Synthesis and biocompatibility evaluation of fluorinated, single-tailed glucopyranoside surfactants

Article abstract of DOI:10.1039/b805015e

Partially fluorinated non-ionic surfactants are of interest for a range of biomedical applications, such as the pulmonary administration of drugs using reverse water-in-perfluorocarbon microemulsions. We herein report the synthesis and characterization of a series of partially fluorinated β-d- glucopyranoside surfactants from the respective alcohols and peracetylated β-d-glucopyranoside using BF₃·Et₂O as catalyst. The surfactant packing parameter of the fluorinated surfactants ranged from 0.472 to 0.534 (MOPAC calculations) or 0.562 to 0.585 (calculated from literature values), which is comparable to surfactants with a similar partially fluorinated tail. Based on an initial biocompatibility assessment, the β-d-glucopyranoside surfactants have low toxicities in the B16F10 mouse melanoma cell line and comparatively low haemolytic activities towards rabbit red blood cells. The fluorinated surfactants appear to be less toxic towards cells in culture and to have a lower haemolytic activity compared to their hydrocarbon analogs. Furthermore, an increasing degree of fluorination appears to reduce both the cytotoxicity and the haemolytic activity. Similar structure-activity relationships have been reported for other partially fluorinated surfactants. Overall, these findings suggest that the surfactants may be useful for biomedical applications, such as novel drug delivery systems. The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2008.

Full text of DOI:10.1039/b805015e

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Synthesis and biocompatibility evaluation of fluorinated, single-tailed
glucopyranoside surfactantsw
a
b
b
b
ˇ
´
´
¨
´
´
´
Xueshu Li, Jaroslav Turanek,* Pavlına Knotigova, Hana Kudlackova,
c
b
c
c
ˇ
Josef Masek, D. Brant Pennington, Stephen E. Rankin, Barbara L. Knutson
and Hans-Joachim Lehmler*a
Received (in Durham, UK) 2nd April 2008, Accepted 31st July 2008
First published as an Advance Article on the web 19th September 2008
DOI: 10.1039/b805015e
Partially fluorinated non-ionic surfactants are of interest for a range of biomedical applications,
such as the pulmonary administration of drugs using reverse water-in-perfluorocarbon
microemulsions. We herein report the synthesis and characterization of a series of partially
fluorinated b-D-glucopyranoside surfactants from the respective alcohols and peracetylated
b-D-glucopyranoside using BF3ꢀEt2O as catalyst. The surfactant packing parameter of the
fluorinated surfactants ranged from 0.472 to 0.534 (MOPAC calculations) or 0.562 to 0.585
(calculated from literature values), which is comparable to surfactants with a similar partially
fluorinated tail. Based on an initial biocompatibility assessment, the b-D-glucopyranoside
surfactants have low toxicities in the B16F10 mouse melanoma cell line and comparatively low
haemolytic activities towards rabbit red blood cells. The fluorinated surfactants appear to be less
toxic towards cells in culture and to have a lower haemolytic activity compared to their
hydrocarbon analogs. Furthermore, an increasing degree of fluorination appears to reduce both
the cytotoxicity and the haemolytic activity. Similar structure–activity relationships have been
reported for other partially fluorinated surfactants. Overall, these findings suggest that the
surfactants may be useful for biomedical applications, such as novel drug delivery systems.
example carnitine,8 (di-)morpholinophosphate,9,10 phospho-
Introduction
choline,11 pyridinium12 and carbohydrate5,13 headgroups,
have been synthesized, and their biocompatibility has been
assessed using in vitro and in vivo approaches. There is
evidence that partially fluorinated surfactants in general dis-
play low to moderate acute toxicity compared to their hydro-
carbon analogues.5 Partially fluorinated surfactants with a
perfluorooctyl or perfluorodecyl group in the hydrophobic tail
have particularly low toxicities. In addition, the haemolytic
activity of partially fluorinated surfactants is low despite their
high surface activity.5
Surfactants with a partially fluorinated tail of the general
structure (CH2)mCnF2n+1 have unique physicochemical pro-
perties, such as high surface activity and weak molecular
interactions.1 Furthermore, perfluorinated tails are larger
and stiffer compared to hydrocarbon tails. For example, the
limiting molecular areas of perfluorinated and hydrocarbon
chains at the air–water interface are 30 and 20 A2, respec-
tively.2,3 This difference in physicochemical properties and
molecular geometry allows (partially) fluorinated surfactants
to self-assemble in supramolecular structures that are distinc-
tively different from those observed for their hydrocarbon
analogues.4 They tend to self-assemble more easily and to
form better organized and more stable systems than their
hydrocarbon counterparts.5
Some fluorinated surfactants form stable, reverse water-in-
perfluorocarbon (micro-)emulsions.14–18 These emulsions are
of particular interest for the perfluorocarbon-assisted pulmo-
nary administration of drugs6,7 because perfluorocarbon-
insoluble drugs can be administered by dissolving them in the
aqueous core of the reverse (micro-)emulsion. For example,
some dimorpholinophosphate surfactants form reverse
(micro-) emulsions that are highly fluid and have a water
content of up to 30 vol% at a surfactant concentration of 2%
(w/v).19 In addition, these reverse (micro-)emulsions appear
to be biocompatible in vitro and in vivo.19,20 However, all other
reverse water-in-perfluorocarbon (micro-)emulsions investi-
gated to date are based on perfluorinated surfactants (e.g.,
poly(oxyethylene) derivatives) that are not biocompatible.15–18
Therefore, there is considerable need for novel partially
fluorinated surfactants that are biocompatible and have the
potential to form stable, reverse water-in-perfluorocarbon
(micro-)emulsions.
Partially fluorinated surfactants are of interest for bio-
medical applications, such as pulmonary drug delivery,6,7
because of these unique properties. A variety of single tailed
partially fluorinated surfactants with different headgroups, for
a Department of Occupational and Environmental Health, University
of Iowa, Iowa City, IA 52242, USA.
E-mail: hans-joachim-lehmler@uiowa.edu; Fax: 3193354290;
Tel: 3193354211
b Veterinary Research Institute v.v.i., Department of Vaccinology and
Immunotherapy, Brno, Czech Republic. E-mail: turanek@vri.cz
c Chemicals and Materials Engineering Department, University of
Kentucky, Lexington, KY 40506, USA
w Electronic supplementary information (ESI) available: Detailed
characterization of compounds 6b,c,e,f, 7b,c,e,f, 8b,c,e,f and 9b,c,e,f.
