Alberta Glycomics Centre and Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, Alberta T6G 2G2, Canada.
Org Biomol Chem. 2018 Mar 14;16(11):1939-1957. doi: 10.1039/c8ob00316e.
Mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce a complex cell wall that is critical for their survival. The largest structural component of the cell wall, the mycolyl-arabinogalactan-peptidoglycan complex, has at its core a galactan domain composed of d-galactofuranose residues. Mycobacterial galactan biosynthesis has been proposed to involve two glycosyltransferases, GlfT1 and GlfT2, which elongate polyprenol-pyrophosphate linked glycosyl acceptor substrates using UDP-galactofuranose as the donor substrate. We here report the first chemical synthesis of GlfT1 and GlfT2 acceptor substrates containing pyrophosphate and polyprenol moieties (compounds 3, 4, 22 and 23). The approach involves chemical synthesis of an oligosaccharide, subsequent phosphorylation at the reducing end and coupling to a polyprenol phosphate. These compounds were shown to be substrates for either GlfT1 (22 and 23) or GlfT2 (3 and 4) and all were substantially more active than the corresponding alkyl glycoside substrates reported previously. Mass spectrometric analysis of the products formed from the reaction of 3, 4, 22 and 23 with the respective cognate enzyme and UDP-galactofuranose provide additional evidence for the galactan biosynthetic model in which GlfT1 adds the first two galactofuranose residues with the remainder being installed via GlfT2. Overall, these results highlight the importance of the pyrophosphate motif in recognition of acceptor substrates by both enzymes and demonstrate a straightforward route for the preparation of such compounds. The work also provides additional support for the process by which this important glycan is biosynthesized using, for the first time, close structural analogs to the natural substrates.
分枝杆菌，包括人类病原体结核分枝杆菌，会产生复杂的细胞壁，这对于它们的生存至关重要。细胞壁的最大结构成分，即 mycolyl-arabinogalactan-peptidoglycan 复合物，其核心是由 d-半乳糖呋喃糖残基组成的半乳糖聚糖结构域。分枝杆菌半乳糖聚糖的生物合成被认为涉及两个糖基转移酶，GlfT1 和 GlfT2，它们使用 UDP-半乳糖呋喃糖作为供体底物，延伸多萜醇焦磷酸连接的糖基受体底物。我们在此报告了含有焦磷酸和多萜醇部分的 GlfT1 和 GlfT2 受体底物的首次化学合成（化合物 3、4、22 和 23）。该方法涉及寡糖的化学合成，随后在还原端进行磷酸化并与多萜醇磷酸偶联。这些化合物被证明是 GlfT1（22 和 23）或 GlfT2（3 和 4）的底物，并且所有化合物都比以前报道的相应烷基糖苷底物的活性高得多。对 3、4、22 和 23 与相应的酶和 UDP-半乳糖呋喃糖反应形成的产物进行质谱分析，为 GlfT1 添加前两个半乳糖呋喃糖残基，其余通过 GlfT2 安装的半乳糖聚糖生物合成模型提供了额外的证据。总的来说，这些结果突出了焦磷酸基序在两种酶识别受体底物中的重要性，并展示了制备此类化合物的直接途径。这项工作还为使用与天然底物的紧密结构类似物首次合成这种重要聚糖的过程提供了额外的支持。
