One‐Pot Synthesis of N‐Acetyl‐ and N‐Glycolylneuraminic Acid Capped Trisaccharides and Evaluation of Their Influenza A(H1 N1) Inhibition

Human lung epithelial cells natively offer terminal N‐acetylneuraminic acid (Neu5Ac) α(2→6)‐linked to galactose (Gal) as binding sites for influenza virus hemagglutinin. N‐Glycolylneuraminic acid (Neu5Gc) in place of Neu5Ac is known to affect hemagglutinin binding in other species. Not normally generated by humans, Neu5Gc may find its way to human cells from dietary sources. To compare their influence in influenza virus infection, six trisaccharides with Neu5Ac or Neu5Gc α(2→6) linked to Gal and with different reducing end sugar units were prepared using one‐pot assembly and divergent transformation. The sugar assembly made use of an N‐phthaloyl‐protected sialyl imidate for chemoselective activation and α‐stereoselective coupling with a thiogalactoside. Assessment of cytopathic effect showed that the Neu5Gc‐capped trisaccharides inhibited the viral infection better than their Neu5Ac counterparts.     

A Water Soluble Pd2L4 Cage for Selective Binding of Neu5Ac

The sialic acid N-acetylneuraminic acid (Neu5Ac) and its derivatives are involved in many biological processes including cell-cell recognition and infection by influenza. Molecules that can recognize Neu5Ac might thus be exploited to intervene in or monitor such events. A key obstacle in this development is the sparse availability of easily prepared molecules that bind to this carbohydrate in its natural solvent; water. Here, we report that the carbohydrate binding pocket of an organic soluble [Pd₂L₄]⁴⁺ cage could be equipped with guanidinium-terminating dendrons to give the water soluble [Pd₂L₄][NO₃]₁₆ cage 7 . It was shown by means of NMR spectroscopy that 7 binds selectively to anionic monosaccharides and strongest to Neu5Ac with K_a=24 M⁻¹. The cage had low to no affinity for the thirteen neutral saccharides studied. Aided by molecular modeling, the selectivity for anionic carbohydrates such as Neu5Ac could be rationalized by the presence of charge assisted hydrogen bonds and/or the presence of a salt bridge with a guanidinium solubilizing arm of 7 . Establishing that a simple coordination cage such as 7 can already selectively bind to Neu5Ac in water paves the way to improve the stability, affinity and/or selectivity properties of M₂L₄ cages for carbohydrates and other small molecules.     

Molecular recognition of N-acetylneuraminic acid with acyclic benzimidazolium- and aminopyridine/guanidinium-based receptors

Acyclic receptors incorporating neutral and cationic recognition sites show effective binding of N-acetylneuraminic acid (Neu5Ac), the most naturally abundant sialic acid, in highly competitive solvents such as dimethyl sulfoxide (DMSO) and water/DMSO. Receptors 6b and 7b are able to form neutral/charge-reinforced hydrogen bonds and ion pairs with Neu5Ac, similar to sialic acid-binding proteins. Syntheses and binding properties of the artificial receptors are discussed.     

Characterization of derivatization of sialic acid with 2-aminoacridone and determination of sialic acid content in glycoproteins by capillary electrophoresis and high performance liquid chromatography--ion trap mass spectrometry.

