Gialih Lin

Microwave-promoted lipase-catalyzed reactions

Abstract Lipase from porcine pancreas is demonstrated to catalyze acylation reactions in organic media under microwave irradiation. Reaction rates and enantioselectivities are significantly enhanced 1–14 and 3–9 fold, respectively.

Ultrasound-promoted lipase-catalyzed reactions

Abstract Lipase from porcine pancreas is first demonstrated to catalyze reactions under ultrasonic condition. Reaction rates are significantly enhanced 7 to 83-fold and enantioselectivities are retained.

Molecular recognition by acetylcholinesterase at the peripheral anionic site: structure-activity relationships for inhibitions by aryl carbamates.

Substituted phenyl-N-butyl carbamates (1-9) are potent irreversible inhibitors of Electrophorus electricus acetylcholinesterase. Carbamates 1-9 act as the peripheral anionic site-directed irreversible inhibitors of acetylcholinesterase by the stop-time assay in the presence of a competitive inhibitor, edrophonium. Linear relationships between the logarithms of the dissociation constant of the enzyme inhibitor adduct (Ki), the inactivation constant of the enzyme-inhibitor adduct (k2), and the bimolecular inhibition constant (k(i)) for the inhibition of Electrophorus electricus acetylcholinesterase by carbamates 1-9 and the Hammett substituent constant (sigma), are observed, and the reaction constants (ps) are -1.36, 0.35 and -1.01, respectively. Therefore, the above reaction may form a positive charged enzyme-inhibitor intermediate at the peripheral anionic site of the enzyme and may follow the irreversible inactivation by a conformational change of the enzyme.

Hammett analysis of the inhibition of pancreatic cholesterol esterase by substituted phenyl-N-butylcarbamate

Substituted phenyl-N-butylcarbamates (1) as active site-directed irreversible inhibitors of pancreatic cholesterol esterase are investigated for values of the dissociation constant (KI), the carbamylation constant (k2), and the bimolecular rate constant (ki). Linear free energy relationships between-logKI, logk2, or logki and substituent constant (σ) are observed.

Linear free energy relationships of the inhibition of pancreatic cholesterol esterase by 4-Nitrophenyl-N-alkylcarbamate

Abstract 4-Nitrophenyl- N -alkylcarbamates ( 1 ) as active site-directed irreversible inhibitors of pancreatic cholesterol esterase are investigated for values of the dissociation constant (K i ), the carbamylation constant (k 2 ), and the bimolecular rate constant (k i ). Linear free energy relationships between −logK i , logk 2 , or logk i and polar substituent constant ( σ ∗ ) are observed. Tafts E s steric constants are not important in these correlationships. Multiple linear relationships of −logK i , logk 2 , or logk i are observed on Charton equation (logk = ασ I + βσ R + ψν + h).

Quantitative structure–activity relationships for the pre-steady-state inhibition of cholesterol esterase by 4-nitrophenyl-n-substituted carbamates

4-Nitrophenyl-N-substituted carbamates (1-6) are the pseudo-substrate inhibitors of porcine pancreatic cholesterol esterase. Thus, the first step of the inhibition (Ki step) is the formation of the enzyme inhibitor tetrahedral adduct and the second step of the inhibition (kc) is the formation of the carbamyl enzyme. The formation of the enzyme inhibitor tetrahedral adduct is further divided into two steps, the formation of the enzyme-inhibitor complex with the dissociation constant, KS, at the first step and the formation of the enzyme-inhibitor tetrahedral adduct from the complex at the second step. The two-step mechanism for the formation of the enzyme-inhibitor tetrahedral adduct is confirmed by the pre-steady-state kinetics. The results of quantitative structure-activity relationships for the pre-steady-state inhibitions of cholesterol esterase by carbamates 1-6 indicate that values of -logKs and logk2/k-2 are correlated with the Taft substituent constant, sigma*, and the rho* values from these correlations are -0.33 and 0.1, respectively. The negative rho* value for the -logKS-sigma*-correlation indicates that the first step of the two-step formation of the enzyme-inhibitor tetrahedral adduct (KS step) is the formation of the positive enzyme inhibitor complex. The positive rho* value for the logk2/k-2 -sigma*-correlation indicates that the enzyme inhibitor tetrahedral adduct is more negative than the enzyme inhibitor complex. Finally, the two-step mechanism for the formation of the enzyme inhibitor tetrahedral adduct is proposed according to these results. Thus, the partially positive charge is developed at nitrogen of carbamates 1-6 in the enzyme-inhibitor complex probably due to the hydrogen bonding between the lone pair of nitrogen of carbamates 1-6 and the amide hydrogen of the oxyanion hole of the enzyme. The second step of the two-step formation of the enzyme-inhibitor tetrahedral adduct is the nucleophilic attack of the serine of the enzyme to the carbonyl group of carbamates 1-6 in the enzyme-inhibitor complex and develops the negative-charged oxygen in the adduct.

