The general procedure for synthesizing compound (CAS:159858-22-7) from compound (CAS:159857-79-1) is as follows: first, Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 equiv; see U.S. Patent No. 6,214,345 by Firestone et al. ) was diluted with DMF (120 mL, 0.2 M). Diethylamine (60 mL) was added to this solution. The progress of the reaction was monitored by HPLC to confirm that the reaction was completed within 2 hours. Subsequently, the reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (~100mL) under sonication for 10 minutes. After addition of ether (200 mL), the precipitate was further sonicated for 5 minutes. The solution was allowed to stand for 30 minutes without stirring, then filtered and dried under high vacuum to give Val-cit-PAB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). Next, Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC-OSu (6.5 g, 21 mmol, 1.0 eq.; see Willner et al. 1993, Bioconjugate Chem. 4: 521). After 2 hours of reaction, the completion of the reaction was confirmed on the basis of HPLC. The reaction mixture was concentrated and the resulting oil was precipitated with ethyl acetate (50 mL). After sonication for 15 minutes, ether (400 mL) was added and the mixture was further sonicated until all large particles were broken. The solution was then filtered and the solid dried to give an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M-H].
