Synthesis, molecular structure, spectral properties and antifungal activity of polymethylene-α,ω-bis(n,n-dimethyl-n-dodecyloammonium bromides)

Article abstract of DOI:10.3390/molecules16010319

Hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) (1), pentamethylene-1,5-bis(N,N-dimethyl-N-dodecylammonium bromide) (2), tetramethylene-1,4-bis(N,N-dimethyl-N-dodecylammonium bromide) (3), trimethylene-1,3-bis(N,N-dimethyl-N-dodecylammonium bromide) (4) and ethylene-1,2-bis(N,N-dimethyl-N-dodecylammonium bromide) (5) have been obtained and characterized by FTIR and NMR spectroscopy. DFT calculations have also been carried out. The optimized bond lengths, bond angles and torsion angles calculated by Hartree-Fock/3-21G(d,p) approach have been presented. MIC values for A. niger, P. chrysogenum, C. albicans have been determined and the relationship between MIC and spacer length has been discussed.

Full text of DOI:10.3390/molecules16010319

Molecules 2011, 16, 319-335; doi:10.3390/molecules16010319
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Molecular Structure, Spectral Properties and
Antifungal Activity of Polymethylene-α,ω-bis(N,N-dimethyl-N-
dodecyloammonium bromides)
1
,
1
2
Bogumil Brycki *, Iwona Kowalczyk and Anna Kozirog
1
Laboratory of Microbiocides Chemistry, Faculty of Chemistry, Adam Mickiewicz University,
Grunwaldzka 6, 60-780 Poznań, Poland
2
Institute of Fermentation Technology and Microbiology, Technical University of Lodz,
Wolczanska 171/173, 90-924 Lodz, Poland
*
Author to whom correspondence should be addressed; E-Mail: brycki@amu.edu.pl.
Received: 22 December 2010; in revised form: 27 December 2010 / Accepted: 4 January 2011 /
Published: 5 January 2011
_
_________________________________________________________________________________
Abstract: Hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) (1),
pentamethylene-1,5-bis(N,N-dimethyl-N-dodecylammonium bromide) (2), tetramethylene-
1
,4-bis(N,N-dimethyl-N-dodecylammonium bromide) (3), trimethylene-1,3-bis(N,N-
dimethyl-N-dodecylammonium bromide) (4) and ethylene-1,2-bis(N,N-dimethyl-N-
dodecylammonium bromide) (5) have been obtained and characterized by FTIR and NMR
spectroscopy. DFT calculations have also been carried out. The optimized bond lengths,
bond angles and torsion angles calculated by Hartree-Fock/3-21G(d,p) approach have been
presented. MIC values for A. niger, P. chrysogenum, C. albicans have been determined and
the relationship between MIC and spacer length has been discussed.
Keywords: polymethylene-α,ω-bis(N,N-dimethyl-N-dodecyloammonium bromides); FTIR
and NMR spectra; DFT calculations; antifungal activity
_
_________________________________________________________________________________
1
. Introduction
Quaternary ammonium salts were introduced as antimicrobial agents by Domagk over seventy years
ago [1]. The first generation of quaternary ammonium compounds (QAC) were standard benzalkonium

