Synthesis and antibacterial activity of aminodeoxyglucose derivatives against Listeria innocua and Salmonella typhimurium

Article abstract of DOI:10.1021/jf901609y

In this study aminodeoxyglucose derivatives were synthesized and evaluated for their antibacterial activity against two food bacteria, Listeria innocua and Salmonella typhimurium. 6-Amino-6-deoxy-α-Dmethylglucopyranose (GSA-6), 3-amino-3-deoxy-D-glucopyranoside (GSA-3), and β-D-glucopyranosylamine (GSA-1) were synthesized and concurrently tested with commercially available D-glucosamine (GSA-2) for antibacterial activity. Results obtained from this study showed a pronounced antagonist effect due to the position of amino groups of aminoglucose derivatives on the antibacterial activity. GSA-3 was the most active compound. At a concentration of 2 × 10 ^-4 mol mL ^-1, it delayed the growth of both bacteria with percentages of inhibition of 29 and 15% for L. innocua and S. typhimurium, respectively. At the same concentration the percentages of inhibition for other aminodeoxyglucoses varied between 5 and 18% and between 2 and 11% for L. innocua and S. typhimurium, respectively. All compounds were characterized by FTIR, ¹H NMR, and ¹³C NMR spectroscopy.

Full text of DOI:10.1021/jf901609y

8770 J. Agric. Food Chem. 2009, 57, 8770–8775
DOI:10.1021/jf901609y
Synthesis and Antibacterial Activity of Aminodeoxyglucose
Derivatives against Listeria innocua and Salmonella
typhimurium
´
´
T
HEONESTE
M
UHIZI, STEPHANE
GRELIER
,
AND VE´ RONIQUE
COMA
*
ꢀ
UMR US2B, Unite
´
des Sciences du Bois et des Biopolymeres, Universite
´
Bordeaux 1, INRA, CNRS, 351
Cours de la Liberation, F-33405 Talence, France
´
In this study aminodeoxyglucose derivatives were synthesized and evaluated for their antibacterial
activity against two food bacteria, Listeria innocua and Salmonella typhimurium. 6-Amino-6-deoxy-R-
methylglucopyranose (GSA-6), 3-amino-3-deoxy- -glucopyranoside (GSA-3), and β- -glucopyranosyl-
amine (GSA-1) were synthesized and concurrently tested with commercially available -glucosamine
D-
D
D
D
(GSA-2) for antibacterial activity. Results obtained from this study showed a pronounced antagonist
effect due to the position of amino groups of aminoglucose derivatives on the antibacterial activity.
GSA-3 was the most active compound. At a concentration of 2ꢀ10-4 mol mL-1, it delayed the growth
of both bacteria with percentages of inhibition of 29 and 15% for L. innocua and S. typhimurium,
respectively. At the same concentration the percentages of inhibition for other aminodeoxyglucoses
varied between 5 and 18% and between 2 and 11% for L. innocua and S. typhimurium, respectively.
1
All compounds were characterized by FTIR, H NMR, and 13C NMR spectroscopy.
KEYWORDS: Antibacterial activity; aminodeoxyglucoses; Salmonella typhimurium; Listeria innocua
INTRODUCTION
polymer could be expanded to its monomer,
D-glucosamine,
and either to its isomers or other small aminoglucose derivatives.
These small sugars are suspected to better enter into cell mem-
branes than their respective polymers. The present work is
principally aimed at the study of these aminodeoxyglucose
derivatives by verifying the impact of amino group features in
biological activity. The impact of amino group position on
glucose in the study of antibacterial activity was the main
objective of this study. For this, we synthesized different amino-
glucose derivatives by changing the position of the amino group
and studied their impact on the development of S. typhimurium
and L. innocua.
Researchfor biocide discovery has longbeenknown. However,
because of their high multiplicity and resistance, total control of
microorganisms is far from being accomplished. Some micro-
organisms are implicated in the contamination of food, water,
and various instruments used by people. Physical changes occur
during the processing, transport, and storage of food, and when
contaminated elements are consumed, these microorganisms can
multiply and cause dangers to humans. Listeria and Salmonella
genera are widely found among those microorganisms, which
necessitate our effort to control them. These two bacteria are
respectively responsible for listeriosis and salmonellosis, two
severe diseases with large numbers of patients and high mortality
rates. The danger of these bacteria is actually due to their capacity
to survive over a wide range of preservation conditions, which
are, for example, low temperatures, low pH, and high concentra-
tions of salt. Therefore, research aimed at the use of new modes of
food conservation is under intense scrutiny. According to some
authors, the use of active packaging can constitute a new method
of achieving the control of bacteria contamination in foods (1-5).
