Synthesis of nickel-chelating fluorinated lipids for protein monolayer crystallizations

Article abstract of DOI:10.1021/jo802651p

Nickel-chelating lipids have been synthesized for use as functionalized templates for 2-D crystallization of membrane proteins. These monolayer-forming lipids have been designed with three distinct components: (i) a branched hydrocarbon tail to confer flu

Full text of DOI:10.1021/jo802651p

Synthesis of Nickel-Chelating Fluorinated Lipids for Protein
Monolayer Crystallizations
Waleed M. Hussein,† Benjamin P. Ross,† Michael J. Landsberg,‡ Daniel Le´vy,§
Ben Hankamer,‡ and Ross P. McGeary*,†,|
The UniVersity of Queensland, School of Molecular & Microbial Sciences, Institute for Molecular
Bioscience, and School of Pharmacy QLD 4072, Australia, and Institut Curie, UMR CNRS 168,
11 rue P.M.Curie, F-75231 Paris, France
r.mcgeary@uq.edu.au
ReceiVed September 23, 2008
Nickel-chelating lipids have been synthesized for use as functionalized templates for 2-D crystallization
of membrane proteins. These monolayer-forming lipids have been designed with three distinct components:
(i) a branched hydrocarbon tail to confer fluidity of the monolayer, (ii) a perfluorinated central core for
detergent resistance, and (iii) a nickel-chelating hydrophilic headgroup to facilitate binding of recombinant,
polyhistidine-tagged fusion proteins. Alkylations of fluorinated alcohols used in these syntheses proceed
in good yields only with the application of prolonged sonication and, in some cases, in the presence of
phase-transfer catalysts. Formation of 2-D crystals of the His-tagged membrane protein BmrA from Bacillus
subtillis is reported.
Introduction
in a single experiment by taking advantage of the high-affinity
interaction between His-tagged proteins and the Ni2+-NTA
ligand.4
Binding and adsorption of proteins on lipid monolayers is
an elegant method to generate high concentrations of oriented
proteins and protein complexes. Bound proteins can be either
imaged directly or, under ideal conditions, induced to form 2-D
crystalline arrays for subsequent structure determination by
either single particle analysis1 or 2-D electron crystallography,2
respectively. 2-D crystals grown by this technique can also be
used as templates for the growth of 3-D crystals for X-ray
diffraction analysis.3 Moreover, it has recently been demon-
strated that purification and monolayer binding can be coupled
Monolayer-forming lipids for 2-D crystallization contain two
domains with distinct properties: a long, branched hydrophobic
tail with the necessary chemistry to impart fluidity of the lipid
monolayer at the air-water interface,5 and a hydrophilic
headgroup which is responsible for orienting the lipid to the
aqueous phase and often incorporates a functional moiety for
specific interaction with target proteins and/or complexes.6-8
Many derivatized lipids have been reported, incorporating
ligands such as ATP,9 biotin,10-14 and steroids,15 and metal ions
† The University of Queensland, School of Molecular & Microbial Sciences.
‡ The University of Queensland, Institute for Molecular Bioscience.
§ Institut Curie.
(4) Kelly, D. F.; Dukovski, D.; Walz, T. Proc. Natl. Acad. Sci. U.S.A. 2008,
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| The University of Queensland, School of Pharmacy.
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Yang, D. S. C.; Edwards, A. M. J. Mol. Biol. 1995, 246, 308–316.
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10.1021/jo802651p CCC: $40.75
Published on Web 01/14/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 1473–1479 1473

Hussein et al.
including Cu2+ and Ni2+.17 These lipids bind proteins through
16
which yielded a projection map at 9 Å resolution.30 Details of
the syntheses of these partially fluorinated lipids, however, were
not reported.
either a natural or conferred affinity for the functionalized head
groups. For example, Ni2+-chelating lipids have been used to
grow 2-D crystals of a number of proteins, including HIV-1
reverse transcriptase,18 S-layer protein sbpA,19 Moloney murine
leukemia virus capsid protein (His-MoCa),20 yeast RNA
polymerase I,21 murine MHC class I,22 FhuA,23,24 F0F1-ATP-
synthase,24 HC-Pro,25 and a VE cadherin fragment.26 The lipid
headgroup is designed to take advantage of the strong binding
interaction between the Ni2+ ion and a sequence of 4-8
consecutive histidine residues (His-tag), often incorporated at
the N- or C-terminus of recombinantly expressed fusion proteins.
