A reduction sensitive cascade biodegradable linear polymer

Article abstract of DOI:10.1002/pola.24180

Cascade degradable linear polymers offer the potential for a high degree of control over the degradation process. They comprise a backbone that is stable in the presence of an end cap, but upon removal of the end cap a cascade of intramolecular reactions

Full text of DOI:10.1002/pola.24180

A Reduction Sensitive Cascade Biodegradable Linear Polymer
MATTHEW A. DEWIT,1 ANNELISE BEATON,1 ELIZABETH R. GILLIES1,2
1Department of Chemistry, The University of Western Ontario, 1151 Richmond St., London, Canada N6A 5B7
2Department of Chemical and Biochemical Engineering, The University of Western Ontario, 1151 Richmond St.,
London, Canada N6A 5B9
Received 23 April 2010; accepted 7 June 2010
DOI: 10.1002/pola.24180
Published online in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: Cascade degradable linear polymers offer the poten-
tial for a high degree of control over the degradation process.
They comprise a backbone that is stable in the presence of an
end cap, but upon removal of the end cap a cascade of intramo-
lecular reactions is initiated that leads of depolymerization of
the polymer backbone. Reported here is a new polymer back-
bone based on N,N0-dimethylethylenediamine and 2-mercapto-
ethanol linked by carbamates and thiocarbamates. A disulfide
end cap was incorporated such that its cleavage under reducing
conditions revealed the thiol of 2-mercaptoethanol, leading to
alternating cyclizations of the 2-mercaptoethanol and N,N0-
dimethylethylenediamine moieties to provide 1,3-oxathiolan-2-
one and N,N0-dimethylimidazolidinone, respectively. The degra-
dation was monitored by 1H NMR and GPC. The expected prod-
ucts were observed, along with a portion of nondegradable
polymer that was likely cyclic species. Overall, the results demon-
strate the promise of this new class of polymers to degrade selec-
tively in reducing environments such as hypoxic tumor tissue or
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V
the intracellular compartments of cells. 2010 Wiley Periodicals,
Inc. J Polym Sci Part A: Polym Chem 48: 3977–3985, 2010
KEYWORDS: biodegradable; biological applications of polymers;
redox-sensitive polymers; self-immolative polymers; stimuli-
sensitive polymers
INTRODUCTION Biodegradable polymers have been of signif-
icant interest in recent years for a wide range of applica-
tions. For example, they can serve as environmentally
friendly substitutes for nondegradable polymers in materials
such as food and beverage containers.1 They have also been
developed for biomedical materials such as sutures,2 stents,3
and tissue engineering scaffolds,4,5 thus allowing the materi-
als to degrade during the natural healing or tissue regenera-
tion process, preventing the need for further interventions to
remove the foreign material. Furthermore, their incorpora-
tion into drug delivery systems such as micelles,6,7 worms,8,9
vesicles,10–12 and nanoparticles13–15 facilitates the release of
encapsulated drug molecules throughout the degradation
process. Thus far, significant progress has been made in
these areas using polymers such as polycaprolactone,16–18
poly(lactic acid),19,20 and poly(glycolic acid).21–23 However,
the ability to ‘‘turn on’’ the degradation of these polymers
under specific physiological conditions has not been demon-
strated as these polymer backbones exhibit gradual degrada-
tion under most physiological conditions.24,25
ical materials or release of drug molecules from the drug
delivery system. Thus far, several polymer backbones contain-
ing acetal26–29 or disulfide30,31 linkages have been developed
to degrade under mildly acidic or reducing conditions respec-
tively. However, the mechanisms of degradation for these
polymers involve random chain scissions throughout the poly-
mer backbone, and many environmentally mediated cleavage
events are required to completely degrade the polymer.
Inspired by elegant work on dendrimer systems that were
designed to degrade by a cascade of reactions upon removal
of a single trigger moiety,32–41 the group of Shabat42,43 as
well as our group44 have recently developed end capped cas-
cade degradable linear polymers. As illustrated in Figure 1,
these polymers comprise backbones that are stable when the
end cap is intact, but upon removal of the end cap via a sin-
gle bond cleavage, a functionality is revealed at the polymer
terminus that initiates a cascade of intramolecular reactions
leading to complete depolymerization from end to end. Like
the dendrimer systems, both of these systems have used self-
immolative linkers previously developed for prodrugs.45–50
Shabat’s group reported the use of a polycarbamate based on
4-aminobenzyl alcohol derivatives, that depolymerized to flu-
orescent monomers via a series of rapid 1,6-elimination reac-
tions in response to an enzyme mediated end cap cleavage,
thus serving as a sensor for the enzyme.42 Our group devel-
oped a polycarbamate that degraded by alternating cyclization
The ability to trigger the degradation of a polymer backbone
under specified conditions such as photochemical or enzy-
matic stimuli, or changes in pH or redox potential offers the
possibility to utilize polymer backbones that will be stable
for extended periods but that will degrade under the desired
conditions, resulting in a controlled disintegration of biomed-
Additional Supporting Information may be found in the online version of this article. Correspondence to: E. R. Gillies (E-mail: egillie@uwo.cas)
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Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 48, 3977–3985 (2010) 2010 Wiley Periodicals, Inc.
