Article pubs.acs.org/Macromolecules
Amphiphilic Random Copolymers with Hydrophobic/HydrogenBonding Urea Pendants: Self-Folding Polymers in Aqueous and Organic Media Kazuma Matsumoto,† Takaya Terashima,*,† Takanori Sugita,† Mikihito Takenaka,†,‡ and Mitsuo Sawamoto*,† †
Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan RIKEN SPring-8 Center, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan
‡
S Supporting Information *
ABSTRACT: Urea and poly(ethylene glycol) (PEG)-functionalized amphiphilic random copolymers served as self-folding polymers in both water and chloroform via hydrophobic and/or hydrogenbonding interactions. For this, a urea-bearing methacrylate (BPUMA) was newly designed as a trigger monomer. Various random, gradient, and block copolymers were synthesized by living radical copolymerization of PEG methyl ether methacrylate (PEGMA) and BPUMA to systematically survey folding/association properties. Importantly, self-folding in both water and chloroform requires the random incorporation of BPUMA along a chain and the control of its composition, while gradient or block counterparts tend to induce multichain aggregation. Typically, 30−40 mol % BPUMA random copolymers effectively fold in water to form compact globular unimer micelles with hydrophobic/hydrogen-bonding core covered by multiple PEG arm chains. The dual functionalization of polymers with hydrophilic PEG and hydrophobic/hydrogen-bonding urea units afforded singlechain compartmentalized polymers in both aqueous and organic media.
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INTRODUCTION Polymer chains can be potentially programmed into the primary structure as encoded information to form specific three-dimensional architectures, i.e., higher order structure, in solutions, and thereby exhibit inherent properties and functions.1 Natural biopolymers such as proteins and enzymes possess precision primary structure (molecular weight, stereoregularity, and monomer sequence) to fold into their own tertiary structure in water via the formation of secondary structure. The self-folding process involves amphiphilic nature of polymer chains and selective hydrogen-bonding interaction; hydrophobic units mainly come inside, and hydrophilic segments are in turn often located in outer surfaces. The folded structure is further stabilized by hydrogen-bonding interaction, in addition to the other physical interactions and covalent bond formation. The resulting polymers carry specific spaces and cavities inside that induce selective functions such as molecular recognition and catalysis. Inspired by these attractive features, single-chain folding technologies of synthetic polymers have been developed to create functional and/or compartmentalized polymeric materials.2−9 This is now one of the most active research areas in polymer science. Importantly, the intramolecular self-assembly (self-folding) of polymers with precision primary structure allows the creation of “single-chain” functional materials with globular, dynamic/reversible, and compartmentalized structures, distinct from intermolecular aggregation of multiple © XXXX American Chemical Society
polymers (e.g., block copolymer micelles). Such self-folding polymers and single-chain polymeric nanoparticles are generally obtained from the two approaches: (1) autonomous self-folding based on the affinity between polymer pendants/main chains and solvents such as conventional unimer micelles without sitespecific interaction,10−14 (2) self-folding via site-specific noncovalent interaction (hydrogen-bonding, coordination, π−π, host−guest, etc.),15−25 and (3) self-folding (intramolecular cross-linking) with chemical reactions.26−32 In any case, on-target design of pendant functional groups is essential for the desired folding process. We have originally designed amphiphilic random methacrylate copolymers bearing hydrophilic poly(ethylene glycol) (PEG) methyl ether pendants as versatile precursors for single-chain folding/cross-linked polymers in various media.13,14,18a,31 Such random copolymers can be designed and synthesized by living radical copolymerization of PEG methyl ether methacrylate (PEGMA) and a series of methacrylates (RMA). The selection of RMA, i.e., its pendant structure, affords the control of the self-folding properties. Typically, copolymers bearing hydrophilic PEG and hydrophobic alkyl pendants fold in water via hydrophobic interaction to form unimer micelles with hydrophobic core (Scheme 1b);13 the Received: August 18, 2016 Revised: September 21, 2016
A
DOI: 10.1021/acs.macromol.6b01702 Macromolecules XXXX, XXX, XXX−XXX
