Peptide-Based Drug Design

The Spot Technique 53
the solutions for synthesis fresh every day, and discard those of the previous day.
Due to the instability of dissolved preactivated arginine derivatives, this solution
must be prepared fresh every day.
3.1.1.2. METHOD 2: PREPARATION OF SOLUTIONS WITH IN SITU
ACTIVATED AMINO ACIDS OR OTHER BUILDING BLOCKS
Prepare a 0.9 M solution of HOBt in amine-free NMP. Dissolve the Fmocamino
acids or desired protected building blocks with the HOBt-solution to a
concentration of 0.45 M. Except for the arginine derivatives, these solutions can
be stored at -20◦C for at least a week. Each day, prepare a 20% mixture of DIC in
amine-free NMP. Use the fresh amino acid/HOBt solution every day and discard
the previous one. To these solutions add 20% DIC/NMP at a ratio of 3:1 (e.g.,
75 �L amino acid solution and 25 �L DIC mixture prepared fresh every day)
(see Note 3).
3.1.2. Preparation of the Membrane
1. Cut a piece of filter paper to the size required so that all peptide spots including
controls can be accommodated. For 0.1 �L of coupling solution the distance
should be at least 2.7 mm, and for 1 �L at least 7 mm.
2. Amine functionalization of filter paper: Dissolve 0.64 g Fmoc-�-alanine in 10 mL
amine-free DMF. Add 374 �L DIC and 317 �L NMI and mix well before transferring
this solution into a chemically resistant box with lid. Place the filter paper
in the box while avoiding formation of air bubbles under the paper, and ensure that
the surface of the membrane is slightly covered by the solution. Close the box.
Allow the reaction to take place for at least 2 h or overnight (see Note 4).
3. Fmoc-deprotection: After the reaction is complete, wash the membrane three times
with DMF for at least 30 s each. For storage, wash the modified membrane at
least twice with methanol or ethanol and dry it in the air stream of a fume hood
or using a hair dryer without heat. For resumption of synthesis after storage, treat
the membrane once with DMF for 20 min (see Note 5). The Fmoc-deprotection is
carried out by treatment of the membrane twice with 20% piperidine in DMF for
at least 5 min each.
4. Staining (optional) (25): Wash the membrane four times with DMF for at least
30 s each, followed by washing at least twice with methanol or ethanol for at least
30 s each. Treat the membrane with staining solution for at least 2 min until the
filter paper shows a homogeneous blue color (see Note 6). After staining wash the
membrane at least twice with methanol or ethanol, until the wash solution remains
colorless and then dry the membrane.
3.1.3. Spot Synthesis of the Peptide Array
1. Definition of the synthesis pattern: Place the dried membrane on an inert
surface (e.g., stainless steel or PP). Deliver the required volume of activated