Oligodeoxynucleoside Methylphosphonates Research Articles

Unexpected High Hybridization Properties of α-Anomeric Oligodeoxynucleoside Methylphosphonates

The Watson-Crick base-pairing properties of oligodeoxynucleoside methylphosphonates containing a-nucleoside units have been evaluated and compared to those of others ionic and non-ionic α- or β-oligonucleotide analogs.

Tissue Distribution and Metabolism of the [32P]-Labeled Oligodeoxynucleoside Methylphosphonate-Neoglycopeptide Conjugate, [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7, in the Mouse

Development of oligodeoxynucleotides (oligo-dNs) and their analogs as therapeutic agents is complicated by their low rate of transport across cellular membranes, which is required for interaction with the intracellular complementary nucleic acid sequences, and the lack of tissue-specific delivery. To overcome these obstacles, bioconjugates between cell surface receptor ligands and oligodeoxynucleoside methylphosphonates (oligo-MPs) have been constructed containing homogeneous, chemically defined covalent linkages. We have previously established that a model conjugate, [32P]-labeled [YEE(ah-GalNAc)3]-SMCC-AET-pUmpT7 (1), is delivered to Hep G2 cells in a ligand-specific manner, reaching a peak value of 26 pmol per 10(6) cells after 24 hours incubation at 37 degrees C (Hangeland et al., 1995). In this work, the in vivo behavior of this conjugate is explored. Administration of this conjugate to mice via tail vein injection demonstrates rapid uptake in liver to the extent of 69.9 +/- 9.9% of the injected dose after 15 minutes. Thereafter, the conjugate and its metabolites are rapidly cleared via the kidney and urine. Polyacrylamide gel electrophoresis analysis of extracts of Hep G2 cells and mouse liver reveal the conjugate 1 to be extensively metabolized. In contrast, the conjugate found in mouse urine is largely intact. These data show that this novel, biodegradable delivery vehicle represents a viable approach for the delivery of antisense oligo-MPs and other oligo-dN analogs to the liver for therapeutic and diagnostic applications.

Transformed and immortalized cellular uptake of oligodeoxynucleoside phosphorothioates, 3′-Alkylamino oligodeoxynucleotides, 2′- o-methyl oligoribonucleotides, oligodeoxynucleoside methylphosphonates, and peptide nucleic acids

Direct quantitative comparisons of cellular uptake across a wide variety of analogs and cell types are necessary for the design of oligonucleotide diagnostic and therapeutic applications. This work reports quantitative cellular uptake and nuclear localization of [ 14C]oligodeoxynucleoside phosphorothioates (PS), 3′-alkylamino Oligodeoxynucleoside phosphodiesters (PO-NH 2), 2′-O-methyl oligoribonucleoside phosphodiesters (2OM), peptide nucleic acids (PNA), and oligodeoxynucleoside methylphosphonates (MP) in several transformed or immortalized cell lines. All analogs demonstrated active cellular uptake in that intracellular concentrations greatly exceeded the extracellular 1 μM concentration within 1–3 hr. However, by 9–24 hr, cellular accumulations of PS exceeded those of PO-NH 2 and 2OM by 3- to 5-fold, PNA by 6- to 7-fold, and MP by 8- to 10-fold. Similar results were observed in two transformed cell lines, HL-60 leukocytes and H- ras transformed fibroblasts, using three different heterogeneous sequences. H- ras and IGF-1R transformed fibroblasts had a 2- to 5-fold higher uptake of all analogs than non-transformed immortalized fibroblasts. Nuclear levels of the PO-NH 2, PS, and MP analogs were approximately 25% of total cellular uptake, while nuclear percentages of 2OM and PNA were less than 20%, suggesting some differences in nuclear localization among the analogs. These observations provide a direct quantitative comparison of cellular uptake as a function of oligonucleotide modification, and imply that transformation enhances cellular uptake. From the perspective of therapy and diagnosis, clear trade-offs were apparent between efficiency of uptake on the one hand, and nuclease resistance and hybridization strength on the other.

Synthesis of oligodeoxynucleoside methylphosphonates utilizing the tert-butylphenoxyacetyl group for exocyclic amine protection.

Oligodeoxynucleoside methylphosphonates were synthesized using methylphosphonamidite monomers incorporating the base labile tert-butylphenoxyacetyl (t-BPA) protecting group on the exocyclic amines of dA, dC, and dG. Synthesis of the oligodeoxynucleoside methylphosphonates required only a small change in oxidation solution from standard DNA synthesis. The increased lability of the t-BPA group over the standard benzoyl and isobutyryl protection permitted the use of milder basic deprotection conditions. Deprotection of the nucleoside bases and release from support was best accomplished by a short treatment with ammonia saturated methanol. This procedure resulted in minimal backgone degradation with no base modifications. Analysis of the resultant oligodeoxynucleoside methylphosphonates by reverse phase HPLC and MALDI-TOF mass spectroscopy are described.

