Hydroxynorleucine as a glycosyl acceptor is an efficient means for introducing amino acid functionality into complex carbohydrates

A new approach to the synthesis of biologically relevant glycosyl amino acids using a non-natural amino acid as the glycosyl acceptor is described. The procedure involves a glycosylation reaction of a suitable carbohydrate donor with Fmoc-l-hydroxynorleucine benzyl ester. This reaction results in the direct incorporation of the amino acid moiety. The acceptor can be used for the preparation of α- or β-O-linked glycosides depending upon the nature of the glycosyl donor. This method has been applied in the synthesis of six different tumor-associated carbohydrate antigens.     

Unusual α-glycosylation with galactosyl donors with a C2 ester capable of neighboring group participation

Glycosylation of 4-methoxyphenyl 2,3,6-tri-O-benzoyl-β-d-glucopyranoside (2) with isopropyl 3-O-allyl-2,4,6-tri-O-benzoyl- (9) or 6-O-allyl-2,3,4-tri-O-benzoyl-1-thio-β-d-galactopyranoside (7) as the donor, afforded an α- and β-linked mixture, whereas with isopropyl 3-O-chloroacetyl-2-O-benzoyl-4,6-O-benzylidene- (13) and isopropyl 3-O-allyl-2-O-benzoyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (15) as the donor, glycosylation of 2 gave α-linked products only, indicating that 4,6-O-benzylidenation led to α-stereoselectivity in spite of the C2 ester capable of neighboring group participation. Using 15 as the donor, glycosylation of mannose derivatives with 2- or 3-OH's, glucose with 2- or 3-OH's, galactose with 2-, or 3-, or 4-OH's, glucosamine and glucuronic acid with a 4-OH, and a lactose derivative with a 4-OH, also furnished α-linked products. However, when using 15 as the donor, glycosylation of aglycon alcohol or sugars with 6-OH's yielded normal β-linked products.     

Oligosaccharide Synthesis via Electrophile-Induced Activation of Glycosyl-N-Allylcarbamates 1

Abstract

Glycosyl-N-allyl carbamates, obtained by reaction of anomerically unprotected saccharides with allyl isocyanate, can be activated by an electrophile-induced cyclisation and reacted with glycosyl acceptors to form the corresponding oligosaccharides By this method the mucin core 2 trisaccharide² has successfully been synthesized. Due to the mild glycosylation conditions even 1-O-acetyl protected glycosyl acceptors can be used. This was demonstrated in the synthesis of a 1,6-linked glucosyl trisaccharide whereby a reptitious glycosylation strategy could be applied.

     

Synthesis of an aryl 2-deoxy-β-glycosyl tetrasaccharide to probe retaining endo-glycosidase mechanism

The synthesis of the 1,3–1,4-β-glucanase substrate analogue 4-nitrophenyl O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→3)-2-desoxi-β-d-glucopyranoside 2 is reported. Starting from the main tetrasaccharide obtained by enzymatic depolymerization of barley β-glucan, the synthetic scheme involves preparation of the corresponding 3-O-substituted glycal which was converted into a 2-deoxy-α-glycosyl iodide as a glycosyl donor. The key glycosylation step was successfully achieved by nucleophilic substitution of the iodide donor with 4-nitrophenolate with high β-selectivity.     

Armed-disarmed effect of remote protecting groups on the glycosylation reaction of 2,3-dideoxyglycosyl donors

The armed-disarmed effect of remote protecting groups at the C-4 and/or C-6 position(s) on the glycosylation reactions of 2,3-dideoxyglycosyl donors was investigated. It was found that under various glycosylation conditions, 4- or 6-O-Bn 2,3-dideoxyglycosyl donors were much more reactive than the corresponding 4,6-di-O-Bz 2,3-dideoxyglycosyl donors. Based on these results, an effective and chemoselective glycosylation reaction using 4,6-di-O-Bn glycosyl acetate and 4-OH-6-O-Bz glycosyl acetate was realized, producing a 2,3-dideoxydisaccharide in good yield with high α-stereoselectivity.     

Synthesis of sulfonic acid-containing maltose-type keto-oligosaccharides by an iterative approach

Two different series of (2→5)-α-linked homologous keto-oligosaccharides up to tri- and tetrasaccharide were synthesized by an iterative approach, using 3,4,7-tri-O-benzyl-5-O-(2-naphthyl)methyl-1-deoxy-1-ethoxysulfonyl-α-d-gluco-hept-2-ulopyranosyl chloride as a key building block. An iterative cycle consisted of a glycosylation step followed by selective cleavage of the (2-naphthyl)methyl group.     

