Synthesis of optically pure diglycerol tetraether model lipids with non-natural branching pattern

Article abstract of DOI:10.1002/ejoc.201100758

Three new, chain-modified, optically pure diglycerol tetraether lipids with one membrane-spanning chain have been synthesised. These lipids contain a different number and constitution of the methyl branches connected to the hydrophobic chains as compared with natural archaeal or other previously synthesised lipids. The correct chirality of the branched alkyl chain was introduced starting from commercially available (S)-citronellyl bromide. For chain elongation the Cu-catalysed Grignard coupling reaction was used. Suitable blocked glycerol ethers were condensed to the tetraether moieties by Grubbs metathesis. The insertion of two or four optically pure methyl branches at the 10- and/or 23-positions of the alkyl chains are sufficient to mimic the main properties of natural tetraether lipids. In this context, it has been shown that these lipids can form closed lipid vesicles.

Full text of DOI:10.1002/ejoc.201100758

FULL PAPER
DOI: 10.1002/ejoc.201100758
Synthesis of Optically Pure Diglycerol Tetraether Model Lipids with
Non-Natural Branching Pattern
Thomas Markowski,[a] Simon Drescher,[b] Annette Meister,[c] Gerd Hause,[d] Alfred Blume,[b]
and Bodo Dobner*[a]
Keywords: Lipids / Synthetic methods / Copper / Grignard reaction / Metathesis
Three new, chain-modified, optically pure diglycerol tetra-
ether lipids with one membrane-spanning chain have been
synthesised. These lipids contain a different number and
the Cu-catalysed Grignard coupling reaction was used. Suit-
able blocked glycerol ethers were condensed to the tetra-
ether moieties by Grubbs metathesis. The insertion of two
constitution of the methyl branches connected to the hydro- or four optically pure methyl branches at the 10- and/or 23-
phobic chains as compared with natural archaeal or other
previously synthesised lipids. The correct chirality of the
branched alkyl chain was introduced starting from commer-
cially available (S)-citronellyl bromide. For chain elongation
positions of the alkyl chains are sufficient to mimic the main
properties of natural tetraether lipids. In this context, it has
been shown that these lipids can form closed lipid vesicles.
tists interested in this field have focused their synthetic work
on simpler model compounds that should mimic the prop-
erties of the natural archaebacterial lipids to a maximal ex-
tent.
Introduction
Diglycerol tetraether lipids, which are found in the cell
membranes of methanogenic and thermoacidophilic ar-
chaebacteria, differ considerably in their chemical structure
from common known lipids. They are composed of two
glycerol-containing hydrophilic head-groups connected to
one or two lipophilic, membrane-spanning alkyl chains
with several methyl branches in an isoprenoid arrangement
as well as a various number of cyclopentane rings. Further-
more, the glycerol-containing head-groups are connected to
the alkyl chains by ether linkages in an sn-2,3-stereochemis-
try and are arranged on both sides of the cell membrane.[1]
The resulting chemical, thermal and enzymatic stability as
well as the tendency to form closed lipid vesicles make these
tetraether lipids interesting for biotechnology, material sci-
ence and pharmacy.[2] However, the isolation of archaebac-
terial lipids is laborious resulting generally in mixtures of
tetraether lipids with different alkyl chain lengths. On the
other hand, their total synthesis has enabled the prepara-
tion of tailor-made lipids.[3] Nevertheless, the assembly of
branched isoprenoid alkyl chains and the macrocyclisation
is a very time-consuming and expensive task yielding only
small amounts of these lipids. Therefore most of the scien-
Yamauchi et al.[4] and also others have prepared an un-
branched model compound with only one membrane-span-
ning chain to avoid the challenge of macrocyclisation.
When the C32 chain had no methyl branches, however, the
compound[4] did not form closed liposomes, but flat ex-
tended sheets, and showed a high transition temperature
(Tm) of 61 °C, which is not observed for archaebacterial
lipids. The insertion of isoprenoid phytanyl residues into
C16 chains led to a decrease in the value of Tm and allowed
the preparation of lipid vesicles.[5] In the following years
other researchers described the use of phytanyl[6] or (R)-
citronellyl residues for even shorter chains.[7] Benvegnu and
co-workers[6c] introduced a cyclopentane ring into the mid-
dle of the membrane-spanning chain to enhance the sta-
bility. In addition, non-symmetric compounds with dif-
ferent head-groups were prepared by using the same struc-
tural concept.[8] However, all model compounds with
branched alkyl chains copied the isoprenoid pattern. In ad-
dition to our investigations on the synthesis of single-chain,
bipolar phospholipids,[9] we were also interested in the syn-
thesis of model compounds with a closer resemblance to
the natural archaebacterial tetraether lipids. Our aim was to
find out whether the isoprenoid arrangement of the methyl
groups in the alkyl chains is necessary to form compounds
with similar behaviour to the archaebacterial lipids or
whether the insertion of only a few methyl branches at defi-
nite positions of the alkyl chains is sufficient to achieve the
desired properties. Therefore we designed three different op-
[a] Institute of Pharmacy, Martin-Luther-University,
Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany
Fax: +49-345-55-27018
E-mail: bodo.dobner@pharmazie.uni-halle.de
[b] Institute of Chemistry, Martin-Luther-University,
von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany
[c] ZIK HALOmem, Martin-Luther-University,
Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany
[d] Biocenter, Martin-Luther-University,
Weinbergweg 22, 06120 Halle (Saale), Germany
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Synthesis of Optically Pure Diglycerol Tetraether Model Lipids
Figure 1. Structures of the synthesised archaebacterial model lipids I–III.
tically pure diglycerol tetraether lipids I–III with two or chromatography in 87% yield, in line with a similar ozon-
four methyl groups at certain positions in the alkyl chains isation product prepared by Kakinuma and co-workers.[3a]
(Figure 1).
We also investigated the oxidative work-up of the ozonides:
As reference compounds we also synthesised bis(phos- instead of sodium borohydride, formic acid and hydrogen
phocholines) consisting of a diglycerol tetraether backbone peroxide were added to the ozonide solution. After heating
with one transmembrane dotriacontanyl and two shorter the solution at reflux for 2 h, the crude carboxylic acid was
hexadecyl chains. The lyotropic behaviour of comparable isolated, dissolved in methanol and heated again with cata-
compounds has been reported previously.[4,5]
lytic amounts of sulfuric acid to yield the corresponding
The methyl branches with R configuration were located methyl ester 3. But in this one-pot procedure, the amount
at the 10-positions of the monopolar hexadecyl chains (I), of ester 3 isolated varied from 62% (chromatography) to
at the 10,23-positions of the transmembrane chain (II) or 77% (distillation), depending on the purification process.
in all of these positions (III; see Figure 1). We present Because the subsequent reduction of ester 3 to the bromo
herein the synthesis of the new, optically pure model lipids alcohol 2 gave only 70% yield, this oxidative synthetic strat-
as well as the first investigations of the lyotropic behaviour egy does not represent an alternative pathway to the re-
of these compounds by differential scanning calorimetry ductive route using sodium borohydride (Scheme 1).
(DSC) and electron microscopy (EM).
Results and Discussion
Synthetic Methods
Within our synthetic strategy, the correct R configuration
of the methyl branching, which is also shown by natural
archaeal membrane lipids, was obtained from a compound
of the chiral pool. Kakinuma and co-workers constructed
the isoprenoid-branched alkyl chains of the archaeal 36-
membered macrocyclic diether lipid[3a] and also of the ar-
chaeal 72-membered tetraether lipid[3b] by using (R)-3-hy-
droxy-2-methylpropionate or (R)-citronellol as the starting
material. After a multistep procedure, the authors isolated
in both cases a selectively blocked octane-1,8-diol with two
methyl branches in the correct stereoconfiguration, which
were subsequently elongated to yield isoprenoid-branched
alkyl chains. The synthesis starting from (R)-citronellol led
to the blocking of the hydroxy group followed by ozon-
isation and reduction to the corresponding alcohol.
In accord with our final compounds I–III and C–C
elongation strategy, respectively, the commercially available
Scheme 1. Synthesis of optically pure methyl-branched alkyl brom-
(S)-citronellyl bromide (1), already containing the bromo ides 8a,b (PPTS: pyridinium p-toluenesulfonate).
atom for the intended Grignard coupling, emerged as the
starting material of choice. Thus, the bromide 1 was allowed
In the next step, the alcohol moiety of the bromo alcohol
to completely react with ozone at –78 °C in methanol. Af- 2 was protected by 3,4-dihydro-2H-pyran and catalytic
terwards, sodium borohydride was added portionwise over amounts of pyridinium p-toluenesulfonate (PPTS) in a
a period of 30 min accompanied by vigorous stirring. Dur- nearly quantitative reaction. The resulting 2-{[(4S)-6-
ing this step the temperature increased to –30 °C. In con- bromo-4-methylhexyl]oxy}tetrahydro-2H-pyran (4) was the
trast, the addition of all the sodium borohydride at –78 °C optically pure starting compound for the chain elongation
followed by slow warming[3a] led to an extreme exothermic reaction. The frequently discussed problems associated with
reaction with a temperature increase up to 50 °C leading to the formation of diastereomers with chiral centres and the
the formation of byproducts. The resulting (4S)-6-bromo-4- tetrahydropyran (THP) ring did not play a role in the sepa-
methylhexan-1-ol (2) was isolated after purification by ration in our case because of the considerable distance be-
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B. Dobner et al.
FULL PAPER
tween the two chiral centres. Only in the 13C NMR spectra bromoundec-1-ene with 2-[(6-bromohexyl)oxy]tetrahydro-
could we find two signals for relevant atoms near the op- 2H-pyran (9) followed by transformation into the bromide
tical centres indicating the formation of two diastereomers. as described above (Scheme 2).
Then compound 4 was coupled with pent-4-enylmagnes-
ium bromide or butylmagnesium bromide in a Grignard re-
action under catalysis with dilithium tetrachlorocuprate-
(II)[10] resulting in the THP-protected alcohols 5a and 5b,
respectively (see Scheme 1). The insertion of the unsatu-
rated residue is necessary for the final coupling (metathesis
reaction) to the tetraether moieties whereas the saturated
butylmagnesium bromide yielded the branched hexadecyl
chain.
