PeptidesPeptidesMartina 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 SynthesisMonitoring 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 test-tube 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!