Cocaine Benzoyl Thioester:  Synthesis, Kinetics of Base Hydrolysis, and Application to the Assay of Cocaine Esterases

Cashman, John R.; Berkman, Clifford E.; Underiner, Gail E.; Kolly, Carrie A.; Hunter, Allen D.

doi:10.1021/tx9800203

Cited by 9 publications

(9 citation statements)

References 21 publications

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“…This conclusion is supported by the specificity constants, k cat / K m = k 2 / K s , for wild‐type and D70G hydrolysis of BzSCh, which are smaller than those for BzCh by 17‐fold and 32‐fold, respectively. These ratios are similar to the ratio ( k cat / K m ) ester/thioester of 52 for the hydrolysis of (–)‐cocaine [31] and (–)‐thiococaine [32] by wild‐type enzyme, at 25 °C. The cocaine esters are the bulkiest benzoyl ester and thioester compounds hydrolyzed by this enzyme.…”

Section: Steady‐state Hydrolysis Of Bzch Vs Bzschsupporting

confidence: 68%

“…With BzSCh, acylation is the rate‐limiting step, whereas deacylation is the rate‐limiting step for hydrolysis of BzCh. This switch in rate‐limiting step is also observed for BuChE‐catalyzed hydrolysis of the bulky benzoyl ester/thioester couple (–)‐cocaine/(–)‐thiococaine [31,32]. The switch in rate‐limiting step appears to be the result of a combination of steric constraints and impaired electronic effects of the ethereal sulfur atom on stabilization of the acylation transition state.…”

Section: Resultsmentioning

confidence: 74%

“…For D70G, k 0 + k −0 = k es + k −es ≈ 0.016 s −1 . K ′ s values should correspond to K s taken from Glaubiger [32], which in Table 3 is 14.1 µ m for wild‐type and 556 µ m for D70G. In the simplest case, if K s = K ′ s , it follows from that k = ( k 0 + k −0 + k es + k −es )/2 when [S] = K s = K ′ s ; then for wild‐type, k = 0.019 s −1 and the dissociation constant of ES and E′S can be estimated graphically from Fig.…”

Section: Dependence Of Hysteresis On Substrate Concentrationmentioning

confidence: 99%

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Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate

Masson

Goldstein

Debouzy³

et al. 2003

European Journal of Biochemistry

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Steady-state kinetics for the hydrolysis of benzoylcholine (BzCh) and benzoylthiocholine (BzSCh) by wild-type human butyrylcholinesterase (BuChE) and by the peripheral anionic site mutant D70G were compared. k cat /K m for the hydrolysis of BzSCh was 17-fold and 32-fold lower than that for hydrolysis of BzCh by wild-type and D70G, respectively. The rate-limiting step for hydrolysis of BzCh was deacylation, whereas acylation was rate-limiting for hydrolysis of BzSCh. Wild-type enzyme and the D70G mutant were found to reach steady-state velocity slowly with BzCh as the substrate. At pH 6, the approach to steady-state for both enzymes consisted of a mono-exponential acceleration upon which a set of damped oscillations was superimposed. From pH 7 to 8.5, the approach to steady-state consisted of a simple exponential acceleration. The damped oscillations were analyzed by both a numerical approximation and simulation based on a theoretical model. BuChE-catalyzed hydrolysis of the thiocholine analogue of BzCh showed neither lags nor oscillations, under the same conditions. The frequency and amplitude of the damped oscillations decreased as the BzCh concentration increased. The apparent induction time for the exponential portion of the lag was calculated from the envelope of the damped oscillations or from the smooth lag. Wild-type BuChE showed a hyperbolic increase in induction time as the BzCh concentration increased (s max ¼ 210 s at pH 6.0). However, the induction time for D70G was constant over the whole range of BzCh concentrations (s max ¼ 60 s at pH 6.0). Thus, the induction time does not conform to a simple hysteretic model in which there is a slow conformational transition of the enzyme from an inactive form E to an active form E¢. No pH-dependence of the induction time was found between pH 6.0 and 8.5 in sodium phosphate buffers of various concentrations (from 1 mM to 1 M). However, increasing the pH tended to abolish the oscillations (increase the damping factor). This effect was more pronounced for D70G than for wild-type. Although the lyotropic properties of phosphate change from chaotropic at pH 6.0 to kosmotropic at pH > 8.0, no effect of phosphate concentration on the oscillations was noticed at the different pH values, suggesting that the oscillations are not related to a pH-dependent Hofmeister effect of phosphate ions. Simulation and theoretical analysis of the oscillatory behaviour of the approach to the steady-state for BuChE led us to propose a model for the hysteresis of BuChE with BzCh. In this model, the substrate-free enzyme is present as an equilibrium mixture of two forms, E and E¢. Substrate binds to E and E¢, but only E¢S makes products. It is proposed that oscillations originate from a time-dependent change in the local concentration, solvation and/or conformation of substrate in the bulk solution. 1 H-NMR measurements provided evidence for a slow equilibrium between two BzCh conformers. Binding of the conformationally preferred substrate conformer leads to products.