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structure is unfolded in organic solvents (e.g., DMF, chloroform) and more dynamic upon heating. Hydrophilic PEG and fluorous perfluoroalkyl pendant copolymers in turn undergo multimode self-folding (Scheme 1c):14b unimer micelles are formed via the self-assembly of the hydrophobic and/or fluorous pendants not only in water but also in N,Ndimethylformamide (DMF) and reverse unimer micelles with hydrophilic PEG core are further obtained in a hydrofluorocarbon (2H,3H-perfluoropentane: 2HPFP). It should be noted that single-chain folding of the PEGMA-based copolymers takes places in water even at relatively high concentration (∼100 mg/mL) owing to the efficient covering of hydrophobic core with PEG pendants. To further push folding technologies with PEGMA-based random copolymers, we focused on hydrogen-bonding urea groups as folding triggers (Scheme 1a). Urea compounds (R1− NHCONH−R2) are well-known to induce self-assembly in solid states or solutions via intermolecular hydrogen-bonding interactions between the nitrogen-bound protons and the carbonyl oxygen atoms. The self-assembly efficiency and the solubility can be tuned by the substituents (R1 or R2). So far, several urea-based compounds are designed to create supramolecular polymers in solutions.33−35 Hydrogen-bonding functional groups have been commonly used to build synthetic
Scheme 1. Single-Chain Folding of Amphiphilic Functional Random Copolymers with (a) Hydrogen-Bonding Urea, (b) Hydrophobic Alkyl, and (c) Fluorous Perfluoroalkyl Pendants in Aqueous and Organic Media
Scheme 2. Synthesis of (a) PEGMA/RMA Random, (b) PEGMA/BPUMA Gradient, and (c) PEGMA/BPUMA Block Copolymers via Ruthenium-Catalyzed Living Radical Polymerization
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DOI: 10.1021/acs.macromol.6b01702 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules Table 1. Synthesis and Characterization of PEGMA/RMA Amphiphilic Copolymersa code P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13
a
RMA
BPUMA BPUMA BPUMA BPUMA BPUMA BPUMA HEMA (BPOMA) PAEMA BPUMA BPUMA (gradient) BPUMA (block) BPUMA (block)
m/n
time (h)
conv (%)c PEGMA/ RMA
Mnd (SEC)
Mw/Mnd (SEC)
m/nobsde (NMR)
Mne (NMR)
Mw,calcd
200/0 190/10 180/20 160/40 140/60 120/80 100/100 140/60
46 49 29 24 23 23 27 52
84/− 75/92 85/91 84/91 92/95 84/88 82/86 81/87
85 400 71 900 69 300 57 200 76 800 57 800 50 600 72 000
1.16 1.11 1.18 1.19 1.26 1.33 1.32 1.17
164/0 173/10 143/18 123/32 129/61 121/72 76/78 112/49
78 100 85 700 74 700 70 400 84 700 85 300 66 300 72 000
140/60 420/180 180/20
46 24 47
81/88 84/88 78/100
59 900 338 000 57 100
1.27 1.49 1.46
n.d. n.d. 137/20
180/20
5
−/56
48 700
1.37
120/80
6
−/77
37 800
1.16
b
Mw,H2Oi (MALLS)
AH2Oj
1.23 1.24 1.07 1.33 1.26 1.35 1.31 1.28
97 100 93 400 118 000 139 000 168 000 526 000 107 000
0.83 0.99 1.05 1.03 1.10 4.57 0.99
99 800 708 000 123 000
n.d. n.d. 1.16
112 000 733 000 1 710 000
1.12 1.04 1.39
116 000
1.10
4 170 000
Mw,DMFg (MALLS)
ADMF
90 600 95 100 88 100 83 800 106 700 113 400 87 500 84 200
112 000 118 000 94 000 112 000 135 000 153 000 115 000 108 000
n.d. n.d. 72 800
n.d. n.d. 106 000
153/12
77 300
106 000
97/70
73 000
84 700
f
h
36.0
a
P1−P7 and P10: [PEGMA]0 + [BPUMA]0/[ECPA]0/[RuCp*Cl(PPh3)2]0/[4-DMAB]0 = 500/2.5/0.5/10 (P1−P7) or 500/0.83/0.33/6.66 (P10) mM in ethanol at 40 °C. P8 (precursor): [PEGMA]0/[HEMA]0/[ECPA]0/[RuCp*Cl(PPh3)2]0/[4-DMAB]0 = 350/150/2.5/0.5/10 mM in ethanol at 40 °C. The precursor was treated with 1-isocyanato-3,5-bis(trifluoromethyl)benzene. P9: [PEGMA]0/[PAEMA]0/[ECPA]0/ [Ru(Ind)Cl(PPh3)2]0/[n-Bu3N]0 = 350/150/2.5/1.0/10 mM in toluene at 80 °C. P11: [PEGMA]0/[BPUMA]0/[ECPA]0/[Ru(Ind)Cl(PPh3)2]0/[n-Bu3N]0 = 450/50/2.5/0.25/2.5 mM in toluene/ethanol (4/1, v/v) at 80 °C. P12 and P13: [BPUMA]0/[PPEGMA-Cl]0/ [RuCp*Cl(PPh3)2]0/[4-DMAB]0 = 62.5 (P12) or 250 (P13)/2.5/0.5/10 mM in ethanol at 40 °C. PPEGMA-Cl for P12: Mn = 63 400, Mw/Mn = 1.19. PPEGMA-Cl for P13: Mn = 40 200; Mw/Mn = 1.16. bm = [PEGMA]0/[ECPA]0; n = [RMA]0/[ECPA]0. cMonomer conversion determined by 1 H NMR (RMA of P8: HEMA). dDetermined by SEC in DMF (10 mM LiBr) with PMMA standard calibration. eDP of PEGMA and RMA (m/ nobsd) and Mn of copolymers determined by 1H NMR. fCalculated weight-average molecular weight (Mw): Mw,calcd = Mn (NMR) × Mw/Mn (SEC). g Absolute weight-average molecular weight (Mw) determined by SEC-MALLS in DMF (10 mM LiBr). hAggregation number in DMF: ADMF = Mw, DMF (MALLS)/[Mn (NMR) × Mw/Mn (SEC)]. iAbsolute weight-average molecular weight (Mw) determined by SEC-MALLS in H2O. j Aggregation number in H2O: AH2O = Mw, H2O (MALLS)/Mw, DMF (MALLS).
