Synthesis, surface properties, and biocompatibility of 1,2,3-triazole- containing alkyl β-D-xylopyranoside surfactants

Article abstract of DOI:10.1016/j.carres.2013.06.020

We are interested in the development of surfactants derived from hemicellulosic biomass, as they are potential components in pharmaceuticals, personal care products, and other detergents. Such surfactants should exhibit low toxicity in mammalian cells. In this study we synthesized a series of alkyl or fluoroalkyl β-xylopyranosides from azides and an alkyne using the copper-catalyzed azide-alkyne (CuAAC) 'click' reaction in 4 steps from xylose. The purified products were evaluated for both their surfactant properties, and for their biocompatibility. Unlike other carbohydrate-based surfactants, liquid-crystalline behavior was not observed by differential scanning calorimetry. The triazole-containing β-xylopyranosides with short (6 carbons) and long (>12 carbons) chains exhibited no toxicity at concentrations ranging from 1 to 1000 μM. Triazole-containing β-xylopyranosides with 8, 10, or 12 carbons caused toxicity via apoptosis, with CC₅₀ values ranging from 26-890 μM. The two longest chain compounds did form stable monolayers at the air-water interface over a range of temperatures, although a brief transition to an the unstable monolayer was observed.

Full text of DOI:10.1016/j.carres.2013.06.020

Carbohydrate Research 379 (2013) 68–77
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis, surface properties, and biocompatibility
of 1,2,3-triazole-containing alkyl b-D-xylopyranoside
surfactants
E. Davis Oldham a, Srivenu Seelam b, Carolina Lema c, Renato J. Aguilera c, Jennifer Fiegel d,e
,
Stephen E. Rankin b, Barbara L. Knutson b, Hans-Joachim Lehmler a,f,
⇑
a Department of Occupational and Environmental Health, The University of Iowa, UI Research Park, IREH, Iowa City, IA 52242-5000, USA
b Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506, USA
c Department of Biological Sciences, Biosciences Research Building, University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, USA
d Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA 52242,USA
e Department of Chemical and Biochemical Engineering, College of Engineering, University of Iowa, Iowa City, IA 52242, USA
f Interdisciplinary Graduate Program in Human Toxicology, University of Iowa, Iowa City, IA 52242, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We are interested in the development of surfactants derived from hemicellulosic biomass, as they are
potential components in pharmaceuticals, personal care products, and other detergents. Such surfactants
should exhibit low toxicity in mammalian cells. In this study we synthesized a series of alkyl or fluoro-
alkyl b-xylopyranosides from azides and an alkyne using the copper-catalyzed azide-alkyne (CuAAC)
‘click’ reaction in 4 steps from xylose. The purified products were evaluated for both their surfactant
properties, and for their biocompatibility. Unlike other carbohydrate-based surfactants, liquid–crystalline
behavior was not observed by differential scanning calorimetry. The triazole-containing b-xylopyrano-
sides with short (6 carbons) and long (>12 carbons) chains exhibited no toxicity at concentrations ranging
Received 26 March 2013
Received in revised form 17 June 2013
Accepted 20 June 2013
Available online 28 June 2013
Keywords:
Click chemistry
Huisgen cycloaddition
Carbohydrate surfactants
Langmuir monolayers
Cytotoxicity
from 1 to 1000
apoptosis, with CC50 values ranging from 26–890
l
M. Triazole-containing b-xylopyranosides with 8, 10, or 12 carbons caused toxicity via
M. The two longest chain compounds did form stable
l
monolayers at the air–water interface over a range of temperatures, although a brief transition to an the
unstable monolayer was observed.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The synthesis and physiochemical characterization of xylose-
based surfactants have been reported in the literature.5–7 These
The broad range of commercial applications of surfactants has
increased the need for ones that are biologically safe.1 As the
demand continues to grow, the importance of surfactants derived
from renewable resources has risen. Carbohydrate-based surfac-
tants often satisfy requirements of low toxicity and sustainable
production, and have been used as pharmaceuticals, detergents,
agrochemicals, and personal care products.1 The most readily
available carbohydrates are glucose and xylose, which, together
with lignin, are the components of lignocellulose. Recent advance-
ments in the field have allowed for the use of materials derived
from cellulose (glucose) and hemicellulose (glucose and xylose).2
The carbohydrate precursors are readily produced using environ-
mentally friendly processes.3,4 Additionally, carbohydrate-based
surfactants often have low toxicities and can be biodegradable.
surfactants were typically synthesized by enzymatic or Fischer
glycosylation reactions, or by hydrolysis of oligosaccharides. The
products, which were unspecified mixtures of anomers, exhibited
typical chemical properties of surfactants. Previously we have
synthesized b-alkyl xylopyranosides as novel surfactants which
exhibited liquid–crystalline behavior and low in vitro toxicity.8
To further expand the available structures of carbohydrate-based
surfactants, we are interested in designing surfactants with linkers
connecting the carbohydrate head group to the hydrophobic tail,
allowing the rapid synthesis of diverse surfactants with different
hydrophobic tails. Here we chose the 1,2,3-triazole, a heterocyclic
moiety readily synthesized by a copper-catalyzed ‘click’ reaction
between an azide and an alkyne, as a versatile linker. This hetero-
cycle has found widespread usage9 in a broad range of disciplines
such as medicinal chemistry and materials chemistry. The 1,2,3-
triazole moiety has been found in bolaamphiphile,10 star-like,11
and fluorocarbon surfactants.12,13 While both xylose head groups
and 1,2,3-triazoles have been used in surfactants, the two have
⇑
Corresponding author. Tel.: +1 (319) 335 4310; fax: +1 (319) 335 4290.
E-mail address: hans-joachim-lehmler@uiowa.edu (H.-J. Lehmler).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.06.020

E. D. Oldham et al. / Carbohydrate Research 379 (2013) 68–77
69
not been used in combination. Triazole-linked glucose-based sur-
factants have been synthesized previously either by the copper-
catalyzed azide alkyne cycloaddition (CuAAC) reaction of a glycosyl
azide and a propargylated fatty alcohol or a propargylated sugar
and an alkyl azide.14–17 In this paper we report the synthesis and
characterization of physiochemical properties of xylose-based car-
bohydrates containing a triazole linker as well as a preliminary
biocompatibility assessment.
2. Results and discussion
2.1. Synthesis of triazole-linked alkyl xylosides
We sought an efficient route to xylose surfactants containing
hydrophobic chains of variable length linked to the polar head
group with a 1,2,3-triazole generated by a ‘click’ reaction. This cop-
per-catalyzed reaction of azides and alkynes proceeds under mild
conditions with excellent regiochemistry; many functional and
protection groups are tolerated.18 Furthermore, this ‘click’ reaction
allows for short, straight-forward syntheses, as few functional
group interconversions and protecting group manipulations are
required. In the context of surfactants, different combinations of
polar head groups and hydrophobic tails can be readily synthesized
from common precursors.
Figure 1. Representative thermograms of 5a–g obtained by differential scanning
calorimetry. Positive peaks indicate endothermic transitions. Only the region of the
thermogram with a phase transition is shown. See under Experimental procedures
for DSC parameters. The thermograms were plotted with relative heat flow on the
y-axis in arbitrary units [a.u.].
The final products were purified by recrystallization in 50–73%
yield and provided satisfactory elemental analysis. In general, the
yield of the final deprotection step increased with increasing chain
length.