For this, chitosan and its derivatives have shown their efficiency
by inhibiting the growth of Listeria innocua, Listeria monocyto-
genes, and Salmonella typhimurium in both solid and liquid
media (6-9). However, the mechanism of action of these com-
pounds is still unknown. It is suspected that there is a close
relationship between amino groups and biological activity of
chitosan (9). In this order, the biological activity from this
MATERIALS AND METHODS
Chemicals. Glucose (Sigma-Aldrich), R-D-methylglucoside 99%
(Janssen Chimica), zinc chloride 98% (Acros Organics), phosphorus acid
85% (Sigma-Aldrich), pyridinium dichromate 98% (Sigma-Aldrich),
acetic anhydride 99% (Sigma-Aldrich), phosphorus dioxide 98% (Acros
Organics), sodium borohydride 98% (Acros Organics), p-toluenesulfonyl
chloride 98% (Sigma-Aldrich), palladium/carbon 5% (Sigma-Aldrich),
sodium hydrogenocarbonate, and sodium sulfate 99% (Acros Organics)
were used in this study. Solvents such as ethanol, methanol, chloroform,
petroleum ether, ethyl acetate, diethyl ether, dichloromethane, toluene,
pyridine99%, N,N-dimethylformamide 99%, and acetone were purchased
from BDH Prolabo and were used without any further purification.
General Methods. Aminodeoxyglucoses GSA-1, GSA-3, and GSA-6
were synthesized and characterized by FTIR, 1H NMR, and 13C NMR
spectroscopy. The synthesis was monitored by thin layer chromatography
realized on aluminum plates (silica gel 60 F254) and developed with a solution
of 2% potassium permanganate in a 0.2 M aqueous solution of NaOH.
Compounds obtained were purified by open column chromatography
*Corresponding author [telephone (33) 5 40 00 2913; fax (33) 5 40 00
64 39; e-mail v.coma@us2b.u-bordeaux1.fr].
pubs.acs.org/JAFC
Published on Web 09/02/2009
© 2009 American Chemical Society

Article
J. Agric. Food Chem., Vol. 57, No. 19, 2009 8771
Figure 1. Synthesis of GSA-3.
(silica gel and with different eluents). FTIR spectra were recorded on a
Perkin-Elmer Paragon 1000 PC spectrophotometer from 300 mg of KBr
pellet disks containing 3 mg of compound. The KBr pellet disks were formed
from Edwards High Vacuum Pump Es 50. Spectra were recorded between
400 and 4000 cm-1 using 50 scans at a resolution of 4.0 cm-1. 1H NMR and
13C NMR spectra were recorded at 300 MHz from a Bruker Avance 300
spectrometer. Chemical shifts are given in parts per million, and the assign-
ments of 1H NMR and13C NMR signals were monitored by COSY, HMBC,
or HMQC NMR spectroscopy.
Brimacombe et al. (14) to give 1,2,5,6-di-O-isopropylidene-3-azido-3-
deoxy-R- -glucofuranose (5): yield, 34.2%; mp, 104-109 °C; FTIR
D
(KBr), νmax (cm-1) 3428, 3019-2839, 2140, 1454-1158, 1081, 702-500;
1H NMR (300 MHz, CDCl3), δH 5.79 (1H, d, H-1), 4.59 (1H, t, H-2), 4.29
(1H, dd, H-4), 4.06-3.90 (3H, m, H-3, H-5, H-6a), 3.85-3.8 (1H, dd, H-
6b), 1.56-1.35 (12H, s, -CH3). Compound 5 was dissolved in methanol
and submitted to hydrogen flow in the presence of Pd/C (5%) to reduce the
azido group. After filtration of catalyst, the solution was concentrated to
half, and hydrochloric acid in aqueous solution (0.2 N) was added. The
mixture was heated at 50 °C for 15 min and washed three times with
Chemical Synthesis. As previously reported (10, 11), β-D-glucopyr-
chloroform, and the solvent was evaporated to give 3-amino-3-deoxy-R-D-
anosylamine (GSA-1) was synthesized from glucose and ammonium
carbamate in methanol to give a desired compound with yield varying
from 98.3 to 100%.
3-Amino-3-deoxy-D-glucose (GSA-3) was synthesized from D-glucose in
seven steps (Figure 1) using the methods previously reported (12-18).
Hydroxyl groups were protected using the method of Blakley et al. (12) to
glucopyranose hydrochloride (15) (GSA-3): yield, 33%; mp, decomposi-
tion at 125 °C; FTIR (KBr), νmax (cm-1) 3428, 3019-2839, 1621, 1454-
1195, 1164, 1081, 1029, 882, 702-500; 1H NMR (300 MHz, D2O), δH 5.21
(1H, d, HR-1), 3.85-3.82 (1H, dd, H-6a), 3.8 (1H, complex, H-5), 3.76 (1H,
t, H-2), 3.76-3.74 (1H, dd, H-6b), 3.65 (1H, dd, H-4), 3.6-3.5 (1H, dd, H-
3); 13C NMR (300 MHz, D2O), δC 97.2 (C-1, isomer R), 92.9 (C-1, isomer
β), 76.2 (C-5), 75.0 (C-2), 73.2 (C-4), 70.1 (C-6), 61.1 and 60.9 (C-3).
