Hybrid double-chain maltose-based detergents: Synthesis and colloidal and biochemical evaluation

Article abstract of DOI:10.1021/acs.joc.9b00873

Four hybrid double-chain surfactants with a maltose polar head were synthesized. The apolar domain consists of a hydrogenated chain, and a partially fluorinated chain made of a propyl hydrogenated spacer terminated by a perfluorinated core of various leng

Full text of DOI:10.1021/acs.joc.9b00873

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Article
Hybrid Double-Chain Maltose-Based Detergents:
Synthesis, Colloidal and Biochemical Evaluation
Christophe Bonnet, Pierre Guillet, Sébastien Igonet, Annette Meister,
Anais Marconnet, Sandro Keller, Anass Jawhari, and Grégory Durand
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00873 • Publication Date (Web): 15 Aug 2019
Downloaded from pubs.acs.org on August 17, 2019
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Hybrid Double-Chain Maltose-Based Detergents: Synthesis, Colloidal and
Biochemical Evaluation
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Christophe Bonnet,a,b Pierre Guillet,a,b Sébastien Igonet,b,c Annette Meister,d Anaïs
Marconnet,a,b Sandro Keller,e Anass Jawhari,b,c Grégory Durand,a,b*
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aInstitut des Biomolécules Max Mousseron (UMR 5247 UM-CNRS-ENSCM) & Avignon University,
Equipe Chimie Bioorganique et Systèmes amphiphiles, 301 rue Baruch de Spinoza – 84916 AVIGNON
cedex 9 (France) ;
bCHEM2STAB, 301 rue Baruch de Spinoza – 84916 AVIGNON cedex 9 (France) ;
cCALIXAR, 60 Avenue Rockefeller – 69008 Lyon (France) ;
dMartin Luther University Halle-Wittenberg, ZIK HALOmem and Institute of Biochemistry and
Biotechnology, Kurt-Mothes-Str. 3a, D-06120 Halle/Saale, Germany ;
eMolecular Biophysics, Technische Universität Kaiserslautern (TUK), Erwin-Schrödinger-Str. 13, 67663
Kaiserslautern, Germany.
*Corresponding Author. Grégory Durand. E-mail: gregory.durand@univ-avignon.fr ; Phone: +33 (0)4
9014 4445.
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Abstract. Four hybrid double-chain surfactants with a maltose polar head were synthesized.
The apolar domain consists of a hydrogenated chain, and a partially fluorinated chain made
of a propyl hydrogenated spacer terminated by a perfluorinated core of various length. Their
water solubility was found to be lower than 1g/L irrespective of the length of both chains. The
self-assembling properties of pure hybrids in water was studied by dynamic light scattering
and transmission electron microscopy, which revealed the formation of two populations of
aggregates with diameters of 8–50 nm diameter and 80–300 nm. When mixed with the
classical detergent n-dodecylmaltoside (DDM), the four hybrids were well soluble and formed
small mixed micelles. DDM/hybrid mixtures were further evaluated for the extraction of the
full-length, wild-type human GPCR adenosine receptor (A2AR) and the bacterial transporter
AcrB. The solubilization of A2AR showed extraction efficiencies ranging from 40% to 70%, while
that of AcrB reached 60% to 90%. Finally, three of the hybrids exhibited significant
thermostabilization when present as additives. The derivative with a C12 hydrogenated chain
and a C4F9 fluorinated chain emerged as the most potent additive exhibiting both good
extraction yields of A2AR and AcrB and thermostabilization of A2AR by ~7°C.
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Introduction.
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Sugar-based amphiphiles have been extensively developed over the past decades.1 The
growing interest arises from several reasons. The growing interest arises from several reasons.
They can be prepared from abundant renewable raw vegetable materials; convenient organic
chemistry allows structural diversity, which, in turn, gives access to different colloidal systems
such as micelles, liposomes, foams, and emulsions; they exhibit low toxicity and high
biodegradability. The diversity in chemical geometry goes from the rather simple “head-and-
tail” alkyl glucosides derivatives that have been used successfully for membrane-protein
solubilization and crystallization2-4 or as hydrogelators,5,6 to more sophisticated gemini
surfactants7 for gene delivery or highly fluorinated8 derivatives for drug delivery, to name but
a few.
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Fluorinated chains present the peculiar property of being both hydrophobic and lipophobic,
which provides to fluorinated surfactants (FSs) numerous industrial and medical
applications.9,10 FSs have also shown promising properties as milder alternatives to classical
detergents for the study of membrane proteins (MPs).11,12 Most of FSs used for the study of
MPs have the same “head-and-tail” architecture as classical hydrogenated detergents, but
they differ from the latter by the presence of several perfluorinated fluorinated carbons within
the tail. More recently, their ability to solubilize artificial membranes13 and to extract MPs has
also been reported,14 making them promising agents for both extraction and stabilization of
membrane target proteins.
Fluorocarbon-hydrocarbon hybrid surfactants have an hydrophobic part made of a fluorinated
and a hydrogenated chain and are referred to as hybrid double-chain surfactants.15 Among
the properties of such hybrid surfactants are the high viscosity of aqueous solutions,
intramicellar phase separation between the two chains, and long micelle lifetimes.15,16 We
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have recently reported the synthesis of two hybrid double-chain surfactants with a di-glucose
polar head whose apolar domain consists of a perfluorohexyl main chain and a butyl
hydrogenated branch as a side chain.17 They were both able to keep the membrane protein
bacteriorhodopsin stable demonstrating the interest of hybrid surfactants for MPs
stabilization.
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We report herein the synthesis of a new series of hybrid double-chain detergents presenting
structural similarity with conventional and widely used n-alkyl maltosides detergents. These
new compounds have a maltose polar headgroup and two alkyl chains, one being fully
hydrogenated while the other one is partially fluorinated. The n-dodecyl (C12) hydrogenated
derivatives LC045-LC047 have a fluorinated chain of various lengths i.e. C2F5, C4F9 or C6F13,
respectively while LC057 has an n-octyl (C8) hydrogenated chain and a C4F9 chain (Figure 1).
OH
O
HO
HO
OH
OH
O
O
HO
O
OH
n-dodecyl -D-maltoside
F
F
F
F
OH
O
F
n
HO
OH
HO
OH
O
O
O
O
HO
OH
m
LC045
LC046
LC047
LC057
: n = 1; m = 5
: n = 3; m = 5
: n = 5; m = 5
: n = 3; m = 1
Figure 1. Chemical structures of n-dodecyl β-D-maltoside (DDM) a widely used detergent for
MPs study and of the four hybrid double-chain surfactants LC045, LC046, LC047 and LC057.
The self-aggregation of these four derivatives was further investigated by means of dynamic
light scattering (DLS) and transmission electron microscopy (TEM). Because of the poor
solubility of these derivatives, the self-aggregation of their mixtures with the classical DDM
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detergent was also investigated. Finally, the ability of such mixtures to solubilize and stabilize
membrane proteins was evaluated.
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Results and Discussion
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Synthesis. The synthesis follows a convergent synthetic route, based on three key steps: (i)
synthesis of the hydrophobic parts; (ii) synthesis of the maltose polar head; and (iii) coupling
of the hydrophobic and hydrophilic parts (Scheme 1). This convergent strategy allows
versatility in chemical structure and would allow the synthesis of a large library of analogs by
varying the nature of both polar head group and fluorinated chain.
OH
R2
O
HO
HO
OH
OH
O
O
O
HO
O
R1
OH
R1 = C6H13 or C10H21
R2 = H, C2F5, C4F9, C6F13
OBz
R2
O
OBz
BzO
BzO
O
OBz
O
O
BzO
HO
O
CCl3
OBz
+
R1
NH
OH
OH
O
HO
HO
HO
OH
OH
O
1,2-octanediol
or
O
HO
OH
OH
Maltose
OH
HO
1,2-dodecanediol
Scheme 1. Retrosynthetic pathway for compounds LC045, LC046, LC047 and LC057 from 1,2-
dodecanediol or 1,2-octanediol.
