Pegylation of peptides

Pegylation of peptides DEFAULT

PEG–Peptide Conjugates

1 Introduction

Attachment of PEG to peptides or proteins, so-called PEGylation, offers improved water solubility and stability as well as reduced clearance through the kidneys, leading to a longer circulation time. (1) Several reviews on polymer–peptide conjugates discuss examples of self-assembling PEG–peptide conjugates, (2-4) and reviews discussing applications of PEGylation of peptides and proteins for applications in biotechnology are available. (5-8)

This review is focused on PEG–peptide conjugates and does not cover methods of protein PEGylation or applications of PEGylated proteins. These topics have been extensively reviewed elsewhere. (9-12) The application of PEGylation in drug delivery has been reviewed, including lists of commercially developed PEGylated proteins for therapeutics. (5, 7, 8, 13) General reviews on polymer–peptide conjugates also feature discussion of PEG–peptide conjugates. (1, 14-16) A book on bioconjugation techniques (17) discusses many chemistries that can be used to prepare PEG–peptide conjugates. This review focuses on the self-assembly of PEG–peptide conjugates, with a brief discussion of the relevant preparation chemistries, as these are reviewed in more detail in the above-mentioned publications.

The following notation is used here for PEG with different molar mass: in PEGxk, x denotes the molar mass in kilograms per mole, and in PEGn, n denotes the average degree of polymerization. Amino acids are abbreviated with single- or three-letter codes.

This review is organized as follows. Methods to synthesize PEG–peptide conjugates are outlined in Section 2. Sections 3 and 4 discuss the self-assembly of PEG–peptide diblock conjugates containing β-sheet or α-helical peptides, respectively. The self-assembly of PEG–peptide conjugates with more complex triblock and multiblock architectures is reviewed in Section 5. In Section 6, the class of PEG–peptides containing synthetic polypeptides such as poly(γ-benzyl l-glutamate) (PBLG) is considered. PEG crystallization effects on self-assembly are examined in Section 7. The final sections introduce selected applications of PEG–peptides. Section 8 concerns enzyme-responsive PEG–peptide conjugates, whereas Section 9 summarizes studies on PEG–peptides for drug delivery. Lastly, a brief summary and outlook are included in Section 10.

2 Synthetic Methods

2.1 Coupling

2.1.1 Coupling Chemistries

Examples of some of the common coupling chemistries are shown in Figure 1. As will become apparent in the following discussion, this is not an exhaustive list of methods used to link PEG polymers and peptides.

Figure 1

Amine groups at the N terminus or on lysine side chains are common sites for PEG attachment using hydroxyl- or aldehyde-functionalized PEG. First-generation methods relied on activated hydroxyl groups on end-functionalized PEGs. (18, 19) A widely used method in current use involves N-hydroxysuccinimide (NHS) esters, which are highly reactive toward amines at physiological pH (Figure 1a). (20) Potential hydrolysis of the ester bond of succinylated PEG can be avoided by use of a linking carbonate group. (1, 21, 22) NHS-terminated PEG was used to prepare alternating multiblock copolymers of PEG and coiled-coil peptides using NHS–PEG3.4k–NHS. (23)

Other methods to activate the reaction of PEG and amines have been reviewed. (1) Rather than preparing polymers with reactive end groups, in situ activation using carbodiimides is possible, (24) and this has been used to couple the carboxylic group of succinylated PEG to various peptides. (1) Aldehyde-terminated polymers may be reacted with N-terminal amines (Figure 1b). (1, 25-27) In one example, poly(oligo-(ethylene glycol) methacrylate) (POEGMA) or PEG was coupled to the 3432 Da peptide hormone salmon calcitonin. (28) POEGMA contains oligomeric ethylene oxide side chains and has some distinct properties to linear PEG while retaining advantages such as biocompatibility. For instance, copolymerization of different oligo-ethylene gycol acrylates leads to the formation of thermosensitive polymers with lower critical solution temperature (LCST) behavior. (29) In the conjugates of PEG or POEGMA with salmon calcitonin, the bioactivity was not influenced by the Mn of POEGMA (containing PEG1.1k) in the range 6.5–109 kDa. (30) Later, this group used dibromomaleimides for site-specific linkage to the disulfide bridge in salmon calcitonin. (31) The same model peptide was also conjugated to poly((monomethoxy ethylene glycol) (meth)acrylates) via the disulfide using water-soluble organic phosphines as catalysts. (32) An amine-terminated polymer may be attached to an aldehyde-functionalized resin, from which peptide synthesis is performed. (33)

Cysteines are an attractive target for conjugation of polymers to proteins because only a few are typically available to react with; (1) however, further discussion of protein conjugation is outside the scope of this review. A number of thiol-reactive groups can be used to couple end-functionalized polymers to cysteine-terminated peptides (Figure 1c,d). One widely used chemistry employs maleimides, which react selectively with the thiols of cysteine residues by Michael addition in the pH range 6.5–7.5 (Figure 1c). (15) To prepare atom-transfer radical polymerization (ATRP) initiators containing maleimides, protection is required. (1) Amine-terminated PEG has been converted to a maleimide by reaction with maleic anhydride. (34) Maleimide–PEGs have been coupled to cysteine flanked silk-like peptide (EG)3EG. (35) In another case, maleimide polymers including POEGMA were coupled to the model thiol-containing peptide reduced glutathione (γ-ECG) as well as the protein bovine serum albumin. (36) The maleimide was incorporated into an initiator for ATRP to produce α-functionalized polymers. Maleimide-modified RAFT chain-transfer agents (CTAs) have also been used to synthesize poly(ethylene glycol) methyl ether acrylate (PEGA) for conjugation to proteins such as lysozyme. (37, 38)

Another chemistry that can be used to link polymers to cysteine residues employs vinyl sulfone-terminated polymers (Figure 1d); for example, this approach has been used to couple PEGA to proteins. (39) A PEGA–PS–PEGA triblock copolymer with pyridyl sulfide end groups, prepared by RAFT using a novel CTA, has been linked to glutathione as a model tripeptide. The triblock forms micelles, and the conjugate forms peptide-decorated micelles. (40)

Thiol-ene chemistry may also be employed to link alkene-terminated polymers and cysteine-containing peptides. For instance, copolymers of di(ethylene glycol) methyl ether methacrylate (DEGMEMA) and allyl methacrylate have been linked to α-keratin using this chemistry. (41)

Diazonium derivatives may be coupled to tyrosine, and this has been exploited to link a diazo-functional PEG (PEG6) to the pentapeptide (d-Ala2)-leucine enkephalin (an endogenous opioid peptide) as well as diazo-functional PEG6 and PEG45 to salmon calcitonin (Figure 2). (42) This method was also employed using POEGMA instead of PEG.

Figure 2

Carboxylated PEG may be linked to oligopeptides via NHS activation with N,N′-dicyclohexylcarbodiimide (DCC). (43) This method was used to prepare conjugates of short PEG chains (PEG350–750) with hydrophobic tetra- and hexapeptides. The conjugates formed micelles with thermosensitive properties because of the LCST behavior.

Oxime formation by reaction of aminooxy end-functionalized polymers and levulinyl-modified proteins or peptides may also be used to prepare conjugates, such as those containing POEGMA. (44) The method has also been used to conjugate branched PEG-based polymers to peptides (small proteins) such as an anti-HIV protein (45, 46) (Figure 3) or synthetic erythropoietic proteins. (47)

Figure 3

On-resin coupling of PEG–CH2–COOH to N-terminal peptide resins has been carried out via acylation, occurring rapidly and with high conversion in the case of lower molar mass PEG (750 g mol–1) but not for PEG10k (PEG5k could be coupled to unhindered N-terminal Gly but not hindered Ile). (48) Di-PEGylation was also possible using C-terminal PEGylated amino acids (ornithine or lysine), and this method was used to prepare di-PEGylated interleukin-2 peptide fragments. Lysine side chain/C-terminal PEG conjugates were also prepared. The authors also showed that PEG end-functionalized with methylnorleucine (Nle) is a good reagent for N-terminal PEGylation using BOP activation. (49, 50) The method was used to prepare a number of PEG–OCHH2CO–Nle(NANP)3 conjugates with PEG5000 (49) as well as a conjugate of PEG5000 and a 13-residue peptide fragment of interleukin-2. (50)

A mixed grafted PLL poly(l-lysine) conjugate was prepared by coupling PEG2k with an NHS end group via the lysine amine as a side chain. (51) In addition, a fraction of PEG chains were functionalized with RGD-based peptides via vinylsulfone groups coupled to cysteine residues. The incorporation of the RGD cell adhesion motif supported the growth of human dermal fibroblasts while blocking adsorption of serum proteins. In a related context, PEG5000 has been grafted via a lysine residue in fluorophore-labeled RGD peptides for applications in bioimaging. (52) PEGylation was found to enhance fluorescence quantum yield while reducing interactions between fluorophores and biomolecules in cells.

2.1.2 Click Coupling

The well-known [3 + 2] cycloaddition click reaction (Figure 1e) between alkynes and azides has been widely used in the synthesis of bioconjugates. (53)

By using click reactions, grafting efficiencies approaching 100% are possible, as demonstrated with poly(γ-propargyl-l-glutamate), to which azide-terminated PEG with molar masses in the range 750–5000 g mol–1 has been attached (Figure 4). (54) The conjugates adopt a α-helical secondary structure. Alkyne–azide click chemistry was used to link PEGA to a GGRGDG peptide. (55)

Figure 4

Peptidomimetics have also been linked via click ligation to PEG, for example, using alkyne-functionalized peptidomimetics and α,ω-diazido-PEG. (56) Other examples of click reactions are provided in the following sections.

2.1.3 Noncovalent Coupling

Reversible coupling can be achieved using noncovalent chemistries. For example proteins (or peptides) may be tagged with the commonly used (in affinity chromatography) hexahistidine motif, which is recognized by the complementary nickel–nitriloacetic acid (Ni-NTA) complex on end-modified PEG. (57) This has been proposed as a facile, reversible PEGylation method for the screening of therapeutic proteins in vivo.

The interaction between heparin and a heparin-binding growth factor (VEGF) has been used to prepare noncovalent hydrogels of heparin-terminated four-arm PEG and VEGF. (58) The presence of VEGF receptors on endothelial cells lead to erosion of the hydrogels, producing a biomaterial responsive to a specific receptor cue.

The biotin–streptavidin complex has been used to couple polymers and proteins noncovalently; (1) however, we are not aware of the use of this high-binding-affinity system to prepare PEG–peptide conjugates.

2.2 Peptide Synthesis from PEG Chains (“Grafting to”)

N-Carboxyanhydride (NCA) polymerization from PEG macroinitiators enables the synthesis of a range of polymer–peptide conjugates. (59, 60) NCA of homopolypeptides such as poly(l-proline) as well as copolymers from PEG macroinitiators has been demonstrated, and self-assembly in the solid state was noted. (61) The polyproline II secondary structure was adopted in water. NCA polymerization has also been used to polymerize l-alanine from amino-terminated PEG. (62) It has also been used to prepare PEG-poly(Z-l-lysine) diblocks with dodecylamine or α-naphthylamine (as a fluorophore) between the two blocks. (63)

A variety of chemistries have been employed using PEG as a cleavable support for peptide synthesis (prior to cleavage, PEG–peptide conjugates are produced). (64) PEGylated resins are available commercially (see below). The PEG chain is connected to the peptide via a linker, of which examples include those that are photocleavable, acid- or base-labile, or thiol-labile. A method to prepare 9-fluorenylmethyl chloroformate (Fmoc)–PEG amino acids to incorporate PEG spacers within peptides, compatible with solid-phase synthesis methods, has been employed by several groups using PEG–amino acids with 3–8 spacers. (65-67) This builds on earlier work in which shorter ethylene glycol spacers were incorporated into disulfide-bridged peptides. (68)

A method to convert synthesized PEG (up to EG29) diols into Fmoc–amino acids compatible with solid-phase peptide synthesis has been reported. (69) These were used to synthesize (on resin) a peptide–PEG17–folate conjugate incorporating a folic acid cysteine-targeting ligand as well as an N-terminal amphipathic peptide with high transfection efficiency. The peptide–PEG17 conjugate was coupled to the cysteine-folate ligand using maleimide coupling chemistry. Monodisperse Fmoc–PEG–COOH with a range of PEG (PEG2–PEG36) are now available commercially (from Quanta Biodesign) (17) and have been used to prepare PEG–peptide conjugates such as PEG–FFKLVFF–COOH. (70)

PEG-functionalized resins are commercially available under the trade name Tentagel PAP (from Rapp Polymere). (71-73) These comprise cross-linked polystyrene beads to which PEG chains are preattached with a labile benzyl ether linkages. A range of PEG molar mass beads is available. The method was originally developed to attach PEG to improve the solubility of hydrophobic peptides or lipopeptides, (71) although recent attention has focused on the interesting self-assembly properties arising from the amphiphilicity of PEG–peptide conjugates.

