Interestingly when comparing the two reactions, the cascade starting from L-glyceraldehyde performed better than with D-glyceraldehyde. One possible explanation for this difference is due to the presence of native E. coli enzymes co-expressed with the EcDERA variants. For example, D-G3P is a substrate for triose phosphate isomerase (TIM), a highly active enzyme that is rate-limited only by diffusion of G3P into the active site . Even small quantities of this enzyme present from E. coli remaining after purification are likely to be sufficent to isomerize a significant proportion of D-G3P into dihydroxyacetone phosphate (DHAP, 20) thus lowering conversions (Scheme 3). Despite this side reaction, the D-ribose-5-phosphate analogues were all generated with conversions ranging from 22-66% for all the substrates screened. Synthesis of 2-Fluoro Pentose-5-Phosphates One modification of particular interest for therapeutic oligonucleotides is 2-fluoro, which is used in several currently approved therapeutic oligonucleotides (lumasiran, inclisiran, etc.) . Synthesis of the 2-F modified R5P requires fluoroacetaldehyde 18 as the donor substrate. In view of the difficulty of handling aldehyde 18 we decided instead to generate it in situ using an oxidase. Initial work showed that fluoroacetaldehyde 18 could be generated from fluoroethanol 17 using methanol oxidase from Pichia pastoris -PpAOWT (supplementary 9.1). This system was then combined with both kinase enzymes, CfDHAK or CsGK, and screened with the EcDERAWT alongside the two best performing variants (Scheme 2). In contrast to the previous aldehyde substrates, with fluoroacetaldehyde 17 the WT enzyme gave higher conversion than both the F76A and L20A/F76A variants presumably due to the small size of the fluorine substituent. The HPLC traces for this cascade (supplementary 9.2.2, 9.3.2) contained multiple peaks, suggesting the presence of a mixture of diastereomers at the 2-position. For 19a ratio = 15:85 for 19b ratio = 50:50).