Abstract: BACKGROUND: A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate alpha-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. CONCLUSIONS/SIGNIFICANCE: These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.
Abstract: Analysis of anomericity is one of the most important issues in the structure elucidation of carbohydrates. Mass spectrometry (MS)-based methods are of particular interest and important to address the issue related to resolving anomericity of monosaccharide units in a glycan. However, direct analysis of hemiacetals has not been possible by MS because of the nonavailability of information regarding the gas-phase behavior of such ion species. We addressed this issue by using stage-discriminated energy-resolved mass spectrometry (ERMS) at the stages of MS(n) and MS(n+1) and showed that such analysis can be made. This was achieved by proving that individual anomers can be identified and that the equilibrium of sodium adducted ion species of alpha- and beta-anomers can be negated in the gas phase under collision-induced dissociation (CID) conditions. On the basis of these results, we could 1) observe the mutarotation of lactose and 2) speculate the hydrolysis mechanism of endo-glycosylceramidase by using mass spectrometry.
Abstract: Described here are the synthesis of five-membered iminocyclitols with galacto-configuration and a pseudodisaccharide, and their inhibitory activities against b-galactosyltransferase, b-galactosidase and a-mannosidase.
Abstract: This paper describes the synthesis of 1,1-linked galactosyl mannosides as sialyl Lewis X mimetics that contain a spiro-ring to position the carboxylate group in a well-defined orientation. It was found that compound 4 is more active as a P-selectin inhibitor (IC50 = 19 microM) than the parent disaccharide 2, which contains a flexible carboxyl group (IC50 = 193 microM). This result is consistent with that observed in the previous NMR study of sialyl Lewis X bound to P-selectin. The chemistry described here should be useful for the development of selective inhibitors of E-, P-, and L-selectins.
Abstract: In an effort to develop new structures as inhibitors of both influenza virus hemagglutinin and neuraminidase a series of sialic acid derivatives, including those with one of the hydrogen atoms at C-3 position replaced by either OH or F, were synthesized. The sialic acid derivative having a 3-eq-OH group was first synthesized via new process and used as the key intermediste for further derivetization at the C-3 position. The stability of these compounds under acid- and sialidase-catalyzed hydrolysis conditions was studied, and the results showed that these compounds exhibit strong resistance toward both conditions when compared with their parent p-nitrophenyl a-sialoside. Further inhibition assay indicated that the 3-ax-OH or F derivatives 4 and 5 and 24, the 4-epimer of 4, are effective specific inhibitors of the sialidase from Clostridium perfringens among other bacterial sialidases tested. The 3-eq-OH derivative 3, however, showed little inhibition. The same tendency was observed for inhibition of human influenza sialidase N1 and N2. Compounds 3-5 and sialic acid were then converted to the distealoylphosphatidylethanolamine conjugates and of these liposome-like compounds, the ones from 4 and 5 showed potent and selective inhibitory activities against the hemagglutinin H3 subtype but resistant to influenza virus neuraminidases N1 and N2.
Abstract: We have developed a new hydrogenolysis method for removal of benzyl protecting groups from solid-bound carbohydrates using palladium nanoparticles as catalysts. Glucose, fucose and lactose were chosen as examples and introduced to various resins through an U.V. active linker, which enabled us to monitor the course of the reaction after cleavage from the solid. Hydrogenolysis reactions were carried out using TentaGel and PEGA resin as the solid support and the result showed that benzyl protecting groups can be used in solid-phase synthesis.
Abstract: A series of iminocyclitols was prepared using a versatile synthetic strategy, and their inhibition of glycosidases was evaluated using capillary electrophoresis. The study has demonstrated that remarkable specificities in enzyme inhibition can be achieved with small modifications on the aglycon side chain and the ring nitrogen. Among the compounds synthesized, (2R,3R,4R,5R)-N-methyl-2-(acetamidomethyl)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidine was found to be very potent; against beta-N-acetylhexosaminidase P with the K-i value of 80 nM.
Abstract: Methods have been developed for the incorporation of hydrophobic groups with and without positive or negative charge to position-6 of the mannose residue in the 1,1-linked disaccharide as mimetics of sialyl Lewis X tetrasaccharide in order to enhance binding affinity to selectins. (C) 1998 Elsevier Science Ltd. All rights reserved
Abstract: Due to high demand to access biologically important oligosaccharide structures, efficient and stereoselective synthetic methods are required. This article addresses these two issues from our recent studies. The first part deals with P-mannosylation and cr-sialylation known as the two most difficult glycosylation reactions which were now made possible by means of an "intramolecular aglycon delivery" system and control by "auxiliary", respectively. In the second part, the remarkably efficient "orthogonal glycosylation strategy" which was developed based on tactical analysis of oligosaccharide synthesis is described.
