Molecular tools for the construction of peptide-based materials.

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Molecular tools for the construction of peptide-based materials B. E. I. Ramakers, J. C. M. van Hest and D. W. P. M. Lo ¨ wik* Proteins and peptides are fundamental components of living systems where they play crucial roles at both functional and structural level. The versatile biological properties of these molecules make them interesting building blocks for the construction of bio-active and biocompatible materials. A variety of molecular tools can be used to fashion the peptides necessary for the assembly of these

Received 15th October 2013

materials. In this tutorial review we shall describe five of the main techniques, namely solid phase

DOI: 10.1039/c3cs60362h

peptide synthesis, native chemical ligation, Staudinger ligation, NCA polymerisation, and genetic engineering, that have been used to great effect for the construction of a host of peptide-based

www.rsc.org/csr

materials.

Radboud University Nijmegen, Institute for Molecules and Materials, Bio-Organic Chemistry, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands. E-mail: [email protected]

materials. Whether they are made up solely of amino acids or are combined with other polymers, peptides are known to self-assemble and so provide an interesting platform for the development of nano-scale, biocompatible materials. Over the last decade this class of materials has been extensively investigated, partially as peptide synthesis is relatively easy but also because a peptide can be conveniently tailored to adopt a desired structure or to suit a particular function.2–8 The expansion of this field has necessitated the development of a diverse number of molecular techniques that facilitate quick, versatile and orthogonal synthesis of these materials. In this tutorial review we highlight various molecular methodologies that have been used for the creation of peptide-based materials

Britta E. I. Ramakers graduated with a Master of Chemistry with honours Medicinal and Biological Chemistry with Industrial Experience from The University of Edinburgh in 2010. The degree course included a yearlong placement in industry which was done in the medicinal chemistry department of Schering-Plough in The Netherlands. In addition to this, a research project was carried out with Dr Jonathan Lowther and B. E. I. Ramakers Dr Dominic Campopiano on the specificity and inhibition of the enzyme serine palmitoyltransferase. Currently she is pursuing a PhD under the supervision of Prof. Jan van ¨wik, which focuses on the synthesis and Hest and Dr Dennis Lo manipulation of peptide-based architectures.

Jan C. M. van Hest conducted his doctoral research on molecular architectures based on dendrimers under the supervision of Prof. Bert Meijer, for which the PhD title was granted in 1996. As a postdoctoral researcher, he investigated the possibilities of protein engineering for the preparation of materials with Prof. David Tirrell. In 1997 he joined the chemical company DSM to work on the development of innovative material concepts. In J. C. M. van Hest 2000 he was appointed as a full professor in bio-organic chemistry at the Radboud University Nijmegen. His research is focused on protein-based materials and bio-inspired processes, with an emphasis on compartmentalization strategies.

Introduction Amino acids, sugars, lipids and nucleic acids are the constituents of nature that facilitate life. They are intrinsically bioactive, biodegradable, and biocompatible, which makes them perfect building blocks for new materials. Peptide-based materials have been the topic of intensive research,1 not only because of their synthetic versatility and their potential in biomedical applications, but also because they show promise as bio-based alternatives to synthetic

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with native peptide bonds. Other useful chemoselective ligation techniques exist, such as oxime and hydrazine ligation; however they yield non-native peptide bonds, and are thus beyond the scope of this tutorial review.9 Techniques that will be covered include solid phase peptide synthesis, native chemical ligation, Staudinger ligation, NCA polymerisation and genetic engineering and for each we will highlight some examples of materials that have been fashioned using that technique.

Solid phase peptide synthesis Pioneered by Merrifield in 1963, solid phase peptide synthesis (SPPS) has rapidly developed into one of the most powerful and versatile molecular tools for the construction of peptides.10 This technique centres on the fact that the growing peptide is covalently attached to an insoluble polymeric resin. The first amino acid residue is attached to the polymeric resin via a linker functionality, and the peptide is then synthesised by repetitive cycles of deprotection of the N-terminus and coupling of the subsequent amino acid interspersed with filtration and washing steps to remove any unreacted reagents (Fig. 1). At the end of the synthesis the desired peptide can be cleaved from the resin. There are two categories of SPPS known as Boc- and FmocSPPS, where the name refers to the N-terminal protecting group of the amino acid. Initially, Boc-SPPS was the main form of SPPS. However, it requires neat trifluoroacetic acid for the removal of the Boc group and neat hydrofluoric acid for the removal of the peptide from the resin. It was for these reasons that a more convenient alternative, Fmoc-chemistry, was developed. Although Boc-SPPS is still used today, it is much less routinely utilised for peptide synthesis than Fmoc-SPPS. In Fmoc-SPPS, the N-terminal protecting group can be removed using piperidine and the peptide can be removed from the resin using trifluoroacetic acid. The relatively safe chemicals

Dennis W. P. M. Lo¨wik, after completing his MSc in organic chemistry in 1994 cum laude, received his PhD in 1998 under the supervision of Prof. Rob Liskamp. This was followed by a post-doctoral position in Cambridge (UK) in the biotechnology group of Prof. Chris Lowe until 2000. After a year’s stay in the supramolecular chemistry group of Prof. Bert Meijer, he was appointed in 2001 at the Radboud University Nijmegen in ¨wik D. W. P. M. Lo the bio-organic chemistry group. His research revolves around peptide-based materials, operating at the interface of chemistry, biology and physics, in order to develop smart materials and drug delivery systems.

