Synthesis, structure, surface and antimicrobial properties of new oligomeric quaternary ammonium salts with aromatic spacers

Article abstract of DOI:10.3390/molecules22111810

New dimeric, trimeric and tetrameric quaternary ammonium salts were accomplished by reaction of tertiary alkyldimethyl amines with appropriate bromomethylbenzene derivatives. A series of new cationic surfactants contain different alkyl chain lengths (C4-C18), aromatic spacers and different numbers of quaternary nitrogen atoms. The structure of the products was confirmed by spectral analysis (FT-IR, ¹H-NMR, ¹³C-NMR and 2D-NMR), mass spectroscopy (ESI-MS), elemental analysis, as well as PM5 semiempirical methods. Compound (21) was also analyzed using X-ray crystallography. Critical micelle concentration (CMC) of 1,4-bis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene dibromides (3-9) was determined to characterize the aggregation behavior. The antimicrobial properties of novel QACs (Quaternary Ammonium Salts) were examined to set their minimal inhibitory concentration (MIC) values against fungi Aspergillus Niger, Candida albicans, Penicillium chrysogenum and bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.

Full text of DOI:10.3390/molecules22111810

molecules
Article
Synthesis, Structure, Surface and Antimicrobial
Properties of New Oligomeric Quaternary
Ammonium Salts with Aromatic Spacers
Bogumił Brycki 1,*, Anna Koziróg 2, Iwona Kowalczyk 1, Tomasz Pospieszny 1, Paulina Materna 1
and Je˛drzej Marciniak 3
1
Laboratory of Microbiocides Chemistry, Faculty of Chemistry, Adam Mickiewicz University in Poznan´,
Umultowska 89b, 61-614 Poznan´, Poland; iwkow@amu.edu.pl (I.K.); tposp@amu.edu.pl (T.P.);
p_materna@wp.pl (P.M.)
Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences,
2
Lodz University of Technology, Wólczan´ska 171/173, 90-924 Łódz´, Poland; anna.kozirog@p.lodz.pl
Department of Materials Chemistry, Faculty of Chemistry, Adam Mickiewicz University in Poznan´,
3
Umultowska 89b, 61-614 Poznan´, Poland; jedrzej.marciniak@amu.edu.pl
*
Correspondence: brycki@amu.edu.pl; Tel.: +48-61-829-1694
Received: 12 October 2017; Accepted: 24 October 2017; Published: 25 October 2017
Abstract: New dimeric, trimeric and tetrameric quaternary ammonium salts were accomplished
by reaction of tertiary alkyldimethyl amines with appropriate bromomethylbenzene derivatives.
A series of new cationic surfactants contain different alkyl chain lengths (C4–C18), aromatic
spacers and different numbers of quaternary nitrogen atoms. The structure of the products was
1
confirmed by spectral analysis (FT-IR, H-NMR, 13C-NMR and 2D-NMR), mass spectroscopy
(ESI-MS), elemental analysis, as well as PM5 semiempirical methods.
was also analyzed using X-ray crystallography. Critical micelle concentration (CMC) of
1,4-bis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene dibromides ( ) was determined to
Compound (21)
3–9
characterize the aggregation behavior. The antimicrobial properties of novel QACs (Quaternary
Ammonium Salts) were examined to set their minimal inhibitory concentration (MIC) values against
fungi Aspergillus niger, Candida albicans, Penicillium chrysogenum and bacteria Staphylococcus aureus,
Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa.
Keywords: quaternary ammonium salts; oligomeric surfactants; rigid spacer; spectroscopy; synthesis;
CMC; antimicrobial properties
1. Introduction
Anionic, cationic, non-ionic and amphoteric surfactants form one of the most useful groups of
chemicals. The hydrophilic-hydrophobic balance (HLB) of these amphiphilic compounds is a crucial
key for their applications in detergents [
natural and synthetic dyes [ ], microbiocides [
leather and paper [ 12 13], agrochemicals [14], fibers [15
enhanced oil recovery [21 23], environmental protection (oil slick dispersant) [24
1
], personal care products [
], organic synthesis [
17], plastics [18], food processing [19
25], and explosives [26].
2
], paint and coatings additives [
10], pharmacy [ 11], textiles,
20],
3],
4,
5
6–
8
9
,
4,
1
,
,
–
,
–
,
Surfactants also play a significant role in the development of high technologies and preparation of
nano- and smart materials [27]. The global surfactant market exceeded 15 million tons in 2015 and
is still growing up a 4.20% CAGR (Compound Annual Growth Rate) [28]. The growing demand on
surfactants is mainly driven by personal care products and detergents (anionics and nonionics) as
well as anticorrosion and biocidal products (cationics). To avoid disadvantageous impacts of such
huge amounts of surfactants on the environment, a new type of more effective amphiphiles should
Molecules 2017, 22, 1810; doi:10.3390/molecules22111810
www.mdpi.com/journal/molecules

