Sakamoto H, Shimohigashi Y, Maeda I, Nose T, Nakashima K, Nakamura I, Ogawa T, Kawano K, Ohno M
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka, Japan.
J Mol Recognit. 1993 Jun;6(2):95-100. doi: 10.1002/jmr.300060207.
A complete series of configurational isomers (L-L, L-D, D-L and D-D) of a dipeptide Leu-Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MHz 1H NMR, the L-D and D-L isomers, but not the L-L and D-D isomers, showed fairly large upfield shifts (0.2-0.4 ppm) of Leu-beta CH2 and gamma CH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe-beta CH2, the NOE enhancement observed between Leu-delta CH3 and Phe-phenyl groups revealed that these groups are in close proximity. These data indicated that L-D and D-L isomers form a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac-Tyr-OEt as a substrate, the L-L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D-L isomer was strongest with Ki of 2.2 x 10(-5) M. It was found that the D-L isomer was only slowly hydrolysed but the L(or D)-D isomer was not. H-D-Phe-L-Leu-OBzl with the inverse sequence of H-D-Leu-L-Phe-OBzl inhibited chymotrypsin more strongly (Ki = 6.3 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)
已合成了二肽亮氨酸 - 苯丙氨酸苄酯的一系列完整构型异构体（L - L、L - D、D - L和D - D），并对其进行了胰凝乳蛋白酶检测。在400 MHz 1H NMR的构象分析中，L - D和D - L异构体（而非L - L和D - D异构体）的亮氨酸β - CH2和γ - CH质子信号出现相当大的高场位移（0.2 - 0.4 ppm），表明存在来自苯环的屏蔽效应。除了苯丙氨酸β - CH2有明显的信号分裂外，亮氨酸δ - CH3和苯丙氨酸苯基之间观察到的核Overhauser效应（NOE）增强表明这些基团距离很近。这些数据表明，L - D和D - L异构体在相邻亮氨酸和苯丙氨酸残基的侧链之间形成了疏水核心。当使用乙酰 - 酪氨酸乙酯作为底物检测二肽对胰凝乳蛋白酶的抑制作用时，L - L异构体没有抑制作用，自身反而成为了底物。然而，其他三种异构体以竞争性方式抑制胰凝乳蛋白酶，其中D - L异构体最强，其抑制常数Ki为2.2×10⁻⁵ M。发现D - L异构体仅缓慢水解，而L（或D） - D异构体不水解。具有与H - D - 亮氨酸 - L - 苯丙氨酸苄酯相反序列的H - D - 苯丙氨酸 - L - 亮氨酸 - OBzl对胰凝乳蛋白酶的抑制作用更强（Ki = 6.3×10⁻⁶ M）。（摘要截短于250字）
