Efficient Catalytic Synthesis of Dendritic Polymers Having Internal

Biomacromolecules 2008, 9, 1745–1754

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Efficient Catalytic Synthesis of Dendritic Polymers Having Internal Fluorescence Labels for Bioconjugation Guanghui Chen,† Philip L. Felgner,‡ and Zhibin Guan*,† Department of Chemistry, 1102 Natural Sciences II, and Department of Medicine, Division of Infectious Diseases, 3052 Hewitt Hall, University of California, Irvine, California 92697-2025 Received December 6, 2007; Revised Manuscript Received February 2, 2008

Here we present an efficient synthesis of functional dendritic polymers carrying internal fluorescence labels for bioconjugation. Specifically, dendritic polymers having pyrene as fluorescence label in the core and Nhydroxysuccinimide (NHS) functional groups at the periphery were synthesized by coupling heterobifunctional PEG to hydroxyl functionalized dendritic polyethylene core. The dendritic polyethylene cores containing one pyrene label per polymer molecule were prepared through a one-step transition-metal-catalyzed polymerization using a pyrene-labeled Pd(II)-R-diimine chain walking catalyst. A series of pyrene-labeled dendritic scaffolds were obtained with different molecular weights and sizes. NHS active end groups were introduced to the periphery of the dendritic scaffolds through end-group functionalization. Those NHS-functionalized dendritic scaffolds were successfully used to conjugate a model protein, ovalbumin, to yield protein-polymer conjugates carrying multiple copies of protein attached to each scaffold.

Introduction One distinctive advantage of dendritic polymers is their capability of displaying desired motifs in a multivalent fashion1–9 and, therefore, providing synergistic enhancement of functions of interest.10–14 In bioconjugation, functional dendritic polymers are of great interest because of their multivalency and comparable size to biomolecules. Due to their biocompatibility and inertness in physiological environment, poly(ethyleneglycol) (PEG)-based polymers are commonly used to functionalize dendritic polymers for biomedical applications.15–22 Unimolecular type PEG functionalized dendritic polymers are of particular interest due to their enhanced stability over the dynamic aggregates of block copolymers and their increased functionalities over star polymers.23–27 Based on the Pd(II)-Rdiimine catalyst initially reported by Brookhart and co-workers,28 our group has developed efficient preparations of dendritic polymers through chain walking polymerizations (CWPs).29–34 Dendritic polymers with hundreds of NHS-activated ester end groups can be prepared with controllable molecular weight and size from CWP, followed by atom transfer radical polymerization (ATRP).34 The core–shell polymers consist of hundreds of polymer arms carrying PEG as side chains. Protein conjugation of these dendritic scaffolds was also demonstrated with high protein conjugation efficiency. Another type of dendritic polymer with a hydrophobic polyethylene core and linear PEG shell was also prepared through CWP, followed by coupling with PEG. These dendritic polymers behave like unimolecular micelles in aqueous solution, which can encapsulate and transport hydrophobic dyes such as Nile Red in aqueous environment.33 For investigation of biological pathway, biodistribution, and the eventual fate of bioconjugates, fluorescent labeling is commonly employed for detection and tracking. Most of the labeling of dendritic polymers is, however, through the peripheral conjugation sites. One major disadvantage of such labeling * To whom correspondence should be addressed. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Medicine.

approach is that the peripheral conjugation sites is also where the functional moieties, such as specific binding ligands or therapeutic molecules, are located. Such peripheral labels may interfere with the interactions of the bioconjugates with targeted systems. A more desirable fluorescence labeling for dendritic polymers is to label internally, preferably at the core of the dendrtic polymers. Here we present an efficient method to label the dendritic polymer scaffolds with a common UV and fluorescence moiety internally and the study of their conjugation behavior with protein (ovalbumin) molecules. The internal labeling of the dendritic polymers can be realized readily through our CWP methodology using a fluorescence-labeled catalyst. To allow for further bioconjugation, heterobifunctional PEG carrying activated functional groups was coupled to the dendritic polymer core. Our design of the dendritic polymer scaffolds is outlined in Scheme 1.

Experimental Section General Procedure. The catalyst’s synthesis and handling were carried out in a Vacuum Atmosphere glove box filled with nitrogen. All other moisture- and air-sensitive reactions were carried out in flamedried glassware using magnetic stirring under a positive pressure of argon or nitrogen. Removal of organic solvents was accomplished by rotary evaporation and is referred to as concentrated in vacuo. Flash column chromatography was performed using forced flow on EM Science 230–400 mesh silica gel. NMR spectra were recorded on Bruker DRX400, Bruker GN500, and Bruker Omega500 MHz FT-NMR instruments. Elemental analysis was preformed by Atlantic Microlabs, Atlanta, GA. Toluene, THF, diethyl ether, dichloromethane, and other solvents were purified by passing through solvent purification columns, following the method introduced by Grubbs.35 Catalyst 1 was synthesized by following literature report.36,37 General Polymerization Procedure. Ethylene homopolymerization and copolymerization with polar comonomers were performed in a Parr pressure reactor equipped with a mechanical stirring and a temperature controller. A typical polymerization procedure is described as follows: The Parr reactor was preheated to 100 °C for 2 h under vacuum and then cooled down under N2 to room temperature before use. A comonomer solution in chlorobenzene/toluene (1:3) was degassed and

10.1021/bm7013476 CCC: $40.75  2008 American Chemical Society Published on Web 06/03/2008

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Biomacromolecules, Vol. 9, No. 7, 2008

Chen et al.

Scheme 1. Design of Functional Dendritic Polymer Scaffolds Carrying Internal Fluorescence Labels

then added into a preprepared Pd(II)-R-diimine catalyst 1 or pyrenelabeled Pd(II)-R-diimine catalyst 2 solution in chlorobenzene/toluene

UV/vis and Fluorescence. UV/vis and fluorescence spectra were recorded on a JASCO FP-750 fluorescence spectrophotometer, which used a 150-W xenon lamp with a shielded lamp house as the light source. Emission spectra were obtained using excitation wavelength at 355 nm for pyrene emission and 490 nm for fluorescein emission.

