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A series of dideoxyribonucleoside methylphosphonate analogues, dNpN and dNpNp, which contain a nonionic 3'--5' methylphosphonyl internucleoside linkage were prepared. The two diastereoisomers, designated isomers 1 and 2, of each dimer differ in configuration of the methylphosphonate group and were separated by column chromatography. The diastereoisomers of each dimer have different conformations in solution as shown by ultraviolet hypochromicity data and their circular dichroism spectra. For example, dApA isomer 1 is more highly stacked than isomer 2, although both isomers are less stacked than the dinucleoside monophosphate, dApA. The circular dichroism spectrum of isomer 1 is very similar to that of dApA, while the CD spectrum of isomer 2 shows a loss of molecular ellipticity, [theta], at 270 nm and a greatly diminished [theta] at 250 nm. These results suggest that the stacked bases of dApA isomer 1 tend to orient in an oblique manner, while those in isomer 2 tend to orient in a parallel manner. This interpretation is verified by the 1H NMR study of these dimers (L. S. Kan, D. M. Cheng, P. S. Miller, J. Yano, and P. O. P. Ts'o, unpublished experiments). Both diastereoisomers of dAaA form 2U:1A and 2T:1A complexes with poly(U) and poly(dT), respectively. The higher Tm (Tm of poly(U)--isomer 1, 15.4 degrees C; Tm of poly(U)--isomer 2, 19.8 degrees C; Tm of poly(dT)--isomer 1, 18.7 degrees C; Tm of poly(dT)--isomer 2, 18.4 degrees C) values of these complexes vs. those of the corresponding dApA--polynucleotide complexes (Tm of poly(U)--dApA, 7.0 degrees C; Tm of poly(dT)--DApA, 9.2 degrees C) result from decreased charge repulsion between the nonionic dimer backbone and the negatively charged polymer backbone. The difference in conformations between dApA isomer 1 and dApA isomer 2 is reflected in the Tm of the isomer 1-poly(U) complex which is 4.4 degrees C lower than that of the isomer 2-poly(U) complex. Since these nonionic oligonucleotide analogues are taken up by cells in culture, they show promise as molecular probes for the function and structure of nucleic acids inside living cells.

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Oligodeoxyribonucleoside methylphosphonates with base sequences complementary to the anticodon loop of tRNALys and to the -ACCA-OH amino acid accepting stem of tRNA were prepared by chemical synthesis. Oligodeoxyadenosine methylphosphonates form stable, triple-stranded complexes with both poly(U) and poly(dT). These analogues selectively inhibit cell-free aminoacylation of tRNALys (E. coli) but have no effect on aminoacylation of tRNALys (rabbit). The extent of inhibition is temperature dependent and parallels the ability of the oligomer to bind to poly(U), which suggests that inhibition occurs as a result of oligomer binding to the -UUUU- anticodon loop of tRNALys (E. coli). The failure of the oligodeoxyadenosine methylphosphonates to inhibit tRNALys (rabbit) amino-acylation suggests that there may be a difference between the structure of tRNALys or its interaction with aminoacyl synthetase in the Escherichia coli and rabbit systems. The oligodeoxyadenosine analogues also effectively inhibit polyphenylalanine synthesis in cell-free translation systems derived from both E. coli and rabbit reticulocytes. The extent of inhibition parallels the Tm values of the oligo(A) phosphonate-poly(U) complexes and suggests that the inhibition is a consequence of complex formation with the poly(U) message. Tritium-labeled oligodeoxyribonucleoside methylphosphonates with a chain length of up to nine nucleotidyl units are taken up intact by mammalian cells in culture. All the oligomer analogues tested inhibited, to various extents, colony formation by bacterial, hamster, and human tumor cells in culture.

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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