Conjugation of ciprofloxacin with poly(2-oxazoline)s and polyethylene glycol via end groups

Article abstract of DOI:10.1021/acs.bioconjchem.5b00393

The antibiotic ciprofloxacin (CIP) was covalently attached to the chain end of poly(2-methyloxazoline) (PMOx), poly(2-ethyloxazoline) (PEtOx), and polyethylene glycol (PEG), and the antimicrobial activity of these conjugates was tested for Staphylococcus

Full text of DOI:10.1021/acs.bioconjchem.5b00393

Article
pubs.acs.org/bc
Conjugation of Ciprofloxacin with Poly(2-oxazoline)s and
Polyethylene Glycol via End Groups
†
†
†
†
‡
‡
Martin Schmidt, Simon Harmuth, Eva Rebecca Barth, Elena Wurm, Rita Fobbe, Albert Sickmann,
†
,†
Christian Krumm, and Joerg C. Tiller*
†Biomaterials and Polymer Science, Department of Bio- and Chemical Engineering, TU Dortmund, Emil-Figge-Straße 66, 44227
Dortmund, Germany
‡
̈
Leibniz-Institut fur Analytische Wissenschaften-ISAS-e.V., Otto Hahn-Straße 6b, 44227 Dortmund, Germany
*
S Supporting Information
ABSTRACT: The antibiotic ciprofloxacin (CIP) was covalently
attached to the chain end of poly(2-methyloxazoline) (PMOx),
poly(2-ethyloxazoline) (PEtOx), and polyethylene glycol (PEG),
and the antimicrobial activity of these conjugates was tested for
Staphylococcus aureus, Streptococcus mutans, Escherichia coli,
Pseudomonas aeruginosa, and Kleisella pneumoniae. Chemical
1
structures of the conjugates were proven by H NMR and
electron spray ionization mass spectrometry. The direct coupling
of PMOx and CIP resulted in low antimicrobial activity. The
coupling via a spacer afforded molecular weight dependent
activity with a molar minimal inhibitory concentration that is even
higher than that of the pristine CIP. The antimicrobial activity of
the conjugates increases in the order of PMOx < PEtOx < PEG.
Conjugation of CIP and a quaternary ammonium compound via PMOx did not result in higher activity, indicating no satellite
group or synergistic effect of the different biocidal end groups.
INTRODUCTION
which are designed without the intention of releasing antibiotics
is another successful concept in drug design but less often used.
Overall, PACs are mostly used for detoxification of anticancer
■
In times of rapid spread of bacterial infections, there is a growing
demand for new antibiotics because the abusive use of antibiotics
afforded the development of numerous resistant bacterial strains.
The latter are becoming a great problem of modern medicine.
Although, there are many developments in finding new
antibiotics, the number of such drugs that reach the market is
21−23
therapeutics such as doxorubicin,
but surprisingly, there are
only few examples of permanently bound polymer antibiotic
conjugates, which focus on the treatment of bacterial
24
infections. Polyethylene glycol (PEG) is commonly used for
conjugation of drug conjugates. Nathan et al. coupled penicillin V
and hydrophilic cephradine to PEG-lysine polyurethane via two
different strategies. Penicillin V was introduced by a hydrolyzable
ester group to the polymer, and cephradine was bound by a
hydrolytically stable amid bond. Whereas the first example is a
nonpermanent PAC, which showed activity after the release of
penicillin V, the second permanently bound PAC showed no
1
,2
rather limited with approximately one new antibiotic per year.
This is partially due to exploding costs for drug development and
approval. Formulation and derivatization of existing antibiotics is
a promising alternative to shorten the time of development for
new antimicrobial agents. Most often, the functional groups of
such drugs are modified to change charge density, solubility,
3
,4
degradability, selectivity, and efficiency.
19
antimicrobial activity at all. PEG was also used for the
conjugation of hydrophilic tobramycin by end group coupling via
amid bonds. Du et al. also reported that the prepared PACs show
a lower antimicrobial activity in solution against Pseudomonas
aeruginosa (P.a.) than free tobramycin. The more remarkable
result was achieved when the PACs were tested against P.a.
biofilms. In this case, the PAC shows a 3.2 fold higher activity
against the resilient biofilms than tobramycin, which indicates the
Moreover, formulations based on macromolecules such as
nanoparticles, liposomes, or molecular micelles are mostly
5
−7
optimized to change bioavailability.
Another very successful
way to prepare efficient derivatives of antibiotics is their
combination with polymers. Macromolecules as carriers for
therapeutics become increasingly important alternatives because
such polymer antibiotic conjugates (PACs) afford lower toxicity,
8
,9
increase solubility, and prolonged activity. In 2013, two of the
25
potential of such permanently bound PACs. Lawson et al.
top 10 selling drugs in the United States were polymer
1
0
therapeutics. In most cases, polymer PACs are used as
temporary antibiotic-carriers, for instance, by using degradable
Received: July 15, 2015
Revised: August 17, 2015
1
1−13
polymer matrices
or hydrolyzable binding to nondegradable
Preparing polymer antibiotic conjugates
1
4−20
polymer matrices.
©
XXXX American Chemical Society
A
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prepared telechelic nondegradable conjugates of PEG and
vancomycin. These conjugates possess an acrylate functionality
as the second terminal end for further surface modification. In
solution, the PEG−PACs revealed 6−75 fold decrease in biocidal
2
6
activity against Staphylococcus epidermidis. After surface
modification with these PACs, the surfaces revealed a 7−8 log
reduction in bacterial colony forming units, which is exceptional
27,28
for an antimicrobial surface.
Turos et al. published a study in
which the covalent conjugation of N-thiolated β-lactam antibiotic
with polyacrylates toward nanoparticles was performed. In all
cases, the nanoparticles show better activity than the antibiotic
containing monomers even with no difference to methicillin-
Figure 1. Ciprofloxacin. The carboxylic acid group is in red and the
piperazine group in green.
29
1
resistant Staphylococcus aureus (MRSA). An innovative concept
for the application of PACs was investigated by the group of
Rimmer. Polymyxin B (PMX) was covalently attached to a highly
branched poly(N-isopropylacrylamid) (PNIPAM). Therefore,
the biocidally active functional group of PMX was removed to
introduce a potential binding site for PNIPAM. The resulting
conjugate is able to precipitate P.a. by binding of the cyclic
peptide of PMX to the lipopolysaccharide in the membrane of
P.a. The caused aggregation decreased the lower critical solution
temperature of PNIPAM below 37 °C, which leads to
precipitation of this aggregates in water solutions. This
represents a very smart method for the detection of Gram-
group of CIP (compare Scheme 1 to the H NMR spectrum in
Figure S3).
The deprotonated carboxylic acid group was used for reaction
with the living end of PMOx. After cleaving the protecting group
with TFA, Me-PMOx-O-CIP was obtained. The structure of the
1
polymer was characterized by H NMR spectroscopy (Figures
S4−S5) and ESI-MS before and after deprotection. The found
m/z values correlate with the expected masses of the polymer
Me-PMOx-O-CIP-Boc and Me-PMOx-O-CIP (Figure 2).
Additionally, a PAC was prepared by modification of the
piperazine group with PMOx. For this purpose, the carboxylic
acid group of CIP was protected by an ethyl ester group,
introduced by esterification with ethanol (Figure S6). This
derivative reacted with the living end of PMOx. It was proven by
30
negative bacteria in aqueous solution. In general, permanent
PACs are a promising way to improve antibiotic performance,
but more examples are needed to truly judge this.
1
H NMR spectroscopy that 70% of the polymer chains are
Poly(2-oxazoline)s (POx) are nontoxic polymers with adjust-
functionalized with CIP (Figure S7). The protecting group was
cleaved by alkaline hydrolysis to get Me-PMOx-N-CIP (Figure
S8).
3
1−33
able hydrophilicity and readily functionalizable end groups.
They are considered as an alternative to PEG.
34,35
So far, POx
have not been investigated as PAC. However, end group
conjugation with quaternary ammonium compounds is a well-
established way of preparing biocidal polymers; sometimes, such
polymeric biocides even surpass the molar activity of their low
In order to introduce a spacer between PMOx and CIP, a
polymer-analogue implementation was developed. The strategy
for the modification of PMOx is pictured in Scheme 2. At first the
active polymer chains were functionalized with ethylene diamine
3
6
40
molecular weight pendants. This is achieved by the
introduction of a second nonbiocidal end group, which is able
(
Figure S9). In a second step, an amino-reactive derivate of CIP
41,42
was synthesized after a protocol of Kerns.
