Synthesis of specific diastereomers of a DNA methylphosphonate heptamer, d(CpCpApApApCpA), and stability of base pairing with the normal DNA octamer d(TPGPTPTPTPGPGPC).

DNA methylphosphonates are candidate derivatives for use in antisense DNA therapy. Their efficacy is limited by weak hybridization. One hypothesis to explain this phenomenon holds that one configuration of the chiral methylphosphonate linkage, Rp, permits stronger base pairing than the other configuration, Sp. To test this hypothesis, four specific pairs of Rp and Sp diastereomers of the DNA methylphosphonate heptamer d(CpCpApApApCpA) were prepared by block coupling of different combinations of individual diastereomers of d(CpCpApA) and d(ApCpA). Each pair of the diastereomers of the heptamer was separated into individual diastereomes using affinity chromatography on a Lichrosorb-NH2 silica column with a covalently attached complementary normal DNA octamer, d(pTpGpTpTpTpGpGpC). The stabilities of complementary complexes of phosphodiester d(TpGpTpTpTpGpGpC) with 8 individual diastereomers of methylphosphonate d(CpCpApApApCpA) were studied by measuring their melting temperatures (Tm). A direct correlation of Tm values with the number of Rp methylphosphonate centers in the heptamer was found: the more Rp centers, the higher the stability of the complex. Tm values for the diastereomers with 6 all-Rp or all-Sp methylphosphonate centers were found to be 30.5 degrees and 12.5 degrees C, respectively, in 100 mM NaCl, 10 mM Na2HPO4, 1 mM EDTA, pH 7.0 with 15 microM of each oligomer. On the average, each substitution of one Rp-center to an Sp-center in the heptamer decreased the Tm by 3 degrees C. Under the same conditions, the Tm of the normal DNA heptamer with its complement was 21 degrees C. These results are consistent with the model that all-Rp methylphosphonate DNAs hybridize much more tightly to complementary normal DNA than do racemic methylphosphonate DNAs, and may therefore exhibit greater potency as antisense inhibitors.     

Preparation of oligodeoxyribonucleoside methylphosphonates on a polystyrene support.

An efficient procedure is described for synthesizing deoxyribonucleoside methylphosphonates on polystyrene polymer supports which involves condensing 5'-dimethoxytrityldeoxynucleoside 3'-methylphosphonates. The oligomers are removed from the support and the base protecting groups hydrolyzed by treatment with ethylenediamine in ethanol, which avoids hydrolysis of the methylphosphonate linkages. Two types of oligomers were synthesized: those containing only methylphosphonate linkages, d-Np(Np)nN, and those which terminate with a 5' nucleotide residue, dNp (Np)nN. The latter oligomers can be phosphorylated by polynucleotide kinase, and are separated by polyacrylamide gel electrophoresis according to their chain length. Piperdine randomly cleaves the oligomer methylphosphonate linkages and generates a series of shorter oligomers whose number corresponds to the length of the original oligomer. Apurinic sites introduced by acid treatment spontaneously hydrolyze to give oligomers which terminate with free 3' and 5' OH groups. These reactions may be used to characterize the oligomers.     

Antisense oligodeoxynucleoside methylphosphonate inhibition of mouse c-myc p65 protein expression in E mu-c-myc transgenic mice.

In transgenic mice bearing a murine immunoglobulin enhancer/c-myc fusion transgene (E mu-myc), it was found that methylphosphonates do not induce acute toxicity following intravenous administration of a 300 nmol dose. In addition, recovery of methylphosphonates from the blood plasma of treated mice indicated that the oligomers remained intact up to 3 hours, while their concentrations decreased rapidly for the first hour, then slowly decreased over the next two hours. Finally, methylphosphonate oligomers targeted against c-myc mRNA inhibited production of c-myc p65 protein in peripheral and splenic lymphocytes, relative to a scrambled sequence oligomer, 4 hours after injection of a 300 nmol dose, as indicated by immunofluorescence of fixed cells stained with an anti-c-myc antiserum.     

