Chapter 1: Synthetic Aspects of Peptide– and Protein–Polymer Conjugates in the Post-click Era Free

The synthesis of peptide– and protein–polymer conjugates offers the possibility to integrate properties of biological macromolecules into synthetic systems, thereby obtaining hybrid materials with unique functions.^1–9  The synthetic polymers within these structures provide a versatile range of properties,⁴  whereas the peptide or protein domain introduces highly specific functions, ranging from enzymatic activity¹⁰  to specific interaction capabilities or recognition,¹¹  and to disease modifying activities (Figure 1.1).¹²  The multidisciplinary field of creating bioconjugates gives access to a variety of materials for application in materials science, biotechnology, or pharmacology, where the bioconjugates act at the interface between biology and synthetic materials.^6–9 

First attempts to combine synthetic polymers with biomacromolecules emerged in the early 1950s, when initial reports concerning the synthesis of peptide–polymer conjugates were published by Jatzkewitz.^14,15  About 20 years later, Abuchowski, Davis, and coworkers described the attachment of a linear poly(ethylene oxide) (PEO) to bovine serum albumin (BSA) and bovine liver catalase.^16,17  Particularly noteworthy was that the extensive modification of these proteins with a synthetic polymer block did not inhibit the functionality of the biological macromolecules while simultaneously reducing their immunogenicity and increasing blood circulation times.^16,17  This widely used strategy of incorporating PEO onto proteins or peptides with the objective of improving the properties of biomacromolecules is termed “PEGylation”, referring to poly(ethylene glycol) or PEG.¹⁸  Due to polymer modification, the stability of the resulting protein–polymer conjugates toward proteolytic digestion and antibody interactions can often be decreased significantly.^19,20  This enhancement in stability and solubility of the modified proteins represents an important improvement for in vivo applications, such as in pharmaceutical research and the development of protein-based drugs.^5,21  Ever since these pioneering works showed the potential of PEGylation, and the combination of proteins or peptides with synthetic polymers in general, many research groups have started to investigate peptide–polymer conjugates as a new class of hybrid materials.^1,5,18,21 

Controlled techniques to connect the building blocks from the synthetic and the biological worlds are indispensable for the preparation of well-defined peptide– and protein–polymer conjugates.²²  In 2001, Sharpless, Kolb, and Finn introduced the concept of “click” chemistry, which describes the most important criteria to attach two molecules to each other in a highly selective and efficient manner.²³  A reaction defined under this term has to result in very high yields and easily isolable products, while generating only non-hazardous side products that can easily be removed afterwards. Besides the copper(i)-catalyzed cycloaddition (CuAAC) between alkynes and azides described independently by Sharpless²⁴  and Meldal,²⁵  a wide range of ligation strategies have since been found to fulfill the criteria of “click” reactions.²³  Of these (in addition to CuAAC), the strain-promoted azide–alkyne cycloaddition (SPAAC), Staudinger ligation, oxime formation, Michael-type additions and other thiol-ene reactions are among the most frequently cited representatives.²⁶  The advent of various highly efficient reactions has also proved advantageous for the preparation of peptide– and protein–polymer conjugates; these synthetic strategies offer versatile opportunities in the fields of materials science and biomedicine, where they are being applied widely.^26,27 

In conjunction with the emerging “click” reactions in this century, scientists have focused on the development of innovative strategies to design more complex structures.^6–9  The rich world of chemical ligation tools, which have traditionally been used for protein modification, has extensively been reviewed.^{1,4,5,10,12,28–32}  This book chapter will mainly provide an overview of the last decade's progress over the preparation of functional bioconjugates, also referred to as “post-click” methods.⁸  After a brief survey of various well-established and widely used ligation techniques, we will focus on novel types of chemistry as well as chemoenzymatic approaches and biotransformations to create functional bioconjugates using enzymatically catalyzed reactions. Finally, recent advances in this field will be described to provide insight into potential future directions for the preparation of functional peptide– and protein–polymer conjugates.

The most widely used method to prepare bioconjugates is based on a convergent strategy – the so-called “grafting to” approach (Figure 1.2a).^22,29  In order to obtain well-defined structures, the peptide or protein, which contains one or more reactive groups, is reacted with a polymer bearing complementary reactive groups. The “grafting to” approach is applied most since the independent synthesis and characterization of both components prior to the ligation enables a facile structural and chemical analysis of the resulting bioconjugates. Some of the disadvantages compared to other methods (vide infra) are that two macromolecules need to be coupled, which is often slow and/or inefficient due to the hindered accessibility of the reactive groups. Additionally, such reactions can only be performed using relatively low concentrations, and separation of the starting materials from the product is often difficult. Therefore, highly efficient coupling reactions are required.²⁸  However, the synthesis and characterization of both components independently enables a facilitated structural and chemical analysis of the resulting bioconjugates.

