Secondary structures of synthetic polypeptide polymers

Colin Bonduelle ab
aCNRS, LCC (Laboratoire de Chimie de Coordination (UPR8241)), 205 route de Narbonne, F-31077 Toulouse, France. E-mail: colin.bonduelle@lcc-toulouse.fr
bUniversité de Toulouse, Université Paul Sabatier, Toulouse, France

Received 11th October 2017 , Accepted 9th November 2017

First published on 14th November 2017


Synthetic peptide-based polymers represent a unique class of macromolecules able to mimic the properties of natural proteins in materials sciences since (1) they present the same macromolecular backbone as do proteins, (2) they can be obtained in large scale and in only one step by using the ROP (Ring-Opening Polymerization) methodology, and (3) they can fold into different secondary structures in the same way as do proteins. The control of this structuring ability paves the way to a wide range of applications in materials science, for which uses of natural proteins remain limited. In this review article, the fundamental principles of polypeptide polymer structuring are summarized. It is also highlighted here, how tuning the polypeptide secondary structure could be a key step to modulate various properties in advanced materials (size, rigidity, self-assembly, etc.).


1. Introduction

Natural proteins are key biomacromolecules derived from 20 naturally occurring amino acids. The linear association of these amino acids defines a primary sequence that has the ability to fold into highly organized structures at the origin of the essential functions of life. The use of proteins as building blocks for materials design holds tremendous promises in many applications,1 but large-scale industrial production is so far challenging: approaches that use genetic engineering are quite promising but require significant development of biological platforms thus still restricting the scale and scope of such preparative methods.2

The challenge of creating synthetic materials with the structural sophistication and complex functions found in biology has been a long-term goal in materials science. The design of synthetic “proteins” by using chemistry has always been an alternative approach to genetic engineering that aims (1) to reproduce the full structure of natural proteins or (2) to prepare simplified analogues that will partially reproduce structural or functional features of the natural model.3 To date, the full reproduction of natural protein structures relies on the synthesis of sequence-controlled peptides by Merrifield's method, the solid phase peptide synthesis (SPPS, Fig. 1).3c,4 Although this technique can be used to generate specific peptide sequences, the process is limited in practice to short peptide sequences that can only be provided at high costs and in small quantities due to low yields, time consuming process steps, and costly purifications.3c,4 To overcome some chemical limitations that rely on the use of amino acids, purely synthetic compounds have also been designed to build foldamer oligomers that evidence structuring properties similar to natural protein systems.4,5 For both classes of sequence-controlled oligomers (peptides and foldamers), multistep processes involved in their preparation rely on iterative coupling methods, the only way to date and using chemistry, to prepare sequence controlled macromolecules with high precision (Fig. 1).


image file: c7py01725a-f1.tif
Fig. 1 Chemical routes to prepare synthetic peptides: SPPS versus ROP methodologies.

Overall, iterative coupling methods afford the preparation of predictable secondary structures depending on the sequence that has been designed. Nevertheless, the use of such methods also implies that (1) cost-effective gram scale production is often a challenge and (2) the preparation of new defined synthetic structures is associated with a new synthetic scheme that has to be optimized. Interestingly, the most economical and efficient process to prepare synthetic polypeptide chains is a one-step polymerization process, the ring-opening polymerization (ROP) of N-carboxyanhydride monomers (ROP, Fig. 1 and Scheme 1).6 This controlled polymerization process involves the simplest reagents, and allows the preparation of polymers made of amino acids in both good yields and large quantities.6i,7 Synthetic polypeptide polymers are simple macromolecules compared to natural proteins, in which an amino-acid is repeated many times, retaining the tendency to adopt ordered secondary conformations such as α-helices or β-sheets, a property that is rare in polymer science.5b,8 Indeed, in materials science, polypeptide polymers that adopt secondary conformations offer a unique way to guide nanoscale structure formation through intermolecular and or intramolecular interactions and therefore, polypeptide polymers have attracted interest in a variety of biomedical applications including nanomedicine.9 Moreover, as compared to natural proteins, polypeptide polymers can easily undergo secondary structure transitions that can be easily implemented and tuned by tailoring amino-acid side chains.6c,10 This article focuses on how the secondary structures of peptide-based polymers can fundamentally change their physico-chemical properties (size, rigidity, degradation, etc) and how these fundamental properties impact their uses in a wide range of applications (drug delivery, gel formation, coatings, etc.) with specific emphasis on smart structuring design.


image file: c7py01725a-s1.tif
Scheme 1 Synthetic polypeptide polymers prepared by the ring-opening polymerization of N-carboxyanhydride (NCA) monomers.

