4-nitrophenyl acetate, catalysed hydrolysis

In general, mechanistic evidence for a reactive intermediate from trapping experiments needs to be linked to arguments against the introduction of an alternative pathway from the reactant, i.e. to show that an intermediate really has been trapped, not the reactant. A classic case is the hydrolysis of 4-nitrophenyl acetate catalysed by imidazole. The mechanism is nucleophile catalysis and the intermediate (N-acetylimidazolium cation) was trapped by aniline (to give acetanilide) with no kinetic effect, i.e. the aniline does not react directly with the substrate [51]. 

Radical polymerization of (14) and the subsequent removal of the benzyl group produces water-soluble polymers containing imidazole-4-carbohydroxamic acid residues. The hydrolysis of 4-nitrophenyl acetate catalysed by this polymer proceeds faster than that catalysed by imidazoles or carbohydroxamic acid but slower than that catalysed by the monomeric analogue. " The hydrolysis of 4-nitrophenyl acetate is catalysed by the bifunctional hydroxamic acid (IS) and... 

Although the addition of hydrophobic ammonium salts has little affect upon the rate of hydrolysis of 4-nitrophenyl acetate catalysed by methacrylic acid-A-methacryloylhistamine co-polymer there is a marked increase when 4-nitrophenyl hexanoate is used as the substrate. It is suggested that the polymer-bound ammonium ions facilitate subsequent binding of the substrate. ... 

Bruice and Sturtevant, (1959) and Bruice, (1959) found extremely facile intramolecular nucleophilic attack by neighbouring imidazole in the hydrolysis of p-nitrophenyl 7-(4-imidazoyl)butyrate [19]. The rate constant for imidazole participation (release of p-nitro-phenolate) in this reaction is nearly identical with the rate constant for a-chymotrypsin catalysed release of p-nitrophenolate ion [190 min in equation (11) at pH 7 and 25°] from p-nitrophenyl acetate. Comparison of the rate constant for intramolecular imidazole participation to that for the analogous bimolecular reaction (imidazole attack on p-nitrophenyl acetate) (s" /m s )... 

Reaction of the m-nitrophenyl ester of pyridine-2,5-dicarboxylic acid with cyclodextrin (see Section 3) gives a picolinate ester [52] of a cyclodextrin secondary hydroxyl group (Breslow, 1971 Breslow and Overman, 1970) which will bind metal ions or a metal ion-pyridine carboxaldoxime complex. Such a complex will catalyse hydrolysis of p-nitrophenyl acetate bound within the cyclodextrin cavity leading to a rate constant approximately 2000-fold greater at... 

A quantitative assessment of the effects of head group bulk on, S k2 and E2 reactions in cationic micelles has been made.148 The kinetics of the acid-catalysed hydrolysis of methyl acetate in the presence of cationic, anionic, and non-ionic surfactants has been reported on.149 The alkaline hydrolysis of -butyl acetate with cetyltrimethylammonium bromide has also been investigated.150 The alkaline hydrolysis of aromatic and aliphatic ethyl esters in anionic and non-ionic surfactants has been studied.151 Specific salting-in effects that lead to striking substrate selectivity were observed for the hydrolysis of /j-nitrophenyl alkanoates (185 n = 2-16) catalysed by the 4-(dialkylamino)pyridine-fimctionalized polymer (186) in aqueous Tris buffer solution at pH 8 and 30 °C. The formation of a reactive catalyst-substrate complex, (185)-(186), seems to be promoted by the presence of tris(hydroxymethyl)methylammonium ion.152... 

Three new macrocyclic ligands (187) when complexed with zinc(II) could promote ester hydrolysis and a kinetic study of the hydrolysis of 4-nitrophenyl acetate in Tris buffer at pH 8.63 in 10% (v/v) MeCN was earned out with these.153 The hydrolysis of lipophilic esters is also catalysed by zinc(H) in a complex of a long alkyl-pendant macrocyclic tetraamine (188) in micellar solution.154 A study with a copper chloride-containing micelle has compared its effectiveness in the hydrolysis of esters and amides.155... 

Fig. 11.6 (A) Spectroscopic detection of acetylimidazole in the imidazole-catalysed hydrolysis of 4-nitro-phenyl acetate at pH 5 (D) and at pH 6 (E) curves are calculated from data in reference [12] and the curve for the acetate ion product (C) is for pH 5. (B) Reaction of imidazole with 4-nitrophenyl acetate (k) and the hydrolysis of acetyl imidazole (k2) curves constructed from data in references [13] and [12].

Figure 11.6A illustrates the effect of a change in the ratio of ki/k2 for the imidazole-catalysed hydrolysis of 4-nitrophenyl acetate. When the pH is changed from 5 to 6, the maximal concentration of the intermediate increases. The pH profile in Fig. 11.6B provides a graphic illustration of the range of pH within which an intermediate can be detected this is between the cross-over pH values of about 5 and 10 for these conditions. The range illustrated is for 0.1 M imidazole and could be extended because k is dependent on the concentration of imidazole whereas k2 is not. 

