Synthesis of Semitelechelic Maleimide Poly(PEGA) for Protein

Jun 9, 2009 - Emmanuelle Bays, Lei Tao, Chien-Wen Chang, and Heather D. Maynard*. Department of Chemistry and Biochemistry and the California ...
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Biomacromolecules 2009, 10, 1777–1781

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Synthesis of Semitelechelic Maleimide Poly(PEGA) for Protein Conjugation By RAFT Polymerization Emmanuelle Bays, Lei Tao, Chien-Wen Chang, and Heather D. Maynard* Department of Chemistry and Biochemistry and the California NanoSystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095-1569 Received February 13, 2009; Revised Manuscript Received April 23, 2009

Maleimide end functionalized polymers for site-selective conjugation to free cysteines of proteins were synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. A furan-protected maleimide chain transfer agent (CTA) was employed in the RAFT polymerization of poly(ethylene glycol) methyl ether acrylate (PEGA). Gel permeation chromatography (GPC) with laser light scattering detection indicated that 20000 and 39000 Da polyPEGA had been made with polydispersity indices of 1.25 and 1.36, respectively. The maleimide group on the polymer chain end was exposed by heating the poly(PEGA)s for 4 h. The deprotection efficiency was estimated to be 80 and 60% for poly(PEGA)20 kDa and poly(PEGA)39 kDa, respectively. Maleimide-poly(PEGA)s were conjugated to V131C T4 lysozyme (T4L), and the resultant polymer-protein conjugates were characterized by size exclusion chromatography and gel electrophoresis.

Introduction Proteins are an important class of therapeutic agents because of their precise biological activity in vivo.1,2 However, clinical applications of these biomolecules have indicated several shortcomings. These limitations include short circulation time, poor stability, and immunogenicity. Consequently, frequent drug administration at high dosage is necessary, which often leads to undesired side effects, in addition to requiring numerous injections.3,4 As a result, alternative strategies to deliver these proteins have been the subject of intense research. PEGylation, the conjugation of the water-soluble polymer, poly(ethylene glycol), to a biomolecule, is an effective way to overcome the limitations associated with native proteins. PEGylation of proteins results in increased therapeutic effects and decreased side-effects. A large number of PEGylated-protein and PEGylated-peptide drugs have been successfully developed by conjugating linear PEG to therapeutic proteins; an example is PEGylated interferon R-2a (Pegasys, Roche).5 In recent years, branched PEG structures such as poly(PEGA) with one protein attachment site have been emerged as an alternative PEGylation agent.6 Compared to linear PEG, poly(PEGA) presents additional benefits in vivo. For example, the greater steric bulk of a multibranched PEG compared to linear PEG leads to a better protection against enzymatic degradation and reduced of immunogenicity.7 Poly(PEGA)s also present advantages regarding their synthesis. There is a greater flexibility to prepare branched PEG, enabling structure optimization that can be tailored to incorporate specific end-groups or other side chains.8 To minimize the loss of biological activity, site-specific conjugation of PEG and poly(PEGA)s to proteins is desired. One promising strategy is conjugation to the thiol group of a free cysteine residue on the protein surface. Free cysteine residues are rare in native proteins and can easily be introduced via protein engineering. Thus, the reaction is site specific and often affords superior bioactivity retention than reaction with lysines and the N-terminus.9 Reaction with maleimides is * To whom correspondence should be addressed. E-mail: maynard@ chem.ucla.edu.