Molecules 2011, 16
320
chlorides, i.e., alkylbenzyldimethyl-ammonium chloride, with specific alkyl distributions, i.e., C12
,
4
0%; C14, 50% and C16, 10% [2]. The second generation of QAC was obtained by substitution of
aromatic rings in alkylbenzyldimethylammonium chlorides by chlorine or alkyl groups to get the
products like alkyldimethylethylbenzylammonium chloride with alkyl distributions of C , 50%; C ,
1
2
14
3
0%; C16, 17% and C18, 3%. The dual quaternary ammonium salts are the third generation of QAC.
These products are a mixture of equal proportion of alkyldimethylbenzylammonium chloride with
alkyl distribution C , 68%; C , 32% and alkyldimethylethylbenzylammonium chloride with alkyl
12
14
distribution C12, 50%; C14, 30%; C16, 17% and C18, 3%. The twin chain quaternary ammonium salts,
like didecyldimethylammonium chloride are the fourth generation of QAC. The concept of the
synergistic combination in the dual QACs has been applied to twin chain quaternary ammonium salts.
The mixture of dialkyldimethylammonium chloride (dioctyl, 25%; didecyl, 25%, octyldecyl, 50%)
with benzalkonium chloride (C , 40%; C , 50%; C , 10%) is the newest blend of quaternary
1
2
14
16
ammonium salts which represents the fifth generation of QACs [2]. Because of the increasing
resistance of microorganisms to commonly used disinfectants, the synthesis of new types of
microbiocides is a very important topic. One of the new groups with good antimicrobial activity are
cyclic quaternary ammonium salts [3]. Some cyclic quaternary ammonium salts have been obtained
previously by intramolecular cyclisation of amine derivatives [4-9]. Another way, i.e., reaction of alkyl
halides with cyclic amines can lead to chiral cyclic quaternary ammonium salts [10].
In recent years the number of applications of quaternary ammonium salts has increased
considerably. They are used as biocides [11-15], and phase-transfer catalysts, especially in
enantioselective reactions [16-21]. Pyrrolidinium salts are analogues of oxotremorine and are used as
muscarinic agonist [5]. Some of quaternary ammonium salts exist as ionic liquids, which can be used
as ”green solvents” [22-26] and electrolytes for liquid batteries [27,28].
In this work we report the synthesis, FTIR and NMR spectroscopy, DFT calculations and
antimicrobial properties of polymethylene-α,ω-bis(N,N-dimethyl-N-dodecyloammonium bromides) 1-
5
. These compounds belong to a new class of quaternary ammonium salts, the so called gemini
surfactants, where two tertiary amines are connected at the nitrogen atoms by a spacer. For symmetric
gemini surfactants the notation [m-s-m] is applicable, where m is the number of carbon atoms in alkyl
chain and s is a number of methylene groups in a spacer. Antimicrobial activity of [12-s-12] against
Staphylococcus aureus depends on the length of spacer, and for s = 4 MIC is four times lower than for
s = 2 [29]. Gemini alkylammonium surfactants are very promising microbiocides.
2
. Results and Discusion
2.1. Synthesis
Polymethylene-α,ω-bis(N,N-dimethyl-N-dodecyloammonium bromides) 1-4 have been obtained by
reaction of N,N-dimethyl-N-dodecylamine with 1,6-dibromohexane, 1,5-dibromopentane, 1,4-dibromo-
butane and 1,3-dibromopropane, respectively. Ethylene-1,2-bis(N,N-dimethyl-N-dodecyloammonium
bromide) (5) has been obtained by reaction of N,N,N’,N’-tetramethylethylenediamine with 1-bromo-
decane. The reaction times were significantly shorter in comparison to procedures described in the
literature, which is due to the more polar solvent that we used. The reaction yields were very high and

Molecules 2011, 16
321
varied from 83 to 92%. The analysis of melting points of gemini surfactants 1-5 shows the relationship
between m.p. and the number of carbon atoms in the spacer (Figure 1).
Figure 1. The relationship between melting points of 1-5 and number of carbon atoms in the spacer.
2
2
2
2
1
1
30
20
10
00
90
80
1
2
3
4
5
6
7
Number of carbon atoms in the spacer
A higher number of carbon atoms in the spacer corresponded to a higher melting point. This clearly
suggests stronger hydrophobic interactions between the hydrocarbon chains with elongation of spacer
and better packaging in the crystal. Solubility in water of polymethylene-α,ω-bis(N,N-dimethyl-N-
dodecyloammonium bromides) also depends on the spacer length. Ethylene-1,2-bis(N,N-dimethyl-N-
dodecyloammonium bromide) (5) is soluble in water below 0.1% wt./wt. while hexamethylene-1,6-
bis(N,N-dimethyl-N-dodecylammonium bromide) (1) is readily soluble in water.
2.2. DFT calculations
The structure and numbering for 1-5 are given in Figure 2. The structures optimized at the
HartreeFock/3-21G(d,p) level of theory are shown in Figure 3. The geometry parameters, energy and
dipole moments computed using the Hartree-Fock/3-21G(d,p) method are given in Table 1. The
calculated energy depends on the number of carbon atoms in the spacer. The relative stabilizing energy
ΔE (a.u.) is a difference between calculated energy for 5, i.e., compound with ethylene group as a spacer
and for 1, i.e., a compound with a hexamethylene spacer (Table 1). According to this assumption the most
stable is hexamethylene-1,6-bis(N,N-dimethyl-N-dodecylammonium bromide) (1, Figure 4).

