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The analysis and polyclonal antibody response for newly synthesized maltose-BSA conjugate neoglycoproteins is described. In this first proof of concept study, a simple carbohydrate antigen, maltose, was linked to BSA by reductive amination. An aglycone spacer was utilized to conserve the intact annular maltose structure and to promote the accessibility of the carbohydrate immunogen hapten during immunization. The neoglycoproteins were investigated by CGE and the number of conjugated maltose residues was determined by MALDI-TOF MS. The neoglycoproteins were then evaluated by immunization of BALB/c mice and the polyclonal antibody response was tested by ELISA as evidence for the presence of sugar-containing epitope-specific antibodies. Selective antibody binding was demonstrated to the synthesized neoglycoproteins with different (low and high) glycosylation degrees suggesting the possible use of this approach to generate antibodies. Moreover, the polyclonal antibody response was not inhibited by maltose or other simple carbohydrates to confirm presence of the neoglycoprotein-specific antibodies.

The fully O-benzylated pentasaccharide glycosyl azide representing the common core structure of N-glycans was synthesized. The β-mannosidic linkage was created by C-2 epimerization of the initially introduced β-d- gluco-unit via DMSO/Ac2O oxidation followed by stereoselective reduction with tetrabutylammonium borohydride.

Mono-, di- and trisaccharide representing the reducing terminal of the core structure of N-glycans were incorporated into Leu-Lys-Asn-Gly-Gly-Pro hexapeptide that is a partial structure of the Trp-cage mini-protein by linear assembly. These studies provide evidence that the used combination of Fmoc and Boc strategy and mild conditions result in glycopeptides in high purity and reasonable yield.

Comparative evaluation of the manno/gluco ratios obtained in the conventional reductions of β-D-glucoside-2-uloses (1−4, 13 and 14) reveals the influence of the substitution pattern: the presence of a 4,6-O-acetal function results in lower stereoselectivity in the monosaccharide-uloside cases and low stereoselectivity in the disaccharide-uloside cases, while the absence of a 4,6-O-acetal group provides distinctly higher stereoselectivity. The 3-O-benzyl and 3-O-allyl ethers vicinal to the carbonyl to be reduced have a similar influence on the steric outcome of the carbonyl reduction. A peculiar effect of acetoxydimethylsulfonium acetate (“activated” DMSO) was observed. In all cases, its presence strongly increased the manno-selectivity of the reduction. A simple, preparatively expedient, commonly suitable protocol has been elaborated for achieving high manno-selectivities and, hence, satisfactory yields.

2,3-Diazido-2,3-dideoxy-beta-D-mannopyranoside derivatives were synthesized in order to prepare beta-glycosides of 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, a rare moiety of bacterial oligosaccharides. A direct glycosyl donor, 4,6-di-O-acetyl-2,3-diazido-2,3-dideoxy-alpha-D-mannopyranosyl bromide, was prepared, and its synthetic capacity was tested in glycosylation reactions. An indirect route was also elaborated: 3-azido-3-deoxy-beta-D-glucopyranosides were converted into beta-D-mannopyranosides. The cis vicinal diazido function successfully tolerated the conditions of mild acidic hydrolysis, tritylation, Jones oxidation, TEMPO oxidation, acetolysis, and bromination with TiBr(4).

For the construction of N-glycoprotein glycan chains, valuable potential glycosyl donors, O-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose octaacetate (19) and O-α-D-mannopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→2)-α-D-mannopyranose undeca-acetate (20) were obtained in gram-scale by the acetylation and subsequent partial acetolysis of baker's yeast, without the isolation of mannan. The acetolytic products were investigated by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Octaacetates of mannobiose (1→3) (11) and (1→6) (17) were chemically synthesized.

Carbohydrate microarrays, comprising hundreds to thousands of different glycan structures on solid surfaces in a spatially discrete pattern, are sensitive and versatile tools for the analysis of glycosylation changes in complex biological samples. Glycoarrays are also suitable for monitoring multiple molecular interactions with biomolecules where sugars are involved, offering a large variety of bioassay options. In this paper we review the most important glycan microarray types currently used with their main applications, and discuss some of the future challenges the technology faces.

Essential components of N-glycoproteins were synthesized in octyl glycoside form starting from 3-O-allyl-D-glucose derivative. The β-mannosidic linkage was formed by the oxidation reduction method. MALDI-TOF mass spectrometry and its post-source decay (PSD) mode were used for identification and structural elucidation of protected synthetic oligosaccharides related to N-glycoproteins. Most fragments, identified in the PSD spectra, were products of the cleavage of glycosidic bonds.