The synthesis of the various hydrophobic parts was achieved following a five-step route
starting either from racemic 1,2-dodecanediol or 1,2-octanediol. The two hydroxyl groups
allow the connection of both fluorinated chain and maltose polar head. We decided to
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introduce the fluorinated chain on the secondary free hydroxyl group of the starting diol in
order to keep the most reactive primary alcohol vacant for the glycosylation step.
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In the first step, the primary alcohol group was selectively protected in the presence of trityl
chloride and Et3N to afford compound 1.18 Then, allylation of the secondary alcohol was
achieved using allyl iodide in the presence of NaH. The insertion of the allyl moiety was
confirmed by 1H NMR with the presence of signals at 5.2 and 5.9 ppm, corresponding to the
CH2 and CH of the double bond, respectively. Deprotection of the trityl group with p-TsOH in
methanol led to compound 3. The addition of the fluorinated chain on the allyl moiety was
achieved by radical addition of perfluoroalkyl iodide. In previous work, direct insertion of
perfluoroalkyl chains was done under inert atmosphere using AIBN as initiator in a sealed
tube.17,19 With the aim to explore milder conditions we used the methodology described by
Takeyama and co-workers, consisting in the use of triethylborane in the presence of oxygen.20
Under these conditions insertion of the fluorinated chain was achieved in moderate to good
yields ranging from 32% for compound 7c to 43% for compound 7a. The insertion of the
fluorinated chain was further confirmed by 1H NMR with the absence of unsaturated protons
at 5.2 and 5.9 ppm and the presence of signals at 1.8 and 2.2 ppm related to the CH2 in α and
β position of the fluorinated segment. Reduction of the C-I bond was also improved compared
to our previous work where we employed stoichiometric quantities of Bu3SnH at 70°C in a
sealed tube under inert condition.17,19,21 Catalytic hydrogenation in the presence of Pd-C was
chosen. This method was performed at room temperature in a hydrogenation reactor and led
to the reduced compounds 8a-c in good yields. This new synthetic route therefore provides a
convenient method for both radical addition of fluorinated chains and reduction of the iodine
group. A similar synthetic route was used for the synthesis of compound 8d with 1,2-
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octanediol as starting material (Scheme 2). Hydrophobic double-chain precursors were
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therefore prepared in 5 steps in 11–16% overall yields.
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Et3N, TrCl,
CH2Cl2 anh.,
rt, 24h
NaH, allyl iodide,
DMF anh.,
rt, 24h
TsOH, MeOH,
rt, 4h to 6h
OH
R1
OH
R1
O
O
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Tr-O
HO
Tr-O
HO
R1
R1
3:
4:
1:
2:
R1 = C10H21 ; 78%
R1 = C6H13 ; 62%
R1 = C10H21 ; 90%
R1 = C6H13 ; 90%
5:
6:
R1 = C10H21 ; 80%
R1 = C6H13 ; 61%
H2/ Pd-C,
I
R2
RF-I, B(Et)3,
hexane, rt,
3h to 24h
CH3CO2H,
MeOH, rt,
overnight
O
R2
O
HO
HO
R1
R1
7a
7b
7c
: R1 = C10H21 ; R2 = C2F5 ; 43%
: R1 = C10H21 ; R2 = C4F9 ; 47%
: R1 = C10H21 ; R2 = C6F13 ; 32%
8a
8b
8c
: R1 = C10H21 R2= C2F5 ; 76%
: R1 = C10H21 R2= C4F9 ; 88%
: R1 = C10H21 R2= C6F13 ; 68%
7d:
R1 = C6H13 ; R2 = C4F9 ; 52%
8d:
R1 = C6H13 R2= C4F9 ; 46%
Scheme 2. Synthesis of the hydrophobic alcohols 8a-d.
In order to obtain high yield and stereoselective O-glycosylation leading to the β anomer,
activation of the anomeric position and protection of the other free hydroxy groups is
preferable. Here, we decided to activate the maltose polar head into trichloroacetimidate and
to use esters protecting groups.22 Hepta-O-benzoyl-maltose-1-O-trichloroacetimidate 15 was
readily prepared on large scale from commercially available maltose following a four steps
synthetic route.23
The coupling between the hydrophilic and the lipophilic parts was next achieved following
Schmidt reaction in the presence of TMSOTf as Lewis acid promoter.24 Preliminary work
showed that TMSOTf compared to BF3.OEt2 led to higher yields of reaction (data not shown).
Under these conditions, hydrophobic alcohol 8a-d were condensed onto the benzoylated
maltose polar head 1523 in good yield in a few hours. Then, benzoyl groups were removed
under Zemplén conditions to afford final hybrid derivatives 17a-d (Scheme 3). Analysis of the
proton NMR spectra showed the formation β anomer witnessed by a doublet between 4.0
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and 4.5 ppm with a coupling constant of ~8 Hz (Jtrans) corresponding to the anomeric proton
H1. The second anomeric proton between the two glucose units appeared at 5.2 ppm with a
coupling constant of ~4 Hz (Jcis).
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OBz
OBz
R2
R2
O
O
TMSOTf (1eq),
CH2Cl2, overnight
BzO
BzO
BzO
BzO
OBz
OBz
OBz
OBz
O
O
O
O
+
O
O
O
O
CCl3
NH
HO
BzO
BzO
R1
OBz
OBz
R1
16a:
R1 = C10H21 ; R2 = C2F5 ; 70%
R1 = C10H21 ; R2 = C4F9 ; 79%
R1 = C10H21 ; R2 = C6F13 ; 76%
R1 = C6H13 ; R2 = C4F9 ; 51%
8a-d
16b:
16c:
16d:
15
OH
R2
O
HO
MeONa, MeOH,
rt, 4h
OH
HO
OH
O
O
O
HO
O
R1
OH
LC045 = 17a:
LC046 = 17b:
LC047 = 17c:
LC057 = 17d:
R1 = C10H21 ; R2 = C2F5 ; 55%
R1 = C10H21 ; R2 = C4F9 ; 100%
R1 = C10H21 ; R2 = C6F13 ; 91%
R1 = C6H13 ; R2 = C4F9 ; 79%
Scheme 3. Glycosylation and deprotection step leading to the formation of compounds LC045,
LC046, LC047 and LC057.
Colloidal characterization. We first evaluated the solubility of the hybrid series in water. At
1g/L and despite sonication and heating at 40°C, aqueous solutions of hybrid derivatives were
opalescent for all derivatives. Centrifugation of the opalescent solutions led to the formation
of a clear supernatant solution and a precipitate. For comparison DDM exhibits a solubility in
water of 200 g/L. This shows that the additional fluorinated segment, regardless of its length,
significantly affects the overall hydrophobicity and significantly reduces the solubility in water.
We next conducted DLS experiments to determine whether the water-soluble fraction of
these hybrid surfactants could form aggregates. The composition of the supernatant solution
of a centrifuged suspension prepared at 1g/L was analyzed. Whatever the hybrid tested,
volume-weighted particle size distributions indicated the presence of two populations of
aggregates within the range of 20 to 300 nm (Figure 2). Number-weighted particle size
distributions showed one main population of aggregates of ∼15 to 50 nm hydrodynamic
diameter, while intensity-weighted particle size distributions were dominated by a population
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of larger aggregates with diameters between 80 and 300 nm (Figure 2). These results suggest
the coexistence of small and large aggregates species in solution, smaller aggregates
accounting for the major part.
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Figure 2. Hydrodynamic size distribution by Contin analysis for aqueous solutions of LC045 (A)
and LC046 (C). TEM images of negatively stained samples LC045 (B) and LC046 (D). The
supernatant of centrifuged suspensions at 1g/L were analyzed.
