Poly(d-glucose carbonate) block copolymers: A platform for natural product-based nanomaterials with solvothermatic characteristics

Article abstract of DOI:10.1021/bm4010832

A natural product-based polymer platform, having the characteristics of being derived from renewable materials and capable of breaking down, ultimately, into natural byproducts, has been prepared through the ring-opening polymerization (ROP) of a glucose-based bicyclic carbonate monomer. ROP was carried out via chain extension of a polyphosphoester (PPE) macroinitiator in the presence of 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) organocatalyst to afford the PPE-b-poly(d-glucose carbonate) (PDGC) block copolymer. This new copolymer represents a functional architecture that can be rapidly transformed through thiol-yne reactions along the PPE segment into a diverse variety of amphiphilic polymers, which interestingly display stimuli-sensitive phase behavior in the form of a lower critical solution temperature (LCST). Below the LCST, they undergo self-assembly to form spherical core-shell nanostructures that display a poorly defined core-shell morphology. It is expected that hydrophobic patches are exposed within the micellar corona, reminiscent of the surface complexity of proteins, making these materials of interest for triggered and reversible assembly disassembly processes.

Full text of DOI:10.1021/bm4010832

Article
pubs.acs.org/Biomac
Poly(D‑glucose carbonate) Block Copolymers: A Platform for Natural
Product-Based Nanomaterials with Solvothermatic Characteristics
Tiffany P. Gustafson,† Alexander T. Lonnecker,† Gyu Seong Heo,† Shiyi Zhang,† Andrew P. Dove,‡
and Karen L. Wooley*,†
†Departments of Chemistry and Chemical Engineering, Texas A&M University, College Station, Texas 77842, United States
‡Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, United Kingdom
S
* Supporting Information
ABSTRACT: A natural product-based polymer platform,
having the characteristics of being derived from renewable
materials and capable of breaking down, ultimately, into
natural byproducts, has been prepared through the ring-
opening polymerization (ROP) of a glucose-based bicyclic
carbonate monomer. ROP was carried out via chain extension
of a polyphosphoester (PPE) macroinitiator in the presence of
1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) organocatalyst to
afford the PPE-b-poly(D-glucose carbonate) (PDGC) block
copolymer. This new copolymer represents a functional
architecture that can be rapidly transformed through thiol-yne reactions along the PPE segment into a diverse variety of
amphiphilic polymers, which interestingly display stimuli-sensitive phase behavior in the form of a lower critical solution
temperature (LCST). Below the LCST, they undergo self-assembly to form spherical core−shell nanostructures that display a
poorly defined core−shell morphology. It is expected that hydrophobic patches are exposed within the micellar corona,
reminiscent of the surface complexity of proteins, making these materials of interest for triggered and reversible assembly
disassembly processes.
Recently we developed a novel glucose-based bicyclic
carbonate monomer that undergoes ring-opening polymer-
ization (ROP) with catalysis by an organobase to yield an
amorphous poly(D-glucose carbonate) (PDGC).17 PDGC is
particularly attractive, as its hydrolytic degradation is expected
to produce carbon dioxide plus the (protected) saccharide
monomer. Development of PDGC constitutes a step toward
the design of potentially biocompatible materials from
renewable resources, yet, PDGC itself lacks the amphiphilic
nature required for supramolecular assembly into nanostruc-
tures.1,2 Thus, we designed a block copolymer of PDGC and
our previously reported polyphosphoester (PPE) system,18,19
which introduces a natural product-based degradable platform
capable of rapid and efficient functionalization. The structural
similarity of the PPE platform19 with natural polymers of
nucleic and teichoic acids20 makes it ideal for design of a block
copolymer whose second block is extended by a monomer
derived from the natural product glucose.
INTRODUCTION
■
As we have been working for many years to develop polymeric
nanomaterials that are capable of broad biomaterials
applications, our focus has shifted recently to their construction
from natural product sources to have the characteristics of
being renewable materials that break down into natural
byproducts. Design of new synthetic biocompatible polymers
for biomedical applications is a rapidly expanding field,1−4
especially in the development of materials that undergo
supramolecular assembly to form nanostructures with broad
applicability in nanomedicine, such as controlled release of
therapeutics and diagnostic imaging.1−3 While this field
continues to grow, it relies heavily on polymeric systems
designed from materials studied for more than 40 years, such as
aliphatic polyesters, poly(hydroxy acids), polyanhydrides, and
so on.5−7 To create the next generation of biomaterials, with
improved biocompatibility and biodegradation characteristics,
the use of renewable resources and natural products has
become of significant interest.8−10 Beyond the potential for
creating more biologically friendly materials, the benefits to
utilizing renewable feedstocks, such as monosaccharides,11−14
menthides,15,16 and so on, are substantial. Their natural
abundance alone will alleviate reliance upon petroleum-based
products made from rapidly dwindling resources, while their
increased biocompatibility and expected lower toxicity provide
added advantages for use in biomedicine.8
Herein, we report the improved synthesis of a glucose-based
bicyclic carbonate monomer 3 and its controlled ROP to form
PDGC 5 in a rapid and efficient manner that is compatible with
the design of a new degradable PPE-b-PDGC block copolymer
architecture. To the best of our knowledge, this is the first
Received: July 23, 2013
Revised: July 29, 2013
Published: August 19, 2013
© 2013 American Chemical Society
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suspended in DMF (100 mL). Methyl-4,6-O-benzylidene-α-D-
glucopyranoside (15 g, 0.074 mol) dissolved in DMF (75 mL) was
added dropwise over 10 min at 0 °C with stirring, and the mixture was
left for 1 h. Iodomethane (23 mL, 0.37 mol) in DMF (50 mL) was
added dropwise over 5 min at 0 °C, and the reaction was allowed to
warm to room temperature and left for 18 h. To quench the reaction,
H2O (50 mL) was added dropwise over 10 min. The mixture was
extracted with DCM and washed with brine. The organic layers were
collected, dried over anhydrous Na2SO4, and removed under vacuum.