Triazole-containing surfactants were synthesized in four steps
from D-xylose (1) as described in Scheme 1; this scheme has been
previously used to synthesize glucose-based triazole-containing
surfactants.15 This strategy was chosen to yield anomerically pure
surfactants, as previous work has suggested the b-alkyl anomers
2.2. Differential scanning calorimetry (DSC) of 5a–g
may be more biocompatible.19,20 Briefly,
D-xylose was peracetylat-
The phase transitions seen in DSC thermograms give information
about thermal stability of compounds. Furthermore, DSC allows a
preliminary assessment of liquid–crystalline properties, a common
characteristic of surfactants, including synthetic carbohydrate-
based surfactants,25–29 and biological surfactants such as cell mem-
branes.30 The liquid–crystalline behavior of these surfactants may
correlate with their biological activity.31 The transitions of the tria-
zole-linked surfactants 5a–g were therefore characterized by differ-
ential scanning calorimetry (DSC). For each triazole-containing
surfactant a single, endothermic peak was observed, as shown in
Figure 1. The phase transitions ranged from 122 to 136 °C and, anal-
ogous to the structurally related triazole-containing alkyl b-gluco-
ed with sodium acetate in acetic anhydride at 100 °C,21,22 produc-
ing 2 as the b-anomer. Glycosylation with propargyl alcohol under
Lewis-acid-promoted conditions afforded 323 as the b-anomer. The
CuAAC reaction between 3 and alkyl azides (prepared from the
corresponding alkyl bromides or iodides)24 was carried out using
0.1 equiv CuSO4 and 0.2 equiv sodium ascorbate in aqueous
tert-butanol18 to generate 4a–g. The coupling reaction was typi-
cally complete in 1 h, with yields ranging from 64% to 80%. In the
last step the acetate protecting groups were removed with sodium
methoxide, followed by neutralization with Dowex 50 W ꢀ 8–100
ion exchange resin8 to yield the triazole-linked surfactants 5a–g.
Scheme 1. Synthesis of triazole-linked alkyl xylopyranosides via a copper-catalyzed azide alkyne cycloaddition (CuAAC) reaction.

70
E. D. Oldham et al. / Carbohydrate Research 379 (2013) 68–77
triazole linkage15 appeared to have a comparable effect on the
melting points in the case of the structurally related alkyl
b-glucopyranosides.32
Table 1
Average transition temperature and enthalpy values for 5a–g, determined by
differential scanning calorimetry
Compound
5a (C6H13
5b (C8H17
5c (C10H21
5d (C12H25
5e (C14H29
5f (C16H33
5g (C2H4C6F13
Transition temperature (°C)
Enthalpy (kJ/mol)
The simple alkyl xylopyranosides with a glycosidic linkage also
exhibited liquid–crystalline behavior in DSC thermograms, as
evidenced by the presence of a second, smaller peak corresponding
to the clearing point in the thermograms.8 This type of peak has
been observed for alkyl glucopyranosides,33 amide-linked glucopyr-
anosides,25 and xylitols.26,34 The previously reported triazole-con-
taining alkyl b-glucopyranosides also exhibited liquid–crystalline
properties.15 However, no second peak indicative of liquid–crystal-
line behavior was observed in the thermograms of any triazole-con-
taining alkyl b-xylopyranosides 5a–e and g; a small peak possibly
representing a clearing point was observed with 5f. Further work,
involving microscopy techniques such as cryo-transmission elec-
tron microscopy (cryo-TEM) or polarized optical microscopy, is
therefore needed to determine the nature of the thermal transitions
of triazole-containing alkyl b-xylopyranosides.
)
)
)
120.1 0.8
126.9 0.8
133.6 0.1
130.1 0.2
134.7 0.4
134.9 0.1
158.8 0.2
27
31
39
34
41
44
45
4
5
2
2
1
2
1
)
)
)
)
2.3. In vitro cytotoxicity of triazole-containing alkyl xyloside
surfactants
Very little is currently known about the toxicity of alkyl glyco-
side surfactants. The toxicities of alkyl b-D-xylopyranosides,
including those containing partially fluorinated hydrophobic tails,
have been investigated in mammalian cell lines.8,19,20,35,36 In the
present study the toxicities of the triazole-linked surfactants
5a–g were investigated in cells in culture using the 3-(4,5-dimeth-
ylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS) assay used previously.8 Spontaneously
immortalized human keratinocytes (HaCaT), Jurkat human T-cell
lymphoblastic leukemia cells, and human Hs27 fibroblasts were
employed as cell lines in this assay. HaCaT cells model potential ef-
fects of toxicants on skin cells;37 this is particularly relevant for
compounds such as surfactants which have potential applications
in personal care products. Jurkat cells have typically been used
for determining the toxicity of a wide range of compounds with
good sensitivity.38,39 Hs27 cells are often used as a control in com-
parison to other cancer cell lines.40 While the triazole unit has been
increasingly used in biological applications,41 it is still important to
determine the effect on biocompatibility that the incorporation of
this linker into a carbohydrate surfactant would have.
Figure 2. Relative transition temperature (Tm/TC6) of triazole-linked surfactants
5a–f (blue diamonds) and the corresponding b-alkyl xylopyranosides8 without the
triazole linker (melting temperature: red squares; clearing point: green triangles)
observed in the corresponding differential scanning calorimetry thermograms. TC6
is the respective transition temperature of the corresponding hexyl derivative. (For
interpretation of the references to color in this figure legend, the reader is referred
to the web version of this book.)
pyranosides,15 increased with increasing chain length (Table 1). The
partially fluorinated surfactant 5g melted at 158 °C, which is consid-
erably higher compared to the corresponding hydrocarbon surfac-
tant 5b. A similar increase in the phase transition temperature of
fluorinated surfactants relative to the corresponding hydrocarbon
surfactant has been previously observed for alkyl glucopyrano-
sides19 and xylopyranosides.8 The phase transition temperature of
the hydrocarbon surfactants began to level off at chain lengths
P10 carbon atoms (Fig. 2). Similarly, the increase in the phase tran-
sition temperature of the structurally analogous triazole-containing
alkyl b-glucopyranosides was less pronounced for surfactants with
tetradecyl and hexadecyl tails.15 The enthalpies associated with
the phase transition were calculated from integration of the thermo-
grams and ranged from 26.6 kJ/mol to 45.1 kJ/mol; these values fol-
lowed the same trend as the transition temperature.
In contrast to triazole-linked surfactants 5a–g, the phase transi-
tions of simple alkyl b-xylopyranosides8, which contain no triazole
linker but otherwise are structurally analogous to 5a–g, were dras-
tically lower (ranging from 68 to 91 °C) and showed a stronger
dependence of melting temperature on tail chain length (Fig. 2).
The analog of the partially fluorinated surfactant 5g also had a
significantly lower melting point of 105 °C. The markedly higher
phase transition temperatures of the triazole-linked surfactants
5a–g, relative to simple alkyl xylopyranosides, indicate that the tri-
azole linker stabilizes the solid-state packing. The addition of the
The chain-length dependence of the toxicities of 5a–g was
determined in vitro. Compounds 5a (hexyl chain), 5e (tetradecyl
chain), 5f (hexadecyl chain), and 5g (partially fluorinated side
chain) were not toxic in all three cell lines at concentrations rang-
ing from 1 to 1000 lM. The compounds with intermediate chain
lengths (5b–d), on the other hand, showed moderate toxicity in
all three cell lines as assessed using the MTS assay (Fig. 3). CC50 val-
ues, corresponding to the concentration at which 50% of the cells
were viable,42 were calculated as described in the Experimental
procedures section, using the data shown in Figure 3, and are sum-
marized in Table 2. In both Jurkat and HaCaT cells, the toxicity in-
creased as the tail length increased from 5b to 5d. Surfactant 5c
was approximately 8-fold more toxic than 5b in both of these cell
lines. Overall, the Jurkat cell line was the most sensitive; 5b–d
exhibited similarly low toxicities in the two non-leukemia cell
lines (HaCaT and Hs27).
Interestingly, surfactants 5b to 5d had toxicities comparable to
alkyl xylopyranosides without the triazole linker.8 For example,
the CC50 values of 5b (containing an octyl side chain) were 890
(HaCaT), 694 M (Hs27), and 580 M (Jurkat); the corresponding
CC50 values for ocyl b-xylopyranoside in the previous study were
850, 760, and 460 M, respectively (Table 2). As in the present study,
lM
l
l
l
the intermediate-length alkyl xylopyranosides were also the most
toxic. The phenomenon in which surfactants of intermediate lengths

E. D. Oldham et al. / Carbohydrate Research 379 (2013) 68–77
71
carbohydrate surfactants in mammalian cell lines8,19,20 and bacte-
ria.44,45 The lack of a linear dependence between chain length and
biological activity is at first surprising, if the activity is assumed to
be a function of membrane permeability: as the length of the hydro-
carbon chain of a surfactant increases, the octanol–water partition
coefficient also increases, which should increase membrane parti-
tioning and, ultimately, activity. Thus, the basis of the cut-off effect
likely is the result of factors other than membrane partitioning, for
example decreasing aqueous and lipid solubility, decreasing critical
micelle concentration, increasing binding to proteins in the culture
medium or decreasing diffusion through the cell membrane with
increasing chain length.43,46
Fluorinated surfactants have been shown to be less toxic than
the corresponding hydrocarbon surfactants.47,36,48 In agreement
with these earlier studies, the partially fluorinated hydrophobic tail
in surfactant 5g appears to have a protective effect, as 5g showed no
measurable inhibition of cell viability in any cell line in the concen-
tration range studied, which is in contrast to the corresponding
hydrocarbon surfactant 5b in HaCaT or Jurkat cell lines. In some
cases, the toxicity of fluorinated surfactants appears to follow a
‘1.5 rule’,49 which states that each CF2 approximates 1.5 CH2 groups.
