Synthesis and properties of surfactants derived from N-acetyl-D-glucosamine

Article abstract of DOI:10.1080/07328300701634804

The synthesis of 2-acetamido-2-deoxy-6-O-octanoyl-D-glucono-1,5-lactone 9 and 2-acetamido-2-deoxy-6-O-octanoyl-α-D-glucopyranose 7 from 2-acetamido-2-deoxy-α-D-glucopyranose is reported. For both targets, the key intermediate was allyl 2-acetamido-3,4-di-

Full text of DOI:10.1080/07328300701634804

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Synthesis and Properties of Surfactants
derived from N‐Acetyl‐D‐Glucosamine
Chadi Khoury a , Michel Minier a , François Le Goffic a & Marie‐Noëlle Rager b
a ENSCP , Laboratoire de Chimie et Biochimie des Complexes Moléculaires,
UMR CNRS , Paris, 7576, France
b ENSCP, Service RMN, Paris, 7576, France
Published online: 07 Nov 2007.
To cite this article: Chadi Khoury , Michel Minier , François Le Goffic & Marie‐Noëlle Rager (2007) Synthesis
and Properties of Surfactants derived from N‐Acetyl‐D‐Glucosamine, Journal of Carbohydrate Chemistry, 26:7,
395-409, DOI: 10.1080/07328300701634804
To link to this article: http://dx.doi.org/10.1080/07328300701634804
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Journal of Carbohydrate Chemistry, 26:395–409, 2007
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300701634804
Synthesis and Properties of
Surfactants Derived from
N-Acetyl-D-Glucosamine
Chadi Khoury, Michel Minier, and Franc¸ ois Le Goffic
´
ENSCP, Laboratoire de Chimie et Biochimie des Complexes Moleculaires, UMR CNRS,
Paris 7576, France
Marie-Noe¨lle Rager
ENSCP, Service RMN, Paris 7576, France
The synthesis of 2-acetamido-2-deoxy-6-O-octanoyl-D-glucono-1,5-lactone 9 and 2-acet-
amido-2-deoxy-6-O-octanoyl-a-D-glucopyranose 7 from 2-acetamido-2-deoxy-a-D-gluco-
pyranose is reported. For both targets, the key intermediate was allyl 2-acetamido-
3,4-di-O-benzyl-2-deoxy-6-O-octanoyl-a-D-glucopyranoside 5. Surface tension measure-
ments (critical micellar concentration of 22.3 mM and 5 mM for 9 and 7, respectively)
showed up the surface activity of both compounds, while enzyme inhibition assays indi-
cated that 9 could inhibit bovine b-N-acetylglucosaminidase (Ki ¼ 6.5 mM) but not
Serratia marcescens chitobiase nor hen egg-white lysozyme. Moreover, 7 was shown
to induce chitinase production of S. marcescens and to be readily metabolized by
these bacteria.
Keywords 2-Acetamido-2-deoxy-6-O-octanoyl-D-glucono-1,5-lactone, Sugar mono-
esterN-Acetyl-D-glucosamine derivatives, Glycosidase inhibition, b-N-
Acetylglucosaminidase, Chitobiase, Surface activity, Affinity surfactant,
Chitinase induction, Serratia marcescens
Received February 20, 2007; accepted June 22, 2007.
´
Address correspondence to Michel Minier, Ecole Nationale Superieure de Chimie de
Paris, UMR CNRS 7576 11 rue Pierre et Marie Curie, F-75231, Paris cedex 05,
France. E-mail: michel-minier@enscp.fr
395

