Synthesis and evaluation of the antimicrobial activity of Dodecyl trimethyl ammonium and N-(N, N-Dimethylamino) propyl glucosylamine

Article abstract of DOI:10.13005/ojc/300239

In this study Dodecyl trimethyl ammonium sulphate (DTA) was obtained through quaternization reaction between dodecylamine and dimethyl sulfate (yield: 51.5%) while Dimethylamino propyl glucosylamine (DAPGA) was successively synthesized from D-glucose and 3-Dimethylamino propylamine (yield: 93.6%). Both compounds were analyzed through NMR spectroscopy. Evaluation of their biological activity against Salmonella Typhimurium, Staphylococcus aureus and Fusarium oxysporum cubens indicated that at a concentration greater than 0.001 10- 4 mole mL -1, DTA significantly inhibited growth of Salmonella Typhimurium and Staphylococcus aureus while DAPGA was not effective. Furthermore, these two compounds showed a pronounced antifungal activity against a toxicogen fungal, Fusarium oxysporum cubens.

Full text of DOI:10.13005/ojc/300239

ISSN: 0970-020 X
CODEN: OJCHEG
2014, Vol. 30, No. (2):
Pg. 687-694
ORIENTAL JOURNAL OF CHEMISTRY
An International Open Free Access, Peer Reviewed Research Journal
www.orientjchem.org
Synthesis and Evaluation of the Antimicrobial Activity
of Dodecyl trimethyl ammonium and N-(N, N-dimethylamino)
Propyl Glucosylamine
THÉONESTE MUHIZI
University of Rwanda, School of Pure and Applied Science,
Department of Chemistry, University avenue 117 Butare, Rwanda.
*Corresponding author E-mail: tmuhizi@nur.ac.rw
http://dx.doi.org/10.13005/ojc/300239
(Received: March 02, 2014; Accepted: March 30, 2014)
ABSTRACT
In this study Dodecyl trimethyl ammonium sulphate (DTA) was obtained through
quaternization reaction between dodecylamine and dimethyl sulfate (yield: 51.5%) while
Dimethylamino propyl glucosylamine (DAPGA) was successively synthesized from D-glucose
and 3-Dimethylamino propylamine (yield: 93.6%). Both compounds were analyzed through NMR
spectroscopy.Evaluation of their biological activity against Salmonella Typhimurium, Staphylococcus
aureus and Fusarium oxysporum cubens indicated that at a concentration greater than 0.001 10-
4
mole mL-1, DTA significantly inhibited growth of Salmonella Typhimurium and Staphylococcus
aureus while DAPGA was not effective. Furthermore, these two compounds showed a pronounced
antifungal activity against a toxicogen fungal, Fusarium oxysporum cubens.
Key words: Dodecyl trimethyl ammonium, Dimethylamino propyl glucosylamine,
Salm. Typhimurium, Staph. aureus, F. oxysporum cubens, biocides.
INTRODUCTION
Furthermore, fungi such us Aspergillus, Penicillium
and Fusarium species essentially found on cereal
are responsible of teratogenic and carcinogenic
toxins which seriously affect human life7-14. Due to
the diversity and ravages of these harmful
microorganisms, human prefer to use antimicrobials
to fight against them. Nevertheless, even if the
importance of these chemicals is no longer
demonstrable, some of them crop up serious eco-
toxicological problems and therefore were
interdicted or limited from the markets15, 16. These
Development of pathogen bacteria and
mycotoxicogen fungi in food constitute a danger
for humanity. These harmful microorganisms can
be major source of food toxi-infection leading to
different human diseases. For instance Salmonella
and
Listeria
species,
very
resistant
mircroorganisms at different conditions used for
food preservation, can lead to salmonellose and
listeriose, two known human fatal diseases
1-6
.