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be investigated and applied in the near future. One of the promising ways is Gemini surfactants.
These compounds contain two hydrophilic head groups and two hydrophobic tails linked by a spacer
at the head groups or close to them. The spacer can be either hydrophobic (polymethylene chain),
or hydrophilic (polymethylene chain with oxygen or nitrogen atoms). From a structural point of view
a spacer can be rigid (aromatic or unsaturated linear hydrocarbons) or flexible (polymethylene chain).
The neutral charge of the cationic Gemini surfactants is retained by the presence of counterions, which
can be organic or inorganic. The Gemini alkylammonium salts show a unique surface and interfacial
properties in aqueous solution. Critical micelle concentrations (CMC) of Gemini surfactants are usually
much lower, even up to 100 times in comparison to CMCs of the corresponding monomeric surfactants.
The effectiveness of dimeric surfactants in lowering the surface tension is also much better than their
monomeric analogs. The values of C20, i.e., surfactant concentration at which the surface tension (γ) is
lowered by 20 mN/m, are a dozen times smaller for Gemini surfactants in comparison to monomeric
surfactants [29].
The cationic Gemini surfactants adsorb very efficiently on metallic and nonmetallic surfaces
and owing to these properties they are used as very effective microbiocides and corrosion inhibitors.
Surface, anticorrosion and antimicrobial activity of Gemini surfactants with flexible polymethylene
spacers and polymethylene spacers modified by heteroatoms have been extensively studied so
far [30–37]. However there are limited data concerning Gemini and oligomeric surfactants with
rigid, benzene spacers [38–40].
Therefore we have undertaken a systematic study of oligomeric N-alkyl-N,N-dimethylbenzene
bromides,
i.e.,
1,4-bis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene
dibromides
17) and
23) in the
(2
–
9), 1,3,5-tris-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene tribromides (10–
1,2,4,5-tetrakis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene tetrabromides (18
–
scope of synthesis, structure, surface properties and antimicrobial activity.
2. Results and Discussion
In this work we report the synthesis, physicochemical properties, antimicrobial activity and
spectroscopic analysis (FT-IR, 1H-NMR, 13C-NMR, 2D-NMR) as well as ESI-MS spectrometry
and crystal structure of di- tri- and tetrameric alkylammonium surfactants with aromatic spacer.
PM5 semiempirical calculations using the WinMopac 2003 program were also performed [41–43].
2.1. Synthesis
Appropriate N,N-dimethyl-1-alkylamines were quaternized with stoichiometric
amounts of bromomethylbenzene derivatives in propanol to give a series of new cationic
surfactants:
1,3,5-tris-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene tribromides (10
tetrakis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene tetrabromides (18–23) (Scheme 1)
1,4-bis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene dibromides
(2–9),
–
17) and 1,2,4,5-
.
Experimental data of synthesized di- tri- and tetrameric alkylammonium surfactants are given in Table
S1 (Supplementary material). 1,2,4,5-tetrakis(bromomethyl)benzene (1c), which is not commercially
available, was prepared by reaction of 1,2,4,5-tetramethylbenzene with N-bromosuccinimide according
to known literature procedure [44]. In general, reaction time significantly extends with the increase of
the degree of oligomerization and the increase of the length of alkyl chain of amine.

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Scheme 1. Structures of di- (2–9) tri- (10–17) and tetrameric (18–23) alkylammonium surfactants with
aromatic spacer.
Melting points of the synthesized compounds in each series rise as alkyl chains lengthen . This
reflects a better packaging in the crystals (Figure 1).
Figure 1. Melting points of dimeric (2–9), trimeric (10–17) and tetrameric surfactants (18–23).

