Stereoselective inhibition of butyrylcholinesterase by enantiomers of exo- and endo-2-norbornyl-N-n-butylcarbamates

Figures & data

Figure 1.  Active sites of human BChE⁴. The enzyme active sites consist of at least five major binding sites: (1) an oxyanion hole (OAH); (2) an esteratic site (ES) or catalytic triad; (3) an anionic substrate binding site (AS); (4) an acyl binding site (ABS); and (5) a peripheral anionic binding site (PAS).

Figure 2.  Structures of (R)-(+)-exo-, (S)-(–)-exo-, (R)-(+)-endo-, (S)-(–)-endo-2-norbornyl-N-n-butylcarbamates, rivastigmine, physostigmine, and carbaryl.

Figure 3.  ¹⁹F-NMR spectra after the reaction of (A) (R)-(–)-exo-2-norborneol with S-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride³⁹ in the presence of pyridine in CDCl₃ and (B) (S)-(–)-exo-2-norborneol with (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride in the presence of pyridine in CDCl₃. For (A), −72.069−ppm was the fluorine chemical shift of unreactive (S)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride. The peaks at −73.948 and −74.113 ppm were assigned to the fluorine chemical shifts of (2R)- and (2S)-exo-norbornyl-(S)-α-methoxy-α-trifluoromethylphenylacetates, respectively (Scheme 3). For (B), −72.089 ppm was the fluorine chemical shift of unreactive S-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride. The peaks at −73.965 and −74.130 ppm were assigned to the fluorine chemical shifts of (2R)- and (2S)-exo-norbornyl-(S)-α-methoxy-α-trifluoromethylphenylacetates, respectively (Scheme 3).

Table 1.  Enantiomeric excess (%) and optical purity (%) for the kinetic resolution of racemic exo-2-norborneol (Scheme 1) and endo-2-norborneol (Scheme 2) by lipase in organic solvent.

Compound	Enantiomeric excess (%)^a	Optical purity (%)^b
(R)-(+)-exo-2-norborneol	80	88
(S)-(–)-exo-2-norboneol	84	90
(R)-(+)-endo-2-norborneol	90	96
(S)-(–)-endo-2-norborneol	92	96

^aEnantiomeric excess (%) was calculated from the ratio of integration of the fluorine chemical shift of their Mosher’s ester derivatives from the ¹⁹F-NMR spectra (Scheme 3 and Figure 3).

^bOptical purity (%) was calculated as 100 × [α]_D²⁵_observed/[α]_D²⁵_literature.

Figure 4.  Nonlinear least-squares curve fittings of (A) the k_app vs. (R)-(+)-exo-2-norbornyl-N-n-butylcarbamate concentration ([I]) plot and (B) the k_app vs. (R)-(+)-exo-2-norbornyl-N-n-butylcarbamate concentration ([I]) plot following Equation (1) for pseudo substrate inhibition of BChE. For Figure 4A, the parameters of the fit were k₂ = 0.0030 ± 0.0001 s⁻¹ and (1 + K_m/[S])K_i = 22 ± 4 nM with R= 0.9963. After calculation, K_i = 11 ± 2 nM and k_i = (270 ± 50) × 10³ M^− 1s⁻¹ (Table 2). For Figure 4B, the parameters of the fit were k₂ = 0.00310 ± 0.00007 s⁻¹ and (1 + K_m/[S])K_i = 100 ± 4 nM with R= 0.9920. After calculation, K_i = 50 ± 2 nM and k_i = (62 ± 3) × 10³ M^− 1s⁻¹ (Table 2).

Scheme 1.  Kinetic resolution of (R)-(+)- and (S)-(–)-exo-2-norborneols from lipase-catalyzed hydrolysis of racemic (±)-exo-2-norbornyl butyrate.

Scheme 2.  Kinetic resolution of (R)-(+)- and (S)-(–)-endo-2-norborneols from lipase-catalyzed acetylation of racemic (±)-exo-2-norborneol with vinyl acetate.

Scheme 3.  Determination of enantiomeric excess and absolute configuration of (R)-(+)- and (S)-(–)-exo-2-norborneols by ¹⁹F-NMR spectra of their Mosher’s ester derivatives.

Scheme 4.  Kinetic scheme for pseudo substrate inhibition of BChE by 2-norbornyl-N-n-butylcarbamate in the presence of substrate. E, enzyme; E-A, acyl enzyme; EI, enzyme–inhibitor Michaelis complex; E-I9, carbamyl enzyme; ES, enzyme–substrate Michaelis complex; I, pseudo substrate inhibitor; k₂, carbamylation constant; k₃, decarbamylation constant; k_2S, formation rate constant of E-A; k_3S, deacylation constant of E-A; K_i, inhibition constant; K_m, Michaelis–Menten constant; P, product, 2-norborneol; P9, product, thiocholine; P99, product, butyrate; Q, product, butylcarbamic acid; S, substrate, BTCh.

Table 2.  The k₂, K_i, and k_i values^a of BChE inhibitions by stereoisomers of 2-norbornyl carbamates.

Inhibitor	Ki (nM)	k2 (10^−3s^−1)	ki (10^3 M^−1s^−1)	Enantio-selectivity^b
(R)-(+)-exo-	11 ± 2	3.0 ± 0.1	270 ± 50	4.35
(S)-(–)-exo-	50 ± 2	3.10 ± 0.07	62 ± 3	1.00
rac-(±)-exo-	30 ± 8	2.9 ± 0.1	100 ± 30	1.61
(R)-(+)-endo-	5.0 ± 0.8	1.00 ± 0.05	200 ± 20	1.00
(S)-(–)-endo-	2.0 ± 0.2	1.00 ± 0.02	500 ± 50	2.50
rac-(±)-endo-	3.0 ± 0.6	0.98 ± 0.04	320 ± 70	1.60

^aThe apparent inhibition constant ((1 + [S]/K_m)K_i) and carbamylation constant (k₂) were obtained from nonlinear least-squares curve fitting of the k_app vs. [I] plot following Equation (1).

^bDefined as ki (more potent enantiomer)/k_i (less potent enantiomer).

Figure 5.  Superimposition of (A) (R)-(+)- and (S)-(–)-exo-2-norbornyl-N-n-butyl- carbamates and (B) (R)-(+)- and (S)-(–)-endo-2-norbornyl-N-n-butylcarbamates at their carbamyl moieties and fitting both enantiomers into the active site of BChE. For (A), an unfavorable repulsion between S-enantiomer and the active serine of the enzyme was observed. For (B), an unfavorable repulsion between R-enantiomer and the active serine of the enzyme was observed.

Nicolet Y, Lockridge O, Masson P, Fontecilla-Camps JC, Nachon F. J Biol Chem 2003; 278:41141–7.

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Dale JA, Mosher HS. J Am Chem Soc 1973; 95:512–19.

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