New series of mixed pronucleotides. Synthesis and anti-HIV activities of mononucleoside phenyl SATE phosphotriesters.

The synthesis and anti-HIV activities of phenyl S-pivaloyl-2-thioethyl (tBuSATE) phosphotriesters of AZT and d4T are reported. These compounds show similar activity compared to bis(tBuSATE) phosphotriesters and appear to be able to deliver the corresponding 5'-mononucleotides inside the cells.     

Studies on the stability of trialkyl phosphates and di-(2'deoxythymidine) phosphotriesters in alkaline and neutral solution. A model study for hydrolysis of phosphotriesters in DNA and on the influence of a beta hydroxyethyl ester group

Various trialkyl phosphates were investigated as model compounds for DNA-phosphotriesters for their stability in neutral or alkaline conditions. The results show that phosphotriesters were highly stable even at strongly alkaline pH, with the exception of diethyl 2-hydroxyethyl phosphate (DHP). The extreme instability of the latter was found to be due to the 2-hydroxy function. In accordance with earlier interpretations the 2-hydroxyethyl group is proposed to participate in the formation of a highly reactive dioxaphospholane ring intermediate which decays rapidly by hydrolysis. Alkylation of 3'- and 5'-deoxythymidine monophosphates with methyl- or hydroxyethylnitrosourea (MNU, HENU) results in practically exclusive phosphate alkylation. In analogy with the model phosphotriesters, di(2'-deoxythymidine) phosphotriesters generated after reaction with MNU or HENU showed extreme dependence of their stabilities on the nature of the alkyl group transferred to phosphate. Whereas the methyl phosphotriester was highly stable, the corresponding hydroxyethyl analogue showed half lives of decay of less than 1 min (pH 12.5), 27 min (pH 9.1) and 60 min (pH 7). Thus the introduction of a 2-hydroxyethyl function into phosphate strongly decreases the stability of the phosphate link of DNA, resulting in DNA single strand breaks, in analogy to RNA phosphotriesters which have been found earlier to be highly unstable because of the presence of the ribose 2'-OH-group.     

New Series of Mixed Pronucleotides. Synthesis and Anti-HIV Activities of Mononucleoside Phenyl SATE Phosphotriesters

Abstract

The synthesis and anti-HIV activities of phenyl S-pivaloyl-2-thioethyl (tBuSATE) phosphotriesters of AZT and d4T are reported. These compounds show similar activity compared to bis(tBuSATE) phosphotriesters and appear to be able to deliver the corresponding 5′-mononucleotides inside the cells.     

Synthesis,Decomposition Pathways and “In Vitro” Evaluation of Bioreversible Phosphotriesters of Azt

Abstract

The synthesis, pharmacokinetic data and biological evaluation of a series of phosphotriesters containing S-acyl-2-thioethyl groups as enzyme-labile phosphate protecting groups and AZT as a model are described. A comparison of pharmacokinetic data and “in vitro” experiments show that such bioreversible phosphotriesters of AZT are able to cross cell membranes and deliver the corresponding nucleoside monophosphate inside the cell. Moreover, kinetic data show that modification of the protecting groups can allow to modulate both the extracellular stability of the parent compond and the delivery of nucleoside monophosphate inside the cell.     

Aryl nucleoside H-phosphonates. Part 16: Synthesis and anti-HIV-1 activity of di-aryl nucleoside phosphotriesters

Di-aryl nucleoside phosphotriesters have been explored as a new type of pronucleotides for the purpose of anti-HIV-1 therapy and efficient synthetic protocols, based on H-phosphonate chemistry, have been developed for the preparation of this class of compounds. It was found that anti-HIV-1 activity of the phosphotriesters bearing an antiviral nucleoside moiety (AZT, ddA) and also ddU was due, at least partially, to intracellular conversion into the corresponding nucleoside 5′-monophosphates, and their efficiency correlated well with the pK_a values of the aryloxy groups present.     

Synthesis and Anti-HIV Activity of Alkyl Steroidal 3′-Azido-3′-deoxythymidin-5′-yl Phosphotriesters as Prodrugs of AZT

Abstract

Alkyl steroidal AZT 5′-monophosphate triesters are designed as lipophilic prodrugs of AZT to improve its therapeutic efficiency. We have synthesized four phosphotriesters of AZT, in one-pot, using phosphoramidite-phosphite triester methodology. This method afforded the desired prodrugs in high yields under mild conditions. The in vitro evaluation of anti-HIV activity of these prodrugs is also reported.     

