Phosphate monoanion

Phosphate esters have a variety of mechanistic paths for hydrolysis. Both C-O and P-0 cleavage are possible depending on the situation. A phosphate monoanion is a reasonable leaving group for nucleophilic substitution at carbon and so 8 2 or SnI reactions of neutral phosphate esters are well known. PO cleavage can occur by associative (by way of a pentacoordinate intermediate), dissociative (by way of a metaphosphate species), or concerted (avoiding both of these intermediates) mechanisms. 

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... 

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). 

The characteristic features of hydrolysis of diaryl phosphate monoanions (pronounced influence of leaving group on the rate of hydrolysis, k o/k = 1.6, and AS = —25 euu7y) also fail to support a metaphosphate mechanism118>. Hydrolysis of the acetyl phenyl phosphate monoanion is likewise far slower than that... 

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. 

Proton transfer may proceed directly or via a six-membered cyclic transition state involving a molecule of water. A calculation of the intermediate zwitter-ionic concentration for the hydrolysis of methyl phosphate monoanion, based on the pKa values for methanol and methyl phosphate dianion, predicts the first-order rate coefficient for zwitterion decomposition to be ca. 10 sec-1 at 100°C. This value is in good agreement with the observed rate of hydrolysis and, considering the assumptions involved, with the rate of P-O bond fission of the presumed zwitterionic intermediate (2) formed in the Hg(II) catalyzed solvolysis of phosphoenolpyruvic acid, a model reaction for pyruvate kinase10. 

The serine group which becomes phosphorylated does not appear to possess any marked nucleophilic reactivity, nor is there any evidence that a histidine group participates as a general acid-general base catalyst. Rate constants for the nonenzymic hydrolysis of alkyl and aryl phosphate monoanions at 25° are in the range HP7 to 10-9 sec-1 (167), while the comparable alkaline phosphatase-catalyzed values (in this case they refer to dianions) are in the range 102 to 103 sec-1. Thus one has to account for a rate enhancement factor of 109 to 1012. Moreover, the... 

This pattern of reactivity at C(l) of aldopyranosyl derivatives was first quantitated by O Connor and Barker (1979), who studied the hydrolyses of aldopyranosyl phosphates at acid pH, the reaction is an SN1 departure of phosphate monoanion. Equatorial/axial rate ratios of 2.3 for glucose (cf. [1] and [2]), 1.9 for L-fucose [10], 2.3 for D-galactose [11] and 4.1 for D-mannose... 

These calculations were, however, performed at a modest level of sophistication. The calculations on dimethyl phosphate monoanion were ab initio, but with iterative geometry optimisation, an STO-3G" basis set, and a Gaussian 70 program package the calculations on the dianionic trigonal bipyramid were similar, but those on the neutral trigonal bypyramid were... 

Linear free-energy relationships (LFER) with monoanionic phosphorylated pyr-idines indicate a loose transition state in which metaphosphate is not an intermediate.16 The hydrolysis of the monoanion of 2,4-dinitrophenyl phosphate is thought to be concerted,39 but the possibility of a metaphosphate intermediate has not been ruled out with esters having less activated leaving groups. A stereochemical study of the hydrolysis of phenyl phosphate monoanion indicates that the reaction proceeds with inversion.21 This result implies either a concerted mechanism, or a discrete metaphosphate intermediate in a pre-associative mechanism. 

The displacement of substituted phenoxide ions from aryl monophosphate monoanions by nicotinamide (Equation 38) has a PLg of -0.95. Reaction of substituted pyridines with 2,4-dinitrophenyl-phosphate monoanion has a 3 of 0.56. Using effective charge data from Scheme 1 construct a combined effective charge map for the... 

Figure 2. Correlation of the rate constants for the reaction of nucleophilic reagents with phosphorylated y-picoline monoanion and methyl 2,4-dinitrophenyl phosphate monoanion. The line has a slope of 0.51 (79).

Fig. 8. Proposed mechanism for the (Na+ + K+)-ATPase and for cation transport (80), based on EPR (80), nuclear relaxation (80, 81) and kinetic studies (80, 86), the detection of phosphoryl transfer (85) to an aspartyl residue (78), yielding a covalent phosphoenzyme intermediate (77, 79). Steps (1—3) show the binding of Mna+, Na+ and the phosphate monoanion to the enzyme. Steps (4—11) represent the ATPase activity. Active cation transport occurs via steps (4—6)...

Na+ compared to K+, since Na+ binds to a phosphate monoanion and K+ binds to a dianion. Recent T1+-NMR data indicates this monovalent cation binds 4.0 A from the enzyme bound Mn2+ and that this distance increases to 5,4 A on binding of phosphate consistent with the mechanism of Fig. 8 (81). However, the possibility of T1+ binding at an inhibitory site rather than its activating site has not been excluded. 

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