Naruchi Kentarou, Hamamoto Tomoki, Kurogochi Masaki, Hinou Hiroshi, Shimizu Hiroki, Matsushita Takahiko, Fujitani Naoki, Kondo Hirosato, Nishimura Shin-Ichiro
Division of Advanced Chemical Biology, Graduate School of Advanced Life Science, Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11, Sapporo 001-0021, Japan.
J Org Chem. 2006 Dec 22;71(26):9609-21. doi: 10.1021/jo0617161.
We have established a facile and efficient protocol for the preparative-scale synthesis of various compound libraries related to lactosaminoglycans: cell surface oligosaccharides composed of N-acetyllactosamine as a repeating disaccharide unit, based on chemical and enzymatic approaches. Substrate specificity and feasibility of a bacterial glycosyltransferase, Neisseria meningitidis beta1,3-N-acetylglucosaminyltransferase (LgtA), were investigated in order to synthesize various key intermediates suited for the construction of mammalian O-glycopeptides and glycosphingolipids containing poly-N-acetyllactosamine structures. Recombinant LgtA exhibited the highest glycosyltransferase activity with strongly basic conditions (pH = 10, glycine-NaOH buffer) and a broad range of optimal temperatures from 20 to 30 degrees C. Interestingly, it was found that LgtA discriminates L-serine and L-threonine and functions both as a core-1 beta1,3-N-acetylglucosaminyltransferase and core-2 beta1,3-N-acetylglucosaminyltransferase toward Fmoc-Ser derivatives, while LgtA showed only core-2 beta1,3-N-acetylglucosaminyltransferase activity in the presence of Fmoc-Thr derivatives. Combined use of LgtA with human beta1,4-galactosyltransferase allowed for controlled sugar extension reactions from synthetic sugar amino acids and gave synthetic lactosaminoglycans, such as a decasaccharide derivative, Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 6)[Galbeta(1 --> 3)]GalNAcalpha1 --> Fmoc-Ser-OH (6), and a dodecasaccharide derivative, Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 6)[Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 3)Galbeta(1 --> 3)]GalNAcalpha1 --> Fmoc-Ser-OH (9). A partially protected pentasaccharide intermediate, GlcNAcbeta(1 --> 3)Galbeta(1 --> 4)GlcNAcbeta(1 --> 6)[Galbeta(1 --> 3)]GalNAcalpha1 --> Fmoc-Thr-OH (11), was applied for the microwave-assisted solid-phase synthesis of a MUC1-related glycopeptide 19 (MW = 2610.1). The findings suggest that this sugar extension strategy can be employed for the modification of lactosyl ceramide mimetic polymers to afford convenient precursors for the synthesis of various glycosphingolipids.
我们基于化学和酶促方法，建立了一种简便高效的方法，用于制备与乳糖胺聚糖相关的各种化合物库：由N-乙酰乳糖胺作为重复二糖单元组成的细胞表面寡糖。为了合成适合构建含有聚N-乙酰乳糖胺结构的哺乳动物O-糖肽和糖鞘脂的各种关键中间体，研究了细菌糖基转移酶脑膜炎奈瑟菌β1,3-N-乙酰葡糖胺基转移酶（LgtA）的底物特异性和可行性。重组LgtA在强碱性条件（pH = 10，甘氨酸-NaOH缓冲液）下表现出最高的糖基转移酶活性，并且在20至30摄氏度的广泛最佳温度范围内均有活性。有趣的是，发现LgtA能够区分L-丝氨酸和L-苏氨酸，并且对Fmoc-Ser衍生物既作为核心-1β1,3-N-乙酰葡糖胺基转移酶又作为核心-2β1,3-N-乙酰葡糖胺基转移酶起作用，而LgtA在Fmoc-Thr衍生物存在下仅表现出核心-2β1,3-N-乙酰葡糖胺基转移酶活性。将LgtA与人类β1,4-半乳糖基转移酶联合使用，可实现从合成糖氨基酸进行可控的糖延伸反应，并得到合成乳糖胺聚糖，例如十糖衍生物Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→6)[Galβ(1→3)]GalNAcα1→Fmoc-Ser-OH（6）和十二糖衍生物Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→6)[Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→3)Galβ(1→3)]GalNAcα1→Fmoc-Ser-OH（9）。一种部分保护的五糖中间体GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→6)[Galβ(1→3)]GalNAcα1→Fmoc-Thr-OH（11）被用于微波辅助固相合成与MUC1相关的糖肽19（分子量 = 2610.1）。这些发现表明，这种糖延伸策略可用于修饰乳糖基神经酰胺模拟聚合物，以提供用于合成各种糖鞘脂的便利前体。
