Peptide-Based Drug Design

214 Singer and Hoffmann
3. Methods
There are two general strategies to synthesize phosphopeptides by Fmoc
chemistry. First, the postsynthetic global phosphorylation approach (7,9) incorporates
an Fmoc-protected Ser, Thr, or Tyr without side-chain protection. An
obvious advantage of this approach is that both the unmodified and phosphorylated
peptides can be obtained from a single synthesis by splitting the resin
after the peptide synthesis prior to phosphorylation. The second approach
incorporates phosphorylated Fmoc amino acid derivatives carrying TFA-labile
protecting groups in the side chain, referred to as the building block strategy.
Both methods have inherent advantages and disadvantages, but usually work
well for singly phosphorylated peptides up to 20 residues in length. For longer
or multiply phosphorylated peptides, the appropriate strategy has to be selected
depending on the peptide sequence and the number of residues to be phosphorylated.
Moreover, it might be necessary in some cases to optimize the heredescribed
standard protocols for “difficult” sequences by using longer reaction
times or a higher reagent excess. Alternatively, the purification strategy has to
be optimized to separate the desired phosphopeptide from by-products.
For the synthesis of monophosphorylated peptides with “easy-to-synthesize”
sequences containing no bulky side chains or protecting groups proximal to the
phosphorylation site, the direct incorporation of monobenzyl protected phosphoserine
(10), phosphothreonine (6), and phosphotyrosine (4) is usually preferred,
as standard peptide synthesis reagents are used and no additional postsynthetic
handling steps are required (see Note 12). For multiply phosphorylated peptides
and especially for proximal phosphorylation sites, we prefer the global phosphorylation
approach. This strategy is also more efficient and cost saving if several
phosphopeptides or the corresponding unmodified peptides are synthesized, for
example, in a parallel peptide synthesis.
3.1. Multiple Solid Phase Peptide Synthesis
1. Swell the resin in DMF for 30 min (e.g., 40 mg Rink-amide MBHA resin,
25 �mol amino groups).
2. Add 1 mL piperidine (40% by vol. in DMF) and discard after 3 min.
3. Add 1 mL piperidine (20% by vol. in DMF) for 10 min and remove.
4. Wash the resin six times with 1 mL DMF for 1 min each.
5. Prepare a solution of 0.5 M HOBt in DMF or in a mixture of NMP and DMF (1:1
by vol.).
6. Dissolve the desired amount of the Fmoc amino acids in the HOBt-solution to a
final concentration of 0.5 M (see Note 13).
7. Add the amino acid solution from step 6 at a 4- to 10-fold molar excess over
the amino groups of the resin (e.g., 200 �mol [8 eq] amino acid derivative corresponding
to 400 �L Fmoc amino acid solution).