Peptides P-4 and P-6 bound to 2-chlorotrityl resin (Additional file 1 : Figure S1) were supplied by PepMic Co., Ltd, China. Drugs (Chlorambucil, Melphalan and Bendamustine) and other chemicals and solvents were purchased from Sigma-Aldrich (Rehovot, Israel). N-Boc Melphalan was prepared by the method described by Xu et al. [20 (link)] and conjugated to the peptide via standard Fmoc-SPPS procedures. The solid phase based synthesis of peptide- Chlorambucil and Bendamustine PDC was performed by following a previously described procedure [21 (link)] The peptide-drug-conjugate were cleaved from resin using 95% TFA 5% DCM mixture, purified on preparative HPLC and dried by lyophilization.
LC/MS analyses were performed on an Agilent Technologies 1260 Infinity (LC) 6120 quadruple (MS) with an Agilent SB-C18 column, 2.1 × 50 mm, column temperature 50 °C, eluent water-acetonitrile (ACN) that contained 0.1% of formic acid. HPLC purifications were carried out on an ECOM preparative system, with dual UV detection at 254 and 230 nm. Phenomenex Gemini® 10 µm RP18 110 Å, LC 250 × 21.2 mm column was used. The column was kept at 25 °C. Eluent A (0.1% TFA in water) and B (0.1% TFA in ACN) were used. A typical elution was a gradient from 100% A to 100% B over 35 min at a flow rate of 25 ml/min.
LC/MS analyses were performed on an Agilent Technologies 1260 Infinity (LC) 6120 quadruple (MS) with an Agilent SB-C18 column, 2.1 × 50 mm, column temperature 50 °C, eluent water-acetonitrile (ACN) that contained 0.1% of formic acid. HPLC purifications were carried out on an ECOM preparative system, with dual UV detection at 254 and 230 nm. Phenomenex Gemini® 10 µm RP18 110 Å, LC 250 × 21.2 mm column was used. The column was kept at 25 °C. Eluent A (0.1% TFA in water) and B (0.1% TFA in ACN) were used. A typical elution was a gradient from 100% A to 100% B over 35 min at a flow rate of 25 ml/min.