Phosphonoethylated Polyglycidols: A Platform for Tunable Enzymatic

May 8, 2013 - The graft copolymers with pendant phosphonate groups are promising candidates for hydrolytically degradable biomaterials, since all buil...
1 downloads 18 Views 1MB Size
Download PDF
Article pubs.acs.org/Macromolecules

Phosphonoethylated Polyglycidols: A Platform for Tunable Enzymatic Grafting Density Jens Koehler, Fabian Marquardt, Helmut Keul,* and Martin Moeller* Institute of Technical and Macromolecular Chemistry, RWTH Aachen University and Interactive Materials Research - DWI at RWTH Aachen e.V., Forckenbeckstr. 50, D-52056 Aachen, Germany S Supporting Information *

ABSTRACT: Polyether graf t polyester copolymers with pendant diethylphosphonatoethyl groups (DEPE), P(GDEPEco-(G-g-εCL)-co-G), were prepared by enzymatic grafting of εcaprolactone from polyglycidols carrying 0, 15.4, and 38.5% of DEPE groups. The graft copolymers were characterized by 1H, 13 C, and 31P NMR spectroscopy and SEC analysis. Despite the steric bulk of the DEPE residues, high initiation efficiencies (IE) of the phosphonoethylated macroinitiators could be achieved by appropriate choice of the reaction parameters such as lipase and monomer concentration. Depending on the degree of functionalization of polyglycidol with phosphonate residues (FDEPE), the IE is tunable in a wide range from 31 to 81%. The graft copolymers with pendant phosphonate groups are promising candidates for hydrolytically degradable biomaterials, since all building blocks are biocompatible and/or biodegradable.

■

INTRODUCTION Enzymes, especially lipases, show great potential to replace commonly used chemical catalysts in synthetic polymer chemistry.1,2 Milder reaction conditions can be used for enzyme-catalyzed reactions, and they are superior to chemical catalysts with respect to stereo-, chemo-, and regioselectivity.3−5 The selectivity of the enzyme can be used for the modification of polymers ending up with architecturally welldefined graft copolymers with tailored microstructures. The preparation of well-defined comb/graft copolymers, such as molecular bottlebrushes, has attracted considerable attention due to the broad scope of structures which can be generated.6,7 Bottlebrush copolymers can be synthesized by three conceptionally different methods, namely by the “grafting through”, “grafting to”, and “grafting from” approach.8,9 The prerequisite for the “grafting from” is a polymer backbone with a definite number of initiation sites. Polyglycidol (PG) fulfills this prerequisite because each repeating unit bears a pendant hydroxyl group which can initiate side-chain growth.10 Additionally, polyglycidols are biocompatible and were approved by the FDA as food and pharmaceutical additives.11 Our group investigated the preparation of cylindrical bottlebrush copolymers prepared by grafting of ε-caprolactone (εCL) from a linear or star-shaped polyglycidol (PG) as macroinitiator.12−14 By appropriate choice of the catalyst, Candida Antarctica Lipase B immobilized on a macroporous acrylic resin (Novozym 435), or zinc(II) octanoate, graft copolymers with identical chemical compositions but with different microstructures were obtained. Through chemical catalyzed grafting nearly all hydroxyl groups of polyglycidol © 2013 American Chemical Society

initiated side-chain growth, whereas with Novozym 435 the efficiency of initiation was below 40%.12 This was ascribed to the site selectivity of the lipase since the hydroxyl end groups of the growing PCL chains become better accessible for the lipase compared to the unreacted hydroxymethyl groups attached directly to the backbone. The distinction of the lipase between different steric situations was reported by others as well. Heise et al. reported a maximum initiation efficiency of 60% in the enzymatic grafting from poly(styrene-co-4-vinylbenzyl alcohol) containing 10% of the hydroxyl-functional monomer.15,16 Howdle et al. investigated the lipase-catalyzed grafting of εCL from a MMA-co-HEMA copolymer and observed that only 30−40% of the hydroxyl groups initiated the grafting. They enhanced the grafting efficiency by using a highly randomized MMA-co-HEMA copolymer up to 80%, suggesting that steric hindrance of the bulky PCL grafts and the proximity of the initiating sites along the polymeric backbone are decisive for the initiation efficiency in enzymatic grafting. Quantitative conversion of the pendant hydroxyl groups was achieved by using poly(ethylene oxide) methacrylate (PEGMA) instead of HEMA. This was attributed to the minimization of the steric hindrance by moving the initiating sites further away from the backbone.17,18 Besides the structural diversity, which can be obtained by the lipase-catalyzed “grafting from” technique, lipases are useful for the preparation of biodegradable polymers. Biodegradable Received: February 4, 2013 Revised: April 26, 2013 Published: May 8, 2013 3708

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

Article

hydride for 24 h, distilled under reduced pressure, and stored under a nitrogen atmosphere over molecular sieves (3 Å). Novozym 435 (Lipase B from Candida Antarctica, immobilized on a macroporous acrylic resin, 10 000 U g−1, Novo Nordisk) was dried in vacuum for 24 h and stored under nitrogen. All reactions were carried out in a nitrogen atmosphere. Nitrogen (Linde 5.0) was passed over molecular sieves (4 Å) and finely distributed potassium on aluminum oxide. Measurements. 1H NMR, 13C NMR, and proton decoupled 31P NMR spectra (31P{1H}) were recorded on a Bruker DPX-400 FTNMR spectrometer at 400, 101, and 162 MHz, respectively. Dimethyl sulfoxide (DMSO-d6) was used as solvent. The residual solvent signal served as internal reference. End-group signals are marked with a superscript E. 31P{1H} NMR spectra were referenced against 85% H3PO4 as external standard. The amphiphilic graft copolymers were analyzed using solvent mixtures (80% DMSO-d6 and 20% DCM (v/ v)). The coupling constants Jxy are given in hertz. Molecular weights (Mn,SEC and Mw,SEC) and molecular weight distributions (Mw/Mn) were determined by size exclusion chromatography (SEC). SEC analyses were carried out with THF or DMF as eluent. For THF SEC a high-pressure liquid chromatography pump (Knauer 6420) with a RI detector (Jasco RI-2031 plus) at 30 °C was used. The eluent was THF (HPLC grade, Carl Roth), and a flow rate of 1.0 mL/min was used. Four columns with MZ SDplus gel were applied. The length of each column was 300 mm and diameter 8 mm, and nominal pore widths of the gel particles were 50, 100, 1000, and 10 000 Å. The calibration was achieved with commercially available poly(methyl methacrylate) standards. For DMF SEC a high-pressure liquid chromatography pump (Knauer K-1001 Wellchrom) with a dual RI-/Visco detector (WGE ETA-2020) at 30 °C was used. The eluent was DMF (optigrade, Promochem) with 1 mg/mL LiBr, and a flow rate of 1.0 mL/min was used. Four columns with PSS GRAM gel were applied. The length of each column was 300 mm and diameter 8 mm, and nominal pore widths of the gel particles were 30, 100, 1000, and 3000 Å. Narrow distributed poly(methyl methacrylate) standards were used for calibration. Results were evaluated using the PSS WinGPC Unity software. Syntheses. Poly(ethoxyethyl glycidyl ether) P(EEGE) (1) and polyglycidol (PG) (2) were synthesized according to literature procedure.12 The results of the chemical analysis for P(EEGE)26 and PG26 are summarized in Table S1 of the Supporting Information. Poly(glycidol diethylphosphonatoethyl-co-glycidol), P(GDEPE-coG) (3a,b). Polyglycidol (PG26) (2) (4.866 g, 65.686 mmol OH) was dissolved in DMF (88 mL) and potassium tert-butoxide (1.64 mL of a 1 M solution in THF, 1.64 mmol) was added over 2 h using a syringe pump. Upon addition of the alkoxide, a small amount of insoluble coagulate was formed, which was identified as potassium salt of polyglycidol. The solution was stirred for 30 min at room temperature. The tert-butanol formed was removed by distillation. Diethyl vinylphosphonate (DEVP) (4.147 g, 25.265 mmol) was added, and the mixture was stirred for 64 h at room temperature. The coagulate formed was filtered, and the solvent was removed under reduced pressure. The product was redissolved in methylene chloride and precipitated in cold pentane to remove traces of unreacted DEVP. A brownish viscous liquid was obtained. P(GDEPE10-co-G16) 3b: yield 81%. P(GDEPE4-co-G22) (3a) was synthesized following the same synthetic protocol. Reagent ratios and reaction conditions are listed in Table S2. 3b: 1H NMR (DMSO-d6): δ 1.23 (tr, 6H, 3JHH = 6.95 Hz, POCH2CH3), 1.78 (quin, 2H, 3JHH = 6.89 Hz, ArCH2CH2), 2.05 (dtr, 2H, 3JHH = 7.12 Hz, 2JHP = 18.2 Hz, CH2OCH2CH2P), 2.62 (tr, 2H, 3 JHH = 7.60 Hz, ArCH2), 3.20−3.75 (m, 14H, ArCH2CH2CH2, OCH2CH(CH2OH)O, OCH2CH(CH2OCH2CH2P)O), 3.99 (quin, 4H, 3JHH = 6.85 Hz, POCH2CH3), 4.53 (br s, OCH2CH(CH2OH)O), 7.15−7.22 (m, 3H, Ar), 7.23−7.31 (m, 2H, Ar). 13C NMR (DMSOd6): δ 16.2 (d, 3JCP = 5.8 Hz, POCH2CH3), 26.0 (d, 1JCP = 136.8 Hz, CH2OCH2CH2P), 31.0 (ArCH2CH2), 31.6 (ArCH2), 61.0 (d, 2JCP = 6.1 Hz, POCH2CH3, OCH2CH(CH2OH)O), 64.7 (CH(CH2OCH2CH2P)O), 68.9−70.2 (ArCH2CH2CH2, OCH2CH(CH2 OCH2 CH2 P)O, OCH2 CH(CH2 OH)O), 78.0 (OCH2 CH-

