We were able to improve the sampling of dif- In the "regular protocol" the LJ-ϵ of the entire molecules was scaled down by multiplying it with a scaling factor of γ = 0.5. In the "restrain dihedral" protocol the rotatable bond shown in yellow (a) was restrained during the free energy calculation and the contribution of the restraint was accounted for by releasing the restraints afterwards. In protocol "R-group ϵ=0.01" the LJ-ϵ of the ethyl methyl ether group was scaled down by a scaling factor of γ=0.01, allowing Hamiltonian lambda exchange to help accelerate sampling of this bond rotation. The grey line shows the experimental binding free energy. Using the regular protocol, transformation from both directions do not converge to the same relative free energy due to sampling problems, while transformations in both directions using the two other protocols converged to the same free energies as well as did transformations within the same protocol.