Glycosylation of internal sugar residues of oligosaccharides catalyzed by alpha-galactosidase from Aspergillus fumigatus

Purified alpha-galactosidase from a thermotolerant fungus Aspergillus fumigatus IMI 385708 was found to catalyze efficiently transgalactosylation reactions using 4-nitrophenyl alpha-D-galactopyranoside as glycosyl donor. Self-transfer reactions with this substrate afforded in low yields several 4-nitrophenyl galactobiosides. Monosaccharides also served as poor glycosyl acceptors. Disaccharides and particularly higher oligosaccharides of alpha-1,4-gluco- (maltooligosaccharides), beta-1,4-gluco- (cellooligosaccharides) and beta-1,4-manno-series were efficiently galactosylated, the latter being the best acceptors that were also doubly galactosylated. With mannooligosaccharides product yields increased with polymerization degree of acceptors reaching 50% at DP of 4-6. Longer oligosaccharide acceptors were galactosylated at internal sugar residues. All galactosyl residues were transferred exclusively to the primary hydroxyl group(s) at C-6 position of oligosaccharide acceptors. This is in accordance with the inability of the enzyme to transfer galactose to beta-1,4-linked xylooligosaccharides. This is the first report of glycosyl transfer reaction to internal sugar residues of oligosaccharides catalyzed by a glycosidase. High affinity to oligosaccharide acceptors also opens a way toward enzymatic glycosylation of polysaccharides, thus modulating their physico-chemical and biological properties.     

Glycosylation of internal sugar residues of oligosaccharides catalyzed by α-galactosidase from Aspergillus fumigatus

Purified α-galactosidase from a thermotolerant fungus Aspergillus fumigatus IMI 385708 was found to catalyze efficiently transgalactosylation reactions using 4-nitrophenyl α-d-galactopyranoside as glycosyl donor. Self-transfer reactions with this substrate afforded in low yields several 4-nitrophenyl galactobiosides. Monosaccharides also served as poor glycosyl acceptors. Disaccharides and particularly higher oligosaccharides of α-1,4-gluco- (maltooligosaccharides), β-1,4-gluco- (cellooligosaccharides) and β-1,4-manno-series were efficiently galactosylated, the latter being the best acceptors that were also doubly galactosylated. With mannooligosaccharides product yields increased with polymerization degree of acceptors reaching 50% at DP of 4–6. Longer oligosaccharide acceptors were galactosylated at internal sugar residues. All galactosyl residues were transferred exclusively to the primary hydroxyl group(s) at C-6 position of oligosaccharide acceptors. This is in accordance with the inability of the enzyme to transfer galactose to β-1,4-linked xylooligosaccharides. This is the first report of glycosyl transfer reaction to internal sugar residues of oligosaccharides catalyzed by a glycosidase. High affinity to oligosaccharide acceptors also opens a way toward enzymatic glycosylation of polysaccharides, thus modulating their physico-chemical and biological properties.     

Syntheses of linear tetra-, hexa-, and octa-saccharide fragments of the i-blood group active poly-(N-acetyl-lactosamine) series. Blockwise methods for the synthesis of repetitive oligosaccharide sequences

N-Phthaloylation of lactosamine gave various glycosyl donors (beta-chloride, beta-trichloroacetimidate) and glycosyl acceptors (3',4'-diol). Coupling of the chloride with a methyl beta-D-glycoside led to the tetrasaccharide fragment, beta-D-Galp-(1----4)-beta-D-GlcpNac-(1----3)-beta-D-Galp-(1----4)- beta-D-GlcpNAcOMe. Acetolysis of the protected tetrasaccharide, followed by treatment with hydrogen chloride, gave a tetrasaccharide chloride which was coupled with the methyl beta-glycoside of lactosamine. A hexasaccharide fragment, [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)]2-beta-D-Galp-(1----4)-bet a- D-GlcpNAcOMe, was thus obtained by this ("n + 1") method. A more efficient ("n + n") method was applied for the synthesis of an octasaccharide fragment, [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)]3-beta-D-Galp- (1----4)-beta-D-GlcpNAcOMe (38), where di- and tetra-saccharide intermediates having a 3,4-O-isopropylidene-beta-D-galactopyranosyl nonreducing terminal group and a benzyl beta-D-glycoside group were precursors, either as glycosyl donors (beta-trichloroacetimidates) or glycosyl acceptors (3,4-diols as nonreducing terminal groups). Thus, doubling the length of the repetitive oligosaccharide sequence could be efficiently accomplished at each glycosylation step.     

