Organocatalytic Synthesis of Quinine-Functionalized Poly(carbonate)s

Article pubs.acs.org/Biomac

Organocatalytic Synthesis of Quinine-Functionalized Poly(carbonate)s Justin A. Edward, Matthew K. Kiesewetter, Hyunuk Kim, James C.A. Flanagan, James L. Hedrick,‡ and Robert M. Waymouth*,† †

Department of Chemistry, Stanford University, Stanford, California 94305, United States IBM Almaden Research Center, 650 Harry Road, California 95120, United States

‡

S Supporting Information *

ABSTRACT: The ring-opening polymerization of substituted cyclic carbonates with 1-(3,5-bis-trifluoromethyl-phenyl)-3cyclohexyl-thiourea (TU)/1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) organocatalysts afford highly functionalized oligocarbonates. The fluorescent alkaloid quinine can be readily incorporated into the oligocarbonates either by initiation from quinine or by ring-opening polymerization of a quininefunctionalized cyclic carbonate (MTC-Q). Copolymerization of MTC-Q with a boc-protected guanidinium cyclic carbonate affords, after deprotection, highly water-soluble cationic copolymers functionalized with both quinine and pendant guanidinium groups. When multiple quinine groups are attached to the oligomers, they exhibit minimal fluorescence due to self-quenching. Upon hydrolysis, the fluorescence intensity increases, providing a potential strategy for monitoring the hydrolysis rates in real time.

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INTRODUCTION Biodegradable poly(esters) and poly(carbonate)s provide a versatile class of biocompatible and biodegradable polymers for biomedical applications.1−9 The development of new synthetic methods for the generation of functionalized poly(esters) and poly(carbonates) has expanded the scope of application of these materials as both the physical and the functional properties of the resulting materials can be tuned10 with the addition of bioactive functional groups.10,11 Aliphatic poly(carbonates)12 are particularly attractive due to the ease of synthesis of functional carbonate monomers13,14 and the facility at which the six-membered carbonates undergo ring-opening polymerization.4,12 We have recently developed a versatile class of organic catalysts15 for the ring-opening polymerization of lactones and cyclic carbonates.6,14,15 Organic catalysts derived from thioureas and neutral bases are particularly useful as they exhibit good activities at room temperature as well as high selectivities and functional group tolerance.6,14−17 The generation of functionalized polyesters or polycarbonates by ring-opening polymerization requires that the functional groups of the monomers and polymers are compatible with the polymerization conditions and the reactivity of the ester bond.18,19 Two general strategies have been employed:1,10 (1) polymerization of monomers bearing a functional group masked by a suitable protecting group,6,8,14,18 and (2) polymerization of monomers containing compatible reactive groups that can be subsequently elaborated after the polymer is generated.5,10,11,20,21 A particular advantage of thiourea catalysts is that they exhibit a high selectivity for ring-opening of carbonates and a very low activity for transesterification of strans esters;15−17 this selectivity provides for facile ring-opening © 2012 American Chemical Society

without redistribution or transesterification of the pendant functional groups (Figure 1).14 Herein, we report a strategy

Figure 1. Monomers and catalysts for ring-opening polymerization.

where an unprotected bioactive molecule is incorporated into a cyclic carbonate monomer and polymerized directly to generate the functionalized polymer. The alkaloid quinine was chosen both for its varied biological activity22,23 and its attractive photophysical properties.24 Quinine has been used as an antimalarial drug dating back to the 17th century;22 recent research suggests that quinine dimers can inhibit drug efflux pumps associated with multidrug resistance.23 In addition, quinine is highly fluorescent and, due to its photostability and resistance to oxygen quenching, it is used as a standard for measuring quantum yields.24−27 Several quinine-containing polymers have been made primarily as supported ligands or catalysts.28−32 Received: May 9, 2012 Revised: July 9, 2012 Published: July 31, 2012 2483

