Dynamic Covalent Diblock Copolymers - American Chemical Society

Oct 29, 2014 - College of Chemistry, Beijing Normal University, Beijing 100875, China. #. Department of Chemistry, University at Buffalo, State Univer...
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Dynamic Covalent Diblock Copolymers: Instructed Coupling, Micellation and Redox Responsiveness Qinglai Yang,† Ling Bai,‡ Yuanqing Zhang,⊥ Fangxia Zhu,† Yuhong Xu,§ Zhifeng Shao,‡ Yu-Mei Shen,*,† and Bing Gong*,∥,# †

Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Bio-ID Center, Shanghai Jiao Tong University, Shanghai 200240, China ‡ School of Biomedical Engineering, Bio-ID Center, Shanghai Jiao Tong University, Shanghai 200240, China § School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China ⊥ Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ∥ College of Chemistry, Beijing Normal University, Beijing 100875, China # Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York 14260, United States S Supporting Information *

ABSTRACT: Instructed by association units that allow reversible and unsymmetrical disulfide bond formation, hydrophilic (PEG) and hydrophobic (PLA) polymer chains are efficiently coupled into amphiphilic diblock copolymers. The desymmetrization of otherwise symmetrical reversible disulfide bond formation is achieved with amide association units that integrate both directional H-bonding and reversible disulfide bond formation, which ensure the connection of different polymer blocks while minimizing self-coupling. The resultant amphiphilic block copolymers self-assemble into longlasting spherical micelles that are responsive to free thiols.

1. INTRODUCTION Stimuli-responsive polymers have many important applications.1,2 For example, redox-responsive amphiphilic block copolymers3 self-assemble into polymeric micelles and vesicles4 that release loaded drugs when triggered by redox potential gradients between extra- and intracellular spaces.5 In known designs, biocompatible blocks such as poly(ethylene glycol) (PEG), sugar-derivatives, and polyglycerol have been used as hydrophilic segments, while biodegradable polyesters, polycarbonates, and polypeptides serve as hydrophobic blocks.6 The naturally occurring disulfide bond, which can reversibly form under mild conditions, has been used to capture and stabilize structures of different origins.7 For example, disulfide bond is often introduced to render a block copolymer with redoxresponsiveness.8 Currently known block copolymers having single disulfide linkages are often prepared by coupling polymer blocks using the irreversible reaction between sulfhydral (SH) and pyridyldisulfide end groups, and have found important applications including drug and gene delivery.9 The synthesis of block copolymers by reversibly forming disulfide linkages is advantageous in that the coupling reaction can be performed efficiently under mild, thermodynamically controlled conditions involving, for example, room temperature and low concentrations. However, coupling based on reversible disulfide bond formation may be complicated by the ready oxidation of SH groups and the reduced activity of SH groups attached to long polymer chains in forming disulfide bonds. Besides, under © XXXX American Chemical Society

reversible conditions, the symmetric nature of reversible disulfide bond formation leads to self-coupled byproducts that seriously compromise the yields of desired block copolymers. The limitations of simple disulfide bonds may be alleviated by controlling the directionality of reversible disulfide bond formation. This approach evolved from a series of hydrogenbonded duplexes we developed over the years.10,11 Our duplexes, consisting of oligoamide strands encoded with Hbonding sequences, i.e., arrays of H-bond acceptors and donors, are featured by tunable stability, programmable sequencespecificity, and ready synthetic availability.10 Our duplexes and related systems developed by others have instructed the formation of β-sheets,12 supramolecular (noncovalent) block copolymers,13−15 the specification of chemical reactions,16 and more recently, as inhibitors for heparin−protein interactions.17 Like most H-bonded complexes,18 our duplexes suffer from instability in polar solvents and thus cannot serve as association units for constructing structures and materials aimed at applications such as those involving aqueous media.13,19 Such a limitation was circumvented by equipping our H-bonded duplexes with the capability of reversibly forming disulfide bonds.20a Along with the reversible formation of disulfide crossReceived: August 19, 2014 Revised: October 15, 2014

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Scheme 1. Strategy for Dynamic Covalent Diblock Copolymers: Instructed Coupling, Micellation and Redox Responsiveness

Scheme 2. (a) Coupling of PEG-A and PLA-B Based on Unsymmetrical, Reversible Disulfide-Bond Formation Instructed by HBonding and (b) Formation of Low-Molecular Weight Compound 1−2 Based on the Same Reversible Disulfide-Bond Formation

linked structures of various sizes to be formed in a selfassembling fashion. Herein, we demonstrate that our Hbonding/dynamic covalent association units, when attached to polymer chains, are very effective in mediating the directional coupling of different polymer strands. Specifically, our study shows that poly(ethylene glycol) (PEG) and polylactic acid (PLA) blocks are coupled into amphiphilic diblock copolymers under the instruction of hydrogen-bonding/dynamic covalent association units (Scheme 1).

links, oligoamide strands having complementary H-bonding sequences were found to pair with each other even in aqueous media, in very high, i.e., nearly quantitative, yields. The integration of H-bonding with reversible disulfide bond formation results in a thermodynamically controlled system in which the predominant, i.e., most stable, products that stand out involve the disulfide-cross-linked duplexes containing strands with fully matched H-bonding sequences. In other words, complementary interstrand H-bonds, which become intramolecular upon the formation of disulfide cross-links, serve to stabilize the cross-linked products. Because of the thermodynamic nature of this system, as few as two H-bonds, when coupled with disulfide bonds, were sufficient to bias the specific pairing of two strands having complementary Hbonding sequences.20b The integration of reversible disulfide bond formation and H-bonding interaction has thus resulted in association units that maintain the sequence-specificity of the original H-bonded duplexes while being covalently cross-linked. These association units, with their efficiency and specificity being demonstrated by low-molecular model compounds,20 should allow covalently

2. RESULTS AND DISCUSSION The specific design is illustrated in Scheme 2a. PEG and PLA chains are end-modified with amide units A and B that bear complementary arrays of H-bond donors and acceptors, along with S-trityl groups capable of reversibly forming disulfide bonds. In the presence of I2,20,21 PEG-A and PLA-B are covalently linked into the corresponding block copolymers PEG-PLA due to reversible but directional disulfide bond formation, with minor self-coupling of the modified PEG and PLA chains. The resultant block copolymers were found to selfB

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assemble into stable polymeric micelles that exhibit responsive behavior. That amide units A and B could indeed lead to unsymmetrical disulfide bond formation was demonstrated by treating amides 1 and 2 with I2 in DMSO/CH2Cl2 (1/1, v/v), a common solvent for both 1 and 2 (Scheme 2b). Analyzing the reaction mixture using HPLC showed that the cross-linked 1−2 formed as the dominant product, along with a very small amount of other byproducts (Figure 1).

Figure 2. Gel permeation chromatography (GPC) traces of the crude products of (a) PEG5000-A (0.5 mM), (b) 1:1 mixture of PEG5000-A (0.5 mM) and PLA1000-B (0.5 mM), and (c) PLA1000-B (0.5 mM), after being treated with I2 (6 mM) in CH2Cl2.