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Fig. 1 General solid phase peptide synthesis scheme, where P-AA-OH represents an N-terminal and side-chain protected amino acid and H-AAOH represents an unprotected amino acid. The first amino acid is loaded onto a solid support, the N-terminal protection group is removed and the subsequent amino acid is coupled. At the end of the synthesis the peptide is cleaved from the solid support and fully deprotected.

used in Fmoc-SPPS meant that special cleavage equipment was no longer required and so SPPS became a more accessible technique. The stepwise elongation inherent to SPPS circumvents the time consuming isolation and purification of intermediate peptides often associated with solution phase synthesis. In addition to this, the coupling reactions can easily be enhanced as, due to the ease with which the reagents can be removed after a reaction, an excess of reagents can be used. Another advantage of this method of peptide synthesis is that it evades any issues caused by a potential lack of solubility of the intermediates. The combination of these features means that SPPS is ideally suited for automation. The preliminary drawbacks of this manner of synthesis included the need for an extremely high coupling efficiency and the chance that the chiral integrity of the peptide may become compromised during the synthesis through oxazoline formation. However, these drawbacks have been extensively addressed through the development of numerous carboxyl activating agents. Nowadays, it is possible to create peptides with absolute control over not only the amino acid sequence but also the C and N terminal functionality, and thus SPPS has become a cornerstone in the construction of an extensive assortment of peptide-based materials.11,12 An excellent example that illustrates the use of SPPS in the development of peptide-based materials is the work carried out by the Schneider group.13 They synthesised a peptide with the sequence VKVKVKVK-VDPPT-KVKVKVKV-NH2 (MAX 1), essentially a tetrapeptide amid two identical fragments containing alternating valine and lysine residues. At an elevated pH or salt concentration and in the cell growth medium, DMEM, this peptide was found to fold into an amphiphilic b-hairpin and subsequently assemble into hydrogels (Fig. 2). Furthermore, these gels could be used for cell culture as they not only


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Fig. 2 Formation of MAX1 hydrogels in cell culture medium. Reproduced with permission from ref. 13.

retained their rigidity during cell proliferation but equally importantly, were also cytocompatible. In an attempt to accelerate hydrogel formation in DMEM a single lysine residue was replaced with a glutamic acid to give a peptide with the sequence VKVKVKVK-VDPPT-KVEVKVKV-NH2 (MAX 8). This minor, site-specific change indeed led to faster gel formation in DMEM than was observed for MAX1. Both MAX 1 and MAX 8 were shown to encapsulate dextrans and protein probes. In addition to this, MAX 8 could be utilized to encapsulate mesenchymal stem cells which, in conjunction with the fact that MAX 8 showed shear-thinning behaviour, made these gels a feasible platform for the delivery of cells in tissue regeneration applications. Small design changes as in the example above can easily be incorporated using SPPS and yet can have a marked effect on the formation of the peptide-based hydrogel.14,15 The work carried out by Pandya et al. also illustrates how easily peptide sequences can be tailored, using SPPS, to encourage the formation of certain tertiary structures.16 They designed a 28 residue peptide in an attempt to induce the formation of a coiled-coil dimer. Coiled-coils are formed when two or more a-helices entwine to give a stable structure (Fig. 3a).5 The a-helices that made up the coiled-coil dimer assemblies were composed of four heptad repeats of amino acids (abcdefg) in which residues a and d were isoleucine and leucine respectively. This clustering of hydrophobic amino acids stabilises the formation of coiled-coil dimers (Fig. 3b). The formation of parallel dimers was reinforced by glutamate and lysine residues that were incorporated at positions e and g. In order to create dimers with sticky ends, and thus realise a-helical coiled-coil fibrils, the isoleucine residues at position a

Fig. 3 (a) Schematic representation of a coiled coil dimer. The letters indicate the positions of the various amino acids. (b) Representation of the parallel coiled-coil fibre, with the interactions between the asparagines highlighted in green. Reproduced with permission from ref. 16.


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in the heptad were replaced with asparagine residues. This gave complementary dimers as the asparagine residues have a preferential interaction with each other. This simple peptide contains a great number of well-reasoned design features that can be easily incorporated using SPPS, which emphasizes the importance of this technique in the construction of peptide based materials. The absolute control over the primary sequence of building blocks afforded by SPPS allows them to be designed in such a way that higher order structures can be obtained. Recently, the Woolfson group reported that they had specifically designed coiled-coils that could self-assemble into cages.17 They devised a 20 residue homodimer (CC-Tri3) and a set of heterodimers: CC-Di-A, an acidic coiled-coil, and CC-Di-B, a basic coiled-coil. The cages are based on the fact that the CC-Tri3 and either the CC-Di-A or the CC-Di-B heterodimer can be covalently linked through disulfide bridges giving rise to two constructs. Upon their introduction to water these constructs form complementary trimeric hubs (hub A and hub B) which when mixed form hexagonal networks, and subsequently fold to form cages (Fig. 4). The modular nature of this approach allows the building blocks to be altered using SPPS to suit specific applications, for example as vehicles for biomolecule delivery or for the development of protocells.18 Lesley et al. showed that SPPS can also be effectively utilised to construct assemblies that closely mimic nature. They achieved this by designing a peptide that contained repeats of the tripeptide sequence, Pro-Hyp-Gly (where Hyp is hydroxyproline), which is distinctive of collagen. The tripeptide was modified at certain positions with lysine and aspartate residues. These residues provide extra stability through the formation of hydrogen bonds and ultimately resulted in the creation of a stable triple helix with sticky-ends. Just like natural collagen it was found that these helices could self-assemble into triple helical nanofibres and subsequently into hydrogels.

Fig. 4 Schematic representation of the self-assembly of coiled coil cages. Left to right: homotrimeric coiled coil (CC-Tri3, green) and heterodimeric coiled coils (CC-Di-A, red, and CC-Di-B, blue). CC-Tri and CC-Di-A/ CC-Di-B are linked via asymmetric disulfide bonds (purple lines) and result in two hubs: hub A (green-red) and hub B (green-blue). Mixing hub A with CC-Di-B or hub B with CC-Di-A produces discrete nine-helix assemblies, whereas mixing the hubs directly produces a hexagonal network, which should close to form a cage. Reproduced with permission from ref. 17.

