Phosphorothioate-modified antisense oligonucleotides (PS-ASOs) interact with a host of plasma, cell-surface and intracellular proteins which govern their pharmacokinetic, pharmacological and toxicological properties. Within cells, PS ASOs interact with ∼60 cellular proteins such as P54nrb which have RNA-recognition motifs (RRMs) as well as chaperone proteins such as HSP90 which lack RNA- or DNA-binding domains and other proteins. Gapmer ASOs have a central gap-region of 7–12 phosphorothioate (PS) modified DNA flanked on either end with modifications which enhance nuclease stability and affinity for complementary RNA. Gapmer ASOs bind their targeted RNA in cells and the resulting RNA/DNA duplexes are substrates for RNaseH1, which selectively cleaves the RNA strand of the heteroduplex. Our recent investigations revealed that toxic ASOs show enhanced binding to cellular proteins as compared to safe ASOs, and cause RNaseH1-dependent nucleolar mislocalization of paraspeckle proteins including P54nrb, nucleolar stress and fragmentation, upregulation of P21 mRNA and activation of caspase activity indicative of apoptosis. Introducing 2′-OMe RNA (OMe) at gap-position 2 from the 5′ wing-gap junction reduced global protein binding, and mitigated cytotoxicity in cells and hepatotoxicity in mice resulting in an improvement in therapeutic index (TI). ASOs with charge-neutral methylphosphonate (MP) linkages have been known almost as long as the PS-modification but have only been used sparingly in the context of ASO drug-discovery . MPs do not support RNaseH1-mediated RNA cleavage near the site of incorporation into an ASO. MPs can be incorporated into nucleic acids using standard chemistry, but MP-modified oligonucleotides are more susceptible to strand cleavage under the basic conditions required to deprotect oligonucleotides after solid-phase synthesis. To address this limitation, we designed the methoxypropylphosphonate (MOP) linkage, which has more steric bulk than MP but similar structural properties as the methoxyethyl group in MOE nucleosides, and examined its potential for enhancing the therapeutic profile of gapmer ASOs.
Synthesis of N,N,N’,N’-tetraisopropyl-1-(3-methoxypropyl) phosphanediamine
Magnesium metal (4.18 g, 171 mmol) was suspended in dry THF (150 ml) in a dry round bottom flask (250 ml capacity) and was purged with dry nitrogen. 1-Bromo-3-methoxypropane (106 mmol, 12 ml) was added in portions via syringe over ∼ 20 min. The reaction was slightly exothermic and was immersed in a water bath (no ice) to control the temperature. The reaction was stirred for an additional 10 min after addition was complete. In a separate dry round bottom flask (1 L capacity), 1-chloro-N,N,N’,N’-tetraisopropylphosphanediamine (93.8 mmol, 25 g) was suspended in dry Et2O (300 ml), and the reaction was cooled in an ice bath (0°C) with stirring under nitrogen. The Grignard reagent (generated above) was transferred to the chlorophosphine suspension via cannula. The reaction was allowed to come to room temperature and was stirred for 1 h. The reaction was monitored by 31P NMR. The reaction mixture was filtered through a plug of celite, and the solids were rinsed with Et2O. The filtrates were pooled, and the solvent was removed on a rotary evaporator. The residue was suspended in dry acetonitrile (∼75 ml). The reaction mixture was transferred to a separatory funnel and was extracted with hexanes (∼250 ml). The hexanes layer was washed with acetonitrile (2 × 150 ml). The hexanes layer was collected and passed thru a plug of cotton to remove particulates and traces of water. The filtrate was concentrated a rotary evaporator and the residue was dried under high vacuum briefly to yield the phosphine reagent as an oil (27.10 g, 84%), which was used immediately for the next reaction. 31P NMR (121 MHz, CDCl3) δ = 47.70.
