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

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-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 N-acetyl-d-galactosamine-cluster with a β-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 β-Ala-H residue resulted in a faster cleavage of the N-(methoxy)oxazolidine-linker at pH 5, being comparable (T0.5 ca 7 h) to hydrazone-based structures.

Improved Synthesis Strategy for N‐Methoxy‐1,3‐oxazinane Nucleic Acids (MOANAs)

“…pH‐dependence of N‐methoxyoxazolidine and N‐methoxyoxazinane hydrolysis was only studied with the 4‐methoxybenzaldehyde derivative between pH 5.05 and 7.00 (Figure 3). Over this narrow range, the reaction was nearly first‐order in hydronium ion, consistent with previous reports on N‐methoxyoxazolidines [37–39] . Modest upward curvature could, however, be seen at the higher end of the pH range, suggesting a minor contribution by a pH‐independent pathway.…”

“…Over this narrow range, the reaction was nearly firstorder in hydronium ion, consistent with previous reports on Nmethoxyoxazolidines. [37][38][39] Modest upward curvature could, however, be seen at the higher end of the pH range, suggesting a minor contribution by a pH-independent pathway. Rate constants of these partial reactions were obtained by non-linear least-squares fitting of the experimental data to Equation 3.…”

Assembly of split aptamers by dynamic pH-responsive covalent ligation

“…Recently we showed that N-methoxyoxazolidines (1) form between 2 0 -deoxy-2 0 -N-(methoxyamino)uridine ( 2) and an aldehyde (Scheme 1) with an equilibrium constant of up to K = 9000 M À1 under aqueous conditions. 30,31 Under acidic conditions the reaction is dynamic, but at pH 7 or above the products are stable and they can be isolated if needed. We also evaluated the applicability of the reaction for dynamic DNA-templated ligation.…”

“…The 5 0 -OH of 5 was acetylated, the 3 0 -O-TBDMS protection was removed and the exposed 3 0 -OH (6) was phosphitylated to obtain phosphoramidite 3, which was used for the automated DNA-synthesis. ON1-ON3 were synthesized on a regular DNA solid support and 2 0 -deoxy-2 0 -N-(methoxyamino)uridine-modified ONs (ON4-ON7) on a customized 30 solid support (7). After the chain elongation, the ONs were released using concentrated aqueous ammonia (55 1C, 16 h) to obtain N-methoxyoxazolidine protected 5 0 -aldehyde-ONs (ON1-ON3), 2 0deoxy-2 0 -N-(methoxyamino)uridine-modified ONs (ON5-ON7) and one bifunctional ON (ON4).…”