Clickable Poly(ethylene glycol)Based Copolymers

Macromolecular Chemistry and Physics

Full Paper

Clickable Poly(ethylene glycol)-Based Copolymers Using Azide–Alkyne Click Cycloaddition-Mediated Step-Growth Polymerization Mehmet Arslan, Ozgul Gok, Rana Sanyal, Amitav Sanyal*

The design, synthesis, and post-polymerization functionalization of linear poly(ethylene glycol) (PEG)-based polytriazole copolymers containing thiol-reactive maleimide functional groups as pendant side chains are described. A copper(I)-catalyzed Huisgen-type 1,3-dipolar cycloaddition reaction is utilized to prepare copolymers through a combination of diazide- and dialkyne-functionalized triethylene glycol derivatives, along with a dialkyne-functionalized furan-protected maleimide-containing monomer. After the polymerization, a microwaveassisted retro-Diels–Alder reaction is utilized to unmask the pendant maleimide groups by removal of the furan moieties. Post-polymerization modifications of these PEG-based copolymers containing the furan-protected maleimide group are accomplished using a photoinitiated radical thiol-ene reaction, whereas polymers containing the reactive maleimide group are functionalized using the Diels–Alder and nucleophilic thiolene reactions. The nucleophilic thiol-ene reaction is utilized to fabricate hydrogels by treatment of the maleimide-containing PEG polymers with dithiol-containing crosslinkers. Facile functionalization of thus-obtained hydrogels under mild conditions is demonstrated through the treatment of the residual maleimide groups within the hydrogel with a thiol-containing fluorescent dye.

1. Introduction Design and synthesis of reactive polymers that allow facile functionalization using post-polymerization modifications have gained increased attention in recent years.[1,2] Polymeric scaffolds with precisely decorated functionalities M. Arslan, O. Gok, Prof. R. Sanyal, Prof. A. Sanyal Department of Chemistry, Bogazici University, Bebek 34342, Istanbul, Turkey E-mail: [email protected] Macromol. Chem. Phys. 2014, 215, 2237−2247 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

have found applications in polymer-based drug delivery,[3] biomolecular immobilization,[4] fabrication of polymeric coatings,[5] and organic–inorganic hybrids.[6] In many applications, the attachment of target molecules on polymers requires fast and clean procedures. Thus, much of the effort has been dedicated to adapt the efficient chemical transformations towards polymer functionalization and postpolymerization modification has emerged as a powerful tool for fabrication of functional materials. Usually, desired post-functionalizable groups are placed on the polymer chain by either employing reactive-group-containing

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DOI: 10.1002/macp.201400210

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initiators or by the incorporation of post-functionalizable monomers during polymerization. Depending on the amount of reactive monomer on the side chain of the polymer, one can tune the density of installed molecules of interest along the polymer backbone. Various functional groups including alkenes, activated esters, alkynes, and azides, pyridyl disulfides, activated dienes, and dienophiles have been incorporated into polymers as pendant groups and these reactive groups have been effectively functionalized by using various efficient chemical transformations.[7] Much of these transformation rely on “click” chemistry-based methodologies. Beside the advancements in post-polymerization modification of polymer obtained via chain-growth polymerization, there is also growing research interest in functionalization of polymers obtained via step-growth polymerization.[8] Efficient chemical transformations based on click strategies serve not only in the synthesis of polymers via a step growth fashion, but they also provide a powerful methodology for post-functionalization of polymers for various applications.[9,10] Click reactions have been utilized for the preparation of polymers via the step-growth methodology using the copper(I)-catalyzed Huisgen-type azide–alkyne cycloaddition.[11] The generally employed strategy is based on the coupling of homobifunctional alkyne and azide monomers in a polyaddition fashion.[12] Due to the orthogonal, selective and highly efficient nature of click reaction, this polymerization technique remains as a powerful method for polymer synthesis. However, in the context of post-polymerization functionalization, there are limited examples of click polymers that allow post-polymerization modification in a simple and direct manner. A click polymerization approach for the preparation of linear poly(ethylene) glycols bearing pendant hydroxyl functionalities was demonstrated by Wang and co-workers.[13] Building blocks including alkyne-terminated Poly(ethylene glycol) (PEG) polymer and a diolcontaining bisazide were polymerized using the CuSO4/ sodium ascorbate catalyst system. After formation of linear polytriazole, hydroxyl groups introduced side chain functionality to the polymer. The approach was extended to synthesize polytriazoles containing alendronate and rhodamine side groups for bone-targeting drug delivery application. However, alendronate and rhodamine were firstly incorporated into separate diazide monomers from one of hydroxyl groups and subsequently used during copolymerization. Recently, Fokin and co-workers demonstrated the synthesis of polytriazoles containing the 5-iodotriazole linkages at polymer backbone.[10] A novel α-azido-ω-iodoalkyne monomer was shown to very reactive in CuAAC-mediated polymerization to yield polymers containing post-functionalizable pendant iodo groups that were efficiently coupled with phenylboronic acid or alkenes by palladium-catalyzed cross-coupling reactions.

