In prokaryotes, peptide synthesis starts with N-formylmethionine (fMet) encoded by the start codon AUG. The formyl group allows the corresponding aminoacyl-tRNA to be accommodated at the P site of the ribosome. N-formylaminoacids have been demonstrated to be prebiotically plausible species on early Earth. Therefore, we studied N-formyl-alanyl (fAla) and N-formyl-glycyl transfer (fGly) (Fig. , Table , Fig. ). First, we demonstrated that both fAla and fGly transferred to the 2',3'diol of a 10-mer acceptor in our system (maximum observed yields 16% and 21% at pH 8.0, Fig. , b Ctrl lines). Next, we tested for peptide formation. With the same 10-mer acceptor strand, an Ala residue was first transferred to the 10-mer 3'-terminus using a free Ala mixed anhydride-tetramer donor (5'-L-Ala-pCUGC). When the yield of 2'/3'-Ala-esters reached their peak, 2 eq. fAla-or fGly-mixed anhydride-pentamer donor (5'-fAla-/fGly-pCUGCG) was added to the system. After the addition of formylaminoacyl mixed anhydride, the peak corresponding to the 2'/3'-Ala-esters was observed to decrease much faster (half-life = 1.9 h for fGly and 3.3 h for fAla) than without the formylaminoacyl mixed anhydride (half-life = 6.6 h). New peaks with similar kinetic profiles to each other were detected (Fig. , S5) as well as peaks identifiable as 2'/3'-formylaminoacyl-esters by comparison with a control reaction (Fig. , b Ctrl lines). After isolation, the species responsible for the new peaks produced MALDI peaks consistent with RNA plus both Ala-and fGly/fAla-residues (Fig. ). Despite our previous assumption that no bis-2',3'-products were formed, we entertained the possibility these new peaks may correspond to the 2',3'-Ala-,fAla-/Ala-,fGly-diesters. We would expect that both 2',3'-Ala-,fAla-and 2',3'-Ala-,fGlydiesters would first hydrolyse to 2'/3'-fAla-or 2'/3'-fGly-esters. However, the corresponding peaks for these species did not increase as the new peaks degraded, supporting formation of 2'/3'-dipeptidyl esters instead.