Ni2+ is usually attached to the lipid headgroup via the quadri-
dentate nitrilotriacetic acid (NTA, cf. compounds 1 and 2)
To extend the compatibility of the monolayer crystallization
technique with membrane proteins, fluorinated lipids 1 and 2
with Ni2+-chelating functional head groups have been designed
to form a fluid yet detergent-resistant, monolayer template.31
These compounds were designed to have fluorinated alkyl chains
coupled to a Ni2+-chelating headgroup for the binding of His-
tagged proteins.32-34 The two lipids contain one (compound 1)
or two (compound 2) branched, partially fluorinated alkyl chains
with the latter linked via a glycerol backbone. We also describe
the synthesis of a “diluting” lipid (compound 18) which, when
mixed with compound 2 and spread at the air-water interface,
is able to act as a template for 2-D membrane protein crystal-
lization.
ligand, which presents an excellent coordination complex for
17
the octahedral coordination sphere of Ni2+
.
The synthesis of compounds containing perfluorinated moi-
eties can be very problematic due to their unusual reactivities,
hydrophobicity, and lipophobicity, which can lead to difficulties
in handling, solubilization, and purification.35,36 Here we report
the synthesis of compounds 1 and 2. One advantage of the
procedures described is that they are modular, allowing for
variation of the heads groups and lipid tails. We also report
several improved techniques for preparing fluoroalkylated
systems with good yields.
The susceptibility of lipid monolayers to detergent solubili-
zation, however, limits their usefulness in the binding and
crystallization of membrane proteins, which often require
significant concentrations of detergent to maintain their solubil-
ity. Some success has been reported in the crystallization of
detergent-solubilized membrane proteins via attachment to
hydrogenated lipid monolayers; however, these successes have,
so far, been limited to membrane proteins solubilized with low
Critical Micelle Concentration detergents and require highly
compressed monolayers. Lipids that are resistant to a broader
range of detergents under more fluid conditions are, therefore,
desirable.27,28 In this respect, it has been demonstrated that
partially fluorinated lipids have vastly improved stability in the
presence of detergents.29 In 2001, Lebeau et al. used partially
fluorinated Ni2+-chelating lipids to grow 2-D crystals of the
plasma membrane proton-ATPase from Arabidopsis thaliana
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Results and Discussion
The branched, saturated methyl ester 4 was prepared by the
conjugate addition of the Grignard reagent derived from
1-bromohexane with methyl non-2-enoate 3 in the presence of
Cu(I) and in situ silylation37 of the intermediate enolate (Scheme
1). The ester 4 was reduced to the corresponding alcohol 5,
either by using NaBH4-LiCl in THF-ethanol (2:1) (64% yield)
or by employing LiAlH4 in THF, and quenching38 with
Na2SO4 ·10H2O (83%). This quench was essential to avoid the
formation of a gelatinous precipitate of Al(OH)3 which hindered
workup and led to low recovery of the desired product. No
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1474 J. Org. Chem. Vol. 74, No. 4, 2009

Protein Monolayer Crystallizations
SCHEME 1
SCHEME 2
reduction of 4 occurred with NaBH4-LiCl in refluxing THF
alone. Conversion of the alcohol 5 to the alkyl iodide 6 was
performed in two steps, via the tosylate intermediate (83%),
and then onto the iodide 6 (68%). A higher yielding, direct
transformation of alcohol 5 to the iodide 6 was achieved using
PPh3, iodine, and imidazole. For this reaction the choice of
solvent was important: in benzene39 the yield of iodide 6 was
50%, in dichloromethane40 75%, and in 1:3 acetonitrile:diethyl
ether 92%.
