The formation of N-methoxyoxazolidines in the preparation of oligonucleotide–peptide conjugates was evaluated. The reaction occurred between unprotected 2′-N-(methoxy)­amino-modified oligonucleotides and peptide aldehydes in reasonable yields when isolated. The reaction is reversible under slightly acidic conditions, and it is pH-responsive. The rate and the equilibrium constant may be varied with structurally different aldehydes, allowing an optimization of the ligation and cleavage rate of the resultant conjugates. Therefore, this concept can be considered a cleavable linker.

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.

Reversible pH-responsive N-methoxyoxazolidine formation is used to ligate split aptamer fragments. Two twice-split models and one thrice-split model of CBA (cocaine-binding aptamer) were examined. The aptamer assembly was dynamic, proportional...

DNA-templated formation and N,O-transacetalization of Nmethoxyoxazolidines have been studied. Compared to the reaction without a DNA-catalyst, the hybridization-driven Nmethoxyoxazolidine formation shows a marked rate acceleration, whereas the rate of corresponding N,O-transacetalization is limited by the rate of decay to aldehyde intermediates. In both cases, the equilibrium yield increases markedly on the DNA template. Different hairpin architectures have been studied to evaluate the role and limits of the template effect. Furthermore, an attention has been paid to stereochemical integrity (R/S) of the N-methoxyoxazolidine linkage. The Nmethoxyoxazolidine formation represents a dynamic pH-responsive DNA-templated ligation that occurs readily in slightly acidic conditions (pH 5).

A detailed protocol for preparation 3′‐glycoconjugated oligonucleotides is described based on one‐pot immobilization of 4,4′‐dimethoxytrityl‐protected carbohydrates to a solid support followed by on‐support peracetylation and automated oligonucleotide assembly. Compared to an appropriate building block approach and post‐synthetic manipulation of oligonucleotides, this protocol may simplify the synthesis scheme and increase overall yield of the conjugates. Furthermore, the immobilization to a solid support typically increases the stability of reactants, enabling prolonged storage, and makes subsequent processing convenient. Automated assembly on these carbohydrate‐modified supports using conventional phosphoramidite chemistry produces 3′‐glycoconjugated oligonucleotides in relatively high yield and purity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 1‐O‐tert‐butyldimethylsilyl‐6‐O‐(4,4′‐dimethoxytrityl)‐β‐D‐glucose Basic Protocol 2: Synthesis of 6‐O‐dimethoxytrityl‐2,3,1′,3′,4′,6′‐hexa‐O‐benzoylsucrose Basic Protocol 3: Synthesis of 6″‐O‐dimethoxytrityl‐N‐trifluoroacetyl‐protected aminoglycosides Basic Protocol 4: Synthesis of 3‐O‐dimethoxytrityl‐propyl β‐D‐galactopyranoside Basic Protocol 5: Synthesis of trivalent N‐acetyl galactosamine cluster Basic Protocol 6: Synthesis of carbohydrate monosuccinates and their immobilization to a solid support Basic Protocol 7: Oligonucleotide synthesis using immobilized carbohydrates