Hydrolysis of / -nitrophenyl acetate

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). 

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. 

The hydrolysis of 4-nitrophenyl acetate in the presence of the macrocyclic complex [Co([15)aneN5)OH)]2+ (41) is somewhat faster201 than the [Co(NH3)5OH]2+-promoted hydrolysis (Table 18). 

Table 18 Rate Constants for the Hydrolysis of 4-Nitrophenyl Acetate by Various Nucleophiles in Water Solvent ...

This enzyme, which occurs in animals, plants and certain microorganisms, is a most effective catalyst of the reversible hydration of C02 and dehydration of HC03. 479-482 It also catalyzes reactions of a number of compounds which undergo hydrolysis or hydration, for example the hydrolysis of 4-nitrophenyl acetate and the hydration of acetaldehyde.480... 

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... 

Haagen, L., and A. Brock. 1992. A new automated method for phenotyping arylesterase (EC 3.1.1.2) based upon inhibition of enzymatic hydrolysis of 4-nitrophenyl acetate by phenyl acetate. Eur J Clin Chem Clin Biochem 30 391. 

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]. 

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. 

Hydrolysis of 4-nitrophenyl acetate (NA) (0.5-2.0 mM) was catalyzed by 11 in 10% volume/volume (v/v) CH3CN aqueous solution under comicellar conditions with 10 mM Triton X-100 at pH 9.2 (20 mM CHES buffer) and 25°C (Scheme 7). The second-order dependence of the rate constant, obsd, on the concentration of NA (10-50 dM) and 11 (0.2-1.0 mM) at pH 10.2 (2 mM CAPS buffer) and 25°C with I = 0.10 (NaN03) fits the kinetic equation (5). No other reaction such as acetate transfer to Triton X-100 was observed, as confirmed by a H NMR experiment with a 10% D20 solution of 2.0 mM NA, 0.2 mM 3, and 10 mM Triton X-100. Since the second-order kinetics held after several catalytic cycles, it was concluded that the NA hydrolysis catalytic. In Equation (5), vobsd is the observed NA hydrolysis rate catalyzed by 3, as derived by subtraction of the buffer-promoted NA hydrolysis rate from total NA hydrolysis rate. 

Theoretical studies on 12 4-substituted phenyl acetates concluded that, in contrast to the proposed interpretation based on 13C NMR chemical shifts and ground-state destabilization calculations, the electrophilicity of the C=0 group increases due to the effect promoted by the electron-withdrawing groups in these systems.11 A DFT investigation of the alkaline hydrolysis of 4-nitrophenyl acetate has shown that a model including seven water molecules proceeds via a concerted transition state.12... 

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). 

Presented in Table 1 is a summary of the second-order rate constants for the hydrolysis of 4-nitrophenyl acetate promoted by various zinc complexes in aqueous solution. A conclusion that may be drawn from this data is that change in the nature of the supporting chelate ligand dramatically influences the second-order rate constant for the carboxy ester hydrolysis reaction. Notably, the slowest rate is found for the four-coordinate zinc hydroxide species [([12]aneN3)Zn(OH)]+ which has three... 

Table 1 Comparison of pKa values of zinc complexes and second-order rate constants k" (M 1s 1) for the hydrolysis of 4-nitrophenyl acetate at 25 °C with that of human CA-II. Adapted from reference 17.

A zinc(II) hydroxo complex supported by a secondary amine-appended phenanthroline ligand (L2, Fig. 18b) has been reported to catalyze the hydrolysis of 4-nitrophenyl acetate with a second-order rate constant of 0.934 M-1 s-1.107 A mechanism involving attack of a terminal zinc hydroxide moiety on the 4-nitrophenyl acetate substrate has been proposed. 

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... 

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. 

The Zn -bound alkoxide complex 8b was isolated as a dimeric form, which dissociates into monomeric species in aqueous solution to make a very reactive nucleophile and catalyzes hydrolysis of 4-nitrophenyl acetate CNA) (see Scheme 3). Through a kinetic study of NA hydrolysis with 8b in 10% (v/v) CH3CN at 25°C and pH 9.3 (20 mM CHES buffer) with I = 0.10 (NaNOs), a second-order rate constant of 0.14 Af sec which is four times greater than the corresponding value of 3.6 x 10 by the Zn -[12]aneN3 complex 9b under the same conditions, was established. 

From the kinetic study o/lb-catalyzed hydrolysis of 4-nitrophenyl acetate inhibited by various aromatic sulfonamides, the apparent 1 1 affinity constants were determined at jH 8.4/ A comparison o/intramolecular Kntra=10 =Wor ll)/i (for la)]ond intermolecular for la with / toluenesulfonamide) contribution of p-toluenesulfonamide anion coordination to the zinc(II)-[12]aneN3 complex gives an effective mo-larity M by the intramolecular location. 

Scheme 2.23 The catalytic cycle for the hydrolysis of 4-nitrophenyl acetate.

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... 

Water pooled in reverse micelles enhances the rate of the imidazole catalysed hydrolysis of 4-nitrophenyl acetate. The rate of acylation of amines in apolar solvents is enhanced more by cationic micelles than by bis(2-ethylhexyl) sodium sulphosuccinate, and may be treated by the pseudophase model. ... 

The first report of a negatively-charged phase-transfer catalyst, sodium tetraphenylborate, claimed to catalyse the acid hydrolysis of 4-nitrophenyl acetate in water-cyclohexane. However, the extension of phase-transfer catalyst to reactions involving cations remains to be demonstrated as the earlier observations are attributable to the decomposition of the tetraphenylborate rather than hydrolysis of the ester. ... 

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. ... 

The [Co([15]aneN5)OH] -promoted hydrolysis of 4-nitrophenyl acetate has also been investigated in some detail ([15]aneNs = 1,4,7,10,13-penta-azacyclopentadecane). This complex has the structure 25. The pKa for the aquo hydroxo equilibrium is 6.3 at 25°C, not... 

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