available. The random copolymers were efficiently synthesized by ruthenium-catalyzed living radical copolymerization of hydrophilic PEGMA (Mn = 475, an average oxyethylene unit of 8.5) and BPUMA. The folding property is controllable by tuning the BPUMA composition and the related comonomers. Selective folding required random sequence distribution of BPUMA units along a chain, while gradient or block sequence of BPUMA induced multichain aggregation. The introduction of BPUMA units into PEGMA-based random copolymers successfully opened the possibility of multimode folding/ unfolding systems controlled by solvents (outer environments), externally added compounds, and temperature.
folding polymers; various supramolecular motifs have been introduced into the polymer side chains and terminals, including ureidopyrimidinone (UPy),16 1,3,5-tricarboxamide (BTA),17,18 thymine/diaminopyridine,19 Hamilton wedge,19 and ureidoguanosine/diaminonaphthyridine.20 For example, chiral BTA-functionalized PEGMA random copolymers18 fold in water via the helical self-assembly of the hydrogen-bonding/ hydrophobic BTA pendants. The resulting nanocompartments are also effective for catalysis in water. We herein report the precision synthesis and folding properties of hydrogen-bonding urea-functionalized amphiphilic random copolymers (Scheme 1a). By introducing both hydrophilic PEG chains and hydrophobic/hydrogen-bonding urea units into the pendants, the random copolymers afford self-folding in both water and chloroform to provide hydrophobic and/or hydrogen-bonding core nanoparticles (unimer micelles) that are covered by hydrophilic PEG arms. The copolymers are reversibly unfolded by changing solvent polarity in water (addition of methanol) or adding trifluoroacid in chloroform. For this, we newly designed a urea-bearing methacrylate (BPUMA) as a hydrophobic/hydrogen-bonding monomer. To improve hydrogen-bonding ability, we introduced an electronwithdrawing trifluoromethyl group (−CF3) into the meta positions of the pendant aromatic ring; this design is often utilized to enhance hydrogen-bonding ability, i.e., Lewis acidity, of urea or thiourea groups as organocatalysts.36 Conveniently, the monomer can be easily obtained from the single-step reaction of 2-isocyanatoethyl methacrylate (IEMA) and 3,5bis(trifluoromethyl)aniline, both of which are commercially
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RESULTS AND DISCUSSION Synthesis of Amphiphilic/Hydrogen-Bonding Copolymers. A series of amphiphilic and hydrogen-bonding methacrylate copolymers (P1−P13) were synthesized by ruthenium-catalyzed living radical polymerization (Scheme 2) to establish molecular design suitable for single-chain folding polymers via intramolecular hydrophobic and/or hydrogenbonding interactions in aqueous and organic media. The molecular design was focused on hydrogen-bonding units (urea, urethane, and ester: no hydrogen bonding), composition, and monomer sequence (random, gradient, and block). BPUMA, a hydrogen-bonding monomer, was efficiently prepared with 2-isocyanatoethyl methacrylate (IEMA) and 3,5-bis(trifluoromethyl)aniline. Random copolymerization of PEGMA and BPUMA was conducted with a ruthenium catalyst [RuCp*Cl(PPh3)2/4dimethylamino-1-butanol (4DMAB)]37 and a chloride initiator C
DOI: 10.1021/acs.macromol.6b01702 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules (ethyl-2-chloro-2-phenylacetate: ECPA) in ethanol at 40 °C (Table 1). The target degree of polymerization [DP = ([PEGMA]0 + [BPUMA]0)/[ECPA]0] was set as 200 or 600, where the BPUMA composition was changed from 5 to 50 mol %. The calculated monomer units per a chain (m = [PEGMA]0/[ECPA]0, n = [RMA]0/[ECPA]0) was given: m/ n = 190/10−100/100 (P2−P7) and 420/180 (P10). PEGMA and BPUMA were simultaneously and smoothly consumed up to 75−90% (Figure 1a and Figure S1) to give well-controlled
4.2−4.0 ppm, d: 3.8−3.4 ppm, e: 3.3 ppm), and a phenyl group of ECPA (7.4−7.2 ppm), in addition to their polymethacrylate backbones [methylene: 2.2−1.5 ppm (b), methyl: 1.2−0.7 ppm (a)] (Figure 2a: P5). DPs of PEGMA and RMA (m/nobsd),
Figure 2. 1H NMR spectra of PEGMA/RMA (140/60) random copolymers (P5, P8, and P9) in acetone-d6 at 25 °C. RMA: (a) BPUMA, (b) BPOMA, and (c) PAEMA.