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MUHIZI, Orient. J. Chem., Vol. 30(2), 687-694 (2014)
decisions come up very rapidly and left questions Synthesis of DAPGA
on the means to be used for replacing them17.
Reported method of Petit (1999)25 was
Furthermore, future effectiveness of antimicrobial adapted to synthesize DAPGA (Fig.2).Ten grams of
therapy is somewhat in doubt since various glucose (0.0555 mole) and 5.7 g (0.0558 mole) of
microorganisms are becoming more resistant to 3-dimethylamino propylamine were dissolved in 66
the existing antimicrobial agents1-6. Therefore, it is mL of methanol. The reaction mixture was refluxed
the responsibility of scientists to work hard and to at 40°C under stirring for 4 hours. The solvent was
find out environment friendly active compounds removed under reduced pressure using a rotary
which can be used in this struggling. Previous evaporator and a viscous yellowish liquid of 9.3 g
reports indicated that amino sugars and ammonium was obtained.
compounds could be one of the good candidates to
reach the goal18-24. This study is our contribution in Microorganisms
this scientists’ task and was enabled to synthesize
two molecules with promised results.
The fungal strain, Fusarium oxysporum
cubens, was isolated and characterized in the
laboratory of University of Rwanda, College of
Science and Technology. Two bacteria Salmonella
Typhimurium (Institut Pasteur 5858) and
EXPERIMENTAL
Chemicals such us glucose (Sigma Staphylococcus aureus (Institut Pateur 25923) were
Aldrich), dodecylamine 98% (Sigma Aldrich), 3- obtained from the Laboratory of US2B, Université
dimethylamino propylamine (Sigma Aldrich), Bordeaux 1, France.
dimethyl sulphate 99% (Aldrich), Sodium sulphate
99% (Aldrich), chloroform (Prolabo), ethanol Antifungal activity assessment
(Schorlau) and methanol (Labosi) were used with
Potato dextrose agar (PDA) medium was
out any further purification. Culture media Mueller used to isolate, identify and revivify F. oxysporum
Hinton agar (Biomérieux), Nutrient broth (Difco) and cubens. The radial growth method17 was used to
Potato dextrose Agar (Himedia RM 007) were used assess the antifungal activity of DTA and DAPGA
to assess biological activity. Azithromycin (Alice against F. oxysporum cubens.Concentrations of 0.4
Pharma PVT, LTD, India) was used as positive × 10-4 and 1.5 × 10-4 mole mL-1 for both compounds
reference.
were evaluated. Distilled water and methanol were
used to dissolve DTA and DAPGA, respectively.Each
essay was done in triplicate and control experiments
General methods
1H NMR spectra of DTA and DAPGA were which contain either distilled water or methanol were
recorded at 300 MHZ on a Bruker Avance 300 done in parallel. The percentage of inhibition from
spectrometer and chemical shifts are given in ppm. the two products was calculated after 8 days of
The melting point of DTA was determined by an incubation using the formula previously reported26.
Electrothermal 9100 Digital Melting Point Apparatus In addition, the diameter of fungi was daily measured
IA 9100.
during 8 days of incubation to evaluate the effect of
the two products on the kinetic growth of fungi.
Synthesis of DTA
1-Dodecylamine (0.0539 mole) was Antibacterial activity assessment on agar media
dissolved in 20 mL of chloroform. The mixture was Mueller-Hinton culture medium was used
refluxed at 50°C and under stirring a small quantity to assess biological activity of DTA and DAPGA
of sodium sulphate was added. After 30 min of against Salm. Typhimurium and Staph. aureus. Disc
reaction, 0.2158 mole of dimethyl sulphate was diffusion method was used to conduct the test17, 27
carefully added to the mixture and reflux was
.
maintained for 6 hours. Solvent was then removed Assessment of antibacterial efficacy of DTA and
under reduced pressure and residue was co- DAPGA in liquid medium
evaporated four times in methanol to recuperate a
white solid which was finally recristallized in ethanol broth, in test tubes were separately contaminated
to obtain 9 grams of DTA (Figure 1). by 1 mL of 18h revivified bacteria strains, Salm.
Nine milliliters of liquid medium, nutrient

