Toward the design of LPEI containing block copolymers: Improved synthesis protocol, selective hydrolysis, and detailed characterization

Article abstract of DOI:10.1002/pola.26261

The synthesis of poly(2-ethyl-2-oxazoline)-b-linear poly(ethylenimine) (PEtOx-b-LPEI) copolymers by selective basic hydrolysis of PEtOx-b-poly(2-H-2- oxazoline) (PEtOx-b-PHOx) is described. For this purpose, an easy method for the preparation of the 2-H-2-oxazoline (HOx) monomer was developed. Based on the microwave-assisted polymerization kinetics for this monomer, PEtOx-b-PHOx copolymers were prepared. Subsequently, the block copolymers were selectively hydrolyzed to PEtOx-b-LPEI under basic conditions. The success of the polymerizations and subsequent post-polymerization reactions was demonstrated by ¹H NMR spectroscopy and MALDI-TOF-MS investigations of the obtained polymers.

Full text of DOI:10.1002/pola.26261

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Toward the Design of LPEI Containing Block Copolymers: Improved
Synthesis Protocol, Selective Hydrolysis, and Detailed Characterization
Lutz Tauhardt,1,2 Kristian Kempe,1,2 Ulrich S. Schubert1,2,3*
1Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich-Schiller-University Jena,
Humboldtstr. 10, 07743 Jena, Germany
2Jena Center for Soft Matter (JCSM), Friedrich-Schiller-University Jena, Philosophenweg 7, 07743 Jena, Germany
3Dutch Polymer Institute (DPI), John F. Kennedylaan 2, 5612 AB Eindhoven, The Netherlands
Correspondence to: U. S. Schubert (E-mail: ulrich.schubert@uni-jena.de)
Received 28 May 2012; accepted 5 July 2012; published online
DOI: 10.1002/pola.26261
ABSTRACT: The synthesis of poly(2-ethyl-2-oxazoline)-b-linear
poly(ethylenimine) (PEtOx-b-LPEI) copolymers by selective ba-
sic hydrolysis of PEtOx-b-poly(2-H-2-oxazoline) (PEtOx-b-PHOx)
is described. For this purpose, an easy method for the prepara-
tion of the 2-H-2-oxazoline (HOx) monomer was developed.
Based on the microwave-assisted polymerization kinetics for
this monomer, PEtOx-b-PHOx copolymers were prepared. Sub-
sequently, the block copolymers were selectively hydrolyzed to
PEtOx-b-LPEI under basic conditions. The success of the poly-
merizations and subsequent post-polymerization reactions was
demonstrated by 1H NMR spectroscopy and MALDI-TOF-MS
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investigations of the obtained polymers. 2012 Wiley Periodi-
cals, Inc. J Polym Sci Part A: Polym Chem 000: 000–000, 2012
KEYWORDS: block copolymer; cationic polymerization; linear poly-
(ethylenimine); poly(2-H-2-oxazoline); ring-opening polymerization
INTRODUCTION Linear poly(ethylenimine)s (LPEIs) are of
high interest in pharmaceutical research. As they can interact
with negatively charged macromolecules like DNA, RNA, and
si-RNA they are investigated as polymeric vectors for gene
delivery.1 However, their in vivo and clinical administrations
are limited due to cytotoxic effects and a low hemocompati-
bility.2 To overcome these drawbacks as well as the poor
water solubility of LPEI, chemical modifications with biocom-
patible compounds have been performed. In this context,
poly(ethylene glycol) (PEG) represents a suitable polymer
with beneficial properties. It has good water solubility and
exhibits the so-called ‘‘stealth effect.’’3 A large number of
PEG-LPEI copolymers are already described in literature.4–6
They can be synthesized, for example, by grafting acryl ter-
minated-PEG to LPEI via Michael addition5 or using tosylated
PEG as initiator for the cationic ring-opening polymerization
(CROP) of poly(2-oxazoline)s (POxs), which are subsequently
hydrolyzed to LPEI.6 As a promising alternative to PEG,
POxs,7–12 particularly poly(2-ethyl-2-oxazoline) (PEtOx)13,14
and poly(2-methyl-2-oxazoline) (PMeOx),15,16 have shown
comparable features like PEG, but have additional advanta-
geous properties such as low viscosity and high stability in
the therapeutically relevant dose. Jeong et al.17 could show
that statistical copolymers of LPEI and PEtOx, obtained by
partial hydrolysis of PEtOx, have a reduced cytotoxicity and
a transfection efficiency similar to branched poly(ethylen-
imine). Hsiue et al.18 reported the synthesis of PEtOx-b-LPEI