Then, we performed TEM experiments to further characterize the aggregates observed by
DLS. The composition of the supernatant of a 1g/L saturated solution was analyzed using the
negative staining method. As shown in Figure 2, LC045 and LC046 solutions allowed the
observation of aggregates with an averaged diameter of ∼15 ± 5 nm (LC045) and ∼25 ± 10 nm
(LC046). The presence of few aggregates with diameter of ∼80 to 100 nm was also noted. This
confirms the coexistence of small and large aggregates as observed by DLS. For the LC047 and
LC057, we also observed the coexistence of larger and smaller aggregates, the latter exhibiting
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40 and 8.0 nm diameter, respectively, in agreement with the hydrodynamic diameters
determined by number analysis (TEM images not shown).
To overcome the poor water-solubility of LC045, LC046, LC047, and LC057 and to allow the
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evaluation of their potency as stabilizing agent for membrane proteins, we next prepared
mixtures with DDM at different molar ratio. On the one hand, DDM is readily soluble in water
and therefore is able to promote the solubilization of our hybrid molecules in water. On the
other hand, the use of DDM/additive mixtures has already been successfully employed in
several biochemical assays.25,26 Below a DDM:LC molar ratio of 100:10, the four hybrids
appeared well soluble in water, as witnessed by transparent solutions. Beyond this ratio,
gradual addition of hybrids promoted the formation of a cloudy solution, the critical
concentration of hybrid molecules at which the transfer from a clear to a cloudy solution
occurred is reported in Table S1. Along the C12 series, LC047 that has the longest fluorinated
chain induced the lowest solubility of the mixture with the appearance of a cloudy solution at
100:20, while for compound LC045 with the shortest fluorinated chain the mixture was still
soluble at 100:50. We next performed hydrodynamic diameter measurements on DDM:LC
mixtures.
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We used the same DDM:LC molar ratios than those applied for membrane solubilization and
stability assays (see below). DDM:LC molar ratios of 100:2 and 100:4 were studied for the C12
series while for LC057 a 100:10 was studied. Contin distribution plots showed the presence of
aggregates with hydrodynamic diameters of about 5 to 8 nm, for all hybrids and all mixing
ratios tested (Figure S59). This indicates that DDM:hybrid mixtures self-assemble into small
micellar aggregates. TEM experiments failed to show any of these aggregates. In general,
aggregates smaller than 10 nm in diameter can only be visualized if the staining gives good
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contrast, if a microscope with high acceleration voltage (300kV) is applied, or if cryo
transmission electron microscopy is performed.
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Solubilization and stabilization of membrane proteins. In order to determine the ability of the
four hybrids compounds to act as solubilizing and stabilizing agents we conducted a
biochemical evaluation on two membrane targets of biological interest: The full length and
wild-type human GPCR adenosine receptor (A2AR), a eukaryotic protein that contains seven
transmembrane domains and the bacterial transporter AcrB, a prokayriotic protein that
contains twelves transmembrane domains. As above, DDM/hybrid mixtures ranging from
100:2 to 100:10 were studied. The solubilization of A2AR showed extraction yields varying from
40% to 70% of the total content of proteins (Figure 3A). These values are close to those
obtained with SDS (not shown) and in DDM/cholesteryl hemisuccinate (CHS) mixture (Figure
3A), two very extracting conditions. The use of DDM/cholesteryl hemisuccinate (CHS) mixture
to efficiently extract and stabilize A2AR has been reported in the literature including our
previous work.27
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Figure 3. Solubilization experiments of human A2AR (A) and of bacterial AcrB (B) in the
presence of DDM:LC045, LC046, LC047, and LC057 mixtures. Biological membranes were
incubated for 2 h in the presence of detergents at 4°C and the solution was subjected to
ultracentrifugation. The soluble fraction was analyzed by Western blotting to quantify the
amount of solubilized protein.
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The solubilization of AcrB indicates good extraction properties for DDM:LC mixtures with high
yields from 60% to 90% close to that of DDM, the standard generally used to extract that
protein (Figure 3B). It is also interesting to note that better extraction yields were obtained
for AcrB compared to A2AR. The solubilizing assays on the two membrane targets A2AR and
AcrB showed that DDM:LC057 and DDM:LC046 mixtures exhibited good extraction potencies
comparable to or even better than that of SDS and DDM/CHS. As for DDM:LC045 and
DDM:LC047 mixtures, the potency remained similar to that of DDM:CHS, thus demonstrating
that their addition does not affect the solubilization efficiency of DDM.
In order to evaluate the stabilizing properties of the DDM/hybrid mixtures, thermal shift (Tm)
measurements were undertaken. The value of Tm corresponds to the temperature at which
half of the proteins in solution are found in a denatured state. Thermostability assays on A2AR
showed that DDM:LC046 and DDM:LC047 mixtures induced a thermal shift of +6.8 and +5.7°C,
respectively, compared to the reference condition (Figure 4), while DDM:LC045, DDM:LC057,
and DDM:CHS mixtures led to similar Tm values (Figure 4). Regarding wild-type, full-length
A2AR receptor, no classical detergent was reported to stabilize the protein. Therefore,
previously reported work describes the removal the C-terminal domain (96 amino acids) and
heavy mutagenesis of the protein to improve its stability (8 mutations).28 Having an
improvement of +6.8 and +5.7°C when using DDM:LC046 and DDM:LC047 mixtures, in
comparison with DDM/CHS, therefore constitutes a significant achievement.
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Thermostability assays in the presence of LC057 were also conducted on AcrB using
DDM:LC057 mixture, indicating a less pronounced thermal shift of +3.2°C in comparison with
the reference (Figure 4).
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*Ref = DDM 0.5% + CHS 1/10.
Figure 4. (A) Thermostability results on the membrane targets A2AR (green) and AcrB (yellow)
in the presence of LC045, LC046, LC047 and LC057. For both proteins DDM is used at 0.5%.
Proteins were submitted to heat treatment (30–80°C), followed by ultracentrifugation before
loading of the supernatant onto SDS-PAGE and Western blotting. (B) A2AR and (C) AcrB
representative gel bands.
Conclusion.
We have synthesized a new series of four fluorocarbon/hydrocarbon hybrid double-chain
surfactants with a maltose polar head. One chain is made of either a n-dodecyl or a n-octyl
hydrogenated chain, while the second chain is made of a propyl hydrogenated spacer
terminated by a perfluorinated core of various length, that is, C2F5, C4F9 and C6F13. Whatever
the length of the chains, these four compounds were found to be poorly soluble in water. The
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supernatant of a 1 g/L saturated solution contains two populations of aggregates of ~8 to 50
nm for the smaller one and of ~80 to 300 nm for the larger one as observed by DLS and TEM.
When mixed with DDM detergent at ratios varying from 2 to 10%, the four hybrids were well
soluble and formed small mixed micelles. The potency of these DDM/hybrid mixtures to
extract two membrane proteins, namely, the full-length, wild-type human GPCR adenosine
receptor (A2AR) and the bacterial transporter AcrB, was further demonstrated without
affecting the potency of DDM itself, thereby indicating that they can be used as additives for
membrane-protein studies. Finally, their ability to thermostabilize the two proteins was also
investigated, and three hybrids exhibited thermostabilization. The derivative with a n-dodecyl
hydrogenated chain and a C4F9 fluorinated chain appeared as the most potent additive
exhibiting both good extraction yields of A2AR and AcrB and thermostabilization of A2AR by
~7°C. The main limitation of the new compounds lies in their low water solubility. With a
convenient synthetic route in hand that allows versatility in the chemical structures, current
work is focused on the synthesis of more water-soluble derivatives.
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Experimental section.
Material & Methods. All starting materials were commercially available and were used
without further purification. All solvents were of reagent grade and used as received unless
otherwise indicated. Anhydrous solvents were dried by simple storage over activated 4-Å
molecular sieves for at least 24h. Molecular sieves were activated by heating in vacuo for 24h.
The progress of the reactions was monitored by thin layer chromatography. The compounds
were detected either by exposure to ultraviolet light (254 nm) or by spraying with sulfuric acid
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(5% ethanol) and/or ninhydrin (5% ethanol), followed by heating at ~150°C. H, C, and F
NMR experiments were performed at 400, 100, and 375 MHz, respectively. Chemical shifts
are given in ppm relative to the solvent residual peak as a heteronuclear reference for 1H and
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13C. The coupling constants J are given in hertz and the chemical shifts in parts-per-million
(ppm). Abbreviations used for signal patterns are: s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; dd, doublet of doublet; dt, doublet of triplet. High-resolution mass spectra were
determined on a Synapt G2-S (Waters) mass spectrometer with a TOF mass analyzer in a
positive ionization mode.
