Synthesis and Antibacterial Activity of Polymerizable Acryloyloxyalkyltriethyl Ammonium Salts

Article abstract of DOI:10.1002/cplu.201700194

This study reports an efficient and practical synthetic approach for the synthesis of a particularly important class of polymerizable quaternary ammonium salts (PQASs), that is, acryloxyalkyltriethylammonium bromides (AATEABs), which may find application as antimicrobial coatings for commercial membranes with antifouling and anti-biofouling properties, to be used for wastewater treatment. The synthetic method is based on a simple two-step procedure from commercially available substrates, entirely carried out under air and without any need for chromatographic purification. All the newly synthesized AATEABs were tested for their antimicrobial activity, and the results showed that AATEABs bearing an alkyl chain of 11 and particularly 12 carbon atoms possessed significant activity against Gram positive bacteria and yeast strains.

Full text of DOI:10.1002/cplu.201700194

Accepted Article
Title: Synthesis and antibacterial activity of polymerizable
acryloyloxyalkyltriethyl ammonium salts
Authors: Bartolo Gabriele, Raffaella Mancuso, Roberta Amuso, Biagio
Armentano, Giuseppe Grasso, Vittoria Rago, Anna Rita
Capello, Francesco Galiano, Alberto Figoli, Giorgio De Luca,
and Jan Hoinkis
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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: ChemPlusChem 10.1002/cplu.201700194
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Synthesis and antibacterial activity of polymerizable
acryloyloxyalkyltriethyl ammonium salts
[a]
[a]
[b]
[a]
[b]
Raffaella Mancuso,* Roberta Amuso, Biagio Armentano, Giuseppe Grasso, Vittoria Rago,
[b]
[c]
[c]
[c]
[d]
Anna Rita Cappello, Francesco Galiano, Alberto Figoli, Giorgio De Luca, Jan Hoinkis, and
Bartolo Gabriele*[a]
Abstract: Polymerizable quaternary ammonium salts (PQASs) are
important starting materials for the development of innovative
polymers with antimicrobial activity. This work reports an efficient and
practical synthetic approach for the synthesis of a particularly
important class of PQASs, that are, acryloxyalkyltriethylammonium
bromides (AATEABs), which may find application as antimicrobial
coatings for commercial membranes with antifouling and anti-
biofouling properties, to be used for wastewater treatment. The
synthetic method is based on a simple two-step procedure from
commercially available substrates, entirely carried out under air and
without any need for chromatographic purification. All the newly
synthesized AATEABs were tested for their antimicrobial activity, and
the results evidenced that AATEABs bearing an alkyl chain of 11 and
particularly 12 carbon atoms possessed significant activity against
Gram +ve bacteria and yeast strains.
membranes for wastewater treatment, characterized by high anti-
fouling and anti-biofouling properties, which have been obtained
by coating a commercial polyethersulfone (PES) membrane with
an antimicrobial polymeric film, able to prevent the growth of
microorganisms on the membrane surface, and thus reducing
fouling and increasing the life-time of the membrane itself.[4] The
antimicrobial surface modification was achieved by in situ radical
polymerization of a polymerizable bicontinuous microemulsion
(PBM) containing acryloyloxyundecyltriethylammonium bromide
(AUTEAB) as a PQAS co-monomer. The potentiality of novel
coated PBM membranes have been successfully proved in
Membrane Bioreactor (MBR) by treating real textile wastewaters.
The results confirmed the features of the novel nanostructured
membranes making them of interest for industrial applications. .
In this work, we report a convenient and practical synthetic
approach for the production of acryloxyalkyltriethylammonium
bromides (AATEABs), bearing an alkyl chain of 6, 9, 11, and 12
carbon atoms, based on a simple two-step procedure from
commercially available substrates, entirely carried out under air
and without any need for chromatographic purification. Our
method allows obtaining AATEABs in an economically convenient
manner, suitable for large-scale and industrial applications. All the
newly synthesized AATEABs were also tested for their
antimicrobial activity against Gram +ve bacteria, Gram -ve
bacteria and yeast strains.
[4]
Introduction
Quaternary ammonium salts (QASs) have been known for a long
time to possess important antimicrobial activities, and have been
extensively used as antiseptics and disinfectants.[1] In particular,
they find application in the textile field as either additives in
different formulations or, when bearing a polymerizable group, as
co-monomers for their chemical incorporation into the textile
structure, so as to prevent leaching from the textile.[2]
Considering the significant antimicrobial activity of QAS,
during the last years there has been a considerable interest in
developing novel polymers with antimicrobial activity, obtained by
the use of polymerizable quaternary ammonium salts (PQASs) as
co-monomers as well as by post-functionalization of the polymer
by quaternization techniques.[3] In this context, our research group
has recently reported the development of novel nanostructured
Results and Discussion
Our synthetic approach for the production of AATEABs 4a-d
(
9 6
4a: AUTEAB, C11 alkyl chain; 4b, C12; 4c, C ; 4d, C ) basically
consists in two steps from commercially available starting
materials: the esterification of -bromo-1-alkanols 1 with acryloyl
chloride 2, carried out in the presence of molecular sieves 3A, to
give -bromoalkyl acrylates 3a-d, followed by quaternization with
Et
3
N (Scheme 1).
[
a]
Dr. R. Mancuso, Miss R. Amuso, Mr. G. Grasso,
Prof. Dr. B. Gabriele
Department of Chemistry and Chemical Technologies
University of Calabria
Via P. Bucci, 12/C, 87036 Arcavacata di Rende (CS), Italy
E-mails: raffaella.mancuso@unical.it (R.M.);
bartolo.gabriele@unical.it (B.G.)
