Structure-property relationships in a series of diglycerol tetraether model lipids and their lyotropic assemblies: The effect of branching topology and chirality

Article abstract of DOI:10.1039/c4ob00048j

Three novel diglycerol tetraether lipids with one membrane-spanning chain have been synthesized. These lipids contain only two or four racemic methyl branches at selected positions of the hydrophobic chains in contrast to natural lipids from archaebacterial membranes with an isoprenoid substitution pattern. The insertion of the methyl moieties was realized starting from either (RS)-citronellyl bromide or the inexpensive methyl malonic acid ethyl ester. For chain elongation the Cu-catalysed Grignard coupling reaction was used. The preparation of diglycerol tetraethers was either performed by condensing suitable blocked monoglycerol diethers by Grubbs metathesis or by reaction of the transmembrane C32-chain with blocked glycerols followed by further alkylation steps. Finally, we could show that the resulting lipids can form closed lipid vesicles comparable to the optically pure counterparts. Therefore, these much simpler lipids compared to the natural lipids from archaebacterial membranes are also suitable for preparation of stable tailored liposomes. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00048j

Organic &
Biomolecular Chemistry
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Structure–property relationships in a series of
diglycerol tetraether model lipids and their
lyotropic assemblies: the effect of branching
topology and chirality†
Cite this: Org. Biomol. Chem., 2014,
12, 3649
Thomas Markowski,a Simon Drescher,*a Annette Meister,b Alfred Blumec and
Bodo Dobner*a
Three novel diglycerol tetraether lipids with one membrane-spanning chain have been synthesized. These
lipids contain only two or four racemic methyl branches at selected positions of the hydrophobic chains
in contrast to natural lipids from archaebacterial membranes with an isoprenoid substitution pattern. The
insertion of the methyl moieties was realized starting from either (RS)-citronellyl bromide or the inexpen-
sive methyl malonic acid ethyl ester. For chain elongation the Cu-catalysed Grignard coupling reaction
was used. The preparation of diglycerol tetraethers was either performed by condensing suitable blocked
monoglycerol diethers by Grubbs metathesis or by reaction of the transmembrane C32-chain with
blocked glycerols followed by further alkylation steps. Finally, we could show that the resulting lipids can
form closed lipid vesicles comparable to the optically pure counterparts. Therefore, these much simpler
lipids compared to the natural lipids from archaebacterial membranes are also suitable for preparation of
stable tailored liposomes.
Received 7th January 2014,
Accepted 31st March 2014
DOI: 10.1039/c4ob00048j
www.rsc.org/obc
membrane) alkyl chains. These bipolar molecules are called
bolaamphiphiles or bolalipids and they are mostly diglycerol
Introduction
The membrane lipids of archaebacteria are different with tetraether lipids with a molecular length representing the
regard to common lipids found in prokaryotes and eukaryotes. thickness of the monolayer membrane formed by these lipids.
This is due to the extreme living conditions under which such This fact in combination with the stability of these bolalipids
extremophiles exist. In detail, the hydrophobic alkyl chains are is therefore of great interest especially in terms of biotechnol-
connected by ether linkages in the inverse sn-2,3-configuration ogy, materials science, and pharmacy.3–7 Since several phos-
to the glycerol backbone making these archaebacterial lipids pholipids proved to be very suitable materials for the
resistant against enzymatic degradation. The fluidity and, preparation of vesicles and also nanocontainers, bolalipids
hence, the adaptation to the milieu conditions are regulated from archaebacteria should also be applicable for this purpose
by the insertion of a number of cyclopentane rings as well as – especially with regard to their stability against decompo-
by the insertion of several methyl branches in an isoprenoid sition enzymes. Liposomes composed of bolalipids are called
substitution pattern resulting from the biosynthesis.1,2
archaeosomes.8 To obtain such bolalipids in considerable
Of special interest are the lipids of the methanogens and amounts, isolation from natural sources is on the one hand a
thermoacidophiles with one or two membrane-spanning (trans- suitable method. However, this method yields only hetero-
geneous lipid mixtures with respect to the composition of the
hydrophobic part of the lipids. On the other hand the total
aMartin-Luther-Universitaet Halle-Wittenberg, Institute of Pharmacy,
synthesis of an archaebacterial lipid with a diglycerol tetra-
Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany.
ether structure, exemplarily shown by Kakinuma et al.,9 is not
E-mail: simon.drescher@pharmazie.uni-halle.de,
practicable on the gram scale. Based on the idea that such
bodo.dobner@pharmazie.uni-halle.de; Fax: +49 (0) 345 5527026, +49 (0) 345 5527018;
bolalipids are suitable for the preparation of tailored lipo-
somes with outstanding possibilities and functionalities,
many researchers interested in this field dealt with simpler
model membranes and, hence, model lipids.
The main problem for the synthesis of archaebacterial
model lipids is the macrocyclisation to the final cycle with 72
Tel: +49 (0) 345 5525196, +49 (0) 345 5525120
bCenter for Structure and Dynamics of Proteins (MZP) Biocenter,
MLU Halle-Wittenberg, Weinbergweg 22, 06120 Halle (Saale), Germany
cMLU Halle-Wittenberg, Institute of Chemistry, von-Danckelmann-Platz 4, 06120
Halle (Saale), Germany
†Electronic supplementary information (ESI) available: ESI-MS, DLS measure-
ments, 1H and 13C NMR spectra. See DOI: 10.1039/c4ob00048j
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carbon atoms. Since tetraether lipids with only one mem-
brane-spanning chain were also found in natural lipids it is
reasonable for simpler model systems to copy this structural
Results and discussion
Synthetic methods
feature. The group of Yamauchi10,11 and then especially The synthetic strategy for the preparation of bolalipids I–III
Benvegnu and co-workers described an effective preparation involves on the one hand the coupling of preformed glycerol
procedure for such simpler compounds.12,13 In a recent triethers including one terminal double bond for the Grubbs
paper14 we published the synthesis of diglycerol tetraethers coupling to the bipolar system. This synthetic pathway was
with one membrane-spanning alkyl chain in the sn-3-position already described previously14 except that we now used a
of the glycerol backbone. In contrast to other archaebacterial benzyl blocking group instead of the trityl residue. On the
model lipids, we introduced only a discrete number of methyl other hand, we developed a novel synthetic procedure for the
branches at certain positions of the chain. We found that preparation of lipids I–III, in which the whole membrane-span-
model lipids with four methyl branches show nearly the same ning alkyl chain was bound to suitable blocked sn-glycerols.
physicochemical behaviour as the natural archaebacterial With respect to our aim to investigate the lipids with racemic
membrane lipids.15 In addition, we could show that these methyl branches, the synthesis of the branched hexadecyl
lipids are able to form liposomes. Hence, these bolalipids rep- chains (14a,b; see Scheme 1) can also be performed by two
resent a remarkable new model system of archaebacterial different strategies: the malonic ester pathway or the citronellyl
lipids applicable for liposomal drug delivery purposes.
Indeed, the preparation of the modified lipids is simpler
bromide pathway described previously in detail.14
The malonic ester pathway starts from a very inexpensive
than total synthesis of natural lipids from archaebacterial starting material: methyl malonic acid ethyl ester (1), which
membranes, but the chiral methyl branching requires greater contains the necessary methyl group and can be easily trans-
synthetic effort for the preparation of these lipids on gram formed into the desired compounds by classic procedures
scale. Whereas optically pure glycerol derivatives are nowadays (Scheme 1). The ester 1 was converted into its carbanion with
comparatively low priced and simple to prepare, the optically sodium hydride in toluene and alkylated with 1-bromohexane
pure methyl branches require more effort starting from chiral and 7-bromohept-1-ene resulting in the formation of hexyl-
precursors. Therefore, we were interested in the influence of (methyl)malonic acid diethyl ether (2a) and hept-6-en-1-yl-
the chirality of the methyl branching at defined positions in (methyl)malonic acid diethyl ether (2b). After the subsequently
the alkyl chain on the aggregation behaviour of the model performed saponification with potassium hydroxide in an
lipids. The loss of the chirality of the methyl branches enabled ethanol–water-mixture, hexyl(methyl)malonic acid (3a) and
us to use inexpensive starting materials and shorter reaction hept-6-en-1-yl(methyl)malonic acid (3b) were isolated. Then,
pathways.
the mono-decarboxylation of compounds 3 resulted in the for-
In this paper, we present the synthesis of three bipolar mation of (2RS)-2-methyloctanoic acid (4a) and (2RS)-2-methyl-
phosphocholines (lipids I–III) with one membrane-spanning non-8-enoic acid (4b), respectively. It is noteworthy that the
alkyl chain of 32 carbon atoms and two shorter alkyl chains decarboxylation of 3b was performed in cumene with catalytic
with 16 carbon atoms in the sn-2-position of the glycerol back- amounts of pyridine in order to avoid side reactions whereas
bone. The alkyl chains contain two or four racemic methyl compound 3a was heated up to 180 °C in a pure manner to
branches, again at the 10-position of the hexadecyl chains forfeit one acid moiety (see Scheme 1, left part).
and/or at the 10- and 10′-position of the transmembrane alkyl
The following conversion of the carboxylic acid into the
chain (Fig. 1). In addition, we present first investigations of hydroxy moiety can be performed either by reduction of the
the lyotropic behaviour of aqueous suspensions of these novel corresponding methyl ester (in the case of 4a) or by direct
tetraether lipids in comparison with the optically pure deriva- reduction of the unsaturated acid 4b with the use of lithium
tives14 by differential scanning calorimetry (DSC), dynamic aluminium hydride finally resulting in the formation of (2RS)-
light scattering (DLS), and transmission electron microscopy 2-methyloctan-1-ol (5a) and (2RS)-2-methylnon-8-en-1-ol (5b).
(TEM).
In the next step, the hydroxy group has to be transformed into
Fig. 1 Chemical structure of the synthesised archaebacterial model lipids I–III including racemic methyl branches.
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dibromide. The advantage of the last mentioned procedure is
that both reaction steps nearly gave quantitative yields.
