Synthetic Glycopolypeptides as Biomimetic Analogues of Natural

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Synthetic glycopolypeptides as biomimetic analogues of natural glycoproteins Colin bonduelle, and Sebastien Lecommandoux Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm4008088 • Publication Date (Web): 22 Jul 2013 Downloaded from http://pubs.acs.org on July 29, 2013

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Synthetic glycopolypeptides as biomimetic analogues of natural glycoproteins Colin Bonduelle a Sébastien Lecommandoux*a a

Université de Bordeaux/IPB, ENSCBP, 16 avenue Pey Berland, 33607 Pessac Cedex, France.

E-mail: [email protected] KEYWORDS Glycoprotein, glycopolypeptide, glycopolymer, amino acid N-carboxyanhydride, self-assembly, stimuli-responsive. Glycoproteins are naturally produced by protein glycosylation and are involved in a wide range of cellular functions. This review aims at summarizing the preparation of well-defined synthetic glycoproteins by using chemical routes as well as to highlight the preparation of ideal polymeric analogues of natural glycoproteins: glycopolypeptides. These macromolecules are simplified models of glycoproteins and are designed with the purpose of both mimicking the properties of natural glycoproteins as well as bringing innovative polymeric structures for materials science applications.

Introduction Carbohydrates represent the first class of bio(macro)molecules in biology and their huge structural diversity is still underestimated.1 Many polysaccharides such as cellulose or chitin are

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ubiquitous in nature forming for example the scaffolds of vegetal and animal cells due to their extraordinary mechanical properties. The 3D structural information encoded in polysaccharides is colossal and after genomics and proteomics, glycomics represents the third challenge of biology in which there is no biological template to control polysaccharide biosynthesis.2 Glycan biopolymers are linear or branched, and there exists a variety of linkages and linkage stereochemistry that create an unprecedented diversity.3 They are often associated with proteins and therefore form glycoproteins, an important class of biomolecules which holds the key to understand fundamental biological processes including cell membrane interactions with bacteria and viruses as well as cell–cell communication mediated by glycoproteins cell receptors (cf figure 1).

Figure 1. Role of glycoprotein cell receptors for the interaction and recognition of cells, bacteria, viruses, toxins, hormones (helical yellow structure represents the peptide and the red structure, the glycan).


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In vivo, glycosylation, i.e. attachment of diverse oligosaccharides (glycan) to proteins, is probably the most complex post-transcriptional process.4 The protein glycosylation depends on a variety of enzymes, which compete to produce adequate mixture of oligosaccharides that biochemists have named “glycoforms”. These “glycoforms” are attached to the peptidic backbone in a controlled fashion creating glycoproteins eventually presenting various complex glycan structures.3,

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It has indeed been estimated that almost half of the human proteins are

glycosylated and it is now well recognized that glycoproteins have potential utility for the development of therapeutics, diagnostics and vaccines.5,

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Nevertheless, and despite the

importance of glycoproteins in the metabolism, their structure and function remain not fully understood, mainly because of the difficulties to obtain them in a homogeneous form.7 While significant advances in genetic engineering have been made to prepare complex proteins, homogeneous expression of glycoproteins is still a challenge because pure “glycoforms” purification is difficult if not impossible to achieve. In that respect, the synthetic preparation of glycopeptides/glycoproteins holds enormous promise and has been extensively studied for several years.7-9 Herein, we aim at describing the different methodologies proposed in the literature to access fully synthetic glycoproteins and emphasizing the emergence of a new class of synthetic analogues inspired by natural glycoproteins which are obtained through smart synthetic polymer design (see figure 2). These biomimetic macromolecules are glycopolymers with pendant carbohydrates on a polypeptide backbone, therefore named glycopolypeptides.10 The preparation of these biomimetic materials involves recent progresses made with N-carboxyanhydride (NCA) controlled ring-opening polymerization (ROP)11 as well as progresses made with efficient coupling reaction (such as the “click chemistry”).12-15 In a complementary way to synthetic


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glycoproteins, the purpose of glycopolypeptides is to mimic the chemical structure of glycoproteins 1) to better understand, by using synthetic models, physicochemical or biological properties of natural glycoproteins; 2) to better extract the fundamental principles of their structure–function relationships; 3) to make these principles available as a useful concept in materials science and engineering to develop innovative applications.

Figure 2. Synthetic glycopolypeptides as biomimetic analogues of natural glycoproteins

1.

