Synthesis of disaccharides containing 6-deoxy-α-L-talose as potential heparan sulfate mimetics.

A 6-deoxy-α-L-talopyranoside acceptor was readily prepared from methyl α-L-rhamnopyranoside and glycosylated with thiogalactoside donors using NIS/TfOH as the promoter to give good yields of the desired a-linked disaccharide (69-90%). Glycosylation with a 2-azido-2-deoxy-D-glucosyl trichloroacetimidate donor was not completely stereoselective (α:ß = 6:1), but the desired a-linked disaccharide could be isolated in good overall yield (60%) following conversion into its corresponding tribenzoate derivative. The disaccharides were designed to mimic the heparan sulfate (HS) disaccharide GlcN(2S,6S)-IdoA(2S). However, the intermediates readily derived from these disaccharides were not stable to the sulfonation/deacylation conditions required for their conversion into the target HS mimetics.

Successful treatment of molybdenum cofactor deficiency type A with cPMP.

Molybdenum cofactor deficiency (MoCD) is a rare metabolic disorder characterized by severe and rapidly progressive neurologic damage caused by the functional loss of sulfite oxidase, 1 of 4 molybdenum-dependent enzymes. To date, no effective therapy is available for MoCD, and death in early infancy has been the usual outcome. We report here the case of a patient who was diagnosed with MoCD at the age of 6 days. Substitution therapy with purified cyclic pyranopterin monophosphate (cPMP) was started on day 36 by daily intravenous administration of 80 to 160 microg of cPMP/kg of body weight. Within 1 to 2 weeks, all urinary markers of sulfite oxidase (sulfite, S-sulfocysteine, thiosulfate) and xanthine oxidase deficiency (xanthine, uric acid) returned to almost normal readings and stayed constant (>450 days of treatment). Clinically, the infant became more alert, convulsions and twitching disappeared within the first 2 weeks, and an electroencephalogram showed the return of rhythmic elements and markedly reduced epileptiform discharges. Substitution of cPMP represents the first causative therapy available for patients with MoCD. We demonstrate efficient uptake of cPMP and restoration of molybdenum cofactor-dependent enzyme activities. Further neurodegeneration by toxic metabolites was stopped in the reported patient. We also demonstrated the feasibility to detect MoCD in newborn-screening cards to enable early diagnosis.

Synthesis of a heparan sulfate mimetic disaccharide with a conformationally locked residue from a common intermediate.

A simple mimetic of a heparan sulfate disaccharide sequence that binds to the growth factors FGF-1 and FGF-2 was synthesized by coupling a 2-azido-2-deoxy-D-glucopyranosyl trichloroacetimidate donor with a 1,6-anhydro-2-azido-2-deoxy-beta-D-glucopyranose acceptor. Both the donor and acceptor were obtained from a common intermediate readily obtained from D-glucal. Molecular docking calculations showed that the predicted locations of the disaccharide sulfo groups in the binding site of FGF-1 and FGF-2 are similar to the positions observed for co-crystallized heparin-derived oligosaccharides obtained from published crystal structures.

Design, synthesis, FGF-1 binding, and molecular modeling studies of conformationally flexible heparin mimetic disaccharides.

Disaccharide mimetics of a heparin sequence that binds to fibroblast growth factors were prepared by coupling a D-galactose donor with a methyl beta-D-gluco- or xylopyranoside acceptor. When fully sulfated, the glucose or xylose moieties exist in solution in equilibrium between the (4)C1 and (1)C4 conformers, as confirmed by 1H NMR spectroscopy, thus mimicking the conformationally flexible L-iduronic acid found in heparin. Docking calculations showed that the predicted locations of disaccharide sulfo groups in the binding site of FGF-1 are consistent with the positions observed for co-crystallized heparin-derived oligosaccharides. Predicted binding affinities are in accord with experimental Kd values obtained from binding assays and are similar to the predicted values for a model heparin disaccharide.

Synthesis and heparanase inhibitory activity of sulfated mannooligosaccharides related to the antiangiogenic agent PI-88.

A stepwise synthetic route to the mannooligosaccharides from the neutral fraction of Pichia holstii phosphomannan hydrolysate, including a tetrasaccharylamine component, was developed using only two or three readily available d-mannose building blocks. These compounds were sulfonated to give the corresponding sulfated oligosaccharides which are closely related to the constituents of the anticancer agent PI-88. The synthetic approach is well suited to the preparation of analogues as demonstrated by the synthesis of a series of (1-->3)-linked mannooligosaccharides. The inhibitory activity of the sulfated oligosaccharides against heparanase was determined using a Microcon ultrafiltration assay. The tetra- and pentasaccharides were potent competitive inhibitors of heparanase (K(i)=200-280nM) whilst the shorter di- and trisaccharides were partial competitive inhibitors and did not completely inhibit the enzyme even at very high concentrations.