dx.doi.org/10.1021/bm300718b | Biomacromolecules 2012, 13, 2483−2489

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and allowed to warm to room temperature overnight. The contents of the flask were filtered, and the solvent was removed under reduced pressure. The solid was washed with ∼50 mL of cold methanol under vacuum filtration, yielding a pure white powder: 0.39 g (30%). 1H NMR (500 MHz, CDCl3) δ 8.79 (d, 1H, H-2′), 8.06 (d, 1H, H-8′), 7.43 (s, 1H, H-5′), 7.42 (m, 1H, H-3′), 7.36 (m, 1H, H-7′), 6.52 (m, 1H, H-9), 5.89 (m. 1H, H-10), 5.05 (m, 2H, H-11), 4.69 (m, 2H, Ha), 4.26 (m, 2H, H-c), 3.98 (s, 3H, H-9′), 3.46 (m, 1H, H-6a), 3.04 (m, 2H, H-8/2a), 2.67 (m, 2H, H-2b/6b), 2.29 (m, 1H, H-3), 2.04 (m, 1H, H-5b), 1.91 (m, 1H, H-4), 1.71 (m, 1H, H-7b), 1.54 (m, 1H, H7a), 1.43 (m, 1H, H-5a), 1.26 (s, 3H, H-b) (Figure S1, Supporting Information). 13C NMR (125 MHz; CDCl3) δ 170.83, 158.38, 147.76, 146.39, 145.13, 141.81, 132.27, 127.29, 122.27, 120.18, 118.84, 115.03, 101.34, 101.31, 73.24, 72.97, 59.59, 59.57, 56.62, 55.94, 42.56, 40.61, 39.81, 28.03, 27.52, 25.41, 17.57. ESI-MS: Experimental: 467.18 m/z (parent), 468.17 m/z (25%), 469.11 m/z (10%); Theoretical: C26H30N2O6 + H+: 467.22 g/mol (parent), 468.22 (30%), 469.22 (5%). Elem. Anal. Calcd: C, 66.94; H, 6.48; N, 6.00. Found: C, 66.77; H, 6.39; N, 5.91. Synthesis of Ethylenediamine-Linked MTC-Q Dimer. To a 5 mL oven-dried vial under N2 flow containing MTC-Q (51.9 mg, 0.111 mmol) was added 0.4 mL of anhydrous dichloromethane. Ethylenediamine (3.6 μL, 0.0535 mmol) was added to the reaction mixture via syringe. The reaction was stirred at rt for 24 h, and solvent was removed under vacuum. The residue (60 mg) was taken up in reagent grade dichloromethane (∼0.5 mL) and placed directly onto a silica gel column, and the product separated by eluting with 80:20 solution of dichloromethane/ethanol. The product fractions were removed and the solvent evaporated to a white powder, a mixture of diastereomers. Final yield: 40 mg (76%). 1H NMR (500 MHz, CDCl3) δ 8.25 (m, 2H), 8.0 (m, 2H), 7.35 (m, 6H), 6.5 (bs, 2H), 5.8 (bs, 2H), 5.0 (m, 4H), 4.3 (m, 4H), 3.95 (s, 6H), 3.7 (m, 4H), 3.5−2.9 (m, 10H), 2.7 (bs, 4H), 2.3 (s, 2H), 1.9−1.5 (m, 10H), 1.25 (m, 6H). 13C NMR (125 MHz; CDCl3) δ 173.51, 158.13, 156.75, 147.22, 144.37, 143.35, 141.33, 131.74, 126.68, 122.19, 118.12, 114.96, 101.25, 70.61, 69.06, 67.27, 66.28, 64.12, 63.86, 63.61, 59.32, 56.45, 55.83, 48.99, 48.48, 42.67, 41.07, 40.92, 39.42, 27.44, 23.90, 17.53, 17.22, 16.76. ESI-MS: Experimental: C54H68N6O12 + H+: 993.49 m/z (parent), C54H68N6O12 + Na+: 1015.47 g/mol (parent); Theoretical: C54H68N6O12 + H+: 993.49 g/mol (parent), C54H68N6O12 + Na+: 1015.48 g/mol (parent; Figures S12−14, Supporting Information). MTC-Q dimer was shown to be pure by HPLC. Retention time of C54H68N6O12 (MTC-Q dimer): 7.580 min. Retention time of C26H30N2O6 (MTC-Q monomer): 7.008 min. Synthesis of MTC-G. The Boc-protected (Boc = tert-butyloxycarbonyl) monomer MTC-G was synthesized as previously reported.3 1 H NMR (500 MHz, CDCl3) δ 11.48 (s, 1H, NH), 8.63 (t, 1H, NH), 4.71 (d, 2H, CH2), 4.33 (t, 2H, CH2), 4.22 (d, 2H, CH2), 3.76 (q, 2H, CH2), 1.50 (s, 18H, CH3), 1.39 (s, 3H, CH3). Polymerization of Benzyl-Functionalized Cyclic Carbonate (MTC-Bn) Initiated by Quinine. TU (8.4 mg, 0.023 mmol), DBU (3.4 mg, 0.022 mmol), and quinine (5.4 mg, 0.017 mmol) were added to a 20 mL oven-dried vial containing a stir bar. To another 20 mL oven-dried vial containing MTC-Bn (103 mg, 0.412 mmol) was added dichloromethane (0.42 mL), and the MTC-Bn solution was transferred to the vial containing catalyst/initiator. The vial was sealed and stirred for 2 h at room temperature. The reaction was quenched by the addition of 10 mg (0.083 mmol) benzoic acid with stirring for 30 min, and solvent was removed under vacuum. The conversion of MTC-Bn was 92%. The crude products were purified by dialysis in MeOH for 1 day. GPC: Mn = 4500 g/mol; Mw = 6300 g/mol; Mw/Mn = 1.38. 1H NMR (500 MHz, CDCl3) δ 8.73 (m, 1H), 8.01 (m, 1H), 7.10−7.60 (br, 157H), 6.31 (m, 1H), 5.80 (m, 1H), 5.14 (br, 56H), 4.28 (m, 110H), 3.94 (s, 3H), 3.71 (m, 4H), 3.33 (m, 1H), 3.11 (br, 2H), 3.05 (m, 1H), 2.68 (br, 1H), 2.60 (m, 1H), 2.48 (br, 2H), 2.28 (br, 1H), 1.85 (br, 1H), 1.68 (m, 14H), 1.23 (m, 87H) (Figure S2, Supporting Information). Polymerization of MTC-G Initiated by Quinine. TU (4.0 mg, 0.011 mmol), DBU (2.0 mg, 0.013 mmol), and quinine (3.0 mg, 0.0092 mmol) were added to a 20 mL oven-dried vial containing a stir

