Versatile Route to Synthesize Heterobifunctional Poly(ethylene Glycol) of Variable Functionality for Subsequent Pegylation

Pegylation using heterotelechelic poly(ethylene glycol) (PEG) offers many possibilities to create high-performance molecules and materials. A versatile route is proposed to synthesize heterobifunctional PEG containing diverse combinations of azide, amine, thioacetate, thiol, pyridyl disulfide, as well as activated hydroxyl end groups. Asymmetric activation of one hydroxyl end group enables the heterobifunctionalization while applying selective monotosylation of linear, symmetrical PEG as a key step. The azide function is introduced by reacting monotosyl PEG with sodium azide. A thiol end group is obtained by reaction with sodium hydrosulfide. The activation of the hydroxyl end group and subsequent reaction with potassium carbonate/thioacetic acid yields a thioacetate end group. The hydrolysis of the thioester end group by ammonia in presence of 2,2′-dipyridyl disulfide provides PEG pyridyl disulfide. Amine terminated PEG is prepared either by reduction of the azide or by nucleophilic substitution of mesylate terminated PEG using ammonia. In all cases, >95% functionalization of the PEG end groups is achieved. The PEG derivatives particularly support the development of materials for biomedical applications. For example, grafting up to 13% of the Na-alg monomer units with α-amine-ω-thiol PEG maintains the gelling capacity in presence of calcium ions but simultaneous, spontaneous disulfide bond formation reinforces the initial physical hydrogel.


Introduction
Pegylation is intensely being used to modify macromolecules, biomolecules, and surfaces 1-4.Poly(ethylene glycol) (PEG) has unique properties indispensable for various biological, chemical, biomedical, and pharmaceutical applications.Advantageous properties are nontoxicity, nonimmunogenicity, biocompatibility, as well as solubility in water and in many organic solvents.The chains of linear PEG are highly flexible and their hydrophilicity can improve the solubility of compounds upon conjugation and so ensure good solubility under physiological conditions.In order to manufacture PEG-modified surfaces, several types of semitelechelic PEG are used.However, PEG chains possessing a functional group at only one end are not suitable for subsequent derivatization/modification, which is frequently crucial for the design of biomaterials or for biomedical applications.For high-performance pegylation, heterotelechelic PEG is required, i.e., PEG molecules having two different reactive functional end groups.Several heterobifunctional PEG derivatives are commercially available but at relatively high costs.
Ring-opening polymerization of ethylene oxide utilizing initiators of appropriate functionality remains the most common way to synthesize heterotelechelic PEG 5-9.However, the polymerization of ethylene oxide can be hazardous.Special care must be taken when working with highly toxic and potentially explosive gases [5].Alternatively, alteration of the terminal hydroxyl groups of commercially available PEG can be performed.Despite the generally milder conditions for the modification of the hydroxyl end groups, preference is given to the ring-opening polymerization because the second approach mostly yields a mixture of mono-, di-, and un-substituted components, which have subsequently to be separated 5-10.Asymmetric activation of the hydroxyl group at one chain end enables the introduction of a series of functional groups in case of not too high molar mass of the PEG.Indeed, many pegylation applications do not need high molar mass PEG.
Biocompatibility, and the ability to form hydrogels when exposed to multivalent cations, favors the use of the biopolymer sodium alginate (Na-alg) for cell microencapsulation 11-13.However, for several applications, hydrogel formation by only electrostatic interaction is insufficient.The modification of Na-alg for subsequent reinforcement of ionically cross-linked network by covalent cross-linking has gained interest and it is being increasingly investigated 14-20.
This paper summarizes the synthesis of a series of heterobifunctional PEG derivatives by alteration of the terminal hydroxyl groups of linear PEG.First, mono-tosyl PEG was synthesized.Subsequently, the tosyl end group was converted into a variety of functional end groups, either directly or via intermediate steps.The suitability of these heterobifunctional PEG derivatives for the pegylation process was demonstrated by successful conjugation of α-amine-ω-thiol PEG to a defined number of monomer units of Na-alg.This conjugation increased the solubility of alginate in aqueous media, but did not affect its ability to form hydrogels in presence of divalent cations.Moreover, the conjugated reactive thiol end groups allowed for simultaneous chemical cross-linking via disulfide bonds yielding a novel type of hybrid hydrogel.This paper discusses the synthetic pathways and potential general applications of the PEG derivatives while showing exemplarily one specific application of broad interest.

