Phosphorothioate Triesters Research Articles

Trans-acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high-throughput microbioreactor system.

A rapid and cost-effective transient transfection method for mammalian cells is essential for screening biopharmaceuticals in early stages of development. A library of 25 amphipathic trans-acting oligodeoxythymidine phosphorothioate triester (dTtaPS) transfection reagents, carrying positively charged and lipophilic groups, has been constructed for this purpose. High-throughput screening of the library, using an imaging cytometer and an automated microbioreactor system, has led to the identification of dTtaPS10+ as a potent transfection reagent. This reagent efficiently delivers a plasmid encoding enhanced green fluorescent protein in adherent HeLa cells while exhibiting low cytotoxicity. The microbioreactor system has been particularly useful for assessing the ability of dTtaPS10+ to deliver a plasmid encoding immunoglobulin IgG1 in a fed-batch serum-free suspension CHO cell culture; dTtaPS10+ -mediated transfection resulted in the production of IgG1 in yields comparable to or better than those obtained with commercial lipid-based transfection reagents under similar conditions. The ability of dTtaPS10+ to deliver plasmids is essentially unaffected by the presence of a silicone-based antifoaming reagent, which is commonly used in bioreactor cell cultures. The transfection efficiency of lyophilized dTtaPS10+ -plasmid complexes has been significantly restored upon aqueous reconstitution when compared to that achieved while using commercial transfection reagent complexes under similar conditions. The results of all experiments underscore the potential of dTtaPS10+ for transient transfection of plasmids into adherent cells and fed-batch serum-free suspension CHO cells and rapid screening of reagents in a microbioreactor system.

Linear correlation between effective charge and bond length sensitivity to electronic effects in phosphoryl, sulfonyl, and sulfuryl compounds

This work presents a linear correlation between bond length sensitivity (Δr/ΔpKlg) and effective charge by comparing a series of the following ROX compounds bearing phosphoryl (XP) and sulfonyl and sulfuryl (XS) moieties: phosphate monoester dianions and monoanions, phosphate diesters, phosphate and phosphorothioate triesters, 4‐Me, 3‐NO2 and 2‐ or 4‐NO2 substituted aryl benzene sulfonates, sulfate monoesters, and a previously determined series of sulfamate and mesylate esters. In every series for ROX compounds, the shortest C―O bond corresponds to the longest O―X bond, showing a linear correlation with the pKlg of the ROH parent compound. The chemical reasons for the extent of bond elongation are discussed, and a linear correlation is found between O―X bond length sensitivity and effective charge. These data show that bond length elongation depends on its strength regarding the equilibrium related to the full bond cleavage. Effective charges for phosphorothioate triesters and sulfonates are estimated, and the effective charge for aryl sulfamate esters is determined on the basis of the available linear free energy relationship data. It is expected that the observations of this paper will illuminate the effect of the electronic demand on the O―X bond lengths during phosphoryl, sulfonyl, and sulfuryl group transfer. Copyright © 2013 John Wiley & Sons, Ltd.

Mechanistic and Computational Study of a Palladacycle-Catalyzed Decomposition of a Series of Neutral Phosphorothioate Triesters in Methanol