A concise practical, large scale-adaptable six-step sequence has been developed for the transformation of diacetone-glucose into 4,6-di-O-benzoyl-3-O-benzyl-alpha-D-arabino-hexopyranos-2-ulosy l bromide (7), a most useful indirect beta-D-mannosyl donor as its blocking group pattern allows the construction of biologically relevant beta-D-mannosides branched at O-3 and O-6. The broad utility of this new ulosyl bromide 7 resides in its high anomeric reactivity, and in the ease and uniformity with which beta-stereocontrol can be achieved over both, glycosidations and carbonyl reduction of the beta-ulosides formed: Koenigs-Knorr conditions exclusively provide beta-glycosiduloses, hydride reduction of their carbonyl functions proceeds with high stereoselectivities (20:1) in favor of the beta-D-mannosides. These preparatively auspicious properties are materialized in an efficient, straightforward synthesis of alpha-D-Manp-(1--6)-[alpha-D-Manp-(1--3)]-beta-D-Manp++ +-(1--O)-Octyl, the 3,6-O-branched core-mannotrioside carrying an octyl spacer instead of the chitobiosyl unit.

Commercially available chemicals, such as diethylene glycol, 1,9-nonanediol, 9-decen-1-ol, 1,2,6-hexanetriol, and p-nitrophenol were used to prepare spacer-armed carbohydrate derivatives. Glycosides of d -glucose, N-acetyl- d -glucosamine and 3-O-methyl- d -glucose have been synthesized, carrying reactive groups at the end of the spacer-arms. These glycosides are capable of forming neoglycoproteins. When bromo sugars were reacted with highly apolar aglycones in the presence of mercuric cyanide or mercuric bromide, a considerable amount of ‘2-OH-compounds’ (such as 9-hydroxynonyl 3,4,6-tri-O-acetyl-β- d -glucopyranoside) were formed. Some spacer-armed derivatives (such as 9,10-epoxydecyl β- d -glucopyranoside) are theoretically a mixture of diastereomers, but this fact does not mirror in the 1H and 13C NMR spectra. © 1997 Elsevier Science Ltd.

The synthesis of the title tetrasaccharide glycoside 38 is reported. p -Nitrophenyl endo -3,4- O -benzylidene-6-deoxy-α- l -talopyranoside ( 4 , 3- O -acetyl-2,4-di- O -benzyl-α- l -rhamnopyranosyl trichloroacetimidate ( 7 ), methyl 3- O -acetyl-4- O -benzyl-2- O -methyl-1-thio-β- l -fucopyranoside ( 15 ) 3- O -acetyl-4- O -benzyl-2- O -methyl-α- l -fucopyranosyl bromide ( 16 ), and ethyl 3- O -acetyl-4- O -benzyl-2- O -methyl-1-thio-α- d -rhamnopyranoside ( 33 ) were prepared as intermediates. Compound 4 was glycosylated with imidate 7 as well as with methyl 3- O -acetyl-2,4-di- O -benzyl-1-thio-α- l -rhamnopyranoside ( 9 ), affording the same disaccharide derivative 8 . Deacetylation of 8 gave crystalline 17 . Condensation of 17 with both fucosyl donors 15 and 16 yielded the same trisaccharide derivative 18 stereoselectively. Compound 18 was also prepared by the coupling of 4 with disaccharide glycosyl donor 20 . After deacetylation of 18 (→ 34 ), methyl triflate-promoted glycosylation with compound 33 resulted in tetrasaccharide 35 . Conversion of the p -nitrophenyl group of 35 into the p -trifluoroacetamidophenyl group (→ 36 ) and removal of the protecting groups gave the title tetrasaccharide glycoside 38 .

Treatment of 2- O -benzoyl ( 1 ) and 2- O -acetyl ( 5 ) derivatives of benzyl 4,6- O -benzylidene-3- O -(2,3,4-tri- O -acetyl-α- l -rhamnopyranosyl)-β- d -galactopyranoside under Zemplen conditions (catalytic amount of sodium methoxide in methanol) gave partially deacylated disaccharides in which the 2- O -acyl groups were retained. Likewise, a similar result was obtained with the β- l -rhamnopyranosyl analogue ( 3 ) of 1 . This anomalous reaction was used in a synthesis of the title trisaccharide ( 17 ) and of methyl 4,6- O -benzylidene-3- O -(2,3:4,6-di- O -isopropylidene-α- d -mannopyranosyl)-α- d -glucopyranoside, an intermediate for the synthesis of 2- O -glycosyl-3- O -(α- d -mannopyranosyl)- d -glucoses.

