Summary: | 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-<span style="font-variant: small-caps;">d</span>-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<sub>0.5</sub> ca 7 h) to hydrazone-based structures.
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