The resulting residue was purified by column chromatography (SiO2,
gradient hexane/ethyl acetate) to yield the methylated derivative 1 (20
report of a block copolymer in which a glucose derivative has
been directly incorporated into the polymer backbone,
although, the use of glucose derivatives as end groups or side
chain functionalities in glycopolymers is widely utilized.21−23
This new block copolymer can be rapidly and efficiently
functionalized, through reactions along the PPE segment, to
form amphiphilic polymers that display interesting stimuli-
sensitive phase behavior and undergo self-assembly to form
core−shell nanostructures.
1
g, 0.063 mol, 86% yield). H NMR (500 MHz, CDCl3) δ 7.52−7.46
EXPERIMENTAL SECTION
■
(m, 2H), 7.41−7.32 (m, 3H), 5.54 (s, 1H), 4.86 (d, J = 3.7 Hz, 1H),
4.28 (dd, J = 10.0, 4.6 Hz, 1H), 3.86−3.77 (m, 1H), 3.77−3.66 (m,
1H), 3.63 (s, 3H), 3.55 (s, 3H), 3.53 (m, 1H), 3.30 (dd, J = 9.2, 3.7
Hz, 1H) ppm; 13C NMR (500 MHz, CD2Cl2) δ 137.35, 129.95,
128.22, 126.08, 101.37, 98.38, 82.14, 81.41, 79.85, 69.07, 62.24, 61.05,
59.37, 55.29 ppm; FTIR (ATR) 2928, 2833, 1449, 1369, 1329, 1175,
1082, 1051, 993, 922, 748, 729 cm−1; HRMS (M + H+; ESI+) Calcd
for C16H22O6H+, 311.1450; found, 311.1501; mp = 119−124 °C.
Synthesis of 2. To a solution of 1 (20 g, 0.064 mol) in methanol
(350 mL) was added H2O (40 mL), and 1 N HCl (70 mL). The
reaction was stirred at 60 °C for 3 h, monitoring by TLC. After 3 h it
was neutralized with saturated aq. NaHCO3. Solvent was removed
under vacuum; the product was left under high vacuum for 12 h to
remove remaining water yielding 2, which was used without
purification (14 g, 0.063 mol, 98% yield). 1H NMR (500 MHz,
CDCl3) δ 4.85 (d, J = 3.5 Hz, 1H), 3.89−3.73 (m, 2H), 3.64 (s, 3H),
3.64 (m, 1H), 3.56−3.39 (m, 2H), 3.50 (s, 3H), 3.43 (s, 3H), 3.22
(dd, J = 9.0, 3.5 Hz, 1H) ppm; 13C NMR (500 MHz, CD2Cl2) δ 97.41,
82.64, 81.82, 70.59, 70.27, 62.31, 61.15, 58.41, 55.15 ppm; FTIR
(ATR) 3040−3760, 2922, 2839, 1445, 1375, 1194, 1155, 1107, 1049,
1024, 983, 958, 905, 731 cm−1; HRMS (ESI+) Calcd for C9H18O6H+,
223.1137; found (M + Li+), 229.1277; mp = 82−87 °C.
Materials. All chemicals and reagents were used as received from
Sigma-Aldrich Co. unless otherwise noted. 2-Chloro-2-oxo-1,3,2-
dioxaphospholane (95%) was used as received from Thermo Fisher
Scientific Inc. Tetrahydrofuran (THF), dichloromethane, and N,N-
dimethylformamide (DMF) were purified by passage through solvent
purification system (JC Meyer Solvent Systems) and used as dried
solvents. 4-Methylbenzyl alcohol and 1,5,7-triazabicyclo[4.4.0]dec-5-
ene (TBD) were dried over CaH2 in THF, dried under vacuum, and
stored in a glovebox under Ar atmosphere. Column chromatography
was performed on CombiFlash Rf4x (Teledyne ISCO) with RediSep
Rf Column (Teledyne ISCO).
1
Characterization. H, 13C, and 31P NMR spectra were recorded
on Varian Mercury 300 or Varian Inova 500 spectrometers. Chemical
shifts were referenced to the solvent resonance signals. IR spectra were
recorded on an IR Prestige 21 system (Shimadzu Corp., Japan),
equipped with an ATR accessory, and analyzed using IRsolution v.
1.40 software.
Gel permeation chromatography (GPC) eluted with DMF was
conducted on a Waters Chromatography, Inc. (Milford, MA) system
equipped with an isocratic pump model 1515, a differential
refractometer model 2414, and a four-column set of 5 μm Guard
(50 × 7.5 mm), Styragel HR 4 5 μm DMF (300 × 7.5 mm), Styragel
HR 4E 5 μm DMF (300 × 7.5 mm), and Styragel HR 2 5 μm DMF
(300 × 7.5 mm). The system was equilibrated at 70 °C in prefiltered
DMF containing 0.05 M LiBr, which served as polymer solvent and
eluent (flow rate set to 1.00 mL/min). Polymer solutions were
prepared at a concentration of about 3 mg/mL and an injection
volume of 200 μL was used. Data collection and analysis were
performed with Empower 2 v. 6.10.01.00 software (Waters, Inc.). The
system was calibrated with S3 polystyrene standards (Polymer
Laboratories, Amherst, MA) ranging from 615 to 442800 Da.