MUHIZI, Orient. J. Chem., Vol. 30(2), 687-694 (2014)
689
Typhimurium and Staph. aureus, separately. Then
a given quantity of DTA or DAPGA was added prior
to have a final concentration of 0.08.10-4 mole mL-1
of these products. The obtained tubes were then
incubated at 37°C for 24 hours. Control tubes
without drugs were concurrently incubated. After
24 hours of incubation, 1 mL of this microbial
suspension was previously 106 times diluted and
100 mL were deposited on the Mueller-Hinton agar
medium and gently spread onto the surface of this
prior to incubation at 37°C for 24h. Forming colony
unities were then counted and number recorded.
The experiment was conducted in triplicate.
protons were characterized by signals found at 3.1,
3.33 and 3.67 ppm respectively, depending on the
type of chemical groups surrounding them.
DAPGA
Yield: 93.6%, 1H NMR (300 MHz, D2O): dH
3.9-4.0 (1H, d, H-1), 3.8-3.9 (1H, dd, H-6a), 3.6-3.7
(1H, dd, H-6b), 3.4-3.5 (1H, t, H-3), 3.3-3.4 (2H,
complex, H-4 and H-5), 3.1-3.2 (1H, t, H-2), 2.78-
2.86 (2H, mm, -NHCH2-), 2.35 (2H, t, -CH2N=), 2.15
(6H, s, -N(CH3)2), 1.54-1.65 (2H, m, -CH2-). The
synthesis of DAPGA was confirmed by H-1 signal
in the form of doublet found at lower chemical shift
of 3.9-4.0 ppm compared to H-1a and H-1b of
glucose which were reported to be around 5.23
and 4.65 ppm29, respectively, and this indicated the
replacement of OH-1 group by -NH- group in
glucosylamine.The same observation was reported
Statistical analysis
Results from antibacterial assays were
statistically analyzed using one-way analysis of
variance (ANOVA). The significant difference
between activities of DTA and DAPGA or between
these drugs and control experiments was obtained
when the probability (p) found was higher than the
significance threshold of 0.05.
in various previous works17, 27, 29, 30
.
Antifungal activity of DTA and DAPGA on F.
oxysporum cubens
Some fungi are harmful either to animals
or plants by intoxicating them. Among these fungi
we chose to work with one toxicogen fungus called
RESULTS AND DISCUSSION
Chemical Synthesis
In this study, both DTA and DAPGA were
successively synthesized and characterized by H
NMR spectroscopy.
Table 1: Inhibition of DTA, methanol and
DAPGA on the radial growth of F. oxysporum
cubens at the 8th day of incubation
1
DTA
1
Yield: 51.5%, melting point: 75.1°C, H
Biocides
Concentration Percentage of
(mol ml-1)10-4 inhibition ±SEM
NMR (300 MHz, D2O, Figure 3): H 3.4 (2H, t, -NCH2-
), 3.19 (9H, s, -N+(CH3)3), 1.7-1.9 (2H, br, -CH2CH3),
1.4-1.5 (18H, -CH2-), 0.9-1 (3H, t, -CH3). The total
synthesis of DTA was indicated by a signal in the
DTA
0.4
1.5
0.4
1.5
1 mL
1 mL
100.0 0.0
100.0 0.0
81.5 1.2
100.0 0.0
0.0 0.0
+
form of singulet at 3.19 ppm representing N(CH3)
DAPGA
protons (Figure 3).
Distilled water
Methanol
Our result was confirmed by previous
reports21, 24, 26, 28 in which quaternary ammonium
11.5 1.4
Table 2: Inhibition diameter values of DTA on Staph. aureus and Salm typhimurium
Inhibition diameter ± SEM (in mm)
Bacteria
Concentrations (mol mL-1)10-4
0.08
0.04
0.02
0.01
0.001
Staph. aureus
Salm. Typhimurium
24.3 0.3
15.3 0.9
20.3 0.5
9.7 0.3
18.3 0.7
8.0 0.0
9.2 0.8
2.1 0.5
0.0 0.0
0.0 0.0