Preparation of trimers and tetramers of mixed sequence oligodeoxynucleoside methylphosphonates and assignment of configurations at the chiral phosphorus

Synthesis of stereoregular DNA methylphosphonates has been accomplished for homo-oligomers, but remains a formidable problem for oligomers of a defined antisense target sequence. In this work, four trimer and tetramer deoxynucleoside methylphosphonates of mixed sequence (dACA, dCCAA, dAGGG, and dGCAT) were prepared by block coupling of diastereomerically pure dimers with either monomers or other diastereomerically pure dimers. These oligomers were separated chromatographically into individual diastereomers, and the configurations of the chiral methylphosphonate linkages were assigned. Three types of methods were used to assign configuration of a new methylphosphonate linkage: preparation of the same diastereomer through multiple synthetic pathways, base hydrolysis, and acid hydrolysis. Hydrolysis of the diastereomerically pure oligomers into component dimers and monomers was followed by chromatographic comparison with control dimers of known configuration. In all cases studied, oligomers with R configurations displayed faster elution from silica gel than did oligomers with the respective S configuration. NMR spectra of individual diastereomers of dACA were studied, revealing characteristic differences in chemical shifts which may prove useful in configurational assignments of longer oligomers. Thus, these data provide a methodological basis for synthesis and configurational assignment of longer methylphosphonate oligomers to use as antisense probes.

Inhibition of acquired immunodeficiency syndrome virus by oligodeoxynucleoside methylphosphonates.

Antisense oligodeoxynucleotides containing internucleoside methylphosphonate linkages were examined for their ability to inhibit human immunodeficiency virus (HIV)-induced syncytium formation and virus expression. HIV inhibitory activity was found to be dependent on both chain length and the number of phosphonate residues. Introduction of 18 phosphonate groups in an oligomer of chain length 20 significantly increased HIV inhibitory activity relative to the parent oligonucleotide, whereas 5 such groups showed little or no increase in the HIV inhibition capacity. Methylphosphonate-linked oligomers are more stable to nuclease degradation and hence could be potentially useful in the treatment of acquired immunodeficiency syndrome.

Methylphosphonamidites: Preparation and Application in Oligodeoxynucleoside Methyl-Phosphonate Synthesis

Abstract The synthesis of methylphosphonamidites (Fig. 1) is described for all four deoxynucleosides. The T-amidte 6 was used on an automated DNA-synthesizer to show the usefulness in the solid phase synthesis of oligodeoxy-nucleoside methylphosphonates.

ChemInform Abstract: Oligodeoxynucleoside Methylphosphonates: Synthesis and Enzymic Degradation.

ChemInformVolume 19, Issue 3 Natural Products ChemInform Abstract: Oligodeoxynucleoside Methylphosphonates: Synthesis and Enzymic Degradation. S. AGRAWAL, S. AGRAWAL Worcester Found. Exper. Biol., Shrewsbury, MA 01545, USASearch for more papers by this authorJ. GOODCHILD, J. GOODCHILD Worcester Found. Exper. Biol., Shrewsbury, MA 01545, USASearch for more papers by this author S. AGRAWAL, S. AGRAWAL Worcester Found. Exper. Biol., Shrewsbury, MA 01545, USASearch for more papers by this authorJ. GOODCHILD, J. GOODCHILD Worcester Found. Exper. Biol., Shrewsbury, MA 01545, USASearch for more papers by this author First published: January 19, 1988 https://doi.org/10.1002/chin.198803337AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume19, Issue3January 19, 1988 RelatedInformation

Oligodeoxynucleoside methylphosphonates: synthesis and enzymic degradation

The automated synthesis of oligodeoxynucleosides containing methylphosphonate linkages has been improved by using nucleoside methylphosphonamidites as starting materials. It was found that two adjacent methylphosphonate linkages protect an oligomer from degradation by snake venom phophodiesterase or spleen phophodiesterase.

A new synthesis of oligodeoxynucleoside methylphosphonates on control pore glass polymer support using phosphite approach

Dimer, trimer and tetramer blocks of deoxynucleoside methylphosphonodiesters d(AC), d(AT), d(ATG) and d(ATTT) were synthesizedon CPG polymer support using deoxynucleoside methylphosphonomonochloridite. Only dinucleotide methylphosphonates werephosphorylated at 5′-OH with T4-polynucleotide kinase. These phosphorylated dimers hydrolized quantitatively with NaOH (0.5 M) in 15 minutes at room temperature.