Department of Chemistry, University of California-Davis, One Shields Avenue, Davis, California 95616, USA.
J Am Chem Soc. 2009 Dec 30;131(51):18467-77. doi: 10.1021/ja907750r.
In order to understand the biological importance of naturally occurring sialic acid variations on disialyl structures in nature, we developed an efficient two-step multienzyme approach for the synthesis of a series of GD3 ganglioside oligosaccharides and other disialyl glycans containing a terminal Siaalpha2-8Sia component with different natural and non-natural sialic acids. In the first step, alpha2-3- or alpha2-6-linked monosialylated oligosaccharides were obtained using a one-pot three-enzyme approach. These compounds were then used as acceptors for the alpha2-8-sialyltransferase activity of a recombinant truncated multifunctional Campylobacter jejuni sialyltransferase CstII mutant, CstIIDelta32(I53S), to produce disialyl oligosaccharides. The alpha2-8-sialyltransferase activity of CstIIDelta32(I53S) has promiscuous donor substrate specificity and can tolerate various substitutions at C-5 or C-9 of the sialic acid in CMP-sialic acid, while its acceptor substrate specificity is relatively restricted. The terminal sialic acid residues in the acceptable monosialylated oligosaccharide acceptors are restricted to Neu5Ac, Neu5Gc, KDN, and some of their C-9-modified forms but not their C-5 derivatives. The disialyl oligosaccharides obtained are valuable probes for their biological studies.
为了理解天然存在的唾液酸变异在自然界中二唾液酸结构中的生物学重要性，我们开发了一种高效的两步多酶法，用于合成一系列 GD3 神经节苷脂寡糖和其他含有末端 Siaα2-8Sia 部分的二唾液酰糖，其中包含不同天然和非天然唾液酸。在第一步中，使用一锅三酶法获得α2-3-或α2-6-连接的单唾液酸化寡糖。然后，这些化合物被用作重组截短多功能弯曲杆菌唾液酸转移酶 CstII 突变体 CstIIDelta32(I53S)的α2-8-唾液酰转移酶活性的受体，以产生二唾液酰寡糖。CstIIDelta32(I53S)的α2-8-唾液酰转移酶活性具有混杂的供体底物特异性，并且可以容忍 CMP-唾液酸中唾液酸 C-5 或 C-9 上的各种取代，而其受体底物特异性相对受限。可接受的单唾液酸化寡糖受体中末端唾液酸残基仅限于 Neu5Ac、Neu5Gc、KDN 和它们的一些 C-9 修饰形式，但不是它们的 C-5 衍生物。获得的二唾液酰寡糖是其生物学研究的有价值的探针。
