- Nanobody-Engineered Natural Killer Cell Conjugates for Solid Tumor Adoptive Immunotherapy
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Cancer immunotherapy based on natural killer (NK) cells is demonstrated to be a promising strategy. However, NK cells are deficient in ligands that target specific tumors, resulting in limited antitumor efficacy. Here, a glycoengineering approach to imitate the chimeric antigen receptor strategy and decorate NK cells with nanobodies to promote NK-based immunotherapy in solid tumors is proposed. Nanobody 7D12, which specifically recognizes the human epidermal growth factor receptor (EGFR) that is overexpressed on many solid tumors, is coupled to the chemically synthesized DBCO-PEG4-GGG-NH2 by sortase A-mediated ligation to generate DBCO-7D12. The NK92MI cells bearing azide groups are then equipped with DBCO-7D12 via bioorthogonal click chemistry. The resultant 7D12-NK92MI cells exhibit high specificity and affinity for EGFR-overexpressing tumor cells in vitro and in vivo by the 7D12-EGFR interaction, causing increased cytokine secretion to more effectively kill EGFR-positive tumor cells, but not EGFR-negative cancer cells. Importantly, the 7D12-NK92MI cells also show a wide anticancer spectrum and extensive tumor penetration. Furthermore, mouse experiments reveal that 7D12-NK92MI treatment achieves excellent therapeutic efficacy and outstanding safety. The authors’ works provide a cell modification strategy using specific protein ligands without genetic manipulation and present a potential novel method for cancer-targeted immunotherapy by NK cells.
- Chen, Ying,Cui, Kaisa,Gong, Liang,Hong, Haofei,Huang, Zhaohui,Li, Dan,Li, Jiuming,Li, Yanchun,Wu, Zhimeng,Yin, Yuan
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- Selective Engineering of Linkage-Specific α2,6-N-Linked Sialoproteins Using Sydnone-Modified Sialic Acid Bioorthogonal Reporters
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The metabolic oligosaccharide engineering (MOE) strategy using unnatural sialic acids has recently enabled the visualization of the sialome in living systems. However, MOE only reports on global sialylation and dissected information regarding subsets of s
- Chinoy, Zoeisha S.,Bodineau, Clément,Favre, Camille,Moremen, Kelley W.,Durán, Raúl V.,Friscourt, Frédéric
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p. 4281 - 4285
(2019/02/28)
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- The Bioorthogonal Isonitrile-Chlorooxime Ligation
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Bioorthogonal reactions are valuable tools for the selective labeling and imaging of natural products and proteins. Here, we present the reaction between isonitriles and chlorooximes as a ligation that proceeds quickly (k ≈ 1 M-1 s-1
- Li, Mao,Monaco, Mattia R.,Rivera-Fuentes, Pablo,Sch?fer, Rebecca J. B.,Tirla, Alina,Wennemers, Helma
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p. 18644 - 18648
(2019/11/28)
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- Novel Liposomal Azido Mannosamine Lipids on Metabolic Cell Labeling and Imaging via Cu-Free Click Chemistry
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In comparison with the popular Ac4ManNAz applied as cell labels via Cu-free click chemistry, two novel azido mannosamine lipids with C6 and C12 esters on anomeric hydroxyl groups were prepared and encapsulated in a liposome delivery system, which enhanced chemical stabilities and showed good cell-metabolizable labeling efficiency on MDA-MB-231 cells with strong fluorescence after the treatment of DBCO-Cy5 by triazole formation via click chemistry.