A simple and highly sensitive capillary electrophoresis (CE) method for determining the content of N-acetylneuraminic acid (Neu5Ac) in glycoproteins was developed. Neu5Ac was derivatized with 2-aminoacridone (AMAC) by reductive amination, and the AMAC-Neu5Ac adduct could be readily separated from the other 11 AMAC-derivatized neutral and acidic monosaccharides usually present in glycoproteins by CE in a 0.3 mol/L borate buffer, pH 10.5, and detected at 260 nm. The derivatization of Neu5Ac was achieved at 55 degrees C for 4 h. AMAC-Neu5Ac was stable at 20 degrees C in the dark for at least 12 h while at room temperature it spontaneously converted into another substance with a lower electrophoretic mobility, which was identified as decarboxylated AMAC-Neu5Ac by high performance liquid chromatography - ion trap mass spectrometry (HPLC-ITMS). Concentration and mass of Neu5Ac as low as 1 micromol/L and 35 fmol could be detected. The linear correlation coefficient between the ratio of peak area to migration time of AMAC-Neu5Ac and the concentration of Neu5Ac ranging from 10 to 120 micromol/L was 0.9978 (n=8). This method was successfully applied to the analysis of sialic acid in human urinary trypsin inhibitor (hu-UTI), bovine alpha1-acid glycoprotein (alpha1-AGP) and recombinant human erythropoietin (rhu-EPO). By combination of CE and HPLC-ITMS we found that N-glycolylneuraminic acid (Neu5Gc) was present in bovine alpha1-AGP in addition to Neu5Ac, with a quantity comparable to that of the latter.     

Preferential Lectin Binding of Cancer Cells upon Sialic Acid Treatment Under Nutrient Deprivation

The terminal monosaccharide of glycoconjugates on a eukaryotic cell surface is typically a sialic acid (Neu5Ac). Increased sialylation usually indicates progression and poor prognosis of most carcinomas. Here, we utilize two human mammary epithelial cell lines, HB4A (breast normal cells) and T47D (breast cancer cells), as a model system to demonstrate differential surface glycans when treated with sialic acid under nutrient deprivation. Under a starved condition, sialic acid treatment of both cells resulted in increased activities of α2→3/6 sialyltransferases as demonstrated by solid phase assay using lectin binding. However, a very strong Maackia amurensis agglutinin I (MAL-I) staining on the membrane of sialic acid-treated T47D cells was observed, indicating an increase of Neu5Acα2→3Gal on the cell surface. To our knowledge, this is a first report showing the utility of lectins, particularly MAL-I, as a means to discriminate between normal and cancer cells after sialic acid treatment under nutrient deprivation. This method is sensitive and allows selective detection of glycan sialylation on a cancer cell surface.     

Total Biosynthesis of Legionaminic Acid,a Bacterial Sialic Acid Analogue

Legionaminic acid, Leg5,7Ac₂, a nonulosonic acid like 5‐acetamido neuraminic acid (Neu5Ac, sialic acid), is found in cell surface glycoconjugates of bacteria including the pathogens Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. The presence of Leg5,7Ac₂ has been correlated with virulence in humans by mechanisms that likely involve subversion of the host's immune system or interactions with host cell surfaces due to its similarity to Neu5Ac. Investigation into its role in bacterial physiology and pathogenicity is limited as there are no effective sources of it. Herein, we construct a de novo Leg5,7Ac₂ biosynthetic pathway by combining multiple metabolic modules from three different microbial sources (Saccharomyces cerevisiae, C. jejuni, and L. pneumophila). Over‐expression of this de novo pathway in Escherichia coli that has been engineered to lack two native catabolic pathways, enables significant quantities of Leg5,7Ac₂ (≈120 mg L^?1 of culture broth) to be produced. Pure Leg5,7Ac₂ could be isolated and converted into CMP‐activated sugar for biochemical applications and a phenyl thioglycoside for chemical synthesis applications. This first total biosynthesis provides an essential source of Leg5,7Ac₂ enabling study of its role in prokaryotic and eukaryotic glycobiology.     

Communication: Synthetic Studies on Sialoglycoconjugates 25: Reactivity of Glycosyl Promoters in α-Glycosylation of N-Acetyl-Neuraminic Acid with the Primary and Secondary Hydroxyl Groups in the Suitably Protected Galactose and Lactose Derivatives

Development of an efficient α-glycoside synthesis of sialic acids is critically significant for the syntheses of sialoglycoconjugates, especially gangliosides which carry important biological functions¹ in biological systems. Previously, we demonstrated² a new α-glycosylation of sialic acids by use of dimethyl(methylthio)sulfonium triflate (DMTST)³ as the glycosyl promoter, the suitably protected glycosyl acceptors and the methyl 2-thioglycoside 1 of N-acetylneuraminic acid (Neu5Ac) as the donor in acetonitrile under kinetically controlled conditions, and accomplished⁴ the syntheses of a variety of gangliosides and their analogs.     