The methanolytic cleavage of a series of O,O-dimethyl O-aryl phosphorothioates (1a−g) catalyzed by a C,N-palladacycle, (2-[N,N-dimethylamino(methyl)phenyl]-C1,N)(pyridine) palladium(II) triflate (3), at 25 °C and sspH 11.7 in methanol is reported, along with data for the methanolytic cleavage of 1a−g. The methoxide reaction gives a linear log k2−OMe vs sspKa (phenol leaving group) Brønsted plot having a gradient of βlg = −0.47 ± 0.03, suggesting about 34% cleavage of the P−OAr bond in the transition state. On the other hand, the 3-catalyzed cleavage of 1 gives a Brønsted plot with a downward break at sspKa (phenol) 13, signifying a change in the rate-limiting step in the catalyzed reaction, with the two wings having βlg values of 0.0 ± 0.03 and −1.93 ± 0.06. The rate-limiting step for good substrates with low leaving group sspKa values is proposed to be substrate/pyridine exchange on the palladacycle, while for substrates with poor leaving groups, the rate-limiting step is a chemical one with extensive cleavage of the P−OAr bond. DFT calculations support this process and also identify two intermediates, namely, one where substrate/pyridine interchange has occurred to give the palladacycle coordinated to substrate through the S═P linkage and to methoxide (6) and another where intramolecular methoxide attack has occurred on the P═S unit to give a five-coordinate phosphorane (7) doubly coordinated to Pd via the S− and through a bridging methoxide linked to P and Pd. Attempts to identify the existence of the phosphorane by 31P NMR in a d4-methanol solution containing 10 mM each of 3, trimethyl phosphorothioate (a very slow cleaving substrate), and methoxide proved unsuccessful, instead showing that the phosphorothioate was slowly converted to trimethyl phosphate, with the palladacycle decomposing to Pd0 and free pyridine. These results provide the first reported example where a palladacycle-promoted solvolysis reaction exhibits a break in the Brønsted plot signifying at least one intermediate, while the DFT calculations provide further insight into a more complex mechanism involving two intermediates.

ChemInform Abstract: Internucleotide Phosphite Sulfurization with Tetraethylthiuram Disulfide. Phosphorothioate Oligonucleotide Synthesis via Phosphoramidite Chemistry.

Abstract A new sulfurizing reagent has been developed for the automated synthesis of phosphorothioate oligonucleotides 1 via the phosphoramidite method. Tetraethylthiuram disulfide (TETD) converts internucleotide cyanoethyl phosphite 2 to the phosphorothioate triester 3 within 15 minutes at room temperature after each coupling step. With an optimized synthesis cycle, the efficiency of phosphorothioate oligonucleotide synthesis is comparable to that of phosphodiester oligonucleotides. Large scale synthesis (200μmole), oligonucleotides up to 100 bases long, and mixed phosphorothioate/phosphodiester DNA are demonstrated.

Mechanistic studies of La3+- and Zn2+-catalyzed methanolysis of aryl phosphate and phosphorothioate triesters. Development of artificial phosphotriesterase systems

The methanolyses of a series of O,O-diethyl O-aryl phosphates (2,5) and O,O-diethyl S-aryl phosphorothioates (6) promoted by methoxide and two metal ion systems, (La3+)2(-OCH3)2 and 4:Zn2+:-OCH3 (4 = 1,5,9-triazacyclododecane) has been studied in methanol at 25 degrees C. Brønsted plots of the logk2 values vs. pKa for the phenol leaving groups give beta(lg) values of -0.70, -1.43 and -1.12 for the methanolysis of the phosphates and -0.63, -0.87 and -0.74 for the methanolysis of the phosphorothioates promoted by the methoxide, La3+ and Zn2+ systems respectively. The kinetic data for the metal-catalyzed reactions are analyzed in terms of a common mechanism where there is extensive cleavage of the P-XAr bond in the rate-limiting transition state. The relevance of these findings to the mechanism of action of the phosphotriesterase enzyme is discussed.

An ortho-palladated dimethylbenzylamine complex as a highly efficient turnover catalyst for the decomposition of PS insecticides. Mechanistic studies of the methanolysis of some PS-containing phosphorothioate triesters

