Phosphorothioate Diester Research Articles

Thermolytic 4-methylthio-1-butyl group for phosphate/thiophosphate protection in solid-phase synthesis of DNA oligonucleotides.

The thermolabile 4-methylthio-1-butyl phosphate/thiophosphate protecting group for DNA oligonucleotides has been investigated for its potential application to a "heat-driven" process for either oligonucleotide synthesis on diagnostic microarrays or, oppositely, to the large-scale preparation of therapeutic oligonucleotides. The preparation of phosphoramidites 10a-d is straightforward, and the incorporation of these amidites into oligonucleotides via solid-phase techniques proceeds as efficiently as that achieved with 2-cyanoethyl deoxyribonucleoside phosphoramidites. The versatility of the 4-methylthio-1-butyl phosphate/thiophosphate protecting group is exemplified by its facile removal from oligonucleotides upon heating for 30 min at 55 degrees C in an aqueous buffer under neutral conditions or within 2 h at 55 degrees C in concentrated NH(4)OH. The deprotection reaction occurs through an intramolecular cyclodeesterification mechanism leading to the formation of sulfonium salt 18. When mixed with deoxyribonucleosides and N-protected 2'-deoxyribonucleosides or with a model phosphorothioate diester under conditions approximating those of large-scale (>50 mmol) oligonucleotide deprotection reactions, the salt 18 did not significantly alter DNA nucleobases or desulfurize the phosphorothioate diester model to an appreciable extent.

Diastereomeric Process Control in the Synthesis of 2′‐O‐(2‐Methoxyethyl) Oligoribonucleotide Phosphorothioates as Antisense Drugs

AbstractFor Abstract see ChemInform Abstract in Full Text.

Thermolytic properties of 3-(2-pyridyl)-1-propyl and 2-[N-methyl-N-(2-pyridyl)]aminoethyl phosphate/thiophosphate protecting groups in solid-phase synthesis of oligodeoxyribonucleotides.

Thermolytic groups may serve as alternatives to the conventional 2-cyanoethyl group for phosphate/thiophosphate protection in solid-phase oligonucleotide synthesis to prevent DNA alkylation by acrylonitrile generated under the basic conditions used for oligonucleotide deprotection. Additionally, thermolytic groups are attractive in the context of engineering a "heat-driven" process for the synthesis of oligonucleotides on diagnostic microarrays. In these regards, the potential application of pyridine derivatives as thermolytic phosphate/thiophosphate protecting groups has been investigated. Specifically, 2-pyridinepropanol and 2-[N-methyl-N-(2-pyridyl)]aminoethanol were incorporated into deoxyribonucleoside phosphoramidites 7a-d and 9, which were found as efficient as 2-cyanoethyl deoxyribonucleoside phosphoramidites in solid-phase oligonucleotide synthesis. Whereas the removal of 3-(2-pyridyl)-1-propyl phosphate/thiophosphate protecting groups from oligonucleotides is effected within 30 min upon heating at 55 degrees C in concentrated NH4OH or in an aqueous buffer at pH 7.0, cleavage of 2-[N-methyl-N-(2-pyridyl)]aminoethyl groups occurs spontaneously when their phosphate or phosphorothioate esters are formed during oligonucleotide synthesis. The deprotection of these groups follows a cyclodeesterification process generating the bicyclic salts 13 and 14 as side products. These salts do not alkylate or otherwise modify any DNA nucleobases and do not desulfurize a phosphorothioate diester model under conditions mimicking large-scale oligonucleotide deprotection.

Diastereomeric Process Control in the Synthesis of 2′-O-(2-Methoxyethyl) Oligoribonucleotide Phosphorothioates as Antisense Drugs

Coupling of 2′-O-methoxyethylsubstituted nucleoside phosphoramidites to 5′-hydroxyl group of a nucleoside or nucleotide on solid support is under stereochemical process control and is independent of scale, concentration, synthesizer, ratio of amidite diastereomers, solid support etc. However, activators and phosphate protecting groups do play a role in influencing the ratio of phosphorothioate diesters obtained by sulfurization of phosphite triesters.