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The utilization of Michael-type addition of thiols on maleimides in polymer functionalization has been extensively exploited in recent years due to their high reaction yields, high selectivity under mild and catalyst-free reaction conditions, this versatile chemistry have provided an important tool for engineering materials. The maleimide functional group provides an effective handle for the modification of polymeric materials via the nucleophilic thiolene and Diels–Alder cycloaddition reaction.[14] Especially in designing polymeric scaffolds for biomolecular immobilization of relevant molecules (enzymes, proteins, peptides), it is important to achieve precisely appointed and well-defined systems for a quantitative assessment of such conjugates. For this purpose, reactive cysteine residues of proteins provide an opportunity due to the presence of thiol groups. Effective conjugation of cysteine thiols to maleimide groups of polymers provides site-specific functionalization of polymeric materials in fast under mild conditions.[15] Maleimide is also an excellent dienophile and readily reacts with electron-rich dienes through the Diels–Alder cycloaddition reaction. The Diels–Alder chemistry of maleimides has been exploited in synthesis and functionalization of various polymeric materials such as dendrimers, dendronized polymers, graft and block copolymers, and hydrogels.[16] Here, we report the synthesis and post-polymerization functionalization of linear PEG-based polytriazoles containing reactive maleimide units as pendant side chains. A furan-protected maleimide-containing diyne monomer was copolymerized with diyne and diazide-functionalized triethylene glycol (TEG) derivatives using the copper(I)catalyzed Huisgen-type 1,3-dipolar cycloaddition reaction. After the polymerization, the masked maleimide groups were activated by employing the retro-Diels– Alder (rDA) cycloreversion reaction to remove the furan protecting groups. Thus obtained side chain maleimide and oxo-norbornene (furan/maleimide cycloadduct)containing copolymers were investigated towards postpolymerization modification by utilizing various chemical transformations. As an extended application, covalent crosslinking of maleimide-containing copolymer with a bis-thiol compound was demostrated. Free maleimide groups in obtained hydrogel were efficiently functionalized using a thiol-containing fluorescent dye molecule.

2. Experimental Section 2.1. Materials and Characterization The reagents and solvents were purchased from commercial sources and used as received unless otherwise noted. TEG and sodium azide were purchased from Merck. Sodium hydride, propargyl bromide, Cu(I) bromide, Cu(I) iodide, bromotris(triphenylphosphine)copper(I), N,N,N′,N′,N′′-pentamethyldiethylenetriamine (PMDTA), 1,8-diazabicyclo[5.4.0]-

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Clickable Poly(ethylene glycol)-Based Copolymers...

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undec-7-ene (DBU), N,N-Diisopropylethylamine (DIPEA), benzyl mercaptan, 2,2-dimethoxy-2-phenylacetophenone (DMPA), 3,6-di-2-pyridyl-1,2,4,5-tetrazine, anthracene, and 2,2′-(ethylenedioxy)diethanethiol were obtained from Aldrich. Homobifunctional TEG monomers with alkyne[17] (diyne-TEG) and azide[18] (diazide-TEG) groups at their termini were synthesized according to reported procedures and confirmed with a spectroscopic analysis. Compound diyne-FuMAL was synthesized according to previously published procedure.[19] The gel permeation chromatography (GPC) measurements of polymers were carried out using a Shimadzu GPC analysis system with PSS WinGPC Unity software. PSS Gram (length/ ID 300 mm × 8 mm, 10 μm particle size) column was calibrated with polymethyl methacrylate standards, using refractive index detector (RID-10A). Dimethylacetamide (DMAc) was used as eluent at a flow rate of 1 mL min−1 at 30 °C. NMR spectra were recorded on a Varian 400-MHz spectrometer. The microwaveassisted reactions were performed using a Biotage Initiator synthesizer. Reactions were carried in single-use 10 mL reaction vessels and septa designed for high temperature/pressure reactions were used. Thermogravimetric analysis (TGA) was carried out on a TA Instruments at a heating rate of 10 °C min−1 under a nitrogen atmosphere.