Mutter et al. developed liquid-phase synthesis of peptides up to 20 residues from linear PEG (5–20k) supports, (74) although the method has yet to find widespread application. (75) These authors prepared PEG with several acid- and photocleavable linkers for the synthesis of a series of test peptides.

PEG chains have been used as supports for liquid-phase peptide synthesis, as reviewed in detail elsewhere. (64) In general, the synthesis has been carried out in organic solvents, although some examples in water have been reported (using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) instead of DCC). Figure 5 shows a direct esterification method to produce, in this case, a bifunctional conjugate. This method has been used with PEG up to PEG20k and for up to 14-residue peptides. (76) Fragments of the peptide secretin have been synthesized on a PEG10k support, which provides a solubilizing protecting group. (77-79) The stepwise synthesis of a fragment of insulin B was performed using a PEG3k support. (80) Liquid-phase methods were used to prepare PEG–peptides incorporating PEG10k and oligo-glycine peptides with 1–9 glycine repeats (β-sheet structures were observed for conjugates with longer glycine sequences at low dilution in chlorinated solvents). (81) Similarly, t-Boc-Xn-G-PEG conjugates were prepared with X = Ala, n = 1–8 or X = Val, n = 1–6. (82)

Figure 5

2.3 Polymer Synthesis from Peptide (“Grafting from”)

In the grafting from method, initiators for polymerization methods such as ATRP may be incorporated at the peptide termini. This method has been widely used to prepare protein–polymer conjugates, (83) although this is outside the scope of the present review. The technique has also been used for polymer–peptide conjugates. (84) In one example, two different ATRP initiators were attached at the termini of a model matrix metalloprotease (MMP) substrate 11-residue peptide, and POEGMA was polymerized from the C terminus and poly(N-isopropylacrylamide) (PNIPAM) was polymerized from the N terminus. (85) This conjugate self-assembled into micelles under suitable conditions in aqueous solution, and enzymatic degradation of the peptide linker was demonstrated.

A reversible addition–fragmentation transfer (RAFT) agent has been appended to a solid-phase-synthesized peptide and used as a macroinitiator for RAFT and also via functionality shift for ATRP, which may be used to polymerize PEG (although this study involved polymerization of n-butyl acrylate). (86)

2.4 Synthesis of PEG Side-Chain Polymer–Peptide Conjugates

As an alternative to the attachment of linear PEG to peptides, it is often synthetically convenient to use PEG-rich polymers bearing PEG or oligo-ethylene glycol side chains such as acrylates or methacrylates, which are conveniently prepared by living radical polymerization methods such as RAFT (and ATRP, although this is also used directly to synthesize PEG). (16) Polymers with PEG “grafts” enable the incorporation of PEG of a defined length at a controlled density along the polymer backbone. Furthermore, certain PEG acrylates show very well-defined LCST behavior (i.e., they undergo a sharp coil–globule transition on heating). Incorporation of PEG in the monomer (“grafting through”) is generally advantageous compared to grafting to, where quantitative functionalization can be hard to achieve.

Short PEG chains have been grafted to PBLG, (87) and PEG chains with Mw = 1k, 2k, and 3k have been grafted to poly(l-lysine), (88) with both showing moderate grafting densities. Poly(l-glutamates) with oligo-ethylene glycol (1, 2, or 3 repeats) have been prepared by NCA polymerization of ethylene glycol-modified amino acid monomers. (89) The PEGylated polymers exhibit LCST behavior in water. The LCST transition can be tuned via the composition (PEGylated monomer content) of the copolymer or the enantiomeric composition. (89) Cysteine-based C and CF peptides have been attached to polybutadiene chains in a PEO–polybutadiene diblock via free-radical addition. (90) Spherical or worm-like micelles or vesicles were observed depending on the hydrophobicity of the conjugate copolymers.

Langer’s group studied polyhistidine polymers with PEG grafts (PEG5k) for gene delivery (plasmid DNA) and compared these conjugates to linear PEG–polyhistidine diblocks. (91) Steric hindrance because of PEG led to a direct relationship between PEG content and the size of the complexes formed. The transfection efficiency was good, and cytotoxicity to macrophages was low.

2.5 Synthesis of Tethered PEG peptides

PEGylated peptides may be tethered to solid supports, for instance, to create functionalized surfaces for cells. Ulijn’s group developed a system comprising amine-terminated PEG tethered to silica surfaces in which initially blocked cell adhesion peptide motifs were subsequently exposed via enzymatic cleavage of the terminal blocking group. (92, 93) In a first example, PEGA-coated surfaces were created with reactive amine groups for the sequential coupling with Fmoc–amino acids to produce PEGA–DGRF–Fmoc. Because proteases such as α-chymotrypsin cleave on the carboxylic side of phenylalanine residues, they can be used to remove the blocking Fmoc unit, as was demonstrated, leading to the spreading of osteoblast cells. (92) In a second example, Fmoc–amino acids were then coupled to the PEG–amine to produce PEG–DGRAAFmoc. (93) The Fmoc unit serves as a blocking group that can be removed by enzymatic cleavage at the alanine residues using elastase, thus exposing the cell adhesion RGD motifs. PEG/RGD-based peptide conjugates grafted to solid substrates have been studied as supports for cell growth and differentiation. (51, 93)

3 Self-Assembly of β-Sheet Peptide–PEG Conjugates

Lynn and co-workers investigated the fibrillization of conjugates of PEG with amyloid β (Aβ) peptide fragments (Figure 6). (94) Specifically, they studied the self-assembly of Aβ(10–35)–PEG3k. In contrast to the Aβ(10–35) peptide itself, conjugation to PEG was found to enhance the solubility and led to concentration-dependent reversible fibrillization. The fibril dimensions (of the peptide core and PEG corona) were determined via contrast matching small-angle neutron scattering (SANS) experiments, (95) and the pH dependence of these parameters was also examined. (96)

Figure 6

Conjugates comprising an N-terminal alkyl chain attached to the amyloid-forming hexapeptide KTVIIE and C-terminal PEG3000 were observed to form fibrils. These could be disassembled by removal of the alkyl chains via a UV-active photolabile nitrobenzyl group. (97)

A diblock conjugate incorporating the peptide QQKFQFQFEQQ and a PEG3.7k block self-assembles into β-sheet fibrils. (98) The peptide is designed as a transglutaminase substrate for enzymatic biofunctionalization of the supramolecular structure. A diblock conjugate containing PEG440 and the truncated peptide KFQFQFQ also formed fibrils (the corresponding peptide–PEG–peptide conjugate was not soluble in water), although the analogous peptide-terminated triblock did not (because of insolubility in aqueous solution). The morphology of fibrils formed by the PEG conjugates differed from those of the parent peptides. (98)

PEGylation can hinder β-sheet formation. This is exemplified by a study on a designed alanine-rich peptide. (99) The peptide self-assembles into helices under ambient conditions at acidic pH but converts into β-sheets at high temperature. PEGylated peptides (conjugated to PEG5k or PEG10k) show similar behavior; however, β-sheet formation at elevated temperature is slowed, and there is reduced cooperativity in the thermally induced unfolding. (99)

Nematic and hexagonal columnar-phase formation in aqueous solution by the peptide–PEG conjugate FFKLVFF–PEG3k was also noted. (73, 100, 101) The peptide is based on a fragment of the amyloid β peptide, KLVFF, Aβ(16–20) extended at the N terminus by two phenylalanine residues. This peptide amphiphile forms core–shell cylindrical fibrils. The PEG coronas around the peptide fibrils are expected to mediate the purely repulsive interactions because attractive interactions can result from interpenetration of coronal chains. It is interesting to consider how the balance of electrostatic and steric (soft elasticity from polymer corona) interactions influence the packing constraints that lead to the nematic and hexagonal columnar phases observed in these PEG–peptide conjugates. Nematic ordering has also been reported for a PEG–peptide amphiphile containing a two-tailed peptide motif attached to a rigid aromatic branch point. (102) This forms tape-like aggregates that show nematic ordering at higher concentration.

The effect of PEG molar mass on the self-assembly of FFKLVFF–PEG with PEG1k and PEG2k (both C-terminal PEG) as well as PEG10k–FFKLVFF (N-terminal PEG) was also investigated. (103) The three FFKLVFF–PEG hybrids form fibrils comprising a FFKLVFF core and a PEG corona. The β-sheet secondary structure of the peptide is retained in the FFKLVFF fibril core. At sufficiently high concentration, FFKLVFF–PEG1k and FFKLVFF–PEG2k form a nematic phase, whereas PEG10k–FFKLVFF exhibits a hexagonal columnar phase. Simultaneous small-angle neutron scattering/shear flow experiments were performed to study the shear flow alignment of the nematic and hexagonal liquid crystal phases. On drying, PEG crystallization occurs without disruption of the FFKLVFF β-sheet structure, leading to characteristic peaks in the X-ray diffraction pattern and FTIR spectra (also see Section 7). The stability of β-sheet structures was also studied in blends of the FFKLVFF–PEG conjugates with poly(acrylic acid) (PAA), which can hydrogen bond to PEG, potentially modifying self-assembly. Although PEG crystallization was observed only up to 25% PAA content in the blends, the FFKLVFF β-sheet structure is retained up to 75% PAA.

The influence of the PEG chain length on the self-assembly of N-terminal PEGylated FFKLVFF–PEG conjugates has been examined. (70) Three (EG)n–FFKLVFF–COOH conjugates were studied, where EG denotes ethylene glycol and n = 5, 11, or 27 is the PEG degree of polymerization. Importantly, these samples are based on commercially available (Quanta biodesign) monodisperse ethylene glycol oligomers. For these model conjugates, where PEG polydispersity effects are eliminated, X-ray diffraction revealed different packing motifs dependent on PEG chain length (Figure 7). This is correlated to remarkable differences in self-assembled nanostructures depending on PEG chain length. The control of strand registry points to a subtle interplay between aromatic stacking and electrostatic and amphiphilic interactions.

Figure 7

The influence of PEG molar mass on the self-assembly of FFFF–PEG has been examined. Nanotubes were observed for a conjugate with a low PEG molar mass (350 g mol–1), (104) whereas at higher PEG molar mass (studied in the range 1.2– 5k), fibrils are observed. (104, 105) The nanotubes comprise antiparallel β-sheets that are stabilized by π–π stacking of the aromatic residues. (106) Soft hydrogels arising from nanotube entanglements are reported at higher concentration. (106)

The self-assembly into β-sheet fibrils of the conjugate DGRFFF–PEG containing the RGD cell adhesion motif (attached N terminally), three F residues to ensure amphiphilicity, and PEG3k in aqueous solution has been observed. (107) The adhesion, viability, and proliferation of human corneal fibroblasts were examined for films of the conjugate on tissue culture plates (TCP) as well as low-attachment plates. On TCP, DGRFFF–PEG3k films prepared at sufficiently low concentration are viable, and cell proliferation was observed. However, on low-attachment surfaces, neither cell adhesion nor proliferation was observed, indicating that the RGD motif was not available to enhance cell adhesion. This was ascribed to the core–shell architecture of the self-assembled fibrils with a peptide core surrounded by a PEG shell, which hinders access to the RGD unit.