Molecules 2011, 16
Figure 2. Structures and numbering of hexamethylene-1,6-bis(N,N-dimethyl-N-dodecyl-
322
ammonium bromide) (1), pentamethylene-1,5-bis(N,N-dimethyl-N-dodecylammonium
bromide) (2), tetramethylene-1,4-bis(N,N-dimethyl-N-dodecylammonium bromide) (3),
trimethylene-1,3-bis-N,N-dimethyl-N-dodecylammonium bromide) (4) and ethylene-1,2-
bis(N,N-dimethyl-N-dodecylammonium bromide) (5).
(1)
(2)
i
g
e
c
N
a
Br
k
5
d
b
h
f
j
l
l
j
k
h
f
d
b
Br
N
g
e 5
a
c
i
(
3)
(4)
i
g
e
c
N
a
Br
5
d
b
h
f
j
k
j
h
f
d
b
Br
N
i
g
e 5
a
c
(5)

Molecules 2011, 16
Figure 3. Structures of 1-5 optimized by the Hartree-Fock/3-21G(d,p) method; (a)
323
hexamethylene-1,6-bis(N,N-dimethyl-N-dodecylammonium bromide) (1), (b) penta-
methylene-1,5-bis(N,N-dimethyl-N-dodecylammonium bromide) (2), (c) tetramethylene-
1
,4-bis(N,N-dimethyl-N-dodecylammonium bromide) (3), (d) trimethylene-1,3-bis(N,N-
dimethyl-N-dodecylammonium bromide) (4) and (e) ethylene-1,2-bis(N,N-dimethyl-N-
dodecylammonium bromide) (5).
a)
b)
c)
d)
e)

Molecules 2011, 16
324
Table 1. Selected parameters of investigated compounds 1-5 calculated by the Hartree-
Fock/3-21G(d,p) method.
Parameters
Energy (a.u)
1
2
3
4
5
-6551.697749
20.4558
-6412.885113 -6474.064183
-6396.071637 -6357.506938
Dipole moment
17.1183
4.7632
7.1234
6.5095
(
Debye)
Bond length (Å)
+
-
N …Br
3.895
3.932
3.902
3.727
3.551
3.862
3.954
3.674
3.477
4.116
4.116
3.297
3.213
1.550
3.638
3.768
3.383
3.111
4
.235
-
-
-
C(i)-H…Br
C(j)-H…Br
C(l)-H…Br
3.681
3.859
3.851
-
C(h)-H…Br
N-C(h)
3.661
1.536
1.501
1,540
1.538
1.535
4.003
1.534
1.503
1.539
1.528
1.533
1.537
1.506
1.520
1.540
1.533
1.550
1.529
1.550
N-C(i)
1.536
1.541
N-C(j)
C(j)-C(k)
C(h)-C(g)
1.541
1.544
o
Bond angle ( )
N-C(h)-C(g)
N-C(j)-C(k)
C(j)-N-C(h)
C(i)-N-C(h)
115.7
115.0
110.0
107.1
116.4
116.7
113.0
108.3
116.2
115.7
111.6
108.1
114.5
110.2
108.2
110.6
114.5
-
110.8
113.4
o
Dihedral angle ( )
N-C(h)-C(g)-C(f)
N-C(j)-C(k)-C(l)
C(i)-N-C(h)-C(g)
C(i)-N-C(j)-C(k)
C(a)-C(b)-C(c)-C(d)
C(j)-N-C(h)-C(g)
167.4
160.0
168.7
43.7
177.8
-158.9
56.6
179.9
-179.9
175.4
148.5
180.0
58.6
-179.4
-
-174.0
-
-69.7
161.4
180.0
172.9
-49.1
-
-176.4
-179.6
172.1
179.7
47
180.0
-166.2
The highest stabilizing energy of 1 is a consequence of intramolecular hydrophobic interactions
between two alkyl chains (Figure 3a). In compound 5 with an ethylene spacer, the distance between two
nitrogen atoms is 3.8 Ǻ, whereas the thickness of the dodecyl group is over 5.1 Ǻ. These distances were
calculated using the data from Table 1. Because of the N···N distance and geometry conditions, the
structure of 5 with an ethylene group in the spacer is much more open and two dodecane chains cannot
interact each other. Thus the stabilizing energy of 5 is low. In general as the spacer becomes longer the
alkyl chains get closer together and compounds are more stabilized. In case of pentamethylene-1,5-
bis(N,N-dimethyl-N-dodecylammonium bromide) (2) the stability of the structure is slightly decreased by
conformational strain (Figure 4). Additionally, bromide anions in 1-4 are engaged in three non-linear
weak intramolecular interactions with carbon atoms (Figure 3, Table 1).

