Chemical synthesis of cyclic galactooligofuranosides isolated from enzymatic degradation products of cell wall arabinogalactan of Mycobacterium tuberculosis

Synthesis of cyclic tetra-, hexa- and octasaccharides containing alternating (1-->5)-beta- and (1-->6)-beta-galactofuranosyl linkages has been achieved by intramolecular cycloglycosylation of corresponding linear sugars and by cyclooligomerization of 1,6-linked and 1,5-linked disaccharides. In particular, cyclooligomerization of the (1-->6)-beta-galactofuranosyl disaccharide provides an efficient way to secure all three cyclic sugars in one operation.     

Specific and nonspecific effects of glycosylation

Glycosylation regulates vital cellular processes and dramatically influences protein folding and stability. In particular, experiments have demonstrated that asparagine (N)-linked disaccharides drive a "conformational switch" in a model peptide. The present work investigates this conformational switch via extensive atomically detailed replica exchange molecular dynamics simulations in explicit solvent. To distinguish the effects of specific and nonspecific interactions upon the peptide conformational ensemble, these simulations considered model peptides that were N-linked to a disaccharide and to a steric crowder of the same shape. The simulations are remarkably consistent with experiment and provide detailed insight into the peptide structure ensemble. They suggest that steric crowding by N-linked disaccharides excludes extended conformations, but does not significantly impact the tetrahedral structure of the surrounding solvent or otherwise alter the peptide free energy surface. However, the combination of steric crowding with specific hydrogen bonds and hydrophobic stacking interactions more dramatically impacts the peptide ensemble and stabilizes new structures.     

Regioselective glycosylation of fully unprotected methyl hexopyranosides by means of transient masking of hydroxy groups with arylboronic acids

Facile, one-pot synthesis was developed for several β(1→2)-, β(1→3)- or β(1→4)-linked disaccharides from fully unprotected methyl hexopyranosides according to the molecular recognition by arylboronic acids. The methodology was successfully applied to facile, short step assembly of the trisaccharide fragment of type II arabinogalactan.     

sp2-Iminosugar O-, S-, and N-glycosides as conformational mimics of α-linked disaccharides; implications for glycosidase inhibition

The synthesis of mimics of the α(1→6)- and α(1→4)-linked disaccharides isomaltose and maltose featuring a bicyclic sp(2)-iminosugar nonreducing moiety O-, S-, or N-linked to a glucopyranoside residue is reported. The strong generalized anomeric effect operating in sp(2)-iminosugars determines the α-stereochemical outcome of the glycosylation reactions, independent of the presence or not of participating protecting groups and of the nature of the heteroatom. It also imparts chemical stability to the resulting aminoacetal, aminothioacetal, or gem-diamine functionalities. All the three isomaltose mimics behave as potent and very selective inhibitors of isomaltase and maltase, two α-glucosidases that bind the parent disaccharides either as substrate or inhibitor. In contrast, large differences in the inhibitory properties were observed among the maltose mimics, with the O-linked derivative being a more potent inhibitor than the N-linked analogue; the S-linked pseudodisaccharide did not inhibit either of the two target enzymes. A comparative conformational analysis based on NMR and molecular modelling revealed remarkable differences in the flexibility about the glycosidic linkage as a function of the nature of the linking atom in this series. Thus, the N-pseudodisaccharide is more rigid than the O-linked derivative, which exhibits conformational properties very similar to those of the natural maltose. The analogous pseudothiomaltoside is much more flexible than the N- or O-linked derivatives, and can access a broader area of the conformational space, which probably implies a strong entropic penalty upon binding to the enzymes. Together, the present results illustrate the importance of taking conformational aspects into consideration in the design of functional oligosaccharide mimetics.     

