Glycosyl Acceptors Research Articles

Block Copolysaccharides from Methylated and Acetylated Cellulose and Starch.

All-glucose block copolysaccharides with alternated α- and β-configured blocks do not exist in nature. Such polysaccharides, and materials made thereof, might exhibit very interesting properties due to the different supramolecular structures of the two α- and β-configured blocks. This would be helical for starch and linear for cellulose in the case of a non-derivatized all-glucose polysaccharide. We propose a synthetic pathway for the preparation of methylated and acetylated building blocks of cellulose and amylose for the regioselective modification at the non-reducing and reducing end to act as glycosyl acceptors and donors, respectively. The glycosyl acceptors with the opposite α- or β-configured blocks were prepared by acid-catalyzed methanolysis of permethylated amylose as well as cellulose. The regioselectively modified glycosyl donors and acceptors were successfully used in the preparation of block copolysaccharides with an α(1 → 4)-configured and a β(1 → 4)-configured oligo- or polysaccharide block, respectively.

Glycosylation of Semi-Synthetic Isoflavene Phenoxodiol with a Recombinant Glycosyltransferase from Micromonospora echinospora ATCC 27932.

Glycosyltransferase (GT)-specific degenerate PCR screening followed by in silico sequence analyses of the target clone was used to isolate a member of family1 GT-encoding genes from the established fosmid libraries of soil actinomycetes Micromonospora echinospora ATCC 27932. A recombinant MeUGT1 was heterologously expressed as a His-tagged protein in E. coli, and its enzymatic reaction with semi-synthetic phenoxodiol isoflavene (as a glycosyl acceptor) and uridine diphosphate-glucose (as a glycosyl donor) created two different glycol-attached products, thus revealing that MeUGT1 functions as an isoflavonoid glycosyltransferase with regional flexibility. Chromatographic separation of product glycosides followed by the instrumental analyses, clearly confirmed these previously unprecedented glycosides as phenoxodiol-4'-α-O-glucoside and phenoxodiol-7-α-O-glucoside, respectively. The antioxidant activities of the above glycosides are almost the same as that of parental phenoxodiol, whereas their anti-proliferative activities are all superior to that of cisplatin (the most common platinum chemotherapy drug) against two human carcinoma cells, ovarian SKOV-3 and prostate DU-145. In addition, they are more water-soluble than their parental aglycone, as well as remaining intractable to the simulated in vitro digestion test, hence demonstrating the pharmacological potential for the enhanced bio-accessibility of phenoxodiol glycosides. This is the first report on the microbial enzymatic biosynthesis of phenoxodiol glucosides.

A Comparison of the Transglycosylation Capacity between the Guar GH27 Aga27A and Bacteroides GH36 BoGal36A α-Galactosidases

The transglycosylation behavior and capacity of two clan GH-D α-galactosidases, BoGal36A from the gut bacterium Bacteroides ovatus and Aga27A from the guar plant, was investigated and compared. The enzymes were screened for the ability to use para-nitrophenyl-α-galactoside (pNP-Gal), raffinose and locust bean gum (LBG) galactomannan as glycosyl donors with the glycosyl acceptors methanol, propanol, allyl alcohol, propargyl alcohol and glycerol using mass spectrometry. Aga27A was, in general, more stable in the presence of the acceptors. HPLC analysis was developed and used as a second screening method for reactions using raffinose or LBG as a donor substrate with methanol, propanol and glycerol as acceptors. Time-resolved reactions were set up with raffinose and methanol as the donor and acceptor, respectively, in order to develop an insight into the basic transglycosylation properties, including the ratio between the rate of transglycosylation (methyl galactoside synthesis) and rate of hydrolysis. BoGal36A had a somewhat higher ratio (0.99 compared to 0.71 for Aga27A) at early time points but was indicated to be more prone to secondary (product) hydrolysis in prolonged incubations. The methyl galactoside yield was higher when using raffinose (48% for BoGal36A and 38% for Aga27A) compared to LBG (27% for BoGal36A and 30% for Aga27A).

