Postpolymerization modification using less cytotoxic activated ester polymers for the synthesis of biological active polymers

Article abstract of DOI:10.1021/bm500902t

Activated ester polymers, pioneered by Ferruti and Ringsdorf in the 1970s, are attractive polymeric materials because they can easily be converted into functional polymers by reacting with amine nucleophiles. In the present study, methyl salicylate acryla

Full text of DOI:10.1021/bm500902t

Article
pubs.acs.org/Biomac
Postpolymerization Modification Using Less Cytotoxic Activated
Ester Polymers for the Synthesis of Biological Active Polymers
Lirong He,† Kristina Szameit,‡ Hui Zhao,† Ulrich Hahn,‡ and Patrick Theato*,†
†Institute for Technical and Macromolecular Chemistry, University of Hamburg Bundesstrasse 45, D-20146 Hamburg, Germany
‡Institute for Biochemistry and Molecular Biology, University of Hamburg Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany
S
* Supporting Information
ABSTRACT: Activated ester polymers, pioneered by Ferruti and
Ringsdorf in the 1970s, are attractive polymeric materials because
they can easily be converted into functional polymers by reacting
with amine nucleophiles. In the present study, methyl salicylate
acrylate, salicyl acrylate, and tert-butyl salicylate acrylate monomers
were polymerized yielding three novel reactive precursors suitable
for the postpolymerization modification with primary and
secondary amines. The reactivities of poly(pentafluorophenyl
acrylate), poly(methyl salicylate acrylic ester), and poly(salicyl
acrylate) toward amines were compared by kinetic studies and
revealed the practical applicability of salicylic acid based derivatives
for efficient postpolymerization modifications. In addition, in vitro cytotoxicity of water-soluble leaving groups,
pentafluorophenol and salicylic acid, as well as water-soluble polymers containing the respective activated ester groups were
investigated using HeLa cells. In short, compared to the frequently used poly(pentafluorophenyl acrylate), poly(salicyl acrylate)
activated ester feature a lower reactivity, but exhibit less cytotoxicity. In this respect, poly(salicyl acrylate) as reactive precursor
polymers may become alternative routes for the synthesis of functional polyacrylamides when it comes to advanced applications
in vivo.
poly(N-methacryloxysuccinimide) (pNMAS), have become the
most frequently used activated polymer precursors for the
preparation of biological relevant functional polymers. How-
ever, a drawback of pNAS and pNMAS is their limited
solubility in organic solvents, except DMSO and DMF and
their hydrolytic instability.1 Activated ester polymers containing
thiazolidine-2-thione are an interesting alternative for pNAS
and pNMAS, because polymers featuring thiazolidine-2-thione
allow aminolysis in aqueous solution,14 thereby enabling the
application in conjugating with the amine groups located on
protein surfaces.15 Reactive polymers based on azlactone are
alternative polymer precursors, with their main advantage being
the addition reaction of nucleophiles, that is, no release of a
leaving group in this case. Recently, thermoresponsive polymers
were derived from postpolymerization modification of poly(2-
vinyl-4,4-dimethylazlactone).8 Later on, Theato et al. intro-
duced activated ester polymers based on pentafluorophenyl
(PFP) esters, providing a powerful tool for synthesizing
functional polymeric materials.16−18 Compared with NHS
ester polymers, polymers bearing pentafluorophenyl (PFP)
ester groups feature a higher reactivity, allowing a practically
quantitative conversion with amines under mild conditions,
while being analytically advantageous due to the use of 19F
INTRODUCTION
■
Postpolymerization modification, also known as polymer
analogous reaction, is a versatile synthetic approach in
obtaining functional materials.1−3 The general concept is the
chemical transformation of reactive polymer precursors into
new functional polymers, thereby enabling the introduction of
functional groups, even those that are not compatible with
common polymerization conditions or inhibit a precise
characterization.4 Early examples of postpolymerization mod-
ification can be traced back to 1840, when Hancock and
Ludersdorf independently reported the vulcanization and
hydrogenation of natural rubber,5−8 generating a tough and
elastic material. With the general acceptance of the concept of
macromolecules, polymer analogous reactions were more and
more applied in engineered synthetic polymers.1 In recent
years, the emergence of click type coupling reactions, including
azide−alkyne and diene−dienophile cycloadditions, thiol−ene
and thiol−yne addition, and substitution reactions based on
activated esters laid the foundation for the intensive use of
postpolymerization modification reactions.1,9−12
Among these, activated ester-amine chemistry has many
characteristics of conventional click chemistries, featuring metal
free and mild reaction conditions. In 1970s, Ferruti and
Ringsdorf proposed activated ester polymers as a novel
synthetic tool to prepare pharmacologically active polymer
drug conjugates.13,1 Since then, N-hydroxysuccinimide deriva-
tives (NHS), such as poly(N-acryloxysuccinimide) (pNAS) and
Received: June 19, 2014
Revised: July 13, 2014
Published: July 14, 2014
© 2014 American Chemical Society
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NMR spectroscopy. Other than that, activated ester polymers
based on PFP are (i) easy to synthesize and purify, (ii) soluble
in most midrange polar organic solvents, and (iii) allow to
monitor the aminolysis process by 19F NMR and FT-IR
spectroscopy.16−18 Due to the wide spectrum of benefits that
PFP ester offers, many multifunctional materials, such as
reactive coating surfaces and stimuli responsive polymers, have
been described in the literature.19−24 Recently, copolymers
containing poly(pentafluorophenyl methacrylate) blocks have
been used to prepare well-defined poly(N-(2-hydroxypropyl)-
methacrylamide) derivatives as drug carriers, enabling precise
tracking of the cell uptake behavior as well as the distribution of
these highly biocompatible polymers.25−29 Nevertheless, due to
the toxic nature of the pentafluorophenol group that is released
during the postmodification step and the possibility of
remaining PFP ester groups in the polymer, the utilization of
these polymers derived from PFP ester in the biological area is
still controversial.