Mr. B. Armentano, Miss V. Rago, Prof. Dr. A. R. Cappello
Department of Pharmacy and Health and Nutritional Sciences
University of Calabria
The reaction conditions of both the esterification and
quaternization steps have been carefully optimized in order to
avoid the use of inert gas environment and the lengthy and costly
purification of intermediate 3. In particular, in the final optimized
conditions, the first esterification step between 1 and acryloyl
chloride 2 was carried out with a 1:1 molar ratio under air in MeCN
at 90 °C for 24 h in the presence of activated molecular sieves 3A
and small amounts of BHT as radical inhibitor.
[b]
[c]
[c]
87036 Arcavacata di Rende (CS), Italy
Dr. F. Galiano, Dr. A. Figoli, Dr. G. De Luca
Institute on Membrane Technology
National Research Council
Via P. Bucci, 17/C, 87036 Arcavacata di Rende (CS), Italy
Prof. Dr. J. Hoinkis
Institute of Applied Research
Karlsruhe University of Applied Sciences
Moltkestreet 30, 76133 Karlsruhe, Germany
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under air
molecular sieves 3A
Scheme 2. Synthesis of acryloxyalkyltriethylammonium bromides (AATEABs)
O
O
4e and 4f from 2-bromoethanol 1e and 3-bromopropanol 1f, respectively.
2 n
HO(CH ) Br
+
Cl
MeCN, 90 °C, 24 h
BHT (0.3 mol %)
2 n
O(CH ) Br
1
3
a (n = 11)
b (n = 12)
c (n = 9)
d (n = 6)
2 (1 equiv)
crude product
Et N (1.1 equiv)
CHCl , 60 °C, 72 h
under air
The antimicrobial activity of the all the newly synthesized
3
AATEABs (4a-f) was then measured against two Gram +ve
Staphylococcus aureus and Streptococcus pyogenes) strains,
3
(
three Gram -ve bacterial strains (Escherichia coli, Klebsiella
pneumoniae and Pseudomonas aeruginosa) and two yeast
strains (Saccharomyces cerevisiae and Candida albicans).
Of all the compounds tested, 4a (C11 alkyl chain) and 4b (C12
alkyl chain) showed the most significant activity against Gram +ve
bacteria and yeast strains, 4b being ca. four times more active (%
inhibition was 50-60% at 32 g/mL for 4b and 40-50% at 128
O
a (n = 11) (63%)
b (n = 12) (70%)
c (n = 9) (61%)
d (n = 6) (75%)
2 n 3
O(CH ) Br
NEt +
4
Scheme 1. Synthesis of acryloxyalkyltriethylammonium bromides (AATEABs)
4
a-d from commercially available -bromo-1-alkanols 1.

g/mL for 4a). Both derivatives also showed a moderate activity
against Gram -ve strains (% inhibition was 40-50% at 256 g/mL
for 4b and 30-40% at 512 g/mL for 4a). On the other hand, 4d
(C alkyl chain) exhibited an antibacterial activity only against the
6
Gram +ve strains tested, and displayed no activity against the
yeast strains Saccharomyces cerevisiae and Candida albicans,
even at the highest concentration tested. Conversely, 4c (C )
9
exhibited a significant activity against Candida albicans (40 %
inhibition at 128 g/mL), though no antibacterial activity was
2 3
measured. AATEABs with shorter alkyl chains 4e (C ) and 4f (C )
exhibited no activity against any of the bacterial or yeast strains
tested (Figures 1-3).
The crude 11-bromoalkyl acrylate 3, obtained in the first step,
was then used as such, without chromatographic purification, for
the next step, consisting in the reaction with Et N (1.1:1 molar
ratio with respect to starting 1), carried out under air in CHCl at
0 °C for 72 h. The desired products 4a-d were eventually
3
3
6
1
recovered in satisfactory purity (> 96%, by H NMR) and with good
overall yields over the two steps (61-75%, based on starting 1a-
d) by simple precipitation with Et O followed by washing with the
2
same solvent (see the Experimental Section for details).
In order to compare the antimicrobial activity of AATEABs
a-d with similar derivatives with shorter alkyl chains, we also
4
The inhibition ratios of the novel salts against pathogenic
strains were calculated from minimum diameter values at 128,
tried
to
synthesize
in
an
analogous
way
acryloyloxyethyltriethylammonium bromide 4e (C
2
alkyl chain)
2
56 and 512 g. The average of three inhibition zone diameter
measurements was compared against the commercially available,
non-polymerizable quaternary ammonium salt DTAB
dodecyltrimethylammonium bromide), using the Kirby-Bauer
and acryloyloxypropyltriethylammonium bromide 4f (C
3
alkyl
chain) starting from 2-bromoethanol 1e and 3-bromopropanol 1f,
respectively. However, the method was not successful in these
cases, since the desired products were obtained in unsatisfactory
yields and purity. Accordingly, for preparing these derivatives, we
explored a different synthetic approach. They were eventually
successfully synthesized by a 3-step procedure, involving (a) the
reaction between 1e or 1f with diethylamine as nucleophile to give
-(diethylamino)ethanol 5e or 3-(diethylamino)propanol 5f; (b)
esterification of the latter with acryloyl chloride to give 2-
diethylamino)ethylacrylate 6e or 3-(dethylamino)propylacrylate
f and (c) quaternization of the latter with ethyl bromide to give 4e
(
method.[ Final interpretation of the measurements enabled all of
the bacteria strains tested to be grouped into three categories
related to the disk diffusion zone diameters (Sensitive: diameter
5]
≥
20 mm; Intermediate: diameter 15-19 mm and Resistant:
diameter ≤ 14 mm). The results are summarized in Tables 1 and
. No inhibition zone was seen in the control (impregnated water
2
2
discs) (data not shown).