Substrate specificity of N-acetylhexosaminidase from Aspergillus oryzae to artificial glycosyl acceptors having various substituents at the reducing ends

The substrate specificity of N-acetylhexosaminidase (E.C. 3.2.1.51) from Aspergillus oryzae was examined using p-nitrophenyl 6-O-sulfo-N-acetyl-beta-D-glucosaminide (6-O-sulfo-GlcNAc-O-pNP) as the glycosyl donor and a series of beta-d-glucopyranosides and N-acetyl-beta-D-glucosaminides with variable aglycons at the anomeric positions as the acceptors. When beta-D-glucopyranosides with methyl (CH(3)), allyl (CH(2)CHCH(2)), and phenyl (C(6)H(5)) groups at the reducing end were used as the acceptors, this enzyme transferred the 6-O-sulfo-GlcNAc moiety in the donor to the location of O-4 in these glycosyl acceptors with a high regioselectivity, producing the corresponding 6-O-sulfo-N-acetylglucosaminyl beta-D-glucopyranosides. However, beta-D-glucopyranose lacking aglycon was a poor substrate for transglycosylation. This A. oryzae enzyme could also accept various N-acetyl-beta-D-glucosaminides carrying hydroxyl (OH), methyl (CH(3)), propyl (CH(2)CH(2)CH(3)), allyl (CH(2)CHCH(2)) and p-nitrophenyl (pNP; C(6)H(4)-NO(2)) groups at their aglycons, yielding 6-O-sulfo-N-acetylglucosaminyl-beta(1-->4)-disaccharide products.     

Glycosynthase activity of Bacillus licheniformis 1,3-1,4-beta-glucanase mutants: specificity, kinetics, and mechanism

Glycosynthases are engineered retaining glycosidases devoid of hydrolase activity that efficiently catalyze transglycosylation reactions. The mechanism of the glycosynthase reaction is probed with the E134A mutant of Bacillus licheniformis 1,3-1,4-beta-glucanase. This endo-glycosynthase is regiospecific for formation of a beta-1,4-glycosidic bond with alpha-glycosyl fluoride donors (laminaribiosyl as the minimal donor) and oligosaccharide acceptors containing glucose or xylose on the nonreducing end (aryl monosaccharides or oligosaccharides). The pH dependence of the glycosynthase activity reflects general base catalysis with a kinetic pK(a) of 5.2 +/- 0.1. Kinetics of enzyme inactivation by a water-soluble carbodiimide (EDC) are consistent with modification of an active site carboxylate group with a pK(a) of 5.3 +/- 0.2. The general base is Glu138 (the residue acting as the general acid-base in the parental wild-type enzyme) as probed by preparing the double mutant E134A/E138A. It is devoid of glycosynthase activity, but use of sodium azide as an acceptor not requiring general base catalysis yielded a beta-glycosyl azide product. The pK(a) of Glu138 (kinetic pK(a) on k(cat)/K(M) and pK(a) of EDC inactivation) for the E134A glycosynthase has dropped 1.8 pH units compared to the pK(a) values of the wild type, enabling the same residue to act as a general base in the glycosynthase enzyme. Kinetic parameters of the E134A glycosynthase-catalyzed condensation between Glcbeta4Glcbeta3GlcalphaF (2) as a donor and Glcbeta4Glcbeta-pNP (15) as an acceptor are as follows: k(cat) = 1.7 s(-)(1), K(M)(acceptor) = 11 mM, and K(M)(donor) < 0.3 mM. Donor self-condensation and elongation reactions are kinetically evaluated to establish the conditions for preparative use of the glycosynthase reaction in oligosaccharide synthesis. Yields are 70-90% with aryl monosaccharide and cellobioside acceptors, but 25-55% with laminaribiosides, the lower yields (and lower initial rates) due to competitive inhibition of the beta-1,3-linked disaccharide acceptor for the donor subsites of the enzyme.     

