Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical

Definitions

• RNA interference is an evolutionarily conserved cellular mechanism of post-transcriptional gene silencing found in fungi, plants and animals that uses small RNA molecules to inhibit gene expression in a sequence-specific manner.
• the RNAi machinery can be harnessed to destruct any mRNA of a known sequence. This allows for suppression (knock-down) of any gene from which it was generated and consequently preventing the synthesis of the target protein.
• Smaller siR A duplexes introduced exogenously were found to be equally effective triggers of RNAi (Zamore, P. D., Tuschl, T., Sharp, P. A., Battel, D. P. Cell 2000, 101, 25-33).
• Synthetic RNA duplexes can be used to modulate therapeutically relevant biochemical pathways, including ones which are not accessible through traditional small molecule control.
• RNA modification of RNA leads to improved physical and biological properties such as nuclease stability (Damha et al Drug Discovery Today 2008, 13(19/20), 842-855), reduced immune stimulation (Sioud TRENDS in Molecular Medicine 2006, 12(4), 167-176), enhanced binding (Koller, E. et al Nucl Acids Res. 2006, 34, 4467-4476), enhanced lipophilic character to improve cellular uptake and delivery to the cytoplasm.
• RNA modifications of RNA have relied heavily on work-intensive, cumbersome, multi-step syntheses of structurally novel nucleoside analogues and their corresponding phosphoramidites prior to RNA assembly.
• a major emphasis has been placed on chemical modification of the 2'-position of nucleosides.
• a rigorous approach to structure-activity-relationship (SAR) studies of chemical modifications will obviously require synthesis and evaluation of all four canonical ribonucleosides [adenosine (A), cytidine (C), uridine (U), guanosine (G)].
• RNA has centered for the most part on simple conjugation chemistry. Conjugation has largely been performed on either the 3' or the 5'-end of the RNA via alkylamine and disulfide linkers. These modifications have allowed conjugation of RNA to various compounds such as cholesterol, fatty acids,
• poly(ethylene)glycols various delivery vehicles and targeting agents such as poly(amines), peptides, peptidomimetics, and carbohydrates.
• This invention relates to the post-synthetic chemical modification of RNA at the 2'-postion on the ribose ring via a silver or copper catalyzed Alkyne, Aldehyde, Amine coupling chemistry [AAA or A 3 or AA 3 coupling ("A A 3 coupling” means alkyne, aldehyde, amine coupling)].
• the invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cieavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
• FIGURE 1 Systematic evaluation of the impact on knockdown of the 2'-0-propargylamine chemical modification along positions 1 through 19 of the guide strand of a ApoB (9514) siRNA seq.
• FIGURE 2 Systematic evaluation of the impact on knockdown of the 2'-0-propargylamine chemical modification along positions 1 through 19 of the guide strand of a ApoB (10162) siRNA seq.
• FIGURE 3 Synthesis of multi AAA 2'-0-propargylamine chemical modification positions 1 through 19 of the guide strand of Luc 80 siRNA sequence.
• This invention relates to the post-synthetic chemical modification of RNA at the 2'-postion on the ribose ring via silver or copper catalyzed alkyne, aldehyede, amine coupling chemistry (AAA or A or AA coupling).
• the invention 1) avoids complex, tedious multi-step syntheses of each desired modified ribonucleoside; 2) allows diverse chemical modifications using high-fidelity chemistry that is completely orthogonal to commonly used alkylamino, carboxylate and disulfide linker reactivities; 3) allows introduction of functional groups that are incompatible with modern automated solid-phase synthesis of RNA and subsequent cieavage-deprotection steps; 4) allows introduction of functional groups useful as targeting ligands; and 5) enables high-throughput structure-activity relationship studies on chemically modified RNA in 96-well format.
• RNA is unstable towards hydrolysis and can undergo auto-catalytic cleavage via
• RNA with alkyne functional group at the 2 '-position RNA with alkyne functional group at the 2 '-position.
• the current invention relates to chemical modification of RNA at the 2' ⁇ position of the ribose ring based on "alkyne, aldehyde, amine coupling" chemistry.
