Synthesis, surfactant properties and antimicrobial activities of methyl glycopyranoside ethers

Article abstract of DOI:10.1016/j.ejmech.2017.01.038

A series of amphiphilic methyl glucopyranoside ethers incorporating various alkyl chain lengths has been synthesized from commercially available methyl glucopyranosides following an acetalisation/hydrogenolysis sequence. The amphiphilic properties of ethers and acetal intermediates were evaluated. Both families exhibit excellent surfactant properties with a maximum efficiency obtained for compounds bearing a linear dodecyl chain (CMC = 0.012 mM, γ_sat.= 30 mN m^?1). Antimicrobial activity studies revealed an efficient activity (0.03 < MIC < 0.12 mM) against Gram-positive bacteria such as Listeria monocytogenes, Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus. More importantly, these compounds were found to be active against multi-resistant strains such as vancomycin-, methicillin- and daptomycin-resistant strains. Finally, it was found that antimicrobial activities are closely related to physicochemical properties and are also influenced by the nature of the carbohydrate moiety.

Full text of DOI:10.1016/j.ejmech.2017.01.038

Accepted Manuscript
Synthesis, surfactant properties and antimicrobial activities of methyl glycopyranoside
ethers
Dorine Belmessieri, Charlotte Gozlan, Marie-Christine Duclos, Valérie Molinier, Jean-
Marie Aubry, Oana Dumitrescu, Gérard Lina, Andreas Redl, Nicolas Duguet, Marc
Lemaire
PII:
S0223-5234(17)30048-X
10.1016/j.ejmech.2017.01.038
EJMECH 9189
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 November 2016
Revised Date: 20 December 2016
Accepted Date: 23 January 2017
Please cite this article as: D. Belmessieri, C. Gozlan, M.-C. Duclos, V. Molinier, J.-M. Aubry, O.
Dumitrescu, G. Lina, A. Redl, N. Duguet, M. Lemaire, Synthesis, surfactant properties and antimicrobial
activities of methyl glycopyranoside ethers, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.01.038.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, Surfactant Properties and Antimicrobial Activities of
Methyl Glycopyranoside Ethers
[
a,c,d]
[a,e]
[a]
[b]
Dorine Belmessieri,
Charlotte Gozlan, Marie-Christine Duclos, Valérie Molinier, Jean-Marie
[
b]
[c,d]
[c,d]
[e]
[a]
Aubry, Oana Dumitrescu,
Gérard Lina,
Andreas Redl, Nicolas Duguet,* and Marc
[
a]
Lemaire*
Keywords: Antimicrobial • Carbohydrate • Ether • Gram-positive bacteria • Surfactant
[
a]
Dr
D.
Belmessieri,
C.
Gozlan,
M.-C.
Duclos,
Dr
N.
Duguet,
Prof.
M.
Lemaire
Univ Lyon, Université Claude Bernard Lyon1, CNRS, INSA-Lyon, CPE-Lyon, Institut de Chimie et Biochimie
Moléculaires et Supramoléculaires, (ICBMS), UMR 5246, Equipe CAtalyse, SYnthèse et ENvironnement (CASYEN), 43
boulevard
du
11
novembre
1918,
F-69622
Villeurbanne
Cedex,
France.
E-mails: nicolas.duguet@univ-lyon1.fr , marc.lemaire.chimie@univ-lyon1.fr
[
[
[
[
b]
c]
d]
e]
Dr V. Molinier, Prof. J.-M. Aubry
Univ. Lille, CNRS, ENSCL, UMR 8181 - UCCS - Unité de Catalyse et Chimie du Solide F-59000 Lille, Cité Scientifique,
France.
Dr D. Belmessieri, Dr O. Dumitrescu, Prof. G. Lina
Univ Lyon, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, CNRS, UMR5308, International
Center for Infectiology Research (CIRI), Inserm U1111, 7 rue Guillaume Paradin, F-69008 Lyon, France
Dr D. Belmessieri, Dr O. Dumitrescu, Prof. G. Lina
Centre National de Référence des Staphylocoques
5
9 boulevard Louis Pinel, F-69677 Bron Cedex, France
Dr A. Redl
Tereos Syral SAS, Z.I et Portuaire, B.P.32, 67390 Marckolsheim, France.

ACCEPTED MANUSCRIPT
Abstract: A series of amphiphilic methyl glucopyranoside ethers incorporating various alkyl chain
lengths has been synthesized from commercially available methyl glucopyranosides following an
acetalization/hydrogenolysis sequence. The amphiphilic properties of ethers and acetal
intermediates were evaluated. Both families exhibit excellent surfactant properties with a maximum
-
efficiency obtained for compounds bearing a linear dodecyl chain (CMC = 0.012 mM, γsat. = 30 mN.m
1
)
. Antimicrobial activity studies revealed an efficient activity (0.03 < MIC < 0.12 mM) against Gram-
positive bacteria such as Listeria monocytogenes, Enterococcus faecalis, Enterococcus faecium and
Staphylococcus aureus. More importantly, these compounds were found to be active against multi-
resistant strains such as vancomycin-, methicillin- and daptomycin-resistant strains. Finally, it was
found that antimicrobial activities are closely related to physicochemical properties and are also
influenced by the nature of the carbohydrate moiety.
Introduction
Antibiotics and other antimicrobials are essential compounds to inhibit or prevent the growth of
[
1]
undesirable microorganisms. In this respect, they are widely used to prevent and cure bacterial
[
2]
infection and they have found many applications in medicine, in food industry, and in other fields
[
3]
[4]
such as packaging and textile. However, the consequence of this extensive utilization is the
emergence of antimicrobial-resistant infectious agents, which represents a major problem for public
[
5]
health. It is becoming particularly alarming since all known antimicrobial chemotherapies are
[
6]
striving to cure superbug’s infections. That is the reason why there is an incentive to seek and
[
7]
develop new effective agents with improved activity against resistant pathogens. Moreover, the
attention should also be paid on the cost of such agents in order to guarantee their accessibility to a
large part of the World’s population. Where is the progress if we have in hands highly sophisticated
antimicrobials that no one can afford?
In this context, cheap antimicrobials based on long-chain fatty acids and their corresponding polyol
ester derivatives have been developed. One of the most active is monolaurin, a linear C12 glycerol
®
monoester, manufactured under the trademark Lauricidin . This compound is used as food additive
[
8]
in order to inhibit the growth of various microorganisms. Carbohydrate-derived antimicrobials have
also attracted a lot of attention. Indeed, carbohydrates are cheap, natural products, available in bulk
quantities from renewable resources. Moreover, they are usually non-toxic and bio-degradable. As a
result, they provide an adequate platform for antimicrobials targeting industrial applications. For
example, sucrose esters are common carbohydrate fatty acid esters used by the food industry as
[
9]
preservatives. Particularly, sucrose monolaurate was found to be active against Listeria