Salicylic acid, derived from plants such as willow bark and
latin salix, is a low-cost drug and is classified as nonsteroidal,
anti-inflammatory drug (NSAIDS).30 It is also the active
component of acetylsalicylic acid, commonly known as aspirin,
one of the most widely used and annually consumed drugs in
the world. Aspirin has proved to be effective in modifying
proteins by chemical transfer of its acetate group to amino acid
side chains.30 It is demonstrated that the acetylation occurs on
the amine of lysine and cysteine residues of cells.31−33 In the
meantime, salicylic acid released during acetylation enabled the
pharmacological effects of aspirin in vivo. In light of this
evidence for the successful substitution reactions of amines with
acetylsalicylic acid, it is reasonable to predict that polymers
featuring salicyl esters can act as activated ester polymer
precursors. The advantages of activated ester polymer
precursors based on salicyl ester would be utilizing a natural
product as starting material, which is a cheap (as provided by
VWR, the price of salicylic acid is 23.7 € per 500 g, that of
pentafluorophenol is 394.6 € per 500g) and enables a green
chemistry approach; salicylic acid is an anti-inflammatory,
anticancer, antifungal, and antibacterial drug, resulting in less
concern about the safety of developing well-defined drug
carriers derived from polymer precursors featuring salicyl ester.
As a matter of fact, acrylates of salicylic acid have been
synthesized and copolymerized with acrylic acid to investigate
the release of the drug by hydrolysis.34 However, to the best of
our knowledge, both acrylates of salicylic acid and its derivatives
have never been considered as activated ester polymer
precursors for postpolymerization modifications.
for the postpolymerization modification of polyacrylates with
amines. To demonstrate the practical applicability of salicyl
ester based derivatives for efficient postpolymerization
modifications, the active ester reactivity of P1, P2, and the
established pentafluorophenyl acrylate (PPFPA) are compared
by kinetic studies. In addition, the cell cytotoxicity of salicylic
acid, P2, PFP, as well as PFP-based active ester polymer
precursors, are investigated using HeLa cells.
EXPERIMENTAL SECTION
■
1. Materials. All chemicals were commercially available and used as
received, unless otherwise stated. Dichloromethane (DCM) and
dioxane were freshly distilled over calcium hydride and used
immediately. Triethylamine (Et3N) was dried over calcium chloride
and distilled previously. Azobis(isobutyronitrile) (AIBN) was
recrystallized from methanol prior to use.
2. Structural and Chemical Composition Characterization.
1H NMR spectroscopy was performed on a Bruker 300 MHz. FT-
NMR spectrometer with deuterated solvents. The chemical shifts (δ)
are given in ppm relative to a standard, tetramethylsilane (TMS). The
molecular weight and corresponding molecular weight distribution
(Mw/Mn) was determined by gel permeation chromatography (GPC)
using polystyrene standards. Unless otherwise stated, the measure-
ments were conducted in THF solution with a flow rate of 1 mL·min−1
at 25 °C. Infrared spectroscopy was performed on a Thermo Fisher
Scientific Nicolet iS10 using ATR unit. Kinetic IR measurements were
conducted on a Reactive IR 45m from Mettler Toledo.
3. Monomer Synthesis. Methyl-Salicylate Acrylate (M1).
Barbosa et al.36 have reported the synthesis of M1 and we synthesized
M1 via a slightly modified synthesis route as follows: A solution of 60
mL of dry DCM containing methyl salicylate (80 mM, 12.16 g) and
TEA (84 mM, 8.5 g) was stirred for 10 min in an ice bath. Acryloyl
chloride (84 mM, 7 mL) was added dropwise to the mixture. The
solution was kept stirring and the formation of product was checked
by TLC analysis using ethyl acetate/hexane (1:9 by volume). After 3 h,
the mixture was filtrated to remove the precipitated TEA hydro-
chloride. The filtrate was washed twice with water (30 mL), using a
separation funnel and dried over sodium sulfate, and the solvent was
removed under reduced pressure. The product was isolated by column
chromatography using petrol ether as eluent and was obtained in the
form of yellow oil (7 g, yield 77%).
1H NMR (300 MHz, δ, ppm, CDCl3) 8.04 (dd, J = 7.8, 1.7 Hz, 1H),
7.58 (ddd, J = 8.1, 7.5, 1.7 Hz, 1H), 7.41−7.29 (m, 1H), 7.15 (dd, J =
8.1, 1.1 Hz, 1H), 6.71−6.60 (m, 1H), 6.49−6.30 (m, 1H), 6.11−5.99
(m, 1H), 3.84 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 164.96, 164.60,
150.38, 133.83, 132.76, 131.89, 127.63, 126.08, 123.77, 52.29; IR
(ATR mode) 2952.6, 1756.2 (CO methyl salicylate ester), 1723.6
(CO methyl ester), 1606.6, 1487.3, 1450.6, 1435.4, 1298.3, 1264.8,
1201.1, 1138.5, 1082.3, 962.2 cm−1; ESI-MS: Calcd for C11H10O4 [M
+ Na+], 229.0579; found, 229.0493.
Salicyl Acrylate (M2). Boudreaux et al. have reported the synthesis
of M2.34 A slightly modified synthesis was conducted as follows: A
solution of 175 mL of dry acetonitrile containing salicylic acid (130
mM, 17.94 g) and TEA (390 mM, 54.29 mL) was stirred for 10 min in
an ice bath. Acryloyl chloride (140 mM, 11.25 mL) was added
dropwise to the mixture. The solution was kept stirring and the
formation of product was checked by TLC analysis using ethyl
acetate/hexane (1:1 by volume). After 6 h, the mixture was filtrated to
remove the precipitated TEA hydrochloride. The solvent was removed
under reduced pressure. The compound was dissolved in 100 mL
DCM and washed four times with water (50 mL) using a separation
funnel. The organic phases were combined and dried over anhydrous
sodium sulfate. The solvent was removed under reduced pressure. The
product was isolated by recrystallization in isopropanol/water (3:1) by
dissolving the compound at 60 °C and cooling at −5 °C. The product
was obtained in the form of a yellow-tinted solid (11 g, yield 28%).