Thus, for 5g, the 1.5 rule would correspond to 2 CH2 groups +1.5 ꢀ 6
CF2 groups = 11 CH2 groups. For example, the CC50 values of the
non-triazole-linked fluoroalkyl xylopyranoside (163–267
lM) and
non-triazole-linked decyl xylopyranoside (123–167 M) were
l
comparable.8 In this study, triazole-linked surfactant 5g does not
obey this rule, as the toxicities of 5g and the structurally analogous
surfactant 5c are not comparable. It is possible that the triazole
should be considered part of the side-chain; further separation of
the rigid, fluorocarbon chain from the headgroup due to the pres-
ence of the triazole likely alters the three-dimensional structure
of the surfactant, which would in turn affect interaction with possi-
ble biological targets.
To further investigate the mechanisms of toxicity of triazole-
linked surfactants, the levels of apoptotic and necrotic cells were
determined with annexin V-FITC staining after exposure of Jurkat
cells to the triazole-linked xylosides 5b–d. The relative amounts
of viable, necrotic, and apoptotic cells are shown in Figure 4;
hydrogen peroxide and Tween-20 were used as positive controls
for apoptosis and necrosis, respectively; DMSO was the solvent
control. Notably, cell death occurred from apoptosis and not necro-
sis. Triazole 5b exhibited a profile comparable with the positive
controls for apoptosis, octyl xylopyranoside, and hydrogen perox-
ide.50 As the chain increased from 8 carbons (5b) to 12 carbons
(5d), the number of apoptotic cells increased, and the number of
viable cells decreased; there was no chain length dependence on
the number of necrotic cells. A similar chain-length dependent ef-
fect on apoptosis was also seen with the xylopyranosides lacking a
triazole linker.8 Thus the mechanism of cell death likely involves
complex signaling pathways; for example, the transmembrane
protein Fas51 has been implicated in the toxicity of perfluoralkyl
acids (PFAAs). Other possible targets include the caspase-mediated
apoptosis pathway. For example, an ionic liquid, containing an
imidazolium headgroup and an octyl side chain, increased reactive
oxygen species and Caspase-3 activities.52
Figure 3. Concentration dependence of cytotoxicity of selected triazoles in HaCaT,
Hs27, and Jurkat cells. After 18 h (HaCaT and Hs27 cells) or 16 h (Jurkat cells) of
treatment with (A) 5b, (B) 5c, or (C) 5d, MTS was added and incubation continued
for 2 h (HaCaT and Hs27 cells) and 4 h (Jurkat cells) before measuring absorbance at
490 nm. Error bars represent the standard deviation of the mean from three
replicates. The chain length of the hydrophobic tail is indicated on each panel.
exhibit higher toxicity than those with shorter or longer chains, is
known as the ‘cut-off’ effect;43 this effect has been seen with
Table 2
CC50 values (
l
M) of 5a–f obtained from the curves in Figure 5
Triazole-containing alkyl xyloside surfactants
Cell line
Control surfactants
5a
5b
5c
5d
5e
5f
5g
b-OXP
b-OTG
a
a
a
a
a
a
a
a
a
a
a
a
a
a
HaCaT
Hs27
Jurkat
890 50
120 30
180 50
850 30
760 50
460 13
490 60
380 30
310 40
694
3
580 60
77
9
26
3
a
Cytotoxicity was <50% at the highest compound concentration tested and therefore their CC50 values could not be determined. b-OXP: b-octyl xylopyranoside; b-OTG: b-
octyl thioglucopyranoside.

72
E. D. Oldham et al. / Carbohydrate Research 379 (2013) 68–77
Figure 4. Percentage of viable, apoptotic, and necrotic Jurkat cells after 20 h of
incubation, as determined by annexin V-FITC staining and flow cytometry. b-OGT
(octyl b-thioglucopyranoside) and b-OXP (octyl b-xylopyranoside) were used for
comparison. H2O2 (300 lM) was used as a positive control for apoptosis, and Tween
20 (0.5%) was used as a control for necrosis. Compounds were added at their CC50
value in DMSO. Error bars represent the standard deviation of the mean from three
replicates. The chain length of the hydrophobic tail is indicated in parentheses.
2.4. Langmuir monolayer studies
The interfacial properties of surfactants are important for
applications in pharmaceuticals, detergents, agrochemicals, and
personal care products.1 We characterized the ability of selected
newly synthesized triazole-linked alkyl xylopyranosides to form
monolayers at the air–water interface. Surface pressure isotherms
were determined using a Langmuir balance at varying tempera-
tures on a 150 mM NaCl/1.5 mM CaCl2 subphase used previously
to study monolayers under physiological conditions.53,54 At 14 °C,
only triazoles 5e and 5f formed stable monolayers. The compres-
sion isotherms of both compounds showed
a single phase
transition from an expanded to a condensed phase (Fig. 5). The
phase transition displayed an ‘overshoot’ in surface pressure; the
magnitude of the ‘overshoot’ does not appear to be temperature
dependent. The ‘overshoot’ was greater for 5e than 5f, and likely
indicates the initial formation of an unstable expanded monolayer,
followed by slow reorganization to a stable monolayer in the
condensed phase (Table 2). This phenomenon has been reported
for different types of monolayers.55–57 In all cases the kinetics of
monolayer formation appear to be the driving force behind the
unique shape of the compression isotherm. Further experiments
are necessary to determine the nature of this phase transition of
5e and 5f.
Figure 5. Representative compression isotherms (from three independent exper-
iments) of (A) 5e (tetradecyl chain) and (B) 5f (hexadecyl side chain). The isotherms
were obtained by symmetrical compression at 10 mm/min (7.5 cm2/min) at the
indicated temperature on a 150 mM NaCl/1.5 mM CaCl2 subphase (pH = 7.0).
(ptrans) versus temperature (Fig. 6B) are extrapolated to zero sur-
face pressure, the ‘lift-off’ temperature can be calculated;61 this
temperature corresponds to the point below which no phase tran-
sition occurs. For 5e and 5f, the values were 9.2 and 20.2 °C,
respectively. Linear alkanoic acids follow the same trend: the
‘lift-off’ temperatures were approximately 7 °C for tetradecanoic
acid (the saturated acid analog of 5e) and 26 °C for hexadecanoic
acid (the analog of 5f).61
The compression isotherms for 5e and 5f were recorded at dif-
ferent temperatures to verify that the breakpoints shown in Fig-
ure 5 are indeed attributable to a phase transition from an
expanded to a condensed phase. For 5e (containing a 14-carbon
chain) the surface pressure at the phase transition, ptrans, increased
from 5.6 mN/m at 14 °C to 26 mN/m at 30 °C (Fig. 4a and Table 3).
