Imidazole, 4-nitrophenyl acetate hydrolysis

As one might expect the rate of p-nitrophenyl heptanoate hydrolysis increased at low ethanol concentrations as a result of apolar binding. The rate of p-nitrophenyl acetate hydrolysis also increased markedly at low ethanol concentration. This finding was explained by a conformational effect on the polymer, that is, lower ethanol concentration brings about a shrinkage of the polymer, which increases concerted interactions of the imidazole residues. The hydrolysis of 3-nitro-4-dodecanoyloxybenzoate was found to be 1700 times faster in the presence of poly[4(5)-vinylimidazole] compared to free imidazole (77). A double-displacement mechanism was demonstrated for this system (75). 

Fig. 6. Reaction mechanism for 4-nitrophenyl acetate hydrolysis by imidazole.

Relative reactivity of imidazole and monohydrogen phosphate tfimidazolel/iKHPOj") is order of unity for general base catalysis, but about 10 for nucleophilic catalysis This ratio is 0,25 for ethyl acetate hydrolysis and 4.7 x I0 for p-nitrophenyl acetate hydrolysis 92... 

The Bronsted coefficient p for imidazole catalysis of p-nitrophenyl acetate hydrolysis is 0.8 [29], and the second-order rate constants of all His residues can therefore be related to that of 4-methylimidazole to determine whether there are effects on reactivity beyond those of differential nudeophilicity and levels of protonation. The reactivity of His residues in the pH independent region may be estimated from rate constants, pH and values. The second-order rate constant of the 4-methylimidazole catalyzed hydrolysis of mono-p-nitrophenyl fumarate at pH 5.85 and 290 K is 1.02 x 10 s. From this value and the pfC of 7.9, the second-... 

These data are for the nucleophilic catalysis of the hydrolysis of p-nitrophenyl acetate by imidazoles and benzimidazoles at pH 8.0. Tbe apparent second-order catalytic rate constants are defined by... 

The most effective catalyst for the hydrolysis of p-nitrophenyl acetate was reported to be a cycloheptaamylose derivative containing approximately two imidazole groups per cycloheptaamylose molecule (Cramer and Mackensen, 1970). At pH 7.5 and 23°, this material accelerates the rate of release of phenol from p-nitrophenyl acetate by a factor of 300 when compared with the hydrolysis of this substrate in the absence of catalyst. However, when compared with an equivalent concentration of imidazole, which is an effective catalyst for ester hydrolysis at neutral pHs, the rate accelerations imposed by this cycloheptaamylose derivative are only two- to threefold. Cramer and Mackensen attributed this rate enhancement to nucleophilic displacement of phenol from the included ester by a cycloheptaamylose hydroxyl group, assisted internally by the attached imidazole group... 

Catalysis by imidazole in aqueous systems has received widespread attention because of its central position as the catalytic group in many hydrolytic enzymes. Many imidazole derivatives with long aliphatic chains have been synthesized and their catalytic role in the presence of detergents has been reported as models of hydrolytic enzymes. Representative examples of the hydrolysis ofp-nitrophenyl acetate (8) are summarized in Table 2. 

For the hydrolysis of p-nitrophenyl acetate at pH 7.7 the most effective catalyst was Gly-His-Gly-Gly-His-Gly. However, this peptide had only 50% of the catalytic activity of imidazole. For the seven peptides the range of catalytic effectiveness was found to be 30-50% that of imidazole. 

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 )... 

The constancy of the values of klm and kBlm, rather than the values of k, in Table I suggests that the basic form of imidazole or benzimidazole is the catalytic species, and that the imidazolium or benzimidazolium ion is not. This interpretation is in agreement with that of Gurd (8) and of Bruice and Schmir (3) on the catalyzed hydrolysis of p-nitrophenyl acetate by imidazole. Bronsted and Guggen-... 