Chemical synthesis of glycoproteins First, it is important to note that methods based on molecular biology or genetic

engineering have been proposed to overcome "glycoforms" mixture production.16 Indeed, it has been shown that only the most sophisticated systems, i.e. eukaryotes, are able to handle glycosylation of proteins.17 The inherent complexity of eukaryotic systems still creates significant limitations for homogeneous glycoproteins preparation, including the fact that different kind of glycans can be obtain depending on the cell system that has been chosen for the


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biosynthesis. Readers are encouraged to consult the review written by Wildt and Gerngross for more information.18 Chemical synthesis represents an alternative to biology to produce well-defined glycoproteins.1, 2 In this respect, chemists have developed several synthetic methodologies which are summarized in the following section. These methodologies have generated two different perspectives. First, and following the challenge of total synthesis, synthetic glycoproteins have been developed to perfectly reproduce the chemical structure and/or functions of natural glycoproteins.19 The preparation of synthetic erythropoietin20 (which has been recently achieved21, 22) is a good example of this prospect. Interestingly, this original purpose of total synthesis has generated new tools in chemistry that can overpass the initial task. Researchers are now able to use unnatural amino-acids and/or unnatural linkages to prepare a new class of material called neoglycoproteins.23 In fact, synthesis of both glycoproteins and neoglycoproteins requires the use of synthetic methods from both carbohydrate and peptides chemistry. The main issue is often to combine these methods to form the crucial glycan-amino acid bond. In Nature, it has been revealed that glycan-amino acid linkages are conserved: glycan moieties are generally attached to proteins via an N-glycosidic bond or an O-glycosidic bond. By using synthetic approaches, it is possible to retrieve natural linkages as well as unnatural ones, these last include among others thioether linkages, oxime linkages, 1,4-disubstituted 1,2,3 triazole linkages and allyl linkages. Methodologies to combine a sugar to an amino acid are already well reviewed elsewhere9, 23 and readers are encouraged to consult these references for more details.

1.1. Glycoproteins through solid-phase peptide synthesis


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The peptidic backbone formation is generally the first step of a synthetic preparation of glycoproteins. When obtained from amino acids, its preparation involves the standard solidphase peptide synthesis (SPPS) technique which is the best recognized method to prepare well defined sequences of amino acids.9, 24 In practice, two different strategies can be envisaged from SPPS: a stepwise/linear strategy and a convergent strategy. The linear approach involves glycosylated amino acid derivatives that are assembled with other amino acids in a stepwise fashion (scheme 1): this approach has been successfully used to prepare complex glycoconjugates but implies the judicious choice of protecting groups.25 On the other hand, the convergent strategy that involves the peptide preparation by SPPS as first step, permits to introduce the glycan in a second step through chemical coupling.26 In all cases, SPPS strongly limits the size of the peptide sequence to a maximum recognized around 50 residues and the final yield obtained by using this methodology is rapidly decreasing with the length of the peptide. Nevertheless, it is important to note that SPPS techniques have been sufficiently effective to perfectly reproduce natural glycopeptides such as HIV-1 V3 domain27 or to prepare neoglycopeptides such as the 17-amino acid fragment of P-selectin glycoprotein ligand-1 (PSGL-1)28 or various antifreeze glycoprotein analogs.29, 30

Scheme 1. Glycopeptide synthesis via SPPS using a linear strategy


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1.2. Glycoproteins through native chemical ligation and other efficient ligations The inherent size and yield limitations of SPPS have generated an extensive development of chemical ligation methods, which may serve to assemble complex biomolecules in a convergent manner. Such convergent strategy implies the assembly of individual peptidyl substrates.26 These subunits would ideally be merged, in an iterative fashion, to ultimately afford the desired biomacromolecule. Among the proposed methodologies to create a convergence, the first successful universal strategy relies on the native chemical ligation (NCL),31 also referred as cysteine-based native chemical ligation (cf scheme 2). This reaction involves the chemoselective condensation between a peptide bearing a cysteine at the N-terminal extremity and another peptide bearing a thioester moiety at the C-terminal extremity. The ligation mechanism occurs in two steps, a thioesterification, which is followed by a S-N acyl transfer. This second step is irreversible and thus is the driving force of the selective coupling. NCL has seen significant improvements during the last 20 years and has been used to fully synthesize several glycoproteins.32-35 This chemical ligation has already been the subject of several reviews that can be consulted for more details.9, 26, 35

