Solution behavior of peptide and peptide-hybrid materials

Peptide based and peptide hybrid materials represent two interesting and challenging classes of biomaterials. Both classes consist of and contain, respectively, one or more polypeptide blocks, endowing the resulting polymers with enhanced self-assembly properties, biocompatibility or novel characteristics. Peptide based materials, solely comprised of amino acids, benefit from their advanced self-assembly properties. In the case of peptide hybrid materials, being conjugates of peptides and synthetic polymers, either the polypeptide or the synthetic polymer block can drive the self-assembly process. Furthermore, the latter class benefits from combining the advantages of peptides and synthetic polymers, thus overcoming limitations related to the use of the individual components. The application of these materials is still challenging because it is not possible to predict their final self-assembly behavior based on their design. In order to facilitate a target-oriented design of novel materials for application such as drug delivery, tissue engineering, or self-healing materials, a detailed understanding of the complex interactions within and between these molecules is essential. The work presented in this thesis addresses these fundamental questions. Over the last decades, a plethora of peptide and peptide hybrid materials has been reported. Chapter 1 gives an overview of selected articles describing different synthesis routes for peptide as well as peptide hybrid materials, showing examples of molecules and their self-assembly into superstructures, and highlighting recent applications. Chapter 2 describes the synthesis and characterization of different peptide hybrid diblock oligomers composed of two classes of peptide sequences covalently attached to a poly(ethylene glycol) (PEG) block. The first class of peptide sequences contains the intrinsic propensity to form coiled coils, whereas the second class is composed of "switch" peptides and is able to adopt both amphiphilic α-helical and β-strand conformations. Upon dissolution, the first class yielded superstructures comprised of dimeric and tetrameric coiled coil aggregates, whereas the second class formed fibrillar assemblies. In both cases, the self-assembly properties were driven by the peptide block, and the final superstructure consisted of a peptidic core wrapped by the synthetic polymer block. Chapter 3 presents a detailed study on the aqueous solution self-assembly of two poly(styrene)n-b-poly(L-lysine)m peptide hybrid oligomers. The aim was to clarify whether the observed rod-like micelles, obtained by direct dissolution of the diblock oligomers in aqueous solution, reflect intrinsic self-assembly properties of the hybrid diblock oligomers or whether they present structures that are energetically trapped due to the glassy nature of the polystyrene block. To address this question, the influence of a variety of sample preparation techniques, including the use of organic solvents, dialysis from an organic solvent and several thermal treatments, was investigated. All treatments resulted in solution aggregates that differed in size and shape from samples prepared by direct dissolution in aqueous solution. Thus, it seems likely that direct dissolution of polystyrene-b-poly(L-lysine) diblock oligomers in aqueous solution results in kinetically trapped, rod-like aggregates, which can be transformed into non-spherical micelles by the appropriate treatment. Chapter 4 contains a comprehensive study on peptide-based materials, investigating four different series of short elastin-like peptides (ELPs) based on the GVGVP motif. The objective was to test whether and how defined changes in the primary structure of short ELPs affect the secondary structure and the lower critical solution temperature (LCST) behavior. Therefore, the number of repeats was altered, and within one peptide of constant length, all valines were successively substituted by the more hydrophobic amino acids isoleucine, leucine and phenylalanine, respectively. In parallel, a theoretical approach based on the partition coefficient log P was used to predict the shift of the LCST as function of chain length and composition. For short ELPs, the LCST is strongly affected by changes in the primary structure. This has also been predicted by calculating log P. An increase in the hydrophobicity resulted in a shift of the LCST towards lower temperatures. Furthermore, it was shown for the first time that the sensitivity of short ELPs towards external factors, such as salt concentration, is determined by the intrinsic propensity of the incorporated amino acids to form either α-helices or β-strands. In summary, the obtained results on peptide based and peptide-hybrid materials provides deeper insights into their self-assembly characteristics and highlights the potential of these materials to be used for technical and medical applications.