C. Khoury et al.
396
INTRODUCTION
Sugar-based surfactants are known for their use in food, cosmetics, drugs,
detergents, and in the biochemical field due to some advantages over other
amphiphiles, such as their dermic compatibility and biodegradability.[1 – 5]
Depending on the properties provided either by the hydrophobic tail or by
the sugar moiety, some are biologically active.[6,7] They may act as specific
inhibitors of the different glycosyl hydrolases belonging to families 18
(chitinases) or 20 (b-hexosaminidases, b-N-acetylglucosaminidases, and
chitobiases),[8] which do receive great attention.[9 – 15] Indeed, the inhibitory
activity of 2-acetamido-2-deoxy-D-gluconolactones and some of their deriva-
tives, on b-N-acetylglucosaminidases, has already been described.[16 – 18] To
a larger extent, lactones derived from chito-oligosaccharides are potential
inhibitors of family 18, 20, and 22 glycosyl hydrolases due to the sp2 hybrid-
ization of the anomeric center and the coplanarity of the C-1, C-2, O-5, and
C-5 atoms, thus mimicking the transition state.[15,18 – 24] Design of new
analogs combining surfactant properties with the biological activity of
the lactone may enlarge their field of applications, for example, by
changing their distribution and persistence features in cells or organisms.
Besides, the synthesis of amphiphilic ligands may allow the development
of affinity liquid-liquid separation processes of several glycosyl hydrolases,
as in the case of concanavalin-A, for which it has been shown that incorpor-
ation of an affinity cosurfactant (octyl b-D-glucopyranoside) in a reversed
micellar system led to a great increase in the yield and the specificity of
protein separation.[25] For these reasons, the synthesis of a new surfactant,
2-acetamido-2-deoxy-6-O-octanoyl-D-glucono-1,5-lactone (9), has been
carried out. Amphiphiles derived from gluconolactone,[18,26,27] glucosamine,
or N-acetyl-D-glucosamine (GlcNAc) units[3,4,7,28] are commonly available in
a few synthetic steps. However, the preparation of 9 from 2-acetamido-2-
deoxy-a-D-glucose was never described and is proposed here in nine steps.
Since the mechanism of b-N-acetylglucosaminidase inhibition by 2-aceta-
mido-2-deoxy-D-gluconolactones involves a flattened conformation at C-1,
2-acetamido-2-deoxy-6-O-octanoyl-a-D-glucopyranose (7) was synthesized
and assayed as a negative control. The surfactant properties of 7 and 9
have been characterized and preliminary inhibition tests have been per-
formed. Besides the interest for finding new glycosyl hydrolase inhibitors,
another field of application concerns the utilization of chitinases for plant
protection.[29] Microbial chitinases are known to be inducible enzymes,
secreted by some microorganism species when they are grown on chitin
(poly-(1 ! 4)-b-N-acetyl-D-glucosamine).[30,31] Since chitin is insoluble in
water, the actual inducers must be soluble oligomers derived after partial
degradation of chitin. However, few results have been published on the
mechanisms of induction and on the efficiency of other potential

Surfactants Derived from N-Acetyl-D-Glucosamine
397
inducers.[30,32] In this context, compounds 7 and 1 (allyl 2-acetamido-2-
deoxy-a-D-glucopyranoside) have been tested for chitinase induction in
Serratia marcescens cultures and compared with chitin and N-acetyl-D-
glucosamine.
RESULTS AND DISCUSSION
Synthesis
Starting from GlcNAc, the key intermediate 3 was synthesized according to
known procedures[33,34] (Sch. 1). Detritylation of 3 using p-toluene sulfonic acid
in dichloromethane/methanol afforded allyl 2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-D-glucopyranoside (4) in 62% yield. The 6-O acylation of 4 in hot
toluene was easily achieved to give 5 in 70% yield. The anomeric allyl group of
5 was isomerized in mild conditions with tris(triphenylphosphine)rhodium
chloride in the presence of diazabicyclo[2.2.2]octane[35] and then hydrolyzed
with mercuric chloride to give hemiacetal 6. Purification by chromatography
of the isomerized (1-propenyl) compound and subsequent hydrolysis gave the
best yield of 6 (67%) containing a minimum of impurities. Oxidation of 6 by
dimethyl sulfoxide-oxalyl chloride was performed at 2708C in dichloro-
methane[36] to give the glucono-1,5-lactone 8 in 94% yield. Classic hydro-
genolysis of the benzyl groups yielded the lactone 9 in 87% from 8, and the
hemiacetal 7 in 93% from 6. The overall yield of 9, resp. 7 from GlcNAc was
about 13%, resp. 15%. The conservation of the a-configuration from 2-aceta-
mido-2-deoxy-a-D-glucose to 7 was proved by the NMR characteristics of the
successive intermediates, for instance, the value of the 1H NMR coupling
constant J1,2 ꢀ 4.0 Hz.
Surface Tension Properties
As shown in Fig. 1, both compounds 7 and 9 show surfactant properties
since the surface tension of water is lowered from 71 mN/m to 39 + 0.2 mN/
m for 7, and to 29.7 + 0.2 mN/m for 9. Their critical micellar concentrations
(CMC) can be estimated to 5 + 0.2 mM for 7 and 22.3 + 0.3 mM for 9.
These CMC values are in the same order of magnitude as those of 3-O-
octanoyl-D-glucopyranose
(1.6 mM)[37]
and
octyl-b-D-glucopyranoside
(25 mM).[38] The higher CMC value of the lactone 9 as compared with 7
seems to indicate that 9 is more hydrophilic than 7. It is generally assumed
that the dissolution in water of gluconolactone derivatives results in a
mixture of 1,5-lactone, 1,4-lactone, and open-chain forms.[16,26] After
dissolution of 9 in D2O and long-time equilibration (pH of the solution
between 4 and 5), NMR spectra show one major component corresponding

C. Khoury et al.
398
Scheme 1: Synthesis of the target compounds 9 and 7.
to the 1,5-lactone form, confirmed by HMBC and NOESY experiments.
However, the possible presence of little amounts of other forms not identified
by NMR, in particular the more hydrophilic 2-acetamido-2-deoxy-6-O-
octanoyl gluconic acid, could explain the higher CMC value observed for the
solution of 9.

