Synthesis of Multifunctional Polymers through the Ugi Reaction for

Article pubs.acs.org/Macromolecules

Synthesis of Multifunctional Polymers through the Ugi Reaction for Protein Conjugation Bin Yang, Yuan Zhao, Shiqi Wang, Yaling Zhang, Changkui Fu, Yen Wei, and Lei Tao* The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China S Supporting Information *

ABSTRACT: The Ugi reaction has been utilized as a multicomponent click reaction to efficiently synthesize a series of multifunctional PEGylation agents, and those PEG derivatives were successfully conjugated on a protein surface to generate corresponding multifunctional protein−polymer conjugates, indicating the promising potential of the Ugi reaction in the field of PEGylation.

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INTRODUCTION PEGylation, the process of covalent attachment of poly(ethylene glycol) (PEG) chains to therapeutic proteins/ peptides, can significantly improve the pharmacological and biological properties of therapeutic proteins/peptides.1−3 For instance, the conjugated PEG can reduce immunogenicity and antigenicity of the protein by masking it from the host’s immune system.4,5 The increased hydrodynamic size of the PEGylated protein prolongs its circulatory half-life by reducing renal clearance and proteolytic degradation.6 PEGylation can also improve water solubility of hydrophobic proteins/peptides, expanding their application scopes.7 In some cases, the PEG chains can also be predictably detached in vivo from the conjugate, implementing the controlled release of native protein therapeutics.8−10 Therefore, PEGylation has been rapidly developed since its birth in the 1970s, resulting in remarkable achievement in therapeutic benefits and market success.7,11−18 Meanwhile, the success of PEGylation also motivates the development of other well-defined synthetic polymers as PEG analogues for protein modification.19−29 By now, the research of PEGylation agents is mainly focused on the end-functional linear PEG derivatives2,30−33 (firstgeneration PEGylation agents) and midfunctional branched PEG derivatives8,10,34−38 (second-generation PEGylation agents), in which PEG derivatives are only employed as the protective clothing to the target protein. With the rapid development of proteins therapeutics in clinical medicine, the concept of third-generation PEGylation agents (multifunctional PEGylation agents) has been coined, the protein and polymer should cooperate mutually as a whole; i.e., the polymer should be not only a cloak but also a partner of the therapeutic protein to promote diagnosis and therapy process. For example, © 2014 American Chemical Society

incorporation of tracing element into PEGylation might make the circulation and working site of therapeutic proteins directly visible, leading to more rapid and efficient clinical trial. However, the third-generation PEGylation agents do suffer from significant barriers as they are normally prepared through laborious multistep organic reactions and onerous purification processes, resulting in few researches about multifunctional PEGylation agents although they represent the future trend of this field. Meanwhile, how to efficiently prepare multifunctional PEG derivatives for protein conjugation through simple synthetic routes is also a challenge to polymer chemists. Click chemistry, first described by Sharpless and co-workers in 2001, is a family of highly efficient and modular reactions.39 As point and shoot coupling reactions, click reactions have been proven powerful tools to synthesize PEGylated conjugates. The highly reliable and selective click reactions can efficiently generate linkages between the PEG chain and the protein reactive groups, such as maleimide, N-hydroxysuccinimide (NHS), pyridyl disulfide (PDS), biotin, aldehyde, and thiazolidine-2-thione, et.al.40,41 However, the widely recognized traditional click reactions are all two-component reactions, limiting their applications to synthesize the third-generation PEGylation agents, which should simultaneously contain both protein reactive group and other functional groups. Therefore, multicomponent click reactions (MCCs) might be a solution to this problem. Since the pioneer research of Meier and coworkers to introduce three-component Passerini reaction into polymer science,42−44 more and more multicomponent Received: July 5, 2014 Revised: July 28, 2014 Published: August 4, 2014 5607