A range of different reactions for convergent ligation have been developed so far.^6–9  On the one hand, different amino acid side chains within the peptides and proteins can be addressed using various ligation strategies. Thiol groups of cysteine residues and primary amine groups at the N–terminus or on lysine side chains are the most common sites for attachment to polymers.³³  On the other hand, more appropriate functional groups can be introduced to improve the coupling efficiency and to enable site-specific conjugation, e.g. phosphine or azide residues for Staudinger ligation, aldehydes for oxime formation, or strained cyclooctynes for SPAAC.²⁶ 

In the “grafting from” approach a moiety with the ability to initiate or mediate polymerization is introduced in the biomacromolecule (Figure 1.2b).^{22,31,35–40}  This divergent strategy often relies on controlled radical polymerization techniques, since these are highly tolerant to the presence of functional groups which are frequently found in peptides and proteins.³¹  Furthermore, protein denaturation, resulting in loss of function, is unacceptable, making polymerization in aqueous media under mild conditions desirable. Controlled radical polymerization techniques such as atom transfer radical polymerization (ATRP),^36,41  reversible addition-fragmentation chain transfer (RAFT),^42–45  or nitroxide-mediated polymerization (NMP),⁴⁶  are compatible with aqueous solvents and avoid adverse side reactions.

One of the first examples following the “grafting from” approach was the preparation of a protein–polymer conjugate composed of BSA and poly(N–isopropylacrylamide) (PNIPAM).⁴⁷  The modification of the protein with a maleimide-functionalized chain transfer agent (CTA) enabled the RAFT polymerization of NIPAM, resulting in a thermoresponsive bioconjugate.

The “grafting through” approach implies the incorporation of a polymerizable group into the biomacromolecule, allowing for copolymerization with synthetic monomer units. This results in a bioconjugate product with a comb-like structure, where a certain number of the polymer side chains contain peptide or protein moieties (Figure 1.2c).^22,31  Compared to the concept of “grafting to”, the strategy avoids side reactions and low coupling efficiencies due to the attachment of peptides or proteins to monomers before the polymerization process, at which stage purification is easier.³¹  By comparison, the “grafting through” method is less modular than the “grafting to” strategy. In the “grafting to” approach, theoretically, any peptide containing a suitable reactive group can be coupled to a well-defined precursor polymer to produce different products with, e.g., different degrees of functionalization or with different peptides, etc. For the “grafting through” method, optimized polymerization conditions have to be found for each new monomer and the average length will not be precisely reproducible.

The first application using the “grafting through” approach was the synthesis of a thermoresponsive antibody–polymer conjugate described in 1987.⁴⁸  A more recent example deals with the incorporation of fibrinogen in a protein–polymer conjugate. Pluronic F127 was end-group-modified with an acrylate moiety on one side, while the other was coupled to fibrinogen.⁴⁹  The acrylate moiety was used for UV-activated free-radical polymerization. Since many of the fibrinogen molecules were functionalized with multiple Pluronic F127 moieties, polymerization triggered cross-linking, yielding a thermoresponsive hydrogel.

Beyond these three main concepts, the inverse bioconjugation approach offers another strategy to connect peptides or proteins with synthetic polymers. Using a solid support, which is preloaded with a polymer block, the biological molecule can be assembled in a stepwise fashion through solid-phase synthesis. Mutter and coworkers first showed the attachment of PEO to a poly(styrene) resin via a benzyl ether linker.⁵⁰  This concept was finally developed further by Bayer and Rapp leading to a commercially available PAP resin, which is widely applied in solid-phase peptide synthesis.⁵¹  In a similar approach, Lutz, Börner, and coworkers demonstrated the preparation of cleavable and non-cleavable soluble polystyrene supports by ATRP for the liquid-phase synthesis of peptide–polymer conjugates.⁵² 

The number of applications for peptide– and protein–polymer conjugates is constantly increasing. While classical synthetic reactions take advantage of naturally occurring amino acid side chains for coupling reactions with polymers, many innovative strategies are based on methods to attach synthetic polymers selectively, and with high efficiency, to improve yields and purity of the conjugation products.^6–9  The following sections will give an overview of the most commonly used techniques, sorted according to the different reactive functional groups. Novel bioconjugation approaches will be discussed in detail.

Exploiting primary amines is attractive since amines are among the most reactive functional groups present in peptides and proteins, and are found in relatively high abundance at the surface of globular proteins.⁵³  Various ligation strategies to attach polymers to them have been established.³¹  Most common is the modification of a synthetic macromolecule with an activated carboxylic acid group in order to address the peptide or protein N–terminus or a lysine side chain, if available.³³  Typically, polymeric derivatives of N–hydroxysuccinimide (NHS)⁵⁴  are used (Figure 1.3a). This reaction allows for the coupling of lysine residues in an almost quantitative manner, resulting in a stable amide linkage. For example, the NHS end group modification of PNIPAM derived from RAFT polymerization enabled the ligation to a cyclic peptide.⁵⁵  The resulting thermoresponsive peptide–polymer conjugate possesses the ability to form channels within phospholipid membranes.

However, the applicability of NHS-esters is limited due to their susceptibility to nucleophilic addition reactions, resulting in hydrolytic instability in aqueous media. To avoid low coupling efficiencies, the integration of pentafluorophenyl-activated carboxylic acid groups into synthetic polymers and their behavior in bioconjugation have been investigated (Figure 1.3b).⁵⁶  An appropriate modification of a collagen-like peptide was used for the site-selective conjugation of a stimulus-responsive poly(diethylene methyl ether methacrylate).⁵⁷  Coupling of two such bioconjugates resulted in the formation of triblock copolymer which showed triple-helix formation and thermoresponsiveness.