SCHEME 3
The next step in the synthesis required the monobenzyl
derivative of the fluorinated diol 7. Performing chemical
transformations on perfluorocarbon derivatives presents a
number of challenges because of their poor solubility in many
common organic solvents, unusual chemical properties, and
reactivities.41 In contrast to a recent report,42 benzylation of diol
7 under usual conditions proved remarkably refractory. After
many unsuccessful alkylation attempts, we showed that treat-
ment of the diol 7 with NaH in THF led to rapid and extensive
decomposition of the reactant. We eventually found that a
moderate yield (41%) of the monobenzyl derivative 8 was
achieved using benzyl bromide and NaH in DMF in the presence
of the phase-transfer agent tetrabutylammonium iodide, and with
prolonged sonication (Scheme 2). A small amount of the
dibenzyl ether 9 (16%) was also obtained and recycled to the
monobenzyl ether 8 by partial hydrogenolysis over palladium
on carbon. Pasquato recently reported that attempted benzylation
of 7, under different conditions, led mainly to the formation of
the dibenzyl ether 9 with low conversion and the formation of
only a small amount of 8.41 Lebeau’s group have also reported
that alkylation of 7 with the very reactive tert-butyl bromoacetate
only occurs in the presence of a THF-HMPA solvent mixture.43
Alkylation of the monobenzyl ether 8 with the iodide 6 under
the usual conditions, again, proved problematic. Alkylation using
NaH in THF gave the corresponding benzyl ether in only 34%
yield. The yield of this reaction was improved, to 47%, by
sonicating the reaction mixture. Hydrogenolysis of the benzyl
ether then yielded the fluorinated alcohol 10 in 83% yield (see
Supporting Information). Alternatively, alcohol 10 was prepared
directly from the diol 7, in 51% yield, using sonication and a
phase-transfer catalyst (Scheme 3). Reaction of the fluorinated
alcohol 10 with bromoacetic acid and NaH in anhydrous THF
gave the carboxylic acid 11 in 69% yield.
The next step involved coupling of the carboxylic acid 11
with the lysine-NTA derivative 15. As shown in Scheme 4, the
triester 15 was prepared by treating ε-Cbz-protected L-lysine
12 with bromoacetic acid under alkaline conditions to give the
triacid44,45 13 in 92% yield. Esterification of 13 with acidic
methanol yielded the triester46 14 in 76% yield, and hydro-
genolysis of 14 in methanolic formic acid yielded the formate
salt46 15 in 92% yield. The NMR spectra of 14 and 15 differed
somewhat from those described by Roy46 but were in agreement
with those reported by Zhou.47 It was necessary to store the
amino triester 15 as its formate salt to prevent self-condensation.
(39) Anan, H.; Seki, N.; Noshiro, O.; Honda, K.; Yasumuro, K.; Ozasa, T.;
Fusetani, N. Tetrahedron 1996, 52, 10849–10860.
(40) Lange, G. L.; Gottardo, C. Synth. Commun. 1990, 20, 1473–1479.
(41) Gentilini, C.; Boccalon, M.; Pasquato, L. Eur. J. Org. Chem. 2008, 330,
3308–3313.
(44) Altin, J. G.; Banwell, M. G.; Coghlan, P. A.; Easton, C. J.; Nairn, M. R.;
Offermann, D. A. Aust. J. Chem. 2006, 59, 302–306.
(45) Du Roure, O.; Debiemme-Chouvy, C.; Malthete, J.; Silberzan, P.
Langmuir 2003, 19, 4138–4143.
(42) Slaughter, J. N.; Schmidt, K. M.; Byram, J. L.; Mecozzi, S. Tetrahedron
Lett. 2007, 48, 3879–3882.
(43) Klein, E.; Kerth, P.; Lebeau, L. Biomaterials 2007, 29, 204–214.