estimated from the area ratio of their pendant units to the initiator (ECPA) terminal phenyl group (7.4−7.2 ppm), were well consistent with DPs calculated from the feed ratio of their monomers to ECPA (m/n) and the monomer conversion. The number-average molecular weight (Mn) of P2−P7 was determined as 66 300−85 700 by 1 H NMR. BPUMA composition in the copolymers proportionally increased with BPUMA in monomers (Table 1), demonstrating that monomer feed ratio is directly reflected to the copolymer composition. In sharp contrast to that in ethanol, ruthenium-mediated copolymerization of PEGMA and BPUMA with ECPA (m/n = 180/20) in toluene/ethanol (4/1, v/v) induced preferential consumption of BPUMA against PEGMA (Figure 1c) to provide a PEGMA/BPUMA gradient copolymer (P11, Mn = 57 100; M w /M n = 1.46). The instantaneous BPUMA composition (Finst,BPUMA) gradually decreased from the initiating α-end to the chlorine ω-end, in sharp contrast to the constant monomer composition along a chain in a random copolymer (P3, Figure 1d). The enhanced reactivity of BPUMA against PEGMA would be due to the inter- or intramolecular hydrogen-bonding interaction of the urea unit in such a less polar solvent containing toluene.40 PEGMA/ BPUMA block copolymers with BPUMA homosegments (P12 and P13) were also prepared by RuCp*Cl(PPh3)2/4DMABcatalyzed polymerization of BPUMA with PPEGMA-Cl macroinitiators (Scheme 2c). P3 (random), P11 (gradient), and P12 (block) have different BPUMA-sequence distribution but almost identical cumulative BPUMA content (∼10 mol %) (Figure 1d). To modulate hydrogen-bonding ability and efficiency, urethane (weaker hydrogen-bonding) and ester (no hydrogen-bonding) groups were also introduced into random copolymers (30 mol % RMA). An ester-functionalized random copolymer (P9: m/n = 140/60; Mn = 59 900; Mw/Mn = 1.27)
Figure 1. Ru-catalyzed living radical copolymerization of PEGMA and BPUMA for P3: (a) a time−conversion curve and (b) SEC curves of samples at 5 h (dash), 10 h (gray), and 29 h (black); [PEGMA]0/ [BPUMA]0/[ECPA]0/[Ru(Cp*)Cl(PPh3)2]0/[4-DMAB]0 = 450/50/ 2.5/0.5/10 mM in ethanol at 40 °C. (c) Ru-catalyzed copolymerization of PEGMA and BPUMA for P11: [PEGMA]0/[BPUMA]0/ [ECPA]0/[Ru(Ind)Cl(PPh3)2]0/[n-Bu3N]0 = 450/50/2.5/0.25/2.5 mM in toluene/ethanol (4/1, v/v) at 80 °C. (d) Instantaneous BPUMA composition (Finst,BPUMA) of PEGMA/BPUMA (180/20) random (P3), gradient (P11), and block (P12) copolymers as a function of normalized chain length.
random copolymers (P2−P7, P10) [by size-exclusion chromatography (SEC) calibrated against PMMA standards: Mn = 50 600−76 800, 338 000; Mw/Mn = 1.1−1.5] (Figure 1b and Figure S1, Table 1). All copolymers were purified by preparative SEC in order to remove unreacted monomers and a ruthenium catalyst residue. P7 with 50 mol % BPUMA and P10 with long DP were further fractionated by preparative SEC to remove low molecular weight fraction from the crude products (Figure S1). It should be noted that the synchronized consumption of two monomers supports the random incorporation of both hydrophilic and hydrogen-bonding urea units into a polymer chain without any biased sequence distribution such as gradient copolymers.38,39 Analyzed by proton nuclear magnetic resonance (1H NMR), P2−P7 exhibited proton signals of BPUMA pendants [methylene: 4.2−4.0 ppm (f); 3.8−3.4 ppm (g), phenyl: 8.2− 8.1 ppm (j); 7.5 ppm (k), and urea units: 8.9−8.6 ppm (i); 6.5−6.2 ppm (h)], oligo(oxyethylene) units of PEGMA (c: D
DOI: 10.1021/acs.macromol.6b01702 Macromolecules XXXX, XXX, XXX−XXX
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PEGMA/BPUMA (180/20) copolymers with different monomer sequence (P3: random; P11: gradient; P12: block) were first analyzed by DLS in water ([polymer] = 10 mg/mL) (Figure 4). P3 (random) mainly had small size distribution
was efficiently obtained from Ru(Ind)Cl(PPh3)2/n-Bu3Ncatalyzed living radical copolymerization of PEGMA and an ester-functionalized methacrylate (PAEMA) with ECPA in toluene at 80 °C (Table 1 and Scheme 2a). A urethanefunctionalized random copolymer (P8: m/n = 140/60; Mn = 72 000; Mw/Mn = 1.17) was prepared by the two steps: (1) RuCp*Cl(PPh3)2/4DMAB-catalyzed living radical copolymerization of PEGMA and 2-hydroxyethyl methacrylate (HEMA) with ECPA in ethanol at 40 °C; (2) the pendant functionalization of the resulting PEGMA/HEMA random copolymer (Mn = 65 500; Mw/Mn = 1.13) with 3,5-bis(trifluoromethyl)phenyl isocyanate (Table 1 and Scheme 2a). The chemical structure of P8 and P9 was characterized by 1H NMR (Figure 2). To evaluate the association properties of the BPUMA urea units, a PEGMA/BPUMA random copolymer (P6: 40 mol % BPUMA) was analyzed by 1H NMR in various solvents (Figure 3). Compared with those in acetone-d6 and DMF-d7, the
Figure 4. DLS intensity size distributions of PEGMA/BPUMA (180/ 20) random (P3), gradient (P11), and block (P12) copolymers in water at 25 °C: [polymer] = 10 mg/mL.