• alkyne, aldehyde, amine coupling Three-component coupling between aldehydes, alkynes and amines is known. Wei et al. Synlett. 20 ⁇ 4, 1472 - 1483.
• the invention provides a process for introducing 2'- modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2'-position on at least one ribose ring; b) creating a solution of RNA in a solvent; and c) adding an aldehyde, an amine and a metal catalyst to the solution to form a reaction and creating a 2 '-modified RNA.
• the process is conducted in high-throughput format.
• the step (a) RNA may be purchased or synthesized.
• the step (b) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DM Ac, NMP and a suitable ionic liquid.
• the step (b) solvent is aqueous DMSO.
• the step (c) metal catalyst is selected from silver, copper, ruthenium, iridium, iron, zinc or gold.
• the step (c) metal catalyst is silver.
• the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state and/or induce asymmetry during amine addition.
• the step (c) reaction is performed at temperatures between - 0-300°C for 0 to 18 h.
• the step (c) reaction is performed at temperatures between 5- 120°C for 0.5 to 18 h.
• step (c) reaction is performed at temperatures between
• step (c) reaction is performed at temperatures between 60-90°C for 0.5 to l8 h.
• the step (c) reaction is performed at temperatures between 65-80 °C for 0.5 to 18 h.
• the invention provides a process for introducing 2 - modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2' ⁇ position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; and c) adding an aldehyde, an amine and a metal catalyst to the solution to form a reaction and creating a 2 '-modified RNA.
• the process is conducted in high-throughput format.
• the step (a) RNA may be purchased or synthesized.
• the step (b) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
• the step (b) solvent is aqueous DMSO.
• the step (c) metal catalyst is selected from silver, copper, ruthenium, iridium, iron, zinc or gold.
• the step (c) metal catalyst is silver.
• the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state and/or induce asymmetry during amine addition.
• the step (c) reaction is performed at temperatures between - 20-300°C for 0 to 18 h.
• step (c) reaction is performed at temperatures between 5- 120°C for 0.5 to l8 h.
• the step (c) reaction is performed at temperatures between 20-100°C for 0.5 to 18 h.
• step (c) reaction is performed at temperatures between 60-90°C for 0.5 to l8 h.
• step (c) reaction is performed at temperatures between
• the invention provides a process for introducing 2'- modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2 '-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an aldehyde, an amine and a metal catalyst to the solution to form a reaction and creating a 2'-modified RNA; and d) purifying the 2'-modified RNA.
• the step (a) RNA may be purchased or synthesized.
• the step (c) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, C3 ⁇ 4CN, DMF, D Ac, NMP and a suitable ionic liquid.
• the step (c) solvent is aqueous DMSO.
• the step (c) metal catalyst is selected from silver, copper, ruthenium, iridium, iron, zinc or gold.
• the step (c) metal catalyst is silver.
• the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state and/or induce asymmetry during amine addition.
• step (c) reaction is performed at temperatures between -
• the step (c) reaction is performed at temperatures between 5- 120°C for 0.5 to 18 h.
• the step (c) reaction is performed at temperatures between 20-100°C for 0.5 to 18 h.
• step (c) reaction is performed at temperatures between 60-90°C for 0.5 to 18 h.
• the step (c) reaction is performed at temperatures between 65-80°C or 0.5 to 18 h.
• the step (d) purification is performed in high-throughput format on 96-well CIS cartridges (solid-phase extraction) or strong-a ion-exchange-HPLC or reverse-phase HPLC or poly(acrylamide) gel electrophoresis (PAGE) or size-exclusion chromatography.
• the invention provides a process for introducing 2'- modifications into RNA, said process comprises a) obtaining RNA with an alkyne functional group at the 2'-position on at least one ribose ring of an internal nucleotide; b) creating a solution of RNA in a solvent; c) adding an aldehyde, an amine and a metal catalyst to the solution to form a reaction and creating a 2'-modified RNA; d) cooling the solution and adding a fluoride source; e) heating the solution; f) cooling the solution and adding a diluent; and g) purifying the 2'-modified RNA.
• the step (a) RNA may be purchased or synthesized.
• the step (c) solvent is selected from aqueous buffer solutions (including phosphate buffers), aqueous DMSO, CH 3 CN, DMF, DMAc, NMP and a suitable ionic liquid.