(46) Roy, B. C.; Mallik, S. J. Org. Chem. 1999, 64, 2969–2974.
(47) Zhou, M.; Haldar, S.; Franses, J.; Kim, J.-M.; Thompson, D. H.
Supramol. Chem. 2005, 17, 101–111.
J. Org. Chem. Vol. 74, No. 4, 2009 1475

Hussein et al.
SCHEME 4
SCHEME 5
SCHEME 7
SCHEME 8
SCHEME 6
made directly in 78% yield by heating a mixture of the alcohol
10, epichlorohydrin, NaOH, and a phase-transfer catalyst at 30
°C followed by sonication. Compound 19 was prepared in 95%
yield by alkylation of the alcohol 18 with bromoacetic acid and
sodium hydride in THF. It could also be prepared in two steps
by alkylation of 18 with tert-butyl bromoacetate and sodium
hydride in DMF (45% yield), followed by deprotection of the
ester with TFA (75% yield).
With the acid 19 in hand, we now turned our attention to its
coupling with a suitably protected NTA derivative. Several
approaches were investigated: as well as using the previously
synthesized trimethyl ester 15, use of the tri-tert-butyl ester48-50
22 (Scheme 7) and the persilylated derivative51,52 24 (Scheme
8) was also examined.
The ε-Cbz-protected L-lysine 12 was converted to its tert-
butyl ester 20 by treatment with isobutylene and sulfuric acid
in a pressure bottle.48 Bisalkylation of 20 with tert-butyl
Coupling of the fluorinated acid 11 with the triester 15 to
give the amide 16 was first attempted, without success, using
peptide coupling agents, such as diisopropylcarbodiimide. A
complex mixture was invariably obtained. After many attempts
we found that the carboxylic acid 11 was converted using thionyl
chloride into its corresponding acid chloride, and that this would
react with 15 in the presence of triethylamine to yield the amide
16 in modest yield (Scheme 5). Hydrolysis of 16 with LiOH in
THF-MeOH for 4 days gave the triacid 17 in 69% yield.
Finally, the Ni-NTA complex 1 was prepared by treatment of
17 with NiSO4 in Tris buffer.
The synthesis of the double-tailed carboxylic acid 19 is shown
in Scheme 6. Conversion of fluorinated alcohol 10 to the
tetraether 18 was accomplished in two steps by reaction with
epichlorohydrin and powdered NaOH to give the corresponding
oxirane (60% yield), followed by further reaction with additional
10 to yield 18 (43% yield). This same compound 18 was also
(48) Barton, J. N.; Piwinski, J. J.; Skiles, J. W.; Regan, J. R.; Menard, P. R.;
Desai, R.; Golec, F. S.; Reilly, L. W.; Goetzen, T.; Ueng, S.-N.; Warus, J. D.;
Schwab, A.; Samuels, A. I.; Neiss, E. S.; Suh, J. T. J. Med. Chem. 1990, 33,
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2753–2763.
(50) Lata, S.; Piehler, J. Nature Protocols 2006, 1, 2104–2109.
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(Dordrecht, Neth) 2003, 10, 53–63.
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1476 J. Org. Chem. Vol. 74, No. 4, 2009

Protein Monolayer Crystallizations
FIGURE 1. (A,C) At low magnification, large planar reconstituted membranes (dark gray) are seen. The membrane in A is broken as a result of
transfer to the carbon grid. (B,D) Mosaic 2-D arrays of BmrA are clearly seen at higher magnification. Since structural analysis of BmrA is beyond
the scope of this paper, we did not perform systematic trials to improve the crystallinity. However, the presence of 2-D arrays is only observed
following binding of the protein to the functionalized fluorinated monolayer, thus demonstrating the effectiveness of compound 2.
SCHEME 9
26, either by treatment of 25 with TFA in chloroform (69%) or
by saponification of 27 with LiOH in MeOH-THF (99%).
