A1 Refereed original research article in a scientific journal
Expanding the Scope of the Cleavable N-(Methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates
Authors: Aho Aapo, Äärelä Antti, Korhonen Heidi, Virta Pasi
Publisher: MDPI
Publication year: 2021
Journal: Molecules
Journal name in source: MOLECULES
Journal acronym: MOLECULES
Article number: ARTN 490
Volume: 26
Issue: 2
Number of pages: 11
eISSN: 1420-3049
DOI: https://doi.org/10.3390/molecules26020490
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/53389499
Abstract
Oligonucleotides modified by a 2 '-deoxy-2 '-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ''switched off-state''. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-beta-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary N-acetyl-d-galactosamine-cluster with a beta-Ala-H unit has been synthesized. These aldehydes were conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19-47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to beta-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T-0.5 ca 7 h) to hydrazone-based structures.
Oligonucleotides modified by a 2 '-deoxy-2 '-(N-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding N-(methoxy)oxazolidine-linked oligonucleotide-conjugates. The reaction is reversible and dynamic in slightly acidic conditions, while the products are virtually stable above pH 7, where the reaction is in a ''switched off-state''. Small molecular examinations have demonstrated that aldehyde constituents affect the cleavage rate of the N-(methoxy)oxazolidine-linkage. This can be utilized to adjust the stability of this pH-responsive cleavable linker for drug delivery applications. In the present study, Fmoc-beta-Ala-H was immobilized to a serine-modified ChemMatrix resin and used for the automated assembly of two peptidealdehydes and one aldehyde-modified peptide nucleic acid (PNA). In addition, a triantennary N-acetyl-d-galactosamine-cluster with a beta-Ala-H unit has been synthesized. These aldehydes were conjugated via N-(methoxy)oxazolidine-linkage to therapeutically relevant oligonucleotide phosphorothioates and one DNA-aptamer in 19-47% isolated yields. The cleavage rates of the conjugates were studied in slightly acidic conditions. In addition to the diverse set of conjugates synthesized, these experiments and a comparison to published data demonstrate that the simple conversion of Gly-H to beta-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T-0.5 ca 7 h) to hydrazone-based structures.
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