PEG Analogs Synthesized by Ring-Opening Metathesis

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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

PEG Analogs Synthesized by Ring-Opening Metathesis Polymerization for Reversible Bioconjugation Emma M. Pelegri-O’Day,†,# Nicholas M. Matsumoto,†,# Kyle Tamshen,† Eric D. Raftery,† Uland Y. Lau,‡ and Heather D. Maynard*,†,‡ †

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Department of Chemistry and Biochemistry and California Nanosystems Institute, University of California, Los Angeles, 607 Charles E. Young Drive East, Los Angeles, California 90095-1569, United States ‡ Department of Bioengineering, University of California, Los Angeles, 410 Westwood Plaza, Los Angeles, California 90095, United States S Supporting Information *

ABSTRACT: Poly(ethylene glycols) (PEGs) with protein-reactive end-groups are widely utilized in bioconjugation reactions. Herein, we describe the use of ring-opening metathesis polymerization (ROMP) to synthesize unsaturated protein-reactive PEG analogs. These ROMP PEGs (rPEGs) contained terminal aldehyde functionality and ranged in molecular weight from 6 to 20 kDa. The polymers were readily conjugated to free amines on the protein hen egg-white lysozyme (Lyz). Biocompatibility of the unsaturated PEGs was assessed in vitro, revealing the polymers to be nontoxic up to concentrations of at least 1 mg/mL in human dermal fibroblasts (HDFs). The resulting unsaturated rPEG-lysozyme conjugates underwent metathesis-based depolymerization, resulting in decreased molecular weight of the conjugate.

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opening polymerization of oxo-crown ethers.6−8 Copolymerization of short PEGs with lactides has been a popular route to obtaining degradable PEG analogs.10−13 For example, Hubbell and co-workers demonstrated the formation of degradable hydrogels from acrylate-terminated copolymers of oligo(ethylene glycol) and α-hydroxy acids.12 Hawker and coworkers prepared hydrolytically degradable PEG analogs by synthesizing a PEG backbone with vinyl ether moieties via chloride elimination in poly(ethylene oxide-co-epichlorohydrin).14 Furthermore, reductively degradable PEG analogs have been prepared by introducing disulfide bridges between oligo(ethylene glycol) units.15,16 In this report, we disclose the use of end-functionalized PEG-like polymers prepared by

INTRODUCTION Covalent conjugation of PEG to proteins, or protein PEGylation, is known to increase stability, solubility, and in vivo circulation lifetimes of proteins.1−3 This strategy has been employed in the development of PEGylated protein therapeutics, which display superior pharmacokinetic properties compared to unmodified proteins.4 PEGylation is well established and remains a mainstay in the field of protein therapeutics4,5 with 14 PEGylated protein therapeutics currently approved for human use in the United States. They are employed to treat a variety of diseases including cancer, chronic gout, and hepatitis. Due to the utility of PEG, there is significant interest in preparing functional or structurally different analogs of the polymer. For instance, PEG-like polymers that contain degradable moieties and hydrolyzable backbone functionalities have been introduced.6−9 In several early examples, Meijer and co-workers incorporated esters into linear PEG analogs via ring © XXXX American Chemical Society

Received: September 7, 2018 Revised: October 10, 2018

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DOI: 10.1021/acs.bioconjchem.8b00635 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Scheme 1. ROMP of Unsaturated Crown Ether 1, to Produce rPEGs of Variable Molecular Weight

Table 1. Characterization Data (conv, Mn, and Đ) for rPEGs Synthesized at Different [Monomer]/[Catalyst] Ratiosa [M]/[C]

Conv.

Predicted MW

Mn (NMR)

10/1 25/1 50/1 75/1 100/1

91% 99% 95% 91% 91%

1.8 kDa 5.0 kDa 9.6 kDa 13.8 kDa 18.4 kDa


% aldehyde (% methylene) 58 57 53 46 53

a

GPC data were obtained using MALS GPC.

