Hydrolysis of / -nitrophenyl phosphate

Zinc(II) and Co(II) are the only cations found to reactivate apophos-phatase to any appreciable extent (120). The Co(II) enzyme follows the same formal mechanism as the native enzyme, but has a lower specific activity (113, 121). It lacks the phosphotransferase activity (113, 119, 121) observed for the native enzyme, for example in Tris buffers. This was taken to imply that the lower activity of the cobalt enzyme is due to a lower rate of phosphorylation, so that this step becomes rate-limiting also below f>H 7 (113). Stopped-flow experiments by Gottesman etal. (121) show, however, that a very fast burst of -nitrophenol occurs in the cobalt alkaline phosphatase-catalyzed hydrolysis of -nitrophenyl phosphate over a wide pH region. These results strongly suggest that a step subsequent to the phosphorylation is rate-limiting in this metal derivative. 

Havinga, E., de Jongh, R. O., Dorst, W., Photochemical Acceleration of the Hydrolysis of Nitrophenyl Phosphates and Nitrophenyl Sulfates, Reel. Trav. Chim. Pays Bas Belg. 1956, 75, 378 383. 

The activity of alkaline phosphatase is measured by hydrolysis of />-nitrophenyl phosphate to yield -nitrophenol and free phosphate. At alkaline pH, -nitrophenol dissociates the / -nitrophenylate ion has a strong yellow colour, with maximum absorbance at 405 nm. 

Figure 1 (38) illustrates that maximal hydrolysis of phenyl phosphate occurred at pH 4.9, 5.0, and 5.0 in acetate, citrate, and tris-HCl buffers, respectively. For p-nitrophenyl phosphate, the corresponding values were 4.9, 4.7, and 5.5. For / -glycerophosphate, the values were 5.5, 5.7, and... 

Robinson, 1969a). It is probable that the hydrophobic nature of the phenyl groups of p-nitrophenyl diphenyl phosphate results in deep penetration of the neutral ester in the Stern layer, thus shielding the phosphoryl group from nucleophilic attack. Unlike other reactions between nucleophiles and neutral substrates catalyzed by cationic micelles (Bunton and Robinson, 1968, 1969a) and the hydrolysis of dinitrophenyl phosphate dianions in the presence of cationic micelles (Bunton et al., 1968), the catalysis of the hydrolysis of -nitrophenyl diphenyl phosphate by CTAB arises from an increase in the activation entropy rather than from a decrease in the enthalpy of activation. The Arrhenius parameters for the micelle-catalyzed and inhibited reactions are most probably manifestations of the extensive solubilization of this substrate. However, these parameters can be composites of those for the micellar and non-micellar reactions and the eifects of temperature on the micelles themselves are not known. Interpretation of the factors which affect these parameters must therefore be carried out with caution. In addition, the inhibition of the micelle-catalyzed reactions by added electrolytes has been observed (Bunton and Robinson, 1969a Bunton et al., 1969, 1970) and, as in the cases of other anion-molecule reactions and the heterolysis of dinitrophenyl phosphate dianions, can be reasonably attributed to the exclusion of the nucleophile by the anion of the added salt. 

A review (91 references) on electrophilic and nucleophilic reactions of trivalent phosphoms acid derivatives, reactions of two-coordinate phosphoras compounds, and miscellaneous reactions has appeared. Earlier in this review we looked at the heavy-atom isotope effects on reactions of Co(III)-bound /j-nitrophenyl phosphate, the uranyl ion hydrolysis of/ -nitrophenyl phosphodiesters (218)-(220), and the Th(IV) hydrolysis of these. 

Some years later, Nigam et al. (N3) undertook a kinetic study with preparations that represented an approximately 20- to 30-fold purification (D3). They first presented the time courses of hydrolysis of phenyl phosphate, nitrophenyl phosphate, and /3-glycerophosphate, the first two at an initial concentration of 0.0043 Af and the last, jS-glycero-phosphate, at a concentration of 0.0028 Af. Although the hydrolyses of phenyl phosphate and nitrophenyl phosphate were of zero order for approximately the first 25% of the reaction, that of j8-glycerophosphate was of this order for only the first 10%, the reaction velocity decreasing... 