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1-(trityloxy)dodecan-2-ol (1).29 To a solution of 1,2-dodecanediol (12 g, 59.3 mmol, 1 eq) in
anhydrous CH2Cl2, was added Et3N (16.5 mL, 119.0 mmol, 2 eq). The solution was stirred for
20 min and trityl chloride (19.8 g, 71.2 mmol, 1.2 eq) was added portion-wise. The reaction
mixture was stirred for 24 h, then concentrated in vacuo. The residue was diluted with
cyclohexane and the precipitate was filtered off. The solution was concentrated in vacuo. The
crude compound was purified by flash chromatography (cyclohexane/AcOEt 90:10 v/v) to
1
yield 1 as a colorless oil (23.7 g, 90 %). Rf = 0.64 (cyclohexane/AcOEt 80:20 v/v). H NMR
(CDCl3, 400 MHz): δ 7.45-7.43 (6H, m); 7.28-7.18 (9H, m); 3.70-3.65 (1H, m); 3.04 (1H, dd, J =
6 Hz, J = 12 Hz); 3.01 (1H, dd, J = 4 Hz, J = 12 Hz); 1.53 (2H, m); 1.25 (16H, bs); 0.88 (3H, t, J =
8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 145.6; 129.9-128.3; 87.7; 71.8; 68.8; 34.9; 33.0; 30.4-
30.7; 26.3; 23.7; 14.4. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C31H40O2Na 467.2926; Found
467.2920.
1-(trityloxy)octan-2-ol (2). To a solution of 1,2-octanediol (20 g, 137 mmol, 1 eq) in anhydrous
CH2Cl2, was added Et3N (38 mL, 274 mmol, 2 eq). The solution was stirred for 20 min, and trityl
chloride (45.7 g, 164 mmol, 1.2 eq) was added portion-wise. The reaction mixture was stirred
for 24 h then concentrated in vacuo. The residue was diluted with cyclohexane and the
precipitate was filtered off. The solution was concentrated in vacuo. The crude compound was
purified by flash chromatography (cyclohexane/AcOEt 95:5 v/v) to yield 2 as a colorless oil
(47.9 g, 90 %). Rf = 0.38 (cyclohexane/AcOEt 90:10 v/v). 1H NMR (CDCl3, 400 MHz): δ 7.45-7.30
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(15H, m); 3.78 (1H, m); 3.20 (1H, dd, J = 4Hz, J = 12 Hz); 3.05 (1H, dd, J = 8 Hz, J = 12 Hz); 2.34
(1H, bs); 1.40 (2H, br); 1.26 (8H, bs); 0.89 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ
143.9; 128.7; 127.8; 127.1; 86.6; 71.0; 67.8; 33.3; 31.7; 29.2; 26.9; 26.9; 25.4; 22.6; 14.0. HRMS
(ESI-TOF) m/z: [M + Na]+ calcd for C27H32O2Na 411.2300; Found 411.2312.
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(((2-(allyloxy)dodecyl)oxy)methanetriyl)tribenzene (3). To a suspension of NaH (0.52 g,
21.6 mmol, 2.4 eq) in anhydrous DMF, was added a solution of 1 (4 g, 9 mmol, 1 eq) in
anhydrous DMF at 0 °C. The solution was stirred at 0 °C for 20 min, and allyl iodide (0.9 mL,
9.9 mmol, 1.2 eq) was added dropwise. The reaction mixture was stirred for 24 h, then water
was added, and the solution was extracted twice with AcOEt. The organic layer was dried over
anhydrous Na2SO4, filtered and concentrated in vacuo. The crude compound was purified by
flash chromatography (cyclohexane/AcOEt 95:5 v/v) to yield 3 as a colorless oil (3.4 g, 78 %).
Rf = 0.58 (cyclohexane/AcOEt 95:5 v/v). 1H NMR (CDCl3, 400 MHz): δ 7.50-7.20 (15H, m); 5.95
(1H, m); 5.27 (1H, d, J = 16 Hz); 5.15 (1H, d, J = 12 Hz); 4.18 (1H, dd, J = 4 Hz, J= 12 Hz); 4.00
(1H, dd, J = 8 Hz, J = 12 Hz); 3.45 (1H, m); 3.17 (1H, dd, J = 6 Hz, J = 10 Hz); 3.10 (1H, dd, J =
13
4 Hz, J = 10 Hz); 1.47 (2H, m); 1.26 (16H, bs); 0.89 (3H, t, J = 8 H). C {1H} NMR (CDCl3,
100 MHz): δ 144.4; 135.7; 128.9; 127.9; 127.0; 116.6; 86.7; 78.7; 71.4; 66.2; 32.4; 32.1; 29.8;
29.7; 29.5; 27.1; 25.6; 22.8; 14.3. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C34H44O2Na
507.3239; Found 507.3238.
(((2-(allyloxy)octyl)oxy)methanetriyl)tribenzene (4). To a suspension of NaH (3.0 g,
123.5 mol, 2.4 eq) in anhydrous DMF, was added a solution of 2 (20 g, 51.5 mmol, 1 eq) in
anhydrous DMF at 0 °C. The solution was stirred at 0 C for 20 min, and allyl iodide (5.8 mL,
61.8 mmol, 1.2 eq) was added dropwise. The reaction mixture was stirred for 24 h, then water
was added, and the solution was extracted twice with AcOEt. The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude compound 4 (13.8 g, 62 %)
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was directly used without further purification. Rf = 0.65 (cyclohexane/AcOEt 95:5 v/v).1H NMR
(CDCl3, 400 MHz): δ 7.55-7.20 (15H, m); 5.96 (1H, m); 5.30 (1H, d, J = 16 Hz); 5.15 (1H, d, J =
12 Hz); 4.20 (1H, dd, J = 4 Hz, J = 12 Hz); 4.02 (1H, dd, J = 8 Hz, J = 12 Hz); 3.48 (1H, qt, J = 8 Hz);
3.20 (1H, dd, J = 4 Hz, J = 12 Hz); 3.15 (1H, dd, J = 8 Hz, J = 12 Hz); 1.55 (2H, m); 1.27 (8H, bs);
0.88 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 144.1; 135.4; 128.6; 127.6; 126.8;
116.2; 86.4; 78.4; 71.1; 66.0; 32.1; 31.7; 29.2; 26.8; 25.3; 22.5; 14.0. HRMS (ESI-TOF) m/z: [M
+ Na]+ calcd for C30H36O2Na 451.2608; Found 451.2604.
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2-(allyloxy)dodecan-1-ol (5). To a solution of 3 (3.4 g, 7.0 mmol, 1 eq) in MeOH was added
portion-wise p-TsOH (85 mg, 0.7 mmol, 0.1 eq). The reaction mixture was stirred for 4 h then
the solution was neutralized by addition of Et3N and concentrated in vacuo. The crude
compound was purified by flash chromatography (cyclohexane/AcOEt 90:10 v/v) to yield 5 as
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a colorless oil (1.37 g, 80 %). Rf = 0.21 (cyclohexane/AcOEt 90:10 v/v). H NMR (CDCl3,
400 MHz): δ 5.90 (1H, m); 5.20 (2H, dd, J = 12 Hz, J = 16 Hz); 4.10 (2H, m); 3.65 (1H, m); 3.50
(1H, m); 3.40 (1H, m); 2.26 (1H, bs), 1.55 (1H, m); 1.45 (1H, m); 1.24 (16H, bs); 0.86 (3H, t, J
= 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 135.0; 116.8; 79.7; 70.4; 64.2; 31.8; 30.8; 29.8; 29.5;
29.3; 25.3; 22.6; 14.0. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C15H30O2Na 265.2143; Found
265.2142.