1H NMR (300 MHz, δ, ppm, CDCl3) 10.66 (d, J = 133.5 Hz, 1H),
8.13 (dd, J = 7.8, 1.7 Hz, 1H), 7.64 (td, J = 7.7, 1.6 Hz, 1H), 7.37 (td, J
In this work, we present the preparation of poly(salicyl
acrylate) P2 and its methyl ester and tert-butyl ester protected
derivatives, poly(methyl-salicylate acrylate) P1 and poly(tert-
butyl salicylate acrylate) P3. P1 was developed because of the
cheap price (methyl salicylate costs 39.1 € per 500 g, provided
by VWR), broad solubility in organic solvents and because it is
easy to be prepared, enabling its applicability when
manufacturing general functional materials. P2 is based on
salicylic acid, which is a drug itself, can be used in preparing
biorelated functional materials. P3 is based on tert-butyl ester
protected salicylic acid. The tert-butyl ester group is stable
toward mild aminolysis and can be cleaved by moderately acidic
hydrolysis,35 allowing P3 not only to be a reactive polymer
precursor itself, but also to be transformed into P2 via acidic
hydrolysis. To the best of our knowledge, it is the first time that
these polymers are investigated as successful reactive precursors
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= 7.6, 0.9 Hz, 1H), 7.19 (dd, J = 8.1, 0.9 Hz, 1H), 6.77−6.55 (m, 1H),
6.38 (dd, J = 17.3, 10.4 Hz, 1H), 6.04 (dd, J = 10.4, 1.2 Hz, 1H); 13C
NMR (75 MHz, CDCl3) δ 169.78 (s), 164.59 (s), 150.98 (s), 134.76
(s), 132.75 (s), 132.46 (s), 127.68 (s), 126.18 (s), 123.97 (s), 122.42
(s); IR (ATR mode) 2972.6, 2635.9, 1756.8 (CO salicyl ester),
1718.8 (CO carboxylic acid), 1607.3, 1578.7, 1487.5, 1451.6,
1309.5, 1247.1, 1199.3, 1137.4, 1079.5, 1046.3, 889.6 cm−1; ESI-MS:
Calcd for C11H8O4 [M + Na+], 215.0423; found, 215.0593.
1H NMR (300 MHz, δ, ppm, DMSO) 13.08 (S, 1H), 7.81 (m, 1H),
7.21 (m, 3H), 3.11 (s, 1H), 1.97 (ddd, 2H); IR (ATR mode): 1756.8
cm−1 (salicyl ester CO), 1718.8 cm−1 (salicylic acid CO).
Poly(tert-butyl salicylate acrylate) P3. Free radical polymerization
of M3 was carried out as follows: 1,4-dioxane (0.5 mL) solution of M3
(0.5 g, 100 equiv) and AIBN (1 equiv) were degassed under argon at
25 °C for 20 min. After filling with argon, the flask was immersed in a
preheated oil bath of 70 °C for 15 h. Afterward, the reaction mixture
was exposed to air to quench the polymerization. After taking a small
portion to check the conversion rate, the solution was diluted with
THF and purified by reprecipitation (THF/hexane) to afford a white
powder polymer. Yield: 200 mg (40%).
tert-Butyl-salicylate Acrylate (M3). 2-methyl-2-propanyl salicylate
(A) was synthesized as follows: 1,1′-carbonyldiimidazole (100.3 mM,
16.28 g) and salicylic acid (100.3 mM, 13.7 g) were dissolved in 120
mL of DMF. After stirring at 50 °C for 30 min, the mixture of tert-
butyl alcohol (200.6 mM, 18.98 mL) and 1,8-diazabicycloundec-7-ene
(200.6 mM, 29.83 mL) was added dropwise. The reaction mixture was
stirred at 50 °C for 24 h. Then the solution was poured into saturated
NaHCO3 (550 mL) and extracted with ethyl acetate (300 mL, 3
times). The organic layers were combined and dried over anhydrous
Na2SO4, filtered, and concentrated. The compound (colorless oil, 14.4
g) was purified by column chromatography with ethyl acetate/hexane
(0.05:1 by volume). Yield: 75%
1H NMR (300 MHz, δ, ppm, CDCl3) 7.69 (s, 1H), 6.98 (d, J = 34.8
Hz, 3H), 3.16 (d, J = 86.0 Hz, 1H), 2.03 (ddd, J = 82.5, 67.5, 10.1 Hz,
2H), 1.55−0.93 (m, 9H). GPC (PS standard, THF) Mn = 1.86 × 104
g/mol, Mw = 3.38 × 104 g/mol, Mw/Mn = 1.81. IR (ATR mode):
1758.9 cm−1 (tert-butyl ester salicylate CO), 1715 cm−1 (tert butyl
CO).
5. Typical Procedure for Deprotection of tert-Butyl Ester
with TFA/Dichloroethane. P3 (0.2 mM, 1 equiv, 50 mg) was
dissolved in 1 mL of dry dichloroethane. TFA (2 mM, 10 equiv, 150
μL) was added and the solution was stirred at 25 °C for 6 h. The
deprotected polymer was purified by precipiatation (THF/hexane)
and obtained in practically quantitative yield.