(
6
The antimicrobial sensitivity profile of the bacterial and yeast
strains showed that all the strains were found to be sensitive to
the commercial ionic non-polymerizable surfactant, DTAB and
that, among the ammonium salts tested, 4a (C11), 4b (C12), and
or 4f (Scheme 3). The use of a lower molar amount of EtBr with
respect to 6 (0.7 equiv) in the quaternization step facilitated the
product purification.
6
4d (C ) displayed antimicrobial activity. In particular, a
comparison of data obtained on the inhibition zones of the
pathogenic microorganisms proved that both the two Gram +ve
bacterial strains and Saccharomyces cerevisiae and Candida
albicans were Sensitive to 128 g 4b (C ) or 256 g 4a (C )
under nitrogen
(
a) HO(CH ) Br
+
Et NH
HO(CH ) NEt
2
2
n
2
2 n
EtOH, 60 °C, 6 h
1
(5 equiv)
5
e (n = 2) (96%)
f (n = 3) (98%)
1
2
11
O
6
(Tables 1 and 2). Compound 4d (C ) exhibited antibacterial
O
under nitrogen
(
b)
c)
+
5
activity only against the Gram +ve bacterial strains, at the highest
concentration tested (512 g). Conversely, among the Gram –ve
strains, E. coli and K. pneumoniae were Sensitive only at 512 g
MeCN, 0 °C (2 h) to rt (15 h)
2 n
O(CH ) NEt2
Cl
6
(
1.0-1.1 equiv)
e (n = 2) (57%)
f (n = 3) (61%)
4b (C12), while P. aeruginosa was found to be Intermediate at this
O
under nitrogen
concentration. None of the three Gram -ve strains tested were
Sensitive to 4a (C11), indeed E. coli and K. pneumoniae were
Intermediate at the highest concentration tested (512 g) (Table
(
6
+ EtBr
0.7 equiv)
NEt +
2 n 3
O(CH ) Br
50 °C, 15 h
(
4
hydroquinone (0.1 mol %)
e (n = 2) (53%)
f (n = 3) (41%)
1). Surprisingly Candida albicans appeared Sensitive to 256 g
4
c (C ) (Table 2).
9
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Figure 1. Effect of the newly synthesized AATEABs (4a-f) against Gram +ve bacterial strains. The results obtained with the commercially available, non-
polymerizable ammonium salt DTAB (dodecyltrimethylammonium bromide) are also reported for comparison. The bars represent the percentage of residual activity
of S. pyogenes (a) and S. aureus (b) in the presence of increasing concentrations of salts, with respect to the control value without salt. The bacteria cells were
grown in Muller-Hinton broth, as described in Experimental Section. The values represent the means ± S.D. of three different experiments, each performed with
triplicate samples. (* p<0.01, ** p<0.001, *** p<0.0001, **** p<0.00001, one-way ANOVA and Student’s t-test calculations).
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Figure 2. Effect of the newly synthesized AATEABs (4a-f) against Gram -ve bacterial strains. The results obtained with the commercially available, non-
polymerizable ammonium salt DTAB (dodecyltrimethylammonium bromide) are also reported for comparison. The bars represent the percentage of residual activity
of E. coli (a), P. aeruginosa (b) and K. pneumoniae (c) in the presence of increasing concentrations of salts, with respect to the control value without salt. The
bacteria cells were grown in Muller-Hinton broth, as described in Experimental Section. The values represent the means ± S.D. of three different experiments, each
performed with triplicate samples. (* p<0.01, ** p<0.001, *** p<0.0001, **** p<0.00001, one-way ANOVA and Student’s t-test calculations).
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Figure 3. Effect of the newly synthesized AATEABs (4a-f) against yeast strains. The results obtained with the commercially available, non-polymerizable ammonium
salt DTAB (dodecyltrimethylammonium bromide) are also reported for comparison. The bars represent the percentage of residual activity of (a) Candida albicans
and (b) Saccharomyces cerevisiae in the presence of increasing concentrations of salts, with respect to the control value without salt. The yeast cells were grown
in appropriated medium for yeast, as described in Experimental Section. The values represent the means ± S.D. of three different experiments, each performed
with triplicate samples. (* p<0.01, ** p<0.001, *** p<0.0001, **** p<0.00001, one-way ANOVA and Student’s t-test calculations).
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COMPOUNDS
DTAB
Table 1. Antimicrobial susceptibility profiles against bacterial strains (S:Sensitive; R:Resistant; I:Intermediate).
g
BACTERIAL STRAINS
Staphylococcus
Streptococcus
Escherichia
Klebsiella
Pseudomonas
aureus
pyogenes
coli
pneumoniae
aeruginosa
ATCC25923
ATCC19615
ATCC25922
ATCC13883
ATCC27853
1
256
28
S
S
S
R
R
R
R
R
R
R
I
S
R
R
R
I
S
S
S
R
R
R
R
R
R
R
I
S
R
R
R
I
S
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
I
S
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
I
I
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
I
512
128
4e (C-2)
256
512
128
4f (C-3)
256
512
128
4
d (C-6)
c (C-9)
256
512
128
4
256
512
128
4a (C-11)
256
S
S
S
S
S
S
S
S
S
S
512
128
R
I
S
R
I
S
4b (C-12)
256
12
5
Table 2. Antimicrobial susceptibility profiles againts yeast strains (S:Sensitive; R:Resistant; I:Intermediate).