polymers, especially poly(ε-caprolactone)-based (PCL) materials, recently received special attention because of their biocompatibility, hydrolytic degradability, good mechanical properties, and ease of manufacture.19−26 They have been considered in a variety of biomedical applications, for example, tissue engineering scaffolds or matrices in controlled drug release systems. Compared to other aliphatic polyesters, such as poly(lactide)s or poly(glycolide)s, the rate of hydrolysis of PCL is much lower, which makes it most suitable for long-term applications.27,28 Phosphorus-containing polymers received increasing interest due to their biodegradability, blood compatibility, and strong interactions with dentin, enamel, and bones.29−31 Hence, multifunctional polymers that are partly phosphonoethylated are of potential interest for many applications in the biomedical field. Recently, we reported on the synthesis of PGs with phosphonate ester side groups through Michael-type addition of the hydroxymethyl side groups of PG to diethyl vinylphosphonate under alkaline conditions.32 The phosphonate moiety is a versatile precursor for the corresponding ethyl phosphonate/phosphonic acids and phosphonic acids (Figure 1).

Figure 1. Diethyl phosphonate residue and acidic derivatives derived thereof.

In this paper we present the enzymatic grafting of εCL from partly phosphonoethylated PGs as multifunctional macroinitiators. The combination of biocompatible PG, biodegradable PCL, and the beneficial properties ascribed to phosphonates opens up new possibilities for the application of polyether graf t polyester copolymers in the biomedical field. In this study, we used PGs with three different concentrations of phosphonate groups ranging from 0 to 38.5% to initiate the grafting catalyzed by Novozym 435. The microstructures of the graft copolymers were investigated with special emphasis on the initiation efficiency in dependence of the degree of prefunctionalization with phosphonate groups. The poly(glycidol diethylphosphonatoethyl-co-(glycidol-graf t-ε-caprolactone)-co-glycidol) copolymers were characterized by means of NMR and SEC analysis. The introduction of phosphonate side groups has the potential to tune the hydrolytic degradation rate of the copolymers, thus opening up new possibilities for biomedical applications.

■

EXPERIMENTAL SECTION

Materials. Potassium tert-butoxide (1 M solution in THF, Aldrich), N,N-dimethylformamide (dry, over molecular sieves, Aldrich), and dichloromethane (p.a., VWR) were used as received. Diglyme was distilled over sodium before use. 3-Phenyl-1-propanol (≥98%, Fluka) was reacted with small amounts of sodium and distilled. Ethoxyethyl glycidyl ether (EEGE) was synthesized from 2,3epoxypropan-1-ol (glycidol) and ethyl vinyl ether according to Fitton et al.,33 purified by distillation, and stored under a nitrogen atmosphere over molecular sieves (3Å). Diethyl vinylphosphonate (95+%, Aldrich) was stirred with calcium hydride for 24 h, distilled under reduced pressure, and stored under a nitrogen atmosphere over molecular sieves (3 Å). ε-Caprolactone (99%, ABCR) was stirred with calcium 3709

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

Article

Figure 2. Overview on the polymer nomenclature used. With G: glycidol; DEPE: diethylphosphonatoethyl; εCL: ε-caprolactone; [CL]/[OH]: ratio of ε-caprolactone to the number of hydroxyl groups of the macroinitiator.

Table 1. Enzyme-Catalyzed Synthesis of Poly(glycidol diethylphosphonatoethyl-co-(glycidol-graf t-ε-caprolactone)-co-glycidol) P(GDEPE-co-(G-g-εCL)-co-G) 5a−f and 6a−f Using 3a and 3b, Respectively, as Macroinitiator (t = 22 h, T = 80 °C): Reagent Ratios, Conversion of εCL, and Yields macroinitiator polymer

no.

g (mmol OH)

εCL g (mmol)

Novozyma g (wt %)

εCLb conv

yieldc (%)

5a 5b 5c 5d 5e 5f 6a 6b 6c 6d 6e 6f

3a 3a 3a 3a 3a 3a 3b 3b 3b 3b 3b 3b

0.223 0.180 0.139 0.185 0.162 0.160 0.215 0.593 0.235 0.424 0.408 0.284

0.867, (7.598) 0.700, (6.133) 0.541, (4.736) 1.439, (12.607) 1.260, (11.040) 1.245, (10.904) 0.440 (3.857) 1.214 (10.638) 0.481 (4.216) 1.736 (15.200) 1.671 (14.639) 1.163 (10.190)

0.035, (4) 0.056, (8) 0.065, (12) 0.058, (4) 0.101, (8) 0.149, (12) 0.018 (4) 0.097 (8) 0.058 (12) 0.069 (4) 0.134 (8) 0.140 (12)

