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1. 发明授权

US09816981B2 Alkynyl sugar analogs for labeling and visualization of glycoconjugates in cells 有权
公开(公告)号：US09816981B2
公开(公告)日：2017-11-14
申请号：US13159339
申请日：2011-06-13
申请人： Masaaki Sawa , Chi-Huey Wong , Tsui-Ling Hsu , Sarah Hanson
发明人： Masaaki Sawa , Chi-Huey Wong , Tsui-Ling Hsu , Sarah Hanson
IPC分类号： C07H15/04 , C07H19/10 , G01N33/50 , G01N33/533
CPC分类号： G01N33/5005 , G01N33/5008 , G01N33/533 , G01N2400/00
摘要： 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.

2. 发明授权

US07960139B2 Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells 有权
标题翻译：炔基糖类似物用于细胞中糖缀合物的标记和可视化
公开(公告)号：US07960139B2
公开(公告)日：2011-06-14
申请号：US12079226
申请日：2008-03-24
申请人： Masaaki Sawa , Chi-Huey Wong , Tsui-Ling Hsu , Sarah Hanson
发明人： Masaaki Sawa , Chi-Huey Wong , Tsui-Ling Hsu , Sarah Hanson
IPC分类号： G01N33/53
CPC分类号： G01N33/5005 , G01N33/5008 , G01N33/533 , G01N2400/00
摘要： 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.
摘要翻译：本公开内容涉及将衍生的含炔烃类似物作为“标签”掺入细胞糖缀合物的代谢寡糖工程方法。所公开的方法将炔基衍生化的Fuc和炔基衍生的ManNAc糖引入细胞糖缀合物中。使用包含叠氮化物基团和视觉探针或荧光探针的化学探针来标记炔衍生的糖标记的糖缀合物。在一个方面，化学探针通过Cu（I）催化的[3 + 2]叠氮炔环加成共价结合到炔基上，并且在细胞表面，细胞内或细胞提取物中可视化。标记的糖缀合物能够通过流式细胞术，SDS-PAGE，Western印迹，ELISA或共聚焦显微镜和质谱检测。

3. 发明申请

US20120149887A1 ALKYNYL SUGAR ANALOGS FOR LABELING AND VISUALIZATION OF GLYCOCONJUGATES IN CELLS 审中-公开
标题翻译：用于在细胞中标记和观察甘油三酯的ALKYNYL糖模拟物
公开(公告)号：US20120149887A1
公开(公告)日：2012-06-14
申请号：US13159339
申请日：2011-06-13
申请人： Masaaki Sawa , Chi-Huey Wong , Tsui-Ling Hsu , Sarah Hanson
发明人： Masaaki Sawa , Chi-Huey Wong , Tsui-Ling Hsu , Sarah Hanson
IPC分类号： C07H15/04 , C07H19/10
CPC分类号： G01N33/5005 , G01N33/5008 , G01N33/533 , G01N2400/00
摘要： 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.
摘要翻译：公开了将衍生的含炔烃类似物作为细胞糖缀合物的“标签”的代谢寡糖工程方法。将炔基衍生的Fuc和炔基衍生的ManNAc糖掺入细胞糖缀合物中。公开了包含叠氮基和视觉或荧光探针并用于标记炔衍生的糖标记的糖缀合物的化学探针。化学探针通过Cu（I）催化的[3 + 2]叠氮炔环加成与炔基共价结合，并在细胞表面，细胞内或细胞提取物中显现。标记的糖缀合物能够通过流式细胞术，SDS-PAGE，Western印迹，ELISA，共聚焦显微镜和质谱检测。