Aryl H-phosphonates. Part 13.1 A new, general entry to aryl nucleoside phosphate and aryl nucleoside phosphorothioate diesters

The reaction of nucleoside H-phosphonate monoesters with phenols in the presence of a condensing agent, followed by oxidation of the in-situ-generated aryl nucleoside H-phosphonate diesters with iodine–water or with elemental sulfur, provides a new, ‘one-pot’, efficient entry to nucleoside phosphate or nucleoside phosphorothioate diesters bearing diverse aryl moieties.

Synthesis and use of DNA containing a 5'-bridging phosphorothioate as a suicide substrate for type I DNA topoisomerases.

AbstractType I DNA topoisomerases change the linking number of supercoiled DNA by carrying out orcheastrated cleavage and ligation reactions involving phosphodiester bonds. Strand cleavage is not the result of phosphodiester hydrolysis, but the result of transesterification of an active-site tyrosine, creating a 3′-DNA covalent intermediate and a 5′-terminus (5′-OH). Strand ligation occurs during a second transesterification event when the 5′-OH displaces the enzyme (see Fig. 1). A change in DNA linking number results when strand unwinding occurs between these cleavage and ligation reactions. Rationale for suicide substrate design. A phosphodiester linkage at the site of strand cleavage/ligation and an active site tyrosine are diagrammed on the left; the cleaved DNA and the 3′-phosphotyrosyl enzyme covalent complex are diagrammed on the right. In the unmodified phosphodiester, X represents oxygen; for the 5′-bridging phosphorothioate (OPS DNA), X represents sulfur. Chiral phosphorothioate diesters, in which one of the nonbridging oxygens is replaced by sulfur (not diagrammed), have been extensively used as mechanistic probes in enzymology and should not be confused with the achiral 5′-bridging phosphorothioate diesters described here. KeywordsStrand CleavageTrityl GroupSuicide SubstratePhenyl Mercuric AcetateRibose SugarThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Fluorine-18 labeling of oligonucleotides bearing chemically?modified ribose?phosphate backbones

We have recently described the labeling of a natural deoxyribose phosphodiester oligonucleotide with fluorine-18 (t1/2 : 109·8 min) and demonstrated its potential for in vivo imaging in a primate PET study. We here report that the methodology employed can be reliably and routinely applied to the most popular chemical modifications: (a) full length internucleosidic phosphorothioate diester bonds deoxyribose oligonucleotides (the modification most favoured by industry for human antisense therapy), (b) hybrid methylphosphonate/phosphodiester internucleosidic bonds deoxyribose oligonucleotides and (c) 2′Methyl modified ribose oligonucleotides. The whole fluorine-18 labeling procedure allows us to obtain 15 to 21 mCi (0·55 to 0·74 GBq) of pure labeled oligonucleotides (regardless the modification of the sugar phosphate backbone) in 180 minutes with a specific radioactivity of 0·8 to 2 Ci/μmol (30 to 70 GBq/μmol) at the end of synthesis. Copyright © 2000 John Wiley & Sons, Ltd.

Oligodeoxyribonucleotides with Internucleotidic or Terminal Phosphorothioate Groups: Different Pathways in the Reaction with Water-Soluble Carbodhmide

Different ‘thiophilicity’ of water-soluble carbodiimides to sulfhydryl anions in mono-and diesters of phosphorothioic acid was demonstrated. The EDC (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide) treatment of oligonucleotides containing phosphorothioate diesters with non-bridging sulfur leads to formation of stable adducts which can be isolated by PAGE or HPLC. In contrast, oligomers with terminal phosphorothioate monoesters form with EDC highly reactive intermediates which react easily with media nucleophiles (e.g. water or methanol, phosphomonoester anions).

