With the structural information in hand, we then sought to construct a detailed molecular map defining the substrate scope of non-natural nucleoside synthesis using the optimised biotransformation conditions as a basis (Figure ). Using either 8 (dC) or thymidine as the 2'deoxyribosyl donor, the scope of nucleobase acceptance was explored using a range of pyrimidine analogues modified at the 5-position (18-22, Figure ). Our structures of LlNDT-2 suggest that the active site can accommodate substituents at this position as they project into a space between the side chains of Y157, F13 and N123. Unexpectedly, the lack of substituents in the 4-position (23) and nucleobase modifications to the 2-(26-27), 3-( ) and 4-positions (25-27) were also tolerated. However, an acceptor nucleobase with a substituent at position 6 (30) was not tolerated as this may clash with the side chain of N123. Nucleobases such as triazine (28), hydrogen bond donor groups in the 2-position (29) or pyridine (31) were not substrates, confirming that the N3 is essential. This is presumably due to the need for the nucleophilic nitrogen to be adjacent to a tautomeric oxygen. The acceptance of purine nucleobase analogues by LlNDT-2 was far more extensive. Purines with modifications at all carbon positions were tolerated, producing non-natural nucleosides 32-51 with moderate to high conversions (Figure ). Purine acceptors with a range of modifications at both C2 and C6 positions (32-40) were accepted, including precursors for the preparation of 6-thioguanosine (36), a known DNA damage adduct O6-methyl guanosine 37, and those including protecting groups used for solid phase DNA synthesis (e.g., 38). The active site of LlNDT-2 in complex with the nucleobase 17 revealed that the mobility of the loop bearing Q46 permits the accommodation of a range of modifications at the C2 and C6