- Shen, Li,Cai, Kaimin,Yu, Jin,Cheng, Jianjun
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p. 2317 - 2322
(2019/09/06)
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- Synthesis of a novel fluorescent ruthenium complex with an appended Ac4GlcNAc moiety by click reaction
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The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells, which plays a fundamental role in the activity of many cells and is associated with pathologies like type II diabetes, Alzheimer’s disease or some cancers. However, the precise connexion between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Confocal microscopy is a powerful and effective tool for in-cell elucidation of the function of biological molecules. Chemical labeling of non-ultraviolet or non-fluorescent carbohydrates with fluorescent tag is an essential step that makes intra-cellular microscopic inspection possible. Here we report a strategy based on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues Ac4GlcNAc (substituted with an azido group) and the corresponding fluorescent tag Ru(bpy)2(Phen-alkyne)Cl2 (4) to synthesize the fluorescent dye Ru(bpy)2(Phen-Ac4GlcNAc)Cl2 (5) under mild and neutral reaction conditions. Moreover, 5 showed good stability, desirable fluorescence characteristics, and exhibited rather low levels of cytotoxicity against sensitive MCF-7 cells. Additionally, we have achieved successful fluorescent imaging of 5 transported in living MCF-7 cells. Cell images displayed that proteins are potentially labelled with 5 in the cytoplasm.
- Cheng, Qi,Cui, Yalu,Xiao, Nao,Lu, Jishun,Fang, Chen-Jie
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- Design, synthesis and cytotoxic activity of N-Modified oleanolic saponins bearing A glucosamine
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A series of N-acyl, N-alkoxycarbonyl, and N-alkylcarbamoyl derivatives of 2′-deoxy-glucosyl bearing oleanolic saponins were synthesized and evaluated against HL-60, PC-3, and HT29 tumor cancer cells. The SAR studies revealed that the activity increased in order of conjugation of 2′ -amino group with carbamate > amide > urea derivatives. Lengthening the alkyl chain increased the cytotoxicity, the peak activity was found to around heptyl to nonyl substitutions. 2′-N-heptoxycarbonyl derivative 56 was found to be the most cytotoxic (IC50 = 0.76 μM) against HL-60 cells. Due to the interesting SARs of alkyl substitutions, we hypothesized that their location in the cell was different, and pursued a location study using 2′-(4″-pentynoylamino) 2′-deoxy-glucosyl OA, which suggested that these compounds distributed mainly in the cytosol.
- Lin, You-Yu,Chan, She-Hung,Juang, Yu-Pu,Hsiao, Hsin-Min,Guh, Jih-Hwa,Liang, Pi-Hui
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supporting information
p. 1942 - 1958
(2017/11/16)
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- Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates
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The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells have proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here, we report on the creation of fluorescent uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT, we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time- and dose-dependent manner. Because no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-color pulse chase experiments and monitoring OGT activity on specific protein substrates in live cells.
- Tan, Hong Yee,Eskandari, Razieh,Shen, David,Zhu, Yanping,Liu, Ta-Wei,Willems, Lianne I.,Alteen, Matthew G.,Madden, Zarina,Vocadlo, David J.
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p. 15300 - 15308
(2018/11/03)
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- TRIGGER-ACTIVATABLE SUGAR CONJUGATES FOR CANCER-SELECTIVE LABELING AND TARGETING
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Disclosed are compounds for the selective labeling of cell-surface sugars in cancer cells. The compounds are activatable by triggers specific to cancer cells, and, when metabolized, label a cancer cell surface sugar with an azide chemical group. Facilitated by a click chemistry reaction, combination of the cell surface-expressed azide with a alkynyl-drug conjugate enables efficient targeted drug delivery to cancer cells with reduced toxicity. Also disclosed are compounds for delivering a drug to an azide-bearing cancer cell, and methods of treating cancer using the compounds.
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Page/Page column 69; 70
(2018/09/08)
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- METHODS FOR PREPARING ANTIBODIES WITH A DEFINED GLYCOSYLATION PATTERN
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The present disclosure relates to a process for preparing antibodies with a defined glycosylation pattern, in particular antibodies with a glycan terminating in an N-acetylglucosamine. The antibodies of the disclosure are suitable for use in a process to conjugate a payload thereto. The disclosure also extends to molecules obtained and obtainable from the process disclosed herein, novel molecules and intermediates, compositions comprising said molecules and uses of the molecules and compositions, particularly in treatment, for example in the treatment of cancer.