Conformational Flexibility and Dynamics of Two (1→6)‐Linked Disaccharides Related to an Oligosaccharide Epitope Expressed on Malignant Tumour Cells

The conformational flexibility and dynamics of two (1→6)‐linked disaccharides that are related to the action of the glycosyl transferase GnT‐V have been investigated. NMR NOE and T‐ROE spectroscopy experiments, conformation‐dependent coupling constants and molecular dynamics (MD) simulations were used in the analyses. To facilitate these studies, the compounds were synthesised as α‐d‐ [6‐¹³C]‐Manp‐OMe derivatives, which reduced the ¹H NMR spectral overlap and facilitated the determination of two‐ and three‐bond ¹H,¹H, ¹H,¹³C and ¹³C,¹³C‐coupling constants. The population distribution for the glycosidic ω torsion angle in α‐d‐ Manp‐(1→6)‐α‐d‐ Manp‐OMe for gt/gg/tg was equal to 45:50:5, whereas in α‐d‐ Manp‐OMe it was determined to be 56:36:8. The dynamic model that was generated for β‐d‐ GlcpNAc‐(1→6)‐α‐d‐ Manp‐OMe by MD simulations employing the PARM22/SU01 CHARMM‐based force field was in very good agreement with experimental observations. β‐d‐ GlcpNAc‐(1→6)‐α‐d‐ Manp‐OMe is described by an equilibrium of populated states in which the ? torsion angle has the exo‐anomeric conformation, the ψ torsion angle an extended antiperiplanar conformation and the ω torsion angle a distribution of populations predominantly between the gauche–trans and the gauche–gauche conformational states (i.e., gt/gg/tg) is equal to 60:35:5, respectively. The use of site‐specific ¹³C labelling in these disaccharides leads to increased spectral dispersion, thereby making NMR spectroscopy based conformational analysis possible that otherwise might be difficult to attain.     

Conformational domino effect in saccharides: a prediction from alkyl beta-(1-->6)-diglucopyranosides

A series of alkyl beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosides, containing nonchiral and chiral aglycons, were synthesized and analyzed by NMR and CD. The results, collected from four sets of disaccharides, demonstrated that the rotational properties of the interglycosidic linkage depend on the structural natures of both the aglycon and the solvent. Stereoelectronic and steric factors explain this rotational dependence, the gauche- trans (gt) rotamer being the most stable. Furthermore, correlations between Taft's steric parameters or between the pKa values of the alkyl substituent (aglycon) versus corresponding rotamer populations were observed. These results point to a natural conformational domino effect in oligosaccharides, where the conformational properties of each (1-->6) interglycosidic linkage will depend on the structure of the previous residue or its aglycon. In addition, a very weak rotational population dependence of the hydroxymethyl group at residue II on the aglycon at residue I was observed. The population of the gauche- gauche (gg) rotamer decreased, and that of gt increased as the Taft's steric parameters of the remote aglycon increased, independently of the disaccharide series and of the solvent.     

Synthesis and conformational analysis of 6-C-methyl-substituted 2-acetamido-2-deoxy-beta-D-glucopyranosyl mono- and disaccharides

Several 6-C-substituted 2-acetamido-2-deoxy-beta-D-glucopyranosides (beta-D-GlcNAc monosaccharides 1a-3a and 1,4-linked disaccharides 1b-3b) were studied by solution NMR spectroscopy. Conformational analysis of the (6S)- and (6R)-C-methyl-substituted beta-d-GlcNAc monosaccharides indicates that the stereodefined methyl groups impose predictable conformational biases on the exocyclic C-5-C-6 bond, as determined by (1)H-(1)H and (13)C-(1)H coupling constants. Variable-temperature NMR experiments in methanol-d(4) were performed to determine DeltaDeltaH and DeltaDeltaS values derived from the two lowest energy conformers. These indicate that while the influence of 6-C-methyl substitution on conformational enthalpy is in accord with the classic principles of steric interactions, conformational preference in solution can also be strongly affected by other factors such as solvent-solute interactions and solvent reorganization.     

Differentiation of stereochemistry of glycosidic bond configuration: Tandem mass spectrometry of diastereomeric cobalt-glucosyl-glucose disaccharide complexes

Configurations of glycosidic linkages (α or β) in a series of 1,3-, 1,4-, and 1,6-glucosyl-glucose disaccharides were differentiated by tandem mass spectrometry. Diastereomeric octahedral complexes, [Co⁺³ (acac)₂/disaccharide]⁺, were generated in situ via fast-atom bombardment ionization. Mass-analyzed, ion kinetic energy spectra of the metastable complexes obtained in the absence of collision gas indicated that the major product ion results from the loss of an acetylacetonate ligand, which thus generates the ion [Co⁺²(acac)/disaccharide]⁺. Kinetic energy release measurements for this dissociation display a consistently greater value for complexes that possess an α-linked disaccharide relative to those that possess β-linked disaccharides, regardless of linkage position.     

