To seek vancomycin analogs with broader antibacterial activity, effects of backbone modifications for the agylcon 2 on binding with D-Ala-D-Ala- and D-Ala-D-Lac-containing peptides were investigated by Monte Carlo/free energy perturbation (MC/FEP) calculations. The experimental trend in binding affinities for 2 with three tripeptides was well reproduced. Possible modifications of the peptide bond between residues 4 and 5 were then considered, specifically for conversion of the OCNH linkage to CH(2)NH(2)(+) (6), FCCH (7), HCCH (8), and HNCO (9). The MC/FEP results did not yield binding improvements for 7, 8, and 9, though the fluorovinyl replacement is relatively benign. The previously reported analog 6 remains as the only variant that exhibits improved affinity for the D-Ala-D-Lac sequence and acceptable affinity for the D-Ala-D-Ala sequence.