Organocatalysis: A recent development on stereoselective synthesis of o-glycosides

ABSTRACT Organocatalysis is the use of non-stoichiometric amounts of low-molecular-weight organic molecules made up of C, H, N, O, S, and P to accelerate stereoselective chemical transformations. Despite having a long history, the use of small organic molecules as chiral catalysts in enantioselective synthesis has only recently intrigued scientists interest. However, in synthetic and medicinal chemistry, there has been a resurgence of interest in performing asymmetric synthesis of a variety of important targets by utilizing an organocatalytic approach as a leading reaction. The development of an asymmetric protocol for the synthesis of enantioenriched molecules of therapeutic relevance is crucial in the development of new drug candidates. Modern asymmetric catalysis is based on three pillars: (i) biocatalysts (i.e., enzymes), (ii) transition metal catalysis, and (iii) organocatalysis. Among these catalytic systems, organocatalysis has become the most flourishing field within the field of a catalytic regime that offers many significant benefits to synthetic and medicinal chemists. In contrast to many metal-based catalytic models, most organocatalysts have a high tolerance capability for air and moisture, easy availability, easy handling, non-toxic and enantiomeric purity, etc. They are often derived from natural resources of high enantiomeric virtue with the desired stereocentre or easily synthesized in the laboratory in some simple synthetic operations. This perspective aims to draw readers’ attention to the power of asymmetric organocatalysis in assembling glycosyl donors and acceptors in the synthesis of biologically significant stereodefined O-glycosides. Glycosylation is a fundamental chemical transformation and is ubiquitous in both nature and chemical laboratories. In nature, the glycosylation processes are carried out by enzyme catalysis. We were interested in portraying the recent impressive progress achieved by the scientific community in the field of chemical glycosylation, specifically O-glycosylation. Poly-functionalized sugar moieties offer many opportunities to discover new drug candidates in which the modification of aglycone through the glycosylation process is a key reaction.

SnCl2‐Catalyzed Acetalation/Selective Benzoylation Sequence for the Synthesis of Orthogonally Protected Glycosyl Acceptors

AbstractBased on SnCl2‐catalyzed acetalation and selective benzoylation, a one‐pot strategy to efficiently synthesize orthogonally protected glycosyl acceptors with 2‐OH/3‐OH was developed. Consequently, 2‐OBz or 3‐OBz 4,6‐O‐benzylidene galactosides and glucosides were efficiently prepared in moderate to high yields starting from free galactosides and glucosides, and were used as valuable glycosyl acceptors for the synthesis of blood group antigens O and B analogues in this study.

1, 2-trans-Stereoselective 7-O-Glycosylation of Flavonoids with Unprotected Pyranoses by Mitsunobu Reaction.

The glycosylation of protecting-group-free pyranoses with flavonoids to generate flavonoid O-glycosides under Mitsunobu conditions was reported. The methodology allows to prepare a wide range of natural 7-flavonoid O-glycosides and their derivatives from commercially available chemicals in good to excellent yields with exclusive 1,2-trans-stereoselectivity regardless the anomeric configuration of employed pyranoses. The highly regioselective glycosylation was also achieved among different types of hydroxyl groups on the glycosyl acceptors.

Practical non-enzymatic synthesis of propargyl sialyl-α-(2-3’)-lactosamine trisaccharide using minimal protecting groups manipulation

The trisaccharide, prop-2-ynyl 5-acetamido-3,5-dideoxy-d-glycero-α-d-galacto-2-nonulopyranosylonic acid-(2 → 3)-β-d-galactopyranosyl-(1 → 4)-2-acetamido-2-deoxy-β-d-glucopyranoside (9) has been efficiently synthesized in a few steps without the need of conformationally constrained glycosyl donors and acceptors or enzymes. First, using the known prop-2-ynyl 2-acetamido-2-deoxy-6-O-tert-butyldiphenylsilyl-β-d-glucopyranoside as acceptor (2) and the peracetylated galactosyl trichloroacetimidate (3) as glycosyl donor, followed by protecting groups manipulation, prop-2-ynyl (6-O-tert-butyldiphenylsilyl-β-d-galactopyranosyl)-(1 → 4)-2-acetamido-2-deoxy-6-O-tert-butyldiphenylsilyl-β-d-glucopyranoside (6) was synthesized with exclusive O-4 regioselectivity due to steric hindrance of the upper face of the acceptor at O-3. Sialylation with the thiophenyl glycosyl donor (7) afforded the desired trisaccharide with the shortest number of steps and in higher overall yield than previously reported methodologies. The direct use of minimally protected N-acetyl-lactosamine acceptor (6) was critical for the efficient synthesis of the title compound. The propargylic aglycone is suitable for chemical ligation using click chemistry as reported for its (2 → 6) sialylated analog.