Glass transition temperatures (Tg) and melting points (mp) were
measured by differential scanning calorimetry (DSC) on a Mettler-
Toledo DSC822 (Mettler-Toledo, Inc., Columbus, OH) under N2.
Measurements of Tg were taken with a heating rate of 5 °C/min and
those for mp were taken with a heating rate of 10 °C/min. The
measurements were analyzed using Mettler-Toledo Stare v. 10.00
software. The Tg was taken as the midpoint of the inflection tangent,
upon the third heating scan. The mp was taken as the temperature
range from the onset to the peak of the transition. Thermogravimetric
analysis (TGA) was performed under Ar atmosphere using a Mettler-
Toledo model TGA/DSC 1, with a heating rate of 10 °C/min.
UV/vis measurements were acquired on a Shimadzu UV-2550
spectrophotometer with an automated temperature controller. Trans-
mission electron microscopy (TEM) images were collected on a JEOL
1200EX operating at 100 kV and micrographs were recorded at
calibrated magnifications using a SIA-15C CCD camera. Samples were
deposited by dipping Formvar carbon film on 300 mesh copper grids
into 0.2 mg/mL polymer samples in PBS 10 times. Excess sample was
wicked of using filter paper and the grids were allowed to dry in air.
Subsequently, the grids were stained with 5 μL of a 1 wt %
phosphotungstic acid (PTA) aqueous solution. Excess stain was
wicked of using filter paper and the samples were left to dry under
ambient conditions
Synthesis of 3. Pyridine (14 mL, 0.17 mol) and 2 (10 g, 0.045 mol)
were combined in DCM (300 mL) with stirring. Triphosgene (16 g,
0.054 mol) was added in DCM (75 mL) dropwise over 10−15 min,
and the reaction was heated to 30 °C for 17 h, monitoring by TLC.
The reaction was cooled to 0 °C, quenched with NaHCO3 (100 mL),
extracted with DCM, and washed with 5% aq. HCl. The organic layers
were combined, dried over anhydrous Na2SO4, and removed under
vacuum. The resulting residue was purified by column chromatog-
raphy (SiO2, gradient hexane/ethyl acetate) to give the product 3,
which was recrystallized from ethyl acetate/hexanes (1:10) to give the
monomer 3 (2.8 g, 0.011 mol, 25% yield), which was used for
1
polymerization. H NMR (500 MHz, CDCl3) δ 4.88 (d, J = 3.6 Hz,
1H), 4.48 (dd, J = 9.8, 5.7 Hz, 1H), 4.22 (dd, J = 10.3, 9.8 Hz, 1H),
4.12−3.94 (m, 2H), 3.65 (m, 1H), 3.64 (s, 4H), 3.55 (s, 3H), 3.48 (s,
3H), 3.27 (dd, J = 9.3, 3.6 Hz, 1H) ppm; 13C NMR (126 MHz,
CD2Cl2) δ 147.42, 98.74, 80.88, 79.69, 79.30, 69.66, 61.33, 59.82,
59.61, 56.12 ppm; FTIR (ATR) 2936, 2835, 1759, 1464, 1400, 1202,
1130, 1090, 1055, 995, 958, 912, 763 cm−1; HRMS (ESI+) Calcd for
C10H22O7H+, 249.0930; found (M + Li+), 255.1042; mp = 74−77 °C.
Polymer Synthesis and Functionalization. Synthesis of PDGC
5. This reaction was carried out in a glovebox. Monomer 3 was dried
under vacuum over P2O5 for 3 d before being transferred to the
glovebox under an inert atmosphere. To a solution of 3 (0.20 g, 0.81
mmol) in DCM (1.2 mL) was added 4-methylbenzyl alcohol (0.016
mmol, 2.2 mg) in DCM (220 μL) with stirring. TBD (2 mol % to 3,
2.2 mg, 0.016 mmol) in DCM (220 μL) was slowly added to the
reaction. After 10 min, Amberlyst 15 H-form resin (20−50 mg) was
added to quench the reaction. The reaction mixture was purified by
precipitation from DCM into ether three times to give 5 (0.13 g, 66%
1
yield). H NMR (500 MHz, CD2Cl2) δ 7.25 (m, Ar(CH)), 7.18 (m,
Ar(CH)), 5.09 (ABq, OCH2Ar, JAB = 12 Hz), 4.84 (m, C1H), 4.63 (m,
C6H), 4.19 (m, C6′H, C4H), 3.88 (m, C3H), 3.45 (m, (OCH3)3), 3.27
(m, C2H), 2.37 (s, CH3Ar) ppm; 13C NMR (126 MHz, CD2Cl2) δ
155.14, 155.87, 154.49, 97.94, 81.69, 80.85, 75.09, 67.56, 66.59, 61.33,
61.15, 59.07, 55.78 ppm; FTIR (ATR) 2936, 2908, 2835, 1751, 1445,
1348, 1236, 1159, 1098, 1022, 989, 783 cm−1; Mn (NMR) = 11500 g/
Synthesis of the Cyclic D-Glucose Monomer 3. Synthesis of 1.