PLA1000-S-S-PLA1000 resulted from self-coupling, constituted 31% and 28%, respectively, of the crude polymer product (Figure S28). Thus, in the absence of H-bonds, coupling based on disulfide bonds under reversible conditions could only result in a roughly statistical distribution of all possible products. Further purified diblock copolymer PEG5000-PLA1000 was analyzed with 1H NMR. The 1H NMR spectrum of PEG5000PLA1000 (Figure 3b) not only reveals signals of the PEG block (3.85−3.41 ppm from the repeating −OCH2CH2O− unit and 3.37 ppm from the terminal CH3O- groups), those of the repeating LA residues (5.29−5.10 ppm from the −CH− groups), the terminal LA residue (4.38−4.31 ppm from the −CH− group), and the CH3- groups of the LA units (1.64− 1.45 ppm), but also those of the amide linking units (2.21−2.07 ppm from −COCH2−, −SCH2−, −CH2−, and 3.11−2.43 ppm from −NCH2−, −SCH2−). The dominant signals of the trityl groups in the spectra of PEG5000-A (Figure 3a) and PLA1000-B (Figure 3c) are absent in the spectrum of PEG5000-PLA1000, indicating that disulfide cross-linking indeed happened. In comparison with the corresponding signals of either PEG5000-A or PLA1000-B, the signals of protons d, e, f, j, and k of the amide association units show noticeable shifts. These observations demonstrate that covalent coupling of the PEG and PLA chains did happen. The molecular weights of PEG5000-PLA1000 and PEG5000PLA5000, along with the corresponding PEG-A and PLA-B strands, were examined with GPC and MALDI−MS, results from which are consistent with the formation of the diblock copolymers (Figures S16−S20). Table 1 summarizes the molecular weights (Mn and Mw), molecular-weight polydispersity (PDI) and MALDI−MS. Thus, association units A and B, which integrate H-bonding and dynamic covalent interactions, could indeed desymmetrize reversible disulfide bond formation and direct the efficient coupling of two different polymer chains. The successful coupling of PEG5000-A and PLA5000-B into diblock copolymer PEG5000-PLA5000 showed that this strategy is effective for coupling both short and long polymer chains. Given that the

Figure 1. HPLC trace of the solution of the 1:1 mixture of 1 and 2 in CH2Cl2/DMSO (1/1, v/v) in the presence of iodine.

The 1:1 (molar ratio was calculated by the number-average molecular weight (Mn) of polymers) mixture of PEG5000-A and PLA1000-B (0.5 mM) was treated with I2 (6 mM) in CH2Cl2 at room temperature for 1 h. After removing solvent, the remaining solid residue was suspended in ethyl ether and then sonicated. The polymer products, being insoluble in diethyl ether, are separated from other low-molecular weight molecules by centrifugation. Repeating this procedure for three times removed low-molecular weight components from the crude product. Analyzing the polymer product using gel permeation chromatography (GPC) external standard method revealed the dominant product to be cross coupling copolymer PEG5000-PLA1000 (82%). At the same time the self-coupling products PEG5000-PEG5000 and PLA1000-PLA1000 appeared only in small proportions (8% and 10% respectively) (Figure 2). The identity of the crude diblock polymer product was also confirmed by comparing with separately prepared self-coupling products and by analyzing with matrix-assisted laser desorption ionization mass spectrometry (MALDI−MS) (Figure S19, Figure S21, and Figure S22, Supporting Information). On the basis of the same conditions, PEG5000-A and PLA5000-B were also coupled into copolymer PEG5000-PLA5000 (Figure S20). The effect of amide units A and B on directing the reversible formation of disulfide cross-links was further demonstrated by comparing the outcome of treating 1:1 mixture of PEG5000SCPh3 and PLA1000-SCPh3 (1 mM), which do not carry end groups capable of H-bonding capability, with I2 (Scheme 3). After removing low-molecular weight components by treating with ethyl ether, the polymer crude product was analyzed with GPC external standard method. It was found that PEG5000-S-SPLA1000 accounted for 41%, and PEG5000-S-S-PEG5000 and C

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Scheme 3. Coupling of PEG5000-SCPh3 and PLA1000-SCPh3

Figure 3. 1H NMR (400 MHz) spectra of PEG5000-A in CDCl3, PLA1000-B in CDCl3, and PEG5000-PLA1000 in CD3OD/CDCl3 (50/50, v/v).

dynamic light scattering (DLS) detected particles with a unimodal size distribution, with an average diameter of 117 nm that is typical of polymer micelles (Figure S34). Transmission electron microscopy (TEM) revealed that the micelles are all spherical, with average diameter of 40 nm (Figure S34). After further purification, the polymer product was again subjected to micellation. On the basis of the molecular weight of diblock copolymer PEG5000-PLA5000, the critical micelle concentration (CMC)23 (Figure S35) was found to be 1.21 × 10−5 M, a value consistent with the high stability characteristic of polymer micelles.24 DLS measurement revealed a unimodal size distribution for the micelles of the purified product with an average diameter of 107 nm that is very close to that revealed on the crude product (Figure 4). Consistent with the DLS results, TEM demonstrated that the micelles are all spherical, with micellar cores having average diameter of 48 nm (Figure 4). TEM imaging demonstrated micellar cores diameter (48 nm) is in dry state, however DLS measurements is the mechanical equivalent of the hydrated radius (107 nm), this means a solvent layer attached to the surface of micellar cores.

Table 1. Characterization Details of PEG-A, PLA-B and PEG−PLA

a b

polymer

Mna,b

Mwa,b

PDIa,b

PEG5000-A PLA1000-A PLA5000-B PEG5000-PLA1000 PEG5000-PLA5000

5700/5600 1900/1600 4000/3400 6300/5900 8900/8900

6200/5800 2300/1800 4800/3800 7300/6500 10500/10000

1.09/1.04 1.20/1.12 1.20/1.12 1.16/1.10 1.18/1.12

Estimated by GPC (THF, 1 mL/min) using polystyrene standards. Estimated by MALDI−MS.

self-coupling of PEG5000-A and PLA5000-B lead to products with the similar molecular weights as that of PEG5000-PLA5000, the identity of PEG5000-PLA5000 was further verified by studying the self-assembly of the crude and further purified polymer products. After removing low-molecular weight components, the crude polymer product from the coupling of PEG5000-A and PLA5000B in DMSO was dialyzed into water (see the Supporting Information).22 Examining the resultant aqueous solution with D

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3. CONCLUSION In summary, amphiphilic block copolymers have been constructed using association units allowing H-bond instructed, directional but reversible disulfide bond formation. The presence of complementary H-bonding sequences in the linking units serves to desymmetrize the otherwise symmetrical disulfide-bond forming process, which ensures the controlled coupling of the PEG and PLA blocks under thermodynamic conditions, while minimizing undesired self-coupling of the same blocks. Results from DLS and TEM measurements confirm that the block copolymers can self-assemble into longlasting, nanosized spherical micelles, consistent with the covalent nature of the resultant block polymers. Free thiols trigger the cleavage of the disulfide linkages, leading to the decomposition of the polymeric micelles. With its thermodynamic (reversible) nature and mild conditions involved, this strategy should not be limited to the PEG−PLA diblock copolymers described here. Instead, the same coupling method should be general to the controlled coupling of many other polymer blocks that otherwise are not compatible. By taking advantage of the sequence-programmability of the H-bonded and disulfide-linked duplexes we developed,10,20 association units having different specificities should allow the efficient coupling of oligomeric or polymeric blocks of desired number and properties into redox-responsive block copolymers under reversible conditions.

Figure 4. TEM image and DLS plot of PEG5000-PLA5000 micelle.

Therefore, the size demonstrated by DLS results is larger than that of TEM imaging. Subjecting the self-coupling product of PEG5000-A or PLA5000-B to the same micellation condition failed to result in the formation of polymer micelles. PEG5000-PEG5000, formed from the self-coupling of PEG5000-A, is soluble in water, while PLA5000-PLA5000, from the self-coupling of PLA5000-B, was simply insoluble in water. These results unequivocally demonstrate that the coupling of PEG5000-A and PLA5000-B has indeed led to amphiphilic diblock copolymer PEG5000PLA5000. ‘ DLS measurements showed that the micelles formed from PEG5000-PLA5000 were stable for weeks at both room and physiological temperature (37 °C). The average diameter of the micelles of PEG5000-PLA5000 remained unchanged for up to 40 days at room temperature and 20 days at 37 °C (Figure S36). Diblock copolymer PEG5000-PLA5000 contains double disulfide cross-links that, as redox-responsive triggers, should allow not only instructed the formation but also the controlled dismantlement of the block copolymer and thus the decomposition of the corresponding polymer micelles. It is well-known that disulfide bonds can be cleaved by free thiols such as dithiothreitol (DTT) that is widely used to cleave disulfide bonds in proteins.25 Shortly after adding DTT, the long-lasting, unimodal size (107 nm) distribution of untreated PEG5000-PLA5000 micelles changed into a broad size distribution (Figure 5). The observed change upon treating with DTT can be explained by the cleavage of disulfide linkages, which results in the detachment of PEG shells and thus the decomposition of the micelles.