Anomeric Thioglycosides Give Different Anomeric Product Distributions under NIS/TfOH Activation.

The reaction of a series of anomeric thioglycosides with various glycosyl acceptors and N-iodosuccinimide/catalytic triflic acid was investigated with respect to reactivity and anomeric selectivity. In general, β-configured donors were found to give a more β-selective reaction outcome compared to their α-configured counterparts. The relative reactivity of various thioglycosides was measured through competition experiments, and the following order was established: phenyl, tolyl, methyl, ethyl, isopropyl, and 1-adamantyl.

Engineering of cyclodextrin glycosyltransferase from Paenibacillus macerans for enhanced product specificity of long-chain glycosylated sophoricosides

Poor water solubility limits the application of genistein in food and pharmaceutical fields. Glycosylation can greatly improve the water solubility of genistein by attaching long chain sugars. Here, glycosylation of sophoricoside (genistein monoglucoside) by cyclodextrin glycosyltransferase from Paenibacillus macerans (PmCGTase) was investigated. Based on molecular docking and hydrolytic activity analysis, saturation mutagenesis was conducted at residues Y195 and S77 of PmCGTase. Variants S77N, Y195S, Y195I, S77N/Y195S, and S77N/Y195I showed enhanced product specificity toward long-chain glycosylated sophoricosides (LCGS). In particular, S77N/Y195I displayed 119% increased LCGS product specificity and 20.6% higher sophoricoside conversion than the wild type. Increased comprehensive activities of Y195S and Y195I contributed to enhanced conversion, and decreased hydrolytic activity of S77N promoted LCGS accumulation. Kinetics results revealed improved catalytic efficiency and substrate affinity of variants toward both the glycosyl donor and acceptor. Furthermore, MD simulation elucidates that the decreased substrate binding energy of variants could lead to improved kinetics, and increased hydrogen binding between residues at -6/-7 subsites and the glycosyl donor is primarily responsible for enhanced LCGS specificity. An “Increased Income-Reduced Expenditure” mechanism was proposed, explaining that the enhanced LCGS product specificity of PmCGTase is closely related to the increased hydrogen bond interactions at -6/-7 subsites and decreased hydrolytic activity.

Blockwise synthesis of polylactosamine fragments and keratan sulfate oligosaccharides comprised of dimeric Galβ(1 → 4)GlcNAc6Sβ

In this paper, the chemical synthesis of polylactosamine fragments up to docosasaccharide (22-mer) via the blockwise synthetic approach is reported. We used suitably protected tetrasaccharide and octasaccharide sequences as key building blocks. The use of such large building blocks as glycosyl donors and acceptors enabled the rapid construction of polysaccharide frameworks. Furthermore, the coupling reaction between these large building blocks facilitated the purification of glycosylated products, for which size exclusion column chromatography is highly effective. Then, we applied the building blocks to the synthesis of keratan sulfate glycan, which is partially sulfated poly-N-acetyllactosamine. Consequently, we achieved the synthesis of the octasaccharide of a keratan sulfate glycan comprised of a repeating Galβ(1 → 4)GlcNAc6Sβ disaccharide unit.

Triflic Imide‐Catalyzed Glycosylation of Disarmed Glycosyl ortho‐Isopropenylphenylacetates and ortho‐Isopropenylbenzyl Thioglycosides

AbstractGlycosylation reaction involving the coupling of a glycosyl donor with a glycosyl acceptor is one of the cornerstones of chemical preparation of pure glycans and glycoconjugates of biological relevance. Catalytic glycosylation of glycosyl ester donors and thioglycosides is an attractive but underexplored topic in carbohydrate chemistry. Herein, triflic imide (Tf2NH)‐catalyzed glycosylation of various O‐, S‐, and C‐nucleophiles has been achieved using disarmed glycosyl ortho‐isopropenylphenylacetates (GIPPAs) and ortho‐isopropenylbenzyl thioglycosides as glycosylating agents. The reactions proceed under mild conditions to give the desired glycosides in good‐to‐excellent yields. Of particular note, the comparable reactivity for the α‐isomers and the β‐ones of GIPPAs is observed. The mechanistic investigation demonstrates that the isopropenyl group is essential for the reaction and its preferential protonation triggers the reaction. This work provides another member to the arsenals of glycosyl ester and thioglycoside donors suitable for acid‐catalyzed glycosylation to create various glycosidic bonds.