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Ellman’s assay14,55 was carried out to calculate the reactivity of the free thiol group on the BSA surface (57.8%, data not presented). Then, PDS with a carboxyl group (PDS−acid) was chosen as the protein reactive group and the carboxyl source to conduct the Ugi reaction while modified dansyl chloride with an aldehyde group (dansyl−aldehyde) was chosen as the fluorescent group and aldehyde source. Amino-terminated methoxypoly(ethylene glycol) (mPEG− NH2, Mn ∼ 5150) was treated with the PDS-acid, the dansylaldehyde and cyclohexyl isocyanide (Figure 1a, molar ratio

reactions (MCRs) have been exploited to prepare multifunctional polymers.45−51 During the study of MCRs, some highly efficient MCRs have been found to fulfill the criterion of click chemistry, leading to the creation of MCCs, in which three or more components can be as rapidly and almost quantitatively compressed in a selective product as click reactions.52 Thus, MCCs can not only work as efficient coupling reactions like traditional two components click reactions, but also add new functions to the target compounds due to their multicomponent nature. Some famous multicomponent reactions, such as the Biginelli reaction, the Ugi reaction, and the mercaptoacetic acid locking imine (MALI) reaction, have been explored their clickable features and have been introduced into polymer chemistry and other fields, demonstrating the vitality of those “old” reactions in the “new” areas outside organic chemistry.52−54 In current research, the new MCC, Ugi fourcomponent reaction has been utilized to simply synthesize a series of multifunctional PEGylation agents for protein conjugation. Then, a list of correlated sophisticated protein− polymer conjugates, fluorescent PEGylated protein, midfunctional PEG-b-poly(N-(2-hydroxypropyl) methacrylamide) (PEG-b-PHPMA) conjugated protein, and fluorescent PEGylated protein dimer, have thereby been successfully prepared, suggesting the potential of the Ugi reaction as the MCC for the synthesis of multifunctional PEGylation agents (Scheme 1). Scheme 1. Synthesis of the Multifunctional PEGs through the Ugi Reactiona

Figure 1. Synthesis of fluorescent monomer PEG via the Ugi reaction, and its application for protein conjugation. (a) Reaction conditions: [mPEG−NH2] = 20 mM, [NH2]/[COOH]/[CHO]/[NC] = 1/2/2/ 3, MeOH as solvent, 25 °C, 2 h. (b) 1H NMR spectra (CDCl3-d, 400 MHz, portion) of the fluorescent monomer PEG. (c) GPC curves of the fluorescent monomer PEG using RID and UV detectors, respectively. (d) MALDI−TOF MS analysis of the BSA−PEG conjugate. (e, f) SDS−PAGE of the BSA−PEG conjugates with Coomassie bright blue staining (e) and under UV ∼312 nm (f) ((A, A′) native BSA; (B, B′) BSA−FITC; (C, C′) BSA−PEG conjugate; (D, D′) reduced BSA; (E, E′): reduced BSA−FITC; (F, F′) reduced BSA−PEG conjugate). a Fluorescent monomer and dimer PEGs, mid-functional PEG-bPHPMA, and their protein conjugation: fluorescent PEGylated protein (a), PEG-b-PHPMA conjugated protein (b), and fluorescent PEGylated protein dimer (c).

NH2/COOH/CHO/NC = 1/2/2/3, 25 °C) in methanol. After 2 h, the resulting polymer was isolated by recrystallization from 2-propanol, and the 1H NMR spectrum of the purified PEG derivative illustrated the desired structure (Figure 1b). The specific −NCHCO− peak of the Ugi structure (∼5.95 ppm) can be clearly observed. Meanwhile, the characteristic peaks of the fluorescent moiety (∼8.52, 8.23, 2.87 ppm) and the PDS moiety (∼8.38 ppm) can also be clearly observed in the purified polymer. The integral ratio between the characteristic peaks of the Ugi structure, fluorescent moiety, PDS moiety and mPEG (I5.95/I8.52/I8.38/I3.36) is 1/1/1/3.2 (theoretical value = 1/1/1/3), indicating ∼94% PEG chain end has been converted through the Ugi reaction. As expected, the fluorescent group integrated polymer has an excitation wavelength at 335 nm and an emission wavelength at 530 nm (Figure S1, Supporting

