Preparation of oligodeoxyribonucleoside methylphosphonates on a polystyrene support

Miller, Paul S.; Agris, Cheryl H.; Murakami, Akira; reddy, P.Malleswara; Spitz, Sharon A.; Ts’o, Paul O. P.

doi:10.1093/nar/11.18.6225

Cited by 66 publications

(25 citation statements)

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“…Methylphosphonate diesters are susceptible to random base catalysed hydrolysis of one of the ester linkages under conditions in which phosphodiesters are unaffected (Murakami et al, 1985;Miller et al, 1983). Consequently, it was possible to characterise the chimeric oligodeoxynucleotides by following the course of 1 M piperidine catalysed hydrolysis at 37°C using SAX hplc to analyse the reaction mixtures (Figures 1 and 3).…”

Section: Resultsmentioning

confidence: 99%

“…In addition, piperidine catalysed hydrolysis of the parent chimeric molecules (Miller et al, 1983;Murakami et al, 1985) provided a convenient source of oligodeoxynucleotides with which to evaluate the protective effects of methylphosphonate monoester end groups (Figure 1). Our results suggest that a 3'-phosphodiesterase activity (oligonucleate 5'-nucleotidohydrolase) plays a predominant role in the breakdown of oligodeoxynucleotides by fetal calf serum and that significant increases in the half lives of intact molecules in tissue culture medium can be achieved by merely protecting the 3' ends with methylphosphonate diester or even monoester groups.…”

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confidence: 99%

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Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups

Tidd

Warenius²

1989

Br J Cancer

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Summary Under certain circumstances sequence-specific inhibition of gene expression may be achieved in intact cells using exogenous anti-sense oligodeoxynucleotides. The efficacy of this approach to investigating gene function is limited in part by the rapid serum nuclease mediated degradation of oligodeoxynucleotides in culture media. In order to determine the relative contributions of 3'-exonuclease, 5'-exonuclease and endonuclease activity in fetal calf serum to oligodeoxynucleotide destruction, we have tested chimeric N-ras anti-sense sequence molecules protected against exonuclease attack with terminal methylphosphonate diester linkages. An 18-mer with two methylphosphonate diester linkages at the 3'-terminus, a 20-mer with two methylphosphonate diester groups at both ends, and the 16-mer 3'-methylphosphonate monoester components of their respective piperidine hydrolysates were totally resistant to venom phosphodiesterase, whereas the 16-mer 3'-hydroxyl components of the hydrolysates were rapidly degraded. Both the chimeric oligodeoxynucleotides and 3'-methylphosphonate monoesters were considerably more stable than normal 3'-hydroxyl oligodeoxynucleotides at 37'C in McCoy's 5A medium containing 15% heat inactivated fetal calf serum. Typically 20-30% of the former (initial concentration 10-100 jM) remained intact at 20 h as compared to the latter which were 88-100% degraded in 4 h and undetectable at 20 h. We conclude that a 3'-phosphodiesterase activity is a predominant nuclease responsible for oligodeoxynucleotide degradation by fetal calf serum, and that for cell culture studies, significant protection of oligodeoxynucleotides may be achieved by incorporating 3'-terminal methylphosphonate diester or even monoester end groups.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups

Tidd

Warenius²

1989

Br J Cancer

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“…At these high concentrations, growth rates were somewhat decreased by 48 hr after seeding (by factors of 1.3 and 1.6 for 300 and 150 ,M, respectively). However, there was no apparent decrease in the growth rates of cells exposed to lower (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) ,M) concentrations of d(TpCCTCCTG) (Fig. 3) (24), the rate of amino acid incorporation into acid-insoluble material in HSV-1-infected cells increased at 4 hpi, reached maximal levels at 6-7 hpi (presumably reflecting the synthesis of viral proteins), and declined thereafter.…”

Section: Methodsmentioning

confidence: 99%

“…d(TpCCTCCTG) is a sequence complementary to the previously described (8) sequence of the 3' acceptor splice junction in HSV-1. It was synthesized by solid-phase techniques on a polystyrene support and purified as described (16). The oligomer, characterized as described by Murakami et Fig.…”

Section: Methodsmentioning

confidence: 99%

Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5.