Total Biosynthesis of Legionaminic Acid,a Bacterial Sialic Acid Analogue

Legionaminic acid, Leg5,7Ac₂, a nonulosonic acid like 5‐acetamido neuraminic acid (Neu5Ac, sialic acid), is found in cell surface glycoconjugates of bacteria including the pathogens Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. The presence of Leg5,7Ac₂ has been correlated with virulence in humans by mechanisms that likely involve subversion of the host's immune system or interactions with host cell surfaces due to its similarity to Neu5Ac. Investigation into its role in bacterial physiology and pathogenicity is limited as there are no effective sources of it. Herein, we construct a de novo Leg5,7Ac₂ biosynthetic pathway by combining multiple metabolic modules from three different microbial sources (Saccharomyces cerevisiae, C. jejuni, and L. pneumophila). Over‐expression of this de novo pathway in Escherichia coli that has been engineered to lack two native catabolic pathways, enables significant quantities of Leg5,7Ac₂ (≈120 mg L⁻¹ of culture broth) to be produced. Pure Leg5,7Ac₂ could be isolated and converted into CMP‐activated sugar for biochemical applications and a phenyl thioglycoside for chemical synthesis applications. This first total biosynthesis provides an essential source of Leg5,7Ac₂ enabling study of its role in prokaryotic and eukaryotic glycobiology.     

Quantitation of cytidine-5′-monophospho-N-acetylneuraminic acid in human leukocytes using LC–MS/MS: method development and validation

The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography–tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol–water mixture and the leukocyte extract was used for LC–MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.     

Monomeric inhibitors of influenza neuraminidase enhance the hemagglutination inhibition activities of polyacrylamides presenting multiple C-sialoside groups

Background: Influenza viruses use hemagglutinin (HA) arrays to bind to sialic acid moieties on the surface of cells; crosslinking of erythrocytes by this mechanism leads to hemagglutination. A number of synthetic polymers containing multiple sialic acid (Neu5Ac) groups as side chains are potent inhibitors of this process. Inhibition may be due to two mechanisms: polyvalent binding of the inhibitor's multiple Neu5Ac side chains to multiple HA sites on the viral surface, or steric stabilization of the viral particle by a layer of the adsorbed, water-swollen polymer, which prevents adhesion to the erythrocyte. The balance between these two effects is not yet known.Results: Polyacrylamides with multiple C-sialosides (PA(Neu5Ac)) were 2–20 fold more effective as inhibitors of virally mediated hemagglutination when assayed in the presence of Neu2en-NH₂, a potent monomeric inhibitor of influenza neuraminidase (NA). The ability of monomeric inhibitors of NA to enhance the inhibition of hemagglutination in this assay correlated with the affinity of the monomer for NA.Conclusions: We propose that inhibitors of NA act by competing with the C-sialosides of PA(Neu5Ac) for binding to the active sites of the NA. Competitive displacement of Neu5Ac causes an expansion of the layer of polymeric gel adsorbed to the virus, enhancing its inhibitory effect. This study provides an example of synergy between two ligands directed toward the active sites of two different proteins, and reinforces the conclusion that steric stabilization is important for the activity of polyvalent inhibitors.     

Synthesis of α- and β-Methyl NEU5Ac α-(2→8) NEU5Ac Disaccharides

ABSTRACT

Acid hydrolysis of colominic acid, an α-(2→8)-linked oligomer of sialic acid, yielded Neu5Ac α-(2→8) Neu5Ac (di-Neu5Ac) 2 as one of the products. Starting from this disaccharide, it was possible to prepare two potential di-Neu5Ac donors, 5 and 8, as their corresponding 2-chloro derivatives. Subsequent reaction of the donor 8 with methanol as a simple acceptor led to the α- and β-methyl Neu5Ac α-(2→8) Neu5Ac glycosides.     

