Hans Paulsen

Complex Carbohydrates Are Not Removed During Processing of Glycoproteins by Dendritic Cells: Processing of Tumor Antigen MUC1 Glycopeptides for Presentation to Major Histocompatibility Complex Class II-restricted T Cells

In contrast to protein antigens, processing of glycoproteins by dendritic cells (DCs) for presentation to T cells has not been well studied. We developed mouse T cell hybridomas to study processing and presentation of the tumor antigen MUC1 as a model glycoprotein. MUC1 is expressed on the surface as well as secreted by human adenocarcinomas. Circulating soluble MUC1 is available for uptake, processing, and presentation by DCs in vivo and better understanding of how that process functions in the case of glycosylated antigens may shed light on antitumor immune responses that could be initiated against this glycoprotein. We show that DCs endocytose MUC1 glycopeptides, transport them to acidic compartments, process them into smaller peptides, and present them on major histocompatability complex (MHC) class II molecules without removing the carbohydrates. Glycopeptides that are presented on DCs are recognized by T cells. This suggests that a much broader repertoire of T cells could be elicited against MUC1 and other glycoproteins than expected based only on their peptide sequences.

Lewissäure-katalysierte synthesen von di- und trisaccharid-sequenzen der O- und N-glycoproteine. Anwendung von trimethylsilyltrifluoromethanesulfonat

Abstract In the presence of trimethylsilyltrifluoromethanesulfonate as Lewis acid catalyst, β-acetates reacted, as glycosyl donors and with neighboring-group participation, with secondary hydroxyl groups of saccharides having low reactivity to give β-glycosidically linked di- and oligo-saccharides in high yields. The protecting groups of both compounds of the reaction had to be stable under acid conditions. Thus, the reaction of 1,2,3,4,6-penta-O-acetyl-β- d -galactopyranose with methyl 2-azido-4,6-di-O-benzoyl-2-deoxy-β- d -galactopyranoside gave, in 83% yield, methyl 2-azido-2,6-di-O-benzoyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β- d -galactopyranosyl)-β- d -galactopyranoside which is a useful building unit. The glycosidic reactions under Lewis acid catalysis and Koenigs—Knorr conditions are compared. The efficiency of the Lewis acid-catalyzed procedure was demonstrated by the further synthesis of di- and tri-saccharides, such as lactosamine and chitobiose derivatives, whereby hydroxyl groups having low reactivity could be glycosylated in good yields.

Synthese der glycopeptide O-β-d-galactopyranosyl-(1→3)-O-(2-acetamido-2-desoxy-α-d-galactopyranosyl)-(1→3)-l-serin und -l-threonin

Abstract In the presence of silver carbonate-silver perchlorate and dichloromethane-toluene as solvent, 3,4,6-tri- O -acetyl-2-azido-2-deoxy-β- d -galactopyranosyl chloride, and derivatives of l -serine and - l -threonine, gave, with high stereoselectivity, the benzyl esters of N -(benzyloxycarbonyl)-3- O -(3,4,6-tri- O -acetyl-2-azido-2-deoxy-α- d -galactopyranosyl)- l -serine ( 7 ) and - l -threonine ( 22 ), which were hydrogenolyzed and deblocked to give 3- O -(2-acetamido-deoxy-α- d -galactopyranosyl)- l -serine and - l -threonine, respectively, corresponding to the hapten of the Tn-antigen. Reduction of the azido group of 7 , followed by selective O -deacetylation and benzylidenation, gave a derivative that was glycosylated with 2,3,4,6-tetra- O -acetyl-α- d -galactopyranosyl bromide to yield a disaccharide. A similar sequence of reactions, starting from 22 , gave the l -threonine analog. Removal of the protecting groups from both compounds afforded O -β- d -galactopyranosyl-(1→3)- O -(2-acetamido-2-deoxy-α- d -galactopyranosyl)- l -serine and - l -threonine, respectively, the hapten of the T-antigen.

