2-Nitrophenyl acetate

Certain molecules that can permit concerted proton transfers are efficient catalysts for reactions at carbonyl centers. An example is the catalytic effect that 2-pyridone has on the aminolysis of esters. Although neither a strong base (pA aH+ = 0.75) nor a strong acid (pJsfa = 11.6), 2-pyridone is an effective catalyst of the reaction of -butylamine with 4-nitrophenyl acetate. The overall rate is more than 500 times greater when 2-pyridone acts... 

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). 

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. 

Fig. 3.7. Two phases in the formation of 4-nitrophenol from 4-nitrophenyl acetate by a-...

Human oxyhemoglobin (oxyHb A) hydrolyzes 4-nitrophenyl acetate at a higher rate than bovine serum albumin [107], It has been proposed that imidazole catalysis by /3-His2 is primarily responsible for the esterase activity, and... 

The indomethacin-hydrolyzing enzyme from pig liver microsomes was purified and partially characterized [60]. The enzyme was found to be different from known pig liver esterases, since it did not hydrolyze naphth-l-yl-acetate and (4-nitrophenyl)acetate, which are typical substrates for these car-boxy lesterases. The amino acid sequence of the enzyme showed high homology with the mouse carboxylesterase isoenzyme ES-male. Human liver car-... 

A few acetates of phenols have been used extensively as probes to investigate esterases, e.g., phenyl acetate (7.15), 4-nitrophenyl acetate (7.16), a-naphthyl acetate (7.17) and 7-acetoxy-4-mc(hyl-27/-[l bcnzopyran-2-onc (4-methylumbelliferyl acetate, 7.18). Such substrates are easy to handle and their phenolic metabolite is readily analyzed, allowing convenient monitoring of the reaction. 

Photodecarboxylations of 3-nitrophenyl acetates and 4-nitrophenyl acetates a-(2,4-dinitroplienyloxy)-acetic acids a-(2-nitrophenylthio)-acetic acids... 

Some interesting solvent effects have been observed as a function of medium change in the acetonitrile-water system. Benzohydroxamate (BHA", pKa = 8.8) has been shown to be more reactive than its reference nucleophile, 3-chlorophenoxide (3-CIC5H4O", pKa = 9.02) toward 4-nitrophenyl acetate (PNPA) as shown in Figure 3 BHA" is ca 160-fold more reactive than S-ClCeFLtO" in pure water but the reactivity ratio,... 

The pKa of Zn H20 in the isoenzymes are determined from the pH dependence of 4-nitrophenyl acetate esterase activity and spectroscopic properties of the metal substituted (essentially Co11) enzyme (142 146). In bovine isoenzyme II, the titration behavior of zinc-H20 and His-64 are interdependent and both have similar pKa values. Similar behavior has been reported for human isoenzyme II (147). 

In isoenzyme I, the titration behavior of zinc H20 is complicated due to the presence of three titratable active-site histidines as described in Section VI.D. Lindskog reports a value of 7.1 for the pKa of zinc-H20 (142), but higher values were obtained by other authors. The pH-rate profile for 4-nitrophenyl acetate esterase activity yields pKa = 7.45 (142). 

Lindskog reported that Cl- is a competitive inhibitor of the CO2-HCO3 exchange reaction for isoenzyme I (157c). H NMR experiments (142,157c) and pH dependence of the SCN- inhibition of the 4-nitrophenyl acetate esterase activity have shown that the affinity constants of SCN- for various free enzyme forms follow the order HEH > EH > HE > E (142). The estimated values of the affinity constants are found to be 5x 104 M-1 and < 2 for HEH and E, respectively. Under inhibitory conditions the CA has a finite residual activity (kcai value) which is large for I-, intermediate for N3 and SCN-, and very small for NCO-. This has been explained on the basis of the relative binding affinities of these anions for the enzyme (190). 