Synthesis of Catalyst and Polymers

(1:3). The whole mixture was transferred into the Parr reactor under N2. After purging the reactor with N2 and ethylene, the system was filled with ethylene to the desired pressure and polymerization was allowed to continue at room temperature for about one to two days. The polymerization was quenched by the addition of an excess amount of triethylsilane under N2. The polymer solution was passed through Celite and neutral alumina gel to remove residual catalyst and then concentrated in vacuo. A small amount of hexanes was added to dissolve the polymer before cold acetone was added under vigorous stirring. The precipitate was redissolved and reprecipitated two more times. The polymer was obtained by drying in vacuo at room termperature. Determination of Comonomer Incorporation Percentage by NMR. The chemical structures of the copolymers were analyzed by 1 H NMR spectra. The polar comonomer incorporation level was calculated from the integrations of peaks of the comonomer and peaks of the polyethylene backbone. The calculation of branching density was based on the integration of the terminal methyl group and the integration of the polyethylene backbone. SEC-MALS Characterization of Copolymers.29,38 All the polymers were characterized by size-exclusion chromatography (SEC) coupled to a multiangle light scattering detector (MALS) for obtaining both the molecular weight (M) and the radius of gyration (Rg). For smaller polymers that are below the detecting limit of MALS, molecular weight calibration was done by using linear polystyrene standards (Aldrich) from the SEC data. Measurements were made on highly dilute fractions eluting from a SEC consisting of a HP Aglient 1100 solvent delivery system/auto injector with an online solvent degasser, temperature-controlled column compartment, and an Aglient 1100 differential refractometer. A Dawn DSP 18-angle light scattering detector (Wyatt Technology, Santa Barbara, CA) was coupled to the SEC to measure both the molecular weights and the sizes for each fraction of the polymer eluted from the SEC column. A 30 cm column was used (Polymer Laboratories PLgel Mixed C, 5 µm particle size) to separate polymer samples. The mobile phase was tetrahydrofuran (THF) and the flow rate was 0.5 mL/min. Both the column and the differential refractometer were held at 35 °C. A total of 60 µL of a 2 mg/mL solution was injected into the column. ASTRA 4.7 from Wyatt Technology was used to acquire data from the 18 scattering angles (detectors) and the differential refractometer. The Mw, Mn, and Rg data were obtained by following classical light scattering treatments using software ASTRA 4.7. The Rg data reported are the weight-averaged radius of gyration in specified solvent. Protein Conjugates Separation. Protein conjugates separation was done by using a Sephacryl S-200 HR column (Sigma-Aldrich). All measurements were done at 25 °C using 1 × PBS buffer (pH ) 7.4) as mobile phase at a flow rate of 1 mL/min.

Pyrene-acrylate 5. At 0 °C, a solution of acryloylchloride (0.36 mL, 4.4 mmol, 1.2 equiv) in 10 mL of DCM was slowly added to a solution of 4-pyrenebutanol (1.0 g, 3.65 mmol, 1.0 equiv) and i-Pr2NEt (0.76 mL, 4.4 mmol, 1.2 equiv) in 10 mL of DCM. The mixture was then allowed to stir at room temperature overnight. After diluting with 50 mL of DCM, an aqueous solution of NaHCO3 was added to quench the reaction. The aqueous layer was separated and washed with DCM three times. Combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The product 5 was obtained (1.1 g, 93% yield) after flash column chromatography (EtOAc in hex: 0-20%). 1H NMR (500 MHz, CDCl3) δ 8.27 (d, J ) 9.24 Hz, 1H), 8.18 (dd, J1 ) 4.3 Hz, J2 ) 7.5 Hz, 2H), 8.12 (dd, J1 ) 3.9 Hz, J2 ) 7.5 Hz, 2H), 8.06–7.99 (m, 3H), 7.87 (d, J ) 7.7 Hz, 1H), 6.40 (d, J ) 17.3 Hz, 1H), 6.13 (dd, J1 ) 10.5 Hz, J2 ) 17.3 Hz, 2H), 5.82 (d, J ) 10.4 Hz, 1H), 4.25 (dd, J1 ) J2 ) 6.6 Hz, 2H), 3.41 (dd, J1 ) J2 ) 6.6 Hz, 2H), 1.97 (m, 2H), 1.88 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 166.54, 136.52, 131.63, 131.09, 130.86, 130.08, 128.82, 128.73, 127.71, 127.50, 127.45, 126.86, 126.04, 125.31, 125.22, 125.12, 125.02, 124.93, 124.49, 64.59, 33.25, 28.82, 28.31. Anal. Calcd for C23H20O2: C, 84.12; H, 6.14. Found: C, 84.21; H, 6.03. Pyrene-Labeled Pd Bisimine Catalyst 2. To a solution of (bisimine)Pd(Me)Cl (1.69 g, 3.0 mmol, 1.0 equiv) and NaBAF (2.66 g, 3.0 mmol, 1.0 equiv) in Et2O was added pyrene acrylate 5 (1.0 g, 3 mmol, 1.0 equiv) at room temperature. The solution was allowed to stir for 48 h before it was filtered through celite and concentrated in vacuo. The solid was then recrystallized from DCM at -35 °C to give pyrene-labeled catalyst 2 (4.2 g) in 80% yield. 1H NMR (500 MHz, CDCl3) δ 8.28–8.08 (m, 8H), 7.86 (d, J ) 7.7 Hz, 1H), 7.78–7.63 (b, 8H), 7.60–7.50 (b, 4H), 7.39–7.31 (m, 3H), 7.13–7.11 (m, 3H), 3.44 (dd, J1 ) 7.0 Hz, J2 ) 13.9 Hz, 3H), 3.34–3.25 (m, 4H), 2.92–2.80 (m, 4H), 2.37 (dd, J1 ) J2 ) 5.8 Hz, 2H), 2.16 (s, 3H), 2.15 (s, 3H), 1.70–1.67 (m, 2H), 1.55–1.45 (m, 4H), 1.42–1.33 (m, 8H), 1.20–1.13 (m, 24H), 0.61 (m, 2H). HR-MS calcd for C52H63N2O2Pd+, 853.39; found, 853.35. Pyrene-Labeled Polyethylene 8.32 Refer to the general polymerization procedure for the synthesis. Ethylene pressure 0.1 atm was used. Polymerization time is 48 h at room temperature. 1 H NMR (500 MHz, CDCl3) δ 1.05–1.40 (broad, 16.4H), 0.80–0.92 (broad, 3H). SEC-MALS: Mn ) 221000 g/mol; Mw ) 329000 g/mol; PDI ) 1.49; Rg ) 9.2 ( 1.2 nm. Average number of branching per polymer is calculated as the following:

Dendritic Polymers for Bioconjugation

B)

NCH3 Ntotal carbonin backbone


) 103

(1)

UV/vis: λmax,1 ) 337 nm; λmax,2 ) 320 nm. Fluorescence: λmax,1 ) 379 nm; λmax,2 ) 398 nm (excitation wavelength: 335 nm). Average number of pyrene molecule per polymer ) 0.9. Polymer 9.32 Refer to the general polymerization procedure for the synthesis. Ethylene pressure 0.1 atm was used. Polymerization time is 48 h at room temperature. 1H NMR (500 MHz, CDCl3) δ 7.55–7.75 (4H), 7.30–7.40 (6H), 3.29 (2H), 1.08–1.43 (broad, 15.8H), 0.80–0.93 (broad, 15.6H). SEC-MALS: Mn ) 248000 g/mol; Mw ) 391000 g/mol; PDI ) 1.58; Rg ) 12.9 ( 1.0 nm. The average number of branching per polymer is calculated to be 156. The average incorporation percentage of olefin comonomer with OTBDPS (r) is calculated as the following:

r)

Ncomonomer NTBDPS ) ) 25.9% Ncomonomer + Nethylene NTBDPS + Nethylene (2)

The average number of chain ends (OTBDPS) per polymer is calculated as the following:

NOTBDPS ) r ·

Mn ) 570 (1 - r) · MWethylene + r · MWcomonomer (3)

Copolymer 10. Copolymer 10 was prepared according to literature procedure.33 1H NMR (500 MHz, CDCl3) δ 3.30 (1.8H), 1.08–1.40 (broad, 15.8H), 0.80–0.92 (broad, 6.6H). SECMALS: Mn ) 101000 g/mol; Mw ) 156000 g/mol; PDI ) 1.55; Rg ) 10.4 ( 1.1 nm. The average number of chain ends (OH) per polymer molecule is 520. The chain end converting efficiency (φ) can be calculated two ways:

φ)

NMRchain ends, product 1.8 ⁄ 2 ) · 100% ) 90% NMRchain ends, reactant 1.0

or

φ)

Nchain ends, product 520 ) · 100% ) 91% (4) Nchain ends, reactant 570

The NMR method was used to determine the chain end reaction efficiency and number of chain ends for the following polymers. Copolymer 11.33 Copolymer 11 was prepared according to literature procedure. 1H NMR (500 MHz, CDCl3) δ 4.25 (broad, 3.7H), 3.50–3.84 (broad, 60H), 3.38 (s, 2.8H), 0.95–1.35 (broad, 15.8H), 0.85–0.95 (broad, 6.6H). SEC-MALS: Mn ) 475000 g/mol; Mw ) 642300 g/mol; PDI ) 1.4; radius of gyration, Rg ) 20.9 ( 0.4 nm.The chain end converting efficiency (φ) from NMR is calculated to be 93%, as defined above. The average number of chain ends (PEG-OMe) per polymer molecule is calculated to be 485. Calculated molecular weight of the polymer based on the number of chain ends, Mn ) Mn,core + NPEG-OMe × MWarm ) 480 × 103 g/mol. Bn-PEG-600 15. PEG diol 14 (Mn ) 600; 3.0 g, 5 mmol, 1.0 equiv) was predried at 120 °C for 3 h under N2 and was dissolved in 50 mL of dry THF. At 0 °C, the above solution was slowly added to the sodium hydride (0.17 g, 7 mmol, 1.4 equiv) dispersion in 50 mL of THF over 30 min. BnBr (0.72 mL, 5.5 mmol, 1.1 equiv) and NH4I (0.0015 g, 0.5 mmol, 0.1

