Detergent-Like Polymerizable Monomers: Synthesis, Physicochemical, and Biochemical Characterization

Article abstract of DOI:10.1002/ejoc.202000540

Three monomers with a maltose polar head, an alkyl hydrogenated chain, and an acrylamide-based polymerizable moiety were synthesized. The self-assembly properties in aqueous solutions of these monomers were studied by means of isothermal titration calorimetry (ITC), surface tension (SFT) measurements, and dynamic light scattering (DLS), which indicated the formation of small micellar aggregates of about 6 nm diameter. The critical micellar concentration (CMC) was found to depend on the length of the alkyl chain and on the nature of the polymerizable moiety, ranging from 0.35 mm to ca. 10 mm. The monomers were found to solubilize phospholipid vesicles and to extract a broad range of proteins from Escherichia coli membranes. Finally, the extraction of two membrane proteins, namely, the full-length, wild-type human G-protein-coupled receptor (GPCR) adenosine A2A receptor (A_2AR) and the bacterial transporter AcrB was demonstrated.

Full text of DOI:10.1002/ejoc.202000540

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Detergent-Like Polymerizable Monomers: Synthesis,
Physicochemical, and Biochemical Characterization
Authors: Christophe Bonnet, Pierre Guillet, Florian Mahler, Sébastien
Igonet, Sandro Keller, Anass Jawhari, and Gregory Durand
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202000540
Link to VoR: https://doi.org/10.1002/ejoc.202000540
01/2020

10.1002/ejoc.202000540
European Journal of Organic Chemistry
FULL PAPER
Detergent-Like Polymerizable Monomers:
Synthesis, Physicochemical, and Biochemical Characterization
Christophe Bonnet,[a],[b] Pierre Guillet,[a],[b] Florian Mahler,[c] Sébastien Igonet,[b],[d] Sandro Keller,[c] Anass
Jawhari, [b],[d] Grégory Durand,* [a],[b]
[a]
Mr. C. Bonnet, Dr. P. Guillet and Dr. G. Durand
Institut des Biomolécules Max Mousseron (UMR 5247 UM-CNRS-ENSCM) & Avignon University, Chimie Bioorganique et Systèmes amphiphiles
301 rue Baruch de Spinoza – 84916 AVIGNON cedex 9 (France)
E-mail: gregory.durand@univ-avignon.fr
http://www.chem2stab.org/
[b]
[c]
[d]
Mr. C. Bonnet, Dr. P. Guillet, Mr. S. Igonet, Dr. Anass Jawhari and Dr. G. Durand
CHEM2STAB
301 rue Baruch de Spinoza – 84916 AVIGNON cedex 9 (France)
Dr. F. Mahler and Dr. S. Keller
Molecular Biophysics, Technische Universitaꢀ Kaiserslautern (TUK)
Erwin-Schrꢁdinger-Str. 13, 67663 Kaiserslautern (Germany)
Dr. S. Igonet and Dr. A. Jawhari
CALIXAR
60 Avenue Rockefeller – 69008 Lyon (France)
Supporting information for this article is given via a link at the end of the document.
Abstract: Three monomers with a maltose polar head, an alkyl
hydrogenated chain, and an acrylamide-based polymerizable moiety
were synthesized. The self-assembly properties in aqueous solutions
of these monomers were studied by means of isothermal titration
calorimetry (ITC), surface tension (SFT) measurements, and dynamic
light scattering (DLS), which indicated the formation of small micellar
aggregates of about 6 nm diameter. The critical micellar concentration
(CMC) was found to depend on the length of the alkyl chain and on
the nature of the polymerizable moiety, ranging from 0.35 mM to ~10
mM. The monomers were found to solubilize phospholipid vesicles
and to extract a broad range of proteins from Escherichia coli
transmembrane surface of membrane proteins thanks to their
alkyl chains and the complex thus formed remains water-soluble
thanks to the hydrophilic groups. MP/amphipols complexes are
indeed very stable even at high dilutions due to the slow dynamic
of the polymer which remains tightly attached to the protein.
Unlike the other heterogeneous systems, SMA and DIBMA can
efficiently recruit MPs and associated lipids directly from natural
or artificial membranes into nanoscale lipid-bilayer patches that
closely mimic the lamellar organization of cellular membranes.
However, one common limitation of A8-35, SMA, and DIBMA lies
in the presence of carboxylic groups along the polymer chain that
membranes. Finally, the extraction of two membrane proteins, namely, result in polymer aggregation at acidic pH or in the presence of
the full-length, wild-type human G-protein-coupled receptor (GPCR)
adenosine receptor (A2AR) and the bacterial transporter AcrB was
demonstrated.
Introduction
Membrane proteins (MPs) perform a wide range of essential
cellular functions and are involved in a large number of
pathologies, which makes them priority drug targets representing
nearly 70 % of therapeutic targets.[1] Because MPs exhibit high
insolubility in water and poor stability outside their native
multivalent cations.[10]
membrane environment, their extraction from the membrane is
highly challenging and may result in denaturation and/or
aggregation of the protein once removed from their native
Figure 1. Structure of (A) A8-35 amphipol, (B) poly(styrene-co-maleic acid)
(SMA) copolymer, (C) poly(diisobutylene-alt-maleic acid) (DIBMA) copolymer,
(D) non-ionic amphipol (NAPol), and (E) its constituting detergent-like monomer
(LC027).
environment. The need of natively isolated, yet stable proteins
has prompted the development of sophisticated chemically well-
defined detergents over the recent years. Among these new,
chemically homogenous systems, one can cite neo-pentyl glycols
derivatives,[2] derivatives with branched[3] or fluorinated chains,[4]
as well as steroid-based and facial derivatives.[5]
This limitation has prompted the development of several
amphipols derivatives including zwitterionic,[11] sulphonated,[12] or
phosphocholine-based amphipols[13] as well as non-ionic APols
called NAPols.[14] A first series of glucose-based NAPols was
obtained either from co-telomerization of hydrophilic and
hydrophobic monomers[14b] or by homo-telomerization of
hydrophilic monomers, followed by hydrophobization of the
polymer.[15] These glycosylated NAPols showed good potency at
Other approaches for handling and studying MPs relate to the use
of heterogeneous systems such as protein-lipid nanodiscs[6], A8-
35 amphipol (APols),[7], and nanodiscs bounded by poly(styrene-
co-maleic acid) (SMA)[8] and poly(diisobutylene-alt-maleic acid)
(DIBMA)[9] copolymers. Amphipols adsorb onto the hydrophobic
1
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10.1002/ejoc.202000540
European Journal of Organic Chemistry
FULL PAPER
keeping various MPs soluble in their native state in the absence
of detergent.[14b, 15] We further designed homopolymeric NAPols
(Figure 1) consisting of an amphiphilic repeating unit[16] (called
LC027 in the current study).[14a] This more convenient synthetic
route offers the advantage of resorting to only one amphiphilic
monomer and to allow better batch-to-batch reproducibility and
higher yields. LC027 due to its amphiphilic chemical structure and
its surface activity belongs to the family of surfmers.[17] Surfmers
combine the functionalities of surface active agents with the
reactivity of monomers. The polymerization of surfmers has been
successfully employed for several applications such as emulsion
stabilization, nanomaterials synthesis, drug-delivery systems, and
hydrogels. The promising results obtained with this most
advanced series of NAPols further confirmed their advantages
over the classical A8-35 for specific applications such as cell-free
synthesis, nuclear magnetic resonance (NMR) spectroscopy, and
mass spectrometry (MS), to name but a few.[18] More recently, the
first cryo-EM structure of the translocase of the outer membrane
(TOM) core complex in NAPol has been reported.[19]
The colloidal properties of the four surfmers were evaluated by
means of isothermal titration calorimetry (ITC), surface tension
(SFT) measurements, as well as dynamic light scattering (DLS).