NaH (a 60% dispersion in mineral oil, 18 g, 0.45 mol) was washed
with THF (100 mL) under N2. THF was removed and NaH was
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Scheme 1. Synthesis of Monomer 3 and PDGC 5
mol; Mn (GPC) = 16100 g/mol; PDI = 1.12; Tg = 123 °C; TGA in Ar:
remove excess thiol and photoinitiator byproducts. The solution was
then lyophilized to give the products as white powders.
Cationic PBYP-b-PDGC 9a, 9b. Yield, 90%; H NMR (500 MHz,
5%
10%
Td = 226 °C, Td = 240 °C.
Synthesis of PBYP 7a, 7b:24 1H NMR (300 MHz, CD2Cl2) δ 4.41−
4.27 (m, OCH2CH2O), 4.27−4.12 (m, OCH2CH2C), 2.74−2.57 (m,
OCH2CH2C), 2.21 (m, CHC) ppm; 31P NMR (121 MHz, CD2Cl2) δ
−1.31; 13C NMR (126 MHz, CD2Cl2) δ 80.28, 70.98, 67.03, 66.31,
21.11, 21.05 ppm; FTIR (ATR) 3289, 2957, 1647, 1456, 1271, 1124,
1
DMSO-d6) δ 7.30 (m, Ar), 7.25 (m, Ar), 5.02 (m, OCH2Ar), 4.90 (br,
C1H), 4.53 (br, C4H), 4.35−3.95 (br, OCH2CH2O, OCH2CH2C,
C6H2), 3.80 (br, C5H), 3.49−3.19 (br, C3H, (OCH3)3, C2H), 3.10−
2.72 (br, CH2SCH2CHSCH2, CH2NH3, OCH2CH2CH), 2.32 (br,
CH3Ar) ppm; 31P NMR (121 MHz, DMSO-d6) δ −1.26 ppm; 13C
NMR (126 MHz, DMSO-d6) δ 154.11, 153.69, 153.29, 96.49, 80.16,
79.73, 73.95, 66.25, 66.20, 66.15, 65.35, 65.31, 59.99, 59.81, 57.60,
54.67, 41.35, 38.74, 33.38, 28.65, 26.86 ppm; FTIR (ATR) 3280−
3680, 2936, 2914, 2837, 2546, 1755, 1712, 1607, 1456, 1352, 1240,
1072, 1013, 974, 843, 804 cm−1; Mn (NMR) = 7500 g/mol; Mn
5%
(GPC) = 10200 g/mol; PDI = 1.12; Tg = −36 °C; TGA in Ar: Td
=
10%
209 °C, Td = 227 °C.
Synthesis of PBYP-b-PDGC 8a. This reaction was carried out in a
glovebox. TBD was dried over CaH2, and PBYP was dried under
vacuum over P2O5, for 3 days before being transferred to the glovebox
under an inert atmosphere. To a solution of 3 (0.13 g, 0.54 mmol) in
DCM (0.40 mL) was added 7 (89 mg, 0.012 mmol) in DCM (0.50
mL) with stirring. TBD (4 mol % to 3, 3.0 mg, 0.022 mmol) in DCM
(0.30 mL) was slowly added to the reaction. After 30 min, Amberlyst
15 H-form resin (20−50 mg) was added to quench the reaction. The
product was precipitated from DCM into ether three times to give 8
1089, 1020, 986, 806, 785, 743 cm−1. (9a) Tg = 27 °C; TGA in Ar:
5%
Td5% = 204 °C, Td10% = 217 °C. (9b) Tg = 27 °C; TGA in Ar: Td
=
10%
210 °C, Td = 221 °C.
Anionic PBYP-b-PDGC 10a, 10b. Yield, 79%; 1H NMR (500 MHz,
DMSO-d6) δ 7.30 (m, Ar), 7.25 (m, Ar), 5.02 (m, OCH2Ar), 4.90 (m,
C1H), 4.53 (m, C4H), 4.35−3.95 (br, OCH2CH2O, OCH2CH2CH,
C6H2), 3.95−3.75 (br, OCH2CH2CH, C5H), 3.49−3.19 (m, C3H,
(OCH3)3, C2H), 2.95−2.51 (m, CH2SCH2CHSCH2, CH2COOH,
OCH2CH2CH), 2.32 (s, CH3Ar) ppm; 31P NMR (121 MHz, DMSO-
d6) δ −1.26 ppm; 13C NMR (126 MHz, DMSO-d6) δ 172.96, 172.90,
154.11, 153.69, 153.29, 96.49, 80.16, 79.73, 73.95, 67.67, 66.45, 65.62,
65.19, 63.61, 59.99, 59.81, 57.60, 54.93, 54.67, 37.58, 34.83, 27.28,
25.31 ppm; FTIR (ATR) 2300−3700, 2938, 2839, 1755, 1713, 1651,
1445, 1398, 1348, 1240, 1190, 1159, 1020, 985, 954, 800, 785, 750
cm−1. (10a) TGA in Ar: Td5% = 153 °C, Td10% = 175 °C. (10b) TGA
1
(0.17 g, 74% yield). H NMR (500 MHz, CD2Cl2) δ 7.34 (m, Ar),
7.25 (m, Ar), 5.09 (ABq, OCH2Ar, JAB = 0), 4.88 (m, C1H), 4.68 (m,
C4H), 4.27 (m, OCH2CH2O, OCH2CH2C, C6H2), 3.92 (C5H), 3.57
(m, C3H), 3.53 (m, (OCH3)3), 3.31 (m, C2H), 2.67 (m, OCH2CH2C),
2.40 (s, CH3Ar), 2.19 (m, CHC) ppm; 31P NMR (121 MHz, CD2Cl2)
δ −1.31 ppm; 13C NMR (121 MHz, CD2Cl2) δ 155.16, 154.84,
154.52, 97.94, 81.68, 80.84, 80.29, 75.08, 70.99, 67.56, 67.04, 66.59,
66.30, 61.33, 61.14, 59.06, 55.78, 21.08 ppm; FTIR (ATR) 3300, 3244,
2954, 2837, 2361, 2250, 1755, 1650, 1452, 1350, 1271, 1255, 1024,
5%
10%
in Ar: Td = 136 °C, Td = 155 °C.
