Peptide-Based Drug Design

210 Singer and Hoffmann
by kinases is not specific for a single position and often not quantitative, it is
necessary to synthesize phosphopeptides to study the underlying mechanisms or
to develop novel drugs, e.g., specific kinase inhibitors.
Currently, two strategies to synthesize multiply phosphorylated peptides by 9fluorenylmethoxycarbonyl
(Fmoc) chemistry are well established and routinely
used in many laboratories (1). The first Fmoc-based phosphopeptide synthesis
on solid phase were reported more than 20 years ago using different reagents to
phosphorylate partially unprotected peptides (i.e., free hydroxyl groups at serine,
threonine, or tyrosine positions to be phosphorylated) after completion of the
synthesis and before cleaving all other side-chain–protecting groups (2). Among
the many reagents tested, phosphoramidites (3) offered the best yields and highest
purities. The building-block strategy to directly incorporate phosphoamino acid
derivatives was introduced first for tyrosine (4,5) which is stable against the basic
conditions during Fmoc cleavage. On the other hand, it was only 10 years ago
when Vorherr and Bannwarth introduced partially protected phosphoserine and
phosphothreonine derivatives compatible to the Fmoc chemistry (6).
Whereas both approaches typically yield the targeted sequences in high
purities for peptide lengths up to 30 residues (7), they face the same limitations
described for some peptide syntheses, i.e., the “difficult sequences.” The lower
coupling efficiencies obtained at certain positions or even truncated sequences
are a result of sterical hindrance or the formation of secondary structures on the
solid phase that lower the efficiency of either the following coupling or deprotection
steps. This is especially true for the bulky phosphoamino acid building
blocks, although the global phosphorylation can also be hampered. In spite of
these limitations, peptides with up to three phosphorylation sites can be synthesized
routinely by the described protocols. In our experience, the analytics is
often more challenging for multiphosphorylated sequences than the synthesis
(8). In RP-HPLC these peptides often elute in broad peaks not separated from
lower phosphorylated versions or deleted sequences. In mass spectrometry, the
high content of negatively charged groups reduces the ionization efficiency in
positive ion mode typically applied to analyze peptides. As a rule of thumb
the signal intensity of phosphopeptides in a mass spectrum decreases about
10 times with each additional phosphate group. Therefore, low signal intensities
of peptides carrying several phosphate groups do not necessarily indicate that
the targeted sequences are obtained in low yields.
2. Materials
2.1. Peptide Synthesis
1. Synthesize peptides with the base-labile Fmoc group to temporarily protect the
�-amino group and acid-labile permanent side-chain protecting groups: tert-butyl