Hydrolysis of fluorophosphates

It is found that the hydrolysis of fluorophosphate esters is also accelerated by transition metal ions and complexes. This would be an observation of little general interest, except for the fact that fluorophosphate esters form one of the more commonly encountered types of nerve gases (Fig. 4-51). The hydrolysis of fluorophosphate esters is increased dramatically in the presence of copper(n) and other transition metal complexes, and this sug-... 

The formation of copper(II) complexes with terpy has been investigated fairly intensively. The interaction is pH dependent, and numerous hydroxy, aqua, and polynuclear species are present in aqueous solution 94, 245,278). In general, an Eigen-Wilkins mechanism appears to be operative, although the kinetics are complicated by ligand-protonation equilibria 263,390,391). In acidic solution, 1 1 complexes predominate (567). A number of substituted terpyridine ligands have been evaluated as potential colorimetric reagents for copper 400). The adsorption behavior of copper(II)-terpy complexes at silica surfaces has been studied 499). Such complexes are reasonably active as catalysts for the hydrolysis of fluorophosphate esters 456). 

S]s lcb-type deprotonation of coordinated ammonia, followed by an intramolecular reaction generating coordinated bidentate amidophosphate. This can then react further with either Ir—N or Ir—O bond cleavage (Scheme 3). A series of copper(II) complexes of general formula [Cu(15)Cl2] has been shown to be effective in the hydrolysis of fluorophosphates. The complexes themselves are prepared in a metal-directed addition of ROH to 2-cyanopyridine. A very mild hydrolysis of coordinated trimethylphosphite to dimethylphosphonate is seen in the conversion of [ P(CH2CH2PPh2)3 Pt P(OMe)3 ] + to [ P(CH2CH2PPh2)3 Pt P(=0)(0Me)2 ]+ upon... 

This enzyme [EC 3.4.16.5] (also known as serine-type carboxypeptidase I, cathepsin A, carboxypeptidase Y, and lysosomal protective protein) is a member of the peptidase family SIO and catalyzes the hydrolysis of the peptide bond, with broad specificity, located at the C-terminus of a polypeptide. The pH optimum ranges from 4.5 to 6.0. The enzyme is irreversibly inhibited by diisopropyl fluorophosphate and is sensitive to thiolblocking reagents. 

The mammalian serine proteases have a common tertiary structure as well as a common function. The enzymes are so called because they have a uniquely reactive serine residue that reacts irreversibly with organophosphates such as diisopropyl fluorophosphate. The major pancreatic enzymes—trypsin, chymotrypsin, and elastase—are kinetically very similar, catalyzing the hydrolysis of peptides... 

The preceding experiments prove that there is an intermediate on the reaction pathway in each case, the measured rate constants for the formation and decay of the intermediate are at least as high as the value of kcat for the hydrolysis of the ester in the steady state. They do not, however, prove what the intermediate is. The evidence for covalent modification of Ser-195 of the enzyme stems from the early experiments on the irreversible inhibition of the enzyme by organo-phosphates such as diisopropyl fluorophosphate the inhibited protein was subjected to partial hydrolysis, and the peptide containing the phosphate ester was isolated and shown to be esterified on Ser-195.1516 The ultimate characterization of acylenzymes has come from x-ray diffraction studies of nonspecific acylenzymes at low pH, where they are stable (e.g., indolylacryloyl-chymotrypsin),17 and of specific acylenzymes at subzero temperatures and at low pH.18 When stable solutions of acylenzymes are restored to conditions under which they are unstable, they are found to react at the required rate. These experiments thus prove that the acylenzyme does occur on the reaction pathway. They do not rule out, however, the possibility that there are further intermediates. For example, they do not rule out an initial acylation on His-57 followed by rapid intramolecular transfer. Evidence concerning this and any other hypothetical intermediates must come from additional kinetic experiments and examination of the crystal structure of the enzyme. 

Alkaline hydrolysis of hexafluorophosphate (PFg ) occurs without accumulation of lower fluorophosphates. The rates have been examined between 160 and 190 °C, with lithium, sodium and potassium hydroxides. At unit ionic strength (nitrates) the rates were dependent on the nature of the cation (K" < Na" " ionic strength, behaviour is more predictable and the rate expression is... 

Wagner-Jauregg, T., Hackley Jr., B.E., Lies, T.A., Owens, O.O., and Proper, R. 1955. Model reactions of phosphoms-containing enzyme inactivators. IV. The catal)Tic activity of certain metal salts and chelates in the hydrolysis of diisopropyl fluorophosphate. Journal of the American Chemical Society, 77 922-929. 