Abstract: The kinetic parameters of a galactosyltransferase-catalyzed reaction were determined for the first time using capillary zone electrophoresis (CZE) using the methylumbelliferyl (MU) glycoside of N-acetylglucosamine as the acceptor molecule. The CZE was performed using borate buffer and the enzymatic transformations were monitored at 214 nm. The kinetic parameters obtained for MU-GlcNAc were Km = 35.9 microM and Vmax = 7.5 micromol/min/mg, and those for UDP-Gal were Km = 115.3 microM and Vmax = 12.4 micromol/min/mg. A representative inhibition assay was also carried out using UDP as an inhibitor to give the Ki value of 83.9 microM against MU-GlcNAc. The structure of the synthetic product was also confirmed using 1H NMR spectroscopies after isolation by simple chromatography.
Abstract: The tumor related enzyme N-acetylglucosaminyltransferase V catalyzes the transfer of GlcNAc to OH-6 of branching Man to give complex oligosaccharide structures, A transition state analog inhibitor of this enzyme was designed and a model compound was synthesized to illustrate the feasibility of the synthetic strategy.
Abstract: The orthogonal glycosylation strategy was applied for the synthesis of extended blood type B determinant (2) of a novel glycolipid 1. Key features in the synthesis are 1) four monosaccharide units were synthesized as either glycosyl fluoride or thioglycoside to be engaged to the orthogonal glycosylation strategy and 2) all necessary manipulations were completed at the monosaccharide level, therefore, manipulations during the elongation of sugar chain were minimized.
Abstract: A single reaction of an unprotected beta-D-GlcNAc glycoside with tetra-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate in dioxane, catalyzed by BF3-etherate, was shown to yield all six possible Gal-GlcNAc disaccharides. This result is surprising not only because significant amounts of alpha-linked disaccharides were formed, despite the presence of a participating group at O-2 of the glycosyl donor, but also because glycosylation of the primary OH-6 is not the dominant reaction. These results suggest 'random-glycosylation' to be a valid strategy for the rapid production of oligosaccharide libraries.
Abstract: Solid phase synthesis of polylactosamine oligosaccharide was performed starting from resin supported lactose 1a,b. Glycosylation of 1a with the lactosamine unit 6 followed by delevulinoylation afforded tetrasaccharides, which were further converted into hexa- and octasaccharide and was cleaved from resin by TrBF(4) in CH2Cl2 to afford 7. Ester linked Ib was converted in a similar manner into hexasaccharide that was liberated under basic conditions to give 8. Subsequent deprotection into 9 was performed in three steps.
Abstract: Random glycosylation has proven remarkably effective for the generation of mixtures of oligosaccharides. Clearly, not all of the possible glycosidically-linked isomers are formed in equal quantity in these reactions. In the instances where product structures have been thoroughly investigated, however, all have been shown to be present. So far, only one random glycosylation step has been performed and the challenge will be to see if two tandem steps can generate a useful oligosaccharide library. Whether or not the present formulation of random glycosylation succeeds as a dominant strategy for the synthesis of oligosaccharide libraries, this important challenge is open to many approaches where creativity in both formulating the problem, as well as experimentally addressing it, warrants a major international effort.
Abstract: N-Acetylglucosaminyltransferase-V (GlcNAc T-V) transfers a beta-linked GlcNAc residue from UDP-GlcNAc to OH-6' (of the alpha Man residue) in oligosaccharides terminating in the sequence beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp(or Manp)-OR (3, R = (CH2)7CH3). It was previously found that OH-4" (of the GlcNAc residue) in 3 was a critical element for substrate recognition by this enzyme. We show here that OH-3" and OH-6" are also key recognition elements. Four analogs of trisaccharide 3 where OH-3" and OH-6" were replaced, independently, by NH2 and NHAc groups, were prepared by multi-step chemical synthesis and kinetically evaluated as substrates for GlcNAc T-V from hamster kidney. These substitutions were selected since they replaced the OH groups with groups probing both hydrogen bonding and steric bulk. The 3"-modified compounds were found to be very poor substrates with Km values more than 50-fold elevated over that for 3 (26 microM) while the 6"-modified compounds were completely inactive. An intact 3,4,6 triol system in the terminal GlcNAc residue therefore appears to be the key polar group system that is recognized by this enzyme.