ACCEPTED MANUSCRIPT
[
10]
monocytogenes and, to a lesser extent, against Staphylococcus aureus. Other carbohydrate fatty
acid esters, such as 6’-O-lauroylmaltose and 6’’-O-lauroylmaltotriose were found to inhibit the
[
11]
growth of Streptococcus sobrinus, hence they are promising additives in oral-hygiene products.
[
12]
Moreover, these molecules revealed to be active against Bacillus spp. and Lactobacillus plantarum.
Monosaccharide fatty acid esters have also shown interesting antimicrobials properties. For example,
[
13]
methyl 6-O-lauroyl-α-D-glucopyranoside displayed activity against S. aureus and Escherichia coli.
Some other analogues were also evaluated and found to be active against L. innocua and L.
[
14]
monocytogenes.
From a mechanistic point of view, carbohydrate fatty acid esters exhibit
[
15]
antimicrobial activity due to their ability to interact with biological membranes. Indeed, their
surfactant properties allow the solubilisation of the lipids constituting the cell membrane. This
[
16]
phenomenon impairs the membrane integrity and causes cellular lysis. However, the ester bond of
these antimicrobials could be cleaved by cellular esterases, thus releasing the corresponding inactive
[
13b]
sugar and free fatty acid.
For example, Ruzin and Novick have shown that monolaurin is rapidly
[
17]
hydrolyzed (t1/2 of about 5 min) in the presence of S. aureus cells.
To prevent the hydrolysis of these antimicrobial agents, the ester linkage can be advantageously
replaced by an ether function, insensitive to esterases. For example, the corresponding ether of
monolaurin, 1-O-dodecylglycerol, has shown greater activity against E. faecium than monolaurin
[
18]
itself, probably due to its greater retention in the cell. Similar observations were made with
carbohydrate fatty ethers such as methyl 6-O-dodecanyl-α-D-glucopyranoside, which exhibits an
[
13]
[14]
enhanced activity against S. aureus and Listeria spp. in comparison to its corresponding ester.
Consequently, carbohydrate fatty ethers seem to be promising candidates for new antimicrobial
agents with improved activity and half-life in cells. However, the synthesis of carbohydrate-derived
ethers usually requires a multi-step route including protection/deprotection strategies for selectivity
and solubility reasons. In addition, the ether function is usually introduced using Williamson
[
19]
conditions requiring a strong base and an alkyl halide or pseudo-halide. As a result, the atom
economy of such routes is usually quite low and large amount of waste is produced. Finally, such
strategies result in a very high production cost which is not suitable for widespread industrial
applications. This could probably explain why, to date, no carbohydrate fatty ether antimicrobial is
available on the market. In this context, our group has recently developed an efficient methodology
to prepare alkyl ethers of carbohydrate derivatives through hydrogenolysis of the corresponding
[
20]
acetals. Herein, we report our efforts to improve and extend the scope of application of this
methodology to a range of methyl glycopyranosides. We also describe the amphiphilic properties and
[
21]
antimicrobial activities of a series of methyl glycopyranoside ethers and their acetal intermediates.

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Results and Discussion
Synthesis of methyl α- -glucopyranoside alkyl ethers
D
The Dunne’s group described the synthesis of methyl 6-O-dodecyl-α-D-glucopyranoside from
commercially available methyl α- -glucopyranoside 1 following traditional carbohydrate chemistry
D
[
13a]
(
Scheme 1, a).
Scheme 1: Comparison of synthetic routes to access methyl 6-O-dodecyl-α-
dimethylformamide, PMB = para-methoxybenzyl, TBAI = tetrabutylammonium iodide, TBAF = tetrabutylammonium
fluoride, CAN = cerium ammonium nitrate.
D-glucopyranoside. TIPS = triisopropylsilyl, DMF
=
This five-step sequence involves 13 elemental reactions including TIPS-protection of the primary
alcohol, PMB-protection of the secondary alcohols, selective TBAF-deprotection of the primary
alcohol followed by the installation of the dodecyl chain using Williamson conditions and final
secondary alcohols deprotection with CAN. The desired product was obtained as a single
[
22]
stereoisomer in 21% overall yield and 6 % atom economy. However, this type of synthetic protocol
[
23]
is no longer in line with the green chemistry principles. Consequently, we have developed a two-
step acetalisation / hydrogenolysis sequence for the preparation of methyl O-alkyl-α-
D-
glucopyranoside directly from unprotected methyl α-D-glucopyranoside 1 (Scheme 1, b). Following
[
20]
our previously reported procedure,
a range of methyl α-D-glucopyranoside 4,6-O-acetals 2-6
bearing different alkyl chain lengths were synthesized in 26-44% isolated yields (Scheme 2).
Subsequent hydrogenolysis of the acetals afforded the corresponding ethers 7-11 as a mixture of 6-
O- and 4-O- regioisomers in up to 75 : 25 selectivity and in 37-81% isolated yields (Scheme 2). Methyl
6
-O-dodecyl-α-D-glucopyranoside 11 was prepared in 10 % overall yield but with an improved 95 %

