Peptide-Based Drug Design

The Spot Technique 55
solution. Ensure that there are no air bubbles under the surface. Do not shake!
Allow the reaction to occur for at least 5 min. Remove the liquid. Add an appropriate
amount of capping solution with 2% DIPEA. Place the membrane face up
in this solution. Allow the membrane to react with the solution for at least 5 min.
8. Building up the peptide chain: Except for the last coupling cycle, repeat steps
3–11. At the last coupling cycle repeat the above steps without capping and
staining!
9. Final Fmoc-deprotection: Wash four times with DMF for at least 30 s each. Treat
with 20% piperidine/DMF twice for 5 min. Wash four times with DMF followed
by three times with DCM for at least 30 s each.
10. Final side-chain deprotection: Treat the membrane with at least 25 mL of
cleavage solution I for 30 mins. The surface of the membrane must be well
covered by the cleavage solution. Keep the box closed. Do not shake. Pour off
the solution very carefully. Wash the membrane five times with DCM for at
least 1 min each. Treat with at least 25 mL of cleavage solution B for 3 h. The
surface of the membrane must be well covered by the cleavage solution. Keep the
box tightly closed. Do not shake. Pour off the solution very carefully. Wash the
membrane five times for at least 1 min each with DCM. After TFA treatment,
the membrane may become very soft. Do not try to lift the membrane out until it
becomes harder and less likely to break apart during the washing steps (around
the last DCM washing step)! Wash the membrane five times with methanol or
ethanol for at least 1 min each. Dry the membrane in the air stream of a fume
hood or with a hair dryer without heat.
11. Sometimes it may be necessary to use the peptides solubilized and unbound to the
cellulose membrane. If it is necessary to release the peptides from the membrane,
one method is to expose the entire dry membrane overnight in a glass desiccator
containing ammonia gas. The strong basic environment leads to a break of the
ester bond to the cellulose by forming a C-terminal amide (see Note 8). The
next day, punch out the spots and transfer the discs into wells of microtiter plates
(MTPs) or into vials in which you can dissolve and test the released peptides (see
Note 9). If a free carboxy terminal is desired on the peptides, then do not treat
the membrane with ammonia gas, punch out the spots and expose them to a basic
aqueous solution, for example, ammonium hydroxide or sodium hydroxide (26).
Another method is to release the peptide by forming a C-terminal diketopiperazine.
In this case, couple Boc-Lys(Fmoc)-OH instead of the �-alanine spacer
(Fmoc-�-Ala-OH), followed by coupling of Fmoc-proline as the first coupled
amino acid. After TFA treatment the spots can be punched out and transferred to
a MTP or vials (see Note 9). The peptides can be released by overnight treatment
with basic aqueous buffers (pH ≥ 7.5) (1).
3.2. Screening Techniques
In this chapter we present some array techniques to screen for active peptides
(see Note 10). Most of these screening techniques were developed before the