Tape structures were observed for a PEG–peptide containing PEO68 and a peptide incorporating a (TV)4–ester–VG “switch” peptide sequence that forms β-sheet tape structures upon increasing the pH to 6.2 in aqueous solution because of an O → N acyl switch. (108) The authors observed fibrils for a related conjugate comprising PEG linked via a dibenzofuran-based linker to two tails containing the VTVT peptide. (109) It was pointed out that short peptide end groups can be used to direct the ordering of PEO into fibrillar structures.

4 Self-Assembly of Coiled-Coil and α-Helical Peptide–PEG Conjugates

Klok and co-workers have observed that the coiled-coil structure of designed peptides can be retained upon conjugation to PEG. (110) The peptides are based on a de novo sequence designed by Hodges et al. (111) and incorporate two heptad LAEIEAK sequences conjugated to PEG with n = 15 or 40. An equilibrium between unimers and dimeric and tetrameric coiled-coil aggregates was proposed, with increases in concentration or temperature favoring the more aggregated state (Figure 8). (110) The close folding of PEG around the coiled-coil structures was indicated by electron paramagnetic resonance (EPR), which also pointed to a parallel alignment of the helices, as least for the dimeric species. (112)

Figure 8

A later study focused on the secondary structure in a related series of coiled-coil peptides with substitutions among the charged residues, which compared PEG conjugates (PEGn, n = 15 and 40) and the parent peptides. (113) The stability of the secondary structure against changes in concentration and pH was analyzed. Substitutions of E and K residues did not influence the ability of the peptides and conjugates to form coiled-coil structures. The biocompatibility of the conjugates was also examined via hemolysis assays. The stability of PEGylated conjugates (compared to the parent peptide) based on this type of coiled-coil peptide against changes in pH and concentration was studied via CD spectroscopy. (114) In addition, several switch peptides, (115) which are designed to switch between α-helical and β-sheet structure depending on pH, were investigated in comparison with their PEG conjugates. (114) The conjugates of the coiled-coil peptides exhibited reduced concentration-dependent changes in α-helix content, although the coiled-coil structure was retained. The switch peptide-PEG conjugates exhibited enhanced stability of the coiled-coil structure against pH increase. Solid-state structures were also examined by TEM and AFM.

The formation of heterodimers between pairs of E- and R-rich coiled-coil peptides was not impaired by their incorporation into alternating multiblock PEG copolymers. (23)

The influence of PEG2k on the secondary structure of a series of peptides (VSSLESK)n with n = 3–6 was examined using CD spectroscopy. (116) The conjugates with longer peptides n = 5 and 6 adopted a two-stranded α-helical structure in aqueous solution. PEG was found not to interfere with the formation of α-helices for the shorter conjugates as well. The thermal stability of the conjugate with n = 6 was higher than that of the corresponding peptide.

The conformation of the PEG chain attached laterally to coiled-coil 3- or 4-helix bundle peptides (30-mers) via cysteine residues using maleimide-functionalized PEG was examined by SANS. (117) The form factor was described by a cylinder (for the coiled-coil structure) and a Gaussian coil for the PEG conformation.

Complexation of PEG–K and PS–E containing α-helical peptides termed E (G(EIAALEK)3) and K ((KIAALKE)3G) with opposite charges led to the formation of heterocoiled coils and thus a noncovalently linked PS–E/K–PEG triblock (Figure 9), which self-assembles into rod-like micelles. (118) In related work, the coiled-coil conjugate K–PEG77 was prepared as part of a study on complexation with diblocks containing oppositely charged coiled-coil sequences (EIAALEK)3 and PBLG. (119) Vesicle formation was observed in the case of complexes formed with lower molar mass (PBLG36) diblocks.

Figure 9

Oligo-ethylene glycol (EG2) has been attached to a protein with a grafted minimal p53 tumor suppressor peptide sequence to improve the solubility of the α-helix-stabilized self-assembled peptide nanostructure. (120)

5 Self-Assembly of Peptide–PEG–Peptide, PEG–Peptide–PEG, and Related Conjugates

Telechelic peptide–PEG–peptide conjugates containing hydrophobic peptides offer the possibility to create hydrogels by physical association of the terminal peptide domains. This has been further enhanced with disulfide cross-linking (Figure 10) of conjugates based on terminal peptides incorporating designed six-heptad leucine zipper coil motifs. (121) The unstructured midblock comprises ((AG3)PEG)10. Intermolecular interactions between these triblock constructs was later probed by AFM force measurements under extension/retraction, and the adhesive interactions between molecules were found to increase upon lowering the pH. (122) The influence of the end blocks on the erosion rate of this type of leucine zipper peptide hydrogel was examined. (123) Erosion was much slower for hydrogels constructed from triblocks with dissimilar end blocks compared to those with the same end groups, an effect ascribed to reduced chain looping in the heterotelechelic constructs. (123) Similar constructs were later developed to create cell adhesive materials by incorporation of the RGDS cell adhesion motif or heparin-binding motifs into the terminal coiled-coil peptide domains. (124) Furthermore, cross-linked coatings could be produced by 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC)-mediated cross-linking of proximal Glu and Lys residues.

Figure 10

A triblock comprising PEG end blocks and a central peptide block of silk-like tandem (AG)3EG repeats forms fibrils in aqueous solution irrespective of PEG with a molar mass in the range 750–5000 g mol–1 is used. (35)

In another example, collagen-mimetic peptides were attached to four-arm PEG. Hydrogels were formed via physical cross-links mediated by thermally reversible triple helical assembly of collagen-mimetic peptides. (125) Hydrogel formation by a peptide–PEG–peptide conjugate containing peptides derived from the coiled-coil region of fibrin has been investigated. (126) The hydrogels may have application in tissue engineering and/or wound healing.

A thermoplastic hydrogel has been produced from a hexablock copolymer containing PEO and PBLG, with a complex architecture comprising four PBLG-bearing arms, two of which also contain PEO. (127) The thermoplastic properties may be due to microphase separation within the concentrated hydrogels.

The self-assembly in aqueous solution of peptide–PEG–peptide conjugates comprising aromatic dipeptides linked telechelically to PEG1.5k has been examined. (128) The role of capping Fmoc (N-fluorenyl-9-methoxycarbonyl) units at one or both termini was also examined. A self-assembled β-sheet fibril-based hydrogel was identified for a conjugate containing dityrosine end groups (and a C-terminal Fmoc unit), which exhibits a gel–sol transition near body temperature (Figure 11). (128) This thermoresponsive PEG-based biofunctional hydrogel is expected to have diverse potential uses in delivery or diagnostics for biomedical applications. Another group later prepared similar telechelic conjugates of desaminotyrosine or desaminotyrosyl-tyrosine with a linear PEG3k midblock and also four-armed–PEG conjugate. A critical aggregation concentration was noted for the linear midblock conjugate, pointing toward self-assembly at high concentration. (129)

Figure 11

As mentioned in Section 2.2, Fmoc–PEG amino acids have been developed that incorporate oligo-ethylene glycol spacers. Boumrah et al. developed the synthesis of these residues, consistent with solid-phase peptide synthesis methods, and incorporated triethylene glycol repeats into their disulfide-bridged atrial natriuretic factor peptide analogues. (68) A tyrosine kinase-based tetrapeptide has been incorporated within pYTGL–(ethylene glycol)n–pYETL conjugates (pY, phosphorylated tyrosine) with n = 4 or 6. (67) The spacer leads to an affinity between the two linked phosphopeptides that is comparable to that between the two sequences in the full native peptide, showing that the PEG spacer can substitute for the intervening amino acids. PEG spacers have been incorporated into multivalent (four-arm dendron) peptide constructs with peripheral cyclo(RGDfE) integrin-targeting units attached via hexaethylene glycol spacers linked to terminal lysine units. (65) Peptides incorporating PEG spacers have been developed for applications in gene delivery. A DNA-binding lysine sequence (K16) was separated from a disulfide-bridged integrin-binding sequence (CRRETAWAC) by oligo-ethylene glycol spacers with 3–8 repeats. (66) The PEG spacer delivered enhanced stability in buffer compared to control (peptide lacking PEG spacer).

A L4K8L4VPRGS–PEO conjugate containing a VPRGS substrate for thrombin self-assembles into β-sheet fibrils in response to addition of the enzyme (in contrast to a peptide containing a scrambled peptide sequence). (130) Heterotelechelic conjugate PS–L4–PEO (PS, polystyrene), a so-called peptide-inserted triblock copolymer with a tetraleucine β-sheet-forming midblock, forms vesicles in aqueous solution. (131) This peptide was prepared starting from a Tentagel PAP PEGylated resin (Section 2.2). ATRP of PS was then performed from a brominated L4–PEG macroinitiator.

The solid-state morphology of a silk-based multiblock ABCD copolymer where A, GAGA; B, EO5; C, AGAG; and D, EO13 has been investigated (P1, Figure 12). (132) In these copolymers, PEG replaces the amorphous domains within the native silk structure (Figure 12a). Microphase separation was noted with 20–50 nm peptide domains within a continuous PEO matrix. In later work, the solid-state structure of ABCD multiblocks containing the Bombyx mori (silk worm) sequence GAGA was compared to that of multiblocks containing the Nephila clavipes spider silk-type sequence AAAAAA (P2 and P3, Figure 12). The formation of antiparallel β-sheets was observed in both cases. The copolymers containing the oligo-alanine sequence exhibited a higher modulus than those of the GAGA copolymers, and this was ascribed to the presence of physical cross-links. (133)

Figure 12

6 Self-Assembly of PEG–Peptide Conjugates Containing Synthetic Homopolypeptides

6.1 Self-Assembly in the Solid State

In the solid state, PBLG–PEG conjugates undergo microphase separation of the components of the block copolymers. This leads to hierarchical ordering because of the additional shorter length scale periodicities resulting from the packing of the PBLG, its secondary structure, and the crystal structure of PEG (if the molar mass is sufficiently large). Floudas et al. reported that the phase behavior of PBLG–PEG–PBLG triblocks depends on the composition, specified as the peptide volume fraction, f. (134) For copolymers with low peptide volume fractions (f < 0.4), microphase separation was noted along with α-helical or β-sheet ordering of PBLG (depending on chain length) and chain-folding of crystalline PEG. (134) In contrast, for f > 0.4, no regular microphase-separated structures were noted, and only an α-helical secondary structure of PBLG was observed. Our group subsequently examined the microphase-separated morphology within the same series of triblock copolymers via SAXS, AFM, and TEM experiments. (135) A phase diagram was presented, including ordered structures that were observed for some samples with f > 0.4. The PBLG adopts a mixture of α-helical and β-sheet structure for low chain lengths (n < 18), but it is purely α-helical when the degree of polymerization is larger.

