Bioorthogonal chemical reporter methodology for visualization, isolation and analysis of glycoconjugates.

Intact and integral glycosylation of membrane-associated as well as secreted glycoproteins has been shown to be essential for many aspects of the proper function of biological systems. Recombinantly expressed glycoproteins, such as antibodies, growth factors, hormones, vaccines, and contrast agents are key elements in medical applications. The quality of these therapeutically administered glycoproteins can be efficiently improved by the incorporation of chemically functionalized monosaccharides into their glycan moieties, a process denoted as metabolic oligosaccharide engineering (MOE). In addition to these pharmaceutical applications, MOE has greatly advanced diagnostics by localizing and visualizing glycans even in living animals. To date, a multitude of chemically modified monosaccharides have been designed for MOE applications. Owing to their terminal position at glycan structures of glycoproteins and relevance for cellular recognition, sialic acids and their metabolic precursor N-acetylmannosamine (ManNAc), are the most prominent targets for MOE. Several ManNAc derivatives with N-acetyl side-chain modifications have been synthesized and metabolically incorporated by the sialic acid biosynthetic pathway into a corresponding sialic acid C5 analogue (Figure 1). This approach was beneficial to extending the understanding of the biological role of the N-acyl side chain of sialic acids, for example, in virus infection or neuronal differentiation. Alternatively, C9 modifications of sialosides have also been achieved by directly administering synthetic sialic acid analogues. Additionally, selective cleavage of the glycol moiety led to a truncated sialic acid equipped glycans with an aldehyde for labeling reactions (Figure 1). All of these modifications address sialylation of both, Nand O-glycosylation of glycoproteins, to almost the same extent. Herein we investigate whether the biosynthetic machinery for sialic acids also tolerates other ManNAc derivatives as substrates, which are modified directly at the six-membered carbohydrate ring. The modification of the C4 position appeared most attractive, because it is not enzymatically modified during cellular glycoprotein production and would deliver previously unknown C7-modified sialic acid containing glycoproteins (Figure 1). To probe the biosynthetic promiscuity, we targeted a C4-modified ManNAc derivative, N-acetyl-4-azido-4-deoxymannosamine (4-azido-ManNAc, 1), in our study to enable postglycosylational conjugation and visualization by bioorthogonal reactions. N-acetyl-(1,3,6-O-acetyl)-4-azido-4-deoxy-mannosamine (Ac3-4-azido-ManNAc) was generated by an optimized literature method (Figure S1 in the Supporting InformaFigure 1. Methods for the structural modification of glycan-bound sialic acids by application of chemically modified ManNAc or direct periodate oxidation of glycan-bound sialic acids (left). Specific modification of the C7 position of sialic acids was achieved by C4-modified ManNAc in this study (right; note that to date these methods were carried individually, resulting in only one modification of a single sialic acid molecule).

Bioorthogonal chemistry reactions occur in physiological conditions without interfering with normal physiological processes. Through metabolic engineering, bioorthogonal groups can be tagged onto cell membranes, which selectively attach to cargos with paired groups via bioorthogonal reactions. Due to its simplicity, high efficiency, and specificity, bioorthogonal chemistry has demonstrated great application potential in drug delivery. On the one hand, bioorthogonal reactions improve therapeutic agent delivery to target sites, overcoming off-target distribution. On the other hand, nanoparticles and biomolecules can be linked to cell membranes by bioorthogonal reactions, providing approaches to developing multi-functional drug delivery systems (DDSs). In this review, we first describe the principle of labeling cells or pathogenic microorganisms with bioorthogonal groups. We then highlight recent breakthroughs in developing active targeting DDSs to tumors, immune systems, or bacteria by bioorthogonal chemistry, as well as applications of bioorthogonal chemistry in developing functional bio-inspired DDSs (biomimetic DDSs, cell-based DDSs, bacteria-based and phage-based DDSs) and hydrogels. Finally, we discuss the difficulties and prospective direction of bioorthogonal chemistry in drug delivery. We expect this review will help us understand the latest advances in the development of active targeting and multi-functional DDSs using bioorthogonal chemistry and inspire innovative applications of bioorthogonal chemistry in developing smart DDSs for disease treatment.