2. Fundamental principles of secondary structuring

In biological systems, the folding of natural proteins is hierarchical and involves various interactions among the amino acids that constitute the primary sequence of the macromolecule. Secondary structure formation corresponds to a local organization of the amino acids that creates structured segments along the peptidic backbone. In natural proteins, this local organization is a chemical leverage to (1) significantly modify polypeptide degradation properties; (2) permit segmental amphiphilicity (amphipathicity); (3) drive the tridimensional folding of the overall protein; (4) guide further supramolecular assemblies of proteins (quaternary structure). In natural proteins and in synthetic polypeptides, macromolecules are indeed constituted by a repetition of two neighboring planes that are connected by one Cα carbon; thus, the torsion angles (ψ, ϕ) are allowed for each amino acid residue, as shown in the Ramachandran plot (Fig. 2).11 A possible region of (ψ, ϕ) corresponding to favorable low-energy regions is therefore limited by the chirality of the Cα carbon and the type of amino acid side chains.12
image file: c7py01725a-f2.tif
Fig. 2 Ramachandran plot (top, dots correspond to (ψ, ϕ) torsion angles data extracted from about 500 PDB protein structures) and molecular modelling of the two main secondary structures found within proteins (down, α-helix and β-sheet) (reproduced with permission from ref. 12, copyright [2017], Wiley).

2.1 Secondary structure formation

The preorganized secondary structure is stabilized by various parameters including intra- or intermolecular hydrogen bonds, electrostatic interactions, and hydrophobic interactions. The approximate location and shape of the most favorable, low-energy regions of the Ramachandran plot define two main regions that correspond to α-helix and β-sheet structuring (see Fig. 2).13

The α-helix is a rod-like structure whose inner section is formed by a tightly coiled main chain, with its side chains extending outward in a helical array (Fig. 2). The α-helix structure takes advantage of the hydrogen bonding network created between the oxygen of the C[double bond, length as m-dash]O of each peptide bond in the strand (i) and the hydrogen of the N–H group of the peptide bond four amino acids below it in the helix (i + 4). This hydrogen bonding network makes this structure more stable when the polymerization degree is higher. The most current angles found in the α-helix geometry are ϕ = −60° and ψ = −45° with the segment forming a compact, rodlike structure, with 3.6 amino acids (1.5 Å) per repeating unit and a radius of 2.3 Å, excluding side chains (Fig. 2). Despite the fact that, based on the Ramachandran plot, both right-handed and left-handed α-helices are among the permitted conformations, the right-handed α-helix is energetically more favorable because of less steric hindrance between the side chains and the main chain.

In a β-sheet, the main chain, called the β-strand, is fully extended rather than tightly coiled, and the side chains of adjacent amino acids point in opposite directions. The dihedral angles of the β-sheets are ϕ = −130° and ψ = 120°, forming an extended structure with some right-handed twist. The hydrogen bonding network in a β-sheet consists of pairs of strands lying side-by-side (Fig. 2). The carbonyl oxygens in one strand allow the creation of hydrogen bonds with the amino hydrogens of the adjacent strand. The two strands can be either parallel or anti-parallel depending on whether the strand directions (C-terminus to N-terminus for polypeptide polymers) are the same or opposite. It is worth noting that the anti-parallel β-sheet is more stable due to a better aligned hydrogen bonding network.

Outside α-helix and β-sheet supramolecular structuring, several other ψ and ϕ torsion angles are allowed and at the origin of other secondary structures (310 helix, β-turn, etc).12 Previous structural studies indicate that the propensity of individual amino acids to form particular secondary structures is the result of a combination of (1) the influence of their lateral chain function and (2) environmental factors (template, solvent, etc.). It is possible to find tables drawn by structural biologists in which are depicted the relative frequencies of amino acid residues that are found, within proteins, in a specific secondary structure.14 Overall, α-helix structuring can be regarded as the default conformation. Branching at the β-carbon atom, as in valine, threonine, and isoleucine, tends to destabilize α-helices because of steric clashes. On the other hand, serine, aspartate, and asparagine tend to disrupt α-helices because their side chains contain hydrogen-bond donors or acceptors in close proximity to the main chain, where they compete for main-chain NH and CO groups. Finally, steric residues are often better accommodated in β-strands, in which their side chains project out of the plane containing the peptidic backbone.