See DOI: 10.1039/b805015e
ꢁc
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Building on earlier studies with partially fluorinated carbo-
hydrate surfactants,5 the present study describes the synthesis
and characterization of a series of partially fluorinated
b-D-glucopyranoside surfactants. In addition, this study
evaluates their toxicity in a cell culture model as well as
their haemolytic activity in comparison to the analogous
hydrocarbon surfactants.
between the glucose moiety and the terminal perfluoroalkyl
group of these surfactants is short, with two,23 three26–28 or
six24,25 methylene groups. To the best of our knowledge,
glucopyranoside surfactants with longer hydrocarbon spacers
have not been synthesized previously. The following section
describes the synthesis of the partially fluorinated b-D-gluco-
pyranosides 8a–f, which were selected based on their surfac-
tant packing parameters.
First, a series of partially fluorinated alcohols with the
general structure CmF2m+1(CH2)nOH (m = 4, 6 and 8, n =
10 and 11) were synthesized from decenol (1) or 10-undecenoic
acid (2), respectively.12 As shown in Scheme 1, the two starting
materials were converted into the corresponding acetate or
methyl ester, and the hydrocarbon precursor was coupled with
a perfluorinated iodide in an AIBN mediated radical reac-
tion.29–32 The resulting secondary iodides were deiodinated
with HI/Zn/EtOH and converted into the respective alcohol 3
either by saponification or by reduction with LAH.
Results and discussion
Surfactant packing parameters of b-D-glucopyranosides
Although it is difficult to predict if a partially fluorinated
surfactant forms reverse water-in-perfluorocarbon micro-
emulsions,5 the molecular geometry of a surfactant is one
important determinant of the self-assembled structures formed
by a surfactant. One possible way to describe the molecular
geometry of a surfactant is the surfactant packing parameter
P = v/(a0lc) (where v is the volume of the hydrophobic chain,
a0 is the area of the polar head group and lc is the length of the
hydrophobic chain).4,21 To guide us with the selection of
promising target molecules, we calculated the surfactant
packing parameter for several b-D-glucopyranosides using
either MOPAC or published values for v, a0 and lc.4,22 In
addition, we calculated the surfactant packing parameters of
two dimorpholinophosphate surfactants which have been
shown to form stable, reverse water-in-perfluorocarbon
(micro-)emulsions.14,19 The results of these calculations are
shown in Table 1.
Subsequently, the fluorinated alcohols 3a–f and the corre-
sponding hydrocarbon analogues 4a–f were reacted with
peracetylated b-D-glucopyranoside (5) to obtain the peracetyl-
ated glucopyranosides 6a–f and 7a–f. This glycosylation
reaction can be performed using a number of Lewis acids,
for example SnCl4,33,34 ZnCl2 or BF3ꢀEt2O24,36–39 as cata-
35
lysts. Initial experiments showed that reaction of the fluori-
nated alcohols 3 with 5 in the presence of ZnCl2 or AlCl3
required long reaction times and high reaction temperatures,
which led to the formation of mixtures of the a- and
b-anomers of the desired peracetylated glucopyranosides
6a–f. In contrast, BF3ꢀEt2O gave the desired b-anomers of
the glucopyranosides 6a–f and 7a–f in 40–55% yield when
low reaction temperatures (o5 1C) and short reaction times
(o3 h) were employed. The glycosylation products obtained
under these conditions typically contained traces of a-anomer
(o1%) as determined by 1H NMR. This small amount of the
a-anomer was considered acceptable for the intended applica-
tion of these compounds as surfactants in biomedical applica-
tions. The thermodynamically more stable a-anomers were
obtained when the reaction temperature was raised to 30 1C
and the reaction time was extended to 10 h (data not shown).
No other products, for example 2-, 3-, 4- or 6-(F-)alkyl
glucopyranosides, were formed according to TLC and 1H
NMR analysis.
The volumes, tail lengths and surfactant parameters calcu-
lated with the two different approaches differed significantly
from each other. Similarly, Giulieri and Krafft reported
significant differences between published and experimental
values for v and lc.4 Despite these discrepancies, the calculated
surfactant packing parameters allowed a comparison of the
molecular geometry of glucopyranoside and dimorpholino-
phosphate surfactants and the identification of trends. Inde-
pendent of the approach used, the surfactant packing
parameters of both types of surfactants were similar, and near
the packing parameter value that would be associated with
ideal cylindrical micelles (P = 0.5).21 For the MOPAC results,
P for the glucopyranosides was slightly larger than the P for
dimorpholinophosphate surfactants, while the converse was
suggested from the experimental data due to the smaller
experimental dimorpholinophosphate headgroup area.4
Furthermore, P of the partially fluorinated glucopyranoside
surfactants increased slightly with increasing degree of fluor-
ination, whereas P for the hydrocarbon glucopyranoside
surfactants did not change with increasing chain length. Over-
all, the surfactant packing parameters shown in Table 1
suggest that partially fluorinated glucopyranoside and dimor-
In the final step of the synthesis, the (F-)alkylated perace-
tylated b-D-glucopyranosides 6a–f and 7a–f were deacetylated
using NaOMe in absolute methanol, followed by neutraliza-
tion with Dowexs 50Wꢂ8-100 ion exchange resin.38 The
crude product was purified by column chromatography on
silica gel followed by recrystallization from acetone. The pure
glucopyranosides 8a–f and 9a–f were obtained with yields
ranging from 92 to 98%.
pholinophosphate surfactants have
a similar molecular
In a preliminary study we also investigated a different route
to the desired glucopyranosides to improve the overall yields
of the synthesis. Analogous to published syntheses that
employ bromo-sugars and an alcohol as starting materials,40,41
we reacted 2,3,4,6-tetraacetyl-a-D-glycopyranosyl bromide
(10) with a partially fluorinated alcohol in the presence of
Ag2CO3 in DCM at 0 1C (Scheme 2). However, instead of
the desired glucopyranoside 6e, we obtained the fluorinated
geometry. Therefore, glucopyranosides 8a–f were synthesized
for further studies because they may also form reverse water-
in-perfluorocarbon microemulsions.