Diastereomeric Process Control in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

The emergence of antisense and antigene oligonucleotides as potential sequenceselective inhibitors of gene expression is evidenced by the growing number of ongoing clinicals trials against a variety of diseases. First generation antisense therapeutics utilize a uniformly modified oligodeoxyribonucleotide phosphorothioate where one non-bridging oxygen atom is formally replaced by sulfur, because natural DNA is unstable towards extra- and intracellular enzymes. Phosphoramidite chemistry has been widely used for the synthesis of phosphorothioate oligonucleotides because of its potential for automation, high coupling efficiency, ease of site-specific thioate linkage incorporation, and ready scalability. The large scale solid-supported synthesis of phosphorothioates is presently carried out by initial formation of the internucleotidic phosphite linkage followed by sulfurization of the phosphite triester to phosphorothioate using the Beaucage reagent. The resulting O,O-linked phosphorothioate diester linkage in the oligonucleotide is a chiral functional group. For a typical 20-mer there are 524,288 (219) possible diastereoisomers. Separation and individual quantification of this number of diastereomers is currently not feasible. In addition, the best reported methods for stereocontrolled synthesis of phosphorothioate oligomers are not presently useful for drug synthesis; that is, since net 100% enantiomeric excess is not achieved in the coupling step, the oligomeric product still consists of the same mixture of Sp and Rp diastereomers, except that the levels of all but one isomer are reduced to low individual levels. As a result, even a small change in the and Sp phosphorothioate diesters, due to racemization during coupling, indicating that the overall synthetic process is stereo reproducible and under inherent process control.

New Methods for the Formation of the P-N and P-F Bonds, their Relevance to Nucleotide and Oligonucleotide Analogues Synthesis

Two approaches have been developed for the formation of phosphoramidates with the nitrogen atom in bridging positions of the internucleotide linkage. One of them makes use of H-phosphonamidates as intermediates and the other is based on the oxidative coupling of H-phosphonate monoesters or their analogues with appropriate amines. Also, a new, convenient entry to nucleoside methylphosphonamidates, consisting of an oxidative coupling of nucleoside 3'-methylphosphinates with 5'-aminonucleosides, was investigated. Finally, iodine promoted desulfurization or deselenization of the appropriate phosphorothioate and phosphoroselenoate diesters in the presence of fluoride anion, was found to provide new means for the formation of the P-F bond.

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.

The pro-oligonucleotide approach. V: Influence of the phosphorus atom environment on the hydrolysis of enzymolabile dinucleoside phosphotriesters

Six dithymidine phosphotriester analogs bearing an enzymolabine phosphate protecting group were synthesized in order to select the most appropriate model for the prooligonucleotide approach. Series 1 and 3 were chosen on the basis of their stability in culture medium and their capacity, when incubated in total cell extract, to deliver selectively dithymidine phosphodiester and phosphorothioate diester respectively.

The synthesis and reactivity of new 2-( N, N-diisopropylamino)-3-methylsulfonyl-1,3,2-benzoxazaphospholes. The utility of the 5-chloro analogue in the one-pot synthesis of oligothiophosphates: [Ap sppA, Ap spppA, ppp5′A2′p s5′A, m 7Gp sppA, Ap spppp, Ap spp