Good coupling yields were obtained, however, using the
persilylated triester 24 with the acid chloride derived from 19
under microwave irradiation. Using this method, a yield of 62%
of the triacid 26 was isolated after chromatography. Treatment
of the triacid 26 with NiSO4 in Tris buffer then gave the nickel
complex 2.
2-D Protein Crystallization
Using our previously published method,24 we investigated
the ability of the Ni2+-chelating lipid 2 to induce 2-D protein
crystallization of the His-tagged membrane protein BmrA,53 an
ABC transporter from Bacillus subtillis. A lipid monolayer
composed of a 1:3 mixture of 2 (the functionalized lipid) and
18 (the diluent lipid) was spread at the air-water interface,
following which detergent solubilized BmrA was injected below
the lipid layer. After removal of the detergent and subsequent
incubation for 4 days at 2 °C, the surface was transferred to a
carbon-coated grid, negatively stained with 2% uranyl acetate,
and examined by electron microscopy (see Supporting Informa-
tion for more details). Figure 1 shows electron micrographs of
the resulting 2-D crystalline arrays, in which oriented binding
and concentration of the protein at the air-water interface can
be seen.
bromoacetate then gave the triester 21, and hydrogenolysis
yielded the deprotected aminotriester 22.
The Cbz protecting group of the triacid 13 was removed by
hydrogenolysis to give the amino-triacid52 23, which was
converted to the soluble silylated derivative51 24, using chlo-
rotrimethylsilane and diisopropylethylamine in toluene.
Conclusions
Two partially fluorinated lipids with nickel-chelating head
groups have been synthesized, for use in 2-D crystallization of
His-tagged proteins and for structure determination by electron
crystallography. The core fluorinated alcohol moieties of these
lipids proved to be very difficult to alkylate under standard
Coupling of the acid 19 with the tri-tert-butyl ester 22 using
EDC and NHS in DMF gave a very poor yield of the desired
amide 25 (Scheme 9). Similarly, coupling of the acid chloride
derived from 19 with the trimethyl ester 15 in the presence of
DIPEA gave only 13% of the desired amide 27. Both of these
products could be carried forward to the corresponding triacid
(53) Steinfels, E.; Orelle, C.; Fantino, J.-R.; Dalmas, O.; Rigaud, J.-L.;
Denizot, F.; Di Pietro, A.; Jault, J.-M. Biochemistry 2004, 43, 7491–7502.
J. Org. Chem. Vol. 74, No. 4, 2009 1477

Hussein et al.
NMR (75 MHz, CDCl3) δ 14.0, 22.7, 26.5, 29.7, 31.9, 33.4, 33.7,
34.3, 60.6 (J 25.5 Hz), 67.8 (J 24.9 Hz), 71.8. Anal. calcd for
C25H36F16O2: C, 44.65; H, 5.40. Found: C, 44.64; H, 5.41.
conditions, leading to little expected product and extensive
degradation of the reactants. These alcohols can, however, be
alkylated in good yields using phase-transfer catalysts and
prolonged sonication. The utility of fluorolipid 2 to act as a
template for binding and orientation of His-tagged proteins has
been demonstrated for the membrane protein BmrA.
2-(2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-Hexadecafluoro-10-(3-hexyl-
nonyloxy)decyloxy)acetic Acid (11). This fluorinated acid was
prepared according to the general method described by Elshani.54
A solution of the fluorinated alcohol (10) (336 mg, 0.50 mmol) in
dry THF (2.5 mL) was added dropwise over a period of 30 min to
a mixture of NaH (60% dispersion in oil, 120 mg, 3.00 mmol) in
dry THF (2.5 mL) under argon. The mixture was stirred for 1 h at
room temperature, and a solution of bromoacetic acid (138 mg,
1.00 mmol) in dry THF (2.5 mL) was added dropwise over a period
of 1 h. The mixture was stirred for 24 h, and then the excess NaH
was destroyed by dropwise addition of water (5 mL). The THF
was evaporated in vacuo, and 6 M HCl (20 mL) was added to the
remaining mixture prior to extraction with CH2Cl2 (2 × 100 mL).