Frey and coworkers presented the squaric acid mediated PEGylation of proteins.⁵⁸  Amine bearing PEO was end-group functionalized with squaric acid diethyl ester, using one of the reactive groups in the squaric acid moiety. The other was still available for the efficient functionalization of lysine residues in proteins like BSA (Figure 1.4).

Despite the efficient coupling reactions available for the functionalization of amines, their high abundance in peptides or proteins prevents selective bioconjugate formation, since a single-site modification is often not possible. Furthermore, the multiple and unspecific conjugation of synthetic polymers can result in loss of function and reduced enzymatic activity, and can potentially even induce toxicity.³³  For these reasons, it is often desirable to apply more specific and directed coupling strategies, e.g. using reactions in which one of the components is rarely found in peptides and proteins.

Cysteine residues are frequently addressed in biological molecules because they are rather rare in peptides and proteins. If accessible thiols are present, they can therefore allow for a site-specific polymer ligation, yielding well-defined bioconjugates.⁵⁹  Classically, thioether formation by Michael addition of the thiol to a suitable electron-deficient alkene in the polymer blocks is used. The Michael addition reaction between thiols and maleimides constitutes the most well-known reaction, and is widely used to prepare bioconjugates of peptides and proteins (Figure 1.5a).⁶⁰ 

For example, the aminolysis of the chain transfer agent end group of PNIPAM prepared by RAFT exposed thiol end groups, which can be reacted with 1,8-bis-maleimidodiethyleneglycol. Ligation of bovine serum albumin and ovalbumin to the maleimide-terminated polymer yielded protein–polymer conjugates.⁶¹ 

Due to its electrophilic properties, maleimides are susceptible to the addition of other nucleophiles besides thiols, such as primary amines. Under physiological conditions, the reaction between a thiol group and a maleimide proceeds more rapidly than with an amine, as is desired.⁶³  However, slight changes in the pH to alkaline conditions can already result in a shift towards more side reactions with e.g. lysine side chains or the N–terminus of the biomacromolecule.⁶⁴  Beyond that, higher pH-values can even effect the hydrolysis of the maleimide group to an open maleamic acid form, which is no longer reactive towards thiols.⁶⁵  After the formation of the thiol-maleimide product, the ring-opening can effect the stabilization of the resulting conjugate, which prevents a possible thiol exchange.⁶⁶ 

These limitations can be avoided by using the free-radical addition of a thiol to a double bond (Figure 1.5b), which is referred to as a thiol-ene reaction⁶²  and can also proceed as a “click” reaction.²³  Initially, radical formation can be induced by heat or light.^67,68  The generated thiyl radical enters the addition reaction with the vinyl group, resulting in a carbon-centered radical. Subsequently, the desired thioether product is generated by hydrogen abstraction from other thiols, yielding further thiyl radicals. To achieve bioconjugates for in vivo applications, a photoinitiator is often added to generate radicals, since this procedure allows for the use of mild, relatively long wavelength (>300 nm) UV irradiation and can be performed in aqueous media under physiologic conditions.⁶⁹ 

The post-polymerization modification of poly(pentafluorophenyl methacrylate) with allylamine to couple a thiol-terminated peptide domain was presented by Klok and coworkers.⁷⁰  The polymeric side chain modification was used later on for the synthesis of polyvalent peptide–polymer conjugates exhibiting HIV-1 inhibitory properties with antiviral activity depending on the bioconjugate length.⁷¹ 

In proteins, cysteine residues are not always readily accessible, since they are often involved in disulfide bridges within the complex three-dimensional biomacromolecular structures. Therefore, only a small number of cysteine residues can be used for bioconjugation reactions.⁵⁹  The ligation of polymer bound dibromomaleimides takes advantage of this circumstance, as it allows for the functionalization of disulfide moieties. Haddleton and coworkers demonstrated the applicability of this reaction for bioconjugation of salmon calcitonin (sCT).^72,73 

Therein, the Mitsunobu reaction was adapted in order to end-group functionalize a linear PEO with a dibromomaleimide moiety. This modified polymer was conjugated to sCT after reduction by tris(2-carboxyethyl)phosphine (TCEP) under mild reaction conditions, yielding disulfide rebridging conjugation (Figure 1.6).⁷²  The efficient ligation reaction of dibromomaleimides with cysteine side chains or disulfide bridges, respectively, enables a site specific protein modification in a fast and equimolar manner. Despite opening a disulfide bridge in a protein, the native structure might be less significantly affected since a covalent bridge between both cysteine residues is reinstalled (cf. Figure 1.6). Furthermore, O'Reilly and coworkers used dibromomaleimide monomers for the preparation of profluorescent polymers, which were capable of chemico-fluorescent responsiveness mediated by thiol conjugation, resulting in a switch of the fluorescence emission.⁷⁵ 

For certain applications, the ligation reactions need to be performed in vivo without interfering with the given biological system. This severely limits the number of reactions that can be used due to, for example, the chemically very complex environment in biological systems and the large variety of functional groups present in a living system. In this context, the term “bioorthogonal” reaction was first coined by Bertozzi in 2003⁷⁶  in order to summarize efficient non-toxic chemical reactions that can be used in such systems, e.g. Staudinger-based ligation techniques, oxime/hydrazone formation, or strain-promoted azide–alkyne cycloaddition reactions.⁷⁷  To be able to specifically address a particular protein target, or other moieties, these reactions use chemical groups that are not typically found in natural systems.

