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

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...

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Bibliographic Details
Published in:Journal of organic chemistry 2022-08, Vol.87 (15), p.9466-9478
Main Authors: Kundu, Jayanta, Ghosh, Atanu, Ghosh, Ujjwal, Das, Arnab, Nagar, Dhriti, Pattanayak, Sankha, Ghose, Aurnab, Sinha, Surajit
Format: Article
Language:English
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Summary: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.
ISSN:0022-3263
1520-6904
DOI:10.1021/acs.joc.2c00265