Their potency to act as solubilizing agents was further evaluated
with model membranes as well as with different membrane
proteins from two cell membranes.
Results and Discussion
Synthesis. The synthesis followed a convergent synthetic
pathway, based on three key steps: (i) synthesis of the
hydrophobic parts; (ii) synthesis of the maltose polar head; (iii)
coupling of the hydrophobic and hydrophilic parts (Scheme 1).
Compounds 8a-b were synthesized following an eight-step
synthetic route starting from 1,2-dodecanediol (Scheme 2).
Selective protection of the primary alcohol group was achieved
using Et3N and 1.2 equivalent of trityl chloride as previously
reported.[20] Mesylation of the secondary alcohol group followed
by nucleophilic substitution in the presence of NaN3 afforded
compound 3 in good yield (65% in three steps). The azide group
was next reduced under hydrogen atmosphere at room
temperature, using catalytic amounts of Pd-C to lead to
compound 4 in high yield. The insertion of the polymerizable
moiety on the amine group was realized using either acryloyl
chloride or methacryloyl chloride at room temperature in the
presence of trimethylamine (TEA). Deprotection of 5a-b in acidic
condition led to compounds 6a-b. In our hands, only moderate
yields of deprotection were observed, that is, 55 and 63%,
respectively.
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 seemed preferable.
We therefore activated maltose into its trichloroacetimidate form
and used benzyl esters protecting groups.[21] Hepta-O-benzoyl-
maltose-1-O-trichloroacetimidate was readily prepared from
commercially available maltose following a four-step synthetic
route[22] and was next condensed onto the hydrophobic parts 6a
and 6b at room temperature following a Schmidt glycosylation[23]
to give compounds 7a-b in very good yields. Hydrolysis of benzoyl
groups using catalytic amount of sodium methoxide in MeOH
afforded compounds 8a-b in good yields also called LC048 and
LC058, respectively. Analysis of the proton NMR spectra showed
the formation β anomer witnessed by a doublet between 4.0 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).
LC049 was synthesized following an eleven-step synthetic route
starting from glycerol (Scheme 3). The two hydroxyl groups were
protected by reaction using 2,2-dimethoxypropane and catalytic
amount of p-TsOH in CH3CN to afford compound 9 also called
solketal.[24] The addition of the hydrogenated chain was achieved
by an SN2 reaction in the presence of sodium hydride (NaH) and
1.2 equivalent of octyl bromide to give compound 10 in 74 % yield.
Then, hydrolysis in acidic conditions gave compound 11 in good
yield, which was next put in reaction with Et3N and 1.2 equivalent
of trityl chloride to yield compound 12. Mesylation of the
secondary alcohol group followed by nucleophilic substitution in
the presence of NaN3, afforded compound 13 in good yield. The
azide group was reduced at room temperature using a catalytic
Scheme 1. Retrosynthetic pathway for compounds LC048 and LC058.
However, like others amphipols, NAPols are unable to solubilize
membrane proteins. It is therefore necessary to proceed in two
distinct steps by doing first the extraction of the protein with a
detergent and then performing the exchange with the polymer. As
part of our long-term project, the work presented herein deals with
the evaluation of the solubilizing properties of detergent-like
monomers that could be further used in the synthesis of new
NAPols. We expected that, while polymeric NAPols are very
efficient stabilizing agents, the constituting monomers could
exhibit solubilizing properties.
We report the synthesis of three detergent-like monomers refer to
as surfmers and called LC048, LC049, and LC058 that present
chemical
similarity
with
conventional
n-dodecyl-β-d-
maltopyranoside (DDM), which so far is considered as the gold
standard for membrane-protein extraction. These new
compounds are characterized by the presence of a maltose polar
head, a hydrogenated alkyl chain, and an acrylamide or
methacrylamide moiety that enables polymerization for further
work. We chose to use acrylamide group instead of acrylester due
to higher stability of the amide bond compared to the ester one.
For the sake of comparison, LC027 was also included in the study.
2
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10.1002/ejoc.202000540
European Journal of Organic Chemistry
FULL PAPER
amount of Pd-C under hydrogen pressure to afford compound 15
in 64 % yield in three steps. Next, the insertion of the
methacrylamide moiety on the amino group was achieved using
MeOH afforded compound 19, also called LC049. The overall
yield for the synthesis of 19 is ~18% while that of 8a and 8b is
respectively, 22 and 19%. Since we started from racemic starting
materials, we expected the formation of mixture of diastereomers
although neither NMR nor HPLC analysis allowed us to
distinguish the presence of such diastereomers. Therefore, the
investigated properties reported below are considered as the
result of a mixture of diastereomers.
triethylamine and methacryloyl chloride, followed by
a
deprotection in acidic conditions to lead to compound 17. Hepta-
O-benzoyl-maltose-1-O-trichloroacetimidate was condensed onto
17 at room temperature following a Schmidt glycosylation[23] to
give compounds 18 in 80% yield. Finally, hydrolysis of benzoyl
groups using catalytic amount of sodium methoxide (MeONa) in
Scheme 2. Synthesis of LC048 (8a) and LC058 (8b).
Scheme 3. Synthesis of LC049 (19)
3
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10.1002/ejoc.202000540
European Journal of Organic Chemistry
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Micellization. The micellization processes of the monomers were
characterized by means of high-sensitivity ITC and surface
tension such as exemplified in Figure 2. CMC values derived from
these two techniques (Table 1) were found to be in very good
agreement between each other. LC027, LC049, and LC058 were
well soluble in aqueous solution; once the compounds were
solubilized, the solutions remained transparent. By contrast,
LC048 tended to precipitate after solubilization. Several attempts
at heating and using sonication failed to keep it soluble for long
periods of time.
The surface activity of surfactants in solution at the air−water
interface was determined by the Wilhelmy plate technique for the
four monomers. The two derivatives LC048 and LC058 exhibited
CMC values of 0.35 and 0.64 mM, respectively. Compared with
that of DDM (0.17 mM, see Table 1), these values indicate a
strong influence of the polymerizable moiety. Indeed, introducing
the acrylamide group contributes to shortening the alkyl chain
from C12 to C10. This effect is likely due to the polarity of the
amide bond directly attached to alkyl chain that can favor
hydration. The CMC of LC048 and LC058 lie in between that of
DDM and of the undecyl derivative UDM (0.65 mM). This indicates
that the double bond of the acrylamide group brings also
hydrophobicity to the molecule, the contribution of the
methacrylamide being obviously more pronounced than that of
the acrylamide. LC049 has a CMC close to 10 mM, which is
~25 times higher than that of LC048, indicating that the position
of the oxygen atom within the chain contributes to shortening it to
a C8 alkyl chain. The CMC of LC049 lies in between that of the n-
nonylmaltoside (6 mM) and that of the octyl derivative (19.5 mM)
in agreement with the slight hydrophobic contribution of the
double bond of the acrylamide group. Since ITC requires good
solubility of the tested compounds for preparation of stock solution
at ~10 times the CMC, only LC049, LC058, and LC027 were
tested, as the rather limited water solubility of LC048 made the
preparation of such stock solutions impossible.
Table 1. Self-Aggregation Properties of DDM, LC027, LC048, LC058, and LC049.