Microphase separation into a lamellar structure was observed in the solid state (solvent cast samples) for a series of PBLG–PEO-PBLG triblocks containing 25–75% PBLG. (136) The WAXS pattern was dominated by the contribution from the α-helical PBLG. This group also observed microphase separation in the films cast from a PCLL–PEO–PCLL (PCLL, poly(ε-benzyloxycarbonyl-l-lysine)) triblock. (137) Lamellar ordering has been reported in the solid state of di- and triblock copolymers of PEO and poly(dl-valine-co-dl-leucine). (138)

Ordering in the solid state has been observed for complexes of a PEG–PLGA (PLGA, poly(l-glutamic acid)) diblock with n-alkylamines (octadecylamine, dodecylamine, and octylamine). (139) Hierarchical ordering was observed, ranging from the length scale associated with PEG crystallization to the PLGA–alkylamine complexes (lamellar structure for the octadecylamine complex) as well as microphase separation of the block copolymer (also lamellar).

The morphology and mechanical properties in the solid state of a PBLA–PEO–PBLA (PBLA, poly(benzyl l-aspartate)) triblock have been examined. (140) The PBLA chains adopted a α-helical conformation, whereas the PEG also crystallized, although an increase in β-sheet content of the PBLA chains was noted upon cooling following heating above the PEO melting temperature.

Microphase separation in the solid state was suggested as a result of the observation of two distinct thermal transitions by DSC for a poly(l-alanine)–PEG conjugates. (62)

6.2 Self-Assembly in Solution

In solution, PEG–polyelectrolyte diblocks based on charged synthetic peptides can self-assemble into vesicles. In one study, the kinetics of the growth of PEG–poly(aspartic acid) unilamellar vesicles via 2D supramolecular polymerization has been monitored by dynamic light scattering. (141) A four-armed H-shaped (PzLL)2–PEG–(PzLL)2 conjugate (PzLL, poly(ε-benzyloxylcarbonyl-l-lysine)) self-assembles into vesicles in aqueous solution. (142) Furthermore, the vesicles can be loaded with the hydrophobic drug doxorobucin and delivered to human breast cancer cells.

PEG–peptide diblock copolymers can form polyion complex micelles (PICs) by pairwise association of diblocks containing oppositely charged blocks. In one example, PICs were observed in aqueous solutions of PEG–poly(aspartic acid) (P(Asp)) and PEG–poly(lysine) (P(Lys)). (143) Vesicle formation by complexation of a pair of diblocks containing oppositely charged blocks has been observed. (144) The vesicles, termed PICsomes, were formed by complexation of PEG–P(Asp) (anionic) with PEG–P(Asp-AE) or PEG–P(Asp-AP), where these cationic blocks are prepared by aminolysis of poly(β-benyl-l-aspartate). These vesicles were shown, by fluorescence imaging, to be semipermeable. Triblock copolymers can self-assemble into three-layer micelles. In one example, micelle-forming PEG–PMPA–PLL copolymers were investigated for DNA condensation. (145) The PLL forms the micelle core, with a poly(3-morpholinopropyl) aspartamide (PMPA) buffering inner layer and a biocompatible PEG outer layer. Enhanced transfection of DNA into HeLa cells was observed for the three-layer micelles compared to those from PEG–PLL.

A thermoresponsive conjugate of PEG and poly(γ-(2-methoxyethoxy)esteryl-l-glutamate) has been prepared by ring-opening polymerization. (146) Extended annealing times drive a transition in peptide secondary structure from helical to β-sheet, leading to a concomitant nanostructure transition from worm-like micelles to nanoribbons.

The conformational properties (α-helical structure) of PBLA in a PEO–PBLA diblock in organic solvents has been examined by NMR and optical rotation measurements. (147)

Charged polypeptide blocks within self-assembling block copolymers can form complexes with oppositely charged species, leading to novel nanostructures. Micelles were observed for PEO–PLL diblocks forming complexes with retinoic acid. The α-helix formed by PLL is stabilized over a wider pH range within the complexes. (148) This was also observed in the solid state and is in contrast to the mixed α-helix/β-sheet structure for the PLL block in the uncomplexed copolymer. The micelle core contains a smectic-like PLL–retinoic acid complex. Nanoparticles were observed upon complexation of a PEO–PGlu (PGlu, poly(l-glutamate)) conjugate with diminazene. (149) Under pH 7.4 conditions where the PGlu adopts a random-coil structure, complexation led to a transition to α-helical structure.

7 PEG Crystallization Effects on Self-Assembly

For PEG of sufficiently high molar mass, crystallization is observed in the dry state. This can influence the nanostructure observed for PEG–peptide conjugates, for example, by TEM, where the specimen is dried. We have found that PEG crystallization can overwhelm peptide fibrillization if the latter is not strong. This was investigated for a series of PEG–peptide conjugates: FFKLVFF–PEG3k, AAKLVFF–PEG3k, and KLVFF–PEG3k. Fibrillization, as characterized in particular by the presence of a cross-β amyloid structure as well as by the macroscopic morphology, was disrupted for the conjugate containing the weak fibrillizer KLVFF, whereas fibrils were retained (on drying) for the conjugates containing the stronger fibrillizers AAKLVFF and FFKLVFF containing additional hydrophobic AA or FF units. (150, 151) The formation of spherulites resulting from PEG crystallization is observed for the KLVFF conjugate but not for the other two (Figure 13). (151) The fibrillization strength depends on the number of hydrophobic, particularly aromatic, residues; indeed, several tables summarizing different assessment methods of β-sheet propensity show that phenylalanine particularly favors this secondary structure. (152-155) For AAKLVFF–PEG and FFKLVFF–PEG, the alignment of peptide fibrils also drives the orientation of the attached PEG chains. (150) These results highlight the importance of the antagonistic effects of PEG crystallization and peptide fibril formation in the self-assembly of PEG–peptide conjugates.

Figure 13

As mentioned in Section 3, the self-assembly and bioactivity of the peptide–polymer conjugate DGRFFF–PEG3k has been investigated. (107) At sufficiently high concentration, self-assembled β-sheet fibrillar nanostructures were observed. The fibrils are observed despite PEG crystallization which occurs on drying. This suggests that DGRFFF has an aggregation tendency that is sufficiently strong not to be hindered by PEG crystallization.

8 Enzyme-Responsive PEG–Peptide Conjugates

An enzyme-responsive hydrogel has been prepared by Cu-catalyzed click chemistry from four- or eight-armed PEG capped with alkyne end groups and a bis-azido-funtionalized protease-sensitive peptide. (156) The peptide sequence d-Ala-Phe-Lys is sensitive to plasmin and trypsin, leading to biodegradable hydrogels.

A PEG–peptide micelle system that can be enzymatically cleaved, leading to the release of unaggregated amyloid-based peptides, has been reported. (157) The βAβAKLVFF–PEG3k conjugate incorporates the βAβAKLVFF peptide based on the amyloid β peptide sequence Aβ(16–20) with two N-terminal β-alanine residues. The enzyme α-chymotrypsin cleaves the conjugate to produce βAβAKLVF and F–PEG3k (Figure 14). (157) The hexapeptide does not aggregate into β-sheet structures, in contrast to the heptapeptide βAβAKLVFF that forms well-defined β-sheet ribbons. (158, 159)

Figure 14

Following a similar concept, it has been shown that PEG–peptide micelles with a peptide substrate (GPLGVRG) for MMP2 can be cleaved enzymatically, thus removing the PEG coating and leaving polyaspartamide-based nanoparticles that can form polyplexes with DNA. (160) These PEG-free polyplex micelles can be taken up by cells (with high endosomal escape) for gene delivery applications.

9 PEG–Peptides for Drug Delivery

It is now well-established that attachment of PEG improves the circulation time of PEGylated molecules as well as reducing renal clearance. Extended circulation lifetimes result from reduced recognition by the host response system (reduced immunogenicity) and reduced enzymatic degradation. This concept has been widely employed in the development of therapeutic materials, a subject that will not be reviewed here; instead, the focus will be solely on PEG–peptides used for drug delivery applications.

A review by Torchilin provides a number of examples where PEG–peptide micelles have been loaded with pharmaceutical compounds for drug delivery applications. (161) Kataoka and co-workers have also reviewed PEGylated block copolymers, including those incorporating polypeptides, in drug delivery and other biological applications. (162-165)

PEG–peptide conjugates containing poly(aspartic acid) (P(Asp)) have been employed by the Kataoka group and others in several studies on delivery of model drugs. These conjugates form PIC micelles that can be used to entrap drugs, enzymes, and other molecules through electrostatic interactions with the polyanionic peptide block. The enzyme lysozyme (positively charged in aqueous solution) was trapped within such micelles. (166) Later, hydrophobic aromatic groups were attached to the N-terminus of PEG–P(Asp) polymers to enhance association between the PIC micelles and lysozyme; this led to enhanced stability against increased NaCl concentration. (167)


Peptide PEGylation

PEGylation is the process of covalently attaching polyethylene glycol (PEG) chains to peptides, proteins or other biomolecules. PEGs are polymers that are nonionic, nontoxic, biocompatible and highly hydrophilic.

PEGylation of peptides can enhance therapeutic properties due to their increased solubility (for hydrophobic peptides), prolonged half-life through reduced renal clearance, and masked antigenicity for minimum immune response in the host. Thus, PEGylated peptides may overcome many of the challenges for peptide drug candidates.

Therefore, PEGylation has evolved into a widely used method for improving the stability and bioavailability of peptides. Regulatory authorities have approved PEGylated drugs of varying PEG chain lengths and with MWs ranging from 5‑40 kDa.

PEGs exceeding 60 atoms in length are generally not well defined and consist of a mixture of different oligomer sizes in broadly or narrowly defined molecular weight (MW) ranges. For this reason they are also referred to as polydispersed PEG. For example ’PEG 10000’ denotes a mixture of PEG molecules (n = 195 to 265) having an average MW of 10,000.

For shorter PEGs, polyethylene glycol derivatives composed of a precisely-defined numbers of PEG units and therefore of defined length (MW) are available. They are referred to as monodispersed PEG. These shorter PEGs are frequently applied as linkers or spacers. PEG spacers have become quite versatile tools to attach all sort of sensitive molecules (e.g. fluorophores) to peptides, or to create homo- or hetero-dimeric peptides.

For PEGylated peptides, the PEG is usually attached to the peptide using standard amide bond formation but other linkage chemistry are possible as well (e.g. thiol-maleimide, oxime ligation, click chemistry).

Pepscan has extensive experience with each of these PEG conjugation chemistries. Contact us for more information about the possibilities to incorporate PEGs into your peptides.


PEG Carrier

PEG Carrier

PEG Spacer

PEG Spacer


PEG2 Spacer or AEEA
(8-amino-3,6-dioxaoctanoic acid)


PEG3 Spacer
(12-amino-4,7,10-trioxadodecanoic acid)


PEG4 Spacer
(15-amino-4,7,10,13-tetraoxapenta-decanoic acid)

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From Synthesis to Characterization of Site-Selective PEGylated Proteins


The binding of proteins, peptides, enzymes, antibody fragments, oligonucleotides, or small synthetic drugs to polymers has become a very useful method for improving therapeutic activity or decreasing the toxicity of these biological agents (Mishra et al., 2016). Among the polymeric materials, polyethylene glycol (PEG) is the most used for these purposes, mainly due to its high biocompatibility, low toxicity, and limited side effects (Gauthier and Klok, 2008). PEGs are water-soluble polymers approved by the Food and Drug Administration for use in oral, topical, and intravenous formulations (D’souza and Shegokar, 2016). It presents a structure of repeated units of polyether diols (either linear or branched) chemically formulated as HO-(CH2CH2O)n-CH2CH2-OH (Figure 1A), where each ethylene oxide residue has a molecular weight (MW) of 44 Da (Roberts et al., 2012). PEGylation refers to the covalent or non-covalent attachment of PEG to different molecules, such as proteins, macromolecular carriers, oligonucleotides, vesicles, and others to improve the pharmacokinetic (Milton Harris et al., 2001; Lee et al., 2013) and pharmacodynamic properties (Abbina and Parambath, 2018). The conjugation to PEG generates an increase in the hydrodynamic volume of the biomolecule of interest, creating a shield around it (Gokarn et al., 2012). This effect enables clearance by the renal system to be reduced, and therefore, the half-life is increased in the bloodstream (Milton Harris and Chess, 2003) concomitant with the increases in PEG molecular weight (Hamidi et al., 2006). Additionally, this approach has been used to improve the stability of some proteins (Yang et al., 2007; Jevševar et al., 2010; Lawrence and Price, 2016; Santos et al., 2019), as well as decrease the immune response against several biomolecules (Soares et al., 2002; Zheng et al., 2012; Sathyamoorthy and Magharla, 2017; Wu et al., 2017).