CASCADE BIODEGRADABLE LINEAR POLYMER, DEWIT, BEATON, AND GILLIES
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
was performed using a PE-Sciex API 365 triple quadrupole
instrument. Gel permeation chromatography was performed
using a Waters 515 HPLC pump, equipped with Wyatt mini-
DawnTREOS and Wyatt Optilab Rex detectors, and two Resi-
Pore 300 ꢀ 7.5 mm, 3 lm particle size columns from Poly-
mer Laboratories. The eluent used was THF and the
calibration was performed using polystyrene standards.
FIGURE 1 Schematic of a cascade biodegradable polymer.
Synthesis of Compound 2
To a solution of imidazole (3.84 g, 56.3 mmol, 2.16 eq.) in
DMF (40 mL) was added a solution of tert-butyldiphenyl-
chlorosilane (7.75 g, 28.2 mmol, 1.08 eq.) in DMF (30 mL)
and the resulting solution was stirred for 10 minutes. 2-Mer-
captoethanol (1) (2.04 g, 26.1 mmol, 1.00 eq.) in DMF
(8 mL) was then added and the reaction mixture was stirred
at room temperature for 24 hours. The DMF was then
removed in vacuo and the crude product was taken up in
CH2Cl2 (30 mL), filtered, and washed with an equal volume
of H2O to remove the imidazole. The organic layer was dried
with MgSO4, filtered, and the solvent was removed in vacuo.
The residue was purified by silica gel chromatography using
97:3 hexanes:ethyl acetate as an eluent to provide 7.74 g
(24.4 mmol) of 2 as a clear colorless oil.
and 1,6-elimination reactions and incorporated this polymer
into an amphiphilic block copolymer by using a poly(ethylene
glycol) derivative as an end cap.44 It was demonstrated that
the cyclization reaction could be used to control the overall
rate of depolymerization and also that the block copolymer
could be assembled into nanoparticles in aqueous solution.
These nanoparticles were capable of encapsulating and
releasing a model drug molecule in a controlled manner, thus
demonstrating the promise of cascade degradable linear poly-
mers in drug delivery applications.
To fully exploit this new class of polymers, it will be neces-
sary to develop a series of polymer backbones with different
depolymerization rates and also a series of end caps that can
be removed under different conditions. This will allow for
the selection of the appropriate backbone and end cap com-
bination for the desired application. Furthermore, it has
been suggested that the quinone methide intermediates
involved in the 1,6-elimination reactions can potentially lead
to toxicity,51–53 so it would be desirable to develop new
backbones that do not involve hydroxybenzyl alcohol or ami-
nobenzyl alcohol. Towards this goal, we report here the first
Yield: 94%. 1H NMR (400 MHz, CDCl3): d 7.71–7.67 (m, 4H,
phenyl CH), 7.48–7.37 (m, 6H, phenyl CH), 3.79 (t, J ¼ 6.35
Hz, 2H, OACH2), 2.68 (dt, J1 ¼ 8.30, J2 ¼ 6.30 Hz, 2H, S-CH2),
1.60 (t, J ¼ 8.30 Hz, 1H, SH), 1.08 (s, 9H, t-butyl). 13C NMR
(100 MHz, CDCl3): 135.2, 133.1, 129.4, 127.4, 65.3, 26.8, 26.5,
18.9. IR (NaCl, thin film, cmꢁ1): 1520 (aryl C¼¼C), 1650 (aryl
C¼¼C), 1685 (aryl C¼¼C), 1770 (CAH bond), 1930 (SAH),
2970 (CAH), 3070 (sp2 CAH) cmꢁ1. HRMS (m/z): calcd for
example of
a cascade degradable linear polymer that
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18H24OSSi, 315.1239; found (ESI), 315.1230 [M-H]þ.