Molecules 2011, 16
325
Figure 4. The relationship between number of carbon atoms in the spacer and relative
energy ΔE (a.u.) of 1-5.
2
1
1
00
50
00
5
0
0
1
2
3
4
5
6
Number of carbon atoms in the spacer
In 5 the bromide atom forms only two non-linear weak intramolecular C-H···Br interactions. Bromide
+
-
anions interact also via Coulombic attractions with positively charged nitrogen atoms. The N ···Br
distances are given in Table 1. The conclusions concerning stability of 1-5 in gas phase are in accordance
with the stability of 1-5 in the solid state expressed by an increasing melting points for compounds with
longer spacer.
2
.3. FTIR spectra study
The FTIR spectra of polymethylene-α,ω-bis(N,N-dimethyl-N-dodecyloammonium bromides) 1-5
were measured in KBr pellets at 20 °C.
Figure 5. FTIR spectrum of ethylene-1,2-bis(N,N-dimethyl-N-dodecylammonium bromide) (5).
1
00
80
60
40
20
0
3
500 3000 2500 2000
1500
1000
500
-1
Wavenumbers/cm

Molecules 2011, 16
326
The spectra show typical bands of stretching asymmetric (νas) and symmetric (ν
a
) vibrations as well
-1
-1
as bands of deformation vibrations (δ), at 2850-2980 cm and 1360-1490 cm , respectively (Figure 5).
No significant changes were observed between the FTIR spectra of 1 with the longest spacer and 5
with the shortest spacer.
1
13
2
.4. H-NMR and C-NMR spectra
The proton chemical shift assignments (Table 2) of polymethylene-α,ω-bis(N,N-dimethyl-N-
dodecyloammonium bromides) 1-5 are based on 2D COSY experiments, in which the proton-proton
connectivity is observed through the off-diagonal peaks in the counter plot (Figure 6). The relations
1
13
between the experimental H and C chemical shifts (δ ) and the GIAO (Gauge-Independent Atomic
exp
Orbitals) isotropic magnetic shielding tensors (σcalc) are shown in Figures 5 and 6. Both correlations
are linear, described by the relationship: δexp = a + b·σcalc The a and b parameters are given in Table 2.
It has been reported in the literature [20] that the correlation between the experimental chemical shifts
and calculated isotropic screening constants are usually better for carbon-13 atoms than for protons.
The protons are located on the periphery of the molecule, thus are more sensitive to solute-solvent
interactions than carbon atoms which are more hidden. For this reason the correlation between the
experimental and calculated data for protons is worse than that for carbon atoms. The differences
between calculated and experimental shifts for protons and carbons are due to different phases. The
calculated shifts describe single molecules in the gas phase, while experimental shifts include all
interactions in the condensed phase.
3
Table 2. Chemical shifts (δ, ppm) in CD Cl calculating GIAO nuclear magnetic shielding
tensors (σcalc) for hexamethylene-1,6-bis(N,N-dimethyl-N-dodecylammonium bromide) (1)
and ethylene-1,2-bis(N,N-dimethyl-N-dodecylammonium bromide) (5). The predicted
GIAO chemical shifts were computed from the linear equation δexp. = a + b·σcalc with a and
b determined from the fit the experimental data ( r is the correlation coefficient).
δexp.
δcalc
σcalc
δexp.
δcalc
σcalc
Hexamethylene-1,6-bis(N,N-dimetyl-N-dodecyldodecylammonium bromide) (1)
Carbon-13
C (a)
C (b)
C (c)
C (d)
C (e)
C (f)
C(g)
C(h)
C(i)
Proton
H (a)
13.84
22.63
31.60
29.17
29.17
26.06
22.38
64.38
50.73
63.81
21.51
24.42
14.98
21.93
30.49
29.62
29.35
24.74
24.60
67.19
49.27
199.37
193.49
186.25
186.98
191.11
191.23
155.19
170.35
159.70
192.31
193.11
0.88
1.25
1.25
1.25
1.25
1.25
1.72
3.52
3.39
3.69
1.98
1.56
1.16
1.27
1.21
1.22
1.24
1.22
2.03
2.92
3.35
32.767
32.644
32.711
32.699
32.676
32.704
31.787
30.790
30.308
29.547
32.068
32.339
H (b)
H(c)
H (d)
H (e)
H (f)
H(g)
H(h)
H(i)
C(j)
C(k)
C(l)
61.86
23.33
22.38
H(j)
H(k)
H(l)
4.03
1.78
1.54
a
250.5599
-1.1816
0.98999
30.40303
-0.892456
0.93883
b
2
r

