DiGalactosyl-Glycero-Ether Lipid: Synthetic approaches and evaluation as SK3 channel inhibitor

Article abstract of DOI:10.1039/c3ob40634b

The recent discoveries of the involvement of SK3 channel in some cell motility mechanisms occurring in cancer disease have opened up the way to the synthesis of inhibitors that could reduce metastasis formation. On the basis of our recent previous works showing that both lactose-glycero-ether lipid (Ohmline) and some phosphate analogues (GPGEL) were efficient compounds to modulate SK3 channel activity, the present study, which found its inspiration in the structure of the natural glycolipid DiGalactosylDiacylGlycerol (DGDG), reports the incorporation of a digalactosyl moiety (α-galactopyranosyl- (1→6)-β-galactopyranosyl-) as the polar head of a glycero ether lipid. For the construction of the digalactosyl fragment, two synthetic approaches were compared. The standard strategy which is based on the use of the benzyl protecting group to produce 1→6 disaccharide unit, was compared with a second method that made use of the trimethylsilyl moiety as a protecting group. This second strategy, which is applied for the first time to the synthesis of (1→6)-disaccharide unit, presents a net advantage in terms of efficacy (better global yield) and cost. Finally, compound 16, which is characterized by a (1→6) DiGalactosyl unit (DG) as the polar head of the amphiphilic structure, was tested as a modulator of the SK3 channel activity. Patch-clamp experiments have shown that compound 16 reduced SK3 currents (-28.2 ± 2.0% at 5 μM) and cell migration assays performed at 300 nM have shown a reduction of cell migration (SK3 + HEK293T) by 19.6 ± 2.7%. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40634b

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DiGalactosyl-Glycero-Ether Lipid: synthetic approaches
and evaluation as SK3 channel inhibitor†
Cite this: DOI: 10.1039/c3ob40634b
Charlotte M. Sevrain,a Jean-Pierre Haelters,*a Aurélie Chantôme,b,c
Hélène Couthon-Gourvès,a Maxime Gueguinou,b,c Marie Potier-Cartereau,b,c
Christophe Vandier*b,c and Paul-Alain Jaffrès*a
The recent discoveries of the involvement of SK3 channel in some cell motility mechanisms occurring in
cancer disease have opened up the way to the synthesis of inhibitors that could reduce metastasis for-
mation. On the basis of our recent previous works showing that both lactose-glycero-ether lipid
(Ohmline) and some phosphate analogues (GPGEL) were efficient compounds to modulate SK3 channel
activity, the present study, which found its inspiration in the structure of the natural glycolipid DiGalacto-
sylDiacylGlycerol (DGDG), reports the incorporation of a digalactosyl moiety (α-galactopyranosyl-(1→6)-
β-galactopyranosyl-) as the polar head of a glycero ether lipid. For the construction of the digalactosyl
fragment, two synthetic approaches were compared. The standard strategy which is based on the use of
the benzyl protecting group to produce 1→6 disaccharide unit, was compared with a second method
that made use of the trimethylsilyl moiety as a protecting group. This second strategy, which is applied
for the first time to the synthesis of (1→6)-disaccharide unit, presents a net advantage in terms of
efficacy (better global yield) and cost. Finally, compound 16, which is characterized by a (1→6) DiGalacto-
syl unit (DG) as the polar head of the amphiphilic structure, was tested as a modulator of the SK3
channel activity. Patch-clamp experiments have shown that compound 16 reduced SK3 currents (−28.2
2.0% at 5 μM) and cell migration assays performed at 300 nM have shown a reduction of cell migration
(SK3 + HEK293T) by 19.6 2.7%.
Received 30th March 2013,
Accepted 10th May 2013
DOI: 10.1039/c3ob40634b
www.rsc.org/obc
lines known for their high metastatic potential.4 Interestingly,
the inhibition of this channel by apamin (peptide consisting
1. Introduction
Most of the cancer chemotherapy approaches aim to reduce of 18-amino acid units) reduced cell migration in the course of
primary tumour growing. Besides the actions on tumor in vitro assays.5 However, apamin which is a neurotoxic is
growth, the limitation of metastasis formation and cancerous therefore not relevant for in vivo studies. Besides, many hetero-
cell spreading constitutes a complementary therapeutic goal.1 cyclic compounds have been identified as SKCa channel
Many cell targets have been explored to develop anti-cancer inhibitors and used for in vitro assays.4 In addition to the
agents2 and more recently, it was shown that some ion chan- studies dedicated to heterocyclic compounds, we found that
nels were also involved in cancer spreading, and can be viewed amphiphilic compounds like edelfosine (Fig. 1) were also able
as new targets.3 Some of us have demonstrated that the SK3 to modulate the activity of SKCa channels and more specifi-
channel (belonging to the small conductance calcium-acti- cally SK3 channel.6 However, the toxicity of edelfosine limited
vated K+ channels family, SKCa) was a key actor of cell its use for in vivo applications.
migration associated with some types of cancers.4 Moreover,
In very recent works, we have synthesized and shown that
the expression of this channel was found in several cancer cell the replacement of the polar head of edelfosine by a sacchar-
ide or disaccharide unit produced very efficient SK3 channel
inhibitors.7 As a consequence of this inhibition, cell migration
was reduced in vitro. Ohmline, which is one of the more
efficient and non-toxic compounds in this series of glyco-ether
lipids, was characterized by a polar head formed by a lactose
unit (Fig. 1). More interestingly, this compound demonstrated
its strong in vivo potential since its administration (systemic
administration) led to an improvement of the metastatic
profile in a murine breast cancer model.7 These results, which
aUniversité de Brest, UEB, CEMCA, CNRS UMR 6521, SFR ScInBIoS, 6 Avenue
Le Gorgeu, 29238 Brest, France. E-mail: jean-pierre.haelters@univ-brest.fr,
pjaffres@univ-brest.fr
bInserm, U1069, Tours, F-37032 France
cUniversité François Rabelais, Tours, F-37032 France.
E-mail: christophe.vandier@univ-tours.fr
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob40634b
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Fig. 2 Retrosynthetic scheme for the synthesis of DiGalactosyl-Glycero-Ether
Lipid 16 from galactosepentaacetate.
important part of this study was dedicated to the comparison of
two synthetic approaches used for the synthesis of the digalacto-
syl unit. Finally, the capacity of compound 16 to modulate SK3
channel activity is reported jointly with migration assays and
toxicity measurements.