Control of ribonucleic acid function by oligonucleoside methylphosphonates

Oligodeoxyribonucleoside methylphosphonates contain nonionic 3'-5' linked methylphosphonate internucleotide bonds in place of the normal charged phosphodiester linkage of natural nucleic acids. These oligomers are resistant to nuclease hydrolysis, can pass through the membranes of mammalian cells in culture and can form stable hydrogen-bonded complexes with complementary nucleotide sequences of cellular RNAs such as mRNA. The oligomers are readily synthesized on insoluble polymer supports. Their chainlength and nucleotide sequence can be determined by chemical sequencing procedures. Oligonucleoside methylphosphonates which are complementary to the 5'-end, initiation codon region, or coding region of rabbit globin mRNA inhibit translation of the mRNA in rabbit reticulocyte lysates and globin synthesis in rabbit reticulocytes. This inhibition is due to the interaction of the oligomers with mRNA and the extent of inhibition is influenced by the secondary structure of the mRNA and the location of oligomer binding site on the mRNA. Oligomers complementary to the initiation codon regions of N, NS and G protein mRNAs of Vesicular stomatitis virus (VSV) inhibit virus protein synthesis in VSV-infected Mouse L-cells. These oligomers do not affect L-cell protein synthesis or growth. Virus protein synthesis and growth can also be selectively inhibited by oligonucleoside methylphosphonates which are complementary to the donor or acceptor splice junctions of virus pre mRNA. An oligomer complementary to the donor splice junction of SV40 large T antigen mRNA inhibits T-antigen synthesis in SV40-infected African green monkey kidney cells but does not inhibit overall cellular protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylphosphonate Modified DNA Hairpin Loops

Abstract

We synthesized and analyzed DNA hairpin molecules with methylphosphonate linkages of defined stereochemistry in the loop region. Dinucleotide building blocks ApA and TpT (p indicating methylphosphonate linkage with either Rp or Sp configuration) were synthesized, separated into the diastereomers, and incorporated at three positions of the tetraloops 5′-CGCAAAAGCG-3′ and 5′-CGCTTTTGCG-3′. The oligonucleotides were analyzed for their melting behavior. With a Tm of 67.5°C the molecule 5′-CGCAAApAGCG-3′ with a Sp configurated methylphosphonate is distinctly more stable than the Rp configurated one (Tm = 60.5 °C) and the unmodified oligonucleotide (Tm = 64.5 °C). In contrast to double helical DNA where the substitution of a phosphorodiester by a Sp configurated methylphosphonate results in a lower Tm, in DNA hairpin the introduction of Sp and Rp methylphosphonates at specific positions can lead to a stabilization of the structure.     

Syntheses of alternating oligo-2'-O-methylribonucleoside methylphosphonates and their interactions with HIV TAR RNA

Oligonucleotide analogues 15-20 nucleotides in length have been prepared, whose sequences are complementary to nucleotides in the upper hairpin of HIV TAR RNA. These alternating oligonucleoside methylphosphonates, mr-AOMPs, contain 2'-O-methylribonucleosides and alternating methylphosphonate and phosphodiester internucleotide linkages. The methylphosphonate and phosphodiester linkages of these oligomers are highly resistant to hydrolysis by exonuclease activity found in mammalian serum and to endonucleases, such as S1 nuclease. The oligomers were prepared using automated phosphoramidite chemistry and terminate with a 5'-phosphate group, which provides an affinity handle for purification by strong anion exchange HPLC. A 15-mer mr-AOMP, 1676, that is complementary to the 5'-side of the TAR RNA hairpin, including the 3-base bulge and 6-base loop region, forms a 1:1 duplex with a complementary RNA 18-mer, mini-TAR RNA. The T(m) of this duplex is 71 degrees C, which is similar to that of the duplex formed by the corresponding all phosphodiester 15-mer. Introduction of two mismatched bases reduces the T(m) by 17 degrees C. The apparent dissociation constant, K(d), for the 1676/mini-TAR RNA duplex as determined by an electrophoretic mobility shift assay at 37 degrees C is 0.3 nM. Oligomer 1676 also binds tightly to the full length TAR RNA target under physiological conditions (K(d) = 20 nM), whereas no binding was observed by the mismatched oligomer. A 19-mer that is complementary to the entire upper hairpin also binds to TAR RNA with a K(d) that is similar to that of 1676, a result that suggests only part of the oligomer binds. When two of the methylphosphonate linkages in the region complementary to the 6-base loop are replaced with phosphodiester linkages, the K(d) is reduced by approximately a factor of 10. This result suggests that interactions between TAR RNA and the oligomer occur initially with nucleotides in the 6-base loop, and that these interactions are sensitive to presence and possibly the chirality of the methylphosphonate linkages in the oligomer. The high affinities of mr-AOMPs for TAR RNA and their resistance to nuclease hydrolysis suggests their potential utility as antisense agents in cell culture.     

Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA

Oligodeoxyribonucleoside methylphosphonates derivatized at the 5' end with 4'-(amino-alkyl)-4,5',8-trimethylpsoralen were prepared. The interaction of these psoralen-derivatized methylphosphonate oligomers with synthetic single-stranded DNAs 35 nucleotides in length was studied. Irradiation of a solution containing the 35-mer and its complementary methylphosphonate oligomer at 365 nm gave a cross-linked duplex produced by cycloaddition between the psoralen pyrone ring of the derivatized methylphosphonate oligomer and a thymine base of the DNA. Photoadduct formation could be reversed by irradiation at 254 nm. The rate and extent of cross-linking were dependent upon the length of the aminoalkyl linker between the trimethylpsoralen group and the 5' end of the methylphosphonate oligomer. Methylphosphonate oligomers derivatized with 4'-[[N-(2-aminoethyl)amino]methyl]- 4,5',8-trimethylpsoralen gave between 70% and 85% cross-linked product when irradiated for 20 min at 4 degrees C. Further irradiation did not increase cross-linking, and preirradiation of the psoralen-derivatized methylphosphonate oligomer at 365 nm reduced or prevented cross-linking. These results suggest that the methylphosphonate oligomers undergo both cross-linking and deactivation reactions when irradiated at 365 nm. The extent of cross-linking increased up to 10 microM oligomer concentration and dramatically decreased at temperatures above the estimated Tm of the methylphosphonate oligomer-DNA duplex. The cross-linking reaction was dependent upon the fidelity of base-pairing interactions between the methylphosphonate oligomers and the single-stranded DNA. Noncomplementary oligomers did not cross-link, and the extent of cross-linking of oligomers containing varying numbers of noncomplementary bases was greatly diminished or eliminated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photochemical cross-linking of psoralen-derivatized oligonucleoside methylphosphonates to rabbit globin messenger RNA

Antisense oligodeoxyribonucleoside methylphosphonates targeted against various regions of mRNA or precursor mRNA are selective inhibitors of mRNA expression both in cell-free systems and in cells in culture. The efficiency with which methylphosphonate oligomers interact with mRNA, and thus inhibit translation, can be considerably increased by introducing photoactivatable psoralen derivatives capable of cross-linking with the mRNA. Oligonucleoside methylphosphonates complementary to coding regions of rabbit alpha- or beta-globin mRNA were derivatized with 4'-(aminoalkyl)-4,5',8-trimethylpsoralens by attaching the psoralen group to the 5' end of the oligomer via a nuclease-resistant phosphoramidate linkage. The distance between the psoralen group and the 5' end of the oligomer can be adjusted by changing the number of methylene groups in the aminoalkyl linker arm. The psoralen-derivatized oligomers specifically cross-link to their complementary sequences on the targeted mRNA. For example, an oligomer complementary to nucleotides 56-67 of alpha-globin mRNA specifically cross-linked to alpha-globin mRNA upon irradiation of a solution of the oligomer and rabbit globin mRNA at 4 degrees C. Oligomers derivatized with 4'-[[N-(2-amino-ethyl)amino]methyl]-4,5',8-trimethylpsoralen gave the highest extent of cross-linking to mRNA. The extent of cross-linking was also determined by the chain length of the oligomer and the structure of the oligomer binding site. Oligomers complementary to regions of mRNA that are sensitive to hydrolysis by single-strand-specific nucleases cross-linked to an approximately 10-30-fold greater extent than oligomers complementary to regions that are insensitive to nuclease hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methylphosphonates as probes of protein-nucleic acid interactions.