Glycosylation Using Methylthioglycosides of N-Acetylneuraminic Acid and Dimethyl(Methylthio)Sulfonium Triflate

Abstract

Recently, great interest has been focussed on the synthesis of oligosaccharides containing N-acetylneuraminic acid (Neu5Ac) because of its important roles in a variety of biological recognitions.² However, many difficulties in the synthesis of naturally occurring α-glycosides still remained.³ We have recently reported the stereo-selective synthesis of a series of α- and β-2-thio-neuraminyl glycosides.^1-4

In the meantime, the utility of thioglycosides in oligosaccharide synthesis has been widely developed.⁵ Particularly noteworthy is the dimethyl(methylthio)sulfonium triflate (DMTST) promoted glyco-sylation method^5k.1 because excellent yields are achieved due to the high thiophilicity of this reagent.     

A new synthesis of N-acetylneuraminic acid

A new synthesis of N-acetylneuraminic acid (Neu5Ac; 28 ) via aldehyde 10 is described. The aldehyde 10 was obtained from N, acetyl-D -glucosamine ( 11 ; 5 steps, overall yield ca. 6%) or from D-glucono-1,5-lactone ( 17 ; 6 steps, overll yield ca 57%). Thus, on the one hand, N-acetyl-D -mannosamine ( 12 ), obtained from 11 , was transformed into the known dithioacetal 14 and hence into the (ethylthio)dihydrooxazole 16 , which was cleaved under weakly acidic conditions to the aldehyde 10 . On the other hand, the known ester 18 , obtained from 17 , was sulfonylated and further transformed via the azide 20 into the N-acetyl-D -mannonate 22 . Reduction of 22 to 23 and oxidation of 23 with ‘periodinane’ again gave 10 . The aldehyde 10 was treated with the organozinc reagent 8 obtained from tert-butyl 2-(bromomethyl)acrylate ( 2 ) to yield predominantly 24 , which was transformed (two steps) into the 2-methylidene-D -glycero-nononic acid 27 and hence into Neu5Ac (28).     

Conformational Study of α‐N‐Acetyl‐D‐Neuraminic Acid by Density Functional Theory

The stable structures of α‐N‐acetyl‐D‐neuraminic acid (Neu5Acα) in the gas phase were studied at the B3LYP level of theory using 6‐31G(d,p) and 6‐31++G(d,p) basis sets. They are classified into five types according to the patterns of the intramolecular hydrogen bond formations. One of the stable structures had intramolecular hydrogen bond network of O₉H_O9 … O₈H_O8 … O?C₁‐O₁H_O1 and O₇H_O7…O?CHN‐C₅ similar to the crystal structure of Neu5Ac‐α‐methyl glycoside methyl ester. The stable structures of Neu5Acα are reasonable for the following sialooligosaccharide ligand studies with respect to the relationship between OH group orientations and intramolecular hydrogen bond formations. The barrier heights for isomerizations between the stable structures were computed to be 2.8 to 6.7 kcal/mol at the B3LYP/6‐31++G(d,p)//B3LYP/6‐31G(d,p) level, which are basic factors for the conformational behavior of Neu5Acα before its interactions with receptors. We also calculated Neu5Acα–4 or 5‐water complexes to take account of the solvent effect on the intramolecular hydrogen bonds in the stable structures. Consequently, the structures of Neu5Acα in the complexes are similar to each other, which is consistent with the known NMR data. Thus, the optimum Neu5Acα‐water complexes are some of the reasonable pseudohydrous Neu5Acα.     

Anomalous binding profile of phenylboronic acid with N-acetylneuraminic acid (Neu5Ac) in aqueous solution with varying pH