4. EXPERIMENTAL SECTION 4.1. General Methods. All chemicals were purchased from Aldrich or Aladdin and were used as received unless otherwise indicated. All reactions were followed by thin-layer chromatography (TLC) (precoated 0.25 mm silica gel plates from Aldrich), and silica gel column chromatography was carried out with silica gel 60 (mesh 200− 400). Dichloromethane, N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) were dried over calcium hydride and then purified by vacuum distillation. Tetrahydrofuran (THF) was dried by refluxing with the fresh sodium-benzophenone complex under N2 and distilled just before use. Amine-terminated poly(ethylene glycol) monomethyl ether (mPEG-NH2) was purchased from Shanghai Yare Bio. Co. Ltd. (number-average molecular weight (Mn) determined by GPC, Mn = 5000) and used as received. Polylactic acid (PLA) was purchased from Jinan Daigang Biology Co. Ltd. (viscosity-average molecular weight (Mv) determined by viscometer, Mv = 1000 and 5000; number-average molecular weight (Mn) determined by GPC, Mn = 800 and 3400) and used as received. The dialysis bag was purchased from Spectrum. Nuclear magnetic resonance (NMR) analyses were recorded on a Bruker Avance III 400 MHz spectrometer with deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD) and deuterated chloroform (CDCl3) as solvents. The number-average molecular weight (Mn), weight-average molecular weight (Mw) and polydispersity index (PDI) were measured by gel permeation chromatography (GPC) and matrix-assisted laser desorption ionization mass spectrometry (MALDI−MS). GPC was performed on Agilent GPC series 1260 infinty, 10 μm PLgel 600 × 7.5 mm column, THF used as the mobile phase at a flow rate of 1.0 mL/min at 35 °C, linear polystyrene calibration, equipped with a refractive index (RI) detector. MALDI−MS was performed on a Shimadzu Biotech MALDI−MS spectrometer, 2,5-dihydroxybenzoic acid (DHB) or 1,8,9-trihydroxyanthracene used as matrix. The purified polymers were spotted on the layer of the matrix on the target or the crude product of disulfide formation reaction was washed with ether and then directly spotted on the layer of the matrix on the target. UPLC−TOF−MS spectra were recorded on Waters UMS Q-Tof Premier, Waters UPLC BEH C18 1.0 × 100 mm column, the traces were recorded with a UV detector at 254 nm. UPLC conditions: 0.4 mL/min, methanol−water gradient

Figure 5. DLS plots of PEG5000-PLA5000 micelle before and after being incubated with DTT (10 mM) in PBS (50 mM, pH 7.4) at 37 °C. E

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Scheme 4. Synthesis of Compounds A, B, 1, 2, and 1−2

product was distilled in vacuo to give 5 (8.1 g, 22.5 mmol, 90%) as white powder. 1H NMR (400 MHz, CDCl3) δ7.43−7.37 (m, 6H, ArH), 7.29−7.23 (m, 6H, ArH), 7.22−7.15 (m, 3H, ArH), 2.38 (t, J = 7.4 Hz, 2H, −CH2COO−), 2.30 (t, J = 7.2 Hz, 2H, −CH2S−), 1.76− 1.73 (m, 2H, −CH2−). Methyl 3,5-Diaminobenzoate (7).26 3,5-Diaminobenzoic acid (6) (1.52 g, 10 mmol) was dissolved in 20 mL of 10% sulfuric acid− methanol solution under an ice water bath, then refluxed at 90 °C for 6 h. After cooling, the solution was concentrated in vacuo, extracted with ethyl acetate, neutralized with saturated sodium bicarbonate solution, the extract was dried over anhydrous Na2SO4, ethyl acetate evaporated, and the residue was distilled in vacuo to give 7 (1.48 g, 8.9 mmol, 89%). 1H NMR (400 MHz, CD3OD) δ6.76−6.73 (m, 2H, ArH), 6.32−6.31 (m, 1H, ArH), 3.82 (s, 3H, CH3O−). Methyl 3,5-bis(4-(tritylthio)butanamido)benzoate (1). To a stirred solution of 5 (1.2 g, 3.3 mmol) and 7 (0.2 g, 1.2 mmol) in 30 mL methylene chloride were added 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI) (1.0 g, 5.2 mmol) at 0 °C. Stirring was continued for 12 h upon the mixture was warmed up to room temperature. Solvent was evaporated, and the residue was dissolved in methanol at 0 °C about 1 h. Recrystallized from methanol afforded 1 (0.63 g, 0.73 mmol, yield 61%). 1H NMR (400 MHz, DMSO-d6) δ10.07 (s, 2H, −NH(CO)−), 8.10 (s, 1H, ArH), 7.91 (s, 2H, ArH), 7.43−7.12 (m, 30H, ArH), 3.84 (s, 3H, −OCH3), 2.29 (t, J = 7.2 Hz, 4H, −CH2CO−), 2.15 (t, J = 7.1 Hz, 4H, −CH2S−), 1.73−1.55 (m, 4H, −CH2CH2CH2−); 13C NMR (100 MHz, DMSOd6) δ171.1, 166.7, 145.0, 140.2, 130.8, 129.5, 128.4, 127.1, 114.9, 114.2, 66.5, 55.2, 52.7, 35.8, 31.4, 24.4. HRMS (ESI): calcd for C54H50N2O4S2Na, 877.3110 (M + Na+); found, 877.3132. 3,5-Bis(4-(tritylthio)butanamido)benzoic Acid (A). 1 (0.3 g, 0.35 mmol) was dissolved in 6 mL of DMSO then heated to 120 °C. To the solution, a solution of 0.3 mL of NaOH (1.0 M) in water was added in several portions. The reaction was monitored by TLC. The

elution with methanol 20% in 1 min followed by methanol 20% to 40% in 1 min, methanol 40% to 60% in 6 min, and finally, methanol 60−95% in 2 min. The identity of each peak was confirmed by collecting the corresponding fraction that was then subjected to analysis by mass spectrometry. The HPLC experiments were carried out on Shimadzu LC-20A. The traces were recorded with a UV detector at 254 nm. The column was a SB-C18 (4.6 × 250 mm, 10 μm) of Agilent. An aliquot (20 μL) drawn from a reaction mixture was directly injected for analysis at ambient temperature. HPLC conditions: 1.0 mL/min, methanol−water gradient elution with methanol 40% in 2 min followed by methanol 40% to 50% in 5 min, methanol 50% to 85% in 36 min, finally, methanol 85−95% in 20 min. Dynamic light scattering (DLS) measurements were performed in aqueous solution using a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ = 633 nm. All samples of 1 mg/mL were measured at 25 °C and at a scattering angle of 173°. Transmission electron microscopy (TEM) studies were performed with a JEM-2010HT instrument operated at 200 kV. The samples were prepared by directly dropping the solution of micelles onto carbon-coated copper grids and dried at room temperature overnight without staining before measurement. 4.2. Synthesis and Characterization. 4-(Tritylthio)butanoic Acid (5).20a 4-Bromobutyric acid 3 (16.48 g, 98 mmol) was refluxed for 3 h with thiourea (8.5 g, 107 mmol) in 30 mL of ethanol. Then 50 mL of aqueous NaOH (4 M, 200 mmol) was added, and refluxing was continued for another 2 h. The solution was concentrated in vacuo, acidified with aqueous HCl (6 M), and extracted with ether, the extract dried over anhydrous sodium sulfate (Na2SO4), evaporated, and the residue distilled in vacuo to give 4 (10.45 g, 87.1 mmol, 87%) as a light pink oil. Then, mixture of 4 (3 g, 25 mmol) and trityl chloride (10 g, 38 mmol) were stirred in 40 mL of DMF for 5 days at room temperature. A 10% sodium acetate solution (350 mL) was then added, and the precipitate was filtered and washed with water. The F