6-Amino-6-deoxy-R-D-methylglucoside (GSA-6). This com-
pound was synthesized in three steps as follows (Figure 2). The modified
method of Kondo et al. (19) was used to tosylate the C-6 of glucose. Then,
a substitution of tosyl by an azide group was realized using the modified
methods of Brimakombe et al. (14) and Li et al. (20), and azide reduction
was followed using the modified method of Jary et al. (15). To synthesize
give 1,2,5,6-di-O-isopropylidene-D-glucofuranose (1): yield, 43.4%; mp,
107-111 °C; 1H NMR (300 MHz, CDCl3), δH 5.96 (1H, d, H-1), 4.52 (1 H,
dd, H-2), 4.31 (2H, complex, H-4, H-5), 4.13 (1H, dd, H-3), 4.03 (1H,
dd, H-6a), 3.92 (1H, dd, H-6b), 2.63 (1H, s, -OH), 1.49-1.31 (12H, s,
-CH3). The oxidation of OH-3 was done using a method described by
Saito et al. (17) and gave 1,2,5,6-di-O-isopropylidene-3-oxo-D-gluco-
furanose (2): yield, 96.7%; mp, 130 °C; FTIR (KBr), νmax (cm-1) 3390,
3000-2856, 1729, 1456-1352, 1278-1215, 1067-1014; 13C NMR (300
MHz, CDCl3), δC 208.7 (C-3, CdO), 129.0 (C-2), 128.2 (C-1), 114.2 (C-8),
110.3 (C-7), 103.0 (C-4), 78.9 (C-5), 64.2 (C-6), 27.4-25.2 (-4CH3). A
carbonyl function of compound 2 was reduced by NaHB4 in ethanol to
give an epimer of 1, which is compound 3 (16): yield, 50.4%; mp, 106 °C;
FTIR, νmax (cm-1) 3390, 3000-2856, 1456-1352, 1278-1215, 1067-
1014. Then, this compound easily reacted with p-toluenesulfonyl chloride
(Ts) in dry pyridine (13) to give 1,2,5,6-di-O-isopropylidene-3-O-(p-
6-(p-toluenesulfonyl)-R-D-methylglucoside, R-D-methylglucoside (10 g,
51.5 mmol) was dissolved in 20 mL of pyridine previously dried on 4A
molecular sieves. At 0 °C, under shaking, a mass of 19.6 g (103 mmol) of p-
toluenesulfonyl chloride was added. The mixture was then kept at 0 °C for
24 h before being shaken at 5 °C for 48 h and at ambient temperature for
48 h. The reaction mixture was diluted with 20 mL of distilled water and
extracted three times with portions of 20 mL of chloroform. The organic
layer was washed successively with a portion of 20 mL of 0.2 N
hydrochloric acid, saturated sodium hydrogenocarbonate, and distilled
water before being dried with sodium sulfate. Solvent was evaporated, and
residue was taken in a small volume of chloroform and submitted to the
open column chromatography (silica gel, ethyl acetate/cyclohexane 7:3).
The product 9 was obtained: yield, 30.5%; Rf, 0.52 (ethyl acetate/
toluenesulfonyl)-R- -glucofuranose (4): yield, 91.6%; mp, 116-118 °C;
D
1H NMR (300 MHz, CDCl3), δH 7.8 (2H, d, H-arom), 7.3 (2H, d,
H-arom), 5.92 (1H, dd, H-3), 4.83 (1 H, d, H-1), 4.79 (1H, dd, H-2),
4.1-3.8 (4H, m, H-4, H-5, H-6a, H-6b), 2.4 (3H, s, -CH3 arom), 1.47-
1.14 (12H, s, -CH3). A substitution of the tosyl group by an azido group
was done in DMF at 120 °C for 2 days using a modified method of

8772 J. Agric. Food Chem., Vol. 57, No. 19, 2009
Muhizi et al.
Figure 2. Synthesis of GSA-6.
cyclohexane 7:3); 1H NMR (300 MHz, CDCl3), δH 7.81-7.75 (2H, dd, H-
arom), 7.35-7.30 (2H, dd, H-arom), 4.57 (1H, d, H-1), 4.25-4.06 (5H, m,
H-3, H-4, H-5, H-6a, and H-6b), 3.19 (3H, s, -OCH3), 2.42 (3H, s, -CH3).
Then, product 9 (4 g, 11.5 mmol) was dissolved in 20 mL of dried DMF,
and 2.9 g of sodium azide (44 mmol) was added. The mixture was refluxed
at 120 °C for 48 h. Solvent was removed. A viscous yellowish residue was
purified on open column chromatography (silica gel, ethyl acetate/
cyclohexane 8:2) and compound 10, 6-azido-6-deoxy-R-D-methylgluco-
side, was obtained: yield, 68%; Rf, 0.9 (ethyl acetate/cyclohexane 8:2);
FTIR (KBr), νmax (cm-1) 3569-3125, 2971-2821, 2105, 1482-1278,
1090, 713-524. Thus, compound 10 (1.2 g; 5.5 mmol) was dissolved in
20 mL of dried methanol, and 0.4 g of Pd/C (5%) was added. The mixture
was submitted under a flow of hydrogen to reduce the azide group for 5 h.
After the catalyst had been filtered and the solution concentrated, the
yellowish residue obtained was purified by open column chromatography
(silica gel, ethyl acetate/cyclohexane 8:2) and 1 g of compound 6-amino-6-
Figure 3. FTIR spectra of Glu, GSA-1, GSA-3, and GSA-6.
