P-nitrophenol acetate

Esterases form a wide family of enzymes that catalyze the hydrolysis of ester bonds. They are ubiquitously expressed in all tissues including the intestine, and are found in both microsomal and cytosolic fractions. Prueksaritonont et al. [6] have studied the metabolism of both p-nitrophenol acetate and acetylsalicylic acid by esterases from human intestinal microsomal and cytosolic systems, and the activity values were 2.76 pmol min-1 mg-1 and 0.96 nmol min-1 mg-1, respectively. Thus, the activity for the hydrolysis of p-nitrophenol acetate in human intestine approaches that in the liver. 

Esterases. Acetyl esterase (EC 3.1.1.6) removes acetyl esters from acetylated xylose and short-chain xylo-oligomers. It s polymeracting counterpart, acetyl xylan esterase (EC 3.1.1.72), has a similar activity, but prefers polymeric xylan.244 In addition to acetate-specific enzyme detection kits, HPLC or GC analysis of acetate release from native extracted xylan and chemically acetylated xylan, colorimetric substrates, such as p-nitrophenol acetate and /3-napthyl acetate, or the fluorometric substrate, 4-methylumbelliferyl acetate are also used to assay acetyl esterases.244,253 The third esterase, ferulic acid esterase (EC 3.1.1.73), hydrolyzes the ester bond between ferulic acid or coumaric acid and the arabinose side chain of arabinoxylan. Assays for this activity are usually carried out using starch-free wheat bran or cellulase-treated gramineous biomass as a substrate and monitoring ferulic or coumaric acid released by HPLC or TLC. When preparing enzyme-treated substrates, care must be taken to employ phenolic-acid-esterase-free cellulases.244 Other substrates include methyl and ethyl esters of the phenolic acids, as well as finely ground plant biomass.240,254,255... 

Fig. 8-6 Log /f - ApiC plot for ideal case, imidazole (pX = 6.95, donors glucose, phenol, HP207, p-nitrophenol, acetic acid) and acetylacetonate (pX = 9.5, donors H2O, glycerine, mannose, glucose, trimethylphenol, phenol, o-chlorophenol, w-nitrophenol, p-nitrophenol, dimedone, acetic acid, benzoic acid, chloraoetic acid, (Data from Eigen and Kruse [12],...

After that, the built-up Zinc-enzyme model (Fig. 6.2(b)) could effectively catalyze the hydrolysis of aryl phosphate. Another better hydrolyzing enzyme model is 4 nitrogen heterocyclic 12 silane cobalt complexes (Fig. 6.2(c)), which is one of the most effective acyl transferring catalysts. If the 4 nitrogen heterocyclic 12 silane cobalt complexes were covalently linked with -CD, the obtained enzyme model could increase 900-fold of the hydrolysis rate of PNPA (p-nitrophenol acetate). If the same group was conjugated onto the second hydroxyl group of f -CD, the obtained model could increase 2,900-3,700-fold of the hydrolysis rate of PNPA [21]. 

Acylaminophenols. A mixture of p-nitrophenol, acetic anhydride, glacial acetic acid, and 5%-Pd-on-carbon hydrogenated under 2 atm. until Hg-uptake is complete after ca. 1 hr. p-hydroxyacetanilide. Y 79. Pd-on-carbon gives purer products in better yields than PtOg. Also o- and m-isomers s. M. Frei-felder, J. Org. Chem. 27, 1092 (1962). 

Leonhardt et al were able to show a specific hydrolytic effect when treating nitrophenyl esters with an MIP imprinted with pyridine derivatives of N-boc-amino acids using DVB and 4(5)-vinylimidazole as monomers in combination with chelated Co ions. Compared to the control polymer, hydrolysis was accelerated by a factor of 4 to 5, while a comparison with MIP imprinted with other pyridine derivatives of N-boc-amino acids only gave an acceleration of the reaction by a factor of 2 to 3. Robinson et al used phosphonates as TSA and showed a catalytic effect of an MIP imprinted with p-nitrophenylmethyl phosphonate, using 4(5)-vinylimidazole and Co ions, on the hydrolysis of p-nitrophenol acetate. The authors— aware of the fact that imidazole containing polymers in general exhibit catalytic effects—could nevertheless demonstrate that the imprinted specimens were of 60% higher activity than the control polymers. 