IR (ATR mode) 3141.5 (O−H), 1667.3 cm−1 (tert-butyl CO);
1H NMR (300 MHz, CDCl3) δ 11.07 (s, 1H), 7.80 (dd, J = 8.0, 1.7
Hz, 1H), 7.43 (ddd, J = 8.7, 7.3, 1.7 Hz, 1H), 6.97 (dd, J = 8.4, 0.9 Hz,
1H), 6.92−6.72 (m, 1H), 1.63 (s, 9H).
6. Postpolymerization Modifications with Amines. The
postpolymerization modifications of P1 with amines were performed
as follows: after dissolving P1 (0.2 mM, 1 equiv) in 1 mL of dry 1,4-
dioxane, 3 equiv of amine and triethylamine were added to the
solution sequentially. The mixture was stirred over 24 h at 50 °C. A
small portion was taken for FT-IR measurements.
The postpolymerization modifications of P2 with amines were
performed as follows: after dissolving P2 (0.2 mM, 1 equiv) in 1 mL of
dry 1,4-dioxane and DMSO (1:1), 3 equiv of amine and triethylamine
were added to the solution sequentially. The mixture was stirred at 50
°C for 24 h. After removing the solvent under vacuum, the remaining
crude produce was redissolved in THF. A small portion was taken for
FT-IR measurements.
tert-Butyl salicylate acrylate (M3) was synthesized as follows: A
solution of 20 mL dry dichloromethane containing 2-methyl-2-
propanyl salicylate (A; 10 mM, 1.94 g) and triethylamine (11 mM,
1.53 mL) was kept stirring for 10 min in an ice bath. Acryloyl chloride
(10 mM, 0.804 mL) was added dropwise to the mixture. The solution
was kept stirring and the formation of product was checked by TLC
analysis using ethyl acetate/hexane (0.1:1 by volume). After 6 h, the
mixture solution was filtrated to remove the precipitated TEA
hydrochloride. The filtrate was washed twice with water (30 mL)
using a separation funnel and dried over anhydrous sodium sulfate and
the solvent was removed under reduced pressure. The product was
isolated by column chromatography using ethyl acetate/hexane (0.1:1
by volume) as eluent and was obtained in the form of colorless oil (1.5
g, yield 60%).
After dissolving P3 (0.1 mM, 1 equiv, 25 mg) in 1 mL of dry 1,4-
dioxane, 3 equiv of hexylamine and triethylamine were added to the
solution sequentially. The mixture was stirred over 24 h at 50 °C. A
small portion was taken for FT-IR measurements.
1H NMR (300 MHz, CDCl3) δ 7.94 (dd, J = 7.8, 1.7 Hz, 1H),
7.60−7.45 (m, 1H), 7.37−7.22 (m, 1H), 7.10 (dd, J = 8.1, 1.2 Hz,
1H), 6.70−6.59 (m, 1H), 6.39 (dd, J = 17.3, 10.4 Hz, 1H), 6.05 (dd, J
= 10.4, 1.4 Hz, 1H), 1.53 (s, 9H); 13C NMR (75 MHz, CDCl3) δ
164.33 (s), 164.19 (s), 149.53 (s), 132.93 (s), 132.66 (s), 131.54 (s),
128.02 (s), 125.91 (d, J = 6.4 Hz), 123.38 (s), 81.76 (s), 28.12 (s); IR
(ATR mode) 1745 cm−1 (tert-butyl ester salicylate CO), 1715 cm−1
(tert-butyl CO); Calcd [M + Na+], 271.1049; found, 271.0956.
4. Typical Free Radical Polymerization Procedure. Poly-
(methyl-salicylate acrylate) P1. Free radical polymerization of M1
was carried out as follows: 1,4-dioxane (1 mL) solution of M1 (4 g,
100 equiv) and AIBN (1 equiv) were degassed under argon at 25 °C
for 20 min. After filling with argon, the flask was immersed in a
preheated oil bath of 70 °C for 15 h. The reaction mixture was
exposed to air to quench the polymerization. After taking a small
portion for checking conversion rate, the solution was diluted with
THF and purified by reprecipitation (THF/hexane) to afford a white
powder polymer. Yield: 3.2 g (80%).
7. Kinetic FT-IR Measurements. A general procedure to
investigate the postpolymerization modification kinetically was as
follows: the polymer was dissolved in dry solvent (dioxane for P1 and
PPFPA; 50:50 mixture of DMSO and dioxane for P2) at a
concentration of 0.2 mol/L. The flask was immersed into an oil
bath which was preheated to 50 °C. The IR probe was inserted into
the solution. Once stable IR spectra could be recorded, 3 equiv of the
corresponding amine and TEA were added to the solution. IR spectra
were then recorded in time intervals of 1 min during the first hour of
the reaction and at 10 min intervals after the first hour. Time-resolved
conversion was calculated by the decrease of the carbonyl peak in the
IR spectrum. The integration of the peak at 0 min was defined as 0%
conversion.
8. Cytotoxicity Tests. Poly((N-2-hydroxypropyl)-methacryla-
mide-co-pentafluorophenyl methacrylate) was synthesized by dissolv-
ing poly(pentafluorophenyl methacrylate) (0.5 mM, 126 mg, 1 equiv)
in 1 mL of dry solvent (50:50 mixture of DMSO and dioxane),
hydroxypropylamine (0.8 mM, 60 μL, 1.6 equiv) was added to the
solution. The mixture was stirred over 39 h at 20 °C. A small portion
was taken to be investigated by 19F NMR spectroscopy. The polymer
was purified twice by precipitation (THF/diethyl ether) and then
dialyzed against deionized water for 48 h.
1H NMR (300 MHz, δ, ppm, CDCl3) 7.73 (d, 1H), 7.10 (m, 3H),
3.78 (m, 3H), 3.30 (s, 1H), 2.30 (t, 2H); IR (ATR mode) 1757.8 cm−1
(methyl salicylate ester CO), 1723.6 cm−1 (methyl ester CO).