ACCEPTED MANUSCRIPT
[
22]
atom economy. Considering the atom efficiency (1.3% vs 11.2%), this strategy is 8.5 times more
[
22]
efficient than Dunne’s synthesis. This cheap and environmentally friendly procedure provides a
remarkable improvement in carbohydrate chemistry as it allows a convenient and rapid access to
alkylated sugars.
a
Scheme 2. Preparation of methyl α-
D
-glucopyranoside ethers by acetalisation / hydrogenolysis sequence. The 6-O : 4-O
regioisomeric ratio was determined by GC chromatography after derivatization. CPME = cyclopentyl methyl ether.
Amphiphilic properties of methyl α-D-glucopyranoside acetals and ethers
The surface tension of aqueous solutions of acetals 2-6 and ethers 7-11 was evaluated by the rod
method, based on the Wilhelmy plate method. The amphiphilic properties of acetals 2-6
(
abbreviated CxAcMeGlu, with x = number of carbons of the alkyl chain, Ac = acetal, MeGlu = methyl
α- -glucopyranoside) were first investigated (Figure 1). All acetals allow a decrease of the surface
D
−
1
tension of water to a plateau of about 30 mN.m . Different tensiometric curve profiles were
observed depending on the alkyl chain length (Figure 1). Neither clear break nor plateau was
[
24]
obtained for C5AcMeGlu 2 and C6AcMeGlu 3, typical of hydrotropes. Indeed, the alkyl chain of
these amphiphiles is too short to promote cooperative association into micelles. Nevertheless, when
increasing the concentration they accumulate at the water/air interface and form small aggregates
within the aqueous solution thus, lowering the superficial tension. Typical surfactant profiles were
yet observed for compounds C8AcMeGlu 4 and C10AcMeGlu 5. The limit of solubility of 4 and 5 in
water revealed to be close to the CMC value implying a very small concentration range for micellar
solubilisation. Expectedly, the increase of the alkyl chain length led to a decrease of the MHC / CMC
values, from 82 mM for C5AcMeGlu 2 to 0.15 mM for C10AcMeGlu 5 (Table 1, entries 1-4). Finally,
acetal 6 (Cx = C12) could not be fully analyzed due to its low solubility in water and the formation of

ACCEPTED MANUSCRIPT
liquid crystals at low concentrations and at room temperature. However, its CMC has been estimated
to be > 0.06 mM (Table 1, entry 5).
8
7
6
5
4
3
2
0
0
0
0
0
0
0
-
6
-5
-4
-3
-2
-1
0
Log C (mol/L)
Figure 1. Surface tension vs concentration of methyl α-
C8AcMeGlu 4, – C10AcMeGlu 5.
D
-glucopyranoside acetals. Æ C5AcMeGlu 2, ° C6AcMeGlu 3, Å
Table 1. Minimal Hydrotropic Concentration (MHC), Critical Micelle Concentration (CMC) and surface tension (γsat) values
of methyl a-
D-glucopyranoside acetals.
[a]
[a]
Methyl α-
D
-
MHC or CMC
γsat
-
1
-1
Entry
glucopyranoside
acetals
(mmol.L )
(mN.m )
1
2
3
4
5
C5AcMeGlu 2
C6AcMeGlu 3
C8AcMeGlu 4
C10AcMeGlu 5
C12AcMeGlu 6
82
6.5
31
31
2.5
28
0.15
> 0.06
32
< 33
(
solubility limit)
[a]
Determined by tensiometry.
The surface tension of the regioisomeric mixtures of methyl α-D-glucopyranoside ethers 7-11
(
abbreviated CxEthMeGlu, with x = number of carbons of the alkyl chain, Eth = ether, MeGlu = methyl
α-D-glucopyranoside) was next investigated (Figure 2). Similarly, all ethers decrease the surface
−
1
tension of water to a plateau around 30 mN.m . Considering that the 6-O- : 4-O-regioisomeric ratio
is similar (around 70 : 30) for all methyl α- -glucopyranoside ethers, the alkyl chain length is the only
parameter influencing the MHC and CMC values. This behavior is commonly observed for other types
D
[
25]
of surfactants. As for acetals, ethers 7-8 bearing a C5 and C6 alkyl chain, exhibit a hydrotropic
character with MHC values of 35.0 and 13.4 mM, respectively (Table 2, entries 1-2). With longer alkyl
chain, C8EthMeGlu 9 and C10EthMeGlu 10 show characteristic surfactant profiles but with CMC