degrades entirely by cyclization reactions. Furthermore, we
describe the first incorporation of a disulfide end cap that
can be cleaved under mildly reducing conditions. Such condi-
tions can be encountered in hypoxic tumor tissue54 where
the concentration of the reducing agent glutathione is at
least fourfold higher than in normal tissues55 or within the
intracellular environment where the concentration of gluta-
thione is approximately 0.5–10 mM relative to 2–20 lM in
the extracellular environment.56,57
Synthesis of Compound 3
Compound 2 (1.01 g, 3.20 mmol, 1.00 eq.) was dissolved in
CH2Cl2 (40 mL). Triethylamine (2.2 mL, 15 mmol, 4.7 eq.)
was added followed by 4-nitrophenyl chloroformate (1.30 g,
6.39 mmol, 2.00 eq.) and the reaction mixture was stirred at
room temperature for 18 hours. The reaction mixture was
then poured onto 1 M HCl (50 mL), and the product was
extracted twice with CH2Cl2. The combined organic layers
were dried over MgSO4, filtered, and the solvent was
removed in vacuo. The product was purified by silica gel
chromatography using 1:1 CH2Cl2:hexanes as an eluent to
provide 1.52 g (3.16 mmol) of 3 as a clear oil.
EXPERIMENTAL
General Procedures and Materials
Solvents used were anhydrous and obtained from a solvent
purification system. Chemicals were obtained from Sigma
Aldrich and Alfa Aesar and were used without further purifi-
cation unless otherwise noted. Glassware used in all reac-
tions was flame dried, evacuated and put under N2 before
being charged with any materials. Silica used for column
chromatography was 70–230 mesh, 0.063–0.200 mm particle
Yield: 99%. 1H NMR (400 MHz, CDCl3): d 8.28 (d, J ¼ 9.38
Hz, 2H, p-nitrophenyl), 7.69 (dd, J1 ¼ 7.9 Hz, J2 ¼ 1.5 Hz,
4H, phenyl CH), 7.37–7.50 (m, 6H, phenyl CH), 7.32 (d, J ¼
9.4 Hz, 2H, p-nitrophenyl), 3.91 (t, J ¼ 6.1 Hz, 2H), 3.18 (t,
J ¼ 6.1 Hz, 2H), 1.06–1.11 (m, 9H, CH3). 13C NMR (100 MHz,
CDCl3): 169.5, 155.6, 135.6, 133.1, 129.8, 127.7, 125.2,
122.0, 62.2, 34.3, 26.8, 19.2. IR (NaCl, thin film, cmꢁ1): 1520
(aryl C¼¼C), 1590 (aryl C¼¼C), 1725 (C¼¼O), 2850 (CAH),
2930 (CAH), 3050 (sp2 CAH). HRMS (m/z) calc’d for
1
size. H and 13C NMR spectra were run in CDCl3 (d 7.27 and
77 ppm). 1H NMR spectra were obtained on a Varian Mer-
cury Instrument at 400 MHz and 13C NMR spectra were
obtained on a Varian Inova Instrument at 100 MHz. Chemical
shifts are reported in ppm. Coupling constants (J) are
reported in Hz. IR samples were dissolved in CH2Cl2 and
cast as films on NaCl plates, then spectra were obtained
using a Bruker Tensor 27 instrument. ESI mass spectrometry
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25H27NO5SSi, 482.1457; found (ESI), 482.1461 [MþH]þ.
Synthesis of Compound 4
To a solution of compound 3 (3.17 g, 6.58 mmol, 1.00 eq.) in
toluene (60 mL) were added 4-(dimethylamino)pyridine
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ARTICLE
(DMAP) (0.077 g, 0.63 mmol, 0.096 eq.), N,N-diisopropyle-
thylamine (DIPEA) (1.72 g, 13.3 mmol, 2.02 eq.), and Boc
protected N,N0-dimethylethlyenediamine44 (1.81 g, 9.63
mmol, 1.46 eq.). The reaction mixture was heated at reflux
for 5 hours. The reaction mixture was then washed 1M HCl,
followed by two washes with saturated Na2CO3 solution. The
organic layer was dried with MgSO4, filtered, and the solvent
was removed in vacuo to provide 3.48 g (6.55 mmol) of 4 as
a clear pale yellow oil. Yield: 99%.
CH2Cl2:ethyl acetate as an eluent to provide 0.60 g (1.31
mmol) of 6 as a clear, pale yellow oil.