Synthesis, structural characterization and applications of hyperbranched polymers based on L-lysine

Markus Scholl

L-lysine is an essential a-amino acid and a necessary building block for all proteins in the body. As an essential amino acid, L-lysine is not synthesized by humans or animals. Industrially produced L-lysine has therefore a big market as food additive, e.g. in pig food. In the body, L-lysine plays a major role in calcium absorption, building muscle proteins, recovering from surgery or sports injuries, and the body's production of hormones, enzymes, and antibodies.1 From a chemical point of view, L-lysine is an AB2 building block that contains two amino groups and one carboxylic acid group, which makes it an interesting monomer for the synthesis of dendrimers and hyperbranched polymers. L-lysine indeed has been extensively used to synthesize dendrimers2-4 or conjugates of dendrimers and linear polymers.5,6 These dendrimers and linear-dendritic hybrid architectures have attracted interest for various medical applications, including gene delivery,3,6-8 as drug carriers,9 for the development of multiple antigen peptide systems,10,11 and as magnetic resonance imaging agents.12 The drawback of these dendrimers and linear-dendritic hybrid architectures is their time consuming synthesis and their expensive large scale production. This thesis evaluates the feasibility of L-lysine as a monomer to synthesize hyperbranched polymers and investigates the potential of these materials as encapsulation agents and for various biomedical applications. As the hyperbranched polylysines are prepared in a single step and can be produced conveniently at a large scale, they may offer an interesting alternative to their widely used dendritic analogues. Including this introduction, this thesis consists of 8 chapters, which are briefly described below. Chapter 2 gives a general overview of dendritic and hyperbranched polymers build up of amide bonds. Their synthesis and applications in medicine and catalysis as well as their self-assembling and encapsulation properties will be discussed. Chapter 3 reports on the synthesis of hyperbranched polylysines via the thermal polymerization of L-lysine hydrochloride.13 Different catalysts were investigated to improve the reaction kinetics. The structure of the polymers was determined by 1H-NMR spectroscopy, and the degree of branching and the average number of branches were calculated. Chapter 4 will discuss the feasibility of different approaches to control polymer architecture during the thermal hyperbranched polymerization of L-lysine hydrochloride.14 The reactivity of the more reactive ε-NH2 group was modulated by introducing