4. 发明申请

US20110257376A1 GLYCOPROTEOMIC PROBES FOR FLUORESCENT IMAGING OF FUCOSYLATED GLYCANS IN VIVO 有权
标题翻译：血液中富含荧光成像的GLYCOPROTEOMIC PROBES
公开(公告)号：US20110257376A1
公开(公告)日：2011-10-20
申请号：US13053192
申请日：2011-03-21
申请人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R. Hanson , Masaaki Sawa
发明人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R. Hanson , Masaaki Sawa
IPC分类号： C07K14/47 , C07H19/207
CPC分类号： G01N33/582 , G01N1/30 , G01N2400/00
摘要： 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.
摘要翻译：提供了通过含有包含炔基的探针的Cu（I）催化的[3 + 2]环加成的荧光标记来标记携带叠氮基的细胞聚糖的方法。通过Cu（I）催化的[3 + 2]环加成的探针的炔基与叠氮改性的糖，从非荧光前体4-乙炔基-N-乙基-1,8-萘二甲酰亚胺生成荧光探针是提供。公开了通过岩藻糖补救途径将叠氮基岩藻糖类似物掺入糖缀合物。公开了通过用用点击激活的荧光探针或生物素化的炔标记的6-叠氮基聚糖处理的细胞的流式细胞术的岩藻糖基化细胞的荧光可视化。公开了通过荧光显微镜观察岩藻糖基化糖缀合物的细胞内位置。

5. 发明授权

US07910319B2 Glycoproteomic probes for fluorescent imaging fucosylated glycans in vivo 有权
标题翻译：用于体内荧光成像岩藻糖基化聚糖的糖蛋白质组学探针
公开(公告)号：US07910319B2
公开(公告)日：2011-03-22
申请号：US12079228
申请日：2008-03-24
申请人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R Hanson , Masaaki Sawa
发明人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R Hanson , Masaaki Sawa
IPC分类号： G01N33/53
CPC分类号： G01N33/582 , G01N1/30 , G01N2400/00
摘要： 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.
摘要翻译：本公开提供了通过基于包含炔基的探针的Cu（I）催化的[3 + 2]环加成（点击活化）的荧光标记技术来标记携带叠氮基的细胞聚糖的方法。该方法需要通过Cu（I）催化的[3 + 2]环加成的探针的炔基与非荧光前体4-乙炔基-N-乙基-1,8-萘酰亚胺的生成荧光探针，改性糖。本公开进一步提供了通过岩藻糖补救途径将含叠氮基岩藻糖类似物掺入糖缀合物的方法。本公开内容提供了当用6-叠氮基聚糖处理的细胞用点击激活的荧光探针或生物素化的炔被标记时，流式细胞仪荧光显示岩藻糖基化细胞的方法。还公开了通过荧光显微镜观察岩藻糖基化糖缀合物的细胞内定位的方法。

6. 发明申请

US20080241856A1 Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo 有权
标题翻译：用于体内岩藻糖基化聚糖荧光成像的糖蛋白质组学探针
公开(公告)号：US20080241856A1
公开(公告)日：2008-10-02
申请号：US12079228
申请日：2008-03-24
申请人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R. Hanson , Masaaki Sawa
发明人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R. Hanson , Masaaki Sawa
IPC分类号： G01N33/53 , C12P19/44 , C12Q1/00
CPC分类号： G01N33/582 , G01N1/30 , G01N2400/00
摘要： 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.
摘要翻译：本公开提供了通过基于包含炔基的探针的Cu（I）催化的[3 + 2]环加成（点击活化）的荧光标记技术来标记携带叠氮基的细胞聚糖的方法。该方法需要通过Cu（I）催化的[3 + 2]环加成的探针的炔基与非荧光前体4-乙炔基-N-乙基-1,8-萘酰亚胺的生成荧光探针，改性糖。本公开进一步提供了通过岩藻糖补救途径将含叠氮基岩藻糖类似物掺入糖缀合物的方法。本公开内容提供了当用6-叠氮基聚糖处理的细胞用点击激活的荧光探针或生物素化的炔被标记时，流式细胞仪荧光显示岩藻糖基化细胞的方法。还公开了通过荧光显微镜观察岩藻糖基化糖缀合物的细胞内定位的方法。