2.2 Secondary structure determination

It is now established that the rough secondary-structure content of polypeptides can be estimated spectroscopically by various methods15 including the use of Raman spectroscopy,16 NMR spectroscopy,17 infrared spectroscopy (IR)18 and far-ultraviolet circular dichroism (CD, 190–250 nm).19 The two last methods are the most commonly used by polymer chemists and generally provide enough structural information with polypeptide polymers. CD is an excellent method for rapidly evaluating the secondary structure in aqueous solutions. Implementation of this method can be achieved in organic solvents or in the solid state but may require significant optimization procedures and the use of specific devices.20 Electronic transitions of the amide group of the peptidic backbone occur in the far-UV region: the nonbonding π orbitals (πnb) of the peptide bond and a lone pair orbital of the carbonyl oxygen atom allow two specific electronic transitions to another antibonding π* orbital (πnb to π* at ∼193 nm and n to π* at ∼222 nm).19b This is why, upon circularly polarized UV light irradiation, a polypeptide electronic structure gives rise to characteristic bands in these specific regions of the CD spectrum, reflecting the two characteristic electronic excitation energies. Secondary structural elements, such as α-helices, β-sheets or random coil structures, all induce CD bands of distinctive shapes and magnitudes that are now well documented (Fig. 3).19b A pronounced double minimum at 208 and 222 nm indicates generally α-helical structures (see Fig. 3), whereas a single minimum at 196 nm or 217 nm reflects random-coil or β-sheet structuring, respectively. It is good to know that the influence of the environment (salt, chromophore, pH, etc) may induce significant changes to these ideal cases.21 For instance, precise evaluation of the CD spectra of poly(L-glutamic acid) has shown that the α-helix conformation minima are indeed found at 210 nm and 224 nm.22 For α-helix secondary structures, helix nanoaggregation tends to decrease ellipticity at 208 nm, and the orientation/solubility of β-sheet structures may generate minima that are found to be higher or lower than 217 nm (see Fig. 3). Readers can find various helpful information regarding CD technique analysis within the literature and are invited to consult other dedicated articles if more information is required.15b,19b,23
image file: c7py01725a-f3.tif
Fig. 3 Secondary structure determination of synthetic polypeptide polymers: (A) characteristic CD curves of the two main secondary structure elements as compared to coil conformation (values were taken from ref. 19b); (B) characteristic IR peak wavelength values corresponding to amide CO stretching (values were taken from ref. 18a).

Another common method used for secondary structure determination is infrared spectroscopy.15b This technique is particularly suitable to probe secondary structures of surfaces or in bulk, but secondary structure can also be determined in solutions if an IR “transparent” solvent is used (for instance, deuterated water). IR spectroscopy detects differences in the bond oscillations of amide groups due to hydrogen-bonding, and generally two CO stretchings (amide I and II) are used for secondary structure determination.18b In the solid state, strong amide I stretching is found at 1656 cm−1, 1650 cm−1 and 1630 cm−1 for coils, α-helix and β-sheets, respectively.18a Corresponding amide II stretching bands are found at 1535 cm−1, 1546 cm−1 and 1530 cm−1 for coils, α-helix and β-sheets, respectively.18a It is recommended to check well both amide stretchings to discriminate alpha helix from coil structuring. It is also worth noting that the given stretching might be influenced by the environment (pH, salts, solvent, etc). Finally, it is important to know that a mixture of different structures can be analyzed by spectroscopic analysis and specific deconvolution gives access to secondary structure content (in %) based on the above presented spectroscopic signatures.19b,23b,24

2.3 Physico-chemical properties influenced by secondary structuring

Tuning polypeptide secondary structures is a key step to modulate a wide scope of physico-chemical properties within peptide-based polymers because each secondary structure has its inherent properties. Outside biology, it is interesting to consider that the intrinsic properties of secondary structures could be a chemical leverage that significantly impacts material properties up to the macroscopic scale. At the molecular level, secondary structure formation, as compared to coil conformation, significantly impacts the overall macromolecular hydrodynamic volume.25 As the polymerization degree is critical to define the propensity to adopt secondary structure,22 hydrodynamic volume change needs to be taken into account for SEC analysis, as has been recently evidenced with poly(L-lysine).25a Tuning this hydrodynamic volume is critical for self-assembly purposes or surface grafting and these two specific topics are specifically detailed later in this review article (sections 4.1 and 4.3). Overall, secondary structures are more rigid (rods) than coil conformations and therefore, glass transition,26 intrinsic viscosity25b,27 and micro- and macrophase separation28 have been shown to be strongly influenced by polypeptide structuring. The different conformations adopted by polypeptide polymers also modulate their thermal stabilities,29 solubility behaviors30 and degradations.31