X-ray crystal structure of Zn2+-[12]aneN3-(NCS)2 revealed a five-coordinate TBP which mimics the structure of the NCS- adduct of CA (225). Kinetic results demonstrated that NCS- and N3 inhibit competitively the catalytic activity of 4-nitrophenyl acetate (NA) hydrolysis by Zn2 + [12]aneN3 (46b). These results lend support to... 

FIG. 4. An overall reaction mechanism for 4-nitrophenyl acetate hydrolysis catalyzed... 

Comparison of Second-Order Rate Constants (M 1sec 1) in Hydrolysis (or Acetyl and Phosphoryl Transfer) of 4-Nitrophenyl Acetate, Methyl Acetate and Bis(4-nitrophenyl) Phosphate... 

Buckingham, D. A., and C. R. Clark, Metal-hydroxyde-promoted hydrolysis of activated esters. Hydrolysis of 2,4-dinitrophenyl acetate and 4-nitrophenyl acetate , Aust. J. Chem., 35,431-436 (1982). 

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). 

The [Co(NH3)sOH]2+-promoted hydrolysis (and that promoted by other exchange-labile anc non-labile metal hydroxide species) of 2,4-dinitrophenyI acetate and 4-nitrophenyl acetate hat been studied in some detail.19 Kinetic studies (25 °C, / = 1.0 M) show that these reactions follov shallow Bronsted slopes (/ = 0.33 and 0.40) for the two substrates which extend over a range o 1010 in nucleophile basicity. A correlation is reported which allows the prediction of reactioi... 

The cleavage of 4-nitrophenyl acetate by [Co(NH3)5OH]2+ and [Co(NH3)5Im]2+ (Im = N-deprotonated imidazole) has been studied in water and DMSO solvents.192 All the reactions are exclusively nucleophilic as demonstrated by the detection of the acetylated products [(NH3)sCo02CMe]2+ and [(NH3)5CoImCoMe]3+. Typical kinetic data are summarized in Table 18. The large difference in the reactivity of the two complexes towards 4-nitrophenyl acetate is closely paralleled by their differences in basicity. In Me2SO the complexes have a similar reactivity towards the ester (fcMIm = 30M 1 s 1, k OH = 0.72 M"1 s-1 at 25 °C), and this increase is largely due to the marked increase in the basicity of [Co(NH3)5OH]2+ relative to that of [Co(NH3)5Im]2+ in the dipolar aprotic solvent. 

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... 

The kinetics of proton transfer from ethyl bis(4-nitrophenyl)acetate to N-bases with guanidine-like character, in acetonitrile, are determined by basicity and steric hindrance in die vicinity of the reacting site of the N-base and also by different distributions of positive charge in protonated A-bases.147... 

Carboxylic acids, acyl chlorides, and sulfonyl chlorides used for deri-vatization of 4-aminophenylalanine and >-4-am i n op h e ny I a I a n i n e are as follows 5-hydantoinacetic acid, / ran, v - 4 - co t i n i n ec a r b o xy I i c acid, isonicotinic acid, 3-pyridinepropionic acid, 4-hydroxyphenylacetic acid, 2-butynoic acid, 2-pyrazinecarboxylic acid, cyclopropanecarboxylic acid, 3-hydroxy-2-qui-noxaline carboxylic acid, 5-bromovaleric acid, propargyl chloroformate, 3,4-dimethoxybenzoyl chloride, 2-thiophenesulfonyl chloride, 3-thiophene-carboxylic acid, 2-thiophenecarboxylic acid, 2-methylbutyric acid, 2-thio-pheneacetyl chloride, benzoic acid, furylacrylic acid, 4-nitrophenyl acetic acid, 2,5-dimethoxyphenylacetic acid, p-toluenesulfonyl chloride, 4-(di-methylamino)phenylacetic acid, 3-indolepropionic acid, phenoxyacetic acid, 3-(dimethylamino)benzoic acid, cyclohexanecarboxylic acid, naphtha-lenesulfonyl chloride, 4-bromophenylacetic acid, 4-bromobenzoic acid, 2-phenoxybutyric acid, 3,4-dichlorophenylacetic acid, (l-naphthoxy)acetic acid. 

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]. 

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