Sialic-acid-binding,i mmunoglobulin-type lectin-7 (Siglec-7) is present on the surface of natural killer cells.Siglec-7s hows preference for disialylated glycans,i ncluding a(2,8)a(2,3)-disialic acids or internally branched a(2,6)-NeuAc,such as disialosylglobopentaose (DSGb5). Herein, DSGb5 was synthesized by ao ne-pot multiple enzyme method from Gb5 by a2,3-sialylation (with PmST1) followed by a2,6-sialylation (with Psp2,6ST) in 23 %o verall yield. DSGb5 was also chemoenzymatically synthesized. The protection of the nonreducing-end galactose of Gb5 as 3,4-O-acetonide,3 ,4-Obenzylidene,a nd 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 ar ange of globo-glycans.S urprisingly,D SGb5 shows al ow affinity for Siglec-7 in ag lycan microarray binding affinity assay. Among the synthesized globo-series glycans, a6a3DSGb4 shows the highest binding affinity for Siglec-7.Globosides,stage-specific embryonic antigen-4 (SSEA4, MSGb5, or a3SGb5 in Scheme 1) and disialosylglobopentaosylceramide (DSGb5-Cer), are expressed in relatively large amounts on the surface of cancer cells,including renal cancer cells (RCC), and they are associated with cancer proliferation and metastasis. [1] Therefore,g lobosides have been used for cancer detection [2] and vaccine development. [3] Thes tructure of DSGb5-Cer was determined in 1994, [4] and it is an interesting target since its total synthesis has not been reported. Theh igh affinity of DSGb5-Cer for Siglec-7 (sialic acid-binding immunoglobulin-type lectin-7) was reported in 2011. [5] Siglec-7 is al ectin present on the surface of natural killer (NK) cells as an inhibitory receptor of NK cell cytotoxicity. [6] Later, glycans with a2,8-a2,3-disialic acid or internally branched a2,6-NeuAc were shown to be potent ligands for Siglec-7 in competitive binding assays. [7] In nature, NK cell cytotoxicity is modulated by cis interactions between Siglec-7 and endogenous sialoconjugates on the cell surface. [6] By treating NK cells with sialidase to remove NeuAc and disrupt the cis interactions,t he unmasked Siglec-7 can interact with DSGb5-Cer on the surface of the human renal cancer cell line ACHN,decreasing the cytotoxicity of the NK cells and promoting the metastasis and invasion of RCC. [8] Obtaining as ufficient amount of pure DSGb5 is key to elucidating its mechanism of action and to further develop cancer immunotherapies.C larifying the binding activities of sialylated globo-series glycans and the effect of the NeuAc position on their interactions with Siglec-7 is also of great interest.Thee nzymatic and chemoenzymatic syntheses of globoseries glycans,such as Gb5, SSEA-4, and Globo H, have been reported by several research groups, [9][10][11] but no method has been reported for the synthesis of DSGb5. DSGb5 has aunique glycan structure with two NeuAcs as a2,3-and a2,6linkages on the nonreducing-end galactose (Gal) and Nacetylgalactosamine (GalNAc), respectively,o fG b5 (Figure 1)....