Synthesis, thermal properties, and cytotoxicity evaluation of hydrocarbon and fluorocarbon alkyl β-d-xylopyranoside surfactants

Article abstract of DOI:10.1016/j.carres.2011.11.020

Alkyl β-d-xylopyranosides are highly surface active, biodegradable surfactants that can be prepared from hemicelluloses and are of interest for use as pharmaceuticals, detergents, agrochemicals, and personal care products. To gain further insights into th

Full text of DOI:10.1016/j.carres.2011.11.020

Carbohydrate Research 349 (2012) 12–23
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis, thermal properties, and cytotoxicity evaluation of hydrocarbon
and fluorocarbon alkyl b-D-xylopyranoside surfactants
Wenjin Xu a, Gifty Osei-Prempeh b, Carolina Lema c, E. Davis Oldham a, Renato J. Aguilera c, Sean Parkin d,
Stephen E. Rankin e, Barbara L. Knutson e, Hans-Joachim Lehmler a,
⇑
a Department of Occupational and Environmental Health, The University of Iowa, UI Research Park, 124 IREH, Iowa City, IA 52242, USA
b Department of Chemical Engineering, WVU Institute of Technology, 405 Fayette Pike, Montgomery, WV 25136, USA
c Department of Biological Sciences, Bioscience Research Building, University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, USA
d Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA
e Department of Chemical and Materials Engineering, University of Kentucky, 177 F.P. Anderson Tower, Lexington, KY 40506, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 October 2011
Received in revised form 17 November 2011
Accepted 22 November 2011
Available online 9 December 2011
Alkyl b-D-xylopyranosides are highly surface active, biodegradable surfactants that can be prepared from
hemicelluloses and are of interest for use as pharmaceuticals, detergents, agrochemicals, and personal care
products. To gain further insights into their structure–property and structure–activity relationships, the
present study synthesized a series of hydrocarbon (–C6H13 to –C16H33) and fluorocarbon (–(CH2)2C6F13
alkyl b- -xylopyranosides in four steps from -xylose by acylation or benzoylation, bromination, Koe-
nigs–Knorr reaction, and hydrolysis, with the benzoyl protecting group giving better yields compared to
the acyl group in the Koenigs–Knorr reaction. All alkyl b- -xylopyranosides formed thermotropic liquid
crystals. The phase transition of the solid crystalline phase to a liquid crystalline phase increased linearly
with the length of the hydrophobic tail. The clearing points were near constant for alkyl b- -xylopyrano-
sides with a hydrophobic tail P8, but occurred at a significantly lower temperature for hexyl b- -xylopy-
ranoside. Short and long-chain alkyl b- -xylopyranosides displayed no cytotoxicity at concentration
below their aqueous solubility limit. Hydrocarbon and fluorocarbon alkyl b- -xylopyranosides with inter-
mediate chain length displayed some toxicity at millimolar concentrations due to apoptosis.
Ó 2011 Elsevier Ltd. All rights reserved.
)
D
D
Keywords:
D
Next-generation surfactants
Renewable precursors
Xylose
Structure–activity relationship
Structure–property relationship
Koenigs–Knorr reaction
D
D
D
D
1. Introduction
ond most abundant natural polysaccharides after cellulose,3 are
receiving increasing attention as sources for carbohydrate starting
materials, such as xylose, for the synthesis of next-generation
surfactants.
Carbohydrate-based surfactants are becoming increasingly
important as substitutes for poly(ethylene oxide)-based surfactants
used in a broad range of industrial and consumer applications, such
as pharmaceuticals, detergents, agrochemicals, and personal care
products.1 They can be synthesized from renewable raw materials
using green chemistry processes, have excellent surface activity,
and are readily biodegradable. Furthermore, carbohydrate-based
surfactants are generally considered to be toxicologically safe. For
example, many carbohydrate-based surfactants display little-to-no
toxicity in cells in culture and have limited hemolytic activity.2 In
particular starch, sugar beet, and sugar cane are sources of glucose,
sorbitol, and sucrose, currently the most important carbohydrate
moieties employed in the production of commercially available car-
bohydrate-based surfactants. In addition to these established
sources of carbohydrate starting materials, hemicelluloses, the sec-
A range of chemical and enzymatic processes have been used to
synthesize xylose-based surfactants. For example, simple alkyl xylo-
pyranosides have been prepared by the acid-catalyzed reaction of
D
-xylose4 or xylan with alcohols.5 Enzymatic reactions that have
been employed for the synthesis of alkyl xylopyranosides include,
among others, xylanase-catalyzed transglycosylation reactions of
xylan6 or Aspergillus niger b-xylosidase transxylosyl reactions of
xylobiose2e,7 with short chain alcohols. The yield of octyl xylopyran-
oside synthesized from xylan and octanol using acetone-dried cells
of Aureobasidium pullulans as the xylanase source has been shown
to increase in supercritical carbon dioxide or supercritical CHF3.8
While the physicochemical properties of the xylose-based sur-
factants synthesized by the various chemical and enzymatic ap-
proaches, including their surface activity and foaming properties,
are well investigated and comparable to other carbohydrate sur-
factants,1a,2e,4,5 systematic studies of the thermotropic properties
⇑
Corresponding author. Address: The University of Iowa, Department of Occu-
pational and Environmental Health, University of Iowa Research Park, 221 IREH,
Iowa City, IA 52242-5000, USA. Tel.: +1(319) 335 4211; fax: +1(319) 335 4290.
E-mail address: hans-joachim-lehmler@uiowa.edu (H.-J. Lehmler).
and the cytotoxicity of alkyl b-D-xylopyranosides have not been re-
ported previously. As part of the present study, a series of simple
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.11.020

W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
13
hydrocarbon and fluorocarbon alkyl b-
D
-xylopyranosides was
(DDQ), is another effective, but less expensive activator of glycosyl-
ation reactions.10 I2 in combination with DDQ also avoids the need
for a potentially toxic heavy metal salt as promoter. Finally, 1,1,3,3-
tetramethyl urea (TMU) was investigated because it efficiently
neutralizes the acid formed in the glycosylation reaction.11 In
agreement with several earlier studies and our initial synthesis
attempts,4,9a,b the major drawback of all reaction conditions inves-
tigated was the unsatisfactory yield of the desired octyl tetra-O-
synthesized and their physicochemical properties and cytotoxicity
were investigated to gain further insights into the usefulness of
these promising next-generation surfactants for pharmaceutical
and consumer product applications.
2. Results and discussion
acetyl-b-D-xylopyranoside (4) (Table 1, entries 1–6). The highest
2.1. Synthesis of alkyl b-D-xylopyranosides
yields, largest product to by-product ratios (i.e., ratios of 4/5) and
the shortest reaction time were obtained with I2, DDQ, and 4 Å
MS (entry 4). With Ag2CO3 as the base, the presence of I2 signifi-
cantly decreases the side reaction but did not improve the yield
of 4 (entry 1 vs 2). Furthermore, CaSO4 as drying agent was more
favorable compared to powdered 4 Å MS (entry 1 vs 3). The use
of AgOTf/TMU, either in the presence or absence of 4 Å MS, also
did not improve the yield of 4 or reduce the formation of 5 (entries
5 and 6).
The chemical synthesis of simple alkyl b-
structurally related alkenyl and alkenoyl derivatives of
poor-to-good yields has been reported previously.4,9 In the present
study, octyl b- -xylopyranoside (9b) was synthesized in a four step
synthesis from -xylose (1) as outlined in Scheme 1. Briefly,
-xylose was converted into the corresponding peracetylated
D
-xylopyranosides and
D
-xylose in
D
D
D
b-D-xylose 2 with acetic anhydride in pyridine. Subsequent bro-
mination with HBr followed by glycosylation with alcohols in the
presence of Ag2CO3 and CaSO4 in anhydrous dichloromethane
Subsequent efforts employed the benzoyl protecting group in
order to optimize the reaction efficiency and to avoid the side-
reaction of the acetyl group. As expected, improved total yields
yielded not only the desired octyl 2,3,4-tri-O-acetyl-b-
oside (4) but also significant amounts of 3,4-di-O-acetyl-1,2-O-(1-
octyloxyethylidene)- -xylopyranose (5) which could not be
purified by column chromatography. The desired product, octyl
b- -xylopyranoside (9b), was obtained by deprotection of 4 with
D-xylopyran-
of octyl b-D-xylopyranoside (9b) were obtained when the benzoyl
a-D
protecting group was used to minimize orthoester formation
(25% for acetyl vs 54% for benzoyl protected 9b) (Scheme 2). This
modified synthesis strategy was subsequently employed to pre-
D
sodium methoxide in methanol and subsequent neutralization
with Dowex 50 W ꢀ 8–100 ion exchange resin.
pare a series of alkyl b-
72% from 1,2,3,4-tetra-O-benzoyl-
approach can also be scaled-up to prepare gram quantities of alkyl
b- -xylopyranosides 9a–f.
In addition to the alkyl b-
fluorinated alkyl b- -xylopyranoside 9g was synthesized because
D
-xylopyranosides in total yields of 54–
a
-D
-xylopyranose (6). This
Several reaction conditions were investigated to further opti-
mize the glycosylation reaction (Table 1). Silver salts, including
Ag2CO3 and AgOTf, were employed because they are not only effi-
cient promoters of glycosylation reactions but also neutralize the
HBr released in the reaction. Iodine (I2), either alone or in combina-
tion with Ag2CO3 and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
D
D-xylopyranosides 9a–f, the partially
D
fluorinated surfactants are highly surface active and, at the same
time, biocompatible.2f–j The short perflourooctyl chain was se-
Scheme 1. Synthesis of octyl b-D-xylopyranoside (9b) via 1,2,3,4-tetra-O-acetyl-b-D-xylopyranose (2).
Table 1
Synthesis of octyl 2,3,4-tri-O-acetyl-b-D-xylopyranoside (4) by reaction of 3 with 1-octanola
Entry
Reaction conditions
Time (h)
Yield (%)b
Ratio of 4 to 5c
Reference
1
2
3
4
5
6
Ag2CO3, CaSO4
Ag2CO3, CaSO4, I2
Ag2CO3, 4 Å MS
I2, DDQ, 4 Å MS
AgOTf, TMU, 4 Å MS
AgOTf, TMU
10
10
10
3
4
4
33
26
8
36
23
17
4.4
9.0
1.7
16
2.8
3.2
43
43
43
10
11
11
MS = molecular sieves; DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; TMU = 1,1,3,3-tetramethyl urea.
a
Reactions in anhydrous dichloromethane (1 mL/0.1 g) were performed at ambient temperature using the following amounts of the reagents: Ag2CO3 (1.05 mol equiv),
CaSO4 (same weight as Ag2CO3), 4 Å MS (0.1 g/mL), AgOTf (2.2 mol equiv), and TMU (3 mol equiv). With exception of entry 4 all reactions were performed in the dark.
b
Isolated yield.
c
Determined by gas chromatography and based on relative peak area.