Synthesis
The synthesis of several fluorinated b-D-glucopyranosides has
been reported in the literature.23–25 The hydrocarbon spacer
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Table 1 Surfactant packing parameter Pa of the glucopyranosides 8a–f and their hydrocarbon analogues 9a–f
MOPAC calculation results
Area, a0/A2
Volume, vb/A3
P
Pd
c
Length, lc /A
Entry
Structure
8a
41
357
18.5
0.472
0.493
0.510
0.488
0.513
0.534
0.410
0.413
0.415
0.415
0.418
0.419
0.419
0.566
0.579
0.589
0.562
0.575
0.585
0.488
0.490
0.492
0.489
0.491
0.492
0.618
8b
8c
8d
8e
8f
41
41
41
41
41
41
41
41
41
41
41
50
426
495
394
469
545
307
350
394
332
376
420
495
21.1
23.7
19.7
22.3
24.9
18.2
20.7
23.2
19.5
21.9
24.4
23.7
9a
9b
9c
9d
9e
9f
10a
(continued on next page)
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Table 1 (continued)
MOPAC calculation results
Area, a0/A2
Volume, vb/A3
545
P
Pd
c
Length, lc /A
Entry
Structure
10b
50
24.9
0.438
0.614
a
Surfactant packing parameter P = v/(a0lc), where a0 is the area of the polar head group, v is the volume of the hydrophobic chain, and lc is the
length of the hydrophobic chain.4,21 b The volume v (A3) from MOPAC correlations was calculated as 22.1nH + 37.7nF (nH odd) or 21.9nH
+
34.5nF (nH even), where nH = the number of hydrogenated and nF = the number of fluorinated carbons. The length lc (A) from MOPAC
c
d
correlations was calculated as 0.86 + 1.24nH + 1.3nF. Surfactant packing parameter P calculated using published values for a0, v and lc.4,22
1,2-orthoacetate 11 in 80% yield. Based on the chemical shifts
of the H-10 proton at 5.70 ppm, the 1,2-orthoacetate 11 is the
exo isomer (exo: d 5.72 ppm vs. endo: d 5.59 ppm42). Similarly,
Tsui and Gorin have reported the formation of 1,2-ortho-
acetates as side products (21–34% yield) under comparable
reaction conditions.42 The lower yield in that study may be due
to the shorter chain length of the alcohols employed (rC-8).
Melting points of (F-)alkyl glucopyranosides
Glucopyranosides are compounds that form both thermo-
tropic liquid crystalline phases upon heating and lyotropic
liquid crystalline phases upon addition of solvents such as
water.43–45 As a result of this complex phase behaviour, a
range of melting points has been reported in the literature for
alkyl b-D-glucopyranosides. To investigate the thermotropic
properties of the partially fluorinated glucopyranosides, the
phase transitions of the glucopyranosides 8a–f and 9a–f were
investigated using differential scanning calorimetry (DSC).
The phase transitions determined by DSC were in agreement
with the melting points measured with a MelTemp apparatus.
As shown in Fig. 1, both hydrocarbon and fluorocarbon
glucopyranosides displayed at least two phase transitions. The
main phase transitions were observed at temperatures ranging
from 69 to 116 1C. A minor phase transition observed at
temperatures from 145 to 190 1C corresponds to the clearing
temperatures reported for long-chain hydrocarbon gluco-
pyranosides43,44 and probably represents a transition from
an anisotropic liquid crystalline phase to an isotropic
liquid phase.
While the phase transition temperatures for the even num-
bered glucopyranosides 8a–b and 9a–b were almost the same,
Scheme 1 Synthesis of perfluoroalkyl and alkyl b-D-glucopyrano-
sides. Reagents and conditions: (a) DMAP, Ac–Cl, pyridine, DCM; (b)
the respective phase transition temperatures for the highly
fluorinated glucopyranoside 8c were clearly higher compared
to the corresponding hydrocarbon analogue 9c (Fig. 1(A)).
Similarly, the partially fluorinated glucopyranosides with an
odd number of carbon atoms in the hydrophobic tail (8d–f)
F(CF2)mI (m = 4, 6 or 8), AIBN; (c). HI (55%), Zn, C2H5OH; (d)
CH3OH, KOH; (e) CH3OH, PTSA, toluene; (f) F(CF2)mI (m = 4, 6 or
8), AIBN; (g) HI (55%), Zn, C2H5OH; (h) LiAlH4, anhydrous ether,
ambient temperature; (i) BF3/OEt2(48%), anhydrous DCM, 0 1C to
ambient temperature, 3 h; (j) MeONa/MeOH, 0 1C to ambient
temperature; (k) Dowexs 50Wꢂ8-100 ion-exchange resin.
Scheme 2 Synthesis of orthoester 11. Reagents and conditions: (a) 3e, Ag2CO3, DCM, 0 1C to ambient temperature.
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from 190 to 600 mM. This is in contrast to the positive control,
octylthioglucoside (OTG), which showed significant cytotoxi-
city over the same concentration range. Similarly, partially
fluorinated surfactants derived from maltose,47 mannitol,48
sorbitan,48 sucrose,49 trehalose,49 and xylitol50 also display
moderate-to-low toxicity in cells in culture, with EC50 typically
4100 mM. In contrast, glucopyranosides with shorter fluori-
nated chains (r12 carbon atoms) appear to be more
cytotoxic than glucopyranosides 8a–f.25 For example,
7,7,8,8,9,9,10,10,11,11,12,12,12-tridecafluorododecyl-b-D-gluco-
pyranoside was cytotoxic at concentrations of 50 mM.
The two glucopyranosides 8c and 8f with the perfluorooctyl
terminus were even less toxic than 8a–b and 8d–e, with EC50
values 41.5 mM. Similarly, the cytotoxicity of fluorinated
pyridinium surfactants has been shown to decrease with an
increasing degree of fluorination.12 One possible explanation
for this decrease in cytotoxicity is a lower cellular uptake due
to the hydrophobic and lipophobic perfluoroalkyl terminus of
the hydrophobic tail. This interpretation is supported by a
recent study that showed no cellular uptake of a highly
fluorinated galactopyranoside in the B16 melanoma cell line.25
However, there is also evidence that fluorinated surfactants
readily partition into model membranes.2,51–53 and, thus,
should be able to enter cells in culture. Therefore, cellular
uptake studies are necessary to determine if the glucopyrano-
sides 8c and 8f can indeed partition though cell membranes.
The results from the MTT assay were further confirmed by
Hoffman modulation contrast microscopy (Fig. 3). In agree-
ment with the results form the MTT assay, treatment of
B16F10 cells with high concentrations of surfactant 8c (24 h,
1.5 mM) surfactant neither altered cell growth nor induced
morphological changes in comparison to the control (Fig. 3(A)
and (B)). In contrast, surfactants 8e and 9e induced retarda-
tion in cell proliferation and caused an abnormal prolongated
morphology of the cells at a concentration close to the EC50
(Fig. 3(C) and (D)). In addition, both surfactants induced
apoptosis in a significant number of cells. No viable cells were
observed at high concentrations (1.5 mM) of 8e and 9e
(Fig. 3(E) and (F)). Only necrotic cells and cellular debris
were evident at these high concentrations. Fluorescence stain-
ing with PI and YO-PRO-1 showed only a small portion of
B16F10 cells in early or late stage of apoptosis in control cells
(Fig. 3(G)) but significant amount of cells treated with 8e at
concentration close to the EC50 were in various stages of
apoptosis (see Fig. 3(H)).