1
Zwitterionic PBYP-b-PDGC 11a. Yield, 56%; H NMR (500 MHz,
DMSO-d6) δ 8.60 (br, CH(NH3)COOH), 7.30 (m, Ar), 7.25 (m, Ar,
5.02 (m, OCH2Ar), 4.90 (m, C1H), 4.53 (m, C6H), 4.57−3.15 (br,
OCH2CH2O, OCH2CH2CH, CH2CH(NH3)COOH, C6H2), 3.80 (m,
C5H), 3.65−2.91 (br, C3H, (OCH3)3, C2H, CH2SCH2CHSCH2,
OCH2CH2C), 2.32 (s, CH3Ar) ppm; 31P NMR (121 MHz, DMSO-d6)
δ −1.26 ppm; FTIR (ATR) 3300−3680, 2400−3680, 2934, 2837,
1755, 1711, 1614, 1503, 1454, 1391, 1348, 1240, 1159, 1091, 1020,
987, 908, 858, 806, 787, 743 cm−1; Mn (NMR) = 16400 g/mol; Mn
5%
(GPC) = 12100 g/mol; PDI = 1.08; Tg = 40 °C; TGA in Ar: Td
228 °C, Td = 239 °C.
=
10%
Kinetics of PBYP-b-PDGC 8. This reaction was carried out in a
glovebox. Compound 7 was dried under vacuum over P2O5 for 3 days
before being transferred to the glovebox under an inert atmosphere.
To a solution of 3 (0.15 g, 0.60 mmol) in DCM (0.37 mL) was added
7 (85 mg, 0.011 mmol) in DCM (0.50 mL) with stirring. TBD (4 mol
%, 3.3 mg, 0.024 mmol) in DCM (0.33 mL) was slowly added to the
reaction. At 10 s, 1, 2, 4, 6, 8, and 10 min, 0.11 mL of the reaction was
removed and quenched over Amberlyst 15 H-form resin in DCM
(10−20 mg in 0.10 mL DCM). The reaction mixture at each time
5%
10%
988, 783, 743 cm−1; Tg = 78 °C; TGA in Ar: Td = 157 °C, Td
=
204 °C
Micelle Formation. Direct Dissolution: Each polymer 9a−11a
was evaluated by direct dissolution of 2 mg polymer into 1 mL water
or PBS (a 0.2 wt % solution) at room temperature with sonication
(45−60 min) at 4 °C (low temperature prevents premature
degradation). Upon removal from sonication the polymer solution
was clear, but as the solution warmed to room temperature, it became
opaque with white precipitates (Figure S10). Maintaining the solution
below the LCST allowed for preparation of TEM samples indicating
micelle formation.
1
point was removed from the resin and evaporated for analysis by H
and 31P NMR spectroscopy and GPC without purification.
General Synthesis of Functionalized PBYP-b-PDGC 9−11. PBYPx-
b-PDGCy (0.077 mmol), functional thiol (0.77 mmol), and DMPA
(0.012 mmol) were dissolved in MeOH (2 mL), degassed under N2
for 15 min, and irradiated with a standard TLC lamp at 365 nm for 2
h. The anionic (3-mercaptopropionic acid) functionalized diblock
copolymer was precipitated from methanol into diethyl ether three
times to remove excess functional thiols and photoinitiator by-
products, to yield the product polymer as a white powder. For cationic
(cysteamine hydrochloride) and zwitterionic (L-cysteine hydrochloride
monohydrate) functionalized diblock copolymers, the polymer
solution was transferred to dialysis tubing and dialyzed against
nanopure water with HCl, pH 3.0, in the cold room for 36 h, to
Solvent exchange triggered self-assembly: Each polymer 9a−11a (2
mg) was dissolved in organic solvent (0.5 mL methanol or DMF) and
water was added at a rate of 0.25 mL/h until the ratio of solvent/water
reached 1:1. The mixture was then dialyzed against nanopure water for
24 h at 4 °C. Upon warming to room temperature precipitation was
observed.
LCST and UCST Measurements of 9a−11a. LCST. Polymers
9a−11a were dissolved at 0.2 wt % into PBS pH 7.4 with sonication
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Scheme 2. Synthesis of PBYP-b-PDGC Block Copolymers 8a and 8b by ROP of 3 from PPE Macroinitiators 7a and 7b
1
Figure 1. H and 31P NMR (upper left inset) spectra (CD2Cl2) of PBYP-b-PDGC diblock copolymer 8a.
on ice for 45−60 min. The solution was transferred to a UV/vis
cuvette at 0 °C. The absorbance was then monitored at 300, 500, and
700 nm every 0.1 °C as the temperature was increased at a ramp rate
of 0.1 °C/min. After heating to 40 °C, the solution was sonicated on
ice for 45−60 min, which leads to resuspension of the polymer,
indicating reversibility of LCST.