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MUHIZI, Orient. J. Chem., Vol. 30(2), 687-694 (2014)
Table 3: Antibacterial activity of DTA and DAPGA in water
Drugs
Antibacterial activity (UFC ± SD)
Salm typhimurium
Staph aureus
Control experiment
DTA
374 13
299 23
0
0
0
0
DAPGA
362 17
277 20
Table 4: Inhibition diameter values of DAPGA, DoGA
and 3-DMAP on Staph aureus and Salm typhimurium
Biocides
Inhibition diameter ± SEM (in mm)
Concentration
(mole mL-1) × 10-4
Staph aureus
Salm typhimurium
DoGA
DAPGA
0.5
0.5
2.0
16.0 0.5
0.0 0.0
6.0 0.3
10.6 0.5
0.0 0.0
8.0 0.6
Fusarium oxysporum cubens. Fusarium species are activity (percentage of inhibition of 81.5 %) when a
known, not only to be implicated in the annually concentration of 0.4 x 10-4 mole mL-1 was tested and
worldwide harvest lost12, but also to produce a completely inhibited the growth of fungal at a
variety of toxins such as fumonisins, zearalenone concentration of 1.5 x 10-4 mole mL-1. Methanol and
and trichothecenes in food mainly cereal, and thus water used as negative control exhibited non
exhibiting chronic toxicity7,8,10,14,31. In this study, both significant antifungal activity.
two products DTA and DAPGA were effective
against the growth of Fusarium oxysporum cubens
Furthermore, the antifungal activity of
(Figure 5 and Table 1). At all concentrations used, these two compounds was observed during a week
0.4 x 10-4 and 1.5 x 10-4 mole mL-1, DTA completely to visualize well what was happened during
inhibited the growth of Fusarium oxysporum cubens incubation of fungus. At concentration of 0.4 x 10-4
while DAPGA exhibited a significant antifungal mole mL-1, DTA completely inhibited the growth of
Fig. 1: Synthesis scheme of DTA

MUHIZI, Orient. J. Chem., Vol. 30(2), 687-694 (2014)
691
fungi while DAPGA significantly affected the kinetic
growth of fungus by strongly changing the shape of
its kinetic growth. Methanol used as negative
control did not show significant effect on the kinetic
growth of fungal but slightly delayed it. All these
observations were done in comparison with the
effect of distilled water used as control (Figure 6).
antibacterial activity it had on a Gram negative
bacterium, Salm. Typhimurium. This may partially
due to the chemical composition of their cell-wall
structures which are different and thus conduct to
their different permeability towards drugs33, 34. In
addition, at a concentration of 0.08 mole mL-1,
Azithromycin, an antibiotic used as positive control,
exhibited comparable antibacterial activity with
DTA. DTA indicated inhibition zones of 24.3 0.3
At all concentrations used DAPGA did not
affect the growth of both bacteria while DTA showed
its efficacy in inhibiting these microbes. At the
concentration of 0.08 x 10-4 mole mL-1, DTA
exhibited higher activity against both bacteria. It
inhibited the growth of Staph. aureus and Salm.
Typhimurium with inhibition zones of 24 and 15
mm respectively. These inhibition zones are too
sufficient to consider DTA as effective against both
bacteria 32. In this study, at a concentration
previously quoted, DTA led to an inhibition diameter
which was closed to 16 mm (viz 15.3 0.9) for
Salm. Typhimurium and thus can be used as the
minimal effective concentration against that
bacterium. Furthermore, a concentration of around
0.02 mole mL-1 can be taken as minimal effective
one for Staph. aureus. As shown in the table 2, DTA
with a concentration greater than 0.001.10-4 mole
mL-1 had higher antibacterial activity on Staph.
aureus, Gram positive bacterium, than the
and 15.3
0.9 for Staph. aureus and Salm.
Typhimurium (Table 2), respectively, while for
Azithromycin the inhibition zones were found to be
26.7 1.2 and 20.3 1.8 mm respectively for Staph.
aureus and Salm. Typhimurium. Comparison of
these results indicated that efficiency of DTA is
closely comparable to that from know antibiotic,
Azithromycin. At concentrations lower than or equal
to 0.08 x 10-4 mole mL-1, DAPGA did not show any
significant antibacterial activity.
To verify the reason of difference between
biological activities of DTA and DAPGA, the efficacy
of these two drugs were also assessed in liquid
media. DTA completely inhibited the growth of both
bacteria while DAPGA didn’t show any significant
inhibition compared to control experiments
(Table 3).
Fig. 2: Synthesis scheme of DAPGA
Fig. 3: 1H NMR spectrum of DTA