Monomers
Molar mass (g/mol)
CMC (mM)a
DDM[25]
510.6
0.147
-35.1
3.8
LC027
LC048
593.7
nd
LC058
LC049
696.8
595.7
579.7
0.55 ± 0.02
-28.3 ± 0.10
-0.22 ± 0.01
-28.6 ± 0.1
0.05 ± 0.02
0.51 ± 0.07
35.1 ± 2.8
-28.8 ± 0.3
5.9
0.70 ± 0.04
-29.0 ± 0.2
0.97 ± 0.1
-28.0 ± 0.1
0.12 ± 0.03
0.64 ± 0.10
35.7 ± 2.2
−28.8 ± 1.0
5.9
10.6 ± 0.04
-25.3 ± 0.1
4.02 ± 0.03
-21.2 ± 0.02
0.86 ± 0.04
10.28f
–TΔS° (kJ/mol)b
nd
mic
ΔH° (kJ/mol)c
nd
mic
ΔG° (kJ/mol)d
-31.30
nd
mic
Δc (mM)e
nd
CMC (mM)a
γCMC (mN/m)g
ΔG°mic (kJ/mol)d
DH (nm)h
0.17
34.7
-30.7
7.2
0.35 ± 0.02
34.4 ± 0.2
-29.7 ± 0.2
6.0
32.9f
−21.1f
5.7
[a] Data are averages of three experiments. ± indicates standard errors from the three experiments. [b] Entropic contribution to micelle formation. ± indicates 95 %
confidence interval boundaries. [c] Enthalpic contribution to micelle formation. ± indicates 95 % confidence interval boundaries. [d] Gibbs free energy of micellization.
± indicates 95 % confidence interval boundaries. [e] Micellization concentration range. ± indicates 95 % confidence interval boundaries. [f] Only one experiment. [g]
Surface tension attained at the CMC. [h] Hydrodynamic diameter at 2×(CMC + 5 mM) in buffer (50 mM Tris, 200 mM NaCl, pH 7.4).
m/aq,°
Det
m/aq,°
The changes in Gibbs free energy ΔG
, enthalpy ΔH
,
Det
m/aq,°
and entropy −푇ΔS
accompanying the transfer of monomers
Det
from the aqueous solution into micelles are summarized in
Table 1 and show that micellization was almost exclusively driven
by entropy, with enthalpy making only a minor contribution that
decreased and changed sign with increasing chain length. The
contribution of the oxygen atom to micellization accounted for
about ~8.0 kJ/mol, as deduced by a comparison between LC049
and LC048, which is in rather good agreement with a reduction of
the chain length of two CH2 (typically -3.0 kJ/mol par CH2 unit).
We next conducted DLS experiments in Tris buffer to determine
the hydrodynamic diameters of the aggregates formed. Whatever
monomer tested, volume size distribution indicated the presence
of one population of aggregates of about 6 nm in diameter as
exemplified in Figure 2C. This is similar to what is observed for
DDM (6.6 nm), indicating that the additional polymerizable moiety
did not substantially affect the morphology of the aggregates.
Figure 2. (A) ITC data for LC058. Shown are an experimental isotherm (open
symbols) and a fit based on a generic sigmoidal function (solid line). (B) Surface
tension versus LC058 concentration. The solid line represents the nonlinear fit
of the experimental points. (C) Normalized volume-weighted particle size
distributions for LC058.
Complementary experiments were done in pure water and led to
similar observation with only one population of aggregates of
about 5 nm in diameter (Figure S40).
4
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10.1002/ejoc.202000540
European Journal of Organic Chemistry
FULL PAPER
Number size distribution also showed unimodal distribution with
aggregates of about 4 nm in diameter while intensity distribution
showed a bimodal distribution with main aggregates of about 5 to
6 nm and a second minor population of bigger aggregates (Figure
S40).
Detergency. Because of the rather limited water solubility of
LC048, this compound was not further studied in the following
biochemical validation. To investigate detergency, that is, the
ability both to solubilize artificial lipid bilayers and to extract
membrane proteins, we first investigated the solubilization of
large unilamellar vesicles (LUVs) composed of monounsaturated
zwitterionic
phospholipid
1-plamitoyl-2-oleyl-sn-glycero-
3-phosphocholine (POPC) into mixed micelles with the aid of light
scattering measurements.[4a] The solubilization of 0.3 mM POPC
in the form of LUVs was complete within a few hours for LC027,
LC049, and LC058 (Figure 3). While LC058 enabled complete
solubilization in less than an hour, LC027 also allowed complete
but slower solubilization over a time period of ~5 h. Finally, LC049
achieved fast but only partial solubilization or formed larger mixed
micelles than these obtained from LC027 or LC058. POPC was
chosen because—under the premise that a complex cellular
membrane can be mimicked by one single phospholipid
species—it is generally considered the most “typical” eukaryotic
lipid.
Next, we investigated the extraction of integral membrane
proteins from E. coli membranes in terms of sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) band
patterns and overall amounts of extracted proteins. We compared
the protein-extraction yields thus obtained with those afforded by
DDM. As can be seen in Figure 4A, the three derivatives were
able to extract MPs spanning a broad range of molar masses.
Figure 4B shows the overall protein-extraction yields relative to
the value obtained using 10 mM micellar DDM in dependence on
the concentration of micellar detergent (i.e., total detergent
concentration minus CMC). As expected, all protein-extraction
yields were concentration-dependent, but this dependence varied
clearly among the derivatives tested. At a concentration of 1 and
2 mM above the respective CMC, the three surfmers performed
better than DDM, with LC027 and LC049 being the most efficient
protein solubilizers. Increasing the concentration further to 5 and
10 mM above the CMC did not enhance the yields for LC027 and
LC058 and slightly reduced that of LC049, whereas the protein-
extraction yield of DDM continued to increase steeply as
previously observed.[26] However, it needs to be pointed out that
the superior performance of DDM at high concentrations was
largely due to its unusually efficient extraction of a single
abundant membrane protein, namely, outer membrane protein A
(OmpA, ~35 kDa), as previously observed in other cases.[27]
Figure 3. Vesicle solubilization by 5.55 mM LC027, 15.56 mM LC049, or
5.70 mM LC058 (i.e., 5 mM above the respective CMC determined by ITC) at
25 °C as monitored in terms of the light scattering intensity recorded at an angle
of 90°. Initially, each sample contained 0.3 mM POPC present in the form of
LUVs.
In order to evaluate if the solubilizing properties of these surfmers
can be extended to other membranes and other membrane
proteins, we applied them to membrane proteins recombinantly
produced in E. coli and insect cells (Sf9), namely, the bacterial
transporter AcrB and the adenosine receptor A2AR. These targets
represent two important but distinct classes of membrane
proteins, that is, transporters and G-protein-coupled receptors
(GPCRs), respectively. Solubilization efficiency was assessed
using stain-free SDS-PAGE and Western blots for total and target
proteins, respectively, for both pure surfmers at 10 times the CMC
and for DDM/monomer mixtures at a molar ratio of [10:1]. As
previously reported, for solubilizing A2AR, a DDM/cholesterol
hemi-succinate [DDM:CHS] mixture was used as a reference
condition, while we used DDM as reference for solubilizing AcrB.
Solubilization of A2AR showed that LC058 exhibited rather good
extraction yields (∼79 %), while that of LC049 remained moderate
(∼49 %). When used as solubilization additives, both
[DDM:LC049] and [DDM:LC058] mixtures outperformed the
reference conditions, with extraction yields of ∼73 % and ∼90 %,
respectively, indicating additive effects (Figure 5A). For the sake
of comparison, LC027 was also tested and showed good
solubilizing properties (~70%) similar to the reference.
Solubilization of AcrB led to lower extraction yields for both LC049
and LC058 monomers than for the reference. However, when
[DDM:LC049] and [DDM:LC058] mixtures were used, extraction
yields were as high as that of the reference (Figure 5B), indicating
that, even if the new compounds failed to
extract significant amounts of AcrB on their own, they did not
preclude extraction by the reference detergent DDM. As has been
observed numerous times before, there appears to be no obvious
correlation between membrane composition, protein structure,
and detergent chemistry on the one hand and their potency of
protein extraction on the other.