by coupling PEtOx with LPEI in a thiol-disulfide exchange
reaction between sulfhydryl and pyridyl disulfide terminated
groups. These polymers also showed a low cytotoxicity and
high transfection efficiency. However, the block copolymer
synthesis via thiol-disulfide exchange includes multiple reac-
tion steps and is rather time-consuming. Conversely, statisti-
cal copolymers of LPEI and PEtOx suffer from the problem
that they are not well-defined, since only an overall degree
of hydrolysis can be determined, but not the exact lengths of
the LPEI blocks. Moreover, some free nitrogens of the LPEI
units might not be usable for DNA complexation, since the
LPEI units are either too short or sterically blocked by the
adjacent ethyl side chains. Hence, well-defined block copoly-
mers of PEtOx and LPEI are of interest for pharmaceutical
research. Unfortunately, the synthesis of well-defined POx-
LPEI block copolymers is demanding. As LPEI is prepared by
acid19–21 or basic22,23 hydrolysis of POx, usually statistical
cleaved copolymers of LPEI and POx are obtained, when POx
homo- or copolymers are hydrolyzed. However, it is known
that the sterical hindrance, and therefore, the type of the
side chain significantly influence the rate of hydrolysis.19,24
The more bulky the rest is the slower the POx side chain is
cleaved. This leads to the following order for the rate of hy-
drolysis: H > Me > Et > Ph. The selective hydrolysis of
PMeOx-b-PPhOx to LPEI-b-PPhOx is already reported.24 Basic
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SCHEME 1 Schematic representation of the synthesis of HOx.
hydrolysis conditions were found to have a much slower hy-
drolysis rate, but also a higher selectivity, so that the poly(2-
phenyl-2-oxazoline) (PPhOx) block remained almost unhy-
drolyzed. Litt et al.25 described the selective hydrolysis of
poly[2-(4-t-butylbenzoyl)-2-oxazoline]-b-PEtOx, where only
the PEtOx block is hydrolyzed to LPEI with the help of meth-
anesulfonic acid. A similar behavior is expected for block
copolymers of 2-H-2-oxazoline (HOx) and 2-ethyl-2-oxazoline
(EtOx). Saegusa et al.23 reported that LPEI can be prepared
However, HOx can also be prepared by dehydration of
N-(2-hydroxyethyl)formamide, heating it with a Lewis acid
under reduced pressure. When Al2O3 and Fe2O3 are used
as catalyst the product yield is low, due to decomposition
to 2-ethanolamine.39 However, when zinc chloride is used
as catalyst the decomposition does not occur, and the
product can be obtained in a high yield accompanied by
the formation of water.40 This method was used for the
synthesis of HOx in multigram scale (Scheme 1). Before
the polymerization the water had to be removed. As HOx
tends to autopolymerize when heated, and has also a high
fugacity, water could not be removed by fractionized distil-
lation under heating as stated by the Kaiser.40 However,
Saegusa described a method to dry the HOx, obtained
from the instable N-(2-chloroethyl)formamide, by distilla-
tion over crushed KOH under reduced pressure into a
cooling trap.23 This procedure turned out to be the most
suitable drying method, since no heating or extraction is
needed, and hence, no autopolymerization or the presence
of impurities from extraction solvents are observed. Also,
the fugacity could be overcome, since the product is col-
lected in a cooling trap. Additional distillation over BaO
gave dry HOx in an excellent purity and good yield (67%),
suitable for the subsequent application in the CROP. By
combining the ZnCl2 catalyzed dehydration method with
Saegusa’s drying method, it is possible to obtain HOx in
only two reaction steps with a high purity. Moreover, the
observed overall yield (67%) starting from N-(2-hydroxy-
ethyl)formamide is much higher than for other synthesis
approaches, for example, 42% for Saegusa’s approach, and
the reaction can be easily upscaled to synthesize multiple
grams of HOx. No instable, expensive, smelly, or toxic
compounds have to be used. The intermediate N-(2-
hydroxyethyl)formamide can be easily prepared by heating
2-ethanolamine with methyl formate, followed by fraction-
ized distillation. It can be stored in a freezer without
decomposition for more than a year.