Biodegradable polyesters and polycarbonates have considerable potential as drug-delivery agents.18 One of the major challenges of drug delivery is the barrier provided by cell membranes,6,33−35 which limit the entry of molecules that lack the appropriate hydrophilic/hydrophobic balance. The discovery of molecular transporters (MoTrs), 33 a functional descriptor for cell-penetrating oligomers inspired by cellpenetrating peptides,36 has inspired new approaches in drugdelivery strategies. We recently reported a facile organocatalytic oligomerization strategy to guanidinylated carbonate oligomers that were shown to readily traverse cell membranes.6 In these studies, fluorophores or luminophores were appended to the terminus of the guanidinylated oligomer.6 Herein, we report that the copolymerization of quinine-functionalized monomers with guanidinylated carbonates provides a strategy for the generation of water-soluble oligomers containing multiple quinines attached to guanidinylated oligomers.

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EXPERIMENTAL METHODS

Materials and General Procedures. All solvents and reagents were purchased commercially and, unless otherwise noted, were used without further purification. Dichloromethane for polymerization was distilled from CaH2 prior to use. Dialysis bags (1000 g/mol cut off) were purchased from SpectraPor. The ring-opening polymerization experiments were performed in a glovebox under nitrogen atmosphere. Gel permeation chromatography (GPC) was performed in tetrahydrofuran (THF) or dimethylformamide (DMF). Those in THF were carried out at a flow rate of 1.0 mL/min on a Waters chromatograph equipped with four 5 μm Waters columns (300 × 7.7 mm) connected in series. A Viscotek S3580 refractive index detector, Visotek VE3210 UV/vis detector, and Viscotek GPCmax autosampler were employed. The system was calibrated using monodisperse polystyrene standards (Polymer Laboratories). Gel permeation chromatography (GPC) in dimethylformamide (DMF) was performed at a flow rate of 1.0 mL/min on an Agilent 1100 Series chromatograph equipped with three 5 μm Agilent columns (300 × 7.7 mm) connected in series. Agilent 1100 series refractive index and UV/vis detectors and autosampler were employed. The system was calibrated using monodisperse polystyrene standards (Polyscience). Electrospray ionization mass spectrometry (ESI-MS) studies of the quinine dimer were conducted by directly infusing 0.1 mM methanol solutions of the compound into the ion source of a hybrid quadrupole (Q) orthogonal time-of-flight (TOF) mass spectrometer (Q-TOF API-US, Waters Corporation). Measurements were conducted in positive-ion mode, with the source temperature and desolvation temperature set to 80 and 100 °C, respectively. Mass spectra were recorded from 100 to 2000 amu, and scans to higher mass ranges did not contain additional peaks. Electrospray ionization (ESI) mass spectra of all other compounds were collected on a ThermoFinnigan LCQ ion trap mass spectrometer operated in positive ion electrospray. Reverse-phase high performance liquid chromatography (RPHPLC) was performed with a Varian ProStar 210/215 HPLC using a preparative column (Alltec Alltima C18, 250 × 22 mm). The products were eluted utilizing a solvent gradient (solvent A = 0.1% TFA/H2O; solvent B = 0.1% TFA/ CH3CN). NMR spectra were recorded on Varian INOVA 500 MHz and Varian Mercury 400 MHz magnetic resonance spectrometers. Fluorescence spectroscopy data was collected on a Horiba Scientific FluoroLog-3 spectrofluorometer. Synthesis of MTC-Q. To a flame-dried 500 mL Schlenk flask under N2 flow containing MTC−OH (0.448 g, 0.0028 mol), which was prepared in situ according to established methods,15 was added 18 mL of anhydrous THF and 2 drops of DMF. A solution of oxalyl chloride (0.362 mL, 0.042 mol) in 20 mL of anhydrous THF was added to the flask over 25 min at 0 °C. The reaction was stirred at RT for 1 h, and then solvent was removed under vacuum. To the flask was added 20 mL of anhydrous THF. A solution of quinine (1.0 g, 0.0031 mol) and triethyl amine (0.86 mL, 0.0062 mol) in 22 mL of anhydrous THF was slowly added to the flask at 0 °C. The reaction was stirred 2484


