When taking a close look at the HPLC trace of the products of one such transfer reaction of alanyl (Ala) residues, (3 eq. 5'-L-Ala-pCUG mixed anhydride as donor strand mixed with 1 eq. 5'-CGCAGUUCCA as acceptor stand, Fig. ) we discovered a new peak with a longer retention time than the 2'/3'-Ala-ester acceptors (Fig. ). The peak reached its maximum yield of 8% after 140 min, compared to 70 min for the known 2'/3'-Ala-esters (Fig. , Table , Fig. ). This new peak decreased in intensity slowly with a half-life of 14 h at 16°C, more than double that of the 2'/3'-Ala-esters. After isolation of species responsible for the new peak, MALDI-TOF characterization was consistent with a 10-mer strand plus two Ala residues (Fig. ). Although it is possible in theory to produce a bis-2',3'-Ala,Ala-diester (with Ala residues both at terminal or internal 2' and terminal 3' positions), we would expect such species to have comparable lability to the 2'/3'-Ala-esters. However, we expected the 2'/3'-dialanyl-esters (Ala-Ala) to be more stable than the monoalanyl esters and therefore tentatively assigned the new peak to result from the formation of the 2'/3'-Ala-Ala-ester acceptor strands.