Results and Discussion
Synthesis of Heterobifunctional PEG Derivatives.A series of heterobifunctional PEG derivatives was synthesized via asymmetric activation of commercially available symmetrical PEG.First, mono-tosyl PEG was synthesized.Subsequently, the tosyl end group was converted into a variety of functional end groups, either directly or via intermediate steps as shown in Scheme I. Synthesis efficiency, final yields and molecular characteristics are summarized in Table 1.
Scheme I. Syntheses pathways to heterobifunctional PEG derivatives.PEG (1).Tosylate groups are known as good substrate for substitution reactions.Their leaving group character enables the conversion to versatile types of functional groups.Thus, a tosylate end group in a PEG molecule is one of the most useful candidates for replacement by another functionality.For the preparation of a series of heterotelechelic PEG derivatives, α-tosyl-ω-hydroxyl PEG (1) was synthesized as precursor (Scheme I).Bouzide and Sauve reported selective monotosylation of symmetrical diols in the presence of silver oxide and a catalytic amount of potassium iodide 21.We adapted this approach to modify commercially available PEG.To achieve selective monotosylation also addition of an excess of symmetric diols was reported 22.However, in the case of PEG having a relatively high molar mass, the separation of non-modified excess from the modified product is difficult.In the present study, the synthesis was carried out at rt avoiding drawbacks such as harsh conditions or the use of an excess of diols.The degree of functionalization of α-tosyl-ω-hydroxyl PEG was calculated from 1 H-NMR using deuterated dimethyl sulfoxide (DMSO) 23.The spectrum shows a triplet at 4.56 ppm corresponding to hydroxyl protons, which does not shift or broaden with variation of the concentration, and which is well separated from the PEG backbone peak (see Appendix, Figure S1).The degree of functionalization was almost quantitative (≈99%).Moreover, only unimodal peaks without shoulders and indicating similar molar mass distributions were observed by GPC for the native PEG and for α-tosyl-ω-hydroxyl PEG (Figure 1(a)).The absence of ditosylate byproduct was confirmed by MALDI-TOF/TOF MS.The main peaks for native PEG and α-tosyl-ω-hydroxyl PEG were detected at 1,449.71 and 1,603.72,respectively (see Appendix, Figure S24).The increase of the molar mass corresponds to the introduction of the tosyl group at only one end.A peak at 1,756.71 corresponding to the ditosylate byproduct was absent.Such selective monotosylation has been attributed to internal hydrogen bonding (IHB) 21.The hydrogen atom involved in IHB becomes less acidic, and therefore, the second hydroxyl group will preferentially be deprotonated by silver oxide.PEG (2).The displacement of the tosylate end group by NaN 3 yielded α-azide-ω-hydroxyl PEG (2).Azide-terminated PEGs have received considerable attention in the field of bioconjugate chemistry, in particular for the highly chemoselective copper (I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction (termed "click chemistry").Because of its specificity, quantitative yield, and good functional group tolerance, click chemistry has shown to be a promising approach for pegylation under mild conditions in aqueous buffers within a wide pH range 24-28.The quantitative introduction of the azide function was confirmed by 1 H-NMR.On the one hand, the spectra did not show any traces of tosylate signals.On the other hand, the integration of the hydroxyl group remained constant, suggesting that no hydrolysis of the tosylate group took place (see Appendix, Figure S3).The 13 C-NMR and FTIR spectra (see Appendix, Figure S4 and Figure 2a) confirm the quantitative introduction of the azide function.The chemical displacement of the carbon adjacent to azide and the antisymmetric stretching vibration band of azide were detected at 50.64 ppm and 2,103 cm −1 , respectively.PEG (3).Besides being efficient precursors for click chemistry, azides are known to serve as synthons for the preparation of amines.The reduction of azides to amines has been widely studied and been found to be highly diverse 29-31.In our approach, a complete reduction of azide was achieved by Staudinger reduction.The reduction mechanism involves the formation of a linear phosphazine intermediate, which yields an iminophosphorane with concomitant loss of N 2 .By spontaneous hydrolysis of iminophosphorane primary amine and the corresponding phosphine oxide are obtained 32,33.The use of PPh 3 as reducing agent in MeOH leads to α-amine-ω-hydroxyl PEG (3).The completeness of the reaction was confirmed by 13 C-NMR.The spectra exhibit peaks at 41.78 and 73.45 corresponding to α-and β-amine carbons, respectively, while no traces of the characteristic peak of azide at 50.64 ppm are visible (see Appendix, Figure S6).Furthermore, FTIR shows absence of the antisymmetric stretching vibration band of azide (Figure 2(a)).
PEG (5).Thiol-terminated PEG is very efficient as pegylation agent.For instance, the functionalization of quantum dots and nanoparticles with PEG-thiol increased their stability and their hydrophilicity, and thus, reduced their toxicity in biological systems 34-36.Furthermore, the ability of thiol-terminated PEG to form hydrogels via Michael-type addition was exploited for cell encapsulation, drug delivery, and tissue engineering 37-39.Here, α-thiol-ω-hydroxyl PEG (5) was prepared by reaction of α-tosyl-ω-hydroxyl PEG (1) with an excess of sodium hydrosulfide hydrate in water.The downfield tosylate aryl peaks were not detectable by 1 H-NMR (SI, Figure S10) confirming the complete displacement of the tosylate group by the hydrosulfide ion.However, the GPC chromatogram presented in Figure 1(b) shows two peaks, one with similar retention time as the starting α-tosyl-ω-hydroxyl PEG and a second peak with shorter retention time corresponding to twice the molar mass.The treatment of the polymer with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) prior to the injection into the GPC column significantly reduces this second peak (data not shown) suggesting that the early peak corresponds to dimer molecules formed by oxidation of the thiol function to a disulfide bond, which is reduced by reaction with TCEP.The formation of disulfide bonds could not completely be avoided using a one-step process although special care was taken to eliminate air.