Synthesis of a Gal-β-(1→4)-Gal disaccharide as a ligand for the fimbrial adhesin UcaD

The disaccharide Gal-β-(1→4)-Gal was recently identified as a ligand for the adhesin UcaD, a fimbrial protein used by Proteus mirabilis to adhere to exfoliated uroepithelial cells and colonise the urinary tract. To facilitate further studies, Gal-β-(1→4)-Gal was synthesised as the α-methyl glycoside via glycosylation of methyl 2,3,6-tri-O-benzoyl-α-d-galactopyranoside with 2,3,4,6-tetra-O-acetyl-d-galactopyranosyl trichloroacetimidate, followed by deprotection. The disaccharide was fully characterised by NMR spectroscopy. Earlier attempts to use a thiogalactoside as the glycosyl acceptor were hindered by intermolecular aglycone transfer side reactions.

Protecting group free glycosylation: one-pot stereocontrolled access to 1,2-trans glycosides and (1→6)-linked disaccharides of 2-acetamido sugars.

Unprotected 2-acetamido sugars may be directly converted into their oxazolines using 2-chloro-1,3-dimethylimidazolinium chloride (DMC), and a suitable base, in aqueous solution. Freeze drying and acid catalysed reaction with an alcohol as solvent produces the corresponding 1,2-trans-glycosides in good yield. Alternatively, dissolution in an aprotic solvent system and acidic activation in the presence of an excess of an unprotected glycoside as a glycosyl acceptor, results in the stereoselective formation of the corresponding 1,2-trans linked disaccharides without any protecting group manipulations. Reactions using aryl glycosides as acceptors are completely regioselective, producing only the (1→6)-linked disaccharides.

D-Allose, a rare sugar. Synthesis of D-allopyranosyl acceptors from glucose, and their regioselectivity in glycosidation reactions.

Although D-allose (D-All) is a sugar with low natural abundance, it has great pharmacological and alimentary potential due to its biological properties. However, its chemistry, regarding the regioselectivity in protective reactions and glycosidations, has been scarcely explored. Glycobiological studies require appreciable quantities of carbohydrates with defined structures and high purity. Thus, the development of efficient strategies for their synthesis is crucial. In this frame, the knowledge of the regioselectivity between different hydroxyl groups of glycosyl acceptors is valuable because it allows minimizing the use of protecting groups. We have long been interested in the relative reactivity of OH-3 and OH-4 of glycosyl acceptors in glycosidation reactions. In this paper we synthesized D-allose glycopyranosyl acceptors with free OH-3 and OH-4 from D-Glc precursors. We assessed glycosidations with galactose trichloroacetimidates as donors and the experimental results were compared with those obtained by molecular modeling. Axial O-3 was the preferred site of glycosylation for α-anomers, whereas equatorial O-4 was the preferred site for a β-anomer. A good correlation between the experimental and modeling results was observed using atomic charges and cationic intermediates, although Fukui indices did not predict adequately the experimental results. The achieved regioselectivities are useful for the efficient design of oligosaccharide synthesis containing D-All moieties.

Slow glycosylation: Activation of trichloroacetimidates under mild conditions using lithium salts and the role of counterions

Glycosylations were carried out with the two glycosyl donors 4-O-acetyl-2,3-O-isopropylidene-1-O-trichloroacetimidoyl-α-l-rhamnopyranose and 2,3,4-tri-O-benzyl-1-O-trichloro-acetimidoyl-α-l-rhamnopyranose in combination with the two alcohols 1-adamantanol and l-menthol as model glycosyl acceptors. As catalysts, the five lithium salts LiNTf2, LiI, LiClO4, LiPF6 and LiOTf were investigated. We demonstrated that both lithium and the respective counterions are playing a role in the activation of trichloroacetimidate glycosyl donors at rt. Under these very mild conditions, the glycosylations are slow and completed in two to eight days. Depending on the counterion, the rate and yield of the reaction differs; however, the selectivity of all investigated lithium salts is deficient.

