Supplementary informations
NMR experiments
NMR spectra were recorded in deuterated DMSO at 300 K on a Bruker AvANCE-600
spectrometer operating at 600.13 MHz for the 1H and a Bruker AvANCE-400 spectrometer
operating at 400.24 MHz for the 1H. The spectrometer was equipped with a 5-mm Z gradient
inversion probe head. Typical parameters used were 10200 Hz and 7200 Hz spectral width for
600 and 400 spectrometer respectively. 64 scans were accumulated using Bruker standard
sequence. Chemical shifts (CSs) were given using an external standard reference
(Tetramethylsilane, TMS).
Mass spectrometry
Chemical structures of surfactants were ascertained by ionization mass spectrometry.
Assignments were made on the basis of calculated m/z ratios.
Surfactant synthesis
Synthesis of lithocholanoyl-DG
Lithocholic acid (2 g, 5.31 mmol, 1 eq) was dissolved in CH2Cl2 (70 mL) and dried THF (50
mL). N, hydroxysuccinimide (NHS) (1,53 g, 13.3 mmol, 2.5 eq), 1,3-dicyclohexylcarbo
diimide (DCC) (0.11 g, 5.84 mmol, 0.1 eq) and 4, dimethylamine pyridine (DMAP) (0.065 g,
0.53 mmol, 0.1 eq) were added under magnetic stirring for 24 h at room temperature (Table 1,
Fig. S1). Then paper filtration was performed and solvents were evaporated under reduced
pressure. The obtention of lithocholanoyl-NHS was confirmed by thin layer chromatography,
1
H NMR and mass spectrometry. Lithocholanoyl-NHS (1.82 g, 3.84 mmol, 1 eq) and
diglutamic acid (1.06 g, 3.84 mmol, 1 eq) were both dissolved in DMSO (75 mL) and Et3N
(1.94 g, 19.2 mmol, 5 eq) under magnetic stirring and nitrogen flow overnight at room
temperature. Lyophilization was then performed. The residue was dissolved in a mixture of
acetonitrile and water (50/50 v/v). After filtration through a membrane filter of 0.45 µm pore
size (polysulfone), the solution was injected onto a preparative reversed phase column
1
chromatography with a 300 g bed of grafted silica (Kromasil, AkzoNobel) as stationnary
phase. UV signal was recorded at 210 nm. Eluting conditions were as follow: 0-15 min 100 %
A, 15-35 min gradient from 100 % A to 100 % B then 35-45 min 100 % B. A, being purified
water with 0.2 % TFA and B CH3CN with 0.2 % TFA. The expected product was collected
after 34 min elution time, concentrated under pressure and freeze-dried (1.16 g, 1.83 mmol,
48 %). Surfactant chemical structure was evidenced with 1H NMR and mass spectrometry.
Lithocholanoyl-DG (C34H54N2O9): 1H NMR (600 MHz, DMSO-d6): δ 0.6 (3H, s), 0.87 (3H,
s), 0.88 (3H, s), 0.89 (1H, td), 0.95-1.40 (18H, m), 1.50 (2H, m), 1.55-2.05 (12H, m), 2.17
(1H, ddd), 2.24 (2H, t), 2.26 (2H, m), 3.36 (1H, m), 4.17 (1H, m), 4.27 (1H, td), 4.44 (1H, sl),
7.93 (1H, d), 8.14 (1H, d), 11.60-13.00 (3H, bs).
MS (ESI-): m/z: 633.4 [M-H]-, 655.4 [M+Na-2H]MS (ESI+): m/z: 635.4 [M+H]+, 657.4 [M+Na]+, 1291.8 [2M+Na]+
Synthesis of arachidonoyl-DG
Diglutamic acid (0.69 g, 2.49 mmol, 1 eq) was dissolved in DMF (70 mL) and Et3N (2.2 mL,
21.8 mmol, 8.8 eq) under magnetic stirring at 70°C (Table 1, Fig. S2). Arachidonoyl-NHS
(0.95 g, 2.37 mmol, 0.95 eq) was dissolved in DMF (35 mL) under magnetic stirring at room
temperature and nitrogen flow. Arachidonoyl-NHS solution was added to the diglutamic acid
one under magnetic stirring at room temperature with an aluminium protection against UV
rays. The reaction was monitored using Ultra-Fast Liquid Chromatography (UFLC) at 210
nm. When arachidonoyl-DG was formed at 95 %, the reaction was stopped. Solvent was then
evaporated under reduced pressure. The residue was then dissolved in a mixture of
acetonitrile and water (50/50 v/v). After filtration through a membrane filter of 0.45 µm pore
size (polysulfone), the product was purified with a preparative reversed phase column
chromatography, as described above. The expected product was collected after 28 min elution
time, concentrated under pressure and freeze-dried (0.78 g, 1.39 mmol, 59 %). Surfactant
chemical structure was evidenced with 1H NMR and mass spectrometry.
