3.1.2 Generation
of Peptide Probes:
Conjugation with Cy5
Maleimide
1. For the synthesis of peptides probes we follow the procedure
described in Subheading 3.1.1 until step 8, were we add a CSG
spacer to the total peptide sequence.
2. The peptide is then cleaved, purified, and lyophilized following
the steps 10–15 described in Subheading 3.1.1
3. Degassed PBS buffer is then used to dissolve the desired
amount of peptide. To degas the buffer seal the flask with a
Septa and place a long syringe in the bottom of the flask with
N 2 or He sparging and leave another small syringe in the top to
allow the excess of N 2 to get removed.
4. 10 eq of Cy5-maleimide is then dissolved in DMSO and added
to the peptide solution in PBS. Leave the reaction for 2 h at
room temperature.
5. The peptide mixture is then purified 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 (see Note 9)
6. Confirm the mass of the final product 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
. 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 .
7. Lyophilize the final product (see Note 10).
3.2 Dimeric PDZ
Peptides and Cell
Penetrating Tags
Compared to monomeric peptides, dimerization of peptides has
several advantages, such as increased binding affinity or enhanced
selectivity against a specific protein. Below we provide a general
approach on how to synthesize dimeric peptide inhibitors based on
the usage of standard Fmoc SPPS as described in Subheading 3.1.1.
Moreover, cell penetrating peptide (CPP) tags facilitate the
cellular uptake of therapeutical binders or “cargo” them inside
cells. These CPPs tags are usually enriched in positively charged
amino acids or have sequences with an alternating pattern of polar/
charge amino acids or nonpolar/hydrophobic combinations.
In the section below we explain how to synthesize dimeric PDZ
peptides and how to include a CPP tag to them.
3.2.1 Synthesis
of Dimeric Symmetrical
PDZ Binders
1. Follow the steps in Subheading 3.1.1 until step 8, until the
pentameric binder is completed on resin and remove the Fmoc
group by using 20% (v/v) piperidine in DMF (see Note 11).
2. The PEG-diacid (0.1 eq) (see Note 12) is preactivated with
HBTU (0.2 eq) and DIPEA (0.4 eq) and added to the peptideresin (1 eq, 0.25 mmol) in DMF (2 mL), and incubated for
166
Dominik J. Essig et al.
of Peptide Probes:
Conjugation with Cy5
Maleimide
1. For the synthesis of peptides probes we follow the procedure
described in Subheading 3.1.1 until step 8, were we add a CSG
spacer to the total peptide sequence.
2. The peptide is then cleaved, purified, and lyophilized following
the steps 10–15 described in Subheading 3.1.1
3. Degassed PBS buffer is then used to dissolve the desired
amount of peptide. To degas the buffer seal the flask with a
Septa and place a long syringe in the bottom of the flask with
N 2 or He sparging and leave another small syringe in the top to
allow the excess of N 2 to get removed.
4. 10 eq of Cy5-maleimide is then dissolved in DMSO and added
to the peptide solution in PBS. Leave the reaction for 2 h at
room temperature.
5. The peptide mixture is then purified 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 (see Note 9)
6. Confirm the mass of the final product 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
. 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 .
7. Lyophilize the final product (see Note 10).
3.2 Dimeric PDZ
Peptides and Cell
Penetrating Tags
Compared to monomeric peptides, dimerization of peptides has
several advantages, such as increased binding affinity or enhanced
selectivity against a specific protein. Below we provide a general
approach on how to synthesize dimeric peptide inhibitors based on
the usage of standard Fmoc SPPS as described in Subheading 3.1.1.
Moreover, cell penetrating peptide (CPP) tags facilitate the
cellular uptake of therapeutical binders or “cargo” them inside
cells. These CPPs tags are usually enriched in positively charged
amino acids or have sequences with an alternating pattern of polar/
charge amino acids or nonpolar/hydrophobic combinations.
In the section below we explain how to synthesize dimeric PDZ
peptides and how to include a CPP tag to them.
3.2.1 Synthesis
of Dimeric Symmetrical
PDZ Binders
1. Follow the steps in Subheading 3.1.1 until step 8, until the
pentameric binder is completed on resin and remove the Fmoc
group by using 20% (v/v) piperidine in DMF (see Note 11).
2. The PEG-diacid (0.1 eq) (see Note 12) is preactivated with
HBTU (0.2 eq) and DIPEA (0.4 eq) and added to the peptideresin (1 eq, 0.25 mmol) in DMF (2 mL), and incubated for
166
Dominik J. Essig et al.