GPC (PS standard, THF) Mn = 1.35 × 104 g/mol, Mw = 4.85 × 104 g/
mol, Mw/Mn = 3.59.
Poly(salicyl acrylate) P2. M2 was polymerized as follows: THF
solution of M2 (2 g, 100 equiv) and AIBN (1 equiv) were placed in a
Schlenk tube. Three freeze−pump−thaw cycles were performed to
degas the solution. The flask was immersed in a preheated oil bath of
65 °C for 20 h. The reaction mixture was exposed to air to quench the
polymerization. After taking a small portion to check the conversion
rate, the solution was diluted with THF and purified by reprecipitation
(THF/diethyl ether) to afford a white powder polymer. Yield: 1.26 g
(63%).
8.1. Cell Culture Tests. The cytotoxicity studies of activated ester
precursors were carried out using a human HeLa cell line, which was
derived from cervix carcinoma cells. The cryopreserved cells were
revived and cultured in RPMI 1640 medium, supplemented with 10%
fetal calf serum (FCS) plus 1% streptomycin. The cells were grown in
a T-25 flask and incubated under 5% CO2 and 95% humidity at 37 °C.
After incubating for 24 h, the cells were harvested using trypsin EDTA
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Scheme 1. Synthesis of Poly(methyl-salicylate acrylate) (P1), Poly(salicyl acrylate) (P2), and Poly(tert-butyl-salicylate acrylate)
(P3) and Their Post-Polymerization Modification with Amines
Table 1. Results and Conditions for Polymerization of Different Active Ester Monomers M1 to M3
polymer
monomer
solvent
t (h)
T (°C)
Mn (g/mol)
Mw (g/mol)
Mw/Mn
P1
P2
P3
M1
M2
M3
dioxane
THF
15
20
15
70
65
70
1.35 × 104
N
1.86 × 104
4.85 × 104
N
3.38 × 104
3.59
N
dioxane
1.81
N: not determined.
and were then used for further experiments. The culture medium was
changed every 5 days.
dioxane, and DMF, but still insoluble in water. All monomers
could be prepared on a large scale and are stable for several
months, when stored at 4 °C.
8.2. Resazurin Fluorimetric Assay. Resazurin Fluorimetric Cell
Viability Assay Kit (Biotium, U.S.A.) was used for cell viability testing
according to the company’s instructions. Briefly, HeLa cells were
seeded at a density of 5000 cells per well in a 96-well plate in 200 μL
of culture medium and incubated overnight. The next day, the growth
medium was removed and 200 μL of fresh medium containing the
desired amounts of compounds and polymers were injected into each
well. In the case of testing the cytotoxicity of polymers, the
corresponding concentrations of P2 and poly((N-2-hydroxypropyl)-
methacrylamide-co-pentafluorophenyl methacrylate) were dissolved in
2 μL of DMSO before they were applied to the cells, which means cell
culture medium containing 1% DMSO was adopted in this case. Thus,
cell viability cultured in medium containing 1% DMSO was tested as
blank control. The cells were incubated for 24 h, after which the
medium was removed and cells were washed with PBS before adding a
mixture of resazurin and RPMI (100 μL) in a volume ratio of 1:10 into
each well. After incubating for 4 h, cell viability was monitored by
measuring the fluorescence with an excitation wavelength at 550 nm
and an emission of 590 nm. Fluorescent intensity generated by the
assay was directly related to the number of living cells in the sample.
Measurements were carried out using Infinite 200 microplate reader
(TECAN, Crailsheim, Germany). Results were presented after
subtracting the fluorescence background. The percentage of survival
of active ester treated cells was calculated based on the control cells.
Correspondingly, cells treated with culture medium containing 1%
DMSO were used as blank control (cell viability 95.7%), cell viability
was calculated based on this control.
2. Polymerization and Postpolymerization Modifica-
tion. Polymerization of M1 to M3 was conducted via free
radical polymerization using AIBN as initiator. The polymer-
ization conditions and the results are summarized in Table 1.
All polymers were characterized by 1H NMR and FT-IR
spectroscopy (Figures S1−S3). GPC data confirmed that P1
was obtained with molecular weights of 1.35 × 104 g/mol and
molecular weight distributions of 3.59. However, the free
carboxylic acid group on the side group of P2 made it difficult
to characterize by GPC. Therefore, P3 was synthesized because
it allows the cleavage of tert-butyl ester under moderately acidic
conditions, yielding the respective P2.35 Deprotection of the
carboxylic acid was conducted in the presence of excessive
TFA. 1H NMR spectroscopy documented the complete
disappearance of the broad peak around 1.42 ppm (Figure
S4), originating from the tert-butyl protons of P3, and thereby
suggesting the successful removal of the tert-butyl ester group.
1H NMR and IR (Figure S5) showed that after deprotection of
P3 using TFA, the polymer featured the same characteristic
spectrum of P2 and, consequently, proved the successful
indirect way to prepare P2. GPC data showed that P3 was
obtained with reasonable molecular weight of 1.86 × 104 g/mol
and molecular weight distribution of 1.81 via free radical
polymerization.
The polymers were synthesized as activated ester polymer
precursors and their postpolymerization modification with
amines can be easily monitored by IR spectroscopy. In initial
studies, P1 was selected out of the three polymers because of its
good solubility in organic solvents and it is the easiest to
prepare. Hexylamine was used to optimize the postpolymeriza-
tion modification conditions. Aminolysis of P1 by adding
hexylamine several times was conducted at 50 °C. As shown in
Figure S6-A, the methyl-salicylate ester peak at 1757.8 cm−1
(peak 1) and the methyl ester peak around 1723.6 cm−1 (peak
2) decreased simultaneously with the addition of hexylamine. A
total of 3 equiv of amine (Figure S6−B) at 50 °C completed
the ester modification, while still providing comparable
reactivity with the other postpolymerization modification
approaches via activated ester polymers.25,37
RESULTS AND DISCUSSIONS
■
1. Monomer Synthesis. Methyl-salicylate acrylate (M1),
salicyl acrylate (M2), and tert-butyl-salicylate acrylate (M3)
were synthesized by alcoholysis of acryloyl chloride in the
presence of an auxiliary base, as shown in Scheme 1. M1 and
M3 were purified by column chromatography and obtained as
colorless liquids. M2 was isolated by recrystallization. The
1
obtained compounds were characterized by H NMR spec-
troscopy (Figures S1−S3) as pure monomers for polymer-
ization.