ꢀ
by basic hydrolysis of PHOx within 3 h at 98 C, whereas the
basic hydrolysis of PEtOx proceeds much slower. Only 12%
21
ꢀ
of the side chains are cleaved after 2 h at 130 C.
Here, we report the preparation of PEtOx-b-LPEI based on a
significant improved and straightforward method for the
large scale synthesis of the HOx monomer. Most of the prep-
aration methods described so far suffer from problems like
low overall yields due to instable intermediates and multiple
reaction steps, or they use expensive or highly toxic starting
material. Moreover, polymerization kinetics of HOx, the syn-
thesis of PEtOx-b-PHOx and its subsequent hydrolysis to
PEtOx-b-LPEI were performed. All polymers were character-
ized by in detail by 1H NMR spectroscopy and MALDI-TOF-
MS investigations.
RESULTS AND DISCUSSION
Synthesis of HOx
For the detailed kinetic studies, the preparation of different
PEtOx-b-PHOx copolymers, as well as for the subsequent hy-
drolysis, larger amounts of HOx are required. HOx was first
synthesized by Wenker in 1938.26 With minor changes,
Wenker’s way is the most common method to obtain HOx.
Starting from N-(2-hydroxyethyl)formamide, N-(2-chloro-
ethyl)formamide is prepared by chlorination with thionyl chlo-
ride. Subsequent addition of a base, for example, KOH,23,26–30
NaHCO3,29 or NaH,27,31 leads to the formation of the oxazoline
ring. However, the intermediate N-(2-chloroethyl)formamide is
very unstable and tends to decompose to carbon monoxide
and chloroethylamine, resulting in a low overall yield and a
contaminated product. By performing the reactions under
inert condition with prepurified educts, and using solvents
such as 1-methyl-2-porrolidone,27,31 triglyme,27 and tertiary
amines or amides,23,27 the yield could be improved. Another
drawback of the ‘‘Wenker method’’ is the ring closure by
means of a base/water system, which increases the chance of
uncontrolled polymerization or the decomposition of the reac-
tive HOx, and therefore decreases the yield.
Polymerization and Characterization of HOx
The CROP of HOx, its polymerization kinetics and polymer-
ization mechanism, was first studied by Saegusa et al. in the
beginning of the 1970s.23,41–44 The polymerization kinetics
were only recorded at 24, 31, and 40 ꢀC, respectively, aꢀnd
polymerizations were performed in general for 5 h at 80 C.
However, no polymerization kinetic for that temperature was
reported. Since Saegusa et al. already showed that methyl
tosylate (MeOTs) is the most suitable initiator, and that
N,N-dimethylformamide (DMF) and acetonitrile are the best
solvents for the CROP of HOx, the polymerization kinetics
have been studied in acetonitrile with MeOTs as initiator.
The polymerization was performed in a microwave synthesizer
at 80 ꢀC, with a [M]/[I] ratio of 20 and a monomer
Other methods to synthesize HOx either use highly toxic
and expensive compounds, like AgCN32 and t-butyl
isocyanide,32,33 or they suffer from bad overall yields.34–38
Therefore, they are not suitable for a large scale synthesis.
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FIGURE 3 Expanded region of the MALDI- (top) and the ESI-
TOF-MS (bottom) spectra as well as structural assignments for
the different distributions of PHOx.
