Glycosyl Acceptors Research Articles

One‐Pot Protection Strategy of Glucosamine to Assemble Building Blocks of Chitosan and Lipid A

This investigation describes a one‐pot reaction to prepare a series of building blocks for glycosylation reactions, such as 3‐alcohol glucosamines, fully protected glucosamines, O‐4 and O‐6 alcohol glucosamines. These reactions readily produce not only glycosyl donors and acceptors, but also different glycosyl units that can be changed based on the needs of the experiment. The synthesis of some molecules containing glucosamines, including saccharide chains of glycopeptide (GP) and precursors of lipid A disaccharide backbone, are also described. GP has good selectivity for tumor angiogenesis and this phenomenon makes the GP a potential target drug. Lipid A has recently been adopted as an adjuvant for human vaccines.

Biocatalytic Transfer of Pseudaminic Acid (Pse5Ac7Ac) Using Promiscuous Sialyltransferases in a Chemoenzymatic Approach to Pse5Ac7Ac-Containing Glycosides

Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni, and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host–pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein, we describe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors, enabling the enzymatic synthesis of Pse5Ac7Ac-containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late-stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot two-enzyme process using biosynthetic enzymes. Finally, screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac-terminated glycosides.

Synthesis and Glycosylation Properties of C6‐Silylated Ido‐ and Gluco‐Pyranosyl Donors

Silyl groups are widely used as alcohol protecting groups and their study has provided insight on some very remarkable structure and reactivity features within carbohydrate chemistry. However, not much work has been put into the effect on the reactivity of silyl groups directly attached to the sugar carbon chain. In this work, we have developed a synthetic methodology to obtain both d‐glucosyl and l‐idosyl donors containing a dimethylphenylsilyl group directly attached to C6. Glycosylation and competition experiments with different glycosyl acceptors have shown that this group is completely stable under glycosylation conditions and enhances reactivity beyond what a benzyl group attached to the sugar oxygen would do. Finally, we found adequate conditions for the protodesilylation of Si‐containing glycosides to yield the 6‐deoxy sugar analog.

Substrate Engagement and Catalytic Mechanisms of N-Acetylglucosaminyltransferase V

α-Mannoside β-1,6-N-acetylglucosaminyltransferase V (MGAT5) is a mammalian glycosyltransferase involved in complex N-glycan formation, which strongly drives cancer when overexpressed. Despite intense interest, the catalytic mechanism of MGAT5 is not known in detail, precluding therapeutic exploitation. We solved structures of MGAT5 complexed to glycosyl donor and acceptor ligands, revealing an unforeseen role for donor-induced loop rearrangements in controlling acceptor substrate engagement. QM/MM metadynamics simulations of MGAT5 catalysis highlight the key assisting role of Glu297 and reveal considerable conformational distortions imposed upon the glycosyl donor during transfer. Detailed mechanistic characterization of MGAT5 will aid inhibitor development to correct cancer-associated N-glycosylation.

Cyanomethyl Ether as an Orthogonal Participating Group for Stereoselective Synthesis of 1,2-trans-β-O-Glycosides.

Stereoselective formation of glycosidic linkages has been the prime focus for contemporary carbohydrate chemistry. Herein, we report cyanomethyl (CNMe) ether as an efficient and effective participating orthogonal protecting group for the stereoselective synthesis of 1,2-trans-β-O-glycosides. The participating group facilitated good to high β-selective glycosylation with a broad range of electron-rich and electron-deficient glycosyl acceptors. Detailed experimental and theoretical studies reveal the involvement of CNMe ether in the formation of a six-membered imine-type cyclic intermediate for the observed stereoselectivity. Rapid incorporation and selective removal of the CNMe ether group in the presence of benzyl ether and isopropylidene acetal protection have also been reported here. The nitrile group provided an opportunity for the glycodiversification through further derivatizations.

Solvent-Dependent Mechanism and Stereochemistry of Mitsunobu Glycosylation with Unprotected Pyranoses.

An SN2 mechanism was proposed for highly stereoselective glycosylation of benzoic acid with unprotected α-d-glucose under Mitsunobu conditions in dioxane, while an SN1 mechanism was indicated for nonstereoselective glycosylation in DMF. The SN2-type stereoselective Mitsunobu glycosylation is generally applicable to various unprotected pyranoses as glycosyl donors in combination with a wide range of acidic glycosyl acceptors such as carboxylic acids, phenols, and imides, retaining its high stereoselectivity (33 examples). Glycosylation of a carboxylic acid with unprotected α-d-mannose proceeded also in an SN2 manner to directly afford a usually less accessible 1,2-cis-mannoside. One- or two-step total syntheses of five simple natural glycosides were performed using the glycosylation strategy presented here using unprotected α-d-glucose.