The bromides 6a,b were further used for alkyl chain elonga-
tion reaction. For this purpose, compounds 6a,b were trans-
ferred into the corresponding Grignard reagents in ethereal
solution and coupled with 2-[(8-bromooctyl)oxy]tetrahydro-2H-
pyran (7a) in THF under catalysis with dilithium tetrachloro-
cuprate-(II),17 yielding the compounds 13a and 13b.
Both compounds (13a,b) can also be synthesised following
the citronellyl bromide pathway (see Scheme 1, right part),
which had been described previously in detail.14 This reaction
route is exemplarily shown for the preparation of compound
13b. It starts from the commercially available (RS)-citronellyl
bromide (8) that was allowed to react with ozone at −78 °C in
methanol. After the reductive work-up using sodium boro-
hydride, (4RS)-6-bromo-4-methylhexan-1-ol (9) can be isolated.
After the protection of the hydroxy moiety using DHP and PPTS,
the resulting compound 10 was coupled with pent-4-enylmagne-
sium bromide in a Grignard reaction under catalysis with
dilithium tetrachlorocuprate-(II).17 Then, the THP-alkane 11 was
transformed into the corresponding bromide, again using the
procedure described by Schwarz et al.16 The (8RS)-11-bromo-8-
methylundec-1-ene (12) obtained was used for the second chain
elongation, again using the copper-catalysed Grignard reaction
and 7b finally resulting in compound 13b.
Comparing the two synthetic routes – the malonic ester and
the citronellyl bromide pathway – with compounds 13, one can
conclude that both pathways resulted in nearly the same
overall yields: 27–58% vs. 35% (46–57%14) with a slight gain
for the malonic ester route although it includes at least one syn-
thetic step more. In addition, the purification of the products
within the first two steps of the malonic ester pathway is not
necessary, as shown for the synthesis of compound 5b – an
additional advantage for this route. Finally, both THP-blocked
methyl-branched alcohols (13a,b) can be transferred into the
bromides (10RS)-1-bromo-10-methylhexadecane (14a) and
(8RS)-17-bromo-8-methylheptadec-1-ene (14b) using again the
protocol of Schwarz et al.16
The synthesis of diglycerol tetraethers can be realised by
two different synthetic strategies: pathway (I), one can use the
olefin metathesis with the Grubbs first-generation catalyst18 in
order to connect pre-built monoglycerol diethers including a
terminal double bond (see Scheme 2) or pathway (II), one can
insert the transmembrane alkyl chain in the first step, fol-
lowed by further alkylation steps (see Scheme 3). Realising the
sn-2,3-stereochemistry of the glycerol backbone of natural
archaebacterial lipids, both synthetic pathways have to start
with the commercially available (S)-1,2-O-isopropylidene-
glycerol (15).
Scheme 1 Reagents and conditions: (i) NaH, toluene, 1-bromohexane
or 7-bromohept-1-ene, reflux; (ii) KOH, EtOH–H2O; (iii) 180 °C; (iv) pyri-
dine, cumene, reflux; (v), H2SO4, MeOH, reflux; then LiAlH4, Et2O, reflux;
(vi) LiAlH4, Et2O, reflux; (vii) MesCl, CHCl3, 0 °C; then LiBr, acetone,
reflux; (viii) DHP, CH2Cl2, PPTS, r.t.; (ix) PPh3, Br2, CH2Cl2, r.t.; (x) Mg,
Et2O, reflux; then 2-[(8-bromooctyl)oxy]tetrahydro-2H-pyran (7a),
Li2CuCl4, THF, 0 °C; (xi) O3, MeOH, −78 °C; (xii) NaBH4, MeOH, r.t.; (xiii)
Mg, R2(CH2)3Br, Et2O, reflux; then Li2CuCl4, THF, 0 °C; (xiv) Mg, 2-[(6-
bromohexyl)oxy]tetrahydro-2H-pyran (7b), 50 °C; then Li2CuCl4, THF,
0 °C.
a bromide. For this purpose two procedures are conceivable:
(2RS)-1-bromo-2-methyloctane (6a) was prepared by a conven-
tional two-step procedure including a reaction with methane-
sulfonyl chloride (MesCl) followed by
a
Finkelstein-
replacement of the sulfonic ester with lithium bromide in
anhydrous acetone. On the other hand, (8RS)-9-bromo-8-
The reaction pathway I (Scheme 2) starts with a first alkyl-
ation step: compound 15 was deprotonated with potassium
hydride in toluene and subsequently alkylated with terminal
unsaturated bromides, namely 14b and 17-bromoheptadec-1-
ene,14 yielding 16a and 16b. Afterwards, the isopropylidene
(IP) blocking group of compounds 16a,b was cleaved using
PPTS in methanol leading to 3-O-alkyl-sn-glycerols 17.
methylnon-1-ene (6b) was prepared using
a procedure
described by Schwarz et al.16 Therefore, the alcohol 5b was
firstly converted into the appropriate acetal with 3,4-dihydro-
2H-pyran (DHP) and a catalytic amount of pyridinium p-toluene-
sulfonate (PPTS). Then, this compound was transformed into
the favoured bromide 6b using triphenylphosphoranediyl
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Scheme 2 Reagents and conditions: (i) KH, toluene, 17-bromoheptadec-1-ene or 14b, reflux; (ii) PPTS, MeOH, reflux; (iii) Bu2SnO, toluene–MeOH,
reflux; then benzyl bromide, 1,2-dimethoxyethane, reflux; (iv) KH, toluene, 14a or 1-bromohexadecane, reflux; (v) RuCl2(vCHPh)(PCy3)2, CH2Cl2,
reflux; (vi) Pd(OH)2/C, H2, EtOH–EtOAc, r.t.
In our previous work,14 the selective introduction of the diyl)-bis(2-O-alkyl-sn-glycerol)s 20 in very good yields (72–88%
C16-chain in the sn-2-position of the glycerol was realised by compared to 19).
regioselective tritylation of the primary hydroxyl group and
The reaction pathway II (Scheme 3) also starts from the gly-
subsequently performed deprotonation and alkylation with cerol compound 15. In contrast to pathway I, the transmem-
1-bromohexadecane or the 10-methyl analogue. Since the brane alkyl chain was inserted in the first alkylation step.
trityl residue is very bulky, the yields during the deprotonation Therefore, the dotriacontane-1,32-diol (21a)20 and the racemic
and alkylation steps are quite moderate and, hence, we (10RS,23RS)-10,23-dimethyldotriacontane-1,32-diol
(21b)21
used a regioselective benzylation of the dibutylstannylene were transferred into the corresponding dibromide 22a and
derivatives of the 3-O-alkyl-sn-glycerols 17 instead.19 However, bis(methanesulfonate) (22b), respectively, by common chemi-
beside the desired 3-O-alkyl-1-O-benzyl-sn-glycerols 18 the 3-O- cal procedures and subsequently used to connect two mole-
alkyl-2-O-benzyl regioisomers were formed. But, both com- cules of compound 15 in a twofold O-alkylation reaction. Both
pounds could be fully separated by column chromatography products (23a,b) were obtained in acceptable yields (43–56%).
using chloroform–diethyl ether as the eluent and the gradient After nearly quantitative cleavage of the IP blocking groups,
technique. The subsequent second alkylations of glycerols 18a resulting in bis(sn-glycerol)s 24a and 24b, the sn-1-position of
and 18b were carried out under nearly the same conditions the glycerol was blocked by tritylation. The reaction of com-
described above using potassium hydride in toluene and pounds 24a,b with trityl chloride was performed in pure pyri-
(10RS)-1-bromo-10-methylhexadecane (14a) or 1-bromohexa- dine with catalytic amounts of 4-dimethylaminopyridine
decane. The yields of the 1-O-benzyl-2,3-O,O-dialkyl-sn-glycerols (DMAP) at room temperature yielding 25a and 25b. It is note-
19a–c are slightly higher (67–74%) compared to the alkylation worthy that despite the bulkiness of the trityl moiety we
of the corresponding 3-O-alkyl-1-O-trityl-sn-glycerol derivatives observed a tris- and tetrakis-tritylation of compounds 24a,b
(55–68%) described previously.14 However, taking into account (see ESI†) in a non-negligible amount, which prohibited a pro-
the more laborious synthesis of the pure 3-O-alkyl-1-O-benzyl- longed reaction time. However, the different tritylation pro-
sn-glycerol 18 using the dibutylstannylene pathway, both ducts can be fully separated from each other by MPLC.
methods benzylation and tritylation at the sn-1-position fol-
lowed by O-alkylation at the sn-2-position of the glycerol hydride, toluene, and bromide 14a in a comparable procedure
moiety are comparable. as described above. Compounds 26a and 26b were obtained in
The second O-alkylation was performed using potassium
The synthesis of the final diglycerol tetraethers 20 based on very good yields (47–57%), particularly with regard to the two
compounds 19a–c – following pathway I – was realised in two O-alkylation reactions performed in this synthetic step. The
steps: as described previously,14 the olefin metathesis with the relatively high amount of the monoalkylated side-product
Grubbs first-generation catalyst18 followed by simultaneous (22–31%; see the ESI† for MS) is due to the steric shielding of
hydrogenation of the double bonds and debenzylation using the sn-2-position of the glycerol and it could not be reduced by
palladium hydroxide on carbon yielded 3,3′-O-(alkane-1,1′- either elongation of the reaction time or increasing the
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Scheme 4 Reagents and conditions: Cl2P(O)O(CH2)2Br, TEA, CHCl3;
then CHCl3, acetonitrile, EtOH, N(CH3)3, 50 °C.
avoids the usage of expensive ruthenium catalysts and it is at
least one step shorter.
Lastly, the methyl-branched bipolar phosphocholines I–III
were prepared from the diglycerol tetraethers 20 by the
method described by Eibl et al.23 with the phosphorylating
reagent of Hirt and Berchthold (Scheme 4).24 The use of 2-bromo-
ethylphosphoric acid dichloride in the phosphorylation step
resulted in the highest yields in comparison with other phos-
phorylating agents, e.g. phospholanes or phosphoroxytrichlor-
ide.25,26 The subsequently performed quarternisation with
trimethylamine provided the diglycerol tetraether lipids I–III
in 28–41% yield after purification and with respect to the
glycerols 20.