The corresponding molecular area, Atrans, decreased from 68 Å2/
molecule to 38 Å2/molecule in this temperature range. The same
The limiting molecular areas, Alim, of 5e and 5f were 0also depen-
dent upon temperature. Alim for 5e increased from 31 ÅA2/molecule
0
at 14 °C to 42 AÅ2/molecule at 30 °C, while Alim for 5f increased from
33 Å2/molecule at 22 °C to 41 Å2/molecule at 37 °C. At 26 °C, Alim for
5e and 5f were 33 and 37 Å2/molecule, respectively. Alim did not
show a strong temperature dependence, indicating that Alim is pri-
marily a function of the carbohydrate head group and/or the tria-
zole linker. Alim of saturated fatty acids are approximately 20 Å2,62
which reflects the cross-sectional area of hydrocarbon chains. For
Gibbs monolayers of selected alkyl glucosides at the air–water
interface, the limiting molecular area at 25 °C has been measured
using the de Nouy method, with values of 42–44 Å2/molecule for
n-octyl, -decyl, and -dodecyl glucopyranosides.63 At 25 °C, the
trends were observed for 5f; the transition surface pressure, ptrans
,
increased from 1.6 to 18 mN/m, the transition area, Atrans, de-
creased from 83 to 51 Å2/molecule, as the subphase temperature
was increased from 17 to 37 °C. The changes in ptrans and Atrans
as a function of temperature are shown in Figure 6. The depen-
dence of phase transitions of insoluble monolayers at the air–water
interface on temperature is well-known; comparable effects on ex-
panded to condensed transitions have been shown for alkyl nicoti-
nates,58 partially fluorinated carboxylic acids,59 and hydroxystearic
acids.60 When the plots of surface pressure at the phase transition

E. D. Oldham et al. / Carbohydrate Research 379 (2013) 68–77
73
Table 3
Temperature-dependence of parameters determined from the isotherms presented in Figure 5 for 5e (tetradecyl chain) and 5f (hexadecyl chain)
Compound
Temperature (°C)
ptrans
Atrans
Alim
31. 2.0
29.6 0.6
29.7 0.7
32.8 1.2
41.6 0.4
pmax
Cmax
5e
14
17
22
26
30
5.6 0.2
11.3 0.2
15.0 0.1
20.9 0.2
27.0 0.5
68.5 0.9
53.6 0.9
47.5 0.2
42.1 0.7
38.0 1.2
64.1 0.5
62.4 1.4
59.4 0.6
53.0 2.0
47.0 3.0
250 20
200 20
233
140
71
1
2
3
5f
22
26
30
33
37
1.6 0.2
6.8 0.1
9.9 0.1
11.6 0.3
18.4 0.4
84.0 2.0
70.0 2.0
65.0 2.0
61.0 2.0
51.2 0.9
32.9 1.1
37.0 2.0
41.0 5.0
41.0 3.0
41.8 0.2
64.0 3.0
58.6 0.8
53.6 1.0
51.6 0.4
48.0 2.0
250 50
239 20
146
5
130 20
102 11
T: temperature of the subphase; Alim: limiting molecular area (Å2/molecule); Atrans: mean molecular area at phase transition (Å2/molecule);
transition (mN/m); max: maximum surface pressure (mN/m); Cmax: maximum compressibility.
ptrans: surface pressure at phase
p
glucopyranoside and 32 Å2 for 6-O-stearoyl galactopyranoside.65
These values are comparable to the limiting molecular areas for
the triazole-linked surfactants 5e and 5f reported here. This obser-
vation suggests that the cross-sectional area of the carbohydrate
moiety may determine the limiting molecular area of 5e and 5f.
The collapse pressure,
pmax, also decreased with increasing
temperature, ranging from 64 to 47 mN/m for both 5e and 5f, as
expected for stable surfactant monolayers. This follows the trend
seen in DPPC monolayers as a function of temperature.66 The col-
lapse pressures for 6-O-stearoyl glucopyranoside and 6-O-stearoyl
galactopyranoside were 43 and 66 mN/m, respectively, at room
temperature,65 which is comparable to the collapse pressures of
5e and 5f. The compressibility modulus was obtained from the
compression isotherms using the equation Cs = ꢁA (d
graphs of compressibility versus surface pressure exhibited a
temperature-dependent peak. The maximum compressibility, Cmax
P/dA); the
,
ranged from 251 to 71 mN/m for 5e and 247 to 102 mN/m for 5f,
and decreased with increasing temperature. This property of sur-
factants has been discussed in the literature, although not for car-
bohydrate-based surfactants;67 the effect is likely due to an
increase in fluidity of the monolayer.
2.5. Conclusions
In this paper we reported the synthesis and characterization of
novel alkyl xylopyranosides, incorporating a 1,2,3-triazole linker.
The compounds were synthesized using ‘click chemistry’ in moder-
ate to excellent yields. Unlike previously reported alkyl xylopyrano-
sides8 and glucose-based triazole-containing surfactants,15 the
triazole-linked surfactants 5a–g did not exhibit liquid–crystalline
properties, as assessed using DSC. Further studies of their liquid–
crystalline properties are therefore warranted. The compounds
did exhibit other properties of surfactants (i.e., they formed stable
monolayers at the air–water interface). The potential biocompati-
bility of the surfactants was tested, as they have potential uses in
various consumer applications. The triazole-linked surfactants with
short or long chains were shown to be non-toxic in several mamma-
lian cell lines. Intermediate length surfactants exhibited low cyto-
toxicity, primarily through apoptosis, with CC values in the high
micromolar range. Overall, triazole-linked surfactants merit further
investigation because they are readily synthesized from renewable
starting materials, and display low toxicity and surface activity.
Figure 6. Temperature dependence of (A) average molecular area, Atrans, and (B)
surface pressure, trans, at the phase transition for 5e (tetradecyl chain) and 5f
(hexadecyl chain). The values presented are the average of at least three
experiments, with standard deviation.
p
3. Experimental procedures
3.1. General methods
limiting molecular areas of n-octyl glucosamine and n-nonyl
glucosamine were 48 and 46 Å2/molecule, respectively.64 These
literature values are consistent with in silico calculations.20 6-O-
Stearoyl hexoses formed insoluble monolayers at the air–water
interface with limiting molecular areas of 44 Å2 for 6-O-stearoyl
The 1H and 13C NMR spectra were recorded on multinuclear
Bruker Avance 300 or Bruker DRX 400 Digital NMR spectrometers.

74
E. D. Oldham et al. / Carbohydrate Research 379 (2013) 68–77
Spectral assignments were determined from COSY experiments.
19F spectra were recorded using a Bruker Avance 300. The mass
spectra were measured at the University of California, Riverside
Mass Spectrometry facility. Elemental analyses were obtained
from Atlantic Micro Lab Microanalysis Service (Atlanta, Georgia,
USA). Melting points were determined using a MelTemp apparatus,
and are uncorrected. All reactions were monitored by thin layer
chromatography, followed by visualization with UV and anisalde-
triazole-N-CH2CH2), 1.17–1.37 (m, 10H, 5 ꢀ CH2), 0.86 (t,
J = 6.7 Hz, 3H RCH3); 13C NMR (100 MHz, CDCl3): d 170.0, 169.8,
169.4, 144.1, 122.3, 99.8, 71.3, 70.7, 68.7, 62.5, 62.0, 50.3, 31.6,
30.2, 29.0, 28.9, 26.4, 22.5, 20.70, 20.66 (2 ꢀ C), 14.0; HRESIMS
calcd for C22H36N3O8 (M+H)+: 470.2497; found: 470.2505; Anal
calcd for C22H35N3O 8: C 56.28; H 7.51; N 8.95; found: C 56.63; H
7.48; N 8.97.
hyde–H2SO4. 1,2,3,4-Tetra-O-acetyl-b-
2,3,4-tri-O-acetyl-b-
-xylopyranoside (3)23 were prepared as de-
scribed previously. Azides were synthesized using known
D
-xylose (2)21 and propargyl
3.2.3. (1-Decyl-1H-1,2,3-triazol-4-yl)methyl 2,3,4-tri-O-acetyl-b-
xylopyranoside (4c)
D
a
Following the general procedure with propargyl xylose
3
method24 and used without further purification. Propargyl alcohol
and DowexÒ 50 W ꢀ 8–100 ion exchange resin were obtained from
Acros. Sodium methoxide was obtained from Alfa Aesar. Sodium
azide and sodium ascorbate were obtained from Sigma–Aldrich.
