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Facile chemoenzymatic synthesis of Lewis a (Lea) antigen in gram-scale and sialyl Lewis a (sLea) antigens containing diverse sialic acid forms.

ABSTRACT: An efficient streamlined chemoenzymatic approach has been developed for gram-scale synthesis of Lewis a angtigen (Lea?ProN3) and a library of sialyl Lewis a antigens (sLea?ProN3) containing different sialic acid forms. Intially, commercially available inexpensive N-acetylglucosamine (GlcNAc) was converted to its N'-glycosyl p-toluenesulfonohydrazide in one step. Followed by chemical glycosylation, GlcNAc?ProN3 was synthesized using this protecting group-free method in high yield (82%). Sequential one-pot multienzyme (OPME) ?1-3-galactosylation of GlcNAc?ProN3 followed by OPME ?1-4-fucosylation reactions produced target Lea?ProN3 in gram-scale. Structurally diverse sialic acid forms was successfully introduced using a OPME sialylation reation containing a CMP-sialic acid synthetase and Pasteurella multocida ?2-3-sialyltransferase 1 (PmST1) mutant PmST1 M144D with or without a sialic acid aldolase to form sLea?ProN3 containing naturally occurring or non-natural sialic acid forms in preparative scales.

SUBMITTER: Tasnima N

PROVIDER: S-EPMC6417886 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Facile chemoenzymatic synthesis of Lewis a (Lea) antigen in gram-scale and sialyl Lewis a (sLea) antigens containing diverse sialic acid forms.

Tasnima Nova N Yu Hai H Yan Xuebin X Li Wanqing W Xiao An A Chen Xi X

Carbohydrate research 20181209

An efficient streamlined chemoenzymatic approach has been developed for gram-scale synthesis of Lewis a angtigen (LeaβProN3) and a library of sialyl Lewis a antigens (sLeaβProN3) containing different sialic acid forms. Intially, commercially available inexpensive N-acetylglucosamine (GlcNAc) was converted to its N'-glycosyl p-toluenesulfonohydrazide in one step. Followed by chemical glycosylation, GlcNAcβProN3 was synthesized using this prot ...[more]

PMID: 30562693

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Project description:Sialyl Lewis(a) (sLe(a)), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe(a) is known to be the ligand for endothelial cell selectins suggesting a role for sLe(a) in cancer metastases and adhesion. For these reasons, sLe(a) may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe(a) is structurally similar to sLe(x) and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe(a) will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe(a) hexasaccharide and its conjugation to KLH to construct a sLe(a)-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe(a)-HSA by ELISA and against sLe(a) positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized with unconjugated sLe(a) plus GPI-0100 or unconjugated sLe(a) mixed with KLH plus GPI-0100 failed to produce antibodies against sLe(a). However, mice immunized with sLe(a)-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLe(a)-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLe(a) by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLe(a) positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related antigens, including Le(y), Le(x), and sLe(x) by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLe(a) positive cancer in clinical settings.

Dataset Information

Facile chemoenzymatic synthesis of Lewis a (Lea) antigen in gram-scale and sialyl Lewis a (sLea) antigens containing diverse sialic acid forms.

Publications

Facile chemoenzymatic synthesis of Lewis a (Le<sup>a</sup>) antigen in gram-scale and sialyl Lewis a (sLe<sup>a</sup>) antigens containing diverse sialic acid forms.

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