Scheme 2. Cysteine-based native chemical ligation


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Although the native chemical ligation paves the way to glycoproteins synthesis, the coupling of partners obtained by recombination has also been envisaged for larger targets. The method of ligation basically involves the same NCL extremities and the two-step mechanism already described. The difference comes from the peptide/protein origin of one of the partners, which can be obtained by molecular engineering using prokaryotic systems.36 NCL in that case is called expressed protein ligation (EPL). The recombination used for EPL implies that the introduction of the glycan takes place through a non-expressed partner.37,38 Significant examples of glycoproteins synthesized using the EPL approach are the 124-amino acid enzyme RNase C39 or the glycoprotein GlyCAM-1.40 NCL and the corresponding semi-synthetic variant EPL appear the best methods so far to prepare glycoproteins by chemical synthesis. Nevertheless these two approaches require that one of the peptide partners present a N-terminal cysteine residue, an amino-acid rare in human protein sequences (estimation 1.5 to 2%). Whereas it is possible to introduce artificially a cysteine residue for the ligation, alternative strategies have been developed to overtake this prerequisite. For example, thiol-based molecules such as 4,5,6-trimethoxy-2-mercaptobenzyl have been used to substitute the cysteine.41 As the side chain of a cysteine residue, the thiol of this “auxiliary” is involved in the chemical ligation and should be easily removed and replaced by a peptidic bond. Other auxiliary-based ligations have been developed to avoid the endcysteine residue: this includes, among others, sugar-assisted ligation,42 native chemical ligation through desulfurisation43 or thiol free ligations.44 Readers are encouraged to consult previous reviews on chemical ligation strategies to obtain more details.9, 26


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1.3. Glycoproteins through enzymatic synthesis By using only chemical routes, complex oligosaccharides introduction onto the protein backbone is still a challenge. In fact, the steric hindrance of amino acids possessing large glycan drastically reduces the efficiency of the peptide coupling reactions during SPPS and, subsequently, limits glycoproteins preparation through peptides chemical ligation. While it has been shown that it is possible to attach efficiently such complex oligosaccharides to the peptide backbone through a non-natural linkage,9 to attach a glycan through a native linkage remains a challenge. A promising methodology consists in taking a convergent approach where a single sugar residue is first introduced onto the peptidic backbone during SPPS.45 Following the peptide synthesis, this sugar serves, in a following step, as a handle for enzymatic reaction using glycosyltransferases.45 For that purpose, endo-β-N-acetylglucosaminidases that catalyze transglycosylation reactions have been used to transfer of an oligosaccharide from a glucosamine residue to another sugar residue. Consequently, if the acceptor sugar is bound to a peptide, the result is a glycopeptide bearing a complex glycan. As an impressive example, this strategy has been applied to prepare an homogeneous glycoform of RNase B.10

1.4. Towards simplified analogues of natural glycoproteins Finally, preparation of synthetic glycoproteins has recently seen significant progresses which are supported by the discovery of several efficient chemical ligations.26 These new methodologies have allowed retrieving natural linkages of glycoproteins and overcoming some


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limitations given by SPPS. Consequently, chemical synthesis has been successfully applied to prepare several synthetic glycoproteins and to reproduce some biological post-translational modification at the origin of the glycosylation of proteins.7, 8 Despite these recent progresses, the preparation of new natural glycoproteins remains a challenge because the majority of these macromolecular structures are highly complex. To tackle this challenge, other complementary approaches are proposed by materials scientists and rely on biomimicry. Biomimetic approaches include synthesizing simplified models of biomolecules capable of mimicking their natural properties. The second part of this review aims at highlighting this complementary approach which has now been successfully used for a decade to prepare simplified analogues of glycoproteins.

2.

Glycopolypeptides as polymeric analogues of natural glycoproteins New advances in drug delivery, tissue engineering, and nanomedicine are demonstrating

that the potential of polymer chemistry to improve health care is stronger than ever.46 The design of sophisticated functional polymers and precise/directed self-assembly has considerably improved during the last years.47-50 In this context, glycosylation of synthetic polymers yielded advanced materials with the ability to produce multivalent interactions with lectins or other biological targets (cells, viruses etc...).51-55 In principle, glycopolymers are produced using two different strategies: the polymerization of carbohydrate-containing monomers or the chemical ligation of the glycan with a polymeric backbone.51,

56,

57

Among glycopolymers,

glycopolypeptides (glycopolymers with pendant carbohydrates on a polypeptide backbone) hold