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RESULTS AND DISCUSSION Preparation of Fluorescent Monomer PEGs and Fluorescent PEGylated Protein. The addition of tracing elements makes it possible to monitor the delivery and work process of therapeutics agents. Therefore, fluorescence element was integrated in the PEGylation agents via the Ugi reaction. Bovine serum albumin (BSA) was chosen as the model protein, and we attempted to modify the free cystein residue (thiol group, Cys-34) on its surface. Before the protein conjugation, 5608

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Information). Through the gel permeation chromatography (GPC) with an UV detector, the polymer showed the absorption at 365 nm (Figure 1c), confirming the polymer chain end has been successfully integrated with a fluorescent group. Therefore, the Ugi reaction can highly efficiently modify a polymer chain-end with different functions. The obtained PEG was subsequently reacted with BSA (2 mg/mL), the conjugation reactions were carried out at pH 7.0 using an excess of polymer ([thiol]:[polymer] = 1:2, 25 °C, 5 h) to guarantee the complete conjugation. After removing the salt in the solution by centrifugal filtration (MWCO: 30 K), the concentrated solution was used for MALDI−TOF MS and gel electrophoresis analyses. From the MALDI−TOF MS spectrum (Figure 1d), the appropriate molecular weight difference between native BSA (∼66.4 kDa) and BSA−PEG conjugate (∼72.3 kDa) can be clearly detected, indicating the successful linkage of PEG on protein surface. Meanwhile, from the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS− PAGE), the BSA−PEG conjugate can be clearly observed at the higher molecular weight position (∼59 kDa, lane C) than the native BSA (∼49 kDa, lane A). After reduction by DTT, the disulfide linkage between polymer and protein was cleaved, thus, the reduced BSA−PEG conjugate (∼62 kDa, lane F) stayed at the same position as the reduced BSA (∼62 kDa, lane D). The gel was also exposed under UV light (312 nm) for visualization using the fluorescein isothiocyanate conjugated BSA (BSA−FITC) as the marker (lane B′). The band attributed to the BSA−PEG conjugate could be clearly observed (∼59 kDa, lane C′), confirming the linkage of fluorescent polymer on protein surface. After reduction, there is no any fluorescent signal observed from the PAGE, suggesting the fluorescence of the protein conjugate comes from the cleaved polymer. Different ratios of [thiol]/[polymer] were also investigated, and there are no obvious differences between those protein conjugates by native eyes, indicating the highly efficient coupling reaction between the cysteine residue and the PDS moiety at the polymer chain end (Figure S2). All the results above confirm the fluorescent polymer has been successfully synthesized and conjugated on protein surface through the reversible disulfide bond. Preparation of Midfunctional PEG−PHPMA and PEG− PHPMA Conjugated Protein. Compared with the linear counterparts, the “umbrella-like” structure of midfunctional PEG derivatives can cover more area on protein surface to offer better protection to the protein, and the protein conjugates reserve higher bioactivity. Therefore, the research and exploitation of branch-structural PEG derivatives are attracting more and more attention.35,36,56,57 However, the study of branched polymer conjugates rarely involves the miktoarm copolymers because of the complex synthesis process. Herein, we simultaneously incorporate a protein reactive group and a polymer-generation group at PEG chain end to finally achieve a midfunctional miktoarm copolymer for protein conjugation. Briefly, an aldehyde functional chain transfer agent (CTA, for reversible addition−fragmentation chain transfer (RAFT) polymerization), the PDS−acid, and the mPEG−NH2 were mixed in the presence of cyclohexyl isocyanide (Figure 2a, molar ratio NH2/COOH/CHO/NC = 1/2/2/3, 25 °C), after 2 h, the target polymer was obtained through recrystallization from 2-propanol. 1H NMR was used to characterize the resulted PEG−PDS-CTA (Figure 2b). The integral ratio between the characteristic peaks of the Ugi structure, CTA moiety, PDS moiety and mPEG (I5.87/I3.96/I8.37/I3.36) is 1/2/1/