ACCEPTED MANUSCRIPT
values poorly defined at low concentrations, i.e. 0.43 and 0.28 mM respectively (Table 2, entries 3-4).
Gratifyingly, further increase of the alkyl chain length to 12 carbons permits a decrease of the CMC to
0
.012 mM (Table 2, entry 5). For a given alkyl chain, the CMC or MHC values of ethers are usually
lower than the acetals implying a higher hydrophilicity for the ether family. This trend was previously
[
20b]
observed for sorbitan derivatives.
8
7
6
5
4
3
2
0
0
0
0
0
0
0
-
8
-7
-6
-5
-4
Log C (mol/L)
-3
-2
-1
0
Figure 2. Surface tension vs concentration of methyl α-
D-glucopyranoside ethers (mixtures of regioisomers). Æ C5EthMeGlu
7
, ° C6EthMeGlu 8, Å C8EthMeGlu 9, – C10EthMeGlu 10, ‹ C12EthMeGlu 11.
Table 2. Minimal Hydrotropic Concentration (MHC), Critical Micellar Concentration (CMC) and surface tension (γ ) values
sat
of methyl α-D-glucopyranoside ethers (mixtures of regioisomers).
[a]
[b]
[b]
Entry
Methyl α-
D
-
Ratio
(%)
MHC or CMC
γsat
-1
-1
glucopyranoside ethers
(mmol.L )
(mN.m )
1
2
3
4
5
C5EthMeGlu 7
C6EthMeGlu 8
C8EthMeGlu 9
C10EthMeGlu 10
C12EthMeGlu 11
[b]
70 : 30
72 : 28
75 : 25
68 : 32
73 : 27
35.0
13.4
33
31
27
30
28
0.434
0.277
0.012
[a]
6
-O : 4-O regioisomeric ratio. Determined by tensiometry.
To probe the influence of regioisomers on amphiphilic properties, physicochemical analyses were
also performed on pure 4-O- and 6-O-C12EthMeGlu 11a and 11b regioisomers (Figure 3). Pure
regioisomers were separated from the mixture by column chromatography on silica gel. Similar
profiles were obtained for both regioisomers, however, 4-O-isomer 11a (brown triangles) showed a
short plateau with a solubilisation limit close to its CMC, while a better surfactant profile was
obtained for 6-O-isomer 11b (green squares). In addition, the CMC and γsat.values of the two pure 4-
-
1
O- and 6-O-isomers are relatively close, with 0.046 and 0.030 mM, and 31 and 29 mN.m respectively
(
Table 3, entries 1-2). Consequently, these results prove that the position of the alkyl chain on the

ACCEPTED MANUSCRIPT
sugar scaffold does not induce major changes on neither the surface tension nor the CMC values.
Interestingly, when using a 73 : 27 mixture of 6-O- and 4-O-isomers 11 (purple crosses), the CMC
decreases to 0.012 mM (Table 3, entry 3). This non-ideal behavior is very common when mixing two
surfactants. It is attributed to an attractive interaction between two surfactants located at the
surface monolayer and within mixed aggregates. This leads to a stabilization of the mixed superficial
[
26]
layer and the co-micelles. We applied the equations of the regular solution theory to this specific
isomeric ratio, which gave an interaction parameter β = - 4.7 and a mole fraction of isomer 6-O in the
mixed micelle X1 = 0.61 (see references 26a and 26b for calculation details). Even if CMC
measurements on other isomeric ratios are required to get a more accurate value of interaction
parameter, this strong negative β value clearly indicates preferential attractive interactions between
the two regioisomers.
8
7
6
5
4
3
2
0
0
0
0
0
0
0
-
8
-7
-6
-5
-4
-3
-2
Log C (mol/L)
Figure 3. Surface tension vs concentration of methyl dodecyl-α-
and ‹ a 73 : 27 mixture of 6-O- and 4-O-isomers 11.
D
-glucopyranoside: Å 4-O-isomer 11a, ° 6-O-isomer 11b,
Table 3. Critical Micelle Concentration (CMC) and surface tension (γ ) values of regioisomers 6-O and 4-O of C12EthMeGlu
sat
1
1.
[
b]
[b]
Methyl α-
D
-
CMC
γsat
[a]
Entry
Ratio
-
1
-1
glucopyranoside ethers
(mmol.L )
(mN.m )
4
6
-O-C12EthMeGlu
1a
1
2
3
0 : 100
100 : 0
73 : 27
0.046
31
1
-O-C12EthMeGlu
1b
0.030
0.012
29
30
1
(
6+4)-O-C12EthMeGlu
1
1
[a]
[b]
6
-O : 4-O regioisomeric ratio. Determined by tensiometry.
In order to evaluate the efficiency of these surfactants, a comparison of methyl 6-O-dodecyl-α-
D-
glucopyranoside 11b with known sugar-based non ionic surfactants was next carried out (Table 4).

ACCEPTED MANUSCRIPT
Only compounds bearing a C12 alkyl chain were selected in order to establish a structure / properties
relationship. First of all, methyl 6-O-dodecyl-α-D-glucopyranoside (CMC = 0.030 mM) was found by
far more efficient than its glucose analog (CMC = 0.5 mM), showing the importance of the
substitution at the anomeric position (Table 4, entries 1-2). Moreover, the replacement of the ester
function in methyl 6-O-dodecanoyl-α-D-glucopyranoside (CMC = 0.052 mM) by an ether link does not
have a significant impact on the CMC (Table 4, entry 3). However, the introduction of a second
carbohydrate unit in 6-O-dodecanoyl-sucrose and in 6’-O-dodecanoyl-maltose leads to an important
increase of the CMC to 0.25 and 0.24 mM, respectively (Table 4, entries 4-5). Higher CMC values
were also obtained with the corresponding commercially available dodecyl α-D-glucopyranoside and
dodecyl maltoside (Table 4, entries 6-7). This means that, in the case of methyl 6-O-dodecyl-α-
D-
glucopyranoside 11, about 12 to 17 times less product is required to obtain the same performances
than these commercial surfactants. Finally, this study highlights the potential of ether 11 as new bio-
sourced non-ionic surfactant with improved robustness in comparison to esters, broadening the
scope of applications of such amphiphilic compounds.
Table 4. Comparison of CMC values (mM) of known sugar-based non-ionic surfactants.
-
1
Entry
Carbohydrate surfactants
CMC (mmol.L )
Ref.
0
.012
(
mixture of
isomers)
1
This work
0
.03 (6-O-isomer)
2
3
0.5
[27]
[28]
0.052
0.25
0.24
4
5
[29]
[29]