The organic layer was washed with water (50 mL), dried over
MgSO4, filtered, and evaporated in vacuo. The crude product was
purified by silica flash column chromatography (a gradient elution
of 10-70% EtOAc in LP) to afford the fluorinated acid (11) as a
pale yellow oil (250 mg, 69%) Rf: 0.30 (70% EtOAc in LP, KMnO4
dip). ESI-MS, m/z: 753 [M + Na]+. HRMS calculated for
Experimental Section
10-(Benzyloxy)-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluoro-
decan-1-ol (8) and 1,10-Bis(benzyloxy)-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-
hexadecafluorodecane (9). Method A. Sodium hydride (60%
dispersion in oil, 1.13 g, 28.2 mmol) was added portionwise to a
solution of the fluorinated diol (7) (10.0 g, 21.8 mmol) in DMF
(100 mL). The reaction mixture was sonicated under argon for 10
min. Benzyl bromide (4.80 g, 28.2 mmol) and TBAI (4.00 g, 10.8
mmol) were added, and the mixture was sonicated for 3 h. The
solvent was evaporated in vacuo, and the residue was taken up in
5% HCl (100 mL). This solution was extracted with EtOAc (3 ×
100 mL). The combined organic extracts were washed with 0.2 M
Na2S2O3 (2 × 50 mL) and brine (50 mL), dried over MgSO4,
filtered, and evaporated in vacuo to afford a yellow oil. The crude
product was purified by silica flash column chromatography (a
gradient elution of 10-20% EtOAc in LP) to afford the monobenzyl
fluorinated alcohol42 (8) as a white solid (4.86 g, 41%), mp 48 °C.
Rf: 0.37 (30% EtOAc in LP, KMnO4 dip). ESI-MS, m/z: 551 [M -
H]-. HRMS calculated for C17H11F16O2- 551.0504, found 551.0518.
HRMS calculated for C17H12F16NaO2+ 575.0480, found 575.0482.
1H NMR (300 MHz, CDCl3) δ 2.52 (1H, br s), 3.93 (2H, t, J 13.9
Hz), 4.04 (2H, t, J 14.0 Hz), 4.67 (2H, s), 7.29 - 7.41 (5H, m).
13C NMR (75 MHz, CDCl3) δ 60.5 (t, J 25.5 Hz), 66.7 (t, J 25.5
Hz), 74.5, 127.9, 128.3, 128.6, 136.4. Anal. calcd for C17H12F16O2:
C, 36.97; H, 2.19. Found: C, 36.87; H, 2.14.
+
1
C27H38F16NaO4 753.2412, found 753.2437. H NMR (300 MHz,
CDCl3) δ 0.86 (6H, t, J 6.9 Hz), 1.20-1.30 (20H, m), 1.36-1.41
(1H, m), 1.52 (2H, q, J 6.8 Hz), 3.60 (2H, t, J 6.9 Hz), 3.90 (2H,
t, J 13.9 Hz), 4.11 (2H, t, J 13.7 Hz), 4.30 (2H, s). 13C NMR (75
MHz, CDCl3) δ 14.1, 22.7, 26.5, 29.7, 31.9, 33.4, 33.6, 34.3, 68.7,
71.7, 172.4.
12,12,13,13,14,14,15,15,16,16,17,17,18,18,19,19,27,27,28,28,29,
29,30,30,31,31,32,32, 33,33,34,34-Dotriacontafluoro-7,39-dihexyl-
10,21,25,36-tetraoxapentatetracontan-23-ol (18). A mixture of the
fluorinated alcohol (10) (482 mg, 0.72 mmol), TBABr (11.60 mg,
0.04 mmol), and powdered NaOH (31.60 mg, 0.79 mmol) was
stirred for 15 min at 30 °C. Epichlorohydrin (16.59 mg, 14 µL,
0.18 mmol) was added to the reaction mixture. The mixture was
stirred for 14 h at 30 °C and then sonicated for another 7 h.