Catalytic and Atom-Economic Glycosylation using Glycosyl Formates and Cheap Metal Salts.

Benzylated glycosyl formates have been synthesized in one step from the corresponding hemiacetal or orthoester with formic acid as the sole reagent. The glycosyl formates are used as glycosyl donors under catalytic conditions with cheap metal catalysts based on iron or bismuth. A 13 C NMR spectroscopic method is developed and evaluated for screening reactions conditions, giving precise information on the selectivity, yield, and byproducts formed. The major side reaction is transesterification, which gives the formylated acceptor and regenerates the hemiacetal. By using this approach, catalyst loadings and solvents are optimized and the scope of the glycosylation is evaluated for a variety of glycosyl donors and acceptors. A proof of concept for a traceless glycosylation, utilizing a dual-purpose iron catalyst that catalyzes both glycosylation and dehydrogenation of formic acid, is also provided.

Synthesis of a Pseudodisaccharide Suitable for Synthesis of Ring I Modified 4,5-2-Deoxystreptamine Type Aminoglycoside Antibiotics.

To facilitate the synthesis of paromomycin and/or neomycin analogues, we describe a cleavage of ring I from paromomycin that proceeds in the presence of azides and affords a glycosyl acceptor for the installation of a modified ring I. A paromomycin 4',6'-diol is oxidized by the Dess-Martin periodinane followed by m-chloroperoxybenzoic acid. Base treatment then affords a protected pseudodisaccharide, which functions as a glycosyl acceptor. The method should also apply to the cleavage of pyranosyl 4,6-diols from oligosaccharides and glycoconjugates.

Azidophenylselenylation of glycals towards 2-azido-2-deoxy-selenoglycosides and their application in oligosaccharide synthesis

Abstract 2-Amino-2-deoxy-pyranosyl units are important structural components of cell-wall polymers in prokaryotes, fungi and mammals. With respect to the need for development of novel and efficient vaccines and tools for serodiagnosis of infectious diseases, of particular interest are the oligosaccharide cell-wall antigens of pathogenic bacteria and fungi, which comprise 2-amino-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-galactopyranose units as α- or β-anomers. Synthesis of N-acylated α-GlcN and α-GalN containing oligosaccharides is a special challenge due to the presence of a participating group at C2 which favors the formation of β- rather than α-glycoside bond. Herein we overview the efficient two-step approach for preparation of 1,2-cis-glycosides of 2-amino-2-deoxy-D-glucopyranose and 2-amino-2-deoxy-D-galactopyranose, which was recently developed in our laboratory. In the first step, an efficient and straightforward azidophenylselenylation procedure of glycals gives phenyl 2-azido-2-deoxy-1-selenoglycosides as versatile glycosyl donors. In the second step, these donors can be efficiently transformed into α- or β-glycosides depending on the choice of the solvent. In acetonitrile, total β-stereocontrol was achieved, and the use of diethyl ether as a solvent favouring α-stereoselectivity of glycosylations with phenyl 2-azido-2-deoxy-1-selenoglycosides. Besides, it was shown, that low reactivity and nucleophilicity of glycosyl acceptors which are glycosylated with phenyl 2-azido-2-deoxy-1-selenogalactosides facilitated the formation of α-GalN derivatives. To date, homogenous azidophenylselenylation of glycals and glycosylation with phenyl 2-azido-2-deoxy-1-seleno-α-D-glycopyranosides can be regarded as most useful tool for introduction of 2-amino-2-deoxy-D-glycopyranoside residues into complex synthetic oligosaccharides.

Regio‐ and stereoselective glycosylation of 2‐(o‐dihydroxyborylbenzyl) thioglucoside and unprotected methyl glycosides

AbstractA highly regio‐ and stereoselective glycosylation of a boronic acid‐containing thioglucoside and unprotected methyl glycosides is described. A boronic acid moiety was installed at the ortho‐position of the 2‐O‐benzyl group of a thioglucosyl donor. This provides transient partial protection for the unprotected glycosyl acceptor upon condensation and concomitantly prearranged the acceptor with respect to the donor for the ensuing intramolecular glycosylation.