M1 and M3 were soluble in many organic solvents such as
THF, acetone, dioxane, diethyl ether, n-hexane, and DMF. In
the case of M2, due to the hydrophobic nature of the benzene
group and the hydrophilic property of the carboxylic group, it is
soluble in organic solvents like THF, DMSO, methanol,
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Figure 1. (A) IR (ATR mode) spectra of during postpolymerization modification of P1 with hexylamine at 50 °C; (B) IR spectra of methyl
salicylate; (C) IR (ATR mode) spectra of P3 before (black solid line) and after postpolymerization modification with hexylamine (red dot line); (D)
IR spectra of tert-butyl salicylate.
1
Figure 2. H NMR spectra in CDCl3 of P1 before (I) and after postpolymerization modification with hexyl amine P13 (II).
1
Figure 3. H NMR spectra in DMSO of P2 before (I) and after post modification with hexylamine P21 in CDCl3 (II).
In terms of P1 and P3, the stability of methyl ester group and
tert-butyl ester group during aminolysis was investigated. The
process of postpolymerization modification with 3 equiv of
hexylamine was followed by in situ IR. As shown in Figure 1A,
the methyl ester group of P1 (1723.6 cm−1) gradually shifted to
1674 cm−1 as the reaction proceeded, which is the signal of
methyl ester group for methyl salicylate (Figure 1B). In the case
of P3, the in situ IR of P3 before and after aminolysis also
showed that tert-butyl ester group (1715 cm−1 for P3) shifted
to 1667.3 cm−1 (signal for tert-butyl ester of tert-butyl
salicylate), which means that tert-butyl salicylate is the leaving
group during the aminolysis process. Thus, it can be concluded
that no side reactions occurred during postpolymerization
modification of P1 and P3 with amines.
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Figure 4. (A) Representative IR of P1 before (solid black line) and after postpolymerization modification with hexylamine P11 (red dashed line)
and isopropylamine (blue dashed-dotted line) P13; (B) Representative IR of P2 before (solid black line) and after (red dashed line)
postpolymerization modification with hexylamine P21.
Table 2. Library of Primary and Secondary Amines Used to Modify P1 and P2
a
Determination by comparison of the intensity of the corresponding characteristic band in the IR spectra of the active ester moiety in the precursor
b
polymer and after modification with amines. The reaction was conducted at 25 °C for 2 days.
In order to provide direct evidence of the successful
aminolysis of the obtained activated ester precursors, 1H
NMR measurements of the purified polymers before and after
postpolymerization modification with hexylamine were carried
out and the results for P1 are shown in Figure 2. The peaks at
7.84, 7.14, and 7.13 ppm owing to the aromatic protons of the
salicylate group and the peak at 3.64 ppm originating from the
methyl ester group disappeared (Figure 2B), while a broad peak
at 6.77 ppm owing to the NH protons of the amide group and a
peak at 1.28 ppm as well as a sharp peak at 0.88 ppm
originating from the hexyl chain were found. This shows that
the methyl salicylate group was fully substituted and that
hexylamine was successfully installed via the activated ester
postpolymerization modification reaction. In the case of P2, as
shown in Figure 3I, a broad peak occurred at 13.1 ppm
originating from the free acid group of P2 and three peaks
appeared at 7.86, 7.24, and 7.05 ppm owing to the aromatic
protons of the salicylic group. After the postpolymerization
modification with hexylamine, the signals for aromatic protons
disappeared and the product P21 (Figure 3II) showed the
expected signals, which were identical to those found for P13
(Figure 2II). Postpolymerization modification of P3 with
hexylamine under the same condition was checked as well, H
NMR (Figure S7) suggests that hexylamine was successfully
installed and the product showed that expected signal in the 1H
NMR spectrum.
1
FT-IR spectroscopy is a very useful tool to determine the
completeness of the postpolymerization modification via
aminolysis. Representative spectra are shown in Figure 4. The
absorbance spectrum of the precursor polymer P1 is plotted in
Figure 4 and shows two strong bands at 1757.8 cm−1 (methyl
salicylate ester CO stretch) and 1723.6 cm−1 (methyl ester
CO stretch) in the carbonyl region as well as a sharp peak
with medium intensity at 1602 cm−1, originating from the
aromatic stretch of the methyl salicylate group. The FT-IR
spectrum of the product of the aminolysis of P1 with
hexylamine is shown in middle plot in Figure 4. P11 did not
show any of the characteristic bands of the precursor polymer,
but rather showed a broad peak around 3295 cm−1, owing to
the N−H stretching of amide. Compared with the starting
material, the absorbance intensity of methylene around 2927
cm−1 increased in the C−H stretch region (3000−2800 cm−1),
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revealing the presence of additional −CH2− groups. Most
notably is the carbonyl region, where the strong ester bands
(1757.8 and 1723.6 cm−1) completely disappeared, suggesting
the complete conversion of the methyl salicylate ester. Instead,
the IR spectrum of P11 showed strong peaks at 1637 and 1545
cm−1, characteristic bands for CO stretching and C−N
bending of amide, respectively. The spectrum of P1 modified
with isopropylamine, P13, which is plotted at the top of Figure
3, showed similar bands in the N−H, CO stretching region
and C−N bending region owing to amides. P13 showed a
lesser intensity band in the C−H stretch region, resulting from
the fact that isopropylamine features less C−H bonds
compared to hexylamine.