Cupric sulfate pentahydrate was obtained from Mallinckrodt. All
organic solvents were reagent grade or higher, and used without
further purification. Flash chromatography was performed using
(438 mg, 1.39 mmol) and n-decyl azide (263 mg, 1.43 mmol), then
column chromatography using EtOAc:hexanes (2:1, v/v), 407 mg
(74%) of 4c was obtained as a clear oil which, solidified upon stand-
ing. 1H NMR (400 MHz, CDCl3): d 7.50 (s, 1H, triazole-CH), 5.17
(pseudo t, J ꢂ8.4 Hz, 1H, H-3), 4.87–5.00 (m, 3H, H-10a, H-2, H-4),
4.78 (d, J = 12.6 Hz, 1H, H-10b), 4.64 (d, J = 6.9 Hz, 1H, H-1), 4.34
(t, J = 7.3 Hz, 2H, triazole-NCH2), 4.15 (dd, J = 11.9, 5.1 Hz, 1H, H-
5e), 3.40 (dd, J = 11.9, 8.8 Hz, 1H, H-5a), 2.06 (s, 3H, OCOCH3),
2.03 (s, 3H, OCOCH3), 2.02 (s, 3H, OCOCH3), 1.83–1.96 (m, 2H, tria-
zole-N-CH2CH2), 1.14–1.40 (m, 14H, 7 ꢀ CH2), 0.88 (t, J = 6.7 Hz, 3H
RCH3); 13C NMR (100 MHz, CDCl3): d 170.0, 169.9, 169.5, 144.1,
122.4, 99.8, 71.3, 70.7, 68.8, 62.6, 62.1, 50.4, 31.8, 30.3, 29.4,
29.3, 29.2, 29.0, 26.4, 22.6, 20.75, 20.70 (2 ꢀ C), 14.1; HRESIMS
m/z calcd for C24H40N3O8 (M+H)+: 498.2810; found: 498.2824;
Anal. calcd for C24H39N3O8: C 57.93; H 7.90; N 8.44; found: C
57.92; H 7.86; N 8.45.
60 Å (40–63
vent mixture.
lm, 230 ꢀ 400 mesh) silica gel with the indicated sol-
3.2. General procedure for the CuAAC reaction
Triacetyl propargyl xylose 3 and n-alkyl azide (1.0–1.1 equiv)
were combined with 2:1 tBuOH/water (0.33 M) at room tempera-
ture. Sodium ascorbate (0.2 equiv, 1.0 M in water) was added,
followed by CuSO4 pentahydrate (0.1 equiv, 75 mg/mL in water),
and the mixture was stirred at room temperature for 90 min. At
this time the reactions often became homogeneous and faint blue.
The reaction mixture was diluted with water and extracted three
times with ethyl acetate. The combined extracts were washed with
brine, dried over MgSO4, and concentrated. The crude residue was
purified by silica gel column chromatography (hexanes/EtOAc).
3.2.4. (1-Dodecyl-1H-1,2,3-triazol-4-yl)methyl 2,3,4-tri-O-
acetyl-b-xylopyranoside (4d)
Following the general procedure with propargyl xylose
3
(431 mg, 1.37 mmol) and n-dodecyl azide (297 mg, 1.40 mmol)
then column chromatography using EtOAc:hexanes (3:2, v/v),
463 mg (64%) of 4d was obtained as a waxy white solid. 1H NMR
(400 MHz, CDCl3): d 7.48 (s, 1H, triazole-CH), 5.15 (pseudo t, J
ꢂ8.5 Hz, 1H, H-3), 4.87–4.98 (m, 3H, H-10a, H-2, H-4), 4.77 (d,
J = 12.4 Hz, 1H, H-10b), 4.64 (d, J = 6.8 Hz, 1H, H-1), 4.33 (t,
J = 7.2 Hz, 2H, triazole-NCH2), 4.14 (dd, J = 11.8, 5.2 Hz, 1H, H-5e),
3.40 (dd, J = 11.8, 8.8 Hz, 1H, H-5a), 2.04 (s, 3H, OCOCH3), 2.01 (s,
3H, OCOCH3), 2.00 (s, 3H, OCOCH3), 1.82–1.94 (m, 2H, triazole-N-
CH2CH2), 1.15–1.39 (m, 18H, 9 ꢀ CH2), 0.87 (t, J = 6.6 Hz, 3H
RCH3); 13C NMR (CDCl3,100 MHz): d 170.0, 169.8, 169.4, 144.1,
122.3, 99.8, 71.4, 70.8, 68.8, 62.6, 62.1, 50.4, 31.8, 30.3, 29.54
(2 ꢀ C), 29.46, 29.33, 29.27, 28.9, 26.4, 22.6, 20.68, 20.64 (2 ꢀ C),
14.1; HRESIMS calcd for C26H44N3O8 (M+H)+: 526.3123; found:
526.3134; Anal. calcd for C26H43N3O8: C 59.41; H 8.25; N 7.99;
found: C 59.67; H 8.14; N 7.96.
3.2.1. (1-Hexyl-1H-1,2,3-triazol-4-yl)methyl 2,3,4-tri-O-acetyl-b-
xylopyranoside (4a)
Following the general procedure with propargyl xylose 3
(427 mg, 1.35 mmol) and n-hexyl azide (172 mg, 1.35 mmol), then
column chromatography using EtOAc/hexanes (1:1, v/v), 462 mg
(78%) of 4a was obtained as a clear oil, which solidified upon stand-
ing. 1H NMR (400 MHz, CDCl3): d 7.49 (s, 1H, triazole-CH), 5.16
(pseudo t, J ꢂ8.5 Hz, 1H, H-3), 4.89–4.99 (m, 3 H, H-10a, H-2, H-
4), 4.78 (d, J = 12.5 Hz, 1H, H-10b), 4.64 (d, J = 6.8 Hz, 1H, H-1),
4.34 (t, J = 7.3 Hz, 2H, triazole-N-CH2), 4.14 (dd, J = 11.8, 5.1 Hz,
1H, H-5e), 3.40 (dd, J = 11.8, 8.8 Hz, 1 H, H-5a), 2.05 (s, 3H,
OCOCH3), 2.02 (s, 3 H, OCOCH3), 2.00 (s, 3H, OCOCH3), 1.83–1.95
(m, 2H, triazole-N-CH2CH2), 1.25–1.36 (m, 6H, 3 ꢀ CH2), 0.88 (t,
J = 6.9 Hz, 3H, RCH3); 13C NMR (100 MHz, CDCl3): d 170.0, 169.8,
169.4, 144.2, 122.3, 100.0, 71.4, 70.8, 68.8, 62.6, 62.1, 50.4, 31.1,
30.2, 26.1, 22.3, 20.67, 20.63 (2 ꢀ C), 13.9; HRESIMS calcd for
3.2.5. (1-Tetradecyl-1H-1,2,3-triazol-4-yl)methyl 2,3,4-tri-O-
20H32N3O8 (M+H)+: 442.2184; found: 442.2184; Anal. calcd for
20H31N3O8: C 54.41; H 7.08; N 9.52; found: C 54.49; H 7.09; N
acetyl-b-
D-xylopyranoside (4e)
C
C
Following the general procedure with propargyl xylose
3
(419 mg, 1.33 mmol) and n-tetradecyl azide (355 mg, 1.48 mmol),
then column chromatography using EtOAc:hexanes (2:1, v/v),
537 mg (73%) of 4e was obtained as a waxy white solid. 1H NMR
(400 MHz, CDCl3): d 7.49 (s, 1H, triazole-CH), 5.16 (pseudo t, J
ꢂ8.2 Hz, 1H, H-3), 4.88–5.00 (m, 3H, H-10a, H-2, H-4), 4.78 (d,
J = 12.5 Hz, 1H, H-10b), 4.65 (d, J = 6.9 Hz, 1H, H-1), 4.33 (t,
J = 7.3 Hz, 2H, triazole-NCH2), 4.15 (dd, J = 11.9, 5.1 Hz, 1H, H-5e),
3.40 (dd, J = 11.9, 8.8 Hz, 1H, H-5a), 2.05 (s, 3H, OCOCH3), 2.02 (s,
3H, OCOCH3), 2.01 (s, 3H, OCOCH3), 1.82–1.95 (m, 2H, triazole-N-
CH2CH2), 1.18–1.39 (m, 22H, 11 ꢀ CH2), 0.88 (t, J = 6.6 Hz, 3H
RCH3); 13C NMR (CDCl3,100 MHz): d 170.0, 169.9, 169.4, 144.2,
122.3, 99.9, 71.4, 70.8, 68.9, 62.6, 62.1, 50.4, 31.9, 30.3, 29.65,
29.61 (2 ꢀ C), 29.57, 29.5, 29.4, 29.3, 29.0, 26.5, 22.7, 20.72, 20.68
(2 ꢀ C), 14.1; HRESIMS calcd for C28H48N3O8 (M+H)+: 554.3436;
9.54.