Deoxydinucleoside methylphosphonates were prepared by chemical synthesis and were introduced stereospecifically into the lac operator at two sites. These sites within d(ApApTpTpGpTpGpApGpCpGpGpApTpApApCpApApTpT), segment I, and d(ApApTpTpGpTpTpApTpCpCpGpCpTpCpApCpApApTpT), segment II, are indicated by p. Each segment containing a chiral methylphosphonate was annealed to the complementary unmodified segment. The interactions of these four modified lac operators with lac repressor were analyzed by the nitrocellulose filter binding assay. Introduction of either chiral phosphonate in segment II had little effect on the stability of the repressor-operator complex. When methylphosphonates were introduced into segment I, the affinity of lac repressor for the modified operators was shown to be dependent on the stereochemical configuration of the methylphosphonate.     

Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system.

Antisense oligonucleotides containing either anionic diester or neutral methylphosphonate internucleoside linkages were prepared by automated synthesis, and were compared for their ability to arrest translation of human dihydrofolate reductase (DHFR) mRNA in a nuclease treated rabbit reticulocyte lysate. In the case of oligodeoxyribonucleotides, tandem targeting of three 14-mers resulted in synergistic and complete selective inhibition of DHFR synthesis at a total oligomer concentration of 25 microM. Hybrid arrest by three or six tandem oligodeoxyribonucleoside methylphosphonates was dramatically less effective. This difference does not result from preferential recognition of hybrids involving oligodeoxyribonucleotides by endogenous RNaseH activity. A ribonuclease protection assay demonstrated that antisense oligodeoxyribonucleoside methylphosphonates bind selectively to target RNA sequences, but with 275 fold lower affinity than the corresponding oligodeoxyribonucleotides. This low binding affinity results in poor arrest of translation, and may be related to the stereochemistry of the methylphosphonate linkage.     

Sequence dependent effects in methylphosphonate deoxyribonucleotide double and triple helical complexes.

Deoxyribooligonucleotides containing 19 repeating bases of A, T or U were prepared with normal phosphodiester (dA19, dT19, dU19) or methylphosphonate (dA*19, dT*19, dU*19) linkages. Complexes of these strands have been investigated at 1:1 and 1:2 molar ratios (purine:pyrimidine) by thermal melting and gel electrophoresis. There are dramatic sequence dependent differences in stabilities of complexes containing methylphosphonate strands. Duplexes of dA*19 with dT19 or dU19 have sharp melting curves, increased Tm values, and slopes of Tm versus log (sodium ion activity) plots reduced by about one half relative to their unmodified 'parent' duplexes. Duplexes of dA19 with either dT*19 or dU*19, however, have broader melting curves, reduced Tm values at most salt concentrations and slopes of less than one tenth the values for the unmodified duplexes. Duplex stabilization due to reduced phosphate charge repulsion is offset in the pyrimidine methylphosphonate complexes by steric and other substituent effects. Triple helical complexes with dA19 + 2dT19 and dA19 + 2dU19, which can be detected by biphasic melting curves and gel electrophoresis, are stable at increased Na+ or Mg+2 concentrations. Surprisingly, however, no triple helix forms, even at very high salt concentrations, when any normal strand(s) is replaced by a methylphosphonate strand. Since triple helical complexes with methylphosphonates have been reported for shorter oligomers, inhibition with larger oligomers may vary due to their length and extent of substitution.     