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equiv) was added to the reaction solution. The reaction mixture was then allowed to warm up to room temperature and stir overnight. After being diluted with 100 mL of DCM, the aqueous solution of NH4Cl was added to quench the reaction. The aqueous layer was separated and washed with DCM three times. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The product 15 was obtained (0.98 g, 28% yield) after flash column chromatography (MeOH in DCM: 0-10% gradient). 1H NMR (500 MHz, CDCl3) δ 7.4–7.2 (m, 5H), 4.55 (s, 2H), 3.65–3.59 (m, 54H), 3.41–3.82 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 138.17, 128.17 (2C), 127.68 (2C), 127.54, 72.65, 70.38–70.35 (mC), 70.08, 69,36, 61.45, 50.25. Polymer 19. To a solution of 15 (0.27 g, 0.4 mmol, 1.0 eqiv) in THF was slowly added to a solution of succinic anhydride (0.049 g, 0.5 mmol, 1.25 equiv.) and i-Pr2NEt (0.084 mL, 0.48 mmol, 1.2 equiv) in THF. The mixture was then allowed to stir at room temperature overnight. After being diluted with THF, the aqueous solution of NaHCO3 was added to quench the reaction. The aqueous layer was separated and washed with DCM three times. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. Compound 16 was obtained (0.3 g, 97% yield) after flash column chromatography (MeOH in DCM: 0-20%). To a solution of compound 16 (0.3 g, 0.4 mmol, 1.0 equiv) in toluene was added phosgene (4 mmol, 10 equiv) at 0 °C. The reaction solution was allowed to stir at room temperature overnight before the solvent was removed in vacuo. The residue was dried under high vacuum to yield compound 17 (99%), which was used without further purification. At 0 °C, the solution of 17 (0.3 g, 0.4 mmol) in THF was added to the polymer 10 solution in THF (0.09 g, 0.9 µmol) in the presence of pyridine (0.032 mL, 0.4 mmol). The reaction mixture was allowed to stir at room temperature for 24 h before being diluted with a large volume of CHCl3 and washed with brine. The organic layer was separated and concentrated. The residue was then redissolved with a small amount of acetone. Copolymer 18 (0.3 g) was obtained in 83% yield by repetitive precipitation of the polymer from the acetone solution by addition of a mixture of hexanes and ethyl acetate (10:1). Compound 18 (0.1 g) was then dissolved in EtOH and transferred to a pressure reactor. After addition of Pd(OH)2 (50 mg), the reactor was sealed and charged with hydrogen to 60 psi. At 60 °C, the reactor was stirred overnight. The reaction mixture was then filtered through celite and concentrated in vacuo and dried under high vacuum. The product 19 was obtained in 88% yield. 1H NMR (500 MHz, CDCl3) δ 4.31 (m, 1.2H), 4.21 (s, 1.2H), 3.40–3.80 (m, ∼40H), 2.24 (b, 2.8H), 0.95–1.35 (broad, 15.8H), 0.85–0.95 (broad, 6.6H). SEC-MALS: Mn ) 287000 g/mol; Mw ) 443300 g/mol; PDI ) 1.5; radius of gyration, Rg ) 17.1 ( 0.8 nm. The chain end converting efficiency (two steps; φ) from 10 by NMR is calculated to be 60%. The average number of chain ends (OH) per polymer molecule is calculated to be 309. The calculated molecular weight of the polymer 19 based on the number of chain ends Mn ) Mn,core + NOH × MWarm ) 311 × 103 g/mol. Scaffold 21. To a solution of 19 (70 mg, 0.25 µmol) in THF was slowly added to a solution of succinic anhydride (100 mg, 1.0 mmol) and i-Pr2NEt (0.18 mL, 1.0 mmol). The mixture was then allowed to stir at room temperature overnight. The reaction was then diluted with MeOH and dialysis was performed against MeOH for 3 days (dialysis tube cutoff molecular weight (MWCO) ) 10000 g/mol). The solution from the dialysis tube was collected, concentrated, and then dried under vacuum to afford 20 (60 mg, 70%). To a solution of 20 in THF was added

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Scheme 2. Mechanism for the Synthesis of Pyrene-Labeled Dendritic Polymer Using a Pyrene-Labeled Pd(II)-R-diimine Catalyst 2

EDC (72 mg, 0.4 mmol), NHS (46 mg, 0.4 mmol), i-Pr2NEt (72 µL, 0.4 mmol), and DMAP (2.5 mg, 0.08 mmol). The mixture was then allowed to stir at room temperature overnight. The reaction was then diluted with MeOH and dialysis was performed against MeOH for 3 days (dialysis tube cutoff molecular weight (MWCO) ) 10000 g/mol). The olution from the dialysis tube was collected, concentrated, and then dried under vacuum to afford 21 (40 mg, 61%). 1H NMR (500 MHz, CDCl3) δ 4.31 (m, 1.2H), 4.21 (s, 1.2H), 3.40–3.80 (m, ∼40H), 2.64 (b, 1.7H), 2.24 (b, 2.8H), 0.95–1.35 (broad, 15.8H), 0.85–0.95 (broad, 6.6H). SEC-MALS: Mn ) 343000 g/mol; Mw ) 531300 g/mol; PDI ) 1.5; radius of gyration, Rg ) 17.2 ( 0.8 nm. The chain end converting efficiency (two steps; φ) from 19 by NMR is calculated to be 71%. The average number of chain ends (NHS) per polymer molecule is calculated to be 221. The calculated molecular weight of the polymer 19 based on the number of chain ends, Mn ) Mn,core + NNHS × MWarm ) 353 × 103 g/mol. The overall chain end converting efficiency to obtain scaffold 21 from hydroxyl polymer 10 is calculated to be 42%. Boc-amino-PEG-3000 23. To a solution of ω-amino-PEG3000 (60 mg, 0.02 mmol, 1 equiv) in methanol was added NaHCO3 (10 mg) and (Boc)2O (10 mg, 2.4 equiv) at 0 °C. The mixture was sonicated for 60 min while keeping the temperature at 25 °C. The reaction mixture was then filtered, and the filtrate was concentrated. After being precipitated by the addition of Et2O, the resulting solid was then dissolved in a very small amount of THF and precipitated with Et2O again. The solid was then dried under vacuum to afford 23 (60 mg, 92%). 1H NMR (500 MHz, CDCl3) δ 4.15 (s, 1H,), 3.40–3.80 (m, 300H), 2.22 (b, 2H), 1.40 (s, 9H). Polymer 27. To a solution of 23 (120 mg, 0.04 mmol, 1 equiv) in toluene was added di-imidazolecarbonate (8.0 mg, 0.05 mmol, 1.25 equiv) in THF (5 mL). The solution was allowed to stir overnight at room temperature before THF was removed under vacuum. The resulting polymer was dried overnight under high vacuum and then dissolved in THF. Toward the above solution, hydroxyl functionalized dendritic polymer 10 (10 mg, 1 equiv) and pyridine (10 µL) were added under N2. The solution was then allowed to stir for 2 days at room temperature before a solution of HCl in dioxane (4 M) was added. After 12 h of treatment of HCl, the above solution was diluted with methanol and submitted to dialysis against methanol for 3 days (dialysis tube cutoff molecular weight (MWCO) ) 10000 g/mol). Solution from the dialysis tube was collected, concentrated, and then dried under vacuum to afford 27 (110 mg, 85%). 1 H NMR (500 MHz, CDCl3) δ 4.41 (m, 1.1H), 4.26 (s, 1.1H), 3.40–3.80 (m, ∼176H), 2.24 (b, 1.1H), 0.95–1.35 (broad, 15.8H), 0.85–0.95 (broad, 6.6H). SEC-MALS: Mn ) 965000 g/mol; Mw ) 1543000 g/mol; PDI ) 1.6; radius of gyration, Rg ) 28.9 ( 0.3 nm. The chain end converting efficiency (two steps; φ) from 10 by NMR is calculated to be 55%. The average number of chain ends (NHS) per polymer molecule is calculated to be 282.