(67) (76) (82) (65) (65)

Mn (GPC)

Đ (GPC)


1.13 1.28 1.54 1.40 1.56

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RESULTS AND DISCUSSION Aldehyde end-functionalized PEGs are commonly used for bioconjugation to surface exposed amines on proteins via mild and efficient reductive amination reactions.25−27 rPEGs of variable molecular weight were prepared by ROMP of the unsaturated crown ether monomer 1 (Scheme 1).23,24 Aldehyde ω-end-group functionality was installed by terminating the ROMP with vinylene carbonate as previously reported by Kilbinger and co-workers for norbornenes.28 Polymerizations were carried out in a glovebox under nitrogen atmosphere at 20−23 °C with ratios of [Grubbs I]: [monomer 1] equal to [1]:[10−100]. Polymerizations were monitored by 1H NMR and after the desired conversion was achieved, terminated by the addition of excess vinylene carbonate. Any residual active ruthenium catalyst was subsequently quenched with ethyl vinyl ether. The crude polymers were then purified by repeated precipitation into cold diethyl ether to yield thick tan oils, indicating the presence of residual ruthenium. Ruthenium could be completely removed after treatment with tris(hydroxymethyl)phosphine29 and aqueous workup, resulting in colorless polymer samples (reduction from 1327.6 ± 37.1 to 136.5 ± 7.0 ng Ru/mg polymer). However, the removal treatment resulted in significant loss of polymer due to partial solubility of rPEG in water and no negative effects due to the residual ruthenium were observed. Therefore, trace ruthenium was not removed in subsequent experiments, including cytotoxicity studies. Polymers were then analyzed by both 1H NMR and gel permeation chromatography (GPC). The number-average molecular weight (Mn) was determined by 1H NMR end-group analysis by comparing the integration of the terminal phenyl protons (7.35−7.40 ppm) to the backbone alkenes (5.90−5.65 ppm). GPC characterization was carried out using either a multiangle light scattering (MALS) detector (MALS GPC) or using PEG standards and refractive index detector (RI GPC) in order to determine Mn and molecular weight dispersity (Đ). A total of five rPEGs were prepared in the typical molecular weight range used for therapeutic PEGs, between 6 and 20 kDa as measured by GPC (Table 1). Dispersities for the polymers were relatively high (1.13 ≤ Đ ≤ 1.56), which was expected for the entropy-driven ROMP of monomer 1 in contrast to typical

ROMP that undergo metathesis depolymerization, resulting in degraded polymer−protein conjugates. ROMP is a functional group tolerant and well-established method for the development of functional and bioactive polymers.17,18 To date, there are only several examples of ROMP polymers for protein−polymer conjugation. Kane and co-workers have reported the preparation of ROMP polymers containing pendant 2-chloromethylester functionality; displacement of the chloride by cysteine residues on bovine serum albumin resulted in protein multimers.19 In another example, Sleiman and co-workers prepared block copolymers with biotin end groups that were capable of binding streptavidin by terminating the ROMP reaction with vinyl ether-functionalized biotin.20 Recently, Pokorski and coworkers reported ROMP-based graf ting from and graf ting to methods for protein−polymer conjugation.21,22 In their graf ting f rom approach, lysozyme (Lyz) modified with norbornene functionality was subjected to a Grubbs-type ruthenium metathesis catalyst and subsequently used as a macro-catalyst for the polymerization of a PEG-modified norbornene.21 In their graf ting to approach, a water-soluble PEG-functionalized norbornene polymer was end-capped with a short block of amine-reactive norbornene dicarboxylic anhydride.22 The resulting diblock copolymer could be conjugated to free amines present on Lyz, as well as the coat protein of bacteriophage Qβ.22 We previously reported the ROMP of unsaturated crown ethers to produce water-soluble unsaturated PEG analogs using Grubbs’ first generation catalyst (Grubbs I).23 Pendant amino acids could also be incorporated into these analogs by copolymerization with unsaturated benzocrown ethers containing amino acids.24 However, these polymers did not have functional end groups for attachment to proteins. In this report, we describe the ROMP synthesis of PEG analogs containing ω-aldehyde end-group functionality, for straightforward conjugation to surface exposed amines via reductive amination. Formation of rPEG-protein conjugates and subsequent depolymerization of the polymer is described. B