14
W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
(Scheme 3).2k,12 The yield was higher for the reaction with the
perbenzoate 6 (56%) than with the peracetylated derivative 2
(33%), as with the Koenigs–Knorr reaction described above. Both
products were readily deprotected with sodium methoxide/Dowex
50 W ꢀ 8–100 ion exchange resin to provide 9g in 62% yield.
Comparable yields for both synthesis steps have been reported
previously by Petrovic et al. for the synthesis of hydrocarbon alkyl
b-
D
-xylopyranosides 9b–e.9c
2.2. Crystal structure of decyl b-
D
-xylopyranoside (9c)
While crystal structures of simple alkyl
ing alkyl 2-deoxy-
-arabino-hexopyranosides13 and alkyl
-glucopyranosides,14 have been extensively studied, only rela-
tively few crystal structures of alkyl b- -pyranosides have been pub-
lished. In the present study, crystals of the hemihydrate of 9c
suitable for crystal structure determination were obtained by slow
evaporation of the solvent from a saturated solution of 9c in ace-
tone/hexane. Crystals of 9c were monoclinic (space group C2) with
a = 13.2 154(6), b = 4.3040(2), c = 29.9491(16) Å, and b = 102.66
5(3)° (see Tables S1 and S2 for additional crystal data, structure
refinement information as well as bond length and bond angles of
9c). The molecular structure of 9c with the atom numbering scheme
a-D-pyranosides, includ-
a-D
a-D
D
Scheme 2. Synthesis of alkyl b-D-xylopyranosides 9a–g from 1,2,3,4-tetra-O-
benzoyl-a-D-xylopyranose (6). DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone;
MS: molecular sieves; DCM: dichloromethane.
lected as hydrophobic tail to obtain a moderately water-soluble
surfactant for a preliminary biocompatibility assessment. The yield
of the partially fluorinated alkyl b- -xylopyranoside 9g synthe-
D
sized from 1,2,3,4-tetra-O-benzoyl- -D-xylopyranose (6) was low-
a
is shown in Figure 1. Only two other crystal structures of alkyl b-
xylopyranosides have been reported. Methyl b- -xylopyranoside
crystallized in the monoclinic space group (P21), with a = 7.893,
b = 6.908, c = 7.709 Å, and b = 113.4°.15 In contrast, heptyl b-
xylopyranoside monohydrate crystallized in the orthorhombic
D-
er than the yield of the corresponding hydrocarbon surfactants,
with a total yield of only 27%. Therefore, the alternate route re-
ported by Petrovic et al. was explored to synthesize larger quanti-
ties of 9g.9c Briefly, boron trifluoride ethyl etherate was used to
D
D-
glycosylate
2
and 6, yielding 8g and 10, respectively
Scheme 3. Synthesis of 9g from 1,2,3,4-tetra-O-acetyl-b-D-xylopyranose (2) or 1,2,3,4-tetra-O-benzoyl-a-D-xylopyranose (6) using BF3–Et2O.
Figure 1. Molecular structure of the hemihydrate of decyl b-D-xylopyranoside (9c). Compound 9c was crystallized from acetone–hexane. Displacement ellipsoids are drawn
at the 50% probability level.

W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
15
space group P212121 (a = 53.215(8), b = 8.8301(9), and
c = 6.5276(7) Å).16
The unit cell of 9c contained two independent molecules that
form alternating bilayers of hydrophilic xyloside groups and
hydrophobic decyl groups parallel to the a–b plane (Fig. S30).
The distance of the layers d was calculated as d = c ꢀ sinb, where
c is the length of the cell axis c and b the monoclinic angle. The dis-
tance of the layers calculated using this formula was 25.3 Å, which
is comparable to the value of the corresponding glucoside of
26.4 Å.14 A relatively large d value of 26.6 Å was observed for the
shorter chain heptyl b-D
-xylopyranoside monohydrate.16
The formation of 9c bilayers in the solid state is not surprising
because interactions between different parts of an organic mole-
cule are usually less favorable than the interactions between sim-
ilar parts of the molecule (i.e., the decyl chains and the
carbohydrate groups of 9c).17 As with other carbohydrate surfac-
tants,13,14,16 the carbohydrate groups and decyl chains of 9c are
packed in a manner that maximizes intermolecular interactions
within the crystal. Specifically, the decyl chains have van der Waals
contact by adopting a tilt angle of 41.7° relative to the a–b plane
Figure 3. Comparison of the thermograms of the hydrocarbon octyl xyloside 9b
and corresponding fluorocarbon octyl xyloside 9g.
Figure 2. Representative thermograms of hydrocarbon alkyl b-
9a–f.
D-xylopyranosides
Figure 4. TGA thermogram of (a) the hydrocarbon octyl xyloside 9b and (b) the
corresponding fluorocarbon octyl xyloside 9g.

16
W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
and the carbohydrate groups and the water molecule are con-
nected by a network of hydrogen bonds. Similarly, the two inde-
pendent molecules in the crystal structure of heptyl b-D-
xylopyranoside monohydrate form an acute angle of 49.2° and
37.4° to the bilayer plane (i.e. the b–c plane).16 This separation into
hydrophobic and hydrophilic regions has been reported for many
phase to a smectic A type phase,23 occurred at 72.5 °C. The clearing
point of 9b was observed at 99.4 °C. Comparable temperatures for
Mp (64.5 °C23; 67.5 °C24) and Cp (96.9 °C23; 103.3 °C24) have been
reported previously for 9b. The putative Mp and Cp for the fluori-
nated b-D-xylopyranoside 9g occurred at much higher tempera-
tures (105.1 °C and 147.0 °C, respectively) due to the
perfluorinated hydrophobic tail (Fig. 3). This increase in the phase
transition temperatures of a fluorinated compound compared to
the respective hydrocarbon analogue is consistent with other fluo-
rinated compounds, for example, perfluorinated carboxylic acids25
carbohydrate
surfactants
displaying
liquid–crystalline
properties.18
2.3. Thermotropic liquid crystalline properties of alkyl b-D-
xylopyranosides 9a–g
or partially fluorinated alkyl b-D
-glucopyranosides,2k and is a result
of the more dense packing of the rigid perfluorinated tail in the so-
lid state.
The liquid crystalline properties of biological lipids play an
important role in biological structures and function, and in the case
of simple surfactants, may correlate with their biological effects.18,19
Many pentose-based surfactants, such as alkyl arabinopyrano-
sides20 and alkyl DL-xylitols,21 also display liquid–crystalline proper-
ties and, as first described by Noller and Rockwell for simple alkyl
The first phase transition observed for the alkyl b-D-xylopyrano-
sides 9a, 9c, 9d, and 9e also corresponds to a change from a solid
crystalline phase to a liquid crystalline phase. One likely explana-
tion for the other two phase transitions is the loss of crystal water
from the sealed DSC pan. As a result, the second, broad phase tran-
sition most likely represents the melting of the anhydrous alkyl
D
-glucopyranosides,22 show ‘double melting transitions’ Mp repre-
senting a change from a solid crystalline phase to a liquid crystalline
phase, followed by the clearing point Cp (i.e., a transition from the li-
quid crystalline phase to a liquid melt phase). The present study uses
DSC and TGA to gain initial insights into the chain length-dependent
thermotropic behavior of hydrocarbon and fluorocarbon alkyl
b-
D-xylopyranosides of 9a, 9c, 9d, and 9e and the third phase tran-
sition is their Cp. A similar effect of the loss of crystal water has
been reported previously for the phase transitions of alkyl
a-D-
glucopyranosides.14 Although the experimental parameters are
quite different, the interpretation of the DSC results is supported
by TGA experiments which suggest the loss of approximately half
b-
D-xylopyranosides 9a–g (Figs. 2–4).
The DSC analysis of the hydrocarbon alkyl b-
D
-xylopyranosides
a water molecule from the alkyl b-
(Table 3). It is interesting to note that the decomposition temper-
ature of the fluorinated alkyl b- -xylopyranoside 9g was lower
compared to the hydrocarbon compounds in the TGA experiments
(Fig. 4 and Table 3). In contrast to this partially fluorinated com-
pound, perfluorocarbons and perfluorinated surfactants are ther-
mally more stable than their hydrocarbon analogues.26
D-xylopyranoside samples
showed two endothermic peaks in the case of 9b and 9f and three
endothermic peaks in the case of 9a, 9c, 9d, and 9e (Fig. 2, and Ta-
ble 2). These phase transitions occurred in a relatively narrow tem-
perature range. Decomposition of the hydrocarbon alkyl
D
b-
9g was observed at temperatures above 150 °C in the DSC experi-
ments. In the case of octyl b- -xylopyranoside (9b), the first phase
D-xylopyranosides 9a–f, but not the partially fluorinated analog
D
Mp of the hydrocarbon alkyl b-D-xylopyranosides 9a–f
transition (Mp), most likely a transition from a solid crystalline
displayed distinctive trends as a function of hydrophobic tail
Table 2
Onset, maximum (Tm) and half-width of the phase transitions observed for the alkyl b-D-xylopyranosides 9a–g (see Figs. 2 and 3 for representative thermograms)
Compound
RF,H
Onset of phase transition (°C)
Tm (°C)
Half-width of phase transition (°)
9a
n-C6H13
66.3 0.1
73.0 0.1
77.9 0.6
71.5 0.1
98.8 0.9
76.5 0.1
86.7 1.4
97.8 0.9
81.8 0.1
91.3 1.3
100.2 1.1
87.0 0.1
93.0 0.1
103.1 0.1
91.4 0.1
100.4 0.1
102.8 0.2
146.8 0.1
68.3 0.4
73.5 0.2
82.4 0.5
72.5 0.3
99.4 0.9
77.6 0.1
91.6 0.8
97.9 0.9
82.5 0.0
94.9 1.0
100.4 1.2
87.6 0.1
96.7 0.1
103.4 0.1
91.9 0.1
101.7 0.8
105.1 0.1
147.0 0.1
2.0 0.3
0.7 0.2
3.9 0.2
1.3 0.3
0.9 0.1
1.1 0.2
3.5 0.3
0.6 0.1
0.9 0.1
2.9 0.1
0.5 0.2
1.0 0.2
2.9 0.2
0.5 0.1
0.7 0.1
2.0 0.4
1.9 0.2
0.3 0.1
9b
9c
n-C8H17
n-C10H21
9d
9e
n-C12H25
n-C14H29
9f
n-C16H33
9g
(CH2)2C6F13
Table 3
Weight loss and combustion temperature of alkyl b-D-xylopyranosides 9a–g
Compound
RF,H
Temperature of solvent loss (°C)
Percent weight loss (%)
Number of water molecules
Decomposition temperature (°C)
9a
9b
9c
9d
9e
9f
n-C6H13
n-C8H17
81.74 0.41
85.32 0.55
96.63 0.44
86.35 0.98
100.1 2.93
103.4 1.95
50.57 1.64
2.50 0.66
2.35 0.91
2.69 0.07
1.56 0.09
1.87 1.08
1.24 0.49
2.64 0.83
0.4
0.5
0.5
0.3
0.5
0.3
0.9
262.8 4.34
267.1 9.15
277.2 11.5
290.4 10.5
294.8 7.15
295.9 2.86
231.6 4.57
n-C10H21
n-C12H25
n-C14H29
n-C16H33
(CH2)2C6F13
9g