2-(allyloxy)octan-1-ol (6). To a solution of 4 (13.8 g, 32.2 mmol, 1 eq) in MeOH was added p-
TsOH (0.38 g, 3.2 mmol, 0.1 eq), and the reaction mixture was stirred for 6 h. Then the solution
was neutralized by addition of Et3N, and concentrated in vacuo. The crude compound was
purified by flash chromatography (cyclohexane/AcOEt 80:20 v/v) to yield 6 as colorless oil
(3.66 g, 61 %). Rf = 0.35 (cyclohexane/AcOEt 80:20 v/v). 1H NMR (CDCl3, 400 MHz): δ 5.90 (1H,
m); 5.25 (1H, d, J = 16 Hz); 5.14 (1H, d, J = 12 Hz); 4.03 (2H, m); 3.63 (1H, m); 3.46 (1H, m); 3.38
(1H, m); 2.27 (1H, bs); 1.50 (2H, m); 1.26 (8H, bs); 0.85 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3,
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100 MHz): δ 135.1; 116.8; 79.7; 70.5; 64.3; 31.7; 30.8; 29.4; 25.3; 22.6; 14.0. HRMS (ESI-TOF)
m/z: [M + H]+ calcd for C11H23O2 187.1693; Found 187.1688.
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2-((4,4,5,5,5-pentafluoro-2-iodopentyl)oxy)dodecan-1-ol (7a). To a solution of 5 (1.4 g,
5.8 mmol, 1 eq) in hexane, was added perfluoroethyl iodide (1.0 mL, 8.7 mmol, 1.5 eq) at -
40 °C and in a sealed tube. The reaction mixture was stirred at -40 °C for 20 min, and
triethylborane (0.5 mL, 3.5 mmol, 0.6 eq) was added dropwise. The reaction mixture was
stirred for 24 h at room temperature then the solution was concentrated in vacuo. The crude
compound was purified by flash chromatography (cyclohexane/AcOEt 90:10 v/v) to yield 7a
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as a yellow oil (1.22 g, 43 %). Rf = 0.28 (cyclohexane/AcOEt 90:10 v/v). H NMR (CDCl3,
400 MHz): δ 4.40 (1H, m); 3.80 (2H, m); 3.70 (1H, m); 3.60 (1H, m); 3.45 (1H, m); 3.10 (1H, m);
2.70 (1H, m); 2.40 (1H, m); 1.55 (1H, m); 1.46 (1H, m); 1.26 (16H, bs); 0.87 (3H, t, J = 8 Hz). 13C
{1H} NMR (CDCl3, 100 MHz): δ 81.1; 73.9; 64.5; 37.5; 31.9; 30.8; 29.7; 29.6; 29.5; 29.4, 29.3;
25.4; 25.3; 22.7; 16.4; 15.8; 13.9. 19F (CDCl3, 375 MHz): δ -85.8; -117.6.
2-((4,4,5,5,6,6,7,7,7-nonafluoro-2-iodoheptyl)oxy)dodecan-1-ol (7b). To a solution of 5
(1.5 g, 6.2 mmol, 1 eq) in hexane was added perfluorobutyl iodide (1.6 mL, 9.3 mmol, 1.5 eq)
then the solution was cooled down to 0°C and triethylborane (0.55 mL, 3.8 mmol, 0.6 eq) was
added dropwise. The mixture was stirred for 10 min at 0°C and for 24h at room temperature
then the solution was concentrated in vacuo. The crude compound was purified by flash
chromatography (cyclohexane/AcOEt 90:10 v/v) to yield 7b as a yellow oil (1.7 g, 47 %). Rf =
0.49 (cyclohexane/AcOEt 80:20 v/v). 1H NMR (CDCl3, 400 MHz): δ 4.40 (1H, m); 3.85-3.70 (2H,
m); 3.65 (1H, m); 3.55 (1H, m); 3.42 (1H, m); 3.04 (1H, m); 2.70 (1H, m); 2.24-2.14 (1H, m); 1.55
(1H, m); 1.45 (1H, m); 1.24 (16H, bs); 0.86 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ
81.2; 74.0; 64.4; 37.8; 37.6; 37.4; 31.9; 30.8; 29.7; 29.6; 29.5; 29.3; 26.9; 25.4; 25.3; 22.6; 16.3;
15.7; 14.0. 19F (CDCl3, 375 MHz): δ -81.19; -114.01; -124.7; -126.0.
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2-((4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluoro-2-iodononyl)oxy)dodecan-1-ol (7c). To a solution
of 5 (0.5 g, 2.07 mmol, 1 eq) in hexane was added perfluorohexyl iodide (1.1g , 2.5 mmol,
1.2 eq) before the solution was cooled down to 0°C and triethylborane (0.4 mL, 0.4 mmol,
0.2 eq) was added dropwise. The mixture was stirred for 10 min at 0°C and for 3h at room
temperature, then the solution was concentrated in vacuo. The crude compound was purified
by flash chromatography (cyclohexane/AcOEt 90:10 v/v) to yield 7c as a yellow oil (0.45 g,
32 %). Rf = 0.34 (cyclohexane/AcOEt 90:10 v/v). 1H NMR (CDCl3, 400 MHz): δ 4.40 (1H, m);
3.90-3.70 (2H, m); 3.63 (1H, m); 3.51 (1H, m); 3.41 (1H, m); 3.10 (1H, m); 2.75 (1H, m); 2.45-
2.36 (1H, m); 1.60 (1H, m). 1.50 (1H, m); 1.25 (16H, bs); 0.89 (3H, t, J = 8 Hz). 13C {1H} NMR
(CDCl3, 100 MHz): δ 81.1; 74.3; 64.4; 37.7; 31.9; 31.0; 30.8; 29.7; 29.6; 29.6; 29.5; 29.5; 29.4;
29.3; 29.3; 25.4; 25.3; 22.6; 16.3; 15.7; 13.9.19F (CDCl3, 375 MHz): -81.2; -113.8; -122.0; -123.1;
-123.9; -126.5. MS (ESI+) m/z = [M + H]+ = 688.84.
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2-((4,4,5,5,6,6,7,7,7-nonafluoro-2-iodoheptyl)oxy)octan-1-ol (7d). To a solution of 6 (1.0 g,
5.36 mmol, 1 eq) in hexane was added perfluorobutyl iodide (1.4 mL, 8.0 mmol, 1.5 eq), then
the solution was cooled down to 0°C and triethylborane (0.47 mL, 3.22 mmol, 0.6 eq) was
added dropwise. The mixture was stirred for 10 min at 0°C and for 24 h at room temperature
then the solution was concentrated in vacuo. The crude compound was purified by flash
chromatography (cyclohexane/AcOEt 90:10 v/v) to yield 7d as a yellow oil (1.5 g, 52 %). Rf =
0.42 (cyclohexane/AcOEt 80:20 v/v). 1H NMR (CDCl3, 400 MHz): δ 4.40 (1H, m); 3.82-3.70 (3H,
m); 3.56 (1H, m); 3.46 (1H, m); 3.05 (1H, m); 2.70 (1H, m); 2.09-1.95 (1H, m); 1.58 (1H, m); 1.46
(1H, m); 1.28 (8H , bs); 0.88 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 81.2; 74.2; 64.5;
37.8; 37.6; 37.4; 31.7; 30.9; 30.8; 29.4; 25.3; 22.5; 16.3; 15.7; 14.0. 19F (CDCl3, 375 MHz): -81.0;
-113.8; -124.6; -125.9. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C15H23F9IO2 533.0599; Found
533.0605.