Yield: 85%. 1H NMR (400 MHz, CDCl3): d 8.33–8.25 (m, 2H,
p-nitrophenyl), 7.46–7.37 (m, 2H, p-nitrophenyl), 4.42 (t, J ¼
6.5 Hz, 2H, carbonate CH2) 3.61–3.50 (m, 2H, (rotamer) dia-
mine CH2), 3.40 (d, J ¼ 6.1 Hz, 2H, carbamate CH2), 3.27 (d,
J ¼ 5.5 Hz, 2H, (rotamer) diamine CH2), 3.04 (s, 3H, carba-
mate CH3), 2.89 (br s, 3H, Boc protected amine CH3), 1.47
(br. s. 9H, boc CH3). 13C NMR (100 MHz, CDCl3): 167.0
(rotamer), 166.8 (rotamer), 166.2 (rotamer), 155.6
(rotamer), 155.4, 155.2 (rotamer), 152.1, 145.3, 125.1, 121.7,
79.7 (rotamer), 79.52 (rotamer), 79.45 (rotamer), 88.0, 53.4,
47.9 (rotamer), 47.5 (rotamer), 46.7 (rotamer), 46.4
(rotamer), 45.6 (rotamer), 35.5 (rotamer), 35.2 (rotamer),
34.7 (rotamer), 34.3 (rotamer), 28.5 (rotamer), 28.3
(rotamer). IR (NaCl, thin film, cmꢁ1): 1650 (C¼¼O), 1690
(C¼¼O), 1770 (C¼¼O), 2920 (CAH), 2964 (CAH), 3070 (sp2
CAH), 3105 (sp2 CAH). HRMS (m/z) calc’d for C19H27N3O8S,
458.1597; found (ESI), 458.1599 [MþH]þ.
1H NMR (400 MHz, CDCl3): d 7.68 (dd, J1 ¼ 7.9 Hz, J2 ¼ 1.7
Hz, 4H), 7.47–7.34 (m, 6H), 7.30–7.14 (m, 3H), 3.82 (m, 2H,
CH2AO), 3.57–3.32 (m, 4H, (rotamer) diamine CH2), 3.18–
3.08 (m, 2H, CH2-S), 3.02 (br s, 3H), 2.94–2.81 (m, 3H), 1.46
(d, J ¼ 8.40 Hz, 9H, Boc CH3), 1.06 (s, 9H, CH3). 13C NMR
(100 MHz, CDCl3): 168.2 (rotamer), 167.5 (rotamer), 155.7,
155.4, 135.5, 133.6, 129.6, 127.6, 125.3, 115.6, 79.7, 63.3,
47.6 (rotamer), 46.7 (rotamer), 45.8 (rotamer), 35.7
(rotamer), 35.3 (rotamer), 34.8 (rotamer), 34.6 (rotamer),
32.99 (rotamer), 32.93 (rotamer), 28.4, 26.8, 19.2. IR (NaCl,
thin film, cmꢁ1): 1650 (C¼¼O), 1690 (C¼¼O), 2850 (CAH),
2925 (CAH), 3060 (sp2 CAH). HRMS (m/z): calcd for
Synthesis of Compound 8
Compound 758 (0.24 g, 1.3 mmol, 1.0 eq.) was dissolved in
CH2Cl2 (6 mL) and pyridine (0.30 mL, 3.8 mmol, 2.9 eq.)
then 4-nitrophenyl chloroformate (0.51 g, 2.5 mmol, 1.9 eq.)
was added. The reaction mixture was stirred for 4 hours.
Triethylamine (0.34 mL, 2.5 mmol, 1.9 eq.) and tri(ethylene
glycol) monomethylether (0.30 mL, 1.9 mmol, 1.5 eq.) were
then added and the reaction mixture was stirred for an addi-
tional 10 minutes. The reaction mixture was then poured
into 1M HCl (5 mL), and the product was extracted twice
with CH2Cl2. The combined organic layers were dried with
MgSO4 and filtered. The solvent was removed in vacuo and
the resultant residue was purified by silica gel chromatogra-
phy using 9:1 hexanes:EtOAc as an eluent to provide 0.33 g
(0.94 mmol) of 8 as a pale yellow oil.
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28H42N2O4SSi, 531.2707. found (ESI), 531.2691 [MþH]þ.
Synthesis of Compound 5
To a solution containing 4 (1.18 g, 2.22 mmol, 1.00 eq.) in
THF (20 mL) was added a 1 M solution of tetrabutylammo-
nium fluoride (TBAF) in THF (4.43 mL, 4.43 mmol, 2.00 eq.)
and the reaction mixture was stirred at room temperature
for 2 hours. The solvent was then removed in vacuo and the
resulting residue was purified by silica gel chromatography
using 85:15 hexanes:ethyl acetate as an eluent to provide
0.50 g (1.73 mmol) of 5 as a clear, pale yellow oil.