W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
17
temperature of this phase transition has been observed for other
carbohydrate surfactants, such as glucopyranosides2k,14 and alkyl
DL-xylitol surfactants;21a,c however, the increase was typically not
as linear as observed for 9a–f.21c A clearing point was observed
in all thermograms, with only 9f being an exception. The respective
transition temperatures were near constant for alkyl b-
anosides with a hydrophobic tail P8, and only occurred at a signif-
icantly lower temperature for hexyl b- -xylopyranoside (9a)
D-xylopyr-
D
(Fig. 5). The clearing temperatures of ether, thioether, and ester-
based xylitol surfactants show a comparable dependence on the
length of the hydrophobic tail.21c
2.4. Cytotoxicity of alkyl b-D-xylopyranosides
Limited studies of the toxicity of carbohydrate surfactants2 sug-
gest that the headgroup and the hydrophobic tail are both impor-
tant structural determinants of surfactant–lipid interactions and,
thus, their toxicity. However, systematic studies of the toxicity of
alkyl b-
been reported. In the present study, the cytotoxicity of the alkyl
b- -xylopyranosides 9a–g was initially investigated in the sponta-
D-xylopyranoside in mammalian cells in culture have not
Figure 5. Chain length-dependent changes in the transition from a solid crystalline
phase to a liquid crystalline phase (Mp) and the transition from the liquid crystalline
phase to a liquid melt phase (Cp) of hydrocarbon alkyl b-D-xylopyranosides 9a–f.
D
neously immortalized human keratinocyte cell line HaCaT using
the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazolium) cell viability assay. This
cell line was selected because it exhibits characteristics of normal
length (Figs. 2 and 5). Mp linearly increased with chain length
(y = 2.53x + 51.22; R2 = 0.999).
A
similar increase in the
9b
9g
120
120
A
C
100
100
80
60
40
20
0
HaCaT
Hs27
Jurkat
HaCaT
Hs27
Jurkat
80
60
40
20
0
0
100
200
300
400
500
600
0
200
400
600
800
1000
1200
Concentration (µM)
Concentration (µM)
9c
OTG
120
100
80
60
40
20
0
120
100
80
60
40
20
0
B
D
HaCaT
Hs27
Jurkat
HaCaT
Hs27
Jurkat
0
100
200
300
400
500
600
0
200
400
600
800
1000
1200
Concentration (µM)
Concentration (µM)
E
9b
9c
9g
OTG
809
530
388
HaCaT
Hs27
580
474
510
165
123
125
183
267
163
Jurkat
Figure 6. Dose–response curves and determination of CC50 values utilizing the MTS assay. Dose–response effect and CC50 were determined in HaCaT, Hs27 and Jurkat cells for
alkyl b- -xylopyranosides 9b (A), 9c (B), 9g (C) and octylthioglucoside (OTG) (D). Cells were exposed for 20 h to increasing concentrations of 9b, 9c, 9g, or OTG. The cellular
viability is shown in the y axis while the concentration of compounds ( M) is shown in the x axis. Data are represented as the mean percentage of viable cells at each
dose SD of four replicates. CC50 values of the experimental compounds for each cell line are summarized in (E).
D
l

18
W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
skin keratinocytes27 and, therefore, provides insights into possible
effects of alkyl b- -xylopyranosides on skin cells. No effect on cell
viability was observed for the shortest chain alkyl b- -xylopyrano-
side 9a (CC50 >1 mM) and the long chain alkyl b- -xylopyranosides
9d–f (CC50 >100 mM for 9d and 9e; CC50 >25 mM for 9f). These four
alkyl b- -xylopyranosides were therefore not included in subse-
quent cell culture experiments with Jurkat and Hs27 cells. The oc-
tyl and decyl b- -xylopyranosides 9b and 9c displayed moderate
toxicity, with the longer chain xylopyranoside 9c being more toxic
in the HaCaT cell line (Fig. 6). The fluorinated octyl b- -xylopyrano-
CC50 values decreased in the order 9b > 9g ꢁ 9c. This trend is com-
parable to the one observed for the HaCaT cell line. A slightly dif-
ferent rank order was observed for the Hs27 cell line, with
9b > 9g > 9c. The three cell lines displayed different sensitivity to-
ward alkyl b-D-xylopyranoside-mediated toxicity. For the three
hydrocarbon surfactants 9b, 9c, and OTG, the HaCaT cells were less
sensitive compared to the other two cell lines and displayed the
highest CC50 values. In the case of the hydrocarbon alkyl b-D-xylo-
pyranosides 9b and 9c, the Hs27 cell line seemed to be more sen-
sitive than the other two cell lines. A different rank order was
D
D
D
D
D
D
sides 9g also displayed moderate toxicity in HaCaT cells and had a
CC50 that was comparable to the CC50 of 9c. All three xylopyrano-
sides were more toxic than octylthioglucoside (OTG), a hydrocar-
bon surfactant that has been used previously as positive control
in cytotoxicity studies with simple, carbohydrate-based
surfactants.2k,12a
observed for the partially fluorinated alkyl b-D-xylopyranoside
9g, with the Hs27 cell line being less sensitive toward 9g compared
to the HaCaT and Jurkat cell lines.
Several studies have reported that fluorinated surfactants are
less toxic than the analogous hydrocarbon surfactant due to a pro-
tective effect of the perfluorinated tail.2f–j In contrast to these ear-
lier findings, surfactant 9g was more cytotoxic compared to the
analogous hydrocarbon compound 9b. One likely explanation for
the comparatively high toxicity of 9g is that the degree of fluorina-
tion is not sufficient to convey a protective effect in the cell lines
under investigation. In contrast, the CC50 values of 9g and 9c were
comparable in the HaCaT and Jurkat cell lines and only slightly dif-
ferent in the Hs27 cell lines (Fig. 6E), despite the difference in the
length of the hydrophobic tail (C8 vs C10). This finding can be ex-
plained with the ‘1.5 rule’ which predicts that fluorinated surfac-
The observation that the short (9a) and long chain alkyl
b-D-xylopyranosides (9d–f) had no effect on HaCaT cell viability
in the concentration range investigated is in agreement with previ-
ous structure–activity studies with other carbohydrate surfactants
in B16F10 cells2k,12a and various anesthetic and antimicrobial com-
pounds in non-mammalian systems.28 The low toxicity of carbohy-
drate surfactants with a short hydrophobic tail, such as 9a, is likely
due to the limited partitioning of these surfactants into the cell
membrane.12a The low toxicity of the long chain alkyl b-
D-xylopyr-
anosides 9d–f appears to be counterintuitive because the cellular
uptake of surfactants and, therefore, their toxicity should increase
with increasing length of the hydrophobic tail. However, a number
of factors (e.g., a decrease in aqueous and lipid solubility, an in-
crease in binding to protein in the culture medium, a decrease in
the critical micelle concentration, and a decrease in the diffusion
through the cell membrane with increasing length of the hydro-
phobic tail) can reduce the cellular uptake and, thus, the toxicity
tants behave like analogous hydrocarbon surfactants with
longer hydrophobic tail, with one CF2 group approximating 1.5
CH2 groups.31 According to this rule, the fluorinated alkyl b-
-xylo-
pyranosides 9g should behave like undecyl b- -xylopyranoside
(i.e., 2 ꢀ CH2 + 1.5 ꢀ 6 ꢀ CF2 = 11) and thus display properties com-
parable to decyl b- -xylopyranoside (9c). Indeed, in the CC50 values
of 9g and 9c are similar, which suggests that physicochemical
parameters, such as the partitioning of alkyl b- -xylopyranoside
a
D
D
D
D
of the long chain alkyl b-
D
-xylopyranosides 9d–f.12a,28
surfactants into the cell membrane, may play an important role
in their cytotoxicity.
Subsequent cell culture experiments investigated the effect of
the carbohydrate surfactants 9b, 9c, 9g, and OTG on the cell viabil-
ity in Jurkat and Hs27 cells using the MTS assay. The Jurkat human
T-cell lymphoblastic leukemia is a transformed cell line that grows
in suspension and was selected because it is highly sensitive to a
variety of chemical treatments.29 Finally, human HS27 fibroblasts
derived from newborn foreskin were studied because they are
commonly used as a normal cell line control when compared to
transformed immortal cancer lines.29b,30 Experiments with these
two cell lines also revealed moderate toxicity for all three alkyl
The reduction in cell viability observed with 9b, 9c, 9g, and OTG
canbe dueto necrosisorapoptosis, thetwomajorformsof celldeath.
There is evidence that the effect of carbohydrate surfactants on cell
viability in mammalian cell lines is not due to a massive disruption
of phospholipid membranes but caused by more specific mecha-
nisms. Previous studies with carbohydrate surfactants demonstrate
that the CC50 values for cell viability in B16G10 cells are significantly
smaller than the corresponding values for hemolytic activity in rab-
bit red blood cells.2k,12a Furthermore, carbohydrate surfactants with
only minor differences in the structure of the head group have
b-D-xylopyranosides and OTG (Fig. 6). In the Jurkat cell line, the
Figure 7. Induction of apoptosis by alkyl b-
D-xylopyranosides 9b, 9c, 9g and OTG. Jurkat cells were evaluated for apoptosis via Annexin-FITC and propidium iodide staining
after exposure for 20 h to alkyl b- -xylopyranosides 9b, 9c, 9g or OTG at their respective CC50. Positive controls for apoptosis and necrosis included 2
D
lM Stauroporine and
0.5% Tween 20, respectively. Untreated cells and DMSO treated cells were used as negative and solvent controls, respectively. Data are represented as the mean SD of three
replicates.

