Sialidases as regulators of bioengineered cellular surfaces.

Human sialidases (NEUs) catalyze the removal of N-acetyl neuraminic acids from the glycome of the cell and regulate a diverse repertoire of nominal cellular functions, such as cell signaling and adhesion. A greater understanding of their substrate permissivity is of interest in order to discern their physiological functions in disease states and in the design of specific and effective small molecule inhibitors. Towards this, we have synthesized soluble fluorogenic reporters of mammalian sialidase activity bearing unnatural sialic acids commonly incorporated into the cellular glycocalyx via metabolic glycoengineering. We found cell-surface sialidases in Jurkat capable of cleaving unnatural sialic acids with differential activities toward a variety of R groups on neuraminic acid. In addition, we observed modulated structure-activity relationships when cell-surface sialidases were presented glycans with unnatural bulky, hydrophobic or fluorinated moieties incorporated directly via glycoengineering. Our results confirm the importance of cell-surface sialidases in glycoengineering incorporation data. We demonstrate the flexibility of human NEUs toward derivatized sugars and highlight the importance of native glycan presentation to sialidase binding and activity. These results stand to inform not only metabolic glycoengineering efforts but also inhibitor design.

Total synthesis of trifluorobutyryl-modified, globally protected sialyl Lewis x by a convergent [2+2] approach.

Structural and quantitative changes in the expression of sialic acid residues on the surface of eukaryotic cells profoundly influence a broad range of biological processes including inflammation, antigen recognition, microbial attachment, and tumor metastasis. Uptake and incorporation of sialic acid analogues in mammalian cells enable structure-function studies and perturbation of specific recognition events. Our group has recently shown that a trifluorobutyryl-modified sialic acid metabolite diminishes the adhesion of mammalian cells to E and P-selectin, presumably by leading to the expression of fluorinated sLex epitopes on cell surfaces, and interfering with the sLex-selectin interactions that are well known in mediating tumor cell migration.1 For studies directed towards understanding the molecular basis of this reduced adhesion, chemical synthesis of trifluorobutyrylated sialyl Lewis x (C4F3--sLex) was crucial. We have developed a highly efficient [2+2] approach for the assembly of C4F3-sLex on a preparative scale that contains versatile protective groups allowing the glycan to be surface immobilized or solubilized as needed for biophysical studies to investigate selectin interactions. This strategy can, in principle, be used for preparation of other N-modified sLex analogues.

Insulin-mimicking bioactivities of acylated inositol glycans in several mouse models of diabetes with or without obesity.

Insulin-mimetic species of low molecular weight are speculated to mediate some intracellular insulin actions. These inositol glycans, which are generated upon insulin stimulation from glycosylphosphatidylinositols, might control the activity of a multitude of insulin effector enzymes. Acylated inositol glycans (AIGs) are generated by cleavage of protein-free GPI precursors through the action of GPI-specific phospholipase C (GPI-PLC) and D (GPI-PLD). We synthesized AIGs (IG-1, IG-2, IG-13, IG-14, and IG-15) and then evaluated their insulin-mimicking bioactivities. IG-1 significantly stimulated glycogen synthesis and lipogenesis in 3T3-L1 adipocytes and rat isolated adipocytes dose-dependently. IG-2 significantly stimulated lipogenesis in rat isolated adipocytes dose-dependently. IG-15 also enhanced glycogen synthesis and lipogenesis in 3T3-L1 adipocytes. The administration of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with normal diets. The administration of IG-1 decreased plasma glucose in STZ-diabetic C57B6N mice. The treatment of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with high fat-diets and db/db mice. The long-term treatment of IG-1 decreased plasma glucose and reduced food intake and body weight in C57B6N mice with high fat-diets and ob/ob mice. Thus, IG-1 has insulin-mimicking bioactivities and improves glucose tolerance in mice models of diabetes with or without obesity.

Fluorogenic sialic acid glycosides for quantification of sialidase activity upon unnatural substrates.

Herein we report the synthesis of N-acetyl neuraminic acid derivatives as 4-methylumbelliferyl glycosides and their use in fluorometrically quantifying human and bacterial sialidase activity and substrate specificities. We found that sialidases in the human promyelocytic leukemic cell line HL60 were able to cleave sialic acid substrates with fluorinated C-5 modifications, in some cases to a greater degree than the natural N-acetyl functionality. Human sialidases isoforms were also able to cleave unnatural substrates with bulky and hydrophobic C-5 modifications. In contrast, we found that a bacterial sialidase isolated from Clostridium perfringens to be less tolerant of sialic acid derivatization at this position, with virtually no cleavage of these glycosides observed. From our results, we conclude that human sialidase activity is a significant factor in sialic acid metabolic glycoengineering efforts utilizing unnatural sialic acid derivatives. Our fluorogenic probes have enabled further understanding of the activities and substrate specificities of human sialidases in a cellular context.

Some natural flavonoids are competitive inhibitors of Caspase-1, -3 and -7 despite their cellular toxicity.

A common feature of both apoptosis and inflammation is the activation of caspases. Caspases are aspartate-directed cysteine proteases that have numerous cellular targets. It has been discovered that several flavonoids are inhibitors of caspases. Flavonoids are members of a family of polyphenolic compounds from plants that have many biological properties, one of which is the ability to induce cell death. Some flavonoids are selective inhibitors of particular caspases. Since some of the inhibitory flavonoids are nevertheless cytotoxic, these results suggest that flavonoid-induced cell death may be occurring through a non-classical apoptosis pathway that is not dependent on caspase activity.

Modulation of cellular adhesion by glycoengineering.