The kinetics of the alkaline hydrolysis of phosphotriesters in DNA.

The degradation in alkali of normal DNA and DNA alkylated with dimethyl sulphate (DMS), N-methyl-N-nitrosourea (MNUA) and N-ethyl-N-nitrosourea (ENUA) has been investigated using analytical ultracentrifugation techniques. For control T7-DNA (w.st. denatured form 12.5 - 10(6) daltons) the rate of degradation at 37 degrees varies from 0.14 breaks/molecule/h in 0.1 M NaOH to 1.2 breaks/molecule/h in 0.4 M NaOH. When DNA is alkylated with reagents known to produce phosphotriesters addition of alkali leads to an initial rapid degradation not observed with control DNA. Ethyl phosphotriesters are hydrolysed at about half the rate of methyl phosphotriesters. Approximately one third of the methyl or ethyl phosphotriesters present hydrolyse to give breaks in the DNA chain.     

Phosphotriesters in rat liver deoxyribonucleic acid after the administration of the carcinogen NN-dimethylnitrosamine in vivo.

After treatment with NN-di[14C]methylnitrosamine, samples of DNA were isolated from rat livers by a conventional phenol procedure and examined for the presence of phosphotriesters. A method of capable of detecting relatively small amounts of 14C-labelled phosphotriesters was developed and used to establish that these products account for 10-12% of the total methylation pattern found after treatment with this agent in vitro. The significance of the presence of phosphotriesters in DNA is discussed.     

Synthesis and properties of some cyclic AMP alkyl phosphotriesters

Cyclic AMP was converted to its phosphotriesters according to the classical approach of phosphate activation with a sulfonyl chloride, followed by esterification with an alcohol. The methyl, ethyl, propyl, butyl and cetyl triesters were prepared, and some of their physical-chemical properties determined. Alkaline hydrolysis of these alkyl phosphotriesters resulted predominantly in ring opening. On the other hand, nucleophilic attack by thiourea led to the formation of cAMP as the main product. The conclusion can be drawn from these results that cAMP phosphotriesters could serve as suitable storage forms of cAMP, and cyclic triesters may be the best vehicle of transporting nucleotides through biological membranes.     

An assay for phosphotriester formation in the reaction of alkylating agents with deoxyribosenucleic acid in vitro and in vivo.

Preliminary studies in vitro using bacteriophage T7-DNA have shown that breaks formed in the DNA on the alkaline hydrolysis of apurinic sites and phosphotriesters can be distinguished from each other by measuring the extent of degradation of the DNA immediately after adding NaOH to 0.1 M and after incubating for 1 h in 0.5 M NaOH. This method has then been applied to the study of the formation and stability of phosphotriesters invivo. Methyl phosphotriesters formed in liver DNA following injection of mice with N-methyl-N-nitrosourea (MNUA) disappear with time (50% in 4-5 days). The concentration of ethyl phosphotriesters in liver DNA formed by injecting mice with N-ethyl-N-nitrosourea (ENUA) does not appear to decrease with time. Results of experiments on injecting methyl methane-sulphonate (MMS), ethyl methanesulphonate (EMS) and dimethyl sulphate (DMS) are also reported. The method described does not require the use of radioactively labelled reagents.     

DFT investigations of phosphotriesters hydrolysis in aqueous solution: a model for DNA single strand scission induced by N-nitrosoureas

DNA phosphotriester adducts are common alkylation products of DNA phosphodiester moiety induced by N-nitrosoureas. The 2-hydroxyethyl phosphotriester was reported to hydrolyze more rapidly than other alkyl phosphotriesters both in neutral and in alkaline conditions, which can cause DNA single strand scission. In this work, DFT calculations have been employed to map out the four lowest activation free-energy profiles for neutral and alkaline hydrolysis of triethyl phosphate (TEP) and diethyl 2-hydroxyethyl phosphate (DEHEP). All the hydrolysis pathways were illuminated to be stepwise involving an acyclic or cyclic phosphorane intermediate for TEP or DEHEP, respectively. The rate-limiting step for all the hydrolysis reactions was found to be the formation of phosphorane intermediate, with the exception of DEHEP hydrolysis in alkaline conditions that the decomposition process turned out to be the rate-limiting step, owing to the extraordinary low formation barrier of cyclic phosphorane intermediate catalyzed by hydroxide. The rate-limiting barriers obtained for the four reactions are all consistent with the available experimental information concerning the corresponding hydrolysis reactions of phosphotriesters. Our calculations performed on the phosphate triesters hydrolysis predict that the lower formation barriers of cyclic phosphorane intermediates compared to its acyclic counter-part should be the dominant factor governing the hydrolysis rate enhancement of DEHEP relative to TEP both in neutral and in alkaline conditions.

