Cocaine Benzoyl Thioester Hydrolysis
Chem. Res. Toxicol., Vol. 11, No. 8, 1998 901
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es could presumably also catalyze the hydrolysis of the
cocaine benzoyl thioester diastereomers examined. Al-
lococaine benzoyl thioester was chosen as a prototypical
substrate because it is efficiently chemically synthesized
in sufficient quantities, it is relatively stable to spontane-
ous hydrolysis (allopseudococaine benzoyl thioester was
hydrolyzed considerably faster than allococaine benzoyl
thioester at pH ) 7.4), and it gave reliable enzyme kinetic
data. The Kmapp values for the hydrolysis of allococaine
benzoyl thioester by PLE, hBuChE, mBuChE, and mAce-
tylChE compares with that of the kinetic constants for
hydrolysis of (-)-cocaine by the same esterases (20) and
this suggests that allococaine benzoyl thioester could be
a convenient surrogate substrate for studying (-)-cocaine
esterases.
Although the configuration at the C-3 position is not
the naturally occurring â configuration, it is possible that,
due to the achievement of the boat-type conformation,
the position of the aromatic group in allococaine benzoyl
thioester is in the same general proximity as it exists in
(-)-cocaine and, as such, provides a substrate that the
enzyme recognizes as not too dissimilar to naturally
occurring (-)-cocaine.
In summary, allococaine benzoyl thioester hydrolysis
by esterases has several advantages over currently used
assays for the study of cocaine esterases. It is sensitive,
rapid, and simple. A further advantage to this assay is
that enzyme activity can be readily determined even in
relatively complex biological matrixes with the proviso
that the medium does not contain high concentrations
of strongly UV-absorbing materials. The assay described
herein does not require any derivatization steps and
avoids the use of radioactive substrates. The assay is
not as sensitive as a radiometric procedure but is less
ambiguous than simply monitoring radioactivity ex-
tracted from an enzyme incubation. For the esterases
examined, it appears that parallel binding avidity and
rate of hydrolysis exists in comparison of allococaine
benzoyl thioester and (-)-cocaine and, thus, allococaine
benzoyl thioester provides an acceptable surrogate for
monitoring cocaine esterases.
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Automation, Inc., Analytical X-ray Instruments, 6300 Enterprise
Lane, Madison, WI 53719-1173.
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Ack n ow led gm en t. We thank R. J ohn Speirs for his
excellent technical assistance. This project was finan-
cially supported (Grants DA08531 and DA00269) by the
National Institute on Drug Abuse. Donations of rare
chemicals from the National Institute on Drug Abuse
Drug Supply System is gratefully acknowledged. We
acknowledge the support for the X-ray diffraction facili-
ties from the National Science Foundation (NSF-RUI
Grant DUE-9403889), the Ohio Board of Regents (1994
OBoR Action Fund CAP-098, 1995 OBoR Investment
Fund CAP-075), and Youngstown State University. C.A.K.
thanks Youngstown State University for a University
Scholarship. A.D.H. thanks Youngstown State University
for Research Professorships for 1993/94 and 1994/95, a
Faculty Improvement Leave 1995/96, and University
Research Grants Numbers 854 and 883.
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Synthesis of an immunogenic template for the generation of
catalytic antibodies for (-)-cocaine hydrolysis. J . Org. Chem. 61,
5686-5689.
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