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tremendous promise because they are an ideal class of biomimetic materials to reproduce physicochemical properties of glycoproteins. In addition, polypeptides have been available for many decades but have mainly been used as structural materials.58 Synthesis of polypeptides has however significantly progressed during the last fifteen years11, 59 and it is now possible to prepare well defined block and hybrid copolypeptides with potential biomedical applications.60, 61 In this context, the development of glycosylated polypeptides is becoming an eminent topic because the preparation of synthetic glycoproteins remains difficult and not cost-effective. As simplified analogues of glycoproteins, glycopolypeptides are easier to prepare and are promising candidates for material applications improving public health. Compared to others glycopolymers, glycopolypeptides conserve the ability to fold into well-defined secondary structures, which is a rare property in polymer science. Moreover, the chemical structure of the overall biomacromolecule (and in particular of the polymer backbone) is similar to the chemical structure of glycoproteins: compared to synthetic glycopolymers coming from radical polymerization,51 glycopolypeptides are biodegradable and potentially more relevant for medical application.

Scheme 3. Polymerization of α-amino acid-N-carboxyanhydrides (NCA) by ring-opening polymerization (ROP)


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The most economical and expedient process to prepare polypeptide chains is the ring-opening polymerization (ROP) of α-amino acid-N-carboxyanhydrides (NCAs, scheme 3).11,

58, 62

This

polymerization involves the simplest reagents, and permits to prepare in both, good yield and large quantity, high molecular weight polymers without racemization. Historically, the limitations of NCA polymerization have been 1) the purification of monomers which has generally to be achieved by crystallization; 2) the presence of side reactions (chain termination and chain transfer) that significantly reduces the control over molecular weight. These two limitations have now been fully overcome. First, several catalysts/strategies have been developed in recent years to avoid side reactions during the polymerization process thus allowing a precise control of the molecular weight and the emergence of living NCA ring-opening polymerization;59,

63, 64

second, the purification of NCA monomers can now be efficiently

achieved through other methods, which permits to purify non crystalline monomers.65 Logically, synthetic glycopolypeptides have been prepared by ring-opening polymerization of sugar substituted NCA monomers (cf figure 3, pathway 1) or by post-polymerization modification which permits the addition of saccharide moieties to the side chains/end extremities of an existing polypeptide scaffold using efficient chemical ligations (cf figure 3, pathway 2 and 3).


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Figure 3. Preparation of synthetic glycopolypeptides. Pathway 1) Ring opening polymerization of glycosylated NCA; Pathway 2) Lateral chains glycosylation of synthetic polypeptides; Pathway 3) Glycosylation at the end extremity of polypeptides. From ref 99. Copyright

2013,

reproduced

by permission

of

the

Royal

Society

of

Chemistry.

http://pubs.rsc.org/en/content/articlelanding/2013/FD/C3FD00082F

2.1. Glycopolypeptides preparation via ring-opening polymerization (ROP) of glycosylated monomers


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Scheme 4. Glycosylated NCA polymerization via living ROP catalysed by (PMe3)4Co66

Pioneers in using glycosylated NCA ring-opening polymerization, Okada and coworkers have been the first to publish the preparation of glycopeptides from O-linked glyco-serine : their process was controlled, but the monomer synthesis was tedious and only polymers with a low degree of polymerization were obtained.67 More recently, living polymerization of glycosylatedL-lysine NCA (glyco-K NCA) have been performed by Deming and coworkers to prepare welldefined glycopolypeptides having high molecular weight (see scheme 4).66 Synthesis of the monomer was achieved in 5 steps (overall yield 25%) by using protected β-D-glucose, β-Dgalactose or β-D-mannose and the polymerization was achieved with (PMe3)4Co. This same catalyst was also used for the preparation of glycopolypeptides from glycosylated-L-cysteine NCA (glyco-C NCA) prepared in 4 steps (overall yield 60%) from alkene-terminated C-linked glycosides thanks to thiol-ene coupling.68 It is interesting to highlight that glycopolypeptides prepared from these glyco-C NCA presented thioether linkages, which were subsequently turned in sulfone linkages, a parameter that drastically impacted the secondary structure of the final glycopolypeptide (see section 3 of this review). Other examples of glycosylated-NCA ringopening polymerization were reported (from O-glycosylated lysine derivatives) by Wenz and coworker69 and Gupta and coworkers.70-72 It is to note that these last authors efficiently prepared


