Biotinylated diaminopyridine: an approach to tagging oligosaccharides and exploring their biology.

Fluorescent tagging of free oligosaccharides by reductive amination permits sensitive detection and fractionation of these molecules. To expand the scope of this approach, we have synthesized a fluorescent reagent, 2-amino-(6-amidobiotinyl)pyridine. This reagent can tag oligosaccharides under nondegradative conditions with high efficiency. The resulting adducts show excellent fractionation by reverse-phase HPLC with sensitive detection in the low picomole range. When combined with sequential exoglycosidase digestion, stepwise sequencing of the sugar chains is possible. The biotinyl group can also be used to recover the sugar chain from reaction mixtures. The high-affinity interaction of the biotinyl group with multivalent avidin or streptavidin can be used to create the functional equivalent of neoglycoproteins carrying multiple copies of oligosaccharides of defined structure. These complexes allow the production of IgG antibodies directed against the oligosaccharide chain. They can also harness the power of (strept)avidin-biotin technology for the detection and isolation of oligosaccharide-specific receptors from native sources of recombinant libraries.

通过还原胺化法对游离寡糖（oligosaccharide）进行荧光标记，可实现这类分子的高灵敏检测与分级分离。为拓展该方法的应用范围，本研究合成了一种荧光试剂：2-氨基-(6-酰胺基生物素基)吡啶（2-amino-(6-amidobiotinyl)pyridine）。该试剂可在非降解条件下高效标记寡糖，所得标记加合物可通过反相高效液相色谱（reverse-phase HPLC）实现优异的分级分离，并在低皮摩尔级别下实现灵敏检测。若结合连续外切糖苷酶（exoglycosidase）消化步骤，则可实现糖链的逐步测序分析。该生物素基团还可用于从反应混合物中回收目标糖链。生物素基与多价亲和素（avidin）或链霉亲和素（streptavidin）之间的高亲和力相互作用，可用于构建携带多份结构明确寡糖的新糖蛋白（neoglycoprotein）功能等效物。此类复合物可用于制备靶向寡糖链的免疫球蛋白G（IgG）抗体，还可借助（链霉）亲和素-生物素技术的优势，从天然样品或重组文库中分离鉴定寡糖特异性受体。