Synthesis and Bioactivity of Polymer-Based Synthetic Mimics of Antimicrobial Peptides (SMAMPs) Made from Asymmetrically Disubstituted Itaconates

Article abstract of DOI:10.1002/chem.201800907

A series of asymmetrically disubstituted diitaconate monomers is presented. Starting from itaconic anhydride, functional groups could be placed selectively at the two nonequivalent carbonyl groups. By using 2D NMR spectroscopy, it was shown that the first functionalization step occurred at the carbonyl group in the β position to the double bond. These monomers were copolymerized with N,N-dimethylacrylamide (DMAA) to yield polymer-based synthetic mimics of antimicrobial peptides (SMAMPs). They were obtained by free radical polymerization, a metal-free process, and still maintained facial amphiphilicity at the repeat unit level. This eliminates the need for laborious metal removal and is advantageous from a regulatory and product safety perspective. The poly(diitaconate-co-DMAA) copolymers obtained were statistical to alternating, and the monomer feed ratio roughly matched that of the repeat unit content of the copolymers. Investigations of varied R group hydrophobicity, repeat unit ratio, and molecular mass on antimicrobial activity against Escherichia coli and on compatibility with human keratinocytes showed that the polymers with the longest R groups and lowest DMAA content were the most antimicrobial and hemolytic. This is in agreement with the biological activity of previously reported SMAMPs. Thus, the design concept of facial amphiphilicity has successfully been transferred, but the selectivity of these polymers for bacteria over mammalian cells still needs to be optimized.

Full text of DOI:10.1002/chem.201800907

A Journal of
Accepted Article
Title: Synthesis and Bioactivity of Polymer-based Synthetic Mimics of
Antimicrobial Peptides (SMAMPs) Made From Asymmetrically
Disubstituted Itaconates
Authors: David Boschert, Alexandra Schneider-Chaabane, Andreas
Himmelsbach, Alice Eickenscheidt, and Karen Lienkamp
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201800907
Link to VoR: http://dx.doi.org/10.1002/chem.201800907
Supported by

10.1002/chem.201800907
Chemistry - A European Journal
DOI: 10.1002/
Full Paper
Synthesis and Bioactivity of Polymer-based Synthetic Mimics of
Antimicrobial Peptides (SMAMPs) Made From Asymmetrically
Disubstituted Itaconates
David Boschert,# Alexandra Schneider-Chaabane,# Andreas Himmelsbach, Alice
Eickenscheidt, Karen Lienkamp*
# contributed equally
–––––––––
D. Boschert, A. Schneider-Chaabane, A. Himmelsbach, Dr. A. Eickenscheidt, Dr. K.
Lienkamp
Freiburg Center for Interactive Materials and Bioinspired Technologies (FIT) and Department
of Microsystems Engineering (IMTEK), Albert-Ludwigs-Universität, Georges-Köhler-Allee
105, 79110 Freiburg, Germany; *E-Mail: lienkamp@imtek.uni-freiburg.de
–––––––––
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.2018xxxxx.
- 1 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
Abstract:
A series of asymmetrically disubstituted diitaconate monomers is presented. Starting from
itaconic anhydride, functional groups could be placed selectively at the two non-equivalent
carbonyl groups. Using 2D NMR, it was shown that the first functionalization step occurred at
the carbonyl in -position to the double bond. These monomers were copolymerized with
dimethylacrylamide (DMAA) to yield polymer-based synthetic mimics of antimicrobial
peptides (SMAMPs). They were obtained by free radical polymerization, a metal free process,
and still maintained the facial amphiphilicity at the repeat unit level. This eliminates the need
for laborious metal removal and is advantageous from a regulatory and product safety
perspective.
The poly(diitaconate-co-DMAA) copolymers obtained were statistical to alternating, and the
monomer feed ratio roughly matched the repeat unit content of the copolymers. Investigations
of varied R group hydrophobicity, repeat unit ratio and molecular mass on the antimicrobial
activity against E. coli and on the compatibility with human keratinocytes showed that the
polymers with the longest R groups and the lowest DMAA content were the most
antimicrobially and the most hemolytic ones. This is in line with the biological activity of
previously reported SMAMPs. Thus, the design concept of facial amphiphilicity was
successfully transferred, yet, the selectivity of these polymers for bacteria over mammalian
cells still need to be optimized.
Keywords: antimicrobial polymer, bioactive polymer, copolymerization parameters, itaconic
acid, monomer synthesis.
FIGURE FOR ToC_ABSTRACT
- 2 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
TEXT FOR ToC_ABSTRACT
Radically facially amphiphilic. Poly(diitaconates) copy the natural blueprint for antimicrobial
peptides, yet can be obtained by simple free radical polymerization and still maintain the
desired facial amphiphilicity of the parent peptides.
- 3 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
1. Introduction
Due to the continuously increasing incidence of bacterial infections caused by antibiotics-
resistant bacteria, the need for new drugs and materials that inhibit bacterial growth is more
pressing than ever.[1] Very few new antibiotic drug classes have been developed in the past
decades. However, it was estimated that by 2050, 10 million people will die every year from
infections with antibiotics-resistant bacteria - making antimicrobial resistance more deadly
than cancer.[2]
Synthetic Mimics of Antimicrobial Peptides (SMAMPs) are a promising substance class that
could help relieve this problem; consequently, research on small molecule SMAMPs or
polymer-based SMAMPs has strongly increased over the last decade.[3] The SMAMP
approach was derived from nature. SMAMPs copy the design of natural antimicrobial
peptides (AMPs, Figure 1a).[4] AMPs can be found in most organisms as part of the innate
immune system and are crucial for the immediate response of these organisms to pathogens
including bacteria; additionally, they activate other parts of the adaptive immune system.[5]
The antimicrobial activity of AMPs results from so-called facial amphiphilicity: as a result of
the secondary structure of the AMP backbone, the residues of the hydrophobic and cationic
amino acids fall onto opposite faces of the molecule (Figure 1a).[3i, 4, 6] This enables AMPs to
attach with their cationic face to the negatively charged outer envelope of bacteria, where they
aggregate and eventually permeate the cell membrane using their hydrophobic groups.[4a, 6] At
the same time, there is no such electrostatic driving force for the interaction of AMPs with
overall charge-neutral mammalian cells, which is why AMPs selectively target bacteria and
not the cells of their host organism.[4a, 6] For the development of SMAMPs, the concept of
facial amphiphilicity was successfully transferred to the design of antimicrobial synthetic
molecules.[3d, 3h, i, 3l, 7] In these studies, it became clear that the most important parameter to be
controlled in SMAMPs was the regular distribution of the hydrophilic, cationic groups and the
hydrophobic groups along the molecular backbone, as this led to SMAMPs which also had a
- 4 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
high activity against bacteria, and selectivity for bacteria over mammalian cells.[3d, 3i, 3l, 8]
Small molecule-based SMAMPs are obtained in step-by-step organic synthesis, thus there is
excellent control over structural precision.[9] However, such syntheses are laborious and
expensive. Polymer-based SMAMPs, on the other hand, can be synthesized in fewer synthetic
steps,[3f, g, 8b, 10] yet often at the expense of molecular precision. This is due to lack of
sequence-control during the polymerization step. How regular two different monomers are
attached to a growing SMAMP chain depends solely on their relative reactivity, i.e. on their
copolymerization parameters. Unless these dictate alternating copolymerization, the
polymerization outcome is random or statistical. In SMAMPs, this may lead to a local
imbalance in the distribution of hydrophobicity, and thus to increased toxicity.[3l, 11] Another
factor that reduces structural precision of synthetic polymers compared to peptides is that they
are typically lacking a secondary structure that pre-defines the orientation of their functional
groups. In spite of this, some polymer-based SMAMPs showed excellent antimicrobial
activity and cell selectivity in vitro.[3f, 8c] It was hypothesized that the amphiphilic polymer-
based SMAMPs could self-assemble at the liquid-bacteria interface into an appropriate
configuration, and thereby become membrane-active.[10c] This hypothesis was substantiated
by several studies that confirm membrane-activity of SMAMPs.[12]
A regular distribution of functional groups and a better bioactivity profile of polymer-based
SMAMPs are more easily achieved with facially amphiphilic monomers, where the
hydrophobic and the cationic, hydrophilic group are attached to the same polymerizable unit.