W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
19
different CC50 values12a and, in the case of several glucopyranosides,
induce apoptosis in B16G10 cells.2k In the present study the number
43–45 °C, lit.9a 52–53 °C (ethanol–water); 1H NMR (CDCl3,
300 MHz):
d
0.85 (t, 3H, J = 6.7 Hz, H-80), 1.24 (br s, 10H,
of apoptotic Jurkat cells after exposure to the alkyl b-
D-xylopyrano-
5 ꢀ CH2), 1.51–1.59 (m, 2H, H-20), 2.01–2.03 (3 ꢀ s, 9H,
3 ꢀ CH3CO), 3.33 (dd, 1H, J5a,5e = 11.7 Hz, J4,5a = 7.5 Hz, H-5a), 3.42
sides 9b, 9c, and 9g as well as OTG was quantified using Annexin V-
FITC, a molecule that binds to the phosphatidylserine present on the
extracellular side of the cell membrane of cells in the early stage of
apoptosis. As shown in Figure 7, all four surfactants decrease cell via-
bility in Jurkat cells by apoptosis and not necrosis. This observation
provides furtherevidencethat carbohydrate surfactantscause toxic-
ityby specificmechanisms, such as a selectiveinteractionwith lipids
rafts in the cell membrane, which ultimately results in a receptor-
independent activation of intracellular signaling pathways, such as
Fas-mediated apoptosis cascades.32
(dt, 1H, J1 a,1 b = 9.6 Hz, J1 a,2 = 6.6 Hz, H-10a), 3.78 (dt, 1H,
0
0
0
0
J1 a,1 b = 9.5 Hz, J1 a,2 = 6.4 Hz, H-10b), 4.09 (dd, 1H, J5a,5e = 11.7 Hz,
J5e,4 = 5.1 Hz, H-5e), 4.43 (d, 1H, J1,2 = 6.9 Hz, H-1), 4.88 (dd, 1H,
J1,2 = 6.8 Hz, J2,3=8.7 Hz, H-2), 4.90–4.96 (m, 1H, H-4), 5.13 (pseudo
t, 1H, J2,3 = J3,4 = 8.6 Hz, H-3); 13C NMR (CDCl3, 75 MHz): d 14.3,
21.0, 22.9, 26.1 (C-30), 29.5–29.7, 32.0, 62.2, 69.2, 70.0, 70.1, 71.7,
100.9, 169.7, 170.1, 170.4; EIMS m/z (relative abundance%): 170
(58), 157 (42), 139 (12), 128 (100), 115 (32), 97 (24), 86 (20), 69
(44), 57 (13).
0
0
0
0
In addition, crude 3,4-di-O-acetyl-1,2-O-(1-octyloxyethylidene)-
2.5. Conclusions
a-D
-xylopyranose (5) was obtained as a colorless oil: 1H NMR (CDCl3,
400 MHz): d 0.85 (t, 3H, J = 7.1, Hz, H-80), 1.26–1.28 (m, 13H, 5 ꢀ CH2
and CH3), 1.57–1.62 (m, 2H, H-20), 2.00 (s, 3H, CH3CO), 2.07 (s, 3H,
CH3CO), 3.39–3.57 (m, 5H, H-10, H-2, H-5), 4.80 (d, 1H, J1,2 = 4 Hz,
H-1), 4.88 (ddd, 1H, J4,5a = 10.6, J3,4 = 9.7, J4,5e = 5.8, H-4), 5.19 (pseu-
do t, 1H J2,3 = J3,4 = 9.7 Hz, H-3); 13C NMR (CDCl3, 75 MHz): d 14.3,
20.9–21.2 (2 ꢀ CH3CO and CH3), 22.8, 26.3, 29.4–29.6, 32.0, 58.8,
68.9, 69.1, 71.2, 73.2, 98.4, 103.2, 170.2, 171.3; mass spectrum m/z
(relative abundance%): 217 (4), 157 (9), 128 (68), 115 (24), 97 (39),
86 (32), 69 (100), 57 (36).
A series of simple alkyl xyloside surfactants was synthesized
from xylose and readily-available alcohols, using an optimized
Koenigs–Knorr reaction as the key glycosylation step. These surfac-
tants were shown to be non-toxic in several established cell lines.
Furthermore, all xylosides exhibited liquid–crystalline behavior
based on DSC experiments. In agreement with the thermotropic
behavior observed in the DSC studies, the hemihydrate of alkyl
xyloside 9b formed bilayers in the solid state. These compounds
have potential as environmentally friendly surfactants due to their
low toxicity and renewable precursors, but further studies are
needed to fully characterize their phase behavior, investigate their
surface properties and assess their toxicity.
3.3. General procedure for the synthesis of alkyl xylosides 8
A solution of the respective alkyl alcohol (12 mmol, 2.0 equiv)
in dichloromethane (30 mL, 1 mL per 0.1 g of 3) was stirred with
I2 (1.45 g, 6 mmol), DDQ (0.65 g, 3.0 mmol) and powdered 4 Å
molecular sieve for 30 min at room temperature. 2,3,4-Tri-O-ben-
3. Experimental
zoyl-a-D-xylopyranosyl bromide (7) (3.0 g, 6.0 mmol) was added
3.1. General methods
and the reaction mixture was stirred at room temperature for
3 h. The reaction mixture was filtered through Celite, washed with
an aqueous solution of Na2S2O4 (2%, w/v), satd NaHCO3
(3 ꢀ 30 mL), and satd NaCl solution (2 ꢀ 30 mL). The crude product
was purified by column chromatography on silica gel to yield 8.
The 1H and 13C NMR spectra were recorded on a multinuclear
Bruker Avance 300 or a Bruker DRX 400 Digital NMR spectrometer
at ambient temperature. 19F spectra were recorded using a Bruker
Avance 300 NMR spectrometer. Mass spectra, including accurate
mass measurements, were recorded by the High Resolution Mass
Spectrometry Facility at the University of California, Riverside. Ele-
mental analyses were obtained from Atlantic Micro Lab Microanal-
ysis Service (Atlanta, Georgia, USA). X-ray diffraction data were
3.3.1. Hexyl 2,3,4-tri-O-benzoyl-b-D-xylopyranoside (8a)
Prepared from 1-hexanol (1.16 g, 11.4 mmol, 2.0 equiv) and 7
(3.0 g, 5.7 mmol). Purified by flash column chromatography as de-
scribed above (hexane–ethyl acetate, 6:1, v/v, then 3:1, v/v) to af-
ford 8a (2.75 g, 88%): Colorless oil; Rf 0.61 (hexane–ethyl acetate,
3:1, v/v); 1H NMR (CDCl3, 400 MHz) d 0.78 (t, 3H, J = 7.0 Hz, CH3),
1.12–1.24 (m, 4H, 2 ꢀ CH2), 1.24–1.32 (m, 2H), 1.52–1.63 (m,
collected at 90.0(2) K using MoK
a X-rays on a Nonius KappaCCD
as described previously.33 Melting points were determined using
a MelTemp apparatus, and are uncorrected. All reactions were
monitored by thin layer chromatography, followed by visualization
2H), 3.53 (dt, 1H, J1 a,1 b = 9.6 Hz, J1 a,2 = 6.8 Hz, H-10a), 3.69 (dd,
0
0
0
0
with UV and anisaldehyde–H2SO4. 1,2,3,4-Tetra-O-acetyl-b-
pyranose (2),34 2,3,4-tri-O-acetyl- -xylopyranosyl bromide (3),35
1,2,3,4-tetra-O-benzoyl-
-xylopyranose (6),36 and 2,3,4-tri-O-
benzoyl-
D-xylo-
0
0
1H, J5a,5e = 12.0 Hz, J4,5a = 7.0 Hz, H-5a), 3.88 (dt, 1H, J1 a,1 b = 9.6 Hz,
J1 b,2 = 6.8 Hz, H-10b), 4.42 (dd, 1H, J5a,5e = 12.0 Hz, J4,5e = 4.4 Hz, H-
5e), 4.81 (d, 1H, J1,2 = 5.4 Hz, H-1), 5.29 (pseudo td, 1H,
J3,4 = J4,5a = 7.0 Hz, J4,5e = 4.4 Hz, H-4), 5.37 (dd, 1H, J2,3 = 7.0 Hz,
J1,2 = 5.4 Hz, H-2), 5.76 (pseudo t, 1H, J2,3 = J3,4 = 7.0 Hz, H-3),
7.30–7.40 (m, 6H), 7.44–7.56 (m, 3H), 7.96–8.00 (m, 6H); 13C
NMR (CDCl3, 100 MHz): d 14.2, 22.7, 25.9, 29.7, 30.1, 31.7, 61.4,
69.4, 69.8, 70.5, 100.3, 128.51, 128.54, 128.6, 129.4, 129.5, 129.6,
130.0, 130.07, 130.1, 133.4, 133.5, 133.54, 165.4, 165.6, 165.8;
HRESIMS m/z: calcd for C32H34O8Na [M+Na]+: 569.2146; found:
569.2134.
a
-D
0
0
a-D
a-D
-xylopyranosyl bromide (7)36 were prepared according
to known literature procedures. Their 1H NMR, 13C NMR, and melt-
ing points matched literature values.
3.2. General method for the glycosylation of 336
1-Octanol (2 mol equiv) in dichloromethane (1 mL per 0.1 g of
3) was stirred with the respective reagents (see Table 1) for
30 min.
2,3,4-Tri-O-acetyl-
a-
D-xylopyranosyl
bromide
(3)
(2–10 mmol) was then added and the reaction mixture was stirred
at room temperature. The reaction mixture was filtered through
Celite and washed with satd NaHCO3 (3 ꢀ 50 mL) and NaCl solu-
tion (3 ꢀ 50 mL). The only exception was the reaction with I2/
DDQ/MS (entry 4), which was extracted first with 2% (w/v) Na2S2O4
solution to remove excess I2. The crude product was purified by
column chromatography on silica gel using hexane:ethyl acetate
(6:1, v/v, then 3:1, v/v) as eluent to yield 4 as a white solid: mp
3.3.2. Octyl 2,3,4-tri-O-benzoyl-b-D-xylopyranoside (8b)
Prepared from 1-octanol (0.50 g, 3.8 mmol, 2.0 equiv) and 7
(1.0 g, 1.9 mmol). Purified by flash column chromatography (hex-
ane–ethyl acetate, 6:1, v/v, then 3:1, v/v) to yield 8b as a colorless
oil (0.82 g, 75%) that was used directly in the next reaction. Recrys-
tallization from ethanol gave a white solid (28%); Rf 0.61 (hexane–
ethyl acetate, 4:1, v/v); mp 47–48 °C; 1H NMR (CDCl3, 300 MHz) d
0.84 (t, 3H, J = 6.9 Hz, CH3), 1.14–1.27 (m, 10H, 5 ꢀ CH2), 1.51–1.62

