Enzymatic Synthesis Of Tumor-Associated Carbohydrate Antigen KH-1

Tumor-associated carbohydrate antigens (TACAs) are a family of unusual carbohydrate motifs which overexpressed on the surface of cancer cells as glycoprotens or glycolipids. This abnormal glycosylation is strongly correlated with tumor metastasis and signal transduction. Immunization with TACAs-based vaccine can induce immune response, generate antibodies that against cancers. However, a highly efficient approach is missing to access structurally well-defined complex TACAs in pure form.The Lewis series antigens, such as Lewis x (Lex), Lewis y (Ley), sialyl Lewis x (SLex), sialyl Lewis a (SLea) and KH-1 are the common TACAs. The KH-1 antigen (adenocarcinoma) was found only overexpressed on the surface of breast and prostate cancer cells. Chemically, this nonasaccharide [Fucα(1â†'2)]Galβ(1â†'4) [Fucα(1â†'3)]GlcNAcβ(1â†'3)Galβ(1â†'4)[Fucα(1â†'3)GlcNAcβ(1â†'3]Galβ(1â†'4)Glc βOCer contains a Ley, a Lex and a lactoside fragment, respectively. KH-1 antigen was firstly isolated from human colonic adenocarcinoma cells. The following study revealed that this antigen has only been found on the surface of adenocarcinomas cells, and has never been isolated from normal colonic tissue, therefor KH-1 is a highly specific marker for colon malignancies. However, the isolation, immunocharacterization, and structural identification of large carbohydrate-based tumor antigens remains a highly complex and challenging undertaking. Owing to the microheterogeneous of TACAs, the availability of isolation from nature resource is quite limited. Hence, artificial synthesis is efficient and available to obtain desired structure. Several group have synthesized the antigen by using different strategies of chemical method, and some groups studied biological activity, revealed that KH-1— KLH vaccines could provoke intense immune response, and have great potential to develop anticancer vaccines. Nevertheless, to sum up previous work, all of them are chemical synthesis. The inevitable protection and deprotection manipulation lead to complicated procedures and low yields, and relied on the sophisticated synthetic abilities of the researchers.The specificity and high efficiency of enzymes shows the superiority of enzyme method in contrast to chemical synthesis. So, the one-pot multiple-enzymes (OPME) system was developed for the sequential enzymatic synthesis of KH-1 antigen.Starting from lactoside as acceptor, two OPME system was applied twice for enzymatic chain elongation to afford the hexasaccharide backbone in 4 steps. The hexasaccharide backbone then used as acceptor for double α1,3-fucosylation and α1,2-fucosylation to introduce two α1,3-linked and one α1,2-linked fucose moieties to provide the KH-1 antigen.This highly efficient one-pot multienzyme strategy was demonstrated for the enzymatic synthesis of complex KH-1 antigen from readily available lactoside and corresponding cheap monosaccharides. The OPME strategy which combines bacterial glycosyltransferases and sugar nucleotide generation enzymes provide easy access to the biologically important complex oligosaccharides.