2.2. General Procedure for the Polymerization of Diyne-TEG and Diazide-TEG Monomers Using Different Copper Catalysts Diyne-TEG (226.20 mg, 0.1 mmol), diazide-TEG (200.20 mg, 0.1 mmol), and copper catalyst/ligand mixture (CuBr/PMDTA:0.1 mmol/0.2 mmol; CuI/DBU:0.1 mmol/1.0 mmol; Cu(PPh3)3/ DIPEA:0.1 mmol/1.0 mmol) were dissolved in dimethylformamide (DMF) (4 mL) in a sealed tube equipped with a magnetic stir bar. The mixture was purged with nitrogen for 15 min and allowed to stir at 25 °C for 12 h. After polymerization, the catalyst was removed by passing the mixture through neutral aluminum oxide. The solvent was removed under reduced pressure and remaining crude polymer was redissolved in minimum amount of dichloromethane before precipitating into cold tetrahydrofuran (THF). After drying in vacuum, polymers were obtained as waxy liquids. 1H NMR (400 MHz, CDCl3, δ, ppm): 7.69 (s, 2H, OCH2C CH), 4.63 (s, 4H, NCCH2O), 4.48 (bt, 4H, NCH2CH2O), 3.79– 3.52 (backbone CH2 groups, 20 H, stack).

2.3. General Procedure for the Polymerization of DiyneFuMAL, Diyne-TEG, and Diazide-TEG Monomers with CuBr Catalyst (Pt-FuMAL) Typical polymerization procedure for Pt-FuMAL-1: Diyne-FuMAL (49.95 mg, 0.1 mmol), diyne-TEG (203.60 mg, 0.9 mmol), diazideTEG (200.20 mg, 1.0 mmol), CuBr (14.30 mg, 0.1 mmol), and PMDTA (41.71 μL, 0.2 mmol) was dissolved in DMF (4 mL) in a sealed tube equipped with a magnetic stir bar. After 15 min nitrogen purge of the solution, reaction mixture was stirred at 25 °C for 12 h. The catalyst was removed by passing the mixture through neutral aluminum oxide. The solvent was removed under reduced pressure and remaining crude polymer was redissolved in minimum amount of dichloromethane


before precipitating into cold THF. The polymer was obtained as waxy liquid (67%). 1H NMR (400 MHz, CDCl3, δ, ppm) 7.67 (s, OCH2C CH, 21H), 7.45 (s, CH2CH2C CH, 2H), 6.47 (s, CH CH, 2H), 5.20 (s, CH bridgehead protons, 2H), 4.63 (s, NCCH2O, 42H), 4.47 (t, NCH2CH2O, 46H), 4.26–4.15 (m, OCH2C ester, 4H), 4.06– 3.95 (m, OCH2CH2 ester, 2H), 3.79–3.51 (backbone CH2s, stack), 2.97 (t, NCCH2, 4H), 2.83 (s, bridge protons, 2H), 2.71 (t, NCCH2CH2, 4H), 1.87 (m, NCCH2CH2, 2H), 1.23–1.19 (two singlets, CH3, 3H).

2.4. General Procedure for Post-Polymerization Activation of Maleimide Functional Groups (Pt-MAL) Polymer Pt-FuMAL-1 (50.0 mg) was dissolved in 1:1 mixture of chloroform/acetonitrile (2 mL) and irradiated in microwave for 60 min (110 °C). On cooling at room temperature, solvent was evaporated in vacuo to yield polymer Pt-Mal-1 in quantitative yield. The obtained polymer was characterized using 1H NMR spectroscopy.

2.5. Post-Polymerization Functionalization of Pt-MAL-1 Polymer via Nucleophilic-Thiol-Ene Reaction Polymer Pt-MAL-1 (50.0 mg, 0.004 mmol) and benzyl mercaptan (6.20 mg, 0.05 mmol) were dissolved in 2 mL of dichloromethane and triethylamine (1.0 μL, 0.007 mmol) was added to the solution. The mixture was stirred at room temperature for 12 h. After reaction, the mixture was precipitated in cold THF affording the thiol appended polymer.

