Synthesis of Modified Oligonucleotides

Oligonucleotides were synthesized using modified (2'-F, 2'-o-Me, locked nucleic acid [LNA]) phosphoramidites with standard protecting groups. Phosphoramidite solid-phase synthesis was done on a MerMade12 (BioAutomation) using modified protocols. Unconjugated oligonucleotides were synthesized on CPG functionalized with a long-chain alkyl amine (LCAA) and unylinker terminus (Chemgenes). Cholesterol CPG was purchased from Chemgenes (Wilmington MA) Product # N-9166–05. GalNAc-conjugated oligonucleotides were grown on custom 3′-GalNAc-CPG (23 (link)); divalent oligonucleotides (DIOs) were synthesized on modified solid support (5 (link)), and VP phosphoramidite was synthesized as described. Phosphoramidites were prepared at 0.1 M in anhydrous ACN, with added dry 15% DMF in the 2′-OMe U amidite. 5-(Benzylthio)-1H-tetrazole (BTT) was used as the activator at 0.25 M. Detritylations were performed using 3% trichloroacetic acid in DCM. Capping was done with non-tetrahydrofuran-containing reagents CAP A, 20% n-methylimidazole in ACN and CAP B, 20% acetic anhydride (Ac₂O) and 30% 2,6-lutidine in ACN (synthesis reagents were purchased at AIC). Sulfurization was performed with 0.1 M solution of 3-[(dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (DDTT) in pyridine (ChemGenes) for 3 min. Phosphoramidite coupling times were 3 min for all amidites used.