Stereoselective glycosylation reactions with chiral auxiliaries.
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Carbohydrates↗
Biomedical subjects
Geert-Jan Boons
Publications and source records attributed to Geert-Jan Boons.
Potent and selective inhibition of class II alpha-D-mannosidase activity by a bicyclic sulfonium salt.
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Cyclization↗
Synthesis and proinflammatory properties of muramyl tripeptides containing lysine and diaminopimelic acid moieties.
The unusual amino acid diaminopimelic acid (DAP) was prepared by cross metathesis of appropriately protected vinyl glycine and allyl glycine derivatives. Catalytic hydrogenation of the cross-coupling product resulted in reduction of the double bond and the removal of protecting groups. The resulting compounds were appropriately protected for the polymer-supported and solution-phase synthesis of muramyl tripeptides 2 and 3, which differ in the amidation of the alpha-carboxylic acids of the isoglutamine and DAP moieties. Muramyl dipeptide (1, MDP), the DAP-containing muramyl tripeptide 3, and the lysine-containing muramyl tripeptides 4 and 5 induced TNF-alpha gene expression without TNF-alpha protein production in a human monocytic cell line. The observed block in translation could be removed by co-incubation with LPS, resulting in an apparent synergistic effect. Compound 2 did not induce TNF-alpha gene expression, neither did it exhibit a synergistic effect with LPS; this indicates that amidation of the alpha-carboxylic acids of the isoglutamine and DAP moieties results in a loss of biological activity. It is proposed that amidation of alpha-carboxylic acids is a strategy that may be used by pathogens to avoid detection by the innate immune system. Furthermore, the pattern recognition receptors Nod1 and Nod2 have been implicated in the possible induction of a synergistic effect of muropeptides with LPS.
Acetylmuramyl-Alanyl-Isoglutamine↗
Crystal structure of a peptidoglycan recognition protein (PGRP) in complex with a muramyl tripeptide from Gram-positive bacteria.
Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind, and in some cases hydrolyse, bacterial peptidoglycans (PGNs). We determined the crystal structure of the C-terminal PGN-binding domain of human PGRP-Ialpha in complex with a muramyl tripeptide representing the conserved core of lysine-type PGNs. The peptide stem of the ligand is buried at the deep end of a long binding groove, with N-acetylmuramic acid situated in the middle of the groove, whose shallow end could accommodate N-acetylglucosamine. Both peptide and glycan moieties are essential for binding by PGRPs. Conservation of key PGN-contacting residues indicates that all PGRPs employ this basic PGN-binding mode. The structure identifies variable residues that likely mediate discrimination between lysine- and diaminopimelic acid-type PGNs. In addition, we propose a mechanism for PGN hydrolysis by Zn2+-containing catalytic PGRPs.
Acetylmuramyl-Alanyl-Isoglutamine↗
Structural basis for peptidoglycan binding by peptidoglycan recognition proteins.
Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern-recognition receptors of the innate immune system that bind and, in some cases, hydrolyze bacterial PGNs. We determined the crystal structure, at 2.30-A resolution, of the C-terminal PGN-binding domain of human PGRP-Ialpha in complex with a muramyl tripeptide representing the core of lysine-type PGNs from Gram-positive bacteria. The peptide stem of the ligand is buried at the deep end of a long binding groove, with N-acetylmuramic acid situated in the middle of the groove, whose shallow end can accommodate a linked N-acetylglucosamine. Although most interactions are with the peptide, the glycan moiety also seems to be essential for specific recognition by PGRPs. Conservation of key PGN-contacting residues shows that all PGRPs employ this basic PGN-binding mode. The structure pinpoints variable residues that likely mediate discrimination between lysine- and diaminopimelic acid-type PGNs. We also propose a mechanism for PGN hydrolysis by Zn(2+)-containing PGRPs.
Acetylmuramyl-Alanyl-Isoglutamine↗
Synthesis and biological evaluation of a lipid A derivative that contains an aminogluconate moiety.