Smith¹,

Aurelian²,

Reddy³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

272

106

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Selective inhibition of regulatory immediate early (IE) genes of herpes simplex virus type 1 (HSV-1) should inhibit virus growth. Treatment of HSV-1-infected cells with the oligo(nucleoside methylphosphonate) d(TpCCTCCTG) (deoxynucleoside methylphosphonate residues in italic), which is complementary to the acceptor splice junction of HSV-1 IE pre-mRNA 4 and 5, before (1-24 hr) or at the time of infection caused a dose-dependent inhibition in virus replication. Virus titers were decreased 50% and 90% in cells treated with 25 ILM and 75 ,uM oligomer, respectively; at 300 tsM, a 99% reduction in virus production was observed. Viral DNA synthesis was reduced 70-75% and there was a 90% reduction in synthesis of viral proteins, including other LE species and viral functional (130-kDa major DNA-binding) and structural (glycoprotein gB) proteins. In the same concentration range, d(TpCCTC-CTG) caused a minimal reduction (0-30%) in protein synthesis and growth rates (<40%) of uninfected cells. The data suggest that oligo(nucleoside methylphosphonate)s may be effective in antiviral chemotherapy.Numerous nucleoside or nucleotide analogues have been screened for antiviral activity (1). Their mode of action exploits differences in the specificity of viral versus host enzymes, thus affecting differentially the rate of viral as compared to host-cell nucleic acid synthesis. However, with rare exceptions, most of them are not effective in experimentally infected animals (2).An alternative approach to antiviral chemotherapy is the use of sequence-specific oligonucleotides or their analogues to selectively inhibit viral gene expression at the level of mRNA processing or translation. For this purpose we have developed sequence-specific nonionic nucleic acid analogues that contain a 3'-5' methylphosphonate group in place of the negatively charged phosphodiester group normally found in oligonucleotides (3). These analogues are resistant to nuclease hydrolysis and penetrate mammalian cells in culture (4). An oligo(deoxyribonucleoside methylphosphonate) complementary to the Shine-Dalgarno sequence of 16S ribosomal RNA inhibits protein synthesis in Escherichia coli, but not in mammalian cells (5). Oligomers complementary to the initiation codon regions ofrabbit globin mRNA inhibit translation in a cell-free system and in rabbit reticulocytes (6), while oligomers complementary to the initiation codon regions of vesicular stomatitis virus mRNAs inhibit viral but not cellular protein synthesis in infected mouse L cells (7).To explore the possibility that control of gene expression by methylphosphonates could be an effective antiviral modality, we synthesized an oligo(nucleoside methylphosphonate) [d(TpCCTCCTG); deoxynucleoside methylphosphonate residues in italic] that is complementary to the acceptor splice junction of herpes simplex virus type 1 (HSV-1) immediate early (IE) mRNAs 4 and 5 (8). The rationale for this choice is based on previous findings indicating that (i) HSV proteins form at least three kinetic groups [IE(a); earl...

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“…Modified oligodeoxynucleotides synthesized to serve as antisense oligomers, a new class of potential chemotherapeutic agents (1)(2)(3)(4)(5), should posses the following properties: good solubility in water, chemical stability, resistance against nucleases, taken up well by cells and stable complex-forming ability with complementary natural target sequence (mRNA or viral RNA). Such oligodeoxynucleotides are those which have (i) modified phosphodiester linkage, like phosphorothioate (6-12), and the 'nonionic' analogues like methylphosphonate (8,9,(12)(13)(14)(15), phosphoramidate (12,16) and phosphotriester (8, 9. 17) or (ii) modified sugar residues, like ca-oligodeoxynucleotides (18)(19)(20)(21) and (iii) the derivatized oligomers (modified as above or not) that are covalently linked to different compounds through terminal 3' or 5'-OH groups, like intercalators to promote hybridization (22)(23)(24)(25), reactive groups to cause selective damage to nucleic acid (26)(27)(28)(29) or poly(L-lysine) to stimulate delivery (30,31).…”

Section: Introductionmentioning

confidence: 99%

Biochemical properties of oligo[(+)]-carbocyclic-thymidylates] and their complexes

et al. 1990

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We report here spectroscopic and biochemical data of a novel series of sugar-modified oligodeoxy-nucleotides, the carbocyclic oligothymidylates, c(dT)3-20. In c(dT)n a methylene group has been substituted for the oxygen atom of the deoxyribose ring of the natural thymidylate unit. c(dT)10-20 form helical structures, in contrast with oligothymidylates or poly(dT), based on absorbance versus temperature melting profiles. Secondary structure of c(dT)n, where n greater than 10 is assumed to be double helix. In addition to this, c(dT)n forms as a stable duplex with complementary poly(dA) as does parent (dT)n. On the other hand, c(dT)n-containing oligo/poly duplex is nearly inactive either as a template or as a primer in various DNA polymerase systems, and c(dT)n inhibits DNA replication as well. c(dT)n can efficiently be extended by terminal transferase and shows an increased nuclease stability compared to (dT)n. Base-pairing ability and nuclease stability of c(dT)n suggest that (+)-carbocyclic nucleoside-containing oligomers could be new potential antisense oligodeoxynucleotides.

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Preparation of oligodeoxyribonucleoside methylphosphonates on a polystyrene support

Abstract: An efficient procedure is described for synthesizing deoxyribonucleoside methylphosphonates on polystyrene polyner supports which involves condensing 5'-dimethoxytrityldeoxynucleoside 3'-methylphosphonates. The

Cited by 66 publications

References 7 publications

Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups

Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups

Antiviral effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5.

Biochemical properties of oligo[(+)]-carbocyclic-thymidylates] and their complexes

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