To a stirred solution of L-prolin-N-(2-(tert-butyloxycarbonylaminomethyl)-5-chlorobenzyl)amide from step 2 (3.90 g, 10.6 mmol, HPLC RT=2.77 min), (2R)-3,3-dimethyl-R-hydroxybutyric acid from step 3 (1.40 g, 10.6 mmol), and HOBT hydrate (1.68 g, 11 mmol) in DMF (60 mL) was added EDC (2.65 g, 13.8 mmol). Diisopropylethylamine was then added slowly in portions (11.5 mL total) to bring the pH of the solution to 6-7 as measured on wetted E. Merck pH indicator strips. The mixture was stirred at ambient temperature for 6 h, at which time HPLC analysis indicated complete consumption of the proline starting material. Water (10 mL) was added and the solvents were removed under reduced pressure. The residue was partitioned between EtOAc (300 mL) and saturated aqueous NaHCO3 (200 mL). The EtOAc layer was separated, dried over anhydrous MgSO4, and filtered. The filtrate solvent was removed under reduced pressure and the residue was purified by flash chromatography using a gradient elution of 2:1, 4:1, and 1:0 EtOAc:hexanes. 1-((2R)-3,3-dimethyl-2-hydroxybutanoyl)-N-(2-(tert-butyloxycarbonylaminomethyl)-5-chlorobenzyl)-L-prolinamide was obtained as a colorless gum (4.65 g, 91%; TLC Rf=0.7 (EtOAc); HPLC RT=3.35 min, method A; LC-MS m/z=482).
