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Abstract Inspired by nature, chemists have spent the last 50 years systematically designing and synthesizing a vast array of sugar-modified nucleic acids, so-called xenonucleic acids (XNAs), collectively forming what we now describe as the XNA alphabet. Within the alphabet, systems can be categorized into two major groups: those capable of interacting with natural nucleic acids and those that do not cross-pair with DNA or RNA. The sugar component of XNAs plays a crucial role in defining their conformational space, which, in turn, influences their hybridization properties and potential applications across biotechnology and synthetic biology. This review provides an overview of sugar-modified XNA systems developed to date as well as the geometric parameters and physicochemical principles that have enhanced our understanding of XNA conformational behavior, particularly in relation to their orthogonality to (i.e. inability to cross-pair with) natural nucleic acids. These insights are essential for developing a more rational approach to key processes such as XNA replication and evolution, ultimately paving the way for applications in areas including synthetic genetics, nucleic acid therapeutics, diagnostics, and nanotechnology. 

Polymerases are among the most powerful tools in the molecular biology toolbox; however, access to large quantities of chemically modified nucleoside triphosphates for diverse applications remains hindered by the need for purification by high-performance liquid chromatography (HPLC). Here, we describe a scalable approach to modified nucleoside triphosphates that proceeds through a P(III)−P(V) mixed anhydride inter-mediate obtained from the coupling of a P(III) nucleoside phosphoramidite and a P(V) pyrene pyrophosphate reagent. The synthetic strategy allows the coupling, oxidation, and deprotection steps to proceed as stepwise transformations in a single one-pot reaction. The fully protected nucleoside triphosphates are purified by silica gel chromatography and converted to their desired compounds on scales exceeding those achievable by conventional strategies. The power of this approach is demonstrated through the synthesis of several natural and modified nucleoside triphosphates using protocols that are efficient and straightforward to perform.