Maeda I, Shimohigashi Y, Ikesue K, Nose T, Ide Y, Kawano K, Ohno M
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
J Biochem. 1996 May;119(5):870-7. doi: 10.1093/oxfordjournals.jbchem.a021324.
The dipeptide benzyl amide H-D-Thr-Phe-NH-CH2-C6H5 was found to inhibit chymotrypsin strongly (K1 = 4.5 x 10(-6) M) in a competitive manner. When a series of phenyl amides H-D-Thr-Phe-NH-(CH2)n-C6H5 (n = 0-4) were tested, inhibitory potency peaked at n = 1 (benzyl amide). Incorporation of a methyl group into the benzyl methylene resulted in formation of stereoisomers, H-D-Thr-Phe-NH-(R or S)-CH(CH3)-C6H5, with considerably different inhibitory potencies. The R-isomer was as active as the benzyl amide, while the S-isomer was about 30-fold less active than the benzyl amide. Furthermore, when a fluorine atom was introduced into the para-position of the amide-benzyl group, the resulting H-D-Thr-Phe-NH-CH2-C6H4(p-F) showed considerably enhanced inhibitory activity (about 5-fold, K1 = 9.1 x 10(-7) M). In conformational analysis by 400 mHz 1H-NMR, all dipeptides having D-Thr-Phe backbone structure showed large upfield shifts of D-Thr-beta OH (shifts in ppm, 0.09-0.17), D-Thr-beta CH (0.23-0.32), and D-Thr-gamma CH3 (0.38-0.53), indicating the presence of shielding effects from the benzene ring. In addition, NOE enhancements between the D-Thr-gamma CH3 and Phe-phenyl groups were evidenced by measurements of two-dimensional NOESY spectra and NOE difference spectra. These observations demonstrated the spatial proximity of these side chains, which is due to side chain-side chain CH/pi interaction. All these results support the idea that the amide-benzyl group binds at the chymotrypsin S1 site, while the hydrophobic core with CH/pi interaction binds at the S2 or S1' site.
发现二肽苄基酰胺H-D-苏氨酸-苯丙氨酸-NH-CH₂-C₆H₅以竞争性方式强烈抑制胰凝乳蛋白酶（K₁ = 4.5×10⁻⁶ M）。测试一系列苯基酰胺H-D-苏氨酸-苯丙氨酸-NH-(CH₂)ₙ-C₆H₅（n = 0 - 4）时，抑制效力在n = 1（苄基酰胺）时达到峰值。在苄基亚甲基中引入一个甲基导致形成立体异构体H-D-苏氨酸-苯丙氨酸-NH-(R或S)-CH(CH₃)-C₆H₅，其抑制效力有很大差异。R-异构体与苄基酰胺活性相当，而S-异构体的活性比苄基酰胺低约30倍。此外，当在酰胺苄基的对位引入一个氟原子时，所得的H-D-苏氨酸-苯丙氨酸-NH-CH₂-C₆H₄(p-F)表现出显著增强的抑制活性（约5倍，K₁ = 9.1×10⁻⁷ M）。在400 mHz ¹H-NMR的构象分析中，所有具有D-苏氨酸-苯丙氨酸主链结构的二肽都显示出D-苏氨酸-β OH（化学位移以ppm计，0.09 - 0.17）、D-苏氨酸-β CH（0.23 - 0.32）和D-苏氨酸-γ CH₃（0.38 - 0.53）的大幅高场位移，表明存在来自苯环的屏蔽效应。此外，通过二维NOESY谱和NOE差谱测量证明了D-苏氨酸-γ CH₃和苯丙氨酸-苯基之间的NOE增强。这些观察结果证明了这些侧链在空间上的接近性，这是由于侧链-侧链CH/π相互作用。所有这些结果支持这样的观点，即酰胺苄基在胰凝乳蛋白酶的S1位点结合，而具有CH/π相互作用的疏水核心在S2或S1'位点结合。
