Dianionic phosphate

In the solid state, X-ray crystallography (Blank et al., 1971) and Raman spectroscopy (Isbrandt and Oertei, 1980) data suggest that the dodecasodium salt of phytic acid adopts the sterically hindered 5 ax/1 eq conformation. Again, the 5 ax/1 eq form may be adopted to minimize electrostatic repulsion between the five contiguous dianionic phosphates in equatorial positions. 

In summary, the electrostatic repulsion due to four or more equatorially oriented dianionic phosphates disfavors the 1 ax/5 eq orientation and induces ring flip to the 5 ax/1 eq form. The proclivity toward ring flip is influenced by the number, position, and orientation of dianionic phosphate substituents on the inositol ring the facility for ring flip is as follows (29) > (27) > (31) > (33) > (35). 

Figure 11. Calculated energetics of proton transfer between the catalytic cysteine and the dianionic phosphate group of the substrate in LMPTP and PTPIB.

Two protein tyrosine phosphatases (PTPases) have been studied with hybrid potentials — the catalytic domain of human PTPIB [89] and the bovine PTPase (BPTP) [90]. These proteins have similar active centers and there is an invariant catalytic cysteine amino acid residue. Hillier et al characterized the transition state for the phosphate hydrolysis by PTPIB (with a dianion phosphate) using a PM3/MM potential but keeping the protein matrix and some of the QM atoms fixed. They found a dissociative mechanism in which the cleavage of the P-0 bond occurred before the formation of the S-P bond. The breaking of the P-O bond was determined to be the rate limiting step in agreement with kinetic... 

These results suggest that the nucleotide binds at around neutral pH in the dianionic ionization state. Thus the 3 -CMP-RNase A complex resonance is shifted upfield less than 0.3 ppm from the free 3 -CMP between pH 6.5 and 7.5, whereas monoprotonation of the free dianion results in a 4 ppm upfield shift. Furthermore, the addition of the first proton to the nucleotide complex (p 2 = 6.0-6.7) must occur mainly on some site other than the dianionic phosphate because the signal is shifted upfield by only 1-2 ppm. The addition of a second proton... 

Evans and Kaplan (1979) have shown that P chemical shifts in a series of 5 -nucleotides and related phosphate monoesters are only modestly sensitive to intramolecular hydrogen bonding between the base RNH and dianionic phosphate. The 0.4-ppm shielding resulting from this hydrogen-bonding interaction correlates nicely with the population of the g,g conformation about the C-4 —C-5 bond. 

Problem 2.9 Organic phosphate groups occur commonly in biological molecules. Calculate formal charges on the four O atoms in the methyl phosphate dianion. 

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). 

A kinetic isotope effect 160/180 of 2% in the spontaneous hydrolysis of the 2,4-dinitrophenyl phosphate dianion, whose ester oxygen is labeled, suggests a P/O bond cleavage in the transition state of the reaction, and thus also constitutes compelling evidence for formation of the metaphosphate 66,67). The hydrolysis behavior of some phosphoro-thioates (110) is entirely analogous 68). 

In the aprotic solvent acetonitrile, the trianion of cyclic trimetaphosphoric acid 115 is formed via the phosphorane 112 and the pyrophosphate dianion 113 protic solvents differ by reacting via the phosphorane 114 to give the phosphates 104 or H2PO . 

Yet another situation is observed in the 2,4-dinitrophenyl phosphate dianion. A significant effect of amines on the rate of decomposition is admittedly observed however, typical 2nd order kinetics, lower enthalpy of activation compared with spontaneous hydrolysis, and strongly negative AS values (see Table 3) indicate an Sn2(P) reaction. Surprisingly, the reaction rate remains unaffected by the basicity of the amine, even when its pKa value changes by 8 units. 

Table 3. Activation parameters of spontaneous hydrolysis and second-order reactions with amines of the 2,4-dinitrophenyi phosphate dianion...

A situation similar to that in acetyl phosphate is also encountered in benzoyl phosphate76 . Electron-attracting substituents on the phenyl ring accelerate the hydrolysis of the dianion (a linear relationship exists between log khydrol and the Hammett a constants with q = 1.2 and the linear log ki,j,drol./pKa relationship is the same as for the phosphoric monoaryl ester dianions65 . On the other hand, hydrolysis of the monoanion is influenced only slightly by substituents in the phenyl ring. These observations can also be rationalized in terms of the decomposition mechanism to the POf ion formulated for 116 and 117. 