Fig. 10.3. Schematic representation of hydrolysis of a phosphate ester (ROP) by APase. Pi represents inorganic phosphate, ROP is substrate and EP is phosphoryl enzyme. Typical values for the various rate constants for the bacterial enzyme in 50 mM Tris-HCl buffer, pH 8.3, at 10°C with p-nitrophenyl phosphate as substrate (Bale et al., 1980) are k,jk, = X, S 1.5 x 10" M k jK = 1.6 x 10 M sec k2 2.4 lO sec k = 78 sec" and k-t = 7.6 sec. ...

On the other hand, phosphorane intermediates are not expected to be involved in the hydrolysis of phosphate monoesters, so the effective observed catalysis by the carboxyl group of salicyl phosphate 3.21 [51] (Scheme 2.26) is presumed to be concerted vith nucleophilic attack. (The hydrolysis reaction involves the less abundant tautomer 3.22 of the dianion 3.21, and the acceleration is >10 -fold relative to the expected rate for the pH-independent hydrolysis of the phosphate monoester dianion of a phenol of pK 8.52.) However, this system differs from the methoxy-methyl acetals discussed above, in that there is a clear distinction between neutral nucleophiles, which react through an extended transition structure similar to 3.16 in Scheme 2.23, and anions, which do not react at a significant rate, presumably because of electrostatic repulsion. This distinction is well-established for the dianions of phosphate monoesters with good leaving groups (p-nitrophenyl [52] and... 

Aldol condensation of pyruvate and L-aspartate - /3-semialdehyde Phosphomonoesters hydrolysis (p-nitrophenyl phosphate, 3.3 x 10 )... 

Morrow and Trogler (109) have studied the hydrolysis of two phosphate diesters by [Cu(bipy)] (bipy = 2,2 -bipyridine) in aqueous solution at 75°C in the pH range 5.8-8.3. For both bis(4-nitrophenyl)phosphate and ethyl-4-nitrophenylphosphate the reaction was proposed to proceed via coordination of the diester to the [Cu(bipy)] moiety followed by attack of a cis coordinated OH ion at the P center. Maximal rate enhancements of 1(P- to- 10 -fold were reported. The reaction was accompanied by incorporation of a single label in the product ethylphosphate when the reaction was conducted in labeled water. Saturation kinetics were observed for the hydrolysis of ENPP (ethyl-4-nitrophenylphosphate). The reaction obeyed Michaelis-Menton kinetics with a for the ENPP ion of... 

An alternative role for metal ions in the hydrolysis of organic phosphorus compounds is the coordination and induced deprotonation of water to create a very reactive nucleophile (Smolen and Stone, 1997). The effect can be further enhanced if the phosphorus compound under attack is proximally coordinated to the hydroxyl ion. For example, Jones et al. (1983) reported a 10 increase in the rate of hydrolysis oipara-nitrophenyl phosphate when it is coordinated to a cobalt (111) complex with a proximal hydroxyl ligand. 

In a later study we examined the ability of cyclodextrin dimers linked by a bipyridyl unit to catalyse the hydrolysis of a phosphate ester, fcw-p-nitrophenyl phosphate. With a bound lanthanum ion the hydrolysis was accelerated by 300 million-fold, a huge acceleration that could well be of practical interest in the hydrolysis of phosphate esters generally. A full paper described this work in some detail. " ... 

The initial rate of hydrolysis of / -nitrophenyl acetate (PNPA) was measured at 25°C in pH 7.2 phosphate buffer (1/15 M) in the presence of dimethyl-/ -cyclodex-trin bearing the imidazolylethyl group (2), dimethyl-j8-cyclodextrin () -DMCD) (1) or imidazole, and in the absence of them [8]. The conditions of large excess of PNPA were used and concentrations of PNPA were changed from 10-fold to 100-fold over those additives except 1. The reaction was followed by monitoring the released p-nitrophenol at 400 nm. 

