A post-modification approach to peptide foldamers

Hydrophilic/hydrophobic patterning is a powerful strategy to control folding in non-natural polymers/oligomers. In this contribution, we present a novel strategy for the preparation of alternating hydrophilic/hydrophobic patterned non-natural peptide foldamers. This strategy relies on the post-modification of a reactive peptide precursor that can be prepared via standard solid phase peptide synthesis without the need for side chain protective groups. The peptide scaffolds consisted of an alternating sequence of L-leucine and L-allylglycine residues. Using thiol-ene chemistry, the double bonds in the side chains of the L-allylglycine units could be post-modified with cysteamine hydrochloride, thioglycolic acid, 1-thioglycerol or 2,3,4,6-tetra-O-acetyl-thio-beta-D-glucopyranose to afford alternating hydrophilic/hydrophobic patterned peptides. In agreement with the alternating hydrophilic/hydrophobic patterned primary structure, cysteamine and thioglycolic acid post-modified octapeptides were found to adopt a beta-sheet secondary structure in basic or acidic aqueous media, respectively. The proposed synthetic approach is not only of interest to generate diverse libraries of peptide foldamers from a limited number of reactive precursor scaffolds, but may also represent an attractive, general strategy for the synthesis of peptides with complex side chain functionalities that are not easily accessible via standard solid phase techniques.

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

Phage Selection of Bicyclic Peptide Ligands and Development of a New Peptide Cyclisation Method

Jeremy Touati

Bicyclic peptides binding to targets of interest can be isolated from combinatorial libraries using a phage display-based approach. In a first project of this thesis, we aimed at generating bicyclic peptide inhibitors of matrix metalloproteinases (MMPs), a family of proteases that share high structural similarities and have been difficult to target in a specific manner. From phage-encoded combinatorial libraries, we isolated a bicyclic peptide that inhibits specifically the gelatinase MMP-2 with a Ki of 2 μM. This inhibitor was less potent than the best peptidic MMP-2 inhibitor, the linear APP-derived inhibitory peptide (Ki = 13 nM). However, due to its conformational constraint, the bicyclic peptide is expected to be significantly more stable and hence more suitable for applications in biological systems. It may be used as a research tool alternatively to the broadly applied monocyclic peptide MMP-2 inhibitor CTT which is less potent (Ki of 142 μM). If affinity matured, the bicyclic peptide MMP-2 inhibitor might even be developed into an anti-cancer therapeutic. In a second project, bicyclic peptide binders to the centriolar protein SAS-6 were generated. SAS-6 plays an important structural role in centrioles and bicyclic peptide binders are currently applied in cellular assays by the laboratory of Professor Pierre Gönczy (EPFL) to study the process of centriole formation. In a third project of this thesis, we established an enzymatic method to selectively cyclise peptides with unprotected side chains using a transglutaminase (TGase). TGases catalyse the formation of stable amide bonds between the side chains of glutamine and primary amines. They have been used extensively in biotechnological applications to cross-link peptides and proteins or to attach labels to proteins. We found that the microbial transglutaminase (MTGase) of Streptomyces mobaraensis can cyclise quantitatively peptides with an N- terminal glutamine-donor sequence and a C-terminal lysine residue in an intramolecular reaction. Experiments with minimised glutamine-donor substrates revealed that peptides with only an Ala-Leu dipeptide N-terminal to the glutamine and a randomly chosen peptide between the glutamine and lysine residues are efficiently cyclised. By combining the MTGase-based cyclisation strategy with a chemical thiol-based cyclisation reaction, we were able to generate tricyclic peptide structures. It remains to be tested if this method can be applied to cyclise combinatorial peptide libraries on the surface of phage. In a fourth and last project, we developed a method to follow qualitatively and quantitatively chemical or enzymatic modifications applied to peptides displayed on phage. In this method, peptides on phage were chemically modified, cleaved off by a protease, purified, concentrated and analysed by mass spectrometry. The method will help to assess new reactions applied to phage peptides such as the MTGase-based enzymatic cyclisation reaction.

EPFL2012

Switch peptides

Richard Alan Mimna

Aspects of conformational transitions, folding, and misfolding of peptides and proteins have recently taken center stage in various domains at the interface of chemistry, biology, and medicine because of their impact on protein misfolding diseases. Due to the intimate relationship between sequence, secondary structure, and physicochemical properties, studies on oligo- and polypeptides exhibiting high propensity for β-sheet formation are severely limited by their intrinsic tendency toward self-association and irreversible aggregation. Consequently, β-sheet and fibril-forming processes related to protein misfolding diseases remain elusive, as delineation of structure nucleation or inhibition is a formidable task, often yielding contradictory observations. With the aim of surmounting these experimental difficulties and of inducing conformational transitions in situ, the present thesis elaborates the novel concept of switch-peptides (see figure). Modular switch-peptides are synthesized comprising three distinct elements: a conformation induction unit (σ), a switch-element (S), and a peptide (P). Of central importance to the switch-peptide concept, the switch-element S dissects the native polyamide backbone of the peptide P by an ester and a flexible C-C bond, thereby preventing the peptide from folding and separating the conformational impact of σ. The triggering of the switch-element by removal of the protecting group Y reestablishes the native polyamide backbone via a spontaneous O → N acyl migration reaction, whereupon peptide folding can then proceed. Detailed herein is the development of the key elements of the concept, σ and S, and their subsequent application to various model peptides and peptides of biological interest (P). In particular, two synthetic routes to S-elements were demonstrated, i.e. the stepwise solid phase synthesis of depsipeptides using a minimal protection strategy and the preparation of protected depsidipeptides for use as building blocks in solution or solid-phase synthesis. Kinetic investigations of their acyl rearrangements reveal half-lives that range from less than one minute to several hours. The possibility to tailor the half-lives by manipulation of experimental conditions is also elaborated. Orthogonal protection strategies including photolytically and enzymatically labile groups were developed. This enabled the incorporation of multiple S-elements into a peptide that could be triggered sequentially. In this way, the mutual conformational impact of peptide segments as well as their role in folding and self-assembly was explored. The design and synthesis of novel, helix-nucleating N-cap compounds (a type of σ element) is described and applied for the in situ nucleation of helical conformations according to the figure. For the first time, a detailed study of a wide variety of original N-caps is realized in which their helix inducing abilities are quantified by CD spectroscopy. When applied to model β-sheet-forming switch-peptides (random coil → β-sheet), the use of an N-cap is shown to overcome the intrinsic β-sheet propensity and instead induce a conformational transition to an α-helix. Furthermore, an hitherto unobserved conformational transition of type β-sheet → α-helix in an amyloid beta-derived switch-peptide is demonstrated and shown by electron microscopy to inhibit its fibrillization. Through a series of designed switch-peptides, the S-element is shown to indeed conformationally decouple segments σ and P and, upon triggering, to induce conformational transitions relevant to protein misfolding. The results obtained firmly establish the capability of employing the switch-peptide concept as a tool to study folding events during the dynamic process of structure onset and evolution. With the methodologies elaborated herein, the stage is set for its further application to the study of structure-function relationships, prodrug design, the design of diagnostic systems for evaluating inhibitors of secondary structure, and to β-breaker peptides.

EPFL2005