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Scheme 5. Synthesis of PEG-A, PLA-B, and PEG−PLA

solution was poured into 30 mL of HCl (1.0 M) solution, and the precipitate was filtered, washed with water, and distilled in vacuo to give compound A (0.25 g, 0.3 mmol, 86%). 1H NMR (400 MHz, DMSO-d6): δ 9.99 (s, 2H, −NH(CO)−), 8.06 (s, 1H, ArH), 7.84 (m, 2H, ArH), 7.31−7.23 (m, 24H, ArH), 7.20−7.15 (m, 6H, ArH), 2.26 (t, J = 7.2 Hz, 4H, −CH2S−), 2.10 (t, J = 7.1 Hz, 4H, −CH2CO−), 1.65−1.54 (m, 4H, −CH2CH2CH2−). 13C NMR (100 MHz, DMSO-d6): δ 171.0, 167.6, 148.3, 145.0, 140.0, 131.8, 129.5, 128.5, 128.3, 127.8., 127.1, 115.2, 66.5, 35.8, 31.4, 24.4. HRMS (ESI): calcd for C53H48N2O4S2Na, 863.2953 (M + Na+); found, 863.2990. 2-(Tritylthio)ethanamine (9).27 Triphenylmethanol (2.29 g, 8.8 mmol) was added to a solution of 2-aminoethanethiol hydrochloride (8) (1.0 g, 8.8 mmol) in 4 mL of trifluoroacetic acid (TFA). The resulted solution was stirred at room temperature for about 40 min. TFA was removed in vacuo. The residue was triturated with ethyl ether. The formed white precipitate was filtered, partitioned with aqueous NaOH (25 mL, 1 M) and extracted with ethyl acetate. The organic layers were dried over anhydrous Na2SO4. After evaporation, the product 9 was obtained as white solid (2.35 g, 7.4 mmol, 84%). 1H NMR (400 MHz, CD3OD): δ 7.46−7.41 (m, 6H, ArH), 7.34−7.27 (m, 6H, ArH), 7.26−7.21 (m, 3H, ArH), 2.54−2.44 (m, 4H, −NCH2−, −SCH2−). 5-Amino-N1,N3-bis(2-(tritylthio)ethyl)isophthalamide (B). To a stirred solution of 5-aminoisophthalic acid (10) (0.36 g, 2.0 mmol) and 9 (1.91 g, 6.0 mmol) in 20 mL of N,N-dimethylformamide (DMF) were added 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCI) (0.95g, 4.8 mmol), and N-hydroxybenzotriazole (HOBt) (0.65 g, 4.8 mmol) at 0 °C. Stirring was continued for 4 h while the mixture was warmed up to room temperature. The solution was poured into 30 mL ice−water solution, the precipitate was filtered and distilled in vacuo to give compound B (0.97g,1.24 mmol, 62%). 1H NMR (CDCl3, 400 MHz) δ7.43−7.34 (m, 12H, ArH), 7.28−7.14 (m, 19H, ArH), 7.06 (s, 2H, ArH), 6.34 (t, J = 5.6 Hz,2H, −NHCH2−), 3.27−3.18 (m, 4H, −NCH2−), 2.48 (t, J = 6.4 Hz, 4H, −SCH2−). 13C NMR (100 MHz, CDCl3): δ 166.9, 147.1, 144.6, 135.9, 129.5, 128.0, 126.9, 116.2, 114.6, 67.9, 38.8, 32.0. HRMS (ESI): calcd for C50H45N3O2S2Na, 806.2851 (M + Na+); found, 806.2824. 5-Acetamido-N1,N3-bis(2-(tritylthio)ethyl)isophthalamide (2). Compound B (0.784g,1.0 mmol) was dissolved in 20 mL methylene chloride (CH2Cl2) solution, the solution was stirred at 0 °C. Then

triethylamine (0.2 g, 2.0 mmol) was added and acetyl chloride (0.156 g, 2.0 mmol) was added dropwise slowly. Stirring was continued for 4 h upon the mixture was warmed up to room temperature. The solution was washed with saturated sodium bicarbonate solution, and dried over anhydrous Na2SO4, evaporated, and the residue was distilled in vacuo to give 2 (0.78 g, 0.95 mmol, 95%). 1H NMR (400 MHz, CDCl3): δ 9.14−9.09 (m, 1H, −NH(CO)CH3), 8.24 (s, 2H, ArH), 7.83 (s, 1H, ArH), 7.46−7.37 (m, 12H, ArH), 7.31−7.24 (m, 12H, ArH), 7.23−7.17 (m, 6H, ArH), 6.73−6.67 (m, 2H, −NHCH2−), 3.34−3.22 (m, 4H, −NCH2−), 2.50 (t, J = 6.6 Hz, 4H, −SCH2−), 2.14 (s, 3H, −(CO)CH3). 13C NMR (100 MHz, CDCl3): δ 169.5, 166.6, 144.6, 139.6, 135.2, 129.5, 128.0, 126.8, 120.9, 120.6, 66.9, 53.5, 39.1, 31.7, 24.6. HRMS (ESI) calcd for C52H47N3O3S2Na (M + Na+), 848.2957; found, 848.2899. Compound 1−2. Compounds 1 (55.51 mg, 0.065 mmol) and 2 (53.69 mg, 0.065 mmol) were dissolved in 30 mL of (DMSO/CH2Cl2, 1/1, v/v) solution. CH2Cl2 was evaporated in vacuo and the residue was dissolved with 50 mL of iodine solution (7.8 mM) in CH2Cl2. The resulting mixture was stirred at room temperature. After 45 min, the reaction mixture was cooled to 0 °C, and a sodium thiosulfate aqueous solution (3 mM) was added until the color of iodine disappeared. The mixture then was extracted with CH2Cl2. The organic layer was washed with brine and dried over anhydrous Na2SO4. Purification was accomplished by TLC on silica gel by using CH2Cl2/CH3OH as eluent. The product distilled in vacuo to give compound 1−2 (24.82 mg, 0.035 mmol, 54%). 1H NMR (400 MHz, DMSO-d6): δ 10.19− 10.13 (m, 3H, −CH3(OC)NH−, −(OC)NH−), 8.70 (t, J = 5.5 Hz, 2H, −CH2NH−), 8.53 (s, 1H, ArH), 8.14 (s, 2H, ArH), 7.94 (s, 1H, ArH), 7.78−7.76 (m, 2H, ArH), 3.83 (s, 3H, CH3O−), 3.57−3.48 (m, 4H, −CH2NH−), 2.90 (t, J = 7.1 Hz, 4H, −CH2S−), 2.81 (t, J = 7.1 Hz, 4H, −CH2(CO)−), 2.43 (t, J = 7.1 Hz, 4H, −CH2S−), 2.05 (s, 3H, CH3(CO)), 2.03−1.92 (m, 4H, −CH2CH2CH2−). 13C NMR (100 MHz, DMSO-d6): δ 171.2, 169.1, 166.4, 166.3, 140.3, 139.9, 135.6, 130.6, 121.1, 120.5, 114.8, 114.7, 52.6, 40.9, 38.2, 37.6, 35.6, 25.4, 24.4. HRMS (ESI) calcd for C30H37N5O7S4Na, 730.1474 (M + Na+); found, 730.1504. PEG5000-A. Common procedure: Under a nitrogen atmosphere, compound A (1.0 mmol), NMM (1 mmol), HATU (1.5 mmol), and PEG5000 (1.0 mmol) in anhydrous DMF (15 mL) were cooled in ice− water bath with stirring by a magnet. After stirring for 1 h, reaction continued at room temperature for additional 18 h. The resulting G

dx.doi.org/10.1021/ma5017083 | Macromolecules XXXX, XXX, XXX−XXX

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Scheme 6. Synthesis of PEG5000-SCPh3, PLA1000-SCPh3, PEG5000-S-S-PEG5000, PEG5000-S-S-PLA1000 and PLA1000-S-S-PLA1000

