Oligonucleotide Synthesis and GalNAc Conjugation

Assembly of the oligonucleotide chain and linker building blocks was performed applying phosphoramidite methodology. Necessary building blocks are depicted in Figure S1A. For the on-column conjugation of the triantennary GalNAc (positive control molecule), the final two synthesis cycles were performed using the necessary trivalent branching amidite (4a or 4b) followed by another round of the synthesis cycle using the C4GN amidite (5). Synthesis of serial GalNAc-conjugated oligonucleotides is also depicted in Figure S1A with the sense strand of siRNA-24 as an example. Synthesis was performed using DMT-(S)-serinol(TFA)-succinate-lcaa-CPG (1) and/or DMT-(S)-serinol(TFA)-CEP (2) in the respective synthesis cycles. Upon completion of the solid-phase synthesis, the crude product was cleaved from the solid support and then purified by AEX-HPLC to yield the pure precursor oligonucleotide (pre-siRNA-24B, Figure S1B, left). Conjugation of the GalNAc synthon was achieved by coupling GalNAc-NHS ester (3) to the serinol-amino function of the respective precursor. AEX analysis indicated full conversion after 30 min (Ac-siRNA-24B, Figure S1B, middle) by peak shift. LC-MS analysis confirmed successful conjugation multiplicity (data not shown). After precipitation of the crude product by addition of 10% volume of 2M NaCl, followed by 10× iPrOH, the crude product pellet was dissolved in 40% aqueous (aq.) MeNH₂ and then further purified by AEX-HPLC to yield the final sense strand siRNA-24B with high purity (Figure S1B, right).

Detailed protocols are found in the Supplemental Information.