Capture‐Release Strategy Facilitates Rapid Enzymatic Assembly of Oligosaccharides

Comprehensive SummaryA convenient and highly efficient strategy was developed for the simplification of enzymatic synthesis and purification of oligosaccharides. Glycosyl acceptors terminated with a thiol tag were extended by enzymatic modular assembly (EMA) in the aqueous phase, and the desired products were captured during solid‐phase workup (SPW) using commercially available thiopropyl Sepharose 6B resin through a reversible disulfide linkage. Finally, the products were released from solid‐phase resin in the presence of dithiothreitol (DTT) as reducing agent and the resin could be recovered and reused. This strategy overcomes the uncertain influence of polymer‐ or ionic‐supports commonly used in enzymatic glycosylation, and showed perfect compatibility with all 10 enzyme modules for the rapid assembly of a series of complex oligosaccharides.

Chemoenzymatic Synthesis of Sialyl Sulfo-Oligosaccharides as Potent Siglec-8 Ligands via Transglycosylation Catalyzed by Keratanase II.

Sialyl type-II sulfo-oligosaccharides are gaining much attention as bioactive ligands for Siglecs. In this study, we have achieved the first synthesis of sialyl type-II sulfo-oligosaccharides chemoenzymatically by utilizing the transglycosylation activity of keratanase II. The oxazoline derivative of α(2→3)-sialylated 6,6'-di-sulfo-LacNAc (3) was newly designed as the glycosyl donor for enzymatic transglycosylation. Keratanase II efficiently catalyzed the transglycosylation of 3 with two kinds of glycosyl acceptors, 6-sulfo-Lewis X and 6,6'-di-sulfo-LacNAc derivatives, providing sialyl sulfo-hexasaccharide (1) and sialyl sulfo-pentasaccharide (2) with 86 and 95% yields, respectively. The products 1 and 2 showed higher affinity to Siglec-8 with KD 70 and 25 μmol·L-1, respectively, compared to the known ligand of the α(2→3)-sialylated 6,6'-di-sulfo-Lewis X with KD 185 μmol·L-1. Thus, this study will advance not only the study of Siglec-8 biology but also the exploration of functions of sialyl sulfo-oligosaccharides having various microstructures.

Streamlined access to carbohydrate building blocks: Methyl 2,4,6-tri-O-benzyl-α-d-glucopyranoside

Presented herein is an improved synthesis of a common 3-OH glycosyl acceptor. This compound is a building block that is routinely synthesized by many research groups to be used in glycosylation refinement studies. The only known direct synthesis by Koto lacks regioselectivity and relies on chromatography separation using hazardous solvents. Our improved synthetic approach relies on Koto's selective benzylation protocol, but it is followed by acylation-purification-deacylation sequence. Although this approach involves additional manipulations, it provides consistent results and is superior to other indirect strategies. Also obtained, albeit in minor quantities, is 4-OH acceptor, another common building block.

Synthesis of a cyclic tetramer of 3-amino-3-deoxyallose with axially oriented amino groups

A linear tetramer of β-(1 → 6)-linked 3-azido-3-deoxy-d-allose containing glycosyl donor and glycosyl acceptor functions in the terminal monosaccharide units was prepared starting from 3-azido-3-deoxy-1,2:5,6-di-O-isopropylidene-α-d-allofuranose. Cyclization of the linear tetramer under glycosylation conditions afforded the corresponding cyclic tetrasaccharide in 77% yield; its deprotection and reduction of the azido groups resulted in the formation of the cyclic tetramer of 3-amino-3-deoxy-d-allose with axial amino groups, a potential scaffold for the synthesis of tetravalent functional clusters.

Fluorous-Tag-Assisted Synthesis of GAG-Like Oligosaccharides.

The classic, solution-phase synthesis of glycosaminoglycan (GAG) oligosaccharides is hampered by the numerous, time-consuming chromatographic purifications required for the isolation of the glycosylation products after each coupling step between sugar building blocks. Here, we present a detailed experimental procedure for a glycosylation reaction involving a glycosyl acceptor unit that is equipped with a perfluorinated tag. The presence of this fluorous tail allows the quick purification of the desired glycosylation product by performing a simple fluorous solid-phase extraction (F-SPE). The described fluorous-tag-assisted glycosylation strategy greatly facilitates the assembly of building blocks, speeding up the preparation of biologically relevant GAG-like oligomers.