Metal-catalysed hydrolysis of / -nitrophenyl picolinate at pH 7.5 was in the order Cu(II) > Ni(II) > Zn(II) > Co(II) > La(III). The probable mechanism is via attack by external HO- on the metal-ion complex (80).80 High catalytic activity in the hydrolysis at pH 7 of p-nitrophenyl picolinate, but not / -nitrophenyl acetate, was displayed by the metal complexes M(2-aminopyridine)2(OAc)2 (M = Zn, Ni), showing that they were good models for hydrolytic metalloenzymes.81... 

The enzyme also catalyses the hydrolysis of various esters such as 4-nitrophenyl acetate and sultones. A number of slightly different enzymes, carbonic anhydrases A, B and C occur in different organisms. The most well characterised enzymes are the bovine and human carbonic anhydrases B, which are monomeric and contain one tightly bound zinc per 30000 molecular weight. 

The effect of added co-solvent on the initial state is also important in more complicated reactions. For example, in the a-chymotrypsin-catalysed hydrolysis of p-nitrophenyl acetate and of N-acetyl-L-tryptophan methyl ester, the difference in the pattern of rates of hydrolysis when the solvent composition is varied can be attributed to the variation in the properties of the initial states of the esters (Bell et al., 1974). 

A great variety of chemical reactions can be advantageously carried out in microemulsions [860-862]. In one of the first papers in this field, Menger et al. described the imidazole-catalyzed hydrolysis of 4-nitrophenyl acetate in water/octane microemulsions with AOT as an anionic surfactant [=sodium bis(2-ethyl-l-hexyl)-sulfosuccinate] [864]. The solubilized water, containing the imidazole eatalyst, is confined in spherical pools encased by surfactant molecules, which have only their anionic head groups (-SOb ) immersed in the aqueous droplets. When the ester, dissolved in water-insoluble organic solvents, is added to this water/octane/AOT/imidazole system, it readily undergoes the catalysed hydrolysis under mild reaction conditions (25 °C). 

If the a-chymotrypsin-catalysed hydrolysis of 4-nitrophenyl acetate [10] is monitored at 400 nm (to detect 4-nitrophenolate ion product) using relatively high concentrations of enzyme, the absorbance time trace is characterised by an initial burst (Fig. 5a). Obviously the initial burst cannot be instantaneous and if one uses a rapid-mixing stopped-flow spectrophotometer to study this reaction, the absorbance time trace appears as in Fig. 5b. Such observations have been reported for a number of enzymes (e.g. a-chymotrypsin [11], elastase [12], carboxypeptidase Y [13]) and interpreted in terms of an acyl-enzyme mechanism (Eqn. 7) in which the physical Michaelis complex, ES, reacts to give a covalent complex, ES (the acyl-enzyme) and one of the products (monitored here at 400 nm). This acyl-enzyme then breaks down to regenerate free enzyme and produce the other products. The dissociation constant of ES is k2 is the rate coefficient of acylation of the enzyme and A 3 is the deacylation rate coefficient. Detailed kinetic analysis of this system [11] has shown... 

Scheme 3.1 Benzamidine-catalysed hydrolysis of p-nitrophenyl acetate...

The first compound described as an artificial enzyme in the literature was the one we reported in which we attached a metal ion binding group to a-cyclodextrin. We found that this would bind p-nitrophenyl acetate into the cavity and a bound nickel ion then catalysed the hydrolysis of the substrate. This was a direct hydrolysis, not an acylation of a cyclodextrin hydroxyl (which is not in reach with the para esters). This type of catalyst then extends metal-catalysed reactions to substrates that do not intrinsically bind to metal ions, which was formerly required for such catalysis. 

One problem with such studies is that p-nitrophenyl acetate is a highly reactive ester, and it is more of a challenge to catalyse the hydrolysis of ordinary esters or of amides. In a move in this direction we showed that an appropriate cyclodextrin dimer with a bound copper ion (8) could indeed catalyse the hydrolysis of an ordinary ester group, not a phenyl ester. " The acceleration was 18,000-fold, certainly a respectable catalytic result... 

The effectiveness of three isomers, 2-, 3- and 4-hydroxyiminomethyl-l-pyridinium iodide (61) as agents to catalyse the hydrolysis in cationic micellar media of p-nitrophenyl acetate (PNPA) and p-nitrophenyl diphenyl phosphate (PNPDPP) were studied at pH 8. The 2-isomer was found to be the most effective for the hydrolysis of the carboxylic ester PNPA, but the 4-isomer for the hydrolysis of the triphosphate ester PNPDPP. ... 

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