A highly convergent strategy for the synthesis of several derivatives of the lipid A of Rhizobium sin-1 has been developed. The synthetic derivatives are 2-aminogluconate 3 and 2-aminogluconolactone 4, both of which lack C-3 acylation. These derivatives were obtained by the preparation of disaccharides in which the two amino groups and the C-3' hydroxy group could be modified individually with acyl or beta-hydroxy fatty acyl groups. Detailed NMR spectroscopy and MS analysis of 3 and 4 revealed that, even under neutral conditions, the two compounds equilibrate. The synthetic compounds lack the proinflammatory effects of Escherichia coli lipopolysaccharide (LPS), as indicated by an absence of tumor necrosis factor production. Although 3 and 4 were able to antagonize E. coli LPS, they were significantly less potent than the synthetic compound 2, which is acylated at C-3, and R. sin-1 LPS; these results indicate that the beta-hydroxy fatty acyl group at C-3 contributes to the antagonistic properties of R. sin-1 LPS. Based on a comparison of the biological responses of the synthetic lipid A derivatives with those of the R. sin-1 LPS and lipid A, the 3-deoxy-D-manno-octulosonic moieties appear to be important for the optimal antagonization of enteric LPS-induced cytokine production.
Gluconates↗
Preparation of a lipid a derivative that contains a 27-hydroxyoctacosanoic acid moiety.
[structure: see text] A general synthetic strategy for long-chain omega-1 hydroxy fatty acids has been developed, which employs as a key reaction step a cross metathesis between omega-unsaturated ester and 3-butene-2-ol. The resulting lipids were used for the preparation of lipid A derivatives of Rhizobium sin-1, which have the ability to inhibit the E. coli LPS-dependent synthesis of tumor necrosis factor by human monocytes.
Escherichia coli↗
Inhibition of Golgi mannosidase II with mannostatin A analogues: synthesis, biological evaluation, and structure-activity relationship studies.
Mannostatin and aminocyclopentitetrol analogues with various substitutions at the amino function were synthesized. These compounds were tested as inhibitors of human Golgi and lysosomal alpha-mannosidases. Modification of the amine of mannostatin had only marginal effects, whereas similar modifications of aminocyclopentitetrol led to significantly improved inhibitors. Ab initio calculations and molecular docking studies were employed to rationalize the results. It was found that mannostatin and aminocyclopentitretrol could bind to Golgi alpha-mannosidase II in a similar mode to that of the known inhibitor swainsonine. However, due to the flexibility of the five-membered rings of these compounds, additional low-energy binding modes could be adopted. These binding modes may be relevant for the improved activities of the benzyl-substituted compounds. The thiomethyl moiety of mannostatin was predicted to make favorable hydrophobic interactions with Arg228 and Tyr727 that would possibly account for its greater inhibitory activity.
Amines↗
Disaccharide mimetics of the aminoglycoside antibiotic neamine.
A highly convergent approach has been employed for the facile synthesis of a library of 24 disaccharides that are alpha(1-3), beta(1-3), alpha(1-4), or beta(1-4) linked and contain 2-4 amino groups. Fourier-transformation ion cyclotron resonance mass spectrometry (FT-ICR MS) has been used to determine dissociation constant (Kd) values for the binding of the disaccharides to a prototypical fragment of 16S ribosomal RNA. Several derivatives bound with affinities similar to that of neamine. Structure-activity relationships have revealed the substitution pattern that is important for high-affinity binding. The compounds described here are unique lead compounds for the design of novel aminoglycoside antibiotics.
Aminoglycosides↗
The immunogenicity of the tumor-associated antigen Lewis(y) may be suppressed by a bifunctional cross-linker required for coupling to a carrier protein.
A Lewis(y) (Le(y)) tetrasaccharide modified by an artificial aminopropyl spacer was synthesized by a highly convergent approach that employed a levulinoyl ester and a 9-fluorenylmethoxycarbonate for temporary protection of the hydroxy groups and a trichloroethyloxycarbonyl as an amino protecting group. The artificial aminopropyl moiety was modified by a thioacetyl group, which allowed efficient conjugation to keyhole limpet hemocyanin (KLH) modified by electrophilic 4-(maleimidomethyl)cyclohexane-1-carboxylate (MI). Mice were immunized with the KLH-MI-Le(y) antigen. A detailed analysis of sera by ELISA established that a strong immunoglobulin G (IgG) antibody response was elicited against the linker region. The use of a smaller and more flexible 3-(bromoacetamido)propionate for the attachment of Le(y) to KLH not only reduced the IgG antibody response against the linker but also led to a significantly improved immune response against the Le(y) antigen. This study shows that highly antigenic linkers suppress antibody responses to weak antigens such as self-antigens.
Antibodies↗
Glycosyltransferase activity can be selectively modulated by chemical modifications of acceptor substrates.
