With the use of HPLC-MS analysis of the reaction mixture from the operation of 1, we did not detect any products corresponding to other peptide compositions (neither different sequences nor peptides with more or less than one Phe, Leu, or Ala residue), indicating that the peptide synthesis occurs overwhelminglywithin the confines of the molecular machine. In contrast, a control reaction carried out under identical conditions but using the nonthreaded strand and macrocycle yielded several products, including strands with one or more amino acid groups cleaved, but there was no evidence for the formation of 7 under these conditions. Thus, the threaded architecture of the molecular machine—encompassing the catalytic site, elongation site, and the building block strand—is essential for the sequential peptide synthesis, and the mode of operation of the molecular machine is consistent with the mechanism shown in Fig. 3. The peptide (9), still bearingm the GlyGlyCys unit at the C terminus, could subsequently be cleaved from the macrocycle by hydrolysis (Fig. 3, iii).