Anomeric Stereochemistry Research Articles

Atom-Economic Synthesis of Unsymmetrical gem-Diarylmethylthio/Seleno Glycosides via Base Mediated C(O)-S/Se Bond Cleavage and Acyl Transfer Approach of Glycosylthio/Selenoacetates.

Herein, we invented the Cs2CO3-mediated atom economic method that streamlines the scission of the C(O)-S/Se bond involving the in situ generation of an anomeric thiolate/selenolate anion, which reacted with p-QMs to yield novel unsymmetrical gem-diarylmethylthio/seleno glycosides while retaining the anomeric stereochemistry. Notably, the key features of this protocol involve unprecedented long-range acyl transfer (from S/Se to O), thus affording acylation of the final product which is not yet reported by classical methods. This straightforward protocol offers a mild, short reaction time, synthetically simple approach, and compatibility with 8 types of sugar along with phenylthio/benzylseleno esters.

Concise synthesis of the tetrasaccharide repeating unit of the O-antigen from Escherichia coli O131 containing N-acetyl neuraminic acid

Synthesis of the tetrasaccharide repeating unit of the O-antigen from E. coli O131 was accomplished through a linear strategy involving rationally protected monosaccharide units derived from commercially available sugars. The challenging α-glycosylation of N-acetyl neuraminic acid was achieved through activation of thioglycoside using NIS-mediated glycosylation strategy. The target tetrasaccharide in the form of its 3-aminopropyl glycoside may be used for further glycoconjugate formation without hampering the anomeric stereochemistry.

Chemical Synthesis of the Linker‐Armed Trisaccharide Repeating Unit of the O‐Antigen from Pseudomonas putida BIM B‐1100

AbstractThe manuscript reports the concise chemical synthesis of the trisaccharide repeating unit of the O‐antigen from Pseudomonas putida BIM B‐1100 in the form of its aminoethyl glycoside through a linear strategy with rational protecting group manipulations. The challenging aspects of this synthesis include the incorporation of the β‐D‐ManpNAcA and the α‐D‐GlcpNAc residues. The introduction of the desired carboxylic acid moiety has been achieved through late‐stage TEMPO‐mediated oxidation of the primary OH group on the protected trisaccharide. 1,2‐cis aminoethyl glycoside with central glucosamine is used at the reducing end to the leave the scope for further glycoconjugate formation with suitable aglycone without hampering the anomeric stereochemistry.

Chemical synthesis of the rare D-Fuc3NAc containing tetrasaccharide repeating unit of the O-antigenic polysaccharide from E. coli O74

Chemical synthesis of the tetrasaccharide repeating unit of the O-antigen from E. coli O74 is accomplished by a convergent [2 + 2] block synthesis strategy. The challenging rare D-Fuc3NAc has been prepared using DTBP and TIPST mediated deoxygenation reaction. Other monosaccharide synthons are prepared through rational protecting group manipulations and the stereoselective glycosylations are achieved either by the activation of thioglycoside or glycosyl trichloroacetimidate. The target tetrasaccharide is made in the form of its 2-aminoethyl glycoside to facilitate further glycoconjugate formation without affecting the anomeric stereochemistry.

Cellular Fucosylation Inhibitors Based on Fluorinated Fucose-1-phosphates*.

Fucosylation of glycans impacts a myriad of physiological and pathological processes. Inhibition of fucose expression emerges as a potential therapeutic avenue for example in cancer, inflammation, and infection. In this study, we found that protected 2‐fluorofucose 1‐phosphate efficiently inhibits cellular fucosylation with a four to seven times higher potency than known inhibitor 2FF, independently of the anomeric stereochemistry. Nucleotide sugar analysis revealed that both the α‐ and β‐GDP‐2FF anomers are formed inside the cell. In conclusion, we developed A2FF1P and B2FF1P as potent new tools for studying the role of fucosylation in health and disease and they are potential therapeutic candidates.

Stereoselective Synthesis of 1,1′‐Disaccharides by Organoboron Catalysis

AbstractThe highly stereoselective synthesis of 1,1′‐disaccharides was achieved by using 1,2‐dihydroxyglycosyl acceptors and glycosyl donors in the presence of a tricyclic borinic acid catalyst. In this reaction, the complexation of the diols and the catalyst is crucial for the activation of glycosyl donors, as well as for the 1,2‐cis‐configuration of the products. The anomeric stereochemistry of the glycosyl donor depends on the employed glycosyl donor. Applications of the produced 1,1′‐disaccharides are also described.

