Synthesis of Xylo‐nucleoside <i>H</i>‐Phosphinates and Insights into Their Conformational Preferences Within a Chimeric Duplex

Abstract An efficient method for the synthesis of xylo‐nucleoside H ‐phosphinate building blocks containing both pyrimidine and purine nucleobases is reported. A DNA‐phoXyloNA‐DNA chimeric sequence containing a central 2′‐5′‐phosphonomethoxy‐linked xylo‐nucleoside uracil residue was chemically synthesized, and its structural properties were examined using advanced spectroscopic techniques and computational simulations. Results outlined herein suggest that the xylose sugar moiety predominantly favors an N ‐type conformation stabilized by intraresidue hydrogen bonding interactions, while the inclusion of an extra methylene group in the backbone introduces significant flexibility within the internucleotide linkage. Within a duplex structure, disrupted hydrogen bonding interactions in the xU5::dA16 base pair, combined with diminished stacking interactions at the site of modification collectively contribute to a reduction in thermal stability compared to the unmodified counterpart. These findings enhance our understanding of the stability and dynamics of orthogonal backbone‐sugar modified nucleic acids (XNAs), aiding in the design of functionally tailored analogues.