30 min, followed by washing with DMF. This procedure is
repeated 5 times.
3. Add 5 mL cleavage cocktail to the reaction tube and incubate at
room temperature on a MiniBlock® at 500 rpm for 2 h.
4. 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.
5. 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.
6. Dissolve pellet with water. If peptide is not soluble use the
minimal amount of DMSO or 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
.
3.2.2 Synthesis of
the Dimeric Peptide Linker,
Ns-NPEG4 Linker
[10-((2-Nitrophenyl)
4,7,13,16-Tetraoxa-10Azanonadecane-1,19-Dioic
Acid] for CPP Conjugation
1. Wash the 2-chlorotrityl chloride resin (3 mmol) with DMF
(15 times, each 10 mL). Finally, swell in 15 mL of DMF for
20 min, drain, and treat with Fmoc-NH-PEG 2 -
CH 2 CH 2 COOH (2 mmol) in dry DMF (8 mL) and DIPEA
(10 mmol). After shaking for 60 min, add MeOH (25 mmol)
(see Fig. 3, step 1).
2. Drain and wash the resin with DMF (5 times, each of 10 mL),
and deprotect the Fmoc group with 20% (v/v) piperidine in
DMF (15 mL) for 5 min and 15 min. Wash in between with
DMF and tetrahydrofuran (THF) (5 times, each of 10 mL) (see
Fig. 3, step 2.1)
3. Swell the resin in DIPEA (12 mmol) and THF (8 mL) for
15 min, and add slowly ortho-nitrobenzenesulfonyl chloride
(NsCl, 8 mmol) in DCM (5 mL) while gently stirring the resin.
Fig. 3 SPPS synthesis scheme of the Ns-NPEG4 linker. (1) Fmoc-NH-PEG 2 -CH 2 CH 2 COOH is loaded into the
resin (2) Removal of Fmoc group and addition of NsCl and PEG 2 -CH 2 CH 2 COOtBu. (3) Removal of tBu group and
cleavage from the resin using TFA/H2O/TIPS to obtain the final Ns-NPEG4 Linker. (Figure adapted from Bach
et al. (2012))
PDZ Domain Peptide Inhibitors
167
repeated 5 times.
3. Add 5 mL cleavage cocktail to the reaction tube and incubate at
room temperature on a MiniBlock® at 500 rpm for 2 h.
4. 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.
5. 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.
6. Dissolve pellet with water. If peptide is not soluble use the
minimal amount of DMSO or 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
.
3.2.2 Synthesis of
the Dimeric Peptide Linker,
Ns-NPEG4 Linker
[10-((2-Nitrophenyl)
4,7,13,16-Tetraoxa-10Azanonadecane-1,19-Dioic
Acid] for CPP Conjugation
1. Wash the 2-chlorotrityl chloride resin (3 mmol) with DMF
(15 times, each 10 mL). Finally, swell in 15 mL of DMF for
20 min, drain, and treat with Fmoc-NH-PEG 2 -
CH 2 CH 2 COOH (2 mmol) in dry DMF (8 mL) and DIPEA
(10 mmol). After shaking for 60 min, add MeOH (25 mmol)
(see Fig. 3, step 1).
2. Drain and wash the resin with DMF (5 times, each of 10 mL),
and deprotect the Fmoc group with 20% (v/v) piperidine in
DMF (15 mL) for 5 min and 15 min. Wash in between with
DMF and tetrahydrofuran (THF) (5 times, each of 10 mL) (see
Fig. 3, step 2.1)
3. Swell the resin in DIPEA (12 mmol) and THF (8 mL) for
15 min, and add slowly ortho-nitrobenzenesulfonyl chloride
(NsCl, 8 mmol) in DCM (5 mL) while gently stirring the resin.
Fig. 3 SPPS synthesis scheme of the Ns-NPEG4 linker. (1) Fmoc-NH-PEG 2 -CH 2 CH 2 COOH is loaded into the
resin (2) Removal of Fmoc group and addition of NsCl and PEG 2 -CH 2 CH 2 COOtBu. (3) Removal of tBu group and
cleavage from the resin using TFA/H2O/TIPS to obtain the final Ns-NPEG4 Linker. (Figure adapted from Bach
et al. (2012))
PDZ Domain Peptide Inhibitors
167