Generally, secondary structure formation is accompanied by a decrease in hydrophilicity and solubility in water. This has served for instance to prepare fluorescence responsive polypeptides with either aggregation induced30c or enhanced30b emissions. Recent progress in chemical design has nevertheless afforded water-soluble ionic poly(L-glutamate) derivatives30a,32 or glycopolypeptides33 with high helical stability. These scaffolds have attracted interest in biomedical applications as described in the last part of this review article (section 4.4). In macromolecular chemistry, secondary structures also bring important features. For instance, Hammond and coworkers have evidenced that post-polymerization modifications are favored specifically by a secondary structure conformation and, for instance, full grafting of α-helical poly(γ-propargyl-L-glutamate) polymers can be achieved with fairly high PEG molecular weights via Huisgen cycloaddition.34 More recently, impressive template effects have been evidenced with bottlebrush polypeptide topologies in which poly(γ-benzyl-L-glutamate) adopting α-helix secondary structures significantly enhanced its own ROP kinetics through cooperativity,35 a feature that was previously evidenced by Higashi and coworkers on gold surfaces.36 It is rather interesting to remember that, in marked contrast, β-sheet structuring generally disfavors the propagation of ROP process by sterically hindering the polymer extremity.37 Finally, an interesting structuring feature concerns specifically α-helices that exhibit large dipole moment.38 This implies that electron transfer through the α-helix axis might occur and that the orientation of α-helix macrodipoles induces tunable electrical conductivity (see Fig. 4)39 that can be at the origin of protein-like piezoelectric properties.40


image file: c7py01725a-f4.tif
Fig. 4 Surface grafting of α-helical-polypeptide brushes is an interesting way to implement tunable electrical conductivity by controlling the orientation of helix macrodipoles (reproduced with permission from ref. 39, copyright [2017], Springer).

3. Early use: secondary structures as tools for biology

Peptide-based polymers are constituted of one (or several) amino-acid(s) that are randomly repeated many times but are still able to adopt conformations found in natural proteins. This feature has attracted synthetic chemists since the beginning of the 20th century, when Leuchs discovered N-carboxyanhydride monomers.37 Early uses of these materials were indeed dedicated to natural protein study at a time when their chemical conformations were not fully understood.41 It is interesting to note that polypeptide polymers are still simplified models used to better understand how natural protein systems work and they have been used recently to better understand glycoprotein multivalency6c,42 or protein system cooperativity.35a

3.1 Secondary structure of natural proteins

Interestingly, polypeptide polymers have historically been the first synthetic models used to shed light on natural protein folding. Well-defined segments, locally organized in secondary structures, organize the complex macromolecular architecture of the 3D structure of a protein. These repetitive motifs have been recognized very early as an important element of protein structural characterization. By using polypeptide polymers, biochemists have successfully characterized individual properties of several secondary structures, for instance rationalizing the shape of the main circular dichroism profile corresponding to α-helix and β-sheet secondary structures.19b Historically, α-helix and β-sheet structuring were discovered by Pauling and Corey at the California Institute of Technology in 1951, two years before DNA structural determination by Crick and Watson.41 Several decades were then necessary to well establish the structural and physical properties of both these conformations: for instance poly(S-carboxyethyl-L-cysteine)43 as well as poly(S-carboxymethyl-L-cysteine)44 have been extensively used since the 70's as a water soluble model of β-sheet structuring and the last article published by Ikeda and coworkers on this topic was published twenty years later in 1989.45 Several polymeric models were also developed to study the α-helix conformation and two water soluble polypeptides have been particularly used for this purpose, poly(L-glutamic acid)46 and poly(L-lysine).47 Both these polymers adopt α-helical conformations, at acidic and basic pH. They have been widely used, since then, for materials science purposes including for instance the preparation of pH-responsive polymers for biomedical applications.6i It is rather interesting to note that these two polymers are also used nowadays in chemical education to teach in practical classes how proteins fold and how to study protein secondary structures.48

