Bio-inspired Polymers: Volume 22 (Polymer Chemistry Series) - Hardcover

Hardcover

ISBN 10: 1782624139 ISBN 13: 9781782624134

Publisher: Royal Society of Chemistry, 2016

Synopsis

This book will provide a comprehensive review of the large field of bio-inspired polymers and is written and edited by leading experts in the field.

About this title

From the Back Cover

Many key aspects of life are based on naturally occurring polymers, such as polysaccharides, proteins, and DNA. Unsurprisingly, their molecular functionalities, macromolecular structures and material properties are providing inspiration for designing new polymeric materials with specific functions for example responsive, adaptive and self-healing materials.

Bio-inspired Polymers covers all aspects of the subject, ranging from the synthesis of novel polymers, to structure-property relationships, materials with advanced properties and applications of bio-inspired polymers in such diverse fields as drug delivery, tissue engineering, optical materials and lightweight structural materials.

Written and edited by leading experts on the topic, the book provides a comprehensive review and essential graduate level text on bio-inspired polymers for biochemists, materials scientists and polymer chemists working in both industry and academia.

From the Inside Flap

Many key aspects of life are based on naturally occurring polymers, such as polysaccharides, proteins, and DNA. Unsurprisingly, their molecular functionalities, macromolecular structures and material properties are providing inspiration for designing new polymeric materials with specific functions for example responsive, adaptive and self-healing materials.

Bio-inspired Polymers covers all aspects of the subject, ranging from the synthesis of novel polymers, to structure-property relationships, materials with advanced properties and applications of bio-inspired polymers in such diverse fields as drug delivery, tissue engineering, optical materials and lightweight structural materials.

Written and edited by leading experts on the topic, the book provides a comprehensive review and essential graduate level text on bio-inspired polymers for biochemists, materials scientists and polymer chemists working in both industry and academia.

Excerpt. © Reprinted by permission. All rights reserved.

Bio-inspired Polymers

By Nico Bruns, Andreas F. M. Kilbinger

The Royal Society of Chemistry

Copyright © 2017 The Royal Society of Chemistry
All rights reserved.

ISBN: 978-1-78262-413-4

Contents

Chapter 1 Synthetic Aspects of Peptide- and Protein-Polymer Conjugates in the Post-click Era Maria Meißler, Sebastian Wieczorek, Niels ten Brummelhuis and Hans G. Börner, 1, 
Chapter 2 Glycopolymers Laura Hartmann and Kira Neuhaus, 31, 
Chapter 3 Synthesis of Non-natural Polymers with Controlled Primary Structures Abdelaziz Al Ouahabi, Raj Kumar Roy and Jean-Francois Lutz, 66, 
Chapter 4 Single-chain Nanoparticles Justin P. Cole, Christopher K. Lyon and Erik B. Berda, 107, 
Chapter 5 Polymeric Tubular Structures Samantha Doninelli, Michael Badoux and Andreas F. M. Kilbinger, 141, 
Chapter 6 Bio-inspired Polymer Membranes Viktoria Mikhalevich, Christina Zelmer, Anja Car, Cornelia Palivan and Wolfgang Meier, 221, 
Chapter 7 Polymeric Ionic Liquids with Micelle-like Topologies and Functions Benjamin Kerscher, Fabian Schüler, AnnaKatharina Evers and Rolf Mülhaupt, 259, 
Chapter 8 Biological and Bio-inspired Heterogeneous Composites: From Resilient Palm Trees to Stretchable Electronics Markus Riiggeberg, Andre R. Studart and Ingo Burgert, 286, 
Chapter 9 Translating Mussel Adhesion: Four Uncertainties about the Interface J. Herbert Waite, 305, 
Chapter 10 Mussel Adhesive-inspired Polymers Bruce P. Lee, 322, 
Chapter 11 Self-reporting Polymeric Materials with Mechanochromic Properties Jose V. Araujo, Omar Rifaie-Graham, Edward A. Apebende and Nico Bruns, 354, 
Chapter 12 Mechanically Adaptive Nanocomposites Inspired by Sea Cucumbers Dafni Moatsou and Christoph Weder, 402, 
Chapter 13 Bio-inspired Polymer Artificial Muscles Sina Naficy, Geoffrey M. Spinks and Ray H. Baughman, 429, 
Chapter 14 Materials for Tissue Engineering and 3D Cell Culture Caitlin R. Langford and Neil R. Cameron, 460, 
Chapter 15 Antimicrobial Polymers and Surfaces – Natural Mimics or Surpassing Nature? Christian Krumm and Joerg C. Tiller, 490, 
Chapter 16 Superwettability of Polymer Surfaces Xi Yao, Jie Ju and Lei Jiang, 523, 
Chapter 17 Bio-mimetic Structural Colour using Biopolymers Rox Middleton, Ullrich Steiner and Silvia Vignolini, 555, 
Subject Index, 586, 

