O-2 Substituted pyranosyl oxacarbenium ions are C-2-O-2 2-fold rotors with a strong syn preference

The substituent at O-2 of glycopyranosides is known to have a pronounced effect on both the formation and the cleavage of glycosides at C-1. This is primarily attributed to stereoelectronic effects on the formation and stability of the related glycopyranosyl oxacarbenium ions. Previous QM studies of 2-O-methyl substituted manno and gluco configured pyranosyl oxacarbenium ions found a preference for the methyl carbon to be syn to the CH-2 methine. This study examines the conformational preference of variously substituted O-2 tetrahydropyranosyl oxacarbenium ions and confirms this syn preference. Neutral analogues are shown to have the expected 3-fold rotation whereas the charged species exhibit 2-fold rotation about C-2-O-2. Natural bond order (NBO) calculations suggest that the dominant stabilizing interaction is a unimodal O-2 lone pair to C-1-O-5 pi-bond hyperconjugative interaction. This syn conformational preference has important implications for mimics of glycopyranosyl oxacarbenium ion transition states. It also suggests a conformational based mechanism that can be exploited to tune the reactivity of glycopyranosyl donors in the glycosylation reaction.     

The two-conformer hypothesis: 2,3,4,6-tetra-O-methyl-mannopyranosyl and -glucopyranosyl oxacarbenium ions

Computational chemistry can give information about the probable conformations of reactive intermediates that are difficult to determine experimentally. Based on density functional theory (DFT) calculations of tetra-O-methyl-D-mannopyranosyl and -glucopyranosyl oxacarbenium ions, two families of conformations, which we call B0 and B1, were found. For the manno configuration, a 4H3 and 3E almost isoenergetic pair were found, whereas for the gluco-configuration a 4H3 and 5S1 pair favouring 4H3 were calculated. These results corroborate earlier results and suggest that this two or more conformer hypothesis is general. Nucleophilic attack on these pairs of cations was modelled with methanol and led to four cases to consider namely alpha- or beta-attack on B0 or B1. The resulting complexes (G0, G1 and F0, F1) demonstrate facial selectivity. The relative energies of these complexes are dominated by intramolecular hydrogen bonding and the conformational consequences to the pyranose ring of changes in the C-5-O-5-C-1-C-2 torsion angle. Constrained variation of the nucleophilic oxygen (methanol) to C-1 distance shows that these ion dipole complexes are the only minima with this constraint.     

DFT studies of the ionization of alpha and beta glycopyranosyl donors

Current attempts at mimicking the transition states (TSs) of glycosyl processing enzymes (GPEs) that proceed through TSs with a high degree of oxacarbenium ion formation suffer from a paucity of data about the conformations of such oxacarbenium ions. Because TSs are maxima, the current models based on minimized structures may need some refinement. As part of studies directed at optimizing chemical glycosylation the ionization of 3,4,6-tri-O-acetyl-alpha/beta-D-glucopyranosyl chlorides and triflates, 2,3,4,6-tetra-O-methyl-alpha/beta-D-glucopyranosyl fluorides, chlorides and triflates, 2,3,4,6-tetra-O-methyl-alpha/beta-D-mannopyranosyl fluorides, 2,3-di-O-methyl 4,6-O-benzylidene alpha/beta-D-mannopyranosyl triflates and 2,3-di-O-methyl 4,6-O-benzylidene alpha/beta-D-glucopyranosyl triflates was studied by a prototypic density functional theory (DFT) procedure. In all cases, the alpha-anomers ionized smoothly to 4H3 half chair conformations or adjacent envelopes. By contrast, all beta-anomers exhibited an abrupt conformational change before ionization was complete. The nature of the conformations sampled depends on both the leaving group and the protecting group. The methods presented can be readily adapted to the study of any GPE or chemical glycosylation and provide a method for initial evaluation of plausible TSs, which in turn can be used in mimetic design.     

Synthesis and characterization of some anomeric pairs of per-O-acetylated aldohexopyranosyl cyanides (per-O-acetylated 2,6-anhydroheptononitriles). On the reaction of per-O-acetylaldohexopyranosyl bromides with mercuric cyanide in nitromethane