Figure 1 A general scheme of protein PEGylation at N-terminus. (A) PEG chemical structure based on linear polyethylene oxide repeating units. (B) Schematic representation of the N-terminal PEGylation reaction. The square represents PEG functional group that covalently binds to the terminal amine of the protein. PEG chain is end-capped with a terminal methoxy group to prevent reactivity and enzymatic attack upon administration in mammals.

Since the 1990s several PEGylated biopharmaceuticals (see Table 1) have been approved by the FDA, and some more are currently undergoing clinical trials (information available at Most of the approved PEGylated proteins were synthesized by non-site-specific chemical conjugation strategies, resulting in heterogeneous mixtures of multi-PEGylated (polydisperse) proteins due to the presence of several reactivity sites on the protein surface (Alconcel et al., 2011), requiring complex separation steps. In addition, protein PEGylation can lead to the loss of protein activity through several mechanisms that include the direct PEGylation of the active site or receptor binding site (Schiavon et al., 2000), the steric entanglement imposed by PEG chains that cause restricted movements (Kubetzko, 2005; Xiaojiao et al., 2016) and conformational changes in proteins (Chiu et al., 2010), among others. Also, recent research has revealed certain shortcomings related to highly PEGylated forms, such as activation of the immune system, non-degradability, and possible accumulations with high molecular weight PEGs. These are strong reasons that support the need to find site-selective PEGylation techniques, yielding homogenous mono-PEGylated products, a field that has garnered considerable in recent years. Although certainly the in vivo potency of therapeutic proteins can be affected by the PEGylation process, this decrease in activity can be largely balanced by their prolonged half-life in the circulation (Oclon et al., 2018). Site-selective PEGylation has been a very useful strategy for introducing PEG at specific amino acid sites in various proteins. Some methods like pH-controlled N-terminal selective acylation (Chan et al., 2006; Chan et al., 2012) or reductive alkylation (Kinstler et al., 1996; Marsac et al., 2006), the use of oxidizing agents (Kung et al., 2013; Obermeyer et al., 2014), the chemo-selective capability of catechol (Song et al., 2016) and transamination reaction (Gilmore et al., 2006) have been used to perform PEGylation at the N-terminus of proteins. Additionally, in recent years there has been a lot of work on using “grafting from” approaches to grow PEG from the surface of proteins via ATRP and RAFT polymerization methods (Quémener et al., 2006; Ameringer et al., 2013; Gody et al., 2015; Tucker et al., 2017). These approaches involve the direct generation of conjugates containing high molecular weight polymers (like PEGs) by directly growing the polymer from the protein surface (Wallat et al., 2014; Obermeyer and Olsen, 2015).

Table 1 PEGylated therapeutic peptides and proteins approved for clinical applications.

An illustrative example of success in chemical site-selective PEGylation is the case of Neulasta®, which is an N-terminally mono-PEGylated granulocyte colony-stimulating factor bearing a 20-kDa PEG (Molineux, 2004). The improved pharmacokinetic behavior of this biopharmaceutical allows administration only once per chemotherapy cycle compared to the first generation, Neupogen®, which is administered daily (Cesaro et al., 2013; Zhang et al., 2015).

Despite their robustness, chemical methods usually involve the use of excessive amounts of reagents and careful working conditions. Site-specific PEGylation of peptides and proteins has been approached successfully not only from the chemical point of view but also enzymatically. Several studies report the use of enzymes to conjugate PEG to peptides, proteins, and oligonucleotides (Sato, 2002; Mero et al., 2009; Da Silva Freitas et al., 2013; Sosic et al., 2014).These enzymes usually catalyze the reaction between the biomolecule of interest and a substrate analog containing a functional group (Dozier and Distefano, 2015), which can be the case of PEG. There are a number of more recent approaches aimed at achieving site-selective modification including the use of the Spytag/Spycatcher system (Schoene et al., 2014; Reddington and Howarth, 2015; Cayetano-Cruz et al., 2018; Kim et al., 2018); however, there is as yet insufficient information focused on these methods, and some studies are being developed in this direction.

The structural changes in protein characteristics after the attachment to PEG influence the subsequent characterization of PEGylated proteins. These changes result in an analytical challenge due to the heterogeneity of the PEGylation products and the degree of PEGylation, coupled with the complex protein structure (Hutanu, 2014). Several studies have reported the use of analytical techniques with differing degrees of difficulty—from colorimetric methods to more complex techniques such as computational approaches—for the characterization of PEGylated peptides and proteins.

In the present review, we have focused on summarizing both classic and novel chemical and enzymatic tools used for the covalent attachment of PEG in site-specific regions of peptides and proteins, as well on the main analytical methods for PEGylated molecule characterization.

Chemical Approaches for Site-Selective Pegylation

For the selective modification of specific amino acids in peptides and proteins, the knowledge of some characteristics about their primary structure is needed. An important physicochemical feature in proteins is the difference in pKa between the amino group of an N-terminal amino acid residue (∼7.6) and the amino groups in the side chains of lysine (∼10.5) and arginine (∼12) (Roberts et al., 2012). This difference allows the selective N-terminal modification of proteins based on pH control and the use of reductive agents like sodium cyanoborohydride. A useful strategy for the specific conjugation of peptides and proteins is based on the amino acid ratio in a protein being variable. Moelbert et al. reported the accessibility index on the surface of the 20 essential amino acids, which makes it possible to know the expression of these amino acids in different areas of the proteins in relation to their natural abundance (Moelbert, 2004). It has also been reported that short peptides/proteins (less than 50 residues) tend to over-represent glutamine and cysteine in the N-terminal region (Villar and Koehler, 2000). It is well known that single-chain proteins possess only one N-terminal residue, having a uniquely reactive site for chemical modification (Rosen and Francis, 2017). Therefore, as virtually all proteins present these functional groups, a number of valuable reactions have been developed for their selective modification (Boutureira and Bernardes, 2015).

The use of potassium ferricyanide as an oxidizing agent in o-aminophenol-performing N-terminal PEGylation has also been shown (Obermeyer et al., 2014). In 2016 Song et al. described an alternative strategy for PEGylation at the N-terminus of several proteins as well as two peptides based on the chemoselectivity of catechol (Song et al., 2016). More recently, Rosen and Francis described classical methods for the selective modification of N-terminal amino group under pH control. These methods include the selective acylation and alkylation of N-terminal amines at low-to-neutral pH and also transamination using pyridoxal-5′-phosphate aldehyde, which undergoes condensation with ε-amines from lysine side chains and N-terminal α amines to form imines (Gilmore et al., 2006; Rosen and Francis, 2017). Chen et al. demonstrated the ability of benzaldehyde to selectively modify native peptides and proteins on their N-termini. Preservation of the positive charge on the N-terminus of the human insulin A-chain through reductive alkylation instead of acylation leads to a 5-fold increase in bioactivity. They showed that under mild conditions, aldehyde derivatives and carbohydrates can site-specifically react with peptide and protein N-termini, providing a universal strategy for site-selective N-terminal functionalization in native peptides and proteins (Chen et al., 2017).

PEG-isocyanate is in the group of PEG reagents used for the site-specific modification of different proteins (Berberich et al., 2005; Sharma et al., 2017). The reaction takes place via the amine group to produce a stable thiourea linkage (Ganesan et al., 2015). For example, in 2009 Cabrales et al. generated PEGylated human serum albumin (PEG-HSA) by conjugating PEG-phenyl-isothiocyanate 3 and 5 kDa at primary amine groups of the HSA, enhancing the hydrodynamic volume of the protein and restoring intravascular volume after hemorrhagic shock resuscitation (Cabrales et al., 2008). Furthermore, Chen and He reported in 2015 the achievement of nanophosphors coated with PEG-isocyanate and polylactic acid (PLA) for paclitaxel delivery, resulting in a significant improvement and serving as a platform in the field of drug development (Chen and He, 2015). Lee et al. synthesized a dual functional cyclic peptide gatekeeper attached on the surface of nanocontainers by using PEG-isocyanate as a linker to enhance dispersion stability and biocompatibility. This allowed the active targeting of cancer cells with high CD44 expression together with the ability of triggered drug release (Lee et al., 2018). It is important to note that specific PEG-reagents like isocyanates have a short half-life in aqueous solutions (Erfani-Jabarian et al., 2012); thus, a stoichiometric excess of these reagents is necessary, causing difficulties in the removal of the remaining PEG.

A relevant report for one-step N-terminus-specific protein modification showed the stable and selective imidazolidinone product at the N-terminus, with 2-pyridinecarboxaldehyde (2PCA) derivatives (Macdonald et al., 2015). The main basis of this reaction is the nucleophilic attack of the neighboring amide nitrogen on the electrophilic carbon of the initially formed N-terminal imine (Koniev and Wagner, 2015). As an example, a 2PCA-functionalized polyacrylamide-based hydrogel has been developed for the immobilization of extracellular matrix proteins through the N-terminus to study their biochemical and mechanical influence on cells (Lee et al., 2016).

In the next section, we provide an overview based on reactions which can be used to selectively modify specific amino acids. Keeping that in mind, in some cases the described modification does not refer to the PEGylation itself, but the concept could be applied if the introduction of PEG reagents is desired. A mechanism corresponding to N-terminal PEGylation has been illustrated in Figure 1B, while general mechanisms of the site-selective chemical reactions are shown in Figure 2.

Figure 2 Schematic representation of chemical reactions described for the selective PEGylation of proteins.

Strategies for the Modification of Specific Amino Acids

Targeting Cysteine

Cysteine residues are interesting targets for residue-specific modification of peptides/proteins due to their low apparition frequency (Harvey et al., 2000). These are often found partially or fully covered within the protein structure, limiting their accessibility to chemical reagents (Thordarson et al., 2006). Proteins with N-terminal cysteine have been successfully modified through native chemical ligation (NCL) when, on the first and reversible step, a thioester intermediate is formed, which then undergoes a spontaneous S-to-N acyl shift and yields an amide bond (Johnson and Kent, 2006; Rosen and Francis, 2017). This methodology has been useful in the preparation of high complexity protein–polymer conjugates. For example, Zhao et al. described a PEGylated human serum albumin (HSA) in a site-specific method by taking advantage of the unusual chemical reactivity of the only one free Cys34 of the HSA molecule and the high specificity of PEG-maleimide for the protein sulfhydryl (– SH) groups. Targeting the distinctive free Cys34 through this site-specific PEGylation could generate a chemically well-defined and molecularly homogeneous product and may be also convenient in preventing dimerization (Zhao et al., 2012). Another technique which plays a major role in modern chemical biology and has been used for many applications is known as expressed protein ligation (EPL) (Mitchell and Lorsch, 2014; David et al., 2015; Liu et al., 2017). EPL constitutes an improvement for NCL, and in this case selectivity over lysine acylation was achieved through pH control, by using benzaldehyde derivatives bearing selenoesters to acylate N-terminal positions through acyl transfer (Raj et al., 2015).