temporary protective groups. The distribution of structural units was analyzed by 1H-NMR spectroscopy and the degree of branching and the average number of branches were calculated. Chapter 5 will describe the dilute solution and solid state structure of hyperbranched polylysines and polyelectrolyte complexes generated from hyperbranched polylysine and various anionic, sodium alkyl sulfate surfactants.15 Structural models for the obtained liquid crystalline phases will be proposed. Chapter 6 will show some preliminary results of the end group modification of hyperbranched polylysine with different hydrophilic and hydrophobic agents. The feasibility of these hydrophobically modified hyperbranched polylysines to encapsulate hydrophilic dyes and transfer them from aqueous to organic media has been investigated. Chapter 7 provides a first insight into the biological properties of hyperbranched polylysine. In this chapter the results from preliminary cytotoxicity and cellular uptake experiments will be discussed. Chapter 8 will give a short summary of the work done in this thesis and an outlook. References Nelson, D. L.; Cox, M. M. "Lehninger, Principles of Biochemistry" 3rd Ed. Worth Publishing: New York, 2000. ISBN 1-57259-153-6. Denkewalter, R. G.; Kolc, J. F.; Lukasavage, W. J. In U.S. Pat. 4289872; Allied Corporation, 1981; Chem. Abstr. 1981, 102, 79324. Ohsaki, M.; Okuda, T.; Wada, A.; Hirayama, T.; Niidome, T.; Aoyagi, H. Bioconjugate Chem. 2002, 13, 510-517. Driffield, M.; Goodall, D. M.; Smith, D. K. Org. Biomol. Chem. 2003, 1, 2612-2620. Lübbert, A.; Nguyen, T. Q.; Sun, F.; Sheiko, S. S.; Klok, H.-A. Macromolecules 2005, 38, 2064-2071. Choi, J. S.; Joo, D. K.; Kim, C. H.; Kim, K.; Park, J. S. J. Am. Chem. Soc. 2000, 122, 474-480. Vlasov, G. P.; Korol'kov, V. I.; Pankova, G. A.; Tarasenko, I. I.; Baranov, A. N.; Glazkov, P. B.; Kiselev, A. V.; Ostapenko, O. V.; Lesina, E. A.; Baranov, V. S. Russ. J. Bioorg. Chem. 2004, 30, 12-20. Choi, J. S.; Lee, E. J.; Choi, Y. H.; Jeong, Y. J.; Park, J. S. Bioconjugate Chem. 1999, 10, 62-65. Sakthivel, T.; Toth, I.; Florence, A. T. Pharm. Res. 1998, 15, 776-782. Tam, J. P. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 5409-5413. Pessi, A.; Bianchi, E.; Bonelli, F.; Chiappinelli, L. J. Chem. Soc., Chem. Commun. 1990, 8-9. Nicolle, G. M.; Tóth, E.; Schmitt-Willich, H.; Radüchel, B.; Merbach, A. E. Chem. Eur. J. 2002, 8, 1040-1048. Scholl, M.; Nguyen T. Q.; Bruchmann, B.; Klok, H.-A. J. Polym. Sci. Part A Polym. Chem. 2007, 45, 5494-5508. Scholl, M.; Nguyen T. Q.; Bruchmann, B.; Klok, H.-A. Macromolecules 2007, 40, 5726-5734. Canilho, N.; Scholl, M.; Mezzenga, R.; Klok, H.-A. Macromolecules 2007, 40, 8374-8383.