The stability of methyl and ethyl phosphotriesters in DNA in vivo

C57BL male mice were injected with N-methyl-N-nitrosourea (MNUA) or N-ethyl-N-nitrosourea (ENUA) and the concentration of alkyl phosphotriesters in the DNA of lung, liver, brain, kidney, spleen and thymus determined from the extent of degradation induced in isolated DNA by alkali. The same total dose of reagent was given either as a single injection (i.p.) or by weekly injections carried out over 5-20 weeks. Methyl phosphotriesters induced in liver, lung and kidney by the single injection were lost with a half-life of about 7 days, in brain the loss was more rapid, t1/2 = 2-3 days. During the multiple injections the observed t1/2 was 16 days. Ethyl phosphotriesters formed in the DNA of lung, liver, kidney and brain were much more stable than the methyl derivatives, t1/2 = 10-15 weeks. Phosphotriesters formed in the DNA of spleen and thymus disappeared very quickly after the single injection presumably as a result of dilution due to DNA replication. No accumulation of phosphotriesters occurred in the DNA of these tissues during the multiple injections. The general pattern of the results suggests that phosphotriesters are not excised by cellular repair systems.     

Neighbouring group participation in the unblocking of phosphotriesters of nucleic acids.

Two examples of neighbouring group participation during the removal of protecting groups from phosphotriesters of partially or fully protected intermediates of nucleic acids are presented. The first example shows that ammonolysis of aryl groups from phosphotriesters of partially protected - 5'- hydroxy free - nucleic acids (e.g., 4b approximately to; Ar=2C1C 6H4) gives rise to the formation of unnatural nucleic acids (e.g., 7 approximately to and 8 approximately to). The second one illustrates that fluoride ion promoted hydrolysis of 2,2,2-trichloroethyl groups from phosphotriesters of fully protected nucleic acids (e.g., 18a approximately to), having t-butyldimethylsilyl groups at the 2'-positions, leads to the formation of a considerable amount of side-products (e.g., 20 approximately to and 21 approximately to).     

Synthesis and properties of oligodeoxyribonucleotides containing an ethylated internucleotide phosphate

Internucleotide phosphotriesters comprise an important class of DNA lesions produced by carcinogenic alkylating agents. To avoid confusion resulting from the presence of other DNA lesions, synthetically prepared oligonucleotides containing ethylated internucleotide phosphates as the sole form of damage were employed to investigate several chemical and biochemical properties of DNA alkyl phosphotriesters. A total of four oligonucleotides were synthesised for this study, the dimers Tp(Et)T and pTp(Et)T and the decamer d-TpTpTp(Et)TpCpTpApTpTpT together with its unmodified analogue. The dimers were characterized by UV and phosphorus NMR spectroscopy and the decamers by two-dimensional homochromatography, alkali hydrolysis, and variable-temperature circular dichroism (CD). Alkali hydrolysis of the ethylated decamer produced strand breaks in approximately 75% of the molecules. This is in close agreement with data previously obtained for dinucleoside ethyl phosphotriesters and triesters in alkylated cellular DNA. Results from the CD study suggest that the ethyl substituent does not disrupt base stacking within the oligomer. The interactions of two enzymes with the alkylated oligonucleotides were examined. First, it was found that ethylation of the internucleotide phosphate renders TpT inactive as a substrate for T4 polynucleotide kinase, implying that a negative charge is required on the 3'-phosphate group of the nucleotide to be phosphorylated. Hence, postlabeling assays of DNA damage that depend upon enzymatic phosphorylation of modified 3'-nucleotides cannot be applied to dinucleoside alkyl phosphotriesters. Second, both decamers, when annealed to a single-stranded plasmid template, were able to prime DNA synthesis, catalyzed by Escherichia coli DNA polymerase I, with equal effectiveness. The use of this reaction as a means of site-specifically incorporating phosphotriesters into viral vectors is recognized.     

