Australian Centre for Research on Separation Science, School of Chemistry, University of Tasmania, GPO Box 252-75, Hobart, Tasmania 7001, Australia.
Anal Chim Acta. 2010 Mar 10;662(2):206-13. doi: 10.1016/j.aca.2010.01.011. Epub 2010 Jan 18.
This study describes the development of a novel fluorescent tag, O-2-[aminoethyl]fluorescein, for the separation of sugars by capillary electrophoresis with fluorescence detection using an argon ion laser. The tag was synthesised using three consecutive steps namely: esterification, alkylation and hydrolysis, specifically designed to offer a flexible way in which to make an assortment of fluorescent tags from cheap and readily available starting reagents (typically less than $1 per g of fluorescent tag). Via this flexible synthetic pathway, O-2-[aminoethyl]fluorescein was designed and synthesised with a spacer group to lower steric effects between the fluorescein backbone and the reducing end of the carbohydrate which were anticipated to improve the reactivity of the tag. The newly synthesised tag, O-2-[aminoethyl]fluorescein was evaluated against structurally similar commercial fluorescent motifs namely fluorescent 5-aminomethylfluorescein and non-fluorescent 5-aminofluorescein. Kinetic studies indicated that O-2-[aminoethyl]fluorescein showed similar labeling efficiencies as 5-aminomethylfluorescein, but were achieved in only 30 min, supporting the notion of improved reactivity of the spacer group. The sensitivity of O-2-[aminoethyl]fluorescein was evaluated using maltoheptaose with a detection limit of 1 nM obtained, which was slightly higher than that of 0.3 nM obtained with 5-aminomethylfluorescein, and was due to its lower quantum yield (0.24) when conjugated to the sugar. The separation performance of the tag was also benchmarked with the two commercial reagents using a range of corn syrup oligosaccharides, from 4 to 10 glucose units, typically found in rice starch. Separations were performed using an electrolyte containing 100 mM boric acid, tris at pH 8.65 as background electrolyte, 30 kV applied voltage, 50 microm I.D. x 40 cm (30 cm effective length) capillary. The novel tag showed better resolution of small oligosaccharides, G3 and G4, than the other two reagents, but slightly worse resolution for the longer oligosaccharides, most likely due to the monovalent charge state of the O-2-[aminoethyl]fluorescein compared to the divalent charge of the other two tags.
本研究描述了一种新型荧光标记物 O-2-[氨基乙基]荧光素的开发，用于通过毛细管电泳与氩离子激光荧光检测分离糖。该标记物通过三个连续步骤合成：酯化、烷基化和水解，专门设计用于提供一种灵活的方式，从便宜且易于获得的起始试剂（通常每克荧光标记物不到 1 美元）中制造各种荧光标记物。通过这种灵活的合成途径，设计并合成了 O-2-[氨基乙基]荧光素，其中带有一个间隔基，以降低荧光素骨架和碳水化合物还原端之间的空间位阻，预计这将提高标记物的反应性。新合成的标记物 O-2-[氨基乙基]荧光素与结构相似的商业荧光基序（即荧光 5-氨基甲基荧光素和非荧光 5-氨基荧光素）进行了评估。动力学研究表明，O-2-[氨基乙基]荧光素的标记效率与 5-氨基甲基荧光素相似，但仅需 30 分钟即可达到，这支持了间隔基反应性提高的观点。使用麦芽七糖评估了 O-2-[氨基乙基]荧光素的灵敏度，得到的检测限为 1 nM，略高于 5-氨基甲基荧光素得到的 0.3 nM，这是由于其与糖结合时量子产率（0.24）较低。还使用一系列玉米糖浆低聚糖（通常存在于大米淀粉中，具有 4 至 10 个葡萄糖单元）对标记物的分离性能与两种商业试剂进行了基准测试。使用包含 100 mM 硼酸、pH 8.65 的 tris 的电解质作为背景电解质，施加 30 kV 的电压，50 微米内径 x 40 厘米（有效长度 30 厘米）毛细管进行分离。与其他两种试剂相比，新型标记物对小寡糖 G3 和 G4 的分辨率更好，但对较长寡糖的分辨率略差，这很可能是由于 O-2-[氨基乙基]荧光素的单价电荷状态与其他两种标记物的二价电荷状态相比所致。
