Hybrid Fluorinated and Hydrogenated Double-Chain Surfactants for Handling Membrane Proteins

Article abstract of DOI:10.1021/acs.joc.5b02137

Two hybrid fluorinated double-chain surfactants with a diglucosylated polar head were synthesized. The apolar domain consists of a perfluorohexyl main chain and a butyl hydrogenated branch as a side chain. They were found to self-assemble into small micel

Full text of DOI:10.1021/acs.joc.5b02137

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Hybrid Fluorinated and Hydrogenated Double-Chain Surfactants
for Handling Membrane Proteins
Frederic Legrand,† Cecile Breyton,‡,§,∥ Pierre Guillet,† Christine Ebel,‡,§,∥ and Gregory Durand*,†
́
́
́
†Institut des Biomolec
́
́ ́
ules Max Mousseron UMR 5247 CNRS-Universite Montpellier-ENSCM & Avignon Universite,
Equipe Chimie Bioorganique et System
̀
es Amphiphiles, 301 rue Baruch de Spinoza BP 21239, 84916 Cedex 9 Avignon, France
‡Universite
́
Grenoble Alpes, IBS, F-38044 Grenoble, France
§CNRS, IBS, F-38044 Grenoble, France
∥CEA, IBS, F-38044 Grenoble, France
S
* Supporting Information
ABSTRACT: Two hybrid fluorinated double-chain surfactants
with a diglucosylated polar head were synthesized. The apolar
domain consists of a perfluorohexyl main chain and a butyl
hydrogenated branch as a side chain. They were found to self-
assemble into small micelles at low critical micellar concen-
trations, demonstrating that the short branch increases the
overall hydrophobicity while keeping the length of the apolar
domain short. They were both able to keep the membrane pro-
tein bacteriorhodopsin stable, one of them for at least 3 months.
stability as experienced for fluorinated surfactants.16 As shown
etergents are used as solubilizing agents for the extrac-
Dtion, purification, and further downstream characteri- for alkyl maltosides, decreasing the length of the aliphatic chain
zation of integral membrane proteins.1 However, many of them
leads to micelles and protein/detergent complexes of smaller
sizes,17 and adding a small aliphatic branch close to the head
lose their native structure and function in the presence of deter-
increased hydrophobicity without affecting the size of the
micelles.18 Therefore, increasing the overall hydrophobicity of
the surfactant while keeping the length of the apolar domain
short is a means for obtaining small, nondenaturing micelles,
essential for membrane protein structural investigations.
gent, often irreversibly. Hence, recent years have witnessed
increasing efforts at developing alternative, gentler solubilizing
agents that could substitute for classical detergents without
interfering with membrane protein structure and activity. There
are currently several approaches including heterogeneous sys-
tems such as bicelles,2 nanodiscs,3 and polymers.4−6 Among
the homogeneous systems, i.e., chemically well-defined
detergent-like molecules, one can cite maltoside−neopentyl
glycols,7 cyclic-based maltosides derivatives,8 as well as tripod
derivatives.9
In this work, we have synthesized two hybrid double-chain
surfactants 1 and 2 that possess a diglucose-based polar head-
group and a six-carbon perfluorinated chain with a branched
hydrogenated chain. The aim was to combine both properties
of hydrogenated and fluorinated chains and to increase the
hydrophobicity of the surfactants. Previously designed hybrid
surfactants exhibit potentially interesting properties for
membrane proteins solubilization as they form small micelles
with long lifetime,19 in which hydrophobic molecules can be
solubilized.20 Due to the low critical micellar concentration
(CMC) of fluorocarbon surfactants compared to their hydro-
genated analogues,21 we used a six-carbon perfluorinated chain.