20
W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
(m, 2H), 3.52 (dt, 1H, J1 a,1 b = 9.6 Hz, J1 a,2 = 6.5 Hz, H-10a), 3.72 (dd,
7.31–7.37 (m, 6H), 7.46–7.64 (m, 3H), 7.95–8.00 (m, 6H); 13C
NMR (CDCl3, 100 MHz): d 14.3, 22.9, 26.2, 29.5–29.9, 32.1, 61.4,
69.4, 69.8, 70.6, 100.4, 128.53, 128.56, 128.63, 129.4, 129.5,
129.7, 130.07, 130.10, 130.11, 133.4, 133.5, 133.6, 165.4, 165.6,
165.8; HRESIMS m/z: calcd for C40H50O8Na [M+Na]+: 681.3398;
found: 681.3380.
0
0
0
0
0
0
1H, J5a,5e = 12.1 Hz, J4,5a = 7.0 Hz, H-5a), 3.88 (dt, 1H, J1 a,1 b = 9.6 Hz,
J1 b,2 = 6.5 Hz, H-10b), 4.44 (dd, 1H, J5a,5e = 12.1 Hz, J4,5e = 4.3 Hz, H-
5e), 4.84 (d, 1H, J1,2 = 5.4 Hz, H-1), 5.32 (pseudo td, 1H,
J3,4 = J4,5a = 7.0 Hz, J4,5e = 4.3 Hz, H-4), 5.40 (dd, 1H, J2,3 = 7.0 Hz,
J1,2 = 5.4 Hz, H-2), 5.79 (pseudo t, 1H, J2,3 = J3,4 = 7.0 Hz, H-3),
7.28–7.35 (m, 6H), 7.43–7.51 (m, 3H), 7.98–8.01 (m, 6H); 13C
NMR (CDCl3, 75 MHz): d 14.1, 22.7, 26.1, 29.2, 29.4, 29.7, 31.8,
61.3, 69.4, 69.6, 70.5, 100.2, 128.38, 128.41, 128.5, 129.36, 129.4,
129.6, 129.91, 129.94, 129.96, 133.2, 133.3, 133.4, 165.2, 165.5,
165.6; HRESIMS m/z: calcd for C34H38O8Na [M+Na]+: 597.2459;
found: 597.2450.
0
0
3.3.6. Hexadecyl 2,3,4-tri-O-benzoyl-b-D-xylopyranoside (8f)
Prepared from 1-hexadecanol (2.77 g, 12.0 mmol, 2.0 equiv) and
7 (3.0 g, 5.7 mmol). Purified by flash column chromatography as
described above (hexane–ethyl acetate, 9:1, v/v, then 6:1, v/v) to
afford 8f (3.54 g, 90%): White solid; Rf 0.63 (hexane–ethyl acetate,
5:1, v/v); mp 67–69 °C; 1H NMR (CDCl3, 400 MHz) 0.86 (t, 3H,
J = 6.8 Hz, CH3), 1.12–1.24 (m, 26H, 13 ꢀ CH2), 1.52–1.57 (m, 2H),
3.3.3. Decyl 2,3,4-tri-O-benzoyl-b-D-xylopyranoside (8c)
0
0
0
0
Prepared from 1-decanol (1.90 g, 12.0 mmol, 2.0 equiv) and 7
(3.0 g, 5.7 mmol). Purified by flash column chromatography as de-
scribed above (hexane–ethyl acetate, 9:1, v/v, then 6:1, v/v) to af-
ford 8c (3.43 g, 99%): White solid; Rf 0.62 (hexane–ethyl acetate,
3:1, v/v); mp 59–60 °C; 1H NMR (CDCl3, 400 MHz) d 0.88 (t, 3H,
J = 7.0 Hz, CH3), 1.10–1.36 (m, 14H, 7 ꢀ CH2), 1.52–1.59 (m, 2H),
3.50 (dt, 1H, J1 a,1 b = 9.6 Hz, J1 a,2 = 6.5 Hz, H-10a), 3.68 (dd, 1H,
0
0
J5a,5e = 12.1 Hz, J4,5a = 7.0 Hz, H-5a), 3.86 (dt, 1H, J1 a,1 b = 9.6 Hz,
J1 b,2 = 6.4 Hz, H-10b), 4.41 (dd, 1H, J5a,5e = 12.1 Hz, J4,5e = 4.3 Hz, H-
5e), 4.79 (d, 1H, J1,2 = 5.4 Hz, H-1), 5.28 (pseudo td, 1H,
J3,4 = J4,5a = 7.0 Hz, J4,5e = 4.3 Hz, H-4), 5.35 (dd, 1H, J2,3 = 7.0 Hz,
J1,2 = 5.4 Hz, H-2), 5.73 (pseudo t, 1H, J2,3 = J3,4 = 7.0 Hz, H-3),
7.31–7.37 (m, 6H), 7.48–7.54 (m, 3H), 7.95–7.99 (m, 6H); 13C
NMR (CDCl3, 100 MHz): d 14.6, 23.1, 26.5, 29.5–30.0, 32.2, 61.4,
69.4, 69.8, 70.6, 100.4, 128.54, 128.57, 128.6, 129.4, 129.5, 129.7,
130.1, 130.11, 130.12, 133.4, 133.5, 133.6, 165.4, 165.6, 165.8;
HRESIMS m/z: calcd for C42H54O8Na [M+Na]+: 709.3711; found:
709.3692.
0
0
3.51 (dt, 1H, J1 a,1 b = 9.5 Hz, J1 a,2 = 6.5 Hz, H-10a), 3.71 (dd, 1H,
0
0
0
0
0
0
J5a,5e = 12.1 Hz, J4,5a = 7.0 Hz, H-5a), 3.88 (dt, 1H, J1 a,1 b = 9.5 Hz,
J1 b,2 = 6.4 Hz, H-10b), 4.42 (dd, 1H, J5a,5e = 12.1 Hz, J4,5e = 4.4 Hz,
H-5e), 4.82 (d, 1H, J1,2 = 5.6 Hz, H-1), 5.29 (pseudo td, 1H,
J3,4 = J4,5a = 7.0 Hz, J4,5e = 4.4 Hz, H-4), 5.37 (dd, 1H, J2,3 = 7.0 Hz,
J1,2 = 5.6 Hz, H-2), 5.76 (pseudo t, 1H, J2,3 = J3,4 = 7.0 Hz, H-3),
7.31–7.39 (m, 6H), 7.46–7.54 (m, 3H), 7.96–8.00 (m, 6H); 13C
NMR (CDCl3, 100 MHz): d 14.3, 22.9, 26.2, 29.5, 29.6, 29.7, 29.72,
29.75, 31.1, 32.1, 61.4, 69.4, 69.8, 70.5, 100.3, 128.51, 128.54,
128.6, 129.4, 129.5, 129.6, 130.0, 130.08, 130.09, 133.4, 133.48,
133.54, 165.4, 165.6, 165.8; HRESIMS m/z: calcd for C36H42O8Na
[M+Na]+: 625.2772; found: 625.2756.
0
0
3.3.7. 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl 2,3,4-tri-O-
benzoyl-b-D-xylopyranoside (8g)
Prepared from 1H,1H,2H,2H-perfluorooctanol (2.1 g, 5.7 mmol,
1.5 equiv) and 7 (2.0 g, 3.8 mmol) to afford 8 g (0.37 g, 9%) after
flash column chromatography followed by recrystallization from
ethanol: White solid; Rf 0.61 (hexane–ethyl acetate, 3:1, v/v); mp
0
3.3.4. Dodecyl 2,3,4-tri-O-benzoyl-b-
D
-xylopyranoside (8d)
119–120 °C; 1H NMR (CDCl3, 400 MHz)
d 2.42 (tt, 2H, J2 ,
0
0
30
Prepared from 1-dodecanol (2.23 g, 12.0 mmol, 2.0 equiv) and 7
(3.0 g, 5.7 mmol). Purified by flash column chromatography as de-
scribed above (hexane–ethyl acetate, 9:1, v/v, then 6:1, v/v) to af-
ford 8d (3.14 g, 87%): White solid; Rf 0.63 (hexane–ethyl acetate,
3:1, v/v); mp 64–66 °C; 1H NMR (CDCl3, 400 MHz) 0.86 (t, 3H,
J = 6.4 Hz, CH3), 1.23–1.28 (m, 18H, 9 ꢀ CH2), 1.54–1.59 (m, 2H),
= 18.8 Hz, J1 ,2 = 6.5 Hz, H-20), 3.74 (dd, 1H, J5a,5e = 12.2 Hz,
0
0
0
0
J4,5a = 6.7 Hz, H-5a), 3.83 (dt, 1H, J1 a,1 b = 10.3 Hz, J1 a,2 = 6.7 Hz, H-
10a), 4.16 (dt, 1H, J1 a,1 b = 10.3, J1 b,2 = 6.5 Hz, H-10b), 4.43 (dd, 1H,
J5a,5e = 12.2 Hz, J4,5e = 4.2 Hz, H-5e), 4.85 (d, 1H, J1,2 = 5.21 Hz, H-
1), 5.28 (pseudo td, 1H, J3,4 = J4,5a = 7.0, J4,5e = 4.2 Hz, H-4), 5.35
(dd, 1H, J2,3 = 7.0 Hz, J1,2 = 5.2 Hz, H-2), 5.73 (pseudo t, 1H,
J2,3 = J3,4 = 7.0 Hz, H-3), 7.30–7.39 (m, 6H), 7.47–7.55 (m, 3H),
7.94–8.01 (m, 6H); 13C NMR (CDCl3, 100 MHz): d 31.5, 61.1, 68.9,
69.87, 69.92, 100.1, 128.30, 128.34, 128.4, 129.1, 129.17, 129.21,
129.83, 129.88, 129.9, 133.3, 133.38, 133.41, 165.2, 165.3, 165.5;
19F NMR (282 MHz, CDCl3): d ꢂ81.3 (CF3), ꢂ113.9 (CF2), ꢂ122.5
(CF2), ꢂ123.4 (CF2), ꢂ124.2 (CF2), ꢂ126.7 (CF2); HRESIMS m/z:
calcd for C34H25O8F13Na [M+Na]+: 831.1234; found: 831.1241.
0
0
0
0
3.50 (dt, 1H, J1 a,1 b = 9.5 Hz, J1 a,2 = 6.5 Hz, H-10a), 3.69 (dd, 1H,
0
0
0
0
0
0
J5a,5e = 12.1 Hz, J4,5a = 7.0 Hz, H-5a), 3.86 (dt, 1H, J1 a,1 b = 9.5 Hz,
J1 b,2 = 6.3 Hz, H-10b), 4.41 (dd, 1H, J5a,5e = 12.1 Hz, J4,5e = 4.4 Hz, H-
5e), 4.80 (d, 1H, J1,2 = 5.5 Hz, H-1), 5.28 (pseudo td, 1H,
J3,4 = J4,5a = 7.0 Hz, J4,5e = 4.4 Hz, H-4), 5.35 (dd, 1H, J2,3 = 7.0 Hz,
J1,2 = 5.5 Hz, H-2), 5.74 (pseudo t, 1H, J2,3 = J3,4 = 7.0 Hz, H-3),
7.31–7.38 (m, 6H), 7.46–7.63 (m, 3H), 7.95–7.99 (m, 6H); 13C
NMR (CDCl3, 100 MHz): d 14.3, 22.9, 26.2, 29.5, 29.6, 29.7, 29.75,
29.8, 29.81, 29.84, 31.1, 32.1, 61.4, 69.4, 69.8, 70.5, 100.3, 128.51,
128.54, 128.6, 129.4, 129.5, 129.6, 130.0, 130.1, 133.4, 133.48,
133.53, 165.4, 165.6, 165.8; HRESIMS m/z: calcd for C38H46O8Na
[M+Na]+: 653.3085; found: 653.3066.
0
0
3.4. General procedure for the synthesis of 9a–g2k,37
A solution of sodium methoxide (0.32 g, 6.0 mmol, 3.0 equiv) in
methanol (20 mL) was added drop wise to a solution of 4 or 8a–g
(2 mmol) in methanol or dichloromethane (10 mL). The mixture
was stirred at ambient temperature for 30 min followed by neu-
tralization with Dowex 50 Wꢀ8–100 ion exchange resin. The ion
exchange resin was filtered off and the solvent was removed under
reduced pressure. The crude product was dissolved in acetone. The
residue was filtered and the crude product was purified by column
chromatography and/or recrystallized to give 9a–g.
3.3.5. Tetradecyl 2,3,4-tri-O-benzoyl-b-D-xylopyranoside (8e)
Prepared from 1-tetradecanol (2.57 g, 12.0 mmol, 2.0 equiv) and
7 (3.0 g, 5.7 mmol). Purified by flash column chromatography as
described above (hexane–ethyl acetate, 9:1, v/v, then 6:1, v/v) to
afford 8e (3.57 g, 95%): White solid; Rf 0.66 (hexane–ethyl acetate,
3:1, v/v); mp 67–68 °C; 1H NMR (CDCl3, 400 MHz) 0.86 (t, 3H,
J = 6.9 Hz, CH3), 1.12–1.24 (m, 22H, 11 ꢀ CH2), 1.52–1.58 (m, 2H),
3.50 (dt, 1H, J1 a,1 b = 9.5 Hz, J1 a,2 = 6.5 Hz, H-10a), 3.68 (dd, 1H,
3.4.1. Hexyl b-D-xylopyranoside (9a)
0
0
0
0
0
0
J5a,5e = 12.1 Hz, J4,5a = 7.0 Hz, H-5a), 3.86 (dt, 1H, J1 a,1 b = 9.5 Hz,
Prepared from 8a (2.3 g, 4.2 mmol) to afford 9a (0.75 g, 76%).
The crude product was purified by recrystallization from acetone
to yield white, flaky crystals (0.45 g, 46%; 72% from 6 without puri-
fication of intermediates): Rf 0.53 (CH2Cl2–MeOH, 8:1, v/v); mp
84–85 °C, lit.37 90–91 °C (acetone); 1H NMR (MeOH-d4,
J1 b,2 = 6.4 Hz, H-10b), 4.41 (dd, 1H, J5a,5e = 12.1 Hz, J4,5e = 4.3 Hz, H-
5e), 4.79 (d, 1H, J1,2 = 5.4 Hz, H-1), 5.28 (pseudo td, 1H,
J3,4 = J4,5a = 7.0 Hz, J4,5e = 4.3 Hz, H-4), 5.35 (dd, 1H, J2,3 = 7.0 Hz,
J1,2 = 5.4 Hz, H-2), 5.74 (pseudo t, 1H, J2,3 = J3,4 = 7.0 Hz, H-3),
0
0