3.2 Smart structuring

Smart polypeptide polymers are ideal candidates to mimic adaptive biological systems such as those involving natural proteins, by undergoing structural or conformational changes in response to various external stimuli (temperature, pH, etc).49 Interestingly, synthetic polypeptides can easily undergo secondary structure transitions that can be easily implemented and tuned by tailoring amino acid side chains.15a This is in marked contrast to natural protein systems produced by genetic engineering and therefore this point represents an important advantage of synthetic peptide materials. For instance, helix-to-coil transitions can be controlled by pH changes for poly(L-glutamic acid)22,46 and poly(L-lysine).47 It has been established that their reversible structuring transition was attributable to the electric repulsion of their respective side chains.50 When charged, the repulsion of lateral chains breaks the hydrogen bonding network at the origin of the α-helix structure. Interestingly, tuning the pKa of the lateral chain function permits tuning the pH around which the structural transition occurs. For instance, poly(imidazoyl-L-lysine) has been found to exhibit an helix-to-coil transition around a neutral pH, in aqueous solutions, according to the pKa of the grafted imidazole moiety (Fig. 5).51 The use of several other lateral chain functions introduced either onto the NCA monomer or by post-polymerization grafting has enabled structuring switching upon response to various stimuli including solvent changes (poly(L-serine) or poly(L-cysteine) derivatives),52 temperature changes (poly(L-lysine) or poly(L-glutamate) derivatives),53 alkylation reactions (poly(L-methionine) derivatives),54 photo-responsiveness (OEGylated azobenzene side chains),55 redox changes (poly(L-cysteine) or poly(L-methionine) derivatives),54,56 coordination to transition metals (poly(L-glutamic acid))57 or even DNA binding (poly(L-glutamate) derivatives).58 So far, one major drawback of secondary structure transitions is the poor water solubility of the ordered conformations, whereas water is a critical solvent to develop biomedical applications.59 To enhance water solubility, a careful design of the polymer side chains allows producing water-soluble structured polypeptides, using for instance ionic polypeptide backbone,32 but the corresponding secondary structures are generally insensitive to structural switches.32,60 It is worth noting in this context that glycopolypeptide polymers that incorporate as few as 10 mol% of sugar units have allowed the preparation of smart pH-responsive systems for which the structured and the non-structured form have fairly good water solubility.33,61 Overall, polypeptide polymers that are able to mimic the adaptive character of biological systems can be useful in various materials science applications, for instance, to trigger the release of therapeutics, or to amplify signals in biosensors. In this context, the development of responsive structuring has received an increasing interest and the current status and future prospects in this research field are depicted in the following paragraphs.
image file: c7py01725a-f5.tif
Fig. 5 Smart poly(imidazoyl-L-lysine) exhibits reversible α-helix-to-coil transition that occurs around a neutral pH in aqueous solution.51

4. Current interests: secondary structure for materials science application

Synthetic polypeptides are of great interest because of their wide scope of potential applications including in biomedicine as tissue engineering scaffolds, drug delivery systems or even as macromolecular therapeutics.6f–i Tuning the secondary structure of polypeptides could be a key step in modulating various properties in advanced materials including specifically the field of nanomaterials, gels, membranes and surface coatings. These different materials are detailed below with specific emphasis on how the secondary structure influences materials properties.

4.1 Aqueous self-assembly

Nanomaterials are more and more studied in academic and industrial research as individual nanostructures (nanowires, nanotubes, nanoparticles, etc) that exhibit unprecedented physico-chemical properties (electric, magnetic and catalytic, among others).62 Self-assembly of polymeric materials is an efficient way to access nanomaterials and this approach has been a “hot topic” during the past two decades. For instance, like surfactants, amphiphilic block copolymers can self-assemble in aqueous solutions into specific nanostructures including polymeric micelles or polymersomes.63 As polypeptide polymers can adopt ordered conformations, hybrid block copolymers composed of a peptide segment (adopting secondary structure conformation) and a synthetic block, possess a rodcoil character.9a,64

Based on this design, amphiphilic polypeptide polymers have been extensively used to prepare a wide range of nanomaterials including polymeric vesicles whose structural properties were found to be close to those of natural viral capsids.59b,d,65 Using diblock copolymers, responsive structuring has been used to create polymeric micelles with pH-responsive diameter66 or with tunable disassembly upon pH changes.67 Generally, for diblock copolymer self-assembly, the rigidity brought by secondary structures offered opportunities to direct nanoscale structure formation, such as lamellar structures68 or amyloid-like fibrils69 and, for amphiphilic copolymers, secondary structured hydrophobic segments favoured polymersome formation.59b,c It is interesting to note that for polymersomes made of poly(L-lysine)-block-poly(butadiene), responsive structuring has been a tool to tune the size and/or the swelling of vesicles in response to external stimuli such as pH or temperature.70 Even more interesting, Deming and co-workers have shown recently that responsive structuring can be used to develop bioactive polymersomes from poly(L-methionine)-block-poly(L-leucine-stat-L-phenylalanine) whose disassembly, upon enzymatic reduction, wass triggered by secondary structure transition (Fig. 6).71 Such advanced design clearly shows how the manipulation of secondary structures can be key to develop innovative nanomaterials. Finally, it is good to mention that the self-assembly of more complex macromolecular structures, such as amphiphilic grafted copolymers, has shown that conformational changes impact the nanomorphologies; however, these systems still need to be rationalized.72


image file: c7py01725a-f6.tif
Fig. 6 Vesicles made of redox responsive polypeptide exhibited enzyme-triggered disassembly via a helix-to-coil transition mechanism (reproduced with permission from ref. 71, copyright [2017], ACS).

