Gapmer oligonucleotides comprising a phosphorodithioate internucleoside linkage
A technology between phosphorothioate nucleosides and phosphorothioate nucleosides, wherein at least one of which is an LNA nucleoside and wherein R is hydrogen or phosphoric acid field, can solve the problem of not yet reported LNA thiophosphoramidite Synthesis etc.
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Embodiment 1
[1100] Embodiment 1: monomer synthesis
[1101] 1.1: S-(2-sulfanylethyl)benzenethiocarboxylate
[1102]
[1103] To a solution of 1,2-ethanedithiol (133.57 mL, 1592 mmol, 1 equiv) and pyridine (64.4 mL, 796 mmol, 0.5 equiv) in chloroform (200 mL) was added chloroform (200 mL ) in benzoyl chloride (92.4 mL, 796 mmol, 0.5 eq) for 1 h, and the reaction was stirred at 0 °C for 1 h. The mixture was washed with water (300 mL) and brine (300 mL). Dilute the organic phase in Na 2 SO 4 dried and concentrated to a yellow oil. The oil was distilled (135-145°C) to provide S-(2-sulfanylethyl)benzenethiocarboxylate (40 g, 202 mmol, 13% yield) as a colorless oil. 1 H NMR (400MHz, CDCl 3 )δ7.97(d, J=7.34Hz, 2H), 7.53-7.64(m, 1H), 7.47(t, J=7.58Hz, 2H), 3.31(t, J=7.34Hz, 2H), 2.77- 2.86 (m, 2H), 1.70 (t, J = 8.56Hz, 1H).
[1104] 1.2: S-[2-[[(1R,3R,4R,7S)-1-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-(5-methyl Base-2,4-dioxo-pyrimidin-1-yl)-2,5-dioxabicyclo[2.2.1]hept-7-yl]oxy-...
Embodiment 2
[1117] Example 2: Oligonucleotide Synthesis
[1118] Oligonucleotides were synthesized by Bioautomation using a MerMade 12 automated DNA synthesizer. Use controlled-pore glass supports with universal adapters Synthesis was carried out on a 1 μmol scale.
[1119] In the standard cycling procedure for coupling DNA and LNA phosphoramidites, use CH 2 Cl 2 3% (w / v) trichloroacetic acid in 200 μL was applied three times for 30 seconds for DMT deprotection. With 100 µL of a 0.1 M solution in acetonitrile (or for LNA- Me C structural unit, for the acetonitrile / CH 2 Cl 2 1:1 solution) and 110 μL of 0.1M 5-(3,5-bis(trifluoromethylphenyl))-1H-tetrazole solution in acetonitrile as activator and coupling time 180 seconds, coupling corresponding phosphoramidites three times. For sulfur oxidation, 0.1 m of a solution of 3-amino-1,2,4-dithiazole-5-thione in acetonitrile / pyridine 1:1 (3x190 μL, 55 sec) was used. Using THF / lutidine / Ac 2 O 8:1:1 (CapA, 75 μmol) and THF / N-methylimidazo...
Embodiment 3
[1139] Example 3: In vitro efficacy test and cell uptake test
[1140] Primary rat hepatocytes were plated in 96-well plates and treated in Williams medium E containing 10% FCS without antibiotics. Cells were treated with LNA solution at the indicated concentrations in complete cell culture medium. After 24 hours and 72 hours of incubation time, the cells were treated with Ca 2+ and Mg 2+ Washed 3 times with PBS and lysed with 165uL PureLink Pro Lysis Buffer. Total RNA was isolated using the PureLink PRO 96 RNA kit from Thermo Fisher according to the manufacturer's instructions, and RT-qPCR was performed using the LightCycler Multiplex RNA Virus Master Mix (Roche) with Primer Probe Set for RnApoB (Invitrogen). The obtained data were normalized to Ribogreen.
[1141] Intracellular concentrations of LNA oligonucleotides were determined using hybridization-based ELISA assays for various compounds. All data points were performed in triplicate and data are presented as their m...
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