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.
2501 hplc system
Manufactured by Knauer
Sourced in Germany
The 2501 HPLC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative applications. It features a dual-piston pump, a manual injection valve, and a UV-VIS detector. The system is capable of generating a precise and stable flow rate for the separation and analysis of a wide range of chemical compounds.
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4 protocols using 2501 hplc system
1
Cyclic NGR Peptide-Daunorubicin Conjugates
2
RP-HPLC Purification of Peptides and Conjugates
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Analogous to the description in [17 (link)] RP-HPLC purification was used for the isolation of pure peptides and conjugates. 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 15% B; 5 min 15% B; 55 min 70% 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 9.5 mL/min. Peaks were detected at 220 nm.
3
Purification and Analysis of Crude Products
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The crude products were purified on a KNAUER 2501 HPLC system (KNAUER, Berlin, Germany) using a semipreparative Phenomenex Jupiter C 18 column (250 mm x 10 mm) with 10 µm silica (300 Å pore size; Gen-Lab Ltd., Budapest, Hungary). Linear gradient elution (10-75% B; 35 min) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile/H 2 O (80:20, v/v)) was used at a flow rate of 4 mL/min. Peaks were detected at 220 nm. Analytical RP-HPLC was performed on an Exformma EX1600 HPLC system (Gen-Lab Ltd., Budapest, Hungary) using an Agilent Zorbax SB-C 18 column (150 mm x 4.6 mm) with 5 µm silica, 80Å pore size (Kromat Ltd, Budapest, Hungary) as a stationary phase.
Linear gradient elution (0-90% B; 20 min) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile/H 2 O (80:20, v/v)) was used at a flow rate of 1 mL/min. Peaks were detected at 220 nm.
Linear gradient elution (0-90% B; 20 min) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile/H 2 O (80:20, v/v)) was used at a flow rate of 1 mL/min. Peaks were detected at 220 nm.
4
Purification and Analysis of Conjugates
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The conjugates were purified on a KNAUER 2501 HPLC system (KNAUER, Germany) using a semipreparative Phenomenex Jupiter C18 column (250 mm × 10 mm) with 10 μm silica (300 Å pore size, Gen-Lab Ltd., Hungary). Linear gradient elution (0-5 min 10% B, 5-75 min 10-80% B) with eluent A (0.1% TFA in water) and eluent B (0.1% TFA in acetonitrile/H 2 O (80:20, V/V)) was used at a flow rate of 4 mL/min. Peaks were detected at 220 nm. Analytical RP-HPLC was performed on an Exformma EX1600 HPLC system (Gen-Lab Ltd., Hungary) using an Agilent Zorbax SB-C18 column (150 mm × 4.6 mm) with 5 μm silica, 100 Å pore size (Kromat Ltd, Hungary) as a stationary phase.
Linear gradient elution (0-10 min 0% B, 10-30 min 0-90% B) was used at a flow rate of 1 mL/min.
Peaks were detected at 220 nm.
Linear gradient elution (0-10 min 0% B, 10-30 min 0-90% B) was used at a flow rate of 1 mL/min.
Peaks were detected at 220 nm.
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