Table 1. FTIR Bands Attribution
wavenumber (cm-1
)
band attributions
deoxy-R-D-methylglucoside (11 or GSA-3) was obtained: yield, 94.5%;
FTIR (KBr), νmax (cm-1) 3569-3125, 3371, 3309, 2971-2821, 1607,
3389
symmetric and asymmetric stretching vibration of O-H
1
1482-1278, 1090, 893, 713-524; H NMR (300 MHz, CDCl3), δH 4.77
3344-3333 (double symmetric and asymmetric stretching vibration of N-H
vibration)
(1H, d, H-1), 3.73-3.67 (2H, t, H-3, H-4), 3.56-3.40 (4H, m, H-5,
H-6a, H-6b, H-2,), 3.47 (3H, -OCH3); 13C NMR (300 MHz, CDCl3),
δC 99.4 (C-1), 74.1 (C-3), 71.9 (C-5), 70.8 (d, C-2, C-4), 55.4 (-OCH3),
51.3 (C-6).
2963-2857
1619
symmetric and asymmetric stretching vibration of CH2
bending vibration in plane scissoring of N-H
bending vibration in plane scissoring, out of plan wagging,
and out of plan twisting of CH2
1457-1253
Microorganisms and Preparation of Inocula. L. innocua (ISTAB,
´
Universite Bordeaux1) and Salmonella typhimurium (Institut Pasteur
5858) were maintained at -70 °C in 20% of glycerol. Overnight pre-
cultures were performed as follows: L. innocua and S. typhimurium were
grown in tryptose broth (Difco 262200) and nutrient broth (Difco 234000)
at 37 °C for 18 h, respectively.
1122
1085
bending vibration of C-O for secondary alcohol
bending vibration out of plan twisting of C-N
stretching vibration of C-O for primary alcohol
bending vibration out of plan twisting of N-H
bending vibration in plan rocking of CH2
1029
875
640-559
Antibacterial Activity Assessment. The antibacterial assessment
of aminodeoxyglucose derivatives was conducted using an agar plate
method. Compounds were tested at concentrations varying in the range
from 0.25 ꢀ 10-4 to 2 ꢀ 10-4 mol mL-1. To do this, 20 mL of culture
medium prepared by mixing tryptose broth (Difco 262200) or nutrient
broth (Difco 234000) with 15% (w/w) agar (Difco 215530) for L. innocua
and S. typhimurium, respectively, was poured into each Petri dish.
After solidification, 2 mL of the tested compound was poured into
each test Petri dish and spread on the surface of the agar medium. Dishes
were then left opened for 1 h in a sterilized chamber to enable solvent to
be evaporated. Thus, about 100-300 cell charges per plate of an 18 h
microbial culture were deposited on the medium prior to incubation at
37 °C for 48 h prior to colony counting. The initial microbial charge of an
18 h culture was evaluated from preliminary numeration experiments
on tryptose and nutrient broth, respectively, for listerial and nonlisterial
strains. Moreover, the initial cell number was confirmed by the control
assays conducted in parallel without biocide. The control experi-
ments without any compounds to be tested were conducted in parallel,
and all experiments were in triplicate. The effectiveness of amino-
deoxyglucoses was determined as percentage of inhibition calculated as
follows:
expressed as
M1 -M2
SD
t ¼
where M1 is the control mean (or the first test experiment mean), M2 is the
test experiment mean (or the second test experiment mean), and SD is the
standard deviation between means. The degree of freedom used to
determine this probability was equal to 4 in our experiment. The standard
σ
n
p
ffiffi
error means (SEM) were calculated as SEM ¼
where n is the size of
sample (n=3) and σ is the standard deviation.
RESULTS AND DISCUSSION
Physicochemical Characteristics of GSA-1, GSA-3, and GSA-6.
GSA-1, GSA-3, and GSA-6 were preliminarily characterized by
FTIR spectra (Figure 3) showing the vibration bands around
1619 cm-1, which were not observed from glucose spectra
and thus indicate the presence of N-H bonding. Furthermore,
GSA-1 and GSA-6 showed other particular double-vibration
bands around 3330 cm-1, suggesting the presence of free amino
groups in both molecules (21). These vibration bands were absent
in the GSA-3 spectrum because of its amino group in the form
of ammonium. Vibration bands observed around 1085 and
875 cm-1 were attributed to the bending vibrations of C-N
and out of plan vibration bands of N-H, respectively. Other
vibration bands observed from Figure 3 were from different
pyranosic ring bonds such as C-H, CH2, C-O, and O-H as
indicated in Table 1.
CFU in control plates -CFU in test plates
percentage of inhibition ¼
CFU in control plates
ꢀ 100
Analysis of Results. Results from the antibacterial assays were
statistically analyzed using Student’s t test. The probability (p) was
determined from the critical t value obtained by comparing results
from control and different experiments, on the one hand, and
those from test experiments on the other hand. The critical t value is

Article
J. Agric. Food Chem., Vol. 57, No. 19, 2009 8773
To complete the FTIR data and characterize GSA-1, GSA-3,
shifts of anomeric proton and C-1 of GSA-1 were taken as
references in NMR spectroscopies to confirm the fixation of
NH2 group, in comparison to glucose. For this, a signal in the
form of doublet of Hβ-1 and another of C-1 were obtained at
chemical shifts of 4.15 and 85.08 ppm despite 4.65 and 92.3 ppm
found for glucopyranose (22, 23). In the synthesis of GSA-3
different melting points obtained from intermediary products
conformed to these reported (12-15, 17, 24) and thus suggest
purity; however, their further characterization using either FTIR
or NMR spectroscopy was needed to complete these literature
data. The structure of product 2 was confirmed by a signal at a
chemical shift of 208.7 ppm in 13C NMR and by a vibration band
at 1729 cm-1, both indicating the presence of a carbonyl group in
FTIR (21, 25); this band was disappeared by the complete
reduction done with NaBH4 to give the product 3. Substitution
of the hydrogen of OH-3 by a tosyl group was indicated by signals
at chemical shifts of 7.8 and 7.3 ppm in the form of doublets,
which was attributed to aromatic protons. These signals dis-
appeared when compound 4 was reacted with sodium azide to
give compound 5, which was confirmed by FTIR spectra showing
a strong vibration band at 2140 cm-1. This group was characteri-
zed in other papers by the vibration bands observed around
2100 cm-1 (4,26). Reduction of the azido group of 5 followed by a
deprotection of the hydroxyl group gave GSA-3, which was
characterized by both FTIR ad NMR spectroscopies. The
FTIR spectrum of this compound showed a vibration band at
1624 cm-1, characterizing a bending vibration in plane scissoring
of N-H. This was confirmed by both 1H and 13 C NMR spectra.
