Gowda D C, Ambekar S Y, Gupta P, Lecchi P, Pannell L K, Davidson E A
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D.C. 20007, USA.
Bioconjug Chem. 1996 Mar-Apr;7(2):265-70. doi: 10.1021/bc960009j.
Benzoylecgonine-horseradish peroxidase conjugate (BE-HRP) can be used as a diagnostic reagent for the detection of cocaine in illicit drug samples and in biological fluids. This paper describes the preparation and characterization of BE-HRP. Two hydrazide derivatives of benzoylecgonine, N-2-(tert-butyloxycarbonyl)benzoylecgonine hydrazide and mono(N-2'-benzoylecgoninoyl)adipic dihydrazide, were synthesized by carbodiimide-activated coupling of benzoylecgonine to N-2-(tert-butyloxycarbonyl) hydrazide and adipic dihydrazide, respectively. Removal of the tert-butyloxycarbonyl protecting group in N-2-(tert-butyloxycarbonyl)benzoylecgonine hydrazide with anhydrous HCl yielded benzoylecgonine hydrazide hydrochloride. NMR and high-resolution mass spectral analyses demonstrated that the benzoyl group of benzoylecgonine remained intact under the conditions of both carbodiimide coupling and anhydrous HCl treatment. By aldehyde-hydrazide condensation, the hydrazides were covalently conjugated to the carbohydrate residues of horseradish peroxidase (HRP). Dot blot analysis of the conjugates employing antibodies specific to benzoylecgonine demonstrated the presence of bound benzoylecgonine in HRP. The stoichiometry of benzoylecgonine residues to HRP was determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Mono(N-2'-benzoylecgoninoyl)adipic dihydrazide gave a 2.5-3-fold higher coupling compared with benzoylecgonine hydrazide. Conjugates were also prepared by the coupling of the carbodiimide-activated benzoylecgonine to HRP that was derivatized with adipic dihydrazide.
苯甲酰爱康宁 - 辣根过氧化物酶偶联物（BE - HRP）可用作诊断试剂，用于检测非法药物样品和生物体液中的可卡因。本文描述了BE - HRP的制备和表征。通过碳二亚胺活化的苯甲酰爱康宁与N - 2 -（叔丁氧羰基）肼和己二酸二肼的偶联反应，分别合成了苯甲酰爱康宁的两种酰肼衍生物，即N - 2 -（叔丁氧羰基）苯甲酰爱康宁酰肼和单（N - 2'-苯甲酰爱康宁酰基）己二酸二肼。用无水HCl去除N - 2 -（叔丁氧羰基）苯甲酰爱康宁酰肼中的叔丁氧羰基保护基，得到苯甲酰爱康宁酰肼盐酸盐。核磁共振（NMR）和高分辨率质谱分析表明，在碳二亚胺偶联和无水HCl处理条件下，苯甲酰爱康宁的苯甲酰基保持完整。通过醛 - 酰肼缩合反应，酰肼与辣根过氧化物酶（HRP）的碳水化合物残基共价偶联。使用对苯甲酰爱康宁特异的抗体对偶联物进行斑点印迹分析，证明HRP中存在结合的苯甲酰爱康宁。通过基质辅助激光解吸/电离质谱（MALDI - MS）测定苯甲酰爱康宁残基与HRP的化学计量比。与苯甲酰爱康宁酰肼相比，单（N - 2'-苯甲酰爱康宁酰基）己二酸二肼的偶联率高2.5 - 3倍。还通过碳二亚胺活化的苯甲酰爱康宁与用己二酸二肼衍生化的HRP的偶联反应制备了偶联物。