To this end, CIP
37,38
to enhance or to weaken the antimicrobial efficiency.
This
was coupled with α,α′-dichloro-p-xylene and obtained with 49%
1
second end group is called satellite group, and in our latest
investigations, we could show that this satellite group can be
chemically or enzymatically altered for switching the antimicro-
bial activity of the macromolecules over several orders of
yield and high purity (>98%) confirmed by H NMR
spectroscopy (Figure S10).
As seen in Figure 3, all signals in the H NMR spectrum can be
1
assigned to the expected polymer structure, indicating that
polymer chains were successfully functionalized with the CIP-
3
9
magnitude. Therefore, the conjugation of poly(2-oxazoline)s
with antibiotics as functional end groups might be a suitable
concept for preparing new biocidally active poly(2-oxazoline)s
PACs. In the present studies, ciprofloxacin (CIP)-PMOx PACS
are synthesized and explored regarding their antimicrobial
activity and hemotoxicity.
1
derivative. The analysis of the obtained H NMR spectrum
results in a degree of polymerization of 32 suggesting quantitative
conversion.
The ESI-MS of the derivative is shown in Figure 4. The found
degree of polymerization of 30 matches that determined in the
1
H NMR. Eighty-six percent of all signals in the ESI-MS could be
RESULTS
■
assigned to macromolecules with the methyl starter and the CIP
end group, respectively. Nine percent of all polymer molecules
were attributed to proton initiated polymers. This is due to chain
transfer reactions that occur during the polymerization caused by
CIP contains two suitable functional groups, the carboxylic acid
and the piperazine group, that might be used in the termination-
step of the cationic ring-opening polymerization of 2-R-2-
oxazolines or other coupling reactions (see Figure 1).
37,43
β-H-cleavage.
Previous works have shown that poly(2-oxazoline)s equipped
First, CIP was reacted with the living cationic polymer chains
1
36,39
of poly(2-methyloxazoline) (PMOx). It was found by H NMR
with antimicrobial end groups,
which were derived from low
spectroscopy and electron ionization mass spectrometry (ESI-
MS) that 80% of the polymer chains carry a CIP (Figure S1,
Supporting Information). H NMR spectroscopy reveals that
molecular biocides such as DTAC show a great potential with
regard to their antimicrobial activity and selectivity. By
modification of these biocidal functionalized polymers with an
antibiotic end group, both antibiotics and biocides would be
combined in a single macromolecule.
1
1
3% of the polymer chains are conjugated via the carboxyl group
of CIP. The other 87% of the PMOx chains are coupled to CIP
via the piperazine ring (Figure S2).
37
The previously described polymer DDA-X-PMOX -EDA
3
0
1
In order to obtain CIP-PACs with more uniform structures,
tert-butyloxycarbonyl (Boc) was chosen to protect the piperazine
( H NMR, Figure S11) was used as precursor for coupling with
CIP as depicted in Scheme 3.
B
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Scheme 1. Synthesis of Me-PMOx−CIP with the Direct Termination Strategy
Figure 2. ESI-MS of polymer Me-PMOx-O-CIP-Boc and Me-PMOx-O-CIP.
1
DDA-X-PMOX -EDA-CIP was characterized by H NMR
species with the CIP-end group (Figure 6). More than 85% of
these polymers contain a DDA-X at the other end. Fourteen
percent of the polymers chains were initiated by protons. This is
similar to the above-described results found for Me-PMOx-EDA-
CIP. The analytical data of all synthesized polymers including the
varied molecular weight species are given in Table 1 and Table 2
30
spectroscopy and ESI-MS. The initiating DDA-X and terminat-
ing CIP-functionalities can be clearly assigned to the signals in
the obtained H NMR spectrum and the degree of functionality
1
could be determined to 99% (see Figure 5).
According to this high degree of modification, more than 99%
of the masses obtained by ESI-MS measurements represent
1
( H NMR spectrum, Figure S12−S16).
C
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Scheme 2. Synthesis of Me-PMOx-EDA, the CIP-Spacer, and the Polymer Analogue Implementation of the CIP-Spacer to Me-
PMOx-EDA-CIP
1
Figure 3. H NMR of Me-PMOx-EDA-CIP in CDCl .
3
1
Additionally, 2-ethyl-2-oxazoline was used for further variation
of the polymer backbone. For this purpose, methyl p-
toluenesulfonate (MeTos) was used as initiating agent, and the
living polymer chains were functionalized with EDA (Figure
S17) and reacted with the CIP-spacer as described above. The
analytical data and the H NMR spectrum of this poly(2-
ethyloxazoline) (Me-PEtOx-EDA-CIP) is given in Figure S18.
1
The H NMR spectrum suggests a degree of modification of
93%, which is similar to the respective PMOx-CIP conjugate.
Further, polyethylene glycol (PEG) was conjugated with CIP.
D
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Article
Figure 4. ESI-MS of Me-PMOx-EDA-CIP.
Scheme 3. Synthesis of DDA-X-PMOx-EDA-CIP
Therefore, PEG was equipped with an EDA end group. This was
achieved by mesylation of the OH end group of PEG and a
subsequent nucleophilic substitution reaction with EDA. This
terminal amino group offers the possibility to use the same
coupling route already applied for the POx derivatives (Scheme
the concentration of an antimicrobial active compound at which
99% of bacteria are inhibited in growth. Direct termination of
1
CIP with PMOx results in a MIC
value of 1250 μg·mL−
S.aureus
−1
(390 μmol·L ). This low activity seems to indicate that
conjugation with PMOx drastically lowers the activity of the
1
1
−1
−1
4
; H NMR spectrum, Figure S19−S21). The H NMR spectrum
antibiotic CIP (MIC
= 0.5 μg·mL (1.29 μmol·L )). As
S.aureus
1
reveals 90% conversion with respect to CIP.
shown by H NMR spectroscopy, the termination occurs at the
carboxylic as well as at the secondary amine group of CIP. It is
known from literature that low molecular derivatives of CIP
The minimal inhibitory concentrations (MIC) of the
synthesized PACs were investigated. This concentration displays
E
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1
Figure 5. H NMR messurement of DDA-X-PMOx-EDA-CIP in CDCl .
3
exhibit reduced biological activity when modified at the
The comparison of the two PACs Me-PMOx -EDA-CIP
30
4
4,45
carboxylic group,
while modification of the piperazine ring
(Figure 8a) and Me-PMOx -EDA-CIP (Figure 8b) reveals the
10
results in an antibacterial activity on the same level as the
unmodified fluoroquinone.
same trend for the S. mutans and for S. aureus. The antimicrobial
4
molar activity Me-PMOx -EDA-CIP is 3 times higher than that
10
−1
The derivative PMOxylated at the carboxylic group (Me-
of the pristine antibiotic (MICS.mutans = 0.3 μmol·L ). A different
picture is found for the Gram negative bacterial strains. In all
cases, the conjugates show a drastically lower activity than CIP
against the respective bacterial strain. While the conjugates show
a very good antimicrobial activity against E. coli after an
PMOx-O-CIP) exhibits a MIC
value of 625 μg·mL− (189
1
S.aureus
−1
μmol·L ), while the respective conjugate modified at the amine
group (Me-PMOx-N-CIP) shows MIC
1250 μg·mL−
1
S.aureus
−1
(
260 μmol·L ). These results show that direct conjugation of
47
CIP with PMOx is in contrast to the literature for low molecular
weight groups not dependent on the modified functional group.
Nevertheless, conjugation drastically lowers the antimicrobial
activity of the antibiotic CIP. The biological activity of CIP is the
evaluation standard of Fortuniak et al., the conjugates are
only moderately active against P. aeruginosa and K. pneumoniae.
In order to judge the influence of the nature of the polymer
backbone on the activity of the CIP-PACs, conjugates with
poly(2-ethyloxazoline) and poly(ethylene glycol) were prepared
accordingly. The MIC values of these conjugates shown in Figure
8c−d are generally lower than those of the respective Me-
PMOx -EDA-CIP but show a similar trend for the different
46
inhibition of the intracellular enzyme gyrase. The reduced
activity of the PMOx-CIP conjugates could be caused by a
reduced affinity to the enzyme or lowered diffusion ability into
the bacterial cell. Thus, alternative PMOx-CIP conjugates that
have a spacer between the antibiotic and the polymer were
synthesized (Me-PMOx-EDA-CIP, see Scheme 2). Several
conjugates with different PMOx lengths were prepared and
tested against S. aureus. As seen in Figure 7, the Me-PMOx-EDA-
CIP conjugates exhibit a strong antimicrobial activity against this
microorganism. The molar activity is linearly increasing with
shorter PMOx chain lengths. The molar MICS.aureus value of Me-
PMOx -EDA-CIP is even 5 times lower than that of CIP. This
30
bacterial strains. Thus, the conjugates exhibit excellent activity
against S. aureus, S. mutans, and E. coli. The very good activity of
Me-PEG -EDA-CIP against K. pneumoniae is rather surprising.