Synthesis of Oligodeoxyribonucleoside Methylphosphonates Containing 2-Aminopurine

Abstract

Oligodeoxyribonucleoside methylphosphonates containing multiple 2-aminopurine bases were synthesized by solid-phase phosphonamidite chemistry for investigating their triple helix formation with natural DNA or other duplex targets and for cellular uptake studies of the methylphosphonate oligomers. The base-labile phenoxyacetyl group was used as the N ²-amino protecting group for 2-aminopurine, allowing the final deprotection of the oligomers to be performed under the standard ethylenediamine condition.     

Solid-phase synthesis of oligo-2-pyrimidinone-2'-deoxyribonucleotides and oligo-2-pyrimidinone-2'-deoxyriboside methylphosphonates.

A synthetic method was developed for the synthesis of oligodeoxyribonucleotides and oligodeoxyribonucleoside methylphosphonates comprised exclusively of the fluorescent 2-pyrimidinone base for the first time. The method utilized the solid-phase 2-cyanoethylphosphoramidite and methylphosphonamidite chemistry for internucleotide couplings and a baselabile oxalyl linkage to anchor the oligomers onto the CPG support. Cleavage of the oligomers from the support was effected by a short treatment of the support with 5% ammonium hydroxide in methanol at room temperature, without any degradation of the base-sensitive 2-pyrimidinone residues or the base-sensitive methylphosphonate backbone.     

Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site

The synthesis and characterization of an octanucleotide, d(GGsAATTCC), containing the recognition sequence of the EcoRI restriction endonuclease with a phosphorothioate internucleotidic linkage at the cleavage site are described. Two approaches for the synthesis of the RP and SP diastereomers of this octamer by the phosphite method are presented. The first consists of the addition of sulfur instead of H2O to the phosphite at the appropriate position during chain elongation. This method results in a mixture of diastereomers that can be separated by high-performance liquid chromatography after 5'-terminal phosphorylation. The second uses the presynthesized and diastereomerically pure dinucleoside phosphorothioate d[Gp(S)A] for the addition to the growing oligonucleotide chain as a block. The products are characterized by digestion with nuclease P1, fast atom bombardment mass spectrometry, 31P NMR spectroscopy, and conversion to d(GGAATTCC) by desulfurization with iodine. Only the RP diastereomers of d(GGsAATTCC) and its 5'-phosphorylated derivative are cleaved by EcoRI endonuclease. The rate of hydrolysis is slower than that of the unmodified octamer. The phosphorothioate octamer will be useful for the determination of the stereochemical course of the EcoRI-catalyzed reaction.     

Interactions of hairpin oligo-2'-O-methylribonucleotides containing methylphosphonate linkages with HIV TAR RNA

Methylphosphonate-modified oligo-2'-O-methylribonucleotides 15-20 nucleotides (nt) in length were prepared whose sequences are complementary to the 5' and 3' sides of the upper hairpin of HIV trans-acting response element (TAR) RNA. These anti-TAR oligonucleotides (ODNs) form stable hairpins whose melting temperatures (Tm) range from 55 degrees C to 80 degrees C. Despite their rather high thermal stabilities, the hairpin oligo-2'-O-methylribonucleotides formed very stable complexes with TAR RNA, with dissociation constants in the nanomolar concentration range at 37 degrees C. The affinities of the hairpin oligomers for TAR RNA were influenced by the positions of the methylphosphonate linkages. The binding affinity was reduced approximately 17-fold by the presence of two methylphosphonate linkages in the TAR loop complementary region (TLCR) of the oligomer, whereas methylphosphonate linkages outside this region increased binding affinity approximately 3-fold. The configurations of the methylphosphonate linkages in the TLCR also affected binding affinity, with the RpRp isomer showing significantly higher binding than the SpSp isomer. In addition to serving as probes of the interactions between the oligomer and TAR RNA, the presence of the methylphosphonate linkages in combination with the hairpin structure increases the resistance of these oligomers to degradation by exonucleases found in mammalian serum. The combination of high binding affinity and nuclease resistance of the hairpin ODNs containing methylphosphonate linkages suggests their potential utility as antisense compounds.     