1
Yield: 78%. H NMR (400 MHz, CDCl3): d 3.75 (t, J ¼ 5.8 Hz,
2H CH2-O), 3.54–3.33 (m, 4H (rotamer) diamine CH2), 3.05
(t, J ¼ 5.6 Hz, 2H, CH2-S), 2.99 (br s, 3H carbamate CH3),
2.90–2.78 (m, 3H, boc protected amine CH3), 2.00 (s, 1H,
OH), 1.42 (s, 9H, boc CH3). 13C NMR (100 MHz, CDCl3):
168.9 (rotamer), 168.7 (rotamer), 155.5, 80.0 (rotamer),
79.7 (rotamer), 79.5 (rotamer), 62.1 (rotamer), 61.8
(rotamer), 48.1 (rotamer), 47.8 (rotamer), 47.4 (rotamer),
47.4 (rotamer), 46.7 (rotamer), 46.5 (rotamer), 45.6
(rotamer), 35.7 (rotamer), 35.4 (rotamer), 35.1 (rotamer),
34.9 (rotamer), 34.7 (rotamer), 34.4 (rotamer), 33.2
(rotamer), 32.8, 28.2. IR (NaCl, thin film, cmꢁ1): 1650
(C¼¼O), 1680 (C¼¼O), 2850 (CAH), 2930 (CAH), 2960 (ACH),
3400 (OAH). HRMS (m/z) calc’d for C12H24N2O4S, 292.1457;
found (ESI), 292.8123 [M]þ.
Yield: 74%. 1H NMR (400 MHz, CDCl3): d 8.53–8.48 (m, 1H,
pyridyl), 8.32–8.26 (m, 2H, p-nitrophenyl), 7.70–7.63 (m, 2H,
pyridyl), 7.42–7.36 (m, 2H, p-nitrophenyl), 7.15–7.12 (m, 1H,
pyridyl), 4.57 (t, J ¼ 6.4, 2H, CH2AO), 3.17 (t, J ¼ 6.4, 2H,
CH2AS). 13C NMR (100 MHz, CDCl3): d 159.1, 155.3, 152.1,
149.8, 145.4, 137.1, 125.3, 121.7, 121.1, 120.2, 66.6, 36.7. IR
(NaCl, thin film, cmꢁ1): 1520 (aryl C¼¼C), 1570 (aryl C¼¼C),
1590 (aryl CAN), 1615 (aryl C¼¼C), 1760 (C¼¼O), 2855
(CAH), 2960 (CAH), 3045 (sp2 CAH), 3080 (sp2 CAH), 3115
(sp2 CAH). HRMS (m/z) calc’d for C14H12N2O5S2, 352.0188;
found (ESI), 352.0184 [M]þ.
Synthesis of Polymer 10
Compound 6 (0.60 g, 1.3 mmol, 1.0 eq.) was dissolved in 1:1
CH2Cl2:trifluoroacetic acid (6 mL) and the reaction mixture
was stirred at room temperature for 2 hours. The solvent
was evaporated in vacuo, and then CH2Cl2 was added and
evaporated five times to remove residual TFA, providing 9.
The residue was dissolved in toluene (1 mL) and triethyl-
amine (0.91 mL, 6.5 mmol, 5.0 eq.), DMAP (0.015 g, 0.12
mmol, 0.09 eq.) and end cap 8 (10.6 mg, 30 lmol, 0.023 eq.)
were added. The resulting solution was stirred at room tem-
perature for 18 hours. The solvent was removed in vacuo
Synthesis of Compound 6
To a solution containing 5 (0.45 g, 1.5 mmol, 1.0 eq.) in
CH2Cl2 (10 mL) was added pyridine (0.37 mL, 4.6 mmol, 3.0
eq.), followed by 4-nitrophenyl chloroformate (0.62 g, 3.1
mmol, 2.0 eq.) and the reaction mixture was stirred for 20
hours. The reaction mixture was then washed with 10 mL of
1M HCl, then the organic layer was dried over MgSO4, fil-
tered, and the solvent was removed in vacuo. The resulting
residue was purified by silica gel chromatography using 1:1
CASCADE BIODEGRADABLE LINEAR POLYMER, DEWIT, BEATON, AND GILLIES
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
FIGURE 2 Design of the cascade degradable polymer: (a) cyclization of 2-mercaptoethanol derivatives to 1,3-oxathiolan-2-one; (b)
undesired cyclization of an activated 2-mercaptoethanol based monomer prohibits polymerization; (c) proposed polymerization of
an activated heterodimer and depolymerization of the resulting polymer; (d) proposed disulfide based end cap.