100 100 100 98 100 100 99 100 100 98 99 100

89 91 87 90 95 94 92 94 96 91 88 91

(1.899) (1.533) (1.184) (1.575) (1.380) (1.363) (0.964) (2.658) (1.053) (1.900) (1.829) (1.273)

Weight percent of Novozym related to the monomer in the feed. bThe conversion of εCL was determined by 1H NMR analysis after 22 h. cYield obtained after purification by precipitation in pentane.

a

(CH2OCH2CH2P)O), 79.8−80.1 (OCH2CH(CH2OH)O), 125.6 (Ar), 128.2 (Ar), 141.7 (Ar). 31P NMR (DMSO-d6): δ 28.6. Enzyme-Catalyzed Grafting from 2: Synthesis of Poly((glycidolgraft-ε-caprolactone)-co-glycidol), P((G-g-εCL)-co-G) (4a−f). The enzymatic grafting of ε-caprolactone (εCL) from PG26 (2) as macroinitiator was performed according to literature.12 Reagent ratios and reaction conditions are listed in Table S3. Enzyme-Catalyzed Grafting from 3a,b: Synthesis of Poly(glycidol diethylphosphonatoethyl-co-(glycidol-graf t-ε-caprolactone)-coglycidol), P(GDEPE-co-(G-g-εCL)-co-G) (5a−f, 6a−f). P(GDEPE4-co-G22) (3a) (0.180 g, 1.533 mmol OH) and εCL (0.700 g, 6.133 mmol) were heated to 50 °C in order to obtain a homogeneous solution. Novozym 435 (0.056 g, 8 wt %) was added, and the mixture was stirred for 22 h at 80 °C. The polymerization was quenched by addition of methylene chloride, and the enzyme was removed by filtration. The product was purified by precipitation in pentane, isolated by decantation of the mother liquor, and dried under reduced pressure. 5b: yield 91%. 5b: 1H NMR (DMSO-d6/DCM): δ 1.25 (tr, 6H, 3JHH = 7.02 Hz, POCH2CH3), 1.27−1.38 (m, 2H, OCOCH2CH2CH2), 1.38−1.48E (m, 2H, CH 2 CH 2 CH 2 OH), 1.48−1.67 (m, 4H, OCOCH 2 CH2CH2CH2), 1.78 (quin, 2H, 3JHH = 6.78 Hz, ArCH2CH2), 1.96− 2.12 (m, 2H, 2JHP = 18.4 Hz, CH2OCH2CH2P), 2.18−2.38 (m, 2H, OCOCH2CH2), 2.56−2.65 (m, 2H, ArCH2), 3.18−3.75 (m, 19H, ArCH 2 CH 2 CH 2 , OCH 2 CH(CH 2 OH)O, OCH 2 CH(CH 2 OCH2CH2P)O, OCH2CH(CH2OCOCH2)O, CH2CH2OH), 3.92−

4.08 (m, 6H, POCH2CH3, CH2CH2OCO), 4.12−4.34 (m, 2H, CHCH2OCO), 4.34−4.55 (br s, CHCH2OH groups), 7.10−7.32 (m, 5H, Ar). 13C NMR (DMSO-d6/DCM): δ 16.1 (d, 3JCP = 5.5 Hz, POCH2CH3), 24.0, 24.4E (COCH2CH2CH2CH2CH2O), 24.9, 25.0E (COCH 2 CH 2 CH 2 CH 2 CH 2 O), 26.1 (d, 1 J CP = 136.5 Hz, CH2OCH2CH2P), 27.8, 32.1E (COCH2CH2CH2CH2CH2O) 31.0 31.7 (ArCH2), 33.4, 33.6E (ArCH2CH2), (COCH 2 CH 2 CH 2 CH 2 CH 2 O), 60.6 E , 63.4 (COCH 2 CH 2 CH2CH2CH2O), 60.9 (POCH2CH3, OCH2CH(CH2OH)O), 64.7 (CH(CH2OCH2CH2P)O), 68.3−70.0 (ArCH2CH2CH2, OCH2CH(CH2OCH2CH2P)O, OCH2CH(CH2OH)O), 76.9 (CH(CH2OCO)O), 78.0 (OCH2CH(CH2OCH2CH2P)O), 80.0, 80.1 (OCH2CH(CH2OH)O), 125.5 (Ar), 128.0 (Ar), 141.6 (Ar), 172.6, 172.7E (COCH2CH2CH2CH2CH2O). 31P NMR (DMSO-d6/DCM): δ 28.4. The graft copolymers 5a, 5c−f were synthesized in analogy to 5b. Polymers 6a−f were synthesized with 3b as macroinitiator following the same synthetic protocol. NMR signals of graft copolymers 6a−f were the same as reported for 5b only differing in relative intensity. The reagent ratios and reaction conditions are listed in Table 1.

■

RESULTS AND DISCUSSION The enzymatic and chemical catalyzed ring-opening polymerization of ε-caprolactone (εCL) using polyglycidol (PG) as multifunctional macroinitiator leads to graft copolymers with 3710

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

Article

Figure 3. Multifunctional macroinitiators prepared within this study: Polyglycidol with 26 repeating units, PG26 (2), phosphonoethylated PGs P(GDEPEx-co-Gy) with x = 4 (3a) and 10 (3b).

Figure 4. 1H NMR spectra of PG26 (2) (top) and P(GDEPE10-co-G16) (3b) (bottom) in DMSO-d6. ∗ = MeOH.

significantly different microstructures.12 Chemical catalysis with zinc(II) octanoate leads to a high conversion of PGs hydroxyl groups (>80%), whereas with Novozym 435 less than 40% of the hydroxyl groups were converted. Enzyme catalysis leads to loosely grafted copolymers with head-to-tail structures, since the majority of PGs hydroxyl groups remained unreacted. The remaining hydroxyl groups were converted chemically to prepare heterografted bottlebrush copolymers.12−14 In the present work we focused on the synthesis of polyether graf t polyester copolymers with pendant phosphonate groups. The synthetic strategy comprises the preparation of phosphonoethylated polyglycidols followed by ring-opening polymerization of εCL by means of enzymatic catalysis. A commercially available lipase, Novozym 435, immobilized on a macroporous resin was chosen as catalyst. The objective of this study was to elucidate the stability of the phosphonate residues toward enzymatic transesterification and to evaluate the influence of the prefunctionalization of polyglycidol with diethyl phosphonatoethyl groups (DEPE) on the density of the ε-caprolactone grafting. For this purpose, polyglycidols with 0, 15.4, and 38.5% of phosphonoethylated hydroxymethyl groups were used as multifunctional macroinitiators. The lipase concentration and the monomer concentration were varied in order to study their influence on the initiation efficiency of the hydroxyl groups of the macroinitiators as well. Syntheses. Synthesis and Characterization of the Phosphonoethylated Macroinitiators (3a,b). Polyglycidol (PG) was obtained in a two-step synthetic procedure through