Surfactants Derived from N-Acetyl-D-Glucosamine
399
Figure 1: Surface tension of aqueous solutions of compounds 9 and 7 versus log C (mol/L)
at 258C.
Enzyme Inhibition Assays
Initial kinetic analysis of b-N-acetylglucosaminidase (NAGase) from
bovine kidney was performed using the synthetic substrate 4-nitrophenyl
2-acetamido-2-deoxy-b-D-glucopyranoside (pNPGlcNAc), an analog of chito-
biose (GlcNAc)2. Lineweaver-Burk plots (Fig. 2) yield Km value
(2.05 + 0.1 mM) and show that 9 behaves as a competitive inhibitor of
NAGase. Its IC50 was 16 + 3 mM for a 2.5 mM pNPGlcNAc concentration.
The inhibition constant Ki was found to be about 6.5 + 1 mM. By comparison,
7, which does not have the flattened conformation of C-1, is a very moderate
inhibitor with an IC50 ¼ 1 + 0.1 mM.
Figure 2: Lineweaver-Burk plots of b-N-acetylglucosaminidase activity in the presence (7 mM)
or the absence of compound 9.

C. Khoury et al.
The lactone 9 has also been assayed against a batch of partially purified chit-
400
inolytic enzymes from a Serratia marcescens culture.[39] Since the substrate used
was again pNPGlcNAc, the assay concerned the chitobiase activity of the prep-
aration.[40] The IC50 value (1.2 mM) indicates weak inhibition. This preliminary
result has to be verified by inhibition assays on each purified enzyme separetely.
However, it is in agreement with the exo-chito-oligosaccharide b-N-acetylgluco-
saminidase character of the chitobiase,[41] which presents a great decrease of
affinity when decreasing chito-oligosaccharide chain length. Tests of 9 against
hen egg-white lysozyme showed no inhibition up to 1.8 mM, which is in agree-
ment with the endoglycosidase activity of this enzyme.[22]
Due to the possible interconversion of the gluconolactone into the corre-
sponding free acid and into its other ring form, it is virtually impossible to
obtain the inhibition constants of each form separately. However, as mentioned
above, at the pH of our assays (pH 4.2) one major form exists (1,5-lactone),
which appears to be mostly responsible of inhibition, in agreement with
previous results.[16]
In conclusion, though exhibiting inhibition features lower than the nonsur-
factant parent 2-acetamido-2-deoxy-D-glucono-1,5-lactone (Ki ¼ 0.55 + 0.1 mM
for bull epididymis NAGase[16]), 9 keeps a strong and selective affinity for
NAGase, among retaining b-glycosidases likely sharing a common catalysis
mechanism.[24] Its surfactant properties make it a potential affinity ligand
for separation processes and could help its vectorization.
Induction of Chitinase Production in Bacterial Culture
Several Serratia marcescens cultures were grown in baffled flasks
using different media comprising a basal medium, in which the only
carbon source is yeast extract 0.5 g/L, and different additives as chitin,
GlcNAc, and compounds 7 and 1 (allyl 2-acetamido-2-deoxy-a-D-glucopyra-
noside). Maximum cell growth and chitinase production usually occur after
4 to 6 days. After sampling, bacterial cell concentration expressed as
colony forming unit (CFU) per mL and chitinase activity were assayed
(Table 1). The results show that 7 is a substrate for the bacteria and
that 7 induces their chitinase production. The consumption of 7 by S. mar-
cescens is evidenced by the increase of cell concentration when it is added
to the basal medium and also by the visual observation of its disappear-
ance as growth is going on, since 7 formed insoluble mixtures at initial
concentrations. This also proves that 7 would be a readily biodegradable
surfactant. Concerning chitinase induction, 7 appears to be less efficient
than chitin but more than GlcNAc. Whatever the concentration tested,
between 1 and 16 g/L, 7 leads to approximately the same chitinolytic
activity (8 + 2 U/mL), which corresponds to about 18% + 3% of chitin
reference assay activity. GlcNAc is known to cause catabolite repression