Indeed, related glycosylated fluorinated surfactants with the
same chain exhibit CMCs in the 0.1−1.0 mM range.21−23 The
linker between the fluorinated chain and the polar headgroup is
either an amide bond for compound 1 or an ether bond for
Fluorinated surfactants are believed to provide a mild
solubilizing environment for membrane proteins.10,11 Indeed,
fluorinated alkyl chains are considerably bulkier than fully
hydrogenated ones and thus insert less easily between protein
transmembrane helices. In addition, van der Waals interactions
between fluorocarbons and hydrocarbons are weaker than those
among hydrocarbons, thereby preserving native interactions
and avoiding delipidation. As a consequence, fluorinated
surfactants do not to exert detergent activity and, thus, do
not allow direct membrane solubilization and protein extraction
excepted in rare cases.12,13
According to the packing parameter theory,14,15 micelle size
and shape are a function of the geometry of the detergent itself,
which in turn alter protein/detergent complex composition and
Received: October 5, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.5b02137
J. Org. Chem. XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of the Amide-Based Double-Chain Intermediate 9
Scheme 2. Synthesis of the Ether-Based Double-Chain Intermediate 15
compound 2, which allowed study of their effect on the overall
hydrophobicity of the surfactant and made the synthetic route
more flexible for further functionalization. Zhang et al. demon-
strated with a series of branch-chained maltoside detergents
that adding two carbons on the branch had a similar effect on
the CMC as extending the main chain by one carbon.18 We
therefore used a butyl-hydrogenated branch as a side chain so
as to maintain sufficient water solubility and to mimic the
addition of two carbons in the main chain. We report herein
their synthesis and their micellar and biochemical properties.
The convergent synthetic route for the synthesis of the
double-chain surfactants 1 and 2 is based on three key steps:
(i) synthesis of the apolar double-chain intermediates through
radical addition of 1-iodoperfluorohexane onto butenoic acid
based derivatives (Schemes 1 and 2); (ii) synthesis of the
diglucose polar headgroup from aminopropanediol (Scheme 3);
and (iii) its condensation onto the apolar double-chain inter-
mediates (Scheme 3).
Scheme 3. Synthesis of the Diglucose-Polar Head Group 19
and Condensation onto the Double-Chain Intermediates 9
and 15
Condensation of 3-butenoic acid onto 3-aminopropane-1,2-
diol was achieved using 2-ethoxy-1-(ethoxycarbonyl)-1,2-
dihydroquinoleine (EEDQ) in ethanol to afford compound 3
in 90% yield. The two hydroxyl groups of compound 3 were
next protected by reaction with 2,2-dimethoxypropane in
CH3CN to lead to compound 4. The connection of the fluo-
rinated chain onto the olefin was achieved by radical addition of
1-iodoperfluorohexane onto compound 4 in the presence of
2,2′-azobis(2-methylpropionitrile) (AIBN) as radical initia-
initiator leading to compound 6 in 86% yield. Hydrolysis of
the 1,3-acetonide under mild acidic conditions yielded the
dihydroxy derivative 7, which was next put in reaction with 1.2
equiv of pentanoic acid in the presence of N,N′-dicyclohex-
ylcarbodiimide (DCC) and 4-(N,N-dimethylamino)pyridine
(DMAP) to give compound 8 in 40% yield. Due to the slight
excess of pentanoic acid, formation of the diester derivative was
also observed in low yield. Succinic anhydride was finally
grafted onto compound 8 under basic condition leading to
compound 9 in 84% yield.
