The question remained, in the case of the fAla transfer onto preformed 2'/3'-Ala-esters why were there two new peaks with significant differences in retention time? When a fAla-residue was transferred from a pentamer donor to the 2',3'-diol of the 10-mer acceptor as a control experiment, two overlapping peaks arose (Fig. ). This could be consistent with racemization of fAla during the chemical synthesis of the mixed anhydride followed by transfer of both D-and L-fAla-residues. Previous experiments had demonstrated free D-Ala mixed anhydrides would only transfer inefficiently whilst D-N-acetylalanyl (D-AcAla) mixed anhydrides transferred equally well as L-across a nicked loop. Alternatively, the two new peaks could be due to a mixture of 2'/3'aminoacyl-esters resolving differently on the column. In the case of fGly transfer to a 2',3'-diol, only one peak was identified, which could suggest either the transfer was regioselective for one alcohol or that both 2'/3'-aminoacyl-esters have equal retention times.