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6
7
0.19
[30]
[31]
0
.15 (α-isomer)
.20 (β-isomer)
0
Antimicrobial activities of methyl α-D-glucopyranoside acetals and ethers
The antimicrobial activities of methyl α-D-glucopyranoside acetals and ethers were first evaluated
®
TM
®
against Gram-negative bacteria such as E. coli (ATCC 8739 ) and Pseudomonas aeruginosa (ATCC
TM
2
7853 ) and their efficacy was determined using the Minimum Inhibitory Concentration values
[
32]
(
MIC).
concentration up to 4.0 mM for neither acetal nor ether derivatives under the conditions used.
Similar results were obtained against this type of microorganism in Dunne’s studies on carbohydrate
Unfortunately, no antimicrobial activity was detected against these bacteria at a
[
33]
[
13a]
fatty esters, with MIC values reaching 10-20 mM.
The bacterial species tested represent two of
the most frequently Gram-negative pathogens isolated in humans, our results suggest that these
compounds cannot be used to target Gram-negative bacterial infections. In fact, one of the structural
feature of Gram-negative bacteria is the presence of a external membrane surrounding each
bacterial cell wall. Thus, one possible explanation would be we hypothesized that the derivatives are
enable to go through the outer lipopolysaccharide membrane, preventing them to penetrate into the
cells.
Consequently, the antimicrobial activities of methyl α-D-glucopyranoside acetals and ethers were
next evaluated against Gram-positive bacteria, namely, L. monocytogenes (CIP 103575), S. aureus
®
TM
®
TM
(
ATCC 29213 ) and E. faecalis (ATCC 29212 ). The results are gathered in Table 5.
Table 5. Antimicrobial activities of methyl α-D-glucopyranoside acetals 2-6 and ethers 7-11 against Gram positive bacteria.
-
1
-1
MICs (Minimum Inhibitory Concentration) in mmol.L . MBCs (Minimum Bactericidal Concentration) in mmol.L are given in
brackets.
Bacteria strains
L. mono-
cytogenes
CIP 103575)
S. aureus
Entry
Compounds
E. faecalis
®
(ATCC
®
TM
(
ATCC 29212
)
TM
29213
(
)
1
C5AcMeGlu 2
> 4
> 4
> 4

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2
3
4
5
6
7
8
9
C5EthMeGlu 7
C6AcMeGlu 3
> 4
> 4
> 4
2
> 4
> 4
> 4
> 4
2
> 4
> 4
> 4
> 4
> 4
C6EthMeGlu 8
C8AcMeGlu 4
C8EthMeGlu 9
C10AcMeGlu 5
C10EthMeGlu 10
C12AcMeGlu 6
C12EthMeGlu 11
C12EthMeGlu 11b
C12EstMeGlu
2
[a]
nd
[a]
nd
[a]
nd
0.5 (0.5)
2 (2)
0.5 (0.5)
[a]
nd
[a]
nd
[a]
nd
1
0
0.03 (0.03)
0.04
0.12 (0.12)
0.04
0.03 (0.03)
[
b]
b]
1
1
2
nd
nd
[
1
0.08
0.32
[a]
[b]
Nd: not determined due to the insolubility of the compounds when mixed with the inoculum.
MIC values from
reference 13.
No significant antimicrobial activity (MIC = 2 or > 4 mM) was detected for acetals and ethers bearing
a short C5, C6 or a medium C8 alkyl chain (Table 5, entries 1-6). Furthermore, the MICs of acetals 5
and 6 could not be determined due to their poor solubility in water (Table 5, entries 7 and 9).
However, increasing the alkyl chain length of methyl α-D-glucopyranoside ethers from C8 to C12 led
to an important decrease of the MIC values (Table 5, entries 6, 8 and 10). The best antimicrobial
activities were obtained for C12EthMeGlu 11 (73 : 27 mixture of regioisomers) with a MIC of 0.03
mM for both L. monocytogenes and E. faecalis and of 0.12 mM for the more virulent S. aureus strain
(
Table 5, entry 10). These results are comparable with those obtained with regioisomerically pure
methyl 6-O-dodecyl-α- -glucopyranoside 11b (Table 5, entry 11). This reveals that the alkyl chain
D
position has a minor impact on the biological activity. Thus, it highlights the interesting properties of
these mixtures obtained through step-economical acetalisation/hydrogenolysis sequence. Finally,
ether derivative C12EthMeGlu 11 was found more active than its corresponding ester C12EstMeGlu
[
13,18]
which is in accordance with the literature
(Table 5, entry 12).
The bactericidal activities of ethers 10 and 11 were then assessed for each bacteria strain by the
[
34]
measurement of the Minimum Bactericidal Concentration (MBC). Indeed, the MBC corresponds to
the lowest concentration of antimicrobial agent required to induce the death of a microorganism,