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Fig. 6 (a) Structure of the spacer amphiphile. (b) Structure of the RGDS amphiphile. (c) 3D model of the cylindrical micelles with the RGDS amphiphiles highlighted in yellow. Adapted with permission from ref. 25.

Fig. 5 3D model of the self-assembly of b-annulus peptide fragments into a b-annulus structure and subsequently into a virus-like nanocapsule. Adapted with permission from ref. 20.

When subjected to collagenase it was discovered that these hydrogels were degraded at a similar rate as is observed for natural collagen.19 Another example of a peptide-based material that closely mimics nature is virus-like nanocapsules made up of 24 mer b-annulus peptide fragments which come from the tomato bushy stunt virus (Fig. 5).20 Hollow capsules, with a diameter of 30–15 nm, were formed upon dissolving the b-annulus peptide in water. The hollow nature of these assemblies was confirmed using small-angle X-ray scattering (SAXS). The cationic interior of the capsules facilitated the encapsulation of small anionic dyes and DNA polyanions to create core–shell nanospheres.21 SPPS can also be utilised to make cyclic peptides. The cyclisation can be achieved either on the solid support or once it has been cleaved off the resin. These cyclic peptides have been widely exploited in the formation of organic nanotubes.22 One of the most well-known examples is the nanotubes formed upon the self-assembly of Ghadiri type peptide rings.23 These peptide rings are comprised of an even number of amino acids, with D- and L-amino acids alternating around the ring. In this way the amide functionalities lie perpendicular to the plane of the nanotube and the amino acid side chains point outwards. Self-assembly was induced using an acidic medium to protonate the glutamic acid residues and so allow hydrogen bonds to form between the rings. All these examples demonstrate that a wide range of materials can be made using SPPS, and only the canonical amino acids. In addition to being a facile technique for constructing peptide-based materials consisting solely of amino acids, SPPS can also be used to construct peptide amphiphiles. By introducing amphiphilicity the self-assembly processes that often lead to the formation of peptide-based materials can be influenced.2 Tirrell reported the formation of cylindrical micelles made up of peptide amphiphiles, synthesised using SPPS, where the peptide component was derived from ovalbumin protein, and

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to which a dipalmitic acid tail was coupled N-terminally.24 The peptide component encompassed a cytotoxic T-cell epitope and it was found that these micelles could not only induce an immune response but also offered in vivo protection from tumours. Webber et al. have also shown that by coupling palmitic acid to the N-terminus of a peptide containing an RGDS domain an amphiphile is created which when mixed with a non RGDS containing spacer amphiphile can self-assemble into cylindrical micelles (Fig. 6).25 It was found that these synthetic scaffolds could be utilised to culture bone-marrow mononuclear cells (BMNCs) in vitro, and they also demonstrated that these fibres could support transplanted BMNCs in vivo. In a similar research study, the Stupp group revealed that the components of such assemblies could be covalently linked by utilising SPPS to introduce specific functionalities such as cysteines, which can form disulfide bridges, or diacetylenes, which can be cross-linked using UV light.26,27 Analogous to this, ¨wik et al. showed that SPPS could be used to construct Lo amphiphiles with a peptide segment based on the CS protein of a malaria parasite and a hydrophobic segment incorporating the diacetylene functionality.28 They found that these amphiphiles formed highly organised materials under the influence of a magnetic field, and that the alignment at a molecular level was transferred to the high molecular weight fibres that formed upon polymerisation of the diacetylene moieties in the alkyl tail of the amphiphiles.29 Furthermore, it was found that if these aligned fibres were irradiated with polarised light only the positions where the polarisation of the incident light was parallel to the fibre orientation were polymerised. This effect offers opportunities with regard to the formation of detailed nano-architectures.30 Cross-linking peptide-based materials adds an additional degree of stability which was demonstrated by Biesalski et al. using amphiphiles composed of a 10,12-tricosadiynoic acid tail and a GRGDSP head group.31 They were able to use diacetylenes to cross-link monolayers comprising these peptide amphiphiles mixed with the bare 10,12-tricosadiynoic acid. These monolayers were stable enough to be transferred onto solid substrates and were suitable for several rounds of cell culture without losing any of their surface functionality.


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Fig. 7 The functionalisation of an aldehyde resin to give a doubly functionalised peptide.

In all the examples mentioned above the alkyl tail is introduced N-terminally; in 2006 ten Brink et al. reported a facile method for the C-terminal functionalisation of peptides using the aldehyde-functionalised resin (BAL resin) developed by Kappel and Barany (Fig. 7).32,33 They utilised a variety of functional and bulky amines which they attached to the resin using reductive amination, and subsequently synthesised a small peptide using the functionalised resin. After cleavage from the resin the peptide was obtained with a functionalised C-terminus. Consequently, they used this methodology to synthesise an assortment of amphiphiles, based on the model peptide Ac-KTVIIE-NH2, in which they varied only the position of the alkyl tail.34 They found that b-sheet type assemblies formed for both amphiphiles with either a C-terminal or an N-terminal alkyl tail. They expanded the approach by introducing alkyl tails at both the C-terminus and the N-terminus of the model peptide; however, due to the dramatic increase in hydrophobicity these amphiphiles failed to selfassemble. It was determined that the approach was also suitable for the synthesis of biohybrid block co-polymers, as was shown by the synthesis of a polystyrene-based peptide-polymer amphiphile.35 Klok and co-workers also reported the synthesis and self-assembly of PEG-b-peptide diblock copolymers, in which the peptide sequences are based on the coiled coil protein folding motif, and the PEG segment was coupled to the resin-bound peptide.36 The versatile nature of SPPS means that additional, nonnative functionalities can be introduced at a predetermined position in the peptide while it is still on the resin. This was demonstrated by Couet and Biesalski, who modified Ghadiri type peptide rings with an initiator for atom transfer radical polymerisation (ATRP).37 These modified rings self-assembled into the anticipated nanotubes and these nanotubes were subsequently used as macro-initiators for ATRP. As the polymerisation reaction progressed the nanotubes disassembled as a result of the increasing steric bulk on the nanotube surface, an asset which was exploited by van Hest and co-workers to induce a colour change in their diacetylene containing peptide amphiphile fibres. They functionalised the peptide component of the amphiphile with an initiator for ATRP and subsequently used the diacetylene moiety in the alkyl tail of the amphiphile to covalently cross-link fibres made up of these amphiphiles. Using these fibres as macro-initiators allowed them to induce a colour change in the diacetylene backbone of these fibres,