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glycopolypeptides through regular amine initiated polymerization by using for example per-Obenzoylated-D-glyco-L-lysine carbamate NCA to yield high molecular weights polymers (up to 70000 g/mol) but the polymerizations did not present a living character. In their case, the synthesis of the monomers was, in the best case, achievable in 3 steps (overall yield of 70%) from stable glycosyl donor73 : propargyl 1,2-orthoester of glucose/mannose or propargyl 1,2orthoester of galactose/lactose. Glycopolypeptides indeed prepared were able to specifically recognize Concanavalin A lectins as verified by many assays including ITC, whatever the secondary structure of the polypeptide backbone was.71

Scheme 5. Synthesis of glycopolypeptides via anionic ring-opening polymerization74

It has to be mentioned here that glycopolypeptides obtained by ring-opening polymerization were also produced via anionic polymerization of β-lactam carbohydrate containing monomers (see scheme 5).74 This mild and high-yielding polymerization method has provided β-glycopolypeptides of controlled molecular weight and narrow dispersities. These biomimetic materials formed helical structures in solution and were also able to specifically recognize lectin proteins.


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2.2. Glycopolypeptides preparation via post-polymerization modification of polypeptides To avoid the inherent difficulties of glycosylated-NCA synthesis, glycopolypeptides preparation was also performed by using methodologies involving a post polymerization modification: the chemical ligation of the glycan to an already formed polypeptide backbone. This alternative pathway to glycopolypeptide involves first the synthesis of the polymeric backbone and is followed by the glycan grafting. This grafting has indeed been envisaged onto polypeptide side chains with small saccharides units (mono-disaccharides, figure 2 pathway 2) or at the end-extremity of the polypeptide with oligosaccharides extracted from the biomass (figure 2, pathway 3). Whereas the polypeptide scaffold is often synthesized from NCA ring-opening polymerization, integration of chemical tools with biological technologies have also been envisaged to design monodisperse synthetic glycopolypeptides having controlled saccharide placement.

2.2.1 Side chains post-polymerization glycosylation Logically, natural amino-acids NCA have been first used to achieve post polymerization glycosylation in order to introduce the saccharides units along the polypeptide backbone. Ringopening polymerization of ε-benzyloxycarbonyl-L-lysine N-carboxyanhydride is a representative example of this approach : after removal of the side chain protective group, free amines have been shown to react with glycosyl isothiocyanate75 or glycosyl lactones76 but the substitution degree was found significantly limited in both cases by the steric hindrance (Scheme 6a and 6b).75 Glycosyl amine additions onto poly(glutamic acid) have alternatively been envisaged by using a regular peptidic coupling promoted by HOBt77-79 or by a less commonly used DMT-MM


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coupling agent (Scheme 6c).80 Higher conversions were obtained through these methodologies (up to 78% for HOBt and up to 89% for DMT-MM) that were strongly influenced by the nature of the glycosyl part or by the stoichiometry. To date, only one example involving the ROP of natural amino-acid NCA has shown quantitative glycan introduction along a polypeptide backbone. Deming and co-workers recently proposed to alkylate poly(L-methionine) by using iodoethyl glycosides or alkyl triflate glycoside (Scheme 6d).81 This chemical ligation took advantage of the thioether groups introduced by the methionine residues and by using this approach, carbohydrates have been introduced onto the side chains with conversion up to 97%.

Scheme 6. Glycosylation onto polypeptide backbones made of natural amino-acids NCA.75-81 On the other hand, protein engineering methods have also been employed to produce artificial protein-based polymers, with controlled conformational properties and varied placement of


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amino acid residues, for materials applications82-84. Among the abundant literature covering the field, Kiick and coworkers have shown precise and specific glycosylation of glutamic acid residues of such artificial protein by using peptidic coupling (HBTU, DIEA) onto divers helical scaffold

including

the

sequence

(AAAQAAQAQAAAEAAAQAAQAQ)6.85

This

multidisciplinary strategy, which involves a biological approach, allowed modulating the saccharide spacing and thus to fully glycosylate all the glutamic acid residues. This strategy also permitted to produce perfect monodisperse glycopolypeptide with a precision that NCA ringopening polymerization cannot afford : these well-defined synthetic analogues were further used to probe the influence of the sugar branching to target divers biological receptors including toxins86 or specific lectins.87

Scheme

7.

