Peptides Martina Zlámalová Peptide Synthesis Methodology • is carried out by FMOC chemistry, using PEG-Polystyrene resins • peptides are cleaved from the resin and de-protected using cleavage cocktail • peptides are precipitated from the cocktail using cold diethyl ether • washed three times with the cold ether and then dissolved in a crude buffer Reversed phase chromatography • Peptides QC is based on reversed phase chromatography on a Supelco Bio Wide Pore column and by MALDI-TOF mass spectrometry. Peptides not meeting the purity criteria are purified by reversed phase chromatography. Peptide Synthesis Monitoring reagents • Colour test of amino acids in SPPS and SPOS • Reaction of ninhydrin with free primary amines (intensive blue) • Test is applied quantitatively and qualitatively • Important to state – NOT yield the typicaly dark blue with serine, asparagine, aspartic acid and proline Structure • The test kit contains 50 ml each of the following solutions: Phenol, ~ 80% in ethanol, KCN in H2O/pyridin, Ninhydrin 6% in ethanol Procedure • 1. Remove a few resin beads from the reaction vessel and wash 3 times with ethanol • 2. Transfer beads into a small glass test tube and add 3 drops of each solution • 3. Mix well and heat the testtube at 120° for 5 minutes • The resin beads and the solution turn dark blue when a free primary amine is present. The resin beads remain their colour and the solution stays yellow when no free primary amine is present (expected result after successful coupling). • A recoupling step is necessary when a slight blue colour is detected in the solution and/or on beads Thank You for Your attention!