Synthesis of phosphinic inhibitor DG013A

The synthesis of phosphinic pseudotripeptide DG013A (43) started from the stereochemically pure building block Cbz-(R)-hPhe[PO(OH)CH₂](S)-Leu-OH (the preparation of this precursor will be reported elsewhere). Removal of the amino terminal Cbz protecting group was performed in acidic conditions by using HBr/AcOH as a deprotection medium. The hydrobromic salt of the resulting pseudodipeptide was protected with the Boc group under basic conditions by using (Boc)2O as a protection reagent. This procedure afforded the building block of the formula Boc-(R)-hPhe[PO(OH)CH₂](S)-Leu-OH in 83% overall yield, starting from the Cbz protected analogue (48). Coupling of H- (S)Trp-NH2 followed a previously reported protocol which allows efficient peptide coupling in the presence of an unprotected phosphinic acid group (49). In particular, excess of N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride was employed as a coupling reagent, in the presence of hydroxybenzotriazole and N,N-Diisopropylethylamine. Purification of the resulting Boc-protected tripeptide and subsequent standard acidic deprotection by dilute trifluoroacetic acid afforded DG013A in 75% yield for the last two synthetic steps. The compound was purified by reversed-phase HPLC on a C18 chromolith column (Merck) using a 10–−50% (vol/vol) acetonitrile gradient in water containing 0.05% (vol/vol) trifluoroacetic acid. A single major peak was characterized by mass spectrometry, lyophilized, and dissolved in deionized water. The inhibitor concentration in the final working stock was calculated using the measured absorbance at 280nm and the extinction coefficient 5,700 M⁻¹·cm⁻¹.