Chitinase-catalyzed synthesis of an alternatingly N-sulfonated chitin derivative

An alternatingly N-sulfonated chitin derivative (2) was synthesized via ring-opening polyaddition of an N-sulfonated chitobiose oxazoline derivative (1) catalyzed by chitinases from Bacillus sp. and Serratia marcescens. The polymerization proceeded homogeneously, providing 2 as a water-soluble polysaccharide in good yields with total control of regioselectivity and stereochemistry. M(n) of 2 reached 1900 and 4180 by use of chitinases from Bacillus sp. and Serratia marcescens, which correspond to 8-10 (n = 4-5) and 18-20 (n = 9-10) saccharide units, respectively. These results indicate that M(n) of 2 is controllable by selecting chitinases from different origins. It is considered that the C-2 position of the nonreducing unit in the oxazoline-type monomer is not deeply involved in the catalysis of chitinase.     

1,6-Anhydro-N-Acetyl- beta-D-glucosamine in oligosaccharide synthesis: II. synthesis of a spacered Ley tetrasaccharide

3-Aminopropyl glycoside of 3,2'-di-O-alpha-L-fucosyl-N-acetyllactosamine (Ley tetrasaccharide) was synthesized. The glycosyl donor, 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-galactopyranosyl bromide, was coupled with glycosyl acceptor, 1,6-anhydro-2-acetamido-2-deoxy-beta-D-glucopyranose or its 3-O-acetyl derivative, to give the corresponding N-acetyllactosamine derivatives in 20 and 71% yields, respectively. The glycosyl donor was synthesized from 1,2-di-O-acetyl-3,4,6-tri-O-benzoyl-D-galactopyranose, which was obtained by the treatment of benzobromogalactose with sodium borohydride to yield 1,2-O-benzylidene derivative and subsequent removal of benzylidene group and acetylation. Acidic methanolysis of the disaccharide derivatives resulted in the selective removal of one or both acetyl groups to give the disaccharide acceptor bearing hydroxy groups at C3 of the glucosamine residue and C2 of the galactose residue. The introduction of fucose residues in these positions by the treatment with tetrabenzylfucopyranosyl bromide resulted in a tetrasaccharide derivative, which was converted into 3,2'-di-O-alphha-L-fucopuranosyl- 1,6-anhydro-N-acetyllactosamine peracetate after substitution of acetyl groups for benzoyl and benzyl groups. Opening of the anhydro ring by acetolysis resulted in peracetate, which was then converted into the corresponding oxazoline derivative in two steps. Glycosylation of the oxazoline derivative with 3-trifluoroacetamidopropan-1-ol and removal of O-acetyl and N-trifluoroacetyl protective groups resulted in a free spacered Ley tetrasaccharide.     

Use of ionically tagged glycosyl donors in the synthesis of oligosaccharide libraries

We have developed a new procedure based on the random glycosyl reaction of a partially benzoylated glycosyl acceptor with a glycosyl donor containing a 4,6-O-(4-methoxycarbonylbenzylidene) protecting group as a masked/caged ion-tag. Glycosylated products are ionically tagged by saponification of the methyl ester and the use of this anion-tag greatly simplifies the separation of the desired oligosaccharides from unreacted or excess glycosyl acceptors as well as from over-glycosylated oligosaccharides. In addition, the use of partially benzoylated acceptors greatly improves their solubility in dichloromethane increasing the yield of product formation and, also, of altering the distribution of positional isomers in favor of products derived by reaction of the donors at hydroxyl groups which otherwise would be considerably less reactive. Using this new approach in random glycosyl reactions, several oligosaccharide libraries were readily prepared in overall yields of 60–70% and the individual positional isomers present in the libraries were identified using the ‘reductive-cleavage’ method.     