Stereoselective Synthesis of 1,1′‐Disaccharides by Organoboron Catalysis

The highly stereoselective synthesis of 1,1'-disaccharides was achieved by using 1,2-dihydroxyglycosyl acceptors and glycosyl donors in the presence of a tricyclic borinic acid catalyst. In this reaction, the complexation of the diols and the catalyst is crucial for the activation of glycosyl donors, as well as for the 1,2-cis-configuration of the products. The anomeric stereochemistry of the glycosyl donor depends on the employed glycosyl donor. Applications of the produced 1,1'-disaccharides are also described.

Synthesis of the tetrasaccharide repeating unit of the O-antigen from Pseudomonas putida BIM B-1100 having rare D-Quip3NAc

Chemical synthesis of the complex tetrasaccharide repeating unit of the O-antigen from Pseudomonas putida BIM B-1100 is accomplished in the form of its 2-aminoethyl glycoside to leave the scope for further glycoconjugate formation without hampering the anomeric stereochemistry. A [2 + 2] strategy is followed to complete the total synthesis and a late stage TEMPO mediated oxidation is used to install the required uronic acid. A radical mediated 6-deoxygenation with subsequent protecting group manipulation strategy is used for the preparation of the rare D-FucpNAc and D-Quip3NAc derivatives from suitable d-glucosamine derivatives.

Concise chemical synthesis of the pentasaccharide repeating unit of the O-antigen from Escherichia albertii O2

Total chemical synthesis of the pentasaccharide repeating unit of the O-antigen from Escherichia albertii O2 is accomplished by following a [3 + 2] strategy. The target pentasaccharide in the form of its 2-aminoethyl glycoside is particularly attractive as the free amine end can be coupled with suitable aglycon to make further glycoconjugate without affecting the anomeric stereochemistry. Phthalimido derivatives were used successfully as the precursor of the desired acetamido glucose moieties and ensured the 1,2-trans linkages.

Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6-anhydro-β-d-glucopyranose) in the presence of Mg2+ -ATP. For the Lipomyces starkeyi LGK, we show here with real-time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α-d-glucose-6-phosphate in a manner reminiscent of an inverting β-glycoside hydrolase. Kinetic characterization revealed the Mg2+ concentration for optimum activity (20-50 mm), the apparent binding of levoglucosan (Km =180 mm) and ATP (Km =1.0 mm), as well as the inhibition by ADP (Ki =0.45 mm) and d-glucose-6-phosphate (IC50 =56 mm). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d-glucose-6-phosphate and ADP was observed under a broad range of conditions. 6-Phospho-α-d-glucopyranosyl fluoride and 6-phospho-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol (6-phospho-d-glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip 4 C1 →1 C4 required for 1,6-anhydro-product synthesis from d-glucose-6-phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.

Synthesis and intramolecular glycosylation of sialyl mono-esters of o-xylylene glycol. The importance of donor configuration and nitrogen protecting groups on cyclization yield and selectivity; isolation and characterization of a N-sialyl acetamide indicative of participation by acetonitrile

The synthesis and cyclization reactions, leading to spirocyclic medium ring-sized diolides, of o-(hydroxymethyl)xylylene monoesters of sialyl thioglycosides is described. Cyclization yields and stereoselectivities are found to vary as a function of the anomeric stereochemistry of the thioglycoside and of the N5 protecting group, and these effects are discussed in terms of the reaction mechanism. Cyclization in the presence of acetonitrile results in the isolation and characterization of a Ritter-type N-sialyl acetamide, which affords strong evidence for the participation of acetonitrile in the form of sialyl nitrilium ions.