efficient and leads to chemoselective conjugation to proteins at solution pHs less than or equal to 7. Pyridyl disulfide is another group that is selective for thiols. Controlled radical polymerizations (CRP) have been developed to synthesize polymers with predesigned molecular weights, narrow polydispersity index (PDI) and precisely controlled architectures. Previously, we have reported use of atom transfer radical polymerization (ATRP) to conjugate thiolreactive polymers to proteins using a pyridyl disulfide group, resulting in reversible conjugates with proteins without the need for post polymerization modification.10 We also used ATRP to form telechelic polystyrene with maleimide end groups.11 Polystyrene was synthesized with a protected maleimide initiator and subjected to atom transfer radical coupling to form the bisfunctionalized polymer. The reactivity of the bis-functionalized polymer was demonstrated by an in situ deprotection and addition with cysteine. Haddleton and co-workers have also synthesized poly(PEG methacrylate)-maleimide and poly(glycerol) methacrylate-maleimide polymers by ATRP.12 Reversible addition-fragmentation chain transfer (RAFT) polymerization is controlled by a reversible transfer reaction between a growing radical and chain transfer agent (CTA).13-15 The CTA contains two components: a Z-group, which stabilizes the trapped radical intermediate, and an R-group, which forms an active radical to create a new polymer chain. This chain transfer reaction continues during the polymer propagation, which leads to uniformity in chain length and consequently in a well-defined polymer with low PDI.16,17 R- or ω-Functionalized polymers can be straightforwardly prepared using appropriately designed CTAs.18-20 RAFT is also amenable to polymerizing from proteins.21,22 Previously, we have used RAFT to synthesize a polymer with a pyridyl disulfide group for reversible biomolecule conjugation.23 One of the advantageous features of RAFT is that it is easy to modify the ω-end of the polymer with a fluorophore24 or other moieties. Thus, we explored for the first time RAFT in the presence of a maleimidefunctionalized CTA to form poly(PEGA), which would allow us to prepare irreversible conjugates. Polymerization and deprotection of the maleimide group were studied, and the

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polymers were characterized by gel permeation chromatography (GPC) and 1H NMR spectroscopy. Finally, the conjugation of poly(PEGA) to a model protein, T4 lysozyme (T4L), was demonstrated.