Figure 2. Synthesis of midfunctional miktoarm copolymer via the Ugi reaction, and its conjugation to protein. (a) Reaction conditions: [NH2]/[COOH]/[CHO]/[NC] = 1/2/2/3, MeOH as solvent, 25 °C, 2 h. (b) 1H NMR spectra (CDCl3-d, 400 MHz, portion) of the mPEG derivative with both CTA and PDS at the chain end. (c) 1H NMR spectra (D2O-d2, 400 MHz, portion) of the midfunctional PEGb-PHPMA. (d) GPC curves of the PEG−PDS-CTA (black) and PEGb-PHPMA (red). (e) SDS−PAGE analysis of the BSA−PEG conjugates with Coomassie bright blue staining. ((A) BSA; (B) BSA−copolymer conjugate; (C) reduced BSA; (D) reduced BSA− copolymer conjugate).

3.2 (theoretical value = 1/2/1/3), indicating the approximate 94% conversion of the Ugi reaction. HPMA was then used as the monomer for subsequent RAFT polymerization with the PEG−PDS−CTA as the macro-CTA ([M]0/[CTA]/[AIBN] = 200/1/0.3; 65 °C; methanol as solvent). When the monomer conversion reached ∼90% in 10 h, the polymerization was quenched, and the polymer was purified by precipitation in toluene/THF mixture (v/v: 1/1). The purified polymer has a narrow PDI and obviously increased molecular weight (Figure 2d, MnGPC ∼ 55 300, PDI = 1.18). From the 1H NMR spectrum of the purified polymer (Figure 2c), the characteristic peaks of the two polymer arms: PEG and PHPMA are clearly visible while the PDS group (∼8.31 ppm) is also retained (Figure 2c, inset), indicating the successful synthesis of the midfunctional miktoarm copolymer. The obtained copolymer (MnNMR ∼ 42 100, MnGPC ∼ 55 300, PDI = 1.18) was treated with BSA (2 mg/mL, [thiol]: [polymer] = 1:2) as previously described. From the SDS− PAGE (Figure 2e), the protein−polymer conjugate appeared at higher molecular weight position (∼95−130 kDa, lane B) than the pristine BSA (∼49 kDa, lane A), and disappeared after reduction (lane D), indicating the successful conjugation between the protein and midfunctional miktoarm PEG-bPHPMA through the disulfide linkage. 5609

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Preparation of Fluorescent PEG Dimer and Fluorescent PEGylated Protein Dimer. The self-association of proteins to form dimers and higher-order oligomers is a very common phenomenon of life.58−61 Recent structural and biophysical studies indicate that the dimerization and oligomerization are necessary forms of many proteins in their physiological process, such as enzymes, ion channels, receptors and transcription factors.58,62−64 Thus, locking proteins into multiple-conjugate is worthwhile for people to simulate multivalent interactions in biological processes, and prepare pharmaceutics with higher activity than monovalent counterparts.65,66 Herein, we employed the Ugi reaction to not only synthesize the protein-reactive telechelic polymer, but also implant fluorescent tracing group into the polymer structure, and finally achieved fluorescent homodimeric protein−polymer conjugate. Diamino terminated poly(ethylene glycol) (PEG−diNH2, Mn ∼ 4300) was treated with the PDS-acid, the fluorescent aldehyde and cyclohexyl isocyanide (Figure 3a, molar ratio: NH2/COOH/CHO/NC = 1/2/2/3, 25 °C) in methanol for 2 h. The resulting polymer was isolated by recrystallization from 2-propanol. In the 1H NMR spectrum (Figure 3b), the integral ratio between the characteristic peaks of the Ugi structure, fluorescent moiety, PDS moiety and PEG (I5.95/I8.47/I8.36/I4.20)

