Christopher D. Warren

The synthesis of allyl 2-acetamido-3,6-DI-O-benzyl- 2-DEOXY-α-d-glucopyranoside and of chitobiose derivatives by the oxazolone procedure

Controlled, partial benzylation of allyl 2-acetamido-3-O-benzyl-2-deoxy-alpha-D-glucopyranoside gave a mixture of the 3,4-di-, 3,6-di- (15), and 3,4,6-tri-O-benzyl derivatives, the major product being 15. Condensation of 15 with 2-methyl-(3,4,6-tri-O-acetul-1,2-dideoxy-alpha-D-glucopyrano)-[2,1-d]-2-oxazoline gave a disaccharide which, after purification, removal of the allyl group, and hydrogenolysis of the benzyl substituents, gave 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-i-deoxy-beta-D-glucopyranosyl)-2-deoxy-alpha-D-glucopyranose. This compound was further converted into di-N-acetyl-hexa-O-acetylchitobiose by acetylation, or into 2-methyl-[4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-3,6-di-O-acetyl-1,2-di-deoxy-alpha-D-glucopyrano]-[2,1-d]-2-oxazoline, a starting material for the preparation of di-N-acetyl-alpha-chitobiosyl phsophate.

The synthesis of O-β-D-mannopyranosyl-(1→4)-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→4)-2-acetamido-2-deoxy-D-glucopyranose. part II

Abstract Allyl 2-acetamido-3,6-di-O-(2-butenyl)-2-deoxy-β- D -glucopyranoside was prepared, and coupled with 2-methyl-(4-O-acetyl-3,6-di-O-benzyl-1,2-dideoxy-α- D -glucopyrano)-[2,1-d]-2-oxazoline. The resulting, protected disaccharide allyl 2-acetamido-4-O-(2-acetamido-4-O-acetyl-3,6-di-O-benzyl-2-deoxy-β- D -glucopyranosyl)-3,6-di-O-(2-butenyl)-2-deoxy-β- D -glucopyranoside was O-deacetylated and the product coupled with 2-O-acetyl-3,4,6-tri-O-benzyl-α- D -glucopyranosyl bromide in the presence of silver trifluoromethanesulfonate and 1, 1,3,3-tetramethylurea, to give the trisaccharide, allyl O-(2-O-acetyl-3,4,6-tri-O-benzyl-β- D -glucopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β- D -glucopyranosyl)-(1→4)-2-acetamido-3,6-di-O-(2-butenyl)-2-deoxy-β- D -glucopyranoside. O-Deacetylation, oxidation with acetic anhydride-dimethyl sulfoxide, and stereoselective reduction with sodium borohydride gave mainly allyl O-(3,4,6-tri-O-benzyl-β- D -mannopyranosyl)-(1→4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-β- D -glucopyranosyl)-(1→4)-2-acetamido-3,6-di-O-(2-butenyl)-2-deoxy-β- D -glucopyranoside. Removal of the 2-butenyl groups was performed by treatment with potassium tert-butoxide in dimethyl sulfoxide, followed by isomerization of the allyl to a 1-propenyl group with tris(triphenyl-phosphine)rhodium chloride. Mild, acid treatment, and catalytic hydrogenation, gave the title trisaccharide.

Ligand recognition by purified human mannose receptor

In this work we examine the carbohydrate binding properties of human placental mannose receptor (HMR) using a rapid and sensitive enzyme-linked immunosorbent microplate assay. The assay is based on the inhibition of binding of highly purified receptor to yeast mannan-coated 96-well plates. The specificity of ligand binding was inferred from the potency of different saccharides in blocking HMR binding to the mannan-coated wells. The relative inhibitory potency of monosaccharides was L-Fuc greater than D-Man greater than D-Glc greater than D-GlcNAc greater than Man-6-P much greater than D-Gal much greater than L-Rha much greater than GalNAc. The inhibitory potency of mannose increased by two orders of magnitude when linear oligomers were used. Oligomers containing alpha-1-3- and alpha-1-6-linked mannose residues were more inhibitory than those containing alpha-1-2- and alpha-1-4-linked mannoses. Linear or branched oligomannosides larger than three units did not have a significant influence on their inhibitory potency; rather, potency was found to decrease in comparison with oligomannosides with three units. Compared to linear oligomers, inhibition of binding was the best using branched mannose oligosaccharides, alpha-D-Man-bovine serum albumin conjugates, or mannan. A model is discussed in which branched ligand is bound to spatially distinct sites on the HMR.

