfated oligosaccharides. Immunoassays using these
oligosaccharides as inhibitors showed the strongest activity by disulfated tetrasaccharide, weak but positive activity by
monosulfated tetrasaccharide at the terminal galactose, and no activity by nonsulfated tetrasaccharides. These results establish
the HMOCC-1 epitope, which should serve as a useful reagent to further characterize ovarian cancer.
Journal of Biological Chemistry 02/2012; 287(9):6592-6602. · 4.77 Impact Factor
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ABSTRACT: A big step towards routine: The title synthesis provides of mannuronic acid alginate fragments featuring up to 12 cis-mannosidic linkages, in multi-milligram quantities (see scheme; Bn=benzyl, Lev=levulinoyl). Mannuronic acid building blocks were used in a second-generation carbohydrate synthesizer to secure the stereoselective introduction of the β-mannosidic bonds in a fully automated fashion.
Angewandte Chemie International Edition 02/2012; 51(18):4393-6. · 13.45 Impact Factor
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ABSTRACT: Malaria is a serious global health issue. Artemisinin combination treatments are the first-line drugs, but supplies are limited because artemisinin is obtained solely by extraction from Artemisia annua. A continuous-flow process that converts dihydroartemisinic acid into artemisinin was shown to be an inexpensive and scalable process that can ensure a steady, affordable supply of artemisinin.
Angewandte Chemie International Edition 02/2012; 51(7):1706-9. · 13.45 Impact Factor
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ABSTRACT: Glycomics and glycoproteomics have become indispensible tools in the study of glycoconjugates. Mass spectrometry based methods are standardly used to study the proteome and/or glycome and these approaches are capable of providing both, qualitative and quantitative information using top down techniques. The human immune system marks a particular area of interest for glycomics and glycoproteomics research since a large number of key proteins in innate and adaptive immunity are glycoproteins. In numerous examples, the crucial influence of glycosylation on critical steps such as receptor interaction and binding has been demonstrated. In this review, we focus on different glycomics and glycoproteomics approaches and their application for studying protein glycosylation in the immune system.
Current opinion in chemical biology 01/2012; 16(1-2):214-20. · 8.30 Impact Factor
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ABSTRACT: Glycan microarrays, carrying hundreds of different sugars on chip surfaces, have become a standard tool for the study of interactions of biomolecules with carbohydrates. The chip-based format offers important advantages, including the ability to screen in parallel several thousand binding events on a single slide, the minimal amount of sample required for one experiment, and the multivalent display of sugars on the chip that mimics the presentation of carbohydrates in nature. This chapter presents recent advances and future challenges in glycan microarray technology. We describe different immobilization and detection methods as well as applications in glycomics, drug discovery, and biomedicine.
Methods in molecular biology (Clifton, N.J.) 01/2012; 808:1-12.
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ABSTRACT: Sialic acid-containing glycans play a major role in cell-surface interactions with external partners such as cells and viruses. Straightforward access to sialosides is required in order to study their biological functions on a molecular level. Here, automated oligosaccharide synthesis was used to facilitate the preparation of this class of biomolecules. Our strategy relies on novel sialyl α-(2→3) and α-(2→6) galactosyl imidates, which, used in combination with the automated platform, provided rapid access to a small library of conjugation-ready sialosides of biological relevance.
Beilstein Journal of Organic Chemistry 01/2012; 8:1601-9. · 2.52 Impact Factor