originating from unimer micelle (Rh = ∼8 nm: >99% volume fraction) though P12 (block) had large single-modal size distribution derived from multichain micelle (Rh = ∼52 nm). Uniquely, P11 (gradient) clearly had bimodal size distribution of small unimer (Rh = ∼6.4 nm: ∼87% volume fraction) and large micelle (Rh = ∼125 nm). These results indicate that random and homogeneous distribution of BPUMA units is important to unimolecularly solubilize PEGMA/BPUMA copolymers in water. Thus, various amphiphilic random copolymers (P2−P10) with different monomer contents (m/n), hydrophobic/hydrogen-bonding units, and chain length (DP) were analyzed by SEC-MALLS. If the copolymers self-fold in water to form compact unimer micelles, the absolute weight-average molecular weight by MALLS in water [Mw,H2O] is identical to that of unimer in good solvents such as DMF [Mw,DMF], while the molecular weight determined by SEC, i.e., hydrodynamic volume, in water would be smaller than that in such a good organic solvent. Absolute weight-average molecular weight [Mw (MALLS)] of P1−P10 was determined by SEC-MALLS in DMF and H2O (Table 1). In DMF, P1−P9 of approximately 200 DP had Mw,DMF (MALLS) ranging from 94 000 to 153 000, respectively, close to the values calculated from their corresponding Mn by 1 H NMR and Mw/Mn by SEC [Mw,calcd = Mn (NMR) × Mw/Mn (SEC)]. P1−P9 thus exist as unimer in DMF (aggregation number: ADMF = Mw,DMF (MALLS)/Mw,calcd = ∼1). In contrast, the copolymers had Mw,H2O (MALLS) widely ranging from 93 400 to 526 000 in H2O. The aggregation number in water (AH2O), defined as AH2O = Mw,H2O (MALLS)/ Mw,DMF (MALLS), depended on the hydrophobic/hydrogenbonding RMA content (Table 1): (1) copolymers with less than 40 mol % BPUMA (P2−P6: m/n = 190/10−120/80) existed as unimer in water (AH2O = ∼1); (2) a copolymer with
Figure 3. 1H NMR spectra of a PEGMA/BPUMA (120/80) random copolymer (P6) in (a) acetone-d6, (b) DMF-d7, (c) CDCl3, and (d) D2O at 25 °C.
proton signals of the bis(trifluoromethyl)phenyl group (j, k, peak assignment: Figure 2) turned extremely broad in D2O. This strongly indicates that the BPUMA pendants self-assemble with hydrophobic and/or hydrogen-bonding interaction in water to have low mobility. Uniquely, in CDCl3, P6 also showed the broad and bimodal proton signals of the pendant urea (h, i) and phenyl (j) groups, suggesting the BPUMA pendants should induce self-assembly via hydrogen bond. On the basis of this preliminary study, we investigated the selffolding properties of P1−P13 in water and chloroform. Single-Chain Folding in Water. Single-chain folding and aggregation properties of P1−P13 in water were systematically investigated by SEC, multiangle laser light scattering coupled with SEC (SEC-MALLS), and dynamic light scattering (DLS). Discussion was directed to the effects of monomer sequence (random, gradient, and block), composition (m/n), hydrogen bond (urea, urethane, and ester), and chain length (DP) on the properties. Owing to multiple PEG pendants, all of the polymers are easily soluble even in water. Thus, a simple protocol was used to prepare homogeneous aqueous solutions for characterization as follows: viscous liquid-like bulk polymers were vigorously mixed with water, followed by sonication for several seconds or minutes and filtration (PTFE membrane filter: 0.45 μm pore). E
DOI: 10.1021/acs.macromol.6b01702 Macromolecules XXXX, XXX, XXX−XXX
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Figure 5. Effects of primary structure (RMA composition and structure, chain length) on the compactness of folded PEGMA/RMA random copolymers. (a−c) SEC curves of PEGMA/BPUMA random copolymers (a: P1; b: P4; c: P6) in DMF (black) and H2O (blue). (d) The ratio of SEC peak-top molecular weight of P1−P10 in H2O and that in DMF [Mp(H2O)/Mp(DMF)] against RMA content. (e) DLS intensity size distribution of P10 in DMF (black), H2O (blue), and CHCl3 (red): [polymer] = 10 mg/mL.
