Expanding the Scope of the Cleavable <i>N</i>-(Methoxy)oxazolidine Linker for the Synthesis of Oligonucleotide Conjugates

• Main Authors: Aapo Aho, Antti Äärelä, Heidi Korhonen, Pasi Virta
• Format: Article
• Language: English
• Published: MDPI AG 2021-01-01
• Series: Molecules
• Subjects: oligonucleotide conjugates; cleavable linker; <i>N</i>-(methoxy)oxazolidine
• Online Access: https://www.mdpi.com/1420-3049/26/2/490

Oligonucleotides modified by a 2′-deoxy-2′-(<i>N</i>-methoxyamino) ribonucleotide react readily with aldehydes in slightly acidic conditions to yield the corresponding <i>N</i>-(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 <i>N</i>-(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-β-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 <i>N</i>-acetyl-d-galactosamine-cluster with a β-Ala-H unit has been synthesized. These aldehydes were conjugated via <i>N-</i>(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 β-Ala-H residue resulted in a faster cleavage of the <i>N</i>-(methoxy)oxazolidine-linker at pH 5, being comparable (T_0.5 ca 7 h) to hydrazone-based structures.