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Synthesis of Block P(MTC-Q/MTC-G). In a glovebox with a N2 atmosphere using flame-dried glassware TU (4.9 mg, 0.011 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 1.6 mg, 0.011 mmol), and benzyl alcohol (35.8 μmol) were charged in a 20 mL glass vial equipped with a stir bar. A small volume of methylene chloride was added, and the formed solution stirred for 10 min. MTC-G (0.095 g, 0.21 mmol) dissolved in enough additional methylene chloride for a final concentration of 1 M monomer was added to the catalyst/ initiator solution, and the resulting solution kept stirring for 1.25 h. MTC-Q (0.1 g, 0.21 mmol) added to reaction mixture and the resulting solution kept stirring for an additional 0.75 h. Benzoic acid (15 mg, 120 μmol) was added to quench the catalyst. The crude reaction solution was transferred into a dialysis bag (1000 g/mol cutoff), the solution was dialyzed against methanol for 48 h, and the methanol solution was changed after 24 h. The remaining solvent was evaporated yielding 0.069 g as a white, sticky solid P(MTC-Q10-bbocG7). GPC: Mn = 4200; Mw = 5500; Mw/Mn = 1.30. 1H NMR of P(MTC-Q10-b-G7) (500 MHz, CDCl3) δ 11.50 (br, 7H, δ), 8.70 (br, 10H, H-2′), 8.64 (br, 7H, γ), 8.01 (d, 10H, H-8′), 7.34−7.51 (br, 34H, 5′/3′/7′/d), 6.49 (br, 10H, 9), 5.81 (br, 10H, 10), 5.15 (s, 2H, c), 4.99 (m, 20H, 11), 4.10−4.45 (br, 82H, a/α), 3.95 (br, 30H, 9′), 3.72 (br, 14H, β), 3.36 (br, 10H, 8), 2.96−3.12 (br, 20H, 6), 2.53−2.70 (br, 20H, 2), 2.20−2.31 (br, 10H, 3), 1.70−1.91 (br, 40H, 5/7), 146−1.53 (br, 126H, ε), 1.17−1.31 (br, 38H, b/4). Synthesis of Block P(MTC-Q/MTC-bocG). TU, DBU, and benzyl alcohol were charged in a 20 mL glass vial equipped with a stir bar. A small volume of methylene chloride was added, and the formed solution was stirred for 10 min. MTC-G dissolved in methylene chloride was added to the catalyst/initiator solution, and the resulting solution kept stirring for 1 h. MTC-Q was added to the reaction mixture and the resulting solution kept stirring for an additional 1 h. Benzoic acid was added to quench the catalyst. The crude reaction solution was transferred into a dialysis bag (1000 g/mol cut-off), and the solution was dialyzed against methanol for 1 day. The remaining solvent was evaporated to afford the product. 1H NMR of P(MTCQ13-b-bocG14) (500 MHz, CDCl3) δ 11.49 (m, 14H, H-δ), 8.70 (d, 13H, H-2′), 8.64 (br, 16H, H-γ), 8.01 (d, 13H, H-8′), 7.34−7.50 (br, 34H, H-5′/3′/7′/d), 6.51 (br, 12H, H-9), 5.80 (br, 12H, H-10), 5.15 (s, 2H, H-c), 4.99 (m, 25H, H-11), 4.10−4.45 (br, 150H, H-a/α), 3.95 (br, 41H, H-9′), 3.72 (br, 28H, H-β), 3.35 (br, 13H, H-8), 3.03 (br, 26H, H-6), 2.60 (br, 24H, H-2), 2.25 (br, 11H, H-3), 1.92 (br, 28H, H-5), 1.82 (br, 24H, H-7), 1.51 (br, 346H, H-ε), 1.28 (br, 114H, H-b/ 4) (Figure S9, Supporting Information). Deprotection of Block P(MTC-Q/MTC-bocG). P(MTC Q10-bbocG7) (0.069 g, 46 μmol) was charged in a 100 mL round-bottom flask equipped with a stir bar and dissolved in 2 mL of methylene chloride. Trifluoroacetic acid (TFA; 0.3 mL) was added and the flask was sealed and left under stirring at ambient temperature for 18 h. Nitrogen gas was bubbled through the solution for 30 min and the remaining solvent was evaporated by rotational evaporation, yielding (0.060 g, 87%) a slightly yellow, waxy solid. 1H NMR of deprotected block P(MTC-Q10-b-G7) (500 MHz, D2O) δ 8.58 (bs, 10H, PMTCQ aromatic), 7.84 (bs, 10H, PMTC-Q aromatic), 7.48 (bs, 30H, PMTC-Q aromatic), 6.81 (bs, 10H, PMTC-Q CH), 5.42 (bs, 10H, PMTC-Q), 4.08 (bs, 94H, PMTC-G CH2 + PMTC−CH2), 3.89 (bs, 30H, PMTC-Q CH3), 3.34−3.71 (m, 40H, PMTC-Q), 3.29 (bs, 18H, PMTC-G CH2), 3.09 (bd, 20H, PMTC-Q), 2.57 (bd, 10H, PMTCQ), 1.46−2.15 (m, 50H, PMTC-Q), 1.19 (bs, 27H, PMTC-G CH3), 1.01 (bs, 30H, PMTC-Q CH3). Fluorimetric Studies of P(MTC-Q16) and Quinine. Into two 3 mL cuvettes, a 0.1 mM aqueous solution of quinine and a 0.1 mM aqueous solution of PMTC-Q were prepared. The quinine concentration in the PMTC-Q solution was 1.6 mM. Degradation of PMTC-Q homopolymer (DP = 16) was measured at pH 7 over a period of two weeks. Solutions that were buffered contained 10 mM HEPES buffer. To account for any changes in intensity due to lamp drift or quinine fluorescence due to pH changes, we developed a series of standards. The [quinine] was determined by comparison to standards under the appropriate conditions (solvent, pH, etc). A calibration curve was prepared at every data collection to control for

