Anomeric Linkage Research Articles

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural β-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and β-anomeric linkages, N-glycosides with β-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

Insightful Improvement in the Design of Potent Uropathogenic E. coli FimH Antagonists

Selective antiadhesion antagonists of Uropathogenic Escherichia coli (UPEC) type-1 Fimbrial adhesin (FimH) are attractive alternatives for antibiotic therapies and prophylaxes against acute or recurrent urinary tract infections (UTIs) caused by UPECs. A rational small library of FimH antagonists based on previously described C-linked allyl α-D-mannopyranoside was synthesized using Heck cross-coupling reaction using a series of iodoaryl derivatives. This work reports two new members of FimH antagonist amongst the above family with sub nanomolar affinity. The resulting hydrophobic aglycones, including constrained alkene and aryl groups, were designed to provide additional favorable binding interactions with the so-called FimH "tyrosine gate". The newly synthesized C-linked glycomimetic antagonists, having a hydrolytically stable anomeric linkage, exhibited improved binding when compared to previously published analogs, as demonstrated by affinity measurement through interactions by FimH lectin. The crystal structure of FimH co-crystallized with one of the nanomolar antagonists revealed the binding mode of this inhibitor into the active site of the tyrosine gate. In addition, selected mannopyranoside constructs neither affected bacterial growth or cell viability nor interfered with antibiotic activity. C-linked mannoside antagonists were effective in decreasing bacterial adhesion to human bladder epithelial cells (HTB-9). Therefore, these molecules constituted additional therapeutic candidates' worth further development in the search for potent anti-adhesive drugs against infections caused by UPEC.

Highly Efficient and Stereoselective Synthesis of 6,7‐Dideoxy‐β‐d‐ido‐octopyranuronates

Abstract6‐Deoxy‐β‐ido‐heptopyranosides are challenging glycosides to synthesize due to their unusual 6‐deoxy‐heptose backbone, rare ido‐configuration and most importantly, the β‐1,2‐cis anomeric linkage. They are found in nature, such as being part of the repeating disaccharide of C. jejuni HS:4 capsular polysaccharides (CPs). Interestingly, the bacterial CPs is found to be partially modified with an O‐methyl phosphoramidate (MeOPN) functionality at the O‐2 or O‐7 position of the 6‐deoxy‐β‐d‐ido‐heptopyranosides. In this work, we report the first synthesis of three analogous of β‐d‐ido‐octopyranosides (1–3) that contain a 6,7‐dideoxy‐functionality and either a terminal methyl ester or carboxylic acid or amide. Since carboxylic acids have been reported as bioisosteres of phosphate group, compound 1–3 can be regarded as carbon‐based bioisosteres of 6‐Deoxy‐β‐ido‐heptopyranoside containing the MeOPN group without a chiral center. Compounds (1–3) were efficiently synthesized from a O‐2 activated β‐d‐galacto‐octopyranuronate (19) that was efficiently converted to the desired β‐d‐ido‐octopyranuronate configuration using an elegant one‐pot process.

Identification of Isomeric N-Glycans by Conformer Distribution Fingerprinting using Ion Mobility Mass Spectrometry.

Glycans possess unparalleled structural complexity arising from chemically similar monosaccharide building blocks, configurations of anomeric linkages and different branching patterns, potentially giving rise to many isomers. This level of complexity is one of the main reasons that identification of exact glycan structures in biological samples still lags behind that of other biomolecules. Here, we introduce a methodology to identify isomeric N‐glycans by determining gas phase conformer distributions (CDs) by measuring arrival time distributions (ATDs) using drift‐tube ion mobility spectrometry‐mass spectrometry. Key to the approach is the use of a range of well‐defined synthetic glycans that made it possible to investigate conformer distributions in the gas phase of isomeric glycans in a systematic manner. In addition, we have computed CD fingerprints by molecular dynamics (MD) simulation, which compared well with experimentally determined CDs. It supports that ATDs resemble conformational populations in the gas phase and offer the prospect that such an approach can contribute to generating a library of CCS distributions (CCSDs) for structure identification.

Minor N-Glycan Mapping of Monoclonal Antibody Therapeutics Using Middle-Down NMR Spectroscopy.