3H per repeating unit, PLA−CH3−). GPC retention time: 6.77 min. GPC: Mn = 1900, Mw = 2300, PDI = 1.20. MALDI−MS: Mn = 1600, Mw = 1800, PDI = 1.12. IR (KBr, cm−1): 3385, 2994, 2941, 1757, 1657, 1599, 1536, 1449, 1379, 1359, 1266, 1189, 1129, 1092, 1047, 951, 866, 803, 746, 702, 622, 507. PLA5000-B. Purified isolated yield: 71%. 1H NMR (400 MHz, CDCl3): δ 8.17−8.11 (m, 2H, ArH), 7.80 (s, 1H, ArH), 7.44−7.37 (m, 12H, ArH), 7.29−7.23 (m, 12H, ArH), 7.22−7.16 (m, 6H, ArH), 6.37 (brs, 2H, −NH−), 5.42−5.02 (m, 56H, 1H per repeating unit, PLA− CH−), 4.37−4.31 (m, 1H, PLA−CH−), 3.30−3.23 (m, 4H, −NCH2−), 2.51 (t, J = 6.4 Hz, 4H, −SCH2−), 1.76−1.36 (m, 171H, 3H per repeating unit, PLA−CH3−). GPC retention time: 5.96 min. GPC: Mn = 4000, Mw = 4800, PDI = 1.20. MALDI−MS: Mn = 3400, Mw = 3800, PDI = 1.12. IR (KBr, cm−1): 3392, 2994, 2943, 1756, 1662, 1600, 1533, 1451, 1379, 1268, 1189, 1129, 1092, 1048, 952, 866, 743, 702, 623, 508. PEG−PLA. Common procedure: PEG-A (20 mL) and PLA-B (20 mL) were dissolved in CH2Cl2 solutions (1.0 mM) separately and mixed together in a 250 mL round-bottom flask. Solvent was evaporated in vacuo and the residue was dissolved with 40 mL of iodine solution (6 mM) in CH2Cl2. The resulting mixture was stirred at room temperature without inert gas protection. After 1 h, the reaction mixture was cooled in ice−water bath and sodium thiosulfate aqueous solution (3 mM) was added until the color of iodine disappeared. The mixture was extracted with CH2Cl2. The combined organic extracts were washed with water and brine, dried over anhydrous Na2SO4. Product was washed with ether or further purified by thin-layer chromatography on silica gel by using CH2Cl2/CH3OH (10/1) as eluent.

mixture was poured into water and extracted with CH2Cl2, the combined organic extracts were washed with water and brine, dried over anhydrous MgSO4 and evaporated in vacuo. The product was dried under vacuum to a constant weight. Purified isolated yield: 76%. 1 H NMR (400 MHz, CDCl3): δ 8.16 (s, 1H, −NH−), 8.05 (s, 1H, ArH), 7.73 (s, 2H, ArH), 7.59 (s, 1H, −NH−), 7.44−7.34 (m, 12H, ArH), 7.29−7.22 (m, 12H, ArH), 7.23−7.13 (m, 6H, ArH), 3.87−3.41 (m, 412H, PEG, 2H per repeating unit, −OCH2CH2O−), 3.37 (s, 3H, PEG, CH3O−), 2.37−2.19 (m, 8H, −(CO)CH2−, −SCH2−), 1.82−1.65 (m, 4H, −CH2CH2CH2−). GPC retention time: 5.57 min. GPC: Mn = 5700, Mw = 6200, PDI = 1.09. MALDI−MS: Mn = 5600, Mw = 5800, PDI = 1.04. IR (KBr, cm−1): 2882, 1755, 1664, 1601, 1553, 1466, 1350, 1302, 1280, 1082, 955,843, 747, 702, 621, 580. PLA-B. Common procedure: Under a nitrogen atmosphere, PLA (1.0 mmol), NMM (1.0 mmol), HATU (1.5 mmol) and compound B (1.5 mmol) in anhydrous DMF (15 mL) were cooled in ice−water bath with stirring by a magnetic. After stirring for 1 h, reaction was continued at room temperature for additional 24 h. The resulting mixture was poured into water and extracted with CH2Cl2, the combined organic extracts were washed with water and brine, dried over anhydrous MgSO4 and evaporated in vacuo. The product was further purified by chromatography on silica gel by using CH2Cl2/ CH3OH (100/1 to 50/1) as eluent. PLA1000-B. Purified isolated yield: 76%. 1H NMR (400 MHz, CDCl3): δ 8.19−8.10 (m, 2H, ArH), 7.80 (s, 1H, ArH), 7.45−7.38 (m, 12H, ArH), 7.31−7.23 (m, 12H, ArH), 7.22−7.16 (m, 6H, ArH), 6.34 (brs, 2H, −NH−), 5.40−5.02 (m, 14H, 1H per repeating unit, PLA− CH−), 4.39−4.31 (m, 1H, PLA−CH−), 3.30−3.22 (m, 4H, −NCH2−), 2.51 (t, J = 6.4 Hz, 4H, −SCH2−), 1.65−1.44 (m, 45H, H

dx.doi.org/10.1021/ma5017083 | Macromolecules XXXX, XXX, XXX−XXX

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(s, 3H, CH3O−), 3.00−2.87 (m, 4H, −SCH2−), 2.81−2.72 (m, 4H, −(CO)CH2−), 2.55−2.41 (m, 4H, −SCH2−),2.15−1.98 (m, 4H, −CH2−), 1.75−1.34 (m, 102H, 3H per repeating unit, PLA−CH3−). GPC retention time: 5.24 min. GPC: Mn = 8900, Mw = 10500, PDI = 1.18. MALDI−MS: Mn = 8900, Mw = 10000, PDI = 1.12. IR (KBr, cm−1): 3449, 2882, 1754, 1639, 1558, 1447, 1383, 1265, 1187, 1109, 957, 841, 799, 619. PEG5000-SCPh3. Under nitrogen atmosphere, 5 (1.0 mmol), NMM (1.0 mmol), HATU (1.5 mmol), and PEG (1.0 mmol) in anhydrous DMF (15 mL) were cooled in ice−water bath with stirring by a magnetic. After stirring for 1 h, reaction was continued at room temperature for additional 12 h. The resulting mixture was poured into water and extracted with CH2Cl2. The combined organic extracts were washed with water and brine, dried over anhydrous MgSO4 and evaporated in vacuo. The product was dried under vacuum to a constant weight. Purified isolated yield: 94%. 1H NMR (400 MHz, CD3OD): δ 7.54−7.09 (m, 15H, ArH), 3.89−3.41 (m, 428H, PEG, 2H per repeating unit, −OCH2CH2O−), 3.36 (s, 3H, PEG, CH3O−), 2.28−2.07 (m, 4H, −(CO)CH2−, −SCH2−), 1.74−1.54 (m, 2H, −CH2CH2CH2−). GPC retention time: 5.69 min. GPC: Mn = 5400, Mw = 5600, PDI = 1.04. MALDI−MS: Mn = 5200, Mw = 5400, PDI = 1.04. IR (KBr, cm−1): 2884, 1962, 1721, 1669, 1600, 1537, 1468, 1358, 1296, 1344, 1280, 1239, 1146, 1114, 1060, 961, 843, 745, 702, 529. PLA1000-SCPh3. Under nitrogen atmosphere, PLA (1.0 mmol), NMM (1.0 mmol), HATU (1.5 mmol) and 9 (1.5 mmol) in anhydrous DMF (15 mL) were cooled in ice−water bath with stirring by a magnetic. After stirring for 1 h, reaction continued at room temperature for additional 18 h. The resulting mixture was poured into water and extracted with CH2Cl2. The combined organic extracts were washed with water and brine, dried over anhydrous MgSO4 and evaporated in vacuo. Product was further purified by chromatography on silica gel by using CH2Cl2/CH3OH (100/1 to 50/1) as eluent. Purified isolated yield: 86%. 1H NMR (400 MHz, CDCl3): δ 7.42− 7.08 (m, 15H, ArH), 6.57−6.20 (m, 1H, −NH−), 5.30−4.88 (m, 14H, 1H per repeating unit, PLA−CH−), 4.41−4.30 (m, 1H, PLA−CH−), 3.20−2.94 (m, 2H, −NCH2−), 2.50−2.26 (m, 2H, −SCH2−), 1.78− 1.29 (m, 45H, 3H per repeating unit, PLA−CH3−). GPC retention time: 7.06 min. GPC: Mn = 1100, Mw = 1400, PDI = 1.27. MALDI− MS: Mn= 1000, Mw= 1200, PDI = 1.20. IR (KBr, cm−1): 2994, 2943, 1756, 1686, 1596, 1527, 1490, 1541, 1381, 1363, 1268, 1188, 1130, 1092, 1050, 954, 865, 746, 702, 620. Formation of Simple Disulfide Bond Conjugated Copolymers. PEG5000-SCPh3 (20 mL) and PLA1000-SCPh3 (20 mL) were dissolved in CH2Cl2 solution (1.0 mM) separately and mixed together in a 250 mL round-bottom flask. Solvent was evaporated in vacuo and the residue was dissolved with 40 mL of iodine solution (6 mM) in CH2Cl2. The mixture was stirred at room temperature without inert gas protection. After 1 h, the reaction mixture was cooled in ice−water bath and sodium thiosulfate aqueous solution (3 mM) was added until the color of iodine disappeared. The mixture was extracted with CH2Cl2. The combined organic extracts were washed with water and brine, dried over anhydrous Na2SO4. Product was washed with ether or purified by chromatography on silica gel by using CH2Cl2/CH3OH (80/1 to 20/1) as eluent. (a). PEG5000-S-S-PEG5000. PEG5000-SCPh3 (0.5 mM) was dissolved in CH2Cl2 in the presence of iodine (6 mM) to give the self-linked product. Purified isolated yield: 95%. 1H NMR (400 MHz, CDCl3): δ 3.93−3.38 (m, 865H, PEG, 2H per repeating unit, −OCH2CH2O−), 3.37 (s, 6H, PEG, CH3O−), 2.72 (t, J = 7.0 Hz, 4H, −(CO)CH2−), 2.09−1.95 (m, 4H, −(CO)CH 2 −), 1.62−1.51 (m, 4H, −CH2CH2CH2−). GPC retention time: 5.13 min. GPC: Mn = 10100, Mw = 11300, PDI = 1.12. MALDI−MS: Mn = 10100, Mw = 10600, PDI = 1.05. IR (KBr, cm−1) 2884, 1651, 1359, 1343, 1279, 1241, 1147, 1113, 1060, 962, 703, 529. (b). PLA1000-S-S-PLA1000. PLA1000-SCPh3 (0.5 mM) was dissolved in CH2Cl2 in the presence of iodine (6 mM) to give the self-linked product. Purified isolated yield: 91%. 1H NMR (400 MHz, CDCl3): δ 5.38−4.92 (m, 28H, 1H per repeating unit, PLA−CH−), 4.46−4.26 (m, 2H, PLA−CH−), 3.73 (t, J = 6.6 Hz, 4H, −NCH2−), 1.93−1.76 (m, 4H, −SCH2−), 1.76−1.33 (m, 88H, 3H per repeating unit, PLA−