The calculated molecular weight of the polymer 27 based on the number of chain ends, Mn ) Mn,core + NNH2 × MWarm ) 924 × 103 g/mol. Scaffold 29. To a solution of 27 (110 mg, 1 equiv) in THF was added disuccinicarbonate (4.3 mg, 1 equiv). The solution was then allowed to stir for 2 days at RT before it was concentrated and precipitated with Et2O to afford scaffold 29 (114 mg, 99%). 1H NMR (500 MHz, CDCl3) δ 4.41 (m, 1.1H), 4.26 (s, 1.1H), 3.40–3.80 (m, ∼180H), 2.68 (b, 1.9H), 2.54 (b, 1.0H), 2.24 (b, 1.1H), 0.95–1.35 (broad, 15.8H), 0.85–0.95 (broad, 6.6H). SEC-MALS: Mn ) 994000 g/mol; Mw ) 1590000 g/mol; PDI ) 1.6; radius of gyration, Rg ) 28.7 ( 0.3 nm. The chain end converting efficiency (one step; φ) from 27 by NMR is calculated to be 83%. The average number of chain ends (NHS) per polymer molecule is calculated to be 225. The calculated molecular weight of the polymer 29 based on the number of chain ends, Mn ) Mn,core + NNHS × MWarm ) 958 × 103 g/mol. The overall chain end converting efficiency to obtain scaffold 29 from hydroxyl polymer 10: 43%.

Results and Discussion Synthesis of Pyrene-Labeled Polymers. For easy detection of the dendritic polymer scaffolds at very low concentrations, we introduced a simple method of labeling the polymers internally with a pyrene chromophore. Pyrene is a common fluorescence dye used for labeling in biological systems.40,41 Our strategy for internally labeling our dendritic polymer is shown in Scheme 2. We first prepared a pyrene-labeled Pd(II)R-diimine catalyst 2. Brookhart and co-worker have shown that the ester chelates of Pd(II)-R-diimine can initiate living polymerization of ethylene under certain conditions.39 It was further shown that chelates in the Pd(II)-R-diimine catalyst became the starting chain ends in polyethylene when using such catalysts for ethylene polymerization.36,37 Based on these results, we first prepared a pyrene-labeled chelate Pd(II)-R-diimine catalyst 2. Upon initiation of ethylene polymerization at low ethylene pressure,29 CWP will commence and the pyrene moiety will be covalently inserted to the starting chain end, which statistically should reside in the core of the polymer (Scheme 2) due to the dendritic topology of the polymer. The synthesis of the pyrene-labeled Pd(II)-R-diimine catalyst 2 is shown in Scheme 3. Pyrene acrylate 5 was first prepared by coupling 4-pyrenebutanol 3 with acryloyl chloride. Activation of the R-diimine-PdII(Me)Cl precursor with NaBAF (please see Scheme 3 for the structure of NaBAF) in the presence of pyrene acrylate 5 yielded activated catalyst 2 in 80% yield after recrystallization from dichloromethane. The activation of the catalyst follows the same mechanism as reported: NaBAF first abstracts the chloride from the catalyst precursor 6, which is followed by coordination of the pyrene acrylate and subsequent migratory insertion of the methyl group. The major migratory insertion pathway is predominantly 2,1-insertion, which is



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Scheme 3. Synthesis of Pyrene-Labeled Pd(II)-R-diimine Catalyst 2

Table 1. Polymerization Results of Ethylene and Comonomers Using Pd(II)-R-diimine Catalyst 1 and the Pyrene-Labeled Pd(II)-R-diimine Catalyst 2a

entry

catalyst

polymer

yield (g)

b

TON

c

3

Mn (SEC; 10 g/mol)

d

3

Mn (LS; 10 g/mol)

d

PDI (LS)

e

Rg (LS; nm)

f

B

# of polymer molecules per catalystg

Ethylene Homopolymerization 1 2

1 2

7 8

0.5 0.5

8640 8592

86 89

220 221

1.49 1.49

9.2 ( 1.8 9.2 ( 1.8

103 103

1.1 1.1

1.58 1.55 1.40

12.9 ( 1.5 10.4 ( 1.6 20.9 ( 0.5

156 N/Ah N/Ah

1.1 N/Ah N/Ah

Ethylene Copolymerization 3 4 5

2 2 2

9 10 11

0.8 0.2 0.7

2799 N/Ah N/Ah

55 43 321

248 101 475

a Catalyst load, entries 1 and 2: 0.3 mmol/L; entries 3–5: 2 mmol/L; solvents: chlorobenzene/toluene (1:3); reaction temperature: room temperature; reaction time: 48 h. b Number of average radius of gyration obtained from SEC-MALS in THF. c Data obtained from SEC in THF calibrated with linear polystyrene standards. d Data obtained from multiangle light scattering coupled SEC (SEC-MALS). e TON is the catalyst turn over number. f Branches per 1000 carbon, data obtained from 1H NMR, branching of copolymers include the number of chain ends with functional groups. g The number of polymer molecules per catalyst is estimated on the basis of the catalyst TON and the molecular weight of PE: # of polymer molecules per catalyst ratio ) [MWethylene · TON]/Mn,PE, where MWethylene is the molecular weight of ethylene monomer (28 g/mol) and Mn,PE is the number of average molecular weight determined by light scattering. h Not applicable.