Darstellung selektiv blockierter 2-azido-2-desoxy-D-gluco- und -D-galactopyranosylhalogenide: reaktivität und 13C-NMR-spektren

Abstract The synthesis of several 2-azido-2-deoxy- D -gluco- and - D -galactopyranosyl halides is described. With tetraethylammonium chloride, α-pyranosyl bromides react to give β-pyranosyl chlorides. This provides a facile method for obtaining selectively blocked halides for the synthesis of α-linked, amino sugar of oligosaccharides. The inversion reaction at the anomeric centre is shown to be of second order, corresponding to an S N 2 mechanism. The rates of the inversion reactions were correlated to the 13C-n.m.r. data of C-1 of α-bromides. Within the gluco series, the 13C-n.m.r. shift of C-1 proves to be proportional to the natural logarithm of the rate constant. An analogous correlation in the galacto series could not be observed.

Synthesis, structure and reactions of glycosyl azides

Abstract Recent progress in synthetic methods and structure determination has resulted in several new developments in the chemistry of glycosyl azides. A review on these synthetically useful intermediates was published by Micheel and Klemer1 more than 30 years ago but newer treatments2,3 of related topics scarcely take notice of these compounds despite their definite importance as precursors to glycosyl amines and heterocyclic derivatives such as 1,2,3-triazoles.

Synthese eines trisaccharides aus N-acetylneuraminsäure und N-acetyllactosamin☆

Abstract The reaction of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5-tri-deoxy- d -glycero-β- d -galacto-2-nonulopyranosonate with benzyl 2-azido-3,6-di-O-benzyl-2-deoxy-4-O-(2,3-di-O-benzyl-β- d -galactopyranosyl)-β- d -glucopyranoside in the presence of mercury cyanide—mercury bromide gave a 1:1 mixture of the two anomeric (2→6)-linked trisaccharides containing N-acetylneuraminic acid. By chromatography, 22% of benzyl O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy- d -glycero-α- d -galacto-2- nonulopyranosylonate)-(2→6)-O-(2,3-di-O-benzyl-β- d -galactopyranosyl)-(1→4)-2-azido-3,6-di-O-benzyl-2-deoxy-β- d - glucopyranoside and 23% of the corresponding β-(2→6)-linked isomer could be isolated. A deblocking sequence, consisting of hydrogen sulfide reduction, acetylation, deacetylation, ester hydrolysis, and hydrogenolysis led to the deblocked O-(5-acetamido-3,5-dideoxy- d -glycero-α- d -galacto-2-nonulopyranosylonic acid)-(2→6)-O-β- d -galactopyranosyl-(1→4)-2-acetamido-2-deoxy- d -glucopyranose and to the corresponding β-(2→6)-linked compound.

Bräunungsreaktionen und fragmentierungen von kohlenhydraten : Teil I. Die flüchtigen abbauprodukte der pyrolyse von d-glucose

Zusammenfassung Volatile products from the thermal degradation of D -glucose at 300° were analysed. 1,4:3,6-Dianhydro- D -glucopyranose is the main product. The most-volatile compounds were separated by repeated gas-liquid chromatography. Fifty-six compounds were identified by mass spectrometry, “syringe-reactions”, and comparison with synthetic, standard substances. Furans are formed as the principal decomposition products, along with aldehydes, ketones, diketones, and aromatic hydrocarbons. Seventeen alkylfurans, some unsaturated, carbonyl-containing compounds, and some aromatic hydrocarbons were isolated for the first time from carbohydrate pyrolysis. Typical degradation products at the start of pyrolysis are vinylfurans, penta-1,3-dien-5-al, and furfural. Some compounds containing more than 6 C-atoms (for instance, 2-methyl-5-vinylfuran) were found. This suggests that furans are formed not from D -glucose itself, but by fragmentation of polymers.

Dynamic epigenetic regulation of initial O-glycosylation by UDP-N-Acetylgalactosamine:Peptide N-acetylgalactosaminyltransferases. site-specific glycosylation of MUC1 repeat peptide influences the substrate qualities at adjacent or distant Ser/Thr positions.