Efficient Strategy for α-Selective Glycosidation of d-Glucosamine and Its Application to the Synthesis of a Bacterial Capsular Polysaccharide Repeating Unit Containing Multiple α-Linked GlcNAc Residues.

An efficient α-selective glycosylation method was developed for the synthesis of 2-deoxy-2-amino-d-glucosides based on synergetic α-directing effects of the TolSCl/AgOTf promotion system and the functional groups at the corresponding azido donor 6-O-position to exert steric β-shielding effect or remote participation in the glycosylation reaction. Its practicability was verified with a wide range of monosaccharide glycosyl acceptors and the first, one-pot synthesis of the challenging pentasaccharide repeating unit of an Acinetobacter baumannii K47 capsular polysaccharide.

Advances in Protecting Groups for Oligosaccharide Synthesis.

Carbohydrates contain numerous hydroxyl groups and sometimes amine functionalities which lead to a variety of complex structures. In order to discriminate each hydroxyl group for the synthesis of complex oligosaccharides, protecting group manipulations are essential. Although the primary role of a protecting group is to temporarily mask a particular hydroxyl/amino group, it plays a greater role in tuning the reactivity of coupling partners as well as regioselectivity and stereoselectivity of glycosylations. Several protecting groups offer anchimeric assistance in glycosylation. They also alter the solubility of substrates and thereby influence the reaction outcome. Since oligosaccharides comprise branched structures, the glycosyl donors and acceptors need to be protected with orthogonal protected groups that can be selectively removed one at a time without affecting other groups. This minireview is therefore intended to provide a discussion on new protecting groups for amino and hydroxyl groups, which have been introduced over last ten years in the field of carbohydrate synthesis. These protecting groups are also useful for synthesizing non-carbohydrate target molecules as well.

Stereoselective synthesis of (+)-5-thiosucrose and (+)-5-thioisosucrose.

(+)-5-Thiosucrose 1, a novel isosteric sulfur analog of sucrose, was synthesized stereoselectively for the first time via indirect β-d-fructofuranosidation involving selective β-d-psicofuranosidation, followed by stereo-inversion of the secondary hydroxy group at the C-3 position on the furanose ring. Glycosidation of protected 5-thio-d-glucose with a d-psicofuranosyl donor provided β-d-psicofuranosyl 5-thio-α-d-glucopyranoside and that with d-fructofuranosyl donor gave α-d-fructofuranosyl 5-thio-α-d-glucopyranoside. Two anomeric stereocenters of the glycosyl donor and acceptor were controlled correctly to provide a single disaccharide among four possible anomeric isomers in the glycosylation. Conversion of the resulting disaccharides afforded (+)-5-thiosucrose 1 and (+)-5-thioisosucrose 2 in excellent yields, respectively. Inhibitory activities of 1 and 2 against α-glucosidase in vitro were also examined.

Regioselectivity of glycosylation reactions of galactose acceptors: an experimental and theoretical study.

Regioselective glycosylations allow planning simpler strategies for the synthesis of oligosaccharides, and thus reducing the need of using protecting groups. With the idea of gaining further understanding of such regioselectivity, we analyzed the relative reactivity of the OH-3 and OH-4 groups of 2,6-diprotected methyl α- and β-galactopyranoside derivatives in glycosylation reactions. The glycosyl acceptors were efficiently prepared by simple methodologies, and glycosyl donors with different reactivities were assessed. High regioselectivities were achieved in favor of the 1→3 products due to the equatorial orientation of the OH-3 group. A molecular modeling approach endorsed this general trend of favoring O-3 substitution, although it showed some failures to explain subtler factors governing the difference in regioselectivity between some of the acceptors. However, the Galp-(β1→3)-Galp linkage could be regioselectively installed by using some of the acceptors assayed herein.

Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation.

Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure–activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.

