Enzymatic synthesis of nucleoside analogues potentially offers a sustainable, cost and stepefficient alternative to purely chemical synthetic approaches. Of the various enzymes used in their biocatalytic synthesis, nucleoside phosphorylases (NPs) have been the most prominent (Figure ). NPs catalyse a 'nucleobase swap' where, typically, the nucleobase of a natural nucleoside feedstock is exchanged for a non-natural nucleobase. Purine NPs show substrate tolerance for a range of purine nucleobases as well as a range of modifications to the ribosugar, whereas pyrimidine NPs can accept modifications to the pyrimidine nucleobase. A mechanistic hallmark of NPs is that nucleobase exchange proceeds via a two-step process involving N-glycosidic cleavage of a requisite nucleoside 5 by phosphate, producing pentose-1-phosphate 6 and a nucleobase. The formation of a nucleoside analogue 7 then involves nucleophilic displacement of the C1' phosphate in 6 with a nucleobase analogue, exclusively forming the desired β anomer (7). One major challenge of NP-catalyzed nucleobase swapping is that the synthesis of the desired nucleoside product (e.g., 7) is in equilibrium with the competitive formation of 6. As such, efforts have focused on developing strategies to mitigate the undesirable reverse reactions by biasing the equilibrium towards formation of the desired nucleoside analogue, building a feedstock of 6, or through the application of flow-based synthetic routes. An alternative to the use of NPs to prepare nucleoside analogues is to use nucleoside 2'deoxyribosyltransferases (NDTs). Whilst these enzymes catalyse the formation of nucleoside analogues via an overall nucleobase exchange akin to NPs, NDTs form a covalent adduct within the active site, thus controlling stereoselective nucleophilic attack of the incoming nucleobase on the β face (Figure ). The mechanistic divergence of NDTs offers potential advantages over NPs as they do not form a phosphate intermediate such as 6. At present, in-depth knowledge of the substrate promiscuity of NDTs has yet to be established, particularly with respect to the ability of these enzymes to be used for scale-up. Herein, we describe a workflow for the gram-scale preparation of nucleoside analogues catalyzed by a Type-2 NDT derived from Lactobacillus leichmannii (LlNDT-2). Structural studies highlight potential sites where modification of the nucleobase could be tolerated. We use this knowledge to map the substrate promiscuity of both purine and pyrimidine substrates. Finally, we use this knowledge to establish sustainable conditions for the cost-effective preparation of prominent nucleoside analogues.