The synthesis and characterization of the anomeric pairs of the per-O-acetylaldohexopyranosyl cyanides of D-galactose, L-fucose, D-glucose, and D-mannose, as well as of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl cyanide, are described. Cyanation of the readily available, per-O-acetylaldohexopyranosyl bromides with mercuric cyanide in nitromethane, and subsequent purification, gave the corresponding, crystalline glycosyl cyanides with a high degree of 1,2-trans stereoselectivity. Thus, per-O-acetylated aldohexopyranosyl cyanides of the 1,2-trans configuration were obtained in yields ranging from 20 to 79%, whereas the corresponding 1,2-cis anomers were obtained in yields of less than or equal to 8.4%, the ratios of the 1,2-trans:1,2-cis anomers so prepared being greater than or equal to 8.5:1. The principal by-products of these irreversible, cyanation reactions were the per-O-acetylated 1,2-O-[1-(exo- and endo-cyano)ethylidene]aldohexopyranoses, obtained in yields of up to 40%. The structural assignments of the per-O-acetylaldohexopyranosyl cyanides were unequivocally established by elemental analysis, chemical transformation, vibrational spectroscopy, and 13C- and 1H-nuclear magnetic resonance spectroscopy. Correlations between the physical properties and the anomeric configurations of these C-aldohexopyranosyl compounds are described.     

Studies on the origin of stereoselectivity in the synthesis of 1,2-trans glycofuranosyl azides

The stereoselectivity of the 1,2-trans directed, Lewis acid-catalysed azidation of peracylated furanoses was found to depend on the reactivity of the azide donor (azide nucleophilicity) and the configuration at the anomeric centre relative to the neighbouring 2-O-acyl group. Reactions of 1,2-trans glycosyl esters with highly nucleophilic azide donors, generated from SnCl4 and Me3SiN3, were stereospecific. The results are interpreted in terms of the rapid reaction of the azide species with bicyclic 1,2-acyloxonium (1,2-O-alkyliumdiyl-D-glycofuranose) ions, which were the primarily formed reactive intermediates. When using 1,2-cis glycosyl esters as starting materials the selectivity was reduced (90-94% de); the same is true with 1,2-trans counterparts if less nucleophilic Me3SiN3 in combination with Me3SiOTf catalyst was used. This occurred due to the appearance of the more reactive but less selective oxocarbenium (glycofuranoxonium) ions either as primarily formed reactive intermediates in the former case or after equilibration with acyloxonium ions in the latter case. Protected 1,2-trans beta-D-glycofuranosyl azides with ribo, xylo and 3-deoxy-erythro-pento configurations were best prepared from the corresponding glycosyl esters using 0.05 equivalents of SnCl4, i.e., under anomerization-free conditions. Azidation of methyl glycofuranosides proceeds with inferior (80-90% de) and less predictable selectivity irrespective of the starting anomeric configuration.     

Recent studies on reaction pathways and applications of sugar orthoesters in synthesis of oligosaccharides

Formation of sugar-sugar orthoesters consisting of a fully acylated mono- or disaccharide donor and a partially protected mono- or disaccharide acceptor is regioselective, and rearrangement of the orthoesters via RO-(orthoester)C bond cleavage gives a dioxolenium ion intermediate leading to 1,2-trans glycosidic linkage. The activity order of hydroxyl groups in the partially protected mannose and glucose acceptors is 6-OH>3-OH>2- or 4-OH. The coupling reactions with acylated glycosyl trichloroacetimidates as the donors usually give orthoesters as the intermediates specially when the coupling is carried out at slowed rates, and this is successfully used in regio- and stereoselective syntheses of oligosaccharides. Mannose and rhamnose orthoesters readily undergo O-2-(orthoester)C bond breaking, and this is used for synthesis of alpha-(1-->2)-linked oligosaccharides. (1-->3)-Glucosylation is special since the rearrangement of its sugar orthoester intermediates can occur with either RO-(orthoester)C bond cleavage with formation of the dioxolenium ion leading to 1,2-trans linkage, or C-1-O-1 bond cleavage leading to 1,2-cis linkage, and this is dependent upon the structures of donor and acceptor that compose the orthoester.     

Synthesis of allyl 2-O-(alpha-L-arabinofuranosyl)-6-O-(alpha-D-mannopyranosyl)-beta-D-mannopyranoside, a unique plant N-glycan motif containing arabinose

The synthesis of the trisaccharide allyl 2-O-(alpha-L-arabinofuranosyl)-6-O-(alpha-D-mannopyranosyl)-beta-D-mannopyra-noside is reported. Stereoselective glycosylation at C-6 of a non-protected allyl beta-mannoside with the acetylated alpha-D-mannosyl bromide gave the alpha-(1 --> 6)-disaccharide as the main product and the crystalline 3,6-branched trisaccharide as minor compound. Further glycosylation of the 2,3 diol (1 --> 6) disaccharide with L-arabinofuranosyl bromide furnished a mixture of 3-O- and 2-O-alpha-L-Ara-trisaccharides from which the title compound was isolated.     

