Squaric acid mediated bioconjugation expanded to polymers prepared by ATRP

The squaric acid mediated bioconjugation reaction is an attractive approach for the amine-selective protein modification with side-chain functional polymers. This manuscript presents 3 squaric acid based atom transfer radical polymerization (ATRP) initiators that significantly expand the scope of this bioconjugation reaction to a broad range of synthetic polymers that can be prepared by ATRP. Two proof-of-principle studies are discussed that demonstrate the feasibility of these novel initiators for the preparation of protein-synthetic polymer conjugates via both the grafting-onto as well as the grafting-from strategy. ATRP of polyethylene glycol methacrylate (PEGMA) with these initiators was used to generate a library of linear, mid-functional and 3-arm squaric acid functionalized PPEGMA derivatives. These PPEGMA polymers could subsequently be grafted onto bovine serum albumin (BSA), which was selected as a model protein. Alternatively, BSA could also be modified with these squaric acid based initiators to afford protein macroinitiators, which were successfully used to mediate ATRP of PEGMA and generate BSAPPEGMA conjugates in a grafting-from process.

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/protein – polymer conjugates: synthetic strategies and design concepts

Marc Gauthier, Harm-Anton Klok

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.

2008

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.