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2-((4,4,5,5,5-pentafluoropentyl)oxy)dodecan-1-ol (8a). To a solution of 7a (1.1 g, 2.3 mmol,
1 eq) in MeOH was added Pd-C (135 mg) and CH3CO2Na (0.60 g, 7.4 mmol, 3.2 eq). The
reaction mixture was stirred overnight under a 6.5 bar pressure of H2 in a hydrogenation
reactor. Then, the solution was filtered through a pad of Celite and concentrated in vacuo. The
crude compound was purified by flash chromatography (cyclohexane/AcOEt 80:20 v/v) to
yield 8a as a colorless oil (0.63 g, 76 %). Rf = 0.41 (cyclohexane/AcOEt 80:20 v/v). 1H NMR
(CDCl3, 400 MHz): δ 3.65 (1H, dd, J = 4 Hz, J = 12 Hz); 3.56 (2H, t, J = 8 Hz); 3.48 (1H, dd, J =
6 Hz, J = 12 Hz); 3.35 (1H, m); 2.15 (2H, m); 1.97 (1H, bs); 1.86 (2H, qt, J = 6 Hz); 1.59 (1H, m);
1.37 (1H, m); 1.25 (16H, bs); 0.87 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 80.4; 67.8;
64.3; 31.9; 30.7; 29.8; 29.6; 29.6; 29.5; 29.3; 27.9; 27.7; 27.5; 25.4; 22.7; 21.4; 14.0. 19F (CDCl3,
375 MHz): δ -85.5; -118.3. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C17H32F5O2 363.2322; Found
363.2321.
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2-((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)dodecan-1-ol (8b). To a solution of 7b (1.6 g,
2.7 mmol, 1 eq) in MeOH was added Pd-C (136 mg) and CH3CO2Na (0.70 g, 8.7 mmol, 3.2 eq).
The reaction mixture was stirred overnight under a 6.5 bar pressure of H2 in a hydrogenation
reactor. Then, the solution was filtered through a pad of Celite and concentrated in vacuo. The
crude compound was purified by flash chromatography (cyclohexane/AcOEt 80:20 v/v) to
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yield 8b as a colorless oil (1.1 g, 88 %). Rf = 0.41 (cyclohexane/AcOEt 80:20 v/v). H NMR
(CDCl3, 400 MHz): δ 3.65 (1H, m); 3.57 (2H, m); 3.48 (1H, m); 3.34 (1H, m); 2.20 (2H, m); 2.08
(1H, bs); 1.88 (2H, qt, J = 6 Hz); 1.59 (1H, m); 1.37 (1H, m); 1.25 (16H, bs); 0.86 (3H, t, J = 8 Hz).
13C {1H} NMR (CDCl3, 100 MHz): δ 80.5; 67.9; 64.3; 31.9; 30.8; 29.8; 29.6; 29.6; 29.6; 29.5;
29.5; 29.3; 28.1; 27.8; 27.6; 25.4; 22.6; 21.2; 14.0. 19F (CDCl3, 375 MHz): δ -81.2; -114.7; -124.6;
-126.2. HRMS (ESI-TOF) m/z: [M + Na]+ calcd for C19H31F9O2Na 485.2078; Found 485.2072.
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2-((4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)oxy)dodecan-1-ol (8c). To a solution of 7c
(1.0 g, 1.5 mmol, 1 eq) in MeOH was added Pd-C (80 mg) and CH3CO2Na (0.39 g, 4.8 mmol,
3.2 eq). The reaction mixture was stirred overnight under a pressure of H2. Then, the solution
was filtered through a pad of Celite and concentrated in vacuo. The crude compound was
purified by flash chromatography (cyclohexane/AcOEt 80:20 v/v) to yield 8c as a colorless oil
(0.57 g, 68 %). Rf = 0.33 (cyclohexane/AcOEt 80:20 v/v). 1H NMR (CDCl3, 400 MHz): δ 3.70 (1H,
m); 3.60 (2H, m) ; 3.45 (1H, m); 3.35 (1H, m); 2.20 (2H, m); 2.00-1.80 (3H, m, CH2); 1.60 (1H,
m); 1.37 (1H, m); 1.25 (16H, bs); 0.86 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 80.5;
67.9; 64.3; 31.9; 30.8; 29.8; 29.7; 29.6; 29.6; 29.5; 29.5; 29.4; 29.3; 28.2; 28.0; 27.7; 26.9; 25.4;
22.7; 21.2; 13.9. 19F (CDCl3, 375 MHz): δ -81.2; -114.6; -122.2; -123.1; -123.7; -126.4. HRMS
(ESI-TOF) m/z: [M + H]+ calcd for C21H32F13O2 563.2195; Found 563.2192.
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2-((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)octan-1-ol (8d). To a solution of 7d (1.5 g,
2.82 mmol, 1 eq) in MeOH was added Pd-C (160 mg) and CH3CO2Na (0.74 g, 9.0 mmol, 3.2 eq).
The reaction mixture was stirred overnight under a 6.5 bar pressure of H2 in a hydrogenation
reactor. Then, the solution was filtered through a pad of Celite and concentrated in vacuo. The
crude compound was purified by flash chromatography (cyclohexane/AcOEt 80:20 v/v) to
yield 8d as a colorless oil (0.53 g, 46 %). Rf = 0.38 (cyclohexane/AcOEt 80:20 v/v). 1H NMR
(CDCl3, 400 MHz): δ 3.63-3.41 (4H, m); 3.29 (1H, m); 2.52 (1H, bs); 2.14 (2H, m); 1.83 (2H, qt, J
= 8 Hz); 1.47 (1H, m); 1.40 (1H, m); 1.24 (8H, bs); 0.83 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3,
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100 MHz): δ 80.5; 67.9; 64.2; 31.8; 31.7; 30.8; 29.4; 27.8; 26.8; 25.3; 22.5; 21.2; 13.8. F
(CDCl3, 375 MHz): δ -81.6; -114.9; -124.8; -126.4. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C15H24F9O2 407.1627; Found 407.1625.
(2-((4,4,5,5,5-pentafluoropentyl)oxy)dodecanyl tri-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzoyl-
α-D-glucopyranosyl)-β-D-glucopyranoside (16a). To a solution of 8a (0.6 g, 1.66 mmol, 1 eq)
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and 15 (2.43 g, 2.0 mmol, 1.2 eq) in anhydrous CH2Cl2, was added dropwise 1M TMSOTf in
CH2Cl2 (1.6 mL, 1.66 mmol, 1 eq) at 0°C. The reaction mixture was stirred 2h at 0°C then
overnight at room temperature. The solution was neutralized by addition of Et3N and
concentrated in vacuo. The crude compound was purified by flash chromatography
(cyclohexane/AcOEt 80:20 v/v) to yield 16a as a white solid (1.64 g, 70 %). Rf = 0.37
(cyclohexane/AcOEt 80:20 v/v). 1H NMR (CDCl3, 400 MHz): δ 8.10-7.15 (35H, m); 6.30 (1H, t, J
= 10 Hz); 6.01 (1H, m); 5. 80 (1H, m); 5.52 (1H, m); 5.08-4.45 (6H, m); 4.01-3.21 (9H, m); 2.13-
1.80 (2H, m); 1.40-1.00 (20H, m); 0.90 (3H, t, J = 8 Hz).13C {1H} NMR (CDCl3, 100 MHz): δ 166.0-
165.0; 133.0-128.0; 100.1; 96.9; 78.3; 75.5; 74.8; 73.4; 72.5; 71.4; 70.9; 69.9; 69.1; 68.8; 68.1;
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63.4; 62.7; 31.7; 29.4; 29.3; 29.2; 29.1; 27.5; 27.4; 26.8; 25.2; 22.5; 21.3; 13.9. F (CDCl3,
375 MHz): δ -85.4; -118.2. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C78H80F5O19 1415.5214;
Found 1415.5193.