Poly(γ-benzyl-L-glutamate)–block-poly(DL-glycosylated

propargylglycine)

preparation via a combination of sequential ROP and click chemistry88


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Besides, and following the emergence of the “click chemistry”, unnatural aminoacids NCA were designed to favor an efficient coupling after polymerization. First, the copper-catalyzed azide-alkyne click chemistry was envisaged through the use of propargylglycine NCA89, γpropargyl-L-glutamate NCA,90 γ-3-chloropropyl-L-glutamate NCA91 or azido containing NCA.92 Subsequent cycloaddition was achieved by using CuBr,91, 92 CuSO490 or Cu(PPh3)Br.89 All these systems allowed the quantitative introduction of carbohydrates functionalized with an azide89-91 or an alkyne moiety.92 It is to note here that Heise and coworkers showed that the control over DL-propargylglycine ring-opening polymerization permitted to achieve the polymerization in a sequential way89 thus allowing access to amphiphilic glycopolypeptides having linear88 or branched structures (see scheme 7).93

Scheme 8. Glycopolypeptide preparation from poly(DL-allylglycine)94

Besides clickable polypeptides based on Huisgen cycloaddition, thiol-ene clickable polypeptides were also developed by Schlaad and coworkers (see scheme 8).94 DL-allylglycine NCA monomers were used for that purpose and polymerized by regular amine initiated ringopening polymerization to afford poly(DL-allylglycine). Radical thiol-ene additions were further performed with 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose and quantitatively achieved for


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poly(ethylene oxide)-block-poly(DL-allylglycine).94 This strategy was even extended very recently to thiol-yne additions by using copolypeptides made of γ-benzyl-L-glutamate NCA and DL-propargylglycine NCA.95

2.2.2 Glycosylation at the end extremity of a polypeptide backbone In an alternative way, one can also take advantage of the initiation step of a regular amine initiated NCA polymerization, to introduce a reactive moiety (alkyne, azide…) at the endextremity of a polypeptide, this end-group functionality being subsequently available for a coupling step involving an efficient reaction58. This functionalization strategy was successfully used to introduce different oligosaccharides by using copper catalyzed Huisgen cycloadditions with an azido end-functionalized poly(γ-benzyl-L-glutamate).96 After coupling, hybrid glycopolypeptides incorporating sophisticated glycan were indeed obtained. The first example of such ligation has involved dextran of a molecular weight of 6600 g/mol already endfunctionalized with an alkyne moiety through reductive amination.97 Interestingly, the resulting copolymers were able to self-assemble in water into glycosylated polymersomes with structures similar to viral capsids.96 The originality of this strategy, which consisted in using a biopolymer moiety that acts as a bioreceptor and as hydrophilic stabilizing agent was also used to prepare linear glycopolypeptides incorporating a biologically active hyaluronan block of a molecular weight of 4000 g/mol,98 or tree-like glycopolypeptides having intriguing self-assembly properties (see figure 4).93, 99


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Figure 4. Oligosaccharide based glycopolypeptides prepared by glycosylation at the endextremity of synthetic polypeptides.93, 96-99 Graphic adapted from ref 99. Copyright 2013, reproduced by permission of the Royal Society of Chemistry. http://pubs.rsc.org/en/content/articlelanding/2013/FD/C3FD00082F 2.3. Towards complex glycopolypeptides structures

Figure 5. Schematic representation of glycopolypeptide−dendron conjugates Reprinted with permission from reference 70. Copyright 2012 American Chemical Society. In marked contrast to synthetic glycoproteins presented in section 1 of this review, the way by which glycopolypeptides are prepared allows modulating easily their macromolecular composition and architecture. This polymer chemistry approach has been the focus of only few examples so far concerning glycopolypeptides but the topic holds many promises. Again, Okada


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and coworkers have been the pioneers to follow such perspective and have prepared complex and hybrid synthetic glycopolypeptides by envisaging the ROP of glycosylated NCA through the use of a polyoxazoline macroinitiator100 to target heterogeneous diblocks or by envisaging the radical polymerization of glycopolypeptides macromonomers to prepare grafted biomacromolecules.101 More recently, Gupta and co-workers studied the conjugation between glycopolypeptides blocks and

hydrophobic dendrons (see figure 5).70 These amphiphilic macromolecules were self-

assembled into nanoparticles able to recognize specifically Concanavalin A lectins due to mannose residues exposed at the surfaces of the aggregates. The same group obtained another hybrid material where natural silk fibroin was conjugated to linear glycopolypeptides by Huisgen cycloaddition.102 After ligation, authors evidenced the formation of a completely water-soluble brush-like polymer displaying very high affinity towards the same Concanavalin A lectins. The preparation of tree-like glycopolypeptides is another example of macromolecular design in which a short poly(γ-benzyl-L-glutamate)–block-poly(DL-propargylglycine) was grafted with oligosaccharides (see figure 4).93 Interestingly and unlike linear counterparts, this unique and nonlinear architecture was capable of driving spontaneous self-assembly by direct hydration in water.