As N-terminal cysteines are rare in nature, they frequently need to be introduced by genetic engineering (Nguyen et al., 2014a; Uprety et al., 2014; Gunnoo and Madder, 2016). Methionine aminopeptidase can take out the first methionine to liberate an N-terminal cysteine (Gentle et al., 2004), and some proteolytic enzymes that specifically cleave in the presence of cysteine residues in a protease recognition sequence (Busch et al., 2008; Wissner et al., 2013) have been used as strategies for the exposure of N-terminal cysteine and its subsequent bioconjugation.

Targeting Serine and Threonine

The presence of an N-terminal serine or threonine offers unique opportunities due to the high susceptibility of 1, 2-aminoalcohols to periodate oxidation, resulting in the formation of a glyoxylyl group, which can be used to form several linkages (Xiao et al., 2005). It has been shown that the extra periodate used to oxidize the N-terminal residues of proteins carries the risk of oxidizing other residues, such as cysteines and methionines, as well as causing unwanted oxidative cleavage of protein glycosyl groups (Huang et al., 2015). This is mainly the approach applied in classical research, based on targeting serines or threonines at the N-terminal position, which uses periodate oxidation to generate a glyoxylyl group. Gaertner et al. performed site-selective PEGylation of an N-terminal serine residue, which was oxidized using sodium periodate followed by subsequent oxime ligation with an aminooxy and hydrazyde PEG derivative (Gaertner and Offord, 1996). The modified proteins, interleukin (IL)-8, granulocyte colony-stimulating factor (G-CSF) and IL-1rα, fully retained their activity after PEGylation (Krall et al., 2016).

Targeting Tyrosine

Francis et al. Have Reported a Number of Efficient strategies where tyrosine residues were modified via a three-component Mannich-type reaction, alkylation of the residue and coupling with diazonium reagents (Tilley and Francis, 2006). However, Jones et al. were the first to describe direct polymer conjugation, including PEGylation, to tyrosine residues. These authors developed a general route to polymer-peptide biohybrid materials by preferentially targeting peptide tyrosine residues using diazonium salt-terminated polymers. Also, aniline derivatives are attractive molecules for tyrosine-targeted protein modifications with 4-aminobenzoyl-N-PEG2000-OMe through either diazonium coupling or three-component Mannich-type reactions (Jones et al., 2012). Recently, the first study to apply Mannich reaction modification and reactive coloration in fibrous proteins was developed, providing promising future applications for the reactive dyeing process of silk (Chen et al., 2019).

Targeting Tryptophan

Peptides containing N-terminal tryptophan residues may be modified using the Pictet-Spengler reaction with aldehydes in glacial acetic acid. The Pictet-Spengler reaction is based on the oxidation of the N-terminal amino group to an imine, where an aldehyde undergoes cyclic condensation with the α-amine and the indole side chain of a tryptophan residue, forming a new stable C–C bond (Agrawal et al., 2013; Mittal et al., 2014). Li et al. applied the Pictet-Spengler reaction to peptide ligation using peptide segments containing an aldehyde at the C-terminal and a Trp at the N-terminal. The main advantage of this reaction is the formation of a product with a stable C–C bond in a single step (Li et al., 2000). Also, Sasaki et al. applied the Pictet-Spengler reaction to the N-terminal labeling of horse heart myoglobin with an N-terminal glycine, employing tryptophan methyl ester and tryptamine as the coupling partners (Sasaki et al., 2008).

As an alternative to chemoselective modification, recombinant methods have also been used to incorporate unnatural amino acids (UAA) into proteins as chemical handles for a bio-orthogonal conjugation reaction (Liu and Schultz, 2010). The transfer of non-natural amino acids with azide and ketone functional groups at the N-terminus of proteins bearing N-terminal arginine residues using leucyl/phenylalanyl (L/F)-tRNA-protein transferase has proven efficient, both in the presence of other peptides and in crude protein mixture (Taki and Sisido, 2007). Although considerable progress has been made, an improvement in the existing N-terminal strategies is needed as none of the methods reported to date offer universal sequence compatibility.

Enzymatic Tools for Selective Pegylation of Proteins

Enzyme-mediated bioconjugation has gained a lot of attention in recent years because of the ability of biocatalysts to modify specific molecular tags under mild conditions. In this section, we briefly explore some enzymatic tools used for selective PEGylation purposes. Among these, sortase A (SrtA) from Staphylococcus aureus has been the most widely applied enzyme for protein bioconjugation in academic research (Tsukiji and Nagamune, 2009; Popp and Ploegh, 2011; Schmidt et al., 2017; Wang et al., 2017). It catalyzes a transpeptidase reaction between an N-terminal amino group derived from glycine and a specific internal amino acid sequence on a protein, usually LPXTG (where X can be any amino acid) (Rosen and Francis, 2017) (Figure 3A). Although the sortase A is applied for labeling the peptides and proteins among them, the approach of sortase-mediated PEGylation has been used to label large macroscopic particles with PEG-stabilized proteins to the surface of cells (Tomita et al., 2013). More recently, Li et al. took advantage of the mutated sortase A enzyme, which can enzymatically ligate the universal α-amino acids to a C-terminal tagged protein, allowing specific modification of the C-terminus of human growth hormone (hGH) with PEG. This site-specific bound PEG-hGH has similar efficacy as wild-type hGH (Shi et al., 2018). Despite there being as yet no approved PEGylated drugs derived from sortagging, it could be a promising advancement for improving the performance of traditional PEGylated drugs.

Figure 3 Enzyme-mediated modification of proteins. The (x) and (a) locked in circles represent hypothetical amino acids.

Microbial Transglutaminases

Microbial transglutaminases (mTGases) are another class of enzymes that has frequently been used for protein conjugation (Figure 3B). Several excellent reviews covering applications of microbial transglutaminase have been published previously (Mariniello and Porta, 2005; Rachel and Pelletier, 2013; Adrio and Demain, 2014; Strop, 2014). In general terms, TGases catalyze the acyl transfer reaction between the c-carboxyamide group of a protein-bound Gln residue and a variety of linear primary amines, such as the amino group of Lys (Griffin et al., 2002). In terms of site selective PEGylation this approach could be ineffective due to promiscuity in the amine substrates for these enzymes (Rachel and Pelletier, 2013). Nevertheless, Pasut et al. examined how the properties of PEGylated human growth hormone (hGH) changed depending on whether it was generated by chemical modification at the N-terminus or enzymatically using transglutaminase. Enzymatic labeling of hGH was carried out using TGase and a PEG reagent incorporating a primary amine. The study shows that although hGH carries 13 glutamine residues, 63.3% of the reaction product was a monoPEGylated form at position 141, showing a certain degree of site selectivity (Da Silva Freitas et al., 2013). Spolaore et al. studied the reactivity of IFN α-2b to microbial mTGase to obtain a site-specific conjugation of this biopharmaceutical. Characterization by mass spectrometry of the conjugates indicated that among the 10 Lys and 12 Gln residues of the protein only Gln101 and Lys164 were selectively conjugated with a PEG-NH2 for Gln101 and a PEG modified with carbobenzoxy--glutaminyl-glycine for Lys164 derivatization, with activity retention and improvements at pharmacokinetic levels (Spolaore et al., 2016). A mono-PEGylated derivative of filgrastim (granulocyte colony-stimulating factor) was also prepared using mTGase. The conjugation yielded an active protein with a single conjugation site (Gln135) that exhibited good in vivo stability (Scaramuzza et al., 2012). Although in the previous examples the PEGylation sites do not correspond to the N-terminal amino acid, they do illustrate a partial selectivity of mTGase despite its tendency toward substrate promiscuity. Also, these results indicate the potential of mTGase in the future of specific PEGylation and the development of innovative biopharmaceuticals. More recently, Braun et al. obtained an insulin-like growth factor 1-PEG (IGF1-PEG) conjugate for release in diseased tissue by using a combination of enzymatic and chemical bio-orthogonal coupling strategies. In this interesting example, mTGase was used for the ligation at the level of the N-terminal lysine of IGF1 to a PEG30 kDa modified protease-sensitive linker (Braun et al., 2018).


Subtiligase is a redesigned peptide ligase based on the modification of the active site of subtilisin. It was engineered by converting catalytic Ser221 to Cys, thereby increasing the ligase activity compared to amidase activity, and Residue Pro225 was converted to Ala to reduce steric assembling (Haridas et al., 2014). Subtiligase facilitates the ligation between a peptide C-terminal ester and a peptide N-terminal α-amine, without requiring a recognition motif at the termini of any reaction partners (Lin and He, 2018) (Figure 3C). The selective modification of the α-amine using subtiligase is a powerful approach in proteomics to enrich new N-termini arising from protease recognition and cleavage (Wiita et al., 2014), because 80% and 90% of wild-type eukaryotic proteins are acetylated at the N-terminal position (Polevoda and Sherman, 2003). This advantage could be exploited for the selective attachment of PEG-modified peptides as an innovative application to improve either the conjugation efficiency or the originality in the development of therapeutics.

Butelase 1

Butelase 1 is an enzyme isolated from the medicinal and ornamental plant Clitoria ternatea, which is a high-yielding asparagine/aspartate-specific cysteine ligase (Nguyen et al., 2014b) (Figure 3D). In spite of being C-terminal-specific for Asx, this enzyme accepts most N-terminal amino acids to mediate intermolecular peptide and protein ligation (Nguyen et al., 2016). Although it was recently discovered, butelase 1 has been used for several purposes, such as protein modification and engineering, peptide/protein ligation and labeling, peptide/protein macrocyclization, and living-cell surface labeling (Lin and He, 2018). No work has yet reported butelase 1 as being used for PEGylation reactions. However, some recent experiences with the enzyme, such as the method developed by Nguyen et al. for butelase-mediated ligation using thiodepsipeptides, have been applied in N-terminal labeling of ubiquitin and green fluorescent protein (GFP). The ligation yield of > 95% could be achieved for the model peptide and ubiquitin with a small substrate excess. This result anticipates a wide-ranging application and the perspectives of using butelase 1 for N-terminal modification of peptides and proteins (Nguyen et al., 2015).

Lipoid Acid Ligase

Lipoid acid ligase (LplA) is an alternative enzyme that has also been exploited for protein bioconjugation. This enzyme is able to recognize a specific LplA acceptor peptide (LAP) and catalyze the attachment of a lipoate moiety to a lysine residue in LAP (GFEIDKVWYDLDA) through an ATP-dependent reaction (Puthenveetil et al., 2009; Zhang et al., 2018) (Figure 3E). Regarding PEGylation, Plaks et al. used LplA for multisite clickable modification based on the incorporation of azide moieties in GFP at the N-terminal and two internal sites. Modification of the ligated azide groups with PEG, α--mannopyranoside and palmitic acid resulted in highly homogeneous populations of conjugates, being a potential approach, for instance, for site-specific multipoint protein PEGylation, among other modifications (Plaks et al., 2015). Additionally, other studies have been conducted using LplA-mediated enzymatic protein labeling followed by subsequent bio-orthogonal reactions (Hauke et al., 2014; Drake et al., 2016; Gray et al., 2016), allowing site-specific labeling of N- or C-terminus, even at the internal regions of a target protein.

There are other enzymes that have also been exploited for protein bioconjugation, including tubulin tyrosine ligase, which catalyzes the ATP-dependent addition of a tyrosine residue to the C-terminus of a-tubulin yielding a peptide bond (Schumacher et al., 2015; Zhang et al., 2018); N-myristoyltransferase, leading the transference of myristate from myristoyl-CoA to the N-terminal glycine of protein substrates, resulting in an amide linkage (Wright et al., 2010; Zhang et al., 2018); and biotin ligase, another ATP-dependent enzyme, catalyzes the conjugation of biotin derivatives onto proteins (Howarth and Ting, 2008; Fairhead and Howarth, 2015).