Chloroanisole and p-nitrophenol, the nitrations of which are susceptible to positive catalysis by nitrous acid, but from which the products are not prone to the oxidation which leads to autocatalysis, were the subjects of a more detailed investigation. With high concentrations of nitric acid and low concentrations of nitrous acid in acetic acid, jp-chloroanisole underwent nitration according to a zeroth-order rate law. The rate was repressed by the addition of a small concentration of nitrous acid according to the usual law rate = AQ(n-a[HN02]atoioh) -The nitration of p-nitrophenol under comparable conditions did not accord to a simple kinetic law, but nitrous acid was shown to anticatalyse the reaction. 

When Jencks reacted hydroxylamine with p-nitrophenyl acetate, p-nitrophenolate ion was released at a rate faster than that at which acetohydroxamic acid was formed. This burst effect is evidence for a two-step reaction. In this case the intermediate is O-acetylhydroxylamine, which subsequently reacts with hydroxylamine to form the hydroxamic acid. 

The importance of the proximity effect in cyclodextrin catalysis has been discussed on the basis of the structural data. Harata et al. 31,35> have determined the crystal structures of a-cyclodextrin complexes with m- and p-nitrophenols by the X-ray method. Upon the assumption that m- and p-nitrophenyl acetates form inclusion complexes in the same manner as the corresponding nitrophenols, they estimated the distances between the carbonyl carbon atoms of the acetates and the adjacent second-... 

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 )... 

Group transfer polymerization (GTP) requires either a nucleophilic or Lewis acid catalyst. Bifluoride (HF2) and fluoride ions, supplied by soluble reagents such as tris(dimethylamino)-sulfonium bifluoride, [(CH3)2N]3SHF2, and (w-C NF, are the most effective nucleophilic catalysts, although other nucleophiles (CN , acetate, p-nitrophenolate) are also useful. Zinc... 

For the assay of a-D-mannosidase, the incubation mixture employed in our laboratory contained 0.5 ml of M acetate buffer at a pH appropriate for the particular enzyme preparation, 1.5 ml of 16 mM p-nitrophenyl a-D-mannopyranoside, 1.5 ml of water (which could be replaced by other additives as required), and 0.5 ml of suitably diluted, enzyme preparation. After 1 hour at 37°, the reaction was terminated, and the color was developed by adding 4 ml of 0.4 M glycine-sodium hydroxide buffer, pH 10.5. The mixture was centrifuged for 15 minutes at 1500 g, and the color intensity of the liberated p-nitrophenol (25-150 fig) in the supernatant liquor was measured on a Spekker photoelectric absorptiometer, with use of Ilford No. 601 violet filters having maximal transmission at 430 nm, and a 1-cm light path. Separate control-experiments for enzyme and sub-... 

In 1952, Hartley and Kilby showed that p-nitrophenyl acetate reacts with chymotrypsin, and advanced a two-step mechanism for the process (Hartley and Kilby, 1952). Two years later Hartley showed that a burst of p-nitrophenol was produced in the reaction (Hartley and Kilby, 1954). That is to say, a graph of the production of p-nitrophenol from the chymotryptic hydrolysis of p-nitrophenyl acetate does not seem to begin at the origin, but instead a small amount of p-nitrophenol is produced very rapidly. Fur-... 