Materials and Methods Materials. 4-(3-Hydroxy-propyl)-10-oxa-4-aza-tricyclo[5,2,1,02,6] dec-8-ene-3,5-dione (1)25 and 2-(ethyl trithiocarbonate)propionic acid26 were synthesized as previously reported. Azobisisobutyronitrile (AIBN, Aldrich, 98%) was recrystallized twice from ethanol. Tetrahydrofuran (THF) was refluxed over sodium pellets and distilled prior to use. PEGA (Aldrich, Mn ∼ 454, g 99%), N,N′-dicyclohexylcarbodiimide (DCC, Fluka, g99%), and 4-dimethylaminopyridine (DMAP, Fluka, g99%) were used as received. All other chemicals were purchased from Sigma or Fisher Scientific and used as received. The T4L plasmid was provided by Professor Wayne Hubbell at UCLA. Analytical Technique. 1H and 13C NMR spectra were acquired on an ARX 400 MHz NMR spectrometer and spectra were processed using Topspin 1.2 NMR software. UV-vis spectra were obtained on a Biomate 5 Thermo Spectronic UV-vis spectrometer using quartz cells. Infrared absorption spectra were recorded using a PerkinElmer FT-IR equipped with an ATR accessory. TLC plates precoated with silica gel 60 F254 were developed in the indicated solvent systems. GPC was conducted on a Shimadzu HPLC system equipped with a refractive index detector RID-10A, one Polymer Laboratories PLgel guard column, and two Polymer Laboratories PLgel 5 µm mixed D columns. Lithium bromide (LiBr; 0.1 M) in dimethylformamide (DMF) at 40 °C was used as the mobile phase (flow rate: 0.80 mL/min). Calibration was performed using near-monodisperse poly(methyl methacrylate) standards from Polymer Laboratories. Chromatograms were processed using the EZStart 7.2 chromatography software. Molecular weights of the polymers were also analyzed on a Viscotek VE 2001 system equipped with a 270 Dual Detector (laser light scattering (LLS) and viscometer) and VE 3580 refractive index detector with three 300 × 7.8 mm Viscotek ViscoGEL I-Series Mixed Bed Mid MW columns (with column guard). DMF containing 0.1% LiBr was used as the mobile phase (60 °C, flow rate 1.0 mL/min). Chromatograms were processed using the OmniGPC 4.5.6 software. Size exclusion chromatography (SEC) was performed on a Shimadzu system equipped with a UV-vis detector SPD-10A VP and a Tosoh Bioscience TSKgel 4 µm Super SW3000 column. Buffer at pH 6.6 (10 mM ammonium acetate and 100 mM sodium chloride) was used as the mobile phase at 23 °C (flow rate: 0.2 mL/min). Chromatograms were acquired at 215 nm. Matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry was performed on an Applied Biosystems Voyager-DE STR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using 4-15% TRIS-glycine precast gradient gels (Invitrogen), and samples were dissolved in TRIS buffer containing SDS, bromophenol blue, and glycerol. Synthesis of CTA (2). Compound 1 (1.17 g, 5.2 mmol), 2-(ethyl trithiocarbonate)propionic acid (1.0 g, 4.8 mmol), and DMAP (58 mg, 0.48 mmol) were dissolved in dry THF (50 mL). DCC (1.18 g, 5.7 mmol) was subsequently added to the solution. The system was maintained at 23 °C under argon with constant stirring for 4 h. The precipitate was removed by filtration and the solvent was removed under reduced pressure. The CTA was purified by silica gel chromatography (ethyl acetate/hexanes ) 1:4). The compound was obtained as a yellow oil (1.0 g, yield: 78%). 1H NMR (400 MHz, CDCl3): δ 6.50 (2H, s, CH)CH), 5.25 (2H, s, CHOCH), 4.81 (1H, q, J ) 7.5 Hz, CHCH3), 4.10 (2H, t, J ) 6.9 Hz, CH2O), 3.62-3.53 (2H, m, NCH2), 3.36 (2H, q, J ) 7.5 Hz, SCH2), 2.84 (2H, s, CHCON), 1.96-1.90 (2H, m, CH2CH2CH2), 1.61 (3H, d, J ) 7.5 Hz, CHCH3), 1.34 (3H, t, J ) 7.5 Hz, CH2CH3). 13C NMR (100 MHz, CDCl3): δ 222.08, 176.51, 171.24, 136.73, 81.14, 36.23, 48.03, 47.59, 35.90, 31.68, 26.78, 18.98, 13.10. IR (cm-1): 2927, 2850, 1776, 1726, 1694, 1573, 1446, 1403, 1368, 1306, 1281, 1249, 1165, 1072, 1021, 994. MALDI-TOF [M + Na]:

Bays et al. obsd, 438.09; calcd, 438.05; [M + K]: obsd, 454.08; calcd, 454.02. UV-vis: λmax ) 306 nm. Synthesis of Poly(PEGA) Using RAFT Polymerization (3). RAFT polymerization was conducted using standard Schlenk techniques. PEGA (4.54 g, 10 mmol), 2 (140 mg, 0.33 mmol), and AIBN (5.5 mg, 0.03 mol; [monomer]/[CTA]/[AIBN] ) 30:1:0.1) were loaded into a Schlenk tube along with 5 mL of DMF as a solvent. The tube was sealed and subjected to three freeze-pump-thaw cycles. The polymerization was then initiated by immersion of the tube into a 60 °C oil bath and eight different samples were removed overtime using a degassed syringe for molecular weight and conversion analysis. The polymerization was stopped by exposure to air and cooling to 23 °C after 6 h (83% conversion). The DMF was removed under reduced pressure and polymer 3 was purified by dialysis against MeOH (molecular weight cutoff, MWCO 6-8000 Da). Polymer conversions were calculated by 1H NMR spectroscopy in deutero-chloroform (CDCl3). The integrations of the proton signals from the vinyl moiety of monomer PEGA were used to estimate unreacted PEGA. The integrations of the proton signals from the methylene protons adjacent to the ester group (CH2OOC) of both the monomer and the polymer was used to estimate total PEGA. Conversion was calculated by comparing these integrations at each time point. Each fraction was subjected to GPC (RI) to obtain the number-average molecular weight (Mn) and the PDI. Fractions were prepared in DMF and filtered through a 0.2 µm membrane prior to injection. 1H NMR (400 MHz, CDCl3): δ 6.51 (2H, s, CH)CH), 5.24 (2H, s, CHOCH), 4.35-4.00 (CH2O), 3.70-3.45 (CH2CH2O), 3.37 (OCH3), 2.84 (2H, s, CHCON), 2.60-2.20 (CHCH2 main chain), 2.00-1.40 (CHCH2 main chain), 1.34 (3H, t, SCH2CH3), 1.20-1.05 (3H, m, CHCH3). The same synthesis procedure was carried out to prepare poly(PEGA) with higher molecular weight. The starting ratio for polymerization was [monomer]/[CTA]/[AIBN] ) 120:1:0.3 and the polymerization was stopped at 62% conversion. Formation of Maleimide Chain End Poly(PEGA) (4). Deprotection of polymer 3 was accomplished by a retro Diels-Alder reaction. Typically, polymer 3 (25 mg) was dissolved in 5 mL of toluene and heated to reflux for 4 h in an oil bath. Toluene was removed under reduced pressure to give the final polymer 4. By comparing peak integration values of the protons of the maleimide alkene to the ester protons of the main chains, the retention was 80% maleimide chain end for poly(PEG)20 kDa. 1H NMR (400 MHz, CDCl3): δ 6.72 (2H, s, CH)CH), 4.35-4.00 (CH2 ester side chain), 3.70-3.45 (CH2CH2O), 3.37 (OCH3), 2.60-2.20 (CHCH2 main chain), 2.00-1.40 (CHCH2 main chain), 1.34 (3H, t, SCH2CH3), 1.20-1.05 (3H, m, CHCH3). The same deprotection procedure was carried out to obtain maleimide poly (PEGA)39 kDa with 60% yield of maleimide. T4 Lysozyme Expression and Purification. T4L was expressed according to a literature procedure.27,28 E. coli host BL21(DE3) served to express the single-cysteine substitution mutant T4L V131C. A single colony was selected and transferred into 10 mL of LB broth with shaking at 250 rpm (37 °C, 12 h). After transferring 2 mL of the bacteria suspension into 300 mL of LB broth, the bacteria were grown to an optical density (OD600) of 0.6 - 0.8. Protein expression was then induced with 1 mM isopropyl-β-D-1-thiogalactopyranosid (IPTG) under shaking at 250 rpm (37 °C, 3 h), and mutant T4L was present intrabacterially. To purify the lysozyme mutant, bacterial cells were then pelleted and resuspended in lysis buffer (50 mM Tris, 2 mM ethylenediaminetetraacetic acid (EDTA), pH 8). Spontaneous bacterial lysis occurred during resuspension due to the proteolytic activity of the lysozyme mutant. Benzoase (0.2 µL/mL) was then added to the solution and incubated at 4 °C for 3 h. Bacterial debris was then separated by centrifugation at 20000 rpm for 30 min. The supernatant containing the water-soluble lysozyme among other bacterial proteins was then filtered through a French Press. Bacterial debris were then separated by centrifugation at 15000 rpm for 40 min at 4 °C and the supernatant was filtered through syringe filters with a pore size of 0.2 µm (Puradisc 25 AS disposable filter Whatman) before purification of T4L by cation exchange chromatography. T4L was loaded onto the

Synthesis of Semitelechelic Maleimide Poly(PEGA)

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Scheme 1. Synthesis of CTA

Scheme 2. Synthesis of Poly(PEGA) End-Functionalized with Maleimide

solved in 25 µL of PBS pH 7.0 and analyzed by SDS-PAGE under reducing conditions. Poly(PEGA)39 kDa-maleimide was also employed to prepare a protein-polymer conjugate using the same procedure.