Saturated ammonium chloride (50 mL) was added, and the mixture
was extracted with Et2O (3 × 50 mL). The combined organic
extracts were dried over Na2SO4, filtered, and evaporated in vacuo
to afford a colorless oil. The crude product was purified by silica
flash column chromatography (a gradient elution of 5-10% EtOAc
in LP) to afford the diglyceride (18) as a colorless oil (196 mg,
78%), Rf: 0.10 (10% EtOAc in LP, KMnO4 dip). ESI-MS, m/z:
1423 [M + Na]+. HRMS calculated for C53H76F32NaO5+ 1423.508,
found 1423.510. 1H NMR (300 MHz, CDCl3) δ 0.86 (12H, t, J 6.8
Hz), 1.20-1.30 (40H, m), 1.38-1.43 (2H, m), 1.53 (4H, q, J 6.6
Hz), 2.30-2.35 (1H, br s), 3.60 (4H, t, J 6.9 Hz), 3.67-3.74 (4H,
m), 3.91 (4H, t, J 9.3 Hz), 3.94-4.04 (5H, m). 13C NMR (75 MHz,
CDCl3) δ 14.1, 22.7, 26.5, 29.7, 31.9, 33.4, 33.6, 34.3, 67.8, 68.4,
69.3, 71.7, 73.2.
2-(12,12,13,13,14,14,15,15,16,16,17,17,18,18,19,19,27,27,28,28,
29,29,30,30,31,31,32,32,33,33,34,34-Dotriacontafluoro-7,39-dihexyl-
10,21,25,36-tetraoxapentatetracontan-23-yloxy)acetic Acid (19).
This fluorinated diglyceride acid was prepared according to the
general method described by Elshani.54 A solution of fluorinated
diglyceride alcohol (18) (1.41 g, 1.00 mmol) in dry THF (5 mL)
was added dropwise over a period of 15 min to a mixture of NaH
(60% dispersion in oil, 240 mg, 6.00 mmol) in dry THF (5 mL)
under argon at 0 °C. The mixture was stirred for 30 min at 0 °C,
and a solution of bromoacetic acid (280 mg, 2.00 mmol) in dry
THF (5 mL) was added dropwise over a period of 10 min. The
mixture was stirred for 24 h at room temperature, and the excess
NaH was destroyed by dropwise addition of water (5 mL). The
THF was evaporated in vacuo, and to the remaining mixture 6 M
HCl (40 mL) was added. The mixture was extracted with CH2Cl2
(3 × 100 mL), dried over Na2SO4, filtered, and evaporated in vacuo.
The fluorinated dibenzyl ether41 (9) also was obtained as a
colorless solid (2.17 g, 16%), mp 30-32 °C. Rf: 0.81 (30% EtOAc
in LP, KMnO4 dip). ESI-MS, m/z: 665 [M + Na]+. HRMS
calculated for C24H18F16NaO2+ 665.0944, found 665.0956. 1H NMR
(300 MHz, CDCl3) δ 3.93 (4H, t, J 9.2 Hz), 4.67 (4H, s), 7.28-7.40
(10H, m). 13C NMR (75 MHz, CDCl3) δ 66.7 (t, J 25.6 Hz), 74.4,
127.8, 128.3, 128.6, 136.4. Anal. calcd for C24H18F16O2: C, 44.87;
H, 2.82. Found: C, 44.53; H, 2.81.
Method B. A mixture of the fluorinated dibenzyl ether (9) (3.44
g, 5.35 mmol) and 5% Pd/C (1.00 g) in THF (50 mL) was stirred
under an atmosphere of hydrogen for 2 days at room temperature.
The reaction mixture was filtered, and the filtrate was evaporated
in vacuo to afford a colorless oil. The crude product was purified
by silica flash column chromatography (10% EtOAc in LP) to afford
the monobenzyl fluorinated alcohol (8) as a white solid (0.47 g,
16%) with spectra in agreement with those described above.
