Enzymatic Synthesis of Trideuterated Sialosides

Analytical and Bioanalytical Chemistry, 2011

In 2007, Martin et al. developed a method for the analysis of sialic acids by HPLC following 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatisation (Martín et al., Anal Bioanal Chem 387:2943-2949. Within the article, the authors noted that lactose interfered with the analysis, giving erroneously high results when lactosecontaining products were analysed. Such an observation is important when analysing milk-based products, yet was contradictory to the observations of Nakamura et al. (Chem Pharm Bull 35(2):687-692, 1987) who demonstrated that DMB was specific for α-keto acids and did not react with simple sugars such as glucose or lactose. In order to clarify the situation, this phenomenon was investigated and it was confirmed that lactose does not interfere with the analysis. However, it was found that most commercial preparations of lactose do contain small amounts of sialic acids, either as the free monosaccharide or bound to lactose in the form of 3′and 6′-sialyllactose.

Journal of the American Society for Mass Spectrometry, 2018

Mass spectrometric analysis of intact glycopeptides can reveal detailed information about glycosite, glycan structural features, and their heterogeneity. Sialyl glycopeptides can be positively, negatively, or neutrally charged depending on pH of their buffer solution and ionization conditions. To detect sialoglycopeptides, a negative-ion mode mass spectrometry may be applied with a minimal loss of sialic acids, although the positively charged or neutral glycopeptides may be excluded. Alternatively, the sialyl glycopeptides can be identified using positive-ion mode analysis by doping a high concentration of sodium salts to the analytes. Although manipulation of unmodified sialoglycopeptides can be useful for analysis of samples, less than optimal ionization, facile loss of sialyl and unfavorable ionization of accompanying non-sialyl peptides make such strategies suboptimal. Currently available chemical derivatization methods, while stabilizing for sialic acid, mask sialic acid linkage configuration. Here, we report the development of a novel approach to neutralize sialic acids via sequentially chemical modification that also reveals their linkage configuration, often an important determinant in biological function. This method utilizes several components to facilitate glycopeptide identification. These include the following: solid phase derivatization, enhanced ionization of sialoglycopeptides, differentiation of sialic acid linkage, and enrichment of the modified glycopeptides by hydrophilic interaction liquid chromatography. This technology can be used as a tool for quantitative analysis of protein sialylation in diseases with determination of sialic acid linkage configuration.

Animals, 2021

Swine are one of the most important agricultural species for human food production. Given the significant disease challenges confronting commercial pig farming systems, introduction of a new feed additive that can enhance animal performance by improving growth and immune status represents a major opportunity. One such candidate is sialic acid (Sia), a diverse family of nine-carbon acidic sugar, present in various organs and body fluid, as well as an essential structural and functional constituent of brain ganglioside of humans and animals. Sias are key monosaccharide and biomarker of sialylated milk oligosaccharide (Sia-MOS’s), sialylated glycoproteins and glycolipids in milk and all vertebrate cells. Sias accomplish many critical endogenous functions by virtue of their physiochemical properties and via recognition by intrinsic receptors. Human milk sialylated glycoconjugates (Sia-GC’s) are bioactive compounds known to act as prebiotics that promote gut microbiota development, gut m...

Analytical Biochemistry, 1990

Glycoconjugate Journal, 2015

Carbohydrates, in addition to their metabolic functions, serve important roles as receptors, ligands, and structural molecules for diverse biological processes. Insight into carbohydrate biology and mechanisms has been aided by metabolic oligosaccharide engineering (MOE). In MOE, unnatural carbohydrate analogs with novel functional groups are incorporated into cellular glycoconjugates and used to probe biological systems. While MOE has expanded knowledge of carbohydrate biology, limited metabolism of unnatural carbohydrate analogs restricts its use. Here we assess metabolism of SiaDAz, a diazirine-modified analog of sialic acid, and its cell-permeable precursor, Ac 4 ManNDAz. We show that the efficiency of Ac 4 ManNDAz and SiaDAz metabolism depends on cell type. Our results indicate that different cell lines can have different metabolic roadblocks in the synthesis of cell surface SiaDAz. These findings point to roles for promiscuous intracellular esterases, kinases, and phosphatases during unnatural sugar metabolism and provide guidance for ways to improve MOE.

Carbohydrate Research, 1999

A novel N-acetylneuraminic acid analogue, 2-S-(5%-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-a-Dgalacto-2-nonulopyranosidonic acid, as well as the thiosialoside 2-S-(2%-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero -a-D-galacto-2-nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group. The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield.

Biochemistry, 2001

Unnatural analogues of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogues bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogues with ketone-containing N-acyl groups that varied in the length or steric bulk was chemically synthesized and tested for metabolic conversion to cell surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

Bioorganic & Medicinal Chemistry Letters, 2009

A convenient chemoenzymatic strategy for synthesizing sialosides containing a C5-diversified sialic acid was developed. The α2,3and α2,6-linked sialosides containing a 5-azido neuraminic acid synthesized by a highly efficient one-pot three-enzyme approach were converted to C5″-amino sialosides, which were used as common intermediates for chemical parallel synthesis to quickly generate a series of sialosides containing various sialic acid forms.

Drug Discovery Today, 2007

Sialic acids are structurally diverse molecules that have important roles in the physiological reactions and characteristics of prokaryotes and eukaryotes. These include the ability to mask epitopes on underlying glycan chains and to repulse negatively charged moieties. Here, we describe the metabolism and immunological relevance of sialic acids and outline how their properties have been exploited by the pharmaceutical industry to enhance the therapeutic properties of proteins such as asparaginase and darbepoetin a.

Carbohydrate Research, 1984