ABSTRACT
Platinum-drug-based chemotherapy in clinics has achieved great success in clinical malignancy therapy. However, unpredictable off-target toxicity and the resulting severe side effects in the treatment are still unsolved problems. Although metabolic glycan labeling-mediated tumor-targeted therapy has been widely reported, less selective metabolic labeling in vivo limited its wide application. Herein, a novel probe of B-Ac3ManNAz that is regulated by reactive oxygen species in tumor cells is introduced to enhance the recognition and cytotoxicity of DBCO-modified oxaliplatin(IV) via bioorthogonal chemistry. B-Ac3ManNAz was synthesized from Ac4ManNAz by incorporation with 4-(hydroxymethyl) benzeneboronic acid pinacol ester (HBAPE) at the anomeric position, which is confirmed to be regulated by ROS and could robustly label glycans on the cell surface. Moreover, N3-treated tumor cells could enhance the tumor accumulation of DBCO-modified oxaliplatin(IV) via click chemistry meanwhile reduce the off-target distribution in normal tissue. Our strategy provides an effective metabolic precursor for tumor-specific labeling and targeted cancer therapies.
Subject(s)
Antineoplastic Agents , Oxaliplatin , Polysaccharides , Prodrugs , Reactive Oxygen Species , Prodrugs/chemistry , Prodrugs/pharmacology , Prodrugs/chemical synthesis , Oxaliplatin/pharmacology , Oxaliplatin/chemistry , Humans , Reactive Oxygen Species/metabolism , Polysaccharides/chemistry , Polysaccharides/metabolism , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Mice , Cell Line, Tumor , Mice, Inbred BALB C , Mice, NudeABSTRACT
O-GlcNAcylation is a ubiquitous post-translational modification governing vital biological processes in cancer, diabetes and neurodegeneration. Metabolic chemical reporters (MCRs) containing bio-orthogonal groups such as azido or alkyne, are widely used for labeling of interested proteins. However, most MCRs developed for O-GlcNAc modification are not specific and always lead to unexpected side reactions termed S-glyco-modification. Here, we attempt to develop a new MCR of Ac34FGlcNAz that replacing the 4-OH of Ac4GlcNAz with fluorine, which is supposed to abolish the epimerization of GALE and enhance the selectivity. The discoveries demonstrate that Ac34FGlcNAz is a powerful MCR for O-GlcNAcylation with high efficiency and the process of this labeling is conducted by the two enzymes of OGT and OGA. Most importantly, Ac34FGlcNAz is predominantly incorporated intracellular proteins in the form of O-linkage and leads to negligible S-glyco-modification, indicating it is a selective MCR for O-GlcNAcylation. Therefore, we reason that Ac34FGlcNAz developed here is a well characterized MCR of O-GlcNAcylation, which provides more choice for label and enrichment of O-GlcNAc associated proteins.
Subject(s)
Protein Processing, Post-Translational , Proteins , Acetylglucosamine/chemistry , Proteins/chemistry , AcylationABSTRACT
Galactose is a naturally occurring monosaccharide used to build complex glycans that has not been targeted for labeling as a metabolic reporter. Here, we characterize the cellular modification of proteins by using Ac46AzGal in a dose- and time-dependent manner. It is noted that a vast majority of this labeling of Ac46AzGal occurs intracellularly in a range of mammalian cells. We also provided evidence that this labeling is dependent on not only the enzymes of OGT responsible for O-GlcNAcylation but also the enzymes of GALT and GALE in the Leloir pathway. Notably, we discover that Ac46AzGal is not the direct substrate of OGT, and the labeling results may attribute to UDP-6AzGlc after epimerization of UDP-6AzGal via GALE. Together, these discoveries support the conclusion that Ac46AzGal as an analogue of galactose could metabolically label intracellular O-glycosylation modification, raising the possibility of characterization with impaired functions of the galactose metabolism in the Leloir pathway under certain conditions, such as galactosemias.
ABSTRACT
A facile and convergent procedure for the synthesis of azobenzene-based probe was reported, which could selectively release interested proteins conducted with sodium dithionite. Besides, the cleavage efficiency is closely associated with the structural features, in which an ortho-hydroxyl substituent is necessary for reactivity. In addition, the azobenzene tag applied in the Ac4GlcNAz-labled proteins demonstrated high efficiency and selectivity in comparison with Biotin-PEG4-Alkyne, which provides a useful platform for enrichment of any desired bioorthogonal proteomics.
Subject(s)
Acetylglucosamine/metabolism , Alkynes/metabolism , Azides/metabolism , Dithionite/metabolism , N-Acetylglucosaminyltransferases/metabolism , Acetylglucosamine/chemistry , Alkynes/chemistry , Azides/chemistry , Cycloaddition Reaction , Dithionite/chemical synthesis , Dithionite/chemistry , Molecular Structure , N-Acetylglucosaminyltransferases/chemistry , ProteomicsABSTRACT
Currently, controllable linker cleavage at the target site will facilitate the clinical treatment of cancer. Dual-functional prodrugs in combination of carbohydrate as targeting group and pH-sensitive cleavable linker are desired in clinical development. Here, a qualified structure of N-phenylcarbamate-d-gluconhydroximo-1,5-lactam was employed and proved to be a potential candidate prodrug in the drug design. To proof this concept, the possible mechanism of Beckmann rearrangement and the degraded products were confirmed by HPLC and LC-MS under the acid condition mimic lysosome. Hence, the strategy of d-gluconhydroximo-1,5-lactam as a prodrug carrier fabricated with interested drugs will provide a great potential approach for chemotherapy.