Short-Step Anodic Synthesis of Color-Tunable Emissive Nucleoside Analogues

Since natural nucleobases are not emissive, emissive nucleoside analogues have been widely used to monitor fundamental biochemical transformations directly. Nowadays, useful libraries of synthetic emissive nucleoside analogues have been constructed and researchers have a wide variety of choices. A typical approach to prepare these synthetic probes is “pre-formed” manner, namely, a glycosylation is carried out using emissive nucleobases under carefully selected conditions to control both stereoselectivity at the anomeric position and regioselectivity of the aglycon attack. Such a glycosylation usually requires a time-consuming trial-and-error procedure, because the reaction selectivities are affected by the interaction of the protected groups of both the sugar and aglycon. On the other hand, the preparation approach based on “post-forming” manner would be effective since a cumbersome glycosylation could be done in an early synthesis stage. We have been developing anodic intermolecular cycloadditions in lithium perchlorate/nitromethane electrolyte solution. In this electrolyte solution, unactivated alkenes can function as effective carbon nucleophiles to trap anodically generated cations or radical cations, leading to the formation of corresponding ring systems. Especially, we previously reported that the anodic oxidation of methoxyphenols in the presence of alkene nucleophiles was able to give highly emissive dihydrobenzofuran derivatives through formal [3 + 2] cycloadditions. These results in hand, we herein demonstrate a short-step “post-forming” synthesis of novel emissive nucleoside analogues based on the anodic cycloadditions. Initially, we tried to attach a methallyl moiety to a ribose scaffold as an alkene nucleophile. After the numerous optimizations, we finally accomplished the stereoselective allylation at the anomeric position to obtain both a- and b-methallyl ribose and we were able to use each corresponding pure form for further transformations. The anomeric stereochemistry was successfully fixed at the first synthesis stage. Methoxyphenols were then anodically oxidized in the presence of the methallyl ribose and we found that emissive dihydrobenzofuran structures were able to be constructed on the sugar moiety in one step. The emission color was significantly affected by the carbonyl substituent, ranging roughly from 400 nm to 500 nm with long stokes shift values. In the presentation, the synthesis details and photophysical properties will be discussed.

Recombinant α-NAcetylgalactosaminidase from Marine Bacterium-Modifying A Erythrocyte Antigens

A plasmid based on pET-40b was constructed to synthesize recombinant α-N-acetylgalactosaminidase of the marine bacterium Arenibacter latericius KMM 426T (α-AlNaGal) in Escherichia coli cells. The yield of α-Al- NaGal attains 10 mg/ml with activity of 49.7 ± 1.3 U at 16°C, concentration of inductor 2 mM, and cultivation for 12 h. Techniques such as anion exchange, metal affinity and gel filtration chromatography to purify α-AlNaGal were applied. α-AlNaGal is a homodimer with a molecular weight of 164 kDa. This enzyme is stable at up to 50°C with a temperature range optimum activity of 20-37°C. Furthermore, its activity is independent of the presence of metal ions in the incubation medium. 1H NMR spectroscopy revealed that α-AlNaGal catalyzes the hydrolysis of the O-glycosidic bond with retention of anomeric stereochemistry and possesses a mechanism of action identical to that of other glycoside hydrolases of the 109 family. α-AlNaGal reduces the serological activity of A erythrocytes at pH 7.3. This property of α-AlNaGal can potentially be used for enzymatic conversion of A and AB erythrocytes to blood group O erythrocytes.

Atomistic simulation studies of the α/β-glucoside and galactoside in anhydrous bilayers: effect of the anomeric and epimeric configurations

Fully atomistic molecular dynamics simulation studies of thermotropic bilayers were performed using a set of glycosides namely n-octyl-β-D-glucopyranoside (β-C8Glc), n-octyl-α-D-glucopyranoside (α-C8Glc), n-octyl-β-D-galactopyranoside (β-C8Gal), and n-octyl-α-D-galactopyranoside (α-C8Gal) to investigate the stereochemical relationship of the epimeric/anomeric quartet liner glycolipids with the same octyl chain group. The results showed that, the anomeric stereochemistry or the axial/equatorial orientation of C1-O1 (α/β) is an important factor controlling the area and d-spacing of glycolipid bilayer systems in the thermotropic phase. The head group tilt angle and the chain ordering properties are affected by the anomeric effect. In addition, the L(C) phase of β-C8Gal, is tilting less compared to those in the fluid L(α). The stereochemistry of the C4-epimeric (axial/equatorial) and anomeric (α/β) centers simultaneously influence the inter-molecular hydrogen bond. Thus, the trend in the values of the hydrogen bond for these glycosides is β-C8Gal > α-C8Glc > β-C8Glc > α-C8Gal. The four bilayer systems showed anomalous diffusion behavior with an observed trend for the diffusion coefficients; and this trend is β-C8Gal > β-C8Glc > α-C8Gal > α-C8Glc. The "bent" configuration of the α-anomer results in an increase of the hydrophobic area, chain vibration and chain disorganization. Since thermal energy is dispensed more entropically for the chain region, the overall molecular diffusion decreases.

Synthesis and Thermotropic Phase Behavior of Four Glycoglycerolipids

Four glycoglycerolipids with different head groups have been synthesized and their physicochemical properties studied. The lengths of the head groups from a mono-saccharide to a trisaccharide, in addition to the anomeric stereochemistry for the smaller glycoglycerolipids, have been modified. The synthesis has been optimized to avoid glycerol epimerization and to allow up-scaling. The physicochemical properties of the glycoglycerolipids were studied and a strong de-mixing of the gel-phase, depending on the head-group, was observed.