2. Results and discussion
Fig. 1 Chemical structure of edelfosine, Ohmline, Glyco-Phospho-Glycero-Ether
The general retrosynthetic scheme to produce the targeting
compounds 16 that possess a polar head formed by a 1→6 con-
nected disaccharide (digalactosyl) unit is based on the glycosy-
lation reaction of compound 5 with a galactosyl derivative
correctly protected (Fig. 2). The synthesis of compound 5 can
be easily achieved in 4 steps from galactosepentaacetate 1 (see
Fig. S1†). For the glycosylation reaction of compound 5, the
choice of orthogonal protecting groups (benzyl/trityl) constitu-
tes the classical synthetic strategy that permits a selective
deprotection of the hydroxy group in position 6 of the galac-
tose unit present in compound 5. This strategy was previously
reported for the construction of 1→6 connected disaccharide
derivatives.12 Herein, we have also tested a second strategy that
was initially proposed for the synthesis of α-galactosylcera-
mides13 and that made use of the trimethylsilyl moiety as a
protecting group: one first advantage of such a strategy for the
synthesis of disaccharide units lies in the number of synthetic
steps, which is reduced when compared to the first approach.
According to the first strategy (benzyl/trityl protecting
groups),14 the hydroxyl group in position 6 of the galactosyl
motif was first protected with a trityl moiety to produce 6
(Fig. 3 – part A). The three remaining hydroxyl groups were
then protected with a benzyl motif to produce the intermediate
7. This compound was then selectively deprotected in position
615 thus affording compound 8 with a global yield of 50% (two
steps from 5). According to the second strategy (trimethylsilyl
as a protecting group), the free hydroxyl groups of the glyco-
lipid 5 were silylated16 to produce 9 (Fig. 3 – part B). Then the
protected primary alcohol was selectively deprotected by reac-
tion with acetic acid17 to produce compound 10 in two steps
from 5 (49% yield – two steps).
Lipid (GPGEL) and DiGalactosylDiacylGlycerol (DGDG).
demonstrated the interest in modulating SK3 channel activity
to reduce metastasis formation, opened up the way to the
design and evaluation of new structures. Toxicity of the new
SK3 modulators constitutes another feature that must be con-
sidered with great attention in order to select compounds
having suitable properties for in vivo studies. In this way we
have very recently reported a first modification of Ohmline by
the introduction of a phosphate functional group that was
placed between the saccharide and the Glycero-Ether-Lipid
moiety (Fig. 1), thus defining a new series of compounds
labelled as Glyco-Phospho-Glycero Ether Lipids (GPGEL).8 This
series of compounds was also efficient in reducing SK3
channel activity without any noticeable toxicity. These results
point out that structural modifications of the polar head were
possible without affecting SK3 inhibition. By a careful study of
the wide structural diversity of natural glyco-lipids, our atten-
tion was focused on DiGalactosylDiacylGlycerol (DGDG)
(Fig. 1); this type of natural glycol-lipids is present in both pro-
karyotic and eukaryotic cells. It was reported that some plants
(e.g. oat) can substitute phosphocholine-based glycerolipids by
DGDG as a consequence of phosphate deprivation.9 These
results suggest that the (1→6) DiGalactosyl group (DG) could
replace the phosphocholine moiety to produce glycerolipids
having a similar physiological function.10 On the basis of the
hypothesis that the phosphocholine moiety (also present in
the structure of edelfosine) can be replaced by a (1→6) digalac-
tosyl fragment, we report herein the synthesis of one ether
lipid possessing a digalactosyl unit thus defining the polar
head region. Following a biomimetic approach, the configur-
ation of the anomeric position of the digalactosyl unit in the
new ether lipid was controlled and was similar to those present
in DGDG (α-galactopyranosyl-(1→6)-β-galactopyranosyl-).11 One
For the next step (glycosylation reaction), the second sac-
charide unit (in our case a galactopyranosyl fragment) must be
previously prepared with the adapted protecting groups. More-
over these protecting groups must be compatible with the
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Fig. 3 Synthesis of the two intermediates 8 and 10 that make use of two
different kinds of protecting groups. (A) Trityl and benzyl protecting groups:
(a) TrCl, DABCO, dry CH2Cl2, 3 hours, R.T. 74%, (b) NaH, BnBr, dry DMF, 15 hours,
R.T. (c) APTS, CH2Cl2/MeOH 20 hours, R.T., 67% (two steps). (B) Trimethylsilyl
(TMS) as a protecting group: (d) TMSCl, Et3N, dry DMF 92%, (e) CH3CO2H,
acetone/MeOH, 48 hours, R.T., 53%.
activation of the anomeric position that will be used to
produce α-glycosylation. First, a combination of benzyl and
acetyl protecting groups was selected (Fig. 4 – Part A). Accord-
ing to this synthetic scheme, the galactosyl compound 12, pro-
tected in positions 1 and 6 with an acetyl moiety and a benzyl
group at positions 2, 3 and 4, was prepared in two steps (56%
yield)18 from the quite expensive commercial compound 11
(1-O-methyl α-D-galactopyranoside). Then, the anomeric posi-
tion was converted in the α-iodide intermediate 13 which is, as
previously reported,13c,19 suitable for α-glycosylation. The glyco-
sylation reaction involving 13 and the glyco-glycero-lipid 8 was
achieved in 65% yield. The protected disaccharide 14 was then
deprotected in two steps. First the acetyl group present in posi-
tion 6 of the terminal galactosyl fragment was removed in 47%
yield followed by the debenzylation of the remaining protected
hydroxyl groups. This debenzylation step required a large
amount of catalyst (Pd/C) for completion of the reaction. After
purification compound 16 was isolated in 56% yield. A second
strategy based on the use of trimethylsilyl as a protecting
group was investigated. This second strategy that was recently
employed for the synthesis of monosaccharide derivatives13
was, to the best of our knowledge, never applied to the syn-
thesis of disaccharide compounds. This sequence starts from
galactose 17 (Fig. 4 – part B). Of note, this starting material is
much cheaper than 1-O-methyl α-D-galactopyranoside 11 (com-
pound 11 is almost 10 times more expensive than galactose
17) used in the first synthetic approach. The per-silylation reac-
tion of galactose 17 produced compound 18 in excellent yield
(92%). Then, the deprotection of the anomeric position and its
one-pot transformation in α-iodide derivative were achieved as
reported by Gervay-Hague et al.13,19 Then, compound 19 was
isolated in good yield (72%, two steps) after a glycosylation
reaction with the previously prepared TMS-protected
Fig. 4 Final steps for the synthesis of 16 following two different synthetic
schemes. (A) Strategy with benzyl and acetyl as protecting groups: (a) TMSI, dry
CH2Cl2, 30 min, 0 °C, (b) compound 8, DIPEA, TBAI, dry benzene, 3 hours, reflux,
65% (two steps), (c) K2CO3, MeOH, 2 hours, 20 °C, 47%, (d) Pd/C, MeOH, 20
bars H2, 48 hours, 20 °C, 56%. (B) Strategy with trimethylsilyl (TMS) as a protect-
ing group: (e) TMSCl, Et3N, dry DMF, 92%, (f ) TMSI, dry CH2Cl2, 2 hours, 0 °C
and then (g) compound 10, DIPEA, TBAI, CH2Cl2 anh., 36 hours, 20 °C, 72%
(two steps), (h) Dowex 50WX8-200®, MeOH, 4 hours, 20 °C, 77%.
monosaccharide 10. Finally, the deprotection of the alcohol
functional groups occurred in only one step to produce com-
pound 16 after reaction with Dowex 50WX8-200® ion exchange
resin (77% yield). Accordingly 101 mg of compound 16 were
isolated and fully characterized (see the Experimental section).