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which could be correlated to the increased steric hindrance on the surface of the fibres as a result of the growing polymer.38 In the examples above ATRP was utilised after the peptidebased material had formed; however ATRP is important in the field of material synthesis in its own right and many reviews have been written that cover this technique.39–41 Since its discovery, SPPS has become an integral molecular tool for the synthesis of numerous functional peptides, proteins and peptide-based materials. The modular nature of the method and the orthogonal protecting strategy it encompasses provide a means of introducing functionality throughout the peptide. This means that a great deal of rational design can be incorporated into peptides made using SPPS, and this in turn affords a great deal of control over the morphology and functionality of the materials formed by these peptides.

Native chemical ligation Native chemical ligation (NCL) is a synthetic approach based on the chemoselective reaction between an N-terminal cysteine and a peptide a-thioester, which leads to the formation of a thioester linked intermediate. This intermediate then spontaneously rearranges to afford a native peptide bond (Fig. 8). Although this reaction was first observed in 1953 by Wieland, it was not until 1994 that Dawson et al. developed it into a potent tool for protein synthesis.42 Since then it has been used to great effect for the synthesis of numerous proteins, including human interleukin and human psoriasin amongst many others.43,44 More recently the native chemical ligation approach has been elaborated so that ligations can be carried out at alanine

Fig. 8 Native chemical ligation is a reaction between a peptide fragment with a C-terminal thioester (1) and a peptide fragment with an N-terminal cysteine (2). The reaction yields a thioester intermediate (3) which rearranges via an intramolecular S,N-acyl shift (4) to give the native peptide bond (5).

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Fig. 10 Synthesis of high molecular weight polymers using native chemical ligation.

Fig. 9 Native chemical ligation at an alanine residue. An N-terminal alanine residue is replaced with a cysteine residue (2) which can be used in native chemical ligation with a C-terminal thioester (1). This yields the peptide with a cysteine in place of an alanine (3). The cysteine residue can then be reduced back to an alanine residue (4) using selective desulfurization.

and even leucine or valine residues. In this extended methodology a residue in the peptide sequence, for example an alanine, is replaced by a cysteine residue. This cysteine can then be employed in the conventional native chemical ligation reaction with a thioester functionalised peptide fragment. A selective desulfurization reaction is subsequently used to convert the non-native cysteine residue back to an alanine residue, and so the native peptide will be obtained (Fig. 9). This methodology has led to the successful synthesis of complex proteins such as erythropoietin and human parathyroid hormonerelated protein.45 NCL has also been used as a molecular tool in the construction of an assortment of peptide-based materials; one example includes collagen-like peptide polymers. These polymers were designed by the Hartgerink group as a chemical and structural mimic for the extracellular matrix.46 They achieved this by polymerising peptides that they had previously synthesised with SPPS, using NCL (Fig. 10). The peptides that were synthesised contained different numbers of the triamino acid repeat proline, hydroxyproline and glycine (Pro-Hyp-Gly), characteristic to collagen, an N-terminal cysteine and a C-terminal thioester. These peptides were polymerised in phosphate buffer in the presence of dithiothreitol to give high molecular weight polymers. The authors chose NCL as a polymerisation technique as the aqueous conditions required for NCL also allowed the growing polymers to adopt their natural conformation, in this case a triple helix which is typical of collagen. NCL has also been utilised for the functionalisation of dendrimers with both oligopeptides and recombinantly expressed GFP.47 This was achieved through the incorporation of cysteine residues on the dendrimer and a thioester on the peptide or protein. The introduction of thioesters on proteins can be realised

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via so-called expressed protein ligation using expression systems based on self-cleavable intein domains.48 Full functionalisation of the dendrimer was attained with both the oligopeptides and protein, and introducing a single copy of the protein and derivatising the remaining cysteines with the oligopeptides was also found to be feasible. Analogous to this, Dirksen et al. described the synthesis of multivalent peptide-based nonsymmetric dendrimers using NCL.49 They fashioned two dendritic wedges, one functionalised with diethylene triamine pentaacetic acid (DTPA) ligands using maleimide chemistry and the other functionalised with the integrin binding domain RGDS using NCL (Fig. 11). The bulk of both wedges was comprised of a poly(lysine) scaffold, with the DTPA wedge incorporating a thioester at its core whereas the RGDS wedge contained a thioproline, which could be converted into a cysteine once the periphery of the wedge had been decorated with the RGDS peptide. These design features allowed the authors to use NCL to generate multivalent peptide-based nonsymmetric dendrimers, which may provide a versatile route to dendrimer-based multivalent target-specific MRI contrast agents.

Fig. 11 Schematic representation of the two dendritic wedges coupled by NCL. The DTPA labels are represented in blue and the RGDS peptides in pink.