The synthesis of 2-( N, N-diisopropylamino)-2,3-dihydro-3-methylsulfonyl-1,3,2-benzoxazaphospholes 22, 23 and 24 is reported. Their reactivities have been investigated using a variety of acid catalyst under conditions normally employed in phosphoramidite chemistry for oligonucleotide synthesis. The rate (k) of activation of 22, 23 and 24 by acid catalysis with N-methylanilinium hydrochloride (MAC) to their protonated species 25A, 26A and 27A, respectively, (Scheme 2) has been estimated to be 6.813 × 10 −7mol −1min −1, 1.237 × 10 −6mol −1min −1 and 1.972 × 10 −7mol −1min −1 at 18°C with a ratio of the rates as 0.56 : 1 : 0.16. The 5-chlorobenzoxazaphosphole 23 selectively activated by MAC gave the intermediate 2-(N-methylanilinium)-5-chlorobenzoxazaphosphole 26A (∼90% by NMR), which was then reacted with alcohols (nucleosides) to generate the reactive 2-alkoxybenzoxazaphosphole 30 (Scheme 4) or 33–35 (Scheme 5) (∼70% by NMR). We have then shown that 33–35 (Scheme 5) react with binucleophilic reagents, ADP, ATP or pyrophosphate, to generate the corresponding P 1-alkoxycyclometatriphosphite intermediate 36, 37, 50, 52 or 54. These intermediates were then sulfurised to form the P 1-alkoxy-1-thiocyclotriphosphate intermediates 38, 39, 51, 53 and 55, which were ring-opened by hydrolysis. Thus these steps constituted a one-pot multicomponent reaction (MCR) leading to the synthesis of R P and S P mixtures of each of the mono-thioanalogues of naturally-occurring oligophosphates: Ap sppA ( 43) (10%), Ap spppA ( 45) (19%), ppp5′A2′p s5′A ( 46) (24%), the cap structure m 7Gp sppA ( 47) (3%), Ap spppp ( 42) (23%), Ap spp ( 41) (33%) and Ap sp ( 40) (7%). The reaction of putative 34 and ADP gave the desired 43 (10%) along with pp5′A2′p s 5′A ( 44) (5%). The reaction sequences from 34 and ATP gave 45 (19%) and ppp5′A2′p s5′A ( 46) (24%). The proposed reaction mechanism for the synthesis of 43, 45 and 47 proceeds via the corresponding (dinucleoside 5′)-cyclometatriphosphite intermediates 50, 54, 52 and the (dinucleoside 5′)-1-thiocyclotriphosphate intermediates 51, 55, 53. The existence of these cyclic P(III) and P(V) intermediates were supported by 31P- and 1H-NMR spectroscopy. We here also demonstrate that 5-chlorobenzoxazaphosphole 23 can be used to synthesise a protected ribonucleoside 2′,3′-cyclic phosphorothioate block 28, a protected ribonucleoside 3′-(O-(4-chloro-2-mesylsulfonamido)phenyl phosphorothioate diester block 29 and bis(2-deoxyadenosine-5′)-thymidine-3′-monophosphate 32. The correct coupling of the nucleoside residues to the oligophosphate chain in 43, 45 & 47 has unequivocally been assessed by 2D 31P- 31P and 1H- 31P correlation spectroscopy.

Stereo-Reproducibility of the Phosphoramidite Method in the Synthesis of Oligonucleotide Phosphorothioates

Stereo-reproducibility of phosphoroamidite-based phosphorothioate oligonucleotide synthesis is examined through synthesis of singly thioate-substituted oligodeoxynucleotide model compounds. Baseline RP-HPLC resolution of resulting Rp and Sp diastereomers allowed accurate determination of any enantiomeric excess at each phosphorothioate linkage. Our results show that phosphorothioate linkage formation is not a stereo-random process. AH investigated stereoisomeric phosphorothioate diester linkages were formed with a small, reproducible excess of the R isomer (2-6% per linkage). Regardless of the synthesized sequence, all Rp/Sp diastereoisomer ratios were between 50:50 and 60:40, indicating that this synthesis process is under inherent stereochemical control.

Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester.

The purpose of this study was to determine whether the gamma-S of nucleoside thiotriphosphates and the non-bridging sulfur of internucleotide phosphorothioate diesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymidyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimide (PM) and the fluorescence intensity was recorded. The observed reactivity of the phosphorothioate nucleotides towards maleimide was used as a basis for preparing covalent protein-nucleotide conjugates of ATPS and of the internucleotide phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorbance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of protein-nucleotide conjugates, with absorbance maxima near 260 nm. The degree of conjugation was 1.69 nucleotides (nt)/BSA molecule for ATPS and 0.44 nt/BSA molecule for dA3(PS)2. The extent of conjugation of the gamma-S of the nucleoside thiotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with maleimide-derivatized protein agreed with their relative reactivity towards PM. Both the gamma-S of the nucleoside thiotriphosphate and the internucleotide phosphorothioate diester were found to possess sufficient thiol character to permit formation of maleimide-mediated protein conjugates.