anionic ring-opening polymerization of ethoxyethyl glycidyl ether with 3-phenyl-1-propanol/10% potassium 3-phenyl-1propanolate as initiator.12 The polymerization was conducted at 100 °C. Finally, the acetal protection group was removed under acidic conditions, and a linear PG with 26 repeating units (PG26 (2)) was obtained as proven by 1H NMR analysis. Partly phosphonoethylated polyglycidols, P(GDEPE-co-G), were obtained by alkaline-catalyzed Michael-type addition of the hydroxymethyl side groups of PG to the vinylic double bond of diethyl vinylphosphonate (DEVP).37 This approach allows the controlled addition of diethylphosphonatoethyl groups (DEPE) to a hydroxyl-functional polymer, since the degree of functionalization with DEPE groups (FDEPE) can be adjusted by appropriate choice of the molar ratio of DEVP to PGs hydroxyl groups. Two different macroinitiators (3a,b) with varying FDEPE were synthesized following the aforementioned route. Figure 3 gives an overview on the chemical composition of the macroinitiators prepared: PG26 (2), P(GDEPE4-co-G22) (3a), and P(GDEPE10-co-G16) (3b). The P(GDEPE-co-G)s (3a,b) were obtained in yields >81%. The phenyl group of 3-phenyl-1-propanol, which was used as initiator for the synthesis of 2, served as internal reference in 1H NMR spectra for the calculation of FDEPE and the absolute molecular weights Mn,NMR of the macroinitiators. The 1H NMR spectra of PG26 (2) and P(GDEPE10-co-G16) (3b) are shown in Figure 4. The number of phosphonate groups which were attached to PG26 was calculated by comparing the signal intensity of the 33711

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

Article

Table 2. Macroinitiators with Varying Phosphonate Concentration PG26 (2) and P(GDEPE-co-G) (3a,b): Degree of Functionalization with Diethylphosphonatoethyl Groups (FDEPE), Percentage of Glycidol Repeating Units, Average Molecular Weight of the Repeating Unit, Number-Average Molecular Weights Determined by 1H NMR, and SEC Analysis and Molecular Weight Distributions P(GDEPEx-co-Gy)

FDEPE a x (%)

glycidola y (%)

Mn,NMRb (g/mol)

Mw,ruc (g/mol)

Mn,SECd (g/mol)

Mw/Mn,SECd

PG26 (2) P(GDEPE4-co-G22) (3a) P(GDEPE10-co-G16) (3b)

0 (0) 4 (15.4) 10 (38.5)

26 (100) 22 (84.6) 16 (61.5)

1926 2583 3567

74.08 99.35 137.21

2600 2900 3600

1.2 1.3 1.4

a According to 1H NMR analysis. bAbsolute molecular weight according to 1H NMR analysis. The accuracy of integration in 1H NMR spectra is ±5%. cAverage molecular weight of the repeating unit (ru), calculated by dividing Mn,NMR through the number of repeating units (26). dMolecular weight and molecular weight distribution determined by size exclusion chromatography (SEC) using narrow distributed poly(methyl methacrylate) standards and DMF as eluent.

prefunctionalized with DEVPthe concentration of εCL per initiating hydroxyl group was kept at constant ratios of [CL]/ [OH] = 4 and 8, respectively. However, the enzyme concentration was varied between 4 and 12 wt % in order to elucidate the influence of the catalyst concentration on the initiation efficiency of the macroinitiators. 31 P NMR Analysis of the Graft Copolymers 5 and 6 Inertness of Phosphonate Groups? The prerequisite for a successful preparation of polyether graf t polyesters with pendant phosphonate groups is the resistance of the phosphonate groups toward lipase-catalyzed transesterification. Since lipases are useful catalysts for inter- and intramolecular transesterifications,34,35 we evaluated the behavior of the lipase toward the DEPE residues on the basis of 31P NMR analysis of the graft copolymers in comparison to the phosphonoethylated macroinitiator 3. Figure 5 shows the 31P NMR spectra of P(GDEPE4-co-G22) (3a) and P(GDEPE4-co-(G-g-εCL7)14-co-G8) (5b) as an example. P(GDEPE4-co-G22) (3a) exhibits a single signal at δ = 28.7 ppm. The spectrum of the graft copolymer 5b shows only one signal, too, which is located at δ = 28.4 ppm. The deviation in the chemical shift can be ascribed to the change in the NMR solvent from DMSO-d6 (3a) to DMSO-d6/DCM (5b). Hence, based on 31P NMR data, the DEPE groups are not involved in transesterification reactions. 1 H NMR and SEC Analysis of the Graft Copolymers 4− 6. The NMR spectra of the graft copolymers 4, 5, and 6 were recorded in a mixture of DMSO-d6 and DCM (v/v, 80/20) to account for the amphiphilic structure of the graft copolymers because the resolution of the hydrophobic PCL grafts in comparison to the hydrophilic PGDEPE backbone depends strongly on the NMR solvent used. In CDCl3 the PCL part is well soluble, whereas the backbone is collapsed. For DMSO-d6 it is vice versa. Thus, we chose an appropriate mixture of DMSO-d6/DCM (80%/20%) as NMR solvent. DCM has a chemical shift of δ = 5.76 ppm36 in DMSO-d6 and does not interfere with any signal of the graft copolymers 4−6. Figure 6 shows the 1H NMR spectra of macroinitiator P(GDEPE4-co-G22) (3a) and P(GDEPE4-co-(G-g-εCL7)14-co-G8) (5b) which was prepared by enzymatic catalysis with 8 wt % of the lipase as an example. The absolute molecular weights (Mn,NMR) of the graft copolymers and the initiation efficiencies (IE) of the macroinitiators were determined by end-group analysis from the corresponding 1H NMR spectrum. The molecular weights of the copolymers are made up of the molecular weight of the parent macroinitiator and the total number of εCL units (v) which were grafted from the macroinitiator. By comparing the signal intensity of the 3phenyl-1-propyl end group (1−3) to the methylene group 19 of

phenyl-1-propyl end group (1, 2, 3) with signal 12, which was assigned to the methylene group adjacent to the phosphorus atom. The degrees of functionalization with DEPE groups were 15.4% (3a) and 38.5% (3b), meaning 4 and 10 phosphonoethylated hydroxyl groups on average, respectively. Thus, the phosphonoethylated PGs possess 84.6% of hydroxymethyl side groups (3a) and 61.5% (3b), which can initiate the grafting of εCL. The results of the chemical analysis of 2 and 3a,b are summarized in Table 2. For PG26 (2) a narrow distributed monomodal elution curve was obtained using DMF as eluent. The SEC analysis of the phosphonoethylated macroinitiators 3a,b showed monomodal elution curves, too, with molecular weight distributions in the range of 1.3 ≤ Mw/Mn ≤ 1.4. The presence of a small shoulder to higher molecular weight in the elution curves of 3a,b can be ascribed to transesterification reactions at the phosphonate residues (Figure S1).32 Enzyme-Catalyzed Grafting of εCL from PG26 (2) and P(GDEPE-co-G) (3a,b). The ring-opening polymerization of εCL using PG26 (2), P(GDEPE4-co-G22) (3a), and P(GDEPE10-co-G16) (3b) as macroinitiators was performed in bulk with Novozym 435 as catalyst at 80 °C (Scheme 1). The macroinitiator and Scheme 1. Preparation of P((G-g-εCL)-co-G) (4a−f), P(GDEPE4-co-(G-g-εCL)-co-G) (5a−f), and P(GDEPE10-co-(Gg-εCL)-co-G) (6a−f)

εCL were heated to 50−60 °C in order to obtain a homogeneous solution prior to the addition of the catalyst. The corresponding polyether graf t polyester copolymers poly((glycidol-graf t-ε-caprolactone)-co-glycidol), P((G-gεCLn)m-co-Gp) (4a−f), poly((glycidol diethylphosphonatoethyl)-co-(glycidol-graf t-ε-caprolactone)-co-glycidol), P(GDEPE4-co(G-g-εCLn)m-co-Gp) (5a−f), and P(GDEPE10-co-(G-g-εCLn)m-coGp) (6a−f) were obtained after precipitation in pentane as waxy solids. Although, the macroinitiators differ in the absolute number of initiating sites (2: 26; 3a: 22; and 3b: 16 OH groups)due to different numbers of PGs hydroxymethyl groups which were 3712

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

Article

Figure 5. 31P NMR spectra of P(GDEPE4-co-G22) (3a) in DMSO-d6 and P(GDEPE4-co-(G-g-εCL7)14-co-G8) (5b) in a mixture of DMSO-d6/DCM.