Ligands closely related to orthophosphate esters include acetylphosphate, acetylphenylphosphate and fluorophosphate. Alkaline hydrolysis of [Co OP(0)20COMe (NH3)5] occurs exclusively at the carbonyl centre, and the acceleration provided by cobalt(III) four atoms removed is minimal (10 times, equation This is a good comparative example of phosphoryl versus acyl hydrolysis,... 

In the balance of this paper, we describe the application of our method of chemical modification to the generation of fluorohydrolases The most common substrate to measure fluorohydrolase activity is diiso-propylphosphorofluoridate (DFP). Although DFP is not found in nature, Mazur reported the enzymatic hydrolysis of this highly toxic organo-fluorophosphate by an enzyme isolated from hog kidney.Since then, diisopropyl phosphorofluoridate fluorohydrolase (DFPase, E.C. 3.8.2.1) has been extensively studied and well characterized. In addition, enzymes with organofluorophosphate hydrolyzing activity have been shown to be widely distributed phylogenetically and sources are known from bacteria, protozoa, invertebrates, and vertebrates. 

FIGURE 59.2 (A) Hydrolysis of paraoxon with aryldiaUcylphos-phatase (paraoxonase, EC 3.1.8.1) and (B) hydrolysis of diisopropyl-fluorophosphate (DFP) with diisopropylfluorophosphatase (DFPase) (EC 3.1.82). 

Phosphoms oxyfluoride is a colorless gas which is susceptible to hydrolysis. It can be formed by the reaction of PF with water, and it can undergo further hydrolysis to form a mixture of fluorophosphoric acids. It reacts with HF to form PF. It can be prepared by fluorination of phosphoms oxytrichloride using HF, AsF, or SbF. It can also be prepared by the reaction of calcium phosphate and ammonium fluoride (40), by the oxidization of PF with NO2CI (41) and NOCl (42) in the presence of ozone (43) by the thermal decomposition of strontium fluorophosphate hydrate (44) by thermal decomposition of CaPO F 2H20 (45) and reaction of SiF and P2O5 (46). 

Reaction of dibenzofuran, ferrocene, and aluminum powder in the presence of aluminum chloride with subsequent hydrolysis and precipitation of the hexa-fluorophosphate salt gives 34 [80JOM(186)265]. When ferrocene and aluminum chloride are in excess, dicationic complexes 35 result. The reaction leading to 36 was also reported [84JOM(260)105]. 

The serine proteases act by forming and hydrolyzing an ester on a serine residue. This was initially established using the nerve gas diisopropyl fluorophosphate, which inactivates serine proteases as well as acetylcholinesterase. It is a very potent inhibitor (it essentially binds in a 1 1 stoichiometry and thus can be used to titrate the active sites) and is extremely toxic in even low amounts. Careful acid or enzymatic hydrolysis (see Section 9.3.6.) of the inactivated enzyme yielded O-phosphoserine, and the serine was identified as residue 195 in the sequence. Chy-motrypsin acts on the compound cinnamoylimidazole, producing an acyl intermediate called cinnamoyl-enzyme which hydrolyzes slowly. This fact was exploited in an active-site titration (see Section 9.2.5.). Cinnamoyl-CT features a spectrum similar to that of the model compound O-cinnamoylserine, on denaturation of the enzyme in urea the spectrum was identical to that of O-acetylserine. Serine proteases act on both esters and amides. 

Subtilisin (1 % by weight) is added, and hydrolysis is allowed to proceed at 40°C for at least 6-8 hr (or overnight). The subtilisin is subsequently inhibited by incubation with 0.1 % (v/v) diisopropyl-fluorophosphate (DFP) for 1 hr at 40°C. In some instances it may be possible to omit the use of subtilisin after pepsin, or pronase or papain may be found to give better results. 

Fig. 14. Amount of hydrolysis given as moles of amino acid liberated (from ninhydrin color) per mole of mercuripapain (80). Molar ratio of enzyme (LAP) to substrate (MP) given as E/S on the basis that the molecular weight of LAP is 15 times that of MP. Extent of hydrolysis was determined after incubation at 40° for 24 hours at pH 8.5 0.1 in the presence of 0.001 M MgCL and 0.005 M Veronal or Tris buffer. Highly purified preparations of LAP were used after treatment with diisopropyl fluorophosphate.

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