If an amine P-NH2 is used in the aqueous solution, one obtains RCONHP instead of RCOOH. Rates of cleavage of three acyl nitrophenyl esters were followed by the appearance of p-nitrophenolate ion as reflected by increased absorbances at 400 nm. The reaction was carried out at pH 9.0, in 0.02 M tris(hydroxymethyl)aminomethane buffer, at 25°C. Rate constants were determined from measurements under pseudo-first-order conditions, with the residue molarity of primary amine present in approximately tenfold excess. First-order rate graphs were linear for at least 80% of the reaction. With nitrophenyl acetate and nitrophenyl caproate, the initial ester concentration was 6.66xlO 5M. With nitrophenyl laur-ate at this concentration, aminolysis by polymer was too fast to follow and, therefore, both substrate and amine were diluted tenfold for rate measurements. 

The complex between 1,10-phenanthroline and picric acid was stated to be a n-7i complex, not the expected n-n type.362 1,10-Phenanthroline also forms molecular complexes with phloroglucinol,363 halogenated p-nitrophenols 364 hexachlorocyclohexanes,365 and iodine.366,367 It forms an adduct with acetic anhydride.368 Electronic spectra of molecular complexes of 1,10-phenanthroline with porphyrins have been recorded.369... 

In 1954, B. S. Hartley and B. A. Kilby1 examined the reaction of substrate quantities of chymotrypsin with excess p-nitrophenyl acetate or p-riitrophenyl ethyl carbonate. They noted that the release of p-nitrophenol did not extrapolate back to zero but instead involved an initial burst, equal in magnitude to the concentration of the enzyme (Chapter 4, Figure 4.10). They postulated that initially the ester rapidly acylated the enzyme in a mole-to-mole ratio, and that the subsequent turnover of the substrate involved the relatively slow hydrolysis of the acylenzyme as the rate-determining step. This was later verified by the stopped-flow experiments described in section B2. 

Chromophoric leaving group.2-4 The original work on p-nitrophenyl acetate has been extended by synthesizing p-nitrophenyl esters of specific acyl groups, such as acetyl-L-phenylalanine, -tyrosine, and -tryptophan. The rate of acylation of the enzyme is determined from the rate of appearance of the nitro-phenol or nitrophenolate ion, which absorbs at a different wavelength from the parent ester. 

The initial evidence for the formation of an acyl-enzyme ester intermediate came from studies of the kinetics with which chymotrypsin hydrolyzed analogs of its normal polypeptide substrates. The enzyme turned out to hydrolyze esters as well as peptides and simpler amides. Of particular interest was the reaction with the ester p-nitrophenyl acetate. This substrate is well suited for kinetic studies because one of the products of its hydrolysis, p-nitrophenol, has a yellow color in aqueous solution, whereas p-nitrophenyl acetate itself is colorless. The change in the absorption spectrum makes it easy to follow the progress of the reaction. When rapid-mixing techniques are used to add the substrate to the enzyme, an initial burst of p-nitrophenol is detected within the first few seconds, before the reaction settles down to a constant rate (fig. 8.8). The amount of p-nitrophe-... 

Several kinds of evidence indicate that the reactions are catalytic rather than stoichiometric. When the reaction is followed to completion, linear first order plots are obtained for at least 90% of the reaction 7>. At the ratio of substrate to polymer employed, about 1 1 by weight, nonlinear first order plots would be predicted for a stoichiometric reaction. When a second aliquot of substrate is added after completion of the reaction, the first order rate constant noted with the second aliquot is essentially identical to that of the original7). The liberation of acetate and p-nitrophenol in equimolar proportions is also consistent with an inference of catalysis 7>. 

D-Xylosidase (EC 3.2.1.37), acetylesterase (EC 3.1.1.6), and a-L-arabinofuranosidase (EC 3.2.1.55) activities were determined using the synthetic substrates 1 mM p-nitrophenyl- 3-D-xylopyranoside, 5 mM p-nitrophenyl-acetate, and 5 mM p-nitrophenyl-a-L-arabinofuranoside (all from Sigma). Release of p-nitrophenol was monitored spectrophoto-metrically at 410 nm using a Jasco V-530 Spectrophotometer. 

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