2-((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)dodecanyl tri-O-benzoyl-4-O-(2,3,4,6-tetra-O-
benzoyl-α-D-glucopyranosyl)-β-D-glucopyranoside (16b). To a solution of 8b (1.1 g,
2.33 mmol, 1 eq) and 15 (2.92 g, 2.4 mmol, 1 eq) in anhydrous CH2Cl2, was added dropwise
1 M TMSOTf in CH2Cl2 (2.4 mL, 2.4 mmol, 1 eq) at 0°C. The reaction mixture was stirred for a
few minutes at 0°C then 2h at room temperature. The solution was neutralized by addition of
Et3N and concentrated in vacuo. The crude compound was purified by flash chromatography
(cyclohexane/AcOEt 80:20 v/v) to yield 16b as a white solid (2.87 g, 79 %). Rf = 0.3
(cyclohexane/AcOEt 80:20 v/v). 1H NMR (CDCl3, 400 MHz): δ 8.16-7.16 (35H, m); 6.30 (1H, t, J
= 10 Hz); 6.10 (1H, m); 5.90-5.70 (2H, m); 5.52-5.28 (2H, m); 5.10-4.25 (7H, m); 4.20 (1H, m);
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3.60-3.15 (4H, m); 2.0 (2H, m); 1.50-1.00 (20H, m); 0.89 (3H, m). C {1H} NMR (CDCl3,
100 MHz): δ 166.0-165.0; 133.0-128.0; 100.0; 96.5; 78.4; 78.3; 74.9; 73.9; 73.3; 72.2; 71.7;
70.9; 69.9; 69.1; 68.4; 63.5; 62.5; 31.8; 29.5; 25.4; 22.7; 14.1. 19F (CDCl3, 375 MHz): δ -81.1; -
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114.6; -124.6; -126.6. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C80H80F9O19 1515.5150; Found
1515.5170.
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2-((4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)oxy)dodecanyl tri-O-benzoyl-4-O-(2,3,4,6-
tetra-O-benzoyl-α-D-glucopyranosyl)-β-D-glucopyranoside (16c). To a solution of 8c (1.0 g,
1.78 mmol, 1 eq) and 15 (3.0 g, 2.5 mmol, 1.4 eq) in anhydrous CH2Cl2, was added dropwise
1 M TMSOTf in CH2Cl2 (1.8 mL, 1.78 mmol, 1 eq) at 0°C. The reaction mixture was stirred 2h at
0°C then overnight at room temperature. The solution was neutralized by addition of Et3N and
concentrated in vacuo. The crude compound was purified by flash chromatography
(cyclohexane/AcOEt 80:20 v/v) to yield 16c as a white solid (2.2 g, 76 %). Rf = 0.34
(cyclohexane/AcOEt 80:20 v/v). 1H NMR (CDCl3, 400 MHz): δ 8.21-7.16 (35H, m); 6.34-6.16
(1H, m); 6.04 (1H, m); 5.82 (1H, m); 5.50 (1H, m); 5.10-4.40 (6H, m); 4.13-3.22 (9H, m); 2.21-
1.84 (2H, m); 1.50-1.00 (20H, m); 0.92 (3H, m). 13C {1H} NMR (CDCl3, 100 MHz): δ 166.0-165.0;
133.4-128.3; 100.7; 97.0; 79.0; 78.4; 75.6; 74.7; 73.4; 72.5; 72.2; 71.4; 70.0; 69.1; 68.9; 63.4;
62.8; 31.9; 31.8; 31.6; 31.6; 29.5; 29.4; 29.3; 25.3; 25.2; 22.6; 14.1. 19F (CDCl3, 375 MHz): δ –
80.9; -114.3; -121.9; -122.8; -123.5; -126.1. HRMS (ESI-TOF) m/z: [M + H]+ calcd for
C82H80F13O19 1615.5086; Found 1615.5267.
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2-((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)octanyl tri-O-benzoyl-4-O-(2,3,4,6-tetra-O-
benzoyl-α-D-glucopyranosyl)-β-D-glucopyranoside (16d). To a solution of 8d (0.53 g,
1.3 mmol, 1 eq) and 15 (1.58 g, 1.3 mmol, 1 eq) in anhydrous CH2Cl2, was added dropwise
TMSOTf (1.3 mL, 1.3 mmol, 1 eq) at 0°C. The reaction mixture was stirred for a few minutes at
0°C then overnight at room temperature. The solution was neutralized by addition of Et3N and
concentrated in vacuo. The crude compound was purified by flash chromatography
(cyclohexane/AcOEt 80:20 v/v) to yield 16d as a white solid (0.97 g, 51 %). Rf = 0.38
(cyclohexane/AcOEt 80:20 v/v). 1H NMR (CDCl3, 400 MHz): δ 8.16-7.16 (35H, m); 6.30 (1H, t, J
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= 10 Hz); 6.10 (1H, m); 5.90-5.70 (2H, m); 5.52-5.28 (2H, m); 5.10-4.25 (7H, m); 4.20 (1H, m);
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70.9; 69.9; 69.1; 68.4; 63.5; 62.5; 33.1; 31.8; 29.5; 25.4; 23.7; 22.7; 14.1.19F NMR (CDCl3,
375 MHz): δ -81.0; -114.5; -124.4; -126.0.
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2-((4,4,5,5,5-pentafluoropentyl)oxy)dodecanyl 4-O-(α-D-glucopyranosyl)-β-D-
glucopyranoside (17a). To a solution of 16a (1.8 g, 1.27 mmol, 1 eq) in MeOH was added
MeONa (0.027 g, 0.5 mmol, 0.4 eq) and the reaction mixture was stirred for 4 h. The solution
was neutralized by addition of IRC-50, filtered and concentrated in vacuo. The crude
compound was purified by flash chromatography (CHCl3/MeOH 90:10 v/v) to yield 17a as a
white solid (480 mg, 55 %). Rf = 0.32 (CHCl3/MeOH 80:20 v/v). 1H NMR (CD3OD, 400 MHz): δ
5.12 (1H, m); 4.25 (1H, m); 3.92-3.16 (17H, m); 2.29-2.11 (2H, m); 1.77 (2H, qt, J = 8 Hz); 1.48
(2H, m); 1.26 (16H, bs); 0.86 (3H, t, J = 8 Hz). 13C {1H} NMR (CD3OD, 100 MHz): δ 105.0; 103.1;
81.4; 80.2; 77.9; 76.7; 75.1; 74.8; 74.2; 73.2; 72.5; 71.5; 69.4; 62.8; 62.2; 33.0; 32.8; 30.7; 30.5;
28.7; 26.6; 23.7; 22.4; 14.4. 19F (CD3OD, 375 MHz): δ - 85.4; -118.1. HRMS (ESI-TOF) m/z: [M +
H]+ calcd for C29H52F5O12 687.3379; Found 687.3373. Anal. Calcd for C29H51F5O12: C, 50.72; H,
7.49; Found: C, 49.44; H, 7.52.
2-((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)dodecanyl 4-O-(α-D-glucopyranosyl)-β-D-
glucopyranoside (17b). To a solution of 16b (2.9 g, 1.91 mmol, 1 eq) in MeOH was added
MeONa (0.041 g, 0.77 mmol, 0.4 eq) and the reaction mixture was stirred for 4 h. The solution
was neutralized by addition of IRC-50, filtered, and concentrated in vacuo. The crude
compound was purified by flash chromatography (CHCl3/MeOH 90:10 v/v) to yield 17b as a
white solid (1.5 mg, 100 %). Rf = 0.38 (CHCl3/MeOH 80:20 v/v). 1H NMR (CD3OD, 400 MHz): δ
5.19 (1H, m); 4.31 (1H, m); 3.96-3.24 (17H, m); 2.38-2.20 (2H, m); 1.85 (2H, qt, J = 8 Hz); 1.54
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(2H, m); 1.31 (16H, bs); 0.91 (3H, t, J = 8 Hz). 13C {1H} NMR (CD3OD, 100 MHz): δ 104.8; 102.9;
81.4; 80.2; 77.8; 76.7; 75.1; 74.8; 74.2; 73.3; 72.6; 71.5; 69.4; 62.8; 62.3; 33.1; 32.9; 32.8; 30.8;
30.7; 30.5; 28.9; 26.7; 23.8; 22.2; 14.5. 19F (CD3OD, 375 MHz): δ -82.58; -115.7; -125.5; -127.2.
HRMS (ESI-TOF) m/z: [M + H]+ calcd for C31H52F9O12 787.3315; Found 787.3314. Anal. Calcd
for C31H51F9O12: C, 47.33; H, 6.53; Found: C, 48.16; H, 7.02.