Reaction of mono-, di-, and trisaccharide derivatives of methyl beta-D- and octyl beta-D-mannopyranosides bearing ester groups at isolated and non-isolated positions on the same molecule, under Zemplen conditions (catalytic amount of sodium methoxide in methanol) gave partially deacylated compounds, in which the O-acyl groups were retained at isolated sites. In the case of one disaccharide, all the benzoyl groups remained intact at the reducing end, while all the acetyl functions were removable from the nonreducing end. In another case, both isolated ester groups at positions 2 and 4 were retained at the reducing end. The isolated 2-O-acyl groups on methyl alpha-D-mannopyranoside compounds were more labile than on the corresponding beta-mannosides under the same conditions. The mechanism of the reaction may be different for ester groups at isolated or non-isolated positions. In the latter case, acyl migration may take place and carry acyl groups into a less hindered position.

The heptakis (2,3-di-O-acetyl-6-bromo-6-deoxy)-β-cyclodextrin has been converted to the 6-azido derivate with 82% yield, then deacetylated by the Zemplen's method (yield 75%) and methylated by methyl-iodide (yield 66%), to heptakis (2,3-di-O-methyl-6-azido-6-deoxy)-β-cyclodextrin, which is an appropriate intermediary toward methylated amino-CD-derivatives. The structure of all compounds were unambiguously proven by NMR spectroscopy. Halogen-Azid-Austausch zur Darstellung einiger symmetrisch substituierter β-Cyclodextrinderivate Heptakis-(2,3-di-O-acetyl-6-bromo-6-desoxy)-β-cyclodextrin wurde umgewandelt zum 6-Azido-Derivat mit einer Ausbeute von 82%, man desacetyliert nach Zemplen (Ausbeute 75%) und methyliert mit Methyliodid (Ausbeute 66%) zu Heptakis-(2,3-di-O-methyl-6-azido-6-desoxy)-β-cyclodextrin, welches ein geeignetes Zwischenprodukt zur Herstellung von methylierten Amino-Cyclodextrin-Derivaten ist. Die Strukturen wurden durch NMR-Spektroskopie bewiesen.

p-Nitrophenyl glycosides of 3-O-Me-β- d -Glcp-(1 → 3)-α- l -Rhap, α- l -Rhap(1 → 2)-6-deoxy-α- l -Talp, and 3-O-Me-β- d -Glcp-(1 → 3)-α- l -Rhap-(1 → 2)-6-deoxy-α- l -Talp have been prepared, related to Mycobacterium avium. Various glycosylation methods have been used for the formation of the interglycosidic linkages. The p-nitrophenyl derivatives were converted into p-isothiocyanatophenyl glycosides, capable of forming neoglycoproteins. © 1997 Elsevier Science Ltd.

Both diastereoisomers of 1-phenylethylidene acetals (acetophenone acetals) of methyl and benzyl β- l -arabinopyranoside and α- l -rhamnopyranoside were prepared. Acetal-exchange reactions gave only the endo -phenyl isomers; their 2- O - and 4- O -acetyl derivatives were isomerised into the exo -phenyl compounds. 1 H-N.m.r. data were used to determine the absolute configuration at the acetal carbon atom in these compounds. The protons of the methyl group of the exo -phenyl isomers resonate at lower field than those of the endo -phenyl isomers. Hydrogenolysis of various methylene, ethylidene, and isopropylidene derivatives gave axial ethers. The endo -phenyl isomers of the acetophenone derivatives also gave axial 1-phenylethyl ethers in two diastereoisomeric forms. The exo -phenyl isomers of the arabinosides were stable towards the reagent (LiAlH 4 AlCl 3 ), whereas the corresponding rhamnopyranosides gave the 2-(1-phenylethyl) ethers, but cleavage required prolonged reaction time and higher temperature.