Sulfation of chondroitin oligosaccharides in vitro. Analysis of sulfation ratios

Microsomal preparations from cultured chick embryo chondrocytes were incubated with 3'-phosphoadenosine 5'-phosphosulfate and oligosaccharides prepared from chondroitin. Rates of 4- and 6-sulfation were measured at pH 6 and 8 in the presence of MnCl2 and Brij 58. Ratios of the overall 6-sulfation to 4-sulfation rates ranged from 40-200 at pH 8 and from 6-35 at pH 6, depending upon the composition of the assay mixture. When saturating concentrations of 3'-phosphoadenosine 5'-phosphosulfate and the oligosaccharide acceptors were used, the resulting products were mixtures of monosulfated oligosaccharides. The compositions of the mixtures formed from oligosaccharides with degrees of polymerization from 4-12 at pH 6 and 8 were analyzed. Sulfate substituents were found at all N-acetyl-D-galactosamine (GalNAc) residues in the acceptors but were not evenly distributed along the oligosaccharide chains. For oligosaccharides with nonreducing terminal D-glucuronic acid (GlcUA) residues, sulfation at the nonreducing terminal GlcUA----GalNAc occurred exclusively at the C6 of the GalNAc residue. However, for oligosaccharides with nonreducing terminal GalNAc residues the rate of 6-sulfation of the nonreducing terminal GalNAc was markedly reduced and was similar to the rate of 4-sulfation at the same position. The rates of sulfation at the reducing ends of the oligosaccharides were relatively high for the shorter oligosaccharide acceptors but decreased with increasing length of the acceptor, suggesting that the sulfotransferases recognized primarily the GalNAc residues in the nonreducing terminal regions.     

Bottom-up synthesis of hyaluronan and its derivatives via enzymatic polymerization: direct incorporation of an amido functional group

This paper reports the synthesis of hyaluronan (HA) and its derivatives via the hyaluronidase-catalyzed polymerization of 2-substituted oxazoline derivative monomers designed as "transition-state analogue substrates". Polymerization of 2-methyl oxazoline monomer from N-acetylhyalobiuronate (GlcAbeta(1-->3)GlcNAc) effectively proceeded at pH 7.5 and 30 degrees C, giving rise to synthetic HA (natural type) in an optimal yield of 78% via ring-opening polyaddition under total control of regioselectivity and stereochemistry. Hyaluronidase catalysis enabled the polymerization of 2-ethyl, 2-n-propyl, and 2-vinyl monomers, affording the corresponding HA derivatives (unnatural type) with N-propionyl, N-butyryl, and N-acryloyl functional groups, respectively, at the C2 position of all glucosamine units in good yields. The 2-isopropyl oxazoline derivative provided the N-isobutyryl derivative of HA in low yields. Monomers of 2-phenyl and 2-isopropenyl oxazoline derivatives were not polymerized. The mechanism of the polymerization is discussed.     