Deciphering the molecular specificity of phenolic compounds as inhibitors or glycosyl acceptors of \u03b2-fructofuranosidase from Xanthophyllomyces dendrorhous

Enzymatic glycosylation of polyphenols is a tool to improve their physicochemical properties and bioavailability. On the other hand, glycosidic enzymes can be inhibited by phenolic compounds. In this work, we studied the specificity of various phenolics (hydroquinone, hydroxytyrosol, epigallocatechin gallate, catechol and p-nitrophenol) as fructosyl acceptors or inhibitors of the β-fructofuranosidase from Xanthophyllomyces dendrorhous (pXd-INV). Only hydroquinone and hydroxytyrosol gave rise to the formation of glycosylated products. For the rest, an inhibitory effect on both the hydrolytic (H) and transglycosylation (T) activity of pXd-INV, as well as an increase in the H/T ratio, was observed. To disclose the binding mode of each compound and elucidate the molecular features determining its acceptor or inhibitor behaviour, ternary complexes of the inactive mutant pXd-INV-D80A with fructose and the different polyphenols were analyzed by X-ray crystallography. All the compounds bind by stacking against Trp105 and locate one of their phenolic hydroxyls making a polar linkage to the fructose O2 at 3.6–3.8 Å from the C2, which could enable the ulterior nucleophilic attack leading to transfructosylation. Binding of hydroquinone was further investigated by soaking in absence of fructose, showing a flexible site that likely allows productive motion of the intermediates. Therefore, the acceptor capacity of the different polyphenols seems mediated by their ability to make flexible polar links with the protein, this flexibility being essential for the transfructosylation reaction to proceed. Finally, the binding affinity of the phenolic compounds was explained based on the two sites previously reported for pXd-INV.

Synthesis of N-acetyllactosamine based branched hexasaccharide

The protected spacer-armed branched tri-N-acetyllactosamine has been synthesized as a building block for the synthesis of biantennary ABH blood group antigens. The key synthetic step is the simultaneous glycosylation of the 3′- and 6′-OH groups of an N-acetyllactosamine glycosyl acceptor by N-Troc-lactosamine trichloroacetimidate.

Thermostable Amylosucrase from Calidithermus timidus DSM 17022: Insight into Its Characteristics and Tetrameric Conformation.

Amylosucrase (EC 2.4.1.4, ASase), a typical carbohydrate-active enzyme, can catalyze 5 types of reactions and recognize more than 50 types of glycosyl acceptors. However, most ASases are unstable even at 50 °C, which limits their practical industrial applications. In this study, an extremely thermostable ASase was discovered from Calidithermus timidus DSM 17022 (CT-ASase) with an optimal activity temperature of 55 °C, half-life of 1.09 h at 70 °C, and melting temperature of 74.47 °C. The recombinant CT-ASase was characterized as the first tetrameric ASase, and a structure-based truncation mutation was conducted to confirm the effect of tetrameric conformation on its thermostability. In addition, α-1,4-glucan was found to be the predominant product of CT-ASase at pH 6.0-8.0 and 30-60 °C.

Direction of Chain Growth and Substrate Preferences of Shape, Elongation, Division, and Sporulation-Family Peptidoglycan Glycosyltransferases.

The bacterial cell wall is composed of peptidoglycan, and its biosynthesis is an established target for antibiotics. Peptidoglycan is assembled from a glycopeptide precursor, Lipid II, that is polymerized by peptidoglycan glycosyltransferases into glycan strands that are subsequently cross-linked to form the mature cell wall. For decades bacteria were thought to contain only one family of enzymes that polymerize Lipid II, but recently, the ubiquitous Shape, Elongation, Division, and Sporulation (SEDS)-family proteins RodA and FtsW were shown to be peptidoglycan polymerases. Because RodA and FtsW are essential in nearly all bacteria, these enzymes are promising targets for new antibiotics. However, almost nothing is known about the mechanisms of these polymerases. Here, we report that SEDS proteins synthesize peptidoglycan by adding new Lipid II monomers to the reducing end of the growing glycan chain. Using substrates that can only react at the reducing end, we also show that the glycosyl donor and acceptor in the polymerization reaction have distinct lipid requirements. These findings provide the first fundamental insights into the mechanism of SEDS-family polymerases and lay the groundwork for future biochemical and structural studies.

α-Selective glycosidation of d-tagatofuranose with a 3,4-O-isopropylidene protection

An α-selective glycosidation reaction of d-tagatofuranose was successfully achieved using 3,4-O-isopropylidene-protected d-tagatofuranose as a glycosyl donor. A variety of glycosyl acceptors, including primary, secondary, and β-amino alcohols, and carbohydrates, can be used for this d-tagatofuranosidation reaction with complete α-selectivities and good yields (57–83%). The stereochemistries at the anomeric positions were determined by nuclear Overhauser effect spectroscopic correlations, as well as comparison of the chemical shifts in the 13C NMR spectra.