Cui Li, Guan Xiao-Qing, Liu Zhang-Min, Fan Liu-Yin, Li Qian, Feng Yan
a State Key Laboratory of Microbial Metabolism, School of Life Science & Biotechnology, and Joint International Research Laboratory of Metabolic & Developmental Sciences , Shanghai Jiao Tong University , Shanghai 200240 , China.
b School of Life Science & Technology , China Pharmaceutical University , Nanjing 210009 , China.
J Asian Nat Prod Res. 2017 Apr;19(4):347-357. doi: 10.1080/10286020.2017.1292257.
Valienamine and β-valienamine are representative C N aminocyclitols with significant glycosidase inhibition activity that have been developed as important precursors of drugs for diabetes and lysosomal storage diseases, respectively. The quantitative analysis of these chiral compounds is crucial for asymmetric in vitro biosynthetic processes for converting valienone into valienamine epimers using aminotransferase. Here, we developed an efficient and sensitive method for separation and quantitative analysis of chiral valienamine using reversed-phase high-performance liquid chromatography (HPLC) through o-phthalaldehyde (OPA) pre-column derivatization of the analytes. The epimers were derivatized by OPA in borate buffer (pH 9.0) at room temperature for 30 s, separated on an Eclipse XDB-C18 (5 μm, 4.6 × 150 mm) column, eluted with 22% acetonitrile at 30 °C for 18 min, and detected by a fluorescence detector using 445 nm emission and 340 nm excitation wavelengths. The average resolution of the epimers is 3.86, and the concentration linearity is in the range of 0.02-20 μg/ml. The method proved to be effective, sensitive, and reliable with good intra- and inter-day precision and accuracy, and successfully evaluated the enantiopreference and catalytic capability of the potential aminotransferases on an unnatural prochiral substrate, facilitating the design of an asymmetric biosynthetic route for optically pure valienamine and β-valienamine.
缬氨霉素和β-缬氨霉素是具有显著糖苷酶抑制活性的代表性C-N氨基环糖醇，它们分别被开发为糖尿病药物和溶酶体贮积症药物的重要前体。对这些手性化合物进行定量分析，对于使用转氨酶将缬氨酮转化为缬氨霉素差向异构体的不对称体外生物合成过程至关重要。在此，我们开发了一种高效、灵敏的方法，通过对分析物进行邻苯二甲醛（OPA）柱前衍生化，利用反相高效液相色谱（HPLC）对手性缬氨霉素进行分离和定量分析。差向异构体在硼酸盐缓冲液（pH 9.0）中于室温下用OPA衍生化30秒，在Eclipse XDB-C18（5μm，4.6×150mm）柱上分离，于30℃用22%乙腈洗脱18分钟，并用荧光检测器在发射波长445nm和激发波长340nm下进行检测。差向异构体的平均分离度为3.86，浓度线性范围为0.02 - 20μg/ml。该方法被证明是有效、灵敏且可靠的，具有良好的日内和日间精密度与准确度，并成功评估了潜在转氨酶对非天然前手性底物的对映体选择性和催化能力，有助于设计光学纯缬氨霉素和β-缬氨霉素的不对称生物合成路线。