The formation of self-linked products PEG5000-PEG5000 and PLA1000-PLA1000 was also applied the common synthetic procedure of PEG−PLA. External Standard Method. The crude products proportion for reaction of PEG5000-A and PLA1000-B was determined by external standard method.28 Common procedure: after the reaction mixture was extracted with CH2Cl2 and solvent was removed. The remaining solid residue was suspended in ethyl ether and then sonicated and centrifuged, repeating this procedure for three times to give crude product. Purified products PEG5000-PEG5000 (a), PEG5000-PLA1000 (b), and PLA1000-PLA1000 (c) was used as external standard, the products was dissolved in THF at the concentration of Cra, Crb, and Crc respectively, and then determined at the same GPC condition (THF used as the mobile phase at a flow rate of 1.0 mL/min at 35 °C, equipped with a refractive index detector), the resulting peak areas were Ara, Arb, and Arc. The crude products for reaction of PEG5000-A and PLA1000-B was also analyzed at the same GPC condition, the crude product solution volume is V. Compared with the retention time of PEG5000-PEG5000 (a), PEG5000-PLA1000 (b), and PLA1000-PLA1000 (c), we can determine the part of component peaks of the crude product, the resulting peak areas were Aa, Ab, and Ac. The content of each component is calculated as follows:

ma = (Cra/Ara)A a V ;

mb = (Crb/Arb)A bV ;

mc = (Crc/Arc)AcV The proportions of each component P are calculated as follows:

Pa (%) = ma /(ma + mb + mc) × 100

Pb (%) = mb /(ma + mb + mc) × 100 Pc (%) = mc /(ma + mb + mc) × 100 (a). PEG5000-PEG5000. PEG5000-A (0.5 mM) was dissolved in CH2Cl2 in the presence of iodine (6 mM). Purified isolated yield: 93%. 1 H NMR (400 MHz, CD3OD) δ8.12(s, 2H, ArH), 7.68−7.64 (m, 4H, ArH), 3.77−3.39 (m, 805H, PEG, 2H per repeating unit, −OCH2CH2O−), 3.32 (s, 6H, PEG, CH3O−), 2.39−2.21 (m, 16H, −(CO)CH2−, −SCH2−), 1.83−1.68 (m, 8H, −CH2CH2CH2−). GPC retention time: 4.72 min. GPC: Mn = 10500, Mw = 11600, PDI = 1.10. MALDI−MS: Mn = 10500, Mw = 11200, PDI = 1.07. IR (KBr, cm−1) 3435, 2882, 1685, 1601, 1552, 1552, 1466, 1359, 1343, 1279, 1242, 1146, 1111, 1060, 946, 841, 746, 702, 622. (b). PLA1000-PLA1000. PLA1000-B (0.5 mM) was dissolved in CH2Cl2 in the presence of iodine (6 mM). Purified isolated yield: 96%. 1H NMR (400 MHz, CDCl3): δ 7.91−7.85 (m, 6H, ArH), 6.38 (brs, 2H, −NH−), 5.31−5.06 (m, 27H, 1H per repeating unit, PLA−CH−), 4.39−4.31 (m, 2H, PLA−CH−), 3.46−3.41 (m, 8H, −NCH2−), 2.97 (t, J = 6.4 Hz, 8H, −SCH2−),1.56−1.40 (m, 87H, 3H per repeating unit, PLA−CH3−). GPC retention time: 6.73 min. GPC: Mn = 2000, Mw = 2400, PDI = 1.20. MALDI−MS: Mn = 1900, Mw = 2200, PDI = 1.16. IR (KBr, cm−1) 2993, 2943, 1755, 1682, 1532, 1492, 1451, 1381, 1301, 1269, 1188, 1130, 1091, 1050, 955, 894, 865, 638. (c). PEG5000−PLA1000. Purified isolated yield: 82%. 1H NMR (400 MHz, (CD3OD/CDCl3, 50/50, v/v)): δ 8.25−7.31 (m, 6H, ArH), 5.42−4.96 (m, 13H, 1H per repeating unit, PLA−CH−), 4.34−4.26 (m, 1H, PLA−CH−), 3.86−3.41 (m, 436H, 2H per repeating unit, −OCH2CH2O−), 3.55−4.50 (m, 4H, −NCH2−), 3.35 (s, 3H, CH3O−), 2.98−2.87 (m, 4H, −SCH2−), 2.85−2.76 (m, 4H, −(C O)CH2−), 2.57−2.48 (m, 4H, −SCH2−), 2.13−2.03 (m, 4H, −CH2−), 1.61−1.31 (m, 42H, 3H per repeating unit, PLA−CH3−). GPC retention time: 5.51 min. GPC: Mn = 6300, Mw = 7300, PDI = 1.15. MALDI−MS: Mn = 5900, Mw = 6500, PDI = 1.10. IR (KBr, cm−1): 3455, 2888, 1752, 1650, 1543, 1461, 1380, 1353, 1278, 1238, 1189, 1113, 954, 843. PEG5000-PLA5000. Purified isolated yield: 41%. 1H NMR (400 MHz, (CD3OD/CDCl3, 50/50, v/v)): δ 9.12 (s, 1H, −NH−), 8.61−7.32 (m, 6H, ArH), 5.29−5.07 (m, 53H, 1H per repeating unit, PLA− CH−), 4.38−4.26 (m, 1H, PLA−CH−), 3.86−3.41 (m, 425H, 2H per repeating unit, −OCH2CH2O−), 3.56−3.51 (m, 4H, −NCH2−), 3.36 I