The two synthetic schemes shown in Fig. 4 allow producing
the expected compound 16. According to this first strategy
29 mg of compound 16 were isolated in 8 steps (Fig. 3 and 4)
from 5 and 11 and with a global yield of 5%. On the other
hand, the second strategy (Fig. 3 and 4, part B) required a
reduced number of steps (5 instead 8) and is characterized by
a higher global yield (25%). Moreover this second way was
economically advantageous since galactose 17 is definitively
cheaper than methyl α-D-galactopyranoside 11.
Next, compound 16 was tested as an SK3 channel inhibitor
on the HEK293T cells expressing homotetramer SK3 channels,
HEK293T-SK3 cells. We evaluated the inhibitory effect of com-
pound 16 on whole cell SK3 current using the patch-clamp
technique (Fig. 5A). It must be noted that testing the com-
pounds at 5–10 μM on the SK3 channel permitted a fast and
robust pre-selection of effective SK3-channel inhibitors that
were then tested, at lower concentration (see below), to reduce
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1 μM Ohmline.7 Based on these results indicating that com-
pound 16 is able to inhibit SK3 channel activity as does
Ohmline, the next experiments were dedicated to evaluating
the capacity of this molecule to inhibit SK3-dependent cell
migration; this type of migration is clearly implicated in
cancer cell spreading. If a 15 h treatment with 100 nM com-
pound 16 has no effect on cell migration, increasing the con-
centration to 300 nM significantly inhibited cell migration of
HEK293T-SK3 (Fig. 5B; reduction of 19.6 2.7%). The capacity
of compound 16 to reduce SK3-dependent cell migration was
lower than that observed with Ohmline (close to 50% with
100 nM Ohmline).8 Finally, the toxicity of compound 16 was
evaluated following MTT assays at 1 and 10 μM for 24 h and
48 h. As reported in Fig. 5C, compound 16 shows only a notice-
able level of toxicity at 10 μM demonstrating that it exhibited
no toxicity at 5 μM or at 300 nM, the concentrations used in
patch-clamp experiments and cell migration assays. These
results invite some conclusions. First, this study clearly shows,
once more,7 the consistency between the patch-clamp experi-
ments and the SK3-dependent migration assays. Herein, the
SK3 current reduction of 28.2 2.0% (patch-clamp) is corre-
lated to a decrease of cell migration by 19.6 2.7%. Second,
the decrease of SK3 current with compound 16 is less impor-
tant than with Ohmline. This difference is likely explained by
the (1→6)-glycosidic link (compound 16) where a (1→4) con-
nection is present for Ohmline and/or to the different anome-
ric configuration of the terminal saccharide unit (α in the case
of compound 16 and β in the case of Ohmline). Finally, it
must be mentioned that compound 16, which belongs to the
family of SK3 inhibitors possessing an amphiphilic structure,
does not likely interact with the pore of the channel but would
modify the biophysical properties of bio-membranes, thus
impacting the SK3 activity. This hypothesis is supported by
our recent work,20 which demonstrates the reduction of SK3
channel activity as a result of the dissociation of the SK3–Orai1
complex, localized in the plasma membrane, which is present
in cancer cell lines. Accordingly, the shape of the amphiphilic
compounds, as previously reported for the activation of K+
channels TREK-1 and TRAAK,21 is a parameter that is worth to
be considered for the design of new SK3 inhibitors.
Fig. 5 Compound 16 inhibited SK3 currents and SK3-dependent cell
migration. (A) Typical example of whole cell K+ currents recorded in HEK293T
cells expressing recombinant SK3 channels before (vehicle: 0.6‰ ethanol/
0.4‰ DMSO) and after the application of 5 μM compound 16 for 4 min. Cur-
rents were generated by ramp protocol from +95 mV to −95 mV in 500 ms
from a holding potential of 0 mV. (B) Histograms showing the effect of 100 and
300 nM compound 16 on the cell migration of HEK293T expressing recombi-
nant SK3 channel. The normalized cell number corresponds to the ratio of the
total number of migrating cells in the presence of compound 16 (100, 300 nM)
to the total number of migrating cells in the presence of vehicle. (C) Cell viability
of compound 16 determined with the tetrazolium salt reduction method (MTT).
HEK293T cells expressing recombinant SK3 channel were either treated with
vehicle or compound 16 (1 and 10 μM) for 24 h or 48 h. Results of three inde-
pendents experiments performed in triplicate are expressed as mean S.E.M.
*significantly different from vehicle application at p < 0.05.
3. Conclusion
The naturally occurring DiGalactosylDiacylGlycerol (DGDG) is
characterized by a (α-galactopyranosyl-(1→6)-β-galactopyrano-
syl-) as the polar head. Previous studies have shown that this
polar moiety can replace the phosphocholine polar head in
some plant submitted to phosphorus deprivation. Previously
we have shown that edelfosine (a phosphocholine-based ether
lipid) and edelfosine’s analogues like Ohmline and Glyco-
Phospho-Glycero-Ether Lipids (GPGEL) were efficient inhibi-
tors of SK3 channel activity. Following a hypothesis based on
the biomimetic approach, we report the incorporation of the
digalactosyl unit present in DGDG in the structure of ether
lipid. To achieve this goal, two synthetic strategies were
SK3-dependent cancer cell migration. The effect of compound
16 on the amplitude of SK3 current was measured at 0 mV (the
current is only carried by SK3 channels under these con-
ditions) for 2–4 min after the application of compound 16.
