Buranaprapuk Apinya, Malaikaew Yaowaluk, Svasti Jisnuson, Kumar Challa V
Department of Chemistry, University of Connecticut, U-3060, 55 N. Eagleville Road, Storrs, Connecticut 06269-3060, USA.
J Phys Chem B. 2008 Jul 31;112(30):9258-65. doi: 10.1021/jp802791c. Epub 2008 Jul 4.
Strong chiral discrimination and site-selective photocleavage of two model proteins, lysozyme and bovine serum albumin (BSA), by new pyrenyl probes are reported here. The enantiomeric pyrenyl probes D-phenylalanine-1(1-pyrene)methylamide (PMA- D-Phe) and L-phenylalanine-1(1-pyrene)methylamide (PMA- l-Phe) were synthesized by coupling the carboxyl function of D-phenylalanine or L-phenylalanine with the amino group of 1(1-pyrene)methylamine. Binding affinities of the two enantiomers with the proteins were quantitated in absorption titrations. BSA indicated 10-fold selectivity for PMA- D-Phe, and the binding constants for the L- and D-enantiomers were 3.8 x 10(5) and 4.0 x 10(6) M(-1), respectively. Lysozyme, similarly, indicated a 6-fold preference for PMA- D-Phe with binding constants of 3.3 x 10 (5) and 2.0 x 10(6) M(-1) for the L- and D-isomers, respectively. Such strong chiral discrimination illustrates the key role of the chiral center of the probe (Phe) in the binding interactions. The enantiomers were tested to examine how the chiral discrimination for their binding influences reactivity toward protein photocleavage. Irradiation of the probe-protein complexes, at 342 nm in the presence of hexammine cobalt(III) chloride, resulted in the cleavage of the protein backbone. Photocleavage did not proceed in the dark or in the absence of the pyrenyl probes. Both enantiomers indicated low reactivity with BSA (<5% yield), while large photocleavage yields ( approximately 57%) have been noted with lysozyme. This lysozyme photocleavage yield is a significant improvement over previous reports. However, both enantiomers cleaved lysozyme at the same location between Trp108-Val109, despite the strong chiral selectivity for binding. H-atom abstraction from Trp 108, accessible from the active site cleft, could initiate the observed peptide bond cleavage.
本文报道了新型芘基探针在两种模型蛋白（溶菌酶和牛血清白蛋白（BSA））上实现的强烈手性识别和位点选择性光裂解。对映体芘基探针D-苯丙氨酸-1（1-芘）甲酰胺（PMA-D-Phe）和L-苯丙氨酸-1（1-芘）甲酰胺（PMA-L-Phe）是通过将D-苯丙氨酸或L-苯丙氨酸的羧基与1（1-芘）甲胺的氨基偶联而合成的。在吸收滴定中对这两种对映体与蛋白质的结合亲和力进行了定量。BSA对PMA-D-Phe表现出10倍的选择性，L-和D-对映体的结合常数分别为3.8×10⁵和4.0×10⁶ M⁻¹。同样，溶菌酶对PMA-D-Phe表现出6倍的偏好，L-和D-异构体的结合常数分别为3.3×10⁵和2.0×10⁶ M⁻¹。这种强烈的手性识别说明了探针（苯丙氨酸）的手性中心在结合相互作用中的关键作用。测试了对映体，以研究它们结合时的手性识别如何影响对蛋白质光裂解的反应性。在六氨合钴（III）氯化物存在下，于342 nm照射探针-蛋白质复合物，导致蛋白质主链裂解。在黑暗中或没有芘基探针时，光裂解不会发生。两种对映体与BSA的反应性都很低（产率<5%），而溶菌酶的光裂解产率很高（约57%）。与之前的报道相比，这种溶菌酶光裂解产率有了显著提高。然而，尽管结合具有很强的手性选择性，但两种对映体都在Trp108-Val109之间的同一位置裂解溶菌酶。从活性位点裂隙可接近的Trp 108上夺取氢原子可能引发观察到的肽键裂解。