45
Altogether, the data clearly show that the nature of the polymer
backbone strongly influences the antimicrobial activity of the
CIP conjugates. Generally, the activity decreases in the order
PEG > PEtOx > PMOx. The greatest influence was found for K.
pneumoniae. The MICK.pneumoniae is 143 μmol·L for Me-PMOx30-
EDA-CIP, 37 μmol·L for Me-PEtOx -EDA-CIP, and 4.47 μmol·
10
shows that conjugation of CIP with PMOx via a spacer leads to
highly active PACs.
30
L for Me-PEG -EDA-CIP.
45
The PACs with PMOx10 and PMOx30 were tested against 4
further clinically relevant ubiquitous bacterial strains, being the
Gram positive, caries causing strain, Streptococcus mutans, and the
three Gram negative bacterial strains Escherichia coli, Pseudomo-
nas aeruginosa, and Klebsiella pneumoniae.
We hypothesize that the reason for the higher molar
antimicrobial activity of the CIP conjugates compared to pristine
CIP is a higher binding constant for bacterial gyrase. Therefore,
the attached polymer chain might additionally hinder ATP
binding to the enzyme.
F
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Figure 6. ESI-MS of DDA-X-PMOx-EDA-CIP.
1
Table 1. Analytic Data of the PACs, Characterized by H NMR Spectroscopy and ESI-MS
a
b
[g·mol−1
Fd
c
Mn,ESI [g·mol−1
Fd
ESI
d
e
polymer
DPset
DPNMR
M ,
n NMR
]
DPesi
]
PDIESI
NMR
Me-PMOx -EDA-CIP
30
30
30
30
30
32
43
36
25
46
3200
> 99%
> 99%
86%
31
31
35
35
34
3100
3400
3200
3400
3300
80%
85%
78%
75%
54%
1.03
1.03
1.03
1.02
1.06
30
DDA-X-PMOx -EDA-CIP
4300
3400
2600
4341
30
Me-PMOx -CIP
30
Me-PMOx -CIP-Boc
54%
30
Me-PMOx -O-CIP
90%
30
a
Degree of polymerization set by the ratio of monomer to initiator according to [initiator] = [monomer]·DPset−1. bDegree of polymerization
1
determined by H NMR spectroscopy via comparison of the respective signals caused by the initiating group and the signals caused by the protons of
the polymer backbone. Degree of functionalization determined by H NMR spectroscopy via comparison of the respective signals caused by the
initiating and the terminal CIP groups. Degree of functionalization determined by ESI-MS. The targeted molecular weights were rated by their
intensity with respect to the intensity sum of all obtained molecular weight peaks. Calculated from the data of the ESI-MS by rating all obtained
c
1
d
e
peaks with their corresponding intensity.
As shown previously, the group distal to the biocidal end
group, the satellite group, of a polymer biocide conjugate is
dramatically influencing the activity of the whole macro-
the distant sites of action in the bacterial cell. The only significant
influence was found for E. coli, where the DDA-X group increased
the activity of the conjugates by a factor of 3 to 16.
Additionally, the hemocompatibility of the prepared PACs was
explored. Therefore, the hemolytic concentration at which 50%
3
9
molecule. In order to investigate if this is also the case for
CIP PACs, the biocidal group DDA-X (compare Scheme 3) was
introduced as satellite group for the PMOx-CIP conjugate. The
MIC values of the DDA-X-PMOx-EDA-CIP diagrammed in
Figure 8e and f show that the DDA-X group does not strongly
influence the antimicrobial activity of the conjugates for most
bacterial strains. This might be due to the fact that CIP is an
enzyme inhibitor and that satellite groups are not influencing
those as previously shown in the example of polymeric
of the porcine red blood cells (HC ) are lysed was determined.
50
All synthesized PACs were found to have HC values above
50
1
5
000 μg·mL− indicating low hemocytotoxicity (Table S.1).
CONCLUSIONS
■
The goal of this work was to investigate permanently bound
polymer antibiotic conjugates of CIP and different hydrophilic
polymers. It could be shown that direct conjugation of CIP by
using the latter as termination reagent of the cationic ring
48
collagenase inhibitors. Further, there might not be a synergistic
effect of quaternary ammonium compounds and CIP because of
G
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Table 2. Analytical Data of PACs, Which Were Characterized
1
by H NMR Spectroscopy
M ,
[g·
]
n NMR
mol
a
b
−1
Fd
NMR
c
polymer
DPset DPNMR
Me-PMOx -CIP-OEt
30
30
10
10
20
40
60
30
45
53
52
12
10
22
39
58
34
46
4900
4800
1300
1600
2400
3800
5400
3800
2600
70%
54%
30
Me-PMOx -N-CIP
30
Me-PMOx -EDA-CIP
>99%
87%
10
DDA-X-PMOx -EDA-CIP
10
Me-PMOx -EDA-CIP
>99%
>99%
87%
20
Me-PMOx -EDA-CIP
40
Me-PMOx -EDA-CIP
60
Me-PEtOx -EDA-CIP
93%
30
Me-PEG -EDA-CIP
90%
45
a
Figure 7. Degree of polymerization against MICS.aureus value [μmol·L−1]
Degree of polymerization was set by the ratio of monomer to initiator
−1
b
according to [initiator] = [monomer]·DP
ization determined by H NMR spectroscopy via comparison of the
respective signals caused by the initiating group and the signals caused
by the protons of the polymer backbone. Degree of functionalization
determined by H NMR spectroscopy via comparison of the respective
.
Degree of polymer-
for Me-PMOx-EDA-CIP.
set
1
length of the macromolecules play a crucial role. So far, a satellite
group effect could not be found when using CIP. Future work
will be dedicated to further optimize this system regarding a more
suited spacer and exploring the properties of the conjugates with
respect to biodegradability, blood plasma half life, and building
up of bacterial resistances. Additional antibiotics will be
conjugated as well.
c
1
signals caused by the initiating and the terminal CIP groups.
opening polymerization of 2-methy-2-oxazoline is successful but
leads to lowly antimicrobial conjugates. Functional group
selective termination of PMOx resulted in poorly active
conjugates as well, indicating that the conjugation of CIP with
hydrophilic polymers might not be a promising approach.
Conjugates of CIP and PMOx prepared via a spacer showed high
antimicrobial activity, which are dependent on the molecular
weight of the polymer, even exceeding the activity of pristine
CIP. Additionally prepared conjugates with poly(2-ethyloxazo-
line) (PEtOx) as well as PEG revealed a strong dependence of
the CIP conjugate activity increasing in the order PMOx <
PEtOx < PEG. The introduction of the biocidal satellite group
DDA-X did not strongly improve activity for most bacterial
strains.
EXPERIMENTAL PROCEDURES
■
Materials. All reactions, purifications, and polymerizations
were carried out under an inert atmosphere. Chloroform
(AppliChem) was distilled under reduced pressure from
aluminum oxide (Merck) and stored in a flask with 4 Å
molecular sieves. N,N-dimethylformamide (VWR) and acetoni-
trile (Merck) were distilled from diphosphorus pentoxide
(VWR), then from potassium carbonate (VWR) and stored
over 3 Å molecular sieves. The water content was determined by
Karl Fischer titration (<0.5 ppm). The monomers 2-methyl-2-
oxazoline and 2-ethyl-2-oxazoline (MOx, EtOx, Sigma-Aldrich)
It could be shown that permanent conjugation of hydrophilic
polymers and antibiotics via the polymer end group is a
promising approach toward antibiotics with different activity
patterns for various bacterial strains. Therefore, the coupling
strategy, including the use of spacers, as well as the nature and the
were distilled from CaH (ABCR). N,N-dimethyldodecylamine
2
(DDA, Sigma-Aldrich) and ethylene diamine (EDA, ABCR)
were distilled under reduced pressure. α,α′-Dibromo-p-xylene
(ACROS, TCI) was recrystallized in chloroform. Ciprofloxacin
(Alfa aesar, Sigma-Aldrich, TCI), α,α′-dichloro-p-xylene (TCI
Scheme 4. Synthesis Route Used for the Preparation of Me-PEG-EDA-CIP
H
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as full scan data in positive mode, by accumulation of 2 μscans in
the mass range m/z 225−2000 with a resolution of 100.000 (at
6
m/z 400), AGC target value 1 × 10 . In the range of m/z 600−
1
400, the obtained mass spectra contained charges up to 12.