Aberrant glycosylation of lipid and protein molecules on cellular surfaces is responsible for many of the pathophysiological events in tumor progression and metastasis. Sialic acids in particular, are overexpressed on the glycocalyx of malignant tumor cells and sialic acid-mediated cell adhesion is required for metastasis. We report here that replacement of sialic acids on cell surfaces with fluorinated congeners dramatically decreases cell adhesion to E- and P-selectin-coated surfaces. Comparison of adhesion of fluorinated cells with those modified with nonfluorinated analogues suggests that both reduce binding of the modified sialosides to their cognate lectins to a similar extent on a per molecule basis. The overall reduction in cell adhesion results from greater cell surface presentation of the fluorinated congeners. This work suggests an avenue for inhibition of metastasis by administration of small molecules and concomitant noninvasive imaging of tumor cells by (19)F MRI before they are visible by other means.

The biological activity of structurally defined inositol glycans.

BACKGROUND: The inositol glycans (IGs) are glycolipid-derived carbohydrates produced by insulin-sensitive cells in response to insulin treatment. IGs exhibit an array of insulin-like activities including stimulation of lipogenesis, glucose transport and glycogen synthesis, suggesting that they may be involved in insulin signal transduction. However, because the natural IGs are structurally heterogeneous and difficult to purify to homogeneity, an understanding of the relationship between structure and biological activity has relied principally on synthetic IGs of defined structure. DISCUSSION: This article briefly describes what is known about the role of IGs in signal transduction and reviews the specific biological activities of the structurally defined IGs synthesized and tested to date. CONCLUSION: A pharmacophore for IG activity begins to emerge from the reviewed data and the structural elements necessary for activity are summarized.

Fluorination of mammalian cell surfaces via the sialic acid biosynthetic pathway.

Metabolic oligosaccharide engineering has been employed to introduce fluorine-containing groups onto mammalian cell surfaces. Incubation of HeLa, Jurkat, and HL60 cells in culture with fluorinated sialic acid and mannosamine analogues resulted in cell-surface presentation of fluorinated glycans. Metabolic conversion of fluorinated precursors was detected and quantified by DMB-derivatization and HPLC ESI-MS analysis. Between 7% and 72% of total membrane-associated sialosides were fluorinated, depending on the precursor used and the cell type. Fluorination of mammalian cell surfaces provides a means for introducing a bioorthogonal surface for modulating noncovalent interactions such as those involved in cell adhesion.

Enantio- and Diastereodivergent Synthetic Route to Multifarious Cyclitols from D-Xylose via Ring-Closing Metathesis.

Short stereoselective syntheses of various cyclitols, including the derivatives of conduritol B, conduritol F, myo-inositol and chiro-inositol, have been accomplished. The key steps in the syntheses are a ring-closing metathesis process and a diastereodivergent organometallic addition to a D-xylose-derived alde-hyde.

Synthesis of Differentially Protected myo- and chiro-Inositols from D-Xylose; Stereoselectivity in Intramolecular SmI(2)-Promoted Pinacol Reactions.

Methods for the enantioselective conversion of D-xylose to differentially protected myo-inositol and L-chiro-inositol have been developed. The key transformation is a highly diastereoselective intramolecular SmI(2)-promoted pinacol coupling. The stereoselectivity was extremely dependent on the conditions, suggesting a change in mechanism. Preliminary mechanistic experiments and possible explanations for this behavior are discussed.

Synthesis of galactosaminyl D-chiro-inositols.

All six isomeric D-galactosaminopyranosyl-D-chiro-inositols have been prepared by glycosylation of appropriate penta-O-benzyl-D-chiro-inositols. The three requisite protected D-chiro-inositols were prepared by SmI2-promoted pinacol coupling of dialdehydes derived ultimately from L-arabinose.

An anionic inositol phosphate glycan pseudotetrasaccharide exhibits high insulin-mimetic activity in rat adipocytes.

Inositol phosphate glycan pseudotetrasaccharides consisting of man-(alpha1-6)-man-(alpha1-4)-glcN-(alpha,beta1-6)-myo-inositol-1,2-cyclic phosphate possessing a sulfate group at either O-6 (compounds 3alpha,beta) or O-2 (compounds 4alpha,beta) of the terminal mannose have been prepared. Compound 4alpha was able to stimulate lipogenesis in native rat adipocytes to 78% of the maximal insulin response (MIR) with an EC50 of 1.1 microM. The other compounds exhibited lower maximal stimulations (47-63% MIR) and higher EC50 values (9.5-10.6 microM).

A fluorescent inositol phosphate glycan stimulates lipogenesis in rat adipocytes by extracellular activation alone.

The chemical synthesis of 2,6-dideoxy-2-amino-6-mercaptoglucopyranosyl-(alpha1-6)-myo-inositol 1,2-cyclic phosphate and its conjugation with a lucifer yellow derivative are reported. The resulting fluorescent IPG analogue was able to stimulate lipogenesis in rat adipocytes despite the fact that it was not internalized into the cell. The results demonstrate that internalization of the IPG is not required for manifestation of its insulin-like effects.

Synthesis of 1,2-diamino-1,2-dideoxy-myo- inositol-derived ligands for the investigation of metal complex reactivity.

A method for the synthesis of chiral 1,2-diamino-1,2-dideoxy-myo-inositol-based bis-pyridyl ligands 3a and 3b from the corresponding myo-inositol precursor is described. These highly functionalized inosamine-bis-pyridyl ligands are expected to provide a useful platform for exploring the relationship between chiral ligand structure and enantioselective olefin oxidation catalyzed by their metal complexes.