The impact of oxacarbenium ion conformers on the stereochemical outcome of glycosylations

The search for stereoselective glycosylation reactions has occupied synthetic carbohydrate chemists for decades. Traditionally, most attention has been focused on controlling the S_N2-like substitution of anomeric leaving groups as highlighted by Lemieux’s in situ anomerization protocol and by the discovery of anomeric triflates as reactive intermediates in the stereoselective formation of β-mannosides. Recently, it has become clear that also S_N1-like reaction pathways can lead to highly selective glycosylation reactions. This review describes some recent examples of stereoselective glycosylations in which oxacarbenium ions are believed to be at the basis of the selectivity. Special attention is paid to the stereodirecting effect of substituents on a pyranosyl ring with an emphasis on the role of the C-5 carboxylate ester in the condensations of mannuronate ester donors.     

Investigations into the role of oxacarbenium ions in glycosylation reactions by ab initio molecular dynamics

We present a constrained ab initio molecular dynamics method that allows the modeling of the conformational interconversions of glycopyranosyl oxacarbenium ions. The model was successfully tested by estimating the barriers to ring inversion for two 4-substituted tetrahydropyranosyl oxacarbenium ions. The model was further extended to predict the pathways that connect the (4)H(3) half-chair conformation of 2,3,4,6-tetra-O-methyl-d-glucopyranosyl cation to its inverted (5)S(1) conformation and the (4)H(3) half-chair conformation of 2,3,4,6-tetra-O-methyl-d-mannopyranosyl cation to its inverted (3)E conformation. The modeled interconversion pathways reconcile a large body of experimental work on the acid-catalyzed hydrolysis of glycosides and the mechanisms of a number of glucosidases and mannosidases.     

Synthesis of capsular polysaccharide of Streptococcus pneumoniae type 14. 3. Synthesis of a monomer for polycondensation

Synthesis of a tritylated tetrasaccharide 1,2-O-(1-cyano) ethylidene derivative is described by glycosylation of 3,6-di-O-benzoyl-4-O-(2,4,6-tri-O-benzoyl-beta- D-galactopyranosyl)-1,2-O-[1-(exo-cyano)ethylidene]-alpha-D- glucopyranose with 6-O-acetyl-3-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzoyl-beta- D-galactopyranosyl)-2-deozy-2-phthalimido-D-glucopyranosyl. bromide followed by selective deacetylation and tritylation.     

A dileucine signal situated in the C-terminal tail of the lysosomal membrane protein p40 is responsible for its targeting to lysosomes

The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone against time. The electronic, steric and hydrophobic effects of the substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain the suicide inactivation, we propose a mechanism in which the enzymatic form E(ox) (oxy-tyrosinase) is responsible for such inactivation. A key step might be the transfer of the C-1 hydroxyl group proton to the peroxide, which would act as a general base. Another essential step might be the axial attack of the o-diphenol on the copper atom. The rate constant of this reaction would be directly related to the strength of the nucleophilic attack of the C-1 hydroxyl group, which depends on the chemical shift of the carbon C-1 (delta(1)) obtained by (13)C-NMR. Protonation of the peroxide would bring the copper atoms together and encourage the diaxial nucleophilic attack of the C-2 hydroxyl group, facilitating the co-planarity with the ring of the copper atoms and the concerted oxidation/reduction reaction, and giving rise to an o-quinone. The suicide inactivation would occur if the C-2 hydroxyl group transferred the proton to the protonated peroxide, which would again act as a general base. In this case, the co-planarity between the copper atom, the oxygen of the C-1 and the ring would only permit the oxidation/reduction reaction on one copper atom, giving rise to copper(0), hydrogen peroxide and an o-quinone, which would be released, thus inactivating the enzyme.     

Studies on the stereoselective synthesis of a protected α-D-Gal-(1→2)-D-Glc fragment

A novel 1,2-cis stereoselective synthesis of protected α-D-Gal-(1→2)-D-Glc fragments was developed. Methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (13), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-glucopyranoside (15), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (17), and methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-D-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-β-D-glucopyranoside (19) were favorably obtained by coupling a new donor, isopropyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (2), with acceptors, methyl 3-O-benzoyl-4,6-O-benzylidene-α-D-glucopyranoside (4), methyl 3,4,6-tri-O-benzoyl-α-D-glucopyranoside (5), methyl 3-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside (8), and methyl 3,4,6-tri-O-benzoyl-β-D-glucopyranoside (12), respectively. By virtue of the concerted 1,2-cis α-directing action induced by the 3-O-allyl and 4,6-O-benzylidene groups in donor 2 with a C-2 acetyl group capable of neighboring-group participation, the couplings were achieved with a high degree of α selectivity. In particular, higher α/β stereoselective galactosylation (5.0:1.0) was noted in the case of the coupling of donor 2 with acceptor 12 having a β-CH(3) at C-1 and benzoyl groups at C-4 and C-6.     