Molecules 2011, 16
327
Table 2. Cont.
Ethylene-1,2-bis-(N,N-dimethyl-N-dodecylammonium bromide) (5)
Carbon-13
C (a)
C (b)
C (c)
C (d)
C (e)
C (f)
C(g)
C(h)
C(i)
Proton
H (a)
14.3
15.84
22.64
30.32
29.62
29.31
24.67
23.77
62.89
50.24
199.21
192.97
185.92
186.56
186.85
191.10
191.93
156.04
167.64
157.57
0.88
1.30
1.25
1.25
1.25
1.25
1.81
3.67
3.43
4.55
1.24
1.35
1.29
1.30
1.31
1.31
1.33
3.69
3.11
32.732
32.568
32.648
32.637
32.620
32.623
32.598
28.976
29.866
27.423
22.99
31.84
29.53
29.53
26.20
22.60
65.54
51.29
56.51
H (b)
H(c)
H (d)
H (e)
H (f)
H(g)
H(h)
H(i)
C(j)
60.78
H(j)
4.71
a
232.9141
-1.0897
0.98592
22.6482
-0.6541
0.96714
b
2
r
Figure 6. COSY NMR spectrum for hexamethylene-1,6-bis(N,N-dimethyl-N-
dodecyldodecylammonium bromide) (1).
3
Figure 7. Experimental chemical shifts (δexp, CD Cl) in hexamethylene-1,6-bis(N,N-
dimethyl-N-dodecylammonium bromide) (1) vs. the isotropic magnetic shielding (σcalc)
13
1
from the GIAO/HF/3-21G(d,p) calculations for molecules δexp = a + b·σcalc : (a) C and (b) H.
5
6
5
4
3
2
1
0
0
0
0
0
0
a)
b)
4
3
2
1
0
1
50 160 170 180 190 200
29
30
31
32
33
σcalc [ppm]
σcalc [ppm]

Molecules 2011, 16
328
3
Figure 8. Experimental chemical shifts (δexp, CD Cl) in ethylene-1,2-bis(N,N-dimethyl-N-
dodecylammonium bromide) (5) vs. the isotropic magnetic shielding (σcalc) from the
1
3
1
GIAO/HF/3-21G(d,p) calculations for molecules δexp = a + b σcalc : (a) C and (b) H.
70
60
50
40
30
20
10
5
4
3
2
1
0
b)
a)
1
50 160 170 180 190 200 210
27
28
29
30
31
32
33
σcalc [ppm]
σcalc [ppm]
2.5. Antimicrobial activity
Minimal inhibitory concentration (MIC) values for all polymethylene-α,ω-bis(N,N-dimethyl-N-
dodecyloammonium) bromides against A. niger ATCC 16404 and A. niger LOCK 0439 are higher
than those against P. chrysogenum LOCK 0531 and C. albicans ATCC 10231 (Table 3).
Table 3. The minimal inhibitory concentration (MIC) (μM/mL) of polymethylene-α,ω-
bis(N,N-dimethyl-N-dodecyloammonium bromides) (1-5) for conidia and vegetative cells.
Strains
Compound
A.niger
ATCC 16404
0.12
P.chrysogenum
LOCK 0531
0.06
A.niger
LOCK 0439
0.12
C.albicans
ATCC 10231
0.015
(
(
(
(
(
1)
2)
3)
4)
5)
0.12
0.15
0.3
0.3
0.06
0.095
0.15
0.15
0.24
0.375
0.4
0.015
0.037
0.075
0.075
0.4
Aspergillus niger is a fungus, which are very resistant to chemical disinfectants, what make them
very difficult to removed from the surface and air. The highest antifungal activity was shown by
hexamethylene-1,6-bis(N,N-dimethyl-N-dodecylammonium bromide) (1) and pentamethylene-1,5-
bis(N,N-dimethyl-N-dodecylammonium bromide) (2). In general for all fungal strains a longer spacer
corresponds to a lower MIC (Figure 9). This phenomenon may result from the fact that polymethylene-
α,ω-bis(N,N-dimethyl-N-dodecyloammonium bromides) with long spacers are more flexible and
connect more easily with the conidial surface. The MIC value of dodecyltrimethylammonium chloride,
which is a monomer analog of polymethylene-α,ω-bis(N,N-dimethyl-N-dodecyloammonium
bromides), against A. niger is 20 times higher than the MIC value for hexamethylene-1,6-bis(N,N-
dimethyl-N-dodecylammonium bromide) (1) [2]. This means that the same biocidal effect can be
obtained using at least ten times less dimeric surfactant instead of a monomeric surfactant. These are
fundamental results for the application of gemini surfactants like polymethylene-α,ω-bis(N,N-
dimethyl-N-dodecyloammonium bromides), as chemical microbiocides.