Scheme 2. Synthesis of 17-bromoheptydec-1-ene (8c).
In addition to acting as a protecting group, the THP resi-
due can be readily substituted in a high-yielding step to
Our synthetic strategy was further pursued by connecting
form bromides, necessary for further Grignard coupling re- the hydrophobic, functionalized alkyl chains to appropri-
actions. According to the procedure described by Schwarz ately blocked glycerol derivatives. According to the sn-2,3-
et al.,[11] compounds 5 were nearly quantitatively trans- stereochemistry of natural archaebacterial lipids of the glyc-
formed into (8R)-11-bromo-8-methylundec-1-ene (6a) and erol backbone, the synthesis started with the commercially
(4R)-1-bromo-4-methyldecane (6b). In a second Grignard available (S)-1,2-O-isopropylideneglycerol (10; Scheme 3).
cross-coupling reaction of 6a,b with 6-[(tetrahydro-2H-pyr- In a first alkylation step, the terminal unsaturated bromides
an-2-yl)oxy]hexyl bromide (9), the THP ethers 7a and 7b, 8a and 8c were inserted after deprotonation of the alcohol
with the alkyl chain lengths required for the precursors, component 10 by using potassium hydride leading to 1,2-
were obtained. A second bromination[11] yielded (8S)-17- O-isopropylidene-3-O-(heptadec-16-en-1-yl)-sn-glycerol (11a)
bromo-8-methylheptadec-1-ene (8a) and (10R)-1-bromo-10- and 1,2-O-isopropylidene-3-O-[(10S)-10-methylheptadec-
methylhexadecane (8b), respectively, in an optically pure 16-en-1-yl]-sn-glycerol (11b), respectively. Performing the
form and were used for the subsequent alkylation of the deprotonation with potassium hydride instead of sodium
appropriately blocked glycerol derivative. The above-men- hydride required less time and gave higher yields. Sub-
tioned strategy has the advantage that, in addition to the sequently, the isopropylidene blocking moiety of com-
methyl substitution pattern described herein, nearly all the pounds 11a,b was cleaved by using PPTS in methanol.
desired positions of the methyl moiety are feasible by varia- Thereafter, the primary hydroxy function of the resulting
tion of the Grignard reagent.
glycerol derivatives 12a,b was selectively blocked with trityl
17-Bromoheptadec-1-ene (8c), necessary for the metathe- chloride in high yields (83–97%; see Scheme 3). Although
sis reaction leading to the unbranched transmembrane alkyl other techniques are available to build up the glycerol back-
chain of bolalipid I, was synthesised by the reaction of 11- bone starting from optically pure glycide ether,[12] the
Scheme 3. Synthesis of the optically pure tetraether model lipids I–III (PPTS: pyridinium p-toluenesulfonate; TEA: triethylamine).
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Synthesis of Optically Pure Diglycerol Tetraether Model Lipids
method described herein using (S)-1,2-O-isopropylidene- branches. Compared with the lipid with no methyl groups,
glycerol was most appropriate for us.
the incorporation of two methyl groups into the shorter
The second alkylations of 3-O-(heptadec-16-en-1-yl)-1- alkyl chains (lipid I) lowered the transition temperature
O-trityl-sn-glycerol (13a) and 3-O-[(10S)-10-methylhepta- from 61 °C for the unbranched tetraether lipid[4] to 17 °C
dec-16-en-1-yl]-1-O-trityl-sn-glycerol (13b), respectively, for lipid I. When the methyl branches were located in the
were carried out under nearly the same conditions as de- membrane-spanning chain (lipid II) the transition tempera-
scribed above, although we had to heat the suspension of ture decreased even further to 9 °C, which is nearly the
the glycerol derivatives 13a,b with potassium hydride to same value as found for model systems with two phytanyl
100 °C to attain a quantitative formation of the potassium residues.[5,6a] For lipid III with four methyl branches in all
salts. However, the yields of the 2,3-O,O-dialkyl-1-O-trityl- the 10- and 23-positions of the alkyl chains, no phase tran-
sn-glycerols 14a–c were in the range of 55–68%, which has sition above 2 °C could be detected. This behaviour is sim-
been attributed to steric hindrance.
ilar to that found for natural archaebacterial lipids.[17] In
In the final C–C bond formation reaction, the olefin me- conclusion, not only the number but also the positions of
tathesis with Grubbs first-generation catalyst[13] followed by the methyl branches within the alkyl chains are important
hydrogenation using palladium hydroxide on carbon, en- for the aggregation and transition behaviour of the tetra-
abling the detritylation as well as the hydrogenation of the ether lipids. It seems that the van der Waals contacts of the
double bonds in one step, provided the 3,3Ј-O-(alkane-1,1Ј- longest unbranched segments of the alkyl chains determine
diyl)-bis(2-O-alkyl-sn-glycerols) 15a–c. In the last step, the the values of the transition temperature and transition en-
methyl-branched bis(phosphocholines) I–III were synthe- thalpy. Similar results have been found before for mono-
sised by the method of Eibl et al.,[14] that is, by esterification polar methyl-branched phospholipids.[18]
of the primary alcohol moiety of the glycerol using the clas-
The ability of these new lipids to form closed lipid vesi-
sic 2-bromoethylphosphoric acid dichloride.[15] Attempts to cles was studied by freeze-fracture replica electron micro-
use 2-chloro-1,3,2-dioxophospholane as the reactive phos- scopy (EM). The samples were prepared by the technique
phorylation reagent for the insertion of the phosphor ester described by Bangham et al.[19] After treatment with ultra-
moiety led to lower yields, in line with previous results.[16] sound the liposomes were extruded through 200 nm poly-
The following quarternisation with trimethylamine in a
mixture of chloroform, acetonitrile and ethanol provided
the diglycerol tetraether lipids I–III in yields of 33–44%
with respect to the glycerols 15a–c.
Physicochemical Characterisation
The lyotropic phase behaviour of the novel, archaebac-
terial diglycerol tetraether phospholipid analogues I–III
were characterized by differential scanning calorimetry
(DSC). The final products were therefore suspended in
aqueous solution (c = 1 mgmL–1) and investigated in the
temperature range between 2 and 95 °C. The DSC thermo-
grams (Figure 2) of the lipids I–III show different peaks
depending on the positions and the number of methyl
Figure 3. Electron microscopic images of freeze-fracture replicas of
Figure 2. DSC heating curves of aqueous suspensions of lipids I–
III (c = 1 mgmL–1, heating rate = 20 Kh–1). The curves are shifted
horizontally for clarity.
liposomes composed of lipids I–III (c = 15–20 mgmL–1) prepared
in water by using the film method of Bangham et al.[19] (A,B) Lipid
I, (C,D) lipid II and (E,F) lipid III.
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B. Dobner et al.
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Co. and used without further purification. 2-Bromoethylphosphoric
acid dichloride was prepared according to the literature.[14] All
solvents were dried and distilled before use. The purity of all com-
pounds was checked by thin-layer chromatography (TLC) using sil-
ica gel 60 F254 plates (Merck). The chromatograms were developed
by using Bromothymol Blue. Silica gel (Merck, 0.063–0.200 mm)
was used for the column chromatography of all products. Melting
points were measured with a Boetius apparatus. Optical rotations
were determined with a Polartronic E (Schmidt und Haensch) in-
carbonate membranes. The resulting suspension was rapidly
quenched from room temperature and freeze-fractured at
–150 °C without etching. The surfaces were shadowed with
platinum and subsequently with carbon to stabilize the ul-
tra-thin metal films. Figure 3 shows the EM images, which
indicate that all three tetraether lipids are capable of form-
ing mostly unilamellar liposomes with diameters ranging
from 80 to 200 nm. Major differences in liposome forma-
tion behaviour between the lipids I–III could not be de-
strument and [α]D values are given in 10–1 degcm2 g–1. Elemental
1
tected. In some cases, however, we also found bi- and multi- analyses were carried out with a Leco CHNS-932 instrument. H
and 13C NMR spectra were recorded with a Varian Gemini 2000
lamellar liposomes (see arrows in part F of Figure 3), but
spectrometer at 400 and 100 MHz, respectively, using CDCl3 or
this is normal as the extrusion procedure does not produce
CD3OD as internal standard. Mass spectrometric data were ob-
solely unilamellar vesicles, as known from other phospho-
tained with a Finnigan MAT SSQ 710 C (ESI-MS) or AMD 402
lipid systems. The fracture surface showed no distinct dif-
(70 eV) spectrometer (EI-MS). High-resolution mass spectra
ference between the inner and outer side of the liposomes.
(HRMS) were recorded with a Thermo Fisher Scientific LTQ-Or-
Therefore one can conclude that cross-fracturing of the
bitrap mass spectrometer with static nano-electrospray ionisation.
membranes occurred as no inner fracture faces could be
Analytical HPLC (Jasco) was performed with a Kromasil column
observed as they appear when bilayer membranes are frac-
tured. In the case of oligolamellar vesicles the fracture
(Si 100–5 μm, 250ϫ 4.6 mm), PU 980 Intelligent HPLC Pump and
an LG-1580-02 Ternary Gradient Unit (Jasco) with an SEDEX 55
seems to run along the outer surface of the inner membrane ELS detector (SEDERE, France) using the following solvents for
elution: 5 min isocratic CHCl3/MeOH/water (45:45:10), 5 min con-
tinuous increase to CHCl3/MeOH/water (42:42:16), 10 min iso-
cratic CHCl3/MeOH/water (45:45:10); flow = 1 mLmin–1.
before cross-fracturing occurs. Based on this observation we
conclude that all three lipids are arranged in a membrane-
spanning fashion and not in a U-shaped form with a bilayer
arrangement, which would lead to a higher energy due to (4S)-6-Bromo-4-methylhexan-1-ol (2): A solution of (S)-citronellyl
bromide (1; 10.96 g, 50 mmol) in methanol (30 mL) was cooled to
–78 °C. At this temperature, a stream of ozone was passed through
the solution for 10–11 h. After compound 1 had disappeared
(TLC), the ozone was replaced by a stream of air for 30 min and
the temperature was maintained at –78 °C. Afterwards, methanol
(6 mL) and sodium borohydride (2.09 g, 0.056 mol) were added in
portions and the solution was brought to –65 °C. A further portion
of sodium borohydride (1.0 g, 27 mmol) was added within 15 min
and the mixture was raised very slowly to room temperature. The
solvent was evaporated and the residue was dissolved in methanol
(40 mL). Sodium borohydride (0.75 g, 20 mmol) was added and the
mixture was stirred overnight. Thereafter, water (50 mL) was added
to the mixture, which was then acidified with hydrochloric acid,
saturated with ammonium chloride and extracted with diethyl ether
(2ϫ 50 mL). The ethereal solution was washed with water (50 mL),
potassium hydroxide solution (50 mL, 5%), water (50 mL) and
dried with sodium sulfate. After evaporation of the solvent the resi-
due was purified by column chromatography; yield 8.52 g (87.3%).