Figure 6. 1H NMR spectra of P(GDEPE4-co-G22) (3a) in DMSO-d6 (top) and P(GDEPE4-co-(G-g-εCL7)14-co-G8) (5b) in DMSO-d6/DCM. # = DCM (bottom).

the PCL side chains, the number of εCL units which were attached to the macroinitiator was calculated. The IE was determined by comparison of the 3-phenyl-1-propyl group with signal 17, which was assigned to the methylene groups adjacent to the converted hydroxymethyl side groups of the macroinitiator. Finally, the degree of polymerization of the side chains (Pn,SC) can be obtained by dividing the number of εCL repeating units (v) through the number of converted hydroxyl groups. Table 3 summarizes the number of εCL repeating units (v) which were grafted from the macroinitiators, molecular weights determined from NMR (Mn,NMR) and SEC analysis (Mn,SEC), and the IEs and Pn,SC of the graft copolymers 4−6. The SEC analysis of the graft copolymers was performed with THF as eluent and narrow distributed PMMA standards. Figure 7 shows the elution curves of 4c, 5c, and 6c prepared

through enzymatic catalysis (12 wt % Novozym) with [CL]/ [OH] = 4 equiv as representative examples. The graft copolymers show monomodal elution curves, with molecular weight distributions ranging between 1.2 ≤ Mw/Mn ≤ 1.6, depending on the applied macroinitiator. With increasing number of DEPE groups attached to the macroinitiator a tailing of the elution curves to lower molecular weights is observed, which leads to a broadening of the molecular weight distribution. This can be attributed to the amphiphilic structure of the graft copolymers. The hydrophobic PCL grafts are well soluble in THF, whereas the hydrophilic macroinitiators themselves cannot be eluted with THF. Thus, the higher the weight fraction of PCL, the better is the solubility of the copolymer in THF. This is further confirmed by the SEC curves of copolymer 6f, which was prepared with 8 equiv of 3713

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

Article

Table 3. Synthesis of P((G-g-εCLn)m-co-Gp) (4a−f), P(GDEPE4-co-(G-g-εCLn)m-co-Gp) (5a−f), and P(GDEPE10-co-(G-g-εCLn)m-coGp) (6a−f): Macroinitiator Used, Lipase Concentration, Ratio of εCL Repeating Units per OH Group of the Macroinitiator, Conversion of εCL, Molecular Weights Obtained from 1H NMR and SEC Analysis, and Molecular Weight Distributions from SEC polymer

initiator

Novozyma (wt %)

[CL]/[OH]b

εCL convc % (v)

Mn,NMRd

Mn,SECe

Mw/Mn,SECe

4a 4b 4c 4d 4e 4f 5a 5b 5c 5d 5e 5f 6a 6b 6c 6d 6e 6f

2 2 2 2 2 2 3a 3a 3a 3a 3a 3a 3b 3b 3b 3b 3b 3b

4 8 12 4 8 12 4 8 12 4 8 12 4 8 12 4 8 12

4 4 4 8 8 8 4 4 4 8 8 8 4 4 4 8 8 8

96 (148) 100 (131) 99 (162) 78 (206) 99 (245) 99 (239) 100 (92) 100 (102) 100 (87) 98 (181) 100 (204) 100 (215) 99 (79) 100, (66) 100 (67) 98 (141) 99 (131) 100 (145)

18 819 16 878 20 417 25 439 29 890 29 205 13 084 14 225 12 513 23 242 25 639 27 123 12 584 11 100 11 214 19 660 18 519 20 117

18 600 18 500 21 700 20 700 24 700 20 800 13 000 14 100 12 500 21 400 22 500 22 800 10 200 9100 9100 18 600 17 800 19 000

1.6 1.4 1.2 1.7 1.5 1.7 1.4 1.3 1.3 1.5 1.6 1.5 1.5 1.6 1.5 1.6 1.4 1.5

Weight percent of enzyme in relation to monomer in the feed. bEquivalents of εCL per hydroxyl group of the macroinitiator in the feed. Conversion of εCL and equivalents of εCL per macroinitiator (v) according to 1H NMR analysis. dAbsolute molecular weight (Mn,NMR) determined by 1H NMR analysis. The accuracy of integration in 1H NMR spectra is ±5%. eNumber-average molecular weight (Mn,SEC) and molecular weight distribution (Mw/Mn) determined by SEC analysis using narrow distributed PMMA standards and THF as eluent.

a c

5c, and 6c revealed that the poly(ε-caprolactone) homopolymers have molecular weights ranging from a few hundred to ∼4000 Da, depending on the monomer and enzyme concentration in the feed (Figures S4−S6). Different series were identified, thus proving the coexistence of cyclic and linear PCL. The presence of the free PCL could affect the calculation of the molecular weights and the degree of polymerization of the PCL side chains. However, the concentration of the free PCL is too low to be of considerable importance, since the inaccuracy of NMR integration of ±5% is higher than the PCL content. Since MALDI-TOF MS proved that the PCL is of rather low molecular weight, we determined the weight fraction of PCL by integration of the low molecular weight region in the SEC curves of the graft copolymers. An overview on the concentrations of free PCL in the products is shown in the Supporting Information (Figure S7 and Table S5). Moreover, the grafting of chains can also occur through condensation of carboxyl-terminated PCL chains to the pendant hydroxyl groups of the macroinitiator instead of ring-opening of εCL. Certainly, the latter is highly favored since it is much more easier for the enzyme to form an activated monomer complex with εCL compared to carboxyl-terminated PCL chains. This is emphasized by the high excess of εCL in the reaction mixture compared to carboxyl-terminated PCL. The analysis of the microstructures of the graft copolymers 4a−f, 5a−f, and 6a−f, initiation efficiencies (IE), and degrees of polymerization of the side chains (Pn,SC) is summarized in Table 4. Factors Influencing the Initiation Efficiency in Enzymatic Grafting of εCL. Lipase Concentration. In a first series of experiments, we investigated the lipase-catalyzed grafting using PG26 (2) (0% DEPE groups), P(GDEPE4-co-G22) (3a) (15.4% DEPE groups), and P(GDEPE10-co-G16) (3b)

Figure 7. THF-SEC traces of selected graft copolymers prepared by enzyme catalysis: P((G-g-εCL12)14-co-G12) (4c), P(GDEPE4-co-(G-gεCL7)12-co-G10) (5c), and P(GDEPE10-co-(G-g-εCL7)10-co-G6) (6c).

