Fluorophosphonate

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

A competitive version of ABPP identifies the target(s) and assesses the selectivity of an enzyme inhibitor in biological systems by gauging how well the inhibitor slows the enzyme s reaction with an ABP. For example, fluorophosphonate ABP 3 was used to profile the selectivity of fatty acid amide hydrolase (FAAH) inhibitors within the serine hydrolase superfamily [27] (FAAH hydrolyzes endocannabinoids such as anandamide). Serine hydrolases that exhibited reduced labeling by the probe in the presence of inhibitor were scored as targets of the inhibitor. Urea FAAH inhibitors exemplified by PF-3845 (5) that covalently modify the active-site serine nucleophile of FAAH were found to be exquisitely selective for FAAH in brain and liver... 

Cook, H. G., B. C. Saunders, and F. E. Smith. "Esters Containing Phosphorus. VIII. Structural Requirements for High Toxicity and Miotic Action of Esters of Fluorophosphonic Acid." Journal of the Chemical Society, Abstracts (1949) 635-638. 

From (IV), chlorosulphonic acid (V) and benzene sulphonic acid (VI) are obtained by the replacement of H by Cl and C6H5 respectively Arguing along similar lines, (VIII) is phosphonic acid and (IX) is fluorophosphonic acid, hence (I) is diethyl chlorophosphonate. Th ... 

Phosphoric acid Phosphonic acid Fluorophosphonic acid... 

Now 4 mol. are required to effect complete hydrolysis to sodium orthophosphate according to equation (c), but only 2 mol. are required either for the removal of fluorine alone and conversion into sodium di-isopropyl phosphate (d) or for conversion into disodium fluorophosphonate (e). To decide between these reactions, half of the above hydrolysis product was rendered acid to bromophenol Hue... 

Dixon and Mackworth, Report no. 13 to Ministry of Supply, Mode of action of fluorophosphonate esters , April 1942 Dixon and Webb, Report no. 27 to Ministry of Supply, May 1944. 

As we have seen (pp. 50 et seq.) the reaction between phosphorus oxydichlorofluoride and alcohols, phenols and thiols, affords dialkyl, dicyctoalkyl, diaryl phosphorofluoridates and dialkyl phosphorodithiolates. In a Report1 to the Ministry of Supply on fluorophosphonates a description was given of a new type of nitrogen fluorophosphonate formed by the action of 4 mol. of aniline on 1 mol. of phosphorus oxydichlorofluoride, the fluorine atom being unaffected ... 

In Report no. 16 on fluorophosphonates to the Ministry of Supply,3 we gave an account of a very useful alternative method of preparation. The method consisted in treating an amine with the calculated quantity of phosphorus oxychloride in ethereal solution P0C13+4NH2.R = P0C1(NH.R)2 + 2.R-NH3C1. (a)... 

Report no. 15 on fluorophosphonates to Ministry of Supply, by McCombie and Saunders, 9 December 1943. 

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. 

There are several such toxic agents that cause considerable medical, public and political concern. Two examples are discussed here the heavy metal ions (e.g. lead, mercury, copper, cadmium) and the fluorophosphonates. Heavy metal ions readily form complexes with organic compounds which are lipid soluble so that they readily enter cells, where the ions bind to amino acid groups in the active site of enzymes. These two types of inhibitors are discussed in Boxes 3.5 and 3.6. There is also concern that some chemicals in the environment, (e.g. those found in industrial effluents, rubbish tips and agricultural sprays), although present at very low levels, can react with enhanced reactivity groups in enzymes. Consequently, only minute amounts concentrations are effective inhibitors and therefore can be toxic. It is suggested that they are responsible for some non-specific or even specific diseases (e.g. breast tumours). 

Symptoms of fluorophosphonate poisoning are difficulty in focusing the eye for near vision and difficulty in breathing, since the poison causes sustained contraction of the smooth muscles of the respiratory tract, which causes death by asphyxiation. 

Insects are very sensitive to fluorophosphonates, so that the compound parathion was synthesised and used as an insecticide soon after the Second World War. However, it entered the food chain and eventually found its way into mammals and caused death. An important breakthrough occurred with the synthesis of malathion, an insecticide which has high toxicity to insects, where it is converted to malaoxon, a potent acetylcholinesterase inhibitor. However, malathion is much less toxic to mammals, since it is readily detoxified (Appendix 3.8). 

Treatment of fluorophosphonate poisoning involves the use of oximes, such as pralidoxime, which bind the phos-phoryl group very strongly so that it is removed from the enzyme. 

S.G. Withers, i.P. Street, M.D. Percivai, Fiuorinated carbohydrates as probes of enzyme specificity and mechanism, ACS Symposium Series 374 (1988) 59-77. W.G. Stirtan, S.G. Withers, Phosphonate and aipha-fluorophosphonate analogue probes of the ionization state of pyridoxai 5 -phosphate (PLP) in glycogen phosphor-ylase. Biochemistry 35 (1996) 15057-15064. 

Fluorophosphonates, fluoro-C-glycosides and difluorodisaccharides can be used as non-hydrolisable and stable mimics of phosphates, sulfates, disaccharides, where the anomeric oxygen atom is replaced by a CHF or a CF2 group. 

Ethyl fluorophosphonate is commercially available it is prepared through a Michael-Arbuzov reaction between triethyl phosphite and ethyl bromofluoroacetate. 

Reaction of a fluorophosphonate with an aldehyde that provides F-a-fluoro-a, jS-esters. 

The radicals generated from esters of halogenodifluoroacetic acid or halogenodi-fluorophosphonic acid add onto olefins and enolates. When these reactions are intramolecular, they afford tetrahydrofurans. In the presence of copper dust, ethyl bromodifluoroacetate can couple with aromatic and vinyl halides or can add onto Michael acceptors (Figure 2.13). ... 

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