2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-Hexadecafluoro-10-(3-hexylnon-
yloxy)decan-1-ol (10). Sodium hydride (60% dispersion in oil, 346
mg, 8.66 mmol) was added to a solution of the fluorinated diol (7)
(4.00 g, 8.66 mmol) in DMF (30 mL). The mixture was sonicated
for 10 min. The alkyl iodide (6) (1.47 g, 4.33 mmol) and TBAI
(80.0 mg, 0.22 mmol) were added, and the mixture was sonicated
under argon for 2 h. The solvent was evaporated in vacuo, and the
residue was taken up with 5% HCl (100 mL). The solution was
extracted with EtOAc (3 × 100 mL). The combined organic extracts
were washed with 0.2 M Na2S2O3 (2 × 50 mL) and brine (50 mL),
dried over Na2SO4, filtered, and evaporated in vacuo to afford a
colorless oil. The crude product was purified by silica flash column
chromatography (a gradient elution of 0 - 30% EtOAc in LP) to
afford the fluorinated alcohol (10) as a colorless oil (1.50 g, 51%),
Rf: 0.22 (10% EtOAc in LP, KMnO4 dip). ESI-MS, m/z: 671 [M -
1
H]-, 707 [M + 35Cl]-, 709 [M + 37Cl]-. H NMR (300 MHz,
CDCl3) δ 0.86 (6H, t, J 6.9 Hz), 1.20-1.28 (20H, m), 1.39-1.44
(1H, m), 1.53 (2H, q, J 6.9 Hz), 1.95-2.00 (1H, br s), 3.59 (2H,
t, J 6.9 Hz), 3.89 (2H, t, J 14.0 Hz), 4.08 (2H, t, J 13.9 Hz). 13C
(54) Elshani, S.; Kobzar, E.; Bartsch, R. A. Tetrahedron 2000, 56, 3291–
3301.
1478 J. Org. Chem. Vol. 74, No. 4, 2009

Protein Monolayer Crystallizations
The crude product was purified by silica flash column chromatog-
raphy (5-30% EtOAc in LP) to afford the fluorinated diglyceride
acid (19) as a colorless oil (1.39 g, 95%), Rf: 0.53 (70% EtOAc in
LP, KMnO4 dip). ESI-MS, m/z: 1481 [M + Na]+. HRMS calculated
for C55H78F32NaO7 1481.5134, found 1481.5173. H NMR (300
MHz, CDCl3) δ 0.86 (12H, t, J 6.9 Hz), 1.20-1.28 (40H, m),
1.35-1.41 (2H, m), 1.53 (4H, q, J 6.8 Hz), 3.58 (4H, t, J 6.9 Hz),
3.75-4.03 (13H, m), 4.29 (2H, s). 13C NMR (75 MHz, CDCl3) δ
14.0, 22.7, 26.5, 29.7, 33.4, 33.7, 34.4, 67.9, 68.1, 68.5, 71.8, 72.5,
78.9, 173.8. Anal. calcd for C55H78F32O7: C, 45.27; H, 5.39. Found:
C, 45.08; H, 5.46.
Blan0420) for financial support and Aurelie di Cicco for
technical help for the 2-D crystallization of BmrA. This work
was supported by an Australian Research Council Project Grant
(DP0556547).
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1
Supporting Information Available: Experimental proce-
dures for compounds 1, 2, 4-6, 13-17, 20-22, and 24-27.
Alternate experimental procedures for compounds 10, 18 and
19. 1H and 13C NMR spectra of compounds 4-6, 8-11, 16-19,
25-27. Detailed description of formation of 2-D crystals of
BmrA using 2 and 18. This material is available free of charge
via the Internet at http://pubs.acs.org.
Acknowledgment. We thank Dr. Tri Lee for assistance with
NMR acquisition, Mr. Graham MacFarlane for accurate mass
measurements, and Mr. George Blazak for elemental analyses.
D.L. thanks The Agence Nationale de la Recherche (ANR-06-
JO802651P
J. Org. Chem. Vol. 74, No. 4, 2009 1479