4-Nitrophenyl acetate hydrolysis

Figure 6-8 is a pH-rate profile for the hydrolysis of p-nitrophenyl acetate. The slopes of the straight-line portions are —1,0, and -L 1, reading in the acid to base direction, and this system can be described by... 

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... 

An artificial metalloenzyme (26) was designed by Breslow et al. 24). It was the first example of a complete artificial enzyme, having a substrate binding cyclodextrin cavity and a Ni2+ ion-chelated nucleophilic group for catalysis. Metalloenzyme (26) behaves a real catalyst, exhibiting turnover, and enhances the rate of hydrolysis of p-nitrophenyl acetate more than 103 fold. The catalytic group of 26 is a -Ni2+ complex which itself is active toward the substrate 1, but not toward such a substrate having no metal ion affinity at a low catalyst concentration. It is appearent that the metal ion in 26 activates the oximate anion by chelation, but not the substrate directly as believed in carboxypeptidase. 

The hydrolysis of p-nitrophenyl acetate and bis(p-nitrophenyl phosphate) are frequently used to probe hydrolytic activity. A problem with some other dinuclear systems is that the Zn units are held together by bridging ligands which can be cleaved on reaction with the substrate.440 This is not the case in a ditopic ligand such as those designed by Lippard and co-workers based on Kemp s triacid imide with a xylyl spacer.441,442 Both zinc dimers and mixed metal dimers were formed and a structure characterized with a bridging phosphodiester (Figure 6). 

Bimetallic zinc complexes formed with hexaazamacrocycles were studied in the hydrolysis of activated carboxyesters. Potentiometric titration demonstrated the dominant presence of a dinuclear hydroxo bridged species at pH >7. /)-Nitrophenyl acetate is hydrolyzed with no loss of catalytic activity for at least 2.7 catalytic cycles 4... 

Hua I, Hochemer RH, Hoffmann MR (1995) Sonolytic hydrolysis of p-nitrophenyl acetate The role of supercritical water. J Phys Chem 99 2335-2342... 

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... 

The rate effects imposed by this derivative, however, are dependent on the structure of the substrate. For example, the hydrolysis of 8-acetoxy-5-quinoline-sulfonate (AQS), a large substrate which cannot be included within the cyclohexaamylose cavity, is not enhanced by this derivative. Moreover, in contrast to the effects of unmodified cycloamyloses on the hydrolyses of nitrophenyl acetates, the rate accelerations imposed by this... 

In a preliminary attempt to improve the catalytic properties of the cycloamyloses Bunting and Bender (1968) and, subsequently, Kice and Bender (1968) replaced, in separate experiments, both a primary and a secondary cyclohexaamylose hydroxyl group with a thiol group which has a pKa closer to neutrality than a hydroxyl group. Unfortunately, neither derivative catalyzed the hydrolysis of m-nitrophenyl acetate to any greater extent than unmodified cyclohexaamylose. 

The hydrolysis of esters by the nickel derivative (271) provided an early example of the use of a metal-capped cyclodextrin as a catalyst (shown here as its p-nitrophenyl acetate inclusion complex) (Breslow Overman, 1970 Breslow, 1971). The synthesis of this host involves the following steps (i) covalent binding of the pyridine dicarboxylic acid moiety to cyclodextrin, (ii) coordination of Ni(n) to this species, and (iii)... 

The use of a lipophilic zinc(II) macrocycle complex, 1-hexadecyl-1,4,7,10-tetraazacyclododecane, to catalyze hydrolysis of lipophilic esters, both phosphate and carboxy (425), links this Section to the previous Section. Here, and in studies of the catalysis of hydrolysis of 4-nitrophenyl acetate by the Zn2+ and Co2+ complexes of tris(4,5-di-n-propyl-2 -imidazolyl)phosphine (426) and of a phosphate triester, a phos-phonate diester, and O-isopropyl methylfluorophosphonate (Sarin) by [Cu(A(A(A/,-trimethyl-A/,-tetradecylethylenediamine)l (427), various micellar effects have been brought into play. Catalysis of carboxylic ester hydrolysis is more effectively catalyzed by A"-methylimidazole-functionalized gold nanoparticles than by micellar catalysis (428). Other reports on mechanisms of metal-assisted carboxy ester hydrolyses deal with copper(II) (429), zinc(II) (430,431), and palladium(II) (432). 

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. 

Photolysis of 4- and 3-nitrophenyl acetates (176 —> 177 178 —> 179) in neutral aqueous solution leads to the corresponding phenols with quantum yields 0.002 and O.OO6105 (equation 84). A greater difference in the photoreactivity (quantum yields of 0.002 and 0.129, respectively) is shown between 2-mcthoxy-4-nitrophenyl acetate 180 and 2-methoxy-5-nitrophenyl acetate 182. The nitro substituent clearly exhibits a meta-activating effect in the hydrolysis of phenyl acetates. 

Serine peptidases can hydrolyze both esters and amides, but there are marked differences in the kinetics of hydrolysis of the two types of substrates as monitored in vitro. Thus, the hydrolysis of 4-nitrophenyl acetate by a-chy-motrypsin occurs in two distinct phases [7] [22-24]. When large amounts of enzyme are used, there is an initial rapid burst in the production of 4-nitro-phenol, followed by its formation at a much slower steady-state rate (Fig. 3.7). It was shown that the initial burst of 4-nitrophenol corresponds to the formation of the acyl-enzyme complex (acylation step). The slower steady-state production of 4-nitrophenol corresponds to the hydrolysis of the acetyl-enzyme complex, regenerating the free enzyme. This second step, called deacylation, is much slower than the first, so that it determines the overall rate of ester hydrolysis. The rate of the deacylation step in ester hydrolysis is pH-dependent and can be slowed to such an extent that, at low pH, the acyl-enzyme complex can be isolated. 

Effect of Various Histidine Peptides on the Rate of Hydrolysis of p-Nitrophenyl Acetate at pH 7.73 ... 

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. 

Apolar binding of substrates has been demonstrated with polymers of vinylimidazole. Overberger etal. (77) studied the hydrolysis ofp-nitrophenyl acetate and p-nitrophenylheptanoate by poly[4(5)-vinylimidazole] in ethanol water mixtures. 

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). 

Manecke and his collaborators have synthesized polymers of vinylimid-azole hydroxamic acid (75) (22, 23, 24). Hydrolysis of p-nitrophenyl acetate... 

The oxamate group has been incorporated into a vinyl polymer by Kirsh and Kabanov 41). These workers prepared a copolymer of 4-vinyl-A -(phenacyloxime)pyridinium bromide and vinylpyridine. For the hydrolysis of p-nitrophenyl acetate the oxamate polymer produced a significant rate enhancement over the monomeric analogs. 

The final intriguing use of CLAs is in the immobilization of enzymes in the soapy shell in order to carry out an enzymatic reaction. Thus the hydrolysis of -nitrophenyl acetate to / -nitrophenol has been demonstrated by immobilizing a lipase into the shell of a CLA. The CLAs were then pumped through a cross-fiow membrane, where they were separated and recycled, with the product appearing in the permeate [70]. 

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... 

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