Prearranged Glycosides,Part 10. Intramolecular Glycosylation with Cellobiosyl,Lactosyl, and Maltosyl Donors

ABSTRACT

Acetyl protected 1,2-O-(1-methoxyethylidene)-disaccharides 1 of maltose, cellobiose, and lactose, respectively were converted via the corresponding benzyl protected couterparts 2, the benzyl protected phenyl 2-O-acetyl- 3 and 2-O-unprotected 1-thio-glycoside disaccharides 4 into 2-O-succinoylated disaccharides 5. The latter were esterified with benzyl 2-O-benzoyl-4,6-di-O-benzylidene-α-D-glucopyranoside (6) to afford succinyl linked derivatives 7 the benzylidene groups of which were regioselectively opened to give prearranged glycoside trisaccharides 8. Intramolecular glycosylation of the latter with N-iodosuccinimide resulted in exclusive formation of the corresponding α-(1→4)-linked trisaccharides 9. No influence of the donor moiety on the diastereoselectivity of the intramolecular glycosylation was observed.     

A highly stereoselective construction of 1,2-trans-β-glycosidic linkages capitalizing on 2-azido-2-deoxy-d-glycosyl diphenyl phosphates as glycosyl donors

The scope of TMSOTf-promoted glycosidation of 2-azido-2-deoxyglycopyranosyl diphenyl phosphates is investigated. The 3,4,6-tri-O-benzyl-protected glucosyl and galactosyl donors and the 4,6-O-benzylidene-protected galactosyl donor each react with a range of acceptor alcohols in the presence of a stoichiometric amount of TMSOTf in propionitrile at −78 °C to afford 1,2-trans-β-linked disaccharides in high yields with α:β ratios ranging from 9:91 to 1:>99, regardless of the anomeric composition of the donor used. The use of propionitrile as a solvent at −78 °C has proven to be among the best choice for the highest levels of β-selectivity reported to date for this type of glycosidation. A plausible reaction mechanism, which features a large equilibrium preference for α-glycosyl-nitrilium ions over β-nitrilium ions, is proposed based on byproducts formed through their intermediacy and accounts for the observed excellent β-selectivities.     

A facile one-pot synthesis of Nα-Z/Boc-protected S-linked 1,3,4-oxadiazole tethered peptidomimetics

A new class of S-linked 1,3,4-oxadiazole-tethered N^α-protected peptidomimetics is designed and synthesized by a reaction of N^α-Z/Boc-protected 1,3,4-oxadiazole-2-thiol with α-bromo ester derived from amino acid. The protocol has also been employed for the synthesis of glycosylated amino acids and N,N′-orthogonally protected dipeptidomimetics bearing S-linked 1,3,4-oxadiazole mimetics as well. The intermediate 5-alkyl amino-1,3,4-oxadiazole-2-thiols have been isolated as stable compounds. Further, the chain extension at both N- and C-termini of N-protected S-linked 1,3,4-oxadiazole tethered dipeptidomimetics was also demonstrated.     

Synthesis of highly branched oligomannosides

Helferich glycosylation of 1,2-O-[(1-cyano)ethylidene]-β-d-mannopyranose with acetobromomannose results in selective 3,6-bis-glycosylation of the acceptor. The peracetylated trisaccharide cyanoethylidene derivative is an efficient glycosyl donor, which is exemplified by preparation of a branched tetrasaccharide.     

Structural elucidation of O-linked glycopeptides by high energy collision-induced dissociation

O-linked glycopeptides that bear a GalNAc core with and without the presence of sialic acid have been analyzed by high energy collision-induced dissociation (CID). We show that the CID spectra from the glycosylated precursor ions contain sufficient information to identify the peptide sequence and to determine the glycosylated site(s). Asialo O-linked glycopeptides, previously prepared from a tryptic digest of bovine fetuin were studied. One of the glycopeptides contained only a single Hex (hexose)-HexNAc (N-acetylhexosamine) substitution at Thr²⁶², whereas the other exhibited Hex-HexNAc moieties at both Thr²⁶² and Ser²⁶⁴. In addition, sialo and asialo fetuin glycopeptides from a pronase digest were derivatized with t-butoxycarbonyl-tyrosine, and characterized by high energy CID analysis. The presence of a Galβ(1,3)GalNAc core structure at Ser²⁶⁴ was confirmed by using the substrate specificity of endo-α-N-acetylgalactosaminidase. These studies revealed the presence of a β-galactosidase specific for β(1,4) linkages in the endo-α-N-acetylgalactosaminidase preparation employed. Finally, the relative stability of N-and O-glycosyl bonds to high energy CID is addressed based upon comparison of the behavior of a synthetic N-linked glycopeptide with analogous O-linked structures.     