The reaction of benzyl exo -3,4-O-benzylidene-β- l -arabinopyranoside 1 with α-acetobromo- d -glucose 3 resulted in a mixture of two disaccharides, 5 and 6 , in which the configuration of the acetal ring was different. The reaction of 1 with α-acetobromo- l -rhamnose 4 gave the desired disaccharide 7 without isomerisation of the dioxolane-type benzylidene ring. The reason for the isomerisation, occuring during the Koenigs-Knorr reaction, is discussed. Similar treatment of benzyl endo -3,4-O-benzylidene-β- l -arabinopyranoside 2 with 4 yielded 8 . Compounds 6 , 7 and 8 were deacetylated and benzylated to obtain 9 , 10 and 11 . Hydrogenolysis (LiAlH 4 AlCl 3 ) of all fully protected disaccharides afforded derivatives with a free OH-3 ( 9 → 13 and 11 → 15 ). Hydrogenolysis of 10 also resulted in 15 , and the desired 14 with a free OH-4 was only the minor product of the reaction. Glucosylation of compounds 14 and 15 resulted in the two trisaccharide derivatives in protected form ( 16 and 17 ). Deprotection of 6 , 7 , 16 and 17 gave the four title compounds ( 22 , 23 , 18 and 19 ). The synthesized compounds were studied by 1 H- and 13 C-NMR spectroscopic methods. In disaccharides having (1 → 2) bonds and in trisaccharide 19 having (1 → 2) and (1 → 3) bonds the arabinose moiety is present in pyranose and furanose forms. The complex spectra of these derivatives were assigned using the methyl ethers of l -arabinose ( 24–29 ) as model compounds. The 13 C-NMR spectrum of 18 was assigned with the aid of 4-O-β- d -glucopyranosyl- l -arabinopyranose. For comparisons, the spectra of all mono- and dibenzyl ethers of benzyl β- l -arabinopyranoside were also recorded and assigned.

The conformation of the methylene, isopropylidene, benzylidene, and 1-phenylethylidene acetals of some rhamnopyranoside and arabinopyranoside derivatives has been investigated by n.m.r. spectroscopy. In each series, the pyranoside ring adopts a distorted chair conformation. For the benzylidene acetals, the pyranoside ring is more distorted in the endo -phenyl isomers, and these isomers are compared to the methylene and isopropylidene acetals. For the 1,3-dioxolane ring, only the coupling constant of the acetal carbon atom with the axial bridgehead proton is measurable; for the benzylidene acetals, it is larger in the exo -phenyl isomer. For the other types of acetals, the coupling constant depends on the nature of the endo substituent at the acetal carbon atom. If it is bulky, the coupling constant and the conformation of the dioxolane ring are similar to those of the corresponding endo -phenyl benzylidene derivative.

Condensation of benzyl 3-O-benzoyl-4,6-O-benzylidene-, benzyl 2-O-benzoyl-4,6-O-benzylidene- (2), and benzyl2,3,6-tri-O-benzyl-⨿-d-galactopyranoside, separately, with tri-O-acetyl-α-l-rhamnopyranosyl bromide gave mainly α-linked disaccharide derivatives. An appreciable proportion of the ⨿-linked disaccharide was also obtained from 2. An anomalous deacylation reaction was found for the (1→3)-linked disaccharide, and the partially benzoylated products were isolated and characterised. The anomeric configuration of each disaccharide was established on the basis of JC-1,H-1 values. The chemical shifts for the galactose moieties of the α- and β-l-rhamnopyranosyl derivatives differed in a systematic way.

Benzyl 2- O -acetyl-4,6- O -benzylidene-3- O -(2,3,4-tri- O -acetyl-α- l -rhamnopyranosyl)-β- d -galactopyranoside ( 11 ) has been synthesised by two routes. Partial deacetylation of 11 and then acid hydrolysis yielded benzyl 2- O -acetyl-3- O -α- l -rhamnopyranosyl-β- d -galactopyranoside, catalytic hydrogenolysis of which gave the first title compound in excellent yield. Benzyl 4,6- O -benzylidene-3- O -α- l -rhamnopyranosyl-β- d -galactopyranoside was benzylated, and hydrogenolysis (LiAlH 4 -AlCl 3 ) of the product gave the disaccharide derivative 16 with only HO-6 unsubstituted. Acetylation of 16 followed by catalytic hydrogenolysis gave the crystalline, second title compound. As model compounds for comparative n.m.r. studies, 2- O -, 3- O -, and 6- O -acetyl- d -galactose were also synthesised.