Surfactants Derived from N-Acetyl-D-Glucosamine
401
Table 1: Comparison of cell growth and chitinase production with and without
additives.
Cell
CNase
activity
(U/mL)
CNase activity
ratio act./
act.Ref
Additive conc. concentration
(g/L); (mM)
ratio C/CaRef
Basal medium
Chitin
0
,0.1
1
0
0
1
10; 45 mM)b
40–70
(Reference)
GlcNAc
GlcNAc
7
7
7
1
2; [9 mM]
10; [45 mM]
1; [3 mM]
4; [11 mM]
16; [46 mM]
4; [15 mM]
0.4
—
0.2
0.7–1.5
2
,0.1
0
,2
6
6–10
11
0
,0.03
0.15
0.15–0.22
0.15
0
0
aCRef ¼ Cell concentration in Reference (chitin 10 g/L) reachs a maximum between 1010 and 2
1010 CFU/mL, after 4–5 days of growth.
bChitin molarity expressed as GlcNaC equivalent.
At initial time (inoculation) cell concentration ranges around 2 108 CFU/mL.
of chitinase synthesis at high concentration (10 g/L) but induction at low
concentration.[30,32] In this way, chitinase production in batch culture would
be maximum when a maximal concentration of microbial cells is maintained
as long as possible at an inducing level of GlcNAc, estimated to 6 mg/L.[32]
The results presented in Table 1 suggest that transmembrane transport
mechanisms allow 7 to enter bacterial cell, where it induces chitinase
synthesis before—or after—being metabolized, while its low aqueous solubi-
lity prevents it from exerting catabolite repression. Thus, the solid reserve
formed by the initial amounts of 7 added to the culture seems to play the
same role as chitin, although it results finally in lower chitinase production.
This difference could come from the presence of more effective inducing
oligomers when chitin is used. On the other hand, it can be noted that 1
behaves neither as a substrate nor as an inducer for Serratia.
EXPERIMENTAL
General Methods
Physicochemical and biological characteristics were measured on crude
1
materials without recrystallisation. H and 13C NMR spectra were recorded
either with a Bruker AC 200 or a Bruker Avance 400 spectrometers. Chemical
shifts are given in parts per million (ppm) and referenced to the residual
solvent signals. Assignments were confirmed by COSY, HMQC, HMBC, or
NOESY experiments. Mass spectra were recorded on a quadrupole Ribermag
R 1010 spectrometer by DCI/NH3. High-resolution mass spectra were conducted

C. Khoury et al.
402
´
at Ecole Normale Superieure (Paris, France) on a JEOL MS 700 spectrometer.
Thin layer chromatography was carried out using aluminium precoated plates
of silica gel 60 F254 (Merck) with the solvent system chloroform-ethanol (19:1).
The spots were detected by UV (254 nm) or by iodine. Column chromatography
was conducted on silica gel (Merck) of 0.040–0.063 mm particle size. Concen-
trations were conducted in vacuo. Microanalyses were performed by the labora-
tory of Microanalysis CNRS (Gif-sur-Yvette, France).
Allyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-glucopyranoside (4). Com-
pound 3 (5 g, 7.3 mmol) was dissolved in a methanol-dichloromethane (30:70)
mixture (125 mL) containing p-toluenesulfonic acid (5 g, 26.3 mmol). The
mixture was stirred at rt for 3 h or until TLC showed complete conversion
of 3 into 4 (RF 0.4). The reaction mixture was neutralized with Na2CO3,
washed with water, and dried (Na2SO4). Solvents were evaporated and the
crude product was chromatographed (19:1 CHCl3-EtOH) to give 4 (2 g, 62%)
as a white amorphous mass. 1H NMR (400 MHz) in CDCl3: d 1.84 (s, 3H,
2
COCH3), 3.65–3.83 (m, 5H, H-3, H-4, H-5, H-6), 3.93 (ddt, 1H, J ¼ 13.0 Hz,
3J ¼ 6.2 Hz, 4J ¼ 1.3 Hz, OCH2 (Allyl)), 4.12 (ddt, 1H, 2J ¼ 13.0 Hz,
3J ¼ 5.3 Hz, 4J ¼ 1.4 Hz, OCH2 (Allyl)), 4.21 (td, 1H, JNH,2 ¼ J2,3 ¼ 9.6 Hz,
J1,2 ¼ 3.6 Hz, H-2), 4.66 (d, 1H, 2J ¼ 11.7 Hz, CH2Ph), 4.68 (d,
1H, 2J ¼ 10.9 Hz, CH2Ph), 4.81 (d, 1H, J1,2 ¼ 3.6 Hz, H-1), 4.87 (d, 1H,
2
2J ¼ 11.7 Hz, CH2Ph), 4.88 (d, 1H, J ¼ 10.9 Hz, CH2Ph), 5.18–5.27 (m, 2H,
CH255CH), 5.34 (d, 1H, JNH,2 ¼ 9.6 Hz, NH), 5.80–5.90 (m, 1H, CH255CH),
7.30–7.36 (m, 10H, Ar). 13C NMR (100 MHz) in CDCl3: d 23.5 (COCH3), 52.6
(C-2), 61.7 (C-6), 68.2 (OCH2 (Allyl)), 71.6 (C-5), 74.9, 75.2 (CH2Ph), 78.2 (C-4),
80.0 (C-3), 96.9 (C-1), 117.7 (CH255CH), 127.9, 128.0, 128.2, 128.2, 128.6,
128.6, (CH Ar), 133.6 (CH255CH), 138.0, 138.4 (Cq Ar), 169.9 (C55O). MS for
C25H31NO6 (M, 441): m/z 442 (M þ H)þ, 459 (M þ NH4)þ. Anal. Calcd for
C25H31NO6: C, 68.01; H, 7.07; N, 3.17. Found: C, 67.94; H, 7.07; N, 3.07.
Allyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-octanoyl-a-D-glucopyrano-
side (5). A mixture of 4 (9.5 g, 21.5 mmol) and toluene (150 mL) was heated
at 90 to 958C and capryloyl chloride (5 g, 30.7 mmol) was slowly added.
Boiling under reflux was then continued for 4 h. The cooled reaction was
washed with water and the product was extracted with more toluene
(3 ꢁ 100 mL). The combined extracts were washed with an aq solution of
NaHCO3 (5% w/v) then with water, dried (Na2SO4), and concentrated.
Column chromatography of the residue (19:1 CHCl3-EtOH) afforded 5 (8.6 g,
70%) as a white solid. 1H NMR (400 MHz) in (CD3)2SO: d 0.82 (t, 3H,
3J ¼ 6.8 Hz, CH2CH3), 1.21–1.25 (m, 8H, (CH2)4CH3), 1.51 (quint., 2H,
3J ¼ 7.1 Hz, COCH2CH2), 1.86 (s, 3H, COCH3), 2.31 (t, 2H, 3J ¼ 7.2 Hz,
COCH2CH2), 3.48 (t, 1H, J3,4 ¼ J4,5 ¼ 9.4 Hz, H-4), 3.72–3.78 (m, 2H, H-3,
H-5), 3.95–4.02 (m, 2H, H-2, OCH2 (Allyl)), 4.12 (dd, 1H, 2J ¼ 13.3 Hz,
3J ¼ 5.1 Hz, OCH2 (Allyl)), 4.18 (dd, 1H, J6,6 ¼ 11.7 Hz, J5,6 ¼ 5.1 Hz, H-6),
0
2
4.26 (dd, 1H, J6,6 ¼ 11.7 Hz, J5,6 ¼ 2.0 Hz, H-60), 4.54 (d, 1H, J ¼11.0 Hz,
0
0

