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.     

Analysis of sialyl oligosaccharides by high-performance liquid chromatography-electrospray ionisation-mass spectrometry with differentiation of alpha2-3 and alpha2-6 sialyl linkages

A method for analysing sialyl oligosaccharides from bovine colostrum using high-performance liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS) is described. Under positive ionisation mode, mass spectra of alpha2-3 and alpha2-6 linkages were different, and the former produced a prominent B2 (or B3 in disialyl lactose) mass fragment. This fragment was absent from mass spectra with alpha2-6 linkages. Two sialyl oligosaccharides, which have not been reported previously, were tentatively identified. One comprises a N-acetyl neuraminic acid (Neu5Ac), two hexoses (Hex), and one N-acetyl hexosamine (HexNAc) residue ((Neu5Ac)1 (Hex)2 (HexNAc)1), and the other comprises one Neu5Ac and one Hex residue ((Neu5Ac)1(Hex)1).     

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.     

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.     

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.     

Determination of sialic acids released from glycoproteins using capillary zone electrophoresis/electrospray ionization mass spectrometry

A simple method for the determination of the two most abundant sialic acids released from glycoproteins based on CZE-MS is presented. Several parameters like BGE with various organic modifiers and sheath liquids were studied with respect to their suitability for the fast and easy analysis of the selected compounds by CZE-MS. Finally, a BGE containing 10 mM ammonium acetate allowed the quantification of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) in glycoproteins as well as human plasma samples. LOD and LOQ were 2 microg/mL and 6 microg/mL, respectively.     

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.     

Stereochemical studies of sialic acid derivatives by vibrational circular dichroism

Systematic VCD studies of N-acetylneuraminic acid (Neu5Ac), a recognition-related unique carbohydrate, were performed for the first time. Two pairs of anomeric isomers regarding a quaternary C2 asymmetric carbon of Neu5Ac derivatives were synthesized. VCD spectral patterns around the ester carbonyl region, as well as other Mid-IR regions, would be practical markers to distinguish the C2 stereochemistry.     

3 beta-Hydroxysialic acid glycosides. I. Calcium-binding ability and chemical and enzymatic stabilities.

Methyl alpha- and beta-glycosides of N-acetylneuraminic acid (Neu5Ac) and N-acetyl-3 beta-hydroxyneuraminic acid (Neu5Ac beta 3OH) (1-4) were prepared to evaluate their calcium-binding ability. (Methyl alpha-glucopyranosidonyl) alpha- and beta-, and 4-methylumbelliferyl alpha-glycosides of Neu5Ac and Neu5Ac beta 3OH (5-10) were also synthesized for the comparison of chemical and enzymatic stabilities, respectively. Methyl beta-glycosides of Neu5Ac and Neu5Ac beta 3OH, 3 and 4, respectively, showed intense calcium-binding abilities, while no such ability was observed in the corresponding alpha-glycosides, 1 and 2. The Neu5Ac beta 3OH glycosides, 6, 8, and 10, showed much stronger resistance to acidic hydrolysis and sialidase digestion than the corresponding Neu5Ac glycosides, 5, 7, and 9.     

Efficient synthesis of a sialic acid alpha(2-->3)galactose building block and its application to the synthesis of ganglioside GM3

Glycosylation of various galactose derivatives with O-acetylated sialic acid N-phenyltrifluoroacetimidate as the donor was investigated. Efficient alpha(2,3)sialylation of galactose, with up to 73% yield and 8.4:1 stereoselectivity, was realized when 2,3,4-unprotected galactose derivatives and TBSOTf were used as acceptors and promoter, respectively. Sialylation of 2-(trimethylsilyl)ethyl 6-O-tert-butyldiphenylsilyl-beta-D-galactopyranoside (3f) gave the best result, and the resultant Neu5Ac alpha(2-->3)Gal disaccharide was successfully used in the synthesis of ganglioside GM3.     

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.     

Expression and distribution of N-acetyl and N-glycolylneuraminic acids in secreted and cell-associated glycoconjugates by two human osteosarcoma cell lines

N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the dominant sialic acids (Sia) in mammals usually found in the non-reducing terminal of oligosaccharide side chains in glycoproteins and glycolipids. Their expression and distribution pattern have been correlated both with the malignant phenotype and tumor grade of human cancers. The aim of the present study was to determine by reversed-phase HPLC method the amounts of Neu5Ac and Neu5Gc as well as their distribution among the culture media and cell surface of MG-63 and Saos-2 human osteosarcoma cell lines of high and low metastatic potential. It was determined that MG-63 cells produce up to 5-fold more total sialic acid as compared with the Saos 2 cells. Neu5Ac accounts for ca 60% of the total sialic acids secreted by MG-63 cells, whereas Neu5Gc is the predominant sialic acid present on the MG-63 cell membrane. Saos 2 cells secrete considerable amounts of Neu5Ac to culture media. The obtained data indicate that the human osteosarcoma cells express both forms of Sia-containing glycoconjugates; the differences in the amounts of each of the two major Sia types and their distribution may be related to their differences in morphology and/or metastatic potentials.     