Molecules 2011, 16
Figure 9. The relationship between number of methylene groups in the spacer of
329
polymethylene-α,ω-bis(N,N-dimethyl-N-dodecyloammonium bromides) 1-5 and MIC
of conidia.
0
0
0
0
.4
.3
.2
.1
0
A. niger ATCC
P. chrysogenum
A. niger ŁOCK
C. albicans
1
2
3
4
5
6
7
Number of methylene groups in the spacer
The minimal concentrations which inhibit 48-hour mycelium development for hexamethylene-1,6-
bis(N,N-dimethyl-N-dodecylammonium bromide) (1) and pentamethylene-1,5-bis(N,N-dimethyl-N-
dodecylammonium bromide) (2) are significantly higher than those for conidia. (Table 4). The higher
MIC values for mycelium in comparison to conidia result from the character of mycelium growth.
Fragments of hypha growing by apical elongation are sensitive to disinfectants, while portions of the
hyphae away from the tips are more resistant [30]. This resistance is due to a thicker cellular wall that
hinders penetration of microbiocide molecules. Another reason of this resistance is wall porosity,
which decreases with age [31].
Table 4. (MIC, μM/mL) of hexamethylene-1,6-bis(N,N-dimethyl-N-dodecylammonium
bromide) (1) and pentamethylene-1,5-bis(N,N-dimethyl-N-dodecylammonium bromide) (2)
for mycelium and pseudomycelium.
Strains
Compound
A.niger
ATCC 16404
0.31
P.chrysogenum
LOCK 0531
0.31
A.niger
LOCK 0439
0.31
C.albicans
ATCC 10231
0.31
1
2
0.31
0.76
0.76
0.45
3
. Experimental
.1. General
The NMR spectra were measured with a Varian Gemini 300VT spectrometer, operating at 300.07
3
1
13
and 75.4614 MHz for H and C, respectively. Typical conditions for the proton spectra were: pulse
width 32°, acquisition time 5s, FT size 32 K and digital resolution 0.3 Hz per point, and for the carbon

Molecules 2011, 16
330
spectra pulse width 60°, FT size 60 K and digital resolution 0.6 Hz per point, the number of scans
1
3
1
varied from 1,200 to 10,000 per spectrum. The C and H chemical shifts were measured in CDCl
3
1
relative to an internal standard of TMS. All proton and carbon-13 resonances were assigned by H
1
3
(
COSY) and C (HETCOR). All 2D NMR spectra were recorded at 298 K on a Bruker Avance DRX
1
13
6
00 spectrometer operating at the frequencies 600.315 MHz ( H) and 150.963 MHz ( C), and
1
31
equipped with a 5 mm triple-resonance inverse probe head [ H/ P/BB] with a self-shielded z gradient
1
13
coil (90° H pulse width 9.0 μs and C pulse width 13.3 μs). Infrared spectra were recorded in the KBr
pellets using a FT-IR Bruker IFS 66 spectrometer. The ESI (electron spray ionization) mass spectra
were recorded on a Waters/Micromass (Manchester, UK) ZQ mass spectrometer equipped with a
Harvard Apparatus syringe pump. The sample solutions were prepared in methanol at a concentration
-5
of approximately 10 M. The standard ESI – MS mass spectra were recorded at a 30 V cone voltage.
3
.2. Computational details
The calculations were performed using the Gaussian 03 program package [32] at the Hartree-Fock
[
33,34] levels of theory with the 3-21 basis set [33]. The NMR isotopic shielding constants were
calculated using the standard GIAO (Gauge-Independent Atomic Orbital) approach [32-35] of
GAUSSIAN 03 program package [36].
3
.3. Antimicrobial study
Fungal strains. The antifungal activity of the gemini surfactants was evaluated against Aspergillus
brasiliensis (previously A. niger) ATCC 16404, Aspergillus niger LOCK 0439, Penicillium
chrysogenum LOCK 0531 and Candida albicans ATCC 10231.
Antifungal activity. Minimal inhibitory concentrations (MIC values) against conidia of moulds were
measured by a tube standard 2-fold dilution method. Malt Extract Broth (Merck) was used for the
antifungal tests. Moulds were preincubated on MEA slant for 5 days at 28 ºC, yeast – for 1 day at
3
7 ºC. Conidia suspensions of each strain were prepared by adding sterile water containing 0.1% (w/w)
Tween 80 to the slant. The yeast cell suspension of Candida albicans was prepared by similar
6
procedure but without Tween 80. The conidia and yeast cells were adjusted to 1-2 × 10 cells/ml by
counting them in Thoma chamber. One mL of conidia suspension was mixed with 1 mL of media
containing the tested compounds and incubated at 28 ºC for 72 h – moulds, 37 ºC for 48 h - yeast. The
MICs were defined as the lowest concentration of the compounds at which there was no visible
growth. Minimal inhibitory concentrations (MIC values) against mycelium of moulds were measured
by the suspension method. Conidia and yeast cells suspensions were prepared in the same way as for
the conidia test. Next, 1.5 mL of inoculum was mixed with 12 mL of MEB medium and incubated for
4
8 h - moulds at 28 ºC, yeast- 37 ºC. After this time, 1.5 mL of tested gemini surfactants in different
concentrations were added and all cultures were incubated another 48 h. The control sample was
culture of mycelium without gemini surfactants. In this case, the MICs were defined as the lowest
concentration of the compounds at which the development of mycelium in comparison to the control