Temperature-dependent aggregation behaviour
The lyotropic phase behaviour of the novel, archaebacterial
diglycerol tetraether phospholipid analogues I–III containing a
different number and position of racemic methyl branches was
characterised by differential scanning calorimetry (DSC) in the
temperature range between 2 and 95 °C in comparison with
the optically pure counterparts described previously.14 Lipid I
with two methyl groups in the shorter alkyl chain shows an
endothermic transition at a temperature (Tm) of 19.1 °C
Scheme 3 Reagents and conditions: (i) DHP, CH2Cl2, PPTS, r.t.; then
PPh3, Br2, CH2Cl2, r.t.; (ii) MesCl, DMAP, CHCl3, r.t.; (iii) KH, toluene, 15;
then 22a or 22b, reflux; (iv) PPTS, MeOH, reflux; (v) trityl chloride, pyri-
dine, DMAP, r.t.; (vi) KH, toluene, 14a, reflux; (vii) BF3·Et2O, MeOH.
amount of bromide 14a. The final cleavage of the trityl block-
ing groups of compounds 26a,b was performed with BF3·Et2O
in methanol in accordance with Hermetter and Paltauf22
leading to the formation of compounds 20a and 20c in nearly
quantitative yields (95%).
The comparison of the two synthetic pathways for the
preparation of the diglycerol tetraethers 20 – starting from IP-
glycerol 15 – shows that both routes gave nearly the same
yields: pathway I, using the Grubbs metathesis reaction, pro-
vides 16–32% (previously 15–30%14) after 6 reaction steps
whereas pathway II, which utilises the insertion of the trans-
membrane alkyl chain, provides 8–20% after 5 steps. Although
the last route resulted in slightly lower overall yields, it is from
the economical point of view our preferred pathway because it
Fig. 2 DSC heating curves of aqueous suspensions of lipids I–III (c =
1 mg mL−1, heating rate = 20 K h−1). The curves are shifted vertically for
clarity. The inset shows the corresponding transition temperatures (Tm
values) of lipids investigated as well as the Tm values of the optically
pure counterparts described previously.14
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(Fig. 2), which is much lower compared to Tm of the
unbranched tetraether lipid described by Yamauchi (Tm
=
61 °C).11 Compared to the optically pure counterpart of lipid
I,14 Tm has increased by 2.4 K. Lipid II with two methyl
branches in the 10 and 10′-position of the transmembrane
alkyl chain shows an endothermic transition at 11.1 °C (with a
small shoulder at 14.3 °C), which is again 2.6 K higher com-
pared to the optically pure analogue14 but 8 K lower compared
to lipid I. For lipid III with four methyl groups, no phase tran-
sition above 2 °C could be detected (Fig. 2), which is in line
with the behaviour of natural archaebacterial lipids15 and
which was also found for the optically pure counterpart.14
It is obvious that the van der Waals contacts of the longest
unbranched segment of the alkyl chains determine the value
of the transition temperature. Similar results have been found
before for monopolar methyl-branched phospholipids.27,28
The differences in Tm values between tetraether lipids with
either racemic or optically pure methyl branches could be
related to small changes in the van der Waals interactions of
the alkyl chains; due to the identical orientation of the methyl
branches with respect to the long alkyl chain in an all-trans
conformation in the optically pure tetraether lipids, close chain
packing could be more difficult compared to the racemic lipids
I–III, which as a consequence would lead to a further decrease
of the transition temperature. In conclusion, not only the
number and the positions but also the stereospecificity of
the methyl branches within the alkyl chains are important for
the aggregation and the lyotropic transitions of the tetraether
lipids.
The ability of compounds I–III to form closed lipid vesicles
was studied by transmission electron microscopy (TEM).
Samples of lipid suspensions (c = 0.03–0.1 mg mL−1) were
stained with uranyl acetate, dried and then imaged. Fig. 3
shows the EM images of lipid samples prepared at two
different temperatures. The images show that all three digly-
cerol tetraether lipids are capable of forming liposomes with
diameters of about 100 nm (Fig. 3B and E) and up to 3 µm
(giant vesicles; Fig. 3C). Major differences between the sample
preparations at temperatures below the transition observed in
DSC (Fig. 3A and B) and preparations at ambient temperature
(Fig. 3C–F) could not be detected. For lipid I we observed large
vesicles, which were disrupted (Fig. 3A) or collapsed and
folded (Fig. 3C and D) during the drying process of sample
preparation. Lipid II (Fig. 3B and E) showed the formation of
smaller vesicles compared to lipid I and the aggregates
seemed to be less structured. For lipid III we observed vesicles
(Fig. 3F) with diameters ranging between 200 nm and 1 µm.
The observations for these racemic tetraether lipids (I–III) are
comparable to those found for the optically pure counterparts
described previously.14
Fig. 3 TEM images of aqueous suspensions of lipids I–III (c
=
0.03–0.1 mg mL−1) prepared at different temperatures: (A, B) 4 °C, (C–F)
22 °C; (A, C, D) lipid I, (B, E) lipid II, and (F) lipid III. Samples were stained
with uranyl acetate.
Fig. 4 DLS measurements of liposomes prepared from aqueous lipid
suspensions (c = 1 mg mL−1) using the technique described by Bangham
et al.29 after different times of storage (n = 3).
To further prove the capability of the tetraether lipids I–III
to form liposomes stable against aggregation or fusion we per-
formed dynamic light scattering (DLS) experiments (Fig. 4). through 100 nm polycarbonate membranes and examined by
DLS. The hydrodynamic diameters obtained were in the range
Liposomes were prepared by the technique described by
Bangham et al.29 After treatment of sample suspensions (c =
of 99–111 nm with a polydispersity index (PdI) between 0.08
1 mg mL−1) with ultrasound, the liposomes were extruded and 0.12, which indicates a relatively narrow size distribution
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of the vesicles (see ESI, Fig. S1 and S2†). The DLS measure-
ments were repeated after several weeks (1–12) of storage in
order to check the stability of liposomes against aggregation or
Experimental section
General
fusion. For lipid I with an unbranched transmembrane alkyl Apart from palladium hydroxide on carbon (20%, Acros Organ-
chain we observed a continuous increase of the z-average value ics) all chemicals were purchased from Sigma Aldrich Co. and
up to 330 nm in diameter after 12 weeks of storage (Fig. 4). were used without further purification. 2-Bromoethylphos-
The corresponding PdI values also increased over time, indi- phoric acid dichloride was prepared according to the litera-
cating an increase in polydispersity (see ESI, Fig. S3†). In con- ture.23 All solvents were dried and distilled before use. The
trast, liposomes composed of lipids II and III, which contain purity of all compounds was checked by thin-layer chromato-
two methyl groups within the long, transmembrane alkyl graphy (TLC) using silica gel 60 F254 plates (Merck). The chro-
chain, are stable for at least 3 months. The reason for this be- matograms were developed by means of bromothymol blue.
haviour remains unclear at this time, but it is conceivable that Silica gel (Merck, 0.063–0.200 mm) was used for column
the additional methyl branches within the long, transmem- chromatography of all products. Melting points were deter-
brane alkyl chain induce a flexibility that is necessary for lipo- mined with Boetius apparatus. Optical rotation was quantified
somal stability. The further enhanced stability of liposomes on a Polartronic E (Schmidt und Haensch) and [α]D values are
made from lipid III with two additional methyl branches in given in 10−1 deg cm2 g−1. Elemental analyses were carried out
1
the short chains supports this assumption. This notion is also on a Leco CHNS-932. H and 13C NMR spectra were recorded
supported by experimental results on membranes composed on a Varian Gemini 2000 spectrometer or a Varian Inova 500
of
1,2-diphytanoyl-sn-glycero-3-phosphocholine
(DPhPC), using CDCl3 or CD3OD as the internal standard. Chemical
where each chain has 4 additional methyl groups. This com- shifts (δ) are reported in parts per million (ppm). The coupling
pound is the lipid of choice for the formation of black lipid constants (J) are reported in Hz. Mass spectrometric data were
membranes (BLM) due to the high stability of the bilayer.30
obtained with a Finnigan LCQ-Classic (ESI-MS) or were
recorded on an AMD 402 (70 eV) spectrometer (EI-MS). High-
resolution mass spectra (HRMS) were recorded on a Thermo
Fisher Scientific LTQ-Orbitrap mass spectrometer with static
nano-electrospray ionisation. Analytical HPLC (Jasco) was
Conclusions
We have developed several synthetic strategies for the prepa- performed on a Kromasil Si 100–5 µm–250 × 4.6 mm, a PU 980
ration of diglycerol tetraether model lipids that enables the Intelligent HPLC-Pump, and LG-1580-02 Ternary
a
insertion of racemic methyl branches at selected positions Gradient Unit (Jasco) with a SEDEX 55 ELS detector (SEDERE
within the alkyl chains. For the synthesis of the bromoalkane France) using the following solvents: 5 min isocratic with
precursors we established a malonic ester pathway – besides the CHCl3–MeOH–water (45/45/10, v/v/v),
5 min continuous
citronellyl bromide pathway described previously14
–
that increase with CHCl3–MeOH–water (42/42/16, v/v/v), 10 min
improves the synthetic handling and the yields obtained. The isocratic with CHCl3–MeOH–water (45/45/10, v/v/v); flow =
subsequent preparation of the diglycerol tetraethers was 1 mL min−1
realised using on the one hand the Grubbs metathesis reac-
.
Synthesis of methyl-branched bromoalkanes – the malonic
tion14 (pathway I) and on the other hand the insertion of the
transmembrane alkyl chain as a whole (pathway II). The latter
ester pathway
ones further improve the synthetic economy circumventing the The synthetic procedures and analytical data of compounds
usage of ruthenium catalysts. For the selective glycerol alkyl- 2a, 3a, 4a, 5a, and 6a can be found in the ESI.†
ation reactions and also for the insertion of the phosphocholine
headgroups, robust and established approaches were used.