Fig. 1 Comparison of the phase transition maxima of perfluoroalkyl
and alkyl b-D-glucopyranosides with an (A) even (8a–c and 9a–c) and
(B) odd number (8d–f and 9d–f) of carbon atoms in the hydrophobic
tail (m and n = fluoroalkyl; ’ and & = alkyl; closed symbols =
major phase transition; open symbols = minor phase transition; E
represents the maximum of a second phase transition observed for
alkyl glucopyranosides 9c and 9e). Values shown are the experimental
mean ꢃ one standard deviation of at least three DSC experiments.
displayed higher phase transition temperatures compared to
their hydrocarbon analogues 9d–f (Fig. 1(B)). In comparison,
the melting points of the partially fluorinated b-D-glucopyrano-
sides 8a–f are lower compared to fluorinated glucopyranosides
with a propylene hydrocarbon spacer (–(CH2)3CmF2m+1, m =
4, 6 and 8).26,28
These differences in the phase transition temperatures bet-
ween hydrocarbon and fluorocarbon b-D-glucopyranosides
are a result of the perfluoroalkyl terminus in the hydrophobic
tail. Due to the larger steric demand of the fluorine atom,
perfluoroalkyl chains are more rigid, which allows a more
efficient packing in the solid state. As a consequence, the
melting point of the glucopyranosides 8c–f was higher
compared to the corresponding hydrocarbon analogues 9c–f.
Similar trends in phase transition temperatures have been
reported for other partially fluorinated compounds, such
as partially fluorinated, long-chain carboxylic acids with
the general structure F2m+1Cm(CH2)10COOH (m = 4, 6
and 8)29 or fluorinated b-D-glucopyranosides with a short
hydrocarbon spacer.26,28
Biological studies
An initial cytotoxicity assessment of the glucopyranoside
surfactants 8a–f and 9a–f was performed using the B16F10
mouse melanoma cell line.46 Haemolytic activities, both in the
presence and absence of 20% serum, were determined using
rabbit red blood cells.12 Octylthioglucoside was used as a
positive control for the cytotoxicity studies and the determina-
tion of haemolytic activities. Cytotoxicities and haemolytic
activities, expressed as EC50 values, are summarized in Table 2
for all compounds under investigation. Representative cyto-
toxicity curves for surfactants 8c, 8e and 9e are presented in
Fig. 2.
In addition to the cytotoxicity experiments, the haemolytic
activity of all surfactants was assessed using rabbit red blood
cells.12 The glucopyranosides 8a–f and 9a–f were haemolytic at
low millimolar concentrations. The haemolytic activity de-
creased for both groups of surfactants in the presence of
serum, with EC50 values 415 mM for 8a–f in the presence
of serum and 45 mM for 9a–f in the absence of serum.
Furthermore, the hydrocarbon glucopyranosides 9a–f had a
higher haemolytic activity compared to their fluorinated ana-
logues 8a–f. Similar observations have been reported for
various other fluorinated surfactants.5,12,47–50 In addition,
the haemolytic activity decreased for structurally related
glucopyranosides (i.e., 8a–d with ten and 8d–f with eleven
methylene groups in the hydrocarbon spacer) with increasing
The partially fluorinated glucopyranosides 8a–b and 8d–e
were moderately toxic, with EC50 values ranging from 179 to
311 mM. These EC50 values are comparable to the EC50 values
of the analogous hydrocarbon derivatives 9a–d, which ranged
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Table 2 Assessment of cytotoxicity and haemolytic activity of partially fluorinated glucopyranosides and their hydrocarbon analogues in the
B16F10 cell line.12,46
Haemolytic activity, EC50a/mM
Entry Structure R(F or H)-b-D-Glu
Fluorocarbon surfactants
Cytoxicity, EC50/mM
Without serum
20% Serum
8a
194
420 (10%)
420 (10%)
8b
8c
8d
8e
8f
311
41500
190
420 (25%)
20
420
420
20
15
179
420
420
420
41500
420
Hydrocarbon surfactants
9a
250
300
190
230
230
600
20
10
15
9b
9c
9d
9e
9f
420 (30%)
420
420
15
420 (10%)
5
15
420 (5%)
420
3
420
0.5
OTG
Octylthioglucoside
a
Data in parenthesis represent the percentage haemolysis at a 20 mM concentration of the respective glucopyranoside.
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Fig. 2 Cytotoxic effect of glucopyranosides 8c, 8e and 9e in the B16F10 mouse melanoma cell line. B16F10 cells were exposed for 24 h to the
respective glucopyranoside at the concentrations shown and assessed for MTT activity as described in the Experimental section. A representative
hydrocarbon surfactant, octylthioglucoside (OTG), is shown for comparison.
Fig. 3 Cytotoxic effect of glucopyranosides 8c, 8e and 9e in the B16F10 mouse melanoma cell line. The B16F10 cells were treated for 24 h with (A)
the vehicle (PBS + 1% DMSO) or with glucopyranoside (B) 8c (1.5 mM), (C) 8e (250 mM, black arrows indicate abnormal spindle-shaped cells),
(D) 9e (250 mM, black arrow indicate abnormal spindle-shaped cell, white arrow indicate typical necrotic cell), (E) 8e (1.5 mM) or (F) 9e (1.5 mM,
black arrows indicate cellular debris, white arrows indicate necrotic cells). The fluorescent markers Yo-Pro-1 (green) and PI (red/yellow) were used
for the visualization of early apoptotic changes and post-apoptotic secondary necrosis in B16F10 cells treated for 24 h with (G) vehicle (green
arrows indicate early stage of apoptosis, orange arrows indicate late stage of apoptosis) or (H) 8e (250 mM, green arrows indicate early stage of
apoptosis, orange arrows indicate late stage of apoptosis, red arrows indicate secondary necrosis). The cells were observed under an epifluorescent
microscope.
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length of the perfluoroalkyl terminus. This anti-haemolytic
effect of an increasing degree of fluorination has been
reported previously for a number of structurally diverse
surfactants.5,12,47–50
were measured at the University of Iowa Mass Spectrometry
Facility. High-resolution mass spectra (HR-MS) were mea-
sured using an Autospec ESI-MS instrument and ESI mass
spectra were recorded using a ThermoFinnigan LCQ Deca
mass spectrometer. Elemental analyses were obtained from
Atlantic Micro Lab Microanalysis Service (Atlanta, Georgia,
USA). Melting points were determined using a MelTemp
apparatus and are uncorrected. In addition, the maxima of
the major phase transitions of all glucopyranosides was
determined using differential scanning calorimetry (DSC)
from 0 to 200 1C with a 10 1C minꢄ1 ramp.29,45 All reactions
were monitored by thin layer chromatography, followed by
visualization with anisaldehyde–H2SO4.55 The detailed
characterization of representative compounds with odd and
even numbered tails is shown in the text below (the detailed
characterization of the remaining compounds is available as
ESIw).