An ortho-palladated complex Pd(dmba)(py)(OTf) (9), or Pd(N,N-dimethylbenzylamine)(pyridine)(trifluoromethanesulfonate), was synthesized and its solution properties in methanol studied as a function of pH. In neutral solution the triflate dissociates from the complex to give a dominant form Pd(dmba)(py)(HOCH3), and in acid the pyridine dissociates to give Pyr-H+ and Pd(dmba)(HOCH3)(HOCH3). Under basic conditions, Pd(dmba)(py)(HOCH3) ionizes to give Pd(dmba)(py)(-OCH3) from which the pyridine can dissociate to yield a mixture of a bis-methoxy-bridged dimer (Pd(dmba)(-OCH3))2 (15-dimer), and its monomer Pd(dmba)(HOCH3)(-OCH3). Kinetic studies under buffered conditions reveal that 9 is an effective catalyst for the methanolysis of fenitrothion and other P=S pesticides. The active form of the catalyst is a basic one having one associated methoxide generated with an apparent (s)(s)pK(a) of 10.8. Analysis of the change in the UV/vis spectrum as a function of pH generates a spectrophotometric (s)(s)pK(a) of 10.8 +/- 0.1. This catalytic system is shown to promote the methanolysis of fenitrothion (3), diazinon (4), quinalphos (5), coumaphos (10) and dichlofenthion (11) at 0.05 mol dm(-3) triethyl amine buffer, (s)(s)pH 10.8, 25 degrees C, under turnover conditions where the [phosphorothioate]/[9] ratio is 48.6, 13.4, 13.4, 18.6, and 48.6 respectively. In all cases, the products were derived from displacement of the leaving group by methoxide, the second-order turnover rate constants being 36.9, 0.45, 0.12, >146.7 and 44.3 dm3 mol(-1) s(-1) respectively. An associative mechanism for the catalyzed methanolysis of the P=S pesticides is proposed where a transiently coordinated S=P substrate is intramolecularly attacked by the Pd(II)-coordinated methoxide.

Formation of 4,4′-dimethoxytrityl- C-phosphonate oligonucleotides

Incomplete sulfurization during solid-phase synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry was identified as the cause of formation of two new classes of process-related oligonucleotide impurities containing a DMTr- C-phosphonate (DMTr = 4,4′-dimethoxytrityl) moiety. Phosphite triester intermediates that failed to oxidize (sulfurize) to the corresponding phosphorothioate triester react during the subsequent acid-induced (dichloroacetic acid) detritylation with the DMTr cation or its equivalent in an Arbuzov-type reaction. This leads to formation of DMTr- C-phosphonate mono- and diesters resulting in oligonucleotides modified with a DMTr- C-phosphonate moiety located internally or at the 5′terminal hydroxy group. DMTr- C-phosphonate derivatives are not detected when optimized sulfurization conditions are employed.

2-Methyl-5-tert-butylthiophenol—An Odorless Deprotecting Reagent Useful in Synthesis of Oligonucleotides and Their Analogs

A solution of 2-methyl-5-tert-butylthiophenol and triethylamine in acetonitrile efficiently removed the methyl protecting groups from phosphate and phosphorothioate triesters and yielded oligonucleotides of high quality. This inexpensive odorless liquid is not toxic and is a suitable replacement for hazardous thiophenol and other reagents often used for this purpose.

Indolyl-oxazaphosphorine Precursors for Stereoselective Synthesis of Phosphite Triesters and Dithymidinyl Phosphorothioates

Several novel chiral indolyl-oxazaphosphorines 7 were synthesized, and their potential as precursors to chiral phosphorothioates was evaluated. Reaction of 7 with a thymidine derivative gave phosphite triester 8 with a large degree of stereoselectivity. Sulfurization with Beaucage's reagent provided phosphorothioate triesters 9. The chiral auxiliary 9b containing a cyano group could be easily removed with aqueous ammonia to form dithymidinyl phosphorothioate in more than 97% diastereomeric excess. The chiral indolyl-oxazaphosphorines 7 are a new class of precursors for stereoselective synthesis of phosphorothioates.

Synthesis of Dimer Phosphoramidite Synthons for Oligodeoxyribonucleotide Phosphorothioates Using Diethyldithiocarbonate disulfide as an Efficient Sulfurizing Reagent

An efficient solution phase synthesis of deoxyribonucleoside phosphorothioate dimers utilizing phosphoramidite approach is described. Diethyldithiocarbonate disulfide (DDD) was found to be an efficient sulfurizing reagent in the conversion of phosphite triesters to phosphorothioate triesters.