Article

Bioconjugate Chemistry

Figure 1. Conjugation of rPEG to lysozyme by reductive amination. A. Reaction scheme of reductive amination conjugation. B. SDS-PAGE of Lyz and Lyz-rPEG showing the appearance of a high molecular weight smear after treatment with rPEG and NaCNBH3 (lane 1: protein marker; lane 2: Lyz; lane 3: Lyz-rPEG conjugate; lane 4: rPEG). C. FPLC chromatogram of Lyz and Lyz-rPEG conjugate using a cation exchange column showing the decreased retention time of the conjugate as well as good separation between polymer and conjugate peaks. Structure of lysozyme from PDB number 1DPX.

strain-promoted polymerizations.30 Larger molecular weight polymers exhibited higher dispersities, potentially due to increased chain transfer during longer polymerization times. Furthermore, the presence of the aldehyde (9.8 ppm) could be confirmed by 1H NMR and ranged between 50% and 60% end-group retention. It should be noted that the aldehyde signals are likely lower than expected. This may be due to hydration of the aldehyde resulting in loss of signal at 9.8 ppm or a side reaction during the polymerization reaction. Therefore, end-group methylene units (2.5 ppm) resulting from the vinylene carbonate installation (proton marked with * in Scheme 1) were also compared as evidence of the aldehyde’s installation, and this provided 65−82% end-group retention. An ω-aldehyde rPEG (Mn = 9.7 kDa, calculated using RI GPC with PEG standards) was conjugated to Lyz via reductive amination (Figure 1A). Lyz and rPEG were incubated in a pH 6.0, 100 mM phosphate buffer (PB) at 22 °C using a ratio [Lyz]:[rPEG] = 1:50. A large excess of polymer was used to ensure complete conjugation to all surface exposed amines on Lyz. After 1 h, sodium cyanoborohydride was added to a final concentration of 20 mM in the reaction solution. The conjugation mixture was allowed to incubate for 26 h at 22 °C and the resulting Lyz-rPEG conjugate was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 1B). Conjugation is evident by a high molecular weight smeared band (Figure 1B, lane 3), which corresponds to a higher molecular weight than unmodified Lyz (Figure 1B, lane 2). The gel was additionally stained using 0.1 N iodine, confirming the presence of polymer in the high molecular weight smear (Figure S1). The conjugate was also

purified and analyzed using a fast protein liquid chromatography (FPLC) system equipped with a cation exchange column (Figure 1C). The uncharged polymers were easily separated from protein-containing materials using a salt gradient. Additionally, PEGylation decreased the observed charge on the conjugate, resulting in decreased conjugate retention time (∼14 min) compared to unmodified Lyz (∼27 min). Based on the SDS-PAGE and FPLC results, Lyz is likely a heterogeneous mixture with a high degree of conjugation to the 7 amines present on the protein surface.31 The unsaturated rPEGs were previously reported to undergo depolymerization via a lithium-templated backbiting metathesis to reform the unsaturated 12-crown-4 monomer in high yield in dichloromethane/tetrahydrofuran.23 Reversible protein−polymer conjugates can be prepared using main-chain degradable polymers to facilitate efficient renal clearance of the polymer after hydrolytic, reductive, or enzymatic degradation, or by using cleavable conjugation linkers.32−38 However, there is far less investigation into the development of polymers for protein conjugation that are cleavable by nonbioavailable chemoselective triggers, such as organometallic reagents. While not feasible for therapeutics in the human body, this strategy may have applications in biotechnology such as biosensing and point-of-care diagnostics. Furthermore, since protein−polymer conjugates are difficult to characterize with regard to number of chains and attachment sites, depolymerization of rPEG to small fragments could be useful for this purpose. We initially explored the depolymerization/degradation of rPEG alone (Mn = 24.1 kDa, calculated using RI GPC with PEG standards) in aqueous conditions. Typical depolymerizaC