FIGURE 1 First order polymerization kinetics for the micro-
wave-assisted polymerization of HOx in acetonitrile at 80ꢀC
using MeOTs as initiator.
lium chloride. Some dissolved the PHOx, but they were too
hygroscopic to be used as polymerization solvent. Because of
dissolution problems and unsuitable columns, on which the
polymer adhered, size exclusion chromatography (SEC) char-
acterization of the samples from the kinetic study could not
be performed.
concentration of 4 M; the monomer conversion was deter-
mined by GC. The linearity of the first order kinetic plots
shows the living character of the polymerization (Fig. 1). A
polymerization rate of kp ¼ 0.0193 L molꢁ1 sꢁ1 was calcu-
lated. As expected, the reactions proceeds much faster com-
pared to Saegusa’s results for 24, 31, and 40 ꢀC.43 In every
case, the product precipitated during the polymerization, as
was already noted by Saegusa. This behavior is ascribed to
the formation of strong hydrogen bonds between the poly-
mer molecules, due to interactions of the oxygen and hydro-
The PHOx was characterized by MALDI-TOF- and ESI-Q-TOF-
MS (Fig. 3), showing similar results. This is the first report
of PHOx mass spectra. Distributions for hydroxyl and ethyle-
nimine-terminated species can be found. The two different
end groups originate from the varying possible ring-opening
reactions when the reaction is quenched with water
(Scheme 2). Either a hydroxyl group or a formyl ester is
formed and subsequently hydrolyzed. The ester formation
also occurs for other POxs.45,46
gen of the formyl side chains. A broad band at 3400 cmꢁ1
,
typical for hydrogen bonds, can be found in the IR spectrum
(Fig. 2). The obtained PHOx is soluble only in water at room
temperature and dimethyl sulfoxide (DMSO) at temperatures
above 120 ꢀC. In DMSO, unfortunately no polymerization
could be observed. To overcome the drawback, different
polar ionic liquids have been investigated as solvent, for
example, 1-ethyl-3-methylimidazolium chloride, 1-butyl-3-
methylimidazolium chloride, and 1-allyl-3-methylimidazo-
Synthesis of PEtOx-b-PHOx
PEtOx-b-PHOx was prepared by the sequential monomer
addition method (Scheme 3). The concentration of EtOx in
the solvent acetonitrile was adjusted to 4 M and MeOTs was
used as initiator. As reference, always an aliquote of the
stock solution was polymerized under the same conditions,
but without addition of the second monomer. Three block
copolymers with EtOx/HOx ratios of 20/5 (1b, 2b) and 40/
10 (3b) were synthesized using this approach (Table 1).
SCHEME 2 Schematic representation of the different ring-
FIGURE 2 ATR-FT-IR spectrum of PHOx.
opening possibilities leading to different end groups of PHOx.
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SCHEME 3 Schematic representation of the synthesis of
PEtOx-b-LPEI.
Characterization by SEC revealed the growth of the polymer
chain and therewith the increase of the molar mass. Com-
pared to the reference PEtOx homopolymer, the SEC curve is
shifted to higher molar masses after the addition of the sec-
ond monomer (HOx, Fig. 4). Furthermore, in the 1H NMR
spectra an additional peak at d ¼ 8.02 ppm, deriving from
the formyl side chain, can be found (Fig. 5). Similar as for
pure PHOx the polymer precipitated during the polymeriza-
tion of the second block. When the PHOx block becomes too
long, the copolymer is only soluble in water. A good ratio for
EtOx and HOx was found to be n/m ¼ 4/1. For this ratio,
the polymer is still soluble in polar solvents like methanol,
DMSO, and N,N-dimethylacetamide.
FIGURE 4 Comparison of the SEC curves of the PEtOx-b-PHOx
3b (dashed) and the reference PEtOx homopolymer 3a
(straight) (SEC eluent: DMAc þ 0.21% LiCl).
spectrum can also be explained with the mass discrimination
occurring in MALDI-TOF-MS.
Hydrolysis of PHOx, PEtOx, and PEtOx-b-PHOx Under
Basic Conditions
As already reported by Saegusa, PHOx can be easily hydro-
23
ꢀ
lyzed under basic conditions at 98 C within 3 h. When the
ꢀ
hydrolysis of PHOx with 3 M NaOH was performed at 100 C
Characterization by MALDI-TOF-MS revealed a PEtOx with
one HOx unit (PEtOxn-b-PHOx1) as main distribution. Also,
other distributions were found, but they were hardly visible.