Fluoridolysis of 5,6-epoxy carbohydrates: application to the synthesis of 5-fluoro lactosamine and isolactosamine glycosides

The synthesis of 6-selenophenyl derivatives of β-1,3 and β-1,4 disaccharides has been explored for the purpose of extending our epoxide fluoridolysis methodology to the synthesis of 5-fluoro analogues of N-acetyl isolactosamine (isoLacNAc, lacto-N-biose) and N-acetyl lactosamine (LacNAc) glycosides. Successful synthesis of the C-6 selenium-containing disaccharides was achieved via Lewis acid-mediated donor and acceptor substrates, the latter containing a selectively protected C-6 hydroxyl group for ultimate conversion to the desired 6-selenophenyl disaccharides. In contrast, the use of selenium-containing acceptor substrates under a variety of conditions failed to yield the desired selenium-containing disaccharides. Oxidation of the 6-selenophenyl derivatives to the corresponding selenoxides followed by thermal elimination yielded the exocyclic olefins, which were converted to the 5,6-epoxides. Epoxide fluoridolysis yielded the desired target compounds, 5-fluoro β-octyl glycoside analogues of type 1 and type 2 glycans. The newly synthesized fluorine-containing disaccharides have potential application as fucosyltransferase substrates, both for mechanistic studies and in the chemoenzymatic synthesis of fluorine-containing oligosaccharides.     

Efficient Synthesis of the Disialylated Tetrasaccharide Motif in N‐Glycans through an Amide‐Protection Strategy

A disialylated tetrasaccharide, Neu5Ac(α2,3)Gal(β1,3)[Neu5Ac(α2,6)]GlcNAc ( 1 ), which is found at the termini of some N‐glycans, has been synthesized. Compound 1 was obtained through an α‐sialylation reaction between a sialic acid donor and a trisaccharide that was synthesized from the glycosylation of a sialylated disaccharide with a glucosaminyl donor. This synthetic route enabled the synthesis of the as‐described disialylated structure. A more‐convergent route based on the glycosylation of two sialylated disaccharides was also established to scale up the synthesis. Protection of the amide groups in the sialic acid residues significantly increased the yield of the glycosylation reaction between the two sialylated disaccharides, thus suggesting that the presence of hydrogen bonds on the sialic acid residues diminished their reactivity.     

Anomeric configuration,glycosidic linkage,and the solution conformational entropy of O-linked disaccharides

Oligosaccharides perform a large number of biological roles, as dictated by their chemical structure and spatial arrangement. While conformational entropies are usually determined in vacuo by computer modeling, molecular recognition processes normally take place in solution. Here I show results of experiments using size-exclusion chromatography (SEC), an entropically driven solution technique. These clearly differentiate the individual contributions of the alpha and beta anomeric configurations and of the (1 --> 4) and (1 --> 6) glycosidic linkages to the solution conformational entropy of O-linked disaccharides. I also distinguish between the members of the epimeric disaccharide pair isomaltose-melibiose and trace the difference to that between their constituent monosaccharides, alpha-glucose and alpha-galactose.     

Efficient synthesis of carbopeptoid oligomers: insight into mimicry of β-peptide

The ready access to a new class of carbohydrate mimetics was demonstrated by the synthesis of tetrameric carbopeptoids, in which glycosidic bonds were replaced with amide linkages. We herein describe the detailed synthetis method of β(1→2)- and β(1→6)-linked carbopeptoids starting from each d-glucosamine and d-glucose derivative. The building blocks were polymerized using BOP reagent and DIEA to form a homooligomer. These produced carbopeptoids are resistant to glycosidases and have interesting biological activity. With conformational analysis by molecular modeling calculation, β(1→2)-linked decamer showed a typical 16-helix form as a mimic of β-peptide. Therefore, our polysaccharide analogues have potential as peptide foldamers.     