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- The synthesis of new fluorinated or nonfluorinated sugar phosphonates and phosphoramidates as building blocks in the synthesis of modified hyaluronic acid subunits
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The synthesis of several new fluorinated or nonfluorinated sugar phosphonates and phosphoramidates as building blocks for the synthesis of modified hyaluronic acid subunits is described. These compounds were prepared from d-glucose and d-glucosamine hydro
- Koroniak-Szejn, Katarzyna,Tomaszewska, Joanna,Koroniak, Henryk
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p. 683 - 694
(2017/06/05)
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- TRIGGER-ACTIVATABLE METABOLIC SUGAR PRECURSORS FOR CANCER-SELECTIVE LABELING AND TARGETING
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Disclosed are compounds for the selective labeling of cell-surface sugars in cancer cells. The compounds are activatable by triggers specific to cancer cells, and, when metabolized, label a cancer cell surface sugar with an azide chemical group. Facilitated by a click chemistry reaction, combination of the cell surface-expressed azide with a alkynyl- drug conjugate enables efficient targeted drug delivery to cancer cells with reduced toxicity Also disclosed are compounds for delivering a drug to an azide-bearing cancer cell, and methods of treating cancer using the compounds of the invention.
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Page/Page column 51; 52
(2017/08/01)
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- Glucosamine- and galactosamine- based monosaccharides with highly fluorinated motifs
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Synthesis of modified monosaccharides, derivatives of glucose and galactose, having a highly fluorinated chain, as a library of synthetic building blocks for hyaluronic acid (HA) modified subunits has been developed. “Click” chemistry has been employed as a strategy for the synthesis of these molecules. 1,2,3-triazole ring derivatives were obtained with good to excellent yields.
- Tomaszewska, Joanna,Kowalska, Karolina,Koroniak-Szejn, Katarzyna
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- Targeted Ultrasound-Assisted Cancer-Selective Chemical Labeling and Subsequent Cancer Imaging using Click Chemistry
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Metabolic sugar labeling followed by the use of reagent-free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N-azidoacetylmannosamine (Ac4ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4ManAz-loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac4ManAz-loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies.
- Wang, Hua,Gauthier, Marianne,Kelly, Jamie R.,Miller, Rita J.,Xu, Ming,O'Brien, William D.,Cheng, Jianjun
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supporting information
p. 5452 - 5456
(2016/05/09)
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- A chemoenzymatic approach toward the preparation of site-specific antibody-drug conjugates
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An efficient chemical synthesis of UDP-N-azidoacetylgalactosamine (UDP-GalNAz) is presented, while the value of this molecule was demonstrated through its attachment to an antibody Fc domain. Thus, the antibody was first degalactosylated, which was followed by loading of the UDP-GalNAz with a recombinant galactosyltransferase. This engineered Azide-Fc-N-glycan antibody was subsequently 'clicked' by a strain-promoted alkyne-azide cycloaddition reaction for site-specific attachment of a fluorescent probe. The principles detailed will allow for the facile preparation of chemically defined homogeneous antibody-drug conjugates (ADCs).
- Cai, Xiaoqing,Janda, Kim D.
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p. 3172 - 3175
(2015/06/02)
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- Synthesis of NAG-thiazoline-derived inhibitors for β-N-acetyl-d-hexosaminidases
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Abstract β-N-Acetyl-d-hexosaminidases are responsible for the metabolism of glycoconjugates in diverse physiological processes that are important targets for medicine and pesticide development. Fourteen new NAG-thiazoline derivatives were synthesized by cyclization and click reaction using d-glucosamine hydrochloride as the starting material. All the compounds created were characterized by NMR and HRMS spectra. A preliminary bioassay, using four enzymes from two β-N-acetyl-d-hexosaminidase families, showed that most of the compounds synthesized exhibit selective inhibition of GH84 β-N-acetyl-d-hexosaminidase. Among the compounds tested, compounds 5a (IC50=12.6 μM, hOGA) and 5e (IC50=12.5 μM, OfOGA) proved to be a highly selective and potent inhibitor.
