5. Flow-wash with DMF for 1 min before continuing with the
coupling of the next amino acid.
6. Dissolve 4 eq Fmoc-protected amino acid in 4 eq HBTU
(0.8 mL) from the stock solution in a 10 mL glass vial. After
complete dissolution, add 8 eq. (0.14 mL) DIEA into the
mixture to preactivate the amino acid for 2 min (see Note
14). Transfer the preactivated amino acid mixture to the reaction vessel containing the deprotected resin-bound peptide (see
Note 15).
7. For the introduction of the phosphorylated amino acid perform step 4, but with 4 eq Fmoc-protected phosphorylated
building block and dissolve them with 4 eq. 1-[Bis
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (152 mg) in
0.8 mL DMF. After complete dissolution, add 8 eq.
(0.14 mL) DIEA, mix and preactivate for 2 min. Transfer the
preactivated amino acid mixture to the reaction vessel containing the deprotected resin-bound peptide.
8. Close the reaction vessel with a septum and incubate under
shaking at RT for 1 h (see Note 16).
9. Flow-wash with DMF for 30 s to remove excess coupling
mixture and by-products (see Note 17).
10. Coupling efficiency can be tested via a Kaiser test (see Note
18). If coupling is incomplete, indicated by a blue color of the
resin beads, repeat coupling. If the Kaiser test is negative
(no blue color change), continue with deprotection and coupling of the next amino acid (see Note 19).
3.7.3 Cleavage
and Global Deprotection
1. Prepare 5 mL cleavage mix (TFA/phenol/H 2 O/thioanisole/
EDT 82.5/5/5/5/2.5) per 0.1 mmol scale synthesis (see Note
20) [30].
2. Add 5 mL cleavage mixture to the deprotected peptide and
incubate for 1–2.5 h while shaking (see Note 21).
3. Transfer the cleavage solution into a 50 mL-conical tube by
filtering it through a reaction vessel, equipped with a filter. To
achieve full recovery, flush subsequently 2Â with TFA/DCM
(0.3–1 mL in total).
4. Precipitate the peptide with 30–40 mL ice-cold diethyl ether
and spin it down by centrifuging 7 min with 3500 Â g at 4
C
(see Note 22). Remove the clear supernatant by decanting and
dissolve the peptide pellet in a solution of 25% acetonitrile,
0.1% TFA in ultrapure water. Lyophilize the peptide to obtain
white solids, which can be stored long-term at À20
C (see
Note 23) until further use.
Synthetic PDZ Domains
209
coupling of the next amino acid.
6. Dissolve 4 eq Fmoc-protected amino acid in 4 eq HBTU
(0.8 mL) from the stock solution in a 10 mL glass vial. After
complete dissolution, add 8 eq. (0.14 mL) DIEA into the
mixture to preactivate the amino acid for 2 min (see Note
14). Transfer the preactivated amino acid mixture to the reaction vessel containing the deprotected resin-bound peptide (see
Note 15).
7. For the introduction of the phosphorylated amino acid perform step 4, but with 4 eq Fmoc-protected phosphorylated
building block and dissolve them with 4 eq. 1-[Bis
(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (152 mg) in
0.8 mL DMF. After complete dissolution, add 8 eq.
(0.14 mL) DIEA, mix and preactivate for 2 min. Transfer the
preactivated amino acid mixture to the reaction vessel containing the deprotected resin-bound peptide.
8. Close the reaction vessel with a septum and incubate under
shaking at RT for 1 h (see Note 16).
9. Flow-wash with DMF for 30 s to remove excess coupling
mixture and by-products (see Note 17).
10. Coupling efficiency can be tested via a Kaiser test (see Note
18). If coupling is incomplete, indicated by a blue color of the
resin beads, repeat coupling. If the Kaiser test is negative
(no blue color change), continue with deprotection and coupling of the next amino acid (see Note 19).
3.7.3 Cleavage
and Global Deprotection
1. Prepare 5 mL cleavage mix (TFA/phenol/H 2 O/thioanisole/
EDT 82.5/5/5/5/2.5) per 0.1 mmol scale synthesis (see Note
20) [30].
2. Add 5 mL cleavage mixture to the deprotected peptide and
incubate for 1–2.5 h while shaking (see Note 21).
3. Transfer the cleavage solution into a 50 mL-conical tube by
filtering it through a reaction vessel, equipped with a filter. To
achieve full recovery, flush subsequently 2Â with TFA/DCM
(0.3–1 mL in total).
4. Precipitate the peptide with 30–40 mL ice-cold diethyl ether
and spin it down by centrifuging 7 min with 3500 Â g at 4
C
(see Note 22). Remove the clear supernatant by decanting and
dissolve the peptide pellet in a solution of 25% acetonitrile,
0.1% TFA in ultrapure water. Lyophilize the peptide to obtain
white solids, which can be stored long-term at À20
C (see
Note 23) until further use.
Synthetic PDZ Domains
209