This is ascribed to a mass discrimination effect, which often
occurs in MALDI-TOF-MS measurements. Hence, the copoly-
mer was fractionated by preparative SEC using a Sepahdex
LH20 column with methanol as eluent. The obtained frac-
tions showed a complex pattern of overlapping peaks in the
expected molar mass region, but allowed a detailed interpre-
tation. In all fractions, both monomers were incorporated,
but with different ratios and chain lengths. An example
MALDI-TOF-MS spectrum and the assignment of all the peaks
is shown below (Fig. 6). Based on the results of 1H NMR
spectroscopy, this fraction contains an EtOx/HOx ratio of
4/1. The difference between the ratio calculated from 1H
NMR spectroscopy and the ratio in the MALDI-TOF mass
in a microwave synthesizer, the degree of hydrolysis was 93%
after 1 min, and 99% after 5 min as determined by H NMR
1
spectroscopy. After 10 min full hydrolysis was observed. This
shows that the formyl side chain can be very easily cleaved off.
Under the same conditions, PEtOx was only hydrolyzed for 2%
after 1 min, 6% after 5 min, and 8% after 10 min, respectively.
When the reaction time was increased to 4 h the degree of hy-
drolysis was about 12%. As the PHOx can be hydrolyzed much
easier and faster than the PEtOx, it is assumed that PEtOx-b-
PHOx can be selectively hydrolyzed to PEtOx-b-LPEI. For the
investigation of the hydrolysis conditions, 25 mg of PEtOx-b-
PHOx were heated with 1 mL of 3 M NaOH in a microwave
synthesizer at 100 ꢀC for different times. The solvent was
removed under vacuum. The degree of hydrolysis was deter-
mined by 1H NMR spectroscopy by correlating the proton sig-
nal of the uncleaved PHOx side chain (d ¼ 8.02 ppm) and the
TABLE 1 SEC Data for the Different PEtOx-b-PHOx Copolymers and Reference PEtOx Homopolymers
DMAc þ 2.1% LiCl
Water þ 0.1% TFA þ 0.05 M NaCl
(PEG calibration)
(PEG calibration)
Sample
EtOx:HOx
Mn (g molꢁ1
)
Mw (g molꢁ1
)
PDI
Mn (g molꢁ1
)
Mw (g molꢁ1
)
PDI
1a
1b
2a
2b
3a
3b
20:0
20:5
20:0
20:5
40:0
40:10
1,950
3,100
1,780
2,280
3,240
4,660
2,180
3,950
1,970
2,680
3,750
5,850
1.12
1.27
1.11
1.17
1.16
1.26
1,870
1,980
1,730
1,850
2,240
2,420
2,170
2,440
2,020
2,390
3,080
3,380
1.16
1.23
1.17
1.29
1.37
1.40
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FIGURE 7 Degree of hydrolysis of 2b (circle) and 3b (square)
against hydrolysis time as determined by 1H MNR
spectroscopy.
FIGURE 5 1H NMR spectra of the reference PEtOx 2a (top),
PEtOx-b-PHOx 2b (middle), and PEtOx-b-LPEI (bottom) (solvent:
CD3OD).
1
resulting formiate (d ¼ 8.53 ppm). The determination of the
removal of the solvent. The comparison of the H NMR spec-
1
degree of hydrolysis of the PEtOx block by H NMR spectros-
tra before and after the hydrolysis shows that the peak of
the formiate side chain of the PHOx block totally disap-
peared (Fig. 5). Instead a new peak for LPEI can be found at
2.76 ppm. Moreover, no signals for formiate or propionate
can be found anymore, showing that extracting the block co-
polymer with dichloromethane represents a suitable purifica-
tion method. SEC was measured on a PSS SUPREMA-MAX
column, which is applicable for cationic polymers and does
not only separate by size, but also by charge. Consequently,
the SEC curve of the PEtOx-b-LPEI is shifted to a lower elu-
tion volume although the molar mass is lower compared to
the starting material PEtOx-b-PHOx (Fig. 8), confirming that
the PHOx block is hydrolyzed and the cationic LPEI is
formed. Unfortunately, there are no suitable available stand-
ards for the determination of the molar masses and PDI
values of the PEtOx-b-LPEI.
copy was challenging since the broad signals of LPEI (d ¼ 2.76
ppm) and CH2 group of PEtOx (d ¼ 2.42 ppm) overlap. Also,
the signals of the CH2 group of the resulting propionate (d ¼
2.20 ppm) and CH2 groups of PEtOx were not clearly sepa-
rated. Nevertheless, reproducible results could be obtained.
