zlamalovapeptides

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Transcript zlamalovapeptides

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!