The first phase of establishing an optimization workflow was to survey reaction conditions for the synthesis of non-natural nucleosides (i.e., 7) where the higher value non-natural nucleobase is the limiting reagent. An excess (5 equiv.) of 2'-deoxycytidine (dC, 8) was used as the corresponding 2'-deoxyribosyl donor relative to 1 equiv. of a non-natural purine 9 or pyrimidine 10 (Figure ). Transglycosylation catalyzed by LlNDT-2 showed remarkable tolerance to a range of organic solvents (20% v/v) and even pure water, producing the desired purine 11 or pyrimidine 12 nucleosides in conversions of up to 98% (11) and 85% (12) (Figure ).