Ligation techniques based on carbonyl chemistry between aldehydes or ketones with nucleophilic amine derivatives, such as aminooxy or hydrazide groups, have been developed for classical bioorthogonal tools such as protein-labeling⁷⁸  and protein–polymer conjugation (Figure 1.7).²⁶  These types of condensation reactions were originally reported in the context of the in vitro assembly of a cytotoxin from non-harmful precursors through hydrazone formation.⁷⁹  Apart from the potential reversibility of the resulting imine bond, the oxime/hydrazone formation enables a highly selective route to prepare protein–polymer conjugates under physiological and additive-free conditions.⁸⁰ 

In a recent example, Maynard and coworkers demonstrated the synthesis of a Boc-protected aminooxy functionalized initiator for copper(i)-mediated ATRP of NIPAM (Figure 1.8).⁸¹  Deprotection of the polymer end groups allows for bioconjugation with the aminooxy moiety. Therefore, N–levulinyl (a ketone containing group)-modified bovine serum albumin can be attached to the aminooxy functionalized PNIPAM rapidly and in a highly selective manner, resulting in a thermoresponsive protein–polymer conjugate.

Furthermore, oxime formation has been used to functionalize proteins; for example, N–terminal oxidation of interleukin 8 with sodium periodate⁸²  and biomimetic transamination of myoglobin with pyridoxal-5-phosphate,⁸³  followed by site-specific protein PEGylation with hydroxylamine-functionalized polymer blocks. The selectivity of this reaction is limited by the fact that other amino acids within the protein can be oxidized as well, and incomplete and unselective transamination can take place.⁵ 

In a first in vitro application, the Staudinger ligation was used for the modification of an azide-functionalized glycoconjugate with a biotinylated triarylphosphine resulting in cell surface labeling.⁸⁴  The ligation technique is a modern adaptation of the classical Staudinger reaction originally published in 1919.⁸⁵  In the first and rate limiting reaction step, a phosphazide is formed under the liberation of nitrogen (Figure 1.9a).⁸⁶  The ester group in ortho-position to the phosphorus group is subsequently involved in an intramolecular cyclization. The cyclization is followed by hydrolysis in aqueous media, and the desired amide bond is generated with simultaneous formation of a phosphine oxide byproduct. An additional feature of this reaction is the possibility of creating a “traceless” product by changing the linker between the ester or thioester and the phosphine moiety, which results in the elimination of the phosphine oxide side product in the final hydrolysis step (Figure 1.9b).^87,88 

The ligation can be performed in aqueous media in the absence of any metal catalyst. In addition, the absence of azides and phosphines in the natural environment renders the Staudinger ligation highly suitable for bioorthogonal labeling in living systems^89,90  and for the conjugation of synthetic polymer blocks to biological macromolecules.⁹¹  Among others, the incorporation of non-natural amino acids in protein biosynthesis,⁹²  the diazo transfer onto primary amines,⁹³  or native chemical ligation strategies⁹⁴  can be used for the introduction of the azide moiety into peptide or proteins.

An interesting example combines the previously mentioned free-radical thiol-ene “click” addition with the Staudinger ligation in order to obtain biodegradable peptide-functionalized poly(lactide)-graft-poly(ethylene oxide) copolymers (Figure 1.10).⁹⁵ 

In this “grafting through” approach, allyl lactide and a bifunctional tri(ethylene glycol) initially react to generate a tri(ethylene glycol)-containing lactide analogue using thiol-ene chemistry. Subsequently, ring-opening polymerization induced by tin octoate yields a well-defined copolymer with a molecular weight of 6×10³ g mol⁻¹ and a dispersity index of 1.6. Finally, azide terminal groups on the copolymer tri(ethylene glycol) chains of the copolymer enable efficient post-polymerization functionalization by Staudinger ligation to install a GRGDS peptide domain.

One of the organic reactions that has recently been most intensively studied to combine two macromolecules is the 1,3-dipolar cycloaddition between an azide and an alkyne resulting in a 1,2,3-triazole linker. Originally reported by Huisgen in 1961,⁹⁶  the reaction has been classified as a paradigm for the “click” reaction by Sharpless at the beginning of the 21st century.²³  Through additional regioselectivity using copper(i) as the catalyst (Figure 1.11),^24,25  this cycloaddition (CuAAC, copper(i)-catalyzed alkyne–azide cycloaddition) reaction became a valuable strategy to prepare peptide– and protein–polymer conjugates for a variety of applications.^8,27,97 