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2.2. Spectroscopic Study
1
2.2.1. H-NMR, 13C-NMR and 2D-NMR Spectra
1H-NMR chemical shifts of compounds 1c
–
23 are given in Tables S2 and S3 (Supplementary
material) whereas 13C-NMR chemical shifts are given in Tables S4 and S5 (Supplementary material).
1
Representative H-NMR and 13C-NMR spectra are shown in Figures S1 and S2 (Supplementary
material). The protons and carbons of terminal methyl groups in alkyl substituents resonate at the
lowest values of chemical shifts while those of methylene group attached to positively charged nitrogen
atoms are strongly deshielded and lie in the region of the higher values of chemical shifts. To resolve
chemical shifts of proton and carbon at methylene groups as well as aromatic carbons the 13C DEPT
spectrum was generated (compound 14, Figure 2). It was observed that the signals derived from
carbon atoms “a” are shifted towards the highest values, while slightly lower values are assigned to
quaternary carbon atoms in benzene “h”.
Figure 2. 13C DEPT spectrum of 1,3,5-tris[(N-(1-dodecyl)-N,N-dimethylammoniummethyl)benzene
tribromide (14).
To get more accurate information about chemical shifts, a correlation spectra COSY and HSQC
were measured. The conjunction of the HSQC and COSY allows correlation of the bonds between the
proton originating from one carbon atom and signals from nuclei of the same element, spaced by not
more than three bonds, respectively. Careful analysis has confirmed the structure of the compounds
and revealed a direct correlation between the atoms of hydrogen-hydrogen and carbon-hydrogen
bonds. Moreover, HMBC technique allowed correlation of the signals from the nuclei of various
elements spaced by 3 or 5 bonds (Figure S3, Supplementary material). Figure 3 shows HSQC spectrum
for compound 21 and Figure 4 shows HMBC spectrum for compound 14.

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Figure 3. HSQC spectrum (CD3OD) of 1,2,4,5-tetrakis[(N-(1-dodecyl)-N,N-dimethylammoniummethyl)
benzene tetrabromide (21).
Figure 4. HMBC spectrum (CD3OD) of 1,3,5-tris[(N-(1-dodecyl)-N,N-dimethylammoniummethyl)benzene
tribromide (14).
2.2.2. FT-IR Spectra
FT-IR spectra of 1,2-di(bromomethyl)benzene and compound
The characteristic features of the FT-IR spectra for dimeric ( ), trimeric (10
18 23) quaternary ammonium salts are a broad intense absorption of stretching asymmetric (ν
9
and 23 are shown in Figure 5.
17) and tetrameric
as) and
2–
9
–
(
–

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−1
νs) vibrations of methyl and methylene groups at 3000–2840 cm and deformation bands,
symmetric (
asymmetric and symmetric vibrations (
−1
δ
,
δ
as) of –CH3 group at 1470–1430 cm and 1395–1365 cm−1
) of –CH2 group at 1475–1450 cm−1. Moreover, absorption
bands of benzene ring, i.e., stretching vibrations of the (=C–H) group (
) at 3050–3010 cm−1, skeletal
vibrations of the C–C group at 1620–1430 cm−1 (four bands), in-plane deformation vibrations (δip) at
s
as well as deformation vibrations (
δ
ν
1250–950 cm−1 and out-of-plane deformation vibrations ( oop) at 900-650 cm−1 (of =C–H group) are
δ
also observed. The disappearance of the stretching vibrations band of C-Br group at 700–500 cm−1
upon quaternization confirm the formation of alkylammonium products (Figure 5a). The strong
absorption at the 3450–3350 cm−1 region in (23) arises from the OH stretching vibrations (Figure 5b).
(a)
100
80
60
40
20
0
4000
3000
2000
1000
-1
Wavenumber [cm ]
(b)
100
80
60
40
20
0
4000
3000
2000
1000
Wavenumber [cm-1]
Figure 5. FT-IR spectra (KBr pellets) of 1,2-di(bromomethyl)benzene (—) and 9 (—) (a) and 23 (—) (b).
2.2.3. PM5 Calculations
PM5 semiempirical calculations were performed using the WinMopac 2003 program.
Representative compounds ( 14 21) are shown in Figure 6. The final heats of formation (HOF)
17) and tetrameric (18 23) quaternary ammonium salts are presented
6,
,
for all dimeric (
2–
9
), trimeric (10
–
–
in Table 1.

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(a)
(b)
(c)
Figure 6. Molecular models of representative compounds
6 (a), 14 (b) and 21 (c) calculated by the
PM5 method.

