Cyclic NGR Peptide-Daunorubicin Conjugates

The cyclic NGR peptide-daunorubicin conjugates were prepared by a combination of solid phase peptide synthesis and chemoselective ligation (oxime bond formation) in solution as described in Tripodi et al. [57 (link)]. The crude peptides and conjugates were purified on a KNAUER 2501 HPLC system (KNAUER, Bad Homburg, Germany) was applied with a semi-preparative Phenomenex Luna C18 column (250 mm × 21.2 mm) with 10 μm silica (100 Å pore size) (Torrance CA). Linear gradient elution (0 min 5% B; 60 min 90% B) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in MeCN-H₂O (80:20, v/v)) was used at a flow rate of 4 mL/min. The resulting fractions were lyophilized. Electrospray Ionization (ESI)-mass spectrometric analyses were carried out on an Esquire 3000+ ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). The freeze-dried bioconjugates were directly used for the in vitro and in vivo studies.