5
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10.1002/ejoc.202000540
European Journal of Organic Chemistry
FULL PAPER
and LC058 can be viewed as DDM molecules onto which were
grafted, respectively, a methacrylamide and an acrylamide unit,
while LC049 differs from LC048 by substitution of one oxygen
atom for a CH2 in the chain. The effect of attaching an amide bond
within the chain contributes to shortening the length of the alkyl
chain to 10 carbon atoms for LC048 and LC058, and to 8 carbon
atoms for LC049. All three monomers formed micellar aggregates
of similar size (~6 nm in diameter), indicating that the attached
polymerizable moiety did not significantly affect the self-
association properties. However, we noticed that the nature of the
polymerizable moiety had a much stronger effect on the water
solubility, the methacrylamide derivative LC048 being poorly
soluble above its CMC, which precluded its use in biochemical
evaluation. LC049, LC058, and LC027 a previously designed
monomer that is used for the synthesis of non-ionic amphipols
(NAPols) showed potency in solubilizing a model membrane
system which is POPC preformed liposomes, and in extracting
integral membrane proteins from E. coli membranes. Further, the
potency of these surfmers to extract the full-length, wild-type
human GPCR adenosine receptor (A2AR) was demonstrated,
while lower solubilization yields were observed for the bacterial
transporter AcrB. Taken together, these findings demonstrate that
the new amphiphilic monomers behave similarly to classical
detergents. This warrants further development of poly-detergent
based polymers that could be used for handling membrane
proteins.
Experimental Section
Figure 4. (A) SDS-PAGE of E. coli extracts upon exposure to different
monomers at increasing micellar concentration (i.e., total detergent
concentration minus CMC). The arrow indicates the position of the abundant
outer-membrane porin OmpA, which was extracted extraordinarily well by DDM.
(B) Relative protein-extraction yields as functions of the micellar concentration
of LC027, LC058, LC049, or DDM. Extraction yields are reported relative to the
yield obtained when DDM was used at 10 mM. Error bars indicate standard
deviations of 2 experiments except for 10 mM micellar LC049.
Materials & Methods. All starting materials were commercially available
and were used without further purification. Racemic 1,2-dodecanediol and
racemic glycerol acetonide were used as starting materials. All solvents
were of reagent grade and used as received unless otherwise indicated.
MeOH was dried over Na under argon atmosphere. 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 (5% ethanol) and/or ninhydrin (5% ethanol),
followed by heating at ~150 °C. 1H and 13C NMR analysis were performed
at 400 and 100 MHz, respectively. Chemical shifts are given in ppm
relative to the solvent residual peak as a heteronuclear reference for 1H
and 13C. 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.
Milli-Q water (resistivity of 18.2 MΩ cm, surface tension of 71.45 mN/m at
25 °C) was employed for all physicochemical experiments.
(((2-azidododecyl)oxy)methanetriyl)tribenzene (3). To a solution of 1
(24 g, 54.0 mmol, 1 eq) in anhydrous CH2Cl2, was added Et3N (15.1 mL,
108.0 mmol, 2 eq). The solution was stirred for 20 minutes then
methanesulfonyl chloride (5.0 mL, 64.8 mmol, 1.2 eq) was added
dropwise. The reaction mixture was stirred for 16 h and the solution was
filtered and concentrated in vacuo. The crude compound was purified by
flash chromatography (cyclohexane/AcOEt 95:5 v/v) to yield compound 2
as a white solid (26.0 g, 92 %) which was directly used in the next step. To
a solution of 2 (22 g, 42.1 mmol, 1 eq) in anhydrous DMF was added NaN3
(6.8 g, 84.2 mmol, 2 eq). The reaction mixture was stirred for 16 h at 100°C.
Then, the solution was diluted with water and 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 100:0 to 95:5 v/v) to yield compound
3 as a colorless oil (15.2 g, 78 %). Rf = 0.7 (cyclohexane/AcOEt 95:5 v/v).
1H NMR (CDCl3, 400 MHz): δ 7.48-7.24 (15H, m); 3.38 (1H, m); 3.29 (1H,
Figure 5. (A) Solubilization of A2AR (Ref1 = DDM 0.5 % + CHS 0.06 %). (B)
Solubilization of AcrB (Ref2 = DDM 0.5 %).
Conclusion
We have synthesized a series of three amphiphilic monomers that
are chemically similar to the conventional detergent DDM. LC048
6
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000540
European Journal of Organic Chemistry
FULL PAPER
dd, J = 4 Hz, J = 10 Hz); 3.16 (1H, dd, J = 8 Hz, J = 10 Hz); 1.40 (2H, m);
40:60 v/v) to yield compound 6b as a white solid (0.8 g, 55 %). Rf = 0.2
(cyclohexane/AcOEt 40:60 v/v). 1H NMR (CDCl3, 400 MHz): δ 6.27 (1H, d,
J = 16 Hz); 6.15 (1H, dd, J = 10 Hz, J = 16Hz); 5.63 (1H, d, J = 12 Hz);
3.98 (1H, m); 3.68 (1H, dd, J = 4H z, J = 12 Hz); 3.57 (1H, dd, J = 8 Hz, J
= 12 Hz) 3.30 (1H, bs) ; 1.51 (2H, m); 1.24 (16H, bs) ; 0.86 (3H, t, J = 8 Hz).
13C {1H} NMR (CDCl3, 100 MHz): δ 166.3; 130.8; 126.7; 65.33; 52.0; 31.9;
31.2; 29.6; 29.5; 29.3; 26.1; 22.7; 14.0. HRMS (ESI-TOF) m/z: [M + H]+
Calcd for C15H30NO2 256.2277; Found 256.2282.
[12]
1.23 (16H, bs); 0.88 (3H, t, J = 8 Hz). 13C
NMR (CDCl3, 100 MHz): δ
143.7; 128.6-127.0; 87.0; 66.3; 62.6; 31.8; 30.8; 29.6; 29.5; 29.4; 29.3;
29.2; 25.9; 22.6; 14.1. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for
C31H39N3ONa 492.2991; found 492.2989.
1-(trityloxy)dodecan-2-amine (4). To a solution of 3 (6 g, 12.8 mmol,
1 eq) in Et2O was added to a suspension of Pd-C (640 mg) in Et2O. 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 40:60 v/v) to yield compound 4 as a colorless oil
(4.9 g, 86 %). Rf = 0.38 (cyclohexane/AcOEt 40:60 v/v). 1H NMR (CDCl3,
400 MHz): δ 7.50-7.21 (15H, m); 3.11 (1H, dd, J = 4 Hz, J = 8 Hz); 2.98
(1H, m); 2.90 (1H, m); 1.55 (2H, bs); 1.24 (18H, bs); 0.89 (3H, t, J = 8 Hz).
13C {1H} NMR (CDCl3, 100 MHz): δ 144.1; 128.7-126.9; 86.3; 68.6; 51.5;
34.2; 31.8; 29.7; 29.6; 29.5; 29.3; 26.0; 22.6; 14.0. HRMS (ESI-TOF) m/z:
[M + Na]+ Calcd for C31H41NONa 466.3086; found 466.3088.
(2R,3R,4S,5R,6R)-2-((benzoyloxy)methyl)-6-(((2R,3R,4S,5R,6R)-4,5-
bis(benzoyloxy)-2-((benzoyloxy)methyl)-6-((2-
methacrylamidododecyl)oxy)tetrahydro-2H-pyran-3-
yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (7a). To a solution of
6a (0.8 g, 3.0 mmol, 1 eq) and hepta-O-benzoyl-maltose-1-O-
trichloroacetimidate[22] (3.6 g, 3.0 mmol, 1 eq) in anhydrous CH2Cl2 was
added dropwise TMSOTf (0.54 mL, 3.0 mmol, 1 eq). The reaction mixture
was stirred for 24 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 compound 7a as
1
a white solid (3.6 g, 90 %). Rf = 0.54 (cyclohexane/AcOEt 60:40 v/v). H
N-(1-(trityloxy)dodecan-2-yl)methacrylamide (5a). To a solution of 4
(4.0 g, 9.0 mmol, 1 eq) in anhydrous CH2Cl2 was added Et3N (2.5 mL,
18.0 mmol, 2 eq). The solution was stirred for 20 minutes then
methacryloyl chloride (1.0 mL, 10.8 mmol, 1.2 eq) was added dropwise.
