One way to influence the stability of peptide conformations is by replacing native residues with unnatural modifications, which can also be used to permit chemoselective cyclization. To examine the effect of modifications on pre-lasso peptide stability, we repeated the simulations from modified isopeptide bond-forming residues with an auxiliary attached to Gly1N (Figure ) and thioester functionalities at Glu8Cδ (Figure ). In both cases, due to the introduction of auxiliary and thioester functionalities, an enlargement of the pre-lasso ring was necessary to mitigate steric hindrance during the minimization and equilibration process. The simulations were started with the peptide in prelasso conformation and run for similar times to the native peptide (Table ). The resulting 2D free energy maps show marked differences for both the pre-lasso and pre-tadpole conformations (Figure ). For MOD1, the proportion of pre-lasso and pre-tadpole configurations sampled from the entire trajectory increased from 4.1% to 13.5% (Table ).