tor.24,22 The reaction monitored by H NMR showed the dis-
1
appearance of vinylic protons and the presence of a char-
acteristic multiplet assigned to CHI at ∼4.70 ppm. After flash
chromatography purification, compound 5 was obtained in
55% yield. In our hands, several attempts at condensating
1-iodoperfluorohexane onto compound 3 failed, likely due to
the presence of free hydroxyl groups. The reduction of C−I
bond was next carried out using tributyltin hydride (Bu3SnH)
in the presence of a catalytic amount of AIBN as radical
Based on the work by Huang et al.,25 a related synthetic
strategy was used for the synthesis of the ether derivative. The
commecially available racemic 1,2-isopropylideneglycerol was
used as starting material and was transformed to its alkoxide
B
DOI: 10.1021/acs.joc.5b02137
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derivative in the presence of sodium hydride in dry THF, which
was then added to allyl bromine to give compound 10 in 84%
yield. The connection of the fluorinated chain onto the olefine
10 was next achieved by radical addition of 1-iodoperfluor-
ohexane followed by reduction of the C−I bond leading to
compound 12 in 59% yield in two steps. Hydrolysis of the 1,3
acetonide and subsequent condensation of pentanoic acid led
to compound 14. The remaning free hydroxyl goup was finally
condensated with succinic anhydride leading to compound 15.
The polar headgroup was prepared from aminopropanediol,
whose amino group was first protected as a benzyloxycarbonyl
group to give compound 16. The free hydroxyl group was next
put in reaction with an excess of 2,3,4,6-tetra-O-acetyl-α-D-
glucopyranosyl bromide in the presence of HgCN2 under
ultrasound activation. After purification by flash chromatog-
raphy, compound 17 was obtained in 35% yield. Removal of the
benzyloxycarbonyl group was achieved by hydrogenolysis in the
presence of palladium over carbon to afford compound 18 in
64% yield, and then the acetyl groups were removed under
Table 1. Self-Aggregation Properties of Compounds 1 and 2
a
b
surfactants
1
2
molar mass (g/mol)
CMC (mM)
1074.8
1047.8
0.079 0.020
84.7 21.3
0.011 0.001
11.1 0.6
CMC (mg/L)
γCMC (mN/m)
24
2.15 0.08
77
2
24
2
c
a
Γmax × 10−12 (mol/mm2)
1.97 0.09
c
a
Amin (Å2)
3
85
4
d
ΔGmS /aq,
°
(kJ/mol)
−33.4 0.5
−37.2 0.6
7.5 0.5
−38.4 0.1
−43.0 1.0
11.2 0.6
e
ΔGaSd,° (kJ/mol)
f
DH (nm)
a
b
Data are averages of three experiments. Data are averages of four
c
experiments unless specified. The surface excess (Γmax) and the
surface area per molecule (Amin) were estimated from the slope of the
surface tension curve. Gibbs free energy of micellization. Free energy
of adsorption. Hydrodynamic diameter distribution by Contin
d
e
f
analysis, data are averages of 10 runs.
́
Zemplen conditions to afford the polar headgroup 19. The
chain on the packing at the air/water interface, in agreement
with limit surface tension values.
polar head was used without any purification and directly
condensated onto compounds 9 and 15 in the presence of
EEDQ to give after purification compounds 1 and 2 in 35% and
31% yield, respectively.
The standard Gibbs free energies of micellization, ΔGmS /aq,°,
are −33.4 and −38.4 kJ/mol for 1 and 2, respectively (Table 1).
Since the incremental Gibbs energy contribution to micellization
typically amounts to ∼−3 kJ/mol per methylene group,28,29
the contribution of the ether linker compared to the amide
one (−5.0 kJ/mol) corresponds roughly to the extension of the
hydrocarbon chain by two methylene groups. The standard free
energy of adsorption ΔGaSd,° values are −37.2 and −43.0 kJ/mol
for 1 and 2, respectively (Table 1). As the ΔGaSd,° per CH2
group usually ranges from −3.0 to −3.5 kJ/mol at 25 °C,28 the
contribution to adsorption of the ether linker compared to the
amide one (−5.8 kJ/mol) corresponds roughly to the addition
of two methylene groups.
From the surface tension curves, the CMC and the limit
surface tension attained at the CMC (γCMC) are directly
obtained (Figure 1). Compound 1 exhibits a CMC of ∼0.08
Dynamic light scattering (DLS) experiments were performed
at 1 g/L, i.e., several times the CMC. Figure 1 shows that both
surfactants self-organize into well-defined assemblies with
apparent hydrodynamic diameters of ∼7 and ∼11 nm,
respectively, suggesting the formation of rather small micelles.