The preparation of a partially protected heptasaccharide-asparagine intermediate for glycopeptide synthesis

The heptasaccharide O-alpha-D-mannopyranosyl-(1----6)-O-[alpha-D-mannopyranosyl-(1----3)]-O- alpha-D-mannopyranosyl-(1----6)-O-[alpha-D-mannopyranosyl-(1----3)]-O-be ta- D-mannopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)- (1----4)-2-acetamido-2-deoxy-D-glucopyranose, isolated from the urine of swainsonine-intoxicated sheep, was peracetylated and was converted into the glycosyl azide by three alternative procedures, the most successful of which was formation of peracetyl oxazoline by treatment with trimethylsilyl trifluoromethanesulfonate, followed by treatment with trimethylsilyl azide. Reduction of the glycosyl azide in the presence of Lindlar catalyst gave the glycosylamine derivative, which was coupled with 1-benzyl N-fluoren-9-ylmethoxycarbonyl-L-aspartate to yield a protected glycosylasparagine. The benzyl ester group was easily removed by hydrogenolysis to form an intermediate suitable for glycopeptide synthesis.

The synthesis and properties of benzylated oxazolines derived from 2-acetamido-2-deoxy-D-glucose

Abstract 2-Methyl-(2-acetamido-3,4,6-tri-O-benzyl-1,2-dideoxy-α- D -glucopyrano)-[2,1-d]-2-oxazoline,2-methyl-(2-acetamido-6-O-acetyl-3,4-di-O-benzyl-1,2-dideoxy-α- D -glucopyrano)-[2,1-d]-2-oxazoline,and 2-methyl-(2-acetamido-4-O-acetyl-3,6-di-O-benzyl-1,2-dideoxy-α- D -glucopyrano)-[2,1-d]-2-oxazoline were synthesized from the allyl 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy- D -glucopyranosides, and from the 3,4-di-O-benzyl or 3,6-di-O-benzyl analogs, respectively, both the α and β anomer being used in each case. The preparation of allyl 2-acetamido-3,4,6-tri-O-benzyl- and 3,6-di-O-benzyl-2-deoxy-β- D -glucopyranoside is also described. Treatment of the tri-O-benzyl oxazoline with dibenzyl phosphate gave a pentabenzylglycosyl phosphate, from which all the benzyl groups were removed by catalytic hydrogenation, giving 2-acetamido-2-deoxy-α- D -glucopyranosyl phosphate. The corresponding β anomer was not detectable. Treatment of the 3,4-, or 3,6-, di-O-benzyl oxazoline with allyl 2-acetamido-3,4-di-O-benzyl-α- D -glucopyranoside readily gave disaccharide products from which the protecting groups were removed, to give the (1→6)-linked isomer of di-N-acetylchitobiose. Under both acidic and basic conditions, this isomer was less stable than the (1→4)-linked compound. Attempts to employ the 3,6-di-O-benzyl oxazoline for the formation of (1→4)-linked disaccharides, by treatment with either anomer of allyl 2-acetamido-3,6-di-O-benzyl-2-deoxy- D -glucopyranoside, were not very successful, presumably owing to hindrance by the bulky benzyl groups.