Analytical Methods for Characterization of PEGylated Proteins

The evidence indicates that the use of PEG to improve the properties of biopharmaceuticals or diagnostic agents will increase. This is supported by the growing number of proposals in clinical evaluation each year. In order to achieve high-quality products, it is necessary to take into account the implementation of accurate methods for the analysis of some parameters that provide a higher level of characterization of the molecule under study. It is important to note that none of the techniques on their own allows for the most complete characterization of the PEGylated proteins, but in many cases the combination of these is necessary to obtain more accurate results. This section provides an overview of the most frequently used analytical methods for the characterization of PEGylated peptides and proteins.

High-Performance Liquid Chromatography–Mass Spectrometry

High-performance liquid chromatography (HPLC) has been used for the separation and quantitation of free PEG and its PEGylated-protein form (PEG-conjugate). Some features of the PEGylated protein such as conjugate molecular weight, polymer mass distribution, or the degree and sites of PEGylation can be measured by HPLC methods. Lee et al., using SEC (size-exclusion chromatography) and RP-HPLC (reversed phase-high-performance liquid chromatography) mapping, assessed N-terminal PEGylated EGF, demonstrating the formation of a PEGylated macromolecule and that PEGylation occurred at the N-terminal position, respectively (Lee et al., 2003). Also, Brand et al. performed the separation of N-terminal PEGylated retargeted tissue factor tTF-NGR by using HPLC-based gel filtration, revealing pure elution fractions with the mono-PEGylated protein, which were represented by one clear band in SDS-PAGE and Western blotting (Brand et al., 2015). Although generally useful, the HPLC conditions and detection method must be improved for each compound based on the specific properties of the conjugated proteins.

To improve HPLC performance in the characterization of PEGylated proteins and to provide a more detailed characterization, the solution of coupling liquid chromatography to mass spectrometry was adopted. For decades, mass spectrometry (MS) has been the technique of choice for PEGylated protein characterization in terms of accurate average molecular weight and degree of PEGylation (Hutanu, 2014). A comparison of PEGylated and un-PEGylated counterparts by MS and peptide mapping is used to identify and quantify PEGylation sites and characterize impurities that occasionally go undetected by simpler techniques (Caserman et al., 2009). Collins et al. performed N-terminal amine PEGylation to stabilize oxytocin formulations for prolonged storage. Conjugation was confirmed by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) MS, where a clear shift in molecular weight was observed in the MALDI-TOF spectrum from the NHS ester polymer to the polymer-peptide conjugate (Collins et al., 2016). In another study conducted by Qin et al. following MALDI-TOF MS, PEG modification sites were determined through comparative analysis of peptide mapping between rhGH (recombinant human growth hormone) and PEG-rhGH. The use of MS makes it possible to discriminate positional isomers, with PEGylation sites potentially located at the N-terminus and nine lysine residues of rhGH (Qin et al., 2017). However, the exact determination of the PEG attachment site(s) continues to be highly challenging, especially in a mixture composed of products with differing degrees of PEGylation (Gerislioglu et al., 2018). On the other hand, ESI-TOF has overcome some disadvantages related to polydispersion and the overlapping protein charge pattern of PEGylated proteins (Forstenlehner et al., 2014). Furthermore, ESI-MS is preferred to MALDI due to automated workflow and reduced sample preparation time (Hutanu, 2014). Several studies have reported applying the approach of the line-up of liquid chromatography to MS (LC-MS) for the sensitive quantitation of free PEG in biological fluid samples (Pelham et al., 2008; Yin et al., 2017) or tissues (Gong et al., 2014), as well as clear detection and identification of the positional isomers formed upon PEGylation (Gerislioglu et al., 2018; Shekhawat et al., 2019), obtaining significant structural information in a heterogeneous sample of PEGylated proteins (Mero et al., 2016; Muneeruddin et al., 2017), among others.

Dynamic Light Scattering

Dynamic light scattering (DLS) is an additional technique also convenient for the molecular weight evaluation of PEGylated proteins, as it can measure the molecular radii of the samples (Kusterle et al., 2008; Gokarn et al., 2012), and discriminate between linear and branched PEGs (Wan et al., 2017). This method, among others, was used by Vernet et al. in 2016 to assess the first large-scale study with the site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total (Vernet et al., 2016). In addition, Khameneh et al. conducted a study in which site-specific PEGylated hGH was prepared by using microbial transglutaminase. Physicochemical properties, size and zeta potentials of native and PEGylated hGH, were evaluated by DLS, indicating that the size and zeta potentials of the protein were increased and decreased respectively by PEGylation, enhancing the stability of the protein (Khameneh et al., 2016). Recently, Meneguetti et al. applied DLS for the characterization of a novel N-terminal PEGylated asparaginase, showing that the PEGylation of ASNase caused an increase in the hydrodynamic diameter of the protein related to the increase in the amount of PEG attached to the protein (Meneguetti et al., 2019). The DLS approach has been used in the characterization not only of PEGylated proteins, but also of PEGylated organic nanotubes, revealing that PEGylation dramatically improves the dispersibility of the nanotubes in saline buffer (Ding et al., 2014). Despite its wide use in the characterization of the hydrodynamic radius of PEGylated proteins, this methodology presents certain disadvantages in its application, such as the presence of large particles that can also be detected during the analysis; low resolution when the populations are close in size or a highly polydispersed sample; light absorption by the dispersant can interfere with detection because of their viscosity as well as the density of the particles. These are important parameters to take into account when carrying out this type of analysis.

Nuclear Magnetic Resonance

1H nuclear magnetic resonance (NMR) spectroscopy is useful to quantify PEGylated species in complex biological fluids with advantages of time and simplicity in the sample preparation (Alvares et al., 2016). The application of this technique for the structural characterization of conjugates with PEG (Kiss et al., 2018) has been being useful in the quantitative determination of the degree of PEGylation (Zaghmi et al., 2019), the assessment of the higher-order structure of PEGylated therapeutic proteins (Cattani et al., 2015; Hodgson and Aubin, 2017) or even the behavior of free PEG in serum samples (Khandelwal et al., 2019). More recently, solid state NMR has been used for the structural characterization of large PEGylated proteins such as asparaginase (Giuntini et al., 2017; Cerofolini et al., 2019). The combination of NMR with other techniques such as LC-MS/MS has enabled the accurate quantification of isobaric glycan structures, even in the picomolar order (Wiegandt and Meyer, 2014), an approach that could be used for a better characterization of high complexity PEGylated molecules.

Immunoenzymatic Assays

Enzyme-linked immunosorbent assay (ELISA) is a powerful tool for measuring the concentration of PEGylated proteins in serum samples (Wang et al., 2012). This technique permits the study of the effects of PEGylation in protein immunogenicity as well as the anti-PEG immune response (Wan et al., 2017). While direct ELISA has the advantage of lacking only one specific antibody for compound detection, it cannot distinguish between PEGylated and unPEGylated proteins (Cao et al., 2009). On the other hand, sandwich or indirect ELISA employs two antibodies: one to capture the analyte on a solid surface and a second to determine the concentration of the captured analyte (Gan and Patel, 2013). Bruno et al. used a quantitative sandwich ELISA to analyze the pharmacokinetics of Pegasys and PEG-Intron using two mouse monoclonal antihuman IFN-α antibodies that recognize different epitopes of IFN-α (Bruno et al., 2004), and a similar ELISA was used for the measurement of Neulasta® (Roskos et al., 2006) and Mircera® (Macdougall et al., 2006). Su et al. produced second-generation monoclonal antibodies attached to PEG (AGP4/IgM and 3.3/IgG) that also bind to the repeating subunits of the PEG backbone, but with greater affinity than those of first-generation AGP3 and E11 (Su et al., 2010). Since then, they have produced a range of specific anti-PEG IgG and IgM monoclonal antibodies for use in ELISA, FACs, IHC, and flow cytometry, which can be found under anti-PEG in the Institute of Biomedical Sciences at Academia Sinica, Taiwan.

Bioinformatics Methods

With the advent of the era of bioinformatics, computational methods have been effectively employed for an easier designing, engineering, and characterization of proteins, which supports experimental methodologies and, in many cases, saves time and materials. At present, computational analysis is highly recommended to select the proper position on the protein for site-selective PEGylation (Rouhani et al., 2018). In 2013 Mu et al. conducted a bioinformatics study in which four forms of PEGylated staphylokinase obtained by site-specific conjugation of PEG to the N- and C-termini of SK, respectively, were structurally evaluated to provide greater molecular insight into the interaction between the PEGylated protein and its receptor (Mu et al., 2013). The results suggested that the PEG polymer wraps around the protein providing steric shield, and this effect depends on the PEG chain length and PEGylation site of the protein (Rouhani et al., 2018b). Also, Mirzaei et al. (2016) applied computational and non-glycosylated systems to define an artless methodology for site-selective (cysteine) PEGylation of erythropoietin analogs. The results showed that using an in silico approach together with the experimental methodologies can be a strategy to optimize the parameters of PEGylation (Mirzaei et al., 2016). Recently, Xu et al. (2018) used interferon (IFN) as a representative model system to characterize the molecular-level changes in IFN introduced by several degrees of PEGylation through molecular dynamics simulations. The simulations generated molecular evidence directly linked to improved protein stability, bioavailability, retention time, as well as the decrease in protein bioactivity with PEG conjugates, providing an important computational approach in the improvement of PEGylated protein drug conjugates and their clinical performance (Xu et al., 2018a). However, and in spite of the advances obtained in this field, there are still some drawbacks that must be solved, such as the computational cost in terms of infrastructure, and many times, it could be hard to explain what the biological or clinical meaning of features identified using bioinformatics analysis.

Recent Approaches in the Site-Selective Conjugation of Proteins

The chemistry of natural amino acids has been a highly exploited approach in the bioconjugation of proteins. However, there is often poor control over the site and various modifications and incompatibilities with complex mixtures or living systems (Reddington and Howarth, 2015). Since the manipulation of proteins is at the core of biochemical research, the search for new strategies in efficient and specific bioconjugation has been an objective developed by the scientific community through protein engineering. These strategies include for example the SpyTag/SpyCatcher system.

The Spytag/Spycatcher System

The SpyTag/SpyCatcher system allows the specific and covalent conjugation of proteins through two short polypeptide tags (Zakeri et al., 2012). The larger partner, the SpyCatcher, adopts an immunoglobulin-like conformation that specifically binds the SpyTag (γ-carbon of Asp-117), leading the formation of an extremely resistant intermolecular bond between two amino acid side chains (Gilbert et al., 2017). In this extremely fast method, no exogenous enzymes need to be added or removed (Fisher et al., 2017) and despite its recent description, this system has already been used in the production of synthetic vaccines (Brune et al., 2016), thermo-stable enzymes (Schoene et al., 2016; Wang et al., 2016), and other applications (Fierer et al., 2014; Dovala et al., 2016; Lakshmanan et al., 2016). Take advantage of this system, Gilbert et al. described how the XynA enzyme was genetically encoded to covalently conjugate in culture media, providing a novel and flexible strategy for protein conjugation exploiting the substantial advantages of extracellular self-assembly (Gilbert et al., 2017). Recently, Cayetano-Cruz et al. published a study in which the α-glucosidase Ima1p enzyme of Saccharomyces cerevisiae was attached to the surface of virus-like particles (VLPs) of parvovirus B19 using the SpyTag/SpyCatcher system. This approach made it possible to obtain a more thermostable enzyme and the modified VLPs were also able to act on glycogen. Hence, these particles may be developed in the future as part of the therapy for the treatment of diseases caused by defects in the human acid α-glucosidase (Cayetano-Cruz et al., 2018). SpyCatcher is large and may be difficult to attach to polymers; therefore, the final product contains a large SpyCatcher protein sequence (Zakeri et al., 2012). It could be a reason why no study to date has been reported using this system to modify proteins with PEGs. However, this is a promising mechanism to create PEGylated proteins, taking advantage of the fact that SpyTag can be placed at the N-terminus, at the C-terminus and at the internal positions of a protein (Zakeri et al., 2012), and previously bound, for instance, to the polymers (PEG) being conjugated.

