Phosphate ester hydrolysis ligands

The last work pertaining to the discovery of new catalysts is perhaps the most novel approach to be reported thus far. In one of the earliest approaches taken toward catalyst development, Menger et al. (61) attempted to find catalysts for phosphate ester hydrolysis. A series of eight functionalized carboxylic acids were attached to polyallylamine in various combinations. Each of these polymers were then treated with one of three metals, Mg2+, Zn2+, or Fe3+. The different members of each library were identified by the relative percentages of each carboxylic acid attached to the polyamine. For example, one polymer possessed 15% Oct, 15% Imi, 15% Phe, and 5% Fe3+. There is no attempt to identify the location of the various carboxylic acids in a given polymer. This approach is novel since each system consists of an ensemble of different ligands with the carboxylic acids positioned in various locations. Each polymer within a given ratio of carboxylic acids consists of a combinatorial library of potential catalysts. 

Zinc complexes of the cyclen ([12]aneN4 = 1,4,7,10-tetraazacyclododecane) ligand have been extensively studied in terms of phosphate ester hydrolysis reactivity. For example, a proposed binuclear Zn(II) hydroxide complex of cyclen was reported to enhance the rate of hydrolysis of ethyl(2,4-dinitrophenyl)-methylphosphonate and diethyl(2,4-dinitrophenyl) phosphate.223,224 It should be noted that the nuclearity of the zinc-cyclen complex in solution was not conclusively identified in this work. 

The dependence of the activity of calcineurin on the redox state of the metal center highlights its importance for catalysis and provides clues to its function in that process. Site-directed mutagenesis studies of PPl, calcineurin, and bacteriophage X protein phosphatase have also provided insights regarding the roles of non-ligand active site residues. Furthermore, the contributions afforded by studies of synthetic model compounds which mimic features of metallophosphatase active sites provide important clues to possible catalytic mechanisms. Indeed many of these models exhibit impressive rate enhancements for phosphate and phosphonate ester hydrolysis [54-62]. In this section we discuss current models regarding the mechanism of phosphate ester hydrolysis by calcineurin and other metallophosphatases in consideration of these studies. 

Figure 17-11 Two-step mechanism for phosphate ester hydrolysis. In the first step, substrate binds to the Fe ion, thereby displacing the bond from the -hydroxo ligand to this metal ion. The resulting Fe -coordinated hydroxide serves as the nucleophile in the hydrolysis reaction. The resulting species is a phosphate bridge, the same species observed in x-rays studies of product-inhibited enzyme.

Fe Fe or Fe Zn forms, despite the 100-fold lower rate of ligand exchange for A1 Fe3+ i 4 Regardless of which mechanism is correct, the ability to replace Fe " " by Zn " " and vice versa, with full retention of activity, suggests that it is likely to be applicable to both the dinuclear iron site in the mammalian PAPs and the FeZn site in the plant PAPs. In all cases, the key feature appears to be the use of a trivalent-divalent dinuclear site for phosphate ester hydrolysis. 

For effective catalytic hydrolysis of lipophilic phosphate esters in aqueous solution, metallomicelles such as 3 [21], 4 [22], 5 [23], 6 [24], 7 [25], and 8 [26] have been designed (Scheme 3). These complexes are essentially metal ions chelated with ligands that are attached with lipophilic long alkyl chains. Some of these were used together with surfactants to make water-miscible solutions. 

Me2pyo[14]trieneN4 (CR) ligand (Fig. 6a) catalyzes the hydrolysis of the triester diphenyl 4-nitrophenyl phosphate in aqueous acetonitrile solution.221 This reaction is first-order in zinc complex and phosphate ester. On the basis of pH-rate studies, which revealed a kinetic pifa value of 8.7,40 the active zinc complex is proposed to be [(CR)Zn-OH]+. A hybrid mechanism in which the zinc center of [(CR)Zn-OH]+ serves to provide the hydroxide nucleophile, and also electrophilically activates the phosphoryl P O bond, is favored for this system. This type of bifunctional mechanism was proposed based on the fact that the second-order rate constant for the [(CR)Zn-OH]+-catalyzed reaction (2.8 x 10 1 M-1 s 1) is an order of magnitude larger than that of free hydroxide ion-catalyzed hydrolysis (2.8 x 10 2M 1 s 1). As OH- is a better nucleophile than the zinc-coordinated hydroxide, Lewis acid activation of the substrate is also operative in this system. 

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