UCST. Polymers 9a−11a were dissolved at 0.2 wt % into PBS pH
7.4 with sonication on ice for 45−60 min. The solution was transferred
to a UV/vis cuvette at 0 °C. The absorbance was then monitored at
300, 500, and 700 nm every 0.1 °C as the temperature was increased at
a ramp rate of 0.2 °C/min. After heating to 80 °C, the increased
potential for degradation of the sample prohibited resuspension by
sonication to prove reversibility.
Preparation of TEM Samples. Polymers 10a and 11a were
dissolved at 0.2 wt % into PBS pH 7.4 with sonication on ice for
45−60 min. The solution was transferred to a UV/vis cuvette at 0 °C.
A TEM grid was prepared at 0, 5, 10, 15, 20, 25, 30, 35, and 40 °C, as
described above. After preparing each TEM sample, transitions to
higher temperatures took place at 1 °C/min.
which undergoes ROP by an alcoholic initiator in the presence
of the catalyst 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to
form poly(2-but-3-yn-1-yloxy)-2-oxo-1,3−2-dioxaphospholane
(PBYP, 7, Scheme 2) and is then efficiently functionalized by
thiol−yne click chemistry.19 Previous studies have shown that
longer polymerization times, greater than several minutes, have
led to an increase in the polydispersity indices (PDIs) of the
resultant polymers.18 The initially reported ROP synthesis of
PDGC, catalyzed by 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),
required 7 h to reach ≥98% conversion.17 Therefore, to ensure
uniform growth of PDGC from PBYP, without allowing for
adverse transesterification reactions, the small molecule
initiated ROP of 3 was optimized such that formation of 5
occurred rapidly. By reducing the monomer concentration from
1.0 to 0.5 M and performing the reaction under an Ar
atmosphere in a glovebox at ∼30−35 °C, conversion of 3 was
complete within <10 min in the presence of the organocatalyst
TBD to afford 5. The product maintained a comparable
number-average degree of polymerization (DPn = 50) and PDI
(1.13) to those originally reported (DPn = 51, PDI = 1.15).
Under these conditions, transesterification was minimized,
allowing for higher DP values to be obtained, up to 100, while
maintaining a low PDI of 1.19 (Figure S.4).
The block copolymer PBYP-b-PDGC 8 was prepared by
conducting the ROP of BYP monomer 6, followed by the use
of PBYP 7 as a macroinitiator for the ROP of 3. Polymerization
of both 6 and 3 proceeds via an organobase-catalyzed ROP, yet
the optimized conditions under which each reaction takes place
are different. To achieve high monomer conversion of 6,
yielding 7 at room temperature, the less reactive base DBU was
utilized.18 The monomer concentration must also be unusually
high (1 g/mL, equivalent to 5.7 M) for ROP of 6, whereas low
monomer concentrations, ≤1 M, are generally employed for
most cyclic ester or carbonate monomers, to avoid trans-
esterification side reactions and ensure that polymer products
RESULTS AND DISCUSSION
■
Before constructing the PPE-b-PDGC block copolymer, the
synthesis of 3 was modified (Scheme 1), such that it could be
prepared quickly and at lower cost.17 To significantly shorten
the overall reaction time, the hydrogenolysis of 1 (69 h) was
replaced by benzylidene deprotection via acid-catalyzed
hydrolysis (3 h). Cost was reduced by replacing bis-
(pentafluorophenyl) carbonate with inexpensive triphosgene
in the condensation of the diol 2 to form the bicyclic monomer
3, for which the overall yield following purification was similar
to the previously reported yield.17
Remarkably, it was observed that the organocatalyzed ROP
of 3 prepared by this new methodology proceeded significantly
more rapidly than had the initially studied ROP,17 and this
finding was important to the success of the PPE-b-PDGC block
copolymer synthesis. The PPE segment was designed based on
2-(but-3-yn-1-yloxy)-2-oxo-1,3,2-dioxaphospholane (BYP, 6),
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are obtained with low PDI.12,14,25,26 Polymerization of 3
necessitates the use of the more reactive catalyst, TBD,17 and
relatively low concentrations (0.5 M). Therefore, after drying
over P2O5 under vacuum, 7 and 3 were combined under an Ar
atmosphere (glovebox) in dichloromethane (DCM) with 4 mol
% (to 3) of TBD as an organocatalyst, such that the final
concentration of 3 was 0.5 M. After 10 min, the reaction was
quenched with Amberlyst 15 H-form resin. From 7a and 7b, 8a
and 8b were obtained in about 75% yield, respectively (Scheme
2), following precipitation three times from DCM into diethyl
ether.
Confirmation of the block copolymer structure of 8 was
made by a combination of data obtained from gel permeation
chromatography (GPC), 1H and 31P NMR spectroscopies
(Figure 1), thermogravimetric analysis (TGA; Figure S.6), and
differential scanning calorimetry (DSC) in comparison to the
macroinitiator 7 and homopolymer 5. GPC analysis showed
decreased retention volumes that correlated with increased
number-average molecular weight (Mn) values, with main-
tenance of low PDIs, upon growth of 8a from 7a (Figures 2 and
S.5), while analysis of 1H NMR spectra revealed the presence of
resonances that correspond to both PBYP and PDGC polymer
blocks. Characterization of 8 by TGA and DSC demonstrated
5
obvious changes in thermal degradation temperatures (Td ,
Td10) and the glass transition temperature (Tg), compared to
the respective homopolymers (Table 1). Interestingly, a single
Tg, intermediate to those of 5 and 7, was displayed for 8,
indicating a lack of phase segregation between the two blocks in
the solid state.