The oligosaccharide components, 3-O-Me-beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-beta-D-Glcp-(1----6)- alpha-D-Glcp-(1----1)-alpha-D-Glcp (1) and beta-D-Glcp-(1----4)-beta-D-Glcp-(1----6)-alpha-D-Glcp-(1----1)-alpha-D- Glcp, of the glycolipid-type antigens isolated from M. smegmatis have been synthesised from 2,3,4,2',3',4',6'-hepta-O-acetyl-alpha,alpha-trehalose and the appropriate glycosyl bromides under Helferich conditions with Hg(CN)2 as the promoter. Condensation of the trisaccharide glycosyl bromide 27 gave an orthoester derivative (28) which could be rearranged, using HgBr2 or boron trifluoride etherate, into the acetylated derivative (29) of 1. The model compound beta-D-Glcp-(1----6)-alpha-D-Glcp-(1----1)-alpha-D-Glcp has also been synthesised.

The oligosaccharide core region, β- d -Glc p -(1→3)-β- d -Glc p -(1→4)-α- d -Glc p -(1↔1)-α- d -Glc p ( 1 ), of the lipo-oligosaccharide-type antigens isolated from M. kansasii has been synthesised from 2,3,2′,3′,4′,6′-hexa- O -benzyl-6- O -(1-phenylethyl)-α,α-trehalose ( 4 ). Compound 4 was obtained by LiAlH 4 -AlCl 3 -type hydrogenolysis of 2,3,2′,3′,4′,6′-hexa- O -benzyl-4,6- O -( S )-(1-phenylethylidene)-α,α-trehalose. The β-laminaribiosyl part of the molecule was built-up by sequential glycosylation steps using 2,4,6-tri- O -acetyl-3- O -allyl-α- d -glucopyranosyl bromide in the presence of HgBr 2 and methyl 2,3,4,6-tetra- O -acetyl-1-thio-β- d -glucopyranoside promoted by methyl triflate. The complete a priori 13 C-n.m.r. spectrum assignment of 1 was achieved by applying 2D methods.

A series of prazosin analogues comprised of N-acyl derivatives of N'-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazine was prepared and the nature of their binding to alpha 1-adrenoceptors was investigated. Derivatives with alpha, beta-unsaturated acyclic acyls had some affinity but no irreversible action at the receptor. Other potent compounds, also without irreversible activity, contained cinnamoyl or (phenylamino)thiocarbonyl residues. High affinity and irreversible binding were obtained with a bicyclo[2.2.2]octa-2,5-dien-2-ylcarbonyl derivative. The conjugated double bond in this compound was in about the same position and distance from the pharmacophore as in some of the above compounds of high affinity but with no irreversible action. Two consecutive recognition steps were thought to be involved in irreversible blockade: reversible binding of the pharmacophore part of the molecule to the binding site of the receptor, followed by reaction of the chemoreactive part with an adjacent nucleophile of the receptor. The present results suggest that for the second step to occur efficiently, some affinity for the receptor must be present even in the chemoreactive part of the molecule; simple spanning of the binding and nucleophile sites of the receptor was insufficient.

The aim of the presentation is to give a comprehensive picture about the different methods used for the synthesis of chemically modified cyclodextrin derivatives with well defined and characterized structures. The efficiency of the different chromatographic and spectroscopic methods used for the structure elucidation of CD-derivatives are treated and estimated.

A photoaffinity probe for α 1 -adrenoceptors was synthesized and its properties examined on rat brain membrane preparations. The binding of 4-amino-2-[4-(4-azidocinnamoyl)piperazino]-6,7-dimethoxyquinazoline (ACP) to these receptors was of high affinity (K D = 1.05 nM) and reversible in the dark. A dose-dependent decrease in the concentration of [ 3 H]prazosin binding sites without a change in K D was observed when membranes were preincubated with ACP, photolyzed, and then extensively washed prior to assay. This reduction in receptor concentration was prevented by α 1 -adrenergic ligands. The specificity of ACP for α 1 -receptors was further demonstrated by its inability to compete with [ 3 H]dihydroalprenolol and [ 3 H]yohimbine binding in these same membranes. Also, the concentrations and affinity constants of β-adrenoceptors and α 2 -adrenoceptors were unaffected in membranes which had been photolyzed after preincubation with ACP. No reduction in concentration of α 1 -adrenoceptors was detected if ACP was photolyzed prior to incubation with receptors or if ACP was maintained in darkness throughout the experiment. The results suggest that ACP is a specific and sensitive photoprobe that may be useful for further studies on α 1 -adrenoceptor coupled systems and that may be particularly suited for use in cell culture work.