εCL, in comparison to 6c. In 6f the weight fraction of PCL is higher, which makes this graft copolymer better soluble in THF, and no tailing to lower molecular weights is observed (Figure S2). Additionally, the DMF-SEC traces of 4c, 5c, and 6c were measured. Since DMF is a good solvent for the macroinitiator as well as the PCL grafts, the aforementioned tailing is not observed (Figure S3 and Table S4). However, traces of free poly(ε-caprolactone), ranging from 2 to 4 wt % were found in the products. Their occurrence is most probably due to traces of enzyme-bound water, which initiated the homopolymerization of εCL.37 The free PCL could not be removed completely from the products by precipitation in pentane. MALDI-TOF MS analysis of the graft copolymers 4c, 3714

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

Article

Table 4. Microstructures of the Graft Copolymers 4a−f, 5a−f, and 6a−f According to 1H NMR Analysis, Initiation Efficiencies, and Degrees of Polymerization of the PCL Side Chains entry PG26 (2)

(3b)

[CL]/[OH]

P(G-g-εCLn)m-co-Gp n; m; p

IEa m (%)

entry

4 8 12 4 8 12 lipase (wt %)

4 4 4 8 8 8 [CL]/[OH]

19; 8; 18 10; 13; 13 12; 14; 12 23; 9; 17 19; 13; 13 20; 12; 14 P(GDEPE4-co-(G-g-εCLn)m-co-Gp) n; m; p

8 (31) 13 (50) 14 (54) 9 (35) 13 (50) 12 (46) IEa m (%)

5a 5b 5c 5d 5e 5f entry

4 8 12 4 8 12 lipase (wt %)

4 4 4 8 8 8 [CL]/[OH]

8; 12; 10 7; 14; 8 7; 12; 10 15; 12; 10 19; 11; 11 20; 11; 11 P(GDEPE10-co-(G-g-εCLn)m-co-Gp) n; m; p

12 (55) 14 (64) 12 (55) 12 (55) 11 (50) 11 (50) IEa m (%)

6a 6b 6c 6d 6e 6f

4 8 12 4 8 12

4 4 4 8 8 8

7; 11; 5 6; 11; 5 7; 10; 6 12; 12; 4 10; 13; 3 12; 12; 4

4a 4b 4c 4d 4e 4f

P(GDEPE4-co-G22) (3a)

P(GDEPE10-co-G16)

lipase (wt %)

11 11 10 12 13 12

(69) (69) (63) (75) (81) (75)

Pn,SCb n 19 10 12 23 19 20 Pn,SCb n 8 7 7 15 19 20 Pn,SCb n 7 6 7 12 10 12

a IE: initiation efficiency of the macroinitiator. bPn,SC: degree of polymerization of the side chains. Calculated from the number of εCL units (v) grafted from the macroinitiator divided by the number of sites which initiated the grafting. Calculation of n, m, and p: n = Pn,SC; m = IE, p = number of initiating sites minus IE. For all calculations an error from NMR analysis of ±5% must be considered.

(38.5% DEPE groups) as macroinitiators for the ring-opening polymerization of εCL with a ratio of monomer to initiator of [CL]/[OH] = 4. The concentration of Novozym 435 was varied from 4 to 12 wt % to investigate the influence of lipase concentration on the IE of the initiator. PG26 (2) (0% DEPE Groups). For the experiments 4a−c performed with the nonfunctionalized polyglycidol PG26 (2) as macroinitiator the IEs ranged from 31 to 54%, depending on the lipase concentration applied. In 4a synthesized with 4 wt % of Novozym, 31% of the hydroxyl groups of 2 were converted. With 8 wt % of Novozym the initiation efficiency increased to 50% in 4b. A further increase in catalyst concentration to 12 wt % did not enhance the grafting density since 54% of PGs hydroxyl groups were converted in 4c, which is within the error of NMR integration comparable to 4b. P(GDEPE4-co-G22) (3a) (15.4% of DEPE Groups). For the grafting of εCL initiated by 3a the IEs obtained ranged from 55% in 5a and 5c to 64% in 5b. With 4 wt % of the lipase 55% of the hydroxyl groups of 3a initiated the grafting. A lipase concentration of 8 wt % as in 5b afforded the highest initiation efficiency of 64% within this series of experiments. As reported for PG26 (2), an increase in lipase concentration to 12 wt % did not lead to an improved IE; on the contrary, the IE drops off to 55% again. P(GDEPE10-co-G16) (3b) (38.5% of DEPE Groups). When P(GDEPE10-co-G16) (3b) is used to initiate the ROP of εCL, the IEs can be increased to 63−69%, for a monomer concentration of 4 equiv of εCL per hydroxyl group of the macroinitiator. Lipase concentrations of 4 and 8 wt % afforded higher IEs (6a, 6b) compared to 12 wt % of the lipase (6c). The grafting results obtained with 3b as macroinitiator show no dependence on the lipase concentration. Monomer Concentration. In proceeding experiments (4d− f, 5d−f, and 6d−f) we raised the εCL concentration to [CL]/ [OH] = 8 equiv and applied the same lipase concentrations as

discussed previously. Here, we analyzed the influence of the monomer concentration on the initiation efficiency of the different macroinitiators. PG26 (2) (0% DEPE Groups). The IEs which were observed for PG26 (2), at a monomer concentration of [CL]/[OH] = 8, ranged from 35 to 50%, depending on the lipase concentration applied. In 4d synthesized with 4 wt % of Novozym, 35% of the hydroxyl groups of 2 were converted. With 8 wt % of Novozym the IE increased to 50% in 4e. By applying 12 wt % of the lipase 46% of PGs hydroxyl groups were converted (4f). These results are comparable to 4a−c prepared at lower monomer concentration of [CL]/[OH] = 4 (compare Table 4). Thus, an increase in the monomer concentration from 4 to 8 equiv does not alter the IEs in the enzymatic grafting of εCL from the nonfunctionalized PG26 (2). P(GDEPE4-co-G22) (3a) (15.4% of DEPE Groups). With 3a as multifunctional macroinitiator 50−55% of the hydroxyl groups initiated the ring-opening polymerization of εCL. At a lipase concentration of 4 wt % (5d), the highest IE of 55% was obtained for the experiments with 8 equiv of εCL. In comparison to 5a−c, where a ratio of monomer to initiator of [CL]/[OH] = 4 was applied, the average IEs decreased from 58 to 52% in 5d−f. Since these efficiencies are comparable, an increase in monomer concentration does not improve the IE of P(GDEPE4-co-G22) (3a). P(GDEPE10-co-G16) (3b) (38.5% DEPE Groups). In contrast to the experiments using PG26 (2) and P(GDEPE4-co-G22) (3a) as initiator, the initiation efficiency of P(GDEPE10-co-G16) (3b) could be increased to 75−81% by raising the monomer concentration to [CL]/[OH] = 8 equiv in 6d−f. With 4 equiv of εCL per hydroxyl group of the initiator IEs of 63−69% (6a− c) were achieved. Graft copolymer 6e, which was synthesized with 8 wt % of Novozym and 8 equiv of εCL, exhibits 81% of esterified hydroxyl groups, the highest IE of all enzymatic grafting experiments conducted. 3715

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

Article

An influence of the monomer concentration on the IEs of the macroinitiators was only observed in the experiments performed with P(GDEPE10-co-G16) (3b) as initiator. This suggests that the prefunctionalization of PG26 (2) with DEPE groups influences the behavior of the macroinitiator, thus leading to improved IEs in the enzymatic grafting. In the following section, the tunability of the grafting density by varying FDEPE will be discussed in detail. Prefunctionalization of PG26 (2) with DEPE Groups. The influence of the FDEPE on the initiation efficiency in the lipase-catalyzed grafting with a monomer concentration of 4 equiv of εCL is shown in Figure 8. The IE was plotted against FDEPE of the macroinitiators for the different lipase concentrations.