Scheme 4. Synthesis of Dendritic Polyethylene Using the Regular Pd(II)-R-diimine Catalyst 1 and the Pyrene-Labeled Pd(II)-R-diimine Catalyst 2

followed by chain walking to form the most stable 6-membraned chelate catalyst, as shown in catalyst 2 (Scheme 3). The catalyst structure was characterized by 1H NMR and high resolution mass spectrometry (HR-MS). The pyrene-labeled Pd(II)-R-diimine catalyst 2 was then evaluated for ethylene polymerization. The molecular weight and size (radius of gyration) of the polymers were measured by SEC-MALS, and the branching density was obtained from the 1H NMR spectra (Table 1). The polymerization results were compared with those obtained from the regular Pd(II)-R-diimine catalyst (1; Scheme 4 and Table 1, entry 1 and 2). Based on the comparison, we concluded that the introduction of the pyrene chromophore to the catalytic center caused no change in the catalytic polymerization behaviors of the catalyst in terms of the molecular weight and size of the resulting polymers, the branching topology of the resulting polymers, and the catalyst activity (turnover numbers and turnover frequency).

Under the polymerization conditions (ethylene pressure: 0.1 atm, room temperature), ethylene polymerizations by both catalysts showed that the number of polymer molecule generated per catalyst is very close to 1. The slightly more than 1.0 value indicates the presence of small extent of chain transfer during polymerization. After each chain transfer, the Pd catalyst will then initiate a new polymer chain in which no pyrene acrylate moiety will be incorporated. Based on the number of polymer chains produced per catalyst center, it is estimated that on average about 91% of the polymer molecules made by pyrenelabeled Pd(II)-R-diimine catalyst 2 have one pyrene molecule covalently attached. Copolymerization of ethylene with the polar comonomer 12 successfully yielded copolymer 9 using the pyrene-labeled Pd(II)-R-diimine catalyst 2 (Scheme 5). Following the removal of the TBDPS protecting groups, pyrene-labeled dendritic polymer bearing hydroxyl groups (10) was obtained. Upon coupling with PEG choroformate 13, pyrene-labeled amphiphilic core–shell polymer 11 was obtained. The molecular weight and size (radius of gyration) of the polymers 9-11 were measured by SEC-MALS, and the branching density was obtained from 1H NMR spectra (Table 1, entries 3–5). The presence of the pyrene label in the polymers was confirmed with the UV/vis and fluorescence spectra of the polymers (Figures 1 and 2). Both the UV/vis and fluorescence spectra of the copolymer solutions were similar to those for the control compound, 1-pyrenebutanol. Concentrations of

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Scheme 5. Synthesis of Pyrene-Labeled Copolymers Using Pyrene-Labeled Pd(II)-R-diimine Catalyst 2

pyrene were determined by the absorbance and extinction coefficient of pyrene (Figure 1) from Beer’s Law:

A ) cl where c is the molar concentration of the molecule, l is the path length of the sample cell (cm), and is the molar extinction coefficient, which was shown to be 54000 L/(mol × cm) in cyclohexane at wavelength of 335 nm.42 Based on the UV/vis absorbance, the pyrene concentrations of PE 8 solution showing in Figure 1 is determined to be 7.6 µM. Comparison with the concentration of the total polymer (PE 8, 8.5 µM) indicates that, on average, about 90% of the polymer molecule is labeled with a pyrene moiety. This agrees well with the number of polymer chains per catalyst data shown in Table 1, where such pyrene per polymer ratio for pyrenelabeled polymer 10 is also determined to be about 90%. Preparation of Pyrene-Labeled Functional Dendritic Copolymers Using Heterobifunctional PEG. For further bioconjugation purpose, we prepared pyrene-labeled dendritic copolymers carrying multiple surface functionalized N-hydroxysuccinimide (NHS) groups. For this purpose, a heterobifunctional PEG was first prepared by using commercially available simple PEG diol. Treating PEG with benzyl bromide in the presence of catalytic amount of ammonium iodide yielded a statistical mixture of monobenzyl-PEG and dibenzyl-PEG from which the monobenzyl-PEG (15) was separated by silica gel column chromatography. Such reaction was also tried with PEGs of higher

Figure 1. UV/vis spectra of pyrene-labeled polymers in various solvents: green, polymer 11 in water; purple, polymer 10 in THF; blue, PE 8 in cyclohexane; brown, pyrenebutanol in THF. Concentrations of the polymer solutions: [8] ) 8.5 µM; [10] ) 13 µM; [11] ) 6.0 µM.