Results and Discussion

cation-exchange column (HiTrap SP HP, 5.0 mL, Amersham Biosciences) and the column was extensively washed with lysis buffer. Two column volumes of lysis buffer with increasing NaCl concentration (0-0.5 M NaCl in 0.1 M steps) were used to elute the T4L. The elution fractions were analyzed by UV-vis at 280 nm and SDS-PAGE to determine which fractions contained T4L. Preparation and Purification of Poly(PEGA)20 kDa- and Poly (PEGA)39 kDa-T4L Conjugates. Poly(PEGA)20 kDa-maleimide (3.16 mg. 1.5 × 10-1 mmol) was dissolved in 0.5 mL of degassed phosphate buffered saline (PBS) containing 10 mM EDTA and 10 mM tris(2carboxyethyl)phosphine (TCEP) at pH 7.5. T4L solution (0.4 mL, 0.399 mg/mL, 1.5 × 10-2 mmol in TRIS buffered saline [TBS, pH 7.6]) was added to the polymer solution in a 1:10 (protein/polymer) ratio. The sample was stirred at 4 °C for 14 h. The conjugation reaction was monitored by SEC at 215 nm. The crude conjugates were purified by SEC. The fractions containing the conjugate were collected together, concentrated by centrifugal filtration (Amicon Ultra, MWCO: 10000) to remove salts and then lyophilized. The dried samples were redis-

Figure 1. Molecular weight and PDI vs conversion plot for RAFT polymerization of PEGA with CTA 2 ([M0]/[CTA]/[AIBN] ) 30:1:0.1, T ) 60 °C). Both the Mn and PDI were determined from GPC with RI detection and DMF/0.1 M LiBr as the mobile phase. The drawn line is the best fit of the data points.

CTA Design and Synthesis. The CTA contained a maleimide in the R group, and the maleimide was protected to prevent reaction during polymerization. A furan group was used so that postpolymerization, the deprotection could be achieved via a simple retro Diels-Alder reaction. The synthetic route is outlined in Scheme 1. Alcohol 1 and 2-(ethyl trithiocarbonate)propionic acid were synthesized following literature procedures and coupled using DCC and DMAP to form 2 in 78% yield. Synthesis of Poly(PEGA) via RAFT Polymerization. Furan-protected maleimide CTA 2 was employed in the polymerization of PEGA to form 3 (Scheme 2). Initial [M]/[CTA]/[AIBN] ratios of 30:1:0.1 were used and the polymerization was stopped at 83% conversion. Time points were taken throughout the reaction. For each sample, the number-average molecular weight (Mn) and PDI were determined by GPC with RI detection. The conversions were determined from the 1H NMR spectra by comparison of the integrations of the vinyl proton peaks of the monomer and the methylene protons adjacent to the ester of both the monomer and polymer. The molecular weight and PDI versus conversion plot (Figure 1) showed that the molecular weight increased linearly with conversion. As the reaction proceeded, the molecular weight distributions remained narrow (PDI < 1.30). This demonstrated that RAFT polymerization of PEGA with this particular CTA results in well-defined polymers. After removing residual monomer and impurities by dialysis against methanol, the polymer was analyzed by 1H NMR and GPC. The 1H NMR spectrum of the polymer is provided as Figure 2. The protons of the oxa-norbornene end group were

Figure 2. 1H NMR spectrum (CDCl3) of furan-protected maleimide poly(PEGA)20 kDa.

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Figure 3. 1H NMR spectrum (CDCl3) of maleimide poly(PEGA)20 kDa.