W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
21
400 MHz): d 0.86 (t, 3H, J = 6.9, CH3), 1.24–1.37 (m, 6H, 3 ꢀ CH2),
3.4.6. Hexadecyl b-D-xylopyranoside (9f)
1.52–1.59 (m, 2H, H-20), 3.08–3.16 (m, 2H, H-2 and H-5a), 3.22–
3.27 (m, 1H, H-3), 3.39–3.50 (m, 2H, H-10a and H-4), 3.72–3.81
(m, 2H, H-10b and H-5e), 4.13 (d, 1H, J1,2 = 7.5, H-1); 13C NMR
(MeOH-d4, 100 MHz) d 14.5, 23.8, 26.9, 30.9, 32.9, 67.0, 71.0,
Prepared from 8f (3.28 g, 4.76 mmol) to afford 9f after recrystal-
lization from acetone (1.36 g, 76%). The product was further puri-
fied by recrystallization from methanol to yield a white, flaky
solid (0.55 g, 31%; 70% from 6 without purification of intermedi-
ates): Rf 0.39 (9:1 CH2Cl2-MeOH); mp 102–103 °C, lit.37 105.5–
107; 1H NMR (MeOH-d4, 400 MHz): d 0.84 (t, 3H, J = 6.7, CH3),
1.23–1.31 (m, 26 H, 13 ꢀ CH2), 1.52–1.57 (m, 2H, H-20), 3.08–
3.15 (m, 2H, H-2 and H-5a), 3.22–3.26 (m, 1H, H-3), 3.39–3.49
(m, 2H, H-10a and H-4), 3.72–3.81 (m, 2H, H-10b and H-5e), 4.13
(d, 1H, J1,2 = 7.2, H-1); 13C NMR (MeOH-d4, 100 MHz) d 14.6, 23.9,
27.2, 30.6, 30.7, 30.8, 30.8–31.0, 33.2, 67.1, 71.1, 71.4, 75.1, 78.0,
105.3; HRESIMS m/z: calcd for C21H46NO5 [M+NH4]+: 392.3371;
found: 392.3369.
71.4, 75.0, 78.0, 105.2; HRESIMS m/z: calcd for
C11H26NO5
[M+NH4]+: 252.1806; found: 252.1801.
3.4.2. Octyl b-D
-xylopyranoside (9b)2k
Synthesized from 8b (1.16 g, 2.02 mmol) and purified by col-
umn chromatography on silica gel (eluent: dichloromethane–
methanol, 8/1, v/v) to yield 9b (0.44 g, 56%; 54% from 6 without
purification of intermediates). Recrystallization from acetone and
acetone–hexane gave a white solid (0.24 g, 31%): Rf 0.71 (6:1
CH2Cl2-MeOH); mp 90–91 °C, lit.9a 91–92 °C; 1H NMR (MeOH-d4,
300 MHz): d 0.87 (t, 3H, J = 6.9, CH3), 1.27 (br s, 10H, 5 ꢀ CH2),
1.53–1.60 (m, 2H, H-20), 3.09–3.18 (m, 2H, H-2 and H-5a), 3.26–
3.28 (m, 1H, H-3), 3.40–3.53 (m, 2H, H-10a and H-4), 3.73–3.84
(m, 2H, H-10b and H-5e), 4.19 (d, 1H, J1,2 = 7.5, H-1); 13C NMR
(MeOH-d4, 75 MHz): d 14.6, 23.9, 27.3, 30.4–31.0, 33.2, 67.1,
71.1, 71.4, 75.1, 78.0, 105.3; HRESIMS m/z: calcd for C13H30NO5
[M+NH4]+: 280.2119; found: 280.2122. Anal. Calcd for C13H26O5:
C, 59.52; H, 9.99. Found: C, 59.72; H, 10.00.
3.4.7. 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl b-D-
xylopyranoside (9g)
Prepared as described in the general procedure from crude 8g
(3.35 g, 4.14 mmol) to afford 9g after crystallization from
acetone/hexane as a white solid (0.54 g, 27% from 6 without puri-
fication of intermediates): Rf 0.49 (CH2Cl2-MeOH, 8:1, v/v); mp
103–104 °C; 1H NMR (MeOH-d4,400 MHz): d 2.44–2.56 (m, 2H,
H-20), 3.10–3.19 (m, 2H, H-2 and H-5a), 3.23–3.28 (m, 1H, H-3),
3.43 (ddd, 1H, J4,5a = 10.2 Hz, J3,4 = 8.7 Hz, J4,5e = 5.3 Hz, H-4),
3.78–3.84 (m, 2H, H-10a and H-5e), 4.05 (dt, 1H, J1 a,1 b = 10.4 Hz,
0
0
3.4.3. Decyl b-D-xylopyranoside (9c)
J1 b,2 = 6.9 Hz, H-10b), 4.19 (d, 1H, J1,2 = 7.5 Hz, H-1); 13C NMR
(MeOH-d4, 100 MHz) d 32.7, 62.7, 67.1, 71.3, 74.9, 77.9, 105.5;
19F NMR (282 MHz, CDCl3): d ꢂ80.8 (CF3), ꢂ112.8 (CF2), ꢂ121.2
(CF2), ꢂ122.2 (CF2), ꢂ123.0 (CF2), ꢂ125.6 (CF2); HRESIMS m/z:
calcd for C13H17NO5F13 [M+NH4]+: 514.0894; found: 514.0890.
0
0
Prepared from 8c (3.0 g, 5.0 mmol) to afford 9c (1.12 g, 78%).
The crude product was purified by recrystallization from acetone
to yield white, flaky crystals (0.83 g, 58%; 64% from 6 without puri-
fication of intermediates): Rf 0.50 (CH2Cl2–MeOH, 8:1, v/v); mp
94–95 °C, lit.37 98.5–99.5 °C (acetone/ethanol); 1H NMR (MeOH-
d4, 400 MHz): d 0.84 (t, 3H, J = 6.6, CH3), 1.24–1.31 (m, 14 H,
7 ꢀ CH2), 1.51–1.58 (m, 2H, H-20), 3.07–3.14 (m, 2H, H-2 and H-
5a), 3.21–3.27 (m, 1H, H-3), 3.39–3.49 (m, 2H, H-10a and H-4),
3.71–3.81 (m, 2H, H-10b and H-5e), 4.12 (d, 1H, J1,2 = 7.5, H-1);
13C NMR (MeOH-d4, 100 MHz) d 14.6, 23.9, 27.2, 30.6, 30.7,
30.83, 30.86, 30.9, 33.2, 67.0, 71.0, 71.3, 75.0, 78.0, 105.2; HRESIMS
m/z: calcd for C15H34NO5 [M+NH4]+: 308.2432; found: 308.2431.
3.4.8. 3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl 2,3,4-tri-O-
acetyl-b-
1,2,3,4-Tetra-O-acetyl-b-
2.08 mmol) and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanol
(544 L, 909 mg, 2.49 mmol, 1.2 equiv) were dissolved in CH2Cl2
(8 mL) at room temperature. The stirring solution was cooled in
an ice-water bath. BF3ꢃEt2O (385 L, 443 mg, 3.12 mmol, 1.5 equiv)
D
-xylopyranoside (10)2k,9c,12
D
-xylopyranose
(2)
(662 mg,
l
l
was added drop wise. The ice-bath was removed and the solution
was allowed to warm to room temperature overnight. The reaction
was quenched with satd NaHCO3 (10 mL). After separation of the
layers the aqueous phase was extracted twice with CH2Cl2
(15 mL). The combined extracts were dried over Na2SO4, filtered,
and concentrated. The crude residue was purified by silica gel flash
column chromatography using hexanes–EtOAc as eluent (5:1, v/v)
to yield 10 (434 mg, 34%) as a white solid. Rf 0.75 (4:1 hexanes:
EtOAc, v/v); mp 94–95 °C; 1H NMR (CDCl3, 400 MHz): d 2.04 (s,
3H, CH3CO), 2.04 (s, 3H, CH3CO), 2.06 (s, 3H, CH3CO), 2.42 (tt, 2H,
3.4.4. Dodecyl b-D-xylopyranoside (9d)
Prepared from 8d (2.64 g, 4.19 mmol) to afford 9d (1.11 g, 84%).
The crude product was purified by recrystallization from acetone
to yield a white flaky solid (0.61 g, 47%; 61% from 6 without puri-
fication of intermediates): Rf 0.47 (CH2Cl2–MeOH, 6:1, v/v); mp
96–97 °C, lit.37 101.5–102 °C (methanol); 1H NMR (MeOH-d4,
400 MHz):
d 0.84 (t, 3H, J = 7.0, CH3), 1.23–1.32 (m, 18 H,
9 ꢀ CH2), 1.47–1.63 (m, 2H, H-20), 3.07–3.16 (m, 2H, H-2 and H-
5a), 3.19–3.30 (m, 1H, H-3), 3.37–3.49 (m, 2H, H-10a and H-4),
3.71–3.81 (m, 2H, H-10b and H-5e), 4.12 (d, 1H, J1,2 = 7.2, H-1);
13C NMR (MeOH-d4, 100 MHz) d 14.6, 23. 9, 27.2, 30.6, 30.7,
30.8–31.0, 33.2, 67.1, 71.0, 71.4, 75.1, 78.0, 105.2; HRESIMS m/z:
calcd for C17H38NO5 [M+NH4]+: 336.2745; found: 336.2743.
J2 ,3 = 18.8 Hz, J1 ,2 = 6.5 Hz, H-20), 3.40 (dd, 1H, J5a,5b = 11.8,
0
0
0
0
0
0
0
0
J4,5a = 8.6 Hz, 5a), 3.79 (dt, 1H, J1 a,1 b = 10.4 Hz, J1 a,2 = 6.7 Hz, H-
10a), 4.07–4.16 (m, 2H, H-10b and H-5e), 4.52 (d, 1H, J1,2 = 6.6 Hz,
H-1), 4.90–4.97 (m, 2H, H2 and H4), 5.16 (pseudo t, 1H,
J2,3 = J3,4 = 8.3 Hz, H-3); 13C NMR (CDCl3, 100 MHz,): d 20.5, 20.6,
20.7, 31.4, 61.3, 62.0, 68.7, 70.3, 71.0, 100.6, 169.4, 169.8, 170.0;
19F NMR (282 MHz, CDCl3): d ꢂ81.3 (CF3), ꢂ113.9 (CF2), ꢂ122.4
(CF2), ꢂ123.3 (CF2), ꢂ124.1 (CF2), ꢂ126.6 (CF2); HRESIMS m/z:
calcd for C19H19O8F13Na [M+Na]+: 645.0765; found: 645.0781;
Anal. Calc for C19H19O8F13: C 37.67; H 3.08; found: C 36.75; H 2.96.
3.4.5. Tetradecyl b-D-xylopyranoside (9e)
Prepared from 8e (3.30 g, 5.01 mmol) to afford 9e (1.58 g, 91%).
The crude product was purified by recrystallization from ethanol to
yield a flaky, white solid (0.77 g, 45%; 72% from 6 without purifica-
tion of intermediates): Rf 0.50 (CH2Cl2–MeOH, 9:1, v/v); mp 100–
101 °C, lit.37 103–104 °C (ethanol); 1H NMR (MeOH-d4, 400 MHz):
d 0.85 (t, 3H, J = 6.7, CH3), 1.23–1.31 (m, 22 H, 11 ꢀ CH2), 1.51–
1.59 (m, 2H, H-20), 3.07–3.15 (m, 2H, H-2 and H-5a), 3.21–3.26
(m, 1H, H-3), 3.39–3.49 (m, 2H, H-10a and H-4), 3.72–3.81 (m,
2H, H-10b and H-5e), 4.12 (d, 1H, J1,2 = 7.2, H-1); 13C NMR
(MeOH-d4, 100 MHz) d 14.6, 23.9, 27.2, 30.6, 30.7, 30.8–31.0,
33.2, 67.1, 71.0, 71.4, 75.1, 78.0, 105.3; HRESIMS m/z: calcd for
3.5. Differential scanning calorimetry
Alkyl b-D-xylopyranosides 9a–g were stored under vacuum to
avoid moisture adsorption. Before starting the DSC experiments
the TA Instruments Q200 instrument (TA Instruments, New Castle,
DE) was calibrated with indium for temperature and enthalpy
accuracy. Samples were prepared by weighing 1.0–1.5 mg of the
C
19H38O5Na [M+Na]+: 369.2611; found: 369.2606.