Figure 9. Plot of initiation efficiencies in dependence of the prefunctionalization of the macroinitiators with DEPE groups for [CL]/[OH] = 8. Average IEs: 4d−f 44%; 5d−f 52%, and 6d−f 77%. ■ = [CL]/[OH] = 8 equiv, 4 wt % CALB, ● = [CL]/[OH] = 8 equiv, 8 wt % CALB; ▲ = [CL]/[OH] = 8 equiv, 12 wt % CALB.

The impact of FDEPE on the initiation efficiency in the lipasecatalyzed grafting of εCL can be demonstrated most effectively by comparing the grafting experiments with 4 wt % of the lipase and 4 equiv (4a, 5a, and 6a) and 8 equiv of εCL (4d, 5d, 6d), respectively. By applying 4 wt % of the lipase the initiation efficiency can be tuned from 31% (4a) to 69% (6a) when a ratio of [CL]/[OH] = 4 is adjusted in the feed and from 35% (4d) to 75% (6d) with 8 equiv of εCL. From these results it can be concluded that a higher selectivity is obtained for lower lipase concentrations (4 and 8 wt %, respectively). The results for the enzyme-catalyzed grafting of εCL from PG26 (2) are in agreement with former studies of our group.12 The rather low initiation efficiencies in enzymatic grafting experiments were explained by steric effects. It is much easier for the bulky activated monomer−enzyme complex to interact with the hydroxyl groups at the chain ends of initially formed PCL grafts compared to an approach of the unreacted hydroxyl groups at the PG backbone. Since the growing chain ends are better accessible, a large number of hydroxyl groups remained unreacted.12,13 Based on the results we obtained for the enzymatic grafting of εCL from partly phosphonoethylated PGs 3a,b the achievable density of the enzymatic grafting depends on steric effects as aforementioned but also on the hydrodynamic radii of the polymer coils. This was estimated from the number-average molecular weights of the macroinitiators 2 and 3a and 3b obtained by SEC measurements with DMF as eluent. For PG26 (2) Mn,SEC = 2600 g/mol was found. The functionalization with four DEPE groups in 3a resulted in an increase in molecular weight to Mn,SEC = 2900 g/mol, and for P(GDEPE10-co-G16) (3b) an Mn,SEC = 3600 g/mol was measured. Since the molecular weights derived from SEC measurements are a function of the hydrodynamic radii of the polymer coils, the Mn,SEC values suggest an opening of the polymer coil with increasing FDEPE (Figure 10). This was further supported by dynamic light scattering experiments of 2, 3a, and 3b in water as well as εCL as solvent (Table S6). In aqueous solution, aggregates of PG26 (2) show the largest hydrodynamic radius (Rh = 278 nm) because of the highest number of pendant hydroxyl groups. The Rh of

Figure 8. Plot of initiation efficiencies in dependence of the prefunctionalization of the macroinitiators with DEPE groups for [CL]/[OH] = 4 equiv. Average IEs: 4a−c 45%; 5a−c 58%, and 6a−c 65%. ■ = [CL]/[OH] = 4 equiv, 4 wt % CALB; ● = [CL]/[OH] = 4 equiv, 8 wt % CALB, ▲ = [CL]/[OH] = 4 equiv; 12 wt % CALB.

On the one hand, the average IE increases with increasing FDEPE from 45% obtained with 2 and 58% with 3a to 65% with 3b. On the other hand, the range of IEs which can be achieved by varying the lipase concentration decreases significantly with increasing FDEPE. For 4a−c which were prepared starting from PG26 (2) IEs of 31−54% were obtained, whereas for 5a−c IEs of 55−64% were achieved. The IEs for the grafting experiments 6a−c with 3b as initiator (63−69%) differ only by one group, and no dependence on the lipase concentration is observed. The IE seems to approach a maximum which cannot be altered by further increasing FDEPE of the macroinitiator while keeping the [CL]/[OH] ratio at 4 equiv. In the grafting experiments 4d−f, 5d−f, and 6d−f where a ratio of monomer to initiator [CL]/[OH] = 8 was applied, the impact of FDEPE on the IE is much more pronounced as shown in Figure 9. The plot of the IEs for [CL]/[OH] = 8 equiv, in dependence of the degree of prefunctionalization of the macroinitiator with DEPE groups (Figure 9), shows an almost linear increase in the IE with increasing FDEPE. In the grafting experiments with PG26 (2) the IE does not exceed 50%, and an average IE of 44% was calculated. The prefunctionalization of the macroinitiator with DEPE groups leads to an average IE of 52% when four DEPE groups are attached to PG26. The functionalization with 10 DEPE groups, as in 3b, enables an average IE of 77%. 3716

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

Article

The hydrophilicity of the graft copolymers carrying DEPE groups can be tuned between 0.26 and 0.47, although the molecular weights are in the same order of magnitude (11.2K− 12.5K). This is a significant increase compared to graft copolymer 4c without DEPE groups. The phosphonoethylated PGs (3) are well suited precursors to achieve a dense grafting by enzymatic means. By appropriate choice of the reaction parameters, the IE could be tuned from 31% up to 81%. Moreover, the prefunctionalization with DEPE groups can be used effectively to tailor the hydrophilicity of the corresponding graft copolymers as shown in Table 5. Thus, it is possible to prepare polyether graf t polyester copolymers with tunable grafting density and hydrophilicity by grafting of εCL from phosphonoethylated polyglycidols by enzymatic means.

■

CONCLUSION For the first time phosphonoethylated polyglycidolswith 15.4% and 38.5% of phosphonate groupswere used as multifunctional macroinitiators for the preparation of polyether graf t polyester copolymers with pendant phosphonate groups. The macroinitiators were synthesized by Michael-type addition of polyglycidols hydroxyl groups to diethyl vinylphosphonate. The copolymers were prepared through ring-opening polymerization of εCl by enzymatic catalysis (Novozym 435) and characterized by NMR and SEC analysis, with special emphasis on the initiation efficiency of the macroinitiators in dependence of lipase and monomer concentration. The results were compared to a nonfunctionalized polyglycidol. It was shown that the initiation efficiency can be tuned effectively by introducing phosphonate groups to the polyglycidol backbone. Depending on the degree of functionalization with phosphonate groups, grafting densities of 31% to 81% were achieved. Thus, we developed a straightforward procedure to prepare densely grafted copolymers by enzymatic means, which is still a challenge in synthetic polymer chemistry.

Figure 10. Sketch of the hydrodynamic volume of the macroinitiators 2 and 3a,b in solution depending on the prefunctionalization with DEPE groups.