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Table 1. Heat of formation (HOF) [kcal/mol] of di-, tri- and tetrabromosubstituted derivatives of
benzene (1a, 1b, 1c) and its quaternary ammonium salts (2–23).
Compound
Heat of Formation [kcal/mol]
∆HOF [kcal/mol]
1a
1b
1c
2
3
4
5
6
7
8
10.6060
5.6268
3.0680
−2.1145
–
–
–
8.4915
−24.5953
−46.7459
−68.8809
−91.0872
−113.3524
−135.6207
−158.0240
−34.9103
−68.3429
−101.8142
−135.2579
−155.2349
−213.0509
−233.2146
−266.9034
−94.7088
−130.8176
−184.1519
−228.7916
−280,4156
−325,1616
−13.9893
−36.1399
−58.2749
−80.4812
−102.7464
−125.0147
−147.4180
−5.6268
9
10
11
12
30
14
15
16
17
18
19
20
21
22
23
−62.7161
−107.4410
−140.8847
−160.8617
−218.6777
−238.8414
−272.5302
−97.7768
−133.8856
−187.2199
−231.8596
−283,4836
−328,2296
∆HOF = HOFcompounds (2–23) − HOFbromobenzene
.
The lowest values of HOF from among all quaternary ammonium salts (
2–23) are
observed for tetrameric (18 23) substituted derivatives. This is consistent with the results
–
obtained for 1,4-bis(bromomethyl)benzene (1a), 1,3,5-tris(bromomethyl)benzene (1b) as well as
1,2,4,5-tetrakis(bromomethyl)benzene (1c), wherein HOF is decreasing from disubstituted to
tetrasubstituted benzene derivative (Table 1). In general the HOF of 1,2,4,5-tetrasubstituted benzene
derivatives were lower than those of 1,3,5-tri- and 1,4-disubstituted benzene derivatives. A similar
relationship was observed in our previous work [45,46]. This is related to symmetry and the convertible
position of long hydrocarbon chains of molecules. Simultaneously, an increase of the length of an
alkyl chain also reduces HOF. These compounds are stabilized by electrostatic interactions that arise
from the quaternary nitrogen atoms as well as bromide anion. In addition, in the semiempirical
calculations we included the London Dispersion Forces between the hydrocarbon chains, which is
illustrated by the increase in melting compounds. Furthermore, the presence of two water molecules
stabilizes the molecule (21) and reduces the HOF to
nearly 100 kcal/mol lower than the isolated molecule (see Table 1).
−
328.4818 kcal/mol (Figure 7b). This value is
2.2.4. X-ray Analysis
1,2,4,5-Tetrakis-[N-(1-dodecyl)-N,N-dimethylammoniummethyl)benzene tetrabromide crystallizes
in the triclinic space group P-1. Molecular aggregation of compound (21) is governed by ionic
interactions between bromine anions and positively charged nitrogen atoms, and by attractive H-H
interactions between C12H25 hydrocarbon chains [47]. The water molecule is hydrogen-bonded to the
bromine anions. The composition of compound (21) molecules is reflected in the molecular packing.
The crystal of tetrameric quaternary ammonium salts with benzene ring (21) in spacer is built of
hydrophilic sub-layers containing the bromine anions and nitrogen cations, and of hydrophobic
sub-layers consisting of –C12H25 hydrocarbon chains. This results in a structure with alternating

Molecules 2017, 22, 1810
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hydrophilic and hydrophobic layers, in which the aggregation is governed by distinct forces. In the
hydrophilic layer, the ionic interaction between bromine and nitrogen is accompanied by eight CH···Br
and two OH···Br hydrogen bonds. Selected details for compound (21) about the crystal structure,
experiment and structure solution and refinement are given in Table 2. Figures 7a and 8 shows
molecular structure and crystal packing of compound (21) respectively. Summary of X-ray data for
(21) are given in CCDC 1554259.
Table 2. Crystal data and structure refinement for compound 21.
Empirical Formula
C66H138Br4N4O2
Formula weight
Temperature
1339,44
293(2) K
Wavelength
0.71073 Å
Crystal system, space group
Triclinic, P-1
a: 8.6269(17)
b: 9.2619(13)
c: 23.787(4)
α: 96.864(13)
β: 99.006(15)
γ: 90.157(14)
1863.3(6) Å3
2; 1194 mg/mm3
Unit cell dimensions
Volume
Z; Dx
−1
µ
2200 mm
F(000)
718
θ Range for data collection
Limiting indices
6.354–53.284
−10 ≤ h ≤ 10, −11 ≤ k ≤ 11, −29 ≤ l ≤ 29
27738/7298 (0,0111)
Reflections collected/unique
0.927
0.1306
0.1437
R(F) I > 2σ (I)
wR(F2)
max/min ∆ρ
−3
0.45/−0.40 e/Å
(a)
Figure 7. Cont.