Unusual Formation of a β-linked Disaccharide in a Nis Glycosylation

Abstract

The N-halosuccinimide glycosylation is a highly selective reaction that leads to trans-configured 1-alkoxy-2-halo-glycosides (halo = bromo, iodo).²³ As an exception to the generally observed exclusive formation of α-linked glycosides in such reactions,² we obtained a 3 / 1 mixture of α- and β-disaccharide 6 and 7, when we treated the silylated glycal 4 with NIS (N-iodosuccinimide) and the glycoside 5.⁴ The similarly protected arabino glycal 9, on the other hand, gave exclusively the expected α-linked saccharide 11, when treated with NIS and the alcohol component 10.⁵ Silylated glycals 4 and 9 were obtained from L-digitoxal 1 ⁶ and L-rhamnal 8 ⁷ by treatment with tert-butyldiphenylchlorosilane⁸ and tert-butyldimethylchlorosilane,⁹ respectively. In both cases the 3-O-silylated derivatives formed in high yields. Only in case of the ribo-configuration minor amounts of a 4-O-silylated product 3 were identified.     

Synthesis of tryptophan N-glucoside

The naturally occurring l-tryptophan N-glucoside was synthesized using 2-O-pivaloylated glucosyl trichloroacetimidate, which gave β-N^In-glucosides. From 2-O-acetylated donors only tryptophan-1-yl-ethylidene compounds (amide acetals) were obtained. The employment of α-azido l-tryptophan benzyl ester facilitated purification and deprotection and improved the yields of the glycosylation step.     

Concise assembly of linear α(1→6)-linked octamannan fluorescent probe

Synthesis of a fluorescently labelled (dansylated) linear α(1→6)-linked octamannan, using glycosyl fluoride donors and thioglycosyl acceptors, is described. A selective and convergent two-stage activation progression was executed to construct di-, tetra, and octa-mannosyl thioglycosides in three glycosylation steps with excellent yield. Further, a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) group was coupled to 1-azidoethyl octamannosyl thioglycoside. Global deprotection of the coupled product afforded the desired dansylated homo-linear α(1→6)-linked octamannan.     

Atropdiastereoisomers of ellagitannin model compounds: configuration,conformation, and relative stability of d-glucose diphenoyl derivatives

Conformational analysis reveals a remarkable rigidity of 2,3-, 4,6-, 3,6-, and 2,4-O-(S)- and (R)-diphenoyl (DP) bridged methyl β-d-glucosides, which were used as model compounds to evaluate the atropisomeric features of the natural ellagitannins, which possess at least one hexahydroxydiphenoyl (HHDP) moiety. The 2,3- and 4,6-O-(S)-DP bridged glucosides with ⁴C₁ pyranose geometries are thermodynamically more stable than their (R)-DP counterparts, whilst in the 3,6- and 2,4-O-linked series with ¹C₄ glucopyranose geometries the (R)-DP configuration is preferred. The chiral scaffold of glucose exerts a strong atropdiastereoselective effect onto the diphenoyl units, which is mediated through 10- to 12-membered rings via ester linkages. The calculated results not only explain the observed (S)-diastereoselectivity of di-esterification reactions of suitably protected racemic hexaoxydiphenic acids with 4,6-unsubstituted d-glucopyranose derivatives, but also correlate the observed configuration of axially chiral HHDP-moieties of natural ellagitannins with conformational parameters.     