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Section: Steady‐state Hydrolysis Of Bzch Vs Bzschsupporting

confidence: 68%

Section: Resultsmentioning

confidence: 74%

Section: Dependence Of Hysteresis On Substrate Concentrationmentioning

confidence: 99%

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Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate

Masson

Goldstein

Debouzy³

et al. 2003

European Journal of Biochemistry

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“…The catalytic efficiency of butyrylcholinesterase to hydrolyze esters is in fact mostly dependent on the size and enantiomeric configuration of the acyl or alcohol moieties. For instance, k cat for (+)cocaine is 7500 min −1 while it is 3.9 min −1 for (−)cocaine [30]. The substrate slides down the gorge to form a first enzyme-substrate complex, ES 1 , that is non productive.…”

Section: Catalytic Issuesmentioning

confidence: 99%

Butyrylcholinesterase for protection from organophosphorus poisons: Catalytic complexities and hysteretic behavior

Masson

Lockridge

2010

Archives of Biochemistry and Biophysics

204

135

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Butyrylcholinesterase is a promiscuous enzyme that displays complex kinetic behavior. It is toxicologically important because it detoxifies organophosphorus poisons (OP) by making a covalent bond with the OP. The OP and the butyrylcholinesterase are both inactivated in the process. Inactivation of butyrylcholinesterase has no adverse effects. However inactivation of acetylcholinesterase in nerve synapses can be lethal. OP-inhibited butyrylcholinesterase and acetylcholinesterase can be reactivated with oximes provided the OP has not aged. Strategies for preventing the toxicity of OP include a) treatment with an OP scavenger, b) reaction of nonaged enzyme with oximes, c) reactivation of aged enzyme, d) slowing down aging with peripheral site ligands, and e) design of mutants that rapidly hydrolyze OP. Option (a) has progressed through phase I clinical trials with human butyrylcholinesterase. Option (b) is in routine clinical use. The others are at the basic research level. Butyrylcholinesterase displays complex kinetic behavior including activation by positively charged esters, ability to hydrolyze amides, and a lag time (hysteresis) preceding hydrolysis of benzoylcholine and N-methyl indoxyl acetate. Mass spectrometry has identified new OP binding motifs on tyrosine and lysine in proteins that have no active site serine. It is proposed, but not yet proven, that low dose exposure involves OP modification of proteins that have no active site serine.

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“…Scaling the synthesis of 3a by as much as 30-fold had no obvious effect on the efficiency of this method, further attesting the robustness of the process. Unexpectedly, the thioester functionality of 3a could not be hydrolyzed through a common base-catalyzed method, likely because of steric hindrance. Oxidative hydrolysis was then realized with the use of alkaline hydrogen peroxide–urea (UHP), and furan-3-carboxylic acid 3aa was obtained in excellent yield.…”

Section: Resultsmentioning

confidence: 99%

Access to Multisubstituted Furan-3-carbothioates via Cascade Annulation of α-Oxo Ketene Dithioacetals with Isoindoline-1,3-dione-Derived Propargyl Alcohols

et al. 2018

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A Brønsted acid-promoted, unprecedented formal (3 + 2) annulation strategy for the synthesis of multisubstituted furan-3-carbothioates is reported. This transformation represents the first regioselective annulation of α-oxo ketene dithio-acetals as 1,3-bis-nucleophiles in a cascade manner. The choice of isoindoline-1,3-dione-derived propargyl alcohols is crucial to the uncommon annulation mode between an alkyne-type bis-electrophile and a 1,3-bis-nucleophile under metal-free conditions. The scale-up of the synthesis and several interesting transformations of an as-synthesized product were further investigated. A Nazarov-like cyclization is proposed for the ring-closure process according to the experimental observations.

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Cocaine Benzoyl Thioester: Synthesis, Kinetics of Base Hydrolysis, and Application to the Assay of Cocaine Esterases

Cited by 9 publications

References 21 publications

Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate

Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate

Butyrylcholinesterase for protection from organophosphorus poisons: Catalytic complexities and hysteretic behavior

Access to Multisubstituted Furan-3-carbothioates via Cascade Annulation of α-Oxo Ketene Dithioacetals with Isoindoline-1,3-dione-Derived Propargyl Alcohols

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