3.2.2. (1-Octyl-1H-1,2,3-triazol-4-yl)methyl 2,3,4-tri-O-acetyl-b-
xylopyranoside (4b)
Following the general procedure with propargyl xylose 3
(462 mg, 1.47 mmol) and n-octyl azide (259 mg, 1.66 mmol), then
column chromatography using EtOAc:hexanes (1:1, v/v), 511 mg
(74%) of 4b was obtained as a clear oil which, solidified upon
standing. 1H NMR (400 MHz, CDCl3): d 7.48 (s, 1H, triazole-CH),
5.15 (pseudo t, J ꢂ8.7 Hz, 1H, H-3), 4.84–5.02 (m, 3H, H-10a, H-2,
H-4), 4.77 (d, J = 12.6 Hz, 1H, H-10b), 4.63 (d, J = 6.8 Hz, 1H, H-1),
4.33 (t, J = 7.3 Hz, 2H, triazole-NCH2), 4.13 (dd, J = 11.9, 5.1 Hz,
1H, H-5e), 3.39 (dd, J = 11.9, 8.8 Hz, 1H, H-5a), 2.04 (s, 3H OCOCH3),
2.01 (s, 3H OCOCH3), 1.99 (s, 3H OCOCH3), 1.82–1.93 (m, 2H,

E. D. Oldham et al. / Carbohydrate Research 379 (2013) 68–77
75
found: 554.3442; Anal. calcd for C28H47N3O8: C 60.74; H 8.56; N
7.59; found C 60.46; H 8.57; N 7.65.
104.4, 77.9, 75.0, 71.3, 67.2, 63.2, 51.5, 32.4, 31.4, 27.3, 23.6,
14.4; HRESIMS calcd for C14H26N3O5 (M+H)+: 316.1867; found:
316.1873; Anal calcd for C14H25N3O5: C 53.32; H 7.99; N 13.32;
found: C 53.08; H 7.97; N 13.17.
3.2.6. (1-Hexadecyl-1H-1,2,3-triazol-4-yl)methyl 2,3,4-tri-O-
acetyl-b-D-xylopyranoside (4f)
Following the general procedure with propargyl xylose 3
(420 mg, 1.34 mmol) and n-hexadecyl azide (369 mg, 1.38 mmol),
then column chromatography using EtOAc:hexanes (2:1, v/v),
621 mg (80%) of 4e was obtained as a waxy white solid. 1H NMR
(CDCl3,400 MHz): d 7.48 (s, 1H, triazole-CH), 5.15 (pseudo t, J
ꢂ8.8 Hz, 1H, H-3), 4.86–4.98 (m, 3H, H-10a, H-2, H-4), 4.76 (d,
J = 11.5 Hz, 1H, H-10b), 4.63 (d, J = 6.8 Hz, 1H, H-1), 4.32
(J = 7.2 Hz, 2H, triazole-NCH2), 4.13 (dd, J = 11.9, 5.1 Hz, 1H, H-
5e), 3.39 (dd, J = 11.9, 8.8 Hz, 1H, H-5a), 2.04 (s, 3H, OCOCH3),
2.01 (s, 3H, OCOCH3), 2.00 (s, 3H, OCOCH3), 1.83–1.94 (m, 2H, tria-
zole-N-CH2CH2), 1.17–1.37 (m, 26H, 13 ꢀ CH2), 0.87 (t, J = 6.6 Hz,
3H RCH3); 13C NMR (CDCl3,100 MHz): d 169.9, 169.8, 169.4,
144.1, 122.3, 99.8, 71.3, 70.7, 68.8, 62.5, 62.1, 50.3, 31.9, 30.2,
29.62, 29.61 (2 ꢀ C), 29.58 (2 ꢀ C), 29.53, 29.46, 29.32, 29.28,
28.9, 26.4, 22.6, 20.66, 20.63 (2 ꢀ C), 14.0; HRESIMS calcd for
3.3.2. (1-Octyl-1H-1,2,3-triazol-4-yl)methyl b-D-xylopyranoside
(5b)
Triazole 4b (441 mg, 0.939 mmol) was deprotected using the
general conditions and purified by recrystallization from ethanol-
hexanes to yield 160 mg (50%) of
(400 MHz, CD3OD): 7.97 (s, 1H, triazole-CH), 4.91 (d,
a
white solid. 1H NMR
d
J = 12.3 Hz, 1H, H-10a), 4.73 (d, J = 12.3 Hz, 1H, H-10b), 4.39 (t,
J = 7.1 Hz, 2H, triazole-NCH2), 4.32 (d, J = 7.5 Hz, 1H, H-1), 3.88
(dd, J = 11.4, 5.3 Hz, 1H, H-5e), 3.49 (m, 1H, H-4), 3.28–3.34 (m,
1H, overlapping with CD3OD, H-5a), 3.16–3.26 (m, 2H, H-2, H-3),
1.79–1.97 (m, 2H, triazole-N-CH2CH2), 1.20–1.39 (m, 10H,
5 ꢀ CH2), 0.89 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD):
d 145.6, 125.1, 104.3, 77.8, 74.9, 71.2, 67.0, 63.0, 51.4, 32.9, 31.3,
30.2, 30.0, 27.5, 23.7, 14.4; HRESIMS calcd for
C16H30N3O5
(M+H)+: 344.2180; found: 344.2179; Anal. calcd for C16H29N3O5:
C 55.96; H 8.51; N 12.24; found: C 55.82; H 8.51; N 12.21.
C
C
30H52N3O8 (M+H)+: 582.3749; found: 582.3761; Anal. Calcd for
30H51N3O8: C 61.94; H 8.84; N 7.22; found: C 61.66; H 8.91; N
6.96.
3.3.3. (1-Decyl-1H-1,2,3-triazol-4-yl)methyl b-D-xylopyranoside
(5c)
3.2.7. (1-(3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl)-1H-1,2,3-
triazol-4-yl)methyl 2,3,4-tri-O-acetyl-b-D-xylopyranoside (4g)
Triazole 4c (400 mg, 0.804 mmol) was deprotected using the
general conditions and purified by recrystallization from acetone
to yield 163 mg (55%) of a white solid. 1H NMR (400 MHz, CD3OD):
d 7.97 (s, 1H, triazole-CH), 4.90 (d, J = 12.3 Hz, 1H, H-10a), 4.73 (d,
J = 12.3 Hz, 1H, H-10b), 4.39 (t, J = 7.1 Hz, 2H, triazole-NCH2), 4.32
(d, J = 7.5 Hz, 1H, H-1), 3.88 (dd, J = 11.4, 5.4 Hz, 1H, H-5e), 3.49
(m, 1H, H-4), 3.27–3.36 (m, 1H, overlapping with CD3OD, H-5a),
3.15–3.27 (m, 2H, H-2, H-3), 1.81–2.01 (m, 2H, triazole-N-CH2CH2),
1.14–1.42 (m, 14H, 7 ꢀ CH2), 0.90 (t, J = 6.7 Hz, 3H, CH3); 13C NMR
(100 MHz, CD3OD): d 145.6, 125.1, 104.3, 77.8, 74.9, 71.2, 67.0,
63.0, 51.4, 33.0, 31.3, 30.6, 30.5, 30.4, 30.1, 27.4, 23.7, 14.4; HRE-
SIMS calcd for C18H34N3O5 (M+H)+: 372.2504; found: 372.2499;
Anal. calcd for C18H33N3O5: C 58.18; H 8.96; N 11.32; found: C
58.17; 8.92; N 11.28.
