Phosphomonoester formation

While there have been a considerable number of structural models for these multinuclear zinc enzymes (49), there have only been a few functional models until now. Czamik et al. have reported phosphate hydrolysis with bis(Coni-cyclen) complexes 39 (50) and 40 (51). The flexible binuclear cobalt(III) complex 39 (1 mM) hydrolyzed bis(4-nitro-phenyl)phosphate (BNP-) (0.05 mM) at pH 7 and 25°C with a rate 3.2 times faster than the parent Coni-cyclen (2 mM). The more rigid complex 40 was designed to accommodate inorganic phosphate in the in-temuclear pocket and to prevent formation of an intramolecular ju.-oxo dinuclear complex. The dinuclear cobalt(III) complex 40 (1 mM) indeed hydrolyzed 4-nitrophenyl phosphate (NP2-) (0.025 mM) 10 times faster than Coni-cyclen (2 mM) at pH 7 and 25°C (see Scheme 10). The final product was postulated to be 41 on the basis of 31P NMR analysis. In 40, one cobalt(III) ion probably provides a nucleophilic water molecule, while the second cobalt(III) binds the phosphoryl group in the form of a four-membered ring (see 42). The reaction of the phosphomonoester NP2- can therefore profit from the special placement of the two metal ions. As expected from the weaker interaction of BNP- with cobalt(in), 40 did not show enhanced reactivity toward BNP-. However, in the absence of more quantitative data, a detailed reaction mechanism cannot be drawn. 

Chu et al. (1983, 1986) and Ghosh et al. (1990) describe modified carbodiimide protocols using the water-soluble reagent EDC instead of DCC. They also incorporate a second reactive intermediate, a phosphorimidazolide, created from the reaction of the phosphomonoester at the 5 -terminus of DNA with EDC in the presence of imidazole. A reactive phosphorimidazolide will rapidly couple to amine-containing molecules to form a phosphoramidate linkage (Fig. 398). The chemical reaction had been used previously to effect the formation of phosphodiester linkages between short DNA strands (Shabarova et al., 1983). 

Phosphate Esters. An ester is formed by elimination of H20 and formation of a linkage between an acid and an alcohol (or phenol) (Fig. III-22). Phosphomonoesters, especially of monosaccharides, are very common (Fig. ffl-23). Because phosphoric acid is a tribasic acid, it can also form di- and triesters (Fig. III-24). Phosphotriesters are rarely found in nature, but diesters are extremely important, particularly as the fundamental linkage of the nucleic acid polymers, which are sequences of ri-bose (or deoxyribose) units linked by 3 —> 5 phos-phodiester bonds (see Fig. III-25). Like phosphoric acid, which has three dissociable protons (Fig. III-26), phosphomono- and phosphodiesters are acidic and typically ionize as shown in Fig. HI-27. Note the similarities between the pvalues for... 

Other Biological Phosphate Compounds. Elimination of water between phosphoric acid and certain other types of compounds results in formation of a variety of phosphate compounds that have properties that are different from simple phosphomonoesters. A phosphate ester of a monosaccharide in which phosphate is linked to the anomeric hydroxyl is called a phosphoacetal. An example is a-D-glucopyranosyl-1 -phosphate (glucose-1-phosphate) (Fig. III-28). A related group of compounds... 

However, the stereochemical results on enzymatic reactions have not led to identifying one of the four possibilities as the general mechanism in enzyme catalysis. First, formation of a metaphosphate intermediate (mechanism A) may not necessarily result in racemization since in the enzyme active site it may not be free to rotate before it is trapped by the acceptor. Racemization did not even occur in the chemical methanolysis of some phosphomonoesters under dissociative conditions (143). Therefore an observed inversion does not rule out pathway A. Second, the two in-line associative pathways B and C may not be distinguishable in enzyme catalysis and may both proceed with inversion. Lastly, stereochemical results can not differentiate between mechanism D and a double displacement mechanism in which each displacement occurs with inversion. 

Structure of the substrate and the reaction conditions determine the transition state for reaction with a particular nucleophile 104, 105). The extreme cases are generally described as the dissociative and associative substitution mechanisms. The fully dissociative mechanism entails the formation of monomeric metaphosphate monoanion as a discrete intermediate and was first formulated by F. H. Westheimer, who pioneered the physical organic chemistry of the hydrolysis of phosphate esters 106, 107). This mechanism is depicted in Eq. (40) and is possible only for phosphomonoesters with good leaving groups, examples of which are shown. 

A bell-shaped pH vs rate profile with a maximum rate at pH 7.8 was seen in NP hydrolysis by 1,3-phenyl-linked dimer 48. The phosphomonoester NP " prefers the two zinc(II) ions to be close, and hence two alternative mechanisms as shown in Scheme 11 (a or b) are proposed for the hydrolysis. The loss of catalytic activity at pH > 12 may be accounted for by the formation of a double OH"-bridged zinc(II)... 

Such specificity for the primary hydroxyl indicates that preparation of desired 3 -phosphomonoesters may be difficult. For example, diphenyl phosphorochloridate will not react with 2, 5 -bis-0-methoxytetrahydropyran-4-yl uridine in a pyridine solvent, at room temperature. The free 3 -hydroxyl is too hindered to react with this bulky phosphorylating agent. However, replacement of pyridine with a more potent nucleophilic catalyst, 5-chloro-1-methylimidazole will allow product formation to occur. Other phos-phorodichloridates have been examined for the purpose of synthesis of nucleoside monophosphates. Perhaps one which has found the most use for the preparation of nucleoside T-phosphates is 2,2,2-trichloroethyl-2-chlorophenyl-phosphorochloridate. While phosphorylation will occur in pyridine, use of 1-methylimidazole provides a quicker reaction rate. This is... 

Figure 36 Neighboring group-catalyzed formation of a nucleoside-phosphomonoester initiated...

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