22
W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
dry surfactant into pre-weighed Tzero aluminum pans. Tzero her-
metic lids were then pressed onto the pan. Empty Tzero pans with
Tzero hermetic lid were used as the reference sample. The thermal
analysis was performed between 20 °C and 250 °C with a ramp rate
of 2.5 °C/min. The phase transitions were analyzed using Universal
Analysis software (TA Instruments).
adjustments were implemented to achieve significant absorbance
changes. For all three cell lines the total period of incubation was
20 h. The colored formazan product was measured by absorbance
at 490 nm with a reference wavelength of 650 nm using a micro-
plate reader (VERSAmax tunable microplate reader, MDS, Inc., Tor-
onto, Canada). Control wells containing the same volumes of
culture medium and MTS reagent were used to subtract back-
3.6. Thermogravimetric analysis
ground absorbance. In addition, 300 lM hydrogen peroxide
(H2O2; Acros Organics, NJ) was used as positive control. DMSO
treated cells as solvent control and untreated cells were included
in each plate. Data were expressed as percentage of cell viability
relative to DMSO treated cells. A series of quadruplicates were pro-
cessed for experimental concentrations that were used to obtain
the mean and standard deviation (SD) values.
A Mettler Toledo TGA/SDTA851e instrument (Mettler-Toledo
Inc., Columbus, OH) was used to perform thermogravimetric anal-
ysis (TGA) on the alkyl b-
calibrated with indium and aluminum before the experiments
were started. Between 2.5 mg to 4.5 mg of each alkyl b- -xylopy-
D-xylopyranosides. The instrument was
D
ranoside were added to the alumina crucible. TGA analysis was
performed by heating the sample from 25 °C to 200 °C at a ramp
rate of 10 °C/min under flowing nitrogen at 2 mL/min. Data were
analyzed by STARe software (Mettler-Toledo Inc.).
3.7.4. Generation of dose–response curves and determination of
CC50
Data derived from the MTS assay were used to create dose–
response curves and determine the 50% cytotoxic concentration
(CC50). CC50 was defined as a concentration of compound causing
loss of cellular viability of 50% of the cell population as com-
pared to solvent treated cells after 20 h of incubation. The CC50
values were obtained as previously described.41 The two cellular
viability percentages closest to the 50% value were plotted with
its associated chemical compound concentration and the equa-
3.7. Cell culture experiments
3.7.1. Stock solutions
One-hundred millimolar stock solutions were prepared from all
compounds and the positive control, octylthioglucoside (OTG;
Thermo Scientific, Rockford, IL), which were readily soluble in di-
methyl sulfoxide (DMSO; Sigma–Aldrich, St. Louis, MO) at that
concentration. To verify the solubility of the compounds at exper-
tion of the regression line was utilized to determine the CC50
.
imental conditions, 2
lL of each stock were added to 200
l
L of cell
3.7.5. Apoptosis assay
culture medium, vortexed for 5 s, and centrifuged at 1400 rpm for
1 min for visual inspection of eventual precipitation. While 9a, 9b
and OTG remained soluble, 9c–g precipitated out of solution and
were further diluted. Therefore, the following stock solutions were
used for cytotoxicity analyses: 9a (100 mM), 9b (100 mM), 9c
(50 mM), 9d (10 mM), 9e (10 mM), 9f (2.5 mM), 9g (50 mM), and
OTG (100 mM).
A day prior to the assay, the viability of Jurkat cells was in-
creased by using Ficoll-Paque™PLUS density gradient centrifuga-
tion.42 Briefly, after centrifugation at 400g for 30 min at room
temperature, live cells at the interface were collected and
washed with cell culture medium and were then expanded by
starting a new culture. The next day, Jurkat cells were seeded
in a flat bottom 96-well MICROTEST™ plates at a cell density
of 25,000 cells per well in 200
with10% FBS and antibiotics as described above. Alkyl
b- -xylopyranosides 9b, 9c, 9g, and OTG were added to the cells
at their respective CC50 values as well as 2 M Staurosporine
(Sigma–Aldrich) and 0.5% Tween 20 (Acros Organics) as positive
controls for apoptosis and necrosis, respectively. In addition,
DMSO and untreated controls were included. All treatments
including controls were run in series of triplicates. After 20 h
exposure to compounds, cells were processed as previously de-
scribed with minor modifications.41 Briefly, cells were collected
in an ice-cold tube, centrifuged at 1,400 rpm for 5 min at 4 °C
lL RPMI medium supplemented
3.7.2. Cell lines and cell culture
Three cell lines were used for the cytotoxicity assays: spontane-
ously immortalized human keratinocytes (HaCaT);38 normal non-
transformed human foreskin fibroblasts (Hs27); and human acute
leukemia T lymphocytes (Jurkat).39 The culture medium for HaCaT
and Hs27 cells was Dulbecco’s Modified Eagle Medium (DMEM;
HyClone, Logan, UT) with 10% heat-inactivated newborn calf serum
(NCS; HyClone), while that for Jurkat cells was Roswell Park
Memorial Institute medium (RPMI; HyClone) with 10% heat inacti-
vated fetal bovine serum (FBS; HyClone). Both media were supple-
D
l
mented with 100 U/mL penicillin, 100
l
g/mL streptomycin, and
and stained by resuspension in 100
HEPES, pH 7.4, 140 mM NaCl, and 2.55 mM CaCl2) containing
L of 25 g/mL Annexin V-FITC and 5 L of 250 g/mL propidi-
lL binding buffer (0.1 M
0.25 g/mL amphotericin B (Lonza, Walkersville, MD). The incuba-
l
tion conditions of the cells were at 37 °C in humidified 5% carbon
1
l
l
l
l
dioxide (CO2) atmosphere, in a regular water jacketed incubator.
um iodide (PI; Beckman Coulter, Miami, FL). After incubation for
15 min on ice in the dark, cell suspensions were added with
3.7.3. MTS assay
200 lL of ice-cold binding buffer, gently homogenized and
HaCaT and Hs27 cells were seeded in flat bottom MICROTEST™
tissue culture 96-well plates (Becton Dickinson Labware, Franklin
Lakes, NJ) at a density of 5000, while Jurkat cells were seeded at
a density of 25,000 cells per well. Compounds were tested at a con-
immediately analyzed using a Cytomics FC 500 flow cytometer
(Beckman Coulter, Miami, FL). For each sample, 3,000 individual
events were collected and analyzed using CXP software (Beck-
man Coulter, Miami, FL). Prior to data acquisition, the flow
cytometer was set up and calibrated utilizing unstained, single-
(PI or Annexin V-FITC) and double- (PI and Annexin V-FITC)
stained cells.
centration range from 1
lM to 1000 lM. After 18 h of incubation,
20 L of the MTS reagent (CellTiter 96 AQueousOne Solution Cell
l
Proliferation Assay; Promega, Madison, WI) were added to each
well of HaCaT and Hs27 cells, which were subsequently incubated
for an additional 2 h. For Jurkat cells, the MTS reagent was added
after 16 h of incubation with the test compounds and was then
incubated for an additional 4 h in the presence of the MTS reagent.
Jurkat cells were seeded at higher density and were incubated
longer in the presence of the MTS reagent since lymphocytes ap-
pear to produce less formazan than other cell types40 and these
Acknowledgments
This work was supported by the National Science Foundation
(CBET-0967381/0967390), the U.S. Department of Agriculture Bio-
mass Research and Development Initiative (Grant Agreement 68-
3A75-7-608) and the Department of Energy Development and