In the case of P2, the carbonyl region shows two strong
broad peaks at 1756.8 cm−1 (salicyl ester CO stretch) and
1718.8 cm−1 (carboxylic acid CO stretch); and it shows a
sharp peak with medium intensity at 1602 cm−1, owing to the
aromatic stretch of the salicyl ester group. The product of the
aminolysis with hexylamine, P21, the upper plot in Figure 4B,
shows the characteristic bands of N−H stretching, CO
stretching and C−N bending of amide. All in all, IR
spectroscopy confirmed the expected structure of the
polyacrylamides after aminolysis and demonstrated the
complete conversion of the methyl-salicylate ester and salicyl
ester, respectively.
In order to show the reactivity of the obtained polymer
precursors, different amines were applied for the postpolyme-
rization modification of P1 and P2. As shown in Table 2, for
primary amines such as hexylamine, octylamine, and
isopropanolamine, both P1 and P2 can be converted
quantitatively. For isopropylamine, P1 can reach around 90%
of conversion and P2 can reach almost 100% of conversion
when reacting with 3 times of amine at 25 °C for 2 days. For
aromatic amines, such as p-anisiline, 50% conversion was
achieved for P1 and a slightly higher conversion of P2 was
realized. None of them reacted with aniline. For the above
tested amines, P2 shows a slightly higher reactivity compared
with P1 and this reactivity difference seems be more prominent
when it comes to secondary amines. For dihexylamine and
diethylamine, only a slight aminolysis (less than 10%)
happened with P1, while P2 could reach 80 and 60%
conversion (Figure S8), respectively. In conclusion, the
activated ester precursors have a reasonable reactivity toward
amines, making them suitable candidates for the synthesis of
functional polyacrylamides via postpolymerization functionali-
zation.
In order to show a direct evidence of the reactivity of the
obtained activated ester precursors, postpolymerization mod-
ification of poly(pentafluorophenyl acrylate), P1 and P2 with
hexylamine were compared by kinetic studies. The reactions
were tracked by in situ IR measurements. During the
postpolymerization modification, the intensity of the carbonyl
group of activated ester (1787 cm−1 for PFP ester, 1757.8 cm−1
for methyl salicylate ester, and 1756.8 cm−1 for salicyl ester)
decreased. Instead, the intensity of carbonyl group of amide at
1682.4 cm−1 increased (Figures S9−13). The time dependence
of conversion was determined by the decrease of the area of
carbonyl peak. The integration of the peak area at 0 min was
defined as 0% conversion. The aminolysis of PPFPA with
hexylamine, as shown in Figure 5 (black star curve), was
completed within 1 min under the same reaction conditions
(polymer concentration 0.2 mM, 3 equiv hexylamine, 3 equiv
TEA, 50 °C). P1 and P2 reached almost 100% of conversion
Figure 5. Reaction time dependence of conversion for the aminolysis
of PPFPA (black solid squares), P1 (red circles) and P2 (blue squares)
with hexylamine in the presence of an auxiliary base.
after 225 min. It is noteworthy that P2 has a slightly higher
reactivity compared with P1, which is in agreement with the
data shown in Table 2. Obviously, PPFPA has an even higher
reactivity, making it the ideal candidate when high reactivity is
required. In all other cases, P1 and P2 are suitable alternative
activated ester precursor polymers.
CYTOTOXICITY ASSAY
■
Due to the high reactivity and quantitative conversion toward
primary amines, activated ester polymer precursors, such as
poly(pentafluorophenyl methacrylate), have been used for the
synthesis of drug carriers in the field of nanomedicine.24,26−29
However, due to the toxic nature of pentafluorophenol, small
amounts of remaining pentafluorophenol after postpolymeriza-
tion modification is still a controversy when it comes to the
application of the drug carrier in vivo. In any case, intensive and
time-consuming purification procedures, such as dialysis, must
be conducted. In this work, the obtained activated ester
precursor P2 is based on salicylic acid, the active component of
aspirin, which is a nonsteroidal anti-inflammtory drug
(NSAID).30 Compared with PFP based polymers, activated
ester precursors made of salicylic acid have the potential to
solve the safety dispute in terms of biorelated application. Thus,
the cytotoxicity of leaving groups and polymers featuring PFP
and salicylic acid were investigated using HeLa cell lines. As live
cells have enzymes that can reduce resazurin, the resazurin
fluorimetric assay is a widely used method in quantifying the
cell viability38,39 and was applied within the present study.
In terms of testing the toxicity of leaving groups, cells were
treated with different concentrations of PFP and salicylic acid,
ranging from 0 to 1000 μg/mL. As shown in Figure 6, when the
concentration was increased from 0 to 1000 μg/mL for samples
treated with salicylic acid, the cell viability decreased to 54%,
whereas when treated with PFP it decreased to 15%. This
shows that PFP exhibited a significantly higher toxicity toward
cells. It is reasonable that salicylic acid exhibits toxicity toward
cells, especially in the tested high concentration. Noteworthy,
after taking Aspirin orally, the normal plasma concentration of
salicylate is in the range of 150−300 μg/mL.40 Higher doses
than 350 μg/mL can induce hyperventilation, reversible
tinnitus even acidosis.40 According to our cytotoxicity tests,
when treated with 300 μg/mL salicylic acid, the cell viability
was around 90%. Whereas when the same amount of PFP was
applied, cell viability decreased to around 60%. In summary,
PFP showed a higher toxicity than salicylic acid. It should be
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to 20%, which is similar to their corresponding leaving groups
(Figure 6). It is obvious that polymers containing salicyl ester
groups (open triangles in Figure 7) are less toxic than those
featuring PFP esters (solid triangles in Figure 7). Noteworthy,
during the application of biocompatible polymers or polymer
drug conjugates derived from activated ester polymer
precursors in vivo, it is the remaining activated ester group in
the polymer that is controversial. For example, take the
normally applied concentration of polymer (1000 μg/mL) in
vivo derived from activated ester polymer precursors. If up to
10% active esters are left after postpolymerization modification,
cell viability showed that cells treated with P2 decreased to
92%, which did not induce serious cytotoxic effect and this
toxicity level is still acceptable as biocompatible gene carriers.43
In contrast, at the same concentration, the cell viability for PPF
ester polymer samples decreased to 78% (Figure 7, gray filled
pattern region). Thus, functional polymers derived from P2
featured a lower cytotoxicity, which makes P2 an ideal activated
ester precursor polymer candidate for the preparation of
biorelated functional materials.
