For the 2′-Me adenosine 27 changing the aldolase from EcDERAWT to EcDERAF76A gave an increase in conversion from 17% to 38%. DOE was used to determine the effects of enzyme loading on conversion for the three main enzymes (DERA, PPM, PNP) (supplementary 13). This revealed that the concentration of PNP was by far the most limiting of the three enzymes. By increasing the concentration of PNP in these reactions an increase in conversions for both 2′-Me (38% to 65%) and 2′-F (6% to 20%) was observed (Table ). Addition of a recently published PNP variant which was engineered to give a small increase in activity to 2′-F adenosine resulted in a further increase in conversion to 28% (Table ). The lower conversions seen for the fluorinated analogues are in part due to a large proportion of this product being converted to 2′-F-inosine (presumably by contaminant adenosine deaminase) this issue is compounded by the relatively large amounts of protein required and highlights the need for further engineering, particularly of the PNP enzyme. In addition to 2′-OH adenosine 2′-OH guanine was synthesized using the optimal conditions from the adenosine reaction (Table ). While some product was generated, the conversion for the synthesis of guanosine is notably lower than for adenosine, this is likely due in part to its poorer solubility under the reaction conditions.