Hept-6-en-1-yl(methyl)malonic acid diethyl ester (2b). The
sodium salt of the methyl malonic acid ester was prepared
Through the syntheses described herein we have shown from 1 (13.92 g, 0.08 mol) and sodium hydride (60%, 3.2 g,
that, beside the usage of a reduced number of methyl 0.08 mol) in dry toluene. Then, 7-bromohept-1-ene (10.0 g,
branches,14 the stereospecificity of those methyl moieties is 85 mmol) was added and the mixture was heated for 8 h under
not of substantial significance for the properties of our archae- reflux. Water (20 mL) was added and the organic phase was
bacterial model lipids; the optically pure14 and also the racemic separated and evaporated. The crude ester was used for the
model lipids – described herein – show an aggregation behav- saponification without further purification.
iour comparable to archaebacterial lipids found naturally, i.e.,
Hept-6-en-1-yl(methyl)malonic acid (3b). The crude 2b was
the formation of stable liposomes. This fact is of particular taken up with EtOH (100 mL). Afterwards, KOH (11.2 g,
interest for a simpler, less expensive, and a straight forward 0.2 mol) dissolved in water (40 mL) was added and the mixture
synthesis of such model lipids. With the insertion of a discrete was refluxed for 8 h. Most of the EtOH was evaporated and the
number of methyl branches at positions other than those residue was diluted with water (100 mL). The solution was
described above, fine-tuning of the aggregation behaviour and, extracted two times with Et2O (40 mL) and the alkaline water
hence, the formation of tailored liposomes seem to be feasible. phase was cooled to 0 °C. At this temperature the water phase
This aspect and the preparation of model compounds with was acidified with ice-cold aqueous HCl and then extracted
different headgroup structures are under investigation.
three times with Et2O (75 mL). The collected ethereal layers
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were washed with brine, dried and evaporated to dryness. The purification the crude product was run on a short column con-
crude acid 3b was used without purification. taining silica gel. Compound 6b was eluted with pure heptane
(2RS)-2-Methylnon-8-enoic acid (4b). The crude hept-6-en-1- yielding colourless oil (5.36 g, 94%). C10H19Br requires C,
1
yl(methyl)malonic acid (3b) was dissolved in cumene (100 mL) 54.80; H, 8.74; found: C, 54.96; H, 8.66%; H NMR (400 MHz;
and pyridine (1 mL). The mixture was heated for 1 h under CDCl3) δ 1.01 (d, J = 6.6 Hz, 3 H, CH3), 1.15–1.51 (m, 8 H, CH2),
reflux. The solvent was evaporated, the oily residue was dis- 1.70–1.86 (m, 1 H, CH), 1.99–2.14 (m, 2 H, H2CvCHCH2),
solved in Et2O (50 mL) and the solution was washed with 3.30–3.42 (m, 2 H, BrCH2), 4.89–5.05 (m, 2 H, H2CvCH), 5.81
diluted HCl and brine. After drying over Na2SO4, the Et2O was (ddt, 2J[Z] = 17.0 Hz, 2J[E] = 10.2 Hz, 3J = 6.7 Hz, 1 H, H2CvCH);
removed under reduced pressure and the residue was purified 13C NMR (100 MHz; CDCl3)
δ
18.8 (CH3), 26.7
by column chromatography yielding colourless oil (5.7 g, 82% (CH2CH2CHCH3), 28.8 and 29.1 (H2CvCHCH2CH2CH2), 33.7,
with respect to compound 1). C10H18O2 requires C, 70.55; H, 34.8, 35.1 (CH), 41.5 (CH2Br), 114.2 (H2CvCH), 139.0
1
10.65; found: C, 70.73; H, 10.88%; H NMR (500 MHz; CDCl3) (H2CvCH); EI-MS m/z 218/220 (2%, M).
δ 1.18 (d, J = 6.9 Hz, 3 H, CH3), 1.22–1.49 (m, 7 H, CH2),
Synthesis of methyl-branched bromoalkanes – the citronellyl
1.65–1.72 (m, 1 H, CH2CH), 2.00–2.06 (m, 2 H, H2CvCHCH2),
bromide pathway
2.42–2.49 (m, 1 H, CHCH3), 4.90–5.03 (m, 2 H, H2CvCH), 5.80
(ddt, 2J[Z] = 16.9 Hz, 2J[E] = 10.2 Hz, 3J = 6.7 Hz, 1 H, H2CvCH); The synthetic procedures and analytical data of compounds
13C NMR (125 MHz; CDCl3)
δ
16.8 (CH3), 26.9 9–12, 13a–b, and 14a–b can be found in the ESI.†
(CH2CH2CHCH3), 28.7 and 28.9 (H2CvCHCH2CH2CH2), 33.4,
33.7, 39.4 (CH), 114.2 (H2CvCH), 138.9 (H2CvCH), 183.6
(COOH); EI-MS m/z 170 (3%, M), 152 (10, M − H2O).
Synthesis of diglycerol tetraethers – reaction pathway I
The synthetic procedures and analytical data of compounds
(2RS)-2-Methylnon-8-en-1-ol (5b). To a slurry of lithium alu- 16a–b and 17a–b can be found in the ESI.†
minium hydride (1.13 g, 30 mmol) in dry Et2O (50 mL) was
added dropwise a solution of compound 4b (5.10 g, 30 mmol)
in dry Et2O (20 mL). The mixture was stirred for 6 h under
General procedure for the synthesis of 3-O-alkyl-1-O-benzyl-sn-
glycerols 18
reflux. Afterwards, the reaction mixture was hydrolysed with In a dried and argon-flashed flask were placed 3-O-alkyl-sn-
ice and acidified using 10% HCl. The layers were separated glycerols 17 (10 mmol), a mixture of dry toluene–MeOH
and the water phase was extracted two times with Et2O (40 mL; 10/1, v/v), a powdered molecular sieve (4 g) and
(50 mL). The combined ethereal phase was washed with 5% dibutyl tin oxide (2.89 g, 11.6 mmol). The mixture was heated
KOH (50 mL), water (50 mL) and brine (50 mL). After drying under reflux for 1 h and then filtered. The filtrate was evapor-
over Na2SO4, the solvent was evaporated and the crude product ated to dryness and the residue was suspended in dry di-
was purified by column chromatography with heptane–Et2O as methoxyethane (40 mL). Benzyl bromide (1.99 mL, 13 mmol)
the eluent using the gradient technique yielding colourless oil dissolved in dry dimethoxyethane (30 mL) was dropped into
(4.4 g, 94%). C10H20O requires C, 76.86; H, 12.90; found: C, the mixture with stirring. After 30 min at r.t., the mixture was
76.75; H, 12.98%; 1H NMR (400 MHz; CDCl3) δ 0.91 (d, J = stirred for 20 h under reflux. For work up the dimethoxyethane
6.7 Hz, 3 H, CHCH3), 1.02–1.16 (m, 1 H, CH2), 1.19–1.42 (m, was removed in vacuo and substituted by dry toluene (60 mL).
7 H, CH2), 1.52–1.76 (m, 1 H, CH2), 1.99–2.10 (m, 2 H, After cooling, the mixture was intensively stirred with ice
H2CvCHCH2), 3.37–3.55 (m, 2 H, CH2OH), 4.88–5.01 (m, 2 H, (40 mL) and phosphate buffer (40 mL; 0.5 M, pH = 5). The
H2CvCH), 5.81 (ddt, 2J[Z] = 16.9 Hz, 2J[E] = 10.2 Hz, 3J = 6.7 Hz, organic phase was separated and the water layer was extracted
1 H, H2CvCH); 13C NMR (100 MHz; CDCl3) δ 16.5 (CH3), 26.8 two times with toluene (20 mL). The combined organic layers
(CH2CH2CHCH3), 28.9 and 29.4 (H2CvCHCH2CH2CH2), 33.1, were dried over K2CO3, filtered and evaporated to dryness. The
33.7, 35.7 (CH), 68.3 (CH2OH), 114.2 (H2CvCH), 139.1 purification was done by column chromatography using
(H2CvCH); EI-MS m/z 156.2 (4%, M), 138.2 (0.4, M − H2O).
(8RS)-9-Bromo-8-methylnon-1-ene (6b). Compound 5b (4.06 g, desired product was eluted with a polarity of 93/7 (v/v).
26 mmol) was dissolved in dry CH2Cl2 (30 mL). 3,4-Dihydro- 1-O-Benzyl-3-O-(heptadec-16-en-1-yl)-sn-glycerol (18a). Fol-
heptane–Et2O as the eluent and the gradient technique. The
2H-pyrane (3.78 g, 45 mmol) and PPTS (50 mg) were added lowing the general procedure, 17a (3.29 g) gave 18a (2.47 g,
and the mixture was allowed to stir overnight. After the reac- 58%), a white crystalline solid. M.p. 28–30 °C; [α]D22 −1.5
tion was complete (TLC control), the mixture was washed (c 0.10 g mL−1, CHCl3); C27H46O3 requires C, 77.46; H, 11.07;
twice with water and the organic layer was dried over Na2SO4. found: C, 77.08; H, 11.18%; 1H NMR (400 MHz; CDCl3)
The solvent was removed and the residue was filtered over a δ 1.25–1.38 (m, 24 H, (CH2)12), 1.52–1.57 (m, 2 H, CH2CH2O),
short column. The isolated and exhaustive dried 2-[(2-methyl- 2.00–2.05 (m, 2 H, CH2CHvCH2), 2.45 (bs, 1 H, OH), 3.36–3.62
non-8-en-1-yl)oxy]tetrahydro-2H-pyran was poured into a solu- (m, 6 H, 3× CH2O), 3.94–3.99 (m, 1 H, CHOH), 4.54 (s, 2 H,
tion of triphenylphosphoranediyl dibromide prepared from CH2C6H5), 4.90–5.01 (m, 2 H, CHvCH2), 5.75–5.85 (m, 1 H,
Br2 (9.2 g, 57 mmol) and triphenylphosphane (14.9 g, CHvCH2), 7.12–7.35 (m, 5 H, C6H5); 13C NMR (100 MHz;
57 mmol) in CH2Cl2 (160 mL) at 0 °C. The dark coloured solu- CDCl3) δ 26.18, 29.03, 29.23, 29.55–29.74, 33.88, 69.60, 71.50,
tion was stirred overnight at r.t. The mixture was washed twice 71.73, 71.87, 73.49, 114.09, 127.70, 128.40, 138.08, 139.21;
with water, dried over Na2SO4 and evaporated to dryness. For ESI-MS m/z 436.9 (M + NH4).