This avoids runs of hydrophobic or hydrophilic repeat units in the polymer, which are often
observed when copolymerizing two monomers carrying a hydrophilic and a hydrophobic
group each ('segregated units').[3g] For example, Gabriel et al. compared poly(norbornenes)
with facially amphiphilic repeat units[8b] to the corresponding segregated polymers,[3g] and
found that the cell selectivity of the facially amphiphilic polymers against Gram-positive and
Gram-negative bacteria was by far higher.[3g]
- 5 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
However, the abundance of monomers that can be independently functionalized with two
groups without becoming unpolymerizable (or of monomers that form strictly alternating
copolymers) is rather limited. Norbornenes are such monomers, and facially amphiphilic
poly(norbornene) SMAMPs can be obtained fast and efficiently by ring-opening metathesis
polymerization (ROMP).[8b, 8d, 13] However, the quantitative removal of the transition-metal
based ROMP initiator is often difficult, expensive or just not possible.[14] For medical
applications (drugs or biomaterials), this can become a regulatory issue and a product safety
problem. While fascinating new concepts that use the ROMP initiator in catalytic amounts are
emerging,[15] these as yet lack the precision and control over molecular weight of ROMP with
stoichiometric amounts, so that alternatives to the ROMP-based platform for SMAMPs are
desirable.
The aim of this work was thus to develop a metal-free platform for the synthesis of SMAMP
polymers from facially amphiphilic itaconic acid derivatives. Itaconic acid was chosen
because it can carry two functional groups, because it is cheap and can be obtained from
sustainable resources, and because it can be polymerized by radical polymerization.[16]
Considering that it is side product in the citric acid cycle,[16] itaconic acid is a promising, non-
toxic starting point for the development of drugs and biomaterials. Unfortunately,
homopolymerization of itaconic acid derivatives by a radical mechanism is rather slow
compared to acrylic or methacrylic acid derivatives because itaconates have a high chain
transfer constant.[17] Diitaconate esters, particularly those with short substituents, can be
driven to quantitative conversion within 48 h, however the molecular weights obtained remain
low due to chain transfer.[18] This situation has been improved when controlled radical
polymerization, particularly RAFT, was used to polymerize diitaconates, as reported by
Barner-Kowolik and also by Kamigaito.[19] In these papers, polymer molecular masses (Mn)
as high as 60 000 g mol-1 were reported. However, even in controlled radical
homopolymerization of diitaconates, high chain transfer was observed.[19b]
- 6 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
However, itaconate derivates can be easily copolymerized, e.g. with styrene, methyl
methacrylate or vinyl chloride[20] to obtain poly(itaconates) with varying backbone polarity.
To our knowledge, unsymmetrically substituted poly(itaconates) in general have so far only
been reported twice, and in these cases the product isolation was rather laborious.[21] We here
describe a straight-forward synthesis of a series of facially amphiphilic, unsymmetrically
disubstituted diitaconates, and their copolymerizion with with N,N-dimethylacrylamide
(DMAA). DMAA was chosen as a co-repeat unit because it had a suitable reactivity, and
because poly(dimethyacrylamide) (PDMAA), the homopolymer of DMAA, is protein
repellent and biologically inert.[22] Thus, the presence of DMAA repeat units in the
poly(diitaconate-co-DMAA) SMAMPs would not compromise their biocompatibility. We
also determined the copolymerization parameters of the diitaconate monomers in free radical
copolymerization with either DMAA or styrene. The results indicate that these
copolymerizations yielded statistical to slightly alternating copolymers with tunable
antimicrobial activity, as detailed below.
2. Results and Discussion
System Design
To obtain facially amphiphilic SMAMPs by metal-free polymerization, poly(itaconates)
derivatives were targeted. The design of the poly(itaconate) SMAMPs here presented is a
direct translation of the design concept of the previously reported, highly selective
poly(oxonorbornene) SMAMPs:[3f, 3k] (Figure 1). In both polymers, the hydrophilic (blue) and
the hydrophobic (green) functional groups are attached via ester groups to the polymer
backbone (black). What is particular about the itaconate system is that its two carbonyl groups
are not equivalent. As a result, they can be selectively functionalized. This was not possible in
the symmetric oxonorbornene monomer, where the direction of the ring opening was random.
- 7 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
Thus, at the monomer level, there is even higher structural control in the diitaconate monomer
(Figure 1b) than in the oxonorbornene diester monomer (Figure 1c).
Figure 1. Synthesis route (1 to 9) and design concept (a to c) for poly(itaconate) SMAMPs.
The molecular structure of the highly selective poly(norbornene) SMAMPs (c) was
transferred to the poly(diitaconate) system (9). Both polymers carry a hydrophobic (green)
and a hydrophilic (blue) group, which are connected to the polymer backbone (black) via ester
bonds.
To obtain polymers with tunable amphiphilicity, the R group was varied from R = methyl to
R = pentyl. Since diitaconates do not homopolymerize well, the target polymers were
copolymers with dimethyl methacrylate (DMAA). While the diitaconate repeat units contain
both functional groups required for bioactivity, their bioactivity profile and tunability will still
depend on the ability to control the distribution of the diitaconate and DMAA repeat units
along the polymer chain, i.e. on their copolymerization parameters 푟 and 푟2. For this reason,
1
the copolymerization parameters for the diitaconates with DMAA (and for comparison also
- 8 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
those of the diitaconates with styrene) were determined. Additionally, the molar ratio of
diitaconate to DMAA was used as a further variable to tune the overall polymer
hydrophilicity, and the molar fractions of diitaconate (with R = methyl to butyl) was varied
from 40 mol% to 60 mol%. The effect of these variations on the bioactivity of the polymers
was determined by testing their antimicrobial activity and cell compatibility.