COMPOUNDS g YEAST STRAINS
Saccharomyces
Candida
cerevisiae
albicans
ATCC Y10000
ATCC 10231
1
28
S
S
S
R
R
R
R
R
R
R
R
R
R
R
I
S
S
S
R
R
R
R
R
R
R
R
R
I
DTAB
256
5
12
28
1
4
4
4
4
4
4
e (C
2
)
256
5
12
28
1
f (C
2
)
256
5
12
28
1
d (C
6
)
256
5
12
28
1
c (C
9
)
256
S
S
I
5
12
28
1
R
I
a (C11
)
256
S
S
S
S
S
5
12
28
S
S
S
S
1
b (C12
)
256
12
5
The disc-diffusion susceptibility test (Kirby-Bauer test) could
only test bacterial susceptibility to various antimicrobial reagents
at pre-designed dosages. Such information as the minimal
inhibitory concentration (MIC) of a specific compound could not
be obtained from this test. Therefore, in order to determine MIC
values for the newly synthetized ammonium salts, the broth
dilution method was performed. As expected, the MIC
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Table 3. MIC (top rows) and MBC (bottom rows) values of the investigated compounds.
MIC
MBC
g/mL)
COMPOUNDS
BACTERIAL STRAINS

Staphylococcus
aureus
ATCC25923
128
Streptococcus
pyogenes
ATCC19615
128
Escherichia
coli
Klebsiella
pneumoniae
ATCC13883
128
Pseudomonas
aeruginosa
ATCC27853
256
ATCC25922
128
MIC
DTAB
MBC
MIC
128
128
128
128
256
>512
>512
>512
>512
512
>512
>2048
>2048
>2048
>2048
>2048
>2048
>2048
>2048
2048
>2048
>2048
>2048
>2048
>2048
>2048
>2048
>2048
2048
>2048
4
4
4
4
4
4
e (C
2
)
MBC
MIC
>512
>2048
>512
>2048
f (C
3
)
MBC
MIC
>512
>2048
512
>2048
d (C
6
)
)
MBC
MIC
>512
>512
>512
512
>512
>2048
>512
>2048
c (C
9
MBC
MIC
>512
>2048
256
>2048
a (C11
)
MBC
MIC
512
512
2048
2048
>2048
64
64
512
512
1024
b (C12
)
MBC
64
64
512
512
1024
Table 4. MIC (top rows) and MFC (bottom rows) values of the investigated compounds.
MIC
MFC
g/mL)
COMPOUNDS
YEAST STRAINS

Saccharomyces
Candida
albicans
ATCC 10231
128
cerevisiae
ATCC Y10000
128
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
DTAB
e (C
f (C
d (C
c (C
128
128
>512
>512
>512
>512
>512
>512
512
>512
512
512
64
>512
>512
>512
>512
512
>512
256
>512
256
256
4
2
)
4
3
)
4
4
6
)
9
)
4a (C11
)
64
64
4b (C12
)
64
determination results were in good agreement with the disc-
diffusion susceptibility test (Tables 3 and 4). For example, in the
disc-diffusion test, S. aureus, S. pyogenes, S. cerevisiae and C.
albicans were found to be Sensitive to 128 g 4b or 256 g 4a; in
the dilution experiment, their MICs were lower than that of the
other ammonium salts tested. Surprisingly, 4b displayed the MIC
values (64 g/mL) lower than those reported for the commercial
non-polymerizable surfactant DTAB (MIC 128 g/mL) (Tables 3
and 4).
against Gram +ve bacteria and yeast than those found for all the
newly synthesized AATEABs (Tables 3 and 4). It is noteworthy
that, in the AAETEAB derivatives tested, the carbon chain
elongation, tended to increase the antimicrobial activity.
We next assessed if the 4b (C12) antimicrobial activity was due
to morphological changes in the cell envelope, followed by an
overall increase in outer membrane permeability. To this end, we
used propidium iodide (PI),
a
membrane-impermeable
fluorescent dye, on S. aureus cells treated with different 4b
concentrations (1/2 MIC, MIC and 2MIC values), in order to detect
a possible permeation of cell membrane.[6] After 30 minutes, the
The minimal bactericidal concentration (MBC) and the
minimum fungicidal concentration (MFC) values were also
determined, in bacterial and yeast cultures, respectively. Similarly, treated cells showed increased PI fluorescence relative to
a (C11) and 4b (C12) displayed lower MBC and MFC values untreated cells, in a dose-dependent manner,
4
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em.620 nm) compared to untreated control sample. (***p < 0,0001,****p <
0.00001; ns: not significant; one-way ANOVA and Student’s t-test calculations).
indicating destabilization of the cellular envelope and increased
membrane permeability (Figure 4). The observed increase was
quantified by detecting red fluorescent signal by a fluorescent
plate reader (Figure 5). Cells treated with 4b (C-12), at MIC
concentration, displayed red fluorescent signal three times
increased, compared to untreated cells.
It is worth noting that the possibility to co-polymerize the C-11
derivative 4a (acryloyoxyundecyl triethylammonium bromide,
AUTEAB) under free-radical conditions (using ammonium
persulfate as radical initiator) was recently successfully
[4]
demonstrated by us. In that work, AUTEAB was prepared by a
classical”approach, involving the purification of the intermediate
a obtained from the first esterification step, which was purified
“
3
by column chromatography and then subjected to quaternization
to give AUTEAB. The latter was then copolymerized together with
other components.[4] The polymeric film was casted on a
commercial membrane, and the novel surface-functionalized
membrane thus obtained showed significant antibiofouling
properties, thus confirming the antimicrobial activity exerted by
polymerized AUTEAB.[4] Further studies aimed at functionalizing
commercial membranes with the polymeric film obtained by
copolymerization of the C-12 derivative prepared in this work are
currently in progress in our laboratories and will be reported in due
course.