Surfactants Derived from N-Acetyl-D-Glucosamine
403
CH2Ph), 4.67 (d, 1H, J1,2 ¼ 3.5 Hz, H-1), 4.68 (d, 1H, 2J ¼ 11.0 Hz, CH2Ph), 4.75
(d, 1H, 2J ¼ 11.0 Hz, CH2Ph), 4.76 (d, 1H, 2J ¼ 11.0 Hz, CH2Ph), 5.19 (dd, 1H,
3
3Jcis ¼ 10.5 Hz, 2J ¼ 1.5 Hz, CH255CH cis), 5.35 (dd, 1H, Jtrans ¼ 17.2 Hz,
2J ¼ 1.5 Hz, CH255CH trans), 5.87–5.97 (m, 1H, CH255CH), 7.24–7.36
(m, 10H, Ar), 8.12 (d, 1H, JNH,2 ¼ 9.2 Hz, NH). 13C NMR (100 MHz) in
(CD3)2SO: d 14.1 (CH2CH3), 22.2, 28.5, 28.6, 31.3 ((CH2)4CH3), 22.7 (COCH3),
24.6 (COCH2CH2), 33.6 (COCH2), 52.8 (C-2), 62.7 (C-6), 67.6 (OCH2 (Allyl)),
69.0 (C-5), 74.2 (CH2Ph), 78.2 (C-4), 80.0 (C-3), 96.6 (C-1), 117.3 (CH255CH),
127.6, 127.7, 127.8, 127.9, 128.4, 128.5 (CH Ar), 134.5 (CH255CH), 138.2,
138.8 (Cq Ar), 169.6 (COCH3), 172.9 (COO). Anal. Calcd for C33H45NO7: C,
69.81; H, 7.98; N, 2.46. Found: C, 69.43; H, 7.89; N, 2.34.
2-Acetamido-3,4-di-O-benzyl-2-deoxy-6-O-octanoyl-a-D-glucopyranose (6).
A solution of 5 (8 g, 14.1 mmol) and diazabicyclo [2.2.2] octane (1.2 g,
10.7 mmol) in 9:1 methanol-water (400 mL) was stirred at 808C and treated
with (PPh3)3RhCl (3 g, 3.2 mmol). After 4 h of reaction and cooling, the
catalyst was filtered off and the filtrate was concentrated to give up a residue
that was taken up in chloroform (250 mL). The resulting solution was succes-
sively washed with aqueous citric acid (5% w/v) and water, and dried
(Na2SO4). Evaporation gave a syrup, which was chromatographed (19:1
CHCl3-EtOH) to give a solid (6 g, 75%), identified as 1-propenyl 2-acetamido-
3,4-di-O-benzyl-2-deoxy-6-O-octanoyl-a-D-glucopyranoside. The latter product
(2 g) was dissolved in 9:1 acetone-water (50 mL) and the solution was treated
with HgCl2 (1 g, 3.7 mmol) and stirred at rt under Ar for 1 h or until TLC
showed complete hydrolysis of the starting material to give 6 (RF 0.3). After
evaporation of most of the acetone, the residue was extracted with 50 mL of
chloroform. The organic layer was washed with a saturated solution of KI
then twice with water (2 ꢁ 100 mL), dried, and concentrated. The crude
product was finally purified by column chromatography (19:1 CHCl3-EtOH)
to give 6 (1.7 g, yield from 5: 67%). 1H NMR (400 MHz) in CDCl3: d 0.87
3
(t, 3H, J ¼ 6.8 Hz, CH2CH3), 1.25–1.30 (m, 8H, (CH2)4CH3), 1.62 (quint, 2H,
3J ¼ 7.4 Hz, COCH2CH2), 1.84 (s, 3H, COCH3), 2.32 (t, 2H, 3J ¼ 7.4 Hz,
COCH2CH2), 3.64 (t, 1H, J3,4 ¼ J4,5 ¼ 9.4 Hz, H-4), 3.80 (dd, 1H,
J2,3 ¼ 10.5 Hz, J3,4 ¼ 9.4 Hz, H-3), 4.04–4.09 (m, 1H, H-5), 4.12–4.18 (m, 1H,
0
H-2), 4.24 (dd, 1H, J6,6 ¼ 12.1 Hz, J5,6 ¼ 3.9 Hz, H-6), 4.38 (dd, 1H,
2
J6,6 ¼ 12.1 Hz, J5,6 ¼ 2.3 Hz, H-60), 4.61 (dd, 1H, J ¼ 11.0 Hz, CH2Ph), 4.67
(dd, 1H, 2J ¼ 11.0 Hz, CH2Ph), 4.87 (dd, 2H, 2J ¼ 11.0 Hz, CH2Ph), 5.18
(t, 1H, J1,2 ¼ J1,OH ¼ 3.5 Hz, H-1), 5.44 (d, 1H, JNH,2 ¼ 9.1 Hz, NH), 7.26–
7.38 (m, 10H, Ar). 13C NMR (50 MHz) in CDCl3: d 14.0 (CH2CH3), 22.5, 28.8,
29.0, 31.6, (CH2)4CH3), 23.2 (COCH3), 24.8 (COCH2CH2), 34.1 (COCH2), 53.0
(C-2), 62.5 (C-6), 69.0 (C-5), 74.9 (CH2Ph), 78.2 (C-4), 79.5 (C-3), 91.7 (C-1),
127.9, 128.0, 128.2, 128.4 (CH Ar), 137.6, 138.1 (Cq Ar), 170.5 (COCH3),
173.7 (COO). Anal. Calcd for C30H41NO7: C, 68.30; H, 7.78; N, 2.65. Found:
C, 68.20; H, 7.51; N, 2.80.
0
0