Conclusions
A series of b-D-glucopyranoside surfactants with (F-)alkyl
chain lengths ranging from 14 to 19 carbon atoms was
synthesized in good yields from the respective alcohols and
peracetylated b-D-glucopyranoside (5) using BF3ꢀEt2O as
catalyst. Similar to other partially fluorinated surfactants, an
increasing degree of fluorination reduced their toxicity and
haemolytic activity. Because of their biocompatibility as well
as their molecular geometry (i.e., the surfactant packing
parameter), the partially fluorinated glucopyranoside surfac-
tants 8a–e are of particular interest for biomedical applica-
tions, such as the pulmonary administration of drugs using
reverse water-in-perfluorocarbon (micro-)emulsions.6,7
General procedure for the synthesis of perfluoroalkyl and alkyl
2,3,4,6-tetra-O-acetyl-b-D-glucopyranosides (6a–f and 7a–f)38
Boron trifluoride diethyl ether complex (10.0 mmol, 48% w/w)
in 5 mL dry dichloromethane was added dropwise to a
solution of pentaacetyl-b-D-glucopyranose 5 (5.0 mmol) and
the corresponding alcohol 3a–f or 4a–f (6.0 mmol) in 15 mL
dry dichloromethane at 0 1C. The reaction mixture was
stirred at 0 1C for 2 h and allowed to slowly warm to ambient
temperature. Dichloromethane (20 mL) was added and
the mixture was washed with saturated NaHCO3 solution
(3 ꢂ 15 mL) and brine (2 ꢂ 15 mL). The organic phase was
dried over anhydrous Na2SO4, filtered, and the solvent was
removed under reduced pressure. The residue was purified by
column chromatography on silica gel with hexane and ethyl
acetate as eluent (hexane–ethyl acetate = 3 : 1). The product
was obtained as a white solid or a yellowish viscous liquid with
moderate yields ranging from 46 to 55%.
Experimental
Calculation of surfactant packing parameters of (F-)alkyl
b-D-glucopyranosides
The geometry of each molecule was optimized using MOPAC
as implemented in Chem3D Ultra 9.0 (CambridgeSoft).54
Molecular volumes were calculated as COSMO volumes,
and tail lengths were measured directly from the optimized
geometry as the length from the O atom to the terminal atom
in the tail. Tail length and volume correlations were developed
for hydrocarbon and fluorocarbon surfactants in the series.
Areas were determined by determining volumes for a series of
at least three surfactants with the same head group and using a
linear correlation between tail length and volume to determine
the head volume (from the intercept). The area was then
calculated assuming a spherical head geometry (which may
not be reasonable for dimorpholinophosphinate surfactants).
The alternative calculations were based on experimental areas
and correlations between carbon number and bond lengths
and tail volumes.4,22
Perfluoroalkyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosides
11,11,12,12,13,13,14,14,14-Nonafluorotetradecyl-2,3,4,6-
tetra-O-acetyl-b-D-glucopyranoside (6a). Viscous liquid; yield,
1
55%; H NMR (300 MHz, CDCl3): d/ppm 1.1–1.3 (m, 12H,
6 ꢂ CH2), 1.45 (m, 4H, H-20 and H-90), 1.8–2.0 (m, 14H, 4 ꢂ
CH3CO and H-100), 3.35 (m, 1H, H-10a), 3.59 (m, 1H, H-5),
3.76 (m, 1H, H-10b), 4.00 (d, 1H, J6a,6b = 12.0 Hz, H-6a), 4.16
(dd, 1H, J6a,6b = 12.0 Hz, J5,6b = 4.7 Hz, H-6b), 4.39 (d, 1H,
J = 8.0 Hz, H-1), 4.84 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.5 Hz,
H-2), 4.96 (‘‘t’’, 1H, J B 9.9 Hz, H-4), 5.09 (‘‘t’’, 1H, J B
9.5 Hz, H-3). 13C NMR (75 MHz, CDCl3): d/ppm 20.0 (C-90),
20.5 (4 ꢂ CH3CO), 25.8 (C-30), 29.0–29.4 (C-20, C-40 to C-80),
30.7 (t, J = 21 Hz, C-100), 62.0 (C-6), 68.5 (C-4), 70.0 (C-10),
71.3 (C-2), 71.7 (C-5), 72.8 (C-3), 100.8 (C-1), 169.1, 169.4,
170.2, 170.5 (4 ꢂ COCH3). 19F NMR (282 MHz, CDCl3):
d/ppm ꢄ81.6 (CF3), ꢄ115.1 (CF2), ꢄ124.9 (CF2), ꢄ126.6
(CF2). Positive-ion ESI-MS peak at m/z 729 ([M + Na]+).
Synthesis of (F-)alkyl b-D-glucopyranosides
The long-chain hydrocarbon starting materials 1 and 2 were
purchased from TCI Chemicals (Portland, Oregon, USA).
Pentaacetyl-b-D-glucopyranose (5), the long chain alkyl alco-
hols 4a–f and anhydrous dichloromethane were obtained from
Fisher Scientific (Fairlawn, New Jersey, USA). Perfluorinated
iodides were purchased from Oakwood Chemical Co. (West
Columbia, South Carolina, USA). The 1H and 13C NMR
spectra were recorded on a multinuclear Bruker Avance 300 or
Bruker DRX 400 Digital NMR Bruker spectrometers at
ambient temperature. Due to their poor solubility, NMR
spectra of long-chain glucopyranosides were recorder at
1
318 K. All H and 13C chemical shifts are reported in parts
12,12,13,13,14,14,15,15,15-Nonafluoropentadecyl-2,3,4,6-
per million (ppm) relative to internal tetramethylsilane. 13C
signals of the glucose moiety were assigned as described
previously.36 19F spectra were recorded using the Bruker
Avance 300 with CFCl3 as internal standard. The mass spectra
tetra-O-acetyl-b-D-glucopyranoside (6d). Viscous liquid; yield,
1
51%; H NMR (300 MHz, CDCl3): d/ppm 1.1–1.3 (m, 14H,
7 ꢂ CH2), 1.44 (m, 4H, H-20 and H-100), 1.8–2.0 (m, 14H,
4 ꢂ CH3CO and H-110), 3.34 (m, 1H, H-10a), 3.60 (ddd, 1H,
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J4,5 = 9.8 Hz, J5,6a = 2.4 Hz, J5,6b = 4.5, H-5), 3.74 (m, 1H,
H-10b), 4.10 (dd, 1H, J6a,6b = 12.2 Hz, J5,6a = 2.3 Hz, H-6a),
4.15 (dd, 1H, J6a,6b = 12.2 Hz, J5,6b = 4.7 Hz, H-6b), 4.38
(d, 1H, J = 8.0 Hz, H-1), 4.84 (‘‘t’’, 1H, J B 8.4 Hz, H-2), 4.95
(dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.5 Hz, H-4), 5.08 (‘‘t’’, 1H, J B
9.4 Hz, H-3). 13C NMR (75 MHz, CDCl3): d/ppm 20.0
(C-100), 20.4 (4 ꢂ CH3CO), 25.7 (C-30), 29.0–29.5 (C-20, C-40
to C-90), 30.6 (t, J = 22 Hz, C-110), 61.9 (C-6), 68.5 (C-4),
70.0 (C-10), 71.3 (C-2), 71.7 (C-5), 72.8 (C-3), 100.8 (C-1),
169.0, 169.3, 170.1, 170.5 (4 ꢂ COCH3). 19F NMR (282 MHz,
CDCl3): d/ppm ꢄ81.6 (CF3), ꢄ115.1 (CF2), ꢄ124.9
(CF2), ꢄ126.6 (CF2). Positive-ion ESI-MS peak at m/z 743
([M + Na]+).