Conclusions
We have developed an efficient and convenient synthetic
approach to the synthesis of
polymerizable quaternary ammonium salt (PQAS), that are,
acryloxyalkyltriethylammonium bromides (AATEABs) 4a-d with a
a very important class of
Figure 4. Compound 4b (C12) increases outer membrane permeability in
Staphylococcus aureus cells. Bright field and fluorescence microscopy of PI-
stained untreated (a1) cells and cells treated for 30 minutes with 32µg/mL (b1),
6 9
relatively long alkyl chain (C , C , C11, and C12). These AATEAB
64 µg/mL (c1), 128 µg/mL (d1) 4b. Bacterial cells were visualized using an
Olympus BX41 microscope and the images were taken with CSV1.14 software
using a CAMXC-30 for image acquisition. The experiment was repeated three
times for each sample. Scale Bars 12.5µm.
derivatives are useful starting material for the preparation of
polymeric membrane coatings with anti-biofouling properties, to
be employed in wastewater treatment. Our method is suitable for
large-scale applications, since it is entirely carried out under air
and avoids lengthy and costly chromatographic purification
procedures. It is based on esterification of commercially available
1
0
8
6
4
2
0

-bromo-1-alkanols 1 with acryloyl chloride 2 in the presence of
activated molecular sieves 3A followed by quaternization of the
ensuing crude 11-bromoalkyl acrylate 3 with Et N. This method
*
***
3
did not work suitably for the synthesis of AATEABs bearing
shorter carbon chains (with two or three carbon atoms, 4e and 4f,
respectively). The latter derivatives, however, could be prepared
by a 3-step approach, based on the reaction of the corresponding
*
**
ns
-bromoalcohols with diethylamine followed by esterification with
acryloyl chloride and quaternization with ethyl bromide.
ctrl 1/2 MIC MIC 2 MIC
All the newly synthesized AATEABs 4a-f were tested for their
antimicrobial activity. Among them, 4a (C11 alkyl chain) showed
interesting antimicrobial activity against the yeast and bacterial
strains tested apart from Pseudomonas aeruginosa. In particular,
it revealed promising results against Streptococcus pyogenes and
Figure 5. Compound 4b (C12) increases outer membrane permeability in
Staphylococcus aureus cells. Quantification of propidium iodide fluorescent
signal intensity in untreated (control) or 4b-treated cells was evaluated
measuring red fluorescent signal by a fluorescent plate reader (ex.530 nm;
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Candida albicans (MIC values 256 g/mL). Similar antimicrobial
behavior was detected for 4c (C ), but against Saccharomyces
9
JASCO FT-IR 4200 spectrometer. Mass spectra were obtained by using a
Shimadzu QP-2010 GC-MS apparatus at 70 eV ionization voltage and by
electrospray ionization mass spectrometry (ESI-MS) by using an Agilent
cerevisiae and Candida albicans, only. On the other hand,
compound 4b (C12) exhibited good antimicrobial activity against
the two yeast strains tested. Regarding the bacteria, 4b was the
most active derivative, especially against Staphylococcus aureus
and Streptococcus pyogenes, with a MIC value of 64 g/mL,
which was approximately four times lower than the MIC value
detected for 4a (C11). In comparison, all the other AATEABs
showed a significantly lower antimicrobial activity.
6540 UHD accurate-mass Q-TOF spectrometer equipped with a Dual AJS
ESI source working in positive mode. N was employed as desolvation gas
2
at 300°C and a flow rate of 8 L/min. The nebulizer was set to 45psig. The
Sheat gas temperature was set at 400°C and a flow of 12 L/min. A potential
of 3.5 kV was used on the capillary for positive ion mode. The fragmentor
was set to 175 V. MS spectra were recorded in the 150–1000 m/z range.
The HPLC system was an Agilent 1260 Infinity. A reversed-phase C18
column (ZORBAX Extended-C18 2.1
x 50 mm, 1.8 mm) with a
Phenomenex C18 security guard column (4mm×3mm) were used. The
flow-rate was 0.4 mL/min and the column temperature was set to 30°C.
The eluents were formic acid–water (0.1:99.9, v/v) (phase A) and formic
acid–acetonitrile (0.1:99.9, v/v) (phase B). The following gradient was
employed: 0–10 min, linear gradient from 5% to 95% B; 10–15 min,
washing and reconditioning of the column to 5% B. Injection volume was
10 mL. The eluate was monitored through MS TIC. Microanalyses were
carried out with a Thermo-Fischer Elemental Analyzed Flash 2000.
Based on these results, we may conclude that the C11 and
especially the C12 AATEAB derivatives, synthesized in this work
through an efficient, practical and easily scalable procedure, are
excellent candidates for the industrial development of novel
antimicrobial polymers and materials, to be employed in different
applicative fields, including the textile and the biomedical fields. In
particular, the use of these derivatives for the development of
novel nanostructured membranes characterized by anti-fouling
and anti-biofouling properties, to be efficiently employed in
wastewater treatment is currently underway in our laboratories.
Preparation of Acryloxyalkyltriethylammonium Bromides (AATEABs)
Experimental Section
4a-d
Chemical syntheses: General methods.