Molecules 2011, 16
331
sample was inhibited. Each experiment was repeated three times and the mean values were used to
compute the MICs.
3.4. Synthesis
Hexamethylene-1,6-bis(N,N-dimethyl-N-dodecyldodecylammonium bromide) (1). N,N-Dimethyl-N-
dodecylamine (18.2 g, 0,09 M) was mixed with 1,6-dibromohexane (10,7 g, 0.04 M) in acetonitrile
(
80 mL). The reaction mixture was heated under reflux for 5 h. The solvent was evaporated under
reduced pressure and the residue was dried over P O and then recrystallized from acetonitrile, yield
4
10
8
4
4%, m.p. 231-232 °C; Elemental analysis: found (calc) %C 60.54 (60.88); %H 11.75 (11.12); %N
+
1
.03 (4.18); ES MS m/z 255 (C H N /2); H-NMR: δ 0.88 [6H, H(a)], 1,25 [4H, H(b)], 1.25 [4H,
3
4
74
2
H(c)], 1.25 [4H, H(c)], 1.25 [4H, H(d)], 1.25 [20H, H(e)], 1.25 [4H, H(f)], 1.72 [4H, H(g)], 3.52 [4H,
13
H(h)], 3.39 [12H, H(i)], 3.69 [4H, H(j)], 1.98 [4H, H(k)], 1.56 [4H, H(l)]; C-NMR: δ 13.84 C(a),
2.63 C(b), 31.60 C(c), 29.17 C(d), 29.17 C(e), 26.06 C(f), 22.38 C(g), 64.38 C(h), 50.73 C(i), 63.81
2
C(j), 21.51 C(k), 24.42 C(l)
Pentamethylene-1,5-bis(N,N-dimethyl-N-dodecyldodecylammonium bromide) (2). N,N-Dimethyl-
dodecylamine (9.3 g, 0.04 M) was mixing with 1,5-dibromopentane (5.0 g, 0.02 M) in acetonitrile
(
80 mL). The reaction mixture was heated under reflux for 4 h. The solvent was evaporated under
reduced pressure and the residue was dried over P 10 and then recrystallized from acetonitrile, yields
6%, m.p. 226-227 °C; Elemental analysis found (calc) %C 59.99 (60.35); %H 11.58 (11.05); %N
4
O
8
4
+
1
72 2
.02 (4.27); ES MS m/z 248 (C33H N /2); H-NMR: δ 0.88 [6H, H(a)], 1,36 [4H, H(b)], 1.25 [4H,
H(c)], 1.25 [4H, H(c)], 1.25 [4H, H(d)], 1.25 [20H, H(e)], 1.25 [4H, H(f)], 1.73 [4H, H(g)], 3.52 [4H,
13
H(h)], 3.38 [12H, H(i)], 3.86 [4H, H(j)], 2.08 [4H, H(k)], 1.62 [2H, H(l)]; C-NMR: δ 13.98 C(a),
2.79 C(b), 31.75 C(c), 29.28 C(d), 29.28 C(e), 26.20 C(f), 22.46 C(g), 64.45 C(h), 50.69C(i), 63.76
2
C(j), 21.73 C(k), 22 53 C(l).
Tetraethylene-1,4-bis(N,N-dimethyl-N-dodecyldodecylammonium bromide) (3). N,N-Dimethyl-
dodecylamine (9.7 g, 0.05 M) was mixing with 1,4-dibromobutane (4.9 g 0.03 M) in acetonitrile
(
80 mL). The reaction mixture was heated under reflux for 5 h. The solvent was evaporated under
reduced pressure and the residue was dried over P 10 and then recrystallized from mixture
acetonitrile:aceton, yields 92%, m.p. 225-226 °C; Elemental analysis found (calc) %C 58.74
4
O
+
1
(
59.80); %H 11.47 (10.98); %N 4.18 (4.36); ES MS m/z 241(C H N /2); H-NMR: δ 0.88 [6H,
3
2
70
2
H(a)], 1,26 [4H, H(b)], 1.26 [4H, H(c)], 1.26 [4H, H(c)], 1.26 [4H, H(d)], 1.26 [20H, H(e)], 1.26 [4H,
1
3
H(f)], 1.76 [4H, H(g)], 3.46 [4H, H(h)], 3.33 [12H, H(i)], 3.85 [4H, H(j)], 2.08 [4H, H(k)]; C-NMR:
δ 13.81 C(a), 22.60 C(b), 31.59 C(c), 29.19 C(d), 29.19 C(e), 26.08 C(f), 22.37 C(g), 64.75 C(h), 50.67
C(i), 63.17C(j), 19.57 C(k).
Threemethylene-1,3-bis(N,N-dimethyl-N-dodecyldodecylammonium bromide) (4). N,N-dimethyl-
dodecylamine (9.7 g, 0.05 M) was mixing with 1,3-dibromopentane (4.6 g, 0.03 M) in acetonitrile
(
80 mL). The reaction mixture was heated under reflux for 6 h. The solvent was evaporated under
reduced pressure and the residue was dried over P 10 and then recrystallized from mixture
4
O