Synthesis of Asymmetrically Disubstituted Itaconates
The asymmetrically disubstituted diitaconates were synthesized in a two-step process
(Figure 1). First, itaconic anhydride 1 was ring-opened with the appropriate alcohol 2 (R =
methyl to pentyl). The ring-opening of the anhydride 1 took place exclusively at the carbonyl
in -position to the double bond, so that mono-itaconate 3 was obtained. This was confirmed
by 2D-NMR spectroscopy (Heteronuclear Multiple Quantum Correlation, HMQC, and
Heteronuclear Multiple Bond Correlation, HMBC; the 2D-spectra of compound 3, with R =
propyl, are shown in the Supporting Information as Figures S1 and S2). The side product of
the reaction was the symmetrically substituted diester 4. Compounds 3 and 4 could be
separated easily during work-up by washing and extraction steps (see Experimental section).
Interestingly, the product ratio of 3 and 4 could be tuned by adding different amounts of acid.
The more acid was used, the more diester 4 was obtained. When the acid quantity was
reduced to catalytic amounts, a yield of up to 80% of product 3 was obtained (Table 1). For
the esterification in the second step, at first Steglich conditions (dicyclohexylcarbodiimide,
DCC and a catalytic amount of N,N-dimethylaminopyridine, DMAP) were applied. However,
these conditions only lead to low yields of the disubstituted diitaconates and a large amount of
a side product (N-acylurea). It is a well-known problem that DCC-activated acids (O-
acylurea) rearranges intramolecularly to the corresponding N-acylurea, which then cannot
react further to the desired product. However, this rearrangement is slow and could therefore
be suppressed by adding stoichiometric amounts of DMAP, which then transfered the O-
- 9 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
acylurea quantitatively to the reactive DMAP-amide. This intermediate reacted rapidly with
the alcohol 5 and gave the desired asymmetrically substituted diitaconates esters 6 in good
yiels (up to 94%, Table 1). Again, the work-up of the product consisted of simple extraction
and filtration steps, and avoided column chromatography.
1
13
The structures of all compounds obtained were confirmed by H-NMR, C-NMR and mass
1
spectrometry. As an example, the H-NMR spectra of the mono-itaconate 3c and the
disubstituted monomer 6c (R = propyl in both cases) are shown in Figure 2. All the peaks in
these spectra could be assigned to the target compounds (Figure 2). When comparing the
spectrum of compound 3c to 6c (Figures 2a and 2b) a slight shift of the double bond signals
(H5 and H6) to lower field strengths was observed, while the methylene peak (H4) was
unaffected. The signal positions and intensities of the other peaks (H1 to H3 and H7 to H9,
respectively) confirmed the assumed structures and thus the attachment of the hydrophobic
1
13
and the hydrophilic groups in each reaction step. The H- NMR, C-NMR spectra and mass
spectra of the other mono- and diitaconates can be found in the Supporting Information
(mono-itaconates: Figures S3 to S8 - NMR spectra, Figures S9 to S13 - mass spectra;
diitaconates: Figures S14 to S19 - NMR spectra, Figures S20 to S24 - mass spectra).
Table 1. Reaction yields for the synthesis of monoitaconates 3a to 3e, the symmetrically
substituted diitaconates 4a to 4e, and the asymmetrically substituted diitaconates 6a to 6e.
mono-itaconate 3 diester 4 diitaconate 6
R group
/ %
/ %
/ %
Methyl a
Ethyl b
Propyl c
Butyl d
Pentyl e
30
25
61
75
80
75
28
2
7
94
91
70
78
14
9
- 10 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
Copolymerization of Diitaconates with DMAA
To obtain the facially amphiphilic target polymers, the asymmetrically substituted
diitaconates 6a-d were copolymerized by free radical polymerization with DMAA (7) at
different monomer ratios using standard free radical polymerization conditions (Figure 1).
The reaction temperature needed to be carefully adjusted, as higher temperatures (80°C and
more) led to polymer cross-linking, and too low polymerization temperatures resulted in long
reaction times and low monomer conversion. The optimum temperature range was 65-70°C.
The polymer was recovered by precipitation from dichloromethane into n-hexane.
1
Figure 2. H-NMR spectra (in CDCl3) of mono-itaconate 3c (a), the asymmetrically
disubstituted diitaconate 6c (b), and the poly(diitaconate-co-DMMA) 8c (c) with a
diitaconate:DMAA ratio of 1:1. The NMR signals (H1 to H11) of each spectrum could be
assigned to the expected protons of the target structures. The signal intensities (numbers
underneath each peak) closely matched the expected proton numbers.
- 11 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
1
The H-NMR spectrum of copolymer 8c with a diitaconate:DMAA ratio of 1:1 is shown in
1
Figure 2c. Further H-NMR spectra of the copolymers 8a-d, with 40-60 mol% diitaconate
content, can be found in the Supporting Information (Figures S25 to S28). The signals of H1
to H3 and H7 to H9 in the diitaconate repeat unit remained at similar chemical shifts as the
corresponding signals of monomer 6c (Figure 2b), albeit with the typical polymer peak
broadening. The signal H4 of the methyl group of the DMAA repeat unit appeared slightly
above 3 ppm. The polymer backbone protons are, expectedly, even broader than the side
chain signals and fall into the typical regions for such aliphatic backbone protons. Gel
permeation chromatography (GPC, calibrated with poly(methyl methacrylate) (PMMA)
standards) was used to determine the molecular weight (number average molar mass, Mn) and
polydispersity index (PDI) of the copolymers 8a-d with varying diitaconate content (40-60
mol% diitaconate). A typical GPC elugram is shown in Figure3a. The GPC elugrams of the
other polymers can be found in the Supporting Information (Figure S30); the data thus
obtained is summarized in Table 2.
Figure 3. Gel permeation chromatography (GPC) elugrams (in chloroform, flow rate:
1 mL min-1, SDV columns, PMMA standards, refractive index detector intensity I vs. elution
volume V)) of a) poly(diitaconate-co-DMAA) 8c, diitaconate:DMAA ratio 1:1, and b)
poly(diitaconate-co-DMAA) 8c, diitaconate:DMAA ratio 1:1, with different molecular
weights.
- 12 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
Table 2. Analytical data for polymers 8 and 9. The number average molecular mass
푀푛 and polydispersity index PDI (= 푀푀 ) of the N-Boc-protected polymers 8 were determined
푤
푛
by gel permeation chromatography (GPC, in chloroform, SDV columns, calibrated with
PMMA standards). The minimum inhibitory concentration (MIC90) against E. coli and the
hemolytic concentration (HC50) of the corresponding deprotected copolymers 9a-d were
determined as described in the Experimental; * = value out of experimental range, n.d. = not
determined.
