Figure 6.

Aminopeptidases LAP3, LTA4H, and RNPEP can hydrolyze melflufen to melphalan and 4-F-Phe-OEt. (A) Reaction pathway of the hydrolysis of melflufen peptide bond by the aminopeptidases LAP3, LTA4H, and RNPEP. (B–D) Incubation buffer and substrates were mixed and incubated without and with (B) LAP3, (C) LTA4H, and (D) RNPEP for 2 h at 37 °C (with duplicate incubations). The incubation was stopped by adding ice-cold acetonitrile. Using LC-HRMS, 4-F-Phe-OEt was then quantified, with concentrations presented as the peak area in the figure. 4-F-Phe-OEt: para-fluoro-L-phenylalanine ethyl ester; LC-HRMS: liquid chromatography–high-resolution mass spectrometry.