bar. To another 20 mL oven-dried vial containing MTC-guanidine-boc (MTC-G) (102 mg, 0.229 mmol) was added dichloromethane (0.25 mL), and the MTC-G solution was transferred to the vial containing catalyst/initiator. The vial was sealed and stirred for 1 h at room temperature. The reaction was quenched by the addition of 10 mg (0.083 mmol) benzoic acid with stirring for 10 min, and solvent was removed under vacuum. The conversion of MTC-G was 93%. The crude products were purified by dialysis in MeOH for 1 day. GPC: Mn = 7300 g/mol; Mw = 10000 g/mol; Mw/Mn = 1.38. 1H NMR (500 MHz, CDCl3) δ 11.49 (m, 24H, δ), 8.77 (d, 1H, H-2′), 8.64 (br, 28H, γ), 8.03 (d, 1H, H-8′), 7.37−7.40 (m, 3H, H-5′/3′/7′), 6.29 (m, 1H, H-9). 5.82 (m, 1H, H-10), 5.01 (m, 1H, H-11), 4.10−4.50 (br, 173H, H-a/α), 3.97 (s, 3H, H-9′), 3.65−3.85 (br, 60H, H-β), 3.33 (s, 1H, H8), 3.11 (m, 2H, H-6), 2.92 (s, 2H, H-2), 2.68 (m, 1H, H-7a), 2.61 (d, 1H, H-5a), 2.29 (s, 1H, H-3), 1.40−1.65 (br, 536H, H-5b/7b), 1.17− 1.36 (br, 89H, H-ε/4) (Figure S3, Supporting Information). Synthesis of PMTC-Q Initiated by Benzyl Alcohol. In a glovebox with a N2 atmosphere using flame-dried glassware, 5 mol % TU/1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and benzyl alcohol were charged in a 20 mL glass vial equipped with a stir bar. A small volume of methylene chloride was added, and the formed solution stirred for 10 min. MTC-Q dissolved in methylene chloride for a final concentration of 0.4 M monomer was added to the catalyst/initiator solution, and the resulting solution kept stirring for 1 h. Benzoic acid (15 mg, 120 μmol) was added to quench the catalyst. The crude reaction solution was transferred into a dialysis bag (1000 g/mol cutoff), and the solution dialyzed against methanol for 24 h. The remaining solvent was evaporated yielding the final product (Figure S4−6, Supporting Information). Synthesis of Random P(MTC-Q/MTC-bocG). In a glovebox with N2 atmosphere using flame-dried glassware TU (4.9 mg, 11 μmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (1.6 mg, 11 μmol), and benzyl alcohol (3.7 μL, 35.8 μmol) were charged in a 20 mL glass vial equipped with a stir bar. A small volume of methylene chloride was added, and the formed solution stirred for 10 min. MTC-G (0.095 g, 0.21 mmol) and MTC-Q (0.1 g, 0.21 mmol) dissolved in enough additional methylene chloride for a final concentration of 1 M monomer was added to the catalyst/initiator solution, and the resulting solution kept stirring for 1 h. Benzoic acid (15 mg, 120 μmol) was added to quench the catalyst. The crude reaction solution was transferred into a dialysis bag (1000 g/mol cut-off), the solution was dialyzed against methanol for 48 h, and the methanol solution was changed after 24 h. The remaining solvent was evaporated, yielding 0.024 g as a white, sticky solid P(MTC-Q10-r-bocG17). GPC: Mn = 3400 g/mol; Mw = 4600 g/mol; Mw/Mn = 1.34. 1H NMR of P(MTCQ10-r-bocG17) (500 MHz, CDCl3) δ 11.49 (m, 10H, H-δ), 8.71 (d, 13H, H-2′), 8.64 (br, 10H, H-γ), 8.00 (d, 12H, H-8′), 7.34− 7.50 (br, 46H, H-5′/3′/7′/d), 6.51 (br, 10H, H-9), 5.80 (br, 11H, H10), 5.15 (s, 2H, H-c), 4.99 (m, 28H, H-11), 4.10−4.45 (br, 146H, Ha/α), 3.95 (br, 42H, H-9′), 3.72 (br, 36H, H-β), 3.35 (br, 13H, H-8), 3.03 (br, 29H, H-6), 2.60 (br, 31H, H-2), 2.25 (br, 18H, H-3), 1.92 (br, 38H, H-5), 1.82 (br, 24H, H-7), 1.51 (br, 303H, H-ε), 1.28 (br, 94H, H-b/4) (Figure S7, Supporting Information). Deprotection of Random P(MTC-Q/MTC-bocG). The bocprotected random copolymer P(MTC-Q10-r-bocG17) (0.069 g, 46 μmol) was charged in a 100 mL round-bottom flask equipped with a stir bar and dissolved in 2 mL of methylene chloride. Trifluoroacetic acid (TFA; 0.3 mL) was added and the flask was sealed and left under stirring at ambient temperature for 18 h. Nitrogen gas was bubbled through the solution for 30 min and the remaining solvent was evaporated by rotational evaporation yielding (0.060 g, 87%) a slightly yellow, waxy solid. 1H NMR of deprotected random P(MTC-Q10-rG17) (500 MHz, D2O) δ 8.58 (bs, 10H, PMTC-Q aromatic), 7.84 (bs, 10H, PMTC-Q aromatic), 7.44 (bs, 30H, PMTC-Q aromatic), 6.81 (bs, 10H, PMTC-Q CH), 4.08 (bs, 112H, PMTC-G CH2 + PMTC−CH2), 3.80 (bs, 30H, PMTC-Q CH3), 3.25−3.67 (m, 40H, PMTC-Q), 3.18 (bs, 24H, PMTC-G CH2), 3.03 (bd, 20H, PMTC-Q), 2.54 (bd, 10H, PMTC-Q), 1.39−2.07 (m, 50H, PMTC-Q), 1.10 (bs, 30H, PMTC-Q CH3), 0.88 (bs, 36H, PMTC-G,Q CH3). 2485