Again, the unique reactivity of azides (as in the previously described Staudinger ligation) often allows for almost quantitative product formation rates and mild reaction conditions.⁹⁸  Because the cycloaddition reaction is preferred over a nucleophilic substitution mechanism, biological macromolecules can be modified in a highly selective manner, whereas side reactions rarely occur, rendering protective groups redundant.³¹ 

Jia and coworkers took advantage of CuAAC to design an elastin-like peptide–polymer conjugate.⁹⁹  The reaction between a bifunctional, azide-terminated PEO block and an alkyne-functionalized peptide composed of two different functional domains yielded a multiblock copolymer (Figure 1.12). While the sequence (AKAAAKA)₂ is abundant in the cross-linking region of natural elastin, the RGD motif in GGRGDSPG mediates cell adhesion. Finally, covalent crosslinking through the lysine residues using hexamethylene diisocyanate resulted in hydrogels with elastin-like mechanical properties.

Unfortunately, utilization of copper(i) to control the regioselectivity yields several problems. Although only catalytic amounts are required, copper can induce cytotoxic effects¹⁰⁰  and protein denaturation,¹⁰¹  which can complicate bioconjugation by CuAAC for applications in vitro and in vivo. In addition, copper salts are often rather difficult to remove from the reaction products. Nevertheless, advantages like high selectivity, mild reaction conditions, and almost quantitative product formation have proven CuAAC highly beneficial for the preparation of site-specific peptide– and protein–polymer conjugates.¹⁰² 

The drawbacks of CuAAC can be circumvented by replacing the alkyne in the copper(i)-catalyzed reaction with a ring-strained cyclooctyne. The spontaneous reaction between an azide and a cyclooctyne (OCT) was first reported in 1953 (Figure 1.13a).¹⁰³  However, until Bertozzi and coworkers took advantage of the strain-promoted azide–alkyne cycloaddition (SPAAC) with enhanced biocompatibility, this reaction had hardly been used.¹⁰⁴  Ever since, many different strained azophiles have been reported in connection with e.g. protein-labeling, cell surface modification, or in vivo imaging.⁷⁸  Besides the classic OCT,¹⁰⁴  derivatives like monofluorinated cyclooctyne (MOFO)¹⁰⁵  or dibenzocyclooctyne (DIBO)¹⁰⁶  have been developed to enhance the reactivity towards azide moieties (Figure 1.13b).

The potential of SPAAC in peptide–polymer conjugation was successfully demonstrated by Chaikof and coworkers using a cyclooctyne-functionalized peptide for surface decoration (Figure 1.14).¹⁰⁷  It was shown that a cytocompatible poly(l-lysine)-graft-poly(ethylene oxide) copolymer bearing azido groups was efficiently modified with a DIBO-functionalized IKVAV peptide, yielding peptide–polymer conjugates with controlled grafting densities that could be used in peptide microarrays.¹⁰⁷ 

Despite the advantages of site-specific bioconjugation, certain limitations of SPAAC have to be considered. Besides reaction kinetics 10–100 times slower than CuAAC,¹⁰⁸  hydrophobicity and instability of the highly reactive cyclooctynes¹⁰⁹  under physiological conditions restrict its application in living systems. For that reason, the scientific community still strives for novel approaches to design peptide– and protein–polymer conjugates in a highly selective and fast manner.

Reactions between a diene and a dienophile yielding cyclohexene derivatives through carbon–carbon or carbon–heteroatom bond formation are referred to as Diels–Alder (DA) reactions. The concerted [4+2] cycloaddition was first described in 1928 by Diels and Alder,¹¹⁰  and can be adapted for the efficient conjugation of peptides and proteins to synthetic polymers.²⁶  Despite being recognized as highly selective and additive-free, DA reactions require elevated temperatures to obtain the desired product. Furthermore, the reversibility of the reaction limits its application in materials science.²³ 

In comparison, the inverse-electron-demand Diels–Alder (IEDA) reaction between a tetrazine and a strained alkene or alkyne followed by retro-Diels–Alder elimination of nitrogen offers a highly selective alternative, yielding stable dihydropyridazine or pyridazine products.¹¹¹  Studies on, e.g., trans-cyclooctene have revealed rate constants up to 2×10³ M⁻¹ s⁻¹, which are much higher than those of any other “click”-type reactions.¹¹²  For that reason, trans-cyclooctene derivatives and other strained alkenes, such as cyclobutene or norbornene derivatives (Figure 1.15a), can be used for bioconjugation purposes.¹¹³ 

An interesting example of this reaction deals with the IEDA between a multifunctional PEO-tetrazine polymer and a dinorbornene peptide.¹¹⁴  In this system, a four-arm amino-functionalized PEO (20×10³ g mol⁻¹) was modified with benzylamino tetrazine. The IEDA reaction with the cross-linking peptide KGPQGIWGQKK containing norbornene moieties triggered the formation of a hydrogel.