Signals were deconvoluted using Xtract RAW. Polymer
distribution was determined based on the monoisotopic peaks.
The calculation of the theoretical molecular masses was
1
2
−1
performed by using the following isotopes: C = 12.000 g·mol ,
1
7
6
9
−1 14
−1 1
−1
O = 15.995 g·mol , N = 14.003 g·mol , H = 1.008 g·mol ,
−1
1
+
−1
19
Br = 78.918 g·mol , H = 1.0072766 g·mol , and F =
8.998 g·mol . The UV/vis measurements were recorded on a
−1
1
double-beam spectrophotometer Specord 210 of the Analytik
Jena company by 25 °C.
Syntheses. 4-(Bromomethyl)-N-dodecyl-N,N-dimethyl-
benzene Ammonium Bromide (DDA-X). A suspension of
α,α′-dibromo-p-xylene (5.00 g, 18.94 mmol, 1 equiv) in
chloroform (15 mL) was stirred, and a solution of N,N-
dimethyldodecylamine (DDA; 4.04 g, 18.94 mmol, 1 equiv) in
chloroform (7.5 mL) was added dropwise at 42 °C for 4 h. The
mixture was stirred for another 30 min at this temperature. The
solvent was concentrated to one-third of its initial volume. The
residue was stored at 0 °C for 6 h, and the unreacted α,α′-
dibromo-p-xylene was crystallized and filtered off. Chloroform
was removed in vacuum, and tetrahydrofuran (150 mL) was
added. The byproduct was precipitated and was removed by
filtration. The solution was concentrated in vacuum to give the
Figure 8. Molar MIC values for the different PACs (a−f), against S.
aureus (S.a.), S. mutans (S.m.), E. coli (E.c.), P. aeruginosa (P.a.), and K.
pneumoniae (K.p.) in comparison to the molar MIC value of CIP against
each strain (white columns). All measurements were performed in
triplicate, and the error bars are the standard deviation. (Compare to
Table 4 in Experimental Procedures).
1
highly viscous product (5.48 g, 13.78 mmol, 77%). H NMR
(
1
3
300 MHz, chloroform-d) δ = 0.69−0.95 (t, J = 6.8 Hz 3 H),
.06−1.46 (m, 18 H), 1.58−1.90 (m, 2 H), 3.28 (s, 6 H), 3.41−
.57 (m, 2 H), 4.57 (s, 2 H), 5.15 (s, 2 H), 7.43 (d, J = 8.42 Hz, 2
Europa), 2,3,5-triphenyltetrazolium chloride (AppliChem),
polyethylene glycol 2000 monomethyl ether (Fluka), mesyl
chloride (Sigma-Aldrich), sodium hydrogen carbonate (Merck),
sodium chloride (Fischer), sodium dihydrogen phosphate
dihydrate (Merck), sodium hydroxide (Merck), sodium citrate
H), 7.68 (d, J = 8.42 Hz, 2 H) ppm.
-(4-(4-(Chloromethyl)benzyl)piperazin-1-yl)-1-cycloprop-
yl-6-fluoro-4-oxo-1,4-dihydroquino-line-3-carboxylic Acid
7
(
CIP-Spacer). α,α′-Dichloro-p-xylene (875 mg, 5.00 mmol, 5
(
Sigma-Aldrich), citric acid monohydrate (Sigma-Aldrich),
glucose monohydrate (Sigma-Aldrich), hydrochloric acid
VWR), acetic acid (VWR), formic acid (Merck), and nutrient
equiv) and NaHCO (168 mg, 2 mmol, 2 equiv) were dissolved
3
in a mixture of N,N-dimethylformamide and acetonitrile (1:1, 4
mL). Ciprofloxacin (331 mg, 1.00 mmol, 1 equiv) was added to
the solution at room temperature. The reaction mixture was
(
broth (ISO, APHA, VWR) were used without further
purifications. The germs Escherichia coli (Gram-negative,
ATCC 25922), Klebsiella pneumoniae (Gram-negative, ATCC
stirred at 80 °C for 24 h. The product was precipitated in Et O,
2
the supernatant was decanted, and the solid residue was purified
1
1
3883), Pseudomonas aeruginosa (Gram-negative, ATCC
7423), Staphylococcus aureus (Gram-positive, ATCC 25323),
by column chromatography (SiO2; CH Cl /MeOH = 20:1) to
2
2
get the product as slightly yellow solid (230 mg, 0.489 mmol,
and Streptococcus mutans (Gram-positive, ATCC 25175) were
4
9%). R = 0.48.
f
provided by the German Resource Center for Biological Material
1
H NMR (300 MHz, chloroform-d) δ = 1.15−1.25 (m, 2 H),
.32−1.44 (m, 2 H), 2.69 (t, J = 4.80 Hz, 4 H), 3.36 (t, J = 4.80
(
DSMZ). Fresh porcine blood was provided by a local butcher
1
shop and immediately implemented to erythrocyte concentrates.
1
Hz, 4 H), 3.52 (td, J = 6.77, 2.93 Hz, 1 H), 3.61 (s, 2 H), 4.61 (s, 2
H), 7.31−7.42 (m, 5 H), 8.02 (d, J = 13.17 Hz, 1 H) 8.77 (s, 1 H)
ppm.
Measurements. H NMR spectra were recorded in
deuterated solvents (CDCl , DMSO-d ) using FT-appliances
3
6
of Bruker, types DPX-300 (300 MHz), DRX-400 (400 MHz),
DRX-500 (500 MHz), or FT-appliances of Varian type Inova 500
7-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-1-cyclopropyl-6-
fluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylic Acid (CIP-
Boc). To a suspension of ciprofloxacin (4.00 g, 12.08 mmol, 1
equiv) in 140 mL of dry dichloromethane, triethylamine (5.02
mL, 36.24 mmol, 3.00 equiv) and di-tert-butyl dicarbonate (3.10
mL, 14.50 mmol, 1.20 equiv) were added at room temperature
and stirred for 12 h. After cooling to −5 °C, the suspension was
filtered. The collected precipitate was dissolved in 200 mL of
dichloromethane and washed with an aqueous solution of acetic
acid (0.3 M, 100 mL). The solution was dried with sodium
sulfate, filtered, and concentrated in vacuum to get the crude
product. The product was purified by recrystallization in a
mixture of dichloromethane and hexane (300:100 mL) to get
CIP-Boc as a yellow solid (1.031 g, 2.39 mmol, 20%).
(
500 MHz). The residual protons of the not fully deuterated
solvents served as an internal standard.
The mass spectrometry measurements were performed on a
Thermo LTQ-FT-ICR-Ultra mass spectrometer (linear iron
trap−Fourier transform ion cyclotron resonance mass spec-
trometer). The polymer samples were dissolved in distilled water
with 30 vol % acetonitrile and 0.1 vol % formic acid. The polymer
samples were measured in a concentration of 10 pmol·μL . For
direct infusion, a 5 μL sample was injected using a Chip-A-384
emitter, (with 5 μm i.d.), resulting in a flow rate of ∼200 nL
min . Nominal nitrogen back pressure and ESI voltage were
adapted to the respective analyte, varying between 0.4 and 1.4 psi
and 1.8 kV, respectively. The profile mass spectra were obtained
−1
−1
I
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Bioconjugate Chemistry
Article
1
H NMR (400 MHz, chloroform-d) δ = 1.19−1.22 (m, 2H),
power was reduced to a basis level maintaining the target
temperature, and the vessels were cooled by compressed air
shocks within the next 2 to 4 min to maintain at the adjusted
temperature. The corresponding reaction times varied with the
DPset between 1 and 5.5 h.
1
1
7
.38−1.42 (m, 2H), 1.50 (s, 9H), 3.29−3.31 (m, 4H), 3.55 (tt,
H, J = 7.1 Hz, J = 3.3 Hz), 3.66−3.67 (m, 4H), 7.35 (d, 1H, J =
.1 Hz), 7.95 (d, 1H, J = 12.9 Hz), 8.70 (s, 1H).
Ethyl 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylate (CIP-OEt). To an ice-cooled
suspension of ciprofloxacin (2.00 g, 6.04 mmol, 1 equiv) and 50
mL of ethanol, thionyl chloride (8.76 mL, 120.80 mmol, 20
equiv) was added dropwise. After 15 h under reflux, the solution
was concentrated under reduced pressure. The resulting solid
was dissolved in an aqueous solution of potassium carbonate and
extracted with dichloromethane three times. The collected
organic phases were dried with sodium sulfate, and the solvent
was removed under reduced pressure. The crude product was
recrystallized in acetonitrile to get CIP-OEt as a white solid (1.41
g, 3.92 mmol, 65%).