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lamp drift; fluorescence intensity was normalized to that of quinine at 0.1 mM.

homopolymerization of quinine-functionalized monomer MTC-Q with TU/DBU in DCM at room temperature in the presence of benzyl alcohol initiators at a variety of different monomer/initiator ratios (M/I = 8−100, Table 1). As shown in

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RESULTS AND DISCUSSION The cyclic carbonate monomers MTC-Bn14 and MTC-bocG6 (Figure 1) and 1-(3,5-bis-trifluoromethyl-phenyl)-3-cyclohexylthiourea (TU)37 were prepared as previously described; the quinine containing carbonate MTC-Q was prepared from quinine and MTC-OH.14 Quinine can be used directly as an initiator in ring-opening polymerization,38 yielding oligocarbonates functionalized with one quinine group at the chain terminus. The ring-opening polymerization (eq 1) of the benzylated cyclic carbonate MTC-

Table 1. Homopolymerization of MTC-Q Initiated by Benzyl Alcohol in the Presence of TU/DBU Catalyst (5 mol %) run

time (h)

[M]o

initial [M]o/[I]o

% conv

Mna

Mw/ Mn

DPb

yield (%)

1 2 3 4

1.25 0.83 0.83 0.83

0.37 0.37 0.36 0.38

8 25 49 100

88 82 72 72

2400a 5400b 6200b 7100b

1.26 1.15 1.16 1.20

8 25 47 92

48 41 43 53

a

GPC analysis of the polymers was performed in THF (run 1) or DMF (runs 2−4) and referenced to polystyrene standards. bDP = degree of polymerization determined by 1H NMR.

Bn was carried out by dissolving MTC-Bn in dichloromethane and adding this solution to a mixture of quinine, the thiourea (TU, Figure 1), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). After two hours at room temperature, analysis by 1H NMR revealed that 92% of the monomer had been consumed to afford a polymer with Mn = 4500 g/mol and Mw/Mn = 1.38. Analysis of the polymer by 1H NMR and gel-permeation chromatography (GPC) with a UV detector confirmed the presence of quinine at the terminus of the polymer. Similarly, the ring-opening polymerization of the guanidinylated monomer MTC-bocG was carried out in dichloromethane with quinine as the initiator to afford the end-functionalized oligomer after 1 h at room temperature (93% conversion, Mn = 7300 g/mol, Mw/Mn = 1.38, eq 2). Analysis of the resulting

Figure 2. Mn (squares) and Mw/Mn (circles) vs % monomer conversion for polymerization of MTC-Q (the dotted line shows linear fitting of Mn values; Figure S8, Supporting Information).

Table 1 and Figure 2, the ring-opening polymerization of MTC-Q exhibited a linear evolution of Mn with increased monomer conversion, characteristic of a living polymerization. Throughout the reaction, the polydispersity remained below 1.3 (Figure 2). Isolation of the polymer after dialysis of the polymer in methanol afforded isolated yields of 41−53%. Analysis of the resultant polymers by comparison of the integration of the 1H NMR resonances originating from the benzyl alcohol initiator group (5.15 ppm) and the methylene moiety on the polymer (5.78 ppm) revealed degrees of polymerization in good agreement with that from the [M]o/[I]o ratio. These results illustrate that the organocatalytic oligomerization of unprotected quinine monomer is well-behaved and provides an expedient synthesis of highly functionalized oligocarbonates.

polymer by 1H NMR indicated an average degree of polymerization (DP) of 28, in good agreement with that predicted from the initial monomer/initiator ratio (M/I = 25). These results reveal the tolerance of the organocatalytic ringopening polymerization to the unprotected alkaloid, quinine. The molecular weight distributions (Mw/Mn = 1.38) of polymers initiated from quinine are slightly broader than those obtained from primary alcohol initiators (Mw/Mn = 1.11−1.16);6,14 this is likely due to the fact that initiation occurs from a secondary alcohol where propagation occurs from a primary alcohol.39 The use of an alcohol-containing bioactive agent/fluorescent probe such as quinine as an initiator provides an expedient synthesis of a biodegradable poly(carbonate) containing one bioactive agent attached to the terminus of the polymer chain.6,38 However, for many applications, it would be useful to have multiple bioactive agents attached to a single chain.40 The direct ring-opening polymerization of a monomer functionalized with an unprotected bioactive agent would provide efficient strategies to such constructs. Thus, we investigated the 2486


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Figure 3. Synthesis of copolymers bearing both quinine and guanidinium groups.