Rapid and efficient conversion of sialyl thioglycosides to sialyl esters via NIS/BF3OEt2-promoted glycosylation

A rapid and efficient one-step conversion of sialyl thioglycosides to sialyl esters was disclosed. Under the promotion of NIS and BF₃OEt₂, the glycosylation of per-acetylated sialyl thioglycoside with a set of carboxylic acids provided β-sialyl esters as the major products in good to excellent yields within 5 min. Compared with the long-chain alkyl-, aryl- and α,β-unsaturated acids, complete β-selectivities were observed when the short-chain alkyl acids were selected as the coupling partners. The resultant β-selectivity for the glycosylation of the per-acetylated sialyl thioglycoside with acetic acid was compromised when the 5-N,4-O-oxazolidinone protected sialyl thioglycoside was employed as the coupling partner.     

A general method for the synthesis of oligosaccharides consisting of α-(1→2)- and α-(1→3)-linked rhamnan backbones and GlcNAc side chains

A general method has been developed for the synthesis of oligosaccharides consisting of (1→2)- and (1→3)-linked rhamnans with GlcNAc side chains. As examples, highly effective and convergent syntheses of two decasaccharides in the O polysaccharide moiety of the lipopolysaccharide of the phytopathogenic bacterium Pseudomonas syringae pv. ribicola NCPPB 1010 were achieved. The two decasaccharides consist of O polysaccharide repeating units I+II and II+I, respectively. Allyl 3-O-acetyl-4-O-benzoyl-α-l-rhamnopyranoside, allyl 2-O-benzoyl-3-O-chloroacetyl-α-l-rhamnopyranoside, 2,4-di-O-benzoyl-3-O-chloroacetyl-α-l-rhamnopyranosyl trichloroacetimidate, and 3-O-acetyl-2,4-di-O-benzoyl-α-l-rhamnopyranosyl trichloroacetimidate, which were obtained by highly regioselective 3-O-acylations, were used as the key synthons to obtain the required α-(1→2)- and α-(1→3)-linked rhamnoocta saccharide acceptors with 3³- and 3⁷-free hydroxyl groups. Therefore, several disaccharides were synthesized, from which tetrasaccharides and hexasaccharides were then synthesized. Coupling of the hexasaccharide donors with the disaccharide acceptors gave the octasaccharide acceptors. Finally, the coupling of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl trichloroacetimidate with the octasaccharide acceptors, followed by deprotection, afforded the two target decasaccharides. A repeating hexasaccharide unit of the cell wall polysaccharide of β-hemolytic Streptococci Group A was also synthesized in a similar way.     

One-pot preparation and activation of glycosyl trichloroacetimidates: operationally simple glycosylation induced by combined use of solid-supported, reactivity-opposing reagents

By the combined use of solid-supported reactivity-opposing reagents, that is, basic PTBD and acidic Nafion^®-SAC resins, sequential reactions consisting of glycosyl trichloroacetimidate formation and glycosylation can be effected in a one-pot operation starting from 1-O-unprotected sugars. The solid-supported reagents can be introduced into the reaction vessel either sequentially or, more conveniently, in a `one-shot' manner in comparable yields.     

Glucuronidase-assisted Transglycosylation for the Synthesis of Highly Functional Disaccharides: β-D-Glucuronyl 6-O-Sulfo-β-D-Gluco- and -β-D-Galactopyranosides

The substrate specificity of snail (Helix pomatia and Helix aspersa), limpet (Patella vulgata), and bovine glucuronidases was examined by using p-nitrophenyl glucuronide (GlcA-O-pNP) and p-nitrophenyl 6-O-sulfo-β-D-glycopyranosides as the glycosyl donor and acceptors, respectively. When the donor was treated with these enzymes in the absence of the acceptors, β (1 → 3) glucuronyl disaccharides were obtained as the major products together with β (1 → 2) isomers as the result of an enzymatic “self-transglycosylation” reaction. When p-nitrophenyl 6-O-sulfo-β-D-glucopyranosides (6-O-sulfo-Glc-O-pNP and 6-O-sulfo-Glc-S-pNP) were applied as acceptor substrates, every glucuronidase transferred the GlcA residue to either the O-3 or O-2 position in 6-O-sulfo-Glc to yield a mixture of GlcAβ (1 → 3)- and GlcAβ (1 → 2)-linked disaccharides in a ratio of 12:1 ～ 1:1. On the other hand, when p-nitrophenyl 6-O-sulfo-β-D-galactopyranosides (6-O-sulfo-Gal-O-pNP and 6-O-sulfo-Gal-S-pNP) were applied, limpet and bovine glucuronidases gave a GlcAβ (1 → 3)-linked disaccharide regioselectively, while the snail enzymes showed no reactivity.     

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

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