Efficient Glycosynthase Mutant Derived from Mucor hiemalis Endo-��-N-acetylglucosaminidase Capable of Transferring Oligosaccharide from Both Sugar Oxazoline and Natural N-Glycan

Endo-M, an endo-β-N-acetylglucosaminidase from Mucor hiemalis, is a family 85 glycoside hydrolase. This enzyme is unique in that it can transfer en bloc the oligosaccharide of various types of N-glycans onto different acceptors, and thereby it enzymatically generates diverse glycoconjugates. In this study, we performed mutational and kinetic studies focusing on a key catalytic asparagine 175 of Endo-M. We have shown that most of the Asn-175 mutants had significantly diminished hydrolysis activity but acted as glycosynthases capable of using synthetic sugar oxazoline for transglycosylation. Our results confirm the critical role of this asparagine residue in promoting the formation of an oxazolinium ion intermediate in the first step of the substrate-assisted catalysis. Interestingly, the N175Q mutant was found to possess dramatically enhanced glycosynthase-like activity with sugar oxazoline in comparison with N175A and a transglycosidase-like activity with “natural” N-glycan as well. These results also implicated the significance of amide side chain in the asparagine 175 of Endo-M for promoting oxazoline transglycosylation in the second step of the catalysis. The highly efficient syntheses of glycopeptides/glycoproteins by N175Q combined with synthetic sugar oxazolines or natural N-glycan substrates were exemplified. In addition, we also identified several previously unknown residues that seem to play a role in the catalysis of Endo-M.     

Synthesis of a library of fucopyranosyl-galactopyranosides consisting of a complete set of anomeric configurations and linkage positions

A library composed of a complete set of fucopyranosyl-galactopyranosides was synthesized. A perbenzylated phenylthio fucopyranoside and a series of tri-O-benzyl-galactopyranosyl fluorides having single hydroxyl groups at the 2-, 3-, 4-, and 6-positions were used as the glycosyl donor and glycosyl acceptors, respectively. The chosen set of functionalities at the anomeric centers enabled rapid access to the oligosaccharides based on chemoselective activation. The first coupling reaction was achieved by the action of dimethyl(methylthio)sulfonium trifluoromethanesulfonate (DMTST). The resulting disaccharide fluoride was readily activated by hafnocene bistrifluoromethanesulfonate [Cp2Hf(OTf)2] and glycosidated with n-octanol.     

Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background

An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.

Methods

We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.

Results

Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.

Conclusions

Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.

General significance

Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.     

Enzymatic synthesis of chondroitin 4-sulfate with well-defined structure

Synthesis of chondroitin sulfate (ChS) with well-defined structure was achieved for the first time by hyaluronidase-catalyzed polymerization. N-Acetylchondrosine (GlcAbeta(1-->3)GalNAc) oxazoline derivatives sulfated at C4 (1a), C6 (1b), and both C4 and C6 (1c) in the GalNAc unit were synthesized as transition state analogue substrate monomers for hyaluronidase (HAase) catalysis. Compound 1a was effectively polymerized by the enzyme, giving rise to synthetic ChS sulfated perfectly at the C4 position in all N-acetylgalactosamine units (Ch4S, 2a) in good yields. Molecular weights (Mn) of 2a ranged from 4000 to 18,400, which were controlled by varying reaction conditions. Compounds 1b and 1c were not catalyzed by the enzyme, affording the corresponding disaccharides through the oxazoline ring-opening without formation of polysaccharides.     

Chitin oligosaccharide synthesis by rhizobia and zebrafish embryos starts by glycosyl transfer to O4 of the reducing-terminal residue

Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.     

Synthesis of beta-D-arabinofuranosides: stereochemical differentiation between D- and L-enantiomers

The glycosylation of 3,5-O-di-tert-butylsilylene-protected d-thioarabinofuranosides with a range of glycosyl acceptors using NIS/AgOTf as promoters proceeded in a stereoselective manner to give the corresponding β-d-arabinofuranosides in high yields.     

Synthesis of a D-rhamnose branched tetrasaccharide, repeating unit of the O-chain from Pseudomonas syringae pv. Syringae (cerasi) 435

The first synthesis of a d-rhamnose branched tetrasaccharide, corresponding to the repeating unit of the O-chain from Pseudomonas syringae pv. cerasi 435, as methyl glycoside is reported. The approach used is based on the synthesis of an opportune building-block, that is the methyl 3-O-allyl-4-O-benzoyl-alpha-D-rhamnopyranoside, which was then converted into both a glycosyl acceptor and two different protected glycosyl trichloroacetimidate donors. Successive couplings of these three compounds afforded the target oligosaccharide. The reported synthesis is also useful to perform the oligomerization of the repeating unit.     