Facile method for the preparation of lyso-GM1 and lyso-GM2

This paper reports a facile method for the preparation of lyso-GM1 [Gal beta1-->3GalNAc beta1--> 4(Neu5Ac alpha2-->3)Galbeta1-->4Glc beta1-->1'-sphingosine] and lyso-GM2 [GalNAc beta1-->4(Neu5Ac alpha2-->3)Gal beta1-->4Glc beta1-->sphingosine], respectively, from GM1 [Galbeta1-->3GalNAc beta1-->4(Neu5Ac alpha2-->3)Galbeta1-->4Glc beta1-->1'-Cer] and GM2[GalNAc beta1-->4(Neu5Ac alpha2-->3)Galbeta1-->4Glc beta1-->1'-Cer], using sphingolipid ceramide deacylase and high performance anion-exchange chromatography (HPAEC). The enzymatically released lyso-GM1 and/or lyso-GM2 was effectively separated from its parent ganglioside by HPAEC using a Mono Q HR 5/5 column with an Amersham Biosciences fast protein liquid chromatography system. The yield was almost quantitative and the separation completed in approximately 3 h. This method is more convenient and effective than the conventional method using alkaline hydrolysis and silicic acid chromatography to generate and purify lyso-gangliosides.     

Intramolecular oximercuration-demercuration reaction: a new stereocontrolled approach to sialic acid containing disaccharides

Methyl 2,3,4-tri-O-benzyl-α,-D-glucopyranoside has been converted into Neu5Ac α(2-6)Glu and Neu5Ac β (2-6)Glu derivatives, using an intramolecular oximercuration-demercuration reaction.     

Preparation of 4-pentenoic acid ester of Neu5Ac and 4-pentenyl glycoside of Neu5Ac and their application to glycosylation

Novel sialosyl donors, 4-pentenoic acid ester of N-acetylneuraminic acids (Neu5Ac) and 4-pentenyl glycoside of Neu5Ac were successfully prepared from the corresponding per-O-acetylated 2-hydroxy and 2-chloro derivatives of Neu5Ac, respectively and applied to the synthesis of O-sialosides.     

Separation of basic drug enantiomers by capillary electrophoresis using chicken alpha1-acid glycoprotein: insight into chiral recognition mechanism

Recombinant chicken alpha(1)-acid glycoprotein (alpha(1)-AGP) was prepared by the Escherichia coli expression system and completely deglycosylated alpha(1)-AGP (cd-alpha(1)-AGP) was obtained by treatments of native alpha(1)-AGP with a mixture of endoglycosidase and N-glycosidase. The average molecular masses of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP were estimated to be about 29 200, 21 700 and 20 700, respectively, by matrix-assisted laser desorption-time of flight-mass spectrometry. We compared the chiral recognition ability of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP using them as chiral selectors in capillary electrophoresis. The chicken alpha(1)-AGP showed higher resolution for eperisone, pindolol and tolperisone than cd-alpha(1)-AGP or recombinant alpha(1)-AGP. Recombinant alpha(1)-AGP still showed chiral recognition for three basic drugs tested. By addition of propranolol as a competitor in the separation solution in CE, no enantioseparations of three basic drugs were observed with chicken alpha(1)-AGP, cd-alpha(1)-AGP or recombinant alpha(1)-AGP. These results reveal that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition ability, and that the chiral recognition site(s) for basic drugs exists on the protein domain.     

Determination and distribution of N-acetyl- and N-glycolylneuraminic acids in culture media and cell-associated glycoconjugates from human malignant mesothelioma and adenocarcinoma cells

Sialic acids containing glycoconjugates are very common in human neoplasias and their expression frequently correlates with malignant phenotype and the tumor grade. The majority of tumor markers containing sialic acids in man involve changes in the amount of total sialic acids and in the presence of the two main sialic acid types, Neu5Ac and Neu5Gc, and their derivatives. The aim of the present study was to examine whether malignant mesothelioma cell lines synthesize sialic acid containing glycoconjugates at both the extracellular and cell membrane levels and particularly whether the type and the content of Neu5Ac and Neu5Gc are of biological importance for mesothelioma cell differentiation and evaluation of its prognosis. The study was performed in three human malignant mesothelioma cell lines, two with a fibroblast like phenotype (STAV-FCS and Vester) and one of epithelial differentiation (STAV-AB), which developed from the pleural effusions of patients with malignant mesothelioma and in one human adenocarcinoma cell line (Wart). Neu5Ac and Neu5Gc were determined following a mild hydrolysis step and a sample clean-up procedure. The determination was performed by reversed-phase HPLC after the NeuAc and NeuGc had been converted to per-O-benzoylated derivatives. It was found that Neu5Gc is the major sialic acid in the culture media of all cell lines examined. Molar ratios of Neu5Ac to Neu5Gc showed that Neu5Gc is the predominant sialic acid in the culture medium of the fibroblast-like mesothelioma cells. Neu5Ac is almost undetectable in the cell membrane, whereas Neu5Gc is present in considerable amounts. The obtained results suggest that the type and the content of Neu5Ac and Neu5Gc in culture media are of biological importance for mesothelioma cell differentiation and may be of value in the evaluation of prognosis.     

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.     

Synthesis of nor-C-linked neuraminic acid disaccharide: a versatile precursor of C-analogs of oligosialic acids and gangliosides

The Neu5Acalpha(2,8)Neu5Ac disaccharide is an important constituent of tumor related antigen, however, the O-linkage is catabolically unstable. Vaccination with a catabolically stable sialic acid C-glycoside analog might enhance immunogenicity. The synthesis of Neu5Ac nor-C-disaccharide 20R/S, corresponding to versatile precursors of C-analogs of oligosialic acid and gangliosides, is reported. The synthesis of the protected acceptor was not straightforward, as ester, silyl ether, and isopropylidene protection failed to afford desired C-linked disaccharide. Allyl ether protection of hydroxyl groups and acetyl protection of the acetamido facilitated the successful synthesis of the 8-aldehyde neuraminyl acceptor. Samarium mediated C-glycosylation afforded the desired nor-C-disaccharide as a mixture of two separable diastereomers.     

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.