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Another interesting peptide-based material in which NCL was utilised to great effect are covalently cross-linked hydrogels covered with the integrin binding domain RGDS.50 By incorporating either thioesters or cysteine residues at the extremities of star-shaped poly(ethylene glycol)s and subsequently allowing these two macromolecules to react with each other, hydrogels were rapidly formed. The thiol on the cysteine side chain was then functionalised with a peptide maleimide containing the RGDS domain and consequently the hydrogels were utilised as a surface for human mesenchymal stem cell growth. Due to the mild, aqueous conditions in which NCL can be carried out the peptide-based materials made using this technique do not require extensive purification and can be used as obtained. However, the aqueous conditions necessary for NCL can also pose a problem when either one or both of the components of the NCL reaction do not dissolve in aqueous medium. A novel approach to NCL was reported by the Boons group, who surmounted the solubility issues posed when trying to ligate lipophilic (lipo)peptides and glycopeptides in the conventional reaction, by encapsulating both components in dodecylphosphocholine liposomes.51 In this way they were able to synthesise lipophilic (glycol)peptides, which serve as three component vaccine candidates. The advantage of using NCL to create these materials is that it provides a modular synthetic route, which in turn provides the flexibility required to optimize the immunological properties of these three component vaccine candidates. Although NCL has been much more widely used in the synthesis of proteins, it is evident from the examples described above that NCL can also be used to great effect for the synthesis of peptide-based materials. Its power lies in the fact that it is a selective yet reliable method for the formation of peptide bonds in aqueous medium and all that is required is a cysteine and an a-thioester.

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for the labelling of a whole array of biomolecules. Both the nontraceless and traceless variant of the Staudinger ligation have been used for the construction of peptide-based materials.55 An interesting example of a peptide-based material synthesised using traceless Staudinger ligation is the RGD functionalised polyamidoamine–DNA nanoparticles described by Parkhouse et al.56 The aim of their endeavours was to increase the uptake efficiency of polyamidoamide–DNA nanoparticles into cells, and consequently increase the efficiency of gene expression, using a decapeptide containing the RGD motif. They realised this by using the Staudinger ligation to attach the phosphinothioester of the peptide to an azide bearing N-acylated PEGylated polyamidoamine both pre- and post-nanoparticle formation. In both instances well defined particles were obtained which gave similar results in cell uptake studies. Remarkable was the fact that the ligation of the peptide to pre-formed nanoparticles was five times more efficient than the ligation of the peptide to the unconfined polymer. The authors hypothesised that this may be due to the fact that the azide functionality is on the surface of the nanoparticles and thus more readily available for the ligation reaction. The Staudinger ligation was also successfully employed by the Higuchi group in the creation of peptide–porphyrin conjugates.57 They made these conjugates in the pursuit of suitable models for heme proteins. They found that using a porphyrin with four azide functionalities and a phosphinothioester functionalised peptide they were able to obtain various different tetra-substituted porphyrin conjugates. Recently, the Weck group reported the use of Staudinger ligation in the convergent synthesis of GRGDS-functionalized poly(lactide)-graft-poly(ethylene glycol) copolymers (Fig. 13).58 The authors employed thiol-ene chemistry to synthesise

Staudinger ligation In addition to native chemical ligation another ligation technique has been developed that can be utilised not only in the semi-synthesis of proteins and the labelling of biomolecules, but also as a means of constructing peptide-based materials. Hermann Staudinger was the first to report the reaction between an azide and phosphine in 1919 (Fig. 12).52 After initially elaborating this reaction into what is now known as the non-traceless Staudinger ligation, Bertozzi and Raines independently reported the development of a traceless Staudinger ligation at the beginning of the millennium.53,54 The main advantage of the traceless Staudinger ligation over nontraceless Staudinger ligation is that phosphine oxide is eliminated as a side product from the product in the final hydrolysis step. The traceless Staudinger ligation has mainly been exploited

Fig. 12

The Staudinger ligation between a phosphine and an azide.


Fig. 13 Schematic representation of the different components of the GRGDS-functionalised poly(lactide)-graft-poly(ethylene glycol) copolymer. The moieties for thiol-ene chemistry and Staudinger ligation are highlighted in purple and green respectively.

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tri(ethylene glycol)-containing lactide derivatives in which the chain end was terminated with an azide. Subsequently ring opening polymerisation was used to afford well-defined PLA-g-PEG copolymers interspersed with azides, which were used in a non-traceless Staudinger ligation to decorate the copolymer with GRGDS peptides. The traceless Staudinger ligation has primarily been used in the synthesis of large peptide fragments, the semi-synthesis of proteins such as RNase A and the modification of surfaces with proteins.59–61 Nevertheless, it has also seen some use as a molecular technique for the construction of peptide-based materials. We believe that it has the potential to be a useful tool; however with native chemical ligation reactions becoming more comprehensive and being constantly improved it seems improbable that Staudinger ligation will become a routinely used technique for the construction of peptide-based materials.

NCA polymerisation NCA polymerisation is a technique that makes use of a-amino acid-N-carboxy anhydrides (NCAs) to generate high molecular weight polypeptides (Fig. 14). The polypeptides are obtained in good yield, while the integrity of the chiral centres is retained. Due to the extensive number of NCAs available numerous polypeptides can be prepared, which makes this an ideal technique for the construction of peptide-based materials. Control over NCA polymerisation has greatly improved by the development of various metal- and organo-catalysts and thus the synthesis of well-defined block copolymers and hybrid copolymers has been facilitated.62 In spite of this, NCA polymerisation still does not afford the same degree of control over the primary structure of the polypeptide as is characteristic of SPPS or genetic engineering. Chen et al. developed an alkyl polypeptide amphiphile, based on a diethylene-glycol-monomethyl-ether-functionalised poly-L-glutamate (poly-L-EG2Glu) component, which they synthesised using NCA polymerisation. This polypeptide can selfassemble into nanoribbons and form a transparent hydrogel.63 The strength of the hydrogel can be tuned by varying the length of the alkyl tail, with a longer alkyl tail giving rise to a stiffer gel. The gels also show shear thinning behaviour which makes them potential candidates for the injectable delivery of biologically relevant molecules. In addition to this, the amphiphiles that form the foundation of these gels have an amine

Fig. 14

NCA polymerisation initiated by a nucleophilic amine.

