Peptide/protein – polymer conjugates: synthetic strategies and design concepts

Marc Gauthier, Harm-Anton Klok
2008
Journal paper

Abstract

This feature article provides a compilation of tools available for preparing well-defined peptide/protein–polymer conjugates, which are defined as hybrid constructs combining (i) a defined number of peptide/protein segments with uniform chain lengths and defined monomer sequences (primary structure) with (ii) a defined number of synthetic polymer chains. The first section describes methods for post-translational, or direct, introduction of chemoselective handles onto natural or synthetic peptides/proteins. Addressed topics include the residue- and/or site-specific modification of peptides/proteins at Arg, Asp, Cys, Gln, Glu, Gly, His, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val residues and methods for producing peptides/proteins containing non-canonical amino acids by peptide synthesis and protein engineering. In the second section, methods for introducing chemoselective groups onto the side-chain or chain-end of synthetic polymers produced by radical, anionic, cationic, metathesis and ring-opening polymerization are described. The final section discusses convergent and divergent strategies for covalently assembling polymers and peptides/proteins. An overview of the use of chemoselective reactions such as Heck, Sonogashira and Suzuki coupling, Diels–Alder cycloaddition, Click chemistry, Staudinger ligation, Michaels addition, reductive alkylation and oxime/hydrazone chemistry for the convergent synthesis of peptide/protein–polymer conjugates is given. Divergent approaches for preparing peptide/protein–polymer conjugates which are discussed include peptide synthesis from synthetic polymer supports, polymerization from peptide/protein macroinitiators or chain transfer agents and the polymerization of peptide side-chain monomers.

Official source

https://infoscience.epfl.ch/record/137288?ln=en

About this result

This page is automatically generated and may contain information that is not correct, complete, up-to-date, or relevant to your search query. The same applies to every other page on this website. Please make sure to verify the information with EPFL's official sources.

Related concepts

Related publications

Marc Gauthier, Harm-Anton Klok
2008
Journal paper

Abstract

Official source

https://infoscience.epfl.ch/record/137288?ln=en

About this result

Related concepts (29)

Related concepts

Related publications (63)

Related publications

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

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.

EPFL2008

Polymer Brushes as Substrates for Microarray Applications

Raphaël Barbey

The development of innovative bioanalytical technologies that enable the discovery of new therapeutics or the creation of systems for rapid, accurate and parallel analyses has gained an extraordinary interest in the biomedical sector in the last 10-15 years. Since proteins are the key-targets for such essential discoveries and developments, a particular emphasis has been placed on proteomics (study of proteins), including the investigation of protein structures, changes of protein expression upon treatment of cellular systems, the role of post-translational protein modifications on cell signaling events and the validation of selected proteins as disease-specific markers or pharmaceutical targets. These studies usually aim at using crude biological samples as the starting materials, which typically contain a large variety of proteins at concentrations covering a wide dynamic range and where the low abundant proteins are often the biologically most relevant. Therefore, the challenge is to create analytical tools which are able to cope with this large protein concentration range and which provide sufficiently high sensitivity to measure low abundant analytes in the presence of a high abundant protein matrix. The information that is expected from such technologies may soon exert a drastic change on the pace of medical research and considerably impact on the care of patients. As protein microarrays offer a powerful tool for the rapid, parallel analysis of complex samples and only require a minimal amount of reagents, they are currently one of the hot topics of life sciences, which strive to provide such analytical solutions. One of the current limiting factors in the field of protein microarrays for achieving the required analytical sensitivity is the amount of proteins that can be immobilized on the proteomic chip surface. Polymer brushes, which consist of an arrangement of polymer chains that are attached by one end to a substrate in a stretched, well-defined chain conformation, can increase the surface area available for protein binding, and thus act as a new category of 3D protein microarray substrates. With the advent of surface-initiated controlled polymerization techniques, extremely precise control over the thickness, composition, architecture and grafting density can be achieved, which allow to finely tune the properties of the polymer brush. In addition, these polymer chains may contain, natively or after specific post-polymerization reaction of their side chains, a high density of functional groups that can react with proteins. The different approaches available for the preparation of such surface-tethered polymer chains, the strategies allowing control over their architecture and properties as well as examples of applications related to protein binding are presented in Chapter 1. This Thesis describes the use of polymer brushes to create a new type of 3D proteomic chips, with the ultimate goal to improve the protein binding capacity compared to existing chip surface, without altering the accessibility of the detection reagents or increasing the intrinsic fluorescence of the background. The objective of this research work is to prepare polymer brush-based substrates that can outperform the sensitivity (i.e. increase the signal-to-noise ratio) that is obtained with a commercial microarray system, which is used as a benchmark in this Thesis. In this regard, functional polymer brushes containing epoxide groups that can react with the nucleophilic moieties of proteins have been chosen as promising candidates. In a preliminary set of experiments, the potential of glycidyl methacrylate-containing brushes to bind (bio)molecules via a post-polymerization modification reaction under mild conditions, i.e. at room temperature in aqueous medium, has been studied (Chapter 2). To this end, poly(glycidyl methacrylate) brushes, as well as several poly(glycidyl methacrylate)-co-poly(2-(diethylamino)ethyl methacrylate) copolymer brushes of different composition have been prepared by surface-initiated atom transfer radical (co)polymerization. The kinetics of the post-polymerization modification reaction of these (co)polymer brushes were followed using various model primary amines, as well as proteins. The introduction of tertiary amine groups, incorporated in the polymer brushes such as 2-(diethylamino)ethyl methacrylate units, was demonstrated not only to enhance the rate of the epoxide ring-opening reaction but also to induce higher protein binding capacities as compared to poly(glycidyl methacrylate) homopolymer brushes. The improved loading capacities of the (co)polymer brushes were evaluated in Chapter 3 under conditions that more closely resemble real microarray applications, such as the use of automated nanospotters to deposit proteins and fluorescence readers to detect protein immobilization. In this regard, commercially-available proteomic chips were coated with the epoxide-containing (co)polymer brushes and the spotting conditions as well as the proteins were varied. These studies revealed that the loading capacity depends on the pH and on the investigated protein, but also that the (co)polymer brush-coated substrates show far higher loading capacities than the standard, unmodified proteomic chips. This investigation also ascertained the supremacy of brushes containing 2-(diethylamino)ethyl methacrylate units compared to homopolymer brushes, as well as the superiority of thick (co)polymer brushes in comparison to their thinner equivalents. These (co)polymer brushes were subsequently used as platforms for real, although simplified, protein assays. The modified proteomic chips were spotted with unlabeled proteins that were further recognized with detection antibodies via either a one-step or a two-step approach. The clear benefits observed for copolymer brushes as compared to the existing technology under these realistic assay conditions nicely support the fact that the interior volume of the brush remains accessible for the detection reagents. In conclusion, the combination of the high loading capacity and the remaining accessibility for the detection partners make these (co)polymer brushes, and particularly the poly(glycidyl methacrylate)-co-poly(2-(diethylamino)ethyl methacrylate) brushes, excellent candidates for the improvement of the sensitivity in protein microarray applications.

EPFL2010