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(b)
Figure 7. Comparison of the X-ray (a) and PM5 (b) structures of compound 21.
Figure 8. Crystal packing of compound 21.
2.2.5. Aggregation Behavior
The variation of the specific conductivity in aqueous solutions of the compounds (
function of the concentration was investigated. Conductivity measurements were carried out at 50 ◦C
6–
9) as a
due to low solubility of compounds in water. Representative conductometric profile of surfactant (
displayed in Figure 9.
7) is
◦
Figure 9. Specific conductivity (κ) versus concentration of 7 in water at 50 C.

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The CMC values of dimeric surfactants decrease with increasing number of carbon atoms in
the hydrophobic chain (Table 3). There was observed no clear dependence between the parameter
β
(counterion binding parameter) value and the length of hydrophobic chain in the molecule (Table 3).
It was calculated that compounds with dodecyl, tetradecyl and hexadecyl chain have the highest
values, which may be caused by a stronger binding counterion by aggregate molecules compared
to compounds with short alkyl chains. The average Gibbs energy takes negative values for all of
the surfactants what indicates that the aggregation process is spontaneous. The value of ∆
G◦mic for
compounds with longer alkyl (C12, C14 and C16) are much smaller what clearly show that these
compounds are less agile than the free ions in solution.
Table 3. Critical micelle concentration (CMC), counterion binding parameter (
of micellization (∆G mic) of compounds (3–9) obtained by conductivity measurements.
β
) and Gibbs free energy
◦
◦
Compound
CMC [mM]
∆G
[kJ/mol]
β
mic
3
4
5
6
7
8
9
12.38
11.67
7.27
1.2
0.92
0.48
0.29
−18.12
−19.15
−19.25
−39.01
−43.88
−38.81
−28.79
0.19
0.22
0.16
0.53
0.62
0.40
0.15
2.2.6. Antimicrobial Properties
Minimal inhibitory concentrations (MIC), i.e., minimum concentration of the preparation
at which further growth of tested microorganisms is stopped, of 1,4-bis-[N-(1-alkyl)-N,
N-dimethylammonium-methyl]benzene dibromides have been determined against bacteria, S. aureus,
B. subtilis, E. coli and P. aeruginosa as well as microscopic fungi, C. albicans, A. niger and P. chrysogenum
(Table 4).
Table 4. Minimal inhibitory concentrations (MIC [mM]) of 1,4-bis-[N-(1-alkyl)-N,N-dimethylammonium
-
methyl]benzene dibromides.
Compound S. aureus
B. subtilis
E. coli
P. aeruginosa
C. albicans
A. niger
P. chrysogenum
2
3
4
5
6
7
8
9
>12.5
6.2500
0.1953
0.0061
0.0122
0.0488
0.3906
0.3906
>12.5
12.5000
0.7812
0.0244
0.0244
1.5625
1.5625
6.2500
>12.5
6.2500
0.3906
0.0244
0.0244
0.7812
1.5625
6.2500
>12.5
12.5000
1.5625
0.0976
0.0976
1.5625
3.1250
3.1250
>12.5
6.2500
0.1953
0.0122
0.0122
0.0488
0.3906
0.3906
>12.5
12.5000
1.5625
0.1953
0.0976
0.1953
0.3906
0.7812
>12.5
6.2500
1.5625
0.0976
0.0488
0.0976
0.1953
0.7812
The MIC values of 1,4-bis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene dibromides for
bacteria range from 0.0122 to 12.5 mM. The shorter hydrocarbon chain is not able to penetrate the cell
wall and their minimal concentration, which inhibits growth of bacteria, is above 6 mM. The highest
antibacterial activity are observed for derivatives that have 10–12 carbon atoms in the alkyl substituent
(Figure 10). The elongation of the hydrocarbon chain to 14–18 methylene group causes increases the
MIC values. The least susceptible bacteria to chemical substances is Pseudomonas aeruginosa. These
Gram-negative rots very often cause the formation of biofilms on many surfaces in cosmetic and food
industry and also in medical environment.