Figure 4. Overlaid GPC traces of furan-protected maleimide poly (PEGA)20 kDa before (solid line) and after (dot line) deprotection. The GPC traces were obtained with RI detection and DMF/0.1 M LiBr as the mobile phase.

observed at 6.51, 5.24, and 2.84 ppm demonstrating that the desired furan protected maleimide was at the polymer chain end. The integrations of these same peaks were compared to the peak integration of the methylene protons next to the ester moiety centered at 4.16 ppm to calculate the molecular weight by 1H NMR (19.6 kDa). The Mn by GPC with RI detection was 14.7 kDa. This value was inaccurate because it was determined by comparison to inauthentic polymer standards. Therefore, the molecular weight was also determined by GPC with laser light scattering detection. The value obtained by this technique was close to that obtained by 1H NMR (20.0 kDa). The PDI of the polymer by GPC (LLS) was 1.25. The maleimide group was deprotected by heating the polymers in refluxing toluene in order to cause a retro Diels-Alder reaction.

Bays et al.

Figure 6. SDS-PAGE analysis of T4L conjugates under reducing conditions. (A) T4L purified by cation exchange column, (B) T4L-poly (PEGA)20 kDa conjugate purified by SEC, (C) T4L-poly(PEGA)39 kDa conjugate purified by SEC, and (D) T4L with furan protected maleimide poly(PEGA)39 kDa.

The 1H NMR spectrum is shown in Figure 3. The peaks corresponding to the protecting group (6.51, 5.24, and 2.84 ppm) disappeared completely, and a new signal at 6.72 ppm corresponding to the maleimide was observed. The deprotection yield was obtained by comparing the integration of this maleimide peak to the ester peak at 4.16 ppm. The deprotection yield was 80% for poly(PEGA)20 kDa. This result indicated that some of the group was removed during the process, possibly due to hydrolysis of the ester bond between the maleimide and the polymer. PEG is known to sequester water, and hydrolysis reactions may be inhibited by careful dehydration of the polymer prior to the retro Diels-Alder step. Alternatively, a higher yield may be obtained by preparing the polymer from an amide linked CTA.29 The GPC retention time, Mn and PDI values were similar before and after deprotection (Figure 4). This indicated that cross-linking between polymer chains or cleavage of the polymer branches did not occur during the heating process. With the successful synthesis of this maleimide-functionalized polymer, a longer chain length was also targeted. PEGA was polymerized with a [M]/[CTA]/[AIBN] of 120: 1:0.3, and the polymerization was stopped at 62% conversion. The larger polymer had a Mn of 39 KDa and a PDI of 1.36 by GPC (LLS). The deprotection yield calculated from the 1 H NMR spectrum (Figure S2) was lower at 60%. Hydrolysis side reactions may again partially explain the yield. However, the PDI value also increased after deprotection (See Figure

Figure 5. (a) SEC traces at 215 nm of (A) T4L, (B) crude T4L-poly(PEGA)20 kDa conjugate, and (C) purified T4L-poly(PEGA)20 kDa conjugate; (b) (A) T4L, (B) crude T4L-poly(PEGA)39 kDa conjugate, and (C) purified T4L-poly(PEGA)39 kDa conjugate.

Synthesis of Semitelechelic Maleimide Poly(PEGA)