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end group. The authors functionalised this amine with FITC, a fluorescent label, and induced gel formation to demonstrate that this does not affect the structure of the hydrogel and thus this amine group can potentially be employed to adorn the gel with bioactive species. Another example in which NCA polymerisation was utilised to create polymers that could form hydrogels is presented by the Sofroniew and Deming groups.64 They found that hydrogels consisting of the diblock copolymers Lys180Leu20 and Glu180Leu20 can be injected into the mouse brain and persist there for a number of weeks. This in combination with the fact that the gels are biocompatible and that they biodegrade makes them ideal contenders for the site-specific delivery of bioactive proteins across the blood brain barrier. To this effect, nerve growth factor (NGF) was encapsulated in the gels and was shown to induce hypertrophy, an enlargement, of the forebrain cholinergic neurons up to a distance of 5 mm from the deposit site, for a minimum of four weeks. This response was not seen for unencapsulated NGF, and so emphasises the importance of peptide-based materials in designing and developing new therapeutic treatments.65 Often peptide-based materials can fulfil a multitude of functions as was demonstrated by the hydrogel system developed by Cheng et al. in 2013.66 By carrying out the NCA polymerisation of g-propargyl-L-glutamate N-carboxyanhydride monomers in the presence of amino-terminated mPEG, they were able to synthesise the block copolymer poly(ethylene glycol)-block-poly(g-propargyl-L-glutamate) or PEG-PPLG, which incorporated alkynyl side chains. These polymers formed thermoresponsive hydrogels that could not only be employed for the 3D cell culture of mouse fibroblasts, but the alkynyl side chains could also be functionalised with bioactive molecules such as biotin and galactose by means of copper catalysed azide alkyne cycloaddition (click chemistry).67 It was found that the galactose functionalised hydrogels, possibly as a result of the absorption of fibronectin in the extracellular matrix, improved cell adhesion to the gels. These properties in combination with the facile synthetic route recommend the gels as platforms for bioadhesive and bioresponsive materials. Recently, novel block copolymers were reported consisting of a poly(ethylene glycol) block and an oligo(tyrosine) block. Polymers with varying compositions were made using NCA polymerisation. It was discovered that the block copolymer with the optimum composition of PEG to peptide was PEG44-Tyr6 as this polymer showed thermoresponsive gelation behaviour between 25 and 50 1C. This behaviour was observed for hydrogels with a concentration of 0.25–0.3 wt% of PEG44-Tyr6. Cryogenic transmission electron microscopy (Cryo-TEM) revealed that the hydrogel was made up of continuous fibres formed by the block copolymers. An explanation of the thermoresponsive nature of these hydrogels is that there is an improvement in the b-sheet packing of the peptide block at an increased temperature. Preliminary studies suggest that the hydrogel is biodegradable and not cytotoxic. In addition to this the hydrogel showed sustained release of the small molecule drug desferrioxamine (DFO). These findings suggest that this type


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Fig. 15 (a) PB40-b-PGA100 vesicles. (b) Freeze-fracture TEM image of the PB40-b-PGA100 aggregates. (c) Structure of the PB40-b-PGA100 block copolymers. Adapted with permission from ref. 70.

of hydrogel has potential as a material for injectable drug delivery.68 This is just a recent example; however there are many more examples in which NCA polymerisation is used in the synthesis of stimuli responsive polypeptides.69 NCA polymerisation has also been utilised to prepare polypeptide diblock copolymers that assemble into other interesting structures, for example water soluble stimuli responsive vesicles. The Klok group used NCA polymerisation to synthesise poly(L-glutamic acid) which was coupled to a hydrophobic polybutadiene block.70 Upon the introduction of these block copolymers to an aqueous solution with a basic pH, welldefined vesicular structures were observed (Fig. 15). The dimensions of the assemblies could be adjusted by altering the secondary structure of the polypeptide segment which was achieved by varying the pH of the aqueous environment. Moreover, the 1,2-vinyl bonds in the hydrophobic polybutadiene block could be cross-linked using UV-light, thereby stabilising these assemblies. The authors hypothesise that these vesicles have a number of potential applications, such as their use as capsules for the release of both hydrophobic and hydrophilic entities and as potential sensor nanodevices. Recently, the Zhang group presented core–shell micelles made up of four-armed poly(e-adamantane-L-lysine)2-blockpoly(ethylene glycol)-block-poly-(e-adamantane-L-lysine)2, which is abbreviated as PLys(Ad)2-b-PEG-b-PLys(Ad)2 (Fig. 16).71 The polymers were made by NCA polymerisation of the corresponding amino acid N-carboxyanhydrides and the micelles were found to form spontaneously upon dissolution of the polymers in aqueous solutions. Interestingly, these micelles disassembled upon the introduction of b-cyclodextrin to the solution. This was due to a host–guest interaction between the b-cyclodextrin and the adamantane moieties in the polymers. This, in addition to the fact that these micelles were non-toxic to and could also be internalised by HeLa cells, makes them ideal candidates for the controlled release of drugs such as doxorubicin. Another type of peptide-based material that offers perspective as a means of controlled drug delivery are thermoresponsive polymersomes made up of well-defined poly(trimethylene carbonate)-bpoly(L-glutamic acid) polymers. These were synthesised from