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Figure 10. Antibacterial activity of compounds 2–9.
The range of MIC values of 1,4-bis-[N-(1-alkyl)-N,N-dimethylammonium-methyl]benzene
dibromides, analogues of benzalkonium chlorides (BAC), for microscopic fungi C. albicans, A. niger
and P. chrysogenum are the same like for bacteria 0.0122–12.5 mM (Table 4). However the lowest
concentrations, above 0.2 mM, have compounds that contain 10–14 carbon atoms in the alkyl
substituent (Figure 11). The highest MIC values are obtained for the derivatives with the shortest
C4–C8 alkyl chains. Among the three tested strains of microscopic fungi the least sensitive to the
effects of derivative BAC was A. niger.
Figure 11. Antifungal activity of compounds 2–9.

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Comparing the MIC values (Table 4) of all tested microorganisms, it was observed that the lowest
concentrations were obtained for 1,4-bis-[N-(1-dodecyl)-N,N-dimethylammoniummethyl]benzene
dibromide (6). Quaternary ammonium salts containing at least one chain of 12 carbon atoms are also
most commonly described in the literature. Among them is benzalkonium chloride (BAC), which
is often used in many industries and medicine as a component of biocidal preparations [48–50].
Received by Fazlara et al. and Merianos MIC values for BAC are generally higher compared
to 1,4-bis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene dibromides presented at work
(Table 5) [49,51]. Exceptions are the results obtained by El Hage et al., which obtained lower
benzalkonium chloride concentrations for S. aureus and partially A. niger [52]. The same species
of bacteria or fungi, but different strains may have different sensitivities for the same compound as
Koziróg and Brycki write in their work [53]. Thus, compounds containing decyl carbon chains
for MIC values for this strain may be ordered in ascending order as 1,4-bis-[N-(1-decyl)-N,N-
dimethylammoniummethyl]benzene dibromide (5) < BAC < Benzyl-N-decyl-dimethylammonium
bromide < N,N-dimethyl-N-(4-methylpyridyl)-N-decyl-ammonium chlorides. However, the MIC
results for compounds containing from 12 to 16 methylene groups in the chain are higher in comparison
with those obtained by El Hage et al., but lower than for pyridyl analogues of BAC [52,54].
Table 5. MIC values for benzalkonium chloride derivatives containing 10–16 carbon atoms in aliphatic
chains—literature data [49–52,54].
Microorganisms
Literature
Alkyl Chain
Chemical Substance
S. aureus
P. aeruginosa
A. niger
10
12
14
16
10
12
14
16
12
12
0.0048 **
0.0024 **
0.0006 **
0.0006 **
1.44 *
0.13 *
0.04 *
0.076 *
0.122 *
0.0028
1.25 *
0.312 *
0.0781 *
0.156 *
3.85
0.35
0.11
0.5
0.183
0.175
0.625 *
0.0781 *
0.0195 *
0.156 *
El Hage et al. [52]
BAC
Merianos [51]
nt
nt
nt
Fazlara et al. [49]
Feder-Kubis et al. [50]
10
12
14
16
1.5625 *
0.1953 *
0.0488 *
0.0244 *
1.5625
0.7812
0.1953
0.0976
12.499 *
1.5625 *
0.3906 *
0.1953 *
N,N-dimethyl-N-(4-
methylpyridyl)-N-alkyl-
ammonium chlorides
Brycki et al. [54]
El Hage et al. [52]
10
12
14
16
0.0195 **
0.0048 **
0.0024 **
0.0012 **
1.25 *
0.156 *
0.078 *
0.312 *
0.625 *
0.0781 *
0.0195 *
0.0781 *
Benzyl-N-alkyl-dimethyl-
ammonium bromide
(* or **) the same strains of the species; BAC benzyl-N-alkyl-dimethylammonium chloride.
3. Materials and Methods
3.1. General Information
All chemicals were commercial products from Sigma-Aldrich (Poznan´, Poland). Infrared spectra of
products were recorded in the KBr (in the frequency range 4000–400 cm−1) pellets in temperature 298 K
using Bruker FT-IR −IF1S 66/s spectrometer (Karlsruhe, Germany), evacuated to avoid water and CO2
absorptions, at 2 cm resolution. The 1H-NMR and 13C-NMR spectra were measured Varian Mercury
300/400 MHz spectrometer (Oxford, UK) in CDCl3 or CD3OD, and TMS was used as an internal
standard. 2D-NMR spectra of products were recorded using spectrometer Bruker Avance 600MHz
spectrometer (Billerica, MA, USA) operating at the frequencies 600.281 MHz and 150.947 MHz for 1H
and 13C respectively, and equipped with a 5 mm triple-resonance inverse probe head [1H/31P/BB]
with a self-shielded z gradient coil (90◦ 1H pulse width 9.0 s and 13C pulse width 13.3
µ µs) with CDCl3