M,26,27 which would suggest that oligomers bearing pendant quinines would exhibit low fluorescence. To test this, we compared the fluorescence intensity of the P(MTC-Q16) oligomer to that of free quinine. As shown in Figure 4, the

The quinine carbonate MTC-Q can also be copolymerized with guanidinylated cyclic carbonates such as MTC-bocG, providing a route to water-soluble guanidinylated oligocarbonates6 containing pendant quinine groups. Both random and block copolymers are readily generated by ring-opening with alcohol initiators (Figure 3). Random copolymers were prepared by ring-opening polymerization of a mixture of MTC-Q and MTC-G with 5 mol % TU/DBU cocatalysts and benzyl alcohol in CH2Cl2 to afford P(MTC-Q10-r-bocG17) in 12% isolated yield after dialysis in MeOH. Removal of the Boc groups by treatment of the copolymer with trifluoroacetic acid afforded the water-soluble copolymer P(MTC-Q10-r-G17). Block copolymers were generated by addition of the MTCbocG monomer to benzyl alcohol in CH2Cl2 in the presence of 5 mol % TU/DBU for 1 h, followed by the addition of MTC-Q and polymerization for another hour (Table 2). Purification of Table 2. Copolymerization of MTC-Q and MTC-bocG target P(MTC-Q10-rbocG17) P(MTC-Q10-bbocG7) P(MTC-Q13-bbocG14)

time (h)

[M]o (M)

[M]o/ [I]o

Mn

Mw/ Mn

yield (%)

1

1

11

3000

1.4

12

2

1

11

3400

1.3

35

3

0.36

15

3900

1.5

69

Figure 4. Emission spectra of an aqueous solution of 0.1 mM P(MTCQ16) compared to that of a 0.1 mM solution of quinine in 0.1 M H2SO4.

the diblock copolymers was carried out by dialysis in MeOH and characterized by 1H NMR and gel permeation chromatography in DMF (Table 2) to give the boc-protected block copolymers P(MTC-Q10-b-bocG7) and P(MTC-Q13-bbocG14) (Figure S9, Supporting Information). Removal of the Boc groups by treatment of the copolymer with trifluoroacetic acid afforded the water-soluble block copolymer P(MTC-Q10-b-G7). Spectroscopic Properties of PP(MTC-Q16) and MTC-Q Dimer. The photophysics of quinine has been extensively investigated.24−27 While a number of oligomers23,32 and polymers28−32 containing quinine are known, few studies have described the fluoroescence behavior of quinine oligomers.41 Early studies had suggested that quinine exhibits self-quenching at solution concentrations greater than 10−3

fluorescence intensity (IP) of a 0.1 mM aqueous solution of quinine oligomer P(MTC-Q16) ([quinine] in oligomer = 1.6 mM) is considerably lower than that of a 0.1 mM aqueous solution of free quinine (ratio of fluorescence intensity IP/IQ ∼ 0.03). These data suggest that the self-quenching42 of quinine fluorescence is very efficient when 16 quinines are constrained into the oligocarbonate. Self-quenching was also observed, but was less efficient43 for the dimer of MTC-Q, prepared by reacting MTC-Q with ethylenediamine. The fluoresence intensity of the dimer (IQD) was approximately half that of quinine (IQ) at a similar concentration of quinine (0.1 mM, IQD/IQ ∼ 0.40, Figure S10). 2487


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The fluorescence spectra of an aqueous solution of the random P(MTC-Q10-r-G17) copolymer was monitored at pH 7 in HEPES buffer at 25 and 37 °C and compared to that of quinine. When the fluorescence intensity of an aqueous solution of oligomer (1 μM oligomer, [Q] = 10 μM) is monitored as a function of time, the fluorescence was observed to increase (Figure S11, Supporting Information), consistent with hydrolysis of the oligocarbonate to release fluorescent quinine-containing fragments. At 37 °C, the fluorescence intensity increased over a period of 5 days and then remained relatively constant. These preliminary results indicate that monitoring the increase in fluorescence might provide a useful means for monitoring the in situ degradation of water-soluble oligocarbonates. Similar strategies utilizing self-quenching fluorophores have been used to monitor enzymatic degradation of proteins and peptides in vitro and in vivo.42−44 Additional studies are underway to assess the merits of this fluorescence assay relative to the HPLC assay used previously6 to monitor oligocarbonate hydrolysis.