C. Khoury et al.
2-Acetamido-3,4-di-O-benzyl-2-deoxy-6-O-octanoyl-D-glucono-1,5-lactone
404
(8). A mixture of dichloromethane (5 mL) and oxalyl chloride (0.16 mL,
1.9 mmol) was stirred at 2708C under argon. Dimethyl sulfoxide (0.26 mL,
3.7 mmol) in dichloromethane (4 mL) was added dropwise to the solution and
stirring was continued for 10 min. A solution of 6 (200 mg, 0.38 mmol) in
dichloromethane (5 mL) was then added and the reaction mixture was
stirred for 45 min. Triethylamine (1 mL, 7.2 mmol) was added and stirring
was continued at 2708C for an additional 30 min. The mixture was allowed
to warm to rt and water (25 mL) was added. The aqueous layer was extracted
twice with dichloromethane and the combined organic layers were washed suc-
cessively with HCl (1M), NaHCO3 (5%, w/v), and water. Drying on Na2SO4 and
1
concentration gave 8 as a clear syrup (185 mg, 94%). H NMR (200 MHz) in
3
CDCl3: d 0.88 (t, 3H, J ¼ 6.8 Hz, CH2CH3), 1.29 (bs, 8H, (CH2)4CH3), 1.61
(quint, 2H, COCH2CH2), 1.87 (s, 3H, COCH3), 2.30 (t, 2H, 3J ¼ 7.6 Hz,
COCH2CH2), 3.79–4.92 (m, 10H, H-2, H-3, H-4, H-5, H-6, CH2Ph), 7.15
(d, 1H, JNH,2 ¼ 6.6 Hz, NH), 7.27–7.40 (m, 10H, Ar).13C NMR (50 MHz) in
CDCl3: d 13.9 (CH2CH3), 22.3, 22.5, 24.7, 28.8, 29.0, 31.5, 33.9 (CH2)6CH3,
COCH3), 55.5 (C-2), 61.7 (C-6), 74.5, 74.7 (CH2Ph), 75.4 (C-5), 76.4 (C-4), 79.9
(C-3), 128.0–128.5 (CH Ar), 137.0, 137.6 (Cq Ar), 168.5 (C-1), 170.8 (COCH3),
173.1 (COO). MS for C30H39NO7 (M, 525): m/z 526 (M þ H)þ and 543
(M þ NH4)þ. Anal. Calcd for (C30H39NO7, 0.5 H2O): C, 67.40; H, 7.54; N,
2.62. Found: C, 67.43; H, 7.43; N, 2.53.
2-Acetamido-2-deoxy-6-O-octanoyl-D-glucono-1,5-lactone (9). To
a
solution of 8 (160 mg, 0.30 mmol) in methanol (10 mL) was added 10% palla-
dium-on-carbon catalyst (180 mg) and the suspension was shaken with
hydrogen overnight. The catalyst was removed by filtration and the filtrate
was passed through a cotton plug. Evaporation of the solvent afforded 9 as a
thick gel (92 mg, 87%). 1H NMR (400 MHz) in D2O: d 0.74 (t, 3H,
3J ¼ 7.0 Hz, CH2CH3), 1.18 (bs, 8H, (CH2)4CH3), 1.47–1.54 (m, 2H,
COCH2CH2), 1.96 (s, 3H, COCH3), 2.32 (t, 2H, 3J ¼ 7.4 Hz, COCH2CH2),
3.56 (dd, 1H, J4,5 ¼ 8.2 Hz, J3,4 ¼ 3.1 Hz, H-4), 3.77–3.82 (m, 1H, H-5), 4.09
0
(dd, 1H, J6,6 ¼ 11.7 Hz, J5,6 ¼ 5.8 Hz, H-6), 4.13 (dd, 1H, J2,3 ¼ 5.0 Hz,
J3,4 ¼ 3.1 Hz, H-3), 4.24 (dd, 1H, J6,6 ¼ 11.7 Hz, J5,6 ¼ 2.7 Hz, H-60), 4.36
(d, 1H, J2,3 ¼ 4.7 Hz, H-2). 13C NMR (100 MHz) in D2O: d 13.3 (CH2CH3),
21.8 (COCH3), 21.9, 28.0, 28.2, 30.9 ((CH2)4CH3), 24.3 (COCH2CH2), 33.8
(COCH2), 56.6 (C-2), 65.5 (C-6), 68.6 (C-5), 69.3 (C-3), 71.4 (C-4), 174.1
(COCH3), 175.1 (C-1), 177.4 (COO). Anal. Calcd for (C16H27NO7, 1.5 H2O):
C, 51.60; H, 8.12; N, 3.76. Found: C, 51.77; H, 7.72; N, 4.08. HRMS (FAB, m/
z) calcd for (C16H27O7 N þ Hþ): 346.1866, found: 346.1872.
0
0
2-Acetamido-2-deoxy-6-O-octanoyl-a-D-glucopyranose (7). To a solution of
6 (230 mg, 0.44 mmol) in absolute ethanol (7 mL) was added 10% palladium-
on-carbon catalyst (90 mg) and the suspension was shaken with hydrogen
overnight. The catalyst was removed by filtration and the filtrate was