푴풏
Diitaconate
content
MIC90
HC50
Polymer
8a
PDI Polymer
selectivity
/ μg mL-1
/ μg mL-1
/ g mol-1
19,100
11,900
12,500
19,500
7,300
3.0
*, >400 *, >8000
*, >400 *, >8000
*, >400 *, >4000
*, >400 *, >8000
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.5
1.6
0.2
0.2
0.06
3.2
1.8
4.6
6
40 mol%
50 mol%
60 mol%
40 mol%
50 mol%
60 mol%
40 mol%
50 mol%
60 mol%
40 mol%
50 mol%
60 mol%
50 mol%
50 mol%
50 mol%
50 mol%
50 mol%
2.6
2.7
2.1
2.6
3.3
2.5
2.9
3.5
3.9
2.8
2.7
3.4
1.6
2.8
2.9
1.8
9a
9b
9c
9d
*, >400
*, >400
200
1050
6100
n.d.
300
160
9
8b
8c
8d
6,900
11,200
12,500
6,600
200
100
24,000
17,700
11,800
1,600
50
25
5
50
3
*, >400
200
1,260
350
230
300
70
4,300
7,200
50
8c
9c
12,500
41,000
50
12.5
0.6
- 13 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
Reaction Conversion and Copolymerization Parameters of Diitaconate 6c and DMAA
1H-NMR spectroscopy was used to determine the monomer conversion during the
copolymerization of diitaconate 6c with DMAA. For this purpose, nine reaction batches with
varying ratios of diitaconate 6c (10-90 mol%) and DMAA (90-10 mol%, respectively) were
prepared. Following a literature method,[23] the conversion of each monomer over time was
determined by calculating the relative intensity of the respective monomer peaks for each time
point (signal at 6.26 ppm for the diitaconate, signal at 5.62 ppm for the DMAA, normalized to
the constant signal of the added N,N-dimethylformamid standard at 8.02 ppm). The results are
summarized in Figure 4. Figure 4a and Figure 4b show the time dependent consumption of
the diitaconate monomer 6c and the total monomer conversion (6c + DMAA), respectively,
for the nine different batches. From one time point with low reaction conversion each, a plot
of the molar fraction of the diitaconate repeat units (X) in the nine copolymers vs. the molar
fraction x of diitaconate in the monomer mixture was composed (Figure 4c). The curve is
slightly S-shaped and bends around the diagonal line on which the repeat unit composition of
the polymer corresponds to the composition of the monomer mixture. Thus, the
polymerization is almost statistical. This is important for the SMAMPs synthesis, as it means
that the monomer feed ratio approximately matches the repeat unit composition of the
resulting polymer over a wide range of compositions. Thus, the monomer feed ratio can be
used to tune the overall hydrophilicity of the resulting polymer. Figure 4a and Figure 4b also
show that the monomer conversion increased with increasing amount of DMAA. This
observation matches the results of the GPC analysis (Figure 4d and Table 3), which indicates
that the isolated polymers also had higher average molecular masses with increasing DMAA
content. This was expected, since DMAA homopolymerizes more easily at 70°C than the
diitaconate. In the case of polymer 8c with 10 mol% diitaconate repeat units, the
polydispersity index was extremely high, most likely due to formation of inter-chain cross
- 14 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
links, as the chosen polymerization time (24 h) was too long for this particular composition.
For the other compositions, the polydispersity index ranged between 1.7 to 3.8 (Figure 4d).
Figure 4. Copolymerization of diitaconate 6c with DMAA. The monomer composition was
varied from 10% diitaconate and 90% DMAA (= 10 mol% in the figure) to 90% diitaconate
and 10% DMMA (= 90 mol% in the figure). a) Conversion of the diitaconate monomer vs.
time and b) total monomer conversion vs. time. Both data sets were determined by 1H-NMR
spectroscopy. c) Plot of molar fraction X of diitaconate in the polymer vs. the molar fraction x
of diitaconate in the monomer mixture at 12-21% total conversion. On the diagonal line, the
repeat unit composition of the polymer corresponds to the composition of the monomer
mixture. d) Gel permeation chromatography (GPC) elugrams (refractive index detector
intensity I vs. elution volume V) of copolymers 8c with different monomer ratios of
diitaconate and DMAA that were isolated at the end point of the reactions (Figure 4a).
- 15 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
Table 3. Gel permeation chromatography (GPC) data (in chloroform, SDV columns,
calibrated with PMMA standards) for polymers 8c (obtained by copolymerization of 6c with
DMAA), and for copolymers obtained by copolymerization of diitaconate 4c with styrene. 푀푛
푀
푤
= number average molecular weight,
= polydispersity index (PDI). The polymer
푀
푛
composition was varied from 10% diitaconate and 90% DMAA (=10 mol% in the table) to
90% diitaconate and 10% DMMA (= 90 mol% in the table).
Mn
Mn
Polymer 8c
PDI Polymer 4c/styrene
PDI
/ g mol-1
/ g mol-1
10 mol%
20 mol%
30 mol%
40 mol%
50 mol%
60 mol%
70 mol%
80 mol%
90 mol%
124,100 12.5
92,000 2.2
32,000 3.8
26,700 2.8
22,100 2.3
20,600 2.2
10,100 2.0
12,000 1.9
10 mol%
20 mol%
30 mol%
40 mol%
50 mol%
60 mol%
70 mol%
80 mol%
90 mol%
23,400 2.3
21,000 2.2
20,200 1.6
17,700 1.6
11,200 1.8
12,100 1.8
8,100
5,200
3,800
1.8
1.6
1.4
8,800
1.7
The copolymerization parameters (r-parameters), i.e. the relative reactivity of the two
1
monomers, were calculated from the individual H-NMR spectra using the Fineman-Ross
plot, the Inverted-Fineman-Ross plot and the Kelen-Tudos plot.[23-24] These plots linearize the
Mayo-Lewis equation, which is used to describe copolymerization kinetics, so that the
copolymerization parameters can be obtained (see Supporting information, Figures S37 to
S38). The r-parameters thus obtained are summarized in Table 4. Copolymerization
parameters are different for each comonomer pair A and B and also temperature-dependent,
푘
푘
푘
퐴퐴
퐴퐵
as they are the ratios of two propagation rate constants (푟 =
and 푟2 = 퐵퐵, respectively,
1
푘
퐵퐴
where 푘퐵퐴 (for example) is the rate constant of the addition of monomer A to a polymer with
a chain end radical B), which are temperature-dependent themselves. Besides the
copolymerization parameters for the reaction of 6c with DMAA, we also determined the
- 16 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
copolymerization parameters for the reaction of 4c with styrene to have a reference data set
for comparison with literature data (Figure S39) For the system 4c/styrene (polymerized at
80°C), both copolymerization parameters were well below one, which indicates that the
propagation rate of the reaction of each monomer with a like chain end (푘퐴퐴, 푘퐵퐵,
corresponding to a 'homopolymerization' step) was slower than the propagation rate with an
unlike chain end (푘퐴퐵, 푘퐵퐴, corresponding to a 'copolymerization' step). This hints at a
tendency to form alternating copolymers rather than strictly statistical copolymers. For the
system 6c/DMMA the polymerization temperature was set to 70°C. This was due to the
higher overall reactivity of DMAA compared to styrene. Nevertheless, the copolymerization
parameters for the system 6c/DMAA were similar to those of the system 4c/styrene. In both
cases, the copolymerization parameters were below one, showing that both monomers slightly
favored alternating copolymerization rather than forming statistical copolymers.