Fig. 16 Schematic representation of micelle formation, their subsequent internalisation by cells and b-cyclodextrin mediated disassembly of the micelles. Reproduced with permission from ref. 71.

g-benzyl-L-glutamate N-carboxyanhydride monomers using NCA polymerisation and amine-functionalised poly(trimethylene carbonate) (PTMC) as a macro-initiator.72,73 The hydrophobic PTMC block has a crystalline nature that is subject to a temperature triggered conformational change. This gave rise to a reversible, temperature triggered change in polymersome size. It was found that these changes in size were a result of the fission/budding or fusion of vesicles. This property should enable the temperaturecontrolled release of drug molecules from the polymersomes.74 NCA polymerisation is a versatile technique and as such provides a facile, scalable route to a large number of block co-polymers and thus a variety of peptide-based materials, provided that absolute control over the primary amino acid sequence is not crucial.

Genetic engineering Genetic engineering can be defined as the manipulation of the molecular circuitry and machinery of a cell in order to manufacture functional compounds, such as peptides and proteins. Cells have evolved sophisticated strategies that allow the synthesis of long, complex polypeptides in high yields with absolute control over the amino acid sequence, and thus the secondary structure, of the polypeptide. This combination is unattainable using techniques such as solid phase peptide synthesis, where the length is a limiting factor, or NCA polymerisation, which lacks control over the amino acid sequence. Due to this, genetic engineering has become instrumental as a molecular tool for the construction of peptide-based materials.75,76 The technique is based on the fact that a gene can be synthetically designed to incorporate the DNA sequence that

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encodes for a certain polypeptide or protein. An expression vector, a circular piece of double stranded DNA also known as a plasmid, carries a cloning site which can be accessed by utilising specific restriction enzymes. The synthetic gene can be ligated into the open plasmid by ligation enzymes, circularising the plasmid once more. The plasmid is then known as the expression vector. The resulting recombinant plasmid is transformed into a non-expression host, and as the vector carries an antibiotic resistance gene, the expression vector can subsequently be selected for by antibiotic resistance. This amplifies the number of copies of the plasmid, and once it has been isolated it can be transformed into the expression host. When the cells reach the log phase of their growth curve expression of the polypeptide can be induced. The Chilkoti group explored an interesting class of recombinant biopolymers known as elastin-like polypeptides (ELPs). Derived from human tropoelastin, ELPs consist of Val-Pro-GlyXaa-Gly repeats, where Xaa may be any residue except proline. They have used this class of biopolymers to create chimeric polypeptides with a cysteine segment that doxorubicin (Dox), a hydrophobic chemotherapeutic, can be conjugated to.77 These ELP–Dox conjugates self-assembled into nanoparticles upon their introduction into water. The degree of drug loading and the size of the nanoparticles could easily be controlled by manipulating the synthetic gene with respect to the number of reactive sites and the molecular weight of the ELP segment. Furthermore, the biodegradability of these conjugates makes them ideally suited as drug delivery vehicles; it was found that when administered to a murine cancer model, the nanoparticles caused almost complete regression of the tumour

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after a single dose. ELPs have an additional, distinguishing trait, namely their ability to undergo a reversible, inverse phase transition. Consequently, the Chilkoti group designed thermally responsive chimeric polypeptides that formed nanoparticles between 38 and 42 1C, a feature that may be exploited in thermally mediated drug delivery.78 Genetic engineering allows a great degree of control to be exercised over the primary structure of the peptide-based material, and accordingly materials can be designed that encompass several different structural elements. A good example of this is the calcium sensitive elastin-like polypeptides (CELPs) designed by Hassouneh et al.79 They interspersed sections of ELP with the calcium binding sequences from the protein calmodulin to create thermally sensitive, calcium binding polypeptides (Fig. 17a). Calmodulin is a protein that undergoes a conformational change upon binding calcium, and this characteristic change was also observed when calcium was added to the CELPs. Furthermore, this property was used to induce nanoparticle formation in ELP–CELP diblock polypeptides (Fig. 17b). A hydrophilic ELP segment was coupled to the calcium binding ELP polymer, and once calcium had bound, the CELP segment became more hydrophobic, resulting in the formation of an amphiphilic polymer. Subsequently, this amphiphilic polymer could assemble into monodisperse nanoparticles between 33 and 46 1C. ELP can also be used to govern the self-assembly process of proteins as was shown in the work carried out by van Eldijk et al.80 A fusion of ELP with the cowpea chlorotic mottle virus (CCMV) capsid protein was produced and afforded a protein block copolymer. This copolymer could be assembled into virus

Fig. 17 (a) Schematic representation of the calcium sensitive, elastin-like polypeptide. In the event of calcium binding the charges are neutralised and the polypeptide collapses. (b) The design of the CELP block copolymer and the formation of micelles upon addition of calcium. Reproduced with permission from ref. 79.

