Finally, to demonstrate the application of this cascade to generate nucleosides functionalized at the 2′-position the aldolase step was combined with phosphopentomutase (EcPPMWT) and purine nucleoside phosphorylase (EcPNPWT) to synthesize guanosine and adenosine analogues in a one pot reaction (Scheme 5). Initial studies showed that the five enzyme cascade (Scheme 5A) was able to generate the ribonucleoside adenosine 25 in good conversions (Table ). Furthermore as CfDHAKWT is enantiospecific, D-glyceraldehyde can also be substituted with 2 equivalents of DL-glyceraldehyde (Scheme 5E). While the 2′-substrate scope of wild-type PPM and PNP is likely to be restricted we found that in addition to 2′-OH both 2′-Me 27 and 2′-F 28 substitutions were accepted, by both enzymes, albeit with lower conversions (Table ). Alongside this, a 10x excess of the D-glyceraldehyde starting material was required to give reasonable conversions. This is presumably due to the lower activity of the PPM and PNP steps, alongside the instability of the phosphorylated intermediates, particularly the corresponding 1' phosphate. The higher activity of the cascade towards the wild-type substrate 2′-OH suggests that, in principle, this excess would not be required for 2′-Me and 2′-F analogues if more active variants of PPM and PNP were obtained.