2.6. Post-Polymerization Functionalization of Pt-MAL-1 Polymer via Diels–Alder Cycloaddition Reaction A solution of polymer Pt-MAL-1 (50.0 mg, 0.004 mmol) and anthracene (2.30 mg, 0.013 mmol) in DMF (1 mL) was heated at 120 °C for 24 h in the dark. The reaction mixture was concentrated in vacuo to remove the solvent and the crude product was dissolved in minimum amount of dichloromethane before precipitating in cold THF.

2.7. Functionalization of Pt-FuMAL-1 Polymer via Radical-Thiol-Ene Reaction Polymer Pt-FuMAL-1 (50.0 mg, 0.004 mmol), benzyl mercaptan (6.20 mg, 0.05 mmol), and DMPA (1.80 mg, 0.007 mmol) were dissolved in the 200 μL of DMF. The mixture was purged with nitrogen for 10 min and then was exposed to UV light at 365 nm for 1 h. After reaction, the resulting polymer was purified by precipitating in cold THF.

2.8. Hydrogel Synthesis via Crosslinking of MaleimideContaining Polymer The polymer Pt-MAL-1 (50.0 mg, 0.004 mmol) was placed in a vial and dissolved in dichloromethane (200 μL). A solution of 2,2′-(ethylenedioxy)diethanethiol (1.64 mg, 0.009 mmol) and triethylamine (0.10 μL, 9 × 10−4 mmol) in dichloromethane (100 μL) was then added to this solution. The mixture was briefly sonicated


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to assist homogeneous gelation. After hydrogel formation, unreacted reagents were removed by washing the gel with THF followed by distilled water several times. The swollen hydrogel sample was frozen and lyophilized to yield dried hydrogel.

2.9. Functionalization of Hydrogels with Thiol-Containing Dyes Hydrogels containing free maleimide groups were functionalized via SAMSA fluorescein thiol conjugation. First, the protecting group of the dye was removed by following the procedure indicated by the supplier. Briefly, 10 mg of SAMSA fluorescein was dissolved in 0.1 M NaOH and incubated at room temperature for 15 min to remove the acetyl protecting group. Then, the solution was neutralized with concentrated HCl and buffered to pH 7 with 0.5 M sodium phosphate. The amount of free maleimide groups in a 10 mg hydrogel sample was calculated and a tenfold excess of activated SAMSA fluorescein thiol solution was added. After incubation at room temperature for 12 h, hydrogel was washed several times with deionized water before analysis of fluorescence microscopy. As a control experiment, a sample of hydrogel was incubated with acetyl protected SAMSA fluorescein thiol dye.

3. Result and Discussion 3.1. Synthesis of Maleimide Side-Chain-Functionalized Click Step-Growth Polymers Cu-catalyzed azide–alkyne cycloaddition polymers comprising reactive side-chain groups were synthesized via utilizing click polyaddition of homobifunctional monomers containing complementary click coupling functionalities. A diyne-functionalized furan-protected maleimidecontaining monomer (diyne-FuMAL) was copolymerized with hydrophilic diyne (diyne-TEG) and diazide (diazide-TEG) triethylene glycol derivatives. The strategy involves a A1–A1/B1–B1/B2–B2-type combination of diazide- and dialkyne-containing monomers. The amount of dialkyne monomer containing reactive maleimide functionality allows one to tune the degree of functional groups in the final polymer. First, in the absence of diyneFuMAL monomer, the polymerizability of diyne-TEG and diazide-TEG monomers was investigated using different copper catalyst systems (Scheme 1A). The polytriazoles with hydrophilic oligo(ethylene glycol) backbones were synthesized using CuBr, CuI, and Cu(PPh3)3 catalyst at room temperature. DMF was chosen as the reaction solvent due to its high solvating ability for the hydrophilic diyne-TEG and diazide-TEG monomers, and the hydrophobic diyne-FuMAL monomer, as well as the obtained polytriazoles. The polymerization results are summarized in Table 1 (Items 1–3). According to SEC analysis, copolymers prepared from diyne-TEG and diazide-TEG monomers using CuBr and CuI catalysts have molecular weights