The degree of hydrolysis of PEtOx is about 5% for n/m ¼ 40/
10 and 14% for n/m ¼ 20/5 when full cleavage of the PHOx
side chain is achieved. This is in good agreement with the
degree of hydrolysis for the basic hydrolysis of the reference
PEtOx homopolymers. It was found that the copolymer with
n/m ¼ 20/5 hydrolyzed much faster than the copolymer with
doubled block lengths; however, the block copolymer with n/
m ¼ 40/10 is hydrolyzed more selectively (Fig. 7).
Unlike LPEI, the PEtOx-b-LPEI copolymer with a m/n ratio of
4/1 is soluble in chloroform and dichloromethane. This
allows the purification of the copolymer, since it can be
easily extracted from the salt residues (NaOH, formiate) after
The MALDI-TOF mass spectrum showed a complex pattern
of overlapping peaks, making the interpretation nearly
FIGURE 6 Expanded region of the MALDI-TOF mass spectrum
of PEtOx-b-PHOx 1b and assignments of the different distribu-
tions (matrix: DHB).
FIGURE 8 SEC curve of PEtOx-b-PHOx 3b before (straight) and
after hydrolysis (dashed) (SEC eluent: water þ 0.1% TFA þ 0.05
M NaCl).
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ture of 30 ꢀC, was used. Molar masses were calculated
against PEG. The MALDI-TOF-MS spectra were recorded
using an Ultraflex III TOF/TOF (Bruker Daltonics GmbH, Bre-
men, Germany), equipped with a frequency-tripled Nd:YAG
laser, operating at a wavelength of 355 nm. All spectra were
measured in the positive reflector mode using 2,5-dihydroxy-
benzoic acid (DHB) as matrix. ESI-Q-TOF-MS measurements
were performed with a micrOTOF Q-II (Bruker Daltonics)
mass spectrometer. The ESI-Q-TOF mass spectrometer was
operating at 4.5 kV at a desolvation temperature of 180 ꢀC.
The mass spectrometer was operating in the positive ion
mode. As solvent methanol/water mixtures were used. The
ESI-Q-TOF-MS instrument was calibrated in the m/z range of
50–3000 using an internal calibration standard (Tunemix so-
lution), which was supplied from Agilent. The electron
impact MS spectra were measured with a Finnigan MAT
SSQ710 spectrometer. The IR spectra were recorded on a FT-
IR spectrometer IRAffinity-1 (Shimadzu).
FIGURE 9 Expanded region of the MALDI-TOF mass spectrum
of PEtOx-b-LPEI and assignments of the different distributions
(matrix: DHB).
impossible. Therefore, analog to the PEtOx-b-PHOx, the copol-
ymer was fractionated by preparative SEC using a Sephadex
LH20 column (eluent methanol). After fractionation, fractions
with different EtOx and EI chain lengths and ratios, but inter-
pretable MALDI-TOF mass spectra were obtained. Both EtOx
and EI repeating units can be assigned, demonstrating that
the PHOx block is successfully hydrolyzed to LPEI (Fig. 9).
Synthesis of N-(2-Hydroxyethyl)formamide
Methyl formate (107 mL, 1.72 mol) was heated with a 25%
excess 2-ethanolamine (130 mL, 2.15 mol) for 8 h under
reflux. The resulting methanol was removed by distillation at
normal pressure and the residue was purified by vacuum
distillation. A colorless liquid was obtained (bp. 110–112 C,
0.01 mbar). Yield: 132.93 g (87%).
ꢀ
1H NMR (300 MHz, CD3OD, d, ppm): 8.09 (s, trans, 1H), 8.01
(s, cis, 1H), 3.57–3.64 (m, 2H), 3.28–3.37 (m, 2H). 13C NMR
(300 MHz, CD3OD, d, ppm): 166.36 (cis), 162.68 (trans),
61.60 (cis), 60.00 (trans), 43.94 (cis), 40.11 (trans). IR (ATR-
FT-IT, cmꢁ1): 3278 (OH/NH), 2877 (CH), 1651 (C¼¼O), 1531
(NH), 1060 (CAO).