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Fig. 18 A virus capsid protein and ELP fusion and its assembly products based on pH-induced and ELP-induced assembly. Reproduced with permission from ref. 80.

capsids in the traditional way for CCMV, using a pH dependent mechanism, but assembly could also be induced by ELP, using elevated temperature or salt concentration (Fig. 18). It was discovered that the ELP-induced assembly gave rise to particles which differed significantly in size compared to particles obtained using the traditional assembly method, 18 nm for the former and 28 nm for the latter. This viral capsid architecture is attainable only when the properties of ELP and the viral capsid protein are combined, a requisite which can easily be satisfied using genetic engineering. A further illustration of how the thermoresponsive nature of ELP can be exploited to form a peptide-based material has recently been reported by the Narutaki group.81 They designed a polypeptide based on two hydrophobic blocks, a VPGXG block sandwiched between two VGGVG blocks. These constructs were found to form nanoparticles, with a high b-turn content, in aqueous solution at 45 1C. Subsequently, beaded nanofibres formed in these solutions, which gained more b-sheet character with time. These sequence dependent constructs may be interesting candidates as scaffolds for tissue engineering or drug delivery systems. The characteristics of two different structural proteins can be combined using genetic engineering. An example of this is silk-elastin like proteins in which the high tensile strength of silk and the resilience of elastin can be combined to give a superior class of material that can even be electrospun into nanofibres and used as a platform for cell growth.82 Another example where two functionalities were combined using genetic engineering is the elastin like recombinamers (ELRs) designed by Punet et al.83 These recombinamers were based on the ELP sequence VPGIG and intermingled with RGDS domains, for cell binding, and VPGKG domains for crosslinking. Functionalisation of poly(lactic) acid surfaces with this recombinamer allowed them to be utilised as surfaces for cell growth. Cell attachment to surfaces modified with ELRs was superior compared to that using short peptides, which, in addition to the fact that unspecific binding of proteins to these surfaces is minimal, makes ELRs good candidates for the functionalisation of surfaces. An alternative, emerging class of peptide-based materials are based on the highly elastic insect protein resilin. Analogous to elastin-like polypeptides, resilin-like polypeptides (RLPs) have been engineered to incorporate the cell binding domain RGDS.84


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These materials were suitable for cell growth and in addition to this they may also be suitable for the delivery of heparin, as both heparin and metalloproteinase sensitive sequences, which promote proteolytic degradation, could be incorporated into the RLP using molecular engineering. In 2011, the Kiick group reported that RLPs could be cross-linked with b-[Tris(hydroxymethyl)phosphino] propionic acid or betaine, and more recently they reported that three different RLPs could be cross-linked with a PEG-vinyl sulfone cross-linker using a Michael addition reaction.85,86 This gave rise to RLP– PEG hydrogels that were suitable for the encapsulation of human aortic adventitial fibroblast cells, which suggests that these materials are suitable for the engineering of cardiovascular tissues. Petka et al. designed a gene that when expressed yielded a material consisting of a flexible, water soluble polyelectrolyte segment amid two leucine zippers. The aggregation of the terminal leucine zippers in a neutral aqueous solution resulted in the formation of a polymeric network. The disassembly of this network was achieved at elevated pH or temperature.87 In a similar study these types of networks were stabilised using templated disulfide bond formation. Cysteine residues were incorporated into leucine zippers that flanked a random coil central segment. These constructs formed networks above a certain concentration, and the aggregation of the leucine zippers in these constructs resulted in the formation of disulfide bonds between the cysteine residues. This stabilised the networks to such an extent that they were no longer prone to dissolution upon addition of excess buffer solution.88 Another interesting peptide-based material are the coiled-coil tetrahedron structures constructed by Gradisˇar et al.89 which can be compared to the coiled-coil cages fashioned by the Woolfson group.17 They employed genetic engineering to synthesise a polypeptide chain containing 12 coiled coil-forming segments

Fig. 19 (a) The sequence of the coiled-coil segments in the polypeptide chain, showing the pairings of the coiled-coil dimers. (b) Tetrahedron structure formed upon assembly of the coiled-coils into dimers. Adapted with permission from ref. 89.

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interspersed with flexible peptide hinges (Fig. 19a). The polypeptide chain was designed so that the coiled-coil segments traversed each edge of the tetrahedron twice, facilitating the formation of dimers between complementary parallel or antiparallel coiled-coils (Fig. 19b). With the combination of absolute control over the amino acid sequence of a peptide and the ability to produce high molecular weight peptides, genetic engineering fills the niche left by SPPS and NCA polymerisation, especially as unnatural amino acids can now be incorporated, and thus the assortment of functionalities that can be integrated into peptides and proteins can be easily expanded.90 Although genetic engineering is perhaps a less accessible technique than SPPS and NCA polymerisation, and relatively low yields of the material are obtained compared with the afore mentioned techniques, it is still an invaluable tool for the construction of peptide-based materials.

Concluding remarks With the demand for new medical technologies growing, peptide-based materials offer an excellent platform for the development of targeted drug delivery systems and as substrates for regenerative medicine. A wide range of synthetic techniques are available for the synthesis of these materials, each technique with different pros and cons. Solid phase peptide synthesis allows precise control over the amino acid sequence of a material; however high molecular weight peptides can only be obtained through the use of native chemical ligation or Staudinger ligation. Conversely, NCA polymerisation yields materials with a high molecular weight but without a primary sequence that is as well-defined as with SPPS. Genetic engineering provides a means of combining the ability to produce high molecular weight peptides with precise control over the amino acid sequence; however obtaining the materials is more labour intensive than the other techniques, the yields are often significantly lower and unless additional efforts are made only the natural amino acids can be used. These molecular tools all complement each other and can be combined in order to incorporate a host of functional moieties or structural design features, as is evident from the countless examples known in the literature. The ability to precisely design peptidebased materials to suit a particular function is possibly one of their biggest strengths. However, as yet there are no examples of these materials being applied in a clinical setting. The molecular tools needed for the construction of these materials, especially genetic engineering, ring opening polymerisation and solid phase peptide synthesis, are well established in industry; however the transition between fundamental research and industry remains elusive. Peptidebased materials have the potential to be of paramount importance in the future; however before this can be realised both the academic sector and the industrial sector must make an effort to bridge the gap that now exists between fundamental research and industrial applications.

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Acknowledgements The authors would like to acknowledge the Dutch Science Foundation (NWO) and the Ministry of Education, Culture and Science (Gravity program 024.001.035) for their financial support. The authors would like to thank Rik Stijnen for the design and realisation of the graphical abstract.

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