CHAPTER 1

Synthetic Aspects of Peptide – and Protein – Polymer Conjugates in the Post-click Era

MARIA MEIßLER, SEBASTIAN WIECZOREK, NIELS TEN BRUMMELHUIS AND HANS G. BÖRNER

1.1 Introduction

The synthesis of peptide- and protein-polymer conjugates offers the possibility to integrate properties of biological macromolecules into synthetic systems, thereby obtaining hybrid materials with unique functions. The synthetic polymers within these structures provide a versatile range of properties, whereas the peptide or protein domain introduces highly specific functions, ranging from enzymatic activity to specific interaction capabilities or recognition, and to disease modifying activities (Figure 1.1). The multidisciplinary field of creating bioconjugates gives access to a variety of materials for application in materials science, biotechnology, or pharmacology, where the bioconjugates act at the interface between biology and synthetic materials.

First attempts to combine synthetic polymers with biomacromolecules emerged in the early 1950s, when initial reports concerning the synthesis of peptide-polymer conjugates were published by Jatzkewitz. About 20 years later, Abuchowski, Davis, and coworkers described the attachment of a linear poly(ethylene oxide) (PEO) to bovine serum albumin (BSA) and bovine liver catalase. Particularly noteworthy was that the extensive modification of these proteins with a synthetic polymer block did not inhibit the functionality of the biological macromolecules while simultaneously reducing their immunogenicity and increasing blood circulation times. This widely used strategy of incorporating PEO onto proteins or peptides with the objective of improving the properties of biomacromolecules is termed "PEGylation", referring to poly(ethylene glycol) or PEG. Due to polymer modification, the stability of the resulting protein-polymer conjugates toward proteolytic digestion and antibody interactions can often be decreased significantly. This enhancement in stability and solubility of the modified proteins represents an important improvement for in vivo applications, such as in pharmaceutical research and the development of protein-based drugs. Ever since these pioneering works showed the potential of PEGylation, and the combination of proteins or peptides with synthetic polymers in general, many research groups have started to investigate peptide-polymer conjugates as a new class of hybrid materials.

Controlled techniques to connect the building blocks from the synthetic and the biological worlds are indispensable for the preparation of well-defined peptide- and protein-polymer conjugates. In 2001, Sharpless, Kolb, and Finn introduced the concept of ("click" chemistry, which describes the most important criteria to attach two molecules to each other in a highly selective and efficient manner. A reaction defined under this term has to result in very high yields and easily isolable products, while generating only non-hazardous side products that can easily be removed afterwards. Besides the copper(I)-catalyzed cycloaddition (CuAAC) between alkynes and azides described independently by Sharpless and Meldal, a wide range of ligation strategies have since been found to fulfill the criteria of "click" reactions. Of these (in addition to CuAAC), the strain-promoted azide-alkyne cycloaddition (SPAAC), Staudinger ligation, oxime formation, Michael-type additions and other thiol-ene reactions are among the most frequently cited representatives. The advent of various highly efficient reactions has also proved advantageous for the preparation of peptide – and protein-polymer conjugates; these synthetic strategies offer versatile opportunities in the fields of materials science and biomedicine, where they are being applied widely.

In conjunction with the emerging "click" reactions in this century, scientists have focused on the development of innovative strategies to design more complex structures. The rich world of chemical ligation tools, which have traditionally been used for protein modification, has extensively been reviewed. This book chapter will mainly provide an overview of the last decade's progress over the preparation of functional bioconjugates, also referred to as "post-click" methods. After a brief survey of various well-established and widely used ligation techniques, we will focus on novel types of chemistry as well as chemoenzymatic approaches and biotransformations to create functional bioconjugates using enzymatically catalyzed reactions. Finally, recent advances in this field will be described to provide insight into potential future directions for the preparation of functional peptide- and protein-polymer conjugates.

1.2 General Concepts for Bioconjugation

The most widely used method to prepare bioconjugates is based on a convergent strategy – the so-called "grafting to" approach (Figure 1.2a). In order to obtain well-defined structures, the peptide or protein, which contains one or more reactive groups, is reacted with a polymer bearing complementary reactive groups. The "grafting to" approach is applied most since the independent synthesis and characterization of both components prior to the ligation enables a facile structural and chemical analysis of the resulting bioconjugates. Some of the disadvantages compared to other methods (vide infra) are that two macromolecules need to be coupled, which is often slow and/or inefficient due to the hindered accessibility of the reactive groups. Additionally, such reactions can only be performed using relatively low concentrations, and separation of the starting materials from the product is often difficult. Therefore, highly efficient coupling reactions are required. However, the synthesis and characterization of both components independently enables a facilitated structural and chemical analysis of the resulting bioconjugates.