Termination with Ethylene Diamine. A 10 fold molar excess
(based on the initiator molarity) of ethylene diamine was added
to the living polymer chains and heated to 45 °C for 72 h.
Termination with CIP or CIP-Derivatives. For functionaliza-
tion with CIP, a 10 fold molar excess (based on the initiator
amount) was used, and the mixture was heated to 70 °C for 48 h.
Purification. The raw polymeric product was precipitated in
diethyl ether and dialyzed against distilled water for 24 h using
benzoylated cellulose membranes (1000 MWCO). The water
was removed by lyophilization, and purified polymers were
obtained in yields of 69%−93%.
1
H NMR (400 MHz, chloroform-d) δ = 1.12−1.16 (m, 2H),
Purifications of Direct CIP Terminated Polymers. After
precipitation of the polymers in diethyl ether, they were
suspended in water and stirred for 12 h. Then, the suspension
was filtered with a 0.2 μm syringe filter, and the filtrate was
1
4
(
.32 (q, 2H, J = 6.4 Hz), 1.41 (t, 3H, J = 7.0 Hz), 3.09−3.11 (m,
H), 3.23−3.26 (m, 4H), 3.43 (dt, 1H, J = 7.0, J = 3.3 Hz), 4.39
dt, 2H, J = 7.2 Hz), 7.26−7.28 (m, 1H), 8.04 (d, 1H, J = 13.3
Hz), 8.54 (s, 1H) ppm.
lyophilized to get the purified polymer in yields of 33%−97%.
1
Methoxy-poly(ethylene glycol)-mesylate (Me-PEG-OMs).
Polyethylene glycol 2000 monomethyl ether (4.00 g, 2.00
mmol, 1.00 equiv) was dried under reduced pressure at 100 °C
for 2 h. The dry polymer was dissolved in 100 mL of
dichloromethane, and triethylamine (2.77 mL, 20.00 mmol,
DDA-X-PMOx30-EDA: H NMR (500 MHz, DMSO-d
) δ =
6
0.72−0.93 (m, 3 H), 1.25 (br. s., 18 H), 1.67−2.16 (m, 117 H,)
2.51−2.72 (m, 6 H), 2.95 (br. s., 6 H), 3.08−3.71 (m, 165 H),
4.37−4.74 (m, 4 H), 7.04−7.84 (m, 4 H) ppm.
1
Me-PMOx30-EDA: H NMR (500 MHz, chloroform-d) δ =
10.0 equiv) was added. The reaction mixture was cooled to 0 °C,
1.74−2.20 (m, 81 H), 2.66−2.89 (m, 4 H), 2.91−3.02 (m, 3H),
and mesyl chloride (1.48 mL, 20.00 mmol, 10.0 equiv) was
slowly added. After stirring for 24 h at room temperature, the
solvent was removed. The residue was dissolved in water and
extracted with dichloromethane (3 × 50 mL). The collected
organic phases were precipitated in diethyl ether. The
precipitating step was performed three times to get Me-PEG-
OMs as a white solid (2.39 g, 1.15 mmol, 58%).
3.10−3.72 (m, 112 H) ppm.
1
Me-PMOx30-CIP: H NMR (400 MHz, chloroform-d) δ =
1.37 (d, J = 6.53 Hz, 3 H) 1.90−2.34 (m, 110 H) 2.96−3.07 (m, 3
H) 3.20−3.62 (m, 141 H) 7.11 (d, J = 7.78 Hz, 1 H) 7.68 (d, J =
8
.03 Hz, 1 H) 8.71 (s, 1 H) ppm.
1
Me-PMOx -CIP-Boc: H NMR (400 MHz, chloroform-d) δ
30
=
1.11 (br. s., 2 H) 1.44 (s, 4 H) 1.82−2.36 (m, 75 H) 2.67 (br. s.,
1
H NMR (500 MHz, chloroform-d) δ = 3.04 (s, 3 H) 3.33 (s, 3
H) 3.42−3.78 (m, 168 H) 4.30−4.36 (m, 2 H) ppm.
Table 3. Used Bacteria Strains and Their Incubation
Conditions
Methoxy-poly(ethylene glycol)-ethylene Diamine (Me-PEG-
EDA). Me-PEG-OMs (208 mg, 0.10 mmol, 1.00 equiv) was
dissolved in 5 mL of chloroform. Ethylene diamine (66.66 μL,
Gram
bacteria strain
DSM
1103
ATCC
25922
straining
incubation conditions
1.00 mmol, 10.0 equiv) was added to the stirred solution, and the
Escherichia coli
negative
nutrient broth, pH 6.8,
37 °C
reaction mixture was stirred for 48 h at 78 °C. The polymeric raw
product was precipitated in diethyl ether and dialyzed against
distilled water for 24 h using benzoylated cellulose membranes
Klebsiella
30104
50078
1104
13883
17423
25923
24178
negative
negative
positive
positive
nutrient broth, pH 7.0,
pneumoniae
37 °C
Pseudomonas
aeruginosa
nutrient broth, pH 7.0,
30 °C
nutrient broth, pH 7.3,
37 °C
nutrient broth, pH 7.3,
37 °C
(1000 MWCO). The water was removed by lyophilization, and
purified Me-PEG-EDA was obtained in a yield of 85% (170 mg,
Staphylococcus
aureus
0
3
3
.083 mmol, 83%).
H NMR (500 MHz, chloroform-d) δ = 2.43−2.98 (m, 6 H)
.36 (s, 3 H) 3.62 (s, 182 H) ppm.
General Procedure for the Polymerization of Polymers with
0 Repeating Units. The required concentration of the initiator
1
Streptococcus
mutans
20523
2 H) 2.97−3.10 (m, 3 H) 3.14−3.78 (m, 102 H) 4.31−4.44 (m, 1
H) 7.11 (d, J = 8.03 Hz, 1 H) 7.67 (d, J = 8.03 Hz, 1 H) 8.43−8.57
(DDA-X or methyl tosylate) was calculated with the start-
concentration of monomer and the desired degree of polymer-
ization DPset according to [initiator] = [monomer]·DPset−1. The
initiator (DDA-X; 0.149 g or methyl tosylate; 0.059 g, 0.375
mmol, 1 equiv) was dissolved in chloroform (5 mL) at room
temperature, and the monomer was added (2-methyl-2-oxazo-
line; 1.00 g or 2-ethyl-2-oxazoline; 1.11 g, 11.25 mmol, 30 equiv)
to the solution. The polymerization was conducted in a CEM
Discover synthesis microwave. The reaction temperature was
monitored with a vertically focused IR temperature sensor. The
vessels were heated (110 °C for DDA-X and 90 °C for methyl
tosylate) using the maximum available power (300 W) under
magnetic stirring. After reaching the desired temperature, the
(m, 1 H) ppm.
1
Me-PMOx -CIP-OEt: H NMR (400 MHz, chloroform-d) δ
30
= 1.04−1.31 (m, 3 H) 1.34 (t, J = 7.03 Hz, 2 H) 1.81−2.29 (m,
162 H) 2.95−3.04 (m, 3 H) 3.13−3.61 (m, 200 H) 4.25−4.36
(m, 1 H) 7.06−7.13 (m, 1 H) 7.56−7.68 (m, 1 H) 8.48 (s, 1 H)
ppm.
1
Me-PEtOx -EDA: H NMR (300 MHz, chloroform-d) δ =
30
1.11 (br. s., 94 H) 2.14−2.49 (m, 61 H) 2.59 (br. s., 6 H) 3.02 (s,
3 H) 3.28−3.78 (m, 120 H) ppm.
General Procedure of CIP-Linking. Ciprofloxacin-derivate
(93.8 mg, 2.00 mmol, 2 equiv) and NaHCO (16.8 mg, 2.00
3
mmol, 2 equiv) were suspended in a mixture of N,N-
J
DOI: 10.1021/acs.bioconjchem.5b00393
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Article
Table 4. MIC Tests in μmol·L− against Different Germs, Two Gram-Positive Strains (S. aureus and S. mutans) and Three Gram-
1
a
Negative Strains (E. coli, P. aeruginosa, and K. pneumonia)
MIC [μmol·L−1]
samples
S.a.
S.m.
E.c.
P.a.
K.p.
