Hydrolysis of phosphotriester

The hope that promiscuity is predictable is also supported by the identification of systematic patterns of promiscuity. For example, lactonases, and in particular lactonases that favor hydrophobic lactones, show a consistent tendency to promiscuously catalyze the hydrolysis of phosphotriesters. This pattern has now been seen in lactonases from three different superfamilies PLLs (TIM-barrels from the amidohydrolase superfamily Table 1, entry 11) PONs, or serum paraoxonases (calcium-dependent six-bladded /3-propellers Table 1, entry 13) and AHA (a lactonase from the metallo-/3-lactamase superfamily Table 1, entry 12). That very different scaffolds and active-sites configurations share the same promiscuity pattern suggests that these reactions share a key feature, probably in the geometry of their transition states. This feature must be distinct, also because many of these lactonases do not hydrolyze esters that are much closer to lactones than phosphotriesters, and should thus be amenable to structural analysis and prediction. 

A zinc(II) complex 22a with an alcohol-pendent polyamine has been synthesized (23). The alcoholic OH deprotonates with pifa of 8.6 (determined by pH-metric titration), yielding 22b. Reaction of 22 (2 mM) with a phosphotriester diethyl(4-nitrophenyl) phosphate (0.1 mM) in 10 mM TAPS buffer (pH 8.6) at 25°C seemed to promote phosphoryl-transfer reactions to 23, just like acyltransferred intermediates 10 and 16a in the reactions between Znn-macrocyclic complexes with an alcohol pendent and NA (see Scheme 4). The pH dependence of the first-order rate constants gave a sigmoidal curve with an inflection point around the pKa value of 8.6. The hydrolysis of the substrate phosphotriester to the phosphodiester product diethyl phosphate thus seemed to... 

Increasing the substrate range of an enzyme could be thought of as improving a promiscuous activity. However, here we will use a more strict definition in which a promiscuous activity involves catalysis of a reaction with a different class of substrate. Many enzymes are promiscuous in the sense that they can catalyze other reactions. This is not surprising considering that the enormous variety of enzymes that exist utilize only a small number of active site chemistries and structural scaffolds. Thus, an almost identical enzyme could catalyze lactone hydrolysis or phosphotriester hydrolysis. Because these activities are often very weak to begin with, directed molecular evolution experiments to improve these activities often result in remarkable improvements. 

Perigaud has extended his work on the synthesis and biological activities of phosphotriester derivatives of AZT bearing a phenyl group or L-tyrosinyl residues. The novel AZT-derivatives (21a-d) also incorporating one S-pivaloyl-2-thioethyl residue were obtained via either P(III) or P(V) chemistry from the appropriate aryl precursors and evaluated for their in vitro anti-HIV activity. ECso values for their ability to inhibit HIV-1 replication in various cell culture experiments ranged between micro- and the nanomolar concentrations. S-Pivaloyl-2-thioethyl aryl phosphotriester derivatives of AZT were able to allow the efficient intracellular delivery of the parent nucleotide via their successive intracellular hydrolysis by an esterase and a phosphodiesterase. ... 

Mononuclear zinc complexes of A, A /,A ///-tris(3-aminopropyl)amine ligands having either hydrogen atoms or methyl groups as the substituents on the coordinating nitrogen atoms exhibit slight catalytic activity for the hydrolysis of the phosphotriester 2,4-dinitrophenyl diethyl phosphate.233 A zinc hydroxide complex is proposed as the reactive species that promotes phosphotriester hydrolysis. 

Associative mechanisms may include the formation of discrete trigonal bi-pyramidal addition intermediates. In the case of the hydrolysis of five-membered ring cyclic phosphodiesters and phosphotriesters, the pentavalent adducts certainly are intermediates (61). The five-membered ring cyclic esters undergo hydrolysis in acid or base much faster than noncyclic esters such as dibenzyl phosphate because the five-membered rings are strained, and this strain is relieved by the formation of trigonal bipyramidal adducts. An example of the intermediate formation of a pentavalent intermediate is the alkaline hydrolysis of ethylene phosphate shown in reaction (41). 

PLLs (PTE-like lactonases) Hydrolysis of quorum sensing Phosphotriester hydrolysis EC 3.1.8.1. Catalytic promiscuity 88... 