PEG (6) and PEG (7).In order to avoid this dimer formation and to synthesize PEG with protected thiol end groups, a two-step synthesis was adopted.The tosylate group was first displaced by reaction with the in situ formed potassium thioacetate in DMF to yield α-thioacetate-ω-hydroxyl PEG (6).The introduction of the thioester end group was confirmed by 1 H and 13 C-NMR (see Appendix, Figure S12  and S13).The complete hydrolysis of the thioester was accomplished under mild conditions using ammonia in MeOH at rt.Furthermore, adding 2,2'-dipyridyl disulfide (2-PDS) as capping agent avoided the formation of dimer molecules during the hydrolysis.The liberated sulfhydryl end group simultaneously reacts with 2-PDS yielding a protected thiol as pyridyldithio. 1 H and 13 C-NMR spectra of α-pyridyldithio-ω-hydroxyl PEG (7) exhibit the characteristic peaks of the pyridyl protons (see Appendix, Figure S14 and S15), while no traces of the thioester bond were detected.GPC confirmed the absence of the dimer byproduct (Figure 1(c)).
PEG (4), PEG (8), and PEG (9).The α-azide-ω-hydroxyl PEG (2) was used as precursor for the synthesis of α-azide-ω-thioacetate PEG (4).The first step involved the activation of the free hydroxyl into tosylate.The reaction proceeded quantitatively as the hydroxyl protons typically observed by 1 H-NMR in DMSO at 4.56 ppm were not detectable (see Appendix, Figure S7).The thioacetate function was introduced by the reaction with the in situ formed potassium thioacetate in DMF yielding α-azide-ω-thioacetate PEG (4). 1 H and 13 C-NMR confirmed the quantitative introduction of the thioacetate function (Figure S8 and S9).Beside the antisymmetric stretching vibration band of azide detected at 2103 cm −1 , the FTIR spectrum of α-azide-ω-thioacetate PEG (4) in Figure 2b exhibited a strong band at 1692 cm −1 corresponding to carbonyl stretching vibration.The reaction of (4) with ammonia in presence of 2-PDS yielded α-azide-ω-pyridyldithio PEG (8).FTIR spectra show complete disappearance of the peak corresponding to carbonyl stretching, Figure 2b.Reduction of α-azide-ω-thioacetate PEG (4) was achieved by PPh 3 . 13C-NMR exhibit the characteristic peaks of α-and β-amine carbons of PEG (9), while no traces of the characteristic peak of α-azide carbon at 50.64 ppm were detected (see Appendix, Figure S19), suggesting complete reduction of the azide function.Furthermore, the cleavage of the thioester bond was confirmed by FTIR.The spectrum shows complete disappearance of both the antisymmetric stretching vibration band of azide at 2,103 cm −1 and the peak corresponding to carbonyl stretching at 1,692 cm −1 (Figure 2(c)).
1 H-NMR spectra confirm the presence of amine and thiol end groups (see Appendix, Figure S18).
The GPC curve of PEG (9) shows a unimodal peak without any shoulder (Figure 1(d)).This is explained by the reductive effect of the PPh 3 , which reduces the formation of disulfide bonds 40.However, disulfide formation becomes obvious after two-weeks storage by a peak at shorter retention time corresponding to the double of the molar mass (Figure 1(e)).This dimer formation confirms the usefulness of protecting the thiol group at the chain end.It could be removed easily by standard reducing agents such as dithiothreitol (DTT) or TCEP before further use for specific pegylation.PEG (10) and PEG (11).Simultaneous introduction of amine and pyridyldithio end groups was accomplished by performing a two-step synthesis using α-thioacetate-ω-hydroxyl PEG (6) as precursor.The hydroxyl group was first converted into mesylate, PEG (10). 1 H-NMR confirmed the quantitative introduction of a mesylate end group exhibiting no traces of hydroxyl groups at 4.56 ppm (Figure S20).The integration of the thioacetate protons remained unchanged suggesting that no hydrolysis of the thioacetate end is taking place during the reaction.The absence of hydrolysis might be explained both by the low concentration of NEt 3 and/or by the short reaction time.The amine function was successfully introduced by displacing the mesylate end group by ammonia yielding PEG (11), as shown in SI (Figures S22 and S23).Simultaneously, the cleavage of the thioester end group was accomplished, and a pyridyldithio end group was obtained by trapping the liberated sulfhydryl by 2-PDS.Because of the low solubility of 2-PDS in water, a mixture of MeOH/H 2 O was used as solvent.
Overall, the syntheses of all heterobifunctional PEG derivatives aimed at almost quantitative end group functionalization and high yields obtainable at mild reaction conditions.Optimization of the reaction conditions, in particular reaction times, seems possible.
Pegylation of Sodium Alginate.The presence of the carboxylic groups and cis-diols in the Na-alg chain units provides numerous approaches for chemical modification 41-45.Here, the modification of Na-alg by pegylation aims at improving the mechanical resistance and the durability of alginate-based hydrogels while not interfering the biocompatibility.In our approach, the conjugation of α-amine-ω-thiol PEG (9) to Na-alg was achieved via amide bonding (Scheme II).The carboxylate groups of Na-alg reacted with (9)  The conjugation of the hydrophilic PEG chains to Na-alg significantly increased the solubility of the resulting Na-alg-PEG.For the same concentration, a homogeneous aqueous solution of Na-alg-PEG is obtained within minutes while the unmodified Na-alg needs hours to dissolve.Thus, especially for higher molar mass Na-alg, a positive impact on the solution preparation for practical applications is achieved.With the aim to extend the materials basis for cell microencapsulation, we prepared novel calcium alginate poly(ethylene glycol) hybrid microspheres (Ca-alg-PEG) from Na-alg-PEG and studied the suitability of these microspheres for cell microencapsulation.Although the focus of this paper is not to investigate the suitability of Na-alg-PEG for cell encapsulation, we briefly show some results of the ongoing studies.It was found that Na-alg-PEG maintains the gelling capacity in presence of divalent cations, while the free thiol end groups allow for simultaneous chemical cross-linking.Stable microspheres were prepared in a one-step process and without incorporation of polycations 46,47.Human hepatocellular carcinoma cells (Huh-7) were successfully encapsulated within Ca-alg-PEG (Figure 3).They maintained their viability, proliferated and continued secreting albumin during a two-weeks study 46,47.