A range of N-acetyllactosamine derivatives, which are modified by a wide range of functionalities at C-2(') and C-6, have been synthesised and the kinetic parameters of transfer catalysed by recombinant alpha-2,6-sialyltransferase and alpha-1,3-fucoyltransferase VI determined. Several of the chemical modifications led to selective modulate the activity the enzymes and offer promising lead compounds for the development of oligosaccharide primers for selective metabolic inhibition of oligosaccharide biosynthesis.
Amino Sugars↗
The design and synthesis of a selective inhibitor of fucosyltransferase VI.
Inversion of configuration of the C-2[prime or minute] hydroxyl of methyl N-acetyllactosamine was accomplished by a two-step procedure involving oxidation to a ketone followed by reduction with NaBH(4). After deprotection, the resulting derivative was examined as a substrate for [small alpha]-(2,6)- and [small alpha]-(2,3)-sialyltransferase and fucosyltransferase III, IV, V and VI. It was found that none of these enzymes could glycosylate. However, it showed exquisite selectivity for inhibition of fucosyltransferase VI. The kinetic data support an unusual mechanism in which the inhibitor can bind to the GDP-fucose complex as well as another enzyme form.
Amino Sugars↗
A convergent strategy for the preparation of N-glycan core di-, tri-, and pentasaccharide thioaldoses for the site-specific glycosylation of peptides and proteins bearing free cysteines.
Mammalian glycoprotein biosynthesis produces heterogeneous ranges of proteins that possess the same peptide backbone but differ in the nature and site of glycosylation. This feature has frustrated efforts to develop therapeutic glycoproteins as well as the elucidation of biological functions of individual glycoforms. We have developed an attractive approach to well-defined glycoforms of glycoproteins by oxidative coupling of thioaldoses to cysteine-containing peptides and proteins to give disulfide-linked neoglycoconjugates. To this end, the chemical synthesis di-, tri-, and pentasaccharide N-glycan thioaldoses was undertaken. A convergent approach was used for the preparation of the pentasaccharide containing a 'synthetically difficult' beta-mannoside linkage. This linkage was installed by forming initially the corresponding beta-glucoside-containing pentasaccharide, followed by inversion of configuration at C-2. This approach exploited a levulonyl ester at C-2 of a glucosyl donor, which directed the coupling to give the beta-glucoside exclusively and could be removed selectively using hydrazine acetate without affecting other base-labile functionalities. The resulting alcohol was converted into a triflate, which was displaced by tri-n-butylammonium acetate to give a beta-mannosidic linkage. The trisaccharide N-glycan was prepared in a similar manner. Thioaldoses were prepared by displacing the peracetylated alpha-glycosyl chlorides with thioacetate to give the peracetylated beta-thioacetates, which upon saponification gave the desired compounds. The incubation of molar excesses of chitobiose thioaldose with cysteine-containing glutathione and BSA resulted in the site-specific formation of a disulfide-linked neoglycopeptide and neoglycoprotein, respectively.
Binding Sites↗
Dendritic cell-based assays, but not mannosylation of antigen, improves detection of T-cell responses to proinsulin in type 1 diabetes.
In vitro detection of T-cell responses to autoantigens in type 1 diabetes is recognized as being technically challenging. We aimed to accurately measure cellular responses to proinsulin in patients with diabetes, and speculated that presentation of antigen by dendritic cells (DCs) would enhance the sensitivity of the peripheral blood assay. Antigen was mannosylated to facilitate uptake through DC surface mannose receptors to further improve the assay. Whole proinsulin, as well as mannosylated peptides of proinsulin, were combined with peripheral T cells and autologous immature DCs in a proliferative assay in a panel of newly diagnosed type 1 diabetic patients. The DC-based assay detected responses to proinsulin in five of 15 diabetic patients compared to one of 15 diabetic patients detected using the standard mononuclear cell assay. When the results of all patients were combined, the DC assay, but not the mononuclear cell assay, had a proinsulin response that was significantly higher than background (P < 0.001). The DC assay was, however, associated with high autologous mixed lymphocyte reactions that possibly masked responses in individual patients. Mannosylated antigen was taken up in larger quantities than non-mannosylated antigen, but not presented any more powerfully. Our data suggest that autologous DC-based assays are more powerful than standard peripheral blood mononuclear cell assays. However, they are compromised by high autologous mixed lymphocyte reactions and this requires addressing before they can be used as a routine readout of in vitro peripheral T-cell responses.
Adolescent↗
New set of orthogonal protecting groups for the modular synthesis of heparan sulfate fragments.
