Phosphorothioates

Hydrolytic decomposition of phosphorothioic acid and its salts yields phosphoric acid and hydrogen sulphide (in at least 79-86 % yield). The rates vary 

Using dissociation constants determined at 25 °C and the same ionic strength, 1 M, (pA i = 1.35, pK2 = 4.95, pKs = 9.8), the rate coefficients at 52.8 °C were determined as = 2.43 x 10 sec, 2 = 16.7 x 10 sec, A 3 = 3.81 x 10 sec, 4 = 1.56 X 10 sec If pA 3 had the value (10.3) found in dilute solution it would be sufficient to write k, = 0. No evidence for a further, acid-catalysed, reaction was found for acid concentrations to 7 M, apart from a change attributable to a salt effect. The relative reactivity of the dianion is large by com-parision with those of other phosphate derivatives. 

In this subclass of OPs, a sulfur atom is double-bonded to the phosphorus atom. Parathion is a typical phosphorothioate. It is an insecticide and acaricide with contact and stomach poison activity for control of foliar insects and mites. It has an oral LD50 in rats of 3.6-10 mg/kg. 

Structure 4.12 C2H5° i /= c2h5ox w Parathion O.O-Diethyl 0-/ -nitrophenyl Phosphorothioate 

Structure 4.13 s II /= ch3o — p—0——no2 och3 Methyl Parathion 0,0-Dimethyl 0-/ -nitrophenyl Phosphorothioate 

Chlorpyrifos is an insecticide with contact and stomach poison activity against a wide range of pests. It has an oral LD50 in rats of 96-270 mg/kg. 

With respect to stereochemistry, substitution of one unbonded oxygen atom of the proch-iral phosphorus linkage in an oligonucleotide by sulfur, nitrogen or borane creates a chiral center leading to diastereomeric mixtures of RP and SP compounds in synthesis. Since the number of diastereomers increases by 2 , this sums up to the formation of about half a million diastereomeric compounds during the synthesis of a 20-mer. Nevertheless, antisense activity remains intact in such diastereomeric mixtures. 

SODNs may be prepared without difficulty using the regular ODN phosphoramidite method. Several reagents used for sulfurization are shown in Table 5. 

Apart from TETD, Beaucage s reagent (3/7-1,2-benzodithiol-3-one-l,1-dioxide) [74] is used most frequently for sulfurization after coupling of the phosphoramidite. The latter is applied in 0.05 molar solution in anhydrous acetonitrile. Sulfurization proceeds within 0.5 min. 

With respect to the dominance of the phosphoramidite method, the formation of phosphorothioates by the phosphite [75] and phosphotriester methods [76] is not outlined here. As mentioned previously, a new chiral center is generated with each sulfurization step of phos- 

5 M solution in anhydrous acetonitrile Sulfurization time 15 min Sulfurization proceeds better in acetonitrile than in dichloromethane or THF. 

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Phosphoric Acid and Phosphorothioic Acid Anhydrides. The aUphatic organophosphoms esters originally developed by Schrader (27) are extremely toxic to mammals and are largely of historic interest. Tetraethyl pyrophosphate [107-49-3] (40) (bp 104—110°C at 10.7 Pa, d 1.185, vp 6.1 mPa at 30°C) is miscible with water and hydrolyzes very rapidly with a half-life of 6.8 h at 25°C. The rat LD qS ate 1.1 (oral) and 2.4 (dermal) mg/kg. 

Aliphatic Phosphorothioate Esters. Many of the eady developments of OP insecticides were simple esters of phosphorothioic acid, (H0)2P(0)SH, and phosphorodithioic acid, (HO)2P(S)SH. 

The rat LD qS are 13, 3.6 (oral) and 21, 6.8 (dermal) mg/kg. Parathion is resistant to aqueous hydrolysis, but is hydroly2ed by alkah to form the noninsecticidal diethjlphosphorothioic acid and -nitrophenol. The time required for 50% hydrolysis is 120 d ia a saturated aqueous solution, or 8 h ia a solution of lime water. At temperatures above 130°C, parathion slowly isomerizes to 0,%diethyl 0-(4-nitrophenyl) phosphorothioate [597-88-6] which is much less stable and less effective as an insecticide. Parathion is readily reduced, eg, by bacillus subtilis ia polluted water and ia the mammalian mmen to nontoxic 0,0-diethyl 0-(4-aminophenyl) phosphorothioate, and is oxidized with difficulty to the highly toxic paraoxon [511-45-5] diethyl 4-nitrophenyl phosphate d 1.268, soluble ia water to 2.4 mg/L), rat oral LD q 1.2 mg/kg. 