Chondroitin Sulfate is the Primary Receptor for a Peptide-Modified AAV That Targets Brain Vascular Endothelium In Vivo

Many adhesion and signaling molecules critical for development, as well as surface markers implicated in diseases ranging from cancer to influenza, contain oligosaccharides that modify their functions. Inside a cell, complex glycosylation pathways assemble these oligosaccharides and attach them to proteins and lipids as they traffic to the cell surface. Until recently, practical technologies to manipulate glycosylation have lagged unlike the molecular biologic and genetic methods available to intervene in nucleic acid and protein biochemistry; now, metabolic oligosaccharide engineering shows promise for manipulating glycosylation. In this methodology, exogenously-supplied non-natural sugars intercept biosynthetic pathways and exploit the remarkable ability of many of the enzymes involved in glycosylation to process metabolites with slightly altered chemical structures. To date, non-natural forms of sialic acid, GalNAc, GlcNAc, and fucose have been incorporated into glycoconjugates that appear on the cell surface; in addition O-GlcNAc protein modification involved in intracellular signaling has been tagged with modified forms of this sugar. Reactive functional groups, including ketones, azides, and thiols, have been incorporated into glycoconjugates and thereby provide chemical 'tags' that can be used for diverse purposes ranging from drug delivery to new modes of carbohydrate-based cell adhesion that can be used to control stem cell destiny. Finally, strategies for further engineering non-natural sugars to improve their pharmacological properties and provide complementary biological activities, such as addition of short chain fatty acids, are discussed in this article.

The Diels-Alder reaction with inverse electron demand (DAinv reaction) of 1,2,4,5-tetrazines with electron rich or strained alkenes was proven to be a bioorthogonal ligation reaction that proceeds fast and with high yields. An important application of the DAinv reaction is metabolic oligosaccharide engineering (MOE) which allows the visualization of glycoconjugates in living cells. In this approach, a sugar derivative bearing a chemical reporter group is metabolically incorporated into cellular glycoconjugates and subsequently derivatized with a probe by means of a bioorthogonal ligation reaction. Here, we investigated a series of new mannosamine and glucosamine derivatives with carbamate-linked side chains of varying length terminated by alkene groups and their suitability for labeling cell-surface glycans. Kinetic investigations showed that the reactivity of the alkenes in DAinv reactions increases with growing chain length. When applied to MOE, one of the compounds, peracetylated N-butenyloxycarbonylmannosamine, was especially well suited for labeling cell-surface glycans. Obviously, the length of its side chain represents the optimal balance between incorporation efficiency and speed of the labeling reaction. Sialidase treatment of the cells before the bioorthogonal labeling reaction showed that this sugar derivative is attached to the glycans in form of the corresponding sialic acid derivative and not epimerized to another hexosamine derivative to a considerable extent.

Higher reactivity of apolipoprotein B-100 and α-tocopherol compared to sialic acid moiety of low-density lipoprotein (LDL) in radical reaction

Salla disease is a lysosomal storage disorder characterized by mental retardation and disturbed sialic acid metabolism. To study endogenous synthesis and breakdown of sialic acid, fibroblasts were incubated for 5 d in the presence and then in the absence of N-[3H]acetylmannosamine. Labeling of free sialic acid was 5-10 times higher in mutant than in normal cells. Radioactivity decreased in 4 d by 75% in normal but only by 30% in mutant fibroblasts. The labeling pattern was not normalized upon coculture of mutant and normal cells. To study the metabolism of extracellular sialic acid, low-density lipoprotein (LDL) was labeled in the sialic acid moiety (periodate-NaB3H4) or in the protein moiety (125I). Binding, internalization, lysosomal degradation, and exit of products of protein catabolism were similar in normal and mutant fibroblasts. Upon incubation with LDL labeled in the sialic acid moiety, mutant cells accumulated 2-3 times more free sialic acid radioactivity than normal fibroblasts, mostly in the lysosomal fraction. After a 24-h chase incubation, radioactivity in free sialic acid decreased by 70-80% in normal but only by 10-30% in mutant cells. In mutant fibroblasts, 40% of the radioactivity remained in lysosomes, whereas no labeled free sialic acid was detected in lysosomes from normal fibroblasts. We conclude that in Salla disease, fibroblast endogenous synthesis of sialic acid and lysosomal cleavage of exogenous glycoconjugates is normal, but free sialic acid cannot leave the lysosome. These findings suggest that the basic defect in Salla disease is deficient transport of free sialic acid through the lysosomal membrane.