P(GDEPE10-co-G16) (3b) is with 184 nm significantly smaller. The reversed situation was observed in εCL as hydrophobic medium. Here, the hydrodynamic radius of 3b is 280 nm, and PG26 (2) exhibits a radius of 242 nm. These results strongly support our hypothesis that the achievable grafting density depends on the interaction of the macroinitiator with the surrounding medium. The opening of the polymer coil enhances the accessibility of the initiating hydroxyl groups and thus the efficiency of the grafting. If sterical hindrance alone was responsible for the low IEs, one would assume that the initiation efficiencies in lipase-catalyzed grafting from 3a,b would be further decreased because of the prefunctionalization with sterical demanding phosphonate groups. Since we observed higher IEs for the P(GDEPE-co-G)s (3a,b) compared to the parent PG26 (2), the efficiency in the enzymatic grafting depends predominantly on the chemical composition and the interaction of the initiator with the reaction medium. The degree of prefunctionalization with DEPE groups influences not only the initiation efficiency but also the hydrophilic/hydrophobic balance of the graft copolymers 4−6, since the number of initiation sites as well as the weight fraction of PCL decreases with increasing FDEPE. Consequently, the hydrophilic/hydrophobic balance (hhb) is steadily shifted more to the hydrophilic side. The graft copolymers 4c, 5c, and 6c were chosen as representative examples, and the weight fractions of PCL as well as the hhbs are given in Table 5.

■

* Supporting Information Reagent ratios for the synthesis of the macroinitiators 2, 3a, and 3b and graft copolymers 4a−f; SEC traces of 2, 3a, and 3b in DMF; SEC traces of 6c and 6f in THF; SEC traces of 4c, 5c, and 6c in DMF; MALDI-TOF MS analysis of 4c, 5c, and 6c; weight fraction of PCL contained in the graft copolymer samples, dynamic light scattering of 2, 3a, and 3b in water as well as εCL. This material is available free of charge via the Internet at http://pubs.acs.org.

■

Table 5. Molecular Weights of P((G-g-εCL12)14-co-G12) (4c), P(GDEPE4-co-(G-g-εCL7)12-co-G10) (5c), and P(GDEPE10-co(G-g-εCL7)10-co-G6) (6c) Obtained from NMR Analysis, Molecular Weight of the PCL Grafts, Weight Fractions of PCL, and Hydrophilic/Hydrophobic Balance entry

initiator

Mn,NMRa (g/mol)

MPCLb (g/mol)

wt % PCL

hhbc

4c 5c 6c

2 3a 3b

20 417 12 513 11 214

18 491 9 930 7 647

90.6 79.4 68.2

0.10 0.26 0.47

ASSOCIATED CONTENT

S

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (H.K.); [email protected] (M.M.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the DFG for financial support (scholarship for J.K., International Research Training Group “Selectivity in Chemoand Biocatalysis” - SeleCa). The authors thank Bjoern Schulte for MALDI-TOF MS measurements and fruitful discussions. Thanks to Richard Meurer for assistance with dynamic light scattering.

The accuracy of integration in 1H NMR spectra is ±5%. bWeight fraction of PCL within the graft copolymers. cHydrophilic/hydrophobic balance was calculated by dividing the molecular weight of the macroinitiator through MPCL. a

3717

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718

Macromolecules

■

Article

REFERENCES

(1) Gross, R. A.; Kumar, A.; Kalra, B. Chem. Rev. 2001, 101, 2097. (2) Varna, I. K.; Albertsson, A.-C.; Rajkhova, R.; Srivastava, R. K. Prog. Polym. Sci. 2005, 30, 949. (3) Duxbury, C. J.; Hilker, I.; de Wildeman, S. M. A.; Heise, A. Angew. Chem., Int. Ed. 2007, 46, 8452. (4) Bisht, K.; Deng, F.; Gross, R. A.; Kaplan, D. L.; Swift, G. J. J. Am. Chem. Soc. 1998, 120, 1363. (5) Cordova, A.; Iversen, T.; Hult, K. Macromolecules 1998, 31, 1040. (6) Zhang, M.; Müller, A. H. E. J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 3209. (7) Rzayev, J. ACS Macro Lett. 2012, 1, 1146. (8) Sheiko, S. S.; Sumerlin, B. S.; Matyjaszewski, K. Prog. Polym. Sci. 2008, 33, 759. (9) Lee, H.; Pietrasik, J.; Sheiko, S. S.; Matyjaszewski, K. Prog. Polym. Sci. 2010, 35, 24. (10) Keul, H.; Möller, M. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 3209. (11) Frey, H.; Haag, R. Rev. Mol. Biotechnol. 2002, 90, 257. (12) Hans, M.; Gasteier, P.; Keul, H.; Möller, M. Macromolecules 2006, 39, 3184. (13) Hans, M.; Keul, H.; Heise, A.; Möller, M. Macromolecules 2007, 40, 8872. (14) Hans, M.; Keul, H.; Möller, M. Biomacromolecules 2008, 9, 2954. (15) Padovani, M.; Hilker, I.; Duxbury, C. J.; Heise, A. Macromolecules 2008, 41, 2439. (16) Duxbury, C. J.; Cummins, D.; Heise, A. Macromol. Rapid Commun. 2007, 28, 235. (17) Villarroya, S.; Zhou, J.; Thurecht, K. J.; Howdle, S. M. Macromolecules 2006, 39, 9080. (18) Villarroya, S.; Dudek, K.; Zhou, J.; Irvine, D. J.; Howdle, S. M. J. Mater. Chem. 2008, 18, 989. (19) Chandra, R.; Rustgi, R. Prog. Polym. Sci. 1998, 23, 1273. (20) Vert, M. Biomacromolecules 2005, 6, 538. (21) Gomes, M. E.; Reis, R. L. Int. Mater. Rev. 2004, 49, 261. (22) Gomes, M. E.; Reis, R. L. Int. Mater. Rev. 2004, 49, 274. (23) Woodruff, M. A.; Hutmacher, D. W. Prog. Polym. Sci. 2010, 35, 1217. (24) Albertsson, A.-C.; Varma, I. K. Biomacromolecules 2003, 4, 1466. (25) Storey, R. F.; Sherman, J. W. Macromolecules 2002, 35, 1504. (26) Kobayashi, S. Macromol. Rapid Commun. 2009, 30, 237. (27) Middleton, J. C.; Tipton, A. J. Biomaterials 2000, 21, 2335. (28) Gunatillake, P. A.; Adhikari, R. Eur. Cells Mater. 2003, 5, 1. (29) Monge, S.; Canniccioni, B.; Graillot, A.; Robin, J.-J. Biomacromolecules 2011, 12, 1973. (30) Marsico, F.; Wagner, M.; Landfester, K.; Wurm, F. R. Macromolecules 2012, 45, 8511. (31) Richards, M.; Dahiyat, B. I.; Arm, D. M.; Brown, P. R.; Leong, K. W. J. Biomed. Mater. Res. 1991, 25, 1151. (32) Köhler, J.; Keul, H.; Möller, M. Chem. Commun. 2011, 47, 8148. (33) Fitton, A. O.; Hill, J.; Jane, D. E.; Millar, R. Synthesis (Stuttgart) 1987, 12, 1140. (34) Kumar, A.; Gross, R. A. J. Am. Chem. Soc. 2000, 122, 11767. (35) Bankova, M.; Kumar, A.; Impallomeni, G.; Ballistreri, A.; Gross, R. A. Macromolecules 2002, 35, 6858. (36) Fulmer, G. R.; Miller, A. J. M.; Sherden, N. H.; Gottlieb, H. E.; Nudelman, A.; Stoltz, B. M.; Bercaw, J. E.; Goldberg, K. I. Organometallics 2010, 29, 2176. (37) Mei, Y.; Kumar, A.; Gross, R. Macromolecules 2003, 36, 5536.

3718

dx.doi.org/10.1021/ma400255n | Macromolecules 2013, 46, 3708−3718