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Glycosphingolipids (GSLs) play essential roles in many important biological processes, making them attractive synthetic targets. In this paper, a viable chemoenzymatic method is described for the synthesis of globo-series GSLs, namely, Gb4, Gb5, SSEA-4, and Globo H. The strategy uses a chemically synthesized lactoside acceptor equipped with a partial ceramide structure that is uniquely extended by glycosyltransferases in a highly efficient one-pot multiple enzyme (OPME) procedure. A direct and quantitative conversion of Gb4 sphingosine to Globo H sphingosine is achieved by performing two-sequential OPME glycosylations. A reduction and N-acylation protocol allows facile incorporation of various fatty acids into the lipid portions of the GSLs. The chemically well-defined lipid-modified Globo H-GSLs displayed some differences in their immunosuppressive activities, which may benefit the structural modifications of Globo H ceramides in finding new types of immunosuppressive agents. The strategy outlined in this work should be applicable to the rapid access to other complex GSLs.

Herein, we applied PmST1 (a sialyltransferase) to achieve acceptor-mediated regioselective sialylation (AMRS) on the nonreducing end GalNH2 or GalAz (2-azido-2-deoxy galactose). Thus, C5 and C8-modified sialic acid was efficiently assembled on GalNH2 (or GalAz) to achieve the synthesis of the GAA-7 (one of the echinodermatous gangliosides with higher neuritogenic activity) glycan moiety.

Herein, we report a bifunctional sulfo-fluorous affinity (SOFA) tag-assisted enzymatic synthesis and purification strategy for the facile preparation of bioactive glycans using fluorous solid-phase extraction (FSPE). The incorporation of a sulfonate moiety onto the heavy fluorous tag significantly increases its water solubility, which allows the broad use of the inherently hydrophobic fluorous tag in aqueous buffers. In addition, the SOFA tag contains a photocleavable linker, enabling the easy release of amino-functionalized oligosaccharides by UV irradiation. The SOFA tag was used in the synthesis of both negatively charged and neutral glycans to demonstrate its broad utility as an acceptor toward six different glycosyltransferases, significantly improving the feasibility of the preparation of complex glycans using FSPE. All the reactions were performed in an aqueous buffer, a minimum amount of methanol was used to purify the products, and the SOFA tag was easily recovered after photo-irradiation. Thus, the entire synthetic process is environmentally benign.

Sialic-acid-binding, immunoglobulin-type lectin-7 (Siglec-7) is present on the surface of natural killer cells. Siglec-7 shows preference for disialylated glycans, including α(2,8)-α(2,3)-disialic acids or internally branched α(2,6)-NeuAc, such as disialosylglobopentaose (DSGb5). Herein, DSGb5 was synthesized by a one-pot multiple enzyme method from Gb5 by α2,3-sialylation (with PmST1) followed by α2,6-sialylation (with Psp2,6ST) in 23 % overall yield. DSGb5 was also chemoenzymatically synthesized. The protection of the nonreducing-end galactose of Gb5 as 3,4-O-acetonide, 3,4-O-benzylidene, and 4,6-O-benzylidene derivatives provided DSGb5 in overall yields of 26 %, 12 %, and 19 %, respectively. Gb3, Gb4, and Gb5 were enzymatically sialylated to afford a range of globo-glycans. Surprisingly, DSGb5 shows a low affinity for Siglec-7 in a glycan microarray binding affinity assay. Among the synthesized globo-series glycans, α6α3DSGb4 shows the highest binding affinity for Siglec-7.