Borates are known to interact with carbohydrate moieties expressed on the surface of biological membranes of a variety of cells, viruses, bacteria, and fungi. This study revealed the anomalous binding profile of borate in aqueous solution with N-acetylneuraminic acid (Neu5Ac, sialic acid) as a potential receptor site on the surfaces of biological membranes using (11)B, (1)H, (13)C, and (15)N nuclear magnetic resonance spectroscopies. 3-(Propionamido)phenylboronic acid (PAPBA) was chosen as the model borate compound. The equilibrium constant (K) for Neu5Ac binding to PAPBA was compared with those for glucose, mannose, and galactose, which are the major carbohydrate constituents of glycoproteins and glycolipids expressed on biological membranes. In the Neu5Ac/PAPBA system, the unusual pH dependency of the K values, a decrease in K with increasing pH, was observed, suggesting the formation of a trigonal-formed complex stabilized by the coordination of an amide group of Neu5Ac at the C-5 position to the boron atom, forming intramolecular B-N or B-O bonding. Furthermore, the anomalously high complexing ability at physiological pH 7.4 was confirmed for this system, with the K value 37.6 which is approximately 7 times higher than that for glucose. This exceptionally high value of K at physiological pH, compared to those of other sugars, strongly suggests that the boronic acid selectively recognizes the Neu5Ac residues of the glycosylated components including glycoproteins and gangliosides existing on the surface of the biological membranes.     

A capillary zone electrophoresis method for determining N-acetylneuraminic acid in glycoproteins and blood sera

A simple capillary zone electrophoresis (CZE) method for the determination of the content of the major sialic acid form N-acetylneuraminic acid (Neu5Ac) in glycoproteins was established. The present method utilizes a simplified hydrolysis-purification procedure consisting of mild acid hydrolysis (25 mM trifluoroacetic acid for 2h at 80 degrees C) to release Neu5Ac and ultrafiltration on Centricon-3 membrane to remove the obtained asialoglycoproteins and other macromolecules present in biologic samples. Derivatization with benzoic anhydride at 80 degrees C for 20 min resulted in complete conversion of Neu5Ac to per-O-benzoylated Neu5Ac. CZE analysis was performed using the operating buffer 25mM phosphate, pH 3.5, containing 50% (v/v) acetonitrile as organic modifier at 30 kV, and detection of the per-O-benzoylated Neu5Ac at 231 nm. The method showed excellent repeatability (RDS<1.98%) and a linearity range from 5 microg/mL to 5mg/mL with a detection limit of 2 microM. Application of the method to microanalysis of human alpha(1)-acid glycoprotein and blood serum samples showed excellent agreement with previously published values, suggesting a high precision for the developed CZE method.     

Synthesis of the 6-C-Methyl and 6-C-(Hydroxymethyl) Analogues of N-Acetylneuraminic Acid and of N-Acetyl-2,3-didehydro-2-deoxyneuraminic Acid

The synthesis of 6-C-methyl-Neu2en5Ac ( 4 ), 6-C-(hydroxymethyl)-Neu2en5Ac ( 5 ), and 6-C-methyl-Neu5Ac ( 6 ) is described. The 4-methylumbellyferyl glycosides 8 and 9 were also prepared but proved unstable. Protection of the previously reported nitro ether 10 (→ 11 ) followed by a Kornblum reaction gave the branched-chain derivative 13 which was transformed into aldehyde 14 and hence via 16 into the-protected 6-C-hydroxymethylated 20 and into the 6-C-methyl-substituted 18 (Scheme 1). Debenzylidenation of 20 and 18 afforded the diols 21 and 19 , respectively. Selective oxydation of 19 followed by esterification (→ 22 ), acetylation (→ 23 ), and elimination led to the protected 6-C-methyl-Neu2en5Ac derivative 24 (Scheme 2). Bromomethoxylation yielded mainly 25 and some 26 , which were reductively debrominated to 27 and 28 , respectively. Attempted deprotection of 27 did not lead to the corresponding acid, but to the 2,7- and 2,8-anhydro compounds 29 and 30 which were characterised as their peracetylated esters 31 and 32 (Scheme 3). The structure of 32 was established by X-ray analysis. Oxydation of 19 and 21 , followed by deprotection, esterification, and acetylation gave 37 and 38 , respectively (Scheme 4). The branched-chain Neu2en5Ac derivatives 4 and 5 were obtained by β-elimination (→ 39 and 40 ) and deprotection. Omission of the esterification after oxydation of 33 and 34 gave the lactones 35 and 36 which were transformed into 37 and 38 , respectively. Bromoacetoxylation of 39 gave 41-43 which were reductively debrominated to 44 (from 41 and 42 ) and 45 (Scheme 5). Bromoacetoxylation of 40 yielded 46 which was debrominated to 47. Glycosidation of the glycosyl chlorides obtained from 44 and 47 led to the α -D-glycosides 48 and 49 and to the elimination products 39 and 40 , respectively (Scheme 6). Transesterification of 48 , followed by saponification gave the unstable glycoside 8 and hence 6-C-methyl-Neu5Ac ( 6 ). The unstable glycoside 9 was obtained by similar treatment of 49 but yielded 50 under acidic conditions. The branched-chain 4 and 5 were weak inhibitors of Vibrio cholera sialidase, and 8 and 9 were very poor substrates.     