Synthesis, structure, and inclusion capabilities of trehalose-based cyclodextrin analogues (cyclotrehalans)

Concise and efficient strategies toward the synthesis of D2h- and D3h-symmetric cyclodextrin analogues alternating alpha,alpha'-trehalose disaccharide subunits and pseudoamide segments (cyclotrehalans, CTs) are reported. The conformational properties of these cyclooligosaccharides are governed by the rigidity of the alpha,alpha'-trehalose disaccharide repeating unit and the partial double-bond character of the N-(C=X) linkages. In contrast to the typical concave-shaped cavity of cyclodextrins (CDs), CTs feature a convex-shaped hydrophobic cavity in which the beta-face of the monosaccharide subunits is oriented toward the inner side, as supported by NMR and modeling (molecular mechanics and dynamics) studies. In the case of cyclodimeric CTs (CT2s), the existence of intramolecular hydrogen bonds results in collapsed cavities, too small to allow the formation of inclusion complexes with organic molecules. Cyclotrimeric CTs (CT3s) display cavity sizes that are intermediate between those of alphaCD and betaCD, ideally suited for the complexation of complementary guests with ternary symmetry such as adamantane 1-carboxylate (AC). The higher flexibility of the pseudoamide bridges as compared with classical glycosidic linkages endow these glyconanocavities with some conformational adaptability properties, making them better suited than CDs for complexation of angular guests, as seen from comparative inclusion capability experiments against the fluorescent probes 6-p-toluidinonaphthalene-2-sulfonate (TNS; linear) and 8-anilinonaphthalene-1-sulfonate (ANS; angular).     

Solution Structure of Mannobioses Unravelled by Means of Raman Optical Activity

Structural analysis of carbohydrates is a complicated endeavour, due to the complexity and diversity of the samples at hand. Herein, we apply a combined computational and experimental approach, employing molecular dynamics (MD) and density functional theory (DFT) calculations together with NMR and Raman optical activity (ROA) measurements, in the structural study of three mannobiose disaccharides, consisting of two mannoses with varying glycosidic linkages. The disaccharide structures make up the scaffold of high mannose glycans and are therefore important targets for structural analysis. Based on the MD population analysis and NMR, the major conformers of each mannobiose were identified and used as input for DFT analysis. By systematically varying the solvent models used to describe water interacting with the molecules and applying overlap integral analysis to the resulting calculational ROA spectra, we found that a full quantum mechanical/molecular mechanical approach is required for an optimal calculation of the ROA parameters. Subsequent normal mode analysis of the predicted vibrational modes was attempted in order to identify possible marker bands for glycosidic linkages. However, the normal mode vibrations of the mannobioses are completely delocalised, presumably due to conformational flexibility in these compounds, rendering the identification of isolated marker bands unfeasible.     

Ramachandran-type plots for glycosidic linkages: Examples from molecular dynamic simulations using the Glycam06 force field

The goals of this article are to (1) provide further validation of the Glycam06 force field, specifically for its use in implicit solvent molecular dynamic (MD) simulations, and (2) to present the extension of G.N. Ramachandran's idea of plotting amino acid phi and psi angles to the glycosidic phi, psi, and omega angles formed between carbohydrates. As in traditional Ramachandran plots, these carbohydrate Ramachandran-type (carb-Rama) plots reveal the coupling between the glycosidic angles by displaying the allowed and disallowed conformational space. Considering two-bond glycosidic linkages, there are 18 possible conformational regions that can be defined by (alpha, phi, psi) and (beta, phi, psi), whereas for three-bond linkages, there are 54 possible regions that can be defined by (alpha, phi, psi, omega) and (beta, phi, psi, omega). Illustrating these ideas are molecular dynamic simulations on an implicitly hydrated oligosaccharide (700 ns) and its eight constituent disaccharides (50 ns/disaccharide). For each linkage, we compare and contrast the oligosaccharide and respective disaccharide carb-Rama plots, validate the simulations and the Glycam06 force field through comparison to experimental data, and discuss the general trends observed in the plots.     