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around 14 kDa. The polymerization with Cu(PPh3)3 catalyst yielded relatively lower molecular weight copolymer. The lower performance of this catalyst was attributed to the side reaction between the PPh3 groups from the copper catalyst and azide groups (via the Staudinger reaction).[20] Polytriazole obtained by CuBr catalyst showed narrower polydispersity index compared with CuI catalyst. A higher polymer conversion was also attained in the case of CuBr/PMDTA system. Obtained polymers were soluble in common organic solvents like dichloromethane, chloroform, DMF, and acetonitrile; however, they are not soluble in THF and dioxane. All copolymers were characterized by 1H NMR spectroscopy, and the specific signal belonging to triazole proton was observed at 7.69 ppm (Figure 1). Furthermore, all the characteristic signals belonging to the oligo(ethylene glycol) backbone were clearly evident and could be assigned to confirm that PEG-based oligomers were connected through triazole linkages. After the brief survey of various catalysts and polymerization conditions, the diazide-TEG and diyne-TEG monomers were copolymerized with varying amounts of the diyne-FuMAL monomer using the CuBr/PMDTA catalyst system (Pt-FuMAL, Table 1, Items 4–6). All polymerizations were carried out in DMF at room temperature to ensure homogeneity during the reaction. After polymerization, purification was carried out by precipitating the polymers in THF to remove the unreacted monomers and low-molecular-weight linear and cyclic oligomeric fractions. Successful incorporation of diyneFuMAL monomer into the copolymers was confirmed from the presence of proton resonances around 6.47, 5.20, and 2.83 ppm belonging to the bicyclic moiety of the furan-maleimide adduct, along with the additional triazole signal at 7.45 ppm in the 1H NMR spectra of the copolymers (Figure 2). The extent of incorporation of the diyne-FuMAL monomer in the final copolymers could be inferred from the comparison of integration values of the two triazole proton resonances. Overall, a slightly lower incorporation of the monomer than aimed in the feed was observed. This could be explained by the steric bulk of the monomer as it differs considerably from the linear diyne-TEG derivatives. Another underlying reason for low contribution might be the formation of AA+BB-type dimerized cyclization products that consumes the monomer to some extent, thus lowering its incorporation.[21] After obtaining the polymers, the side-chain-masked maleimide moieties were deprotected by utilization of the rDA reaction. First attempts to remove the furan protecting group in refluxing toluene or dioxane were unsuccessful due to limited solubility of polymers, whereas heating of polymers without any solvent in vacuo generated insoluble materials. As an alternative strategy, the polymers were subjected to microwave heating at 110 °C in a chloroform–acetonitrile mixture.





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Scheme 1. A) Syntheses of polytriazoles from diyne-TEG and diazide-TEG monomers with different copper catalysts (CuX: CuBr, CuI or Cu(PPh3)3). B) Synthesis of maleimide side-chain-functionalized PEG-based copolymer.

Within an hour, complete removal of the furan protecting groups was achieved as revealed by the 1H NMR analysis (Figure 3). The disappearance of proton resonances of bridgehead bicyclic structure and formation of a new peak at 6.68 ppm belonging to the maleimide double bond confirmed the successful cycloreversion. The weight


loss due to the removal of furan moieties during rDA reaction was also determined by TGA (Figure 4). The polymers showed an initial weight loss between temperatures of 90 to 150 °C due to the loss of the furan protecting groups. As expected, an increasing weight loss was observed for polymers with increasing amounts of the furan-protected


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Table 1. Properties of copolymers obtained by Cu-catalyzed polyaddition of diyne and diazide monomers.

Item

Polymer

Catalyst

[M]1:[M]2:[M]3a)

Fb) [%]

M n ,SECc)

c)

M w /M n

Conv. [%]

mol−1]

[g

1

Pt-1

CuBr/PMDTA

1:1:0

–

14 400

1.72

72.4

2

Pt-2

CuI/DBU

1:1:0

–

14 200

2.23

66.3

3

Pt-3

Cu(PPh3)3/DIPEA

1:1:0

–

5600

1.94

51.7

4

Pt-FuMAL-1

CuBr/PMDTA

1:0.9:0.1

8.73

11 700

1.60

66.7

5

Pt-FuMAL-2

CuBr/PMDTA

1:0.8:0.2

12.80

9400

1.68

58.9

6

Pt-FuMAL-3

CuBr/PMDTA

1:0.7:0.3

21.20

7700

1.76

69.2

a)Mol equivalent; [M] : diazide-TEG, [M] : diyne-TEG, [M] : diyne-FuMAL; b)F = [M] /([M] + [M] ) Determined by 1H NMR analysis; c) Esti1 2 3 3 2 3 mated by SEC eluted with DMAc, using polymethyl methacrylate standards.

maleimide-containing monomer. Importantly, no detrimental effect of the thermal rDA reaction on the polymer structure was observed since the molecular weight and its distribution did not change significantly, as inferred from the SEC analysis (Figure 5). 3.2. Functionalization of Polymers Containing Pendant Reactive Groups

Figure 1. 1H NMR spectrum of the polytriazole Pt-1 obtained by the polymerization of diazide-TEG and diyne-TEG monomers.