W. Xu et al. / Carbohydrate Research 349 (2012) 12–23
23
Rowan, A. S.; Nicely, N. I.; Cochrane, N.; Wlassoff, W. A.; Claiborne, A.;
Hamilton, C. J. Org. Biomol. Chem. 2009, 7, 4029–4036.
13. Singh, M. K.; Swain, D.; Guru, R. T. N.; Jayaraman, N. Carbohydr. Res. 2009, 344,
1993–1998.
14. Hoffmann, B.; Milius, W.; Voss, G.; Wunschel, M.; van Smaalen, S.; Diele, S.;
Platz, G. Carbohydr. Res. 2000, 323, 192–201.
15. Brown, C. J.; Cox, G.; Llewellyn, F. J. J. Chem. Soc. A 1966, 922–927.
16. Ishigami, Y.; Goto, M.; Takizawa, Y.; Shinoyama, H. J. Oleo Sci. 2001, 50, 555–
560.
17. Lehmler, H.-J.; Parkin, S.; Brock, C. P. Acta Crystallogr., Sect. B 2004, 60, 325–332.
18. Goodby, J. W.; Goertz, V.; Cowling, S. J.; Mackenzie, G.; Martin, P.; Plusquellec,
D.; Benvegnu, T.; Boullanger, P.; Lafont, D.; Queneau, Y.; Chambert, S.;
Fitremann, J. Chem. Soc. Rev. 2007, 36, 1971–2032.
19. Singh, M. K.; Jayaraman, N. J. Indian Inst. Sci. 2009, 89, 113–135.
20. van Doren, H. A.; van der Heijden, A. M.; de Goede, A. T. J. W.; van Rantwijk, F.;
van Bekkum, H. Liq. Cryst. 2000, 27, 63–68.
21. (a) Goodby, J. W.; Haley, J. A.; Watson, M. J.; Mackenzie, G.; Kelly, S. M.;
Letellier, P.; Gode, P.; Goethals, G.; Ronco, G.; Harmouch, B.; Martin, P.; Villa, P.
Liq. Cryst. 1997, 22, 497–508; (b) Harmouch, B.; Gode, P.; Goethals, G.; Goodby,
J.; Haley, J.; Kelly, S.; Letellier, P.; Mackenzie, G.; Martin, P.; Ronco, G.; Watson,
M.; Villa, P. J. Carbohydr. Chem. 1997, 16, 479–497; (c) Goodby, J. W.; Haley, J.
A.; Watson, M. J.; Mackenzie, G.; Kelly, S. M.; Letellier, P.; Douillet, O.; Gode, P.;
Goethals, G.; Ronco, G.; Villa, P. Liq. Cryst. 1997, 22, 367–378.
22. Noller, C. R.; Rockwell, W. C. J. Am. Chem. Soc. 1938, 60, 2076–2077.
23. Martin, P.; Gode, P.; Villa, P.; Goethals, G. J. Therm. Anal. Calorim. 2001, 63, 339–
344.
Independence, Energy and Environment Cabinet of The Common-
wealth of Kentucky. The cell culture work was supported by Grant
5G12RR008124 to the Border Biomedical Research Center (BBRC),
granted to the University of Texas at El Paso from the National Cen-
ter for Research Resources (NCRR) of the NIH.
Supplementary data
Complete crystallographic data for the structural analysis of
compound 9c have been deposited with the Cambridge Crystallo-
graphic Data Centre, CCDC No. 854184. Copies of this information
may be obtained free of charge from the director, Cambridge Crys-
tallographic Data Centre, 12 Union Road, Cambridge, CB2 1EZ, UK
(fax: +44-1223-336033, e-mail: deposit@ccdc.cam.ac.uk or via:
www.ccdc.cam.ac.uk). Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.carres.2011.11.020.
References
24. Harmouch, B.; Gode, P.; Goethals, G.; Martin, P.; Ronco, G.; Villa, P. Comun. J.
Com. Esp. Deterg. 1997, 27, 379–390.
25. Lehmler, H.-J.; Oyewumi, M. O.; Jay, M.; Bummer, P. M. J. Fluorine Chem. 2001,
107, 141–146.
26. Kissa, E. Fluorinated surfactants and repellents In Surfactant Science Series;
Marcel Dekker: New York, 2001; Vol. 97,
1. (a) Hill, K.; LeHen-Ferrenbach, C. Sugar-based surfactants for consumer
products and technical applications In Surfactant Sci. Ser; Ruiz, C. C., Ed.; CRC
Press: Boca Raton, 2009; Vol. 143, pp 1–20; (b) Savic, S.; Tamburic, S.; Savic, M.
M. Expert Opin. Drug Delivery 2010, 7, 353–369; (c) Rojas, O. J.; Stubenrauch, C.;
Lucia, L. A.; Habibi, Y. Interfacial properties of sugar-based surfactants; AOCS
Press, 2009. pp 449–472.
27. Deyrieux, A. F.; Wilson, V. G. Cytotechnology 2007, 54, 77–83.
28. Balgavy, P.; Devinsky, F. Adv. Colloid Interf. Sci. 1996, 66, 23–63.
29. (a) Shaik, N.; Martinez, A.; Augustin, I.; Giovinazzo, H.; Varela-Ramirez, A.;
Sanau, M.; Aguilera, R. J.; Contel, M. Inorg. Chem. 2009, 48, 1577–1587; (b)
Martinez, A.; Rajapakse, C. S. K.; Sanchez-Delgado, R. A.; Varela-Ramirez, A.;
Lema, C.; Aguilera, R. J. J. Inorg. Biochem. 2010, 104, 967–977.
30. Pavillard, V.; Drbal, A. A. A.; Swaine, D. J.; Phillips, R. M.; Double, J. A.; Nicolaou,
A. Biochem. Pharmacol. 2004, 67, 1587–1599.
2. (a) Söderlind, E.; Wollbratt, M.; von Corswant, C. Int. J. Pharm. 2003, 252, 61–71;
(b) Söderlind, E.; Karlsson, L. Eur. J. Pharm. Biopharm. 2006, 62, 254–259; (c)
Isomaa, B.; Haegerstrand, H.; Paatero, G.; Engblom, A. C. Biochim. Biophys. Acta
1986, 860, 510–524; (d) Isomaa, B.; Haegerstrand, H.; Paatero, G. Biochim.
Biophys. Acta 1987, 899, 93–103; (e) Shinoyama, H.; Gama, Y.; Nakahara, H.;
Ishigami, Y.; Yasui, T. Bull. Chem. Soc. Jpn. 1991, 64, 291–292; (f) Milius, A.;
Greiner, J.; Riess, J. G. New J. Chem. 1991, 15, 337–344; (g) Zarif, L.; Greiner, J.;
Riess, J. G. J. Fluorine Chem. 1989, 44, 73–85; (h) Zarif, L.; Greiner, J.; Pace, S.;
Riess, J. G. J. Med. Chem. 1990, 33, 1262–1269; (i) Kasuya, M. C. Z.; Cusi, R.;
Ishihara, O.; Miyagawa, A.; Hashimoto, K.; Sato, T.; Hatanaka, K. Biochem.
Biophys. Res. Commun. 2004, 316, 599–604; (j) Kasuya, M. C. Z.; Ito, A.; Cusi, R.;
Sato, T.; Hatanaka, K. Chem. Lett. 2005, 34, 856–857; (k) Li, X.; Turánek, J.;
Knötigová, P.; Kudlácková, H.; Mašek, J.; Pennington, D. B.; Rankin, S. E.;
Knutson, B. L.; Lehmler, H.-J. New J. Chem. 2008, 32, 2169–2179.
3. Sun, R.; Lawther, J. M.; Banks, W. B. Ind. Crops Products 1995, 4, 127–145.
4. Damez, C.; Bouquillon, S.; Harakat, D.; Henin, F.; Muzart, J.; Pezron, I.;
Komunjer, L. Carbohydr. Res. 2007, 342, 154–162.
5. Bouxin, F.; Marinkovic, S.; Le, B. J.; Estrine, B. Carbohydr. Res. 2010, 345, 2469–
2473.
6. Matsumura, S.; Sakiyama, K.; Toshima, K. Biotechnol. Lett. 1999, 21, 17–22.
7. Shinoyama, H.; Kamiyama, Y.; Yasui, T. Agric. Biol. Chem. 1988, 52, 2197–2202.
8. Nakamura, T.; Toshima, K.; Matsumura, S. Biotechnol. Lett. 2000, 22, 1183–
1189.
9. (a) de Bruyne, C. K.; Loontiens, F. G. Nature 1966, 209, 396–397; (b) Satge, C.; Le
Bras, J.; Henin, F.; Muzart, J. Tetrahedron 2005, 61, 8405–8409; (c) Petrovic, Z.
D.; Andjelkovic, D.; Spasojevic, A. Indian J. Chem. 2006, 45B, 272–275.
10. Kartha, K. P. R.; Aloui, M.; Field, R. A. Tetrahedron Lett. 1996, 37, 8807–8810.
11. Hanessian, S.; Banoub, J. Carbohydr. Res. 1977, 53, C13–C16.
31. (a) Mukerjee, P. J. Am. Oil Chem. Soc. 1982, 59, 573–578; (b) Shinoda, K.; Hato,
M.; Hayashi, T. J. Phys. Chem. 1972, 76, 909–914.
32. Mollinedo, F.; Gajate, C. Drug Resistance Updates 2006, 9, 51–73.
33. Lehmler, H.-J.; Parkin, S.; Robertson, L. W. Chemosphere 2002, 46, 485–488.
34. (a) Son, J. B.; Hwang, M.-H.; Lee, W.; Lee, D.-H. Org. Lett. 2007, 9, 3897–3900;
(b) Camponovo, J.; Hadad, C.; Ruiz, J.; Cloutet, E.; Gatard, S.; Muzart, J.;
Bouquillon, S.; Astruc, D. J. Org. Chem. 2009, 74, 5071–5074.
35. Mitchell, S. A.; Pratt, M. R.; Hruby, V. J.; Polt, R. J. Org. Chem. 2001, 66, 2327–
2342.
36. Fletcher, H. G., Jr.; Hudson, C. S. J. Am. Chem. Soc. 1947, 69, 921–924.
37. Hori, R. Yakugaku Zasshi 1958, 78, 523–526.
38. Boukamp, P.; Petrussevska, R. T.; Breitkreutz, D.; Hornung, J.; Markham, A.;
Fusenig, N. E. J. Cell Biol. 1988, 106, 761–771.
39. Schneider, U.; Schwenk, H. U.; Bornkamm, G. Int. J. Cancer 1977, 19, 621–626.
40. Chen, C. H.; Campbell, P. A.; Newman, L. S. Int. Arch. Allergy Appl. Immunol. 1990,
93, 249–255.
41. Varela-Ramirez, A.; Costanzo, M.; Carrasco, Y. P.; Pannell, K. H.; Aguilera, R. J.
Cell Biol. Toxicol. 2011, 27, 159–168.
42. Boyum, A. Scand. J. Clin. Lab. Invest., Suppl. 1968, 21, 77–89.
43. Kasuya, M. C.; Ikeda, M.; Hashimoto, K.; Sato, T.; Hatanaka, K. J. Carbohydr.
Chem. 2005, 24, 705–715.
12. (a) Li, X.; Turanek, J.; Knoetigova, P.; Kudlackova, H.; Masek, J.; Parkin, S.;
Rankin, S. E.; Knutson, B. L.; Lehmler, H.-J. Colloids Surf., B 2009, 73, 65–74; (b)