EPFL2007

Combating HIV Type 1 with Peptide - Synthetic Polymer Conjugates

Maarten Danial

Polymer therapeutics is a class of (bio)hybrid materials that include a plethora of biologically active (synthetic) macromolecules that either consist of polymeric drugs, drug-polymer conjugates, peptide/protein-polymer conjugates or of drug, peptide or proteins covalently linked to self-assembled entities, such as polymer micelles and liposomes. Interest in polymer therapeutics has increased in the last decades particularly as anti-cancer therapeutics, influenza therapeutics, non-viral alternatives to gene delivery and in the advent of biological terrorism as anthrax inoculates. The human immunodeficiency virus (HIV) is one of the most deadly diseases known and despite improvement in education, preventative methods and drug therapies, the virus still affects approximately 33 million people worldwide. Classical therapeutics against HIV consist of low molecular weight drugs that inhibit various viral enzymes in the cell, including reverse transcriptase, integrase and protease. More recently, drugs that target the entry and fusion of the virus have formed a new class of inhibitors, some of which are effective (candidate) drugs. One of the fusion inhibitors that were approved is the drug T-20. T-20 is a 36-amino acid peptide derived from the heptad repeat 2 (HR2) of the HIV-1 glycoprotein gp41and acts as a competitive inhibitor by preventing the fusion of the viral and host cell membrane. Despite the effectiveness of T-20, one major drawback is associated with this drug namely low plasma lifetime. One of the goals of this Thesis is to design and synthesize effective HR2 derived fusion inhibitors through conjugation with synthetic polymers, while extending the lifetime. In addition, a novel concept for macromolecular HIV entry inhibition known as polyvalent inhibition is assessed in this Thesis. This Thesis consists of five chapters in which polymer therapeutics as carriers and polyvalent entities are explored against HIV-1. With the state of the art of HIV-1 therapeutics including polymer therapeutics summarized in Chapter 1, the remainder of the Thesis expands and defines the terrain with regard to novel (bio)hybrid materials as therapeutics against HIV-1. Very few polymer therapeutics that target HIV before cellular entry have been described. Even fewer polymer-based therapeutics inhibit HIV-1 entry via the specific interaction to the HIV-1 envelope proteins. In Chapter 2, novel polyvalent copolymers that specifically target the HIV-1 envelope protein gp120 were generated using a living polymerization technique known as ‘reversible addition-fragmentation chain transfer’ (RAFT), followed by a post-polymerization modification strategy. The strategy involves two synthetic steps, namely an ester/amide exchange followed by a thiol-ene addition of peptide ligands to generate a library of polyvalent copolymers, with varying peptide ligand, 2-hydroxypropyl methacrylamide and allyl methacrylamide content. These novel polyvalent polymers were shown to be effective HIV-1 entry inhibitors. Fusion inhibitors derived from the HR2 domain of gp41 such as T-20, act as competitive inhibitors by binding to the HR1 domain on the envelope glycoprotein gp41, which is involved in HIV-1 – host cell membrane fusion. However, the peptidic nature of these fusion inhibitors restricts their lifetime because they are prone to proteolytic degradation. Chapter 3 explores the effect of attaching synthetic polymer poly(ethylene glycol) (PEG) site-specifically to a HR2 derived peptide fusion inhibitor. The PEGylated peptides were assessed for their biophysical properties, efficacy as membrane fusion inhibitors and stability towards trypsin that was used as a model to show the effectiveness of the PEG to protect the peptide from proteolytic degradation. Although PEGylation causes a reduction in efficacy, this Chapter demonstrates that judicial PEGylation of the peptide can minimize the detrimental effects on inhibitory efficacy, while increasing lifetime against a protease. Conjugation of PEG and the site of PEG conjugation both have a profound effect on the efficacy of HR2 derived membrane fusion inhibitors. However, the effects on the mechanism of HIV-1 infectivity inhibition by polymer conjugated HR2 peptide inhibitors are not known. Chapter 4 investigates the effect of architecturally different synthetic linear PEG, v-shaped PEG or branched poly(glycerol) polymers conjugated to HR2 derived peptides. The peptide-polymer conjugates were then assessed for their inhibition efficacy, affinity and kinetics of binding towards a model of gp41, known as 5-Helix. The results indicate that attachment of a synthetic linear or branched polymer, despite causing a reduction in HIV-1 infectivity inhibition, does not significantly affect affinity towards 5-Helix. However, the rates of association and dissociation towards and from 5-Helix were reduced. Furthermore, synthetic polymer conjugation to HR2 derived peptides prolong their lifetime bound to gp41 and thus potentially reduces HIV-1 inhibition escape. Therefore, this Chapter demonstrates a previously unknown advantage of polymer conjugation to HIV-1 fusion peptide inhibitors. Chapter 5 systematically explores the modification of hydrophilic residues on the hydrophobic face of a HR2 derived peptide. The peptide mutants were assessed for their efficacy against membrane fusion inhibition as well as against fusion of HIV-1 wild type and T-20 resistant variants. Although the peptide mutants did not exhibit any improvement towards the inhibition of wild type membrane fusion, selected peptide mutants with point mutations were shown to be effective inhibitors towards T-20 resistant HIV-1 strains. In conclusion, peptide-polymer hybrid materials can make excellent candidates as long lasting therapeutics against wild type HIV-1 but also against emergent HIV-1 mutant variants. The knowledge from these Chapters will aid the design of innovative, next generation of peptide-polymer hybrid therapeutics.