1st Step: Esterification of -bromoalkan-1-ols 1a-d with acryloyl
chloride 2
MeCN was purchased from Hypersolv (purity ≥ 99.9%) and was freshly
distilled over activated molecular sieves 4A before use. CHCl
purchased from Sigma-Aldrich (purity 99.0-99.4 %) and was distilled over
activated molecular sieves 4A before use. Et O, stabilized with
dibutylhydroxytoluene (BHT, 1 ppm) was purchased from Sigma-Aldrich
3
was
To a stirred solution of the -bromoalkan-1-ol (11-bromo-1-undecanol 1a:
5.0 g, 19.9 mmol; 12-bromo-1-dodecanol 1b: 1.0 g, 3.8 mmol; 9-bromo-1-
2
nonanol 1c: 1.1 g, 5.3 mmol; 6-bromo-1-hexanol 1d: 1.0 g, 5.5 mmol) in
anhydrous MeCN (100 mL for 1a; 19.1 mL for 1b; 26.6 mL for 1c; 27.7 mL
for 1d), maintained at room temperature in a Schlenk flask, was added
BHT (13.9 mg, 0.063 mmol, for 1a; 2.6 mg, 0.012 mmol, for 1b; 3.7 mg,
(
(
purity ≥ 99.9%) and used as received. EtOH was purchased from Panreac
purity 99.8%) and was used as received. CH Cl was purchased from
2
2
Sigma-Aldrich purity ≥ 99.8%) and was used as received. Cyclohexane
was purchased from Panrec (purity 99.5%) and was used as received.
0
.017 mmol, for 1c; 3.8 mg, 0.017 mmol for 1d), followed by acryloyl
chloride (dropwise; 1.62 mL, 1.80 g, 19.9 mmol, for 1a; 310 L, 344 mg,
.8 mmol, for 1b; 430 L, 480 mg, 5.3 mmol, for 1c; 450 L, 500 mg, 5.5
mmol, for 1d), and activated sieves 3 Å (10 g for 1a, 1.9 g for 1b, 2.7 g for
3
6
-Bromo-1-hexanol (purity >95%), 9-bromo-1-nonanol (purity >95%), 11-
bromo-1-undecanol (purity > 98%), 12-bromo-1-dodecanol (purity > 98%),
-bromoethanol (purity ≥ 95%) were purchased from TCI Europe and used
as received. 3-Bromo-1-propanol was purchased from Fluorochem (purity
7%) and used as received. BHT was purchase from Fluka (purity ≥ 99%)
1c, 2.8 g for 1d). The flask was sealed, and the mixture allowed to stir at
2
90 °C for 24 h. After cooling, the mixture was filtered in order to remove
the molecular sieves and the solvent evaporated. The crude products 3a-
d thus obtained were used as such for the next step without purification.
9
and used as received. Acryloyl chloride (stabilized with 400 ppm
phenothiazine) (purity ≥ 97%), triethylamine (purity ≥ 99%), diethylamine
2nd Step: Quaternization of crude -bromoalkyl acrylates 3a-d with
triethylamine
(
purity 98%), ethyl bromide (purity 98%), and hydroquinone (purity ≥ 99%)
were purchased from Sigma-Aldrich and used as received. NaHCO was
purchased from Panreac (purity 98-100 %) and used as received. Na SO
3
2
4
To a solution of the crude -bromoalkyl acrylate 3 deriving from the
previous step in anhydrous CHCl (9.6 mL for 3a; 2.0 mL for 3b; 2.6 mL
3
was purchased from Analar (purity 99.2%) and used as received.
Molecular sieves 4 A (pellets, 1.6 mm diameter) were purchased from
Sigma-Aldrich and activated for in oven at 300 °C for 4 h. Molecular sieves
for 3c; 2.7 mL for 3d), at room temperature in a Schlenk flask, was added
triethylamine (3.06 mL, 2.22 g, 21.9 mmol, for 3a; 580 L, 423 mg, 4.18
mmol, for 3b; 810 L, 590 mg, 5.83 mmol, for 3c; 840 L, 613 mg, 6.05
mmol, for 3d) dropwise. The flask was sealed, and the mixture allowed to
stir at 60 °C for 72 h. After cooling, diethyl ether (75 mL for 3a; 15 mL for
3
3
A (powder) were purchased from Alfa Aesar and activated for in oven at
00 °C for 4 h.
All reactions were analyzed by TLC on silica gel 60F254 (Merck) and by
GLC by using a Shimadzu GC-2010 gas chromatograph and capillary
columns with polymethylsilicone+5% phenylsilicone (HP-5) as the
stationary phase. Evaporation refers to the removal of solvent under
reduced pressure. Melting points are uncorrected. H NMR and 13C NMR
3b; 20 mL for 3c and 3d) was added dropwise and the solution was
maintained under stirring for 10 min. The resulting white precipitate was
filtered and washed with diethyl ether, to give the pure
acryloxyalkyltriethylammonium
bromide
(AATEAB):
1
acryloyloxyundecyltriethylammonium bromide (AUTEAB, 4a) was
a
spectra were recorded at 25 °C on
Spectrometer in CDCl solutions at 300 and 75 MHz, respectively, with
Me Si as internal standard. Chemical shifts () and coupling constants (J)
are given in ppm and in Hz, respectively. IR spectra were taken with a
a Bruker DPX Avance 300
colorless solid (mp = 40-41 °C; yield: 5.10 g, 63% based on starting 1a);
acryloyloxydodecyltriethylammonium bromide (4b) was a colorless solid
3
4
(mp = 65-67 °C; yield: 1.11 g, 70% based on starting 1b);
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acryloyloxynonyltriethylammonium bromide (4c) was a colorless solid (mp
Saccharomyces cerevisiae strain Y10000, wild-type, was provided by
EUROFAN resource center EUROSCARF (Frankfurt, Germany). The cells
were grown in rich medium containing 2% bactopeptone and 1% yeast
extract (YP), supplemented with fermentable 2% glucose as carbon
source.[7] The final pH was adjusted to 4.8. Candida albicans strain (ATCC
=
25-26 °C; yield: 1.22 g, 61% based on starting 1c);
acryloyloxyhexyltriethylammonium bromide (4d) was a colorless gummy
low-melting solid (yield: 1.39 g, 75% based on starting 1d).