[α]3D2 = +7.40 (c = 1.16 gmL–1, pure). 1H NMR (400 MHz, CDCl3):
bending of the alkyl chain. The thickness of this lipid layer
was determined to be 5–8 nm, which roughly corresponds
to the length of one lipid molecule. Further investigations,
including by X-ray diffraction, exploring the exact arrange-
ment of the lipids are currently under way.
Conclusions
We have developed a new synthetic pathway for the prep-
aration of diglycerol tetraether model lipids that enables the
insertion of methyl branches into different positions and
configurations. The Cu-catalysed Grignard coupling reac-
tion has proved to be a suitable method for alkyl chain
elongation. The coupling of glycerol diethers to form sym-
metric diglycerol tetraethers was successfully realised by
using the Grubbs metathesis reaction. For selective glycerol
alkylation and also for the insertion of the phosphocholine
head-group, established and robust approaches were the
methods of choice. Through the syntheses described herein
we have shown that it is possible to reduce the number of
methyl branches in archaebacterial model lipids without
losing the main properties of the natural lipids. This fact is
of particular interest with regard to a simpler and less ex-
pensive synthesis of such lipids and also in view of the prep-
aration of stable liposomes. With the insertion of methyl
branches into positions other than those described above, a
fine-tuning of the physicochemical parameters seems to be
feasible. This aspect and the preparation of model com-
pounds with different head groups are under investigation.
3
δ = 0.90 (d, JH,H = 6.4 Hz, 3 H, CH3), 1.16–1.26 [m, 1 H, CHH-
CH(CH2)2Br], 1.33–1.42 (m, 2 H, HOCH2CH2), 1.47–1.72 (m, 3
H, CHH-CH-CHH-CH2Br), 1.82–1.91 (m, 1 H, CHH-CH2Br),
3
3.35–3.48 (m, 2 H, CH2Br), 3.62 (t, JH,H = 6.6 Hz, 2 H,
HOCH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 19.04 (CH3),
30.00, 31.58, 32.00, 32.55, 40.02 (CH2CH2Br), 63.29
(HOCH2) ppm. MS (EI): m/z (%) = 176/178 (0.5) [M – H2O]+.
C7H15BrO (195.10): calcd. C 43.09, H 7.75; found C 43.15, H 7.72.
Methyl (4S)-6-Bromo-4-methylhexanoate (3): (S)-Citronellyl bro-
mide (1; 21.9 g, 0.1 mol) was dissolved in dry methanol (100 mL)
and the mixture was cooled to –78 °C. Afterwards, a stream of
ozone gas was passed through the solution and the exothermic re-
action led to a slight increase of temperature. After the reaction
temperature had reached –78 °C again, the stream of ozone was
stopped and the mixture was stirred for a further 1 h at this tem-
perature. After the addition of formic acid (75 mL) at –60 °C and
Experimental Section
General: Apart from palladium hydroxide on carbon powder (20%; hydrogen peroxide (36 mL, 35%) at –40 °C, the mixture was
Acros Organics), all chemicals were purchased from Sigma Aldrich brought to room temperature. After heating the solution for 2 h at
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Synthesis of Optically Pure Diglycerol Tetraether Model Lipids
reflux, dry sodium acetate (10 g) was added at room temperature
and the mixture was extracted with diethyl ether (2ϫ 100 mL). The
combined ethereal extracts were dried with sodium sulfate, filtered,
evaporated to dryness and the residue was dissolved in dry meth-
anol (80 mL). After addition of concentrated sulfuric acid (2 mL)
the solution was heated at reflux for 3 h. After removing the ma-
jority of the methanol under vacuum, water (50 mL) and diethyl
ether (75 mL) were added and the organic layer was separated. The
aqueous layer was extracted with diethyl ether (2ϫ 30 mL) and the
collected ethereal phases were dried and the solvents evaporated.
The residue was distilled or purified by column chromatography;
yield 17.18 g (77% by distillation), 13.83 g (62% by chromatog-
raphy), colourless liquid, b.p. 72 °C/0.3 kPa. [α]2D4 = +4.66 (c =
1.25 gmL–1, pure). 1H NMR (400 MHz, CDCl3): δ = 0.88 (d, 3JH,H
= 6.2 Hz, 3 H, CH3), 1.37–1.44 (m, 1 H, COCH2CH2), 1.56–1.68
(m, 3 H, CHH-CH-CHH-CH2Br), 1.77–1.86 (m, 1 H, CHH-
CH2Br), 2.20–2.34 (m, 2 H, COCH2), 3.31–3.43 (m, 2 H, CH2Br),
3.61 (s, 3 H, H3CO) ppm. MS (EI): m/z (%) = 223/225 (0.3) [M]+.
C8H15BrO2 (223.10): calcd. C 43.07, H 6.78; found C 42.94, H 7.02.
The data are in agreement with published values.[20]
prepared dilithium tetrachlorocuprate solution (10 mL, 0.1 m). The
mixture was stirred for 2–3 h at –5 to 0 °C. Afterwards, the mixture
was poured into an ice-cold saturated solution of ammonium chlo-
ride. The organic layer was separated and the aqueous phase was
extracted with diethyl ether (2ϫ 50 mL). The combined ethereal
phases were washed with water and brine, dried with sodium sulfate
and the solvents evaporated. The oily residues were purified by col-
umn chromatography.
2-{[(4R)-4-Methylundec-10-en-1-yl]oxy}tetrahydro-2H-pyran (5a):
Yield 5.2 g (77%), colourless liquid. [α]2D2 = +0.27 (c = 0.95 gmL–1,
1
3
pure). H NMR (400 MHz, CDCl3): δ = 0.85 (d, JH,H = 6.4 Hz,
3 H, CH3), 1.06–1.40 [m, 11 H, CH2CH(CH2)4], 1.48–1.62 (m, 6
H, 2 CH2CH2O, CH2CH2CHO), 1.67–1.73 (m, 1 H, CHHCHO),
1.77–1.85 (m, 1 H, CHHCHO), 1.99–2.04 (m, 2 H, CH2CH=CH2),
3.32–3.38 (m, 1 H, CHOCHH), 3.45–3.51 (m, 1 H, CHOCHH),
3.66–3.73 (m, 1 H, CHOCHH), 3.83–3.88 (m, 1 H, CHOCHH),
4.55–4.56 (m, 1 H, OCHO), 4.89–4.99 (m, 2 H, CH=CH2), 5.74–
5.84 (m, 1 H, CH=CH2) ppm. 13C NMR (100 MHz, CDCl3): δ =
19.78 and 19.79 (CH3, diast.), 19.87, 25.69, 27.01, 27.43, 27.45,
29.12, 29.59, 30.96, 32.76, 33.09, 33.51, 33.92, 37.02, 37.04, 62.41
[(CH2)3CH2O], 68.09 and 68.12 [C(CH3)(CH2)2CH2OCH, diast.],
98.85 and 98.89 (OCHO, diast.), 114.07 (CH=CH2), 139.14
(CH=CH2) ppm. MS (EI): m/z (%) = 267 (0.9) [M – H]+. C17H32O2
(268.43): calcd. C 76.06, H 12.02; found C 75.79, H 11.74.
(4S)-6-Bromo-4-methylhexan-1-ol (2) (via ester 3): Compound 3
(36.7 g, 0.165 mol), dissolved in dry diethyl ether (50 mL), was
added very slowly whilst stirring to a suspension of lithium alumin-
ium hydride (3.75 g, 0.1 mol) in dry diethyl ether (130 mL) at 0 °C.
The mixture was stirred for a further 4 h at this temperature. The
excess lithium aluminium hydride was destroyed with ice water and
the white precipitate was dissolved with sulfuric acid (10%). After-
wards, the mixture was extracted with diethyl ether (3ϫ 60 mL).
The combined ethereal phases were washed with water (100 mL)
and brine (100 mL), and dried with sodium sulfate. After evapora-
tion the crude alcohol 2 was purified by column chromatography
(yield 22.53 g, 70%). The analytical data are in accordance with
the data described above.
2-{[(4R)-4-Methyldecyl]oxy}tetrahydro-2H-pyran (5b): Yield 5.5 g
(86%), colourless liquid. [α]2D2 = +0.09 (c = 0.88 gmL–1, pure). H
1
3
NMR (400 MHz, CDCl3): δ = 0.85 (d, JH,H = 6.4 Hz, 3 H,
CHCH3), 0.87 (t, 3JH,H = 6.8 Hz, 3 H, CH2CH3), 1.08–1.38 [m, 13
H, CH2CH(CH2)5], 1.48–1.61 (m,
6
H,
2
CH2CH2O,
CH2CH2CHO), 1.67–1.72 (m, 1 H, CHH-CHO), 1.80–1.83 (m, 1
H, CHH-CHO), 3.34–3.37 (m, 1 H, CHOCHH), 3.46–3.49 (m, 1
H, CHOCHH), 3.67–3.71 (m, 1 H, CHOCHH), 3.83–3.86 (m, 1 H,
CHOCHH), 4.55–4.57 (m,
1
H, OCHO) ppm. 13C NMR
(100 MHz, CDCl3): δ = 14.23 (CH2CH3), 19.79 and 19.80 (CH3,
diast.), 19.87, 22.81 (CH2CH3), 25.70, 27.15, 27.44, 27.46, 29.78,
30.96, 32.06, 32.78, 33.53, 37.10, 37.12, 62.41 [(CH2)3CH2O], 68.10
and 68.13 [C(CH3)(CH2)2CH2OCH, diast.], 98.84 and 98.89
(OCHO, diast.) ppm. MS (EI): m/z (%) = 255 (1.4) [M – H]+.