Article


Figure 2. Depolymerization/degradation of rPEG in aqueous conditions. A. Reaction scheme of aqueous depolymerization of polymer alone with structure of Grubbs III. B. Reaction scheme of polymer depolymerization/degradation of Lyz-rPEG conjugate. C. SDS-PAGE of Lyz and Lyz-rPEG conjugate showing decrease in molecular weight of the conjugate smear after treatment of conjugate with Grubbs III (lane 1: protein marker; lane 2: Lyz; lane 3: Lyz-rPEG conjugate; lane 4: depolymerized Lyz-rPEG conjugate; lane 5: rPEG). Structure of lysozyme from PDB number 1DPX.

cross-metathesis on the polymer backbone, which effectively cleaves the rPEG into short oligomers. Depolymerization/degradation of the Lyz conjugates with Grubbs III was investigated (Figure 2B). The Lyz-rPEG conjugate was incubated with 5 mM Grubbs III in buffer solution containing 100 mM MgCl2 and 20% t-BuOH. The solution was allowed to incubate at 23 °C for 17 h, after which the sample could be purified by initial crude pelleting of the catalyst via centrifugation, followed by centrifugal filtration and desalting using a Sephadex resin to remove any residual small molecules. The depolymerized/degraded conjugate was then analyzed by SDS-PAGE (Figure 2C). Successful depolymerization was evident by the decrease in the molecular weight of the conjugate (Figure 2C, lane 4) compared to before treatment (Figure 2C, lane 3), as visualized by SDS-PAGE using Coomassie staining for protein bands. Staining the gel using a 0.1 N iodine solution enabled visualization of the polymer and additionally confirmed depolymerization, with a decrease in molecular weight compared to both the conjugate and the polymer alone (Figure S3). PEG alternatives for applications in protein−polymer drug development must be nontoxic. rPEG cytotoxicity was evaluated through a LIVE/DEAD viability/cytotoxicity assay using human dermal fibroblasts (HDFs) as a primary noncancerous cell line. We found that the polymer (Mn = 20.4 kDa, RI GPC with PEG standards) was noncytotoxic at concentrations of 0.1, 0.5, and 1 mg/mL (Figure 3A).

tions/degradations were carried out in modified conditions reported by Davis and co-workers for cross-metathesis on proteins.39 rPEG (3 mg/mL) was dissolved in a phosphate buffer containing 20% tert-butyl alcohol (t-BuOH), 100 mM MgCl2, and 5 mM of Grubbs’ third generation catalyst (Grubbs III) (Figure 2A). Our previous report detailed the importance of lithium in the backbiting depolymerization process to reform cyclic monomers,23 and thus the aqueous depolymerization was also conducted in the presence of 100 mM LiCl. However, as expected, the interaction of lithium ion with the polymer in water is not strong enough to template the reaction as in the case of organic solvents and there was no difference in depolymerization efficiency or products the presence or absence of lithium salts. After incubating the solution for 20 h at 20 °C, the reaction was stopped via the addition of ethyl vinyl ether. The reaction solvent and volatile components were then removed under reduced pressure, followed by lyophilization to remove water. The crude reaction mixture was dissolved in dimethylformamide (DMF) and analyzed by GPC in DMF (Figure S2). Comparison of the GPC chromatograms of the polymer and depolymerized/ degraded products shows major degradation of the polymer when exposed to Grubbs III in aqueous solution containing tBuOH. However, unsaturated crown ether monomer 1 was never recovered from the reaction of rPEG with Grubbs III; we expect that the main mode of polymer degradation is random D