7. 发明授权

US08329413B2 Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo 有权
标题翻译：用于体内岩藻糖基化聚糖荧光成像的糖蛋白质组学探针
公开(公告)号：US08329413B2
公开(公告)日：2012-12-11
申请号：US13053192
申请日：2011-03-21
申请人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R Hanson , Masaaki Sawa
发明人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R Hanson , Masaaki Sawa
IPC分类号： G01N33/53
CPC分类号： G01N33/582 , G01N1/30 , G01N2400/00
摘要： 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.
摘要翻译：提供了通过含有包含炔基的探针的Cu（I）催化的[3 + 2]环加成的荧光标记来标记携带叠氮基的细胞聚糖的方法。通过Cu（I）催化的[3 + 2]环加成的探针的炔基与叠氮改性的糖，从非荧光前体4-乙炔基-N-乙基-1,8-萘二甲酰亚胺生成荧光探针是提供。公开了通过岩藻糖补救途径将叠氮基岩藻糖类似物掺入糖缀合物。公开了通过用用点击激活的荧光探针或生物素化的炔标记的6-叠氮基聚糖处理的细胞的流式细胞术的岩藻糖基化细胞的荧光可视化。公开了通过荧光显微镜观察岩藻糖基化糖缀合物的细胞内位置。

8. 发明授权

US07943330B2 Tailored glycoproteomic methods for the sequencing, mapping and identification of cellular glycoproteins 有权
标题翻译：用于测序，测定和鉴定细胞糖蛋白的定制糖蛋白学方法
公开(公告)号：US07943330B2
公开(公告)日：2011-05-17
申请号：US12079157
申请日：2008-03-24
申请人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R Hanson
发明人： Chi-Huey Wong , Tsui-Ling Hsu , Sarah R Hanson
IPC分类号： G01N33/53 , G01N31/00
CPC分类号： G01N33/582 , G01N33/6842 , G01N2400/00
摘要： 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-MS2). LC-MS2 may be used to identify cellular glycans, and more specifically cancer-related glycoproteins.
摘要翻译：本公开涉及定制的糖蛋白学方法，更具体地涉及使用糖选择性生物正交探针测序，测定和鉴定细胞糖蛋白的方法。公开了糖选择性糖蛋白鉴定（ID）和聚糖测绘（GIDmap）的方法，其生成可以选择性分离的生物递归标记的炔基糖定制的糖蛋白，允许通过质谱蛋白质组学进行糖蛋白ID和聚糖测定，包括液相色谱 - 坦德质谱（LC-MS2）。 LC-MS2可用于鉴定细胞聚糖，更具体地是癌症相关糖蛋白。

9. 发明授权

US08460669B2 Compositions and methods for identifying response targets and treating flavivirus infection responses 有权
标题翻译：用于鉴定反应目标和治疗黄病毒感染反应的组合物和方法
公开(公告)号：US08460669B2
公开(公告)日：2013-06-11
申请号：US13106046
申请日：2011-05-12
申请人： Shie-Liang Hsieh , Chi-Huey Wong , Tsui-Ling Hsu , Szu-Ting Chen
发明人： Shie-Liang Hsieh , Chi-Huey Wong , Tsui-Ling Hsu , Szu-Ting Chen
IPC分类号： A61K39/395 , A61K39/285
CPC分类号： C07K16/2851 , A61K2039/505 , C07K2317/56 , C07K2317/76 , C07K2319/30 , G01N2333/18 , G01N2333/185 , G01N2500/02 , Y02A50/386 , Y02A50/388 , Y02A50/39 , Y02A50/394
摘要： Cellular receptors are identified that induce plasma leakage and other negative effects when infected with flaviviruses, such as dengue virus or Japanese encephamyelitis virus. Using fusion proteins disclosed herein, the receptors to which a pathogen, such as flavivirus, binds via glycan binding are determined. Once the receptors are determined, the effect of binding to a particular receptor may be determined, wherein targeting of the receptors causing a particular symptom may be targeted by agents that interrupt binding of the pathogen to the receptor. Accordingly, in the case of dengue virus and Japanese encephamyelitis virus, TNF-α is released when the pathogen binds to the DLVR1/CLEC5A receptor. Interrupting the DLVR1/CLEC5A receptor with monoclonal antibodies reduced TNF-α secretion without affecting secretion of cytokines responsible for viral clearance thereby increasing survival rates in infected mice from nil to around 50%.
摘要翻译：鉴定当感染黄病毒如登革热病毒或日本脑炎病毒时诱导血浆渗漏和其它负面影响的细胞受体。使用本文公开的融合蛋白，确定病原体（例如黄病毒）通过聚糖结合结合的受体。一旦确定了受体，就可以确定与特定受体结合的作用，其中导致特定症状的受体的靶向可能被阻断病原体与受体结合的试剂所靶向。因此，在登革热病毒和日本脑炎病毒的情况下，当病原体与DLVR1 / CLEC5A受体结合时，TNF-α被释放。用单克隆抗体中断DLVR1 / CLEC5A受体降低TNF-α分泌，而不影响负责病毒清除的细胞因子分泌，从而将感染小鼠的存活率从零增加到约50％。