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1-O-Benzyl-3-O-[(10RS)-10-methylheptadec-16-en-1-yl]-sn- (CH2)7CH(CH2)4, (CH2)13CH3), 1.51–1.59 (m,
4
H, 2×
glycerol (18b). Following the general procedure, 17b (3.43 g) CH2CH2O), 1.99–2.03 (m, 2 H, CH2CHvCH2), 3.41 (t, J =
gave 18b (3.3 g, 76%), slight yellow oil. [α]D22 −2.0 (c 0.04 g 6.6 Hz, 2 H, CH2O), 3.47–3.59 (m, 7 H, 3× CH2O, CHO), 4.54 (s,
mL−1, CHCl3); C28H48O3 requires C, 77.72; H, 11.18; found: C, 2 H, CH2C6H5), 4.89–4.99 (m, 2 H, CHvCH2), 5.75–5.85 (m,
77.39; H, 11.28%; 1H NMR (400 MHz; CDCl3) δ 0.82 (d, J = 6.6 Hz, 1 H, CHvCH2), 7.24–7.32 (m, 5 H, C6H5); 13C NMR (100 MHz;
3 H, CH3), 1.03–1.38 (m, 23 H, (CH2)7CH(CH2)4), 1.51–1.58 (m, CDCl3) δ 14.26, 19.87, 22.84, 26.28, 26.31, 27.07, 27.25, 29.15,
2 H, CH2CH2O), 2.00–2.06 (m, 2 H, CH2CHvCH2), 2.44 (d, J = 29.51–29.85, 30.19, 30.29, 32.07, 32.90, 33.96, 37.17, 37.25,
4.2 Hz, 1 H, –OH). 3.41–3.56 (m, 6 H, 3× CH2O), 3.94–4.00 (m, 70.49, 70.69, 70.89, 71.75, 73.45, 78.06, 114.07, 127.44, 127.53,
1 H, CHOH), 4.55 (s, 2 H, CH2C6H5), 4.89–5.00 (m, 2 H, 128.26, 138.48, 139.16; EI-MS m/z 656 (2%, M − H).
CHvCH2), 5.75–5.85 (m, 1 H, CHvCH2), 7.19–7.35 (m, 5 H,
1-O-Benzyl-3-O-[(10RS)-10-methylheptadec-16-en-1-yl]-2-O-
C6H5); 13C NMR (100 MHz; CDCl3) δ 19.77, 26.17, 26.97, 27.13, [(10RS)-methylhexadecyl]-sn-glycerol (19c). Following the
29.05, 29.55–29.71, 30.07, 32.81, 33.87, 37.08, 37.15, 69.61, general procedure, 18b (1.06 g) and 14a (2.43 g) gave 19c
71.47, 71.74, 71.83, 73.50, 114.07, 127.69, 128.40, 138.07, (1.10 g, 67%), colourless oil. [α]D22 −0.6 (c 0.1 g mL−1, CHCl3).
139.23; EI-MS m/z 432.2 (1%, M − H).
C45H82O3 requires C, 80.53; H, 12.32; found: C, 80.24; H,
12.40%; H NMR (400 MHz; CDCl3) δ 0.82 (d, J = 6.4 Hz, 6 H,
2× CHCH3), 0.87 (t, J = 6.8 Hz, 3 H, CH2CH3), 1.05–1.38 (m, 48
H, (CH2)7CH(CH2)4, (CH2)7CH(CH2)5CH3), 1.51–1.63 (m, 4 H,
1
General procedure for the synthesis of 1-O-benzyl-2,3-O,O-
dialkyl-sn-glycerols 19
Compounds 19a–c were synthesised according to the prepa- 2× CH2CH2O), 2.00–2.05 (m, 2 H, CH2CHvCH2), 3.41 (t, J =
ration of compounds 16a,b. KH (2.45 mmol, 0.3 mL 30% sus- 6.6 Hz, 2 H, CH2O), 3.44–3.61 (m, 7 H, 3× CH2O, CHO), 4.54 (s,
pension in mineral oil) was separated from the oil and 2 H, CH2C6H5), 4.89–5.00 (m, 2 H, CHvCH2), 5.75–5.85 (m,
suspended under argon in dry toluene (5 mL). A solution of 1 H, CHvCH2), 7.23–7.32 (m, 5 H, C6H5); 13C NMR (100 MHz;
18a or 18b (2.45 mmol) in dry toluene (15 mL) was added CDCl3) δ 14.29, 19.88, 19.90, 22.87, 26.30, 26.32, 27.08, 27.23,
slowly at r.t. and the mixture was stirred for 20 h at this temp- 27.26, 29.16, 29.66–29.85, 30.20, 30.30, 32.12, 32.91, 32.93,
erature. Afterwards, the bromide 14a and 1-bromohexadecane 33.97, 37.19, 37.26, 37.28, 70.47, 70.70, 70.88, 71.76, 73.45,
(7.6 mmol), respectively, dissolved in dry toluene (10 mL), were 78.06, 114.08, 127.45, 127.54, 128.27, 138.47, 139.17; EI-MS
added and the mixture was refluxed for 12 h. Subsequently, m/z 670 (5%, M − H).
the cooled reaction mixture was stirred vigorously with water
(30 mL). Then, brine (20 mL) was added and the organic layer
was separated. The water phase was extracted two times with
General procedure for the synthesis of 3,3′-O,O-(alkane-1,1′-
diyl)bis(2-O-alkyl-sn-glycerols) 20
toluene (20 mL) and the combined organic layers were dried The olefins 19a–c (1 mmol) were dissolved in dry CH2Cl2
over Na2SO4 and evaporated. The crude products were purified (30 mL) under an argon atmosphere. Then a solution of
by column chromatography using heptane–CHCl3 as the Grubbs first-generation catalyst {[RuCl2(vCHPh)(PCy3)2],
solvent and the gradient technique.
29 mol%, 0.18 g} in dry CH2Cl2 (25 mL) was added dropwise.
1-O-Benzyl-3-O-(heptadec-16-en-1-yl)-2-O-[(10RS)-10-methyl- The mixture was stirred for 24 h under reflux. Afterwards, the
hexadecyl]-sn-glycerol (19a). Following the general procedure, solvent was removed under reduced pressure and the crude
18a (1.02 g) and 14a (2.43 g) gave 19a (1.0 g, 62%), colourless residue was purified by column chromatography using a
oil. [α]D22 +0.4 (c 0.094 g mL−1, CHCl3); C44H80O3 requires C, heptane–CHCl3 gradient. The subsequently performed hydro-
80.42; H, 12.27; found: C, 80.29; H, 12.11%; 1H NMR genation of the double bonds and the removal of the benzyl
(400 MHz; CDCl3) δ 0.82 (d, J = 6.4 Hz, 3 H, CHCH3), 0.87 (t, J = blocking groups were realised in one step. Therefore, the
7.0 Hz, 3 H, CH2CH3), 1.05–1.38 (m, 49 H, (CH2)12, (CH2)7CH- olefins were dissolved in EtOH–EtOAc (50 mL, 1/1, v/v). After
(CH2)5), 1.51–1.64 (m, 4 H, 2× CH2CH2O), 1.99–2.05 (m, 2 H, the addition of Pd(OH)2 (33 mol%, 20% on carbon) the
CH2CHvCH2), 3.41 (t, J = 6.6 Hz, 2 H, CH2O), 3.44–3.64 (m, mixture was stirred under hydrogen (2 atm) at r.t. for 18 h. The
7 H, 3× CH2O, CHO). 4.55 (s, 2 H, CH2C6H5), 4.89–5.00 catalyst was removed by filtration and washed with CHCl3
(m, 2 H, CHvCH2), 5.75–5.85 (m, 1 H, CHvCH2), 7.25–7.32 several times. The combined organic solutions were evapor-
(m, 5 H, C6H5); 13C NMR (100 MHz; CDCl3) δ 14.29, 19.89, ated. The residue was purified by column chromatography
22.87, 26.29, 26.31, 27.22, 27.26, 29.13, 29.32, 29.68–29.85, using the gradient technique and CHCl3–Et2O as the eluent to
30.20, 30.29, 32.12, 32.93, 33.96, 37.27, 70.47, 70.70, 70.88, give the methyl-branched diols 20a–c.
71.75, 73.45, 78.05, 114.07, 127.45, 127.54, 128.26, 138.46,
139.17; EI-MS m/z 656 (1%, M − H).
3,3′-O,O-(Dotriacontane-1,32-diyl)bis{2-O-[(10RS)-10-methyl-
hexadecyl]-sn-glycerol} (20a). Following the general procedure,
1-O-Benzyl-2-O-hexadecyl-3-O-[(10RS)-10-methylheptadec-16- 19a (0.66 g) gave 20a (0.49 g, 88%), a white solid. M.p. 49 °C;
en-1-yl]-sn-glycerol (19b). Following the general procedure, [α]D22 +5.5 (c 0.110 g mL−1, CHCl3). C72H146O6 requires C, 78.05;
18b (1.06 g) and 1-bromohexadecane (2.32 g) gave 19b (1.19 g, H, 13.28; found: C, 78.34; H, 13.26%; 1H NMR (400 MHz;
74%), slight yellow oil. [α]D22 +1.1 (c 0.070 g mL−1, CHCl3). CDCl3) δ 0.81 (d, J = 6.4 Hz, 6 H, 2× CHCH3), 0.86 (t, J = 7.0 Hz,
C44H80O3 requires C, 80.42; H, 12.27; found: C, 80.11; H, 6 H, 2× CH2CH3), 1.04–1.35 (m, 106 H, (CH2)28, 2× (CH2)7CH-
1
12.12%; H NMR (400 MHz; CDCl3) δ 0.82 (d, J = 6.4 Hz, 3 H, (CH2)5CH3), 1.50–1.57 (m, 8 H, 4× CH2CH2O), 2.14 (bs, 2 H,
CCH3), 0.87 (t, J = 7.0 Hz, 3 H, CH2CH3), 1.05–1.38 (m, 49 H, 2× OH), 3.39–3.71 (m, 18 H, 8× CH2O, 2× CHO); 13C NMR
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(100 MHz; CDCl3) δ 14.12, 19.74, 22.72, 26.15, 27.08, 27.11, product was purified by column chromatography using
29.50–29.73, 30.04, 30.13, 31.97, 32.79, 37.13, 63.10, 70.39, heptane–CHCl3 as the eluent giving compound 22b (1.2 g,
70.92, 71.84, 78.32; ESI-MS m/z 1130.3 (M + Na).