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Article

MATERIALS AND METHODS

Materials. All chemicals were purchased from SigmaAldrich, Fisher Scientific, and Acros and used as received unless otherwise noted. Lyz was obtained from Sigma-Aldrich and used as received. Dichloromethane (DCM) was distilled over CaH2 and stored under argon. Tetrahydrofuran (THF) was distilled over sodium/benzophenone and stored under argon. Unsaturated crown ether 1 was prepared as previously described,23 ruthenium catalyst was removed as described,29 and the monomer was distilled under reduced pressure before use. As a precaution, LiClO4 templated ring-closing metathesis was performed on small scale behind a blast shield. Polymerizations were carried out in a Vacuum Atmospheres Genesis stainless steel glovebox under nitrogen (Airgas, 99.998%) atmosphere. Analytical Techniques. NMR spectra were obtained on Bruker AV 500 and DRX 500 MHz spectrometers. Proton NMR spectra were acquired with a relaxation delay of 2 s for small molecules and 30 s for all polymers. GPC was conducted on one of two systems. System one is 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 with a column oven set at 40 °C and LiBr (0.1 M) in DMF as an eluent (flow rate: 0.60 mL/min). Calibration was performed using nearmonodisperse PEG standards from Polymer Laboratories. System two is a Multi-Angle Light Scattering GPC (MALSGPC) and was conducted on a Shimadzu HPLC Prominence-i system equipped with a UV detector, Wyatt DAWN Heleos-II Light Scattering detector, Wyatt Optilab T-rEX RI detector, one MZ-Gel SDplus guard column, and two MZ-Gel SDplus 100 Å 5 μm 300 × 8.0 mm columns with a column oven set at 40 °C and THF as eluent. The flow rate was set at 0.70 mL/ min as a compromise between backpressure and method time. A dn/dc value of 0.067 mL/g for PEG in THF was used (Polymer Source). SDS-PAGE was performed using Bio-Rad Any kD Mini-PROTEAN-TGX gels. SDS-PAGE protein standards were obtained from Bio-Rad (Precision Plus Protein Prestained Standards). For SDS-PAGE analysis, approximately 5 μg of protein was loaded into each lane. Conjugates were purified and analyzed by FPLC on a Bio-Rad BioLogic DuoFlow chromatography system equipped with a 1 mL GE Healthcare HiTrap Heparin HP column (used as a cation exchange column) employing a method of 0 to 1 M NaCl in 10 mM PB, pH 6.0 (Buffer A: 10 mM PB, pH 6.0; Buffer B: 10 mM PB, pH 6.0 + 1 M NaCl; 0.4 mL/min; 3 × 0.33 mL injections; 2 CV 0% Buffer B, then 8 CV 0−100% Buffer B, then 2 CV 100% Buffer B). For inductively coupled plasma mass spectrometry (ICP-MS) analysis, residual ruthenium in the polymer samples was measured after overnight nitric acid digestion using a PerkinElmer NexION 2000. Samples were measured in triplicate and compared to a blank nitric acid digestion. Methods. Representative ROMP of Cyclic Monomer 1. In a nitrogen-filled glovebox, Grubbs I (8 mg, 0.1 mmol) was dissolved in DCM (495 μL) and added to a dram vial containing monomer 1 (100 mg, 0.49 mmol) and a stir bar. The polymerization solution was stirred rapidly at room temperature for 4 h before addition of vinylene carbonate (75 μL) and stirring for an additional 60 min. The reaction vial was then removed from the box and any residual active catalyst was further quenched with ethyl vinyl ether (150 μL) and stirred

Figure 3. Cytotoxicity of the rPEG on HDF cells. A. Percent cell viability at 0.1, 0.5, and 1 mg/mL rPEG. B. LIVE/DEAD assay fluorescent images at varying concentrations of rPEG (0, 0.1, 0.5, and 1 mg/mL). All samples are statistically equivalent (ANOVA).