Following the general procedure with propargyl68 xylose 3
(430 mg, 1.27 mmol) and 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl
azide (550mg, 1.41 mmol), then column chromatography using
EtOAc:hexanes (2:1, v/v), 743 mg (77%) of a waxy white solid
was obtained. 1H NMR (CDCl 3,400 MHz): d (ppm): 7.58 (s, 1H, tri-
azole-CH), 5.17 (pseudo t, J ꢂ8.5 Hz, 1H, H-3), 4.90–5.01 (m, 3H, H-
10a, H-2, H-4), 4.79 (d, J = 13.1 Hz, 1H, H-10b), 4.68 (t, J = 7.2 Hz, 2H,
triazole-NCH2), 4.64 (d, J = 6.6 Hz, 1H, H-1), 4.16 (dd, J = 11.9,
5.1 Hz, 1H, H-5e), 3.41 (dd, J = 11.9, 8.7 Hz, 1H, H-5a), 2.84 (tt,
J = 17.9, 7.5 Hz, 2H, triazole-N-CH2CH2), 2.05 (s, 3H, OCOCH3),
2.03 (s, 3H, OCOCH3), 2.01 (s, 3H, OCOCH3); 13C NMR (100 MHz,
CDCl3): d 170.0, 169.9, 169.5, 144.8, 123.1, 99.9, 71.3, 70.8, 68.8,
62.4, 62.1, 42.5, 31.7, 20.70, 20.65, 20.62; 19F NMR (282 MHz,
CDCl3): d ꢁ81.3, ꢁ114.6, ꢁ122.3, ꢁ123.3, ꢁ123.9, ꢁ126.6; HRE-
SIMS calcd for C22H23 F13N3O8 (M+H)+: 704.1272; found:
704.1297; Anal. Calcd for C22H22F13N3O8: C 37.57; H 3.15; N
5.97; found: C 37.56; H 3.01; N 5.91.
3.3.4. (1-Dodecyl-1H-1,2,3-triazol-4-yl)methyl b-D-
xylopyranoside (5d)
Triazole 4d (566 mg, 1.08 mmol) was deprotected using the
general conditions and purified by recrystallization from acetone
to yield 278 mg (65%) of a white solid. 1H NMR (400 MHz,
DMSO-d6): d 8.07 (s, 1H, triazole-CH), 5.03 (d, J = 5.0 Hz, 1H, OH),
4.95 (pseudo d, J = 2.7 Hz, 2H, 2 ꢀ OH), 4.76 (d, J = 12.1 Hz, 1H, H-
10a), 4.58 (d, J = 11.3 Hz, 1H, H-10b), 4.32 (t, J = 7.1 Hz, 2H, tria-
zole-NCH2), 4.22 (d, J = 7.5 Hz, 1H, H-1), 3.73 (dd, J = 11.3, 5.3 Hz,
1H, H-5e), 3.23–3.30 (m, 1H, H-4), 2.91–3.14 (m, 3H, H-2, H-3, H-
5a), 1.71–1.85 (m, 2H, triazole-N-CH2CH2), 1.11–1.35 (m,
9 ꢀ CH2), 0.85 (t, J = 6.7 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): d
143.6, 123.9, 102.8, 76.6, 73.2, 69.6, 65.7, 61.5, 49.3, 31.3, 29.7,
29.0 (2 ꢀ C), 28.95, 28.86, 28.7, 28.4, 25.8, 22.1, 13.9; HRESIMS
calcd for C20H38N3O5 (M+H)+: 400.2806; found: 400.2806; Anal
calcd for C20H37N3O 5: C 60.11; H 9.34; N 10.52; found: C 60.08;
H 9.41; N 10.49.
3.3. General procedure for acetate removal8
Triazole peracetates 4 were stirred in dry methanol. NaOMe (3–
4 equiv) was added and the solution stirred at room temperature
for two hours. DowexÒ 50 W ꢀ 8–100 ion exchange resin was
added and the reaction mixture stirred for another 30 min. The re-
sin was filtered and the solvent concentrated. The crude residue
was purified by recrystallization to yield pure 1-alkyl-1H-1,2,3-
triazol-4-yl)methyl b-D-xylopyranosides 5.
3.3.1. (1-Hexyl-1H-1,2,3-triazol-4-yl)methyl b-D-xylopyranoside
(5a)
Triazole 4a (443 mg, 1.00 mmol) was deprotected using the
general conditions and purified by recrystallization from chloro-
form to yield 160 mg (51%) of a white solid. 1H NMR (CD3-
OD,400 MHz): d 7.98 (s, 1H, triazole-CH), 4.90 (d, J = 12.4 Hz, 1H,
H-10a), 4.73 (d, J = 12.4 Hz, 1H, H-10b), 4.39 (t, J = 7.1 Hz, 2H, tria-
zole-NCH2), 4.32 (d, J = 7.5 Hz, 1H, H-1), 3.88 (dd, J = 11.4, 5.3 Hz,
1H, H-5e), 3.49 (m, 1H, H-4), 3.28–3.34 (m, 1H, overlapping with
CD3OD peak, H-5a), 3.17–3.26 (m, 2H, H-2, H-3), 1.88–1.93 (m,
2H, triazole-N-CH2CH2), 1.21–1.43 (m, 6H, 3 ꢀ CH2), 0.90 (t,
J = 6.9 Hz, 3H, CH3); 13C NMR (100 MHz, CD3OD): d 145.8, 125.2,
3.3.5. (1-Tetradecyl-1H-1,2,3-triazol-4-yl)methyl b-D-
xylopyranoside (5e)
Triazole 4e (435 mg, 0.786 mmol) was deprotected using the
general conditions and purified by recrystallization from acetone
to yield 218 mg (65%) of a white solid. 1H NMR (400 MHz,
DMSO-d6): d 8.07 (s, 1H, triazole-CH), 5.01–5.07 (br s, 1H, OH),
4.94–5.00 (m, 2H, 2 ꢀ OH), 4.76 (d, J = 12.1 Hz, 1H, H-10a), 4.58
(d, J = 12.1 Hz, 1H, H-10b), 4.32 (t, J = 7.1 Hz, 2H, triazole-NCH2),
4.22 (d, J = 7.6 Hz, 1H, H-1), 3.73 (dd, J = 11.3, 5.3 Hz, 1H, H-5e),

76
E. D. Oldham et al. / Carbohydrate Research 379 (2013) 68–77
3.20–3.33 (m, 1H, H-4), 2.96–3.12 (m, 3H, H-2, H-3, H-5a), 1.66–
1.91 (m, 2H, triazole-N-CH2CH2), 1.06–1.40 (m, 11 ꢀ CH2), 0.85 (t,
J = 6.6 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): d 143.6,
123.9, 102.8, 76.6, 73.2, 69.6, 65.8, 61.5, 49.3, 31.3, 29.7, 29.0,
29.0 (2 ꢀ C), 29.0, 28.9, 28.9, 28.7, 28.4, 25.8, 22.1, 13.9; HRESIMS
calcd for C22H42N3O5 (M+H)+: 428.3119; found: 428.3118; Anal.
calcd for C22H41N3O5: C 61.78; H 9.57; N 9.83; found: C 61.52; H
9.41; N 9.69.
3.5.2. Cell lines and cell culture
Spontaneously immortalized human keratinocytes (HaCaT),
normal non-transformed human foreskin fibroblasts (Hs27) and
human acute leukemia T lymphocytes (Jurkat) were used to evalu-
ate the cytotoxicity of 5a–g. HaCaT and Hs27 cells were main-
tained in Dubelcco’s Modified Eagle Medium (DMEM; HyClone,
Logan, UT) with 10% heat-inactivated newborn calf serum (NCS;
HyClone). Jurkat cells were maintained in RPMI media with 10%
heat-inactivated FBS (HyClone). Penicillin (100 U/mL), streptomy-
cin (100 lg/mL) and amphotericin B (0.25 lg/mL) were added to
the media to prevent microbial contamination. Cells were incu-
bated at 37 °C with 5% CO2.
