Aqueous coupling reactions are used in modern medicinal chemistry and biotechnological applications. As an example, DNA-templated synthesis (DTS) is employed to generate massive chemical libraries up to trillions of compounds, which can be utilized for screening of new active pharmaceutical ingredients (APIs). Furthermore, high-affinity receptors and ligands can be discovered by performing the synthesis and screening in a single process called target-directed dynamic combinatorial chemistry (tdDCC), which is based on settling of a thermodynamic equilibrium directed by the targets of interests – the templates. Reversible template-induced assembly is also attractive for detection as chemical sensors. tdDCC with biological target requires reversible aqueous coupling reactions. These kinds of coupling reactions have also been used as linkers for conjugation of APIs with various cell-specific or membrane-penetrating molecular agents to increase the efficacy of the API by more efficient cellular uptake.

In this thesis, formation of N-methoxyoxazolidines is presented as a potential pH-responsive coupling chemistry suitable for tdDCC and conjugation of unprotected oligonucleotides. By studying a model reaction between 2´-deoxy-2´-N-methoxyuridine and aldehydes, it was found out that N-methoxyoxazolidines are obtained in high yields under aqueous conditions. The reaction is reversible, and its rate is dependent on the acidity of the reaction solution. In practice, the reaction is dynamic under slightly acidic conditions (pH 4–6) while the obtained products (N-methoxyoxazolidines) are stable at pH 7 or above. The N-methoxyoxazolidine formation was applied for the preparation of various oligonucleotide conjugates (including carbohydrates and peptides). The conjugates were shown to be cleavable in acidic conditions perceived to early endosomes. Based on these studies, N-methoxyoxazolidines could be used as dynamic cleavable linkers for therapeutic oligonucleotide conjugates. In addition, the applicability of the reaction for DNA- and small-molecule-templated syntheses were studied. This was demonstrated for the assembly of dynamic pH-responsive split-aptamers and G-quadruplexes, induced by a small molecule substrate and metal ions, respectively. Therefore, N-methoxyoxazolidine was proven to be a suitable tool for tdDCC.