Table 4. Copolymerization parameters 푟 and 푟2 for the reaction of diitaconates 4c and 6c with
1
styrene and DMMA respectively.
Reaction
Comonomer 1
Comonomer 2
Temperature Method
/ °C
r1
r2
80
Fineman-Ross
0.22 0.46
0.32 0.45
Inverted Fineman-
Ross
styrene
80
4c
80
70
Kelen-Tudos
0.38 0.58
0.47 0.53
Fineman-Ross
Inverted Fineman-
Ross
DMAA
70
70
0.49 0.49
0.55 0.58
6c
Kelen-Tudos
Tate et al.[20] found copolymerization parameters for the reactions of various itaconic acid
derivatives with different comonomers (Table 5). The r-parameters determined in this work
were in good agreement with this data: the 푟 values of the diitaconates were below 1 when
1
- 17 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
copolymerized with either styrene or methyl methacrylate. The r-parameters 푟2 of styrene in
these systems were also below 1, indicating a statistical to alternating incorporation of both
monomers. The literature values of 푟2 for reactions of diitaconates with methyl methacrylate
were mostly above 1, which indicates a preference of methyl methacrylate for the
homopolymerization step in these systems. For the copolymerization of (di)itaconates with
DMAA, no copolymerization parameters were reported so far, yet the copolymerization
parameters found for the comonomer pair 8c and DMAA in this work are reasonable. In the
context of SMAMPs, the fact that 푟 and 푟2 were below 1 for the investigated diitaconate-
1
comonomer systems, which indicates a statistical to alternating monomer incorporation, is an
important finding. It means that there is no intrinsic tendency to an irregular repeat unit
distribution in these polymers. Thus, there is sufficient reaction control in the polymerization
step, and the global as well as the local molecular amphiphilicity and charge density of the
SMAMP copolymers can be tuned by varying the R groups on the facially amphiphilic repeat
units, and by adjusting the molar fractions of the repeat units.
Table 5. Copolymerization parameters r1 and r2 for the reaction of various itaconic acid
derivatives with different comonomers.
Comonomer 1
Comonomer 2
styrene
n-butylacrylate
풓ퟏ
0.41
0.94
풓ퟐ
0.25
Lit.
[25]
4c
4a
4a
4d
[20]
[20]
[20]
0.40
1.3
methyl methacrylate 0.3
methyl methacrylate 0.4
0.8
Copolymer Activation and Bioactivity
The protected poly(diitaconate-co-DMAA) 8 copolymers were activated by removing their
tert-butyloxycarbonyl (Boc) protective group with hydrochloric acid. This gave
poly(diitaconate-co-DMAA) SMAMPs 9 with primary ammonium groups. They were
- 18 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
precipitated from methanol into diethyl ether and then dried. The removal of the Boc groups
1
was confirmed by H-NMR spectroscopy (see Experimental section and Figure S35). The
antimicrobial activities of the copolymers 9a-d with varying DMAA content and of polymers
9c with varying molecular mass (at constant diitaconate:DMAA ratio of 1:1) were determined
using the Minimum Inhibitory Concentration (MIC) assay. In this assay (described in the
Experimental section), the MIC90 value is determined, which corresponds to the SMAMP
concentration at which 90% bacterial growth is inhibited - the smaller this value, the more
active the polymer. The toxicity of the copolymers was estimated by determining their
propensity to lyse human red blood cells using and the Hemolysis assay (described in the
Experimental section). In this assay, the hemolytic concentration HC50 (the concentration at
which 50% of human red blood cells are lysed by the polymer) was determined - the higher
this value, the less hemolytic the polymer. The curves thus obtained are shown in Figure 5,
and the corresponding MIC and HC50 values are listed in Table 2. In line with the results
obtained for the parent poly(oxanorbornene) system,[3f] the highest antimicrobial activity was
found for the more hydrophobic copolymers (with longer alkyl side chains at R, Figure 5a).
The impact of hydrophobicity on antimicrobial activity was also seen in the series of
polymers which had the same R groups, but different molar ratios of diitaconate and DMAA.
As all diitaconate repeat units were more hydrophobic than DMAA, the antimicrobial activity
of these polymers also increased with increasing diitaconate content. This was particularly
apparent for polymer 9c (Figure 5a). A similar effect was observed in the HC50 data (Figure
5b), which also increased with increasing hydrophobicity. These results are consistent with
antimicrobial data of many other polymer series, where a general increase in antimicrobial
activity with increased molecular hydrophobicity (at constant overall molecular weight) has
been observed.[3h, 8c, 26] Polymers that deviate from this trend are typically found to be less
soluble in aqueous media, and thus less bioavailable than their more hydrophilic
counterparts.[3h, 8c, 26] The most hydrophobic copolymers (particularly 9d, with R = butyl)
- 19 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
were also the most toxic ones. The shorter the alkyl group R and the higher the DMAA
content (at constant molecular mass), the lower was their hemolytic activity. This is also
consistent with the trends observed for previously reported polymers.[3h, 8c, 26] Thus, the design
concept of the facially amphiphilic poly(oxonorbornenes) was successfully translated to
poly(diitacoante-co-DMAA) copolymers mit facially amphiphilic repeat units, which were
antimicrobially active and had tunable properties.
For the copolymer series with different molecular weights (9c, diitaconate:DMAA ratio 1:1,
푀푛 = 1,500 - 41,000 g mol-1), the antimicrobial activity increased significantly with increasing
molecular mass of the samples tested (Figure 5c). A similar trend was also found in their
hemolytic activity (Figure 5d). This was somewhat unexpected, as an increase of
antimicrobial activity in this molecular weight range is typically not observed. For example,
for amphiphilic poly(norbornene) SMAMPs, antimicrobial activity was either unaffected by
molecular weight, or was found to decrease.[8b] Likewise, the hemolytic activity of these
polymers was independent of their molecular mass.[8b] For another poly(oxonorbornene)
SMAMP, both antimicrobial activity and hemolytic activity decrease with increasing 푀푛.[3h,
12c]
However, the poly(methacrylates) reported by of Kuroda and DeGrado were consistently
more hemolytic with increasing molecular weight, although only a narrow molecular range
from 1,600 g mol-1 to 8,700 g mol-1 was investigated in these studies.[26] For the same polymer
series, antimicrobial activity decreased with increasing 푀푛 for the more hydrophobic
polymers (R = propyl, butyl, benzyl), while it slightly increased for with increasing 푀푛 for the
corresponding methyl and ethyl copolymers. For the nylon-3 random copolymers described
by Gellman, hemolysis strongly increased with increasing polymer chain length.[8c] Thus, the
molecular weight effect on these properties, in contrast to the hydrophobicity effect, is not yet
fully understood.