3.3.6. (1-Hexadecyl-1H-1,2,3-triazol-4-yl)methyl b-D-
xylopyranoside (5f)
Triazole 5f (447 mg, 0.763 mmol) was deprotected using the
general conditions and purified by recrystallization from chloro-
form to yield 259 mg (74%) of a white solid. 1H NMR (400 MHz,
DMSO-d6): d 8.07 (s, 1H, triazole-CH), 5.03 (d, J = 5.1 Hz, 1H, OH),
4.95 (dd, J = 4.8, 3.1 Hz, 2H, 2 ꢀ OH), 4.76 (d, J = 12.2 Hz, 1H, H-
10a), 4.58 (d, J = 12.0 Hz, 1H, H-10b), 4.32 (t, J = 7.1, 2H, triazole-
NCH2), 4.22 (d, J = 7.6 Hz, 1H, H-1), 3.73 (dd, J = 11.2, 5.4 Hz, 1H,
H-5e), 3.21–3.32 (m, 1H, H-4), 2.91–3.15 (m, 3H, H-2, H-3, H-5a),
1.68–1.88 (m, 2H, triazole-N-CH2CH2), 1.08–1.34 (m, 13 ꢀ CH2),
0.85 (t, J = 6.6 Hz, 3H, CH3); 13C NMR (100 MHz, DMSO-d6):
d = 143.6, 123.9, 102.8, 76.5, 73.2, 69.6, 65.7, 61.5, 49.3, 31.3,
29.7, 29.0 (4 ꢀ C), 29.0 (2 ꢀ C), 28.9, 28.9, 28.7, 28.4, 25.8, 22.1,
13.9; HRESIMS calcd for C24H46N3O5 (M+H) +: 456.3432; found:
456.3434; Anal. calcd for C24H45N3O5: C 63.25; H 9.96; N 9.23;
found: C 63.01; H 9.83; N 9.12.
3.5.3. MTS assay
Cells were seeded in flat bottom MICROTEST™ tissue culture
96-well plates (Becton Dickinson Labware, Franklin Lakes, NJ) at
a density of 5000 (HaCaT and Hs27 cells) or 25,000 (Jurkat cells).
Compounds 5a–g in DMSO were added to the cells at concentra-
tions of 1–1000
kat cells), MTS (20
l
M. After 18 h (HaCaT and Hs27 cells) or 16 h (Jur-
L, CellTiter 96 AQueousOne Solution Cell
l
Proliferation Assay; Promega, Madison, WI) was added to each
well. The cells were further incubated for 2 h (HaCaTand Hs27
cells) or 4 h (Jurkat cells). The absorbance of formazan at 490 nm
was measured using a microplate reader (VERSAmax tunable micro-
plate reader, MDS, Inc., Toronto, Canada). Absorbance at 650 nm
was used as a reference. The absorbance was corrected by subtract-
ing the background absorbance of control wells (medium and MTS
reagent). DMSO treatment alone and untreated cells were used as
3.3.7. (1-(3,3,4,4,5,5,6,6,7,7,8,8,8-Tridecafluorooctyl)-1H-1,2,3-
triazol-4-yl)methyl b-D-xylopyranoside (5g)
negative controls, while 300 lM hydrogen peroxide (Acros Organ-
ics, NJ) was used as a positive control. Cell viability is expressed as
a percentage of DMSO-treated cells. Each data point represents the
average of three replicates.
Triazole 4g (400, mg, 0.597 mmol) was deprotected using the
general conditions and purified by recrystallization from chloro-
form to yield 192 mg (56%) of a white solid. 1H NMR (400 MHz,
DMSO-d6): d 8.21 (s, 1H, triazole-CH), 5.02 (d, J = 5.1 Hz, 5H, OH),
4.95 (d, J = 4.9 Hz, 2H, 2 ꢀ OH), 4.78 (d, J = 12.2 Hz, 1H, H-10a),
4.72 (t, J = 7.0 Hz, 2H, triazole-NCH2), 4.60 (d, J = 11.3 Hz, 1H, H-
10b), 4.23 (d, J = 7.6 Hz, 1H, H-1), 3.73 (dd, J = 11.3, 5.3 Hz, 1H, H-
5e), 3.25–3.31 (m, 1H, H-4), 2.97–3.09 (m, 5H, H-2, H-3, H-5a, tri-
azole-N-CH2CH2); 13C NMR (100 MHz, DMSO-d6): d 143.8, 124.5,
102.8, 76.6, 73.2, 69.5, 65.7, 61.3, 41.6, 30.3; 19F NMR (282 MHz,
3.5.4. Generation of dose–response curves and determination of
CC50
The data from the MTS assay were plotted as dose–response
curves. The 50% cytotoxic concentration (CC50) was determined
from a linear regression of the data points surrounding 50% cell
viability as described previously.42 The data were normalized as
described above. Briefly, data were normalized by subtracting from
each experimental value the average percentage of dead cells from
six wells treated with 1% DMSO (solvent control). The two cellular
viability percentages closest to the 50% value were plotted with its
DMSO-d6):
d
ꢁ79.76, ꢁ113.02, ꢁ121.26, ꢁ122.17, ꢁ122.66,
ꢁ125.29. HRESIMS calcd for C16H17F13N3O5 (M+H)+: 578.0955;
found: 578.0969; Anal. calcd for C16H16F
13N3O5 (M+H)+: C 33.29;
H 2.79; N 7.28; found: C 33.51; H 2.62; N 7.16.
associated chemical compound concentration and the equation of
42
the regression line was utilized to determine the CC50
.
3.4. Differential scanning calorimetry
3.5.5. Apoptosis assay
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 pre-
pared by weighing 1.0–1.5 mg of the dry surfactant into pre-
weighed Tzero aluminum pans. Tzero hermetic lids were then
pressed onto the pan. Empty Tzero pans with Tzero hermetic lid
were used as the reference sample. The thermal analysis was per-
formed between 25 and 200 °C with a ramp rate of 2.5 °C/min. The
phase transitions were analyzed using Universal Analysis software
(TA Instruments).
For apoptosis assays, Ficoll-Paque™PLUS density gradient cen-
trifugation50,69 was used to increase the viability of Jurkat cells;
this was done one day prior to the assay. Cells were centrifuged
at 400ꢀg for 30 min at room temperature and live cells at the
interface were then collected and washed with cell culture med-
ium. The resulting Jurkat cells were seeded in flat-bottomed 96-
well MICROTEST™ plates at a cell density of 25,000 cells per well
in 200
l
L RPMI medium supplemented with 10% FBS and antibiot-
M, apoptosis
ics as described above. 5b, 5c, 5g, OTG, H2O2 (300
l
positive control), and Tween 20 (0.5%, necrosis positive control)
were added to cells in triplicate at their CC50. DMSO vehicle control
and untreated cells, also in triplicate, were used as negative con-
trols. Following 20 h of incubation, the cells were prepared for flow
cytometry as previously described with minor modifications 39).
Briefly, ice-cold cells were centrifuged at 1400 rpm (357ꢀg) for
5 min at 4 °C. The cells were then stained by resuspension in
3.5. Cell culture experiments
3.5.1. Stock solutions
All compounds were dissolved in DMSO at 100
under experimental conditions was verified by adding 2
solution to 200 L of cell culture medium, vortexed for 5 s, centri-
lM. Solubility
lL of each
l
100
2.55 mM CaCl2) containing 1
l of 250 g/ml PI (Beckman Coulter, Miami, FL). Following 15
ll binding buffer (0.1 M HEPES, pH 7.4, 140 mM NaCl, and
fuged at 1,400 rpm (357ꢀg) for 1 min before inspection for
l
l of 25 g/ml annexin V-FITC and
l
precipitation.
5
l
l

E. D. Oldham et al. / Carbohydrate Research 379 (2013) 68–77
77
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FITC) and double- (annexin V-FITC and PI) stained cells. For each
sample, 3000 individual events were collected and analyzed using
CXP software (Beckman Coulter, Miami, FL, USA).
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This work was supported by the National Science Foundation
(CBET-0967381/0967390), the U.S. Department of Agriculture
Biomass Research and Development Initiative (Grant Agreement
68-3A75-7-608) and the Department of Energy Development and
Independence, Energy and Environment Cabinet of The Common-
wealth of Kentucky. The cell culture work was performed at the
Cell Culture Core of the Border Biomedical Research Center (BBRC)
supported by grant 8G12MD007592 from the National Center for
Research Resources (NCRR) of the NIH, granted to the University
of Texas at El Paso.
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