C16H32O2 (256.42): calcd. C 74.94, H 12.58; found C 74.67, H
12.80.
2-{[(4S)-6-Bromo-4-methylhexyl]oxy}tetrahydro-2H-pyran
(4):
Compound 2 (7.8 g, 0.04 mol) was dissolved in dichloromethane
(50 mL), 3,4-dihydro-2H-pyran (5.04 g, 60 mmol) and PPTS (0.1 g)
were added and the mixture was stirred for 18 h at room tempera-
ture. Afterwards, the solution was washed with water (50 mL),
dried with sodium sulfate, evaporated and the residue was purified
by column chromatography by using heptane/diethyl ether as elu-
ent; yield 10.50 g (94%), colourless liquid. [α]2D2 = +3.26 (c =
1.17 gmL–1, pure). 1H NMR (400 MHz, CDCl3): δ = 0.89 (d, 3JH,H
= 6.2 Hz, 3 H, CH3), 1.16–1.26 [m, 1 H, CHH-CH(CH2)2Br], 1.32–
1.43 [m, 1 H, CHH-CH(CH2)2Br], 1.47–1.73 [m, 9 H, 2 CH2CH2O,
(CH2)2CHO, CHCH3], 1.77–1.91 (m, 2 H, CH2CH2Br), 3.33–3.50
(m, 4 H, CH2Br, 2 CHOCHH), 3.67–3.73 (m, 1 H, CHOCHH),
3.82–3.87 (m, 1 H, CHOCHH), 4.54–4.56 (m, 1 H, OCHO) ppm.
13C NMR (100 MHz, CDCl3): δ = 19.04 and 19.05 (CH3, diast.),
19.87, 25.67, 27.19, 30.93, 31.65, 31.69, 32.07, 33.01, 33.04, 40.09
and 40.11 (CH2CH2Br, diast.), 62.47 [(CH2)3CH2O], 67.79
[C(CH3)(CH2)2CH2OCH], 98.91 and 98.96 (OCHO, diast.) ppm.
MS (EI): m/z (%) = 277/279 (3.0) [M – H]+. C12H23BrO2 (279.21):
calcd. C 51.62, H 8.30; found C 51.82, H 8.14.
Methyl–Branched Bromoalkanes 6a and 6b: Triphenylphosphane
(7.87 g, 30 mmol) was dissolved in dry dichloromethane (70 mL)
and bromine (2.4 g, 30 mmol), diluted in dichloromethane (10 mL),
was added dropwise into the solution whilst stirring at 0 °C. Com-
pound 5a or 5b (19 mmol) was added and the mixture was stirred
overnight at room temperature. Afterwards, the organic layer was
washed with water (100 mL) and the crude bromides 6a and 6b
were purified by column chromatography with heptane as eluent.
(8R)-11-Bromo-8-methylundec-1-ene (6a): Yield 4.6 g (98%),
colourless liquid. [α]2D2 = –2.06 (c = 0.98 gmL–1, pure). 1H NMR
3
(400 MHz, CDCl3): δ = 0.86 (d, JH,H = 6.6 Hz, 3 H, CH3), 1.07–
1.46 [m, 11 H, CH2CH(CH2)4], 1.77–1.91 (m, 2 H, BrCH2CH2),
2.00–2.05 (m, 2 H, CH2CH=CH2), 3.35–3.39 (m, 2 H, BrCH2),
4.90–5.01 (m, 2 H, CH=CH2), 5.74–5.85 (m, 1 H, CH=CH2) ppm.
13C NMR (100 MHz, CDCl3): δ = 28.34, 28.91, 29.11, 29.29,
29.57–29.78, 33.01, 33.93, 34.05, 114.04 (CH=CH2), 139.19
(CH=CH2) ppm. MS (EI): m/z (%) = 246/248 (0.2) [M]+. C12H23Br
(247.22): calcd. C 58.30, H 9.38; found C 57.96, H 9.21.
2-{[(4R)-4-Methylalk-1-yl]oxy}tetrahydro-2H-pyrans 5a and 5b: 5-
Bromopent-1-ene or 1-bromobutane (50 mmol), respectively, dis-
solved in dry diethyl ether (45 mL), were slowly added to magne-
sium turnings (1.46 g, 60 mmol). The mixture was stirred for 2 h at
reflux. The Grignard solution was decanted from excess magne-
sium under a stream of argon. After removing the diethyl ether in
vacuo the oily residue was diluted in dry THF (50 mL) and cooled
to –5 °C. Afterwards, compound 4 (7.0 g, 25 mmol), dissolved in
dry THF (10 mL), was added in one portion followed by a freshly
(4R)-1-Bromo-4-methyldecane (6b): Yield 4.16 g (93%), colourless
1
liquid. [α]2D2 = –2.29 (c = 1.02 gmL–1, pure). H NMR (400 MHz,
CDCl3): δ = 0.84–0.88 (m, 6 H, 2 CH3), 1.06–1.44 [m, 13 H,
CH2CH(CH2)5], 1.77–1.89 (m, 2 H, BrCH2CH2), 3.35–3.39 (m, 2
Eur. J. Org. Chem. 2011, 5894–5904
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5899

B. Dobner et al.
FULL PAPER
H, BrCH2) ppm. 13C NMR (100 MHz, CDCl3):
δ
=
14.23
(100 MHz, CDCl3): δ = 19.86, 25.70, 26.40, 29.11, 29.29, 29.64–
29.92, 30.96, 33.93, 62.39 [(CH2)3CH2O], 67.77 [(CH2)14CH2OCH],
98.86 (OCHO), 114.03 (CH=CH2), 139.19 (CH=CH2) ppm. MS
(CH2CH3), 19.74 (CHCH3), 22.82 (CH2CH3), 27.09, 29.74, 30.71,
32.04, 32.38, 34.39, 35.66, 37.01 ppm. MS (EI): m/z (%) = 234/236
(0.2) [M]+. C11H23Br (235.20): calcd. C 56.17, H 9.86; found C (EI): m/z (%) = 337 (4.3) [M – H]+. C22H44O2 (338.57): calcd. C
56.38, H 9.96. The analytical data are comparable to the published
78.05, H 12.50; found C 77.98, H 12.67.
values of the 4S enantiomer.[21]
Methyl-Branched Bromoalkanes 8a–c: The bromides 8a–c were pre-
2-[(10-Methylalk-1-yl)oxy]tetrahydro-2H-pyrans 7a and 7b: The pared from triphenylphosphoranediyl dibromide (14.55 g,
Grignard reagent was prepared from magnesium (0.88 g, 36 mmol)
and 2-[(6-bromohexyl)oxy]tetrahydro-2H-pyran (9; 8.0 g, 30 mmol)
in dry THF (30 mL) according to the procedure described for com-
pounds 5a,b. The resulting Grignard solution was coupled with
compound 6a or 6b (15 mmol) under catalytic conditions with di-
lithium tetrachlorocuprate (3 mL, 0.1 m) at 0 °C. After work-up as
described for compounds 5a,b the crude product was purified by
column chromatography.
25.40 mmol) and compounds 7a–c (12.0 mmol) according to the
method described above for compounds 6a,b.
(8S)-17-Bromo-8-methylheptadec-1-ene (8a): Yield 3.61 g (91%),
colourless liquid. [α]2D2 = +0.03 (c = 0.98 gmL–1, pure). H NMR
1
3
(400 MHz, CDCl3): δ = 0.82 (d, JH,H = 6.7 Hz, 3 H, CH3), 1.05–
3
1.42 [m, 23 H, (CH2)7CH(CH2)4], 1.84 (quint., JH,H = 7.0 Hz, 2
3
H, BrCH2CH2), 2.00–2.04 (m, 2 H, CH2CH=CH2), 3.39 (t, JH,H
= 7.0 Hz, 2 H, BrCH2), 4.90–4.99 (m, 2 H, CH=CH2), 5.76–5.84
(m, 1 H, CH=CH2) ppm. 13C NMR (100 MHz, CDCl3): δ = 19.86
(CH3), 27.05, 27.19, 28.34, 28.92, 29.14–30.09, 32.88, 33.01, 33.94,
34.06, 37.15, 37.20, 114.05 (CH=CH2), 139.19 (CH=CH2) ppm.
MS (EI): m/z (%) = 330/332 (1.8) [M]+. C18H35Br (331.37): calcd.
C 65.24, H 10.65; found C 65.41, H 10.80.
2-{[(10S)-10-Methylheptadec-16-en-1-yl]oxy}tetrahydro-2H-pyran
(7a): Yield 4.39 g (83%), colourless liquid. [α]2D2 = +0.08 (c =
0.88 gmL–1, pure). 1H NMR (400 MHz, CDCl3): δ = 0.82 (d, 3JH,H
= 6.2 Hz, 3 H, CH3), 1.01–1.38 [m, 23 H, (CH2)7CH(CH2)4], 1.46–
1.60 (m, 6 H, 2 CH2CH2O, CH2CH2CHO), 1.65–1.73 (m, 1 H,
CHH-CHO), 1.78–1.85 (m, 1 H, CHH-CHO), 1.99–2.04 (m, 2 H,
CH2CH=CH2), 3.33–3.39 (m, 1 H, CHOCHH), 3.45–3.50 (m, 1 H,
CHOCHH), 3.67–3.73 (m, 1 H, CHOCHH), 3.82–3.87 (m, 1 H,
CHOCHH), 4.54–4.56 (m, 1 H, OCHO), 4.89–5.00 (m, 2 H,
(10R)-1-Bromo-10-methylhexadecane (8b): Yield 3.64 g (95%),
colourless liquid. [α]2D2 = –0.19 (c = 0.94 gmL–1, pure). 1H NMR
3
(400 MHz, CDCl3): δ = 0.82 (d, JH,H = 6.6 Hz, 3 H, CHCH3),
0.87 (t, 3JH,H = 7.0 Hz, 3 H, CH2CH3), 1.05–1.42 [m, 25 H, (CH2)7-
CH=CH2), 5.74–5.84 (m,
1
H, CH=CH2) ppm. 13C NMR
3
CH(CH2)5], 1.84 (quint., JH,H = 7.0 Hz, 2 H, BrCH2CH2), 3.37–
(100 MHz, CDCl3): δ = 19.86 (CH3), 25.70, 26.40, 27.05, 27.21,
29.14, 29.63–30.13, 30.96, 32.89, 33.93, 37.16, 37.22, 62.39
[(CH2)3CH2O], 67.77 [C(CH3)(CH2)8CH2OCH], 98.87 (OCHO),
114.03 (CH=CH2), 139.18 (CH=CH2) ppm. MS (EI): m/z (%) =
351 (0.6) [M – H]+. C23H44O2 (352.59): calcd. C 78.35, H 12.58;
found C 78.25, H 12.95.