50 mol % BPUMA (P7) formed a large aggregate (AH2O = ∼5); and (3) copolymers (P5, P8, and P9) with 30 mol % RMA units were unimer in water, independent of the pendant functional groups (urea, urethane, and ester). Similar to P5, P10 of long DP (30 mol % BPUMA) was also unimer in water. These results suggest that the local concentration of RMA is critical to control the intra- or intermolecular association of the copolymers in water.10b,13,14b,18a Compactness of P1−P10 in water was evaluated with the comparison between SEC peak-top molecular weight (Mp) in H2O and that in DMF [Mp,H2O/Mp,DMF] (Figure 5 and Figure S2). Here, SEC is calibrated against poly(ethylene oxide) standards in both water and DMF. Mp,H2O/Mp,DMF indexes the compactness of the folded structures in water, normalized by the structure in DMF. As typically shown in Figure 5a−c, ureafunctionalized polymers (P2−P7) had Mp,H2O’s smaller than corresponding Mp,DMF’s. Importantly, the Mp,H2O/Mp,DMF values gradually decreased with increasing BPUMA composition up to 40 mol % [m/n: 200/0−120/80 (P1−P6)] to be a minimum of 0.43 (P6) and again increased with increasing BPUMA up to 50 mol % (P7) (Figure 5d). This result reveals that PEGMA/ BPUMA random copolymers gradually self-fold with increasing BPUMA units to form the most compact unimer micelle at 40 mol % BPUMA, while they induce multichain aggregation over 40 mol % BPUMA. Urethane or ester-functionalized random copolymers (P8: urethane; P9: ester) also efficiently self-folded in water (Figure S2). Judging from Mp,H2O/Mp,DMF, P8 (Mp,H2O/Mp,DMF = 0.43) was as compact as P5 (urea), while P9 (Mp,H2O/Mp,DMF = 0.52) was slightly larger than P5 (Figure 5d). This suggests that the intramolecular hydrogen-bonding interaction of urea or urethane pendants efficiently promotes the self-folding of polymer chains in water. Confirmed by 1H NMR, the folded polymethacrylate backbone of urea-functionalized P5 was less mobile than that of ester-functionalized P9: i.e., the proton signals of polymethacrylate backbone of P5 were much broader than those of P9 (Figure S3). Such low mobility for P5 also implies the efficient stabilization of the folded structure by hydrogen-bonding interaction of the urea pendants in water, where the hydrogen atoms (N−H) interact with carbonyl oxygen atoms (CO) of urea33 and/or methacrylate esters. In
the identical BPUMA content (∼30 mol %), P10 with a longer main chain (Mw,DMF = 708 000) self-folded more compact than P5 [Mp,H2O/Mp,DMF = 0.21 (P10), 0.43 (P5)] (Figure 5d and Figure S2). Rh of P10 in H2O (16 nm) by DLS was clearly smaller than that in DMF (27 nm) (Figure 5e). This revealed that long polymer chains form more shrunk structure in water than short counterparts. Single-chain folding of PEGMA/BPUMA random copolymers in water is triggered by the hydrophobic and/or hydrogen-bonding interaction of the urea pendants and polymethacrylate backbones in water. The folded structure would be dynamic and variable by changing temperature or solvents. We thus examined temperature-dependent 1H NMR measurements of a PEGMA/BPUMA (160/40) random copolymer (P4) in D2O at the temperature range between 25 and 80 °C (Figure 6), focusing on the phenyl protons of the BPUMA pendants (j, k) and the tip methyl protons of hydrophilic PEG chains (e). The phenyl protons (j, k) turned sharp upon heating from 25 to 80 °C (Figure 6a): the halfwidth gradually became narrow. However, the half-width of the PEG methyl groups was almost constant (Figure 6b). This result demonstrates that the mobility of the hydrophobic/ hydrogen-bonding core increased with temperature. Methanol was also effective to unfold P4 by disruption of hydrophobic and/or hydrogen-bonding interaction in water. The proton signals of the BPUMA phenyl groups (j, k) turned sharp by adding methanol into the D2O solution of P4 (Figure 6c). PEGMA-based copolymers often show lower critical solution temperature (LCST)-type phase separation in water.13,14b The phase separation behavior of P6 in water was investigated by the temperature-dependent turbidity measurements of the aqueous solution between 30 and 80 °C, monitored at 660 nm with the heating/cooling rate of 1 °C/min. As shown in Figure 7a, P6 sharply and reversibly showed LCST-type phase separation in water. The cloud point, defined as 90% transmittance upon heating, was 52 °C. P5 of 30 mol % BPUMA, more hydrophilic than P6 (40 mol % BPUMA), had a higher cloud point of 62 °C. To further evaluate the stability of the self-folded structure in water, the aqueous solution of P6 after LCST phase separation was analyzed by DLS at 25 °C (Figure 7b, [polymer] = 10 mg/ mL). P6 kept small Rh (∼8 nm: >99% volume fraction) even F