Chen et al.

molecular weight (Mn ) 1000 and 1500 g/mol), which afforded monobenzylation products in lower yields (6–28%; the higher the molecular weight of the PEG diol, the lower the separation yield). To synthesize heterobifunctional PEG, alcohol 15 was converted to acid 16, which was then activated with thionyl chloride to give acetochloride 17 (Scheme 6). PEG 17 was then coupled to the hydroxyl dendritic core 10 to yield the core–shell polymer 18. Exposure of 18 to hydrogen in the presence of palladium hydroxide catalyst yielded polymer 19 with free hydroxyl groups, which were subsequently converted to acid groups with maleic anhydride. Treating polymer 20 with EDC and N-hydroxysuccinimide (NHS) yielded the NHS-activated polymer 21. The Mn of polymer 21 is 343000 g/mol and the radius of gyration is 17.2 nm in THF, both determined by size exclusion chromatography coupled with multiangle light scattering detectors (SEC-MALS) using THF as mobile phase. For the final product 21, it was determined that 42% of the total chain ends/ surface functionality was converted to NHS groups. Because of difficulty in purification, the above method is only applicable to PEG glycol with molecular weight less than 2000 g/mol. A larger NHS-functionalized polymer was prepared from a commercially available heterobifunctional PEG: the ω-aminoPEG 3000 22, which was only available with a molecular weight of 3000 g/mol. After the amino group in PEG 22 was protected with tert-butyloxycarbonyl (Boc), di-imidazolcarbonate (24) was used to activate the hydroxyl group in 23 to yield compound 25 (Scheme 7). Coupling of 25 to the hydroxyl dendritic core 10 yielded polymer 26. Exposure of 26 to 4 M HCl afforded amino terminated polymer 27, which was activated by excess amount of disuccinamidecarbonate to yield NHS-activated polymer 29. SEC-MALS characterization data for the NHS-functionalized polymers were summarized in Table 2. Molecular weights of the NHS-functionalized dendritic polymers increase with the increase of the molecular weight of PEG unit used. From 1H NMR of the polymers, the number of end groups can be calculated (e.g., Bn groups in polymer 18, Boc groups in polymer 26, and NHS groups in 21 and 29; Table 2). The number of NHS groups in the final products (21 and 29) is about 220 per polymer, which is about 43% of the total end groups in their precursors (polymer 10) after multiple steps of reaction. The calculated molecular weights of the polymers (Mn,cal (polymer) ) Mn (core) + Nnumber of arms × Mn (PEG arm)) based on the molecular weight of each arm, the number of arms, and the core moiety molecular weight are listed in Table 2, which are consistent with the SEC-MALS experimental values. The Rg of the polymers also increases consistently with the increase of the molecular weight of the PEG used. Protein Conjugation with the NHS-Activated Polymer Scaffolds. Ovalbumin (Sigma-Alrich, source: chicken egg white), a relatively small protein (MW ) 44287 g/mol), was chosen as a model protein for bioconjugation studies.43 The ovalbumin used in the study was labeled with a fluorescent dye, fluorescein, which will enable detection of protein in a very small quantity. A control experiment was conducted by mixing ovalbumin with polymer 11, in which the chain ends are not reactive (Scheme 8). The mixture was then diluted with PBS buffer and submitted to a gel filtration column chromatography using Sephecryl HR S200 resin as solid phase and 1×PBS buffer (pH 7.4) as mobile phase. Fluorescence intensity of the fractions was recorded at two excitation wavelengths (355 nm for pyrene and 490 nm for fluorescein). Because of the contrast in



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Figure 2. Emission spectra of pyrene-labeled polymers (excitation wavelength 355 nm): green, polymer 11 in water; purple, polymer 10 in THF; blue, PE 8 in cyclohexane; brown, pyrenebutanol in THF. Concentration of the polymer solutions: 1.0 µM. Scheme 6. Synthesis of NHS-Functionalized Dendritic Polymer Scaffold Having a Pyrene Label

molecular size (Rg ∼ 21 nm for 11 and an estimated size of 5 nm for ovalbumin), polymer 11 came out right after the column void volume, while ovalbumin was eluted much later. The baseline separation of nonreactive scaffold 11 and ovalbumine mixture also indicated that ovalbumin does not physically complex to the polymer scaffold (Figure 3). To quantify the protein molecules attached to the polymer scaffold, ovalbumin was injected for gel filtration chromatography separation at a series of concentrations and the maximum fluorescence intensity of each elution was recorded. When injected protein concentration is below 12 µM, a linear correlation between maximum fluorescence intensity and ovalbumin concentration was obtained (Figure 4), which allowed us to use gel filtration chromatography coupled with fluorescence monitoring to quantify the amount of protein being conjugated to polymer scaffold.

Ovalbumin conjugate 30 and 31 were obtained by treating ovalbumin (2 mg, 1.0 equiv protein) solution in PBS buffer with NHS-functionalized polymer scaffold 21 (0.08 mg, 1.1 equiv NHS-functionalized end groups) and 29 (0.2 mg, 1.0 equiv NHS-functionalized ends), respectively, at 4 °C for 12 h. The ratio of ovalbumin molarity to the NHS-functionalized end group molarity was about 1:1 in both reactions. An excess amount (10 equiv to the amount of NHSfunctionalized end groups) of ω-aminoethyleneglycol was added at the end of the reactions to quench any unreacted NHS-functionalized end groups on the polymer chain ends (Scheme 9). The above reaction mixtures were then diluted with 1×PBS buffer and subjected to gel filtration chromatography for separation. Protein fluorescence signal (green curve, excitation 490 nm) from both reaction mixtures were shown to be separated

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Chen et al.

Scheme 7. Synthesis of NHS-Activated Dendritic Polymer Scaffold Using the Amino-PEG (Molecular Weight: 3000 g/mol)

Table 2. Molecular Weight, Radius of Gyration, and Number of Functional Groups in the Functional Dendritic Polymer Scaffolds

entry

polymer sample

Mn of precursor PEGc (103 g/mol)

Mn by LS (10 g/mol)

PDI

Rg (nm)

1 2 3

10 21a 29b

N/A 0.6 3.0

101 343 994

1.6 1.5 1.6

10.4 ( 1.4 17.2 ( 0.8 28.7 ( 0.3

d

3

e

Mn calcd (10 g/mol)

# of functional groups in the precursorsg

# of NHS groupsg

N/A 353 958

520h 309i 305j

N/A 221k 225k

f

3

a PEG diol was used. b ω-Amino-PEG-3000 was used. c Number-averaged molecular weight of PEG provided by Aldrich. d Number-averaged molecular weight obtained from multiangle light scattering coupled SEC (SEC-MALS) using THF as mobile phase. e Number-averaged radius of gyration obtained from SEC-MALS in THF. f Mn (polymer) ) Mn (core) + Nnumber of arms × Mn (PEG arm). g Number of functional groups was calculated from 1H NMR by using the integrations of the peaks for the end group and the polymer backbone. h The functional groups in the precursors are CH2OH groups in 10. i The functional groups in the precursors are OH groups (calculated from CH2COO groups using 1H NMR) in 19. j The functional groups in the precursors are NH2 groups (calculated from CH2COO groups using 1H NMR) in 27. k The functional groups in the precursors are C(O)CH2CH2C(O) groups in the NHS units.