Molecules 2017, 22, 1810
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as solutions. The ESI (electron spray ionization) mass spectra were recorded on a Waters/Micromass
(Manchester, UK) ZQ mass spectrometer equipped with a Harvard Apparatus (Saint Laurent, QC,
Canada), syringe pump. The standard ESI-MS mass spectra were recorded at the cone voltage 30 V.
Melting point was determined using Stuart SMP10 apparatus. Single-crystal diffractometer Xcalibur
EOS CCD with Mo Kα X-ray source was used for diffraction measurements. The sample solutions were
prepared in methanol at the concentration of approximately 10−5 M. Determination of the UB-matrices
and initial data reduction were performed using the CrysAlisPro program suite [54]. CCDC 1554260
contains the supplementary crystallographic data for this paper. These data can be obtained free of
charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk). All structures
were solved by direct methods with SHELXS and refined with anisotropic displacement parameters
for non-H atoms by program SHELXL-97, using the OLEX2 interface [55,56]. Hydrogen atoms were
located in the ideal position from molecular geometry and refined according the “riding” model.
3.2. Synthesis
1,2,4,5-tetrakis(bromomethyl)benzene (1c) was obtained by the reaction of 1,2,4,5-tetramethylbenzene
(2.8 g; 20 mmol) with NBS (14.4 g; 80 mmol) and benzoyl peroxide (1 g; 5.9 mmol) in tetrachloromethane
(50 mL). Reagents were heated at 77 ◦C for 50 min. The precipitate was filtered off under reduced
pressure and washed with hot tetrachloromethane. The filtrate was concentrated to small volume and
cooled to 4 ◦C. Crude product was filtrated and purified by crystallization from methanol. Rt< 1 h,
◦
white solid (15%), m.p. 159–160 C. 1H-NMR (CDCl3)
δ
: 7.37 ppm (2H, a), 4.60 ppm (8H, b). 13C-NMR
max: 3026, 2980, 2933, 1504, 1456,
(CDCl3) : 137.6 ppm (a), 133.6 ppm (h), 28.7 ppm (b). FT-IR (KBr)
δ
ν
1210, 911, 797, 678, 598.
Series of dimeric quaternary ammonium salts (2–9).
1,4-bis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene dibromides (2–9) were synthesized
of 1 equivalent of 1,4-di(bromomethyl)benzene (0.5 g; 1.89 mmol) (1a) with 2 equivalents (3.78
mmol) of N-butyl-N,N-dimethylamine (0.38 g), N-hexyl-N,N-dimethylamine (0.49 g), N,N-dimethyl-N-
octylamine (0.59 g), N-decyl-N,N-dimethylamine (0.7 g), N-dodecyl -N,N-dimethylamine
(0.81 g), N,N-dimethyl-N-tetradecylamine (0.91 g), N-hexadecyl -N,N-dimethylamine (1.02 g),
N,N-dimethyl-N-octadecylamine (1.13 g), respectively, by heating in n-propanol from 4 h to 10 h.
White solids were obtained. The crude products were purified by recrystallization from a mixture of
acetone/methanol (10:1) (Supplementary material).
Series of trimeric quaternary ammonium salts (10–17).
1,3,5-tris-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene tribromides
(10–17) were
synthesized of 1 equivalent of 1,3,5-tri(bromomethyl)benzene (0.5 g; 1.4 mmol) (1b) with 3
equivalents (4.2 mmol) of N-butyl-N,N-dimethylamine (0.43 g), N-hexyl-N,N-dimethylamine
(0.54 g), N,N-dimethyl-N-octylamine (0.66 g), N-decyl-N,N-dimethylamine (0.77 g), N-dodecyl-N,
N-dimethylamine (0.9 g), N,N-dimethyl-N-tetradecylamine (1.03 g), N-hexadecyl-N,N-dimethylamine
(1.13 g), N,N-dimethyl-N-octadecylamine (1.25 g), respectively, by heating in n-propanol from 4 h
to 15 h. White solids were obtained. The crude products were purified by recrystallization from a
mixture of acetone/methanol (10:1) (Supplementary material).
Series of tetrameric quaternary ammonium salts (18–23).
1,2,4,5-tetrakis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene tetrabromides (18–23) were
synthesized of 1 equivalent of 1,2,4,5-tetra(bromomethyl)benzene (0.5 g; 1.12 mmol) (1c) with 4
equivalents (4.48 mmol) of N-hexyl-N,N-dimethylamine (0.58 g), N,N-dimethyl-N-octylamine (0.7 g),
N-decyl-N,N-dimethylamine (0.83 g), N-dodecyl-N,N-dimethylamine (0.95 g), N,N-dimethyl-N-
tetradecylamine (1.08 g), N-hexadecyl-N,N-dimethylamine (1.23 g), respectively, by heating in