Synthesis, biological activity, and preliminary pharmacokinetic evaluation of analogues of a phosphosulfomannan angiogenesis inhibitor (PI-88).

The phosphosulfomannan 1 (PI-88) is a mixture of highly sulfated oligosaccharides that is currently undergoing clinical evaluation in cancer patients. As well as its anticancer properties, 1 displays a number of other interesting biological activities. A series of analogues of 1 were synthesized with a single carbon (pentasaccharide) backbone to facilitate structural characterization and interpretation of biological results. In a fashion similar to 1, all compounds were able to inhibit heparanase and to bind tightly to the proangiogenic growth factors FGF-1, FGF-2, and VEGF. The compounds also inhibited the infection of cells and cell-to-cell spread of herpes simplex virus (HSV-1). Preliminary pharmacokinetic data indicated that the compounds displayed different pharmacokinetic behavior compared with 1. Of particular note was the n-octyl derivative, which was cleared 3 times less rapidly than 1 and may provide increased systemic exposure.

The synthesis of phosphorylated disaccharide components of the extracellular phosphomannan of Pichia (Hansenula) holstii NRRL Y-2448.

Methods for the stereoselective synthesis of alpha-(1-->2)- and alpha-(1-->3)-linked 6(II)-O-phosphomannobiosides were developed. Two strategies were successfully employed: a D-mannosyl acceptor was coupled with a phosphorylated D-mannosyl trichloroacetimidate donor, or alternatively with a differentially 6-O-protected D-mannosyl trichloroacetimidate donor which, after glycosylation, was selectively deprotected and phosphorylated. Two target phosphomannobiosides intended for use in SAR studies of the antiangiogenic drug candidate PI-88, 2-O-(6-O-phospho-alpha-D-mannopyranosyl)-D-mannopyranose and methyl 3-O-(6-O-phospho-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, were synthesized. The former is a minor component of the side-chain repeating unit of the extracellular phosphomannan of Pichia (Hansenula) holstii NRRL Y-2448, whilst the latter represents a nonreducing end fragment of the phosphomannan.

The development of inhibitors of heparanase, a key enzyme involved in tumour metastasis, angiogenesis and inflammation.

Heparanase is an endo-beta-glucuronidase that degrades the glycosaminoglycan heparan sulfate, a major component of the extracellular matrix and basement membranes, and has been implicated in such processes as inflammation, angiogenesis and metastasis. The identification of inhibitors of heparanase is an attractive approach towards developing new therapeutics for metastatic tumours and chronic inflammatory diseases. This review focuses on heparanase inhibitors that have been isolated or synthesised to date. More recent developments in the understanding of heparanase structure and function that may ultimately aid in the future design of inhibitors with improved potency and specificity, are also discussed.

Structural characterization by (13)C-NMR spectroscopy of products synthesized in vitro by polysaccharide synthases using (13)C-enriched glycosyl donors: application to a UDP-glucose:(1-->3)-beta-D-glucan synthase from blackberry (Rubus fruticosus).

A simple and sensitive method for the characterization of products synthesized in vitro by polysaccharide synthases is described. It relies on the use of (13)C-enriched nucleotide sugars as substrates and on the analysis of the newly synthesized polysaccharides by (13)C-nuclear magnetic resonance (NMR) spectroscopy. The method was validated with a (1-->3)-beta-d-glucan synthase from blackberry, but it may be applied to the study of any glycosyltransferase. The chemical synthesis of UDP-d-[U-(13)C]glucose was achieved in a classical procedure with an overall yield of 50%. A uniformly labeled (1-->3)-beta-d-glucan was synthesized from this substrate, using detergent extracts of blackberry cell membranes as a source of synthase. One hundred micrograms of product was sufficient for liquid and solid-state (13)C-NMR spectroscopy analyses. The method is at least 100 times more sensitive than in the case of nonenriched polysaccharides. It allows the unequivocal identification and direct structural characterization of the products synthesized in vitro, as opposed to conventional methods that rely on the use of radioactive substrates and enzymatic hydrolysis of the polysaccharides with specific glycoside hydrolases. The method proves that the glycan analyzed was synthesized de novo because the final product is enriched in (13)C. Information on the 3D organization of the polymer may also be obtained by solid-state NMR spectroscopy.

Probing the interactions of phosphosulfomannans with angiogenic growth factors by surface plasmon resonance.

The binding interactions of the phosphosulfomannan anticancer agent PI-88 (1) with the angiogenic growth factors FGF-1, FGF-2, and VEGF were studied by surface plasmon resonance (SPR) on a BIAcore 3000 biosensor. Compared with heparin, PI-88 has at least 11-fold higher affinity for FGF-1 and at least 3-fold higher affinity for VEGF, but at least 13-fold lower affinity for FGF-2. To define the structural features of PI-88 that are important for growth factor binding, several analogues, such as dephosphorylated PI-88 and a sulfated pentasaccharide, were prepared. The binding interactions of these analogues with FGF-1, FGF-2, and VEGF were similarly studied by SPR, and structure-activity relationships were determined.