Synthesis of phosphorothioate esters of L-phenyl-lactic acid as transition-state inhibitors of carboxypeptidase A

Diastereoisomeric phosphorothioate diesters and the phosphorothioate monoester of L-phenyl-lactic acid have been prepared and their inhibitory properties with carboxypeptidase A investigated. All the phosphorothioates have Ki-values in the micromolar range, suggesting that neither the stereochemistry nor additional negative charge on the phosphorothioate moiety has a major impact on binding.

Sequencing of oligonucleotide phosphorothioates based on solid-supported desulfurization.

We described a solid-supported desulfurization procedure allowing easy access to the sequence analysis of oligonucleotide phosphorothioates. The described method is based upon selective removal of the 2-cyanoethyl phosphate protecting groups, followed by iodine-promoted desulfurization of the resulting phosphorothioate diesters. Automatic oxidation of oligonucleotide phosphorothioates, anchored via an ester linkage to a standard solid support (LCAA/CPG), is combined with Maxam-Gilbert solid-support sequencing. The overall procedure allows rapid simultaneous sequence analysis of several oligonucleotide analogs.

Site-specific labeling of DNA sequences containing phosphorothioate diesters

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSite-specific labeling of DNA sequences containing phosphorothioate diestersJacqueline A. Fidanza, Hiroaki Ozaki, and Larry W. McLaughlinCite this: J. Am. Chem. Soc. 1992, 114, 14, 5509–5517Publication Date (Print):July 1, 1992Publication History Published online1 May 2002Published inissue 1 July 1992https://doi.org/10.1021/ja00040a004RIGHTS & PERMISSIONSArticle Views674Altmetric-Citations73LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

The covalent attachment of multiple fluorophores to DNA containing phosphorothioate diesters results in highly sensitive detection of single-stranded DNA

DNA fragments containing multiple internucleotidic phosphorothioate diesters, prepared by either chemical or enzymatic syntheses, are amenable to labeling with the fluorophore monobromobimane. With the incorporation of phosphorothioate diesters at each internucleotidic site, multiple fluorophores, ideally one for each nucleotide residue, can be covalently attached to the DNA fragment. The presence of multiple labels can be expected to interfere with analysis techniques, such as polyacrylamide gel electrophoresis. To avoid such problems, the fluorophores are introduced in a "postassay" fashion, that is, while the fragments are still embedded within the gel matrix. The detection limit (to the naked eye) for multiply labeled single-stranded DNA containing hundreds of base residues is in the low femtomole range. DNA containing greater than 1000 base residues can be visualized in some cases in the subfemtomole range without the use of sophisticated electronic instrumentation.

Binding and cleavage of nucleic acids by the "hairpin" ribozyme

The "hairpin" ribozyme derived from the minus strand of tobacco ringspot virus satellite RNA [(-)sTRSV] efficiently catalyzes sequence-specific RNA hydrolysis in trans (Feldstein et al., 1989; Hampel & Triz, 1989; Haseloff & Gerlach, 1989). The ribozyme does not cleave DNA. An RNA substrate analogue containing a single deoxyribonucleotide residue 5' to the cleavage site (A-1) binds to the ribozyme efficiently but cannot be cleaved. A DNA substrate analogue with a ribonucleotide at A-1 is cleaved; thus A-1 provides the only 2'-OH required for cleavage. These results support cleavage via a transphosphorylation mechanism initiated by attack of the 2'-OH of A-1 on the scissile phosphodiester. The ribozyme discriminates between DNA and RNA in both binding and cleavage. Results indicate that the 2'-OH of A-1 functions in complex stabilization as well as cleavage. The ribozyme efficiently cleaves a phosphorothioate diester linkage, suggesting that the pro-Rp oxygen at the scissile phosphodiester does not coordinate Mg2+.