P-chiral oligonucleotides. 2D Roesy NMR assignment of absolute configuration at phosphorus and conformational analysis of 5'-O-monomethoxytrityl-(2'-O-deoxyribonucleoside) 3'-O-[O-(4-nitrophenyl)]methanephosphonates

Fast and simple methodology for the assignment of the absolute configuration at the phosphorus atom in diastereomerically pure Rp and Sp 5'-O-monomethoxytrityl-2'-O-deoxynucleoside 3'-O-(O-4-nitrophenyl) methanephosphonate (3) was established. The method utilizes 2D ROESY NMR and can be used for the stereochemical analysis of other P-chiral mononucleotides. Configurational analysis shows that the major conformation of the sugar residue in 3 is of the S (South) type. This study will facilitate synthesis of stereoregular methylphosphonate oligonucleotide analogues via the transesterification method.     

Phosphoramidites of (Rp) and (Sp)-Configurated Adenosine Methylphosphonate Dimers

Abstract

The synthesis of adenosine methylphosphonate dimers, the separation of the diastereomers and the conversion into phosphoramidites is presented. These dimer building blocks were tested in the solid phase DNA synthesis of the hairpin decanucleotide 5′-CGCAAAAGCG-3′.     

Use of EDTA derivatization to characterize interactions between oligodeoxyribonucleoside methylphosphonates and nucleic acids

EDTA-derivatized oligonucleoside methylphosphonates were prepared and used to characterize hybridization between the oligomers and single-stranded DNA or RNA. The melting temperatures of duplexes formed between an oligodeoxyribonucleotide 35-mer and complementary methylphosphonate 12-mers were 4-12 degrees C higher than those of duplexes formed by oligodeoxyribonucleotide 12-mers as determined by spectrophotometric measurements. Derivatization of the methylphosphonate oligomers with EDTA reduced the melting temperature by 5 degrees C. Methylphosphonate oligomer-nucleic acid complexes were stabilized by base stacking interactions between the terminal bases of the two oligomers binding to adjacent binding sites on the target. In the presence of Fe2+ and DTT, the EDTA-derivatized oligomers produce hydroxyl radicals that cause degradation of the sugar-phosphate backbone of both targeted DNA and RNA. Degradation occurs specifically in the region of the oligomer binding site and is approximately 20-fold more efficient for single-stranded DNA than for RNA. In comparison to the presence of one oligomer, the extent of target degradation was increased considerably by additions of two oligomers that bind at adjacent sites on the target. For example, the extent of degradation of a single-stranded DNA 35-mer caused by two contiguously binding oligomers, one of which was derivatized by EDTA, was approximately 2 times greater than that caused by the EDTA-derivatized oligomer alone. Although EDTA-derivatized oligomers are stable for long periods of time in aqueous solution, they undergo rapid autodegradation in the presence of Fe2+ and DTT with half-lives of approximately 30 min. This autodegradation reaction renders the EDTA-derivatized oligomers unable to cause degradation of their complementary target nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivity of oligonucleotide derivatives, containing a methylphosphate group. Affinity modification of target DNA by 4-N-(methyl-N-2-chlorethylamino)benzyl 3'- and 5'-phosphamide derivatives of octathymidylate containing methylphosphonate residues

Modification of 5'-32P-labelled octadecadeoxyribonucleotide d(pC5A8C5) (III) with octathymidylate methylphosphonate derivatives bearing both 3'- and 5'-terminal alkylating 4-(N-2-chloroethyl-N-methylamino)benzylphosphoamide residue has been investigated. Yield in the modification depends on configuration of methylphosphonate fragment, in case of Rp-isomer it may amount to 90%. Specificity of alkylation of nucleic acide target (III) by reagents based on the oligonucleotide methylphosphonates is almost the same as by reagents based on the oligonucleotides having phosphodiester internucleotide bonds.