Partial hydrolysis of the meso-oxyphosphorane obtained when butanedione reacts with the 1,3,2-dioxaphospholene (364) gave two cyclic phosphotriesters [e.g. (365)] that differ in the configuration at the phosphorus atom. Further... 

Cleavage of the trityl ether from the Manlll with formic acid followed by the phos-phitylation with phosphoramidite 25 and meto-chloroperoxybenzoic acid (m-CPBA) oxidation at —40°C gave phosphotriester 39 as a 1 1 mixture of diastereomers. Upon removing the cyclohexanone ketal from the inositol, the corresponding diol was converted into an orthoester using the trimethyl orthoester of myristic acid. Hydrolysis of the orthoester in the presence of Yb(OTf)3 installed the myristate ester... 

The mechanism of phosphate ester hydrolysis by hydroxide is shown in Figure 1 for a phosphodiester substrate. A SN2 mechanism with a trigonal-bipyramidal transition state is generally accepted for the uncatalyzed cleavage of phosphodiesters and phosphotriesters by nucleophilic attack at phosphorus. In uncatalyzed phosphate monoester hydrolysis, a SN1 mechanism with formation of a (POj) intermediate competes with the SN2 mechanism. For alkyl phosphates, nucleophilic attack at the carbon atom is also relevant. In contrast, all enzymatic cleavage reactions of mono-, di-, and triesters seem to follow an SN2... 

Various esterases exist in mammalian tissues, hydrolyzing different types of esters. They have been classified as type A, B, or C on the basis of activity toward phosphate triesters. A-esterases, which include arylesterases, are not inhibited by phosphotriesters and will metabolize them by hydrolysis. Paraoxonase is a type A esterase (an organophosphatase). B-esterases are inhibited by paraoxon and have a serine group in the active site (see chap. 7). Within this group are carboxylesterases, cholinesterases, and arylamidases. C-esterases are also not inhibited by paraoxon, and the preferred substrates are acetyl esters, hence these are acetylesterases. Carboxythioesters are also hydrolyzed by esterases. Other enzymes such as trypsin and chymotrypsin may also hydrolyze certain carboxyl esters. 

Treatment of 2,6-f>w(bromomethyl)pyridine with 2-(methylamino)ethanol afforded 2,6-6is[(2-hydroxyethyl)methylaminomethyl]pyridine, which with 4-nitrophenylphosphoro dichloride under high dilution conditions gave rise to the desired cyclic phosphotriester 24 divalent metal ions dramatically enhance the rate of hydrolysis [95TL4031],... 

Phosphorus oxychloride is a suitable reagent for preparation of the symmetrically substituted phospho-triesters of type (RO)3PO. The preparation is easily achieved by treatment of phosphorus oxychloride with 3 equiv. of alcohols or their metal salts. The reaction is generally promoted by a base or acid. Titanium trichloride is a particularly effective catalyst for the reaction. Conversion of POCI3 to unsymmetri-cally substituted phosphotriesters is achievable with difficulty. Phosphorochloridates and phosphorodichloridates have been used for the preparation of mixed tertiary phosphoric esters of type (ROlmPOfOROn (ffi = 1, n = 2, or m = 2, n = 1) in a very wide variety. Reaction of phosphorus oxychloride and 1 or 2 equiv. of alcohols followed by hydrolysis forms phosphomonoesters or phosphodi-esters, respectively. The hydrolysis may be generally effected by dilute aqueous alkali. Some phosphoFodichlori te intermediates are easily hydrolyzed by water. For example, the phosphorylation of a ribonucleoside (1 equation 4) with phosphorus oxychloride in an aqueous pyridine-acetonitrile mixture furnishes the nucleoside S -monophosphate (2) in excellent yield. ... 

At this pH, the phosphotriesters formed mainly lose alcohol to re-form the phosphodiester, but at higher pH, hydrolysis with chain scission takes place. Alkylation also proceeds at nucleophilic centres on the base and sugar moieties, and the modification likely to cause most damage in vivo has not been defined. Studies involving aflatoxin Bi and benzanthracene derivatives have tended to concentrate on the modification of the bases, to the exclusion of possible effects at the phosphate group, but there is good evidence that the latter should not be ignored. ... 

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