Molecules 2011, 16
332
acetonitrile:aceton, yields 86%, m.p. 199-200 °C; Elemental analysis found (calc) %C 58.81
+
1
(
59.22); %H 10.56 (10.90); %N 4.17 (4.46); ES MS m/z 234 (C31
H
68
N
2
/2); H-NMR: δ 0.88 [6H,
H(a)], 1,35 [4H, H(b)], 1.26 [4H, H(c)], 1.26 [4H, H(d)], 1.26 [20H, H(e)], 1.26 [4H, H(f)], 1.79 [4H,
1
3
H(g)], 3.55 [4H, H(h)], 3.42 [12H, H(i)], 3.82 [4H, H(j)], 2.77 [4H, H(k)]; C-NMR: δ 13.87 C(a),
2.73 C(b), 31.66 C(c), 29.27 C(d), 29.27 C(e), 26.13 C(f), 22.42 C(g), 66.00 C(h), 50.99, C(i), 60.64
2
C(j), 18.55 C(k).
Ethylene-1,2--bis (N,N-dimethyl-N-dodecyldodecylammonium bromide) (5). N,N,N’,N’-Tetra-
methylethylamine (2 g, 0.02 M) was mixing with bromododecane (4.6 g, 0.03 M) in acetonitrile
(
30 mL). The reaction mixture was heated under reflux for 4 h. The solvent was evaporated under
reduced pressure and the residue was dried over P O10 and then recrystallized from mixture
4
ethanol:ethyl acetate, yields 83%, m.p. 186-187 °C; Elemental analysis found (calc) %C 58.49
+
1
(
58.62); %H 10.83 (10.82); %N 4.57 (4.56); ES MS m/z 227 (C H N /2); H-NMR): δ 0.88 [6H,
3
0
66
2
H(a)], 1,30 [4H, H(b)], 1.25 [4H, H(c)], 1.25 [4H, H(d)], 1.25 [20H, H(e)], 1.25 [4H, H(f)], 1.81 [4H,
1
3
H(g)], 3.67 [4H, H(h)], 3.43 [12H, H(i)], 4.55 [4H, H(j)]; C-NMR (CDCl ): δ 14.03 C(a), 22.99 C(b),
3
3
1.84 C(c), 29.53 C(d), 29.53 C(e), 26.20 C(f), 22.60 C(g), 65.54 C(h), 51.29, C(i), 56.51 C(j).
. Conclusions
4
Polymethylene-α,ω-bis(N,N-dimethyl-N-dodecyloammonium bromides) 1-4 have been obtained
with good yield after short reaction times by reaction of N,N-dimethyl-N-dodecylamine with 1,6-
dibromohexane, 1,5-dibromopentane, 1,4-dibromobutane and 1,3-dibromopropane. Ethylene-1,2-bis-
(
N,N-dimethyl-N-dodecyloammonium bromide) (5) has been prepared in good yield by reaction of
N,N,N’,N’-tetramethylethylenediamine with 1-bromodecane. The structures of the title compounds
have been analyzed by FTIR and NMR spectroscopy, as well as by DFT calculations. Properties of
polymethylene-α,ω-bis(N,N-dimethyl-N-dodecyloammonium bromides) 1-5, including energy, melting
points, solubility and antifungal activity, depend strongly on the length of the spacer. The longer the
spacer the better the water solubility, stability and antifungal activity. Hexamethylene-1,6-bis(N,N-
dimethyl-N-dodecyloammonium bromide) shows the best antifungal activity and can be used as an
efficient microbiocide.
Acknowedgments
Authors thank to Zofia Żakowska from Institute of Fermentation Technology and Microbiology,
Technical University of Lodz for helpful comments and advice which helped to develop ideas put
forward. This work was supported by the funds from Adam Mickiewicz University, Faculty of
Chemistry. The microbiological study were supported by KBN grant “Fungicidal properties of new
gemini surfactants” N N401 027736.

Molecules 2011, 16
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©
2011 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 license
http://creativecommons.org/licenses/by/3.0/)
(