When compared to the popular detergent, n-dodecyl-β-D-
maltoside (DDM) that forms globular micelles of ∼7 nm
hydrodynamic diameter, its perfluorinated analogue (F6OM)
forms rodlike micelles of 30 nm hydrodynamic diameter with
60 nm maximal length.23,30 Upon dilution, no significant
difference in the Contin distribution was observed for both
compounds (data not shown). The slightly larger micelles
observed for compound 2 may arise from the lower polarity of
the ether bond, which would make the hydrogenated chain
closer to the main chain leading to a bulkier hydrophobic
moiety as compared to compound 1. Moreover, with com-
pound 1, stronger hydrogen bonding may occur between water
molecules and the amide linker facilitating the penetration of
water molecule. This would make the polar headgroup bulkier
in agreement with the higher CMC and higher surface area per
molecule.
Figure 1. Plots of surface tension vs concentration for compounds 1
and 2 at 25 °C (left). Hydrodynamic diameter distribution plots for
compounds 1 (full line) and 2 (dash line) at 1 g/L (right). Inset shows
autocorrelation functions.
mM, while that of 2 appears to be 8 times lower with a value of
∼0.01 mM (Table 1). These low values, compared to those of
single chain perfluorohexane-based surfactants, such as the
commercial octylfluorinated maltoside F6OM (∼0.70 mM)23
or our structurally related F6DigluM (0.38 mM)26 and
F6H3DigluM (0.79 mM)27 derivatives, are indicative of a
significant contribution of the butyl branch on the overall
hydrophobicity in agreement with the findings by Zhang et al.,
who demonstrated that adding two carbons on the branch had
a similar effect on the CMC as extending the main chain by one
carbon.18 Moreover, the nature of the linker between the
fluorinated chain and the polar head also has an effect with a
lower CMC value for the ether derivative compared to the
amide one. Both compounds 1 and 2 revealed close values
of surface excess (Γ) of ∼2.15 and 1.97 × 10−12 mol/mm2,
which correspond to surface areas (Amin) of ∼77 and 85 Å2,
respectively (Table 1). These values are comparable to those
reported for the single-chain structurally related F6DigluM
(∼2.24 × 10−12 mol/mm2) and F6H3DigluM (∼1.7 ×
10−12 mol/mm2), suggesting only a slight effect of the second
To test the biochemical relevance of compounds 1 and 2,
their potential to keep the model membrane protein
bacteriorhodopsin (bR) soluble and in its native form was
investigated. bR is composed of seven transmembrane α-helices
and binds a covalent cofactor, a retinal molecule, whose visible
absorption spectrum is very sensitive to its local environment
and is an excellent marker of the integrity and oligomeric state of
the protein.31 The insets of Figure 2 show the results of sucrose
C
DOI: 10.1021/acs.joc.5b02137
J. Org. Chem. XXXX, XXX, XXX−XXX

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of ∼7 and ∼11 nm hydrodynamic diameter, respectively.
This demonstrates that the butyl branch significantly increases
hydrophobicity while keeping the length of the apolar domain
short. Compound 1 was found to form rather homogeneous
complexes with the protein bacteriorhodopsin, which remained
stable for a month. Complex with compound 2 was
heterogeneous, but the protein remained stable for more than
three months.
EXPERIMENTAL SECTION
■
Figure 2. Spectral time course of bR in compounds 1 (left) and 2
(right). Samples incubated at 4 °C in the dark, and UV−vis spectra
were recorded at the indicated time, in days. Inset: migration of bR in
10−30% sucrose gradients containing 6 mM of compounds 1 or 2.
Black arrows indicate the position of the main absorption peak and of
the shoulder, and gray arrows and line indicate the position of the
protein in the gradient.