An anomeric proton signal was observed at 5.21 ppm in 1H
NMR, which confirmed a total deprotection of OH-1, whereas
other protons of GSA-3 were represented by signals in the zone of
chemical shifts of 3.85-3.50 ppm, which was confirmed by the
finding of Milner et al. (27). Furthermore, in 13C NMR, signals of
C-1 and C-3 were found at chemical shifts of 97.2 and 60.9 ppm,
respectively, and this corroborated those described for this
compound (27,28). For the GSA-6 synthesis, the characterization
of intermediary products, compounds 9 and 10, was conducted as
done in the case of GSA-3. For compound 9, two signals of
aromatic protons were found at 7.81 and 7.35 ppm in 1H NMR,
whereas the formation of compound 10 was justified by FTIR
spectra that showed a vibration band at 2105 cm-1 characteristic
of an azide group. Reduction of compound 10 was confirmed by
the FTIR spectrum of GSA-6, which showed, on the one hand,
the disappearance of the vibration at 2105 cm-1 and, on the other
hand, the appearance of a new absorption at 1607 cm-1, indicat-
1
and GSA-6, H NMR and 13C NMR spectroscopy were used
(Figures 4-6). As reported in our recent paper (11) the chemical
Figure 4. NMR spectra of GSA-1.
1
ing the bending vibration in plane scissoring of N-H. In H
NMR, the signal of H-1 was found at a chemical shift of 4.8 ppm.
Figure 5. NMR spectra of GSA-3.
Its shifted displacement at low parts per million in comparison of
that of R-D-glucopyranose, which was reported to be at 5.23
ppm (23), was due to the replacement of OH by OMe in this
compound. In addition, protons H-6a and H-6b resonated at
lower chemical shifts compared to those of the same protons of
GSA-3 or GSA-1, which confirmed the substitution of OH-6 by
an NH2 group in GSA-6 (Table 2). Furthermore, in 13C NMR
C-6 resonated at a chemical shift of 51.28 ppm, and this
corroborated the finding of Murphy et al. (29), who characterized
a C-6 of 1-methyl-6-deoxy-6-azido-β-D-glucopyranoside, a com-
pound quite similar to GSA-6.
Antibacterial Activities of GSA-1, GSA-2, GSA-3, and GSA-6.
To determine the effect of amino group position on the anti-
bacterial activity of aminoglucose derivatives, four compounds,
GSA-1, GSA-2, GSA-3, and GSA-6, were comparatively tested
on two food bacteria, L. innocua and S. typhimurium. Concentra-
tions ranging from 0.25 ꢀ 10-4 to 2 ꢀ 10-4 mol mL-1 were used
in this study.
Figure 6. NMR spectra of GSA-6.

8774 J. Agric. Food Chem., Vol. 57, No. 19, 2009
Muhizi et al.
Table 2. Different Chemical Shifts of C-6, H-6a, and H-6b for GSA-1, GSA-2,
and GSA-6
Table 3. Antibacterial Activities of GSA-1, GSA-2, GSA-3, and GSA-6 against
the Growth of L. innocua at Different Concentrations
chemical shifts (ppm)
percentage of inhibition (ꢀ10-4 ( SD) at a concentration of
aminoglucose 0.25 mol mL-1 0.5 mol mL-1 1 mol mL-1 2 mol mL-1
type of signal
GSA-1
GSA-3
GSA-6
C-6
60.8
3.95
3.76
60.9
3.86-3.82
3.76-3.74
51.3
e3.56
e3.56
GSA-1
GSA-2
GSA-3
GSA-6
0.0 ( 0.0
2.2 ( 1.0
10.0 ( 0.4
3.6 ( 1.0
1.1 ( 0.7
7.8 ( 3.0
19.3 ( 1.5
10.0 ( 7.0
3.3 ( 1.0
8.9 ( 4.0
22.8 ( 1.3
12.8 ( 3.0
5.1 ( 3.0
10.6 ( 1.1
29.1 ( 2.5
17.7 ( 1.4
H-6a
H-6b
Results showed that GSA-3 was significantly (p < 0.05) more
anti-L. innocua than the other compounds tested (Table 3).
However, there is no significant difference between the anti-S.
typhimurium activities of GSA-3 and GSA-6, and these two
compounds are significantly (p < 0.05) more anti-S. typhimurium
than GSA-1 and GSA-2 (Table 4).