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2-((4,4,5,5,6,6,7,7,8,8,9,9,9-tridecafluorononyl)oxy)dodecanyl 4-O-(α-D-glucopyranosyl)-β-
D-glucopyranoside (17c). To a solution of 16c (2.5 g, 1.55 mmol, 1 eq) in MeOH was added
MeONa (0.033 g, 0.6 mmol, 0.4 eq), and the reaction mixture was stirred for 4 h. The solution
was neutralized by addition of IRC-50, filtered, and concentrated in vacuo. The crude
compound was purified by flash chromatography (CHCl3/MeOH 90:10 v/v) to yield 17c as a
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white solid (1.25 g, 91 %). Rf = 0.41 (CHCl3/MeOH 80:20 v/v). H NMR (CD3OD, 400 MHz): δ
5.18 (1H, m); 4.30 (1H, m); 3.96-3.23 (17H, m); 2.39-2.22 (2H, m); 1.86 (2H, qt, J = 8 Hz); 1.52
(2H, m); 1.23 (16H, bs); 0.91 (3H, t, J = 8 Hz). 13C {1H} NMR (CD3OD, 100 MHz): δ 104.8; 102.9;
81.4; 80.1; 77.8; 76.6; 75.1; 74.7; 74.1; 73.2; 72.5; 71.5; 69.3; 62.7; 62.2; 33.0; 32.9; 32.7; 30.7;
30.4; 28.9; 26.5; 23.7; 22.2; 14.4.19F (CD3OD, 375 MHz): δ - 82.4; -115.5; -122.9; -123.9; -124.5;
-127.3. HRMS (ESI-TOF) m/z: [M + H]+ calcd for C33H52F13O12 887.3251; Found 887.3273. Anal.
Calcd for C33H51F13O12: C, 44.70; H, 5.80; Found: C, 44.01; H, 6.11.
2-((4,4,5,5,6,6,7,7,7-nonafluoroheptyl)oxy)octanyl 4-O-(α-D-glucopyranosyl)-β-D-
glucopyranoside (17d). To a solution of 16d (0.97 g, 0.66 mmol, 1 eq) in MeOH was added
MeONa (0.014 g, 0.26 mmol, 0.4 eq), and the reaction mixture was stirred for 4 h. The solution
was neutralized by addition of IRC-50, filtered, and concentrated in vacuo. The crude
compound was purified by flash chromatography (CHCl3/MeOH 90:10 v/v) to yield 17d as a
white solid (0.38 g, 79 %). Rf = 0.43 (CHCl3/MeOH 80:20 v/v). 1H NMR (CD3OD, 400 MHz): δ
5.12 (1H, m); 4.26 (1H, m); 3.91-3.17 (17H, m); 2.24 (2H, m); 1.80 (2H, qt, J = 8 Hz); 1.48 (2H,
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m); 1.27 (8H, bs); 0.86 (3H, m). 13C {1H} NMR (CD3OD, 100 MHz): δ 104.8; 102.8; 81.3; 80.1;
77.8; 76.6; 75.0; 74.7; 74.1; 73.2; 72.5; 71.4; 69.3; 62.7; 62.2; 33.0; 32.9; 30.4; 28.8; 26.6; 23.6;
22.2; 14.4. 19F (CD3OD, 375 MHz): δ -82.6; -115.6; -125.5; -127.2. HRMS (ESI-TOF) m/z: [M +
Na]+ calcd for C27H43F9O12Na 753.2509; Found 753.2515. Anal. Calcd for C27H43F9O12: C, 44.39;
H, 5.83; Found: C, 43.49; H, 5.94.
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Water solubility. Pure compounds. Stock solution of hybrid were prepared at 1g/L. The
solution was sonicated in a sonic bath at 40°C for 15 min. Centrifugation for 10 min at 5000
rpm of the cloudy opalescent solutions obtained led to the formation of a clear supernatant
solution and a precipitate. The supernatant solutions was further used for DLS and TEM
experiments. DDM:LC mixtures. The mixtures were prepared using molar ratio DDM:LC 100:4
and 100:2 for LC045-LC047 and 100:10 for LC057. The hybrid compounds were solubilized in
a 1.7 mM solution of DDM (10 CMC). Mixtures were sonicated using a sonication bath for a
few minutes then solutions were centrifuged at 5000 rpm for 10 min. The resulting solutions
were further used for DLS and TEM experiments.
DLS measurements. Hydrodynamic particle size distributions were determined on a Zetasizer
Nano-S model 1600 (Malvern Instruments, UK) equipped with a He−Ne laser (λ = 633 nm,
4.0 mW). All measurements were performed at (25 ± 0.5)°C. The time dependent correlation
function of the scattered light intensity was measured at an angle of 173° (backscattering
detection). The hydrodynamic diameter (DH) of the particles was estimated from their
diffusion coefficient (D) using the Stokes−Einstein equation, D = kBT/3πηDH, where kB is
Boltzmann’s constant, T absolute temperature, and η the viscosity of the solvent. Contin
analysis was used for evaluating autocorrelation functions. All measurements were performed
at (25 ± 0.5)°C.
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TEM. The negatively stained samples were prepared by spreading 5 µL of the surfactant
solution (0.5 mM) onto a Cu grid coated with a Formvar-film (PLANO, Wetzlar, Germany). After
1 min excess liquid was blotted off with filter paper and 5 µL of 1% aqueous uranyl acetate
solution were placed onto the grid and drained off after 1 min. The dried specimens were
examined with an EM 900 transmission electron microscope (Carl Zeiss Microscopy GmbH,
Oberkochen, Germany). Micrographs were taken with a SSCCD SM-1k-120 camera (TRS,
Moorenweis, Germany).
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Solubilization. The experiments were performed as previously described.27 Briefly, membrane
fractions were incubated for 2h at 4°C at a final concentration of 5mg/mL in 50mM Hepes
buffer pH 7.4, 200 mM NaCl, 1X protease inhibitor cocktail, and with 10-fold the CMC of DDM
in combination with CHS (100:10 ratio) or LC compounds (100:2, 100:4 or 100:10 ratios). After
solubilization samples were centrifuged at 100000g for 45min at 4°C and an aliquot of the
total extract, the pellet and the supernatant from each solubilization condition was analyzed
by SDS-PAGE and western-blot using an antibody A2AR (7F6-G5-A2) and against the his-tag for
A2AR and AcrB, respectively.
Thermostability assay. Membranes of A2AR or AcrB (4mg/mL total protein) were solubilized
in different conditions (see solubilization method above) for 2 hours at 4°C. Solubilized
fractions were obtained after 100,000g ultracentrifugation for 1h at 4°C. Solubilized fraction
serves to make 50μL aliquots to be submitted to one temperature each as part of a gradient
of temperature ranging from 25 to 72°C using PCR thermal cycler (PeqSTAR 2x gradient;
Peqlab). Samples were then centrifuged 40min at 20000g and supernatants were analyzed by
SDS-PAGE and western-blot using anti- A2AR antibody (7F6-G5-A2). The relative intensity of
the target protein at each temperature was quantified on Western-blot using Image Lab
software 4.1 from Bio-rad. Each condition was performed twice. Intensity was plotted as a
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function of the temperature, normalized and fit to the Boltzmann equation with the least
square method using Solver Adds-in of Excel software. The method is described by Ashok et
al.30
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Acknowledgements. This work was supported by the Agence Nationale de la Recherche (ANR)
through grants no. ANR-14-LAB7-0002 and by the Deutsche Forschungsgemeinschaft (DFG)
through grant no. KE 1478/7-1. Christophe Bonnet was the recipient of a PhD fellowship from
“Région Provence Alpes Côte d’Azur”. We thank Dr Nasreddine Kanfar for his assistance in the
revision of the manuscript.
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Supplementary material available. H, C {1H} and F NMR spectra of compounds; Water-
solubility of DDM:LC mixtures; Contin distribution plots for DDM:LC mixtures in water.
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