Custom Complex N-linked Oligosaccharides Synthesis
CD BioGlyco has extensive experience in the synthesis of custom complex N-linked Oligosaccharides. The unique technology platforms and professional researchers of our company ensure that we offer high-efficient services at a competitive price for our clients. We have the confidence to help our clients accelerate their research on glycobiology by reducing project costs and timelines.
CD BioGlyco employs state-of-the-art chemo-enzymatic synthesis, a powerful approach that merges the flexibility of chemical synthesis with the unparalleled regio- and stereoselectivity of enzymatic reactions. This synergy allows for the precise construction of complex N-glycans, overcoming limitations often encountered with purely chemical or enzymatic methods.
Our core technologies revolve around:
These enzymes are central to our synthesis, catalyzing the formation of glycosidic bonds by transferring saccharides from activated donors to specific acceptors. The increasing availability and characterization of recombinant GTs allow for the installation of diverse glycoside linkages, enabling the preparation of specific N-glycan epitopes and the creation of complex, multi-antennary structures. Our capabilities extend to working with both commercially available and in-house expressed recombinant GTs.
While glycosidases typically hydrolyze glycosidic bonds, engineered variants known as glycosynthases retain the ability to form new glycosidic bonds while suppressing unwanted hydrolysis reactions. This innovative technology is crucial for efficient glycan assembly and for tailoring existing glycan structures. For instance, we utilize glycosynthases, such as engineered endo-β-N-acetylglucosaminidases (ENGases), to precisely attach structurally defined homogeneous N-glycans to glycoproteins, a vital step in producing homogeneous therapeutic proteins like monoclonal antibodies (mAbs).
The availability and stability of activated sugar donors, such as nucleotide sugars (e.g., UDP-GlcNAc, GDP-Fuc, CMP-Sia), are critical for efficient glycosylation. We employ strategies to overcome challenges associated with expensive or unstable sugar donors, including the use of multi-enzyme catalyzed one-pot reactions and in situ sugar nucleotide regeneration systems. We also explore the use of non-natural sugar donors and analogs to expand the scope and flexibility of N-glycan synthesis, enabling the creation of novel glycan structures with enhanced properties.
For increased efficiency and reproducibility, we integrate automated solid-phase synthesis strategies where appropriate. This approach simplifies purification steps and accelerates the synthesis of complex oligosaccharides, ensuring high-throughput capabilities for diverse project requirements.
N-linked oligosaccharide is characterized by the attachment of glycans to an asparagine side chain of the protein. The glycosylation site of N-linked oligosaccharides is Asn-X-Ser/Thr, where X represents any amino acid except proline. N-linked oligosaccharides have important biological significance, including enhancing solubility and stability of protein, acting as signal molecules in protein folding and transportation, determining the specificity of membrane receptors in cell-to-cell interactions or innate immunity, and changing the properties of hormones and secreted proteins.
The biosynthesis of N-linked glycans occurs through 3 major steps: synthesis of dolichol-linked precursor oligosaccharides, en bloc transfer, and initial trimming of precursor oligosaccharides in the endoplasmic reticulum (ER), and Subsequent processing in the Golgi apparatus. At present, multi-step Chemical or biological synthesis of N-linked oligosaccharides with various structures is the main effective way to obtain N-linked oligosaccharides by artificial strategy, which is of great significance to study the relationship between saccharides and disease.
Fig.1 Different stages of N-glycan complexity. (Fisher, 2016)
Equipped with advanced technology platforms and professional research teams in the field of glycobiology, CD BioGlyco has established effective chemical or chemoenzymatic methods to synthesize high-purity N-linked oligosaccharides for our customers. The services we provide include but are not limited to:
Our custom complex N-linked oligosaccharide synthesis follows a meticulously designed workflow to ensure the highest quality and precision for your research and development needs. Our process is adaptable to diverse project requirements, from initial design to final product delivery.
The process begins with an in-depth consultation to understand your specific requirements. Our expert team collaborates with you to define the target N-glycan structure, purity specifications, quantity, and any specific applications. We leverage our extensive knowledge of N-glycan structures and synthesis strategies to design the most efficient and effective chemo-enzymatic route for your custom oligosaccharide.
Depending on the target glycan, we either chemically synthesize key N-glycan modular structures or prepare N-glycan precursors from natural sources through enzymatic digestion.
This is the core of our chemo-enzymatic approach. Using a carefully selected panel of highly specific GTs and engineered glycosidases, we sequentially add monosaccharide units to the precursor. This step allows for precise control over glycosidic linkages and branching patterns, enabling the creation of diverse and complex N-glycan structures, including those with sialylation and fucosylation.
Following synthesis, the custom N-glycans undergo rigorous purification processes to achieve the desired purity level. This often involves techniques such as high-performance liquid chromatography (HPLC) and other advanced chromatographic methods. Subsequently, comprehensive characterization is performed using techniques like mass spectrometry and nuclear magnetic resonance (NMR) to confirm the exact structure, purity, and integrity of the synthesized oligosaccharide.
Each batch of custom N-glycans undergoes stringent quality control checks to ensure it meets the highest standards and client specifications. Upon successful validation, the product is prepared for delivery, accompanied by a detailed certificate of analysis.
DOI.: 10.3390/biom10040624
Journal: Biomolecules
IF: 4.8
Published: 2020
Results: This review article explores the structural mechanisms of N-linked glycosylation catalyzed by the enzyme OST across different domains of life. It highlights how high-resolution structures (particularly recent cryo-EM structures of yeast and human OST complexes) reveal that the catalytic subunit Stt3/STT3 (and its bacterial/archaeal counterparts PglB/AglB) employs a conserved "carboxamide twist" mechanism. In this mechanism, two key acidic residues (e.g., D56 and E319 in bacterial PglB) activate the acceptor asparagine side chain by forming hydrogen bonds with its amide protons, facilitating nucleophilic attack on the lipid-linked oligosaccharide (LLO) donor. The review contrasts the simple single-subunit OST in prokaryotes with the complex multi-subunit OST assemblies in eukaryotes (yeast OST and mammalian OST-A/OST-B isoforms), discussing the roles of non-catalytic subunits and the distinct functions of human OST-A (primary co-translational glycosylation) versus OST-B (proofreading missed sites). Overall, the structures provide insights into sequon recognition, catalytic metal ion coordination, and the evolutionary conservation of the core glycosylation machinery.
To further support your research and development in glycobiology, we offer a suite of complementary services and related products that can seamlessly integrate with our custom complex N-linked oligosaccharide synthesis offerings.
Glycoprotein Analysis Services
Beyond synthesis, we provide comprehensive analytical services for glycoproteins, including glycan profiling, site-specific glycosylation analysis, and glycopeptide mapping.
Cell-based N-Glycan Array
For high-throughput screening of glycan-binding proteins or antibodies, We assist in the development of custom glycan arrays utilizing our diverse library of synthesized oligosaccharides.
CD BioGlyco has successfully synthesized a variety of complex N-linked oligosaccharides through multi-step synthesis. We have the confidence to offer custom complex types of N-glycans synthesis service with high quality for our clients. If you are interested in our services, please contact us for more detailed information.
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