Highly Efficient Chemoenzymatic Synthesis of Naturally Occurring and Non‐Natural α‐2,6‐Linked Sialosides: A <i>P. damsela</i> α‐2,6‐Sialyltransferase with Extremely Flexible Donor–Substrate Specificity

Yu, Hai; Huang, Shengshu; Chokhawala, Harshal A.; Sun, Mingchi; Zheng, Huanhuan; Chen, Xi

doi:10.1002/anie.200600572

Cited by 238 publications

(221 citation statements)

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“…Preparation of Sialylated GAEABs-We chose a synthetic strategy that combined two earlier developments, the bifunctional fluorescent tag AEAB (27) and the one-pot three-enzyme sialylation reaction (19,20,25) (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…Sialyl glycans containing various natural modifications of sialic acid linked ␣2-3 or ␣2-6 to the three different fluorescent glycans described above were synthesized using a highly efficient one-pot three-enzyme system similar to that reported previously (19,20,25,28). The fluorescent glycan-AEAB conjugates (GAEABs) of LNT, LNnT, or NA2 (130 -200 g) were incubated with three enzymes, including Escherichia coli sialic acid aldolase (28) (10 -15 g), Neisseria meningitidis CMPsialic acid synthetase (28) (10 -15 g), and Photobacterium damselae ␣2-6-sialyltransferase (25) (10 -15 g; used for the synthesis of ␣2-6-linked sialosides) or Pasteurella multocida ␣2-3-sialyltransferase PmST1 (19) (3-6 g; used for the synthesis of ␣2-3-linked sialosides) in a reaction mixture of 25 l containing 100 mM Tris-HCl buffer, pH 8.5.…”

Section: Methodsmentioning

confidence: 99%

“…acetyl or lactyl group). The following were added to this mixture: (a) sialic acid precursors (20,25,28,29) (e.g. mannose, N-acetylmannosamine, or their derivatives) at 2.0 or 4.0 eq relative to LNT or LNnT and NA2 (biantennary-AEAB conjugate), respectively; (b) sodium pyruvate (5 eq for LNT or LNnT, 10 equiv.…”

Section: Methodsmentioning

confidence: 99%

“…in chemoenzymatic synthesis of sialosides containing natural and non-natural sialic acid residues using multiple promiscuous sialoside biosynthetic enzymes (5,(15)(16)(17)(18)(19)(20).…”

mentioning

confidence: 99%

“…To further explore the recognition and function of modified sialic acid residues, we exploited the development of new chemoenzymatic methods to synthesize modified sialic acids (5), along with recent developments of glycan microarray technology, which provide an innovative approach to studying glycan recognition and function by glycan-binding proteins. We adapted our recent development of natural glycan microarrays (21)(22)(23)(24) to the production of a diverse array of sialylated glycans using fluorescent derivatives of glycans that terminate with ␤-linked galactose, which are sialyltransferase acceptors, in a one-pot three-enzyme approach for the microscale chemoenzymatic synthesis of sialosides (19,20,25). The resultant microarray consists of 77 sialylated glycans incorporating 16 modified sialic acids in ␣2-3 and ␣2-6 linkages to different underlying structures.…”

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confidence: 99%

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A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

Song

Chen

et al. 2011

Journal of Biological Chemistry

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Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in ␤-linked galactose at the nonreducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate ␣2-3-and ␣2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting ␣2-6-and ␣2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either ␣2-6-linked Neu5Ac9Lt or ␣2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…in chemoenzymatic synthesis of sialosides containing natural and non-natural sialic acid residues using multiple promiscuous sialoside biosynthetic enzymes (5,(15)(16)(17)(18)(19)(20).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

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A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

Song

Chen

et al. 2011

Journal of Biological Chemistry

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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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One‐Pot Multienzyme Synthesis of Lewis x and Sialyl Lewis x Antigens

Lau

et al. 2012

CP Chemical Biology

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L‐Fucose has been found abundantly in human milk oligosaccharides, bacterial lipopolysaccharides, glycolipids, and many N‐ and O‐linked glycans produced by mammalian cells. Fucose‐containing carbohydrates have important biological functions. Alterations in the expression of fucosylated oligosaccharides have been observed in several pathological processes such as cancer and atherosclerosis. Chemical formation of fucosidic bonds is challenging due to its acid lability. Enzymatic construction of fucosidic bonds by fucosyltransferases is highly efficient and selective but requires the expensive sugar nucleotide donor guanosine 5′‐diphosphate‐L‐fucose (GDP‐Fuc). Here, we describe a protocol for applying a one‐pot three‐enzyme system in synthesizing structurally defined fucose‐containing oligosaccharides from free L‐fucose. In this system, GDP‐Fuc is generated from L‐fucose, adenosine 5′‐triphosphate (ATP), and guanosine 5′‐triphosphate (GTP) by a bifunctional L‐fucokinase/GDP‐fucose pyrophosphorylase (FKP). An inorganic pyrophosphatase (PpA) is used to degrade the by‐product pyrophosphate (PPi) to drive the reaction towards the formation of GDP‐Fuc. In situ generated GDP‐Fuc is then used by a suitable fucosyltransferase for the formation of fucosides. The three‐enzyme reactions are carried out in one pot without the need for high‐cost sugar nucleotide or isolation of intermediates. The time for the synthesis is 4 to 24 hr. Purification and characterization of products can be completed in 2 to 3 days. Curr. Protoc. Chem. Biol. 4:233‐247 © 2012 by John Wiley & Sons, Inc.

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Highly Efficient Chemoenzymatic Synthesis of Naturally Occurring and Non‐Natural α‐2,6‐Linked Sialosides: A P. damsela α‐2,6‐Sialyltransferase with Extremely Flexible Donor–Substrate Specificity

Cited by 238 publications

References 68 publications

A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

Enzymatic Synthesis of Carbohydrate‐Containing Biomolecules

One‐Pot Multienzyme Synthesis of Lewis x and Sialyl Lewis x Antigens

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