The reaction mixture was stirred for 2 h and the solution was filtered and
concentrated in vacuo. The crude compound was purified by flash
chromatography (cyclohexane/AcOEt 95:5 v/v) to yield compound 5a as a
NMR (CDCl3, 400 MHz): δ 8.07-7.24 (35H, m); 6.18 (1H, t, J = 10 Hz); 5.92
(1H, m); 5.73 (1H, t, J = 10 Hz); 5.61 (1H, s); 5.41 (1H, dd, J = 4 Hz, J =
10 Hz); 5.35 (1H, s); 4.95 (1H, t, J = 10 Hz); 4.85 (1H, m); 4.65-4.40 (5H,
m); 4.10-3.80 (5H, m); 3.50 (1H, m); 1.87 (3H, s); 1.40 (2H, m); 1.25 (16H,
bs); 0.90 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 167.9-165.1;
140.1; 133.5-128.3; 119.1; 100.2; 97.0; 78.5; 78.2; 75.4; 75.3; 74.7; 72.6;
71.5; 71.4; 70.5; 69.9; 68.9; 63.4; 62.7; 48.7; 31.9. 31.5; 31.3; 29.5; 29.3;
26.0; 22.6; 18.5; 14.0. HRMS (ESI-TOF) m/z: [M+H]+ Calcd for C77H80NO19
1322.5319; Found 1322.5350.
1
colorless oil (4.1 g, 90 %). Rf = 0.40 (cyclohexane/AcOEt 90:10 v/v). H
NMR (CDCl3, 400 MHz): δ 7.45-7.20 (15H, m); 5.99 (1H, d, J = 8 Hz); 5.64
(1H, s); 5.30 (1H, s); 4.10 (1H, m); 3.18 (2H, m); 1.95 (3H, s); 1.65 (2H,
m); 1.24 (16H, bs); 0.88 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz):
δ 167.7; 143.8; 140.4; 128.6-127.1; 118.9; 86.31; 64.4; 49.4; 32.2; 31.9;
29.6; 29.5; 29.3; 26.0; 22.7; 18.7; 14.1. HRMS (ESI+) m/z: [M + Na]+ Calcd
for C35H45NO2Na 534.3348; Found 534.3353.
(2R,3R,4S,5R,6R)-2-(((2R,3R,4S,5R,6R)-6-((2-
acrylamidododecyl)oxy)-4,5-bis(benzoyloxy)-2-
((benzoyloxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)-6-
((benzoyloxy)methyl)tetrahydro-2H-pyran-3,4,5-triyl tribenzoate (7b).
To a solution of 6b (0.75 g, 2.94 mmol, 1 eq) and hepta-O-benzoyl-
maltose-1-O-trichloroacetimidate[22] (3.57 g, 2.94 mmol, 1 eq) in
anhydrous CH2Cl2 was added dropwise TMSOTf (0.54 mL, 2.97 mmol,
1 eq). The reaction mixture was stirred for 24 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 compound 7b as a white solid (3.57 g, 93 %). Rf = 0.7
(cyclohexane/AcOEt 60:40 v/v). 1H NMR (CDCl3, 400 MHz): δ 8.16-7.19
(35H, m); 6.08 (2H, m, CH); 5.78 (2H, m); 5.69 (1H, t, J = 10 Hz); 5.55 (1H,
m); 5.32 (3H, m); 4.97 (1H, m); 4.75 (2H, m, CH); 4.86 (3H, m); 4.31 (1H,
m); 4.10 (2H, m); 3.92 (1H, m); 3.62 (1H, m); 1.25 (18H, bs); 0.89 (3H, t, J
= 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 169.7-164.9; 133.6-126.0;
101.4; 96.5; 74.5; 73.4; 73.0; 72.7; 72.4; 71.9; 70.9; 69.9; 63.4; 62.5; 48.9;
31.9; 31.6; 31.2; 29.4; 26.9; 26.0; 22.7; 14.1. HRMS (ESI-TOF) m/z:
[M+H]+ Calcd for C76H78NO19 1308.5181; Found 1308.5189.
N-(1-(trityloxy)dodecan-2-yl)acrylamide (5b). To a solution of 4 (4.1 g,
9.2 mmol, 1 eq) in anhydrous CH2Cl2 was added Et3N (2.6 mL, 18.5 mmol,
2 eq). The solution was stirred for 20 minutes then acryloyl chloride
(0.9 mL, 11.1 mmol, 1.2 eq) was added dropwise. The reaction mixture
was stirred for 2 h and the solution was filtered and concentrated in vacuo.
The crude compound was purified by flash chromatography
(cyclohexane/AcOEt 90:10 v/v) to yield compound 5b as a colorless oil
(4.3 g, 95 %). Rf = 0.40 (cyclohexane/AcOEt 80:20 v/v). 1H NMR (CDCl3,
400 MHz): δ 7.41-7.20 (15H, m); 6.22 (1H, d); 6.02 (1H, dd); 5.60 (1H, dd);
4.11 (1H, m); 3.18 (2H, m); 1.60 (2H, m); 1.24 (16H, bs); 0.86 (3H, t, J =
8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 164.9; 143.8; 131.2; 128.6-
126.1; 86.4, 64.5; 49.4; 32.2; 31.9; 29.6; 29.5; 29.4; 29.3; 26.0; 22.7; 14.1.
HRMS (ESI+) m/z: [M + Na]+ Calcd for C34H43NO2Na 520.3186; Found
520.3193.
N-(1-hydroxydodecan-2-yl)methacrylamide (6a). To a solution of 5a
(3.6 g, 7.0 mmol, 1 eq) in MeOH was added p-TsOH (121 mg, 0.7 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
60:40 v/v) to yield compound 6a as a colorless oil (1.17 g, 62 %). Rf = 0.17
(cyclohexane/AcOEt 60:40 v/v). 1H NMR (CDCl3, 400 MHz): δ 6.15 (1H, d,
J = 8 Hz); 5.68 (1H, s); 5.30 (1H, s); 3.95 (1H, m); 3.66 (1H, dd, J = 4 Hz,
J = 12 Hz, CH2O); 3.56 (1H, dd, J = 8 Hz, J = 12 Hz, CH2O); 1.93 (3H, s,
CH3); 1.55 (1H, m, CH2); 1.45 (1H, m, CH2); 1.22 (16H, bs, CH2); 0.85 (3H,
t, J = 8 Hz, CH3). 13C {1H} NMR (CDCl3, 100 MHz): δ 169.1; 139.8; 119.7;
65.2; 51.8; 31.8; 31.2; 29.5; 29.4; 29.2; 26.1; 22.6; 18.6; 14.0. HRMS (ESI-
TOF) m/z: [M + H]+ Calcd for C16H32NO2 270.2433; Found 270.2438.