Synthesis of 5-aza-7-deazapurine and 3,7-dideazapurine 2'-deoxyribofuranosides by solid-liquid phase-transfer glycosylation

The 2'-deoxyguanosine isostere 1 as well as the pyrrolo [3,2-c] pyridine 2'-deoxyribofuranosides 2a,b and 3a-d have been synthesized in high yield by solid-liquid phase-transfer glycosylation. It was shown that only the strongly nucleophilic pyrrolo [3,2-c] pyridines formed exclusively beta-2'-deoxyribofuranosides, whereas weakly nucleophilic imidazo [1,2-a]-s-triazines gave mixtures of anomers.     

Antigenic bacterial polysaccharides. 30. The structure of the polysaccharide chain of lipopolysaccharide of Pseudomonas syringae pv. syringae 281 (serogroup I)

Anomeric methyl 3-O-(D-mannopyranosyl- and L-rhamnopyranosyl)-beta-D-talopyranosides were synthesised by the stereoselective 1,2-cis- and 1,2-trans manno- and rhamnosylation of methyl 2,4,6-tri-O-acetyl-beta-D-talopyranoside, which has been prepared from methyl beta-D-galactopyranoside by a synthetic scheme including conversion of the C2 configuration. From 13C-NMR spectra of the disaccharides obtained the spectral alpha- and beta-effects of O3-glycosylation of talopyranose were determined.     

Synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 14. 2. Synthesis of methyl-6-O-acetyl-4-O-(2,3,46-tetra-O-benzoyl -beta-D-galactosylpyranosyl)-2-deoxy-2-phthalimido-beta-D-gluco- pyranoside--a lactosamine precursor in the monomer synthesis for polycondensation

Glycosylation of methyl 6-O-acetyl-3-O-benzoyl-2-deoxy-phthalimido-beta-D-glucopyranoside and its 4-trityl ether by benzobromogalactose, 1-O-acetyl-2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranose, 1,2-[(alpha-p-tolylthio)benzylidene]- and 1,2-O-[(alpha-cyano)benzylidene]-3,4,6-tri-O-benzoyl-alpha-D- galactopyranoses proceeds non-stereospecifically. The best yield of beta-linked disaccharide was obtained upon glycosylation by benzobromogalactose in the presence of silver triflate and tetramethylurea in nitromethane.     

A facile formation of 2,5-anhydro sugars by ring contraction in methyl hexopyranoside 2-triflates under conditions of nucleophilic displacement

Reaction of methyl 3,4-O-isopropylidene-2-O-(trifluoromethylsulfonyl)-alpha-L-fucopyr anoside and its beta anomer with a variety of nucleophilic reagents under mild conditions led to displacement of the triflic ester group, with migration of the ring-oxygen atom to C-2 and entry of the nucleophile at C-1, to give good yields of 2,5-anhydro sugar derivatives. Reagents employed were hydrogen fluoride-triethylamine complex, methanol in the presence of sodium hydrogencarbonate, sodium benzoate, sodium azide, potassium thiocyanate, and sodium borohydride. The same type of substitutive ring-contraction also occurred in methyl 4-O-benzyl-3-O-(4-methoxybenzyl)-2-O-(trifluoromethylsulfonyl)-alp ha-L- fucopyranoside and methyl 4,6-dideoxy-3-O-(4-methoxybenzyl)-2-O-(trifluoromethylsulfonyl)-al pha-L-xylo- hexopyranoside. The reaction is discussed in light of a literature survey of the chemical behavior of hexopyranoside 2-sulfonates in general, and 2-triflates in particular. Direct SN2 displacements, eliminations, and such different kinds of rearrangement as have previously been observed with structural analogs were not encountered in the present study. However, there is some precedent, in hexopyranoside 2-triflates, for the facile rearrangement that the four representatives here investigated have been found to undergo. The synthesis of these triflates from L-fucose is described.     