Conclusions
Efficient synthesis of heterobifunctional PEG is challenging due to the potential of these molecules to add novel, advantageous functionality to other molecules and surfaces upon pegylation.With the ultimate goal to provide tools for the modification of molecules and surfaces, versatile synthesis of heterobifunctional PEG derivatives via selective tosylation of commercially available symmetrical PEG was demonstrated.Addressed were the functionalities, which are most frequently used for pegylation technology, including amine, thiol, and azide.Because the synthesis pathway is based on the successive activation of hydroxyl end groups, the chemistry presented here may be applied to introduce other desired functionalities.The reactions provide heterotelechelic PEG at least in the multi-gram scale.As proof of concept for the development of novel materials, amine-terminated PEG was grafted onto Na-alg as an example of pegylation technology.The reactive thiol end groups remaining after the pegylation were efficient to spontaneously form a hydrogel via disulfide bonds.Simultaneous fast ionic gelation and slow chemical cross-linking of the pegylated Na-alg allowed for producing a novel type of hybrid hydrogel microspheres suitable for cell microencapsulation.The molar masses of heterobifunctional PEG were estimated from 1 H-NMR spectra, according to previously published method [23].Values are given in Table 1.

Figure 3 .
Figure 3. Huh-7 encapsulated within Ca-alg-PEG hybrid microspheres.The cells were observed at day 3 using optical (left) and fluorescence (right) microscopy.Viable cells fluorescence green, while dead cells fluorescence bright red.Scale bar: 150 µm.

Table 1 .
Summary of syntheses efficiency and molecular characteristics.

) a Molar Mass (g/mol) PDI b GPC 1 H-NMR
a Final yield, after one or more steps; b PDI: Polydispersity index from GPC.