and the crude polymer was dissolved in 2 mL of DMF. The
solution was dialyzed against DMF (200 mL, 1 solvent
change) using a regenerated cellulose membrane (Spectrum
Laboratories Spectra/Por, 3500 Mw cutoff). The DMF
was then removed in vacuo to provide 0.10 g of polymer 10.
itol (DTT) was added at the beginning and subsequently
every 7 days to maintain reducing conditions. The extent of
depolymerization was quantified using 1H NMR by integrat-
ing the methylene peak corresponding to the N,N0-dimethyli-
midazolidinone degradation product (3.4 ppm) relative to
the peak corresponding to the methylene group adjacent to
the oxygen in the polymer (4.3 ppm). A control sample was
monitored under the same conditions as above, but without
DTT. For SEC samples, a 0.25 mL aliquot was dried and the
resulting residue was taken up in THF. The salts were
removed by filtration through a 0.2 lm filter.
1
Yield: 34%. H NMR (400 MHz, CDCl3): d 8.48 (d, J ¼ 4.7 Hz,
1H, pyridyl), 8.28 (d, J ¼ 9.0 Hz, 1H, p-nitrophenyl) 7.74–
7.61 (m, 2H, pyridyl), 7.41 (d, J ¼ 9.0 Hz, 1H, p-nitrophenyl),
7.10 (br s, 1H, pyridyl), 4.39–4.28 (m, 2H, CH2AO terminal),
4.27–4.07 (m, 76H, CH2AO), 3.61–3.34 (m, 155H, CH2AN),
3.23–3.10 (m, 77H, CH2AS), 3.02 (br S, 114H, CH3AN), 2.98–
2.89 (m, 120H, CH3AN). SEC: Mn ¼ 1800 g/mol, Mw ¼ 2950
g/mol, PDI ¼ 1.6.
RESULTS AND DISCUSSION
Design
Degradation Study
Buffer Preparation
NaH2PO4ꢂH2O (0.069 g, 0.5 mmol) was dissolved in 5 mL of
D2O. To this, a saturated solution of NaOH in D2O was added
dropwise with stirring, while monitoring with a pH meter
until the desired pH of 7.4 was obtained.
A diverse array of intramolecular cyclization reactions have
been reported, potentially allowing the rate of depolymeriza-
tion to be controlled by the choice of the cyclization
reaction.48,59,60 In this particular work, the cyclizations of
2-mercaptoethanol derivatives to the corresponding cyclic
thiocarbonate [Fig. 2(a)] were of interest as they have been
recently reported as components of traceless self-immolative
spacers in fluorescent protease sensors.59
Degradation of Polymer 10
Fifteen milligrams of polymer 10 was dissolved in 1 mL of
0.1 M phosphate buffered D2O:acetone-d6 (3:2), and the so-
lution was incubated at 37ꢃC. Three milligrams of Dithiothre-
The development of monomers capable of undergoing poly-
merization to form cascade degradable polymers requires
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ARTICLE
SCHEME 1 Synthesis of the protected monomer.
careful design. In particular, in the preparation of polymers
designed to degrade by cyclization mechanisms, cyclization
of the activated monomer [Fig. 2(b)] must be avoided. In our
previous work, it has been found that the synthesis and poly-
merization of activated dimers is an effective approach, as
the activated leaving group is distant from the nucleophilic
moiety such that the resulting ring size is not particularly
favorable for cyclization.44 This allows polymerization to be
a highly competitive reaction at high concentrations. In par-
ticular, the use of alternating monomers, and thus the prepa-
ration of activated heterodimers as polymerizable ‘‘mono-
mers’’ has been found to be an effective strategy for
overcoming the challenges associated with the synthesis of
both the activated monomers and their corresponding poly-
mers.44 Therefore, an activated heterodimer based on 2-mer-
captoethanol and N,N0-dimethylethylenediamine units was
proposed. Carbamate derivatives of N,N0-dimethylethylenedi-
amine are known to spontaneously cyclize to form N,N0-
dimethylimidazolidinone49 and this spacer has been incorpo-
rated into our previously reported linear cascade degradable
polymer44 as well and some of the previously reported cas-
cade degradable dendrimers.32,36,39,40 As shown in Figure
2(c), polymerization of this activated heterodimer in the
presence of an end cap should lead to a polymer based on
2-mercaptoethanol and N,N0-dimethylethylenediamine with
alternating carbamate and thiocarbamate linkages. Removal
of an end cap would lead to alternating cyclization reactions
resulting in end to end depolymerization with the release of
N,N0-dimethylimidazolidinone and 1,3-oxathiolan-2-one.