97%) as a white solid. M.p. 57–58 °C; C36H74O6S2 requires C,
3,3′-O,O-[(10RS,23RS)-10,23-Dimethyldotriacontane-1,32-diyl]- 64.82; H, 11.18; S, 9.61; found: C, 64.85; H, 11.00; S, 9.48%; 1H
bis(2-O-hexadecyl-sn-glycerol) (20b). Following the general pro- NMR (400 MHz; CDCl3) δ 0.81 (d, J = 6.6 Hz, 6 H, 2× CHCH3),
cedure, 19b (0.66 g) gave 20b (0.45 g, 82%), a white solid. 1.02–1.38 (m, 54 H, CH2 and CH), 1.69–1.76 (m, 4 H,
M.p. 25–26 °C; [α]D22 +7.2 (c 0.110 g mL−1, CHCl3); C72H146O6 CH2CH2O), 2.98 (s, 6 H, SCH3), 4.20 (t, J = 6.6 Hz, 4 H, CH2O);
requires C, 78.05; H, 13.28; found: C, 77.71; H 13.18%; 1H 13C NMR (100 MHz; CDCl3)
δ 19.71 (CHCH3), 25.42
NMR (400 MHz; CDCl3) δ 0.82 (d, J = 6.4 Hz, 6 H, 2× CHCH3), (CH2(CH2)2O), 27.06 and 27.10 (CH2CH2CH(CH3)CH2CH2),
0.87 (t, J = 6.8 Hz, 6 H, 2× CH2CH3), 1.02–1.36 (m, 106 H, 29.04, 29.14, 29.44, 29.56, 29.71, 29.74, 29.96 and 30.05 (CH2),
(CH2)7CH(CH2)12CH(CH2)7, 2× (CH2)13CH3), 1.51–1.58 (m, 8 H, 32.77 (CH), 37.10 and 37.11 (CH2CH(CH3)CH2), 37.38 (SCH3),
4× CH2CH2O), 1.73 (bs, 2 H, 2× OH), 3.40–3.73 (m, 18 H, 8× 63.09 (CH2O); ESI-MS m/z 689.43 (M + Na), 1355.19 (2M + Na).
CH2O, 2× CHO); 13C NMR (100 MHz; CDCl3) δ 14.26, 19.86,
General procedure for the synthesis of 3,3′-O,O-(alkane-1,1′-
22.84, 26.26, 27.25, 29.50–29.89, 30.17, 30.20, 30.24, 32.07,
diyl)bis(1,2-O-isopropylidene-sn-glycerols) 23
32.92, 37.26, 63.16, 70.47, 70.99, 71.92, 78.40; ESI-MS m/z
1130.6 (M + Na).
The suspension of KH (10 mmol, 30%) was separated from
3,3′-O,O-[(10RS,23RS)-10,23-Dimethyldotriacontane-1,32-diyl]- paraffin oil by washing with dry toluene under argon. The
bis{2-O-[(10RS)-10-methylhexadecyl]-sn-glycerol} (20c). Follow- residue of KH was suspended in dry toluene (10 mL). A solu-
ing the general procedure, 19c (0.67 g) gave 20c (0.41 g, 72%), tion of 1,2-O-isopropylidene-sn-glycerol (15; 1.32 g, 10 mmol)
colourless oil. [α]D22 +4.1 (c 0.025 g mL−1, CHCl3). C74H150O6 in dry toluene (10 mL) was dropped into the slurry whilst stir-
requires C, 78.24; H, 13.31; found: C, 78.01; H, 13.08%; ring. The mixture was stirred for a further 18 h at r.t. until the
1H NMR (400 MHz; CDCl3) δ 0.82 (d, J = 6.4 Hz, 12 H, 4× K-salt formation was complete. Afterwards, compounds 22
CHCH3), 0.87 (t, J = 7.0 Hz, 6 H, 2× CH2CH3), 1.05–1.38 (m, 104 (2.5 mmol), dissolved in dry toluene (10 mL), were added and
H, (CH2)7CH(CH2)12CH(CH2)7, 2× (CH2)7CH(CH2)5CH3), the mixture was heated for 20 h under reflux. After cooling to
1.52–1.57 (m, 8 H, 4× CH2CH2O), 3.40–3.72 (m, 18 H, 8× CH2O, r.t., the suspension was poured into a cold saturated NH4Cl
2× CHO); 13C NMR (100 MHz; CDCl3) δ 14.10, 19.71, 22.69, solution (30 mL). The organic layer was separated and the
26.12, 27.06, 27.10, 29.49, 29.63–30.10, 31.96, 32.77, 37.12, aqueous phase was extracted two times with CHCl3 (20 mL).
63.01, 70.37, 70.88, 71.80, 78.36; ESI-MS m/z 1158.1 (M + Na).
The combined organic layers were dried over Na2SO4, evapor-
ated and purified by middle pressure liquid chromatography
(MPLC) using CHCl3–Et2O as the eluent and the gradient tech-
Synthesis of diglycerol tetraethers – reaction pathway II
1,32-Dibromodotriacontane (22a). Compound 22a was syn- nique to give the compounds 23a and 23b.
thesised according to the preparation of 6b from dotriacon-
3,3′-O,O-(Dotriacontane-1,32-diyl)bis(1,2-isopropylidene-sn-
tane-1,32-diol (21a)20 using 21a (2.0 g, 4.1 mmol), 3,4-dihydro- glycerol) (23a). Following the general procedure, 15 (1.32 g)
2H-pyrane (1.25 g, 14.9 mmol) and PPTS. The crude and dried and 22a (1.52 g) gave 23a (1.0 g, 56%), a white solid.
bis(tetrahydro-2H-pyranyl) ethers (85% yield) were sub- M.p. 74–76 °C; C44H86O6 requires C, 74.31; H, 12.19; found: C,
1
sequently reacted with triphenylphosphoranediyl dibromide 73.77; H, 12.19%; H NMR (400 MHz; CDCl3) δ 1.25–1.33 (m,
prepared from Br2 (1.8 g, 11.3 mmol) and triphenylphosphane 56 H, O(CH2)2(CH2)28(CH2)2O), 1.36 and 1.42 (2 s, 2× 6 H,
(2.96 g, 11.3 mmol). The crude product was purified by CH3), 1.53–1.60 (m,
4
H, OCH2CH2(CH2)28CH2CH2O),
column chromatography and heptane as the eluent yielding 3.39–3.53 (m, 8 H, CH2OCH2(CH2)30CH2OCH2), 3.73 (dd, 2J =
22a (2.04 g, 81% compared to 21a, 95% compared to bis(thp) 8.2 Hz, 3J = 6.4 Hz, 2 H, 2× OCH2CH), 4.06 (dd, 2J = 8.2 Hz, 3J =
ether) as a white solid. M.p. 85–86 °C; C32H64Br2 requires C, 6.4 Hz, 2 H, 2× OCH2CH), 4.23–4.29 (m, 2 H, CH); 13C NMR
63.14; H, 10.60; Br, 26.26; found: C, 62.89; H, 10.50; Br, (100 MHz; CDCl3) δ 25.43, 26.06 and 26.78 (CH3, O(CH2)2CH2),
24.25%; 1H NMR (400 MHz; CDCl3) δ 1.24–1.32 (m, 52 H, CH2), 29.47, 29.56, 29.60, 29.61, 29.67, 29.69 and 29.70 (CH2), 66.96
1.37–1.44 (m,
4 H, BrCH2CH2CH2), 1.80–1.87 (m, 4 H, (CH2CHCH2O-alkyl), 71.82 and 71.89 (CH2OCH2(CH2)30CH2
BrCH2CH2), 3.39 (t, J = 6.9 Hz, 4 H, BrCH2f); 13C NMR OCH2), 74.79 (CH), 109.34 (C); ESI-MS m/z 733.51 (M + Na),
(100 MHz; CDCl3) δ 28.19 (Br(CH2)2CH2), 28.78 (Br(CH2)3CH2), 693.50 (M – IP + Na), 653.49 (M – 2IP + Na).
29.45, 29.55, 29.62, 29.66, 29.68, 29.69 and 29.70 (CH2), 32.86
(BrCH2CH2), 34.03 (BrCH2).
3,3′-O,O-[(10RS,23RS)-10,23-Dimethyldotriacontane-1,32-diyl]-
bis(1,2-isopropylidene-sn-glycerol) (23b). Following the general
(10RS,23RS)-10,23-Dimethyldotriacontane-1,32-diylbismethane- procedure, 15 (1.32 g) and 22b (1.67 g) gave 23b (0.79 g, 43%),
sulfonate
(22b). (10RS,23RS)-10,23-Dimethyldotriacontane- pale yellow oil. C46H90O6 requires C, 74.74; H, 12.27; found: C,
1,32-diol21 (21b, 1.0 g, 1.9 mmol) was dissolved in dry CHCl3 74.58; H, 12.21%; 1H NMR (400 MHz; CDCl3) δ 0.81 (d, 3J =
(50 mL). Methanesulfonyl chloride (0.89 g, 7.8 mmol) and 6.8 Hz, 6 H, 2× CHCH3), 1.01–1.10 and 1.20–1.31 (2 m, 54 H,
DMAP (0.358 g, 7.8 mmol) were added and the mixture was CH2, CHCH3), 1.34 and 1.40 (2 s, 2× 6 H, 4× CCH3), 1.51–1.58
stirred at r.t. After complete conversion of compound 21b, (m,
4 H, OCH2CH2-alkyl-CH2CH2O), 3.37–3.51 (m, 8 H,
water was added and the organic phase was separated, washed CH2OCH2-alkyl-CH2OCH2), 3.70 (dd, 2J = 8.4 Hz, 3J = 6.8 Hz,
with water, dried over Na2SO4 and evaporated. The crude 2 H, 2× OCH2CH), 4.03 (dd, 2J = 8.4 Hz, 3J = 6.8 Hz, 2 H,
3658 | Org. Biomol. Chem., 2014, 12, 3649–3662
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2× OCH2CH), 4.21–4.27 (m, 2 H, OCH); 13C NMR (100 MHz; found: C, 81.96; H, 9.38%; 1H NMR (400 MHz; CDCl3)
CDCl3) δ 19.71 (CHCH3), 25.43, 26.06 and 26.77 (CCH3, 1.24–1.31 (m, 56 H, CH2), 1.49–1.56 (m, H,
δ
4
O(CH2)2CH2), 27.08 and 27.10 (CH2CH2CH(CH3)CH2CH2), OCH2CH2(CH2)28CH2CH2O), 2.39–2.40 (bm, 2 H, 2× OH),
29.47, 29.56, 29.59, 29.60, 29.65, 29.71, 29.74, 30.01 and 30.04 3.14–3.21 (m, 4 H, 2× CH2O), 3.37–3.53 (m, 8 H, 4× CH2O),
(CH2), 32.76 (CHCH3), 37.10 (CH2CH(CH3)CH2), 66.96 3.89–3.96 (m, 2 H, OCH), 7.19–7.30 and 7.40–7.43 (2 m, 30 H,
(CH2CHCH2O-alkyl-OCH2CHCH2), 71.82 and 71.89 (CH2OCH2- 6× C6H5); 13C NMR (100 MHz; CDCl3) δ 26.11 (O(CH2)2CH2),
alkyl-CH2OCH2), 74.76 (OCH), 109.32 (C); ESI-MS m/z 761.69 29.51, 29.61, 29.64, 29.67, 29.69, 29.70 and 29.72 (CH2), 31.88
(M + Na).