In search of possible epigenetic regulatory mechanisms ruling the initiation of O-glycosylation by polypeptide:N-acetylgalactosaminyltransferases, we studied the influences of mono- and disaccharide substituents of glycopeptide substrates on the site-specific in vitroaddition of N-acetylgalactosamine (GalNAc) residues by recombinant GalNAc-Ts (rGalNAc-T1, -T2, and -T3). The substrates were 20-mers (HGV20) or 21-mers (AHG21) of the MUC1 tandem repeat peptide carrying GalNAcα or Galβ1–3GalNAcα at different positions. The enzymatic products were analyzed by MALDI mass spectrometry and Edman degradation for the number and sites of incorporated GalNAc. Disaccharide placed on the first position of the diad Ser-16-Thr-17 prevents glycosylation of the second, whereas disaccharide on the second position of Ser-16-Thr-17 and Thr-5-Ser-6 does not prevent GalNAc addition to the first. Multiple disaccharide substituents suppress any further glycosylation at the remaining sites. Glycosylation of Ser-16 is negatively affected by glycosylation at position −6 (Thr-10) or −10 (Ser-6) and is inhibited by disaccharide at position −11 (Thr-5), suggesting the occurrence of glycosylation-induced effects on distant acceptor sites. Kinetic studies revealed the accelerated addition of GalNAc to Ser-16 adjacent to GalNAc-substituted Thr-17, demonstrating positive regulatory effects induced by glycosylation on the monosaccharide level. These antagonistic effects of mono- and disaccharides could underlie a postulated regulatory mechanism.

Cyclic Acyloxonium Ions in Carbohydrate Chemistry

Publisher Summary The neighboring-group reactions studied most intensively have been those of acyloxy groups, which constitute the complex neighboring-groups. The intermediate formation of a dioxolanylium ion is invoked. The formation of such a ring between C-1 and C-2 of aldoses determines the stereochemical outcome of glycosylation reactions from glycosyl halides. Neighboring-group reactions of acyloxy groups involve a synchronous process whereby the nucleophilic oxygen atom of the carbonyl group becomes attached at the same time as the nucleophilic leaving group is split off. The intermediate dioxolanylium ion is exceedingly reactive. The chapter presents reactions that can generate the dioxolanylium and dioxanylium ring-systems selectively in carbohydrate derivatives, in the absence of a nucleophile and in the presence of a difficultly polarizable complex anion. The various rearrangement reactions such as rearrangement of acyloxonium ions in polyol systems and monosaccharides, rearrangement of esters of polyols and monosaccharides in liquid hydrogen fluoride, rearrangement of cyclitols with acetic acid-sulfuric acid, and rearrangement of saccharides with other lewis acids are studied in the chapter.

Synthese der pentasaccharid-kette des forssman-antigens

Abstract The pentasaccharide chain of the Forssman antigen, O-(2-acetamido-2-deoxy-α- d -galactopyranosyl)-(1→3)-O-(2-acetamido-2-deoxy-β- d -galactopyranosyl)-(1→3)-O-α- d - galactopyranosyl-(1→4)-O-β- d -galactopyranosyl-(1→4)- d -glucopyranose (46) was synthesized by a block synthesis in which an α- d -glycoside linkage between two d -galactose residues was formed. The trisaccharide O-(6-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-α- d -galactopyranosyl)- (1→3)-O-(6-O-acetyl-4-O-benzyl-2-deoxy-2-phthalimido-β- d -galactopyranosyl)-(1→3)-6-O-acetyl-2,4-di-O-benzyl- α- d -galactopyranosyl bromide (40) (this was obtained through acetolysis of O-(6-O-acetyl-2-azido-3,4-di-O-benzoyl-2-deoxy-α- d -galactopyranosyl)- (1→3)-O-(6-O-acetyl-4-O-benzyl-2-deoxy-2-phthalimido-β- d -galactopyranosyl)-(1→3)-1,6-anhydro-2,4-di-O-benzyl-β- d - galactopyranose to the acetyl derivative, followed by reaction with titanium tetrabromide under anhydrous conditions) was condensed with benzyl-4-O-(6-O-benzoyl-2,3-di-O-benzyl-β- d -galactopyranosyl)-2,3,6- tri-O-benzyl-β- d -glucopyranoside were in the presence of silver carbonate and perchlorate. The resulting pentasaccharide was deprotected to give 46.