For the transformations starting from L2 (L2 à L* in Figure ), the average free energy change from TIES calculations agrees well with the experimental data (Figure ). The transformation L30 à L2, however, overestimates the difference by 1.28 kcal/mol. The two-step transformation for all 17 compounds (L30 à L2 à L*) generates an averaged overestimation of 1.17 kcal/mol, similar to that observed in L30 à L2 transformation. Compounds L* and L2 (Figure ) have the same/similar alkyl isopropyl substituent at the hydrophobic position (Figure ), while L30 does not; transformations L30 à L* therefore share the same change as that in L30 à L2 transformation. There is a similar overestimation in L30 à L* (-0.99 kcal/mol) as that in L30 à L2, which is likely to be rooted in the same alchemical change of growing an alkyl group from L30.