The N-glycosylation pattern of Asn-297 may have impacts on monoclonal antibody (mAb) drug plasma clearance, antibody-dependent cell mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). Notably, the changes in the relative abundance of certain minor glycans, like the afucosylation, high-mannose, or galactosylation are known to change mAb properties and functions. Here, a middle-down NMR spectroscopy based analytical procedure was applied to assess the composition and structure of glycans on adalimumab and trastuzumab without glycan cleavage from the mAbs. The anomeric 2D 1H-13C spectra showed distinct patterns that could be used to profile and differentiate mAb glycan compositions. Specifically, the anomeric C1/H1 resonances from N-acetylglucosamine (GlcNAc2 and -5) and mannose (Man4) were identified as characteristic peaks for key glycan anomeric linkages and branching states. They were also utilized for measuring the relative abundance of minor glycans of total afucosylation (aFuc%), high mannose (HM%), and branch specific galactosylation (Gal1-3% and Gal1-6%). The obtained total aFuc% value of 11-12% was similar between the two mAbs; however, trastuzumab had significantly lower level of high mannose and a higher level of galactosylation than adalimumab. Overall, the 2D-NMR measurements provided functionally relevant mAb glycan composition and structure information. The method was deemed fit-for-purpose for assessment of these mAb quality attributes and involved fewer chemical preparation steps than the classical approaches that cleave glycans prior to making measurements.

Synthesis and Antitumor Activity of C-7-Alkynylated and Arylated Pyrrolotriazine C-Ribonucleosides.

A number of biologically active nucleoside analogues contain the adenine isostere 4-amino-pyrrolo[2,1-f][1,2,4]triazine connected to various sugar moieties through a C-C anomeric linkage. We employed palladium-catalyzed cross-coupling chemistry to promptly functionalize the 7-position of such a heterocyclic scaffold with various alkynyl and aryl groups starting from a common 7-iodo-pyrrolotriazine C-ribonucleoside intermediate. Analogues bearing a 7-cyclopropyl-, 7-propyl-, and 7-butylacetylene moiety displayed potent cytotoxic activity, with the latest being the most selective of this series toward cancer cells. Further insights revealed that such C-nucleosides could exert their antiproliferative action by causing dose-dependent DNA damage.

New Enzymatic Approach to Distinguish Fucosylation Isomers of N-Linked Glycans in Tissues Using MALDI Imaging Mass Spectrometry.

Specific alterations in N-linked glycans, such as core fucosylation, are associated with many cancers and other disease states. Because of the many possible anomeric linkages associated with fucosylated N-glycans, determination of specific anomeric linkages and the site of fucosylation (i.e., core vs outer arm) can be difficult to elucidate. A new MALDI mass spectrometry imaging workflow in formalin-fixed clinical tissues is described using recombinant endoglycosidase F3 (Endo F3), an enzyme with a specific preference for cleaving core-fucosylated N-glycans attached to glycoproteins. In contrast to the broader substrate enzyme peptide-N-glycosidase F (PNGaseF), Endo F3 cleaves between the two core N-acetylglucosamine residues at the protein attachment site. On tissues, this results in a mass shift of 349.137 a.m.u. for core-fucosylated N-glycans when compared to N-glycans released with standard PNGaseF. Endo F3 can be used singly and in combination with PNGaseF digestion of the same tissue sections. Initial results in liver and prostate tissues indicate core-fucosylated glycans associated to specific tissue regions while still demonstrating a diverse mix of core- and outer arm-fucosylated glycans throughout all regions of tissue. By determining these specific linkages while preserving localization, more targeted diagnostic biomarkers for disease states are possible without the need for microdissection or solubilization of the tissue.

