5. Add 4 eq DIPEA to the amino acid solution and vortex it. Incubate 2–3 min to preactivate the building block for efficient
coupling.
6. Transfer amino acid solution to the reaction tube, close with a
septum and Incubate at room temperature on the MiniBlock®
at 500 rpm for 1 h (see Fig. 2, step 2).
7. Check completion of the coupling reaction using Kaiser test,
and proceed to step 8 only when coupling is complete (see
Note 5). If coupling is incomplete repeat steps 4–7
8. Wash the resin extensively with 20 mL of DMF. Repeat steps
2–7 (see Fig. 2, steps 3–6) for the next amino acid until all
building blocks have been coupled (see Note 6).
9. After extensive washing with 20 mL of DMF and 20 mL of
DCM dry the resin under vacuum
10. Add 5 mL cleavage cocktail to the reaction tube and incubate at
room temperature on a MiniBlock® at 500 rpm for 2 h (see
Note 7) (see Fig. 2, steps 7 and 8).
11. Filter resin by using air to push cleavage mixture through the
disposable reaction tubes and collecting flow-through, which
contains peptide and cleavage cocktail, in a sterile 50 mL
Falcon tube.
12. Precipitate peptide by adding 50 mL ice-cold diethyl ether, and
centrifuge for 10 min at 3500 Â g, in 4
C. Discard supernatant. Repeat this step 3 times.
13. Dissolve pellet with water. If peptide is not soluble use the
minimal amount of dimethyl sulfoxide (DMSO) (see Note 8)
or acetonitrile (MeCN), and purify using preparative
RP-HPLC with a linear gradient of a binary solvent system of
H 2 O–MeCN–TFA (A: 95/5/0.1; B: 5/95/0.1) at a flow rate
of 20 mL min
À1 .
14. Confirm final peptide mass with LC-MS using reverse-phase
C18 column for peptide analysis, operating at a linear gradient
binary solvent system of H 2 O–MeCN–formic acid (A: 95/5/
0.1; B: 5/95/0.086) at a flow rate of 1 mL min
À1 .
15. Analyze peptide purity with RP-UPLC using the C18 reverse
phase column for peptide analysis and a linear gradient of the
binary solvent system of H 2 O–MeCN–TFA (A: 95/5/0.1; B:
5/95/0.1) at a flow rate of 1 mL min
À1 .
16. Lyophilize the final product.
ä
Fig. 2 (continued) subsequent amino acid additions (steps 4–6) and final Fmoc
deprotection and cleavage from the resin (steps 7–8). Fmoc group, temporary
protecting groups (TPG) and resin linker are highlighted in red, blue and green
respectively. AA n and R n represent an amino acid and its side chain in a specific
n position
PDZ Domain Peptide Inhibitors
165
coupling.
6. Transfer amino acid solution to the reaction tube, close with a
septum and Incubate at room temperature on the MiniBlock®
at 500 rpm for 1 h (see Fig. 2, step 2).
7. Check completion of the coupling reaction using Kaiser test,
and proceed to step 8 only when coupling is complete (see
Note 5). If coupling is incomplete repeat steps 4–7
8. Wash the resin extensively with 20 mL of DMF. Repeat steps
2–7 (see Fig. 2, steps 3–6) for the next amino acid until all
building blocks have been coupled (see Note 6).
9. After extensive washing with 20 mL of DMF and 20 mL of
DCM dry the resin under vacuum
10. Add 5 mL cleavage cocktail to the reaction tube and incubate at
room temperature on a MiniBlock® at 500 rpm for 2 h (see
Note 7) (see Fig. 2, steps 7 and 8).
11. Filter resin by using air to push cleavage mixture through the
disposable reaction tubes and collecting flow-through, which
contains peptide and cleavage cocktail, in a sterile 50 mL
Falcon tube.
12. Precipitate peptide by adding 50 mL ice-cold diethyl ether, and
centrifuge for 10 min at 3500 Â g, in 4
C. Discard supernatant. Repeat this step 3 times.
13. Dissolve pellet with water. If peptide is not soluble use the
minimal amount of dimethyl sulfoxide (DMSO) (see Note 8)
or acetonitrile (MeCN), and purify using preparative
RP-HPLC with a linear gradient of a binary solvent system of
H 2 O–MeCN–TFA (A: 95/5/0.1; B: 5/95/0.1) at a flow rate
of 20 mL min
À1 .
14. Confirm final peptide mass with LC-MS using reverse-phase
C18 column for peptide analysis, operating at a linear gradient
binary solvent system of H 2 O–MeCN–formic acid (A: 95/5/
0.1; B: 5/95/0.086) at a flow rate of 1 mL min
À1 .
15. Analyze peptide purity with RP-UPLC using the C18 reverse
phase column for peptide analysis and a linear gradient of the
binary solvent system of H 2 O–MeCN–TFA (A: 95/5/0.1; B:
5/95/0.1) at a flow rate of 1 mL min
À1 .
16. Lyophilize the final product.
ä
Fig. 2 (continued) subsequent amino acid additions (steps 4–6) and final Fmoc
deprotection and cleavage from the resin (steps 7–8). Fmoc group, temporary
protecting groups (TPG) and resin linker are highlighted in red, blue and green
respectively. AA n and R n represent an amino acid and its side chain in a specific
n position
PDZ Domain Peptide Inhibitors
165