Glycoscience has been recognized as an important area in biomedical research. Currently, a major obstacle for glycoscience study is the lack of diverse, biomedically relevant, and complex glycans in quantities sufficient for exploring their structural and functional aspects. Complementary to chemoenzymatic synthesis, natural glycans could serve as a great source of biomedically relevant glycans if they are available in sufficient quantities. We have recently developed oxidative release of natural glycans (ORNG) for large-scale release of N-glycans as free reducing glycans. While free reducing glycans can be readily derivatized with ultraviolet or fluorescent tags for high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis, it is difficult to remove tags for the regeneration of free reducing glycans without affecting the structural integrity of glycans. To address this inconvenience, we explored the use of a cleavable tag, O-benzylhydroxylamine (BHA). Free reducing glycans are easily and efficiently labeled with BHA under mild conditions, enabling UV detection during HPLC purification. Individual glycan-BHA conjugates can then be separated using multidimensional HPLC and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MS/MS. The BHA tag can then be easily removed by palladium-on-carbon (Pd/C)-catalyzed hydrogenation to efficiently regenerate free reducing glycans with little effect on glycan structures. This procedure provides a simple and straightforward way to tag free reducing glycans for purification at a preparative scale using multidimensional HPLC and subsequently recover purified free reducing glycans.

中文翻译：

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O-苄基羟胺 (BHA) 作为用于分离和纯化还原聚糖的可切割标签。

糖科学已被公认为生物医学研究的重要领域。目前，糖科学研究的一个主要障碍是缺乏数量足以探索其结构和功能方面的多样化、生物医学相关和复杂的聚糖。作为化学酶合成的补充，如果天然聚糖的数量充足，它们可以作为生物医学相关聚糖的重要来源。我们最近开发了天然聚糖 (ORNG) 的氧化释放，用于大规模释放 N-聚糖作为游离还原聚糖。虽然游离的还原聚糖可以很容易地用紫外线或荧光标签进行衍生，用于高效液相色谱 (HPLC) 和质谱 (MS) 分析，在不影响聚糖的结构完整性的情况下，很难去除用于游离还原聚糖再生的标签。为了解决这种不便，我们探索了可切割标签 O-苄基羟胺 (BHA) 的使用。游离还原聚糖在温和条件下可以轻松有效地用 BHA 标记，从而在 HPLC 纯化过程中进行 UV 检测。然后可以使用多维 HPLC 分离单个聚糖-BHA 缀合物，并通过基质辅助激光解吸/电离飞行时间质谱 (MALDI-TOF-MS) 和 MS/MS 进行表征。然后可以通过钯碳 (Pd/C) 催化氢化轻松去除 BHA 标签，以有效地再生游离还原聚糖，而对聚糖结构几乎没有影响。