Me-PMOx -EDA-CIP
0.2 ± 0.1
2.2 ± 0.2
0.7 ± 0.3
1.7 ± 0.6
0.4 ± 0.1
0.2 ± 0.1
1.3 ± 0.1
1.2 ± 0.1
0.3 ± 0.02
0.9 ± 0.2
0.2 ± 0.05
2.6 ± 0.7
0.6 ± 0.2
0.6 ± 0.2
0.9 ± 0.01
4.3 ± 0.9
17 ± 2
78 ± 19
105 ± 5
41 ± 1.5
143 ± 6.1
39 ± 11.7
38 ±. 11.9
37 ± 5.9
4.5 ± 2
10
Me-PMOx -EDA-CIP
5.3 ± 1.5
1.0 ± 0.1
1.5 ± 0.1
2.4 ± 0.6
1.5 ± 0.4
0.1 ± 0.01
5.1 ± 2.4
30
DDA-X-PMOx -EDA-CIP
60 ± 2.5
96 ± 4.1
44 ± 2.5
35.0 ± 0.01
0.7 ± 0.2
10 ± 0.2
10
DDA-X-PMOx -EDA-CIP
30
Me-PEtOx-EDA-CIP
Me-PEG-EDA-CIP
CIP
0.6 ± 0.2
12 ± 5
CIP-spacer
aAdditional MIC values for all other synthesized polymers in μmol·L−1 and in μg·mL−1 are given in Tables S2−S3.
dimethylformamide and acetonitrile (1:1, 4 mL). The polymer
DDA-X-PMOx -EDA; 300 mg, Me-PMOx -EDA; 219 mg,
dried polymers were suspended in CHCl and filtered by a 0.45
3
(
μm PTFE filter and precipitated in Et O. The polymers were
30
30
2
Me-PEtOx x-PMOx -EDA; 305 mg, Me-PEG -EDA; 205 mg,
resolved in CHCl , and the solvent was evaporated.
30
30
45
3
1
0.10 mmol, 1 equiv) was added to the suspension at room
H NMR (400 MHz, chloroform-d) δ = 1.13−1.39 (m, 2 H)
2.01 (m, 163 H) 2.98 (m, 3 H) 3.39 (m, 204 H) 7.08 (m, 1 H)
7.59 (m, 1 H) 8.69 (s, 1 H) ppm.
temperature. The reaction mixture was stirred at 80 °C for 24 h.
The product was precipitated in Et O, and the supernatant was
2
decanted. The residue was dissolved in water, and polymers were
dialyzed in membranes of Roth (ZelluTrans) with a molecular
Bacterial Susceptibility: Minimal Inhibitory Concentration
(MIC). Stock solutions of the respective bacterial strains were
prepared from a stock pellet from DSMZ. The stock pellet of the
respective bacterial strain was suspended in 50 mL of nutrient
both (standard 1, 25 g in 1 L distilled water) and incubated under
the conditions given in Table 3 for 24 h. The confluent medium
was centrifuged (3000 rpm, 10 min), and the supernatant was
decanted. The residue was suspended in 50 mL of sterile PBS
buffer (8.77 g NaCl, 1.56 g NaH PO 2H O, adjusted to pH 7.0
with 0.1 M NaOH) and then centrifuged (3000 rpm, 10 min)
again. This step was repeated three times. In the last step, the
bacterial residue was suspended in 10 mL of sterile PBS buffer
and mixed with 10 mL of a sterile-filtered 50% glycerin-solution.
The stock solution was stored at −20 °C.
−1
cutoff of 2000 g·mmol . Polymers with a lower molecular
weight were dialyzed two times in membranes with a cutoff of
−
1
1
000 g·mmol . The water was removed by lyophilization, and
the purified polymers were obtained in yields of 45%−85%.
DDA-X-PMOx -EDA-CIP: H NMR (400 MHz, chloro-
1
30
form-d) δ = 0.80 (t, J = 6.78 Hz, 3 H), 0.99−1.25 (m, 22 H),
1
4
8
.65−2.28 (m, 128 H), 2.60 (br. s., 17 H), 3.00−3.76 (m, 206 H),
.39−5.39 (m, 6 H), 7.27 (s, 8 H), 7.56 (m, 1H), 7.85 (m, 1 H)
.56 (m, 1 H) ppm.
2
4
2
1
Me-PMOx -EDA-CIP: H NMR (400 MHz, chloroform-d) δ
30
=
1.18 (br. s., 2 H), 1.35 (br. s., 2 H), 1.70−2.30 (m, 79 H), 2.54−
2
.86 (m, 8 H), 3.01−3.05 (m, 2 H), 3.24−3.72 (m, 107 H),
7.23−7.33 (m, 6 H), 7.89−7.99 (m, 1 H) 8.65−8.74 (m, 1 H)
Fifty microliters of the respective stock solution was added to
25 mL of nutrient broth (standard 1). The solution was
incubated for 24 h (For conditions, see Table 3). The bacterial
concentration of the suspension was controlled by UV/vis-
spectroscopy at 541 nm/25 °C and diluted with nutrient broth to
ppm.
1
Me-PEtOx -EDA-CIP: H NMR (500 MHz, chloroform-d) δ
30
=
0.90−1.29 (m, 98 H) 1.36 (d, J = 6.50 Hz, 2 H) 2.07−2.47 (m,
7
0 H) 2.65 (br. s., 8 H) 2.96−3.09 (m, 3 H) 3.22−3.79 (m, 135
7
−1
H) 7.19−7.41 (m, 6 H) 7.98 (d, J = 13.00 Hz, 1 H) 8.73 (s, 1 H)
∼10 cells mL .
ppm.
A sample of the polymer (5.00 mg) was dissolved in pure
nutrient broth (4.00 mL), and a dilution series was prepared with
halved concentrations in every following sample. Every probe
was incubated with 20 μL of the diluted bacterial stock solution at
37 or 30 °C (see Table 3) for 24 h. Positive and negative controls
were incubated with the polymer samples. The MIC was visually
observed, and then 100 μL of a 2,3,5-triphenyltetrazolium
1
Me-PEG -EDA-CIP: H NMR (500 MHz, chloroform-d) δ =
45
1
3
7
.10−1.20 (m, 2 H) 1.32−1.41 (m, 2 H) 2.55−2.72 (m, 8 H)
.28−3.36 (m, 4 H) 3.37 (s, 3 H) 3.46−3.80 (m, 173 H) 7.29 (s,
H) 7.84−8.01 (m, 1 H) 8.65−8.75 (m, 1 H) ppm.
Deprotection of the Boc-Group. The protecting group was
cleaved of from Me-PMox -CIP-Boc (260 mg, 0.076 mmol, 1
3
0
−1
equiv) with a mixture of dichloromethane and trifluoroacetic acid
chloride (TCC)-solution in water (1 mg mL ) was added. After
a further 3 h of incubation, the samples with a bacterial cell
concentration >1% showed a red coloration. The MIC was the
sample with the lowest polymer concentration without red
coloration. The effective molar concentration of the CIP
conjugate was calculated from the average molecular weight
(TFA) (1:1 vol %, 4 mL). The raw polymeric product was
purified as described above. The raw polymeric product was
precipitated in diethyl ether and dialyzed against distilled water
for 24 h using benzoylated cellulose membranes (1000 MWCO).
The water was removed by lyophilization.
1
1
H NMR (400 MHz, chloroform-d) δ = 1.02−1.14 (m, 2 H)
determined by H NMR. The concentration of unmodified CIP
1
(
(
.30 (br. s., 2 H) 1.75−2.27 (m, 135 H) 2.95−3.08 (m, 3 H) 3.41
was less than 0.05 mol % in all cases according to the respective
ESI MS.
m, 168 H) 4.28−4.46 (m, 1 H) 7.11 (d, J = 7.78 Hz, 1 H) 7.64
d, J = 7.78 Hz, 1 H) 8.43−8.60 (m, 1 H) ppm.
Hemocompatibility (HC ). Sodium citrate (26.30 g, Sigma
5
0
Hydrolysis of Ethyl Ester. Two hundred milligrams (0.041
Aldirch), citric acid monohydrate (3.27 g, Sigma-Aldrich),
sodium dihydrogen phosphate dihydrate (2.51 g, Merck), and
glucose monohydrate were dissolved in 1.00 L of distilled water
and sterile filtered (0.2 μm). The pH was adjusted to 7.38 with a
sterile sodium hydroxide solution (1 M) to give the CPD buffer.
mmol, 1 equiv) of the respective polymer was dissolved in 10 mL
of 0.015 M aqueous sodium hydroxide solution and stirred for 12
h at 50 °C. After cooling to room temperature, the reaction
mixtures were neutralized with 1 M HCl and lyophilized. The
K
DOI: 10.1021/acs.bioconjchem.5b00393
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Fresh, nonsalted porcine blood (10 mL) was centrifuged (4000
rpm) for 15 min. The supernatant plasma was then rejected, and
the obtained crude erythrocyte sediment was washed (six times)
with a sterile, isotonic sodium solution (6.00 g·L ). The
erythrocyte sediment was suspended in a CPD buffer solution
(3) Craig, W. A. (2004) Overview of newer antimicrobial formulations
for overcoming pneumococcal resistance. American Journal of Medicine
Supplements 117, 16−22.
