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 journalArticle

5 Citations (Scopus)

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
StatePublished - Dec 1 1996

Fingerprint

Horseradish Peroxidase
Carbohydrates
Aldehydes
Antibodies
Stoichiometry
Ionization
Mass spectrometry
Condensation
Desorption
Nuclear magnetic resonance
Derivatives
Fluids
Lasers
Carbodiimides
benzoylecgonine
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Street Drugs
Cocaine

All Science Journal Classification (ASJC) codes

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

Cite this

Channe Gowda, D., Ambekar, S. Y., Gupta, P., Lecchi, P., Pannell, L. K., & Davidson, E. A. (1996). Benzoylecgonine hydrazides: Synthesis, coupling to horseradish peroxidase, and characterization of the conjugates. Bioconjugate Chemistry, 7(2), 265-270.
Channe Gowda, D. ; Ambekar, Sarvottam Y. ; Gupta, Priyadarshan ; Lecchi, Paolo ; Pannell, Lewis K. ; Davidson, Eugene A. / Benzoylecgonine hydrazides : Synthesis, coupling to horseradish peroxidase, and characterization of the conjugates. In: Bioconjugate Chemistry. 1996 ; Vol. 7, No. 2. pp. 265-270.
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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.",
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Channe Gowda, D, Ambekar, SY, Gupta, P, Lecchi, P, Pannell, LK & Davidson, EA 1996, 'Benzoylecgonine hydrazides: Synthesis, coupling to horseradish peroxidase, and characterization of the conjugates', Bioconjugate Chemistry, vol. 7, no. 2, pp. 265-270.

Benzoylecgonine hydrazides : Synthesis, coupling to horseradish peroxidase, and characterization of the conjugates. / Channe Gowda, D.; Ambekar, Sarvottam Y.; Gupta, Priyadarshan; Lecchi, Paolo; Pannell, Lewis K.; Davidson, Eugene A.

In: Bioconjugate Chemistry, Vol. 7, No. 2, 01.12.1996, p. 265-270.

Research output: Contribution to journalArticle

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T1 - Benzoylecgonine hydrazides

T2 - Synthesis, coupling to horseradish peroxidase, and characterization of the conjugates

AU - Channe Gowda, D.

AU - Ambekar, Sarvottam Y.

AU - Gupta, Priyadarshan

AU - Lecchi, Paolo

AU - Pannell, Lewis K.

AU - Davidson, Eugene A.

PY - 1996/12/1

Y1 - 1996/12/1

N2 - 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.

AB - 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.

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