3.40 (m, 2 H, BrCH2) ppm. 13C NMR (100 MHz, CDCl3): δ =
14.25 (CH2CH3), 19.87 (CHCH3), 22.84 (CH2CH3), 27.19, 28.34,
28.92, 29.58–30.09, 32.09, 33.01, 34.06, 37.22, 37.24 ppm. MS (EI):
m/z (%) = 318/320 (0.6) [M]+. C17H35Br (319.36): calcd. C 63.93,
H 11.05; found C 63.95, H 11.36.
17-Bromoheptadec-1-ene (8c): Yield 3.77 g (99%), colourless liquid.
1H NMR (400 MHz, CDCl3): δ = 1.24–1.43 [m, 24 H, Br(CH2)2-
(CH2)12CH2], 1.84 (quint., 3JH,H = 7.0 Hz, 2 H, BrCH2CH2), 2.00–
2.04 (m, 2 H, CH2CH=CH2), 3.39 (t, 3JH,H = 7.0 Hz, 2 H, BrCH2),
4.90–5.00 (m, 2 H, CH=CH2), 5.76–5.84 (m, 1 H, CH=CH2) ppm.
13C NMR (100 MHz, CDCl3): δ = 19.73, 26.95, 29.09, 29.54, 30.69,
32.35, 33.91, 34.38, 35.63, 36.93, 114.12 (CH=CH2), 139.10
(CH=CH2) ppm. MS (EI): m/z (%) = 316/318 (10) [M]+. C17H33Br
(317.35): calcd. C 64.34, H 10.48; found C 64.55, H 10.67. The data
are in agreement with published values.[22]
2-{[(10R)-10-Methylhexadecyl]oxy}tetrahydro-2H-pyran (7b): Yield
4.04 g (79%), colourless liquid. [α]2D2 = –0.12 (c = 0.84 gmL–1,
1
3
pure). H NMR (400 MHz, CDCl3): δ = 0.82 (d, JH,H = 6.2 Hz,
3
3 H, CHCH3), 0.85–0.88 (t, JH,H = 7.1 Hz, 3 H, CH2CH3), 1.02–
1.35 [m, 25 H, (CH2)7CH(CH2)5], 1.48–1.73 (m, 7 H, 2 CH2CH2O,
CH2-CHH-CHO), 1.77–1.85 (m, 1 H, CHH-CHO), 3.33–3.39 (m,
1 H, CHOCHH), 3.45–3.51 (m, 1 H, CHOCHH), 3.68–3.74 (m, 1
H, CHOCHH), 3.82–3.88 (m, 1 H, CHOCHH), 4.55–4.56 (m, 1 H,
OCHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.24 (CH2CH3),
19.86, 19.87 (CHCH3), 22.84 (CH2CH3), 25.70, 26.40, 27.19, 27.22,
29.64–30.96, 32.09, 32.91, 37.24, 62.39 [(CH2)3CH2O], 67.77
[C(CH3)(CH2)8CH2OCH], 98.86 (OCHO) ppm. MS (EI): m/z (%)
= 339 (4.5) [M – H]+. C22H44O2 (340.58): calcd. C 77.58, H 13.02;
found C 77.45, H 13.02.
3-O-Alkyl-1,2-O-isopropylidene-sn-glycerols 11a and 11b: Potassium
hydride (14.7 mmol, 1.7 mL, 30% suspension in paraffin) was sepa-
rated from the oil and suspended under argon in dry toluene
(5 mL). A solution of 1,2-O-isopropylidene-sn-glycerol (10; 1.94 g,
14.7 mmol) in dry toluene (15 mL) was added slowly at room tem-
perature and the mixture was stirred for 18 h at this temperature.
The alkyl bromides 8a,c (9.8 mmol) were dissolved in dry toluene
(10 mL), added and the mixture was stirred for 10 h at reflux.
Thereafter, water (30 mL) was added at room temperature and the
mixture was stirred vigorously. After phase separation, the organic
layer was washed with water (50 mL), saturated ammonium chlo-
ride solution (50 mL), dried with sodium sulfate, evaporated to dry-
ness and purified by chromatography using heptane/diethyl ether
as eluent and the gradient technique.
2-(Heptadec-16-en-1-yloxy)tetrahydro-2H-pyran (7c): The Grignard
reagent was prepared from magnesium (0.88 g, 36 mmol) and 11-
bromoundec-1-ene (7.0 g, 30 mmol) in dry THF (30 mL) according
the procedure described for compounds 5a,b. A solution of 2-[(6-
bromohexyl)oxy]tetrahydro-2H-pyran (9; 5.3 g, 20 mmol) in dry
THF (10 mL) and a freshly prepared dilithium tetrachlorocuprate
solution (5 mL, 0.1 m) were added and the mixture was stirred for
3 h at temperatures between –5 and 0 °C. After work-up as de-
scribed for compounds 5a,b, the crude product was purified by col-
umn chromatography; yield 3.71 g (73%), colourless liquid. 1H
NMR (400 MHz, CDCl3): δ = 1.24–1.38 [m, 24 H, (CH2)12-
1,2-O-Isopropylidene-3-O-(heptadec-16-en-1-yl)-sn-glycerol (11a):
CH2CH=], 1.48–1.60 (m, 6 H, 2 CH2CH2O, CH2CH2CHO), 1.67– Yield 2.6 g (72%), colourless oil. [α]2D2 = +12.82 (c = 0.86 gmL–1,
1.72 (m, 1 H, CHH-CHO), 1.79–1.84 (m, 1 H, CHH-CHO), 2.00– pure). 1H NMR (400 MHz, CDCl3): δ = 1.23–1.32 [m, 24 H,
2.04 (m, 2 H, CH2CH=CH2), 3.34–3.38 (m, 1 H, CHOCHH), 3.46– (CH2)12], 1.34 (s, 3 H, CH3), 1.40 (s, 3 H, CH3), 1.55 (quint., 3JH,H
3.50 (m, 1 H, CHOCHH), 3.68–3.73 (m, 1 H, CHOCHH), 3.83–
3.87 (m, 1 H, CHOCHH), 4.55–4.56 (m, 1 H, OCHO), 4.89–4.99 3.37–3.51 (m, 4 H, 2 CH2O), 3.68–3.72 [m, 1 H, CHH-OC-
(m, 2 H, CH=CH2); 5.75–5.83 (m, 1 H, CH=CH2) ppm. 13C NMR
(CH3)2O], 4.01–4.05 [m, 1 H, CHH-OC(CH3)2O], 4.23 (quint.,
= 7.0 Hz, 2 H, CH2CH2O), 1.99–2.04 (m, 2 H, CH2CH=CH2),
5900
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 5894–5904

Synthesis of Optically Pure Diglycerol Tetraether Model Lipids
3JH,H = 6.0 Hz, 1 H, CHO), 4.88–4.99 (m, 2 H, CH=CH2), 5.74–
5.84 (m, 1 H, CH=CH2) ppm. 13C NMR (100 MHz, CDCl3): δ =
25.89, 26.22, 26.94, 29.10, 29.30, 29.60–29.80, 33.94, 67.07
was separated, the aqueous layer was extracted with chloroform
(2ϫ 50 mL) and the combined organic phases were washed with
water (50 mL). After drying with sodium sulfate and removing the
[CH2OC(CH3)2O], 71.91 (CH2O), 71.96 (CH2O), 74.85 (OCH), solvent, the residue was purified by column chromatography using
109.32 [OC(CH3)2O], 114.05 (CH=CH2), 139.18 (CH=CH2) ppm.
MS (EI): m/z (%) = 368 (8.6) [M]+. C23H44O3 (368.59): calcd. C
74.95, H 12.03; found C 75.07, H 12.24.
chloroform/diethyl ether and 0.1% triethylamine as eluent.
3-O-(Heptadec-16-en-1-yl)-1-O-trityl-sn-glycerol (13a): Yield 2.21 g
(97%), white waxy substance, m.p. 42–43 °C. [α]2D2 = –1.9 (c =
0.064 gmL–1, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 1.20–1.38
[m, 24 H, (CH2)12], 1.48–1.56 (m, 2 H, CH2CH2O), 2.00–2.05 (m,
2 H, CH2CH=CH2), 2.40 (d, J = 3.6 Hz, 1 H, OH), 3.15–3.22 [m,
2 H, CH2OC(C6H5)3], 3.39–3.54 (m, 4 H, 2 CH2O), 3.92–3.94 (m,
1 H, CHOH), 4.90–5.00 (m, 2 H, CH=CH2), 5.77–5.84 (m, 1 H,
CH=CH2), 7.20–7.43 [m, 15 H, C(C6H5)3] ppm. 13C NMR
(100 MHz, CDCl3): δ = 26.20, 29.04, 29.24, 29.59–29.75, 33.88,
64.69, 69.89, 71.66, 72.06, 86.64 [C(C6H5)3], 113.99 (CH=CH2),
126.94, 127.15, 127.71, 127.83, 128.59, 139.15 (CH=CH2),
143.79 ppm. MS (ESI): m/z = 593.6 [M + Na]+. C39H54O3 (570.70):
calcd. C 82.06, H 9.53; found C 82.24, H 9.15.