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corresponding to a decrease of the initial bacterial inoculum by at least 99.9%. The MBC values
obtained for C10EthMeGlu 10 and C12EthMeGlu 11 were identical to their corresponding MICs close
enough to MIC values in order to place them in the same serial dilution interval (Table 5, entries 8
and 10, results in brackets). This observation suggests clearly indicates that these compounds are not
only efficient to stop the bacterial growth but also induce the death of the microorganisms.
The antimicrobial activities of methyl α-D-glucopyranoside ethers were further compared to their
amphiphilic properties (Table 6).
Table 6. Comparison of the Critical Micelle Concentration (CMC) and Minimum Inhibitory Concentration (MIC) of methyl α-
-glucopyranoside ethers 7-11.
D
Methyl α-D-
CMC
MIC
Entry
glucopyranoside
ethers
-
1
-1
(
mmol.L )
(mmol.L )
1
2
3
4
5
C5EthMeGlu 7
C6EthMeGlu 8
C8EthMeGlu 9
C10EthMeGlu 10
C12EthMeGlu 11
35
> 4
> 4
13.4
0.434
0.277
0.012
2 – 4
0.5 – 2
0.03 – 0.12
An increase of the alkyl chain length (from C5 to C12) on methyl α-D-glucopyranoside ethers induce
both, a decrease of the CMC from 35 to 0.012 mM and of the MIC from >4 to about 0.03-0.12 mM.
Noticeably, the antimicrobial activities correlate quite well with the amphiphilic properties of
compounds 7-11 with MICs about 2 to 10 times the CMC values (Table 6, entries 1-5). These
amphiphilic compounds might interact with the cell bi-layer membrane, exerting a variety of
[
35]
effects, thus leading to the cell lysis. Having identified that 12 carbons was the optimal alkyl chain
length to reach efficient antimicrobial activity, the effects of the monosaccharide core and the
anomeric carbon configuration were next probed. For this purpose, methyl α-
-galactopyranoside, β- -rhamnopyranoside and β- -glucopyranoside were subjected to acetalisation
with dodecanal to give the corresponding acetals 12-15 with unoptimized 11-49% isolated yields.
The hydrogenolysis of these acetals gave three new alkylated sugars 16-18 with unoptimized 27-34%
D-mannopyranoside, α-
D
L
D
[
36]
[
36]
isolated yields (Figure 4). Unfortunately, all attempts to reduce acetal 15 to the desired ether
failed under our conditions. This could be explained by the difference of adsorption energies of α-

ACCEPTED MANUSCRIPT
[
37]
and β-isomers on palladium during the hydrogenolysis step. As a result, only β-C12AcMeGlu 15
was considered for further tests.
10
O
O
O
OMe
HO
OH
-
C12AcMeGlu 15
OH
OH
OH
O
10
O
O
10
O
HO
O
OMe
HO
HO
OH
Me
O 10
OMe
C12EthMeMan 16
OH
OMe
-
-C12EthMeGal 17
-C12EthMeRh 18
Figure 4. Range of acetal and ether derivatives obtained from acetalisation / hydrogenolysis sequence.
With these molecules in hands, antimicrobial activity studies were next carried out on a wide range
of Gram-positive bacteria. The efficacy of the derivatives was first studied against L. monocytogenes
strains such as the reference (CIP 103575) and three clinical isolated strains (LM1: 015189074801,
LM2: 015170199001, LM3: 015181840701) using amoxicillin as control (Table 7). The glucose
derivative 11 gave the best activities against all these strains with MICs of 0.02 mM while mannose-
based compound 16 was slightly less active (Table 7, entries 1-2). However, galactose derivative 17
was by far less efficient with a MIC of 0.18 mM on all strains (Table 7, entry 3). Interestingly,
rhamnose derivative 18 was also active but to a lesser extent (Table 7, entry 4). Finally, acetal 15 was
also quite active against these L. monocytogenes strains, proving that this class of compounds could
also been interesting as antimicrobials, provided their solubility (Table 7, entry 5).
Table 7. Antimicrobial activities of compounds 11, 15-18 against L. monocytogenes strains. MICs (Minimum Inhibitory
-1
Concentration) in mmol.L .
L. monocytogenes strains
Entry
Compounds
CIP 103575
LM1
LM2
LM3
1
2
3
4
C12EthMeGlu 11
C12EthMeMan 16
C12EthMeGal 17
C12EthMeRh 18
0.02
0.09
0.18
0.09
0.02
0.02
0.18
0.09
0.02
0.04
0.18
0.09
0.02
0.04
0.18
0.09

ACCEPTED MANUSCRIPT
5
6
β-C12AcMeGlu 15
0.04
0.04
0.04
0.18
amoxicillin
0.002
0.002
0.004
0.002
®
TM
The activity of compounds 11, 15-18 was next studied against E. faecalis (ATCC 29212 and
0
0
15206179901) and E. faecium strains (CIP 103510, Van A 0151850763, 015205731401 and
15205261801) using amoxicillin as control (Table 8). A good level of activity was observed for sugar
derivatives 11, 16 and 18 against the six Enterococcus strains studied (MIC ≤ 0.09 mM), particularly
for glucose derivative 11 with a MIC of 0.02 mM for most strains (Table 8, entries 1, 2 and 4). More
importantly, this compound was also active against a vancomycin-resistant strain at 0.02 mM (Van
A). Once again, galactose derivative 17 was the least active indicating that the nature of the
carbohydrate could affect the antimicrobial efficacy. However, great differences could be observed
between strains as the MIC varies from 0.02 to > 0.71 mM (Table 8, entry 3). Similar phenomenon is
observed with acetal 15 with MICs ranging from 0.02 to 0.18 mM (Table 8, entry 5)
Table 8. Antimicrobial activities of compounds 11, 15-18 against E. faecalis and E. faecium strains. MICs (Minimum
-1
Inhibitory Concentration) in mmol.L .
E. faecalis strains
E. faecium strains
Entry
Compounds
0
1520573140
ATCC 29212
015206179901
CIP
Van A
015205261801
1
1
2
3
4
5
6
C12EthMeGlu 11
C12EthMeMan 16
C12EthMeGal 17
C12EthMeRh 18
β-C12AcMeGlu 15
amoxicillin
0.02
0.04
0.18
0.05
0.18
0.002
0.04
0.09
0.18
0.05
0.09
0.002
0.02
0.09
0.02
0.04
0.35
0.05
0.09
0.5
0.02
0.04
0.09
0.02
0.04
0.25
0.02
0.04
0.02
0.05
0.02
0.25
> 0.71
0.05
0.09
0.06
The activity of compounds 11, 15-18 was next studied against a wide range of S. aureus strains using
oxacillin as control. Two reference strains were considered as well as six methicillin-resistant and
three daptomycin-resistant strains (Table 9). Indeed, the development of resistance during therapy is
[
38]
becoming a major health issue highlighted in numerous reports. Similar activities were observed
for glucose 11, mannose 16 and rhamnose 18 derivatives with MIC values from 0.04 to 0.09 mM for

