3. Close the reaction vessel with the stopcock and add 2 mL neat
TFA. Close the reaction vessel with the Teflon screw cap and
allow deprotection for 1 min.
4. Repeat the deprotection (step 2) to ensure complete
Boc-deprotection.
5. Flow wash the resin with DMF for 30 s.
6. Weigh 4 eq of Boc-protected amino acid into a 10 mL glass vial
and add 4 eq of HBTU (0.8 mL) from the 0.5 M stock
solution. Dissolve the amino acid by shaking.
7. After complete dissolution, add 8 eq DIEA (0.14 mL) to the
amino acid–HBTU mixture. Allow preactivation for 2 min and
transfer the solution to the resin.
8. Close the reaction vessel with the Teflon screw cap and shake
the mixture in a wrist arm shaker. An incubation time of
10–20 min is needed for complete coupling efficiency. After
10–20 min, the coupling efficiency can be tested with a Kaiser
test (see Note 18). If the coupling is shown to be incomplete by
a blue color change of the resin beads, repeat the coupling step.
In case of complete coupling (no blue color change), continue
with the deprotection step and the coupling of the next
amino acid.
3.8.3 Cleavage
and Global Deprotection
Cleavage and deprotection of peptides synthesized by the Boc/Bzl
is most efficiently done by the anhydrous hydrogen fluoride, a
strong acid that is volatile and toxic and must be handled in
specialized equipment. The protocol for HF cleavage is beyond
the scope of this chapter, but extensively described elsewhere [32].
3.9 Expressed
Protein Ligation
For both, C- and N-terminal modifications, the same ligation
protocol is used. If a modification is implemented in the
N-terminal peptide fragment, this fragment bears a thioester in
case of Boc-SPPS, or a hydrazine linker in case of Fmoc-SPPS.
For the latter option, activate the hydrazide via oxidation preliminary to the ligation step (see Subheading 3.9.1).
3.9.1 Oxidation
of Hydrazide Peptide
to Generate an Active
Thioester
1. Weight out the hydrazide peptide in a reaction tube and dissolve it to a final concentration of 4 mM in ligation buffer 1.
2. Add 5 eq of the 0.2 M NaNO 2 solution to the hydrazide
peptide and cool the reaction mixture to 0
C, while stirring
(see Note 26). Complete oxidation takes 20 min (see Note 27).
3.9.2 Ligation
1. Weigh out the fragments with a proportion of 1/1.2 of
C-terminal fragment to the N-terminal fragment.
2. Dissolve the N-terminal thioester peptide to a final concentration of 2 mM in ligation buffer 2 and adjust pH to 6.0 with
NaOH (see Note 28).
Synthetic PDZ Domains
211
TFA. Close the reaction vessel with the Teflon screw cap and
allow deprotection for 1 min.
4. Repeat the deprotection (step 2) to ensure complete
Boc-deprotection.
5. Flow wash the resin with DMF for 30 s.
6. Weigh 4 eq of Boc-protected amino acid into a 10 mL glass vial
and add 4 eq of HBTU (0.8 mL) from the 0.5 M stock
solution. Dissolve the amino acid by shaking.
7. After complete dissolution, add 8 eq DIEA (0.14 mL) to the
amino acid–HBTU mixture. Allow preactivation for 2 min and
transfer the solution to the resin.
8. Close the reaction vessel with the Teflon screw cap and shake
the mixture in a wrist arm shaker. An incubation time of
10–20 min is needed for complete coupling efficiency. After
10–20 min, the coupling efficiency can be tested with a Kaiser
test (see Note 18). If the coupling is shown to be incomplete by
a blue color change of the resin beads, repeat the coupling step.
In case of complete coupling (no blue color change), continue
with the deprotection step and the coupling of the next
amino acid.
3.8.3 Cleavage
and Global Deprotection
Cleavage and deprotection of peptides synthesized by the Boc/Bzl
is most efficiently done by the anhydrous hydrogen fluoride, a
strong acid that is volatile and toxic and must be handled in
specialized equipment. The protocol for HF cleavage is beyond
the scope of this chapter, but extensively described elsewhere [32].
3.9 Expressed
Protein Ligation
For both, C- and N-terminal modifications, the same ligation
protocol is used. If a modification is implemented in the
N-terminal peptide fragment, this fragment bears a thioester in
case of Boc-SPPS, or a hydrazine linker in case of Fmoc-SPPS.
For the latter option, activate the hydrazide via oxidation preliminary to the ligation step (see Subheading 3.9.1).
3.9.1 Oxidation
of Hydrazide Peptide
to Generate an Active
Thioester
1. Weight out the hydrazide peptide in a reaction tube and dissolve it to a final concentration of 4 mM in ligation buffer 1.
2. Add 5 eq of the 0.2 M NaNO 2 solution to the hydrazide
peptide and cool the reaction mixture to 0
C, while stirring
(see Note 26). Complete oxidation takes 20 min (see Note 27).
3.9.2 Ligation
1. Weigh out the fragments with a proportion of 1/1.2 of
C-terminal fragment to the N-terminal fragment.
2. Dissolve the N-terminal thioester peptide to a final concentration of 2 mM in ligation buffer 2 and adjust pH to 6.0 with
NaOH (see Note 28).
Synthetic PDZ Domains
211