Lysosomal accumulation of the glycosphingolipid globotriaosylceramide Gb3 is linked to the deficient activity of the α-galactosidase A in the Anderson-Fabry disease and an elevated level of deacylated Gb3 is a hallmark of this condition. Localization of Gb3 in the plasma membrane is critical for studying how the membrane organization and its dynamics are affected in this genetic disorder. Gb3 analogs containing a terminal 6-azido-functionalized galactose in its head group globotriose (αGal1, 4βGal1, and 4Glc) are attractive chemical reporters for bioimaging, as the azido-group may act as a chemical tag for bio-orthogonal click chemistry. We report here the production of azido-Gb3 analogs employing mutants of galactokinase, UTP-glucose-1-phosphate uridylyltransferase, and α-1,4-galactosyltransferase LgtC, which participate in the synthesis of the sugar motif globotriose. Variants of enzymes galactokinase/UTP-glucose-1-phosphate uridylyltransferase generate UDP-6-azido-6-deoxy-d-galactose, which is the galactosyl-donor used by LgtC for transferring the terminal galactose moiety to lactosyl-acceptors. Residues at the galactose-binding site of the 3 enzymes were modified to facilitate the accommodation of azido-functionalized substrates and variants outperforming the wild-type enzymes were characterized. Synthesis of 6-azido-6-deoxy-d-galactose-1-phosphate, UDP-6-azido-6-deoxy-d-galactose, and azido-Gb3 analogs by variants GalK-E37S, GalU-D133V, and LgtC-Q187S, respectively, is 3-6-fold that of their wild-type counterparts. Coupled reactions with these variants permit the production of the pricy, unnatural galactosyl-donor UDP-6-azido-6-deoxy-d-galactose with ~90% conversion yields, and products azido-globotriose and lyso-AzGb3 with substrate conversion of up to 70%. AzGb3 analogs could serve as precursors for the synthesis of other tagged glycosphingolipids of the globo-series.

中文翻译：

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GalK、GalU 和 LgtC 酶的半乳糖结合位点的单突变能够有效合成 UDP-6-叠氮基-6-脱氧-d-半乳糖和叠氮基功能化的 Gb3 类似物。

鞘糖脂球三糖基神经酰胺 Gb3 的溶酶体积累与安德森-法布里病中 α-半乳糖苷酶 A 的活性缺陷有关，脱酰化 Gb3 水平升高是该病的标志。Gb3 在质膜中的定位对于研究这种遗传性疾病中膜组织及其动力学如何受到影响至关重要。Gb3 类似物在其头部基团球三糖（αGal1、4βGal1 和 4Glc）中含有末端 6-叠氮基功能化半乳糖，对于生物成像来说是有吸引力的化学报告分子，因为叠氮基可以充当生物正交点击化学的化学标签。我们在这里报道了使用半乳糖激酶、UTP-葡萄糖-1-磷酸尿苷酰转移酶和α-1,4-半乳糖基转移酶LgtC的突变体生产叠氮基-Gb3类似物，这些突变体参与糖基序球三糖的合成。半乳糖激酶/UTP-葡萄糖-1-磷酸尿苷酰转移酶的变体产生 UDP-6-叠氮基-6-脱氧-d-半乳糖，它是 LgtC 用于将末端半乳糖部分转移到乳糖基受体的半乳糖基供体。对 3 种酶的半乳糖结合位点的残基进行了修饰，以促进叠氮基功能化底物的容纳，并表征了优于野生型酶的变体。通过变体 GalK-E37S、GalU-D133V 和 LgtC- 合成 6-叠氮基-6-脱氧-d-半乳糖-1-磷酸、UDP-6-叠氮基-6-脱氧-d-半乳糖和叠氮基-Gb3 类似物Q187S 分别是其野生型对应物的 3-6 倍。与这些变体的偶联反应可以产生昂贵的非天然半乳糖基供体 UDP-6-叠氮基-6-脱氧-d-半乳糖，转化率约为 90%，并且产物叠氮基-球三糖和溶血-AzGb3 的底物转化率高达至 70%。AzGb3 类似物可以作为 globo 系列其他标记鞘糖脂合成的前体。