Abstract: Development of inhibitors specific for the glycosyltransferases involved in the biosynthesis of asparagine-linked sugar chains has been undertaken in the hopes that these compounds may serve as tools to elucidate the roles of complex carbohydrates in biological recognition events. We report here the first example of a glycosyltransferase acceptor analog in which strategic replacement of a nonreacting hydroxyl group with a larger substituent produces a molecule which is recognized by the enzyme but does not react because of a steric block to the glycosyl transfer reaction. N-Acetylglucosaminyltransferase V catalyzes the transfer of GlcNAc from the sugar nucleotide donor UDP-GlcNAc to the 6-OH group of mannose in the synthetic trisaccharide acceptor beta GlcNAc(1-->2)alpha Man(1-->6)beta Glc-O(CH2)7CH3 (Km = 23 +/- 2 microM; Vmax = 116 +/- 3 pmol/h) to form the tetrasaccharide beta GlcNAc(1-->2)(beta GlcNAc(1-->6))alpha Man(1-->6)beta Glc-O(CH2)7CH3. The acceptor analog produced by replacement of the adjacent nonreacting 4-OH group of the mannose residue with an O-methyl group was not a substrate for the enzyme but was found to be a good competitive inhibitor of GlcNAc transferase V with Ki = 14 +/- 2 microM. To test the theory that it was the presence of the large methyl group which prevented the glycosyl transfer reaction the 4'-deoxygenated analog was synthesized. It was found to be a good substrate with Km = 74 +/- 6 microM and an almost 5-fold higher kcat (Vmax = 535 +/- 13 pmol/h). NMR data show no evidence of important conformational differences between the trisaccharide analogs, and kinetic experiments detected no differences for the binding of UDP-GlcNAc in their presence. The conclusion was therefore reached that the large methyl group introduced on O-4' sterically prevented the formation of product even though both potential substrates were bound by the enzyme. This "steric exclusion" strategy offers potential for the design of inhibitors for that class of glycosyltransferases in which the reactive hydroxyl group is also an essential recognition element.
Abstract: The enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) transfers GlcNAc from UDP-GlcNAc to the OH-6' group of oligosaccharides terminating in the sequence beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp (or Manp)-OR (5, R = (CH2)7CH3) to yield the sequence beta-D-GlcpNAc-(1-->2)-[beta-D-GlcpNAc-(1-->6)]-alpha-D-Manp-(1--> 6)- beta-D-Glcp (or Manp)-OR. Biosynthetically, if beta-(1-->4)-galactosyltransferase acts first on 5, the product beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-be ta-D-Glcp (or Manp)-OR (7) is no longer a substrate for GlcNAcT-V even though it retains the active OH-6' group. The reason for this loss in activity is examined in this paper. Six analogues of the acceptor trisaccharide 5, all with the reducing-end D-gluco configuration, were chemically synthesized. A key feature of the synthetic scheme is the use of 1,2-diaminoethane for the efficient removal of N-phthalimdo protecting groups. In these analogues OH-4 of the terminal sugar unit, the site of galactosylation by GalT in the normal GlcNAc-terminating trisaccharide 5, was systematically replaced by OMe, F, NH2, NHAc, and H, as well as inverted to the galacto configuration. The interactions of the resulting trisaccharide analogues with GlcNAcT-V from hamster kidney were then evaluated kinetically. All six compounds were found to be essentially inactive either as acceptors or as inhibitors of GlcNAcT-V. The conclusion is reached that galactosylation of natural acceptors for GlcNAcT-V destroys acceptor activity, not by introduction of the steric bulk of an added sugar residue, but by destroying an important hydrogen-bonding interaction of terminal OH-4 of the GlcNAc residues with the enzyme. This OH-4 group is therefore designated as a key polar group for GlcNAcT-V.
Abstract: Random glycosylation has proven remarkably effective for the generation of mixtures of oligosaccharides. Clearly, not all of the possible glycosidically-linked isomers are formed in equal quantity in these reactions. In the instances where product structures have been thoroughly investigated, however, all have been shown to be present. So far, only one random glycosylation step has been performed and the challenge will be to see if two tandem steps can generate a useful oligosaccharide library. Whether or not the present formulation of random glycosylation succeeds as a dominant strategy for the synthesis of oligosaccharide libraries, this important challenge is open to many approaches where creativity in both formulating the problem, as well as experimentally addressing it, warrants a major international effort.