Ring Opening Polymerization

Ring opening polymerization (ROP) is a reaction, in which the terminal end of a polymer chain acts as a reactive center where additional cyclic monomers can react by opening its ring system, forming a longer polymer chain (Jenkins et al., 1996) with the occurrence of two main reactions: initiation and growth (Penczek and Pretula, 2016). In 2013, Spears et al. used the approach of ROP for first time for the in situ controlled branching of polyglycidol and formation of BSA-glycidol bioconjugates with “PEG-like” arms (Spears et al., 2013; Qi and Chilkoti, 2015). Since then, ROP has been used as a methodology to modify various molecules as well as to obtain different varieties of polymers. Ma et al. prepared a cross-linked fluorescent polymer through ROP and performed a subsequent ring opening PEGylation with 4-arm PEG-amine, yielding polymeric nanoparticles in aqueous solution with hydrophilic PEG groups covered at the surface (Ma et al., 2015). Also, Tian et al. (2018) developed smart polymeric materials based on biomimetic PEGylated polypeptoids by combining ring-opening polymerization and a post-modification strategy (Tian et al., 2018). Furthermore, the usefulness of this approach has also been established in the preparation of PEGylated and fluorescent nanoprobes for biomedical applications (Wan et al., 2015; Xu et al., 2018b) and the development of polymeric gene vectors with high transfection efficiency and improved biocompatibility (Xiao et al., 2018). All this demonstrates the potential that ROP could have in the design of PEGylated proteins of biopharmaceutical interest or other molecules used in the diagnosis of different diseases.

Click Chemistry

Click chemistry is another method widely used for PEG attachment to proteins for different purposes (Jølck et al., 2010; Leung et al., 2012; Li et al., 2012; Xu et al., 2015; Huang et al., 2018; Lou et al., 2018). Here, azide and alkyne groups react selectively with each other in the presence of Cu1+ as the catalyst (Rostovtsev et al., 2002) through the initial reaction of reduced thiols with a maleimide compound containing a click-reactive alkyne moiety. Then, a large PEG molecule containing a complementary click-reactive azide moiety is selectively conjugated to the click-tagged thiols (van Leeuwen et al., 2017). This method is versatile, fast and simple to use, easy to purify, site-specific, and gives high product yields (Hein et al., 2008); however, its drawback is related to the toxicity of copper, even in small amounts. This could limit the development of pharmaceuticals using this methodology; as a result, PEGylation via copper-free click reaction has gained more attention nowadays (Debets et al., 2010; Koo et al., 2012; Lou et al., 2018). The reaction conditions are extremely mild and do not cause protein denaturation, nor are any metals, reducing agents or ligands required.

Non-Covalent PEGylation

Non-covalent PEGylation is an innovative approach in which a chemical reaction between protein and PEG is avoided (Reichert and Borchard, 2016). It is based on the mechanisms of hydrophobic interactions (Mueller et al., 2011a; Mueller et al., 2011b; Mueller et al., 2012), ionic interactions (Khondee et al., 2011), protein polyelectrolyte complex (Kurinomaru and Shiraki, 2015; Kurinomaru et al., 2017), or chelation (Mero et al., 2011). The main advantage of this technique is that it eliminates a potential loss of product due to additional purification processes (Reichert and Borchard, 2016). However, the release of the protein during storage is an important shortcoming for this approach (Santos et al., 2018).

Concluding Remarks

The covalent attachment of peptides and proteins to polyethylene glycol remains a preferred method for modifying the pharmacokinetic and immunological properties of therapeutic molecules, supported not only by the introduction of PEGylated drugs on the market but also by the increasing number of currently ongoing clinical studies. The chemical versatility of polyethylene glycol derivatives enables the synthesis of various PEGylated protein structures, with a trend to target-specific amino acid residues located at the terminal ends (N or C-terminus) of the peptides or protein of interest, which contributes to obtaining homogeneous and well-defined conjugates. These site-selective modifications must preserve the biological activity of the PEGylated molecule. As part of the development of the science of PEGylation, new methods continue to be implemented based on new approaches, as well as faster and more efficient techniques, such as enzymatic ligation or the development of bio-orthogonal chemistry. As the number and location of PEG chains attached to a protein can affect its activity, it is critical to uncover these important structural details. Thus, strong analytical methods must be developed, allowing for a qualitative and quantitative characterization with a greater degree of robustness and accuracy. In this sense, the computational tools (predictive models based on molecular dynamics) are a great help in clarifying interactions, binding sites or stability of PEGylated proteins in the unending search for and design of new, more effective biopharmaceuticals.

Author Contributions

LB and CR-Y: writing of the topic related to the chemical reactions of site-specific pegylation. JBL and ML-E: writing of the topic related to enzymatic pegylation. BE and RC: writing of the topic related to the characterization of pegylated proteins. AP and JF: critical revisions and corrections of the manuscript.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We would like to acknowledge the support provided by the National Commission for Scientific and Technological Research (CONICYT) Fellowship for Doctoral Studies in Chile (No. 21170061), research grants from the National Fund for Scientific and Technological Development (FONDECYT) Project (No. 3180765), the São Paulo Research Foundation (FAPESP, Grant No. 2016/22065-5) and DIUFRO Projects (DI12-PEO1; EXE12- 0004; DIE14-0001) of Universidad de La Frontera.


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Evaluation of Nanoparticle PEGylation: Quantitative and Qualitative Determination

An Overview of PEGylation of Peptide

Release date:2020/6/10 10:09:04

Peptide therapeutics have played a notable role in medical practice since the advent of insulin therapy in the 1920s. Over 60 peptide drugs are approved in the United States and other major markets, and peptides continue to enter clinical development at a steady pace. Peptide drug discovery has diversified beyond its traditional focus on endogenous human peptides to include a broader range of structures identified from other natural sources or through medicinal chemistry efforts. We maintain a comprehensive dataset on peptides that have entered human clinical studies that include over 150 peptides in active development today and an additional 260 tested in human clinical trials. 

Limitations of Peptide Drugs

But, enthusiasm for peptide therapeutics was subsequently tempered by certain limitations of native peptides, such as short plasma half-life and negligible oral bioavailability. The short half-life of many peptide hormones is explained by the presence of numerous peptidases and excretory mechanisms that inactivate and clear peptides. This lability allows the body to rapidly modulate hormone levels to maintain homeostasis but is nonetheless inconvenient for many therapeutic development projects. 

Another obstacle for peptide drug development is oral bioavailability: digestive enzymes designed to break down amide bonds of ingested proteins are effective at cleaving the same bonds in peptide hormones, and the high polarity and molecular weight of peptides severely limit intestinal permeability. As oral delivery is often viewed as attractive for supporting patient compliance, the need for injection made peptides a less appealing option for indications that required chronic, outpatient therapy. (In September 2019, FDA approved the first oral peptide drug Semaglutide tablets.)

Modification and Performance

Conjugation has emerged as a popular mechanism to alter or enhance the properties of peptide and protein drug candidates. Chemical modification of the peptide using polyethylene glycol (PEG) can improve multiple physiochemical and pharmacokinetic performance with minimal increase in manufacturing cost. PEG is a highly investigated polymer that is used in covalent modification of biopolymers as proteins and peptides. It is incorporated into the manufacturing process of the bulk API in a technique known as PEGylation. The effects of PEGylation on peptide pharmacokinetics include avoidance of reticuloendothelial (RES) clearance, mitigation of immunogenicity, and reduction of enzymatic proteolysis and of losses by renal filtration, with potentially beneficial changes in biodistribution. These effects can dramatically increase the half-life of a peptide in vivo, with potential collateral improvement in bioavailability but without adversely affecting binding and activity of the peptide ligand.

Peginesatide is a synthetic peptide, attached to polyethylene glycol ("PEGylated"). It was approved by the U.S. Food and Drug Administration for the treatment of anemia associated with chronic kidney disease (CKD) in adult patients on dialysis.  But, On June 16, 2014, Affymax and Takeda issued a press release stating that Takeda will work with the FDA to withdraw the peginesatide New Drug Application.
Peptide nameConjugated moietyRationale for conjugationCurrent status
PeginesatidePEGHalf-life extensionApproved, then withdrawn
PEG's most common form is a linear or branched polyether with terminal hydroxyl groups synthesized by anionic ring-opening polymerization - HO-(CH2CH2O)n-CH2CH2 -OH.

Monofunctional methoxy-PEG (mPEG) is preferred for peptide modification - CH3O-(CH2CH2O)n-CH2CH2 -OH, as it can be derivatized with a number of linkage moieties, yielding methoxyPEG-amines, -maleimides, or -carboxylic acids (Fig. 2).


Four general factors affect the performance of PEGylated peptides:

1. Molecular weight and structure - whereas PEGs of <1,000 Da can be broken down into subunits that can have some toxicity, PEGs of >1,000 Da have not demonstrated any toxicity in vivo. PEGs of up to 40-50,000 Da have been used in clinical and approved pharmaceutical applications.
2. Number of PEG chains - two or more lower-weight chains can be added to increase the total molecular weight of the PEG complex
3. Site of attachment - for each peptide, the location of the PEGylation sites has to be carefully engineered experimentally to retain the highest possible binding efficiency and activity of the peptide ligand.
4. PEGylation chemistry - the type of linkage for attaching PEG to the peptide as well as the purity of raw materials, intermediates and final product.

The latter is the most important factor determining the yield of the PEGylation process and the scalability of the manufacturing protocol. Peptide and linker have to be very pure and very stable during the conjugation reaction to yield a pure conjugate with high efficiency.

Biochempeg offers PEGylation as a cost-effective modification of peptides which has the potential to improve bioavailability compared to the unmodified molecule.

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Types of PEGylation of Peptides

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Of peptides pegylation

Pegylated peptides. II. Solid-phase synthesis of amino-, carboxy- and side-chain pegylated peptides

General procedures are presented for the site-specific pegylation of peptides at the NH2-terminus, side-chain positions (Lys or Asp/Glu) or COOH-terminus using solid-phase Fmoc/tBu methodologies. A model tridecapeptide fragment of interleukin-2, IL-2(44-56)-NH2, was chosen for this study since it possesses several trifunctional amino acids which serve as potential sites for pegylation. The pegylation reagents were designed to contain either Nle or Orn, which served as diagnostic amino acids for confirming the presence of 1 PEG unit per mole of peptide. NH2-Terminal pegylation was carried out by coupling PEG-CH2CO-Nle-OH to the free NH2-terminus of the peptide-resin. Side-chain pegylation of Lys or Asp was achieved by one of two pathways. Direct side-chain pegylation was accomplished by coupling with Fmoc-Lys(PEG-CH2CO-Nle)-OH or Fmoc-Asp(Nle-NH-CH2CH2-PEG)-OH, followed by solid-phase assemblage of the pegylated peptide-resin and TFA cleavage. Alternatively, allylic protective groups were introduced via Fmoc-Lys(Alloc)-OH or Fmoc-Asp(O-Allyl)-OH, and selectively removed by palladium-catalyzed deprotection after assemblage of the peptide-resin. Solid-phase pegylation of the side-chain of Lys or Asp was then carried out in the final stage with PEG-CH2CO-Nle-OH or H-Nle-NH-(CH2)2-PEG, respectively. COOH-Terminal pegylation was achieved through the initial attachment of Fmoc-Orn(PEG-CH2CO)-OH to the solid support, followed by solid-phase peptide synthesis using the Fmoc/tBu strategy. The pegylated peptides were purified by dialysis and preparative HPLC and were fully characterized by analytical HPLC, amino acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry.

Peptides, Proteins and Other Biopharmaceuticals

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