N-(1-(((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-
(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-
pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)dodecan-2-
yl)methacrylamide (8a). To a solution of 7a (3.6 g, 2.72 mmol, 1 eq) in
MeOH was added catalytic amount of MeONa (58 mg, 1.1 mmol, 0.4 eq)
and the reaction mixture was stirred for 16 h. Then IRC-50 was added, and
the solution was filtered and concentrated in vacuo. The crude compound
was purified by flash chromatography (CHCl3/MeOH 80:20 v/v) to yield
compound 8a as a white solid (1.3 g, 80 %). Rf = 0.71 (CHCl3/MeOH 70:30
v/v). 1H NMR (CD3OD, 400 MHz): δ 5.67 (1H, d, J = 4 Hz); 5.36 (1H, d, J
= 4 Hz); 5.16 (1H, d, J = 4 Hz); 4.28 (1H, d, J = 8 Hz); 4.12 (1H, m); 3.95-
3.20 (14H, m); 1.92 (3H, s); 1.60 (2H, m); 1.29 (16H, bs); 0.90 (3H, t, J =
8 Hz). 13C {1H} NMR (CD3OD, 100 MHz): δ 169.5; 139.6; 119.1; 103.1;
101.2; 79.7; 75.7; 74.7; 73.2; 72.7; 72.6; 72.1; 71.7; 71.3; 69.7; 61.2; 60.4;
49.4; 49.1; 31.3; 30.7; 29.0; 28.9; 28.8; 25.6; 25.5; 22.1; 17.8; 13.3. HRMS
(ESI-TOF) m/z: [M + H]+ Calcd for C28H52NO12 594.3490; Found 594.3493.
N-(1-hydroxydodecan-2-yl)acrylamide (6b). To a solution of 5b (3.0 g,
6.0 mmol, 1 eq) in MeOH was added p-TsOH (0.1 g, 0.6 mmol, 0.1 eq) and
the reaction mixture was stirred for 6 h. The solution was neutralized by
addition of Et3N and the solution was concentrated in vacuo. The crude
compound was purified by flash chromatography (cyclohexane/AcOEt
7
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202000540
European Journal of Organic Chemistry
FULL PAPER
N-(1-(((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-
(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-
pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)dodecan-2-
flash chromatography (cyclohexane/AcOEt 95:5 v/v) to yield compound 13
as a colorless oil (10.0 g, 92 %) which was directly used without further
characterization. To a solution of 13 (10.0 g, 19.0 mmol, 1 eq) in
anhydrous DMF was added portion wise NaN3 (2.47 g, 38.0 mmol, 2 eq).
The reaction mixture was stirred for 16 h at 80 °C. Then the solution was
diluted with water and extracted twice with AcOEt. The organic layer was
dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The
yl)acrylamide (8b). To a solution of 7b (3 g, 2.29 mmol, 1 eq) in MeOH
was added catalytic amount of MeONa (49 mg, 0.92 mmol, 0.4 eq) and the
reaction mixture was stirred for 16 h. Then IRC-50 was added, and the
solution was filtered and concentrated in vacuo. The crude compound was
purified by flash chromatography (CHCl3/MeOH 80:20 v/v) to yield
compound 8b as a white solid (0.93 g, 70 %). Rf = 0.55 (CHCl3/MeOH
crude
compound
was
purified
by
flash
chromatography
(cyclohexane/AcOEt 100:0 to 95:5 v/v) to yield compound 14 as a colorless
oil (8.3 g, 96 %). Rf = 0.7 (cyclohexane/AcOEt 95:5 v/v). 1H NMR (CDCl3,
400 MHz): δ 7.46-7.24 (15H, m); 3.65 (1H, m); 3.55 (2H, m); 3.41 (2H, m);
3.26 (1H, dd, J = 4 Hz, J = 12 Hz); 3.21 (1H, dd, J = 8 Hz, J = 12 Hz); 1.55
(2H, m); 1.27 (10H, bs); 0.89 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3,
100 MHz): δ 143.6; 128.6-127.1; 87.0; 71.6; 70.4; 63.3; 61.2; 31.8; 29.6;
29.4; 29.2; 25.9; 22.6; 14.1. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for
C30H38N3O2 472.2959; Found 472.2957.
1
70:30 v/v). H NMR (CD3OD, 400 MHz): δ 6.22 (2H, m); 5.62 (1H); 5.13
(1H, dd); 4.25 (1H, d, J = 8 Hz); 4.07 (1H, m); 3.90-3.20 (14H, m); 1.55
(2H, m); 1.25 (16H, bs); 0.86 (3H, t, J = 8 Hz). 13C {1H} NMR (CD3OD,
100 MHz): δ 167.9; 132.2; 126.7; 104.8; 102.8; 81.2; 77.6; 76.6; 74.9; 74.7;
74.6; 74.5; 74.0; 72.9; 71.5; 62.7; 62.1; 50.9; 50.6; 32.9; 32.3; 30.6; 30.5;
30.4; 27.0; 23.7. HRMS (ESI-TOF) m/z: [M + Na]+ Calcd for C27H49NO12Na
602.3152; Found 602.3153.
2,2-dimethyl-4-((octyloxy)methyl)-1,3-dioxolane (10) To a suspension
of NaH (3.0 g, 127.2 mol, 2.4 eq) in anhydrous DMF, was added a solution
of 9[24] (7 g, 53.0 mmol, 1 eq) in anhydrous DMF. The solution was stirred
for 20 minutes and 1-bromooctane (11 mL, 63.6 mmol, 1.2 eq) was added
dropwise. The reaction mixture was stirred for 24 h. Then water is added
and the solution was extracted twice with AcOEt. The organic layer was
dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The
1-(octyloxy)-3-(trityloxy)propan-2-amine (15). To a solution of 14 (8.3 g,
18.2 mmol, 1 eq) in Et2O was added Pd-C (910 mg). 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 40:60 v/v) to
yield compound 15 as
a
colorless oil (6.9 g, 85 %). Rf
=
0.51
1
(cyclohexane/AcOEt 40: 60 v/v). H NMR (CDCl3, 400 MHz): δ 7.47-7.20
(15H, m); 3.49 (1H, dd, J = 4 Hz, J = 12 Hz); 3.44-3.31 (3H, m); 3.15 (2H,
m); 3.05 (1H, m); 1.50 (4H, m); 1.28 (10H, bs); 0.89 (3H, t, J = 8 Hz). 13
C
crude
compound
was
purified
by
flash
chromatography
(cyclohexane/AcOEt 95:5 v/v) to yield compound 10 as a colorless oil
(9.5 g, 74 %). Rf = 0.6 (cyclohexane/AcOEt 90:10 v/v). 1H NMR (CDCl3,
400 MHz): δ 4.24 (1H, qt, J = 8 Hz); 4.03 (1H, dd, J = 6 Hz, J = 8 Hz); 3.71
(1H, dd, J = 6 Hz, J = 8 Hz); 3.45 (4H, m); 1.55 (2H, m); 1.40 (3H, s); 1.34
(3H, s); 1.25 (10H, bs); 0.86 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100
MHz): δ 109.3; 74.8; 71.9; 71.8; 66.9; 31.8; 29.5; 29.4; 29.2; 26.7; 26.0;
25.4; 22.6; 14.0. HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C14H20O3
245.2112; Found 245.2118.
{1H} NMR (CDCl3, 100 MHz): δ 144.1; 128.7-126.9; 86.5; 73.3; 71.5; 65.7;
51.4; 31.8; 29.7; 29.4; 26.9; 26.2; 22.6; 14.1. HRMS (ESI-TOF) m/z: [M +
Na]+ Calcd for C30H39NO2Na 468.2878; Found 468.2869.
N-(1-(octyloxy)-3-(trityloxy)propan-2-yl)methacrylamide (16). To
a
solution of 15 (6.9 g, 15.5 mmol, 1 eq) in anhydrous CH2Cl2 was added
Et3N (4.3 mL, 31.0 mmol, 2.0 eq). The solution was stirred for 20 minutes
then methacryloyl chloride (1.8 mL, 18.6 mmol, 1.2 eq) was added
dropwise. The reaction mixture was stirred for 2h and the solution was
filtered and concentrated in vacuo. The crude compound was purified by
flash chromatography (cyclohexane/AcOEt 90:10 v/v) to yield compound
16 as a yellow oil (7.32 g, 92 %). Rf = 0.51 (cyclohexane/AcOEt 80:20 v/v).