Synthesis. Mercury cyanide Hg(CN)2 was dried overnight on
P2O5 under vacuum. All of the solvents were of reagent grade, distilled,
and dried according to standard procedures prior to use. The progress
of the reactions was monitored by thin-layer chromatography (TLC,
silica plates), and the compounds were detected either by exposure to
ultraviolet light (254 nm) or by spraying with a 5% sulfuric acid
solution in ethanol and with a 2% ninhydrin solution in ethanol
following heating at ∼150 °C. Ultrasonication was performed with a
sonicator equipped with a 13 mm diameter titanium probe. Flash
chromatography purification was carried out on silica gel (40−63 μm
granulometry). Water was deionized with a Milli-Q water purification
system (resistivity of 18.2 MΩ cm). The 1H, 13C, and 19F NMR
experiments were performed at 250, 62.86, and 235 MHz, respectively.
Chemical shifts are given in ppm relative to the solvent residual peak
gradient experiments. For bR solubilized by compound 1, two
species can be distinguished within the sharp-colored band: an
upper dark purple one and a lower more pinkish one (indicated
by two arrows), in agreement with the absorbance spectrum,
which presents a main visible absorption peak at 555 nm with
a shoulder at 570 nm (Figure 2, left panel, D0, red trace).
The position and the closeness of the two species within the
gradient suggest that they correspond to homogeneous
monomer and dimer.10 These species, and their relative
amount, appear stable over 1 month. After 3 months, however,
the spectrum shows diffusion, witness of aggregation of the
protein, with the 555 and 570 nm absorbing species remaining
nondenatured (Figure 2, left panel, D91, blue trace). In
compound 2, bR migrates as a broad, diffuse band. This may be
related to the fact that compound 2 forms larger micelles
(Figure 1) and/or reflects the presence of a mixture of different
oligomeric states of the protein. Indeed, the spectrum of the
collected band shows a major peak at 555 nm, with a slight
shoulder around 590 nm (Figure 2, right panel), suggesting that
more than one oligomeric state of the protein is present. These
species are very stable over time, as the spectrum does not
show any sign of neither denaturation nor aggregation after
three months, which is quite remarkable. When solubilized in
n-octyl-β-D-thioglucopyranoside (OTG), bR is monomeric and
is completely denatured in only 3 days (not shown). The
presence of higher molecular weight oligomers thus confirm
that these hybrid surfactants are milder than detergents and that
protein−protein interactions can be favored over surfactant−
protein ones. This is particularly interesting when working with
proteins composed of several subunits and/or that oligomerize.
It is interesting to note that when solubilized in compounds
1 or 2, the protein displays a maximum of absorption in the
555−590 nm range and does not appear blue (λmax = 615 nm),
as when handled in fluorinated surfactants.16,27 This suggests
that compounds 1 and 2 organize so that the hydrogenated part
of their hydrophobic moiety interacts with the hydrophobic
domain of the protein. This is coherent with the fact that, as
mentioned in the introduction, van der Waals interactions are
weaker between fluoro- and hydrogenated groups than among
hydrogenated ones.
1
as a heteronuclear reference for H and 13C. Abbreviations used for
signal patterns are s, singlet; b, broad; d, doublet; t, triplet; q, quartet;
qt, quintet; sext, sextet; m, multiplet; dd, doublet of doublet. HR-MS
spectra were recorded on a mass spectrometer equipped with a TOF
analyzer for ESI+ experiments.