Chemical Remodeling of Cell‐Surface Sialic Acids through a Palladium‐Triggered Bioorthogonal Elimination Reaction

We herein report a chemical decaging strategy for the in situ generation of neuramic acid (Neu), a unique type of sialic acid, on live cells by the use of a palladium‐mediated bioorthogonal elimination reaction. Palladium nanoparticles (Pd NPs) were found to be a highly efficient and biocompatible depropargylation catalyst for the direct conversion of metabolically incorporated N‐(propargyloxycarbonyl)neuramic acid (Neu5Proc) into Neu on cell‐surface glycans. This conversion chemically mimics the enzymatic de‐N‐acetylation of N‐acetylneuramic acid (Neu5Ac), a proposed mechanism for the natural occurrence of Neu on cell‐surface glycans. The bioorthogonal elimination was also exploited for the manipulation of cell‐surface charge by unmasking the free amine at C5 to neutralize the negatively charged carboxyl group at C1 of sialic acids.     

A mechanistic study of sialic acid mutarotation: implications for mutarotase enzymes

The mutarotation of N-acetylneuraminic acid (Neu5Ac) proceeds by four kinetically distinct pathways: (i) the acid-catalyzed reaction of neutral Neu5Ac; (ii) the spontaneous reaction of the carboxylic acid (the kinetically equivalent acid-catalyzed reaction on the anion being ruled out by the solvent deuterium kinetic isotope effect of 3.74 ± 0.68); (iii) a spontaneous, water-catalyzed, reaction of the anion; and (iv) a specific-base catalyzed reaction of the anion. The magnitude of the solvent kinetic isotope effect, k(H2O)/k(D2O) = 4.48 ± 0.74 is consistent with a ring-opening transition state in which a water molecule is deprotonating the anomeric hydroxyl group in concert with strengthening solvation of the ring oxygen atom. The mechanistic implications for Neu5Ac mutarotases are discussed.     

Molecular recognition of sialic acid end groups by phenylboronates

A multinuclear NMR study of the interaction between phenylboronic acid (PBA) and sialic acid (Neu5 Ac) has been performed. The latter compound is known to be overexpressed on the cell surface of tumor cells. The results of this investigation suggest that the binding of PBA to sialic acid is pH dependent. 17O NMR experiments with glycolic acid as the model compound prove that an interaction at the alpha-hydroxycarboxylate occurs at pH < 9, while a study with threonic and erythronic acids shows that the PBA group interacts selectively with the vicinal diol functions at higher pH. Similarly, Neu5 Ac binds PBA through its alpha-hydroxycarboxylate at low pH (< 9) and through its glycerol side chain at higher pH values. The conditional stability constant of the phenylboronate ester at pH 7.4 is 11.4. On cell surfaces, sialic acid is connected to the neighboring sugar unit through the 2-hydroxy group. To mimic this the 2-alpha-O-methyl derivative of Neu5 Ac was included in this study. The erythro configuration of the hydroxy substituents prevents stable-complex formation at positions C7 and C8 and, consequently, the strongest interaction is observed at positions C8 and C9, leading to a five-membered 2-boron-1,3-dioxalate. In addition, a relatively small amount of the C7-C9 six-membered complex was observed. Molecular modeling studies confirm that the C8-C9 boronate complex has the lowest energy.