Convergent synthesis of the tetrasaccharide repeating unit related to the O-antigenic polysaccharide of Escherichia coli 78

A convergent synthesis of the tetrasaccharide repeating unit of the O-antigenic cell wall polysaccharide of Escherichia coli 78, as the corresponding methyl glycoside (I), is being reported. It involved stereoselective glycosidation of a β-linked mannodisaccharide acceptor with a β-linked glucosamine based disaccharide thioglycoside donor, which were prepared from the corresponding functionalised monosaccharide based glycosyl donors and acceptors. The resulting tetrasaccharide derivative was finally converted to (I) by selective deprotection and also by global protection and deprotection techniques.     

An improved method for synthesizing antennary β-d-mannopyranosyl disaccharide units

The merits of an indirect protecting method for hydroxyl groups using allyl groups via allyloxycarbonyl groups in the synthesis of antennary β-d-mannopyranosyl disaccharides from β-d-galactopyranosyl disaccharides were studied. Regioselective allyloxycarbonylation and conversion reactions involving simultaneous double S_N2 nucleophilic substitution at C-2′ and C-4′ of benzyl O-[β-d-galactopyranosyl]-(1-4)-3,6-di-O-benzyl-2-deoxy-2-N-phthalimido-β-d-glucopyranoside were examined for comparison with the direct allylation method. The required β-d-mannopyranosyl disaccharide having proper protecting groups was obtained using this indirect method in 52% yield. In contrast, the reported direct allylation method using methyl O-(β-d-galactopyranosyl) disaccharide gave the corresponding β-d-mannopyranosyl disaccharide in only 7.5% yield.     

Hydroxymethyl rotamer populations in disaccharides

Sixteen methyl glucopyranosyl glucopyranoside disaccharides (methyl beta-d-Glcp(p-Br-Bz)-(1-->x)-beta/alpha-d-Glcp) containing beta-glycosidic linkages (1-->2, 1-->3, 1-->4, and 1-->6) were synthesized and analyzed by means of CD and NMR spectroscopy in three different solvents. For each of these four types of disaccharides, a correlation was observed between the hydroxymethyl rotational populations around the C5-C6 bond of the glucopyranosyl residue II with the substituents and the anomeric configuration of the methoxyl group in residue I, as well as with the solvent. Nonbonded interactions, the stereoelectronic exo-anomeric effect, and hydrogen bonding were found to be responsible for the observed rotameric differences. Whereas the rotational populations of the (1-->6)-linked disaccharides are mainly dependent on the exo-anomeric effect, the (1-->2)-bonded disaccharides are strongly dependent on the anomeric configuration at C1, and the (1-->3)- and (1-->4)-linked disaccharides are mainly dependent on the substituents and the solvent. The population of the gt rotamer decreases as nonbonded interactions increase but increases as the exo-anomeric effect becomes greater, as well as in the presence of intramolecular hydrogen bonding to the endocyclic oxygen O5'. Comparison of the hydroxymethyl rotational preferences between our model disaccharides revealed a dependence on the glycosidic linkage type. Thus the population of the gg and gt rotamers decreases/increases from (1-->2)- (beta series), to (1-->6)-, to (1-->2)- (alpha series), to (1-->4)-, and to (1-->3)-bonded disaccharides respectively, while the tg rotamer population remains almost constant (around 20%), except for the (1-->3)- and (1-->4)-linked disaccharides with the intramolecular hydrogen bonding to O5', where this population decreases to 10%.     

Synthesis of Disaccharides Containing 6-Deoxy-a-L-talose as Potential Heparan Sulfate Mimetics

A 6-deoxy-a-L-talopyranoside acceptor was readily prepared from methyl a-L-rhamnopyranoside and glycosylated with thiogalactoside donors using NIS/TfOH as the promoter to give good yields of the desired a-linked disaccharide (69-90%). Glycosylation with a 2-azido-2-deoxy-D-glucosyl trichloroacetimidate donor was not completely stereoselective (a:b = 6:1), but the desired a-linked disaccharide could be isolated in good overall yield (60%) following conversion into its corresponding tribenzoate derivative. The disaccharides were designed to mimic the heparan sulfate (HS) disaccharide GlcN(2S,6S)-IdoA(2S). However, the intermediates readily derived from these disaccharides were not stable to the sulfonation/deacylation conditions required for their conversion into the target HS mimetics.