A range of different reactions for convergent ligation have been developed so far. On the one hand, different amino acid side chains within the peptides and proteins can be addressed using various ligation strategies. Thiol groups of cysteine residues and primary amine groups at the N-terminus or on lysine side chains are the most common sites for attachment to polymers. On the other hand, more appropriate functional groups can be introduced to improve the coupling efficiency and to enable site-specific conjugation, e.g. phosphine or azide residues for Staudinger ligation, aldehydes for oxime formation, or strained cyclooctynes for SPAAC.

In the "grafting from" approach a moiety with the ability to initiate or mediate polymerization is introduced in the biomacromolecule (Figure 1.2b). This divergent strategy often relies on controlled radical polymerization techniques, since these are highly tolerant to the presence of functional groups which are frequently found in peptides and proteins. Furthermore, protein denaturation, resulting in loss of function, is unacceptable, making polymerization in aqueous media under mild conditions desirable. Controlled radical polymerization techniques such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT), or nitroxide-mediated polymerization (NMP), are compatible with aqueous solvents and avoid adverse side reactions.

One of the first examples following the "grafting from" approach was the preparation of a protein-polymer conjugate composed of BSA and poly(N-isopropylacrylamide) (PNIPAM). The modification of the protein with a maleimide-functionalized chain transfer agent (CTA) enabled the RAFT polymerization of NIPAM, resulting in a thermoresponsive bioconjugate.

The "grafting through" approach implies the incorporation of a polymerizable group into the biomacromolecule, allowing for copolymerization with synthetic monomer units. This results in a bioconjugate product with a comb-like structure, where a certain number of the polymer side chains contain peptide or protein moieties (Figure 1.2c). Compared to the concept of "grafting to", the strategy avoids side reactions and low coupling efficiencies due to the attachment of peptides or proteins to monomers before the polymerization process, at which stage purification is easier. By comparison, the "grafting through" method is less modular than the "grafting to" strategy. In the "grafting to" approach, theoretically, any peptide containing a suitable reactive group can be coupled to a well-defined precursor polymer to produce different products with, e.g., different degrees of functionalization or with different peptides, etc. For the "grafting through" method, optimized polymerization conditions have to be found for each new monomer and the average length will not be precisely reproducible.

The first application using the "grafting through" approach was the synthesis of a thermoresponsive antibody-polymer conjugate described in 198 7. A more recent example deals with the incorporation of fibrinogen in a protein-polymer conjugate. Pluronic F127 was end-group-modified with an acrylate moiety on one side, while the other was coupled to fibrinogen. The acrylate moiety was used for UV-activated free-radical polymerization. Since many of the fibrinogen molecules were functionalized with multiple Pluronic F127 moieties, polymerization triggered cross-linking, yielding a thermoresponsive hydrogel.

Beyond these three main concepts, the inverse bioconjugation approach offers another strategy to connect peptides or proteins with synthetic polymers. Using a solid support, which is preloaded with a polymer block, the biological molecule can be assembled in a stepwise fashion through solid-phase synthesis. Mutter and coworkers first showed the attachment of PEO to a poly(styrene) resin via a benzyl ether linker. This concept was finally developed further by Bayer and Rapp leading to a commercially available PAP resin, which is widely applied in solid-phase peptide synthesis. In a similar approach, Lutz, Börner, and coworkers demonstrated the preparation of cleavable and non-cleavable soluble polystyrene supports by ATRP for the liquid-phase synthesis of peptide-polymer conjugates.

1.3 Chemical Synthesis of Peptide- and ProteinPolymer Conjugates

The number of applications for peptide- and protein-polymer conjugates is constantly increasing. While classical synthetic reactions take advantage of naturally occurring amino acid side chains for coupling reactions with polymers, many innovative strategies are based on methods to attach synthetic polymers selectively, and with high efficiency, to improve yields and purity of the conjugation products. The following sections will give an overview of the most commonly used techniques, sorted according to the different reactive functional groups. Novel bioconjugation approaches will be discussed in detail.

1.3.1 Coupling with Amines

Exploiting primary amines is attractive since amines are among the most reactive functional groups present in peptides and proteins, and are found in relatively high abundance at the surface of globular proteins. Various ligation strategies to attach polymers to them have been established. Most common is the modification of a synthetic macromolecule with an activated carboxylic acid group in order to address the peptide or protein N-terminus or a lysine side chain, if available. Typically, polymeric derivatives of N-hydroxysuccinimide (NHS) are used (Figure 1.3a). This reaction allows for the coupling of lysine residues in an almost quantitative manner, resulting in a stable amide linkage. For example, the NHS end group modification of PNIPAM derived from RAFT polymerization enabled the ligation to a cyclic peptide. The resulting thermoresponsive peptide-polymer conjugate possesses the ability to form channels within phospholipid membranes.