(5) Ong, Z. Y.; Fukushima, K.; Coady, D. J.; Yang, Y.-Y.; Ee, P. L. R.; Hedrick, J. L. J. Controlled Release 2011, 152, 120−126. (6) Cooley, C. B.; Trantow, B. M.; Nederberg, F.; Kiesewetter, M. K.; Hedrick, J. L.; Waymouth, R. M.; Wender, P. A. J. Am. Chem. Soc. 2009, 131, 16401−16403. (7) Albertsson, A. C.; Varma, I. K. Biomacromolecules 2003, 4, 1466− 1486. (8) du Boullay, O. T.; Saffon, N.; Diehl, J.-P.; Martin-Vaca, B.; Bourissou, D. Biomacromolecules 2010, 11, 1921−1929. (9) Bourissou, D.; Moebs-Sanchez, S.; Martin-Vaca, B. C. R. Chim. 2007, 10, 775−794. (10) Seyednejad, H.; Ghassemi, A. H.; van Nostrum, C. F.; Vermonden, T.; Hennink, W. E. J. Controlled Release 2011, 152, 168−176. (11) Tempelaar, S.; Mespouille, L.; Dubois, P.; Dove, A. P. Macromolecules 2011, 44, 2084−2091. (12) Rokicki, G. Prog. Polym. Sci. 2000, 25, 259−342. (13) Sanders, D. P.; Fukushima, K.; Coady, D. J.; Nelson, A.; Fujiwara, M.; Yasumoto, M.; Hedrick, J. L. J. Am. Chem. Soc. 2010, 132, 14724−14726. (14) Pratt, R. C.; Nederberg, F.; Waymouth, R. M.; Hedrick, J. L. Chem. Commun. 2008, 114−116. (15) Kiesewetter, M. K.; Shin, E. J.; Hedrick, J. L.; Waymouth, R. M. Macromolecules 2010, 43, 2093−2107. (16) Lohmeijer, B. G. G.; Pratt, R. C.; Leibfarth, F.; Logan, J. W.; Long, D. A.; Dove, A. P.; Nederberg, F.; Choi, J.; Wade, C.; Waymouth, R. M.; Hedrick, J. L. Macromolecules 2006, 39, 8574− 8583. (17) Dove, A. P.; Pratt, R. C.; Lohmeijer, B. G. G.; Waymouth, R. M.; Hedrick, J. L. J. Am. Chem. Soc. 2005, 127, 13798−13799. (18) Jerome, C.; Lecomte, P. Adv. Drug Delivery Rev. 2008, 60, 1056−1076. (19) Williams, C. K. Chem. Soc. Rev. 2007, 36, 1573−1580. (20) Xu, J. W.; Prifti, F.; Song, J. Macromolecules 2011, 44, 2660− 2667. (21) Jiang, X.; Vogel, E. B.; Smith, M. R.; Baker, G. L. Macromolecules 2008, 41, 1937−1944. (22) Achan, J.; Talisuna, A. O.; Erhart, A.; Yeka, A.; Tibenderana, J. K.; Baliraine, F. N.; Rosenthal, P. J.; D’Alessandro, U. Malar. J. 2011, 10 (144), 1−12. (23) Pires, M. M.; Emmert, D.; Hrycyna, C. A.; Chmielewski, J. Mol. Pharmacol. 2009, 75, 92−100. (24) Pant, D.; Tripathi, H. B.; Pant, D. D. J. Lumin. 1992, 51, 223− 230. (25) Joshi, N. K.; Rautela, R.; Joshi, H. C.; Pant, S. J. Lumin. 2011, 131, 1550−1555. (26) Schaefer, F. P.; Roellig, K. Z. Phys. Chem. 1964, 40, 198−222. (27) Mishra, H.; Pant, D.; Pant, T. C.; Tripathi, H. B. J. Photochem. Photobiol., A 2006, 177, 197−204. (28) Ye, Q.; Wang, X. S.; Zhao, H.; Xiong, R. G. Chem. Soc. Rev. 2005, 34, 208−225. (29) Kobayashi, N.; Iwai, K. J. Am. Chem. Soc. 1978, 100, 7071−7072. (30) Obaleye, J.; Caira, M.; Tella, A. J. Chem. Crystallogr. 2007, 37, 707−712. (31) Kim, B. M.; Sharpless, K. B. Tetrahedron Lett. 1990, 31, 3003− 3006. (32) Rowan, S. J.; Sanders, J. K. M. J. Org. Chem. 1998, 63, 1536− 1546. (33) Wender, P. A.; Galliher, W. C.; Goun, E. A.; Jones, L. R.; Pillow, T. H. Adv. Drug Delivery Rev. 2008, 60, 452−472. (34) Kolonko, E. M.; Kiessling, L. L. J. Am. Chem. Soc. 2008, 130, 5626−5627. (35) Tezgel, A. O.; Telfer, J. C.; Tew, G. N. Biomacromolecules 2011, 12, 3078−3083. (36) Torchilin, V. P. Adv. Drug Delivery Rev. 2008, 60, 548−558. (37) Pratt, R. C.; Lohmeijer, B. G. G.; Long, D. A.; Lundberg, P. N. P.; Dove, A. P.; Li, H.; Wade, C.; Waymouth, R. M.; Hedrick, J. L. Macromolecules 2006, 39, 7863−7871.

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CONCLUSIONS We have developed an expedient organocatalytic method for the generation of quinine-functionalized oligocarbonate polymers and copolymers with pendant guanidinium groups. These results highlight the functional group tolerance of the thiourea/ amine catalysts for the direct ring-opening polymerization of functionalized monomers. The oligocarbonate platform has the added benefit of being hydrolytically degraded under physiological conditions. Incorporation of the quinine moieties allows for the fluorescence monitoring of the decomposition of the oligomers in real time.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details; 1H and 13C NMR spectra; mass spectrometry and degradation data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Erika Geihe and Paul Wender for the use of the HPLC, Andrew Ingram for his technical assistance, and Professor Richard Zare for the use of the mass spectrometer. We gratefully acknowledge support from the NSF (GOALI CHE-0957386). This work was also supported by the NRF (NRF-2010-C00059) of the Korean Ministry of Education, Science and Technology (H.K.).

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REFERENCES

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