50Wꢂ8-100 ion exchange resin (2.0 g). The ion exchange resin
was filtered off and the solvent was removed under reduced
pressure. The crude product was purified by recrystallization
from acetone or column chromatography on silica gel (eluent:
DCM–MeOH = 8 : 1), followed by recrystallization from
acetone, to give the products as white solids in high yields
ranging from 92 to 98%.
Perfluoroalkyl b-D-glucopyranosides
11,11,12,12,13,13,14,14,14-Nonafluorotetradecyl-b-D-gluco-
pyranoside (8a). White solid; mp 59–61 1C; mp (DSC) 58.43 ꢃ
1
1.76 1C, 166.19 ꢃ 1.51 1C; yield, 95%; H NMR (300 MHz,
CD3OD): d/ppm 1.3–1.5 (m, 12H, 6 ꢂ CH2), 1.6–1.7 (m, 4H,
H-20 and H-90), 2.18 (m, 2H, H-100), 3.22 (‘‘t’’, 1H, J B
8.3 Hz, H-2), 3.3–3.4 (m, 3H, H-3, H-4 and H-5), 3.57 (m, 1H,
H-10a), 3.70 (dd, 1H, J6a,6b = 11.9 Hz, J5,6b = 5.1 Hz, H-6a),
3.8–4.0 (m, 2H, H-6b and H-10b), 4.29 (d, 1H, J1,2 = 7.7 Hz,
Alkyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosides
Tetradecyl-2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside (7a)38,56
.
1
White solid; mp 63–64 1C (lit.: 57–84 1C56,57); yield, 51%; H
13
H-1). C NMR (75 MHz, CD3OD) d/ppm 21.3 (C-90), 27.1
NMR (300 MHz, CDCl3): d/ppm 0.88 (t, 3H, J = 7.0 Hz,
H-140), 1.1–1.4 (m, 22H, 11 ꢂ CH2), 1.56 (m, 2H, H-20), 1.9–2.1
(C-30), 30.2–30.9 (C-20, C-40 to C-80), 31.7 (t, J = 22 Hz,
C-100), 62.8 (C-6), 70.9 (C-10), 71.8 (C-4), 75.1 (C-2), 78.0
(C-5), 78.1 (C-3), 104.4 (C-1). 19F NMR (282 MHz, CD3OD)
d/ppm ꢄ81.0 (CF3), ꢄ114.1 (CF2), ꢄ124.0 (CF2), ꢄ125.7
(CF2). Anal. Calc. for C20H31F9O6: C 44.61, H 5.80. Found:
C 44.39, H 5.68%. HR-MS of [M + Na]+ m/z: Calc.
561.1875, Found. 561.1880.
0
0
0
0
(4 ꢂ s, 12H, 4 ꢂ CH3CO), 3.48 (dt, 1H, J1 a,1 b = 9.6 Hz, J1 a,2
= 6.6 Hz, H-10a), 3.69 (ddd, 1H, J4,5 = 9.8 Hz, J5,6a = 2.5 Hz,
0
0
0
0
J5,6b = 4.7, H-5), 3.86 (dt, 1H, J1 a,1 b = 9.6 Hz, J1 a,2 = 6.6 Hz,
H-10b), 4.13 (dd, 1H, J6a,6b = 12.2 Hz, J6a,5 = 2.5 Hz, H-6a),
4.26 (dd, 1H, J6a,6b = 12.2 Hz, J5,6b = 4.7 Hz, H-6b), 4.48
(d, 1H, J1,2 = 8.0 Hz, H-1), 4.98 (dd, 1H, J1,2 = 8.0 Hz, J2,3
=
9.5 Hz, H-2), 5.09 (‘‘t’’, 1H, J B 9.6 Hz, H-4), 5.21 (‘‘t’’, 1H, J B
9.4 Hz, H-3). 13C NMR (75 MHz, CDCl3): d/ppm 14.3 (C-140),
20.8–20.9 (4 ꢂ CH3CO), 22.9 (C-130), 26.1 (C-30), 29.5–29.9
(C-20, C-40 to C-110), 32.1 (C-120), 62.2 (C-6), 68.7 (C-4), 70.4
(C-10), 71.6 (C-2), 71.9 (C-5), 73.0 (C-3), 101.0 (C-1), 169.4,
169.6, 170.5, 170.9 (4ꢂCOCH3). Positive-ion ESI-MS peak at
m/z 567 ([M + Na]+).
12,12,13,13,14,14,15,15,15-Nonafluoropentadecyl-b-D-gluco-
pyranoside (8d). White solid; mp 98–99 1C; mp (DSC) 101.57 ꢃ
1
0.88 1C, 174.57 ꢃ 0.07 1C; yield, 98%; H NMR (400 MHz,
CD3OD): d/ppm 1.3–1.4 (m, 14H, 7 ꢂ CH2), 1.6–1.7 (m, 4H,
H-20 and H-100), 2.18 (m, 2H, H-110), 3.22 (‘‘t’’, 1H, J B
8.0 Hz, H-2), 3.3–3.4 (m, 3H, H-3, H-4 and H-5), 3.58 (m, 1H,
H-10a), 3.70 (dd, 1H, J6a,6b = 11.9 Hz, J5,6b = 5.3 Hz, H-6a),
3.8–4.0 (m, 2H, H-6b and H-10b), 4.29 (d, 1H, J1,2 = 7.8 Hz,
H-1). 13C NMR (100 MHz, CD3OD) d/ppm 21.3 (C-100), 27.1
(C-30), 30.2–30.9 (C-20, C-40 to C-90), 31.8 (t, J = 22 Hz,
C-110), 62.9 (C-6), 70.9 (C-10), 71.8 (C-4), 75.2 (C-2), 77.9
(C-5), 78.3 (C-3), 104.4 (C-1). 19F NMR (282 MHz, CD3OD)
d/ppm ꢄ81.0 (CF3), ꢄ114.1 (CF2), ꢄ124.0 (CF2), ꢄ125.7
(CF2). Anal. Calc. for C21H33F9O6: C 45.65, H 6.02. Found:
C 44.97, H 6.11%. HR-MS of [M + Na]+ m/z: Calc.