Figure 6. Viability of HeLa cells (human cell line derived from cervix
carcinoma cells, adherent) as a function of PFP (solid black circles)
and salicylic acid (open red circles) with error bars. Cell viability was
determined by resazurin fluorimetric assay.
noted that a suitable concentration of salicylic acid (less than
300 μg/mL) in vivo is acceptable.40
Cytotoxicity of polymers containing salicyl and PFP esters
were tested using HeLa cell lines as well. In order to improve
the solubility of PFP ester polymer in the cell culture medium,
hydroxypropylamine was used to partially modify polyPFPMA,
yielding poly(N-(2-hydroxypropyl)-methacrylamide)
(pHPMA), which is known to be a nontoxic drug carrier that
has already entered clinical trials.27,41 Cell toxicity tests also
showed that pHPMA did not exhibit any cell toxicity, even up
to a dose of 2 mg/mL.42 Postpolymerization modification of
polyPFPMA was conducted using hydroxylpropylamine at 25
°C for 39 h. Retaining PFP ester groups after the
postpolymerization modification were proved by 19F NMR
and IR spectroscopy (Figure S8), suggesting that the resulting
poly((N-2-hydroxypropyl)-methacrylamide-co-pentafluoro-
phenyl methacrylate) poly(HPMA-co-PFPMA) contained
19.5% PFP groups (molar ratio). The amount of the copolymer
applied to cell test was calculated based on the neat weight of
polyPFPMA (Table S1). As known from pharmaceutical
studies, an overdose of aspirin is harmful to the body. Doses
higher than 350 μg/mL lead to hyperventilation and result in
acidosis at 450 μg/mL.40 Thus, the cytotoxicity of P2 below
500 μg/mL, instead of going higher to 1000 μg/mL, was
checked in this work. As shown in Figure 7, when the
concentration of polymers was increased from 0 to 500 μg/mL,
the cell viability treated with P2 decreased to around 50%,
whereas when applying a poly(HPMA-co-PFPMA), it decreased
CONCLUSION
■
In conclusion, we have successfully synthesized three novel
activated ester polymer precursors: Poly(salicyl acrylate) and its
methyl ester and tert-butyl ester protected derivatives, poly-
(methyl-salicylate acrylate), and poly(tert-butyl-salicylate acryl-
ate). In general, poly(methyl salicylate acrylate) is cheap, has a
broad solubility in organic solvents, and can be prepared easily,
enabling its applicability in manufacturing general functional
materials. Poly(salicyl acrylate) is based on a cheap drug,
salicylic acid, the active component of aspirin and can be used
in preparing biorelated functional materials. Poly(tert-butyl-
salicylate acrylate) is not only a reactive polymer precursor
itself, but it can also be transformed into poly(salicyl acrylate),
allowing an alternative synthesis of poly(salicyl acrylate). With
primary amines, all of the three activated esters allowed an
almost quantitative functionalization. For secondary amines,
poly(salicyl acrylate) reached partial conversion up to 80%. As
demonstrated by kinetic IR, poly(salicyl acrylate) and poly-
(methyl-salicylate acrylate) showed a similar ester reactivity,
which was not as high as that of PFP ester. Cytotoxicity tests of
leaving groups using HeLa cell lines suggested that salicylate
has a lower toxicity than pentafluorophenol. Cell viability tests
of polymers containing these activated ester groups also
showed that salicyl ester based polymers possess a lower
toxicity than PFP based active esters, making these polymers
ideal activated ester precursor polymer candidates for the
preparation of biorelated functional materials due to their
significantly reduced cytotoxicity.
ASSOCIATED CONTENT
■
S
* Supporting Information
Supplementary spectroscopic data of monomers, poly(tert-
butyl-salicylate acrylate) and poly((N-2-hydroxypropyl)-meth-
acrylamide-co-pentafluorophenyl methacrylate), as well as
kinetic IR spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
Figure 7. Viability of HeLa cells as a function of the different
concentrations of neat polyPFPMA (closed triangles) and P2 (open
triangles); Cell viability was determined by resazurin fluorimetric
assay.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: theato@chemie.uni-hamburg.de.
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(32) College, J. M.; Jefferson, T. J. Pharmacol. Exp. Ther. 1989, 1,
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Notes
The authors declare no competing financial interest.
(33) Rainsford, K. D.; Schweitzer, A.; B, K. Biochem. Pharmacol.
1983, 32, 1301−1308.
ACKNOWLEDGMENTS
■
(34) Boudreaux, C. J.; Bunyard, W. C.; McCormick, C. L. J.
Controlled Release 1997, 44, 171−183.
(35) Bertrand, O.; Schumers, J.-M.; Kuppan, C.; Marchand-Brynaert,
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(36) Barbosa, T. P.; Sousa, S. C. O.; Amorim, F. M.; Rodrigues, Y. K.
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We thank Dr. Felix Scheliga for help with reactive IR
measurements and data analysis. L.H. gratefully acknowledges
the China Scholarship Council (CSC, Grant 201206240006)
for partial support of this work.
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