Dianions phosphomonoesters

As a reference to this intramolecular hydrolysis, ethyl (4-nitrophenyl) phosphate (NEP ) was intermolecularly hydrolyzed by 15b. In this case, the second-order rate constant A(NEP") was 7.9 x 10" M" sec at 35°C with I = 0.10. Thus, the intramolecular hydrolysis is 45,000 times faster than the intermolecular NEP" hydrolysis with 1 mM 15b. That is to say, the effective molarity is 45 M (=A(PDE)/ (NEP")) in favor of the intramolecular phosphate of 25b. The final phosphomonoester product 26 was completely inert and was not hydrolyzed even at high pH (up to ca. 12). Therefore, one could not use the Zn -alkoxide in 24 as a catalyst for phosphate hydrolysis, as was the case for carboxyester hydrolysis. The Zn -cyclen complex 12 was shown to strongly bind to dianionic phosphomonoesters (e.g., K = 10 M" for 1 1 NPP -Zn -cyclen). It is highly probable that the pendent phosphomonoester in 26 itself is too strong a competitive inhibitor, by occupying the Zn catalytic site, to generate Zn —OH" species. 

The iron(II)-iron(III) form of purple acid phosphatase (from porcine uteri) was kinetically studied by Aquino et al. (28). From the hydrolysis of a-naphthyl phosphate (with the maximum rate at pH 4.9) and phosphate binding studies, a mechanism was proposed as shown in Scheme 6. At lower pH (ca. 3), iron(III)-bound water is displaced for bridging phosphate dianion, but little or no hydrolysis occurs. At higher pH, the iron(III)-bound OH substitutes into the phosphorus coordination sphere with displacement of naphthoxide anion (i.e., phosphate hydrolysis). The competing affinity of a phosphomonoester anion and hydroxide to iron(III) in purple acid phosphatase reminds us of a similar competing anion affinity to zinc(II) ion in carbonic anhydrase (12a, 12b). 

It was found that the polymer exhibited selectivity towards phosphomonoester dianions. Less polar compounds were found to bind non-specifically to the polymer. The polymer was then used as a stationary phase for a HPLC column. A mixture containing dA, 5 -dAMP and 3, 5 -cAMP was thus separated. As expected, the retention time of 5 -AMP was larger than those for dA and 3, 5 -cAMP. The same was tme for other nucleotides compared to the corresponding nucleosides. When the Zn2+-free control polymer was used, all compounds were immediately eluted. The possibility to use polymer-anchored recognition units to separate biologically important phosphates was thus proved. 

The pKa values of phosphomonoesters are 1 and 6 therefore, there will be two ionic species at pH 7, with the dianion predominating ... 

Fig. 1 From left to right, the structures of a phosphate monoester, diester, and triester. Depending upon pH, monoesters may be neutral, monoanionic, or dianionic diesters may be neutral or anionic. The first pKa of an alkyl phosphomonoester, and the pKa of a dialkyl diester, is 2. The second pKa of an alkyl monoester is 6.8. Oxygen atoms bonded to ester groups (OR) are called bridging oxygen atoms the other oxygen atoms are nonbridging. Thus, a triester has one nonbridging oxygen atom, an ionized diester has two, and a fully ionized monoester has three.

Fig. 2 The dissociative, associative, and concerted mechanistic pathways of phosphoryl transfer. The dissociative and concerted mechanisms are shown for the dianion form of a phosphomonoester. The associative mechanism is shown for a phosphotriester.

The data and mechanistic conclusions summarized above come from work with aryl phosphomonoesters as predicted by the steep jSlg value, alkyl ester dianions react at very slow rates. A recent study of methyl phosphate found the rate of the dianion hydrolysis to be below the threshold of detectability, with an estimated rate constant of 2 x 10 20 s 1 at 25 °C.3 Since this value is close to the rate predicted from an extrapolation of the Bronsted plot of aryl phosphomonoester dianions, a similar mechanism is likely to be followed for alkyl and aryl esters. 

This value is close to the rate predicted from an extrapolation of the Br0nsted plot of aryl phosphomonoester dianions, suggesting that the alkyl and aryl esters likely follow a similar hydrolysis mechanism. 

Lad C, Williams H, Wolfenden, R. The rate of hydrolysis of phosphomonoester dianions and the exceptional catalytic proficiencies of protein and inositol phosphatases. Proc Natl Acad Sci U S A 2003 100 5607-5610. 

Phosphomonoester Also called a phosphate monoester. A derivative of phosphate in which one of the oxygen atoms is bonded to an alkyl group. For example, methyl phosphate, in the dianion form, is CHsOPOs. ... 

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