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CH3−). GPC retention time: 6.81 min. GPC: Mn = 1700, Mw = 2000, PDI = 1.18. MALDI−MS: Mn = 1200, Mw = 1300, PDI = 1.08. IR (KBr, cm−1) 2994, 2944, 1755, 1381, 1361, 1318, 1268, 1188, 1130, 1091, 1049, 955, 638. (c). PEG5000-S-S-PLA1000. 1:1 mixture of PEG5000-SCPh3 (0.5 mM) and PLA1000-SCPh3 (0.5 mM) were dissolved in CH2Cl2 in the presence of iodine (6 mM) to give three products, they are crosslinked copolymer and the other two self-linked products, PEG5000-S-SPEG5000: yield (Determined by GPC) 31%. PLA1000-S-S-PLA1000: yield (determined by GPC) 28%. PEG5000-S-S-PLA1000: yield (determined by GPC) 41%. 1H NMR (400 MHz, CDCl3): δ 5.27− 4.98 (m, 15H, 1H per repeating unit, PLA−CH−), 4.39−4.25 (m, 1H, PLA−CH−), 3.92−3.38 (m, 418H, PEG, 2H per repeating unit, −OCH2CH2O−), 3.35 (s, 3H, PEG, CH3O−), 2.74−2.64 (m, 2H, −NCH2−), 2.45−2.36 (m, 2H, −(CO)CH2−), 2.33−2.22 (m, 2H, −(CO)CH2−), 2.06−1.93 (m, 2H, −SCH2−), 1.74−1.66 (m, 2H, −CH2CH2CH2−), 1.64−1.33 (m, 48H, 3H per repeating unit, PLA− CH3−). GPC retention time: 5.62 min. GPC: Mn = 5600, Mw = 6300, PDI = 1.12. MALDI−MS: Mn = 5400, Mw = 5900, PDI = 1.09. IR (KBr, cm−1): 2872, 1758, 1675, 1381, 1353, 1300, 1269, 1187, 1097, 952, 637. 4.3. Preparation and Characterization of Micelles. Briefly, the diblock polymer (10 mg) was dissolved in 1 mL of DMSO and stirred at room temperature for 15 min. Then, the solution was slowly added to 8 mL of deionized water and stirred for another 1 h. Subsequently, the solution was dialyzed against deionized water for 24 h (MWCO = 2000 g·mol−1, and the deionized water was exchanged every 5 h for 2 days, and the dialysate through the 0.45 μm filter membrane to give micelles solution. The critical micelle concentration (CMC) was determined using diphenylhexatriene (DPH) as a UV probe by monitoring the absorbance at 313 nm. The concentration of block copolymer was varied from 0.5 × 10−4 to 0.5 mg mL−1 and the DPH concentration was fixed at 5 × 10−6 M. The absorbance spectra of all solutions were recorded using BioTek Synergy 2. 4.4. Stability of PEG5000-PLA5000 Micelles. The size change of micelles in response to 10 mM of DTT in phosphate-buffered saline (PBS) (50 mM, pH 7.4) was determined by DLS measurement. In brief, DTT was added to 10 mL of solution of PEG5000-PLA5000 micelles (1 mg.mL−1) in PBS (50 mM, pH 7.4) to achieve a final concentration at 10 mM, which was degassed in advance with nitrogen for 15 min. Then, the solution was mildly stirred at 37 °C. At different time intervals, the size was measured by DLS.

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REFERENCES

(1) For recent reviews, see: (a) Allen, T. M.; Cullis, P. R. Science 2004, 303, 1818. (b) Alarcón, C. H.; Pennadam, S.; Alexander, C. Chem. Soc. Rev. 2005, 34, 276. (c) Stuart, M. A. C.; Huck, W. T.; Genzer, J.; Müller, M.; Ober, C.; Stamm, M.; Sukhorukov, G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M. Nat. Mater. 2010, 9, 101. (d) Wojtecki, R. J.; Meador, M. A.; Rowan, S. J. Nat. Mater. 2011, 10, 14. (e) Cheng, Z.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Science 2012, 338, 903. (f) Mura, S.; Nicolas, J.; Couvreur, P. Nat. Mater. 2013, 12, 991. (2) For specific examples, see: (a) Bellomo, E. G.; Wyrsta, M. D.; Pakstis, L.; Pochan, D. J.; Deming, T. J. Nat. Mater. 2004, 3, 244. (b) Capadona, J. R.; Shanmuganathan, K.; Tyler, D. J.; Rowan, S. J.; Weder, C. Science 2008, 319, 1370. (c) Wang, C.; Chen, Q.; Wang, Z.; Zhang, X. Angew. Chem., Int. Ed. 2010, 122, 8794. (3) (a) Koo, A. N.; Lee, H. J.; Kim, S. E.; Chang, J. H.; Park, C.; Kim, C.; Park, J. H.; Lee, S. C. Chem. Commun. 2008, 6570. (b) Yan, Q.; Yuan, J.; Cai, Z.; Xin, Y.; Kang, Y.; Yin, Y. J. Am. Chem. Soc. 2010, 132, 9268. (c) Tang, L.-Y.; Wang, Y.-C.; Li, Y.; Du, J.-Z.; Wang, J. Bioconjugate Chem. 2009, 20, 1095. (d) Nakahata, M.; Takashima, Y.; Yamaguchi, H.; Harada, A. Nat. Commun. 2011, 2, 511. (e) Wang, F.; Klaikherd, A.; Thayumanavan, S. J. Am. Chem. Soc. 2011, 133, 13496. (4) (a) Zhou, K.; Wang, Y.; Huang, X.; Luby-Phelps, K.; Sumer, B. D.; Gao, J. Angew. Chem., Int. Ed. 2011, 50, 6109. (b) Jin, Y.; Song, L.; Su, Y.; Zhu, L.; Pang, Y.; Qiu, F.; Tong, G.; Yan, D.; Zhu, B.; Zhu, X. Biomacromolecules 2011, 12, 3460. (c) Chen, W.; Cheng, Y.; Wang, B. Angew. Chem., Int. Ed. 2012, 51, 5293. (5) (a) Saito, G.; Swanson, J. A.; Lee, K. D. Adv. Drug. Delivery. Rev. 2003, 55, 199. (b) Balendiran, G. K.; Dabur, R.; Fraser, D. Cell Biochem. Funct. 2004, 22, 343. (c) Son, S.; Namgung, R.; Kim, J.; Singha, K.; Kim, W. J. Acc. Chem. Res. 2011, 45, 1100. (6) (a) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Nature 1997, 388, 860. (b) Bhattarai, N.; Gunn, J.; Zhang, M. Adv. Drug. Delivery. Rev. 2010, 62, 83. (c) Danhier, F.; Ansorena, E.; Silva, J. M.; Coco, R.; Le Breton, A.; Préat, V. J. Controlled Release 2012, 161, 505. (7) (a) Hartgerink, J. D. Curr. Opin. Chem. Biol. 2004, 8, 604. (b) Leclaire, J.; Vial, L.; Otto, S.; Sanders, J. K. Chem. Commun. 2005, 1959. (c) Khakshoor, O.; Nowick, J. S. Org. Lett. 2009, 11, 3000. (d) Seo, J.; Barron, A. E.; Zuckermann, R. N. Org. Lett. 2010, 12, 492. (e) Lista, M.; Areephong, J.; Sakai, N.; Matile, S. J. Am. Chem. Soc. 2011, 133, 15228. (f) Almeida, A. M.; Li, R.; Gellman, S. H. J. Am. Chem. Soc. 2011, 134, 75. (g) Deng, G.; Li, F.; Yu, H.; Liu, F.; Liu, C.; Sun, W.; Jiang, H.; Chen, Y. ACS Macro Lett. 2012, 1, 275. (f) Miller, S. E.; Watkins, A. M.; Kallenbach, N. R.; Arora, P. S. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 6636. (8) (a) Yuan, C.; Raghupathi, K.; Popere, B. C.; Ventura, J.; Dai, L.; Thayumanavan, S. Chem. Sci. 2014, 5, 229. (b) Thambi, T.; Yoon, H. Y.; Kim, K.; Kwon, I. C.; Yoo, C. K.; Park, J. H. Bioconjugate Chem. 2011, 22, 1924. (9) (a) Cerritelli, S.; Velluto, D.; Hubbell, J. A. Biomacromolecules 2007, 8, 1966. (b) Ghosh, S.; Basu, S.; Thayumanavan, S. Macromolecules 2006, 39, 5595. (c) Klaikherd, A.; Ghosh, S.; Thayumanavan, S. Macromolecules 2007, 40, 8518. (d) Klaikherd, A.; Nagamani, C.; Thayumanavan, S. J. Am. Chem. Soc. 2009, 131, 4830. (e) Ge, Z.; Liu, S. Chem. Soc. Rev. 2013, 42, 7289. (f) Huo, M.; Yuan, J.; Tao, L.; Wei, Y. Polym. Chem. 2014, 5, 1519. (10) (a) Gong, B. Polym. Int. 2007, 56, 436. (b) Gong, B. Acc. Chem. Res. 2012, 45, 2077. (11) (a) Gong, B.; Yan, Y. F.; Zeng, H. Q.; Skrzypczak-Jankunn, E.; Kim, Y. W.; Zhu, J.; Ickes, H. J. Am. Chem. Soc. 1999, 121, 5607. (b) Zeng, H. Q.; Miller, R. S.; Flowers, R. A.; Gong, B. J. Am. Chem. Soc. 2000, 122, 2635. (c) Zeng, H. Q.; Ickes, H.; Flowers, R. A.; Gong, B. J. Org. Chem. 2001, 66, 3574. (12) Zeng, H. Q.; Yang, X. W.; Flowers, R. A.; Gong, B. J. Am. Chem. Soc. 2002, 124, 2903. (13) Yang, X. W.; Hua, F. J.; Yamato, K.; Ruckenstein, E.; Gong, B.; Kim, W.; Ryu, C. Y. Angew. Chem., Int. Ed. 2004, 43, 6471.