As for the acetyl phosphate monoanion, a metaphosphate mechanism has also been proposed 78) for the carbamoyl phosphate monoanion 119. Once again, an intramolecular proton transfer to the carbonyl group is feasible. The dianion likewise decomposes in a unimolecular reaction but not with spontaneous formation of POf as does the acetyl phosphate dianion, but to HPOj and cyanic acid. Support for this mechanism comes from isotopic labeling proof of C—O bond cleavage and from the formation of carbamoyl azide in the presence of azide ions. 

The mechanism of hydrolysis of o-carboxyaryl phosphates, whose dianions also hydrolyze much faster then, e.g., the phenyl phosphate monoanion 79,80) (maximum rate at about pH 4.8 and 25 °C 81)), was long a point of mechanistic contention. Thorough investigations81 led to proposal of a fast initial transprotonation... 

A mechanism proposed 87) for the alkaline hydrolysis of tetraethyl pyrophosphate, which is markedly accelerated by HPO e ions, has been substantiated by isotopic labeling 88). The nucleophilic attack by HPOJp on the symmetrical pyrophosphate 131 is considered to lead initially to the unsymmetrical P P1-diethyl pyrophosphate dianion 132 which decomposes spontaneously under the conditions of reaction to give the diethyl phosphate anion and POf 102. The latter reacts with water to form inorganic phosphate and with alcohols suclj as methanol and ethylene glycol to produce alkyl phosphates. 

Direct synthesis of P. P1-diethyl pyrophosphate 89> was accomplished later. It is stable at pH 3, but hydrolyzes rapidly at pH 7/room temperature. Thus in this case it is the dianion which hydrolyzes faster than the monoanion, in contrast to the behavior of alkyl phosphates or pyrophosphate monoesters (see below). 

The highly electrophilic character of the POf ion would suggest a very unselective phosphorylation behavior. For example, the ratio of alkyl phosphate to inorganic phosphate obtained in hydrolyses of phosphoric esters in water/alcohol mixtures should reflect the molar ratio of water and alcohol. This is indeed found in numerous cases, e.g. in the hydrolysis of phenyl and 4-nitrophenyl phosphate monoanions 97) or of 4-nitrophenyl phosphate dianions 65) at 100 °C in methanol/ water mixtures of various compositions, as also in the solvolysis of the acetyl phosphate dianion at 37 °C 97) or of phosphoenol pyruvate monoanions 82). Calculations of the free energy of the addition reactions of water and ethanol to the POf ion support the energetic similarity of the two reactions 98) (Table 4). 

Full details on the phosphorylation of water and alcohols by 4-nitrophenyl dihydrogen phosphate and the NfC H ) - and N(CH3) -salts of its mono- and dianion have been published 146>. Phosphoryl group transfer from the monoanion and dianion is thought to proceed via the monomeric POf ion. Addition of the sterically unhindered amine quinuclidine to an acetonitrile solution containing the phosphate monoanion and tert-butanol produces t-butyl phosphate at a faster rate than does the addition of the more hindered diisopropylethylamine. This nucleophilic catalysis of the phosphorylation reaction is also explained by the intermediacy of the POf ion. 

When monomeric metaphosphate anion POf (102) is generated form the phos-phonate dianion 170 in the presence of the hindered base 2,2,6,6-tetramethylpiper-idine, it undergoes reaction with added carbonyl compounds147), Thus, it phosphoryl-ates acetophenone to yield the enol phosphate, whereas in the presence of acetophenone and aniline the Schiff base is formed from both compounds, probably by way of the intermediate C6H5—C(CH3) (OPO e) ( NH2C6HS). This reactivity pattern closely resembles that of monomeric methyl metaphosphate 151 (see Sect. 4.4.2). 

Chemical shift and line width are related and can be used to describe conformational changes. The 831P value of 5 ppm and the line width of 20 Hz indicate that dianionic 5 phosphate is very tightly bound to the active site and tumbles with the protein. Lower values of chemical shift indicate more loosely bound phosphate. Higher values of the line width indicate the presence of two conformers in equilibrium. If the open and close form undergo slow interconvertion, two signals are observed in the 31P NMR spectra. 

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