Geometric Attributes of Retaining Glycosyltransferase Enzymes Favor an Orthogonal Mechanism

Retaining glycosyltransferase enzymes retain the stereochemistry of the donor glycosidic linkage after transfer to an acceptor molecule. The mechanism these enzymes utilize to achieve retention of the anomeric stereochemistry has been a matter of much debate. Re-analysis of previously released structural data from retaining and inverting glycosyltransferases allows competing mechanistic proposals to be evaluated. The binding of metal-nucleotide-sugars between inverting and retaining enzymes is conformationally unique and requires the donor substrate to occupy two different orientations in the two types of glycosyltransferases. The available structures of retaining glycosyltransferases lack appropriately positioned enzymatic dipolar residues to initiate or stabilize the intermediates of a dissociative mechanism. Further, available structures show that the acceptor nucleophile and anomeric carbon of the donor sugar are in close proximity. Structural features support orthogonal (front-side) attack from a position lying ≤90° from the C1-O phosphate bond for retaining enzymes. These structural conclusions are consistent with the geometric conclusions of recent kinetic and computational studies.

Synthesis of Glycans and Glycopolymers Through Engineered Enzymes

The synthesis of defined glycans enables us to further understand their roles in a biological context. Although useful chemical methods have been developed for the synthesis of glycans, these typically require complex protection and deprotection steps along with challenging control of anomeric stereochemistry. Enzymatic methods offer an attractive alternative to chemical synthesis. In particular, the use of glycosynthases and thioglycoligases, classes of engineered glycoside hydrolases, offers an enticing approach to the stereo- and regioselective synthesis of glycans without the need for protecting groups. Herein, we describe recent progress in the use of glycosynthases and thioglycoligases for the synthesis of glycans and glycopolymers.

Synthesis, characterization and X-ray structure of glycosyl-1,2-isoxazoles and glycosyl-1,2-isoxazolines prepared via 1,3-dipolar cycloaddition

A convenient preparative method of a series of glycosyl-1,2-isoxazoles (6–11) and glycosyl-1,2-isoxazolines (15–20) by a simple and efficient 1,3-dipolar cycloaddition of a series of aryl nitrile oxide, generated in situ from aryl oximes (4–5), with a variety of O-propargyl glycosyles (1–3) or O-allyl glycosyles (12–14) respectively, is reported. The carbohydrate-containing 1,2-isoxazoles and 1,2-isoxazolines compounds were isolated in excellent yields (81–91%) and they were fully characterized by 1H, 13C NMR and mass spectrometry. The relative stereochemistry of the glycosyl-1,2-isoxazole 10 was confirmed by single crystal X-ray analysis. The molecular structure of 10 confirms the retention of both, the anomeric stereochemistry of the D-fructose as well as the placement of the acetal groups.

Synthesis of the complete series of mono acetates of N-acetyl-d-neuraminic acid

The short syntheses of each of the mono-acetates of N-acetyl-D-neuraminic acid are reported. These are important molecules for studying the mechanism and function of enzymes which utilise Neu5Ac as a substrate. However, until now these molecules were not available as pure compounds and instead had to be studied as mixtures. Neu4,5Ac(2) and Neu5,8Ac(2) were synthesised from a common precursor in 2 and 4 steps respectively, while Neu2,4Ac(2) and Neu5,7Ac(2) were synthesised in 3 and 4 steps respectively from another common precursor. Both precursors could be easily prepared in 3 steps from Neu5Ac itself. Importantly, no scrambling of the anomeric stereochemistry was detected throughout the course of these syntheses.

Semisynthesis of fuscoside B analogues and eunicosides, and analysis of anti-inflammatory activity

A small library of semisynthetic analogues of fuscol and eunicol have been prepared and evaluated for in vivo topical anti-inflammatory activity using the mouse-ear edema assay. The first glycosylation of fuscol and eunicol has been achieved using a modified Koenigs–Knorr glycosylation to synthesize new fuscosides and eunicosides, a novel structural class of diterpene glycosides. The availability of adequate glycosylation methods for this synthesis was limited owing to the instability of the glycosyl acceptors. Glycosyl donor protecting group type had a pronounced effect on overall glycosylation yields of a model glycosyl acceptor. This synthesis provided access to the unnatural β-glycosides allowing for an evaluation of the effect of differing anomeric stereochemistry on anti-inflammatory activity. The PEGylated derivatives of fuscol and eunicol were also synthesized by a convenient acid-promoted solvolysis of the natural product aglycones. This work highlights the importance of the glycan portion of fuscoside B, notably the stereochemical configuration of the glycosidic linkage, in the observed anti-inflammatory activity.