Phosphorofluoridate activity

The esters of monofluorophosphoric acid are of great interest because of their cholinesterase inhibiting activity which causes them to be highly toxic nerve gases and also gives them medical activity (see Enzyme inhibitors). The most studied is the bis(l-methylethyl)ester of phosphorofluoridic acid also known as diisopropyl phosphorofluoridate [155-91 DFP (5), and as the ophthalmic ointment or solution Isoflurophate USP. It is used as a... 

DeFrank JJ, WE White (2002) Phosphorofluoridates biological activity and biodegradation. Handbook Environ Chem 3N 295-343. 

Brief notes are added on phosphorofluoridates even though their destruction by microbial activity— though clearly possible—is limited by their toxicity to the requisite microorganisms. One of the motivations for their inclusion is the fact that the hydrolytic enzyme(s) responsible for defluorination—organophosphorus acid anhydrase (OPA)—is widespread, and is found in a number of bacteria (Landis and DeFrank 1990). The microbial hydrolysis of organophosphorus pesticides and cholinesterase inhibitors is accomplished by several distinct enzymes, which are collectively termed organophosphorus acid anhydrases (OPAs). These have been reviewed (DeFrank 1991), so that only a few additional comments are necessary. 

Concurrently with experiments on animals, the action of the phosphorofluoridates on enzymes was investigated in Cambridge.3 It was shown in 1942 that esters of phosphorofluoridic acid inhibit4 the action of the enzyme cholinesterase, which is present in tissue fluids and hydrolyses acetylcholine to the much less active choline. 

To test this possibility, they first examined the effect of phosphorofluoridates on isolated rabbit s intestine. On such a preparation the action of drugs, like acetylcholine, which act directly on the muscle differs characteristically from the action of those, like eserine, which act by inhibition of cholinesterase activity. The directly acting drugs produce an immediate contraction which proceeds rapidly to a maximum, and after the drug has been washed out the muscle again quickly relaxes. The contraction produced by cholinesterase-inhibiting drugs, such as... 

The cholinesterase-inhibiting activity of the phosphorofluoridates was compared quantitatively with that of eserine sulphate thus. To 0-2 ml. of heparinized human plasma was added 05 ml. of a solution containing either eserine or the phosphorofluoridate in varying concentrations then the mixture was kept at room temperature for 10 min. before 1 /tg. of acetylcholine in 1 c.c. saline solution was added. After 5 min. at room temperature, the mixture was made up to 10 ml. with frog saline containing eserine 1/100,000, which at once stopped the action of any cholinesterase not yet inactivated. The solution was then assayed for acetylcholine on the frog rectus-muscle preparation. 

Reversibility. It is known that the effect of eserine on cholinesterase can be completely reversed by prolonged dialysis against water. On the other hand, it proved impossible to obtain any reversal of the poisoning by the phosphorofluoridate esters (see table below). The enzyme solution (5 ml.) was treated with the inhibitor for 15 min. at 38° 1 ml. was used at once for activity estimation, and the remainder dialysed against running water for 24 hr. in the case of the eserine experiment, 36 hr. in the others. It was clear that the combination between the phosphorofluoridate esters and the enzyme is much firmer than that between eserine and the enzyme. 

Effect of dialysis against water on activity of cholinesterase poisoned with eserine and phosphorofluoridate... 

Fig. 13. Effect of substrate concentration on inhibition of horse-serum cholinesterase.1 Enzyme activity was estimated by titration with 0-01 n NaOH at pH 7-4 and 20°. — , control, no inhibitor x — x, 2x 10 7 m eserine 0—O, 5 x 10 8 m di-isopropyl phosphorofluoridate.

True and pseudo-cholinesterase. The above serum preparations contained both the true and pseudo- cholinesterases of Mendel and Rudney.1 The effect of di-isopropyl phosphorofluoridate on these components was examined separately by means of the specific substrates described by Mendel, Mundel and Rudney,2 using the titration method described above. Phosphorofluoridate (5 x 10 8m) gave an inhibition of 57 per cent of the activity towards 00045m acetylcholine, 30 per cent of the activity towards 0-0005 m acetyl-/ methyl-choline, and 40 per cent of that towards 0-005 m benzoylcholine, after incubating the enzyme with the poison for 5 min. Thus in these experiments there appeared to be no appreciable difference in sensitivity of the true and pseudo-cholinesterases of horse serum to phosphorofluoridates. 

The active trichloride was converted into di-isopropyl hydrogen phosphite and thence through the phosphorochloridate into the phosphorofluoridate essentially according to the scheme... 

Results. Phosphorus (1 g.) and phosphorus trichloride (1 ml.) were used. The weight of di-isopropyl phosphorofluoridate obtained was 5-5 g. (62 per cent). The purity, determined by fluorine analysis1 on 50 mg. of the active product, was 98-5 per cent. 

The radioactive phosphorus used had an activity of 28,000 counts/min./mg. (counter efficiency, ca. 1 per cent, i.e. specific activity ca. 1 mc./g.), whilst the radioactive di-isopropyl phosphorofluoridate had an activity of 2200 counts/min./mg. (corrected to zero time). 

It was suggested,1 on the basis of kinetic measurements, that the phosphorofluoridates inhibit esterases by virtue of a highly specific affinity for the active centres of this group of enzymes. Preliminary experiments by Boursnell and Webb2 with diisopropyl phosphorofluoridate containing 32P gave results which were in accordance with this view. 