10. 发明授权

US07998482B2 Methods and reagents for the analysis and purification of polysaccharides 有权
标题翻译：用于多糖分析和纯化的方法和试剂
公开(公告)号：US07998482B2
公开(公告)日：2011-08-16
申请号：US12533086
申请日：2009-07-31
申请人： Chi-Huey Wong , Shie-Liang Hsieh , Tsui-Ling Hsu , Shih-Chin Cheng , Szu-Ting Chen
发明人： Chi-Huey Wong , Shie-Liang Hsieh , Tsui-Ling Hsu , Shih-Chin Cheng , Szu-Ting Chen
IPC分类号： A61K39/395 , C12N7/00 , A61K39/285
CPC分类号： C07K16/2851 , C07K2317/76 , C07K2319/30 , G01N33/56972 , G01N33/56983 , G01N2333/18 , G01N2500/02 , Y02A50/53
摘要： The disclosure provides fusion proteins comprising a carbohydrate recognition domain of an innate immunity receptor and a heterologous polypeptide. The fusion proteins of the disclosure may be used, for example, to fingerprint polysaccharide compositions and to purify polysaccharide compositions. Polysaccharide compositions include those isolated from Ganoderma lucidum (Reishi). The methods and reagents of the disclosure may also be used to identify innate immunity receptors and cell types that bind to polysaccharide compositions (including polysaccharide compositions associated with pathogens), whereupon modulators of the identified receptors can then be obtained. The fusion proteins also may be used to inhibit the interaction between a polysaccharide composition and an innate immunity receptor on a cell surface. The methods and reagents of the disclosure are used in one example to determine that the DLVR1 innate immunity receptor on macrophages interacts with Dengue virus (DV), and that DLVR1 is responsible for DV-mediated secretion of proinflammatory cytokines from macrophages. The disclosure also provides DVLR1 antibodies that prevent the secretion of proinflammatory cytokines by DV-infected macrophages.
摘要翻译：本公开提供了包含先天免疫受体的碳水化合物识别结构域和异源多肽的融合蛋白。本公开的融合蛋白可以用于例如指纹多糖组合物和纯化多糖组合物。多糖组合物包括从灵芝（Reishi）分离的那些。本公开的方法和试剂也可用于鉴定结合多糖组合物（包括与病原体相关的多糖组合物）的先天免疫受体和细胞类型，于是可以获得鉴定的受体的调节剂。融合蛋白也可用于抑制多糖组合物和细胞表面上的先天免疫受体之间的相互作用。在一个实例中使用本公开的方法和试剂来确定巨噬细胞上的DLVR1先天免疫受体与登革热病毒（DV）相互作用，并且DLVR1负责来自巨噬细胞的DV介导的促炎细胞因子的分泌。本公开还提供DVLR1抗体，其防止DV感染的巨噬细胞分泌促炎细胞因子。

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