Peptide-Based Drug Design

268 Otvos
3. Methods
The main goal of this chapter is to describe the synthesis details of complex,
orthogonally protected peptide constructs. Thus, major emphasis is placed on the
peptide chain assembly design and practice and the alterations from the solidphase
synthesis of simple, nonmodified peptides. The technology for peptide
purification and quality control is not significantly different from that of other
peptides, and these methods will be just briefly described. Many chapters of
this book focus on the optimization of HPLC and MALDI-MS procedures for
peptide separation and analysis and illustrate the expected and/or acceptable
quality control parameters.
3.1. Construct Assembly
The basic idea is to build the construct on an oligolysine–oligoglycine
scaffold where the active peptides are attached to the side chains of the scaffold
lysine amino groups (Fig. 3). The two T-helper cell epitopes are built individually
during the assembly of the scaffold, while the four identical M2e fragments
are synthesized simultaneously after the scaffold assembly is completed. For this
strategy we need to use four different amino protecting groups.
While the amino acid coupling and deprotection steps used here are standard
for Fmoc-N-terminal protection-based peptide synthesis, the sheer size of the
construct and the fact that for the M2e fragment four peptide side chains are built
concomitantly require special considerations. To complete the coupling cycles,
the resin has to be loaded with relatively low amounts of reactive amino groups,
the reaction times have to be extended along with occasional double coupling
and deprotection cycles. The large number of acid-labile protecting groups to
be removed at the end of the synthesis requires overnight cleavage from the
resin even if a �-alanine moiety is incorporated between the resin and the
peptide backbone to spatially separate the growing peptide chain from the solid
support.
The three orthogonally removable lysine protecting groups we use here are
Fmoc (9-fluorenyl methoxy carbonyl), cleavable with 20% base, preferably with
20% piperidine or 3% DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) (18), Dde(1-
(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl), cleavable with 2% hydrazine,
and Aloc (allyloxy carbonyl), cleavable with palladium. Hydrazine also removes
Fmoc, and thus it can be applied only if no Fmoc groups are present on the
growing peptide chain. Acid-sensitive amino protecting groups available are
Boc (tert-butyloxy carbonyl), cleavable with 90% TFA, and Mtt (4-methyltrityl),
cleavable with 1% TFA. We use Boc for the protection of the N-terminal
moieties of N-terminal amino acids in each peptide chain as well as at the Nterminus
of the scaffold.