4.2 Rheology

It is well known that secondary structures of natural proteins can form hydrogels via two different mechanisms: “hydrophobically driven” coiling of α-helices (Fig. 7 arrow 1) and association of β-strand segments (Fig. 7 arrow 2).73 Polypeptide polymers bring an extra mechanism of hydrogel formation, which reproduces the features of β-strand association using long α-helix segments (Fig. 7 arrow 3).73,74 This third mechanism allows unprecedented tuning of macroscopic hydrogel properties simply and intuitively via the tuning of simple macromolecular parameters (secondary structure switching, polymerization degree, etc).
image file: c7py01725a-f7.tif
Fig. 7 Hydrogel formation with polypeptides occurs via three different mechanisms. The third one is specific to polypeptide polymers with α-helix conformation.

Overall, secondary structure can be used to drive hydrogel formation but can also tune hydrogel rheological properties. For instance, thermosensitivity was tuned by varying helicogenic hydrophobic residue content, for instance, leucine or alanine, to prepare smart hydrogels whose formation can be reversed by temperature changes.75 As another representative example, Hammond and coworkers used α-helix-to-coil structural transition in hydrogels made of poly(L-glutamate) derivatives to strongly influence hydrogel stiffness without changing swelling or permeability properties.76 Similarly, poly(L-alanine) blocks with temperature triggered helix-to-beta sheet transitions were used to prepare advanced hydrogels whose transition temperatures “did not follow the simple rule that a more hydrophobic polymer has a lower transition temperature”.77 It is important to note that polypeptide polymers can also reproduce the natural protein gelation mechanism. Hydrogel formation through β-strand association has, for instance, been achieved with poly(L-tyrosine) blocks to prepare biocompatible PEO-based hydrogels.78 When β-sheet structured polypeptide polymers are used to prepare hydrogels, an increase of β-sheet structure content has been correlated to a decrease in sol-to-gel transition temperatures.79 Tuning directly this β-sheet content via smart structuring, i.e. using segments with coil-to-β-sheet transition permitted preparing smart hydrogels with reverse thermal gelation and degradation properties.80

Rheological properties are also deeply influenced by secondary structure formation when polypeptide polymers are used for organogel formation purposes. In THF, PEO–poly(L-Z-lysine) conjugates have shown a tendency to organogelation, a tendency that increases with the following polypeptide conformation order: coil < α-helix < β-sheet.81 In toluene, organogels incorporating poly(γ-benzyl-L-glutamate) blocks have shown that secondary structure content was correlated to a significant increase in gelation temperature82 and gel strength.82,83 In various other solvents, simple diblock copolymers based on hydrophilic PEO and hydrophobic racemic homopolypeptides were able to form gels whose gelation was driven by β-sheet supramolecular assembly.84 Interestingly, sonication responsive organogels were prepared in DMF by Wooley and co-workers with polypeptide copolymers composed of γ-benzyl-L-glutamate and glycine units: stiffness of these organogels was easily tuned by simply varying the ratios of both units and therefore of α-helix to β-sheet structures.85 An interesting aspect of how secondary structure influences organogel rheological properties has been presented by Nakanishi and co-workers who developed cross-linked hydrogels, with poly(L-glutamate) derivatives, exhibiting anisotropic swelling and shrinking behavior (Fig. 8).86 This impressive property was attributed to solvent-triggered helix-to-coil transition but this result paves the way to a wide range of smart gel systems. It is indeed interesting to note that cross-linked gels made of α-helical polypeptides have been used as a tool for structural biology to create an enantiodiscriminating alignment medium for NMR experiments.87


image file: c7py01725a-f8.tif
Fig. 8 Crosslinked polypeptide hydrogels could exhibit anisotropic swelling and shrinking behaviors that were controlled by helix-to-coil transition (reproduced with permission from ref. 86, copyright [2017], ACS).

4.3 Surface coating and membrane sciences

The formation of surface-grafted polypeptide films and polymeric membranes via NCA ring-opening polymerization affords the preparation of responsive materials with excellent biocompatibility properties and advanced chemical functionalities.88 Polymeric films hold many promises for applications in chemical sensing, optical devices, and enantioselective separation. Ultrathin polypeptide films can be formed by various versatile techniques including approaches involving grafting-to and from a substrate surface (also commonly referred as polypeptide brushes).88b,89 In most cases, covalent anchoring is preferred as it provides greater film stability under various conditions. For instance, the use of silanization to create a 3-aminopropyl-triethoxysilane (APS) monolayer represents an easy access to a poly(L-lysine)20a brush monolayer with film thicknesses ranging from a few nm to more than 100 nm. It is interesting to note that changing the secondary structure of the poly(L-lysine) segment significantly modifies the thickness of the layer.20a It has also been evidenced that dielectric property changes can be observed that are attributable to the nature of the secondary structure,20a or, with α-helices, to the supramolecular organization of the helices (see section 2.3).39 Interesting smart polypeptide brush monolayers have also been designed from poly(L-aspartate) derivatives with rare opposite α-helicity, in the vertical direction, triggered by solvent changes (Fig. 9).90 The same monolayers also exhibited temperature triggered irreversible helix-to-beta sheet transitions (Fig. 9).90
image file: c7py01725a-f9.tif
Fig. 9 Secondary structure formation of smart poly(aspartate) derivative brush monolayers could exhibit unique helix-to-helix transition upon solvent changes (reproduced with permission from ref. 90, copyright [2017], ACS).

