1-Bromopinacolone, an active site-directed covalent inhibitor for acetylcholinesterase.

1-Bromopinacolone, BrPin, acts initially as a reversible competitive inhibitor for acetylcholinesterase, KI = 0.18 mM in hydrolysis of acetylcholine. Unlike bromoacetone, with time it acts as an irreversible covalent inhibitor. BrPin has a hydrolytic half-life of 30 h at the pH of incubation, 7.8. The enzyme-BrPin complex is 50% inactivated in 2 h. First order kinetics are observed; the rate constant is proportional to the concentration of complex. Retardation by cationic inhibitors of the inactivation is consistent with inactivation occurring as a result of binding of BrPin to the active site. Efficiency of irreversible inhibition by BrPin is essentially the same for hydrolysis of cationic and uncharged substrates, acetylcholine, 3,3-dimethylbutyl acetate, phenyl acetate, n-butyl acetate, and indophenyl acetate. In contrast, a cationic alkylating agent, N,N-dimethyl-2-phenylaziridinium ion, DPA, acts noncompetitively; it inactivates completely toward cationic, and partially toward uncharged substrates, and does so slightly more rapidly than BrPin, but less than would be commensurate with its greater intrinsic reactivity. Enzyme first treated with DPA is inactivated by BrPin toward hydrolysis of 3,3-dimethylbutyl acetate. It is proposed that BrPin, and not DPA, binds and reacts in, and may be a useful labeling agent for, the active site.

Cationic and uncharged substrates and reversible inhibitors in hydrolysis by acetylcholinesterase (EC 3.1.1.7). The trimethyl subsite.

Structurally related cationic and uncharged compounds have been studied as inhibitors of hydrolysis by acetylcholinesterase of acetylcholine and its uncharged carbon analog, 3,3-dimethylbutyl acetate. Similar effects of the inhibitors on hydrolysis of the two substrates indicate that the quaternary ammonium group of acetylcholine and the neopentyl group of 3,3-dimethylbutyl acetate bind at the same subsite. Comparison of (CH3)3+NCH2CH2CH2COCH3 (Compound I), Ki = 0.02 mM, and its tertiary homologue, (CH3)2-+NHCH2CH2CH2COCH3 (Compound V), Ki = 0.75 mM, with a secondary isomer of Compound I, 3-oxo-(N-tert-butyl)-butanaminium, (CH3)3C+NH2CH2CH2COCH3 (Compound II), Ki = 0.15 mM, and its lower homologue, (CH3)2CH+NH2CH2CH2COCH3 (Compound IX), Ki = 2 mM, attests to the importance of the branched trimethyl structure and the smaller effect of hydrophobicity of the quaternary ammonium structure. This is supported by competitive inhibition by tert-butyl ammonium, (CH3)3C+NH3 (Compound IV), Ki = 0.45 mM, compared with mixed inhibition by its quarternary isomer, (CH3)4+N (Compound VII), Ki = 1.5 mM, and choline (Compound VI), Ki = 1.0 mM. Uncharged analogues of Compound II, 4-tert-butylthio-2-butanone, (CH3)3CSCH2CH2COCH3 (Compound III), Ki = 0.4 mM, and 4-tert-butoxy-2-butanone, (CH3)3COCH2CH2COCH3 (Compound VIII), Ki = 1.6 mM, and of Compound VI, 3,3-dimethylbutanol (Compound XI), Ki = 7.5 mM, indicate that positive charge contributes factors of 3 to 10 to binding. This may be attributed to peripheral negative charges, present at pH 7-8 in the enzyme of isoelectric point approximately 5, indicating that the binding subsite may be explored more specifically by tert-butyl than by charged reagents.

Quantitative analysis of degradation products in pilocarpine hydrochloride ophthalmic formulations.

The presence of isopilocarpine, an epimer of pilocarpine, and of pilocarpinic acid, a hydrolytic degradation product of pilocarpine, was established and all three substances were assayed in various commercial ophthalmic formulations of pilocarpine hydrochloride by 13C-Fourier transform spectroscopy. Assay was based upon integrated intensities of selected resonances from any formulation calibrated against the intensity of tetramethylammonium bromide, used as a common external reference. The normalized intensities were then related to those of a reference solution of pilocarpine hydrochloride, thereby eliminating any factor arising from variability of 13C-relaxation times. The 13C-resonance for the N-methyl group, being common to all products, provides a convenient basis for the assay of the total alkaloid content whereas the C-8 resonances are best suited for assaying residual pilocarpine and its degradation products. This procedure, estimated as accurate to +/- 5%, constitutes the first comprehensive analytical method to differentiate between pilocarpine and its degradation products.