• the step (c) solvent is aqueous DMSO.
• the step (c) metal catalyst is selected from silver, copper, ruthenium, iridium, iron, zinc or gold.
• the step (c) metal catalyst is silver.
• the step (c) metal catalyst is copper with a suitable ligand to stabilize the Cu(I) oxidation state and/or induce asymmetry during amine addition.
• the step (c) reaction is performed at temperatures between - 20-300°C for 0 to 18 h.
• the step (c) reaction is performed at temperatures between 5- 120°C for 0.5 to 18 h.
• the step (c) reaction is performed at temperatures between 20-100°C for 0.5 to 18 h.
• step (c) reaction is performed at temperatures between 60-90°C for 0.5 to 18 h.
• step (c) reaction is performed at temperatures between 65-80°C for 0.5 to l8 h.
• the step (e) fluoride source is Et3N-3HF
• the step (e) fluoride source is ammonium fluoride.
• the step (f) diluent is NaCl.
• the step (g) purification is performed in high-throughput format on 96-well CI 8 cartridges (solid-phase extraction) or strong-anion-exchange-HPLC or reverse-phase HPLC or poly(acrylamide) gel electrophoresis (PAGE) or size-exclusion chromatography.
• the instant invention also discloses a method for attaching targeting ligands to RNA utilizing the process described herein.
• the instant invention further discloses a method for attaching targeting ligands to internal nucleotides in RNA utilizing the process described herein.
• 2'-modified RNA means a RNA wherein at least one ribose ring is modified at the 2'-position.
• Alkyne functional group means any chemical compound containing an alkyne functional group.
• the preferred alkyne functional group is propargyl.
• High-throughput format means that several operations are run in parallel fashion such as for example in 96-well plate chemical synthesis, 96-well plate purification, 96- well plate chromatographic analysis and 96-well plate mass spectrometric analysis.
• Internal nucleotide means a nucleotide in an RNA molecule that is not at the 3'- or 5'-end.
• the internal nucleotides in a 21mer siRNA occur at positions 2-20.
• RNA means a chemically modified or unmodified ribonucleic acid molecule (single stranded or double stranded) comprising at least 3 nucleotides, including but not limited to miRNA and siRNA. In another embodiment, “RNA” means miRNA. In another
• RNA means siRNA.
• Chemical modifications include, for example,
• the base can be a canonical base (A, G, T and U) or a modified or universal base (including but not limited to inosine and nitroindole). See US2006/0240554.
• Aldehyde means any chemical compound containing an aldehyde functional group.
• Amine means any chemical compound containing an amine functional group.
• Metal catalyst means any chemical form of silver, copper, iridium, ruthenium, iron, zinc or gold. Including solid-supported variants.
• metal catalyst include Agl, CuBr, C Br-Me2S, Cul, CuSC>4 or CuOAc and a suitable reducing agent such as sodium ascorbate.
• Ribose ring means the ribose moiety in a ribonucleotide.
• Targeting ligand means a conjugate delivery moiety capable of delivering the RNA to a target cell of interest.
• Targeting ligands include, but are not limited to, lipids (cholesterol), sugars (NAG), proteins (transferrin), peptides, poly(ethylene)glycols and antibodies. See Juliano et al., Nucleic Acids Research, 2008, 1-14, doi:10.1093/nar/gkn342.
• the present invention provides a process for introducing chemical modifications into RNA at the 2'-position on the ribose ring. It is well known in the art that RNA are useful for therapeutic and research purposes.
• RNA The synthesis of RNA is well known in the art.
• a suitable 2'-O-propargyl nucleoside phosphoramidite is incorporated into RNA using modern techniques based on the phosphoramidite approach.
• the crude, solid-support bound protected oligonucleotide is then treated with aqueous methylamine to remove nucleobase and phosphate protecting groups.
• the crude product is then lyophilized to remove volatiles.
• the crude product is dissolved in DMSO:H 2 0, treated with a suitable aldehyde, a suitable amine and silver or copper catalyst (scheme 1). After aging an appropriate amount of time, the reaction mixture is treated with fluoride to remove the 2 r -0-teri-butyldimethylsilyl protecting groups.
