Synthesis of non-natural ManNAc analogs for the expression of thiols on cell-surface sialic acids

Sampathkumar, Srinivasa-Gopalan; Li, Adrienne V.; Yarema, Kevin J.

doi:10.1038/nprot.2006.319

Cited by 30 publications

(34 citation statements)

References 20 publications

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“…Compound synthesis Ac 4 ManNAz synthesis was performed following the protocol described by Sampathkumar [19] (see the electronic supplementary material) [19]. Biotin-LC-alkyne has been described as a probe to detect azido-modified glycoconjugates [16].…”

Section: Methodsmentioning

confidence: 99%

Cell surface glycoprotein profiling of cancer cells based on bioorthogonal chemistry

et al. 2012

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Bioorthogonal chemistry refers to chemical reactions that can occur within a living system without altering native biochemical processes. Applications of this concept extend to studies on a group of biomolecules that includes glycans, proteins, and lipids. In this study, a strategy for isolating cell surface glycoproteins and based on bioorthogonal chemistry was employed to identify new cancer-related glycoproteins. A novel alkyne reagent containing one disulfide bond was synthesized for the enrichment of glycoproteins metabolized with peracetylated N-azidoacetylmannosamine, which was applied on three different cancer cell lines, and all isolated proteins were analyzed by high-performance liquid chromatography-tandem mass spectrometry. The strategy of purifying cell surface glycoproteins introduced in this article was shown to be reliable, and a total of 56 cell surface glycoproteins were identified. Neuronal cell adhesion molecule was found uniquely expressed in A549 lung adenocarcinoma, and its expression in non-small-cell lung carcinomas was detected by immunohistochemistry. Furthermore, a significant increase of neuronal cell adhesion molecule expression was identified in non-small-cell lung adenocarcinoma compared with adjacent noncancerous tissues, and could be a novel potential target and marker in cancer treatment and detection.

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Section: Methodsmentioning

confidence: 99%

Cell surface glycoprotein profiling of cancer cells based on bioorthogonal chemistry

et al. 2012

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“…This is generally achieved by using NHS-activated acids [38,39], or alternatively, can also be achieved by treatment of isobutyl chloroformate with an acid, yielding an amine-reactive anhydride [36,40]. Sampathkumar et al reported the synthesis of peracetylated ManNAc analogues by treatment with naphthaldehyde for N-activation using ManN hydrochloride as starting material [41]. However, the high price of mannosamine is a major limitation in the synthesis of sialic acid analogues.…”

Section: Chemoenzymatic Synthesis Of Mannosamine and Sialic Acid Derimentioning

confidence: 99%

N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism

Wang

Laborda

et al. 2016

Catalysts

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Abstract:The control of cellular N-acetylmannosamine (ManNAc) levels has been postulated to be an effective way to modulate the decoration of cell surfaces with sialic acid. N-acetylglucosamine 2-epimerase catalyzes the interconversion of N-acetylglucosamine (GlcNAc) and ManNAc. Herein, we describe the cloning, expression, purification and biochemical characterization of an unstudied N-acetylglucosamine 2-epimerase from Pedobacter heparinus (PhGn2E). To further characterize the enzyme, several N-acylated glucosamine derivatives were chemically synthesized, and subsequently used to test the substrate specificity of PhGn2E. Furthermore, NMR studies of deuterium/hydrogen exchange at the anomeric hydroxy group and C-2 positions of the substrate in the reaction mixture confirmed for the first time the postulated epimerization reaction via ring-opening/enolate formation. Site-directed mutagenesis of key residues in the active site showed that Arg63 and Glu314 are directly involved in proton abstraction and re-incorporation onto the substrate. As all mechanistically relevant active site residues also occur in all mammalian isoforms, PhGn2E can serve as a model N-acetylglucosamine 2-epimerase for further elucidation of the active site mechanism in these enzymes.

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“…Other analogs can be prepared following published protocols (e.g., see Sampathkumar et al, 2006d) that contain recipe-type instructions for synthesizing Ac 5 ManNGc and Ac 5 ManNTGc.…”

Section: Strategic Planningmentioning

confidence: 99%

Experimental Design Considerations for In Vitro Non‐Natural Glycan Display via Metabolic Oligosaccharide Engineering

Tan

Almaraz

Khanna

et al. 2010

CP Chemical Biology

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Metabolic oligosaccharide engineering (MOE) refers to a technique where non‐natural monosaccharide analogs are introduced into living biological systems. Once inside a cell, these compounds intercept a targeted biosynthetic glycosylation pathway and in turn are metabolically incorporated into cell‐surface‐displayed oligosaccharides where they can modulate a host of biological activities or be exploited as “tags” for bio‐orthogonal and chemoselective ligation reactions. Undertaking a MOE experiment can be a daunting task based on the growing repertoire of analogs now available and the ever increasing number of metabolic pathways that can be targeted; therefore, a major emphasis of this article is to describe a general approach for analog design and selection and then provide protocols to ensure safe and efficacious analog usage by cells. Once cell‐surface glycans have been successfully remodeled by MOE methodology, the stage is set for probing changes to the myriad cellular responses modulated by these versatile molecules. Curr. Protoc. Chem. Biol. 2:171‐194 © 2010 by John Wiley & Sons, Inc.

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Synthesis of non-natural ManNAc analogs for the expression of thiols on cell-surface sialic acids

Cited by 30 publications

References 20 publications

Cell surface glycoprotein profiling of cancer cells based on bioorthogonal chemistry

Cell surface glycoprotein profiling of cancer cells based on bioorthogonal chemistry

N-acetylglucosamine 2-Epimerase from Pedobacter heparinus: First Experimental Evidence of a Deprotonation/Reprotonation Mechanism

Experimental Design Considerations for In Vitro Non‐Natural Glycan Display via Metabolic Oligosaccharide Engineering

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