−1
́ ́ ́
(4) Vila, J., Sanchez-Cespedes, J., Sierra, J. M., Piqueras, M., Nicolas, E.,
Freixas, J., and Giralt, E. (2006) Antibacterial evaluation of a collection
of norfloxacin and ciprofloxacin derivatives against multiresistant
bacteria. Int. J. Antimicrob. Agents 28, 19−24.
(
10 mL) after the final washing step. The thus-prepared
erythrocyte suspensions were stored at 4 °C for a maximum of
(5) Cheng, Y., Qu, H., Ma, M., Xu, Z., Xu, P., Fang, Y., and Xu, T.
5
days. The polymer samples (40−80 mg) were dissolved in
(2007) Polyamidoamine (PAMAM) dendrimers as biocompatible
CPD buffer solution (800 μL), and a dilution series was prepared.
A small amount of the erythrocyte concentrate was slowly
brought to room temperature and added (200 μL) to every
carriers of quinolone antimicrobials: An in vitro study. Eur. J. Med.
Chem. 42, 1032−1038.
(
6) Lop
́
ez-Cebral, R., Romero-Caaman
̃
o, V., Seijo, B., Alvarez-
́
Lorenzo, C., Martín-Pastor, M., Concheiro, A., Landin, M., and
sample. The final mixtures have a concentration of approximately
8
5
× 10 red blood cells per mL based on counting with a
Sanchez, A. (2014) Spermidine Cross-Linked Hydrogels as a Controlled
Release Biomimetic Approach for Cloxacillin. Mol. Pharmaceutics 11,
2358−2371.
Neubauer-Improved counting chamber; the test series was
incubated for 1 h at 37 °C and then centrifuged (13500 rpm, 5
min). The supernatant liquid was further diluted with CPD
buffer (1:20), and the absorbance of the released hemoglobin
was measured in a UV/vis spectrometer at 541 nm and 25 °C.
The concentration at which 50% of the erythrocytes were
destroyed (HC ) was determined against a positive control that
(
7) Ghosh, S., Chakraborty, P., Saha, P., Acharya, S., and Ray, M.
2014) Polymer based nanoformulation of methylglyoxal as an
antimicrobial agent: efficacy against resistant bacteria. RSC Adv. 4,
3251−23261.
8) Hoste, K., De Winne, K., and Schacht, E. (2004) Polymeric
prodrugs. Int. J. Pharm. 277, 119−131.
9) Duncan, R., Gac-Breton, S., Keane, R., Musila, R., Sat, Y. N., Satchi,
(
2
(
50
was treated with 2 μL of Triton X and a negative buffer control
containing no active substance.
(
R., and Searle, F. (2001) Polymer−drug conjugates, PDEPT and PELT:
basic principles for design and transfer from the laboratory to clinic. J.
Controlled Release 74, 135−146.
ASSOCIATED CONTENT
■
(10) Duncan, R. (2014) Polymer therapeutics: Top 10 selling
*
S
Supporting Information
pharmaceuticals  What next? J. Controlled Release 190, 371−380.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconj-
chem.5b00393.
(11) Woo, G. L. Y., Mittelman, M. W., and Santerre, J. P. (2000)
Synthesis and characterization of a novel biodegradable antimicrobial
polymer. Biomaterials 21, 1235−1246.
ESI-MS and NMR spectra as well as tables with the
antimicrobial and hemotoxical evaluation of different
PACS (PDF)
(12) Woo, G. L. Y., Yang, M. L., Yin, H. Q., Jaffer, F., Mittelman, M. W.,
and Santerre, J. P. (2002) Biological characterization of a novel
biodegradable antimicrobial polymer synthesized with fluoroquino-
lones. J. Biomed. Mater. Res. 59, 35−45.
(13) GAC, S., Coudane, J., Boustta, M., Domurado, M., and Vert, M.
AUTHOR INFORMATION
(1999) Synthesis, Characterisation and In Vivo Behaviour of a
Norfloxacin-Poly(L-Lysine Citramide Imide) Conjugate Bearing
Mannosyl Residues. J. Drug Targeting 7, 393−406.
■
Corresponding Author
E-mail: joerg.tiller@udo.edu.
*
(
Zoł
14) Sobczak, M., Nałęcz-Jawecki, G., Kołodziejski, W. L., Gos,
́
P., and
towska, K. (2010) Synthesis and study of controlled release of
ofloxacin from polyester conjugates. Int. J. Pharm. 402, 37−43.
15) Parwe, S. P., Chaudhari, P. N., Mohite, K. K., Selukar, B. S., Nande,
S. S., and Garnaik, B. (2014) Synthesis of ciprofloxacin-conjugated poly
L-lactic acid) polymer for nanofiber fabrication and antibacterial
evaluation. Int. J. Nanomed. 9, 1463.
16) Namazi, H., and Kanani, A. (2009) Investigation diffusion
Notes
̇
́
The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
■
(
We thank the Deutsche Forschungsgemeinschaft (DFG) for
financing this project (KR 4700/1-1: Selbst-deaktivierende biozide
Polymere). All polymers were synthesized using CEM Discover
microwaves, which were kindly provided by CEM for ungraduate
student education. We thank Dr. Wolf Hiller and his team from
the department of chemistry for recording the NMR spectra at
the TU Dortmund. We thank the students Helene Wall, Livia
Bast, Sebastian Uppenkamp, and Shinthujah Selvarasa for lab
assistance. We also thank the butcher shop Vollmer, Dortmund
for providing fresh porcine blood. This work was supported by
(
mechanism of β-lactam conjugated telechelic polymers of PEG and β-
cyclodextrin as the new nanosized drug carrier devices. Carbohydr.
Polym. 76, 46−50.
(17) Kugel, A., Chisholm, B., Ebert, S., Jepperson, M., Jarabek, L., and
Stafslien, S. (2010) Antimicrobial polysiloxane polymers and coatings
containing pendant levofloxacin. Polym. Chem. 1, 442−452.
(18) Greenwald, R. B., Zhao, H., Xia, J., and Martinez, A. (2003)
Poly(ethylene glycol) Transport Forms of Vancomycin: A Long-Lived
Continuous Release Delivery System. J. Med. Chem. 46, 5021−5030.
the Ministerium fu
Landes Nordrhein Westfalen and by grants from the
Bildungsministerium fur Bildung und Forschung.
̈
r Innovation, Wissenschaft und Forschung des
(19) Nathan, A., Zalipsky, S., Ertel, S. I., Agathos, S. N., Yarmush, M. L.,
and Kohn, J. (1993) Copolymers of lysine and polyethylene glycol: a
̈
new family of functionalized drug carriers. Bioconjugate Chem. 4, 54−62.
(
́
20) Pichavant, L., Bourget, C., Durrieu, M.-C., and Heroguez, V.
REFERENCES
(
2011) Synthesis of pH-Sensitive Particles for Local Delivery of an
■
Antibiotic via Dispersion ROMP. Macromolecules 44, 7879−7887.
(
1) Boucher, H. W., Talbot, G. H., Bradley, J. S., Edwards, J. E., Gilbert,
̌
̌
(
21) Ríhova,
́
B., Jelínkova,
́
M., Strohalm, J., Subr, V., Plocova,
́
D.,
D., Rice, L. B., Scheld, M., Spellberg, B., and Bartlett, J. (2009) Bad Bugs,
No Drugs: No ESKAPE! An Update from the Infectious Diseases
Society of America. Clin. Infect. Dis. 48, 1−12.
2) Spellberg, B., Powers, J. H., Brass, E. P., Miller, L. G., and Edwards,
J. E. (2004) Trends in Antimicrobial Drug Development: Implications
Hovorka, O., Novak
́
, M., Plundrova,
́
D., Germano, Y., and Ulbrich, K.
(2000) Polymeric drugs based on conjugates of synthetic and natural
macromolecules.: II. Anti-cancer activity of antibody or (Fab′)2-
targeted conjugates and combined therapy with immunomodulators. J.
Controlled Release 64, 241−261.
(
for the Future. Clin. Infect. Dis. 38, 1279−1286.