N-(2,3-Dihydroxypropyl)but-3-enamide (3). To a solution of 3-
aminopropane-1,2-diol (1.98 mL, 0.026 mol) and 3-butenoic acid
(1.97 mL, 0.023 mol) in ethanol (120 mL) was added EEDQ (6.890 g,
0.028 mol) portionwise under stirring. The reaction mixture was
heated at 60 °C for 18 h. After the mixture was cooled to rt, acidic
resin IRC-50 was added, and the reaction mixture was filtered and
concentrated in vacuo. The resulting crude compound was purified by
flash chromatography (9:1 EtOAc/MeOH) to afford compound 3
1
(3.291 g, 90%) as a white powder. H NMR (CDCl3): δ 6.85 (1H, t,
J = 6.0 Hz); 6.00−5.79 (1H, m); 5.24−5.14 (2H, m); 4.37 (1H, m);
4.22 (1H, m); 3.74 (1H, m); 3.61−3.44 (2H, m); 3.43−3.22 (2H, m);
3.03−2.99 (2H, d, J = 7.1 Hz). 13C NMR (CDCl3): δ 172.9; 130.9;
120.5; 71.2; 63.8; 42.4; 41.4. HRMS (ESI+) for C7H14NO3 m/z:
[M + H]+ = 160.0974 (calcd); [M + H]+ = 160.0974 (found).
N-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)but-3-enamide (4).
To a solution of 3 (12.000 g, 0.075 mol) in CH3CN (110 mL)
were added dimethoxypropane (10.2 mL, 0.082 mol) and a catalytic
amount of APTS under stirring. The reaction mixture was stirred at rt
for 4 h. Et3N was added dropwise until the pH reached ∼9, and the
reaction mixture was concentrated in vacuo. The resulting crude
compound was purified by flash chromatography (2:1 cyclohexane/
EtOAc) to afford compound 4 (10.500 g, 70%) as a colorless oil.
1H NMR (CDCl3): δ 6.20−5.75 (2H, m); 5.36 (1H, m); 5.11 (1H,
m); 4.24 (1H, m); 4.01 (1H, dd, J = 1.9 Hz, J = 6.5 Hz); 3.60 (1H, dd,
J = 2.0 Hz, J = 6.4 Hz); 3.55−3.25 (2H, m); 3.04 (2H, d, J = 7.5 Hz);
1.42 (3H, s); 1.34 (3H, s). 13C NMR (CDCl3): 171.0; 131.3; 119.8;
109.3; 74.4; 66.7; 41.4; 26.8; 25.3. HRMS (ESI+) for C10H18NO3 m/z:
[M + H]+ = 200.1287 (calcd); [M + H]+ = 200.1285 (found).
N-((2,2-Dimethyl-1,3-dioxolan-4-yl)methyl)-5,5,6,6,7,7,8,8,9,-
9,10,10,10-tridecafluoro-3-iododecanamide) (5). To a solution of 4
(0.200 g, 1.0 mmol) in anhydrous THF (5 mL) were added C6F13I
(0.282 mL, 1.3 mmol) and AIBN (0.099 g, 0.6 mmol) under stirring
and argon atmosphere. The reaction mixture was heated at 70 °C for
18 h in a sealed tube. After being cooled to rt, the reaction mixture was
concentrated in vacuo. The resulting crude compound was purified by
flash chromatography (2:1 cyclohexane/EtOAc) to afford compound 5
(0.355 g, 55%) as a white powder. 1H NMR (CDCl3): δ 5.90 (1H, bs);
4.70 (1H, m); 4.28 (1H, m); 4.05 (1H, dd, J = 1.9 Hz, J = 6.5 Hz);
3.79 (1H, dd, J = 2.0 Hz, J = 6.5 Hz); 3.52−3.21 (2H, m); 3.07−2.76
(4H, m); 1.46 (3H, s); 1.36 (3H, s). 13C NMR (CDCl3): δ 169.6;
We have synthesized two novel fluorinated surfactants whose
apolar domain comprises a perfluorohexane group in the main
chain and a butyl-hydrogenated side chain. The two com-
pounds 1 and 2 have rather low CMCs, ∼0.08 and ∼0.01 mM,
respectively, and they self-assemble into small aggregates
D
DOI: 10.1021/acs.joc.5b02137
J. Org. Chem. XXXX, XXX, XXX−XXX