1,2-O-Isopropylidene-3-O-[(10S)-10-methylheptadec-16-en-1-yl]-
sn-glycerol (11b): Yield 2.66 g (71%), colourless oil. [α]2D2 = +11.4
1
(c = 0.84 gmL–1, pure). H NMR (400 MHz, CDCl3): δ = 0.82 (d,
3JH,H = 6.4 Hz, 3 H, CHCH3), 1.05–1.38 [m, 23 H, (CH2)7CH-
(CH2)4], 1.34 (s, 3 H, CCH3), 1.40 (s, 3 H, CCH3), 1.52–1.59 (m, 2
H, CH2CH2O), 1.99–2.05 (m, 2 H, CH2CH=CH2), 3.38–3.52 (m,
4 H, 2 CH2O), 3.69–3.73 [m, 1 H, CHH-OC(CH3)2O], 4.02–4.05
3
[m, 1 H, CHH-OC(CH3)2O], 4.24 (quint., JH,H = 6.2 Hz, 1 H,
CHO), 4.89–4.99 (m,
2
H, CH=CH2), 5.74–5.84 (m,
1
H,
CH=CH2) ppm. 13C NMR (100 MHz, CDCl3):
δ
=
19.77
(CHCH3), 25.50, 26.13, 26.84, 26.97, 27.13, 29.05, 29.53–30.06,
31.93, 32.79, 33.87, 37.06, 37.12, 66.67 [CH2OC(CH3)2O], 71.82
(CH2O), 71.86 (CH2O), 74.75 (OCH), 109.22 [OC(CH3)2O], 113.96
(CH=CH2), 139.05 (CH=CH2) ppm. MS (EI): m/z (%) = 382 (5)
[M]+. C24H46O3 (382.62): calcd. C 75.34, H 12.12; found C 75.25,
H 12.20.
3-O-[(10S)-10-Methylheptadec-16-en-1-yl]-1-O-trityl-sn-glycerol
(13b): Yield 1.94 g (83%), colourless oil. [α]2D2 = –2.5 (c =
0.26 gmL–1, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 0.83 (d,
3JH,H = 6.4 Hz, 3 H, CH3), 1.04–1.39 [m, 23 H, (CH2)7CH-
(CH2)4], 1.51–1.57 (m, 2 H, CH2CH2O), 1.99–2.04 (m, 2 H,
CH2CH=CH2), 2.39 (d, J = 3.7 Hz, 1 H, OH), 3.15–3.22 [m, 2 H,
CH2OC(C6H5)3], 3.37–3.53 (m, 4 H, 2 CH2O), 3.92 (br. s, 1 H,
CHOH), 4.90–5.00 (m, 2 H, CH=CH2), 5.75–5.85 (m, 1 H,
CH=CH2), 7.16–7.43 [m, 15 H, C(C6H5)3] ppm. 13C NMR
(100 MHz, CDCl3): δ = 19.87 (CH3), 26.27, 27.07, 27.23, 29.14,
29.65–29.82, 30.17, 32.89, 33.96, 37.17, 37.24, 64.77, 69.97, 71.73,
72.14, 86.71 [C(C6H5)3], 114.07 (CH=CH2), 127.01, 127.22, 127.78,
127.89, 128.67, 139.20 (CH=CH2), 143.86 ppm. MS (ESI): m/z =
608.3 [M + Na]+. C40H56O3 (584.87): calcd. C 82.14, H 9.65; found
C 81.76, H 10.02.
3-O-Alkyl-sn-glycerols 12a and 12b: Compound 11a or 11b
(6.0 mmol) and PPTS (0.1 g) were poured into dry methanol
(35 mL) and heated for 10 h at reflux. Afterwards, the solvent was
evaporated and the residue was dissolved in chloroform (50 mL).
After washing with water (50 mL), drying over sodium sulfate, the
solvent was evaporated and the crude product was purified by col-
umn chromatography.
3-O-Heptadec-16-en-1-yl-sn-glycerol (12a): Yield 1.68 g (85%),
white solid, m.p. 56 °C. [α]2D2 = –0.6 (c = 0.1 gmL–1, CHCl3). 1H
NMR (400 MHz, CDCl3): δ = 1.19–1.37 [m, 24 H, (CH2)12], 1.52–
1.59 (m, 2 H, CH2CH2O), 1.99–2.04 (m, 2 H, CH2CH=CH2), 2.29
(br., 2 H, 2 OH), 3.42–3.53 (m, 4 H, 2 CH2O), 3.61–3.72 (m, 2 H,
CH2OH), 3.81–3.86 (m, 1 H, CHOH), 4.89–5.00 (m, 2 H,
2,3-O,O-Dialkyl-1-O-trityl-sn-glycerols 14a–c: Compounds 14a–c
were synthesized according to the synthesis of compounds 11a,b.
Potassium hydride (1.51 mmol, 0.2 mL, 30% suspension in paraf-
fin) was separated from the oil and suspended under argon in dry
toluene (5 mL). A solution of 13a or 13b (1.51 mmol) in dry tolu-
ene (10 mL) was added slowly at room temperature. The mixture
was stirred for 18 h at this temperature and then for 1 h at 100 °C.
Afterwards, the alkyl bromide 8b or hexadecyl bromide
(4.51 mmol), dissolved in dry toluene (10 mL), was added and the
mixture was stirred for 10 h at reflux. Thereafter, water (30 mL)
was added at room temperature and the mixture was stirred vigor-
ously. After phase separation, the organic layer was washed with
water (30 mL) and ammonium chloride solution (30 mL). The or-
ganic solution was dried with sodium sulfate, evaporated and puri-
fied by chromatography using heptane/diethyl ether and the gradi-
ent technique.
CH=CH2), 5.74–5.84 (m,
1
H, CH=CH2) ppm. 13C NMR
(100 MHz, CDCl3): δ = 26.14, 29.01, 29.21, 29.52–29.71, 33.85,
64.27 (CH2OH), 70.59 (CHOH), 71.88 (CH2O), 72.46 (CH2O),
114.05 (CH=CH2), 139.20 (CH=CH2) ppm. MS (EI): m/z (%) =
328 (8.9) [M]+. C20H40O3 (328.53): calcd. C 73.12, H 12.27; found
C 73.28, H 11.94.
3-O-[(10S)-10-Methylheptadec-16-en-1-yl]-sn-glycerol (12b): Yield
1.58 g (90%), colourless oil. [α]2D2 = +0.2 (c = 0.18 gmL–1, CHCl3).
3
1H NMR (400 MHz, CDCl3): δ = 0.81 (d, JH,H = 6.4 Hz, 3 H,
CH3), 1.02–1.37 [m, 23 H, (CH2)7CH(CH2)4], 1.51–1.58 (m, 2 H,
CH2CH2O), 1.99–2.04 (m, 2 H, CH2CH=CH2), 2.30–2.51 (br., 2
H, 2 OH), 3.42–3.52 (m, 4 H, 2 CH2O), 3.59–3.70 (m, 2 H,
CH2OH), 3.81–3.86 (m, 1 H, CHOH), 4.89–5.00 (m, 2 H,
CH=CH2), 5.74–5.84 (m,
1
H, CH=CH2) ppm. 13C NMR
3-O-(Heptadec-16-en-1-yl)-2-O-[(10R)-10-methylhexadecyl]-1-O-
(100 MHz, CDCl3): δ = 19.73 (CH3), 26.11, 26.93, 27.09, 29.01,
29.48–29.66, 30.02, 32.76, 33.83, 37.04, 37.10, 64.29 (CH2OH),
70.50 (CHOH), 71.86 (CH2O), 72.48 (CH2O), 114.03 (CH=CH2),
139.18 (CH=CH2) ppm. MS (EI): m/z (%) = 342 (5) [M]+.
C21H42O3 (342.56): calcd. C 73.63, H 12.36; found C 73.41, H
12.39.
trityl-sn-glycerol (14a): Yield 0.83 g (68%), colourless oil. [α]2D2
=
1
–5.4 (c = 0.14 gmL–1, CHCl3). H NMR (400 MHz, CDCl3): δ =
0.82–0.84 (d, 3JH,H = 6.4 Hz, 3 H, CHCH3), 0.88 (t, 3JH,H = 7.0 Hz,
3 H, CH2CH3), 1.08–1.35 [m, 49 H, (CH2)12, (CH2)7CH(CH2)5],
1 . 4 9 – 1 . 5 7 ( m , 4 H , 2 CH2 CH2 O), 2.01–2. 06 (m, 2 H,
CH2CH=CH2), 3.13–3.20 [m, 2 H, CH2OC(C6H5)3], 3.34–3.41 (m,
2 H, CH2O), 3.46–3.57 (m, 5 H, 2 CH2O, CHO), 4.90–5.01 (m, 2
H, CH=CH2), 5.74–5.86 (m, 1 H, CH=CH2), 7.16–7.42 [m, 15 H,
C(C6H5)3] ppm. 13C NMR (100 MHz, CDCl3): δ = 14.18
(CH2CH3), 19.80 (CHCH3), 22.77 (CH2CH3), 26.20, 26.26, 27.13,
27.18, 29.04, 29.23, 29.59–29.77, 30.12, 30.24, 32.04, 32.85, 33.88,
37.20, 63.74 (CH2O), 70.75 (CH2O), 71.28 (CH2O), 71.67 (CH2O),
3-O-Alkyl-1-O-trityl-sn-glycerols 13a and 13b: Trityl chloride
(1.34 g, 4.8 mmol) and the glycerol 12a or 12b (4.0 mmol) were dis-
solved in a mixture of dry chloroform and dry pyridine (30 mL,
1:1). The solution was stirred for 16 h at room temperature. After-
wards, water (20 mL) was added to hydrolyse the excess trityl chlo-
ride. After the addition of further water (20 mL), the organic phase
Eur. J. Org. Chem. 2011, 5894–5904
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5901

B. Dobner et al.
FULL PAPER
78.39 (OCH), 86.57 [C(C6H5)3], 114.08 (CH=CH2), 126.87, 127.70,
128.78, 139.24 (CH=CH2), 144.20 ppm. MS (ESI): m/z = 831.9 [M
+ Na]+. C56H88O3 (809.26): calcd. C 83.11, H 10.96; found C 83.32,
H 11.27.
CHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.17 (CH2CH3),
19.78 (CHCH3), 22.75 (CH2CH3), 26.17, 27.11, 27.14, 29.54–29.77,
30.73, 30.16, 32.02, 32.83, 37.17, 63.17 (CH2OH), 70.44 (CH2O),
70.97 (CH2O), 71.90 (CH2O), 78.31 (OCH) ppm. MS (ESI): m/z =
1108.7 [M + H]+. C72H146O6 (1107.93): calcd. C 78.05, H 13.28;
found C 77.69, H 13.16.
2-O-Hexadecyl-3-O-[(10S)-10-methylheptadec-16-en-1-yl]-1-O-
trityl-sn-glycerol (14b): Yield 0.67 g (55%), colourless oil. [α]2D2
=
1
–5.6 (c = 0.086 gmL–1, CHCl3). H NMR (400 MHz, CDCl3): δ =
3,3Ј-O-[(10R,23R)-10,23-Dimethyldotriacontane-1,32-diyl]bis(2-
3
3
0.83 (d, JH,H = 6.4 Hz, 3 H, CHCH3), 0.87 (t, JH,H = 7.0 Hz, 3 O-hexadecyl-sn-glycerol) (15b): Yield 0.224 g (27%), white waxy so-
H, CH2CH3), 1.06–1.39 [m, 49 H, (CH2)7CH(CH2)4, (CH2)13CH3], lid, m.p. 27 °C. [α]2D2 = +8.7 (c = 0.050 gmL–1, CHCl3). H NMR
1
3
1 . 4 7– 1 . 5 6 (m , 4 H , 2 CH2 CH2 O), 2.00–2. 05 (m, 2 H,
CH2CH=CH2), 3.14–3.16 [m, 2 H, CH2OC(C6H5)3], 3.36–3.40 (m,
(400 MHz, CDCl3): δ = 0.81 (d, JH,H = 6.6 Hz, 6 H, 2 CHCH3),
3
0.86 (t, JH,H = 7.0 Hz, 6 H, 2 CH2CH3), 1.02–1.35 [m, 106 H,
2 H, CH2O), 3.47–3.56 (m, 5 H, 2 CH2O, CHO), 4.90–5.00 (m, 2 (CH2)7CH(CH2)12CH(CH2)7, 2 (CH2)13CH3], 1.50–1.58 (m, 8 H, 4
H, CH=CH2), 5.75–5.83 (m, 1 H, CH=CH2), 7.18–7.46 [m, 15 H, CH2CH2O), 2.20 (br., 2 H, 2 OH), 3.37–3.77 (m, 18 H, 8 CH2O, 2
C(C6H5)3] ppm. 13C NMR (100 MHz, CDCl3): δ = 14.21 CHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.09 (CH2CH3),
(CH2CH3), 19.81 (CHCH3), 22.78 (CH2CH3), 26.21, 26.26, 27.01,
27.19, 29.08, 29.44–29.79, 30.13, 30.24, 32.01, 32.84, 33.90, 37.11,
37.19, 63.70 (CH2O), 70.72 (CH2O), 71.24 (CH2O), 71.64 (CH2O),
78.35 (OCH), 86.52 [C(C6H5)3], 114.00 (CH=CH2), 126.78, 127.60,
128.68, 139.14 (CH=CH2), 144.08 ppm. MS (ESI): m/z = 832.7 [M
+ Na]+. C56H88O3 (809.26): calcd. C 83.11, H 10.96; found C 83.20,
H 11.30.
19.69 (CHCH3), 22.67 (CH2CH3), 26.09, 27.08, 27.09, 29.35–30.07,
30.42, 31.91, 32.76, 37.11, 63.07 (CH2OH), 70.39 (CH2O), 70.89
(CH2O), 71.84 (CH2O), 78.28 (OCH) ppm. MS (ESI): m/z = 1108.0
[M]+. C72H146O6 (1107.93): calcd. C 78.05, H 13.28; found C 77.71,
H 13.34.
3,3Ј-O-[(10R,23R)-10,23-Dimethyldotriacontane-1,32-diyl]bis{2-O-
[(10R)-10-methylhexadecyl]-sn-glycerol} (15c): Yield 0.298 g (35%),
3-O-[(10S)-10-Methylheptadec-16-en-1-yl]-2-O-[(10R)-10-methyl-
colourless oil. [α]2D2 = +6.4 (c = 0.123 gmL–1, CHCl3). 1H NMR
3
hexadecyl]-1-O-trityl-sn-glycerol (14c): Yield 0.72 g (59%), colour- (400 MHz, CDCl3): δ = 0.81 (d, JH,H = 6.4 Hz, 12 H, 4 CHCH3),
less oil. [α]2D2 = –4.6 (c = 0.06 gmL–1, CHCl3). 1H NMR (400 MHz,
0.86 (t, JH,H = 7.0 Hz, 6 H, 2 CH2CH3), 1.05–1.38 [m, 104 H,
3
CDCl3): δ = 0.83 (d, 3JH,H = 6.6 Hz, 6 H, 2 CHCH3), 0.88 (t, 3JH,H (CH2)7CH(CH2)12CH(CH2)7, 2 (CH2)7CH(CH2)5CH3], 1.52–1.56
= 6.8 Hz, 3 H, CH2CH3), 1.06–1.39 [m, 48 H, (CH2)7CH(CH2)4, (m, 8 H, 4 CH2CH2O), 2.24 (br., 2 H, 2 OH), 3.38–3.71 (m, 18 H,
(CH2)7CH(CH2)5], 1.48–1.57 (m, 4 H, 2 CH2CH2O), 2.01–2.04 (m, 8 CH2O, 2 CHO) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.09
2 H, CH2CH=CH2), 3.15–3.17 [m, 2 H, CH2OC(C6H5)3], 3.37–3.40 (CH2CH3), 19.68 (CHCH3), 22.67 (CH2CH3), 25.74, 26.08, 27.03,
(m, 2 H, CH2O), 3.47–3.55 (m, 5 H, 2 CH2O, CHO), 4.90–5.00 (m, 27.06, 27.07, 27.08, 29.34–30.06, 31.94, 32.73, 32.75, 37.09, 37.10,
2 H, CH=CH2), 5.77–5.81 (m, 1 H, CH=CH2), 7.18–7.46 [m, 15
63.03 (CH2OH), 70.37 (CH2O), 70.87 (CH2O), 71.82 (CH2O),
H, C(C6H5)3] ppm. 13C NMR (100 MHz, CDCl3): δ = 14.21
78.29 (OCH) ppm. MS (ESI): m/z = 1136.1 [M]+. C74H150O6
(CH2CH3), 19.81 (CHCH3), 19.82 (CHCH3), 22.79, 22.97, 26.22, (1135.98): calcd. C 78.24, H 13.31; found C 78.53, H 13.15.
26.28, 26.44, 26.53, 27.01, 27.15, 27.20, 29.09, 29.59–29.82, 30.14,
Bis(phosphocholines) I–III: 2-Bromoethylphosphoric acid dichlo-
30.25, 31.04, 31.96, 32.04, 32.84, 32.87, 33.89, 35.52, 37.12, 37.20,
ride (0.38 g, 1.6 mmol) was poured into dry chloroform (10 mL)
63.73 (CH2O), 70.73 (CH2O), 71.26 (CH2O), 71.64 (CH2O), 78.36
whilst cooling with ice/water. A mixture of dry triethylamine
(OCH), 86.53 [C(C6H5)3], 114.00 (CH=CH2), 126.78, 127.15,
(0.38 mL, 2.8 mmol) in dry chloroform (10 mL) was added slowly
127.60, 127.82, 128.69, 139.12 (CH=CH2), 144.09 ppm. MS (ESI):
with stirring, which was continued for 30 min at 0 °C. The glycerols
m/z = 846.4 [M + Na]+. C57H90O3 (823.32): calcd. C 83.15, H
15a,b (in solid form) or 15c [dissolved in chloroform (5 mL)]
11.02; found C 83.03, H 11.24.
(0.2 mmol) were poured in one portion into the mixture. To dis-
3,3Ј-O-(Alkane-1,1Ј-diyl)bis(2-O-alkyl-sn-1-glycerols) 15a–c: The solve the solid glycerols 15a,b, the reaction mixture was gently
olefins 14a–c (0.75 mmol) were dissolved in dry dichloromethane
(30 mL) under argon. A solution of Grubbs first-generation cata-
lyst {[RuCl2(=CHPh)(PCy3)2], 0.18 g, 29 mol-%} in dry dichloro-
methane (18 mL) was added dropwise. The resulting mixture was
heated at reflux for 24 h. The solvent was removed under reduced
pressure and the crude residue was purified by silica gel column
chromatography using a heptane/chloroform gradient. A mixture
of the resulting light-brown oil in ethanol/ethyl acetate (50 mL, 1:1)
and palladium(II) hydroxide (48 mg, 20% on carbon) was stirred
under hydrogen (2 atm) at room temperature for 18 h. The catalyst
was removed by filtration and washed with chloroform several
times. The combined organic solutions were evaporated. The resi-
due was passed through a silica gel column using the gradient tech-
nique and chloroform/diethyl ether as eluent to give the methyl-
branched diols 15a–c.
warmed until a clear solution disappeared. Afterwards, the mixture
was stirred at room temperature for 24 h. After the complete con-
version of the glycerols, crushed ice (20 mL) was added to the solu-
tion and the mixture was stirred vigorously for a further 2 h. The
organic layer was separated and the aqueous phase was extracted
with chloroform (3ϫ 20 mL). The combined organic layers were
evaporated and the residue was dissolved in THF/water (10 mL,
1:1). After 1 h, ammonium chloride solution (10 mL, 5%) was
added and the mixture was extracted with a chloroform/methanol
solution (5:1, 3ϫ 25 mL). The chloroform fraction was evaporated
to dryness. The crude bromo esters were transferred into a mixture
of chloroform (10 mL), acetonitrile (10 mL), and an alcoholic solu-
tion of trimethylamine (2 mmol, 4.2 m). The mixture was kept in a
closed tube while warming up to 50 °C for 10 h. The clear solution
was allowed to stand 3–4 d at room temperature. Afterwards, the
reaction mixture was evaporated to dryness and the residue was
purified by chromatography using chloroform/methanol/water and
the gradient technique.
