Tous G, Bush A, Tous A, Jordan F
Department of Chemistry, Rutgers, State University, Newark, New Jersey 07102.
J Med Chem. 1990 Jun;33(6):1620-34. doi: 10.1021/jm00168a015.
A series of O'-(epoxyalkyl)tyrosines and a carboxy terminal (epoxyalkyl)tyrosine and -phenylalanine were synthesized as potential serine protease inhibitors. N-Acetyl derivatives showed irreversible inactivation vis-a-vis subtilisin, while the N-benzoyl ones were specific toward chymotrypsin. The most potent inactivation of chymotrypsin was achieved by a O'-(3,4-epoxybutyl)-L-tyrosine derivative. The inactivation was shown to be stereospecific since a D derivative led to no irreversible inactivation. Placement of the epoxyalkyl group at the carboxy terminus led to potent rapid inactivation. Under these conditions some of the activity was later recovered. The two classes of inactivators (O'-epoxyalkyl and carboxy-epoxyalkyl) appear to operate by different mechanisms. Most importantly, it was found that irreversible inactivation by O'-(epoxyalkyl)-L-tyrosine only resulted if the carboxy terminus was a substrate (i.e. a compound with free carboxy terminus did not lead to inactivation). The ultimate activity kinetic assay (Daniels, S. B.; et al. J. Biol. Chem. 1983, 258, 15046-15053.) indicated that the epoxyalkyl group on the phenolic oxygen had an optimal length of four carbons with respect to the turnover ratio (the ratio of molecules undergoing turnover compared to those that inactivate the enzyme) for chymotrypsin. A different kinetic assay (Ashani, Y.; Wins, P.; Wilson, I. B. Biochim. Biophys. Acta 1972, 284, 427-434.) demonstrated that substratelike turnover was proceeding at considerably slower rates than for the corresponding true substrates and with rate-limiting deacylation of the acyl-enzyme. Amino acid analysis subsequent to acid hydrolysis demonstrated that Met had been selectively alkylated by the O'-(epoxyalkyl)tyrosine derivative. By contrast, alpha-chymotrypsin inactivated with N-benzoyl-L-Phe-2,3-epoxypropyl ester then subjected to amino acid analysis showed no change in the content of any amino acid that would serve as a potential nucleophile to the inhibitor. Yet, the L-Phe content increased, indicating that a covalent bond had been formed between the inhibitor and the enzyme. Either the bond between the inhibitor and the enzyme did not withstand the hydrolytic conditions and/or there was less than 10% decrease in the amino acids with nucleophilic side chains upon inactivation. Finally, two tripeptides containing O'-(epoxyalkyl)-L-tryosines were synthesized [N-(tert-butoxycarbonyl)-L-alanyl-L-alanyl-O'-(2,3-epoxypropyl)-L-tyrosi ne ethyl ester and N-(trifluoroacetyl)-L-valyl-O'-(2,3-epoxypropyl)-L-tyrosyl-L-valine methyl ester] as potential elastase inhibitors and were found to reversibly and competitively inhibit porcine pancreatic elastase.
合成了一系列O'-（环氧烷基）酪氨酸以及一个羧基末端（环氧烷基）酪氨酸和-苯丙氨酸，作为潜在的丝氨酸蛋白酶抑制剂。N-乙酰基衍生物对枯草杆菌蛋白酶表现出不可逆失活，而N-苯甲酰基衍生物对胰凝乳蛋白酶具有特异性。O'-（3,4-环氧丁基）-L-酪氨酸衍生物对胰凝乳蛋白酶的失活作用最强。由于D型衍生物不会导致不可逆失活，所以这种失活具有立体特异性。将环氧烷基置于羧基末端会导致高效快速失活。在这些条件下，部分活性后来得以恢复。两类失活剂（O'-环氧烷基和羧基-环氧烷基）似乎通过不同机制起作用。最重要的是，发现只有当羧基末端是底物时，O'-（环氧烷基）-L-酪氨酸才会导致不可逆失活（即具有游离羧基末端的化合物不会导致失活）。最终活性动力学分析（Daniels, S. B.；等人，《生物化学杂志》，1983年，258卷，15046 - 15053页）表明，酚氧上的环氧烷基相对于胰凝乳蛋白酶的周转比（发生周转的分子与使酶失活的分子之比）具有四个碳原子的最佳长度。另一种动力学分析（Ashani, Y.；Wins, P.；Wilson, I. B.，《生物化学与生物物理学报》，1972年，284卷，427 - 434页）表明，类似底物的周转速率比相应的真实底物慢得多，且酰基酶的脱酰化是限速步骤。酸水解后的氨基酸分析表明，甲硫氨酸已被O'-（环氧烷基）酪氨酸衍生物选择性烷基化。相比之下，用N-苯甲酰基-L-苯丙氨酸-2,3-环氧丙酯失活后进行氨基酸分析的α-胰凝乳蛋白酶，其作为抑制剂潜在亲核试剂的任何氨基酸含量均未发生变化。然而，L-苯丙氨酸含量增加，表明抑制剂与酶之间形成了共价键。要么抑制剂与酶之间的键无法承受水解条件，和/或失活后具有亲核侧链的氨基酸减少量不到10%。最后，合成了两种含有O'-（环氧烷基）-L-酪氨酸的三肽[N-（叔丁氧羰基）-L-丙氨酰-L-丙氨酰-O'-（2,3-环氧丙基）-L-酪氨酸乙酯和N-（三氟乙酰基）-L-缬氨酰-O'-（2,3-环氧丙基）-L-酪氨酰-L-缬氨酸甲酯]作为潜在的弹性蛋白酶抑制剂，发现它们能可逆且竞争性地抑制猪胰弹性蛋白酶。