Self-assembled monolayers (SAM) on gold substrates represent an efficient grafting option to generate densely bound polypeptide brush monolayers, uniform at the angstrom level.36 Such molecular precision was obtained by taking advantage of (1) the α-helix secondary structures of poly(γ-benzyl-L-glutamate) and (2) the resulting α-helix macrodipole interactions that create an unprecedented cooperative effect. This cooperative effect was also recently observed when grafting-from approaches were used from the polymeric backbone.35

A non-covalent polypeptide coating was also achieved by the physisorption of polyelectrolyte multilayers constructed from polypeptides and made by successive electrostatic interactions (layer by layer or Lbl). They have presented significant propensity to adopt secondary structure conformations that played an important role in governing both the adsorption process and the final film properties.91 Secondary structures within Lbl films have made polypeptide films particularly stable to various other external stimuli including temperature and pH.92 Smart structuring has also been implemented in Lbl coatings with simple polypeptides: poly(L-lysine)–poly(L-glutamic) films assembled at neutral pH had a predominantly β-sheet character but structural transition to α-helix by decreasing the pH resulted in a more open and loose film morphology.93 It is rather interesting that this film process was able to be used for optically active separation via the use of polypeptide secondary structuring.94

Polypeptide grafting onto permeation membranes has also led to enantioselective separation processes. For instance, porous poly(tetrafluoroethylene) membranes grafted with poly(L-glutamic acid) exhibited a smart permeation rate upon pH changes due to helix-to-coil transitions.95 A gold-coated porous membrane, also made of poly(L-glutamic acid), presented a nanometer-sized signal-responsive gate that was visualized in water by atomic force microscopy;96 this gating was regulated by helix-to-coil transitions of the monolayer.96 Interestingly, chemical modification of PVDF ultrafiltration membranes with poly(L-glutamate) derivatives even afforded enantioselective permeation that was directly correlated to α-helix content.97 Following the same idea, Kinoshita and co-workers have prepared functional molecular membranes incorporating poly(L-leucine) segments that further exhibited enantiospecific permeation for L amino-acids, a result that was attributed to β-sheet copolymer structuring.98

Finally, it is important to note that polypeptide surface grafting has been intended onto various nanoparticle surfaces.88a For instance, helical poly(L-glutamic) acid has been grafted onto inorganic99 or organic100 core nanoparticles to control nanoparticle dispersion via the secondary structure of the shell. It is interesting to mention here that the β-sheet structure was also used by Wooley and coworkers as an anchoring segment to stabilize carbon nanotubes, allowing an efficient noncovalent incorporation of these nanoparticles in an organogel matrix without disrupting their Raman band emission spectrum.101

4.4 Bioactive macromolecular materials

New advances in drug delivery, tissue engineering, and nanomedicine demonstrate that the potential of polymer chemistry to improve health care is stronger than ever.102 The design of sophisticated functional polymers and precise/directed self-assembly has considerably improved during the past few years.103 Polypeptide polymers have been available for many decades but have mainly been used as structural materials.6b Recent progress in the design, regulation and applications of synthetic polypeptide polymers paves the way to obtain functional mimics of natural proteins to further develop a bioactive platform of biomedical interest in materials science.6c,f,104

Typically, cationic polypeptides such as poly(L-lysine) or poly(L-arginine) are unable to adopt α-helical conformations at physiological pH because of charge disruption with the side chains.50 The helical structure of cationic polypeptides can indeed be stabilized by increasing the distance between the charged groups of the side chains and the backbone of the polypeptides.32 By following this general strategy, Cheng and coworkers have generated numerous interesting polypeptides104a including a new class of polypeptide materials that are sufficiently large and positively charged to bind and condense DNA.105 Such macromolecular design has been further optimized to give birth to α-helical cationic polypeptides with non-viral gene delivery capacity that outperformed commercial transfection reagents such as Lipofectamine® 2000 or poly(ethylene imine) by up to 2 orders of magnitude.105b