3,3Ј-O-(Dotriacontane-1,32-diyl)bis{2-O-[(10R)-10-methylhexa-
decyl]-sn-glycerol} (15a): Yield 0.258 g (31 %), white solid, m.p.
49 °C. [α]2D2 = +10.7 (c = 0.023 gmL–1, CHCl3). 1H NMR
3
(400 MHz, CDCl3): δ = 0.82 (d, JH,H = 6.4 Hz, 6 H, 2 CHCH3), 3,3Ј-O-(Dotriacontane-1,32-diyl)bis({2-O-[(10R)-10-methylhexa-
3
0.86 (t, JH,H = 6.8 Hz, 6 H, 2 CH2CH3), 1.02–1.32 [m, 106 H, decyl]-sn-glycer-1-yl}-2-(trimethylammonio)ethyl phosphate) (I):
(CH2 )2 8 , 2 (CH2 )7 CH(CH2 )5CH3 ], 1.50–1.57 (m, 8 H, 4 Yield 0.124 g (43%), white solid, m.p. 217–219 °C. [α]2D2 = –10 (c =
CH2CH2O), 1.89 (br., 2 H, 2 OH), 3.40–3.72 (m, 18 H, 8 CH2O, 2
2 mgmL–1, DMSO*). 1H NMR (400 MHz, CDCl3/CD3OD): δ =
5902
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 5894–5904

Synthesis of Optically Pure Diglycerol Tetraether Model Lipids
3
3
0.82 (d, JH,H = 6.4 Hz, 6 H, 2 CHCH3), 0.87 (t, JH,H = 6.9 Hz, followed by warming, vortexing and treatment with ultrasound.
6 H, 2 CH2CH3), 1.03–1.31 [m, 106 H, (CH2)28, 2 (CH2)7CH-
(CH2)5CH3], 1.48–1.55 (m, 8 H, 4 CH2CH2O), 3.26 [s, 18 H, 2
N(CH3)3], 3.39–3.61 (m, 14 H, 6 CH2O, 2 CHO), 3.66–3.69 (m, 4
The sample solution and the water reference were degassed under
vacuum whilst stirring. The measurements were performed at a
scan rate of 20 °Ch–1 from 2 to 95 °C in three consecutive runs. To
H, 2 NCH2CH2OP), 3.87–3.90 (m, 4 H, 2 POCH2CH), 4.25–4.30 eliminate any variation due to the cells a water/water baseline was
(m, 4 H, 2 NCH2CH2OP) ppm. 13C NMR (100 MHz, CDCl3/
subtracted. The DSC scans were evaluated by using the MicroCal
CD3OD): δ = 13.82 (CH2CH3), 19.46 (CHCH3), 22.50 (CH2CH3), ORIGIN 8.0 software.
25.89, 25.93, 26.87, 26.93, 29.36, 29.39, 29.50–29.53, 29.87, 29.91,
Electron Microscopy: The liposomes were prepared for electron mi-
2
31.77, 32.60, 36.94, 54.05 [t, J = 3.7 Hz, N(CH3)3], 58.59 (d, JC,P
croscopy by using the film method. The bolalipids I–III (15–20 mg)
were dissolved in chloroform/methanol (1:1), the solvent was evap-
orated and the samples were dried overnight in vacuo. The lipid
films were then incubated with water (1 mL) at 50 °C for 1 h,
treated with ultrasound for 30 min at the same temperature and
extruded 41 times through a polycarbonate membrane (200 nm).
The liposomes were freeze-fixed by using a JFD 030 (BAL-TEC,
Balzers, Lichtenstein) propane jet-freeze device. Thereafter, the
samples were freeze-fractured at –150 °C without etching by using
a BAF 060 (BAL-TEC, Balzers, Liechtenstein) freeze-fracture/
freeze-etching system. The surfaces were shadowed with platinum
to produce good topographic contrast (2 nm layer, shadowing angle
45°) and subsequently with carbon to stabilise the ultra-thin metal
film (20 nm layer, shadowing angle 90°). The replicas were floated
in sodium chloride (4%, Roth, Karlsruhe, Germany) for 30 min,
rinsed in distilled water (10 min), washed in 30% acetone (Roth,
Karlsruhe, Germany) for 30 min and rinsed again in distilled water
(10 min). Thereafter the replicas were mounted on copper grids,
coated with formvar film and observed with a transmission electron
microscope (EM 900, Carl Zeiss SMT, Oberkochen, Germany) op-
erating at 80 kV. Pictures were taken with a Variospeed SSCCD
SM-1k-120 camera (TRS, Moorenweis, Germany).
2
= 4.6 Hz, NCH2CH2O), 64.90 (d, JC,P = 5.4 Hz, POCH2CH),
66.41 (br., NCH2CH2O), 70.38 (CH2O), 70.41 (CH2O), 71.53
(CH2O), 77.79 (d, 3JC,P = 8.1 Hz, POCH2CH) ppm. HRMS: calcd.
for C82H170N2O12P2 [M + 2H]2+ 719.6187; found 719.6200. HPLC:
tR = 3.65 min, purity: 99.0%.
3,3Ј-O-[(10R,23R)-10,23-Dimethyldotriacontane-1,32-diyl]bis[(2-
O-hexadecyl-sn-glycer-1-yl)-2-(trimethylammonio)ethyl phosphate]
(II): Yield 0.095 g (33%), white waxy solid, m.p. 212–214 °C. [α]2D2
= –10 (c = 2 mg mL–1, DMSO*). 1H NMR (400 MHz, CDCl3/
3
CD3OD): δ = 0.81 (d, JH,H = 6.6 Hz, 6 H, 2 CHCH3), 0.86 (t,
3JH,H = 6.9 Hz, 6 H, 2 CH2CH3), 1.04–1.37 [m, 106 H, (CH2)7-
CH(CH2)12CH(CH2)7, 2 (CH2)13CH3], 1.49–1.55 (m, 8 H, 4
CH2CH2O), 3.20 [s, 18 H, 2 N(CH3)3], 3.35–3.56 (m, 14 H, 6
CH2O, 2 CHO), 3.58–3.61 (m, 4 H, 2 NCH2CH2OP), 3.87–3.91
(m, 4 H, 2 POCH2CH), 4.22–4.27 (m, 4 H, 2 NCH2CH2OP) ppm.
13C NMR (100 MHz, CDCl3/CD3OD): δ = 14.02 (CH2CH3), 19.70
(CHCH3), 22.65 (CH2CH3), 26.06, 26.09, 27.01, 27.06, 29.32,
29.51, 29.54, 29.58–29.68, 29.94, 30.00, 30.10, 31.89, 32.71, 37.04,
2
54.40 [t, J = 3.4 Hz, N(CH3)3], 58.75 (d, JC,P = 5.0 Hz,
2
NCH2CH2O), 65.02 (d, JC,P = 5.8 Hz, POCH2CH), 66.70 (br.,
NCH2CH2O), 70.50 (CH2O), 70.60 (CH2O), 71.67 (CH2O), 77.95
3
(d, JC , P = 5.4 Hz, POCH2 CH) ppm. HRMS: calcd. for
C82H170N2O12P2 [M + 2H]2+ 719.6187; found 719.6201. HPLC: tR
Acknowledgments
= 3.94 min, purity: 98.8%.
3,3Ј-O-[(10R,23R)-10,23-Dimethyldotriacontane-1,32-diyl]bis({2-
O-[(10R)-10-methylhexadecyl]-sn-glycer-1-yl}-2-(trimethyl-
ammonio)ethyl phosphate) (III): Yield 0.129 g (44%), white waxy
This work was supported by grants for post-graduate studies of the
German Federal State Sachsen-Anhalt (to T. M.).
solid, m.p. 195–200 °C. [α]2D2 = –10 (c = 2 mgmL–1, DMSO*). H
1
[1] a) T. A. Langworthy, Biochim. Biophys. Acta 1977, 487, 37–50;
b) M. De Rosa, E. Esposito, A. Gambacorta, B. Nicolaus, J. D.
Bu’Lock, Phytochemistry 1980, 19, 827–831.
3
NMR (400 MHz, CDCl3/CD3OD): δ = 0.82 (d, JH,H = 6.4 Hz, 12
3
H, 4 CHCH3), 0.87 (t, JH,H = 6.9 Hz, 6 H, 2 CH2CH3), 1.07–1.25
[m, 104 H, (CH2)7CH(CH2)12CH(CH2)7, 2 (CH2)7CH(CH2)5CH3],
1.46–1.56 (m, 8 H, 4 CH2CH2O), 3.26 [s, 18 H, 2 N(CH3)3], 3.38–
3.60 (m, 14 H, 6 CH2O, 2 CHO), 3.68–3.71 (m, 4 H, 2
NCH2CH2OP), 3.89–3.91 (m, 4 H, 2 POCH2CH), 4.28–4.32 (m, 4
H, 2 NCH2CH2OP) ppm. 13C NMR (100 MHz, CDCl3/CD3OD):
δ = 14.00 (CH2CH3), 19.63 (CHCH3), 19.65 (CHCH3), 22.63
(CH2CH3), 26.02, 26.06, 27.00, 27.05, 27.07, 29.50, 29.52, 29.59–
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*For polarimetry it was not possible to obtain a clear and concen-
trated solution of the lipids I–III in chloroform/methanol/water
mixtures. Even in dry DMSO only concentrations of 2 mgmL–1
could be achieved. Therefore, the stated [α]D values only give an
estimation of the optical rotation.
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with a Microcal VP-DSC differential scanning calorimeter (Micro-
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Eur. J. Org. Chem. 2011, 5894–5904
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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Published Online: August 16, 2011
5904
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© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 5894–5904