The regioselectivity for the hydrolysis of nitrophenyl acetate isomers by 2 was demonstrated. The reactions were carried out under the conditions of excess of substrate in pH 8.2 phosphate buffer (1/15 M) at 25°C. Kinetic parameters are shown in Table II. Both KJ m for PNPA are larger than for the others. 

Table II. Kinetic parameters for hydrolysis of nitrophenyl acetate isomers at 25°C in pH 8.2 phosphate buffer by 2 (1.75 x 10 M)...

It has to be pointed out that, with a few exceptions, the acceleration by polyelectrolytes was associated with decreases and Table IV gives the thermodynamic parameters for the aquation reactions of Co(NH3)5Br induced by Ag". Similar decreases in and AS were found for various reactions the Hg -induced aquation of Co(NH3)5Br , the SpjAr reaction of dinitrochlorobenzoic acid with OH [51], the hydrolysis of 2,4-dinitrophenyl phosphates [reaction (E)] [33], the outer-sphere electron-transfers between Co-complexes [Co(NH3)5N3, Co(NH3)5Br , Co(en)2Cl2 ] and Ru(NH3)6 or [8, 20] [en ethylenediamine], the polyvinyl-imidazole-accelerated solvolysis of p-nitrophenylacetate [52], the coupling reactions of dinitrofluorobenzene with aminoacids [53], dipeptides [53] and aniline [54], the lignin sulfonic acid-accelerated hydrolysis of methyl acetate [55], and the hydrolysis of nitrophenyl esters [37]. The opposite tendency (acceleration caused by increases... 

Theoretical studies of the hydrolysis of methyl phosphate anion have found that the dissociative mechanism involving the formation of metaphosphate is favoured over the associative mechanism involving a pentacoordinated intermediate.Phytic acid, myo-inositol hexakis(dihydrogen phosphate) (127), was completely hydrolysed to inositol in an aqueous polybasic alcohol at 150 °C a reaction mechanism was proposed. The reactions of p-nitrophenyl diethyl phosphate (128) and ethyl p-niuophenyl ethylphosphonate (129) with a wide range of oximate ions (p/(a = 7-13) in 10% aqueous ethanol at 25 °C have been studied. For oximate ions with p a 9.0, the reactivity of the oximes tends towards that of alcoholate ions and their Q -effect disappears (as had been previously shown with / -niuophenyl acetate). The reason for this is the unfavourable solvation effects of the solvent. A review (253 references) has appeared on the synthesis and reaction of Q -aminophosphonates. ° ... 

Deming and Pardue studied the kinetics for the hydrolysis of p-nitrophenyl phosphate by the enzyme alkaline phosphatase. The progress of the reaction was monitored by measuring the absorbance due to p-nitrophenol, which is one of the products of the reaction. A plot of the rate of the reaction (with units of pmol mL s ) versus the volume, V, (in milliliters) of a serum calibration standard containing the enzyme yielded a straight line with the following equation... 

The rat LD qS are 13, 3.6 (oral) and 21, 6.8 (dermal) mg/kg. Parathion is resistant to aqueous hydrolysis, but is hydroly2ed by alkah to form the noninsecticidal diethjlphosphorothioic acid and -nitrophenol. The time required for 50% hydrolysis is 120 d ia a saturated aqueous solution, or 8 h ia a solution of lime water. At temperatures above 130°C, parathion slowly isomerizes to 0,%diethyl 0-(4-nitrophenyl) phosphorothioate [597-88-6] which is much less stable and less effective as an insecticide. Parathion is readily reduced, eg, by bacillus subtilis ia polluted water and ia the mammalian mmen to nontoxic 0,0-diethyl 0-(4-aminophenyl) phosphorothioate, and is oxidized with difficulty to the highly toxic paraoxon [511-45-5] diethyl 4-nitrophenyl phosphate d 1.268, soluble ia water to 2.4 mg/L), rat oral LD q 1.2 mg/kg. 

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