Thunecke F, Kálmán A, Kálmán F, Ma S, Rathore A S, Horváth C
Department of Chemical Engineering, Yale University, New Haven, CT 06520, USA.
J Chromatogr A. 1996 Sep 13;744(1-2):259-72. doi: 10.1016/0021-9673(96)00241-5.
The equilibrium and kinetic parameters of cis-trans interconversion of dipeptides containing peptidyl-proline moiety were investigated using the in-column incubation method with both CZE and HPLC and the ad hoc dissolution method. The use of the latter was possible because the conformational make-up of the solid peptides, and consequently of their ad hoc solution, was sufficiently different from that of the solution at equilibrium. This method with 1H-NMR and CZE analyses yielded very similar results for the cis-trans isomerization of Phe Pro in aqueous sodium borate, pH 8.4, at 10 degrees C with an average value of 0.34 and 6.6 x 10(-5) s-1 for the equilibrium and rate constant, respectively. The in-column incubation method is performed by CZE or HPLC so that the conformers are separated in the first half of the column and then incubated in column where they interconvert and reach equilibrium. Subsequent separation in the second half of the column yielded four peaks. Thus by measuring the conformer composition as a function of the reaction time, the rate constant can be evaluated. The forward rate constant of 1.42 x 10(-4) s-1 determined by the CZE in-column incubation method for Phe-Pro isomerization at 10 degrees C was twice of the value obtained by the ad hoc dissolution method. It is believed that the inner wall of fused-silica capillaries has a catalytic effect on the isomerization. Computer simulation was also employed to gain further insight on the catalytic activity of the capillary inner wall on such isomerization. Whereas the experimental and simulation profiles of Phe-Pro in aqueous borate buffer, pH 8.4, with a 37 cm long capillary were in excellent agreement, a four times faster interconversion rate had to be used to match the experimental profile obtained with a 57 cm long capillary under otherwise identical conditions. The catalytic effect of the octadecyl silica stationary phase on the isomerization was confirmed by the in-column incubation method with HPLC. The overall rate of the cis-trans isomerization of Phe-Pro, which entails the reaction both on the stationary phase and in the mobility phase, was about six times faster at 0 degree C than the rate measured by NMR in free solution using the mobile phase containing 65% (v/v) sodium phosphate, pH 6.5, and 35% (v/v) methanol. The results presented here serve as a caveat that the effect of the wall in CZE or the stationary phase in HPLC on the reaction cannot be ignored.
采用柱内孵育法结合毛细管区带电泳（CZE）和高效液相色谱（HPLC）以及特制溶解法，研究了含有肽基脯氨酸部分的二肽顺反异构化的平衡和动力学参数。之所以能够使用后者，是因为固体肽的构象组成，以及由此形成的特制溶液的构象组成，与平衡状态下的溶液有足够大的差异。该方法结合1H-NMR和CZE分析，对于在10℃、pH 8.4的硼酸钠水溶液中苯丙氨酸-脯氨酸（Phe-Pro）的顺反异构化，分别得到了非常相似的结果，平衡常数和速率常数的平均值分别为0.34和6.6×10-5 s-1。柱内孵育法通过CZE或HPLC进行，使得构象异构体在柱的前半部分分离，然后在柱内孵育，在那里它们相互转化并达到平衡。随后在柱的后半部分进行分离得到四个峰。因此，通过测量构象异构体组成随反应时间的变化，就可以评估速率常数。在10℃下，通过CZE柱内孵育法测定的Phe-Pro异构化的正向速率常数为1.42×10-4 s-1，是特制溶解法所得值的两倍。据信，熔融石英毛细管的内壁对异构化有催化作用。还采用计算机模拟来进一步深入了解毛细管内壁对这种异构化的催化活性。在pH 8.4的硼酸盐缓冲水溶液中，对于37 cm长的毛细管，Phe-Pro的实验和模拟曲线吻合得非常好，但在其他条件相同的情况下，必须使用快四倍的相互转化率才能与57 cm长的毛细管得到的实验曲线相匹配。通过HPLC柱内孵育法证实了十八烷基硅胶固定相对异构化的催化作用。Phe-Pro顺反异构化的总速率，包括在固定相和流动相中的反应，在0℃时比在含有体积分数65%的磷酸钠（pH 6.5）和体积分数35%的甲醇的流动相的自由溶液中通过NMR测量的速率快约六倍。此处给出的结果警示人们，CZE中的管壁或HPLC中的固定相对反应的影响不能被忽视。
