Activation and transfer of novel synthetic 9‐substituted sialic acids

Gross, Heinz; Bünsch, Almuth; Paulson, James C.; Brossmer, Reinhard

doi:10.1111/j.1432-1033.1987.tb13458.x

Cited by 108 publications

(40 citation statements)

References 36 publications

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“…These sites were measured in terms of galactose content of the asialo-al-acid glycoprotein or antifreeze glycoprotein as described previously [8].…”

Section: Galactose Acceptor Sitesmentioning

confidence: 99%

“…One unit is defined as the amount of enzyme catalyzing the production of 1 pmol CMP-NeuAc/min under the assay conditions described previously IS]. Synthase assay was perfornied essentially as outlined earlier [8] with the following modifications. The reaction mixture (0.1 ml) contained 16 pmol Tris/HCl pH 9, 4 pmol MgC12, 50 pg bovine serum albumin, 1 pmol CTP, 0.1 pmol dithioerythritol and varying amounts of NeuAc or 9-fluoresceinyl-NeuAc.…”

Section: Cmpsialic Acid Synthase Assaymentioning

confidence: 99%

“…Fluorescein isothiocyanate (isomer I) was obtained from Serva (Heidelberg). Cytidine-5'-monophospho-N-acetylneuraminic acid (CMPNeuAc) was prepared enzymatically as described previously 584 [8] and contained 3.5% CMP. al-Acid glycoprotein was a generous gift from Dr Karl Schmid (Boston) and antifreeze glycoprotein (VIII) from Dr Robert Feeney (Davis).…”

Section: Muter Iulsmentioning

confidence: 99%

“…Analytical HPLC system (7,81 CMP and CMP-glycosides were measured at 275 nm [7,8] using an aminopropyl-phase column (0.4 ern x 5.0 cm, Serva Heidelberg). NeuAc and 9-fluoresceinyl-NeuAc were measured at 200 nm [7, 81 using a Spherisorb NH2 column (0.4 cm x 25 cm, Zinsser) while 9-fluoresceinyl-NeuAc was additionally detected at 240 nm.…”

Section: Galactose Acceptor Sitesmentioning

confidence: 99%

“…We have recently reported that a number of synthetic sialic acid analogues modified at position C-9 and C-4 were suitable substrates for CMP-sialic acid synthase and sialyltransferase [6 -91. Interestingly, substrate specificity of both enzymes with respect to C-9 of sialic acid was low [8,91. These results stimulated the synthesis of 5-acetamido-9-(3-fluoresceinylthioureido)-3,5,9-trideoxy-neuraminic acid which differs from the parent molecule only in the, albeit bulky, substituent at C-9.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins

Lijnen¹,

Nelles²,

Hoef³

et al. 1988

European Journal of Biochemistry

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Aiming at the introduction of a fluorescent sialic acid into glycoconjugates, 5-acetamido-9-(3-fluoresceinylthioureido)-3,5,9-trideoxy-2-nonulosonic acid (9-fluoresceinyl-NeuAc) was synthesized which has an intact carbon chain.a) Despite the space-filling substituent at C-9, the fluorescent NeuAc analogue was activated to the corresponding CMP-glycoside by CMP sialic acid synthase from bovine brain. Whereas the K, value of the synthase was little affected by the modification (K, = 2.1 mM, for NeuAc K , = 1.4 mM), the Vvalue decreased to 7.5%.b) CMP-9-fluoresceinyl-NeuAc was synthesized on a preparative scale (1 7% overall yield), and characterized by analytical HPLC, absorption and fluorescence spectra. c) 9-Fluoresceinyl-NeuAc was transferred onto asialo-al -acid glycoprotein by both GalPl, 4GlcNAc ct2, 6sialyltransferase and GalPl,4(3)GlcNAc a2,3sialyltransferase (rat liver), and onto antifreeze glycoprotein by GalNAc a2,6-sialyltransferase (porcine submaxillary glands). Using analytical HPLC, transfer was confirmed after release of the fluorescent sialic acid by Vibrio chalerae sialidase. d) Initital rate studies indicated a low K, value of GalP-l,4GlcNAc ~2,6sialyltransferase, and GalNAc ct2,6sialyltransferase (specific for 0-linked oligosaccharide chains) for CMP-9-fluoresceinyl-NeuAc.Though sialic acids are ubiquitous and, in general, terminal constituents of oligosaccharide chains of soluble or cell-surface-bound glycoconjugates, in many cases their exact biological significance is not well established [l, 21. Some evidence has been accumulated that these acids are involved in various phenomena of 'recognition' and 'masking'. To study biological functions, different labeling procedures have been applied. Labeling of sialoglycoconjugates in the sialic acid moiety with radioactive, fluorescent or biotinylated compounds requires chemical pretreatment [3 -51 (and earlier references therein). The method most frequently used for this purpose is periodate oxidation of glycolipids or glycoproteins under controlled conditions, which yields a shortened sialic acid side chain and produces a reactive aldehyde group.Dedicated to Professor Friedhelm Schneider on the occasion of his 60th birthday.Correspondence to R. Brossmer, Institut fur Biochemie I1 der Universitat Heidelberg, Im Neuenheimer Feld 328, D-6900 Heidelberg, Federal Republic of Germany Abbreviations. NeuAc, 5-acetamido-3,5-dideoxy-~-~-neuraminic acid; 9-fluoresceinyl-NeuAc, 9-fluoresceinylthioureido-NeuAc, 5-acetamido-9-(3-fluoresceinylth~oure~do)-3,5,9-t~eoxy-~-~-g/~cer~-~-galacto-2-nonulosonic acid; 9-amino-NeuAc, 5-acetamido-9-amino-3,5,9-trideoxy-2-nonulosonic acid; 9-azido-NeuAq 5-acetamido-9-azido-3,5,9-trideoxy-2-nonulosonic acid; CMP-NeuAc, cytidine-5'-monophospho-N-acetylneuraminic acid; CMP-9-fluoresceinyl-NeuAc, cytidine-5'-monophospho-9-fluoresceinylthioureido-NeuAc.Enzymes. CMPsialic acid synthase (EC 2.7.7.43); sialidase, acylneuraminylhydrolase (EC 3.2.1.18); Galpl,4GlcNAc ct2,6sialyltrans-ferase (EC 2.4.99.1); Gal,% ,4(3)GlcNAc a2,3sialyl...