Surfactants Derived from N-Acetyl-D-Glucosamine
405
passed through a cotton plug. Evaporation of the solvent afforded 7 as a white
solid (141 mg, 93%). 1H NMR (400 MHz) in (CD3)2SO: d 0.85 (t, 3H,
3J ¼ 6.8 Hz, CH2CH3), 1.25 (bs, 8H, (CH2)4CH3), 1.50–1.53 (m, 2H,
COCH2CH2), 1.82 (s, 3H, COCH3), 2.29 (t, 2H, 3J ¼ 7.2 Hz, COCH2CH2),
3.07–3.13 (m, 1H, H-4), 3.47–3.52 (m, 1H, H-3), 3.56–3.62 (m, 1H, H-2),
0
3.76–3.81 (m, 1H, H-5), 4.01 (dd, 1H, J6,6 ¼ 11.7 Hz, J5,6 ¼ 6.2 Hz, H-6),
4.28 (dd, 1H, J6,6 ¼ 11.7 Hz, J5,6 ¼ 1.6 Hz, H-60), 4.89 (t, 1H,
J1,2 ¼ J1,OH ¼ 4.0 Hz, H-1), 7.66 (d, 1H, JNH,2 ¼ 8.2 Hz, NH). 13C NMR
(50 MHz) in (CD3)2SO: d 14.2 (CH2CH3), 22.3, 28.6, 28.6, 31.4, ((CH2)4CH3),
22.9 (COCH3), 24.7 (COCH2CH2), 33.7 (COCH2), 54.4 (C-2), 64.0 (C-6), 69.5
(C-5), 70.5 (C-3), 71.4 (C-4), 90.9 (C-1), 169.7 (COCH3), 173.0 (COO). Anal.
Calcd for (C16H29NO7, 0.2 H2O) C, 54.75; H, 8.44; N, 3.99. Found: C, 54.73;
H, 8.05; N, 3.96. HRMS (FAB, m/z) calcd for (C16H29O7 N þ Hþ): 348.2022,
found: 348.2033.
0
0
Determination of Critical Micellar Concentration
The surface tension (g) of 9 and 7 solutions were measured at 258C with a
Wilhelmy tensiometer (Tensimat n83 - Prolabo). The measurements were
repeated three times and the respective mean value was considered. The
critical micellar concentration was determined from the break point of each
surface tension vs. concentration (on log-scale) curve.
Bacterial Culture and Chitinase Induction Experiments
The bacterial strain Serratia marcescens BCCM 18541 was grown at 328C,
in baffled Erlenmeyer flasks containing basal medium (g/L): yeast extract 0.5,
.
(NH4)2SO4 1, MgSO4 7H2O 0.3, KH2PO4 1.36. The pH was adjusted to 8.0 with
2 M NaOH before sterilization (1218C—30 min). Each compound to be tested
was added to this medium before inoculation. Purified chitin[39] was used as
reference. Cell concentration (CFU) was determined by spread plate technique
on LB/agar.[39]
Chitinase Activity Assay
The activity of S. marcescens chitinases was determined by following the
amount of reducing ends released by the cleavage of colloidal chitin (100–
170 mesh).[39] The reaction mixture, consisting of trial solution (0.5 mL) and
colloidal chitin (20 mg) in 1.5 mL of 100 mM phosphate buffer (pH 6.6), was
shaken for 1 h at 508C. The reaction was stopped by adding 0.1 mL 10% (m/
v) trichloroacetic acid to 0.6 mL of the mixture. After centrifugation, 0.5 mL
supernatant was heated for 3 min at 1008C with 1.5 mL dinitrosalicylic acid
(DNS) reagent (0.69 g DNS, 1.18 g NaOH, 20 g sodium potassium tartrate

