Roach Timothy A, Macdonald Victor W, Hosmane Ramachandra S
Laboratory for Drug Design and Synthesis, Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, USA.
J Med Chem. 2004 Nov 18;47(24):5847-59. doi: 10.1021/jm030645k.
Bis[2-(4-phosphonooxyphenoxy)carbonylethyl]phosphinc acid (BPPCEP) was prepared and evaluated as a site-directed affinity reagent for cross-linking human hemoglobin. It was synthesized in four steps starting from 4-benzyloxyphenol and was converted to its pentasodium salt so as to afford efficient cross-linking in an aqueous medium. The reagent was found to specifically cross-link human hemoglobin A(0) in the beta-cleft chains under oxygenated reaction conditions at neutral pH. The amino acid residues involved in the cross-linking were determined by mass spectral analyses of tryptic digest fragments of cross-linked hemoglobin, employing a MALDI-TOF mass spectrometer. The MS analyses suggested that the most likely amino acids involved in the cross-links are Val-1 or Lys-82 present on one of the beta subunits and Lys-82 or Lys-144 on the other. Molecular modeling studies performed on the reagent-HbA(0) complex corroborated the conclusions reached by MALDI-MS analyses. The oxygen equilibrium measurements of the three major BPPCEP-cross-linked Hb products, isolated and purified by preparative cation exchange chromatography, exhibited oxygen affinity (P(50)) values of 14.5, 12.1, and 15.5 Torr as compared with the P(50) of 13.1 Torr for cell-free hemoglobin. The oxygen-binding cooperativity of the modified products, as determined by the Hill coefficient generated from the Hill plots of the respective P(50) values, coupled with the absence of sigmoidal shape of the O(2) equilibrium curves, was considerably lower than that of the native hemoglobin.
制备了双[2-(4-膦酰氧基苯氧基)羰基乙基]膦酸（BPPCEP），并将其作为一种定点亲和试剂用于交联人血红蛋白进行评估。它以4-苄氧基苯酚为起始原料，经四步合成，然后转化为其五钠盐，以便在水性介质中实现高效交联。发现该试剂在中性pH值的氧化反应条件下，能特异性地交联β-裂隙链中的人血红蛋白A(0)。通过使用基质辅助激光解吸电离飞行时间质谱仪对交联血红蛋白的胰蛋白酶消化片段进行质谱分析，确定了参与交联过程中的氨基酸残基。质谱分析表明，参与交联的最可能的氨基酸是一个β亚基上的缬氨酸-1或赖氨酸-82，以及另一个β亚基上的赖氨酸-82或赖氨酸-144。对试剂-HbA(0)复合物进行的分子模拟研究证实了基质辅助激光解吸电离质谱分析得出的结论。通过制备型阳离子交换色谱法分离和纯化得到的三种主要BPPCEP交联的血红蛋白产物的氧平衡测量结果显示，其氧亲和力（P(50)）值分别为14.5、12.1和15.5托，而无细胞血红蛋白的P(50)值为13.1托。根据各自P(50)值的希尔图生成的希尔系数所确定的修饰产物的氧结合协同性，以及O(2)平衡曲线不存在S形，均明显低于天然血红蛋白。