Facile Synthesis of a Phosphotriester Intermediates for Solution-Phase Preparation of Oligonucleotide Phosphorothioates

Abstract Fully protected 2′-deoxynucleoside phosphorothioate triester derivatives, which are key starting materials for the large-scale solution-phase preparation of phosphorothioate oligonucleotides, were easily synthesized by a new method starting from common, fully protected 2′-deoxynucleosides.

Stereocontrolled Synthesis of Dithymidine Phosphorothioates by Use of a Functionalized 5′- Protecting Group Bearing an Imidazole Residue†

Diastereoselective formation of internucleotidic phosphorothioate triester bonds was achieved by use of 3-(imidazol-1-ylmethyl)-4′,4″-dimethoxytrityl (IDTr) as a 5′-hydroxyl protecting group in the phosphotriester approach. After removal of all the protecting groups, stereochemistry of the major product was determined as the Spconfiguration by enzymatic digestion. This paper is dedicated to the memory of the late Professor Tsujiaki Hata.

Solution Phase Synthesis of an Oligodeoxyribonucleotide Phosphorothioate for Therapeutic Applications

Solution phase synthesis of an oligodeoxyribonucleotide phosphorothioate Octamer (5′-TTGGGGTT) using phosphorothioate triester method is reported.

Phosphoester Hydrolysis and Intramolecular Transesterification of Ribonucleoside 2'- and 3'-Phosphoromonothioate Triesters: Kinetics and Mechanisms for the Reactions of 5'-O-Methyluridine 2'- and 3'-Dimethylphosphoromonothioates.

The hydrolytic reactions of the monothioate analogs of 5'-O-methyluridine 2'- and 3'-dimethylphosphates have been followed over a wide acidity range, H(0) = -1.7 ([HCl] = 5 mol L(-)(1)) to pH 9. Two reactions were found to compete: mutual interconversion of the 2'- and 3'-isomers and phosphoester hydrolysis to a mixture of phosphorothioate diesters, viz., the R(P) and S(P) diastereomers of 2',3'-cyclic thiophosphate and 2'/3'-monomethylthiophosphates (i.e., three pairs of diastereomers). No marked desulfurization could be observed. The interconversion and hydrolysis both show first-order dependence of rate on acidity at pH < 0, the isomerization being 3-4 times as fast as the phosphoester hydrolysis. Under less acidic conditions, the hydrolysis remains pH-independent up to pH 7, while the isomerization becomes hydroxide-ion-catalyzed (first-order in [OH(-)]) already at pH 2. The hydrolysis is susceptible to general base catalysis in carboxylic acid buffers, the Brönsted beta value being 0.8. In contrast, no conclusive evidence for buffer-catalyzed isomerization could be obtained. All these reactions are suggested to proceed via a pentacoordinated thiophosphorane intermediate, obtained at pH < 1 by an attack of the neighboring hydroxy function on a protonated (monocationic) thiophosphate group and at pH > 2 by an attack of a deprotonated hydroxy function (oxyanion) on a neutral thiophosphate. The monocationic intermediate (pH < 1) may collapse to hydrolysis and isomerization products without further catalysis (departure of alcohol). The monoanionic thiophosphorane (pH > 2) also gives isomerization products without catalysis (departure of 2'/3'-oxyanion), whereas breakdown to the hydrolysis products needs either a specific or a general acid catalysis process (departure of methanol). Accordingly, the observed general-base-catalyzed hydrolysis most likely consists of consecutive specific base/general acid catalysis. The phosphorothioate triesters studied are, under very acidic conditions, more than 2 orders of magnitude more stable than their oxyphosphate counterparts, whereas the rate-retarding "thio effect" (k(P)(=)(O)/k(P)(=)(S)) is much smaller with the hydroxide ion-catalyzed reactions (ca. 4) and almost negligible with the pH-independent hydrolysis.