Anti-norovirus activity of C7-modified 4-amino-pyrrolo[2,1-f][1,2,4]triazine C-nucleosides

Synthetic nucleoside analogues characterized by a C–C anomeric linkage form a family of promising therapeutics against infectious and malignant diseases. Herein, C-nucleosides comprising structural variations at the sugar and nucleobase moieties were examined for their ability to inhibit both murine and human norovirus RNA-dependent RNA polymerase (RdRp). We have found that the combination of 4-amino-pyrrolo[2,1-f][1,2,4]triazine and its 7-halogenated congeners with either a d-ribose or 2′-C-methyl-d-ribose unit resulted in analogues with good antiviral activity against murine norovirus (MNV), albeit coupled with a significant cytotoxicity. Among this series, 4-aza-7,9-dideazaadenosine notably retained a strong antiviral effect in a human norovirus (HuNoV) replicon assay with an EC50 = 0.015 μM. This study demonstrates that C-nucleosides can be used as viable starting scaffolds for further optimization towards the development of nucleoside-based inhibitors of norovirus replication.

A minimalist approach to stereoselective glycosylation with unprotected donors

Mechanistic study of carbohydrate interactions in biological systems calls for the chemical synthesis of these complex structures. Owing to the specific stereo-configuration at each anomeric linkage and diversity in branching, significant breakthroughs in recent years have focused on either stereoselective glycosylation methods or facile assembly of glycan chains. Here, we introduce the unification approach that offers both stereoselective glycosidic bond formation and removal of protection/deprotection steps required for further elongation. Using dialkylboryl triflate as an in situ masking reagent, a wide array of glycosyl donors carrying one to three unprotected hydroxyl groups reacts with various glycosyl acceptors to furnish the desired products with good control over regioselectivity and stereoselectivity. This approach demonstrates the feasibility of straightforward access to important structural scaffolds for complex glycoconjugate synthesis.

Regulation of Notch signaling by O‐glucosylation: Notch‐modifying xylosyltransferase‐substrate complexes support an SNi‐like retaining mechanism

A major question remaining in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xyloside α‐1,3‐xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi‐like reaction as the retaining mechanism for XXYLT1. Unexpectedly, the epidermal growth factor–like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Importantly, XXYLT1 is amplified in a number of squamous cell carcinomas and may be a target for therapeutics. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations in which Notch dysregulation is known to cause cancer or developmental disorders.Support or Funding InformationThe work is supported by NIH grants GM061126 (to R.S.H) and AG029979 (to H.L.), SBU‐BNL Seed Grant (to R.S.H. and H.L.), DFG grant BA4091/5‐1 (to H.B.) and NSF grant DMR 1404985 (to E.L.).

The influence of the carbohydrate anomeric linkage on the free radical scavenging activity of enzymatically-synthesized phenolic glycosides

Phenolic glycosides, widely recognized for their nutraceutical properties exist in nature as α- and β-glycosides.

Structural Insights into the Binding of Sugar Receptors (Lectins) to a Synthetic Tricyclic Tn Mimetic and Its Glycopeptide Version

AbstractCycloadduct 1 is a conformationally constrained mimetic of the tumor‐associated Tn antigen. It maintains the 4C1 conformation and the α‐O‐glycosidic linkage of the natural epitopes. Due to its tricyclic structure, the anomeric linkage is constrained and its conformation “frozen”. Using saturation transfer difference NMR spectroscopy experiments, epitope mapping for binding by three lectins [i.e., from Erythrina cristagalli (ECL), human macrophages (MGL), and from Helix pomatia (HPA)] was carried out. Striking differences in the epitope viewed from the ligand's perspective were revealed by this comparison. Evidently, the structural change around the C‐2 position has a major impact, if the contact pattern to the ligand is not mostly restricted to galacto configuration with the axial hydroxyl group at C‐4, in combination with C‐3/C‐6. These measurements thus provide insights into actual contacts, which help predict applicability as an inhibitor for distinct lectins, and as an elicitor of specific antibodies.

Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism

A major remaining question in glycobiology is how a glycosyltransferase (GT) that retains the anomeric linkage of a sugar catalyzes the reaction. Xylosideα1–3 Xylosyltransferase (XXYLT1) is a retaining GT that regulates Notch receptor activation by adding xylose to the Notch extracellular domain. Here, using natural acceptor and donor substrates and active Mus musculus XXYLT1, we report a series of crystallographic snapshots along the reaction, including an unprecedented natural and competent Michaelis reaction complex for retaining enzymes. These structures strongly support the SNi-like reaction as the retaining mechanism for XXYLT1. Unexpectedly the Epidermal Growth Factor-like repeat acceptor substrate undergoes a large conformational change upon binding to the active site, providing a structural basis for substrate specificity. Our improved understanding of this retaining enzyme will accelerate the design of retaining GT inhibitors that can modulate Notch activity in pathological situations where dysregulation of Notch is known to cause cancer or developmental disorders.