C. Khoury et al.
406
per 100 mL water). After dilution, the absorbance was measured at 530 nm.
The amount of reducing moieties released, expressed as GlcNAc equivalent,
was determined from a calibration curve. One unit (U) of chitinase activity
was defined as the amount of enzyme required to produce 1 mmol GlcNAc
equivalent in 1 h at 508C and pH 6.6.
Enzyme Inhibition Experiments
4-Nitrophenyl 2-acetamido-2-deoxy-b-D-glucopyranoside (pNPGlcNAc)
from Acros (22941-1000) and b-N-acetyl-b-glucosaminidase (EC 3.2.1.52)
from bovine kidney from Sigma (A-2415) were used. The commercial prep-
aration of the enzyme, a suspension in 3.2 M (NH4)2SO4, was centrifuged
(13,000 rpm, 5 min) and the pellet was dissolved in water (1 mL). b-N-acetyl
glucosaminidase solution diluted 10 times (100 mL), citrate buffer 0.5 M, pH
4.2 (100 mL), and inhibitor solution (or water for controls) (300 mL) were incu-
bated at 378C for 5 min in a 1 mL spectrophotometer cuvette.[42] After addition
of pNPGlcNAc (5 mM or 2.5 mM in water, 500 mL), incubation was continued
for 8 min at 378C and the amount of 4-nitrophenol released was followed vs.
time by measurement of UV/vis absorption at 400 nm. Absorbance calibration
curve (linear) was set from 4-nitrophenol standard solutions maintained in the
same buffer and temperature conditions as the enzyme reaction pool. IC50 was
defined as the concentration of inhibitor needed to cause 50% decrease of
activity. The Ki value was obtained from comparison of KM0 and KM affinity con-
stants calculated in the presence or absence of a known concentration of inhibi-
tor and by plotting 1/v vs. I for two concentrations of substrate (2.5 and
1.25 mM), where v is the rate of 4-nitrophenol release and I the concentration
of inhibitor (Dixon graphical method). One unit of enzyme activity will hydro-
lyze 1 mmol of pNPGlcNAc per minute.
Chitinolytic enzymes were obtained from S. marcescens cultures and were
partially purified on a FPLC anion-exchange column Mono P 5/20 (Pharma-
cia). Stock enzyme preparation contained 35 U/mL or 50 U/mg of albumine
equivalent. The substrate used was pNPGlcNAc at 2.5 mM in order to point
up chitobiase activity. Enzyme solution was diluted 4 times and the inhibition
study was performed as for b-N-acetylglucosaminidase. A solution of hen egg-
white lysozyme from Sigma (L-6876) at 0.5 g/L and a suspension (170 g/L) of
lyophilized cells of Micrococcus lysodeikticus (Sigma, M-3770) were prepared
in a phosphate buffer (100 mM, pH 6.5). Lysozyme solution (100 mL) and
inhibitor solution (400 mL) were incubated at rt for 5 and 10 min. Forty
microliters of the mixture were added to 3 mL of the substrate supension in
a spectrophotometer cuvette. Lytic activity was determined by monitoring at
450 nm the decrease in absorbance, which occured during the lysis of the
suspension.

Surfactants Derived from N-Acetyl-D-Glucosamine
407
ACKNOWLEDGEMENTS
The authors thank Dr. Bruno Kokel for helpful discussions. This work was sup-
ported by a grant awarded to C. Khoury by the Agence De l’Environnement et
ˆ
de la Maıtrise de l’Energie (ADEME).
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