Additionally, HDFs exposed to rPEG proliferated normally in the presence of polymer (Figure 3B). The depolymerized products were also nontoxic up to 0.5 mg/mL, with slightly reduced (91%) cell viability at 1 mg/mL (Figure S4). The results show that aldehyde-functionalized rPEG is a possible alternative to PEG because it is noncytotoxic up to at least 1 mg/mL in HDFs and readily conjugates to proteins. However, unlike PEG it is readily degraded away from the protein, with only short fragments of the polymer still attached. This may provide an interesting strategy for removing PEGlike polymers from proteins that are used for nontherapeutic applications and may be particularly useful for characterization of therapeutic protein−polymer conjugates. Furthermore, the flexibility of the ROMP based polymerization method may allow for copolymerization with other monomers and modification of both ends of the polymer, for example, by use of functionalized catalysts.18 This would in turn allow for greater functionalization of PEGylated protein−polymer conjugates.

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CONCLUSIONS A series of unsaturated rPEGs with protein-reactive aldehyde ω-end-group functionality have been prepared. Subsequent conjugation via reductive amination to the model protein lysozyme was demonstrated. The polymer could be removed by ruthenium-catalyzed depolymerization of the rPEGs in aqueous solution. The polymers were shown to be noncytotoxic to HDFs in high concentrations, up to 1 mg/mL and are therefore interesting alternatives to PEG for protein conjugation. E


Article


incubated at 37 °C and 5% CO2. After 24 h, the culture media was replaced with 200 μL of the working medium containing known compounds at concentrations of 0.1, 0.5, and 1 mg/mL. After incubation for 24 h, the cells were gently washed with prewarmed Dulbecco’s phosphate buffered saline (D-PBS), and stained with the LIVE/DEAD reagent (2 μM calcein AM and 4 μM ethidium homodimer-1). Fluorescent images of each well were captured on an Axiovert 200 microscope with an AxioCam MRm camera and FluoArc mercury lamp. The number of live and dead cells were counted, and percent cell viability was calculated by dividing the number of live cells by the total number of cells. Percent viability was determined using the formula 100 × (number of live cells/total number of cells). All experiments were conducted in quadruplicate. Each experimental set was normalized to a control of blank medium.