shown that the incorporation of disulfide linkages into gene
and drug delivery systems can provide a selective release of
the cargo under the reducing conditions within cells, leading
to enhanced therapeutic efficacy.61–66 In addition, because of
the incorporation of the 2-mercaptoethanol cyclization reac-
tion in the degradation cascade, the disulfide was a natural
choice for an end cap as the thiol moiety can be readily con-
verted to a disulfide which upon cleavage can directly initi-
ate the depolymerization cascade.
Synthesis
As shown in Scheme 1, the synthesis of the target activated
heterodimer began by the selective protection of the alcohol
group on 2-mercaptoethanol (1) using tert-butyldiphenyl-
chlorosilane in the presence of imidazole to provide the tert-
butyldiphenylsilyl (TBDPS) protected derivative 2. The thiol
of 2 was then treated with 4-nitrophenylchloroformate to
provide the activated thiocarbonate 3. The mono tert-butyl-
carbamate (Boc) protected derivative of N,N0-dimethylethyle-
nediamine44 was reacted with 3 using 4-(dimethylamino)-
pyridine (DMAP) as a catalyst and N,N-diisopropylethylamine
(DIPEA) as a base to give the thiocarbamate 4, and then the
TBDPS protecting group was removed using tetrabutylammo-
nium fluoride (TBAF) in tetrahydrofuran (THF) to provide
the alcohol 5. The alcohol was then converted to the acti-
vated carbonate 6 by reaction with 4-nitrophenyl chlorofor-
mate, providing the protected version of the polymerization
monomer.
For the synthesis of the target end cap, the alcohol group of
the previously reported thiopyridyl derivative 758 was treated
with 4-nitrophenyl chloroformate to provide the activated
carbonate 8 as shown in Scheme 2. This activated carbonate
The end cap selected for the target polymer was a disulfide
[Fig. 2(d)]. Disulfide linkages are known to be cleaved by bi-
ological reducing agents such as glutathione, and it has been
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
D2O:acetone-d6 (3:2). DTT was added to cleave the disulfide
end cap, thus initiating the degradation cascade and the sam-
ple was incubated at 37 ꢃC. The reducing conditions were
maintained by periodic additions of DTT. The degradation
1
was monitored by H NMR spectroscopy. As shown in Figure
3, over time, characteristic peaks appeared corresponding to
N,N0-dimethylimidazolidinone at 2.8 and 3.4 ppm and 1,3-
oxathiolan-2-one at 3.2 and 3.7 ppm. The presence of these
products is a strong indicator that the degradation proceeds
by the proposed cascade of cyclization reactions as random
chain scissions would lead to N,N0-dimethylethylenediamine
and 2-mercaptoethanol, products that were not detected in
the NMR spectra. In addition, a control sample incubated
under the same conditions except in the absence of DTT did
not reveal the appearance of any degradation products, thus
indicating the end cap cleavage was required to initiate the
degradation (Supporting Information). Furthermore, an addi-
tional control polymer having a Boc end cap was also pre-
pared and was demonstrated to undergo depolymerization
in pH 7.4 phosphate buffered D2O:acetone-d6 (3:2) following
prior treatment with TFA/CH2Cl2 to remove the Boc group.
This confirmed that the polymer degradation could not be
attributed to random polymer backbone cleavage by the DTT
(Supporting Information).
SCHEME 2 Synthesis of the end cap.
allows for incorporation of the end cap onto the polymer.
Tri(ethylene glycol) monomethyl ether (TEGMME) was used
to quench the excess chloroformate in this reaction as it was
otherwise chromatographically inseparable from the product.
In preparation for the polymerization, the Boc group was
removed from compound 6 using trifluoroacetic acid (TFA),
providing the corresponding amine 9, isolated as its TFA salt
(Scheme 3). This molecule was sufficiently stable that as long
as the deprotection was performed within hours before the
polymerization reaction it could be isolated and transferred
to the polymerization conditions without premature polymer-
ization or cyclization. In contrast, upon deprotonation 9 is ca-
pable of self-condensing via reaction of the amine with the
activated carbonate to form a polycarbamate, releasing p-
nitrophenol. The polymerization was carried out by reacting 9
with 0.02 equivalents of the end cap 8 in toluene in the pres-
ence of DMAP as a catalyst and triethylamine as a base.