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Having these observations, decomposition process, with H2O used to prepare liquid medium in
of DAPGA in D2O was followed using 1H-NMR. Both which both compounds was tested. After 24 h in
disappearance of –NH(CH2)- and decreasing D2O, the initial DAPGA decreased of 50% while at
intensity of H-1 signals were served as references 48 h, only 37.5% of DAPGA was remained in the
(Figure 8). We chose to work with D2O as solvent, medium without any decomposition (Figure 8).
because of its quite same properties, in hydrolysis
Fig. 4: 1H NMR spectrum of DAPGA
Fig. 5: Illustration of antifungal activity of DTA and DAPGA on
Fusarium oxysporum cubens at a concentration of 0.4 x 10-4 mole.mL-1
Fig. 6: The effect of synthesized products on the kinetic growth
of F. oxysporum cubens at the concentration of 0.4 x 10-4 mole mL-1

MUHIZI, Orient. J. Chem., Vol. 30(2), 687-694 (2014)
693
This remarked rapid decomposition of
DAPGA could be the reason of its poor antibacterial
activity. As previously reported17, the lack of activity
of glucosylamines against bacteria, may be partially
explained by their rapid hydrolysis during the first
24 hours.To verify this assumption, the antibacterial
activity of DAPGA was compared to that of Dodecyl
glucosylamine (DoGA), a molecule which was
previously synthesized and found to be not highly
hydrolyzed17. At a concentration of 0.5 x 10-4 mole
mL-1, both Salm. Typhimurium and Staph. aureus
were exactly sensible to DoGA but not to DAPGA
(Table 4).
DAPGA started to exhibit antibacterial
activity at higher concentration of 2.0 x 10-4 mole
mL-1 compared to DoGA and this observed activity
was even not significant.
Note: S.t.: Salm. Typhimurium, S.a: Staph. aureus, C1, C2, C3 and C4: concentrations of drugs which are equal to: 0.04,
0.02, 0.01 and 0.001 (x 10-4) mole mL-1 respectively, w: water
Fig. 7: Illustration of antibacterial activity of DTA on Salm typhimurium and Staph aureus
Fig. 8: Decomposition of DAPGA in D2O for 48 h
In conclusion, results revealed that at all
concentrations used, DTA and DAPGA were
effectiveness to inhibit the growth of F. oxysporum
cubens. Furthermore, Dodecyl trimethyl ammonium
(DTA) showed significant antibacterial activity
against the growth of Salm. Typhimurium and Staph.
aureus. The lack of significant antibacterial of N-
(N,N-Dimethylamino) propyl glycosylamine against
Staph. aureus and Salm. Typhimurium could be due
to its remarked decomposition in D2O and research
on its possible stabilization should be of interest.
confirmed: satifying antimicrobial activity was
obtained from small biodegradable molecule,
dodecyl trimethyl ammonium sulphate. It is however
to note that the toxicity assessment of this
compound should be carefully realized before any
application of this compound in various domains.
ACKNOWLEDGMENTS
We thank the University of Rwanda-
Swedish International Development Cooperation
Agency (UR-SIDA) Project and UR Research
Commission for funding this work. Many thanks to
Mr Nestor Ishimwe for technical assistance.
Through our findings, the reported antibacterial
24, 28
efficacy of ammonium compounds21,
was

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