575.2031, Found. 575.2041.
Pentadecyl-2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside (7d)6.
1
White solid; mp 68–69 1C; yield, 53%; H NMR (300 MHz,
CDCl3): d/ppm 0.88 (t, 3H, J = 7.0 Hz, H-150), 1.1–1.4
(m, 24H, 12 ꢂ CH2), 1.56 (m, 2H, H-20), 2.0–2.1 (4 ꢂ s,
0
0
0
0
=
12H, 4 ꢂ CH3CO), 3.47 (dt, 1H, J1 a,1 b = 9.7 Hz, J1 a,2
6.7 Hz, H-10a), 3.69 (ddd, 1H, J4,5 = 9.9 Hz, J5,6a = 2.5 Hz,
0
0
0
0
J5,6b = 4.7, H-5), 3.86 (dt, 1H, J1 a,1 b = 9.7 Hz, J1 a,2 =
6.4 Hz, H-10b), 4.13 (dd, 1H, J6a,6b = 12.3 Hz, J6a,5 = 2.5 Hz,
H-6a), 4.26 (dd,1H, J6a,6b = 12.3 Hz, J5,6b = 4.6 Hz, H-6b),
4.48 (d, 1H, J1,2 = 7.9 Hz, H-1), 4.98 (dd, 1H, J1,2 = 7.9 Hz,
J2,3 = 9.6 Hz, H-2), 5.08 (‘‘t’’, 1H, J B 9.7 Hz, H-4), 5.21 (‘‘t’’,
1H, J B 9.4 Hz, H-3). 13C NMR (75 MHz, CDCl3): d/ppm
14.3 (C-150), 20.8–20.9 (4 ꢂ CH3CO), 22.9 (C-140), 26.0 (C-30),
29.5–29.9 (C-20, C-40 to C-120), 32.2 (C-130), 62.3 (C-6), 68.7
(C-4), 70.5 (C-10), 71.6 (C-2), 72.0 (C-5), 73.1 (C-3), 101.1
(C-1), 169.5, 169.6, 170.5, 170.8 (4 ꢂ COCH3). Positive-ion
ESI-MS peak at m/z 581 ([M + Na]+).
Alkyl b-D-glucopyranosides
Tetradecyl-b-D-glucopyranoside (9a)38,44. White powder; mp
69–71 1C (lit.: 64.8 1C44); mp (DSC) 66.96 ꢃ 2.52 1C, 151.47 ꢃ
3.97 1C; yield, 95%; 1H NMR (300 MHz, CD3OD): d/ppm
0.90 (m, 3H, J = 6.9 Hz, H-140), 1.3–1.4 (m, 22H, 11 ꢂ CH2),
1.63 (m, 2H, H-20), 3.31 (‘‘t’’, 1H, J B 8.9 Hz, H-2), 3.2–3.4
(m, 3H, H-3, 4 and 5, overlapped with residue proton of
CD3OD), 3.57 (m, 1H, H-10a), 3.71 (dd, 1H, J6a,6b = 12.0 Hz,
J5,6a = 5.0 Hz, H-6a), 3.8–4.0 (m, 2H, H-6b and H-10b), 4.28
(d, 1H, J1,2 = 7.7 Hz, H-1). 13C NMR (75 MHz, CD3OD)
d/ppm 14.4 (C-140), 23.7 (C-130), 27.1 (C-30), 30.4–30.8 (C-20,
C-40 to C-110), 33.0 (C-120), 62.9 (C-6), 71.0 (C-10), 71.8 (C-4),
75.2 (C-2), 77.9 (C-5), 78.2 (C-3), 104.4 (C-1). Anal. Calc.
for C20H40O6: C 63.80, H 10.71. Found: C 63.97, H 10.67%.
HR-MS of [M + Na]+ m/z: Calc. 399.2723, Found. 399.2735.
General procedure for the synthesis of perfluoroalkyl and
alkyl b-D-glucopyranosides (8a–f and 9a–f)8. A solution of
sodium methoxide (5 mmol) in methanol (5 mL) was added
dropwise to a solution of the respective tetraacetylated gluco-
pyranoside 6a–f or 7a–f (2 mmol) in methanol (10 mL) and the
mixture was stirred at ambient temperature for 1 h. The
reaction mixture was neutralized by addition of Dowexs
ꢁc
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Pentadecyl-b-D-glucopyranoside (9d). White powder; mp
75–77 1C; mp (DSC): 72.27 ꢃ 2.85 1C, 154.32 ꢃ 7.84 1C;
yield, 95%; 1H NMR (300 MHz, CD3OD): d/ppm 0.90
(m, 3H, J = 7.0 Hz, H-150), 1.2–1.4 (m, 24H, 12 ꢂ CH2),
1.63 (m, 2H, H-20), 3.19 (‘‘t’’, 1H, J B 8.9 Hz, H-2), 3.2–3.4
(m, 3H, H-3, 4 and 5, overlapped with residue proton of
CD3OD), 3.57 (m, 1H, H-10a), 3.71 (dd, 1H, J6a,6b = 12.0 Hz,
J5,6a = 5.2 Hz, H-6a), 3.8–4.0 (m, 2H, H-6b and H-10b), 4.27
(d, 1H, J1,2 = 7.7 Hz, H-1). 13C NMR (75 MHz, CD3OD)
d/ppm 14.4 (C-150), 23.7 (C-140), 27.1 (C-30), 30.4–30.8 (C-20,
C-40 to C-120), 33.0 (C-130), 62.9 (C-6), 71.0 (C-10), 71.8 (C-4),
75.2 (C-2), 77.8 (C-5), 78.2 (C-3), 104.4 (C-1). Anal. Calc. for
C21H42O6: C 64.58, H 10.84. Found: C 64.06, H 10.64%.
HR-MS of [M + Na]+ m/z: Calc. 413.2879. Found. 413.2870.
2.5–3.0 ꢂ 104 mLꢄ1, 100 mL per well, and allowed to grow
for 16–24 h in culture medium. The tested compounds were
first dissolved in DMSO (Sigma, Czech Republic) and than in
sterile PBS. Final concentrations of DMSO in samples were
below 1%. PBS with DMSO (1 and 5%) served as control.