is 1/1/1/2.2 (theoretical value = 1/1/1/2), indicating the approximate 91% conversion of the the Ugi reaction. Through the GPC with an UV detector, the fluorescent polymer dose have absorption at 365 nm (Figure 3c), confirming the successful integration of fluorescent groups at the polymer chain ends. Conjugation reaction between the fluorescent PEG dimer and BSA was carried out in PBS buffer (pH 7.0, [thiol]: [polymer] = 2:1) at 25 °C for 10 h. After removing salts by centrifugal filtration (MWCO 30 K), the concentrated mixture was used for MALDI−TOF MS and SDS−PAGE analyses. From the MALDI−TOF MS spectrum (Figure 3d), the signal corresponding to the dimer conjugate (BSA−PEG−BSA, ∼138.5 kDa) can be clearly observed. SDS−PAGE analysis also supported the formation of that dimeric BSA−PEG−BSA conjugates (Figure 3e,f). The Coomassie bright blue stained PAGE showed two new higher molecular weight spots at ∼59 kDa and ∼110 kDa (lane B), respectively, which are attributed to the monomer and dimer protein−polymer conjugates. After reduction, those two spots were not visible (lane F) as expected due to the cleavage of disulfide linkage between protein and polymer. When the PAGE was exposed under the UV light (312 nm), the spots corresponding to the monomer and dimer conjugates were observed (lane B′), and disappeared after reduction by DTT, confirming the successful linkage of fluorescent polymer on protein surface. Combining the SDS−PAGE and MALDI− TOF MS data, it can be concluded that the desired dimeric protein−polymer conjugate has been successfully generated. Meanwhile, different ratios of [thiol]/[polymer] were investigated, and higher [thiol]/[polymer] ratios were found no obvious promotion to form the dimeric BSA−PEG−BSA conjugate (Figure S3). Bioactivity Evaluation of the Multifunctional PEGylated Proteins. The bioactivity of the obtained fluorescent PEGylated protein, PEG−PHPMA conjugated protein, and fluorescent PEGylated protein dimer has been evaluated, and the apparent bioactivity of the mixture (BSA− polymer conjugates and unreacted BSA) exhibited almost the same bioactivity conservation as the native BSA (Figure 4), qualitatively suggesting the protein remained intact structure during the conjugation. All the other BSA conjugates obtained under different thiol/polymer ratios have also been tested and no obvious bioactivity loss observed, indicating the excellent

Figure 3. Synthesis of fluorescent PEG dimer via the Ugi reaction, and its application in protein polymer conjugation. (a) Reaction conditions: [NH2]/[COOH]/[CHO]/[NC] = 1/2/2/3, MeOH as solvent, 25 °C, 2 h. (b) 1H NMR spectra (CDCl3-d, 400 MHz, portion) of the fluorescent PEG dimer. (c) GPC curves of the fluorescent PEG dimer via RID and UV detectors. (d) MALDI−TOF MS analysis of the fluorescent BSA−PEG−BSA dimer. (e, f) SDS− PAGE of the protein conjugates with Coomassie bright blue staining (e) and under UV ∼312 nm (f) ((A,A′) native BSA; (B,B′) BSA− FITC; (C,C′) BSA−PEG−BSA conjugate; (D,D′) reduced BSA; (E,E′) reduced BSA−FITC; (F,F′) reduced BSA−PEG−BSA conjugate).

Figure 4. Structural integrity test of BSA conjugates with different multifunctional PEG agents (fluorescent monomer PEG (thiol/ polymer: 1/2); fluorescent dimer PEG (thiol/polymer: 2/1); branched Y-type PEG−PHPMA (thiol/polymer: 1/2)). 5610

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protons and the methine protons). GPC was used to analyze the PDI and molecular weight of the final polymer. 1 H NMR (400 MHz, D 2 O-d 2 , δ/ppm): 3.96−3.80 (m, CHOHCH 3 ), 3.70−3.61 (m, OCH 2 CH 2 O), 3.25−2.95 (m, NHCH 2 CHOH), 2.10−1.52 (m, CH 2 CCH 3 ), 1.23−0.77 (m, CH2CCH3, CHOHCH3). MnGPC ∼ 55 300, MnNMR ∼ 42 100, and PDI = 1.18. Conjugation between the Polymer and BSA. All samples were conducted through same method, typically: Freshly prepared BSA solution (1.0 mL, 1.7 × 10−5 mmol active thiol group, 2.0 mg/mL in PBS buffer, pH 7.0) was added to a small plastic vial, followed by the addition of different volume multifunctional PEG agents solution (5 mg/mL in PBS buffer, pH 7.0). The vial was incubated at 37 °C with gentle shaking for 4 h. Then, 250 μL of BSA/PEG mixture was transferred into another vial for bioactivity analysis. The salts in remained mixture were removed by centrifugal filtration (MWCO: 30k, 7 times, 8000 rpm, 10 min per time), and the concentrated solution was used directly for SDS−PAGE analysis and MALDI−TOF mass spectrometry. Bioactivity Evaluation of the PEGylated Protein. All samples were tested through same method, typically: 250 μL of BSA/polymer mixture was put in a vial, and then, freshly prepared 4-nitrophenylacetate solution in acetonitrile (1 M, 5 μL) was added. The mixture was diluted to 1.0 mL with water, then incubated at 25 °C for 3 min prior to the analysis by UV (405 nm) for five times. The data was given as mean ± SD. Native BSA was tested for in the same way, and the value was defined as 100%.