A new and efficient entry to D-xylo-hexos-4-ulose and some derivatives thereof through epoxidation of the 3,4-hexeno derivative of diacetone-D-glucose

A new preparation of D-xylo-hexos-4-ulose (1) and of its 3-m-chlorobenzoate (2) has been devised using the epoxidation of 3-deoxy-1,2:5,6-di-O-isopropylidene-D-erythro-hex-3-enofuranose (6) as the key step. The epoxidation of 6 in CH2Cl2 furnished with high yield 1,2:5,6-di-O-isopropylidene-3-O-m-chlorobenzoyl-4-C-hydroxy-D-xylo-hexos-4-ulo-1,4-furanose as a mixture of C-4 hemiacetal anomers (7a,b), which, on acid hydrolysis, gave a tautomeric mixture of 3-O-m-chlorobenzoyl-D-xylo-hexos-4-ulose (2) with an overall 60% yield from 6. The formation of 4-C-methoxy-diacetone-D-glucose derivatives (11a,b) through epoxidation-methanolysis of 6, took place with reduced yield because of the competition between m-chlorobenzoic acid (MCBA) and methanol to the opening by attack at C-4 of the intermediate epoxide and the formation of acyclic products arising from the alternative nucleophilic attack at C-1. Acid hydrolysis of derivatives 11 gave D-xylo-hexos-4-ulose (1) with a 35% overall yield from 6. NMR analysis showed that 2 is composed, in CD3CN, mainly by a 7:3 mixture of 4-keto-alpha- and beta-pyranose forms, while 1, in D2O, is present as a more complex mixture constituted mainly by 4-keto-alpha- and beta-pyranoses and their respective hydrates in a 17:15:34:34 ratio.     

Synthesis of 2'-deoxy-6,3'-methano-cyclo-pyrimidine nucleosides

The nucleophilic attack of lithiated pyrimidines to a suitably-protected 3-ketosugar, afforded the 3-branched sugar stereospecifically, followed by intramolecular glycosylation to give 6,3'-methano-cyclo-pyrimidine nucleosides. The 2'-deoxygenation was achieved by sequential protection, deprotection and radical reduction.     

Syntheses of an alpha-D-Gal-(1-->6)-beta-D-Gal diglyceride, as lipase substrate

Two different routes were explored to afford 3-O-(6-O-alpha-D-galactopyranosyl-beta-D-galactopyranosyl)-1,2-di-O-dodecanoyl-sn-glycerol. In the first one, the key step was the glycosylation of the 3-O-(2,3,4-tri-O-benzyl-beta-D-galactopyranosyl)-1,2-O-isopropylidene-sn-glycerol acceptor with 2-pyridyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside as the donor. In the second one, the key step was the coupling of 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-D-galactopyranosyl trichloroacetimidate donor with 1,2-O-isopropylidene-sn-glycerol. Even though the number of steps was the same in both pathways, the first one afforded a better overall yield (12.4%) than the second one (6.5%). This eight-step synthesis allowed the preparation of the expected glycolipid, which was used as substrate for recombinant GPLRP2 galactolipase using the monomolecular film technique.     

The C-4 hydroxyl group of galactopyranosides is the major determinant for ligand recognition by the lactose permease of Escherichia coli.

Binding specificity in lactose permease toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by nonspecific hydrophobic interactions with the non-galactosyl moiety [Sahin-Tóth, M., Akhoon, K. M., Runner, J., and Kaback, H. R. (2000) Biochemistry 39, 5097-5103]. To characterize the contribution of individual hydroxyls, binding of structural analogues of p-nitrophenyl alpha-D-galactopyranoside (NPG) was examined by site-directed N-[(14)C]ethylmaleimide (NEM) labeling of the substrate-protectable Cys148 in the binding site. NPG blocks NEM alkylation of Cys148 with an apparent affinity of approximately 14 microM. A deoxy derivative at position C-2 binds with 25-fold lower affinity (K(D) 0.35 mM), and the deoxy analogue at C-3 exhibits ca. 70-fold decreased binding (K(D) 1 mM), while binding of 6-deoxy-NPG is at least 130-fold diminished (K(D) 1.9 mM). Remarkably, the C-4 deoxy derivative of NPG binds with almost 1500-fold reduced affinity (K(D) approximately 20 mM). No significant substrate protection is afforded by NPG analogues with methoxy (CH(3)-O-) substitutions at positions C-3, C-4, and C-6. In contrast, the C-2 methoxy analogue binds almost normally (K(D) 26 microM). The results confirm and extend the observations that the C-2, C-3, C-4, and C-6 OH groups of galactopyranosides participate in important H-bonding interactions. Moreover, the C-4 hydroxyl is identified as the major determinant of ligand binding, suggesting that sugar recognition in lactose permease may have evolved to discriminate primarily between gluco- and galactopyranosides.