Why Do We Produce GM3 Ganglioside?

GM3 is a major ganglioside that plays a role in the formation of other complex gangliosides. It has been associated with cancer and is considered a significant cancer antigen. Levels of GM3 gangliosides in human serum have been linked to metabolic disease risk factors. Increased GM3 synthesis is connected to insulin resistance and impaired wound healing in patients with type 2 diabetes. GM3 inhibits the activities of receptor tyrosine kinases, such as the epidermal growth factor receptor. There is a variant of GM3 called Neu5Gc-GM3 that has been found in different types of cancer. In clinical trials, Neu5Gc-GM3 has been used in the treatment of breast cancer. Developing efficient methods for synthesizing diverse GM3 gangliosides is a priority.

Fig.1 Structure of GM3 ganglioside. (Labrada, et al., 2023)

Tumor-Associated GM3 Ganglioside Antigen Production Service at CD BioGlyco

We provide a simplified synthesis of GM3 by employing strategic reactions and reducing purification steps.

• The N(Boc)-protected amino alcohol acetonide is a starting material that is protected using BF₃·Et₂O, generating acetonide. For the synthesis of LacβSph, acetonide is directly reacted with lithium dimethyl methylphosphonate to form β-ketophosphonate. The E-olefin geometry in the sphingoid base skeleton is established via the Horner-Wadsworth-Emmons reaction. β-Ketophosphonate reacts with myristyl aldehyde to exclusively yield an E-olefin derivative.
• Column purification enhances the diastereomeric excess. We perform the Luche reduction to produce a mixture of R and S diastereomers. Diastereomers are deprotected using p-TsOH/MeOH, followed by the formation of a six-membered ring acetal leads to a clean separation of isomers. The desired acetal-protected d-erythro isomer is obtained by crystallization from the hexane/EtOAc mixture.
• The isomer is then converted to diBoc-protected sphingosine by selectively opening the acetal with AlH₃. The diBoc-protected sphingosine acts as an acceptor for glycosylation and couples with lactosyl trichloroacetimidate, resulting in a β-linked product. Global deprotection produces LacβSph serves as the acceptor substrate for enzymatic synthesis of GM3 glycosphingosines and their derivatives.

Our synthetic route offers a more efficient and economical approach to the production of sphingosine and related compounds. Subsequently, we synthesize GM3 gangliosides with different forms of sialic acid by first performing OPME synthesis of GM3 sphingosines, followed by chemically acylating the sphingosine chain.

• We successfully installed a fatty acyl chain onto GM3βSph by coupling palmitoyl chloride with the amine in sphingosine. This method is robust, and the acylation reactions of compounds with stearoyl chloride are completed within only 2 hours under the aforementioned conditions. Notably, GM3 gangliosides, which encompass various sialic acid forms, are synthesized with nearly quantitative yields.
• Moreover, we achieve high yields in introducing fatty acyl chains of different lengths, with or without cis- or trans-double bonds. The reactions of GM3βSph with myristoyl chloride, palmitoyl chloride, 10-undecenoyl chloride, and (Z)-9-octadecenoyl chloride results in the production of GM3 gangliosides with different fatty acyl chains.

These compounds are tested for their effects on the proliferation of HCT-116 cells, a human colon cancer cell line, and highly metastatic B16-F10 cells, a murine melanoma cell line, using the MTT assay. Additionally, an in vitro wound healing experiment is performed to study the compounds’ influence on the migration ability of B16-F10 cells. These tests provide valuable insights into the potential therapeutic effects of the compounds in inhibiting cell proliferation and migration. By evaluating their impact on these fundamental cellular processes, these compounds demonstrate promising prospects for further research and potential application in the field of cancer treatment.

We also provide GM3 conjugate vaccine with carriers, including outer membrane protein complex from Neisseria meningitidis and very low-density serum lipoproteins (VLDL). We will culture melanoma cells that are inoculated into the mice. Subsequently, serological assays are performed to evaluate the effect of our vaccine. We evaluate whether the tumor protection induced by the GM3 vaccine is specific, adjuvant-dependent, and non-transient.

Purification of GM3 gangliosides

Fig.2 The synthesis process. (CD BioGlyco)

Applications

• Anti-ganglioside antibodies (AGAs) to GM3 can be used as diagnostic and prognostic markers for autoimmune diseases, neurodegenerative disorders, malignancy, diabetes, and inflammation. Increased levels of serum IgG anti-GM3 antibodies have been observed in patients with secondary progressive MS, throat cancer, elderly people with diabetes, and animal models subjected to lead intoxication.
• Early-stage multiple sclerosis (MS) patients show a significant decrease in serum GM3 levels, which correlates with early damage and severe destruction of the blood-brain barrier. Monitoring serum GM3 levels may aid in early therapy initiation.
• Imbalances in specific GM3 species, especially in conditions like obesity and metabolic syndrome, have been linked to disease progression. Further research is needed to fully understand the implications and applications of GM3 in these conditions.

Advantages

• Our approach minimizes the need for column purification steps, making it suitable for large-scale chemical synthesis of LacβSph from cost-effective l-serine derivatives.
• The clean separation of d-erythro and d-three sphingosine derivatives by forming six-membered acetals and subsequent recrystallization enhances the reliability and adaptability of the process for large-scale synthesis.
• The hydrophobic tag of sphingosine in water-soluble LacβSph enables easy purification using a simple C18-cartridge purification process, facilitating efficient purification from enzymatic reaction mixtures.
• The OPME sialylation system with in situ generation of CMP-sialic acids allows for the convenient incorporation of different sialic acid forms, including natural sialic acids Neu5Ac and Neu5Gc, as well as sialic acid derivatives with F, N₃, or diazirine substitutions.
• The installation of desired fatty acyl chains in the final step of the process yields high product yields, offering a streamlined synthesis and purification procedure for the production of GM3 gangliosides with diverse fatty acyl chains.
• The chemoenzymatic method shows high efficiency in the total synthesis of a diverse library of GM3 gangliosides, encompassing different sialic acid residues.
• The method’s combination of highly efficient OPME sialylation and downstream chemical acylation processes can serve as a general strategy for producing diverse GM3 gangliosides on a preparative scale.

CD BioGlyco specializes in the production of tumor-associated GM3 ganglioside antigens. Our focus is on delivering high-quality carbohydrate antigens through our advanced Glyco™ Vaccine Development Service Platform. With state-of-the-art technologies and stringent quality control measures, we ensure the structural integrity and immunogenicity of our antigens. If you have any inquiries about our services, please don’t hesitate to contact us.

Reference

1. Labrada, M.; et al. GM3 (Neu5Gc) ganglioside: an evolution fixed neoantigen for cancer immunotherapy. Seminars in oncology. 2018, 45(1-2): 41-51.