DOI: 10.1021/acs.macromol.6b01702 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
single polymer chain that is effectively isolated by multiple PEG pendants. This result further implies that P6 maintains folded structure in the macroscopic aggregates that are derived from the dehydration of PEG segments. In contrast, a block copolymer (P12) micelle after LCST phase separation showed bimodal size distribution mainly containing quite large aggregates (Rh = ∼350 nm; ∼70% volume fraction, Figure S4), distinct from the original size distribution (Rh = ∼50 nm, Figure 4). Thus, “intramolecular” hydrophobic/hydrogenbonding interaction of BPUMA units is effective to stably maintain the folded structure. Single-Chain Folding in Chloroform. To evaluate folding properties in chloroform, PEGMA/BPUMA random copolymers (P5 and P6: 30 and 40 mol % BPUMA) were analyzed by DLS in chloroform in the absence or presence of trifluoroacetic acid (TFA) at 25 °C ([polymer] = 10 mg/mL, [TFA]/[urea] = ∼10/1, molar ratio). Here, bulk polymers were simply mixed with chloroform, followed by sonication for several seconds and filtration (PTFE membrane filter: 0.45 μm pore), to immediately give homogeneous chloroform solutions. The size of P5 and P6 was compared with that of an esterfunctionalized random copolymer (P9) or a PEGMA/BPUMA block copolymer (P13). TFA was employed to disrupt hydrogen-bonding interaction of the urea units. P5 and P6 showed unimodal DLS intensity size distribution (Figure 8a,c), while Rh with TFA was larger than that without
Figure 6. Effects of temperature and methanol on a folded P4 in water. (a) 1H NMR spectra of P4 in D2O/CD3OD (100/0, 40/60, and 0/100, v/v) at 25−80 °C. Effects of (b) temperature (25−80 °C) and (c) CD3OD on the half-width of the proton signals.
Figure 8. DLS intensity distributions of (a) P6, (a) P13, (a) P5, and (a) P9 in CHCl3 (black) and in CHCl3 with TFA (red) at 25 °C: [polymer] = 10 mg/mL.
TFA (P6: Rh = ∼10 nm with TFA, ∼6 nm without TFA; P5: Rh = ∼13 nm with TFA, ∼10 nm without TFA). In contrast, an ester-functionalized P9 (without hydrogen-bonding urea units) had identical DLS size distribution, independent of TFA (Rh = ∼13 nm, Figure 8d). These results importantly suggest that the polymer chains of P5 and P6 unfold by disrupting the hydrogen-bond of the BPUMA urea units in the presence of TFA; namely, PEGMA/BPUMA random copolymers efficiently self-fold to form compact unimer micelles in chloroform via the pendant hydrogen bonding. The disruption of the hydrogen bond was also confirmed by 1H NMR in CDCl3 (Figure 9c,d). By adding TFA ([TFA]/[urea] = ∼10/1), the unique bimodal and broad signals of BPUMA phenyl (j, j′) and urea (h, i) protons turned unimodal and sharp. Monomer sequence and composition of the copolymers also affected the folding/association properties in chloroform. In
Figure 7. (a) Turbidity measurements of the aqueous solution of P6 (5 mg/mL, heating/cooling = 1 °C/min, λ = 670 nm). (b) DLS intensity size distribution of P6 in water at 25 °C (10 mg/mL) before and after LCST-type phase separation.
after a LCST phase separation process; the light scattering intensity size distribution was almost identical to that of original P6 (Rh = ∼7 nm: >99% volume fraction). Thus, P6 reversibly and stably form folded structure in water even after the macroscopoic aggregation and precipitation at high temperature, though the mobility of hydrophobic/hydrogen-bonding units gradually increased upon heating. This is owing to the “intramolecular” association of BPUMA pendants within a G
DOI: 10.1021/acs.macromol.6b01702 Macromolecules XXXX, XXX, XXX−XXX
Article
Macromolecules
Figure 9. 1H NMR spectra of (a) P3, (b) P5, (c) P6, (d) P6 with TFA, (e) P12, and (f) a MMA/BPUMA (120/80) random copolymer in CDCl3 at 25 °C.
Figure 10. TEM images of (a) P10 and (b) P5 cast on carbon coat grids from the aqueous solutions (1 mg/mL).