EXPERIMENTAL SECTION
Chemicals and Instrumentation
Dry acetonitrile and methyl formate were purchased from
Acros Organics and 2-ethanolamine from Sigma Aldrich. EtOx
and MeOTs were obtained from Acros Organics, distilled to
dryness over barium oxide (BaO), and stored under argon.
Synthesis of the HOx Monomer
The Initiator Sixty single-mode microwave synthesizer from
Biotage, equipped with a noninvasive IR sensor (accuracy:
62%), was used for polymerizations and hydrolyses under
microwave irradiation. Before usage, the microwave vials
were heated to 110 ꢀC overnight and allowed to cool down
to room temperature under an argon atmosphere. Proton
(1H) and carbon (13C) nuclear magnetic resonance (NMR)
spectra were recorded on a Bruker AC 300 MHz at 298 K.
Chemical shifts are reported in parts per million (ppm, d
scale) relative to the residual signal of the deuterated sol-
vent. SEC of PEtOx and PEtOx-b-PHOx was measured on an
Agilent Technologies 1200 Series gel permeation chromato-
graphy system equipped with a G131A isocratic pump, a
G1329A autosampler, a G1362A refractive index detector,
and both a PSS Gram 30 and a PSS Gram 1000 column
placed in series. A 0.21% LiCl solution in N,N-dimethylaceta-
mide (DMAc) was used as eluent at 1 mL minꢁ1 flow rate
and a column oven temperature of 40 ꢀC. Molar masses
were calculated against PEG. SECs of PEtOx-b-PHOx and
A flask equipped with a Claisen condenser was charged with
ZnCl2 (1.412 g, 10 mmol) and N-(2-hydroxyethyl)formamide
(10.026 g, 112 mmol). The reaction mixture was heated at
ꢀ
180 C and 30 mbar. The product was collected in a cooling
trap together with the resulting water. Crushed KOH (8.38 g)
was added and the crude product was distilled into a cooling
trap at 30 mbar. To the distillate, crushed KOH (6.24 g) was
added, and the product was distilled again under reduced
pressure (30 mbar) into a cooling trap. Repeated addition of
KOH (3.22 g) was followed by distillation under reduced
pressure (30 mbar) into a cooling trap. At last, the product
was distilled over BaOH under reduced pressure (30 mbar)
into a cooling trap. The product was stored under argon
over mol sieve 4 Å. Yield: 5.38 g (67%).
1H NMR (300 MHz, CD3OD, d, ppm): 6.71 (s, 1H), 4.08 (t,
3
3J ¼ 9.71 Hz, 2H), 3.71 (td, J ¼ 9.71 Hz, 4J ¼ 1.88 Hz, 2H).
13C NMR (300 MHz, CDCl3, d, ppm): 155.16 (CH), 65.39
(CH2), 53.37 (CH2). (ATR-FT-IT, cmꢁ1): 2978 (CH), 1627
(C¼¼N), 1080 (CAOAC), 918 (¼¼CH). MS (EI, m/z, %): 71 (41)
[Mþ], 41 (45) [MACH2O]þ, 40 (12) [MAC2H3N]þ, 32 (100).
PEtOx-b-LPEI were measured on
a Jasco SEC system
equipped with a PU-980 pump, a AS-1555 autosampler, a
DG-980-50 degasser, a RI930 refractive index detector, and a
PSS SUPREMA-MAX column. As eluent, a solution of 0.1%
trifluoroacetic acid (TFA) and 0.05 M NaCl in water, running
at a flow rate of 1 mL minꢁ1 and a column oven tempera-
Kinetic Investigation of the Polymerization of HOx
For the kinetic studies, a stock solution containing MeOTs
(initiator), HOx (monomer), and acetonitrile (solvent) was
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prepared. The monomer concentration was adjusted to 4 M,
and a monomer to initiator ratio ([M]/[I]) of 20 was used.
The stock solution was divided over six microwave vials that
were capped under argon. For the calculation of the conver-
was removed, and the ꢀresulting polymer was dried under
reduced pressure at 40 C.