bioactivity reservation of the multifunctional PEGylated BSA (Figure S4).

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EXPERIMENTAL SECTION

Synthesis of Fluorescent Monomer PEG. Amino-terminated methoxypoly(ethylene glycol) (mPEG−NH2, Mn ∼ 5150, 205 mg, 0.04 mmol), 2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethyl 4-formylbenzoate (34 mg, 0.08 mmol), 3-(pyridin-2-yldisulfanyl)propanoic acid (18 mg, 0.08 mmol), and cyclohexyl isocyanide (13 mg, 0.12 mmol) were charged into a dry EP tube along with methanol (2 mL). The EP tube was put into a thermo-shaker (25 °C) for 2 h. The resulting polymer was isolated by recrystallization from 2propanol and then dried under vacuum to obtain the pure polymer (177 mg, 75%) for further use and characterizations. 1 H NMR (400 MHz, CDCl3, δ/ppm): 8.52 (d, 1H, J = 8.4 Hz, CHCCN), 8.38 (d, 1H, J = 4.6 Hz, CHNCSS), 8.23 (m, 2H, CHCCSO 2 , CHCSO2 ), 7.68−7.00 (m, 14H, CHCHCNCH3 , CHCHCSO2, CHCHCCOO, CHCHCNCO, CHCHCHCSS), 5.95 (s, 1H, NCHCO), 5.74 (d, 1H, J = 8.0 Hz, CONH), 5.37 (m, 1H, SO2NH), 4.27−4.13 (m, 4H, CH2COOCH2, ArCOOCH2), 3.83−3.40 (m, OCH2CH2O), 3.36 (s, 3H, OCH3), 3.27 (m, 2H, NHCH2), 3.01 (m, 2H, SSCH2), 2,87 (s, 6H, NCH3), 2.48 (m, 2H, SSCH2CH2). Synthesis of Fluorescent PEG Dimer. Diamino-terminated poly(ethylene glycol) (PEG−diNH2, Mn ∼ 4300, 86 mg, 0.02 mmol), 2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethyl 4-formylbenzoate (34 mg, 0.08 mmol), 3-(pyridin-2-yldisulfanyl)propanoic acid (18 mg, 0.08 mmol), and cyclohexyl isocyanide (13 mg, 0.12 mmol) were charged into a dry EP tube along with methanol (2 mL). The EP tube was put into a thermo-shaker (25 °C) for 2 h. The resulting polymer was isolated by recrystallization from 2-propanol and then dried under vacuum to obtain the pure polymer (82 mg, 70%) for further use and characterizations. 1 H NMR (400 MHz, CDCl3, δ/ppm): 8.47 (d, 2H, J = 8.4 Hz, CHCCN), 8.36 (d, 2H, J = 4.6 Hz, CHNCSS), 8.22 (m, 4H, CHCCSO 2 , CHCSO2 ), 7.66−6.96 (m, 28H, CHCHCNCH3 , CHCHCSO2, CHCHCCOO, CHCHCNCO, CHCHCHCSS), 5.95 (s, 2H, NCHCO), 5.82 (d, 2H, J = 8.0 Hz, CONH), 5.55 (m, 2H, SO2NH), 4.20 (m, 4H, CH2COOCH2), 4.15 (m, 4H, ArCOOCH2), 3.82−3.38 (m, OCH2CH2O), 3.25 (m, 4H, NHCH2), 2.98 (m, 4H, SSCH2), 2,83 (s, 12H, NCH3), 2.46 (m, 4H, SSCH2CH2). Synthesis of PEG−PDS−CTA. Amino terminated methoxypoly(ethylene glycol) (mPEG−NH2, Mn ∼ 5150, 205 mg, 0.04 mmol), 3(4-formylphenoxy)propyl 4-cyano-4-(((ethylthio)carbonothioyl)thio)pentanoate (34 mg, 0.08 mmol), 3-(pyridin-2-yldisulfanyl)propanoic acid (18 mg, 0.08 mmol), and cyclohexyl isocyanide (13 mg, 0.12 mmol) were charged into a dry EP tube along with methanol (2 mL). The EP tube was put into a thermo-shaker at 25 °C for 2 h. The resulting polymer was isolated by recrystallization from 2-propanol and then dried under vacuum to obtain the pure polymer (185 mg, 78%) for further use and characterizations. 