S1 for the GPC traces), indicating that some chain coupling reactions had occurred. This could have resulted from hydrolysis of the omega-trithiocarbonate chain end to the thiol. The thiol could dimerize or react with the newly formed maleimide, which would broaden the molecular weight. The latter would also lower the overall amount of maleimide chain end. Although the synthesis of the maleimide poly(PEGA) with 39 kDa molecular weight was not as high yielding, the maleimide group for conjugation was observed in the NMR. Therefore, both of the polymers were subsequently employed for conjugation to T4L containing a free thiol group. Conjugation of Poly(PEGA)20 kDa and Poly(PEGA)39 kDa to T4L. The reaction between poly(PEGA)20 kDa or poly (PEGA)39 kDa and T4L with one free cysteine was performed at 4 °C for 14 h. The conjugation was confirmed by SEC at 215 nm (Figure 5) and SDS-PAGE (Figure 6). Figure 5a shows the SEC traces of T4L (A), T4L-poly (PEGA)20 kDa conjugate (B), and the conjugate after purification (C). Trace A had a peak at 48 min attributed to T4L after purification. Impurities were detected at longer retention time, which corresponded to what was observed on the gel (lane A of Figure 6). Trace B showed two peaks at 43 and 48 min, which corresponded to T4L-poly(PEGA)20 kDa and T4L, respectively. The conjugation efficiency was roughly estimated at 50% by comparison of conjugated T4L and free T4L peaks. Trace C was the isolated T4L-poly(PEGA)20 kDa conjugate (42 min) after purification by SEC. Removal of the majority of unreacted T4L was confirmed by the lack of a peak at 48 min. Figure 5b are the SEC traces at 215 nm of T4L, T4L-poly(PEGA)39 kDa conjugate, and the conjugate after purification. Trace B displayed two peaks at 41 and 48 min, which correspond to T4L-polyPEG39 kDa conjugate and T4L, respectively. The conjugation efficiency was roughly estimated at 80%. Due to the higher molecular weight of the T4L-poly(PEGA)39 kDa conjugate, the retention time (41 min) was shorter than for the T4L-polyPEG20 kDa conjugate (42.5 min). Trace C was the purified T4L-poly(PEGA)39 kDa. The conjugates were also analyzed by SDS-PAGE (Figure 6). Lane A contained T4L. The control experiment result is displayed in lane D. No reaction was observed between the furan-protected maleimide poly(PEGA)39 kDa and T4L. This indicated that there was no physical absorption between the polymers and protein and that the maleimide was necessary for conjugation. When the maleimide chain-end polymer was employed for the conjugation to T4L (lanes B and C), spots with increased molecular weights (25-50 kDa, 60-120 kDa) appeared with broadening characteristic of polymer conjugates. The conjugates were purified by SEC prior to SDS-PAGE analysis. However, residual free T4L was still visible in lane B. Because of the larger difference in retention time between T4L-poly(PEG)39 kDa and T4L, this conjugate could be obtained pure (lane C). Together, these results demonstrated that T4L-poly(PEGA)20 kDa and T4L-poly(PEGA)39 kDa were successfully obtained.

Conclusion We have developed a straightforward method to synthesize poly(PEGA) with a maleimide end group for conjugating proteins in a site-specific manner. Furan-protected maleimide poly(PEGA)s were synthesized by RAFT polymerization using a functionalized CTA. The resulting polymers were deprotected via a retro Diels-Alder reaction. Successful conjugation of T4L to poly(PEGA) was observed. This route to branched PEGs for site-specific conjugation to proteins should be useful for synthesizing therapeutic protein conju-