SYNTHESIS OF PHOSPHOROTHIOATE ANALOGS OF PHOSPHATIDYLINOSITOL 3,4,5-TRISPHOSPHATE

Abstract Phosphorothioate analogs of phosphatidylinositol 3,4,5-trisphosphate have been synthesized using Me3N-Me3SiCl reagent in a critical exhaustive cleavage of O-benzyl phosphorothioate triester intermediates. © 1997 Elsevier Science Ltd.

A stereoselective synthesis of dinucleotide phosphorothioate triesters through a chiral indol-oxazaphosphorine intermediate

(S)-1-(indol-2-yl)-propan-2-ol was used as a chiral auxiliary to form a dinucleotide phosphorothioate triester in 97% ee.

Solution-phase synthesis of phosphorothioate oligodeoxynucleosides by the phosphotriester method.

A "phosphorothioate triester method" was investigated for the solution-phase synthesis of phosphorothioate oligonucleosides. Using fully protected 3'-phosphorothiolate thymidine bearing O-cyanoethyl and S-2,4-dichlorobenzyl groups as phosphorothioate protecting groups, decathymidine nonaphosphorothioate was efficiently assembled through a blockwise procedure. Two side reactions occurred during the deprotection steps: breakage of internucleoside linkages (1.8% per linkage) and formation of phosphate diester linkages (0.9%). Substitution of the dichlorobenzyl group by the more labile 4-nitrobenzyl S-protecting group reduced the extent of internucleoside bond breakage by one-half.

New efficient sulfurizing reagents for the preparation of oligodeoxyribonucleotide phosphorothioate analogues

A set of new sulfurizing agents representing disulfides of arylsulfonic acids has been developed for the automated synthesis of phosphorothioate oligonucleotide analogues via the phosphoramidite method. These reagents, such as bis(benzenesulfonyl)disulfide, bis(p-toluenesulfonyl)disulfide, bis(p-methoxybenzensulfonyl)disulfide, and bis (p-chlorobenzenesulfonyl) disulfide, are easily prepared crystalline solid compounds. They are relatively inexpensive, easy to handle, and efficiently convert internucleotide cyanoethyl phosphite to the phosphorothioate triester within 1-2 min. The efficiency of phosphorothioate oligonucleotide synthesis with the use of these reagents is comparable to that of phosphodiester oligonucleotides.

Internucleotide phosphite sulfurization with tetraethylthiuram disulfide. Phosphorothioate oligonucleotide synthesis via phosphoramidite chemistry

A new sulfurizing reagent has been developed for the automated synthesis of phosphorothioate oligonucleotides 1 via the phosphoramidite method. Tetraethylthiuram disulfide (TETD) converts internucleotide cyanoethyl phosphite 2 to the phosphorothioate triester 3 within 15 minutes at room temperature after each coupling step. With an optimized synthesis cycle, the efficiency of phosphorothioate oligonucleotide synthesis is comparable to that of phosphodiester oligonucleotides. Large scale synthesis (200μmole), oligonucleotides up to 100 bases long, and mixed phosphorothioate/phosphodiester DNA are demonstrated.

Synthesis of potential UDP‐glucuronosyltransferase inhibitors containing a diphosphate function

AbstractThe synthesis of potential inhibitors of UDP‐glucuronosyltransferase, in which the β‐phosphate moiety of uridine 5′‐diphosphate is linked to phenolic or alcoholic hydroxyl groups, is described. Key intermediates in the formation of the diphosphate function are S‐(4‐methylphenyl) 2‐cyanoethyl phosphorothioate triesters which, after conversion into the corresponding S‐(4‐methylphenyl) phosphorothioate diesters, react with phosphate monoesters, in the presence of iodine, to give the target molecules.