10231) was provided by REMEL. This yeast strain was grown in
Preparation of acryloxyalkyltriethylammonium bromides (AATEABs)
Sabouraud dextrose broth containing 10g/ L peptone, 40g/ L dextrose. The
final pH was adjusted to 5.6.
4e and 4f
1st Step: Reaction of -bromoalkan-1-ols 1e and 1f with Et
2
NH
Disc-diffusion susceptibility testing
To a solution of 2-bromoethanol 1e (1.0 g, 8.0 mmol) or 3-bromo-1-
propanol 1f (2.0 g, 14.4 mmol) in EtOH (14.5 mL for 1e; 26.0 mL for 1f),
The antimicrobial susceptibilities of the novel synthetic ammonium salts
were evaluated using the Kirby-Bauer disc-diffusion method,[5,8] performed
according to CLSI guidelines and results were interpreted using CLSI
breakpoints.[9] DTAB discs were used as positive controls. The stock
solutions of DTAB and of all salts were prepared by dissolving them in
distillated water and were utilized to impregnate the Blank Antimicrobial
Susceptibility Disks (Oxoid™). Specifically, overnight cultures of
microorganisms tested were adjusted to a turbidity of 0.5 McFarland
standards (106 CFU/mL) before inoculation onto agar plates with sterile
cotton swabs. A cotton swab dipped in the cell culture was streaked onto
an agar plate surface in such a way as to obtain a uniform layer of bacteria
across the whole surface. After 10–15 min, DTAB or novel salts discs were
laid on the inoculated surface of the agar plates; then, all agar plates were
incubated at 37°C (bacteria) or 30°C (yeast), overnight. The diameters of
inhibition were measured and susceptibility was expressed in terms of
resistance (R), moderate susceptibility (I) and susceptibility (S). Agar
plates inoculated with microorganism tested with impregnated water discs
were used as controls. Analysis for each combination of microorganism
tested and discs with positive controls and with several new compounds
were repeated three times. Each experiment was performed with triplicate
samples.
maintained under nitrogen with stirring, was added dropwise Et
mL, 2.91 g, 39.8 mmol for 1e; 7.4 mL, 5.25 g, 71.6 mmol, for 1f). The
reaction mixture was heated at 60°C for 6 h. After cooling, CH Cl (30 mL
for 1e; 54 mL for 1f) was added, and the mixture was washed with a 5%
w/w solution of NaHCO (3 x 10 mL for 1e;3 x 20 mL for 1f) followed by
water (5 mL for 1e; 10 mL for 1f). Phases were separated, and the organic
phase dried over Na SO . After filtration and elimination of the solvent by
2
NH (4.1
2
2
3
2
4
distillation at atmospheric pressure, the products were obtained as
yellowish oils [2-(dimethylamino)ethanol 5e: 688.0 mg, 96%; 3-
(dimethylamino)propanol 5f: 1.85 g, 98%].
2nd Step: Esterification of 2-(diethylamino)alkanol 5e and 5f with
acryloyl chloride 2
To a solution of the 2-(diethylamino)alkanol [2-(diethylamino)ethanol 5e:
688 mg, 7.7 mmol; 3-(diethylamino)propanol 5f: 1.85 g, 14.1 mmol] in
anhydrous CH CN (7.7 mL for 5e; 14.1 mL for 5f), maintained at 0 °C under
3
nitrogen with stirring, was added dropwise acryloyl chloride (626 L, 697
mg, 7.7 mmol for 5e; 1.15 mL, 1.28 g, 14.1 mmol for 5f). The solution was
kept at 0°C for 2 h and then allowed to stir for 15 h at room temperature.
The reaction mixture was then neutralized with a 5 % w/w NaHCO
50 mL for 5e; 90 mL for 5f) and finally extracted with CH Cl (7 x 10 mL
for 5e; 7 x 20 mL for 5f). Phases were separated, and the organic phase
dried over Na SO . After filtration and elimination of the solvent by
distillation at atmospheric pressure, the products were obtained as
yellowish oils [(2-diethylamino)ethyl acrylate 6e: 750 mg, 57%; (3-
diethylamino)propyl acrylate 6f: 1.6 g, 61%)].
3
solution
Determination of MIC, MBC and MFC values
(
2
2
The minimal inhibitory concentration (MIC) of antibacterial compounds
was determined by broth dilution method, according to CLSI
recommendations. Briefly, a solution of each compound was diluted,
serially, with MHB or with the appropriated medium for yeast strains. Then,
the suspensions of the microorganisms, prepared from overnight cultures,
2
4
6
3rd Step: Quaternization of (diethylamino)alkyl acrylates 6e and 6f
with ethyl bromide
at a concentration of 10 cfu/mL, were added to each dilution in a 1:1 ratio.