The hazards of human poisoning by the parathions have stimulated the development of safer analogues. Two chlorinated derivatives have gready reduced mammalian toxicides. Dicapthon [2463-84-5], 0,0-dimethyl 0-(2-chloro-4-nitrophenyl) phosphorothioate (63) (mp 53°C), has rat LD qS of 400, 330 (oral) and 790, 1250 (dermal) mg/kg. Chlorthion [500-20-8], 0,0-dimethyl 0-(3-chloro-4-nitrophenyl) phosphorothioate (64) (mp 21°C, <71.437), has rat LD qS of 890, 980 (oral) and 4500, 4100 (dermal) mg/kg. These compounds have been used as household insecticides. 

The reactivity of the individual O—P insecticides is determined by the magnitude of the electrophilic character of the phosphoms atom, the strength of the bond P—X, and the steric effects of the substituents. The electrophilic nature of the central P atom is determined by the relative positions of the shared electron pairs, between atoms bonded to phosphoms, and is a function of the relative electronegativities of the two atoms in each bond (P, 2.1 O, 3.5 S, 2.5 N, 3.0 and C, 2.5). Therefore, it is clear that in phosphate esters (P=0) the phosphoms is much more electrophilic and these are more reactive than phosphorothioate esters (P=S). The latter generally are so stable as to be relatively unreactive with AChE. They owe their biological activity to m vivo oxidation by a microsomal oxidase, a reaction that takes place in insect gut and fat body tissues and in the mammalian Hver. A typical example is the oxidation of parathion (61) to paraoxon [311-45-5] (110). 

Phosphorothioates. All three synthetic approaches appHcable to unmodified oligonucleotides can be adapted for synthesis of phosphorothioates (11) (33,46). If all of the phosphodiester linkages in an oligonucleotide are to be replaced with phosphorothioates, the ff-phosphonate method for coupling, followed by oxidation with Sg in carbon disulfide and triethylamine in the final step, is the most straightforward method. 

Chirahty at the phosphoms is an unavoidable problem in all phosphorothioate syntheses. The phosphoramidite method produces a mixture of both the and the diastereomers having a small excess of the isomer (53). Although some progress has been made in the chiral synthesis of dinucleoside phosphorothioates, low yields have limited the utility of these approaches. The chiral center may be eliminated by replacing the other, nonbridging oxygen with sulfur. Avoidance of the chirahty problem is one reason for the interest in phosphorodithioates. 

Phosphorothioates generally protect normal tissues more than tumors. Tumor protection reported in some animal studies can pardy be explained by physiological effects of the particular dmgs, which are specific to rodents (4). WR-2721 does not appear to protect human and most animal tumors, apparentiy because of the low availabiUty of the dmg to tumor cells (4). Many tumors appear to have a reduced capillary density (44), which may mean that these tumors have altered levels of alkaline phosphatase, the enzyme that converts WR-2721 to WR-1065. A reduced abiUty of thiols to protect the hypoxic cells characteristic of many tumors may also contribute to their selectivity for normal tissues. The observation that WR-1065 protects cultured normal human fibroblasts, but not fibrosarcoma tumor cells, suggests that additional factors may contribute to the selectivity of radioprotection by WR-2721 m vivo (18). 

Diethyl 0-(3-methyl-5-pyrazolyl) phosphate (722) and 0,0-diethyl 0-(3-methyl-5-pyrazolyl) phosphorothioate (723) were prepared in 1956 by Geigy and they act, as do all organophosphates in both insects and mammals, by irreversible inhibition of acetylcholinesterase in the cholinergic synapses. Interaction of acetylcholine with the postsyn-aptic receptor is therefore greatly potentiated. 0-Ethyl-5-n-propyl-0-(l-substituted pyrazol-4-yl)(thiono)thiolphosphoric acid esters have been patented as pesticides (82USP4315008). 

Phosphorothioic acid, o, o-dlethyl-o- 4-nitrophenyl) ester) Pentachlorophenol (PCP)... 

Phosphorothioate 0,0-DiethylO-2-Isopropyl-4-Methyl-6-Pyrimidyl Thiophosphate Diethyl 2-Isopropyl-4-Methyl 6-Pyrimidyl Thionophosphate Alpha-Tox Saralex Spectracide Cherrucal Formula C]2H2iNj03PS. 

These groups, along with a number of other trialkylsilylethyl derivatives, were examined for protection of phosphorothioates. Only the phenyl-substituted silyl derivative was useful, because simple trialkylsilyl derivatives were prone to acid-catalyzed thiono-thiolo rearrangement. Other trialkylsilylethyl derivatives also suffer from inherent instability upon storage,but the trimethylsilylethyl group has been used successfully in the synthesis of the very sensitive agrocin 84 and for intemucleotide phosphate protection with the phosphoramidite approach. 

Na, ammonia. These conditions also remove cyanoethyl- and benzyl-protective groups. Phosphorothioates are similarly deprotected. 

Cleavage of an 5-2-nitrobenzyl phosphorothioate is achieved with thiophen-... 

From a phosphorothioate TFMSA, m-cresol, thiophenol, TFA. These conditions minimized the migration of the benzyl group to the thione. ... 

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