(OCH2CH2), 64.64 (CH2OTr), 69.87, 71.65 and 72.04
(CHCH2OCH2), 86.64 (C), 127.03 (C-4, C6H5), 127.81 and
128.67 (C-2, C-3, C-5, C-6, C6H5), 143.89 (C-1, C6H5); ESI-MS
m/z 1113.79 (M – H), 1137.78 (M + Na).
General procedure for the synthesis of 3,3′-O,O-(alkane-1,1′-
diyl)bis(sn-glycerols) 24
The IP-protected glycerols 23 and catalytic amounts of PPTS
3,3′-O,O-[(10RS,23RS)-10,23-Dimethyldotriacontane-1,32-diyl]-
were dissolved in dry MeOH (50–100 mL) and heated for 3 h at bis(1-O-trityl-sn-glycerol) (25b). Following the general pro-
reflux. The products crystallised while cooling the suspension. cedure, 24b (0.20 g, 0.30 mmol), trityl chloride (0.21 g,
After filtration, compounds 24 were purified by recrystalliza- 0.76 mmol), and DMAP (5 mg) in pyridine (7 mL) gave 25b
tion from heptane–MeOH.
(0.24 g, 70%), colourless oil. C78H110O6 requires C, 81.91; H,
1
3,3′-O,O-(Dotriacontane-1,32-diyl)bis(sn-glycerol) (24a). Fol- 9.695; found: C, 81.84; H, 9.71%; H NMR (400 MHz; CDCl3)
lowing the general procedure, 23a (1.23 g) gave 24a (1.02 g, δ 0.82 (d, J = 6.6 Hz, 6 H, 2× CHCH3), 1.02–1.10 and 1.17–1.38
93%), a white solid. M.p. 117–119 °C; C38H78O6 requires C, (2 m, 54 H, CH2, CHCH3), 1.49–1.58 (m, 4 H, OCH2CH2-alkyl-
72.33; H, 12.46; found: C, 72.34; H, 12.82%; 1H NMR (400 MHz; CH2CH2O), 2.38–2.40 (bm, 2 H, 2× OH), 3.14–3.21 (m, 4 H, 2×
CDCl3) δ 1.23–1.29 (m, 56 H, O(CH2)2(CH2)28(CH2)2O), 1.45–1.58 CH2O), 3.38–3.53 (m, 8 H, 4× CH2O), 3.89–3.96 (m, 2 H, OCH),
(m, 4 H, OCH2CH2(CH2)28CH2CH2O), 3.43–3.54 (m, 8 H, 7.19–7.31 and 7.40–7.43 (2 m, 30 H, 6× C6H5); 13C NMR
2
3
CH2OCH2(CH2)30CH2OCH2), 3.63 (dd, J = 11.4 Hz, J = 5.4 Hz, (100 MHz; CDCl3) δ 19.72 (CHCH3), 26.11 (O(CH2)2CH2), 27.10
2 H, 2× HOCH2CH), 3.70 (dd, 2J = 11.4 Hz, 3J = 4.0 Hz, 2 H, (CH2CH2CH(CH3)CH2CH2), 29.47, 29.52, 29.60, 29.62, 29.66,
2× HOCH2CH), 3.81–3.86 (m, 2 H, CH); ESI-MS m/z 653.55 29.67, 29.69, 29.72, 29.75, 30.02, 30.04 and 30.06 (CH2), 32.78
(M + Na).
(CH(CH3)), 37.13 (CH2CH(CH3)CH2), 64.64 (CH2OTr), 69.87,
3,3′-O,O-[(10RS,23RS)-10,23-Dimethyldotriacontane-1,32-diyl]- 71.65 and 72.05 (CHCH2OCH2), 86.64 (C), 127.03 (C-4, C6H5),
bis(sn-glycerol) (24b). Following the general procedure, 23b 127.81 and 128.67 (C-2, C-3, C-5, C-6, C6H5), 143.89 (C-1,
(480 mg) gave 24b (360 mg, 85%), a white solid. M.p. 50–51 °C; C6H5); ESI-MS m/z 1141.88 (M – H), 1166.15 (M + Na).
C40H82O6 requires C, 72.89; H, 12.54; found: C, 72.41; H,
1
General procedure for the synthesis of 3,3′-O,O-(alkane-1,1′-
diyl)bis(2-O-alkyl-1-O-trityl-sn-glycerols) 26
12.87%; H NMR (400 MHz; CDCl3) δ 0.81 (d, J = 6.8 Hz, 6 H,
2× CHCH3), 1.02–1.10 and 1.18–1.33 (2 m, 54 H, CH2, CHCH3),
1.52–1.59 (m, 4 H, OCH2CH2-alkyl-CH2CH2O), 3.42–3.55 (m, Compounds 26a,b were synthesised according to the synthesis
8 H, CH2OCH2-alkyl-CH2OCH2), 3.63 (dd, 2J = 11.4 Hz, 3J = of compounds 16a,b – a second O-alkylation on the glycerol
2
3
5.2 Hz, 2 H, 2× HOCH2CH), 3.70 (dd, J = 11.4 Hz, J = 3.6 Hz, moiety. KH (2.5 fold excess, 30% suspension in mineral oil)
2 H, 2× HOCH2CH), 3.82–3.86 (m, 2 H, OCH); ESI-MS m/z was separated from the oil and suspended under argon in dry
657.70 (M – H), 659.52 (M + H), 681.61 (M + Na).
toluene (2 mL). A solution of 25a or 25b in dry toluene (10 mL)
was added at r.t. and the mixture was stirred for 20 h at this
temperature and, in addition, for 2 h at 40 °C. Afterwards, the
bromide 14a (2.5 fold excess), dissolved in dry toluene (2 mL),
General procedure for the synthesis of 3,3′-O,O-(alkane-1,1′-
diyl)bis(1-O-trityl-sn-glycerols) 25
Trityl chloride (2.5 equiv.), glycerol 24a or 24b (1 equiv.), and was added and the mixture was refluxed for 12 h. Sub-
DMAP (10 mol%) were suspended in dry pyridine (7–20 mL). sequently, saturated NH4Cl solution (15 mL) was added and
The suspension was stirred for at least 3 d at r.t. After complete the organic layer was separated. The water phase was extracted
conversion of the glycerol compounds, water (10–20 mL) was two times with Et2O (15 mL) and the combined organic layers
added to hydrolyse the excess trityl chloride. After the addition were dried over Na2SO4 and evaporated. The crude products
of further water (∼20 mL), the organic layer was separated, the were purified by MPLC using heptane as the solvent to give
aqueous phase was extracted with CHCl3 and the combined compounds 26a and 26b.
organic layers were washed with brine. After drying with
3,3′-O,O-(Dotriacontane-1,32-diyl)bis{2-O-[(10RS)-10-methyl-
Na2SO4 and removing the solvent, the raw product was purified hexadecyl]-1-O-trityl-sn-glycerol} (26a). Following the general
by MPLC using CHCl3–heptane and 0.5% TEA as the eluent procedure, 25a (150 mg, 0.134 mmol), KH (13.4 mg,
and the gradient technique to give compounds 25a and 25b.
0.335 mmol, 50 µL), and 14a (107 mg, 0.335 mmol) gave 26a
3,3′-O,O-(Dotriacontane-1,32-diyl)bis(1-O-trityl-sn-glycerol) (100 mg, 47%), colourless oil. C110H174O6 requires C, 82.96; H,
1
(25a). Following the general procedure, 24a (0.92 g, 11.01; found: C, 82.87; H, 10.96%; H NMR (500 MHz; CDCl3)
1.46 mmol), trityl chloride (1.02 g, 3.64 mmol), and DMAP δ 0.86 (d, J = 6.5 Hz, 6 H, 2× CHCH3), 0.90 (t, J = 6.9 Hz, 6 H, 2×
(18 mg) in pyridine (20 mL) gave 25a (0.81 g, 50%), a white CH2CH3), 1.07–1.13 and 1.23–1.37 (2 m, 106 H, CH2, CH),
solid. M.p. 68–70 °C; C76H106O6 requires C, 81.82; H, 9.58; 1.49–1.61 (m, 8 H, 4× OCH2CH2-alkyl), 3.17–3.22, 3.40–3.43
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and 3.50–3.60 (3 m, 18 H, CH2O, OCH), 7.22–7.31 and quarternisation with trimethylamine (1.5 mmol) as described
7.47–7.49 (2 m, 30 H, 6× C6H5); 13C NMR (125 MHz; CDCl3) previously in detail.14,20 The crude bis(phosphocholines)
δ 14.12 (CH2CH3), 19.73 (CHCH3), 22.71 (CH2CH3), 26.13 and were purified by column chromatography using CHCl3–
26.19 (O(CH2)2CH2-alkyl-CH2(CH2)2O), 27.07 and 27.11 (O MeOH–water as the eluent and the gradient technique to give
(CH2)2CH2(CH2)6CH(CH3)(CH2)5CH3), 29.54, 29.57, 29.65, lipids I–III.