The search for new amphiphiles: synthesis of a modular, high-throughput library.

Amphiphilic compounds are used in a variety of applications due to their lyotropic liquid-crystalline phase formation, however only a limited number of compounds, in a potentially limitless field, are currently in use. A library of organic amphiphilic compounds was synthesised consisting of glucose, galactose, lactose, xylose and mannose head groups and double and triple-chain hydrophobic tails. A modular, high-throughput approach was developed, whereby head and tail components were conjugated using the copper-catalysed azide–alkyne cycloaddition (CuAAC) reaction. The tails were synthesised from two core alkyne-tethered intermediates, which were subsequently functionalised with hydrocarbon chains varying in length and degree of unsaturation and branching, while the five sugar head groups were selected with ranging substitution patterns and anomeric linkages. A library of 80 amphiphiles was subsequently produced, using a 24-vial array, with the majority formed in very good to excellent yields. A preliminary assessment of the liquid-crystalline phase behaviour is also presented.

Mushroom Dietary Fiber from the Fruiting Body of Pleurotus tuber-regium: Fractionation and Structural Elucidation of Nondigestible Cell Wall Components

The cell wall of mushroom fruiting body is constituted of nondigestible macromolecules that are a rich source of dietary fiber with biological functions that are beneficial to human health. The cell wall components of an edible mushroom fruiting body from Pleurotus tuber-regium (PTR) were fractionated, and their chemical structures were investigated by chemical, physicochemical, and microscopic analyses. The present results suggest that the cell wall of the PTR mushroom fruiting body contains four main fractions: an outer fraction of polysaccharide and protein complex, which can be extracted using boiling water; a cold alkali-soluble fraction of heteropolysaccharides associated with a small amount of proteins; a hot alkali-soluble fraction of hyper-branched glucans; and an alkali-insoluble fraction of glucan-chitin complex with a normalized relative percentage of 3.6:21.9:55.7:18.8. The anomeric linkage of all the glucans was revealed by infrared spectroscopy to be β type. The structure of the major mushroom fruiting body cell wall polysaccharide (the hot alkali-soluble one, FHA-I) was elucidated by the methylation analysis to be composed of →1)-Glcp-(4→ linkages as the backbone with a 52% degree of branching consisting of →1)-Glcp-(6→ linkages in the side chains, whereas some →1)-Glcp-(3→ linkages might exist in the backbone or side chains. Size exclusion chromatography coupled with multiangle laser light scattering analysis revealed that FHA-I had a molecular weight of 4.224 × 10(6) g/mol and a root-mean-square radius of 30.4 nm. Both scanning electron and atomic force microscopy further showed the highly branched microstructure of FHA-I when dispersed in an aqueous sodium dodecyl sulfate solution.

Serogroup-Specific Interaction of Neisseria meningitidis Capsular Polysaccharide with Host Cell Microtubules and Effects on Tubulin Polymerization

We have previously shown that during late stages of the infectious process, serogroup B meningococci (MenB) are able to escape the phagosome of in vitro-infected human epithelial cells. They then multiply in the cytosolic environment and spread intracellularly and to surrounding cells by exploiting the microtubule cytoskeleton, as suggested by results of infections in the presence of microtubule inhibitors and evidence of nanotubes connecting neighboring cells. In this study, by using microtubule binding assays with purified microtubule asters and bundles and microtubule bundles synthesized in vitro, we demonstrate that the MenB capsule directly mediates the interaction between bacteria and microtubules. The direct interaction between the microtubules and the MenB capsular polysaccharide was confirmed by coimmunoprecipitation experiments. Unexpectedly, serogroup C meningococci (MenC), which have a capsular polysaccharide that differs from that of MenB only by its anomeric linkage, α(2→9) instead of α(2→8), were not able to interact with the microtubules, and the lack of interaction was not due to capsular polysaccharide O-acetylation that takes place in most MenC strains but not in MenB strains. Moreover, we demonstrate that the MenB capsular polysaccharide inhibits tubulin polymerization in vitro. Thus, at variance with MenC, MenB may interfere with microtubule dynamics during cell infection.