The percentage of degradation was determined by the rela-
tive integration of the peak at 4.3 ppm assigned to the meth-
ylene group adjacent to the oxygen in the polymer and the
peak at 3.4 ppm corresponding to the methylene unit of
N,N0-dimethylimidazolidinone. As shown in Figure 4, the deg-
radation reached 80% completion after 10–14 days. How-
ever, no significant further degradation was observed, even
after 30 days. Size exclusion chromatograms were also
obtained at different time points during the degradation pro-
cess. As shown in Figure 5, before degradation, the chromat-
ogram exhibited a peak at an elution volume of 16.7 mL as
well as a distinct shoulder at 18.5 mL. As the degradation
progressed, the peak at 16.7 mL decreased in intensity, con-
sistent with the degradation progress observed by 1H NMR
spectroscopy. On the other hand, no change in the intensity
of the peak at 18.5 mL was observed. Therefore, it is possi-
ble that the peak at 18.5 mL corresponds to cyclic polymers.
These cyclic species would not be end capped and thus deg-
radation would only be initiated by a random chain scission
of the polymer backbone. As observed for the control sample
such cleavages are extremely slow under the degradation
conditions. This may explain why the depolymerization did
The resulting polymer 10 was purified by dialysis in N,N-
dimethylformamide (DMF) using a regenerated cellulose
membrane with a molecular weight (MW) cut-off of 3500
g/mol to remove small molecule byproducts. The material
isolated from the dialysis was pure and free of low MW
impurities as determined by 1H NMR and size exclusion
chromatography (SEC). In the analysis of the dialysate, cycli-
zation products were not detected (Supporting Information),
indicating that the cyclization of the monomer was not a
competing reaction during the polymerization. In addition,
no monomer was detected, indicating that the polymeriza-
tion proceeded to completion. However, some polymeric
material was lost into the dialysate. It should be noted that
the MW cut-off of 3500 g/mol is an estimate as it depends
on the macromolecule’s size and shape. In addition, the MW
cut-off corresponds to aqueous conditions and is likely lower
in DMF due to the decreased swelling of the membrane in
DMF. However, we have routinely observed that linear poly-
mers above the MW cut-off can pass through the membrane,
likely via reptation.67 Although the yield for this polymer
following dialysis was relatively low, ꢄ35%, this method is
much less labor intensive than preparative SEC, which was
previously used to purify our cascade degradable linear poly-
mers. Using 1H NMR spectroscopy a ratio of end cap to
monomer of ꢄ35:1 was determined (Supporting Informa-
tion). Using SEC the polymer was found to have a number
average MW (Mn) of 1800 g/mol, a weight average MW (Mw)
of 2950 g/mol, and a polydispersity index (PDI) of 1.6 rela-
tive to polystyrene standards.
Polymer Degradation
To study the depolymerization initiated by end cap cleavage,
polymer 10 was dissolved in pH 7.4 phosphate buffered
SCHEME 3 Polymerization.
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ARTICLE
FIGURE 3 1H NMR spectra of polymer 10 and its degradation products in pH 7.4 phosphate buffered D2O:acetone-d6 (3:2) at differ-
ent time points following the addition of DTT: (a) immediately following DTT addition; (b) after 4 days; (c) after 8 days.
not reach 100% completion according to 1H NMR
spectroscopy.
It should also be noted that the cyclic polymers would not
be distinguishable from the linear end capped polymers by
NMR spectroscopy. Therefore, based on the degradation pla-
teau at 80% completion, it is possible that the ratio of mono-
mer to end cap in the linear polymers is closer to 30:1 than
the 35:1 ratio mentioned earlier. Altering the concentration
of the polymerization reaction did not appear to change the
content of the possible cyclic species significantly, nor the
polymer MW. Interestingly, such nondegradable species were
not observed in our previously reported cascade degradable
polymers based on N,N0-dimethylethylenediamine and 4-
hydroxybenzyl alcohol.44 This may be explained by the
increased rigidity imparted by the aromatic groups of 4-
hydroxybenzyl alcohol, making cyclization less favorable.
Nevertheless, this somewhat unexpected result provides
additional evidence of the polymer backbone’s inherent sta-
bility and the specificity of the degradation process mediated
FIGURE 4 Kinetics of depolymerization of polymer 10, as meas-
ured by 1H NMR spectroscopy in pH 7.4 phosphate buffered
D2O:acetone-d6 (3:2), following addition of DTT.
CASCADE BIODEGRADABLE LINEAR POLYMER, DEWIT, BEATON, AND GILLIES
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JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
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