Currents generated before and after the application of mole-
cules using the ramp protocol from +95 mV to −95 mV are
illustrated in Fig. 5. Compound 16, at a concentration of 5 μM,
decreased the amplitude of SK3 current to 28.2 2.0% of inhi-
bition (n = 5). This value is lower than the inhibitory capacity
of 10 μM Ohmline, −73%, and close to that observed with
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explored. The first one was based on the use of benzyl motif as DABCO (569 mg, 5.07 mmol). The mixture was stirred for
a protecting group. Following this classical strategy, the 2 hours at room temperature, filtered on Celite and concen-
expected DiGalactosyl-Glycero-Ether Lipid was isolated but in a trated. The crude product was purified by chromatography on
modest global yield (5%). The second strategy, based on the silica gel (ethyl acetate) and the pure compound was obtained
use of trimethylsilyl as a protecting group, was applied for the as a colorless oil (74%). Rf (ethyl acetate–petroleum ether: 2/1):
3
first time to produce disaccharide units. This new method is 0.27. 1H NMR (300.131 MHz, CDCl3): 0.88 (3H, t, JHH = 7.5,
more efficient since the final compound 16 was isolated in CH3); 1.24–1.40 (26H, m, CH2 fatty chain); 1.51–1.60 (2H, m,
better global yield (25%; 5 steps) starting from a cheaper start- CH2 β fatty chain); 3.34–3.71 (14H, m, CH2 α fatty chain +
ing material (galactose).
OCH3 + CH2 sn-1 + CH2 sn-3 + CH sn-2 + H3 + H5 + H6);
The inhibitory effect of compound 16 on SK3 channel 3.93–4.02 (2H, m, H2 + H4); 4.21–4.25 (1H, m, H1); 7.20–7.44
activity was first evaluated by the patch-clamp technique. It (15H, m, CH (Ph)). 13C NMR (75.475 MHz, CDCl3): 14.1 (CH3
was found that this compound was able to inhibit SK3 current fatty chain); 22.7(CH2 fatty chain); 26.1 (CH2 fatty chain); 29.4
albeit to a lower extant than Ohmline. These results point out (CH2 fatty chain); 29.5 (CH2 fatty chain); 29.7 (CH2 fatty chain);
that the structure of the disaccharide directly impacts the SK3 31.9 (CH2 fatty chain); 57.9 (OCH3); 62.6 (C6); 69.0 (C4); 69.2
inhibition process. Based on this inhibition effect of SK3 current, (CH2 sn-3); 70.2 (CH2 sn-1); 71.7 (C2); 71.9 (CH2 α fatty chain);
the SK3-dependent cell migration was measured and this com- 73.6 (C3); 73.8 (C5); 79.3 (CH sn-2); 86.9 (O-C-(Ph)3); 103.7 (C1);
pound was found to reduce cell migration as expected by an 127.1 (CHpara); 127.9 (CHmeta); 128.7 (CHortho); 143.7 (Cipso).
inhibitor of SK3 channel activity. Since the inhibitory capacity of
4.3. Synthesis of 3-(hexadecyloxy)-2-methoxypropyl-2,3,4-tri-O-
benzyl-β-D-galactopyranoside 8
compound 16 on the SK3 channel was lower than that observed
for Ohmline, it was not surprising to find the same lower capacity
to reduce SK3-dependent cell migration. In addition to methodo- To a stirred solution of 6 (1.0 g, 1.50 mmol) in dry DMF
logical aspects, this study points out the importance of the glyco- (20 mL) was added NaH (162 mg, 6.73 mmol). The mixture
sidic link of the disaccharide group incorporated in the glycero- was stirred for 2 hours at room temperature and then cooled
ether-lipid (1→6 versus 1→4 and α versus β glycosidic bond) on to 0 °C. Benzyl bromide (0.81 mL, 6.73 mmol) was added drop-
the SK3-dependent cell migration. The synthesis of other disac- wise and the mixture was stirred for 15 hours at room tempera-
charide-based glycero-ether-lipids (the current work) will be ture. Excess of NaH was neutralized by addition of a few mL of
achieved with the aim to further study the consequences of the MeOH. The solution was diluted with Et2O (30 mL) and
glycosidic link for SK3-dependent cell migration.
washed with water (15 mL). The organic layer was dried over
MgSO4, filtered and concentrated to give compound 7. The
product was dissolved in CH2Cl2–MeOH (1/2) (30 mL) and
p-toluenesulfonic acid (36 mg, 0.21 mmol) was added. The
mixture was stirred for 20 hours at room temperature and then
concentrated. The residue was dissolved in CH2Cl2 (30 mL)
and washed twice with water (10 mL), then with an aqueous
10% sodium carbonate solution (10 mL) and finally with brine
(10 mL). The organic layer was dried over MgSO4, filtered and
concentrated. The crude product was purified by chromato-
graphy on silica gel (ethyl acetate–petroleum ether: 1/2) and
the pure compound was obtained as a white powder (67%). Rf
(ethyl acetate–petroleum ether: 1/5): 0.21. 1H NMR
4. Experimental
4.1. General
Solvents were dried with a solvent purification system MBraun-
SPS (Methanol, CH2Cl2, DMF) or freshly distilled on appropri-
ate driers. All compounds were fully characterized by 1H
(500.13, 400.133 or 300.135 MHz) and 13C (125.773 or
75.480 MHz) NMR spectroscopy (Bruker AC 300, Avance DRX 400
and Avance DRX 500 spectrometers). Coupling constants J are
given in hertz. The following abbreviations were used: s for
singlet, d doublet, t triplet, q quadruplet, qt quintuplet, m for
multiplet and dt for doublet of triplets. When needed, 13C hetero-
nuclear HMQC and HMBC were used to unambiguously establish
molecular structures. Mass spectroscopy analyses were performed
by the “Service commun de spectrometrie de masse” (Brest) on a
Bruker Autoflex MALDI TOF-TOF III LRF200 CID. Fourier Trans-
form Infra-Red (FTIR) spectra were recorded on a Bruker Vertex
70 FT-IR (ATR) spectrometer. Commercial compounds were used
as received except DIPEA which was distilled over KOH and TMSI
which was distilled over CaCl2. Optical rotation was measured on
a Jasco P-1010 polarimeter using a 100 mm cell.
3
(400.012 MHz, CDCl3): 0.89 (3H, t, JHH = 7.0, CH3); 1.22–1.32
(26H, m, CH2 fatty chain); 1.53–1.56 (2H, m, CH2 β fatty
chain); 3.36–3.42 (3H, m, CH2 α fatty chain + H5); 3.42 (3H, s,
OCH3); 3.44–3.67 (6H, m, Ha-CH sn-1 + CH2 sn-3 + CH sn-2 +
H3 + H6a); 3.75–3.79 (2H, m, H4 + H6b); 3.83–3.87 (1H, m, H2);
3
3.97–4.10 (1H, m, Hb-CH sn-1); 4.39 (1H, t, JHH = 7.5, H1);
4.65–4.98 (6H, m, CH2-Ph); 7.29–7.38 (15H, m, CH (Ph)).
