Department of Analytical Chemistry and Food Chemistry, University of Vienna, Vienna, Austria.
Chirality. 2010 May 15;22(5):463-71. doi: 10.1002/chir.20764.
Direct enantiomer separation of hypericin, pseudohypericin, and protohypericin was accomplished by high-performance liquid chromatography (HPLC) using immobilized polysaccharide-type chiral stationary phases (CSPs). Enantioselectivities up to 1.30 were obtained in the polar-organic elution mode whereby for hypericin and pseudohypericin Chiralpak IC [chiral selector being cellulose tris(3,5-dichlorophenylcarbamate)] and for protohypericin Chiralpak IA (chiral selector being the 3,5-dimethylphenylcarbamate of amylose) gave favorable results. Enantiomers were distinguished by on-line electronic circular dichroism detection. Optimized enantioselective chromatographic conditions were the basis for determining stereodynamic parameters of the enantiomer interconversion process of hypericin and pseudohypericin. Rate constants delivered by computational simulation of dynamic HPLC elution profiles (stochastic model, consideration of peak tailing) were used to calculate averaged enantiomerization barriers (DeltaG(enant)(#)) of 97.6-99.6 kJ/mol for both compounds (investigated temperature range 25-45 degrees C). Complementary variable temperature off-column (i.e., in solution) racemization experiments delivered DeltaG(enant)(#) = 97.1-98.0 kJ/mol (27-45 degrees C) for hypericin and DeltaG(enant)(#) = 98.9-101.4 kJ/mol (25-55 degrees C) for pseudohypericin. An activation enthalpy of DeltaH(#) = 86.0 kJ/mol and an activation entropy of DeltaS(#) = -37.7 J/(K mol) were calculated from hypericin racemization kinetics in solution, whereas for pseudohypericin these figures amounted to 74.1 kJ/mol and -82.6 J/(K mol), respectively. Although the natural phenanthroperylene quinone pigments hypericin and pseudohypericin as well as their biological precursor protohypericin are chiral and can be separated by enantioselective HPLC low enantiomerization barriers seem to prevent the occurrence of an excess of one enantiomer under typical physiological conditions--at least as long as stereoselective intermolecular interactions with other chiral entities are absent.
高效液相色谱法（HPLC）使用固定化多糖型手性固定相（CSP）实现了金丝桃素、伪金丝桃素和原金丝桃素的直接对映体分离。在极性有机洗脱模式下，对映选择性高达 1.30，其中金丝桃素和伪金丝桃素用 Chiralpak IC（手性选择体为纤维素三（3,5-二氯苯甲酰基）），而原金丝桃素用 Chiralpak IA（手性选择体为 3,5-二甲基苯甲酰基淀粉）得到了有利的结果。对映异构体通过在线电子圆二色性检测进行区分。优化的对映选择性色谱条件是确定金丝桃素和伪金丝桃素对映体相互转化过程的立体动力学参数的基础。通过动态 HPLC 洗脱曲线的计算模拟（随机模型，考虑峰拖尾）得到的速率常数用于计算两种化合物的平均对映体异构化势垒（DeltaG(enant)(#)）为 97.6-99.6 kJ/mol（研究温度范围为 25-45°C）。补充的变温离线（即在溶液中）外消旋实验得到金丝桃素的 DeltaG(enant)(#) = 97.1-98.0 kJ/mol（27-45°C）和伪金丝桃素的 DeltaG(enant)(#) = 98.9-101.4 kJ/mol（25-55°C）。从金丝桃素在溶液中的外消旋动力学计算得到活化焓 DeltaH(#) = 86.0 kJ/mol 和活化熵 DeltaS(#) = -37.7 J/（K mol），而对于伪金丝桃素，这些值分别为 74.1 kJ/mol 和 -82.6 J/（K mol）。尽管天然的菲并苯醌类色素金丝桃素、伪金丝桃素及其生物前体原金丝桃素是手性的，可以通过对映体选择性 HPLC 分离，但低的对映体异构化势垒似乎可以防止在典型的生理条件下出现一种对映体过量，至少在没有与其他手性实体的立体选择性分子间相互作用的情况下是这样。