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“…These sites were measured in terms of galactose content of the asialo-al-acid glycoprotein or antifreeze glycoprotein as described previously [8].…”

Section: Galactose Acceptor Sitesmentioning

confidence: 99%

Section: Cmpsialic Acid Synthase Assaymentioning

confidence: 99%

Section: Muter Iulsmentioning

confidence: 99%

Section: Galactose Acceptor Sitesmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins

Lijnen¹,

Nelles²,

Hoef³

et al. 1988

European Journal of Biochemistry

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Colon carcinoma glycoproteins carrying α 2,6-linked sialic acid reactive withSambucus Nigra agglutinin are not constitutively expressed in normal human colon mucosa and are distinct from sialyl-tn antigen

et al. 1997

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In human colon carcinoma, increased amounts of sialic acids have been found and correlated with tumor progression. Further, the degree of O-acetylation of sialic acid residues in normal mucosa is higher than in colon carcinoma. Thus, tumor-associated sialylated antigens may be constitutively expressed in O-acetylated form in normal mucosa unreactive with the respective monoclonal antibodies. We have earlier demonstrated a colon carcinoma-associated expression of a 2,6-linked sialic acid residues with the Sambucus nigra agglutinin (SNA). We report now that de-acetylation of normal and transitional colonic mucosa, in contrast to sialyl-Tn antigen, does not result in SNA binding. Further, the a 2,6-linked sialic acid recognized by SNA is distinct from that of sialyl-Tn antigen. This is confirmed by Northern blotting detecting transcripts for a 2,6 sialyltransferase of N-glycoproteins and measurement of activity for this sialyltransferase. Blot analysis by SNA of colon carcinoma cells revealed few reactive glycoproteins. Quantitative differences in lectin labeling and sialyltransferase activity were found in HCT116 colon carcinoma cell sub-lines. Our data suggest that SNA binding in human colon carcinoma is due to de novo expression of a specific sialic acid present on selected glycoproteins. Int.

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Enzymatic Synthesis of Carbohydrate‐Containing Biomolecules

Chen

2008

Wiley Encyclopedia of Chemical Biology

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As important biomolecules in living organisms, carbohydrates have received increasing attention in recent years. Their important roles in biologic events are being continuously unraveled. The development of synthetic methodologies, including both chemical and enzymatic methods, contributes greatly to the advance of the field of glycoscience. The involvement of regio‐ and stereo‐selective enzymes in the synthesis of complex carbohydrate‐containing molecules has become an indispensable approach. Many enzymes involved in the biosynthesis and biodegradation of carbohydrates have been characterized and have been applied for the production of carbohydrate‐containing biomolecules, including oligosaccharides, polysaccharides, glycoproteins, glycolipids, and glycosylated natural products. A range of strategies for enzymatic synthesis have also been developed, such as protein engineering of glycosidases and glycosyltransferases by site‐directed mutagenesis or directed evolution, one‐pot multiple‐enzyme synthesis, sugar nucleotide regeneration, solid‐phase enzymatic synthesis, synthesis using immobilized enzymes, and cell‐based synthesis. Enzymatic synthesis will continue to play critical roles in obtaining complex carbohydrate‐containing biomolecules. Future efforts should focus on identifying synthetically useful enzymes such as those with flexible or novel substrate specificity and those that can form new bonds. This identification can be achieved by functional genomics and mutagenesis studies. Development of novel enzymatic synthetic methods is also critical to access diverse naturally occurring and non‐natural derivatives of carbohydrates and glycoconjugates.

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Activation and transfer of novel synthetic 9‐substituted sialic acids

Cited by 108 publications

References 36 publications

Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins

Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins

Colon carcinoma glycoproteins carrying α 2,6-linked sialic acid reactive withSambucus Nigra agglutinin are not constitutively expressed in normal human colon mucosa and are distinct from sialyl-tn antigen

Enzymatic Synthesis of Carbohydrate‐Containing Biomolecules

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