Another remarkable strategy is based on the addition of heterodienophiles, such as imines, aldehydes, or thiocarbonyl compounds, to suitable dienes (Figure 1.15b).¹¹⁵  These reactions are summarized under the term hetero Diels–Alder (HDA) reactions. Barner-Kowollik and coworkers explored HDA reactions for the preparation of peptide-functionalized polymers using cellulose as the polymer scaffold (Figure 1.16).¹¹⁶  As a model reaction, a thioamide-functionalized peptide (Phos-GGFPWWG) was coupled to poly(methylmethacrylate) containing a cyclopentadienyl terminal group, yielding PMMA-b-GGFPWWG bioconjugates. The strategy was thereafter used for the attachment of Phos-GGFPWWG to cyclopentadienyl functional cellulose. Remarkably, only small amounts of acid were required to obtain conversions near 90%.

Even azide moieties are capable of entering DA cycloaddition reactions with oxanorbornadiene derivatives, yielding triazole products. For example, the tandem [3+2]/Diels–Alder cycloreversion (CrDA) was used for the PEGylation of an N–terminally azido functionalized peptide (GGRGDG).¹¹⁷  Furthermore, labeling of oxanorbornadiene-modified hen egg-white lysozyme with fluorescent coumarine was demonstrated.¹¹⁷  The release of furan offers a versatile and efficient route to prepare peptide–polymer conjugates at moderate temperatures.

Another interesting strategy to design polymer–peptide–polymer conjugates based on the HAD reaction was demonstrated by Barner-Kowollik, Börner, and coworkers. In the first step, the formation of a peptide–PEO conjugate based on maleimide-magainin and a modified PEO bearing a tetrazole and a photoenol moiety was induced by light. Repeated irradiation in the presence of maleimide terminal polylactide lead to the formation of a hybrid triblock macromolecule.¹¹⁸ 

Recent developments concerning fast and highly selective ligation methods use 1,2,4-triazoline-3,5-dione (TAD) as one of the most remarkable and versatile reagents in “post-click” chemistry.⁸  Within the heterocyclic compound, the arrangement of the azo moiety and the electron-withdrawing carbonyls offers a significant reactivity towards a variety of functional groups such as enes or dienes.¹¹⁹  Therefore, Diels–Alder and other addition-type reactions take place, resulting in the formation of stable bonds as well as reversible connections depending on the reaction partner. The reaction proceeds in the absence of a catalyst under moderate conditions, and its orthogonality makes several reactions with TAD highly interesting for the preparation of bioconjugates.²³ 

Du Prez and coworkers presented a relevant approach concerning TAD-based chemistry described as a novel “click” and “trans-click” strategy (Figure 1.17a).¹²⁰  The reaction of 4-butyl-1,2,4-triazoline-3,5-dione (BuTAD) with 2,4-hexadiene-1-ol (HDEO) yielded the Diels–Alder adduct TAD–HDEO in an extremely fast, traceless, and irreversible manner. The occurrence of a retro-Diels–Alder reaction can be excluded based on reference experiments with competing 2,4-hexadiene-1,6-diol. Furthermore, an addition-type adduct (TAD–indole) was prepared using a substituted indole, e.g., tryptophan in proteins. To demonstrate the “trans-click” reactivity, HDEO was added under elevated temperatures resulting in TAD–HDEO (TAD–diene). The ability of TAD to form both reversible and non-reversible covalent linkages offers novel opportunities for polymer and materials science.⁸  The potential of TAD as an extremely fast and highly selective reacting moiety has already been used in a variety of applications such as polymer end-group modification,¹²⁰  polymer–polymer conjugation,¹²⁰  the preparation of polymer networks,¹²⁰  and orthogonal protein labeling.¹²¹ 

The previously described orthogonal strategies, such as ligation by oxime/hydrazone formation,⁸⁰  Staudinger ligation,⁸⁷  or SPAAC,^121,122  etc., require chemical modification of the peptide and proteins for chemoselective polymer conjugation. To circumvent this, Barbas and coworkers studied TAD derivatives possessing azide, alkyne, and ketone groups. They demonstrated the reactivity of the TAD moiety by addressing tyrosine moieties in peptides and proteins (Figure 1.17b).¹²³  As an example of the potential of TAD-based chemistry in the creation of peptide– and protein–polymer conjugates, TAD-functionalized PEO was used for the PEGylation of chymotrypsinogen, which contains four tyrosine residues for addition-type reactions.⁹¹  An alkyne-PEO (5×10³ g mol⁻¹) was modified with a urazole-bearing azide moiety by CuAAC, where the urazole served as a precursor to the reactive TAD. Upon oxidation to TAD–PEO, the reaction with chymotrypsinogen resulted in addition products bearing multiple PEO chains. Taking into consideration that TAD-reactions based on Diels–Alder reactions are even more efficient than those based on addition reactions,¹²⁰  this work demonstrated that TAD chemistry can be used effectively for the development of novel bioconjugation strategies for materials science applications.

Strategies for the preparation of bioconjugates taking advantage of enzymes have been investigated with the objective of addressing functional groups in peptides or proteins in a highly selective manner. From a formal point of view, enzymes can be separated into two categories, by which site-specific modification of biological molecules can be realized.⁶  On the one hand, a number of enzymes are capable of introducing appropriately functionalized synthetic polymers onto biomacromolecular substrates.¹²⁴  On the other hand, enzymes can be used for the modification of a single moiety.¹²⁵  Transglutaminase (TGase) and sortase (Srt) are representatives of the first-mentioned class of enzymes, whereas formylglycine transferase and lipoic acid ligase have been used for the transformation of functional groups.⁶  The potential of these enzymes for bioconjugate synthesis and protein modification will be discussed in the following sections.