contrast to a random P6, a block P13 formed multichain micelle to have relatively large Rh (∼21 nm) (Figure 8b), whereas P13 turned unimer with small Rh (∼8 nm) in the presence of TFA. This indicates that random monomer sequence is important for self-folding in chloroform, similar to that in water. Additionally, folded structure in chloroform became more compact with increasing BPUMA contents: a folded P6 of 40 mol % BPUMA was smaller against unfolded state (with TFA) than a folded P5 of 30 mol % BPUMA. This is because the physical cross-linking of polymers is enhanced with increasing BPUMA contents. Hydrogen-bonding efficiency of the urea pendants was further investigated by 1H NMR measurements of P3, P5, and P6 (10, 30, and 40 mol % BPUMA) in CDCl3 (Figure 9). The bimodal proton signals of BPUMA phenyl groups (j, j′) at around 7.9 ppm were dependent on BPUMA content: one signal j gradually decreased and another j′ in turn increased with increasing BPUMA from 10 to 40 mol % (Figure 9a−c). Importantly, the bimodal signal is independent of the total polymer concentration ([polymer] = 5, 10, and 20 mg/mL) and chain length (DP = 200 or 600) (Figure S5). These results suggest that the bimodal signals are attributed to the difference
of hydrogen-bonding mode, dependent on the local concentration of BPUMA. The urea units of BPUMA pendants potentially allow the three kinds of hydrogen-bonding structures between (1) urea proton (N−H) and adjacent PEG oxygen (PEGMA side chains), (2) urea proton and carbonyl oxygen (CO) of methacrylate (ester), and (3) urea proton and urea carbonyl oxygen. Judging from the increase with PEGMA content, signal j would originate from BPUMA pendants whose urea protons interact with PEG pendant oxygen atoms [mode (1)], while another j′ would be derived from BPUMA whose urea protons interact with carbonyl oxygen atoms of ester and/or urea [mode (2) or (3)]. To clarify the proposed hydrogen-bonding structures, we further analyzed a PEGMA/BPUMA (180/20) block copolymer (P12, micelle) and a MMA/BPUMA (120/ 80) random copolymer by 1H NMR in CDCl3 (Figure 9e,f). The former urea protons cannot virtually interact with PEG oxygen atoms owing to the BPUMA position spatially far from PEGMA, while the latter can only interact with carbonyl oxygens of urea or ester groups. The both copolymers exhibited broad phenyl protons of BPUMA at around 7.9−7.8 ppm; the chemical shift was close to the signal j′ of PEGMA/BPUMA H
DOI: 10.1021/acs.macromol.6b01702 Macromolecules XXXX, XXX, XXX−XXX
Article
Macromolecules
Figure 11. SAXS profiles of P5 (red), P9 (blue), and a PEGMA/DMA random copolymer (40 mol % DMA, DP = 200, green) in (a) water and (b) DMF at 25 °C: [polymer] = 10 mg/mL.
smaller than that in DMF (0.96). These results support that P10 formed self-folded, compact, and globular structure in water, in sharp contrast to unfolded random-coil structure in DMF (confirmed by the slope value of 0.55 in logarithm plots of Rg against molar mass). The three-dimensional structure of urea-functionalized P5 in water was investigated by small-angle X-lay scattering (SAXS) compared with that of non-hydrogen bonding P9 and PEGMA/DMA random copolymer (DMA: 40 mol %, DP 200) (Figure 11: scattering profiles are vertically offset). The SAXS profiles supported that all of the copolymers formed globular structure in water (Figure 11a); however, the details are dependent on the pendant functional groups. P5 and P9 had elongated (anisotropic) globular structure though PEGMA/DMA random copolymer was sphere. Determined by Guinier plots (Figure S9), radius of gyration (Rg) of PEGMA/DMA random copolymer13c and P5 in water (3.3 nm, 4.5 nm) was smaller than that in DMF (5.0 nm, 5.6 nm), respectively, while Rg of P9 in water (6.5 nm) was in turn larger than that in DMF (5.6 nm). The unique structure of P5 and P9 in water is probably due to the ordered self-assembly of the hydrogen-bonding urea and/or planar phenyl pendants. Similar elongated structure has been also reported in the self-folding of chiral BTA-functionalized PEGMA random copolymers, where the pendant BTAs induce helical self-assembly along a polymer chain.18d These results indicate that the design of pendant functional groups allows us to modulate folded/self-assembly structures of polymers.
random copolymers (P3, P5, and P6). Similarly, the phenyl protons of BPUMA (monomer) also shifted to downfield in the presence of poly(ethylene oxide) compared with that those alone or in the presence of MMA (Figure S6). This result is consistent with the proposed hydrogen-bonding modes of BPUMA pendants. Thus, folding into compact structure in chloroform most likely arises from the hydrogen-bonding between the urea protons and carbonyl oxygens of urea and/or methacrylate ester groups (signal j′) (Figure 9). Stability of self-folded P6 by the BPUMA hydrogen bond was further evaluated by 1H NMR in CDCl3 in conjunction with polar compounds such as methanol, acetone, and acetic acid as well as TFA (Figure S7). The hydrogen bond of the BPUMA pendants was maintained in the presence of 10 equiv of methanol, acetone, and acetic acid against the urea units though that was disrupted with 10 equiv of TFA as a strong acid (Figure 9c,d). Surprisingly, P6 efficiently kept the hydrogen-bonding interaction up to 100 equiv of acetic acid. These results reveals that PEGMA/BPUMA random copolymers can stably self-fold in chloroform in the presence of polar compounds by controlling the acidity and amounts. Folded Structure in Water. Folded structures of PEGMA/ BPUMA random copolymers (P5 or P10) with 30 mol % BPUMA in water were further investigated by transmission electron microscopy (TEM) and light scattering (MALLS, DLS). Figures 10a and 10b show the TEM images of P10 and P5 cast on carbon coat grids from the aqueous solutions (1 mg/mL), stained with phosphotungstic acid. Both samples clearly showed small black dot particles (