1H NMR (300 MHz, CD3OD, d, ppm): 3.54 (m, NACH2), 3.08
(m, CH3AN), 2.76 (NHACH2), 2.42 (m, CACH2AC), 1.11 (m,
CACH3).
ꢀ
sion, t0 samples were taken. The vials were heated to 80 C
for different times in the microwave synthesizer. After cool-
ing, the reaction was quenched and GC samples were
prepared to determine the monomer conversion. The poly-
merization solvent was used as internal standard.
CONCLUSION
A cheap and easy method for the synthesis of the monomer
HOx was developed, and its polymerization kinetics were
investigated. The obtained PHOx was characterized by 1H
NMR, MALDI-TOF-MS, and ESI-Q-TOF-MS. The monomer
(HOx) was further used to synthesize PEtOx-b-PHOx copoly-
mers in a sequential monomer addition approach. During the
reaction, the polymer precipitated, due to hydrogen bond
formation between the formyl side chains. If the HOx block
became too long the polymer was only soluble in water,
making the characterization challenging. However, if an
EtOx/HOx ratio of 4:1 is used, the copolymer was soluble in
polar solvents like methanol, DMF, and DMAc. Block copoly-
mers with two different block lengths and an EtOx/HOx ratio
of 4:1 were synthesized and characterized by 1H NMR spec-
troscopy, SEC, and MALDI-TOF MS, proving that the block
copolymer was formed. Subsequently, the basic hydrolysis to
PEtOx-b-LPEI was performed. 1H NMR spectroscopy and
MALDI-TOF-MS demonstrated that the HOx block was suc-
cessfully hydrolyzed to LPEI, and SEC indicated the forma-
tion of a cationic species, due to a different column interac-
tion of the product and educt. The obtained PEtOx-b-LPEI
copolymers represent interesting materials for pharmaceuti-
cal studies, with promising features for a high transfection
efficiency and a ‘‘stealth effect.’’ Their potential is currently
under investigation.
1H NMR (300 MHz, D2O, d, ppm): 7.90 (m, NACHO), 3.42
(m, NACH2), 2.84 (m, CH3AN). IR (ATR-FT-IR, cmꢁ1): 2939
(CH), 2870 (CH), 1651 (C¼¼O), 1427 (CH2/CH3), 1400 (CH2/
CH3), 1153 (CAOAC), 1053 (CAOAC).
Synthesis of the PEtOx-b-PHOx Copolymers
A stock solution containing MeOTs (initiator), 2-ethyl-2-oxa-
zoline (monomer), and acetonitrile (solvent) was prepared.
The EtOx concentration was adjusted to 4 M, with a mono-
mer to initiator ration of [M]/[I] ¼ 20 and [M]/[I] ¼ 40,
respectively. An aliquot of the stock solution was taken and
polymerized as reference for the PEtOx block. Both, the
remaining stock solution and reference sample were heated
in a microwave synthesizer at 140 ꢀC for a predetermined
time. After cooling to room temperature, the second mono-
mer (HOx) was added to the polymerization mixture through
the septum with a syringe and heated in a microwave syn-
ꢀ
thesizer at 80 C for a predetermined time. The reaction mix-
ture was cooled to room temperature. The precipitate was
dissolved in methanol, precipitated in ice-cold diethyl ether
ꢀ
and dried under reduced pressure at 40 C.
1H NMR (300 MHz, CD3OD, d, ppm): 8.02 (m, NACHO), 3.54
(m, NACH2), 3.08 (m, CH3AN), 2.42 (m, CACH2AC), 1.11
(m, CACH3).
ACKNOWLEDGMENTS
Kinetic Investigations of the Basic Hydrolysis of PHOx
and PEtOx
The authors thank the Thuringian Ministry for Education, Sci-
ence and Culture (grant no. B514-09051, B515-07008, and
B715-08011) and the Dutch Polymer Institute (DPI, technology
area HTE) for financial support of this study.
PHOx and PEtOx (40 6 1 mg), respectively, were dissolved
in 3 M NaOH (2 mL) inꢀa microwave vial. The reaction mix-
ture was heated at 100 C for different times in a microwave
synthesizer. After cooling to room temperature, the solvent
was removed under reduced pressure. The residue was dis-
solved in deuterated methanol and the degree of hydrolysis
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