Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry in an Automated Oligo Synthesizer

Abstract

Phosphorodiamidate morpholino oligonucleotides (PMOs) constitute 3 out of the 11 FDA-approved oligonucleotide-based drugs in the last 6 years. PMOs can effectively silence disease-causing genes and modify splicing. However, PMO synthesis has remained challenging for a variety of reasons: inefficient deprotection and coupling methods and instability of monomers. Here, we report the development of a suitable combination of resin supports, deblocking and coupling reagents for synthesizing PMOs using either trityl or Fmoc-protected chlorophosphoramidate monomers. The synthesized PMOs using both the methods on a solid support have been validated for gene silencing in a zebrafish model. The protocol was successfully transferred into an automated DNA synthesizer to make several sequences of PMOs, demonstrating for the first time the adaptation of regular PMOs in a commercial DNA synthesizer. Moreover, PMOs with longer than 20-mer sequences, including FDA-approved Eteplirsen (30-mer), were achieved in >20% overall yield that is superior to previous reports. Hybridization study shows that PMOs exhibit a higher binding affinity toward complementary DNA relative to the DNA/DNA duplex (>6 °C). Additionally, the introduction of Fmoc chemistry into PMOs opens up the possibility for PMO synthesis in commercial peptide synthesizers for future development.

From green innovations in oligopeptide to oligonucleotide sustainable synthesis: differences and synergies in TIDES chemistry

“…Please do not adjust margins Please do not adjust margins An interesting demonstration of the strong interconnection between the SPS of both TIDES was reported a few months ago by Sinha and co-workers, who performed the synthesis of phosphoramidite morpholino oligonucleotides (PMOs) by using trityl/Fmoc chemistry. 67 The selected solid-support to be used on an automatized synthesizer was Ramage ChemMatrix, while 5-(ethylthio)-1H-tetrazole (ETT) and N-methylpyrrolidone were selected as the activating agent and solvent, for the activation of the phosphoramidites, respectively. The innovation stands in the introduction of trityl (Trt) or Fmoc as the N-protecting group on the morpholino unit, which is removed respectively by 3cyanopyridine trifluoroacetic acid (CYPTFA) in DCM or 20% piperidine in DMF, similarly to SPPS.…”

Synthesis and Biophysical Properties of Phosphorodiamidate Piperidino Oligomers

“…We next synthesized the chlorophosphoramidate monomers necessary for the synthesis of PPOs (Table 1). Our initial attempts to synthesize the chlorophosphoramidate monomers using LiBr/1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) 12 or 5-(ethylthio)-1H-tetrazole (ETT) 13 methods were not successful, with products obtained in less than 10% yield. We therefore performed the reaction in the presence of strong bases at −20 °C to prevent product decomposition.…”

Synthesis of 5′-Thiol Functionalized Morpholino Oligo-Nucleotide and Subsequent Conjugation with IGT to Improve Delivery and Antisense Efficacy In Vitro

“…An internal oligo-guanidinium transporter containing hexafluoro-isopropyl group (HF-IGT) was chosen for this, as its activity on conjugation with Nanog PMO at 3′ is already known . Even though cationic PMOs − have the advantage of at-a-stretch synthesis, a separate conjugation scheme avoids the usage of modified monomers and allows standard synthesis protocols of amidates − as well as the full length PMO. , …”

“…Synthesis commenced with the reduction of commercially available Fmoc-Cys(Trt)-OH to the corresponding alcohol via NaBH 4 mediated reduction of the activated anhydride with isobutyl chloroformate (Scheme ). This alcohol was then loaded on to the LinkPoly support (containing double 6-aminohexanoic acid linkers as reported earlier) via succinic anhydride to give the PMO Linker A ( PLA ). However, PLA showed poor loading with G chlorophosphoramidate after Fmoc deprotection.…”

“…Our previous attempts toward 5′-SH terminated PMOs from disulfide modified polystyrene support were unsuccessful because of the incomplete cleavage of the 30-mer PMO with dithiothreitol (DTT). Multiple treatments of the solid support with large excess of DTT or Tris­(2-carboxyethyl)­phosphine (TCEP) lead to partial cleavage only (Figure ).…”