1H NMR (CDCl3, 400 MHz): δ 7.50-7.20 (15H, m); 6.15 (1H, d, J = 8 Hz);
5.65 (1H, s); 5.30 (1H, s); 4.30 (1H, m); 3.71 (1H, dd, J = 4 Hz, J = 8 Hz);
3.58 (1H, dd, J = 6 Hz, J = 10 Hz); 3.42 (2H, t, J = 8 Hz); 3.39 (1H, dd, J =
4 Hz, J = 12 Hz); 3.14 (1H, dd, J = 8 Hz, J = 12 Hz); 1.93 (3H, s); 1.55 (2H,
m); 1.27 (10H, bs); 0.89 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz):
δ 167.7; 143.9; 140.0; 128.6-127.0; 119.5; 86.5; 71.3; 69.1; 61.8; 60.3;
48.8; 31.8; 29.7; 29.4; 29.2; 26.1; 22.6; 18.6; 14.1. HRMS (ESI-TOF) m/z:
[M + Na]+ Calcd for C34H43NO3Na 536.3141; Found 536.3136.
3-(octyloxy)propane-1,2-diol (11) To a solution of 10 (9.3 g, 38.0 mmol,
1 eq) in MeOH was added p-TsOH (0.65 g, 3.8 mmol, 0.1 eq), and the
reaction mixture was stirred for 6 h at 80 °C. Then the solution was
neutralized by addition of Et3N and concentrated in vacuo. The crude
compound was purified by flash chromatography (cyclohexane/AcOEt
60:40 v/v) to yield compound 11 as a colorless oil (6.5 g, 84 %). Rf = 0.35
(cyclohexane/AcOEt 40:60 v/v). 1H NMR (CDCl3, 400 MHz): δ 3.88 (1H,
m); 3.71 (1H, dd, J = 4 Hz, J = 12 Hz); 3.64 (1H, dd, J = 8 Hz, J = 12 Hz);
3.55-3.45 (4H, m); 2.50 (2H, bs); 1.56 (2H, qt, J = 8 Hz); 1.26 (10H, bs);
0.87 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 400 MHz): δ 72.4; 71.8; 70.5;
64.2; 31.8; 29.5; 29.4; 29.2; 26.0; 22.6; 14.0. HRMS (ESI-TOF) m/z: [M +
H]+ Calcd for C11H25O3 205.1804; Found 205.1804.
1-(octyloxy)-3-(trityloxy)propan-2-ol (12). To a solution of 11 (5.2 g,
21.3 mmol, 1 eq) in anhydrous CH2Cl2, was added Et3N (6.0 mL,
42.6 mmol, 2 eq). The solution was stirred at rt for 20 min, and trityl
chloride (7.2 g, 25.6 mmol, 1.2 eq) was added portion-wise. The reaction
mixture was stirred for 24 h and the solution was filtered and concentrated
in vacuo. The crude compound was purified by flash chromatography
(cyclohexane/AcOEt 90:10 v/v) to yield compound 12 as a colorless oil
(9.3 g, 98 %). Rf = 0.32 (cyclohexane/AcOEt 90:10 v/v). 1H NMR (CDCl3,
400 MHz): δ 7.47-7.20 (15H, m); 3.96 (1H, m); 3.54 (1H, dd, J = 4 Hz, J =
12 Hz); 3.48 (1H, dd, J = 8 Hz, J = 12 Hz); 3.44 (2H, m); 3.20 (2H, m); 2.45
(1H, m); 1.55 (2H, m); 1.28 (10H, bs); 0.89 (3H, t, J = 8 Hz). 13C {1H} NMR
(CDCl3, 100 MHz): δ 143.8; 128.6-127.0; 86.6; 72.0; 71.6; 69.8; 64.6; 31.8;
29.6; 29.4; 29.2; 26.0; 22.6; 14.0. HRMS (ESI-TOF) m/z: [M + H]+ Calcd
for C30H38O3Na 469.2713; Found 469.2709.
N-(1-hydroxy-3-(octyloxy)propan-2-yl)methacrylamide (17). To
a
solution of 16 (0.92 g, 1.8 mmol, 1 eq) in MeOH was added p-TsOH
(0.03 g, 0.18 mmol, 0.1 eq) and the reaction mixture was stirred for 2 h.
Then the solution was neutralized by addition of Et3N and concentrated in
vacuo. The crude compound was purified by flash chromatography
(cyclohexane/AcOEt 50:50 v/v) to yield compound 17 as a colorless oil
(0.35 g, 80 %). Rf = 0.24 (cyclohexane/AcOEt 50:50 v/v). 1H NMR(CDCl3,
400 MHz): δ 6.54 (1H, d, J = 8 Hz); 5.71 (1H, s); 5.32 (1H, s); 4.08 (1H,
m); 3.80-3.50 (5H, m); 3.41 (2H, m); 1.94 (3H, s); 1.52 (2H, m); 1.23 (8H,
bs); 0.84 (3H, t, J = 8 Hz). 13C {1H} NMR (CDCl3, 100 MHz): δ 168.5; 139.6;
120.0; 71.6; 70.7; 64.0; 50.7; 31.7; 29.4; 29.3; 29.1; 26.0; 22.5; 18.5; 14.0.
HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C15H30NO3 272.2226; Found
272.2234.
(2R,3R,4S,5R,6R)-2-((benzoyloxy)methyl)-6-(((2R,3R,4S,5R,6R)-4,5-
bis(benzoyloxy)-2-((benzoyloxy)methyl)-6-(2-methacrylamido-3-
(octyloxy)propoxy)tetrahydro-2H-pyran-3-yl)oxy)tetrahydro-2H-
pyran-3,4,5-triyl tribenzoate (18). To a solution of 17 (0.35 g, 1.29 mmol,
1 eq) and hepta-O-benzoyl-maltose-1-O-trichloroacetimidate[22] (1.57 g,
1.29 mmol, 1 eq) in anhydrous CH2Cl2 was added dropwise TMSOTf
(0.23 mL, 1.29 mmol, 1 eq). The reaction mixture was stirred for 24 h.
((2-azido-3-(octyloxy)propoxy)methanetriyl)tribenzene (14). To
a
solution of 12 (9.3 g, 20.8 mmol, 1 eq) in anhydrous CH2Cl2, was added
Et3N (5.8 mL, 41.6 mmol, 2 eq). The solution stirred for 20 minutes then
methanesulfonyl chloride (1.9 mL, 25.0 mmol, 1.2 eq) was added
dropwise. The reaction mixture was stirred for 16 h and the solution was
filtered and concentrated in vacuo. The crude compound was purified by
8
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10.1002/ejoc.202000540
European Journal of Organic Chemistry
FULL PAPER
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 compound 18 as a white solid
(1.37 g, 80 %). Rf = 0.49 (cyclohexane/AcOEt 60:40 v/v). 1H NMR (CDCl3,
400 MHz): δ 8.10-7.20 (35H, m); 6.20 (1H, t, J = 10 Hz); 5.95 (1H, m); 5.75
(1H, t, J = 10 Hz); 5.50 (1H, s); 5.44 (1H, dd, J = 4 Hz, J = 12 Hz); 5.13
(1H, s); 4.95 (1H, t, J = 10 Hz); 4.86 (1H, d, J = 12 Hz); 4.70-4.40 (5H, m);
4.25 (1H, m); 4.10-3.90 (4H, m); 3.65 (1H, dd, J = 6 Hz, J = 10 Hz); 3.45
(1H, dd, J = 4 Hz, J = 10 Hz); 3.35-3.20 (3H, m); 2.90 (1H, d, J = 4 Hz);
1.88 (3H, s); 1.46 (2H, m); 1.25 (10H, bs); 0.90 (3H, t, J = 8 Hz). 13C {1H}
NMR (CDCl3, 100 MHz): δ 167.8-165.1; 139.6; 133.4-128.2; 119.5; 100.6;
96.9; 78.2; 75.4; 74.7; 72.6; 71.4; 71.2; 69.9; 69.1; 68.9; 68.4; 68.1; 67.7;
63.4; 62.7; 48.2; 31.7; 29.4; 29.3; 29.2; 25.9; 22.6; 18.3; 18.4; 14.0. HRMS
(ESI-TOF) m/z: [M + H]+ Calcd for C76H78NO20 1324.5117; Found
1324.5132.