General method for the synthesis of mop phosphoramidites
The 5′-O-DMTr-protected nucleoside (1 equiv.) was dissolved in dry DMF (0.1 M) followed by addition of 1H-tetrazole (0.8 equiv.) and 1-methyl imidazole (0.44 equiv.). The MOP phosphorylation reagent from above (1.5 equiv.) was added and the reaction was allowed to stir at room temperature for 12–16 h under nitrogen. Upon completion the solvent was removed using a rotary evaporator (keep bath temperature under 35°C). The residue was diluted with EtOAc (100 ml) and transferred to a separatory funnel. The organic layer was washed with H2O/brine (1:1, 200 ml) mixture, followed by H2O/saturated aqueous NaHCO3 (1:1, 2 × 200 ml), and finally with saturated NaCl (1 × 100 ml). The organics were pooled, dried over MgSO4, filtered and concentrated. The residue was purified by silica gel chromatography to yield the desired phosphoramidite as a foam.
Oligonucleotide synthesis and purification
Oligonucleotides were synthesized on a 40 μmol scale using Nittophase UnyLinker support (317 μmol/g) on an AKTA 10 Oligopilot. Fully protected nucleoside phosphoramidites were incorporated using standard solid-phase oligonucleotide synthesis, i.e. 15% dichloroacetic acid in toluene for deblocking, 1 M 4,5-dicyanoimidazole 0.1 M N-methylimidazole in acetonitrile as activator for amidite couplings, 20% acetic anhydride in THF and 10% 1-methylimidazole in THF/pyridine for capping and 0.1 M xanthane hydride in pyridine:acetonitrile 1:1 (v:v) for thiolation. MOP couplings were oxidized instead of thiolated using 20% t-BuOOH in ACN. Amidites were dissolved to 0.1 M in acetonitrile:toluene 1:1 (v:v) and incorporated using 6 min. coupling recycling time for DNA amidites and 10 min for all other amidites. At the end of the solid phase synthesis cyanoethyl protecting groups were removed by a 30 min treatment with 20% diethylamine in toluene. ASOs with a single MOP incorporation were deprotected and cleaved using conc. aq. ammonia at room temperature for 48 h. For ASOs with multiple MOP incorporations, the following mild deprotection conditions were used: Protected oligonucleotide on support (400 mg) was suspended in dry THF (5 ml) and stirred for 5 min in a glass pressure vial. Ethylenediamine (EDA, 5 ml) was added via syringe with stirring at room temperature. The reaction was heated 55°C with stirring (oil bath) for 15 min. The reaction was cooled in an ice bath, and was diluted with THF (5 ml). The reaction was centrifuged (300 rpm, 5 min), and the solvent was removed via pipette. The residue was washed with dry THF (2 × 5 ml). The pellet was suspended in 50% EtOH and the spent resin was removed by filtration. The filtrate was concentrated under reduced pressure. Oligonucleotides were purified by ion-exchange chromatography on a GE Akta Explorer 100 HPLC using aqueous buffers with 100 mM NH4OAc and up to 2 M NaBr. The DMT group was removed on-column by treatment with 6% dichloroacetic acid in water. Pure fractions were desalted on a C18 reverse phase column, eluted in 50% acetonitrile in water (v:v) and lyophilized. Despite the use of the modified deprotection conditions, the final yields were lower for MP versus MOP modified ASOs. Presumably, the smaller size of the methyl group in MP allows for more efficient attack of the phosphorus atom by nucleophiles resulting in strand cleavage.
Replacing one or two PS linkages with a neural alkyl phosphonate had a minimal effect on the thermal stability of the ASO/RNA duplexes suggesting that the phosphonate modifications do not adversely affect RNA-binding properties. ASOs with one or two MOP modification showed very similar potency in primary hepatocytes when delivered by free-uptake suggesting that the modification does not impede the ability of the ASO to interact with cell-surface proteins which are responsible for ASO uptake into hepatocytes. However, ASOs with four MOP linkages showed reduced tissue accumulation suggesting that reducing the number of PS linkages below a certain threshold can affect tissue accumulation, presumably be modulating interactions with plasma proteins which facilitate distribution of the ASO from the injection site to peripheral tissues. Site-specific incorporation of neutral alkyl phosphonate linkages can also be useful for modulating the therapeutic properties of nucleic acids which utilize alternate antisense mechanisms such as splice correction, siRNA, miRNA and CRISPR, as they can be combined with sugar modifications used to improve the drug-like properties of therapeutic nucleic acids.