- 20 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
Figure 5. Bioactivity of poly(diitaconate-co-DMAA) SMAMPS. a) Antimicrobial activity
against E. coli bacteria and b) hemolytic activity of copolymers 9a-d with different ratios of
diitaconate to DMAA; c) antimicrobial activity against E. coli bacteria and d) hemolytic
activity of copolymers 9c (diitaconate:DMAA ratio 1:1) with different molecular masses. The
data point with a growth percentage below the straight lines in a) and c) indicates the MIC90
value; the intercept of the curves and the straight lines in b) and d) the location of the HC50
value.
3. Conclusions
We here presented the synthesis and copolymerization of a series of diitaconate monomers
from which facially amphiphilic, antimicrobial synthetic mimics of antimicrobial peptides
(SMAMPs) could be obtained. Their design concept was adapted from facially amphiphilic
poly(oxonorbornenes), yet they had the advantage that they could be obtained by free radical
- 21 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
polymerization, a metal free polymerization technique, and still maintained the facial
amphiphilicity at the repeat unit level.
Starting from itaconic anhydride, two distinct functional groups could be placed at the two
non-equivalent carbonyl groups of that building block. Using 2D NMR techniques, it was
shown that the functionalization of the itaconic anhydride with the first functional group
selectively occurred at the carbonyl in -position to the double bond. By determining the
copolymerization parameters of the diitaconate monomers with DMAA, it was shown that the
poly(diitaconate-co-DMAA) copolymers obtained were statistical to alternating, and that the
monomer feed ratio roughly matched the repeat unit content of the copolymers. By varying
the overall hydrophobicity of the polymers using the length of the alkyl chain R and the
DMAA content as parameters, it could be shown that the polymers with the longest R groups
and the lowest DMAA content were the most antimicrobially active ones, yet they were also
the most hemolytic ones. This is in line with the biological activity of the previously reported
poly(oxonorbornes) and other polymer series.[3h, 3l] Thus, the design concept of the facially
amphiphilic poly(oxonorbornenes) was successfully transfered to the facially amphiphilic
poly(diitaconate-co-DMAA) copolymers. So far, however, the selectivity of these polymers
for bacteria over mammalian cells (Table 2) remains low due to the multitude of parameters
that need to be further optimized in this polymer system. However, the data shows that once
the design rules for one facially amphiphilic SMAMP system are known, these design rules
can be transferred from one polymer system to another, provided that each system is
synthesized with sufficient structural control. Importantly, the here described diitaconate-
based SMAMPs can be synthesized by metal free initiator systems. This eliminates the need
for laborious metal removal during work-up and is thus advantageous for drug and
biomaterials applications from a regulatory and product safety perspective. Additionally, it
makes these polymers much cheaper than the corresponding poly(norbornenes), which is of
particular importance for materials applications, where larger polymer amounts might be
- 22 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
needed. However, to be truly useful as drugs and materials, their hemolytic activity has still to
be optimized. This can be achieved by further structural optimization, particularly by using
other co-repeat units than DMAA. In future work we will strive for even more control in the
polymerization of these systems. In the light of the molecular weight dependency of the
biological activities of these polymers, the next variable to be optimized will be the
polydispersity, using controlled radical polymerization. We will report on our progress in this
project in due course.
4. Experimental Section
General
All chemicals, e.g. itaconic anhydride, methanol, ethanol, n-propanol, n-butanol, n-pentanol,
4-dimethylaminopyridine (DMAP), N,N’-dicyclohexylcarbodiimide (DCC), N-Boc-
ethanolamine, 2,2’-azobis(2-methylpropionitrile) (AIBN) and HPLC grade solvents, e.g.
dichloromethane (DCM), acetone, chloroform, hexane or diethyl ether, were obtained as
reagent grade from Sigma Aldrich (Darmstadt, Germany) or Carl Roth (Karlsruhe, Germany)
and used as received, unless otherwise indicated. N,N-dimethylacrylamide (DMAA), DMF
and styrene were freshly distilled before use. AIBN was recrystallized from methanol. Gel
permeation chromatography (GPC, in chloroform, flow rate 1 ml min-1, calibrated with
poly(methyl methacrylate) (PMMA) standards) was measured on a non-polar GPC column set
(PSS SDV analytical (5 µm, 100 Å and 10.000 Å), PSS, Mainz, Germany). NMR spectra
were recorded on a Bruker 250MHz spectrometer (Bruker, Madison, WI, USA).
Synthesis of Asymmetrical Diitaconates
Synthesis of 4-Alkyl Itaconates 3a to 3e
- 23 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
To obtain the 4-alkyl itaconates 3a to 3e with R = methyl to pentyl, itaconic anhydride (4.3 g,
38 mmol) was dissolved in DCM (10 mL) and the required primary alcohol with the
appropriate R group (40 mL, excess) was added. Next, H2SO4 (conc., 0.5 mL) was added, and
the solution was stirred overnight at room temperature. DCM (50 mL) was added to the
reaction mixture to facilitate phase separation. The organic phase was then extracted with
aqueous K2CO3 (10 w%, 3 x 50 mL) and the aqueous phase was washed with
DCM (3 x 50 mL). The combined organic phases were dried over Na2SO4. The solvent was
evaporated under reduced pressure to yield the side product 4 as a yellow oil. To the aqueous
phase, HCl (conc., 40-50 mL) was added until the pH value was 2. The aqueous phase was
then extracted with DCM (3 x 100 mL). The organic phases were combined and dried over
Na2SO4. The solvent was evaporated under reduced pressure and the product was dried at
high vacuum overnight. The product was obtained as a colorless solid.
4-Methyl itaconate 3a
1H-NMR (250 MHz, CDCl3, δ): 6.50 (s, 1H), 5.88 (s, 1H), 3.74 (s, 3H, CH3), 3.39 ppm (s, 2H,
CH2); 13C NMR (63 MHz, CDCl3, δ): 171.97 (C=O), 171.51 (C=O), 133.60 (s, C=C), 131.33
(s, C=C), 52.56 (C-O), 37.43 ppm (C3); MS (APCI) m/z: [M + H]+ calcd. for C6H8O4, 143.13;
found, 143.03.
4-Ethyl itaconate 3b
1H NMR (250 MHz, CDCl3, δ): 6.49 (s, 1H), 5.86 (s, 1H), 4.20 (td, J = 7.10 Hz, 2H, CH2),
3.37 (s, 2H, CH2), 1.29 ppm (t, J = 7.11 Hz, 3H, CH3); 13C NMR (63 MHz, CDCl3, δ): 172.03
(s, C=O), 171.07 (C=O), 133.76 (C=C), 131.10 (C=C), 61.44 (C-O), 37.70 (C3), 14.43 ppm
(s, C6); MS (APCI) m/z: [M + H]+ calcd. for C7H10O4, 157.15; found, 157.05.
