Goolcharran C, Borchardt R T
The Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66047, USA.
J Pharm Sci. 1998 Mar;87(3):283-8. doi: 10.1021/js970325m.
The intramolecular aminolysis of Phe-Pro-p-nitroaniline (Phe-Pro-pNA) to Phe-Pro-diketopiperazine (Phe-Pro-DKP) was studied as a function of pH, temperature, buffer concentration, and buffer species using an HPLC assay that permits simultaneous analysis of the disappearance of the starting material and the appearance of degradation products. The degradation followed pseudo-first-order kinetics and showed significant dependence on pH. Phosphate (pH 5-8) and glycine (pH 9-10) buffers exhibit general base catalysis. The pH-rate profile suggested that the rate of Phe-Pro-DKP formation depends on the degree of ionization of the N-terminal amino group, with the unprotonated reactant being more reactive than the protonated form. The pKa value of 6.1, determined kinetically, and three microscopic rate constants were adequate to describe the shape of the pH-rate profile. In the pH range studied, Phe-Pro-DKP was the only product generated upon degradation of Phe-Pro-pNA. At pH values between 3 and 8, Phe-Pro-DKP was stable, while at pH less than 3 and greater than 8 it undergoes hydrolysis to the dipeptide, Phe-Pro-OH. Sequence inversion, a reaction normally associated with DKP formation, was not observed. The influence of primary sequence on the formation of DKP was also investigated using X-Pro-pNA analogues, where X = Gly, Ala, Val, Phe, beta-cyclohexylalanine, and Arg. Changing the amino acid preceding the proline residue had a significant effect on the rate of DKP formation at pH 7.0.
使用一种能够同时分析起始原料的消失和降解产物出现的高效液相色谱法，研究了苯丙氨酸 - 脯氨酸 - 对硝基苯胺（Phe - Pro - pNA）分子内氨解生成苯丙氨酸 - 脯氨酸 - 二酮哌嗪（Phe - Pro - DKP）与pH、温度、缓冲液浓度和缓冲液种类的关系。降解遵循准一级动力学，且对pH有显著依赖性。磷酸盐（pH 5 - 8）和甘氨酸（pH 9 - 10）缓冲液表现出一般碱催化作用。pH - 速率曲线表明，Phe - Pro - DKP的形成速率取决于N端氨基的电离程度，未质子化的反应物比质子化形式更具反应性。通过动力学测定的pKa值为6.1，以及三个微观速率常数足以描述pH - 速率曲线的形状。在所研究的pH范围内，Phe - Pro - pNA降解时仅生成Phe - Pro - DKP这一种产物。在pH值为3至8之间，Phe - Pro - DKP是稳定的，而在pH小于3和大于8时，它会水解生成二肽Phe - Pro - OH。未观察到通常与DKP形成相关的序列反转反应。还使用X - Pro - pNA类似物（其中X = Gly、Ala、Val、Phe、β - 环己基丙氨酸和Arg）研究了一级序列对DKP形成的影响。改变脯氨酸残基之前的氨基酸对pH 7.0时DKP的形成速率有显著影响。