for an additional 75 min. The polymer was then precipitated 2× into cold ether (20 mL) and dried in vacuo to yield a tan oil (76 mg, 76% yield). Mn (1H NMR) = 11.6 kDa, Mn (GPC) = 11.5 kDa, MW (GPC) = 17.8 kDa, Đ= 1.54. 1H NMR (500 MHz in CD3CN) δ: 9.67 (t, J = 2 Hz, 1H), 7.42 (d, J = 7.5 Hz, 2H), 7.32 (t, J = 7.5 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 6.61 (d, J = 16 Hz, 1H), 6.32 (dt, J = 16, 5.8 Hz, 1H), 5.89−5.55 (m, 112H), 4.14−3.89 (m, 226H), 3.71−3.42 (m, 690H), 2.58 (dt, J = 1.8, 5.9 Hz, 2H). Representative Conjugation of rPEG to Lyz. Lyz (1.0 mg, 0.07 μmol) was dissolved in pH 6.0 100 mM PB (0.50 mL) in a 1.5 mL LoBind tube. rPEG (Mn = 9.7 kDa, 35.0 mg, 3.50 μmol) was dissolved in pH 6.0, 100 mM PB (0.40 mL) in a separate 1.5 mL tube. The polymer solution was then added to the Lyz solution and placed on a laboratory rocker for 1 h at 23 °C. A 200 mM stock solution of sodium cyanoborohydride was prepared in pH 6.0, 100 mM PB, and 0.10 mL of the sodium cyanoborohydride solution was added to the solution containing Lyz and the polymer for a total volume of 1 mL. The reaction was gently rocked for 26 h at 23 °C. The crude reaction was then filtered through a 0.22 μm syringe filter and purified by FPLC to obtain the conjugate. Aqueous Depolymerization of rPEG. rPEG (Mn = 24.1 kDa calculated using RI GPC with PEG standards, 3.0 mg, 0.12 μmol) was weighed into a 2 mL vial and dissolved in pH 8.0 50 mM PB containing 100 mM MgCl2 (0.80 mL) with or without 100 mM LiCl. A 50 mM saturated stock solution of Grubbs III was prepared in tert-butyl alcohol and 0.20 mL of the catalyst solution was added to the polymer solution. The total concentration of catalyst in the reaction was 5 mM. A magnetic stir bar was added and the solution was sealed under argon. The reaction was stirred for 20 h at 20 °C. After 20 h, the reaction was stopped with the addition of ethyl vinyl ether (0.10 mL) and stirred for 30 min. The ethyl vinyl ether was removed by rotary evaporation, and the crude reaction was lyophilized to remove water. After the water was removed, the remaining solids and oils were dissolved in DMF and analyzed by GPC. A Typical Depolymerization of Lyz-rPEG Conjugate. 100 μL of 200 mM MgCl2 was added to a 1.5 mL LoBind tube containing 100 μL of Lyz-rPEG conjugate (0.51 μg/μL; 51 μg). Next, a saturated solution of Grubbs III catalyst was prepared in t-BuOH (1.1 mg in 50 μL t-BuOH; 25 mM stock) and the entire catalyst suspension was added to the tube, bringing the concentration of catalyst to approximately 5 mM and the concentration of t-BuOH to 20% for a total reaction volume of 250 μL. The reaction mixture was then incubated for 17 h at 23 °C. After 17 h, any precipitate in the solution was pelleted by centrifugation and discarded. The remaining crude mixture was then analyzed by SDS-PAGE. The gel was first incubated in 0.1 N iodine solution (EMD Millipore) for 20 min until polymer could be visualized, then imaged and washed in water for 60 min or until stain was completely removed. The gel was then treated with Coomassie stain to visualize protein. Polymer Cytotoxicity Studies. Cytotoxicity of rPEG (Mn = 20.4 kDa calculated using RI GPC with PEG standards) and rPEG depolymerization products was evaluated using a LIVE/ DEAD viability/cytotoxicity assay (Invitrogen). HDF cells were cultured in fibroblast growth medium containing 2% fetal calf serum, 1 ng/mL basic fibroblast growth factor, 5 μg/mL insulin, and 1% penicillin-streptomycin. The cells were seeded in 48-well plates at a density of 6 × 103 cells per well and

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00635.

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Polymer 1H NMR spectra, GPC chromatograms, and supplementary SDS-PAGE and cytotoxicity studies (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Heather D. Maynard: 0000-0003-3692-6289 Author Contributions #

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.D.M. thanks the National Science Foundation (CHE1507735) for funding. E.M.P.-O. thanks the NSF for a Graduate Research Fellowship (DGE-1144087), UCLA for a Dissertation Year Fellowship and the Christopher S. Foote Graduate Research Fellowship in Organic Chemistry. N.M.M. thanks the NSF for a Graduate Research Fellowship (DGE0707424) and UCLA for a Dissertation Year Fellowship. U.Y.L. thanks the NIH Chemistry Biology Interface Training Fellowship (T32 GM 008496) and UCLA Graduate Division for funding. The authors thank Dr. Paul Chang in the California NanoSystems Institute at UCLA for carrying out the ICP-MS studies. H.D.M. thanks Professor Robert H. Grubbs for mentorship and support while she carried out initial cytotoxicity studies on the rPEGs at the California Institute of Technology.

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REFERENCES

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PEG Analogs Synthesized by Ring-Opening Metathesis

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