ASSOCIATED CONTENT

S Supporting Information *

Figures showing NMR spectra, MALDI−MS spectra, GPC traces, DLS traces, relationship of the absorbance intensity of DPH, and TEM images. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(B.G.) E-mail: bgong@buffalo.edu. *((Y.-M.S.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by Grants 81071250, 91129000, and 91227109 from NSFC, Grant 2011ZX09501001-05 from Major Projects in National Science and Technology, “Creation of Major New Drugs”, Grant CHE1306326 from the US NSF, and Grant 51048-ND7 from the donors of the Petroleum Research Fund, administered by the American Chemical Society. J

dx.doi.org/10.1021/ma5017083 | Macromolecules XXXX, XXX, XXX−XXX

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(14) (a) Brunsveld, L.; Folmer, B.; Meijer, E.; Sijbesma, R. Chem. Rev. 2001, 101, 4071. (b) Wilson, A. J. Soft Matter 2007, 3, 409. (c) Yang, S. K.; Zimmerman, S. C. Isr. J. Chem. 2013, 53, 511. (15) (a) Sijbesma, R. P.; Beijer, F. H.; Brunsveld, L.; Folmer, B. J.; Hirschberg, J. K.; Lange, R. F.; Lowe, J. K.; Meijer, E. Science 1997, 278, 1601. (b) Todd, E. M.; Zimmerman, S. C. J. Am. Chem. Soc. 2007, 129, 14534. (c) Yang, S. K.; Ambade, A. V.; Weck, M. J. Am. Chem. Soc. 2010, 132, 1637. (d) Yan, X.; Li, S.; Pollock, J. B.; Cook, T. R.; Chen, J.; Zhang, Y.; Ji, X.; Yu, Y.; Huang, F.; Stang, P. J. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 15585. (e) Gooch, A.; Murphy, N. S.; Thomson, N. H.; Wilson, A. J. Macromolecules 2013, 46, 9634. (16) Yang, X. W.; Gong, B. Angew. Chem., Int. Ed. 2005, 44, 1352. (17) Montalvo, G. L.; Zhang, Y.; Young, T. M.; Costanzo, M. J.; Freeman, K. B.; Wang, J.; Clements, D. J.; Magavern, E.; Kavash, R. W.; Scott, R. W. ACS Chem. Biol. 2014, 9, 967. (18) (a) Murray, T. J.; Zimmerman, S. C. J. Am. Chem. Soc. 1992, 114, 4010. (b) Yang, J.; Fan, E. K.; Geib, S. J.; Hamilton, A. D. J. Am. Chem. Soc. 1993, 115, 5314. (c) Sessler, J. L.; Wang, R. Z. J. Am. Chem. Soc. 1996, 118, 9808. (d) Vreekamp, R. H.; van Duynhoven, J. P. M.; Hubert, M.; Verboom, W.; Reinhoudt, D. N. Angew. Chem., Int. Ed. 1996, 35, 1215. (e) Beijer, F. H.; Kooijman, H.; Spek, A. L.; Sijbesma, R. P.; Meijer, E. W. Angew. Chem., Int. Ed. 1998, 37, 75. (f) Wu, A. X.; Chakraborty, A.; Fettinger, J. C.; Flowers, R. A.; Isaacs, L. Angew. Chem., Int. Ed. 2002, 41, 4028. (g) Fenniri, H.; Deng, B. L.; Ribbe, A. E. J. Am. Chem. Soc. 2002, 124, 11064. (h) Zhan, C. L.; Leger, J. M.; Huc, I. Angew. Chem., Int. Ed. 2006, 45, 4625. (i) Chu, W. J.; Yang, Y.; Chen, C. F. Org. Lett. 2010, 12, 3156. (j) Gooch, A.; McGhee, A. M.; Pellizzaro, M. L.; Lindsay, C. I.; Wilson, A. J. Org. Lett. 2011, 13, 240. (k) Mudraboyina, B. P.; Wisner, J. A. Chem.Eur. J. 2012, 18, 14157. (19) Examples of stabilizing H-bonding interaction in polar media: (a) Nowick, J. S.; Chen, J. S. J. Am. Chem. Soc. 1992, 114, 1107. (b) Kato, Y.; Conn, M. M.; Rebek, J. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1208. (c) Torneiro, M.; Still, W. C. J. Am. Chem. Soc. 1995, 117, 5887. (d) Brunsveld, L.; Vekemans, J.; Hirschberg, J.; Sijbesma, R. P.; Meijer, E. W. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4977. (20) (a) Li, M. F.; Yamato, K.; Ferguson, J. S.; Gong, B. J. Am. Chem. Soc. 2006, 128, 12628. (b) Li, M. F.; Yamato, K.; Joseph, S.; Ferguson; Singarapu, K. K.; Szyperski, T.; Gong, B. J. Am. Chem. Soc. 2008, 130, 491. (21) Kamber, B.; Hartmann, A.; Eisler, K.; Riniker, B.; Rink, H.; Sieber, P.; Rittel, W. Helv. Chim. Acta 1980, 63, 899. (22) Tuzar, Z.; Kratochvil, P. Micelles of block and graft copolymers in solution. In Surface and Colloid Science; Matijevic, E., Ed.; Plenum Press: New York; 1993; Vol. 15, pp 1−83, Chapter 1. (23) Alexandridis, P.; Holzwarth, J. F.; Hatton, T. A. Macromolecules 1994, 27, 2414. (24) (a) Lukyanov, A. N.; Torchilin, V. P. Adv. Drug. Delivery. Rev. 2004, 56, 1273. (b) Adams, M. L.; Lavasanifar, A.; Kwon, G. S. J. Pharm. Sci. 2003, 92, 1343. (25) Cleland, W. W. Biochemistry 1964, 3, 480. (26) Yoshiizumi, K.; Nakajima, F.; Dobashi, R.; Nishimura, N.; Ikeda, S. Bioorg. Med. Chem. Lett. 2004, 14, 2791. (27) Guo, W. Z.; Li, J. J.; Wang, Y. A.; Peng, X. G. J. Am. Chem. Soc. 2003, 125, 3901. (28) (a) Zhang, C.; Zhang, H.; Yang, J.; Liu, Z. Polymer Degrad. Stab. 2004, 86, 461−466. (b) Cazes, J. Encyclopedia of Chromatography (Print); CRC Press: Boca Raton, FL, 2001.

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