Kinetic studies of the ROP of 3 from the macroinitiator 7
forming 8 confirmed that the reaction proceeded in a
controlled manner. The polymerization was conducted at a
monomer:initiator ([M]0/[I]0) ratio of 45:1. Following
addition of TBD, 100 μL aliquots of the reaction mixture
were removed at regular time intervals and quenched over
Amberlyst 15 H-form resin. The % conversions of monomer to
polymer were determined by integration of 1H NMR signals of
the bicyclic monomer protons 6 and/or 5 and 4 (Figure S.1),
normalized against the methylene protons of the butynyl side
chain groups along the PPE segment (labeled as 10 in Figure 1)
originating from 7, which allowed for calculation of the
consumption of monomer. The DPn of 8 was calculated from
1
integration of H NMR signals of the PDGC segment protons
4 and 5 normalized against the methylene protons from the
PPE macroinitiator 7. The resulting ratio of protons 4 and 5 of
the new PDGC segment to the methylene protons of 7 was
used to calculate the DPn and, hence, the Mn of 8, which was
also obtained from GPC. The linear relationship between Mn
and monomer conversion suggests a well-controlled polymer-
ization, wherein the number of macromolecules remains
constant to ≥98% monomer conversion (Figure 2A). The
PDI values were all below 1.17, decreasing to 1.11 until the
reaction reached 80% monomer conversion, where the PDI
remained constant. Plots of ln([M]0/[M]) versus time show a
linear correlation, which indicates that the polymerization
displays first order kinetics, characteristic of ROP (Figure 2B).
Both the PBYP and the PDGC blocks of 8 are of a
hydrophobic nature; as such, 8 displayed a lack of phase
segregation and an inability to undergo self-assembly in
aqueous medium. Fortunately, the system design facilitates
rapid functionalization of the macroinitiator segment 7, via
thiol-yne click chemistry, an efficient mechanism for generation
of amphiphilic polymers. We have previously reported that the
radical-mediated thiol-yne reaction was compatible with the
PPE backbone.18,19 Therefore, to induce phase segregation and
formation of nanostructures in aqueous solution, the cationic
(9), anionic (10), and zwitterionic (11) variants of 8 were
prepared via the reported methodology (Figure 3) using
cysteamine hydrochloride, 3-mercaptopropionic acid, and L-
cysteine hydrochloride monohydrate, respectively. Formation
of the functionalized amphiphilic polymers was confirmed by
disappearance of the terminal acetylene protons (Figure 1,
protons 11) and the appearance of proton resonances related to
the new functional groups. Further analysis by infrared
spectroscopy (IR) confirmed the loss of the terminal alkynes
and introduction of the desired amine (cationic and
zwitterionic) and carbonyl (anionic and zwitterionic) groups
(Figure S.8 for reaction of 8a to 9a, 10a, and 11a). DSC and
TGA (Figure S.7) analyses revealed changes to the Tg and Td
for all functionalized block copolymers, as compared to 8a and
8b (Table 2). Surprisingly, each functionalized polymer
continued to display a single Tg in the solid state, which
indicates that phase segregation in the solid state remained
elusive, despite addition of ionic groups.
Figure 2. (A) Plot of Mn and PDI vs monomer conversion (%) for
polymerization of 3 using TBD as the catalyst and 7a as the
macroinitiator. The ratio of 3:7a:TBD was 45:1:0.55. The inset
illustrates chain extension of 3 onto 7a to afford 8a, monitored by
GPC at 0.17, 4, and 10 min. (B) Kinetic plots of the extent of
monomer conversion (ln([M]0/[M])) vs time, obtained from 1H
NMR spectroscopy.
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Table 1. Properties of PBYPx-b-PDGCy in Comparison to the Macroinitiator 7 and Homopolymer 5
a
b
c
d
e
f
g
g
10
5
polymer
conv (%)
PDI
Mn
Mn
Mn
Tg (°C)
Td (°C)
Td (°C)
5
99
96
98
85
99
1.13
1.12
1.15
1.10
1.12
12400
8600
16100
10200
16900
12100
19000
11500
7500
123
−36
−39
5
h
282
209
211
228
237
300
227
227
239
250
7a
7b
8800
8600
8a
17000
19700
16400
21500
8b
7a + 5
−37 + 95
a
b
c
Estimated by 1H NMR spectroscopy. PDI measured by GPC (DMF eluent) calibrated with polystyrene standards. Calculated: MWI + MWM
([M]0/[I]0) × conv. (%). Calculated by GPC. Calculated from H NMR spectroscopy. Determined by DSC. Determined by TGA, Td = 5%
mass loss and Td = 10% mass loss. Tg not observed.
×
d
e
f
g
1
5
h
10
Figure 3. Preparation of amphiphilic polymers 9−11 from 8 (Top). TEM of micelles assembled from 10a; (A) 0 °C, Davg = 20 4 nm, (B) 15 °C,
Davg = 16 4 nm + larger aggregates, (C) 20 °C, Davg = 13 4 nm + larger aggregates, and (D) 35 °C, only microscopic aggregates are visualized.
process, the solvent was removed by dialysis against nanopure
water at 4 °C. Upon warming to room temperature,
precipitation was again observed. This result led to our
investigation of temperature-sensitive phase behavior, displayed
as a lower critical solution temperature (LCST).