Six strategically chosen monosaccharide building blocks, which are protected by a novel set of four orthogonal protecting groups (Lev, Fmoc, TBDPS, and All), can be employed for the efficient synthesis of the 20 disaccharide moieties found in heparan sulfate. The properly protected disaccharide building blocks can be converted into glycosyl donors and acceptors, which can be used for the modular synthesis of a wide range of well-defined oligosaccharides that differ in sulfation pattern. [structure: see text]
Carbohydrate Sequence↗
Chemo-enzymatic synthesis of conformationally constrained oligosaccharides.
N-Acetyllactosamine derivative 4, which has a methylene amide tether between C-6 and C-2', was enzymatically glycosylated using rat liver alpha-2,6-sialyltransferase (ST6GalI) or recombinant human fucosyltransferase V (FucT-V) to give conformationally constrained trisaccharides 5 and 6, respectively. The methylene amide linker of 4 was installed by a two-step procedure, which involved acylation of a C-6 amino function of a LacNAc derivative with chloroacetic anhydride followed by macrocyclization by nucleophilic displacement of the chloride by a C-2' hydroxyl. The conformational properties of 4 were determined by a combination of NOE and trans-glycosidic heteronuclear coupling constant measurements and molecular mechanics simulations and these studies established that the glycosidic linkage of 4 is conformationally constrained and resides in only one of the several energy minima accessible to LacNAc. The apparent kinetic parameters of transfer to LacNAc and conformationally constrained saccharides 3 and 4 indicates that fucosyltransferase V recognize LacNAc in its A-conformer whereas alpha-2,6-sialyltransferase recognizes the B-conformer of LacNAc.
Amides↗
Synthesis of oligosaccharides on soluble high-molecular-weight branched polymers in combination with purification by nanofiltration.
[structure: see text] An efficient approach for polymer-supported oligosaccharide synthesis is described whereby branched and high-molecular-weight PEG derivatives are used in combination with purification by nanofiltration. This methodology was applied to the preparation of a tetraglucoside and the tumor-associated antigen Le(x).
Antigens, Tumor-Associated, Carbohydrate↗
Glycosyltransferase activity can be modulated by small conformational changes of acceptor substrates.
A range of N-acetyllactosamine derivatives (compounds 4-7) that have restricted mobilities around their glycosidic linkages have been employed to determine how small changes in conformational properties of an oligosaccharide acceptor affect catalytic efficiencies of glycosylations by alpha-2,6- and alpha-2,3-sialyltransferases and alpha-1,3-fucosyltransferases IV and VI. Restriction of conformational mobility was achieved by introducing tethers of different length and chemical composition between the C-6 and C-2' hydroxyl of LacNAc. Compound 4 is a 2',6-anhydro derivative which is highly constrained and can adopt only two unusual conformations at the LacNAc glycosidic linkage. Compound 5 is modified by a methylene acetal tether and can exist in a larger range of conformations; however, the Phi dihedral angle is restricted to values smaller than 30 degrees, which are not entirely similar to minimum energy conformations of LacNAc. The ethylene-tethered 6 can attain conformations in the relatively large energy plateau of LacNAc that include syn conformations A and B, whereas compound 7, which is modified by a methylamide tether, can only reside in the B-conformer. 2',6-Dimethoxy derivative 2 was employed to determine the effect of alkylation of the C-6 and C-2' hydroxyls of 5 and 6 whereas 3 was used to reveal the effects of the C-6 amide and C-2' alkylation of 7. The apparent kinetic parameters of transfer to the conformationally constrained 4-7 and reference compounds 1-3 catalyzed by alpha-2,6- and alpha-2,3-sialyltransferases and alpha-1,3-fucosyltransferases IV and VI were determined, and the results correlated with their conformational properties. The data for 4-6 showed that each enzyme recognizes N-acetyllactosamine in a low minimum energy conformation. A small change in conformational properties such as in compound 5 resulted in a significant loss of catalytic activity. Larger conformational changes such as in compound 4 abolished all activity of the sialyltransferases whereas the fucosyltransferases showed some activity, albeit very low. The kinetic data for compounds 4 and 5 demonstrate clearly that different glycosyltransferases respond differently to conformational changes, and the fucosyltransferases lost less activity than the sialyltransferases. Correlating apparent kinetic parameters of conformationally constrained 6 and 7 and their reference compounds 2 and 3 further supports the fact that different enzymes respond differently and indicates that sialyltransferases and fucosyltransferases recognize N-acetyllactosamine in a different conformation. Collectively, the data presented here indicate that small conformational changes of an oligosaccharide acceptor induced by, for example, the protein structure can be employed to modulate the patterns of protein glycosylation.
Animals↗