However, the applicability of NHS-esters is limited due to their susceptibility to nucleophilic addition reactions, resulting in hydrolytic instability in aqueous media. To avoid low coupling efficiencies, the integration of pentafluorophenyl-activated carboxylic acid groups into synthetic polymers and their behavior in bioconjugation have been investigated (Figure 1.3b). An appropriate modification of a collagen-like peptide was used for the site-selective conjugation of a stimulus-responsive poly(diethylene methyl ether methacrylate). Coupling of two such bioconjugates resulted in the formation of triblock copolymer which showed triple-helix formation and thermoresponsiveness.

Frey and coworkers presented the squaric acid mediated PEGylation of proteins. Amine bearing PEO was end-group functionalized with squaric acid diethyl ester, using one of the reactive groups in the squaric acid moiety. The other was still available for the efficient functionalization of lysine residues in proteins like BSA (Figure 1.4).

Despite the efficient coupling reactions available for the functionalization of amines, their high abundance in peptides or proteins prevents selective bioconjugate formation, since a single-site modification is often not possible. Furthermore, the multiple and unspecific conjugation of synthetic polymers can result in loss of function and reduced enzymatic activity, and can potentially even induce toxicity. For these reasons, it is often desirable to apply more specific and directed coupling strategies, e.g. using reactions in which one of the components is rarely found in peptides and proteins.

1.3.2 Coupling with Thiols

Cysteine residues are frequently addressed in biological molecules because they are rather rare in peptides and proteins. If accessible thiols are present, they can therefore allow for a site-specific polymer ligation, yielding well-defined bioconjugates. Classically, thioether formation by Michael addition of the thiol to a suitable electron-deficient alkene in the polymer blocks is used. The Michael addition reaction between thiols and maleimides constitutes the most well-known reaction, and is widely used to prepare bioconjugates of peptides and proteins (Figure 1.5a).

For example, the aminolysis of the chain transfer agent end group of PNIPAM prepared by RAFT exposed thiol end groups, which can be reacted with 1,8-bis-maleimidodiethyleneglycol. Ligation of bovine serum albumin and ovalbumin to the maleimide-terminated polymer yielded protein-polymer conjugates.

Due to its electrophilic properties, maleimides are susceptible to the addition of other nucleophiles besides thiols, such as primary amines. Under physiological conditions, the reaction between a thiol group and a maleimide proceeds more rapidly than with an amine, as is desired. However, slight changes in the pH to alkaline conditions can already result in a shift towards more side reactions with e.g. lysine side chains or the N-terminus of the biomacromolecule. Beyond that, higher pH-values can even effect the hydrolysis of the maleimide group to an open maleamic acid form, which is no longer reactive towards thiols. After the formation of the thiol-maleimide product, the ring-opening can effect the stabilization of the resulting conjugate, which prevents a possible thiol exchange.

These limitations can be avoided by using the free-radical addition of a thiol to a double bond (Figure 1.5b), which is referred to as a thiol-ene reaction and can also proceed as a "click" reaction. Initially, radical formation can be induced by heat or light. The generated thiyl radical enters the addition reaction with the vinyl group, resulting in a carbon-centered radical. Subsequently, the desired thioether product is generated by hydrogen abstraction from other thiols, yielding further thiyl radicals. To achieve bioconjugates for in vivo applications, a photoinitiator is often added to generate radicals, since this procedure allows for the use of mild, relatively long wavelength (>300 nm) UV irradiation and can be performed in aqueous media under physiologic conditions.

The post-polymerization modification of poly(pentafluorophenyl methacrylate) with allylamine to couple a thiol-terminated peptide domain was presented by Klok and coworkers. The polymeric side chain modification was used later on for the synthesis of polyvalent peptide-polymer conjugates exhibiting HIV-1 inhibitory properties with antiviral activity depending on the bioconjugate length.

In proteins' cysteine residues are not always readily accessible' since they are often involved in disulfide bridges within the complex three-dimensional biomacromolecular structures. Therefore, only a small number of cysteine residues can be used for bioconjugation reactions. The ligation of polymer bound dibromomaleimides takes advantage of this circumstance' as it allows for the functionalization of disulfide moieties. Haddleton and coworkers demonstrated the applicability of this reaction for bioconjugation of salmon calcitonin (sCT).

(Continues...)

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Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2016
• Language: English
• ISBN 10: 1782624139
• ISBN 13: 9781782624134
• Binding: Hardcover
• Number of pages: 632
• Editor: Nico Bruns