Synthesis of covalently immobilized phospholipid analogues

Cyclic 1-O-acyl-2-O-alkyl-glycero-3-phosphotriesters and 1-O-acyl-2-O-alkyl-glycero-3-bromoethylphosphate with a free acyl moiety in position 1 of the glycerol backbone were synthesized. These phospholipid intermediates were covalently bound to AH-Sepharose via the carbodiimide method. After immobilization the corresponding phosphatidylethanolamine analogues were obtained by acid hydrolysis of the cyclic phosphotriesters and by direct amination of the bromoethylphosphate. Thus, in a short, stepwise synthesis including minimum use of protecting groups, a variety of immobilized phospholipid analogues are available as affinity adsorbents for the purification of enzymes related to phospholipid metabolism.     

The formation and stability of methyl phosphotriesters in the DNA of rat tissues after treatment with the carcinogen N,N-dimethylnitrosamine

Following the injection i.p. of N,N-dimethylnitrosamine (DMN) into Chester Beatty (CB) hooded, female rats (2 mg/kg) measurable concentrations of methyl phosphotriesters were found in the DNA of liver, lung and kidney but not in spleen, thymus or brain. In lung and kidney these lesions were stable for at least 14 days but in liver there was a steady loss (t 1/2 9-11 days). Administering the same total dose in 10 weekly injections produced the same concentration of phosphotriesters in lung and kidney DNA as the single injection but in liver only half of the concentration induced by the single injection was found. It was calculated that the half-life of methyl phosphotriesters in the liver DNA of animals given repetitive injections was of the order of 6 weeks.     

Stereospecific removal of methyl phosphotriesters from DNA by an Escherichia coli ada+ extract.

The ada+ gene product, a DNA methyltransferase present in extracts from an Escherichia coli strain constitutive for the adaptive response, removes only half of the methyl phosphotriesters from alkylated DNA. Since DNA phosphotriesters occur in two isomeric configurations (denoted Rp and Sp), we examined whether this reflects a stereospecific mode of repair by the methyltransferase. Analysis by reverse-phase HPLC, phosphorus NMR and circular dichroism established that only triesters in the Sp configuration are acted upon by the E. coli extract.     

Synthesis and properties of photolabile caged phosphotriester derivatives of dinucleoside phosphates

Dinucleoside phosphates that harbor phosphate groups transiently blocked (caged) by o-nitrobenzyl or o-nitroveratryl residues were synthesized. It was shown that the conditions of the UV-induced deprotection largely depend on the nature of the protective group. The phosphotriesters obtained were resistant toward snake venom phosphodiesterase and nucleases of the cellular extract. The synthesis of the dinucleoside phosphates containing a photolabile group preceded the incorporation of the modified blocks into extended oligonucleotides by the phosphoramidite method.     

The reaction of alkylating agents with bacteriophage R17. Biological effects of phosphotriester formation

The extent of biological inactivation and of the degradation of the RNA after reaction of bacteriophage R17 with ethyl methanesulphonate, isopropyl methanesulphonate and N-ethyl-N-nitrosourea was studied. Formation of breaks in the RNA chain probably results from hydrolysis of phosphotriesters formed in the alkylation reactions. Near neutral pH the ethyl and isopropyl phosphotriesters are sufficiently stable for the kinetics of the hydrolysis reaction to be followed. Results indicate that the rate of hydrolysis increases rapidly as the pH is raised. The evidence shows that a phosphotriester group does not itself constitute a lethal lesion. The extent of phosphotriester formation by the different agents is discussed in terms of reaction mechanism.     

Methyl phosphotriesters in alkylated DNA are repaired by the Ada regulatory protein of E. coli.

The E. coli ada+ gene product that controls the adaptive response to alkylating agents has been purified to apparent homogeneity using an overproducing expression vector system. This 39 kDa protein repairs 0(6)-methylguanine and 0(4)-methylthymine residues in alkylated DNA by transfer of the methyl group from the base to a cysteine residue in the protein itself. The Ada protein also corrects one of the stereoisomers of methyl phosphotriesters in DNA by the same mechanism, while the other isomer is left unrepaired. Different cysteine residues in the Ada protein are used as acceptors in the repair of methyl groups derived from phosphotriesters and base residues.