Academia Sinica

The Scripps Research Institute

Verenium Corporation

Sarah R Hanson

Chi-Huey Wong

Tsui-Ling Hsu

Masaaki Sawa

Peter G Schultz

Mark Joseph Burk

Toby Howard Richardson

David Paul Weiner

Zhiwen Zhang

Grace DeSantis

Lishan Zhao

Tim S Hitchman

William H Greenberg

Aileen Milan

Patti Kretz

Ran Xu

Chi-Heuy Wong

Methods for metabolic oligosaccharide engineering that incorporates derivatized alkyne-bearing sugar analogs as 'tags' into cellular glycoconjugates are disclosed. Alkynyl derivatized Fuc and alkynyl derivatized ManNAc sugars are incorporated into cellular glycoconjugates. Chemical probes comprising an azide group and a visual or fluorogenic probe and used to label alkyne-derivatized sugar-tagged glycoconjugates are disclosed. Chemical probes bind covalently to the alkynyl group by Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition and are visualized at the cell surface, intracellularly, or in a cellular extract. The labeled glycoconjugate is capable of detection by flow cytometry, SDS-PAGE, Western blot, ELISA, confocal microscopy, and mass spectrometry.

Alkynyl sugar analogs for labeling and visualization of glycoconjugates in cells

Methods are provided for labeling cellular glycans bearing azide groups via fluorescent labeling comprising Cu(I)-catalyzed [3+2] cycloaddition of a probe comprising alkynyl group. Generation of fluorescent probes from a nonfluorescent precursor, 4-ethynyl-N-ethyl-1,8-naphthalimide, by Cu(I)-catalyzed [3+2] cycloaddition of the alkyne group of the probe to an azido-modified sugar are provided. Incorporation of azido-containing fucose analog into glycoconjugates via the fucose salvage pathway are disclosed. Fluorescent visualization of fucosylated cells by flow cytometry of cells treated with 6-azidofucose labeled with click-activated fluorogenic probe or biotinylated alkyne is disclosed. Visualization of intracellular location of fucosylated glycoconjugates by fluorescence microscopy are disclosed.

Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo

The invention provides biocatalytic methods for the manufacture of pure single enantiomer compounds. This invention provides methods of screening for enzymes which are highly enantioselective or enzymes that can provide any desired stereoisomer of a compound. The invention provides the use of single enantiomer substrates in performing a growth screen of a clonal library to identify highly stereoselective enzymes. In one aspect, methods for screening and identification of enzymes, e.g., transaminases, nitrilases, aldolases, epoxide hydrolases are provided. Methods for the production and screening of gene libraries generated from nucleic acids isolated from more than one organism for enzyme, e.g., transaminase, activities are also provided.

Methods for the manufacture of pure single enantiomer compounds and for selecting enantioselective enzymes

The present disclosure relates to a method for metabolic oligosaccharide engineering that incorporates derivatized alkyne-bearing sugar analogs as 'tags' into cellular glycoconjugates. The disclosed method incorporates alkynyl derivatized Fuc and alkynyl derivatized ManNAc sugars into a cellular glycoconjugate. A chemical probe comprising an azide group and a visual probe or a fluorogenic probe is used to label the alkyne-derivatized sugar-tagged glycoconjugate. In one aspect, the chemical probe binds covalently to the alkynyl group by Cu(I)-catalyzed [3+2] azide-alkyne cycloaddition and is visualized at the cell surface, intracellularly, or in a cellular extract. The labeled glycoconjugate is capable of detection by flow cytometry, SDS-PAGE, Western blot, ELISA or confocal microscopy, and mass spectrometry.

Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells

The present disclosure relates to tailored glycoproteomic methods, and more particularly to methods for the sequencing, mapping and identification of cellular glycoproteins using saccharide-selective bioorthogonal probes. A method is disclosed for saccharide-selective glycoprotein identification (ID) and glycan mapping (GIDmap) that generates glycoproteins tailored with bioorthogonally tagged alkynyl saccharides that can be selectively isolated, allowing for glycoprotein ID and glycan mapping via mass spectromic proteomics, including liquid chromatography-tandmen mass spectroscopy (LC-MS). LC-MSmay be used to identify cellular glycans, and more specifically cancer-related glycoproteins.

Tailored glycoproteomic methods for the sequencing, mapping and identification of cellular glycoproteins

The disclosure provides a method of labeling of cellular glycans bearing azide groups via a fluorescent labeling technique based on Cu(I)-catalyzed [3+2]cycloaddition (click activation) of a probe comprising an alkynyl group. The method entails generating a fluorescent probe from a nonfluorescent precursor, 4-ethynyl-N-ethyl-1,8-naphthalimide, by Cu(I)-catalyzed [3+2]cycloaddition of the alkyne group of the probe with an azido-modified sugar. The disclosure further provides a method of incorporating an azido-containing fucose analog into glycoconjugates via the fucose salvage pathway. The disclosure provides a method of fluorescent visualization of fucosylated cells by flow cytometry when cells treated with 6-azidofucose are labeled with the click-activated fluorogenic probe or biotinylated alkyne. A method of visualizing the intracellular localization of fucosylated glycoconjugates by fluorescence microscopy is also disclosed.

Glycoproteomic probes for fluorescent imaging fucosylated glycans in vivo

Methods and compositions for making glycoproteins, both in vitro and in vivo, are provided. One method involves incorporating an unnatural amino acid having a N-acetylgalactosamine moiety into a protein; optionally, the N-acetylgalactosamine-containing unnatural amino acid can be further modified with additional sugars.

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San Marcos, CA, United States of America

Sarah R Hanson