In the area of catalysis, the esterolysis reactions of imidazole-containing polymers have been investigated in detail as possible models for histidine-containing hydrolytic enzymes such as a-chymotrypsin (77MI11104). Accelerations are observed in the rate of hydrolysis of esters such as 4-nitrophenyl acetate catalyzed by poly(4(5)-vinylimidazole) when compared with that found in the presence of imidazole itself. These results have been explained in terms of a cooperative or bifunctional interaction between neighboring imidazole functions (Scheme 19), although hydrophobic and electrostatic interactions may also contribute to the rate enhancements. Recently these interpretations, particularly that depicted in Scheme 19, have been seriously questioned (see Section 1.11.4.2.2). 

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]. 

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. 

Acidic proteinoids accelerate the hydrolysis of the unnatural substrate, p-nitrophenyl acetate 7,8). P-Nitrophenyl acetate has been used as a substrate for both natural esterases and esterase models. The imidazole ring of histidine is involved in the active site of a variety of enzymes, including hydrolytic enzymes. Histidine residues of proteinoid play a key role in the hydrolysis, the contribution to activity of residues of lysine and arginine is minor, and no activity is observed for proteinoid containing no basic amino acid 7). 

Fig. 18 Reaction rate of hydrolysis of p-nitrophenyl acetate as a function of inverse temperature. Thermosensitive imidazole-containing copolymers (PVCL-Vim, PNIPA-Vim), 1-methylimidazole and poly(l-vinylimidazole) act as catalysts. Numbers in the copolymer abbreviations denote the Vim content (in mole percent). Vim 1-vinylimidazole. (Adapted from Ref. [18])...

Finally, an aspartic acid residue is necessary for full catalysis and this residue is thought to use its CO2 group as a general base. A chemical model shows that the hydrolysis of p-nitrophenyl acetate in aqueous acetonitrile containing sodium benzoate and imidazole follows the rate law ... 

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). 

Several examples have shown that the degree of activity resulting from synthesis is reproducible, as is the amino acid composition. In other cases, e.g., with p-nitrophenyl acetate, activity was quite variable. Nearly total inactivation by heat in aqueous solution has been demonstrated for some pyropolyamino acids other such systems are heat-stable in aqueous solution. In the p-nitrophenyl acetate system, the nature of the heat inactivation, if not the mechanistic reason for enhanced activity, is understood to involve both imide and imidazole residues. Differing interactions of these residues to produce loci of varying degrees of efficiency could help to explain the quantitative nonreproducibility of activity in separate syntheses. With OAA, selectivity of action was strict, in that several a-keto acids were not measurably acted upon under controlled conditions. The identification of the active locus for hydrolysis of the substrate p-nitrophenyl acetate supports the general inference of specificities, inasmuch as similarly prepared polymers have been shown not to be operative for other reactions, e.g., decarboxylation of OAA (17). 

An often occurring mechanistic problem is the diagnosis of general base or nucleophilic catalysis which give identical kinetics. Imidazole is a well known catalyst for the hydrolysis of 4-nitrophenyl acetate in water and it is known to involve nucleophilic attack because iV-acetylimidazole has been observed from ultraviolet spectral work [17]. The absence of a solvent deuterium isotope effect confirms the operation of the nucleophilic pathway (Table 7) because a primary isotope effect is expected for the general base mechanism. 

M. L. Bender (Illinois Institute of Technology, Chicago, III.) We have found that imidazole, a constituent of chymotrypsin catalyzes the hydrolysis of p-nitrophenyl acetate at 25° and pH 7. The reaction occurs in a stepwise manner, as does the enzymatic reaction, with the intermediate formation of acetylimidazole, which is subsequently hydrolyzed by water. While a serine hydroxyl of chymotrypsin, as proposed by Dr. Gutfreund (Lecture 29), could be converted to acetylserine, there is no obvious way in which it could hydrolyze, whereas this possibility occurs straightforwardly with the imidazole-catalyzed sequence. 

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