13C NMR (100.592 MHz, CDCl3): 14.2 (CH3 fatty chain); 22.7
(CH2 fatty chain); 26.1 (CH2 fatty chain); 29.4 (CH2 fatty chain);
29.5 (CH2 fatty chain); 29.7 (CH2 fatty chain); 32.0 (CH2 fatty
chain); 58.0 (OCH3); 62.1 (C6); 68.7 (CH2 sn-3); 70.3 (CH2 sn-1);
71.8 (CH2 α fatty chain); 73.0 (C4); 73.5 (CH2-Ph); 74.2
(CH2-Ph); 74.7 (C5); 75.1 (CH2-Ph); 79.3 (C2); 79.6 (CH sn-2);
82.3 (C3); 104.4 (C1); 127.4 (CH (Ph)); 127.9 (CH (Ph)); 128.5
4.2. Synthesis of 3-(hexadecyloxy)-2-methoxypropyl-6-O-trityl-
β-D-galactopyranoside 6
To a stirred solution of 5 (1.0 g, 2.03 mmol) in dry CH2Cl2 (CH (Ph)); 129.1 (CH (Ph)); 138.3 (Cipso (Ph)); 138.5 (Cipso (Ph));
(10 mL) was added tritylchloride (1.4 g, 5.07 mmol) and 138.7 (Cipso (Ph)); 138.8 (Cipso (Ph)).
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4.4. Synthesis of 3-(hexadecyloxy)-2-methoxypropyl-2,3,4,6-
tetrakis-O-(trimethylsilyl)-α-D-galactopyranoside 9
77.0 mmol) was added dropwise and the mixture was stirred
for 2 hours at 0 °C and 15 hours at room temperature. Excess
of NaH was neutralized by addition of a few mL of MeOH. The
solution was diluted by Et2O (200 mL) and washed with water
(150 mL). The aqueous layer was extracted twice with Et2O
(100 mL) and the organic layer was dried over MgSO4, filtered
and concentrated. The crude product was purified by chrom-
atography on silica gel (ethyl acetate–petroleum ether: 1/4) and
the pure α-methyl benzylated compound was obtained as a col-
orless oil. Rf (ethyl acetate ethyl–petroleum ether (1/4)): 0.30.
This compound (5.9 g, 10.6 mmol) was dissolved in acetic
acid–acetic anhydride (1/1) (70 mL) at 0 °C and H2SO4 (1.3 mL,
23.4 mmol) was added dropwise. The mixture was stirred for
5 hours at 0 °C, washed twice with a cold saturated sodium
bicarbonate solution (60 mL) and twice with water (30 mL).
The aqueous layer was extracted twice with CH2Cl2 (100 mL)
and then the organic layer was dried over MgSO4, filtered and
concentrated. The crude product was purified by chromato-
graphy on silica gel (ethyl acetate–petroleum ether: 1/4) and
the pure compound was obtained as a colorless oil (56%).
Rf (ethyl acetate–petroleum ether: 1/3): 0.14. 1H NMR
(400.012 MHz, CDCl3): 1.99 (3H, s, CH3); 2.13 (3H, s, CH3);
3.71–4.20 (6H, m, H2 + H3 + H4 + H5 + H6); 4.64–5.02 (6H, m,
CH2-Ph); 6.40–6.46 (1H, m, H1); 7.26–7.41 (15H, m, CH (Ph)).
13C NMR (75.475 MHz, CDCl3): 20.8 (OCH3); 21.1 (OCH3); 63.1
(C6); 70.8 (C5); 73.4 (CH2-Ph); 74.2 (C4); 74.7 (CH2-Ph); 75.4
(C2); 78.6 (C3); 90.7 (C1); 127.4 (CH (Ph)); 127.7 (CH (Ph));
127.8 (CH (Ph)); 128.1 (CH (Ph)); 128.4 (CH (Ph)); 137.9 (Cipso);
138.0 (Cipso); 138.5 (Cipso); 169.4 (CO); 170.6 (CO).
To a stirred solution of compound 5 (1.0 g, 2.0 mmol) and
Et3N (1.0 g, 10.0 mmol) in dry DMF (10 mL) cooled at 0 °C was
added TMSCl (1.1 g, 10.0 mmol) dropwise. The mixture was
stirred at room temperature overnight. Et2O (40 mL) was
added and the solution was placed on ice. The aqueous layer
was extracted twice with Et2O (20 mL). The organic layer was
washed with ice-water (10 mL) and brine (10 mL), dried over
MgSO4, filtered and concentrated. The expected product was
obtained as a colorless oil (92%). Rf (ethyl acetate–petroleum
ether: 1/5): 0.42. 1H NMR (400.012 MHz, CDCl3): 0.10–0.15
3
(36H, m, CH3 (OTMS)); 0.87 (3H, t, JHH = 6.8, CH3); 1.20–1.35
(26H, m, CH2 fatty chain); 1.53–1.58 (2H, m, CH2 β fatty chain);
3.36–3.44 (5H, m, CH2 α fatty chain + CH2 sn-1 + CH sn-2); 3.43
(3H, s, OCH3); 3.45–3.52 (6H, m, H3 + H5 + H6 + CH2 sn-3);
3
3.80–3.95 (2H, m, H2 + H4); 4.17 (1H, t, JHH = 8.4, H1). 13C
NMR (125.816 MHz, CDCl3): −0.45 (CH3Si); 0.45 (CH3Si); 0.7
(CH3Si); 14.1(CH3); 22.7 (CH2 fatty chain); 26.1 (CH2 fatty
chain); 26.2 (CH2 fatty chain); 29.4 (CH2 fatty chain); 29.5 (CH2
fatty chain); 29.7 (CH2 fatty chain); 31.4 (CH2 fatty chain); 57.8
(OCH3); 67.2 (C6); 68.7 (CH2 sn-3); 70.7 (CH2 sn-1); 71.7 (CH2 α
fatty chain); 71.5; 71.6; 75.2; 75.4 (C2 + C3 + C4 + C5); 79.5 (CH
sn-2); 101.3 (C1).
4.5. Synthesis of 3-(hexadecyloxy)-2-methoxypropyl-
2,3,4-tris-O-(trimethylsilyl)-α-D-galacto-pyranoside 10
To a stirred solution of compound 9 (1.0 g, 1.3 mmol) in a
mixture of acetone–methanol (3 : 4) (14 mL) at 0 °C was added
acetic acid (153 mg, 2.6 mmol). The solution was stirred for 4.7. Synthesis of 3-(hexadecyloxy)-2-methoxypropyl-6-O-
36 hours at room temperature. Sodium hydrogenocarbonate (6-O-acetyl-2,3,4-tri-O-benzyl-α-D-galactopy-ranosyl)-2,3,4-tri-O-
(450 mg, 5.4 mmol) was added to neutralize the acid. The benzyl-β-D-galactopyranoside 14
mixture was filtered and concentrated. The residue was puri-
To a stirred solution of 12 (700 mg, 1.31 mmol) in dry CH2Cl2
fied by chromatography on silica gel (ethyl acetate–petroleum
(4 mL) under argon was added TMSI (178 μL, 1.31 mmol) at
ether: 1/2) to give the pure product as a colorless oil (53%).
