Chemistry Reference
In-Depth Information
15.2 SOLID PHASE AND COMBINATORIAL SYNTHESIS
15.2.1 SPPS of Peptide Dendrimers and Their Combinatorial Libraries
Solution phase synthesis and convergent syntheses are usually preferred for den-
drimer assembly to minimize sterical hindrance and purification problems. Contrary
to these principles, we found that peptide dendrimers up to the fourth generation can
be obtained in good yields and purities by a divergent synthesis protocol on solid
support under the standard conditions of solid phase peptide synthesis with Fmoc-
protected amino acid building blocks (Fmoc-SPPS) [11,12]. The dendrimer core is
constituted by the C-terminal amino acids which are coupled first to the solid support.
Branching proceeds as diamino acids are added along the sequence. A typical
dendrimer of third generation with branches, 2 amino acids in length is assembled
in 11 coupling steps for a total of 37 amino acids and an average molecular weight
of 4400 Da, as shown for the esterase dendrimer
C11
(AcHisSer)
8
(DapHisLeu)
4
(DapLysVal)
2
DapIleValNH
2
(Figure 15.1).
The isolated yields of peptide dendrimer obtained from Fmoc-SPPS after side-
chain deprotection, cleavage from the solid support, and purification by reverse-phase
HPLC are comparable to the yields for linear peptides of similar size. As for linear
peptides, the isolated yields are influenced by the amino acid sequence and by the
physico-chemical properties of the product. In particular strongly hydrophobic
sequences may be difficult or impossible to purify due to their lack of solubility in
any solvent. Overall, many “well-behaved” peptide dendrimers comprising a good
balance of charged, polar, and hydrophobic amino acids, are readily accessible by
SPPS. Peptide dendrimer synthesis is also facilitated by the commercial availability of
amino acid building blocks for Fmoc-SPPS at reasonable prices, which renders the
synthesis quite affordable and potentially scalable using the existing industrial
infrastructure for SPPS.
The split-and-mix protocol for assembling peptide libraries by SPPS [13], which
was a founding experiment in combinatorial chemistry, can be applied to prepare the
corresponding peptide dendrimer libraries on solid support [14]. In these so-called
“one-bead-one-compound” (OBOC) libraries, each bead of the solid support carries
only one possible sequence, yet a large number of different sequences are assembled
simultaneously in the course of the synthesis. The number of different sequences
accessible in a split-and-mix synthesis is limited by the number of beads used in the
SPPS synthesis batch. A typical synthesis batch of 500mg of 90
m diameter beads
contains approximately one million beads, which imposes a limit of 100,000
sequences for the library size to ensure 10-fold coverage of sequence space.
m
15.2.2 Activity Screening
OBOC libraries are typically tested for activity using a bead-staining assay [15]. Such
assays include binding assays with a labeled or colored ligand, for example, vitamin
B
12
, and catalysis assays looking for conversion of a chromogenic or fluorogenic
substrate adsorbed on the beads, such as the hydrolysis of acyloxypyrene trisulfonate