• the crude product is then purified to obtain the chemically modified RNA.
• RNA (-50 nmol) containing at least one alkyne functional group (shown below) in 96-well format was dissolved in DMSO;water (75:25, 40 ⁇ ,).
• the "alkyne, amine, aldehyde coupling" reaction can be utilized to introduce multiple chemical modifications in one synthetic operation.
• the A 3 coupling reaction was performed to introduce four units of propargylamines on RNA
• RNA oligomers with the first nucleotide, Adenine (A), replaced with 2'-0-propargyl-Adenine. Then, a second sequence, in which the second nucleoside (U) was replaced with 2'-0 ⁇ propargyI -uridine was synthesized, keeping all other nucleotides unchanged.
• L 2'OmeUridine
• L 2'-0-Propargyl Adenine
• M 2'-0-Propargyl Cytidine
• W 2'-0- Propargyl Guanosine
• Y 2'-0-Propargyl Uridine.
• Hepal-6 cells were transfected with 10 nM of either the unmodified, modified, or negative control siRNA using a commercial lipid transfection reagent.
• the target mRNA was assessed for degradation using standard Taqman procedures. Modified Multiplex luciferase report assay for i vitro duration study
• Multiplex luciferase assay for in vitro duration study is modified from the manufacturer's instruction using HeLa-luc cell line. Briefly, the cell viability and the luciferease expression at the same well are determined by CellTiter-FluorTM (Promega, Cat# G6082) and Bright-GloTM (Promega Cat# E2620) sequentially.
• HeLa-luc cell line is a stable firefly luciferase reporter expression cell line.
• Bright-GloTM luciferase assay system contains the stable substrate - luciferin and assay buffer.
• the luminescent reaction of luciferease and luciferin has high quantum yield and can be detected as luminescence intensity, which represents the luciferase expression level.
• Target siRNAs containing luciferase coding region is designed to be transfected into the HeLa-luc cells. Once the target is effected, the luciferase expression is reduced accordingly. Therefore, the siRNA silencing efficacy can be determined by the relative luminecence intensity of treated cells.
• CellTiter-fluor kit measures the conserved and constitutive protease activity within live cells and therefore serves as a marker of cell viability, using a fluorogenic, cell-permeable peptide substrate (glycyl- phenylalanyl-aminofluorocoumarin; GF-AFC).
• Luciferase stable expressed HeLa-luc cell cells are plated in 96-well plates at density of 4,500 cells per well in 100 ⁇ DMEM media without antibiotics 24 hours prior to transfection.
• si NA transfection is performed using the RNAiMAXTM (Invitrogen). Briefly, 0,05 ⁇ siRNA are mixed with Opti-MEMmedia and RNAiMAX and incubated at room temperature for 15 rnin. The mix is then added to the cells. The final siRNA concentration is 1 nM. Cell plates for all time points are transfected at same time with a medium change at 6 hours post-transfection into 100 ⁇ of fresh completed DMEM (DMEM + 10% FBS +
• In vitro duration is determined by the luciferase expression post-transfection at four time points: day 1, day 2, day 5 and day 7. Addition medium changes are performed at day 2 and day 5 into 100 ⁇ , of fresh completed DMEM (DMEM + 10% FBS + Penn/strep). Luciferase levels are determined using the Bright- Glo Luminescence Assay (Promega) and measuring the wells on an Envison instrument (Perkin Elmer) according to manufacturer's instructions.
• the cell viability of the same treatment wells is measured using CellTiter-fluor kit (Promega) according to manufacturer's instructions.
• This assay measures the conserved and constitutive protease activity within live cells and therefore servers as a marker of cell viability, using a fluorogenic, cell-permeable peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC).
• the fluorescence was measured on the Envision using exciton filter at 405 nm and emission filter at 510 nm.
• the luciferase expression was normalized to cell viability. The log of this number was calculated to determine the luciferase protein that was degraded (knockdown). A non-targeting siRNA was subtracted from this value to account for non-specific background.
• RNAs made by the process of the invention are useful in high-throughput structure-activity relationship studies on chemically modified R A in 96-well format.

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