0 °C. The mixture was stirred for 30 minutes at 0 °C and a few
mL of freshly distilled toluene were added. The solvent was
Rf (ethyl acetate–petroleum ether: 1/3): 0.29. 1H NMR
(400.012 MHz, CDCl3): 0.09–0.14 (27H, m, CH3 (OTMS)); 0.88
removed at 0 °C and several azeotropes have been made until
3
(3H, t, JHH = 6.8, CH3); 1.15–1.32 (26H, m, CH2 fatty chain);
1.48–1.65 (2H, m, CH2 β fatty chain); 3.33–3.46 (5H, m, CH2 α
fatty chain + CH2 sn-3 + H5); 3.41 (3H, s, OCH3); 3.46–3.68 (5H,
m, H3 + H4 + H6 + Ha-CH sn-1); 3.69–3.72 (1H, m, H4);
3.77–3.89 (2H, m, H2 + Hb-CH sn-1); 4.21 (1H, t, 3JHH = 8.2, H1).
13C NMR (75.475 MHz, CDCl3): 0.46 (CH3Si); 0.75 (CH3Si); 14.2
(CH3); 22.7 (CH2 fatty chain); 26.1 (CH2 fatty chain); 29.4 (CH2
fatty chain); 29.5 (CH2 fatty chain); 29.7 (CH2 fatty chain); 32.0
(CH2 fatty chain); 57.8 (OCH3); 62.9 (C6); 69.0 (CH2 sn-3); 70.7
(CH2 sn-1); 71.9 (CH2 α fatty chain); 71.8; 72.5; 75.2; 75.3 (C2 +
C3 + C4 + C5); 79.7 (CH sn-2); 104.6 (C1).
1
the total disappearance of the H NMR TMSOAc signal. Com-
pound 13 was obtained as a brown oil which was characterized
as follows and used without further purification. Rf (ethyl
acetate–petroleum ether: 1/5): 0.47. 1H NMR (400.012 MHz,
3
3
CDCl3): 2.23 (3H, s, CH3); 3.48 (1H, dd, JHH = 9.8, JHH = 4.0,
H2); 4.07–4.12 (1H, m, H3); 4.15–4.22 (1H, m, H4); 4.35–4.50
3
(3H, m, H5 + H6); 4.80–5.19 (6H, m, CH2-Ph); 7.12 (1H, d, JHH
= 3.7, H1); 7.34–7.57 (15H, m, CH (Ph)). To a stirred solution of
8 (170 mg, 0.22 mmol) in benzene (3 mL) was added TBAI
(411 mg, 1.11 mmol), DIPEA (72 mg, 0.56 mmol) and mole-
cular sieves 4 Å. The mixture was stirred for 30 minutes at
room temperature and the intermediate 13 (154 mg,
0.67 mmol) was added. The mixture was stirred at reflux for
3 hours, filtered and concentrated. The crude product was pur-
4.6. Synthesis of 1,6-di-O-acetyl-2,3,4,-tri-O-benzyl-α-D-
galactopyranose 12
To a stirred solution of methyl-α-D-galactopyranose 11 (3.0 g, ified by chromatography on silica gel (ethyl acetate–petroleum
15.0 mmol) in dry DMF (60 mL) was added NaH (2.2 g, ether: 0/1 to 1/1) and the pure compound 14 was obtained as a
93.0 mmol). The mixture was stirred for 30 minutes at room colorless oil (65%). Rf (ethyl acetate–petroleum ether: 1/5):
3
temperature and then cooled to 0 °C. Benzyl bromide (9.2 mL, 0.47. 1H NMR (400.012 MHz, CDCl3): 0.90 (3H, t, JHH = 7.0,
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CH3); 1.22–1.30 (26H, m, CH2 fatty chain); 1.52–1.63 (2H, m, was obtained as a colorless oil (29 mg; 56%). [α]2D0 +51 (c 0.83;
CH2 β fatty chain); 1.95 (s, 3H, OCH3); 3.39–3.45 (2H, m, CH2 α MeOH). Rf (CH2Cl2–MeOH: 8/2): 0.27. IR (ATR, cm−1): 3337
fatty chain); 3.43 (3H, s, OCH3); 3.53–3.61 (6H, m, CH2 sn-1 + (O–H), 2917–2850 (C–H). 1H NMR (500.113 MHz, CD3OD): 0.89
3
CH2 sn-3 + CH sn-2 + H5); 3.75–4.14 (11H, m, H2 + H3 + H4 + H6 (3H, t, JHH = 7.0, CH3); 1.26–1.38 (26H, m, CH2 fatty chain);
3
3
+ H2′ + H3′ + H4′ + H5′ + H6′); 4.39 (1H, t, JHH = 7.5, H1); 1.55 (2H, qn, JHH = 7.0, CH2 β fatty chain); 3.39–3.50 (5H, m,
4.61–4.96 (13H, m, CH2-Ph + H1′); 7.25–7.40 (30H, m, CH (Ph)). OCH3 + CH2 α fatty chain); 3.51–3.73 (8H, m, CH2 sn-1 + CH2
13C NMR (75.475 MHz, CDCl3): 14.1 (CH3 fatty chain); 20.8 sn-3 + CH sn-2 + H3 + H4′ + H5′; 3.75–3.92 (9H, m, H2 + H4 + H5
(OCH3); 22.7 (CH2 fatty chain); 26.1 (CH2 fatty chain); 29.4 + H6 + H2′ + H3′ + H6′); 4.23 (1H, dd, 3JHH = 18.0, 3JHH = 9.0, H1);
3
(CH2 fatty chain); 29.5 (CH2 fatty chain); 29.7 (CH2 fatty chain); 4.86 (1H, d, JHH = 9.0, H1′). 13C NMR (75.475 MHz, CDCl3):
31.9 (CH2 fatty chain); 58.1 (OCH3); 63.4 (C6′); 66.9 (C6); 68.6 14.4 (CH3 fatty chain); 23.7 (CH2 fatty chain); 27.2 (CH2 fatty
(C5′); 69.5 (CH2 sn-3); 70.6 (CH2 sn-1); 71.8 (CH2 α fatty chain); chain); 30.4 (CH2 fatty chain); 30.6 (CH2 fatty chain); 30.7 (CH2
73.0 (C4′); 73.2 (CH2-Ph); 73.6 (CH2-Ph); 74.0 (C4); 74.4 (C2′); fatty chain); 30.8 (CH2 fatty chain); 33.0 (CH2 fatty chain); 58.1
74.5 (CH2-Ph); 75.0 (CH2-Ph); 76.2 (C5); 79.1 (C2); 79.3 (C3′); (OCH3); 62.7 (C6′); 67.7 (C6); 69.7 (CH2 sn-3); 70.0 (C4); 70.2
79.4 (CH sn-2); 82.1 (C3); 98.1 (C1′); 104.2 (C1); 127.4 (CH (Ph)); (C2′); 71.0 (C4′); 71.3 (CH2 sn-1); 71.4 (C3′); 72.2 (C2); 72.5 (C5′);
127.5 (CH (Ph)); 127.6 (CH (Ph)); 127.8 (CH (Ph)); 128.0 (CH 72.7 (CH2 α fatty chain); 74.4 (C3); 74.7 (C5); 80.6 (CH sn-2);
(Ph)); 128.3 (CH (Ph)); 128.4 (CH (Ph)); 138.2(Cipso); 138.7 100.5 (C1′); 105.2 (C1); MS (MALDI-TOF): m/z calcd for
(Cipso); 170.5 (CO). MS (MALDI-TOF): m/z calcd for C76H100O14 C32H62O13 (M + Na) 677.408; found 677.389.