The most common synthetic polymers displaying LCSTs are
poly(N-isopropylacrylamide) (PNIPAM) and its copolymers
and block copolymers,27−29 although it has also been shown
that naturally derived polymers, such as methyl cellulose (MC),
may exhibit LCSTs as well.30 Additionally, there are several
reports of PPEs and polycarbonates prepared by ROP that
display LCSTs, which can be correlated to the ratio of the
hydrophobic/hydrophilic block lengths.31−33 However, our
previously reported PBYP polymers have not displayed
temperature-sensitive phase transitions.18,19
Table 2. Thermal Properties of Functionalized Polymers 9−
11
a
b
b
c
polymer
Tg (°C)
Td (°C)
Td (°C)
LCST* (°C)
e
8a
5
228
204
153
157
237
210
136
236
217
175
204
250
221
155
9a
27
d
9.3 1.6
10a
11a
8b
13.3 2.7
78
d
9.1 5.0
e
e
e
9b
27
d
10b
a
b
Determined by DSC. Determined by TGA, Td5 = 5% mass loss and
c
10
Td = 10% mass loss. LCST turbidity measurements at 0.2 wt % of
polymer, the cloud point equals 10% loss of transmission. Tg not
observed. LCST not observed.
d
e
The LCST behaviors of 9a−11a were investigated by
turbidimetry upon direct dissolution into PBS, whereby
solutions of 2 mg/mL (0.2 wt %) were sonicated at 4 °C for
1 h. The resulting clear solutions were transferred to UV/vis
cuvettes at 0 °C and cloud point assays were performed. The
absorbance of each solution was monitored at 300, 500, and
700 nm every 0.1 °C at a temperature ramp rate of 0.1 °C/min
from 0 to 40 or 0.2 °C/min from 0 to 80 °C (Figure S.9);
visible precipitates were observed above the cloud point.
Polymer samples that had precipitated while being heated to 40
°C were resuspended by sonication at 4 °C (when the
temperature was raised to 80 °C, degradation was a concern),
The characteristics of solvated polymers can vary from those
found in the solid state. Hence, the supramolecular self-
assembly behaviors of polymers 9a−11a were evaluated by
direct dissolution into water with sonication (45−60 min) at 4
°C (low temperature prevented premature degradation). Upon
removal from sonication, the polymer solutions were clear, but
as they were allowed to warm to room temperature, they
became opaque with white precipitates (Figure S.10). Solvent
exchange-triggered self-assembly of 9a−11a from organic
solvent (methanol or DMF) into water was evaluated to
confirm precipitation was not method dependent. In this
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demonstrating the reversibility of the LCST behavior. The
cloud points for 9a−11a were determined where the relative
loss of transmission equaled 10% (Table 2).33,34 Measurements
for all three functionalized polymers are within error, which
indicates that the phase behavior is independent of the
functional group, and suggests that the PDGC segment is
driving the thermal response. Further investigation of the
LCST led to the synthesis of 9b and 10b (cationic and anionic
PPE48-b-PDGC48), respectively, which turned out to be water
insoluble polymers at the longer PDGC chain length. These
results support the hypothesis that the hydrophobicity of the
PDGC block may lead to a significant lowering of the LCST of
the PPE; the lowering effects of saccharides in solution on
LCST are well-known.22,35,36 Another rationale for the
relatively low cloud points for 9a−11a could be incomplete
packaging of the PDGC block segment within a core domain
and incomplete coverage of micellar corona by PPE; however,
PDGC homopolymer was observed to be water insoluble over
the full temperature range evaluated.
applicability in nanomedicine. This new architecture constitutes
a step toward the next generation of biomaterials, designed
from renewable resources, with potentially improved bio-
compatibility and biodegradation characteristics.
ASSOCIATED CONTENT
* Supporting Information
Experimental results in regard to characterization. This material
is available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: wooley@chem.tamu.edu.
■
Funding
We gratefully acknowledge financial support from the National
Heart Lung and Blood Institute (HHSN268601000046C), the
National Science Foundation (CHE-1057441), and the Welch
Foundation (A-0001). A.P.D. is grateful to the Royal Society of
Chemistry for a J.W.T. Jones Traveling Fellowship.
To determine whether functionalized PPE-b-PDGC materi-
als self-assembled into micellar-type nanostructures in aqueous
PBS solutions, they were evaluated below their cloud points by
transmission electron microscopy (TEM). Copolymers 10a and
11a were suspended in PBS at 0 °C and TEM samples were
prepared as the solution temperature was increased, in a
stepwise fashion, at 5 °C intervals. TEM revealed micellar
structures of number-average diameter (Dav) ca. 20 nm below
the cloud point (10a, Figure 3A and 11a Figure S.11). Analysis
of TEM images (Figure 3A−D, Figure S.11A−D) indicates that
samples of micellar assemblies transformed to heterogeneous
mixtures of micelles plus large aggregates and further to mainly
microscopic assemblies with increasing temperature until the
polymer precipitated macroscopically. These observations
indicate that the micelles had poorly defined core−shell
morphology, which is being studied in greater detail with
broader ranges of compositions and structures.
Notes
The authors declare no competing financial interest.
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