Table 4. Antibacterial Activities of GSA-1, GSA-2, GSA-3, and GSA-6 against
the Growth of S. typhimurium at Different Concentrations
percentage of inhibition (ꢀ10-4( SD) at a concentration of
aminoglucose 0.25 mol mL-1 0.5 mol mL-1 1 mol mL-1 2 mol mL-1
For example, at the concentration of 2 ꢀ 10-4 mol mL-1 GSA-
3 affected the growth of L. innocua with a percentage of inhibition
of 29%, whereas GSA-1, GSA-2, and GSA-6 exhibited inhibi-
tions close to 5, 11, and 18%, respectively. The same observation
was made for S. typhimurium, for which GSA-3 altered the
microbial growth with 15% of inhibition, whereas GSA-1,
GSA-2, and GSA-6 exhibited activities of only 2, 7, and 11%
of inhibition, respectively. Thus, the biocide efficiency found in
this study decreased in the order GSA-3 g GSA-6 > GSA-2 >
GSA-1.
The mechanism of action of these compounds was not deter-
mined in this study. However, according to different reports done
on aminosugars and similar compounds (30, 31), these types of
biocide may exhibit their activity by inhibiting the cell wall and
membrane synthesis of bacteria and also the synthesis of proteins
or DNA . According to Llewellyn et al. (31), aminoglucosides
bond at the 30S subunit, which includes 16S NRA, and blocked
all of its biological mechanisms and altered the growth of
GSA-1
GSA-2
GSA-3
GSA-6
0.0 ( 0.0
0.0 ( 0.0
0.0 ( 0.0
0.0 ( 0.0
0.3 ( 1.0
0.0 ( 0.0
5.8 ( 2.0
4.9 ( 1.0
1.0 ( 1.0
3.7 ( 1.0
10.0 ( 3.0
8.4 ( 2.0
2.3 ( 1.0
7.2 ( 1.0
14.6 ( 1.1
10.5 ( 3.0
kanosamine (GSA-3) and the amino group at C-6 in antibiotic
structures is reported to be essential in cell recognition and in
the diversification of binding sites, respectively (36). These pre-
vious reports are in agreement with our results, where GSA-3 and
GSA-6 exhibited better antibacterial activity against L. innocua
and S. typhimurium compared to GSA-1 and GSA-2. Further-
more, in this study GSA-3 was synthesized and isolated as an
ammonium compound and biologically tested as well. This
chemical characteristic of GSA-3 may increase its biological
effects on bacteria growth because the impact of ammonium
compounds on the growth of bacteria has already been re-
ported (9, 37). The impact of quaternary ammonium on the
growth of bacteria is due to its high facility to bind to the cell
membrane of bacteria and thus to modify their permeability (37).
However, even if GSA-3 and GSA-6 are more active than GSA-2
and GSA-1, their biological activity is still to be ameliorated. One
way to achieve this is to N-alkylate these molecules or to manage
other modifications by multiplying the number of glucosyl and
amino groups introduced. The nature and position of pharma-
cophore groups to be introduced may be of interest because both
the position of C-3 and the ammonium form of the amino group
have shown high efficiency in the antibacterial activity assess-
ment. GSA-2 is actually the essential element in the biosynthesis
of peptidoglycan found in the cytoplasm walls of bacteria (34,35).
These essential physiological roles of GSA-2 may explain its
insignificant antibacterial activity against both L. innocua and
S. typhimurium growth. In comparison to chitosan, a known
bacterial inhibitor, these monomers exhibited lower antibacterial
activity against selected microorganisms. In fact, according to
some authors, chitosan exhibited higher antibacterial activity
against both strains at the concentration close to 10-4 mol mL-
1, and at 5 ꢀ 10-4 mol mL-1 this compound quite completely
inhibited the growth of both bacterial strains (9). The higher
antibacterial activity of chitosan compared to small aminosugars
was not studied here. However, its high viscosity and high number
of ammonium groups may explain its antibacterial efficacy.
In conclusion, GSA-1, GSA-2, GSA-6, and GSA-3 have shown
differentantibacterial activities. Accordingto theseresults, further
modifications of these molecules may be envisaged to improve
their antibacterial activity as well.
bacteria. Furthermore, other studies done on
D-glucosamine
showed that its N-alkyl derivatives had high affinity with 16S
NRA, which played a key role in the translation mechanism (32).
