Overview

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.

Key Technologies

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:

Glycosyltransferases (GTs)

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.

Glycosidases (GHs) and Glycosynthases

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).

Optimized Sugar Donors

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.

Automated Synthesis Platforms

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.

Background

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)

Services

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:

• Characterization and profiling of N-linked oligosaccharides.
• Synthesis of various N-linked oligosaccharides by effective and concise chemical methods.
• Synthesis of complex N-linked oligosaccharides through the chemoenzymatic method according to clients' demands.
• Various modifications of N-linked oligosaccharides according to customers' scientific requirements.
• Purity and quality detection of customized synthesized N-linked oligosaccharides.

Workflow

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.

Consultation and Design

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.

Precursor Preparation

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.

Enzymatic Elongation and Modification

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.

Purification and Characterization

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.

Quality Control and Delivery

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.

Publication Data

Advantages

• Our chemo-enzymatic approach ensures exceptional regio- and stereoselectivity, delivering structurally defined homogeneous N-glycans.
• By leveraging optimized enzymatic reactions, our methods often circumvent the need for extensive protecting group strategies and harsh reaction conditions typically associated with chemical synthesis.
• We offer highly customized synthesis services, working closely with clients to meet their exact specifications for N-glycan structures, quantities, and purity levels.

Application

• Our homogeneous N-glycans are essential for detailed investigations into the biological roles of glycans, including their involvement in protein folding, cell-cell recognition, and signal transduction pathways.
• Access to defined N-glycan structures is crucial for the development of glycan-based therapeutics, vaccines, and diagnostics.
• Custom N-glycans serve as invaluable standards and probes for the discovery and validation of novel glycan biomarkers for early disease detection, diagnosis, and prognosis.

Associated Services

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.

Frequently Asked Questions

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.