Cationic polypeptide polymers have also the capacity to mimic cell-penetrating peptides (CPP). For instance, poly(L-arginine) blocks have been used efficiently to prepare amphiphilic copolymers whose nanoassemblies in aqueous media were able to penetrate mammalian cells via an endosome escape mechanism.106 As mentioned above, poly(L-arginine) blocks do not adopt secondary structure conformation. Elongating the polypeptide backbone length and increasing side chain hydrophobicity have permitted preparing α-helical poly(L-arginine) analogues that showed superior cell membrane permeability up to two orders of magnitude higher than that of natural CPP (HIV-TAT peptide).107 Following this chemical design, other positively charged secondary structures have also been designed for cell internalization purposes.104a Poly(L-glutamate) derivatives, for instance bearing quaternary phosphonium side-chains, exhibited stable, α-helical conformations and have presented very good cell penetrating properties.108 It is good to note, in this case, that the polypeptide structuring was stable in aqueous solutions against pH and ionic strength changes.108 Poly(L-glutamate) derivatives were also used recently to combine secondary structure enhanced cell penetration to intracellular multivalent targeting.109 Finally, it is worth mentioning that Cheng and coworkers also developed non-helical cationic poly(L-serine) derivatives that have shown cell-penetrating properties correlated to their β-sheet structuring ability.110

On another front, interest in building polymeric analogues of antimicrobial peptides (AMP) has generated several polypeptide polymer analogues having membrane interactive properties.111 Even if the antimicrobial activities of such polymeric analogues seem to not necessarily require secondary structuring,112 some impressive results implementing α-helical scaffolds need to be presented. Pioneering work in this field involved random copolypeptides made of L-lysine residues mixed with one of the five following hydrophobic residues, L-leucine, L-phenylalanine, L-isoleucine (amino acids favouring α-helix conformation), L-valine and L-alanine (amino acids favouring β-sheet conformation) showed that chain conformation played a key role in determining selective membrane disrupting ability (liposomes).113 Much later, an impressive input in the field of antimicrobial polypeptides has been published by Cheng and coworkers who presented a new class of radially amphiphilic (RA) polypeptides, mimicking amphipathic AMP (Fig. 10).114 These RA polypeptides adopted stable α-helical conformation with a hydrophobic interior and a charged exterior shell around the helical surface (Fig. 10B), affording efficient antimicrobial activities that were closely associated with their structuring ability.114 Further design of this scaffold implied the preparation of copolymers incorporating phosphorylated tyrosine to generate bacterial-responsive polypeptides having strong membrane disruptive capabilities.115


image file: c7py01725a-f10.tif
Fig. 10 Radially amphiphilic (RA) polypeptides can mimic amphipathic AMP properties (A: amphipathic AMP; B: RA polypeptide; C: RA polypeptide chemical structure exhibiting antibacterial activity). Reproduced with permission from ref. 114, copyright 2017, PNAS.

Finally, glycopolypeptide polymers have also been good candidates to prepare bioactive polymeric analogues of natural glycoproteins6c although the secondary structure modestly influenced sugar recognition by lectins.116 Nevertheless, when charged, α-helical glycopolypeptides were also capable of cell permeation.117

5. Conclusions and perspectives

Synthetic polypeptides can be seen as simplified protein analogues: they are ideal materials to design protein mimics, able to reproduce some properties of natural proteins and to create innovative polymeric structures for materials science applications. In particular, polypeptides can adopt natural protein secondary structures such as α-helix or β-sheets and this rare feature is, in polymer chemistry, at the origin of fascinating properties. The use of polymer chemistry to build simplified models adopting secondary structure conformations can be summarized as a “bio-simplification approach” that aims at (1) better understanding physico-chemical properties of natural protein secondary structures; (2) extracting fundamental principles from these properties and (3) making these principles useful for materials science applications. From a materials perspective, the excellent control in polypeptide polymer synthesis available nowadays brings new tools for advanced chemical design. This includes tuning polypeptide secondary structures to prepare advanced nanoparticles, gels, membranes and surface coatings. Recent studies indicate that polypeptide polymers adopting secondary structure conformation can also be at the origin of unprecedented classes of materials such as self-assembled polymeric supramolecular frameworks,118 unique nanoscale coacervate-core micelles119 or even porous spherical shells made of amino-acids.120 Overall, the secondary structure of synthetic polypeptide polymers has the potential to significantly facilitate the development of the next generation of peptidomimetic materials that will find uses at the edge between chemistry and biology.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial support from the REGION Midi-Pyrénées SR 15050412 is gratefully acknowledged. The author thanks E. Piedra-Arroni and G. Pratviel for helpful discussions.

Notes and references

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