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

An efficient streamlined chemoenzymatic approach has been developed for gram-scale synthesis of Lewis a angtigen (Le(a)beta ProN(3)) and a library of sialyl Lewis a antigens (sLe(a)beta ProN(3)) 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 beta ProN(3) was synthesized using this protecting group-free method in high yield (82%). Sequential one-pot multienzyme (OPME) beta 1-3-galactosylation of GlcNAc beta ProN(3) followed by OPME alpha 1-4-fucosylation reactions produced target Le(a)beta ProN(3) 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 alpha 2-3-sialyltransferase 1 (PmST1) mutant PmST1 M144D with or without a sialic acid aldolase to form sLe(a)beta ProN(3) containing naturally occurring or non-natural sialic acid forms in preparative scales.