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Solid-phase Peptide Synthesis

Amphiphilic molecules, which consist of a hydrophilic water-soluble part and a hydrophobic water-insoluble part, can self-organize into a variety of supramolecular structures. This is not only true for polymeric amphiphiles as was discussed above (Section 6.6.2) [12], but applies also to low-molecular-weight amphiphiles. The size and shape of the supramolecular structures formed by these molecules in aqueous solution depend, among other factors, on the relative sizes of the hydrophilic and hydrophobic parts, the charge on the hydrophilic headgroup, and the geometry of the molecules [18]. Amphiphiles consisting of a hydrophilic peptide headgroup and a hydrophobic alkyl tail are a particularly interesting, although relatively unexplored, class of molecules [19]. The ability of solid phase peptide synthesis (SPPS, Box 25) to prepare peptides with well-defined a-amino acid sequences and chain lengths, however, offers the possibility of precise control over the size and charge of the hydrophilic peptide segment and also facilitates the incorporation of bio-

active peptide sequences. Thus, SPPS affords unprecedented opportunities to tailor the structure and properties of supramolecular architectures and materials composed of peptide amphiphiles.

In two recent publications, Stupp and coworkers reported the synthesis and supramolecular organization of a series of novel peptide amphiphiles [20]. The molecules consisted of a hydrophilic peptide headgroup containing 8-12 a-amino acids and alkyl tails comprising 6-22 carbon atoms. The hydrophilic headgroup contained both ionizable a-amino acid residues to enable pH-control of the self-assembly process, and cysteine residues, which were explored to covalently capture supramolecular structures formed in aqueous solution. Because disulfide bonds can be reversibly formed and broken on oxidation and reduction, respectively, su-pramolecular aggregates can be transformed into covalently captured nano-objects, and vice versa. The self-assembly of the peptide amphiphiles was studied in detail with transmission electron microscopy (TEM) and is schematically illustrated in Figure 6.6.3. Acidification of aqueous solutions of the peptide amphiphiles resulted in the formation of precipitates with, according to TEM, a nanofibrillar structure. Oxidation of the thiol groups resulted in cross-linking of the supramolecular fibers and led to covalently captured nano-objects. Interestingly, these cross-linked nano-fibers could direct the mineralization of hydroxyapatite to form a composite material structurally similar to the mineralized collagen fibrils found in bone [20]. At peptide amphiphile concentrations above 0.25 wt%, acidification resulted in the formation of pH-sensitive hydrogels, which could be reversibly formed and disassembled by adjusting the pH.

SPPS is not only useful for preparation of peptide amphiphiles based on unnatural a-amino acid sequences, as discussed above, but can also be used to generate o o o

Fig. 6.6.3. Schematic representation of the self-assembly of a peptide amphiphile into cylindrical micellar nanofibers. (Adapted from Ref. [20a]).

hybrid block copolymers of synthetic macromolecules and a-amino acid sequences derived from protein-folding motifs. Protein-folding motifs, or supersecondary structures, consist of a small number of secondary structural elements such as a-helices or ¿S-strands packed in close proximity in a well-defined geometric arrangement [3]. The specificity of the folding process, which essentially involves transformation of a linear unordered polypeptide chain into a protein with a well-defined three-dimensional structure and very specific properties, is in great contrast to the self-assembly of synthetic amphiphilic block copolymers. Polyalkyl-b-poly(ethylene oxide) and poly(propylene oxide)-b-poly(ethylene oxide) copolymers can, for example, form micellar type structures and hydrogels in aqueous media and are being intensively investigated for biomedical applications including drug delivery and tissue engineering [21]. The self-assembly of such block copolymers is exclusively driven by non-specific hydrophobic interactions. Synthetic block co-polymers are usually polydisperse and do not contain well-defined monomer sequences. Consequently, the structure and properties of the resulting micelles and hydrogels can only be controlled and tailored to a limited extent.

In an attempt to overcome these limitations, we and others have recently started to explore peptide sequences derived from protein-folding motifs to enhance control of the supramolecular organization and association behavior of synthetic poly(ethylene oxide) (PEO)-based block copolymers [22]. If the capacity of peptide sequences to assemble into protein-folding motifs is retained on conjugation to PEO the supramolecular organization and association behavior of the block co-polymers might be very precisely controlled. Improving control over the association behavior and supramolecular organization of PEO-based block copolymers might be of great interest in a variety of biomedical applications and could enable the development of micellar drug carriers and hydrogels whose formation and dissociation can be triggered and controlled with unprecedented precision. The coiled-coil motif, a supersecondary structure consisting of two or more a-helices wound around each other in a superhelical fashion, was explored in preliminary experiments [23]. Coiled-coils are among the most abundant folding motifs and are characterized by a primary structure consisting of a repetitive heptad repeat pattern (-[a-b-c-d-e-f-g]n-). Within this heptad repeat pattern, positions a and d are usually occupied by hydrophobic a-amino acids.

PEO-b-peptide block copolymers were prepared as outlined Scheme 6.6.3. The primary structure of the peptide block was not derived from a natural protein-folding motif but was based on a de-novo designed coiled-coil [24]. Whereas the a-amino acid sequence of natural proteins can be rather irregular, de novo protein-folding motifs are based on a repetitive sequence of heptad repeat units containing a minimum number of different a-amino acids. The regular and repetitive nature of the a-amino acid sequence of such de-novo coiled-coils greatly facilitates systematic variation of block length. The association behavior and supramolecular organization of the PEO-b-peptide copolymers were investigated by means of circular dichroism (CD) and analytical ultracentrifugation (AUC). These experiments indicated that the capacity of the peptide blocks to assemble into well-defined higher-order structures is retained on conjugation to PEO and can be used to direct

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