Synthesis of complex oligosaccharides by using a mutated (1,3)-beta-D-glucan endohydrolase from barley.

Complex oligosaccharides with newly formed (1,3)-beta-glycosidic linkages were obtained in good to excellent yields when substituted or unsubstituted alpha-laminaribiosyl fluorides, acting as donors, were condensed onto mono- and disaccharide beta-D-hexopyranoside acceptors by using a (1,3)-beta-D-glycosynthase. These linear and branched (1,3)-beta-linked oligosaccharides could prove to be important in a range of medical, pharmaceutical, and agricultural applications. Furthermore, the observation that the (1,3)-beta-D-glucan glycosynthase accommodates (1,3)-, (1,4),- and (1,6)-beta-oligosaccharides in its acceptor subsites suggests novel, yet unexpected physiological roles for the wild type (1,3)-beta-D-glucan endohydrolase from higher plants.

Specificity studies of bacillus 1,3-1,4-beta-glucanases and application to glycosynthase-catalyzed transglycosylation.

Bacillus 1,3-1,4-beta-glucanases hydrolyze 1,3-1,4-beta-gluco-oligosaccharides with a retaining mechanism. The binding-site cleft of these endoglycosidases is composed of six subsites (-4 to +2) of which subsite -3 makes the largest contribution to transition state stabilization. The specificity of this subsite is here analyzed for both glycosidase and glycosynthase activities in the wild-type and the nucleophile-less E134A mutant Bacillus licheniformis enzymes. A D-galactosyl residue on the nonreducing end of a trisaccharide substrate is accepted by the enzyme and binds at subsite -3 in the productive enzyme-substrate complex. The wild-type enzyme catalyzes the hydrolysis of the substrate Glcbeta4Glcbeta3GlcbetaMU (Glc=glucosyl, MU=4-methylumbelliferyl) with a k(cat)/K(M) value only 1.3-fold higher than for the Galbeta4Glcbeta3GlcbetaMU (Gal=galactosyl) substrate. The corresponding alpha-fluorides act as good donors for the glycosynthase condensation reaction with mono- and disaccharide acceptors catalyzed by the E134A mutant. Whereas self-condensation and elongation products are also obtained as minor compounds with the Glcbeta4Glcbeta3GlcalphaF donor, nearly quantitative yields of single condensation products are obtained with the Galbeta4Glcbeta3GlcalphaF donor, in which the axial configuration of the 4-OH group on the nonreducing end prevents self-condensation and elongation reactions.

Mutated barley (1,3)-beta-D-glucan endohydrolases synthesize crystalline (1,3)-beta-D-glucans.

Barley (1,3)-beta-D-glucan endohydrolases (EC ), inactivated by site-directed mutagenesis of their catalytic nucleophiles, show autocondensation glucosynthetic activity with alpha-laminaribiosyl fluoride and heterocondensation glycosynthetic activity with alpha-laminaribiosyl fluoride and 4'-nitrophenyl beta-D-glucopyranoside. The native enzyme is a retaining endohydrolase of the family 17 group and catalyzes glycosyl transfer reactions at high substrate concentrations. Catalytic efficiencies (k(cat) K(m)(-1)) of mutants E231G, E231S, and E231A as glycosynthases are 28.9, 0.9, and 0.5 x 10(-4) m(-1) s(-1), respectively. Glycosynthase reactions appear to be processive and proceed with pH optima of 6-8 and yields of up to 75%. Insoluble products formed during the glycosynthase reaction appear as lamellar, hexagonal crystals when observed by electron microscopy. Methylation, NMR, and matrix-assisted laser desorption ionization time-of-flight analyses show that the reaction products are linear (1,3)-beta-D-glucans with a degree of polymerization of 30-34, whereas electron and x-ray diffraction patterns indicate that these (1,3)-beta-D-glucan chains adopt a parallel, triple helical conformation. The (1,3)-beta-D-glucan triple helices are orientated perpendicularly to the plane of the lamellar crystals. The barley (1,3)-beta-D-glucan glycosynthases have considerable potential for tailored and high efficiency synthesis of (1,3)-beta-D-linked oligo- and polysaccharides, some of which could have immunomodulating activity, or for the coupling of (1,3)-beta-D-linked glucosyl residues onto other oligosaccharides or glycoproteins.

Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases.

Family 3 beta-D-glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of beta-D-glucosyl residues from nonreducing termini of a range of beta-D-glucans and beta-D-oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley beta-D-glucan glucohydrolase in complex with nonhydrolyzable S-glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite -1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent beta-D-glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development.