• We provide a highly efficient one-pot three-enzyme chemoenzymatic approach to prepare α2-3- or α2-6-linked mono-sialylated oligosaccharides in a streamlined manner. This innovative system utilizes N-acetylmannosamine (ManNAc), mannose (Man), or their modified derivatives as substrates, which are then coupled with pyruvate to form sialic acid derivatives through the catalysis of a sialic acid aldolase enzyme. These sialic acid derivatives are subsequently activated by a CMP-sialic acid synthetase enzyme and transferred to an acceptor molecule through the action of a suitable sialyltransferase enzyme, resulting in the formation of sialosides.
  • Our chemoenzymatic approach possesses the ability to produce α2-3- or α2-6-linked sialosides efficiently and in a single pot, without the need for intermediate purification steps.
  • The selectivity of the sialyltransferase enzyme determines whether α2-3- or α2-6-linked sialosides are synthesized, offering versatility in the preparation of mono-sialylated glycans.
  • The sialic acid aldolases from E. coli K12 and Pasteurella multocida, the CMP-sialic acid synthetase from N. meningitidis, the multifunctional sialyltransferase from P. multocida (PmST1) for α2-3-linked sialosides, and the sialyltransferase from Photobacterium damsela (Pd2,6ST) for α2-6-linked sialosides prove to be excellent catalysts due to their high expression levels, robust activity, and broad substrate specificity.
• With the α2-3- and α2-6-linked mono sialylated oligosaccharides in hand, we further expand the synthetic scope by incorporating α2-8-linked disialyl oligosaccharides using a one-pot multienzyme process.
• In this step, the α2-8-sialyltransferase CstIIΔ32I53S and the NmCSS enzyme, with or without the E. coli K12 sialic acid aldolase, are utilized.
• The method is successfully applied in the synthesis of targeted GD3-type disialyl glycans, which belong to two major groups: one group with a penultimate α2-3-linked Neu5Ac and different forms of terminal α2-8-linked sialic acid, and the other group with a terminal α2-8-linked Neu5Ac and different forms of α2-3-linked penultimate sialic acid.
• Additionally, we also synthesize GD3-type disialyl glycans containing the combination of two other common sialic acid forms, Neu5Gc and KDN.

Fig.2 Two-step multi-enzyme chemoenzymatic synthesis. (CD BioGlyco)

Besides, we generate GD3 conjugate as a vaccine against malignant melanoma. GD3 is altered through the process of ozone cleavage of the double bond in the ceramide backbone, resulting in the introduction of an aldehyde group. Subsequently, we conjugate GD3 with ε-amino-lysyl groups of proteins using reductive amination. We offer the following substances for coupling.

• Keyhole limpet hemocyanin (KLH)
• Outer membrane proteins (OMP) of Neisseria meningitidis
• Cationized bovine serum albumin
• Polylysine

Applications

• GD3 can be used as a cell-surface biomarker for identifying NSCs in the embryonic, postnatal, and adult brains.
• GD3 is considered a tumor-specific antigen, making it a target for tumor treatment. Researchers have developed anti-GD3 monoclonal antibodies or immune cell therapies for the treatment of various types of tumors, including melanoma, pancreatic cancer, and neuroblastoma.
• GD3 has been investigated as a diagnostic marker for certain types of cancer. Its expression levels or presence on tumor cells may serve as a biomarker to aid in the identification and monitoring of cancer progression.

Advantages

• The method uses a one-pot three-enzyme chemoenzymatic approach, allowing for the efficient synthesis of α2-3 or α2-6 linked mono-sialylated oligosaccharides in a single reaction system. The absence of intermediate purification steps simplifies the synthesis process and improves overall efficiency.
• The method employs enzymes from different sources as catalysts, including bacterial sialic acid aldolase, CMP-sialic acid synthetase, and sialyltransferase. These enzymes can be expressed in large amounts with high activity and substrate specificity, making the method easily scalable for larger-scale synthesis.
• The sialyltransferase used in this method, CstIIΔ32I53S, exhibits promiscuous donor substrate specificity. It can catalyze the transfer of different sialic acids from CMP-sialic acid derivatives synthesized by NmCSS, with or without E. coli K12 sialic acid aldolase, to form mono-sialylated glycans with different terminal sialic acid forms. This versatility allows for the synthesis of a wide range of targeted glycans.

CD BioGlyco is a leading provider of tumor-associated GD3 ganglioside antigens. We are dedicated to delivering carbohydrate antigens of the highest quality through our cutting-edge Glyco™ Vaccine Development Service Platform. For any inquiries about our services, please do not hesitate to contact us. We are always there and ready to assist you.

Reference

1. Giussani, P.; et al. Sphingolipids: key regulators of apoptosis and pivotal players in cancer drug resistance. International journal of molecular sciences. 2014, 15(3): 4356-4392.