Figure 2. Representative 1H NMR spectrum of copolymer Pt-FuMAL-1 containing furan-protected maleimide functional groups as side chains.

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The polymers carrying maleimide and furan-maleimide oxanorbornene groups as side chains along the copolymer backbone were functionalized with small model molecules by employing various click reactions (Figure 6). Functionalization of the maleimide groups was explored using the nucleophilic thiol-ene and the Diels–Alder cycloaddition reactions. As an alternative strategy for the conjugation of thiol-containing molecules to these polymers, the radical thiol-ene reaction was examined for the copolymers containing oxanorbornene moieties. Post-polymerization functionalization efficiency of Pt-MAL-1 polymer with maleimide side chain functionalities was investigated by subjecting them to conjugation with benzyl mercaptan. A fivefold excess equivalent of benzyl mercaptan compared with the maleimide groups on the polymer Pt-MAL-1 was used to ensure complete conversion. Catalytic amount of triethylamine was added to the reaction since the Michael-type addition is generally





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diene. Copolymer Pt-MAL-1 was reacted with a slight excess of anthracene (1.3 eq. per maleimide unit) at 120 °C in DMF for 24 h. Complete functionalization was achieved as indicated by the loss of the proton resonances belonging to the vinylic protons on the maleimide ring, along with the appearance of new aromatic proton resonances around 7.10– 7.40 ppm arising from the flanking aromatic rings in the anthracene-maleimide cycloadduct in the 1H NMR spectrum of the copolymer (Figure 7B). Although our main aim was to synthesize thiol-reactive polymers that contain maleimide-based side chains but it was also apparent that the precursor Figure 3. 1H NMR spectrum of copolymer Pt-MAL-1 containing activated maleimide func- polymer containing the furan-protected tional groups at side chains. maleimide adduct was also amenable to functionalization via the radical thiolene reaction. It is well established that strained alkenes catalyzed by a non-nucleophilic base (Figure 7A). Quanthat are present in the norbornene moiety are excellent titative conjugation of the thiol was clearly evident from candidates for thiol-ene addition reactions. Benzyl merthe 1H NMR spectrum of the functionalized copolymer captan was used as a model compound to evaluate the due to the complete disappearance of the proton resoefficiency of functionalization reaction. After UV irradianance around 6.7 ppm arising from the vinylic protons tion (λ = 365 nm) of the polymer in the presence of excess on the maleimide functional group. Additionally, proton resonances in the aromatic region arising due to the benzyl mercaptan (5 eq. per furan-maleimide group), DMPA phenyl ring of benzyl mercaptan were also visible. as photoinitiator, the crude mixture was precipitated in cold THF to get rid of the excess reagents. Analysis of thus Besides being susceptible towards conjugate addition obtained polymer with 1H NMR spectroscopy revealed comreaction with thiols, maleimides are also known to be excellent dienophiles for addition to various electronplete disappearance of the furan-maleimide cycloadduct rich dienes through the Diels–Alder cycloaddition reacdouble bond peak at 6.47 ppm along with the signal arising tion. In order to probe the functionalization of the penfrom the bridgehead protons at 5.20 ppm, and revealed the dant maleimide groups via the Diels–Alder reaction, appearance of new proton resonances belonging to the aroanthracene was chosen as a representative electron-rich matic protons of benzyl mercaptan (Figure 8).

Figure 4. Thermogravimetric analysis plots of copolymers Pt-FuMAL(1–3).


Figure 5. SEC traces of copolymer Pt-FuMAL-1, before and after retro-Diels–Alder reaction.


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3.3. Synthesis of Thiol-Reactive Hydrogels via Michael-Type Thiol-ene Reaction

Figure 6. General representation of post-functionalization reactions for copolymers carrying side chain maleimide and oxo-norbornene groups.

