Benzoylecgonine hydrazides: Synthesis, coupling to horseradish peroxidase, and characterization of the conjugates

D. Channe Gowda, Sarvottam Y. Ambekar, Priyadarshan Gupta, Paolo Lecchi, Lewis K. Pannell, Eugene A. Davidson

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

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-butylorycarbony] 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′-benzoylecgonmoyl) 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.

Original languageEnglish (US)
Pages (from-to)265-270
Number of pages6
JournalBioconjugate Chemistry
Volume7
Issue number2
DOIs
StatePublished - 1996

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

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