4-Propyl itaconate 3c
1H NMR (250 MHz, CDCl3, δ): 6.49 (s, 1H), 5.87 (s, 1H), 4.10 (t, J = 6.71 Hz, 2H, CH2),
3.38 (s, 2H, CH2), 1.68 (tq, J = 7.10 Hz, 2H, CH2), 0.96 ppm (t, J = 7.42 Hz, 3H, CH3);
- 24 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
13C NMR (63 MHz, CDCl3, δ): 171.98 (C=O), 171.14 (C=O), 133.81 (C=C), 130.99 (C=C),
67.04 (C-O), 37.71 (C3), 22.24 (C6), 10.63 ppm (C7). MS (APCI) m/z: [M + H]+ calcd for
C8H12O4, 171.18; found, 171.06. Anal. calcd. for C8H12O4: C 55.81, H 7.02; found: C 55.47,
H 6.58.
4-Butyl itaconate 3d
1H NMR (250 MHz, CDCl3, δ): 6.48 (1H), 5.86 (1H), 4.14 (t, J = 6.56 Hz, 2H, CH2), 3.37 (s,
2H, CH2), 1.54 - 1.76 (m, 2H, CH2), 1.40 (tq, J = 7.30 Hz, 2H, CH2), 0.95 ppm (t, J = 7.27
13
Hz, 3H, CH3); C NMR (63 MHz, CDCl3, δ): 172.05 (C=O), 171.10 (C=O), 133.81 (C=C),
130.98 (C=C), 65.30 (C5), 37.71 (C3), 30.90 (C6), 19.40 (C7), 13.97 ppm (C8); MS (APCI)
m/z: [M + H]+ calcd. for C9H14O4, 185.21; found, 185.08.
4-Pentylitaconate 3e
1H NMR (250 MHz, CDCl3, δ): 6.49 (1H), 5.86 (1H), 4.13 (t, J = 6.56 Hz, 2H, CH2), 3.37 (s,
2H, CH2), 1.60 - 1.71 (m, 2H, CH2), 1.28 – 1.37 (m, 4H, CH2, CH2), 0.92 ppm (t, J = 7.27 Hz,
3H, CH3); 13C NMR (63 MHz, CDCl3, δ): 170.60 (C=O), 171.13 (C=O), 133.84 (C=C),
130.71 (C=C), 65.48 (C5), 37.61 (C3), 28.49 (C6), 28.27 (C7), 22.55 (C8), 14.14 ppm (C9);
MS (APCI) m/z: [M + H]+ calcd. for C10H16O4, 200.23; found, 200.11.
Synthesis of 1-(N-Boc-2’-aminoethyl) 4-alkyl diitaconate 6a to 6e
The reaction was performed under nitrogen atmosphere. N-Boc-ethanolamine (1.3 g,
7.9 mmol, 1.2 eq) and DMAP (1.2 g, 9.9 mmol, 1.5 eq) were added to a solution of the
appropriate 4-alkylitaconate 3a to 3e (6.6 mol, 1 eq) in DCM (10 mL). The solution was
cooled with ice for 10 min, then DCC (1.5 g, 7.3 mmol, 1.5 eq) in DCM (10 mL) was added
dropwise. The reaction vessel was stirred overnight at room temperature. The reaction
- 25 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
mixture was then filtered and washed with aqueous KHSO4 (10 w%, 3 × 100 mL) and
aqueous NaHCO3 (saturated, 2 × 100 mL). The organic phase was dried over Na2SO4, and the
solvent was removed under reduced pressure. The resulting product was diluted in DCM
(5 mL) and stored in the freezer overnight to get rid of left-over urea side product, which
precipitated. After the cold liquid phase was filtered to remove the precipitate, the solvent was
evaporated under reduced pressure and the product was dried at high vacuum overnight.
1-(N-Boc-2’-aminoethyl) 4-methyl diitaconate 6a
1H NMR (250 MHz, CDCl3, δ): 6.35 (s, 1H), 5.72 (s, 1H), 4.94 (br. s, 1H), 4.22 (t, J = 5.21
Hz, 2H, CH2), 3.71 (s, 3H, CH3), 3.41 (tq, J = 5.20 Hz, 2H, CH2), 3.35 (s, 2H, CH2), 1.44 ppm
(s, 9H, (CH3)3); 13C NMR (63 MHz, CDCl3, δ): 171.68 (s, C=O), 166.23 (C=O), 156.15
(C=O), 133.92 (s, C=C), 129.47 (C=C), 79.83 (C-(CH3)3), 64.74 (C4’), 52.45 (C5), 39.92
(C5’), 37.96 (C3), 28.72 ppm (C-(CH3)3); MS (ESI) m/z: [M + Na]+ calcd. for C13H21NO6,
310.31; found 310.12.
1-(N-Boc-2’-aminoethyl) 4-ethyl diitaconate 6b
1H NMR (250 MHz, CDCl3, δ): 6.39 (s, 1H), 5.75 (s, 1H), 4.92 (br. s., 1H), 4.26 (t, J = 5.30
Hz, 2H, CH2), 4.20 (q, J = 7.20 Hz, 2H, CH2), 3.46 (td, J = 5.30 Hz, 2H, CH2), 3.37 (s, 2H,
13
CH2), 1.47 (s, 9H, (CH3)3), 1.30 ppm (t, J = 7.19 Hz, 3H, CH3); C NMR (63 MHz, CDCl3,
δ): 171.25 (C=O), 166.30 (C=O), 156.16 (C=O), 134.06 (C=C), 129.31 (C=C), 79.81
(C-(CH3)3), 64.76 (C4’), 61.37 (C5), 39.94 (C5’), 38.23 (C3), 28.73 (C (CH3)3), 14.51 ppm
(C6); MS (ESI): m/z = [M+Na]+ calcd. for C14H23NO6, 324.34; found 324.14.
1-(N-Boc-2’-aminoethyl) 4-propyl diitaconate 6c
- 26 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
1H NMR (250 MHz, CDCl3, δ): 6.38 (s, 1H), 5.75 (s, 1H), 4.92 (br. s., 1H), 4.25 (t, J =
5.20 Hz, 2H, CH2), 4.10 (t, J = 6.70 Hz, 2H, CH2), 3.46 (td, J = 5.20 Hz, 2H, CH2), 3.39 (s,
2H, CH2), 1.69 (tq, J=7.10 Hz, 2H, CH2), 1.47 (s, 9H, (CH3)3), 0.96 ppm (t, J = 7.35 Hz, 3H,
CH3);
13C NMR (63 MHz, CDCl3, δ): 171.35 (C=O), 166.33 (C=O), 156.18 (C=O), 134.08 (C=C),
129.32 (C=C), 79.84 (C-(CH3)3), 66.99 (C5), 64.81 (C4’), 39.93 (C5’), 38.24 (C3), 28.73 (s,
C-(CH3)3), 22.27 (C6), 10.69 ppm (C7); MS (APCI) m/z: [M + H]+ calcd. for C15H25NO6,
316.37; found, 316.17. Anal. calcd for C15H25NO6: C 57.13, H 7.99, N 4.44; found: C 57.16,
H 7.45, N 4.43.