Growth (or lack thereof) of the microorganisms was determined visually
after incubation for 24
h at 37°C (bacteria) or 30°C (yeast).
Dodecyltrimethylammonium Bromide (DTAB) was also included as
positive control. The lowest concentration at which there was no visible
growth (turbidity) was considered as MIC value. In order to determine
minimal bactericidal concentration (MBC) and minimum fungicidal
concentration (MFC), 10 µL aliquots of dilution representing MIC value,
and at least three of the more concentrated dilutions of all new synthesized
compounds and DTAB, were plated on Muller–Hinton agar or Sabouraud
dextrose agar or rich supplemented YP agar. Then the plates were
incubated for 24 h, at 37°C (bacteria) or 30°C (yeast). The MBC and MFC
values were determined as the lowest concentration that causes at least
The 2-(diethylamino)alkyl acrylate (6e: 750 mg, 4.38 mmol; 6f: 1.6 g, 8.64
mmol) was allowed to react with pure EtBr (0.22 mL, 318 mg, 2.92 mmol,
for 6e; 0.43 mL, 628 mg, 5.76 mmol, for 6f) in the presence of
hydroquinone (1.0 mg, 9.1×10-3 mmol, for 6e; 2.0 mg, 0.018 mmol, for 6f)
at 50 °C for 15 h. The crude product was purified after several washings
under stirring with cyclohexane (10 x 15 mL) followed by drying under
vacuum pump for 24-30 h. Products were tarry yellow oils
(acryloyloxyethyltriethylammonium bromide 4e: 650 mg, yield 53%;
acryloyloxypropyltriethylammonium bromide 4f: 1.1 g, 43%).
9
9.9% decrease in cfu/mL relative to the initial concentration [10]. The
Antimicrobial Tests
negative control tubes did not contained bacterial inoculum while the
positive control tubes (or turbid) were free of the compounds and they
contained only water. The tests were repeated three times in triplicate.
Bacterial and yeast strains
We used five bacterial strains: Staphylococcus aureus (ATCC 25923),
Streptococcus pyogenes (ATCC 19615), Escherichia coli (ATCC 25922),
Klebsiella pneumoniae (ATCC 13883), Pseudomonas aeruginosa (ATCC
Evaluation of the functional status of the bacterial cell wall
Propidium iodide (PI) was used to monitor the permeability of the
2
(
7853), provided by REMEL. The cells were grown in Muller Hinton Broth
MHB; Difco, Detroit, MI, USA) containing 2g/L beef infusion solids, 17.5
g/ L casein hidrolysate, 1.5 g/ L starch. The final pH was adjusted to 7.4.
membrane. The PI fluorescence increasing is related to the increasing of
the membrane permeability.[6] Staphylococcus aureus overnight cultures
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were diluted in MHB in order to reach OD~ 0.6 at the wavelength of 600
nm, then they were incubated at 37° C by shaking at 300 rpm. After 30
minutes of treatment, 500 L samples were collected, centrifuged at 3.500
rpm, and the medium was removed and replaced with 1 mM PI in PBS.
The samples were incubated in the dark at room temperature for 15 min
and then centrifuged at 3.500 rpm. The supernatant was removed and
replaced with 1X PBS. Bacterial cells were visualized using an OLYMPUS
BX 41 microscope; the images were taken with CSV1.14 software using a
CAMXC-30 for image acquisition. Fluorescent signal intensity was
assessed using a fluorescent plate reader (ex. 530 nm; em.620 nm).
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independent experiments, with ≥ 3 technical replicates per experiment,
unless otherwise stated. Statistical significance was measured using the
analysis of variance (ANOVA) test or student t-test. p ≤ 0.05 was
considered significant and all statistical tests were two sided.
1
14; c) S. A. Deowan, F. Galiano, J. Hoinkis, D. Johnson, S. A. Altinkaya,
G. Gabriele, N. Hilal, E. Drioli, A. Figoli, J. Membr. Sci. 2016, 510, 524-
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The work performed has received funding from the European
Union’s Horizon 2020 research and innovation programme under
grant agreement No 689427 for the project VicInAqua.
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O. I. Parisi, M. Fiorillo, A. Caruso, A. R. Cappello, C. Saturnino, F. Puoci,
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Keywords: antimicrobial polymers • antifouling membranes •
organic synthesis • polymerizable quaternary ammonium salts •
quaternary ammonium salts
p
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Entry for the Table of Contents
FULL PAPER
Acrylate surfactant monomers with
antimicrobial activity: This work
reports novel efficient synthetic
approaches to polymerizable
quaternary ammonium salts with
antimicrobial activity
Raffaella Mancuso,*,a Roberta Amuso,a
Biagio Armentano, Giuseppe Grasso,
O
b
a
HO(CH
2
)
n
Br
+
2 n 3
O(CH ) NEt Br
b
b
ADTEAB (n =12) increase outer
membrane permeability in
Vittoria Rago, Anna Rita Cappello,
1
0
c
c
Staphylococcus aureus cells
Francesco Galiano, Alberto Figoli,
Giorgio De Luca, Jan Hoinkis and
Bartolo Gabriele*,a
8
6
4
2
0
(ctrl: control; MIC: minimal
c
d
nhibitory concentration) ****
(
acryloxyalkyltriethylammonium
***
bromides, AATEABs), which are
useful precursor for the developing of
nanostructured membranes with anti-
biofouling properties in wastewater
treatment.
Page No. – Page No.
ns
Synthesis and antibacterial activity of
polymerizable acryloyloxyalkyltriethyl
ammonium salts
ctrl 1/2 MIC MIC 2 MIC
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