3.

Synthetic glycopolypeptides as biofunctional materials For decades, polymer chemists have progressively increased the complexity of synthetic

polymers by incorporating an increasing number of functionalities into their materials. The relevance of this multifunctionality is currently an important topic in materials science where the preparation of sophisticated polymers is used to target well-defined properties including self-


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organization, environmental adaptations or bioactivity.103 In this context, bioinspired materials60, 104

have been the subject of an increasing interest because methodologies involved in their

preparation allows to introduce high levels of functionality. Fundamentally, glycoprotein mimetic structures make them suitable model compounds for glycomics research. As biomaterial, these biomacromolecules provide novel solutions for disease diagnostics and treatment.105 In addition, their ability to self-assemble into vesicles, micelles and hydrogels holds promise for drug delivery applications as well as for tissue engineering.62

3.1. Smart glycopolypeptides polymers As already mentioned, like proteins, polypeptides conserve the ability to fold into welldefined secondary structures, which is a rare property in polymer science and which has important consequences in biomaterials design and properties.60 It is well known that ROP of NCA lead to various secondary structures if the monomer is enantiomerically pure. Moreover, this secondary structure is defined by the amino acid units.58, 60 The secondary structure content of a polypeptide can be estimated spectroscopically by far UV circular dichroism. With glycopolypeptides when carbohydrates are attached to enantiomerically pure polypeptides, αhelical structures are often formed in solution.66,

89

Even if it is still difficult to predict this

secondary structure conformation, its formation seems to be influenced by the grafting density and by the linkages that combine the glycan to the amino-acid.71, 75, 89, 94 In this context, it is interesting to note that some studies have mentioned structural transition of the glycopolypeptide that was induced in response to an external stimulus. In that respect, Kramer and Deming reported the impressive redox-triggered helix-to-coil transition of


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poly(glycosylated-L-cysteine).68 These glycopolypeptides predominantly formed an α-helix structures in solution, which was disrupted by the complete oxidation of the thioethers linkages using hydrogen peroxide/acetic acid. Even if the process was not reversible, this example of stimuli-responsive glycopolypeptide highlighted the possibility to drastically control the overall secondary structure of the polypeptide and thus the possibility to switch the carbohydrate display with the same peptidic backbone. On their side, Li and co-workers have reported pH-responsive glycopolypeptides which also underwent a helix-to-coil transition.75 These authors used poly(Llysine) partially functionalized with mannose and were able to form, depending on the pH, micellar or vesicular nanometric assemblies in solution. Interestingly, these authors have noticed that the addition of an anionic surfactant (SDS) also induced secondary structure transitions, in their case a coil-to-β sheet transition (see figure 6).75 More recently, Krannig and Schlaad have reported other pH-responsive glycopolypeptides, which underwent similar helix-to-coil transitions in solution and in a reverse fashion.95, 106 In particular, statistical copolypeptides of poly(L-glutamate-co-glycosylated DL-allylglycine) have been found to adopt a random-coil conformation in neutral and basic media and an α-helical conformation in acidic media which was influenced by the allylglycine conformation and/or content.95

Figure 6. α-helix-to-coil transition observed with poly(glycosylated-L-cysteine). Reproduced with permission from reference 68. Copyright 2012 American Chemical Society.


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3.2. Self-assembly

of

glycopolypeptides

:

towards

biomimetic

and

bioactive

nanoparticles Amphiphilic block copolymers are able to self-assemble into nanoscale structures, both in solution or on surfaces.48 Self-assembly of polypeptide-based block copolymers has been the subject of an increasing interest, in particular because the secondary structure of the polypeptide is a crucial parameter, which control both the self-assembly and the biological properties.60, 107 The self-assembly of amphiphilic glycopolypeptides is becoming a strong and emergent topic. First, it has been shown that this class of glycopolypeptides may form in solution vesicular structures which can mimic the structure of the viral capsid made of glycoproteins.96 In that respect, glycopeptosomes have been efficiently prepared from oligosaccharides-block-poly(γbenzyl-L-glutamate).96, 98, 108 The α-helical structure of the polypeptide have been used to favor the formation of lamellar morphologies96 and the oligosaccharides displayed at the surface of the nanoparticles used to target specifically cancer cells overexpressing the CD44 receptor.108 Glycopeptosomes were also observed after self-assembly with glycopolypeptides bearing glycan moieties onto the side chains. For example, by using a solvent replacement method, poly(γbenzyl-L-glutamate)–block-poly(DL-galactosylated propargyl glycine) has been found to form small vesicles having a diameter around 60 nm (see figure 7). These polymeric nanoparticles displayed galactose units at their surface able to specifically recognize RCA120 lectins.88 The same polypeptide poly(γ-benzyl-L-glutamate)–block-poly(DL-propargylglycine) backbone was also used recently to prepare tree-like glycopolypeptides incorporating oligosaccharides and having the property to form monodispersed micelles with sizes below 50 nm by direct hydration in water.93 As already mentioned in the section 2 of this review, amphiphilic glycopolypeptidesdendrons conjugates have been also the focus of self-assembly studies: these amphiphilic


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macromolecules were self-assembled in nanorods or in micelles depending on the size of the dendritic part and both assemblies were able to recognize specifically Concanavalin A lectins due to the mannose residues exposed at the surfaces of the nanoparticles.70 Very recently, the same group evidenced that amphiphilic homoglycopolypeptides were also able to self-assemble into bioactive spherical nanoparticles in water having specific lectin recognition ability for the same reasons.109

Figure 7. Biologically active glycopeptosome obtained by self-assembly of Poly(γ-benzyl-Lglutamate)–block-poly(DL-galactosylated propargylglycine). Reproduced with permission from reference 88. Copyright 2011American Chemical Society.


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The different examples highlighted in this section clearly show that it is possible to take advantage of the simplified macromolecular structure of these synthetic glycopolypeptide to precisely introduce or control different properties: the stimuli-responsiveness of the secondary structure, the self-assembly behavior in solution or the way by which carbohydrate are presented to biological targets such as lectins. It is important here to remember that the introduction of such properties is very difficult to achieve or to modulate in the case of synthetic glycoproteins.

Conclusions and future perspective This review aims at describing the “de novo” synthesis of glycoproteins, an important class of macromolecules in nature that remains not fully understood, together with highlighting the design of simplified analogues of these glycoproteins which are now accessible through the use of polymer chemistry (see figure 8). In fact, the full reproduction of the structures of natural glycoproteins has seen significant progresses recently but the complexity of the biological models still limits this approach. From a materials perspective, the excellent control in polymer synthesis that is available nowadays brings new tools for advanced chemical design. Crossfertilization between advanced polymer synthesis and efficient organic coupling chemistry indeed offer now several methodologies to access synthetic glycopolypeptides, simplified analogues of glycoproteins, and present new opportunities to create fully synthetic systems capable of interactions with living systems (see figure 8). Synthetic glycopolypeptides pave the way to the development of innovative glycoprotein mimics that will impact significantly glycomics research in which simplified analogues will help to better understand the properties of natural glycoproteins. Moreover, synthetic glycopolypeptides are innovative materials which are


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expected to bring important breakthrough in applications belonging to fields that merge materials science and biology. Drug delivery is a good example of such application where glycopolypeptides have been used very recently to prepare artificial viral capsid showing “in vivo” antitumor targeting.98,

108

One can also anticipate a growing interest in regenerative

medicine because glycopolypeptides combine the mechanical and signaling properties of peptides and glycans.


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Figure 8. Synthetic glycopolypeptides as biomimetic analogues of natural glycoproteins


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AUTHOR INFORMATION Colin Bonduelle, Université de Bordeaux/IPB, ENSCBP, 16 avenue Pey Berland, 33607 Pessac Cedex, France. E-mail: [email protected] Sébastien Lecommandoux*, Université de Bordeaux/IPB, ENSCBP, 16 avenue Pey Berland, 33607 Pessac Cedex, France. E-mail: [email protected]

ACKNOWLEDGMENT Financial support from the IUPAC glycopeptides project (AC-POL-09-11-25) is gratefully acknowledged. CNRS and ESF “Precision Polymer Materials” P2M RNP programme are also gratefully acknowledged.

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Biomacromolecules

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Glycopolypeptides are synthetic biomacromolecules acting as simplified models of glycoproteins that are designed with the purpose of both mimicking the properties of natural glycoproteins as well as bringing innovative polymeric structures for materials science. 419x122mm (96 x 96 DPI)


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Synthetic Glycopolypeptides as Biomimetic Analogues of Natural

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