Conformationally restricted carbamate inhibitors of horse serum butyrylcholinesterase

Conformationally restricted carbamate inhibitors, exo-2-norbornyl-N-butylcarbamate (1), endo-2-norbornyl-N-butylcarbamate (2), l-adamantyl-N-butylcarbamate (3), and 2-adamantyl-N-butylcarbamate (4) as active site-directed irreversible inhibitors of horse serum butyrylcholinesterase are investigated for values of the dissociation constant (KI), the carbamylation constant (k2), and the bimolecular rate constant (ki). Compound 1 is the most potent inhibitor of the enzyme and the values of KI and ki are 20 nM and 1.1 x 10(5) M-1sec-1, respectively.

Ortho effects in quantitative structure-activity relationships for acetylcholinesterase inhibition by aryl carbamates

Ortho-substituted phenyl-N-butyl carbamates (1-9) are characterized as “pseudo-pseudo-substrate” inhibitors of acetylcholinesterase. Since the inhibitors protonate at pH 7.0 buffer solution, the virtual inhibition constants (Ki′s) of the protonated inhibitors are calculated from the equation, −logKi′=−logKi−logKb. The logarithms of the inhibition constant (Ki), the carbamylation constant (kc), and the bimolecular inhibition constant (ki) for the enzyme inhibitions by carbamates 1-9 are multiply linearly correlated with the Hammett para-substituent constant (σp), the Taft-Kutter-Hansch ortho steric constant (ES), and the Swan-Lupton ortho polar constant (F). Values of ρ, δ, and f for the −logKi-, logkc-, and logki-correlations are −0.6, −0.16, 0.7; 0.11, 0.03, −0.3; and −0.5, −0.12, 0.4, respectively. The Ki step further divides into two steps: 1) the pre-equilibrium protonation of the inhibitors, Kb step and 2) formation of a negatively charged enzyme-inhibitor Michaelis-Menten complex—virtual inhibition, Ki′ step. The Ki step has little ortho steric enhancement effect; moreover, the kcstep is insensitive to the ortho steric effect. The f value of 0.7 for the Ki step indicates that ortho electron-withdrawing substituents of the inhibitors accelerate the inhibition reactions from the ortho polar effect; however, the f value of −0.3 for the kcstep implies that ortho electron-withdrawing substituents of the inhibitors lessen the inhibition reactions from the ortho polar effect.

Comparison of active sites of butyrylcholinesterase and acetylcholinesterase based on inhibition by geometric isomers of benzene-di-N-substituted carbamates.

We have reported that benzene‐1,2‐, 1,3‐, and 1,4‐di‐N‐substituted carbamates (1–15) are characterized as the conformationally constrained inhibitors of acetylcholinesterase and mimic gauche, eclipsed, and anti‐conformations of acetylcholine, respectively (J Biochem Mol Toxicol 2007;21:348–353). We further report the inhibition of butyrylcholinesterase by these inhibitors. Carbamates 1–15 are also characterized as the pseudosubstrate inhibitors of butyrylcholinesterase as in the acetylcholinesterase catalysis. Benzene‐1,4‐di‐N‐n‐hexylcarbamate (12) and benzene‐1,4‐di‐N‐n‐octylcarbamate (13) are the two most potent inhibitors of butyrylcholinesterase among inhibitors 1–15. These two para compounds, with the angle of 180° between two C(benzene)O bonds, mimic the preferable anti CO/CN conformers for the choline ethylene backbone of butyrylcholine during the butyrylcholinesterase catalysis. The second n‐hexylcarbamyl or n‐octylcarbamyl moiety of inhibitors 12 and 13 is proposed to bind tightly to the peripheral anionic site of butyrylcholinesterase from molecular modeling. Butyrylcholinesterase prefers para‐carbamates to ortho‐ and meta‐carbamates, whereas acetylcholinesterase prefers para‐ and meta‐carbamates to ortho‐carbamates. This result implies that the anionic site of butyrylcholinesterase is relatively smaller than that of acetylcholinesterase because meta‐carbamates, which may bind to the anionic sites of both enzymes, are not potent inhibitors of butyrylcholinesterase.

Cage amines as the stopper inhibitors of cholinesterases.

Cage amines 1-4 are potent peripheral anionic site-bound reversible inhibitors of both acetylcholinesterase and butyrylcholinesterase. Cage amines 1-3 are selective butyrylcholinesterase inhibitor versus acetylcholinesterase. For both enzymes, the -log K(i) values linearly correlate with the difference of substituted phenyl radius of cage amines (-log K(i)=5.4+3.4Deltagamma for acetylcholinesterase, -log K(i)=5.9+3.2Deltagamma for butyrylcholinesterase). Moreover, the relationship between the enzymes and cage amines mimics that between bottles and stoppers.