L
DOI: 10.1021/acs.bioconjchem.5b00393
Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
(
22) Haag, R., and Kratz, F. (2006) Polymer Therapeutics: Concepts
and Applications. Angew. Chem., Int. Ed. 45, 1198−1215.
23) Zhao, Y.-J., Wei, W., Su, Z.-G., and Ma, G.-H. (2009) Poly
ethylene glycol) prodrug for anthracyclines via N-Mannich base linker:
Design, synthesis and biological evaluation. Int. J. Pharm. 379, 90−99.
24) Ferguson, E. L., Azzopardi, E., Roberts, J. L., Walsh, T. R., and
(40) Konieczny, S., Leurs, M., and Tiller, J. C. (2015) Polymer Enzyme
Conjugates as Chiral Ligands for Sharpless Dihydroxylation of Alkenes
in Organic Solvents. ChemBioChem 16, 83−90.
(
(
(41) Kerns, R. J., Rybak, M. J., Kaatz, G. W., Vaka, F., Cha, R., Grucz, R.
G., and Diwadkar, V. U. (2003) Structural features of piperazinyl-linked
ciprofloxacin dimers required for activity against drug-resistant strains of
Staphylococcus aureus. Bioorg. Med. Chem. Lett. 13, 2109−2112.
(42) Kerns, R. J., Rybak, M. J., Kaatz, G. W., Vaka, F., Cha, R., Grucz, R.
G., Diwadkar, V. U., and Ward, T. D. (2003) Piperazinyl-linked
fluoroquinolone dimers possessing potent antibacterial activity against
drug-resistant strains of Staphylococcus aureus. Bioorg. Med. Chem. Lett.
13, 1745−1749.
(43) Litt, M., Levy, A., and Herz, J. (1975) Polymerization of Cyclic
Imino Ethers. X. Kinetics, Chain Transfer, and Repolymerization. J.
Macromol. Sci., Chem. 9, 703−727.
(44) Chu, D. T., and Fernandes, P. B. (1989) Structure-activity
relationships of the fluoroquinolones. Antimicrob. Agents Chemother. 33,
(
Thomas, D. W. (2014) Dextrin−Colistin Conjugates as a Model
Bioresponsive Treatment for Multidrug Resistant Bacterial Infections.
Mol. Pharmaceutics 11, 4437−4447.
(
25) Du, J., Bandara, H. M. H. N., Du, P., Huang, H., Hoang, K.,
Nguyen, D., Mogarala, S. V., and Smyth, H. D. C. (2015) Improved
Biofilm Antimicrobial Activity of Polyethylene Glycol Conjugated
Tobramycin Compared to Tobramycin in Pseudomonas aeruginosa
Biofilms. Mol. Pharmaceutics 12, 1544−1553.
(
26) Lawson, M. C., Shoemaker, R., Hoth, K. B., Bowman, C. N., and
Anseth, K. S. (2009) Polymerizable Vancomycin Derivatives for
Bactericidal Biomaterial Surface Modification: Structure−Function
Evaluation. Biomacromolecules 10, 2221−2234.
1
(
31−135.
45) Tillotson, G. S. (1996) Quinolones: structure-activity relation-
ships and future predictions. J. Med. Microbiol. 44, 320−324.
46) LeBel, M. (1988) Ciprofloxacin: Chemistry, Mechanism of
(
27) Bieser, A. M., and Tiller, J. C. (2011) Mechanistic Considerations
on Contact-Active Antimicrobial Surfaces with Controlled Functional
Group Densities. Macromol. Biosci. 11, 526−534.
(
(
Action, Resistance, Antimicrobial Spectrum, Pharmacokinetics, Clinical
Trials, and Adverse Reactions. Pharmacotherapy: The Journal of Human
Pharmacology and Drug Therapy 8, 3−30.
28) Siedenbiedel, F., Fuchs, A., Moll, T., Weide, M., Breves, R., and
Tiller, J. C. (2013) Star-Shaped Poly(styrene)-block-Poly(4-vinyl-N-
methylpyridiniumiodide) for Semipermanent Antimicrobial Coatings.
Macromol. Biosci. 13, 1447−1455.
(
47) Fortuniak, W., Mizerska, U., Chojnowski, J., Basinska, T.,
Slomkowski, S., Chehimi, M., Konopacka, A., Turecka, K., and Werel, W.
2011) Polysiloxanes With Quaternary Ammonium Salt Biocidal
(
29) Turos, E., Shim, J.-Y., Wang, Y., Greenhalgh, K., Reddy, G. S. K.,
(
Dickey, S., and Lim, D. V. (2007) Antibiotic-conjugated polyacrylate
nanoparticles: New opportunities for development of anti-MRSA
agents. Bioorg. Med. Chem. Lett. 17, 53−56.
Functions and Their Behavior When Incorporated Into a Silicone
Elastomer Network. J. Inorg. Organomet. Polym. Mater. 21, 576−589.
(48) Fik, C. P., Konieczny, S., Pashley, D. H., Waschinski, C. J., Ladisch,
(
30) Sarker, P., Shepherd, J., Swindells, K., Douglas, I., MacNeil, S.,
R. S., Salz, U., Bock, T., and Tiller, J. C. (2014) Telechelic Poly(2-
oxazoline)s with a Biocidal and a Polymerizable Terminal as
Collagenase Inhibiting Additive for Long-Term Active Antimicrobial
Dental Materials. Macromol. Biosci. 14, 1569−1579.
Swanson, L., and Rimmer, S. (2011) Highly Branched Polymers with
Polymyxin End Groups Responsive to Pseudomonas aeruginosa.
Biomacromolecules 12, 1−5.
(
31) Tauhardt, L., Frant, M., Pretzel, D., Hartlieb, M., Bucher, C.,
Hildebrand, G., Schroter, B., Weber, C., Kempe, K., Gottschaldt, M.,
et al. (2014) Amine end-functionalized poly(2-ethyl-2-oxazoline) as
promising coating material for antifouling applications. J. Mater. Chem. B
2
(
, 4883−4893.
32) Weber, C., Becer, C. R., Baumgaertel, A., Hoogenboom, R., and
Schubert, U. S. (2009) Preparation of Methacrylate End-Functionalized
Poly(2-ethyl-2-oxazoline) Macromonomers. Des. Monomers Polym. 12,
1
(
49−165.
33) Krumm, C., Konieczny, S., Dropalla, G. J., Milbradt, M., and
Tiller, J. C. (2013) Amphiphilic Polymer Conetworks Based on End
Group Cross-Linked Poly(2-oxazoline) Homo- and Triblock Copoly-
mers. Macromolecules 46, 3234−3245.
(
34) Farrugia, B. L., Kempe, K., Schubert, U. S., Hoogenboom, R., and
Dargaville, T. R. (2013) Poly(2-oxazoline) Hydrogels for Controlled
Fibroblast Attachment. Biomacromolecules 14, 2724−2732.
(
35) Adams, N., and Schubert, U. S. (2007) Poly(2-oxazolines) in
biological and biomedical application contexts. Adv. Drug Delivery Rev.
9, 1504−1520.
36) Waschinski, C. J., Herdes, V., Schueler, F., and Tiller, J. C. (2005)
5
(
Influence of Satellite Groups on Telechelic Antimicrobial Functions of
Polyoxazolines. Macromol. Biosci. 5, 149−156.
(
37) Fik, C. P., Krumm, C., Muennig, C., Baur, T. I., Salz, U., Bock, T.,
and Tiller, J. C. (2012) Impact of Functional Satellite Groups on the
Antimicrobial Activity and Hemocompatibility of Telechelic Poly(2-
methyloxazoline)s. Biomacromolecules 13, 165−172.
(
38) Waschinski, C. J., Barnert, S., Theobald, A., Schubert, R.,
hter, K., and Tiller, J. C. (2008)
Kleinschmidt, F., Hoffmann, A., Saalwac
Insights in the Antibacterial Action of Poly(methyloxazoline)s with a
̈
Biocidal End Group and Varying Satellite Groups. Biomacromolecules 9,
1
(
764−1771.
39) Krumm, C., Harmuth, S., Hijazi, M., Neugebauer, B., Kampmann,
A.-L., Geltenpoth, H., Sickmann, A., and Tiller, J. C. (2014)
Antimicrobial Poly(2-methyloxazoline)s with Bioswitchable Activity
through Satellite Group Modification. Angew. Chem., Int. Ed. 53, 3830−
3
834.
M
DOI: 10.1021/acs.bioconjchem.5b00393
Bioconjugate Chem. XXXX, XXX, XXX−XXX