Table 2. Computed Relative Free Energies of Binding (kcal/mol) Using Standard FEP and REST^a.

	standard26		REST		EC5026
R	WT	Y181C	WT	Y181C	WT	Y181C
Me	0.00	0.00	0.00	0.00	0.011	0.210
Et	–1.60	–2.90	–0.74	–2.28	0.0013	0.0069
Pr	–0.23	−4.46	0.69	−1.60	–	–
i-Pr	–0.82	–5.45	0.14	–4.40	0.0052	0.0072
OEt	0.97	–1.35	3.15	0.01	0.028	0.048
CH2OMe	2.67	0.78	1.72	1.02	0.0036	0.690
t-Bu	0.21	–4.15	0.73	–3.39	1.3b	–

^aDiscrepancies larger than 1.5 kcal/mol between the two methods are highlighted in bold. Note that isopropyl REST results are averages over the two mutation pathways and four runs described in Table 1; tert-butyl results are averages over three mutation pathways (Table 3). The estimated uncertainties in the REST results range from 0.08 (Et) to 0.22 kcal/mol (OEt). The estimated uncertainties in the standard results are listed in full elsewhere.²⁶ Also shown for comparison are experimental EC₅₀ data (μM).²⁶

^bThe 4-tert-butyl-2,6-dichlorobenzyl analog of 1b.