Protein Final molarity of active phosphorofluoridate Time of reaction at 18° C. Radiophosphorus (g.atoms) fixed per 10s g. protein... 

We found that the toxicity and myotic activity of di-isopropyl phosphorofluoridate (XI) were far greater than that of di- propyl phosphorofluoridate. In Report no. 6 on fluoro-phosphonates to the Ministry of Supply3 we described the preparation of di aec.-butyl phosphorofluoridate (XII) by the hydrogen phosphite method (p. 6). The compound was found to be very toxic and to produce severe myosis in man and animals. The symptoms displayed during and after exposure were identical with those produced by di-isopropyl phosphorofluoridate. The L.c. 50 for di-sec.-butyl phosphorofluoridate for mice for deaths within 2 hr. was 0-6 mg./l., and that for deaths within 48 hr. was 0-54 mg./l. 

It was important to determine whether the fluorine atom must be attached directly to the phosphorus atom in order to produce phosphorofluoridate-like activity. For this purpose we treated the toxic di-sec.-butyl phosphorofluoridate1 with diazomethane and obtained a compound which was undoubtedly di- ec.-butyl fluoromethylphosphonate (XIX). Unlike the parent phosphorofluoridate, the fluoromethylphosphonate was only slightly toxic and produced negligible myosis. (It may be mentioned here that thionyl chloride and carbonyl chloride were converted by means of diazomethane into bis-(chloromethyl)-sulphoxide and S-dichloroacetone respectively.)... 

It was obviously of interest to determine whether other esters of fluoroacetic acid would prove to be more or less toxic than the methyl ester. In the phosphorofluoridate series, for example, we found that esters of secondary alcohols were far more potent than those of primary alcohols for instance, di-isopropyl fluorophosphonate (I) was a compound of considerable activity. Accordingly ethyl, ra-propyl and isopropyl fluoroacetates were prepared by heating the corresponding esters of chloroacetic acid in the rotating autoclave with potassium fluoride. The toxicity figures of these esters were very similar to those of methyl fluoroacetate. 

Combination of fluoroacetate activity and certain other recognizable physiological effects have been successfully combined in fluoroaspirin (drugged sleep), triethyl-lead fluoroacetate (sternutation), difluoroethyl phosphorofluoridate (myosis, but not powerful). In general, quaternary ammonium... 

It has been reported4 that cholinesterase inhibitors (such as di-isopropyl phosphorofluoridate) increase the permeability of squid giant axons towards sodium and potassium. There is also an indication that the erythrocyte requires, among other factors, an adequate acetylcholine-cholinesterase system to prevent a gain of sodium or a loss of potassium.5 The conclusion that permeability is dependent on cholinesterase activity, however, seems to be contested by Strickland and Thompson.6... 

The mechanism by which A-esterases hydrolyze organophosphates is not completely understood. Involvement of a phosphorylated active-site cysteine and displacement of an activated H20 molecule are two possible hypotheses (see Sect. 3.7.1) [56], A-Esterases comprise enzymes that hydrolyze aryl esters, paraoxon (2.2) and related organophosphate pesticides, and diisopropyl-fluorophosphate (DFP, diisopropyl phosphorofluoridate, 2.3) and related compounds, including nerve gases. These enzymes are found in the current nomenclature listed under arylesterases, aryldialkylphosphatase, and diisop-ropyl-fluorophosphatase. 

Other serine hydrolases such as cholinesterases, carboxylesterases, lipases, and fl-lactamases of classes A, C, and D have a hydrolytic mechanism similar to that of serine peptidases [25-27], The catalytic mechanism also involves an acylation and a deacylation step at a serine residue in the active center (see Fig. 3.3). All serine hydrolases have in common that they are inhibited by covalent attachment of diisopropyl phosphorofluoridate (3.2) to the catalytic serine residue. The catalytic site of esterases and lipases has been less extensively investigated than that of serine peptidases, but much evidence has accumulated that they also contain a catalytic triad composed of serine, histidine, and aspartate or glutamate (Table 3.1). 

The syntheses of dithymidylyl-3, 5 -phosphorofluoridate and phosphorothiof-luoridate (87) and (88) have been described. These involved the fluorinolysis of the P-Se bond in the bis-dimethoxytrityl selenomethyl esters (89) and (90). Compounds (87) and (88) were reported to be hydrolytically unstable, with no inhibitory activity on the snake venom and spleen phosphodiesterases and alkaline phosphatases. Finally, neither was considered as a highly toxic dinuc-leotide. ... 

Murachi T (1963). A general reaction of diisopropyl phosphorofluoridate with proteins without direct effect on enzymic activities. Biochim BiophysActa, 71, 239-241. 

However, Borden and Smithi have found that treatment of the p-nitro-phenyl esters of 2 -deoxyribonucleoside 3 - and 5 -phosphates and of ribo-nucleoside 5 -phosphates with potassium fert-butoxide in methyl sulfoxide affords the corresponding nucleoside 3 5 -cyclic phosphates in excellent yields. Other phosphorus-activated nucleotides, such as nucleoside 5 -phosphorofluoridates, 5 -(2,4-dinitrophenyl phosphate)s and 5 -(PS-P " diphenyl pyrophosphate) s, were also cyclized to nucleoside 3 5 -cyclic... 

---