Considering the high similarity of these compounds with the
aminoglucose derivatives used in our study, it is possible that the
same mechanism may be considered for the antibacterial activity
found in this paper. Comparing the biological activities obtained
from GSA-1, GSA-2, GSA-6, and GSA-3 and having in mind
that aminoglucosides mainly act by altering the function of
ribosomes (31), this study suggests that GSA-3 may have more
affinity with ribosome of bacteria than other aminoglucoses
tested. According to these results, an amino group fixed at
position 3 of glucose may increase the biological effects of
aminosugar on the growth of bacteria. These findings on the
activity of GSA-3 are supported by the literature, where the
biological activity of this compound against bacteria was already
demonstrated (27,28,33). According to Milner et al. (27), GSA-3
delayed the growth of both Gram-positive and Gram-negative
bacteria, as, for example, Lactobacillus acidophilus, Staphylo-
coccus aureus, Erwinia herbicola, and Cytophaga johnsonae. Some
authors (34, 35) have suggested that the compound may act by
inhibiting the cell wall synthesis through an inhibition of
D-frucose-6-phosphate incorporation in the cell wall and, thus,
by avoiding the synthesis of 6-phosphate glucosamine, known as
a key product in cell wall reconstruction. The reason for the effect
of the amino group position on antibacterial activity is still
unknown. However, it is reported that the number and the
position of amino groups on sugar rings of antibiotics play
important roles in their antibacterial activity (30). In fact, a higher
activity of kanamycin A, an antibiotic with a 6-aminohexose
group, relative to that of kanamycin C, with a 2-aminohexose
group, was reported (30, 36). In addition, the presence of
ACKNOWLEDGMENT
We thank Fabrice Azemar (student in training in US2B,
University of Bordeaux 1) for technical assistance.

Article
J. Agric. Food Chem., Vol. 57, No. 19, 2009 8775
LITERATURE CITED
(20) Li, S. C.; Bao, X.; Cai, M. S.; Li, Z. J. Optimazed procedure for
synthesis of 6-azido-6-deoxy-galactopyranosides from 6-octosyl-
galactopyranosides. Synth. Commun. 2006, 36 (5), 637–643.
(1) Altieri, C.; Siniganglia, M.; Corbo, M. R.; Buonocor, G. G.;
Falcone, P.; Del Nobile, M. A. Use of entrapped microorganisms
as biological oxygen scavengers in food packaging applications.
Lebensm.-Wiss. -Technol. 2004, 37, 9–15.
(2) Vartiainen, J.; Skytta, E.; Enqvist, J.; Ahvenainen, R. Properties of
antimicrobial plastics containing traditional food preservatives.
Packag. Technol. Sci. 2003, 16, 223–229.
(3) Soares, F. F.; Rutishauser, D. M.; Melo, N.; Cruz, R. S.; Andrade,
N. J. Inhibition of microbial growth in bread through active
packaging. Packag. Technol. Sci. 2002, 15, 129–132.
(4) Lee, C. H.; An, D. S.; Park, H. J.; Lee, D. S. Wide-spectrum
antimicrobial packaging materials incorporating nisin and citosan
in the coating. Packag. Technol. Sci. 2003, 16, 99–106.
(5) Vermeiren, L.; Devlieghere, F.; van Beest, M.; de Kruijf, N.;
Debevere, J. Developments in the active packaging of foods. Tends
Food Sci. Technol. 1999, 10, 77–86.
(21) Silverstein, R. M.; Basler, G. C.; Morill, T. C. Identification Spectro-
´
´
metrique de Composes Organiques; De Boeck Université: Bruxelles,
Belgium, 1998; 420 pp.
(22) Lubineau, A.; Auge, J.; Drouillat, B. Improved synthesis of glyco-
´
sylamines and a straight forward preparation of N-acylglycosyl-
amines as carbohydrate-based detergents. Carbohydr. Res. 1995, 266,
211–219.
(23) Blasko, A.; Bunton, A. B.; Bunel, S.; Ibarra, C.; Moraga, E.
Determination of acid dissociation constants of anomers of amino
sugars by 1H NMR spectroscopy. Carbohydr. Res. 1997, 298, 163–
172.
(24) Trnika, T.; Cerny, M.; Budesinsky, M.; Pacak, J. The synthesis of
3-amino-3-deoxy-
vative conformation of amino derivatives of 1,6-anhydro-β-
D
-glucose (kanosamine) and its 1,6-anhydro deri-
-hexo-
D
pyranoses. Collect. Czech. Chem. Commun. 1975, 40, 3038–3045.
(25) Pretsch, E.; Clerc, T.; Seibl, J.; Simon, W. Tablas para la Elucidacion
Estructural de Compuestos Organicos por Metodos Espectroscopicos;
Springer Verlag: Berlin, Germany, 1989; pp C95-U155.
(6) Coma, V.; Martial-Gros, A.; Garreau, S.; Copinet, A.; Salin, F.;
Deschamps, A. Edible antimicrobial films based on chitosan matrix.
J. Food Sci. 2002, 67, 1162–1169.
(7) Coma, V.; Deschamps, A.; Martial-Gros, A. Bioactive packaging
materials from edible chitosan polymer-antimicrobial activity assess-
ment on dairy-related contaminants. J. Food Sci. 2003, 68, 2788–
2792.
(26) Borjihan, G.; Zhong, G.; Baigude, H.; Nakashima, H.; Uryu, T.
Synthesis and anti-HIV activity of 6-amino-6-deoxy-(1f3)-β-
curdlan sulfate. Polym. Adv. Technol. 2003, 14, 326–329.
D-
(27) Milner, J. J.; Silo-Suh, L.; Lee, J. C.; He, H.; Clardy, J.; Handelsman,
J. Production of kanosamine by Bacillus cereus UW85. Appl.
Environ. Microbiol. 1996, 62 (8), 3061–3065.
(28) Dolak, L. A.; Castle, T. M.; Dietz, A.; Laborde, A. L. 3-Amino-
3-deoxyglucose produced by a Streptomyces sp. J. Antibiot. 1980, 33
(8), 900–901.
(29) Murphy, P. V.; O’Brien, J. L.; Gorey-Feret, L. J.; Smith, A. B.
