When N-acyl-amino acids are activated at their C termini they form oxazolones which are reactive electrophiles. It is possible the formylaminoacyl-mixed anhydrides could be reversibly cleaved to from 5(4H)-oxazolones, which then subsequently react intermolecularly with the nucleophilic amine of the 2'/3'-aminoacyl-ester. To test this hypothesis we incubated the 10-mer acceptor strand and a pentamer (pCUGCG) containing a 5'-phosphate then added the water-soluble carbodiimide EDC and fGly/fAla to provide in situ formation of oxazolone (Fig. ). This failed to produce any acyl transfer products, mixed anhydride or other RNA esters. Repeating the reaction instead using preformed Ala 5(4H)-oxazolone up to 2000 eq. also failed to produce any peaks where we would expect transfer products nor mixed anhydride, in contrast to previous studies with nucleosides (Fig. ). Similar addition of Ala 5-(4H)-oxazolone to preformed 2'/3'-Ala-esters did not produce new peaks with the correct retention time or stability (Fig. ). As no transfer to the 10-mer strand is observed at such high concentrations of oxazolone and given the relative stability of the formylaminoacyl-mixed anhydrides under these conditions, we think it unlikely that 5-(4H)-oxazolone is implicated in the formation of N-formyldipeptidyl-2'/3'-esters or Nformylaminoacyl-2'/3'-esters presented above. These experimental results confirmed our hypothesis that the strand replacement must occur, even though the experiment is performed below the melting temperature of the RNA duplex.