Chemoenzymatic Synthesis Of Floumnated Thomsen-Friedenreich (T)Antigens And Their Sialylated Derivatives

Overexpression of tumor-associated carbohydrate antigens (TACAs) on cell surfaces is a common phenomenon for cancer progression. In the last two decades, there has been a great effort invested in the synthesis and development of TACA-based vaccines for anti-cancer therapy. Despite some promising results in clinical trials, cancer vaccines based on natural TACA structures were not able to induce sufficient T cell-mediated immunity (IgG). This is because many natural TACAs are also presented on normal human tissues at a lower level and are considered as "self" by the human immune system. In addition, natural TACA-based cancer vaccines may be degraded by glycosidases or transglycosidases in vivo and lose the essential carbohydrate recognition elements. This will dramatically affect the specificity of the antibodies elicited by the vaccines. To overcome these obstacles, chemically modified TACA analogs, including fluorosugars, C-glycosides, and S-glycosides, have been developed and tested for vaccine design.The Thomsen-Friedenreich antigen (TF or T-antigen, Galβ1,3GalNAcaSer/Thr) is one of the most common TACAs. T-antigen-presenting mucins are overexpressed on about90%of human carcinoma cells, including those of breast cancer, prostate cancer, ovarian cancer, and lung carcinomas. Recently. T-MUC1glycopeptide analogs containing one or two fluorine substituents on the sugar were synthesized. They have been conjugated to a tetanus toxoid carrier protein and the conjugate vaccines elicited strong and specific immune responses in mice. These "foreign" fluorinated TACA-based vaccines not only provided enhanced immunogenicity and metabolic stability but also improved bioavailability. Despite the broad application of fluorine substitution in medicinal chemistry for generating several blockbuster drugs in the market (such as LipitorTMnd ProzacTM, etc.), only a few fluorinated T-antigens and derivatives have been synthesized so far. The chemical synthesis of fluorinated oligosaccharides including T-antigens and other Gal(31,3-GalNAc-containing O-glycans is challenging. It involves multiple protection and deprotection processes and has to deal with the low reactivity of fluorinated acceptors or donors during glycosylation. It usually requires harsh conditions and leads to low yields. Therefore, a more practical and highly efficient synthetic approach is needed to provide a sufficient amount of fluorinated T-antigens for further vaccine development.Recently, we developed a highly efficient one-pot two-enzyme system containing a novel Bifidobacterium infantis D-galactosyl-β1,3-N-acetyl-D-hexosamine phosphorylase (BiGalHexNAcP) and a recombinant galactokinase (EcGalK) cloned from E. coli K-12for the synthesis of diverse β1,3-linked galactosides. Compared to galactosyltransferase-catalyzed reaction, the BiGalHexNAcP-catalyzed synthesis uses galactose-1-phosphate (Gal-1-P) as the donor substrate and does not require multiple enzymes for the in situ generation or regeneration of expensive UDP-galactose (the sugar nucleotide for galactosyltransferases). In addition, both recombinant BiGalHexNAcP and EcGalK have high expression levels in the E. coli expression system. Furthermore, BiGalHexNAcP has been shown to tolerate some C6-modified GlcNAc as acceptor substrates. Therefore, the one-pot two-enzyme system offers an efficient and simplified approach for the large-scale synthesis of (31,3-linked galactosides and analogs. Moreover, the sialylated derivatives of fluorinated T-antigens were also successfully synthesized through another one-pot two-enzyme system in up to90%yields.The main achievements of our project include the following aspects.(1) A highly efficient "two-step’one-pot’oligosaccharide synthesis strategy" was successfully developed for the assembly of naturally occurring T-antigens and their fluorinated derivatives. This novel chemoenzymatic approach provides a robust large scale production of fluorinated T-antigens for further immulogical evalution;(2) This is the first succeful application of chemoenzymatic synthesis of fluorinated T antigens;(3) For the first time, the sialylated fluorinated T-antigens was achieved by employing "one-pot two enzyme" synthesis;(4) It’s the first time to synthesize the sialylated fluorinated T antigens in the world;(5) The efficient synthesis of fluorinated T antigens illustrates that the BiGalHexNAcP well tolerates with the C6-fluorinanted of donors and acceptors;(6) The results of using Ga12F-l-P as substrate for theBiGalHexNAcP-catalyzed reaction provided an exqusited, accurate and reliable example to the hypothesis that C2-fluorinated sugar has a broad inhibition to various carbohydrate processing enzymes, especially the phosphorylase.