(M + Na) 1259.701; found 1259.698.
4.10. Synthesis of 1,2,3,4,6-pentakis-O-(triméthylsilyl)-α-D-
4.8. Synthesis of 3-(hexadecyloxy)-2-methoxypropyl-2,3,4-tri-O-
benzyl-6-O-(2,3,4-tri-O-benzyl-α-D-galactopyranosyl)-β-D-
galactopyranoside 15
galactopyranose 18
To a solution of D-galactose (1.80 g, 10.0 mmol) and Et3N
(5.56 g, 55.0 mmol) in dry DMF (50 mL) at 0 °C is added
Compound 14 (180 mg, 0.15 mmol) was dissolved in MeOH TMSCl (5.97 g, 55.0 mmol) and the mixture is stirred for
(10 mL) and K2CO3 (10 mg, 0.08 mmol) was added. The solu- 4 hours at room temperature. n-Pentane (150 mL) is added
tion was stirred for 2 hours at room temperature, then Amber- and the mixture is poured into crushed ice. The organic layer
lite IR-120H was added and the mixture was stirred for is washed three times with ice-water (50 mL), dried over
30 minutes at room temperature. The solution was filtered MgSO4 and concentrated to give the expected compound as a
through Celite and concentrated. The crude product was puri- colourless oil (92%). Rf (CH2Cl2–MeOH: 5/1): 0.87. 1H NMR
fied by chromatography on silica gel (acetate–petroleum ether: (400.045 MHz, CDCl3): 0.10–0.16 (45H, m, CH3 (OTMS));
0/1 to 1/1) and the pure compound 15 was obtained as a color- 3.51–3.65 (2H, m, H6); 3.79–3.92 (4H, m, H2 + H3 + H4 + H5);
1
3
less oil (47%). Rf (ethyl acetate–petroleum ether: 1/2): 0.42. H 5.04 (1H, d, JHH = 2.4, H1). 13C NMR (125.816 MHz, CDCl3):
3
NMR (400.012 MHz, CDCl3): 0.89 (3H, t, JHH = 7.0, CH3); −0.45 (CH3Si); 0.2 (CH3Si); 0.3 (CH3Si); 0.5 (CH3Si); 0.7
1.22–1.35 (26H, m, CH2 fatty chain); 1.52–1.62 (2H, m, CH2 β (CH3Si); 61.2 (C6); 70.0 (C5); 70.5 (C3); 71.2 (C2); 72.3 (C4); 94.6
fatty chain); 1.95 (s, 3H, OCH3); 3.36–3.42 (2H, m, CH2 α fatty (C1).
chain); 3.43 (3H, s, OCH3); 3.48–3.62 (8H, m, CH2 sn-1 + CH2
4.11. Synthesis of 3-(hexadecyloxy)-2-methoxypropyl-6-O-
sn-3 + CH sn-2 + H3 + H4′ + H5′); 3.75–3.92 (8H, m, H2 + H4 + H5
3
[2,3,4-tris-O-(trimethylsilyl)-α-D-galacto-pyranosyl]-2,3,4-tris-O-
(trimethylsilyl)-α-D-galactopyranoside 19
+ H6 + H3′ + H6′); 4.02–4.06 (1H, m, H2′); 4.38 (1H, dd, JHH
=
3
18.0, JHH = 9.0, H1); 4.58–4.98 (13H, m, CH2-Ph + H1′);
7.20–7.33 (30H, m, CH (Ph)). 13C NMR (75.475 MHz, CDCl3): To a solution of compound 10 (454 mg, 0.84 mmol) in dry
14.1 (CH3 fatty chain); 22.7 (CH2 fatty chain); 26.0 (CH2 fatty CH2Cl2 (5 mL) at 0 °C is added TMSI (131 μL, 0.92 mmol). The
chain); 29.3 (CH2 fatty chain); 29.7 (CH2 fatty chain); 31.9 (CH2 mixture is stirred for 20 minutes at 0 °C and benzene (3 mL) is
fatty chain); 58.1 (OCH3); 61.6 (C6′); 67.6 (C6); 69.4 (CH2 sn-3); added. The solution is concentrated at 0 °C to give the
70.4 (CH2 sn-1); 70.6 (C5′); 71.8 (CH2 α fatty chain); 73.1 α-iodide. The residue is dissolved in dry CH2Cl2 (5 mL) and
(CH2-Ph); 73.2 (C4′); 73.6 (CH2-Ph); 74.0 (C4); 74.4 (CH2-Ph); stored in an argon atmosphere. In another reactor a solution
74.5 (CH2-Ph); 74.8 (C5); 75.0 (CH2-Ph); 76.3 (C2′); 79.1 (C2); of TBAI (465 mg, 1.26 mmol), DIPEA (163 mg, 1.26 mmol) and
79.2 (C3′); 79.3 (CH sn-2); 82.1 (C3); 98.1 (C1′); 104.0 (C1); 127.4 compound 18 (200 mg, 0.28 mmol) in dry CH2Cl2 (5 mL) is
(CH (Ph)); 127.5 (CH (Ph)); 127.8 (CH (Ph)); 128.1 (CH (Ph)); stirred at room temperature in an argon atmosphere. The solu-
128.2 (CH (Ph)); 128.4 (CH (Ph)); 138.5 (Cipso); 138.6 (Cipso).
