2- phenol phosphate, hydrolysis

The application of fluorogenic labeling to the determination of some organophosphate insecticides has been attempted [178,179]. Fenthion (0,0-dimethyl 0-[(4-metiiylthio)-m-tolyl] phosphorothioate), Ruelene (0-2-chloro-4-ferf.-butylphenyl O-methyl methyl-phosphoramidate), GC 6506 [dimethyl p-(methylthio)phenyl phosphate] and several other compounds which yield phenols on hydrolysis have been examined. The limits of detection for some of these labeled derivatives have been reported to be in the low nanogram range. The organophosphate Proban [0,0-dimethyl 0-(p-sulphamoylphenyl) phosphorothioate] has been determined directly without hydrolysis by dansylation of the free amino group of the molecule [180]. The derivative exhibited blue fluorescence, as compared to yellow for phenol and alkylamine dansyl derivatives. 

According to a patent of the Sumitomo Co. (1965), tris-phenol phosphate is prepared from phenol by esterification with phosphorus oxychloride, which is then chlorinated, yielding after hydrolysis a chlorophenol mixture with an ortho para ratio of 8 1. Using sulfuryl chloride (SO2CI2) as chlorinating agent, a chlorophenol mixture of ortho para ratio 4.1 5.7 is formed in the presence of Lewis acid catalysts. 

Esters of phosphorous acid derived from aUphatic alcohols and unhindered phenols, eg, tris(nonylphenyl)phosphate (24), hydrolyze readily and special care must be taken to minimize decomposition by exposure to water or high humidity. The phosphorous acid formed by hydrolysis is corrosive to processing equipment, particularly at high temperatures. 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

The phosphoryl group in phenylphosphate is derived from the -phosphate group of ATP. The free energy of ATP hydrolysis obviously favors the trapping of phenol K, 0.04 mM), even at a low ambient substrate concentration. The reaction is stimulated several fold by another protein, subunit 3 (24kDa). The molecular and catalytic features of phenylphosphate synthase resemble those of phosphoenolpyruvate synthase, albeit with interesting modifications. ... 

Phenols with an appropriate leaving group in the benzylic position such as fluoride may form QMs by spontaneous hydrolysis, possibly catalyzed by a basic amino acid residue as shown in Scheme 10.2c. Evidence for this process was obtained with 4-(fluoromethyl)phenyl phosphate involving initial enzymatic hydrolysis of the phosphate followed by nonenzymatic formation of a QM.11 Similarly, several lines of evidence demonstrate nonenzymatic QM formation from 4-trifluoromethylphenol under physiologic conditions.12... 

As is apparent from Fig. 1, the dianions of monoalkyl phosphates normally resist hydrolysis. However, for leaving groups whose conjugate acids exhibit a pKa < 5 in water, hydrolysis of the dianion becomes faster than that of the monoanion. Fig. 2 shows a pH profile characteristic of this situation. Whereas the hydrolysis rate of 2,4,6-trichlorophenyl phosphate (pKa of the phenol 6.1) still shows the typical monoanion preference as seen for methyl phosphates (Fig. 1), the dianion of 2,4-dinitrophenyl phosphate (pKa of the phenol 4.09) is hydrolyzed far faster than the monoanion 2-chloro-4-nitrophenyl phosphate represents an intermediate case (pKa of the phenol 5.45)6S). 

These compounds contain the fragment R as an alkyl or aryl moiety. In other words, they result from the esterification of an alcohol or a phenol with nitrous acid, nitric acid, phosphoric acid, sulfuric acid, or sulfamic acid, respectively. Many of the esters to be examined in this chapter must be activated prior to eliciting their effects, e.g., the organic nitrites and nitrates, which act as donors of nitric oxide or an analogous molecule, and phosphates, which are activated by hydrolysis or even by phosphorylation (antiviral agents). Sulfates are very seldom active or used as prodrugs, but they have significance as metabolites and as industrial xenobiotics. 

Fig. 9.8 presents another, more complex type of phosphate prodrugs, namely (phosphoryloxy)methyl carbonates and carbamates (9-26, X = O or NH, resp.) [84], Here, the [(phosphoryloxy)methyl]carbonyl carrier appears quite versatile and of potential interest to prepare prodrugs of alcohols, phenols, and amines. The cascade of reactions leading from prodrug to drug as shown in Fig. 9.8 involves three steps, namely ester hydrolysis, release of formaldehyde, and a final step of carbonate hydrolysis (X = O) or A-decar-boxylation (X = NH). Three model compounds, a secondary alcohol, a primary aliphatic amine, and a primary aromatic amine, were derivatized with the carrier moiety and examined for their rates of breakdown [84], The alcohol, indan-2-ol, yielded a carrier-linked derivative that proved relatively... 