No cytotoxicity of 1% DMSO in PBS was observed. Gluco-
pyranosides dissolved in sterile PBS (total volume of 20 mL) were
added to each well and the cytotoxic effect was evaluated after
24 h of exposure over a concentration range from 6 mM to
1.5 mM using the MTT assay. Octylthioglucoside (Roche) was
used as a positive control.
MTT (Sigma Chemical Co., Czech Republic) was dissolved
in PBS at a concentration of 5 mg mLꢄ1 and sterilized by
filtration. MTT solution was added into all wells of 96-well
flat-bottom microplates with cells in a dose of 20 mL per well.
The plates were incubated for 3 h. To enhance the dissolution
of dark-purple crystals of formazan, 110 mL of 10% SDS in
PBS (final pH 5.5) were added to all wells. The microtitre
plates were stored in a light-tight box at room temperature,
evaluated on the next day using a well-plate spectro-
photometer reader EL 800 (BioTek, USA) at 540 nm and
the EC50 (i.e. the molar concentration which produces 50% of
the maximum possible inhibitory response) values were calcu-
lated from the dose response curves. All experiments were
performed in triplicate and EC50 values were calculated using
GraphPad PRISM V.4.00 (GraphPad Software Inc., San
Diego, CA).
Synthesis of orthoester 11
Ag2CO3 (5 mmol) was added to a solution of 2,3,4,6-tetra-
acetyl-a-D-glycopyranosyl bromide (10)58 in anhydrous DCM
(10 mL) at 0 1C under a nitrogen atmosphere. The mixture was
stirred for 10 min and alcohol 3e (5 mmol) in anhydrous DCM
(10 mL) was added slowly. After 3 h, additional DCM (20 mL)
was added, the reaction mixture was filtered and the solvent
removed under reduced pressure. The yellowish residue was
purified by column chromatograph on silica gel with hexane
and ethyl acetate (3 : 1, v/v) as eluent.
3,4,6-Tri-O-acetyl-1,2-[1-(12,12,13,13,14,14,15,15,16,16,17,17,-
17-tridecafluoroheptadecyloxy)ethylidene]-a-D-glucopyranose).
White powder; mp 82–83 1C; yield, 80%; 1H NMR (300 MHz,
CDCl3): d/ppm 1.27 (m, 14H, 7 ꢂ CH2), 1.56 (m, 4H, H-20 and
H-100), 1.71 (s, 3H, CH3), 2.04–2.11 (m, 11H, 3 ꢂ CH3CO and
H-110), 3.46 (t, 2H, J = 6.6 Hz, H-10), 3.96 (m, 1H, H-5), 4.19
(m, 2H, H-2 and H-6aHH), 4.30 (m, 1H, H-6b), 4.90 (m, 1H,
H-4), 5.17 (‘‘t’’, 1H, J = 2.7 Hz, H-3), 5.70 (d, 1H, J = 5.2 Hz,
The results from the MTT assay were further confirmed by
Hoffman modulation contrast microscopy (epifluorescent
inverted microscope T200, Nikon, Japan) exposing morpho-
logical changes of the cells treated with various surfactants.
Propidium iodide (PI) and YO-PRO-1 (Molecular Probes,
Oregon, USA) were used to distinguish dead or apoptotic
cells from vital living ones.61
13
H-1). C NMR (75 MHz, CDCl3) d/ppm 20.3 (C-30),
20.9–20.1 (3 ꢂ CH3CO and CH3), 26.3 (C-100), 29.3–29.9
(C-20, C-40 to C-90), 31.1 (t, J = 22 Hz, C-110), 63.3 (C-6),
63.9 (C-10), 67.1 (C-5), 68.4 (C-4), 70.3 (C-3), 73.2 (C-2), 97.1
(C-1), 121.5 (QC(CH3)OR), 169.4, 169.9, 170.9 (3 ꢂ COCH3).
19F NMR (282 MHz, CDCl3): d/ppm ꢄ81.3 (CF3), ꢄ114.9
(CF2), ꢄ122.5 (CF2), ꢄ123.4 (CF2), ꢄ124.1 (CF2), ꢄ126.7
(CF2). Anal. Calc. for C31H41F13O10: C 45.37, H 5.04. Found:
C 45.40, H 5.05%.
Haemolytic activity. Rabbit red blood cells (2% in PBS)
were used to perform standard haemolytic tests in both PBS
and PBS containing 20% fetal bovine serum (Gibco). The
surfactants were tested in concentration range extending from
40 mM to 20 mM. The tested compounds were dissolved in
DMSO and added into PBS. Maximal final concentration of
DMSO in PBS was 5% for 20 mM concentration of tested
compound. This concentration of DMSO did not cause any
haemolysis in control red blood cells. After 2 h incubation of
red cells with a particular compound at 37 1C, the released
haemoglobin was separated from red blood cells by centrifu-
gation (700 g, 10 min, three washes) and quantified using a
well-plate spectrophotometer reader EL 800 (BioTek, USA) at
540 nm. Data are expressed as the lowest concentration of
surfactant causing 50% haemolysis.
Assessment of cytotoxicity
Cancer cell line. The B16F10 mouse melanoma cell line
(ECACC) was selected from a panel of cancer cell lines used
for testing in our laboratory. The cell line was grown in
D-MEM medium (Sigma, Czech Republic) supplemented with
10% of fetal calf serum (Gibco, Czech Republic), 50 mg Lꢄ1
penicillin, 50 mg Lꢄ1 streptomycin, 100 mg Lꢄ1 neomycin, and
300 mg Lꢄ1 L-glutamine as reported previously.12,46 Cultures
were maintained in a humidified incubator (Shellab, Sheldon,
OR, USA) at 37 1C and 5% CO2.
Acknowledgements
The authors thank Dr Paul Bummer (University of Kentucky)
for access to the DSC instrument. This work was supported by
grants from the National Institute of Environmental Health
Sciences (ES12475), the National Science Foundation (NIRT
0210517), the US Department of Agriculture Biomass
Research and Development Initiative (Grant Agreement
MTT-based cytotoxicity test. The MTT assay59,60 was
used to assess the cytotoxicity of the glucopyranosides in
cells in the exponential growth phase. In short, cells were
seeded on 96-well flat-bottom microplates at the density
ꢁc
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2178 | New J. Chem., 2008, 32, 2169–2179

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68-3A75-7-608) and the Department of Energy Development
and Independence, Energy and Environment Cabinet of The
Commonwealth of Kentucky. Additional support was pro-
vided by grants from the Ministry of Agriculture of the Czech
Republic (grant No. MZE-0002716201) and NAZV QF-3115
to J. T. Its contents are solely the responsibility of the authors
and do not necessarily represent the official views of the
funding agencies.
27 M. Hein and R. Miethchen, Tetrahedron Lett., 1998, 39, 6679.
28 M. Hein, R. Miethchen and D. Schwaebisch, J. Fluorine Chem.,
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