1 H NMR (400 MHz, CDCl3, δ/ppm): 8.37 (d, 1H, J = 3.7 Hz, CHNCSS), 7.65−7.02 (m, 7H, CHCHCHCSS, CHCHCNCO), 6.99 (d, 2H, J = 8.4 Hz, CHCCONH), 6.67 (d, 2H, J = 8.4 Hz, CHCOCH2), 5.87 (s, 1H, NCHCO), 5.53 (d, 1H, J = 8.0 Hz, CONH), 4.30−4.17 (m, 4H, ArCH2COOCH2, ArOCH2), 3.96 (t, 2H, J = 6.0 Hz, CH2CH2COOCH2), 3.84−3.40 (m, OCH2CH2O), 3.36 (s, 3H, OCH3), 3.32 (q, 2H, J = 7.4 Hz, SCH2CH3), 3.01 (m, 2H, SSCH2), 2.61 (m, 2H, SSCH2CH2). Synthesis of Midfunctional PEG-b-PHPMA. HPMA (0.72 g, 5 mmol), PEG−PDS-CTA (macro-CTA, 118 mg, 0.02 mmol) and AIBN (1.0 mg, 0.006 mmol) were charged into a dry Schlenk tube along with methanol (2 mL). The Schlenk tube was sealed with a rubber septum and purged with nitrogen flow for 20 min. The tube was then put into a 65 °C oil bath for 10 h. The crude was precipitated from methanol to toluene/THF mixture (v/v = 1:1) for 3 times, and then dried under vacuum to obtain the pure polymer for further use and characterizations. The polymerization conversion (∼90%) was calculated by 1H NMR of the crude (comparing the peaks of the vinyl

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CONCLUSIONS In summary, the Ugi reaction has been applied in the field of protein modification as a reliable method to synthesize a series of multifunctional PEGylation agents, and those PEG derivatives were successfully conjugated on protein surface to generate corresponding multifunctional protein−polymer conjugates. Compared to traditional PEGylation technology, the method in current research incorporates new functions to the PEG derivatives and subsequent protein−polymer conjugates, demonstrating its potential to prepare the third-generation PEGlytion agents. Given the easily available starting materials and simple operation, this method appears to be general to synthesize sophisticated multifunctional polymers for biology application. Currently, preparing new multifunctional polymers through other MCC reactions for protein conjugation and preparing protein conjugates using existing multifunctaionl PEG derivatives and mutant protein therapeutics (cysteine residue at suitable position) for further in vivo experiments are under study.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Detailed experimental procedures, fluorescence spectrum of fluorescent monomer PEG, SDS−PAGE and bioactivity evaluation of different protein−polymer conjugates mentioned above. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author

*(L.T.) E-mail: [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interests. 5611

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ACKNOWLEDGMENTS This research was supported by the National Science Foundation of China (21104039) and the National 973 Project (2011CB935700).

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dx.doi.org/10.1021/ma501385m | Macromolecules 2014, 47, 5607−5612