ACCEPTED MANUSCRIPT
reference strains (Table 9, entries 1, 2 and 4). However, slighly lower activities (0.18-0.37 mM) were
observed with acetal 15 (Table 9, entry 5). In accordance with our previous results on Listeria and
Enterococcus strains, galactose derivative 17 gave no significant activity with MIC > 0.71 mM for all
strains studied (Table 9, entry 4). Gratifyingly, good antimicrobial activities (0.04-0.09 mM) were
obtained with glucose derivative 11 against methicillin- and daptomycin-resistant strains (Table 9,
entry 1). Interestingly, a similar level of activity was displayed against sensitive and resistant strains
for all compounds.
These results clearly indicate that carbohydrate fatty ethers are promising candidates for the
treatment of resistant bacteria. They could find various applications in the food industry, in the
medical field and could also be incorporated in various formulations.
Table 9. Antimicrobial activities of compounds 11, 15-18 against S. aureus strains (references), methicillin- and daptomycin-
-1
resistant S. aureus strains. MICs in mmol.L .
S. aureus references
Methicillin resistant S. aureus
Daptomycin resistant S. aureus
Entry
Compounds
ATCC
ATCC
LAC
HT 2004-
0012
LY 199-
0053
HT 2002-
0417
HT 2006-
1004
ST 2015-
0188
ST 2014-
1288
ST 2015-
0989
MU3
25923
29213
USA 300
1
2
3
4
5
6
C12EthMeGlu 11
C12EthMeMan 16
C12EthMeGal 17
C12EthMeRh 18
β-C12AcMeGlu 15
oxacillin
0.04
0.09
0.09
0.09
0.09
0.09
0.18
0.09
0.09
0.04
0.09
0.04
0.09
0.09
0.18
0.09
0.18
0.09
0.09
0.09
0.18
0.09
> 0.71
0.09
> 0.71
0.05
> 0.71
0.09
> 0.71
0.18
> 0.71
0.05
> 0.71
0.09
> 0.71
0.09
> 0.71
0.09
> 0.71
0.18
> 0.71
0.09
> 0.71
0.18
0.18
0.18
0.18
0.18
0.18
0.18
0.37
0.18
0.18
0.18
0.18
0.05
0.001
0.16
0.64
0.08
0.16
0.16
0.32
0.16
0.16
0.32
Mechanistic considerations
We hypothesize that the antimicrobial activity arises from the ability of carbohydrate alkyl ethers to
form micelles and thus to solubilize the lipids constituting the cell membrane. However, important
MIC differences were observed between C12EthMeGlu 11 and C12EthMeGal 17, indicating that the
carbohydrate moiety can also play a crucial role on antimicrobial activity. Consequently, a
recognition phenomenon might also be operating between the carbohydrate core and the cell walls
of bacteria. Alternatively, modification of the OH group configuration (from equatorial in glucose to

ACCEPTED MANUSCRIPT
axial in galactose) could induce a drastic change of the CMC also impacting the activity. In order to
verify this hypothesis, the CMC value of C12EthMeGal 17 was measured and compared to
C12EthMeGlu 11 (Figure 5).
8
7
6
5
4
3
2
0
0
0
0
0
0
0
-
8
-7
-6
-5
-4
-3
-2
Log C(mol/L)
Figure 5. Surface tension of methyl α-
Å C12EthMeGal 17.
D-glucopyranoside ether vs methyl α-D-galactopyranoside ether ‹ C12EthMeGlu 11,
Table 10. Antimicrobial activities vs CMC of compounds 11 and 17.
[a]
[b]
MIC
CMC
Entry
Compounds
-
1
-1
(
mmol.L )
(mmol.L )
1
2
C12EthMeGlu 11
C12EthMeGal 17
0.02-0.09
0.02-0.71
0.012
0.47
[a]
MIC (Minimum Inhibitory Concentration) range including results against against Staphylococcus, Enterrococcus and
[b]
Listeria strains. Determined by tensiometry.
The CMC of galactose derivative 17 is 40 times higher than glucose 11 (Table 10). Galactoside 17 is
therefore more hydrophilic than 11 which is in accordance with previous work on HPLC separation of
[
39]
methyl glycosides.
This phenomenon is directly linked to the inversion of the 4-OH group
[
40]
configuration (from equatorial for 11 to axial in 17). Based on Simon’s DFT calculations and
[
41]
Hüneberger’s conformational analyses, we postulate that the lower hydrophilicity of 11 is due to
the high occurrence of hydrogen-bond networks (Figure 6, left). In the contrary, these interactions
are not favored in the case of galactoside 17 due to the axial 4-OH group, therefore increasing the
sugar polarity (Figure 6, right).

