By targeting the active site of EcDERAWT, simple mutations (F76A, L20A) have been shown to significantly increase donor substrate scope and allow for the synthesis of a broad range of D-pentose-5-phosphate analogues functionalized at the 2-position. Addition of a kinase to generate glyceraldehyde-3-phosphate in situ resulted in the syn-thesis of 2-functionalised D-ribose-5-phosphate and L-lyxose-5-phosphate analogues via a two-step cascade. Furthermore, addition of an alcohol oxidase led to the formation of fluoroacetaldehyde in situ and subsequent generation of 2-F analogues via a three step one pot reaction. To characterize the various pentose-5-phosphate products semi-preparative biotransformations were carried out. This resulted in the synthesis of 2-OMe, MOE, OBn and F analogues in good conversions and isolated yields. With the exception of fluorine, all products were generated as single diastereomers at 2-highlighting the excellent stereoselectivity of DERA.