Transglutaminase (TGase) enzymes are capable of catalyzing an acyl transfer between two proteins involving the glutamyl group of a glutamine (acyl donor) and a primary amine (acyl acceptor), usually the ε-amino group of a lysine (Figure 1.18).¹²⁶  In the catalytic cycle, first an acyl-enzyme intermediate is formed, simultaneously releasing ammonia. The presence of an acyl acceptor leads to the transamidation product, whereas TGase is regenerated.¹²⁷  Using a PEO-amine derivative as an acyl acceptor, site-specific TGase-mediated PEGylation of proteins is feasible.

In a recent example, the recombinant human growth hormone (hGH) was modified with an amine-functionalized PEO-block (20×10³ g mol⁻¹) using microbial TGase (mTGase) to mediate the PEGylation (Figure 1.19). Performed in a water/ethanol mixture, mTGase allowed the selective ligation at hGH Gln₁₄₁, yielding the site-specific conjugate PEO–Gln₁₄₁–hGH.¹²⁸ 

Due to the substrate selectivity of the enzyme, one or two glutamine residues within a protein can be addressed selectively in most cases.^129,130  Taking into account that two large molecules are covalently connected, the site-specific and nearly quantitative product formation is particularly noteworthy.¹²⁸  A disadvantage of TGase-mediated and other enzyme catalyzed reactions is that product purification, in particular enzyme separation, can be rather tedious. The purification can be simplified by involving thermoresponsive polymers such as PNIPAM in protein–polymer conjugates, exploiting the lower critical solution temperature behavior of the polymer to obtain two immiscible phases.¹³¹ 

Sortases are transpeptidases, which use the linear epitope LPXT↓G as a recognition site within a target peptide or protein.¹³²  The cleavage of the amide bond between the threonine and glycine residue (↓) results in an acyl-enzyme intermediate. Subsequently, the thioester intermediate is attacked by a nucleophilic co-substrate comprised of an N–terminal glycine residue in order to form a native peptide bond. Sortase A (SrtA) derived from Staphylococcus aureus is commonly used for site-specific N– and C–terminal protein labeling,^133,134  the synthesis of protein–lipid conjugates,¹³⁵  or intramolecular peptide cyclization.¹³⁶  Furthermore, SrtA enables the site-specific PEGylation of cytokine with an exogenous oligo-glycine functionalized-PEO.¹³⁷  The enzyme-based ligation strategy results in the extension of the cytokine half-life and biological activity.

Unfortunately, the transpeptidation mediated by SrtA is subject to reversibility, since the newly formed ligation product is a potential substrate for another transpeptidation cycle.¹³⁸  In order to prevent the reverse reaction and thus selectively obtain the desired product, the amide bond between the threonine and glycine residues within the recognition site can be replaced by an ester.¹³⁹ 

Besides transglutaminases and sortases, a variety of enzymes are able to catalyze site-specific protein modifications in order to introduce reactive groups for further functionalization and ligation.¹⁴⁰  Biotin ligase, for example, catalyzes the modification of recombinant cell-surface proteins with biotin-mimicking ketones. The ketones can subsequently be used in ligation with hydrazide- or hydroxylamine-functionalized molecules (Figure 1.20a).¹⁴¹  Microbial lipoic acid ligase is also used for protein labeling by attaching an alkyl–azide onto a cell-surface engineered acceptor peptide.¹⁴²  The introduction of the azide group permits the conjugation of a cyclooctyne-containing probe via SPAAC (Figure 1.20b).

Protein farnesyltransferase recognizes a short tetramer peptide motif and can be applied for chemoselective functionalization of proteins with synthetic azido-farnesyl analogs, followed by CuAAC for chemical labeling (Figure 1.20c).¹⁴⁴  Similarly, protein expression vectors can be tagged with CXPXR sequences, which act as recognition sites for formylglycine transferase.¹⁴³  The enzyme catalyzes cysteine oxidation to an aldehyde-bearing formylglycine, which enables site-specific ligation with α–nucleophilic moieties.

The variety of possibilities to address functional groups in biological molecules provided by chemoenzymatic approaches offers a wide range of opportunities for protein modification and bioconjugate synthesis with regard to pharmaceutical and materials science applications.¹²⁴ 

Besides the chemoenzymatic approaches described above, where functional groups are added to allow for conjugation with other macromolecules, enzymes demonstrate other possibilities to obtain peptide– and protein–polymer conjugates, which are normally difficult to obtain by common chemical synthetic strategies. Enzyme responsiveness can be used to modulate bioconjugate properties in order to create hybrid materials with more complex functions.¹⁴⁵  In living systems, enzymes catalyze a broad range of reactions to stimulate biological processes such as protein expression, cellular adhesion, muscle contraction, etc.¹⁴⁶  Some of these processes have been adapted for a variety of biotransformation reactions with the objective of integrating novel properties into bioconjugates. As a result, enzymatically catalyzed reactions are able to affect self-assembly,¹⁴⁷  to induce controlled release of, for example, therapeutic drugs,¹⁴⁸  or to initiate adhesion to diverse surfaces.^149,150 