Molecules 2017, 22, 1810
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n-propanol from 7 h to 14 h. White solids were obtained. The crude products were purified by
recrystallization from a mixture of acetone/methanol (10:1) (Supplementary material).
3.3. Critical Micellization Concentration
Measurements of conductivity were obtaining using a CO300 conductivity meter (VWR, Gdansk,
Poland) with graphite electrode in conjunction with a thermostat. CMC values of compounds 3–9 and
14 were obtained from conductometric titrations conducted in double distilled water in 50 ◦C.
3.4. Antimicrobial Properties
The MIC values of 1,4-bis-[N-(1-alkyl)-N,N-dimethylammoniummethyl]benzene dibromides
(from to 18) against bacteria and microscopic fungi (yeast and molds) were measured at the Institute
4
of Fermentation Technology and Microbiology, Technical University of Lodz in Poland. The MICs
are defined as the lowest concentration of the compounds at which there was no visible growth
of microorganisms. MIC values were determined by a standard tube 2-fold dilution method [29].
The experiments were carried out on the Gram-positive bacteria: Staphylococcus aureus ATCC 6538,
Bacillus subtilis NCAM01644, Gram-negative bacteria: Escherichia coli ATCC 10536, Pseudomonas
aeruginosa ATCC 85327, yeast: Candida albicans ATCC 10231, and molds: Aspergillus niger ATCC 16404,
Penicillium chrysogenum ATCC 60739. Antifungal properties were checked on a Malt Extract Broth
medium (Merck, Darmstadt, Germany) at a density of inoculum of 1–2
×
106 cfu/mL; antibacterial
107 cfu/mL. All tests were
activity—on Trypticase Soy Broth (Merck), density of inoculum 1–2
repeated three times.
×
4. Conclusions
Three series of dimeric, trimeric and tetrameric quaternary alkylammonium salts with benzene
spacers were synthesized by SN2 reaction of bromomethylbenzene derivatives with tertiary
alkyldimethylamines with good yield. The structure and purity of new surfactants were confirmed
1
by FT-IR, H-NMR, 13C-NMR and 2D-NMR, mass spectroscopy (ESI-MS) and elemental analysis.
The structure of one dimeric surfactant (9) was confirmed by X-ray analysis. The PM5 semi-empirical
method proved that tetrameric surfactants are more stable than trimeric and dimeric ones. Moreover,
it has been shown that molecules of water additionally stabilize the structure of the surfactant.
Aggregation behavior of the synthesized compounds depends on the substituent, and CMC
values decrease as alkyl chain lengthens. Dimeric surfactants, which are soluble in water, exhibit
strong antimicrobial activity against bacteria (Staphylococcus aureus, Bacillus subtilis, Escherichia coli,
Pseudomonas aeruginosa) and fungi (Aspergillus niger, Candida albicans, Penicillium chrysogenum).
The biocidal efficacy depends on the length of alkyl chain and is the strongest for decyl and
dodecyl derivatives.
Supplementary Materials: Supplementary materials are available online. Figure S1: 2D NMR spectra of
compound 21: COSY (a), HSQC (b) and HMBC (c), Table S1: Experimental data of synthesized di- tri- and
tetrameric alkylammonium surfactants with aromatic spacer and with different chain lengths, Table S2: The 1H
NMR chemical shifts (ppm) of compounds 1c-10
11-23 Table S4: The 13C NMR chemical shifts (ppm) of compounds 1c-10
shifts (ppm) of compounds 11-23.
,
Table S3: The 1H NMR chemical shifts (ppm) of compounds
,
,
Table S5: The 13C NMR chemical
Acknowledgments: This work has been supported by the National Centre for Research and Development (Poland;
TANGO1/266340/NCBR/2015).
Author Contributions: B.B. conceived and designed the experiments; A.K. performed, analyzed and interpreted
the microbiological experiments; I.K., T.P. performed semiempirical calculations; P.M. carried out the synthetic
work, CMC and spectroscopic measurements; J.M. solved the crystal structure; B.B., A.K., I.K., T.P., P.M., J.M.
wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2017, 22, 1810
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©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).