The synthesis of P1-2-acetamido-4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2-deoxy-α-d-glucopyranosyl P2-dolichyl pyrophosphate, (P1-di-N-acetyl-α-chitobiosyl P2-dolichyl pyrophosphate)

Abstract 2-Acetamido-4-O-(2-acetamido-2-deoxy-β- d -glucopyranosyl)-2-deoxy-α- d -glucopyranosyl phosphate, pure according to thin-layer and gas—liquid chromatography, optical rotation, and treatment with alkaline phosphatase and 2-acetamido-2-deoxy-β- d -glucosidase, was prepared by treatment of 2-methyl-[4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β- d -glucopyranosyl)-3,6-di-O-acetyl-1,2-dideoxy-α- d -glucopyrano]-[2,1-d]-2-oxazoline with dibenzyl phosphate, followed by the removal of the benzyl groups by catalytic hydrogenolysis, and O-deacetylation. In contrast, a sample prepared by the phosphoric acid procedure was shown to consist mainly of the β anomer. 2-Acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β- d -glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-α- d -glucopyranosyl phosphate was treated wit P1-diphenyl P2-dolichyl pyrophosphate to give a fully acetylated pyrophosphoric diester, which was O-deacetylated to give P1-2-acetamido-4-O-(2-acetamido-2-deoxy-β- d -glucopyranosyl)-2-deoxy-α- d -glucopyranosyl P2-dolichyl pyrophosphate. This compound could be separated from the β anomer by t.l.c., and its behavior under dilute acid and alkaline conditions was investigated.

Biosynthesis of a P1-2-acetamido-2-deoxy-D-glucosyl P2-polyisoprenyl pyrophosphate by calf pancreas microsomes

Abstract Incubation of UDP-[14C]- N -acetylglucosamine with calf pancreas microsomes in the presence of Mn++ and potassium thiocyanate gave a labeled glycolipid, tentatively identified as P 1-2-acetamido-2-deoxy- D -glucosyl P 2-dolichyl pyrophosphate on the basis of cochromatography with synthetic P 1-2-acetamido-2-deoxy-α- D -glucopyranosyl P 2-dolichyl pyrophosphate, similar chemical and enzymic hydrolyses of the biosynthetic and synthetic compounds, and stimulation of the biosynthesis by addition to the incubation mixture o dolichyl phosphate or a crude lipid fraction extracted from microsomes.

Synthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Man2GlcNAc2 and Man1GlcNAc2 are transferred from dolichol to protein in vivo

Transfer of truncated oligosaccharides to protein in vivo and the structure of Man2GlcNAc2 synthesized by intact yeast (Saccharomyces cerevisiae) were investigated in the alg2 mutant. At the nonpermissive temperature the alg2 mutant accumulates lipid-linked oligosaccharides that migrate on Bio-Gel P4 in the range expected for Man2GlcNAc2 and Man1GlcNAc2 (T.C. Huffaker and P.W. Robbins (1983) Proc. Natl. Acad. Sci. USA 80, 7466-7470). We characterized the oligosaccharides, derived from protein and lipid, by comigration with standards on HPLC and by Smith degradation followed by HPLC. Man2GlcNAc2 and Man1GlcNAc2 are found on protein in alg2, since their release from a protein-containing precipitate of alg2 cells is N-glycanase (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) dependent. Transfer also occurred in alg2/pAC3 cells, which carry ALG2 on a multicopy plasmid that confers partial correction of the oligosaccharide phenotype. The alg2/pAC3 cells are viable at 36 degrees C. Two isomers of Man2GlcNAc2, Man1----3ManGlcNAc2 and Man1----6ManGlcNAc2, were present on lipid and protein. The transfer of Man2GlcNAc2 and Man1GlcNAc2 to protein by intact cells supports topological models that postulate access by early intermediates to the lumen of the endoplasmic reticulum.

Oligosaccharides from placenta: early diagnosis of feline mannosidosis

High‐pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of α‐mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N‐acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.

Application of lectin histochemistry and carbohydrate analysis to the characterization of lysosomal storage diseases

In lysosomal storage diseases that involve a defect in the catabolism of glycoconjugates, lectin histochemistry adds a new dimension to the characterization of stored carbohydrates as it identifies sugar residues in situ in the affected cells and, thus, determines which cell types are affected by storage. It may be combined with chemical and biochemical analysis by h.p.l.c. The present review summarizes recent results for a variety of storage diseases and presents new data for GM1-gangliosidosis.