BENZOYLECGONINE HYDRAZIDES - SYNTHESIS, COUPLING TO HORSERADISH-PEROXIDASE, AND CHARACTERIZATION OF THE CONJUGATES

Benzoylecgonine-horseradish peroxidase conjugate (BE-HRP) can be used as a diagnostic reagent for the detection of cocaine in illicit drug s amples and in biological fluids. This paper describes the preparation and characterization of BE-HRP. Two hydrazide derivatives of benzoylec gonine, N-2-(tert-butyloxycarbonyl)benzoylecgonine hydrazide and mono( N-2'-benzoylecgoninoyl)adipic dihydrazide, were synthesized by carbodi imide-activated coupling of benzoylecgonine to N-2-(tert-butyloxycarbo nyl) hydrazide and adipic dihydrazide, respectively. Removal of the te rt-butyloxycarbonyl protecting group in N-2-(tert-butyloxycarbonyl)ben zoylecgonine hydrazide with anhydrous HCl yielded benzoylecgonine hydr azide hydrochloride. NMR and high-resolution mass spectral analyses de monstrated that the benzoyl group of benzoylecgonine remained intact u nder the conditions of both carbodiimide coupling and anhydrous HCl tr eatment. By aldehyde-hydrazide condensation, the hydrazides were coval ently conjugated to the carbohydrate residues of horseradish peroxidas e (HRP). Dot blot analysis of the conjugates employing antibodies spec ific to benzoylecgonine demonstrated the presence of bound benzoylecgo nine in HRP. The stoichiometry of benzoylecgonine residues to HRP was determined by matrix-assisted laser desorption/ionization mass spectro metry (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-acti vated benzoylecgonine to HRP that was derivatized with adipic dihydraz ide.