Arachidonoyl-DG (C30H46N2O8): 1H NMR (400 MHz, DMSO-d6): δ 0.86 (3H, m), 1.15-1.35
(6H, m), 1.54 (2H, m), 1.65-2.00 (4H, m), 2.02 (4H, q), 2.13 (2H, m), 2.24 (2H, t), 2.27 (2H,
t), 2.78 (4H, t), 2.81 (2H, t), 4.18 (1H, m), 4.29 (1H, td), 5.33 (8H, m), 7.90 (1H, d), 8.12 (1H,
d), 12.24 (3H, bs).
MS (ESI-): m/z: 280.1 [M-2H]2-, 561.3 [M-H]-, 583.3 [M+Na-2H]-, 605.3 [M+2Na-3H]-
2
Synthesis of linoleoyl-DG
Linoleic acid (1 g, 3.74 mmol, 1 eq) was dissolved in SOCl2 (31 mL) and refluxed for 2 h at
80°C, under magnetic stirring and under N2 atmosphere, in order to obtain linoleoyl chloride
(Table 1, Fig. S3). Then SOCl2 was evaporated under reduced pressure in a water bath at
35°C. The residual was dissolved in dry DMF (30 mL). Diglutamic acid (1 g, 3.69 mmol,1
eq) was dissolved in dry DMF (100 mL), Et3N (3.3 mL, 23.5 mmol, 6 eq) under magnetic
stirring at 80°C and under a nitrogen flow. After diglutamic acid solubilization, linoleoyl
chloride was added under nitrogen flow and under magnetic stirring at room temperature. The
reaction was protected from light with aluminium. DMF was then evaporated under reduced
pressure. The residual was dissolved in CH3CN/water (50/50 v/v) (10 mL) and filtered
through a membrane filter of 0.45 µm pore size (polysulfone). The product was purified using
a preparative reversed phase column chromatography, as described below. The expected
product was collected after 28 min elution time, concentrated under pressure and freeze-dried
(0.315 g, 0.58 mmol, 16 %). Surfactant chemical structure was evidenced with 1H NMR and
mass spectrometry.
Linoleoyl-DG (C28H46N2O8): 1H NMR (400 MHz, DMSO-d6): δ 0.86 (3H, m), 1.15-1.35
(14H, m), 1.47 (2H, qt), 1.65-2.00 (4H, m), 2.02 (4H, q), 2.10 (2H, m), 2.24 (2H, t), 2.26 (2H,
t), 2.74 (2H, t), 4.18 (1H, m), 4.29 (1H, td), 5.32 (4H, m), 7.89 (1H, d), 8.08 (1H, d), 12.30
(3H, bs).
MS (ESI-): m/z: 268.2 [M-2H]2-, 537.3 [M-H]-, 559.3 [M+Na-2H]+, 581.3 [M+2Na-3H]-
Synthesis of stearoyl-DG
Stearic acid (28 g, 98.2 mmol, 1 eq) and N-hydroxysuccinimide (16.9 g, 147.3 mmol, 1.5 eq)
were dissolved in CH2Cl2 (700 mL) followed by the addition of EDC, HCl (28.6 g, 147.3
mmol, 1.5 eq) (Table 1, Fig. S4). The mixture was placed under magnetic stirring at room
temperature for 24 h and then diluted in water (700 mL). Organic and aqueous phases were
then separated. The aqueous phase was extracted with CH2Cl2 (3 x 250 mL). Organic phases
were collected, washed with a brine (250 mL), dried with Na2SO4, filtered and concentrated
under reduced pressure. The product was finally obtained, as a white powder after
recrystallisation from hot EtOAc (700 mL). Stearoyl-NHS (3.5 g, 9.17 mmol, 1 eq),
diglutamic acid (2.5 g, 9.17 mmol, 1 eq) and Et3N (9 mL, 64.2 mmol, 7 eq) were dissolved in
3
DMF (50 mL) under magnetic stirring at room temperature for 16 h. Solvent was then
evaporated under pressure. The residue was taken in water (150 mL) and acidified with HCl
1N until precipitation occurred. The obtained suspension was stirred at room temperature for
30 min and filtered to yield the product as a white powder after drying under vacuum (4.2 g,
7.7 mmol, 84 %).