ACCEPTED MANUSCRIPT
Figure 6. Model proposed to explain the polarity difference between C12EthMeGlu 11 and C12EthMeGal 17 (only one
conformer showed).
As shown above, minor structural modifications could have significant impacts on the antimicrobial
activity. Consequently, we also envisioned to investigate the role of the methyl group in compound
1
1 by preparing the corresponding demethylated compound. Unfortunately, all attempts to remove
[
42]
the methyl group from compound 11 following standard conditions (TMS-OTf / Ac O in CH Cl )
2
2
2
failed and gave a complex mixture of products. However, based on our previous analysis and the
CMC values (Table 4, entries 1-2), we can assume that the antimicrobial activity of 6-O-dodecyl-
D-
glucose should be considerably lower than the corresponding methyl dodecylglucopyranosides.
Further works will be necessary to confirm this hypothesis.
Finally, this study demonstrates that the CMC is the crucial parameter influencing the MIC, however
the sugar recognition can still not be ruled out. Further structural/activity studies of sugar-based
surfactants and computational analyses are currently on going.
Conclusions
In conclusion, a series of amphiphilic methyl glycopyranoside alkyl ethers were prepared from the
corresponding
unprotected
methyl
glycopyranosides
following
a
two-step
acetalization/hydrogenolysis sequence. This route provides a straightforward access to carbohydrate
alkyl ethers with high-atom economy and low waste production in comparison to traditional routes
involving protection/deprotection steps. The amphiphilic properties of these ethers (but also the
acetal intermediates) were measured and a C12 alkyl chain has the optimum length to decrease the
-
1
surface tension of water (CMC = 0.012 mM, γsat. = 30 mN.m ). The antimicrobial activities were also
evaluated on Gram-negative and Gram-positive bacteria. No activity was found against Gram-
negative bacteria (MIC > 4 mM). On the contrary, interesting antimicrobial activities (MIC ≥ 0.02 mM)
were obtained on Gram-positive bacteria such as L. monocytogenes, E. faecalis, E. faecium and S.
aureus for methyl glycopyranosides bearing a C12 alkyl chain. More importantly, some of these

ACCEPTED MANUSCRIPT
compounds are also active against antibiotic-resistant bacteria which represent a major public health
problem. These compounds have a great potential to be used as additives in pharmaceutical and/or
cosmetic formulations and can find applications in food industry, in hospital and in other non-
medical fields.
Experimental Section
General procedure A: acetalisation of methyl pyranosides
In a dry two-necked round bottom flask, the corresponding methyl glycopyranoside (2.0 equiv) and
sodium sulfate (1.5 equiv) were added in dry THF under argon. Dodecanal (1.0 equiv) was added
portionwise over a 1-min period, followed by Amberlyst 15 (20wt%/aldehyde). The mixture was
stirred at 66°C for the time stated. After cooling down to room temperature, the reaction mixture
was filtered, washed with CH Cl (2×25 mL) and the filtrate was concentrated under reduced
2
2
pressure. The residue was purified by flash chromatography (Cyclohexane : EtOAc) to give the
corresponding methyl pyranoside acetals 6, 12-15. See supporting information for characterization
details.
General procedure B: reductive cleavage of methyl pyranoside acetals
In a 100-mL stainless steel autoclave, the corresponding methyl dodecylidene pyranoside (1.0 equiv)
was dissolved in dry CPME (cyclopentyl methyl ether) and 5%-Pd/C (5 mol% in Pd) was added. The
reactor was tightly closed, purged three times with hydrogen and pressurized at 40 Bar. The solution
was then heated at 130°C for 16 hours. After cooling to room temperature, hydrogen pressure was
released and the reaction mixture was dissolved in absolute EtOH (100 mL) and filtered (Millipore
Durapore filter 0.01 µm). The filtrate was evaporated under reduced pressure and the residue was
purified by flash chromatography (Cyclohexane : EtOAc) to give the corresponding methyl pyranoside
ethers 11, 16-18. See supporting information for characterization details.
Physico-chemical studies
The surface tensions were measured at (25.0 ± 0.1)°C with a K100MK2 Krüss tensiometer using a
platinum rod as the probe, allowing to carry out experiments in small vessels. A total of 2.5 mL of
water was first introduced in the vat. The solution was gradually concentrated with a stock surfactant
solution (a manual dilution keeping volume constant was performed) and for each concentration the
surface tension was measured until a stable value was obtained (standard deviation of the 5 final
-
1
values of 0.2 mN.m ).

ACCEPTED MANUSCRIPT
Antimicrobial assays
The minimal inhibitory concentration experiments were realized on Gram-positive strain in
th
accordance with the Clinical-Laboratory-Standards-Institute (6 ed. Approved standard M100-S17.
CLSI, Wayne, PA, 2007) recommendations.
Acknowledgements
The authors would like to thank Jesús Fermin Ontiveros for his contribution to the physico-chemical
analyses. We also acknowledge the FINOVI foundation for financial support (AO9-07) to D.B. Finally
we thank the company TEREOS and the Association Nationale de la Recherche et de la Technologie
(
ANRT) for their financial support through a CIFRE grant (2011/1660) to C.G.
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Highlights
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Amphiphilic
acetalization/hydrogenolysis sequence
Best surfactant properties for sugar derivatives bearing a linear dodecyl chain (CMC = 0.012
carbohydrate
ethers
were
prepared
through
step-economical
-
1
mM, γsat. = 30 mN.m )
The compounds exhibit antimicrobial activity against Gram-positive bacteria (0.03 < MIC <
0
.12 mM)
Antimicrobial activities against multi-resistant strains such as vancomycin-, methicillin- and
daptomycin-resistant strains