Robert S. McDonald

Carbamates with Differential Mechanism of Inhibition Toward Acetylcholinesterase and Butyrylcholinesterase

Most carbamates are pseudoirreversible inhibitors of cholinesterases. Phenothiazine carbamates exhibit this inhibition of acetylcholinesterase but produce reversible inhibition of butyrylcholinesterase, suggesting that they do not form a covalent bond with the catalytic serine. This atypical inhibition is attributable to pi-pi interaction of the phenothiazine moiety with F329 and Y332 in butyrylcholinesterase. These residues are in a helical segment, referred to here as the E-helix because it contains E325 of the catalytic triad. The involvement of the E-helix in phenothiazine carbamate reversible inhibition of butyrylcholinesterase is confirmed using mutants of this enzyme at A328, F329, or Y332 that show typical pseudoirreversible inhibition. Thus, in addition to various domains of the butyrylcholinesterase active site gorge, such as the peripheral anionic site and the pi-cationic site of the Omega-loop, the E-helix represents a domain that could be exploited for development of specific inhibitors to treat dementias.

Differential binding of phenothiazine urea derivatives to wild-type human cholinesterases and butyrylcholinesterase mutants.

A series of N-10 urea derivatives of phenothiazine was synthesized and each compound was evaluated for its ability to inhibit human cholinesterases. Most were specific inhibitors of BuChE. However, the potent inhibitory effects on both cholinesterases of one sub-class, the cationic aminoureas, provide an additional binding mechanism to cholinesterases for these compounds. The comparative effects of aminoureas on wild-type BuChE and several BuChE mutants indicate a binding process involving salt linkage with the aspartate of the cholinesterase peripheral anionic site. The effect of such compounds on cholinesterase activity at high substrate concentration supports ionic interaction of aminoureas at the peripheral anionic site.

Intramolecular catalysis of amide hydrolysis by the carboxy-group. Rate determining proton transfer from external general acids in the hydrolysis of substituted maleamic acids

The highly efficient intramolecular catalysis by the carboxy-group of the hydrolysis of simple dialkylmaleamic acids is itself subject to external general acid catalysis. The kinetic characteristics of the general acid catalysed reaction are those expected for a diffusion-controlled proton transfer. At high concentrations of general acid, external catalysis disappears. This is shown to result from a change in rate-determining step, and is thus evidence for an intermediate on the reaction pathway. The intermediate can only reasonably be a tetrahedral addition intermediate. Kinetic evidence is now available for all the major steps on the reaction pathway, and the requirements for an enzyme catalyst carrying out the reaction can be specified in detail. The full mechanism specifically implicates the O-protonated amide as the reactive species in dilute acid.

Intramolecular catalysis of amide hydrolysis by two carboxy-groups

In di-isopropylmaleamic acids derived from β-amino-acids (i) the rate constant for hydrolysis of the unactivated amide group is comparable with Kcat for pepsin hydrolysing good syntheic substrates; (ii) a neighbouring carboxy-group participates as a nucleophilic catalyst, but requires a specific structural relationship with the amide group to reach high efficiency; (iii) there is a requirement for a second carboxy-group, also in a special structural relationship with the amide group, but this time in the ionised form, for full catalytic efficiency to be maintained. Remarkably, the ionised carboxy-group acts as a general base to catalyse the interconversion of neutral and zwitterionic forms of the tetrahedral carbinolamine intermediate, a reaction which is catalysed by external general acids. As a result we have developed a system showing a bell-shaped pH–rate profile for hydrolysis, of the sort shown by many enzyme-catalysed reactions, and for the same reasons.

Kinetics and mechanism of the reversible ring-opening of thiamine and related thiazolium ions in aqueous solution

Kinetic studies of the ring-opening and reclosure reactions of thiamine and three other thiazolium ions (Q+) in aqueous solution, in the pH range 0–13, have been carried out by stopped-flow and conventional UV–VIS spectrophotometry. At high pH, ring-opening of thiamine exhibits a temporary diversion to the well-known ‘yellow form’. Otherwise, the ring-opening reactions are simply first-order in [OH–], consistent with rate-limiting attack of hydroxide ion at C(2) of the Q+ ring, producing a pseudobase, T°, which rapidly consumes a second equivalent of hydroxide ion to form the ring-opened enethiolate, ETh–. In contrast, ring closure of the enethiol in acidic solution exhibits rather complex kinetic behaviour; two processes are observed for most enethiols, including that derived from thiamine. Both the fast process (a) and the slower process (b) produce the thiazolium ion Q+ and they exhibit pH- and buffer-independent rate plateaux at low pH. Rapid, repetitive UV spectral scans and NMR spectral studies show that the two processes arise from the independent formation of Q+ from the two amide rotamers of the enethiol which do not equilibrate under the reaction conditions. The major amide rotamer (∼75%) gives rise to the fast process (a) and the minor rotamer to the slow reaction (b). The pH–rate profile and buffer catalysis studies reveal that the reclosure reaction undergoes a change in rate-limiting step from uncatalysed formation of T° at low pH to its general acid catalysed breakdown at higher pH. The latter process is characterized by a Bronsted α value of 0.70. Additionally, for process (b), a general base catalysed pathway for formation of T° can be observed, for which the Bronsted β value is 0.74. The mechanistic details of the ring-opening and reclosure pathways are discussed.

Trifluoromethyl ketone hydration. Substituent effects of amino-groups and the hydrating properties of aqueous dimethyl sulphoxide

The equilibrium constants for hydration of the 4-amino-, 4-methylamino-, and 4-dimethylamino-derivatives of ααα-trifluoroacetophenone have been determined in mixtures of dimethyl sulphoxide (DMSO) and D2O (or H2O) using 19F n.m.r. spectroscopy. The dimethylamino-compound follows the pattern observed earlier for the 4-methoxy-compound, i.e. in mixtures up to 80 mol % DMSO these compounds are more highly hydrated than in pure water. The extent of hydration of the amino- and methylamino-compounds, on the other hand, decreases steadily as the water content of the mixture is decreased. The difference in behaviour of the two groups of compounds is attributed to the presence of acidic protons in the unhydrated forms of the amino- and methylamino-compounds, which are able to form strong hydrogen bonds to DMSO. The results indicate the strong solvent dependence to be expected for σ+ substituent constants for the three kinds of amino-group. A sampling of other carbonyl compounds that undergo hydration revealed a considerable variation in the response of such compounds, equilibrium constants to changes in DMSO–water composition, with only one compound, 2-nitrobenzaldehyde, showing the sort of response that is shown by the 4-dimethylamino- and 4-methoxy-derivatives of trifluoroacetophenone.