Synthesis of novel HIV-1 protease inhibitors based on carbohydrate
scaffolds. Tetrahedron 2003, 58, 2259–2271.
(30) Benveniste, R.; Davies, J. Structure-activity relationships among
the aminoglycoside antibiotics: role of hydroxyl and amino groups.
Antimicrob. Agents Chemother. 1973, 4 (4), 402–409.
(8) El-Tahlawy, K. F.; El-Bendary, M. A.; El-Hendawy, A. G.;
Hundson, S. M. The antimicrobial activity of cotton fabrics with
different crosslinking agents and chitosan. Carbohydr. Polym. 2005,
60, 421–430.
(9) Belalia, R.; Grelier, S.; Benaissa, M.; Coma, V. New biocative
biomaterials based on quaternized chitosan. J. Agric. Food Chem.
2008, 56, 1582–1588.
(10) Likhosherstov, L. M.; Novikova, O. S.; Shibaev, V. N. New efficient
synthesis of β-glucosylamines of mono- and disaccharides with
the use of ammonium carbamate. Dokl. Chem. 2002, 383 (4), 500–
503.
(11) Muhizi, T.; Coma, V.; Grelier, S. Synthesis and evaluation of N-
alkyl-β-
D-glucosylamines on the growth of to wood fungi, Coriolus
(31) Llewellyn, N. M.; Spencer, J. B. Biosynthesis of 2-deoxystreptamine
containing aminoglycoside antibiotics. Nat. Prod. Rep. 2006, 23,
864–874.
versicolor and Poria placenta. Carbohydr. Res. 2008, 343, 2369–2375.
(12) Blakley, E. R. 6-Deoxy-6-fluoro- -glucose. Biochem. Prep. 1960,
D
7 (39), 40–44.
(32) Wu, B.; Yang, J.; Robinson, D.; Hofstadler, S.; Griffey, R.; Swayze,
E. E.; He, Y. Synthesis of linked carbohydrates and evaluation of
their binding for 16S RNA by mass spectrometry. Bioorg. Med.
Chem. Lett. 2003, 13, 3915–3918.
(33) Janiak, A. M.; Milewski, S. Mechanism of antifungal action of
kanosamine. Med. Mycol. 2001, 39 (5), 401–408.
(13) Williams, D. T.; Jones, J. K. N. A new synthesis of 3-acetamido-3-
deoxy- -glucose. Can. J. Chem. 1967, 45 (7), 7–9.
D
(14) Brimacombe, J. S.; Bryan, J. G. H.; Husain, A.; Stacey, M.; Tolley,
M. S. The oxidation of some carbohydrate derivatives, using acid
anhydride methyl sulphoxide mixtures and the Pfitzner-Moffatt
reagent. Facile synthesis of 3-acetamido-3-deoxy-
D-glucose and 3-
(34) Iwai, Y.; Tanaka, H.; Oiwa, R.; Shimizu, S.; Omura, S. Studies on
amino-3-deoxy- -xylose. Carbohydr. Res. 1967, 3, 318–324.
D
bacterial cell wall inhibitors. III. 3-Amino-3-deoxy-D-glucose, an
(15) Jary, J.; Kefurtova, Z.; Kovar, J. Aminosugars XX. Synthesis of 3-
amino- and 6-amino-deoxyhexoses of the gluco and allo configura-
tion. Collect. Czech. Chem. Commun. 1969, 34, 1452–1458.
inhibitor of bacterial cell wall synthesis. Biochim. Biophys. Acta 1977,
498, 223–228.
(35) Tanaka, H.; Shimizu, S.; Oiwa, R.; Iwai, Y.; Omura, S. The site of
(16) Yu, Y.; Russell, R. N.; Thorson, J. S.; Liu, L.; Liu, H. Mechanistic
studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseu-
dotuberculosis. J. Biol. Chem. 1992, 267 (9), 5868–5875.
inhibition of cell wall synthesis by 3-amino-3-deoxy-D-glucose in
Staphylococcus aureus. J. Biochem. 1979, 86 (1), 155–159.
(36) Le Goffic, F.; Capmau, M.; Tangy, F.; Baillarge, M. Mechanism of
action of aminoglycoside antibiotics. Eur. J. Biochem. 1979, 102, 73–81.
(37) Kim, C. H.; Choi, J. W.; Chun, H. J.; Choi, K. S. Synthesis of
chitosan derivatives with quaternary ammonium salt and their
antibacterial activity. Polym. Bull. 1997, 38, 387–393.
(17) Saito, Y.; Zevaco, T. A.; Agrofoglio, L. A. Chemical synthesis of 13
C
labeled anti-HIV nucleosides as mass-internal standards. Tetrahe-
dron 2002, 58, 9593–9603.
(18) Guo, J.; Frost, J. W. Kanosamine biosynthesis: a likely source of the
aminoshikimate pathway’s nitrogen atom. J. Am. Chem. Soc. 2002,
124, 10642–10643.
Received December 19, 2008. Accepted July 17, 2009. We are very
grateful to the Ministry of Foreign Affairs of the French government for
funding.
(19) Kondo, Y. Partial tosylation of methyl R- and β-D-galactospyrano-
sides. Agric. Biol. Chem. 1988, 52 (5), 1313–1315.