Two approaches to the chemoenzymatic synthesis of 2-fluorocordycepin and 2-chlorocordycepin were studied: (i) the use of 3′-deoxyadenosine (cordycepin) and 3′-deoxyinosine (3′dIno) as donors of 3-deoxy- d -ribofuranose in the transglycosylation of 2-fluoro- (2FAde) and 2-chloroadenine (2ClAde) catalyzed by the recombinant E. coli purine nucleoside phosphorylase (PNP), and (ii) the use of 2-fluoroadenosine and 3′-deoxyinosine as substrates of the cross-glycosylation and PNP as a biocatalyst. An efficient method for 3′-deoxyinosine synthesis starting from inosine was developed. However, the very poor solubility of2ClAde and2FAde is the limiting factor of the first approach. The second approach enables this problem to be overcome and it appears to be advantageous over the former approach from the viewpoint of practical synthesis of the title nucleosides. The 3-deoxy-α- d -ribofuranose-1-phosphate intermediary formed in the 3′dIno phosphorolysis by PNP was found to be the weak and marginal substrate of E. coli thymidine (TP) and uridine (UP) phosphorylases, respectively. Finally, one-pot cascade transformation of 3-deoxy- d -ribose in cordycepin in the presence of adenine and E. coli ribokinase, phosphopentomutase, and PNP was tested and cordycepin formation in ca. 3.4% yield was proved.