Vesicle solubilization. POPC in powder form was suspended in
phosphate buffer (10 mM NaH2PO4/Na2HPO4 and 150 mM NaCl at pH 7.4).
To obtain large unilamellar vesicles (LUVs), the suspension was extruded
35 times through two stacked polycarbonate membranes with a nominal
pore diameter of 100 nm using a LiposoFast extruder (Avestin, Ottawa,
Canada). Unimodal size distribution was confirmed by DLS. A 0.6 mM
stock solution of POPC LUVs and detergent were mixed in a 3 mm × 3 mm
quartz glass cuvette (Hellma, Müllheim, Germany) before the light
scattering intensity was monitored at 25 °C using a Nano Zetasizer ZS90
(Malvern) equipped with a 633-nm He–Ne laser and a detection angle of
90°. To ensure quantitative comparability of scattering intensities, the
attenuator was fixed to the maximum open position.
Extraction of MPs from E. coli membranes. E. coli BL21(DE3) cells
were transformed with an empty pET-24 vector and thus selected by
kanamycin resistance. After incubation in lysogeny broth overnight at
37 °C under permanent agitation, cells were harvested by centrifugation
and washed twice with saline (154 mM NaCl). Cell pellets were
N-(1-(((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-
(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-
pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-(octyloxy)propan-2-
yl)methacrylamide (19). To a solution of 18 (1.3 g, 0.98 mmol, 1 eq) in
MeOH was added catalytic amount of MeONa (21 mg, 0.39 mmol, 0.4 eq)
and the reaction mixture was stirred for 16 h. Then IRC-50 was added, and
the solution was filtered and concentrated in vacuo. The crude compound
was purified by flash chromatography (CHCl3/MeOH 80:20 v/v) to yield
compound 19 as a white solid (0.51 g, 88 %). Rf = 0.28 (CHCl3/MeOH
80:20 v/v). 1H NMR (CD3OD, 400 MHz): δ 5.70 (1H, d, J = 4 Hz); 5.37 (1H,
d, J = 4 Hz); 5.15 (1H, d, J = 4 Hz); 4.28 (2H, m); 4.10-3.20 (18H, m); 1.94
(3H, s, CH3); 1.54 (2H, qt, J = 8 Hz); 1.28 (10H, bs); 0.89 (3H, t, J = 8 Hz).
13C {1H} NMR (CD3OD, 100 MHz): δ 171.3; 141.4; 120.5; 104.7; 102.9;
81.2; 77.7; 76.7; 75.0; 74.7; 74.6; 74.1; 72.3; 71.5; 70.4; 70.2; 69.8; 62.7;
62.2; 50.8; 33.0; 30.7; 30.5; 27.3; 23.7; 18.8; 14.4. HRMS (ESI-TOF) m/z:
[M + H]+ Calcd for C27H50NO13 596.3282; Found 596.3283.
resuspended in ice-cold buffer (100 mM Na2CO3, pH 11.5) to
a
concentration below ~0.1 g mL–1 and ultrasonicated twice for 10 min in an
S-250A sonifier (Branson Ultrasonics, Danbury, USA). To remove cell
debris, the lysate was centrifuged at 4 °C for 30 min at 1000 g. The
supernatant was centrifuged at 4 °C for 1 h at 100,000 g to separate
membrane fragments from soluble and peripheral proteins. Membrane
fragments were resuspended in buffer (50 mM Tris, 200 mM NaCl, pH 7.4)
to a final concentration of 100 mg wet-weight pellet per 1 mL of buffer and
mixed in a 1:1 volume ratio with stock solutions of DDM or monomers in
buffer. Surfactant concentrations were chosen based on the CMC values
determined in this study to ensure comparable extraction conditions. All
samples were incubated for 16 h at 20 °C under gentle agitation. After
ultracentrifugation at 4 °C for 1 h at 100,000 g, the supernatant containing
micelles was analyzed using SDS-PAGE. Extraction yields were then
determined densitometrically using ImageJ gel analysis.[30]
Isothermal titration calorimetry. High-sensitivity microcalorimetry was
performed at 25 °C on a VP-ITC (Malvern Instruments, Malvern, UK) for
LC027 and on an iTC200 (Malvern Instruments) for LC049 and LC058. All
solutions were prepared in Phosphate buffer (10 mM NaH2PO4/Na2HPO4
and 150 mM NaCl at pH 7.4). For Demicellization experiments 10-µL
aliquots of 7 mM LC027 and 1.5-µL aliquots 8 mM LC058 were titrated into
buffer, whereas 1-µL aliquots of 70 mM LC049 were titrated into a cell
preloaded with 3 mM monomer. Time spacings between consecutive
injections were chosen long enough to allow for complete re-equilibration.
Baseline subtraction and peak integration were performed using
NITPIC.[28] All reactions heats were normalized with respect to the molar
amount of detergent. Non-linear least-squares fitting was performed using
D/STAIN.[29]
Solubilization of A2AR and AcrB. Adenosine receptor (A2AR) was
expressed in insect cells as described.[31] AcrB was expressed in E.coli as
previously reported.[32] 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 or LC compounds. 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 A2A
R
(7F6-G5-A2) and against the his-tag for AcrB, respectively. Solubilization
efficiency was evaluated by comparing the band intensity (in western blot)
of the Soluble (S) to the insoluble (P for Pellet) fractions.
Surface tension measurements. The surface activity of detergents in
Supporting Information available. 1H and 13C NMR spectra of
compounds; HPLC chromatograms of LC027, LC048, LC049, and LC058.
Contin distribution plots in pure water for LC027, LC048, and LC058.
solution at the air/water interface was determined using
a K100
tensiometer (Kruss, Hamburg, Germany). Surface tensions were
determined by dilution of stock solutions (~5×CMC) using the Wilhelmy
plate technique. In a typical experiment, 20–30 concentration steps were
used with ~5–10 min between each concentration step. All measurements
were performed at (25.0 ± 0.5) °C.
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 “Rꢂgion Provence Alpes Cꢃte d’Azur”.
Dynamic light scattering. Hydrodynamic particle size distributions were
determined on a Nano Zetasizer ZS90 (Malvern Instruments, UK)
equipped with a He−Ne laser (λ = 633 nm). Except for LC048 all
measurements were performed at (25 ± 0.5) °C. Measurements for LC048
were performed at 35 °C to keep the compound in solution. The
concentration for each measurement was 2×(CMC + 5 mM) in buffer (50
mM Tris, 200 mM NaCl, pH 7.4). The time dependent correlation function
of the scattered light intensity was measured at an angle of 90°.The
hydrodynamic diameter (DH) of the particles was estimated from their
Keywords: Detergents • Monomers • Surfmers • Extraction •
Membrane proteins
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.
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10.1002/ejoc.202000540
European Journal of Organic Chemistry
FULL PAPER
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Surfmers for Membrane Proteins extraction
Surfmers were synthesized and were found to self-assemble in water into micelles of ~ 6 nm in diameter at a critical micellar
concentration that depends on the length of the alkyl chain. They were found to solubilize phospholipid vesicles and to extract a broad
range of proteins from Escherichia coli membranes as well as the human adenosine receptor A2AR and the bacterial transporter AcrB.
11
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