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gates. The ω chain end is also available for further reaction, for example, with fluorophores or other tagging molecules. We are currently exploring these and other possibilities. Acknowledgment. This research was supported by NSF (CHE-0809832). E.B. thanks the Swiss Government for a fellowship. C.-W.C. thanks the California NanoSystems Institute Postdoctoral Award and H.D.M. thanks the Alfred P. Sloan Foundation for additional funding. Supporting Information Available. 1H NMR spectrum and GPC traces of larger polymer. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) Roberts, M. J.; Bentley, M. D.; Harris, J. M. AdV. Drug DeliVery ReV. 2002, 54, 459–76. (2) Harris, J. M.; Chess, R. B. Nat. ReV. Drug DiscoVery 2003, 2, 214–21. (3) Nicolas, J.; Mantovani, G.; Haddleton, D. M. Macromol. Rapid Commun. 2007, 28, 1083–1111. (4) Duncan, R.; Ringsdorf, H.; Satchi-Fainaro, R. J. Drug Target. 2006, 14, 337–341. (5) Bailon, P.; Palleroni, A.; Schaffer, C. A.; Spence, C. L.; Fung, W. J.; Porter, J. E.; Ehrlich, G. K.; Pan, W.; Xu, Z. X.; Modi, M. W.; Farid, A.; Berthold, W.; Graves, M. Bioconjugate Chem. 2001, 12, 195–202. (6) Lecolley, F.; Tao, L.; Mantovani, G.; Durkin, I.; Lautru, S.; Haddleton, D. M. Chem. Commun. 2004, 2026–2027. (7) Veronese, F. M.; Monfardini, C.; Caliceti, P.; Schiavon, O.; Scrawen, M. D.; Beer, D. J. Controlled Release 1996, 40, 199–209. (8) Ryan, S. M.; Mantovani, G.; Wang, X.; Haddleton, D. M.; Brayden, D. J. Expert. Opin. Drug DeliVery 2008, 5, 371–83. (9) Duncan, R. Nat. ReV. Drug DiscoVery 2003, 2, 347–360. (10) Bontempo, D.; Heredia, K. L.; Fish, B. A.; Maynard, H. D. J. Am. Chem. Soc. 2004, 126, 15372–15373. (11) Tolstyka, Z. P.; Kopping, J. T.; Maynard, H. A. Macromolecules 2008, 41, 599–606. (12) Mantovani, G.; Lecolley, F.; Tao, L.; Haddleton, D. M.; Clerx, J.; Cornelissen, J. J. L. M.; Velonia, K. J. Am. Chem. Soc. 2005, 127, 2966– 2973. (13) Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H. Macromolecules 1998, 31, 5559–5562. (14) Moad, G.; Chong, Y. K.; Postma, A.; Rizzardo, E.; Thang, S. H. Polymer 2005, 46, 8458–8468. (15) Perrier, S.; Takolpuckdee, P. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 5347–5393. (16) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2006, 59, 669– 692. (17) Barner-Kowollik, C.; Buback, M.; Charleux, B.; Coote, M. L.; Drache, M.; Fukuda, T.; Goto, A.; Klumperman, B.; Lowe, A. B.; Mcleary, J. B.; Moad, G.; Monteiro, M. J.; Sanderson, R. D.; Tonge, M. P.; Vana, P. J. Polym. Sci., Part A: Polym. Chem. 2006, 44, 5809–5831. (18) Zhao, Y. L.; Perrier, S. Chem. Commun. 2007, 4294–4296. (19) Boyer, C.; Liu, J.; Wong, L.; Tippett, M.; Bulmus, V.; Davis, T. P. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 7207–7224. (20) Li, M.; De, P.; Gondi, S. R.; Sumerlin, B. S. Macromol. Rapid Commun. 2008, 29, 1172–1176. (21) Boyer, C. B.; Bulmus, V.; Liu, J.; Davis, T. P.; Stenzel, M. H.; BarnerKowollik, C. J. Am. Chem. Soc. 2007, 129, 7145–7154. (22) De, P.; Li, M.; Gondi, S. R.; Sumerlin, B. S. J. Am. Chem. Soc. 2008, 130, 11288–11289. (23) Heredia, K. L.; Nguyen, T. H.; Chang, C. W.; Bulmus, V.; Davis, T. P.; Maynard, H. D. Chem. Commun. 2008, 3245–7. (24) Scales, C. W.; Convertine, A. J.; McCormick, C. L. Biomacromolecules 2006, 7, 1389–1392. (25) Neubert, B. J.; Snider, B. B. Org. Lett. 2003, 5, 765–768. (26) Wood, M. R.; Duncalf, D. J.; Rannard, S. P.; Perrier, S. Org. Lett. 2006, 8, 553–556. (27) Mchaourab, H. S.; Lietzow, M. A.; Hideg, K.; Hubbell, W. L. Biochemistry 1996, 35, 7692–7704. (28) Columbus, L.; Kalai, T.; Jeko, J.; Hideg, K.; Hubbell, W. L. Biochemistry 2001, 40, 3828–3846. (29) Heredia, K. L.; Grover, G. N.; Tao, L.; Maynard, H. D. Macromolecules 2009, 42, 2360–2367.

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