29.68, 29.70 and 29.74 (CH2), 30.03 and 30.17
(OCH2CH2(CH2)7 CH(CH3)(CH2)5CH3), 31.97 (OCH2CH2-alkyl- hexadecyl]-sn-glycer-1-yl}-2-(trimethylammonio)ethyl
3,3′-O-(Dotriacontane-1,32-diyl)bis({2-O-[(10RS)-10-methyl-
phos-
CH2CH2O), 32.78 (CHCH3), 37.13 (CH2CH(CH3)CH2), 63.66 phate) (I). Following the general procedure, 20a (166 mg) gave
(CH2OTr), 70.71, 71.22 and 71.62 (CH2O), 78.34 (CHO), 86.52 lipid I (88 mg, 41%), a white solid. M.p. 217–219 °C;
(C), 126.87 (C-4, C6H5), 127.70 and 128.77 (C-2, C-3, C-5, C-6, C82H170N2O12P2 × 2H2O requires C, 66.81; H, 11.90; N 1.90;
C6H5), 144.19 (C-1, C6H5); ESI-MS m/z 818.87 (M + 2Na), found: C, 66.47; H, 11.91; N, 1.89%; 1H NMR (400 MHz;
1615.15 (M + Na).
CDCl3–CD3OD) δ 0.79 (d, J = 6.6 Hz, 6 H, 2× CHCH3), 0.83 (t,
3,3′-O,O-[(10RS,23RS)-10,23-Dimethyldotriacontane-1,32-diyl]- J = 7.0 Hz, 6 H, 2× CH2CH3), 1.00–1.33 (m, 106 H, (CH2)28,
bis{2-O-[(10RS)-10-methylhexadecyl]-1-O-trityl-sn-glycerol} (26b). 2× (CH2)7CH(CH2)5CH3), 1.45–1.52 (m, 8 H, 4× CH2CH2O), 3.21
Following the general procedure, 26a (210 mg, 0.184 mmol), (s, 18 H, 2× N(CH3)3), 3.35–3.57 (m, 14 H, 6× CH2O, 2× CHO),
KH (18.4 mg, 0.459 mmol, 60 µL), and 14a (147 mg, 3.60–3.62 (m, 4 H, 2× NCH2CH2OP), 3.83–3.86 (m, 4 H,
0.459 mmol) gave 26b (170 mg, 57%), colourless oil. 2× POCH2CH), 4.18–4.25 (m, 4 H, 2× NCH2CH2OP); 13C NMR
C
112H178O6 requires C, 83.00; H, 11.07; found: C, 82.67; H, (100 MHz; CDCl3–CD3OD) δ 14.15, 19.75, 22.74, 26.14, 27.10,
1
11.20%; H NMR (500 MHz; CDCl3) δ 0.88 (d, J = 6.5 Hz, 12 H, 27.18, 29.52–29.77, 30.12, 30.20, 31.99, 32.82, 37.16, 37.17,
4× CHCH3), 0.92 (t, J = 6.9 Hz, 6 H, 2× CH2CH3), 1.09–1.13 and 54.52, 59.11, 59.15, 65.16, 65.21, 66.54, 70.54, 71.73, 77.94,
1.24–1.39 (2 m, 104 H, CH2, CH), 1.52–1.63 (m, 8 H, 4× 78.02; ESI-MS m/z 1438.0 (M + H), 1460.9 (M + Na); HRMS m/z
OCH2CH2-alkyl), 3.18–3.24, 3.40–3.47 and 3.51–3.62 (3 m, calcd for C82H170N2O12P2Na2 (M + 2Na) 741.6007; found:
18 H, CH2O, OCH), 7.23–7.32 and 7.49–7.51 (2 m, 30 H, 6× 741.6034; HPLC tR 3.92 min, purity 99.0%.
C6H5); 13C NMR (125 MHz; CDCl3) δ 14.14 (CH2CH3), 19.74
and 19.75 (CHCH3), 22.72 (CH2CH3), 26.15 and 26.20 bis[(2-O-hexadecyl-sn-glycer-1-yl)-2-(trimethylammonio)ethyl
(O(CH2)2CH2-alkyl-CH2(CH2)2O), 27.08 and 27.13 phosphate] (II). Following the general procedure, 20b
3,3′-O-[(10RS,23RS)-10,23-Dimethyldotriacontane-1,32-diyl]-
(O(CH2)2CH2(CH2)6CH(CH3) (CH2)5CH3), 29.57, 29.59, 29.68, (166 mg) gave lipid II (60 mg, 28%), a white waxy solid.
29.71, 29.73, 29.75 and 29.79 (CH2), 30.08 and 30.18 M.p. 215–218 °C; 1H NMR (400 MHz; CDCl3–CD3OD) δ 0.82 (d,
(OCH2CH2(CH2)7CH(CH3)(CH2)5CH3), 31.99 (OCH2CH2-alkyl- J = 6.4 Hz, 6 H, 2× CHCH3), 0.87 (t, J = 7.0 Hz, 6 H, 2×
CH2CH2O), 32.80 and 32.81 (CHCH3), 37.15 and 37.17 CH2CH3), 1.04–1.30 (m, 106 H, (CH2)7CH(CH2)12CH(CH2)7,
(CH2CH(CH3)CH2), 63.68 (CH2OTr), 70.72, 71.23 and 71.63 2× (CH2)13CH3), 1.49–1.55 (m, 8 H, 4× CH2CH2O), 3.23 (s, 18 H,
(CH2O), 78.36 (CHO), 86.54 (C), 126.88 (C-4, C6H5), 127.71 and 2× N(CH3)3), 3.37–3.65 (m, 18 H, 6× CH2O, 2× CHO, 2×
128.78 (C-2, C-3, C-5, C-6, C6H5), 144.21 (C-1, C6H5); ESI-MS NCH2CH2OP), 3.89–3.92 (m, 4 H, 2× POCH2CH), 4.24–4.29 (m,
m/z 832.91 (M + 2Na), 1643.18 (M + Na).
4 H, 2× NCH2CH2OP); 13C NMR (100 MHz; CDCl3–CD3OD) δ
14.05, 19.74, 22.66, 26.08, 26.97, 27.05, 29.33, 29.54–30.08,
31.89, 32.67, 37.01, 54.38, 59.44, 65.42, 66.56, 70.40, 70.55,
General procedure for detritylation
The detritylation reaction is based on a procedure described 71.70; 77.44; ESI-MS m/z 1438.1 (M + H); HRMS m/z calcd for
by Hermetter and Paltauf:22 the trityl blocked compounds were C82H172N2O12P2 (M + 2H) 719.6187; found: 719.6197, calcd for
suspended in dry MeOH. After the addition of methanolic C82H171N2O12P2 (M + H) 1438.2302; found: 1438.2336; HPLC tR
solution of BF3–Et2O, the mixture was stirred at r.t. until the 3.92 min, purity 99.2%.
educt disappeared (TLC). For the work-up, Et2O and water
3,3′-O-[(10RS,23RS)-10,23-Dimethyldotriacontane-1,32-diyl]-
were added, the aqueous phase was neutralised, and the bis({2-O-[(10RS)-10-methylhexadecyl]-sn-glycer-1-yl}-2-(trimethyl-
organic phase was separated, washed with water, dried over ammonio)ethyl phosphate) (III). Following the general pro-
Na2SO4 and evaporated. The crude products were purified by cedure, 20c (170 mg) gave lipid III (84 mg, 38%), a white waxy
column chromatography using heptane–CHCl3 as the eluent solid. M.p. 205–209 °C; 1H NMR (400 MHz; CDCl3–CD3OD)
and the gradient technique.
δ 0.82 (d, J = 6.4 Hz, 12 H, 4× CHCH3), 0.86 (t, J = 7.0 Hz, 6 H,
Following the general procedure, compounds 20a and 20c 2× CH2CH3), 1.02–1.34 (m, 104 H, (CH2)7CH(CH2)12CH(CH2)7,
were synthesised from 26a and 26b in a quantitative manner 2× (CH2)7CH(CH2)5CH3), 1.49–1.55 (m, 8 H, 4× CH2CH2O),
(>95% yield). The analytical data of 20a and 20c are in accord- 3.23 (s, 18 H, 2× N(CH3)3), 3.38–3.64 (m, 18 H, 6× CH2O, 2×
ance with data described above.
CHO, 2× NCH2CH2OP), 3.87–3.89 (m, 4 H, 2× POCH2CH),
4.22–4.26 (m, 4 H, 2× NCH2CH2OP); 13C NMR (100 MHz;
CDCl3–CD3OD) δ 14.09, 19.70, 19.76, 22.70, 26.09, 26.12,
27.03, 27.07, 27.09, 27.14, 29.56–30.15, 31.97, 32.73,
General procedure for the synthesis of bis(phosphocholines)
I–III
For the synthesis of the final bis(phosphocholines) I–III we 32.80, 37.05, 37.08, 37.14, 37.15, 54.47, 58.91, 58.96, 65.06,
used phosphorylation of the diols 20 (0.15 mmol) with 2-bromo- 65.11, 66.59, 70.58, 70.62, 71.76, 78.00 78.08; ESI-MS
ethylphosphoric acid dichloride (1.2 mmol) and a subsequent m/z 1466.3 (M + H); HRMS m/z calcd for C84H176N2O12P2
3660 | Org. Biomol. Chem., 2014, 12, 3649–3662
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(M + 2H) 733.6344; found: 733.6356, calcd for C84H175N2O12P2 and Bioanalytics, Martin-Luther-Universitaet Halle-Wittenberg)
(M + H) 1466.2615; found: 1466.2658; HPLC tR 3.91 min, for the high-resolution mass spectrometry analysis.
purity 97.7%.
DSC. DSC measurements were performed using a MicroCal
VP-DSC differential scanning calorimeter (MicroCal Inc. North-
ampton, MA, USA). The samples were prepared by mixing
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