Cenker and coworkers demonstrated the enzyme responsiveness of a PEO-peptide micelle system that can be proteolytically cleaved, leading to the release of non-aggregated amyloid-based peptides.¹⁵¹  The peptide–polymer conjugate incorporating the βAβAKLVFF sequence was cleaved by α–chymotrypsin to produce βAβAKLVF and F–PEO. Whereas the hexamer did not aggregate into β–sheet structures, the heptapeptide βAβAKLVFF formed well-defined β–sheet ribbons, which have been associated with Alzheimer's disease.¹⁵² 

Börner and coworkers used acid phosphatase to induce microstructure formation.¹⁵³  The peptide-segment contained three phosphothreonine residues in the (TV)₅ peptide motif of the peptide–PEO conjugate, which prevented β–sheet formation. Enzymatic dephosphorylation of the threonine side chains therefore induced self-assembly of the bioconjugate.

A more recent example of a biotransformation of peptide–polymer conjugates combines biocombinatorial procedures with enzymatic activation processes in order to realize mussel-glue inspired adhesion.¹⁵⁴  Phage-display biopanning incorporating tyrosinase to transform l-tyrosine residues to l-dopa enabled the direct selection of enzymatically activable 12-mer peptide adhesion domains for aluminum oxide (Figure 1.21).

After identification of activable adhesive domains, the 12-mer peptides were synthesized as peptide–PEO conjugates by the inverse bioconjugation approach. Switching of these conjugates from weak to strong binders was successfully triggered by tyrosinase. The activated systems revealed effective adsorption even under saltwater conditions. Furthermore, the coatings from enzymatically activated conjugates possessed antifouling properties and effectively suppressed interactions between blood plasma protein cocktails and bacteria with the coated surface, emphasizing the potential for medical as well as industrial applications.

Within the last decade, a wide range of synthetic and chemoenzymatic strategies have been developed to allow for the preparation of complex hybrid materials combining peptides or proteins with synthetic polymers.^6–9,124  Of the four main strategies for the creation of protein– or peptide–polymer conjugates, i.e. “grafting from”, “through”, “to”, and through inverse bioconjugation, the “grafting to” method is the most widely used, although advances in controlled radical polymerization techniques including ATRP,⁴¹  RAFT,⁴²  and NMP⁴⁶  have significantly contributed to the feasibility of the other methods. The main challenges in the preparation of well-defined conjugates are the site-specific and bioorthogonal modification of peptides and proteins and highly efficient product formation. Since the copper(i)-catalyzed azide–alkyne cycloaddition (CuAAC)^18,19  reaction was proven to be cytotoxic, a variety of ligation techniques that can be considered “click” reactions, such as the ligation by oxime/hydrazone formation,⁸⁰  Staudinger ligation,⁸⁷  and strain-promoted azide–alkyne cycloadditions (SPAAC),¹²³  have been established. Recent advances in bioconjugation by inverse-electron-demand Diels–Alder (IEDA) reactions between tetrazine and strained alkenes, for example, have provided insight into highly efficient and specific potential future reactions for the preparation of functional peptide– and protein–polymer conjugates.¹¹⁵  Furthermore, the reactivity towards strained alkenes can be modulated by varying the electron withdrawing groups using nitrones, nitrile oxides, diazoalkanes, and syndones, as alternative 1,3-dipoles.⁹  Equally, chemistry with 1,2,4-triazoline-3,5-dione (TAD) based on Diels–Alder reactions can be suggested for the development of novel bioconjugation strategies.¹²⁰ 

Some other highly efficient cycloaddition reactions have been used for polymer end-group modification and polymer–polymer conjugation.¹⁵⁵  These include an additive-free version of the hetero Diels–Alder (HDA) reaction¹⁵⁶  or the nitrile-imine-mediated tetrazole-ene cycloaddition (NITEC), the latter only requiring photoirradiation.¹⁵⁷  Both techniques meet the criteria to be considered bioorthogonal reactions and can be performed under mild reaction conditions. As such, these reactions should also be applicable for the preparation of bioconjugates.

As discussed, a wide range of ligation reactions can be (potentially) used for bioconjugate synthesis,¹⁵⁸  and new reactions are being investigated to achieve even higher reactivity, more efficient product formation, and to circumvent side reactions. Increased reactivity and specificity are often incompatible properties though. Therefore, it is necessary to validate the applicability of each system individually. Chemoenzymatic approaches offer a great alternative to prepare peptide– and protein–polymer conjugates. Enzymes are capable of addressing functional groups occurring in peptides or proteins in a highly selective manner: most of the appropriate enzymes can be used for the modification of single (or very few) moieties in a biopolymer, either with small molecules that can be used in further ligation strategies or for the direct attachment of appropriately functionalized synthetic polymers onto biomacromolecular substrates. Though a large body of work exists on the (site-specific) modification of peptides and proteins (e.g. for labeling) using enzymes,^130,131  the exploitation of these reactions for the creation of well-defined functional protein– and peptide–polymer conjugates is still in its infancy.