Treatment and Follow-up Data for Young Patients with a Gne Defect and Congenital Thrombocytopenia

Human apolipoprotein E isoforms are differentially sialylated and the sialic acid moiety in ApoE2 attenuates ApoE2-Aβ interaction and Aβ fibrillation

Separation of anionic oligosaccharides by high-performance liquid chromatography

As prepared nanomaterials of metals, semiconductors, polymers and carbon often need surface modifications such as ligand exchange, and chemical and bioconjugate reactions for various biosensor, bioanalytical, bioimaging, drug delivery and therapeutic applications. Such surface modifications help us to control the physico-chemical, toxicological and pharmacological properties of nanomaterials. Furthermore, introduction of various reactive functional groups on the surface of nanomaterials allows us to conjugate a spectrum of contrast agents, antibodies, peptides, ligands, drugs and genes, and construct multifunctional and hybrid nanomaterials for the targeted imaging and treatment of cancers. This tutorial review is intended to provide an introduction to newcomers about how chemical and bioconjugate reactions transform the surface of nanomaterials such as silica nanoparticles, gold nanoparticles, gold quantum clusters, semiconductor quantum dots, carbon nanotubes, fullerene and graphene, and accordingly formulate them for applications such as biosensing, bioimaging, drug and gene delivery, chemotherapy, photodynamic therapy and photothermal therapy. Nonetheless, controversial reports and our growing concerns about toxicity and pharmacokinetics of nanomaterials suggest the need for not only rigorous in vivo experiments in animal models but also novel nanomaterials for practical applications in the clinical settings. Further reading of original and review articles cited herein is necessary to buildup in-depth knowledge about the chemistry, bioconjugate chemistry and biological applications of individual nanomaterials.

15] Synthesis of Sialyl Lewis X ganglioside and analogs

BackgroundGenetic and epigenetic mechanisms that may contribute to lupus susceptibility in humans and mouse models are of interest especially if they provide enzymes that could function as potential therapeutic targets....

The surface coat of syncytial trophoblast from term human placentas was studied using cytochemical methods (colloidal iron, alcian blue-lanthanum nitrate, dialyzed iron) in coordination with tissue enzyme digestions (trypsin, neuraminidase) and sialic acid analyses. The presence of at least two highly acidic anionic components that contribute significantly to the surface negativity of trophoblast has been demonstrated. The first of these, sialic acid, was removed with neuraminidase. Tissue digestion with this glycosidase was accompanied by a decrease in trophoblast surface staining with colloidal iron, a decrease in tissue sialic acid, and an increase in the concentration of sialic acid in the incubating medium. Results from methylation experiments were consistent with the presence of sialic acid. The second anionic component(s) was identified by removal with trypsin of a glycocalyx constituent that stained with both colloidal iron and lanthanum. After trypsinization, tissue sialic acid levels were not significantly different from control values, and no detectable sialic acid was present in the incubating medium. The identity of this anionic component has not been established. Both sialic acid and nonsialic acid acidic components are distributed in higher density on membrane of microvilli than on intermicrovillous surface membrane. In addition, the sialic acid moieties appear to be clustered in the glycocalyx.

The Asn-linked oligosaccharides from bovine lutropin (bLH(Pit] are predominantly dibranched complex-type structures with the terminal sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha. Recombinant bLH expressed in Chinese hamster ovary cells (bLH(CHO] bears di- (60%) and tribranched (30%) complex-type oligosaccharides; however, these terminate in the sequence Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,2Man alpha. In contrast to the limited spectrum of oligosaccharide structures present on recombinant bLH(CHO), the endogenous glycoproteins synthesized by CHO cells bear a heterogeneous array of Asn-linked oligosaccharides with 0, 1, 2, 3, or 4 sialic acid moieties. The sialic acid moieties on the Asn-linked oligosaccharides of both endogenous glycoproteins and recombinant bLH(CHO) are exclusively alpha 2,3-linked, suggesting that the alpha 2,6-sialyl-transferase is not active in CHO cells. The bioactivities of bLH(Pit) and bLH(CHO) were compared using MA-10 cells following sequential digestion with neuraminidase and beta-galactosidase. Neither the ED50 (dose producing 50% of the maximum response) for progesterone production (7.2 ng/ml) nor the Pmax (maximum level of progesterone produced) (470 ng/ml) was altered for bLH(Pit) by these treatments, consistent with the absence of either sialic acid or Gal on bLH(Pit). The ED50 for progesterone production by recombinant bLH(CHO) (16.4 ng/ml) was significantly greater than for bLH(Pit) but was reduced to 5.3 ng/ml following removal of terminal sialic acid. Removal of the subterminal Gal was without further effect. The Pmax for bLH(CHO) (180 ng/ml) was not altered by these treatments. The reduction in bLH(CHO) bioactivity caused by the presence of terminal sialic acid suggests that the presence of terminal sulfate on bLH(Pit) oligosaccharides may also reduce its bioactivity and may play a modulatory role in regulating hormone bioactivity.