EPFL2011

Synthesis of sequence-defined oligomers and polymers

Nadja Franz

The preparation of synthetic polymers with exact control over chain length and monomer sequence is one of the long-standing challenges in polymer science. Nature perfectly masters the synthesis of high molecular weight, perfectly monodisperse polypeptides (proteins), which outperform synthetic polymers, both in terms of structural complexity as well as with respect to functional properties. The availability of synthetic strategies that would allow the preparation of polymers with a defined primary structure and chain length, and consequently, a controlled folding behavior in solution, may lead to polymeric materials with new or improved properties and functions. Over the past decades, several "living" or controlled polymerization techniques have been developed, which significantly enhanced the ability to control chain length, monomer sequence and composition: nowadays, synthetic polymers can be prepared with relatively narrow chain length distributions. However, in spite of all these advances, these methods still do not allow the preparation of polymers with perfectly uniform chain lengths and strictly defined monomer sequences, which would represent the synthetic counterpart of the proteins produced by Nature. An alternative approach towards precisely-defined polymers relies on multistep synthetic protocols and has already allowed the successful preparation of a wide variety of uniform oligomers. The synthesis of monodisperse oligomers with the propensity to fold (so called "foldamers") provides a useful stepping-stone for the synthesis and establishment of structure-property relationships with the corresponding higher molecular weight polymers. This Thesis describes the synthesis of oligomers and polymers with strictly defined monomer sequences, with the ultimate aim to direct and control their folding behavior, supramolecular organization and properties in solution. The design of the proposed oligomers/polymers is based on the principle of hydrophilic/hydrophobic patterning, which is a well-established and powerful strategy to guide secondary structure formation of both natural as well as non-natural oligomers and polymers. For example, alternating sequences of hydrophilic and hydrophobic residues are expected to lead to β-sheet type secondary structures. Three approaches have thus been investigated to obtain strictly alternating hydrophilic/hydrophobic oligomers and polymers. The different synthetic strategies that have been used for the preparation of these non-natural oligomers and polymers are reviewed in Chapter 1. The first approach explores the feasibility for the synthesis of uniform, sequence-defined, hydrophilic/hydrophobic patterned oligo(α-hydroxy acid)s (Chapter 2). This strategy is based on post-modification of a reactive oligoester scaffold composed of an alternating sequence of hydrophobic ((2S)-2-hydroxy-4-methylpentanoic acid) and masked hydrophilic ((2S)-2-hydroxypent-4-enoic acid) α-hydroxy acids. In a subsequent post-modification step, the allyl side chains could be quantitatively modified via free-radical addition of different ω-functional thiols to afford hydrophilic/hydrophobic patterned oligoesters. This synthetic route provides an interesting method to generate libraries of foldamers with identical chain length and monomer sequence but different side chain functionalities. The second approach relies on the preparation of the corresponding oligopeptide analogues (Chapter 3). Alternating hydrophilic/hydrophobic patterned peptide foldamers were obtained via post-modification of a reactive peptide precursor, synthesized via solid phase peptide synthesis (SPPS). The peptide scaffolds consisted of an alternating sequence of L-leucine and L-allylglycine residues. Thiol-ene coupling chemistry allowed the post-modification of the allylglycine residues with cysteamine hydrochloride, thioglycolic acid, 1-thioglycerol and 2,3,4,6-tetrα-O-acetyl-thio-β-d-glucopyranose. The dilute solution folding properties of the cysteamine and thioglycolic acid post-modified octapeptides were studied by circular dichroism (CD) spectroscopy. In agreement with the alternating hydrophilic/hydrophobic patterned primary structure, these peptides were found to adopt a β-sheet secondary structure in basic and acidic aqueous media, respectively. The preparation of high molecular weight polydepsipeptides, which are structurally related to oligoesters and oligopeptides, is discussed in Chapter 4. Polydepsipeptides were obtained via tin(II) 2-ethylhexanoate catalyzed ring-opening polymerization (ROP) of morpholine-2,5-dione derivatives. To allow flexibility in the choice of the side chain functional groups, morpholine-2,5-dione derivatives were constructed using L-allylglycine. Introduction of diverse functional groups in the side chains was achieved via radical addition of ω-functional thiols. This approach can be regarded as a convenient and rapid method for the synthesis of high molecular weight functional polymers, with strictly alternating monomer sequences.

EPFL2009