Stearoyl-DG (C28H50N2O8): 1H NMR (600 MHz, DMSO-d6): δ 0.85 (3H, m), 1.15-1.30 (28H,
m), 1.46 (2H, m), 1.70 (1H, m), 1.78 (1H, m), 1.87 (1H, m), 1.95 (1H, m), 2.10 (2H, m), 2.23
(2H, t), 2.26 (2H, t), 4.17 (1H, m), 4.28 (1H, td), 7.93 (1H, d), 8.13 (1H, d), 12.17 (2H, vbs),
12.57 (1H, vbs).
MS (ESI+): m/z: 291.2 [M+H+K]2+, 543.4 [M+H]+, 565.3 [M+Na]+, 581.3 [M+K]+
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O O
O
OH
H
H
HO
H
O N
NHS
DCC,DMAP
CH2Cl2, THF
H
H
HO
H
O
H
H
Lithocholanoyl-NHS
Lithocholic acid
Alpha-glutamylglutamic acid
DMSO, Et3N
O
OH
O
Lithocholanoyldiglutamic acid
H
HO
H
N
N
H
H
O
Fig. S1 Synthesis scheme of lithocholanoyl-DG.
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OH
H
O
H
O
OH
O
O
O
O N
O
Alpha-glutamylglutamic acid
H
N
N
H
DMF
O
O
Arachidonic acid-NHS
OH
O
O
OH
OH
Arachidonoyl-diglutamic acid
Fig. S2 Synthesis scheme of arachidonoyl (C20:4)-DG.
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O
OH
Linoleic acid
SOCl2, 80°C
O
OH
O
Cl
+
O
H
N
H2N
OH
O
Linoleoyl chloride
O
OH
DMF
O
OH
O
H
N
N
H
O
Linoleoyl-diglutamic acid
O
O
OH
OH
Fig. S3 Synthesis scheme of linoleoyl (C18:2)-DG
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Alphaglutamylglutamic acid
O
OH
Stearic acid
NHS CH2Cl2
EDC RT, 24h
O
O
O
N
O
Stearoyl-NHS
Alpha-glutamyl- DMF
glutamic acid
RT, 16h
NEt3
O
OH
O
H
N
N
H
O
Stearoyl-diglutamic acid
O
O
OH
OH
Fig. S4 Synthesis scheme of stearoyl (C18:0)-DG.
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Fig. S5 Pyrene fluorescence ratio I1/I3 versus surfactant concentration for polysorbate 80 (■), lithocholanoyl-DG
(∆), arachidonoyl-DG (▲), stearoyl-DG (○) and linoleoyl-DG (●). Data are mean ± S. D., n = 3.
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Fig. S6 Viability of HUVEC treated by polysorbate 80 (■), lithocholanoyl-DG (∆),
arachidonic acid-DG (▲), stearoyl-DG (○) and linoleoyl-DG (●).The surfactant concentration
responsible for a 50 % cell viability decrease, IC50, was determined from the sigmoidal fit. All
data are mean ± S. D., n = 8.
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Fig. S7 LDH release assessment after treatment with polysorbate 80 (■), lithocholanoyl-DG (∆), arachidonoylDG (▲), stearoyl-DG (○) and linoleoyl-DG (●), using LDH release as the toxicity biomarker in HUVEC after an
incubation time of 4h. All data are mean ± S. D., n = 8.
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Fig. S8 Haemolytic activity of polysorbate 80 (■), lithocholanoyl-DG (∆), arachidonoyl-DG (▲), stearoyl-DG
(○) and linoleoyl-DG (●) surfactant aqueous solutions with rat RBCs after an incubation time of 1h at 37°C. All
data are mean ± S. D., n = 3.
* denotes a decrease of haemolysis percentage explained by the rapid aggregation of RBCs.
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