Structure–Activity Relationships of Glucosamine‐Derived Glycerolipids: the Role of the Anomeric Linkage, the Cationic Charge and the Glycero Moiety on the Antitumor Activity

The potent antitumor activity of 1-O-hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-β-D-glucopyranosyl)-sn-glycerol (1) was previously shown to arise through an apoptosis-independent pathway. Here, a systematic structure-activity study in which the effects of the anomeric linkage, the cationic charge and the glycero moiety on the antitumor activity is described. Eight analogues of 1 were synthesized, and their antitumor activity against breast (JIMT1 and BT549), pancreas (MiaPaCa2) and prostate (DU145, PC3) cancer was determined. 1-O-Hexadecyl-2-O-methyl-3-O-(2'-amino-2'-deoxy-α-D-glucopyranosyl)-sn-glycerol (2) consistently displayed the most potent activity against all five cell lines with CC(50) values in the range of 6-10 μM. However, replacement of the O-glycosidic linkage by a thioglycosidic linkage or replacement of the amino group by an azide or guanidino group leads to a threefold or greater decrease in potency. The glycero moiety also contributes to the overall activity of 1 and 2 but its effects are of lesser importance. Investigation into the mode of action of this class of compounds revealed that, in agreement with previous findings, the cytotoxic effects arise through induction of large acid vacuoles.

Synthesis of Carba-NAD and the Structures of Its Ternary Complexes with SIRT3 and SIRT5

Carba-NAD is a synthetic compound identical to NAD except for one substitution, where an oxygen atom adjacent to the anomeric linkage bearing nicotinamide is replaced with a methylene group. Because it is inert in nicotinamide displacement reactions, carba-NAD is an unreactive substrate analogue for NAD-consuming enzymes. SIRT3 and SIRT5 are NAD-consuming enzymes that are potential therapeutic targets for the treatment of metabolic diseases and cancers. We report an improved carba-NAD synthesis, including a pyrophosphate coupling method that proceeds in approximately 60% yield. We also disclose the X-ray crystal structures of the ternary complexes of SIRT3 and SIRT5 bound to a peptide substrate and carba-NAD. These X-ray crystal structures provide critical snapshots of the mechanism by which human sirtuins function as protein deacylation catalysts.

Sulfated Hexasaccharides Attenuate Metastasis by Inhibition of P-selectin and Heparanase

Development of compounds that target both heparanase and selectins is emerging as a promising approach for cancer therapy. Selectins are vascular cell adhesion molecules that mediate tumor cell interactions with platelets, leukocytes, and the vascular endothelium. Heparanase is an endoglycosidase that degrades heparan sulfate in the tumor microenvironment, cell surfaces, and vessel wall. Acting together, these molecules facilitate tumor cell arrest, extravasation, and metastasis. Here, we report the preparation of novel semisynthetic sulfated tri mannose C-C-linked dimers (STMCs) endowed with heparanase and selectin inhibitory activity. The P-selectin specificity of the STMC was defined by the anomeric linkage of the C-C bond. This STMC hexasaccharide is an effective inhibitor of P-selectin in vivo. We show that selective inhibition of heparanase attenuates metastasis in B16-BL6 melanoma cells, expressing high levels of this endoglycosidase, but has no effect on the metastasis of MC-38 carcinoma cells that express little or no heparanase activity. P-selectin-specific STMC attenuated metastasis in both animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. Thus, the small size, the stability of the C-C bond, and the chemically defined structure of the newly generated STMCs make them superior to heparin derivatives and signify STMCs as valuable candidates for further evaluation.

A unique approach to the synthesis of a dengue vaccine and the novel tetrasaccharide that results

An approach to the development of a dengue vaccine by synthesizing the hexasaccharide epitope on the viral surface is examined. The stereochemical and structural challenges include the synthesis of a β-mannoside bond. Synthesis of this bond is approached via a trisaccharide analogue portion of the epitope. A novel tetrasaccharide with a mannose–mannose anomeric linkage results in the course of the synthetic attempts.