PUBLICATION

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

Authors
Kundu, J., Ghosh, A., Ghosh, U., Das, A., Nagar, D., Pattanayak, S., Ghose, A., Sinha, S.
ID
ZDB-PUB-220716-9
Date
2022
Source
The Journal of organic chemistry   87(15): 9466-9478 (Journal)
Registered Authors
Ghose, Aurnab, Pattanayak, Sankha, Sinha, Surajit
Keywords
none
MeSH Terms
  • RNA Splicing
  • DNA
  • Oligonucleotides, Antisense*
  • Zebrafish*
  • Animals
  • Morpholinos/genetics
(all 6)
PubMed
35839125 Full text @ J. Org. Chem.
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.
Genes / Markers
Figures
No images available
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping
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Citation
Kundu, J., Ghosh, A., Ghosh, U., Das, A., Nagar, D., Pattanayak, S., Ghose, A., Sinha, S. (2022) Synthesis of Phosphorodiamidate Morpholino Oligonucleotides Using Trityl and Fmoc Chemistry in an Automated Oligo Synthesizer. The Journal of organic chemistry. 87(15):9466-9478.