Scheme 8. Control Experiment of Nonactivated Polymer 11 with Ovalbumin

into two peaks: one at the elution volume of free protein and the other one overlapping with the polymer scaffolds (Figures 5 and 6). Because ovalbumin does not physically complex to the polymer scaffolds, the presence of both fluorescein and

Figure 3. Fluorescence intensity of gel filtration chromatography fractions of control experiment (polymer 11 + ovalbumin) using Sephecryl S200 resin. Green curve, maximum emission intensity at excitation wavelength 490 nm; emission wavelength, 520 nm; red curve, maximum emission intensity at excitation wavelength 355 nm; emission wavelength, 395 nm.

Figure 4. Calibration of injected protein concentration with maximum fluorescence intensity in gel filtration chromatography. Injected sample volume, 1 mL; maximum emission intensity at excitation wavelength, 490 nm; emission wavelength, 520 nm.

pyrene in the earlier fractions of the gel filtration chromatography indicates the chemical conjugation of ovalbumin to the polymer scaffolds. Based on the fluorescence calibration curve (Figure 4), the amount of free protein was obtained from the maximum fluorescence intensity of fluorescein for the protein peak (retention volume of 13 mL) in gel filtration chromatograph. Subtracting the free protein quantity from the initial total protein quantity used in conjugation, the amount of conjugated protein was calculated. The average number of ovalbumin per polymer scaffold can then be obtained from the polymer concentrations. The number of ovalbumins per polymer scaffold was estimated to be 13 for ovalbumin conjugate 30 and 17 for ovalbumin conjugate 31, respectively. The maximum number of available attaching sites (NHS-activated ester groups, calculated from 1H NMR and SEC-MALS data) is about 220. However, the number of protein molecules conjugated per



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Scheme 9. Synthesis of Ovalbumin Conjugates with the NHS-Functionalized Scaffolds

Conclusions

Figure 5. Fluorescence intensity of gel filtration chromatography fractions from ovalbumin conjugate 30. Injected sample volume: 1 mL. Green curve, maximum emission intensity at excitation wavelength 490 nm; emission wavelength, 520 nm; red curve, maximum emission intensity at excitation wavelength 355 nm; emission wavelength, 395 nm.

Figure 6. Emission of gel filtration chromatography fractions from ovalbumin conjugate 31. Injected sample volume: 1 mL. Green curve, maximum emission intensity at excitation wavelength 490 nm; emission wavelength, 520 nm; red curve, maximum emission intensity at excitation wavelength 355 nm; emission wavelength, 395 nm.

Incorporation of a pyrene moiety onto the Pd(II)-R-diimine catalyst has resulted in an otherwise identical catalyst as the regular Brookhart’s Pd(II)-R-diimine catalyst in terms of CWP behavior. Using this pyrene-labeled Pd(II)-R-diimine catalyst, we successfully prepared a series of dendritic polyethylene and its copolymers that are internally labeled with a pyrene moiety as UV/fluorescence probe. UV/vis studies showed that the polymers prepared from the pyrene-labeled catalyst carry almost quantitatively (90%) one pyrene label per polymer molecule. Furthermore, NHS-functionalized dendritic polymers carrying internal pyrene labels were successfully obtained by coupling heterobifunctional PEG to the hydroxyl dendritic core followed by NHS ester formation. The molecular weight and size of such NHS-activated dendritic polymers were successfully tuned by the PEG unit used in the synthesis. On average, there are more than 200 NHS activated ester sites per scaffold that can be used for conjugation. Incubation of fluorescein-labeled ovalbumin with the NHS-functionalized dendritic polymers resulted in the formation of protein-polymer conjugates that carry two orthogonal types of fluorescent labels: internally there is a pyrene label for the scaffold molecule and peripherally there is a fluorescein label for the protein molecule. Both labels were used for detection purpose in this study and it was found that, in average, about 13–17 copies of ovalbumin protein were conjugated to one dendritic scaffold. Such double orthogonal labeling system should find potential uses in many biomedical applications. Acknowledgment. We thank the National Science Foundation (DMR-0135233, DMR-0703988, CHE-0456719), NIH/ NIAID (AI056464, AI061363, AI065359, AI063728), and the Innovation Fund from UCI School of Physical Sciences for partial financial support. Z.G. gratefully acknowledges a Camille Dreyfus Teacher-Scholar Award and a Humboldt Bessel Research Award.

References and Notes scaffold is much smaller (13 and 17). The reasons could be (1) the size of the polymer scaffolds are only 20-28 nm, due to steric hindrance, the maximum number of protein molecules that can be conjugated to the scaffolds is much less that 220. If we use a close packing model treating the scaffold as a 20 nm hard sphere and ovalbumin as a 5 nm one,13 the maximum number of ovalbumins that can be conjugated to one scaffold surface is estimated to be around 72. (2) Ovalbumin has about 20 surface lysine residues, therefore, one protein molecule may react with more than one copy of NHS-activated chain ends. (3) Some of the chain ends could be “buried” inside and thus not available for protein conjugation.

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Efficient Catalytic Synthesis of Dendritic Polymers Having Internal

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