Acceptor-dependent regioselectivity of glycosynthase reactions by Streptomyces E383A beta-glucosidase

The nonnucleophilic mutant E383A beta-glucosidase from Streptomyces sp. has proven to be an efficient glycosynthase enzyme, catalyzing the condensation of alpha-glucosyl and alpha-galactosyl fluoride donors to a variety of acceptors. The enzyme has maximal activity at 45 degrees C, and a pH-dependence reflecting general base catalysis with an apparent kinetic pKa of 7.2. The regioselectivity of the new glycosidic linkage depends unexpectedly on the acceptor substrate. With aryl monosaccharide acceptors, beta-(1-->3) disaccharides are obtained in good to excellent yields, thus expanding the synthetic products available with current exo-glycosynthases. With xylopyranosyl acceptor, regioselectivity is poorer and results in the formation of a mixture of beta-(1-->3) and beta-(1-->4) linkages. In contrast, disaccharide acceptors produce exclusively beta-(1-->4) linkages. Therefore, the presence of a glycosyl unit in subsite +II redirects regioselectivity from beta-(1-->3) to beta-(1-->4). To improve operational performance, the E383A mutant was immobilized on a Ni2+-chelating Sepharose resin. Immobilization did not increase stability to pH and organic solvents, but the operational stability and storage stability were clearly enhanced for recycling and scaling-up.     

Synthesis of Neu5Ac- and Neu5Gc-alpha-(2-->6')-lactosamine 3-aminopropyl glycosides

In order to prepare 3-aminopropyl glycosides of Neu5Ac-alpha-(2-->6')-lactosamine trisaccharide 1, and its N-glycolyl containing analogue Neu5Gc-alpha-(2-->6')-lactosamine 2, a series of lactosamine acceptors with two, three, and four free OH groups in the galactose residue was studied in glycosylations with a conventional sialyl donor phenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (3) and a new donor phenyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-butoxycarbonylacetamido)-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (4), respectively. The lactosamine 4',6'-diol acceptor was found to be the most efficient in glycosylation with both 3 and 4, while imide-type donor 4 gave slightly higher yields with all acceptors, and isolation of the reaction products was more convenient. In the trisaccharides, obtained by glycosylation with donor 4, the 5-(N-tert-butoxycarbonylacetamido) moiety in the neuraminic acid could be efficiently transformed into the desired N-glycolyl fragment, indicating that such protected oligosaccharide derivatives are valuable precursors of sialo-oligosaccharides containing N-modified analogues of Neu5Ac.     

Synthesis of methyl glycosides of beta-(1----6)-linked D-galactobiose, galactotriose, and galactotetraose having a 3-deoxy-3-fluoro-beta-D-galactopyranoside end-residue

Methyl 2,4-di-O-acetyl-3-deoxy-3-fluoro-beta-D-galactopyranoside was synthesized by sequential tritylation, acetylation, and detritylation of methyl 3-deoxy-3-fluoro-beta-D-galactopyranoside, and used as the initial nucleophile in the synthesis of methyl beta-glycosides of (1----6)-beta-D-galacto-biose, -triose (20), and -tetraose (22) having a 3-deoxy-3-fluoro-beta-D-galactopyranoside end-residue. The extension of the oligosaccharide chains, to form the internal units in 20 and 22, was achieved by use of 2,3,4-tri-O-acetyl-6-O-bromoacetyl-alpha-D-galactopyranosyl bromide as a glycosyl donor, and mercuric cyanide or silver triflate as the promotor. While fewer by-products were formed in the reactions involving mercuric cyanide, the reactions catalyzed by silver triflate were stereospecific and yielded only the desired beta (trans) products.