P. A. Benkovic, M. Hegazi, B. A. Cunningham and S. J. Benkovic, Biochemistry 18, 830 (1979) use continuous monitoring of inorganic phosphate product of enzyme catalyzed hydrolysis of fructose biphosphate. The pH change monitored by phenol red results from an acid, base adjustment of the liberated phosphate. This is a detailed valuable, if complicated, account. 

The aq. soln. of trisodium phosphate reacts alkaline to methyl orange, phenol-phthaleln, and litmus and neutral to Porrier s blue. The aq. soln. of trirubidium and tricsesium phosphates also react alkaline. J. Shields, J. M. van Bemmelen, and A. Kossel found that in aq. soln., trisodium phosphate is almost completely hydrolyzed by water into disodium hydrogen phosphate and sodium hydroxide. E. Salm calculated that the degree of hydrolysis is about 23 per cent, of the sodium or 70 per cent, of theP04 E. Blanc that 34 1 per cent, of a O O A-soln. VOL. II. 3 i... 

It is commonly assumed that the base-catalyzed hydrolysis of substituted dialkyl (i.e., dimethyl or diethyl) phenyl phosphates occurs by nucleophilic attack of OH" at the phosphorus with the phenolate being the leaving group (see also Section 13.4) ... 

A rate enhancement of about 107 is observed over the slow spontaneous hydrolysis of the corresponding di-3-nitrophenyl phosphate. The specific displacement of the exocyclic phenolate may be rationalized in terms, of a pentacovalent intermediate (56)... 

In the author s own laboratory the Cu(II)-catalyzed hydrolysis of the phosphate ester derived from 2-[4(5)-imidazolyl] phenol recently has been investigated146. The pertinent results are (a) the pre-equilibrium formation of a hydrolytically labile Cu(II)-substrate complex (1 1), (b) the occurrence of catalysis with the free-base form of the imidazolyl and phosphate moieties and (c) the extraordinary rate acceleration at pH 6 (104) relative to the uncatalyzed hydrolysis146. The latter recalls the unusual rate enhancement encountered above with five-membered cyclic phosphates and suggests a mechanism in which the metal ion, at the center of a square planar complex or a distorted tetrahedral complex, might induce strain in the P-O ester bonds (60). viz. 

Poly(ribitol phosphate) synthetase has been found in particulate fractions from Staphylococcus aureus H, and Lactobacillus plantatrum.lt ll-m The bulk of the activity in Lactobacillus plantarum was in crude, cell-wall preparations, and the enzyme is apparently located in the membrane, although intimate association with the wall itself has been suggested. Unlike the natural teichoic acid, the enzymically synthesized ribitol phosphate polymer was readily extracted with phenol hydrolysis by acid and by alkali gave the expected products, and oxidation with periodate indicated a chain length of 5-9 units, a value which compares well with that of 8 units for the natural polymer in the walls of this organism. 

The incubation digest (7.0 ml) contained 1 ml of 0.022 M phenyl phosphate 2.5 ml of 0.1 M acetate buffer, pH 5.0 0.5 ml of test enzyme solution and 3.0 ml of solutions of acceptors giving a final concentration as shown in the third column. Incubation time, 30 min. Digests were inactivated by 3.0 ml of 10% trichloroacetic acid solution and were analyzed for phenol and inorganic phosphate. In the case of the standard acceptor, 1,4-butanediol, the expected transfer product, 1,4-butanediol phosphate, was isolated in a yield of 35% from a large-scale experiment. The hydrolysis of this phosphate ester by prostatic acid phosphatase liberated approximately equimolar amounts of 1,4-butanediol and inorganic phosphate. 

Relative rates of hydrolysis were determined with 0.5 ml reaction mixtures in 0.1 M sodium acetate buffer, pH 5.0, at 37°. Liberated phosphate was measured by the method of C. H. Fiske and Y. SubbaRow [JBC 66, 375 (1925)]. The amounts of enzyme used were 0.22 unit of crystalline enzyme and 0.24 unit of peak II enzyme. The concentration of substrate and inhibitor was 1.0 mM. For inhibitor study, 1.0 mM p-nitrophenyl phosphate was used as substrate. Inhibition was calculated from the amount of p-nitro-phenol released and expressed as fractional inhibition. 

Scheme 1 Hydrolysis of diphenyl-carbonate (1) to give 4-nitro-phenol (2), 4-acetamido-phenol and carbon dioxide. The diphenyl-phosphate (4) was used as TSA to mimic the tetrahedral transition state formed during the hydrolysis...

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