- Kong, Hanchu,Chen, Wei,Lu, Huizhe,Yang, Qing,Dong, Yanhong,Wang, Daoquan,Zhang, Jianjun
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p. 135 - 144
(2015/07/07)
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- Peptoids and polyamines going sweet: Modular synthesis of glycosylated peptoids and polyamines using click chemistry
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Sugar moieties are present in a wide range of bioactive molecules. Thus, having versatile and fast methods for the decoration of biomimetic molecules with sugars is of fundamental importance. The glycosylation of peptoids and polyamines as examples of such biomimetic molecules is reported here. The method uses Cu-catalyzed azide alkyne cycloaddition to promote the reaction of azidosugars with either polyamines or peptoids. In addition, functionalized nucleic acids were attached to polyamines via the same route. Based on a modular solid-phase synthesis of peralkynylated peptoids with up to six alkyne groups, the latter were modified with azidosugar building blocks by using copper-catalyzed azide alkyne cycloadditions. In addition, the up-scaling of some particular azide-modified sugars is described.
- Fuerniss, Daniel,Mack, Timo,Hahn, Frank,Vollrath, Sidonie B. L.,Koroniak, Katarzyna,Schepers, Ute,Braese, Stefan
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- Modified GM3 gangliosides produced by metabolic oligosaccharide engineering
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Metabolic oligosaccharide engineering is powerful approach to altering the structure of cellular sialosides. This method relies on culturing cells with N-acetylmannosamine (ManNAc) analogs that are metabolized to their sialic acid counterparts and added to glycoproteins and glycolipids. Here we employed two cell lines that are deficient in ManNAc biosynthesis and examined their relative abilities to metabolize a panel of ManNAc analogs to sialosides. In addition to measuring global sialoside production, we also examined biosynthesis of the sialic acid-containing glycolipid, GM3. We discovered that the two cell lines differ in their ability to discriminate among the variant forms of ManNAc. Further, our data suggest that modified forms of sialic acid may be preferentially incorporated into certain sialosides and excluded from others. Taken together, our results demonstrate that global analysis of sialoside production can obscure sialoside-specific differences. These findings have implications for downstream applications of metabolic oligosaccharide engineering, including imaging and proteomics.
- Whitman, Chad M.,Yang, Fan,Kohler, Jennifer J.
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p. 5006 - 5010
(2011/10/09)
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- Chemoselective attachment of small molecule effector functionality to human adenoviruses facilitates gene delivery to cancer cells
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We demonstrate here a novel two-step "click" labeling process in which adenoviral particles are first metabolically labeled during production with unnatural azido sugars. Subsequent chemoselective modification allows access to viruses decorated with a broad array of effector functionality. Adenoviruses modified with folate, a known cancer-targeting motif, demonstrated a marked increase in gene delivery to a murine cancer cell line.
- Banerjee, Partha Sarathi,Ostapchuk, Philomena,Hearing, Patrick,Carrico, Isaac
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supporting information; scheme or table
p. 13615 - 13617
(2010/12/19)
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- O-GlcNAc peptide epoxyketones are recognized by mammalian proteasomes
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(Chemical Equation Presented) Cytosolic and nuclear proteins may be subject to both O-GlcNAcylation and proteasomal degradation. By means of activity-based profiling, we demonstrate that O-GlcNAc serinecontaining peptide epoxyketones bind to the proteasome catalytic active sites and thus provide the first clear evidence that proteasomes recognize peptides post-translationally modified with a GlcNAc moiety.
- Witte, Martin D.,Florea, Bogdan I.,Verdoes, Martijn,Adeyanju, Oloruntosin,Van Der Marel, Gijs A.,Overkleeft, Herman S.
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supporting information; experimental part
p. 12064 - 12065
(2010/01/30)
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- Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation
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The structure of sialic acid on living cells can be modulated by metabolism of unnatural biosynthetic precursors. Here we investigate the conversion of a panel of azide-functionalized mannosamine and glucosamine derivatives into cell-surface sialosides. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. The reaction provided a means to label the glycoconjugate-bound azidosugars with biochemical probes. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes.
- Saxon, Eliana,Luchansky, Sarah J.,Hang, Howard C.,Yu, Chong,Lee, Sandy C.,Bertozzi, Carolyn R.
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p. 14893 - 14902
(2007/10/03)
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