1-(N-Boc-2’-aminoethyl) 4-butyl diitaconate 6d
1H NMR (250 MHz, CDCl3, δ): 6.26 (s, 1H), 5.64 (s, 1H), 5.05 (br. s., 1H), 4.13 (t, J =
5.29 Hz, 2H, CH2), 4.03 (t, J = 6.63 Hz, 2H, CH2), 3.33 (td, J = 5.20 Hz, 2H, CH2), 3.27 (s,
2H, CH2), 1.45 - 1.62 (m, 2H, CH2), 1.36 (s, 9H, (CH3)3), 1.19 - 1.34 (m, 2H, CH2), 0.84 ppm
(t, J = 7.20 Hz, 3H, CH3); 13C NMR (63 MHz, CDCl3, δ): 171.20 (C=O), 166.24 (C=O),
156.15 (C=O), 134.08 (C=C), 129.09 (C=C), 79.62 (C-(CH3)3), 65.13 (C5), 64.65 (C4’),
39.84 (C5’), 38.10 (C3), 30.87 (C6), 28.65 (C-(CH3)3), 19.34 (C7), 13.93 ppm (C8); MS
(ESI): m/z = [M+Na]+ calcd. for C16H27NO6, 352.39; found 352.17.
1-(N-Boc-2’-aminoethyl) 4-pentyl diitaconate 6e
1H NMR (250 MHz, CDCl3, δ): 6.37 (s, 1H), 5.74 (s, 1H), 4.94 (br. s., 1H, NH), 4.24 (t, J =
5.29 Hz, 2H, CH2), 4.12 (t, J = 6.63 Hz, 2H, CH2), 3.46 (td, J = 5.20 Hz, 2H, CH2), 3.37 (s,
2H, CH2), 1.46 (s, 9H, (CH3)3), 1.28 - 1.36 (m, 6H, 3 × CH2), 0.92 ppm (t, J = 7.20 Hz, 3H,
13
CH3); C NMR (63 MHz, CDCl3, δ): 171.34 (C=O), 166.33 (C=O), 156.18 (C=O), 134.08
(C=C), 129.29 (C=C), 79.89 (C-(CH3)3), 65.59 (C5), 64.83 (C4’), 38.25 (C5’), 35.30 (C3),
- 27 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
28.75 (C6), 28.36 (C-(CH3)3), 22.65 (C7), 14.68 (C8), 14.31 ppm (C9); MS (ESI): m/z =
[M+Na]+ calcd. for C17H29NO6, 366.42; found 366.19.
Copolymerizations
Copolymerization of 1-(N-Boc-2’-aminoethyl) 4-alkyl diitaconates) 6a to 6e with DMAA at
different ratios.
The itaconate monomers were copolymerized under nitrogen atmosphere. The different
reagent amounts for the synthesis of each polymer with different comonomer ratios or
different molecular weights are listed in Table S1. In a typical reaction, the diitaconate
monomer 6, e.g. 6c, and DMMA were dissolved in DMSO to obtain a total monomer
concentration of 0.5 g mL-1. Next, the initiator AIBN was added. The reaction mixture was
degassed by three freeze-pump-thaw cycles and placed into a pre-heated oil bath (bath
temperature: 70°C) for 24 hours. The polymerized mixture was then precipitated into n-
hexane (150 mL). The precipitate was re-dissolved in DCM (15 mL) and again precipitated
into n-hexane (150 mL). This was repeated until no more monomer peaks were observed in
the 1H-NMR spectrum. The thus obtained copolymer 8 (8c in this case) was dried under high
vacuum.
Poly(1-(N-Boc-2’-aminoethyl) 4-propyl diitaconate) 8c (50 mol% 6c, 50 mol% DMAA):
1H NMR (250 MHz, CDCl3, δ): 4.01 (br. s, 4H, CH2, CH2), 3.39 (br. s, 2H, CH2), 3.08 (br. s,
2H, CH2), 2.23 (br. s, 2H, CH2), 2.90 (br. s, 3H, CH2, CH), 1.70 (s, 2H, CH2), 1.46 (s, 9H,
(CH3)3), 0.95 ppm (br. s, 3H, CH3).
1
For the other molar ratios, the chemical shifts of the peaks in the H-NMR spectra were
1
identical, only the peak ratios were different. The H-NMR spectra of these polymers are
shown in Figures S25 to S28 of the Supporting Information. The chemical shifts of the peaks
- 28 -
This article is protected by copyright. All rights reserved.

10.1002/chem.201800907
Chemistry - A European Journal
1
in the H-NMR spectra of copolymer 8c with different molecular weight were identical to
copolymer 8c which is shown in Figure S27. The Gel permeation chromatography elugrams
for these polymers are summarized in Table 2 and shown in Figures S30, S33).
Deprotection of the Copolymers
The removal of the tert-Butyloxycarbonyl protecting group was carried out under nitrogen
atmosphere. The respective polymer was diluted in MeOH (anhydrous, 3 mL). Then HCl (4M
in dioxane, 3 mL) was added. The solution was stirred overnight, and the solvent was
removed under reduced pressure. The copolymer 8 was dissolved in DCM (10 mL) and
repeatedly precipitated into n-hexane (100 mL). The copolymer was then dried in high
vacuum overnight to yield the deprotected copolymers 9a-d.
1H-NMR (250 MHz, CDCl3, δ): 8.63 (br. s., 1 H), 4.01 (br. s., 1 H), 2.92 (br. s., 7 H), 2.20 (s,
1 H), 1.99 (br. s., 4 H), 1.66 (br. s., 3 H), 1.28 (s, 2 H), 0.95 ppm (br. s., 3 H). The 1H-NMR
spectra of the deprotected polymers 9a-d with 50 mol% diitaconate are shown in Figure S29
1
of the Supporting Information. For the other molar ratios, the position of the peaks in the H-
NMR spectra were identical, only the peak ratios differ. The chemical shifts of the peaks in
1
the H-NMR spectra of copolymer 8c with different molecular weight were identical to
copolymer 8c which is shown in Figure S29.
Determination of the r-parameters
To determine the r-parameters, the itaconate monomers were copolymerized under nitrogen
atmosphere in DMSO-d6, so that the crude reaction mixture could be directly used to
determine the monomer conversion by 1H-NMR spectroscopy. DMF was added as a standard
to determine the conversion of the comonomers at different time points. The different
amounts of reagents used for these polymerizations can be found in Tables S2 and S3 in the
- 29 -
This article is protected by copyright. All rights reserved.