tion containing the iodide compound is added dropwise and
the mixture is stirred for 36 hours at room temperature. The
solution is concentrated and the residue is dissolved in dry
diethyl ether (20 mL) to precipitate salts, filtered and concen-
4.9. Synthesis of 3-(hexadecyloxy)-2-methoxypropyl-6-O-α-D-
galactopyranosyl-β-D-galacto-pyranoside 16
Compound 15 (100 mg, 0.08 mmol) was dissolved in MeOH trated. The residue is purified by chromatography on silica gel
(5 mL) and palladium (10% on carbon) (150 mg, 0.14 mmol) (ethyl acetate–petroleum ether: 1/9) to give a colourless oil
was added. The solution was stirred for 3 days at room temp- (72%). Rf (ethyl acetate–petroleum ether: 2/1): 0.27. 1H NMR
erature under 20 bars of pressure of H2. The solution was fil- (400.012 MHz, CDCl3): 0.06–0.17 (m, 63H, CH3 (OTMS)); 0.87
3
tered through Celite and concentrated. The pure compound 16 (t, 3H, JHH = 6.8, CH3); 1.15–1.32 (m, 26H, CH2 fatty chain);
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1.51–1.58 (m, 2H, CH2 β fatty chain); 3.35–3.58 (m, 10H, OCH3 endogenous potassium currents. Potassium SK3 currents were
+ CH2 α fatty chain + CH2 sn-1 + CH2 sn-3 + CH sn-2); 3.59–3.90 analysed at 0 mV to minimize chloride currents (ECl− = 0 mV).
(m, 12H, H2 + H3 + H4 + H5 + H6 + H2′ + H3′ + H4′ + H5′ + H6′);
4.18 (dd, 1H, 3JHH = 7.5, 3JHH = 9.6, H1); 4.69 (d, 1H, 3JHH = 3.6,
4.15. In vitro cell viability and cell migration assay
H1′). 13C NMR (125.816 MHz, CDCl3): −0.9 (CH3 (OTMS)); −0.1
(CH3 (OTMS)); 0.1 (CH3 (OTMS)); 0.2 (CH3 (OTMS)); 0.3 (CH3
(OTMS)); 13.6 (CH3 fatty chain); 22.2 (CH2 fatty chain); 25.6
(CH2 fatty chain); 28.9 (CH2 fatty chain); 31.4 CH2 (fatty chain);
57.3 (OCH3); 61.0 (C6′); 64.8 (C6); 68.1 (CH2 sn-3); 70.3 (CH2 sn-
1); 71.2 (CH2 α fatty chain); 68.7; 70.2; 70.9; 71.0; 71.3; 71.9;
72.6; 74.8 (C2 + C3 + C4 + C5 + C2′ + C3′ + C4′ + C5′); 78.8 (CH sn-
2); 99.6 (C1′); 103.9 (C1).
Cell viability was determined using the tetrazolium salt
reduction method (MTT) as previously described.20 Briefly,
cells were seeded onto 24-well plates at a density of 4 × 104
cells per well and measurements were performed in triplicate
for 24 h and 48 h after implantation. The cell migration assay
was performed in 24-well plates with polyethylene terephthal-
ate membrane cell culture inserts with a pore size of 8 μm
(Becton Dickinson, France) coated with fibronectin from
bovine plasma (1.6 μg cm−2) (Sigma-Aldrich). Briefly, 8 × 104
4.12. Synthesis of 3-(hexadecyloxy)-2-methoxypropyl-6-O-α-D-
galactopyranosyl-β-D-galacto-pyranoside 16
cells were seeded into the upper compartment and the sup-
plemented medium culture was added in the upper and lower
To a stirred solution of 20 (235 mg, 0.20 mmol) in methanol
(15 mL) was added Dowex 50WX8-200® ion exchange resin
(300 mg). The mixture was stirred for 4 hours at room tempera-
ture, filtered and concentrated. The residue was purified by
chromatography on silica gel (CH2Cl2–MeOH: 8/2) to give the
expected product as a white solid (101 mg; 77%). The spectral
characteristics were similar to those reported in section 4.10.
compartments. When a compound was evaluated, it was
added into both compartments. After 15 h, stationary cells
were removed from the top side of the membrane, whereas
migrated cells at the bottom side of the inserts were fixed, the
nuclei were stained with DAPI (Sigma-Aldrich) and then
counted. Three independent experiments were performed in
parallel and these were done in triplicate.
4.13. Cell culture
4.16. Statistics
HEK293T cells were purchased from ATCC, maintained in Dul-
becco’s Modified Eagle’s Medium, supplemented with 10%
(v/v) fetal bovine serum (Lonza, France), 1 mM pyruvate
sodium (Sigma-Aldrich, France), and 1% non-essential amino
acid (Sigma-Aldrich, France) and were grown in a humidified
atmosphere at 37 °C (95% air, 5% CO2). HEK293T cells were
transduced with lentiviral vector containing rat SK3 cDNA to
obtain stable expression of homotetramer SK3 channels fol-
lowing the same protocol as previously described.7,22
Data were expressed as means SEM (n = the number of experi-
ments). Statistical analyses were conducted using Kruskal–
Wallis one-way analysis of variance followed by a Dunn’s test.
Differences were considered significant when p < 0.05 (Sigma-
Stat, Systat Software).
Acknowledgements
This work was funded by ‘Région Centre’, ‘Région Bretagne’,
‘Ligue contre le Cancer’, ‘CNRS’, INSERM, Cancéropole Grand
Ouest and FEDER (Fond européen de développement
regional). We thank the ‘Services Communs’ of the University
of Brest.
4.14. Patch-clamp experiments
Experiments were performed in the whole cell configuration of
the patch-clamp technique. The extracellular solution con-
tained 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
10 mM HEPES, 0.33 mM NaH2PO4, and 11.5 mM D-glucose.
pH was adjusted to 7.4 with NaOH. The intracellular solution
contained 145 mM KCl, 1 mM MgCl2, 1 mM Mg-ATP, 0.7 mM
CaCl2, 1 mM EGTA and 10 mM HEPES (pCa 6). pH was
adjusted to 7.2 with KOH. Patch-clamp experiments were con-
ducted using an Axopatch 200B patch-clamp amplifier (Axon
Instrument, USA) and data, digitized with a 1322-A Digidata
converter (Axon Instrument, USA), were stored on a computer
using the software Clampex of pClamp 9.2 (Axon Instrument,
USA). An extracellular solution containing compound 16 was
superfused into the bath chamber near the cell using the Bio-
Logic MEV-9 superfusion system. The patch-clamp data were
analyzed using both Clampfit 9.2 and Origin 7.0 software
(Microcal Inc., Northampton, MA, USA). The effects of com-
pound 16 on HEK293T-SK3 cells were measured for 2 to 4 min
using a ramp protocol from +95 mV to −95 mV with a holding
potential of 0 mV (500 ms duration; 4 s intervals) to inactivate
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