Free energies of hydration were calculated using GROMACS 86 due to its ability to include extra sites during alchemical perturbation. All input files were generated using QUBEKit which writes OPLS FF style GROMACS .top and .gro files. The virtual sites were all constructed by hand using the simplest method available for each molecule, with a connection being added between the site and parent to again make the 1-4 interaction lists consistent with OpenMM and BOSS. Each molecule of the test set was annihilated from a cubic box containing approximately 1500 TIP4P water molecules using a two-step approach over 21 λ-windows, first turning of the charges followed by the L-J terms. The solute-solvent nonbonded interactions were switched off via coupling to the λ reaction parameter using soft-core potentials with settings α = 0.5, p = 1 and σ = 0.3. The charges were decoupled using λ values of (0.00 0.25 0.50 0.75 1.00) and van der Waals using λ values of (0.00 0.05 0.10 0.20 0.30 0.40 0.50 0.55 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00). The simulations were again run in the NPT ensemble at 1 atm and 25 • C. All solvent-solute and solvent-solvent non-bonded interactions were truncated at 10 Å and smoothed over the last 0.5 Å. PME was used with a long-range correction applied to the total energy and pressure. Each λ-window was run using Langevin dynamics and a two femtosecond time step with bonds involving hydrogen constrained using the LINCS algorithm. The starting configurations at each λ-window were first minimized before being equilibrated twice. The first was a 100 ps run in the canonical ensemble (NVT) followed by a 200 ps run in the NPT ensemble. Finally, the production stage was run for 1 ns and the free energy of hydration was calculated using Bennett's acceptance ratio as implemented in the GROMACS BAR module. All uncertainties for the calculations were found to be less than 0.3 kcal/mol.