Fabrication of hydrogels via the covalent crosslinking of PEG-based copolymers containing pendant reactive maleimide groups using a bis-thiol bearing crosslinker was investigated. The copolymer Pt-MAL-1 and a bis-thiolbased crosslinker 2,2′-(ethylenedioxy)diethanethiol were dissolved in dichloromethane in separate vials. Mixing the two solutions produced a hydrogel via crosslinking due to the Michael-type addition of thiols to the maleimide groups (Figure 9a). In the presence of a catalytic amount of triethylamine, the gel formation was rapid and

Figure 7. Synthetic transformations and 1H NMR spectra of functionalized copolymer Pt-MAL-1 bearing pendant maleimide functionalities. A) Functionalization via nucleophilic thiol-ene reaction. B) Functionalization via the Diels–Alder reaction.

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Figure 8. Functionalization using the radical thiol-ene reaction and 1H NMR spectrum of the functionalized copolymer Pt-FuMAL-1.

after 60 s there was no flow of sample. After the hydrogel formation, residual reagents were removed by washing the gel several times with copious amounts of THF, followed by distilled water. Hydrogel samples equilibrated to swelling were frozen and freeze-dried in vacuo to yield dried hydrogels. The obtained hydrogels was characterized with scanning electron microscopy (SEM) to reveal a microstructure

characteristic of a rubber-like randomly crosslinked polymeric material (Figure S3, Supporting Information). As expected, the PEG-based hydrogel exhibits excellent water uptake (1200% by weight) and reaches swelling equilibrium within 30 min (Figure 9b). Functionalization of the residual-free maleimide groups in the hydrogel was investigated by conjugation of a thiol-containing fluorescent

Figure 9. a) General reaction scheme of hydrogel formation. b,c) Swelling profile of hydrogel (b) and fluorescence microscopy image (c) of dye-functionalized hydrogel.



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dye, namely, SAMSA fluorescein. After incubation of the hydrogel with the thiol-containing dye in aqueous media, residual unbound dye was removed by washing the hydrogel with copious amounts of THF and water. Fluorescence microscopy analysis revealed efficient covalent conjugation of the thiol-containing dye to hydrogel (Figure 9c). As a control experiment, the maleimide-containing hydrogels were treated with acetyl-protected sulfhydryl-bearing fluorescein dye; and as expected, no fluorescence was observed due to the lack of any covalent conjugation of the dye to the hydrogel. Thus the pendant maleimide groups in these PEG-based copolymers could serve the dual role of enabling fabrication of hydrogels under mild conditions as well as their facile functionalization with other thiolcontaining molecules of interest.

4. Conclusion Thiol-reactive copolymers based on linear polytriazoles with oligo(ethylene glycol) backbone appended with maleimide functional groups as side chains were prepared using the step-growth polymerization. The copper(I)-catalyzed Huisgen-type 1,3-dipolar azide–alkyne cycloaddition reaction was utilized for the copolymerization of diazide- and dialkyne-functionalized monomers. Copolymerization of TEG derivatives was first investigated using different copper(I) catalysts to determine optimum reaction conditions and catalyst system. Thereafter, a diyne-functionalized furan-protected maleimide-containing monomer was incorporated into copolymers with varying feed ratios. The maleimide groups along the side chain of the copolymers were unmasked via the rDA reaction. Obtained copolymers were post-functionalized via nucleophilic thiol-ene and Diels–Alder reactions. Additionally, the furan-protected copolymer could be functionalized using photo-initiated radical thiol-ene reaction. These copolymers could be covalently crosslinked using bis-thiol based crosslinkers under mild conditions to yield hydrogels. By adjusting the relative stoichiometry of the thiol and maleimide functional groups during the crosslinking, hydrogels containing residual maleimide groups can be obtained. These maleimide-containing hydrogels undergo facile functionalization using thiol-containing small molecules such as fluorescent dyes. The approach outlined in this work can be extended to obtain various reactive PEG-based polytriazole-containing copolymers for appropriate post-polymerization functionalization through judicious choice of monomer building blocks.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

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Acknowledgements: R.S. would like to thank Turkish Academy of Sciences for TUBA-GEBIP fellowship. The authors also thank the state planning commission of Turkey for financial assistance under DPT grant no. 09K120520. Received: April 24, 2014; Revised: May 22, 2014; Published online: June 25, 2014; DOI: 10.1002/macp.201400210 Keywords: click chemistry; Diels–Alder reaction; hydrogels; stepgrowth polymerization; thiol-maleimide conjugation

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